Study On Germline Mutations Of BRCAL/2,PALB2 And RECQL Gene In The Chinese Patients With Breast Cancer

Background and objectiveBreast cancer has become the most common malignant tumor among Chinese women.In China,the incidence rate of breast cancer is the highest among the gynecologic oncology and the mortality rate is second only to lung cancer.It seriously jeopardizes the women's health and lives in China.Among all breast cancer patients,approximately 5-10%of them have genetic characteristics,while 15-20%of them have family histories.To reduce the morbidity and mortality of breast cancer to some extent,we can detect susceptibility genes of breast cancer,identify and screen the high-risk groups,and finally take appropriate preventive and therapeutic measures.The gene BRCA1 and BRCA2 are the earliest discovered and the most important breast cancer susceptibility genes whose high penetrance are closely related to the occurrence of hereditary breast cancer.With the development and cost reduction of second-generation sequencing technologies,more and more low-and middle-penetrance breast cancer susceptibility genes have been discovered,among which PALB2(partner and localizer of BRCA2)gene,especially RECQL(ATP-dependent DNA helicase Q1)gene was been found in recent years.On the one hand,because the PALB2 gene and the RECQL gene are rarely studied in Chinese populations,the mutational lineage is not yet clear.On the other hand,for oncologists and clinicians,we should clarify the influence of the mutation status of breast cancer susceptibility genes on the prognosis of breast cancer,the clinical management and treatment strategies for mutation carriers can be optimized.However,whether the BRCA1/2 mutation status affects the prognosis of breast cancer carriers has been controversial at home and abroad.Some studies believe that the survival rate of BRCA1/2 mutation-associated breast cancer is better than that of the control group,while others think that the prognosis is worse or no difference.In addition,although more and more genes have been shown to be related to the susceptibility of breast cancer,up to now,there is limited study on the relationship between the mutation status of other susceptibility genes such as PALB2 and prognosis in large-scale unselected breast cancer patients.This study aims to understand more comprehensively the mutation specturm of germline mutations of BRCA1/2,PALAB2,and RECQL gene in patients with breast cancer in China,and combine the clinicopathological features of mutation-associated breast cancers to elucidate the relationship between the mutation status and the prognosis of patients with breast cancer,which can then enrich the database of breast cancer susceptibility gene mutations in Chinese population and provide reference for the development of screening models and genetic counseling for breast cancer susceptibility genes suitable for Chinese population.Study population and methodsA total of 2769 consecutive breast cancer patients were treated at the Second Affiliated Hospital of Zhejiang University from June 1993 to September 2017,excluding the patients whose pathological data could not be obtained and the ones whose blood samples could not meet gene analyses.The cohort 2769 of breast cancer patients were unselected for age at diagnosis,molecular type and family history of breast cancer.All cases were proved to be primary breast cancer by histopathology.This study is divided into two parts.In the first part,peripheral blood was used to extract genomic DNA in all cases,and the susceptibility genes BRCA1/2,PALB2,and RECQL were sequenced using the full-exome sequencing method in the next generation sequencing technology.The sequencing results were compared with the gene database.At the same time,all the mutations detected were confirmed by Sanger sequencing.To identify the mutation lineage,the mutation site,mutation frequency,mutation distribution and mutation type of these genes were analyzed.Three different online algorithms were used to predict the function of variant of unknown significance.In the second part,the clinical pathological features of germline mutations of BRCAl/2,PALB2 and RECQL gene were analyzed in combination with the clinicopathological data and follow-up of the participants.The Kaplan-Meier method was used to analyze the survival of breast cancer patients.Cox proportional hazard model was used to analyze the relationship between germline mutations and prognosis of patients with breast cancer.ResultsPart OneAmong the 2769 unselected breast cancer patients,of which 2.7%(n=74)of patients carried a BRCA1 pathogenic mutation,2.7%(n=76)carried a BRCA2 mutation,0.9%(n=24)carried a PALB2 mutation and 0.07%(n=2)carried a RECQL mutation,respectively.In addition,patients with variant of unknown significance only in BRCA1,BRCA2,PALB2,and RECQL gene accounted for 2.7%(n=75),4.7%(n=130),3.9%(n=107),and 2.3%(n=64),respectively.33 novel pathogenic mutations(BRCA1 n=13,BRCA2 n=15,PALB2 n=5)were found,and 105 novel variants of unknown significance were detected(BRCA1 n=26,BRCA2 n=40,PALB2 n=23,RECQL n=16).The hotspot mutation regions of BRCA1 and BRCA2 gene are both exon 11.The mutations of PALB2 are concentrated in exon 4,and the distribution of germline mutations of RECQL gene are dispersed and most of them are relatively concentrated in exon 9.Among all the pathogenic mutations,the major mutation types of BRCA1 and BRCA2 genes were both frameshift mutations,whereas the major mutation type of PALB2 gene was a nonsense mutation.For 2 mutations of RECQL gene,one was a kind of splicing site mutation and the other was a kind of nonsense mutation.Part TwoIn familial breast cancer patients,BRCA1,BRCA2,and PALB2 gene had the highest prevalence of pathogenic mutations,were 5.2%(n=20),6.3%(n=24),and 1.3%(n=5),respectively,followed by early-onset breast cancer patients,the frequency of mutations in these three genes was 4.7%(n=25),5.3%(n=28),and 1.1%(n=6),respectively.However,the frequency of mutations was lowest in sporadic breast cancer patients,accounting for 1.5%(n=27),1.2%(n=22),and 0.7%(n=13),respectively.In addition,BRCA1 gene has the highest frequency of pathogenic mutations in patients with triple-negative breast cancer,which is 9.6%(n=42),while BRCA2 gene has the highest mutation frequency in Luminal breast cancer patients,which is 3.2%(n=58).BRCA1 and BRCA2 gene had the lowest mutation frequency in HER2-positive breast cancer patients,accounting for 0.8%(n=3)and 0.3%(n=l),respectively.The mutation prevalence of PALB2 gene detected in HER2-positive and triple-negative breast cancer patients was equal,both were 1.1%(n=4 and n=5,respectively).Two patients carried pathogenic mutations of RECQL gene were both from sporadic breast cancer patients and both molecular types of breast cancer were Luminal.Compared with non-carriers,BRCA1 pathogenic mutation carriers had a higher negative rate of ER,PR,and HER2,exhibited a higher histological grade of the tumor,and were more inclined to TNBC,while BRCA2 mutation carriers whose ER-positive rate and HER2-negative rate were higher,and they were more tended to Luminal B.In addition,the disease-free survival(DFS)of BRCA1 mutation carriers was significantly lower than that of non-carriers(unadjusted HR=2.48,95%CI=1.53-4.01,p<0.001;adjusted HR=2.42,95%CI=1.30-4.48,p=0.005).In addition,whether BRCA1 pathogenic mutation carriers in triple negative breast cancer(TNBC)patients or in non-triple negative breast cancer(Non-TNBC)patients,their DFS were both significantly decreased(TNBC subgroup:unadjusted HR=2.46,95%CI=1.22-4.97,p=0.012;adjusted HR=2.36,95%CI=1.05-5.32,p=0.038;Non-TNBC subgroup:unadjusted HR=2.24,95%CI=1.04-4.81,p=0.039;adjusted HR=2.94,95%CI=1.04-8.26,p=0.041).The pathogenic mutation status of PALB2 gene were significantly associated with the decline of overall survival(OS)in the PALB2 mutation carriers(unadjusted HR=7.58,95%CI=2.74-21.01,p<0.001;adjusted HR=10.37,95%CI=2.59-41.42,p=0.001).However,no significant difference was found between BRCA2 pathogenic mutation carriers and non-carriers,of regardless of DFS or OS.Additionally,there were no significant differences in clinical pathological features and prognosis between breast cancer patients carrying only variants of unknown significance of BRCA1,BRCA2,PALB2,and RECQL genes.ConclusionsScreening for germline mutations of BRCA1,BRCA2,and PALB2 gene in Zhejiang area of China is feasible and is recommended in familial breast cancer patients,early-onset breast cancer patients,triple-negative breast cancer patients,and Luminal B-type breast cancer patients.Examining exon 11 of the BRCA1 and BRCA2 genes and exon 4 of the PALB2 gene can improve efficiency and reduce costs.Due to the low frequency of pathogenic mutations of RECQL gene,it is not recommended as a routine indicator of hereditary breast cancer screening in Zhejiang.The novel mutations detected in this study have further enriched the mutation spectrum of breast cancer susceptibility genes in China,providing a certain reference for the development of specific screening patterns and genetic counseling in Chinese populations.In addition,compared with non-carriers,the disease-free survival of BRCA1 pathogenic mutation carriers and the overall survival of PALB2 pathogenic mutation carriers were worse,and the prognosis between BRCA2 pathogenic mutation carriers and non-carriers was similar.There were no significant differences in clinical pathological features and prognosis between breast cancer patients carrying only variants of unknown significance of BRCA1,BRCA2,PALB2,and RECQL genes.To clarify whether they had pathogenicity,require longer-term follow-up and more many other evidence,such as conducting functional study.