The prognosis of hepatocellular carcinoma (HCC) is closely associated with recurrence and metastasis which has been proposed to be initiated by circulating tumor cells (CTCs). Yet, the transcriptomic plasticity and immune evasion mechanism of CTCs during circulation are not well defined.
Blood was drawn from 4 different vascular sites, including hepatic vein (HV), peripheral artery (PA), peripheral vein (PV) and portal vein (PoV) of 10 localized HCC patients. Single CTCs were isolated by negative enrichment and robotic micromanipulator, followed by single-cell RNA sequencing (scRNA-seq). After filtering, 113 CTCs with qualified data were subjected to bioinformatics analysis. The scRNA-seq results were further validated in three independent HCC cohorts.
Our scRNA-seq data revealed remarkable intra- and inter-vascular heterogeneity among CTCs from four vascular sites. We determined CTC transcriptional dynamics during transportation through consecutive vascular sites and revealed their adaptation mechanisms under biomechanical stress. We further classified CTCs from different vascular sites into two subsets, namely dormant and activated CTCs. Dormant CTCs were associated with a non-cycling state and upregulation of EMT/angiogenic signatures and showed stronger prognostic ability for early relapse than activated CTCs. Furthermore, we discovered an immune escape mechanism by which CTCs recruited regulatory T cells (Tregs) via expression of CCL5, consequently promoting the formation of an immunosuppressive microenvironment favorable for their survival in bloodstream and distant colonization.We proved that MAX, activated through the p38 pathway, was the key transcriptional factor regulating CCL5 overexpression, which was validated by ChIP, luciferase reporter gene and in vitro/vivo knockdown assays. And we further determined that Tregs-derived TGF-β1 can heighten MAX expression, thus amplifying the CCL5 expression.
Collectively, our results reveal a previously unappreciated spatial heterogeneity of CTCs and a CTC immune-escape mechanism, which may aid in designing new anti-metastasis therapeutic strategies in HCC.
Jia Fan.
The State Key Program of National Natural Science of China.
All authors have declared no conflicts of interest.
Up to date, there isn’t current method or parameter that allows identifying long survivors in treatment with immunotherapy (IT) in a simple and accessible way. We made a prospective study of the usefulness of quantifying circulating tumor cells (CTCs) and CTCs/PDL1+ in patients treated with immunotherapy.
Patients, diagnosed with non-small cell lung cancer and in second-line treatment with IT were analyzed prospectively. CTCs from peripheral blood samples were isolated by double density gradient and immunomagnetic separation with AutoMACS equipment (M.Biotec). Quantification of CTCs was performed by Cytometry and Confocal Microscopy. The combination of both methodologies allows greater sensitivity and specificity. Samples were acquired in a MACSQuant cytometer (M.Biotec) and TCS SP5 Confocal Microscope (Leica).Data analysis was performed using MACSQuantify and LASF Lite. Determination of CTCs was made at the beginning of the treatment and every 3 months and up to 12 months during IT, together with radiological evaluation in each extraction. We selected those patients who had no progression of the disease for at least 12 months.
Determination of CTCs alone did not allow us to obtain any pattern of recurrence or response. However, we were able to identify 7 patients in the group of long survivors (> 12 months of treatment with IT) using the marker PDL1. The study of their CTCs showed that none of them had circulating CTCs+/PD-L1+. Two of these patients were PDL1+ in tissue sample but negative CTC/PDL1 in blood. We also observed the presence of non-tumor WBC (white blood cells)/PDL1+, but their meaning is not clear in patients with relapse. To emphasize, another patient PDL1 + in tissue, CTC/PDL1+ in serum and low number of WBC, showed positive imaging (PET) of the progression of the disease and its CTC number increased in later determinations.
The absence of circulating CTCs/PDL1 + can predict a sustained response to long-term IT. Isolated CTCs without quantifying their associated expression of PDL1 are not associated with a particular pattern nor appear to be useful in identifying long survivors. WBCs that express PDL1 were associated with the appearance of relapse. Larger studies are needed to validate our results.
Hospital Universitario Puerta de Hierro Majadahonda.
Has not received any funding.
All authors have declared no conflicts of interest.
The PD-1 inhibitors nivolumab and pembrolizumab are currently widely used as treatment of advanced melanoma. While being tolerated very well by some patients, they cause severe and even fatal immune related adverse events (irAEs) in others. Factors associated with increased risk of toxicity have barely been identified. With the perspective of long-term survival in stage IV disease, and increasing numbers of patients receiving adjuvant immunotherapy in stage III melanoma, being able to predict severe (≥grade 3) toxicity becomes more and more important. In this study, we aimed to assess factors associated with severe irAEs in clinical data and routine peripheral blood tests.
Patients with advanced (stage III-IV) melanoma, treated with nivolumab or pembrolizumab with at least 3 months follow-up were included. Demographical parameters together with lactate dehydrogenases (LDH), C-reactive protein (CRP), leukocytes, eosinophils, neutrophils and lymphocytes at baseline and at last measurement before toxicity were retrieved retrospectively. IrAEs were graded according to CTCAE 4.03. Non parametric tests were used to assess the difference between groups.
60 patients treated with anti-PD1 monotherapy were included, of whom 7 developed ≥grade 3 irAEs. Median CRP at baseline was significantly higher in patients that experienced severe irAEs (25mg/L) compared to patients who did not (3 mg/L; p = 0,038). Furthermore, in patients with severe irAEs, median eosinophil level at last measurement before toxicity (0,27x109/L) was significantly increased compared to baseline (0,15x109/L; p = 0,047).
Our data indicate that baseline CRP is a potential predictor of severe anti-PD1 induced toxicity. If confirmed in a larger study, CRP could play a role in clinical decision making at the start of treatment. Furthermore, our observation that increase of eosinophils precedes ≥grade 3 toxicity underpins its possible role in the pathogenesis of irAEs and could help identify and treat irAEs at an early stage.
University Medical Center Utrecht.
Has not received any funding.
K. Suijkerbuijk: Consulting/advisory relationship: Bristol-Myers Squibb, MSD; Honoraria (paid to institution): Novartis, Roche. All other authors have declared no conflicts of interest.
Despite the overall survival (OS) benefit, only 18-20% of aNSCLC patients respond to immune-checkpoint inhibitors (ICI) as second-line therapy with a median progression-free survival (mPFS) of 2-4 months. The identification of predictive and prognostic biomarkers to select patients most likely to respond to ICI is greatly needed in guiding clinical practice.
We conducted a retrospective monocentric analysis of 154 aNSCLC patients receiving single-agent Nivolumab or Pembrolizumab as second-line (68%) and >3rd line (32%). We collected complete blood cell count at baseline and evaluated LDH, absolute neutrophil count (ANC), lymphocyte count (ALC), monocyte count (AMC) and eosinophil count (AEC) and their ratio such as neutrophil-lymphocyte ratio (NLR), derived-NLR (dNLR) and lymphocyte-monocyte ratio (LMR). Univariate and multivariate analyses were performed to identified indipendent predictors factors for immunotherapy (using Kaplan–Meier and Cox Progression analyses).
The multivariate analysis on clinical factors showed the negative predictive role of ECOG PS 2 and liver metastasisand the positive predicitive role of smoking status. The multivariate analysis for PFS showed the negative predictive role of higher ANC (>6000/mL) and LDH (>400 mg/dl) and positive predictive role of higher ALC (>2200/mL). Also, according to stepwise regression analyses, NLR>4 playsa negative predictive and prognostic role at baseline. Finally, five predictive clinical and blood biomarkers at baseline (smoking status, ECOG PS, liver metastases, LDH and NLR), were used to create a predictive score for immunotherapy. Three predictive groups were defined as high, intermediate and low with a mPFS of 10.2 vs 4.9 vs 1.7 months respectively (HR 4.18 95% IC 2.64–6.62, p < 0.001).Predictive Factor Assessment Point ECOG PS 0-1 2 0 1 Smoking (pack-years) > 43 < 43 0 1 Liver metastases No Yes 0 1 LDH (mg/dl) < 400 > 400 0 1 NLR < 4 > 4 0 1 Predictive groups (Points): 1 = 0 2 = 1-2 3 = 3-5 PFS (months): 10.2 4.9 1.7 HR 4.18 95% IC (2.64 – 6.62) p < 0.001
In advanced NSCLC patients treated with second-line immunotherapy, the identification of five and predictive clinical and blood biomarkers at baseline, combined in a predictive score, may help identify patients most likely to benefit from immunotherapy.
Clinical Cancer Center Giovanni Paolo II, Bari, Italy.
Has not received any funding.
G. Domenico: Advisory board: Bristol-Myers Squibb. All other authors have declared no conflicts of interest.
Tumor microenvironment (TME) is a pitch for multiple players, where the crosstalk between tumor and immune cells determines the fate of tumor progression. In fact, immune system may either destroy or paradoxically promote cancer growth, by recruiting immunosuppressive and inflammatory cells. MLR and LDH levels could be dynamic biomarkers that provide indirect information about TME and are associated with poor prognosis in many tumors. Therefore, we evaluated their prognostic impact in mCRC pts.
We conducted a retrospective cohort study evaluating consecutive data of 165 mCRC pts treated in 2004-2017 at the Unit of Medical Oncology and Oncology Prevention, CRO of Aviano. The prognostic impact of MLR and LDH levels on overall survival (OS) was analyzed through uni- and multivariate Cox regression analysis.
At median follow-up of 61.87 months, median OS was 30.74 months. Overall, 100 pts (62%) were aged <65, 63 pts (39%) had a left tumor, 9 (6%) had a BRAF mutation and 45 (28%) had a KRAS mutation, underestimated for the missing data. High levels of LDH (>480 U/L) and MLR (>0.44) were discovered in 35 (21%) and 40 pts (24%), respectively. By univariate analysis, resection (HR 0.25, P < 0.001, 95% CI 0.14-0.45), metastasectomy (HR 0.46, P < 0.001, 95%CI 0.30-0.70) and sidness (left tumors: HR 0.58, P = 0.035, 95%CI 0.36-0.96) were associated with better OS. Conversely, older age (HR 1.63, P = 0.014, 95%CI 1.10-2.42), KRAS mutation (HR 1.77, P = 0.017, 95%CI 1.19-2.85), LDH or MLR high (HR 2.92, P < 0.001, 95% CI 1.77-4.79) or both (HR 4.02, P < 0.001, 95%CI 1.93-8.37) were associated with worse OS. By multivariate analysis, metastasectomy (HR 0.53, P = 0.04, 95%CI 0.29-0.97), KRAS mutation (2.10, P = 0.014, 95% CI 1.16-3.79), LDH or MLR high (HR 3.05, P < 0.001, 95%CI 1.68-5.55) or both (HR 2.65, P = 0.039, 95%CI 1.05-6.68) confirmed their impact on OS. Interestingly, high MLR was associated with right and rectum tumors P = 0.007).
We showed that high baseline LDH, MLR or both are poor prognostic factors in mCRC pts adding further evidence of the interlink between immune system, inflammation and cancer. However, further prospective and translational studies are needed to better deepen this topic.
CRO, IRCCS, National Cancer Institute of Aviano.
Has not received any funding.
All authors have declared no conflicts of interest.
Anti-PD1 therapy has proven to be effective in various cancer types, but the overall impact of the treatment on the immune system is not yet fully understood. Therefore, we aimed in this study to discover the effects of anti-PD1 therapy on the immune system, especially on NK and NKT cells, which are less studied, but known to be involved in antitumor immune events.
Peripheral blood from immuno-oncology naive metastatic melanoma patients (n = 19) were obtained before the first infusion of anti-PD1, then 1 and 3 months later. From each time-point, complete blood counts (CBC) were obtained, and comprehensive immunophenotyping of NK, NKT, and T cells was performed. Also, 92 serum cytokines were measured using the Olink inflammation panel.
Patients were categorized into two cohorts: responders (R, n = 6, PFS=17.0 months) and progressive disease (PD, n = 9, PFS=5.0 months). 4 patients were excluded due to challenging clinical evaluation of response. CBC indicated a significant decrease in the mean frequency of lymphocytes in PD but not in the R cohort. The responders had also higher frequency of lymphocytes at 1- and 3-month time points and lower frequency of neutrophils before initiation and after 1 and 3 months of treatment. The CBC absolute counts revealed that the responders had less neutrophils and monocytes after 3 months of treatment when compared to PD. Immunophenotyping revealed no changes in the frequency of T and NK cells during treatment, however the frequency of NKTbrightcells increased significantly. Moreover, R had more NKTdimcells before and after 3 months of therapy. Also, cytotoxic CD56dimNK cells expressed increased CD25 and CD45RO after 1 month of therapy. The cytokine assay indicated that anti-PD1 significantly increased the levels of CXC-family cytokines in the serum; CXCL9, CXCL11, CXCL10. Also, IL-12B and TNFRSF9 levels were increased. Further comparison of the two cohorts showed that CXCL9 was only increased in R cohort.
Anti-PD1 therapy increases the levels of NKT cells, inflammation related chemokines in blood and the expression of markers linked to enhanced cytotoxicity of NK cells. Therefore, further studies to investigate the role of NK and NKT cells in anti-PD1 induced responses are warranted.
Anna Kreutzman.
Academy of Finland, Cancer Society of Finland, Sigrid Juselius Foundation.
M. Hernberg, S. Mäkelä, L. Kohtamäki: Trial funding: MSD, BMS, Amgen. S. Mustjoki: Honoraria, research funding: Novartis, Bristol-Myers Squibb, Ariad, Pfizer. All other authors have declared no conflicts of interest.
Recent studies have reported that serum microRNAs (miRNAs) are potentially useful cancer biomarkers. In patients with gastric cancer (GC), the efficacy of nivolumab (Nivo), a PD-1 inhibitor, was shown in a phase III study, but predictive biomarkers of Nivo for gastric cancer have not been found. We investigated whether serum miRNAs could be predictive markers of the efficacy of Nivo in GC.
The subjects of this study were 20 patients who were enrolled in the phase III study (ONO-4538-12) and received Nivo 3 mg/kg IV Q2W at our institution. Expressions of 2565 miRNAs in the serum samples before and during treatment were analyzed using “3D-Gene” Human miRNA Oligo Chip (Toray Industries, Inc.). We explored miRNAs that were significantly associated with the treatment response using receiver operating characteristic analysis and Cox regression analysis.
Median progression-free survival (PFS) was 2.3 month, and partial response was achieved in four of the 20 patients who received Nivo. Serum samples before (n = 20) and 4 weeks after (n = 17) the treatment of Nivo were available. Two different miRNAs, one before and the other after treatment, were identifiedwhich were related to response to Nivo. AUC of miR-A identified before treatment was 0.88, whereas that of miR-B after the first treatment was 0.85. The overall response rate (ORR) was 44% (4/9) in miR-A-high patients and 0% (0/11) in miR-A-low patients. The ORR was 50% (4/8) in miR-B-high patients and 0% (0/9) in miR-B-low patients. Median PFS was 14.3 m (95%CI: N/A-35.1) in miR-A-high patients and 1.6 m (95%CI: 0.9-2.3) in miR-A–low patients (HR = 0.19, log rank: p = 0.01), and those were 5.6 m (95%CI: N/A-35.1) and 1.6 m (95%CI: 0.9-2.3) in miR-B-high patients (HR = 0.21, log rank: p = 0.01), respectively.
The two miRNAs in GC pts may be a predictive marker for identifying pts who derived greater benefit from Nivo therapy.
Ken Kato.
Has not received any funding.
S. Takizawa: Personal fees: Toray Industries, Inc., outside the submitted work. O. Takahiro: Grants: Kewpie Corporation, Takeda, BioMimetics Sympathies, Rohto Pharmaceutical Co., Ltd., Inter Stem, Japan Atherosclerosis Research Foundation, Ono Pharmaceutical Co., Ltd., Daiichi-Sankyo Company. All other authors have declared no conflicts of interest.
Treatment with immune checkpoint inhibitors (ICI) prolongs overall survival (OS) and confers long-term disease control in 15-20% of non-small cell lung cancer (NSCLC) patients. Patient selection currently depends on the levels of PD-L1 expression, but correlation with outcome is weak.
We retrospectively analyzed the clinical course of ICI-treated stage IV NSCLC patients at our institution.
A total of 453 patients were identified with a median age of 64 years having received nivolumab (57%), pembrolizumab (35%), PD-L1 inhibitors (7%) or PD1 blockade in combination with chemotherapy or CTLA4 inhibitors (1%). Progression-free survival (PFS) under ICI was significantly longer for patients receiving ICI in the first (21%, 7 months in median) compared to second (47%, 4 months in median) and later treatment lines (2 months in median, p < 0.001), for current and ex-smokers (91% of cases, p = 0.034), in case of adenocarcinoma (66%) compared to squamous cell carcinoma (28%) and other histologies (9%, p < 0.001), while age, sex and ECOG status at initial diagnosis had no influence. The total number of metastatic sites and the presence of liver metastases at start of IO treatment were associated with shorter ICI responses (p = 0.018 and p = 0.005, respectively), while metastases to other organs, especially brain, did not play a role. Blood markers, like the Lymphocyte-to-Neutrophile-Ratio (LNR) as well as CRP and LDH as indicators of inflammation and tumor load, had the highest discriminatory value (≥ 2x longer median PFS for cases with higher LNR or lower CRP or LDH, p < 0.0001 for each), while the occurrence of immune-related adverse events (irAE) conferred longer PFS (p < 0.001) as well as overall survival (OS) from the start of IO treatment (p < 0.01). PFS under ICI was similar for cases with PD-L1 <1% vs. 1-49% (14% and 34%, respectively), while cases with PD-L1 expression >50% had an ICI-PFS twice as long (p = 0.011).
Several clinical and blood parameters appear to correlate with ICI benefit better than tissue PD-L1 expression levels in NSCLC patients and could be used along with molecular markers to improve predictive tools for lung cancer immunotherapy.
Thoraxklinik Heidelberg.
Has not received any funding.
F. Bozorgmehr: Research funding: BMS; Travel grants: BMS, MSD. J. Kuon: Research funding: AstraZeneca, Celgene. C. Heussel: Consultation, lecture and other fees: Novartis, Siemens, Chiesi, Intermune, MEDA Pharma, Bracco, Pfizer, MSD, Roche, Lilly, AstraZeneca, Schering-Plough, Essex, Gilead; Ownership of GSK stocks. F. Herth: Advisory board fees and honoraria: Lilly, Roche, AstraZeneca, Novartis, Boehringer, Chiesi, Teva, Pulmonx BTG, Olympus; Research funding: Lilly, Roche, AstraZeneca, Novartis, Boehringer, Chiesi, Teva. T. Muley: Research funding, patents: Roche. A. Stenzinger: Advisory board honoraria: BMS, AstraZeneca, ThermoFisher, Novartis; Speaker’s honoraria: BMS, Illumina, AstraZeneca, Novartis, ThermoFisher, MSD, Roche; Research funding: Chugai. M. Thomas: Advisory board honoraria: Novartis, Lilly, BMS, MSD, Roche, Celgene, Takeda, AbbVie, Boehringer; Speaker’s honoraria: Lilly, MSD, Takeda; Research funding: AstraZeneca, BMS, Celgene, Novartis, Roche. All other authors have declared no conflicts of interest.
Cumulated clinical experience with checkpoint inhibitors (CPIs) points to a strong need for the identification of predictive biomarkers. Surprisingly, the potential role of the host has not been advocated so far. We developed a custom designed panel of single nucleotide polymorphisms (SNPs) from genes potentially implicated in the response to CPIs.
We studied 94 patients treated in Centre Antoine Lacassagne (Nice, France) with CPI (anti PD-1/PD-L1). High-throughput genotyping of germinal DNA was performed by MassARRAY ImmunoCarta (AGENA Bioscience®) using a custom-panel of 173 SNPs across 90 selected genes (minor allelic frequency ≥5% in the Caucasian population). All tested SNPs were in Hardy-Weinberg equilibrium, and linkage disequilibrium analyses were performed (r2>0.8). A Ridge-penalized logistic regression with 5-fold cross validation was used for the SNP identification to predict objective response (complete or partial response) to treatment.
Median age of patients was 68 (range: 32-85), 67% were male, 51% had non-small cell lung carcinoma (NSCLC), 14% had head and neck squamous cell carcinoma (HNSCC), 15% had renal cell carcinoma (RCC), 13% had melanoma and 7% had another cancer type. Median follow-up was 16.3 months (95%CI: 12.5-18.3). The following SNPs’ decreasing intrinsic weight according to response were selected by the multivariate modeling approach (CCR2: rs1799864, FAS: rs2234767, CD3G: rs3753058, CTLA4: rs5742909, CCL2: rs13900, TNXB: rs12153855, Il1RN: rs419598, PD1: rs11568821, IL17A: rs2275913, IL12B: rs3212227, TLR3: rs7668666, CXCR3: rs2280964, IL10: rs1800871, IL6: rs2069837, TRAF3: rs7145509, VEGFR3: rs307821). In the training set, the accuracy was 0.87 (95%CI: 0.76-0.95; p < 0.001) associated with a sensitivity and a specificity of 0.90 and 0.83, respectively, with a ROC curve AUC at 0.93 (95%CI: 0.87-0.99). In the validation set, the accuracy decreased to 0.71 (95%CI: 0.52-0.85; p < 0.02) associated with a sensitivity and a specificity of 0.82 and 0.60, respectively, and with a ROC curve AUC of 0.85 (95%CI: 0.72-0.98).
These preliminary results point to the feasibility of a signature based on host characteristics for predicting response to CPI.
Centre Antoine Lacassagne, Nice, France.
Has not received any funding.
G. Milano: Paid scientific consultant: Agena Bioscience. S. Shell, R. Everts: Employee of Agena Bioscience. All other authors have declared no conflicts of interest.
Only a fraction of patients with metastatic disease respond to immune checkpoint inhibitors (ICI). Responders are more likely to be defective in mismatch repair genes (MSI+). MSI+ patients are over-represented in the group of tumors with high densities of CD8 T-cells. Patients with High T-cell infiltration have a higher expression of PD-1 and PDL1, and are more likely to respond to ICI. These observations suggest that the response to ICI is strongly dependent on the presence of an established in situ adaptive immune reaction (Mlecnik Immunity 2016). MSI + colon cancer stage IV patients are eligible to ICI. However this only concerns a minority of patients. Taking into account the evaluation of the in situ immune reaction in the primary tumor together with MSI status could clarify and extend the range of patients eligible to ICI.
Immunoscore is a robust and validated IVD test measuring the host immune reaction (CD3+ and CD8+ cells) at the tumor site. Immunoscore classifies patients’ tumors into High, Intermediate or Low. We investigated the Immunoscore (IS) distribution of primary tumors of UICC-TNM stage I-III patients who experienced metachronous metastase(s) from the Immunoscore international validation cohort (Pagès The Lancet 2018). MSI status was assessed with the molecular new Bethesda panel and by IHC.
We recorded 1579 UICC-TNM stage I/II/III patients with available MSI status and IS. 318 patients (20%) experienced a relapse. Among those patients, 36 patients (11%) were MSI+ whereas the vast majority of them (89%; 282 patients) were MSS. 43 patients (13%) were IS High, 136 patients (43%) were IS Intermediate and 139 patients (44%) were IS Low. Importantly, 33 patients (12%) were classified IS High in MSS tumors. Combining MSI+ status with IS high to select patients for ICI extends from 11% to 22% the patients eligible to such immunotherapy.
The proportion of metastatic colon cancer patients eligible for ICI could be refined and extended by the characterization of their primary tumor immune infiltrate based on the Immunoscore status, as suggested by Le et al (Cancer Immunol Res 2017). Interventional trials are now needed to validate the predictive value of Immunoscore.
Society for Immunotherapy of Cancer SITC.
French National Institute of Health and Medical Research, LabEx Immuno-oncology, Transcan ERAnet Immunoscore European project, Association pour la Recherche contre le Cancer, CARPEM, AP-HP, Institut National du Cancer, Italian Association for Cancer Research, national grants, Society for Immunotherapy of Cancer.
J. Galon: HalioDx Co-founder /SAB; Consult/ Grts: PE, IObiotech, MedImmune, Janssen, BMS, AstraZeneca, Novartis, Definiens, Merck Serono, IObiotech, Nanostring, Illum., Northwest, Actelion, Amgen, Kite Pharma, Roche, GSK, Compugen, Mologen. F. Hermitte: Employee and co-founder: HalioDx. M. Roehrl: Consulting/Advisory roles: Member, Scientific advisory board: Proscia, Baltimore, MD - Member, Scientific advisory board, Trans-Hit, Montreal, QC - Advisor, Gerson Lehrman Group F.M. Marincola: Employee: Abbvie. B. Fox, F. Pagès: Immunoscore Patent (INSERM) co-inventor. All other authors have declared no conflicts of interest.
Lung cancer (LC) cells frequently express programmed death-ligand 1 (PD-L1). Although this expression grossly correlates with likelihood of response to checkpoint inhibitors, prediction of response is rather imperfect and, thus, more accurate predictive biomarkers are mandatory. The aim of this study was to investigate the association of immune checkpoint PD-L1 and DNA methylation status of DNA repair genes (RAD51B and XXRC3) as well as vimentin (VIM) expression in non-small cell lung cancer (NSCLC), correlating with patients' outcome.
A cohort of NSCLC patients diagnosed between August 2014 and June 2017 were enrolled after informed consent. Expression of PD-L1 was determined by IHQ. Evaluation of the methylation status of DNA repair genes (RAD51B and XRCC3) and VIM expression was performed by quantitative methylation-specific PCR (qMSP) using SybrGreen methodology. Predictors of PD-L1 expression were determined using logistic regression multivariable models. Impact on overall survival was determined using Cox analysis.
A total of 75 patients with NSCLC were assessed for PD-L1 immunoexpression, 60 (80%) were male and the median age was 64 years old (range: 29-88). Fifty patients (66.7%) presented adenocarcinoma, 24 (32%) squamous cell carcinoma and one NSCLC NOS. Thirty-nine (52%) cases depicted positivity for PD-L1. RAD51B promoter methylation levels and VIM expression were significantly higher in PD-L1 positive cases compared to negative group (p = 0.01). This significant association was maintained in multivariable analyses: per each unit increase in RAD51B promoter methylation level and VIM expression, the OR for PD-L1 expression was 20.4 (CI95%: 1.5-275.2) and 1.23 (CI95%: 1-1.4), respectively. Association between XXRC3 promoter methylation and PD-L1 expression was not found. None of the analyzed markers associated with patients’ overall survival.
Herein, higher RAD51B methylation levels and VIM expression are independently associated with PD-L1 immunoexpression. Further studies with an extended cohort and follow-up period are warranted to validate these results.
Instituto Português de Oncologia do Porto.
Instituto Português de Oncologia do Porto.
All authors have declared no conflicts of interest.
Very few data exist regarding the immune profile of gliomas. Our aim was to study different immunological parameters according to IDH1 mutational status and histologic grade.
Patients with grade II to IV gliomas were prospectively enrolled. Immunohistochemistry (IHC) was used to quantify CD4+ and CD8+ T cells and to study IDH1 and PDL1 (> 5%) expression on tumor cells. These parameters were correlated to mRNA expression of 20 immune-related genes (CSF1R, CTLA4, CXCL9, CXCL10, CXCL13, FOXP3, GZMA, HLADRA, IDO1, INFG, PD1, PDL1, PDL2, PRF1, STAT1, STAT3, TIGIT, TIM3, VISTA, TLR7) according to IDH1 status and histologic grade.
Between February 2017 and July 2018, 20 patients (pts) were enrolled, 14 glioblastomas (GB: 9 IDHWT, 5 IDH1MUT) and 6 lower-grade gliomas (LGG: 2 IDHWT, 4 IDHMUT). Among 18 pts: PDL1 IHC was positive (PDL1IHC+) in 10 GB (3 IDH1MUT) and 1 LGG (IDH1MUT); CD4+ T cell infiltration (intratumoral (IT)/perivascular(PV)) in 5 GB (2 IDH1MUT); CD8+ T cell infiltration in 12 GB (8 IT+PV, 3 IT, 1 PV) and 5 LGG (1 IT, 4 IT+PV). PDL1IHC+ more frequent in GB + LGGIDH1wt vs LGGIDH1mut (P = 0.06). PDL1IHC+ tumors shoed lower expression of STAT1 (P = 0.042), TIM3 (P = 0.02) and TLR7 (P = 0.06). Tumors with > 20 CD8+ T cells/HPF showed higher expression of PD1 (P = 0.03), PRF1 (P = 0.03) and STAT1 (P = 0.03).
Histologic grade and IDH1 status identify glioma groups with different immunophenotypes. This results should be confirmed in a larger sample.
Santiago Cabezas.
GEINO (Grupo Español de Investigación en Neuro-Oncología).
All authors have declared no conflicts of interest.
Pancreatic ductal adenocarcinoma (PDAC) is characterized by an excessive amount of desmoplastic stroma seeded with inflammatory cells and it is one of the most aggressive forms of cancer with no current specific therapies. Tumor-associated macrophages (TAMs) are a major component of the tumor microenvironment (TME), and in most solid cancers increased TAM infiltration is associated with a poor prognosis. TAMs can be described as classically activated M1 types with pro-inflammatory antitumor functions, versus alternatively activated M2 types with immunosuppressive pro-tumor functions. The immunosuppressive functions of M2 TAMs can be exerted through release of cytokines and growth factors as well as via direct recruitment of T regulatory cells (Tregs), a subset of lymphocytes responsible for immune tolerance of the system to the tumor. While the differentiation from M1 to M2 in PDAC has been shown to be associated with a worse prognosis, little is known about PDAC TAM polarization and its potential correlation to Treg recruitment.
For this study we have used MultiOmyx™, a multiplexed immunofluorescence (IF) assay allowing up to 60 protein biomarkers to be interrogated from a single FFPE section. The MultiOmyx™ assay utilizes a pair of directly conjugated Cyanine dye-labeled (Cy3, Cy5) antibodies per round of staining. Each round of staining is imaged and followed by novel dye inactivation chemistry, enabling repeated rounds of staining and deactivation.
Using the pan macrophage marker CD68 in combination with either M1 marker HLA-DR or M2 marker CD163 we confirmed the presence of M1 and M2 TAM populations in 9 stage IIB PDAC FFPE samples and found a positive significant correlation (Pearson’s correlation p < 0.05) between the presence of M2 TAMs and Tregs, but not between M1 TAMs and Tregs. Moreover, in a spatial nearest neighbor analysis we found M2 type TAMS to be in closer proximity to Tregs compared to M1 type TAMs.
We demonstrate a positive significant correlation between the presence of, as well as the distance between, M2 TAMs and Tregs in the TME of PDAC, suggesting a possible pathway in which TAM polarization plays an immunosuppressive function by recruiting Tregs.
NeoGenomics.
NeoGenomics.
A. Juncker-Jensen, J. Fang, R. Padmanabhan, E. Parnell, J. Kuo, Q. Au, E. Leones, F. Sahafi, M. Nagy, N. Hoe, J. William: Employed by NeoGenomics.
Disbalance between the activities of different immunosuppressive and immunostimulating populations of regulatory lymphocytes of innate and adaptive immune compartments (such as CD4 Treg, CD8 Treg, NKreg, NK-like T, Tgammadelta) may occur as a result of chronic infection and be the cause of an altered expression of immune checkpoints which control the level of activation and apoptosis in effector lymphocytes. Immune checkpoints and apoptosis are being increasingly recognized as crucial mechanisms of tumor-immunity interaction during cervical cancer progression; however, their involvement in the induction of cervical neoplasia invasion is not well-characterized.
Peripheral blood (PB) and fresh cervical tissue samples were obtained from women with high-grade cervical intraepithelial neoplasia (CIN) or cervical cancer stage IA; PB samples from healthy women were used as control. Subsets of PB lymphocytes were phenotyped using multicolor flow cytometry.
Coordinated changes of expression pattern of early apoptotic markers (CD95/CD95L, Annexin V, caspase-3) and immune checkpoint molecules (CD279, CD366, CD223) in PB populations of regulatory and conventional/effector CD4 and CD8 T lymphocytes distinguished by the level CD25 and CD127 expression were observed in patients microinvasive cancer and preinvasive CINs. Analysis of differentiation markers NK-like T, Tgammadelta and NK cells within CD3CD56 subsets indicated the involvement of the innate arm in growing immune disbalance during microcarcinoma development. Interestingly, analysis of transcriptomic data showed no significant increase in the expression of immune checkpoint-related and T/NK cell-activation pathways in samples of frankly invasive cancer compared to preinvasive neoplasia, while Treg differentiation pathway was upregulated.
The data suggest that markers and PB lymphocytes analyzed may coordinately contribute to the formation of invasive capacity of cervical neoplastic lesions.
Petrozavodsk State University.
Russian Science Foundation (project no: 17-75-10027).
All authors have declared no conflicts of interest.
The phosphorylation of the androgen receptor (AR) at various sites has been associated with diverse functions and clinical outcomes in prostate cancer and the processes underlying phosphorylation at S81 (pAR81) and S213 (pAR213) may possibly be targeted therapeutically. In addition, inflammation upregulates AR and cancer-promoting pathways, and high CD3+ and CD8+ T-cells in the tumour microenvironment have been associated with poorer outcomes. Therefore, this study aims to assess the prognostic value of tumour markers – AR, immune cell infiltrate and proliferation – and develop a combined prognostic score.
Immunohistochemistry was performed for 243 patients with prostate cancer to assess AR, pAR81, pAR213, Ki67, CD3 and CD8. AR expression was evaluated separately based on localisation: tumour or stromal cell, nuclear or cytoplasmic, with nuclear/cytoplasmic ratio (N/C) calculated. These tumour markers were assessed with overall survival and other biomarkers.
High Ki67 (p = 0.003), CD8 (p = 0.022), CD3 (p = 0.006), and tumour cytoplasmic AR (p = 0.001) and pAR81 (p = 0.049), were associated with lower survival. Conversely, associated with higher survival were high tumour nuclear AR (p = 0.027) and N/C for AR (p = 3 × 10−4), pAR81(p = 0.032) and pAR213 (p = 0.010). In the stroma, high nuclear AR (p = 0.002), AR N/C (p = 0.041) and cytoplasmic pAR81 (p = 0.039) were associated with better survival. The Phosphorylated Androgen Receptor and Lymphocyte (PARL) score was derived by adding 1 for each of: high CD8, high tumour cytoplasmic pAR81, low tumour pAR213 N/C and low stromal nuclear AR. Patients are stratified into low (0-1), medium (2) and high (3-4) with worsening five-year survival of 88%, 79% and 50% respectively. The PARL score was a significant predictor of survival (p = 0.001), independent of Gleason score, prostate-specific antigen, perineural invasion and serum albumin.
CD3+ and CD8+ T-cell infiltration are negative prognostic markers, while the AR has different roles and prognostic values depending on localisation and phosphorylation. The PARL score could be a useful immunohistochemistry-based measure of tumour biomarkers for prognostication and adds value to current clinico-pathological markers.
NHS Greater Glasgow and Clyde.
Prostate Cancer UK Wolfson Foundation.
All authors have declared no conflicts of interest.
HRR-deficient tumors are sensitive to PARP inhibitors (PARPi) and exhibit high levels of cytosolic DNA that can result in the activation of innate immune signaling as well as upregulation of PD-L1, which has the potential to suppress the immune response. We aimed to study if PARPi-induced PD-L1 upregulation limits the antitumor activity of PARPi by inhibiting the antitumor immune response, and if this is counteracted by anti-PD-L1 treatment.
30 Patient-Derived breast (BrC) and ovarian cancer (OvC) Xenografts (PDXs) were implanted in NMRI mice and tested for the PARPi olaparib sensitivity (5 BRCA2-, 23 BRCA1-, 2 PALB2-mutated). Their response was categorized according to the modified RECIST criteria as Complete (CR), Partial (PR) Response, Stable (SD) or Progressive (PD) Disease. RNASeq analyses were performed in PDX samples. We quantified PD-L1, CD45 (leucocyte marker), CD56 (NK cell marker) and CD11b (myeloid cell marker) positive cells by IHC in PDXs and clinical samples. Patient-derived tumor cell models (PDCs) were established (n = 17).
7 PDXs were classified as responders (CR/PR) and 23 as non-responders (SD/PD) to PARPi. At baseline, non-responder tumors expressed several pro-inflammatory genes (TNF, IL1A, IL33 and CXCL11) and exhibited a negative regulation of genes related to leukocyte proliferation by RNASeq compared to responders. In PARPi-sensitive tumors, olaparib treatment significantly increased infiltration of non-NK/non-myeloid-CD45+ immune cells, while in non-responders there was a marked increase in PD-L1 expression. Ex vivo cultures and PDXs recapitulated the patient’s PARPi response and PD-L1 expression, and are being prospectively used to investigate whether the PARPi-induced PD-L1 activation sensitizes to anti-PD-L1 therapy by enhancing the antitumor immune response.
In experimental models, olaparib elicits an antitumor immune response in PARPi-sensitive tumors that is not observed in PARPi-resistant tumors, which upregulate PD-L1. In these cases, the combination with anti-PDL1 therapy could enhance PARPi response.
This Research Project was supported by ESMO with the aid of a grant from Roche. Any views, opinions, findings, conclusions, or recommendations expressed in this material are those solely of the authors and do not necessarily reflect those of ESMO or Roche.
Vall d\'Hebron Institute of Oncology.
AstraZeneca.
J. Balmaña: Advisory board member: Clovis, Tesaro, Medivation; Speaker bureau honoraria: AstraZeneca. M.J. O\'Connor: Employee of AstraZeneca. V. Serra Elizalde: Non-commercial research agreement: AstraZeneca. All other authors have declared no conflicts of interest.
Lately, there has been a great evolution of immunotherapy in non-small cell lung cancer (NSCLC) due to its relationship with inflammation. There is also a growing interest in the changes produced in immune system with age (immunosenescence). However, it is unclear if these differences also exist among elderly patients with cancer compared to adult patients with the same diagnosis and this is the aim of our study.
We retrospectively studied patients diagnosed of NSCLC during January-December 2017 at the time of diagnosis, excluding patients with chronic infections or autoimmune diseases. Included cases were divided into two groups depending on age. Lymphocyte count by flow cytometry was gathered at baseline and we studied if there were statistically significant differences in lymphocyte populations between young (<70) and elderly patients (≥70). The non-parametric Mann-Whitney test was used.
81 patients were analysed; 32 in the young group and 49 in the elderly group. Regarding the innate immunity, the median value of NK lymphocytes showed to be higher in the cohort of patients ≥70 years (295 vs 191 cells/mm3; p = 0.0014). Oppositely, the median value of B-lymphocytes was lower in elderly patients (99 vs 128 cells/mm3; p = 0.0282). The Spearman coefficient for the correlation between age and B-lymphocyte count supports this data: ρ = 0.36 (moderate association). However, there was no difference between the median values of T-lymphocytes (p = 0.142). Interestingly, analysing T-lymphocyte subsets we found that median CD8 value was higher in the elderly group (464 vs 309.5 cells/mm3; p = 0.0226) but there was no difference in the CD4-lymphocyte subset (p = 0.4928).
Our results are similar to the ones reported in non-oncologic population, such as an increased number of NK-lymphocytes in elderly patients and a lower count of B-lymphocytes. Therefore, signs of immunosenescence can be seen in patients with NSCLC. However, subpopulations of lymphocytes show more accurately cell functionality. This is why we are performing deeper research regarding the changes in immune system produced with age in oncologic patients.
Doctor Peset University Hospital.
Has not received any funding.
All authors have declared no conflicts of interest.
Percutaneous radiofrequency ablation (RFA) is one of the main treatments of small hepatocellular carcinoma (HCC). However, it remains unclear whether this local treatment can induce systemic immune variations.
We conducted a prospective study in a tertiary center including consecutive cirrhotic patients with unifocal HCC<5cm treated by a first RFA between 2010 and 2014. Peripheral blood mononuclear cells were isolated before (D0), day after (D1) and month after RFA (M1). Frequencies and phenotypes of myeloid cells, T cells and NK cells were compared between timepoints. Overall recurrence and associated variables were estimated using Kaplan-Meier, log-rank and Cox proportional-hazards models.
80 patients were included (69% male, median age: 67 years). Main etiologies of HCC were alcohol (51%), hepatitis C virus (45%), non-alcoholic steato-hepatitis (36%) and hepatitis B virus (9%). Median overall survival was 55M; median progression-free survival was 29.5M. Among innate immune populations, we observed variations between D0, D1 and M1 in NKp30+ NK cells (p < 0.0001) and in plasmacytoid dendritic cells (pDC, p = 0.0009). Concerning adaptive immunity, we observed variations in CD8 Central Memory (p = 0.006) and CD28+ CD8 Central Memory (p = 0.002). An early dynamic (D0/D1) of activated NKp30+ NK cells was associated with a decreased overall recurrence (log-rank, p = 0.016, median delay 25.1 vs 40.6 months). In contrast, a late dynamic (D1/M1) of immature NK cells (CD56bright) and altered myeloid DC (PDL1+) was associated with an increased overall recurrence (log-rank, p = 0.011 and p = 0.0044, respectively). In multivariate analysis, variation of immature NK cells predict tumor recurrence independently of classical clinical prognostic features (HR = 2.41, CI95%(1.15-5.057), p = 0.019).
Percutaneous RFA of small HCC leads to systemic modifications of innate and adaptive immunity closely linked with overall tumor recurrence.
Service d’Hépatologie, Hopital Jean Verdier.
Grant FAR from SNFGE (Société Nationale Française de Gastro-Entérologie).
N. Ganne-Carrié: Personal fees: Bayer Schering Pharma. D. Olive: Founder of Imcheck Therapeutics. All other authors have declared no conflicts of interest.
Pembrolizumab (PEMBRO) improves survival in patients (pts) with advanced melanoma (MEL). Baseline (BL) parameters that predict long-term benefit for PEMBRO treatment are under investigation.
Outcome data of pts with advanced MEL treated with PEMBRO at our institution were collected as part of a prospective therapeutically non-interventional trial. Objective responses were evaluated using the immune-related response criteria. Total metabolic tumor volume (TMTV) was assessed by 18-fluorodeoxyglucose positron emission tomography (18FDG-PET/CT) using MIM Encore Software®. TMTV was defined as the sum of all tumor-associated voxels with a standardized uptake value (SUV) higher than the mean SUV measured in a reference region in normal liver tissue + 3 standard deviations.
BL 18FDG-PET/CT disease staging results were available for 69 pts. Median progression-free survival (mPFS) was 19 w (95% CI 9-29); median overall survival (mOS) was 130 w. A cut-off value of 90 mL of BL TMTV defined a subpopulation with significantly worse PFS (mPFS 7 w [95% CI 4-9] vs 56 w [95% CI 0-118]; HR 19.10, p < 0.001) and OS (mOS 21 w [95% CI 2-41] vs not reached; HR 46.14, p < 0.001). Additionally, a history of brain metastases (HBM), C-reactive protein (CRP) >5 times upper limit of normal (>5xULN), lactate dehydrogenase (LDH) >1xULN, WHO Performance Status (WHO PS) ≥1 and number of metastatic sites ≥2 were associated with significantly shorter PFS and OS in univariate analysis (log rank p < 0.05). In multivariate analysis (Cox multivariate logistic regression), a BL TMTV >90 mL (HR 3.70 [95% CI 1.79-7.69]), HBM (HR 2.08 [95% CI 1.11-3.85]) and WHO PS ≥ 1 (HR 2.08 [95% CI 1.12-3.85]) were significantly associated with shorter PFS; BL TMTV >90 mL (HR 14.29 [95% CI 5.26-33.33]) and HBM (HR 2.56 [95% CI 1.23-5.26]) were significantly associated with shorter OS.
BL TMTV >90 mL and HBM independently correlate with worse PFS and OS in pts with advanced MEL treated with PEMBRO. Elevated BL CRP and LDH values overlap with, but are inferior to TMTV as predictive biomarkers for outcome of PEMBRO treatment. Confirmation of these results is under investigation in an independent second cohort.
Universitair Ziekenhuis Brussel, Brussels, Belgium.
Has not received any funding.
All authors have declared no conflicts of interest.
Antibodies that block the interaction between programmed death ligand 1 (PD-L1) and PD-1 have shown impressive responses in subgroups of cancer patient. PD-L1 expression in the tumor seems to be a pre-requisite for treatment response. However, it is heterogeneously expressed within tumor lesions and may change upon disease progression and treatment. Imaging of PD-L1 could aid in patient selection.
Previously, we showed the feasibility to image PD-L1 positive tumors in immunodeficient mice. However, PD-L1 is also expressed on immune cell subsets. Therefore, the aim of this study was to assess the potential of PD-L1 microSPECT/CT using radiolabeled anti-PD-L1 antibodies to 1) measure PD-L1 expression in two immunocompetent tumor models (syngeneic mice and humanized mice harboring PD-L1 expressing immune cells) and 2) monitor therapy induced changes in tumor PD-L1 expression levels.
We showed that radiolabeled anti-PD-L1 antibodies accumulated preferentially in PD-L1 positive tumors, despite considerable uptake in certain normal lymphoid tissues (spleen and lymph nodes) and non-lymphoid tissues (duodenum and brown fat). Moreover, PD-L1 microSPECT/CT imaging could distinguish between high and low PD-L1 expressing tumors. Notably, presence of PD-L1 positive immune cells did not compromise tumor uptake of the anti-human PD-L1 antibodies in humanized mice. Most importantly, we demonstrated that radiotherapy induced up-regulation of PD-L1 expression in murine tumors which could be monitored with microSPECT/CT imaging.
Together, these data demonstrate that PD-L1 microSPECT/CT is a sensitive technique to detect varying levels of tumor PD-L1 expression and in the future, this technique may enable patient selection for PD-1/PD-L1 targeted therapy.
Radboud UMC.
Netherlands Organisation for Scientific Research and Dutch Cancer Society.
All authors have declared no conflicts of interest.
Tumor microenvironment (TME) is often hypoxic and characterized by diverse cell populations (tumor cells and lymphocytes). An intranuclear architectural protein termed high mobility group box chromosomal protein 1 (HMGB-1) is enriched in hypoxic environment, where it acts as a chemokine to promote recruitment of inflammatory cells. We studied the effect of tumor derived HMBG1 on B cell migration and phenotype differentiation in esophageal squamous cell carcinoma (ESCC).
Immunohistochemistry (IHC) staining for HMGB-1 was performed on tissue microarray (TMA) of ESCC and correlated with survival outcome. An immunostaining scoring system corresponding to total staining intensity as follows; (strong staining score =3; moderate staining score=2; weak staining scores= 1; no staining scores= 0). Co-staining with CD20+B was also performed to study B cells distribution in the tumor. ESCC cell lines were transfected with HMGB-1 and transwell migration of B cells were studied.
TMA containing 78 paired primary tumors and normal tissue from ESCC patients was analyzed by IHC using an anti-HMGB1 antibody. We found that HMGB1 was expressed at a higher level as compared to paired normal tissue. Survival curves were estimated by the Kaplan-Meier method and compared with log-rank test to evaluate the relationship between biomarker HMGB1 and survival outcomes. Patients were divided into two groups based on the optimal cutoffs of low (score 0-1), high (score 2-3) staining of HMGB1, we found out that HMGB1 staining were not correlated to patient survivals (p = 0.363). We then performed double-staining and demonstrated that B cells were located in the stroma along HMGB1-stained tumor. Stable HMGB1 ESCC cell lines were established where boyden chamber and wound-healing assays demonstrated that B cells migrated and proliferated at higher rates towards HMGB1-overexpressing cell lines.
Our findings indicate that HMGB1 was over-expressed in tumor compared with normal tissue. HMGB1 expression per se was not correlated to survival in our log-rank analysis. HMGB1 expression has probable role in B cells recruitment, migration/proliferation.
Dora LW Kwong.
Has not received any funding.
All authors have declared no conflicts of interest.
Known as co-stimulatory molecule, programmed death ligand-2 (PD-L2) contributes to T-cell exhaustion by interaction with programmed death-1 (PD-1) receptor, but its tumor cell intrinsic signal effects has been little investigated.
PD-L2 expression was detected by immunohistochemistry in 18 pairs of primary osteosarcoma tissues and matching lung metastasis tissues. We also investigated the effects of PD-L2 knockdown on osteosarcoma both in vitro and in vivo.
In our study, PD-L2 expression was elevated in lung metastases compared with primary osteosarcoma according to an immunohistochemistry assay. Wound healing and transwell assays revealed that PD-L2 knockdown leads to inhibition of migration and invasion of human osteosarcoma cells in vitro. Mechanistically, we demonstrated that PD-L2 knockdown attenuated migration and invasion by inactivating RhoA-ROCK-LIMK2 signaling, suppressing epithelial-mesenchymal transition (EMT), and inhibiting autophagy by decreasing beclin1 expression. In support of these observations, beclin1 knockdown also inhibited activation of the RhoA-ROCK-LIMK2 pathway, leading to autophagy inhibition-induced blockade of migration and invasion. Depletion of PD-L2 in KHOS cells markedly weakens pulmonary metastatic potential in vivo by orthotopic transplantation of nude mice.
Our study reveals a pro-metastatic functional mechanism for PD-L2 in osteosarcoma. Furthermore, we demonstrate a regulatory role for PD-L2 on autophagy as well as a relationship between autophagy and metastasis in osteosarcoma, which may represent a potential therapeutic target for osteosarcoma.
Tingting Ren.
Has not received any funding.
All authors have declared no conflicts of interest.
Heterogeneous expression of immune checkpoint molecules by tumor-infiltrating lymphocytes (TIL) and within tertiary lymphoid structures (TLS) was previously demonstrated in breast cancer (BC). This study analyzed primary breast tumors for: TIL and TLS; LAG3 and TIM3 expression; and other clinicopathological parameters.
A cohort of untreated primary BC (N = 95) was prospectively analyzed. Hierarchical unsupervised clustering was performed on: the % of CD4+ and CD8+ TIL expressing LAG3 and TIM3 in fresh BC tissues (flow cytometry, FC); the % of stromal (str), intratumoral and global TIL, CD3+, CD4+, CD8+, CD20+ cells, and number of TLS scored on matched tissue sections dual-stained by immunohistochemistry (IHC; CD3/CD20; and CD4/CD8); age; estrogen receptor (ER) status; and BC clinical subtype. FC data was analyzed using Kaluza™ software. IHC stained tissues were evaluated by two pathologists blinded to the clinical data. Tumors were categorized as TILneg (<10% str-TIL), TILint (>10% and <50% str-TIL) and TILhi (>50% str-TIL) on IHC slides. Continuous marker data was scaled and z-scores were computed on log2transformed data. Clustering was based on Euclidian distances and Wald criteria.
Tumors in this cohort contain a majority of luminal BC (66%). Analysis of all parameters using the gap statistical method identified two optimal clusters, which were classified as TIL-poor and TIL-rich BC. The TIL-rich group (24%) contains the majority of TILhi tumors with increased numbers of TLS and fewer patients >70 years old, compared to the predominant TIL-poor group. LAG3 and TIM3 are expressed on T cells in a small fraction of BC in both TIL-rich and TIL-poor clusters, but rarely co-expressed by T cell subpopulations in the same tumors. LAG3 expression tends to be more frequently associated with TILint triple negative BC (TNBC). A higher proportion of TIL-rich tumors was found to express TIM3.
Our study identified TIL-poor and TIL-rich immune clusters in primary BC. TIL-rich BC has heterogeneous TIM3 and sporadic LAG3 expression. TILint TNBC from the TIL-poor cluster frequently express LAG3. These data suggest that LAG3 and TIM3 screening may be important for BC immunotherapy.
Molecular Immunology Unit, Institut Jules Bordet (Brussels).
Les Amis de l\'Institut Jules Bordet.
All authors have declared no conflicts of interest.
Lymphotoxin alpha (LTa) is a pro-inflammatory cytokine expressed by inflammatory cells with a role in the regulation of tumor microenvironment (TM). A putatively functional genetic polymorphism in LTA, at locus +80 (Thr26Asn, rs1041981), has been associated with altered expression of LTa. Here, we sought to evaluate whether this polymorphism associates with 2 main endpoints (progression-free survival (PFS) and overall survival(OS)) in colorectal cancer (CRC).
This retrospective cohort study was conducted in 166 CRC patients from a single tertiary hospital. Whole blood was used to isolate genomic DNA. Genotyping of LTA +80 C > A was performed through real time-PCR allelic discrimination using specific Taqman probes, and confirmed by sequencing. Retrospective data and long-term outcomes were reviewed. Age and gender-adjusted logistic regression analyses were undertaken. Analyses were conducted after stratification by baseline lymphocyte count (LC) (LC < 1x103/µL vs > 1x103/µL). Kaplan-Meier curves with Log-Rank test were used. Subsequent multivariate Cox regression proportional hazards models were calculated (P for retention > 0.1).
Participants’ median age was 65.9 (IR, 57.5-74.3) years, the majority were males (63%), and 58% with colon cancer. At diagnosis, 45,2% were stage III and 18,1% stage IV. The median follow up time was approximately 4 years (IQR, 25.5-67.0 months). We found a significant association in carriers of the A-allele to have lower LC on linear trend analysis (P for trend = 0.013). Multivariate comparisons between LTA genotypes of additive model showed an independent protective effect for heterozygous compared with C-homozygous in the association with disease progression (HR = 0.5, 95CI = 0.3-0.97, P = 0.041), only in subjects with baseline LC > 1x103/µL. In this group, a significant independent protective effect for all-cause mortality was observed in A-carriers (HR = 0.4, 95CI = 0.1-0.97, P = 0.042).
This LTA genetic variant in subjects with baseline LC > 1x103/µL is associated with PFS and OS in CRC. The understanding of the impact of this polymorphisms on the evolution of the disease can bring us new information on TM regulation.
Ricardo Ribeiro.
Has not received any funding.
All authors have declared no conflicts of interest.
In the emerging field biomarker analysis the need for testing of tumor mutation burden in routine diagnostic becomes more and more important. Currently most analysis is done by exome sequencing but regarding time for diagnosis and limitation of tissue material for testing the applicability of large gene panels becomes of great value. To test the feasibility of targeted next generation sequencing using formalin-fixed paraffin-embedded tissue for assessing tumor mutation burden statuswe applied the 1.95 Mb Illumina TSO500 panel covering an exonic region of about 1.24 Mb.
A three-phase approach was applied: 1. Validation of the panel using DNA derived from 8 different cell lines (mutation high vs. mutation low). 2. Comparison of data from whole exome sequencing (mutation high vs. mutation low) vs.the TSO500 panel from 36 FFPE tumor samples. 3. Testing of ten samples with a) high PD-L1, or b) high MSI status or c) POLE mutation. Sequencing was performed on a NextSeq® system with the NextSeq® 500 hi-Output Kit v2 (300 cycles) and 40ng DNA as input. Data analysis was performed using a bioinformatics pipeline (“TSO500“) provided by Illumina with a limit of detection of 5% allele frequency.
The mutation burden status of all validation samples (phase 1) were confirmed applying our NGS panel approach achieving a concordance of 100%. In phase 2 we were able to confirm the mutation burden status high or low derived from exome sequencing for all except for two cases achieving a concordance of 94% between the different approaches. In further testing a UDG pre-treatment of DNA samples will be performed to decrease the amount of fixation artefacts and therefore improve the quality of the tested samples. Analysis of the samples from phase 3 confirmed a high mutation status for the MSI high and the POLE mutated cases all revealing a mutation burden values whereas the PD-L1 cases showed only borderline values.
We here show that data generated by targeted NGS data approaches can be used to determine the mutation burden status in tumor samples of different entities in high concordance with exome sequencing. Furthermore, additional genetic events (e.g. MSI) that may be clinically exploitable can be identified using a single assay.
Nicole Pfarr.
Illumina.
N. Pfarr: Member of the immune oncology Consortium (Thermo Fisher Scientific). H. Glimm: Clinical studies: Pfizer, AstraZeneca; Honoraria and travel: Roche, Lilly, Amgen. S. Fröhling: Clinical studies: Pfizer, PharmaMar, AstraZeneca; Honoraria and travel: PharmaMar, Roche, Lilly, Amgen. W. Weichert: Advisory board and/or speakers bureau: Lilly, BMS, Merck/Sharpe, Dome, Novartis, Pfizer, Roche, AstraZeneca, Boehringer, Merck; Research funding: BMS, Roche, MSD. All other authors have declared no conflicts of interest.
Immune checkpoint blockade represents a major breakthrough in cancer therapy with recent approvals of PD-1 or PD-L1 antagonists in a range of indications. Phase 3 studies have demonstrated response rates varying from 13% (head and neck squamous cell carcinoma [HNSCC]) to 40% (melanoma) in sensitive diseases, and impressively durable responses in individual patients. The overall rate of response remains relatively low however. Even in immune-sensitive diseases such as melanoma the majority of tumours do not respond to immunotherapy alone and most tumours will eventually develop resistance to the treatment. Furthermore, although PD-1/PD-L1 antagonists are generally well tolerated, a small but significant number of patients experience severe immune-related toxicity, the risk factors of which are poorly understood. Identifying which patients will most benefit from PD-1/PD-L1 antagonist therapy and the mechanisms of resistance are of critical importance for clinicians. The CHECK’UP trial aims to address this question by studying approved PD-1/PD-L1 antagonist treatment across three cancer indications: melanoma, non-small cell lung cancer (NSCLC) and HNSCC.
This prospective, multicentre cohort trial will study 670 patients in 3 parallel groups set to receive one of the single agent PD-1 or PD-L1 antagonists authorised for use in France as standard care for melanoma, NSCLC or HNSCC. Clinical and biological sample data (tumour, blood, microbiome) collected at study entry and throughout the treatment period will be analysed to identify potential response biomarkers. Patients will be followed for 5 years. A penalized logistic regression model will be used to identify associations between different parameters and response to treatment in a training cohort, made up of the first patients included, and to develop a predictive response signature for each indication. The performance of this signature will then be tested in an independent validation cohort comprised of the remaining patients. Mechanisms of primary and acquired resistance, occurrence of long-term treatment related toxicity and a cost/benefit assessment of treatment in a real-life setting will also be explored.
UNICANCER.
Fondation ARC pour la recherche sur le cancer.
All authors have declared no conflicts of interest.
Nivo demonstrated survival benefit and a manageable safety profile in previously treated patients (pts) with GC or gastroesophageal junction (GEJ) cancer in a phase III trial (ATTRACTION-2), providing an objective response rate (ORR) of 11% and a disease control rate (DCR) of 40% (Kang YK, et al. Lancet 2017). Alternatively, about 60% of the pts did not respond to Nivo, raising the necessity of its predictive biomarkers. Routy B, et al. have demonstrated that the efficacy of anti-PD-1-based immunotherapy was associated with composition of gut microbiome in various types of cancers (Science 2017), but little is known about GC. Also, immune-related genetic polymorphisms have been shown to correlate with survival in GC pts (Sunakawa Y, et al. Pharmacogenomics J 2016). We therefore investigate whether host-related immune-factors will serve as predictors for Nivo in GC.
This is an observational/translational study to evaluate efficacy and safety of Nivo for advanced GC in real world, and to discover novel immune-related biomarkers for Nivo. We will enroll 500 pts treated with Nivo alone in any lines, and follow them for at least 2 years. Eligible pts must have: adenocarcinoma of the stomach or GEJ; recurrent or metastatic disease; ECOG PS 0-2; willing to undergo 2 collections of stool and blood before and at termination of the treatment. ORR, DCR, progression-free survival, overall survival, tumor shrinkage rate, and tumor progression rate are evaluated as the efficacy. Translational approach will be performed to identify host immune-related factors (gut microbiome, genetic polymorphism, gene expression, and metabolome in plasma) as predictors for efficacy and safety of Nivo, using fecal and blood samples. The samples will be collected before and after Nivo treatment. Candidate factors will be explored in first 200 pts and then validated in last 300 pts. An association of gut microbiome with efficacy of Nivo, primary endpoint of the approach, will be investigated by metagenomics analyses. Secondary endpoints include associations between immune-biomarkers and clinical outcomes of Nivo. Accrual is starting in March 2018.
UMIN000030850.
Wataru Ichikawa.
Ono Pharmaceutical and Bristol-Myers Squibb.
Y. Sunakawa: Honoraria: Merck Serono, Taiho Pharmaceutical, Chugai Pharma, Takeda, Yakult Honsha, Sanofi, Bayer Yakuhin, Ono Pharma, Bristol-Myers Squibb. K. Muro: Honoraria: Takeda, Chugai Pharma, Yakult Honsha, Merck Serono, Taiho Pharmaceutical, Lilly, Ono Pharma; Research funding: Ono Pharma, MSD, Daiichi Sankyo, Shionogi, Kyowa Hakko Kirin, Gilead Sciences. T.E. Nakajima: Consulting role: Taiho; Honoraria: Taiho, Lilly, Chugai, Kyowa Hakko Kirin, Takeda, Bristol-Myers Squibb, Ono, Bayer, Merck Serono; Research funding: Taiho, Lilly, Chugai, Kyowa Hakko Kirin, Takeda, Ono, MSD, Merck Serono, Yakult Honsha. H. Kawakami: Honoraria: Chugai Pharma, MSD, Lilly, Yakult Honsha, Ono Pharma, Taiho Pharma, Teueda, Merck Serono; Consulting role: Taiho Pharma, Teueda. E. Inoue: Speakers\' bureau: Merck Serono. R. Matoba: Leadership, stock and other ownership interests: DNA Chip Research. Y. Sato: Employment: DNA Chip Research. W. Ichikawa: Consulting role: Ono Pharma; Honoraria: Merck Serono, Taiho Pharma, Chugai Pharma, Takeda, Ono Pharma, Lilly. All other authors have declared no conflicts of interest.
Immune-targeted monoclonal antibodies blocking inhibitory immune checkpoints such as CTLA-4, PD-1 and PD-L1 have shown durable responses in multiple tumour types. These drugs generate a new type of complications in oncology: immune-related adverse events (ImAE). There is no established predictive biomarker to identify patients (pts) who are likely to develop severe ImAE. The hypothesis is that imAEs could be due to host related pre-existing factors: Activation of self-reactive T and B cells, genetic predisposition, metabolic factors, enhanced cross reactivity between cancer cells, healthy cells and resident microbiota flora, and co-morbidity, concomitant treatments and patients’ environment. Response to immunotherapy could also be associated with pre-existing factors and tumor microenvironment features.
Immune-targeted monoclonal antibodies blocking inhibitory immune checkpoints such as CTLA-4, PD-1 and PD-L1 have shown durable responses in multiple tumour types. These drugs generate a new type of complications in oncology: immune-related adverse events (ImAE). There is no established predictive biomarker to identify patients (pts) who are likely to develop severe ImAE. The hypothesis is that imAEs could result from host related pre-existing factors: Activation of self-reactive T and B cells, genetic predisposition, metabolic factors, enhanced cross reactivity between cancer cells, healthy cells and resident microbiota flora, and co-morbidity, concomitant treatments and patients’ environment. Response to immunotherapy could also be associated with pre-existing factors and tumor microenvironment features.
The study is a French ancillary study of a phase 3b, open-Label, multi-Centre, safety study of fixed-dose durvalumab + tremelimumab combination therapy or durvalumab monotherapy in advanced solid malignancies (STRONG) (NCT03084471). By collecting additional blood and tumour samples (table 1) the aim of this ancillary study is to define the immune phenotype, microbiome analysis of patients prior to an immunotherapy and who are subsequently developing an imAE or who achieve response to identify and characterize predictive factors of the toxicity and of efficacy. For both efficacy and safety objectives, respectively 85 and 300 patients will be required.
The STRONG study currently includes pts with an urothelial and nonurothelial carcinoma of the urinary tract, treated with durvalumab as single agent at progression on or after of chemotherapy. As of July 2018, the IOPREDI ancillary study has been opened ,18 centres have been activated and 9 patients have been enrolled.
Table 1
a) definition of AESI see section 6, b) only at baseline for polymorphisms, c) within 72 hours prior to the first treatment administration
d) within 72 hours of occurrence of AESI
| Visit | Screening | Treatment period Baseline | Treatment period C2D1 | Treatment period In case of AESIa ≥ grade 2 | Treatment period C3D1 | At progression |
| Week | Weeks -4 to Week -1 | Week 0 | Week 4 | Week 8 | ||
| Autoantibodies Polymorphisms (serum) | Xb | X | X | X | ||
| Cytokines and soluble factors / Metabolic factors (plasma) | X | X | X | |||
| Cell-free DNA | X | X | ||||
| Immune cells RNA (whole blood) | X | |||||
| Hb1Ac, glycaemia, T3 and T4 | X | X | X | |||
| Stools sample | X c | X d | ||||
| Telomerase, commensal and NYESO-1 T cells specific immune response (frozen PBMC) | X | X | X | X | X | |
| Epithelial mesenchymal transition (tumor sample) | X |
The study is a French ancillary study of a phase 3b, open-Label, multi-Centre, safety study of fixed-dose durvalumab + tremelimumab combination therapy or durvalumab monotherapy in advanced solid malignancies (STRONG) (NCT03084471). By collecting additional blood and tumour samples (table 1) the aim of this ancillary study is to define the immune phenotype, microbiome analysis of patients prior to an immunotherapy and who are subsequently developing an imAE or who achieve response to identify and characterize predictive factors of the toxicity and of efficacy. For both efficacy and safety objectives, respectively 85 and 300 patients will be required. The STRONG study currently includes pts with an urothelial and nonurothelial carcinoma of the urinary tract, treated with durvalumab as single agent at progression on or after of chemotherapy. As of July 2018, the IOPREDI ancillary study has been opened, 18 centres have been activated and 9 patients have been enrolled. Table 1 a) definition of AESI see section 6, b) only at baseline for polymorphisms, c) within 72 hours prior to the first treatment administration d) within 72 hours of occurrence of AESI.
EudraCT: 2016-005068-33.
AstraZeneca.
AstraZeneca.
A. Marabelle, F. Ghiringhelli: Honoraria for scientific boards: AstraZeneca. M. Ayyoub, O. Adotevi, Y. Loriot, O. Lambotte: Honoraria for advisory boards: AstraZeneca. D. Cupissol, D. Damotte, N. Chaput: Honoraria for scientific committee meetings: AstraZeneca. J. Adam: Honoraria for advisory boards: AstraZeneca. B. Petre Lazar, M. Licour: Employee of AstraZeneca. All other authors have declared no conflicts of interest.Visit Screening Treatment period Baseline Treatment period C2D1 Treatment period In case of AESIa ≥grade 2 Treatment period C3D1 At progression Week Weeks -4 to Week -1 Week 0 Week 4 Week 8 Autoantibodies Polymorphisms (serum) Xb X X X Cytokines and soluble factors / Metabolic factors (plasma) X X X Cell-free DNA X X Immune cells RNA (whole blood) X Hb1Ac, glycaemia, T3 and T4 X X X Stools sample X c X d Telomerase, commensal and NYESO-1 T cells specific immune response (frozen PBMC) X X X X X Epithelial mesenchymal transition (tumor sample) X
Acute Myeloid Leukemia (AML) is a clonal disease of the hematopoietic system. Disease relapses are common with current treatment approaches. As an alternative, immunological eradication of leukemic cells by adoptively transferred chimeric-antigen receptor T-cells (CAR T-cells) might be considerably more efficient. To date, however, the search for AML-specific surface antigens has remained largely elusive. To circumvent this problem, we propose to target the stem cell antigen c-Kit (CD117) that is expressed by physiological HSPC as wells as by leukemic blasts in > 90% of AML patients.
A lentiviral vector was generated expressing a second generation CAR targeting human CD117 followed by a T2A sequence and RQR8 as selection marker and depletion gene (surface expression of CD34 and CD20 epitopes).
In vitro, CAR T-cells eliminated over 90% of CD117high leukemia cell lines within 24 hours. In long–term cytotoxicity assays (45d), only CD117low cells were able to escape CAR-mediated killing compared to CD117high and CD117intermediate cells which were not able to survive. Anti-CD117 CAR T-cells effectively depleted >90% of lin-CD117+CD34+CD38+ and >70% of lin-CD117+CD34+CD38- cells from healthy bone marrow in vitro. Similarly, >70% of patient derived leukemic blasts were eliminated by autologous anti-CD117 CAR T-cells within 48 hours. In vivo, immunodeficient mice were engrafted with umbilical cord blood derived CD34+ cells. A single injection of 2x106 anti-CD117 CAR T-cells resulted in > 90% depletion of CD117+ cells in the bone marrow within 6 days. Finally, humanized mice transplanted with bone marrow from AML patients were treated with autologous CAR T-cells. At 6 weeks after injection of CAR T-cells, >98% of hu-CD45 CD117+ cells were depleted in the bone marrow.
We provide proof of concept for the generation of highly-potent CAR T-cells against CD117. Anti-CD117 CAR T-cells exhibit high cytotoxic activity against CD117+ cell lines as well as primary healthy HSPC and patient AML cells in vitro and in vivo in murine xenograft models. Strategies for the complete elimination of CAR T-cells (immunologic or small molecule based) are required before translation of this approach to the clinical setting.
University Hospital Zurich.
PROMEDICA stiftung, URPP University of Zuric, Krebsliga Schweiz.
All authors have declared no conflicts of interest.
Despite the immense potential of CAR-T cell therapy, there are several drawbacks that limit its scope, including high manufacturing costs, on-target off-tumor toxicities, induction of cytokine release syndrome, and poor efficacy against solid tumors. In order to overcome these limitations, alternative cellular platforms could be used as carriers of CARs. Here, we modified NK cell lines to express chimeric antigen receptors. Additionally, to potentiate the antitumor activity of such CAR-NK cell lines, we co-transduced them with cassettes encoding soluble CD47/SIRPa-blocking agents. This modification should eliminate the transmission of protective “don’t eat me” signal from tumor cells to macrophages thereby enhancing cancer cell phagocytosis. We refer to this universal allogeneic antitumor platform as ECAR-NK.
We designed lentiviral constructs encoding a second-generation CAR and secreted chimeric anti-CD47 chimeric mAb, scFv, as well as the extracellular domain of CD47. NK cell lines were cotransduced with the constructs coding the CAR and one of the CD47/SIRPa blockers. Transduction efficiency was assessed by flow cytometry. In vitro cytotoxic activity of ECAR-NKs against target cells was measured by FACS and using iCelligence cell viability monitoring system. To assess the effect of CD47/SIRPa blockade on the phagocytic activity, in vitro phagocytosis assay was performed.
ECAR-NK cells obtained demonstrate pronounced cytotoxicity in vitro against NK-resistant target cells. We show that ECAR-NKs produce both soluble CD47-specific scFv and chimeric mAb which were able to significantly increase phagocytic activity of macrophages in vitro. Analysis of in vivo activity of ECAR-NK cells in NOD/SCID mice bearing xenografts is in progress.
Our findings suggest that NK cell lines may serve as a promising platform for developing allogeneic CAR-NK cell products. We believe that coaction of CD47/SIRPa blocking and CAR-mediated cytotoxicity should result in enhanced antitumor activity and help mitigate the issue of antigen escape. Supported by the RSF grant #16-14-10237.
Russian Ministry of Science.
Russian Science Foundation.
All authors have declared no conflicts of interest.
Chimeric Antigen Receptors (CARs) consist of a target binding moiety fused to an extracellular spacer, a transmembrane region and an intracellular signaling tail comprising a tandem alignment of co-stimulatory and activatory domains. This linear configuration displays rigid spatial orientation and ratio of co-stimulation to activation domains. We have developed a novel mix and match approach (CARpool) where the costimulatory signal is provided in trans on accessory proteins that associate with the antigen binding chain via transmembrane interactions.
Several CD3ζ-containing CAR chains were designed using the transmembrane and cytoplasmic domains of NKG2D or NKp44, associating with DAP10 and DAP12 respectively. Each CAR contained a B7H6-targeting scFv and was co-expressed with corresponding accessory protein using a 2A site. Primary human T cells engineered with the diverse constructs were screened for CAR expression, phenotype and in vitro function.
NKG2D-based CAR complexes were moderately expressed at the cell surface but bound B7H6 and led to potent cells. Modification of the position of the charged residue within the transmembrane domain of the CAR is being used to modulate the surface expression and thus their potency. NKp44-based CAR complexes were more frequently expressed on primary T cells and bound B7H6, though functionality appears to be dependent on the nature of the extracellular spacer.
These studies provide proof-of-concept for a novel CAR design where it is possible to incorporate or interchange costimulatory domain(s) in a stoichiometrically controlled way. Recapitulating physiological TCR activation by providing co-stimulation in trans within the CARpool may result in optimal downstream signaling, thereby enhancing anti-tumoral activity.
Celyad SA.
Celyad SA.
L. Springuel, J. Bolsée, A. Velghe, S. Agaugué, D. Gilham: Employee of Celyad SA.
CD8 T cells are critical to immunotherapeutic intervention in cancer. However, the ability to efficiently target antigens for MHC-I presentation has limited the efficacy of immunization strategies. Here, we use microfluidics-based SQZ cell therapy platform to deliver antigen to the cytosol of target APCs, resulting in enhanced presentation on MHC-I. Given that no expansion time is required to engineer these APCs, this approach allows for rapid manufacturing of billions of non-conventional APCs capable of priming CD8+ T cell responses.
Protein and peptide antigens were delivered to murine or human T cells with the SQZ cell therapy platform. The response to in vivo immunization of C57BL/6 mice was assessed by flow cytometry. Tumor experiments were conducted with the TC-1 cell line. Human T cells were co-cultured with epitope-reactive human responder CD8+ T cells, and interferon gamma production was quantified to assess antigen-specific responses.
In murine systems, we demonstrate that therapeutic immunization with T cells loaded with E7 using the SQZ platform are capable of eliciting strong anti-tumor effects. These anti-tumor responses correlate with an increase in antigen-specific CD8+ tumor infiltrating lymphocytes that is dependent on the co-administration of an adjuvant. In human cells, we demonstrate that primary T cells loaded with CMV and HPV16 antigens using SQZ can stimulate antigen-specific CD8+ T cell responses in vitro. Importantly, delivery to T cells scales from millions up to billions of cells per minute and antigen presentation is retained even after cryo-preservation.
Through the direct cytosolic delivery of antigen, the SQZ cell therapy platform enables the use of T cells, a readily accessible and abundant cell type, to function as APCs for immunization. This strategy has demonstrated the ability to generate CD8+ T cell responses in both murine and human systems and has been scaled up for clinical implementation. With the ease and speed of manufacturing, these results have the potential to translate into a cellular immune therapy for oncology patients.
SQZ Biotechnologies.
SQZ Biotechnologies.
S.M. Loughhead, M.G. Booty, K. Hlavaty, A. Vicente-Suarez, K. Blagovic, M. Myint, B. Stokes, D. Yarar, H. Bernstein, A. Sharei: Employee of SQZ Biotech.
Ex vivo manipulation of primary cells is critical to the success of cell-based therapies, however, limitations of existing ex vivo delivery approaches may dramatically restrict the use of cell engineering to treat disease.
We used a genome-wide approach to study optimized electroporation treatment and microfluidic cell squeezing to identify the impact of delivery technique on gene expression profiles in human T cells. To validate the microarray results, we used a multiplex cytokine analysis comprised of 42 key T cell cytokines to assess perturbation of cytokine secretion. Finally, in a direct comparison of therapeutic functionality, the efficacy of T cells edited for PD-1 via electroporation and cell squeezing was assessed using therapeutic treatment of the Eg.7 OVA tumor model.
We identified striking disruptions in transcript expression after treatment with electroporation (17% of genes mis-regulated, FDR q < 0.1), whereas cells treated with microfluidic squeezing had similar expression profiles to untreated control cells (0% of genes mis-regulated, FDR q < 0.1). These genetic disruptions result in concomitant perturbation of cytokine secretion including a 648-fold increase in IL-2 secretion (p < 0.01) and a 30-fold increase in IFNγ secretion (p < 0.05) in electroporated cells. Squeezing cells did not result in the non-specific secretion of any of the 42 cytokines tested. Ultimately, the effects at the transcript and protein level resulted in functional deficiencies in vivo with electroporated T cells failing to demonstrate sustained antigen-specific effector responses and tumor control.
This work demonstrates that the delivery mechanism used to insert biomolecules affects functionality and warrants further study. The significant differences in outcomes from the two techniques underscores the importance of understanding the impact of intracellular delivery methods on cell function for research and clinical applications. Hence, for both research and therapeutic applications, the functional and safety consequences of the selected intracellular delivery technique and its impact on cell phenotype should be carefully evaluated.
SQZ Biotechnologies.
SQZ Biotechnologies.
J. Hanson, J. Cole, L. Cassereau, J. Bugge, T. Ditommaso, J. Gilbert, H. Bernstein, A. Sharei: Employed by SQZ Biotech.
The use of engineered T cells has proven to be successful in the field of cancer therapy. In vivo persistence of these genetically redirected T cells, which depends on their differentiation profile, plays a central role in the achievement of treatment efficacy. Indeed, less differentiated T cells proliferate and persist longer in vivo compared to their more differentiated counterparts, while having functional antitumor capacities. In this view, cord blood derived T cells display a more naïve phenotype compared to peripheral blood derived T cells. Hence, cord blood could be used as a source of T cells in order to generate gene-modified cancer-targeting cells able to persist in vivo, with the ability to ensure immunosurveillance in treated patients.
Cord blood derived T cells were isolated, activated and cultured with IL-2 or IL-7 and IL-15, before a retroviral transduction with a vector encoding a TCR targeting HPV16-E7/HLA-DR4. Transgenic T cell functional capacities were then evaluated by flow cytometry. The differentiation profile of cord blood derived T cells, genetically redirected or not and cultured in different conditions, were also analyzed.
The IL-7 and IL-15 culture condition resulted in the generation of gene-modified T cells maintaining a higher transgenic TCR expression over time compared with the IL-2 culture condition. Functional assays revealed that genetically redirected T cells cultured in both conditions were reactive against HLA-DR4+ BLCL pulsed with an E7-derived peptide. Both T cell products lead to comparable interferon-γ, tumor necrosis factor-α and interleukine-2 secretion. These results are consistent with the fact that no outstanding difference was detected between both culture conditions regarding transduced and untransduced T cell differentiation profile.
In conclusion, we were able to generate cord blood derived transgenic T cells which were specific for an HPV16-E7 peptide, with a higher TCR expression over time when cultured with IL-7 and IL-15. Our study supports that cord blood units could be used as a source of T cells to develop cost-reducing and easy to access adoptive cancer immunotherapy.
UMR1098: INSERM, EFS BFC, Univ. Bourgogne Franche-Comté.
This work was supported by the Ligue Nationale Contre le Cancer, and by the MiMedi project funded by BPI France (grant No. DOS0060162/00) and the European Union through the European Regional Development Fund of the Region Bourgogne-Franche-Comte (grant No. FC0013440). C.M. has benefited from a fellowship from Nancy Regional University Hospital.
All authors have declared no conflicts of interest.
Dendritic cells (DCs) are unique antigen presenting cells that are used in cancer immunotherapy, with more than 20 years of clinical trials in this field. The production of clinical grade monocyte-derived DCs is highly desired and globally performed, with a large space for improvement and development of clinical standard operating procedures (CSOP). This process is highly dependent on three constraints: used cytokines for cell differentiation, maturating agents and culture medium. There is a clear lack of studies focusing on how the used culture medium and its composition and components affect the process of ex-vivo generation of monocyte-derived DCs. This study aims to compare the effect of 4 culture media (3 GMP media and 1 medium widely used in research) during monocyte-derived DCs differentiation.
We characterized DC viability, differentiation, maturation, internalization of tumor lysates, cytokines production and autologous T cell stimulatory capacity, as well as metabolomic profiles. Commercially available culture media for clinical use were tested, namely DendriMACS, AIM-V and X-VIVO 15. RPMI was also tested as a comparative term given that it is largely used in pre-clinical research.
In terms of differentiation, maturation, DC uptake capacity, and metabolic profiles, AIM-V and X-VIVO 15 present similar results. However, the use of X-VIVO 15 shows an enhanced DC production of IL-12. DCs cultured in X-VIVO 15 and AIM-V media are able to induce a superior stimulation of T cells, mainly CTLs and Th1 responses, while DCs cultured in DendriMACS are more prone to induce Treg polarization.
Our data show that X-VIVO 15 and AIM-V culture media are preferable to support the differentiation of DC to be used in immunostimulatory approaches such as in cancer cell therapy. Overall, this study highlights the need of previously and carefully defining the culture medium to be used in DC cancer immunotherapy, for a better aim to the therapeutic goal and potentiating the clinical experiment and final patient output.
Grupo Tecnimede.
This study received funding from the project ImmunoDCs@CancerStemCells: Cellular Immunotherapy towards the elimination of cancer stem cells (Ref.: POCI-01-0247-FEDER-033532), co-funded by the European Regional Development Fund (FEDER), Competitiveness and Internationalization Operational Program (COMPETE2020) and Own Revenues of the University of Coimbra. João Calmeiro is supported by the Portuguese Science and Technology Foundation (FCT) through an individual PhD fellowship (PD/BDE/135076/2017).
All authors have declared no conflicts of interest.
Intratumoral (IT) myDC play a pivotal role in initiating anti-tumor immune responses and in "re-licensing” of anti-tumor cytotoxic T-lymphocytes within the tumor microenvironment. IT injection of anti-PD-L1 IgG1 mAb AVE and anti-CTLA-4 IgG1 mAb IPI may reduce the number of regulatory T cells and lyse PD-L1+ tumor cells increasing the release of tumor antigens that can be captured and processed by IT co-administered CD1c (BDCA-1)+ myDC, reinvigorating the cancer immunity cycle.
Patients (pts) with advanced solid tumors who failed standard-of-care treatment were eligible for this phase I trial with IT injections of ≥ 1 non-visceral metastasis with IPI (max total dose of 10 mg) and AVE (max total dose of 40 mg) plus IV NIVO (10 mg) on day 1 followed by IT injection of autologous, non-manipulated CD1c (BDCA-1)+ myDC on day 2. Administration of AVE, IPI, and NIVO was repeated every 14 days thereafter. Primary endpoints were safety and feasibility. Clinical responses were defined according to iRECIST criteria.
In this ongoing trial, 4 pts (3 primary melanoma, 1 epithelial ovarian carcinoma) were treated with IT injection of a median of 30.5x106 (range 10-43x106) CD1c (BDCA-1)+ myDC. A median of 5.5 (range 4-7) study drug administrations were performed. One confirmed partial response was documented in a melanoma patient who previously progressed on immune checkpoint inhibitors. In 2 additional melanoma pts regression of the injected subcutaneous metastases coincided with progression of non-injected metastases. Overall, study treatment was well tolerated and adverse events consisted of transient grade 2 local pain at the injection site in 2 pts, grade 1 pruritus in 2 pts, grade 2 pneumonitis in 1 patient, and pruritus and redness of the skin overlaying the injected lesion in 1 patient.
IT injection of autologous, non-manipulated CD1c (BDCA-1)+ myDC with IT co-injection of AVE and IPI plus IV low-dose NIVO is feasible and tolerable and resulted in encouraging early signs of anti-tumor activity in injected as well as non-injected lesions.
EudraCT: 2017-003280-35.
Universitair Ziekenhuis Brussel.
Kom op Tegen Kanker.
All authors have declared no conflicts of interest.
Glioblastoma multiforme (GB) is the most common type of primary brain tumors, that virtually always relapses, despite initial treatment with surgical resection, radio- and chemotherapy. Nowadays new promising approaches are developed in treating GB based on dendritic cell (DC) vaccines for the enhancement of the anti-tumor immune response. IFNα-induced monocyte-derived DCs possess properties of myeloid DCs, plasmacytoid DCs and NK cells. The present study focuses on the role of death-inducing mechanisms in DC cytotoxicity against GB cells.
The study was conducted in 26 donors and 21 GB patients. DCs were generated by culturing of plastic-adherent peripheral blood mononuclear cells in the presence of GM-CSF and IFN-α followed by the addition of LPS. The tumor cell lines were obtained from tissues of 14 GB patients. DC cytotoxicity against tumor cells was studied using MTT-assay.
Donor DCs induced death of GB cell lines (min-max cytotoxicity 11-80%). The lysis of GB cells was accompanied with the increase of Annexin V+ apoptotic tumor cells. GB cells expressed death receptors TNF-R1, TRAIL-R2 and Fas. To examine death receptor-mediated killer activity, donor DC were pretreated with soluble forms of TNF family receptors. The blocking of TNF-R1-signaling pathway decreased DC cytotoxicity against GB cells by 25% on average. rhTRAIL-R2 or rhFas pretreatment slightly abolished DC cytotoxicity (with an average of 10%). The most blocking effect on DC cytotoxicity against GB cells was observed if DCs were pretreated with concanamicin A, an inhibitor of granule-dependent killing (Δ up to 60%). GB patient DCs were characterized by reduced cytotoxicity against autologous GB cell lines compared to donor values. Low cytotoxicity of patient DCs was associated with decreased level of membrane TNFα (mTNFα), but intact FasL, TRAIL, perforin and granzyme B molecule expression. The addition of IL-2 into patient DCs enhanced cytotoxicity of DCs towards autologous GB cells and increased mTNFα expression on DCs.
Thus, DCs with tumoricidal activity and ex vivo regulation of DC cytotoxic function may become a new approach to obtain effective anti-tumor DC-vaccines.
Elena Chernykh.
Grant of the Foundation of the President of the Russian Federation.
All authors have declared no conflicts of interest.
Viral infection is a major cause of disease and mortality after allogeneic hematopoietic stem cell transplantation (AHSCT) in hematologic malignancies. It has been shown that adoptive transfer of expanded virus-specific T cells (VSTs) was capable of treating infections that are resistant to conventional therapies. Recently, it has been shown that PD-L1 expression by activated T cells plays a major role on their survival and activity. Here, we investigated PD-L1 expression during VSTs expansion and the effect of PD-L1 blockade on the phenotype and functional activity of these VSTs.
VSTs were generated from healthy donors PBMCs with or without the addition of PD-L1 antibody after stimulation in G-Rex-10 flasks with a pool of 11 overlapping peptides libraries spanning the 5 most immunodominant viral antigens. All VSTs were collected at day 14. We used ELISpot assay to measure IFN-γ production by VSTs and flow cytometry analysis for phenotyping.
Blockade of PD-L1 has induced 1.8 folds higher proliferation rate and 2 folds increase in IFN-γ production against the viral antigens. After 6 days of expansion, 78% of CD4+ and 60% of CD8+ VST subsets expressed the PD-L1 molecule. At the end of expansion, CD4+ PD-L1+ VSTs were reduced to 6.8% whereas the CD8+ PD-L1+ VSTs were increased to 87.6%. Interestingly, PD-L1 blockade resulted in a further reduction in the CD4+ PD-L1+ population (2.2%), without affecting the CD8+ PD-L1+ VSTs (89.5%). After PD-L1 treatment, the cytotoxicity marker CD107 was slightly increased (1.2 folds) in the CD4+ cells but decreased by 2 folds in the CD8+ VSTs and CD4+ CD45RO+ memory T cells were decreased from 46.1% to 33%.
We have standardized an ex vivo protocol for rapid expansion of VSTs against major viruses causing infection after AHSCT. These VSTs were shown to highly express PD-L1. Blocking of PD-L1 improved the expansion, the anti-viral specificity and the cytotoxicity of these VSTs. This should promote a long-lasting antiviral activity of VSTs in patients undergoing AHSCT. Further investigations are being carried out to confirm these results.
Medical Research Center, Hamad Medical Corporation, Doha, Qatar.
Medical Research Center, Hamad Medical Corporation, Doha, Qatar.
All authors have declared no conflicts of interest.
The outcomes for patients with metastatic or locally recurrent Epstein–Barr virus (EBV)-positive nasopharyngeal carcinoma (NPC) remain poor. We aimed to improve the outcomes for NPC refractory to conventional treatments with autologous EBV and oncogene–targeted Cytotoxic T Lymphocytes (CTL) therapy.
Patients with stage IV NPC who cannot tolerate chemotherapy received autologous EBV and oncogene–targeted CTL expanded ex vivo from peripheral blood lymphocytes through dendritic cells (DCs) transfected by adenovirus-based vector encoded multiple epitopes from LMP1, CK19, survivin, SCC, CEA, SPANX, MAGE-A3, PMSA. Clinical tumor responses, survival rate and toxicity were evaluated.
In total 16 patients received multiple oncogenes-targeted CTL infusion. Among them, 2 patients had locally advanced NPC (LANPC) with high-risk distance metastasis, 5 patients had nasopharyngeal and/or cervical recurrence after re-irriadiation or surgery, 7 patients had distant metastasis and 2 patients had both distant metastasis and nasopharyngeal recurrence. Three patients showed CR, one patient showed PR, and nine patients exhibited SD after CTL infusion. During a median follow-up of 15 months, the median progression-free survival (PFS) was 282 days (range 28-503 days), the mean overall survival is 484 days (range 28–552 days) after autologous CTL infusion. We did not observe severe or chronic adverse reactions related to autologous EBV and oncogene–targeted CTL infusions.
In our study, our findings show that therapy with autologous Epstein-Barr Virus and oncogene–Targeted Cytotoxic T Lymphocytes is safe and well tolerated and may offer clinical benefit to patients with NPC.
Sun Yat-Sen University Cancer Center.
Has not received any funding.
All authors have declared no conflicts of interest.
Recent studies have shown that for optimal immune suppression regulatory T cells (Tregs) are required to adopt a transcriptional profile similar to that of the type of T cells they aim to suppress. For instance, Foxp3+ Tregs upregulate the Th1-associated transcription factor Tbet to control a type 1 cytokine-mediated inflammatory response in order to prevent unwanted tissue destruction and immunopathology. However, little is known about the existence and function of such Treg cells in cancer patients.
To study the presence and potential impact of Tbet-expressing Foxp3+ (Foxp3+Tbet+) Tregs in human cancer, we applied three-color immunofluorescence staining and 12-parameter flow cytometry on the tumor microenvironment (TME) of human papilloma virus (HPV)-driven oropharyngeal squamous cell carcinoma (OPSCC) patients.
Our data revealed that Foxp3+Tbet+ Tregs accumulate and dominate in the tumor cell nests of patients with a concomitant HPV-specific and type 1-oriented intratumoral T cell infiltrate. Moreover, these CD4+CD25+CD127–Foxp3+Tbet+ Tregs exhibited an activated phenotype and co-expressed high levels of CTLA4 and Helios. Assessment of the methylation status of the FoxP3 gene locus TSDR of flow cytometry-sorted Foxp3+Tbet+ and Foxp3+Tbet - Tregs revealed that it was maximally demethylated, indicating that these Tregs have the full capacity to suppress immune cells. Interestingly, OPSCC patients with high intratumoral frequencies of Foxp3+Tbet+ Tregs, but not Foxp3+Tbet– Tregs, displayed prolonged disease-specific survival, suggesting that the presence of Foxp3+Tbet+ Tregs is a reflection of a strong and clinically favorable local tumor-specific type 1 immune response.
In conclusion, bona fide Foxp3+Tbet+ regulatory T cells accumulate in HPV16-driven tumors that are highly infiltrated with type 1 tumor-specific T cells, at levels enough to impede full spontaneous immune-mediated tumor control.
Sjoerd H. van der Burg.
The Dutch Cancer Society.
All authors have declared no conflicts of interest.
Tumour infiltrating lymphocyte (TIL) therapy extracts CD8+ and CD4+ T cells from a tumour mass, expands and re-infuses them back into the patient. TIL therapy can lead to substantial complete, durable responses, particularly in refractory melanoma. TIL therapy is more effective if more T cells are infused, if they are less differentiated and if they target tumour antigen. Radiotherapy (RT) prior to TIL extraction may have immunostimulatory effects and so increase T cell infiltration into tumours, alter the T cell phenotype or increase number of tumour specific T cells in the expanded TIL.
Two murine melanoma models (B16 and 4434) were grown in CD57Bl/6 mice. Treated tumours were irradiated with single dose (8 Gray) local radiotherapy, harvested 72 hours later and TIL were enriched. TIL were expanded for 9-10 days ex vivo using the allosensitised allogeneic lymphocyte expansion protocol (AEP), which uses soluble growth factors and allogeneic feeder cells to stimulate T cell growth. Live expanded TIL were counted using a haemocytometer, phenotyped using fluorescence-activated cell sorting and assayed for tumour reactivity by interferon gamma production.
CD8 + (≤ 61 fold) and CD4 + (≤ 16 fold) murine T cells expanded using the AEP in both murine tumour lines. 4434-derived and B16-derived TIL expanded to similar numbers with (n = 4) and without (n = 4) prior RT to tumour mass. When B16-derived TIL were analysed further, there were no changes in differentiation status or in the tumour reactivity (p = 0.48) of TIL from RT treated tumours. However, B16-derived TIL expanded from RT treated tumours had significantly greater expression of PD1 (as measured by percentage positive cells (p = 0.020) and geometric mean fluorescence intensity (p = 0.030)).
This is the first study to expand murine or human TIL treated with prior RT. The AEP expanded tumour reactive murine melanoma TIL to numbers (1-5 x 106 cells) which might effectively treat in vivo murine models. Increased PD1 expression on TIL from RT-treated tumours might predict increased in vivo effectiveness, particularly if combined with PD1 inhibitors. The effect of different RT regimens on TIL expansion and effectiveness could be investigated with this approach.
The University of Manchester.
The University of Manchester.
All authors have declared no conflicts of interest.
Adoptive cell therapy with TIL has demonstrated durable complete responses in immunogenic tumors with high mutational burden in patients who had not received prior checkpoint therapy (>22% CR). Pembrolizumab is an approved agent for treatment of metastatic melanoma and head and neck cancers. Further, checkpoint inhibitors have been reported to possibly enhance the efficacy of TIL therapy. One aim of this study is to improve on the efficacy response for early line patients by combining TIL with anti-PD1 in metastatic melanoma and head and neck cancers (Cohorts 1 and 2). In Cohort 3, TIL alone is offered to non-small cell lung cancer patients who have received prior systemic therapy including checkpoint inhibitors.
IOV-COM-202 is a Phase 2 multicenter, open-label, nonrandomized study in which patients are enrolled in the combination arms (Cohorts 1 and 2) or LN-145 therapy only (Cohort 3) based upon study disease (see above). Planned enrollment is N = 36 (12/cohort). Tumors resected at local institutions are processed at centralized GMP facilities using a 22-day manufacturing process to generate the final cryopreserved infusion product (LN-144/LN-145) that is shipped to the sites. All patients receive TIL therapy consisting of 1 week of a preconditioning cyclophosphamide/fludarabine lymphodepletion regimen, followed by a single infusion of LN144/LN-145 (Day 0) and up to 6 doses of intravenous IL-2 (600,000 IU/kg). Patients in Cohorts 1 and 2 also receive pembrolizumab on Day 1 and again every 3 weeks for up to 2 years. Eligibility includes: ≥ 18 years of age; 1-3 lines of prior systemic therapy (exclusions apply); ≥ 1 resectable lesion(s) yielding ≥ 1.5 cm in diameter of viable tumor and a remaining RECIST-measurable lesion; and ECOG PS of 0-1. For each cohort, the primary endpoint is ORR per RECIST v1.1 and safety. Secondary endpoints are CR rate, DOR, DCR, PFS, and OS. Exploratory objectives include tumor response per irRECIST, immune correlates of response, and HRQoL. NCT03645928
Iovance Biotherapeutics, Inc.
Iovance Biotherapeutics, Inc.
A. Cacovean, D. Barton: Employee of Iovance Biotherapeutics, Inc. M. Fardis: Chief Executive Officer of Iovance Biotherapeutics, Inc. All other authors have declared no conflicts of interest.
Up to 60% of patients treated with cancer immunotherapy develop severe or life threatening immune-related adverse events (irAEs). Immunosuppression with high doses of corticosteroids or, in refractory cases, with tumor necrosis factor (TNF) antagonists, are the mainstay of treatment for irAEs. It is currently unknown what is the impact of corticosteroids and anti-TNF on the activity of tumor-specific T cells.
The influence of clinically relevant doses of dexamethasone (corresponding to an oral dose of 10 to 125 mg oral prednisolone) and infliximab (anti-TNF, corresponding to the steady-state concentration for the infusion dose of 5mg/kg) on the activation and killing capacity of tumor-infiltrating lymphocytes (TILs) or tumor-specific peripheral blood lymphocytes (PBLs) was tested in vitro. The activation of lymphocytes following recognition of autologous tumor cells (percentage of CD8+ T cells staining positive for the activation marker 4-1BB/CD137) was evaluated with flow cytometry at different time points. The tumor-specific killing ability of TILs versus autologous tumor cells was evaluated in the impedance-based xCELLigence system.
Overall, dexamethasone at low or intermediate/high doses impaired the activation (respectively -46% and -62% in n = 8) and tumor-killing ability (respectively -48% and -53% in n = 6) of TILs. However, even high doses of dexamethasone did not abrogate TILs activity. In contrast, a standard clinical dose of infliximab only had a minor effect on the activation and tumor-killing ability of TILs (respectively -20% in n = 8 and -10% in n = 6). Similar results were obtained comparing the activation of tumor-specific CD8+ PBLs. A 72-hour resting after withdrawal of dexamethasone was sufficient to rescue the in vitro activity of TILs, while a short withdrawal did not result in a full rescue.
Clinically-relevant doses of infliximab only influenced to a lesser extent the activity of tumor-specific T cells in vitro, whereas even low doses of corticosteroids markedly impaired their antitumor functions. These data indirectly support steroid-sparing strategies and early initiation of anti-TNF for the treatment of irAEs in immuno-oncology.
Herlev and Gentofte Hospital.
These works were supported by a research grant to Inge Marie Svane from The Danish Cancer Society, Knæk Cancer campaign.
T.H. Borch: Honoraria for lectures: Bristol-Myers Squibb; Financial support for attending symposia: Bristol-Myers Squibb, Roche. I-M. Svane: Honoraria for lectures: Novartis, Roche, Merck, Bristol-Myers Squibb; Research grant: Novartis; Financial support for attending symposia: Bristol-Myers Squibb, Merck, Novartis, Pfizer, Roche. M. Donia: Honoraria for lectures: Bristol-Myers Squibb, Merck, AstraZeneca, Genzyme; Financial support for attending symposia: Bristol-Myers Squibb, Merck, Novartis, Pfizer, Roche. All other authors have declared no conflicts of interest.
The incidence of any and of severe grade immune-related adverse events (irAEs) with second-line Nivolumab (N) monotherapy is 26% and 6% respectively. While potentially serious and even fatal, in the absence of appropriate therapy, such events might be an indicator of the activation of the immune system and, potentially, of efficacy.
We collected the records of 1.959 NSCLC patients (pts) including those with Squamous (S) and non-Squamous (non-S) histology, treated with N in the Italian expanded access programme and we recorded the appearance of any and of severe grade irAEs. We then retrospectively searched for potential correlations between this type of toxicity and efficacy parameters by using cox regression analysis.
A total of 1.585 and 374 pts had non-S and S cell carcinoma respectively and 57% received N as second-third line of therapy. Overall 342 (17.8%) developed an irAE of any grade. We observed that pts developing any grade irAE achieved a significantly higher response rate (RR 27.2% vs 15.2%; p < 0.0001), disease control rate (DCR 60.5% vs 40.2%; p < 0.0001), median progression-free survival (mPFS 6.0 months [95% CI 4.9-7.1] vs 3.0 [95% CI: 2.8-3.2], p < 0.0001) and median overall survival (mOS 16.7 months [95% CI: 13.5-19.9] vs 9.4 [95% CI: 8.4-10.4], p < 0.00001) compared to pts who did not. IrAEs correlate with clinical outcomes in both non-S and S histology. At multivariate analysis the development of an irAE remained an independent indicator of N efficacy (HR 1.44[95% CI: 1.22-1.71] p < 0.0001).
This is the first report performed in a large series of Caucasian NSCLC pts showing that the activation of the immune system induced by N and documented by the appearance of irAEs correlates with outcome. A careful management of pts with such an event could lead to a maximum clinical benefit.
Editta Baldini.
Has not received any funding.
All authors have declared no conflicts of interest.
Programmed cell death 1 (PD-1) inhibitor-related diarrhea is increased due to the rising administration of PD-1 inhibitor across different tumor types. This adverse event is potentially life-threatening, thus requiring appropriate management. However, its incidence among various tumor types is barely known yet.
Thirty-three original articles of PD-1 inhibitor monotherapy trials were identified based on a PubMed search completed on October 6, 2017. The incidences of all-grade and grade ≥3 diarrhea were collected.
Thirty-three studies containing 7322 patients were included in the meta-analysis. The overall incidence of diarrhea during PD-1 inhibitor monotherapy was 11.3% (95% CI, 9.7%-12.9%) for all-grade and 0.8% (95% CI, 0.6%-1.1%) for grade ≥3 diarrhea. The incidence was higher in renal cell carcinoma (RCC) for both all-grade (14.4% vs 9.4%) and grade ≥3 diarrhea (1.3% vs 0.7%) compared with non-small-cell lung cancer (NSCLC) but only for all-grade diarrhea (14.4% vs 8.5%) compared with urothelial carcinoma. The incidence in melanoma was higher than in NSCLC (15.7% vs 9.4%) and urothelial carcinoma (15.7% vs 8.5%) for all-grade diarrhea but not for grade ≥3 diarrhea. No significant differences were noted between Nivolumab and Pembrolizumab for allgrade diarrhea (11.7% vs 10.7%) or grade ≥3 diarrhea (0.9% vs 0.8%).
The incidence of PD-1 inhibitor-related diarrhea was higher in RCC and melanoma. There was no significant difference in PD-1 inhibitor-related diarrhea between two types of PD-1 inhibitors. These results contribute to heighten clinicians’ awareness of potential adverse events in diverse tumor types.
Radiation Oncology, Chongqing Cancer Institute & Chongqing Cancer Hospital & Chongqing Cancer Center & Chongqing University Cancer Hospital, Chongqing, CN.
Has not received any funding.
All authors have declared no conflicts of interest.
Nintedanib (Vargatef®), an oral triple angiokinase inhibitor of VEGF-, PDGF- and FGF-receptors, is approved in the EU and other countries for treatment of locally advanced or metastatic NSCLC of adenocarcinoma histology in combination with docetaxel after 1st line chemotherapy. Data are scarce regarding efficacy and safety of nintedanib in NSCLC pts who had been pre-treated with ICIs.
This interim analysis is part of this ongoing trial and included 22 treated pts with locally advanced or metastatic lung adenocarcinoma who received nintedanib and docetaxel in 3rd line following ICIs in 2nd line.
Median age was 58 years (range: 45 – 76); 15/22 pts (68.2%) were men, 9/22 pts (40.9%) were ECOG PS0/1, 4/22 pts (18.2%) had brain metastases, and 19/22 pts (86.4%) were current or former smokers. 1st line chemotherapy included pemetrexed (15/22 pts, 68.2%), carboplatin (12/22 pts, 54.6%), cisplatin (12/22pts, 54.6%), bevacizumab (6/22 pts, 27.3%), vinorelbine (4/22 pts, 18.2%), paclitaxel (2/22 pts, 9.1%), and docetaxel (1/22 pts, 4.4%). 2nd line treatment included nivolumab (17/22 pts, 77.3%) and pembrolizumab (5/22 pts, 22.7%). Under nintedanib and docetaxel, 7/12 pts (58.3%) showed a partial response, and 3/12 pts (25.0%) showed stable disease, resulting in a DCR of 83.3% (10/12 pts). Median PFS was 5.5 months (95%CI 1.9 – 8.7). Treatment emergent adverse events (TEAEs) grade ≥3, serious TEAEs, and TEAEs leading to discontinuation occurred in 13/22 pts (59.1%), 11/22 pts (50.0%), and 7/22 pts (31.8%), respectively.
Nintedanib, in combination with docetaxel, showed clinically meaningful efficacy and an acceptable safety profile in a limited number of stage IIIB/IV lung adenocarcinoma patients following chemotherapy and ICIs. Larger studies are warranted to further explore the potential of nintedanib and docetaxel in this novel setting supporting anti-angiogenesis plus docetaxel as a subsequent therapeutic principle in patients progressing under ICI therapy.
NCT02392455.
Boehringer Ingelheim Pharma GmbH & Co. KG.
Boehringer Ingelheim Pharma GmbH & Co. KG.
W. Gleiber: Membership on an advisory board. J. Atz, R. Kaiser: Employee of Boehringer Ingelheim Pharma GmbH & Co KG. All other authors have declared no conflicts of interest.
Older pts with cancer require special consideration with respect to cancer treatment due to multiple comorbidities and an altered immune status. However, older pts are often under-represented in clinical trials, so information about the use of ICIs in elderly pts is marginal.
We retrospectively identified cancer pts aged over 70 yrs treated with any ICI between 04/2012 and 04/2018. Tumor entities, laboratory parameters, steroid use, adverse events (AE) as well as survival and therapy data were compared.
176 pts were included (120 M, 56 F), the median age was 75 yrs (range 70-89) with 89 pts aged 75 or younger (%) and 87 pts over 75 (%). 38 pts (22%) had ECOG 0, 100 pts (57%) had ECOG 1, 23 pts had ECOG 2-3 (13%), in 15 pts (8%) data was not available. 175 pts (99%) had preexisting medical problems. Assessed by the Cumulative Illness Rating Scale for Geriatrics (CIRS-G), 100 pts (57%) had severe comorbidities in at least one, 43 pts (24%) in at least two organ systems. 76 pts (43%) had no severe comorbidities. 89 pts (51%) had malignant melanoma, 62 (35%) non-small cell lung cancer and 8 (5%) renal-cell carcinoma. 155 pts (93%) were in a metastasized stage. 81 pts (46%) received pembrolizumab, 72 (41%) nivolumab, 35 (20%) ipilimumab, 3 (2%) atezolizumab, 1 (0.6%) durvalumab and azacitidin and 6 pts (3%) were treated with a combination of anti-PD(L)1- and anti-CTLA4-antibodies. 19pts (11%) received various consecutive ICIs. An average immunotherapy consisted of 10.2 doses (range 1-52 doses) and took 6.5 months (range 1 day – 3.9 yrs). 152 pts (86%) had AEs. 59 (34%) had severe AEs (CTCAE grad 3-4) and 63 pts (36%) required systemic corticosteroids due to AEs. We observed common described immune related AEs in 82 pts (47%). Therapy discontinuations due to AEs were in 35 pts (20%), in 23 pts (13%) the ICI was definitely stopped. We note a disease control rate (DCR) of 60% (21pts CR; 60pts PR, 67 pts SD) in the follow-up CT scans. The median survival under ICI was 11.8 months (range 5 days – 4.6 yrs).
Our data encourage that the use of ICI in elderly is beneficial even though AEs are commonly reported and potentially harmful.
Department of Hematology, Oncology and Stem Cell Transplantation, Freiburg University Medical Center, Freiburg, Germany.
Has not received any funding.
All authors have declared no conflicts of interest.
Immune checkpoint inhibitors (ICIs) have become standard of care in the management of advanced melanoma. While some evidence suggests that rechallenge in the setting of toxicity with prior ICIs is safe, there is rather limited evidence on the efficacy and safety of rechallenge with alternative ICI in the context of disease progression on initial ICI.
We retrospectively analysed all patients with advanced melanoma who received ICIs between March 2016 and August 2017 at our centre. We used i) RECIST criteria to estimate objective overall response rate (ORR) & ii) Kaplan-Meier method to calculate the progression-free survival (PFS) and overall survival (OS).
Overall, 60 melanoma patients received ICIs, 31 patients discontinued initial ICI due to toxicity (N = 7) or disease progression (N = 24). 17 patients (54.8%) were rechallenged with an alternative ICI and three (17.6%) were rechallenged again with a third ICI. Of the 17 rechallenged patients, their initial treatment was discontinued due to either disease progression (14/17, 82%) or toxicity (3/17, 18%). The types of ICIs in rechallenged patients were as follows: first ICI; ipilimumab [anti-CTLA4] (9), combination ipilimumab and nivolumab [anti-PD-1] (1) and pembrolizumab [anti-PD-1] (7) and subsequent ICI; pembrolizumab (10), ipilimumab (6) and nivolumab (1). The three patients rechallenged for a second time all received nivolumab. The ORR in rechallenged patients was 23.5% vs 51.2% in the non-rechallanged patients. Rates of adverse events were not significantly different compared to first ICIs (Grade 3 irAE: 17.5% vs 23.5%). The median PFS and OS of rechallenged patients were 20 months [95% CI: 3.9-36.0] and 29.4 months [95% CI: 14.6-44.3] respectively, compared to 4 months [95% CI: 1.1-6.8] and 8.4 months [95% CI: 1.9-14.8] for patients who discontinued but not rechallenged. The authors note that comparing these groups should be interpreted with caution as they may constitute different patient cohorts.
In the setting of disease progression or toxicity in the management of advanced melanoma with an initial ICI, rechallenge with alternative ICIs shows some ORR and reasonable PFS and OS. This strategy maybe a viable plan for selected patients.
Guy Faust.
Has not received any funding.
G. Faust: Educational sponsorship to conferences, advisory board: MSD, BMS. All other authors have declared no conflicts of interest.
As the indications and licencing of oncological checkpoint inhibitors (CPI) expands rapidly there are increasing challenges. The need for increased capacity and delivery, support and toxicity management for patients receiving immunotherapy (IO) has been steadily increasing. It is not the numbers alone that are causing a shift in patient management but also the novelty of immune related adverse events (irAEs), the complexity of their management and longditudinaity of treatment.
This study retrospectively reviewed the number of patients receiving IO and assessed the impact of IO on treatment delivery, acute and inpatient services at the Clatterbridge Cancer Centre (CCC), a UK tertiary cancer centre, between June 2016 and June 2018.
A total of 624 patients received licenced CPIs at CCC over the 2 year period. Over the study period there was a 3.7 fold increase in new patients commencing on treatment in the initial and final 6 month period. Five tumour groups were represented. CCC administered 1408 individual treatments in 2016/2017 rising to 3140 in 2017/18. An on-treatment review service (OTR) commenced in June 2017. The OTR activity has increased from 87 individual encounters in July 2017 to 244 in June 2018. Within acute services the centralised telephone triage service saw in increase in IO related calls from 84 calls in Q3 2016/2017 to 316 Q4 2017/2018. This has translated into increased face-to-face patient review with triage attendances rising from 13 to 166. Furthermore IO related admissions have increased from 15 to 120, equating to an 8 fold increase, between the first and last 6 months of the reviewed period.
Immunotherapy has significantly altered the oncological treatment landscape in a very short timeframe. This has left cancer centres ill equipped for the vast impact IO has had in terms of delivery demands, support and acute services. Whilst acute management protocols are plentiful they are alone insufficient to manage the increasing impact of IO. These novel and expanding needs require specific and innovative approaches to allow effective managment. In response to this CCC has developed a comprehensive IO strategy adopting a systems approach and incorporating education development, multilevel management protocols, collaborative working and IO specific services.
Clatterbridge Cancer Center.
Has not received any funding.
A.C. Olsson-Brown: MRC Clinical Fellow at the University of Liverpool; Medical Research Council Fellowship in Clinical Pharmacology and Therapeutics, funded by the MRC(MR/N025989/1), Roche Pharma, Eli Lilly, UCB Pharma, Novartis, the Universities of Liverpool & Manchester. R. Lord: Recipient of travel bursary: AstraZeneca. M. Pirmohamed: Programme Director: Medical Research Council Fellowship in Clinical Pharmacology and Therapeutics, funded by the MRC(MR/N025989/1), Roche Pharma, Eli Lilly, UCB Pharma, Novartis, the Universities of Liverpool & Manchester. J.J. Sacco: Grants and personal fees: Bristol Myers Squibb; Personal fees: Immunocore; Grants: AstraZeneca; Other: MSD Merck. All other authors have declared no conflicts of interest.
Nivolumab (nivo) is a salvage option in relapsed/refractory (r/r) Hodgkin lymphoma (HL) and non-Hodgkin lymphoma. Patients with HIV-related lymphoma may benefit not only anticancer activity of nivo, but also from its potential anti-HIV effect [1]. Just a few cases of HIV-related lymphoma treated with nivo were reported [2,3]. We describe a case series of r/r HIV-related lymphoma receiving nivo in First Pavlov State Medical University of Saint-Petersburg.
Six male patients with r/r HIV-related lymphoma were treated with nivo in 2017. The primary end point was response to therapy, secondary – toxicity, relapse incidence and overall survival (OS) at 12 months. CTCAE v4.03 for the toxicity and immune-related adverse effects have been used. LYRIC criteria for assessing FDG-PET/CT were applied.
The underlying diseases: HL n = 4 (66%) and diffuse large B-cell lymphoma (DLBCL) n = 2 (34%). Median number of prior lines of therapy was 2 (range, 2-3). Three patients received nivo as a bridge to auto-SCT (ASCT). Relapse after ASCT was treated in one patient, two patients didn’t proceed to ASCT. Two patients received nivo as mono, 4 patients in combi with bendamustine and gemcitabine. The median dose of nivo was 1 mg/kg (range, 0,5-1,5 mg/kg), number of nivo doses – 8,5 (range, 2-12). The median of CD4+ was 382 c/mcl (range, 45-490). One patient didn’t receive cART due acute renal failure. Only 5 patients were available for estimating response, one – died before PET/CT was performed. Overall response rate was 100%. Two – partial remission and three (60%) patients had complete metabolic responses. Toxicity according to CTCAE and an immune-related adverse effect was not registered. Relapse of the underlying disease was diagnosed in 2 patients, time to progression were 485 and 266 days. OS was 83,3%. Patient who didn’t receive cART died from undetermined cause. Summary of patients are outlined in the table. Pts – patients, Ds – diagnose, HL – Hodgkin lymphoma, DLBCL – diffuse large B-cell lymphoma, BeGeR – bendamustine, gemcitabine, rituximab, Nivo – nivolumab, CR – complete remission, PR – partial remission, auto-HSCT – hematopoietic stem cell transplantationPts Ds Age HIV load CD4+ cells/mcl cART Nivo N of nivo Response Followed therapy Outcome Follow up 1 HL 33 <40 140 + mono 10 CR Auto-HSCT Remission; Alive; 499 days 2 HL 41 <40 45 + BeGe 12 PR Continued Relapse; 485 days Alive; 506 days 3 HL 36 <40 362 + mono 10 PR Continued Remission; Alive; 528 days 4 HL 40 <40 490 + BeGe 7 CR Auto-HSCT Remission; Alive; 427 days 5 DLBCL 33 3381 410 - BeGe 2 - - - Died; 45 days 6 DLBCL 38 <40 473 + BeGeR 7 CR Auto-HSCT Relapse; 266 days Alive; 397 days
Overall response rate to nivo in patients with HIV-related lymphomas was 100%, one-year OS – 83,3%. Toxicity and immune-related adverse effects were not registered. Preliminary data provide that nivo is effective and safety treatment option for r/r HIV-related lymphoma.
Guihot A, Marcelin AG, Massiani MA, et al. Drastic decrease of the HIV reservoir in a patient treated with Nivolumab for lung cancer. Ann Oncol. -2018.-Vol. 29.-P.517-518
Chang E., Sabichi A.L., Kramer J.R., et al. Nivolumab Treatment for Cancers in the HIV-infected Population. Journal of Immunotherapy. -2018-Vol.41.-P.379–383
Sandoval-Sus J.D., Mogollon-Duffo F., Patel A., et al. Nivolumab as salvage treatment in a patient with HIV-related relapsed/refractory Hodgkin lymphoma and liver failure with encephalopathy. Journal for ImmunoTherapy of Cancer. -2017-Vol.20.-P.49
Raisa Gorbacheva Institute of Children Oncology, Hematology and Transplantation, First Pavlov State Medical University of St. Petersburg, St. Petersburg, Russian Federation.
Has not received any funding.
All authors have declared no conflicts of interest.
In the phase 3 OPTiM trial, intratumoral T-VEC (an oncolytic immunotherapy) significantly improved the overall response rate (ORR) vs GM-CSF in pts with unresectable Stage IIIB–IVM1c melanoma (26% vs 6%; p < 0.001). Treatment options for pts with unresectable or metastatic melanoma who have previously received CPI therapy are limited. This retrospective analysis evaluated response to T-VEC in pts who had received prior CPI therapy.
This analysis included data from two phase 2, single arm clinical trials of T-VEC monotherapy (Study 324 and Study 325). Both studies enrolled pts with unresectable or metastatic, stage IIIB–IVM1c melanoma, who were treated with T-VEC at the approved dose. Pts from these studies were included in the analysis if they had received prior pembrolizumab, nivolumab and/or ipilimumab (alone, sequentially or in combination).
Baseline characteristics of pts who had prior CPI therapy in Study 324 (N = 17)/Study 325 (N = 28), respectively, were: stage IIIB-IVM1a disease 71%/75%; BRAF wild-type 59%/61%; LDH ≤ULN 82%/75%. ORRs with T-VEC in Study 324 and Study 325 were 24% (4/17) and 21% (6/28), respectively (9 partial responses; 1 complete response; see Table). T-VEC-related adverse events (AEs) in CPI-experienced pts in Study 324/Study 325, respectively, were: Grade ≥3 AEs 18%/21%; serious AEs 18%/11%; AEs leading to permanent T-VEC discontinuation 12%/4%. Response rates in pts who received ≥1 dose of T-VEC according to type of prior CPI therapy Tumor response, according to modified WHO criteria using both clinical assessment and radiological imaging, was assessed at Weeks 12, 24 and then every ≤3 months; Pembrolizumab, nivolumab and/or ipilimumab (alone, sequentially or in combination). Only one pt in Study 324 received prior combination ipilimumab/nivolumab and was unevaluable.Response with T-VEC, Study 324 Study 325 Prior pembrolizumab (N = 5) Prior nivolumab (N = 2) Prior ipilimumab (N = 15) Any prior CPI** (N = 17) Prior pembrolizumab (N = 7) Prior nivolumab (N = 9) Prior ipilimumab (N = 22) Any prior CPI** (N = 28) Objective response 1 (20) 0 3 (20) 4 (24) 1 (14) 1 (11) 5 (23) 6 (21) Complete response (CR) 0 0 1 (7) 1 (6) 0 0 0 0 Partial response (PR) 1 (20) 0 2 (13) 3 (18) 1 (14) 1 (11) 5 (23) 6 (21) Disease control (CR/PR/stable disease) 3 (60) 0 8 (53) 9 (53) 3 (43) 2 (22) 9 (41) 11 (39)
Although this retrospective analysis has limitations, the ORR with T-VEC in pts previously treated with a CPI was consistent with the ORR observed with T-VEC in the phase 3 OPTiM trial. Types and incidence of AEs were consistent with the known safety profile of T-VEC. These data suggest that T-VEC demonstrates measurable activity for melanoma pts who have received a prior CPI and have an injectable metastasis.
NCT02366195; NCT02014441.
Amgen Inc.
Amgen Inc.
H. Gogas: Advisor or consultant: Amgen, BMS, MSD, Novartis, Pierre Fabre, Roche. R. Gutzmer: Research support: J&J, Novartis & Pfizer; Consultant/served on speaker’s bureau: Almirall Hermal, Amgen, BMS, GSK, MSD, Novartis & Roche; Speaker’s bureau: Boehringer, Janssen, Merck-Serono, Pfizer. J. Malvehy: Consultant/advisor, honoraria: Amgen, Bristol-Myers Squibb, GlaxoSmithKline, Merck Sharp & Dohme, Roche, Swedish Orphan Biovitrum. J.M. Mehnert: Research grants: Sanofi, Merck, EMD Serono, Novartis, Polyoma, AstraZeneca, Immunocore; Consulting fees: Merck; Advisory board: Boehringer Ingelheim. K. Liu: Employee of Amgen. C.A. Pickett, W. Snyder: Employee and stock holder: Amgen. J. Chesney: Consulting fees/honoraria, travel/accommodation expenses: Amgen; Scientific advisory board member and speakers bureau member: Amgen.
Between 2010 and 2015, pivotal trials with strict enrolment criteria led to the approval of several new immunotherapies for metastatic melanoma (MM). We sought to determine the impact of these treatments in the “real-world” of a population-based study.
The Danish MM database contains data on the entire unselected population diagnosed with MM within a nationwide area. To evaluate the impact of novel treatments, all 843 MM cases (excluding ocular) diagnosed in the three non-consecutive years marked by major changes in the availability of 1st line treatments (2012: i.v. IL-2 and BRAFi; 2014: anti-CTLA-4; 2016: anti-PD-1 and MEKi) were retrieved. Patients were grouped into “trial-like” and “trial-excluded” based on seven predefined eligibility criteria used in all MM registration immunotherapy clinical trials, including CNS metastases and PS ≥ 2.
The baseline characteristics of patients diagnosed in 2012, 2014 and 2016 were similar. In the “trial-like” population (39% of all MM), the median overall survival (OS) was not yet reached in the 2016 group versus 18.8 months in 2014 (hazard ratio [HR] for death 0.52, 95% CI 0.36-0.75; p = 0.0005) and 16.5 months in 2012 (HR 0.41, 95% CI 0.27-0.62; p < 0.0001). In the “trial-excluded” population (61% of all MM), 75% of patients had known CNS metastases and/or PS ≥ 2. Here, the median OS was improved to 6.9 months in the 2016 group versus 5.2 months in 2014 (HR 0.66, 95% CI 0.52-0.84; p = 0.0007) and 4.3 months in 2012 (HR 0.65, 95% CI 0.52-0.83; p = 0.0005). To isolate the effects of immunotherapy, the BRAF wild-type population of 2014 and 2016 was analyzed. Here, “trial-like” patients diagnosed in 2016 had an improved overall survival (median OS not reached in 2016 vs 13.3 months in 2014, HR 0.33, 95% CI 0.20-0.55; p < 0.0001), while “trial-excluded” patients had a 1-year improved survival rate in 2016 (35.9% vs 18.8% in 2014, p = 0.0153).
The introduction of modern treatments has led to an improved survival of real world patients with MM, regardless of their eligibility to clinical trials and BRAF status. These data support indirectly the application of modern treatments, including anti-PD-1, to patient populations which are not represented in pivotal trials.
Herlev and Gentofte Hospital.
Herlev and Gentofte Hospital Research Council.
M. Donia: Honoraria for lectures: Bristol-Myers Squibb, MSD, Sanofi Genzyme, AstraZeneca. H. Schmidt: Honoraria for consultancies and lectures: Novartis, Merck, Bristol-Myers Squibb, Incyte; Restricted research grants: MSD. L. Bastholt: Advisory board: Bristol-Myers Squibb, Roche, Novartis, Merck, Eisai, Bayer Healthcare. I-M. Svane: Honoraria for consultancies and lectures: Novartis, Roche, Merck, Bristol-Myers Squibb, Incyte; Restricted research grants: Novartis, BMS. All other authors have declared no conflicts of interest.
Immuno-oncology (IO) has changed treatment (Tx) landscape of non-small cell lung cancer (NSCLC). IO is efficacious and a less toxic alternative to standard chemotherapy in treating NSCLC. This study analysed 1st line (1L) Tx patterns of patients with stage IIIb/IV NSCLC in real-world setting.
IQVIA Oncology Dynamics (OD) database was used to identify stage IIIb/IV NSCLC patients who initiated and received 1L Tx between June 2017 and March 2018 in the UK. IQVIA OD is a syndicated database collecting longitudinal, anonymized, patient-level oncology data through a quarterly physician panel survey. The UK panel characteristics were: 94% public, 65% university, 12% Scotland/Northern Ireland - 6% Wales - 82% England. The response rate ranged 13-72% among circa 225 physicians per quarterly pulse. Data on patient/disease characteristics, Tx, and biomarkers were extracted and analysed.
In total, data from 738 stage IIIb/IV NSCLC patients receiving 1L cancer Tx were analysed. At questionnaire completion, 57% of patients were male and 68% were aged >60 years. Most patients had cancers with non-squamous histology (79%); fewer had squamous (20%) and other (1%). ECOG performance status was assessed as 0 in 14% and 1 in 76% of patients, and metastases were mostly found in the lung (49%), bone and liver (34% each), lymph nodes local (36%) and distant (25%). COPD was the most common comorbidity (28%); 40% of patients reported none. 49% were former smokers and 22% current smokers. Chemotherapy and targeted therapies were the most frequent 1L Tx received by 301 (41%) and 219 (30%) patients, respectively. 30% of patients (n = 218) received IO Tx in 1L; pembrolizumab was the most common IO therapy (n = 216, 29%). PD-L1 testing was performed among 76% of the latest quarter patients (n = 195/255); 106 (54%) were positive (PD-L1+; ≥50%), 82 (42%) negative (PD-L1-; <50%) and 7 awaiting results. Most PD-L1+ patients received pembrolizumab in 1L (86%), whereas PD-L1- patients were mostly treated with chemotherapy (73%) and one third received targeted therapies.
In the current UK treatment landscape for stage IIIb/IV NSCLC, only 30% of patients were treated with IO Tx in 1L. A high proportion of 1L patients is still treated with traditional chemotherapy.
Editorial assitance was provided by Eleonora Morais from IQVIA.
Outcome Sciences LLC (an IQVIA company).
Bristol-Myers Squibb Pharmaceuticals LTD.
M. Wang: Employee of Bristol-Myers Squibb Pharmaceuticals Ltd. S. Menon, B. Martindale: Employee of IQVIA (IQVIA Oncology Dynamics, a syndicated database collecting oncology data).
For frontline NSCLC patients, multiple immune checkpoint inhibitor (ICI) based regimens have shown promising effects on survival outcomes. However, there are currently no studies with direct comparison between the various regimens. The aim of this study was to establish the most effective treatment using a network meta-analysis (NMA) based on all the available randomized controlled trials.
We conducted an independent review for the RCTs evaluating ICIs in frontline advanced NSCLC. A meta-analysis was performed on the primary outcome of OS using the inverse variance heterogeneity method. The Cochrane Q test for heterogeneity was computed and the I2 coefficient was used to quantify the extent of between-study heterogeneity. A threshold for statistical significance of 0.10 was used for the Cochrane Q test and heterogeneity was considered to be substantial if I2> 60%. Since the heterogeneity was found to be both statistically significant and substantial for the primary outcome, it was judged that a pooled analysis would not be representative of the true treatment effect hence a NMA was computed instead. The NMA was computed by utilizing a frequentist approach with generalized pairwise modeling.
Network estimates of effects on OS showed the superiority of pembro+platinum doublet compared to all the other regimens: pembro (HR = 1.39, 95%CI 1.02-1.89); atezo+platinum doublet (HR = 1.72, 95%CI 1.31-2.26); ipi+platinum doublet (HR = 1.62, 95%CI 1.28-2.04), nivo (HR = 1.82, 95%CI 1.35-2.47) and platinum doublet (HR = 1.79, 95%CI 1.49-2.14). The superiority of pembro+platinum doublet was also consistent on PFS except when compared to pembro (HR = 1.78, 95%CI 0.72-4.42) and ORR except when compared to ipi+platinum doublet (HR = 0.33, 95%CI 0.07-1.44), where the comparisons did not reach the threshold of statistical significance.
Our results support the superiority of pembrolizumab + platinum doublet in the frontline treatment of NSCLC. However, the main limitation of this study is that, due to the limited available data, differences in patient and tumor (pathology and PDL-1 status) characteristics could not be accounted for.
The authors.
Has not received any funding.
All authors have declared no conflicts of interest.
The aim of the present study is to investigate the efficacy of first treatment received after nivolumab in a cohort of patients affected by advanced non-small cell lung cancer (NSCLC).
This is a prospective, multicentre trial conducted between September 2015 and August 2018 at ICS Maugeri Hospital of Pavia (Italy) and Luigi Sacco Hospital in Milan (Italy) on patients who discontinued nivolumab due to disease progression or adverse events. The primary end-point was progression-free survival (PFS) to the first treatment given after nivolumab. Secondary endpoints were response rate (RR) to post–progression therapy and overall survival (OS).
Eighty-four patients have been evaluated with the following baseline characteristics: median age 69 years (range 54 – 83); 66% males; 74% had an ECOG performance status (PS) 0-1; 82% had a metastatic disease; 45% received nivolumab as second–line treatment. Among the 38 patients eligible for post-nivolumab treatment analysis, 75% had and ECOG PS of 0-1; 75% had a metastatic disease; all patients received single agent chemotherapy (CT) (43% docetaxel, 27% vinorelbine, 30% gemcitabine). Median PFS of first-line post-nivolumab treatment was 4.5 months (range 3–16); RR was 33% and OS was 8 months (range 2-17). Patients who received nivolumab in earlier line, had higher PFS and RR to post–nivolumab treatments than patients who received nivolumab as > = 3 line (6 vs 3.5 months, 38% vs 28%, respectively, p < 0.05); OS was 9.5 vs 5.8 months, respectively. Moreover, patients who had a PFS > = 6 months to nivolumab treatment, had a better PFS and RR to the subsequent treatment than patients with shorter benefit to nivolumab (8.1 vs 3.3 months and 45% vs 26%, respectively, p < 0.05). This data is irrespective of the type of CT received.
This trial elicits a better performance of post-nivolumab treatments in patients who received nivolumab in earlier line and who get greater benefit to nivolumab. Some patients (about 20%) experienced also prolonged survival times under CT. This could be due to a long-term effect of immunotherapy on immune memory cells which might be boosted by the further CT lines.
Medical Oncology, ICS Maugeri Pavia.
Has not received any funding.
All authors have declared no conflicts of interest.
GLS-010 is a novel recombinant fully human anti-programmed cell death protein 1 (PD-1) monoclonal antibody. This phase Ia study was to primarily evaluate the safety and tolerance profile of GLS-010 and to identify a proper dose.
A 3 + 3 dose escalation design was applied. GLS-010 was given at 1mg/kg, 4mg/kg and 10mg/kg respectively once every 2 weeks. An exploratory cohort with 240mg fixed dosage was also conducted along with the 10mg/kg cohort.
Until August 2018, 18 subjects were enrolled, including 5 gastric cancer, 4 esophageal cancer, 2 Hodgkin’s lymphoma, 2 cervical cancer, and 1 each for rectum cancer, endometrial cancer, penile cancer, leiomyosarcoma and fibrosarcoma patients. The median dosing number was 5 (range: 1-12). No DLT was observed[JK1], and MTD was not reached. Related treatment emergent adverse event (TEAE)s included haemoglobin decreased (61.1%), blood bilirubin increased (22.2%), fatigue (16.7%), diarrhea (11.1%), decreased appetite (11.1%), neutropenia (11.1%), protein urine present (11.1%), blood creatine kinase increased (11.1%), leukopenia (11.1%) and blood TSH increased (11.1%). Possibly related Grade 3-4 TEAEs was observed in 2 subjects, one subject with haemoglobin decreased, leucopenia and thrombocytopenia, and the other one with haemoglobin decreased only. Pharmacokinetic analysis indicated the drug exposure was increased dose-dependently, with a half-life of 8.2 to 10.1 days. The T cell PD-1 receptor occupancy (RO) rate was over 80% and no anti-drug antibody (ADA) was detected for all subjects. Partial response (PR) was observed in 5 subjects (2 Hodgkin’s lymphoma, 1 gastric cancer, 1 cervical cancer and 1 penile cancer patients). Stable disease (SD) was observed with 1 subject (endometrial cancer). The objective response rate (ORR) was assessed to be 27.8%, and the disease control rate (DCR) was 33.3%.
GLS-010 demonstrated acceptable safety and tolerance, and preliminary efficacy in patients with advanced cancer. A 240mg fixed dosing regimen is currently recommended for further clinical study.
Guangzhou Gloria Biosciences Co., Ltd.
Guangzhou Gloria Biosciences Co., Ltd.
All authors have declared no conflicts of interest.
CAR T-cell therapies (CART) hold out the potential for a paradigm shift in cancer treatment. However, underlying regulatory and manufacturing processes delay hospitals from offering an optimal patient coverage. This study provides an overview of French hospitals regarding CAR T implementation potential and addresses the unmet requirements for a coverage of all French CAR T eligible NHL (Non-Hodgkins Lymphoma) patients.
This study used the PMSI French claim database, which exhaustively lists hospital stays and associated diagnosis, along with a scoring approach applied to main criteria for CAR T hospital eligibility. NHL patient volume was assessed for each hospital and CAR T eligibility was then defined as confirmed NHL with a relapse/refractory disease and non-eligiblility to a Stem Cell Transplantation. The inclusion period was 2015 to 2016. Based on the CAR T implementation guidelines, a score was then attributed to each hospital based on four eligibility criteria: accreditation by the European JACIE committee (score 2); accreditation by the regional health agency for the collection of peripheral Hematopoietic Stem Cells (HSC) (score 1); whether the hospital was referenced by the French Society of Hematology for HSC collection (score 0.5) and for the presence of a leukapheresis unit (score 0.5). “Guidelines from the Francophone Society of Bone Marrow Transplantation and Cellular Therapy (SFGM-TC)” Yakoub-Agha I. et al. (2017). DOI: 10.1016/j.bulcan.2017.10.017.
33 out of 948 hospitals with NHL patients have at least one of the eligibility criteria. Eight hospitals cumulate a score ≥ 2.5, considered as the minimum to be able to integrate CAR T technology within the next 2 years. Seven are University Public Hospitals, one is a Public/Private Oncology center. These eight centers cover only 11% from the calculated CAR T-eligible population.
The current gap between the centers qualified to perform CAR T and the actual size of the population eligible for this therapy suggests that significant adaptations might be necessary, which could directly impact the healthcare structure.
IQVIA.
Has not received any funding.
All authors have declared no conflicts of interest.
In previous reports it has been demonstrated that the 18F-FDG PET/CT performs better than contrast-enhanced CT in assessing the response to immunotherapy treatment in patients affected by metastatic melanomas. Aim of the present study was to evaluate if the first 18F-FDG PET/CT scan performed after the completion of 4 cycles of Ipilimumab treatment was predictive of patient clinical outcome.
26 patients who performed a PET/CT scan before treatment (PET0), after 4 cycles of Ipilimumab (PET1) and at least an additional later evaluation without starting new therapeutic regimen (PET2), were retrospectively evaluated. Response to treatment was evaluated according to PERCIST criteria for PET1 and PET2 and compared with patient clinical outcome. A total of 69 metastatic lesions were also singularly analyzed.
PET1 were performed after a mean of 6 weeks from the end of IPI and PET2 after a mean of 12 weeks from PET1. Patients were classified at PET1 as having progressive metabolic disease (PMD) in 15 cases, stable metabolic disease (SMD) in 6, partial metabolic response (PMR) in 2 and complete metabolic response (CMR) in 3. Discordant results between PET2 and PET1 were observed in 6 patients (from SMD to PMD in 4 cases, from SMD to CMR in 1 and from PMD to CMR in 1).
The results showed that the first PET/CT evaluation after IPI was not always representative for the definitive response to treatment. The immediate start of a new therapeutic line can be proposed for PMD, in all other cases and in particular for SMD an early PET/CT restaging should be performed to confirm the patient clinical outcome.
Ethical Committee.
Has not received any funding.
All authors have declared no conflicts of interest.
Cemiplimab (REGN2810) demonstrated a positive risk/benefit profile and produced antitumour activity in patients (pts) with advanced CSCC in the primary analysis, by independent central review, of a phase 1 CSCC expansion cohorts (ECs). We now report longer follow-up data from the CSCC ECs of the phase 1 study (NCT02383212).
Pts with distantly metastatic or locally/regionally advanced CSCC who were not candidates for surgery were enrolled in ECs 7 and 8, respectively. All pts received cemiplimab 3 mg/kg by intravenous infusion over 30 minutes every 2 weeks for up to 48 weeks. Tumour measurements were performed every 8 weeks by RECIST 1.1 to determine overall response rate (ORR; complete response [CR] + partial response [PR]) according to intention to treat. The data cut-off date was Jan 20, 2018. Tumour response in this report was by investigator assessment.
A total of 26 pts were enrolled (21 M/5 F; 10 in EC 7, 16 in EC 8; median age: 72.5 years [range: 55–88]; ECOG performance status was 1 in 16 pts and 0 in 10 pts). Median duration of follow-up was 11.9 months (range: 1.1–18.2). Median duration of cemiplimab exposure was 36.0 weeks. The most common treatment-emergent adverse event (TEAE) of any grade was fatigue (26.9%). The only TEAEs of grade ≥3 that occurred in more than one pt were hypercalcaemia and skin infection (each 7.7%). ORR was 50.0% (95% CI: 29.9–70.1), with 2 CRs and 11 PRs; 5 patients had stable disease (SD), 6 had progressive disease, and 2 were not evaluable for response. Durable disease control rate (SD or response for ≥105 days) was 57.7% (95% CI: 36.9–76.6). The median time to response was 1.9 months (range: 1.7–7.5). The median duration of response has not been reached, and as of the data cut-off date, for pts with CR or PR, the observed duration of response exceeded 6 months in 9 pts and 12 months in 5 pts.
The increasing duration of response in this analysis provides further evidence of a positive risk/benefit profile for cemiplimab in pts with advanced CSCC.
Medical writing support under the direction of the authors was provided by Emmanuel Ogunnowo, PhD, of Prime (Knutsford, UK) and funded by Regeneron Pharmaceuticals, Inc. and Sanofi according to Good Publication Practice guidelines.
NCT02383212.
Regeneron Pharmaceutical, Inc. and Sanofi.
Regeneron Pharmaceutical, Inc. and Sanofi.
T.K. Owonikoko: Fees for consulting or advisory role: Novartis, Celgene, Lilly, Sandoz, Abbvie, Eisai, G1 Therapeutics, Takeda, Seattle Genetics, Bristol-Myers Squibb, MedImmune. A.K. Salama: Fees for consulting or advisory and speakers’ bureau roles: Bristol-Myers. E. Calvo: Research funding: Boehringer Ingelheim, Roche/Genentech, BMS, Novartis, PsiOxus, Nanobiotix, Janssen, Abbvie, PharmaMar, PUMA, Sanofi, Lilly, Pfizer, Merck, Nektar, Amcure, Amgen, AstraZeneca, Principia, Bayer, CytomX, H3, Incyte, Kura, Loxo, Macrogenics, Menarini, Merck, Serono, Merus, Millenium, Rigontec, Tahio, TesaroReceipt; Honoraria or consultation fees: Novartis, Nanobiotix, Janssen-Cilag, PsiOxus Therapeutics, Seattle Genetics, Pierre Fabre, Boehringer Ingelheim, Cerulean Pharma, EUSA, Abbvie, Celgene; Speaker’s bureau fees: Novartis. N.S. Yee: Institutional research funding: Boston Biomedical Inc., Merck and Co. Inc., Pharmacyclics, Regeneron Pharmaceuticals Inc; Travel/accommodation expenses: Daiichi Sankyo, Foundation Medicine, Caris Life Sciences. D. Mahadevan: Speakers’ bureau and travel, accommodation expenses: Abbvie. J. Niu: Consulting or advisory role fees: Genentech, Biodesix, Paradigm, Ignyta, AstraZeneca and Teueda. K. Kal Mohan: Employee and shareholder of Regeneron Pharmaceuticals, Inc. J. Li: Employee and shareholder of Regeneron Pharmaceuticals, Inc. and Novartis. E. Stankevich: Employee of Regeneron Pharmaceuticals, Inc., a shareholder of Regeneron Pharmaceuticals, Inc., Celgene, Bristol-Myers Squibb, and Merck. M. Mathias: Shareholder and an employee of Regeneron Pharmaceuticals, Inc. I. Lowy: Employee, shareholder, fees for travel and accommodation expenses, leadership: Regeneron Pharmaceuticals, Inc. M.G. Fury: Employee and shareholder of Regeneron Pharmaceuticals, INC.; patents, royalties, other intellectual property: Regeneron Pharmaceuticals, Inc. H.M. Babiker: Honoraria: Bayer, Sirtex; Consulting or advisory role fees: Celgene, Endocyte. All other authors have declared no conflicts of interest.
Cemiplimab (REGN2810), a human monoclonal antibody to PD-1, has exhibited substantial antitumour activities in patients (pts) with advanced malignancies in a Phase 1 study. Most patients with advanced NSCLC do not respond to PD-1 inhibitor monotherapy. Here we report results of the Phase 1 EC 2, a combination regimen of cemiplimab plus RT in advanced NSCLC (NCT02383212).
Pts with advanced NSCLC who had relapsed after, or were refractory to at least first-line therapy and for whom palliative RT was clinically indicated, received cemiplimab 3 mg/kg every 2 weeks intravenously for up to 48 weeks plus RT (9 Gy × 3 times/week given 1 week after first dose of cemiplimab) to a single lesion. The co-primary objectives were to evaluate the safety, tolerability, and efficacy of cemiplimab plus RT. Tumour measurements (of non-irradiated target lesions) were performed by RECIST 1.1 every 8 weeks.
As of 1 Sept, 2017, 33 pts (22 M/11 F; median age 67.0 years [range, 47–82]) were enrolled; 66.7% and 30.3% had an ECOG performance status of 1 and 0, respectively; the status of one pt was unknown. Overall response rate (ORR; complete response [CR] + partial response [PR]) was 18.2% (0 CR and 6 PRs) with a median duration of response of 14.9 months (95% CI: 5.5–14.9). Disease control rate (ORR + stable disease [SD]) was 72.7% (6 PRs + 18 SDs). The most common treatment-emergent adverse events (TEAEs) of any grade were decreased appetite (30.3%), fatigue (27.3%), and cough (24.2%). Grade ≥3 TEAEs occurring in ≥ 2 patients include anaemia (12.1%), hypophosphataemia, and urinary tract infection (each 6.1%). One patient had a TEAE of pneumonitis, considered related to study drug, with an outcome of death.
Cemiplimab plus RT demonstrated antitumour activity in pretreated pts with NSCLC. The safety profile is comparable with other anti-PD-1 agents and RT. The combination therapy regimen did not produce greater efficacy above that which can be achieved with PD-1 inhibitor monotherapy for advanced NSCLC.
Medical writing support under the direction of the authors was provided by Emmanuel Ogunnowo, PhD, of Prime (Knutsford, UK) and funded by Regeneron Pharmaceuticals, Inc. and Sanofi according to Good Publication Practice guidelines.
NCT02383212.
Regeneron Pharmaceutical Inc. and Sanofi.
Regeneron Pharmaceutical Inc. and Sanofi.
M.L. Johnson: Consulting or advisory role: Astellas Pharma, Otsuka. M. Crittenden: Research funding: Jounce, Nanobiotix. S. Jabbour: Research funding grants: Merck, Nestle, outside the submitted work. L. Rosen: Research funding: Regeneron Pharmaceuticals, Inc. P. Garrido: Personal fees: Roche, BMS, MSD, Pfizer, Lilly, Abbvie, Regeneron, AstraZeneca, Novartis, Boerinhger-Ingelheim, outside the submitted work. P. Rietschel: Shareholder and employee, honoraria: Regeneron Pharmaceuticals, Inc. K.K. Mohan, J. Li: Shareholder and employee: Regeneron Pharmaceuticals, Inc. E. Stankevich: Shareholder and employee: Regeneron Pharmaceuticals, Inc.; Shareholder: Celgene, Bristol-Myers Squibb, Merck. M. Feng: Shareholder and employee: Regeneron Pharmaceuticals, Inc.; Shareholder: Bayer. I. Lowy: Shareholder and employee, fees for travel and accommodation expenses, leadership: Regeneron Pharmaceuticals, Inc. M.G. Fury: Shareholder and employee, patents, royalties, other intellectual property: Regeneron Pharmaceuticals, Inc. All other authors have declared no conflicts of interest.
For pts with unresectable HCC, systemic therapy options are limited. Sorafenib and lenvatinib are approved in the US and Europe for HCC treatment. For pts who progress on sorafenib, regorafenib and nivolumab are approved as second-line therapy. Cemiplimab (REGN2810) has demonstrated encouraging efficacy and safety profile in a Phase 1 dose escalation study in pts with advanced malignancies (NCT02383212). We present results of the Phase 1 HCC EC.
HCC pts who are not candidates for surgery and had progressed on, could not tolerate, or refused first-line systemic therapy received cemiplimab 3 mg/kg Q2W IV for up to 48 weeks. The objectives were to evaluate the safety and tolerability (primary) and antitumour activity (secondary) of cemiplimab.
As of 1 Sept 2017, 26 pts were enrolled (25 M/1 F; median age: 65 [range: 40–78] years; ≥1 prior systemic therapy: 24 pts [92.3%]; ECOG PS: 1 in 19 pts [73.1%], 0 in 6 [23.1%], and missing in 1). Median duration of follow-up was 7.2 (range: 1.8–15.5) months. By investigator assessment, 5 pts (19.2%) had partial response, 14 (53.8%) had stable disease, 6 (23.1%) had progressive disease and 1 was not evaluable. Median progression-free survival was 3.7 months (95% CI: 2.3–9.1). Five pts (19.2%) completed the planned 48-week treatment, and 21 (80.8%) discontinued prematurely, primarily due to disease progression (65.4%). Three of the 5 pts who completed planned treatment remain without disease progression at the last response assessment. The most common treatment-emergent adverse events (TEAEs) of any grade were fatigue (26.9%), decreased appetite, increased aspartate aminotransferase (AST), abdominal pain, pruritus, and dyspnoea (each 23.1%). Grade ≥3 TEAEs occurring in ≥ 2 pts were hyponatraemia (3 pts), autoimmune hepatitis (2 pts) and increased AST (2 pts). Two pts (7.7%) had a TEAE resulting in death: 1 with pulmonary embolism that was considered unrelated to treatment and another with hepatic failure considered possibly related to treatment.
Cemiplimab demonstrated evidence of antitumour activity in pts with advanced or metastatic HCC. The safety profile is comparable with that of other anti-PD-1 inhibitors.
Medical writing support under the direction of the authors was provided by Emmanuel Ogunnowo, PhD, of Prime (Knutsford, UK) and funded by Regeneron Pharmaceuticals, Inc. and Sanofi according to Good Publication Practice guidelines.
NCT02383212.
Regeneron Pharmaceutical Inc. and Sanofi.
Regeneron Pharmaceutical Inc. and Sanofi.
G.J. Weiss: Personal fees and ownership interest: Circulogene; Ownership interest: Angiex; Personal fees: Paradigm, Igynta, IDEA Pharma, GLG Council, Guidepoint Global; Travel/lodging fees: Cambridge Health tech Institute, Tesaro; Patent PCT/US2011/020612 issued. G. Falchook: Funding for trial for submitted work. N.S. Yee: Research, travel, accommodation and expenses: Daiichi Sankyo, Foundation Medicine, Caris Life Sciences. S. Shahda: Advisory board fees: Ipsen, Bayer; Research grants: Incyte, Apexian. M. Crittenden: Research funding: Jounce, Nanobiotix. R. Al-Rajabi: Grants: Regeneron Pharmaceuticals Inc., during the conduct of the study. E. Stankevich: Shareholder and employee: Regeneron Pharmaceuticals, Inc.; Shareholder: Celgene, Bristol-Myers Squibb, Merck. M. Feng: Shareholder and employee of Regeneron Pharmaceuticals, Inc.; Shareholder: Bayer. J. Li, M. Mathias, G. Kroog: Shareholder and employee: Regeneron Pharmaceuticals, Inc. I. Lowy: Shareholder and employee, fees for travel and accommodation expenses, leadership: Regeneron Pharmaceuticals, Inc. M.G. Fury: Shareholder, employee, patents, royalties, other intellectual property: Regeneron Pharmaceuticals, Inc. All other authors have declared no conflicts of interest.
Most pts with R/M HNSCC do not respond to PD-1 inhibitor monotherapy. Cemiplimab is a human monoclonal anti-PD-1. An expansion cohort in the phase 1 study (NCT02383212) combined cemiplimab with other potential immune-supportive treatments for pts with R/M HNSCC.
Pts with R/M HNSCC who were refractory to at least first-line therapy and for whom palliative RT is clinically indicated received cemiplimab 3 mg/kg Q2W IV for up to 48 weeks plus RT (9 Gy × 3 times/week beginning 6–8 days after first dose of cemiplimab), cyclophosphamide (200 mg/m2 every 14 days for 4 doses), and GM-CSF (200 μg daily for 7-days after each of the first 4 doses of cemiplimab). The co-primary objectives were to characterise the safety, tolerability, and efficacy of cemiplimab in combination with RT, cyclophosphamide and GM-CSF in 15 pts with R/M HNSCC. Tumour assessments were performed by RECIST 1.1 Q8W.
As of 1 Sept, 2017, 15 pts (9 M/6 F) had been enrolled. Median (range) age was 62.0 (45–78) years; ECOG performance status was 1 in 12 pts (80%), and 0 in 3 (20%); and 14 (93.3%) had received prior RT. The primary site of cancer was upper aerodigestive tract of head and neck. With a median (range) duration of follow-up of 3.3 (0.5–10.2) months, treatment is ongoing in 3 pts (20.0%) and 12 (80%) had discontinued, mainly due to disease progression/recurrence (53.3%). The most common treatment-emergent adverse events (TEAEs) of any grade were fatigue (40.0%), constipation (26.7%), asthenia, dyspnoea, maculo-papular rash and pneumonia (each 20%). The only grade ≥3 TEAE that occurred in > 1 pts was pneumonia (13.3%). By investigator-assessment, there was 1 partial response (6.7%); disease control rate was 40.0% (95% CI: 16.3–67.7; 5 stable disease), 7 pts had progressive disease and 2 were not evaluable. Median progression-free survival by investigator-assessment was 1.8 months (95% CI: 1.7–4.7).
The combination therapy regimen did not demonstrate efficacy above that which can be achieved with PD-1 inhibitor monotherapy for R/M HNSCC.
Medical writing support under the direction of the authors was provided by Emmanuel Ogunnowo, PhD, of Prime (Knutsford, UK) and funded by Regeneron Pharmaceuticals, Inc. and Sanofi according to Good Publication Practice guidelines.
NCT02383212.
Regeneron Pharmaceutical Inc. and Sanofi.
Regeneron Pharmaceutical Inc. and Sanofi.
H.M. Babiker: Honoraria: Bayer, Sirtex; Consulting or advisory role: Celgene, Endocyte. D. Mahadevan: Speakers’ bureau, travel, accommodation expenses: Abbvie. T.K. Owonikoko: Fees for consulting or advisory role: Novartis, Celgene, Lilly, Sandoz, Abbvie, Eisai, G1 Therapeutics, Takeda, Seattle Genetics, Bristol-Myers Squibb, MedImmune. E. Calvo: Research funding: Boehringer Ingelheim, Roche/Genentech, BMS, Novartis, PsiOxus, Nanobiotix, Janssen, Abbvie, PharmaMar, PUMA, Sanofi, Lilly, Pfizer, Merck, Nektar, Amcure, Amgen, AstraZeneca, Principia, Bayer, CytomX, H3, Incyte, Kura, Loxo, Macrogenics, Menarini, Merck, Serono, Merus, Millenium, Rigontec, Tahio, TesaroReceipt; Honoraria/consultation fees: Novartis, Nanobiotix, Janssen-Cilag, PsiOxus Therapeutics, Seattle Genetics, Pierre Fabre, Boehringer Ingelheim, Cerulean Pharma, EUSA, Abbvie, Celgene; Speaker’s bureau fees: Novartis. D. Rischin: Research funding: Genentech/Roche, Merck, Amgen, Regeneron, Bristol-Myers Squibb. M. Crittenden: Research funding: Jounce, Nanobiotix. P. Garrido: Personal fees: Roche, BMS, MSD, Pfizer, Lilly, Abbvie, Regeneron, AstraZeneca, Novartis, Boerinhger-Ingelheim, outside the submitted work. K.K. Mohan, J. Li, M. Mathias: Employee and shareholder: Regeneron Pharmaceuticals, Inc. M.G. Fury: Shareholder, employee, patents, royalties, other intellectual property: Regeneron Pharmaceuticals, Inc. I. Lowy: Shareholder, employee, fees for travel and accommodation expenses, leadership: Regeneron Pharmaceuticals, Inc. E. Stankevich: Shareholder, employee: Regeneron Pharmaceuticals, Inc.; Shareholder: Celgene, Bristol-Myers Squibb, Merck. M. Feng: Shareholder, employee: Regeneron Pharmaceuticals, Inc.; Shareholder: Bayer. All other authors have declared no conflicts of interest.
For pts who progress after first-line platinum based therapy for recurrent/metastatic cervical cancer, there are no therapies available that have been demonstrated to improve survival or quality of life. Cemiplimab (REGN2810), a human monoclonal antibody to PD-1, exhibited encouraging efficacy and acceptable tolerability in a phase 1 dose escalation study. The present report focuses on interim data from the phase 1 cervical cancer expansion cohorts (ECs) of cemiplimab as a monotherapy (EC 23) or in combination with hypofractionated radiotherapy (hfRT) (EC 24) (NCT02383212).
Pts with recurrent or metastatic cervical cancer resistant to or intolerant of platinum and taxane doublet therapy received cemiplimab 3 mg/kg Q2W IV for up to 48 weeks, in ECs 23 and 24, and hfRT (9 Gy x 3 times/week given 1 week after first dose of cemiplimab) in EC 24. The co-primary objectives were to evaluate the safety, tolerability, and efficacy of cemiplimab monotherapy or in combination with hfRT. Tumour response assessments (in non-irradiated target lesions) were performed by RECIST 1.1 Q8W.
As of 1 Sept, 2017, these ECs were fully enrolled with 20 pts (EC 23, n = 10, EC 24, n = 10). Median (range) age was 55.0 (31–76) years (EC 23) and 51.5 (29–65) years (EC 24). ECOG performance status 1 vs. 0 was 60% vs. 40% and 80% vs. 20%, respectively, for EC 23 and EC 24. Investigator-assessed overall response rate (ORR; complete response [CR] + partial response [PR]) was 10.0% (0 CR and 1 PR) in each of EC 23 and EC 24. At the time of data cut-off, both responses were ongoing with durations of 3.7+ months. The most common treatment-emergent adverse events (TEAEs) of any grade were diarrhoea (40.0%), fatigue, hypokalaemia and pain in extremity (each 30.0%) in EC 23, and diarrhoea and urinary tract infection (each 30.0%) in EC 24. There was no grade ≥3 TEAE reported in > 1 patient in either cohort.
Cemiplimab as monotherapy and in combination with hfRT demonstrated antitumour activity with an acceptable safety profile in pts with metastatic or recurrent cervical cancer. Cemiplimab monotherapy vs. chemotherapy in ≥ 2nd line cervical cancer is currently being evaluated in a global randomised phase 3 study (NCT03257267).
Medical writing support under the direction of the authors was provided by Emmanuel Ogunnowo, PhD, of Prime (Knutsford, UK) and funded by Regeneron Pharmaceuticals, Inc. and Sanofi according to Good Publication Practice guidelines.
NCT02383212.
Regeneron Pharmaceutical Inc. and Sanofi.
Regeneron Pharmaceutical Inc. and Sanofi.
D. Rischin: Research funding: Genentech/Roche, Merck, Amgen, Regeneron, Bristol-Myers Squibb. A. González-Martin: Consulting, advisory, speakers’ bureau, travel accommodation expenses: AstraZeneca, Tesaro, Roche, Pharmamar. J.Y. Hou: Consultant and fees: Foundation Medicine, Massive Bio, Inc. D. Cho: Consulting fees: Pfizer, BMS, Exelixis, Genentech, Prometheus. G. Falchook: Funding for trial for submitted work. S. Jabbour: Research funding grants: Merck, Nestle outside of the submitted work. K. Moore: KInstitutional consultancy (honorarium and ad board) fees: Tesaro, Genentech Roche, Clovis, AstraZeneca (for agents not involved in the SOLO-1 study), Immunogen, VBL Therapeutics, Janssen. M.G. Fury: Employee and shareholder: Regeneron Pharmaceuticals, Inc.; Patents, royalties, other intellectual property: Regeneron Pharmaceuticals, Inc. M. Feng: Employee and shareholder: Regeneron Pharmaceuticals, Inc., Bayer. J. Li: Employee and shareholder: Regeneron Pharmaceuticals, Inc., Novartis. I. Lowy: Employee, shareholder, fees for travel and accommodation expenses, leadership: Regeneron Pharmaceuticals, Inc. M. Mathias: Employee and shareholder: Regeneron Pharmaceuticals, Inc. All other authors have declared no conflicts of interest.
Tislelizumab, a humanized IgG4 mAb with high affinity and specificity for PD-1, was engineered to minimize binding to FcɤR on macrophages, thus abrogating antibody-dependent phagocytosis, a potential mechanism of T-cell clearance and resistance to anti-PD-1 therapy. Previous reports from this first-in-human study (NCT02407990), and other early phase studies, suggest tislelizumab was generally well tolerated and had antitumor activity in pts with advanced solid tumors.
Patients with UC received tislelizumab at doses of 2, 5, or 10 mg/kg Q2W or Q3W, and 200 mg Q3W. Tumor cell (TC) and immune cell (IC) PD-L1 expression were retrospectively assessed with the VENTANA PD-L1 (SP263) assay. Adverse events (AEs) were assessed per NCI-CTCAE 4.03 and tumor assessments were performed every 9 wks using RECIST v1.1.
A total of 17 pts with UC (median age, 71 yr [range 39–79]) received tislelizumab, the majority of which received 5 mg/kg Q3W (n = 11). All pts were Caucasian and 14 were male; median number of prior systemic anticancer therapies was 1 (range 0–4). Treatment-related AEs (TRAEs) occurring in ≥ 3 pts included fatigue (n = 5), infusion-related reaction (n = 3), and rash (n = 3). Grade ≥3 TRAEs were fatigue, hyperglycemia, and type 1 diabetes mellitus (T1DM; n = 1 each). Three pts experienced serious TRAEs (infusion-related reaction [n = 1], hyperglycemia and T1DM [n = 1], and pneumonitis [n = 1]). As of 27 Apr 2018, median duration of follow up was 8.8 mo (range 0.9–29.1) and 2 pts remained on treatment. All pts were evaluable for response. Confirmed CR (n = 1) and PR (n = 4) were observed; SD was achieved in 3 pts. ORR and DCR were 29% (95% CI 10.3, 55.9) and 47% (95% CI 22.9, 72.1), respectively. Sixteen samples were available for PD-L1 evaluation. Responses were observed in 4 (n = 1 CR; n = 3 PR) of 10 pts with PD-L1+ tumors (defined as ≥ 25% TC or IC expressing PD-L1 by IHC), while 1 (PR) in 6 pts with PD-L1– tumors responded.
Tislelizumab was generally well tolerated in pts with UC and responses were observed in both PD-L1+ and PD-L1– diseases. Tislelizumab is currently being investigated in China as monotherapy for pts with PD-L1+ UC (CTR20170071).
Editorial and medical writing assistance were provided by Regina Switzer, PhD (SuccinctChoice Medical Communications, Chicago, IL).
NCT02407990.
BeiGene, Ltd.
BeiGene, Ltd.
S. Sandhu: Honoraria: Amgen, Bristol-Myers Squibb, Merck; Consulting or advisory role: Amgen; Speakers\' bureau: Bristol-Myers Squibb, Merck. A. Hill: Stock and other ownership interests: Tasman Oncology; Research funding: Tasman Oncology; Travel, accommodations, expenses: Bristol-Myers Squibb. H. Gan: Consulting or advisory role: AbbVie, Merck Serono; Speakers\' bureau: Abbvie, Bristol-Myers Squibb, Ignyta; Research funding: AbbVie; Travel, accommodations, expenses: AbbVie, Ignyta, Merck Sharp & Dohme. M. Friedlander: Honoraria: AstraZeneca, MSD; Consulting or advisory role: AstraZeneca, MSD; Research funding: BeiGene (Inst). M. Voskoboynik: Travel, accommodations, expenses: Bristol-Myers Squibb. P. Barlow: Travel, accommodations, expenses: MSD, Roche.J. Song, Y. Zhang, L. Liang: Employee: BeiGene. J. Desai: Consulting or advisory role: Amgen, Beigene, Bionomics, Eisai, Lilly, Novartis; Research funding: Bionomics (Inst), GlaxoSmithKline (Inst), Novartis (Inst), Roche (Inst). All other authors have declared no conflicts of interest.
AB122 is a fully human monoclonal antibody targeting PD-1. It binds tightly to a different PD-1 epitope than other currently approved antibodies in this class. Here we report preliminary data from an ongoing, open-label, dose-escalation (3 + 3 design) study evaluating the safety, tolerability, immunogenicity, pharmacokinetics, pharmacodynamics, and antitumor activity of AB122 monotherapy in patients (pts) with select advanced solid tumors.
Eligible pts had pathologically confirmed select advanced solid tumors, received ≤5 prior lines of systemic therapies, and had measurable disease per RECIST v1.1. AB122 was administered intravenously (IV) every 2 wks (Q2W) at escalating doses (80, 240, 720 mg). Intermediate Q2W doses and other schedules (every 3 wks [Q3W] or 4 wks [Q4W]) were also evaluated. Adverse events (AEs) were graded according to NCI CTCAE v4.03 and antitumor activity was assessed using RECIST v1.1.
As of 15Aug2018, 19 pts with advanced ovarian (n = 7), endometrial (n = 3), colorectal (n = 3), gastroesophageal (n = 2), head and neck, breast, prostate, and lung (n = 1 each) cancer were treated at doses of 80 mg Q2W (n = 3), 240 mg Q2W (n = 6), 360 mg Q2W (n = 1), 360 mg Q3W (n = 4), and 480 mg Q4W (n = 5). Most pts were female (74%) and White (89%); median age was 68 yrs (51-80). The number of doses received ranged from 1 to 18; 8 pts remain on study. The most common treatment-emergent AEs were fatigue (42%), diarrhea (21%), urinary tract infection, back pain, nausea, and anemia (16% each). 16% of pts each had ≥Grade 3 AEs and serious AEs but none were considered treatment-related by investigators. There were no dose-limiting toxicities or discontinuations due to AEs. Data from pts in the 80 and 240 mg Q2W cohorts showed that AB122 serum concentrations ≥1.5 μg/mL (equivalent to 10 nM) are associated with full receptor occupancy. Among 12 efficacy-evaluable pts, the disease control rate was 42%.
These preliminary results indicate that AB122 monotherapy was well tolerated and active in various advanced solid tumor types. The dose and schedule of 240 mg IV Q2W were selected for further evaluation as monotherapy and in combination with other agents.
Arcus Biosciences, Inc.
Arcus Biosciences, Inc.
L.C. Seitz, W. Berry, D. Ashok, D. Direnzo, L. Jin, S.J. Lee, A. Park, D. Piovesan, J. Karakunnel: Employee of Arcus Biosciences, Inc. A. Rieger, J.B.L. Tan, M.J. Walters: Employee of Arcus Biosciences, Inc.
CTLA4 (cytotoxic T-lymphocyte-associated protein 4) is homologous to the co-stimulatory protein CD28, and both receptor binds to CD80 and CD86. Cancer cells can be recognized and destructed by the host’s immune system. In this setting, CTLA4 functions as a brake to dampen anti-tumor T cell responses, which promote cancer progression. Antibodies targeting CTLA4 was expected to subvert T cell inhibition. Moreover, regulatory T cells residing in the tumor microenvironment can be killed by anti-CTLA4 antibody since these cells have higher expression level of CTLA4 comparing with the activated T cells. Yervoy, an anti-CTLA4 antibody, was the first FDA-approved antibody targeting the immune checkpoint family, and it protects a fraction of cancer patients when either used alone or in combination with other drugs. We asked whether the efficacy of anti-CTLA4 antibodies could be evaluated in humanized mouse models. And if so, whether we can use in vivo assay to guide the development of potent antibodies targeting CTLA4.
Here, we generated humanized CTLA4 knock-in mouse (B-hCTLA4) with a chimeric PD-1 receptor and this mouse were validated by Yervoy. Then, we utilized humanized B-hCTLA mice and implanted syngeneic tumors subcutaneously, followed by treating mice with purified testing CTLA4 antibodies and Yervoy. Via this approach, we are able to discern several clones that have better efficacy than Yervoy. These clones were further selected for humanization. The cohort of recombinant humanized CTLA4 antibodies were screened in B-hCTLA4 mice.
We successfully generated B-hCTLA4 mouse for anti-human CTLA4 antibody in vivo efficacy evaluation. A cohort of CTLA4 specific antibodies were successfully generated and purified. The clones with better efficacy than Yervoy were screened using Biocytogen’s humanized mouse platform. These clones were humanized and screened by using B-hCTLA4 mice. Both the Fv and Fc portion of the lead antibody were optimized using the B-hCTLA4 mice, leading to the humanized form of the antibodies.
Using the platform established at Biocytogen, we successfully discovered two antibodies whose efficacy is equivalent or exceed that of Yervoy. The clinical evaluation of these new entities are wanted.
Yi Yang.
Beijing Biocytogen Co., Ltd.
All authors have declared no conflicts of interest.
Avelumab—a human anti–PD-L1 IgG1 monoclonal antibody—showed favorable efficacy and safety in pts with mMCC in the phase 2 JAVELIN Merkel 200 trial (NCT02155647), leading to its approval in multiple countries. Here, we describe real-world experience with avelumab in European pts with mMCC.
European pts participating in the EAP (NCT03089658) had stage IV mMCC and progressive disease (PD) on/after chemotherapy or were ineligible for either chemotherapy or participation in clinical trials. In contrast to JAVELIN Merkel 200, pts could have ECOG PS ≥ 2, treated brain metastases, or immunosuppressive conditions. Pts received a 3-mo supply of avelumab (administered 10 mg/kg IV Q2W until PD or unacceptable toxicity); resupply was allowed for pts with complete response (CR), partial response (PR), stable disease, or clinical benefit per physician assessment. No central imaging was obtained.
As of April 30, 2018, of 521 requests for avelumab across 37 countries, 343 were received in Europe: 305 were approved (including 20 for immunocompromised [IC] pts), 29 were medically rejected, and 9 were withdrawn. Most requests were from France (n = 96) and Italy (n = 87). 275 European pts received avelumab. Median age was 73 y (range, 28-95 y), and 69% of pts were male. Of 250 pts on treatment >3 mo, 145 (58%) had either unevaluable tumors or no data reported (including 11 IC pts). Of 105 evaluable pts, physician-assessed objective responses were observed in 54.3% (57 pts; including 3 IC pts [2 CR and 1 PR]) with 25.7% CR (27 pts) and 28.6% PR (30 pts). Median duration of treatment in pts with response was 195 d (range, 30-570 d). The disease control rate in evaluable pts was 75%. No new safety signals were reported. The EAP is ongoing but closing in 2018 as required postapproval.
The avelumab EAP provides an alternative treatment option for pts with mMCC with PD on/after chemotherapy or who are ineligible for either chemotherapy or clinical trials. In a real-world setting, avelumab showed efficacy and safety consistent with JAVELIN Merkel 200. Results in this abstract to be presented in part at ESMO 2018 by Dr. Paul Nathan, 19-23 Oct 2018, Munich, Germany, and published as abstr 4657. Dr. Paolo Ascierto to present at ESMO-IO.
Editorial assistance was provided by ClinicalThinking, a Nucleus Global Company, Hamilton, NJ.
Merck KGaA, Darmstadt, Germany.
Funding was provided as part of an alliance between Merck KGaA, Darmstadt, Germany and Pfizer, Inc.
P.A. Ascierto: Financial interest: Bristol Myers Squibb, Roche-Genentech, MSD, Array, Novartis, Amgen, Merck-Serono, Pierre-Fabre, Incyte, Genmab, Newlink Genetics, Medimmune, Syndax, AstraZeneca. P. Nathan: Financial interest: AstraZeneca, Bristol Myers Squibb, Novartis, Immunocore, Roche, Pfizer. V. Kasturi, M. Hennessy, J. Reed: Employment by EMD Serono, Billerica, MA. A. Engelsberg: Employment by Pfizer Pharma GmbH, Berlin, Germany. S. Hariharan: Employment by and financial interest in Pfizer, Inc., New York, NY. C. Lebbe: Financial interest: Bristol Myers Squibb, MSD Merck, Amgen, Roche, Novartis. All other authors have declared no conflicts of interest.
Anti-GITR (glucocorticoid-induced TNFR-related protein) agonist and gemcitabine (gem) combination improves anti-tumor activity pre-clinically compared to either therapy alone. We investigated the effect of this combination as part of a multi-center phase 1b multi-dose escalation and expansion trial in patients (pts) with advanced solid cancers.
Part C of the phase 1b trial enrolled adult pts with advanced solid cancers for which gemcitabine was clinically appropriate. Pts had failed at-least one prior systemic therapy, had measurable disease and were ECOG PS 0-1 at baseline. All pts received gem (1000mg/m2) IV on D1 and D8 and TRX518 on D2 of a 21-day cycle in two escalation cohorts (2mg/kg or 4mg/kg load[L] in C1 followed by 1mg/kg maintenance[M] from C2). The highest tested safe dose identified in escalation was further evaluated in expansion. Primary endpoint was safety. Secondary and exploratory endpoints included response (RECIST v1.1), PK and PD.
One patient was replaced as patient discontinued before completion of C1 and DLT assessment and the other patient withdrew consent before staging scan but was evaluable for DLT assessment. One patient had clinical progression and did not have a staging scan. Patients have not had C3 scans at the time of this report $ Four of 6 pts have not had C3 scans at the time of this report.Part C: TRX518 + Gemcitabine (1000mg/m2) N Response- Evaluable SD PD NE Cohort/TRX518 dose Cohort 1 - 2mg/kg L/1mg/kg M 4 2 0 2 2 Cohort 2 - 4mg/kg L/1mg/kg M 6 5 4 1 1 Expansion - Cohort 2 dose 16 7 4 3 9 PBC 16 10 6 4 6$ Overall 26 14 8 6 12
From January to September 2018, 26 pts were dosed;16/26 had pancreaticobiliary cancers (PBC) of which14/16 had prior gem. There was 1 treatment-related SAE (G3 anemia, G4 lymphopenia, G3 hypoalbuminemia [DLT] and G3 hypokalemia [DLT]). No treatment-related deaths occurred. Of the 14 response-evaluable pts in cohorts 1 (n = 2), 2 (n = 5) and expansion (n = 7), 57.1% (8/14) had SD. At the 4mg/kg L/1mg/kg M TRX518 dose, 66.7% (8/12) of evaluable pts had SD. 60% of PBC had clinical benefit of which 87.5% had prior gem.
In pts with heavily pre-treated advanced cancer, for which gem is clinically appropriate, TRX518 plus gem was well tolerated with no new safety signals. Preliminary evidence of clinical benefit was observed including in pts with PBC previously treated with gem. This study is ongoing.
NCT02628574.
Leap Therapeutics.
Leap Therapeutics.
V. Velcheti: Advisory/Consultant Role: Merck, BMS, AstraZeneca, Genentech, Celgene, Takeda, Foundation Medicine, Nektar Therapeutics. T. Bauer: Employment - Sarah Cannon Research Institute; Tennessee Oncology Consulting or advisory role - Guardant Health; Ignyta (Inst); Loxo; Moderna Therapeutics (Inst); Pfizer Research funding - Abbvie (Inst); Aileron Therapeutics (Inst); Amgen (Inst). J. Luke: Scientific advisory board: 7 Hills, Actym, Alphamab Oncology, Array, BeneVir, Mavu Consultancy: Aduro, AstraZeneca, Bristol-Myers Squibb, Castle, CheckMate, Compugen, EMD Serono, Ideaya, Janssen, Merck, NewLink, Novartis, RefleXion. O. Rixe: Research Funding: Bexion, Kyowa Hakko Kirin, Leap Therapeutics, Medimmune, Newlink Genetics, Pfizer, Regeneron, Rgenix, Seattle Genetics; Travel, accommodations, expenses: Bexion. D. Bajor: Research funding: Apexigen, Roche/Genentech, Seattle Genetics, Tesaro. G.S. Naik: Former employee: Biocon, current employee: Leap Therapeutics. C. Sirard: Current employee: Leap Therapeutics. D. Davar: Consulting or advisory role: Incyte, Merck; Research funding: Bristol-Myers Squibb, Checkmate Pharmaceuticals, Incyte, Merck.
4-1BB, also called TNF receptor super family member 9 or CD137, is mainly expressed on the surface of activated T, NK, and mononuclear cells. 4-1BB is activated by its ligand 4-1BBL or an activating 4-1BB monoclonal antibody, and stimulates T cell activation, proliferation and cytokine production. This co-stimulatory signal can also multiply antigen presenting cells and result in enhanced cell factor secretion. Experiments show that 4-1BB co-stimulation regulates T cell and antigen presenting cell function for antitumor immunity, providing new targets for tumor immunological therapies.
Here, we developed a cohort of 4-1BB specific antibodies using the classic hybridoma technology. Then, we utilized humanized 4-1BB mice (B-h-4-1BB) and implanted syngeneic tumors subcutaneously, followed by treating mice with purified testing antibodies. Via this approach, we are able to discern several clones that effectively inhibited tumor growth without prior knowledge of their in vitro activities. These clones were further selected for humanization. The cohort of recombinant humanized 4-1BB antibodies were screened in B-h4-1BB mice.
A cohort of 4-1BB specific antibodies were successfully generated and purified. These purified antibodies were screened by their efficacy to stimulate anti-tumor activity in live animals using Biocytogen’s humanized mouse platform. Top clones were humanized and screened by using B-h4-1BB mice. Both the Fv and Fc portion of the lead antibody were optimized using the B-h4-1BB mice, leading to the humanized form of the antibodies.
We adopted an in vivo screen approach to discover candidates of 4-1BB specific antibodies that have potent anti-tumor activity. We discovered the leads with the most potent anti-tumor activity.
Biocytogen Boston Corp.
Biocytogen Boston Corp.
All authors have declared no conflicts of interest.
Over the past years, monoclonal antibodies have been successfully utilized in clinical trials to block or activate key mediators of immune checkpoint pathways, including CTLA-4, PD-1, PD-L1 and OX40 et.al. Although significant benefits including complete regression and long-term survival were reported among the majority of participants, not all patients respond. In order to optimize the efficacy and minimize toxicities of anticancer immunotherapies, immune checkpoint antibodies are being tested in combination with other conventional therapeutics and newer immunotherapies. However, along the IO drug development process, in vivo efficacy models have always been a rate-limiting step, especially for the combination therapy of two IO antibodies.
Here, we generated human PD-1 knock-in (B-hPD-1) mice with a chimeric PD-1 receptor, which is recognizable by human PD-1 antibodies, and can be used to test human PD-1 antibody. Mouse colon cancer MC38 were genetically modified with over-expression of hPD-L1 and knock-out of mPD-L1 (MC38-hPD-L1) and used to evaluate the in vivo efficacy of human PD-L1 antibodies. RNA and protein expression of human PD-1 were evaluated by RT-PCR and flow cytometry. We utilized this humanized PD-1 mice and implanted MC38-hPD-L1 subcutaneously, followed by treating mice with Keytruda/Tecentriq and/or Cisplatin. In this way, we also generated double humanized PD-1/OX40 mouse (B-hPD-1/hOX40) to evaluate the in vivo efficacy of combination therapy. The synergistic effect of Keytruda and human OX40 Ab were evaluated by using humanized double Knock-in B-hPD-1/hOX40 mice pairing with three syngeneic tumor models respectively.
Both the FDA approved human PD-1 antibody Keytruda and human PD-L1 antibody Tecentriq showed a synergistic effect with chemotherapy Cisplatine in B-hPD-1 mice inoculated with MC38-hPD-L1 cells. Keytruda and anti-human OX40 mAb shown clear combination effect in B-hPD-1/OX40 mouse with not only hot tumor MC38, but also moderate tumor EL4 and cold tumor B16F10.
B-hPD-1 mouse and B-hPD-1/hOX40 mouse, represent useful in vivo efficacy models for evaluation of the combination effects of IO/Chemotherapy drugs and IO/IO.
Beijing Biocytogen Co., Ltd.
Beijing Biocytogen Co., Ltd.
All authors have declared no conflicts of interest.
PD-1/L1 axis blockade shows durable responses and extended overall survival across cancer types in a subset of patients. Tumour Necrosis Factor Receptor (TNFR) superfamily activation is also being tested clinically to improve patient responses. Current interventions using therapeutic CD137 agonists to activate T cells are limited by adverse safety effects and poor efficacy as monotherapies. The generation of a bispecific agonist of CD137 following PD-L1 crosslinking allows a greater therapeutic window with improved safety and efficacy.
An anti-CD137/PD-L1 mAb2 was generated by introducing a CD137-binding specificity into the Fc-region of a human IgG1 targeting PD-L1 mAb. FcgR binding was decreased by introducing a LALA mutation. Binding characterisation was assessed and in vitro activity measured in a mouse primary OT-1 CD8+ T cell assay. The anti-tumour activity and PK/PD of anti-CD137/PD-L1 mAb2 was tested in mouse tumour models.
An anti-CD137/PD-L1 mAb2 was developed, which binds to mouse PD-L1 enabling CD137 agonism (in vitro EC50: 3 pM in primary antigen-specific OT-1 assay). The mAb2 significantly reduced tumour growth in 3 syngeneic mouse tumour models (MC38, CT26 and B16-F10) with dose-dependent inhibition seen in CT26, resulting in a significant survival benefit at concentrations of 0.3 mg/kg or above. This growth inhibition was coincident with increases in tumour and peripheral activated CD8 T cells. Liver pharmacology was minimal as defined by histopathology.
We report intra-tumoural and peripheral PD changes leading to an increase in the proliferative CD8+ T cell response following dosing with an anti-CD137/PD-L1 bispecific mAb2. These changes were dose dependent and coincident with tumour growth inhibition. In in vitro T cell activation assays the bispecific was superior to control antibodies and relevant combinations. Minimal liver pharmacology and no toxicity was observed with the anti-CD137/PD-L1 mAb2 unlike with other monoclonal antibodies targeting CD137. This warrants the development of a first-in-class anti-human CD137/PD-L1 bispecific antibody with a novel mode of action and improved therapeutic index for the treatment of human cancer.
The authors.
Has not received any funding.
All authors have declared no conflicts of interest.
NOX-A12 is an inhibitor of the chemokine CXCL12 for treatment of solid tumors. Binding of CXCL12 by NOX-A12 prevents receptor engagement and blocks the ability of CXCL12 to form a chemotactic concentration gradient. The Opera study (NCT03168139) is a Phase 1/2 study to evaluate pharmacodynamic effects and safety of monotherapy with NOX-A12 as well as safety and efficacy of a combination of NOX-A12 with pembrolizumab in metastatic microsatellite-stable colorectal (CRC) and pancreatic (PaC) cancer.
Patients received 300 mg NOX-A12 twice weekly during the two-week monotherapy phase. Biopsies were taken from liver metastases before treatment and after NOX-A12 monotherapy for analysis of immune cell infiltration and cytokine signature. In the combination phase, patients received repeated 21-day cycles of 300 mg NOX-A12 and 200 mg pembrolizumab.
20 patients were recruited, thereof 11 with CRC and 9 with PaC. 15 of the patients (75%) are male, with a median age of 62 (CRC) and 68 years (PaC). Patients were heavily pretreated with a median of 5 (CRC) and 3 lines (PaC) of prior treatment. Known best responses to last prior treatment was PD for 16 out of 20 patients. The AE profile was comparable with the pembrolizumab profile or typical for the underlying diseases. Thus far, 5 of the patients (25%) achieved a stable disease (3 CRC and 2 PaC). Interestingly, some of the patients with SD or clinical benefit also showed a favorable tissue cytokine response upon NOX-A12 monotherapy. Higher changes of CXCL12 levels in tissue observed upon monotherapy – indicating more complete CXCL12 neutralization – correlated with a favorable cytokine profile.
NOX-A12 alone and combined with pembrolizumab was safe and well tolerated. Changes in the cytokine signature in tumor tissue suggest that NOX-A12 modulates the tumor microenvironment and induces an immune-stimulatory Th1-like signature in multiple patients of which some show signs of disease stabilization or clinical benefit. The observed time of treatment compares favorably with the expected clinical course of such end-stage and heavily pre-treated patient population.
EudraCT: 2016-003657-15.
Noxxon Pharma.
Noxxon Pharma AG.
N. Halama, U. Prüfer: Research funded by Noxxon Pharma AG. A. Frömming, D. Beyer, D. Eulberg, J. Jungnelius, A. Mangasarian: Employee of Noxxon Pharma AG.
5-Fluorouracil plus irinotecan or oxaliplatin with target therapy are standard 1st line therapy for metastatic colorectal cancer (mCRC). 5-Fluorouracil plus oxaliplatin are known to present immunogenic properties. Durvalumab (D) is a human monoclonal antibody (mAb) that blocks programmed cell death ligand 1 (PD-L1) binding to its receptor (PD-1). Tremelimumab (T) is a mAb directed against the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). This study is designed to evaluate whether the addition of PD-L1 and CTLA-4 inhibition to FOLFOX increases treatment efficacy.
After a phase I with 9 patients, this phase II study (ClinicalTrials.gov NCT03202758) will assess the efficacy and safety of FOLFOX/D/T association in patients (pts) with good performance status pts (ECOG < 2) with untreated, RAS mutational status mCRC. Prior adjuvant therapy is allowed provided recurrence is > 6 months post-completion. Assuming no safety concerns the study will go on to include 39 additional pts. Pts will receive folinic acid/5-fluorouracil (400mg/m2 as bolus followed by 2400mg/m2 as a 46h infusion)/Oxaliplatin (85mg/m2) q14 days with D (750 mg) D1 q 14 days and T (75 mg) D1q 28 days. After 6 cycles of FOLFOX only, D/T will continue until disease progression, death, intolerable toxicity, or patient/investigator decision to stop. Primary endpoint is safety and efficacy according to PFS; secondary endpoints include overall response rate and quality of life.
9 patients were enrolled in the phase I. Grade 3-4 treatment-related adverse events occurred in 3 of 9 patients. The most common treatment-related grade 3-4 adverse events were neutropenia and diarrhea. No patients discontinued due to a drug-related adverse event. First clinical outcomes have shown 4 patients with partial response, 3 patients with stable disease and 2 patients with progression disease. Ancillary analysis were performed.
FOLFOX/D/T was tolerable with manageable toxicity. Preliminary results have shown encouraging clinical outcome. The results of this phase I study have led to phase II study which are ongoing.
NCT03202758.
Nicolas Isambert.
AstraZeneca.
All authors have declared no conflicts of interest.
Cabozantinib (C) is a tyrosine kinase inhibitor against VEGFR2, RET, MET and AXL that has demonstrated clinical activity in RCC and 2L HCC and has been shown to promote an immune-permissive environment. C is undergoing clinical trials in combination with immune checkpoint inhibitors (ICI). The objectives of this preclinical study were to determine systemic cytokine profile changes induced by the combination with αPD1 and to assess cooperativity in anti-tumoral effects.
Syngeneic colon (CT26, Colon38) and bladder (MBT2) tumor models were established in BALB/C, C57Bl6 or C3H/HEJ mice. C (10 or 30 mg/kg po qd) and αPD1 (10 mg/kg ip 2x weekly) were given for up to 30 days simultaneously or with a delay for αPD1, and serum was collected.
Serum cytokines & chemokines were assessed after coadministration of C (30 mg/kg) and αPD1 for 30 days, or when αPD1 was administered 14 days after C. VEGFA, IL3, GM-CSF, MIP1beta, IL17, CCL5, IFNγ, IL10 and IL5 were elevated in the former with a median of 72x (range 39-1207x), as compared to αPD1 or C administered alone with a median of 3.7x (1.4-135x) and 1.0x (0.5-1.9x), respectively, relative to vehicle-treated animals. Delayed treatment of αPD1 did not exhibit the same increase in analyte levels as seen with the simultaneous administration (median of 1.74x (1-9.9x). For anti-tumor efficacies, two C doses were explored in CT26 (T/C10/30 67/38% - tumor growth inhibition T/C ratios at 10 or 30 mg/kg), MBT2 (T/C10/30 37/14%) and Colon38 (T/C10/30 21/12%) models. C at 30 mg/kg led to strong anti-tumor efficacy in MBT2 and Colon38, limiting any ICI combinatorial impact to the post-treatment tumor expansion. For CT26 (30 mg/kg) and MBT2 (10 mg/kg), simultaneous dosing of C and αPD1 showed greater anti-tumor effects than single agents despite only limited effects of αPD1 alone. C + αPD1 more than doubled the treatment-specific time that tumors required to reach 1000mm3 compared to C alone.
Together, these results indicate that simultaneous treatment of C with αPD1 lead to strong systemic increases in key cytokines & chemokines and can result in robust anti-tumor efficacy. Our results support the clinical exploration of C + ICI combinations and the potential of this TKI for use in further clinical settings.
Ipsen Innovation SAS.
Ipsen Innovation.
S. Rolland, J. Nahkle, R. Delille: Employee of Ipsen. S.G. Klinz, F. Meyer-Losic: Employee and stock owner: Ipsen. All other authors have declared no conflicts of interest.
DPX-Survivac is a novel T cell activating therapy containing a mix of HLA class I peptides designed to evoke a T cell response against survivin, previously optimized for immunogenicity when delivered with low dose CPA. Epacadostat (E) is a potent, selective IDO-1 inhibitor that may reverse tumour-associated immune suppression. This Phase 1b/2 is evaluating if a de novo tumour-specific T cell response, combined with the alteration of immune suppression will result in clinical benefit.
Ovarian cancer subjects with disease progression were treated with DPX-Survivac (2x 0.25 mL q3w, up to 6x 0.1 mL q8w), low dose CPA (50 mg BID, alternating weeks), and E (up to 300 mg BID). Primary endpoints include safety and immunogenicity. Secondary endpoints include objective response by RECIST 1.1. PBMCs were collected at specific timepoints for immune analysis. Tumour biopsies were collected pre-treatment and at day 56 for analysis of infiltrating immune populations.
Enrollment to the Phase 1b is now closed; clinical and immunological analysis is ongoing for treated subjects. Treatments have been well tolerated. Evaluable subjects have shown strong and sustained survivin-specific systemic immune responses by IFN-γ ELISPOT of PBMCs. Survivin-specific T cells cloned from the blood of a subject with tumour regression were also identified in her on-treatment tumour using TCR-β sequencing. Analysis of subpopulations suggests a direct correlation between tumour size and clinical benefit- subjects with sum of target lesion ≤5 cm showing increased tumour regression. Six of 13 subjects with a target lesion sum ≤5 cm showed tumour decrease during treatment. Four partial responses have been observed in this subgroup of subjects and disease control was observed in 9 of 13 subjects.
Combination of DPX-Survivac, low dose CPA, and E has demonstrated so far, robust systemic survivin-specific T cell responses and evidence of survivin-specific T cells in the tumour, supporting the mechanism of action of DPX-Survivac. The tumour burden at the time of treatment may correlate with clinical outcome. The combination is well-tolerated.
NCT02785250.
IMV Inc.
IMV Inc. Incyte Corporation.
O. Dorigo: Advisory board: Merck, Geneos, Tesaro; Speaker: AstraZeneca, Tesaro. A.M. Oza: IIT with [IMV Inc.] J. Strauss: Consulting: Tempus; Stock ownership: Abbvie, Abbott Laboratories, Bristol-Myers Squibb, Intuitive Surgical, Johnson & Johnson, Merck. S. Ghamande: Speaker: Tesaro; Consultant: Advaxis, no bearing on this trial. S. Fiset, L.D. MacDonald, G.M. Weir, M.M. Stanford, O. Hrytsenko: Employee of IMV Inc. and own stock in the company. H. Torrey: Employee of IMV Inc. R. Newton, L. Leopold: Employee and shareholder of Incyte corporation. G.N. Rosu: Chief Medical Officer of IMV Inc. and holds stock options for the company. All other authors have declared no conflicts of interest.
This trial is based on a combination strategy with two immunostimulatory agents in the search of synergism that may induce responses with long term clinical benefit in ABC pts. Here, we report the results from the run-in-phase of the study (ClinicalTrials.gov Identifier: NCT03025880).
HER2-negative ABC pts previously treated with anthracyclines and taxanes (unless contraindicated) and ≥ 2 lines of hormone therapy, if hormone receptor (HR)-positive disease, were eligible. Treatment consisted of 21-day cycles (cy) of P 200mg on day 1 and Gem on days 1 and 8. A 6 + 6 design was used with 2 dose levels (DL) of Gem: 1250mg/m2 (DL0) and 1000mg/m2 (DL1). The primary objective was to define the Recommended Phase II Dose (RP2D) based on dose limiting toxicities (DLT) during the first cy (<33% of pts with a DLT); secondary objectives included evaluation of safety and efficacy. Pts were treated until progression, or unacceptable toxicity whatever occurs first.
Fourteen pts were included in DL0; 3 pts were replaced due to early progressive disease (PD). DL1 was not explored as DL0 was considered the RP2D . Median age was 48 years (range 32-61), and 9 pts had triple negative disease. The majority of pts had an ECOG performance status ≤ 1 (n = 13) and visceral involvement (n = 12); 8 pts had ≤2 involved sites. Median number of prior lines (any therapy) for metastatic disease was 3 (range 1-7). Pts received a median of 4.5 cy of Gem (range 1-11) and 3 cy of P (range 1-11). One DLT was observed, Gem dose omission on day 8 due to grade (G) 2 thrombocytopenia. G ≥ 3 adverse events (AEs) related to the treatment were reported on 5 pts; G4 AEs included thrombocytopenia and lymphopenia (7.1% each); G3 AEs included neutropenia (14.3%), anemia, leukopenia, thrombocytopenia, diarrhea and transaminases increase (7.1% each). The best overall tumor response was stable disease on 3 pts at the time of this analysis; 9 pts discontinued treatment due to PD, 1 pt due to respiratory failure, and 2 pts died of breast cancer. Twenty-two pts have been included on the ongoing phase II part.
P can be safely combined with Gem. P 200mg and Gem 1250mg/m2 was declared as the RP2D.
NCT03025880.
GEICAM, Spanish Breast Cancer Group.
Merck Sharp & Dohme Corp (MSD).
J. Cortes Castan: Honoraria: Roche, Novartis, Eisai, Celgene, Pfizer. Consulting/advisor: Roche, Celgene, AstraZeneca, Cellestia Biotech, Biothera, Merus, Seattle Genetics. E.M. Carrasco: Husband: Honoraria: Pfizer. All other authors have declared no conflicts of interest.
CD47 is a ubiquitously expressed cell surface marker responsible of maintaining self-tolerance under physiological conditions. Upon binding to its receptor signal regulatory protein alpha (SIRPα) on myeloid cells, CD47 triggers a negative signal that ultimately inhibits phagocytosis. The upregulation of CD47 is an escape mechanism frequently used by tumor cells to gain immune resistance. Thus, the blockade of the CD47-SIRPα interaction by high affinity monoclonal antibodies (mAbs) is a promising strategy to restore phagocytosis and enhance tumor cell clearance. However, CD47 expressed on healthy cells may act as potential site of toxicity and antigen sink for high affinity CD47 mAbs. We developed bifunctional antibody derivatives, herein referred to as local inhibitory monoclonal antibodies (licMABs), to restrict the blockade of the CD47-SIRPα innate immune checkpoint to tumor cells and therefore reduce systemic toxicities.
LicMABs are SIRPα-antibody fusion proteins that target a tumor antigen with high affinity and block the CD47-SIRPα interaction with the endogenous SIRPα domain, which has a physiologically low affinity to CD47. For proof or principal studies, we developed licMABs targeting CD33, a validated antigen for Acute Myeloid Leukemia (AML). The anti-tumor efficacy of SIRPα-αCD33 licMABs via antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) mechanisms was evaluated in vitro. Furthermore, the potential toxicity of SIRPα-αCD33 licMABs on CD33-negative cells was addressed by preferential binding assays and ex vivo cytokine release studies in whole blood.
SIRPα-αCD33 licMABs mediated specific lysis of AML cell lines and primary, patient-derived AML cells by ADCC and induced a higher phagocytosis of AML cells than CD33 mAbs. Most importantly, SIRPα-αCD33 licMABs showed a potentially safe cytokine profile and preferentially bound to tumor cells co-expressing CD33 and CD47 even in an excess of healthy CD47-positive CD33-negative cells.
Our results demonstrate the high efficacy and potentially low toxicity of SIRPα-αCD33 licMABs and qualify these molecules as a novel and promising immunotherapy for the treatment of AML.
AG Hopfner, Gene Center, LMU.
DFG CRC1243, m4-Award of the Bavarian Ministry of Economic Affairs and Graduate School of Quantitative Biosciences Munich.
All authors have declared no conflicts of interest.
The curative potential of hematopoietic cell transplantation in the treatment of acute myeloid leukemia (AML) suggests that the immune system is involved in the disease outcome. One of the major obstacles in the development of immunotherapeutic protocols is the lack of specific antigens. Mutations in NPM1 gene are characteristic for AML, they are found in about a third of AML patients and are relatively stable during the disease course. Patients with these mutations form a specific group with better prognosis which might be associated with an immune response against the mutated protein or against its interaction partners.
We evaluated a set of known markers of immune suppression in a cohort of 30 AML patients at diagnosis, with special focus on possible differences between patients with wild-type and mutated NPM1 (NPMwt versus NPMmut). Positive cell fraction and the mean expression levels were determined for each marker using flow cytometry. NPM1 mutations were detected by PCR and by immunofluorescence (cytoplasmic signal).
We found decreased HLA class I expression in a subgroup of NPMmut, but not in NPMwt group. Independently of NPM1 mutations, a part of patients had increased expression of PD-L1, CLIP or Tim-3. These markers were partly mutually correlated. We have also detected increased expression of CD47 and decreased expression of HLA-DR on leukemia blasts in NPMmut group compared to NPMwt. Furthermore, NPMmut patients had higher percentage of CD27-positive B-cells. On the other hand, no difference between NPMmut and NPMwt were found on T-lymphocytes.
Our results confirm that patients with mutated NPM1 form an immunologically specific AML subgroup with increased markers of immune suppression at diagnosis.
Institute of Hematology and Blood Transfusion.
Ministry of Health of the Czech Republic (grant No 16-30268A).
All authors have declared no conflicts of interest.
4-1BB (CD137; TNFRSF9) binds 4-1BBL, which triggers subsequent proliferation and activation of immune cells, T and NK cells in particular. We designed and generated PVR-4-1BBL, a hexameric protein of 4-1BBL fused to PVR (CD155), the major ligand for TIGIT that marks exhausted T cells. This fusion protein efficiently bind to its targets TIGIT and 4-1BB on cells as well as in soluble form.
To evaluate the in vitro efficacy of PVR-4-1BBL, T cells were isolated from PBMCs of healthy individuals or AML patients and then stimulated with anti-CD3 alone or in combination with PVR-4-1BBL. The percentages and numbers of CD25+CD69+ T cells were measured by flow cytometry. Secreted INF-γ and TNF-α were measured by ELISA. In order to test the activity of 4-1BB signaling induced by the fusion protein, we performed 4-1BB-stimulated- NFκB reporter assay.
As a result, PVR-4-1BBL activated human T cell to induce cellular proliferation and activation as well as production of cytokines, such as INF-γ and TNF-α. The fusion protein increased T cell activation particularly under suboptimal conditions of TCR stimulation. Functional activity of this fusion protein on 4-1BB-mediated cellular NF-κB signaling was more potent than a 4-1BB agonist antibody in the presence of TIGIT-positive cells in close proximity. Our fusion protein showed target-specific potency in T cell activation in the presence of both TIGIT and 4-1BB. Therefore it is expected to have a lower side effect than 4-1BB agonist antibody which may affect peripheral T cells. According to a recent report, TIGIT was up-regulated on CD8+ T cells of AML patients. We confirmed that 4-1BB was also induced in T cells from AML patients’ PBMCs along with TIGIT when stimulated by anti-CD3. The fusion protein increased T cell activation and IFN-γ production much more than agonistic anti-4-1BB antibody in AML patients’ PBMCs.
In conclusion, PVR-4-1BBL confers a dual targeting function, effectively enhancing T cell proliferation, activation and cytokine production. Therefore, we propose that the dual-targeting fusion protein, PVR-4-1BBL is a promising drug candidate for AML treatment designed by a novel immunotherapeutic approach.
MOGAM Institute for Biomedical Research.
MOGAM Institute for Biomedical Research.
All authors have declared no conflicts of interest.
The synergistic effect of concomitant chemoradiation (cRTCT) as standard of care in a broad range of cancers has been widely described. Recently, the combination of chemoradiation and immune checkpoint inhibitors raised great interest for the treatment of cancer patients. Although, previous studies have demonstrated the ability of chemotherapy (CT) or radiotherapy (RT) to improve host anti-tumor immunity, the immunological effects of concomitant chemoradiation are still poorly investigated. The main goal of this study was to understand how chemoradiation interplays with host anti-tumor T cell immunity.
Mouse lung carcinoma and colorectal cancer models were used. Concomitant chemoradiation consisting in the combination of cisplatin (5mg/kg) plus 5-fluorouracil (25mg/kg) and radiation, at a single dose of 8Gy was delivered when tumors reached 90mm2. High throughput immunological analysis was performed on tumor infiltrating T lymphocytes (TILs) .
cRTCT promoted higher tumor infiltration by CD8 T cells as compared to CT or RT used as monotherapy. RNAsequencing analysis performed on TILs isolated from mice treated by cRTCT or CT or RT revealed that cRTCT promote immunological signatures characterized by strong Th1 polarization (T-bet, IFNg…) and high expression of T cells cytotoxicity markers (granzyme and perforin) as compared to CT or RT alone. Notably, immune checkpoint receptors such as CTLA-4, PD-1, TIM-3, TIGIT, and LAG-3 expression were highly upregulated by cRTCT. Kinetic monitoring of immune checkpoint receptors showed an early induction on TILs starting at day 3 and persisting over 15 days post cRTCT. This was concomitantly associated to PD-L1 induction on tumor cells and the tumor microenvironment and demonstrates the ability of cRTCT to promote a more suitable inflammatory tumor microenvironment than CT or RT alone. The association of anti-CTLA-4 and anti-PD-1 therapy with chemoradiation induced a strong tumor regression as compared to the immunotherapy combination alone.
Our results demonstrate the ability of cRTCT to promote a high inflammatory tumor microenvironment and strongly support the rational to combine cRTCT with immune checkpoint inhibitors.
UMR1098.
Ligue contre le Cancer.
All authors have declared no conflicts of interest.
Chemokines and their receptors influence many hallmark processes in cancer. C-C Chemokine receptor 4 (CCR4) and its ligands are highly expressed in many types of human tumors, and are often associated with poor prognosis. CCR4 antagonism has been demonstrated to reduce tumor growth in various mouse tumor models. Here we have assessed a small molecule inhibitior of CCR4 as a therapeutic agent to potentiate the effects of anti-CTLA-4 in the CT26 and KCM tumor models.
The subcutaneous CT26 colon cancer model and the orthotopic KCM pancreatic cancer model were used to assess the effects of CCX6239, a CCR4 inhibitor, in combination with anti-CTLA-4 antibody. Mice implanted with CT26 cells were randomized into study groups on day 7 based on the pre-treatment tumor sizes. KCM cells were implanted directly into the pancreas. Dosing of CCX6239 and anti-CTLA-4 began on day 7. CCX6239 was dosed orally twice daily at 30mg/kg, and anti-CTLA-4 was dosed intraperitoneally on days 7, 11, and 15 at 100μg/mouse.
Combined treatment with anti-CTLA-4 and CCR4 inhibitor significantly decreased tumor size and increased the proportion of long-term survivors in the CT26 model. Mice with tumor regression exhibited a high proportion of CD8 T cells that recognized a CT26-specific neoantigen, and these mice were resistant to re-inoculation with CT26 cells (without further dosing of either drug), while another type of tumor grew well in the same mice. In the KCM model, anti-CTLA-4 alone provided substantial tumor growth inhibition, which was further enhanced by CCX6239. Interestingly, CCX6239 alone also significantly reduced tumor burden in this model.
A specific CCR4 inhibitor reduces tumor growth either alone or in combiniation with anti-CTLA-4 in two preclinical models, suggesting CCR4 is a potential new target for cancer treatment.
ChemoCentryx Inc.
ChemoCentryx Inc.
C. Li, J.J. Campbell, L.S. Ertl, Z. Miao, V. Chhina, A. Kumamoto, S. Yau, T. Dang, P. Zhang, T.J. Schall, R. Singh: Full time employee and stock holder of ChemoCentryx Inc.
Combined used of virotherapy and immune checkpoint inhibitors (ICI) has arisen as a promising treatment for cancer. VCN-01 is a selective oncolytic adenovirus (OAd) with hyaluronidase activity that can enhance the activity of ICIs as it can potentially increase their extravasation. The aims of this project are: to demonstrate that VCN01 is able to increase both ICI extravasation and antitumour activity, and to generate new OAds with potentiated hyal-related mechanisms of action.
OAds were i.v. administered in NP-18 (PDL1+; human pancreatic cell line) xenograft male athymic mouse model. Fluorescently labelled anti-PD-L1 was used to assess its extravasation. TRAMP-C2 tumours were subcutaneously implanted in C57BL/6J. VCN01 was injected i.t alone or in combination with 5 doses i.p. of anti-PD-L1 antibody. New OAds were generated with two different hyalorunidase genes (A and B). Different locations within the viral genome and different splicing acceptors to express these hyaluronidases were tested.
VCN-01 significantly increased the extravasation of anti-PD-L1 antibodies into NP-18 tumours compared to a control oncolytic adenovirus ICOVIR15K and to control group, due to VCN-01’s capability to express hyaluronidase. Administration of VCN-01 within subcutaneous TRAMP-C2 tumours, alone or in combination with anti-PD-L1 antibody showed anti-tumour activity when compared with PBS or anti-PD-L1 treated tumours. The combined treatment showed increased anti-tumour activity, but not significant, when compared to VCN-01 alone. Most of the new hyal-OAds presented attenuated tumour cytotoxicity in vitro compared to VCN-01 except from VCNA and VCNB. Moreover, both candidates presented significantly higher hyalorunidase activity in vitro at 24h and 72h post infection compared to VCN-01.
VCN-01 enhances the extravasation of anti-PD-L1 to tumours. Combination of VCN-01 with anti-PD-L1 antibody significantly delayed tumour growth over time when compared with PBS or anti-PD-L1 treated tumours. VCNA and VCNB maintain tumour cytotoxicity and show higher hyaluronidase activity compared to VCN-01.
VCN Biosciences S.L.
VCN Biosciences S.L.
M. Farrera, A. Mato-Berciano, S. Morgado, S.L. M. Bazan-Peregrino: Employed by VCN Biosciences S.L. and presenting a company\'s product. R. Alemany: Founder and part of advisory board of VCN Biosciences. All other authors have declared no conflicts of interest.
Oncolytic virus (OV)-based therapies have an emerging role in the treatment of solid tumors, involving both direct cell lysis and immunogenic cell death. Nonetheless, tumor-associated stroma limits the efficacy of OVs by forming a barrier that blocks efficient viral penetration and spread. Moreover, the stroma plays a critical role in progression and immunosuppression of cancer. Fibroblast activation protein-α (FAP) is highly overexpressed in cancer-associated fibroblasts (CAFs), the main cellular component of tumor stroma, and in this study we assessed whether arming oncolytic adenovirus (OAd) with a FAP-targeting Bispecific T-cell Engager (FBiTE) could retarget infiltrated lymphocytes towards CAFs, enhancing viral spread and anti-tumor immunity to improve therapeutic activity.
The BiTE against FAP was constructed using an anti-human CD3 single-chain variable fragment (scFv) linked to an anti-murine and human FAP scFv. This FBiTE was inserted in the oncolytic adenovirus ICOVIR15K under the control of the major late promoter, generating the ICO15K-FBiTE. ICO15K-FBiTE replication and potency were assessed in HT1080 and A549 tumor cell lines. The expression of the FBiTE and the activation and proliferation of T cells that induced along with the T-cell-mediated cytotoxicity of CAFs were evaluated by flow cytometry in vitro. In vivo T-cell biodistribution and antitumor efficacy studies were conducted in NOD/scid/IL2rg-/- (NSG) mice.
FBiTE expression from the OAd ICO15K did not decrease the infectivity and replication potency in vitro. T-cells activation and proliferation, leading to T-cells-mediated cytotoxicity of FAP-positive cells was successfully achieved after FBiTE-mediated binding of both CD3+cells and FAP+ cells. In vivo, FBiTE expression increased intratumoral accumulation of T-cells and decreased the level of FAP, a marker of CAFs, in tumors. The antitumor activity of the FBiTE-armed OAd was superior to the parental virus.
Combination of viral oncolysis of cancer cells and FBiTE-mediated cytotoxicity of FAP-expressing CAFs might be an effective strategy to overcome a key limitation of oncolytic virotherapy, encouraging its further clinical development.
IDIBELL.
Ministerio de Economía y Competividad and Generalitat de Catalunya.
All authors have declared no conflicts of interest.
HER2 is dysregulated in a wide range of solid tumors, including breast cancer, and is an attractive target for tailored oncologic treatment. GBR 1302 is a HER2xCD3 bispecific antibody that redirects cytotoxic T-cells to kill HER2 overexpressing cancer cells. This unique mode of action is anticipated to result in superior antitumor activity in HER2-positive tumors by harnessing the cytotoxic capabilities of patients’ existing T-cells.
This ongoing, phase 1, first-in-human, open-label, multicenter, dose-escalation study is evaluating GBR 1302 in adults with progressive HER2-positive solid tumors for which no standard or curative treatment is available. Subjects receive intravenous GBR 1302 on Day 1 and Day 15 in 28-day treatment cycles at escalating dose levels, starting at 1 ng/kg. The first 4 cohorts consisted of a single subject; subsequent cohorts are being enrolled using a 3 + 3 design. Blood samples were collected for pharmacokinetic (PK) and anti-drug antibody (ADA) analyses (secondary endpoints). Quantification of GBR 1302 serum concentrations (for PK) and detection/confirmation of anti GBR 1302 antibodies (for immunogenicity) were performed using validated LC/MS/MS and ELISA methods, respectively. PK parameters were evaluated using standard non-compartmental methods.
As of 21 August 2018, PK data were available from 31 subjects over dose range of 1 ng/kg to 750 ng/kg. Serum concentrations were less than the lower limit of quantification of 50 pg/mL at the first dose (1 ng/kg), and only transient concentrations were observed at 3 and 10 ng/kg dose levels. Evaluable PK profiles were observed from 30 ng/kg onwards. GBR 1302 showed maximum serum concentration (Cmax) around the end of infusion, after which serum concentrations declined bi-exponentially with a mean terminal half-life of around 4 to 7 days. Both Cmax and area under the curve (AUC0-t) showed a near dose-proportional increase up to 750 ng/kg (maximum evaluated dose). None of the samples collected from subjects up to cohort 5 showed positive ADA response.
Per ongoing analysis, GBR 1302 showed a favorable, linear PK. None of the subjects evaluated so far showed positive ADA response.
Editorial assistance was provided by Jacqueline Benjamin, PhD of Prescott Medical Communications Group, Chicago, IL.
NCT02829372.
Glenmark Pharmaceuticals SA.
Glenmark Pharmaceuticals SA.
G. Gudi, E. Fluhler: Employee of Glenmark Pharmaceuticals Inc., V. Ca, S. Gn: Employee of Glenmark Pharmaceuticals, Ltd., C. von Gunten, J. Back: Employee of Glenmark Pharmaceuticals SA.
Colorectal cancer (CRC) is the third most diagnosed cancer among men and second among women globally. Generally, with age, the risk of this cancer grows and is happened by various genetic alterations. The immune system plays an important role against CRC and that provides a new means to CRC therapy. Our research is focused on the relative expression level of ten different immune genes namely IFNγ, CD 273, CD274, PD-1, β2M, CD3e, CD28, HLA-A, ICAM 1, and CTLA 4 which are known to play important role in immune regulation during different cancers. We hypothesize that the expression of the above-mentioned genes will be altered during CRC and therapy will promote the expression of genes involved in the activation of immune system.
RNA was extracted from the whole blood sample of CRC patient (n = 50) and normal (n = 34) volunteers. Then cDNA was synthesized, and RNase inhibitor was used. Specific primers for all these genes were designed. Later, RT-qPCR has been done using SYBR® green master mix. Patient demographics were also recorded. All tested genes have been normalized to a housekeeping gene, RPL11. Values of relative expression for all genes were calculated using REST 2009 software.
Among screened genes, CD 273, CD274, and CTLA 4 were not expressed while, IFNγ, PD-1, β2M, CD3e, CD28, HLA-A, and ICAM 1 found expressed in CRC. After comparing the gene expression of advanced CRC patients with normal control, PD-1 found upregulated (P < 0.045). Clinicopathological correlation revealed PD-1 is highly expressed (P < 0.001) in advance stage of CRC patients upon treatment with both chemotherapy and immunotherapy (capecitabine, oxaliplatin, irinotecan hydrochloride, folinic acid, fluorouracil, and bevacizumab).
Our study indicates significant induction of PD-1 at the advanced stage of CRC and during therapy. It suggests that conventional therapies for CRC must be modulated and/or a combination of therapy with anti PD-1 drug should be conducted for the better clinical outcome of CRC.
Cancer and Mutagenesis Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia.
King Abdulaziz City for Science and Technology.
All authors have declared no conflicts of interest.
Several novel and highly promising immunotherapeutic approaches are now in development, such as vaccination with synthesized long peptides or vaccines that encode relevant neoepitope sequences, as well as activated T cell therapies that recognize neoepitopes. Neoantigens (aka neoepitopes) are derived from tumor-specific somatic mutations accumulated during cancer progression. The discovery of patient specific high affinity neoepitopes is challenging, as several genomics attributes of the tumor and germline cells need to be measured and integrated to derive accurate predictions. Most of the existing bio-informatics pipelines for neoepitope prediction are complex sets of command line scripts that are complicated to operate, difficult to run efficiently on cloud or High-Performance Computing (HPC) environments and difficult to validate for the use of clinical trials and submissions.
We established a bio-informatics pipeline that allows calling of somatic variants, the estimation of the expression levels of the mutated proteins and the calling of patient specific MHC genotypes from paired tumor-normal tissue samples. The information can be used for predicting and selecting the most specific patient neoepitopes. We have ensured that the pipeline can run directly on most scalable cloud and HPC environments. The pipeline also includes the required regulatory features so that it can be used in clinical trials, and the results can be directly submitted to regulatory authorities.
We present the automated neoepitope prediction pipeline and show that it includes all compliance features required for operating in regulated clinical environments. We also demonstrate the scientific validation of the pipeline by reproducing and confirming the results from a published medical case of a metastatic breast cancer patient that was successfully treated with tumor-infiltrating lymphocytes (TILs) reactive against 4 patient specific mutated proteins.
We provide a standardized bio-informatics pipeline for neoepitope prediction that can be automated, runs highly efficient on the most scalable HPC environments and can be validated to support clinical trials and decisions.
Genedata AG.
Has not received any funding.
N. Masloboeva-Siwach: Employee of Genedata Inc. S. Ribi, H. Lempiäinen, T. Rujan: Employee of Genedata AG. M. Flesch: Employee of Genedata GmbH.
Copper is elevated in tumors and the use of copper-chelating agents is under intense investigation. It has been reported that copper plays a key role in the immune system, but its activity is unclear. Recent advances in immunotherapies have shown great potential for treating several cancers. However, tumors express molecules, such as the Programmed Death Ligand 1 (PD-L1), to prevent immune cell activity. Several anti PD-1/PD-L1 therapies have been approved by the FDA, but concerns have been raised about their safety. Therefore, there is a need for different approaches to target this pathway.
Experiments were performed in 3 neuroblastoma (NB) cell lines SK-N-FI, SH-SY5Y, SK-N-BE(2)C and 2 glioblastoma (GBM) cell lines U87MG, MO59J. PD-L1, T cells markers and Copper transporter1 (CTR1) protein levels were determined using western blot and flow cytometry. Intracellular copper was measured by ICP-MS analysis. In vivo activity of copper-targeting drugs was assessed in NB syngeneic mouse models.
Tissue microarrays from NB and GBM patient tumors showed a significant correlation between CTR1 and PD-L1 expression (p = 0.00014 & p = 0.012, respectively). Suppression of CTR1 using siRNAs caused a decrease of intracellular copper which in turn led to a downregulation of PD-L1 expression in NB cells, whereas, addition of copper into the media clearly induced PD-L1 mRNA and protein upregulation. Dextran-Catechin (DC) and TEPA, drugs dysregulating copper homeostasis, downregulated PD-L1 expression in both NB and GBM cells. In vivo studies showed that DC prolonged mouse survival by decreasing tumor size in the NB mouse model. Ex vivo immunohistochemistry staining confirmed that downregulation of CTR1 is associated with reduced PD-L1 expression. In addition, DC treatments showed an increase of tumor-infiltrating CD4+ and CD8+ lymphocytes and activated NK cells in the NB immune-competent mouse model.
There is a strong association between PD-L1 expression and intracellular copper levels. Copper dysregulating agents reduce PD-L1 in vitro and in vivo and in turn, promote a significant increase of tumor-infiltrating lymphocytes. This study highlights the potential to enhance tumor immune surveillance by targeting intracellular copper levels.
Children\'s Cancer Institute Australia.
The National Health and Medical Research Council (NHMRC).
All authors have declared no conflicts of interest.
CD47 is a transmembrane protein found on the surface of many cells in the body. Its expression is often up-regulated in many cancer cells. The receptor of CD47 was identified as SIRPa, which is expressed on phagocytic cells. Engagement of SIRPa by CD47 serves as “do not eat me” signal, thus inhibiting the phagocytic activity of macrophages. Cancer cells often hijack this pathway by boosting CD47 expression on their surface, in order to prevent innate cell-mediated phagocytosis. Molecules blocking the CD47-SIRPa interaction will unleash such inhibition and promote tumor destruction. Accordingly, anti-CD47 antibodies offer new hope to successful cancer treatment. Despite people are enthusiastic about CD47 as a potential target, the side effects associated with CD47 blockade have emerged as a major concern. For example, treatment with anti-CD47 antibody greatly reduced the number of circulating red blood cells and platelets that also express CD47. Thus, the class of CD47 antibodies that stimulate tumor cell killing while sparing normal cells in vivo is desirable for the cancer patients.
We are interested in understanding whether humanized mouse models could aid the evaluation of a variety of monoclonal CD47 antibodies we generated. We first screened them using the CDX platform established at Biocytogen. Indeed, most antibodies were able to clear human tumor cells in B-NDG mice. Then we screened the toxicity of these antibodies using humanized knocked-in mice. Body weight and blood were collected and analyzed.
Most antibodies were able to clear human tumor cells in B-NDG mice. However, when we screened the toxicity of these antibodies using humanized knocked-in mice, many antibodies caused rapid death of animals upon the first administration. Interestingly, we found two clonal antibodies bearing the least toxicity since the treated animals exhibited minimal weight loss on Day 2 and then recovered.
We concluded that humanized mice could expedite the development of safer CD47 antibodies that can be advanced to human clinical trials.
Chaoshe Guo.
Beijing Biocytogen Co., Ltd.
All authors have declared no conflicts of interest.
Tumor hypoxia promotes the Warburg effect, decreases anti-tumor immune responses and increases malignant behavior, thereby fostering disease progression. The novel anti-hypoxic molecule myo-inositoltrispyrophosphate (ITPP) acts as an allosteric effector of hemoglobin and efficiently counteracts hypoxia. In preclinical models, ITPP normalized tumor-associated vasculature, enhanced chemotherapy efficacy, decreased tumor burden and increased survival.
The present study is a first in-human exploratory, prospective, open-labelled, mono-centric Phase IB study according to a classical 3 + 3 dose escalation scheme in patients with non-resectable hepatopancreatobiliary tumors. The study intervention consists of 9 infusions of ITPP over 3 weeks, followed by administration of conventional chemotherapy. Primary endpoints are the determination of safety and tolerability of ITPP, while secondary endpoints aim at assessing tumor responses via imaging.
29 patients were included between 04/2015 and 07/2018, with 1 patient withdrawn from the study and 28 assessed for the primary endpoints. ITPP administration was safe and well tolerated. Dose limiting toxicity in the form of Hypercalcemia CTCAE grade 4 occured in the highest cohort tested (Cohort 8: 14’500mg/m2). 7 serious adverse events were recorded without relation to ITPP. 48 adverse events occurred, with 30 judged to be possibly or definitively related to ITPP. Hypercalcemia (19x), Hypomagnesemia (5x), Hypophosphatemia (4x) were the most freequently observed treatment emerged toxicities. Of the ITPP-treated patients, 10 had radiological disease stabilization, 4 experienced a decrease in tumor markers. 4 patients had stable disease with subsequent chemotherapy, while 5 benefited from a strong partial response.
Administration of ITPP before chemotherapy is safe and well tolerated with acceptable side-effects. Further exploration of ITPP's potential in phase II/III trials is warranted.
NCT02528526.
Pierre-Alain Clavien.
Has not received any funding.
All authors have declared no conflicts of interest.
Dendritic cells (DC) are a heterogeneous cell population, with specialist subtypes driving specific immune responses. In mice, the cDC1 subset (also referred to as Batf3-dependent DC, XCR1+ DC, CD8+ DC in lymphoid tissues and CD103+ DC in peripheral tissues) is essential for the induction of tumour immune responses and for the efficacy of checkpoint inhibitor blockade and adoptive T cell immunotherapies. Vaccines that can deliver antigens (Ag) directly to DCs in vivo are more effective than cell-based therapies in mouse models and are promising approaches to translate to humans. CD141+ DC are the human cDC1 equivalent and specifically express the C-type lectin-like receptor CLEC9A, that facilitates cross-presentation of dead cell Ag. Targeting tumour-associated Ag (TAA) to human CD141+ DC using CLEC9A antibody (Ab) is therefore an attractive strategy to induce or boost tumour immune responses.
NYESO1 and WT1 are well characterised, highly immunogenic TAA expressed by a broad array of tumour types. We developed recombinant human chimaeric IgG4 Ab specific for human CLEC9A genetically fused to NYESO1 or WT1. For comparison we developed TAA fusions with chimaeric IgG4 Ab specific for human DEC-205, which is expressed by many human leukocytes, and β-galactosidase as an irrelevant isotype control. CLEC9A-NYESO1 and CLEC9A-WT1 Abs retained their binding specificity for CD141+ DC. Following uptake of CLEC9A-WT1, CD141+ DC cross-presented a WT-1 HLA-A24-restricted epitope for recognition by specific CD8+ cytotoxic T cells. Likewise, a HLA-A2-restricted NYESO1 epitope was cross-presented Ag specific CD8+ T cells by CD141+ DC following uptake of CLEC9A-NYESO1.
For both TAA, the CLEC9A Abs were more efficient at delivery of Ag for cross-presentation than DEC-205 or isotype control Abs. Moreover, using a humanised mouse model in which functional human CD141+ DC and Ag-specific T cells develop, CLEC9A-TAA Ab induced priming of Ag-specific T cells.
Our data advocate further development of human CLEC9A targeting Abs as cancer vaccines.
Mater Research Institute.
Department of Defense, USA.
All authors have declared no conflicts of interest.
NY-ESO-1 is a highly immunogenic cancer-testis antigens and it is a potential candidate for immunotherapy. Cellular expression of NY-ESO-1 gene is heterogeneous depending on the demethylation status of its promoter. Exposure to 5-aza-2'-deoxycytidine (5-aza-CdR) has been reported to enhance NY-ESO-1 protein expression. Our aim is to study the effect of 5-aza-CdR on NY-ESO-1 expression in lung cancer cell lines.
Three human lung cancer cell lines were treated with 5µM of 5-aza-CdR. NY-ESO-1 protein expression was analyzed using cellular ELISA. NCI-H1975 and NCI-H522 were tested for their dose response to increasing doses of 5-aza-CdR (2.5 to 10μM). Expression of NY-ESO-1 mRNA was assessed using qRT-PCR. Cellular ELISA, western blot (WB) and flow cytometry (FACS) techniques were used to assess NY-ESO-1 protein expression. Epigenetic modifications of CpG islands in NY-ESO-1 gene were analyzed using bisulfate sequencing. The proteomic profiling of the cells was carried out using label-free mass spectrometry analysis.
NCI-H1975 and NCI-H522 cells had significantly enhanced expression (11 and 1.6 folds) of NY-ESO-1 protein after exposure to 5µM of 5-aza-CdR. Cellular ELISA showed a dose-dependent increase of NY-ESO-1 protein in both cell lines with the highest expression for NCI-H1975 (16 folds vs. control). qRT-PCR data showed a dose-dependent increase in NY-ESO-1 mRNA expression in both cells, with peak expression in NCI-H1975 cells (128 folds) at 10μM of 5-aza-CdR. FACS and WB analysis revealed increased protein expression with increasing concentration of 5-aza-CdR. Moreover, the CpG islands in the NY-ESO-1 gene were significantly hypomethylated in treated cells especially at 5μM. Proteomic profiling resulted in identification of various proteins with differential expression and functions.
We have shown that treatment with 5-aza-CdR enhances the expression of epigenetically silenced NY-ESO-1 antigen in lung cancer cells and this should trigger T cells immune responses against the NY-ESO-1 antigen resulting in tumor suppression. Further in vivo studies are required to apply such treatment to be as an innovative immunotherapeutic approach to treat lung cancer patients.
Medical Research Center, Hamad Medical Corporation, Doha, Qatar.
Medical Research Center, Hamad Medical Corporation, Doha, Qatar.
All authors have declared no conflicts of interest.
Among the crucial proteins having dual role in cancer and immunity is suppressor of cytokine-signaling3 (SOCS3) protein regulating JAK/STAT and IL 6/STAT3/NF-κB pathways, respectively. Another pivotal membrane anchoring protein; PIG-C, that anchors several proteins involved in carcinogenesis. Previous studies proved that miR-34a regulates PDL1. Also, our previous work proved that lncRNAs XIST and TSIX were able to manipulate PDL1 expression in triple negative breast cancer (TNBC). Therefore, this study aims at studying the impact of miR-34a, XIST/TSIX on SOCS3 and PIGC as novel targets to be added to the picture of molecular targeted immunotherapy.
Twenty pairs of BC tissues and adjacent non-BC tissues were collected from patients undergoing tumor resection surgery. MDA-MB-231 cell line was cultured, transfected for manipulation of gene expression purposes. Total RNA was extracted using Biozol followed by reverse transcription cDNA synthesis, amplification and quantification using quantitative-real-time PCR.
PIG-C was up-regulated in BC compared to controls (p = 0.0017). MiR-34a decreased PIG-C expression in BC cells lines (p < 0.0001) while anti-miR-34a increased PIG-C (p = 0.0259). MiR-34a increased SOCS3 (p = 0.0192) while anti-miR-34a decreased SOCS-3 (p = 0.0003). Upon XIST knockdown, PIG-C was up-regulated (p = 0.0243) and surprisingly SOCS3 was also up-regulated (p = 0.0092). Oppositely, siTSIX decreased PIG-C (p = 0.0320) and it significantly decreased SOCS3 (p = 0.0005) ensuring the previous results with siXIST. Co-transfection of miR-34a and siXIST decreased PIGC (p < 0.0001). Similarly, PIG-C decreased upon co-transfection of miR-34a and siTSIX (p < 0.0001). SOCS3 increased upon co-transfection of miR-34a with siTSIX (p = 0.0017). Synergistically, SOCS3 increased in the cells co-transfected with miR-34a and siXIST (p = 0.0201).
Here, PIG-C and SOCS3 are introduced as new immunotargets in TNBC. In the context of epigenetic regulation, the cross-talk between non-coding RNAs revealed miR-34a to have the lead over XIST and TSIX in regulating PIG-C and SOCS3. These data pave the road for establishing new proteins for molecular targeted immunotherapy along with their key upstream non-coding RNA regulators.
German University in Cairo, Egypt.
Has not received any funding.
All authors have declared no conflicts of interest.
The outcome of peritoneal carcinomatosis, often occurring due to appendix or colorectal cancer, has dramatically improved with the combination of cytoreductive surgery (CRS) and hyperthermic (43 °C) intraperitoneal chemotherapy (HIPEC). Nevertheless, recurrence of the disease presumably due to remnant cancer cells limits survival of the patients suggesting further optimization in HIPEC treatment. Therefore, it is important to understand mechanisms operating behind HIPEC treatment. Since chemotherapy may induce immunogenic changes, we analyzed effects of MitomycinC/Doxorubicin (M/D), widely used chemotherapeutics in HIPEC settings, on the immunogenicity of colorectal cancer cells in-vitro.
Colorectal cell-lines were treated with M/D for 30 minutes with and without hyperthermia (430C). After the treatment, cells were further incubated at 370C for 72 hours. The expression of immunogenic cancer-testig antigens (CTA) was analyzed using qRT-PCR and western blot. To assess DC maturation, we set up a co-culture between differentially treated colorectal cells and monocyte-derived DC`s. We analyzed surface markers such as HLA-DR, CD 86 and CD 83 to assess DC maturation using flow cytometry. Further, the activation of cytotoxic T-cells was measured by intracellular IFN-y staining after co-culture with DC`s that were pre-incubated with treated and untreated colorectal cancer cells.
Initial qRT-PCR screening of CTA revealed that two namely, Cyclin A1 and SSX-4 were upregulated after HIPEC treatment. Compared to the control condition (no drug, 370C), M/D in HIPEC condition led to an increase of Cyclin A1 up to 53 folds and SSX-4 to 30 folds. The amount of Cyclin A1 protein was doubled compared to the control treatment (p = 0.0015, CI mean volume intensity 0.1989 – 0.4233). After co-culturing with HIPEC treated colorectal cancer cells, we noticed significant expression in CD 83, a DC activation marker. DCs that were activated upon incubation with HIPEC-treated cancer cells were able to prime cytotoxic T-cells leading to enhance IFN-y production.
HIPEC treatment can cause immunogenic changes in colorectal cancer cells. This could explain a part of the mechanism, how HIPEC treatment may work and inclusion of an immunotherapy may improve outcome of this treatment.
Kuno Lehmann.
Has not received any funding.
All authors have declared no conflicts of interest.
The therapeutic effect of oncolytic virotherapy is mediated largely by two mechanisms; direct oncolysis due to tumor-selective viral replication and the simultaneous activation of innate and adaptive immune responses with the potential of long-lasting tumor remissions. The chimeric vesicular stomatitis virus pseudotyped with LCMV glycoprotein (VSV-GP) has been previously reported to have both a rapid lytic cycle and a broad tumor tropism. In this study, we demonstrate its therapeutic potential in the syngeneic lung cancer model LLC1.
To address the effect of IFN sensitivity of LLC1 cells to VSV-GP mediated oncolysis, we generated interferon receptor deficient cells (LLC1-IFNAR1-/-) using TALENs. Therapeutic efficacy of VSV-GP was assessed in vivo in syngeneic C57BL/6 mice and athymic nude mice bearing subcutaneous tumors. The mechanisms of VSV-GP treatment effect were investigated using bio-luminescent imaging (BLI), immunohistochemistry, multiplex ELISA and Nanostring® technology.
The ability of VSV-GP to infect and lyse LLC1 cancer cell-lines in vitro was abrogated by exogenously applied interferon (IFN) type I indicating a dependence of the oncolytic effect on defects in the IFN response of cancer cells. Interferon resistance of LLC1-IFNAR1-/- cells correlated with prolonged intratumoral viral replication and improved therapeutic outcome in vivo, as demonstrated by using a matched pair of LLC1 wildtype and LLC1-IFNAR-/- tumors. Additionally, BLI revealed successful tumor-to-tumor spread of viral progeny in bilateral tumor models. VSV-GP therapy was associated with enhanced T cell infiltration and upregulation of various immune-associated genes. Interestingly, the efficacy of VSV-GP therapy in treating LLC1-IFNAR-/- tumors was not diminished by the absence of CD8+ T cells and cured mice were not immune to tumor rechallenge indicating a predominant lytic effect.
The treatment effect of VSV-GP in LLC1-IFNAR-/- lung cancer model is primarily lytic with negligible contribution of adaptive immunity despite strong activation of both innate and adaptive immune signatures.
Medical University of Innsbruck.
ViraTherapeutics GmbH, Innsbruck, Austria.
G. Wollmann: Scientific advisor: Boehringer Ingelheim GmbH & Co. KG. B. Spiesschaert: Part time employment: ViraTherapeutics GmbH; Part of Christian Doppler Laboratory. F. Heinemann, B. Stierstorfer, P. Müller: Employment: Boehringer Ingelheim GmbH & Co. KG. M. Petersson, P. Erlmann: Employment: ViraTherapeutics GmbH. D. von Laer: Inventor of VSV-GP; Minority shares: ViraTherapeutics GmbH (which holds the intellectual property rights for VSV-GP); Scientific advisor: Boehringer Ingelheim. All other authors have declared no conflicts of interest.
ADU-1604 is a humanized IgG1 monoclonal antibody in development for use as monotherapy or in combination with other anti-cancer therapies. It targets a novel epitope on the validated inhibitory receptor, CTLA-4. ADU-1604 was characterized in vitro and shown to bind to human CTLA-4, block binding of CD80 and CD86 to CTLA-4, and stimulate IL-2 production by activated lymphocytes. ADU-1604 enhanced T cell dependent hepatitis B surface antigen vaccine-induced antibody responses in cynomolgus monkeys and demonstrated anti-tumor activity in a non-small cell lung cancer patient-derived xenograft humanized mouse model. The primary objective of the first-in-human study is to determine the recommended phase 2 dose (RP2D) by evaluating the safety, pharmacokinetics (PK), and pharmacodynamics (PD) of ADU-1604 administered as an intravenous (IV) infusion.
This first-in-human, open-label, multicenter, dose-escalation study is conducted in adults with metastatic melanoma without further established treatment options. The study includes two parts: 1) Dose Escalation starts with 0.3 mg/kg of ADU-1604 IV infusion in 3-6 subjects and dosing is escalated until the RP2D is defined (represented by the dose tolerated while not exceeding the maximum tolerated dose or maximum dose (10 mg/kg)). 2) In Dose Confirmation, 7-10 additional subjects receive ADU-1604 at the RP2D until the maximum number of planned doses are administered (4 treatment cycles), disease progression is confirmed, or consent is withdrawn, whichever occurs first. The end of the study is defined as the date when all subjects have completed the final protocol-specified safety assessment and/or discontinued study participation. Primary endpoints include incidence of dose limiting toxicity, treatment-emergent adverse events (TEAEs), and changes from baseline in safety parameters. Secondary endpoints include severity of TEAEs, serious adverse events, changes from baseline in safety assessments, serum concentration-time profiles and PK parameters (including Cmax, AUC), and incidence of anti-ADU-1604 antibodies. This study is designed to provide the RP2D of ADU-1604 based on the totality of PK-PD, as well as clinical responses and safety.
NCT03674502.
Aduro Biotech Europe.
Aduro Biotech Europe.
M. Hendriks, E. de Cock, K. Maplestone, H. Namini, A. van Elsas: Employee of and holds stock in Aduro Biotech Europe at the time of this work.
Most patients (pts) with NSCLC present with advanced disease at diagnosis. Systemic therapy with platinum-based doublet chemotherapy regimens has been the standard first-line treatment for pts with advanced NSCLC whose tumours do not have EGFR, ALK, or ROS 1 mutations, but there is a need for effective treatments to improve long-term survival. With the recognition that NSCLC tumours express PD-L1, checkpoint inhibitors are being investigated in several clinical trials. There is currently only one PD-1 inhibitor approved as monotherapy in first-line treatment of NSCLC with PD-L1 expression ≥50%. In a phase 1 dose escalation and NSCLC expansion cohort, cemiplimab (REGN2810), a human monoclonal anti-PD-1, has demonstrated antitumour activity with an acceptable safety profile in anti-PD-1 naïve, pre-treated pts with NSCLC.
This is a randomised (1:1), multicentre, open-label, phase 3 study of cemiplimab versus platinum-based doublet chemotherapy in systemic treatment-naïve pts (≥18 years) with stage IIIB, IIIC or IV squamous or non-squamous NSCLC whose tumours express PD-L1 in ≥ 50% of tumour cells (NCT03088540). Pts will be stratified by histology and geographic region. Pts will receive cemiplimab 350 mg every 3 weeks intravenously (for up to 108 weeks) or 4–6 cycles chemotherapy with (i) paclitaxel + cisplatin or carboplatin, (ii) pemetrexed + cisplatin or carboplatin with or without pemetrexed maintenance, (iii) or gemcitabine + cisplatin or carboplatin. The primary objective is to evaluate progression-free survival (PFS) as determined by blinded independent review committee. Key secondary objectives include assessment of overall survival and overall response rate. Assuming duration of study enrolment and follow-up of 28 months and 10 months, respectively, approximately 700 randomised pts are required to obtain 525 PFS events to yield approximately 90% power to detect a statistically significant change in median PFS between treatment arms, with the 2-sided type 1 error limited to 5%. An independent data monitoring committee will monitor safety data during study conduct.
Medical writing support under the direction of the authors was provided by Emmanuel Ogunnowo, PhD, of Prime (Knutsford, UK) and funded by Regeneron Pharmaceuticals, Inc. and Sanofi according to Good Publication Practice guidelines.
NCT03088540.
Regeneron Pharmaceutical Inc. and Sanofi.
Regeneron Pharmaceutical Inc. and Sanofi.
V. Sriuranpong: Honoraria for advisory board and institutional study support grants: Regeneron Pharmaceuticals, Inc. P. Clingan: Participation in clinical trial work: MSD, AbbVie and Checkpoint Therapeutics. N. Rizvi: Personal fees: Roche, AstraZeneca, BMS, Merck, Novartis, Merck KGaA, Pfizer, outside the submitted work. O. Aren Frontera: Personal fees (advisory board & lecturing): Bristol Myers Squibb, Roche; Personal fees (lecturing): Novartis, outside the submitted work. S. Lee, S. Li, P. Snodgrass: Employee and shareholder: Regeneron Pharmaceuticals, Inc. P. Rietschel: Employee and shareholder, honoraria: Regeneron Pharmaceuticals, Inc. All other authors have declared no conflicts of interest.
Immune checkpoint blockade represents a major breakthrough in cancer therapy with recent approvals of PD-1 or PD-L1 antagonist therapy in France for a range of cancer indications. To generate high evidenced-based knowledge and to prevent off-label use, the French National Cancer Institute (INCa) launched the AcSé Immuno-therapy Program: two exploratory phase II trials, to allow for a nationwide safe and controlled access to nivolumab or pembrolizumab outside of their current marketing approvals for selected rare cancer indications where the literature suggests a potential benefit for patients, but where the difficulties of development render individual experimental studies unattractive to the pharmaceutical industry.
The two trials, AcSé Nivolumab and AcSé Pembrolizumab are Phase 2, single-arm, national, multicentre trials investigating the efficacy and safety of nivolumab and pembrolizumab, respectively, in adult patients with specific rare cancers who have unresectable locally advanced or metastatic disease which is resistant or refractory to standard therapy and for whom no alternative approved or experimental treatment options exist. Up to 650 patients will be enrolled across the two trials and assigned to one of 13 cohorts (max. 50 patients/cohort) according to their indication (see table).AcSé nivolumab AcSé pembrolizumab Cohort 1: Non-clear cell RCC Cohort 1: Rare sarcoma Cohort 2: Rare head and neck cancer Cohort 2: Rare ovarian cancer Cohort 3: Rare skin cancer Cohort 3: Primary CNS lymphoma Cohort 4: MSI-H cancer (other than CRC) Cohort 4: Rare thyroid cancer Cohort 5: Penile cancer Cohort 5: Rare malignant neuroendocrine cancer Cohourt 6: POLE exonuclease domain mutated cancer Cohort 6: Germ-cell cancer Cohort 7: NK/T-cell lymphoma
The trials will use a two-stage Bayesian enrichment design to identify potentially sensitive indications and assess treatment efficacy per cohort. Toxicity will also be assessed per cohort and biological samples collected to explore the predictive factors of response and mechanisms of acute and acquired resistance to anti-PD-1 therapy in these populations.
UNICANCER.
Institut National du Cancer (INCa), La Ligue contre le Cancer, Bristol-Myers Squibb (BMS), MSD.
All authors have declared no conflicts of interest.
In situ immunization is a strategy where immunomodulatory products are injected into one tumor site in order to use the tumor as its own vaccine and to trigger a systemic anti-tumor immune response. The Pexa-Vec (JX-594, Transgene) is an oncolytic virus genetically modified to secrete GM-CSF. We formulate the hypothesis that IT treatment with this oncolytic virus could synergize with anti-CTLA4 therapy via oncolytic virus-induced tumor cell death & tumor-antigen release, GM-CSF-induced recruitment/maturation/activation of antigen presenting cells, and anti-CTLA4-induced Treg blockade/depletion and reversion of T effectors inhibition.
ISI-JX is a multicentric, Phase I dose escalation trial followed by an extension part aiming to evaluate the safety, feasibility, and anti-tumor effects of an in situ immunization strategy with IT injections of ipilimumab with Pexa-Vec in adults patients (pts) with solid tumors. The trial is conducted in pts with at least one injectable lesion (≥2 and ≤8 cm) and one distant non-injected measurable target site. Pts are treated with an IT injection of Pexa-Vec alone (1 x 109pfu) at Week 1 (W1) Day 1 (D1) followed by 3 IT injections of Pexa-Vec (1 x 109pfu) + ipilimumab (4 dose levels: 2.5, 5, 7.5, 10 mg/injection) at W3 D1, W5 D1 and W9 D1. Primary endpoint for escalation part is the occurrence of Dose Limiting Toxicities (DLT) defined as the toxicities occurring during the DLT assessment window (the first 5 weeks) related to Pexa-Vec, Ipilimumab or both; and the objective response rate as per immune related Response Criteria after 3 months of treatment for extension part. Secondary endpoints include the disease control rate, duration of response, progression free survival and overall survival. The dose escalation part follows a classical 3 + 3 design with 3 to 6 pts at each DL depending of the number of DTL observed (maximum of 24 pts). In the extension part, according to on the first stage of a Gehan design, 12 patients per cohort (3) will be enrolled (maximum of 36 pts). Up to date, the dose escalation part is ongoing: 8 pts were enrolled (DL1 n = 3; DL2 n = 3; DL3 n = 2).
Bidaux As & Garin G; Centre Léon Bérard; DRCI Promotion Phase Précoce.
EudraCT: 2014-001692-32; NCT02977156.
Centre Léon Bérard DRCI Promotion/Essais Précoces.
Centre Léon Bérard & Transgene S.A.
All authors have declared no conflicts of interest.