Preliminary Study Of SET-iFISH Detection Technology In Detecting Circulating Tumor Cells In Newly Diagnosed Breast Cancer

Research Purpose:Identification of circulating tumor cells(CTCs)by surface marker expression and ploidy analysis(SET-iFISH)has been proven to be a highly sensitive method in the identification of some solid cancers.However,few researches have been published in breast cancer especially in newly diagnosed breast cancer.So we performed SET-iFISH analysis to identify CTCs in newly diagnosed non-metastatic breast cancer,and characterized the distribution of CTC subtypes based on cytokeratin(CK)expression and ploidy status to analysis CTCs and their characterizations in breast cancer,so that to provide a more accurate and powerful detection mean in early detection,cancer molecular classification,evaluation of curative effect,prognosis guidance,cancer risk assessment,individualized treatment and a series of other processes in breast cancer.Research methods:One hundred and eighty-four patients with newly diagnosed breast cancer,twenty-six patients with benign breast tumors,and ten healthy donors were enrolled at Changhai Hospital,from February 2014 to June 2015.Peripheral blood samples of the enrolled patients with no prior treatment for breast cancer and healthy donors were collected and evaluated in this study.Written consent was totally provided by all subjects.The study was approved by the Ethics Committee of Changhai Hospital and was performed according to the Declaration of Helsinki Principles.Studies were generally performed as previously published assays and according to manufacturer instructions(Cytelligen,San Diego,CA,USA)[3,15].Briefly,patient blood samples were collected into 7.5 ml tubes containing ACD anti-coagulant(Becton Dickinson,Franklin Lakes,NJ,USA),followed by thorough mixing and addition of 3 ml of hCTC separation matrix.The solution was centrifuged at 450 × g for 5 min at room temperature.Supernatants were collected and incubated with immunomagnetic particles conjugated to anti-leukocytes monoclonal antibodies including anti-CD45 at room temperature for 10 min with gentle shaking.The solution was subsequently subjected to magnetic separation using a magnetic stand(Promega,Madison,WI)to remove leukocytes.The magnetic particle-free solution was spun down at 500 × g for 2 min at room temperature.Cell pellets were thoroughly resuspended in cell fixative and applied onto the coated CTC slides for subsequent iFISH analysis.For CK-iFISH,samples were immunostained with a cocktail of Alexa Fluor 594-conjugated monoclonal anti-CD45 and Alexa Fluor 488-conjugated anti-PanCK(CK4,5,6,8,10,13 and 18)or anti-HER2 for 1 hour in the dark,followed by FISH performed with Centromere Probe(CEP)8 SpectrumOrange(Vysis,Abbott Laboratories,Abbott Park,IL,USA)using a S500 StatSpin ThermoBrite Slide Hybridization/Denaturation System(Abbott Molecular,Des Plaines,IL,USA).CTCs were identified as DAPI+/ CD45-/PanCK+ or-or HER2+ with aneuploid chromosome 8.Statistical analyses were performed with SPSS 19.0 software(IBM,NY,USA).Differences in CTC number between cancer patients,benign tumor patients and healthy donors were compared by Mann-Whitney U test.Positive rates of CTC with or without CK expression were compared using Fisher’s exact test.Graphical plots were generated using OriginPro 8 SR0,ver 8.0(Northampton,MA,USA).All the P values were two-sided,and P values of less than 0.05 were considered statistically significant.Research results 1.CTCs in breast cancerA total of 184 newly diagnosed breast cancer patients,26 benign breast tumor patients,and 10 healthy individuals were recruited at Changhai hospital of Shanghai(China)from February 2014 to June 2015.Aneuploid CTCs were detected in 167 of 184(90.76%)breast cancer patients with the number of CTCs detected ranging 0-19 /7.5 ml(median number = 2).The total number of CTCs detected was 676 cells with triploidy found in 387(57.25%)cells derived from 147 patients(147/167,79.89%).Among the CTCs,CK positivity was detected in only 33(4.88%)cells from 18 patients(18/167,10.78%).Aneuploid CTCs were detected in 8 of the 26 benign tumors(30.77%),which was significantly less than that of breast cancer patients(p =0).Furthermore,all CTC counts in benign tumors were ≤ 3/7.5ml.No CTCs were identified in healthy donor blood samples.2.CTC and tumor sizeOf the 184 total breast cancer patient samples examined,158 had tumor size data available.Amongst this cohort,80 were ≤ 2cm(T1),68 were >2 cm and ≤ 5cm(T2),and 10 were > 5cm(T3).Despite the small sample size of T3 patients,the highest CTC counts were detected in this group(0-18,median = 5.5).Triploid CTC counts were notably different between T3 and T1/T2 patients with a median number of 4 in T3 patients and a median number of 1.5 and 1 in T1 and T2 patients,respectively(P = 0.048 and P = 0.006).CTC subtypes based on CK expression were not significantly different among T1,T2 and T3 patients.3.CTC and molecular subtyping of breast cancerOf the 184 total breast cancer patients,166 had hormone receptor status data available revealing 30 were luminal A(ER+ and / or PR+ and HER2-,and Ki-67<14%=,89 were luminal B(ER+ and / or PR+ and HER2+;ER+ and / or PR+ and HER2-,and Ki-67≥14%),35 were HER2+(ER-and PR-and HER2+),and 12 were triple negative(ER-and PR-and HER2-).CTCs were found in twenty-four of the 30 luminal A patients(80%)ranging 0-18 CTCs/7.5 ml(median number = 2),80 of 89 luminal B patients(89.9%)ranging 0-19 CTCs/7.5 ml(median number = 2),34 of 35 Her2-positive patients(97.1%)ranging 0-15 CTCs/7.5 ml(median number = 4),and 12 of 12 triple negative patients(100%)ranging 0-12 CTCs/7.5 ml(median number = 2.5).Patients with Her2-positive or triple negative breast cancers showed the highest frequency and total number of CTCs compared with luminal A or luminal B patients.CTC subtypes based on karyotype,including triploid,teteraploid and multiploid(≥5),showed a similar distribution in breast cancer patients with differing hormone receptor status.However,CK+ CTCs were detected in luminal A and luminal B tumors.Research conclusion:In patients with non-metastatic breast cancer,aneuploid CTCs independent of CK expression status can be detected by the SET-iFISH system.Presence of CTCs and CTC counts correlated with tumor size and hormone receptor status in breast cancer patients.Triploid CTCs constituted the majority of CTCs detected in all of the breast cancer patients evaluated regardless of hormone receptor status and tumor size.In addition,CK positive CTCs were detected in a small cohort of patients and found at a low rate in CTC counts,demonstrating the existence of EMT.Notably,CK expression was rare in CTCs from HER2+ or triple negative patients,supporting the suggestion that lack of ER and PR may promote EMT and enhance tumor aggression.