Dataset Information

Low-abundance mutations in colorectal cancer patients and healthy adults.

ABSTRACT: Detecting low-abundance mutations is very important for cancer diagnosis and treatment. Here we describe an improved targeted sequencing analysis that dramatically increases sequencing depth. Seven colorectal cancer (CRC) patients and seven healthy adults were enrolled in this study. We examined genetic mutations in tissue samples from the central and peripheral regions of tumors from the CRC patients and in blood cells from the healthy adults. We observed that each CRC carried larger numbers of mutations more than previously estimated. These included numerous deletion mutations in the tumor tissue. While the cellular morphology in the surrounding normal colonic tissues was healthy, these cells also carried many mutations. Similarly, the blood cells from the healthy donors carried numerous mutations. These findings shed new light on the processes of tumorigenesis and aging, and also present a potentially effective method for detecting low-abundance mutations for cancer diagnosis and targeted treatments.

SUBMITTER: Li Y

PROVIDER: S-EPMC6977685 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Low-abundance mutations in colorectal cancer patients and healthy adults.

Li Yanfei Y Dai Zhengsheng Z Huang Gang G Jin Yueling Y Ning Zhongping Z Shen Junwei J

Aging 20200112 1

Detecting low-abundance mutations is very important for cancer diagnosis and treatment. Here we describe an improved targeted sequencing analysis that dramatically increases sequencing depth. Seven colorectal cancer (CRC) patients and seven healthy adults were enrolled in this study. We examined genetic mutations in tissue samples from the central and peripheral regions of tumors from the CRC patients and in blood cells from the healthy adults. We observed that each CRC carried larger numbers of ...[more]

PMID: 31927530

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Project description:Constitutively active KRAS mutations have been found to be involved in various processes of cancer development, and render tumor cells resistant to EGFR-targeted therapies. Mutation detection methods with higher sensitivity will increase the possibility of choosing the correct individual therapy. Here, we established a highly sensitive and efficient microfluidic capillary electrophoresis-based restriction fragment length polymorphism (µCE-based RFLP) platform for low-abundance KRAS genotyping with the combination of µCE and RFLP techniques. By using our self-built sensitive laser induced fluorescence (LIF) detector and a new DNA intercalating dye YOYO-1, the separation conditions of µCE for ?X174 HaeIII DNA marker were first optimized. Then, a Mav I digested 107-bp KRAS gene fragment was directly introduced into the microfluidic device and analyzed by µCE, in which field amplified sample stacking (FASS) technique was employed to obtain the enrichment of the RFLP digestion products and extremely improved the sensitivity. The accurate analysis of KRAS statuses in HT29, LS174T, CCL187, SW480, Clone A, and CX-1 colorectal cancer (CRC) cell lines by µCE-based RFLP were achieved in 5 min with picoliter-scale sample consumption, and as low as 0.01% of mutant KRAS could be identified from a large excess of wild-type genomic DNA (gDNA). In 98 paraffin-embedded CRC tissues, KRAS codon 12 mutations were discovered in 28 (28.6%), significantly higher than that obtained by direct sequencing (13, 13.3%). Clone sequencing confirmed these results and showed this system could detect at least 0.4% of the mutant KRAS in CRC tissue slides. Compared with direct sequencing, the new finding of the µCE-based RFLP platform was that KRAS mutations in codon 12 were correlated with the patient's age. In conclusion, we established a sensitive, fast, and cost-effective screening method for KRAS mutations, and successfully detected low-abundance KRAS mutations in clinical samples, which will allow provision of more precise individualized cancer therapy.

Project description:BackgroundColorectal cancer (CRC) has been ranked as the second most deadly cancer and the third most diagnosed cancer cases for the year 2020. Specifically for Romania, the number of CRC-related deaths in 2019 was estimated at 6307 people, with a standardized mortality rate of 33.8 per 100,000 inhabitants. Although the tumor protein 53 (TP53) gene is intensively studied, there are few data on TP53 mutations in Romanian CRC. Furthermore, since genetic alterations may show geographical differences, our study aimed to analyze the clinical status and TP53 somatic variation in Romanian CRC patients.Subjects and methodsDNA from 40 randomly selected cases of CRC was extracted from formalin-fixed paraffin-embedded tissues and sequenced using direct Sanger sequencing techniques, and variants were annotated according to the recommendations of the Human Genome Variation Society. Novel variants were analyzed using MutationTaster2021 to predict their effects.ResultsThe mean age was 63.6 years (range 33-85 years) with a male to female ratio of 2.3. More than 45% (18/40) had an advanced cancer stage (≥ stage III). Mutations were found in 21/40 cases (52.5%), with one case having two mutations, giving a total of twenty-two mutations in the TP53 coding DNA. These mutations include 3 (13.6%) insertion-deletion mutations, two of which are novel frameshift mutations: c.165delT (in exon 4) and c.928_935dup (in exon 9), both of which are predicted to lead to nonsense-mediated mRNA decay and are classified as deleterious. The remaining 19 (86.36%) were substitution mutations: 1 nonsense and 18 (81.8%) missense mutations, with G > A (n = 7/19; 36.8%) and C > T (n = 6/19; 31.5%) transitions being the most common. The G > T transversion was found in 21.05% (4/19) of the substitution mutations.ConclusionWe have described two novel frameshift mutations in TP53. The discovery of novel mutations following the efforts of The Cancer Genome Atlas and other large-scale cancer genome sequencing projects may be further evidence of the heterogeneous nature of mutations in cancer and may indicate that the identification of carcinogenic mutations is not yet saturated. Further sequencing is therefore needed, especially in less studied populations. Importantly, consideration of their geographical environment will shed light on population-specific carcinogenesis.

Dataset Information

Low-abundance mutations in colorectal cancer patients and healthy adults.

Publications

Low-abundance mutations in colorectal cancer patients and healthy adults.

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