Background

Metastatic castration-resistant prostate cancer (mCRPC) is the most challenging stage in prostate cancer. Patient with visceral metastasis have the poorest outcome amoung them. In this retrospective study, we analyzed the clinical outcome of lutetium-177 prostate-specific membrane antigen (¹⁷⁷Lu-PSMA) in mCRPC patients with visceral metastasis.

Methods

Ten patients of mCRPC with visceral metastasis were enrolled for one cycle of ¹⁷⁷Lu-PSMA therapy. Number of efficacy and safety parameters, e.g., prostate-specific antigen (PSA), visual analog scale (VAS) and analgesic quantification scale (AQS), hemoglobin (Hb), total leukocytes counts (TLC), platelets, creatinine, & total bilirubin, were assessed and compared with Wilcoxon signed-rank test. The progression-free survival (PFS) curve was computed by the Kaplan-Meier method. The receiver operating characteristic curve (ROC) was also plotted for ¹⁷⁷Lu-PSMA dose. P≤0.05 was considered significant.

Results

Liver (80%), lung (30%), adrenal (10%), and peritoneum (10%) were the sites of visceral metastasis in our study. On PSA response assessment, 10%, 60%, and 30% of the patients had partial response, stable disease, and progressive disease, respectively. Forty percent of the patients had improvement in the VAS, while 50% had improvement in the AQS score. Median PFS was 24 weeks in our study. A cut-off of 4.88GBq of ¹⁷⁷Lu-PSMA was the best-predicted progression with 66.67% sensitivity and 100% specificity on ROC analysis. Thirty percent of the patients showed grade 3 anemia. No other significant toxicity was seen.

Conclusions

Lutetium-177-PSMA was a reasonable palliative treatment option with limited toxicity for these end-stage mCRPC patients with visceral metastasis with adequate PSA stabilization.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

Claudin18.2 (CLDN18.2), a member of tight junction protein family, is strictly limited to differentiated epithelial cells of gastric mucosa and multiple tumor types, such as gastric, esophageal and pancreatic cancers. We have generated a novel species cross-reactive CLDN18.2 specific antibody, and labeled it with “Next Generation” radionuclide I-124 (¹²⁴I-18B10).

Methods

I-124 was produced by the medical cyclotron using ¹²⁴Te (p, n) ¹²⁴I reaction. In the cell-based assay, the uptake of ¹²⁴I-18B10 in MKN45-CLDN18.2 (CLDN18.2+ cell line) and MKN45 (CLDN18.2- cell line) were detected at 10, 30, 60 and 120 min, and the blocking group using cold 18B10 antibody to block uptake was also evaluated. PDX-bearing mice, which were selected by immunohistochemical (IHC) method and assessed as CLDN18.2+ or CLDN18.2-, were injected with either 18.5 MBq ¹⁸F- fluorodeoxyglucose (¹⁸F-FDG), or ¹²⁴I-18B10, or ¹²⁴I-hIgG via the tail vein, and Micro-PET/CT images were taken at 2, 60 and 120h post injection.

Results

The specific activity of ¹²⁴I-18B10 was 0.62 mCi/mg antibody and the labeling rate was higher than 95%. The cell-based assay showed that specific uptake of it by the MKN45-CLDN18.2 cells was significantly higher than that of by the MKN45 cells (23.51±0.47 % vs 8.69±0.35 % at 2 h, P<0.05). Both uptake assay and competitive binding assay in the MKN45-CLDN18.2 cells showed that cold 18B10 antibody could significantly reduce the uptake and binding of ¹²⁴I-18B10 (15.33±0.82 % at 2 h, P<0.05). As expected, the uptake of ¹²⁴I-hIgG was low (5.21±0.29 % at 2 h). In PDX bearing mice, the uptake of ¹⁸F-FDG in tumor sites was low. The distribution of ¹²⁴I-18B10 in CLDN18.2+ PDX bearing mice was increasingly enriched in the tumor sites over time. The uptake signals of ¹²⁴I-18B10 in CLDN18.2- PDX bearing mice in all tissues and tumors remained similar at different time points.

Conclusions

The ¹²⁴I-18B10 antibody has good radio-chemical characteristics and stability. The cell uptake assay and competitive binding assay demonstrated that the probe is highly specific to CLDN18.2. Micro-PET images of PDX bearing mice demonstrated that ¹²⁴I-18B10 was enriched in the lesion of CLDN18.2 positive tumors rather than negative tumors or normal tissues.

Legal entity responsible for the study

Peking University Cancer Hospital & Institute.

Funding

Has not received any funding.

Disclosure

F. Teng, Y. Gu, X. Qian: Full/Part-time employment: Research, Mabspace Biosciences (Suzhou) Co. Ltd., Suzhou, China. All other authors have declared no conflicts of interest.

Background

Breast cancer is the most common and frequent cause of death in women in all types of cancer. Current treatment protocols do not provide a complete cure and targeting therapy can provide an important avenue for successful treatment of breast cancer. In this study, we aim to determine the therapeutic effects of the drug-conjugated carrier system with the conjugation of peptide sequence and antibody on HER2-positive breast cancer cells.

Methods

The selectivity of single nanocarriers were compared with doxorubicin (DOX) loaded-HER2 targeting peptide (LTVSPWY) and DOX loaded-monoclonal antibody (Herceptin®) on HER2-positive and HER2-negative breast cells. After defining the physicochemical characterization of micelle-based nanocarriers, the cytotoxic effects of micelles on healthy breast epithelial cells (MCF-10A, HER2-negative) and breast cancer cells (SKBR3- HER2-positive) were determined by MTT cell proliferation assay. Next, apoptotic and genotoxic effects of micelles (IC50 doses of DOX loaded-peptide conjugated) were determined by using JC-1 assay and Western Blotting (Bax and Bcl-2 proteins) and Commet assay, respectively. Also, drug-releasing was analyzed by flurosance microscopy with imaging processes. Lastly, cytostatic effects of micelles were investigated with cell cycle analysis.

Results

DOX loaded-HER2 targeting peptide (DOX-Pep-HER-2-NCs) and DOX loaded-monoclonal antibody micelles (DOX-Anti-HER-2-NCs) had significant differences. DOX-Pep-HER-2-NCs had more toxic effects on SKBR-3 cells than DOX-Anti-HER-2-NCs. However, there is no significant change after the application of these micelles on MCF-10A cells. Besides, the intracellular amounts of doxorubicin with the application of DOX-Pep-HER-2-NCs were detected as higher in HER-2 positive breast cancer cells after measured by fluorescence imaging. Additionally, DOX-Pep-HER-2-NCs had more apoptotic, cytostatic and genotoxic effects on HER2 overexpressed SKBR-3 cells.

Conclusions

The targeting and therapeutic efficiency of DOX-Pep-HER-2-NCs were compared to DOX-Anti-HER-2-NCs on SKBR-3 cells. DOX-Pep-HER-2-NCs was more effective than DOX-Anti-HER-2-NCs on SKBR-3 cells in terms of targeting and therapeutic effects.

Legal entity responsible for the study

Prof. Dr. Sevil Dinçer İşoğlu & Prof. Dr. Alper İşoğlu.

Funding

This project was supported by The Scientific and Technological Research Council of Turkey (1003 Project-216S991).

Disclosure

All authors have declared no conflicts of interest.

Background

Treatments of various forms of cancer with in vitro prepared dendritic cells of various modalities have been shown to be safe but have not led to the desired efficacy. In this study, we examined the safety and efficacy of an autologous vaccine based on the fusion of patient tumor and dendritic cells (aHyC) in the treatment of chemotherapy-naive patients with castration-resistant prostate cancer (CRPC).

Methods

A randomized placebo-controlled cross-over trial was conducted between June 2013 and November 2016 and followed up for survival until September 2020. Twenty-two adult men with CRPC, asymptomatic or minimally symptomatic were enrolled and consecutively allocated to aHyC-first (12 patients) and placebo-first (10 patients) group according to previous randomization. Two patients were excluded during the trial. Autologous monocyte-derived dendritic cells and prostate tumor cells were electrofused to yield hybridomas and injected subcutaneously four times. The primary endpoints were safety, feasibility and quality of life assessments; the secondary endpoints were patients’ clinical and immune responses and overall survival.

Results

Twenty patients were analyzed. There were no serious adverse events (AEs) with aHyC treatment; mild AEs were observed in five patients in the aHyC arm (42%) and in three patients in the placebo arm (38%; P=0.78). The aHyC treatment preserved quality of life in the observed period of 4 months after the first application of aHyC vaccine. An increase in CD4⁺ cell subpopulations, an increase in cytotoxic T cells (CD8⁺), and a decrease in total NK cells were detected only in the aHyC arm compared to baseline. Moreover, the natural killer cell subpopulation remained at basal level, but increased in the placebo arm (P=0.004). The median overall survival from the first aHyC application was 58.5 months (95% CI, 45.4 to 71.7; n=19) and 65.2 months from CRPC diagnosis. The median time to next-in-line therapy (docetaxel, enzalutamide, abiraterone acetate) in patients receiving aHyC (n=19) was 28.0 months.

Conclusions

Treatment with aHyC is safe and effective and may represent a new personalized therapeutic option for patients with CRPC.

Clinical trial identification

EMA: 2012-005498-29.

Legal entity responsible for the study

Robert Zorec.

Funding

This work was supported by grants P3 310, J3 6790, J3 6789, and J3 9266 from the Slovenian Research Agency; by CipKeBip, COST Action BM1002, EU COST Action CM1207-GLISTEN, and EU COST Action CM1207 EuroCellNet.

Disclosure

All authors have declared no conflicts of interest.

Background

Ewing’s sarcoma (ES) is an aggressive and rare malignancy that primarily afflicts children and young adults. Patients with metastases have the worst prognosis. We have previously demonstrated that locally delivered mesenchymal stromal/stem cells (MSCs) can control ES growth releasing tumor necrosis factor-related apoptosis inducing ligand (TRAIL). However, considering the nature of the disease, new MSC strategies for metastatic ES are needed, accounting that ES can express high levels of the disialoganglioside GD2. To optimize the MSC affinity for tumors, we recently developed a bi-functional (BF) strategy where MSCs expressing TRAIL were further modified by a truncated anti-GD2 chimeric antigen receptor (GD2 tCAR). Here, anti-GD2 BF MSCs delivering a soluble variant of TRAIL (sTRAIL) were challenged in several in vitro and in vivo models, including a metastatic ES xenotransplant.

Methods

Co-expression in MSCs of GD2 tCAR together with sTRAIL was obtained by lentiviral vectors. The BF MSC binding to GD2-positive ES cells was verified in a cell-to-cell interaction assay. Cytotoxicity by BF MSCs was assessed by 2D and 3D cocultures. Tumor targeting and killing by BF MSCs was then investigated in a metastatic ES xenotransplant by in vivo imaging and ddPCR.

Results

In vitro data demonstrated both tumor affinity and killing of BF MSCs. The in vivo model closely mimicking the disseminated ES, with lung and liver as the main metastatic sites, demonstrated that MSCs were able to counteract ES growth in the lung with a significant reduction in tumor signal. As for the liver, a slight though not significant antitumor effect was observed. Evidence on engraftment of BF MSCs into ES metastatic sites was also provided indicating that GD2 tCAR ameliorated the tumor targeting of BF MSCs.

Conclusions

Our work represents the first attempt to target metastatic ES by MSCs delivering an anticancer molecule. With the limitation of a monotherapy approach, BF MSCs promise to pave the way for an improved therapeutic delivery of TRAIL to treat metastatic ES and other deadly GD2-positive malignancies.

Legal entity responsible for the study

University of Modena and Reggio Emilia.

Funding

This work was supported in part by grants from the Association ASEOP and from MIUR “Dipartimenti Eccellenti” 2017.

Disclosure

G. Golinelli: supported by an AIRC fellowship for Italy Grant. G. Grisendi, C. Spano, G. Casari: Honoraria (self): Rigenerand Srl. M. Dominici: Honoraria (self), Advisory/Consultancy, Leadership role: Rigenerand Srl. All other authors have declared no conflicts of interest.

Background

Therapeutic resistance to PARP inhibitors (PARPi) is still a clinical hurdle. We already demonstrated that co-treatment with AsiDNA prevents and reverses PARPi-acquired resistance. However, the mechanisms underlying resistance inhibition remain unknown. Here, we describe that AsiDNA is able to target specifically Drug Tolerant Cells (DTCs), the deadly survivors responsible of acquired resistance to PARPi, and therefore represents a therapeutic opportunity to impede tumor progression or relapse.

Methods

AsiDNA is a double-stranded (DS) DNA molecule that mimics DS DNA breaks to interfere with DNA repair, by over-activating a false DNA damage signaling especially through DNA-PK (decoy agonist). We used continuous treatment protocols, to select resistance to PARPi and assessed the impact of AsiDNA addition on resistance prevention. We also addressed the mechanisms underlying the survival and evolution of the residual DTCs under PARPi treatment, and how AsiDNA could impede their outgrowth.

Results

Long-term treatment of cancer cells harboring a BRCA1/2 mutation led to the emergence of acquired resistance to PARPi, via de novo evolution of tumor cells from a drug-tolerant persister state. These DTCs are characterized by senescence-associated phenotypic hallmarks including proliferation restriction and inflammatory secretome, a down-regulation of their DNA repair, and energy production especially through fatty acid metabolism. Addition of AsiDNA to PARPi completely and irreversibly abolished resistance emergence. Long term treatment with AsiDNA induced a down-regulation of major DNA repair pathways and fatty acid metabolism, and a decrease of GPX4 expression, a robust anti-oxidant protein required by DTCs to counteract the overproduction of superoxides during fatty acid metabolism. In line with this, we showed that DTCs are hyper-sensitive to AsiDNA compared to parental cells. AsiDNA significantly delayed PARPi resistance emergence also in vivo using a breast cancer xenografted model BRCA1-mutated.

Conclusions

Our results provide the evidence that resistance to PARPi can evolve from DTCs, and that AsiDNA could be a therapeutic strategy to specifically address these deadly survivors and overcome tumor progression or relapse.

Legal entity responsible for the study

The authors.

Funding

Onxeo.

Disclosure

W. Jdey, V. Hayes, C. Doizelet, M-C. Lienafa, V. Trochon-Joseph, J. Greciet: Full/Part-time employment: Onxeo.

Background

Epigenetic dysregulation contributes to cancer progression and resistance to treatment. Some data suggest that the kinetics of response to epigenetic drugs (ED) may differ from conventional chemotherapy. Investigating whether this operates in ED phase I (P1) trials is crucial, in order to avoid stopping prematurely an efficient treatment. Here, we assessed the kinetics and prognostic factors of response in patients (pts) enrolled in an ED P1 trial.

Methods

All consecutive pts with solid tumors or lymphoma, enrolled in a P1 trial evaluating an ED as monotherapy at the Gustave Roussy Drug Development Department between Jan 2010 and Jun 2020 were included. Pts and trial characteristics, response to treatment and outcome were retrospectively collected. Statistical associations were tested using a Fisher’s exact test and survival distributions were compared using the log-rank test.

Results

Overall, 290 pts (median age of 60 yo, 61% male, 67% with solid tumors, median of 3 previous lines of therapy) enrolled in 22 ED monotherapy P1 trials were included. Median duration of treatment, progression free survival (PFS) and overall survival (OS) were 2.3, 2.1 and 10.1 months (mo), respectively. Disease control rate (DCR) was 52% and overall response rate (ORR) was 11% (32 pts). Median time to response was 2.3 mo (0.7-7.2); 53% of responses occurred at the first evaluation, 19% at the second and 28% beyond. Median duration of response was 4.6 mo (0.4-71.3). Baseline lactate dehydrogenase (LDH) < upper normal limit normal (p<0.001), RMH score 0-1 (p=0.01), GRIm score 0-1 (p=0.029) and absence of liver metastasis (p=0.02) were significantly associated with better DCR, but not ORR. A ≥ 25% increase of baseline LDH at C2D1 was associated with shorter PFS (1.3 vs 3.0 mo, p<0.001) and shorter OS (4.3 vs 13.7 mo, p<0.001), while a ≥ 25% decrease was associated with higher ORR (OR=6.57; 95% CI 2.35-17.95, p<0.001). The association between changes in tumor growth rate and outcome will be available at the time of the congress.

Conclusions

In ED phase I trials, nearly 50% of responses occurred beyond the first evaluation. Baseline LDH, GRIm and RMH scores are predictive of DCR but not ORR, whereas a ≥ 25% LDH variations at C2D1 correlated with responses.

Legal entity responsible for the study

Gustave Roussy Institute.

Funding

Has not received any funding.

Disclosure

J-M. Michot: Non-remunerated activity/ies: Amgen; Non-remunerated activity/ies: Astex; Non-remunerated activity/ies: AstraZeneca; Non-remunerated activity/ies: MedImmune; Non-remunerated activity/ies: Roche; Non-remunerated activity/ies: Sanofi; Non-remunerated activity/ies: Xenecor. L. Verlingue: Honoraria (self): Adaptherapy. V. Ribrag: Advisory/Consultancy, Research grant/Funding (self), Non-remunerated activity/ies: Epizyme; Research grant/Funding (self): ArgenX; Advisory/Consultancy, Non-remunerated activity/ies: Servier; Advisory/Consultancy: NanoString; Advisory/Consultancy: Gilead; Advisory/Consultancy: PharmaMar; Advisory/Consultancy: BMS; Advisory/Consultancy: MSD; Advisory/Consultancy: Incyte; Advisory/Consultancy: Roche; Advisory/Consultancy: Infinity. J-C. Soria: Shareholder/Stockholder/Stock options, Full/Part-time employment, Sept 2017-Dec 2019: AstraZeneca; Officer/Board of Directors: Hookpipa; Shareholder/Stockholder/Stock options: Daiichi Sankyo; Shareholder/Stockholder/Stock options: Gritstone. C. Massard: Non-remunerated activity/ies: Amgen; Non-remunerated activity/ies: Kusajili; Non-remunerated activity/ies: GSK; Non-remunerated activity/ies: Lilly; Non-remunerated activity/ies: PharmaMar. S. Postel-Vinay: Research grant/Funding (self), Non-remunerated activity/ies: Boehringer Ingelheim; Research grant/Funding (self), Non-remunerated activity/ies: AstraZeneca; Research grant/Funding (self): Roche; Research grant/Funding (self): Merck; Non-remunerated activity/ies: Archimaid. All other authors have declared no conflicts of interest.

Background

Hepatocellular carcinoma (HCC) is the most frequent primary tumor of the liver and the third cause of cancer-related deaths. The identification of candidate molecular targets and biomarkers in HCC clinical practice are needed. The signatures of aberrant long non-coding RNAs (lncRNAs) expression in HCC tissues, their extracellular release and stability had led to their exploration as diagnostic and prognostic tools as well as potential therapeutic targets for HCC. Telomeric-Repeat Containing RNA (TERRA) consists of 100nt-9Kb subtelomeric-derived transcripts able to base-pair with TERC RNA, acting as telomerase allosteric inhibitor. Little is known on the role of lncRNA TERRA in HCC.

Methods

By qPCR we measured TERRA expression in tumor and peritumoral (PT) tissues of HCC patients, as well as in plasma and in HCC cells. HCC patients (n.25) did not receive any treatment before surgical resection. By nickel-based isolation method (NBI) we isolated from the conditioned medium (CM) of HA22T/VGH cells the extracellular vesicles (EVs), subsequently analyzed by qNANO instrument.

Results

Global TERRA expression was significantly downregulated in HCC vs PT tissues (p=0.025) and ROC analysis revealed a significant ability to distinguish HCC from PT (p=0.03). Extracellular TERRA transcripts were significantly higher in plasma of HCC patients compared with healthy subjects and logistic regression model strongly evidenced the potential diagnostic ability of circulating TERRA (AUC=0.76; 95% CI=0.624-0.873; p=0.0004). HA22T/VGH cells expressed TERRA, but most of the transcripts are released into the CM, also encapsulated in EVs (mean diameter=185nm, concentration=163x10⁶/ml). Treatment of HCC cells with the multi-kinase inhibitor (KI) sorafenib significantly increased TERRA expression (p=0.001) and decreased (p=0.01) its release in EVs (mean diameter=252nm, concentration=209x10⁶/ml).

Conclusions

Our results provide evidence on TERRA dysregulation in tissues and liquid biopsy of HCC patients, thus focusing on a novel potential non-invasive biomarker of diagnosis and downstream target of the KI. TERRA detected in the EVs of HCC cells open a new field of cancer research to comprehend its role at the extracellular level.

Legal entity responsible for the study

The authors.

Funding

Lega Italiana Lotta ai Tumori (LILT), Italian Ministry of University and Research (FFRB), University of Brescia.

Disclosure

All authors have declared no conflicts of interest.

Background

Breast cancer (BC) is one of the major biomedical research priorities worldwide. Luminal B and triple negative BC patients represents the worst prognosis and highest incidence of recurrence. Among those patients, metastasis is the missing piece in the puzzle. Consequently, extensive research is urgently required to identify specific molecular targets responsible for luminal B and TNBC induced risk of metastasis. ICAM-1 (known as CD54) and PVR (known as CD155) are transmembrane glycoproteins that have been emerged as novel molecular targets that are markedly up-regulated in BC patients. Our group is recently focusing on unraveling the role of Long non-coding RNAs in regulating vital molecular targets in BC patients. LncRNAs regulating ICAM-1 and PVR is still a virgin field. Therefore, the aim of this study is to identify a lncRNA that could dually target ICAM-1 and PVR simultaneously.

Methods

Forty BC patients were recruited. Bioinformatic analysis was performed to identify a lncRNA that could directly or indirectly target ICAM-1 and PVR. MDA-MB-231 and MCF-7 cells were cultured. Cells were transfected using Scrambled and H19 siRNAs by lipofection Technique. Total RNA was extracted using biozol from tissues and cell lines. Reverse transcription and qRT-PCR were performed. MTT, scratch and colony forming assays were performed. Student t-test was performed for statistical analysis.

Results

In silico, H19 lncRNA was found to indirectly target ICAM-1 and PVR through STAT3 axis. H19, ICAM-1 and PVR were found to be upregulated in BC patients. Upon patients stratification, H19 and PVR were found to be specifically upregulated in luminal B BC patients while ICAM-1 was found to be specifically upregulated in TNBC patients. Upon Knocking down of H19, transfection effeciency was evaluated and H19 siRNAs resulted in paradoxical impacts on ICAM-1 and PVR. Yet, H19 siRNAs resulted in a repression of cellular viability, colonogenicity and migration capacity.

Conclusions

H19 inhibitory impact on the metastatic mediator ICAM-1 overrides its inductory effect on PVR in BC cells. Therefore, this study highlights H19 as an oncogenic lncRNA in BC and a novel molecular target for resistant luminal B and TNBC patients.

Legal entity responsible for the study

German University in Cairo.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

The hormonal therapy is among the most effective treatment of the hormone-dependent breast cancer however its efficiency is limited by the acquired resistance to the hormonal drugs. Previously we have shown the exosomes involvement in the transferring of the hormonal resistance from the resistant to the sensitive cells, and here the study of the features of the exosomes of the resistant cells was performed.

Methods

Experiments were performed on the MCF-7 breast cancer cells, MCF-7/T resistant subline developed under long-term tamoxifen treatment, and MCF-7/exoT resistant subline developed under resistant exosomes treatment. Exosomes were prepared from the conditioned medium by the differential ultracentrifugation, and exosome imaging was carried out by transmission electron microscope. The analysis of exosomal microRNAs was performed by HiSeq2500 and at least 5 million reads per samples were obtained. Library preparation was carried out with NEBNext® Small RNA Library Prep Set for Illumina® (E7330S). More than 2500 miRNAs were identified in the exosomal samples. DNA methylation was evaluated by the RRBS (Reduced Representation Bisulfite Sequencing) method.

Results

The analysis of the exosomal microRNAs revealed 471 microRNAs over-expressed in the exosomes of the resistant cells. Among them, three DNMT1/3-targeting microRNAs - miR-148b-3р, miR-193a and miR-383, were identified. The subsequent analysis of the cellular proteins revealed the decreased expression of DNMT1/ DNMT3 both in the primary-resistant MCF-7/T cells and in the exosome-treated MCF-7/exoT cells. The suppression of DNMT1/3 correlated with the hypomethylation of particular CpG islands in DNA of the resistant cells.

Conclusions

Taken together, the results obtained demonstrate the important role of the DNA (de)methylation in the exosome-mediated transferring and maintaining of the hormone resistance in the breast cancer cells.

Legal entity responsible for the study

The authors.

Funding

The Russian Science Foundation, project 19-15-00245.

Disclosure

All authors have declared no conflicts of interest.

Background

Over the past decades, accumulating research evidences revealed that abnormal expressions of long non-coding RNAs (lncRNAs) are associated with tumour initiation, progression, metastasis, and resistance to cancer therapies. Therefore, lncRNAs are considered to be potential biomarkers for many cancer types. In the current study, we examined the expression and molecular mechanisms of a newly identified lncRNA called RNA associated with metastasis 11 (RAMS11) and its association with the development of colorectal cancer (CRC).

Methods

Quantitative RT-PCR was used to determine the expression of RAMS11 in 4 CRC cell lines (DLD-1, HT-29, HCT-116, and SW480) and normal colon cells CCD-112-CoN. To evaluate the biological and physiological functions of RAMS11 in CRC cells, CCK-8 cell proliferation assay, colony formation assay, and wound healing migration assay were performed after RAMS11 knockdown. The expressions of autophagy/apoptosis/mTOR/EMT pathway proteins were determined by Western blotting to evaluate the molecular mechanisms of RAMS11 in CRC cells.

Results

We found that RAMS11 was significantly upregulated in CRC cell lines compared to the normal cells. The knockdown of RAMS11 reduced CRC cells proliferation, and migration through mTOR dependent induction of autophagy, promotion of apoptosis, and inhibition of epithelial-mesenchymal transition (EMT) process.

Conclusions

Overall, our results suggested that RAMS11 is an important oncogenic regulator of CRC initiation and progression whereas, targeting RAMS11 and the related molecular pathways could be used as potential therapeutic strategies for CRC management.

Legal entity responsible for the study

The authors.

Funding

This project is partially supported by: (1) Research grant to HKL including Departmental Seeding Fund and Internal Institutional Research Fund (P0031318-UAHS); (2) Postgraduate studentship from The Hong Kong Polytechnic University for ZIK.

Disclosure

All authors have declared no conflicts of interest.

Background

Recently, hydrogen sulphide (H₂S) has been acknowledged as a pivotal gasotransmitter altering several oncogenic signaling pathways in breast cancer (BC). Our research group has recently identified H₂S and its synthesizing enzyme cystathionine-γ-lyase (CSE) as promising targets for BC treatment especially that their expression pattern was closely associated with aggressive tumor phenotypes. However, the motor engines responsible for such high expression pattern of CSE/H₂S in BC patients are yet to be explored. Transcriptomic analysis for CSE promoter region has revealed STAT-3 as a direct regulator for CSE. Long non-coding RNAs (lncRNAs) have recently been identified as vital post-transcriptional regulators. Yet, lncRNAs regulating H₂S production is still a virgin field. MALAT-1 is an oncogenic lncRNA tuning self-renewal capacity of BC cells. However, the role of MALAT-1 in modulating STAT-3-regulated CSE has never been probed in BC. Therefore, our aim is to investigate the role of MALAT-1 in regulating STAT-3/CSE/H₂S axis.

Methods

BC biopsies and their normal counterparts were collected from 20 BC patients. MDA-MB-231 cells were cultured and transiently transfected using MALAT-1 and CSE siRNAs by lipofection method. Total RNA was extracted using Biozol reagent, reverse transcribed and quantified using qRT-PCR. Western blot analysis was performed.

Results

Screening showed an upregulation of MALAT-1, STAT-3 and CSE in aggressive BC tissues. Upon knocking down of MALAT-1, a marked repression of STAT-3 and CSE was observed nominating MALAT-1 as a novel upstream lncRNA regulating STAT-3/CSE axis. On the other hand, CSE siRNAs resulted in the induction of MALAT-1 expression level, STAT-3 transcript and phosphorylated forms in attempt to restore H₂S levels in BC cells.

Conclusions

MALAT-1 was identified as the first lncRNA regulating STAT-3/CSE/ H₂S in BC. Furthermore, this study underscores the significance of dual targeting of MALAT-1 and CSE for efficient repression of H₂S levels in BC cells by-passing the compensatory mechanism exercised by CSE.

Legal entity responsible for the study

German University in Cairo.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

Platinum-based therapy (PL) remains the standard of care in MPM, despite encouraging emerging data from immunotherapy (IO) trials. Recently, prognostic MPM clusters with potential implications for targeted-treatments were identified by multiplatform profiling but we are still far from translating these data in therapeutic opportunities for pts. In this scenario, we are leading an international multicenter projecton new biomarkers in MPM.

Methods

We used preclinical models to compare PL-naïve and PL-resistant MPM. Protein expression of biomarkers of interest from cells were analyzed by FACS and Western blot at Università degli Studi della Campania Luigi Vanvitelli (Naples, IT). Further, specimens of MPM pts treated with PL and IO will be used for an IHC-based pivotal analysis on known and emerging biomarkers as BAP1, AURKA and CDKN2A. VISTA and PD-L1 will be also analyzed and correlated to other results.

Results

In PL-resistant MPM H2452 cells, generated treating parental cell line with PL dose-escalation, FACS analysis revealed an increased PD-L1 expression (1% in PL-naïve vs 39.1% in PL-resistant). Western blot protein expression analysis confirmed this result, suggesting a PL-driven increased IO-sensitivity, independent from response, as shown in other cancer types. Sixty-two MPM pts treated at Hospital Clínico Universitario de Valencia (Valencia, ES) from 2001 to 2020 were selected for the translational part of the project so far. Mean age was 67.5 years (36-89), 90% (n=56) of pts were male, 80% (n=50) exposed to asbestos and 61% (n=38) ECOG PS<2. The majority (69%, n=43) with epithelioid MPM; 52% (n=33) stage III-IV at diagnosis. Of 58 pts included in the analysis, 4 were exposed to IO and 45 (77%) to PL. 62% (n=28) PL-sensitive and 38% (n=17) PL-resistant. IHC analysis on tissue specimens is ongoing.

Conclusions

Our preliminary results confirmed the activation of PD-L1 by PL, also in resistant setting, showing a biological rationale for IO in these pts. Ongoing analysis will validate on a proteomic level other proposed biomarkers as BAP1, AURKA, CDKN2A and VISTA, to build a cost-effective IHC-based MPM classification, useful for future targeted-treatment strategies.

Legal entity responsible for the study

The authors.

Funding

Università degli Studi della Campania Luigi Vanvitelli, Naples, Italy.

Disclosure

F. Ciardiello: Advisory/Consultancy, Research grant/Funding (institution): Roche; Advisory/Consultancy, Research grant/Funding (institution): Amgen; Advisory/Consultancy, Research grant/Funding (institution): Merck; Advisory/Consultancy: Pfizer; Advisory/Consultancy: Sanofi; Advisory/Consultancy, Research grant/Funding (institution): Bayer; Advisory/Consultancy: Servier; Advisory/Consultancy: BMS; Advisory/Consultancy: Celgene; Advisory/Consultancy: Lilly; Research grant/Funding (institution): AstraZeneca; Research grant/Funding (institution): Ipsen. F. Morgillo: Advisory/Consultancy: MSD; Advisory/Consultancy: Lilly; Research grant/Funding (institution): AstraZeneca. All other authors have declared no conflicts of interest.

Background

Gaucher's disease is characterized by intralysosomal storage of glucosylceramide due to a genetic deficiency of the enzyme glucosylceramidase. Gaucher’s disease can be treated using eliglustat and miglustat, two inhibitors of the glucosylceramide synthase (GSL). The conversion of ceramide to glucosylceramide is one of the first steps in the synthesis of glycosphingolipids which can be therefore inhibited by eliglustat and miglustat. Glycosphingolipids play a major role in brain development and have been involved in the pathology of brain tumors. Here we analyzed the glycosphingolipids composition and the effect of eliglustat in diffuse midline glioma (DMG), one of the deadliest cancers in the pediatric population.

Methods

We established and characterized primary cells from two pediatric DMG patients. Glycosphingolipids were analyzed by thin layer chromatography, liquid chromatography-coupled tandem mass spectrometry and flow cytometry. The effect of eliglustat on the cell proliferation was examined.

Results

Immunohistochemistry analysis of DMG primary cells of both patients revealed glial origin (GFAP), high proliferative activity (Ki67) as well as the presence of H3 K27M mutant protein. The ganglioside GD2 was highly expressed. Neutral glycosphingolipids with 1 to 4 monosaccharides were also present in high concentration. Eliglustat completely abrogated the proliferation of the primary cells. Based on this promising data, treatment with miglustat of one patient has been started.

Conclusions

We show for the first time that inhibition of GSL effectively affects the survival of H3K27M-mutant DMG. Eliglusat and miglustat are released for the treatment of pediatric patients with Gaucher's disease and miglustat accumulates in the brain. Thus, targeting the glycosphingolipids metabolism with miglustat may accelerate the development of new therapies for brain tumors.

Legal entity responsible for the study

The authors.

Funding

Kinderkrebshilfe Mainz.

Disclosure

All authors have declared no conflicts of interest.

Background

Triple-negative breast cancer (TNBC) accounts for 15–20% of breast cancers (BC) and is characterized by high aggressiveness and lack of specific therapeutic targets. The intratumor existence of cancer stem cells (CSCs) represents one of the main causes of tumor relapse and resistance to treatments. Among the signaling pathways responsible for maintaining CSC activity, the Notch pathway plays a role in regulating self-renewal and in promoting BC development and resistance to chemo- and radiotherapy. B-cell CLL/lymphoma 6 (BCL6) is a transcriptional repressor that has a role in germinal center formation. Recent studies demonstrated its involvement in supporting BC cell survival, proliferation, invasiveness and in inducing an epithelial-to-mesenchymal transition program.

Methods

TCGA data from RNA-seq (HiSeq counts) and somatic copy number (CN) alterations were accessed using the Xena browser. METABRIC-processed data were retrieved from cbioportal. Normalized data and clinical information including TNBC data sets were retrieved from GEO Omnibus. Mammosphere formation efficiency percentage (MFE%) was calculated following BCL6 inhibition. Immuno Precipitation (IP) and Chromatin Immuno Precipitation (ChIP) experiments demonstrated the functional cooperation between BCL6 and EZH2.

Results

Through a bioinformatics-based analysis of benchmark BC data we evidenced the existence of a BCL6 altered expression in TNBC subtype. Further, we show that this feature plays a role in the maintenance/expansion of CSCs as showed by the significantly higher levels of BCL6 transcript in TNBC cells cultured under 3D versus 2D conditions. At the contrary, its loss of function significantly decreased MFE% by preferentially targeting CD44-positive cells versus ALDH-positive ones. Moreover, by taking advantage of the small molecule FX1, a BCL6 specific inhibitor, we revealed the existence of a functional interplay between BCL6 and EZH2 that triggers the activation of Notch signaling via Numb transcriptional repression.

Conclusions

Our findings provide insights towards the feasibility of pharmacological targeting of BCL6/Notch axis in combination with standard agents, to eradicate both the CSC compartment and the bulk of the tumor.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

Prevention and early detection of distant metastasis are critical for improving the outcome of patients with lung cancer. Previously, we showed that the presence of isolated basal cell hyperplasia (BCH) in the small bronchi distant from the tumor is associated with a high risk of distant metastasis in patients with lung squamous cell carcinoma (LUSC) and adenocarcinoma. However, mechanisms underlying this phenomenon are unknown. Here we aimed to assess the mutational landscape of metastatic LUSC associated with isolated BCH.

Methods

The study included 10 (48 to 77 years old) patients with LUSC (T_1-4N_0-2M₀) divided into three groups: 1) patients with isolated BCH and metastases; 2) patients with isolated BCH but without metastases; and 3) patients without premalignant bronchial lesions and metastases. Premalignant lesions were analyzed in hematoxylin and eosin-stained sections of formalin-fixed paraffin-embedded samples of small bronchi distant (3-5 cm) from the tumor. Tumor samples were sequenced on a NextSeq 500 (Illumina) using SureSelect XT Human All Exon v7 kit (Agilent).

Results

LUSC patients with BCH and metastases showed 1.6-times more mutations than cases without premalignant bronchial lesions and metastases. Interestingly, among patients with BCH, mutations were 1.8-times more in metastatic tumors than in non-metastatic tumors confirming that the presence of hyperplasia is not absolute for distant metastasis. Metastatic LUSC more often also harbored mutations in genes involved in cell-cell adhesion (CDH8, CDH9, ITGAL, SDK1, SDK2, et al.) and signal pathways of carcinogenesis (TP53, RB1, MMP1, MMP2, EGFR, et al.). Importantly, the most frequently mutated genes in metastatic tumors were TP53 (100% of cases) and PTPRT (75% of cases).

Conclusions

Taken together, our findings indicate that metastatic LUSC associated with isolated BCH shows a highly mutable phenotype.

Legal entity responsible for the study

Cancer Research Institute, Tomsk National Research Medical Center.

Funding

Russian Science Foundation, #20-75-10060.

Disclosure

All authors have declared no conflicts of interest.

Background

IFNα2b elicits potent anti-tumor antiproliferative and immunostimulatory activity; but with systemic toxicity. Delivering IFNα2b targeted to CD20⁺ lymphomas may lower systemic toxicity and increase therapeutic index. IGN002 is a novel recombinant protein comprised of anti-CD20 antibody (rituximab) fused to human IFNα2b by a peptide linker. Here, we report IGN002 stability in serum and tumor as well as efficacy in vivo in a B-cell non-Hodgkin lymphoma (NHL) xenograft model.

Methods

SCID mice bearing subcutaneous CD20⁺ tumors were administered i.v. rituximab at 10 mg/kg or IGN002 at 5, 10 or 15 mg/kg. Serum and tumors were collected at 1, 4, 24 or 48 hours (hrs) to assess IGN002 stability and tumor uptake using sandwich ELISA. In the same model, we examined tumor growth inhibition (TGI) and survival in mice treated with rituximab up to 5 mg/kg or IGN002 at doses up to 6 mg/kg.

Results

IGN002 increased in serum in a dose-related manner with the peak levels achieved at 1-4 hrs (5mg/kg: 33±3; 10mg/kg: 155±5; 15mg/kg: 177±11 μg/ml) and a half-life of 24 hrs. At 48 hrs, IGN002 was still detectable in the circulation (5mg/kg: 25±2; 10mg/kg: 44±6; 15mg/kg: 45±3 μg/ml). Increases in IGN002 were also observed in the tumor at similar levels to rituximab. In the tumor, as hypothesized, IFNα2b increased in as dose-related manner with C_max levels at 4 hrs of 41±12, 57±14 and 92±22 ng/mg of tumor tissue at 5, 10 and 15 mg/kg dose groups, respectively. At 48 hrs, IFNα2b levels were still detected (range: 18-38 ng/mg of tumor tissue). IGN002 improved survival at all doses investigated and increased inhibition of tumor growth when compared to rituximab with no adverse clinical signs.

Conclusions

IGN002 demonstrated adequate stability in the tumor resulting in the reduction in tumor mass and increased survival when compared to rituximab. Thus, demonstrating that the targeted delivery of IFNα2b to CD20⁺ tumor warrants further investigation in NHL patients.

Legal entity responsible for the study

Spectrum Pharmaceuticals.

Funding

Spectrum Pharmaceuticals.

Disclosure

S. Lakshmikanthan, P.S. Kolli, M. Tugnait, F. Lebel, J.A. Barrett: Shareholder/Stockholder/Stock options, Full/Part-time employment: Spectrum Pharmaceuticals. All other authors have declared no conflicts of interest.

Background

ICI have become key in the treatment armamentarium for pts with advanced cancer. Besides PD-L1, numerous biomarkers are being tested to enhance clinicians' ability to predict responses and prognosis. The polycomb repressive complex 2 (PKC2) is a histone methtyltransferase family that plays a major role in chromatin silencing. Preclinical evidence implicates PKC2 components such as EZH2 in immune resistance. This study aimed to assess the clinical relevance of PKC2 mutations (mts) in outcomes of ICI-treated pts.

Methods

NGS data from tumor samples of pts treated with ICI (anti-PD-1/PD-L1, anti-CTLA4 or both) were queried (cbioportal.org: MSKCC, Nat Genet 2019) for mts in PKC2-related genes, including EZH1, EZH2, EED, and SUZ12. The Kaplan Meier method was used to assess the association between mutated and unmutated PKC2 genes with OS (mos). All results were reported at the 0.05 significance level.

Results

1662 pts were examined (350 NSCLC,321 melanoma,214 bladder, 151 RCC,138 head neck-HN,126 esoph/gastr-EG, 117 glioma,110 CRC,90 unknown primary-CUP, 45 breast). The majority received a PD-1 or PD-L1 inhibitor: nivo, pembro, atezo, ave or durva (n=1256), 146 were treated with anti-CTLA4 (ipi or treme) and rest with combinations (n=260). 70 pts (4%) harbored truncating or missense mts in EZH2 (2.4%), EZH1 (1.2%), SUZ12 (0.9%) or EED (0.7%). Presence of these mts was more frequent in CRC (8.2%), followed by bladder (6.2%), melanoma (5.6%), EG (4.8%), glioma (4.3%), HN (3.6%), CUP (3.4%), RCC (3.3%), and NSCLC (1.7%). No significant mt co-occurence or mutual exclusivity among PRC2 genes was found. Pts carrying mts in PRC2 genes had a significantly longer median OS (44 mos) compared to those without (18 mos, log-rank P=0.0174).

Conclusions

Inactivating mts in the PRC2 chromatin silencing machinery, although rare, may predict favorable outcomes in ICI-treated pts with metastatic cancers. This warrants prospective confirmation and suggests that epigenetic regulators could serve as surrogate markers to guide ICI treatment decisions. Additionally, it justifies the rational for combinations of ICI with epigenetic modulators (e.g. EZH2 inhibitors) for unmutated tumors representing the majority of cases.

Legal entity responsible for the study

The author.

Funding

Has not received any funding.

Disclosure

The author has declared no conflicts of interest.

Background

The number of early clinical trials evaluating immunotherapies (IT) has increased exponentially over the last decade. These agents expose to adverse events (AE), which have been largely described with immune checkpoint blockers (ICB) as a monotherapy. However, the safety profile of IT other than ICB or in combination, have been poorly characterized. Here, we comprehensively analyze the safety profile of IT in phase I/II (P1/2) trials when used as a monotherapy (M) or in combination (C) with other anti-cancer agents, and compare it to the one of other anticancer agents assessed in the same patient (pt) population.

Methods

All consecutive pts treated in a P1/2 trial at the Gustave Roussy Drug Development Department between Jan 2008 and Aug 2020 were included. Pt and trial characteristics, AEs of any grade (G) and any type occurring at any cycle on trial, dose administered and treatment modifications were described in the following subgroups: IT as a M (IT-M), targeted therapy (TT) as a M (TT-M), IT in C (IT-C), TT in C (TT-C), other.

Results

1922 inclusions (1815 pts; median age of 56 years; 10% of hematological malignancies; 11300 treatment cycles) were performed in 195 P1/2 trials; 731 pts were enrolled in trials assessing IT (144 IT-M, 587 IT-C) and 1229 pts in trials of TT (523 TT-M, 706 TT-C). Nine toxic death occurred. Pts presented 8835 AE (7,3 % at cycle 1 and 92,7 % beyond cycle 1) including 1694 G3-4 AEs (3% of pts; 11,1 %, 31,5 %, 54,4 % of pts in trials evaluating IT-M, IT-C, TT-M and TT-C, respectively); 5 (10%), 39 (21%), 83 (16%), 135 (15%) G3-4 AEs occurred at cycle 1 in trials of IT-M, IT-C, TT-M and TT-C, respectively. G1-2 AEs occurred in 85.5 %, 88.3%, 76% and 79.8% of pts in these subgroups; 47,8 % of AEs resolved, of which 1/3 required co-medications. Median AE duration was 12.4, 18.1, 9.3 and 9.7 days in trials of IT-M, IT-C, TT-M and TT-C, respectively. The most common G3-4 AE type was neutropenia / anemia in trials evaluating IT-M and TT-C / TT-M and IT-C, respectively.

Conclusions

Severe AEs do not occur more frequently in P1/2 trials evaluating IT in combination. Only 10 – 21% of severe AEs, potentially scoring for dose-limiting toxicities, occur at cycle 1 in trials of IT or TT in this patient series.

Clinical trial identification

PDF file of trials name attached.

Legal entity responsible for the study

Drug Development Department, Institut Gustave Roussy.

Funding

Has not received any funding.

Disclosure

P. Martin-Romano, C. Baldini, A. Varga, A. Hollebecque, J-M. Michot, A. Gazzah, R. Bahleda, S. Champiat, A. Italiano, V. Ribrag, E. Angevin, J-P. Armand, A. Marabelle, C. Massard, S. Postel-Vinay: Non-remunerated activity/ies, As part of the Drug Development Department (DITEP)= Principal/sub-Investigator of Clinical Trials: AbbVie, Adaptimmune, Aduro Biotech, Agios Pharmaceuticals, Amgen, Argen-X Bvba, Arno Therapeutics, Astex Pharmaceuticals, AstraZeneca Ab, Aveo, Basilea Pharmaceutica International Ltd., Bayer Healthcare Ag, Bbb Technologies Bv, Beigene, Blueprint Medicine. C. Helissey: Non-remunerated activity/ies, As part of the Drug Development Department (DITEP)= Principal/sub-Investigator of Clinical Trials: Principal/sub-Investigator of Clinical Trials for AbbVie, Adaptimmune, Aduro Biotech, Agios Pharmaceuticals, Amgen, Argen-X Bvba, Arno Therapeutics, Astex Pharmaceuticals, AstraZeneca Ab, Aveo, Basilea Pharmaceutica International Ltd., Bayer Healthcare A. E. Deutsch: Non-remunerated activity/ies, AbbVie, Adaptimmune, Aduro Biotech, Agios Pharmaceuticals, Amgen, Argen-X Bvba, Arno Therapeutics, Astex Pharmaceuticals, AstraZeneca Ab, Aveo, Basilea Pharmaceutica International Ltd., Bayer Healthcare Ag, Bbb Technologies Bv, Beigene, Blueprint Medicines, Boehringer Ingelheim, Boston Pharmaceuticals, Bristol-Myers Squibb, Ca, Celgene Corporation, Chugai Pharmaceutical Co, Clovis Oncology, Cullinan-Apollo, Daiichi Sankyo, Debiopharm, Eisai, Eisai Limited, Eli Lilly, Exelixis, Faron Pharmaceuticals Ltd., Forma Therapeutics, Gamamabs, Genentech, GlaxoSmithKline, H3 Biomedicine, Hoffmann La Roche Ag, Imcheck Therapeutics, Innate Pharma, Institut de Recherche Pierre Fabre, Iris Servier, Janssen Cilag, Janssen Research Foundation, Kura Oncology, Kyowa Kirin Pharm. Dev, Lilly France, Loxo Oncology, Lytix Biopharma As, Medimmune, Menarini Ricerche, Merck Sharp & Dohme Chibret, Merrimack Pharmaceuticals, Merus, Millennium Pharmaceuticals, Molecular Partners Ag, Nanobiotix, Nektar Therapeutics, Novartis Pharma, Octime: AbbVie, Adaptimmune, Aduro Biotech, Agios Pharmaceuticals, Amgen, Argen-X Bvba, Arno Therapeutics, Astex Pharmaceuticals, AstraZeneca Ab, Aveo, Basilea Pharmaceutica International Ltd., Bayer Healthcare Ag, Bbb Technologies Bv, Beigene, Blueprint Medicine. J-C. Soria: Leadership role, Research grant/Funding (self), Full/Part-time employment, Senior Vice President (Sept 17 to Dec 19): AstraZeneca; Research grant/Funding (institution): AbbVie; Research grant/Funding (institution): Bayer; Research grant/Funding (institution): Blend Therapeutics; Research grant/Funding (institution): Boehringer Ingelheim; Research grant/Funding (institution): Cytomix; Research grant/Funding (institution): Daiichi; Research grant/Funding (institution): Eli Lilly; Research grant/Funding (institution): Genmab; Research grant/Funding (institution): Inivata; Research grant/Funding (institution): Merck; Research grant/Funding (institution): Netcancer; Research grant/Funding (institution): PharmaMar; Research grant/Funding (institution): Roche; Research grant/Funding (institution): Servier; Research grant/Funding (institution): Tarveda. All other authors have declared no conflicts of interest.

Background

Atezolizumab plus nanoparticle albumin-bound (nab)-paclitaxel prolonged progression-free survival (PFS) and overall survival (OS) among patients with unresectable locally advanced TNBC but its use is hampered by the lack of reliable predictors of tumor response. The study evaluates whether PD-L1 mRNA copies per ml in plasma-derived exosomes may predict response to anti-PD-L1 antibodies early in the course of therapy.

Methods

Seventy-seven consecutive patients with unresectable locally advanced TNBC treated with atezolizumab plus nab-paclitaxel were studied by blood draws at baseline, 28 days and 56 days after initiation of treatment. Exosomal PD-L1 mRNA in plasma was determined using Bio-Rad QX100 digital droplet PCR system and exoRNeasy kit. Objective responses were defined following the RECIST criteria v.1.1.

Results

At baseline, patients with complete and partial response (CR+PR, n=28) displayed a significantly higher number of PD-L1 mRNA copies per ml compared to ones with stable or progressive disease (SD+PD, n=49) (mean value: 785.6±121.1 vs 114.7±31.4, p<0.001). In patients with CR and PR mean PD-L1 copies/ml were 747.6±121.1 and 125.4 at baseline vs 2 months, respectively (p=0.001). In patients with stable disease the mean ± s.e.m. values were 270±71.1 vs 217.5±17.3 copies per ml (p=0.614) while in progressive disease PD-L1 mRNA levels were 122.1±31.2 vs 494.3±46.2 copies per ml at baseline vs 2 months, respectively (p<0.001). Patients with an increase of PD-L1 mRNA copies per ml were also characterized by significantly shorter PFS (p=0.007) and shorter OS (p=0.001) (OS: 5 months (range 2-7 months, 95%CI 1.1-6.1) vs more than double (range 8-15 months).

Conclusions

Despite the study’s limitations, our data suggest exosomal PD-L1 is significantly associated with outcome and response to atezolizumab plus nab-paclitaxel.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

Natural killer (NK) cells are the first native invaders against malignancy. NK cells activity is regulated by an array of activating and inhibitory receptors present on their surface. UL-16 binding proteins (ULBP 1-6) are ligands expressed by malignant cells to be detected by NKG2D activating receptor and consequent eradication of transformed cells by NK cells. Breast cancer (BC) patients experience a shedding of ULBP2 ligand in an attempt to evade immune surveillance process exhibited by NK cells. Yet, the mechanism behind the de-regulation of ULBP2 in BC is still to be explored. Crosstalk between different classes of non coding RNAs (ncRNAs) is the current research hotspot. Our research group has recently highlighted a harmonic interplay between several microRNAs and long ncRNAs regulating the immunogenic profile of BC cells. However, the ncRNAs circuit regulating ULBP2 in BC has yet to be identified. Therefore, the aim of this study is to identify the ncRNA circuit regulating ULBP2 in BC cells.

Methods

BC patients were recruited (n=30). Bioinformatic analysis was performed. MDA-B-231 cells are cultured and transfected by different oligonucleotides using lipofection technique. RNA was extracted using biozol, reverse transcribed and amplified and quantified using q-RT-PCR. BC cells transfected with ncRNAs are functionally analyzed using MTT, colony forming and migration assays. Statistical analysis was done using GraphPad 9.0.

Results

In silico analysis revealed a potential targeting capacity of miR-17-5p to STAT3, H19. Moreover, miR-17-5p and H19 were found to target ULBP2. Screening showed an under-expression of miR-17-5p in BC tissues and an up-regulation of STAT3 and H19. miR-17-5p mimics and H19 siRNAs repressed BC hallmarks. Ectopic expression of miR-17-5p mimics resulted in a repression of STAT3, H19 and induction of ULBP2 levels. Knocking down of STAT3 repressed H19 levels and induced ULBP2. Consquently, H19 siRNAs induced ULBP2 levels.

Conclusions

This study showed miR-17-5p/STAT3/H19 as a vital regulatory axis for ULBP2 in BC cells. The current evidence also supports the nomination of miR-17-5p as a potential therapeutic option for BC patients counteracting the immune intolerance phenomena exhibited by BC cells.

Legal entity responsible for the study

German University in Cairo.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

Natural compounds are recently booming as promising immunomodulatory anticancer agents in several cancers including breast cancer (BC). Recently, our group has shown the anticancer activity of several phytoconstiuents through repressing several oncogenic signals. Yet, unraveling their regulatory effect at the immune synapse is still a virgin field. The immune suppressive tumor microenvironment (TME) represents the rate limiting step in the success of any immunotherapeutic approach. Thus our aim is to identify a natural compound to trim the immune suppressive TME through its regulatory effect on a recent class of complex regulators known as competing endogenous RNAs. Ursolic acid (UA) is a phytoconstituent that is abundant in rosemary. Its anticancer activity has been confirmed. Yet, the molecular mechanisms behind it is still unraveled. In this study, we intended to investigate the anti-cancer and immunological effects of UA on BC through the regulation of a pool of ceRNAs and immune suppressive cytokines at the TME of BC cells.

Methods

Computational target prediction analysis was performed. UA was isolated from rosemary. MDA-MB-231 cells were treated with different concentrations of UA (20-100μM). IC50 value was calculated using dose response curve. MTT, colony forming assay and Scratch assay were performed. Total RNA was extracted and quantified by qRT-PCR. IL-10 anf TNF-α Elisa Kits were used.

Results

In-silico, miR-17-5p, MALAT-1 and H19 were found to target the immune suppressive cytokines IL-10 and TNF-α with high binding scores. UA resulted in a dose- and time-dependent repression of several BC hallmarks such as cellular viability, clonogenicity and migration capacity. Molecularly, UA was found to alter the ceRNAs network through inducing miR-17-5p and MALAT1 levels in treated cells compared to vehicle control BC cells. Yet, H19 was found to be repressed. At the Immune synapse, UA was found to repress IL-10 and TNF-α mRNA and protein levels.

Conclusions

This study crystallizes a novel mechanism of Rosemary and UA as promising immunoregulatory anticancer agents at the BC immune synapse through altering H19/MALAT-1/miR-17-5p circuit and the immune suppressive cytokines: IL-10 and TNF-α.

Legal entity responsible for the study

German University in Cairo.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype. At present, TNBC patients do not have an approved targeted therapy. Therefore, patients primarily depend on systemic chemotherapy that has inevitable detrimental side effects and show inadequate therapeutic outcomes leading to a high mortality rate. Hence, there is an urgent need to develop targeted therapies for the TNBC population. Emphasizing new nanotherapeutics approaches to combinational therapy could be an effective alternative tactic.

Methods

We designed a self-assembly strategy to design a highly biocompatible organic polyaniline (PANi) smart polymer nanotherapeutic (NPs) doped hyaluronan (HA) converting the PANi emeralidine base (EB) to emeralidine salt (ES) for strong near infrared (NIR)-mediated photothermal cancer treatment (PTCT). Therefore, smartly designed NPs at 808 nm exhibited a high extinction coefficient 8.23 x 108 M^-1 cm^-1, and adequate photothermal conversion efficiency (PCE) (η=41.6 %) made it an efficient photothermal agent (PTA), highly beneficial for selective CD44-mediated photothermal ablation of TNBC tumors. Furthermore, we have co-encapsulated imiquimod (R837-Toll-like receptor 7 agonist) immunoadjuvant molecules (HA-PANi/R837 NPs) to trigger a strong immune response against post-PTCT tumor.

Results

Encouragingly, targeted HA-PANi/R837 NPs selectively destroyed the tumor under NIR-illumination then released tumor-associated antigens. PTCT also triggered R837 release for the unprecedented role NPs-based immune therapy producing immunological cell death (ICD) of residual tumor cells, efficiently protecting the mice from tumor relapse and metastasis.

Conclusions

The specific high-performance of tumor-targeting homotypic ablation in targeted TNBC models was achieved without noticeable adverse effects on normal cells. Therefore, our smart biocompatible potential nanoplatform could serve as a photothermal immunotherapy modality for future utilization of chemotherapeutics-free clinical treatment. Thus, photothermally activated ICD has the greatest potential of dual-modal cancer therapy, preventing future relapse by the activation of the immune system to recognize and kill the tumor cells.

Legal entity responsible for the study

The author.

Funding

Department of Biotechnology, Government of India.

Disclosure

The author has declared no conflicts of interest.

Background

MiRNAs have been thoroughly studied for their vast roles in oncology, however, recent immune-related miRNAs have been recognized as prospective modulators in the tumor immune surveillance process. The high immunogenicity of TNBC along with its stubbornness towards effective targeted therapy shifted therapeutic interests towards immunotherapy. Accordingly, a promising approach would be targeting the immune suppressive tumor microenvironment (TME). Such an approach could be manifested in a dual manner through blocking prominent checkpoint proteins and regulating Natural Killer (NK) cells and thus modifying the TME. MiR-939-5p is a scarcely explored miRNA especially in TNBC. Therefore, our aim is to identify the expression profile of miR-939-5p in BC tissues and the role it plays in tumor suppression as well as the immunomodulatory role on checkpoint proteins and NKG2D ligands in TNBC.

Methods

Breast biopsies were collected from 40 BC patients. MDA-MB-231 cells were cultured and transfected with different oligonucleotides. Total RNA was extracted and quantified by qRT-PCR. Cellular viability, colony forming ability and migration were measured using MTT, colony forming and scratch assays, respectively.

Results

MiR-939-5p was down-regulated in TNBC tissues compared to normal tissues and other BC subtypes. Functionally, forced expression of miR-939-5p ensued a decline in cellular viability, colony forming ability and migration capacity of MDA-MB-231 cells thus highlighting miR-939-5p as a tumor suppressive miRNA in TNBC. Immunomodulation wise, miR-939-5p overexpression resulted in down-regulation of the checkpoint protein PD-L1 as well as upregulation of the shedded NKG2D ligands MICA/B in TNBC cells. Lastly, drastic repression of the immune inhibitory cytokines, TNF-α and IL-10 was detected upon miR-939-5p upregulation.

Conclusions

The present study categorized miR-939-5p as a tumor suppressing miRNA under expressed in TNBC patients and cell lines. Furthermore, it institutes the first major stride in unraveling the immunomodulatory role of miR-939-5p in advancing NK cells cytotoxicity and trimming TME in favor of TNBC eradication, thus proposing miR-939-5p as a novel therapeutic module in TNBC.

Legal entity responsible for the study

German University in Cairo.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

The hot tumor nature of triple-negative breast cancer (TNBC) tumors serves as a key player in the prognosis of such patients. Giving a strong rational to support the immune-based therapeutic approaches for this hard-to-treat group of patients. Immune checkpoint blockades (ICBs) have been extensively studied especially PDL1/PD1 blockers. Yet, the number of resistant cases started to escalate. Therefore, it became crucial to probe for molecular engines regulating PDL1 expression. Currently, unveiling novel networks between non-coding RNAs (ncRNAs) and their roles in regulating TNBC has become a research hotspot. Our group has recently reported that miR-17-5p represses the immune suppressive IL-10 production. However, its role in regulating PDL1 is still unprobed in TNBC. CCAT1 is an oncogenic long ncRNA. CCAT1 acts as a sponge for several tumor suppressor miRNAs affecting it is downstream targets. Therefore, the aim of this study is to investigate part of the interactive ncRNA network regulating PDL1 in TNBC patients.

Methods

BC patients (n=35) were recruited. Bioinformatics analysis was executed. TNBC cell lines were cultured and transfected with different oligonucleotides using HiPerFect Transfection Reagent. Total RNA was extracted, reverse transcribed and quantified using qRT-PCR. Cellular viability, colony forming ability and migration were measured using MTT, colony forming and scratch assays, respectively.

Results

In-silico analysis revealed that PDL1 is a potential target for miR-17-5p and CCAT1. miR-17-5p was found to be under-expressed in PDL1⁺/CCAT⁺ TNBC tissues compared to its normal counterparts. Ectopic expression of miR-17-5p and/or knocking down of CCAT1 resulted in a prominent reduction PDL1. On the other hand, a mutual interactive loop was observed between miR-17-5p and CCAT1 in TNBC cells; miR-17-5p mimics repressed CCAT1 levels while CCAT1 siRNAs resulted in an induction of miR-17-5p levels. Functionally, CCAT1 siRNAs and miR-17-5p mimics markedly halted TNBC hallmarks.

Conclusions

This study highlights a novel ncRNA circuit regulating PDL1 in TNBC patients. Thus, targeting CCAT1/miR-17-5p axis might provide a potential solution in overcoming the ICBs resistance arises in some TNBC patients.

Legal entity responsible for the study

German University in Cairo.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

Lung squamous cell carcinoma (LUSC) patients suffer from less targetable onco-drivers and potentially acquire clinical benefit from immune checkpoint inhibitors (ICIs). DNA content aberrations contribute to genomic instability and are initial events of tumorigenesis. We established a non-diploidy related prognostic molecular signature (NPMS) based on differentially expressed genes (DEGs) and investigated the immune features of it.

Methods

We conducted a retrospective analysis based on data downloaded from TCGA database. By integrating CNA and SNV via ABSOLUTE algorithm, we obtained the DNA ploidy status and divided patients into near-diploid and non-diploid group. DEGs were selected and gene functional enrichments were carried out. NPMS was established by all subset multivariate Cox regression to further stratify patients and validated by integrative analysis of GSE73403, GSE41271, GSE4212. Gene sets enrichment analysis was applied to further figure out the mechanism behind NPMS.

Results

Non-diploidy coincided with higher TMB and intratumor heterogeneity. Functional enrichment indicated that DEGs mainly participated in genomic instability. NPMS containing HOXB5, TINAGL1, POLR3GL, APOB, FABP6, SCARF1 was established. Patients with low score had better OS than those with high score (HR 0.60, 95%CI 0.45-0.79, p-value=0.000321). This was validated in the GEO cohorts (HR 0.57, 95%CI 0.37-0.88, p-value=0.0121). Dysregulation of DNA repair and cell cycle checkpoints were higher enriched in high score group. The immune phenotype of high score tended to be "immunologically hot", which was characterized as high density but low activity immune cells infiltrated, accompanied by expression of higher immunosuppressive factors, such as PD-1, CTLA4, IDO1. Meanwhile, the upregulation of IFN-γ signaling pathway, lipid alternation and the high correlation between them were observed.

Conclusions

High NPMS score corresponded with an "immunologically hot" feature thereby led to shorter OS. The crosstalk of upregulated IFN-γ signaling and aberrations of tumor metabolism mignt participate. It's rational to deduced that patients with high score might be the potential ICIs benefited subpopulation.

Legal entity responsible for the study

Z. Hu.

Funding

This work was supported by Horizontal Scientific Research Project of China-Japan Friendship Hospital (Grants No. 2018-HX-26).

Disclosure

All authors have declared no conflicts of interest.

Background

Use of immune checkpoint inhibitors in cancer therapy has significantly improved response and survival rates of patients with various cancer types including NSCLC. However, beneficial therapeutic effects are not achieved in all patients and predictive as well as prognostic biomarkers associated with response at a timepoint are often failing when outcomes such as progression free survival (PFS) or overall survival are applied. Yet, the use of novel tools oriented towards precision medicine like high-throughput data independent acquisition mass spectrometry (DIA-MS) could address some of these problems.

Methods

Proteins from plasma samples were extracted and analyzed by capillary flow liquid chromatography coupled to DIA-MS. Proteins were identified and quantification was done with Spectronaut^TM (Biognosys). Univariate statistical approaches were used to identify significantly changing proteins based on patient response status and progression free survival. Relationships between proteins identified as significant and common for both outcomes were analyzed further using publicly available bioinformatics tools.

Results

125 plasma samples from late-stage NSCLC patients treated with immunotherapy regimens - 75 baseline and 50 after 8-weeks treatment - were analyzed and more than 850 proteins were quantified. Protein signatures associated with response to anti-PD-1 treatment were combined with signatures associated to PFS. In total, 12 common proteins were identified which exhibited long lasting systemic changes on the proteome level. Most of the identified candidates had prognostic characteristics. However, one candidate - TMEM198 (Transmembrane Protein 198), showed both predictive and prognostic capabilities. TMEM-198 is a potential novel biomarker with known association to SWI/SNF (SWItch/Sucrose Non-Fermentable) chromatin remodeling complex and REST (RE1 silencing transcription factor) which acts as an oncogene or cancer suppressor.

Conclusions

High dimensional proteomic data can provide dynamic information which is tightly related to clinical features. Here we presented a way how such data can be used for selecting biomarker candidates across multiple clinical outcomes.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

K. Sklodowski, V. Dozio, A. Lanzós, K. Beeler: Full/Part-time employment: Biognosys AG. All other authors have declared no conflicts of interest.

Background

Immune check-point inhibitors (ICPi) are routinely used in cancer treatment in the United Kingdom and are associated with immune-related adverse events (irAE). Most patients with autoimmune disease (AID) are often excluded from clinical trials investigating ICPi, due to hypothetical increased risks of AID flare or severe irAE, but these risks have never been conclusively verified. We present experience using ICPi at a UK tertiary cancer centre, comparing the irAE profile and efficacy in patients with pre-existing AID to those without.

Methods

This was a single centre retrospective analysis of electronic records of solid tumour patients who received treatment with an ICPi from January 2017 to December 2020. From a total of 562 patients, 15 had pre-existing AID. These were compared to those without AID in a 1:3 matched cohort for tumour-type and treatment.

Results

The AID cohort had inflammatory bowel disease (N=5), psoriasis (N=3), rheumatoid arthritis (N=2), polymyalgia rheumatica (N=2), autoimmune endocrinopathies (N=4), lupus (N=1) and sarcoidosis (N=1). 3 patients had 2 co-existing AID. All 15 patients had well controlled AID. 9 were on hormone replacement, immunosuppressive (prednisolone <10mg, n=2, mesalazine, n=1), or topical agents. 6 were not on AID treatment. The table below lists patient demographics, irAE and outcomes. 3 out of 15 AID patients had irAE possibly linked to their underlying AID (1 psoriasis, 1 RA, 1 colitis; no G3 or higher). No significant differences were found in the number of G3 or higher irAE, treatment discontinuation due to irAE, response to treatment, or survival between cohorts. Table: 31P

Patient demographics, irAE and outcomes

Conclusions

Exacerbation of pre-existing AID was infrequent and did not result in severe toxicity. Patients with well-controlled AID could be considered for entry in clinical trials involving ICPi.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

FGFR2 molecular changes was observed in gastric cancer at a frequency of 4-15% and associated with shorter progression-free survival (PFS) and overall survival (OS). Alofanib (RPT835) is a novel selective inhibitor that binds allosterically to the extracellular domain of FGFR2.

Methods

The aim of this phase Ib study was to determine dose-limiting toxicities (DLTs), maximum tolerated dose (MTD), safety, preliminary efficacy and pharmacokinetics (PK) of alofanib administered intravenously daily for 5 days weekly. Patients with advanced or metastatic gastric adenocarcinoma resistant to standard therapy were enrolled in 5 dose levels: 50, 100, 165, 250, and 350 mg/m2, using a 3 + 3 design.

Results

To date, 13 patients have been enrolled in the trial. The MTD was not reached. All patients have not experienced any DLT within the 28-day DLT-assessment window. Intravenous alofanib was safe. There were no correlations between dose level and toxicity. Three grade 3 adverse events (ALT/AST increased at 50 mg/m2, diarrhea at 165 mg/m2, and hyponatremia at 350 mg/m2) were reported. One patient discontinued treatment due to drug related grade 3 uncontrolled diarrhea. Grade 1-2 adverse events included fatigue, diarrhea, nausea, anemia, thrombocytopenia, increased alkaline phosphatase, and reactions immediately after intravenous injections (facial flushing, dizziness, weakness, sweating, and sinus tachycardia). Grade 1 hyperphosphatemia was founded in 25% of cases. Of the 12 assessed patients, 1 (8%) partial response at 50 mg/m2 and 8 (67%) stable diseases at 50-250 mg/m2 were recorded. After a median follow-up of 4.5 months, the median PFS and OS was not reached. PK parameters have increased with dose. PK values (Cmax, AUC, and t1/2) did not correlate with response, PFS and OS (all P>0.1).

Conclusions

Administration of alofanib by intravenous route as single agent was safe and demonstrated promising antitumor activity in heavily pretreated patients with metastatic gastric cancer. The MTD has not been reached up to 350 mg/m2. PK profiles did not correlate with toxicity and efficacy of alofanib. The study is ongoing.

Clinical trial identification

NCT04071184.

Legal entity responsible for the study

Ministry of Health, Russian Federation.

Funding

Skolkovo Foundation.

Disclosure

N. Dragun, D. Reznikov, E. Gavrilova: Full/Part-time employment: Ruspharmtech LLC. All other authors have declared no conflicts of interest.

Background

This study was conducted to evaluate the tolerability of selinexor and to establish the recommended phase II dose in an Asian population.

Methods

We investigated 4 dosing schedules. Schedule 1: twice a week dosing at 40mg/m2 in a 28-day cycle. Schedule 2: once weekly at 50mg/m2 in a 28-day cycle. Schedule 3: twice a week dosing at 40mg/m2, 2 out of 3 weeks in a 21-day cycle. Schedule 4: 3 times a week dosing at 20mg/m2 in a 28-day cycle. Adverse events were graded with the NCI Common Terminology Criteria for Adverse Events v 4.03. Pharmacodynamic assessments: nuclear-cytoplasmic localisation of p27, XPO1 cargo proteins including ki67, apoptag, cleaved caspase 3, and c-myc pre and post selinexor. Pharmacokinetic assessments were conducted in 19 individuals at doses between 40-60mg/m2.

Results

The safest dosing was 40mg/m2 (equivalent 60mg) with no DLTs. The schedule with a 1-week drug holiday was the most tolerable for our patients. G3 adverse events included fatigue (8%), hyponatremia (23%), vomiting (5%), thrombocytopenia (5%), anemia (2%). Selinexor had a rapid oral absorption with median T_max of 2 h with no PK accumulation after multiple doses of tested regimens. Complete responses were noted in 2 lymphoma patients. Partial responses were noted in 3 DLBCL patients, 1 Hodgkins lymphoma and thymic carcinoma. Stable disease was seen in 12 colorectal cancer patients, 3 pancreatic cancer patients, 2 thymic carcinoma patients, 1 each for ovarian cancer, hepatocellular carcinoma, cholangiocarcinoma, lung cancer, breast cancer, tongue cancer and grey zone lymphoma. An exploratory analysis on 36 colorectal cancer cases with known RAS pathway mutational status showed a median progression free survival of 86 vs 50 days, p=0.09, log-rank in RAS mutant compared to wildtype patients.

Conclusions

Selinexor is tolerated by Asian patients at 60mg twice a week. A 1-week drug holiday was needed as our patients could not tolerate the continuous dosing regimens because of persistent G3 fatigue, anorexia and hyponatremia. Exploratory analyses of colorectal cancer patients alluded to a clinical benefit that selinexor may have significant activity in RAS pathway activated tumors.

Legal entity responsible for the study

The authors.

Funding

This study was supported by grant funding from Karyopharm and the National Medical Research Council Singapore, National Research Foundation Singapore and the Singapore Ministry of Education under its Research Centers of Excellence initiative.

Disclosure

H. Xu: Shareholder/Stockholder/Stock options: Karyopharm. All other authors have declared no conflicts of interest.

Background

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease harbouring the most frequent hotspot mutations in the differentiation genes. Disruption of epithelial differentiation acts as a primary driver of HNSCC development and correlates with a poor patient's prognosis. To date, in addition to the non-selective conventional HNSCC treatments, such as chemotherapies and surgeries, the only FDA-approved targeted therapy Cetuximab, an epidermal growth factor receptor inhibitor, has a low response rate with considerable toxicity. Therefore, determining whether functional differentiation can serve as a molecular vulnerability and/or a predictor of targeted therapy in HNSCC is an area of valuable clinical need.

Methods

A multi-omic approach integrating whole-genome and whole-transcriptome sequencing with drug sensitivity screening was employed in tumours from a spontaneous HNSCC murine model, HNSCC patient’s tumours, and established human cell lines to reveal potential predictive biomarkers for targeted therapies. CRISPR-Cas9-mediated gene knockout and activation validated candidate predictors in HNSCC cell lines treated with inhibitors of the PI3K/mTOR, c-MYC and STAT3 pathways, the key signalling in HNSCC oncogenesis.

Results

We identified a novel Grainyhead-like 3 - Filaggrin (GRHL3-FLG) differentiation axis as a predictive biomarker to targeted therapy response in HNSCC. A subset of HNSCC with functional GRHL3-dependent differentiation was the most sensitive to inhibitors of PI3K/mTOR, c-MYC and STAT3 signaling. Furthermore, we identified the GRHL3 transcriptional target gene FLG as a novel tumour differentiation gene and, more importantly, stratified HNSCC subsets as treatment-resistant based on their FLG mutational profile. Moreover, the loss of FLG in sensitive HNSCC cells resulted in a dramatic resistance to targeted therapies while the GRHL3^hi-FLG^wt signature predicted a favourable patient's prognosis.

Conclusions

Functional differentiation (GRHL3^hi-FLG^wt) provides the first example of differentiation-dependent therapy response and establishes a rationale for clinical investigation of differentiation-paired targeted therapy that may improve outcomes in HNSCC and other heterogeneous cancers.

Legal entity responsible for the study

The authors.

Funding

Australian National Health and Medical Research Council, Victorian Cancer Agency.

Disclosure

All authors have declared no conflicts of interest.

Background

Patient-reported adverse events may be a useful adjunct for assessing a drug’s tolerability in DPOT. A review of DPOT registered on clinicaltrials.gov showed increasing use of PROs over time, but overall use remained limited. Little is known about the reasons for limited PRO use. We conducted a global online survey of key stakeholders to understand attitudes towards PROs in DPOT.

Methods

A 35-question survey of clinicians, trial managers, statisticians, funders and regulators involved in DPOT in hospitals, academia or industry distributed via professional bodies. Questions focussed on prior experience of designing, conducting and reporting DPOT with PROs, attitudes towards benefits and barriers to PRO use and their potential role in defining tolerable doses. Adaptive questioning was used. No identifiable data was collected.

Results

112 responses from 15 September to 30 November 2020; 103 trialists [48 clinicians (42.9%), 38 statisticians (34.0%), 17 trial managers (15.2%)], 7 regulators (6.3%) and 2 funders (1.8%). Most had worked in DPOT for 6-10 years (35, 31.3%). Most worked in the United Kingdom (65, 58.0%) or United States (22, 19.6%). Most trialists had no prior experience designing (73, 70.9%), conducting (52, 50.5%) or reporting (88, 85.4%) PROs in DPOT. Respondents strongly agreed/ agreed that PROs could identify new toxicities (75, 67.0%), provide data regarding frequency (86, 76.8%) and duration (81, 72.3%) of toxicities, especially in agents with moderate, chronic or delayed toxicities (77, 68.8%). Respondents agreed that PROs should be reviewed when making dose-escalation decisions (61, 54.5%), when determining the maximum tolerated dose (63, 56.3%) and recommended phase II dose (76, 67.9%). Top 5 concerns were: lack of guidance regarding PRO selection (73/103, 70.9%), missing PRO data (79/112, 70.5%), overburdening staff (68/103, 66.0%) or patients (57/103, 55.3%), and analysis and publication (58/112, 51.8%).

Conclusions

Key stakeholders reported minimal experience collecting and analysing PROs in DPOT. However, there was broad support for using PROs to inform selection of tolerable doses. Guidelines are needed to standardise selection, analysis and reporting, and increase efficiency of PRO collection in DPOT.

Legal entity responsible for the study

Christina Yap.

Funding

Has not received any funding.

Disclosure

A. Minchom: Honoraria (institution), Advisory/Consultancy: Merck; Honoraria (institution), Advisory/Consultancy: Faron; Honoraria (institution), Advisory/Consultancy: Novartis; Honoraria (institution), Advisory/Consultancy: Bayer; Honoraria (institution), Advisory/Consultancy: Janssen. All other authors have declared no conflicts of interest.

Background

The canonical BRAF V600E (class I) mutation is a potent oncogene which is uniquely active as a RAS-independent monomer, and which has been successfully targeted by several FDA-approved inhibitors. While active against monomeric BRAF V600E, these first generation BRAF inhibitors induce paradoxical activation of RAS-driven BRAF dimers in cells expressing wild-type RAF, and this can lead to secondary malignancies. More recently, numerous non-canonical BRAF oncogenic mutations including BRAF-fusions have been described as oncogenes that drive RAS-independent (class II) or RAS-dependent (class III) dimers. These non-canonical dimeric BRAF oncogenes are resistant to the first-generation drugs, effective only against the monomeric BRAF V600E mutation. Discovery of an inhibitor directed against the family of dimeric BRAF oncogenic mutations which avoids paradoxical activation is a major unmet need.

Methods

We applied our proprietary Mutation-Allostery-Pharmacology (MAP) platform technology developed by Black Diamond Therapeutics to identify and validate a group of previously uncharacterized non-canonical oncogenic class II and class III BRAF mutation clusters. We further demonstrate that this ensemble of both novel and previously validated non-canonical oncogenic BRAF mutants can form the basis of a differentiated drug discovery program aimed at identifying small molecules that potently and selectively target this family of dimeric BRAF mutations.

Results

Herein, we describe a series of small molecule inhibitors with potent anti-proliferative activity directed against tumor cells harboring dimer-inducing BRAF oncogenic mutations and which are devoid of paradoxical RAF activation. Leading exemplars of BDTX compounds are orally available inhibitors that achieve target engagement of BRAF dimer oncogenes in vivo and robust anti-tumor efficacy in xenograft models in mice.

Conclusions

These data support continued development of rationally designed molecules targeting a broad range of non-canonical BRAF dimer-promoting mutations to extend the prospect of precision medicine in patients with BRAF mutant tumors.

Legal entity responsible for the study

Black Diamond Therapeutics.

Funding

Black Diamond Therapeutics.

Disclosure

Y-C. Han, P.Y. Ng, R. Schulz, S.N. Yang, A. Lelo, L.S. Ogawa, M. O'Connor, N. Ishiyama, I. Jewett, D. Romashko, A. Salomatov, S. Thakur, S. Smith, E. Buck, C. Roberts, M. Lucas, T-A. Lin: Shareholder/Stockholder/Stock options, Full/Part-time employment: Black Diamond Therapeutics.

Background

Cancer is the second leading cause of death globally and the rise in treatment-resistant cancer cells make treatments for cancer difficult. Several studies have showed the importance of cationic peptides as a potential candidate for anticancer therapeutics. More recently, scorpion venom has emerged to be an important source of peptide candidates targeting human diseases including cancer. The peptide of mauripon produced from Androctonus mauritanicus scorpion, showing a promising candidates anticancer property was explored.

Methods

A list of 34 overlapping short peptides (MDs) was generated by taking 15 amino acids from the N-terminal of mature mauriporin. Among these MDs, M1 with the most potent predicted anticancer activity with low toxicity and haemolytic activities, was selected for hydrophobic modification to produce a series of improved anticancer peptide (M1.1-M1.6). The in-vitro validation of antiproliferation activity of these M1.1-M1.6 peptides on different metastatic cancer cells was studied. The interaction of M1.1-M1.6 peptides with different anti-apoptotic Bcl-2 family proteins was investigated to elucidate its potential to induce apoptotic cell death.

Results

The selective antiproliferation activity of M1.2 (with Ala substitution) was observed by targeting only metatstatic cancer cells without interupting normal cells’ viability. As comparison to other peptides and apoptotic-inducer drug (venetoclax) in protein-peptide studies, M1.2 showed greatest binding efficacy towards Bcl-2, Bcl-xL and Mcl-1 thus inhibiting their anti-apoptotic functions. Moreover, the activation of p53-independent intrinsic apoptosis by M1.2 was observed by the activation of caspase-9 in p53-null and mutant cell lines, as well as low LDH leakage at 24 hours.

Conclusions

In this study, the hydrophobic modification in peptides resulted in an increased anticancer activity and induction of apoptosis that is distinct from the necrotic effect of parental mauriporin peptide. Together, this study provided insights about the development and design strategy of peptide fragment-based drug from animal venom, which can be further exercised in screening potential anticancer peptide targeting cancer.

Legal entity responsible for the study

The authors.

Funding

Taylor’s Research Grant Scheme.

Disclosure

All authors have declared no conflicts of interest.

Background

Matrix metalloproteinases are family of zinc dependent endoproteinases degrade the extracellular matrix, have been identified as poor prognosis markers and therapeutic targets for breast cancer patients. Effect of apigenin was observed on mRNA expression on different MMPs in MDA MB- 231 breast cancer cells including its effect on cancer cell migration and invasion.

Methods

MDA MB- 231 cells were cultured with different concentrations (0, 10, 20 and 40 μM) of apigenin for 48 hrs. mRNA expressions of MMP-1, MMP-3, MMP-7, MMP-9 amd MMP- 11 were analyzed by real time PCR. For cell migration wound healing assay was done at 0, 6, 24, and 48 h periods. To asses the cell invasion MDA MB-231 cancer cells were seeded on collagen based matrigel chamber and treated with 10, 20 and 40μM apigenin, whereas untreated cells considered as control. After 48hrs cells invaded through matrigel quantified by colorimetric method.

Results

Our results showed that MMP-1 and MMP-11 were significantly downregulated upto 0.36 folds and 0.55 folds respectively at 40 μM apigenin treatment in MDA MB-231 cells in comparision to cells without drug. In comparision to untreated cancer cells the MMP-3 gene expressions were 0.46, 0.27 and 0.12 fold in 10μM, 20μM and 40μM apigenin treated MDA MB-231 cells. The apigenin significantly decreased MMP-7 gene expression level upto 0.34 fold and 0.05 fold at 20 μM and 40 μM concentrations in MDA MB-231 cells. Apigenin significantly downregulated MMP-9 mRNA expression level in dose dependent manner in MDA MB-231 cancer cells as compared to untreated cells. Wound healing assay results exhibited that apigenin significantly reduce the migration of cancer cells. In cells treated with 20μM and 40μM apigenin 16.64% and 25.81% of the wound remains unresolved after 48 hrs. The cell invasion assay showed that apigenin significantly inhibited the migration of cells, 40 μM of apigenin showed only 73.52% invasion, as compared to the untreated cells.

Conclusions

In conclusion, apigenin reduced the mRNA expressions of MMP-1, MMP-3, MMP-7, MMP-9 and MMP-11 in MDA-MB-231 cells. Apigenin also inhibited the invasion and cell migration. Therefore, we suggest that apigenin may be used as a candidate drug for the inhibition of metastasis of human breast cancer.

Editorial acknowledgement

This is the part of my Ph. D. degree, done at Animal Biochemistry Division, ICAR- Indian Veterinary Reasech Institute, Izatnagar, Bareilly, U.P., India

Legal entity responsible for the study

Dr. Shweta Rajoriya.

Funding

ICAR-Indian Veterinary Research Institute, Bareilly, U.P., India (Indtitutional Funding).

Disclosure

The author has declared no conflicts of interest.

Background

We have previously reported that hormone-receptor positive (HR+) breast cancer patients with the heterogeneous distribution of ERα expression in tumor tissue had poor prognosis on tamoxifen treatment. There is strong evidence that the response to endocrine therapy involves molecular crosstalk between ERα and TGF-β signaling. In this study we investigated the TGF-β family member’s expression (TGF-β, TGF-βRI, and TGF-βRII) as well as their association with distribution pattern of ERα expression in predicting tamoxifen response and survival in HR+ patients.

Methods

Immunohistochemical staining for TGF-β, TGF-βRI, TGF-βRII, cyclin D1 and ERα was conducted for formalin-fixed paraffin-embedded specimens of 27 HR+ breast cancer patients with progression (distant metastases or relapse) and 95 patients with no progressive disease during adjuvant tamoxifen therapy. The primary endpoint was progression-free survival (PFS).

Results

Tamoxifen-sensitive tumors showed elevated TGF-βRI expression levels compared with tamoxifen-resistant tumors (p=0.030). TGF-βRI-positive tumors were more often associated with the homogeneous ERα expression (p=0.007). There was a significant correlation between the pattern of ERα expression and TGF-βRI expression (r=0.30; p=0.007) as well as cyclin D1 expression (r=0.46; p=0.026). TGF-βRI-negative tumors with the heterogeneous ERα expression were less sensitive to tamoxifen treatment than TGF-βRI-positives with the homogeneous ERα expression pattern (p=0.049). In addition, there was a significantly higher progression rate for TGF-βRI-negative patients with whose tumors demonstrated heterogeneous ERα staining (log-rank p=0.019).

Conclusions

These data indicate that the expression TGF-βRI is linked with the distribution pattern of ERα expression in the tumor. TGF-βRI and the pattern of ERα expression may potentially be useful biomarkers to predict the effectiveness of tamoxifen adjuvant treatment in HR+ breast cancer.

Legal entity responsible for the study

The authors.

Funding

The Russian Scientific Foundation, grant #19-75-30016.

Disclosure

All authors have declared no conflicts of interest.

Background

Photopharmacology is actively developing and bringing new opportunities for the design of chemotherapy drugs. Several photoswitchable active compounds have been developed to inhibit microtubules in cancers. Analogues of combretastatin A-4 (CA4) are the most promising representatives of α-tubulin inhibitors. It is now clear that monotherapy with such photoswitchable agents has very limited applications in oncology. Aim of the study: to discover effective combinations of CA4 analogues with chemotherapy drugs and metformin.

Methods

The MTT test was used to evaluate A431 cell viability. Analogs of combretastatin A-4 based on quinazoline stilbenes were obtained by an alternative method - the Wittig reaction by condensation of the corresponding aldehydes with quinazoline phosphorus ylides in good yields (65-80%). The latter were synthesized by the reaction of quinazoline chloromethyl derivatives with triethyl phosphite. All quinazoline analogs of CA-4 were obtained in the e-form, which is confirmed by the constant of the spin-spin bond of olefinic protons (15.8-16.2 Hz).

Results

Screening of new CA4 analogues showed that hit compounds can reduce the viability of A431 cells with IC50 values below 10 μM. Irradiation of hit compound ma-6595 ((E) -2- (3,4,5-trimethoxystyryl) quinazolin-4 (3H) -one) at λ=365 nm resulted in isomerization giving Z-isomer (ratio of E and Z isomers was 1.25:1); irradiation of ma-6595 caused an increase in antiproliferative activity by 5.4 times. Several effective combinations of ma-6595 photoproduct with chemotherapy drugs have been identified. ma-6595 photoproduct enhanced the effect of cisplatin and 5-fluorouracil on A431 cells. The strongest synergistic effect on cell viability was found for the combination of 5 mM metformin with 1 μM ma-6595 photoproduct (combination index <1).

Conclusions

A new highly active series of CA4 analogues have been provided. The hit compound enhanced the effect of cisplatin and 5-fluorouracil on epidermoid carcinoma cells. Moreover, the combination of the CA4 analogue with the cancer cell metabolism inhibitor metformin was very effective. Novel analogues of CA4 can be used to develop combination therapies and to reduce the dosage of chemotherapy drugs.

Legal entity responsible for the study

The authors.

Funding

The Russian Science Foundation (project 18-13-00308, chemistry) and Minobrnauki of Russia (project 075-15-2020-789, experiments with cisplatin).

Disclosure

All authors have declared no conflicts of interest.

Background

Sorafenib is the only therapy to treat hepatocellular carcinoma (HCC), yet offers limited survival benefits due to drug resistance. Advanced oncotherapeutic research demands a thorough insight into pathological cascades involved in the progression of preneoplastic lesions to HCC. Hence, target mining to unearth key genetic players of HCC followed by screening drug databases against the identified targets will be a rational way forward.

Methods

GSE6764 gene expression profile dataset encompassing microarray data of 10 normal, 10 cirrhosis, 17 dysplastic nodules, 18 early and 17 advanced HCC was analyzed using GEO2R. Subsequently, a protein-protein interaction network was constructed using the Search Tool for the Retrieval of Interacting Genes and visualized through Cytoscape. Crucial targets were identified based on the overall survival (OS) and hazard ratio (HR > 1) of hub genes from the Kaplan-Meier plotter. The identified targets were screened against all FDA-approved drugs by molecular docking studies through extra precision mode (XP) in the Schrodinger drug design suite. Further, the free energy of binding of shortlisted drugs was evaluated by MM/GBSA analysis.

Results

STAT1 and MX1 are substantially overexpressed in cirrhosis while, CCL19 and IL7R are significantly downregulated between dysplastic nodule and cirrhosis. HMMR overexpression is linked with poor prognosis in the evolution of dysplastic nodule to early HCC. Furthermore, overexpression of pathological hallmarks of poor prognosis such as CDK1, CDC20, BUB1, MAD2L1, CCNB2, CENPF, TPX2, TOP2A and PBK was identified in the furtherance from early and advanced HCC. Based on OS with significant HR, CDC20 was shortlisted and subjected to molecular docking and MM-GBSA analysis. Labetalol, a beta-blocker was spotlighted as a hit due to its highest docking score of -7.075, ΔG value -54.08 kcal/mol alongside its stable and stronger ligand-protein complex.

Conclusions

This study reveals a series of key cross-talk genetic underpinnings from pre-neoplastic lesions to HCC and suggests the pertinence of labetalol as a potential repurposable drug in the treatment for HCC.

Legal entity responsible for the study

Gouri Nair.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.