Low Tumor Mutation Burden Group Research Articles

A novel panel based on immune infiltration and tumor mutational burden for prognostic prediction in hepatocellular carcinoma.

Tumor mutation burden (TMB) has been associated with prognosis in various malignancies, but it has yet to be elucidated in hepatocellular carcinoma (HCC). We aimed to investigate the prognostic effects of TMB and its relationship with immune infiltration through multiple databases and whole-exome sequencing, so as to establish a panel model capable of predicting prognosis. The results demonstrated that the prognosis of high TMB group was worse than that of low TMB group, with a cutoff TMB value of 4.9. Enrichment analysis demonstrated that differentially expressed genes were mainly related to T cell activation, cell membrane localization and matrix composition. Tumor immune infiltration analysis revealed the infiltrations of Th2, Th17, and Tgd were up-regulated in high TMB group, while those of Tr1, MAIT, and DC were up-regulated in low TMB group. TMB-Infiltration model fit well with the actual survival observation, with a C-index 0.785 (0.700-0.870), which verified in ICGC-LIRI-JP was 0.650 (0.553-0.747). Additionally, these screened immune genes performed well in predicting tumor vascular invasion with a C-index of 0.847 (0.778-0.916). Overall, these results indicated that patients with high mutation frequency of immune-related genes and high TMB were prone to have worse prognosis and relapse after radical treatment.

Identification of Tumor Mutation Burden and Immune Infiltrates in Hepatocellular Carcinoma Based on Multi-Omics Analysis.

We aimed to explore the tumor mutational burden (TMB) and immune infiltration in HCC and investigate new biomarkers for immunotherapy. Transcriptome and gene mutation data were downloaded from the GDC portal, including 374 HCC samples and 50 matched normal samples. Furthermore, we divided the samples into high and low TMB groups, and analyzed the differential genes between them with GO, KEGG, and GSEA. Cibersort was used to assess the immune cell infiltration in the samples. Finally, univariate and multivariate Cox regression analyses were performed to identify differential genes related to TMB and immune infiltration, and a risk prediction model was constructed. We found 10 frequently mutated genes, including TP53, TTN, CTNNB1, MUC16, ALB, PCLO, MUC, APOB, RYR2, and ABCA. Pathway analysis indicated that these TMB-related differential genes were mainly enriched in PI3K-AKT. Cibersort analysis showed that memory B cells (p = 0.02), CD8+ T cells (p = 0.09), CD4+ memory activated T cells (p = 0.07), and neutrophils (p = 0.06) demonstrated a difference in immune infiltration between high and low TMB groups. On multivariate analysis, GABRA3 (p = 0.05), CECR7 (p < 0.001), TRIM16 (p = 0.003), and IL7R (p = 0.04) were associated with TMB and immune infiltration. The risk prediction model had an area under the curve (AUC) of 0.69, suggesting that patients with low risk had better survival outcomes. Our study demonstrated for the first time that CECR7, GABRA3, IL7R, and TRIM16L were associated with TMB and promoted antitumor immunity in HCC.

Correlations Between Tumor Mutation Burden and Immunocyte Infiltration and Their Prognostic Value in Colon Cancer.

BackgroundColon cancer has a huge incidence and mortality worldwide every year. Immunotherapy could be a new therapeutic option for patients with advanced colon cancer. Tumor mutation burden (TMB) and immune infiltration are considered critical in immunotherapy but their characteristics in colon cancer are still controversial.MethodsThe somatic mutation, transcriptome, and clinical data of patients with colon cancer were obtained from the TCGA database. Patients were divided into low or high TMB groups using the median TMB value. Somatic mutation landscape, differentially expressed genes, and immune-related hub genes, Gene Ontology and KEGG, gene set enrichment, and immune infiltration analyses were investigated between the two TMB groups. Univariate and multivariate Cox analyses were utilized to construct a prognostic gene signature. The differences in immune infiltration, and the expression of HLA-related genes and checkpoint genes were investigated between the two immunity groups based on single sample gene set enrichment analysis. Finally, a nomogram of the prognostic prediction model integrating TMB, immune infiltration, and clinical parameters was established. Calibration plots and receiver operating characteristic curves (ROC) were drawn, and the C-index was calculated to assess the predictive ability.ResultsMissense mutations and single nucleotide polymorphisms were the major variant characteristics in colon cancer. The TMB level showed significant differences in N stage, M stage, pathological stage, and immune infiltration. CD8+ T cells, activated memory CD4+ T cells, activated NK cells, and M1 macrophages infiltrated more in the high-TMB group. The antigen processing and presentation signaling pathway was enriched in the high-TMB group. Two immune related genes (CHGB and SCT) were identified to be correlated with colon cancer survival (HR = 1.39, P = 0.01; HR = 1.26, P = 0.02, respectively). Notably, the expression of SCT was identified as a risk factor in the immune risk model, in which high risk patients showed poorer survival (P = 0.04). High immunity status exhibited significant correlations with immune response pathways, HLA-related genes, and immune checkpoint genes. Finally, including nine factors, our nomogram prediction model showed better calibration (C-index = 0.764) and had an AUC of 0.737.ConclusionIn this study, we investigated the patterns and prognostic roles of TMB and immune infiltration in colon cancer, which provided new insights into the tumor microenvironment and immunotherapies and the development of a novel nomogram prognostic prediction model for patients with colon cancer.

Predictive value of plasma tumor mutation burden (TMB) in the CCTG PA.7 trial: Gemcitabine (GEM) and nab-paclitaxel (Nab-P) vs. GEM, nab-P, durvalumab (D) and tremelimumab (T) as first line therapy in metastatic pancreatic ductal adenocarcinoma (mPDAC).

411 Background: PA.7 evaluated whether combining PD-L1 and CTLA-4 inhibition with GEM and Nab-P increases efficacy as first line therapy in mPDAC. High TMB is associated with immunotherapy sensitivity, with a threshold of ≥10 mut/Mb receiving FDA accelerated approved for pembrolizumab in a tissue agnostic setting. We assessed the predictive value of plasma TMB in the PA.7 trial. Methods: This randomized phase II study (ClinicalTrials.gov NCT02879318) assessed the efficacy and safety of GEM, Nab-P, D, and T (arm A) vs. GEM and Nab-P (arm B) in patients (pts) with mPDAC (n = 180). The primary endpoint was overall survival (OS). Pre-treatment plasma was sequenced with the CLIA-certified PredicineATLAS cfDNA next generation assay (600 genes, 2.4 Mb panel). A pre-specified cut point of 5 mut/MB was selected based on distribution of TMB in the trial. 2-sided alpha set at 0.1. Results: 180 pts were randomized (119 to arm A and 61 to arm B). There was no significant difference in OS (9.8 months in arm A vs. 8.8 months in arm B, p-value 0.72) or PFS (5.5 months and 5.4 months respectively, HR 0.98, p-value 0.91). Plasma TMB analysis was performed on 174/180 pts with available samples, and tumor derived variants were detected in 173/174 pts (99.4%). 172 pts were microsatellite stable and 1 pt was microsatellite high (MSI-H) (plasma TMB 52.9 muts/Mb). Using the pre-specified cut-point of 5 mut/Mb there was no significant predictive value from plasma TMB (interaction p = 0.91). Using a minimum p-value approach, a cut-point of 9 mut/MB appeared predictive (p-interaction = 0.064; significant at pre-specified p = 0.1). 8/174 (4.6%) pts had a plasma TMB ≥9 mut/Mb (5/115 (4.4%) in arm A and 3/59 (5%) in arm B). GEM, Nab-P, D+T was associated with improved OS in patients with plasma TMB ≥9 mut/Mb (HR 0.30, 90% CI 0.06-1.37) while no activity was seen in pts with &lt; 9 mut/Mb, (HR 0.97, 90% CI 0.73-1.29). TMB cut-point analysis revealed a clear trend for a decreasing HR favoring the GEM, Nab-P, D and T arm above the selected cut point, with no benefit in the low TMB group. Conclusions: Plasma TMB analysis was successful in over 99% of pts with available samples. Plasma TMB ≥9 mut/Mb was predictive of benefit from the addition of dual immune checkpoint inhibitors (D and T) to Gem and Nab-P, with a significant interaction p-value. While only present in a subgroup of pts (4.6%), this data defines a group beyond MSI-H PDAC that may benefit from immunotherapy. The optimal cut-point for high TMB in this setting requires validation. A clinical trial specifically assessing the role of chemotherapy combined with immune checkpoint inhibition in high TMB mPDAC is warranted. Clinical trial information: NCT02879318.

ADRB1 was identified as a potential biomarker for breast cancer by the co-analysis of tumor mutational burden and immune infiltration

Breast cancer (BRCA) has traditionally been considered as having poor immunogenicity and is characterized by relatively low tumor mutational burden (TMB). Improving immunogenicity may improve the response to clinical immunotherapy of BRCA. However, the relationship between TMB, immune infiltration, and prognosis in BRCA remains unclear. We aimed to explore their interrelations and potential biomarkers. In this study, based on somatic mutation data of BRCA from The Cancer Genome Atlas (TCGA), patients were categorized into high and low TMB groups utilizing the TMB values. CIBERSOFT algorithm indicated significant infiltration of activated partial immune cells in high TMB group. Besides, ADRB1 had been identified as a prognosis-related immune gene in the mutant genes by the combination of the ImmPort database and the univariate Cox analysis. ADRB1 mutation was associated with lower TMB and manifested a satisfactory clinical prognosis. Various database applications (Gene Set Enrichment Analysis, Tumor IMmune Estimation Resource, Connectivity Map, KnockTF) supported the selection of treatment strategies targeting ADRB1. In conclusion, TMB was not an independent prognostic factor for BRCA and high TMB was more likely to activate a partial immune response. ADRB1 was identified as a potential biomarker and may provide new insights for co-therapy of BRCA.

Multi-omics analysis of tumor mutation burden combined with immune infiltrates in melanoma

BackgroundIn multiple malignancies, whether tumor mutation burden (TMB) correlated with increased survival or promotion of immunotherapy remained a debate. Our aim was to analyze the prognosis of TMB and the possible connection with immune infiltration of the skin cutaneous melanoma (SKCM). MethodsWe gathered somatic mutation data from the 472 SKCM patients using the Cancer Genome Atlas (TCGA) database and analyzed the mutation profiles using ““maftools” package. TMB was determined and samples were divided into high and low TMB groups. We undertook differential analysis to determine the profiles of expression between two groups using the “limma” package and established the 10 Hub TMB signature from a batch survival study. Gene ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and Gene Set Enrichment Analysis (GSEA) were performed in order to test considerably enriched pathways between the two groups. The connections of 10 TMB-related signature mutants with immune infiltration in SKCM were further assessed based on the TIMER database. We used the CIBERSORT package to measure the amount of 22 immune fractions between low and high TMB groups, and Wilcoxon's rank-sum amounts estimated the significant difference. In addition, the Cox regression model and survival analysis were used to determine the prognostic importance of immune cells. Finally, we estabilished a multivaried Cox results Tumor Mutation Burden Prognostic Index (TMBPI) and built a Receiver Operating Characteristic (ROC) curve to check the predictive accuracy. ResultsSingle nucleotide polymorphism (SNP) was more frequent than insertion or deletion and C > T was SKCM’s most frequently single nucleotide variants (SNV). Higher TMB levels provided poor survival outcomes, associated with tumor stage, age, and gender. In addition, 224 differentially expressed genes were obtained and Venn diagram established the top 25 immune-related genes. GSEA observed that patients in high TMB groups associated with nucleotide excision repair, pyrimidine metabolism, basal transcription factors, spliceosome, RNA polymerase, and RNA degradation in cancers. 10 hub TMB-related immune genes were also established and 10 signature mutants were correlated with lower immune infiltrates. In addition, the infiltration levels of macrophages M1 and macrophages M2 in the low-TMB group were lower. Eventually, the TMBPI was developed and the AUC of ROC curve was 0.604. ConclusionsHigh TMB contributed to low survival outcomes and may prevent SKCM immune infiltration. The 10 hub immune signature TMB-related mutants conferred lower immune cell infiltration that required further confirmation.

Abstract 6616: Functional hotness score generated by representative functional cytotoxic T-lymphocytes predicts long-term survival of triple-negative breast cancer independently to the tumor mutational burden

Abstract Background: Cytotoxic T-lymphocytes (CTLs) infiltration in a bulk tumor is a positive prognostic factor in breast cancer. CTL recognition is believed to react to mutation-induced neoantigens, thus, higher tumor mutation burden (TMB) is considered as an important predictor of tumor immunogenicity and response to immunotherapy. Although majority of breast cancer have low tumor mutation burden (TMB), triple-negative breast cancer (TNBC) has higher TMB compared to other breast cancer subtypes. However, it is unclear if the prognostic value high CTL infiltration depends on the TMB as the primary driver of enhanced anti-cancer immunity or represents an independent prognostic marker which can be used as a complement to TMB to predict patients' outcomes. CTLs are identified by CD8 marker, is encoded by CD8A. Granzyme B (GZMB) is a serine protease that is secreted by functionally-active CTLs to induce apoptosis of the target cells. CXCL10 is a chemokine which selectively attracts activated CTLs into subsets of other cancers; but its role in breast cancer remains unknown. We investigated if TNBC infiltrated with high levels of functional CTLs, so called “hot tumors”, have improved survival independently of their TMB. Methods: Utilizing The Cancer Genome Atlas (TCGA) breast cancer cohort, we established Functional Hotness Score (FHS), based on the CD8A, GZMB and CXCL10 gene expression levels of bulk tumors. Total of 4149 breast cancer patients from publicly available multiple cohorts were analyzed to assess FHS and breast cancer patient prognosis as well as distinct immunity markers. Results: FHS predicted the breast cancer patient survival better than each consisting gene. The breast cancer patients with high FHS tumors showed significantly better survival in the testing cohort (TCGA), which was further confirmed in the validation cohort (METABRIC). FHS was higher in the primary breast cancer tissues with metastasis compared to that without metastasis in TCGA. Further, FHS was higher in the metastatic tumors compared to the primary tumors in GSE110590. Among breast cancer subtypes, TNBC showed highest FHS compared to other subtypes in testing TCGA, which was validated in the METABRIC cohort. The patients with high FHS tumor demonstrated significantly better long-term survival than that with low FHS only in the hormone receptor (HR)-negative breast cancers, but not in the HR-positive tumors. The high FHS TNBCs showed higher not only CD8-positive T cell infiltration, but also a broader type-1 anti-cancer immunity. To investigate if FHS predicts patient survival independent of TMB, the patients were divided into high and low TMB groups. Interestingly, the higher FHS patients showed better prognosis not only in the high TMB group but also in the low TMB among TNBC patients. The combination of high TMB with high FHS identified the unique subset of the patients who did not recur over time. Conclusion: In conclusion, TNBC with higher FHS based on the expression levels of CD8A, GZMB and CXCL10 showed improved prognosis with higher anti-cancer immunity regardless of TMB, and constituting an independent prognostic marker of survival, particularly when combined with TMB. Citation Format: Eriko Katsuta, Li Yan, Mateusz Opyrchal, Pawel Kalinski, Kazuaki Takabe. Functional hotness score generated by representative functional cytotoxic T-lymphocytes predicts long-term survival of triple-negative breast cancer independently to the tumor mutational burden [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6616.

Exploration of immune-related genes in high and low tumor mutation burden groups of chromophobe renal cell carcinoma.

Renal cell carcinoma (RCC) is one of most common cancers with gradually increasing incidence and high mortality. Chromogenic RCC (chRCC) is the third most common histological subtype of RCC, accounting for approximately 5–7% of RCC. In our study, the transcriptome expression profile data (n=89) of chRCC, corresponding clinical data (n=113) and the somatic mutation data (n=66) were obtained from the TCGA database. We first analyzed the mutation data of chRCC patients and divided chRCC patients into high and low tumor mutation burden (TMB) groups based on the median TMB. We found that high TMB was significantly associated with worse prognosis and could promote tumor metastasis and development. Moreover, four different immune-related genes (BIRC5, PDGFRL, INHBE, IL20RB) were also identified. We found that BIRC5 was significantly overexpressed in the high TMB group and correlated with worse prognosis. The results of univariate and multivariate COX analyses demonstrated that BIRC5 (hazard ratio (HR) = 2.094) may serve as a prognostic indicator for patients with chRCC with high TMB. In addition, we identified the possible functional pathways of BIRC5 through gene set enrichment analysis (GSEA) enrichment. A positive correlation was obtained between BIRC5 and the abundance of CD4+ T cells. The results of our study revealed their correlation between the immune-related genes and clinicopathologic features as well as potential functional pathways as well as immune infiltrating cells, which may provide more data about the development of chRCC immunotherapy.

Exploration of gene expression profiles and immune microenvironment between high and low tumor mutation burden groups in prostate cancer

BackgroundTumor mutation burden (TMB) has been established as a biomarker for response to immune therapy and prognosis in various cancers. However, the association between TMB and prognosis of prostate cancer (PCa) remains unclear. This study aimed to investigate the impact of TMB in biochemical recurrence (BCR) and the immune microenvironment in high and low TMB groups. MethodsMutation data, gene expression, clinicopathological information were downloaded from The Cancer Genome Atlas (TCGA). Mutation types and TMB values were identified. All samples were divided into high and low TMB groups with median TMB value as the cutoff point. The BCR-free survival rates, Differentially expressed genes (DEGs) and immune cells infiltrations in different TMB groups were identified. ResultsThe most common variant type and SNV were single nucleotide polymorphism and C > T. respectively. High TMB level was significantly associated with older age, positive lymph node, higher International Society of Urological Pathology (ISUP) grade, advanced stage and poor BCR-free survival. 132 DEGs were identified and involved in receptor ligand activity and hormone activity. High expression of six core genes UBE2C, PLK1, CDC20, BUB1, CDK1 and HJURP were associated with worse BCR-free survival. The analysis of immune cells infiltration revealed that the amount of activated CD4+ memory T cells was significantly different in high and low TMB groups. ConclusionsThe current study comprehensively described the summary of mutation and TMB related DEGs in PCa. TMB was associated with BCR-free survival and the infiltration of activated CD4+ memory T cells in the immune microenvironment.

HLA-corrected tumor mutation burden and homologous recombination deficiency for the prediction of response to PD-(L)1 blockade in advanced non-small-cell lung cancer patients

Immune checkpoint inhibitors (ICIs) have been shown to be beneficial for some patients with advanced non-small-cell lung cancer (NSCLC). However, the underlying mechanisms mediating the limited response to ICIs remain unclear. We carried out whole-exome sequencing on 198 advanced NSCLC tumors that had been sampled before anti-programmed cell death 1 (anti-PD-1)/programmed death-ligand 1 (PD-L1) therapy. Detailed clinical characteristics were collected on these patients. We designed a new method to estimate human leukocyte antigen (HLA)-corrected tumor mutation burden (TMB), a modification which considers the loss of heterozygosity of HLA from conventional TMB. We carried out external validation of our findings utilizing 89 NSCLC samples and 110 melanoma samples from two independent cohorts of immunotherapy-treated patients. Homology-dependent recombination deficiency was identified in 37 patients (18.7%) and was associated with longer progression-free survival (PFS; P= 0.049). Using the HLA-corrected TMB, non-responders to ICIs were identified, despite having a high TMB (top 25%). Ten patients (21.3% of the high TMB group) were reclassified from the high TMB group into the low TMB group. The objective response rate (ORR), PFS, and overall survival (OS) were all lower in these patients compared with those of the high TMB group (ORR: 20% versus 59%, P= 0.0363; PFS: hazard ratio= 2.91, P=0.007; OS: hazard ratio= 3.43, P= 0.004). Multivariate analyses showed that high HLA-corrected TMB was associated with a significant survival advantage (hazard ratio= 0.44, P= 0.015), whereas high conventional TMB was not associated with a survival advantage (hazard ratio= 0.63, P= 0.118). Applying this approach to the independent cohorts of 89 NSCLC patients and 110 melanoma patients, TMB-based survival prediction was significantly improved. HLA-corrected TMB can reconcile the observed disparity in relationships between TMB and ICI responses, and is of predictive and prognostic value for ICI therapies.

Comprehensive Analysis of CDC27 Related to Peritoneal Metastasis by Whole Exome Sequencing in Gastric Cancer.

IntroductionThe peritoneum is the most common metastatic site of gastric cancer and is associated with a dismal prognosis. However, there is no reliable biomarker for predicting peritoneal metastasis (PM).Materials and MethodsWhole-exome sequencing (WES) was performed on formalin-fixed, paraffin-embedded (FFPE) samples from 63 patients with stage I–III gastric cancer and circulating tumor DNA (ctDNA) samples from 10 patients with stage IV gastric cancer. Differentially expressed genes (DEGs) were identified between the PM and non-PM groups and analyzed by multiple bioinformatics analyses. Univariate and multivariate Cox regression analyses were used to identify the risk factors for PM and a risk score model was developed.ResultsThe number of mutant genes and the tumor mutation burden (TMB) in the PM group were higher than those in the non-PM group (p < 0.05). There was a significant positive correlation between the number of mutant genes and the TMB (R2 = 0.9997). The risk of PM was significantly higher in the high TMB group than in the low TMB group (p = 0.045). Forty-nine DEGs were identified as associated with PM in gastric cancer. CDC27 mutations were associated with a higher risk for PM and poor survival. The CDC27 mutations were located in the Apc3 region, the TPR region, and the phosphorylation region, and new mutation sites were not included in the TCGA database. Multivariable Cox regression analysis demonstrated that pathological T stage, poor tumor differentiation, Borrmann type, and CDC27 mutations were independent predictive factors of PM. A risk score model was constructed that demonstrated good performance.ConclusionThrough WES, we identified 49 DEGs relevant to PM in gastric cancer. CDC27 mutations were independently associated with PM by statistical and bioinformatics analyses. A risk score model was built and was demonstrated to effectively discriminate gastric cancer patients with and without PM.

Advanced Non–Small Cell Lung Cancer Patients With Low Tumor Mutation Burden Might Derive Benefit From Immunotherapy

The aim of this study is to investigate the association between tumor mutation burden (TMB) and survival in non-small cell lung cancer (NSCLC) patients with anti-programmed cell death protein 1 and anti-programmed death-ligand 1 blockade. Two retrospective cohorts and The Cancer Genome Atlas NSCLC data set were included in this study. The restricted cubic spline analysis was used to explore the association between TMB and survival. The cutoff values for TMB were determined by X-tile software. Primary outcomes were overall survival (OS). The associations between TMB and intratumor heterogeneity, number of segments, fraction of genome alterations, aneuploidy score, and T-cell populations were also investigated. In the restricted cubic spline plots, TMB showed an inverted U-shaped curve with OS. The median OS in the low TMB group was significantly longer than those in the medium TMB group. In The Cancer Genome Atlas NSCLC data set, low TMB was also associated with longer OS in comparison with medium TMB. Furthermore, NSCLC patients with low TMB had significantly lower intratumor heterogeneity, number of segments, fraction of genome alterations, aneuploidy score, T-helper type 2 (Th2) cells, and CD8 T cells, but higher levels of Th1 and Th17 cells. Low TMB might be a prognostic factor for NSCLC patients receiving anti-programmed cell death protein 1/programmed death-ligand 1 immunotherapy.

The prognostic value of tumor mutational burden and immune cell infiltration in esophageal cancer patients with or without radiotherapy.

Growing evidence highlighted the tumor mutational burden (TMB) as an important feature of carcinogenesis and therapeutic efficacy in esophageal cancer (EC). Our study aimed to explore the genomic landscape and the correlation between TMB and immune cell infiltration in EC patients with or without radiotherapy. The EC patients were categorized into high TMB (TMB-H) and low TMB (TMB-L) groups by the ESTIMATE algorithm, and subgroup analysis was performed based on receiving radiotherapy or not. Univariate regression analysis indicated TMB and TNM stages as high-risk prognostic factors (Hazard ratio > 1 and P < 0.05). Multivariate regression analysis suggested TMB as an independent prognostic factor (Hazard ratio = 1.051, P = 0.003). Kaplan-Meier analysis showed no significant difference of the overall survival (OS) between TMB-H and TMB-L groups (P = 0.082). However, EC patients without radiotherapy in the TMB-H group had significantly decreased OS (P = 0.038) and increased Tregs cell infiltration (P = 0.033). These results suggested TMB as a prognostic marker for EC patients. Especially for patients who did not receive radiotherapy, the prognosis of TMB-H patients was significantly poorer than that of TMB-L patients, which might result from the different regulatory T cell infiltration.

Correlations analysis between HCC mutation burden and patients’ prognosis based on data mining

Objective To study the correlations between tumor mutation burden (TMB) and the prognosis of hepatocellular carcinoma (HCC) patients, and to investigate the effect of TMB on differential expression genes of HCC and the proportion of invasive immune cells in tumor tissues. Methods The somatic variation data, gene transcriptional expression data and clinical information of HCC patients were obtained from the cancer genome atlas (TCGA) database. The R program language (version 3.6.1) maftools function package was used to analyze the gene mutation data characteristics of the samples. The TMB value of each sample was calculated using the full-exon sequencing data of patients with hepatocellular carcinoma on the VarScan2 platform, sorted by TMB value, and the median value was used to divide all samples into high TMB and low TMB groups. Kaplan-Meier method was used to draw the survival curves of two groups of patients and log-rank test was performed to determine the correlation between tumor mutation load and prognosis. The Limma function package of R language was used to screen the differentially expressed genes between the two groups (FDR=0.05 and logFC=1), and the clusterProfiler function package of R language was used to perform gene ontology (GO) enrichment analysis of the differential genes and kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis (screening criteria were all P<0.05). Then the CIBERSORT tool was used to compare and analyze the difference in the proportion of invasive immune cells between the two groups. Results A total of 364 patients with HCC from TCGA database were included in the study. Mutations were found in 327(84%) samples, and there was a synergistic correlation between OBSCN and FLG mutations (P 0.05). A total of 198 with differentially expressed genes (28 up-regulated genes and 170 down-regulated genes) were screened between the high TMB group and the low TMB group. In GO analysis, it was found that the differentially expressed genes were mainly enriched in extracellular matrix tissues, extracellular structural tissues, extracellular matrix, extracellular matrix containing collagen, extracellular matrix structural components and other functions. In KEGG analysis, differential genes were highly enriched in extracellular matrix receptor interaction pathway and adhesive plaque pathway. In the correlation analysis of the proportion of infiltrating immune cells, CD4+ memory T cells were more infiltrating in the low TMB group (P<0.05). Monocytes showed a higher degree of infiltration in the high TMB group (P<0.05). Conclusion There was no correlation between TMB and the prognosis of HCC patients. TMB has significant influence on the differential expression genes of HCC and the proportion of invasive immune cells in tumor tissues. Key words: Carcinoma, hepatocellular; Tumor mutation burden; Prognosis

Identification of gene expression profiles and immune cell infiltration signatures between low and high tumor mutation burden groups in bladder cancer.

Bladder cancer is one of the most commonly diagnosed tumors and is results from the accumulation of somatic mutations in the DNA. Tumor mutation burden (TMB) has been associated with cancer immunotherapeutic response. In this study, we attempted to explore the correlation between TMB and cancer prognosis. Identify the different expressed genes and immune cell infiltration signatures between low and high TMB group. Mutation data, gene expression profiles and clinical data were downloaded from The Cancer Genome Atlas (TCGA) database. Patients were divided into high and low TMB groups, allowing differentially expressed genes (DEGs) to be identified. Functional enrichment and protein-protein interaction (PPI) network analysis were used to identify the functions of the DEGs. And immune cell infiltration signatures were evaluated by CIBERSORT algorithm. These results shown that high TMB was significantly associated with prognosis. We obtained a list of TMB related genes which may influence the infiltrations of immune cells. We also found a higher proportion of CD8 T cells, CD4 T cells and NK cells in the high TMB group. Our data suggest that higher TMB tends to promote the infiltrations of T cells and NK cells and patients with higher TMB may achieve a more favorable prognosis in bladder cancer.

Tumor Mutational Burden and Efficacy of Immune Checkpoint Inhibitors: A Systematic Review and Meta-Analysis.

Tumor mutational burden (TMB) is a genomic biomarker that predicts favorable responses to immune checkpoint inhibitors (ICIs). Here, we set out to assess the predictive value of TMB on long-term survival outcomes in patients undergoing ICIs. We systematically searched PubMed, Embase, CENTRAL and clinicaltrials.gov from inception to 6 August 2019. We included retrospective studies or clinical trials of ICIs that reported hazard ratios (HRs) for overall survival (OS) and/or progression-free survival (PFS) according to TMB. Data on 5712 patients from 26 studies were included. Among patients who received ICIs, high TMB groups showed better OS (HR 0.53, 95% CI 0.42 to 0.67) and PFS (HR 0.52, 95% CI 0.40 to 0.67) compared to low TMB groups. In patients with high TMB, those who received ICIs had a better OS (HR 0.69, 95% CI 0.50 to 0.95) and PFS (HR = 0.66, 95% CI = 0.47 to 0.92) compared to those who received chemotherapy alone, while in patients with low TMB, such ICI benefits of OS or PFS were not statistically significant. In conclusion, TMB may be an effective biomarker to predict survival in patients undergoing ICI treatment. The role of TMB in identifying patient groups who may benefit from ICIs should be determined in future randomized controlled trials.

The prognostic value of tumor mutation burden in EGFR-mutant advanced lung adenocarcinoma, an analysis based on cBioPortal data base.

Tumor mutation burden (TMB) is novel biomarker of promising predict value in prediction of immune checkpoint inhibitors (ICPis) in non-small cell lung cancer (NSCLC). However, the distribution of TMB in epidermal growth factor receptor (EGFR)-mutant advanced lung adenocarcinoma (LUAD) patients and the impact on overall survival (OS) time are not well demonstrated. Information regarding gene mutations and patients' survival time in advanced LUAD was downloaded from The Cancer Genome Atlas (TCGA) database. The diversity of TMB in different EGFR-mutant types was observed and the predicted value of TMB for OS as well as other co-mutations were analyzed. The diversity of TMB was also observed in another Chinese cohort of advanced LUAD patients. The median TMB values of EGFR wild-type, other types of EGFR mutations, exon 19 deletions and L858R were 6.12, 5.66, 3.77 and 4.72, differences between wild-type and EGFR sensitive mutations (exon 19 deletion or L858R) were significant (P<0.001 and P<0.01). OS time of high TMB group was inferior to that of the low TMB group (24.03 months vs. not reached, P=0.0020). TMB and TP53 together will make more accurate prediction of OS in EGFR-mutant advanced LUAD patients. Distribution of TMB in another Chinese cohort had the same trend. In advanced LUAD patients, TMB was lower in patients with EGFR-mutant group than EGFR wild group. TMB was a negative prognostic biomarker of OS in EGFR-mutant LUAD patients, especially when TP53 was mutated together.

Epigenetic alterations are associated with tumor mutation burden in non-small cell lung cancer

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

CCTG CO.26: Updated analysis and impact of plasma-detected microsatellite stability (MSS) and tumor mutation burden (TMB) in a phase II trial of durvalumab (D) plus tremelimumab (T) and best supportive care (BSC) versus BSC alone in patients (pts) with refractory metastatic colorectal carcinoma (rmCRC).

3512 Background: Targeting both PD-L1 and CTLA-4 may be synergistic immunotherapy approaches. CO.26 evaluated if dual inhibition leads to improved pt survival vs BSC alone in rmCRC. Methods: rmCRC pts were randomized 2:1 to D+T vs BSC. Treatment consisted of D (1500 mg) D1 q 28 days and T (75 mg) D1 for first 4 cycles, and supportive measures. Primary endpoint was overall survival (OS). Two-sided p &lt; 0.10 was considered statistically significant. Cell-free (cf)DNA sequencing for MSI and TMB used GuardantOMNI panel and baseline plasma. Results: From 08/2016-06/2017, 180 pts were enrolled. Pt characteristics were balanced between arms. At median follow-up of 15.2 months (mos), median OS was 6.6 mos for D+T and 4.1 mos for BSC (p = 0.07; Hazard ratio (HR): 0.72, 90% confidence interval (CI): 0.54 – 0.97). Progression free survival (PFS) was 1.8 mos vs 1.9 mos, respectively (HR 1.01, 90% CI 0.76 – 1.34). Disease control rate (DCR) was 22.6% for D+T and 6.6% for BSC (p = 0.006). cfDNA analysis was successful in 168/169 pts (99.4%). Two pts were MSI-high. In 166 MSS pts, OS HR was 0.66 (p=0.024; 90% CI 0.49-0.89). Excluding the MSI-H cases (TMB of 74.7 and 247.1 mts/Mb), mean TMB was 20.4 ± 16.3 mts/Mb (range: 0.96 – 114.0). In MSS pts, a pre-specified cutpoint of 20 mts/Mb stratified pts into high and low TMB groups but was not predictive for OS , PFS, or DCR (interaction p-values &gt; 0.7). Using a minimum p-value approach, pts with TMB &gt;28 mts/Mb (21% of MSS pts) had the greatest OS benefit (HR 0.34, 90% CI 0.18-0.63) for D+T (interaction p = 0.07). High TMB was associated with a trend in worse prognosis for OS in the BSC arm using both 20 mts/Mb (HR 1.26, 90% CI 0.76-2.12) and 28 mts/Mb (HR 2.59 90% CI 1.46-4.62) cutpoints. Conclusions: D+T significantly prolonged OS in pts with rmCRC. High TMB may select a group of MSS pts who benefit from D+T. Plasma TMB appeared prognostic in the BSC arm. This is the first study showing combined PD-L1 and CTLA-4 inhibition prolongs survival in pts with MSS rmCRC. Updated results based on deaths in more than 90% of pts will be presented. Clinical trial information: NCT02870920.

Tumor mutational burden (TMB) and BCG responsiveness in high-risk non-muscle invasive bladder cancer (NMIBC).

442 Background: Despite optimal management of NMIBC with transurethral resection of bladder tumor (TURBT) and intravesical BCG therapy, a significant proportion of patients (pts) will eventually present with disease recurrence or progression. To date, there is no validated predictive biomarker to guide patient selection for the most appropriate therapy in this setting. Methods: We retrospectively identified pts with high-risk NMIBC treated with TURBT, repeat TUR and intravesical BCG (≥ 6 instillations) from 2009 to 2016. Patients were classified as BCG-responsive (BCG-R) and BCG-unresponsive (BCG-UR) based on the International Bladder Cancer Group criteria. Whole exome sequencing was performed using archival FFPE tumor tissue from pre-BCG TURBT samples. Association of genomic variables and outcomes was evaluated by Cox proportional hazards survival analysis and likelihood ratio tests. Results: Thirty-five patients were included (BCG-R = 17, BCG-UR = 18). Median follow-up was 46 months for BCG-R and 52 months for BCG-UR pts. The majority of pts was male (91.4%), former smoker (60%), and presented with high-grade urothelial carcinoma (85.7%) and/or T1 staging (71.4%). Median time for relapse and progression was 10.5 and 19 months, respectively, in the BCG-UR group. In this cohort, TMB was significantly different in BCG-R and BCG-UR groups, with a median TMB of 5.53 +- 4.60 and 3.17 +- 1.82 mutations/Mb, respectively ( P= 0.045). TMB was also associated with relapse-free survival (RFS), with a median RFS of 38 and 15 months in high versus low TMB groups, respectively ( P= 0.0092). Intratumoral genetic heterogeneity assessed by mutant-allele tumor heterogeneity (MATH) was not statistically different between the groups, with a median MATH score of 31.8 and 21.9 for BCG-R and BCG-UR ( P= 0.14), respectively. On multivariate analysis, age and TMB were independently associated with RFS. Conclusions: In this exploratory biomarker study, high TMB was associated with benefit from immunotherapy with BCG for NMIBC. The identification of predictive biomarkers in this setting is an important unmet need and integrative analysis of TMB with other potential predictive biomarkers should be assessed in larger datasets.