Pretreatment Tumor Biopsies Research Articles

Multicenter validation of an RNA-based assay to predict anti-PD-1 disease control in patients with recurrent or metastatic head and neck squamous cell carcinoma: the PREDAPT study.

Despite advances in cancer care and detection, >65% of patients with squamous cell cancer of the head and neck (HNSCC) will develop recurrent and/or metastatic disease. The prognosis for these patients is poor with a 5-year overall survival of 39%. Recent treatment advances in immunotherapy, including immune checkpoint inhibitors like pembrolizumab and nivolumab, have resulted in clinical benefit in a subset of patients. There is a critical clinical need to identify patients who benefit from these antiprogrammed cell death protein 1 (anti-PD-1) immune checkpoint inhibitors. Here, we report findings from a multicenter observational study, PREDicting immunotherapy efficacy from Analysis of Pre-treatment Tumor biopsies (PREDAPT), conducted across 17 US healthcare systems. PREDAPT aimed to validate OncoPrism-HNSCC, a clinical biomarker assay predictive of disease control in patients with recurrent or metastatic HNSCC treated with anti-PD-1 immune checkpoint inhibitors as a single agent (monotherapy) and in combination with chemotherapy (chemo-immunotherapy). The test used RNA-sequencing data and machine learning models to score each patient and place them into groups of low, medium, or high. The OncoPrism-HNSCC prediction significantly correlated with disease control in both the monotherapy cohort (n=62, p=0.004) and the chemo-immunotherapy cohort (n=50, p=0.01). OncoPrism-HNSCC also significantly predicted progression-free survival in both cohorts (p=0.015 and p=0.037, respectively). OncoPrism-HNSCC had more than threefold higher specificity than programmed death-ligand 1 combined positive score and nearly fourfold higher sensitivity than tumor mutational burden for predicting disease control. Here, we demonstrate the clinical validity of the OncoPrism-HNSCC assay in identifying patients with disease control in response to anti-PD-1 immune checkpoint inhibitors. NCT04510129.

The Innate Immune Landscape of dMMR/MSI Cancers Predicts the Outcome of Nivolumab Treatment: Results from the Drug Rediscovery Protocol.

The treatment efficacy of nivolumab was evaluated in patients with advanced, treatment-refractory solid mismatch repair deficiency/microsatellite-instable (dMMR/MSI) tumors, and in-depth biomarker analyses were performed to inform precision immunotherapy approaches. Patients with dMMR/MSI tumors who exhausted standard-of-care treatment options were enrolled in the Drug Rediscovery Protocol, a pan-cancer clinical trial that treats patients with cancer based on their tumor molecular profile with off-label anticancer drugs (NCT02925234). Patients received nivolumab (four cycles of 240 mg every 2 weeks, thereafter 480 mg every 4 weeks). The primary endpoint was clinical benefit (CB: objective response or stable disease ≥16 weeks). Whole-genome sequencing and RNA sequencing were performed on pretreatment tumor biopsies. A total of 130 evaluable patients were enrolled with 16 different cancer types. CB was observed in 62% [95% confidence interval (CI), 53-70], with an objective response in 45% (95% CI, 36-54). After a median follow-up of 14.5 months (95% CI, 13-19), the median progression-free survival was 18 months (95% CI, 9-not reached), and the median overall survival was not reached. Whereas CB was not, or only weakly, associated with markers of adaptive immune cell infiltration, CB was strongly associated with expression of a broad set of innate immune receptors/ligands. This clearly contrasted findings in melanoma, in which markers of adaptive immunity dominated the biomarker landscape. Nivolumab proved highly effective in advanced dMMR/MSI tumors. Expression of key innate immune receptors/ligands was the main predictor of a good treatment outcome, contrasting findings in melanoma and strengthening the rationale for tumor type-specific biomarkers for guiding immunotherapy.

Eganelisib combined with immune checkpoint inhibitor therapy and chemotherapy in frontline metastatic triple-negative breast cancer triggers macrophage reprogramming, immune activation and extracellular matrix reorganization in the tumor microenvironment

BackgroundTriple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with a poor prognosis particularly in the metastatic setting. Treatments with anti-programmed cell death protein-1/programmed death-ligand 1 (PD-L1) immune...

Epigenetic agents plus anti-PD-1 reprogram the tumor microenvironment and restore antitumor efficacy in Hodgkin lymphoma

AbstractDNA methyltransferase inhibitor decitabine plus anti–programmed cell death 1 (DP) therapy was effective in relapsed/refractory classic Hodgkin lymphoma (cHL). However, a subset of patients experienced primary resistance or relapse/progression after DP therapy. In this study, we evaluated the efficacy and safety of a triplet regimen consisting of the histone deacetylase inhibitor chidamide, decitabine, and anti–PD-1 camrelizumab (CDP) in 52 patients who previously received DP therapy. CDP treatment was well tolerated and resulted in an objective response rate of 94% (95% confidence interval [CI], 84-99), with 50% (95% CI, 36-64) of patients achieving complete response (CR). Notably, all patients who were recalcitrant to previous DP treatment exhibited therapeutic responses after CDP therapy, although their CR rate was lower than patients responsive to prior DP. Overall, the median progression-free survival was 29.4 months. Through single-cell RNA sequencing of pretreatment and on-treatment cHL tumor biopsy samples, we observed the heterogeneity of rare malignant Hodgkin Reed/Sternberg (HRS)–like cells. The classical CD30+ HRS-like cells interacted with abundant immunosuppressive IL21+CD4+ T helper cells, forming a positive feedback loop that supported their survival. While the CD30– HRS-like cell population showed potential resistance to anti–PD-1 immunotherapy. CDP treatment promoted the activation of diverse tumor-reactive CD8+ T cells and suppressed the proliferation of IL21+CD4+ T cells by inhibiting STAT1/3 signaling, thereby alleviating their immunosuppressive effects. These findings provide insights into the cHL microenvironment that contributes to anti–PD-1 resistance and highlight the therapeutic effectiveness of dual epi-immunotherapy in overcoming immunotherapy resistance. This trial was registered at www.clinicaltrials.gov as #NCT04233294.

A Phase I/II Study of Valemetostat (DS-3201b), an EZH1/2 Inhibitor, in Combination with Irinotecan in Patients with Recurrent Small-Cell Lung Cancer.

Recurrent small-cell lung cancer (SCLC) has few effective treatments. The EZH2-SLFN11 pathway is a driver of acquired chemoresistance that may be targeted. This phase I/II trial investigated valemetostat, an EZH1/2 inhibitor, with fixed-dose irinotecan in patients with recurrent SCLC. Phase I primary objectives were to assess safety, tolerability, and a recommended phase II dose (RP2D). The phase II primary objective was overall response rate (ORR), with secondary objectives of determining duration of response (DoR), progression-free survival (PFS), and overall survival (OS). Correlative analyses included immunohistochemistry of pretreatment and on-treatment tumor biopsies and pharmacokinetics analysis. Twenty-two patients were enrolled (phase I, n = 12; phase II, n = 10); one withdrew consent prior to treatment. Three dose-limiting toxicities (DLT) in dose-escalation resulted in valemetostat 100 mg orally daily selected as RP2D. Among 21 evaluable patients, the most frequent (≥20%) treatment-related adverse events were diarrhea, fatigue, nausea, and rash; three patients discontinued treatment for toxicity. Three of the first 10 patients in phase II experienced DLTs triggering a stopping rule. The ORR was 4/19 or 21% [95% confidence interval (CI), 6%-46%]. The median DoR, PFS, and OS were 4.6 months, 2.2 months (95% CI, 1.3-7.6 months), and 6.6 months (95% CI, 4.3 to not reached), respectively. SLFN11/EZH2 expression and SCLC subtyping markers did not correlate with response, but MHC-I expression did increase with treatment. Two responders demonstrated subtype switching on treatment. Combination valemetostat and irinotecan was not tolerated but demonstrated efficacy in recurrent SCLC. Valemetostat, combined with agents without overlapping toxicity, warrants further investigation in SCLC.

Targeting replication stress in pancreatic cancer: A phase II trial investigating azenosertib in combination with gemcitabine in second-line advanced pancreatic cancer.

TPS4211 Background: A major unmet need in pancreatic cancer (PDAC) is molecularly targeted treatment options for patients who develop resistance to first-line platinum chemotherapy. While combined gemcitabine and nab-paclitaxel is commonly used after progression with first-line therapy, the efficacy in the second-line setting is fair, with recent phase clinical trial data demonstrating a median progression-free survival (PFS) of 2.7 months and a median overall survival of 6.6 months (1). In recent years, preclinical work has identified that replication stress is a potential PDAC vulnerability. The WEE1 kinase regulates the checkpoint governing the G2/M transition of the cell cycle to compensate for replication stress. The G2/M checkpoint is critically important in malignancies like PDAC where over 75% of tumors have p53 pathway deficits. Therapies that inhibit cancer cells’ ability to compensate for replication stress are particularly attractive in cancers that have genomic alterations that generate replication stress. It has been demonstrated that in vitro and in vivo PDAC models were exquisitely sensitive to WEE1 inhibition in combination with gemcitabine. Azenosertib (Zn-c3) is an orally available, highly selective, nanomolar inhibitor of the WEE1 kinase and when combined with gemcitabine in PDAC cell lines, synergistic cytotoxicity is observed. Methods: NCT06015659 is an ongoing phase II clinical trial with safety lead-in that is testing the azenosertib WEE1 inhibitor combined with gemcitabine in patients with advanced pancreatic cancer after progression or intolerance with FOLFIRINOX. Subjects are dosed once daily, days 1-5, 8-12, and 15-19 with azenosertib and receive gemcitabine 800 mg/m2 on days 1 and 8 of 21-day cycles. Up to 12 subjects are expected to enroll in the safety lead-in portion based on a typical 3 + 3 design. Once the recommended phase 2 dose is confirmed, we will use a Simon two-stage minimax design to evaluate efficacy where the null hypothesis is that the true 6-month PFS rate is ≤ 24% (unacceptable historical control data) and the alternative hypothesis is that the true 6-month PFS is ≥ 44%. Pretreatment and on-treatment tumor biopsies are incorporated into the trial to identify potential biomarkers of treatment response. Subjects must be able to swallow oral tablets and have measurable disease by RECIST v1.1; prior exposure to gemcitabine is not allowed. Enrollment began November 21, 2023, and is ongoing. 1. Huffman BM, Basu Mallick A, Horick NK, et al. JAMA Netw Open. 2023;6(1):e2249720. Clinical trial information: NCT06015659 .

Hormone Receptor Expression and Activity for Different Tumour Locations in Patients with Advanced and Recurrent Endometrial Carcinoma.

Response to hormonal therapy in advanced and recurrent endometrial cancer (EC) can be predicted by oestrogen and progesterone receptor immunohistochemical (ER/PR-IHC) expression, with response rates of 60% in PR-IHC > 50% cases. ER/PR-IHC can vary by tumour location and is frequently lost with tumour progression. Therefore, we explored the relationship between ER/PR-IHC expression and tumour location in EC. Pre-treatment tumour biopsies from 6 different sites of 80 cases treated with hormonal therapy were analysed for ER/PR-IHC expression and classified into categories 0-10%, 10-50%, and >50%. The ER pathway activity score (ERPAS) was determined based on mRNA levels of ER-related target genes, reflecting the actual activity of the ER receptor. There was a trend towards lower PR-IHC (33% had PR > 50%) and ERPAS (27% had ERPAS > 15) in lymphogenic metastases compared to other locations (p = 0.074). Hematogenous and intra-abdominal metastases appeared to have high ER/PR-IHC and ERPAS (85% and 89% ER-IHC > 50%; 64% and 78% PR-IHC > 50%; 60% and 71% ERPAS > 15, not significant). Tumour grade and previous radiotherapy did not affect ER/PR-IHC or ERPAS. A trend towards lower PR-IHC and ERPAS was observed in lymphogenic sites. Verification in larger cohorts is needed to confirm these findings, which may have implications for the use of hormonal therapy in the future.

Abstract PO3-05-06: Targeting PAM50 HER2-Enriched intrinsic subtype with enzalutamide in hormone receptor-positive/HER2-negative (HR+/HER2-) advanced breast cancer: results of the SOLTI-1502 ARIANNA trial

Abstract Background Pre-clinical evidence and retrospective studies suggest that PAM50 HER2-Enriched (HER2-E), HR+/HER2- tumors have estrogen receptor (ER) independence and poor prognosis but seem to have androgen receptor (AR)-addiction. Enzalutamide (ENZ) is a potent inhibitor of AR signaling. We hypothesized that ENZ may induce a significant proliferative arrest in PAM50 HER2-E, HR+/HER2-negative advanced breast cancer (ABC), leading to clinical benefit in this poor prognosis population. Methods Eligible patients (pts), males or pre/post-menopausal women with advanced HR+/HER2- ABC resistant to endocrine therapy, received ENZ 160 mg/day until disease progression. Pts were assigned to different cohorts according to PAM50 intrinsic subtypes identified in the pre-treatment tumor biopsy: Cohort A included pts with HER2-E and Cohort B pts with Luminal A/B tumors. Centralized molecular assessment of pre-treatment and C1D15 tumor biopsies were required for the primary endpoint evaluation. After C1D15 biopsy, exemestane 50mg QD could be added to ENZ at physician’s discretion. Tumor assessments were performed every 8 weeks. Primary efficacy endpoint was the relative change in the PAM50 11-gene proliferation-related signature in Cohort A. Secondary objectives included anti-proliferative effect of ENZ in Cohort B, overall response rate (ORR), Clinical benefit rate (CBR; partial response [PR] + stable disease ≥ 24 weeks) progression-free survival (PFS), and safety. Results From June 2020 to October 2022, 47 tumors were screened, and 6 PAM50 HER2-E tumors were identified (13%). A total of 34 pts were enrolled: 6 pts in Cohort A and 28 pts in Cohort B. Among them, 4 pts from Cohort A received ENZ plus exemestane and 23 pts in Cohort B. Mean age was 59 (range 39-76), 89% of pts were postmenopausal, 74% had liver disease, and 59% received (neo)adjuvant treatment. The median number of lines for ABC was 4 (1-8). Mean Ki67 (central assessment) in Cohort A was 37% (5%-70%), while in Cohort B it was 30% (3%-90%). Mean central AR in Cohort A was 66% (30-100), while in Cohort B it was 55% (1-100). The mean suppression of PAM50 11-gene proliferation-signature after 2 weeks of treatment with ENZ was 3.7% (p=0.848) in Cohort A and 1.2% (p=0.909) in Cohort B. The median change in Ki67 levels in Cohort A was -0.43% (p=0.812), while in Cohort B it was +3.86% (p=0.081). No statistically significant differentially expressed genes or changes in intrinsic subtypes were observed. Overall, the median PFS was 1.8 months (95% CI 1.6-1.8). In Cohort A, median PFS was 1.6 months (95% CI 1.0-1.9), and in Cohort B, it was 1.8 months (95% CI 1.6-1.8). CBR was 0% and 11% (n=3, 95% CI 2.8-25.9%) in Cohorts A and B, respectively. A PR with 16 months duration in cohort B was observed in a patient with an AR-mutated tumor (AR c. 689C >T). Regarding safety, grade 1-2 and 3-4 toxicities occurred in 94.1% and 35.3% of patients, respectively. ENZ dose reduction and discontinuation occurred in 2 (5.9%) and 3 (8.8%) pts, respectively. The most common toxicities related to ENZ were nausea (n=8, 24%), decreased appetite (n=8, 24%), AST increase (n=6, 18%) and vomiting (n=6, 18%). Conclusions The hypothesis that ENZ could induce proliferation arrest in HR+/HER2-, PAM50 HER2-E tumors was not confirmed, and ARIANNA was prematurely closed due to the lack of efficacy. Additional research is needed to explore if ENZ may benefit specific populations of pts with breast cancer (e.g. AR-mut tumors) and whether alternative AR modulators are more effective in blocking AR signaling in HR+/HER2-, PAM50 HER2-E tumors Citation Format: Mafalda Oliveira, Tomás Pascual, Sonia Pernas, Mireia Margelí, Salvador Blanch, Barbara Adamo, Francisco Javier Salvador Bofill, Xavier Gonzalez-Farré, Samyukta Chillara, Patricia Galván, Esther Sanfeliu, Paula Blasco, Valeria Sirenko, Alejandra Espinosa, Charles M Perou, Aleix Prat, Luis Manso. Targeting PAM50 HER2-Enriched intrinsic subtype with enzalutamide in hormone receptor-positive/HER2-negative (HR+/HER2-) advanced breast cancer: results of the SOLTI-1502 ARIANNA trial [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-05-06.

Abstract PO2-15-08: Exploring Essential Immune Genes Linked to Pathological Complete Response and Survival in Early-Stage Triple-Negative Breast Cancer (TNBC) after Chemotherapy Treatment

Abstract Background: De-escalation and escalation of systemic therapy in early-stage TNBC will require the implementation of biomarkers to guide therapy. To date, high expression of a variety of immune genes has been associated with higher pathological complete response (pCR) rates following neoadjuvant chemotherapy or with improved survival, or both, in early-stage TNBC. However, it is currently unknown which are the key genes influencing survival (i.e., the Core Immune Genes [CIG]) and if combinations of these genes can improve their association with both clinical endpoints. Methods: The expression of up to 185 genes was evaluated on pre-treatment tumor biopsies from 3 independent publicly available datasets of early-stage TNBC: CALGB40603 (n=388) (NCT00861705), BrighTNess (n=482) (NCT02032277), and SCAN-B (n=498). Patients in CALGB40603 and BrighTNess were treated with neoadjuvant chemotherapy-based therapy and had response data. Patients in CALGB40603 and SCAN-B had survival outcome data (event-free survival [EFS] or relapse-free interval [RFi]). Each individual gene was associated with each clinical endpoint within each dataset using Cox model and logistic regression analyses, adjusting for treatment arm. Genes found in common across datasets were selected as CIG, except for PDCD1, which was not available in BrighTNess. Using the CIGs, we evaluated whether combinations of 2 genes enhance their association with each clinical endpoint using different methods: adding, subtracting, multiplying, and ratio. Likelihood ratio testing was used to evaluate the added contribution of each gene in 2 combined cohorts evaluating pCR (i.e., CALGB40603 and BrighTNess) and survival (i.e., CALGB40603 and SCAN-B). Results: 10 CIGs related to B-cells and T-cells (i.e., IRF4, LAX1, POU2AF1, CD274, CD79A, TNFRSF17, PIM2, PDCD1, CXCR6, and SLAMF1) were associated with higher pCR rates and improved survival outcome across datasets. Of note, 5 (50%) genes are known components of the 14-gene immunoglobulin/B-cell (IGG) signature. The correlation coefficient among the 10 CIG genes was 0.63 (moderate). In general, the sum of 2 CIGs provided more prognostic information than a single gene (p< 0.001). Specifically, the sum of 2 CIGs provides more prognostic information than a single gene in 30 of 45 (66.6%) cases. Two-gene subtraction, multiplication or ratio decreased the amount of prognostic information (p< 0.001). The top 10 2-gene CIGs associated with prognosis (p< 0.001) were CD274_TNFRSF17, IRF4_CD274, PDCD1_TNFRSF17, CD79A_CD274, CD274_POU2AF1, CXCR6_TNFRSF17, LAX1_TNFRSF17, SLAMF1_TNFRSF17, CD274_LAX1 and CD79A_CXCR6. In general, the sum of 2 CIGs provided more information about pCR than a single gene (p< 0.001). Specifically, the sum of 2 CIGs provided numerically more information about pCR than 1 gene in 17 of 36 (47.2%) cases. Two-gene subtraction, multiplication or ratio decreased the amount of information about pCR (p< 0.001). The top 10 2-gene CIGs associated with pCR were CD274_PIM2, CD274_POU2AF1, CXCR6_PIM2, CD274_CXCR6, IRF4_CD274, CXCR6_POU2AF1, CD274_LAX1, IRF4_CXCR6, CXCR6_LAX1, and POU2AF1_SLAMF1. Conclusion: The expression of CIGs associated with B-cell and T-cell adaptive immune response is closely linked to pCR and survival outcomes following (neo)adjuvant chemotherapy. When combining the expression of 2 specific CIGs through addition, we found that this method provides more informative insights into pCR and survival outcomes compared to other combination methods. This approach enhances the predictive power of CIGs, enabling a better understanding of treatment response and patient prognosis, ultimately contributing to improved clinical decision-making and patient outcomes. Citation Format: Fara Brasó-Maristany, Laia Paré, Benedetta Conte, Laura Angelats, Ana Vivancos, Joel S Parker, Charles M. Perou, Patricia Villagrasa, Aleix Prat. Exploring Essential Immune Genes Linked to Pathological Complete Response and Survival in Early-Stage Triple-Negative Breast Cancer (TNBC) after Chemotherapy Treatment [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-15-08.

Context matters: Tumor microenvironments impact cellular therapy success

In a recent publication, Locke etal. present data from pretreatment tumor biopsies taken on the ZUMA-7 trial. Their results identify tumor microenvironment (TME) contexts and level of CD19 expression as prognostic indicators for responses to axicabtagene ciloleucel (axi-cel).

Immunological characterization of a long-lasting response in a patient with metastatic triple-negative breast cancer treated with PD-1 and LAG-3 blockade

In patients with advanced triple-negative breast cancer (TNBC), translational research efforts are needed to improve the clinical efficacy of immunotherapy with checkpoint inhibitors. Here, we report on the immunological characterization of an exceptional, long-lasting, tumor complete response in a patient with metastatic TNBC treated with dual PD-1 and LAG-3 blockade within the phase I/II study CLAG525X2101C (NCT02460224) The pre-treatment tumor biopsy revealed the presence of a CD3+ and CD8+ cell infiltrate, with few PD1+ cells, rare CD4+ cells, and an absence of both NK cells and LAG3 expression. Conversely, tumor cells exhibited positive staining for the three primary LAG-3 ligands (HLA-DR, FGL-1, and galectin-3), while being negative for PD-L1. In peripheral blood, baseline expression of LAG-3 and PD-1 was observed in circulating immune cells. Following treatment initiation, there was a rapid increase in proliferating granzyme-B+ NK and T cells, including CD4+ T cells, alongside a reduction in myeloid-derived suppressor cells. The role of LAG-3 expression on circulating NK cells, as well as the expression of LAG-3 ligands on tumor cells and the early modulation of circulating cytotoxic CD4+ T cells warrant further investigation as exploitable predictive biomarkers for dual PD-1 and LAG-3 blockade.Trial registration: NCT02460224. Registered 02/06/2015.

Abstract B005: A phase 1 expansion cohort study evaluating the safety and efficacy of the CHK1 inhibitor LY2880070 with low-dose gemcitabine in metastatic pancreatic adenocarcinoma

Abstract Background: Combining gemcitabine with CHK1 inhibition has shown promise in preclinical models of pancreatic ductal adenocarcinoma (PDAC). Here, we report the findings from a phase I expansion cohort study (NCT02632448) investigating low-dose gemcitabine combined with the CHK1 inhibitor LY2880070 in patients with previously treated advanced PDAC. Methods: Patients with metastatic PDAC were treated with gemcitabine intravenously at 100 mg/m2 on days 1, 8, and 15, and LY2880070 50 mg orally twice daily on days 2–6, 9–13, and 16–20 of each 21-day cycle. Pre-treatment tumor biopsies were obtained from each patient for correlative studies and generation of organoid cultures for drug sensitivity testing and biomarker analyses. Results: Eleven patients with PDAC were enrolled in the expansion cohort between August 27, 2020 and July 30, 2021. Four patients (36%) experienced drug-related grade 3 adverse events. No objective radiological responses were observed, and all patients discontinued the trial by 3.2 months. In contrast to the lack of efficacy observed in patients, organoid cultures derived from biopsies procured from two patients demonstrated strong sensitivity to the gemcitabine/LY2880070 combination and showed treatment-induced upregulation of replication stress and DNA damage biomarkers, including pKAP1, pRPA32, and gamma-H2AX, as well as induction of replication fork instability. Conclusions: No evidence of clinical activity was observed for combined low dose gemcitabine and LY2880070 in this treatment refractory PDAC cohort. However, the gemcitabine/LY2880070 combination showed in vitro efficacy, suggesting that drug sensitivity for this combination in organoid cultures may not predict clinical benefit in patients. Citation Format: Brandon M. Huffman, Hanrong Feng, Kalindi Parmar, Junning Wang, Kevin Kapner, Bose Kochupurakkal, David Martignetti, Thomas Abrams, Leah Biller, Marios Giannakis, Kimmie Ng, Anuj Patel, Kimberly Perez, Harshabad Singh, Douglas Rubinson, Benjamin Schlechter, Elizabeth Andrews, Alison Hannigan, Stanley Dunwell, Zoe Getchell, Srivatsan Raghavan, Brian Wolpin, Alan D'Andrea, Andrew Aguirre, Geoffrey Shapiro, James Cleary. A phase 1 expansion cohort study evaluating the safety and efficacy of the CHK1 inhibitor LY2880070 with low-dose gemcitabine in metastatic pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B005.

Abstract B138: Pharmacodynamics-driven phase 2 trial of talazoparib in patients with advanced solid tumors and aberrations in genes involved in DNA damage response

Abstract Background: Talazoparib is a poly (ADP-ribose) polymerase inhibitor (PARPi) with increased trapping of PARP-DNA complexes compared to other PARPi in clinical development. Talazoparib has single-agent synthetic lethality in BRCA1/2- and PTEN-deficient cell lines, has potent antitumor activity in animal models of tumors harboring mutations in DNA repair pathways, and has FDA-approved indications in advanced BRCA-mutated and HER2-negative breast cancer and in homologous recombination repair gene-mutated metastatic castration-resistant prostate cancer in combination with enzalutamide. This pilot study evaluated the pharmacodynamic (PD) effects of talazoparib on DNA damage response (DDR) markers in tumor biopsy tissue in both patients (pts) who are PARPi-naïve and pts who have received prior PARPi other than talazoparib to investigate for potential differential PARPi PD responses in DNA replication stress. Methods: We are conducting a multicenter phase 2 trial in adult pts with eligible deleterious germline or somatic mutations in BRCA1/2 or genes involved in DDR including ATM, ARID1A, IDH1, BAP1, ATR, CHK2, and PALB2. Pts were enrolled and assessed in two cohorts: 1) pts who were PARPi-naïve, and 2) pts who were treated with and had documented progression on a PARPi other than talazoparib. Talazoparib is administered orally at a dose of 1000 µg/day (D) in 28 D cycles (C). The primary objective is determining the replication stress PD effect of talazoparib as evaluated by activation of Rad51 combined with a lack of γH2AX activation. Paired tumor biopsies (baseline and C2D1) will be analyzed for DDR markers (γH2AX, pNBS1, and Rad51) by IFA. Responses and adverse events (AEs) are defined by RECIST 1.1 and CTCAE v5.0, respectively. Results: The PARPi-naïve cohort had 15 pts enrolled with a median age of 58 years (range 38-76) and median prior lines of systemic therapy of 2 (range 1-5). The PARPi-treated cohort had 8 pts enrolled with a median age of 72 years (range 35-85) and median prior lines of systemic treatment of 5.5 (range 1-10). A total of 11 sets of pre-treatment and post-treatment tumor biopsies have been received to date and analyses of DDR markers are ongoing. Of 11 pts in the PARPi-naïve cohort evaluable for response, 6 pts had stable disease (SD) as best response with 2 pts (nasal cavity cancer with ARID1A alteration, bile duct cancer with IDH1 alteration) who received talazoparib for ≥ 6 C. Of 7 pts in the PARPi-treated cohort evaluable for response, 4 pts had SD as best response. Treatment-related AEs of ≥ grade 2 with ≥ 5% incidence included anemia (n=10, 43.5%), leukopenia (n=7, 30.4%), lymphopenia (n=6, 26.1%), thrombocytopenia (n=6, 26.1%), and neutropenia (n=4, 17.4%). Conclusions: Study enrollment and analyses of PD DDR markers in paired tumor biopsies are both ongoing with data to be presented at the conference. No responses have been observed to date, which may be due to prior therapies, tumor histologies, mutational selection or dose delivered with further characterization anticipated via PD DDR marker data. Citation Format: Brian Ko, Geraldine H O'Sullivan Coyne, Abdul Rafeh Naqash, Thomas J George, David DeRemer, Larry Rubinstein, Jennifer Zlott, Deborah Wilsker, Donna Ketchum, Stephanie Runkle, Katherine V Ferry-Galow, Ralph E Parchment, Naoko Takebe, Sarah Shin, Andrea R Voth, Jiuping Ji, S. Percy Ivy, James H Doroshow, Alice P Chen. Pharmacodynamics-driven phase 2 trial of talazoparib in patients with advanced solid tumors and aberrations in genes involved in DNA damage response [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B138.

Abstract A160: The FGFR axis is a potential radiosensitization target

Abstract Background: Establishing methods that sensitize cancers to photons is important for improving the treatment outcome of radiotherapy. Although sequencing has been widely used to identify druggable mutations in various cancers, this strategy have not been used extensively to identify new targets for radiotherapy sensitization. Thus, we performed clinical sequencing of radiotherapy-treated patient samples and investigated whether inhibition of the identified candidate targets resulted in radiosensitization in vitro and in vivo. Material and Methods: Ninety-eight patients with newly diagnosed and pathologically confirmed cervical cancer who had been treated with definitive radiotherapy were analyzed. DNA was extracted from formalin fixed and paraffin embedded pre-treatment tumor biopsy specimens. The exons of 409 cancer-related genes were sequenced using a next generation sequencer. Identified mutations were analyzed for correlations with clinical outcome. The radiosensitization induced by inhibition of the candidate target was investigated in in vitro clonogenic assays and nude mouse xenograft models using radioresistant cancer cell lines and small molecule inhibitors. The in vitro sensitizer enhancement ratio (SER) was calculated as the ratio of radiation dose required to reduce clonogenic cell survival by 50% without drug to that with drug. Results: Recurrent mutations were identified in PIK3CA (35.7%), ARID1A (25.5%), NOTCH1 (19.4%), FGFR3 (16.3%), FBXW7 (19.4%), TP53 (13.3%), EP300 (12.2%), and FGFR4 (10.2%). The prevalence of mutations in FGFR family genes combined (24.5%) was almost as high as that for well-studied PIK3CA and ARID1A. Fifty-five percent (21/38) of the identified FGFR mutations were located in the tyrosine kinase domain. Five-year progression-free survival (PFS) rates for FGFR mutation-positive patients (n = 24) were significantly worse than those for FGFR mutation-negative patients (n = 74) (43.9% vs. 68.5%). Multivariate analysis identified FGFR mutations as significant predictors of worse 5 year PFS, independent of clinicopathological variables. To investigate whether the FGFR axis can be targeted by drugs to increase radiosensitization, the radiosensitizing effect of a human-administrable selective pan-FGFR inhibitor LY2874455 was investigated. LY2874455 (at 100 nM) showed a potent in vitro radiosensitizing effect (SER: 1.62, 1.53, and 1.31 for A549, H1299, and H1703 cells, respectively). In A549 xenograft models, the combination of X-rays (10 Gy on day 1) and LY2874455 (3 mg/kg body weight, once daily, on Day 1–7) led to significantly greater suppression of tumor growth than either treatment with X-rays or LY2874455 alone, with no evidence of toxicities. LY2874455 treatment inhibited ERK signaling, the main target of the FGFR axis, both in vitro and in vivo. Conclusion: These data indicate that targeting the FGFR axis with LY2874455 could potentially be used to sensitize cancers to radiotherapy, which warrants clinical validation. Citation Format: Takahiro Oike, Eisuke Horigome, Akiko Adachi, Narisa Dewi Maulany Darwis, Atsushi Shibata, Tatsuya Ohno. The FGFR axis is a potential radiosensitization target [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A160.

Prediction Modeling of CAR-T Cell Therapy for Diffuse Large B-Cell Lymphoma Using Artificial Neural Networks on Tumor Vascular Phenotype

Chimeric antigen receptor T (CAR-T) cell therapy targeting CD19 only results in complete remission in approximately 30-40% of relapsed/refractory disease patients with diffuse large B-cell lymphoma (DLBCL). Failure may result from lack of CAR-T entry into the tumor. Endogenous T cell homing is initiated by binding to post-capillary venular endothelial cells (ECs). Naïve and central memory T cells use L-selectin binding to peripheral node addressin (PNad) on high endothelial venules (HEVs) of the lymph node while circulating effector memory T cells, lacking L-selectin, use other molecules to bind cytokine-induced endothelial proteins E-selectin, ICAM-1 and VCAM-1 on flat venules of inflamed tissues. Both of these venular EC types are present in inflamed lymph nodes. We hypothesized that the expression patterns of adhesion molecules on venular ECs in pre-treatment tumor biopsies could predict CAR-T efficacy. To test this, we applied cyclic immunofluorescence to 18 archived pre-treatment DLBCL patient FFPE biopsies (8 responders and 10 non-responders, defined by relapse at 12 months). We detected venular ECs using a combination of anti-CD31 and UEA lectin on larger diameter vessels and assessed expression for PNad, E-selectin, ICAM-1, VCAM-1. We detected endogenous CD8+ T cells and evaluated their expression of L-selectin, alpha 4 integrin and PSGL-1. See table 1. DAPI nuclear staining was included in each cycle. Images of each cycle were combined in ImageJ and imported into QuPath for analysis. Using Warpy, affine transformations were applied to DAPI staining to create overlays between imaging cycles. After alignment, representative images of several samples were randomly chosen for image training using a combination of Artificial Neural Networks (ANNs) and classifiers to perform identification and mask tissue regions of vessels and T-cells within the tissue. Positive and negative cells for each marker of interest (EC PNad, ICAM-1, E-Selectin, VCAM-1, and T cell alpha 4 integrin, L-Selectin, and PSGL-1) were used to create classification algorithms to determine marker expression for ECs and T-cells within the tissue. Results were analyzed by unpaired t-test and one-way ANOVA conducted in Graph-Pad Prism 9 software. Preliminary results from quantitative analysis of pre-treatment biopsies show a significant increase in CD31+/PNAD- venular ECs compared to non-responders that were double positive for ICAM1 and VCAM-1 (p=0.0273). This analysis also revealed a significant increase in CD31+/PNAD+ HEV ECs from responders that were triple positive for E-Selectin, ICAM-1, and VCAM-1 (p=0.0242). See figure 1. Our initial semi-quantitative analysis showed a trend toward elevated numbers of tissue-infiltrating CD3 + CD8 + T cells in the pre-treatment biopsies of responders. T cells expressing PSGL-1, a glycoprotein that may carry carbohydrate moieties recognized by E-Selectin and linked to T-cell exhaustion, was increased in the non-responder cohort. These data suggest an increased extent and activation state of intra-tumoral venular ECs and increased endogenous T-cell infiltrates, which may predict the outcome of CAR-T therapy. Analysis of T-cells in the pre-treatment biopsies leveraging machine learning algorithms is in progress and both complete quantitative findings and prognostic value of this approach will be presented. While our findings require further validation thru additional biopsy analysis, they provide emerging evidence for a rationale involving vascular readiness in DLBCL responder differences to CART therapy.

Molecular Determinants of Response to Mosunetuzumab Plus CHOP in Patients with Previously Untreated (1L) Diffuse Large B Cell Lymphoma (DLBCL)

Co-lead authors: Habib Hamidi and Patrick Kimes Background: Standard induction therapy cures ~60% of patients (pts) with DLBCL; however, most remaining non-responders die from lymphoma, highlighting a need for improved first-line treatments. Biomarkers that can reliably assess the efficacy of new treatments are lacking in clinical practice. Mosunetuzumab (Mosun) is a CD20xCD3 T cell engaging bispecific antibody that redirects T cells to eliminate malignant B cells. In a Phase Ib/II trial (NCT03677141), Mosun combined with CHOP chemotherapy (M-CHOP) induced high response rates in pts with 1L DLBCL (ORR 87.5%, CR 85%; Phillips et al. ASH 2020). Here, we sought to identify biomarkers associated with M-CHOP response. Methods: Pts with 1L DLBCL receiving M-CHOP (six cycles) in NCT03677141 and with available biomarker data were included. Bulk RNASeq data from pre-treatment tumor biopsies were analyzed using gene set variation for tumor intrinsic pathways and xCell cell-type enrichment for immune and stromal cell types. High or low expression of each gene signature was determined by median cut-off. Cox regression examined associations between gene signatures and PFS. To assess specificity to M-CHOP, gene signatures were examined in pre-treatment biopsies from pts treated with rituximab + CHOP chemotherapy (R-CHOP) in GOYA (NCT01287741; Sehn et al. J Hematol Oncol 2020). Peripheral blood biomarkers were evaluated by whole blood flow cytometry and plasma cytokines by ELISA. Circulating tumor (ct) DNA levels were measured at baseline and on treatment using the AVENIO NHL CAPP-Seq assay (Stokowski et al. ASH 2022). Results: In M-CHOP-treated pts (n=33), gene expression analysis revealed two types of signatures associated with PFS: tumor immune microenvironment signature (TIMS) and tumor intrinsic proliferation signature (TIPS; Figure). High TIMS expression (e.g., CD4+ T cells) was associated with significantly longer PFS than low expression. Conversely, pts with high TIPS expression (e.g., MYC targets, DNA repair) had shorter PFS than those with low expression. These biological associations were specific to M-CHOP and were not observed in R-CHOP-treated pts. Consistent with the tumor-gene expression analysis, immune profiling of peripheral blood suggested that in pts treated with M-CHOP, a higher T cell baseline count was associated with a trend for prolonged PFS compared with pts with a lower baseline count. A higher count of circulating suppressive T cells was also associated with shortened PFS. Early on-treatment pharmacodynamic (PD) changes were observed following the first Mosun dose, including: induction of TNFα and IL-6, transient margination of CD4+ and CD8+ T cells, and activation of CD4+ and CD8+ T cells. The magnitude of the PD effects were similar to observations with Mosun monotherapy in pts with relapsed or refractory DLBCL (Hernandez et al. ASH 2019), confirming that Mosun maintains its immune activation effect when combined with CHOP in pts with 1L DLBCL. High baseline ctDNA levels were associated with high IPI score (n=32, Wilcoxon rank-sum p=0.007), ABC/non-GCB DLBCL subtype (n=30, p=0.033), and high lactate dehydrogenase (n=32, p=0.004). Pts with baseline ctDNA levels < median had a modest trend for improved PFS compared with pts > median (unadjusted hazard ratio [HR]=0.57 [95% CI 0.16, 1.75]; IPI-adjusted HR=0.72 [95% CI 0.16, 3.20]). The proportion of pts with undetectable ctDNA increased with each M-CHOP cycle (C) (C2 day [D]1, 25% [8/32]; C3D1, 69% [20/29]; C5D1, 88% [22/25]). Pts with undetectable ctDNA at any on-treatment time point (C2D1, C3D1, C5D1) had superior PFS compared with pts with detectable ctDNA (unadjusted HR=0.12 [95% CI 0.03, 0.52]; IPI adjusted HR=0.14 [95% CI 0.03, 0.61]). Conclusions: Consistent with the Mosun mode of action, pts with high expression of TIMS and those with high levels of circulating T cells had improved PFS versus pts with low expression. In contrast, there was no association between immune cell signatures and PFS in pts treated with R-CHOP, suggesting that the different mode of action of rituximab versus Mosun may influence this. Future studies are warranted to confirm the potential clinical utility of immune cell signatures as biomarkers associated with CD20xCD3 bispecifics' activity.

CD19 Intron Retention Is Mechanism of CAR-T Treatment Resistance in Non-Hodgkin Lymphoma

Background: Chimeric antigen receptor T-cell (CAR-T) therapy has become a viable treatment option for patients (pts) with relapsed or refractory diffuse large B-cell lymphoma (R/R DLBCL). Transcriptomic markers for CD19 directed CAR-T failure have been characterized in pre-treatment B-cell acute lymphoblastic leukemia (B-ALL) but not in DLBCL. Here, we report the identification of RNA alternative splicing (AS) events associated with CD19 directed CAR-T response in pre-treatment DLBCL pts and of RNA binding proteins (RNABP) regulating these AS events. Methods: To identify putative AS markers for CAR-T treatment outcome, we performed differential AS on bulk RNA-seq samples of pre-treatment tumor biopsies in pts that had a durable response (DR), defined via remission up to 9 months post-treatment, and pts that had a non-durable response (NDR) with rMATS-turbo (Shen et al. PNAS. 2014). Intron retention (IR) events within CD19 were quantified with a secondary, more robust algorithm to ensure reproducibility and accuracy. Survival analyses were done using the survival and survminer libraries in R. Validation assays for the downstream effects CD19 IR were conducted in EJ1, Jeko-1, OCI-Ly3, SU-DHL-6, and Toledo cell lines. The surface protein expression of CD19 was assayed via flow cytometry (FC) followed by Quantibrite PE (BD Biosciences, San Jose, CA) quantification. Total CD19 protein expression was measured via western blot (WB) in the aforementioned cell lines. Analyses of CD19 IR was done via RT-PCR followed by electrophoresis, and bands were quantified with iBright Analysis Software (Thermo Fisher Scientific, Carlsbad, CA). The effect of RNABP was assessed via the creation of CRISPR/Cas9 knockout (KO) cell lines followed by WB of the target gene and quantification of CD19 surface protein expression by FC. Results: Introns 2 and 6 of CD19 are more highly expressed in NDR pts. A combined metric estimating the proportion of normal CD19 isoforms that do not express introns 2 or 6 (normal CD19) was created and DR pts expressed normal CD19 isoforms significantly higher than NDR pts (Figure 1). Normal CD19 isoforms classification performance was assessed via leave one out cross validation and achieved an overall accuracy of 67.6%. Sensitivity (classifying NDR) was 61.1% and specificity (classifying DR) was 81.3%. The expression of normal CD19 relative to the median level within the cohort was significantly associated with progress free survival (Figure 2). The expression of CD19 intron 6 was negatively correlated with CD19 surface protein expression in DLBCL cell lines. Cell lines were sorted by cells that exhibited high CD19 surface expression (bright) and low expression (dim). Cell lines sorted into the dim category expressed higher levels of intron 6 than bright cells. The correlation of RNABP expression with CD19 intron 6 expression was done in this patient cohort and the NCICCR-DLBCL cohort. Six RNABP were significantly correlated in both cohorts (r 2 > 0.2 and p-value <0.01). NOP2, one of the RNABP that was positively correlated with intron 6 expression, was knocked out in Jeko-1 cell lines to validate the putative correlation with CD19 protein expression. The resulting knock out cells exhibited lower levels of intron 6 retention and higher CD19 protein expression by FC and WB than non-transformed cells. Conclusion: Pre-treatment tumors of R/R DLBCL patients who relapse before 9 months following CD19 directed CAR-T therapy have higher expression of CD19 isoforms that retain introns 2 and 6. The upregulated expression of the CD19 isoforms retaining these introns is correlated with CD19 surface protein expression and is a marker for sustained remission following CAR-T treatment. NOP2 is correlated with CD19 intron retention and surface protein expression and could be a target for modulating CD19 expression in conjunction with CAR-T treatment. Figure 1: The expression of normal CD19 isoforms in pre-treatment tumor samples of DR (red) and NDR patients (blue). ΔPSI denotes the difference in medians between the two groups. P-value yielded by a Wilcoxon test between the two groups is reported. Figure 2: Progress free survival (PFS) stratified by expression of normal CD19 isoforms. Red - above cohort median expression of normal CD19 isoforms. Blue - below cohort median expression of normal CD19 isoforms. P-value yielded via log-rank test.

Characterization of the tumor microenvironment and identification of spatially predictive biomarkers associated with beneficial neoadjuvant chemoradiotherapy in locally advanced rectal cancer

Neoadjuvant chemoradiotherapy (nCRT) has become the standard treatment for patients with locally advanced rectal cancer (LARC). However, 20–40% of patients with LARC show little to no response to nCRT. Thus, comprehensively understanding the tumor microenvironment (TME), which might influence therapeutic efficacy, and identifying robust predictive biomarkers is urgently needed. Pre-treatment tumor biopsy specimens from patients with LARC were evaluated in detail through digital spatial profiling (DSP), public RNA sequencing datasets, and multiplex immunofluorescence (mIF). DSP analysis revealed distinct characteristics of the tumor stroma compared to the normal stroma and tumor compartments. We identified high levels of human leukocyte antigen-DR/major histocompatibility complex class II (HLA-DR/MHC-II) in the tumor compartment and B cells in the stroma as potential spatial predictors of nCRT efficacy in the Discovery cohort. Public datasets validated their predictive capacity for clinical outcomes. Using mIF in an independent nCRT cohort and/or the total cohort, we validated that a high density of HLA-DR/MHC-II+ cells in the tumor and CD20 + B cells in the stroma was associated with nCRT efficacy (all p ≤ 0.021). Spatial profiling successfully characterized the LARC TME and identified robust biomarkers with the potential to accurately predict nCRT response. These findings have important implications for individualized therapy.

The oesophageal adenocarcinoma tumour immune microenvironment dictates outcomes with different modalities of neoadjuvant therapy - results from the AGITG DOCTOR trial and the cancer evolution biobank.

A plateau in treatment effect can be seen for the current 'one-size-fits-all' approach to oesophageal adenocarcinoma (OAC) management using neoadjuvant chemoradiotherapy (nCRT) or chemotherapy (nCT). In OAC, the tumour microenvironment (TME) is largely immunosuppressed, however a subgroup of patients with an immune-inflamed TME exist and show improved outcomes. We aimed to understand the overall immune-based mechanisms underlying treatment responses and patient outcomes in OAC, and in relation to neoadjuvant therapy modality. This study included 107 patients; 68 patients were enrolled in the Australian Gastro-Intestinal Trials Group sponsored DOCTOR Trial, and 38 patients were included from the Cancer Evolution Biobank. Matched pre-treatment and post-treatment tumour biopsies were used to perform multi-modality analysis of the OAC TME including NanoString mRNA expression analysis, multiplex and single colour immunohistochemistry (IHC), and peripheral blood mononuclear cell analysis of tumour-antigen specific T cell responses. Patients with the best clinicopathological outcomes and survival had an immune-inflamed TME enriched with anti-tumour immune cells and pathways. Those with the worst survival showed a myeloid T regulatory cell enriched TME, with decreased CD8+ cell infiltration and increased pro-tumour immune cells. Multiplex IHC analysis identified that high intra-tumoural infiltration of CD8+ cells, and low infiltration with CD163+ cells was associated with improved survival. High tumour core CD8+ T cell infiltration, and a low tumour margin infiltration of CD163+ cells was also associated with improved survival. nCRT showed improved survival compared with nCT for patients with low CD8+, or high CD163+ cell infiltration. Poly-functional T cell responses were seen with tumour-antigen specific T cells. Overall, our study supports the development of personalised therapeutic approaches based on the immune microenvironment in OAC. Patients with an immune-inflamed TME show favourable outcomes regardless of treatment modality. However, in those with an immunosuppressed TME with CD163+ cell infiltration, treatment with nCRT can improve outcomes. Our findings support previous studies into the TME of OAC and with more research, immune based biomarker selection of treatment modality may lead in improved outcomes in this deadly disease.

A Phase I Expansion Cohort Study Evaluating the Safety and Efficacy of the CHK1 Inhibitor LY2880070 with Low-dose Gemcitabine in Patients with Metastatic Pancreatic Adenocarcinoma.

Combining gemcitabine with CHK1 inhibition has shown promise in preclinical models of pancreatic ductal adenocarcinoma (PDAC). Here, we report the findings from a phase I expansion cohort study (NCT02632448) investigating low-dose gemcitabine combined with the CHK1 inhibitor LY2880070 in patients with previously treated advanced PDAC. Patients with metastatic PDAC were treated with gemcitabine intravenously at 100 mg/m2 on days 1, 8, and 15, and LY2880070 50 mg orally twice daily on days 2-6, 9-13, and 16-20 of each 21-day cycle. Pretreatment tumor biopsies were obtained from each patient for correlative studies and generation of organoid cultures for drug sensitivity testing and biomarker analyses. Eleven patients with PDAC were enrolled in the expansion cohort between August 27, 2020 and July 30, 2021. Four patients (36%) experienced drug-related grade 3 adverse events. No objective radiologic responses were observed, and all patients discontinued the trial by 3.2 months. In contrast to the lack of efficacy observed in patients, organoid cultures derived from biopsies procured from two patients demonstrated strong sensitivity to the gemcitabine/LY2880070 combination and showed treatment-induced upregulation of replication stress and DNA damage biomarkers, including pKAP1, pRPA32, and γH2AX, as well as induction of replication fork instability. No evidence of clinical activity was observed for combined low-dose gemcitabine and LY2880070 in this treatment-refractory PDAC cohort. However, the gemcitabine/LY2880070 combination showed in vitro efficacy, suggesting that drug sensitivity for this combination in organoid cultures may not predict clinical benefit in patients.