The Exploratory Study Of Trastuzumab Resistant Mechanism In Human Epidermal Growth Factor Receptor 2 Positive Breast Cancer Using Circulating Tumor Cells

Part I: Circulating tumor cells detecting in patients with breast tumors by a novel device(Cellcollector): a multicenter clinical trial in ChinaBackground.Circulating tumor cells have become an important part of liquid biopsy,which have undergone a process from counts to molecular typing and genotyping.To this end,we used Cellcollector to verify the effectiveness and safety of in the detection of CTCs in patients with breast tumor.Methods.We included patients from six leading Chinese cancer centers between April to August in 2016.Patients with benign breast tumor were required to receive one CTC testing by Cellcollector,while patients with metastatic breast cancer were required to receive CTC testing before receiving her new line therapy.After 3-4 weeks of new line therapy,they were required to receive the second CTC testing.The positive rate of CTC detecting and its safety would be compared with two groups.Results.A total of 190 patients were included.Among which,127 patients were diagnosed as metastatic breast cancer,and the other 63 patients as benign breast tumors.For these with metastatic breast cancer,74.8%(95/127)were CTC positive.A total of 117 patients in MBC groups received the second detection,among which 44.4%(68/117)were CTC positive patients.Patients with benign tumor had no positive CTC detected.Thus the sensitivity of Cellcollector was 74.8%,specificity was 100%,the area under the curve using receiver operating characteristic curve was 0.832(95%CI:0.784-0.879).The incidence of adverse events in MBC was 66.9%(85/127).39.8%(53/127)were diagnosed as severe adverse events,none of these adverse events were related to Cellcollector.One case of hematoma caused by improper operation were improved after timely treatment.Concludsions.In vivo isolation of CTCs overcomes blood volume limitations of other approaches.From this study,we set up an effect and safe CTC collecting platform for the further application of molecular typing and gene typing.Part II: Disparities of trastuzumab use in different regions and its survival benefit on HER2 positive breast cancer: a real-world study from multicenterBackground.Trastuzumab is a key component of therapy for human epidermal growth receptor 2(HER2)positive breast cancer.Because real-world data are lacking,we aimed to assess the actual use of trastuzumab and to evaluate potential efficacy from trastuzumab in real-world research.Methods.Inpatients with HER2 positive invasive breast cancer from 13 hospitals in Eastern China(2010–2015)were included in this study.The primary outcome was receipt of trastuzumab;The secondary outcomes were DFS and PFS of first-line therapy,time of trastuzumab use.We compared trastuzumab use with different periods and regions using Pearson's test.We also used Kaplan-Meier and Cox proportional hazards regression to estimate hazard ratio(HR)and 95% confidence interval(CI)for the relationship between trastuzumab and DFS or PFS in first line.Results.A total of 1139 inpatients were included.Of 1,017 patients with early stage breast cancer(EBC),40.5%(412/1,017)received trastuzumab therapy.Patients with EBC in resource-abundant regions(gross domestic product per capita >$15,000 and trastuzumab included in Medicare)are more likely to receive trastuzumab than those in resource-limited regions(37.3% vs.13.0%,p<.05).After metastasis,50.8%(366/720)patients received trastuzumab as their first-line therapy.More than 10% of patients with metastatic breast cancer(MBC)continued trastuzumab therapy after twice progression in resource-abundant regions,whereas more than 40% of patients never received any trastuzumab therapy during the whole course of therapy in resource-limited regions.Overall,the median time of trastuzumab use in EBC was 12 months(1-31months)and 10 months(1-74 months)in MBC.The improvement in survival for trastuzumab versus non-trastuzumab was substantial in EBC(HR=0.609,95% CI: 0.505-0.744)and in MBC(HR=0.541,95% CI: 0.418-0.606).This association was greater for patients with MBC who had never received trastuzumab(HR=0.493,95% CI: 0.372-0.576)than for those who had received adequate trastuzumab therapy in EBC stage(H=0.878,95% CI: 0.506-1.431)after a median DFS with 17 months.Conclusion.This study showed great disparities in trastuzumab use in different regions and different treatment stages.Both EBC and MBC patients can benefit from trastuzumab,as the survival data show;however,when trastuzumab is adequate in the early stage,a further trastuzumab-based therapy in firstline treatment of MBC patients will be ineffective,especially for those with short disease-free survival,and a second line of anti-HER2 therapy will be recommended.Part III: HER2 staining and sequencing of CTCs using Cellcollector in HER2 positive metastatic breast cancerBackground.Previous works have shown its efficiency and safety of Cellcollector capturing CTC.Thus,we aimed to use this novel device to capture CTCs in HER2 positive metastatic breast cancer patients.Afterwards,we aimed to use these CTCs to explore the feasibility of HER2 staining and single cell sequencing.Methods.We included HER2 positive MBC patients in our department between April 2016 to September 2017.Patients were required to receive CTC testing before receiving their new line therapy,and to receive the second testing after their disease progression.We selected part of CTCs to have HER2 staining.Besides,we chose patients with long term or short term benefit from trastuzumab to have CTC sequencing and to explored the probable mechanism of trastuzumab resistance.Results.A total of 50 HER2 positive MBC patients were involved.46.0% of patients underwent the second CTC testing.Positive rate of first CTC testing was 72.0%(36/50),these patients showed the same progression free survival than patients with negative CTC(HR=2.539,95% CI: 0.800-8.066).112 CTCs had HER2 staining,of which,57 were CTC HER2 positive.We selected 8 patients had CTC sequencing and found 3179 single nucleotide polymorphisms(SNP)from 44 genes.These including nonsynonymous single nucleotide variants(SNV,41.0%),synonymous SNV(23.5%)and unknown(33.9%).We also found 617 INDEL from 42 genes including frameshift(36.6%)and unknown(58.2%).We also found statistic significance(p<0.05)of SNP or INDEL in four genes(ATM,ESR1,IRAK4 and BRCA2)between two groups with different response to trastuzumab.Conclusions.We can use Cellcollector to capture CTC from HER2 positive breast cancer.We can also use these CTC to have HER2 staining and single cell sequencing which will be of importance to explore the mechanism of trastuzumab resistance.