Cancer carrier screening in the general population using whole-genome sequencing.

BACKGROUND: Cancer is a major cause of death, and its early identification and intervention have potential for clinical actionability and benefits for human health. The studies using whole-genome sequencing (WGS) and large samples analysis of cancer-related genes have been rarely done. METHODS: We performed WGS to explore germline mutations in coding and non-coding areas of cancer-related genes and non-coding driver genes and regulatory areas. Structural variants (SVs) was also analyzed. We used several tools and a subgrouping method to analyze the variants in 1491 healthy participants. Moreover, 275 cancer-related genes sequencing was carried out in 125 cancer patients. RESULTS: The incidence of familial cancer in the Taiwanese general population is 8.79% (131/1491). Cancer carrier rate of cancer-related genes is about 7.04% (105/1491) for pathogenic/likely pathogenic variants (P/LP) on ClinVar database only, and 28.24% (421/1491) for P/LP and loss of function variants. The carrier frequencies of cancer-related genes P/LP on ClinVar database were as follows: 8.40% (11/131), 7.11% (28/394), and 6.83% (66/966) in FC, 1MC, and nMC, respectively. The SVs and non-coding driver gene variants are uncommon. There are 1.54% (23/1491) of actionable cancer genes in American College of Medical Genetics and Genomics (ACMG), and the germline mutation rate of 275 cancer-related genes is 7.2% (9/125) in cancer patients including 4.0% (5/125) of actionable cancer genes in ACMG. After analyzing the frequencies of P/LP variants on GJB2 and SLC25A13 genes, we suggest that these two genes may not be cancer-related genes and need be re-evaluated. CONCLUSIONS: WGS analysis can completely detect germline mutations in cancer carriers. This study use subgrouping approach for samples provides a strategy to study whether a gene or variant is a cancer-related gene or variant in the future studies.

Integrated genomic analyses of hepatocellular carcinoma.

BACKGROUND: Genomic alterations play important roles in the development of cancer. We explored the impact of protein-coding genes and transcriptomic changes on clinical and molecular alterations in Taiwanese hepatocellular carcinoma (HCC) patients. METHODS: We analyzed 147 whole-exome sequencing and 100 RNA sequencing datasets of HCC and compared them with The Cancer Genome Atlas (TCGA)-Liver Hepatocellular Carcinoma cohort and develop a panel of 81 apoptosis-related genes for molecular classification. RESULTS: TERT (50%), TP53 (25%), CTNNB1 (14%), ARID1A (12%), and KMT2C (11%) were the most common genetic alterations of cancer-related genes. ALDH2 and KMT2C mutated at much higher frequencies in our cohort than in TCGA, whereas CTNNB1 was found only in 14% of our Taiwanese patients. A high germline mutation rate of ALDH2 in the APOBEC mutational signature and herb drug-related aristolochic acid-associated signature was also observed. Groups A and B of HCC were identified when we used apoptosis-related genes for molecular classification. The latter group, which had poorer survival outcomes, had significantly more aDC, CD4+ Tem, macrophages M2, NKT, plasma cells, and Th1 cells, and less CD4+ memory T cells, CD8+ Tcm, cDC, iDC, and Th2 cells, as well as more inter-chromosome fusion genes. Metatranscriptomic analysis revealed 54 cases of HBV infection. Moreover, we found that the main target gene of HBV integration is ALB. CONCLUSIONS: Unique genomic alterations were observed in our Taiwanese HCC patients. Molecular classification using apoptosis-related genes could lead to new therapeutic approaches for HCC.

Mutation Analysis of Second Primary Tumors in Oral Cancer in Taiwanese Patients through Next-Generation Sequencing.

Head and neck cancer has poor overall survival. Patients with head and neck cancer more frequently develop second primary tumors than do patients with other cancers, leading to a poor prognosis. In this study, we used next-generation sequencing to analyze and compare mutations between first tumors and second tumors in oral cancer. We retrieved tumor tissues collected from 13 patients who were diagnosed twice as having cancer. We used driver gene and trunk mutations to distinguish between recurrent cancer and primary cancer in oral cancer. We observed unique driver gene mutations in three patients with an initial clinical diagnosis of recurrent cancer; hence, we believe that the corresponding patients had primary cancer. Four patients with an initial clinical diagnosis of primary cancer were found to actually have recurrent cancer according to our results. Genetic testing can be used to enhance the accuracy of clinical diagnosis.

Comparison of multiple imputation algorithms and verification using whole-genome sequencing in the CMUH genetic biobank.

A genome-wide association study (GWAS) can be conducted to systematically analyze the contributions of genetic factors to a wide variety of complex diseases. Nevertheless, existing GWASs have provided highly ethnic specific data. Accordingly, to provide data specific to Taiwan, we established a large-scale genetic database in a single medical institution at the China Medical University Hospital. With current technological limitations, microarray analysis can detect only a limited number of single-nucleotide polymorphisms (SNPs) with a minor allele frequency of >1%. Nevertheless, imputation represents a useful alternative means of expanding data. In this study, we compared four imputation algorithms in terms of various metrics. We observed that among the compared algorithms, Beagle5.2 achieved the fastest calculation speed, smallest storage space, highest specificity, and highest number of high-quality variants. We obtained 15,277,414 high-quality variants in 175,871 people by using Beagle5.2. In our internal verification process, Beagle5.2 exhibited an accuracy rate of up to 98.75%. We also conducted external verification. Our imputed variants had a 79.91% mapping rate and 90.41% accuracy. These results will be combined with clinical data in future research. We have made the results available for researchers to use in formulating imputation algorithms, in addition to establishing a complete SNP database for GWAS and PRS researchers. We believe that these data can help improve overall medical capabilities, particularly precision medicine, in Taiwan.

Pathway Mutations in Breast Cancer Using Whole-Exome Sequencing.

The genomic landscape of breast cancer (BC) is complex. The purpose of this study was to decipher the mutational profiles of Taiwanese patients with BC using next-generation sequencing. We performed whole-exome sequencing on DNA from 24 tumor tissue specimens from BC patients. Sanger sequencing was used to validate the identified variants. Sanger sequencing was also performed on paired adjacent nontumor tissues. After genotype calling and algorithmic annotations, we identified 49 deleterious variants in canonical cancer-related genes in our BC cohort. The most frequently mutated genes were PIK3CA (16.67%), FKBP9 (12.5%), TP53 (12.5%), ATM (8.33%), CHEK2 (8.33%), FOXO3 (8.33%), NTRK1 (8.33%), and NUTM2B (8.33%). Seven mutated variants (ATR p.V1581fs, CSF1R p.R579Q, GATA3 p.T356delinsTMKS, LRP5 p.W389*, MAP3K1 p.T918fs, MET p.K1161fs, and MTR p.P1178S) were novel variants that are not present in any gene mutation database. After grouping the samples according to molecular subtype, we found that the cell cycle, MAPK, and chemokine signaling pathways in the luminal A subtype of BC; the focal adhesion, axon guidance, and endocytosis pathways in the luminal B subtype; and amyotrophic lateral sclerosis in the basal-like subtype were exclusively altered. Survival curve analysis showed that the presence of the MAPK signaling pathway and endocytosis mutations were correlated with a poor prognosis. These survival data were consistent with cBioPortal analyses of 2,051 BC cases. We discovered novel mutations in patients with BC. These results have implications for developing strategic, adjuvant, and gene-targeted therapies.

Molecular characterization of colorectal cancer using whole-exome sequencing in a Taiwanese population.

Next-generation sequencing (NGS) technology is currently used to establish mutational profiles in many heterogeneous diseases. The aim of this study was to evaluate the mutational spectrum in Taiwanese patients with colorectal cancer (CRC) to help clinicians identify the best treatment method. Whole-exome sequencing was conducted in 32 surgical tumor tissues from patients with CRC. DNA libraries were generated using the Illumina TruSeq DNA Exome, and sequencing was performed on the Illumina NextSeq 500 system. Variants were annotated and compared to those obtained from publicly available databases. The analysis revealed frequent mutations in APC (59.38%), TP53 (50%), RAS (28.13%), FBXW7 (18.75%), RAF (9.38%), PIK3CA (9.38%), SMAD4 (9.38%), and SOX9 (9.38%). A mutation in TCF7L2 was also detected, but at lower frequencies. Two or more mutations were found in 22 (68.75%) samples. The mutation rates for the WNT, P53, RTK-RAS, TGF-ß, and PI3K pathways were 78.13%, 56.25%, 40.63%, 18.75%, and 15.63%, respectively. RTK-RAS pathway mutations were correlated with tumor size (P = 0.028). We also discovered 23 novel mutations in NRAS, PIK3CA, SOX9, APC, SMAD4, MSH3, MSH4, PMS1 PMS2, AXIN2, ERBB2, PIK3R1, TGFBR2, and ATM that were not reported in the COSMIC, The Cancer Genome Atlas, and dbSNP databases. In summary, we report the mutational landscape of CRC in a Taiwanese population. NGS is a cost-effective and time-saving method, and we believe that NGS will help clinicians to treat CRC patients in the near future.

Correlation of genomic alterations between tumor tissue and circulating tumor DNA by next-generation sequencing.

PURPOSE: Analysis of circulating tumor DNA (ctDNA) offers an unbiased and noninvasive way to assess the genetic profiles of tumors. This study aimed to analyze mutations in ctDNA and their correlation with tissue mutations in patients with a variety of cancers. METHODS: We included 21 cancer patients treated with surgical resection for whom we collected paired tissue and plasma samples. Next-generation sequencing (NGS) of all exons was performed in a targeted human comprehensive cancer panel consisting of 275 genes. RESULTS: Six patients had at least one mutation that was concordant between tissue and ctDNA sequencing. Among all mutations (n = 35) detected by tissue and blood sequencing, 20% (n = 7) were concordant at the gene level. Tissue and ctDNA sequencing identified driver mutations in 66.67% and 47.62% of the tested samples, respectively. Tissue and ctDNA NGS detected actionable alterations in 57.14% and 33.33% of patients, respectively. When somatic alterations identified by each test were combined, the total proportion of patients with actionable mutations increased to 71.43%. Moreover, variants of unknown significance that were judged likely pathogenic had a higher percentage in ctDNA exclusively. Across six representative genes (PIK3CA, CTNNB1, AKT1, KRAS, TP53, and MET), the sensitivity and specificity of detection using mutations in tissue sample as a reference were 25 and 96.74%, respectively. CONCLUSIONS: This study indicates that tissue NGS and ctDNA NGS are complementary rather than exclusive approaches; these data support the idea that ctDNA is a promising tool to interrogate cancer genetics.