scholarly journals A next-generation sequencing-based strategy combining microsatellite instability and tumor mutation burden for comprehensive molecular diagnosis of advanced colorectal cancer

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Jian Xiao ◽  
Wenyun Li ◽  
Yan Huang ◽  
Mengli Huang ◽  
Shanshan Li ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Microsatellite Instability ◽  
Molecular Diagnosis ◽  
Checkpoint Inhibitors ◽  
The Cancer Genome Atlas ◽  
Immune Microenvironment ◽  
Mutational Burden ◽  
Immune Signatures ◽  
Mutation Burden ◽  
Tumor Mutational Burden

Abstract Background Mismatch repair (MMR)/microsatellite instability (MSI) and tumor mutational burden (TMB) are independent biomarkers that complement each other for predicting immune checkpoint inhibitors (ICIs) efficacy. Here we aim to establish a strategy that integrates MSI and TMB determination for colorectal cancer (CRC) in one single assay. Methods Surgical or biopsy specimens retrospectively collected from CRC patients were subjected to NGS analysis. Immunohistochemistry (IHC) and polymerase chain reaction (PCR) were also used to determine MMR/MSI for those having enough tissues. The NGS-MSI method was validated against IHC and PCR. The MSI-high (MSI-H) or microsatellite stable (MSS) groups were further stratified based on tumor mutational burden, followed by validation using the The Cancer Genome Atlas (TCGA) CRC dataset. Immune microenvironment was evaluated for each subgroup be profiling the expression of immune signatures. Results Tissues from 430 CRC patients were analyzed using a 381-gene NGS panel. Alterations in KRAS, NRAS, BRAF, and HER2 occurred at a significantly higher incidence among MSI-H tumors than in MSS patients (83.6% vs. 58.4%, p = 0.0003). A subset comprising 98 tumors were tested for MSI/MMR using all three techniques, where NGS proved to be 99.0 and 93.9% concordant with PCR and IHC, respectively. Four of the 7 IHC-PCR discordant cases had low TMB (1.1–8.1 muts/Mb) and were confirmed to have been misdiagnosed by IHC. Intriguingly, 4 of the 66 MSS tumors (as determined by NGS) were defined as TMB-high (TMB-H) using a cut-off of 29 mut/Mb. Likewise, 15 of the 456 MSS tumors in the TCGA CRC cohort were also TMB-H with a cut-off of 9 muts/Mb. Expression of immune signatures across subgroups (MSS-TMB-H, MSI-H-TMB-H, and MSS-TMB-L) confirmed that the microenvironment of the MSS-TMB-H tumors was similar to that of the MSI-H-TMB-H tumors, but significantly more immune-responsive than that of the MSS-TMB-L tumors, indicating that MSI combined with TMB may be more precise than MSI alone for immune microenvironment prediction. Conclusion This study demonstrated that NGS panel-based method is both robust and tissue-efficient for comprehensive molecular diagnosis of CRC. It also underscores the importance of combining MSI and TMB information for discerning patients with different microenvironment.

scholarly journals MicroRNAs Expression Patterns Predict Tumor Mutational Burden in Colorectal Cancer

2021 ◽  
Vol 10 ◽  
Author(s):  
Jiahao Huang ◽  
Haizhou Liu ◽  
Yang Zhao ◽  
Tao Luo ◽  
Jungang Liu ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Mirna Expression ◽  
Checkpoint Inhibitors ◽  
Expression Patterns ◽  
The Cancer Genome Atlas ◽  
Training Set ◽  
Test Set ◽  
Internal Validation ◽  
Mutational Burden ◽  
Tumor Mutational Burden

BackgroundTumor mutational burden (TMB) could be a measure of response to immune checkpoint inhibitors therapy for patients with colorectal cancer (CRC). MicroRNAs (miRNAs) participate in anticancer immune responses. In the present study, we determined miRNA expression patterns in patients with CRC and built a signature that predicts TMB.MethodsNext generation sequencing (NGS) on formalin-fixed paraffin-embedded samples from CRC patients was performed to measure TMB levels. We used datasets from The Cancer Genome Atlas to compare miRNA expression patterns in samples with high and low TMB from patients with CRC. We created an miRNA-based signature index using the selection operator (LASSO) and least absolute shrinkage method from the training set. We used an independent test set as internal validation. We used real-time polymerase chain reaction (RT-PCR) to validate the miRNA-based signature classifier.ResultsTwenty-seven samples from CRC patients underwent NGS to determine the TMB level. We identified four miRNA candidates in the training set for predicting TMB (N = 311). We used the test set (N = 204) for internal validation. The four-miRNA-based signature classifier was an accurate predictor of TMB, with accuracy 0.963 in the training set. In the test set, it was 0.902; and it was 0.946 in the total set. The classifier was superior to microsatellite instability (MSI) for predicting TMB in TCGA dataset. In the validation cohort, MSI status more positively correlated with TMB levels than did the classifier. Validation from RT-qPCR showed good target discrimination of the classifier for TMB prediction.ConclusionTo our knowledge, this is the first miRNA-based signature classifier validated using high quality clinical data to accurately predict TMB level in patients with CRC.

scholarly journals Mutational Analysis of Patients With Colorectal Cancer in CALGB/SWOG 80405 Identifies New Roles of Microsatellite Instability and Tumor Mutational Burden for Patient Outcome

2019 ◽  
Vol 37 (14) ◽  
pp. 1217-1227 ◽  
Author(s):  
Federico Innocenti ◽  
Fang-Shu Ou ◽  
Xueping Qu ◽  
Tyler J. Zemla ◽  
Donna Niedzwiecki ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Microsatellite Instability ◽  
Metastatic Colorectal Cancer ◽  
Mutational Analysis ◽  
Gene Mutations ◽  
Phase Iii ◽  
First Line ◽  
Mutational Burden ◽  
Significant Difference ◽  
Tumor Mutational Burden

PURPOSE CALGB/SWOG 80405 was a randomized phase III trial that found no statistically significant difference in overall survival (OS) in patients with first-line metastatic colorectal cancer treated with chemotherapy plus either bevacizumab or cetuximab. Primary tumor DNA from 843 patients has been used to discover genetic markers of OS. PATIENTS AND METHODS Gene mutations were determined by polymerase chain reaction. Microsatellite status was determined by genotyping of microsatellites. Tumor mutational burden (TMB) was determined by next-generation sequencing. Cox proportional hazard models were used, with adjusting factors. Interaction of molecular alterations with either the bevacizumab or the cetuximab arms was tested. RESULTS Patients with high TMB in their tumors had longer OS than did patients with low TMB (hazard ratio [HR], 0.73 [95% CI, 0.57 to 0.95]; P = .02). In patients with microsatellite instability–high (MSI-H) tumors, longer OS was observed in the bevacizumab arm than in the cetuximab arm (HR, 0.13 [95% CI, 0.06 to 0.30]; interaction P < .001 for interaction between microsatellite status and the two arms). Patients with BRAF mutant tumors had shorter OS than did patients with wild-type (WT) tumors (HR, 2.01 [95% CI, 1.49 to 2.71]; P < .001). Patients with extended RAS mutant tumors had shorter OS than did patients with WT tumors (HR, 1.52 [95% CI, 1.26 to 1.84]; P < .001). Patients with triple-negative tumors (WT for NRAS/ KRAS/ BRAF) had a median OS of 35.9 months (95% CI, 33.0 to 38.8 months) versus 22.2 months (95% CI, 19.6 to 24.4 months ) in patients with at least one mutated gene in their tumors ( P < .001). CONCLUSION In patients with metastatic colorectal cancer treated in first line, low TMB, and BRAF and RAS mutations are negative prognostic factors. Patients with MSI-H tumors benefited more from bevacizumab than from cetuximab, and studies to confirm this effect of MSI-H are warranted.

scholarly journals DACH1 mutation frequency in endometrial cancer is associated with high tumor mutation burden

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0244558
Author(s):  
McKayla J. Riggs ◽  
Nan Lin ◽  
Chi Wang ◽  
Dava W. Piecoro ◽  
Rachel W. Miller ◽  
...  
Keyword(s):  
Endometrial Cancer ◽  
Microsatellite Instability ◽  
Information Exchange ◽  
P Value ◽  
Tumor Mutation Burden ◽  
Prognostic Features ◽  
Oncology Research ◽  
Mutational Burden ◽  
Mutation Burden ◽  
Tumor Mutational Burden

Objective DACH1 is a transcriptional repressor and tumor suppressor gene frequently mutated in melanoma, bladder, and prostate cancer. Loss of DACH1 expression is associated with poor prognostic features and reduced overall survival in uterine cancer. In this study, we utilized the Oncology Research Information Exchange Network (ORIEN) Avatar database to determine the frequency of DACH1 mutations in patients with endometrial cancer in our Kentucky population. Methods We obtained clinical and genomic data for 65 patients with endometrial cancer from the Markey Cancer Center (MCC). We examined the clinical attributes of the cancers by DACH1 status by comparing whole-exome sequencing (WES), RNA Sequencing (RNASeq), microsatellite instability (MSI), and tumor mutational burden (TMB). Results Kentucky women with endometrial cancer had an increased frequency of DACH1 mutations (12/65 patients, 18.5%) compared to The Cancer Genome Atlas (TCGA) endometrial cancer population (25/586 patients, 3.8%) with p-value = 1.04E-05. DACH1 mutations were associated with increased tumor mutation count in both TCGA (median 65 vs. 8972, p-value = 7.35E-09) and our Kentucky population (490 vs. 2160, p-value = 6.0E-04). DACH1 mutated patients have a higher tumor mutation burden compared to DACH1 wild-type (24 vs. 6.02, p-value = 4.29E-05). DACH1 mutations showed significant gene co-occurrence patterns with POLE, MLH1, and PMS2. DACH1 mutations were not associated with an increase in microsatellite instability at MCC (MSI-H) (p-value = 0.1342). Conclusions DACH1 mutations are prevalent in Kentucky patients with endometrial cancer. These mutations are associated with high tumor mutational burden and co-occur with genome destabilizing gene mutations. These findings suggest DACH1 may be a candidate biomarker for future trials with immunotherapy, particularly in endometrial cancers.

scholarly journals Prolonged Response to Anti–PD-1 Antibody Therapy in Chemotherapy-Refractory Cholangiocarcinoma With High Tumor Mutational Burden

2019 ◽  
Vol 17 (6) ◽  
pp. 644-648 ◽  
Author(s):  
Olumide Gbolahan ◽  
Neda Hashemi-Sadraei ◽  
Bert O’Neil
Keyword(s):  
Overall Survival ◽  
Clinical Symptoms ◽  
Checkpoint Inhibitors ◽  
Antibody Therapy ◽  
Tumor Mutation Burden ◽  
Mutational Burden ◽  
Mutation Burden ◽  
Tumor Mutational Burden ◽  
Tumor Genome ◽  
Prolonged Response

Management of advanced intrahepatic cholangiocarcinoma (iCCA) is challenging and overall survival is poor. Progress in the development of new therapeutic options for metastatic cholangiocarcinoma (CCA) has been slow; hence, to date, there are no approved second-line agents in this setting. Although the development of immune checkpoint inhibitors has significantly improved overall survival in a variety of malignancies, there has not been a clinically important impact in CCA. This report presents a 66-year-old patient with chemotherapy-refractory iCCA who experienced a prolonged response to immunotherapy. Tumor genome profiling revealed a high tumor mutation burden of 17 mutations per megabase in the absence of microsatellite instability. He was started on immunotherapy with nivolumab and has experienced an ongoing response for 16 months without clinical symptoms and only minimal radiologic disease.

26 ctDNA clearance and radiographic resolution of lymph node metastasis in a patient with metastatic microsatellite stable colorectal cancer on immunotherapy

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A25-A25
Author(s):  
Charles Schneider ◽  
Michael Krainock ◽  
Meenakshi Malhotra ◽  
Paul Billings ◽  
Alexey Aleshin
Keyword(s):  
Colorectal Cancer ◽  
Lymph Node ◽  
Lymph Node Metastasis ◽  
Microsatellite Instability ◽  
Metastatic Colorectal Cancer ◽  
Mismatch Repair ◽  
Node Metastasis ◽  
Mutational Burden ◽  
Tumor Mutational Burden ◽  
Time Point

BackgroundHigh microsatellite instability (MSI-H) in metastatic colorectal cancer (mCRC) is associated with a beneficial response to immunotherapy. Additionally, within MSI-H cancers, tumor mutational burden (TMB) is independently predictive of immunotherapy responsiveness.1 Durable responses to therapy have been demonstrated in patients with MSI-H mCRC treated with Nivolumab and Ipilimumab.2 However, less is known about treatment responsiveness in patients with high mutational burden mCRC that demonstrates microsatellite stability (MSI-L).MethodsWe report on a 55-year-old female with a PALB-2 germline mutation who presented with a right-sided colonic adenocarcinoma with the involvement of the omentum and liver. The patient received 6 cycles of neoadjuvant FOLFOX, followed by an extended right hemicolectomy, omentectomy, and partial liver resection. The surgical specimen revealed a moderately differentiated adenocarcinoma in the cecum demonstrating a poor response to chemotherapy, 0/23 lymph nodes positive, one focus of adenocarcinoma in the liver with clear margins, and focal omental involvement with adenocarcinoma. The patient subsequently underwent 6 cycles of ‘adjuvant’ FOLFOX, with Oxaliplatin omitted after 3 cycles secondary to peripheral neuropathy. Soon after the patient experienced a recurrence that involved the anterior abdominal wall, between the peritoneum, and stomach, which was subsequently resected with negative margins. Molecular profiling of this metastatic focus revealed a TMB of 15.4 mutations per megabase, proficient Mismatch Repair (pMMR), a PDL1 CPS score of 26, and microsatellite stable (MSS) status. First, ctDNA analysis was performed at the time of recurrence and was found to be positive. Based on the TMB score of 15.4 and an elevated PDL1 score, the patient was initiated on Nivolumab and Ipilimumab. ctDNA measurements were obtained at the patient‘s request.ResultsDNA assessment performed after surgery and prior to initiation of immunotherapy revealed an approximate doubling of ctDNA levels, measured in mean tumor molecules (MTM) per mL of plasma, every month. During this period of time and correlating with the rise in ctDNA levels, the patient developed a new and enlarging FDG avid cardiophrenic lymph node. Following 2 cycles of Nivolumab and Ipilimumab, the FDG avid lymph node completely resolved and ctDNA clearance was observed (figure 1).Abstract 26 Figure 1ctDNA time-course demonstrating ctDNA kineticsTime-point A represents the initial ctDNA assay, performed at the time of resection of peritoneal metastasis. An additional time-point (B) drawn a month later reveals a further increase in ctDNA. Time-point C represents a peak in ctDNA levels, concomitant with the new emergence of a PET avid cardiophrenic lymph node. Combination Immunotherapy (IO) was begun shortly after time-point C. Time-point D represents ctDNA clearance and radiographic resolution of lymph node metastasis after two cycles of IO. MTM/mL - mean tumor molecules/milliliter of plasmaConclusionsHere we present a case of ctDNA clearance correlating with a radiographic resolution of metastatic disease in a patient with MSS mCRC. The data is provocative and suggests a possible contributory role of ctDNA-based testing as an additional monitoring parameter to measure disease-responsiveness to immunotherapy. Further investigation is warranted.Ethics ApprovalN/AConsentN/AReferencesSchrock AB, Ouyang C, Sandhu J, Sokol E, Jin D, Ross J8, Miller VA, Lim D, Amanam l, Chao J, Catenacci D, Cho M, Braiteh 7, Klempner SJ, Ali 8M, Fakih M. Tumor mutational burden is predictive of response to immune checkpoint inhibitors in MSl-high metastatic colorectal cancer. Ann Oncol 2019;30(7):1096–1103Overman MJ, et al. Durable Clinical/Benefit With Nivolumab Plus lpilimumab in DNA Mismatch Repair-Deficient/Microsatellite Instability-High Metastatic Colorectal Cancer. Clin Oncol 2018;36(8):773–779.

Crosstalk between the MSI status and tumor microenvironment in colorectal cancer.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e15236-e15236
Author(s):  
Peng Luo ◽  
Anqi Lin ◽  
Jian Zhang
Keyword(s):  
Colorectal Cancer ◽  
Tumor Microenvironment ◽  
Microsatellite Instability ◽  
Immune Cell ◽  
The Cancer Genome Atlas ◽  
Cell Infiltration ◽  
Immune Cell Infiltration ◽  
Immune Microenvironment ◽  
Mutation Data ◽  
Immune Related Genes

e15236 Background: In recent years, cancer immunotherapy has been extensively studied, and colorectal cancer (CRC) patients have also derived clinical benefits from immunotherapy, especially CRC patients with mismatch repair deficiency (dMMR)/microsatellite instability-high (MSI-H), whose sensitivity to immune checkpoint inhibitors (ICIs) is significantly higher than that of patients with microsatellite-stable (MSS)/microsatellite instability-low (MSI-L) disease. This study suggests that patients with MSI-H CRC have a higher mutational burden and more immune cell infiltration than those with MSS/MSI-L disease. However, most studies have not systematically evaluated the immune characteristics and immune microenvironments of MSI-H and MSS/MSI-L CRC. Methods: A published CRC cohort with mutation and immunotherapy-related prognostic data was collected. We analyzed the relationship between the MSI status and prognosis of ICI treatment in an immunotherapy cohort. We then further used mutation data for the immunotherapy and The Cancer Genome Atlas (TCGA)-CRC (colon adenocarcinoma (COAD) + rectum adenocarcinoma (READ) cohorts. For mRNA expression, mutation data analysis of the immune microenvironment and immunogenicity under different MSI status was performed. Results: Compared with MSS/MSI-L CRC patients, patients with MSI-H CRC significantly benefited from ICI treatment. We found that MSI-H CRC had more immune cell infiltration, higher expression of immune-related genes and higher immunogenicity than MSS/MSI-L disease. The MANTIS score used to predict the MSI status was positively correlated with immune cells, immune-related genes, and immunogenicity. In addition, subtype analysis showed that COAD and READ might have different tumor immune microenvironments. Conclusions: MSI-H CRC may have an inflammatory tumor microenvironment and increased sensitivity to ICIs. Unlike those of MSI-H READ, the immune characteristics of MSI-H COAD may be consistent with those of MSI-H CRC. Furthermore, the possible mechanism underlying the prognostic differences among CRC patients receiving ICIs in relation to the immune microenvironment were elucidated to provide theoretical guidance for further improving the curative effect of ICIs treatment on MSI-H CRC patients in the future and solve the problems underlying why MSS/MSI-L CRC patients do not benefit from ICIs treatment.

Identifying GNG4 might play an important role in colorectal cancer TMB

2021 ◽  
pp. 1-16
Author(s):  
Hongcan Zhao ◽  
Sheng Danli ◽  
Ze Qian ◽  
Sunyi Ye ◽  
Jianzhong Chen ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Liver Metastases ◽  
Genomic Analysis ◽  
Recurrence Risk ◽  
Computational Method ◽  
The Cancer Genome Atlas ◽  
Immune Microenvironment ◽  
Tumor Mutation Burden ◽  
Mutation Burden ◽  
Tumor Immune Microenvironment

BACKGROUND: Colorectal carcinoma (CRC) is one of the most leading cause of cancer death all over the world. The tumor immune microenvironment is illustrated to be necessary for the progress of CRC. And the accumulating evidence indicated that tumor mutation burden (TMB) is effective in differentiating responding population of immune checkpoint inhibitor (ICI) therapies in various cancers. In this study, we aimed to evaluated the potential relationship between TMB and the recurrence risk of CRC. METHODS: The transcriptomic and clinical data of CRC patients were collected from The Cancer Genome Atlas (TCGA) database (n= 382). Then the genomic analysis of tumor mutation burden and tumor purity were conducted by a computational method based on transcriptomic data. RESULTS: Firstly, we accessed the distribution of TMB and preferences at the gene and mutation level using somatic mutation data from TCGA data about CRC. We identified that high TMB predicted better prognosis of CRC patients. Secondly, the differentially expressed genes (DEGs) between the low TMB and high TMB group was clarified. Then the protein-protein interaction (PPI) analysis was performed, and the results confirmed ten hub genes among the DEGs. Utilizing the GEPIA web-tool, we discovered that GNG4 was up-regulated in tumor tissues, and GNG4 was related to the overall survival (OS) and tumor free survival (TFS) of CRC patients. Therefore, we considered GNG4 was essential for the tumor immune microenvironment of CRC. Furthermore, we also accessed the protein level of GNG4 in CRC and liver metastases from CRC. CONCLUSIONS: In this study, GNG4 was demonstrated to be the key element of the CRC TMB, which will be essential for the ICI therapy of CRC. Besides, GNG4 was up-regulated in CRC and liver metastases from CRC tissues. Thus, we thought that GNG4 might play an important role in colorectal cancer TMB and induce its metastasis in liver.

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