Abstract B044: Distributed Transcriptomic Modeling for Biomarker Discovery in Immuno-Oncology

654 Background: MSI and TMB correlate with ICI efficacy. We assessed the association between TMB and MSI with outcomes in pts with aUC treated with anti-PD1/L1 in a ‘real-world’ cohort. We hypothesize that pts with high TMB and MSI-high (H) have better outcomes with ICI. Methods: In this retrospective study we included pts from 13 sites treated with ICI for aUC. We calculated overall response rate (ORR), median overall and progression-free survival (mOS, mPFS) using KM method from ICI start. TMB and MSI were assessed by NGS. TMB was analyzed as dichotomous (≥10 vs <10 mut/Mb) and continuous variable. The analysis was stratified by therapy line (1L/upfront, 2+L). Multivariable models were adjusted by Khaki factors for 1L/upfront and Bellmunt factors for 2+L. We separately analyzed pts on maintenance avelumab (mAV). MSI is reported with descriptive statistics only. Results: 342 pts were treated with ICI 1L/upfront or 2+L (69% pure UC, median age at ICI start 69, 77% men, 90% White, 16% upper tract primary, 12% liver metastases [mets], 78% ECOG PS 0-1). Median f/u from ICI start was 34 months (mo). ORR was 36% (95%CI 26-46%) in pts with TMB≥10 vs 30% (95%CI 23-36%) with TMB<10 (OR=1.4 [95%CI 0.8-2.5] p=0.3). ORR was 75% (6/8) in pts with MSI-H and 30% (60/200) with MSI stable (MSI-S). mPFS was similar between TMB≥10 and <10 groups (6 vs 4 mo; HR=0.88 [95%CI 0.61-1.26] p=0.48) and between MSI-H and MSI-S (5 mo). mOS was numerically longer (not significant) in pts with TMB≥10 vs TMB<10: 25 vs 21 mo; (HR=0.7 [95%CI 0.50-1.09] p=0.12) and with MSI-H and MSI-S (NR and 20 mo). A significant association was found between TMB (continuous variable) and OS (HR=0.97 [95%CI 0.95-0.99] p=0.01). TMB analyses by therapy line are shown in Table. In the 1L/upfront setting (N=144), ORR 71% (5/7) vs 33% (45/135), mPFS 5 and 4 mo, mOS NR and 22 mo for MSI-H and MSI-S, respectively. Pts on mAV (n=77) were 77% men, 92% White, 16% upper primary, 13% liver mets, 84% ECOG PS 0-1 with median f/u 15 mo from avelumab start; mOS was NR for MSI-H and 24 mo for MSI-S; mPFS was 4 mo for both groups. Of 3 pts on mAV with MSI-H: 1 CR, 2 SD; of 58 pts with MSI-S: 4CR, 4PR, 24 SD, 21 PD, 5 unknown. Conclusions: MSI-H had numerically higher ORR and higher TMB was associated with longer OS with ICI, but further validation is needed. Limitations: retrospective design, small sample size for MSI-H cases, lack of randomization and central scan review, selection/confounding. [Table: see text]

5064 Background: FoundationOneCDx (F1CDx) supports two FDA-approved biomarkers to guide treatment decisions for ICI for patients with mCRPC: MSI status and TMB. MSI-H and TMB-H (10+ mut/MB) have strongly overlapping prevalence. We sought to better characterize ICI outcome associations of TMB-H / non-MSI-H population and relative effectiveness of taxanes & ICI among patients who received these agents in sequence. Methods: Following a prespecified analysis plan, this study used the nationwide (US-based) de-identified Flatiron Health-Foundation Medicine mCRPC clinico-genomic database (FH-FMI CGDB), with data originating from ~280 US cancer clinics (~800 sites of care). Inclusion criteria included patients with mCRPC treated with single-agent anti-PD(L)1 therapy in the FH network between 1/1/2011 - 3/30/2024. This study used the MSI and TMB algorithms from the tissue based F1CDx. Time to next treatment (TTNT) and OS were assessed with Kaplan-Meier plots and in multivariable Cox models adjusted for ECOG performance score, socioeconomic status, prior treatment history, and baseline PSA. Among patients who received taxanes in a prior line of therapy, the effectiveness (TTNT1 vs. TTNT2) of taxane and ICI were compared. Results: Among 2995 prostate cancer tissue specimens in the database, 95 (3.1%) were MSI-H and 142 (4.7%) were TMB-H. 94 (3.1%) were MSI-H & TMB-H, 1 was MSI-H & TMB-L, and 48 (1.6%) were TMB-H & not MSI-H. Among these, 84 patients with mCRPC were treated with ICI and met inclusion criteria, including MSI-H & TMB-H (n = 30), non-MSI-H & TMB-H (n = 8), and non-MSI-H and TMB-L (n = 46). The respective median TTNT on ICI was 8.0 vs. 9.6 vs. 3 months. The respective median OS from initiation of ICI was 10.9 vs. not reached vs. 4.4 months. In multivariable models evaluating ICI only, compared to non-MSI-H & TMB-L, the MSI-H & TMB-H group had more favorable TTNT (HR: 0.20, 95%CI: 0.10 – 0.41, p < 0.001) and OS (HR: 0.33, 95%CI: 0.16 – 0.70, p = 0.004), and the non-MSI-H & TMB-H group also had more favorable TTNT (HR: 0.13, 95%CI: 0.04 – 0.45, p = 0.002) and OS (HR: 0.20, 95%CI: 0.05 – 0.75, p = 0.017). 50 of the 84 (60%) patients treated with ICI had prior mCRPC taxane treatment. Better TTNT2 on subsequent ICI vs. prior taxane was observed for MSI-H (HR: 0.49, 95%CI: 0.23 – 1.01, p = 0.051) and TMB-H (0.54, 95%CI: 0.30 – 0.98, p = 0.044), but the opposite was true for non-MSI-H and TMB-L subgroups, with significant treatment interactions for each (p = 0.0018, p = 0.00052). Conclusions: TMB-H (4.7%) is more prevalent than MSI-H (3.1%) by F1CDx in mCRPC. Non-MSI-H / TMB-H (1.6%) in the routine practice cohort have similar outcome associations on ICI to MSI-H. Both MSI-H and TMB-H by F1CDx are predictive of differential benefit for ICI vs. taxanes in later mCRPC treatment lines.

Background: Gastric adenocarcinoma (GAC) is a deadly disease in need for new therapies. Recently, the most exciting developments in oncology are immune checkpoint inhibitors (ICIs) such as inhibitors of programmed cell death protein 1 (PD-1) which has shown response rates of 11-16% in unselected GAC patients. This study aimed to characterize the distributions of immunotherapy-related biomarkers in Chinese patients with gastric adenocarcinoma. Methods: Formalin-fixed, paraffin-embedded (FFPE) tumor samples and matched peripheral blood samples of 497 Chinese gastric adenocarcinoma patients were collected for YuansuTM NGS-panel assay (OrigiMed, Shanghai). Genomic alterations (GAs) including single nucleotide variations, short and long insertions/deletions, copy number variations, and gene rearrangements were assessed. Microsatellite instability (MSI), PD-L1 expression and tumor mutational burden (TMB) were assessed in 467(94%), 208(42%) and 467(94%) patients, respectively. Results: There were 334 males (67.2%) and 163 females (32.8%) with a median age of 60 (range 23-87) years in this cohort, including 440 primary tumors and 57 metastases. MSI-H was detected in 39 samples (7.8%). The rate of MSI-H in the ACVR2A mutation group was significantly higher than that in the ACVR2A wild-type group (82.1% vs 17.9%, p<0.001). In addition, we found that patients with mutations of K437Rfs*5 (54.1%) or D96Tfs*54 (11.4%) of ACVR2A were all MSI-H. The frequency of ERBB2 amplification was 7.5%, which was mutually exclusive to MSI-H. PD-L1 expression levels by CPS of negative, 1-9 and ≥10 were 54%, 33% and 13%, respectively. The median value of TMB was 6.2 (0-99.3) muts/Mb and TMB ≥10 muts/Mb was seen in 27.4% tumors. The patients with ACVR2A mutations had a significantly higher TMB (ACVR2A: 5.7 vs 2.6 muts/Mb, respectively, p < 0.01). In addition, the biomarkers associated with hyperprogression (HP) to immune checkpoint inhibitors (ICIs) included 11q13 amp, MDM2/MDM4 amp, and CDKN2A/B gene loss, which were identified in 4%, 2% and 2% of patients, respectively. Conclusions: We analyzed the distribution of microsatellite status, PD-L1 expression and TMB in Chinese patients with GAC. Interestingly, our data showed that mutated ACVR2A was significantly associated with MSI-H and TMB-H. In addition, approximately 8% of patients were identified harboring hyperprogression-related gene alterations. Therefore, the application of comprehensive genomic profiling might be necessary in guiding ICI treatments in GAC. Citation Format: Qun Zhao, Yuntong Guo, XiaoLong Cui, LiangBiao Peng, Junying Li, Fei Wang, Lin Zhang. Comprehensive analysis of immunotherapy-related biomarkers in Chinese patients with gastric adenocarcinoma [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 2375.

Background: BRCA1 and BRCA2 are involved in homologous recombination (HR) DNA repair. They have been found to promote both tumor initiation and progression. BRCA1/2 mutations lead to genomic instability and higher tumor mutation burden (TMB). Therefore, we hypothesized that BRCA1/2 mutations may be a predictor of immune checkpoint inhibitors (ICIs) efficacy. In the present work, we explored the correlation of BRCA1/2 mutations with TMB, high microsatellite instability (MSI-H), PD-L1 expression, tumor-infiltrating lymphocytes (TILs), and ICIs efficacy in colorectal cancer. Methods: A total of 509 Chinese colorectal cancer patients were enrolled from January 2017 to January 2020 in our center. Targeted next-generation sequencing and PD-L1 immunohistochemistry (22C3 antibody) were performed in the tissues from patients. TCGA colorectal cancer cohort was used for characterizing TILs with CIBERSORT. The ICIs treatment cohort from the Memorial Sloan Kettering Cancer Center (MSKCC) was selected to analyze the association of BRCA1/2 mutations with ICIs efficacy. Results: In our cohort, the mutational frequency of BRCA1/2 genes was 9.04%. The median TMB was significantly higher in the BRCA1/2 mutations (Mut) group compared to the wild type (WT) group (52.4 vs 5.7 muts/Mb, P < 0.0001). About 41.30% of patients have MSI-H in the Mut group, while 3.67% of patients display MSI-H in the WT group (P < 0.0001). There was no association of BRCA1/2 mutations with PD-L1 expression (P = 0.172). In TCGA cohort, BRCA1/2 mutations can down-regulate regulatory T (T reg) cells (P = 0.0176) and up-regulate T follicular helper cells (P = 0.0042) and macrophages M1 (P <0.0001). In MSKCC cohort, patients in the Mut group had significantly longer median overall survival after ICIs therapy (34 vs 13 months, HR: 0.37 [95%CI: 0.19-0.72], P = 0.0234). Conclusions: Our data indicated that BRCA1/2 mutations are associated with increased TMB and MSI-H, but not with PD-L1 expression. And BRCA1/2 mutations can regulate TILs. Furthermore, BRCA1/2 mutations are associated with better response to ICIs, suggesting that they may be a useful predictor for ICIs efficacy in colorectal cancer. Citation Format: Dong-Dong Jia, Yanling Niu, Tonghui Ma, Min Shi. Correlation of BRCA1/2 mutations with response to immune checkpoint inhibitors in colorectal cancer [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 1681.

272 Background: Microsatellite instability (MSI) is linked to hypermutability and response to ICI(s), especially in colorectal cancer. The predictive impact of tumor mutational burden (TMB) in ICI-treated MSI/dMMR metastatic colorectal cancer remains controversial, with some studies suggesting low TMB as a potential biomarker of resistance to ICI(s). We aimed to investigate the biological relevance of TMB in MSI/dMMR mCRC and its impact on survival outcomes in ICI(s)-treated patients (pts). Methods: MSI/dMMR mCRC pts treated with ICI(s) from the NIPICOL trial (NCT03350126) and the immunoMSI prospective monocentric cohort with available data from whole exome sequencing (WES) and RNA-sequencing (RNA-seq) were included. All cases were centrally assessed for MSI and dMMR status. TMB was calculated as described (Dupain et al., BMC, 2024). The search for a cutpoint able to optimize the difference in survival between 2 groups of patients with low and high-TMB was applied. The main endpoint was progression free survival (PFS) by iRECIST criteria. The cohort (NIPICOL, immunoMSI) was included as a covariate in the analysis of differential gene expression and pathway enrichment, with various tumor mutational burden (TMB) thresholds evaluated, specifically 10 and 30 mutations per megabase (mut/Mb). Results: A total of 99 pts were analyzed, with 4 (4%) excluded after central MSI/dMMR status review. Of these, 52 received anti-PD1 plus anti-CTLA4 therapy, while 43 received anti-PD1 alone. Median follow-up was 25 months. Median TMB was 51.8 mut/Mb (IQR: 35.9-74.9). TMB was not predictive of patient PFS, whatever the TMB threshold applied, including cutpoints already reported in the literature, the 2-year PFS rates being 80% versus 80% for TMB ≤ 10 and > 10 mut/Mb, and 79% versus 82% for TMB ≤ 30 and > 30 mut/Mb, respectively. Multivariate analysis including treatment type confirmed no association between TMB and PFS (HR 1.00, 95% CI [0.97–1.04]). TMB levels were not linked with specific signalling pathways related to immune checkpoints or antigen presentation as evaluated by RNA-seq with Gene Set Enrichment Analysis (GSEA) at a TMB threshold of 10 or 30 mut/Mb. However pathways involved in cell proliferation (e.g., “cell cycle”, “DNA replication”) were increased in TMB high (adjusted p-value <0.05). RNA-seq signature quantification and deconvolution analysis revealed similar cell populations (e.g., fibroblasts, B cells, T cells) between high and low TMB groups at a TMB threshold of 10 and 30 mut/Mb. Conclusions: TMB has no survival impact for patients with ICI(s)-treated dMMR/MSI mCRC and does not discriminate two biologically distinct subpopulations. Other biomarkers should be developed for personalized medicine amongst these patients.

BackgroundThere is uncertainty around clinical applicability of tumor mutational burden (TMB) across cancer types, in part because of inconsistency between TMB measurements from different platforms. The KEYNOTE 158 trial supported...

Background: Tumor mutational burden (TMB) has emerged as a predictive biomarker of response to immune checkpoint inhibitor (ICI) therapy. Current panel-based TMB algorithms aggregate signal from certain types of somatic variants (e.g. non-synonymous coding SNVs); however, delineating the contributions of these and other types of mutations may refine TMB calculation from gene panels. Moreover, early studies suggest other possible genomic correlates of patient outcome to ICI which may be complementary to TMB. Here, we explore the landscape of mutations comprising TMB and other genomic features correlating with TMB on a subset of several thousand late-stage plasma samples run on GuardantOMNITM (OMNI), a highly sensitive 500-gene cfDNA sequencing platform. Methods: We developed a cfDNA-based TMB algorithm which is robust to variable tumor shedding levels and presence of clonal hematopoiesis. We assessed cfDNA-based TMB in over 1,000 plasma samples across six tumor types, including lung and prostate. We examine the contribution of nonsynonymous, synonymous, intronic SNVs, and indels to TMB score. We investigate correlations between TMB and additional genomic features, including chromosomal instability, loss of HLA-bearing chromosome 6p, microsatellite instability (MSI), and common oncogenic and resistance mutations. Results: We found that the distribution of WES-calibrated TMB scores across this cohort of samples is consistent with TCGA, with median 10 mutations/Mb and upper-tertile of 14 mutations/Mb across tumor types. The number of non-synonymous coding SNVs per sample correlated highly with synonymous coding SNV and intronic SNV counts (Pearson’s r > 0.7 for each). Including this additional signal in TMB calculation improves clinical sensitivity by up to 5%. In MSS samples, indels were highly correlated with SNVs, indicating that both likely arise from a similar underlying mechanism. We found no clear correlation between high TMB and chromosomal instability, with high TMB samples exemplifying a range of tumor ploidies. TMB association with oncogenic drivers is consistent with existing literature, with lower median TMB in EGFR-driven lung tumors (p < 0.01), but little to no correlation between TMB and KRAS or PIK3CA driver status, or STK11 loss of function (p > 0.05), suggesting these latter events could be independent clinical biomarkers to TMB. Conclusions: Panel-based TMB scores can leverage synonymous and non-coding mutations to strengthen the signal of exome-wide mutation load. As more patient outcome data becomes available, TMB algorithms and orthogonal biomarkers of tumor genome immunogenicity will evolve further for improved guidance of patient response to immunotherapy. Sequencing panels with high sensitivity for TMB, via large panel space, and the ability to detect copy-number variations and MSI-status, will be important for biomarker development and clinical applications. Citation Format: Katie Quinn, Elena Helman, Tracy Nance, Jennifer Yen, John Latham, Kristin Gleitsman, Ravi Vijaya-Satya, Carlo Artieri, Alex Artyomenko, Marcin Sikora, Darya Chudova, Richard B. Lanman, AmirAli Talasaz. Landscape and genomic correlates of ctDNA-based tumor mutational burden across six solid tumor types [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 3404.

Tumor mutational burden.

Background: Penile cancer is a rare malignant tumor characterized by invasive growth and early metastasis. Several risk factors related to penile cancer have been identified, such as human papillomavirus status (HPV), circumcision status, smoking, poor hygiene. Current scientific evidence and guidelines on the management and treatment of penile cancer are rarely updated. In particular, there is insufficient evidence for targeted therapy and immunotherapy for penile cancer. Here, we analyzed features of gene profiling of penile cancer and some biomarkers of immunotherapy such as tumor mutation burden (TMB), PD-L1 expression, and status of microsatellite. Methods: Totally 26 tumor samples of Chinese patients with penile cancer were consecutively collected in China from January 2018 to June 2020, Next generation sequencing (NGS) was used to detect gene mutation, microsatellite instability (MSI) or microsatellite stable (MSS), and tumor mutational burden (TMB) analyzed by a 733 gene panel by 3D Medicines, a laboratory accredited by College of American Pathologists (CAP) and Clinical Laboratory Improvement Amendments (CLIA). PD-L1 expression detected by using Dako PD-L1 IHC 22C3 pharmDx, Tumor Proportion Score (TPS) was used to determine expression of PD-L1. TPS was arranged into the following intervals: strong positive ≥50%, moderate positive ≥5% and <50%, weak positive ≥1% and <5%, and negative <1%. Results: Among these 26 samples, 20 samples were available for TMB analysis and MSI/MSS analysis, and 15 samples were available for analysis of PD-L1 expression. The average TMB level of these penile cancer was 7.4 mutants/Mb, and 85% (17/20) of them with TMB level under 10 mutants/Mb. Analysis of PD-L1 expression results showed that 26.7% (4/15) with PD-L1 negative, 6.7% (1/15) weak positive, 53.3% (8/15) moderate positive, 13.3%(2/15) with PD-L1 strong positive. In addition, none of these sample were MSI. Furthermore, NGS results show that a total of 176 gene mutations have been identified in these 26 Chinese patients with penile cancer, and 43.2% (73/176) were germline mutation. The main mutated gene were TP53 (65.4%,17/26), CDKN2A (50.0%,13/26), AKT (46.2%,12/26), FAT1 (38.5%,10/26), PIK3CA/MSH2/POLD1/VEGFA (34.6%, 9/26), BRIP1/MDM2/ATM/XPC/TERT (30.8%,8/26). The top ten of germline mutated genes were AKT (42.3, 11/26), XPA (38.5%,10/26), POLD1 (34.6%, 9/26), ERCC2/TP53/RAC1/MSH2/MSH3/ATM/VEGFA/PRKDC/MGMT/BLM/MDM2/BRIP1/PRSS1/ERCC5 (30.5%,8/26). Conclusion: Patients with penile cancer have rich spectrum of gene mutations, especially, high percentage of germline mutation was displayed in penile cancer, it may suggest that genetic/Familial high-risk should be put into consideration in the management of penile cancer. What's more, immunotherapy may also have certain potential because of the expressing of PD-L1. Citation Format: Bin Zhao, Hushan Zhang, Guoying Zhang, Jian Xu, Maolin Yang. Analysis of oncologic characteristics of penile cancer [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 2193.

Purpose: To date no biomarker has been identified that predicts response to ICI in mTNBC. This study aimed to explore if tumor genomic alterations correlate with efficacy of PD-1/PD-L1 inhibition in patients (pts) with mTNBC. Methods: Demographic, treatment response, and long-term outcome data were collected on patients with mTNBC treated at Dana-Farber Cancer Institute (DFCI) under several clinical trials incorporating PD-1/PD-L1 inhibitors, given as monotherapy or combined with chemotherapy (CT). Pts included in this analysis had available results of targeted exon sequencing performed using Oncopanel, our institutional gene sequencing panel, on archival tumor tissue. TMB was calculated by determining the number of non-synonymous somatic mutations that occur per megabase of exonic sequence data across all genes on the panel. High TMB was defined as 310 mutations/megabase. TMB and gene alterations were correlated with objective response rate (ORR) per RECIST 1.1, progression-free (PFS) and overall survival (OS). Results: A total of 50 pts with mTNBC were included in this analysis. At baseline, the median age was 55.9 years (31.8–75.9), 60% had ECOG 0 and 40% had ECOG 1, 72% had visceral metastasis, and 46% had received 31 prior lines of systemic therapy in the metastatic setting. While 26% of pts received monotherapy [pembrolizumab (n=7, NCT02447003); atezolizumab (n=6; NCT01375842)], 74% received combination with CT [pembrolizumab plus eribulin (n=31; NCT02513472); atezolizumab plus nab-paclitaxel (n=6; NCT01633970)]. PTEN alterations were present in 30% of pts (mutations = 7; one copy number loss = 7; two copy number loss = 1). Median follow-up was 14 months (1–40). Pts with tumors harboring PTEN alterations had lower ORR (7% vs 57%; P<0.001), shorter median PFS (2.3 vs 6.3 months; P=0.027), and shorter median OS (8.1 vs 20.1 months; P=0.012) compared to pts without PTEN alterations. The median TMB was 6.6 mut/Mb (1.2–50.8), and 23% of pts had a high TMB. While high TMB was not associated with higher ORR (P=0.56), it was associated with better median PFS (16.5 vs 2.4 months; P=0.017), and better median OS (not reached vs 13.5 months; P=0.026). Both PTEN status and TMB remained significantly associated with PFS in the multivariable model. Only PTEN status remained significantly associated with OS in the multivariable analysis with the same covariables. Ongoing analysis to better understand if these predictors are specific for predicting benefit to immunotherapy and/or a marker of chemotherapy resistance will be presented at the symposium. Conclusion: PTEN genomic alterations and TMB may impact benefit from PD-1/PD-L1 inhibitors largely administered with chemotherapy in mTNBC. These observations warrant prospective validation and may inform the importance of stratifying pts according to these characteristics in future randomized studies with ICI. Table 1.Multivariable analysis for PFS Hazard ratioConfidence Intervalp-valueCombination therapy0.420.16 – 1.130.009Visceral metastasis1.310.63 – 2.770.46Previous lines of therapy1.020.09 – 0.700.85ECOG 12.11.06 – 1.280.034PTEN altered3.741.65 – 8.440.002Hypermutated tumors0.850.75 – 0.970.011 Citation Format: Barroso-Sousa R, Tyekucheva S, Pernas-Simon S, Exman P, Jain E, Garrido-Castro AC, Hughes M, Bychkovsky B, Di Lascio S, Umeton R, Files J, Lindeman NI, MacConaill LE, Hodi FS, Krop IE, Dillon D, Winer EP, Wagle N, Lin NU, Mittendorf EA, Tolaney SM. PTEN alterations and tumor mutational burden (TMB) as potential predictors of resistance or response to immune checkpoint inhibitors (ICI) in metastatic triple-negative breast cancer (mTNBC) [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 P5-12-02.

e16079 Background: ICIs have revolutionized treatment for mRCC; however there are limited predictive biomarkers for response to ICIs. PD-L1 status is still controversial, demonstrating little predictive utility in mRCC. TMB is predictive for response to ICIs in melanoma and non-small cell lung cancer (NSCLC), but has not been validated in mRCC. Here, we assess the correlations between TMB and PD-L1 status with outcomes to ICI treatment in mRCC. Methods: 34 patients (pts) with mRCC who had previously received ICIs at Duke Cancer Institute were identified. Tumor samples were retrospectively evaluated using a Personal Genome Diagnostics Assay for somatic variants across > 500 genes, as well as TMB and microsatellite status. PD-L1 status was tested via the Dako 28-8 PD-L1 IHC assay. Deidentified clinical information was extracted from the medical record and tumor response was evaluated based on RECIST criteria. Results: Pts were grouped by overall response following ICI therapy into either progressive disease (“PD”, n = 18) or disease control group (“DC”, n = 16), defined as either stable disease, partial response, or complete response. Pts displayed a TMB range from 0.36 to 12.24 mutations/Mb with a mean score of 2.83 muts/Mb, with no significant difference between the PD and DC groups (mean 3.01 muts/Mb vs. 2.63 muts/Mb, p > 0.05). 9 of 32 evaluable samples were PD-L1 positive, with 4 in the PD group and 5 in the DC group. Notably, the DC group displayed a significant enrichment of mutations in genes affiliated with DNA repair (including BRCA1, BRCA2, FANCA, FANCB, FANCG, FANCM, MSH3, MSH6, RAD50, RAD51C, RAD51D, RAD54B, RECQL4, and SLX4; p = 0.0444). DNA damage gene mutations were found in 8/10 (80%) metastatic tumor specimens and 14/24 (58%) primary tumors. Conclusions: Overall, in this mRCC cohort, neither TMB nor PD-L1 correlated with patient outcomes or with ICI response. Furthermore, high TMB was not significantly associated with PD-L1 expression within the samples. The higher frequency of mutations in DNA repair genes in the DC group suggests potential use as a predictive signature for ICI response, warranting future prospective studies. Further studies with matched primary-metastatic samples would be beneficial to determine if DNA repair mutations occur more frequently in metastatic versus primary tumor specimens.

<div>Abstract<p>Tumor mutational burden (TMB) is considered a prototypic feature of tumor foreignness and has been established as a tumor-agnostic FDA-approved biomarker at a threshold of 10 mut/Mb for immune checkpoint inhibitors (ICI). Despite its clinical utility as a companion diagnostic for ICI across cancers, a high TMB does not consistently predict response due to technical and biological limitations. Tumor heterogeneity and purity, blood versus tissue sampling, variation in next-generation sequencing, and algorithmic evaluation attenuate the predictive value of TMB. In addition to technical standardization and moving beyond TMB as a numeric or binarized value, it is of paramount importance to consider the underlying biology and the differential contribution of mutation subsets to tumor foreignness and immunogenicity. The importance of consideration of mutations within the overall TMB that are unlikely to be immunoedited together with the density of immunogenic “quality” mutation-associated neoantigens introduces the concept of biological calibration of TMB that may enhance its clinical utility. Mutagenic processes such as microsatellite instability and ultra-mutation and cancer lineage–dependent co-mutation patterns also represent biological modifiers that enable the interpretation of the overall TMB in different contexts. In this perspective, we dissect TMB on a biological and technical level, followed by a critical assessment of the predictive role of TMB in capturing ICI response in the setting of clinical trials across human cancers. The standardization of technical methodologies, together with the interpretation of TMB on the basis of the tumor genomic landscape, represents key steps toward maximizing the predictive value of TMB for cancer immunotherapy.</p></div>

Background: The clinical implementation of immune-checkpoint inhibitors (ICIs) targeting CTLA4, PD-1, and PD-L1 has revolutionized the treatment of cancer. However, the majority of patients do not derive clinical benefit. Further development is needed to optimize the approach of ICI therapy. Immunotherapy combined with other forms of treatment is a rising strategy for boosting antitumor responses. CD93 was found to sensitize tumors to immune-checkpoint blocker therapy after the blockade of its pathway. However, its role in immune and ICB therapy across pan-cancer has remained unexplored. Methods: In this study, we provide a comprehensive investigation of CD93 expression in a pan-cancer manner involving 33 cancer types. We evaluated the association of CD93 expression with prognosis, mismatch repair, tumor mutation burden, and microsatellite instability, immune checkpoints, tumor microenvironment, and immune using multiple online datasets, including The Cancer Genome Atlas, Cancer Cell Line Encyclopedia, Genotype Tissue-Expression, cBioPortal, Tumor Immune Estimation Resource database, and Tumor Immune Single-cell Hub. Results: CD93 expression varied strongly among cancer types, and increased CD93 gene expression was associated with poor prognosis as well as higher immune factors in most cancer types. Additionally, the level of CD93 was significantly correlated with MMR, TMB, MSI, immune checkpoints, TME, and immune cell infiltration. Noticeably, our results mediated a strong positive contact between CD93 and CAFs, endothelial cells, myeloid dendritic cells, hematopoietic stem cells, mononuclear/macrophage subsets, and neutrophils while a negative correlation with Th1, MDSC, NK, and T-cell follicular helper in almost all cancers. Function analysis on CD93 revealed a link between itself and promoting cancers, inflammation, and angiogenesis. Conclusion: CD93 can function as a prognostic marker in various malignant tumors and is integral in TME and immune infiltration. Inhibition of the CD93 pathway may be a novel and promising strategy for immunotherapy in human cancer. Further explorations of the mechanisms of CD93 in the immune system may help improve cancer therapy methods.

Background: With the recent FDA approvals of atezolizumab and pembrolizumab for the treatment of unresectable locally advanced or metastatic triple-negative breast cancer, immuno-oncology (IO) therapy has become feasible for the first time in breast cancer, and PD-L1 immunohistochemistry (IHC) has become an essential assay in breast cancer diagnostics. However, relatively little is known about the molecular profile of PD-L1+ breast cancers compared to other tumors, such as lung cancer. Furthermore, although the initial data have shown the most benefit in triple-negative patients, it is unclear whether additional subgroups of patients with advanced breast cancer enriched for PD-L1 expression can be identified, and, therefore, might benefit from IO therapy. Methods: We identified 60 women with locally advanced or metastatic breast cancer who underwent next generation sequencing (NGS) of tumor samples obtained at our institution as part of their clinical care and performed PD-L1 testing using the Ventana SP142 platform. Samples were considered positive for PD-L1 if ≥1%. The NGS assay was designed to detect single nucleotide variants, insertions and deletions, copy number variants, and specific translocations in approximately 600 genes. Associations between PD-L1 status, molecular alterations, and tumor histopathologic features were assessed, with P < 0.05 considered statistically significant. Results: Our cohort consisted of 60 women at a median age of 56 years (range 31-81). 50% of samples were from primary tumors within the breast, 15% were from regional lymph node metastases, and 35% were from distant metastatic sites. Most tumors showed ductal histology (80%) and were either grade 2 (38%) or grade 3 (57%), while half showed focal (27%) or extensive (23%) necrosis. Based on IHC, most tumors were Luminal B (54%), followed by triple-negative (25%), Luminal A (13%), and HER2-enriched (8%) tumors. About one third of tumors had either moderate (25%) or marked (8%) TILs, and about half showed positivity for PD-L1 (48%). Most tumors had a low tumor mutational burden (TMB), with 83% having <5, 14% having 5-9, and 3% having ≥10 mutations per Mb. The median number of genes with reportable somatic alterations was 2 (IQR 1-3). The most common genes with somatic alterations were TP53 (51%), PIK3CA (23%), ERBB2 (16%), ESR1 (14%), FGFR1 (9%), AKT1 (7%), CDH1 (7%), GATA3 (7%), MYC (7%), and CCND1 (5%). 50% of luminal A, 38% of luminal B, 50% of HER2-enriched, and 75% of triple-negative tumors were PD-L1+, although this difference was not statistically significant (P = 0.22). PD-L1 positivity was more likely in grade 3 tumors (62% vs 31% grade 1-2, P = 0.02), and in tumors with extensive necrosis (85% vs 37% absent/focal, P = 0.004). Of all the genes tested, only TP53 was significantly associated with being PD-L1+ (69% TP53-mutant vs 32% TP53-wild-type; P = 0.008). Neither number of somatic alterations (P = 0.60) or TMB (P = 0.08) were significantly associated with PD-L1+ tumors. Of note, 53% of non-triple-negative PD-L1+ tumors had mutations in TP53, and 60% of non-triple-negative tumors with TP53 mutations were PD-L1+. Conclusions: Our data suggest that PD-L1 positivity is relatively common in patients with locally advanced or metastatic breast cancer, not only in triple-negative breast cancer but in all breast cancer subtypes. In addition, TP53 mutation is significantly associated with an increased likelihood of positivity for PD-L1, including in non-triple-negative tumors. This correlation could potentially be a useful marker for selecting non-triple-negative breast cancer patients for PD-L1 testing and, if positive, potentially offer them IO options for optimizing their response to therapy. Citation Format: Brian S Finkelman, Massimo Cristofanilli, Luis Z Blanco, Jr, Amir Behdad, Leonidas C Platanias, William J Gradishar, Kalliopi P Siziopikou. Somatic alterations and PD-L1 positivity in advanced breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-02-11.

Background: Tumor mutational burden (TMB) is a genomic biomarker that can predict favorable responses to immune checkpoint inhibitors (ICIs). Although we have better understanding of TMB in cancer immunity and cancer immunotherapy, the relationship between TMB and the clinical efficacy of ICIs remains unknown in the treatment of melanoma patients. Here, we conduct a systematic review and meta-analysis to evaluate the predictive value of TMB on the efficacy of ICIs in patients with melanoma. Methods: We systematically collected data from PubMed, Embase, Cochrane Library, CNKI, China Biomedical Database (CBM), and Wanfang Database. The end date was set to 26 June 2021. We included retrospective studies or clinical trials of ICIs that reported hazard ratios (HRs) for overall survival and/or progression-free survival according to TMB. Data for 1,493 patients from 15 studies were included. In addition, pooled effect size, heterogeneity analysis, sensitivity analysis, publication bias detection, and subgroup analysis were performed based on the included data. Results: Patients with high TMB showed significantly improved OS (HR = 0.49, 95% CI: 0.33, 0.73; p = 0.001) and PFS (HR = 0.47, 95% CI: 0.33, 0.68; p < 0.001) compared with patients with low TMB. This association was very good in patients treated with monotherapy, that is, anti-CTLA-4 or anti-PD-(L)-1 inhibitors, but not for the patients treated with a combination of the two drugs. The subgroup analysis results showed that heterogeneity was substantial in the targeted next-generation sequencing (NGS) group. Publication bias was detected, and the results were visualized using the funnel chart. And sensitivity analysis and trim-and-fill method analysis showed that our results were stable and reliable. Conclusion: High TMB is associated with improved OS and PFS in melanoma patients treated with mono-drug ICIs. TMB determined by NGS should be standardized to eliminate heterogeneity. Therefore, the role of TMB in identifying melanoma patients who may benefit from ICI should be further determined in more randomized controlled trials in the future.