Circulating-Free DNA Mutation Associated With Response Of Targeted Therapy In HER2-positive Metastatic Breast Cancer

Objective The addition of anti-HER2 targeted drug,like Trastuzumab,Lapatinib and Pertuzumab,to chemotherapy significantly improved prognosis of HER2 positive breast cancer patients.Despite effectiveness,it is confusing that different metastatic patients vary in the response to those targeted therapies.What is more,it is inevitable for them to develop resistant to anti-HER2 treatment.Therefore,methods of accurately predicting drug response were really needed.In order to overcome the spatial and temporal limitations of biopsies,we aimed to develop a more sensitive and less invasive method of detecting bio-markers associated with anti-HER2 therapeutic response via cf DNA.Methods From March 6,2014 to December 10,2014,twenty patients with HER2-positive metastatic breast cancer who received systemic anti-HER2 therapy were eligible.We used a panel for detection of hot-spot mutations from 50 oncogenes and tumor suppressor genes,and then used amplicon next-generation sequencing(NGS)to identify somatic mutation of these samples in those 50 genes.Samples taken before their first Trastuzumab administration and subsequently proven with clinical benefit were divided into sensitive group.The others were collected after disease progression of the Trastuzumab based therapy and were grouped into the resistant group.Out of the 24 plasma samples,four blood samples were collected twice from the resistant cases.They were collected after either the disease progression of Trastuzumab or both Trastuzumab and Lapatinib.We collected the clinical details of those patients and then analysis the correlations with the identified SNVs.Results 1.A total of 20 women with HER2-positive breast cancer were eligible in this study.The median age was 45(range 27~71)year-old.Fifteen samples were hormone receptor positive(62.5%)and nine plasma were hormone receptor negative(37.5%).For the therapy regimen after plasma collected,15 were received the regimen containing Trastuzamab,seven were the regimen containing Lapatinib,one was Trastuzamab and Lapatinib and the last was ado-trastuzumab emtansine(T-DM1).2.The covering property of the 24 peripheral blood leucocytes(PBL)and circulating-free samples was 100%.The mean depth of PBL and cf DNA were 1870×(1340~2640×)and 10800×(4910~12860×),respectively.And the mean DNA concentrations were 14.95ng/?L(3.45~45.44 ng/?L)and 2.22 ng/?L(0.59~6.53 ng/?L),respectively of the plasma and PBL library preparation.3.A total of 486 somatic non-synonymous mutations of 46 genes were detected in the 24 blood samples.No mutations were identified in GNA1,CSF1 R,ALK or NPM1 genes.There were 26 genes which were detected containing less than 10 SNV(2%).These 26 genes contained FGFR1,GNAQ,RB1,ABL1,BRAF,FGFR3,GNAS,HRAS,FLT3,AKT,RET,CDH1,CDKN2 A,ERBB2,EZH2,IDH1,IDH2,MET,MLH1,NRAS,CTNNB1,JAK2,NOTCH1,PEPN11,JAK2 and MPL.4.All samples harbored at least one mutation(mean 20,range 1 to 51)and the mean detected AF was 3.9%(range 2%-31.66%).The most frequent alteration identified were PIK3 CA,followed by KIT and TP53.The mutational prevalence of 50 genes identified varied widely from 100% to 0% and reflected a long tail of bio-markers showing substantial genetic differentiation.5.Comparing the SNV from sensitive group with that from resistant groups,we found seven genes' mutations that only occurred in the resistant group.These 7 resistance-associated genes included EGFR,GNAS,HRAS,MLH1,CDH1,NRAS and NOTCH1.Those SNV containing: eleven EGFR mutations in exon 7,exon 18,exon 19 and exon 21;seven GNAS mutations in exon 7 and exon 8;seven HRAS mutations in exon 2 and exon 3;four different CDH1 mutations in exon 8 and exon 3;four MLH1 mutations in exon 11;three NOTHC1 mutations in exon 26,and exon 27 and four NRAS mutations in exon 2 and exon 3.6.In addition,four samples harbored three different HER2 mutations and among those mutational samples two had the same p.S855 I mutation which was never reported previously.It was encouraging to see that these two patients received longest time of effective therapy combined with anti-HER2 targeted agents in this trial,of which free-progress survival(PFS)were 48 and 35 months respectively.7.The correlation of the 46 mutational genes detected in our study and the outcome of the 24 patients was analyzed.Kaplan-Meier analysis revealed that ATM,KDR and PDGFRA genes were significant associated with the PFS of those 24 samples next line anti-HER2 target therapy.Twelve samples harbored SNV in ATM gene(50%),Kaplan-Meier survival analysis revealed that the mutation in this gene was significantly associated with the longer PFS,with the mean PFS were 10.5 and 6.5 months(P=0.04,HR 0.33,95%CI 0.11-0.97)respectively in ATM mutation type and ATM wildtype.Eight and seven samples had mutations in KDR(33.3%)and PDGFRA(29%),Kaplan-Meier survival analysis revealed that the mutation in those two genes were significantly associated with the shorter PFS,with the mean PFS were 5.5 and 12.0 months(P=0.015,HR 4.56,95%CI 1.34~15.53)in KDR mutation type and KDR wildtype and 5.0 and 9.0 months(P=0.036,HR 3.77,95%CI 1.09~13.05)in PDGFRA mutation type and PDGFRA wildtype.Conclusion 1.Circulating-free DNA detected using amplicon NGS had high depth and sensitivity and was more receivable for breast cancer cf DNA identifying.As interested in 50 genes' hot-spots SNV which was be related to the development of tumors,the cost of cf DNA detection has decreased exponentially and deserved the clinical expansion.2.The mutational prevalence of 50 genes identified varied widely from 100% to 0% and reflected a long tail of biomarkers showing substantial genetic differentiation.The most frequent alteration identified were PIK3 CA,followed by KIT and TP53.There were 26 genes which were detected containing less than 10 SNV(2%)may be the driven genes for HER2 positive breast cancer.3.There were seven genes included EGFR,GNAS,HRAS,MLH1,CDH1,NRAS and NOTCH1 that only occurred in the resistant group were associated with anti-HER2 target therapy resistance.In addition,HER2 p.S855 I mutation may predict the continuous response to anti-HER2 therapy.In summary,amplicon NGS of cf DNA has potential clinical utility to detect biomarkers from HER2 targeted therapies.4.The three genes,ATM,KDR and PDGFRA,may predict the sensitivity of anti-HER2 therapy and directed the practice of anti-HER2 therapy.