The Application Study Of Liquid Biopsy In The Individualized Therapy Of HER2 Positive Breast Cancer Patients

Background and Purpose:Breast cancer is one of the most commonly seen malignant tumor,which respectively ranking as No.1 and No.2 in morbidity and mortality for female tumor in USA,and the corresponding data in China is No.1 and No.6.Therefore,breast cancer represents a big problem for the healthcare of female.HER2 is a well-recognized biomarker for breast cancer,and about 20% new diagnosed patients were HER2 positive,who are difficult to treat among all breast cancer subtypes.Target therapy that focuses on HER2 signal pathway is one of the most significant treatments for HER2 positive breast cancer patients,and it's also the highlights for basic and clinical research in breast cancer.For the individualized therapy of breast cancer patients,the clinical problems are including: 1.Some patients may not able to provide the qualified samples for HER2 testing;2.Tools that can timely evaluate therapeutic effect is lacking;3.When patients are resistant to the therapy,tumor tissue is usually unavailable,which making the mechanism analysis of drug resistance and later drug selection are hard to perform.Under such circumstances,the concept of liquid biopsy has become the focus of clinic.In contract the traditional tissue biopsy,the merits of liquid biopsy is less invasive and acceptable for the patients,therefore is useful in real-time dynamic monitoring.Meanwhile,under the liquid status,the elements that originated from tumor are relatively homogeneous and may easily reflect the temporal and spatial heterogeneity from an overall perspective.Peripheral blood is the most widely used liquid samples,among it,Circulating Tumor DNA(ctDNA),Circulating Tumor Cells(CTCs)and Extracellular Vesicles(EVs)are the materials that could be used in liquid biopsy.In present study,we mainly analyzed CTC and ctDNA in the peripheral blood of HER2 positive breast cancer patients,hoping we may know more clearly about liquid biopsy,and then we may fully applies it into clinical practice and effectively direct the individualized therapy for the patients.Material and method:1.Real-time HER2 Status on CTC and patients' HER2 Target Therapy:For HER2 positive and advanced breast cancer patients,HER2 expression on CTCs was detected using a CellSearch system within 7 days before a new line of anti-HER2 therapy was begun.According to the criterion proposed in our previous report,patients were defined as CTC HER2-positive or-negative.After close follow-up,the correlation between CTC HER2 status and the outcome of the treatment was evaluated by statistical analysis.2.The Selection and Comparison between Different CTC TechnologiesTwo CTC enriching methods that used filter membrane were selected and compared with Cellsearch system to evaluating their performance.Meanwhile,the enriched CTC by the two methods were subjected for immunohistochemistry(IHC)or fluorescence in situ hybridization(FISH)and direct sequencing,in ordering to explore their feasibility for real-time characteristic of CTC.3.Next-Generation Sequencing for Circulating Tumor DNActDNA in peripheral blood of the patients were analyzed by next-generation sequencing,we hoping firstly to establish the technical feasibility and secondly to find other useful biomarkers from it,therefore direct patients' individualized therapy more effectively.Results:1.Real-time HER2 Status on CTC and Patients' HER2 Target Therapy:CTCs were detected in 57.4 %(58/101)of the patients.Notably,62.1 %(36/58)of these patients had an inconsistent HER2 status between their tissue and CTCs.The discordant rate may correlate with the time interval between histological and CTC HER2 testing and is more likely to occur in the subgroup of patients with an interval of > 1 year than in those with an interval < 1 year(70.7 % vs.41.2 %,P=0.043).For PFS,positive HER2 status on CTCs was shown to be a valuable predictor,both in univariate(HR=0.321,95%CI,0.156–0.62,P=0.0011)and multivariate(HR=0.383,95%CI,0.166–0.831,P=0.019)Cox regression analysis.Meanwhile,Kaplan-Meier survival curves revealed that the median PFS of CTC HER2-positive patients was significantly longer than CTC HER2-negative ones(8.5 vs.3.5 months,P < 0.001).2.The Selection and Comparison between Different CTC TechnologiesThe detection rate of CTC ?1 for the two new technologies was 100%(13/13)and 95.5%(21/22),respectively,better than the Cell Search system.Among them,the method of Youzhiyou could dectect more CTM and show more advantage in IHC and FISH assay.In the method of Junhui,glucose metabolism dectection was introduced,which could indentify CTC functionally.Meanwhile,the precise locating and automatic sampling enable the single cell sequencing of CTC.The two methods both have their merits and shortcomings,and if being optimized further,they may provide substaintianl help in the patient's indivisual therapy.3.Next-Generation Sequencing for Circulating Tumor DNAEach procedure of sequencing,including nuclear extraction,library construction and sequencing analysis,was optimized based on 12 patients' samples.Finally,sequencing was successfully completed in 38 HER2 positive and advanced breast cancer patients.Several mutations with relatively higher frequency were found and these patients could be divided into different subgroup according to the clustering analysis of the sequencing results.Among them,16 patients had the comparison between 2 time points,and 2 patients had the comparison between 3 time points,such dynamic change may be useful in monitoring and directing the adjustment of the treatment.Besides,based on the experiment in patient #27,the technical details for whole genome DNA methylation analysis was established,and it formed the foundation of our later research.Conclusion:1.HER2 status on CTCs was different from that of tumor tissues and predicted a different outcome of the patients' anti-HER2 therapy.This difference may be correlated with the time interval between tissue and CTC HER2 testing,indicating the necessity of real-time HER2 analysis for histologically HER2-positive MBC patients.2.CTC technologies that using filter membrane have the merits of high capture efficiency and easy to handle and observe.If been optimized and standardized in some technical details,these methods may play more significant roles in the guidance of individualized therapy for HER2 positive breast cancer patients.3.Mutation detection and whole genome DNA methylation analysis were all technically feasible,and the dynamic change of these status may be useful in monitoring and directing the adjustment of the treatment.With the emerging of more clinical evidence in the future,the analysis of ctDNA would bring great help in patients' individualized therapy.