Mechanisms Of Treatment Resistance Research Articles

Abstract 1837: MUC1 confers sensitivity to STAT-3 inhibitor napabucasin in pancreatic ductal adenocarcinoma cells

Abstract Introduction: Pancreatic cancer is the third leading cause of cancer-related deaths in the US and 95% of pancreatic cancers are Pancreatic Ductal Adenocarcinomas (PDACs). Napabucasin is a small molecule inhibitor that targets the signal transducer and activator of transcription 3 (STAT-3) pathway in cancer stem cells. Napabucasin in combination with nab-paclitaxel and gemcitabine, was under phase III clinical trials for gastroenterological cancers including Pancreatic Cancer. The trials have been stopped recently due to futility. Therefore, it is important to find subpopulations of tumor cells that are likely to respond better to Napabucasin treatment alone or as a combination. Mucin1 or MUC1 is a transmembrane glycoprotein found in almost all glandular epithelial cells, and is overexpressed and aberrantly glycosylated in greater than 80% of PDACs. The tumor form of MUC1 is associated with poor prognosis. In PDAC cells, MUC1 expression is critical for tumor cell survival, oncogenic signaling, metastasis, and conferring stemness. Phosphorylated STAT-3 is known to regulate MUC1 expression. Thus, we hypothesize that 1) the STAT-3-MUC1 survival pathway is constitutively activated in MUC1-high PDAC cells and 2) Napabucasin will be more effective in MUC1-high compared to MUC1-low PDAC cells. Methods: Human PDAC cell lines with varying levels of MUC1 (CFPAC, Capan 2, Capan 1, HPAF II, HS766T, BxPc3.MUC1 and BxPC3.Neo) and a normal pancreatic ductal epithelial cell line (HPDE) were treated with increasing doses of Napabucasin (0.05 uM, 0.1uM, 0.2uM, 0.4uM, 0.8uM, 1.6uM, 3.2uM, 6.4uM, 12.8uM and 25.6uM) for 48 and 72 hours. Following treatment, MTT and colony formation assays were performed to determine survival and growth respectively. Results: The cells with high MUC1, CFPAC and Capan 1 showed increased susceptibility to Napabucasin at lower doses (IC50 - 2.088uM and 0.371uM respectively) compared to cells with low MUC1, Capan 2 and HS766T (IC50 - 6.885uM and 7.464uM respectively). MUC1-high HPAF II cells were resistant even at high doses. Data further confirmed that the IC50 of Napabucasin was significantly lower for Bx.Pc3.MUC1 (1.86uM) compared to Bx.Pc3.Neo cells (23.19uM). MUC1-high expressing cell lines lost their colony forming potential at lower doses compared to MUC1-low expressing cell lines. To test if oncogenic signaling linked to MUC1 dimerization confers sensitivity to Napabucasin, the PDA cells were pre-treated with GO-203, a small molecule that inhibits dimerization of MUC1 which renders signaling through the MUC1 cytoplasmic tail non-functional. Conclusion: MUC1 induces susceptibility to Napabucasin in PDACs. Napabucasin has shown anti-tumor activity in preclinical models and early clinical trials of gastroenterological cancers. However, lack of specificity led to increased toxicities and decreased efficacy. It is important to identify and validate biomarkers that better identify patients who have upregulated stemness pathways. MUC1 is one such specific biomarker that could be incorporated in future clinical trials to help identify patient subpopulations that may most likely benefit from combination therapies with stemness-inhibiting drugs. Further study of the crosstalk between stemness signaling pathways is necessary to understand mechanisms of synergistic effects and treatment resistance. Citation Format: Mukulika Bose, Aabha Vora, Taylor Colleton, Pinku Mukherjee. MUC1 confers sensitivity to STAT-3 inhibitor napabucasin in pancreatic ductal adenocarcinoma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1837.

Abstract 232: Comprehensive metabolic profiling and vulnerabilities to metabolic inhibitors among small cell lung cancer subtypes

Abstract Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumor that constitutes approximately 14% of all lung cancers diagnosed in the United States. Despite recent advances in treatment options, recurrence is a major challenge and long-term survival remains poor underscoring the importance of investigating mechanisms of treatment resistance and therapeutic strategies. Interestingly, our laboratory has recently uncovered evidence for the classification of SCLC into distinct subtypes, each with unique gene expression patterns and therapeutic vulnerabilities. These subtypes are characterized by differential expression of the transcription factors ASCL1, NEUROD1, or POU2F3. A fourth subtype is negative for all three transcription factors and has prominent expression of immune genes, thus is termed the Inflamed subtype. These subtypes are associated with differences in drug sensitivity, biomarker signatures, and mechanisms of cellular growth. One facet of tumor biology that has not been explored across the subtypes, or in SCLC in general, is the metabolic mechanisms of energy generation. Although metabolic reprogramming from mitochondrial respiration to glycolysis is considered a major hallmark of cancer, increasing evidence suggests that mitochondrial or amino acid dependencies may also contribute to treatment resistance. Therefore, understanding the metabolic demand, particularly among the subtypes, is critical for the development of new therapies. To address this, gene set enrichment analysis was applied to 81 human SCLC tumors categorized by subtype, and revealed differential gene expression correlating to pathways such as fatty acid metabolism in POU2F3 and reactive oxygen species in Inflamed that was confirmed by reverse phase protein analysis (RPPA). Further, we evaluated nutrient requirements, glycolysis dependence, and mitochondrial function in human SCLC cell lines and found differences in glucose uptake and subsequent lactate generation between the subtypes suggesting that glycolysis inhibition may be effective in particular subsets. Based on this, we then tested the efficacy of PFK-158 (glycolysis inhibitor) on cellular proliferation and metabolic function in these cell lines, which revealed variations in susceptibility dependent on subtype classification. Together, this data serves to enhance the current understanding of SCLC mechanisms of growth and provides evidence for metabolic inhibition as a potential therapeutic opportunity. Citation Format: Kasey R. Cargill, Carl M. Gay, Robert J. Cardnell, You-Hong Fan, Qi Wang, Lixia Diao, Jing Wang, Lauren A. Byers. Comprehensive metabolic profiling and vulnerabilities to metabolic inhibitors among small cell lung cancer subtypes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 232.

Abstract 2998: Linear confined space migration induces treatment resistance and gene expression changes in glioblastoma multiforme

Abstract Background: Glioblastoma Multiforme (GBM), an aggressive brain cancer, is known for its treatment resistant recurrence and lethality. The diffuse migration and invasion of GBM cells commonly occurs along brain white matter tracts in linear confined spaces between axons and has significant clinical implications as these cells survive initial therapies forming the main source of recurrent tumors resistant to therapies. In this context, this project aims to analyze the treatment resistance mechanism associated with linear confined space migrating cells (LCSM) to devise a better treatment strategy for GBM. Methods: We used a combination of microfluidic technology (5 µM wide channels in PDMS block) emulating linear white matter tracts in brain and standard molecular analysis (confocal microscopy, high content imaging, western blot, and flow cytometry) to investigate LCSM cells compared to Standard Monolayer Culture cells (SMCs) for the mechanism of drug resistance. Both established and patient derived GBM cell lines were used in the study. Results: LCSM cells were more resistant to chemo-drugs (Temozolomide and Doxorubicin) than SMCs as observed in cell viability assays. Enhanced drug efflux was observed in LCSM cells compared to SMC cells indicating the activation of drug efflux pumps. Protein expression analysis indicated that ABC drug efflux transporters (ABCB1, ABCC1, ABCG2) were activated or enhanced at varying levels in different LCSM cells compared to SMCs. Pharmacological inhibition of the prominently expressed ABCG2 protein using KO143 resulted in enhanced cell death by chemo drugs, suggesting that chemo resistance in LCSM, at least in part, is conferred by ABCG2 or other efflux transporters. In addition, CD133 (a marker of treatment resistant stem cell population in GBM) and AQP4 (Aquaporin 4; pro-migratory marker in GBM) were also enhanced in LCSM compared to SMC suggesting their possible roles in treatment resistance. Conclusion: Preliminary studies reveal that the enhanced expression ABC transporters, prominently ABCG2, might be responsible for enhanced drug efflux and low therapeutic sensitivity in GBM- LCSM cells. The over expression of AQP4 and CD133, and differential expression of other genes suggest that more complex mechanisms might be involved, which are currently under investigation. Citation Format: Anish Babu, Patrick McKernan, Xue Cai, James Battiste. Linear confined space migration induces treatment resistance and gene expression changes in glioblastoma multiforme [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2998.

The Role of Liquid Biopsies in Pediatric Brain Tumors.

Early detection and serial therapeutic monitoring for pediatric brain tumors are essential for diagnosis and therapeutic intervention. Currently, neuropathological diagnosis relies on biopsy of tumor tissue and surgical intervention. There is a great clinical need for less invasive methods to molecularly characterize the tumor and allow for more reliable monitoring of patients during treatment and to identify patients that might potentially benefit from targeted therapies, particularly in the setting where diagnostic tissue cannot be safely obtained. In this literature review, we highlight recent studies that describe the use of circulating tumor DNA, circulating tumor cells, circulating RNA and microRNA, and extracellular vesicles as strategies to develop liquid biopsies in pediatric central nervous system tumors. Liquid biomarkers have been demonstrated using plasma, urine, and cerebrospinal fluid. The use of liquid biopsies to help guide diagnosis, determine treatment response, and analyze mechanisms of treatment resistance is foreseeable in the future. Continued efforts to improve signal detection and standardize liquid biopsy procedures are needed for clinical application.

Therapeutic Strategies for Overcoming Immunotherapy Resistance Mediated by Immunosuppressive Factors of the Glioblastoma Microenvironment.

Various mechanisms of treatment resistance have been reported for glioblastoma (GBM) and other tumors. Resistance to immunotherapy in GBM patients may be caused by acquisition of immunosuppressive ability by tumor cells and an altered tumor microenvironment. Although novel strategies using an immune-checkpoint inhibitor (ICI), such as anti-programmed cell death-1 antibody, have been clinically proven to be effective in many types of malignant tumors, such strategies may be insufficient to prevent regrowth in recurrent GBM. The main cause of GBM recurrence may be the existence of an immunosuppressive tumor microenvironment involving immunosuppressive cytokines, extracellular vesicles, chemokines produced by glioma and glioma-initiating cells, immunosuppressive cells, etc. Among these, recent research has paid attention to various immunosuppressive cells—including M2-type macrophages and myeloid-derived suppressor cells—that cause immunosuppression in GBM microenvironments. Here, we review the epidemiological features, tumor immune microenvironment, and associations between the expression of immune checkpoint molecules and the prognosis of GBM. We also reviewed various ongoing or future immunotherapies for GBM. Various strategies, such as a combination of ICI therapies, might overcome these immunosuppressive mechanisms in the GBM microenvironment.

Targeting NAD+ Biosynthesis Overcomes Panobinostat and Bortezomib-Induced Malignant Glioma Resistance

To improve therapeutic responses in patients with glioma, new combination therapies that exploit a mechanistic understanding of the inevitable emergence of drug resistance are needed. Intratumoral heterogeneity enables a low barrier to resistance in individual patients with glioma. We reasoned that targeting two or more fundamental processes that gliomas are particularly dependent upon could result in pleiotropic effects that would reduce the diversity of resistant subpopulations allowing convergence to a more robust therapeutic strategy. In contrast to the cytostatic responses observed with each drug alone, the combination of the histone deacetylase inhibitor panobinostat and the proteasome inhibitor bortezomib synergistically induced apoptosis of adult and pediatric glioma cell lines at clinically achievable doses. Resistance that developed was examined using RNA-sequencing and pharmacologic screening of resistant versus drug-naïve cells. Quinolinic acid phosphoribosyltransferase (QPRT), the rate-determining enzyme for de novo synthesis of NAD+ from tryptophan, exhibited particularly high differential gene expression in resistant U87 cells and protein expression in all resistant lines tested. Reducing QPRT expression reversed resistance, suggesting that QPRT is a selective and targetable dependency for the panobinostat-bortezomib resistance phenotype. Pharmacologic inhibition of either NAD+ biosynthesis or processes such as DNA repair that consume NAD+ or their simultaneous inhibition with drug combinations, specifically enhanced apoptosis in treatment-resistant cells. Concomitantly, de novo vulnerabilities to known drugs were observed. IMPLICATIONS: These data provide new insights into mechanisms of treatment resistance in gliomas, hold promise for targeting recurrent disease, and provide a potential strategy for further exploration of next-generation inhibitors.

Overcoming Platinum and PARP-Inhibitor Resistance in Ovarian Cancer.

Platinum chemotherapy remains the cornerstone of treatment for epithelial ovarian cancer (OC) and Poly (ADP-ribose) polymerase inhibitors (PARPi) now have an established role as maintenance therapy. The mechanisms of action of these agents is, in many ways, complementary, and crucially reliant on the intracellular DNA Damage Repair (DDR) response. Here, we review mechanisms of primary and acquired resistance to treatment with platinum and PARPi, examining the interplay between both classes of agents. A key resistance mechanism appears to be the restoration of the Homologous Recombination (HR) repair pathway, through BRCA reversion mutations and epigenetic upregulation of BRCA1. Alterations in non-homologous end-joint (NHEJ) repair, replication fork protection, upregulation of cellular drug efflux pumps, reduction in PARP1 activity and alterations to the tumour microenvironment have also been described. These resistance mechanisms reveal molecular vulnerabilities, which may be targeted to re-sensitise OC to platinum or PARPi treatment. Promising therapeutic strategies include ATR inhibition, epigenetic re-sensitisation through DNMT inhibition, cell cycle checkpoint inhibition, combination with anti-angiogenic therapy, BET inhibition and G-quadruplex stabilisation. Translational studies to elucidate mechanisms of treatment resistance should be incorporated into future clinical trials, as understanding these biologic mechanisms is crucial to developing new and effective therapeutic approaches in advanced OC.

Patient-Derived Urothelial Cancer Xenograft Models: A Systematic Review and Future Perspectives

BACKGROUND: Lack of appropriate models that recapitulate the diversity, heterogeneity, and tumor microenvironment of urothelial cancer (UC) is a limitation to preclinical models. Patient-derived xenograft (PDX) models are a promising tool to overcome some of these issues, and thus we present an up-to-date and comprehensive overview of UC PDX models to aid in their future use. OBJECTIVE: To provide an overview on methodology, applications and limitations as well as future perspectives on bladder cancer PDX models. METHODS: Literature searches using PubMed and Web of Science databases were performed for relevant articles according to the following MeSH terms: “urothelial carcinoma(s)” OR “urothelial cancer” OR “urothelial tumor” OR “bladder cancer(s)” OR “bladder carcinoma(s)” OR “transitional cell carcinoma(s)” AND “xenograft(s)” OR “xenotransplant” at December 6th, 2019. We followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. RESULTS: Of the 49 studies extracted, 41 studies after the year 2000 were finally analyzed. Published studies show that (1) UC PDX platforms retained the histology and genomic characteristics of the corresponding patient tumors. (2) UC PDX can be applied to ask various questions including to study the mechanisms of disease progression and treatment resistance, to develop novel drugs and biomarkers, as well as to potentially realize personalized drug selection. Recent topics of research using PDX have included the development of humanized mice as well as the use of 3D culture to complement some of the limitations of PDX models. CONCLUSIONS: UC PDX models serve as tools for understanding cancer biology, drug development and empowering precision medicine. The improvement of experimental systems using humanized mice to recapitulate the immune microenvironment of tumors will optimize UC PDX to study future questions in the field of immunotherapy.

Transcriptomic silencing as a potential mechanism of treatment resistance.

Next-generation sequencing (NGS) has not revealed all the mechanisms underlying resistance to genomically matched drugs. Here, we performed in 1417 tumors whole-exome tumor (somatic)/normal (germline) NGS and whole-transcriptome sequencing, the latter focusing on a clinically oriented 50-gene panel in order to examine transcriptomic silencing of putative driver alterations. In this large-scale study, approximately 13% of the somatic single nucleotide variants (SNVs) were unexpectedly not expressed as RNA; 23% of patients had ≥1 nonexpressed SNV. SNV-bearing genes consistently transcribed were TP53, PIK3CA, and KRAS; those with lower transcription rates were ALK, CSF1R, ERBB4, FLT3, GNAS, HNF1A, KDR, PDGFRA, RET, and SMO. We also determined the frequency of tumor mutations being germline, rather than somatic, in these and an additional 462 tumors with tumor/normal exomes; 33.8% of germline SNVs within the gene panel were rare (not found after filtering through variant information domains) and at risk of being falsely reported as somatic. Both the frequency of silenced variant transcription and the risk of falsely identifying germline mutations as somatic/tumor related are important phenomena. Therefore, transcriptomics is a critical adjunct to genomics when interrogating patient tumors for actionable alterations, because, without expression of the target aberrations, there will likely be therapeutic resistance.

Abstract IA19: Multiple uses of circulating tumor cells in lung cancer?

Abstract Liquid biopsies are increasingly being used in clinical studies as prognostic, predictive, and pharmacodynamics biomarkers, as surrogates of tumor response, to detect minimal residual disease after treatment with curative intent and to inform on mechanisms of treatment resistance and treatment switching. There is also steady progress in the development of liquid biopsies for early detection of cancers. The advantages of liquid biopsies are that they are minimally invasive and readily repeatable. The liquid biopsy that has been most widely adopted is circulating tumor DNA (ctDNA) as it is readily measured without specialist equipment in most molecular biology laboratories. Circulating tumor cells (CTCs) are technically more challenging and have yet to realize their full potential as biomarkers in clinic. However, CTC technology is constantly improving, and CTCs have several exciting applications beyond ctDNA that, once optimized, will assist personalized cancer medicine, including their use as cultures for real-time therapy testing and their utility, in some cancer types, to derive mouse models. I will discuss the use of CTCs from patients with lung cancer in this presentation. CTCs in both non-small cell lung cancer (NSCLC) and SCLC enumerated by CellSearch (EpCAM and cytokerin positive, CD45 negative) hold prognostic information. However, while CellSearch CTCs are scarce in NSCLC, most likely due to epithelial-to-mesenchymal transition (EMT) that leads to downrgulation of the EpCAM surface marker used to capture them for enumeration, they are prevalent in SCLC. Single CTC copy number analysis has led to the development of classifier that, with further validation studies, could predict response to chemotherapy alongside ctDNA analysis for therapy monitoring. We have exploited SCLC CTC prevalence to derive mouse models in immune-incompetent mice (termed CDX) that allow us to explore biology and test new therapeutics. I will also update on our research using our biobank of 46 CDX models, exploring inter and intratumoral heterogeneity. The most important aspect of this approach is the ability to generate pre- and post-therapy CDX models allowing interrogation of therapy resistance mechanisms and the biology of progressing disease in a tumor type where tumor evolution is rapid and where serial tumor biopsies are rarely obtained. I will also describe our approaches to study mechanisms of tumor dissemination, including vasculogenic mimicry and new models of brain metastasis. More recently, we are developing direct CTC cultures that may allow real-time therapy testing with reporting to the clinic. I will report on our NSCLC CTC studies within the UK TRACERx consortium to study tumor evolution. We have explored the potential of CTCs found in the pulmonary draining vein of stage I-IIIa patients at surgery with curative intent to predict risk of disease recurrence and shown in a case study that a lethal subclone that gives rise to metastasis 10 months later was identified in the pulmonary vein at surgery. In summary, this presentation will exemplify utility of CTCs in lung cancer that are distinct from but complement the implementation of ctDNA. Citation Format: Caroline Dive. Multiple uses of circulating tumor cells in lung cancer? [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr IA19.

Abstract IA09: The promise of liquid biopsy in the early detection of cancer: Hope or hype?

Abstract Noninvasive technologies and methodologies are urgently needed for a population-based early detection and screening. Liquid biopsy continues to draw the attention of patients and physicians alike. Liquid biopsies have the potential to help clinicians screen for disease, stratify patients to the best treatment, and monitor treatment response and resistance mechanisms in the tumor. A liquid biopsy can be used for molecular characterization of the tumor, and its noninvasive nature allows repeat sampling to monitor genetic changes over time without the need for a tissue biopsy. Liquid biopsy generally refers to measuring molecular changes, e.g., genomic, proteomic, metabolomics, epigenetic, and other “omics”-derived changes, in circulating tumor cells (CTCs), cell-free DNA (cfDNA), and exosomes. However, the real success of liquid biopsy continues to evade us. There are several challenges in detecting tumor-derived mutations in plasma or in other body fluids. This is partly due to the fact that the amount of circulating DNA usually is very low. Recent technological developments and the analytics being applied to liquid biopsies are now capable of reproducibly detecting mutations at very low allelic frequencies. Advances have also been made in droplet digital PCR (ddPCR), next-generation sequencing (NGS), beads, emulsion, amplification and magnetics (BEAMing), and amplification of refractory mutation system (ARMS), to name but a few. Ultimately, the choice of platforms and required detection limit will depend on the clinical sample being analyzed, as the most sensitive methods are reported to detect allelic frequencies of as little as 0.01%; if achieved in a liquid biopsy setting, this level of detection would be sufficient to gain knowledge of a tumor genotype and rule out the need for a tissue biopsy. Despite these challenges, the advantage of liquid biopsies over other traditional tissue-based methodologies is very promising, including the ability for longitudinal monitoring, which could help clinical oncologists gain a broader molecular understanding of the disease. Additionally, liquid biopsy could also facilitate cancer progression and treatment monitoring in follow-up visits. Cancer detection in liquid biopsy achieves repetitive sampling that is limited in histologic examination, and it provides a more economical and practical noninvasive detection compared to radiation-based imaging methods such as computed tomography. Furthermore, liquid biopsy may also shed light on tumor heterogeneity and may provide a more comprehensive picture of the genetic and proteomic alternations of the tumor. The National Cancer Institute is supporting a number of programs that are addressing some of the challenges described above and pursuing careful selection of candidate markers (mutations, epigenomic changes, proteomic alterations, etc.), developing better analytical methods and sensitive molecular technologies that may contribute to early cancer detection and monitoring. The speaker will describe the objectives of these programs and provide updates. Citation Format: Sudhir Srivastava. The promise of liquid biopsy in the early detection of cancer: Hope or hype? [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr IA09.

Abstract PR08: Validation and clinical implementation of MSK-ACCESS, an ultra-deep sequencing assay for noninvasive somatic mutation profiling

Abstract Introduction: Circulating cell-free DNA (cfDNA) is a source of tumor-derived DNA to interrogate somatic alterations when tissue is not available or of insufficient quantity for analysis. At MSKCC, we have developed and validated MSK-ACCESS (Analysis of Circulating cfDNA to Evaluate Somatic Status), a targeted next-generation sequencing assay that can detect ultra-low frequency somatic variants in select exons and introns of 129 genes. MSK-ACCESS can identify mutations, copy number alterations, gene fusions, and MSI status in plasma and was recently approved by the NYS-DOH for clinical testing. Here, we present the results of the validation study and our clinical experience with MSK-ACCESS since June 2019. Methods: Target regions from 129 genes were selected to maximize coverage of actionable, oncogenic, and hotspot mutations based on the first 25,000 tumors sequenced using MSK-IMPACT, our institutional clinical sequencing assay. Plasma cfDNA and buffy-coat DNA were extracted from whole blood collected in cell-stabilizing tubes (STRECK BCT cell-free DNA tube). Unique molecular indexes were introduced during DNA library construction, allowing for error suppression from consensus reads collapsed by Marianas, an in-house-developed algorithm. These consensus reads enable variant calling at low allelic frequency (AF) based on a 10−6 background error rate. Results: Analytical validation of MSK-ACCESS demonstrated 93% accuracy (n=100 variants), 99% precision (n=153 variants), and 100% sensitivity based on an assay limit of detection of 0.5% AF (n=19 variants). Variants were detected down to 0.1% AF. To date, 240 clinical cfDNA and matched normal DNA pairs have been sequenced, analyzed for somatic alterations, and clinically reported to guide patient management. Most clinical cases were from lung (55%) or prostate (13%) cancers and submitted for diagnostic purposes (71%). Median raw coverage was 18,367X, and median consensus coverage was 1411X. Mutations were detected in 180 (75%) samples with a median variant AF of 1.8% (0.02% - 95%). Comparison of concurrent commercial plasma testing results to MSK-ACCESS revealed multiple variants that were of clonal hematopoiesis or germline origin incorrectly reported as somatic variants. In the lung cohort, 48 patients had tissue testing with MSK-IMPACT; among 32 patients with a driver alteration detected by MSK-ACCESS, 91% had the identical driver alteration reported by MSK-IMPACT. Additionally, MSK-ACCESS identified a MET exon 14 alteration in one lung cancer patient that led to protocol enrollment and partial response. Conclusions: Liquid biopsy testing using MSK-ACCESS reliably detected clinically actionable mutations, reducing the need for multiple biopsies. These results also illustrate the importance of analyzing a matched normal sample when interpreting cfDNA results and highlight the potential of using cfDNA analysis to guide treatment selection, assess for treatment response, and identify mechanisms of treatment resistance. This abstract is also being presented as Poster A20. Citation Format: A. Rose Brannon, Gowtham Jayakumaran, Monica Diosdado, Yu Hu, Anna Razumova, Fanli Meng, Emily Lebow, Juber Patel, Ian Johnson, Preethi Srinivasan, Maysun Hasan, Jenna-marie Dix, Aijazuddin Syed, Brian Houck-Loomis, Bob T. Li, Charles Rudin, David Solit, Marc Ladanyi, Maria Arcila, Dana Tsui, Ahmet Zehir, Michael Berger, Ryma Benayed. Validation and clinical implementation of MSK-ACCESS, an ultra-deep sequencing assay for noninvasive somatic mutation profiling [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr PR08.

Elucidating resistance mechanism to PARP inhibitors for the development of novel therapeutic approaches in high-grade serous ovarian cancer.

6078 Background: PARP inhibitors (PARPi) have been established as a targeted therapeutic approach not only in patients with high-grade serous ovarian cancer (HGSOC) that have genetic loss of function of BRCA1/2-associated DNA repair. However, treatment efficacy varies and neither BRCA mutation, nor homolog recombination deficiency (HRD) status seem to be optimal predictors. Moreover, mechanisms of treatment resistance are poorly understood and novel approaches are urgently required. Methods: Here we created gene expression data of HGSOC patients (n = 52) before PARPi treatment to elucidate key signaling pathways of resistance to increase their efficacy in combinatorial therapeutic strategies. We performed a comprehensive bioinformatics analysis of the differentially expressed genes between the 25% extreme responders (n = 26; 13 each group), including gene set enrichment analysis (GSEA) and causal inference analysis with the CARNIVAL pipeline to elucidate the underlying molecular and regulatory mechanisms governing treatment efficacy and resistance. Results: In accordance with recent publications, we found higher levels of MYC activity in non-responders and deregulation of the Wnt/ß-catenin signaling pathway resulting in PARPi treatment resistance. The pathway enrichment analysis also revealed specific pathways especially PDGFR, FGFR, PI3K/mTOR and MAPK signaling pathway associated with resistant phenotype. Furthermore, we have identified key kinases, particularly JAK1/2 and SRC that might mediate resistance to PARP inhibition. In addition, differential gene expression analysis revealed folate receptor 1 (FOLR1) to be significantly higher expressed in non-responders (logFC = 2.66; p < 0.0026) with the potential as a serum-based biomarker not only for ovarian cancer, as it correlates closely with CA125, but also PARPi treatment efficacy. Conclusions: In conclusion, these findings define a network of pathways, that are crucial to mediate mechanism of PARPi resistance and identified key signaling kinases as therapeutic targets in ovarian cancer.

Circulating tumor DNA to monitor treatment response and investigate resistance mechanism in BRAF inhibitor treated colorectal cancer patients.

e16112 Background: BRAF mutations are found in about 10% of colorectal cancer (CRC) patients and is a poor prognosis factor in standard chemotherapy. Combination of BRAF and EGFR inhibition exerts a better therapeutic effective, whereas the resistance develops through undefined mechanisms. Circulating tumor DNA (ctDNA) is a non-invasive approach to assess the genetic evolution of tumors and prognosis in response to therapy, which would help better understanding the treatment response and resistance mechanism in BRAF inhibitor treated colorectal cancer patients. Methods: We performed panel next-generation sequencing of 425 cancer-related genes in 39 serial plasma samples collect from 19 patients who have BRAF V600E mutation CRC to track the resistance during the Vemurafenib treatment in combination with Irinotecan and Cetuximab (VIC) and evaluate the treatment response. Tumor responses were assessed radiologically every two months and are used to determine patients with innate or acquired resistance Results: A total of 19 patients were enrolled into the vemurafenib treatment with irinotecan and cetuximab. By January 20, 2020, treatment had been discontinued in 12 (63.2%) of the patients due to disease progression, while the other 7 cases were still under treatment. Among them, four patients with innate resistance (n = 4, 21.1%) were defined as those with PFS of less than 2 months, while patients (n = 15, 78.9%) with acquired resistance were defined as those with PFS of greater than 2 months. The VIC regimen demonstrated efficacy in patients with BRAF-mutant mCRC, with overall response rates was 63.2% (n = 12). Changes in levels of ctDNA at 4 weeks predicts therapeutic responses. CBLB, TP53 and APC baseline mutations were enriched in baseline samples of innate resistant patients. In contrast, MYC and Cell-cycle pathway alterations were enriched in baseline samples of patients with acquired resistance. Acquired RAS and MAPK mutations, known to confer resistance to BRAF inhibitors, were identified in 60% of acquired resistance patients. Moreover, acquired TGFBR2 and SMAD4 loss-of-function mutations were identified as novel resistant mechanism to the combination of BRAF and EGFR inhibition. Experimental validation is ongoing. Conclusions: Longitudinal analysis of ctDNA in BRAF CRC patients provides insights of molecular difference between innate and acquired resistance and gene alternation during resistance requiring. Serial ctDNA monitoring provides early indication of response to BRAF inhibitor in CRC patients.

Clinical and genomic analysis of non-small cell lung cancer (NSCLC) patients with MET exon14 skipping (METex14) mutations and responses to anti-MET therapy.

9613 Background: MET is a validated oncogene for molecular targeted therapy in non-small-cell lung cancer (NSCLC), and METex14 mutations result in MET overexpression. Studies with MET-targeting therapies have demonstrated high objective response rates and prolonged disease control. There are few clinical and genomic analyses of patients with METex14-positive NSCLC in the community setting. We herein characterize key clinical and genomic findings for patients harboring METex14 mutations. Methods: Sarah Cannon provides clinical research to partnering medical oncology practices who order broad-based NGS, from both tissue and blood, as a part of standard of care. Genospace, a clinico-genomic software tool, was used for identification of patients for clinical trials and analysis of clinical and genomic data. Results: Of 6521 lung cancer patients with NGS results, 66 (1.01%) harbored METex14 mutations (45.5% from blood and 54.5% from in tissue). The mean age at diagnosis was 75.7 and 21.2% developed brain metastases. Of the 66 patients with METex14 mutations, 69.5% are current/former smokers. Nineteen percent of former/current smokers and 7.6% of never smokers had PD-L1 scores of > 50%, respectively. The majority of METex14-positive patients either received standard of care (66.7%) or were unable to take (19.7%) 1st-line therapy. Patients who received chemotherapy (Chemo), immunotherapy (IO), and Chemo/IO in the first and second line settings responded to SOC treatment, and patients receiving anti-MET therapy benefited from therapy even after front-line SOC (table). Genomic analysis revealed the most common co-occurring mutations to be EGFR, MET, NF1, KRAS, and BRCA2 (Freq = 8.7%, 8.7%, 8.7%, and 7.6%, respectively). Conclusions: Genospace enables real-time patient identification of METex14-positive NSCLC cases and analysis of these cases indicates that anti-MET therapy may be effective at any line of treatment. Genospace’s clinico-genomic database was used to analyze treatment history, clinical correlates, and co-occurring mutations that may reveal novel treatment combination strategies or resistance mechanisms. [Table: see text]

PCN163 IMPROVEMENT OF THE INNOVATION & VALUE INITIATIVE OPEN-SOURCE COST-EFFECTIVENESS MODEL FOR NON-SMALL-CELL LUNG CANCER BASED ON PUBLIC FEEDBACK

In January 2019, the Innovation & Value Initiative presented a flexible open-source simulation model for EGFR+ non-small-cell lung cancer (NSCLC). Our aim was to collect feedback on this initial version of the model to set priorities for the development of the next model iteration. Following the public release of the model, we invited feedback and suggestions for improvements during a public comment period (January 2019 – April 2019). Subsequently, a panel of subject matter experts (n=5; patient, oncologists, and methodologists) determined which of the received suggestions should be implemented by means of a modified Delphi process. Two rounds of surveys were administered using a web-based survey tool. Panel surveys focused on improvements that either require substantial resources to implement (such as elaborate programming or additional evidence collection) or are likely to have a substantial impact on the findings of model analysis. A total of 98 comments were received. These suggestions related to: the incorporation of additional patient characteristics; palliative care; mechanism of treatment resistance; adverse events; model output; elements of value; addition or removal of specific treatment combinations; impact of prior treatment on future treatment efficacy; and estimation of preference weights for the multi-criteria decision analysis (MCDA) module. The Delphi survey covered 28 items and the following were ranked as most important to include in the next model iteration: add nausea, vomiting, and immune-related adverse events; add out of pocket costs and probability of unemployment; elicit preference weights for the MCDA; and provide output by line of treatment in addition to the total sequence. The current study illustrates areas of improvement for the IVI-NSCLC model based on public feedback. Version 2 of the model is planned to be released in early 2021.

Granzyme B PET imaging of immune-mediated tumor killing as a tool for understanding immunotherapy response

BackgroundCancer immunotherapy research is expanding to include a more robust understanding of the mechanisms of treatment response and resistance. Identification of drivers of pro-tumor and anti-tumor immunity during treatment offers...

SynergyFinder 2.0: visual analytics of multi-drug combination synergies.

SynergyFinder (https://synergyfinder.fimm.fi) is a stand-alone web-application for interactive analysis and visualization of drug combination screening data. Since its first release in 2017, SynergyFinder has become a widely used web-tool both for the discovery of novel synergistic drug combinations in pre-clinical model systems (e.g. cell lines or primary patient-derived cells), and for better understanding of mechanisms of combination treatment efficacy or resistance. Here, we describe the latest version of SynergyFinder (release 2.0), which has extensively been upgraded through the addition of novel features supporting especially higher-order combination data analytics and exploratory visualization of multi-drug synergy patterns, along with automated outlier detection procedure, extended curve-fitting functionality and statistical analysis of replicate measurements. A number of additional improvements were also implemented based on the user requests, including new visualization and export options, updated user interface, as well as enhanced stability and performance of the web-tool. With these improvements, SynergyFinder 2.0 is expected to greatly extend its potential applications in various areas of multi-drug combinatorial screening and precision medicine.

Abstract P1-09-01: ESR1 mutations confer radiation resistance in breast cancer

Abstract Background: Acquired mutations in oncogenic drivers in response to systemic therapies is a common mechanism of treatment resistance. How such mutations alter response to radiation therapy is poorly understood. The increasing adoption of radiation therapy in the treatment of oligometastatic disease further amplifies the need to define the role of such mutations in modifying response to radiation therapy. Over 70% of breast cancers are Estrogen Receptor (ER)-positive. Endocrine therapies, which block ER signaling, are the mainstay in the treatment of such cancers. Acquired mutations in the gene encoding for ER (ESR1) are a common mechanism of acquired resistance to endocrine therapies in metastatic breast cancer. In this study, we define the role of ESR1 mutations in altering response to radiation therapy in pre-clinical models of ER-positive breast cancer. Methods: To test if ESR1 mutations influence response to radiation therapy, we employed ER-positive MCF7 and T47D cell lines which have been genome edited to knockin two of the most common ESR1 mutations found in breast cancer patients (Y537S and D538G). Clonogenic survival assays were carried out to characterize differences in radiation sensitivity of these cells. γ-H2AX and Comet assays were employed to study the differences in residual unrepaired DNA damage following radiation in these cell lines. MCF7 cell line-derived xenografts harboring the wild type or mutant ESR1 were employed to study differences in radiation resistance of these tumors in vivo. Results: ER-positive cell lines harboring ESR1 mutations exhibited profound resistance to radiation therapy compared to cell lines harboring the wild type ESR1. These radioresistant cell lines also demonstrated significantly less unrepaired DNA damage following radiation therapy. Mice harboring ESR1 mutant xenografts showed remarkable resistance to radiation therapy. Conclusion: ESR1 mutations, which confer resistance to endocrine therapies, also confer profound resistance to radiation therapy. ESR1 mutations have been primarily reported in metastatic ER-positive breast cancer patients with prior history of treatment with endocrine therapies. Our findings suggest that oligometastatic ER-positive breast cancer patients who are being treated with radiation therapy (such as those enrolled in NRG BR002 trial) may show significant differences in response to radiation therapy based on their ESR1 mutation status. Recent studies are also exploring the use of neo-adjuvant endocrine therapy in non-metastatic ER-positive breast cancer patients. It is conceivable that some of these patients may acquire ESR1 mutations under selective pressure of neoadjuvant endocrine therapy and may respond poorly to subsequent adjuvant radiation therapy. Finally, our findings support potential personalization of radiation treatments in breast cancer patients based on ESR1 mutation status, heralding an era of precision radiation oncology. Citation Format: Nashir Udden, Qian Wang, Prasanna G Alluri. ESR1 mutations confer radiation resistance in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-09-01.

Abstract GS2-05: Microscaled proteogenomic methods for precision oncology

Abstract Cancer proteogenomics combines genomics, transcriptomics and mass spectrometry-based proteomics to gain insights into cancer biology and treatment responsiveness. While proteogenomics analyses have already shown great potential to deepen our understanding of cancer tissue complexity and signaling, how a patient’s tumor changes upon treatment has largely been the province of genomics. This is due to technical difficulties associated with doing proteogenomic analysis on clinic-derived core-needle biopsies. To address this critical need, we have developed a “microscaled” proteogenomics approach for tumor-rich OCT-embedded core needle biopsies. Tissue-sparing specimen processing (“Biopsy Trifecta EXTraction”, BioTExt) and microscaled proteomics (MiProt) methodologies allowed generation of deep-scale proteogenomics datasets, with copy number and transcript information for >20,000 genes and mass spectrometry-based identification and quantification of nearly all expressed proteins in a tumor (>10,000 proteins) and more than >20,000 phosphosites starting with just 25 micrograms of protein per sample. In order to understand the capabilities and limitations our our approach relative to more conventional deepscale proteomics requiring >10X more starting material, we compared preclinical patient derived xenograft (PDX) models at conventional scale with data obtained by core-needle biopsy of the same tissues. Comparable depth and biological insights were obtained from the cores relative to surgically resected tumors. As a proof-of-concept for implementation in clinical trials, we applied microscaled proteogenomic methods to a small-scale clinical study where biopsies were accrued from patients with ERBB2+ locally advanced breast cancer before and 48 to 72 hours after the first dose of neoadjuvant Trastuzumab-based chemotherapy. Multi-omics comparisons were conducted between samples associated with residual disease versus samples associated with complete pathological response. Integrative, microscaled proteogenomic analyses efficiently diagnosed the molecular bases of diverse candidate treatment resistance mechanisms including: 1) absence of ERBB2 amplification (false-ERBB2+); 2) insufficient ERBB2 activity for therapeutic sensitivity despite ERBB2 amplification (pseudo-ERBB2+); 3) resistance features in true-ERBB2+ cases including androgen receptor signaling, mucin expression and an inactive immune microenvironment; 4) lack of acute phospho-ERBB2 down-regulation in non-pCR cases. In summary, we have developed a robust proteogenomics pipeline well suited for large-scale cancer clinical studies to identify potential resistance mechanism in patients. We conclude that microscaled cancer proteogenomics could improve diagnostic precision in the clinical setting. Citation Format: Shankha Satpathy, Eric Jaehnig, Krug Karsten, Beom-Jun Kim, Alexander Saltzman, Doug Chan, Kimberly Holloway, Meenakshi Anurag, Chen Huang, Purba Singh, Ari Gao, Noel Namai, Yongchao Dou, Bo Wen, Suhas Vasaikar, David Mutch, Mark Watson, Cynthia Ma, Foluso Ademuyiwa, Mothaffar Rimawi, Jeremy Hoog, Samuel Jacobs, Anna Malovannaya, Terry Hyslop, D.R Mani, Charles Perou, George Miles, Bing Zhang, Michael Gillette, Steven Carr, Matthew Ellis. Microscaled proteogenomic methods for precision oncology [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr GS2-05.