High Tumor Mutational Burden Research Articles

Current management of neurotrophic receptor tyrosine kinase fusion-positive sarcoma: an updated review.

In recent years, pembrolizumab has demonstrated significant efficacy in treating tumors characterized by a high tumor mutational burden and high microsatellite instability. Tropomyosin receptor kinase (TRK) inhibitors have shown considerable efficacy against tumors harboring neurotrophic receptor tyrosine kinase (NTRK) fusion genes, highlighting the growing importance of personalized medicine in cancer treatment. Advanced sequencing technologies enable the rapid analysis of numerous genetic abnormalities in tumors, facilitating the identification of patients with positive biomarkers. These advances have increased the likelihood of providing effective, tailored treatments. NTRK fusion genes are present in various cancer types, including sarcomas, and the TRK inhibitors larotrectinib and entrectinib have been effectively used for these malignancies. Consequently, the treatment outcomes for NTRK fusion-positive tumors have improved significantly, reflecting a shift toward more personalized therapeutic approaches. This review focuses on NTRK fusion-positive sarcomas and comprehensively evaluates their epidemiology, clinical features, and radiological and histological characteristics. We also investigated the treatment landscape, including the latest methodologies involving TRK inhibitors, and discussed the long-term efficacy of these inhibitors, and their optimal order of use. Notably, larotrectinib has demonstrated a high response rate in infantile fibrosarcoma, and its efficacy has been confirmed even in advanced cases. However, further research is warranted to optimize treatment duration and subsequent management strategies. The accumulation of clinical cases worldwide will play a pivotal role in refining the treatment approaches for tumors associated with NTRK fusion genes.

Exploring the role of immunotherapy in the management of follicular cell-derived thyroid cancer.

Anaplastic and poorly differentiated thyroid carcinomas are the two most aggressive forms of thyroid cancers and carry significant morbidity and mortality despite multimodal therapeutic approaches. Anaplastic thyroid carcinoma (ATC) and, to a lesser degree, poorly differentiated thyroid carcinoma (PDTC) have a high tumor mutation burden, and immunologically hot tumor microenvironment when compared to well-differentiated thyroid carcinomas. As such, immunotherapy, and in particular immune checkpoint inhibitors, have been hypothesized to be effective against these cancers. This review aims to explore the biological rationale for immunotherapy in dedifferentiated thyroid carcinomas and to summarize the current evidence underlying this treatment modality.

Assessment of prognosis and responsiveness to immunotherapy in colorectal cancer patients based on the level of immune cell infiltration

ObjectiveTo build a new prognostic risk assessment model based on immune cell co-expression networks for predicting overall survival and evaluating the efficacy of immunotherapy for colon cancer patients.MethodsThe Cancer Genome Atlas (TCGA) database was used to obtain mRNA expression profiling data, clinical information, and somatic mutation data from colorectal cancer patients. The degree of tumor immune cell infiltration of the samples was analyzed using the CIBERSORT algorithm. Co-expression of immune-related genes was analyzed using weighted correlation network analysis (WGCNA) and gene modules were identified. Prognosis-related genes were screened and models were constructed using LASSO-Cox analysis. The models were validated by survival analysis. The prognostic potential of the models was quantitatively assessed using Cox regression analysis and the development of column line plots. Immunotherapy sensitivity analysis was performed using CIBERSORT and TIMER algorithms. Gene biofunction analysis was performed using Gene set enrichment analysis (GSEA) and Gene set variation analysis (GSVA). And the chemotherapeutic response to different drugs was assessed.ResultsWe established a novel prognostic model utilizing the WGCNA method, which demonstrated robust predictive accuracy for patient survival. The high-risk subgroup in our model exhibited elevated immune cell infiltration coupled with a higher tumor mutation burden, but the difference in response to immunotherapy was not significant compared to the low-risk group. Furthermore, we identified distinct chemotherapy responses to 39 drugs between these risk subgroups.ConclusionThis study revealed a significant correlation between high levels of immune infiltration and unfavorable prognosis in patients with colon cancer. Furthermore, an accurate prognostic risk prediction model based on the co-expression of relevant genes by immune cells was developed, enabling precise prediction of survival of colon cancer patients. These findings offer valuable insights for accurate prognostication and comprehensive management of individuals diagnosed with colon cancer.

Molecular profiling of a gastroesophageal adenocarcinoma (GEA) multicohort using next-generation sequencing (NGS).

494 Background: The mutational landscape of GEA is rapidly evolving with the identification of promising molecular targets. However, histological subtypes are insufficient to capture the molecular heterogeneity in these patients (pts). We aim to assess the histopathological and molecular features in a GEA cohort using NGS techniques, and to evaluate the mutational frequency and its prognostic correlation. Methods: We conducted a retrospective study on GEA from 2019 to 2024. Immunohistochemistry (IHC) was used to evaluate HER2 expression, microsatellite instability (MSI), Epstein-Barr virus (EBV) and PD-L1 status. In-house and commercial NGS platforms were used to detect molecular alterations from tumor samples. Cox regression model was used to analyze overall survival (OS) based on the clinicopathological and molecular subgroups. Results: Using NGS techniques, we identified the following alterations with potential clinical relevance in the 115-pts included: pathogenic mutations in PIK3CA (5.2%), BRCA1/2 (3.5%) and POLD1 (0.9%), along with copy number alterations (CNA) in ERBB2 (10.4%), EGFR (8.7%), MET (5.2%) and FGFR2 (4.3%) genes, among others. Additionally, a high tumor mutational burden (≥ 13 Mut/Mb) was observed in 6 pts (5.2%). Clinical characteristics are detailed in the table. We detected a higher rate of alterations in CDH1 (10.42% vs 4.48%; p=0.22) and PIK3CA (8.33% vs 2.99%; p=0.20) genes in early (<50y) vs late-onset (≥50y) GEA. Additionally, we identified a significantly higher mutation frequency in diffuse vs intestinal tumors; ARID1A (17.6% vs 2.2%; p=0.01) and CDH1 (11.8% vs 0%; p=0.02) genes. Also, pts with peritoneal involvement showed a greater prevalence of mutations in CDH1 (12% vs 0%; p=0.04) and RHOA (10% vs 0%; p=0.06) compared to those with liver disease. We observed a significantly worse OS in pts with diffuse vs intestinal tumors (15.7m vs 19.1m; p=0.04) and a trend toward shorter survival in pts with CDKN2A mutations (11.97m vs 17.83m; p=0.42). Although not significantly, CNA in ERBB2 were associated with a better prognosis (22.2m vs 16.2m; p=0.09). Conclusions: Our results support the utility of NGS platforms in detecting novel molecular targets with prognostic and clinical impact, beyond ERBB2 . Additionally, we observed poorer prognosis in diffuse subtype tumors, which exhibited a higher frequency of mutations in ARID1A and CDH1 genes. Clinicopathological characteristics of the 115-pts with GEA. N=115 (%) AgeMedian years [range] 54 [18-83] Sex Male 69 (60.0) Tumor site Gastric 72 (62.6) Gastroesophageal junction 37 (32.2) Esophagus 6 (5.2) Histological subtype Diffuse or pooly cohesive 51 (44.3) Intestinal or tubular 46 (40.0) Others 18 (15.7) Stage at diagnosis Advanced 74 (66.9) Localized 41 (33.1) Metastatic site Peritoneum 53 (46.1) Liver 34 (29.6) Molecular profile (IHC) HER2 (+) 20 (17.4) MSI 5 (4.3) EBV (+) 2 (1.8) PDL1 (CPS) ≥1 35 (30.4)

Immunotherapy outcomes in dMMR/MSI-H and/or TMB-H metastatic colorectal cancer (CRC) with peritoneal metastases (PM).

163 Background: CRC with PM has a poor prognosis and limited treatment options. Immunotherapy has shown promise in improving outcomes for patients with microsatellite instability-high (MSI-H), mismatch repair-deficient (dMMR), and high tumor mutational burden (TMB-H) tumors. However the peritoneal niche has a unique microenvironment, which can influence the effectiveness of immune check point inhibitors (ICIs). We assessed the impact of ICI on progression-free survival (PFS) and overall survival (OS) in CRC patients (pts) and PM, considering key clinical and molecular characteristics. Methods: A retrospective review aiming to identify CRC pts with MSI-H/dMMR and PM treated with immunotherapy between 2015 and 2023 was performed. Data collected included pt demographics, tumor characteristics, and treatment details. Responses were evaluated using RECIST v1.1 criteria. Results: 37 pts were included, 9 (24%) had Lynch syndrome. The median age of diagnosis was 61 years and most pts were females (65%). Most pts had MSI-H/dMMR tumors, while one pt was treated based on ultra-hypermutated phenotype (TMB 202) and POLE mutation. Among those with available TMB data, the median TMB was 52 mutations/Mb (0-202). Common mutations were PMS2 (51%), MLH1 (38%), MSH6 (16.2%), and MSH2 (16.2%). Right-sided tumors were in 54%, mucinous histology in 62%, and ascites in 54%. Pts received a median of 11 cycles of ICI (2-52). Pembrolizumab was the most frequently used ICI (78%), while Nivolumab was used either alone (11%) or in combination with Ipilimumab (11%). Median PFS in our cohort was 7.8 months and median OS was 29.9 months. The disease control rate (DCR) was 81%, with an objective response rate (ORR) of 51.3% (9 complete responses, 12 partial responses). At the time of data collection, 30% of pts were off systemic therapy and 5-FU-based chemotherapy combined with a biologic agent was the most common subsequent treatment in 32% who had PD. Univariate analysis showed no significant differences in PFS or OS based on tumor sidedness, isolated PM, liver metastases, mucinous vs. non mucinous histology or BRAF / RAS status (WT vs mutant). Malignant ascites was associated with worse OS(HR 0.3; p= 0.03). Conclusions: Treatment with ICIs in pts with MSI-H/dMMR and/or TMB-H CRC and PM improved disease control in CRC pts with PM, however, as compared to historical controls for patients with MSI-H/d-MMR disease treated with ICI, this group showed less favorable outcomes. Malignant ascites was associated with poorer overall survival. Further research of the immune response and tumor microenvironment in the peritoneal cavity is warranted.

Personalized oncogenomic analysis of metastatic squamous cell carcinoma of the anus: Utilizing whole-genome sequencing to guide clinical decision-making.

11 Background: Metastatic squamous cell carcinoma of the anus (SCCA) is associated with significant morbidity and mortality. Due to its relative rarity, there is limited evidence to support systemic therapy regimens informed by genomic data. This study aims to utilize whole-genome and transcriptome sequencing to enhance the understanding of the genetic alterations driving metastatic SCCA and to identify potentially actionable therapeutic targets. Methods: This report examines nine cases of metastatic SCCA in patients enrolled in the Personalized Oncogenomics (POG) Program at the BC Cancer Agency. Comprehensive genomic profiling, including whole-genome and transcriptome analysis (WGTA) was conducted on fresh tumor biopsy or formalin fixed paraffin embedded (FFPE) tissue. Tumor genomic alterations were analyzed in detail including: SNVs, indels, copy number alterations, structural variants, mutation signatures, viral presence, tumor mutation burden (TMB), expression outliers, and immune cell scores. The somatic alterations were integrated with existing knowledge of drug-target interactions to identify actionable therapeutic targets. Results: Of the nine patients, eight were found to be HPV-positive, with one exhibiting high tumor mutational burden (TMB>10mut/Mb). Mutation signatures included seven cases with AID/APOBEC signatures (commonly seen with HPV), and one case with an ID2 DNA replication slippage signature. The PI3K/AKT/mTOR pathway was the most commonly affected signaling pathway (n=4), followed by FGFR amplification and overexpression (n=3), and RAS/RAF alterations ( BRAF mutation and KRAS overexpression, n=2). EGFR amplification was identified in two cases, one of which also exhibited EGFR overexpression. Additionally, FBXW7 mutations were present in two cases. Immunotherapy was recommended for all nine patients: eight based on HPV positivity, either alone or in combination with other alterations, such as high TMB, SWI/SNF complex alterations, and presence of immune infiltrating cells, and the remaining case based on a SWI/SNF complex mutation. Only two patients ultimately received immunotherapy, both of whom derived clinical benefit. One patient, treated with atezolizumab as part of the phase II CAPTIV-8 trial (NCT04273061), demonstrated progression-free survival (PFS) of 10 months (ongoing), while the other, treated with nivolumab, achieved a PFS of 6 months. Conclusions: Genomic analysis using WGTA supported the recommendation of immunotherapy for all patients in this cohort. Although only two patients received immunotherapy, both experienced clinical benefit, underscoring the potential of personalized genomic-guided treatment in metastatic SCCA.

Use of gene expression profiling to examine association between MUC1 and claudin 18 in gastrointestinal cancers: Implications for targeted therapies.

838 Background: Gastrointestinal tract cancers exhibit considerable molecular heterogeneity, which requires a detailed understanding of their distinct genetic profiles. Understanding the gene expression of other genes along with Claudin 18 (CLDN18) could provide insights into their interactions. Methods: We conducted a retrospective analysis of NGS profiling on 1,043 GI cancer tissue samples from three cohorts: Upper and Lower Gastrointestinal Cancer Cohort (GICC; n=651), Pancreaticobiliary Cancer Cohort (PBCC; n=316), and Hepatocellular Carcinoma Cohort (HCC; n=76). Immunohistochemistry data for PD-L1, Her2 and MMR proteins, NGS based Tumor mutation burden (TMB) and gene expression profiling (GEP) of 20,802 genes including CLDN18 was examined in a subset by targeted transcriptome profiling. We explored associations of these biomarkers with expression of CLDN18. Results: In GICC, frequent mutations were seen in TP53 (68%; 442), APC (33.5%; 218) and KRAS (33.3%; 217). PBCC, showed mutations in TP53 (62%; 197), KRAS (47%; 149) and CDKN2A (16%; 51). HCC showed mutations in TP53 (42%; 32), ARID1A (18%; 14) and KRAS (9%; 7). Amplifications were predominantly observed in MYC (13%; 120), ERBB2 (5%; 49) and CCND1 (5%; 44). Fusions were infrequent. All MSI-H cases (11/462; 2.4%) were observed in GICC. High TMB (≥10 muts/mb) was seen in 23% (111/481), stratified as 28% (90/318) in GICC, 13% (18/139) in PBCC and 10% (3/30) in HCC. Out of 321 samples analyzed for gene expression profiling, CLDN18 was significantly upregulated in 65 (20%) samples. The expression of CLDN18 did not show statistically significant correlation with Her2 (n=184), PD-L1 (n=231), TMB (n=228), or MSI (n=229); (p>0.05). In subset where both CLDN18 and MUC1 expression were analyzed (n=118), all the CLDN18 positive tumors showed overexpression of MUC1. This finding was statistically significant (p<0.05). Increased expression of MUC1 promotes cancer cell mobility promoting metastases. Expression of MUC1 can play an important role in the development of resistance to chemotherapy. MUC1 expression in cancer cells is associated with avoidance of immune defenses. Thus, this finding of CLDN18 and MUC1 association is especially relevant as the CLDN18 targeted therapies may be the potential avenue for the aggressive and chemo-resistant MUC1 expressing tumors. Conclusions: This study identifies potential association between MUC1 and CLDN18. GEP could enhance biomarker detection and support personalized treatment approaches. Future research should integrate these findings with clinical data to improve precision in GI cancer care.

Molecular and clinical profiling of gastroenteropancreatic (GEP) neuroendocrine tumors (NETs): An analysis of the Oncology Research Information Exchange Network database.

669 Background: The incidence of neuroendocrine tumors (NETs) is approximately 7 per 100,000 persons and rising. By location, tumors in the gastroenteropancreatic (GEP) region, particularly midgut NETs, are most common. The Oncology Research Information Exchange Network (ORIEN) database contains complementary clinical, genomic, and transcriptomic profiling, providing opportunities to identify novel associations between molecular features and clinical outcomes in GEP-NETs. Methods: Survival analyses were performed using the log-rank testing, and clinical features were evaluated using Wilcoxon and chi-squared tests. Mutational analyses utilized sample-level enrichments from whole exome sequencing data, and statistical tests were performed using the one-sided Fisher Exact test. Transcriptomic analyses utilized a student’s t-test. We reviewed 240 samples from 226 patients, with 124 of pancreatic origin, 90 small bowel, 16 gastric, and 10 colorectal. All specimens were well-differentiated; further grading information was unavailable on the ORIEN platform. A p-value <0.05 was considered significant. Results: Patients with metastatic disease were significantly younger at diagnosis (58 vs. 61 years, p=0.0242) and had significantly higher tumor mutational burden (TMB) (0.48 vs. 0.32 mut/Mb, p=0.001). Among the 100 most common alterations across the entire cohort, BPTF (p=0.03) and PRKCA (p=0.007) mutations were more prevalent in pancreatic NETs than in other primary sites. The most frequently mutated genes identified included TTN (27.7%), MUC16 (25.1%), CCDC168 (22.1%), KIR3DL1 (20%), TGIF2LX (20%), MUC17 (19.1%). TTN mutation was significantly associated with higher TMB (p<0.0001). The most common copy number alteration was MUC3a amplification (7q22.1) in 61.5% of samples. This amplification was associated with more prolonged overall survival (OS) with a significance of p=0.0501. Patients with NETs harboring a mutation in CSNK2A2 (p=0.0149), WDR74 (p=0.0386), or NCMAP (p=0.0248) experienced significantly shorter OS compared to the cohort as a whole. Conclusions: We report the first clinical, genomic, and transcriptomic analysis of ORIEN GEP-NET cases. These findings create multiple avenues for further investigation and reinforce the value of multi-institutional consortia such as ORIEN in deepening our knowledge of well-differentiated NETs. Most prevalent mutations in GEP-NETs from ORIEN database. Mutation Prevalence (Percentage of Samples) TTN 27.7% MUC16 25.1% CCDC168 22.1% KIR3DL1 20.0% TGIF2LX 20.0% MUC17 19.1%

Biological and therapeutic impact of tumor mutational burden in microsatellite instability/mismatch repair-deficient (MSI/dMMR) metastatic colorectal cancer (mCRC) treated with immune checkpoint inhibitors (ICI).

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.

Clinical and genetic drivers of oligo-metastatic disease in colon cancer.

Oligo-metastatic disease (OMD) in colon cancer patients exhibits distinct clinical behavior compared to poly-metastatic disease (PMD), with a more responsive and indolent course. This study aims to identify clinical and biological factors uniquely associated with oligo-metastatic behavior. Metastatic colon cancer patients from an academic center underwent genetic characterization. OMD was defined as ≤3 lesions per organ, each with a total diameter <70 mm and none exceeding 25 mm. Tumor DNA sequencing by NGS utilized the TruSight Oncology 500 kit. Overall survival (OS) was determined from metastasis diagnosis until death using Kaplan-Meier analysis. Multivariate Cox regression examined prognostic links between clinicopathological and genetic factors. Associations with metastatic patterns were evaluated using Chi-square. The analysis involved 104 patients (44 with OMD, 60 with PMD). OMD was more prevalent in males (P = 0.0299) and with single organ involvement (P = 0.0226). Multivariate analysis adjusted for age (>70 vs. <70 years), gender (male vs. female), tumor side (right vs. left), metastatic involvement (more than one site vs. one site), response to first-line therapy (disease control vs. no disease control), and RAS/BRAF variants (wild-type vs. mutated) identified OMD vs. PMD as the strongest independent predictor of survival (HR: 0.14; 95 % CI: 0.06-0.33; P<0.0001). OMD patients exhibited distinct molecular characteristics, including lower frequencies of BRAF p.V600E (P=0.0315) and KRAS mutations (P=0.0456), as well as a higher frequency of high tumor mutational burden (P=0.0127). Additionally, by integrating data from public datasets and our case study, we hypothesize that some gene alterations (i.e.: BRAF, SMAD4, RAF1, and mTOR) may prevent OMD occurrence. OMD, characterized by male predominance, single-site involvement, and distinct molecular features in colon cancer, suggests the need for tailored management strategies.

Pan-cancer microsatellite instability testing using a multiplex assay including long mononucleotide repeats.

290 Background: Microsatellite instability (MSI)/mismatch repair deficiency (dMMR) testing is a critical component of molecular testing for gastrointestinal cancers, among others. These analyses are important for the identification of patients with lynch syndrome and eligibility for treatment with immunotherapy. The current standard of care testing can identify most cases, however improved identification of MSI-H status is needed for non-colorectal cancers and for cancers with alterations outside MLH1/PMS2. A recent analysis indicated the potential for improved MSI-H detection with a multiplex assay including long mononucleotide repeats (LMR). Methods: Patients were selected based on MSI status, MMR gene variants, or the presence of a high tumor mutation burden and consented for participation as part of an IRB-approved protocol (UW15068). Formalin-fixed tissue slides were annotated by a pathologist for tumor and normal tissue. Slides were scraped and DNA isolated. The LMR MSI Analysis System (Promega, Madison, WI) and the OncoMate MSI Dx Analysis System (Promega) were performed on cancer DNA and normal tissue DNA when available per the manufacturer’s protocols. Instability of microsatellites and the MSI score were reported and correlated with clinical MMR immunohistochemistry (IHC) and commercial next-generation sequencing (NGS). Results: A total of 96 cancer samples were tested across 94 patients with over 20 different cancer types, including lung, colon, rectal, gynecologic, and esophagogastric cancers. Tumors from 20 patients were known to have microsatellite instability based on clinical NGS testing. 27 cases were found to be MSI-H based on the OncoMate assay. All of these cases were also MSI-H per the LMR assay and an additional 3 cases were found to be MSI-H using only the LMR assay. Of these 3 cases all were microsatellite stable by NGS. This number includes 1 colorectal and 2 esophagogastric cancer cases. One of the cases was found to have loss of MSH6 by IHC with an MSH6 mutation (MSH6 L1061fs). One of the cases was MMR intact by IHC, though did have a MSH6 exon 7 splice acceptor alteration that failed NGS calling quality control. The last case did not have MMR IHC performed, no detected MMR gene alterations, and a tumor mutation burden of 6.3 mutations per megabase. Conclusions: In this limited dataset, the utility of various methods for determination of MSI, dMMR, and TMB including PCR, IHC, and NGS were demonstrated using a patient cohort spanning solid tumor types. Promising results were observed with the LMR MSI Analysis System. These findings need to be further validated in a larger dataset and correlated with the response to immunotherapeutics.

Evaluating Tumour Mutational Burden as a Key Biomarker in Personalized Cancer Immunotherapy: A Pan-Cancer Systematic Review

Background: Tumour mutational burden (TMB) is an emerging biomarker for predicting the efficacy of immune checkpoint inhibitors (ICIs) in cancer therapy. While its role is well established in lung cancer and melanoma, its predictive value for breast and prostate cancers remains unclear. Objective: This systematic review aimed to assess the predictive value of TMB for ICI therapy across four major cancer types—lung, melanoma, breast, and prostate—and to explore factors contributing to the variability in its effectiveness as a biomarker. Methods: A systematic search and a review of the literature were conducted in accordance with PRISMA guidelines. Studies examining the relationship between TMB levels and clinical outcomes following ICI therapy in the specified cancers were analyzed. The data were synthesized to evaluate TMB’s predictive value and identify gaps in the current research. Results: High TMB consistently correlated with improved outcomes in lung cancer and melanoma, confirming its predictive utility in these cancers. Conversely, the findings for breast and prostate cancers were inconclusive. The variability in TMB’s predictive value for these cancers suggests the need for complementary biomarkers or refined criteria to enhance its reliability. Methodological inconsistencies in TMB evaluation were also noted as a significant limitation. Conclusions: TMB serves as a robust biomarker for predicting ICI response in lung cancer and melanoma, but demonstrates limited predictive utility in breast and prostate cancers. Future research should prioritize standardizing TMB assessment protocols and investigating additional biomarkers to improve treatment personalization for these cancer types.

Genomic profiles of patients with skin melanoma in the era of immune checkpoint inhibitors.

The use of immune checkpoint inhibitors (ICIs) for treating melanoma has dramatically improved patient prognosis. The genomic profiles of patients receiving ICI therapy would provide valuable information for disease management and treatment. We investigated the genomic profiles of patients with melanoma who had received ICI therapy and explored associations with clinical features and outcomes via a large-scale nationwide database in Japan (the C-CAT database). We identified 339 patients eligible for this study. The most frequent genetic mutations were found in the BRAF (27%), TERT (24%), and NRAS (19%) genes, and the most common copy number variations (CNVs) were in the CDKN2A (36%), CDKN2B (26%), and MTAP (19%) genes. Associations with high tumor mutational burden (TMB-high) status were significant for TERT (p < 0.001), NF1 (p < 0.001), ROS1 (p = 0.015), POLE (p = 0.045), and POLD1 (p = 0.008) mutations, along with older age (≥65 years, p = 0.036). Patients with multiple metastases (two or more) were more likely to have NOTCH3 mutations (p = 0.017) and be younger than 65 years (p = 0.024). In particular, as well as younger age, patients with brain metastases were more likely to harbor BRAF mutations (p < 0.001), while those with liver metastases were more likely to harbor NOTCH3 mutations (p < 0.001) but not CDKN2B CNVs (p = 0.041). Patients with NRAS mutations were less likely to respond to ICI therapy (p = 0.014) and exhibited shorter overall survival (p = 0.006). In this population, the frequency of BRAF mutations was lower than that in fair-skinned populations, but the associations between genomic profiles, clinical features, and outcomes were similar to those previously reported in fair-skinned populations.

Establishment and verification of a prognostic signature associated with fatty acid metabolism in endometrial cancer.

Endometrial carcinoma (EC) is one of the leading causes of mortality in women. Metabolic disorders, such as abnormal fatty acid metabolism (FAM), are considered to be indicators of tumorigenesis. However, to the best of our knowledge, the relationship between EC and FAM remains unclear. The process of FAM is associated with the function of immune cells, thus samples from The Cancer Genome Atlas were grouped according to immune infiltration levels. Subsequently, prognostic gene signatures were constructed based on selected FAM‑associated genes. The signature effect was validated, and enrichment analyses were conducted based on sample classification. Nomograms were used to predict survival, merging clinical data and the gene signature. Samples were divided into high‑ and low‑risk groups based on the gene signature. The survival status, clinical characteristics, enrichment analysis and immune infiltration were significantly different between high‑ and low‑risk groups. According to the nomogram, low microsatellite instability‑high as well as a high tumor mutation burden can be observed in the low‑nomo‑score group. Immune checkpoint inhibitor‑associated genes were differentially expressed between groups and 35 sensitive compounds were identified. Comprehensive bioinformatics analysis in EC revealed potential roles of FAM in tumorigenesis, the tumor microenvironment and prognosis, suggesting that FAM‑associated signatures are promising biomarkers for EC. These findings may improve the understanding of FAM in EC and pave the way for a more accurate assessment of prognosis and immunotherapy outcomes.

Mutation Analysis of TMB-High Colorectal Cancer: Insights Into Molecular Pathways and Clinical Implications.

Colorectal cancer (CRC) is well characterized in terms of genetic mutations and the mechanisms by which they contribute to carcinogenesis. Mutations in APC, TP53, and KRAS are common in CRC, indicating key roles for these genes in tumor development and progression. However, for certain tumors with low frequencies of these mutations that are defined by tumor location and molecular phenotypes, a carcinogenic mechanism dependent on BRAF mutations has been proposed. We here analyzed targeted sequence data linked to clinical information for CRC, focusing on tumors with a high tumor mutation burden (TMB) in order to identify the characteristics of associated mutations, their relations to clinical features, and the mechanisms of carcinogenesis in tumors lacking the major driver oncogenes. Analysis of overall mutation frequencies confirmed that APC, TP53, and KRAS mutations were the most prevalent in our cohort. Compared with other tumors, TMB-high tumors were more frequent on the right side of the colon, had lower KRAS and higher BRAF mutation frequencies as well as a higher microsatellite instability (MSI) score, and showed a greater contribution of a mutational signature associated with MSI. Ranking of variant allele frequencies to identify genes that play a role early in carcinogenesis suggested that mutations in genes related to the DNA damage response (such as ATM and POLE) and to MSI (such as MSH2 and MSH6) may precede BRAF mutations associated with activation of the serrated pathway in TMB-high tumors. Our results thus indicate that TMB-high tumors suggest that mutations of genes related to mismatch repair and the DNA damage response may contribute to activation of the serrated pathway in CRC.

Clinical, Morphologic, and Genomic Differences in Deep Penetrating Nevi and Deep Penetrating Nevus-like Melanomas.

Deep penetrating nevi (DPNs) are characterized by activating mutations in the MAP kinase and Wnt/beta-catenin pathways that result in large melanocytes with increased nuclear atypia, cytoplasmic pigmentation, and often mitotic activity. Together with a lack of maturation, this constellation of findings creates challenges for pathologists to distinguish deep penetrating nevus (DPN) from DPN-like melanoma. To assess the utility of next generation sequencing (NGS) in resolving this diagnostic dilemma, we performed NGS studies on 35 lesions including 24 DPNs and 11 DPN-like melanomas to characterize the specific genomic differences between the two groups and elucidate the genetic events involved in malignant transformation of DPNs. Compared to DPNs, DPN-like melanomas were clinically larger in size (1.1 vs 0.6 cm, p=0.02) and on histopathologic examination more frequently showed high grade nuclear atypia (11/11 vs 9/24; p=0.00052), increased mitotic activity (mean 3.9 vs 1.3 per mm2; p=0.0004), sheet-like growth pattern (5/11 vs 2/24; p=0.01), and involvement of the subcutis (4/5 vs 3/13; p=0.026). From a genomic standpoint, DPN-like melanomas had a higher tumor mutation burden (mean 37.1 vs 7.8 mutations/megabase; p=0.002) than DPNs and more frequently harbored NRAS mutation (3/11 vs 1/24; p=0.046) whereas MAP2K1 in frame deletions were only identified in DPNs (0/11 vs 5/24). There was no statistically significant difference in the frequency or type of CTNNB1 or APC mutations between the two groups. Within progression genes, DPN-like melanomas were more frequently found to have pathogenic variants in TERT promoter (7/11 vs 0/24; p<0.00001), CDKN2A (4/11 vs 0/24; p=0.0008), and protein subunits of the SWI/SNF complex (7/11 vs 3/24; p=0.02) compared to DPNs. Our findings provide a framework for employing NGS in the evaluation of deep penetrating melanocytic tumors.

VACCIMEL, an allogeneic melanoma vaccine, efficiently triggers T cell immune responses against neoantigens and alloantigens, as well as against tumor-associated antigens.

VACCIMEL is a therapeutic cancer vaccine composed of four irradiated allogeneic human melanoma cell lines rationally selected to cover a wide range of melanoma tumor-associated antigens (TAA). We previously demonstrated that vaccination in the adjuvant setting prolonged the distant-metastasis-free survival of cutaneous melanoma patients and that T cells reactive to TAA and the patient's private neoantigens increased during treatment. However, immune responses directed to vaccine antigens that may arise from VACCIMEL's somatic mutations and human polymorphisms remain unexplored. To study these immunogens, we performed whole-exome sequencing of paired tumor and germinal samples from four vaccinated patients and the vaccine cells. VACCIMEL variants were called by comparing the vaccine and the patient's exomes, and non-synonymous coding variants were used to predict T cell epitopes. Candidates were ranked based on their mRNA expression in VACCIMEL, predicted peptide-HLA (pHLA) presentation, and pHLA stability. Then, the immune responses to prioritized epitope candidates were tested using IFNγ ELISpot assays on vaccinated patients' PBMC samples. The comparison of the vaccine with the patients' germinal exomes revealed on average 9481 coding non-synonymous variants, suggesting that VACCIMEL offers a high number of potential antigens. Between 0,05 and 0,2% of these variants were also found in the tumors of three vaccinated patients; however, one patient with a high tumor mutational burden (TMB) shared 19,5% somatic variants. The assessment of T cell responses showed that vaccinated patients mounted highly diverse responses against VACCIMEL peptides. Notably, effector T cells targeting the patient's tumor antigens, comprising neoantigens and TAA, were found in higher frequencies than T cells targeting VACCIMEL-exclusive antigens. On the other hand, we observed that the immunogenic epitopes are not conserved across patients, despite sharing HLA and that immune responses fluctuate over time. Finally, a positive correlation between VACCIMEL antigen expression and the intensity of the T cell responses was found. Our results demonstrate that the immune system simultaneously responds to a high number of antigens, either vaccinal or private, proving that immune responses against epitopes not expressed in the patient's tumors were not detrimental to the immune recognition of neoantigens and TAA.

Evolving Strategies in the Management of Microsatellite Instability-High/Mismatch Repair Deficient Esophagogastric Adenocarcinoma.

This review addresses the current treatment paradigm and new advancements in the management of microsatellite instability-high/mismatch repair deficient (MSI-H/dMMR) esophagogastric cancer (EGC). While chemotherapy and surgery remain the cornerstone of EGC treatment, MSI-H/dMMR tumors harbor high tumor mutational burden and represent a subset of patients who benefit from immune checkpoint inhibitors (ICI). ICI has been incorporated in the front line setting with and without chemotherapy for advanced disease. Recently, ICI has been studied in the perioperative setting for resectable disease. Though perioperative ICI results in improved response rates, it is not yet clear whether this translates to a survival benefit. Despite high response rates with ICI in this patient population, many do not respond to therapy, representing a major challenge in treatment. Preclinical studies have highlighted potential mechanisms of resistance which will guide drug development and clinical trials.

Ovarian squamous cell carcinoma: clinicopathological features, prognosis and immunotherapy outcomes.

To explore the characteristics and survival outcomes of ovarian squamous cell carcinoma (SCC) and the treatment effectiveness of immune checkpoint inhibitors (ICIs). Patients diagnosed with ovarian SCC at Peking Union Medical College Hospital between January 2000 and September 2023 were included. Overall survival (OS) and progression-free survival (PFS) were analyzed using the Kaplan-Meier method. Univariate and multivariate analysis of OS were performed using the Cox proportional hazards model. A total of 42 patients were included, with a median age of 51.5 years (range, 23-74). The majority had SCC arising from teratomas (54.8%), followed by endometriosis (14.3%) and Brenner's tumor (2.4%). Patients undergoing molecular testing exhibited a median tumor mutation burden (TMB) of 10.00 mutations/Mb (range, 7.28-46.86), predominantly featuring PIK3CA mutations. Thirty-eight patients (90.5%) received adjuvant chemotherapy. The median OS was 42.0 months, with the 1- and 5-year OS rates were 73.7% and 48.7%, respectively. And the median PFS was 26.9 months, with the 1- and 5-year PFS rates were 57.5% and 43.8%, respectively. Five patients underwent first-line postoperative adjuvant therapy combining ICIs with chemotherapy. During the 9.5 to 25.1 months follow-up, 4 patients showed no evidence of disease, while 1 relapsed and received treatment. Late-stage disease and younger age at diagnosis were associated with worse survival outcomes. The prognosis for ovarian SCC remains unfavorable. The stage and age were prognostic predictors for survival. ICIs may be beneficial for patients with ovarian SCC, particularly those with a high TMB.

Efficacy of pembrolizumab in microsatellite-stable, tumor mutational burden-high metastatic colorectal cancer: genomic signatures and clinical outcomes.

Pembrolizumab, an immune checkpoint inhibitor (ICI), shows significant survival benefits in patients with microsatellite instability-high (MSI-H) metastatic colorectal cancer (mCRC), but its efficacy in microsatellite-stable (MSS) mCRC is limited. Although ICIs are effective in tumor mutational burden-high (TMB-H) solid tumors, the impact on MSS-TMB-H mCRC, a rare subset within MSS mCRC, remains unclear. We conducted a retrospective analysis using clinical and genomic data from the Center for Cancer Genomics and Advanced Therapeutics (C-CAT) repository in Japan. Patients with MSS-TMB-H mCRC who underwent tissue-based comprehensive genomic profiling and were treated with pembrolizumab or other later-line therapies were included. Pembrolizumab's efficacy was compared with that of trifluridine/tipiracil (FTD/TPI) and regorafenib. Genomic profiles of MSS-TMB-H, MSI-H-TMB-H, and MSS-TMB-low (TMB-L) CRCs were analyzed across 71 cancer-related genes. Among 127 TMB-H mCRC cases treated with pembrolizumab in the C-CAT repository, 77 were MSS and 50 were MSI-H. Pembrolizumab showed significantly shorter time to treatment failure (TTF) and overall survival (OS) in patients with MSS-TMB-H mCRC compared with those with MSI-H-TMB-H mCRC [median TTF 2.0 versus 10.6 months; hazard ratio (HR) 4.79, 95% confidence interval (CI) 2.65-8.64, median OS 4.5 versus 33.6 months; HR 9.86, 95% CI 3.93-24.77, both P < 0.0001]. Among MSS-TMB-H mCRC patients, 19 received pembrolizumab, 73 received FTD/TPI (±bevacizumab), and 18 received regorafenib as their first later-line therapy. Pembrolizumab showed significantly shorter TTF and OS compared with FTD/TPI (median TTF 1.6 versus 4.1 months; HR 2.66, 95% CI 1.41-5.02, P= 0.0017, median OS 5.4 versus 13.8 months; HR 2.42, 95% CI, 1.09-5.38, P= 0.025). Genomic analysis of 6737 CRCs revealed that MSS-TMB-H CRCs harbored fewer pathogenic alterations than MSI-H-TMB-H CRCs but had a profile similar to MSS-TMB-L CRCs. Pembrolizumab may be less effective than FTD/TPI in later-line treatment of MSS-TMB-H mCRC, potentially due to genomic similarities between MSS-TMB-H and MSS-TMB-L CRC, suggesting the need for alternative therapeutic strategies in this subgroup.