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Pancreatic ductal adenocarcinoma (PDAC) is projected to be the second leading cause of cancer mortality in the United States by 2030. Given that resistance to cytotoxic therapy is pervasive, there is a critical need to elucidate salient gene expression programs and spatial relationships among malignant and stromal cells in the tumor microenvironment (TME), particularly in residual disease. We developed and applied a single-nucleus RNA-seq (snRNA-seq) technique to 43 banked frozen primary PDAC specimens that either received neoadjuvant therapy (n=25) or were treatment-naïve (n=18). We discovered expression programs across malignant cell and fibroblast profiles that formed the basis for a refined molecular taxonomy, including a novel neural-like progenitor (NRP) malignant program enriched with neoadjuvant treatment in tumors and organoids, and associated with the worst prognosis in bulk profiles from independent cohorts. To elucidate how neoadjuvant treatment and cancer cell- and fibroblast-intrinsic programs modulate the composition of multicellular neighborhoods, we performed spatial profiling with the GeoMx[1] platform (NanoString) on 21 formalin-fixed paraffin-embedded sections using the human whole transcriptome atlas (WTA). Each tumor showed intra-tumoral heterogeneity in tissue architecture and regions of interest (ROIs) with diverse patterns of neoplastic cells, cancer-associated fibroblasts (CAFs), and immune cells were selected for profiling. We deconvolved the WTA data with our snRNA-seq cell type signatures and mapped expression programs onto the tumor architecture to reveal three distinct multicellular neighborhoods, which we annotated as classical, squamoid-basaloid, and treatment-enriched. The observed enrichment in post-treatment residual disease of multiple spatially-defined receptor-ligand interactions and a neighborhood featuring the NRP program, neurotropic CAF program, and CD8+ T cells may open new therapeutic opportunities. Next, we mapped malignant/CAF programs and immune cell subsets at single-cell spatial resolution by performing spatial molecular imaging (SMI[2]; NanoString CosMx) using a panel of 960 RNA targets on a subset of seven tumors (2 untreated, 5 treated) and captured over 200,000 cells with an average of more than 450 transcripts detected per cell. Correlating ROIs from whole-transcriptome DSP to matched fields of view in kiloplex SMI enabled further dissection of PDAC architecture and treatment-associated remodeling of cell type distributions and receptor-ligand interactions. Ongoing functional studies have begun to elucidate the key regulatory elements underlying the distinct treatment-associated NRP malignant program and its interactions with the TME. Overall, the complementary combination of snRNA-seq, whole-transcriptome DSP, and kiloplex SMI provides a high-resolution molecular framework that can be harnessed to augment precision oncology efforts in pancreatic cancer. [1] GeoMx DSP is for Research Use Only and not for use in diagnostic procedures. [2] CosMx SMI is for Research Use Only and not for use in diagnostic procedures. Citation Format: William L. Hwang, Karthik A. Jagadeesh, Jimmy A. Guo, Hannah I. Hoffman, Carina Shiau, Jennifer Su, Payman Yadollahpour, Jason W. Reeves, Youngmi Kim, Sean Kim, Mark Gregory, Prajan Divakar, Eric Miller, Michael Rhodes, Sarah Warren, Erroll Rueckert, Kit Fuhrman, Daniel R. Zollinger, Robin Fropf, Joseph M. Beechem, Arnav Mehta, Toni Delorey, Cristin McCabe, Jaimie L. Barth, Piotr Zelga, Cristina R. Ferrone, Motaz Qadan, Keith D. Lillemoe, Rakesh K. Jain, Jennifer Y. Wo, Theodore S. Hong, Ramnik Xavier, Orit Rozenblatt-Rosen, Andrew J. Aguirre, Carlos Fernandez-Del Castillo, Andrew S. Liss, Mari Mino-Kenudson, David T. Ting, Tyler Jacks, Aviv Regev. Multicellular spatial community featuring a novel neuronal-like malignant phenotype is enriched in pancreatic cancer after neoadjuvant chemotherapy and radiotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr SY12-04.

Neurotrophic tyrosine receptor kinase genes (NTRK1/2/3) act as oncogenic drivers across a range of tumors. NTRK fusions occur at low frequency (90% of secretory breast carcinomas. Furthermore, up to 30% of patients (pts) with breast cancer will develop central nervous system (CNS) metastases (mets). Entrectinib is a potent, oral TRKA/B/C, ROS1 and ALK inhibitor, specifically selected for its CNS penetration properties. Entrectinib was evaluated in 3 global phase 1/2 clinical trials (ALKA-372-001 [EudraCT 2012-000148-88], STARTRK-1 [NCT02097810], and STARTRK-2 [NCT02568267]), where it demonstrated strong and durable systemic and intracranial efficacy in pts with NTRK-fp solid tumors, including those with breast cancer. We present updated data from this integrated analysis (data cut-off: 31 October 2018) focusing on pts with breast cancer. The entrectinib trials were conducted at >150 sites in 15 countries, and enrolled pts with locally advanced/metastatic NTRK-fp tumors (with or without baseline CNS mets) confirmed by nucleic acid-based methods. Tumor assessments were performed at the end of cycle 1 (4 weeks) and every 8 weeks thereafter, and evaluated by blinded independent central review (BICR) using RECIST v1.1. Primary endpoints were objective response rate (ORR) and duration of response (DoR) by BICR. Secondary endpoints included progression-free survival (PFS), overall survival (OS), and safety. At clinical cut-off, the overall efficacy-evaluable population included 74 adults from the 3 trials, with 12 different NTRK-fp tumor types and >25 histopathologies. In these pts, ORR by BICR was 63.5% (95% CI 51.5-74.4), including five complete responses (CR) and 42 partial responses (PR). Median (95% CI) DoR, PFS and OS were 12.9 (9.3-not estimable [NE]), 11.2 (8.0-15.7) and 23.9 (16.0-NE) months, respectively. The efficacy-evaluable NTRK-fp breast cancer cohort included 6 pts with a median age of 63 (range 36-67) years; most had an Eastern Cooperative Oncology Group performance status of 0 (3/6; 50%) or 1 (1/6; 17%). Breast cancer tumors were classified as secretory (4/6; 67%; all NTRK3-fp) or non-secretory (2/6; 33%; all NTRK1-fp). Pts had received 0 (3/6; 50%), 1 (1/6; 17%) or ≥4 (2/6; 33%) lines of prior therapy for metastatic disease. At data cut off, the median survival follow-up was 17.4 (range 1.7-23.9) months. ORR was 100% (2 CR, 2 PR; 95% CI 39.8-100.0) in pts with secretory and 50% (1 PR, 1 missing/unevaluable; 95% CI 1.3-98.7) in pts with non-secretory histology. Median (95% CI) DoR, PFS, and OS were 12.9 (4.2-NE), 10.1 (5.1-NE), and 23.9 (5.1-23.9) months, respectively. At baseline, 2 pts had CNS mets per investigator assessment; 1 of these pts had missing response data. CNS mets were confirmed by BICR in the other pt (non-secretory); this pt had received whole brain radiotherapy 2-6 months before starting entrectinib treatment, and had systemic PR and intracranial non-CR/non-progressive disease (non-measurable CNS lesion). The overall integrated safety-evaluable population comprised 504 pts with any gene fusion who received ≥1 dose of entrectinib. Most treatment-related adverse events (TRAEs) were Grade ≤3 (96.1%); the most frequently reported TRAEs were dysgeusia (39.7%) and fatigue (31.5%). Seven breast cancer pts were evaluated for safety, of whom six (85.7%) reported TRAEs; all were Grade ≤3. The most frequently reported TRAEs (each occurring in 3/7 pts; 42.9%) were nausea, anemia, and increased alanine or aspartate aminotransferase. Dose reductions and interruptions due to TRAEs were each reported in 3/7 pts (42.9%); no discontinuations or deaths due to TRAEs were recorded. In this updated integrated analysis, entrectinib induced objective responses in all pts with NTRK-fp breast cancer who had data available, and was generally well tolerated with no discontinuations due to TRAEs. Citation Format: Janice Lu, Collin M. Blakely, Christophe Le Tourneau, Saiama N. Waqar, Todd M. Bauer, Filippo de Braud, David S. Hong, Alexander Spira, Tira Tan, Samuel McCallum, Stuart Osborne, Brian Simmons, Minal Barve. Efficacy and safety of entrectinib in NTRK fusion-positive (NTRK-fp) breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS11-28.

Background: SHP2 is an oncogenic tyrosine phosphatase that transduces receptor tyrosine kinase signaling to the RAS/MAPK pathway via its phosphatase-mediated regulation of guanine nucleotide exchange factors. ERAS-601 is a potent, selective, and orally bioavailable allosteric inhibitor of SHP2. In combination with cetuximab, an antibody that targets epidermal growth factor receptor (EGFR), ERAS-601 has demonstrated robust nonclinical activity in human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) and RAS/RAF wild-type colorectal cancer (CRC) tumors. Method: FLAGSHP-1 is the first-in-human trial of ERAS-601 administered as monotherapy and in combination with other cancer therapies in patients with advanced or metastatic solid tumors. The primary objectives are to characterize the safety profile, determine the maximum tolerated dose (MTD)/recommended dose (RD), and characterize the pharmacokinetics (PK) profile of ERAS-601 as a monotherapy and in combination with other cancer therapies. Secondary objectives include tolerability and antitumor activity in solid tumors. Presented here are results from the combination dose escalation cohorts in which patients received ERAS-601 twice a day for three weeks followed by a one-week break (BID 3/1) in combination with cetuximab (500mg/m2) administered every 2 weeks (Q2W) on a 28-day cycle. Results: As of October 31, 2022, a total of 15 patients with previously treated advanced or metastatic solid tumors received ERAS-601 BID 3/1 at the following dose levels: 20 mg BID 3/1 (n=4), 40 mg BID 3/1 (n=8), or 60 mg BID 3/1 (n=3) in combination with cetuximab. Combination therapy MTD was determined​ to be 40 mg BID 3/1. ERAS-601 Treatment Related Adverse Events (TRAEs) at or below the MTD were all Grade 1 and 2. TRAEs occurring in ≥20% of patients included diarrhea (27%), AST increase (27%), ALT increase (20%), dermatitis acneiform (20%). Grade ≥3 TRAEs included Grade 4 hypokalaemia, Grade 3 diarrhea, platelet count decreased anemia (each 7%); high grade TRAEs were only observed at 60 mg BID 3/1 (above the MTD). Dose limiting toxicities (DLTs) were only observed at the 60mg BID 3/1 dose levels and included Grade 3 platelet count decreased (n=1) and Grade 4 hypokalemia (n=1). Pharmacokinetics of ERAS-601 and cetuximab in combination were generally comparable to historical monotherapy PK values, suggesting lack of drug-drug interaction. The evaluation of clinical activity is still ongoing. Conclusions: ERAS-601 in combination with cetuximab in patients with previously treated advanced or metastatic solid tumors shows promising preliminary safety and tolerability with reversible and manageable TRAEs. Further evaluation in relevant tumor types are ongoing. Citation Format: Meredith McKean, Ezra Rosen, Minal Barve, Tarek Meniawy, Judy Wang, David S. Hong, Jennifer Yang, Zhengrong Li, Roxana Picard, Les Brail, Daniela Vecchio, Thomas John, Ezra Cohen, Gregory Obara, Aparna Parikh. Preliminary dose escalation results of ERAS-601 in combination with cetuximab in FLAGSHP-1: a phase I study of ERAS-601, a potent and selective SHP2 inhibitor, in patients with previously treated advanced or metastatic solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT184.

BACKGROUND: Intra-tumoral Clostridium novyi-NT (non-toxic) is an attenuated strain of C. novyi lacking alpha toxin replication within hypoxic tumor regions, causing tumor cell lysis and inflammation. Immunotherapy (IO) augments anticancer activity with intratumoral agents in prior preclinical and clinical studies. This phase 1b dose escalation study assessed safety and potential synergistic effects of pembrolizumab and C. novyi-NT in advanced solid tumors. METHODS: We enrolled patients with percutaneous injectable, solid tumors to receive single intratumoral injection of C. novyi-NT administered on Day 8 across 4 dose cohorts (3 × 10^4 to 100 × 10^4 spores, 3+3 design) with pembrolizumab 200mg IV Q3weekly starting on Day 0 up to 24 months. Primary objectives: Safety, tolerability and maximum tolerated dose (MTD) of combination. Secondary objectives: anti-tumor activity of combination in the injected tumor lesion and overall response by iRECIST 1.1. RESULTS: At data cutoff on October 24, 2022, 16 patients were enrolled and evaluable for toxicity and efficacy. It was well tolerated across all cohorts without dose limiting toxicities with median number of 5 cycles administered (1-34). 10 patients (63%) experienced progression; 1 withdrew consent; 1 completed therapy; 1 came off trial due to toxicity (immune related dermatitis) and 3 patients remain on trial. Median age was 62.5 years (40-71) while all patients (100%) had performance status of 1. 8 (50%) received prior IO and 7 (44%) had > 4 lines of prior therapies. Confirmed overall objective response rate (ORR) was 25% in 4 patients [non-keratinizing undifferentiated nasopharyngeal squamous carcinoma (NPC), human papilloma virus positive squamous cell carcinoma of base of tongue, vulvar melanoma and chordoma] with 3 partial responses (PR) and 1 complete response (CR). Among responders, median duration of response was 8.18 months and 1/4 (25%) had prior IO. NPC patient completed 24 months on trial to completion with best response at PR (-87%) while vulvar melanoma patient remains on trial with best response at CR (-100%). Median injected tumor size was 2.65cm (1-11) with confirmed ORR in injected lesions at 19% with 2 PRs and 1 CR. Most prevalent grade 1 and 2 adverse events were injection site reaction (25%), pyrexia (19%), pruritus (13%), leukopenia (13%) and anemia (13%). No grade 3 or 4 treatment related adverse events noted. Signs and symptoms of C. novyi-NT germination (infection) including fever, injection site pain, erythema, swelling, tenderness, and in some cases, ulceration, spontaneous drainage, tissue sloughing, bleeding, and malodor were observed in 5 patients. CONCLUSIONS: Intratumoral C. novyi-NT with pembrolizumab demonstrates clinical activity with favorable tolerability in patients regardless of tumor histology. This study is ongoing to define the recommended phase 2 dose (NCT03435952). Citation Format: Blessie Elizabeth Nelson, Filip Janku, Siqing Fu, Ecaterina I. Dumbrava, David S. Hong, Daniel Karp, Aung Naing, Jordi Rodon, Apostolia Tsimberidou, Rodabe N. Amaria, Anthony Conley, Senthil Damodaran, Kanwal P. Raghav, Brent L. Kreider, David Tung, Mary Varterasian, Khashayarsha Khazaie, Sarina Piha-Paul. Phase Ib Study of Pembrolizumab in Combination with Intratumoral Injection of Clostridium novyi-NT in Patients with Advanced Solid Tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT107.

A Phase I clinical trial is currently underway to test Nelmastobart (hSTC810), an antibody targeting butyrophilin 1A1 (BTN1A1), a novel immune checkpoint protein. This Phase I clinical trial is using a standard 3 + 3 escalation design to (i) explore safety, tolerability, dose-limiting toxicities, and pharmacokinetics, (ii) define a recommended Phase II dose, and (iii) evaluate preliminary efficacy in patients with advanced solid tumors. The current dose range for the clinical trial is 0.3 mg/kg to 15 mg/kg. Currently, all the dose levels have been completed without any dose-limiting toxicity having been observed. Here, we provide an update on the clinical trial data shown previously. First, we provide the results of the pharmacokinetics study of these patients for the selected doses and dosing intervals, including the peak plasma concentration (Cmax), the time to reach Cmax (tmax), and the average plasma concentration of hSTC810 over the dosing interval in steady state (Cavg). The change in cytokine expression and immune cell concentrations in patient blood samples were also analyzed. Specifically, a panel of cytokines was measured including IFN- γ, IL-2, IL-4, IL-6, IL-10, TNF-α, MCP-1, and TGF- β, while flow cytometry was used to detect a panel of immunological marker proteins including CD3, CD4, CD8, CD19, CD14, CD16, CD56, CD25, CD45. For pharmacokinetic studies, the concentrations of hSTC810 and anti-hSTC810 antibodies were quantified. Immunohistochemistry was performed on patient tumor biopsies that were stained for PD-L1, CD3, CD4, CD8, and BTN1A1 expression. The resultant slides were analyzed for PD-L1 tumor proportional score (TPS), PD-L1 combined positive score (CPS), BTN1A1 scoring, the percentage of CD3+ lymphocytes, CD4+ lymphocytes, CD8+ lymphocytes, tumor-infiltrating lymphocytes, and necrosis. Citation Format: Stephen Sunghan Yoo, Soohyeon Lee, Sangjoon Shin, Hyunjin Jung, David S. Hong. Pharmacokinetic analyses and immunophenotyping of patients treated with Nelmastobart (hSTC810), a novel immune checkpoint inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT238.

Background: NTRK gene fusions are oncogenic drivers in various tumor types. Laro, a highly selective and central nervous system (CNS)-active tropomyosin receptor kinase (TRK) inhibitor, is approved for the treatment of patients (pts) with TRK fusion cancer. Laro demonstrated an objective response rate (ORR) of 69% in 244 pts with solid (non-primary CNS) tumors (Drilon et al. ASCO 2022). Here, we report on the concordance between baseline local tissue-based testing with central tissue-based and liquid biopsy-based ctDNA testing to identify NTRK gene fusions. Methods: Pts with non-primary CNS TRK fusion cancer who underwent independent review committee assessments by July 15, 2019, in three laro clinical trials (NCT02122913, NCT02576431, and NCT02637687) were included. Tumor NTRK gene fusion status was determined locally by a variety of methods, and centrally by next-generation sequencing in both tissue (using Illumina TruSight™ Oncology Comprehensive) and ctDNA (using Guardant360® or GuardantOMNI®) at baseline. Data cut-off: July 20, 2021. Results: Of the 164 pts with NTRK fusions identified by local testing, 117 had evaluable tissue samples for central testing, and 99 had evaluable liquid biopsy samples. In total, 81.2% (95/117) of tumors had the fusion confirmed by central tissue testing. ctDNA testing detected NTRK gene fusions in 33.3% (33/99) of patients. Positive predictive values for each NTRK gene fusion are shown in the Table. The ORR for pts with NTRK gene fusions determined by local and central testing will be presented. Conclusions: A high proportion of tumors with locally identified NTRK gene fusions were confirmed centrally. At present, analysis of ctDNA is significantly less sensitive at detecting NTRK gene fusions. A negative ctDNA result requires next-generation sequencing testing of a tissue biopsy. Further research is needed to improve the sensitivity of ctDNA gene fusion detection. Table NTRK gene fusion Tissue-based LT Tissue-based CT ctDNA-based CT with LB Fusion confirmed, n Fusion confirmed, n Fusion not detected, n Unavailable samples, n† PPV‡: CT vs tissue-based LT, % Fusion confirmed, n Fusion not detected, n Unavailable samples, n† PPV‡: LB vs tissue-based LT, % NTRK1 68 35 14 19 71.4 20 34 14 37.0 NTRK2 4 3 1 0 75.0 0 4 0 0 NTRK3 92§ 57 7 28 89.1 13 28 51 31.7 †Includes no sample available and sample QC failed. ‡Calculated as a percentage of the number of confirmed fusions over the number of available samples. §Includes 9 inferred NTRK3 fusions. CT, central testing; ctDNA, circulating tumor DNA; LB, liquid biopsy; LT, local testing; NTRK, neurotrophic tyrosine receptor kinase; PPV, positive predictive value; QC, quality control. Citation Format: Erin R. Rudzinski, Marc Ladanyi, Lauren L. Ritterhouse, Felix Sahm, Ricarda Norenberg, Hong Zheng, Chi Chen, Vadim Bernard-Gauthier, Nicoletta Brega, Cornelis M. van Tilburg, Jessica J. Lin, Marcia S. Brose, Ulrik N. Lassen, Ray McDermott, Theodore W. Laetsch, David S. Hong, Alexander Drilon, Sinchita Roy-Chowdhuri. Concordance between tissue and circulating tumor DNA (ctDNA) testing for neurotrophic tyrosine receptor kinase (NTRK) gene fusions in larotrectinib (laro) clinical trials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT228.

Background: Irreversible allosteric KRAS p.G12C inhibitors (KG12Ci) such as sotorasib and adagrasib have revolutionized the therapeutic landscape of advanced KG12C-mutant NSCLC, however individual responses are heterogeneous and curtailed by innate and adaptive/acquired resistance. Molecular determinants of KG12Ci efficacy in NSCLC are poorly defined. We dissected the impact of major KG12C co-mutations and explored the effects of less prevalent co-alterations on the clinical activity of KG12Ci in the largest treated cohort to date of patients (pts) with advanced NSCLC. Key findings were validated in preclinical KG12C NSCLC models. Methods: Baseline clinico-genomic features and clinical outcome data from pts with stage IV KG12C NSCLC (ECOG PS 0-2) treated with single-agent KG12Ci were collected retrospectively from 20 centers in the US and Europe. The Kaplan-Meier method was used to estimate PFS and OS and differences were assessed with the log-rank test. Hazard ratios (HR) and their 95% CI were estimated using a Cox proportional hazards model stratified for clinical co-variates. The impact of selected co-alterations on sotorasib efficacy was assessed in syngeneic (C57BL/6) KG12C NSCLC models. Results: 411 eligible pts were included in the study. Median age was 68 years, 77% of pts had received both platinum-based chemotherapy and PD-(L)1 inhibitors and 35% had brain metastases. 83% of pts received sotorasib. ORR with KG12Ci was 32.4% (95% CI, 27.9-37.1), PFS was 5.1m (95% CI, 4.5-5.6) and OS was 10.2m (95% CI, 8.4-12.1). Co-alterations in KEAP1, SMARCA4 and CDKN2A/B were each associated with significantly shorter PFS (KEAP1: 2.8m vs 5.5m, HR 2.50, P Conclusions: Co-mutations in KEAP1, SMARCA4 and CDKN2A/2B define subgroups of KG12C NSCLC pts with markedly distinct outcomes with KG12Ci monotherapy. Tailoring of KG12C inhibitor-anchored therapeutic strategies and patient stratification should take into account the co-mutation status of individual tumors. Citation Format: Marcelo V. Negrao, Haniel A. Araujo, Giuseppe Lamberti, Alissa J. Cooper, Teng Zhou, Neal Akhave, Lukas Delasos, J Kevin Hicks, Mihaela Aldea, Gabriele Minuti, Jacobi Hines, Jacqueline V. Aredo, Michael J. Dennis, Turja Chakrabarti, Susan Scott, Paolo Bironzo, Matthias Scheffler, Petros Christopoulos, So Yeon Kim, Sarah Goldberg, Ying Ni, Blerina Resuli, Lorenza Landi, Shu-Chi Tseng, Mizuki Nishino, Dwight Owen, Collin Blakely, Giannis Mountzios, Catherine A. Shu, Christine Bestvina, Marina Garassino, Kristen Marrone, Jhanelle Gray, Sandip Pravin Patel, Amy L. Cummings, Heather A. Wakelee, Jurgen Wolf, Giorgio V. Scagliotti, Federico Cappuzzo, Fabrice Barlesi, Pradnya Patil, Don L. Gibbons, Funda Meric-Bernstam, J Jack Lee, John V. Heymach, David S. Hong, Rebecca S. Heist, Mark M. Awad, Ferdinandos Skoulidis. Molecular determinants of KRAS p.G12C inhibitor efficacy in advanced NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3431.

Introduction: Clinical trials are exploring ATR inhibitors (ATRi) in genomically selected contexts. However, dose-dependent anemia has limited the therapeutic index of this class. We hypothesized that monocytes and reticulocytes are more vulnerable to ATRi due to their lack of base excision repair and high levels of oxidative stress, respectively. We sought to examine the kinetics of decline and recovery of red blood cell (RBC) and reticulocyte indices, in relation to hemoglobin (Hb), in the first 28 days after ATRi initiation. Methods: We retrospectively retrieved peripheral blood cell indices from complete blood count (CBC) reports of patients (pts) pre- and during treatment with an oral ATRi on phase I/II trials at our center. Pts received ATRi monotherapy or in combination with a PARP inhibitor (ATRi+PARPi) in dose-escalation and expansion cohorts, which included ATRi at potentially toxic doses. We applied linear mixed effect models to evaluate the joint evolution of Hb and other RBC or reticulocyte indices over time via bivariate analysis. A random intercept was included in the model to account for the longitudinal nature of the data and a variance component option for the covariance structure was specified to model a different variance component for each index of interest. To assess if Hb decline was preceded by decline in the index of interest from baseline, piecewise regression models were used to estimate break points, defined as the time point where the fitted functions intersect, of each index. Log transformed data with a base of 2, were noted to have more symmetric distributions, and were used in the analyses. The time variable assessed was days from cycle 1 day 1 of ATRi. A two-sided p value Results: 35,007 indices from 1,843 CBC of 119 pts treated with an ATRi from 10/2017 to 1/2022 were analyzed. 110 (92.4%) pts received ATRi, 9 (7.6%) received ATRi+PARPi. Monocytes (-0.120 vs -0.022, p Conclusions: Charting the kinetics of CBC index evolution in relation to Hb identified peripheral monocytes and reticulocytes as relevant blood indices that herald Hb decline. Our findings may inform patient monitoring strategies to mitigate hematologic toxicity on future ATRi trials. Citation Format: Natalie Y. Ngoi, Heather Y. Lin, Ecaterina Dumbrava, Siqing Fu, Daniel D. Karp, Aung Naing, Shubham Pant, Sarina A. Piha-Paul, Jordi Rodon, Vivek Subbiah, Apostolia M. Tsimberidou, Erick Campbell, Samuel Urrutia, David S. Hong, Funda Meric-Bernstam, Ying Yuan, Timothy A. Yap. Dynamic changes in monocyte and reticulocyte counts predict mechanism-based anemia development and recovery during ATR inhibitor treatment in phase I/II trials. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6181.

Introduction: HER2 protein expression by immunohistochemistry (IHC) has been the main testing method for breast and gastric cancers. Much remains unknown about the frequency of HER2-low (IHC 1+ or 2+) expression and the change of HER2 status from primary to metastatic tumor samples. There is limited data available about the HER2-low frequency in solid tumors beyond breast and gastric/gastroesophageal (GEJ) cancer. Methods: HER2 IHC was performed in 923 patients (pts) with metastatic breast and 51 pts with gastric/GEJ cancers in CLIA-certified labs. We evaluated the HER2 IHC in paired primary and metastatic samples in breast cancer. We also assessed the HER2 status in non-breast, non-gastric/GEJ histologies. In available cases, concurrent HER2 genomic alterations were evaluated. Results: A total of 867 pts with breast cancer had a definitive HER2 IHC score for their metastatic sample: 101 pts (11.6%) were 3+, 171 pts (19.7%) were 2+, 252 pts (29%) were 1+ and 343 (39.6%) had no HER2 expression. The HER2 IHC scores in metastatic samples as compared to primary was in concordance in 54.9% of cases, while HER2 3+ remained unchanged in 87.2%, kappa=0.85 with a 95% CI = (0.79, 0.91). In 48 cases with metastatic gastric/GEJ cancer, HER2 was: 3+ in 2 pts (4.2%), 2+ in 5 pts (10.4%), 1+ in 9 pts (18.8%) and 32 (66.7%) had no HER2 expression. In non-breast and non-gastric/GEJ cancers, HER2-low was found in 631 pts (33.5%) as shown in the table below. A total of 1,552 pts who had HER2 IHC also had genomic testing: 36 had HER2 activating mutations, 48 had HER2 amplification (amp) and 3 had HER2 amp and mutations. 22 of 33 pts with HER2 mutations (without amp) had HER2 IHC expression: 10 pts (30%) were 1+, 10 (30%) were 2+ and 2 (6%) were 3+. Conclusion: Our results show that HER2-low expression is frequently found across tumor types and HER2 activating mutations are enriched in cancers where HER2 is expressed. These findings suggest that a high number of pts with solid tumors with HER2-low expression could potentially benefit from HER2 directed therapies. HER2 expression in solid tumors Cancer Types HER2 expression levels (IHC) Total* 3+(N pts) 3+(%) 2+(N pts) 2+(%) 1+(N pts) 1+(%) 0 (N pts) 0 (%) Appendiceal 0 0 3 6 8 16 39 78 50 Cancer of Unknown Primary 2 4 8 15 15 28 29 54 54 Cholangiocarcinoma 5 4 15 12 35 29 67 55 122 Colorectal 19 3 46 8 118 20 394 68 577 Endometrial 16 13 49 39 10 8 52 41 127 Gallbladder 3 9 8 24 7 21 15 45 33 HNSCC 2 9 1 4 4 17 16 70 23 Salivary 6 13 10 21 14 29 18 38 48 Hepatocellular Carcinoma 0 0 8 25 6 19 18 56 32 Non-small cell lung cancer 6 3 31 15 68 33 100 49 205 Ovarian/Fallopian Tube 4 5 17 20 11 13 53 62 85 Pancreas 1 1 5 7 16 24 45 67 67 Prostate 0 0 0 0 4 13 27 87 31 Renal Cell Carcinoma 0 0 0 0 1 5 18 95 19 Sarcoma 0 0 1 3 1 3 35 95 37 Small Bowel Cancer 2 9 2 9 1 4 18 78 23 Thyroid 0 0 0 0 2 4 54 96 56 Urothelial Carcinoma 38 22 48 27 32 18 57 33 175 *more than 15 samples available; HNSCC=head and neck squamous cell carcinoma Citation Format: Burak Uzunparmak, Cara Haymaker, Gabriela Raso, Chen Zhu, Serena Masciari, Lei Wang, Bryce Kirby, Heather Lin, Aysegul Gorur, Ann-Marie Cimo, Amber Kennon, Qingqing Ding, Andrea Palmieri, Gabriele Elizabeth Urschel, Ying Yuan, Guoxin Feng, Yasmeen Rizvi, Aisha Hussain, Vivek Subbiah, Timothy Anthony Yap, Jordi Rodon-Ahnert, Sarina Anne Piha-Paul, David S. Hong, Funda Meric-Bernstam, Ecaterina E. Dumbrava. HER2-low expression in patients with advanced or metastatic solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 961.

Background: Combinations of HER2 and aromatase or mTOR inhibitors demonstrated activity in the clinical setting. We hypothesized that the triple combination of HER2 targeted therapy, aromatase and mTOR inhibitor has increased anticancer activity. Methods: We designed a 3+3 dose escalation phase I study of the aromatase inhibitor letrozole 2.5mg PO daily, mTOR inhibitor everolimus 2.5-10mg PO daily and HER2 antibody trastuzumab 4-8mg loading dose followed by 2-4mg maintenance dose IV on day 1 of 21-day cycle in patients with hormone-receptor positive, HER2-positive/amplified or mutant advanced cancers (confirmed by immunohistochemistry and/or FISH and/or next-generation sequencing) with preplanned expansion cohort at for patients with metastatic breast cancer to determine maximum tolerated dose (MTD) and/or recommended phase 2 dose (RP2D), dose limiting toxicities (DLT), overall safety and response (NCT02152943). Results: A total of 32 patients (men, 1; women, 31; HER2 amplification, 28; HER2 mutation, 4; breast cancer, 26; other cancers, 6), median age 55.5 years, median of 5 prior therapies (including letrozole [13] or other aromatase inhibitor [10]; everolimus [3]; trastuzumab [25] or other HER2 targeted therapy [2]) were enrolled in the planned 6 dose levels. The MTD has not been reached and letrozole 2.5mg PO daily, everolimus 10mg PO daily and trastuzumab 8mg loading dose followed by 4mg maintenance dose IV on day 1 of 21-day cycle was declared as RP2D. DLTs included grade 3 (G3) mucositis (1 patient) at dose level 3, and G3 thrombocytopenia, neutropenia (1 patient) at dose level 4. Other G3 or G4 treatment-related toxicities included G4 hyperglycemia in 1 patient, G3 hyperglycemia in 3 patients, G4 anemia in 1 patient, G3 anemia in 2 patients, G3 thrombocytopenia in 1 patient, G3 transaminitis in 1 patient, G3 mucositis in 1 patient and G3 headache in 1 patient. Of 32 patients, 5 (16%) had a partial response (all with heavily-pretreated breast cancer with HER2 amplification [4] or HER2A775_G776insYVMA mutation [1]), 23 (72%) stable disease (SD) including 5 (16%) patients with SD > 12 months (all with heavily-pretreated breast cancer) and 4 (13%) progressed. The median change in size of target lesions per RECIST 1.1. was -5% (-91% to +47%). Median time to treatment failure (TTF) was 4.3 months (95% CI 0.0-9.6). A total of 14 patients had serial plasma collection to assess dynamics of circulating tumor DNA and clonal evolution and the data will be presented at the meeting. Conclusions: The combination of letrozole, everolimus and trastuzumab is well tolerated with encouraging activity in heavily-pretreated patients with HER2-amplified or mutant advanced breast cancer. Citation Format: Alexej Ballhausen, Jennifer J Wheler, Daniel D Karp, Sarina A Piha-Paul, Siqing Fu, Shubham Pant, Apostolia M Tsimberidou, David S Hong, Vivek Subbiah, Veronica R Holley, Helen J Huang, Abeena M Brewster, Kimberly H Koenig, Nuhad K Ibrahim, Funda Meric-Bernstam, Filip Janku. Everolimus, letrozole and trastuzumab in hormone receptor-positive, HER2-positive/amplified or mutant metastatic cancer: Evaluating synergy and overcoming resistance [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 P1-19-18.

Pancreatic ductal adenocarcinoma lethality can be attributed to a combination of rapid metastatic dissemination and intrinsic resistance to conventional therapies. Our prior studies using single cell RNA-seq in pancreatic circulating tumor cells (CTCs) revealed that these “seeds of metastasis” had a biphenotypic state of both epithelial and mesenchymal features. This suggested that CTCs are highly plastic cells that exist in an intermediate state along epithelial (E) to mesenchymal (M) transition (EMT). Analysis of CTC markers in primary PDAC tumors revealed that this subpopulation of cancer cells was concentrated at the interface of tumor cells and stromal cancer associated fibroblasts (CAFs). To evaluate the contribution of CAFs in PDAC EMT plasticity, we utilized patient derived PDAC-CAF co-culture preclinical models demonstrating that PDAC EMT heterogeneity was modulated by the density of CAFs and partially driven by TGF-b secretion. Moreover, analysis of patient derived PDAC tumor spheres treated with FOLFIRINOX (FFX) demonstrated selection or induction of EMT changes that were also found in patient tumors that were resected after neoadjuvant FFX. Altogether, our collective work along with others demonstrates the importance of EMT plasticity in PDAC cell metastatic propensity and the ability to resist cytotoxic chemotherapy. A multi-institutional randomized Phase II trial supported by SU2C-Lustgarten Foundation evaluating the role of the TGF-b modulating activities of losartan on locally advanced PDAC response to chemotherapy and suppression of metastatic dissemination has neared completion. This 3 arm (n=40 per arm) trial of neoadjuvant FFX, FFX + losartan, or FFX+ losartan + nivolumab (anti-PD1). Here, we have performed EMT RNA in situ hybridization in all post-treatment resection specimens to determine if there is a difference in EMT proportions between the different arms of the study. As an orthogonal unbiased approach, we have utilized the NanoString GeoMx Digital Spatial Profiler whole transcriptome assay (18,000+ protein coding genes) on each of the resection specimens with analysis of the tumor cells, CAFs, and immune cell separately for multiple regions of interest in these specimens. This provides deeper molecular insight of the PDAC tumor cells that resisted neoadjuvant therapy, the changes in the surrounding CAFs, and the modulation of immune infiltrates that might differ between each arm of the study. We anticipate the results of the trial will be completed before the conference and initial correlative analysis of spatial transcriptomics and RNA-ISH will be presented. Citation Format: Amaya Pankaj, Michael J. Raabe, Bidish Patel, Evan R. Lang, Joshua Kocher, Katherine H. Xu, Linda T. Nieman, Alec C. Kimmelman, David P. Ryan, Theodore S. Hong, William L. Hwang, Martin Aryee, David T. Ting. Characterizing the effects of neoadjuvant therapy in PDAC [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR004.

Purpose: Adding losartan to FOLFIRINOX (FFX) chemotherapy followed by chemoradiation (CRT) resulted in 61% R0 surgical resection in our phase II trial in patients with locally advanced pancreatic cancer. Here we identify potential mechanisms of benefit by assessing the effects of neoadjuvant losartan+FFX+CRT versus FFX+CRT on the stromal tumor microenvironment. Experimental Design: We performed a gene expression analysis of RNA extracted from pancreatic cancer tissue sections and immunofluorescence for cancer cells and immune cells using archived surgical samples from patients treated with losartan+FFX+CRT (NCT01591733), FFX+CRT (NCT01591733) or surgery upfront, without any neoadjuvant therapy. We then assessed whether certain gene sets could stratify the overall survival of patients. Results: Neoadjuvant losartan+FFX+CRT and FFX+CRT increased the expression of genes linked to vascular normalization, transendothelial migration of leukocytes, T cell activation and cytolytic activity, and dendritic cell related genes versus no neoadjuvant treatment. In comparison to FFX+CRT, losartan+FFX+CRT downregulated pro-invasion, immunosuppression, and M2 macrophages related genes, and upregulated genes associated with tumor suppression, including the p53 pathway. Furthermore, immunostaining revealed significantly less residual disease in lesions treated with losartan+FFX+CRT versus FFX+CRT. Losartan+FFX+CRT also reduced CD4+FOXP3+ regulatory T cells in pancreatic cancer lesions with a complete/near complete response. Overall survival was associated with dendritic cell and antigen presentation genes for patients treated with FFX+CRT, and with immunosuppression and invasion genes or dendritic cell- and blood vessel-related genes for those treated with losartan+FFX+CRT. Conclusions: Adding losartan to FFX+CRT reduced pro-invasion and immunosuppression related genes, which were associated with improved treatment outcomes in patients with locally advanced pancreatic cancer. Citation Format: Yves Boucher, Jessica M. Posada, Sonu Subudhi, Spencer R. Rosario, Liqun Gu, Ashwin S. Kumar, Heena Kumra, Mari Mino-Kenudson, Nilesh P. Talele, Dan G. Duda, Dai Fukumura, Jennifer Y. Wo, Jeffrey W. Clark, David P. Ryan, Carlos Fernandez-Del Castillo, Theodore S. Hong, Mikael J. Pittet, Rakesh K. Jain. Addition of losartan to FOLFORINOX and chemoradiation downregulates pro-invasion and immunosuppression-associated genes in locally advanced pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C043.

Intratumoral nerves play important and versatile roles in cancer initiation, progression, recurrence, treatment-resistance, metastasis, morbidity, and mortality for many malignancies but the diverse molecular mechanisms underlying tumor-nerve crosstalk remain largely unknown. One of the differentiating hallmarks of pancreatic ductal adenocarcinoma (PDAC) is an exceptionally high frequency of perineural invasion (PNI), a histopathologic manifestation of tumor-nerve crosstalk whereby cancer cells recruit, migrate towards, and envelop or invade peripheral nerves. Evidence for some neurochemicals/neurotrophins involved in PNI have been uncovered, but most of the underlying work was limited by a lack of cell-type specificity, spatial context, and fragmented focus on individual pathways. To address these shortcomings, we set out to comprehensively identify cell-type specific genes spatially linked to PNI in patient tumors and then dissect the functional roles of these genes through live imaging of dorsal root ganglia (DRG) sensory neurons incubated in conditioned media from cancer cell organoids overexpressing candidate genes via CRISPR activation (CRISPRa). First, we performed whole transcriptome digital spatial profiling (NanoString GeoMx) on twelve custom tissue microarrays (n=288 cores) derived from intratumorally-matched malignant regions with and without PNI in primary resected PDAC specimens (n=31 patients). Differential gene expression (DE) analysis (FDR < 0.001) for PNI demonstrated that for malignant cells there were 271 enriched and 65 depleted genes, and for fibroblasts there were 16 enriched and 27 depleted genes. We further evaluated associations between PNI and expression of malignant subtypes previously identified from single-nucleus RNA-seq applied to 43 primary resected PDAC specimens. We found that malignant cells engaged in PNI were enriched in the mesenchymal, basaloid and neural-like progenitor (NRP) subtypes and depleted in the classical subtype. To test these associations functionally, we generated isogenic murine organoid lines (KrasG12D/+;Trp53FL/FL;R26-dCas9-VPR) overexpressing subtype-driving transcription factors and collected conditioned media. DRG sensory neurons demonstrate enhanced and suppressed growth kinetics when grown in NRP and classical conditioned media, respectively; mesenchymal and basal-like conditioned media do not appear to influence growth kinetics. These results suggest that while mesenchymal, basaloid, and NRP cells likely all play a role in cancer cell invasion of nerves, NRP cells may have an additional role in tumor-nerve tropism. Additional experiments exploring the functional effects of the top enriched and depleted genes from the DE analysis are ongoing. We anticipate that this study will provide a high-resolution understanding of critical intercellular interactions in the PDAC tumor microenvironment that facilitate PNI and tumor-nerve crosstalk more broadly to guide novel therapeutic strategies. Citation Format: William L. Hwang, Jennifer Su, Jimmy A. Guo, Carina Shiau, Jaimie L. Barth, Hannah I. Hoffman, Prajan Divakar, Jason W. Reeves, Eric Miller, Grissel Cervantes-Jaramillo, William Freed-Pastor, Vanessa Funes, Jennifer Y. Wo, Theodore S. Hong, Carlos Fernandez-del Castillo, Lei Zheng, Andrew J. Aguirre, David T. Ting, Mari Mino-Kenudson, Tyler Jacks. Identifying mediators of perineural invasion in pancreatic cancer using spatial transcriptomics [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C052.

Background: Tropomyosin receptor kinase (TRK fusions) involving the genes NTRK1, NTRK2, and NTRK3 occur in a broad range of malignancies including breast cancers (BC) with secretory features. Larotrectinib, the first selective TRK inhibitor in clinical development, has demonstrated an overall response rate of 75% by independent radiology review across various solid tumors, with a favorable safety profile. Here we report on a case series of TRK fusion BC patients treated with larotrectinib alone or in combination. Methods: Patients were treated with oral larotrectinib in the NAVIGATE global phase II study or in single-patient compassionate-use protocols. All patients received the Phase 2 dose of 100mg BID on a continuous 28d schedule. Efficacy was evaluated using RECIST v1.1. Results: As of June 2018, 5 TRK fusion BC patients had been treated, all with secretory characteristics, but diverse ER/PR/HER2 positivity (Table); 2 patients were triple negative. Four patients harbored ETV6-NTRK3 fusions. Larotrectinib treatment yielded a response rate of 80% (4/5 PRs). All responses occurred within the first 2 cycles of therapy and cancer-related symptoms resolved rapidly. No treatment-related Grade ≥3 adverse events have been reported. Patient 2 (14 yr) presented with a recurrent fungating mass (10.4x8.5 cm) having failed multiple rounds of chemotherapy. Significant reduction in tumor size was noted after one week of larotrectinib treatment with near complete resolution after 2 months.1 Patient 3 (37 yr) had extensive involvement of the lung and pleura, bilateral pleural effusions, peritoneal infiltration with ascites, severe dyspnea with PS=3. There was rapid improvement with larotrectinib treatment and the patient was PS=1 after 2 weeks. At 6 weeks, there was >80% reduction in tumor size.2 Patient 5 had a synchronous, locally advanced TRK fusion positive, ER+/HER2- secretory BC and TRK fusion negative, ER+/HER2- metastatic lobular BC. Prior to TRK inhibition, this patient received 2-months of palbociclib/letrozole to which the TRKfusion negative lobular cancer responded, while the TRK fusion SBC did not. The patient tolerated the larotrectinib/letrozole combination with no notable toxicities and experienced a PR in both sites, now ongoing for 11 months. Conclusions: We provide the first evidence that larotrectinib is effective in the treatment of BC harboring NTRK gene fusions. Assays capable of identifying NTRK gene fusions should be considered when profiling patients with BC, especially for patients with secretory BC. Table:Subject summarySubject IDSubtypeRegimenNTRK gene fusionER/PR/HER2 statusBest ResponseProtocol1Invasive ductal carcinoma (NOS)larotrectinibTPM3-NTRK1ER+/PR+/HER2-PDNAVIGATE Ph II2SecretorylarotrectinibETV6-NTRK3ER-/PR-/HER2-PRCompassionate use3Invasive ductal with secretory featureslarotrectinibETV6-NTRK3ER-/PR-/HER2-PRCompassionate use4Invasive ductal with secretory featureslarotrectinibETV6-NTRK3ER+/PR+/HER2+PRCompassionate use5*Secretorylarotrectinib + letrozoleETV6-NTRK3ER+/PR-/HER2-PRCompassionate use*- synchronous TRK fusion negative invasive lobular 1. Shukla et al., JCO Precis Oncol 2017, epub 2. Landman et al., Clin Breast Cancer, 2018, 18:e267-e270. Citation Format: Meric-Bernstam F, Shukla N, Peled N, Landman Y, Onitilo A, Montez S, Ku NC, Hyman DM, Drilon A, Hong DS. Activity of larotrectinib, a highly selective inhibitor of tropomyosin receptor kinase, in TRK fusion breast cancers [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-20-02.

Autologous T-cells engineered with T-cell receptors (TCRs) targeting tumor antigens are promising therapies for metastatic solid cancers.1 Specific peptide enhanced affinity receptor (SPEAR) T-cell therapies are T-cells with engineered HLA-restricted TCRs that precisely target tumor cells with specific antigens, such as MAGE-A4 (a cancer testis antigen), presented on the surface by HLA molecules. In SPEAR T-cell clinical trials targeting MAGE-A4, a 2-step prescreen is done before enrolment. 1) Patients undergo HLA typing via a high-resolution (allelic, 4-digit) sequence-based assay, and those who are positive for the inclusion alleles (HLA-A*02:01P, 02:02P, 02:03P, 02:06P) and not positive for the exclusion allele A*02:05P are eligible. 2) Tumor MAGE-A4 testing is done via an immunohistochemical clinical trial assay (MAGE-A4+ cutoff: ≥30% tumor cell staining at ≥2+ intensity) in HLA-eligible patients. A screening protocol (NCT02636855) has been used in Phase 1 trials of first- and next-generation SPEAR T-cells targeting MAGE-A4 (NCT03132922, NCT04044859) with responses in multiple MAGE-A4+ tumors. As of November 19, 2021, 6,168 patients with 9 solid tumor types were screened at 32 sites across North America and Europe in this screening protocol; among which, 2,744 were HLA-eligible (eligibility rate: 45%, range per tumor type: 42%-55%). HLA-A*02:01 was the most frequent HLA-A*02 allele. Of these HLA-eligible patients, 1,549 had tumor tissues evaluable for MAGE-A4 expression; among which, 313 were MAGE-A4+ (MAGE-A4+ rate: 20%, range: 8%-54%) (Table). MAGE-A4 showed highest prevalence in synovial sarcoma and myxoid/round cell liposarcoma but was seen across all tumor types investigated. Our results will be discussed in the context of tumor histopathology, disease status, and demography. HLA and MAGE-A4 biomarker data will inform the therapeutic opportunities for SPEAR T-cells targeting MAGE-A4 in metastatic solid cancers. 1. D’Angelo et al. Cancer Discov. 2018;8:944. HLA eligibility and MAGE-A4 prevalence in a screening protocol (NCT02636855) Indication Esophageal cancer Esophagogastric junction cancer Gastric cancer Head and neck squamous cell carcinoma Non-small cell lung cancer Melanoma Ovarian cancer Urothelial cancer Synovial sarcoma and myxoid/round cell liposarcoma HLA screened (N) 284 228 271 601 3189 668 539 270 118 HLA eligible (%) 46 45 43 42 43 50 49 43 55 MAGE-A4 evaluable (N) 104 91 73 200 457 245 225 93 61 MAGE-A4 positive (%) 22 25 8 22 14 16 24 32 54 Citation Format: Tianjiao Wang, Jean-Marc Navenot, Stavros Rafail, Mark Carroll, Ruoxi Wang, Cheryl McAlpine, Swethajit Biswas, Francine Brophy, Erica Elefant, Paige Bayer, Sandra McGuigan, Dennis Williams, George Blumenschein, Marcus Butler, Jeffrey M. Clarke, Justin F. Gainor, Ramaswamy Govindan, Victor Moreno, Janet Tu, David S. Hong. Identifying MAGE-A4-positive tumors for SPEAR T-cell therapies in HLA-A*02-eligible patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB001.

Background: Adoptive cellular therapies (ACT) have met with significant success in hematologic malignancies. The number of ACT trials in solid tumors has been increasing, however data are limited on the overall safety and efficacy of ACT in this population. We aimed to conduct a systematic review and meta-analysis looking specifically at TCR and CAR-T in solid tumors and rates of cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) relative to the current approved agents in hematologic malignancies. Methods: We conducted a systematic review of published studies/abstracts from January 2015 up to and including July 2021, selecting those that included ≥ 5 patients. Endpoints included rates and differences in CRS and ICANS. Response rates were evaluated. Statistical tests in the pooled data included χ2 and multivariable logistic regression. Results: This meta-analysis included a total of 1,250 patients [566 patients with solid tumors who had received ACT in phase I, II or I/II clinical trials; a comparison was made with five phase 2 clinical trials in hematologic malignancies used for the first FDA approvals of these agents (JULIET, ZUMA-1, ZUMA-2, TRANSCEND and KARMMA), encompassing 684 heme malignant patients]. Rates of CRS of any grade, CRS ≥ grade 3, ICANS of any grade, and ICANS ≥ grade 3 in solid tumors were 33.0%, 3.8%, 2.3% and 2.1%, respectively. Fever, hypotension, and hypoxia were experienced by 37.4%, 18.5%, and 8.5% of solid tumor patients, respectively. CRS, ICANS, fever and hypoxia in solid tumor patients did not differ by whether the ACT received was TCR or CAR T. Objective response (ORR = CR + PR) in patients with solid tumors was higher with TCR compared with CAR-T [odds ratio (OR) = 3.1, P=0.001] and with NY-ESO-1 than other targets (OR=2.0, P=0.031). Compared to hematologic patients treated with ACT, solid tumor patients treated with adoptive cellular therapies were significantly less likely to experience CRS of any grade (62.4% vs. 33.0%, P Conclusions: CRS and ICANs were less prevalent in solid tumor cellular therapies. Based on this analysis, T-cell therapies in solid tumors are relatively safe, but further studies with larger populations and longer follow up are needed to address the role of these treatments in cancer. Citation Format: Mirella Nardo, Ji Y. Son, Goldy G. George, David S. Hong. Safety and efficacy of CAR T and TCR therapies in solid tumors: A systematic review and meta-analysis, including a comparison with five phase II trials in hematologic malignancies used for the first FDA approvals of these agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2764.

Introduction: Sotorasib, a first-in-class KRASG12C inhibitor, has been FDA-approved for adults with KRAS p.G12C-mutated locally advanced or metastatic NSCLC who received prior systemic therapies based on the Phase 1/2 global, single-arm CodeBreaK100 trial. We will report the longest follow-up for a KRASG12C inhibitor, including 2-year survival, safety, and genomic profiles associated with durable clinical benefit. Methods: Sotorasib was administered orally at 960 mg once daily to patients who progressed on prior therapies and had KRAS p.G12C-mutated locally advanced, metastatic NSCLC. Primary endpoint was objective response rate (ORR) assessed by central review. Key secondary endpoints included duration of response (DOR), progression-free survival (PFS), overall survival (OS), and safety. In an exploratory analysis, baseline tumor tissue and/or plasma samples were collected and analyzed for genomic alterations. Tumor samples were analyzed for PD-L1 levels to evaluate correlates with prolonged tumor response, defined as patients with PFS ≥ 12 months, in comparison with those with PFS ≤ 3 months. Results: In the first analysis of the combined Phase 1 and Phase 2 study (N=174), the median number of prior lines of therapy was 2.0 (range 1-4+). 90.2% received prior anti-PD1 or anti PD-L1 treatment; 82.8% received both prior platinum-based chemotherapy and anti-PD1/PD-L1. Updated ORR by central review was 40.7% (95% CI: 33.2, 48.4) and median DOR was 12.3 months (7.1, 14.6). Median PFS and OS was 6.3 months (95% CI: 5.3, 8.2) and 12.5 months (10.0, 17.8). One and two-year OS was 50.8% and 30.3%. Sotorasib was well-tolerated in the long-term with mild and manageable toxicities, and no new safety signals emerged. Prolonged tumor response was observed across PD-L1 expression, including in tumors with low PDL-1 expression and STK11 co-mutation that may derive less benefit from immunotherapy. Conclusions: In the longest follow-up of any KRASG12C inhibitor, sotorasib continued to demonstrate a favorable safety profile and durable efficacy, including a 2-year OS observed in 30% of patients. Current analyses continue to support long-term clinical benefit across subgroups in patients with KRAS p.G12C-mutated NSCLC, and additional biomarker data will be presented. Citation Format: Grace K. Dy, Ramaswamy Govindan, Vamsidhar Velcheti, Gerald S. Falchook, Antoine Italiano, Juergen Wolf, Adrian G. Sacher, Toshiaki Takahashi, Suresh S. Ramalingam, Christophe Dooms, Dong-Wan Kim, Alfredo Addeo, Jayesh Desai, Martin Schuler, Pascale Tomasini, Qui Tran, Simon Jones, Agnes Ang, Abraham Anderson, Antreas A. Hindoyan, David S. Hong, Bob T. Li. Long-term outcomes with sotorasib in pretreated KRASp.G12C-mutated NSCLC: 2-year analysis of CodeBreaK100 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT008.

Introduction: Neurotrophic tyrosine receptor kinase (NTRK) gene fusions are oncogenic drivers in various tumor types. Gene fusions arise from inter- and intrachromosomal rearrangements involving the 3’ region of the NTRK gene and the 5’ end of a fusion partner gene, leading to the expression of a constitutively active tropomyosin receptor kinase (TRK) fusion protein. Larotrectinib is a highly selective and central nervous system (CNS)-active TRK inhibitor approved for the treatment of adult and pediatric patients with TRK fusion cancer. Larotrectinib has previously demonstrated an objective response rate (ORR) of 75% in 206 patients with non-primary CNS solid tumors (Hong et al, ASCO 2021). Here, we report on the TRK fusion exon junction organization in patients with TRK fusion cancer receiving larotrectinib. Methods: Patients with non-primary CNS TRK fusion cancer treated with larotrectinib were identified from three clinical trials (NCT02122913, NCT02576431, and NCT02637687). NTRK gene fusion data were determined by local molecular testing. The testing method, NTRK gene fusions, and exon structure were extracted from the molecular pathology reports from 212 patients. The data cut-off was July 20, 2020. Results: TRK fusion exon junction data were available for 80 patients from 14 different tumor types. Median age was 47 years (range 0.5-84 years). The ORR for these patients was 82.5% (95% confidence interval [CI] 72.4-90.1). Patients had gene fusions involving NTRK1 (40%), NTRK2 (4%), or NTRK3 (56%), a pattern similar to that seen in the larger dataset. We detected 20 different NTRK gene fusions; the most common were ETV6-NTRK3, TPM3-NTRK1, and LMNA-NTRK1 detected in 39 (49%), ten (13%), and seven (9%) patients, respectively. There were 12, three, and five different fusion partners associated with NTRK1, NTRK2, and NTRK3, respectively. The majority (12/20; 60%) of fusions were intrachromosomal rearrangement events: 8/12 (67%) related to NTRK1, 2/3 (67%) related to NTRK2, and 2/5 (40%) related to NTRK3. There were 35 different 5’/3’ gene breakpoint combinations: 3’ fusion points were located at NTRK1 exon 8 (n = 2), NTRK1 exon 9 (n = 5), NTRK1 exon 10 (n = 15), NTRK1 exon 11 (n = 6), NTRK1 exon 12 (n = 3), NTRK1 exon 13 (n = 1), NTRK2 exon 15 (n = 2), NTRK2 exon 16 (n = 1), NTRK3 exon 14 (n = 21), and NTRK3 exon 15 (n = 24). For ETV6-NTRK3 fusions, the NTRK3 fusion point was at exon 14 in ten of 11 thyroid carcinomas, but in only three of 13 salivary gland tumors. Conclusions: Among 80 patients with TRK fusion cancer, we detected 20 different NTRK gene fusions, of which 55% occurred in only one patient each. There were 36 different fusion variants. Despite the variability of the fusion structure, larotrectinib was equally efficacious. The large number of potential different fusion partners and involvement of various breakpoints highlight the need for validated and appropriate testing methodologies that are agnostic of 5’ partners and breakpoints. Citation Format: Marc Ladanyi, Sinchita Roy-Chowdhuri, Lauren Ritterhouse, Felix Sahm, Ricarda Norenberg, Kui Shen, Chi Chen, Marc Fellous, Nicoletta Brega, Cornelis M. van Tilburg, Jessica J. Lin, Marcia S. Brose, Ulrik N. Lassen, Ray McDermott, Theodore W. Laetsch, David S. Hong, Alexander Drilon, Erin R. Rudzinski. Variability in NTRK gene fusions does not appear to impact response to larotrectinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT545.

Purpose: A phase II trial in patients with locally-advanced pancreatic cancer (LAPC) revealed unprecedented rates of complete surgical resection after adding losartan (L) to FOLFIRINOX (FFX) chemotherapy followed by chemoradiation (CRT) (NCT01821729). The aim of this study was to identify potential mechanisms of benefit by assessing the effects of FFX-L+CRT and FFX+CRT on the stromal tumor microenvironment. Experimental Design: We performed a gene analysis of RNA extracted from pancreatic cancer (PC) tissue sections (NanoString nCounter PanCancer Immune Profiling Panel of 730 genes) and immunohistochemistry using surgical samples from patients treated with FFX+CRT (N=15), FFX-L+CRT (N=17) or underwent surgery upfront, without any neoadjuvant therapy (N=9). Results: In comparison to untreated PC, we found 314 and 243 differentially expressed genes (DEGs, adjusted p-value < 0.05) in FFX-L+CRT and FFX+CRT, respectively, and 54 DEGs between FFX-L+CRT and FFX+CRT. PCs from both neoadjuvant FFX-L+CRT and FFX+CRT had increased expression of genes linked to blood vessel maturation (CDH5, THBS1), transvascular migration of leukocytes (JAM3, PECAM1, MCAM, ICAM2), T cell activation (CD6, ALCAM, NFATC1), cytolytic activity of NK cells and T cells (GZMA, GZMB, GZMH, KLRB1) and dendritic cell (DC) related genes (CD209, CD1C, IL3RA). The FOXP3 gene — encoding for a transcription factor that regulates the activity of CD4+ regulatory T cells (Tregs)— was down-regulated in FFX-L+CRT versus untreated samples. Direct comparison of FFX-L+CRT versus FFX+CRT showed increased expression of genes involved in lymphocyte activation (NFATC4, DPP4, STAT5B), and reduced expression of genes that regulate B cell activity (CD22, TRAF3, MS4A1) and CEACAM6, which promotes PC invasion. We also analyzed the correlation of each gene (n=730) with overall survival (OS). For patients treated with FFX-L+CRT, improved OS was negatively correlated with genes that promote invasion in PC (PBK), B cell development and signaling (SYK, BLNK), infiltration of monocytes and macrophages in tumors (CCR2), immune checkpoint (BTLA) and inhibit angiogenesis (TNFSF15). In patients treated with FFX+CRT, OS was positively correlated with genes that stimulate inflammation (IL32), T cell and DC activation (CD48), presentation of glycolipid antigens (CD1E) and antigen processing and presentation by DCs (CD209). Immunohistochemistry studies revealed significantly less residual disease and a higher infiltration of CD8+CD3+ T cells in FFX-L+CRT than FFX+CRT treated tumors. In addition, in patients treated with FFX-L+CRT we found significantly fewer Tregs in PC lesions with a complete/near complete versus poor/no response, confirming our transcriptomic findings. Conclusions: Our findings suggest that FFX-L+CRT can normalize the vasculature, and reduce invasion and the immunosuppressive effects of B cells, CCR2-positive macrophages and Tregs, and thus improve treatment outcome in patients with LAPC, although additional studies are needed. Citation Format: Yves Boucher, Jessica M. Posada, Sonu Subudhi, Ashwin S. Kumar, Ivy X. Chen, Mei R. Ng, Mari Mino-Kenudson, Nilesh Talele, Dan G. Duda, Dai Fukumura, Janet E. Murphy, Jeffrey W. Clark, David P. Ryan, Carlos Fernandez-Del Castillo, Theodore S. Hong, Rakesh K. Jain. Addition of losartan to FOLFIRINOX and chemoradiation reduces the expression of pro-invasive and immunosuppressive genes in locally-advanced pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-097.

Lung cancers with activating KRAS mutations are characterized by treatment resistance and poor prognosis. In particular, the basis for their resistance to radiation therapy is poorly understood. Here, we describe a radiation resistance phenotype conferred by a stem-like subpopulation characterized by mitosis-like condensed chromatin (MLCC), high CD133 expression, invasive potential, and tumor-initiating properties. Mechanistic investigations defined a pathway involving osteopontin and the EGFR in promoting this phenotype. Osteopontin/EGFR–dependent MLCC protected cells against radiation-induced DNA double-strand breaks and repressed putative negative regulators of stem-like properties, such as CRMP1 and BIM. The MLCC-positive phenotype defined a subset of KRAS-mutated lung cancers that were enriched for co-occurring genomic alterations in TP53 and CDKN2A. Our results illuminate the basis for the radiation resistance of KRAS-mutated lung cancers, with possible implications for prognostic and therapeutic strategies. Cancer Res; 77(8); 2018–28. ©2017 AACR.

Introduction: Neurotrophic tyrosine receptor kinase (NTRK) gene fusions are oncogenic drivers in various tumor types. Larotrectinib is a first-in-class, highly selective, central nervous system (CNS)-active TRK inhibitor approved in over 40 countries, including the US, for the treatment of adult and pediatric patients with TRK fusion cancer. Larotrectinib was approved by the FDA in 2018 based on efficacy and safety outcomes in a primary analysis cohort of 55 patients with TRK fusion cancer from 3 clinical trials (Drilon A et al. NEJM. 2018). Here, we report long-term efficacy follow-up for larotrectinib and molecular data on co-occurring mutations in this primary analysis cohort. Methods: Patients with non-primary CNS TRK fusion cancer treated with larotrectinib were identified from 3 clinical trials (NCT02122913, NCT02576431, NCT02637687). NTRK gene status was determined by local molecular testing. Adult and pediatric patients received larotrectinib 100 mg and 100 mg/m2 twice daily, respectively. The primary endpoint was investigator-assessed objective response rate (ORR) (per RECIST v1.1). Next-generation sequencing was performed on pre-treatment tumor samples from a subset of patients with evaluable tumor material (n=31) using the FoundationOne® CDx assay. The data cut-off was July 15, 2019. Results: A total of 55 patients with 17 different tumor types were included in the primary analysis cohort. Median age was 45 years (range 0.3-76 years). Patients had gene fusions involving NTRK1 (45%), NTRK2 (2%), or NTRK3 (53%). For 31 patients the detailed genomic profile, including co-occurring mutations, will be presented. Forty-four (80%) patients had received prior systemic therapy, with a median of 2 prior therapies (range 0-10). ORR with larotrectinib was 80% (95% confidence interval [CI] 67-90%); 13 (24%) patients had a complete response (CR), 31 (56%) patients had a partial response (PR), 5 (9%) patients had stable disease, and 6 (11%) patients had progressive disease. With longer follow-up, best response for 4 patients improved from PR to CR. Median duration of response and progression-free survival were 35.2 months (95% CI 19.8-not estimable [NE]) and 25.8 months (95% CI 9.9-NE), respectively. Median overall survival (OS) was not reached (95% CI 44.4-NE) over a median follow-up of 32.5 months. The 36-month OS rate was 76% (95% CI 63-89%). In an expanded safety population (n=279), larotrectinib-related adverse events were mainly Grade 1-2. Conclusions: Larotrectinib demonstrated a robust response rate with long durability and extended survival benefits in adult and pediatric patients with TRK fusion cancer. These data highlight the importance of identifying NTRK gene fusions in patients with cancer. Citation Format: Alexander Drilon, Shivaani Kummar, Catherine M. Albert, Ramamoorthy Nagasubramanian, Jaclyn F. Hechtman, John A. Reeves, Georg Beckmann, Marion Rudolph, Justyna A. Wierzbińska, Laura Dima, Nicoletta Brega, Theodore W. Laetsch, David S. Hong. Long-term outcomes of patients with TRK fusion cancer treated with larotrectinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT020.

Highly multiplexed imaging technologies are increasingly applied in biomedical research to identify underlying patterns of disease. As part of an IMI-funded project, the IMMUcan (Integrated immunoprofiling of large adaptive cancer patients cohorts) consortium will generate single-cell resolved molecular profiles for thousands of cancer patients from hospitals across Europe. Patient tissues will be profiled using two imaging modalities, multispectral immunofluorescence (mIF) and Imaging Mass Cytometry (IMC), in junction with data from different omics techniques. IMC enables highly multiplexed tissue imaging, but its current throughput only enables to image a fraction of large/whole-slide tumor tissue sections. The selection of regions of interest for IMC analysis is of critical importance with respect to the cells present in an area and the antibody panel used for the analysis of such an area. To target areas of interest in tumor tissue sections, e.g. with immune cell infiltration or tertiary lymphoid structures, we have developed a data driven approach based on mIF imaging analyses from consecutive sections to select such regions of interest for analysis with IMC. A mIF panel was designed to stain T cells, B cells, macrophages, neutrophils, dendritic cells and tumor cells to have an overview of immune cell populations within the whole tissue. After quantification, mIF images and data are hosted on a webserver and regions that contain cell-types of interest are annotated with the help of a web-based tool. mIF images are used for alignment with brightfield whole slide scans of the consecutive FFPE sections which are used for IMC. After alignment, the annotated regions of interest are acquired in IMC. The mean positional offset from IF to IMC for regions of interest was roughly 120 µm and the overall cell-type numbers across both technologies were well correlated. The approach presented here has the great advantage to allow us to reproducibly target similar areas for IMC across thousands of samples based on mIF within the IMMUcan consortium and will enable single-cell analysis for whole tumor sections based on mIF and IMC to gain a deeper understanding of the underlying tumor biology. Funding: IMI2 JU grant agreement 821558, supported by EU's Horizon 2020 and EFPIA. Citation Format: Daniel Schulz, Stephanie Renaud-Tissot, Robin Liechti, Nils Eling, Michelle Daniel, Jonas Windhager, Yervand Karapetyan, Thomas Mrowiec, Sylvie Rusakiewicz, Henoch S. Hong, Marie Morfouace, Bernd Bodenmiller. A toolbox enabling a data-driven selection of regions of interest in tumor tissue sections for imaging mass cytometry analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2756.

A molecular classification of pancreatic ductal adenocarcinoma (PDAC) that informs clinical management remains elusive. Previously identified bulk expression subtypes in the untreated setting were influenced by contaminating stroma whereas single cell RNA-seq (scRNA-seq) of fresh tumors under-represented key cell types. Two consensus subtypes have arisen from these prior efforts: (1) classical-like, and (2) basal-like. Basal-like tumors were associated with worse survival in the metastatic setting but attempts to refine this binary classification have failed to further stratify patient survival. Here, we developed a robust single-nucleus RNA-seq (snRNA-seq) technique for banked frozen PDAC specimens and studied a cohort of untreated resected primary tumors (n ~ 20). Gene expression programs learned across malignant cell and cancer-associated fibroblast (CAF) profiles uncovered a clinically-relevant molecular taxonomy with improved prognostic stratification compared to prior classifications. Digital spatial profiling revealed an association between malignant cells expressing basal-like programs and greater immune infiltration with relatively fewer macrophages, whereas those exhibiting classical-like programs were linked to inflammatory CAFs and macrophage-predominant microniches. Recent clinical trials have supported the increasing adoption of neoadjuvant therapy to aggressively address the risk of micro-metastatic spread and to circumvent concerns of treatment tolerance in the postoperative setting. There is an urgent need to understand how preoperative treatment impacts residual tumor cells and their interactions with other cell types in the tumor microenvironment to identify additional therapeutic vulnerabilities that can be exploited. Towards this end, we performed snRNA-seq on an unmatched cohort of neoadjuvant-treated resected primary tumors (n ~ 25) with most cases involving FOLFIRINOX chemotherapy followed by chemoradiation. Remarkably, the quality of single-nucleus mRNA profiles was comparable between heavily pre-treated and untreated specimens. We identified differentially expressed genes between treated and untreated samples to infer cell-type specific reprogramming in the residual tumor. This analysis revealed that in the neoadjuvant treatment context, there was lower expression of classical-like phenotypes in malignant cells in favor of basal-like phenotypes associated with TNF-NFkB and interferon signaling as well as the presence of novel acinar and neuroendocrine classical-like states. Our refined molecular taxonomy and spatial resolution may help advance precision oncology in PDAC through informative stratification in clinical trials and insights into compartment-specific therapies. Citation Format: William L. Hwang, Karthik A. Jagadeesh, Jimmy A. Guo, Hannah I. Hoffman, Payman Yadollahpour, Jason Reeves, Eugene Drokhlyansky, Nicholas Van Wittenberghe, Samouil Farhi, Denis Schapiro, George Eng, Jason M. Schenkel, William A. Freed-Pastor, Orr Ashenberg, Clifton Rodrigues, Domenic Abbondanza, Toni Delorey, Devan Phillips, Jorge Roldan, Debora Ciprani, Marina Kern, Jaimie L. Barth, Daniel R. Zollinger, Kit Fuhrman, Robin Fropf, Joseph Beechem, Colin Weekes, Cristina R. Ferrone, Jennifer Y. Wo, Theodore S. Hong, Orit Rozenblatt-Rosen, Andrew J. Aguirre, Mari Mino-Kenudson, Carlos Fernandez-del- Castillo, Andrew S. Liss, David T. Ting, Tyler Jacks, Aviv Regev. Multi-compartment reprogramming and spatially-resolved interactions in frozen pancreatic cancer with and without neoadjuvant chemotherapy and radiotherapy at single-cell resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 94.

Background: Mesothelin overexpression is present in over 80,000 patients/year with either malignant mesothelioma (MPM), ovarian cancer, cholangiocarcinoma, or non-small cell lung cancer (NSCLC) in the U.S. alone. We are testing a novel genetically engineered anti-mesothelin TRuC™ T cell therapy called gavo-cel (TC-210) in a Phase 1 study in treatment refractory patients with any of the four aforementioned cancers (NCT03907852). Methods: Gavo-cel was engineered by transducing T cells with a lentiviral vector encoding for an anti-mesothelin llama-derived single domain antibody fused to the CD3epsilon subunit using a flexible glycine serine sequence. Upon translation, gavo-cel TRuCs integrate and reprogram intact TCR complexes to recognize tumor surface mesothelin in an HLA-independent manner. Eligibility criteria required demonstration of mesothelin overexpression on tumors (≥50% of tumor cells expressing 2+ or 3+ staining by IHC) at a central laboratory. Gavo-cel dose escalation proceeded following a modified 3+3 schema. TC-210 expansion and serum cytokine levels were serially measured. Results: Seven patients (6 MPM, 1 ovarian) received a single gavo-cel intravenous infusion at the initial dose of 5x107/m2 either alone (dose level [DL] 0, n=1) or following lymphodepletion (LD) (DL1, n=6) with fludarabine (30mg/m2/day x4) and cyclophosphamide (600mg/m2/day x3). The median age was 69 years (range, 36-84) and the median number of prior therapies was 5 (range, 2-9). Four patients had received ≥4 lines of therapy, including immune checkpoint inhibitors (n=5), the anti-mesothelin ADC anetumab ravtansine (n=1), and anti-mesothelin mRNA CAR-T (n=1). Four patients required bridging therapy. No on-target/off-tumor toxicities or neurotoxicity events of any grade were observed. Grade (gr) ≥3 treatment emergent AEs occurred in 2 (29%) patients with MPM treated at DL1: 1 with gr3 CRS, and 1 with gr3 CRS, gr3 pneumonitis and gr5 fungal sepsis (unrelated to gavo-cel). CRS and pneumonitis events resolved with tocilizumab and corticosteroids. Grade 3 pneumonitis was considered a DLT, resulting in DL1 cohort expansion to 6 patients. At data cut-off date (November 8, 2020), 6 patients have been followed for >3 months, including 4 followed through month 6. The disease control rate was 100%. All patients experienced target lesion regression by CT: median 61% (range, 5-75%). Three (43%) patients (2 with mesothelioma and 1 with ovarian cancer) had a partial response by RECIST v1.1 criteria, including 1 achieving a complete metabolic response. After a median follow-up of 6.5 months, the median OS at 6 months was (86%). The addition of LD increased TC-210 peak expansion and persistence, with cells being detected up to 30 days post infusion in peripheral blood. Conclusion: Intravenous systemic administration of gavo-cel was generally safe and resulted in three of seven patients having objective partial response. Dose escalation is ongoing at 1x108/m2. Updated clinical and translational data will be presented. Citation Format: David S. Hong, Melissa Johnson, Janos L. Tanyi, Lauren MacMullen, Robert Tighe, Lizzy Jalbert, Viera P. Muzithras, Kevin Zikaras, Alfonso Quintas Cardama, Raffit Hassan. Preliminary safety and efficacy of gavocabtagene autoleucel (gavo-cel, TC-210), a T cell receptor fusion construct (TRuC™), in patients with treatment refractory mesothelin overexpressing solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT105.

Innovative molecular diagnostics deployed in the clinic enable new ways to stratify patients into appropriate treatment regimens. These approaches may resolve a major challenge for early-phase clinical trials, which is to recruit patients who, while having failed previous treatments, may nevertheless respond to molecularly targeted drugs. We report the findings of a prospective, single-center study conducted in patients with diverse refractory cancers who underwent comprehensive genomic profiling (CGP; next-generation sequencing, 236 genes). Of the 500 patients enrolled, 188 (37.6%) received either matched (N = 122/188, 65%) or unmatched therapy (N = 66/188, 35%). The most common reasons that patients were not evaluable for treatment included insufficient tissue, death, or hospice transfer. The median number of molecular alterations per patient was five (range, 1–14); median number of prior therapies, four. The most common diagnoses were ovarian cancer (18%), breast cancer (16%), sarcoma (13%), and renal cancer (7%). Of the 339 successfully profiled patients, 317 (93.5%) had at least one potentially actionable alteration. By calculating matching scores, based on the number of drug matches and genomic aberrations per patient, we found that high scores were independently associated with a greater frequency of stable disease ≥6 months/partial/complete remission [22% (high scores) vs. 9% (low scores), P = 0.024], longer time-to-treatment failure [hazard ratio (HR) = 0.52; 95% confidence interval (CI) = 0.36–0.74; P = 0.0003], and survival (HR = 0.65; 95% CI = 0.43–1.0; P = 0.05). Collectively, this study offers a clinical proof of concept for the utility of CGP in assigning therapy to patients with refractory malignancies, especially in those patients with multiple genomic aberrations for whom combination therapies could be implemented. Cancer Res; 76(13); 3690–701. ©2016 AACR.

Molecular subtyping of pancreatic ductal adenocarcinoma (PDAC) remains in its nascent stages and does not currently inform clinical management or therapeutic development. Previously identified bulk expression subtypes in the untreated setting were influenced by contaminating stroma whereas single cell RNA-seq (scRNA-seq) of fresh tumors under-represented key cell types. Two consensus subtypes have arisen from these prior efforts: (1) classical-pancreatic, encompassing a spectrum of pancreatic lineage precursors, and (2) basal-like/squamous/quasi-mesenchymal, characterized by loss of endodermal identity and aberrations in chromatin modifiers. Basal-like tumors were associated with poorer responses to chemotherapy and worse survival in the metastatic setting but attempts to refine this binary classification have failed to further stratify patient survival. Recent clinical trials have supported the increasing adoption of neoadjuvant therapy to aggressively address the risk of micro-metastatic spread and to circumvent concerns of treatment tolerance in the postoperative setting. There is an urgent need to understand how preoperative treatment reprograms residual tumor cells to identify additional therapeutic vulnerabilities that can be exploited in combination with neoadjuvant CRT. Here, we developed a robust single-nucleus RNA-seq (snRNA-seq) technique for frozen archival PDAC specimens and used it to study both untreated tumors (n = 15) and those that received neoadjuvant CRT (n = 11). Gene expression programs learned across malignant cell and fibroblast profiles uncovered a clinically relevant molecular taxonomy with improved prognostic stratification (median survival: 11.2 months in highest risk group to 44.7 months in lowest risk group) compared to prior classifications. Moreover, in the neoadjuvant treatment context, there was lower expression of classical-like phenotypes in malignant cells in favor of basal-like phenotypes associated with TNF-NFkB and interferon signaling as well as the presence of novel acinar and neuroendocrine classical-like states, which may be more resilient to cytotoxic treatment. These results suggest that differentiated endodermal phenotypes are only prevalent enough to be detected under treatment selection pressure and when observed in treatment-naïve bulk studies, may reflect normal cell contamination. Spatially-resolved transcriptomics revealed an association between malignant cells expressing basal-like programs and higher immune infiltration with increased lymphocytic content, whereas those exhibiting classical-like programs were linked to sparser macrophage-predominant microniches, perhaps pointing to distinct therapeutic susceptibilities. Our refined molecular taxonomy and spatial resolution may help advance precision oncology in PDAC through informative stratification in clinical trials and insights into differential therapeutic targeting leveraging the immune system. Citation Format: William L. Hwang, Karthik A. Jagadeesh, Jimmy A. Guo, Hannah I. Hoffman, Eugene Drokhlyansky, Nicholas Van Wittenberghe, Samouil Farhi, Denis Schapiro, Jason Reeves, Daniel R. Zollinger, George Eng, Jason M. Schenkel, William A. Freed-Pastor, Clifton Rodrigues, Domenic Abbondanza, Debora Ciprani, Jennifer Y. Wo, Theodore S. Hong, Andrew J. Aguirre, Orit Rozenblatt-Rosen, Mari Mino-Kenudson, Carlos Fernandez-del Castillo, Andrew S. Liss, Tyler E. Jacks, Aviv Regev. Single-nucleus and spatial transcriptomics of archival pancreatic ductal adenocarcinoma reveals multi-compartment reprogramming after neoadjuvant treatment [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PR-007.

Advances in our understanding of tumor immune biology and development of cancer immunotherapies have led to improved outcomes for patients who suffer from aggressive cancers such as melanoma. Despite the clinical success of immune checkpoint blockade, a majority of patients still fail to respond, and the underlying mechanisms that drive resistance remain unclear. To understand why a subset of tumors fail to respond to immunotherapy, we established a novel murine model of melanoma that is fully resistant to immune checkpoint blockade. By in vivo passaging nonresponding B16 melanoma tumor cells, we selected for a resistant variant that fails to respond to the combination of CTLA-4, PD-1, and PD-L1 blockade. In comparing gene expression of parental versus resistant tumor cells and analyzing the corresponding immune infiltrate, we determined the adaptations associated with resistance to therapy. We found that evasion of immunotherapy was associated with a “hypermetabolic” phenotype, characterized by an upregulation of glycolytic, oxidoreductase, and mitochondrial oxidative phosphorylation pathways to establish a hypoxic, metabolically hostile microenvironment. Enforced expression of two key genes associated with these pathways in parental tumor cells was sufficient to mediate resistance to triple checkpoint blockade. Flow cytometry assays determined that T cells infiltrating resistant tumors had diminished glycolytic capacity and effector function, indicating a metabolic disadvantage. Consistent with our findings, melanoma patients who failed dual checkpoint blockade exhibited similar metabolic alterations as seen in our resistant variant. Using a novel MRI-based imaging approach, we observed distinct metabolic changes that stratified responding versus nonresponding tumors in live mice. Applying this method to patients could provide insight into predicting response rates to checkpoint modulation. Overall, our data indicate that resistant melanoma tumor cells acquire a “hypermetabolic” phenotype to establish a hostile microenvironment that is capable of inhibiting the antitumor immune response. This abstract is also being presented as Poster A24. Citation Format: Ashvin R. Jaiswal, Arthur J. Liu, Shivanand Pudakalakatti, Prasanta Dutta, Priyamvada Jayaprakash, Todd Bartkowiak, Casey Ager, Zhiqiang Wang, Alex Reuben, Zachary Cooper, Cristina Ivan, Zhenlin Ju, Felix Nwajei, Jing Wang, Michael A. Davies, R. Eric Davis, Jennifer A. Wargo, Pratip K. Bhattacharya, David S. Hong, Michael A. Curran. Melanoma evolves complete immunotherapy resistance through acquisition of a hypermetabolic phenotype [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr PR02.

Background: Immunotherapy has produced durable responses in advanced cancers with microsatellite instability. However, response rates remain very low for microsatellite stable (MSS) adenocarcinomas, and biomarkers predictive of response are urgently needed. PI3K pathway alterations are known to modulate anti-tumor immune microenvironment. We previously reported increased cytotoxic T cell infiltration and greater clinical benefit from immunotherapy among PIK3CA mutant MSS colorectal cancer patients as compared to PIK3CA wild-type patients. Here, we investigated the immune repertoire and treatment outcomes with anti-PD-L1 checkpoint inhibitors in PIK3CA mutant MSS metastatic adenocarcinoma patients. Methods: We retrospectively identified metastatic adenocarcinoma patients treated with anti-PD-L1 antibody either as monotherapy or in combination with anti-PD-1 antibody in early phase clinical trials at the MD Anderson Center for Targeted Therapy. Molecular profiling was performed on archived primary or metastatic tumor tissue using CLIA certified targeted next generation sequencing (FoundationOne or Oncomine). PIK3CA mutations were annotated for functional significance by the Precision Oncology Decision Support Team. Density of cytotoxic T cells was determined using immunohistochemistry (IHC) on biopsy or resection specimens. Microsatellite status was assessed using IHC or polymerase chain reaction. Median time to treatment failure (TTF) was estimated using Kaplan and Meier method. Groups were compared using Fischer's exact, Student's T test or Log-rank test, as appropriate. Results: Twenty-seven patients with MSS metastatic adenocarcinoma (female, 20; male, 7) from 12 different sites were treated with anti-PD-L1 antibody. Breast, colon and esophageal cancers were the most common cancers with 7, 5 and 3 patients, respectively. Activating PIK3CA mutations were present in 7/27 (26%) patients (H1047R, 3; E545K, 2; E542K, 1; co-occuring R88Q and K111N, 1). Patients with activating PIK3CA mutations had trend towards longer median TTF as compared to PIK3CA wild-type patients (6.6 vs. 3.1 months, P=0.1). Similarly, 3/7 (42.9%) patients with activating PIK3CA mutations had stable disease (SD) > 6 months as compared to 2/20 (10%) patients with wild type PIK3CA (P=0.09). CD8 densities were available for 8 patients (5 PIK3CA mutant, 3 PIK3CA wild). Among PIK3CA mutant patients, mean CD8 density was higher in patients who had SD > 6 months as compared to those who did not (630.3 vs 305.1 cells/mm3; P=0.06). Conclusion: Activating PIK3CA mutations are enriched among MSS metastatic adenocarcinoma patients with prolonged SD (> 6 months) on anti-PD-L1 treatment. PIK3CA mutant patients with prolonged SD have higher CD8 densities, possibly due to high-affinity neopeptide-HLA interactions. Larger studies to validate our observations are warranted, including assessment of neopeptide load and HLA types, which may help identify an immunotherapy sensitive subset of PIK3CA mutant MSS adenocarcinoma. Citation Format: Maliha Nusrat, Coya Tapia, S. Greg Call, David S. Hong, Sarina A. Piha-Paul, Vivek Subbiah, Jordi Rodon, Apostolia M. Tsimberidou, Abha Adat, Yan Wang, Funda Meric-Bernstam, Michael J. Overman, Scott Kopetz, Filip Janku. Evaluation of cytotoxic T cell infiltration and clinical benefit from PD-L1 checkpoint inhibition among PIK3CA mutant microsatellite stable (MSS) metastatic adenocarcinoma patients [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 3172.

The development of next generation therapeutics for cancer patients requires an improved understanding of the underlying mechanisms of resistance to existing therapies as well as novel biomarkers that can help identify patients most likely to benefit from the treatments. Most studies addressing these questions have predominantly relied on a limited set of tumor profiling techniques or have been hampered by relatively small patient numbers. In response to this need, the IMMUcan consortium has been formed. Co-led by the EORTC (Brussels, Belgium), SIB (Lausanne, Switzerland), Eli Lilly (Indianapolis, IN, USA) and Merck KGaA (Darmstadt, Germany), IMMUcan is an EU-funded, public-private partnership of expert immunologists, cell biologists, technology developers and clinical key opinion leaders. The mission of IMMUcan is to generate broad molecular and cellular profiling data of the tumor microenvironment from up to 3,000 patients derived from five indications: non-small cell lung cancer, head and neck squamous cell carcinoma, renal cell carcinoma, colorectal cancer and breast cancer. Most of these patients will be treated under standard of care and recruited via the EORTC-SPECTA infrastructure. All acquired tumor samples will undergo detailed and standardized analyses applying diverse profiling modalities including whole exome sequencing to determine the mutational landscape, total RNA sequencing for transcriptome analysis, multiplex immunofluorescence (IF) and imaging mass cytometry (IMC) facilitating the assessment of approx. 80 proteins at single cell resolution. To avoid inter-lab variations, each profiling method (sequencing, IF or IMC) will be performed by a single expert laboratory, which will process all IMMUcan samples. These data will be integrated with clinical information and centralized in a Knowledge Management System. Once published, the entire IMMUcan dataset will be made available to the scientific community for further study and interrogation. IMMUcan also acquires blood samples, including PBMCs, saliva as well as fresh-frozen stool samples. In addition, we have established workflows to generate reports documenting clinically actionable molecular alterations, which are returned to the treating physicians within 6-8 weeks. Thus far, 31 patients have been recruited and first tumor profiling data from these patients will be presented. Funding: IMI2 JU grant agreement 821558, supported by EU's Horizon 2020 and EFPIA. Citation Format: Henoch S. Hong, Robin Liechti, Christoph Reinhard, Marie M. Morfouace, IMMUcan consortium. IMMUcan: Broad cellular and molecular profiling of the human tumor microenvironment [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 1699.

Background: Systemic disease control is the principle driver of prognosis in patients with pancreatic cancer (PDAC). Currently, there are no predictive biomarkers of clinical response to guide selection of an optimal treatment approach. Our work explores the logistics and feasibility of generating patient tumor-derived models to serve as biomarkers. Methods: Patients with borderline resectable or locally advanced PDAC enrolled to participate in a multi-institutional prospective, randomized, trial were eligible. Pretreatment core needle biopsies from diagnostic endoscopy were shipped via commercial post to a centralized organoid laboratory. Patient-specific organoids (PDO) were established as previously described by our group. Putative predictive biomarkers of clinical treatment response, including whole-exome sequencing (WES), RNA-sequencing, and pharmacotyping (chemotherapeutic sensitivity testing) were performed prospectively. Putative predictive biomarkers will be compared to clinical outcomes when approved by the trial's Data Safety Monitoring Board. Results: Approximately 40% (42/98) of patients participated in tissue acquisition for organoid development. Culture maturation can be described in three relevant phases: establishment, expansion, and characterization. The establishment phase describes the emergence of viable ductal organoid development in the setting of a culture being cleared of other components of the tumor microenvironment. During these first 3-5 passages, there are numerous potential pitfalls, including technical and logistical challenges. Out of 42, 26 (62%) PDOs were successfully established. Notably, the establishment phase appears to be an area of the greatest improvement, as the rates of successful establishment improve while the collaborative team builds experience. Expansion involves biomass accumulation in clean culture. Of those that succeeded in establishment, 77% (20/26) entered in expansion phase. We can currently report 15 PDOs having reached the characterization phase (WES, RNA-seq with subtyping, and/or pharmacotyping). Based on RNA expression profile, 10/14 were classified as classical and 4 as basal-like subtype. Mean time to pharmacotyping was 179 days. The PDOs showed a variable population distribution of sensitivity to standard-of-care chemotherapeutics (gemcitabine, paclitaxel, irinotecan, 5-FU, oxaliplatin). Conclusions: Developing a living organoid biobank from PDAC biopsies in a multi-center trial setting is a tractable approach in precision medicine initiatives. Amongst a collaborative team of physician scientists, the establishment phase is critical. PDOs from pre-treatment biopsies can be individually characterized within a clinically relevant time frame and unique tumor-specific ex vivo-derived therapeutic sensitivities can be assessed. Citation Format: Toni T. Seppälä, Jacquelyn W. Zimmerman, Noah Rozich, Alex Blair, Ammar Javed, John L. Cameron, William R. Burns, Jin He, David Tuveson, Christopher L. Wolfgang, David P. Ryan, Alec Kimmelman, Joseph M. Herman, Wells Messersmith, Theodore S. Hong, David T. Ting, Richard Andrew Burkhart. Patient-derived organoids may facilitate precision medicine in pancreatic cancer: Demonstrating feasibility in the context of a multi-center clinical trial [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 LB-011.

Introduction: Larotrectinib, a highly selective Food and Drug Administration- and European Medicines Agency-approved TRK inhibitor, achieved an objective response rate (ORR) of 79% and a median duration of response (DoR) of 35.2 months across various cancers (Hong et al. Lancet Oncol. In press). In this data set, outcomes by the number of lines of prior therapy or baseline performance status have not previously been reported. Methods: Data were pooled from three clinical trials of patients with TRK fusion cancer treated with larotrectinib (NCT02122913, NCT02576431, NCT02637687). Patients were stratified based on the number of lines of prior therapy (0, 1, 2, or ≥3) and baseline Eastern Cooperative Oncology Group (ECOG) performance status (0, 1, 2, or 3). A post hoc analysis of ORR, assessed by investigators using Response Evaluation Criteria in Solid Tumors 1.1, DoR, progression-free survival (PFS), and overall survival (OS) was performed (data cut-off was February 19, 2019). Results: A total of 159 patients were enrolled. Stratified efficacy outcomes are shown in the table. The incidence of Grade 3/4 adverse events was similar across lines of therapy. Number of prior lines of therapy012≥3Total(n=35)(n=48)(n=34)(n=42)(N=159)ORR, % (95% CI)91 (76, 98)70 (54, 82)73 (54, 87)85 (70, 94)79 (72, 85)DoRNENENE35.235.2(19.8, NE)(15.0, NE)(7.6, NE)(22.8, NE)(22.8, NE)1.9+, 37.1+1.6+, 40.2+1.9+, 44.2+1.9+, 38.2+1.6+, 44.2+PFS25.8NENE28.328.3(15.2, NE)(9.9, NE)(7.0, NE)(16.6, NE)(22.1, NE)0.03+, 38.8+0.03+, 42.1+0.03+, 45.2+0.03+, 40.0+0.03+, 45.2+OSNENE36.544.444.4(NE, NE)(33.4, NE)(36.5, NE)(NE, NE)(36.5, NE)0.03+, 39.9+0.5+, 43.1+0.3+, 47.2+1.0+, 44.40.03+, 47.2+ECOG performance status0123Total(n=76)(n=61)(n=19)(n=3)(N=159)ORR, % (95% CI)91 (81, 96)69 (56, 81)71 (44, 90)33 (1, 91)79 (72, 85)DoR35.226.3NENE35.2(22.8, NE)(19.8, NE)(7.2, NE)(22.8, NE)1.8+, 40.2+1.6+, 44.2+1.9+, 29.5+1.6+, 44.2+PFS36.825.8NENE28.3(24.6, NE)(9.0, NE)(2.7, NE)(1.1, NE)(22.1, NE)0.03+, 42.1+0.03+, 45.2+0.03+, 30.4+0.03+, 1.7+0.03+, 45.2+All data values show median (95% CI) and range in months, unless otherwise stated. CI, confidence interval; DoR, duration of response; ECOG, Eastern Cooperative Oncology Group; NE, not estimable; ORR, objective response rate; OS, overall survival; PFS, progression-free survival. Conclusion: While response rates were highest in patients who were treatment-naïve or with an ECOG performance status of 0, larotrectinib benefited patients across varying degrees of pre-treatment or performance status. Citation Format: Alexander Drilon, Cornelis M. van Tilburg, Anna F. Farago, Shivaani Kummar, Jordan Berlin, Catherine M. Albert, Ray McDermott, Ulrik N. Lassen, John A. Reeves, Nicoletta Brega, Barrett H. Childs, Theodore W. Laetsch, David S. Hong. Larotrectinib in TRK fusion cancer patients: Outcomes by prior therapy and performance status [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 CT199.

Our group recently demonstrated that IDH1/2 mutated gliomas exhibit intrinsic homologous recombination (HR) defects mediated by the oncometabolite, 2HG. These defects render IDH1/2 mutated cells exquisitely sensitive to PARP inhibitors (PARPi's), which suggests a novel therapeutic strategy. Here, we studied the potential in vitro and in vivo efficacy of a CNS penetrant PARPi, pamiparib, in combination with radiation therapy (RT) and temozolomide (TMZ) against IDH1/2 mutated gliomas. Our central hypothesis is that multi-modality therapy using agents that are active against 2HG producing cells will increase efficacy, while allowing lower doses of RT and TMZ as a means to mitigate normal tissue toxicity. We performed a comprehensive series of DNA repair functional studies, short and long term viability assays, as well as in vivo studies, using engineered and patient-derived IDH1/2 mutant glioma models. We tested unique combinations of pamiparib, TMZ and RT in these experiments. In vitro short term viability assays and long-term clonogenic survival assays in model IDH1 wild-type (WT) and R132H mutated glioma cells demonstrated that the mutation confers enhanced TMZ sensitivity (IC50 602 vs. 9.5 uM, respectively). A similar differential sensitivity was observed with RT. We also demonstrated significant, mutant IDH1 selective pamiparib sensitivity in vitro (IC50 0.67 vs. 141 uM). Western blot analysis of PARylation, a functional readout of PARPi activity, confirmed PARylation inhibition by pamiparib in a dose dependent manner. Subsequently, we demonstrated synergistic interactions between TMZ and RT combined with pamiparib, particularly at low doses of both DNA damaging agents, in an IDH1 selective manner. Finally, we demonstrated the activity of PARPi's against IDH1 mutant tumors in vivo, including pharmacodynamic and pharmacokinetic properties of pamiparib on target activity within the brain. Pamiparib is a promising, CNS penetrant PARP inhibitor that may selectively target IDH1/2 mutated gliomas, based on intrinsic HR defects associated with these tumors. Pamiparib appears to act synergistically with TMZ and RT, which are the backbone of GBM therapies. These data lay the groundwork for a future trial testing “trimodality therapy” (i.e., pamiparib, RT and TMZ) against IDH1/2 mutant gliomas. Citation Format: Christopher Hong, Amrita Sule, Jason Beckta, Ranjini Sundaram, Ranjit Bindra. Exploiting inherent DNA damage repair defects in IDH1/2 mutated gliomas with the CNS penetrant PARP inhibitor, pamiparib [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 1389.

Introduction: A variety of alterations in NTRK genes have been identified in various cancers, including amplifications, rearrangements, deletions, splice variants as well as fusions. Larotrectinib is a highly selective TRK inhibitor that is active in patients (pts) with TRK fusion cancer. Here, we report post hoc efficacy and safety outcomes of larotrectinib treatment in pts with cancer and NTRK gene fusions (Fusion) vs those with Non-fusion alterations. Methods: Data were pooled from three clinical trials of adult and pediatric pts with TRK fusion cancer treated with larotrectinib (NCT02122913, NCT02576431, NCT02637687). NTRK gene status was assessed using local molecular testing; two studies (NCT02122913 and NCT02637687) initially enrolled pts regardless of TRK fusion status. Efficacy outcomes included objective response rate (ORR), assessed by investigators using RECIST 1.1, duration of response (DOR), progression-free survival (PFS), and overall survival (OS). Adverse events (AEs) were also assessed. The data cut-off was February 19, 2019. Results: This analysis included 159 pts (153 evaluable for efficacy) with NTRK fusions, with 17 different tumor types, and 73 Non-fusion pts with various genomic alterations, with 25 different tumor types. NTRK alterations reported in the Non-fusion pts included point mutations in 8 pts, amplifications in 7, rearrangements in 4 and deletions in 1. The ORR was 79% (95% confidence interval [CI] 72-85) in the Fusion group, with complete responses (CR) in 24 (16%), partial responses (PR) in 97 (63%), stable disease (SD) in 19 (12%), and progressive disease (PD) in 9 (6%). Median DOR was 35.2 months (95% CI 22.8-not estimable [NE]). In the Non-fusion group treated with larotrectinib, there were no responses except for one pt with NTRK1 amplification who had a PR of short duration (3.7 months); 17 Non-fusion pts (23%) had SD and 48 (66%) had PD as best response. Similarly, differences were seen in survival endpoints between pts in the overall Fusion and Non-fusion groups: median PFS 28.3 mo vs 1.8 mo and median OS 44.4 mo vs 10.7 mo, respectively. Median time of treatment was 7.9 months in the Fusion group and 1.7 months in the Non-fusion group. Incidences of Grade 3 or 4 AEs were lower in the Fusion group (49%) vs the Non-fusion group (58%). Discontinuation due to AEs occurred in 6% of pts in the Fusion group vs 25% in the Non-fusion group. Conclusion: Larotrectinib is highly efficacious in pts with TRK fusion cancer and has demonstrated durable responses. Pts with other NTRK alterations, including point mutations and amplifications, had only limited benefit from larotrectinib. Citation Format: David S. Hong, Afshin Dowlati, Howard Burris, Edward Chu, Marcia S. Brose, Anna F. Farago, Cornelis M. van Tilburg, Shivaani Kummar, Leo Mascarenhas, John A. Reeves, Marion Rudolph, Patricia Maeda, Barrett H. Childs, Theodore W. Laetsch, Alexander Drilon. Efficacy and safety of larotrectinib in patients with cancer and NTRK gene fusions or other alterations [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 CT062.

Background: Approximately 30% of TNBCs are characterized by microarray as claudin-low, mesenchymal or mesenchymal stem cell-like and, unlike basal TNBCs, these tumors frequently harbor aberrations in the PI3K/AKT/mTOR axis, raising the possibility of targeting this axis to enhance chemotherapy response. Assays to clinically identify mesenchymal TNBCs are under development, but published results confirm that up to 30% are metaplastic breast cancers (MpBCs), a chemo-refractory group of tumors that contain a mixture of epithelial and mesenchymal components, making them identifiable by microscopy. As such, MpBCs serve as surrogates of response for potential regimens to treat mesenchymal TNBC. Methods: Patients (pts) with advanced TNBC (N=64) were treated with liposomal doxorubicin (D), bevacizumab (A) and the mTOR inhibitors temsirolimus (T) or everolimus (E). D and A were administered IV on day 1 with T (IV on days 1, 8 and 15) or E (continuous daily oral administration) using 21 day cycles. Response was assessed every 6 weeks using RECIST. When available, archived tissue was evaluated for aberrations in the PI3K pathway using standard assays. Results: Fifty-two MpBC pts were treated with DAT (N=39) or DAE (N=13). Median age was 58 (range 37-79); median # of prior regimens for metastatic disease was 1 (range 0-5). The objective response rate (ORR) was 21% [complete response (CR)=4 (8%); partial response (PR)=7 (13%)] and 10 (19%) pts had stable disease (SD)≥6 months for a clinical benefit rate (CBR) of 40%. Tissue was available for testing in 43 pts and 32 (74%) had a PI3K pathway activating aberration (Table 1). Response According to PI3K Pathway AberrationPI3K Pathway AberrationN (%)CRPRSD≥6monthsCBRORRAny PI3K Pathway Aberration*32 (74)46444%31%PIK3CA Mutation19 (59)23447%26%p.H1047R12 (38)21350%25%p.E545K6 (19)02150%33%p.G1007R1 (3)010100%100%p.E545A1 (3)0000%0%p.H1047Y1 (3)0000%0%p.K111E1 (3)0000%0%p.E542K1 (3)0000%0%PIK3CA Amplification1 (3)010100%100%PTEN Mutation5 (16)0000%0%PTEN Loss5 (16)02040%40%AKT1 p.E17K Mutation2 (6)0000%0%AKT2 Amplification1 (3)100100%100%PIK3R1 Mutation2 (6)01050%50%NF2 Mutation1 (3)100100%100%No PI3K Pathway Aberration11 (26)00545%0%*Some tumors had >1 aberration detected PI3K pathway activation was associated with a significant improvement in ORR (31 vs 0%; P=0.043) but not CBR (44 vs 45%; P=1.000) or progression-free survival (median 5.1 vs 2.9 months; P=0.352). A pt with 5 year+ durable CR (on maintenance everolimus) had a mutation in NF2. To emphasize the importance of pt selection, it is notable that 12 pts with non-metaplastic TNBC were also treated with DAT, and only 1 pt had a response (CR/PR=1; SD≥6 months=0), for a CBR that was significantly worse than pts with MpBC (8 vs 40%; P=0.045). Conclusions: Using MpBC as a surrogate of response, DAT/DAE has significantly better activity in mesenchymal compared to non-selected TNBC. Response is enhanced in pts with PI3K pathway activation. DAT/DAE should be tested in non-metaplastic, mesenchymal TNBC once a diagnostic assay is available. Citation Format: Basho RK, Gilcrease M, Murthy RK, Helgason T, Booser DJ, Karp DD, Meric-Bernstam F, Wheler JJ, Valero V, Albarracin C, Litton J, Chavez-MacGregor M, Ibrahim NK, Murray JL, Koenig KB, Hong D, Subbiah V, Kurzrock R, Janku F, Moulder S. Targeting the PI3K/AKT/mTOR pathway for the treatment of mesenchymal triple-negative breast cancer (TNBC): Evidence of efficacy and proof of concept from a phase I trial with dose expansion of mTOR inhibition in combination with liposomal doxorubicin and bevacizumab. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-14-02.

Background Combining anti-estrogen therapy with HER2 and mTOR inhibitors may be synergistic and overcome resistance to hormonal and anti-HER2 therapies in patients with estrogen receptor (ER)-positive and HER2 receptor amplified and/or mutant advanced cancers. Patients and Methods We evaluated the mTOR inhibitor everolimus, aromatase inhibitor letrozole and anti-HER2 antibody trastuzumab in patients with ER-positive, HER2-positive and/or mutant, solid tumors (confirmed by IHC, FISH, and/or HER2 sequencing). The primary objectives were to determine maximum tolerated dose (MTD) and toxicity and to evaluate response. Secondary objectives were to correlate activity with genomic and proteomic signatures. Next-generation sequencing (NGS) was performed either using a 50-gene panel or externally by Foundation Medicine (Cambridge, MA, USA). Results Nine of 10 patients enrolled are currently evaluable for toxicity and response including 7 patients with breast cancer, one patient with ovarian cancer, and one patient with cervical cancer. The median age was 57 years (range, 33-66 years) and the median number of prior therapies in the metastatic setting was 5 (range, 2-7). Of these nine patients, six were HER2+ (by IHC and/or FISH) and three had HER2 mutations (in the absence of HER2 overexpression or amplification). Dose Level (DL) 1 consisted of trastuzumab loading dose of 4mg/kg IV and maintenance dose of 2 mg/kg IV every 21 days, everolimus 5 mg PO daily and letrozole 2.5 mg PO daily. DL2 consisted of trastuzumab loading dose of 6mg/kg IV and maintenance dose of 4 mg/kg IV every 21 days, everolimus 5 mg PO daily and letrozole 2.5 mg PO daily. DL3 consists of trastuzumab loading dose of 6 mg/kg IV and maintenance dose of 4 mg/kg IV every 21 days, everolimus 7.5 mg PO daily and letrozole 2.5 mg PO daily. Currently patients are being enrolled in DL3 and no DLTs have been observed. Additional dose levels are planned with increased trastuzumab and everolimus to the FDA approved doses or until MTD is reached. The most common grade 1 and 2 treatment-related toxicities were fatigue (6 patients), anemia and constipation (2 patients each). No grade 3 or 4 toxicities have been reported. Median time to treatment failure (TTF) was 7.4 months (95% CI 5.2-9.6). One patient with breast cancer and a HER2 mutation experienced a partial response (PR) with a 32% decrease in measurable disease. This patient had no prior HER2-targeted therapy and previously progressed while on letrozole and while on exemestane and everolimus. Six additional patients experienced stable disease (SD) ≥ 6 months, 5 with breast cancer and 1 with cervical cancer. Of these 6 patients, 1 had a HER2 mutation and another had a PIK3CA mutation. Conclusions Combination treatment with everolimus, letrozole and trastuzumab is well tolerated and active in ER+, HER2+ and/or mutant solid tumors, including breast and cervical cancer. Clinical benefit (PR/SD≥6 months) was seen in 7 of 9 patients (78%), including patients with HER2 mutations in the absence of overexpression and/or amplification and patients with no genomic alterations in the PI3K/AKT/mTOR pathway. Enrollment is ongoing. Citation Format: Janku F, Hong DS, Atkins JT, Karp DD, Tsimberidou AM, Piha-Paul SA, Subbiah V, Naing A, Fu S, Moulder SL, Tripathy D, Meric-Bernstam F, Wheler JJ. Everolimus, letrozole and trastuzumab in estrogen receptor and HER2-positive patients with metastatic breast cancer and other solid tumors: Evaluating synergy and overcoming resistance. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-13-11.

Background Combining aromatase inhibitors with PI3K/AKT/mTOR inhibitors in patients with hormone receptor (HR)-positive metastatic breast cancer has demonstrated clinical efficacy. There is limited data on associations between molecular signatures and activity. Patients and Methods We evaluated the combination of anastrozole and everolimus in 56 patients with HR-positive, metastatic breast cancer. The primary objective was to establish safety and maximum tolerated dose (MTD). Dose limiting toxicities (DLTs) were defined as serious grade 3 or 4 toxicities related to treatment that occurred during cycle 1. Dose level 1 was anastrozole 1mg PO QD and everolimus 5 mg PO QD and dose level 2 was anastrozole 1 mg PO QD and everolimus 10 mg PO QD (a dose level -1 included everolimus 2.5 mg PO QD). Secondary endpoints included evaluation of antitumor activity and molecular associations with response. When tissue was available, Next Generation Sequencing (NGS) was performed using genomic libraries selected for all exons of 236 (or 182) cancer-related genes sequenced to average depth of >500× in a CLIA laboratory (Foundation Medicine, Cambridge, MA, USA). An analysis was then performed for all classes of genomic alterations. Results The median age was 59 (range, 37-82) and the median number of prior therapies in the metastatic setting was 3 (range, 0-13). The initial oral daily dose of anastrozole 1 mg oral and everolimus 10 mg PO daily was well tolerated. Five dose-limiting toxicities (DLTs) were seen at full doses, including grade 3 thrombocytopenia (1 patient), grade 3 neutropenia (1 patient), grade 3 increased liver enzymes (1 patient), grade 3 hyperglycemia (1 patient) and, grade 3 mucositis (1 patient). The most common grade 3 or 4 treatment-related toxicities were neutropenia (5%), increased liver enzymes (5%), and hyperbilirubinemia (3%). Of the 56 patients on study, 36 were tested for at least one molecular alteration in the PI3K/AKT/mTOR pathway. Twelve of these 36 patients had NGS analysis of their tumor tissue. Eighteen of 36 patients (50%) tested had at least one alteration in the pathway, including mutations in PIK3CA (n=16), PIK3R1 (n=1), and AKT1 (n=2); PTEN protein loss (n=1); and, AKT3 amplification (n=1). Sixteen of 56 evaluable patients (29%) achieved stable disease (SD) /partial response (PR)/complete response (CR) ≥ 6 months (n = 3 (5%) with PR/CR). Thirteen of the 16 patients who achieved SD/PR/CR ≥ 6 months were tested for a genetic alteration in PI3K/AKT/mTOR pathway and 7 of these patients (54%) had at least one alteration in the pathway, including mutations in PIK3CA (n=6), PIK3R1 (n=1), and AKT1 (n=1); PTEN loss (n=1); and AKT3 amplification (n=1). Conclusions Combination anastrozole 1 mg and everolimus 10 mg is well tolerated and is active in heavily-pretreated patients with HR-positive breast cancer. The presence of a molecular alteration in the PI3K/AKT/mTOR pathway did not predict for clinical activity of this combination. Citation Format: Jennifer J Wheler, Filip Janku, Stacy L Moulder, Aung Naing, Sarina A Piha-Paul, Gerald S Falchook, Ralph G Zinner, Apostolia M Tsimberidou, Siqing Fu, David S Hong, Johnique T Atkins, Roman Yelensky, Vince Miller, Philip J Stephens, Vincente Valero, Funda Meric-Bernstam, Razelle Kurzrock. Anastrozole and everolimus in hormone receptor-positive metastatic breast cancer: Safety profile, activity and associations of molecular alterations in the PI3K/AKT/mTOR pathway [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-05-03.

For many solid tumors, surgical resection remains the gold standard and tumor-involved margins are associated with poor clinical outcomes. Near-infrared (NIR) fluorescence imaging using molecular agents has shown promise for in situ imaging during resection. However, for cancers with difficult imaging conditions, surgical value may lie in tumor mapping of surgical specimens. We thus evaluated a novel approach for real-time, intraoperative tumor margin assessment. Twenty-one adult patients with biopsy-confirmed squamous cell carcinoma arising from the head and neck (HNSCC) scheduled for standard-of-care surgery were enrolled. Cohort 1 (n = 3) received panitumumab-IRDye800CW at an intravenous microdose of 0.06 mg/kg, cohort 2A (n = 5) received 0.5 mg/kg, cohort 2B (n = 7) received 1 mg/kg, and cohort 3 (n = 6) received 50 mg. Patients were followed 30 days postinfusion and adverse events were recorded. Imaging was performed using several closed- and wide-field devices. Fluorescence was histologically correlated to determine sensitivity and specificity. In situ imaging demonstrated tumor-to-background ratio (TBR) of 2 to 3, compared with ex vivo specimen imaging TBR of 5 to 6. We obtained clear differentiation between tumor and normal tissue, with a 3-fold signal difference between positive and negative specimens (P < 0.05). We achieved high correlation of fluorescence intensity with tumor location with sensitivities and specificities >89%; fluorescence predicted distance of tumor tissue to the cut surface of the specimen. This novel method of detecting tumor-involved margins in surgical specimens using a cancer-specific agent provides highly sensitive and specific, real-time, intraoperative surgical navigation in resections with complex anatomy, which are otherwise less amenable to image guidance. Significance: This study demonstrates that fluorescence can be used as a sensitive and specific method of guiding surgeries for head and neck cancers and potentially other cancers with challenging imaging conditions, increasing the probability of complete resections and improving oncologic outcomes. Cancer Res; 78(17); 5144–54. ©2018 AACR.

The resistance to therapy in pancreatic ductal adenocarcinoma (PDAC) is due in part to carcinoma-associated fibroblasts (CAFs) that produce a highly desmoplastic and proinflammatory tumor microenvironment (TME). Our laboratory has shown that the angiotensin receptor blocker (ARB) losartan reduces the CAF-density and desmoplasia, and improves vascular perfusion, and the uptake and efficacy of drugs in animal models of PDAC (Diop-Frimpong et al., PNAS 2011; Chauhan et al., Nature Comm 2013). These preclinical findings led to a phase II trial at Massachusetts General Hospital in locally advanced PDAC patients, which showed that losartan combined with neoadjuvant-FOLFIRINOX chemotherapy followed by chemoradiation more than doubled the resection rate (Murphy et al., JAMA Oncology 2019). Moreover, in a retrospective analysis, we found that the chronic use of angiotensin system inhibitors was independently associated with longer overall survival in nonmetastatic PDAC patients (Liu et al., Clin Cancer Res 2017). However, how ARBs affect CAFs and desmoplasia in human PDAC remains unknown. Here we performed immunohistochemistry in formalin-fixed, paraffin-embedded sections to examine the effect of chronic use of ARB on CAF phenotypes and the extracellular matrix (ECM) in resected PDAC samples from untreated patients and patients treated with ARBs to control their hypertension. We found that the chronic use of ARBs did not reduce the density of CAFs expressing the fibroblast activation protein (FAP), α-smooth muscle actin (α-SMA), or platelet-derived growth factor receptor-beta alone. However, ARBs increased the fraction of CAFs expressing both the angiotensin receptor 1 (AT1) and α-SMA and decreased the fraction of CAFs expressing both AT1 and FAP. ARBs also reduced the intratumoral levels of hyaluronan, suggesting that ARBs reduce desmoplasia in human PDAC. To reveal the underlying mechanisms, we performed a transcriptomic analysis of the bulk tumor as well as α-SMA+ cells isolated from mice bearing orthotopic PDAC tumors and treated with losartan. Gene expression profiling revealed that in both the tumor bulk and α-SMA+ cells, losartan “normalized” the ECM/cytoskeleton interaction, glycosaminoglycan metabolism and platelet-derived growth factor gene sets, and significantly reduced gene transcripts ofS100A8 and S100A9 . Gene products of S100A8 and S100A9 are S100 proteins that stimulate the development of myeloid-derived suppressor cells. In α-SMA+ cells—but not in tumor bulk—losartan reduced cell cycle and muscle contraction gene sets. These results suggest that ARBs can reprogram α-SMA+ cells and normalize the TME in PDAC. Citation Format: Hao Liu, William W. Ho, Kamila Naxerova, Jelena Grahovac, Hadi Nia, Ivy Chen, Jessica M. Posada, Daniel H. Schanne, Matthias Pinter, Jonathan Crain, Xialong Qi, Jeffrey W. Clark, Theodore S. Hong, David P. Ryan, Peigen Huang, Keith D. Lillemoe, Carlos Fernández-Del Castillo, Cristina R Ferrone, Michael Downes, Ronald M. Evans, Vikram Deshpande, Yves Boucher, Rakesh K. Jain. Angiotensin receptor blockers normalize the pancreatic ductal adenocarcinoma stroma by reprogramming carcinoma-associated fibroblasts [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A18.

Objective: Pancreatic ductal adenocarcinoma (PDAC) remains a treatment-refractory disease as existing molecular subtypes are insufficient and do not currently inform clinical decisions. Rare cell types, including those responsible for resistance, are difficult to detect with bulk transcriptomic profiling. Indeed, several previously identified transcriptomic subtypes of PDAC are unintentionally driven by “contaminating” stromal components. Single-cell transcriptomics provides an unprecedented degree of resolution into the properties of individual cells. However, RNA extraction from RNase- and stroma-rich pancreatic tissue is difficult and prior single-cell efforts have been limited by suboptimal dissociation/RNA quality. We developed a robust single-nucleus RNA-seq (sNuc-seq) technique compatible with frozen archival PDAC specimens and computational techniques to identify the transcriptomic programs driving tumor subtypes and therapeutic resistance. Methods: Patients with localized PDAC undergoing surgical resection with or without neoadjuvant chemoradiotherapy were consented for this IRB-approved study. Specimens were screened for RNA Integrity Number >6. Single nuclei suspensions were extracted from flash-frozen primary PDAC specimens and organoids. Approximately 8,000 nuclei were loaded on the 10x Genomics Chromium platform per sample to generate and sequence 3’ gene expression libraries (Illumina HiSeq 2500, 125 bp paired-end reads). sNuc-seq derived reads were processed using the 10X CellRanger v3.0.2 pipeline. Unsupervised clustering was utilized to identify different cell populations and known marker genes from literature were used to label cell types. Results: Both treatment-naïve (n=12) and treatment-resistant (n=11) specimens yielded high-quality sNuc-seq data (>1,000 nuclei per sample, >1,000 median genes per nucleus). In each tumor, distinct clusters with gene expression profiles consistent with ductal, fibroblast, endothelial, endocrine, lymphocyte, and myeloid cell populations were identified. Malignant cells were confirmed by inferred copy number variation analysis (InferCNV v3.9) and segregated into several distinct clusters for each individual patient highlighting intratumoral heterogeneity. While some malignant clusters corresponded to previously identified basal-squamous and classical-progenitor bulk subtypes, others featured expression profiles distinct from known subtypes, including cells with upregulation of hypoxia-associated or cytoskeletal genes. Conclusions: Applying sNuc-seq to treatment-naïve and pretreated PDAC specimens, we uncovered significant intratumoral heterogeneity in the malignant and stromal compartments and identified malignant cells featuring transcriptomic programs that do not fit previously identified bulk subtypes. Characterization of therapeutic resistance programs, spatial relationships among cell types, and association with clinical outcomes is ongoing. Citation Format: William L. Hwang, Karthik A. Jagadeesh, Orr Ashenberg, Eugene Drokhlyansky, George Eng, Nicholas Van Wittenberghe, William Freed-Pastor, Clifton Rodriguez, Danielle Dionne, Julia Waldman, Michael Cuoco, Alexander Tsankov, Connor Lambden, Caroline Porter, Jason Schenkel, Laurens Lambert, Debora Ciprani, Andrew J. Aguirre, Mari Mino-Kenudson, Theodore S. Hong, Orit Rozenblatt-Rosen, Carlos Fernandez-del Castillo, Andrew S. Liss, Aviv Regev, Tyler E. Jacks. Molecular subtypes and resistance programs in pancreatic ductal adenocarcinoma elucidated with single-nucleus RNA-seq [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A22.

Background: Preclinical data from our team demonstrated that MEK inhibition increases antitumor efficacy of PARP inhibitors in tumors with Ras pathway alterations. We sought to identify dose limiting toxicities (DLTs) and maximum tolerated dose (MTD) of olaparib and selumetinib combination in solid tumors with Ras pathway alterations. Methods: Olaparib and selumetinib were given orally twice daily. Dose escalation with three planned dose levels (DL) was performed using the modified toxicity probability interval 2 design with toxicity assessed by CTCAE v4.03. RECIST criteria (v1.1) were used to assess response. Clinical benefit was defined as partial (PR) or complete response, or stable disease for 4 months or longer. Results: 14 patients are evaluable for toxicity in the dose escalation phase, and 12 patients are evaluable for response. Median age was 56.5 years, and 10/14 (71%) patients were female. Median number of prior treatments was 4 (1-9). Of the 14 patients enrolled, 11 had KRAS mutations, 1 amplified KRAS, 1 NRAS mutation and 1 amplified NRAS. The majority of patients had gynecologic cancers. 4 and 3 patients were treated at DL1 and DL2, respectively, and 7 patients at DL3. No DLTs were observed (Table 1). MTD was not reached. DL3 (olaparib 300mg; selumetinib 75mg) was confirmed as the recommended Phase II dose (RP2D). Of 12 evaluable patients, ORR was 17%, and CBR 33%. 2 patients had a PR: 1 patient with KRAS mutant primary peritoneal cancer, and 1 with NRAS mutant ovarian cancer. 2 patients remained on treatment for more than 15 months: 1 with KRAS mutant primary peritoneal cancer (on DL-1), and 1 with KRAS mutant non-small cell lung cancer (DL1). All patients with clinical benefit were BRCA wildtype. Conclusions: The combination of olaparib and selumetinib is well-tolerated at the identified RP2D. This combination also shows promising preliminary anti-tumor activity in patients with mutant RAS. Enrollment to expansion cohorts is ongoing. Table 1Grade 3/4 and Most Common Adverse Events. NOTE: No Grade 4 Events Occurred.Adverse EventGrade 3Any GradeAbdominal pain7%7%Acneiform Rash7%71%Anemia79%Anorexia29%Constipation29%Decreased ejection fraction7%14%Decreased white blood cell count7%36%Diarrhea50%Dizziness29%Dry mouth43%Dry skin29%Dysgeusia36%Edema29%Elevated aspartate aminotransferase7%50%Elevated bilirubin7%7%Elevated CPK7%36%Elevated creatinine29%Fatigue7%64%Hepatic pain7%7%Hypophosphatemia79%Nausea57%Neutropenia7%21%Oral mucositis50%Other skin effects36%Thromboembolic event7%7% Citation Format: Katherine C. Kurnit, Funda Meric-Bernstam, Kenneth Hess, Robert L. Coleman, Priya Bhosale, Katerina Savelieva, Filip Janku, David Hong, Aung Naing, Shubham Pant, Jordi Rodon, Timothy A. Yap, Anil K. Sood, Pamela T. Soliman, David M. Gershenson, Gordon B. Mills, Shannon N. Westin. Phase I dose escalation of olaparib (PARP inhibitor) and selumetinib (MEK Inhibitor) combination in solid tumors with Ras pathway alterations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT020.

Background: Despite the rising incidence of sarcoma in the geriatric population, there is a paucity of older adults enrolled on clinical trials. Whether older sarcoma patients treated on early phase clinical trials have any meaningful differences in clinical benefit or survival outcomes compared to younger patients remains unknown. Methods: We analyzed clinical and next generation sequencing data from sarcoma patients treated on phase 1 trials at MD Anderson Cancer Center (MDACC) and performed logistic and Cox proportional hazards regression analyses to compare response rate (RR), median time to progression (mTTP), clinical benefit rate (CBR; defined as CR, PR, or SD > 6 months), and median overall survival (mOS) between patients younger and older than 65 years of age. Results: Among the 406 patients with advanced sarcomas (321 soft tissue sarcoma, 85 bone sarcomas) treated on phase 1 trials at MDACC from May 2006 to May 2018, median age was 53 (range 11-84), 48% were female, and patients had a median 3 prior lines of therapy (range 0-9). The most commonly treated soft tissue sarcoma subtypes included leiomyosarcoma (n=66; 16%), liposarcoma (n=52; 13%), GIST (n=44; 11%), UPS (n=14; 3%), and synovial sarcoma (n=11; 3%) and most commonly treated bone sarcomas included osteosarcoma (n=34; 8%), chondrosarcoma (n=28; 7%), and Ewing’s sarcoma (n=25; 6%). 25% (n=102) of sarcoma patients treated on phase 1 protocols were age 65 or older. RR in patients younger than 65 years was 7% compared to 8% in those 65 or older, odds ratio 1.21 (95% CI 0.52, 2.83), p=0.66. mTTP for patients younger than 65 was 2.7 months compared to 3.7 months in those 65 or older, hazard ratio 0.90 (95% CI 0.72, 1.14), p=0.39. CBR in patients younger than 65 years was 22% compared to 31% in those 65 or older, odds ratio 1.65 (95% CI 1.00, 2.72), p=0.05. mOS in patients younger than 65 years was 16.1 months compared to 20.0 months in those 65 years or older, hazard ratio 1.08 (95% CI 0.80, 1.46), p=0.61. Conclusion: Heavily pretreated, metastatic sarcoma patients aged 65 or older enrolled on phase 1 studies at MDACC had no significant differences in response rate, time to progression, and overall survival compared to their younger counterparts. In fact, older sarcoma patients demonstrated an improved clinical benefit rate compared to younger patients. Older patients should be encouraged to enroll on early phase clinical trials when clinically appropriate and broad eligibility criteria for such studies are needed to ensure older sarcoma patients are not excluded from these trials. Citation Format: Shiraj Sen, Roberto C. Pestana, Kenneth Hess, David S. Hong, Ishwaria M. Subbiah, Anthony Conley, Gerald S. Falchook, Robert S. Benjamin, Shreyas Patel, Funda Meric-Bernstam, Vivek Subbiah. Outcomes of geriatric sarcoma patients enrolled in phase 1 clinical trials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3997.

The inevitable emergence of acquired resistance is a major limitation to the clinical benefit of precision medicine strategies. Single-lesion tumor biopsies have long been the mainstay of understanding acquired resistance, but recent data suggest tumor biopsies may under-represent the molecular heterogeneity of acquired resistance. Alternatively, studies have suggested that liquid biopsy approaches analyzing cell-free DNA (cfDNA) may offer significant advantages, but extensive prospective comparisons of matched liquid vs. tumor biopsies obtained at the time of acquired resistance are lacking. Here, we assess systematic liquid biopsy upon acquired resistance to targeted therapy in 44 patients across seven molecularly defined subtypes of gastrointestinal cancers. Liquid biopsy at disease progression identified at least one functionally validated molecular mechanism of resistance in 75% of patients, wherein 52% exhibited >1 resistance alteration (range 2-9, median 3 per patient). In 23 patients in whom a matched post-progression tumor biopsy could be obtained, tumor biopsy was less effective than liquid biopsy in identifying resistance mechanisms, with resistance alterations detected in only 48% of patients, and multiple resistance mechanisms detected in only 9% of cases. In matched cases, liquid biopsy detected at least one resistance alteration not detected in tumor biopsy in 78% of cases. Targeted analysis and whole-exome sequencing of serial cfDNA, multiple post-progression biopsies, and rapid autopsy specimens from select cases revealed key insights into the geographic and complex characteristics of heterogeneity captured by liquid biopsy in the setting of acquired resistance. These data illustrate that acquired resistance is characterized by frequent and profound tumor heterogeneity, and suggests that liquid biopsy may more effectively identify heterogeneous clinically relevant resistance alterations compared to standard tumor biopsy. Citation Format: Aparna R. Parikh, Ignaty Leshchiner, Liudmila Elagina, Lipika Goyal, Chaya Levovitz, Giulia Siravegna, Dimitri Livitz, Kahn Rhrissorrakrai, Liz Martin, Emily E. Van Seventer, Megan Hanna, Kara Slowik, Filippo Utro, Christopher J. Pinto, Alicia Wong, Brian P. Danysh, Ferran Fece de la Cruz, Isobel J. Fetter, Brandon Nadres, Heather A. Shahzade, Jill N. Allen, Lawrence S. Blaszkowsky, Jeffrey W. Clark, Bruce Giantonio, Janet E. Murphy, Ryan D. Nipp, Eric Roeland, David P. Ryan, Colin D. Weekes, Eunice L. Kwak, Jason E. Faris, Francois Aguet, Ipsita Guha, Mehlika Hazar-Rethinam, Dora Dias-Santagata, David T. Ting, Andrew X. Zhu, Theodore S. Hong, Todd R. Golub, A J. Iafrate, Viktor Adalsteinsson, Alberto Bardelli, Laxmi Parida, Dejan Juric, Gad Getz, Ryan B. Corcoran. Liquid biopsy versus tissue biopsy to assess acquired resistance and tumor heterogeneity in gastrointestinal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-257.

Background: Larotrectinib, a selective TRKi, is now FDA approved for pediatric and adult TRK-fusion solid tumors, regardless of tumor origin. Emergent TRK kinase mutations are a common mechanism of resistance to TRKis. LOXO-195, a selective TRKi, was developed to maintain potency against multiple TRK kinase domain mutations. Methods: Patients (pts) received LOXO-195 via a Phase I study (NCT03215511, n=20) or FDA expanded access single patient protocol (SPP, n=11). Eligible pts were ≥4-weeks old with a locally identified TRK fusion and had progressed or were intolerant to at least 1 priorTRKi. Parallel 3+3 dose escalations were pursued in adults and children, with intra-patient dose escalation permitted based on tolerance and pharmacokinetics. Pts aged Results: As of 03-DEC-2018, a total of 31 TRK-fusion pts (7 children, 24 adults) with 11 cancer types had been treated. Median duration on last prior TRKi was 9.5 months (range, 2-30). In the Phase 1, doses of 32 mg QD to 150mg BID were explored, and TEAEs (all grades/cause, in >3 pts) were dizziness/ataxia (65%), nausea/vomiting (50%), anemia (30%), myalgia, abdominal pain, fatigue, & lymphopenia (all 20%). Five Phase I pts (all adults) had DLTs: ataxia/dizziness (4), and ataxia/vomiting (1). For the SPPs: 1 pt dose-reduced and none discontinued for a TEAE. Cmax at doses ≥50 mg exceeded the predicted IC50 for TRK kinase mutations. Pretreatment tissue and/or plasma, as available, defined TRK kinase mutation status. Preliminary efficacy overall, and by TRK kinase mutation status, is shown in the Table. Discussion: LOXO-195 had preliminary efficacy in pts with resistance to prior TRKi mediated by TRK kinase mutations. The subset of pts who develop TRK-independent resistance are unlikely to benefit from LOXO-195. Dose selection is ongoing in both children and adults. Confirmed best overall response, all dose levels, investigator assessed per RECIST 1.1 (n=29≠)Patient CohortTotal Patients,nCR/PR, nStable Disease, nPD, nNon- evaluable, †nORRTRK Kinase Mutation20963245% (9/20)Solvent Front14742150% (7/14)Gatekeeper4111125% (1/4)xDFG2110050% (1/2)Identified bypass300210% (0/3)Other/ Unknown*61#31117% (1/6)Overall291096434% (10/29)≠ 2 pts still on study drug and awaiting 1st response assessment not included in Table.† 4 pts non-evaluable: 1 discontinued drug for unrelated new cancer diagnosis Citation Format: David Hyman, Shivaani Kummar, Anna Farago, Birgit Geoerger, Morten Mau-Sorensen, Matthew Taylor, Elena Garralda, Ramamoorthy Nagasubramanian, Michael Natheson, Lucy Song, Michael Capra, Mette Jorgensen, Alan Ho, Neerav Shukla, Steve Smith, Xin Huang, Brian Tuch, Nora Ku, Theodore W. Laetsch, Alexander Drilon, David Hong. Phase I and expanded access experience of LOXO-195 (BAY 2731954), a selective next-generation TRK inhibitor (TRKi) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT127.

Background: The RET receptor tyrosine kinase can be oncogenically activated by gene fusions or point mutations. Multikinase inhibitors such as cabozantinib, lenvatinib, and vandetanib have demonstrated activity in RET dependent (RET+) malignancies. Recent development of selective RET inhibitors is poised to alter their treatment paradigm. However, the responsiveness of RET+ malignancies to immune checkpoint inhibition (ICI) is unknown. We compared the time to progression (TTP) on ICI with non-ICI therapy in patients with malignancies harboring activating RET mutations/fusions. Methods: A retrospective chart review of all RET+ patients who were referred to the phase 1 clinical trials program at MD Anderson Cancer Center between September 2014 and October 2018 was conducted. Patients who had not discontinued treatment or had discontinued treatment for reasons besides disease progression were censored. Time to progression was estimated using Kaplan-Meier analysis. Results: Out of 72 patients who had received systemic therapy for RET+ malignancies, 39 (54.2%) harbored RET mutations (RETm) and 33 (45.8%) harbored RET fusions (RETf). Nineteen patients (26.4%) received ICI and 53 (73.6%) received non-ICI therapy. Thirty four patients (47.2%) had medullary thyroid cancer, 29 (40.3%) had NSCLC and 9 (12.5%) had other types of cancer. PD-L1 expression was only available for 7 patients of which 5 (71.4%) were positive (≥ 1%). Patients had received a median of 1 (1-7) prior lines of therapy. Overall median TTP (mTTP) was 10.5 months (95% CI 6.7-30.1). Hazard ratio for ICI vs. non-ICI therapy was 1.73 (0.70, 4.26) p=0.25 in patients with RETf and 8.0 (2.27, 28.2) p=0.0025 in patients with RETm. Conclusions: In patients with RET+ malignancies, mTTP was shorter with ICI compared to non-ICI therapy. The effect of type of therapy was dependent on the type of RET pathway aberration, with a statistically significant higher risk of progression in RETm malignancies treated with ICI compared to non-ICI therapy. Time to ProgressionRETTherapyNmTTP (months)Freedom from Progression at 6 months (%)Freedom from Progression at 12 months (%)FusionNon-ICI218.36335FusionICI123.03624MutationNon-ICI3231.98273MutationICI75.6430 Citation Format: Aparna Hegde, Le Huang, Shuang Liu, Kenneth Hess, Maria Cabanillas, Mimi Hu, Naifa Busaidy, Steven Sherman, George Simon, George Blumenschein, Vassiliki A. Papadimitrakopoulou, David S. Hong, Funda Meric-Bernstam, John Heymach, Vivek Subbiah. Responsiveness to immune checkpoint inhibitors in RET dependent cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4997.

Background: Metaplastic breast cancers are considered a chemorefractory subset of triple negative breast cancers. Molecular profiling has demonstrated that metaplastic tumors are enriched for epithelial-to-mesenchymal transition (EMT), frequently express myoepithelial differentiation, make up a component of the ‘claudin-low’ subtype, and harbor relatively high rates of mutations/activation of the PI3kinase pathway (Hennessy, Cancer Research, 2009; Prat, Breast Cancer Res, 2010). Methods: Data from pts with myoepithelial/metaplastic breast cancer treated within the Center for Targeted Therapy using regimens with known inhibitors of the PI3K pathway were evaluated to determine response to therapy. Mutational analyses were performed in archived tumor samples when available. Results: 23 pts have been treated using 6 different therapy regimens and one pt was treated on two separate clinical trials for a total of 24 analyzable outcomes. Patients were treated with liposomal doxorubicin, bevacizumab and the mTOR inhibitor, temsirolimus (DAT, n=17); liposomal doxorubicin and temsirolimus (DT, n=1); paclitaxel, bevacizumab and temsirolimus (TAT, n=3); paclitaxel and temsirolimus (TT, n=1), paclitaxel in combination with an experimental PI3K inhibitor (TEx, n=1), or temsirolimus alone (tem, n=1). Response was measured every two cycles using RECIST criteria. Most pts had received prior chemotherapy, median of 2 prior regimens (range 0–7). Three patients were not evaluated for response, one who died of pneumonia during cycle 2 (DAT) and two who have not yet completed 2 cycles of therapy (DAT, TEx). Response rate (CR+PR) was 35% (CR = 2, PR=4, SD≥6 months=2, SD Conclusion: Activating mutations in the PI3K pathway are common in metaplastic breast cancers, a tumor subtype that shares molecular features with claudin-low and mesenchymal/mesenchymal-stem cell like triple negative breast cancers. DAT has demonstrated activity in myoepithelial/metaplastic breast cancer including two durable CRs to therapy. This regimen should be explored in larger, randomized trials to test superiority to chemotherapy alone. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-20-09.

Background: Observational studies and randomized trials have demonstrated an association between hormone replacement therapy (HRT) use and increased risk for breast cancer. However, this risk may vary by age and other factors, including breast density and race/ethnicity. Using data from the Breast Cancer Surveillance Consortium (BCSC), we investigate whether self-reported HRT use in the peri-menopausal and immediate post-menopausal age groups is associated with breast cancer, and also whether risk associated with HRT use differs by breast density and race. Subjects and Methods: The BCSC is a longitudinal registry of mammography screening in seven U.S. centers. In the breast cancer risk prediction model developed by Barlow et al (J Natl Cancer Inst 2006 98:1204-14), HRT use and ten other features were identified as risk factorsfor breast cancer. Data for our study consists of 1,642,824 screens and 9,300 breast cancers among the post-menopausal women aged > 44 in this cohort. Logistic regression models were used to estimate odds ratios for breast cancer associated with HRT use within race, age, and density strata, adjusting for other variables from the Barlow model. Multiple imputation methods were used to accommodate missing data for HRT (14%) and other predictors. Results: Breast cancer risk for HRT use varied by age, with no risk excess among women aged 45-49 and risk excess of 20% or greater for women aged 50-64 (Table 1). Women >64 had a smaller excess risk with HRT use. There was no significant excess risk at any age for Black women, while White women age 50-64 had > 20% excess risk for HRT use. White women with higher breast density had increased risk with HRT use, while Black women did not. Table 1: Odds ratios (95% confidence interval) for HRT use (yes/no) Conclusions: In this large cohort of mammography-screened women, HRT use did not increase breast cancer risk in the peri-menopausal period (age 45-49). Women with low breast density had smaller risk increments associated with HRT than those with high density. For Black women, increased breast cancer risk due to HRT was not apparent at any age or breast density category. Data collection for the BCSC was supported by a NCI-funded Breast Cancer Surveillance Consortium co-operative agreement (U01CA63740, U01CA86076, U01CA86082, U01CA63736, U01CA70013, U01CA69976, U01CA63731, U01CA70040). Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-09-02.

Melanoma-associated antigens-A (MAGE-A) are members of an extensive family of cancer/testis (CT) antigens. Although emerging studies highlight the potential role of MAGE-A family members in promoting cancer growth, the biological function of MAGE-A4 remains poorly understood. MAGE-A4 is an attractive target because it has a high frequency of expression in multiple solid tumors according to The Cancer Genome Atlas (https://cancergenome.nih.gov/). In an attempt to further characterize its expression in solid tumors, immunohistochemistry analysis was performed in 534 resected non-small cell lung carcinoma (NSCLC) cases, stage I to IV with clinicopathological information including overall survival and recurrence. MAGE-A4 expression was observed in 24% of all NSCLC cases, with higher frequency observed in squamous cell carcinoma (SCC) (51%) versus adenocarcinoma (8%). MAGE-A4 expression was observed in 23% of the primary tumors and 48% of the recurrent tumor samples. Log-rank (Mantel-Cox) analysis showed that histology diagnosis, pathological stage, and MAGE-A4 expression are independent prognostic factors in NSCLC. However, no MAGE-A4 prognostic value was observed when analyzing the SCC subtype. An HLA-A*02-restricted MAGE-A4 TCR specific for MAGE-A4230-239 peptide GVYDGREHTV was identified from a pool of parental TCRs and engineered to optimize specificity and activity (MAGE-A4c1032). TCR fine specificity was assessed initially by mapping the response of the MAGE-A4c1032T-cells to panels of synthetic peptides with combinatorial substitutions at each amino acid with every other possible amino acid (X-Scan), to identify potentially cross-reactive peptides in the human and common pathogen proteomes. Secondly, MAGE-A4c1032T-cells were screened against a wide panel of normal primary cells from multiple organ systems, induced pluripotent stem cell-derived cells (iCells), and autologous whole blood to test for off-target reactivity, and against a panel of EBV-derived B-lymphoblastic cell lines expressing a wide range of HLA molecules to assess the risk of alloreactivity. Alloreactivity was observed in antigen negative cells expressing HLA-A*02:05, and therefore this allele is exclusionary. MAGE-A4c1032T-cell potency was assessed by a variety of in vitro assays, including proliferation, IFN-γ release and cytotoxicity in response to antigen-positive tumor lines in 2D and 3D culture, and cytokine release in response to freshly prepared antigen-positive primary tumor material. In conclusion, MAGE-A4 is a promising target for SPEAR T-cell therapy, and clinical trial opened this year to treat patients with inoperable or metastatic (advanced) NSCLC (SCC, adenosquamous, or large cell carcinoma), ovarian, head and neck SCC, gastric, esophageal (SCC or adenocarcinoma), urothelial tumors and melanoma (NCT03132922). Citation Format: Joana Senra, Pamela Villalobos, Barbara Mino, Luisa Solis, Carmen Behrens, Joseph P. Sanderson, Manoj Saini, Andrew B. Gerry, Nicholas J. Pumphrey, Miguel Maroto, Boris Sepesi, Patrick Hwu, David S. Hong, Elizabeht A. Mittendorf, George S. Blumenschein, Ignacio I. Wistuba, Gwendolyn Binder-Scholl, Rafael Amado. Affinity-enhanced T-cell receptor (TCR) for adoptive T-cell therapy targeting MAGE-A4 [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 2562.

Background: Overexpression of P-cadherin, a calcium-dependent cell-to-cell adhesion glycoprotein, correlates with increased tumor cell invasiveness in breast, colon, lung and pancreatic tumors. FF-21101 is a human-mouse chimeric monoclonal antibody directed against P-cadherin, conjugated with 111In for dosimetry and biodistribution and 90Y for therapy. We report the biodistribution and preliminary anti-cancer activity of FF-21101(90Y) in a first-in-human study. Methods: Patients (pts) received 5 mCi/5mg FF-21101(111In) 1 week before the FF-21101(90Y) therapeutic dose to assess biodistribution and ensure 90Y radiation dose estimates did not exceed the maximum tolerated dose (MTD) for each organ. Single therapeutic dose cohorts were planned for FF-21101(90Y) (8 mCi/mg) at 5, 10, 15, 20 or 25 mCi/m2 (3+3 dose escalation schema), with repeat doses allowed every 4 months in pts demonstrating clinical benefit. Disease assessments were based on RECIST V1.1. Pre-treatment tumor samples were assessed for P-cadherin expression by immunohistochemistry (IHC). Pharmacokinetics (PK) of FF-21101 were also assessed. Results: Fourteen pts (6M, 8F) with advanced primary solid tumors and loco-regional metastases were treated in the 5 dose cohorts. Median (range) values: age 58 years (31 – 69), number of prior systemic treatments 3 (0 – 9), tumor types: soft-tissue sarcoma (3), ovarian carcinoma (CA) (2), and vaginal CA, pancreatic neuroendocrine tumor (PNET), desmoplastic small round cell tumor, colorectal CA, appendiceal CA, cholangiocarcinoma, squamous cell CA (SCC) of the anus, papillary thyroid CA and carcinoid (1 each). Primary and/or metastatic tumors from 12 of 14 pts (85.7%) demonstrated uptake of FF-21101(111In) with highest uptake seen in epithelial tumors, consistent with P-cadherin targeting. Median (range) time on study following the therapeutic dose was 21 (2 - 62) weeks, with 3 pts on study > 1 year (2 ovarian CA, 1 carcinoid). Four pts have received multiple treatments. One ovarian CA pt achieved a partial response (65% reduction); this pt also had the highest H score by IHC staining. High H scores (≥ 100) were demonstrated in 5 of 13 pts: 2 ovarian, 1 PNET, 1 SCC of the anus, 1 cholangiocarcinoma; 4 remained on study for 49 - 62 weeks, thus demonstrating the potential predictive utility of pre-treatment tumor P-cadherin expression. FF-21101(90Y) has been well-tolerated; the MTD has not been reached. The most frequently reported drug-related AEs include lymphopenia (n = 8), thrombocytopenia (n = 4) and leukopenia (n = 3); Gr 3 lymphopenia was observed in 3 pts, all reversible. There have been no drug-related serious AEs. Mean FF-21101 Cmax and AUC0-t increased with dose, suggesting linear PK. Conclusions: Tumor P-cadherin overexpression provides an attractive target for RIT. Radiolabeled FF-21101 exhibits favorable dosimetry and biodistribution, good tolerability and preliminary evidence of anti-cancer activity. Pre-treatment tumor P-cadherin expression may be an important biomarker for patient selection. Citation Format: Vivek Subbiah, William Erwin, Osama Mawlawi, Carlos Gonzalez-Lepera, Masahiko Tokura, Michael Kurman, Holly Liu, David S. Hong, Funda Meric-Bernstam, Shubham Pant, Mary Johansen, Timothy Madden, Elmer Santos, Gregory Ravizzini. Phase 1 study of FF-21101(90Y), a radioimmunotherapeutic (RIT) targeting P-cadherin, in 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 CT128.

Metastatic “seeding” is an early event initiated when cancer cells are shed from the primary tumor, enter into the blood stream as circulating tumor cells (CTCs), and travel to distant organs where they can remain dormant as disseminated cancer cells (DTCs) for indefinite lengths of time. The organ serving as the foremost reservoir for DTCs is the bone marrow (BM). Despite widespread knowledge that bone marrow-resident breast cancer cells (BMRCs) affect tumor progression, signaling mechanisms of BMRCs implicated in maintaining long-term dormancy have not been characterized. This is caused by the lack of biomarkers that can identify and isolate viable but dormant BMRCs not expressing neoplastic and/or proliferation markers. To overcome these hurdles, we developed a patient-derived CTC model of clinical dormancy. We hypothesized that a significant distinction exists between signaling pathways of BM-homing vs metastasis-competent CTCs upon transplantation in xenografts. The model was established by three sequential steps: (a) isolation of CTC-enriched cell populations from the peripheral blood of patients with metastatic breast cancer, followed by CTC implantation in NOD-SCID-gamma mice; (b) in vivo depletion of “normal” human cell lineages over 4-8 months (before onset of overt metastasis), and (c) capture and characterization of viable human CTCs from blood and BM of mice via multi-parametric flow cytometry. This strategy was implemented on a set of breast cancer patients stratified according to their primary tumor subtype (ER+/PR+, HER2+, and ER-/PR-/HER2-), and gene-signatures of putative ex vivo CTCs and BMRCs were obtained by whole genome transcriptomic arrays. First, comparative analyses of ex vivo vs de novo CTC-gene signatures identified increased mTORC2 along with decreased mTORC1 signaling activity as the most significant characteristic of human BM-resident CTCs. mTORC2/mTORC1 represent the two complementary arms of mTOR signaling - a critical pathway frequently dysregulated in breast cancer and implicated in cell survival and dormancy. Second, heightened mTORC2 downstream targets augmented quiescent CTC populations (Ki67-/RBL2+ cells) in paired metastatic vs primary breast cancer tissues. Third, IHC analyses of breast cancer CTC xenograft tissues showed that solitary BM and tissue-resident breast cancer CTCs had high mTORC2 activity. Finally, shRNA knockdown of Rictor-the essential component of mTORC2 actions, increased Ki67/PCNA expression and proliferation. Collectively, these findings suggest that the balance between mTORC1 vs mTORC2 signaling pathways regulate CTC-associated mitotic and/or dormancy characteristics. Further elucidation of mTOR-mediated CTC dormancy will provide novel strategies for therapeutic interventions in breast cancer patients having metastatic “seeding” but yet to develop overt metastasis. Citation Format: Debasish Boral, Haowen N. Liu, Wei Yin, Monika Vishnoi, Antonio T. Scamardo, David S. Hong, Dario Marchetti. Targeting bone marrow-resident dormant CTCs to overcome metastasis [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 3264.

Background: Dysregulation of the PI3K-AKT signaling pathway is associated with a number of cancers. and plays a critical role in cancer initiation and progression. AKT can be activated through activated receptor tyrosine kinases, gain-of-function mutations of PIK3CA, PTEN deficiency, and AKT amplification or activating mutations such as AKT1E17K. As the second generation of allosteric AKT inhibitor, ARQ 751 potently inhibits AKT1, 2 and 3 with biochemical IC50 values of 0.55 nM, 0.81 nM and 1.31 nM receptively. In addition, ARQ 751 is very selective; it does not inhibit any other kinase (out of the 245 tested) by greater than 50% at 5 µM. The objective of this study is to determine maximum tolerated dose in patients with advanced solid tumors. Material and Methods: This is Phase 1 Dose Escalation Study to assess the safety and tolerability of ARQ 751 in subjects with advanced solid tumors with AKT1, 2, 3 genetic alterations, activating PI3K mutations, PTEN-null, or other known actionable PTEN mutations. Treatment emerging adverse events (TEAE) were assessed per NCI CTCAE v. 4.03. Tumor response were evaluated per RECIST 1.1. Blood samples were collected for PK. Results: A total of 15 pts have been enrolled [73% female; median age 61 years; 5 Breast (5), endometrial (2), and others (9); activating PI3K mutation (9), PTEN null/other known actionable PTEN mutations (5), AKT 1 mutation (1)] and treated at dose levels of 5 mg QD, 10 mg QD, 20 mg QD, 25 mg QOD and 25 mg QD. There have been no DLTs reported so far. ARQ 751 related TEAEs included nausea (27%), stomatitis, vomiting, fatigue, mucosal inflammation, white blood cell count decreased, hyperkalaemia, cough, oropharyngeal pain, sinus congestion, pain of skin, puritus and hot flushes (7% each). All these TEAEs were grade 1 or 2. There was no ≥ Grade 3 drug related TEAEs or any grade drug-related SAEs. Four pts achieved a best response of stable disease (SD) including 1 with breast cancer treated at 25 mg QOD for 42+ weeks, 1 with head and neck cancer treated at 20 mg QD for 16 weeks, 1 with breast cancer treated at 25 mg QD for 12+, and 1 with endometrial cancer treated at 25 mg QD for 10+ weeks. 3 of 4 SD pts are currently on therapy. Conclusions: ARQ 751 demonstrated a manageable safety profile at Dose level up to 25 mg QD. The dose escalation is ongoing. PK and updated safety, efficacy data will be presented. Citation Format: Shubham Pant, Vivek Subbiah, Jordi Rodon, Filip Janku, David Hong, Daniel Karp, Sarina Piha-Paul, Apostolia M. Tsimberidou, Aung Naing, Siqing Fu, Ron E. Savage, Feng Chai, Yi Yu, Brian Schwartz, Funda Meric-Bernstam, Tim Yap. Results of a phase I dose escalation study of ARQ 751 in adult subjects with advanced solid tumors with AKT1, 2, 3 genetic alterations, activating PI3K mutations, PTEN-null, or other known actionable PTEN mutations [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 CT024.