Mutation Screening And Clinical Evaluation Of Susceptibility Genes Sequencing For Triple Negative Breast Cancer

Objective: 1. To investigate the clinical pathological characteristics in triple negative breast cancer. 2. To analyze the pathogenic variants in BRCA1 and BRCA2 of TNBCs and the relationship with the clinical pathological characteristics. 3. To investigate the pathogenic variants in other genes of TNBCs and the relationship with the clinical pathological characteristics. 4. To evaluate the cancer susceptibility genes' variants of uncertain significance in TNBC patients and the relationship with the clinical pathogenic characteristics. 5. The primary goals of this study were to investigate the relationship of the multiple-gene germline mutations and prognostic of TNBC patients. Methods: 1. This research selected 100 triple negative breast cancer patients with definite pathological diagnosis. 5ml peripheral blood was collected, samples were frozen at-80?. A retrospective review of medical records was conducted to determine clinical characteristics. Patients were told to have an examination every half year and were followed up by telephone. 2. DNA extraction and next-generation sequencing: For each patient, genomic DNA was extracted from peripheral-blood leukocytes samples and DNA was purified, quality was assessed. Library construction and next-generation sequencing?NSG? were performed in a 115 genes panel was designed mainly included 21 breast cancer susceptibility genes and 94 genes known to cause other cancer syndromes or DNA repair pathways. Sequencing the entire coding region, exon-intron boundaries, and all known pathogenic variants in other regions for 115 genes. 3. Gene mutation analysis: Variants with a frequency more than 0.1 in normal controls or previously reported to be rare polymorphism or obviously conflicted with inheritance rules were considered to be polymorphism. Variants that lead to protein truncation are considered to be pathogenic, including nonsense, splicing mutation as well as insertions or deletions that lead to frameshift mutations. CNVs and any other variants previously reported to be deleterious were considered pathogenic, too. Variants of uncertain significance were use Poly Phen-2 program to predict their pathogenicity. Results: 1. Median age at onset was 51.5 years(range 20–75 years. 9 of the 100 patients reported a first-degree family history of breast cancers, 13 of them with a first-degree family history of other cancer, including gastric cancer, rectal carcinoma, colon cancer, hepatic carcinoma, lung cancer and osteosarcoma. 2. Among 100 triple negative breast cancer patients, 9 participants were tested to carry pathogenic variants in BRCA1/2, five pathogenic mutations of BRCA1 in 100 patients?5/100,5%?and fore pathogenic mutations of BRCA2 in 100 patients?4/100,4%?. BRCA1 frameshift mutations c.3296 del C and c.5470₅477del ATTG GGCA were detected, the two pathogenic mutations are novel. A CNV mutation of BRCA1 EX3-8 was detected. BRCA2 frameshift mutations c.7679₇680del TT and c.1856 del A were detected and the two mutations are novel. BRCA2 nonsense mutations c.8970G>A and c.5959C>T were detected. Of the 91 remained, 11 patients were detected but interpreted as a VUS BRCA1/2 mutation. The remaining 80 TNBC participants had tested negative for BRCA1/2 mutations. 3. Thirteen pathogenic variants were detected. Deleterious mutations in 9 other predisposition genes were detected in 12% of patients, including PALB2?3%?, MUTYH?3%?, ATM, GALNT12, HMMR, MSR1, NTRK1, SDHC and VHL?1%?. Eight variants were previously reported in the literature, and five were novel. Pathogenic mutation carries have a high clinical nodal stage?P=0.040?. 4. A total of 183 VUS were identified in 115 genes among 100 participants. Per participant, the average number of VUS across all genes was 1.83. Among all the 115 genes, 49 genes were detected with VUS. Per gene, the median number of VUS detected across all 100 participants was 3, ranging from one?CTNNA1, PIK3 CA, SDHC, etc.? to 13?BRIP1, AXIN2?. The Poly Phen-2 program predicted that 104 were benign?56.8%?, 40 were probably damaging?21.9%?, 28 were possibly damaging?15.3%?, and 11 unknown?6.0%?. Compared with wild type patients, BRCA1 and VHL gene mutation carries tended to be early onset. MUTYHgene mutation carries tended to be late onset. 5. During the study period, in the 100 TNBC patients, there are 40 patients have at least years survival data. We defined different scores for different gene mutations. Five-year DFS in patients with gene mutation score<5 was significantly higher than in gene mutation score?5?P=.0439?. A trend toward improved OS was observed for<5 group compared with?5, but this was not significant?P=0.0615?. Conclusion: 1. DNA germline mutation is more prevalent among triple negative breast cancer, in which BRCA1/2 mutations are highest, BRCA1 and BRCA2 mutation rates are 5% and 4% respectively. Deleterious mutations in 9 other predisposition genes were detected, including PALB2,MUTYH,ATM,GALNT12,HMMR,MSR1,NTRK1,SDHC and VHL. 2. The average number of VUS across all genes was 1.83. Per gene, the median number of VUS detected across all 100 participants was 3, BRIP1 and AXIN2 gene variants of uncertain significance are the most common. 3. The development of triple negative breast cancer is a cumulative effect of multi-gene, the low-penetrance gene polymorphism accumulative effect may be the key to the development of breast cancer. After added up of muti-genes mutation scores, we found that high mutation group has relative poor prognosis, multiple-gene mutation is an prognostic factor for 5-year DFS in triple negative breast cancer patients.