Autophagy promotes growth of tumors with high mutational burden by inhibiting a T-cell immune response

Background. High-TMB (HTMB) is an emerging promising agnostic biomarker for predicting benefit from immune-checkpoint inhibitors, independently of tumor type. At ASCO 2019, the TAPUR trial reported an interesting 21% ORR in heavily pretreated metastatic BC patients with very HTMB [vHTMB, ≥9 mutations/megabase (Muts/Mb)]. We aimed to define the differential gene expression and methylation landscape between low and high TMB in each BC subtype. Methods. In TCGA, we identified 848 patients with WES data available for TMB estimation. [ER+/HER2- (LumA by PAM50)) n = 364; ER+/HER2- (LumB by PAM50), n = 147; HER2+, n = 158; and TN, n = 179]. High TMB was defined according to two different cut-offs: ≥9 (vHTMB) and≥5 Muts/Mb (HTMB). The second arbitrary cut-off was used to define a larger group allowing to better characterize the different molecular landscapes associated with high and low TMB in each BC subtype. The HTMB group was compared with an equal number of tumors with low TMB. We assessed the differential RNA expression and methylation of single genes and pathways (defined using Gene Ontology - GO). “Common” genes and pathways were defined as recurrently associated with TMB (p<0.05) in all subtypes and with a combined p value ≤0.00001. Results. The overall prevalence of vHTMB (≥9) was 4.5%, with no substantial differences across subtypes (4.4%, LumA; 4.8% LumB; 5.7% HER2+; 3.3% TN). The prevalence of HTMB (≥5) was 13.7%, but it was different across BC subtypes (p=8.0E-07) (8.2%, LumA; 12.9% LumB; 13.3% HER2+; 25.7% TN). We found more “common” genes down-regulated (n=70) than up-regulated (n=3) in HTMB group. Two of these three genes (HSPE1 and FEZF1.AS1) have been associated with poor prognosis in BC. When we considered the “common” pathways, only 3 were up-regulated in HTMB, all implicated in post-transcriptional repression of gene expression (gene silencing by miRNA and mRNA binding involved in post-transcriptional gene silencing). Conversely, 66 were significantly down-regulated (including transcription coregulator and coactivator activity, protein serine/threonine kinase activity and ubiquitin-protein transferase activity and binding). Some genes and pathways were associated with TMB only in a specific BC subtype (p ≤0.00001). For instance, 16 pathways were down-regulated in the HTMB group of TNBC. These inlcuded 12 pathways implicated in immune response. Conversely, in LumB, 11 pathways were up-regulated in HTMB group and implicated in immune response. Intrestingly, these pathways were all significantly down-regulated in the HTMB group of LumA and TN. In HTMB group, we found 7 and 4 “common” genes hypermetylated and hypomethylated, respectively. Four pathways were commonly hypermethylated (chromatin silencing at rDNA, telomere organization and positive regulation of G1/S transition of mitotic cell cycle) and five were hypomethilated (including mitotic sister chromatid segregation). Considering private alterations, in TNBC, 23 of 27 significant pathways were hypermetylated in HTMB group including double-strand break repair via nonhomologous end joining, epigenetic negative regulation of gene expression, and regulation of gene silencing by miRNA. Conclusions. Very-high TMB which is considered potentially druggable (≥9 Muts/Mb) is rare in BC, and equally frequent in all subtypes. Instead, HTMB (≥5 Muts/Mb) is more frequent in TNBC. BCs with HTMB had a different molecular landscape. Overall, several genes are recurrently down-regulated in HTMB group, and this is at least partly due to miRNA regulated post-trascriptional silencing, which might rapresents a new mechanism of immune escape. The positive association between TMB and immune genes in LumB, as well as the negative association in TN and LumA, suggest that immune editing and surveillance might be dependent on the molecular context. Citation Format: Luca Licata, Barbara Galbardi, Balázs Győrffy, Thomas Karn, Lorenzo Sica, Alessia Rognone, Patrizia Zucchinelli, Daniela Aldrighetti, Stefania Zambelli, Luca Gianni, Giampaolo Bianchini. Molecular differences between high and low tumor mutational burden (TMB) across breast cancer (BC) subtypes [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 P4-05-09.

Background: Urothelial bladder carcinomas had traditionally been difficult to treat cancers, with high morbidity and mortality rates when invasive and metastatic. In recent years, immunotherapy with immune checkpoint inhibitors has improved outcomes in several cancers, including bladder carcinomas. Despite positive overall results, many bladder cancer patients do not respond to immunotherapies. Validated predictive biomarkers of response would advance the selection of patients for these treatments. Tumor mutation burden (TMB) has been suggested as an immunotherapy biomarker and thus delineation of attributes of tumors with a high TMB is clinically relevant. Methods: Publicly available genomic and clinical data from the urothelial bladder carcinoma cohort of The Cancer Genome Atlas (TCGA) project are used to analyze characteristics and molecular alterations of the subset of cancers with an increased tumor mutation number compared with those with lower number of mutations. The cut-off for the high mutation burden in the analysis was set at 10 mutations per Megabase (MB). Results: In addition to their sensitivity to immune checkpoint inhibitors, urothelial carcinomas with high TMB possess several molecular defects that could be exploited for combinatorial treatments. Compared with bladder carcinomas with low TMB, carcinomas with high TMB display higher prevalence of mutations in tumor suppressor TP53, PIK3CA, in FAT4 cadherin and in genes encoding for several epigenetic modifier enzymes. The frequency of mutations in mismatch repair and DNA damage response genes is higher in cancers with high TMB. The group of urothelial carcinomas with high TMB has a better prognosis than the group with low TMB. This improved Overall Survival (OS) stems from improved survival of stage III cancers with high TMB compared with stage III cancers with low TMB, while stage II and stage IV cancers have similar OS, independently of their TMB. Conclusion: Differences of the landscape of high and low TMB urothelial cancers provides leads for further pathogenesis investigations and may prove useful for development of combination therapies including immunotherapies with targeted inhibitors.

BackgroundTo profile genomic and epigenomic of a naïve Chinese non-small cell lung cancer (NSCLC) cohort and investigate the association between tumor mutation burden (TMB) and DNA methylation (DNAm) to explore potential alternative/complimentary biomarkers for NSCLC immunotherapies.MethodsA total of 89 tumor tissues with matched normal tissues from Chinese NSCLC patients were collected and subjected to whole exome sequencing (WES). From comparison, each patient was evaluated for the TMB value and divided into high, medium and low TMB based on TMB tertile distribution and then relatively high and low TMB samples were selected and subjected to DNAm profiling.ResultsPatients in the low (n = 30), medium (n = 29), and high (n = 30) TMB tertiles had 1.1–2.5, 2.5–4.1, and 4.2–13.9 mutations/Mb, respectively. A statistical directly association between differential methylation probes (DMPs) and TMB level was observed in our cohort (r = 0.63, P value =0.0003) and this was confirmed by using TCGA NSCLC dataset (r = 0.43, P value =0.006). Relatively high TMB group (n = 16, 7.5–13.9 mutations/Mb) harbors more differential DMPs while less in relatively low TMB group (n = 13, 1.1–2.4 mutations/Mb). Eight hundred fifty-eight differential methylation regions (DMRs) were found in relatively high TMB group. In addition, 437 genes show DNAm aberrance status in high TMB patient group and 99 have been reported as its association with lung cancer.ConclusionTo our knowledge, this is the first report for direct link between the methylome alterations and TMB in NSCLCs. High TMB NSCLCs had more DNAm aberrance and copy number variations (CNVs). In addition, the TMB distribution of Chinese NSCLCs population is lower than that of TCGA.

4146 Background: Currently, ICI therapy is recommended for patients with metastatic PDAC whose tumors exhibit dMMR, MSI-H, or high TMB (≥10 mutations/Mb). However, due to the low prevalence of high TMB in PDAC (~1%), few studies have evaluated the role of ICI therapy in this subpopulation. This study aimed to compare GA between PDAC patients with high TMB and low TMB ( < 10 mutations/Mb) and to evaluate the effectiveness of ICIs in real-world PDAC patients by TMB status. Methods: This study included PDAC patients who underwent genomic testing using Foundation Medicine tissue comprehensive genomic profiling (CGP) assays. GA were compared between tissue specimens with high and low TMB by chi-squared, adjusted for multiple comparisons. Patient clinical data was obtained by the US-wide de-identified Flatiron Health and Foundation Medicine real-world clinicogenomic pancreatic database (CGDB), originated from ~280 US cancer clinics between 01/2011 and 09/2022. Real-world overall survival (OS) and time to treatment discontinuation (TTD) were compared between patients receiving an ICI (high TMB versus low TMB) and between patients with high TMB (ICI versus other therapies) by Cox models. Results: We included 21,932 patients with PDAC with tissue CGP data available; 98.7% with low TMB and 1.3% with high TMB. Among actionable alterations, patients with high TMB had higher prevalence of mutations in BRCA2 (p < 0.0001), BRAF (p < 0.0001), PALB2 (p < 0.0001), and genes of the mismatch repair pathway ( MSH2, MSH6, MLH1, and PMS2, p < 0.0001), but lower prevalence of KRAS mutations (p < 0.0001). The most common KRAS mutation in both groups of patients was G12D. In CGDB, 51 patients received an ICI (10 with high TMB and 41 with low TMB) and 17 patients with high TMB received other therapies. Among patients receiving an ICI, those with high TMB had more favorable median OS compared to those with low TMB (25.7 versus 5.2 months, hazard ratio (HR) 0.27, 95% confidence interval (CI) 0.09 - 0.76, p = 0.01) and median TTD (20.7 versus 3.0 months, HR 0.33, 95% CI 0.13 - 0.82, p = 0.02). Among patients with high TMB, those receiving an ICI had more favorable OS compared to those receiving other therapies (25.7 versus 6.6 months, HR 0.31, 95% CI 0.10-0.96, p = 0.043), nominally favoring ICI use in this small cohort (n = 10 versus 17). Conclusions: There is no randomized clinical trial evaluating ICI versus other therapies in PDAC. To our knowledge, this is the largest cohort to date comparing ICI effectiveness in PDAC. We observed more favorable OS among PDAC patients with high TMB receiving ICI versus those with low TMB who received an ICI and more favorable to those with high TMB receiving other therapies. This study supports the FDA-approved use of ICIs in patients with PDAC and high TMB and demonstrates the importance of TMB assessment for patients with PDAC.

6548 Background: Baseline tumor and germline biomarkers in R/M HNSCC were analyzed for predictive potential in pts benefitting from D or D+T. Methods: In HAWK (NCT02207530), 112 pts (PD-L1 tumor cells [TC]≥25%) received D (10 mg/kg Q2W for ≤12 m); in CONDOR (NCT02319044), 67 pts (PD-L1 TC < 25%) received D (10 mg/kg Q2W for ≤12 m), 133 pts received D+T (D 20 mg/kg Q4W, T 1 mg/kg Q4W for ≤12 m), and 67 pts received T (10 mg/kg Q4W [7 doses] then Q12W [2 doses] for ≤12 m) VENTANA PD-L1 (SP263) Assay determined PD-L1 status. Paired FFPE archival tumor and PBMC samples (as germline control) in the HAWK and CONDOR trials were evaluated by whole exome sequencing (WES). Tumor mutation burden (TMB) was number of somatic mutations/megabase. HLA class I types were obtained via WES of PBMCs (CONDOR only). HPV and neutrophil-to-lymphocyte ratio (NLR) were tested locally in CONDOR. Wilcoxon, log-rank tests, and COX-PH models were used. Pooled D & D+T data were analyzed unless noted. Results: 153 pts had paired evaluable FFPE tumor and PBMC samples (HAWK, n = 48; CONDOR, n = 105). TMB distributions were similar between studies ( P= 0.43). TMB correlated with smoking ( P= 0.02) but not HPV ( P= 0.24), NLR ( P= 0.66), or PD-L1 status ( P= 0.43). Overall, high TMB (≥upper tertile) trended with longer OS vs low TMB in all evaluable pts (N = 153; 9.0 vs 5.6 m; HR = 0.70; 95% CI = 0.48-1.01); P= 0.06). In HAWK, there was no association of TMB with OS. In CONDOR, pts (D and D+T arms) with high TMB vs low had significantly longer OS (N = 76; 16.3 vs 5.3 m; HR = 0.53; 95% CI = 0.31-0.92). TMB and OS association was further assessed by increasing TMB cutoffs. Improved HRs trended with higher cutoffs; cutoffs ≥upper quartile significantly linked to OS.TMB was not associated with PFS or ORR. Pts with low PD-L1 and low TMB had worse OS compared to pts with high PD-L1 or high TMB. Pts with high NLR (≥median) and low TMB had significantly worse OS than pts with low NLR and high TMB (HR = 2.63, P< 0.001). Analysis of germline HLA alleles revealed significantly poorer survival for carriers of the HLA-B*15:01 allele (9.4%) (HLA-B variant status did not affect TMB and OS association in CONDOR). Germline HLA heterozygosity did not impact OS. Pts with mutations in ATM (5%), a DNA damage repair gene, also trended with prolonged OS. Conclusions: TMB is a possible predictive biomarker of IO HNSCC therapy. Combined analysis of NLR and TMB may provide additional PD-L1 data in assessing pts most likely to have long-term benefit. Clinical trial information: NCT002207530, NCT02319044 .

Prognostic Value of Tumor Mutational Burden in Follicular Lymphoma Patients Treated with Immunochemotherapy

e15090 Background: Tumor Mutational Burden (TMB) is a critical biomarker for predicting immunotherapy response in oncology. However, targeted panels, limited by narrow genomic coverage, often yield inconsistent TMB estimates, either underestimating or overestimating due to design variability and extrapolation challenges. Whole Genome Sequencing (WGS) offers a more comprehensive and accurate approach, providing genome-wide mutation detection and reliable TMB assessment. Methods: We analyzed 202 tumor samples from lung, breast, colorectal, ovarian, and fallopian tube cancer patients. TMB was evaluated using the validated large targeted panel tests (TP), such as TruSight Oncology (TSO500) and GeneseeqPrime Pan-Solid Tumor, and CancerVision’s target-enhanced WGS (TE-WGS). TP define TMB as non-synonymous somatic mutations per megabase (mut/Mb) of the panel’s genomic regions, with high TMB classified as ≥10 mut/Mb. In contrast, TE-WGS calculates TMB across the effective genome size (2.9Gb) without limiting to coding regions. TE-WGS was analytically validated, demonstrating 99.8% sensitivity for single nucleotide variants (SNVs), 99.2% for indels, and high positive predictive values (SNVs: 99.3%, indels: 98.7%). Results: TMB values were compared between TE-WGS and TP. The TE-WGS assay showed strong concordance with TP for high (≥10 mut/Mb) and low ( < 10 mut/Mb) TMB classification: Sensitivity 79.3% (95% CI: 61.6–90.2%) and Specificity 95.4% (95% CI: 91.1–97.6%). Of the 202 samples, 23 (11.4%) were concordantly classified as high TMB, and 165 (81.7%) as low TMB. Discordance was observed in 14 cases (6.9%): 8 high TMB by TE-WGS but low by TP, and 6 low TMB by TE-WGS but high by TP. Analytical evaluation revealed that TE-WGS quantification was robust across varying tumor cellularity levels, demonstrating consistent performance independent of tumor purity. Discordant cases highlighted the advantage of TE-WGS in capturing mutations outside the genomic regions covered by TP, underscoring the importance of genome-wide assessment for accurate TMB quantification. Conclusions: TE-WGS offers a more comprehensive and reliable approach to TMB assessment compared to targeted panels, minimizing limitations of narrow genomic coverage and enhancing clinical utility.

Despite advances in care in many cancer types, patients with glioblastoma (GBM) have a grim prognosis of 15 to 21 months and unchanged standard therapy since 2005. These tumors have a profoundly immunosuppressive tumor immune microenvironment. Immunotherapy, including the checkpoint inhibitors (ICIs) targeting PD-1 and CTLA-4, has transformed the treatment of many cancers but has failed in trials of both newly diagnosed and recurrent GBM. Tumor mutational burden (TMB) has been proposed as a predictor of response to ICI treatment, presumably through an increase in neoantigens that can be recognized by cytotoxic CD8+ T cells leading to tumor rejection. GBM typically has a low TMB, a feature speculated as a contributor to poor response to ICI treatment. However, cases with an extremely high TMB (i.e., patients with the biallelic mismatch repair deficiency syndrome) do show response to immunotherapy. To begin to investigate the impact of mutational burden on immune response in GBM, we created a murine syngeneic tumor model using CRISPR-Cas9 to knockout (KO) the mismatch repair protein Msh2, the lynchpin to mismatch recognition, in the SB28 GBM model that has a low TMB (108 mutations) at baseline. Msh2 KO increased the TMB from 2 to 12-fold depending on single cell sorted clone, resulting in between 150 and 200 predicted strong MHC-I binding neoantigens estimated by the NetMHC algorithm. Preliminary studies have demonstrated that in vitro proliferation is similar amongst low TMB and high TMB SB28 clones. Flank implantation of high TMB SB28 clones revealed slower growth compared to the low TMB SB28 tumors. Furthermore, treatment with ICIs did provide a survival advantage in this flank model. However, no survival advantage was noted when the high TMB clones were implanted intracranially either with or without ICI treatment. These results suggested that either there is poor immune cell trafficking to the intracranial tumors, or a robust immune response was causing increased intracranial pressure that is prematurely causing death. Use of dexamethasone starting at day 11 to control cerebral edema has shown some survival improvement with ICI treatment, supporting that an inflammatory response is at least partially responsible for the lack of improved survival in early studies. We are currently evaluating CD4+ and CD8+ T cell clonality, earlier timepoints to evaluate the immune infiltrate and anti-tumor immune response, and alterations in chemokines/cytokines in the tumor microenvironment. By creating high mutation burden clones from a GBM with a low mutational burden, we will be able to interrogate potential mechanisms of heightened immunogenicity that has been reported in other cancers. Citation Format: Kevin Breen, Tuesday Haynes, Masashi Watanabe, Mark Gilbert. Determining the role of tumor mutational burden in a pre-clinical model of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2663.

Background: Cytotoxic T-lymphocytes (CTLs) infiltration in a bulk tumor is a positive prognostic factor in breast cancer. CTL recognition is believed to react to mutation-induced neoantigens, thus, higher tumor mutation burden (TMB) is considered as an important predictor of tumor immunogenicity and response to immunotherapy. Although majority of breast cancer have low tumor mutation burden (TMB), triple-negative breast cancer (TNBC) has higher TMB compared to other breast cancer subtypes. However, it is unclear if the prognostic value high CTL infiltration depends on the TMB as the primary driver of enhanced anti-cancer immunity or represents an independent prognostic marker which can be used as a complement to TMB to predict patients' outcomes. CTLs are identified by CD8 marker, is encoded by CD8A. Granzyme B (GZMB) is a serine protease that is secreted by functionally-active CTLs to induce apoptosis of the target cells. CXCL10 is a chemokine which selectively attracts activated CTLs into subsets of other cancers; but its role in breast cancer remains unknown. We investigated if TNBC infiltrated with high levels of functional CTLs, so called “hot tumors”, have improved survival independently of their TMB. Methods: Utilizing The Cancer Genome Atlas (TCGA) breast cancer cohort, we established Functional Hotness Score (FHS), based on the CD8A, GZMB and CXCL10 gene expression levels of bulk tumors. Total of 4149 breast cancer patients from publicly available multiple cohorts were analyzed to assess FHS and breast cancer patient prognosis as well as distinct immunity markers. Results: FHS predicted the breast cancer patient survival better than each consisting gene. The breast cancer patients with high FHS tumors showed significantly better survival in the testing cohort (TCGA), which was further confirmed in the validation cohort (METABRIC). FHS was higher in the primary breast cancer tissues with metastasis compared to that without metastasis in TCGA. Further, FHS was higher in the metastatic tumors compared to the primary tumors in GSE110590. Among breast cancer subtypes, TNBC showed highest FHS compared to other subtypes in testing TCGA, which was validated in the METABRIC cohort. The patients with high FHS tumor demonstrated significantly better long-term survival than that with low FHS only in the hormone receptor (HR)-negative breast cancers, but not in the HR-positive tumors. The high FHS TNBCs showed higher not only CD8-positive T cell infiltration, but also a broader type-1 anti-cancer immunity. To investigate if FHS predicts patient survival independent of TMB, the patients were divided into high and low TMB groups. Interestingly, the higher FHS patients showed better prognosis not only in the high TMB group but also in the low TMB among TNBC patients. The combination of high TMB with high FHS identified the unique subset of the patients who did not recur over time. Conclusion: In conclusion, TNBC with higher FHS based on the expression levels of CD8A, GZMB and CXCL10 showed improved prognosis with higher anti-cancer immunity regardless of TMB, and constituting an independent prognostic marker of survival, particularly when combined with TMB. Citation Format: Eriko Katsuta, Li Yan, Mateusz Opyrchal, Pawel Kalinski, Kazuaki Takabe. Functional hotness score generated by representative functional cytotoxic T-lymphocytes predicts long-term survival of triple-negative breast cancer independently to the tumor mutational burden [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 6616.

Background: Metaplastic breast cancers (MPBC) are rare, typically triple negative aggressive tumors composed of both, adenocarcinoma and metaplastic elements. Recent evidence that TNBC and MPBC can respond vigorously to immune checkpoint inhibitor therapy (Adams et al, ASCO 2017 and npj Breast Cancer 2017) has prompted the following comprehensive genomic profiling (CGP) and histopathologic assessment of tumor infiltrating lymphocytes (TIL) designed to uncover potential biomarkers of immunotherapy response for MPBC, including mutational burden, Microsatellite Instability (MSI) status and gene amplification of 9p21.4 (or CD274, which includes the PD-L1 locus). Methods: 12,214 locally aggressive, relapsed and metastatic breast malignancies (mBM) were subjected to CGP using DNA extracted from 40 µm of FFPE sections and adaptor ligation-based libraries to a mean coverage depth >650X for up to 315 cancer-related genes. The results were analyzed for all classes of genomic alterations (GA) including base substitutions, insertions and deletions, select rearrangements, and copy number changes. Tumor mutational burden (TMB) was determined on 1.1 Mbp of sequenced DNA. MSI status was determined by an algorithm based on the sequencing results. TIL were assessed on archived H&E tumor sections and enumerated per guidelines established by the TIL Working Group (Salgado, Ann Oncol 2015) in a subset of MPBC with the highest TMB and compared with low TMBC cases. Results: 165 of mBM cases were MPBC (1.4%) and are included in this study. All patients were female with a median age of 60 (range 24-86). 165 of the MPBC cases (100%) harbored a wide variety of GA involving more than 100 individual genes. The most common GA were identified in TP53 (65%), followed by PIK3CA (37%). No cases of MSI hi status (0/103) and only one case with amplification of 9p21.4 (1/165, 0.6%) were observed. Most MPBC had a low mutational burden, with a median TMB of 2.7 mutations/Mb (range 0-39.6). Only 11/165 tumors (6.7%) were found to have a TMB over 10 mutations/Mb, including 3 cases (1.8%) with TMB >20. Tumor sections were available for TIL review from 9/11 cases with highest TMB, as well as 11 control cases with lowest TMB. TIL were more frequently observed in high versus low TMB MPBC, with median TIL percentage of 40 and 20 (range 10-80 and range 10-60), respectively, although this difference was not statistically significant (Wilcoxon rank-sum test, p=0.15). Conclusions: Genomic profiling in the largest cohort of MPBC reported to date confirms that MPBC is enriched for TP53 and PIK3CA mutations and many tumors harbor targetable GA. The frequently observed tumoral PD-L1 expression in MPBC is not based on gene amplification as amplification of 9p21.4 is rare. Most tumors had a low mutation burden, and no significant association of TIL with TMB was observed, suggesting additional processes underlying MPBC immunogenicity. Citation Format: Taff J, Suh J, Singh B, Denkert C, Troxel AB, Ross JS, Adams S. Metaplastic breast cancers: Genomic profile, mutational burden and TILs [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-05-03.

Tumor mutational burden.

530 Background: GI cancers are generally insensitive to immune checkpoint inhibitors (ICIs). Response to ICIs has been shown to correlate with TMB. Herein, we attempt to quantify TMB in GI cancers and its correlation with PD-1/PDL-1 expression. Methods: Tumor from various GI sites: right-sided and left-sided colon cancers (RT and LT), rectal cancer (RC), small bowel adenocarcinoma (SBA), gastric adenocarcinoma (GA), anal cancer (SCCA), hepatocellular carcinoma (HCC), esophageal adenocarcinoma and squamous cell carcinoma (EA and E-SCC), biliary cancer (BC), pancreatic adenocarcinoma (PA), and pancreatic neuroendocrine tumors (PNET), were analyzed by NextGen sequencing. TMB was calculated using only somatic nonsynonymous missense mutations sequenced with a 592-gene panel. MSI was assessed by fragment analysis. Correlation of PD-1/PD-L1 expression with TMB was calculated by student’s t test. Results: In total, 1375 tumors were examined. Among the different GI cancer types, RT and LT had the highest TMB (mean: 11.6 and 9.9 mutations [mut]/megabase [MB]), whereas BC and PA had the lowest levels (mean: 5.7 and 4.9 mut/MB) (Table). Overall primary tumors had higher TMB than metastases (mean: 8.3 vs. 6.5 mut/MB, p = 0.037). Using a cut-off of 17 mut/MB to define high vs. low TMB, high TMB was seen in all 24 MSI-H and 2 MSS colon tumors with POLE mutations, but not in other MSS colon tumors (n = 325, p < 0.0001). Similarly, among 6 GA tumors tested for MSI, high TMB was seen in 2 MSI-H while low TMB was seen in the 4 MSS tumors. Overall high TMB was seen most frequently in RT (12%), GA (11%), and SCCA (8%), and least frequently in PA (1.3%) and E-SCC (0%). PD-1 correlated with TMB in some tumor types (RT and RC), as did PD-L1 (RT and HCC). Conclusions: TMB varies among GI cancers. Forthcoming prognostic analysis to assess the correlation between TMB and response to ICIs in GI cancers is underway. [Table: see text]

3039 Background: Higher levels of tumor mutational burden (TMB) can predict sensitivity to immunotherapies (IO), which are FDA approved to treat NSCLC, melanoma, and urothelial carcinoma (Ca). TMB may be a biomarker for sensitivity to IO, irrespective of tumor type. TMB has not been explored widely for tumors of unknown primary site, but may reveal additional treatment options. Methods: Comprehensive genomic profiling of DNA from FFPE tissue samples was performed using hybrid-capture, next-generation sequencing. TMB was calculated by counting all coding short variant alterations (base substitutions and indels), including synonymous alterations, and subtracting from this functionally oncogenic or germline alterations (per ExAC, dbSNPT, or internal algorithmic analysis). To calculate the TMB per Mb, the total number of relevant mutations is divided by the coding region of the bait set (0.8 Mb, 1.1 Mb, or 1.2 Mb). High, intermediate, and low TMB were defined as ≥20 mut/Mb, ≥6 and <20 mut/Mb, or <6 mut/Mb, respectively. Tumor types with >100 samples were analyzed. Results: From a database of 102,878 samples sequenced during routine clinical care, 6116 samples for which the primary tumor site was unclear at sequencing were identified. Table shows TMB metrics (mut/Mb) and median patient age for these cohorts. Conclusions: Significant numbers of patients with each tumor type have high TMB that may indicate benefit from IO, excepting GIST. As expected, urothelial tumors have higher than average TMB and more patients have high TMB. SCC tumors are commonly TMB high (23%), as are tumors difficult to define histologically (15%). For ACUP or CUP, the most common tumors, 8-11% have high TMB. Analysis of responses to treatment with IO are ongoing. [Table: see text]

534 Background: ICIs are frequently used as therapy in mUC, but only a minority of patients (pts) respond to treatment. High TMB is associated with improved outcomes to ICIs. However, much is unknown about biomarkers associated with ICI outcomes in pts with high and low TMB respectively. Methods: We retrospectively identified mUC pts with known TMB status and available next generation sequencing (NGS) results treated with ICI monotherapy at our institution. TMB high was defined as ≥ 10 mutations/Mb, with the rest being TMB low. Somatic alterations present in ≥10% pts ( ARID1A, CCND1, CDKN2A, CDKN2B, ERBB2, FGF3, FGF4, FGF19, FGFR3, KDM6A, MDM2, MLL2, PIK3CA, RB1, TERTp, TP53, TSC1), and presence of DNA damage response (DDR) alterations were assessed as biomarkers of interest. Within the TMB-high and TMB-low pt groups we separately assessed patients based on the presence or absence of these somatic alterations, APOBEC mutational signature and high PD-L1 expression. Log rank test was used to determine differences in overall survival (OS) and progression free survival (PFS) among these groups. P-value ≤0.05 was considered significant. Results: Among 107 mUC pts treated with ICI monotherapy between 12/2014 and 3/2022 who had NGS data (UCSF500, FoundationOne, Strata), 85 pts had TMB data, including 47 TMB high pts and 38 TMB low pts. Among 85 pts with known TMB status, median age was 76 yrs, the majority were male (55, 65%), Caucasian (57, 67%), had pure urothelial histology (46, 55%) and were treated with ICIs in frontline setting (47, 55%). Median OS was 17.2 mos and median PFS was 3.42 mos. In TMB high pts, presence of DDR , MLL2, KDM6A, PIK3CA and TERTp alterations were each associated with improved outcomes, while presence of CDKN2B alterations was associated with inferior outcomes (Table). Among TMB low pts, those with RB1 alterations had shorter mOS (11.3 months vs 17.2 months; p=0.04) compared to wild-type pts. Conclusions: In this single-center retrospective analysis of mUC pts, we identified somatic alterations that were predictive of outcomes with ICI treatment in TMB high and TMB low pts respectively. Further exploration of biomarkers in patients stratified by TMB status is warranted in larger cohorts. [Table: see text]

MA06.07 Genetic and Epigenetic Alterations are Associated with Tumor Mutation Burden in Non-Small Cell Lung Cancer