Detection And Clinical Significance Of Metastatic Breast Cancer Related Genes In ErbB Signaling Pathway By Targeted Exon Deep Sequencing

Background:Breast cancer is the most common cancer and the leading cause of cancer related death in women in both developed and developing countries.The ErbB signaling pathway plays a crucial role through PI3K/Akt and KRAS/RAF/MEK/ERK process.Such as proliferation,invasion,differentiation,migration,and apoptosis.ErbB signaling pathway plays a crucial role in the development and progression of metastatic breast cancer.by using of bioinformatic and data mining technologies,we found that 12 ERBB Signaling Pathway related genes are highly associated with breast cancer.although individual genes in ErbB signaling pathway have been widely investigated,there have been a limited number of Systematic studies that comprehensively investigated mutations in ErbB pathway genes.Here,targeted multiple exons sequencing method can reduce blindness and make the detection technology achieve broad patient coverage at a reasonable cost.Objective:In the present study,We capture and sequence 25 MBC-associated exons of 12 ERBB Signaling Pathway related genes based on qPCR and High throughput sequencing platform.We then conducted targeted amplicon next-generation sequencing to detected mutations in plasma DNA from 40 metastatic breast cancer.We then analyzed their relationship with clinicopathologic features and prognosis.Methods:A prospective cohort study was designed to include 40 breast cancer patients with measurable metastatic lesions.We conducted targeted amplicon next-generation sequencing using the MiSeq Genome Machine to detected mutations of 12 ERBB Signaling Pathway related genes based on QPCR and High throughput sequencing platform.We quantify circulating cell-free DNA,using the Beta-actin gene as the detection target.We quantify ctDNA copy number of metastatic breast cancer patientstogether with the peripheral circulating total cell-free DNA and ctDNA fraction,The relationship between ctDNA mutations and ctDNA copy and the clinico-pathological features and prognosis of breast cancer patients was investigated.Results:Overall,The median follow-up was 106 days(range:67-113 days).Including complete remission(CR),partial remission(PR),stable disease(SD)and progress disease(PD)were 2.5.0%and 42.5%,22.5%and 32.5%,respectively.30 out of 40 patients(75%)harbored at least one mutation.Mutational gene and mutational site were different in progress group and remission group.The alteration of mutational abundance after the first course of treatment in metastatic breast cancer patients was shown in heat maps.The alteration of mutational abundance in 25398281,25398284 and 178936093 were different in progress group and remission group.In Correlation analysis of raised ctDNA mutational abundance after treatment and short term disease,25398284 and 178936093 mutational abundance rise after treatment was more frequent in progress group than in remission group(p=0.04)But significant difference of 25398281 mutational abundance rise after treatment was not observed between progress group and remission group(P=0.004,P=0.05 and P=0.93,respectively).The median circulating cell-free DNA copies in the plasma for all patients pre-and post-treatment were 100.64(23.3-256.9)GE/mL and 161.5(35.4-305.3)GE/mL,respectively(Paired Wilcoxon test,Z=-1.96,P=0.05).However,no significant difference of circulating ctDNA levels could be found between subgroups by demographic and clinical characteristics(all P value from Mann-Whitney U test>0.05).With cutoff points of 297.1 GE/mL and220.7GE/mL,sensitivity and specificity of circulating ctDNA copy of before and after the first course of treatment in distinguish progressed patients from other cases(AUC of ROC were 0.74(95%CI:0.56-0.87,z=2.33,P=0.002)and 0.86(95%CI:0.70-0.95,z=4.51,P=0.003).Then the 40 breast cancer patients were divided into high plasma circulating ctDNA copy group(>297.1 GE/mL)and low DNA copy group(≤297.1 GE/mL).The PFS for patients with ≤297.1 GE/mL of the median circulating cell-free plasma DNA copies in the plasm after the first cours of treatment was superior to those with more than297.1 GE/mL(126 days vs.110 days,92%vs.27%,Log-rank(Mantel-Cox)test x2=4.95,P=0.008).The PFS for patients with ≤220.1 GE/mL of the median circulating cell-free plasma DNA copies in the plasm after the first cours of treatment was superior to those with more than220.1 GE/mL(89%vs.18.9%,Log-rank(Mantel-Cox)test X2=8.98,P=0.005).Conclusions:These results suggest that no significant relationship was found between the plasma circulating ctDNA copy and the clinicopathological features.However,This proof-of-concept analysis suggested that circulating tumor DNA is an informative,inherently specific,and highly sensitive biomarker of metastatic breast cancer.Our results demonstrate that targeted identifying and Detection of ctDNA in the plasma may be useful for monitoring tumor burden of metastatic breast cancer cancer.