Background

Treatment with the checkpoint inhibitors ipilimumab (IPI) and nivolumab (NIVO) are associated with durable remissions in patients (pts) with solid tumors. We describe the durable remissions associated with these agents. There were 19 pts treated with IPI and 25 pts treated with NIVO and 1 pt was treated with a combination of IPI and NIVO.

Methods

The purpose of this analysis is to describe the durable remissions associated with IPI and NIVO on a variety of solid tumors treated at a single centre. This is a retrospective data analysis from 45 pts treated either in an expanded access program, clinical trial setting or post-registration protocol.

Results

A total of 45 pts (30 males, 15 females) were analyzed. Three pts with metastatic malignant melanoma (MMM), 18 with non-small cell lung cancer (NSCLC), 2 with renal cell carcinoma (RCC) and 2 with Hodgkin’s disease (HD) were treated with NIVO and 19 with MMM received IPI. One NSCLC pt received a combination of IPI and NIVO (1 cycle). In total 167 cycles of NIVO (median = 4, range 1-16), and 64 cycles of IPI (median = 4 cycles, range 1-4) were administered. In the MMM group, there were 5 responses out 20 pts (25%) treated with IPI including 3 pts with durable complete response (CR) of 74+, 46+ and 34+ months. One MMM pt treated with NIVO has an ongoing partial response (PR) of 23 months. Among the pts with NSCLC 6 responses were documented among the 18 pts treated with NIVO (33%). Two of these pts had very good and durable PRs of 10+ and 18+ months. One RCC pt treated with NIVO has an ongoing PR of 13+ months. Two heavily pretreated pts with HD treated with NIVO have very good PRs of 14+ and 13+ months. Additionally, a very PR was documented in the NSCLC pt treated with the combination IPI and NIVO.

Conclusions

Anti-PD1 and anti-CTLA4 antibody treatment are associated with durable remissions in pts with a variety of solid tumours. Among our pts durable remissions and durable responses were documented in pretreated pts with RCC, NSCLC, HD and MMM.

Legal entity responsible for the study

Prof Bernardo L. Rapoport

Funding

None

Disclosure

B.L. Rapoport: MSD: Adboards, Speaker engagements, Consultancy, Contract Research; BMS: Adboards, Speaker engagements, Consultancy, Contract Research, Research grant. All other authors have declared no conflicts of interest.

Background

Ipilimumab is a human monoclonal IgG1 antibody against CTLA-4 that has been shown to prolong the overall survival of patients with advanced pretreated melanoma. In 2015, a retrospective, multi-centre, non-interventional analysis was per- formed on data collected from the ipilimumab expanded access programme in South Africa, with last follow-up date (or death) in December 2014. The current study extends this analysis by follow-up on the long-term survival of pre-treated metastatic patients up to September 2016.

Methods

Follow-up questions were sent to participating investigators, who had patients who were still alive (29) or for whom it was not known whether they were still alive (11) following the last ipilimumab infusion. Investigators had to confirm whether patients were still alive, the date of death or last contact, clinical response at last contact, and whether the patient was still responding to ipilimumab.

Results

Of the 108 patients, 84 (78%) had cutaneous melanoma and 24 patients (22%) had non-cutaneous melanoma, including uveal, mucosal, and melanoma of unknown primary. Twenty patients previously received two or more lines of treatment for metastatic melanoma. The median age was 59 years (range 27 – 86) and there were 73 (68%) males and 35 (32%) females. Baseline ECOG PS was 0 in 33%, PS 1 in 58% and PS 2 in 6% of patients. The longest follow-up time available was 5.4 years. The median OS was 9.36 months (95% CI 7.48 – 11.84). One-year survival was 39% (95% CI 29% - 48%), 2-year survival was 22% (95% CI 15% - 30%), 3-year survival was 19% (95% CI 12% - 27%), 4- and 5-year survival was 15% (95% CI 8% - 21%). In the group of cutaneous melanoma patients, the 4- and 5-year survival was 17% (95% CI 9% - 25%) while in the non-cutaneous group the 4- and 5-year survival was 6% (95% CI 0% - 16%).

Conclusions

Ipilimumab at a dose of 3mg/kg is an effective treatment for patients with pre-treated advanced (unresectable or metastatic) melanoma and is associated with durable remissions and long-term survival.

Clinical trial identification

CA184-515 Ethics approval extended on protocol REC 2/21/05/14

Legal entity responsible for the study

Prof Bernardo L. Rapoport

Funding

Investigator Sponsored Research (ISR) trough Bristol-Myers Squibb South Africa

Disclosure

B.L. Rapoport: BMS South Africa: Advisor, Speaker Bureau, Contract Research and Funded Research; MSD: Advisor, Speaker Bureau and Contract Research; AstraZeneca: Advisor, Speaker Bureau and Contract Research; Roche South Africa: Advisor, Speaker Bureau and Contract Research. H. Duvenhage: BMS: Medical Director. All other authors have declared no conflicts of interest.

Background

Renal Cell Carcinoma (RCC) has always been considered an immunogenic tumour with immunostimulatory therapeutic approaches like interleukins and interferons being cornerstone in the 1990s. Clinical data supports the hypothesis that the PD-1/PDL1 interaction is an important regulator in tumour immune tolerance and tumour growth in RCC. No prognostic or predictive biomarkers have previously been identified leaving testing an unknown in RCC. The objective is to evaluate current real-world treatment patterns including PD-L1 expression testing in RCC.

Methods

This study used a QuintilesIMS oncology specific cross-sectional survey collecting anonymized patient-level oncology data in EU4 (France, Germany, Spain, UK) and Asia (China, Japan, Korea) between January – June 2017.

Results

Of the 2,413 surveyed patients, 79% were prescribed Tyrosine Kinase Inhibitors (TKIs), 7% mTOR inhibitors, 2% interferons/interleukins, 9% anti-PD-1/PD-L1 treatments (4% Asia vs 10% EU4). The majority of patients were prescribed anti-PD-1/PD-L1 treatments as monotherapy with under 1% prescribed in combination with TKIs. 97% of patients receiving an anti-PD-1/PD-L1 drug had completed another treatment prior to receiving checkpoint inhibitors while other 3% were new to treatment. The most common previous therapies were TKIs (82%) followed by mTOR inhibitors (26%), and the reason for discontinuation was primarily disease progression (86%).In 70% of patients treated with anti-PD1 treatments the disease metastasised to two or more organs. 22% of RCC patients were tested for PD-1/PDL1 expression in EU4 while none were tested in Asia.

Conclusions

PD-1/PD-L1 inhibitors are mainly being prescribed to patients with RCC that have completed at least one line of treatment and who have manifested disease progression. The precise role of PD-L1 positivity remains to be defined within this patient segment as testing rates for expression remain really low or non-existent in some countries. Further analysis will be required to understand the time to PD-1/PDL1 expression testing and to understand difference in typical patient profile of patients treated with standard of care vs anti-PD-1/PD-L1 treatments.

Legal entity responsible for the study

N/A

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

Ipilimumab was the first immune checkpoint inhibitor to demonstrate a survival benefit for metastatic melanoma patients and has been available in Northern Ireland (NI) since 2012. With the advent of more novel checkpoint inhibitor options and combinations, single-agent Ipilimumab use has declined, but evidence indicates it may continue to have a role after PD-1 inhibitor monotherapy failure. We investigated our clinical outcomes in the earlier era of this treatment.

Methods

Ipilimumab-treated patient data were retrospectively collected using electronic record systems and clinical notes. Descriptive and inferential statistics using Microsoft Excel® and SPSS® were employed to evaluate outcomes, including objective response rates (RR) according to RECIST criteria, disease control rates (DCR), progression free survival (PFS) and overall survival (OS).

Results

70 patients were treated with Ipilimumab in NI between November 2012 and December 2015. 57 patients had a cutaneous or unknown primary, and of these, 40.3% harboured a BRAF mutation. 56.4% of all patients had received prior systemic therapy. Median follow-up duration was 12 months (range 0.63 to 51.2 months), and median number of cycles administered was 4, with 65.7% completing 4 cycles. RR and DCR were 10.1% and 31.9% respectively, and the median PFS was 2.9 months and OS 11.7 months. 50.7% of patients with disease progression received subsequent systemic therapy but 52% of deaths occurred prior to access to PD-1 inhibitor and BRAF/MEK targeted therapy in NI. Grade 3-4 toxicities occurred in 31.4% of patients. The 30-day mortality was 5.7% (4 patients), with only 1 suspected treatment-related death. Serum LDH>upper limit of normal, serum neutrophil:lymphocyte ratio>4, M1c disease, uveal primary, presence of brain metastases and ECOG performance status 2 were identified as clinical factors which correlate with worse PFS and OS, and all early mortality cases had two or more of these factors.

Conclusions

Efficacy and toxicity data, and prognostic indicators for our cohort, are similar to other published data. Ipilimumab is deliverable in a non-clinical trial setting with acceptable outcomes, and may remain relevant as a viable treatment option. Patient selection is key to optimal results.

Legal entity responsible for the study

Belfast Health and Social Care Trust

Funding

None

Disclosure

O. Oladipo: Served on advisory boards for MSD and BMS and supported for travel to educational meetings by both companies. V. Coyle: Almac Diagnostics, Craigavon, UK, Corporate Research Collaboration. All other authors have declared no conflicts of interest.

Background

Nivolumab showed an overall survival(OS) benefit compared with docetaxel (12.2 vs 9.4 months(m), p = 0.002) in pts with non-sqamous NSCLC and a better progression-free survival(PFS) and OS(42% vs 24%, at 1 year, p < 0.001) for sqamous NSCLC after platinum-based chemotherapy(ch). Pembrolizumab showed longer PFS(10.3 vs 6.0 m, p < 0.001) and OS(80.2% vs 72.4% at 6 m, p = 0.005) vs platinum-based ch as first line treatment in pts with stage IV NSCLC and PD-L1 expression on at least 50% of tumor cells.

Methods

Retrospective review of 70 consecutive pts with stage IIIB and IV NSCLC treated with nivolumab or pembrolizumab outside of clinical trials from June 2015 until August 2017 at the University Hospital of Zürich.OS and PS were calculated with Kaplan-Meier function, while differences between subgroups were assessed with log-rank test.

Results

63(90%) pts treated with nivolumab, 56(88.9%) of them in 2nd line, and 7(10%) with pembrolizumab, 5(71.4%) of them in 1st line. Median number of cycles was 11(range(r): 1-50) for nivolumab and 3(r: 1-10) for pembrolizumab. Median age was 64.5 years (r: 39.0-89.4). In the nivolumab cohort: 48(76.2%)pts had non-squamous and 15(23.8%) squamous NSCLC, 25 (39.7%) had brain metastases(mts); median OS based on 22 events was 16.5m(95% CI: 11.5-21.4), 14.2m(95%CI:11.2-17.2) without brain mts vs not reached with brain mts, p = 0.86; median PFS, based on 42 events was 5.5 m(95% CI: 3.0-8.0). In the pembrolizumab cohort: 6 pts(85.7%) had non-squamous NSCLC, 3(42.9%) brain mts; median OS was not reached; median PFS, based on 5 events was 1.4 m(95% CI:0.1-3.1). Immune-mediated adverse effects in 8 (11.4%)pts. Grade 3 pneumonitis in 2 pts, one with treatment discontinuation after 2 cycles and complete response 20m later, other with partial response after 38 cycles.

Conclusions

OS of this pts' cohort treated with nivolumab is superior to previous published data (16.5 vs 12.2 m). Almost 40% of pts had brain mts, indicating that also in this cohort the treatment is effective. The cohort of pts treated with pembrolizumab is still too small for evaluation. Radiological and molecular analysis is ongoing.

Legal entity responsible for the study

University Hospital Zurich

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

Ipilimumab (IPI), nivolumab (NIVO) and pembrolizumab (PEMBRO) can induce immune-related adverse events (IrAEs). We describe the IrAE’s associated with 64 patients (pts). There were 20 pts treated with IPI and 25 pts treated with NIVO, 18 pts received PEMBRO alone or in combination with either epacadostat or Talimogene Laherparepvec (T-VEC), and 1 pt was treated with combination of IPI and NIVO.

Methods

Retrospective data from 64 pts were used treated either in an expanded access programme, clinical trial setting or post-registration protocol.

Results

A total of 64 pts (33 males, 31 females) were analyzed. Three pts with metastatic malignant melanoma (MMM), 18 with non-small cell lung cancer (NSCLC), 2 with renal cell carcinoma and 2 with Hodgkin’s disease were treated with NIVO and 20 with MMM received IPI. The PEMBRO group consisted of 3 breast cancer, 2 ovarian, 1 stomach and 12 MMM pts. One NSCLC pt received combination of IPI and NIVO (1 cycle). In total 167 cycles of NIVO (median = 4, range 1-16), 123 cycles of PEMBRO (median = 4, range 1-26), and 64 cycles of IPI (median = 4 cycles, range 1-4) were administered. Seven IrAEs are described in 15 IPI treated pts. These included endocrinopathy in 3 pts, colitis in 3 pts (1 required infliximab) and hepatitis in 1 pt. Among the pts treated with NIVO, 7 IrAEs were documented. These included pneumonitis in 2 pts, skin rash in 3 pts, mild diarrhea in 1 pt and mild uveitis in 1 pt. One pt developed autoimmune thrombocytopenia, nephritis, and PRES (posterior reversible encephalopathy syndrome). Three chest infections were documented including pulmonary tuberculosis in a NSCLC pt. Among the pts treated with PEMBRO, 10 IrAE were documented. These included 4 dermatological, 1 Bell’s palsy, 3 mild diarrhea and 2 hypothyroidism. The pt receiving combination IPI and NIVO had grade 4 skin toxicity. No IrAE related deaths were document.

Conclusions

A plethora of irAEs are described with anti-PD1 and anti-CTLA4 antibodies. Colitis was more common with IPI while pneumonitis more common with NIVO. Prompt diagnosis of IrAE’s will result in decreased morbidity and mortality.

Legal entity responsible for the study

Prof Bernardo L. Rapoport

Funding

None

Disclosure

B.L. Rapoport: MSD (advisory board, consultancy, speaker engagements, and contract research); BMS South Africa (advisory board, consultancy, speaker engagements, research grant and contract research); Amgen South Africa (advisory board, consultancy, speaker engagements, and contract research). All other authors have declared no conflicts of interest.

Background

Cancer immunotherapy prompted a paradigm shift in oncology, in which therapeutic agents are used to target immune cells rather than cancer cells. Sooner or later, immune oncology (IOs) drugs will be standard of care in multiple indications and with the use of these drugs it comes a unique set of possible side effects named immune related side effects (IRSE) not encountered previously. On the top of this, the usage of non-harmonized terminologies to report and describe IRSE add even more complexity.

Methods

This study used QuintilesIMS syndicated cross sectional surveys collecting anonymized patient-level data. Data collected in Germany, France, UK and Spain between July 2016 and June 2017 was used to describe SE of 2,998 patients currently IOs treated. Their reporting relies exclusively on the usage of secondary data (a predefined list) and no primary data is generated or collected.

Results

Of the 2,998 patients (sample number) IOs currently treated populations, 7.9% patients are under clinical trials. The split by mechanism of action is 2% vs. 92% vs. 16% vs. 0, 2% for PDL1 vs. PD1 vs. CTLA-4 and OTHER (KIR2DL1 inhibitor, anti CD137), respectively. IO-IO, IO-CHEMO combination and IO mono are received by 10%, 2% and 88% of patients, respectively. 70% of patients are not displaying any side effects when treated with IO mono or IO-CHEMO (n = 2686), but this percentage drops at 51% when treated with IO-IO combination (n = 312). With 41%, gastrointestinal disorders are most encountered SE during IO treatments (diarrhea counts for 56% from this group), followed by skin and subcutaneous tissue disorders with 27%. Metabolism and nutrition disorders counts for 20% while general disorders and administrative site conditions counts for 15%. Vascular and blood/lymphatic system counts for 11% each. Other side effects includes hepatobiliary disorders (5%); eye disorders (1%), investigations like weight change and cachexia (9%), nervous system disorders (5%) and others (11%).

Conclusions

Higher SE profile for IO-IO combinations might limit their usage, despite their benefit. Additional investigations are required to reveal whether sequence rather than combination is the solution for an improved ratio benefit/risk.

Clinical trial identification

NONE

Legal entity responsible for the study

QuintilesIMS

Funding

QuintilesIMS

Disclosure

All authors have declared no conflicts of interest.

Background

There is no approved second line treatment for MPM, and chemotherapy used in this setting (vinorelbine, gemcitabine) has limited efficacy. Based on promising early results of the phase Ib KEYNOTE-028 trial, pembro has been used off-label in CH and AUS. Outcomes from patients receiving pembro for MPM outside a clinical trial have been reported independently from AUS and CH cohorts, but conclusions were hampered by small numbers. Here we report on the combined and extended analyses from both cohorts.

Methods

Data from respective registries on pembro for relapsed MPM or patients unable to receive chemotherapy in AUS and CH were pooled. Patient characteristics, including age, gender, histology and previous treatments were entered into separate databases. Outcomes were assessed by the local investigators using standard RECIST v1.1 criteria. PD-L1 expression was determined centrally in each country and categorized as negative (<5%) intermediate (5-49%) and high (≥50%). Statistical analyses used country as strata.

Results

A total of 95 patients were treated (48 from CH, 47 from AUS). Median age was 67.8 years (range, 25-94). The majority of patients (73%) had epithelioid MPM. Sixty-seven patients (72%) were ECOG performance status of 0-1. Pembro was used as second line treatment in 48 patients (51%). PD-L1 results were available for 67 patients (71%). Most were PD-L1 negative (67%), while 18% were intermediate and 15% were PD-L1 high. In the full cohort, the ORR was 19%, median PFS (mPFS) 3.1 months and mOS 7.2 months. In patients with ECOG 0-1 and only one previous systemic treatment (n = 35), the ORR was 37%, with a mPFS of 4.4 months and a mOS of 10.2 months. PD-L1 expression ≥5% was associated with non-epithelioid histology (p = 0.003), and with higher ORR (46%) and longer mPFS (5.6 months).

Conclusions

Compared to reported outcomes in early phase trials, PFS and OS in this real-life cohort were inferior. However, patients with good PS (0-1) and receiving pembro as second line therapy achieved better outcomes. Despite enriching for non-epithelioid histology, PD-L1 positivity was associated with improved ORR and PFS. Details on outcomes in subgroups by histology and PD-L1 expression will be presented at the meeting.

Legal entity responsible for the study

Yannis Metaxas, Thomas

Funding

Krebsliga Graubünden, Switzerland Academic Funds, Australia

Disclosure

L. Mauti: Advisory boards for MSD, BMS, Pfizer, Roche, AstraZeneca. S. Kao: Honoraria: BMS, Roche, Astra-Zeneca; Research Funding: MSD; Travel expenses: BMS, Boehringer Ingelheim, AstraZeneca. B. Hughes: Consulting/Advisory role: MSD, Roche, BMS; Travel expenses: MSD. N. Pavlakis: Honoraria: Specialized Therapeutics, Pfizer, Novartis, Amgen, Bayer, Boehringer Ingelheim, Roche, Merck-Serono, AstraZeneca; Consulting/Advisory role: Specialized Therapeutics, Pfizer, Novartis, Amgen, Bayer, Boehringer Ingelheim, Roche, Sanofi-Aventis, Merck-Serono, AstraZeneca. K. O'Byrne: Honoraria: MSD, BMS, Roche Genentech, Boehringer Ingelheim, AstraZeneca; Consulting/Advisory role: MSD, BMS, Roche Genentech, Boehringer Ingelheim, AstraZeneca; Speaker’s Bureau: MSD, BMS, Roche Genentech, Boehringer Ingelheim, AstraZeneca; Travel expenses: MSD, BMS, Boehringer Ingelheim, AstraZeneca. S. Rothschild: Honoraries (to the institution) for advisory boards of MSD. S. Savic Prince: MSD: participation in advisory boards and speakers honoraria. M. Pless, R. von Moos: MSD: advisory board. T. John: Honoraria: Pfizer, BMS, Roche, Astra-Zeneca, Novartis. All other authors have declared no conflicts of interest.

Background

Up to 40% of pts with MM develop brain metastases (mts). As recent drug development is prolonging survival, an increasing number of cases with brain involvement is going to be observed. Limited data suggest that RT may unmask cancer antigens and potentiate the effects of IT, even on distant non-irradiated sites (so called abscopal effect). We aimed to investigate this topic in a single Institution cohort of pts with MM.

Methods

We retrospectively identified all pts with MM treated with IT and RT at our Institution between 06/12 and 11/16. Permitted IT included either antiCTLA4 or antiPD1. RT could be either stereotactic (ST) or whole brain (WB) and had to be administered within 6 months (mos) since the beginning of IT, in absence of concomitant oncologic treatments (txs). Progression free and overall survival (PFS and OS, respectively) were estimated according to Kaplan-Meier method.

Results

36 pts were identified. 23 were treated with antiCTLA4, 13 with antiPD1. 50% of pts received ST RT, 50% WB RT. IT was administered in 1^st, 2^nd and 3^rd line in 21, 22 and 7 pts, respectively. 17 pts presented neurologic symptoms at brain relapse; 27 received steroid tx. 14 pts presented systemic progression, with a median PFS of 3 mos (range 0-14). Brain progression occurred in 15 cases, with a median PFS of 4 mos (range 1-14). Median PFS was 4 mos in 1^st line, 2 mos in 2^nd line, not evaluable in 3^rd line due to very few data. Median OS was 7 mos in 1^st line, 4 mos in 2^nd line, again not evaluable in 3^rd line. No significant differences in outcome were observed among either tx lines (p .47 for PFS, p .37 for OS), or type of IT (p .59 for PFS, p .89 for OS). Symptomatic pts had significantly worse PFS (2 versus 3 mos, p .05) but not OS (p .27) than asymptomatic ones.

Conclusions

Our experience did not seem to confirm synergistic effect of IT and RT. Pts’ outcome through different tx lines appeared poor. No significant impact on prognosis of either specific drug or RT schedule could be evidenced. Neurologic symptoms at brain relapse were the only predictors of PFS. The high prevalence of steroid prescription may have weakened long term effects of IT and masked potential synergistic effects. More data are needed to clarify this topic.

Legal entity responsible for the study

Fondazione IRCCS Istituto Nazionale dei Tumori

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

The IFN-ɣ+CD4 Th1 response plays a critical role in anticancer immunity and has been extensively studied from tumor microenvironment in many cancers. Evidence support that blood represents a crossroads site in which the cancer immunity also takes place. Here we aimed to characterize the natural history of a tumor-specific Th1 immunity in blood that can provide relevant information about cancer evolution.

Methods

Peripheral blood mononuclear cells were collected from 170 patients with non-small cell lung cancer (NSCLC) before any treatment. The presence of IFN-ɣ+ CD4 T cell response against telomerase (TERT) was used as surrogate marker of the antitumor Th1. The anti-TERT Th1 response was measured by ELISpot after stimulation of PBMC with promiscuous epitopes from TERT. Flow cytometry and multiplex beads assay were used to measure multiple immune cells and soluble factors in blood.

Results

59 pts (35%) exhibited a pre-existing anti-TERT Th1 immunity. The frequency of anti-TERT Th1 responders decreased from localized to metastatic NSCLC (44.8% vs 24%, p < 0.05). The magnitude of anti-TERT Th1 response influenced the overall survival (OS), especially in metastatic patients: median OS of 16 vs 7 months in high and low anti-TERT Th1 responders respectively (p = 0.023). Among the blood immune parameters measured, T cells expressing PD-1 and/or TIM-3 were found inversely correlated to this response, so that blocking these receptors restored the anti-TERT Th1 cycle. Interestingly, the levels of anti-TERT Th1 combined to PD-1+/TIM-3+ CD4 T cells predict distinct immune profile. Patients with anti-TERT Th1^hi/CD4-PD-1/TIM-3^hi showed better OS as compared to in anti-TERT Th1^lo/CD4-PD-1/TIM-3^hi group (median OS: not reached vs 12 months respectively, p = 0.006). Thus, a strong anti-TERT Th1 immunity may counteract the poor prognosis associated with high rate of PD-1/TIM-3+ CD4 T cells.

Conclusions

Our data demonstrate a striking link between the systemic anti-TERT Th1 response with tumor burden and clinical outcome. It also provides evidence that the association of exhausted T cells and anti-TERT Th1 immunity monitoring could be used as a relevant blood-based dynamic immune biomarker.

Legal entity responsible for the study

Olivier Adotévi

Funding

Assistance Publique des Hopitaux de Paris (AP-HP), FEDER-FUI, Ligue Nationale contre le Cancer

Disclosure

All authors have declared no conflicts of interest.

Background

Predicting patient-specific responses to anticancer therapy is the holy grail of therapy-selection. Response or resistance to therapy depends on the heterogeneous tumor microenvironment. This is particularly true for emerging anticancer drugs, such as immune checkpoint inhibitors, which recalibrate the body’s immune defense by modulating exhaustion of cytotoxic lymphocytes, T cells and natural killer (NK) cells. Clinical response to therapy varies greatly and there is a critical gap in our understanding of the mechanisms that drive response or resistance to therapy at the individual patient level.

Methods

We used a patient-autologous, clinically-validated ex-vivo tumor model that recreates the native tumor microenvironment (CANscript^TM) and uses an algorithm-driven method to predict clinical response to therapy (M-Score). Using tissue from luminal, HER2 positive, and triple-negative (ER- PR- HER2-) breast cancer patients (N = 10), we studied phenotypic alterations in the tumor-immune contexture under either standard-of-care regimens or immunotherapies, ex-vivo. We used a comprehensive panel of immunological assays to evaluate changes in cytotoxic lymphocytes by flow cytometry and multiplex immunohistochemistry.

Results

We identified that tumor response, predicted by M-Score, correlated with increased infiltration of NK cells and a pro-inflammatory cytokine signature from the tumor microenvironment. This coincided with induction of MICA/B, known to attract and recruit active NK cells. We also determined that therapy-induced protein biomarkers associated with NK cell exhaustion inversely correlated to the expression of cytotoxic granzyme B in the tumor microenvironment.

Conclusions

These data show that NK cells contribute to the antitumor effect of conventional and immuno-modulatory drugs and how a novel ex-vivo platform can be used to study the mechanisms of response and resistance, which wouldn’t be possible in a drug naïve state. Such an advance in our preclinical methods to study anticancer drugs at the individual patient level can help guide treatment decisions for clinicians while also functioning as a platform to study clinical efficacy of novel and emerging agents.

Legal entity responsible for the study

Mitra RxDx

Funding

Mitra RxDx

Disclosure

M. Smalley, B. Shanthappa, H. Gertje, M. Lawson, B. Ulaganathan, A. Thayakumar, L. Maciejko, P. Radhakrishnan, M. Biswas, S. Thiyagarajan, B. Majumder, A. Goldman: Employee of Mitra RxDx. All other authors have declared no conflicts of interest.

Background

To predict the response to immunotherapy, tissue and circulating cytotoxic lymphocytes, potentially targeted by anti-PD-1/PD-L1 drugs, were simultaneously assessed in NSCLC.

Methods

Twenty-six advanced NSCLC patients receiving nivolumab were included. Tissue samples were immunohistochemically analyzed to quantify PD‐L1 (H‐score) and CD3, CD8, CD4 and PD-1 positive TILs. Peripheral blood (PB) immune profile at baseline (T0) and after 2 (T1) and 4 (T2) cycles of bi-weekly nivolumab was performed by FACS analysis of CD3, CD8, CD4, NK (CD56), Treg (FOXP3) and MDSC. Changes in their number, and functional (PD-1, Granzyme B, Perforin) and proliferative (Ki67) hallmarks were determined. Integrated tissue and circulating parameters were correlated to treatment response (RECIST 1.1).

Results

At tissue level, in association with variable PD-L1 degrees, low PD-1 expression in CD8^pos TILs was present in 100% of patients with clinical benefit (CB, n 9) compared to only 20% of non-responders (NR, p < 0.001). At baseline, PB of CB patients showed higher number of NK, which tended to increase at T1 and T2, while in NR a progressive decline in NK, CD3, CD8 and CD4 was observed (p < 0.05). Moreover, a significant 2.5-fold increase in the incidence of circulating CD8^pos/PD-1^pos cells was detected at baseline in CB vs NR (p < 0.001). Saturation of PD-1 sites was apparent in PB of both groups following nivolumab. Cycling Ki67^poscytotoxic lymphocytes were higher in CB at baseline (12.4%), T1 and T2 while in NR proliferating CD8 (T0: 7.7%) progressively decreased to halve at T2 (p < 0.001). Intriguingly, one of the two patients with a complete response had the highest baseline value of both PD-1^pos (176.3/μl) and Ki67^pos CD8 cells (52%); conversely NR patients with rapidly progressive disease displayed the lowest range of both Ki67 labelling (1.3-3.9%) and PD-1 expression (18.3-22.4/μl).

Conclusions

The inhibitory and activating PD-1 pathways, respectively translated in low tissue PD-1^posCD8^pos TILs, to escape PD-L1 pressure and high peripheral blood PD-1^posCD8^pos, rescued by PD-1 targeting, may identify a specific immune profile predictive of the response to immunotherapy.

Legal entity responsible for the study

University Hospital of Parma

Funding

AIRC project grant

Disclosure

All authors have declared no conflicts of interest.

Background

Tumor-infiltrating lymphocytes (TILs) are associated with pathological complete response (pCR) and survival after neoadjuvant chemotherapy in early breast cancer. Studies on the impact of tumor-associated macrophages (TAMs) or antigen HLA class 1 (HLA-1) expression in this context, however, are limited. Therefore, we investigated their prognostic and predictive role in relation to neoadjuvant chemotherapy with or without zoledronic acid (NEOZOTAC trial).

Methods

On baseline tumor biopsies of the NEOZOTAC patients, immunohistochemical stainings were performed for CD8 (cytotoxic T-cells), FoxP3 (regulatory T-cells), CD68 (macrophages) and for HLA class 1 (HLA-1): HLA-A (HCA2) and HLA-B/C (HC10). Markers were associated with pCR and disease-free survival (DFS).

Results

Patients with strong (above median) intratumoral CD8+ infiltration (pCR: 18.4% vs. 5.2%; p = 0.011) or expression of HLA-1 (≥5% of tumor cells expressing HLA-A or HLA-B/C; pCR: 14.2% vs. 3.4%; p = 0.031) displayed a higher pCR rate compared to the other patients. The presence of HLA-1 expression was a requirement for a pCR when tumors were CD8+ infiltrated (pCR: 21.8% vs. 6.7%; p = 0.035) as they had no impact when HLA-1 expression was downregulated (pCR: 5.9% vs. 0%; p = 0.194). There was no direct association between CD8+, FoxP3+, CD68+ or HLA-1 expression status and DFS, however, patients with a strong CD8+ infiltrated tumor displayed a better DFS when tumors expressed HLA-1 (HR: 0.41, 95% CI 0.15-1.10, p = 0.08). More in depth analyses (e.g. on marker ratio’s and the effect of CD68+ and Foxp3+ infiltrate in HLA-1 expressing tumors) are awaited and will be available before the meeting.

Conclusions

A strong infiltration with CD8+ cells and expression of HLA-1 is associated with a higher pCR rate and better DFS after neoadjuvant chemotherapy.

Clinical trial identification

EudraCT number 2009-016932-11 NL30600.058.09 NCT01099436

Legal entity responsible for the study

Leiden University Medical Center

Funding

Dutch Cancer Society, Amgen, Novartis, Sanofi

Disclosure

J.W.R. Nortier, J.R. Kroep: Research grant Novartis, Amgen and Sanofi. All other authors have declared no conflicts of interest.

Background

We recently found that gastric cancer patients with H. pylori (HP) infection had a better overall survival (OS) than those without. In HP-co-cultured lymphoma cells, we found that HP cytotoxin-associated gene A (CagA) up-regulated expression of phospho (p)-SHP-2 and p-ERK, and immune molecules of PD-L1, and FOXP3. In this study, we assessed whether CagA expression is associated with the expression of PD-L1 or FOXP3 in tumor cells, and clinical outcomes of patients with gastric cancer.

Methods

HP strains were cultured from patients with HP-dependent gastric lymphoma. We co-cultured gastric epithelial cells (GECs) with HP strains and further evaluated the expression patterns of CagA, p-SHP-2, p-ERK, PD-L1, and FOXP3 in GECs. The association between CagA expression and expression patterns of PD-L1 and FOXP3 in tumor cells by immunohistochemistry, clinical stage, and OS were further evaluated in 112 patients with stage I to III gastric cancer.

Results

In HP-co-cultured GECs, we revealed that CagA translocated into nucleus and further up-regulated the expression of p-SHP-2, p-ERK, PD-L1, and FOXP3 in GECs. In tumor samples of gastric cancers, we found that CagA was detected in tumor cells of 43 (38.4%) cases. The CagA expression was closely associated with the stage I/II (p = 0.05) and the expression of PD-L1 (p < 0.001) and FOXP3 (p < 0.001) in tumor cells of gastric cancer. After a median follow-up of 119.9 months, patients with CagA expression had a better 8-year OS than those without (53.1% vs. 26.9%, p = 0.001). Among CagA-positive cases, PD-L1 expression was associated with a better 8-year OS (PD-L1-positive vs. PD-L1-negative; 64.6% vs. 30.8%, p = 0.069), whereas FOXP3 expression was associated with a marginally better 8-year OS (60.3% vs. 41.2%, p = 0.137). Of CagA-negative cases, PD-L1 expression was associated with a worse 8-year OS (PD-L1-positive vs. PD-L1-negative; 19.2% vs. 31.8%, p = 0.088).

Conclusions

CagA can up-regulate the expression of PD-L1 and tumor-infiltrating FOXP3 and thus contribute to the better survival of HP-positive gastric cancer. Further investigation of the association between tumor PD-L1 expression and aggressive behavior of HP CagA-negative gastric cancer is warranted.

Clinical trial identification

Not applicable.

Legal entity responsible for the study

The Ministry of Science and Technology, Taiwan.

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

The checkpoint inhibitor Ipilimumab induces long-lasting clinical responses in a proportion of patients with metastatic melanoma. However, most patients do not benefit from therapy. A desmoplastic stroma (tumour fibrosis), characterized by excessive extracellular matrix (ECM) remodeling, can result in reduced drug delivery and T-cell infiltration into the tumour and has been associated with poor prognosis and lack of therapy response. Here we address non-invasive biomarkers reflecting ECM remodeling in the tumour microenvironment, for associations with clinical response to Ipilimumab in patients with metastatic melanoma.

Methods

Serum biomarkers of collagen type III formation (Pro-C3), MMP-degraded type I collagen (C1M) and MMP-degraded type IV collagen (C4M) were studied with competitive ELISAs in pretreatment serum from 66 patients with metastatic melanoma. Results were correlated with objective response rate (ORR) and survival outcomes (OS) by univariate Cox analysis and Kaplan Meier plots.

Results

Pro-C3, C1M and C4M were significantly elevated in patients with progressive disease (PD) compared to the combined group of patients with stable disease (SD), partial response (PR) and complete response (CR) at baseline. Moreover, in the subgroup of patients with baseline biomarker levels higher than the 75^th percentile (Q4), only 6-13% responded (CR+PR+SD) compared to 46-48% in the group of patients with low biomarker levels (Q1-Q3). The median OS was 285, 161 or 198 days in biomarker high patients versus 596, 592 or 621 days in biomarker low patients for Pro-C3 (HR 2.13, 95%CI 1.12-4.04), C1M (HR 1.70, 95%CI 0.85-3.38) and C4M (HR 2.43, 95%CI 1.26-4.70), respectively.

Conclusions

High baseline levels of Pro-C3, C1M and C4M were associated with progressive disease and decreased overall survival in metastatic melanoma patients receiving Ipilimumab. This suggest the ECM biomarkers ability to predict response to checkpoint inhibitors at baseline. Future studies are needed to investigate the biomarkers applicability in other types of immunotherapies and cancers.

Legal entity responsible for the study

Nordic Bioscience

Funding

None

Disclosure

C. Jensen, M.A. Karsdal, N. Willumsen: Employee of Nordic Bioscience involved in biomarker development.

All other authors have declared no conflicts of interest.

Background

Immunotherapy is used to treat multiple solid tumor malignancies but has a great response in a few. Challenges remain as to the best predictive test for response to checkpoint inhibitors. While PD-L1 expression has been the best correlative marker to date, others such as Tumor Mutation Burden and Tumor Infiltrating Lymphocytes are on the horizon as an adjunct. We studied patients with solid tumors who had both PD-L1 as well as TMB tested and we analyzed the group that received immunotherapy to assess correlation to response.

Methods

We analyzed a total of 110 patients of whom 45 patients had treatment response information available. Patients received either Pembrolizumab, Nivolumab or Atezolizumab. 27 of those patients had either a favorable or unfavorable response to these drugs. PD-L1 was classified as negative, low or high. TMB was categorized as low, intermediate or high per Foundation One. PD-L1 was measured by Neogenomics assay with anti-PD-L1 22C3 antibody. These data were collected as an extension to a previous study that found no correlation between PD-L1 expression and TMB values.

Results

When comparing TMB and PD-L1 between groups of pts with favorable and unfavorable response, TMB was significantly associated with response (p = 0.03, Wilcoxon rank sum test) and PD L1 was not (p = 0.06). Regression models suggested that TMB and PD L1 were significantly associated with response (p = 0.008 and p = 0.03 resp). Higher TMB and PD L1 were associated with better response. Age, gender, diagnosis and TMB class were not significant. PD L1 category was significantly associated with response (p = 0.002).

Conclusions

While our first study concluded that PD L1 and TMB are independent features of solid tumors, this study suggests that both tests may be needed to identify patient subsets that may benefit from immunotherapy. Higher TMB and PD-L1 levels were independently associated with better response to checkpoint inhibitors in solid tumor malignancies. Microsatellite instability and Tumor infiltrating lymphocytes are two additional predictive markers that need further exploration.

Legal entity responsible for the study

Cancer Treatment Centers of America

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

Immunotherapy has been a beacon of hope for many cancers. Recent trials have suggested that PD-L1 expression measured by IHC may predict response to anti-PD 1 (PD-1 & PDL-1) therapy. Response to immunotherapy may have other markers such as Tumor Mutation Burden. Understanding the correlation of these biomarkers may have implications for the application of anti-PD-1 therapies.This study sought to determine TMB and its relation with PD-L1 expression, or lack thereof, in metastatic solid tumors to better define therapeutically relevant patient subgroups.

Methods

We evaluated the relationship between PD-L1 expression and TMB levels among patients with metastatic solid tumors being treated at 5 community-based cancer centers. PD-L1 expression was measured by Neogenomics using their immunohistochemistry assay with anti-PD-L1 22C3 antibody. Tumor Mutational Burden (measuring the number of non-synonymous DNA coding sequence changes per megabase of sequenced DNA) was calculated through Foundation One genomic reports. Both tests had low, intermediate & high values assessed. A retrospective chart review was performed of patients with solid tumor malignancies who had both of these tests performed.

Results

A Spearman’s correlation test was used to assess the relationship between TMB and PD-L1 using a sample of 93 patients. There was no strong correlation between TMB and PD-L1, rz = 0.186, p = 0.08 which was not statistically significant.

Conclusions

There is no correlation between PD-L1 expression level and TMB load. These appear to be independent predictors of possible benefit from checkpoint inhibitors. Studies have also shown Mismatch repair deficiency or Microsatellite instability (MSI-high) to be another independent predictor of benefit from these agents. More studies to evaluate the correlation of these with response from immunotherapy agents are warranted. Immunotherapy specific response data, survival, and predictive biomarkers of response will be reported in a future analysis. This study highlighted the importance to test for both of these markers in order to identify a patient subset that may benefit from immunotherapy.

Legal entity responsible for the study

Shayma M. Kazmi

Funding

None

Disclosure

S.M. Kazmi: Speaker for Merck and Eisai.

Background

Retinoblastoma (RB), malignant tumor of the sensory retina, is the most common eye cancer of childhood due to the defect of RB gene. The immune system plays an important role in controlling and eradicating cancer. Recent studies suggest that programmed death-1/programmed death ligand-1 (PD-1/PD-L1) pathway in T-cell activation plays a major role in tumor escape mechanism from host immunity. Therefore, we investigated the expression of PD-1 and PD-L1 in human retinoblastoma cancer to define their clinical significance in patient’s prognosis after enucleation.

Methods

We prospectively evaluate the expression of PD-1 and PD-L1 protein in 62 cases of primary enucleated retinoblastoma specimens by immunohistochemistry and further validated by immunoblotting. mRNA levels of PD-1 and PD-L1 genes were quantified in normal retina and tumor samples by quantitative real time PCR (qPCR). Expression of PD-1 and PD-L1 proteins were then correlated with clinicopathological parameters and patient survival by statistical analysis.

Results

Immunohistochemistry showed cytoplasmic PD-1 expression in (12/62) 19.3% cases, whereas PD-L1 expressed in 80.6% (50/62) cases. Immunostaining was found in stromal and tumor infiltrating lymphocytes. Immunoreactivity results of target protein were validated by western blotting on representative cases. Protein and mRNA level by IHC and real time qPCR was found to be closely correlated. Expression of PD-L1 showed significant correlation with poor tumour differentiation and tumor invasion (p < 0.05). There was also a significant difference in the overall survival of patients with PD-L1 expression.

Conclusions

These data suggest that PD-L1 expression may be a new therapeutic immunomarkers for patients with retinoblastoma. This study provides first data for the role of immune checkpoints in retinoblastoma patients. Further translational studies are necessary to confirm this observation and evaluate the predictive value of PD-1 and PD-L1 in retinoblastoma in the context of PD-1 inhibition.

Clinical trial identification

NIL

Legal entity responsible for the study

Department of Science and Technology (DST), India

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

Immune checkpoint blocking therapies have yielded positive clinical data in a series of human malignancies, including microsatellite instability in colorectal carcinoma (CRC). Programmed cell death 1 (PD1) and its ligand (PDL1) are key suppressors of the cytotoxic immune response. Previous study found that PDL1 expression was regulated by p53 gene. This study aims to investigate association of PDL1 expressions with TP53, KRAS mutations and microsatellite instability (MSI) in colorectal cancer patients.

Methods

Sixty one samples from CRC patients were immunohistochemically stained using antibodies to detect PDL1 and P53 expressions. Moreover, antibodies against mismatch repair or MMR (MLH1, MSH2, PMS2, and MSH6) proteins were used to assess MSI. The same samples were also tested for KRAS mutations in exons 2 (codon 12 and 13) using PCR high resolution melt (HRM) and direct DNA sequencing. The study was approved by Ethics Committee of Medistra Hospital.

Results

High tumor PDL1 expressions were found in 25% (15/61) patients. Meanwhile MSI was detected in 9 out of 61 patients (15%). Most MSI patients (7 of 9) had positive PDL1 expressions. In contrary only 8 out of 52 MSI negative patients showed PDL1 expressions suggesting significant association of PDL1 expressions with MSI status (p = 0,0004). PDL1 expressions tend to be higher in P53 wildtype (38%) than P53 mutant (18%, p = 0.17). Similarly PDL1 expression tend to be higher in KRAS wildtype (30%) than KRAS Mutant (11%, p = 0.19).

Conclusions

PDL1 expression is strongly associated with MSI in Indonesian CRC patients. MSI screening may be useful to predict patients with high PDL1 expressions and eligibility to anti PD1 immunecheckpoint therapy.

Legal entity responsible for the study

PT Kalbe Farma Tbk.

Funding

PT Kalbe Farma Tbk, Stem Cell and Cancer Institute (SCI)

Disclosure

All authors have declared no conflicts of interest.

Background

In non-small cell lung cancer (NSCLC) TNM staging remains gold standard for prognostic assessment and therapy decisions. Nevertheless, stage-specific outcomes vary significantly, indicating a need for additional prognosticators. Lymphocytic infiltrates in NSCLC patients are associated with a significant increase in disease-free and overall survival (OS). In our project we want to assess the role of cytokines using the example of interleukin-22 (IL-22) and interleukin-17 (IL-17) at the invasive margin (IM) and tumor center (CT) and link these results with prognosis, survival, therapy response and recurrence.

Methods

Tissue microarrays (TMAs) were generated from formalin-fixed paraffin embedded tissue (FFPE) of more than 200 curatively resected patients with stage IA-IIIA NSCLC. TMAs included each 3 cores from CT and IM, selected from areas with most dense lymphocytic infiltrates. IL-22 and IL-17 expression was analyzed by immunohistochemistry (double-staining: IL-22, CD3 resp. IL-17, CD3).

Results

In NSCLC IL-17 is known to promote metastasis. Interestingly we found that in squamous cell carcinoma (SCC) a high IL-17 expression is associated with longer survival, both in IM and CT. For adenocarcinoma the same tendency is visible, but not that pronounced. The IL-17 CT/IM ratio seems to be significant for adenocarcinoma: A high ratio corresponds to a prolonged survival. In SCC a similar tendency is visible, but not statistically significant.

For IL-22 a high CT/IM ratio is cleary linked with longer OS in adenocarcinoma, this cannot be seen in SCC.

Conclusions

Assessment of IL-17 and IL-22 performed on “hot-spots” NSCLC shows a clear correlation with clinical outcome: High IL-17 expression status seems to be linked to prolonged survival, similar results can be seen for IL-22 in adenocarcinoma. We validate these findings in a larger cohort of patients and correlate them with immune infiltrates.

Legal entity responsible for the study

Prof. Dr. R. M Huber

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

IL-33 is a proinflammatory cytokine and involved in the development of chronic inflammation and cancer. sST2 acts as a “decoy” receptor for IL-33 thereby regulating its action. Tumor development results in downregulation of IL-33 in epithelial cells but upregulation of IL33 in the tumor stroma and serum in many types of cancer. IL-33 expression was shown in PDAC cells and activated pancreatic stellate cells. We therefore explored IL-33 and sST2 as a potential predictive biomarker in 14 pts with advanced PDAC undergoing sCTX.

Methods

Plasma levels of IL-33 and sST2 of pts with locally advanced, metastatic or relapsing PDAC that underwent sCTX were determined with specific ELISAs. Serial measurements of IL-33 and sST2 were performed at baseline and 8 weeks after the start of sCTX. The baseline concentrations were compared with the values at week 8. Computertomography as efficacy assessment according to RECIST 1.1 was performed at baseline and at week 12.

Results

Plasma levels of IL-33 decreased more than 50% and sST2 increased more than 15% in all 3 pts (21%) with a partial response. The Cut-off point (COP) for predicting a stable disease (SD) was a 10% increase for IL-33 levels and a 10% decrease for sST2 levels. Of the 6 pts (43%) that had a SD, IL-33 was not measurable in 1 pt. PLasma levels of IL-33 did not increase over the COP in the course of treatment of 4 pts with a SD. In 1 pt with a SD IL-33 levels increased over the COP. sST2 levels were not measurable in 1 pt. In 4 pts sST2 levels did not decrease over the COP. In 1 pt the levels of sST2 decreased under the COP. From the 5 pts (36%) that progressed under sCTX, IL-33 levels increased in 2 pts at least over 50%, but in 3 pts the levels decreased more than 20%. Plasma levels of sST2 decreased in 2 but increased in 3 pts.

Conclusions

Serial monitoring of IL-33 and sST2 plasma levels predicted treatment response in 10 of 14 pts (71%) within 8 weeks of sCTX and have a potential role as predictive biomarker in PDAC pts. With the anticipated introduction of immune modulating drugs in the treatment of PDAC, such biomarkers might be of enormous interest.

Legal entity responsible for the study

Medical University of Vienna

Funding

None

Disclosure

M. Kieler: Travel support from Merck, Bayer, Bristol-Myers Squibb, Roche and participated in advisory board meetings from Bayer. G. Prager: Speaker’s fee from Bayer, Roche, Merck-Serono, Amgen, Servier, Celgen, Shire, MSD, Lilly, Sanofi-Aventis. All other authors have declared no conflicts of interest.

Background

NSCLC accounts for about 85% of all diagnosed lung cancers. As most patients need systemic treatment and immunotherapy is now current standard in second line treatment and will also establish in first line therapy, many patients receive immunotherapy. Pretherapeutic markers to identify patients who will respond to immunotherapy are required, also regarding to the immense costs. Accordingly, this study aims at examining the pretherapeutic inflammation status as well as the cytokine profile of patients who receive immunotherapy with Nivolumab.

Methods

We analyzed 37 patients with NSCLC treated with Nivolumab at a German lung cancer center between 2015 und 2016. We extracted data sets from electronic patient records and compared anthropometric data, pretherapeutic blood values and prognostic scores (neutrophil to lymphocyte ratio (NLR), Systemic Inflammation Index (SII), Prognostic Nutritional Index (PNI) and the Glasgow Prognostic Score (GPS)). Blood samples of 16 patients were analyzed using a cytokine ELISA (IL-4, IL-6, IL-10 and IFNγ).

Results

Patients, who did not respond to Nivolumab treatment showed significant elevated pretherapeutic markers of acute inflammation (NLR p = 0.006; CRP p = 0.001; SII p = 0.017; GPS p = 0.011; PNI p = 0.004). Examination of cytokine profile revealed that most patients with primary progression had at least one elevated marker of (i) acute inflammation (IL-6) or (ii) suppressed T-cell response (IL-10). Elevated IFNγ was associated with response to immunotherapy, whereas in combination with increased IL-6, INFγ showed to characterize patients with primary progression. Patients who progressed after initial response, showed increasing IL-6, IL-10 or decreasing IFNγ values.

Conclusions

NSCLC patients with pretherapeutic acute inflammation status or T-cell suppressing cytokine profile showed significant worse response to immunotherapy with Nivolumab. Pretherapeutic assessment of inflammation status could cost-effectively identify patients who benefit from immunotherapy. Thus, monitoring of cytokine profile under Nivolumab treatment could discriminate between stable disease, pseudoprogression or real progressive disease.

Legal entity responsible for the study

Diego Kauffmann-Guerrero

Funding

Universität München (LMU)

Disclosure

All authors have declared no conflicts of interest.

Background

TP53 somatic mutations have been suspected to induce an autoantibody immune response against p53. The aim of this study was to determine if a specific TP53 tumor mutational status, is associated with the presence of circulating p53-autoantibodies (p53-AAbs) in patients with advanced high grade ovarian cancer (HGOC).

Methods

A retrospective study was carried out in 117 patients with stage III-IV and high grade (G2-G3) tumors, who were enrolled at CRO Institute and provided adequate tissue sample. All patients underwent surgical debulking and received platinum-based regimen. Matched tumor and blood DNA samples were analyzed for TP53 genetic mutations using a MiSeq platform. Commercially available ELISA kit was used to detect p53-AAbs in plasma or serum.

Results

Median age of the patients was 58 years (range 31-82). Median follow-up time was 40 months (IQ range 20-67). TP53 somatic mutations were found in 74,4% (87/117) of HGOC patients and 26,5% (31/117) were positive for p53-AAbs. Among mutated patients, 28.7% (25/87) was tested positive for presence of p53-AAbs and 71.3% (62/87) was p53-AAbs negative (Fisher’s exact test; p=0.473). In p53-AAbs positive patients with at least a TP53 mutation in matched tumor (81%, 25/31), missense mutations were strongly associated with the presence of p53-AAbs while insertion/deletion, splice/intronic and nonsense variants were associated with p53-AAbs negative patients (p<0.001). Gain of function (GOF) missense mutations were prevalent in p53-AAbs positive patients, while insertion/deletion mutations with loss of function (LOF) properties prevailed in p53-AAbs negative patients (p=0.0162).

Conclusions

This study for the first time compared data from the genetic TP53 tumor status through an NGS approach with the presence of circulating p53-AAbs. In most of HGOC, the presence of p53-AAbs was associated with tumors harboring TP53 missense mutations that share GOF effects. These new findings suggest that a specific mutational status in the HGOC tumor is associated with presence of p53-AAbs, but not completely explain the amount of p53-AAbs appearance. Both markers should be further explored and evaluated for impact on clinical outcome.

Legal entity responsible for the study

CRO-National Cancer Institute

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

Blockade of immune checkpoint Programmed cell death protein-1, PD-1/PD-L1 became a principle approach in cancer therapy. MicroRNAs(miR) and Long-noncoding RNAs(LncRNAs) were described to have a critical role in carcinogenesis but not extensively studied in cancer immunotherapy. Our previous data revealed that overexpression of LncRNA X inactive–specific transcript(XIST) downregulates PDL-1 in breast cancer (BC). The study aims to investigate the impact of miR-182 on PD-L1 and to analyze the differential expression of PD-L1 and non coding RNAs in different BC subtypes.

Methods

BC as well as lymph node (LN) biopsies were collected from 21 BC patients (60% Triple negative(TNBC) and 40% Invasive Ductal Carcinoma(IDC)). Expression profiling of PDL-1, MALAT-1, XIST and miR-182 was quantified by Taqman Real Time qPCR and normalized by Beta-2-microglobulin and RNU6B. Manipulation of miR-182 was performed in MDA-MB-231 cells followed by quantifying the target genes by RT-qPCR.

Results

MiR-182, MALAT-1 and XIST were selected as regulators for PD-L1. The relative expression of miR-182, PD-L1 and MALAT-1 were markedly increased in all BC than controls (P = 0.0172, P = 0.0237 and P = 0.0255). MiR-182, PD-L1 and MALAT-1 expression was positively correlated (p = 0.026, p = 0.033 and p = 0.031) and higher in TNBC by 15, 7 and 79.5 folds, respectively, than IDC. However, XIST expression was inversely correlated to miR-182, PD-L1 and MALAT-1 (p = 0.043, p = 0.041 and p = 0.035) and lower in TNBC by 10 folds than IDC. Unexpectedly, PDL-1 was undetectable in IDC LNs, however, it was markedly overexpressed in TNBC LNs compared to control-LN (P = 0.0065). MALAT and XIST expression in LN was non-significant. Overexpression of miR-182 in MDA-MB-231 cells resulted in significant increase in PD-L1 and MALAT-1 expression (P = 0.0057 and P = 0.0468, respectively) and dramatic decrease in XIST (P = 0.0351). Anti-miR-182 reversed the effect of the mimics.

Conclusions

This study sheds the light on the differential expression of miR-182, MALAT-1 and XIST with a significant correlation to PD-L1 in TNBC and IDC. The data suggests a crucial role for these genes in the extent of evasion of cancer immunity in different BC subtypes.

Legal entity responsible for the study

German University in Cairo, Egypt

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

Despite the success of immunotherapies, patients still respond differently. Thus, there is an urge to explore novel predictive biomarkers to understand the complex interactions within cancer immunity. Long non-coding RNAs (lncRNAs) were found to act as biomarkers in breast cancer (BC). X inactive –specific transcript (XIST) has been proved to be an indicator of poor outcome in BC. TSIX is the antisense of XIST and both LncRNAs were shown to have crucial role in breast carcinogenesis. However, no evidences were reported about their role in immune evasion. Our previous data proved that XIST and TSIX were able to paradoxically manipulate PD-L1 expression in BC cell lines. This study aims at investigating the potential role of XIST and TSIX as cancer-immune biomarkers in relation to the PD-L1 expression in different body compartments of BC patients.

Methods

BC biopsies, Lymph nodes (LN), whole blood, serum and nipple discharge(ND) were collected from 35 BC patients (15.7% triple negative BC (TNBC), 36.8% luminal B, 21.05% luminal A, 10.5%Her2+ and 15.78% luminal B Her2+). PBMCs were isolated using Ficoll-Hypaque method. Total RNA extraction was performed using BIOZOL reagent, then the expression profiling of PD-L1, XIST & TSIX was quantified by Taqman Real Time qPCR and normalized to Beta-2-microglobulin.

Results

The relative expression of XIST and TSIX was markedly increased in tumor biopsies (p = 0.0493 and p = 0.0173), PBMCs (p = 0.0414 and p = 0.029), Serum (p = 0.0144 and p = 0.0336) and ND (p = 0.0197 and p < 0.0001) of BC patients compared to their paired controls. XIST and TSIX expression was positively correlated to PD-L1 mRNA expression in these 4 body compartments (p = 0.0468 and p = 0.0362). PD-L1, XIST and TSIX showed a higher expression in TNBC and Luminal B subtypes compared to other subtypes (p = 0.0001, p = 0.0467 and p = 0.0115). In contrast, XIST and TSIX showed a dramatic down regulation in metastatic LNs compared to non-metastatic LNs (p = 0.0061 and P = 0.0028). PD-L1 was abscent in metastatic LNs.

Conclusions

This study introduces XIST and TSIX as novel immune biomarkers that are highly correlated with the expression profile of PD-L1 in different body compartments of BC.

Legal entity responsible for the study

German University in Cairo, Egypt

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

Peripheral T cell lymphoma (PTCL) accounts 15% of NHL, characterized for poor survival. IPI and PIT scores are prognostic factors in survival, but they have limitations. Markers of inflammation: neutrophil/lymphocyte ratio (NLR), monocyte/lymphocyte ratio (MLR), and absolute lymphocyte count/monocyte score (ALC/AMC score), are being evaluated as prognostic tools in different malignancies. The aim of this study is to demonstrate if inflammation markers could be used as prognostic factors in survival in PTCL.

Methods

Data of patients diagnosed with PTCL during years 2005-2015 at Rebagliati Hospital was retrospectively reviewed. Patients with cutaneous T-cell lymphoma, CNS lymphoma, ATLL and incomplete data were excluded. OS was determinate with Kaplan-Meier method, comparison of survival curves was made with Log-rank test. Multivariate analysis, prognostic factors for OS were determined using Cox regression model. Statistical evaluations were performed at a significance of 5%.

Results

A total of 68 patients were included. Median age was 61 years, 60.3% were male, 52.9% had ECOG 0-1 and 79% had B symptoms at the time of diagnosis. 58.2% had primary lymph node involvement and the 73.1% had extra-nodal compromise. The 70.6% of patients had clinical stage III-IV and 44.1% had bone marrow infiltration. The 79.2% of patients had a PTCL-NOS subtype, 13.2% Anaplastic, 5.8% NK/T cell Lymphoma and 2.9% an Angioimmunoblastic T- cell lymphoma. OS was 28.4% in 5 years. NLR> 4 was associated with increased risk of mortality (p = 0.001). There was no statistical significance regarding MLR (p = 0.17) and ALC/AMC score (p = 0.49). Multivariate analysis, High-intermediate and High IPI score (HR = 3.02, p = 0.002) were related to NLR> 4 (HR = 2.312, p = 0.016), and High- Intermediate and High PIT score (HR = 2.41, p = 0.001) were related to NLR> 4 (HR = 3,041, p = 0.001).

Conclusions

NLR has proven its prognostic value in patients with PTCL, which is an easily accessible and low-cost tool. This work can serve to address future designs of more efficient scores that allow selection of groups of patients at greater risk and thus lead more individualized therapy.

Legal entity responsible for the study

Denisse Castro MD

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

Interleukin 10 (IL-10) is considered an immune modulator cytokine, showing both antitumor and pro-tumor characteristics. Its role in the pathogenesis and progression of colorectal cancer depends on microenvironmental milieu.

Methods

A case–control study with 195 newly diagnosed colorectal cancer (CRC) patients, and 195 healthy individuals was conducted to compare the serum IL-10 levels between patients and controls and investigate the involvement of the polymorphism (-1082) A/G of the promoter region of the anti-inflammatory cytokine IL10 in the development of CRC. Serum levels of IL10 were measured by Enzyme amplified sensitivity immunoassay (EASIA) method; genotype and allele frequencies of IL10 (-1082) A/G were assessed using amplified Refractory Mutation System (ARMS_PCR).

Results

Mean serum IL-10 levels were significantly higher in CRC patients than in controls (27.56 +/- 13.59 versus 2.39 +/- 1.4 pg/ml; P = 0.001). CRC patients with worse prognosis at the time of diagnosis tend to have higher levels of circulating IL-10 than those with better prognosis (34.77 +/- 12.37 pg/ml). This study has demonstrated also that the minor allele G was a protective factor against the occurrence of CRC (OR = 0.52; p < 0.001) and the genotype GG and AG were more protective than AA in the occurrence of CRC (p < 0.001 OR = 0.27; p = 0.02 OR = 0.6 and OR = 1 respectively).

Conclusions

Our results demonstrated that IL-10 levels in the serum of CRC patients can be used as a prognostic biomarker in CRC patients and show a negative association of the minor allele G and GG genotype with CRC, and the AG genotype has a more protective role against the occurrence of CRC than does the AA genotype.

Legal entity responsible for the study

Military Hospital

Funding

Military Hospital

Disclosure

All authors have declared no conflicts of interest.

Background

Inflammation has a multifaceted role in cancer progression including initiation, promotion and invasion by affecting the immune surveillance and associated signalling pathways. The aim of this study was to measure circulating pro- and anti-inflammatory cytokines (IL 6, TNF-α, and IL10) and their correlation with prognostic factors and progression in breast cancers in Tunisian patients.

Methods

Serum samples were prospectively collected from a cohort of sixty breast cancer patients after surgery. Circulating levels of the pro-inflammatory cytokines TNF-α and IL6 were measured with the technique of a solid-phase, two-site chemo-luminescent enzyme immune-metric assay (Immulite 1000, Simens, USA). Serum levels of the anti-inflammatory cytokine IL10 were measured by the ELISA sandwich method.

Results

The mean age of patients was 47 years (20 – 80 years), 17 patients were metastatic (32% in liver). The mean level of cytokines Il6, IL10 and TNF-α were, respectively, 3.31 +/- 4.07 pg/ml (min 1, max 29.30 pg/ml); 6.560+/- 3.50 pg/ml (min 0.880, max 17.925 pg/ml) and 6.90 +/- 2.99 pg/ml (min 3, max 20.30 pg/ml). We found a significant correlation between high level of IL6 and metastatic disease (p = 0.04) especially with liver metastasis (p = 0.003). We found also a significant correlation between high level of TNF-α and lymph node involvement (p = 0.0011). On the other hand there was a significant correlation between high level of IL10 and high SBR grade (p = 0.01).

Conclusions

Our results highlight the role of circulating cytokines of inflammation IL6, TNF-α and IL10 as a potential prognostic biomarkers in breast cancer patients which could contribute to tumor growth and progression. So there was a rationale for the use of cytokine and chemokine blockade, and further investigation of anti-inflammatory drugs in the chemoprevention and treatment of malignant diseases should be carried out.

Legal entity responsible for the study

Military Hospital

Funding

Military Hospital of Tunis

Disclosure

All authors have declared no conflicts of interest.

Background

EnAd is a tumor-selective oncolytic adenovirus with highly selective replication and cell killing in a broad range of carcinoma cell lines and little replication in normal and non-carcinoma cells. Following intravenous (IV) dosing in clinical studies, uptake and replication of EnAd, associated with CD8+ cell infiltration, has been shown in various carcinomas.

Methods

EVOLVE was a Phase 1 study to primarily explore the safety and efficacy of EnAd following IV administration of doses from 1e10 to 1e13 viral particles. Treatment cycles comprised IV infusions on Days (D) 1, 3 and 5, repeated every 3 weeks. Initial safety data have been reported elsewhere (ASCO 2014, abstr 3103). Here we present an analysis of the incidence and timing of adverse events (AEs) alongside the cytokine response data.

Results

During dose escalation, DLTs of acute lung injury, dyspnoea and hypoxia were reported at the highest dose tested, occurring on Cycle 1 Day 1 (C1D1). At doses up to the maximum tolerated dose (MTD), a higher incidence of AEs was reported following dosing on C1D1 compared with D3 & D5. Following D1 administration in Cycle 2, the frequency of AEs was lower than that reported on C1D1. Dose-dependent transient increases in MCP-1 and IL-6 were observed in all patients following C1D1 dose, with lower average increases following D3 & D5 administration, or on D1 of subsequent cycles. This pattern of cytokine release tends to mirror that of reported AEs, and appears independent of the anti-EnAd antibody response.

Conclusions

Tolerability of EnAd is primarily determined by C1D1, correlating with the cytokine response across the patient cohort. This supports the hypothesis that tolerability of the C1D1 dose of EnAd is limited by the activation of innate immune cells (for example, Kuppfer cells in the liver) with associated cytokine release. The MTD of EnAd has been determined primarily based on AEs occurring on C1D1, however higher subsequent doses may be tolerable. Manipulation of dosing regimens may allow administration of higher doses of EnAd on D3 & D5, and/or more doses per cycle, to increase EnAd delivery to tumor and optimize its immunotherapeutic potential.

Clinical trial identification

EUDRACT: 2012-001067-79

Legal entity responsible for the study

PsiOxus Therapeutics Ltd.

Funding

PsiOxus Therapeutics Ltd.

Disclosure

H. McElwaine-Johnn, S. Alvis, R. Brown, K. Fisher, C. Morris, B. Champion: Paid employee and stockholder of PsiOxus Therapeutics Ltd. G. Pover: Paid consultant of PsiOxus Therapeutics Ltd. J. Beadle: Paid employee, director and stockholder of PsiOxus Therapeutics Ltd.

Background

Combined tumor immunotherapy interventions have the potential to achieve additive or synergistic effects. Combined local injection of dsRNA analogues (mimicking viral RNA) and repeated vaccination with tumor-lysate loaded dendritic cells shows efficacy against a mouse model of established colon cancer.

Methods

In a pilot phase I clinical trial 16 advanced (stage IV) cancer patients received two cycles of a course consisting of four intradermal daily doses of monocyte-derived dendritic cells preloaded with autologous tumor lysate and matured for 24h with polyICLC (Hiltonol), TNF-α and IFN-α. On days +8 and +10 of each of each cycle, patients received intratumoral image-guided injections of 0.25 mg of Hiltonol. Each cycle was given 3-4 weeks apart and was preceded on day -7 by 600 mg/m² of cyclophosphamide. The last six patients were given SABR (stereoatactic ablative radiotherapy) in some of the lesions including that injected with Hiltonol. Expression of a series of 25 inmune-relevant genes was sequentially monitored by RT-PCR on circulating PBMCs and serum concentrations of a cytokine panel were sequentially determined.

Results

Combined treatment was feasible, safe and well tolerated without >grade 2 side effects. No objective responses by RECIST1.1 criteria were observed while nine patients experienced stabilization of disease (five of them in the six-patient radiotherapy cohort). A heavily pretreated castration-resistant prostate cancer patient experienced a remarkable mixed abscopal response to radiotherapy with responding lesions located far from the irradiated region. Response was associated to an increase of CD8 T cells infiltrating the tumor. In this series of patients post-treatment elevated IFNβ and IFNα mRNA in circulating PBMC following intratumoral Hiltonol was detected, in addition to increases of serum IL-12 and IL-1β following DC vaccination, that occurred more prominently in stable disease cases.

Conclusions

This combination strategy aimed to resembling viral infection in tumor tissue, while on a dendritic-cell vaccine approach and added to radiotherapy is safe for advanced cancer patients and shows indications of immune-associated activity.

Clinical trial identification

NTC01734564

Legal entity responsible for the study

Clinica Universidad de Navarra

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

Therapeutic vaccination with tumor antigen-encoding RNAs by local administration is currently successfully employed in various clinical trials. Advancing from local to more efficient systemic targeting of antigen-presenting cells (APCs), we developed a novel class of RNA-lipoplex (RNA_(LIP)) immunotherapeutics for intravenous (i.v.) application. RNA_(LIP) is a nanoparticulate formulation of lipid-complexed RNA which was engineered to selectively deliver the antigen-encoding RNA to APCs in lymphoid compartments body-wide and to preserve RNA integrity after i.v. injection. Efficient RNA uptake and expression of the encoded antigen by the APCs results in the synchronized induction of both potent adaptive as well as type-I-IFN-mediated innate immune responses.

Methods

The first-in-human phase I/II dose escalation Lipo-MERIT trial (NCT02410733) assesses the safety, tolerability, and biological efficacy of RNA_(LIP) immunotherapy in patients with stage IIIB/C and IV melanoma in four German study centers. This is the first example of an i.v. administered, clinically applicable RNA-based cancer vaccine. Following selective antigen stratification on routinely collected tumor samples, eligible patients are treated with increasing doses of the tetravalent Lipo-MERIT vaccine composed of a fixed set of RNA_(LIP) products encoding the shared melanoma-associated antigens NY-ESO-1, tyrosinase, MAGE-A3, and TPTE that are administered successively within each treatment cycle.

Results

Preliminary data obtained from >40 patients of this ongoing trial confirm the safety and tolerability of the universally applicable RNA_(LIP) immunotherapy platform with patients experiencing mostly mild to moderate adverse drug reactions typically associated with immune activation.

Conclusions

Preliminary results from immunological assessments show a high rate of vaccine-induced immunity and indicate that multiple applications of the Lipo-MERIT vaccine lead to the de novo induction of antigen-specific immune responses and potent expansion of pre-existing immunity.

Clinical trial identification

NCT02410733 March 2015

Legal entity responsible for the study

BioNTech RNA Pharmaceuticals

Funding

BioNTech RNA Pharmaceuticals

Disclosure

L. Heesen, R. Jabulowsky, E. Derhovanessian, A. Kuhn, D. Schwarck-Kokarakis: Employee of BioNTech AG. C. Loquai: Advisory board: Roche, Novartis, Pierre Fabre, MSD, BMS, Leo, Amgen, Biontech; Speekers fee: Roche, Novartis, Pierre Fabre, MSD, BMS, Amgen; Travel Reimbursement: Roche, Novartis, MSD, BMS, Amgen. J. Utikal: Advisory boards and on speakers bureaus of Amgen, BMS, GSK, MSD, Novartis, and Roche. C. Gebhardt: Advisor, honoria and/or travel reimbursements (BMS, MSD, Novartis, Pierre Fabre, Roche). J. Hassel: Corporate-sponsored research (BioNTech AG, BMS, Roche, Novartis, Pierre Fabre, Philogen, Amgen, ImmunoCore); membership on an advisory board (MSD, Amgen). S. Attig: Employee of TRON gGmbH (Mainz, Germany) and does not have any financial relation to the study. D. Jäger: Membership on an advisory board (BMS, Roche/F. Hoffmann La Roche, Amgen, MSD, Bayer, Definiens, CureVac, Oryx). S. Grabbe: Membership on an advisory board (AbbVie, BMS, MSD, Novartis, Merck Serono, Sanofi-Pasteuer-MSD, Roche); travel reimbursement (AbbVie, BMS, MSD, Novartis, Merck Serono, Sanofi-Pasteuer-MSD, Roche, OnkoZert, Takeda, MedConcept, Beiersdorf, L'Oréal). Ö. Türeci: Co-founder (Ganymed Pharmaceuticals GmbH); patent ownership & patent applications. M. Diken: Inventor on patents and patent applications related to this study; Contract with BioNTech AG. Consultant contract with BioNTech AG. L. Kranz, H. Haas: Inventor on patents and patent applications related to this study; Employee of BioNTech AG. P. Langguth: QP contract with BioNTech AG. U. Sahin: BioNTech AG (patent ownership & patent applications; stock owner; management board member and co-founder), Ganymed Pharmaceuticals GmbH (membership on an advisory board; co-founder), TRON gGmbH (co-founder and chair). All other authors have declared no conflicts of interest.

Background

Tumor vaccines are a specific modality of cancer immunotherapy. They are based on the administration to a cancer patient of tumor antigens (TAs) or dendritic cells (DCs) previously pulsed with known TAs. In spite of significant development and testing, DC-based tumor vaccines have largely delivered unsatisfactory clinical results. Extracellular vesicles (EVs) released by cancer cells can potentially deliver TAs to DCs, so as to promote the initiation of anti-tumor immune responses. However, clinical translation of this procedure requires the isolation and ex vivo manipulation of both tumor-derived EVs and patient-derived DCs, which impose significant hurdles.

Methods

We here describe an engineered receptor, called extracellular vesicle-internalizing receptor (EVIR), which enables the selective uptake and processing of endogenous, cancer cell-derived EVs from the patient’s body, thereby circumventing the need of exposing DCs to tumor-derived material ex vivo. EVIRs encompass a truncated low-affinity nerve growth factor receptor fused to an extracellular antibody domain specific to a cancer protein. We developed EVIRs that selectively internalize EVs derived from various cancer cell types, including breast cancer cells that overexpress HER2 and melanoma cells that overexpress diasialoganglioside (GD2).

Results

Lentiviral vector-mediated transduction of the EVIR into primary DCs efficiently and specifically promoted macropinocytosis-mediated uptake of cancer cell-derived EVs and greatly enhanced the presentation of EV-associated TAs to naïve CD8+ T cells. EVIR-engineered DCs effectively inhibited the growth of HER2+ tumors and promoted the expansion of tumor-specific cytotoxic T cells. By employing CRISPR technology for disrupting the expression of either H-2Kb or B2M MHCI components in cancer cells, we found that the EVIR promotes T-cell activation largely through cross-dressing, a process that involves the acquisition by DCs of MHCI/antigen complexes shed by other cells.

Conclusions

EVIR-engineered DCs may be employed to foster the acquisition and presentation of a broad repertoire of cancer-specific TAs by antigen-presenting cells, enabling personalized DC-vaccination protocols for cancer immunotherapy.

Legal entity responsible for the study

EPFL

Funding

European Research Council (ERC) Consolidator Grant 2017

Disclosure

All authors have declared no conflicts of interest.

Background

Immune checkpoint inhibitors have been delivering promising results in an increasing number of indications. This approach proved the potential benefits of the use of the immune system as a tool against malignant cells. However, there is still room from improvement as there is a considerable number of patients not benefiting from these therapies. One of the biggest obstacles to overcome for those unresponsive patients is the lack of immune presence in the tumor. To overcome that limitation, we hypothesize that viruses are an appealing tool, as they are inherently able to attract the attention of the immune system and to express different transgenes. In our case, the viruses were armed with tumor necrosis factor alpha (TNFα) and interleukin-2 (IL-2), two cytokines to improve the trafficking to the tumor and enable T-cell mediated responses in the tumor.

Methods

To study the previously described scenario, an in vivo model of subcutaneous melanoma (B16.OVA) was used. Three different experiments were carried out (n = 48, 75 and 94) in order to assess the benefits and to understand the effect in the tumor microenvironment after the treatments.

Results

When both therapies where given together, they delivered significantly better results than single treatment groups in term of overall survival and tumor growth control. From all the tested conditions, the treatments delivered better results when virotherapy was initiated before checkpoint blockade, in a “prime and boost” approach. In this set up, a 100% complete response was achieved (HR = 0.026 [0,005; 0,139] when compared with checkpoint blockade alone and HR = 0.069 [0,015; 0,327] when compared with virotherapy alone). The biological samples obtained through the study revealed a shift towards a Th1 cytokine status in the tumor while profile of the immune cell population also showed a significant change towards to the antitumor subsets.

Conclusions

The purpose of this study was to try to overcome poor immune infiltration in order to enable checkpoint blockade therapies, based on the results, the objectives were achieved after the use of a viral platform delivering immunostimulatory cytokines in the tumor that helped to redesign the tumor microenvironment.

Legal entity responsible for the study

Cancer Gene Therapy Group

Funding

TILT Biotherapeutics Ltd.

Disclosure

V. Cervera-Carrascon, J.M. Santos, M. Siurala, R. Havunen and S. Sorsa: Employee of TILT Biotherapeutics. A. Hemminki: Shareholder in Targovax ASA (Oslo, Norway) and in TILT Biotherapeutics Ltd. (Helsinki, Finland). Employee of TILT Biotherapeutics.

Background

Modern treatment modality of chronic myeloid leukemia is dependent on tyrosine kinase inhibitors prototyped by Imatinib and other new TKIs, however it is difficult to eradicate the disease at the molecular level by use of TKIs only. Studies show that deeper molecular response is associated with longer survival. In an attempt to investigate the role of active immunotherapy (vaccination) to reduce the molecular burden at least one-log in patients with detectable molecular disease while receiving and continuing TKI, we designed a small trial of a multipeptide vaccine against bcr-abl p210 protein junctional peptide on 10 patients with CML-CP receiving Imatinib 400 mg/day or more for at least 12 months and followed them for 5 years.

Methods

10 adults with chronic myeloid leukemia in chronic phase receiving Imatinib of at least 400 mg/day with defined HLA Class I molecules were injected GM-CSF and IL-12 plasmids 100 micrograms intradermally once and eight 3-weekly apart multipeptide injections subcutaneously beginning one day after the adjuvant treatment. The usual TKI dose was continued or changed in the discretion of treating physician. We sampled peripheral blood and bone marrow just before and 2 months after the vaccination trial to compare the molecular burden by bcr-abl RT-PCR technique.

Results

10 adults with CML-CP were enrolled. There were 9 males and one female. They were diagnosed with CML-CP and were taking Imatinib at least 400 mg/day for more than 12 months. All patients had one of the HLA Class I molecules of A02, A03 or B08. At least one of the nine bcr-abl p210 derived small peptide in the vaccine could adhere to one of the HLA Class I molecules modestly and elicit a cytotoxic T cell response as was published by BIMAS and SYFPEITHI epitope prediction softwares. At the final analysis, 2-months after the last step, laboratory investigation disclosed 1-log decrease in peripheral blood molecular burden in 2 of 10 patients. The active immunotherapy was not accompanied by systemic side effects, however mild local reaction. Long term(5-year) follow up demonstrated undetectable disease in 1 patient and 1-log decrease in 6 others compared to early post-vaccination assay. One of the early responded patient, had discontinued taking Imatinib for 12 months and increased molecular burden that we observed might be attributed to interruption of TKI treatment and the other one did not participate in follow up study, however he was clinically well. We observed 1-log decrease in molecular burden in six and a case of undetectable disease among eight initially nonresponded patients after 5-years.

Conclusions

Active immunotherapy in CML against bcr-abl derived p210 protein junctional peptide, considering immunology of the peptides and the patients could be an attractive and safe treatment that adds another first to the CML firsts.

Legal entity responsible for the study

The Hematology-Oncology Research Center and Stem Cell Transplantation (HORCSCT; formerly HORCBMT) is affiliated The Hematology-Oncology Research Center and Stem Cell Transplantation at Shariati Hospital affiliated to Tehran University of Medical Sciences, Tehran, Iran.

Funding

Hematology-Oncology Research Center and Stem Cell Transplantation at Shariati Hospital affiliated to Tehran University of Medical Sciences, Tehran, Iran.

Disclosure

All authors have declared no conflicts of interest.

Background

The expression of mutated proteins by tumour cells (neoepitopes) and the detection of tumour-specific cytotoxic T-lymphocytes against these antigens in cancer patients support the interest of using vaccines as antitumor treatments. The aim of this study is to assess the possibility of using oncolytic adenovirus (OAds) coding for a patient’s specific neoepitopes as an “oncolytic personalized vaccine”.

Methods

A murine melanoma cell line (B16) was exome-sequenced to select tumor’s neoepitopes. They were selected and prioritized according to their expression levels and predicted MHC-I affinity. Five 27mer peptides including the selected amino acid change in position 14 and a control OVA257 were set up in a Tandem-Minigene (TMG) configuration with or without Gly-Ser linkers. The correspondent mRNA sequence for TMG was incorporated by recombineering as a transgene after the fiber in an OAd (ICO15 with an E3 6.7/19K deletion) regulated by cytomegalovirus promoter (CMV).

Results

The OAd expressing tumour neoepitopes with (ICO15-del6.7/19K-CMV-SpB16TMG.Linkers) or without linkers (ICO15-del6.7/19K-CMV-SpB16TMG) via TMG were successfully generated and purified. We confirmed that they retain the oncolytic potency of its parental virus and that the control OVA257 included in the TMG could be presented via H2-Kb (murine MHC-I) by murine dendritic cell line. Mice were injected with 3e10 viral particles intravenously and ELISPOT was performed 7 days later to assess the capability to induce an immune response against the encoded neoepitopes. Positive peptide controls E1B and OVA257 confirmed a good virus immunization and transgene expression respectively by both viruses. Tumour neoepitopes expressed with linkers presented fewer capacity of inducing immune responses compared to the TMG without linkers. In fact, a clear immune response was detected against a previously published neoepitope (TNPO3, G504A) with similar levels as E1B and OVA257 when expressed by the OAd in the absence of linkers. In vivo efficacy will be assessed.

Conclusions

OAds expressing tumour neoepitopes via TMG induce an immune response against those neoepitopes in vivo. Linkers in a TMG reduces the intensity of the immune response against encoded neoepitopes compared to TMG without linkers expressed by OAds.

Legal entity responsible for the study

Bellvitge Biomedical Research Institute (IDIBELL) and VCN Biosciences S.L.

Funding

Bellvitge Biomedical Research Institute (IDIBELL) and VCN Biosciences S.L.

Disclosure

M. Farrera Sal: Industrial phD student, perfoming it in academic group (Virotherapy and Immunotherapy group, IDIBELL) and biotechnological company (VCN Biosciences S.L.)

All other authors have declared no conflicts of interest.

Background

Adoptive cell transfer (ACT) has shown promising yet suboptimal results in clinical trials for melanoma. We aimed to enhance the efficacy of ACT by coupling it with adenoviruses coding for tumor necrosis factor alpha (TNFa) and interleukin-2 (IL-2).

Methods

Non-replicating adenoviruses (Ad5-CMV-mIL2 and Ad5-CMV-mTNFa) were constructed and confirmed to produce biologically active murine cytokines in vitro and in vivo, and subsequently used in combination with ovalbumin-specific (OT-I) T-cell transfer. Oncolytic adenoviruses expressing human TNFa and/or human IL-2 (Ad5/3-E2F-D24-hTNFa-IRES-hIL2 also known as TILT-123) were also constructed and used in combination with tumor-infiltrating lymphocytes (TIL) transfer in adenovirus-permissive Syrian hamsters.

Results

The non-replicating virus vectors used in combination with OT-I T-cells effectively controlled tumor growth of established murine melanoma (B16.OVA) tumors. In further experiments, a triple combination of Ad5-CMV-mIL2 + Ad5-CMV-mTNFa (1:1 ratio) and OT-I T-cells improved antitumor efficacy over double combinations. Mechanistic studies revealed that intratumoral virus injections induce trafficking of adoptively transferred T-cells to tumors. Further, the cytokine-coding adenoviruses caused favorable alterations in the tumor microenvironment. In hamsters, established pancreatic cancer tumors were eradicated with TILT-123 injections coupled with TIL transfer. The cured animals were protected from tumor rechallenge. In addition, we have demonstrated that adenovirally delivered IL-2 is superior to systemically administered recombinant IL-2 with regard to safety and efficacy in the preclinical setting.

Conclusions

The combinatorial approach studied here highlights the potential of adenovirotherapy to enable T-cell therapies. TILT Biotherapeutics is in the process of confirming the results in clinical trials.

Legal entity responsible for the study

TILT Biotherapeutics Ltd

Funding

TILT Biotherapeutics Ltd, Finland; Jane and Aatos Erkko Foundation, Finland; European Commission Marie Curie Innovative Training Network (ITN) grant VIRION (H2020-MSCA-ITN-2014 project number 643130); Doctoral Program in Clinical Research, University of Helsinki, Finland; Helsinki University Central Hospital (HUCH) Research Funds, Finland; Sigrid Juselius Foundation, Finland; Biocentrum Helsinki, Finland; Biocenter Finland, Finland; Finnish Cancer Organizations, Finland.

Disclosure

J.M. Santos, M. Siurala, R. Havunen, V. Cervera-Carrascon, S. Sorsa: Employee in TILT Biotherapeutics Ltd. A. Hemminki: Shareholder of Targovax SA and employee in TILT Biotherapeutics Ltd.

Background

We intend to develop DNA- and protein-based vaccines targeting immune checkpoints in order to augment the anti-tumor immune responses of radiation therapy.

Methods

Murine CTLA-4 and PD-L1 DNA sequences were PCR amplified from a mouse cDNA library. The PCR products were fused with a transmembrane domain of placental alkaline phosphatase into an expression plasmid, pVAC-1, forming CTLA-4-PD-L1 DNA vaccine (DNA vaccine). CTLA-4 and PD-L1 fusion protein vaccine (protein vaccine) was acquired using E. coli expression system. Mouse tumor models were established by inoculating CT26 colorectal cells on Balb/c mice, and LLC Lewis lung cancer cells on c57BL/6 mice, respectively (on day 0). The mice of each tumor model were divided into 3 groups, each with 5 mice. Group 1 was treated with radiation therapy (RT) alone, group 2 with RT plus DNA vaccine (coupled with liposome), and group 3 with RT plus protein vaccine. Vaccination was done via subcutaneous injections on day 1, 8 and 15. RT was given on day 14 and 21 with 10 Gy each. Sera from the mice was subjected to ELISA assay for the titers of antibodies against CTLA-4 and PD-L1. The sizes of tumors were recorded. After the mice were sacrificed, the tumor samples were subjected to IHC staining for immune profiles, and the splenocytes to cytotoxicity and ELISpot assays.

Results

In the CT26 model, combination treatment with either RT plus DNA vaccine or RT plus protein vaccine (group 2 or 3, respectively) showed better tumor suppression than RT alone (group 1). This superior tumor control in combination treatment groups paralleled the enhanced cytotoxicity of their splenocytes when co-cultured with CT26 cells. Also, the splenocytes from the combination treatment groups showed activated T cell responses as indicated by increased interferon-γ production. The immunohistochemistry staining demonstrated less PD-L1-positive cells in the tumor samples from the combination groups. In the LLC model, however, only the treatment with RT plus CTLA4-PDL1 DNA vaccine showed better tumor suppression than with RT alone.

Conclusions

We have developed potent DNA and protein vaccines targeting CTLA-4 and PD1 immune checkpoints, enhancing the tumor control effects of radiation in the colorectal and lung cancer animal models. This combination treatment strategy show the potential to enhance tumor-specific immune responses and hence could confer systemic anti-tumor ability on radiation therapy, namely, abscopal effect.

Legal entity responsible for the study

Keng-Hsueh Lan

Funding

Ministry of Science and Technology, R.O.C.

Disclosure

All authors have declared no conflicts of interest.

Background

The EnanDIM® family consists of linear single-stranded DNA molecules containing non-methylated CG-motifs for TLR9 activation and L-deoxyribonucleotides at their 3’-ends to prevent degradation. They initiate a broad activation of the innate and adaptive immune system. The conversion of non-immunogenic (“cold”) tumors into immunogenic (“hot”) tumors, characterized by their T cell infiltration, is a pre-requisite for a response to checkpoint inhibitors. This may be achieved by EnanDIM^® due to induced IP-10/CXCL10 secretion. Furthermore, the mode-of-action of EnanDIM^® starts upstream of the initiation points of checkpoint inhibitors (CPI). Therefore EnanDIM^® likely provides the relevant immune activation required for effectiveness of CPI and thus resulting in an enhanced anti-tumor effect in combination approaches.

Methods

The colon carcinoma model CT26 was used for evaluation of the influence of EnanDIM^® on the tumor microenvironment (TME) and to investigate the anti-tumor effect of EnanDIM^® in combination with anti-PD-1. In addition, the impact of EnanDIM^® on T cell responses was analyzed employing an in vitro assay with human peripheral blood mononuclear cells (PBMC) stimulated with CEF-peptides recognized by recall-antigen-specific CD8+ T cells (CMV, EBV, Flu).

Results

An increased infiltration of T cells, especially CD8+ T cells, into the tumor was associated with an anti-tumor response after intratumoral injection of EnanDIM^® in the CT26 model. The moderate anti-tumor effect of aPD-1 was substantially augmented by a combination with EnanDIM^®. After stimulation with CEF-peptides, human PBMC were treated with either EnanDIM^® or aPD-1. Resulting IFN-gamma secretion was higher for EnanDIM^® than for aPD-1 and was further enhanced by the combined treatment.

Conclusions

TLR9 agonist EnanDIM^® activated CD8+ cytotoxic T cells and initiated their infiltration into the tumor complementing the mode-of-action of CPI. Indeed EnanDIM^® enhanced the limited anti-tumor effect of aPD-1 in a murine colon carcinoma model in vivo. These data show the promising potential of EnanDIM^® for the combination with CPI in clinical trials.

Legal entity responsible for the study

Mologen AG

Funding

Mologen AG

Disclosure

K. Kapp, B. Volz, D. Oswald, M. Schmidt: Employee at Mologen AG. B. Wittig: Stock ownership, consults Mologen

Background

Cancer immunotherapy directs the immune system to attack tumor cells by targeting tumor-associated antigens. We manufacture fully personalized monocyte-derived dendritic cell-based vaccines (DC-based vaccines) that are evaluated in investigator-initiated clinical trial “Combined antitumor therapy with ex vivo manipulated dendritic cells producing interleukin-12 in children, adolescents and young adults with progressive, recurrent or primarily metastatic high-risk tumors”.

Methods

DC-based vaccine, called MyDendrix, is manufactured under GMP in Clean rooms of the Department of Pharmacology Masaryk University Brno. Mononuclear cells are collected by apheresis. Monocytes cultivated with IL-4 and GM-CSF differentiate into DC and are exposed to autologous tumor lysate antigens. Semi-maturated DC are aliquoted and cryopreserved. Quality control includes viability, IL-12, IL-10 production, surface markers expression, stimulation of allogenic and autologous T-cells. Vaccines are administered intradermally every 2-4 weeks, up to 35 doses. Detailed immunomonitoring of peripheral blood leucocytes subsets at the baseline and during vaccination is performed. That includes routine examined peripheral blood parameters, monocyte subsets, MDSC, specific lymphocyte subsets including Tregs, effector T-cells, NKT-like cells. Moreover, ex vivo functional immunomonitoring based on autologous T-cell stimulation by DC vaccine is performed.

Results

The primary endpoint of the clinical trial is the assessment of safety by the analysis of occurrence of AESI (adverse events of special interest) in adaptive 5 + 5+5 + 5 design. As of to date, 11 patients have been treated. No AESI was detected. Local skin reaction is usually mild and self-limiting. De facto size and redness of induration at vaccination sites can be reflected by T-cells stimulation after vaccination. Ex vivo T-cell reactivity was enhanced upon vaccination.

Conclusions

Intradermal treatment with the DC-based anti-cancer vaccine is, according to interim analysis, safe and well-tolerated. Ex vivo assessment of pre- and post-vaccination DC-stimulated autologous T-cell activation has shown that anti-cancer vaccine-enhanced pre-existing T-cell response to tumor antigens.

Clinical trial identification

EudraCT 2014-003388-39

Legal entity responsible for the study

Czech Ministry of Health

Funding

This work was supported by Czech Ministry of Health, projects LO1413, LM2015090, DRO (MMCI, 00209805). Academic Clinical Trial.

Disclosure

All authors have declared no conflicts of interest.

Background

Split vaccines are allowed during treatment with immune-checkpoint inhibitors (CKI) and flu vaccine is recommended in cancer patients, despite it seem to amplify immune-related adverse events of CKI and it may be not necessary, since immunotherapy enhances cellular and humoral immunity. We planned a retrospective study at 24 Italian centers, to compare the occurrence of flu syndrome in advanced cancer patients treated with CKI receiving or not flu vaccine. Overall survival (OS) was also evaluated.

Methods

For this preliminary analysis, consecutive data from patients undergoing treatment with CKI at 14 Italian centers from November 2016 to May 2017 were analyzed. Only patients who started CKI after 1^th of January 2016 were enrolled.

Results

203 patients (I-VII treatment line; 77 renal carcinoma, 73 lung cancer, 40 melanoma, 13 others) were enrolled. Median OS was not reached at the median follow-up of 10.2 months (172 censored). 50 patients received flu vaccine. Median time from CKI starting to vaccine was 2 months (mean 3 months). Overall, 29 patients developed flu syndrome: its incidence among vaccinated patients was 30% vs 9.2% of unvaccinated (OR 3.28 95%CI 1.70-6.33, p 0.001). Median time from vaccine to flu syndrome was 2 months (mean 3 months). At 18 months, OS of vaccine group was 83% vs 63% of unvaccinated (p 0.134). Patients who developed flu syndrome had a non-statistically significant trend for better OS compared with unaffected patients (92% vs 63% at 18 months, p 0.127). The 64 patients who had vaccine and/or influenza had significantly better OS (OR 0.52, p 0.049) compared with those not vaccinated nor infected (85% vs 59% at 18 months). At multivariate analysis only treatment response was significantly related to OS (OR 7.3, 95%CI 3.9-13.5, p < 0.001).

Conclusions

With the limit of retrospective study and immature data, it seems that the incidence of influenza among advanced cancer patients treated with CKI is boosted by flu vaccine. Nevertheless, to receive flu vaccine or to develop flu syndrome may prolong survival. Flu vaccine seems to be ineffective in these patients, but it might have positive effect on outcome.

Clinical trial identification

Not applicable.This retrospective observational study has been approved by the local Ethic Committee.

Legal entity responsible for the study

Medical Oncology Unit of the University Hospital of Parma, Italy, Principal Investigator Dr. Sebastiano Buti

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

The therapeutic effect of oncolytic virotherapy is largely based on a two-pronged approach - the direct action of tumor-selective infection, viral replication, and cell killing and the associated activation of innate and adaptive immune responses with the potential of long-lasting tumor remission. Using a chimeric vesicular stomatitis virus variant VSV-GP, we addressed the direct oncolytic effects on the syngeneic mouse lung cancer model LLC1 and the activation of an antitumor immune response in the B16 melanoma model.

Methods

LLC1 lung cancer tumors and B16 melanoma were established in syngeneic C57bl/6 mice. LLC1 tumors were also grafted in immune-incompetent nu/nu mice. Luciferase expressing VSV-GP was used for live in-vivo imaging to study the quantity and kinetics of virus activity in the tumor. Immune fluorescence histological analysis and FACS analysis were used to assess virus-mediated lysis and immune activation.

Results

In vitro, VSV-GP was found to efficiently infect and lyse most cancer cell-lines. Exogenously applied interferon type 1 revealed a dependence of the oncolytic effect on defects in the IFN response of cancer cells. Using a matched pair of LLC1 wildtype and interferon receptor knockout tumors in vivo, interferon insensitivity of cancer cells correlated with prolonged intratumoral viral replication and improved therapeutic outcome. Luciferase imaging also revealed successful tumor-to-tumor spread of viral progeny in bilateral tumor models. Histological analysis revealed widespread and rapid infection and cell killing within the tumor. In contrast, viral replication and tumor-cell killing was limited in the B16-OVA melanoma model, despite showing a therapeutic response in subcutaneous as well as in systemic metastatic settings. FACS analysis revealed an induction of an OVA-specific antitumoral CD8 response as well as an increase of the CD8 to Treg ratio.

Conclusions

The direct oncolytic action of VSV-GP correlates with defects in the innate immune defense of tumor cells. On the other hand, VSV-GP can induce an antitumoral immune-response even in tumors with limited permissiveness for prolonged viral replication.

Legal entity responsible for the study

Guido Wollmann, Medical University Innsbruck

Funding

ViraTherapeutics GmbH

Disclosure

G. Wollmann: Part of the presented research was supported by research grants from ViraTherapeutics GmbH and Boehringer Ingelheim via the Christian Doppler Research Foundation. No other financial relationships exist. M. Petersson: Employed by ViraTherapeutics GmbH. D. von Laer: Founder of and serves as Scientific Advisor for ViraTherapeutics GmbH. Minority shareholder of Viratherapeutics GmbH.

All other authors have declared no conflicts of interest.

Background

Immunization against Epidermal Growth Factor (EGF) has demostrated clinical efficacy in a phase III trial including unselected non-small cell lung cancer (NSCLC) patients. We analyzed if anti-EGF antibodies generated by vaccination (anti-EGF VacAbs) showed antitumor activity in EGFR-mutant, Kras-mutant and Anaplastic Lymphoma Kinase (ALK)-translocated NSCLC cell lines, alone or in combination with tyrosine kinase inhibitors (TKI).

Methods

The EGFR-mutant NSCLC cell line H1975 and osimertinib-resistant cell line (PC9-OR4), Kras-mutant cells A549 and H23 and Alk-translocated cells H3122 and H2228 were treated with anti-EGF VacAbs and the EGFR-mut cells treated also in combination with EGFR TKIs Erlotinib or Afatinib. Cell viability was analyzed by MTT, and apoptosis and cell cycle by fluorescence-activated cell sorting analysis (FACS). Changes of total and phosphorylated proteins were determined by Western blot.

Results

Anti-EGF VacAbs suppressed EGF-induced cell viability and inhibited downstream EGFR signaling. In combination, in EGFR-mutant cells, the anti-EGF VacAbs significantly enhanced the antitumor activity of all EGFR-TKIs tested, suppressed Erk 1/2 phosphorylayion, blocked the activation of STAT3 and downregulated the expression of proteins related to EGFR resistance, such as AXL. Also, they arrested cell cycle progression and increased apoptosis. Finally, anti-EGF VacAbs significantly delayed the emergence in vitro of EGFR TKI resistant clones.

Conclusions

Anti-EGF VacAbs decreased cell viability and inhibited the expression of several EGFR-pathway related proteins. Also, they potentiate the effects of EGFR TKIs and prevent the emergence of resistance in EGFR-mut NSCLC cells. A Phase III clinical trial of anti-EGF vaccination in EGFR wt NSCLC patients is currently ongoing. In addition, a Phase I trial of the EGF vaccine, in combination with EGFR TKIs, will be initiated.

Legal entity responsible for the study

Pangaea Oncology

Funding

Bioven

Disclosure

All authors have declared no conflicts of interest.

Background

Oncolytic adenovirus (OAd) -based therapies face various challenges that may weaken their efficacy. Generating OAd arming FAP-targeting BiTE might improve antitumor efficacy by re-directing T-cells to kill stroma.

Methods

FAP-BiTE (FBiTE) comprises two single chain variable fragments (ScFvs) joined by a flexible Gly-Ser linker. One scFv arm binds human CD3epsilon on the T cell receptor (TCR), while the other binds mouse and human Fibroblast Activation Protein (m/hFAP). The FBiTE gene was incorporated by recombineering in bacteria into the OAd (ICO15K) genome under two different splice acceptors: IIIa and 40SA. The OAds obtained were amplified and purified. HT1080 and 293 FAP- and m/hFAP+ cell lines were used for the in vitro experiments, and A549 cell line and NSG mice for in vivo experiments.

Results

OAds ICO15K-IIIa-FBiTE and ICO15K-40SA-FBiTE were properly generated. Both viruses conserved their cytotoxic properties compared to the parental virus ICO15K and had the capability to infect specifically the tumour cells. FBiTEs were synthesised by the infected cells and secreted to the extracellular media. In vitro, the FBiTE molecules bound simultaneously to FAP+ and CD3+ cells, inducing a catalytic immunological synapsis. This reaction provoked the activation and proliferation of the T-cells specifically against FAP+ cells, inducing significantly more cytotoxicity compared to the parental virus. Moreover, ICO15K-FBiTE in combination with preactivated human T-cells enhanced antitumor efficacy in vivo.

Conclusions

We generated an OAds expressing FAP-targeting BiTE which is expressed and secreted from infected cells. These FBiTEs are able to activate and re-direct T-cells to FAP+ cells, which leads to enhance cytotoxicity in vitro and improve antitumor efficacy in vivo. Further in vivo studies are underway in additional tumour models.

Legal entity responsible for the study

Virotherapy and Immunotherapy group

Funding

MINECO

Disclosure

All authors have declared no conflicts of interest.

Background

Cervical cancer is the second most common cancer in women worldwide and is highly associated with high-risk human papillomavirus (HPV) infection, most often HPV16. Infection caused by HPV requires therapeutic vaccine with the induction of cellular immune responses. However, DNA vaccine alone have limited immunogenicity and therefore, these strategies are needed for increasing their potency such as using an adjuvant. In this study, we used two HPV-16 therapeutic DNA vaccines, pcDNA3.1-E7 and pcDNA3.1-L1, assisted with Brucella abortus RB51 lipopolysaccharide (LPS) as an adjuvant that has less toxicity and no pyrogenic properties in comparison to other bacterial LPS. The aim of this study was the comparison of the efficacy of Brucella abortus RB51 along with two DNA vaccines, pcDNA3.1-E7 and pcDNA3.1-L1, in induction of CTL responses.

Methods

In this study, TC-1 cells were injected subcutaneously to the back of C57BL/6 mice in a volume of 100 μL. For DNA vaccine immunizations, mice were immunized intradermally with a total of 50 μg HPV DNA vaccine containing HPV-16 E7 and HPV-16 L1 DNA alone, or along with B.abortus RB51 LPS (R-LPS). To determine T-cell immune responses, different cytokines such as IFN-γ and IL-4 were evaluated.

Results

Based on the results immunization with pcDNA3.1-E7 or pcDNA3.1-L1 alone could induce strong cellular immune responses, but comparative assessments of these vaccines show that co-administration of HPV-16 E7 and HPV-16 L1 DNA vaccines assisted with R-LPS induced a stronger antigen-specific cellular immune responses and protect mice against TC-1 tumor cell challenges.

Conclusions

The administration of B.abortus RB51 LPS (R-LPS) along with E7&L1 gene plasmid induced HPV16-specific cellular immune responses and protect against TC-1 induced tumor in vivo. We showed that this vaccine was immunogenic, and protective in the TC-1 tumor model. Furthermore, B.abortus RB51 LPS (R-LPS) may be an adequate alternative to enhancing of therapeutic effect of HPV-16 E7 and HPV-16 L1 DNA vaccine.

Clinical trial identification

Legal entity responsible for the study

Tarbiat Modares University

Funding

Tarbiat Modares University

Disclosure

All authors have declared no conflicts of interest.

Background

Researchers have reported a link between several autoimmune reactions and viral infections. In fact, viral antigens can activate cross-reactive T-cells. We hypothesize that the similarity of a tumor peptide with a viral one can be used as a novel selection criteria when designing cancer vaccine therapies in order to engage a wider range of antigen-specific lymphocytes.

Methods

The EpitOME is a novel in silico platform that can compare a tumor protein sequence to an extensive library of viral peptides evaluating HLA-affinity, T-cells cross reactivity and sequence homology. We selected viral sequences that were predicted to be very similar with melanoma epitopes and used them to immunize mice. Animals were then challenged with B16 melanomas and tumor growth was fallowed. ELISPOT assays were used to evaluate the cross-reactivity between the viral and tumor-specific peptides.

Results

To test if viral epitopes can cross-react with tumor ones, we immunized mice with viral peptides resembling TRP2, TYR1 or gp100 antigens. We observed strong cross-reactivity by ELISPOT assays, thus confirming the underlying immunological mechanism. When we challenged these mice with melanoma cells, we observed a slower growth in immunized mice compared to the näive group. Interestingly, targeting TRP2 or gp100 antigens with viral sequences resulted in a remarkable control of the tumor growth.

Conclusions

Taken together, we demonstrated that the degree of similarity between tumor and viral antigens is an important aspect to take into account. The possibility to evoke strong anti-tumor responses by using viral peptides is important for the engagement of anti-tumor T-cells. In addition, the degree of “foreignness” of tumor epitopes can be used as selection criteria when screening peptides list from immunopeptidome analysis, helping in the selection of candidates for cancer vaccines.

Legal entity responsible for the study

Cristian Capasso

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

PDC*vac is a patented technology relying on the use of PDC*line, a HLA-A*02:01+ plasmacytoïd Dendritic Cell (PDC) line, as a potent antigen presenting cell to strongly boost T cell responses both in vitro and in vivo. PDC*vac is composed of PDC*line pulsed with any desired HLA-A*02:01 restricted peptide, relevant for targeting a given cancer. Importantly, PDC*line can be also engineered with mRNA or viral vectors to endogenously express any desired antigens. PDC*vac superior potency and modularity represents a unique solution to overcome all the limitations of conventional therapeutic cancer vaccines. A strong body of preclinical data has demonstrated its unique ability to induce dramatic T cell expansions from naïve and memory CD8+ cells, against multiple antigens in the context of melanoma or lung cancer, both ex vivo from cord blood mononuclear cells, patients’ PBMC or Tumor Infiltrating lymphocytes (TILs), and in vivo in an innovative CD34+ humanized mouse model. Moreover, PDC*vac is being evaluated in a first-in-human phase I clinical trial in advanced melanoma.

Methods

Here, we aimed at exploiting PDC*vac properties for priming and expanding anti-neoantigen (neoAg) T cell responses from healthy donor naïve CD8+ T-cells.

Results

We demonstrated in this study that weekly stimulation by neoAg-pulsed PDC*line leads to sizeable expansion of antigen specific CD8+ T-cells from naïve precursors as soon as 14 days of co-culture, with a powerful expansion as day 21 (Fold increase: 50 to 150 compared to baseline). These results were obtained with cells purified form healthy donors’ mononuclear cells and 2 HLA-A*02:01 neoAg selected from the literature and identified in lung cancer or melanoma. This expansion is specific as no other antigen-specific T-cells were expanded. Moreover, these neoAg-specific T cells display functional activity as revealed by the expression of CD107 and IFNg secretion upon stimulation. Importantly, these cells are specific for the mutated form of the peptide and not the wild type form.

Conclusions

Altogether these data demonstrate that PDC*vac represents a powerful tool for assessing the immunogenicity of neo-epitopes in vitro as well as a powerful vaccine platform for personalized cancer immunotherapies.

Legal entity responsible for the study

PDC*line Pharma

Funding

PDC*line Pharma

Disclosure

J. Plumas: Co-founder, CSO and employee of PDC*line Pharma. P. Alvez, D. Hannani: Employed by PDC*line Pharma. All other authors have declared no conflicts of interest.

Background

Despite originally considered an immunologically silent malignancy, recent studies are encouraging research of breast cancer immunogenicity to evaluate the applicability of immunotherapy as a treatment strategy. The epitope landscape in breast cancer is minimally described. Consequently, this project investigates four proteins commonly upregulated in breast cancer and thus probable tumor associated antigens (TAAs). Aromatase, prolactin, never in mitosis a related kinase 3 (NEK3), and protein inhibitor of activated STAT3 (PIAS3) contribute to increased growth, survival, and motility of malignant cells.

Methods

Aspiring to uncover novel epitopes for cytotoxic T cells, a reverse immunology approach was applied. In silico screening via NetMHC was used to predict binding of peptides within the proteins to HLA-A*0201 and -B*0702. Next, an MHC ELISA was applied to experimentally confirm which of the peptides are indeed HLA-A*0201 and -B*0702 binders. Hereafter, a novel method for high throughout detection of antigen specific T cells was applied. Via DNA barcode labeled MHC multimer technology, parallel screening for T cell recognition of all predicted MHC binding peptides was performed.

Results

415 peptides were predicted in silico as HLA-A*0201 and -B*0702 binders. Subsequent in vitro binding analysis confirmed binding for 147 of the 415 predicted binders. The 147 peptides were evaluated for T cell recognition utilizing DNA barcode labeled MHC multimers to screen peripheral blood lymphocytes from breast cancer patients and healthy donor samples. Significantly more TAA specific T cell responses were detected in breast cancer patients compared to healthy donors for both HLA-A*0201 (p < 0.0039) and -B*0702 (p< .A-0ealthy donors for both HLA-for both HLA A*0201 and B*0702 ses were detected in breast cancer patients 0.001) restricted peptides. Importantly, several of the identified responses were directed towards peptides that were predicted as poor or intermediate affinity binders. This is indicative of the importance of inclusion of low-affinity binders in the search for epitopes within shared TAAs, as these might be less subject to immune tolerance mechanisms.

Conclusions

Thus, the inspected proteins; aromatase, prolactin, NEK3 and PIAS3, indeed contain targets for T cell reactivity.

Legal entity responsible for the study

Sine Reker Hadrup

Funding

Danish Cancer Society, Danish Council for Independent Research

Disclosure

All authors have declared no conflicts of interest.

Background

Lymphodepleting preconditioning is a standard method used to increase the clinical efficacy of adoptive cell therapy (ACT) of solid tumors using tumor-infiltrating lymphocytes (TILs) or gene-engineered T cells. While it boosts the antitumor efficacy of transferred T cells by, increasing the availability of supporting cytokines and reducing the immunosuppression in the tumor, systemic lymphodepleting preconditioning is highly toxic for patients. Conversely, oncolytic adenovirus therapy is well tolerated and, when designed to express tumor necrosis factor alpha (TNFa) and interleukin-2 (IL-2), can improve the potency of ACT by local immune modulation.

Methods

Here, we compare the safety and efficacy of an ACT protocol using, an adenovirus coding for TNFα and IL-2 or, a cyclophosphamide and fludarabine lymphodepleting regimen. Since hamsters allow us to study the oncolytic capability of a human adenovirus, we used a subcutaneous pancreatic tumor model (HapT1) in a syngeneic golden Syrian hamster infused with TILs. Moreover, to identify the immune cells responsive to the TNFa and IL-2, we used a non-replicating adenovirus in a subcutaneous melanoma model (B16.OVA) established in a syngeneic mouse infused with ovalbumin-specific T cells.

Results

Both models showed that adenovirus therapy or lymphodepleting preconditioning improve the efficacy and tumor control of ACT. Yet, in hamsters, lymphodepletion caused a decline in survival compared with adenovirus therapy. Flow cytometry analysis of mouse tumors from both therapies revealed, an upregulation of IL-2, TNFa and interferon gamma (IFNg) and, high infiltration of T cells (CD3+) and mature dendritic cells (CD11c+CD86+). Also, M2-like macrophages (CD11b+F4/80+CD206+) were markedly reduced in adenovirus therapy. Of note, and similar to humans, degeneration in the heart and lungs was observed in animals that received lymphodepleting chemotherapy. Toxicity was barely observed in adenovirus treated groups.

Conclusions

Overall, this data suggests that our oncolytic adenovirus can replace high dose preconditioning chemotherapy in conventional ACT protocols. This rationale will be further explored in a Phase I clinical trial.

Legal entity responsible for the study

Cancer Gene Therapy Group, TILT Biotherapeutics Ltd

Funding

TILT Biotherapeutics Ltd

Disclosure

J.M. Santos, V. Cervera-Carrascon, R. Havunen, M. Siurala, S. Sorsa: Employee in TILT Biotherapeutics Ltd. A. Hemminki: Shareholder in Targovax ASA. Employee and shareholder in TILT Biotherapeutics Ltd.

All other authors have declared no conflicts of interest.

Background

Adoptive T cells transfer is a promising cancer treatment. However, despite the impressive success of CD19-CAR T cells, its transposition to solid tumor is challenging. The selection of safe targets and the requirement of tumor infiltration are two main hurdles. In HPV related cancers, an oncoviral protein such as HPV-E7 is an attractive and safe target, and there are clinical evidences of the effectiveness of E7 targeted therapies (Kenter GG et al. 2009). TIL trials in melanoma have revealed that in vivo persistence of infused cells is required to achieve treatment efficacy. In this view, less differentiated T cells such as stem memory T cells or central memory T cells have a superior in vivo persistence. Cord blood is a source of more naive T cells compared to peripheral blood and could be used to develop novel immunotherapeutic strategies.

Methods

A E7/HLA-DR4 specific TCR has been isolated from a cancer patient and retroviral particles encoding this TCR has been produced. Cord blood and peripheral blood T cells were isolated and activated before the retroviral transduction with a vector encoding for this TCR. We then evaluated by flow cytometry the differentiation profile and functions of cord blood and peripheral blood transgenic T cells.

Results

Functional assays revealed that both transduced CD4 and CD8 T cells were reactive against HLA-DR4⁺ BLCL pulsed with the E7-derived peptide. Transduction efficiency was slightly lower for cord blood T cells. As expected, cord blood transduced T cells show a higher rate of CD4 and CD8 T memory stem cells (CD45RA⁺CD45RO^lowCD95⁺CCR7⁺) than peripheral blood T cells. This more naive phenotype is associated with lower effector functionalities (interferon-γ secretion and degranulation).

Conclusions

In conclusion, cord blood T cells could be redirected with a TCR targeting E7/HLA-DR4 complex and are less differentiated than their peripheral blood counterparts. This profile could allow them to proliferate and persist longer in vivo, while enhancing their antitumor capacities. These results point out the interest of cord blood as a source of cells for adoptive cancer immunotherapy using receptor-engineered T cells.

Legal entity responsible for the study

UMR1098

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

The immunotherapeutic approach, adoptive cell transfer (ACT) have in malignant melanoma studies showed clinical durable responses in more than 50% of patients. However, the expansion of tumor infiltrating lymphocytes (TILs) requires extensive ex vivo culturing often at the cost of T cell differentiation and functional capacity. Most current strategies involve non-specific expansion of bulk TILs, often providing growth preference to co-infiltrated virus specific T cells and driving an exhausted phenotype of the T cell product.

Methods

It is aimed to develop a new technology to expand tumor reactive T cells, through use of Major histocompatibility complex (MHC)-loaded artificial antigen-presenting scaffolds (Ag-scaffold) to provide the T cells with specific functional stimulation to obtain phenotypic and functional properties to mediate tumor regression. These scaffolds will be build using a dextran-based polysaccharide backbone associated with streptavidin molecules where biotinylated peptide-MHC class I molecules are attached to govern the specific interaction with a specific T cell, and a combination of biotinylated cytokines and co-stimulatory molecules are co-attached to provide stimulation to the T cell to achieve increased functional properties. The Ag-scaffolds interacts specifically with T cells based on recognition of the peptide-MHC molecule and effectively expand and functionally stimulate specific T cells, while leaving all other T cell specificities untouched.

Results

from in vitro experiments have showed that antigen specific CD8 T cells stimulated with these Ag-scaffolds has high CD28 expression and low PD-1 expression, associated with high proliferation potential and enhanced antitumor effect in vivo. Furthermore, this expansion strategy provides a high frequency of multifunctional antigen specific CD8 T cells expressing IFN-, TNF-α, and CD107a upon target recognition.

Conclusions

This expansion technology could with great advantage be used in ACT, to increase the anti-tumor effect of the transferred T cell product, as all of the achieved T cell characteristics are of significant importance for in vivo tumor cell recognition following ACT of expanded T cell products.

Legal entity responsible for the study

Sine Reker Hadrup

Funding

Lundbeck foundation

Disclosure

All authors have declared no conflicts of interest.

Background

The promise of cancer immunotherapy has been translated into clinical success with the recent approval of adoptive cell transfer of T cells expressing chimeric antigen receptors (CARs) as a therapy for B cell malignancies. However, the progress of using CAR T cells in the context of solid cancers has been limited so far. We propose that use of “armored” CAR NK-cells secreting an anticancer peptide lactaptin may help deliver this agent in its highly active form into the tumor stroma thereby increasing its local concentration and boosting antitumor effects of the CAR.

Methods

We designed lentiviral constructs allowing stable transduction of human cell lines with cassettes encoding two secreted forms of lactaptin that differ in their leader sequences.

Results

Lactaptin was successfully produced in HEK293T and YT cell lines. Its in vitro activity in the conditioned media was measured against a panel of sensitive cancer cells: MDA-MB-231 breast adenocarcinoma, PC3 prostate cancer and T98G glioblastoma. We evaluated that lactaptin from conditioned media showed greater than 50-fold increase in cytotoxicity compared to the recombinant lactaptin produced in E. coli.

Conclusions

This opens an opportunity to “armor” CAR YT cells with a cassette allowing secretion of lactaptin in situ, which in turn should improve the anticancer activity of adoptively transferred CAR YT cells. Experiments to test this approach in vitro and in vivo are underway.

Legal entity responsible for the study

Institute of Chemical Biology and Fundamental Medicine

Funding

Russian Ministry of Education and Science, Agreement 14.604.21.0169 (unique project identifier RFMEFI60417X0169)

Disclosure

All authors have declared no conflicts of interest.

Background

CT26 tumors are heavily infiltrated by tumor-specific CD8 T cells but nevertheless grow rapidly in Balb/c mice. These tumors are partially responsive to anti-PD-1 monoclonal antibody therapy, suggesting an active suppression of the tumor-specific cytotoxic T cells. As CCR2 is expressed by a potentially suppressive leukocyte subset within these these tumors, we aimed to test whether CCR2 blockade could enhance the anti-tumor effects of anti-PD-1.

Methods

Five days after subcutaneous CT26 implantation into the flanks of 9wk female Balb/c mice (2.5x10⁵/mouse), the recipients were randomized based on tumor size and treatment was begun. Mice received anti-PD-1 by IP injection on days 7, 10, 17 and 21 (200μg/mouse), and received CCR2 antagonist CCX598 (30 or 60mg/kg) or vehicle by oral gavage every 24 hours until day 60.

Results

We have found that the therapeutic effects of anti-PD-1 therapy are appreciably enhanced by specific blockade of chemokine receptor 2 (CCR2) via a small molecule antagonist. This combined anti-PD-1/CCR2i approach significantly decreases tumor size and increases the proportion of long-term survivors, with more than 50% of the mice (up to 73%) showing complete regression of a previously established tumor. The effects of this combined therapy are dependent on the presence of CD8⁺ T cells, as tumors do not respond to the therapy in CD8-depleted mice. The anti-CT26 tumor response is specific: long term survivors are resistant to re-inoculation with the CT26 tumor (even without further dosing of either drug) but are not resistant to the 4T1 breast tumor. CCR2 antagonism alters the tumor microenvironment by reducing the number of mMDSC per gram of tumor (a CCR2^hi population phenotypically defined as CD11b⁺/Ly6G^-/Ly6C^hi). Reduction in tumor size is inversely proportional to the ratio of CD8 T cells to mMDSC.

Conclusions

These data are consistent with a hypothesis that CCR2 antagonism enhances anti-PD-1 therapy by preventing mMDSC from accumulating within the tumor, thus reducing their suppressive effects on cytotoxic T cells.

Legal entity responsible for the study

ChemoCentryx, Inc.

Funding

ChemoCentryx, Inc.

Disclosure

J. Campbell, C. Janson, L. Ertl, C. Li, Z. Miao, V. Chhina, M. Vilalta, A. Kumamoto, T. Dang, S. Liu, S. Yao, P. Zhang, T.J. Schall, R. Singh: Full time employee of ChemoCentryx, Inc.

Background

New approach for cancer therapy using checkpoint inhibitors confirmed that reactivation of antitumor immunity can lead to strong clinical benefits including full regression. However, only a fraction of patients achieved long-lasting therapeutic effects prompting efforts to target additional effectors of antitumor immunity. Evidence from preclinical and clinical studies demonstrated that interference with multiple immune checkpoints provides a superior efficacy. Arginase promotes the immune escape of cancer cells by decreasing the level of arginine and inhibiting proliferation and activation of T cells. High arginase activity has been found in patients with a wide spectrum of cancers, both in plasma and in tumors and correlated with a poor prognosis.

Methods

IC₅₀ was determined against the recombinant arginase 1 and 2 (ARG1/2). M2-polarized, bone marrow derived murine macrophages or CHO cells transfected with human ARG1 or ARG2 were used to assess the cellular activity. The in vivo antitumor efficacy was evaluated in syngeneic mouse models. The levels of arginine and OATD-02 (OAT-1746) in plasma and tumors were determined by LC/MS analysis.

Results

We have developed OATD-02 - a potent, selective, orally active inhibitor of ARG1 and ARG2. The compound is a low nanomolar ARG1/2 inhibitor with < 50 nM cellular activity. In vivo, OATD-02 showed good pharmacological properties and significant antitumor efficacy as a monotherapy in multiple tumor models. Combining OATD-02 with PD-L1 and IDO inhibitors exhibited greatly increased antitumor efficacy. The efficacy correlated with sustained pharmacodynamic effects: 3-6 fold increase in plasma and tumor arginine levels and suppression of tumor arginase activity. The arginine plasma levels exceeded several fold concentration required for the maximal stimulation of T cell proliferation. Induction of inflammatory markers (IFN-gamma, CD94, CD3) in tumors confirmed reversal of immunosuppression.

Conclusions

The results provide a rationale for the clinical development of OATD-02 as a cancer immunotherapy.

Legal entity responsible for the study

OncoArendi Therapeutics SA

Funding

OncoArendi Therapeutics SA

Disclosure

M.M. Grzybowski, P.S. Stańczak, J. Pe¸czkowicz-Szyszka, P. Wolska, A.M. Zdziarska, M. Mazurkiewicz, J. Brzezińska, R. Blaszczyk, A. Gołębiowski, P. Dobrzański, K. Dzwonek: Employee of OncoArendi Therapeutics SA.

Background

The involvement of Nrp1 in the formation of the immunological synapse was first described by our research team, when demonstrating an increased expression of Nrp1 on the surface of activated CD8+Tcells and dendritic cells. Presently, we thus investigated the role of ldCSA in the modulation of CD8+T cells activation and its antitumor effect along to the negative regulation of Nrp1 expression while preserving the T cells activation.

Methods

In vivo, C57BL6 mice were challenged with B16F10GFP murine melanoma for tumor monitoring and phenotypic analysis. Cohort’s treatments comprised: ldCSA (CSA serum 25ng/ml), high dosage of CSA (CSA serum 100ng/ml) and control group (vehicle only). Phenotypic analysis was assessed by flow cytometry for tumor-infiltrating cells, activation markers, exhaustion marker (PD1) and Nrp1 expression on CD8+Tcells. NSG mice were challenged with B16F10GFP for tumor growth monitoring. In vitro, mice and human CD8+T cells sorted received no, low or high dose of CsA for 48h and assessed by flow cytometry towards activation markers, PD1 and Nrp1.

Results

For C57BL6 mice, the cohort treated with ldCSA had a slowed tumor growth (p = 0.05). For NSG mice devoid of T Cells, there was no effect of ldCSA suggesting that the effect observed depends on immune cells instead of a direct effect of CSA on tumor cell growth. C57BL6 experiment showed a lower infiltration of CD8+Tcells in tumor bed, while these cells were more activated when compared to the no dose or high dose treated cohorts. For the ldCSA cohort, CD8+Tcells infiltrating the tumor exhibited a reduction of PD1/Nrp1 expression. For in vitro experiments, upon treatment with ldCSA, it was observed a decreased expression of PD1 and NRP1 on sorted human and mice activated CD8+T cells (p = 0.01) and the upkeep IFNγ production.

Conclusions

Our results support an innovative effect of ldCSA, demonstrating an efficacy in amplifying the immune response of CD8+Tcells. It suggests an effect linked to negative regulation of Nrp1 and PD1. Experiments using NRP-1KO CD8+T cells are ongoing to confirm the CSA mechanism of action. The identification of ldCSA as new long-term therapeutic antitumor strategy deserves further larger studies.

Legal entity responsible for the study

Institut Imagine

Funding

INSERM U 1163/CNRS ERL 8254

Disclosure

All authors have declared no conflicts of interest.

Background

Checkpoint inhibitors including the CTLA-4 blocking antibody ipilimumab have become the new standard therapy of many metastatic cancers. Development of anti-drug antibodies (ADAs) after treatment with other biopharmaceuticals has been thoroughly investigated, but induction of ADAs after treatment with checkpoint inhibitors is poorly investigated. In this retrospective study, we measured ipilimumab serum levels and anti-ipilimumab antibody levels in patients with unresectable or metastatic melanoma (MM), and related the findings to clinical outcome.

Methods

Serum samples from 31 patients with MM were analyzed for ADAs against ipilimumab and serum levels of ipilimumab at baseline, after 1^st infusion and 3^rd infusion using an in-house developed bead-based assay. Data were correlated with progression-free survival (PFS) and overall survival (OS).

Results

Low serum levels of ipilimumab after 1^st infusion correlated significantly to a shorter OS (p = 0.024) but not PFS, and this correlation was not observed after 3^rd infusion. Seven patients (23%) were ADA-positive after 1^st infusion, three of whom were excluded before the 3^rd infusion due to disease progression. Five patients (19%) were ADA-positive after the 3^rd infusion. Patients with a positive ADA status after 1^st infusion had shorter PFS (p = 0.0186) and OS (p = 0.0143) than ADA-negative patients, while ADA-positivity after the 3^rd infusion did not correlate significantly with PFS or OS. ADA status did not correlate with serum levels of ipilimumab.

Conclusions

Low serum levels of ipilimumab after 1^st infusion is correlated with shorter OS, while ADA-positivity is associated with shorter OS as well as PFS in patients with MM.

Legal entity responsible for the study

Inge Marie Svane

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

Ciclopirox Olamine (CPX) is an antifungal agent that has displayed anti-neoplastic activity in solid tumors. Antifungal agents appear to have immunomodulatory effects. These prompted us to assess the antitumor effect of CPX alone and in combination with anti-PD-1 antibodies Pembrolizumab (P), Nivolumab (N) and/or anti-CTL-4 antibody Ipilimumab (I), in a pancreatic cancer xenograft mouse model.

Methods

We assessed the in vivo anti-tumor action of CPX alone and in combination with immunoglobulin G (IgG) or (P) or (N) and/or (I), in C57BL/6 mice (n = 60) bearing tumor mouse cell line Panc02. On day 15, following a hemisplenectomy, livers and spleens were collected for flow cytometry analysis (FACS) of infiltrating lymphocytes (TILs). We also assessed PD-L1 expression of tumor cells. Mouse IFNγ enzyme-linked immunosorbent assay was used to examine IFNγ production by CD8⁺T cells in splenocytes and TILs. A Kaplan-Meier method was conducted and a log-rank test was applied.

Results

We showed that CPX significant upregulated PD-L1 expression in tumors and microenvironment compared to anti-PD-1 or anti-CTL-4 antibodies which exhibited minor PD-L1 expression. Triple combination [(CPX with (P) or (N) plus (I))] robustly increased effector CD4⁺, CD8⁺ T cells, dramatically decreased CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) and exhibited significant IFNγ production in CD8⁺T cells compared to monotherapy [(IgG) or (P) or (N) or (I) or CPX)] and to dual combination [CPX with (IgG) or (P) or (N) or (I)] within the tumor microenvironment. A trend towards enhanced survival was observed with triple combination therapy (OS:91 days) compared to CPX (69 days, p = 0.13) or to (P) or (N) or (I) (approximately OS: 39 days, p = 0.33) or to (I) plus (P) or (N) therapy (OS:52 days, p = 0.225) vs control (IgG) (OS:21 days). The triple combination therapy cured a larger percentage of mice (75%) compared to CPX (38.5%), (P)(12,75%) or (N) (13%) or (I) (20%) single agent or to anti-PD-1 plus (I) combination (22%).

Conclusions

Our findings demonstrate that the triple combination (anti-PD-1 with anti-CTL-4 plus CPX) deplete Tregs and increase proliferation of CD8⁺ and CD4⁺. This strategy may be used to overcome immunosuppressive resistance in pancreatic cancer.

Legal entity responsible for the study

Karamouzis Michalis

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal malignancy with an extremely poor prognosis due to the lack of an efficient diagnostic tool and any radical treatments, except surgical removal. Therefore, it is extremely important to understand its pathology including the host inflammatory immune response of PDAC for development of novel diagnostic tool as well as treatment alternative. In this study, we investigated the pathological feature of PDAC in the context of CSTA, a cytein protease inhibitor, excamining the blood and PDAC tissues.

Methods

We assessed transcriptional expression of CSTA in peripheral blood cells of 41 patients with PDAC and 20 healthy volunteers by quantitative real-time PCR. Next, serum CSTA concentrations in 36 patients with PDAC and those in 37 healthy volunteers were measured, and its correlation with PDAC clinical parameters was analysed. Furthermore, we measured cytokine profiles of sera from 6 PDAC patients with elevated CSTA concentration in sera and 9 PDAC patients without elevation. We also examined the expression of CSTA, its substrate, cathepsin B, and cytokines, in tumor tissues in 20 surgically resected PDAC tissues by immunohistochemical staining.

Results

We observed increment of CSTA mRNA expression in CD4+ cells of peripheral blood of 41 patients with PDAC compared with that in 20 healthy volunteers. Correspondingly, serum CSTA concentrations in 36 patients with PDAC were higher than those in 37 healthy volunteers, and this increase was correlated with PDAC clinical stage. We found that IFN-g, TNF-a, and IL-1b concentrations in sera were significantly correlated with those of CSTA in sera of PDAC patients. As for tumor tissue analysis, CSTA expression was detected in some tumor tissues and many tumor-infiltrating immune cells, particularly neutrophils. Cathepsin B expression was observed in most tumor tissues and tumor-infiltrating immune cells, particularly macrophages.

Conclusions

CSTA expression was involved in the PDAC inflammatory condition in local tumor microenvironment. The CSTA concentration in peripheral blood of PDAC patients have a possibility of potential role as a PDAC immunopathological biomarker.

Legal entity responsible for the study

Kanazawa University Ethics Committee

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

STAT3 (Signal Transducers and Activators of Transcription- 3) transcription factor participates in inflammation, suppression of anti-tumor immune responses, and also cancer cell proliferation invasion, and even maintenance of embryonic stem cells. In the present study we sought to evaluate the inhibition of STAT3 activation on self-renewal ability and immune modulation in gastric cancer.

Methods

Human gastric cancer cell lines (MKN-45 and AGS), as well as patient samples were cultured in low attachment flasks and serum free media supplemented with bFGf, EGF and B27 until gastro-spheres appeared. Following characterization of the gastro-spheres, their effects on T cell response were evaluated by MLR assay as well as T cell culture. Finally the parental cells as well as spheres treated by Sttatic (an inhibitor of STAT3 activation) at IC50 and the self-renewal properties as well as T cell activation and differentiation was evaluated by different in vitro methods.

Results

Gastro-Spheroids had higher potential of colony and sphere formation, higher ability of resistance to drugs, increased expression of genes involved in pluripotecy and EMT. So, spheroids were identified as structures enriched for CSCs. They have higher level of activated STAT3 at mRNA and Protein.STAT3 inhibition by Stattic decreased stemness properties including spheroid formation capacity and pluripotency gene expression of gastric cancer. Moreover, gastro-spheres demonstrated potent immunosuppressive effects on T lymphocyte proliferation, VEGF and TGF-β cytokine expression and regulatory T cell differentiation. Furthermore, STAT3 inhibition in cancer cells decreased immunosupression due to cancer cells and Th17/Treg shift towards Th17.

Conclusions

The present findings could be useful in therapeutic processes directed at CSCs and give better understanding of tumor cross talk with its niche. However, in vivo investigation of anti-tumor and immunomodulatory effects of STAT3 inhibition is desirable.

Legal entity responsible for the study

Royan Institute Ethical Committee

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

Intracellular adhesion molecule-1 (ICAM-1) acts as a double-edged sword in breast cancer (BC) due to its impact on metastasis and its emerging role as a co-stimulatory ligand. The latter effect occurs by stimulating the cytotoxic players of the immune system; cytotoxic T lymphocytes and natural killer cells, to eradicate BC cells. To date, epigenetic regulation of ICAM-1 by microRNAs has never been investigated in BC. miR-486-5p expression is tumor-specific. It acts as a tumor-suppressor in liver cancer and oncomiR in colorectal cancer. However, its role in BC has been rarely investigated. Thus, this study aimed at investigating the impact of miR-486-5p on ICAM-1 in BC cells.

Methods

Tissues were collected from 17 BC patients. In-silico analysis was performed to predict a miRNA that could potentially target ICAM-1 with high scores. MDA-MB-231 and MCF-7 cells were cultured. MDA-MB-231 cells were transfected by miR-486-5p oligonucleotides using lipofection technique. Total RNA was extracted, reverse transcribed and quantified using qRT-PCR. Cellular viability and anchorage independent growth of MDA-MB-231 cells were measured using MTT and colony forming assays, respectively.

Results

miR-486-5p targets ICAM-1 mRNA at 2 binding sites with high scores. In contrast to ICAM-1, miR-486-5p was downregulated in BC tissues compared to normal tissues. ICAM-1 showed higher expression in TNBC cell lines, MDA-MB-231, compared to hormone receptor positive, MCF-7 cells. Paradoxically, miR-486-5p was found to be downregulated in MDA-MB-231 compared to MCF-7 cells. Therefore, upon forcing miR-486-5p expression in MDA-MB-231 cells, (more than 900-fold increase), ICAM-1 levels were ectopically reduced. Furthermore, miR-486-5p led to a significant inhibition of BC cell growth and colony forming ability, while anti-miR-486-5p reversed those effects.

Conclusions

miR-486-5p decreases the metastatic property of ICAM-1. In addition, miR-486-5p acts as a tumor suppressor in BC. Thus, increasing the levels of the under-expressed miR-486-5p in TNBC tissues can be a possible therapeutic approach for halting BC progression. Moreover, the potent inhibition of ICAM-1 by miR-486-5p suggests that it is a possible target for BC therapy.

Legal entity responsible for the study

Molecular Genetics Research Team

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

Some tumor molecules circulate in the blood, and in some cases, they are used as markers of the disease (CEA, PSA). Several authors have reported the efficacy of some of these molecules for immunotherapy, which is the case of the Autologous Hemoderivative Cancer Vaccine (AHCV) that uses a thermostable molecular fraction of autologous blood as intradermal immunogen, and an intradermal way for immunization. We are searching adjuvant procedures to improve such immunotherapy. In this study, we tested AHCV in human hormone-refractory prostate cancer, and as adjuvant, we used metformin that has been identified as a geroprotector with regenerative activity of immunity, depressed by the immunosenescence phenomenon prevalent in aged patients.

Methods

From the data included in published trials that tested AHCV, 45 records were reviewed for this analysis, all patients with hormone-resistant prostate cancer in PSA progression, 60-85 years old, diabetes type 2, treated with metformin. These were divided into3 groups of 15 records each, patients receiving 0, 5 and 10 months of metformin 850 to 1000 mg/day, respectively. All groups had received monthly for one year vaccination with AHCV as was reported. Follow-up of records was for 12 months. Assessments: At the beginning and at the end of the 1-yr follow-up, evaluations were performed: PSA in blood by standard immunochemical method, glycosylated hemoglobin and ferritin, two biomarkers of aging mechanisms (glycation and inflammation), and diameter of intradermal papular response to vaccination measured 48 hours after inoculation. The variations of such assessments were registered.

Results

Shown in Table.Table: 78P

Conclusions

In hormone-resistant prostate cancer, the previously reported immunogenic activity of AHCV increases with metformin pretreatment in association with geroprotection, as estimated by biomarkers of aging.

Clinical trial identification

Records review.

Legal entity responsible for the study

Cooperative Research Group Interdoctors-Telemedical Organization

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

Immunotherapy is revolutionizing the field of oncology and specifically Breast Cancer (BC). Immune exclusion phenomenon that is orchestrated by immune-suppressive cytokines acts as a formidable barrier towards efficient harnessing of tumor cells. Interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) play a pivotal role in shaping the tumor microenvironment and the magnitude of immune resistance. Finding molecular weapons to efficiently harness BC progression, enhance immune cells cytotoxicity and most importantly alleviates the tumor-induced immune suppressive microenvironment was our main goal. microRNA-4317 is a novel miRNA that was reported to have an alerted expression pattern in several malignancies. Thus our aim was to unravel the mechanistic role of miR-4317 in BC patients and to investigate its impact on the tumor microenvironment.

Methods

Breast tissues were collected from 40 BC patients. MDA-MB-231 and MCF7 cells were cultured and transfected with miR-4317 oligonucleotides. Total RNA was extracted and quantified by qRT-PCR. Cellular proliferation and anchorage independent growth were measured using BrdU and colony forming assays respectively. Cell culture supernatants were screened for IFN-γ and TNF-α using Human IFN-γ and TNF-α Elisa kits, respectively.

Results

miR-4317 was found to be specifically down-regulated in BC tissues compared to its normal counterparts. TNBC patients showed the lowest levels of miR-4317. In parallel, the aggressive TNBC cells, MDA-MB-231 showed more reduced expression levels of miR-4317 compared to hormone receptor positive BC cells, MCF7. Mechanistically, ectopic expression of miR-4317 resulted in a significant decrease in cellular proliferation rate and anchorage independent ability of BC cell lines. Additionally, a potent repression of TNF-α release in cellular supernatant was observed together with an increase in IFN-γ levels thus restoring the immune-stimulating conditions at the tumor microenvironment.

Conclusions

miR-4317 acts as a novel tumor suppressor miRNA. Moreover, it has a potent impact in alleviating the tumor induced immune-suppressive microenviroment. Thus suggesting miR-4317 as a novel therapeutic agent in BC.

Legal entity responsible for the study

German University in Cairo

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

In a single-arm, open-label phase 2 trial (NCT02155647 Part A) of avelumab 10 mg/kg q2w, pts with stage IV MCC pretreated with chemotherapy were followed for HRQoL and clinical outcomes. The primary analysis (6 months after enrolment of the last pt) showed nonprogression during avelumab treatment was associated with clinically meaningful improvements in HRQoL. Here we report interim results 12 months after enrollment of the last pt.

Methods

HRQoL was assessed using FACT-Melanoma (FACT-M) and EQ-5D questionnaires at baseline (BL), week 7, every 6 weeks thereafter until disease progression (PD), and at end of treatment (EOT). Linear mixed models (LMM) assessed change from BL until week 49 for FACT-M scales, including covariates of time and PD vs. non-PD. Minimally important differences (MIDs) derived from MCC populations were used to interpret the meaningfulness of changes. Sensitivity analyses (pattern-mixture models) provided estimates assuming missing values were nonrandom. Health utility was assessed by EQ-5D using similar methods.

Results

Overall, no meaningful changes from BL were seen for FACT-M scales during treatment visits up to week 49 in 70 analyzed pts. However, at the final HRQoL assessment before EOT and at EOT, meaningful worsening in HRQoL was observed for all scales, primarily associated with PD at EOT (63.6%). LMM showed significant differences between PD vs non-PD pts in the expected directions in the range of MIDs for (mean change) physical well-being (1.40), emotional well-being (1.52), melanoma (3.07), TOI (5.68), FACT-G total (4.37) and FACT-M total (7.03). The models showed improvement in emotional well-being (1.70) for non-PD pts and worsening in physical well-being (-1.68), TOI (-4.01), FACT-G total (-4.44) and FACT-M total (-5.64) for PD pts. Sensitivity analyses showed outcomes did not differ by timing of dropout. Mean health utility EQ-5D scores based on the US (UK) value set was 0.8024 (0.8269) for non-PD pts and 0.7352 (0.7415) for PD pts.

Conclusions

Consistent with previous findings, nonprogression during avelumab treatment contributed to statistically and clinically meaningful improvements in HRQoL in this longer-term 12-month follow-up analysis.

Clinical trial identification

NCT02155647 Part A

Legal entity responsible for the study

N/A

Funding

This study was funded by and is part of an alliance between Merck KGaA and Pfizer, Inc, NY, USA.

Disclosure

M. Bharmal, M. Schlichting: employee of Merck KGaA, Darmstadt, Germany, P. Williams, M. Hunger, A. Marrel: Mapi employees, consultant for Merck KgaA, M. Hennessy: employee of EMD Serono, H. Kaufman: received research grants from Amgen, Merck. Merck, provided consulting for Amgen, Celldex, EMD Serono, Merck, Prometheus, Sanofi, Turnstone Biologics participated in Speaker’s buereau’s for Merck. Merck, has received honoraria from Amgen, Celldex, EMD Serono, Merck, Prometheus, Sanofi, Turnstone Biologics

Background

The optimal sequence strategy of BRAF/MEK inhibitors, anti-PD-1/PDL-1 and anti-CTLA-4 in metastatic BRAF-mutated melanoma patients (pts) is unknown and no treatment guidelines exist. Therefore, we report a single-institution experience of different treatment approaches using targeted therapy (TT) and immunotherapy and its impact on outcomes.

Methods

BRAF-mutated metastatic melanoma pts treated with TT and immunotherapy from 2012 to2017 were analyzed. Six groups were identified based on treatment strategy. All time-to-event analyses were calculated using the Kaplan-Meier method and Wilcoxon test.Table: 82P Treatment characteristics by group

Results

Forty-four pts were identified. The median age at diagnosis was 49 years (range 21-73), 54% pts were females and 43% developed brain metastases during disease course. The most common approach strategy was immunotherapy followed by TT, the median duration of treatment was 11 and 19 weeks, respectively. Time-to-next therapy (TTNT) following 1^st line treatment was similar in pts treated with TT (median 23 weeks [95% CI: 15-31]) or immunotherapy (median 26 weeks [95% CI: 10-33], p = 0.94). A trend towards better overall survival (OS) was seen in pts who received immunotherapy followed by TT (p = 0.09); patients who received salvage chemotherapy (carboplatin/paclitaxel) had significantly longer OS (median 7 years [95% CI: 3.2-7.08]) (p = 0.03).

Conclusions

No differences in TTNT were seen with immunotherapy, TT or combined (triple therapy) when used as 1^st or 2^nd line. The significant longer OS benefit with 1^st line immunotherapy was only seen in patients who received chemotherapy later in their treatment course.

Legal entity responsible for the study

Mayo Clinic

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

Release of biologically active soluble PD-L1 (sPD-L1) from tumor cells has been causally linked to aggressive pathologic features. Current data support that sPD-L1 is released through proteolytic cleavage of the membrane-bound protein.

Methods

CD274 genomic sequence and corresponding expressed sequence tag (EST) data were downloaded from the National Center for Biotechnology Information (NCBI) portal. CD274 sequence was scanned for the presence of integrated transposable elements by RepeatMasker software. CD274 high-throughput sequencing of polyadenylated RNA (RNA-Seq) data in several normal somatic tissues were analyzed via the NCBI and GTEx Portals. CD274 ribosome profiling data across various cancer tissues were analyzed via the GWIPS‐viz Portal.

Results

CD274 alternatively spliced variant 4 (v4), only recently annotated from NCBI (NM_001314029), represents an aberrant mRNA which was evolutionarily derived from the “exaptation” of an L2a retrotransposon. The truncated peptide encoded by CD274 v4 comprises exclusively PD-L1 extracellular domains and is likely to subsist in soluble form. Expression data of CD274 v4 are scarce in normal somatic tissues, indicating the rarity of its expression under normal conditions. Vice versa, various ESTs support the expression of CD274 v4 in ovarian cancer. Furthermore, ribosome-protected fragments of CD274 v4 are present in breast cancer, cervical cancer, and osteosarcoma cells as well as in TLR2-stimulated macrophages. Recently, Kataoka et al. (Nature, 2016) interrogated 10,210 cancer samples for structural variations (SVs) converging on CD274 3'-untranslated region (3'-UTR). Whilst not reported, in 11 of the 31 SV(+) samples identified, the aberrant transcripts detected are likely to encode for soluble PD-L1 isoforms.

Conclusions

Either generated via retrotransposon “exaptation” or produced via 3'-UTR SVs, aberrant CD274 transcripts are likely to directly encode for sPD-L1 in various cancer cell types and activated immune cells. Whether quantification of these transcripts via PCR-based assays could be efficiently used in predicting sensitivity to treatment with immune checkpoint inhibitors represents an intriguing question.

Legal entity responsible for the study

Spyros I. Papamichos

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

Telomerase (TERT) emerges as an attractive target for immunotherapy in glioblastoma (GBMs), the most common primary brain tumor in adults. High prevalence (∼80%) of activating mutations in TERT promoter was found in GBMs. Our aim was to analyze the spontaneous CD4 Th1 response against TERT in patients with GBMs.

Methods

Thirty-seven GBM patients were analyzed in this study. The anti-TERT Th1 response was measured by IFN-ɣ ELISPOT assay after in vitro stimulation of blood lymphocytes with Th1 epitopes derived from TERT. Peptides mixture derived from Wilms tumor 1 (WT1) was used as second model of GBM-associated tumor antigen. The recall of the antiviral response was also evaluated for each patients by IFN-ɣ ELISPOT assay. Regulatory T cell (Treg) [CD4⁺CD25⁺ CD127^lowFoxP3⁺] and monocytic myeloid-derived suppressor cells (M-MDSC) [Lin^- HLA-DR^-/low CD11b⁺CD14⁺CD33⁺] were measured in blood by flow cytometry. TERT promoter mutations (C228T and C250T) were analyzed on brain tumor using DNA sequencing.

Results

The spontaneous anti-TERT Th1 response was detected in 43.2% (16/37) of GBM patients, and 58.1% patients had anti-WT1 Th1 response. A strong correlation was observed between these two antitumor immune responses. However, there was no correlation between the antitumor immunity and the anti-viral response detected in most patients (91%). We found that the frequency of anti-TERT Th1 response decreased in patients exhibiting high circulating level of Treg and M-MDSC. In contrast to anti-WT1 Th1 response, the anti-TERT Th1 response appeared to be positively associated with patients’ overall survival (OS). Interestingly, this association was more pronounced, but non-significant, when focusing on GBM patients exhibiting TERT promoter mutations (median OS: 33.6 vs 16.3 months, in anti-TERT Th1 responders and non-responders respectively).

Conclusions

We report the presence of spontaneous anti-TERT Th1 response in blood of patients with GBM. This response tends to increase patients’ OS suggesting its involvement in GBM immunosurveillance. These results strongly support the rational to develop immunotherapy targeting telomerase in glioblastoma.

Legal entity responsible for the study

University Hospital Jean Minjoz of Besancon

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

In the rapidly changing landscape of immuno-oncology, matching right drugs with patients remains a critical challenge for informed treatment decision. Colorectal cancer (CRC) is one of the few cancers that maintain a robust tumor immune phenotypic signature, therefore, making the targeting with checkpoint inhibitors (CPI) more tractable. Although high mutational load usually orchestrates a successful response to multiple CPIs, the immune phenotypic underpinning of response profile needs further delineation of complex and dynamic microenvironment.

Methods

We evaluated thirty-one clinically advanced CRC in CANScriptTM platform that preserves the heterogeneity of dynamic tumor-immune interface in a personalized ex vivo setting (Majumder B et al Nature Commun, 2015). Freshly collected tumors from CRC patients were cultured in this platform for 3 days in presence of autologous PBMC. Tumor slices were treated with anti CTLA4 and PD1 inhibitors either as single agents or in combination. Comparative efficacy was evacuated based on a predictive score that informs response status using a panel of kinetic (viability, glucose, LDH release) and phenotypic changes. Modulation of critical markers like CD8, CD4, CD68, ICOS, PD1, PDL1, FOXp3, Ki67, IFN-g & granzymes were measured by IHC. The 3D mechanistic profiling of the On-Tx phenotypic modulation was performed using 7 color multispectral imaging technology.

Results

The mechanistic learning of tumor response to CPI revealed a preferential modulation of multiple effector functions and linked phenotypes, of which, increase in CD8 in tumor core and augmentation of granzymes were found to be more profound. M1 (CD8, Granzyme, IFN-g) and M2 paradigms (FOXP2, CD68) at baseline and post treatment also showed interesting shift, explaining the underlying response to specific inhibitors at individual levels.

Conclusions

Development of immune driven ex vivo functional phenotypic platforms that preserve clinical landscape of drug efficacy in multiple mechanistic fronts may redefine the selection of rational combinations based on individual profile of a complex, dynamic immune milieu in clinically challenging CRC.

Clinical trial identification

Not applicable

Legal entity responsible for the study

Mitra RxDx India Pvt Ltd

Funding

Mitra RxDx India Pvt Ltd

Disclosure

All authors have declared no conflicts of interest.

Background

Besides initiation of tumor-specific T cell immunity, dendritic cells (DCs) are endowed with direct tumoricidal activity. DC cytotoxicity can facilitate tumor antigen uptake by DCs and results in earlier induction of anti-tumor immune response. Previously, we showed monocyte-derived DCs of high-grade glioma patients generated in the presence of IFNα (IFN-DCs) have impaired cytotoxic activity against TNFα-sensitive tumor HEp-2 cells, that is associated with poor survival. The present study focuses on TNFα-dependent tumoricidal activity of glioma patient DCs.

Methods

The study was conducted in 28 donors and 45 high-grade glioma patients (Grade III-IV). 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 11 patients with Grade IV. DC cytotoxicity against tumor cells was studied using MTT-assay. mTNFα expression was determined by flow cytometry, TNFα gene expression – by RT-PCR, TNFα-converting enzyme (TACE) activity – by spectrofluorimetric analysis of DC lysates.

Results

The impairment of cytotoxic activity of patient IFN-DCs against TNFα-sensitive HEp-2 cells was associated with low level of membrane TNFα (mTNFα) and mRNA TNFα expression and tendency to high activity of TACE. Blocking TACE with TAPI-0 enhances mTNFα expression on patient IFN-DCs and significantly increases their cytotoxicity against HEp-2 cells. As for tumor cell lines obtained from glioma patient tissues, donor IFN-DC lysis of glioma cells had high values (≥ 40%). Blocking of TNFα/TNF-R1-signaling pathway by treating of donor IFN-DCs with soluble rhTNFR1 receptor led to partial decrease of DC cytotoxicity against most glioma cell lines (Δ up to 24-40%). Cytotoxic activity of patient IFN-DCs with decreased mTNFα expression against autologous tumor cells sensitive to TNFα/TNF-R1-signaling pathway was lower on average by 30% compared with control (donor) values.

Conclusions

The current study shows an important role of mTNFα as mediator of DC tumoricidal activity and as molecular targets for the regulation of DC cytotoxicity.

Legal entity responsible for the study

Ethics Committees of Institute of Fundamental and Clinical Immunology, Institute of Traumatology and Orthopedics, Federal Neurosurgical Center and Institute of Cytology and Genetics

Funding

Russian Foundation for basic research

Disclosure

All authors have declared no conflicts of interest.

Background

Prognosis of primary brain tumors is poor with a 5-year overall survival (OS) raging between 10 to 27%. The prognostic significance of T-cells in these tumors has been a matter of debate with conflicting evidence. Data on other immune cell populations is scarce and at times methodologically questionable. The goal of this work was to characterize the different immune cell populations in primary brain tumors and in patients’ peripheral blood and to investigate its prognostic value.

Methods

Brain tumors and blood samples were collected from 35 patients with primary brain tumors (23 glioblastoma multiforme (GBM), 5 grade 3 (G3) gliomas and 7 grade 2 (G2) gliomas). PBMC's and dissociated tumor samples were stained with an 8 color panel. Samples were assayed by flow cytometry and analyzed using the FlowJo Tristar software. OS and progression free survival (PFS) data were collected from patients’ files. ripheral blood and to investigate its prognostic value.

Results

Statistically significant differences between tumor infiltrating lymphocytes (TILs) were demonstrated with GBM tumors having a 15-fold higher level of T-helper (Th) cells than G2 and G3 gliomas. Cytotoxic T-cells (CTL) levels’ in GBM were 8-fold higher than in G2 gliomas. ɣα T-cells occurred in all gliomas in similar frequencies. Natural killer (NK) cells were rare, representing approximately one cell per million in gliomas. There was a positive correlation between Th levels and CTL levels in G2 gliomas (R = 0.8), G3 gliomas (R = 0.8) and in GBM (R = 0.89). NK cells did not correlate with any other lymphocytic subset. Positive correlations were also identified between Th levels in the tumor and the peripheral blood in GBM tumors (R = 0.67). A weaker correlation (R = 0.84) between NK cells in G3 gliomas and the peripheral blood was also identified. No correlation between OS or PFS and TILs levels was demonstrated. Similarly, no correlation between age and TIL levels was found, though age was negatively correlated with OS and PFS.

Conclusions

The data suggests to a correlation between tumor grade and T-cells infiltration in gliomas with only a partial correlation between infiltration in the primary tumor and the peripheral blood. TILs were not found to have a prognostic value.

Legal entity responsible for the study

Cancer immunotherapy lab, Tel-Aviv Sourasky Medical Center

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

Plasmacytoid dendritic cells (pDC) are the most important IFN type I producing cells. They are studied in context of many malignancies and data show their negative effect on patient prognosis in breast, ovarian, skin and oral cancer. It is assumed, that tumor infiltrating pDC are dysfunctional and support immunosuppressive environment in comparison to blood derived cells. Hence, there is only a few data on functional state of pDC in head and neck cancer (HNSCC).

Methods

Single cell suspensions derived from tumor tissue and PBMC were analysed by flow cytometry. Cytokine levels in cell culture supernatants were detected using Luminex (IFNa, IFNg, IL-10, IL-17a, IL-3, IL-4, IL-6, TNFa). Plasmacytoid DCs were identified as CD45+, Lineage -, CD4+, BDCA2+, CD123+ cells.

Results

We detected higher number of IFNa+ pDC using intracellular staining after Imiquimod stimulation in comparison to CpG (tumor: 18.6% vs. 1.9%, PBMC: 6.2% vs. 2.6%). However, there were higher levels of IFNa in cell culture supernatants after CpG stimulation (tumor: 722.1 vs 208.3 pg/ml, PBMC: 590.8 vs. 23.3 pg/ml). Moreover we found, that IFNa production is highly dependent on a delay between collecting and processing of samples. There was decrease in number of IFNa+ pDC when measured 5h (IMQ: 72.2%, CpG: 53%) and 10h after collection (IMQ: 97.9%, CpG: 84.5%) in healthy controls. The decrease was bigger in samples stored at room temperature. Also, we observed difference when comparing blood samples collected before and after surgery. Non-oncology patients showed stronger reaction to stimulation in samples collected after surgery (IMQ: 13.6% vs 10% IFNa+ pDC, CpG: 6.5% vs 3.2% IFNa+ pDC). On the contrary, preoperative samples of oncology patients showed high number of IFNa+ pDC even in unstimulated cultures. After surgery we observed decrease especially in unstimulated (90.6%) and IMQ stimulated cells (69.2%).

Conclusions

Plasmacytoid DCs might play an important role in regulation of tumor microenvironment in patients with HNSCC. Our data confirm that functionality of pDC is highly dependent on time spent ex vivo. Indeed, there are factors during sample processing which can significantly influence data analysis and so the precise standardization of working protocols is crucial.

Legal entity responsible for the study

Sotio

Funding

Sotio

Disclosure

All authors have declared no conflicts of interest.

Background

Oral squamous cell carcinoma (OSCC) is one of the major cancers affecting Asian countries. The main causative factor has been the tobacco habit. It has been reported that immune dysfunction in these patients is one of the major factors for tumor growth and dissemination that affects disease-free survival of the patients.

Methods

We assessed the phenotypic and functional characteristics of Regulatory T (Treg) CD4+CD25+FoxP3+subsets in patients (n = 53) with OSCC and healthy individuals (n = 30) by multicoloured flow cytometry. Subsequently we investigated the effects their inhibition via TDG on growth of OSCC cell lines in vitro.

Results

An increased (p < 0.05) prevalence of Treg phenotypes (CD4+CD25+, CD4+FoxP3+, CD8+FoxP3+, CD4+CD25+FoxP3+) was observed in the peripheral circulation of OSCC patients that positively correlated with clinicopathological features. The increased frequency of CD4+CD8+CD25+FoxP3+, a unique T cell subset, CTLA4+, GITR+, NrP1+ and granzyme B + (GzmB) Tregs also showed a significantly higher prevalence in OSCC patients. Functionally CD4+FoxP3+ Tregs showed skewed expression of IL2, IL10 and IL35 in patients as compared with the normal controls. Higher expression of TGFβ in tumor tissues suggests their dominant role in the up regulation of differentiation of Tregs from naive T cells in the tumor bearing host. Further, enhanced expression of CCR5 and CCR7 on Tregs with up regulation of their ligands (CCL5, CCL19 and CCL21) in tumor cells indicates efficient recruitment and trafficking of Tregs to the tumor site. Treatment with βGBP showed growth promoting effects on Tregs and oral cancer cells. However, the treatment with its inhibitor TDG resulted in inhibition of Treg subsets and also decreased the frequency of IL10+ and IL35+ Tregs indicating its immunomodulatory effects.

Conclusions

Hence, it seems reasonable to assume that modulation of functional dynamics of selective Treg subsets may be useful in enhancing anti-tumor immunity and developing immunotherapeutic strategies for patients with oral squamous cell carcinoma.

Legal entity responsible for the study

Sadhna Aggarwal

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

Immunophenotypic markers play significant role in prognostication in different cancers. Recently identified one such marker is Leukocyte-associated Ig-like receptor (LAIR-1) which is an inhibitory immuno-receptor. Few studies done worldwide have shown controversial expression of this marker in different leukemias.

Methods

We measured the LAIR-1 expression in paediatric ALL patients (n-42) by flow cytometry gating of CD45 and CD3/19 positive lymphoblasts. Median Fluorescence intensities were calculated after appropriate compensation using controls and isotype samples. Demographic, clinical variables and early treatment outcome parameters of patients were noted and correlated with expression level of LAIR-1.

Results

The age of ALL cohort 1 - 11 y & M:F ratio 2.5:1. 64% had WBC count <50x109/L and 15 (36%) >50x109/L. 52% were standard risk and 48% high risk. 6 were T-ALL and 36 B-ALL. AML1-TEL, E2A-PBX, BCR-ABL & MLL-AF4 transcripts were noted in 3, 6, 2 and 1 patient respectively. Day 8 ABC was <1000 in 31 & >1000 in 8 cases. 30 had low MRD (<0.01) and 7 high (>0.01) at day 35 of treatment. The median of MFIs of LAIR-1 expression in control cases was 8.2 (range of 7.76-11.69) & in ALL cases 4.02 (range of 0.56 to 11.87). 74% (n-31) of ALL cases showed lower expression while 26% (n-11) had normal expression. However, on correlating the standard ALL risk factors and MRD, no significant correlation was found. Out of 42 patients, 4 patients died during induction treatment and one left therapy. 60% (n-3/5) with above event had low expression of LAIR-1. Also ALL patients with low LAIR-1 expression had t(12;21), t(1;19) and t(4;11) translocations in 2, 4 and 1 samples respectively but none had t(9;22). While patients with high LAIR-1 expression, 2 had t(9;22) (MFIs-14.43 & 11.87).

Conclusions

This is one of the first studies on LAIR-1expression in ALL and suggests low expression of the inhibitory molecule on leukemic cells. However, the findings need to be confirmed in larger cohort along with study of pathophysiological role in leukemic clone survival and immune system escape.

Legal entity responsible for the study

Dr. Prateek Bhatia

Funding

PGIMER Chandigarh

Disclosure

All authors have declared no conflicts of interest.

Background

PRAME was discovered as a tumor-associated antigen in melanoma, capable of eliciting a cellular immune response. Re-expression of PRAME has been found in various cancers, including breast cancer where it is associated with poor prognosis. Given its restricted expression pattern in tumors and its immunogenic nature, PRAME is considered a potential target for immune-based interventions. Very little is known about its function in cancer. Hence, this study aims to investigate its role and potential as a target in triple negative breast cancer, an aggressive subtype of breast cancer.

Methods

We manipulated the PRAME expression in the triple negative breast cancer cell line BT549 using siRNA to evaluate the effect of ablation on epithelial-to-mesenchymal transition, cell adhesion, cancer cell migration and invasion.

Results

Silencing of PRAME significantly reduced cell migration as observed in a 40% reduction in wound closure compared to control treated cells. We did not find any significant effect on epithelial-to-mesenchymal transition. PRAME silencing did not change the protein expression and/or localization of the epithelial and mesenchymal markers E-cadherin or vimentin respectively. Nor did we observe collective up- or downregulation of the epithelial-to-mesenchymal transcription factors Snail, Twist and Zeb1. In the absence of PRAME, BT549 cancer cells exhibited a more invasive behavior though Matrigel but not collagen I, accompanied by an increase in MMP-2 and MMP-9. We are currently investigating the effect of PRAME on cell matrix adhesion in order to gain more insight into its role in cell migration and invasion. In addition, we observed enlarged nuclei after PRAME silencing, a well-known characteristic of advanced/metastatic malignancy. Since the mechanisms controlling nuclear size remain unclear, it is of great interest to explore how PRAME is involved in this process and how this affects cellular function.

Conclusions

To conclude, our findings collectively suggest that PRAME may exert different, opposing effects on cell migration and invasion in triple negative breast cancer. This might indicate that PRAME plays different roles in localized versus metastatic disease, requiring a different treatment approach for each disease setting.

Legal entity responsible for the study

Dr Julie Decock

Funding

QBRI-HBKU

Disclosure

All authors have declared no conflicts of interest.

Background

CD4⁺CD25⁺FOXP3⁺ regulatory T (Treg) cells are immune suppressive cells and are prominent with tumor progression. In opposite, CD4⁺IL-17⁺ inflammatory T helper 17 (Th17) cells play a critical role in autoimmune disease along with Th1. It is demonstrated that cancer stem-like cells, a rare pluripotent population among tumor cells mediate immune suppressive effects and are responsible for the most tumor progression. Although Treg and Th17 cells have been reported in the most advanced solid tumors, their interaction with gastro-spheres have been elusive. The object of this study was to find the effect of gastro-spheres on differentiation of Treg and Th17 cells.

Methods

Gastro-spheres were used as the model for enriched cancer stem like cells. The mixed leukocyte reaction was performed in the presence of MKN-45 cells and its derived gastro-spheres conditioned medium. The cultures were continued for 5 days at 37˚C with stimulation three times. Expansion rate of responder cells was evaluated by CFSE and the percentage of CD4⁺CD25⁺FOXP3⁺ cells and CD4⁺IL-17⁺ cells were analyzed by flow cytometry.

Results

Flow cytometry phenotypic analysis revealed that there were increases in percentages of CD4⁺CD25⁺FOXP3⁺ Tregs (4.67 vs 1.53) and CD4⁺IL-17⁺ Th17 (3.11 vs 0.34) cells in peripheral blood mononuclear cells (PBMCs) treated with gastro-sphere conditioned medium in comparison with non-gastro-sphere conditioned medium. It was also demonstrated that T cell expansion was not significantly decreased in gastro-sphere conditioned medium treatment compared with the parental (non-gastrospheres) and control group.

Conclusions

We concluded that gastro-sphere-soluble factors increase the percentage of Treg and Th17 cells more than its parental soluble factors. This suggested the possible existence of differentiation factors such as key cytokines among tumor cell secretions, which contribute to T cell differentiation. While it has been shown that gastro-sphere secretions can be possibly more immunomodulator than its parental cell secretions and lead to limited T cell expansion.

Legal entity responsible for the study

Royan Institute for Stem Cell Biology and Technology and Hamadan University of Medical Sciences

Funding

Royan Institute for Stem Cell Biology and Technology and Hamadan University of Medical Sciences

Disclosure

All authors have declared no conflicts of interest.

Background

The TLR9 agonist lefitolimod is a covalently-closed dumbbell-like immune surveillance reactivator with a broad immunomodulatory potential. After promising data from the phase 2 IMPACT trial as maintenance therapy after 1st line induction chemotherapy in patients with metastatic colorectal cancer (mCRC) lefitolimod is now evaluated in the phase 3 IMPALA trial in mCRC patients.

Trial design

The international, multicenter, randomized, open-label phase 3 IMPALA trial is conducted in collaboration with the AIO, TTD and GERCOR cooperative groups (NCT02077868). IMPALA’s primary endpoint is OS; secondary endpoints include PFS, response rates, and safety. Induction treatment, CEA and activated NKT are stratification factors for prospective assessment. Patients having achieved an objective tumor response following 1st line induction therapy receive lefitolimod switch maintenance monotherapy or local standard of care (control). In case of relapse, patients will reintroduce induction treatment, whenever feasible, with those in the experimental arm continuing to receive lefitolimod therapy in the weeks without infusion of chemotherapy. Recruitment took 32 months from Sep 2014 until May 2017 to include 549 of 630 patients screened; with unresectable mCRC and ‘fit for chemotherapy’. Sites from 8 European countries participated and accrued 274 patients in the lefitolimod and 275 in the control arm (Austria, 6 patients; Belgium, 8; Estonia, 34; France, 87; Germany, 191; Italy, 54; Spain, 132; UK, 37). 116 female (42.3%) and 158 male (57.7%) were included into the lefitolimod and 92 female (33.5%) and 183 male (66.5%) into the control arm. The median age is 65 years both in the lefitolimod and the control arm. Age group distribution showed also a similar pattern: 12 vs 7 patients from 24-43 yo, 112 vs 114 from 44-63 yo, 56 vs 55 from 64-68 yo, 37 vs 46 from 69-73 yo, 35 vs 37 from 74-78 yo, 19 vs 15 from 79-83 yo and 3 vs 1 from 84-88 yo in the lefitolimod vs the control arm as pre-requisite to guide maintenance therapy in the future.

Clinical trial identification

NCT02077868

Legal entity responsible for the study

Mologen AG

Funding

Mologen AG

Disclosure

D. Cunningham, E. Van Cutsem, R. Salazar, M. Ducreux, W. Scheithauer, C. Tournigand, A. Sobrero, D. Arnold: Member of the IMPALA Steering Committee. F. Sclafani: Investigator of the IMPALA trial. E. Wiegert, M. Schmidt: Employee of MOLOGEN AG.

Background

Huge advances have been made in the treatment of metastatic melanoma (MM); especially immunotherapy is showing impressive results. Combination of Ipilimumab and Nivolumab is to date the most effective treatment strategy, but over 50% of the patients experience grade 3/4 adverse events. New effective combinations with less toxicity are strongly needed. It is known that cancer cells induce a state of tolerance against the immune system. Two well-known mechanisms are brought through overexpression of PD-L1 and IDO on the tumor cells, which inhibits activation of cytotoxic T-cells. We have identified spontaneous T-cell reactivity against PD-L1 and IDO in the tumor microenvironment and in the peripheral blood of patients with MM and healthy donors. Both IDO and PD-L1 reactive CD8 T-cells are cytotoxic and can kill cancer cells and immune regulatory cells in vitro. Thus boosting specific T-cells that recognize immune regulatory proteins such as IDO and PD-L1 may directly modulate immune regulation. Due to distinct mechanisms of action, the combination of treatment with a monoclonal antibody targeting PD-1 and a vaccine with peptides against PD-L1 and IDO may have a synergistic effect. We recently reported a phase I trial where the IDO peptide was tested in 15 patients with MM in combination with Ipilimumab leading to induction of immunity without increased toxicity. The PD-L1 peptide is currently being tested in patients with multiple myeloma, so far without severe toxicity.

Trial design

A two-step clinical phase I/II trial design is used. 6 patients with MM will be included in a pilot study to test feasibility and tolerability. If the treatment is feasible 24 patients will further be included. The objectives are to describe anti-tumor immune responses in both blood as well as tumor tissue samples and objective responses using RECIST 1.1. Patients are treated with Nivolumab, involving outpatient IV infusions biweekly as long as there is clinical benefit. The PD-L1/IDO (IO102/IO103) peptide vaccine is given from start of Nivolumab and biweekly for the first 6 vaccines and thereafter every fourth week up to 1 year. Patients will be followed with clinical controls and diagnostic imaging every 12 weeks.

Clinical trial identification

NCT03047928

Legal entity responsible for the study

Inge Marie Svane

Funding

IO Biotech

Disclosure

All authors have declared no conflicts of interest.

Background

The role of immunotherapy in multiple myeloma is well established – from the use of thalidomide and its analogs over the recent approval of anti-CD38- and anti-SLAMF7-antibodies to ongoing trials involving checkpoint blocking antibodies. The checkpoint molecule programmed death-1 (PD-1) ligand (PD-L1) is upregulated on myeloma cells and the surrounding microenvironment. The PD-1/PD-L1 interaction renders T cells exhausted, and blockade of the PD-1/PD-L1 antagonizes this exhaustion. Recent trials indicate that antibodies against PD-1 have activity against multiple myeloma, but trials combining anti-PD-1 antibodies and standard therapies have recently come to a halt due to adverse events. We have recently described the presence of spontaneously occurring PD-L1 specific cytotoxic T-cells, which recognize both PD-L1 expressing immune cells and malignant cells. We have further shown that these “anti-regulatory” T cells improve the function of other T cells, in addition to their ability to kill PD-L1 positive cancer cells directly. Boosting PD-L1 specific T cells thus leads to cytotoxicity against both immune-regulatory cells and cancer cells. The ability of the induced PD-L1 specific T cells to release pro-inflammatory cytokines in the tumor microenvironment supports additional antineoplastic immunity.

Trial design

As such PD-L1 may be targeted by a vaccine comprised of PD-L1 peptide and we have initiated a first-in-human phase I study with peptide vaccination against PD-L1 in patients with multiple myeloma following high dose melphalan with autologous stem cell transplantation. We have included 8 patients and plan to include a total of 10 patients. Primary end points will be safety and toxicity and we will concurrently conduct immune monitoring of the patients to investigate the possible phenotypic and functional changes inferred by the vaccine. So far no adverse events above grade 2 have been found.

Clinical trial identification

EudraCT number: 2016-000990-19; ClinicalTrials.gov number: NCT03042793

Legal entity responsible for the study

Dr.Med. Lene Meldgaard Knudsen

Funding

The Danish Cancer Society

Disclosure

N.G. Jørgensen: The peptide vaccine has been patented by the region of Copenhagen and the commercial rights have been transferred to the company spun-out of the region, IO Biotech. The presenting author is not employed by this company, but funded via the Danish Cancer Society. M.T. Lundsager: Industrial PhD-student at IO Biotech. I.M. Svane: One of the founders of IO Biotech. M.H. Andersen: A founder and a member of the board of directors of IO Biotech.

All other authors have declared no conflicts of interest.

Background

CRT with cisplatin is the standard of care for patients with LA-HNSCC not treated by surgery. Preclinical data in murine cancer models show improved tumor growth control and survival when RT is combined with a PD-1 inhibitor. Pembrolizumab has proven effective for the treatment of recurrent/metastatic HNSCC, and initial results from a phase 1b study suggest that pembrolizumab plus CRT is tolerable in patients with LA-HNSCC. KEYNOTE-412 (NCT03040999) is a phase 3, randomized, placebo-controlled, double-blind trial to determine the efficacy and safety of pembrolizumab given concomitantly with CRT and as maintenance therapy versus placebo plus CRT in LA-HNSCC.

Trial design

Eligibility includes age ≥18 years; newly diagnosed, treatment-naive, oropharyngeal p16 positive (any T4 or N3), oropharyngeal p16 negative (any T3-T4 or N2a-N3), or larynx/hypopharynx/oral cavity (any T3-T4 or N2a-N3) SCC; evaluable tumor burden (RECIST v1.1); and ECOG PS 0-1. Patients will be randomly assigned (1:1) to receive pembrolizumab 200 mg every 3 weeks plus cisplatin-based CRT or placebo plus cisplatin-based CRT. Treatment will be stratified by RT regimen (accelerated RT [56-70 Gy, 6 fractions/week for 6 weeks] or standard RT [56-70 Gy, 5 fractions/week for 7 weeks]), tumor site/p16 status (oropharynx p16 positive vs p16 negative or larynx/hypopharynx/oral cavity), and disease stage (III vs IV). A priming dose of pembrolizumab or placebo will be given 1 week before CRT, followed by 2 doses during CRT and an additional 14 doses after CRT, for a total of 17 pembrolizumab or placebo infusions. Treatment will be discontinued at centrally confirmed disease progression, unacceptable toxicity, or patient/physician decision to withdrawal. Response will be assessed by computed tomography or magnetic resonance imaging 12 weeks after CRT, every 3 months for 3 years, then every 6 months for years 4 and 5. Safety will be monitored throughout the study and for 30 days after treatment end. The primary end point is event-free survival. Secondary end points include overall survival, safety, and patient-reported outcomes. Recruitment will continue until ∼780 patients are enrolled.

Clinical trial identification

NCT03040999

Legal entity responsible for the study

Merck & Co., Inc.

Funding

Merck & Co., Inc.

Disclosure

J-P. Machiels: Advisory board: MSD (uncompensated), INNATE, AstraZeneca, Nanobiotix, DEBIO Research funding: Bayer, Janssen, Novartis. L. Licitra: Travel expenses, including accommodations: Merck-Serono, Debiopharm, Jobi, Bayer, Amgen Consulting or Advisory Role: Eisai, BMS, MSA, Merck-Serono, BMS, Debiopharm, Jobi, Novartis, AstraZeneca, Bayer, Roche, Amgen. D. Rischin: Research funding: Genentech/Roche, Merck, Threshold Pharmaceuticals. B. Burtness: Advisory board: Merck, Boehringer Ingelheim, Celgene, Astra Zeneca, BMS, Amgen Research funding: Merck, Advaxis, Innate. S. Aksoy: Advisory board: Pfizer, Abdi İbrahim AŞ, MSD, Honoraria: Novartis, Roche, MSD, Abdi İbrahim AŞ, Astellas. J.Y. Ge: Employment and stock ownership: Merck Sharp & Dohme. H. Brown, B. Bidadi: Employment and Stock Ownership: Merck. L. Siu: Advisory board: Merck, AstraZeneca/MedImmune, Boehringer Ingelheim, Celgene, and Pfizer Research funding: AstraZeneca/MedImmune, Bayer, Boehringer Ingelheim, Bristol-Myers Squibb, Celgene, Genentech/Roche, GlaxoSmithKline, Merck, Novartis, and Pfizer. O. Peeters: MSD Employee.

All other authors have declared no conflicts of interest.

Background

There are no approved treatments and no current standard of care for recurrent or metastatic cSCC. Regimens that are effective for squamous cell carcinoma of the head and neck (HNSCC) may also be effective for cSCC. Cisplatin- and cetuximab-based regimens are commonly used in recurrent or metastatic cSCC; however, supporting evidence for their efficacy is limited. Pembrolizumab is a PD-1 inhibitor that directly blocks the interaction between PD-1 and its ligands, PD-L1 and PD-L2. Pembrolizumab has demonstrated evidence of efficacy and safety in patients with recurrent or metastatic HNSCC in the phase 1b KEYNOTE-012 study. This single-arm, open-label phase 2 trial will evaluate the efficacy and tolerability of pembrolizumab in pts with previously treated recurrent or metastatic cSCC (NCT03284424).

Trial design

120 pts will be enrolled. Key inclusion criteria are: age ≥18 years; histologically-confirmed cSCC as the primary site of malignancy; metastatic disease and/or locally recurrent disease not curable by surgery, radiation, or systemic therapy; previously treated with a platinum- or cetuximab-based regimen; measurable disease per Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1); and Eastern Cooperative Oncology Group performance status 0-1. Pts will be treated with pembrolizumab 200 mg every 3 weeks by intravenous infusion, continued for 35 doses (∼2 years) or until disease progression, unacceptable toxicity, intercurrent illness, noncompliance, or investigator or pt decision to withdraw. The primary endpoint is the objective response rate per RECIST 1.1 assessed by blinded independent central review. Secondary endpoints include duration of response, disease control rate (complete or partial response or stable disease for ≥12 weeks), and progression-free survival per RECIST 1.1, and overall survival, safety and tolerability. Pharmacokinetics, biomarkers, and health-related quality of life will be evaluated as exploratory endpoints.

Clinical trial identification

NCT03284424

Legal entity responsible for the study

Merck & Co., Inc.

Funding

Merck & Co., Inc.

Disclosure

L. Licitra: Travel expenses, including accommodations: Merck-Serono, Debiopharm, Jobi, Bayer, Amgen Consulting or Advisory Role: Eisai, BMS, MSA, Merck-Serono, BMS, Debiopharm, Jobi, Novartis, AstraZeneca, Bayer, Roche, Amgen. L. Siu: Advisory board member: Merck Research funding: Merck (clinical trial). E. Cohen: Advisory board member/Consulting: Eisai; Pfizer; AstraZeneca; Bristol-Myers Squibb; Human Longevity, Inc. P. Zhang, R. Swaby: Employment and stock ownership: Merck. B. Gumuscu: Employment and stock ownership: Merck Sharp & Dohme Travel expenses, including accommodations: Merck Sharp & Dohme. K. Harrington: Advisory board member: Amgen, AZ, BMS, Merck-Serono, MSD, Pfizer Speakers’ bureau: Amgen, AZ, BMS, Merck-Serono, MSD Research funding: AZ, MSD Honoraria: Amgen, AZ, BMS, Merck-Serono, MSD, Pfizer.

Background

Adoptive T-cell therapy (ACT) with tumor infiltrating lymphocytes (TIL) has proven to be a powerful treatment option for patients with metastatic melanoma with response rates of approximately 50% and durable complete responses in about 15%. However, there is still a need for improving TIL efficacy and a promising strategy is combination with immunomodulating agents. One such is vemurafenib (vem), a selective BRAF inhibitor, which induces objective responses in about 50% of melanoma patients with tumors expressing BRAF^V600E/K. In addition to the direct anti-cancer effect, vem has been shown to increase T-cell infiltration into tumors, upregulate melanoma antigen expression and increase the frequency of TIL recognizing autologous melanoma cells. This trial was previously presented at the Society for Immunotherapy of Cancer annual meeting (1,2). Updated data on clinical responses and preliminary immunological analyses will be presented.

Trial design

A total of 12 patients will be included in this open phase II non-randomized trial primarily to investigate safety when combining ACT and vem (ClinicalTrials.gov ID NCT02354690). Secondarily, clinical responses will be evaluated according to RECIST and extensive immune monitoring will be performed. Patients are treated with vem orally 960 mg BID one week prior to excision of tumor material for T-cell generation and continue vem until hospital admission (4-7 weeks). During hospitalization patients will receive a preparative lymphodepleting regimen consisting of cyclophosphamide 60 mg/kg for 2 days and fludarabine 25 mg/m² for 5 days. TIL infusion consists of 5-10 x 10¹⁰ T-cells and patients are subsequently treated with continuous interleukin-2 infusion following the decrescendo-regimen for 5 days. Patients are evaluated for up to 5 years or until progression. 1. Borch TH, Andersen R et al. T cell therapy in combination with Vemurafenib in BRAF mutated metastatic melanoma patients. J Immunother Cancer. 2014;2 (Suppl 3:P67). 2. Borch TH, Andersen R et al. T cell therapy in combination with Vemurafenib in BRAF mutated metastatic melanoma patients. Journal for ImmunoTherapy of Cancer 2016, 4(Suppl 1):P131.

Clinical trial identification

NCT02354690

Legal entity responsible for the study

Inge Marie Svane

Funding

Danish Cancer Society The Research Foundation of the Capital Region of Denmark

Disclosure

T.H. Borch: Honorarium for lecture from BMS. M. Donia: Honorarium from Genzyme, MSD, BMS. Received travel suppoer from Novartis, MSD, BMS, Roche and Pfizer. I.M. Svane: Advisory board memberships and lectures: Roche, Novartis, MSD, Celgene, Incyte, TILT bio, Pfizer, BMS, AstraZeneca. Institution has received limited grants for translational research from BMS, Roche, Novartis. All other authors have declared no conflicts of interest.

Background

The treatment of triple negative breast cancer (TNBC) is hampered by the lack of established therapeutic targets like hormonal receptors or HER-2. Chemotherapy and radiotherapy is the standard of care and survival rates in TNBC remain poor. Approaches tailored to the patient’s individual tumor signature may bring improvement. The Mutanome Engineered RNA Immuno-Therapy (MERIT) consortium is validating an innovative, individualized mRNA-based vaccine for the treatment of TNBC. MERIT is a collaboration project of five European partners from academia and industry dedicated to realize a personalized approach for treatment of TNBC. The consortium has set up a clinical workflow, covering drug development from target discovery and validation to GMP manufacturing and drug release for each individual patient. Moreover, the consortium has generated a pre-synthesized mRNA vaccine warehouse containing the most frequent shared tumor antigens in TNBC for drug supply.

Trial design

A phase I trial in 4 European countries assesses the feasibility, safety and biological efficacy of this personalized immunotherapy. TNBC patients (pT1cN0M0 – T_xN_xM0) after surgery and adjuvant chemotherapy will be allocated to one of two study arms. Patients in ARM1 receive 8 vaccination cycles with a personalized set of shared tumor antigens from pre-synthesized mRNA vaccine warehouse that correspond to the patient tumor’s antigen-expression profile. Patients in ARM2 are first treated with the personalized WAREHOUSE vaccine approach followed by 8 vaccination cycles of an on-demand manufactured MUTANOME vaccine encoding unique mutation signature of the individual patient identified by NGS. The mRNAs are administered intravenously as a nanoparticulate lipoplex formulation, which protects RNA from degradation, activates innate immunity, transfects APCs and consequently induces highly potent antigen-specific T cell responses. Four clinical sites are open for recruitment; >10 patients have been screened and the vaccinations with pre-manufactured warehouse RNAs have started. We give insights into features of the established process and present first stratification data. MERIT is funded by the EU Commission’s FP7 and led by BioNTech AG.

Clinical trial identification

EudraCT No.: 2014-002274-37

Legal entity responsible for the study

BioNTech AG

Funding

European Commission’s FP7

Disclosure

K. Frenzel: Employee at BioNTech AG. L. Heesen, S. Bolte, V. Bukur, E. Derhovanessian: Employee of BioNTech AG. M. Diken: Employee of TRON gGmbH. S. Kreiter: BioNTech Group Mainz Advisor. A. Kuhn: Employee of BioNTech Group. K. Kuehlcke: Employee of BioNTech Group/Eufets. M. Löwer: Employee of TRON, Consultant for BioNTech Group. Ö. Türeci: Co-founder of Ganymed Pharmaceuticals AG. Inventor on 530 patents and patent applications. Member of Scientific Advisory Board of BioNTech AG. U. Sahin: Co-founder and CEO BioNTech AG, Mainz. Inventor of Licensed Patents related to Cancer Immunotherapy. Head of the Scientific Advisory Board of Ganymed Pharmaceuticals. T. Sjöblom: Founder, board member and shareholder of ExScale Biopecimen Solutions AB which markets CE/IVD solutions for nucleotide extraction in molecular pathology. K. Thielemans: eTheRNA Immunotherapies: receipt of grants, honoraria, consultation fees, Stock shareholder.

All other authors have declared no conflicts of interest.