Search

Patient-derived tumor xenograft (PDX) mouse models have emerged as an important oncology research platform to study tumor evolution, mechanisms of drug response and resistance, and tailoring chemotherapeutic approaches for individual patients. The lack of robust standards for reporting on PDX models has hampered the ability of researchers to find relevant PDX models and associated data. Here we present the PDX models minimal information standard (PDX-MI) for reporting on the generation, quality assurance, and use of PDX models. PDX-MI defines the minimal information for describing the clinical attributes of a patient's tumor, the processes of implantation and passaging of tumors in a host mouse strain, quality assurance methods, and the use of PDX models in cancer research. Adherence to PDX-MI standards will facilitate accurate search results for oncology models and their associated data across distributed repository databases and promote reproducibility in research studies using these models. Cancer Res; 77(21); e62–66. ©2017 AACR.

A figure showing the process of generating and using PDX models.

Introduction Tumor mutational burden (TMB) has emerged as a clinically relevant biomarker that may be associated with immune checkpoint inhibitor efficacy. Standardization of TMB measurement is essential for implementing diagnostic tools to guide treatment. Objective Here we describe the in-depth evaluation of bioinformatic TMB analysis by whole exome sequencing (WES) in formalin-fixed, paraffin-embedded samples from a phase III clinical trial. Methods In the CheckMate 026 clinical trial, TMB was retrospectively assessed in 312 patients with non-small-cell lung cancer (58% of the intent-to-treat population) who received first-line nivolumab treatment or standard-of-care chemotherapy. We examined the sensitivity of TMB assessment to bioinformatic filtering methods and assessed concordance between TMB data derived by WES and the FoundationOne® CDx assay. Results TMB scores comprising synonymous, indel, frameshift, and nonsense mutations (all mutations) were 3.1-fold higher than data including missense mutations only, but values were highly correlated (Spearman’s r = 0.99). Scores from CheckMate 026 samples including missense mutations only were similar to those generated from data in The Cancer Genome Atlas, but those including all mutations were generally higher. Using databases for germline subtraction (instead of matched controls) showed a trend for race-dependent increases in TMB scores. WES and FoundationOne CDx outputs were highly correlated (Spearman’s r = 0.90). Conclusions Parameter variation can impact TMB calculations, highlighting the need for standardization. Encouragingly, differences between assays could be accounted for by empirical calibration, suggesting that reliable TMB assessment across assays, platforms, and centers is achievable.

KRAS is the most common oncogenic driver in lung adenocarcinoma (LUAC). We previously reported that STK11/LKB1 (KL) or TP53 (KP) comutations define distinct subgroups of KRAS-mutant LUAC. Here, we examine the efficacy of PD-1 inhibitors in these subgroups. Objective response rates to PD-1 blockade differed significantly among KL (7.4%), KP (35.7%), and K-only (28.6%) subgroups (P < 0.001) in the Stand Up To Cancer (SU2C) cohort (174 patients) with KRAS-mutant LUAC and in patients treated with nivolumab in the CheckMate-057 phase III trial (0% vs. 57.1% vs. 18.2%; P = 0.047). In the SU2C cohort, KL LUAC exhibited shorter progression-free (P < 0.001) and overall (P = 0.0015) survival compared with KRASMUT;STK11/LKB1WT LUAC. Among 924 LUACs, STK11/LKB1 alterations were the only marker significantly associated with PD-L1 negativity in TMBIntermediate/High LUAC. The impact of STK11/LKB1 alterations on clinical outcomes with PD-1/PD-L1 inhibitors extended to PD-L1–positive non–small cell lung cancer. In Kras-mutant murine LUAC models, Stk11/Lkb1 loss promoted PD-1/PD-L1 inhibitor resistance, suggesting a causal role. Our results identify STK11/LKB1 alterations as a major driver of primary resistance to PD-1 blockade in KRAS-mutant LUAC. Significance: This work identifies STK11/LKB1 alterations as the most prevalent genomic driver of primary resistance to PD-1 axis inhibitors in KRAS-mutant lung adenocarcinoma. Genomic profiling may enhance the predictive utility of PD-L1 expression and tumor mutation burden and facilitate establishment of personalized combination immunotherapy approaches for genomically defined LUAC subsets. Cancer Discov; 8(7); 822–35. ©2018 AACR. See related commentary by Etxeberria et al., p. 794. This article is highlighted in the In This Issue feature, p. 781

Patient-derived tumor xenograft (PDX) mouse models have emerged as an important oncology research platform to study tumor evolution, mechanisms of drug response and resistance, and tailoring chemotherapeutic approaches for individual patients. The lack of robust standards for reporting on PDX models has hampered the ability of researchers to find relevant PDX models and associated data. Here we present the PDX models minimal information standard (PDX-MI) for reporting on the generation, quality assurance, and use of PDX models. PDX-MI defines the minimal information for describing the clinical attributes of a patient's tumor, the processes of implantation and passaging of tumors in a host mouse strain, quality assurance methods, and the use of PDX models in cancer research. Adherence to PDX-MI standards will facilitate accurate search results for oncology models and their associated data across distributed repository databases and promote reproducibility in research studies using these models. Cancer Res; 77(21); e62–66. ©2017 AACR.

Current understanding of the mutation spectrum of relapsed/refractory (RR) tumors is limited. We performed whole exome sequencing (WES) on 47 diffuse large B cell lymphoma (DLBCL) tumors that persisted after R-CHOP treatment, 8 matched to primary biopsies. We compared genomic alterations from the RR cohort against two treatment-naïve DLBCL cohorts (n=112). While the overall number and types of mutations did not differ significantly, we identified frequency changes in DLBCL driver genes. The overall frequency of MYD88 mutant samples increased (12% to 19%), but we noted a decrease in p.L265P (8% to 4%) and increase in p.S219C mutations (2% to 6%). CARD11 p.D230N, PIM1 p.K115N and CD79B p.Y196C mutations were not observed in the RR cohort, although these mutations were prominent in the primary DLBCL samples. We observed an increase in BCL2 mutations (21% to 38% of samples), BCL2 amplifications (3% to 6% of samples) and CREBBP mutations (31% to 42% of samples) in the RR cohort, supported by acquisition of mutations in these genes in relapsed compared to diagnostic biopsies from the same patient. These increases may reflect the genetic characteristics of R-CHOP RR tumors expected to be enriched for during clinical trial enrollment. These findings hold significance for a number of emerging targeted therapies aligned to genetic targets and biomarkers in DLBCL, reinforcing the importance of time-of-treatment biomarker screening during DLBCL therapy selection.

Matthew B Schabath,1,2 Tapashi B Dalvi,3 Hongyue A Dai,4 Alan L Crim,4 Anita Midha,5 Norah Shire,3 Nicholas T Gimbrone,1 Jill Walker,6 Danielle M Greenawalt,7 David Lawrence,8 James R Rigas,9 Robert Brody,9 Danielle Potter,9 Naveen S Kumar,4 Shane A Huntsman,4 Jhanelle E Gray2 1Department of Cancer Epidemiology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA; 2Department of Thoracic Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA; 3Oncology R&D, AstraZeneca, Gaithersburg, MD, USA; 4M2Gen, Tampa, FL, USA; 5Department of Personalised Healthcare and Biomarkers, AstraZeneca, Cambridge, UK; 6Department of Precision Medicine Oncology, AstraZeneca, Cambridge, UK; 7Department of iMED Oncology Informatics, AstraZeneca, Waltham, MA, USA; 8Department of Global Medicines Development, AstraZeneca, Cambridge, UK; 9Department of Global Medical Affairs Oncology, AstraZeneca, Gaithersburg, MD, USACorrespondence: Matthew B SchabathH. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USATel +1 813 745 4150Fax +1 813 745 6525Email matthew.schabath@moffitt.orgPurpose: To characterize programmed cell death ligand-1 (PD-L1) expression in relation to survival and gene mutation status in patients with advanced NSCLC. The study also explored the influence of tumor mutational burden (TMB) on PD-L1 expression and patient characteristics.Patients and methods: Adult patients with histologically or cytologically documented Stage IIIB/Stage IV/recurrent/progressive NSCLC, Eastern Cooperative Oncology Group performance status 0 to 3, and >2 lines of prior systemic treatment regimens were included in this retrospective analysis. Patients were treated from 1997 to 2015 at H. Lee Moffitt Cancer Center and Research Institute, Tampa, or at 7 community centers across the United States. PD-L1 expression level was determined using the VENTANA PD-L1 (SP263) Assay. EGFR and KRAS mutation status and ALK rearrangements were determined by targeted DNA sequencing; these were obtained from clinical records where targeted DNA sequencing was not performed. TMB was calculated as the total number of somatic mutations per sample.Results: From a total of 136 patients included in the study, 23.5% had tumors with high PD-L1 expression (≥25%). There were no significant differences in patient characteristics, overall survival (OS), and progression-free survival (PFS) between patients with high PD-L1 expression (median OS: 39.5 months; median PFS: 15.8 months) vs low PD-L1 expression (

IntroductionTumor mutational burden (TMB) has emerged as a clinically relevant biomarker that may be associated with immune checkpoint inhibitor efficacy. Standardization of TMB measurement is essential for implementing diagnostic tools to guide treatment.ObjectiveHere we describe the in-depth evaluation of bioinformatic TMB analysis by whole exome sequencing (WES) in formalin-fixed, paraffin-embedded samples from a phase 3 clinical trial.MethodsIn the CheckMate 026 clinical trial, TMB was retrospectively assessed in 312 patients with non-small cell lung cancer (58% of the intent-to-treat population) who received first-line nivolumab treatment or standard-of-care chemotherapy. We examined the sensitivity of TMB assessment to bioinformatic filtering methods and assessed concordance between TMB data derived by WES and the FoundationOne®CDx assay.ResultsTMB scores comprising synonymous, indel, frameshift, and nonsense mutations (all mutations) were 3.1-fold higher than data including missense mutations only, but values were highly correlated (Spearman’s r = 0.99). Scores from CheckMate 026 samples including missense mutations only were similar to those generated from data in The Cancer Genome Atlas, but those including all mutations were generally higher. Using databases for germline subtraction (instead of matched controls) showed a trend for race-dependent increases in TMB scores. WES and FoundationOne CDx outputs were highly correlated (Spearman’s r = 0.90).ConclusionsParameter variation can impact TMB calculations, highlighting the need for standardization. Encouragingly, differences between assays could be accounted for by empirical calibration, suggesting that reliable TMB assessment across assays, platforms, and centers is achievable.Key PointsTumor mutational burden (TMB) is a clinically relevant biomarker for efficacy of immunotherapy in patients with cancerVariations in TMB assessment parameters can shift the final TMB value. Harmonization and standardization are important to the successful clinical implementation of TMB testingTMB values assessed by different methods are highly correlated. Harmonization of TMB testing in patients with cancer is therefore achievable

Bromodomain and extraterminal (BET) proteins recognize acetylated lysine residues for the purpose of transcriptional regulation of genes, including those involved in stem cell renewal and oncogenes such as MYC. Inhibition of BET proteins BRD2, BRD3, BRD4, and BRDT represents a promising treatment option for patients with cancer. However, little is known about the consequence of BET inhibition on normal stem cell renewal processes, such as hematopoiesis. This study explored the mechanistic effects of BET inhibition on bone marrow (BM) hematopoiesis. Rats treated with BET inhibitors JQ1, BMS-X, or BMS-986158 for 4 days in vivo showed dose-dependent pan-cellular BM atrophy and reduction of hematopoietic progenitors of myeloid and erythroid lineage (combination of anti-rat CD45, CD11b, anti-granulocyte, CD71, anti-erythroid, and CD90 via flow cytometry) and consequential reductions in circulating platelet and reticulocyte counts, with complete reversibility within 10 days of stopping treatment with BET inhibitors. Primary rat BM stem and progenitor cells treated with BET inhibitors in vitro were evaluated with the colony-forming unit assay and resulted in dose-dependent reduction of multiple lineage progenitor colonies, especially the erythroid and megakaryocyte lineages. To further elucidate pathways involved in BET-related BM atrophy, erythropoiesis and thrombopoiesis genes regulated by GATA1, a BRD-associated transcription factor, from rat BM, as well as rat and human whole-blood samples exposed to BET inhibitor(s) were evaluated via RNAseq and RT-PCR. Dose-dependent responses in genes involved in erythropoiesis (Alas2, ABCme, PBG-D, HMBS) and thrombopoiesis (NFE2, PF4, GP1Bb, MPL) were observed after 4 days of treatment with BMS-986158. In a clinical trial (NCT02419417), patients with solid tumors treated with BMS-986158 demonstrated reversible thrombocytopenia and downregulation of NFE2, PF4, and HMBS expression, similar to that observed in rats. GATA1 was also downregulated in rat BM, with target engagement in rat and human demonstrated by the induction of HEXIM1 transcription, a pharmacodynamic (PD) biomarker of growth inhibition and apoptosis induced by BRD4 inhibition. Overall, our results suggest inhibition of BET signaling causes target-related, dose-dependent repression of hematopoietic progenitors through alterations of GATA1-associated erythropoiesis and thrombopoiesis regulation in rat BM and human blood samples, and these effects are reversible on cessation of BET inhibitor treatment. This is the first in vivo study demonstrating the mechanism of BET inhibition resulting in GATA1-associated repression of hematopoietic progenitors that is correlated to clinical pharmacokinetics and PD (Chen X, et al. AACR 2020) and is translatable from preclinical evaluation to clinical experience. Citation Format: Cindy Zhang, Ke Xu, Julie Panzica-Kelly, Jennifer Price, Denise Bounous, Shodeinde Coker, Kezi Unsal-Kacmaz, Danielle Greenawalt, Ashvinikumar Gavai, Ronald Fleming, Karen Augustine-Rauch, Richard Westhouse. Inhibition of BET signaling leads to reversible GATA1-associated repression of hematopoietic progenitors: translation from preclinical assessment to clinical development [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 1742.

Agents targeting the PD1-PDL1 axis have transformed cancer therapy. Factors that influence clinical response to PD1-PDL1 inhibitors include tumor mutational burden, immune infiltration of the tumor and local PDL1 expression. To identify peripheral correlates of the anti-tumor immune response in the absence of checkpoint blockade, we performed a retrospective study of circulating T cell subpopulations and matched tumor gene expression in melanoma and non-small cell lung carcinoma (NSCLC) patients. Notably, both melanoma and NSCLC patients whose tumors exhibited increased inflammatory gene transcripts presented high CD4+ and CD8+ central memory T cell (CM) to effector T cell (Eff) ratios in blood. Consequently, we evaluated CM/Eff T cell ratios in a second cohort of NSCLC. The data showed that high CM/Eff T cell ratios correlated with increased tumor PDL1 expression. Furthermore, of the 22 patients within this NSCLC cohort who received nivolumab, those with high CM/Eff T cell ratios, had longer progression-free survival (PFS) (median survival: 91 vs. 215 days). These findings show that by providing a window into the state of the immune system, peripheral T cell subpopulations inform about the state of the anti-tumor immune response and identify potential blood biomarkers of clinical response to checkpoint inhibitors in melanoma and NSCLC.

Background Linrodostat mesylate is a potent, selective, oral, once-daily IDO1i being evaluated in combination with NIVO. The kynurenine (KYN) pathway, mediated through the enzymatic activity of IDO1 and TDO2, reflects an immunosuppressive mechanism that may limit clinical response to immune checkpoint blockade. In the phase I/IIa CA017-003 study (NCT02658890), linrodostat + NIVO decreased serum and tumoral KYN levels and increased tumoral CD8+ T-cell proliferation in pts with advanced cancers (Luke et al. SITC 2017). Here we describe an RNA-based composite biomarker that may enrich for clinical benefit with linrodostat + NIVO. Methods Tumor and serum samples were obtained from pts with solid tumors (bladder, cervical, melanoma, lung, pancreatic, renal cell, head and neck) and lymphoma treated with linrodostat + NIVO during the expansion phase in CA017-003. KYN and tryptophan (TRP) levels were measured in frozen serum (n = 400) and tumors (n = 45) obtained before and during treatment (cycle 1, day 15) using liquid chromatography–mass spectrometry. RNA sequencing was done on baseline formalin-fixed, paraffin-embedded solid tumor samples (n = 108). Results In serum and tumors, decreases in KYN and KYN/TRP were consistently observed; however, baseline and change from baseline in KYN or KYN/TRP did not correlate with response. Overall, in the tumor-based gene expression cohort, high IFN-γ gene signature (Ayers et al. J Clin Invest. 2017) was associated with response and improved progression-free and overall survival; nonresponders had higher expression of TDO2. In the non-melanoma subset (n = 56), a composite biomarker of IFN-γ signature and TDO2 gene expression was more significantly associated with response (P = 0.021) than the IFN-γ signature alone (P = 0.063). Conclusions IFN-ɣ signature and TDO2 gene expression may be a potential composite biomarker to identify pts with certain tumor types more likely to respond to linrodostat mesylate + NIVO. Future studies are needed to clarify the underlying mechanisms and validate the potential of this composite biomarker. Clinical trial identification NCT02658890. Release date January 20, 2016. Editorial acknowledgement Writing and editorial assistance was provided by Jillian Brechbiel, PhD, of Chrysalis Medical Communications, Inc, funded by Bristol-Myers Squibb. Legal entity responsible for the study Bristol-Myers Squibb. Funding Bristol-Myers Squibb. Disclosure J. Luke: Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Bristol-Myers Squibb; Advisory / Consultancy, Shareholder / Stockholder / Stock options: Actym Therapeutics; Advisory / Consultancy: 7 Hills Pharma; Advisory / Consultancy: Aduro Biotech; Advisory / Consultancy: Alphamab Oncology; Advisory / Consultancy, Travel / Accommodation / Expenses: Amgen; Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Array BioPharma; Advisory / Consultancy, Travel / Accommodation / Expenses: AstraZeneca/MedImmune; Research grant / Funding (institution): Abbvie; Advisory / Consultancy, Travel / Accommodation / Expenses: Benevir; Research grant / Funding (institution): Boston Biomedical; Advisory / Consultancy, Travel / Accommodation / Expenses: Castle Biosciences; Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Checkmate Pharmaceuticals; Research grant / Funding (institution): Celldex; Advisory / Consultancy, Research grant / Funding (institution): Compugen; Research grant / Funding (institution): Corvus Pharmaceuticals; Advisory / Consultancy, Travel / Accommodation / Expenses: EMD Serono; Research grant / Funding (institution): Delcath Systems; Advisory / Consultancy, Travel / Accommodation / Expenses: Gilead Sciences; Research grant / Funding (institution): Evelo Therapeutics; Advisory / Consultancy, Travel / Accommodation / Expenses: Ideaya Biosciences; Advisory / Consultancy, Travel / Accommodation / Expenses: Janssen; Advisory / Consultancy, Travel / Accommodation / Expenses: Jounce Therapeutics; Advisory / Consultancy: Mavu Pharmaceutical; Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Merck; Advisory / Consultancy: Newlink Genetics; Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Novartis; Advisory / Consultancy, Travel / Accommodation / Expenses: Reflexion Medical; Advisory / Consultancy: Spring Bank; Advisory / Consultancy: Syndax; Advisory / Consultancy: Tempest Therapeutics; Advisory / Consultancy: TTC Oncology; Advisory / Consultancy: Vividion; Advisory / Consultancy: WntRx; Research grant / Funding (institution): Five Prime Therapeutics ; Research grant / Funding (institution): FLX Bio ; Research grant / Funding (institution): Genentech/Roche ; Research grant / Funding (institution): Immunocore ; Research grant / Funding (institution): Incyte ; Research grant / Funding (institution): Macrogenics ; Research grant / Funding (institution): MedImmune ; Research grant / Funding (institution): Palleon Pharmaceuticals ; Research grant / Funding (institution): Pharmacyclis; Research grant / Funding (institution): Tesaro; Research grant / Funding (institution): Xencor ; Licensing / Royalties: Serial #15/612,657 (Cancer Immunotherapy); Licensing / Royalties: Serial #PCT/US18/36052 (Microbiome Biomarkers for Anti-PD-1/PD-L1 Responsiveness: Diagnostic, Prognostic and Therapeutic Uses Thereof. L.L. Siu: Advisory / Consultancy, Research grant / Funding (institution): Merck; Advisory / Consultancy, Research grant / Funding (institution): Pfizer; Advisory / Consultancy, Research grant / Funding (institution): Celgene; Advisory / Consultancy, Research grant / Funding (institution): AstraZeneca/MedImmune; Advisory / Consultancy: Morphosys; Advisory / Consultancy: Roche; Advisory / Consultancy: GeneSeeq; Advisory / Consultancy: Loxo; Advisory / Consultancy: Oncorus; Advisory / Consultancy, Research grant / Funding (institution): Symphogen; Advisory / Consultancy: Seattle Genetics; Research grant / Funding (institution): Novartis; Research grant / Funding (institution): Bristol-Myers Squibb; Research grant / Funding (institution): Boerhinger-Ingelheim; Research grant / Funding (institution): GlaxoSmithKline; Research grant / Funding (institution): Roche/Genentech; Research grant / Funding (institution): Karyopharm; Research grant / Funding (institution): Astellas; Research grant / Funding (institution): Bayer; Research grant / Funding (institution): Abbvie; Research grant / Funding (institution): Amgen; Research grant / Funding (institution): Intensity Therapeutics; Research grant / Funding (institution): Mirati; Research grant / Funding (institution): Shattucks. J. Santucci-Pereira: Full / Part-time employment: Bristol-Myers Squibb. D. Nelson: Shareholder / Stockholder / Stock options, Full / Part-time employment: Bristol-Myers Squibb. E.H. Kandoussi: Shareholder / Stockholder / Stock options, Full / Part-time employment: Bristol-Myers Squibb. B. Fischer: Shareholder / Stockholder / Stock options, Full / Part-time employment: Bristol-Myers Squibb. M. Wind-Rotolo: Shareholder / Stockholder / Stock options, Full / Part-time employment: Bristol-Myers Squibb. D. Greenawalt: Shareholder / Stockholder / Stock options, Full / Part-time employment: Bristol-Myers Squibb. Y. Ishii: Shareholder / Stockholder / Stock options, Full / Part-time employment: Bristol-Myers Squibb.

Murine syngeneic tumor models are critical to novel immuno-based therapy development, but the molecular and immunologic features of these models are still not clearly defined. The translational relevance of differences between the models is not fully understood, impeding appropriate preclinical model selection for target validation, and ultimately hindering drug development. Across a panel of commonly used murine syngeneic tumor models, we showed variable responsiveness to immunotherapies. We used array comparative genomic hybridization, whole-exome sequencing, exon microarray analysis, and flow cytometry to extensively characterize these models, which revealed striking differences that may underlie these contrasting response profiles. We identified strong differential gene expression in immune-related pathways and changes in immune cell–specific genes that suggested differences in tumor immune infiltrates between models. Further investigation using flow cytometry showed differences in both the composition and magnitude of the tumor immune infiltrates, identifying models that harbor “inflamed” and “non-inflamed” tumor immune infiltrate phenotypes. We also found that immunosuppressive cell types predominated in syngeneic mouse tumor models that did not respond to immune-checkpoint blockade, whereas cytotoxic effector immune cells were enriched in responsive models. A cytotoxic cell–rich tumor immune infiltrate has been correlated with increased efficacy of immunotherapies in the clinic, and these differences could underlie the varying response profiles to immunotherapy between the syngeneic models. This characterization highlighted the importance of extensive profiling and will enable investigators to select appropriate models to interrogate the activity of immunotherapies as well as combinations with targeted therapies in vivo. Cancer Immunol Res; 5(1); 29–41. ©2016 AACR.

Background TMB is a clinically relevant biomarker associated with response to immune checkpoint inhibitors in patients with NSCLC. Tumor TMB (tTMB) can be assessed by next-generation sequencing (NGS) gene panels; however, obtaining sufficient tumor tissue can be challenging, and NGS methods have been developed for TMB assessment from blood (bTMB). We compared bTMB values using 3 commercial NGS assays that differ in gene number, depth of coverage, and variant allele cutoff. Methods bTMB was assessed in 25 commercial NSCLC plasma samples with 3 NGS assays, and values were compared by Spearman’s correlation. For assay A, 500 genes, sequenced to a depth of ∼ 1500x. To determine concordance between bTMB and tTMB we assessed 86 commercial NSCLC matched plasma and tumor samples using bTMB assays A and C and a clinically validated tTMB gene panel assay, and performed subgroup analysis by disease stage. Results bTMB in stage I–III NSCLC samples assessed by assays B and C showed greater correlation (r = 0.78) than by assays A and B (r = 0.59) and A and C (r = 0.53). Across 86 matched samples, tTMB and bTMB concordance was lower for assay A (r = 0.24) than assay C (r = 0.51) and was higher among stage IV NSCLC samples (A, r = 0.38; C, r = 0.72) than stage I–III (A, r = 0.24; C, r = 0.40). Using clinically relevant cutoffs, high bTMB was observed in 5/30 patients with assay A (range: 10.5–21.1), and 19/30 patients with assay C (range: 12.4–67.6); high tTMB ranged from 10.0 to 36.6. Conclusions In patients with NSCLC, bTMB was concordant (r > 0.5) between 3 NGS assays. Concordance between bTMB and tTMB varied and was improved for bTMB assays with increased coverage and sequencing depth, and in patients with higher stage metastatic disease. Assay parameters impact accurate and reproducible TMB assessment, with lower coverage and sequencing depth, and differing variant allele cutoff, risking false-negative results that may affect outcomes in response to immune checkpoint inhibitor therapy. These data underscore the need for demonstrating clinical utility of bTMB assays and for assessment of bridging analytical performance between assays. Editorial acknowledgement Amrita Dervan, PhD, and Jay Rathi, MA, of Spark Medica Inc, funded by BristolMyers Squibb. Legal entity responsible for the study Bristol-Myers Squibb. Funding Bristol-Myers Squibb. Disclosure J. Baden: Shareholder/Stockholder/Stock options, Full/Part-time employment: Bristol-Myers Squibb; Shareholder/Stockholder/Stock options: Johnson & Johnson. H. Chang: Shareholder/Stockholder/Stock options, Full/Part-time employment: Bristol-Myers Squibb. D.M. Greenawalt: Shareholder/Stockholder/Stock options, Full/Part-time employment: Bristol-Myers Squibb. S. Kirov: Shareholder/Stockholder/Stock options, Full/Part-time employment: Bristol-Myers Squibb. S. Pant: Shareholder/Stockholder/Stock options, Full/Part-time employment: Bristol-Myers Squibb. A. Seminara: Shareholder/Stockholder/Stock options, Full/Part-time employment: Bristol-Myers Squibb. S. Srinivasan: Shareholder / Stockholder / Stock options, Full/Part-time employment: Bristol-Myers Squibb. G. Green: Shareholder/Stockholder/Stock options, Full/Part-time employment: Bristol-Myers Squibb.

Background: Activation of OX40, a costimulatory protein in the tumor necrosis factor receptor super family, enhances T effector cell activation and inhibits T regulatory (Treg) cell-mediated suppression. Preclinical data showed that combination of an OX40 agonist with checkpoint blockade (anti-PD-1 or anti-CTLA-4) enhanced antitumor activity vs checkpoint blockade alone. BMS-986178 is a fully human IgG1 agonist monoclonal antibody that binds with high affinity to OX40 and was well tolerated ± NIVO (anti-PD-1; Olszanski AJ, et al. J Immunother Cancer 2017;5(suppl2) [abstract O17]). The relationship between receptor modulation and the activity of agonist OX40 agents in the clinic is not clear. Here we present findings on the regulation of OX40 receptor expression and T-cell activation by BMS-986178 ± NIVO or IPI (anti–CTLA-4) from the phase 1/2a study in patients with advanced solid tumors (NCT02737475). Methods: Patients with ≥ 1 prior therapy were treated in this open-label, dose-escalation and -expansion study. During escalation, patients received BMS-986178 monotherapy 20-320 mg IV Q2W, BMS-986178 20-320 mg + NIVO 240 mg IV Q2W, or BMS-986178 20-320 mg + IPI 1 mg/kg IV Q3W. Pharmacokinetics (PK) and systemic pharmacodynamics (PD) were evaluated, including analysis of OX40 receptor occupancy (RO), CD4+ T cell- and Treg-cell surface OX40 expression, soluble OX40 (sOX40) levels, cytokines, and proliferation of effector memory T cells in circulation. Results: BMS-986178 PK was linear from 20 to 320 mg when administered as monotherapy (n = 20), + NIVO (n = 38), or + IPI (n = 32). Moreover, BMS-986178 ± NIVO or IPI stimulated the production of IFN-γ and increased proliferating (Ki-67+) effector memory T cells. Concomitant downregulation of OX40 expression on the cell surface of CD4+ T cells and Tregs was observed as OX40 RO approached saturation at BMS-986178 doses of ≥ 40 mg. Furthermore, BMS-986178 doses of 20-160 mg ± NIVO or IPI increased sOX40 levels, while BMS-986178 doses of ≥ 160 mg ± NIVO or IPI led to sOX40 plateau. Conclusions: Clinical PK/PD findings showed efficient BMS-986178 target engagement and confirmed preclinical observations that BMS-986178 can modulate RO and OX40/sOX40 expression. Furthermore, peripheral T-cell activation was observed in patients treated with BMS-986178 ± NIVO or IPI. Coupled with our preclinical observations, these findings highlight a complex dose-response relationship between BMS-986178 receptor saturation, receptor modulation, and induction of soluble receptor. These data support further clinical investigation of a broader dose range and optimal schedules for BMS-986178 ± NIVO or IPI. Citation Format: Rui Wang, Yan Feng, Ed Hilt, Xiling Yuan, Chan Gao, Xiao Shao, Yongliang Sun, Michael D'silva, Katherine Yang, Becky Penhallow, Goce Bogdanoski, Rajesh Anand, Irene Pak, Danielle Greenawalt, Anke Klippel, Nataly Manjarrez-Orduno, Robert Neely, Michael Quigley, Michael Hedrick, Praveen Aanur, Z Cao. From bench to bedside: Exploring OX40 receptor modulation in a phase 1/2a study of the OX40 costimulatory agonist BMS-986178 ± nivolumab (NIVO) or ipilimumab (IPI) in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-127.

Background JAK1 LoF mutations were reported as a mechanism of resistance to anti-PD-1 therapy in patients with metastatic melanoma (n = 2) or mCRC (n = 1).1,2 Here, we report an exploratory analysis of CheckMate-142 (NCT02060188) investigating whether JAK1 LoF mutations were present in patients with dMMR/MSI-H mCRC who achieved clinical benefit with nivolumab + ipilimumab treatment. Methods Patients received nivolumab 3 mg/kg + ipilimumab 1 mg/kg Q3W × 4 doses and then nivolumab 3 mg/kg Q2W. Whole-exome sequencing was performed on pretreatment biopsies preserved in RNAlater and germline DNA from peripheral blood mononuclear cells. Single nucleotide variants and insertions/deletions were called by 2 variant callers in paired normal and tumor samples, and the union of mutations was included in the downstream analysis as previously described.3 Best overall response was assessed by investigators (RECIST v1.1). Results JAK1 LoF mutations were identified in 4 of 40 patients evaluated in this analysis. Of these 4 patients, 1 had an ongoing partial response of 9.7 months at data cutoff, and 3 had stable disease (Table); 2 patients with stable disease were still on treatment (12 and 17 months) at data cutoff, and 1 discontinued treatment after 2.9 months due to an unrelated adverse event and had not progressed after 10 months off treatment. Conclusions Clinical benefit was achieved with nivolumab + ipilimumab in patients with dMMR/MSI-H mCRC who harbored a JAK1 LoF mutation. Additional analyses are needed to assess the effect of concurrent JAK1 loss of heterozygosity and other genetic alterations in patients with dMMR/MSI-H mCRC. 1. Zaretsky JM et al. N Engl J Med. 2016;375:819-829. 2. Shin D et al. Cancer Discov. 2017;7:188-201. 3. Carbone DP et al. N Engl J Med. 2017;376:2415-2426. Table. Best overall response in patients with dMMR/MSI-H mCRC identified as having wild-type JAK1 or a JAK1 LoF mutationJAK1 LoF mutant n = 4JAK1 wild type n = 36Best overall response, nComplete response01Partial response1a18Stable disease3b11Progressive disease04Not evaluable02fs, frameshift. a JAK1 mutation: I975*. b JAK1 mutations: D899Gfs*10, K860Nfs*16, and K142Rfs*26. Citation Format: Scott Kopetz, Theirry Andre, Michael J. Overman, Vittorina Zagonel, Sara Lonardi, Massimo Aglietta, Fabio Gelsomino, Ray McDermott, Ka Yeung Mark Wong, Alain Hendlisz, Pilar Garcia Alfonso, Heinz-Josef Lenz, Alice Walsh, Rebecca A. Moss, Danielle Greenawalt, Z. Alexander Cao. Exploratory analysis of Janus kinase 1 (JAK1) loss-of-function (LoF) mutations in patients with DNA mismatch repair-deficient/microsatellite instability-high (dMMR/MSI-H) metastatic colorectal cancer (mCRC) treated with nivolumab + ipilimumab in CheckMate-142 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2603.

Introduction: In the phase 3 study CheckMate 141 (NCT02105636), patients with platinum-refractory head and neck squamous cell carcinoma treated with nivolumab (NIVO) had longer median overall survival (OS) (7.5 vs 5.1 months; P=0.01) and a higher objective response rate (all: 13.3 vs 5.8%; tumor PD-L1 ≥1%: 17 vs 1.6%) compared with investigator’s choice (IC) (Ferris et al. NEJM. 2016). This exploratory analysis evaluated the immune profile of patients from CheckMate 141, in context of tumor PD-L1 expression, and assessed the relationship with treatment (txt) outcomes. Methods: PD-L1 expression in tumor and tumor-associated immune cells (TAIC) was analyzed at baseline (n=252) and assessed for association with clinical outcome. Tumor PD-L1 expression was quantitatively assessed using Dako IHC 28-8 pharmDx assay. TAIC PD-L1 abundance (numerous/intermediate, rare) and location (intra/intra-peritumoral, peritumoral) were qualitatively assessed (unvalidated). Peripheral blood (n=36) at baseline and day 43 was assessed for immune cell biomarkers by flow cytometry and analyzed by 2-way ANOVA with Sidak’s multiple comparisons test correction. Results: Abundant PD-L1+ TAICs (numerous/intermediate) were associated with greater median OS with NIVO vs IC in tumors with PD-L1 ≥1% (abundant: 8.7 vs 4.4 months, hazard ratio [HR] and 95% CI 0.44 [0.27, 0.71] and rare: 6.7 vs 4.9 months, HR 0.88 [0.42, 1.86]) and PD-L1 Conclusion: In this exploratory, qualitative immune profile analysis, abundance of PD-L1+ TAICs was associated with higher median OS and greater likelihood of response to NIVO vs IC. Response to NIVO may be associated with higher circulating CD8+ T cells and lower Tregs at baseline, and abundant PD-L1+ TAICs in the tumor microenvironment. Citation Format: Robert L. Ferris, George Blumenschein, Kevin Harrington, Jérôme Fayette, Joël Guigay, A. Dimitrios Colevas, Lisa Licitra, Stefan Kasper, Caroline Even, Francis Worden, Nabil F. Saba, Everett Vokes, Cheryl Ho, Fernando Concha-Benavente, Danielle Greenawalt, Chelsea Jin, Mark Lynch, Makoto Tahara, Robert Haddad, Manish Monga, Henry Kao, Maura Gillison. Tumor-associated immune cell PD-L1 expression and peripheral immune profiling: Analyses from CheckMate 141 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT021. doi:10.1158/1538-7445.AM2017-CT021

Our current understanding of the mutation spectrum of relapse/refractory patients is limited. Public sequencing efforts have focused by design, on primary tumors, while re-biopsy of patients who are refractory to first line therapy or who relapse on treatment is not standard protocol. We have performed whole exome sequencing on post standard of care biopsies from 3 patient cohorts, 47 DLBCL patients post-relapse following R-CHOP, 49 triple negative breast (TNBC) following taxol alone or in combination and 41 lung adenocarcinoma (LUAD) following platinum alone or in combination. Comparative analysis of the mutation spectrum of these cohorts was performed against available clinically matched primary tumor cohorts. Comparison of the mutational signatures, somatic variant frequencies and copy number variation between matched pre- and post-treatment samples, and treated-primary tumor cohorts were performed. Significant differences were identified in the frequency of oncogenic drivers including PIK3CA in TNBC and KRAS in LUAD. We also observed mutational signature differences between cohorts that could impact treatment regimens post standard of care. Collectively these data further highlight the value of re-assessing the tumor genetic landscape at the time of treatment rather than relying on primary tumor samples, which may not be representative of the cohort to be treated. Citation Format: Danielle Greenawalt, Austin Dulak, Manasa Ramakrishna, Zhongwu Lai, Justin Johnson, Hongyue Dai, Melissa Mitchell, Carl Barrett, Stephen Fawell, Jonathan Dry. Your targeted population might not be what you predict: Changes in tumor genetic landscapes post standard of care. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3168.

The pre-clinical assessment of immuno-oncology (IO) therapies can be enabled by the use of murine syngeneic tumors established in immuno-competent mice. With the aims of selecting relevant models and of minimizing animal experimentation by reducing the number of models tested, the full characterisation of syngeneic models at the transcriptomic and genomic level is a key objective for pre-clinical scientists. Model characterisation includes global aCGH, exon array analysis and FACS profiling alongside exome sequencing. The model data is undergoing hypothesis free and driven analyses which are already generating valuable insights. Comparison of in vivo tumor samples with their in vitro equivalents has highlighted enrichment for a number of immune pathways; as has the comparison of different tumor lines. The genomic, transcriptomic and ‘proteomic’ model data are being integrated to give a functional output which will act as a ‘Syngenomic Fingerprint’ for each model. The resulting Syngenomic fingerprints will help pre-clinical scientists to refine their in vivo plans through an improved understanding of the limits and advantages as well as the clinical relevance of some of our preclinical models. It is also supporting the targeted modification of models to better match specific human cancer types. Citation Format: John E. Prime, Suzanne Mosely, Jens-Oliver Koopmann, Dennis YQ Wang, Danielle Greenawalt, James Harper, Miika J. Ahdesmaki, Rebecca Leyland, Olivia Harris, Ross Stewart, Philip Brohawn, Brandon Higgs, Bryony Langford, Athula Herath, Robert Kozarski, Jane Coates-Ulrichsen, Judith Anderton, Michelle Morrow, Richard C. A Sainson, Robert W. Wilkinson. Syngenomic fingerprint: the biomic characterization of the mouse syngeneic tumor models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4186.

Our current understanding of the mutation spectrum of relapse/refractory patients is limited. Public sequencing efforts have focused, by design, on diagnostic (pre-treatment) biopsies, while re-biopsy of patients who are refractory to first line therapy or who relapse on treatment is not standard protocol. Many gold standard algorithms used to call mutations perform poorly in ultra-deep sequencing data necessary for sensitive detection of resistance mutations emerging in circulating tumor DNA. They can also miss a wide range of complex genetic aberrations prevalent in cancer genomes. We have performed whole exome sequencing on post treatment core needle biopsies from 47 DLBCL patients post-relapse following anthracycline-based therapy such as R-CHOP, 8 with matched diagnostic biopsies. Similarly we have profiled 88 post-treatment solid tumor biopsies from triple negative breast and lung adenocarcinoma patients recurrent following platinum or multi-agent chemotherapy, 12 with matched diagnostic biopsies. We compared the somatic variant frequencies and copy number variation between matched pre- and post-treatment samples, and with published data for cohorts of primary (diagnostic) samples matched by patient and tumor characteristics. NGS data from all samples were processed using the same mutation caller, VarDict http://github.com/AstraZeneca-NGS/VarDict, optimized to call large InDels and complex variants in ultra-deep sequencing. For DLBCL, the average mutation rate and founding driver mutation profile were similar, however a number of oncogenic driver mutations were found to have emerged in the post-treatment biopsies. Collectively these data further highlight the value of re-assessing the tumor genetic landscape at the time therapeutic choices are made. Citation Format: Dry R. Jonathan, Danielle Greenawalt, Zhongwu Lai, Carl Barrett. Tumor genetic shift post standard of care therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B51.

Bladder cancer is a disease with high unmet need, with no new treatments being registered in the last two decades. Intravesical delivery is an attractive option for organ-confined bladder cancer; indeed, non-muscle invasive disease is currently treated by intravesical administration of agents such as bacillus Calmette-Guerin (BCG) and mitomycin C, and neoadjuvant intravesical therapy has the potential to preserve the bladder and improve survival in M0 muscle invasive disease. Intravesical delivery has the potential to increase the therapeutic index compared with systemic dosing routes, particularly if the duration of exposure can be extended. In order to explore this concept, we have developed an orthotopic model of bladder cancer and a drug delivery method to administer gemcitabine into the nude rat bladder for one week. An orthotopic model of bladder cancer was established by serial passaging of MGH-U3 human bladder cancer cells into the muscle wall. Tumor take-rate and growth was monitored by ultrasound imaging. Serial ex vivo - in vivo passaging increased growth and take rate of the tumors in both orthotopic and subcutaneous locations. Orthotopic implantation of cells following two previous passages in vivo resulted in reproducible tumor growth and take rates of >90%. Immunohistochemistry analysis showed that the orthotopic tumors were stably epithelial in phenotype after three serial in vivo passages. Principal component analysis of gene expression (GE) data from RNA sequencing of the parental cell line, sequentially passaged cells growing ex vivo, and both subcutaneous and orthotopically implanted tumours after various numbers of passages revealed three clusters; two distinct but closely related clusters representing the orthotopic and subcutaneously growing tumors, and another distinct cluster representing the various cell lines. This analysis showed that in vitro/in vivo location of growth is a more important determinant of global GE than the process of serial passaging in vivo. We have used the iPRECIO programmable infusion pump system to continuously infuse gemcitabine into the bladders of rats with orthotopic bladder tumors. Infusion of 2.5 mg/day at a flow rate of 5 ul/h for 7 days resulted in urine concentrations in the range of 1- 10 mM (∼ 1000-fold greater than systemic plasma drug concentrations), a 4-fold increase of γH2AX and induction of both necrosis and apoptosis (10-fold induction of cleaved casapse 3) in the orthotopic tumors. This model system holds considerable potential for evaluation of intravesical small molecule therapies that are potentially translatable to patients. Citation Format: Leigh J. Williams, Stephanie K. Klein, Danielle Greenawalt, Dawn Trueman, Helen Musgrove, Helen Jones, Linette Ruston, Barry R. Davies. Establishment of an orthotopic bladder cancer model to evaluate continuous intravesical delivery of small molecule inhibitors in the nude rat. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5146. doi:10.1158/1538-7445.AM2015-5146

Mutations of MAPK1 E322K are observed in ∼8% of cervical ∼1% and head and neck cancer but there is currently little evidence of their impact on the ERK pathway. This study evaluated the impact of the E322K mutation on the ERK pathway output and feedback using an inducible system pTRIPZ in MCF10A immortalised cells. Expression of E322K MAPK1 following doxycycline induction reduced RSK phosphorylation on T359/S363 but increased pELK1 S383. DUSP6 protein levels were also increased in MCF10A expressing E322K mutant compared to WT. Pathway output was determined using the mRNA MEK signature (Dry et al. 2010). Following Dox induction, MCF10A E322K showed a 4-fold increase in DUSP2 mRNA and a 2-fold increase in DUSP6 mRNA while levels were unchanged in MCF10A expressing WT MAPK1. Conversely, >2-fold decrease in ETV5 levels was observed in E322K MCF10A compared to WT cells. Exposure of WT and E322K MCF10A cells to an ATP competitive ERK inhibitor showed a greater reduction in pRSK T359/S363 levels in E322K cells compared to WT cells (likely due to lower baseline levels) but abrogation of pELK1 required higher concentrations of the inhibitor in mutant compared to WT cells. Consistent with its mechanism of action, the ERK inhibitor increased pERK levels. Interestingly, the combination of the MEK inhibitor selumetinib with the ERK inhibitor completely abrogated pERK in WT but not in E322K mutant MCF10 cells. Overall, the results suggest that the E322K mutation increases ERK-regulated genes such as DUSP2 which may only partially de-phosphorylate ERK due to the E322K mutation affecting substrates’ binding to the D site. The impact of E322K on the sensitivity to ERK and MEK inhibition is currently under evaluation. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C144. Citation Format: Nin Guan, Oona Delpuech, Danielle Greenawalt, Kelly Jacques, Farzin Gharahdaghi, Michael Zinda, Paul J. Smith, Sylvie M. Guichard. MAPK1 mutation E322K modulates ERK pathway output and feedback. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C144.