Translational Medical Research, Using The Peripheral Tumor Molecular Diagnostics

Background and Purpose:Malignant tumor is the major source of disease-related deaths in our country, nomatter in city or rural area. Unlike other disease, the clinical outcome of sametreatment is totally different among different cancer patients. The main reason for thisphenomenon is that the underling molecular characteristics among the patients arenot the same. Consequently, the conception of individual therapy through moleculardiagnostics has become an agreement in cancer therapy. The molecular characteristicof the member of HER family has always been one of the biggest concerns in cancertherapy, since they play important role in tumor evolvement and development.However, it is impossible to obtain the patient's tumor tissue and monitor theirgenotype changing dynamically, which interfere the individualized therapeuticdecision making. On the contrary, there are many tumor originated nuclear acid andcirculating tumor cells in cancer patient's peripheral blood, which are considered tobe an ideal sample source for molecular diagnostics since the sampling processes isusually easy, less invasive, and repeatable. Nevertheless, the testing procedure stillneeds to be optimized, standardized and validated.This study focused on lung cancer and breast cancer, trying to answer or resolvethe problems arising in clinical practice through nuclear acid analysis and CTCdetection or characterization, hoping to improve and optimize the individualizedtherapy in cancer patients.Material and method:1. EGFR mutation analysis with body fluids (pleural fluid and plasma) ofNSCLC patients:Patients with TKIs therapy experience among the patients who joined the EGFRmutation analysis using body fluids were selected. EGFR mutation status of the extracted DNA was re-evaluated by a more sensitive method (ARMS) and thepatient's clinical outcome of TKIs was analyzed retrospectively.2. CTC detection and characterization in lung cancer patientsCTCs of NSCLC and SCLC patients were enumerated using CellSearch system. For NSCLCpatients, the DNA of CTC was extracted for EGFR mutation detection. For SCLC patients, theexpression of IGF-1R on CTC was analyzed by adding FITC-labeled anti-body of IGF-1R intoCellSearch system.3. Prognostic significance of CTC enumeration for breast cancer patientsCTCs of MBC patients were enumerated using CellSearch system. Kaplan-Meier survival plotswere generated based on CTC levels at baseline, and the curves were compared using log-rank testing. Theprognostic significance of CTC enumeration was evaluated by univariate and multivariate Cox regressionanalysis.4. CTC HER2status for the prediction of HER2targeting therapyCTCs of HER2-positive MBC patients were enumerated and characterized using CellSearchsystem. Kaplan-Meier survival plots were generated based on CTC levels and CTC HER2status atbaseline, and the curves were compared using log-rank testing. McNemar's test was used to determinewhether a statistically significant difference existed regarding variations in HER2status between CTCs andhistological results.Results:1. EGFR mutation analysis with body fluids (pleural fluid and plasma) ofNSCLC patients:As compared with direct sequencing,16positive and23negative patients were confirmed byARMS, and the other11former negative patients (6pleural fluids and5plasmas) were redefinedas positive, with a fairly well clinical outcome (7PR,3SD, and1PD). The objective responserate (ORR) of positive patients was significant,81.3%(direct sequencing) and72.7%(ARMS)for pleural fluids, and80%(ARMS) for plasma. Notably, even reclassified by ARMS, the ORRfor negative patients was still relatively high,60%for pleural fluids and46.2%for plasma. 2. CTC detection and characterization in lung cancer patientsCTC detection rate for first line and second line of NSCLC patients is44.6%(25/56) and55.8%(24/53), respectively.1patients finished EGFR mutation analysisand the results was19exon deletion. Up to81.3%(26/32) of the SCLC patientsdetected CTC, but IGF-1R expression on CTC was not detected at any time.3. Prognostic significance of CTC enumeration for breast cancer patients294MBC patients finished the test at base line, with a CTC detection rate of61%(179/294), and39%(115/294) of the patients with a CTC count≥5. CTC enumeration couldpredict PFS and OS of the patients very well, when ever been tested at baseline,3-4weeks or6-8weeks. The dropping of CTC count during therapy was demonstrated having relation withclinical benefit. Univariate and multivariate Cox regression analysis both demonstrated thatCTC was an independent prognostic factor for PFS and OS.4. CTC HER2status for the prediction of HER2targeting therapyAt least1CTC was detected in45%(27/60) of the patients, most of them (59.3%) with CTCs numberranged from1to4. Significant difference of the median PFS was found in the groups that defined bycut-off of≥1CTCs (2.5VS7.5months, P=0.0125). Furthermore, we proposed a HER2positive criteriondefined as>30%CTCs over-expressing HER2(3+). Consequently, for the patients with targeting therapy(n=18), only those with positive CTC HER2could get benefit (8.8VS2.5months, P=0.002). Meanwhile,among the patients with positive CTC HER2(n=13), the median PFS for those receiving targeting therapywas significantly longer than others (8.8VS1.5months, P=0.001). Notably, up to51.9%(14/27) of thehistological HER2-positive patients were CTC HER2-negative, and anti-HER2therapy didn't significantlyimprove the median PFS (2.5VS0.9months, P=0.499).Conclusion:1. When using body fluids for EGFR mutation analysis, positive result was consistently a goodindicator for TKIs therapy, and the predictive effect was no less than that of tumor tissue, nomatter what method was employed. However, we should recognize that the correlation betweennegative results and clinical outcome of TKIs was still unsatisfied, even been reclassified by ARMS.2. It is feasible to realize the detection of CTC and EGFR mutation analysis on CTCin NSCLC patients, but the CTC detection rate is relative low, genotyping throughCTC in NSCLC patients may need more sensitive method for CTC enrichment. Onthe contrary, CTC detection rate and CTC count is higher in SCLC patients, it couldbe used as a real-time method for clinical efficacy evaluation. Nevertheless, theexpression of IGF-1R was not detected on CTC of SCLC patients.3. CTC enumeration by CellSearch system predicts PFS and OS of Chinese MBC patients verywell. The detection performance and clinical value of this system was same as that of westernpopulations. Meanwhile, the changing of CTC count during therapy was demonstrated havinggood relations with clinical benefit. CTC detection possesses with the advantage of early,dynamic and quantify, as compared with traditional radiographic method.4. CTC detection rate was lower for HER2-positive patients. CTC enumeration with a cut-offadjusted from≥5to1/7.5ml of blood could offer superior prognostic information for HER2positive MBC patients. In addition, CTC HER2status was different from that of tumor tissue, anda positive criterion defined as>30%CTCs over-expressing HER2(3+) may predict an improvedresponse to targeting therapy. Our findings strongly suggest the importance and urgency of CTCanalysis in HER2-positive MBC patients.