Elevated Tumor Mutational Burden Research Articles

Abstract PO5-06-09: Metastatic triple negative breast cancer has distinct tumor immune landscape

Abstract Background: Triple negative breast cancer (TNBC) is considered the most immunogenic breast cancer subtype due to higher levels of tumor infiltrating immune cells (TILS), elevated tumor mutational burden and PD-L1 expression, providing a rationale for immunotherapy. Here we aimed to investigate the differences in the immune microenvironment of primary vs. metastatic sites in TNBC vs. non-TNBC and their impact on survival. Methods: Comprehensive immune profiling, including PD-L1 IHC and the expression of 395 immune genes, was performed on 147 real-world FFPE breast cancer samples (32 primary, 115 metastatic). 37 samples were, by definition, triple negative for ER, PR, and HER2 overexpression. PD-L1 (CPS positive ≥1% and CPS positive ≥10%) was determined using immunohistochemistry (22C3). mRNA expression signatures of tumor inflammation (TIGS, weak/moderate/strong) and cell proliferation (CP, poor/moderate/high) were determined by RNA-sequencing. Demographic and clinicopathologic variables were collected. Statistical comparisons of biomarkers between groups were calculated using the Wilcoxon Rank-Sum test for continuous variables and the proportions test for categorical variables (p≤0.05 for significance). Survival differences were quantified by Cox proportional hazards analysis (p≤0.05 for significance). Results: Triple negative status was associated with current use of alcohol [p=0.03]. Black patients were also more likely to have TNBC, constituting 24% of TNBC patients while only making up 14% of the total cohort [p=0.04]. Conversely, white patients were less likely to have TNBC, constituting 73% of TNBC patients while making up 84% of the total cohort [p=0.04]. Comparing TNBC and non-TNBC cases, TNBC cases were observed to have significantly higher TIGS [p=0.014] and PD-L1 expression [p=4.4 × 10−7]. Additionally, TNBC cases trended towards exhibiting greater cell proliferation [p=0.069]. These differences were found to be driven primarily by metastatic tumors, among which TNBC tumors showed significantly higher TIGS [p=0.015], cell proliferation [p=0.021], and PD-L1 expression [p=2.9 × 10−7] than non-TNBC tumors, while none of these significant associations were observed among primary tumors. Triple negative status was significantly associated with PD-L1 expression (assessed by IHC), with a majority of PD-L1 positive cases determined to be triple negative [70% of CPS≥1% cases, p=5 × 10-5; 59% of CPS≥10% cases, p=6 × 10−4]. Several immune-related genes and immune checkpoint molecules were over-expressed in triple negative breast cancer, including CD8, GZMB, PRF1, CCL5, IFNG, TIGIT and CXCL10. A number of genes were also significantly overexpressed in metastatic tumors: KRT5 [p=0.02], TFRC [p=0.04], ABCF1 [p=0.04], FCRLA [p=0.05], LAMP3 [p=0.05], and underexpressed in metastatic tumors: ENTPD1 [p=0.05], IGSF6 [p=0.03], and ITGA1 [p=0.03]. Conclusions: Our comprehensive biomarker analyses showed that metastatic TNBC has a more inflamed tumor microenvironment and higher checkpoint target expression compared to non-TNBC. Further analysis of immune expression by site-specific metastases and correlation with immunotherapy outcomes is warranted to guide clinicians in selection of the ideal metastatic site to biopsy for therapeutic decision making. However, in a collective TNBC context, these data support the use of immunotherapy in metastatic TNBC. Citation Format: Robert Seager, Heidi Ko, Sarabjot Pabla, Maria-Fernanda Senosain, Erik Van Roey, Shuang Gao, Kyle Strickland, Rebecca Previs, Mary Nesline, Stephanie Hastings, Shengle Zhang, Jeffrey Conroy, Taylor Jensen, Marcia Eisenberg, Brian Caveney, Eric Severson, Shakti Ramkissoon, Shipra Gandhi. Metastatic triple negative breast cancer has distinct tumor immune landscape [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 PO5-06-09.

DNA methylation regulator-based molecular subtyping and tumor microenvironment characterization in hepatocellular carcinoma.

Although recent studies have reported the regulation of the immune response in hepatocellular carcinoma (HCC) through DNA methylation, the comprehensive impact methylation modifications on tumor microenvironment characteristics and immunotherapy efficacy has not been fully elucidated. In this research, we conducted a comprehensive assessment of the patterns of DNA methylation regulators and the profiles of the tumor microenvironment (TME) in HCC, focusing on 21 specific DNA methylation regulators. We subsequently developed a unique scoring system, a DNA methylation score (DMscore), to assess the individual DNA methylation modifications among the three distinct methylation patterns for differentially expressed genes (DEGs). Three distinct methylation modification patterns were identified with distinct TME infiltration characteristics. We demonstrated that the DMscore could predict patient subtype, TME infiltration, and patient prognosis. A low DMscore, characterized by an elevated tumor mutation burden (TMB), hepatitis B virus (HBV)/hepatitis C virus (HCV) infection, and immune activation, indicates an inflamed tumor microenvironment phenotype with a 5-year survival rate of 7.8%. Moreover, a low DMscore appeared to increase the efficacy of immunotherapy in the anti-CTLA-4/PD-1/PD-L1 cohort. In brief, this research has enhanced our understanding of the correlation between modifications in DNA methylation patterns and the profile of the tumor microenvironment in individuals diagnosed with HCC. The DMscore may serve as an alternative biomarker for survival and efficacy of immunotherapy in patients with HCC.

Abstract 6469: The association of mismatch repair gene alterations with elevated tumor mutational burden in microsatellite stable gastrointestinal cancers

Abstract Background: Benefit from single-agent immune checkpoint inhibitor has been observed in 5-10% of the patients with microsatellite stable (MSS) gastrointestinal (GI) cancers. However, a reliable predictive marker to immune checkpoint inhibitor has not been discovered in this patient population. We sought to assess the incidence of mismatch repair (MMR) gene alterations and their association with tumor mutational burden (TMB) in MSS GI cancers. Methods: This is a retrospective cohort study of patients with advanced GI cancer who had molecular profiling of the tumor using either Guardant360 blood-based or Tempus tissue next generation sequencing (NGS) platforms between 2020 and 2023. We assessed four MMR genes including MLH1, MSH2, MSH6 and PMS2 for alterations. Continuous variables were summarized as median and interquartile range (IQR) and compared using Mann-Whitney test. Categorical variables were summarized as counts and proportions and compared using Fisher’s exact test. Results: A total of 2920 patients with Guardant360 were included with a median age of 65 (IQR: 56-73) years. 870 (30%) patients had pancreas cancer; 846 (29%) patients had colorectal cancer; 688 (24%) patients had biliary tract cancers; and the rest had other GI cancers. Among patients evaluable for microsatellite instability (MSI) status, 58 patients (2%) had MSI-high GI cancers, 31 (53%) of whom had MMR gene alterations. 2445 (98%) patients had MSS GI cancers, 130 (5.3%) of whom had MMR gene alterations. Patients with MSS GI cancers bearing MMR gene alterations had a median age of 70 (IQR: 62-75) years, significantly older than those without MMR gene alterations (p<0.001). In addition, patients with MMR gene alterations had significantly higher median blood TMB of 11 (IQR: 7.4-16) mut/Mb compared to 7.7 (IQR: 4.8-11) mut/Mb in patients without MMR gene alterations. In MSS GI cancers, MMR gene alterations are more commonly associated with the presence of alterations in APC (p=0.03), TP53 (p=0.01), CTNNB1 (p<0.001) and SMAD4 (p=0.01). 1453 patients with GI cancer and Tempus tissue NGS data were included as a validation cohort. They had comparable demographics with the Guardant360 cohort. 14 (1.6%) of 896 patients with MSS GI cancers had MMR gene alterations, significantly fewer (p<0.001) than those found in the Guardant360 cohort. Patients with MMR gene alterations and wild type POLE had significantly higher (p=0.001) median TMB of 9.8 (IQR: 4.8-14) mut/Mb compared to 4.7 (IQR: 3.2-6.3) mut/Mb in patients without MMR gene alterations. Conclusions: MMR gene alterations can be identified by clinical NGS platforms in a small proportion of patients with MSS GI cancers. They are associated with elevated TMB, which may suggest a hypermutated profile serving as a basis for potential role of immune checkpoint inhibitor in MSS GI cancers. Citation Format: Conor D. O'Donnell, Mojun Zhu, Hao Xie. The association of mismatch repair gene alterations with elevated tumor mutational burden in microsatellite stable gastrointestinal cancers [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 6469.

Abstract 6751: In situ vaccination with Flt3l gene modified CD103+ type 1 conventional dendritic cells synergizes with anti-PD-1 checkpoint blockade in murine models of non-small cell lung cancer

Abstract A major hurdle in treatment of Non-Small Cell Lung Cancer (NSCLC) with anti-PD-1 immune checkpoint blockade (ICB) therapy is a lack of response (primary resistance) and relapse after an initial response (acquired resistance). Recent studies reveal that responses to PD-1/PD-L1 blockade are associated with high tumor mutational burden (TMB), increased CD8+ T cell infiltration and high baseline PD-L1 expression within the tumor microenvironment (TME), while impaired tumor antigen presentation and the immunosuppressive TME have been associated with resistance to ICB. One approach to overcome anti-PD-1 resistance is to intratumorally vaccinate NSCLC tumors with gene modified conventional dendritic cells (cDC), specifically the type I conventional DC (cDC1) lineage. Recent studies have established that generation of an anti-tumor immune response driven by CD8+ T cells requires the cross presentation of tumor associated antigens and that cDC1s are the primary cross presenting APC subtype in vivo, which can license CD8+ T cells to initiate an adaptive anti-tumor immune response. In addition, previous studies have shown that intratumoral administration of the FMS-like tyrosine kinase 3 ligand (FLT3L) protein can expand endogenous CD103+ cDC1s in the TME and augment anti-tumor immune responses to ICB therapy. Here, we engineered murine CD103+ cDC1s to constitutively secrete soluble FLT3L (FLT3L_cDC1) and performed in situ vaccination studies on anti-PD1 resistant murine models of NSCLC with LKB1-deficiency and elevated TMB that better represents human disease. In situ vaccination with FLT3L_cDC1 enhances anti-tumor efficacy compared to non-modified cDC1 vaccination and synergizes with anti-PD-1 ICB to inhibit tumor growth. FLT3L_cDC1 + anti-PD-1 combination therapy induces significant activation and expansion T cells and cDC1s within the TME. Furthermore, combination therapy significantly increases DC progenitor numbers within the tumor draining lymph node, including DC progenitors that are committed to the cDC1 lineage. Our data suggests in situ vaccination with FLT3L_cDC1 may represent a promising strategy to potentiate the efficacy of ICB and improve outcomes for patients with primary resistance PD-1/PD-L1 monotherapy. Citation Format: Jensen W. Abascal, Raymond J. Lim, Ramin Salehi-Rad, Zhe Jing, Michael S. Oh, William P. Crosson, Bitta P. Kahangi, Edgar Perez Reyes, Camelia Dumitras, Diana Reyimjan, Jessie Zhu, Linh M. Tran, Manash Paul, Kostyantyn Krysan, Bin Liu, Steven M. Dubinett. In situ vaccination with Flt3l gene modified CD103+ type 1 conventional dendritic cells synergizes with anti-PD-1 checkpoint blockade in murine models of non-small cell lung cancer [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 6751.

Abstract 7612: Genetic predictors of anti-PD(L)-1 therapy response in hepatocellular carcinoma

Abstract Introduction: Immunotherapy targeting PD-1/PD-L1 shows promise in treating hepatocellular carcinoma (HCC), but standard predictors like PD-L1 expression are unreliable for gauging treatment response. RNA sequencing has identified a BMS 4-gene inflammatory signature that could more accurately forecast clinical outcomes. However, genomic alterations and tumor mutational burden (TMB) haven't consistently predicted anti-PD(L)-1 therapy response in HCC. Therefore, the aim of this research is to identify potential genetic alternation that could serve as predictive marker for anti-PD(L)-1 therapy responses in advanced HCC. Methods: Formalin-fixed paraffin-embedded (FFPE) tumor tissues from 38 HCC patients were analyzed using targeted next-generation sequencing with the ACTOncoTM panel before anti-PD(L)-1 therapy. This analysis identified somatic variants across 440 genes and calculated TMB. In silico tools like NetMHC and IEDB predicted neo-peptide-HLA class I binding affinity. Additionally, peripheral blood mononuclear cells provided germline data for paired TMB calculations, with tumor responses assessed by RECIST criteria. Results: Within a cohort of 167 patients with HCC undergoing ICI therapy, 35 (20.9%) responded to treatment, while 132 (79.1%) did not. Available formalin-fixed paraffin-embedded (FFPE) samples from this population included those from 14 responders and 30 non-responders. After conducting quality control measures, which assessed tumor purity, DNA quantity and integrity, as well as sequencing quality, 13 samples from responders and 25 from non-responders qualified for further analysis. Gene alteration analysis identified mutations in TP53 (47%), MUC16 (34%), CTNNB1 (32%), USH2A (16%), ARID1A (13%), NOTCH3 (13%), and ADAMTS16 (11%) of the cases. A TP53 mutation was linked to poor overall survival, whereas mutations in CTNNB1 and the RTK/RAS/RAF pathway did not correlate with clinical outcomes. The median TMB, predicted at 3.2 ± 4.3 mutations per megabase (muts/Mb), was nominally higher in patients who achieved disease control compared to non-responders (4.6 vs. 3.7 muts/Mb, p=0.53). Utilizing paired TMB analysis, those with disease control exhibited a higher TMB trend than non-responders (6.0 vs. 2.2 muts/Mb, p=0.06). Notably, the 13 cases with MUC16 mutations were associated with a significantly higher predicted TMB (10.0 vs. 3.3 muts/Mb, p=0.0015). Conclusions: This study suggests that certain genetic alterations, especially TP53 mutations, may have predictive value for the response to anti-PD(L)-1 therapy in HCC. Moreover, an elevated TMB, particularly when associated with MUC16 mutations, seems to be correlated with improved disease control. Citation Format: San-Chi Chen, Nai-Jung Chiang, MingHuang Chen, Muh-Hwa Yang. Genetic predictors of anti-PD(L)-1 therapy response in hepatocellular carcinoma [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 7612.

Abstract 3929: Comparative genomics of breast cancer in indigenous African and western populations first results from the E-Predict study

Abstract Breast cancer (BC) stands as the foremost female malignancy globally and is the most prevalent cancer in numerous developing countries, particularly in sub-Saharan Africa (SSA). Over the past decade, significant strides in comprehending the pathobiology of the disease and tumour dynamics have greatly enhanced treatment options, particularly in industrialized nations. Regrettably, SSA countries have witnessed only marginal, if any, improvements, with mortality rates surpassing incidence rates. Unfavourable outcome, early onset and high-grade tumours are predominant in SSA. Despite these challenging clinical phenotypes, there is currently a limited understanding of the primary molecular drivers, primarily due to a dearth of reliable data. Furthermore, there is a paucity of research conducted in, and pertinent to, the challenges of the disease in low-income countries (LICs), despite a rising incidence and higher mortality-to-incidence ratios. We thus established an international translational cancer research consortium in five distinct African countries. This initiative has contributed to the creation of a high-quality repository of biomaterials. Additionally, we have conducted whole exome sequencing on the initial 100 cases of breast cancer, comparing the molecular architecture of these tumors with data from The Cancer Genome Atlas (TCGA). We examining and compared the clinical disease phenotypes between indigenous African women and Western patients. A higher proportion of grade III tumors was observed in African patients aged between 30 to 59 years as compared to other patients within the same age group. In comparative molecular analyses, we found a significantly elevated tumor mutational burden in African patients (median of >15/MB) in contrast to other populations within the TCGA (median ~ 0.7/MB). Gene sets featuring co-occurring mutations, predicted to be associated with survival using the TCGA dataset, showed high mutation rates in the African cohort. Notably, co-occurring mutations between PIK3CA and genes linked to genomic instability, such as BIRC6 (HR: 11.7, p = 2.7e-05), KMT2C (HR: 4.7, p = 4.01e-04), NEB (HR: 3.84, p = 0.48), and PCLO (HR: 4.9, p = 0.016), among others, strongly correlated with poor overall survival and were highly mutated within the African cohort. Interestingly, within the TCGA, patients with early breast cancer onset exhibit twice as many mutations in KMT2C, a gene known for its association with DNA damage repair, genomic instability, and showed an improved response to immune checkpoint inhibition. These initial findings suggest that breast cancer genomics in indigenous African populations may exhibit substantial differences, potentially warranting consideration for distinct therapeutic modalities for these patients. Citation Format: Smiths Sengkwawoh Lueong, Pamela Derliche Tonouo Derliche Tonouo, Arnol Auvaker Tiofack, Rovaldo Nguims Kenfack, Jules Roger Kuiate, Esther Dina Mbassi, Sidonie Noa Ananga, Etienne Okobalemba Atenguena, Gustave Simo, Abdel Jelil Njouendou, Walters Ndaka, Zacharie Sando, Emmanuella Mayemi, Rachel Tayou, Gilles Gael Aghoagni, Anne Sango, Benjamin Pokam, Augustin Tozoula Bambara, Jens T. Siveke. Comparative genomics of breast cancer in indigenous African and western populations first results from the E-Predict study [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 3929.

Tertiary lymphoid structures and B cells determine clinically relevant T cell phenotypes in ovarian cancer

Intratumoral tertiary lymphoid structures (TLSs) have been associated with improved outcome in various cohorts of patients with cancer, reflecting their contribution to the development of tumor-targeting immunity. Here, we demonstrate that high-grade serous ovarian carcinoma (HGSOC) contains distinct immune aggregates with varying degrees of organization and maturation. Specifically, mature TLSs (mTLS) as forming only in 16% of HGSOCs with relatively elevated tumor mutational burden (TMB) are associated with an increased intratumoral density of CD8+ effector T (TEFF) cells and TIM3+PD1+, hence poorly immune checkpoint inhibitor (ICI)-sensitive, CD8+ T cells. Conversely, CD8+ T cells from immunologically hot tumors like non-small cell lung carcinoma (NSCLC) are enriched in ICI-responsive TCF1+ PD1+ T cells. Spatial B-cell profiling identifies patterns of in situ maturation and differentiation associated with mTLSs. Moreover, B-cell depletion promotes signs of a dysfunctional CD8+ T cell compartment among tumor-infiltrating lymphocytes from freshly isolated HGSOC and NSCLC biopsies. Taken together, our data demonstrate that – at odds with NSCLC – HGSOC is associated with a low density of follicular helper T cells and thus develops a limited number of mTLS that might be insufficient to preserve a ICI-sensitive TCF1+PD1+ CD8+ T cell phenotype. These findings point to key quantitative and qualitative differences between mTLSs in ICI-responsive vs ICI-irresponsive neoplasms that may guide the development of alternative immunotherapies for patients with HGSOC.

Integrating molecular subtype and CD8+ T cells infiltration to predict treatment response and survival in muscle-invasive bladder cancer

BackgroundLuminal and Basal are the primary intrinsic subtypes of muscle-invasive bladder cancer (MIBC). The presence of CD8+ T cells infiltration holds significant immunological relevance, potentially influencing the efficacy of antitumor responses. This study aims to synergize the influence of molecular subtypes and CD8+ T cells infiltration in MIBC.MethodsThis study included 889 patients with MIBC from Zhongshan Hospital, The Cancer Genome Atlas, IMvigor210 and NCT03179943 cohorts. We classified the patients into four distinct groups, based on the interplay of molecular subtypes and CD8+ T cells and probed into the clinical implications of these subgroups in MIBC.ResultsAmong patients with Luminal-CD8+Thigh tumors, the confluence of elevated tumor mutational burden and PD-L1 expression correlated with a heightened potential for positive responses to immunotherapy. In contrast, patients featured by Luminal-CD8+Tlow displayed a proclivity for deriving clinical advantages from innovative targeted interventions. The Basal-CD8+Tlow subgroup exhibited the least favorable three-year overall survival outcome, whereas their Basal-CD8+Thigh counterparts exhibited a heightened responsiveness to chemotherapy.ConclusionsWe emphasized the significant role of immune-molecular subtypes in shaping therapeutic approaches for MIBC. This insight establishes a foundation to refine the process of selecting subtype-specific treatments, thereby advancing personalized interventions for patients.

TP53 disruptive mutation predicts platinum-based chemotherapy and PD-1/PD-L1 blockade response in urothelial carcinoma.

TP53 mutation is one of the most common genetic alterations in urothelial carcinoma (UrCa), and heterogeneity of TP53 mutants leads to heterogeneous clinical outcomes. This study aimed to investigate the clinical relevance of specific TP53 mutations in UrCa. In this study, a total of eight cohorts were enrolled, along with matched clinical annotation. TP53 mutations were classified as disruptive and nondisruptive according to the degree of disturbance of p53 protein function and structure. We evaluated the clinical significance of TP53 mutations in our local datasets and publicly available datasets. The co-occurring events of TP53 mutations in UrCa, along with their therapeutic indications, functional effects, and the tumor immune microenvironment, were also investigated. TP53 mutations were identified in 49.7% of the UrCa patients. Within this group, 25.1% of patients carried TP53Disruptive mutations, a genetic alteration correlated with a significantly poorer overall survival (OS) when compared to individuals with TP53Nondisruptive mutations and those with wild-type TP53. Significantly, patients with TP53Disruptive mutations exhibit an increased probability of responding favorably to PD-1/PD-L1 blockade and chemoimmunotherapy. Meanwhile, there was no noteworthy distinction in OS among patients with varying TP53 mutation status who underwent chemotherapy. Samples with TP53Disruptive mutations showed an enriched APOBEC- and POLE-related mutational signature, as well as an elevated tumor mutation burden. The sensitivity to immunotherapy in tumors carrying TP53Disruptive mutation may be attributed to the inflamed tumor microenvironment characterized by increased CD8+ T cell infiltration and interferon-gamma signaling activation. In conclusion, UrCa patients with TP53Disruptive mutations have shown reduced survival rates, yet they may respond well to PD-1/PD-L1 blockade therapy and chemoimmunotherapy. By distinguishing specific TP53 mutations, we can improve risk stratification and offer personalized genomics-guided therapy to UrCa patients. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Methylthioadenosine Phosphorylase Genomic Loss in Advanced Gastrointestinal Cancers

Abstract Background One of the most common sporadic homozygous deletions in cancers is 9p21 loss, which includes the genes methylthioadenosine phosphorylase (MTAP), CDKN2A, and CDKN2B, and has been correlated with worsened outcomes and immunotherapy resistance. MTAP-loss is a developing drug target through synthetic lethality with MAT2A and PMRT5 inhibitors. The purpose of this study is to investigate the prevalence and genomic landscape of MTAP-loss in advanced gastrointestinal (GI) tumors and investigate its role as a prognostic biomarker. Materials and Methods We performed next-generation sequencing and comparative genomic and clinical analysis on an extensive cohort of 64 860 tumors comprising 5 GI cancers. We compared the clinical outcomes of patients with GI cancer harboring MTAP-loss and MTAP-intact tumors in a retrospective study. Results The prevalence of MTAP-loss in GI cancers is 8.30%. MTAP-loss was most prevalent in pancreatic ductal adenocarcinoma (PDAC) at 21.7% and least in colorectal carcinoma (CRC) at 1.1%. MTAP-loss tumors were more prevalent in East Asian patients with PDAC (4.4% vs 3.2%, P = .005) or intrahepatic cholangiocarcinoma (IHCC; 6.4% vs 4.3%, P = .036). Significant differences in the prevalence of potentially targetable genomic alterations (ATM, BRAF, BRCA2, ERBB2, IDH1, PIK3CA, and PTEN) were observed in MTAP-loss tumors and varied according to tumor type. MTAP-loss PDAC, IHCC, and CRC had a lower prevalence of microsatellite instability or elevated tumor mutational burden. Positive PD-L1 tumor cell expression was less frequent among MTAP-loss versus MTAP-intact IHCC tumors (23.2% vs 31.2%, P = .017). Conclusion In GI cancers, MTAP-loss occurs as part of 9p21 loss and has an overall prevalence of 8%. MTAP-loss occurs in 22% of PDAC, 15% of IHCC, 8.7% of gastroesophageal adenocarcinoma, 2.4% of hepatocellular carcinoma, and 1.1% of CRC and is not mutually exclusive with other targetable mutations.

POLE variant phenotypic classification to enable identification of patients with colorectal cancer (CRC) who may benefit from immune checkpoint inhibition (ICI).

194 Background: Pathogenic POLE (p POLE) variants interfere with the exonuclease activity of POLε during DNA replication, resulting in somatic ultramutation and conferring susceptibility to ICI. Historical classification of p POLE relies on localization to the exonuclease domain (ED). We present the landscape of p POLE variants in CRC according to a proposed phenotypic classification model. Methods: Comprehensive genomic profiling was performed on tissue (TBx) and liquid (LBx) biopsies during the course of clinical care. A POLE-specific variant classification model including tumor mutational burden (TMB), mutational signatures, and germline frequency was used to differentiate p POLE from benign POLE (b POLE) variants. Results: POLE variant status was evaluated in 455,965 TBx and 41,804 LBx pan-tumor. POLE alterations, including p POLE and variants of unknown significance, were detected in 3.9% of samples with 7,404 discrete alleles observed. After applying the classification model, 30 variants were classified as p POLE, representing 3.6% (751/20,601) of observed POLE variants. All p POLE were localized to the ED and a majority were missense substitutions (29/30). There were an additional 620 ED variants categorized as b POLE. CRC had the second highest prevalence of p POLE variants (0.5%, 302/60,547). p POLE variants were associated with ultramutation in CRC where the median tissue TMB was 183.0 mut/Mb, 1-2 orders of magnitude higher than for CRC harboring b POLE variants (7.8 mut/Mb) or without POLE variants (3.5 mut/Mb). 32.2% of p POLE CRC also had evidence of high microsatellite instability (MSI-H). Combined p POLE + MSI-H was associated with markedly elevated TMB compared to either signature alone (344.4 versus 53.1 and 151.3 mut/Mb for b POLE + MSI-H and p POLE + MS-Stable, respectively). A small minority of CRC tumors with p POLE variants (7/298, 2.3%) had low TMB (<10 mut/Mb). However, this was associated with a low p POLE variant allele frequency (VAF; median 2.8% versus 24.0% for tumors w/ TMB ≥10, p < .0001) pointing to low tumor purity and likely an underestimation of TMB. While targetable alterations (e.g., homologous recombination repair [HR] gene variants) were enriched in p POLE tumors, these tumors had a low rate of HR deficiency (HRD) as determined by a computational algorithm (HRDsig), suggesting these are likely incidental monoallelic variants. Conclusions: A POLE phenotype-specific classification model identified p POLE variants associated with ultramutation in CRC. Co-occurrence of p POLE and MSI-H in CRC is common and has a synergistic effect leading to even higher TMB. We hypothesize that p POLE detection predicts ultramutation and response to ICI even when TMB is under-detected due to low tumor purity. Caution is advised for therapy selection based on co-alterations in the setting of p POLE due to an abundance of incidental variants.

Development of a novel disulfidptosis-related lncRNA signature for prognostic and immune response prediction in clear cell renal cell carcinoma

Disulfidptosis, a novel form of regulated cell death, occurs due to the aberrant accumulation of intracellular cystine and other disulfides. Moreover, targeting disulfidptosis could identify promising approaches for cancer treatment. Long non-coding RNAs (lncRNAs) are known to be critically implicated in clear cell renal cell carcinoma (ccRCC) development. Currently, the involvement of disulfidptosis-related lncRNAs in ccRCC is yet to be elucidated. This study primarily dealt with identifying and validating a disulfidptosis-related lncRNAs-based signature for predicting the prognosis and immune landscape of individuals with ccRCC. Clinical and RNA sequencing data of ccRCC samples were accessed from The Cancer Genome Atlas (TCGA) database. Pearson correlation analysis was conducted for the identification of the disulfidptosis-related lncRNAs. Additionally, univariate Cox regression analysis, Least Absolute Shrinkage and Selection Operator Cox regression, and stepwise multivariate Cox analysis were executed to develop a novel risk prognostic model. The prognosis-predictive capacity of the model was then assessed using an integrated method. Variation in biological function was noted using GO, KEGG, and GSEA. Additionally, immune cell infiltration, the tumor mutational burden (TMB), and tumor immune dysfunction and exclusion (TIDE) scores were calculated to investigate differences in the immune landscape. Finally, the expression of hub disulfidptosis-related lncRNAs was validated using qPCR. We established a novel signature comprised of eight lncRNAs that were associated with disulfidptosis (SPINT1-AS1, AL121944.1, AC131009.3, AC104088.3, AL035071.1, LINC00886, AL035587.2, and AC007743.1). Kaplan–Meier and receiver operating characteristic curves demonstrated the acceptable predictive potency of the model. The nomogram and C-index confirmed the strong correlation between the risk signature and clinical decision-making. Furthermore, immune cell infiltration analysis and ssGSEA revealed significantly different immune statuses among risk groups. TMB analysis revealed the link between the high-risk group and high TMB. It is worth noting that the cumulative effect of the patients belonging to the high-risk group and having elevated TMB led to decreased patient survival times. The high-risk group depicted greater TIDE scores in contrast with the low-risk group, indicating greater potential for immune escape. Finally, qPCR validated the hub disulfidptosis-related lncRNAs in cell lines. The established novel signature holds potential regarding the prognosis prediction of individuals with ccRCC as well as predicting their responses to immunotherapy.

Potential impact of cuproptosis-related genes on tumor immunity in esophageal carcinoma.

Cuproptosis involves a direct interaction with the tricarboxylic acid (TCA) lipid acylation components. This process intricately intersects with post-transcriptional lipid acylation (LA) and is linked to mitochondrial respiration and LA metabolism. Copper ions form direct bonds with acylated DLAT, promoting DLAT oligomerization, reducing Fe-S cluster proteins, and inducing a protein-triggered toxic stress response that culminates in cell demise. Simultaneously, the importance of immune contexture in cancer progression and treatment has significantly increased. We assessed the expression of cuproptosis-related genes (CRGs) across TCGA and validated our findings using the GEO data. Consensus clustering divided esophageal cancer (ESCA) patients into two clusters based on the expression of 7 CRGs. We evaluated the expression of immune checkpoint inhibitor (ICI) targets and calculated the elevated tumor mutational burden (TMB). Weighted gene co-expression network analysis (WGCNA) identified genes associated with the expression of CRGs and immunity. Cluster 1 exhibited increased immune infiltration, higher expression of ICI targets, higher TMB, and a higher incidence of deficiency in mismatch repair-microsatellite instability-high status. WGCNA analysis identified 14 genes associated with the expression of CRGs and immune scores. ROC analysis revealed specific hub genes with strong predictive capabilities. The expression levels of SLC6A3, MITD1, and PDHA1 varied across different pathological stages; CCS, LIPT2, PDHB, and PDHA1 showed variation in response to radiation therapy; MITD1 and PDHA1 exhibited differences related to the pathological M stages of ESCA. CRGs influence the immune contexture and can potentially transform cold tumors into hot tumors in ESCA patients.

Genomic Profiles and Clinical Outcomes of Penile Squamous Cell Carcinoma With Elevated Tumor Mutational Burden.

Tumor mutational burden (TMB) is a putative biomarker of efficacy for immune checkpoint inhibitor (ICI) therapies of solid tumors, but not specifically for penile squamous cell carcinoma (PSCC). To characterize biomarker features and ICI therapy outcomes associated with high TMB in PSCC in the routine clinical practice setting. In this cohort study, 397 PSCC cases were analyzed to identify genomic alterations in more than 300 cancer-associated genes and genomic signatures, including TMB, using a hybrid capture-based comprehensive genomic profiling assay. Tumor mutational burden was categorized as low (<10 mutations per megabase [mut/Mb]), high (10-19 mut/Mb), or very high (≥20 mut/Mb). Germline status of genetic alterations was predicted using a validated somatic-germline computational method. Clinical outcomes of patients with metastatic PSCC receiving first-line ICI were abstracted using the deidentified nationwide Clinico-Genomic Database (CGDB) from January 1, 2011, through December 31, 2022. Comprehensive genomic profiling was performed using FoundationOne and FoundationOne CDx assays from Foundation Medicine Inc. The spectrum of genetic alterations by TMB level in PSCC, the percentage of germline genetic alterations, and the outcome (overall survival with routine clinical treatment) by TMB of chemotherapy-naive patients with PSCC who received ICI treatment up front were assessed in this descriptive study. Among 397 patients (median [IQR] age, 65 [54-73] years; 266 [67.0%] of European, 83 [20.9%] of admixed American, and 34 [8.5%] of African or other genomic ancestry), the median (IQR) age (eg, 65 [53-73] years for low TMB vs 68 [61-78] years for TMB ≥10 mut/Mb) and genomic ancestry distribution (eg, European 228 of 339 [67.3%] for low TMB vs 38 of 58 [65.5%] for TMB ≥10 mut/Mb) were similar between TMB subgroups. There were 339 PSCC cases (85.4%) with low TMB, 40 cases (10.1%) with high TMB, and 18 cases (4.5%) with very high TMB. Comparisons of TMB of 10 mut/Mb or higher vs low TMB showed an enrichment of genetic alterations in PIK3CA (48.3% vs 18.3%; P < .001) and KMT2D (29.3% vs 7.7%; P < .001) and less frequent genetic alterations in CDKN2A (25.9% vs 45.7%; P = .05). Most genetic alterations did not co-occur. Human papillomavirus identification was more frequent as TMB increased: 28.3% for low TMB, 50.0% for high, and 72.2% for very high. In total, 95 of 1377 genetic alterations (6.9%) were germline. Of 10 patients identified from the CGDB receiving frontline ICIs, median (IQR) follow-up was 9.9 months. Four patients had overall survival with clinical treatment of more than 12 months, including 2 of 3 patients with TMB of 10 mut/Mb or higher. In this cohort study of advanced metastatic PSCC based on TMB levels, significant differences were observed for biomarkers in nearly 15% of patients with a TMB of 10 mut/Mb or higher. Germline testing and ICI-based therapy should be integrated into the management of selected PSCC cases.

Challenges and Future Directions in the Management of Tumor Mutational Burden-High (TMB-H) Advanced Solid Malignancies.

A standardized assessment of Tumor Mutational Burden (TMB) poses challenges across diverse tumor histologies, treatment modalities, and testing platforms, requiring careful consideration to ensure consistency and reproducibility. Despite clinical trials demonstrating favorable responses to immune checkpoint inhibitors (ICIs), not all patients with elevated TMB exhibit benefits, and certain tumors with a normal TMB may respond to ICIs. Therefore, a comprehensive understanding of the intricate interplay between TMB and the tumor microenvironment, as well as genomic features, is crucial to refine its predictive value. Bioinformatics advancements hold potential to improve the precision and cost-effectiveness of TMB assessments, addressing existing challenges. Similarly, integrating TMB with other biomarkers and employing comprehensive, multiomics approaches could further enhance its predictive value. Ongoing collaborative endeavors in research, standardization, and clinical validation are pivotal in harnessing the full potential of TMB as a biomarker in the clinic settings.

PATH-23. CLINICOPATHOLOGIC ANALYSIS OF NOVEL METHYLATION CLUSTERS OF IDH-WILDTYPE DIFFUSE GLIOMAS

Abstract Methylation profiling of brain tumors has revealed novel clusters whose biological and clinical significance is unknown. These include three entities termed by the 12.5 version of the Heidelberg methylation classifier as adult-type diffuse high grade glioma, IDH-wildtype, subtype B (HGG_B), subtype E (HGG_E), and subtype F (HGG_F). We have identified 75 cases belonging to these groups: HGG_B (25 cases, 32% female), HGG_E (40 cases, 42% female), and HGG_F (18 cases, 33% female). Although originally characterized as “adult-type,” we find that 70% of HGG_E cases are pediatric, with a median age at diagnosis of 11 years. HGG_B cases are predominantly seen in young adults, and HGG_F in older adults. The histology varies among methylation classes, with HGG_B cases demonstrating high-grade, predominantly astrocytic, morphology, while HGG_E cases demonstrate high-grade glial histology with distinct nuclear features, and HGG_F cases show highly infiltrative but lower grade glial morphology. Patient outcome data obtained to date shows a collective median survival exceeding 5 years, suggesting re-consideration of the proposed grading for these tumors. MGMT promoter methylation differs across the HGG_B (0%), HGG_E (12%), and HGG_F (0%) subtypes. Mutational analyses show enrichment of TERT promoter mutations in the HGG_F group, while HGG_E samples are enriched for tumors with POLE mutations and elevated tumor mutation burden. HGG_E tumors show epigenetic similarity to pediatric high-grade diffuse gliomas (pedHGG_RTK1A); we therefore propose that HGG_E be considered a pediatric-type HGG. HGG_B tumors show demographic and genomic similarities to high-grade astrocytomas with piloid features, including a subset of cases with CDKN2A/2B loss and ATRX mutation. HGG_F separate into two groups dictated by brain location, and despite the presence of TERT promoter mutations, appear distinct from GBMs. Overall, our work represents an initial effort to understand the morphologic, genomic and clinical characteristics of these previously uncharacterized subtypes of gliomas.

Comprehensive assessment of base excision repair (BER)-related lncRNAs as prognostic and functional biomarkers in lung adenocarcinoma: implications for personalized therapeutics and immunomodulation.

Lung adenocarcinoma (LUAD) is the most prevalent subtype of lung cancer, and comprehending its molecular mechanisms is pivotal for advancing treatment efficacy. This study aims to explore the prognostic and functional significance of base excision repair (BER)-related long non-coding RNAs (BERLncs) in LUAD. A risk score model for BERLncs was developed using the least absolute shrinkage and selection operator regression and Cox regression analysis. Model validation and prognostic evaluation were performed using Kaplan-Meier and receiver-operating characteristic curve analyses. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were conducted to elucidate the potential biological functions of BERLncs. Comparative analyses were carried out to investigate disparities in tumor mutation burden (TMB), immune infiltration, tumor immune dysfunction and exclusion (TIDE) score, chemosensitivity, and immune checkpoint gene expression between the two risk groups. A predictive risk score model comprising 19 BERLncs was successfully developed. Patients were divided into high-risk and low-risk groups according to the median risk score. The high-risk subgroup exhibited significantly inferior overall survival. Functional enrichment analysis revealed pathways associated with lung cancer development, notably the neuroactive ligand-receptor interaction pathway. High-risk patients demonstrated elevated TMB, diminished TIDE scores, and an immunosuppressive tumor microenvironment, while low-risk patients displayed potential benefits from immunotherapy. Additionally, the risk model identified potential anticancer agents. The risk score model based on BERLncs shows promise as a prognostic biomarker for LUAD patients, providing valuable insights for clinical decision-making, therapeutic strategies, and understanding of underlying biological mechanisms.

Adhesion-regulating molecule 1 (ADRM1) can be a potential biomarker and target for bladder cancer

Adhesion-regulating molecule 1 (ADRM1) has been implicated in tumor development, yet its specific role in bladder cancer (BC) remains undefined. This study aimed to elucidate the function of ADRM1 in BC through a combination of bioinformatics analysis and immunohistochemical analysis (IHC). Utilizing R version 3.6.3 and relevant packages, we analyzed online database data. Validation was conducted through IHC data, approved by the Institutional Ethics Committee (Approval No. K20220830). In both paired and unpaired comparisons, ADRM1 expression was significantly elevated in BC tissues compared to adjacent tissues, as evidenced by the results of TCGA dataset and IHC data. Patients with high ADRM1 expression had statistically worse overall survival than those with low ADRM1 expression in TCGA dataset, GSE32548 dataset, GSE32894 dataset, and IHC data. Functional analysis unveiled enrichment in immune-related pathways, and a robust positive correlation emerged between ADRM1 expression and pivotal immune checkpoints, including CD274, PDCD1, and PDCD1LG2. In tumor microenvironment, samples with the high ADRM1 expression contained statistical higher proportion of CD8 + T cells and Macrophage infiltration. Meanwhile, these high ADRM1-expressing samples displayed elevated tumor mutation burden scores and stemness indices, implying potential benefits from immunotherapy. Patients with low ADRM1 expression were sensitive to cisplatin, docetaxel, vinblastine, mitomycin C, and methotrexate. According to the findings from bioinformatics and IHC analyses, ADRM1 demonstrates prognostic significance for BC patients and holds predictive potential for both immunotherapy and chemotherapy responses. This underscores its role as a biomarker and therapeutic target in BC.

Prognostic and tumor microenvironmental feature of clear cell renal cell carcinoma revealed by m6A and lactylation modification-related genes.

Both lactylation and m6A modification have important implications for the development of clear cell renal cell carcinoma (ccRCC), and we aimed to use crosstalk genes of both to reveal the prognostic and immunological features of ccRCC. Our first step was to look for lactylation-related genes that differed between normal and tumor tissues, and then by correlation analysis, we found the genes associated with M6A. Following that, ccRCC subtypes will be identified and risk models will be constructed to compare the prognosis and tumor microenvironment among different subgroups. A nomogram was constructed to predict the prognosis of ccRCC, and in vitro, experiments were conducted to validate the expression and function of key genes. We screened 100 crosstalk genes and identified 2 ccRCC subtypes. A total of 11 prognostic genes were screened for building a risk model. we observed higher immune scores, elevated tumor mutational burden, and microsatellite instability scores in the high-risk group. Therefore, individuals classified as high-risk would derive greater benefits from immunotherapy. The nomogram's ability to predict overall survival with a 1-year AUC of 0.863 demonstrates its significant practical utility. In addition, HIBCH was identified as a potential therapeutic target and its expression and function were verified by in vitro experiments. In addition to developing a precise prognostic nomogram for patients with ccRCC, our study also discovered the potential of HIBCH as a biomarker for the disease.

Validity and utility of blood tumor mutational burden (bTMB) is dependent on circulating tumor DNA (ctDNA) shed: SCRUM-Japan MONSTAR-SCREEN

BackgroundThe tumor mutational burden (TMB) is a genomic biomarker associated with the benefits from immune checkpoint inhibitors (ICIs) cancer therapy. An elevated blood TMB (bTMB) in circulating tumor DNA (ctDNA) represents a compelling non-invasive diagnostic approach; however, the validity and utility of this emerging biomarker across cancer types have not been examined. Patient and methodsThe blood and tissue TMB was measured in a large pan-tumor clinical cohort and the MONSTAR-SCREEN observational study (UMIN000036749) using the FoundationOne Liquid CDx and FoundationOne CDx assays. A subset of the MONSTAR-SCREEN cohort was used to evaluate the association between the bTMB and the efficacy of ICIs therapy. ResultsThe majority of cancer types showed similar prevalence of TMB≥10 mutations/megabase and bTMB≥10. There was high concordance between bTMB and TMB in blood and tissue biopsy from the same patient when the plasma tumor fraction (TF) was at least 1% (Spearman's coefficient 0.74 and > 80% sensitivity to detect TMB-high). High microsatellite instability (MSI-H) was detected by ctDNA with 79% sensitivity when TF was at least 1%, but only 6% sensitivity when TF was <1%. Among patients with bTMB≥14 and elevated TF≥10% treated with ICIs, there was a trend toward a longer progression-free survival and overall survival compared with patients with bTMB<14 and elevated TF≥10% (HR, 0.62 [95%CI, 0.39–0.98]; p = 0.04 and HR, 0.60 [95%CI, 0.34-1.1]; p = 0.05). ConclusionsOur findings suggest that an elevated bTMB is correlated with elevated TMB and represents a pragmatic biomarker for assessing ICIs benefits. The utility of this biomarker is likely to be associated with high TF levels, informing future prospective investigations.