Dataset Information

Extensive subclonal mutational diversity in human colorectal cancer and its significance.

ABSTRACT: Human colorectal cancers (CRCs) contain both clonal and subclonal mutations. Clonal driver mutations are positively selected, present in most cells, and drive malignant progression. Subclonal mutations are randomly dispersed throughout the genome, providing a vast reservoir of mutant cells that can expand, repopulate the tumor, and result in the rapid emergence of resistance, as well as being a major contributor to tumor heterogeneity. Here, we apply duplex sequencing (DS) methodology to quantify subclonal mutations in CRC tumor with unprecedented depth (104) and accuracy (<10-7). We measured mutation frequencies in genes encoding replicative DNA polymerases and in genes frequently mutated in CRC, and found an unexpectedly high effective mutation rate, 7.1 × 10-7 The curve of subclonal mutation accumulation as a function of sequencing depth, using DNA obtained from 5 different tumors, is in accord with a neutral model of tumor evolution. We present a theoretical approach to model neutral evolution independent of the infinite-sites assumption (which states that a particular mutation arises only in one tumor cell at any given time). Our analysis indicates that the infinite-sites assumption is not applicable once the number of tumor cells exceeds the reciprocal of the mutation rate, a circumstance relevant to even the smallest clinically diagnosable tumor. Our methods allow accurate estimation of the total mutation burden in clinical cancers. Our results indicate that no DNA locus is wild type in every malignant cell within a tumor at the time of diagnosis (probability of all cells being wild type, 10-308).

SUBMITTER: Loeb LA

PROVIDER: S-EPMC6936702 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Extensive subclonal mutational diversity in human colorectal cancer and its significance.

Loeb Lawrence A LA Kohrn Brendan F BF Loubet-Senear Kaitlyn J KJ Dunn Yasmin J YJ Ahn Eun Hyun EH O'Sullivan Jacintha N JN Salk Jesse J JJ Bronner Mary P MP Beckman Robert A RA

Proceedings of the National Academy of Sciences of the United States of America 20191205 52

Human colorectal cancers (CRCs) contain both clonal and subclonal mutations. Clonal driver mutations are positively selected, present in most cells, and drive malignant progression. Subclonal mutations are randomly dispersed throughout the genome, providing a vast reservoir of mutant cells that can expand, repopulate the tumor, and result in the rapid emergence of resistance, as well as being a major contributor to tumor heterogeneity. Here, we apply duplex sequencing (DS) methodology to quantif ...[more]

PMID: 31806761

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Project description:Purpose: This study aimed to identify a novel biomarker or a target of treatment for colorectal cancer (CRC). Experimental Design: The expression profiles of cancer cells in 104 patients with CRC were examined using laser microdissection and oligonucleotide microarray analysis. Overexpression in CRC cells especially in patients with distant metastasis was a prerequisite to select candidate genes. The mRNA expression of candidate gene was investigated by quantitative reversetranscription polymerase chain reaction (RT-PCR) in 77 patients as a validation study. We analyzed the protein expression and localization of the candidate gene by immunohistochemical study, and investigated the relationship between the expression and the clinicopathological feature in 274 CRC patients. Results: We identified 6 genes as candidates related to distant metastasis in CRC patients by microarray analysis. Among these genes, Osteoprotegerin (OPG) is known to have an association with aggressiveness in several cancers through inhibiting apoptosis by neutralizing the function of tumor necrosis factor related apoptosis inducing ligand (TRAIL). The mRNA expression of OPG in cancer tissues was significantly higher in patients with distant metastasis than those without metastasis. The overexpression of OPG protein was significantly associated with worse overall survival and relapse free survival (RFS). Moreover, the overexpression of OPG protein was an independent risk factor for recurrence of CRC. Conclusion: The overexpression of OPG would be a predictive biomarker of recurrence and a target of the treatment for CRC. Total of 148 microarray datasets obtained from LCM and homogenized tissues of colorectal cancer patients were normalized using robust multi-array average (RMA) method under R 2.6.2 statistical software with affy package from BioConductor. Normalization was separately performed for LCM dataset and homogenized tissue dataset. The normalized gene expression levels were presented as log2-transformed values by RMA.

Project description:ObjectiveThe mesentery is a potential site of residual tumor in patients with colorectal cancer (CRC). However, the mesenteric immune microenvironment remains unclear. In this study, we investigated the immune landscape of the mesentery, particularly the role of lymphocytes and its association with the clinicopathological characteristics of CRC.MethodsFlow cytometry was used to detect lymphocytes in the paired mesenteric tissue specimens adjacent to the colorectal tumors and normal mesenteric tissue specimens 10 cm away from the colorectal tumor edge and preoperative peripheral blood samples obtained from patients with CRC who underwent surgery. T-distributed stochastic neighbor embedding was utilized to analyze multiparameter flow cytometry data. Multiplex immunohistochemistry was performed to evaluate T cells subsets in the paired mesentery adjacent to the colorectal tumors and normal mesentery. The Fisher's exact test and non-parametric Wilcoxon's matched-pairs tests were used for statistical analysis. The non-parametric Mann-Whitney U test was used to determine associations between percentage data and clinical parameters of patients with CRC.ResultsWe found that immune cells in the normal mesentery were mainly of lymphoid lineage. Compared with peripheral blood, the normal mesentery showed decreased NK cells and the CD4/CD8 ratio and increased CD3+ CD56+, memory CD4+ T, memory CD8+ T, CD4+ tissue-resident memory T (TRM), and CD8+ TRM cells. Compared with the normal mesentery, the mesentery adjacent to the colorectal tumor showed increased B and regulatory T cells and decreased NK, CD3+ CD56+, CD4+ TRM, and CD8+ TRM cells. Moreover, memory CD8+ T cells and plasmablasts are negatively correlated with the depth of invasion of CRC. Increased memory CD4+ T cells are associated with distant metastasis of CRC and high preoperative serum carcinoembryonic antigen levels.ConclusionThe mesentery shows a specific immune microenvironment, which differs from that observed in peripheral blood. CRC can alter the mesenteric immune response to promote tumor progression.

Dataset Information

Extensive subclonal mutational diversity in human colorectal cancer and its significance.

Publications

Extensive subclonal mutational diversity in human colorectal cancer and its significance.

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