Nikos Xenidis

Different Prognostic Value of Cytokeratin-19 mRNA Positive Circulating Tumor Cells According to Estrogen Receptor and HER2 Status in Early-Stage Breast Cancer

PURPOSE To examine the prognostic value of cytokeratin-19 (CK-19) mRNA-positive circulating tumor cells (CTCs) in early-stage breast cancer patients focusing on clinically relevant subgroups based on estrogen receptor (ER) and HER2 expression. PATIENTS AND METHODS CK-19 mRNA-positive CTCs were detected by real-time reverse transcriptase polymerase chain reaction in the blood of 444 consecutive, stage I-III, breast cancer patients before initiation of adjuvant chemotherapy. The association between detection of CK-19 mRNA-positive CTCs and clinical outcome was analyzed for patients with ER-positive, ER-negative, triple-negative, HER2-positive, and ER-positive/HER2-negative tumors. RESULTS CK-19 mRNA-positive CTCs were detected in 181 (40.8%) of 444 patients; 109 (41.9%) of 260 patients with ER-positive tumors; 71 (40.6%) of 175 patients with ER-negative tumors; 27 (35%) of 77 patients with triple-negative tumors; 35 (39.8%) of 88 patients with HER2-positive tumors; and 82 (44.1%) of 186 patients with ER-positive/HER2-negative tumors. After a median follow-up of 53.5 months, patients with CK-19 mRNA-positive CTCs experienced reduced disease-free survival (DFS; P < .001) and overall survival (OS; P < .001); this was mainly observed in patients with ER-negative (P < .001 and P < .001, respectively) but not ER-positive tumors (P = .172 and P = .425, respectively) and in patients with triple-negative (P = .008 and P = .001, respectively) and HER2-positive (P = .023 and P = .040, respectively) but not ER-positive/HER2-negative tumors (P = .210 and P = .578, respectively). In multivariate analysis, the interaction between CK-19 mRNA-positive CTCs and ER status was the strongest independent prognostic factor for reduced DFS (hazard ratio [HR], 3.808; 95% CI, 2.415 to 6.003; P < .001) and OS (HR, 4.172; 95% CI, 2.477 to 9.161; P < .001). CONCLUSION Detection of CK-19 mRNA-positive CTCs before adjuvant chemotherapy predicts poor clinical outcome mainly in patients with ER-negative, triple-negative, and HER2-positive early-stage breast cancer.

Detection of cytokeratin-19 mRNA-positive cells in the peripheral blood and bone marrow of patients with operable breast cancer

Background:To compare detection rates and evaluate the clinical relevance of cytokeratin-19 (CK-19) mRNA-positive cells in the peripheral blood (circulating tumour cells, CTCs) and bone marrow (disseminated tumour cells; DTCs) of patients with early breast cancer.Methods:Paired samples of peripheral blood and bone marrow were obtained from 165 patients with stage I–II breast cancer before the initiation of adjuvant chemotherapy. In 84 patients, paired blood and bone marrow samples were also available after chemotherapy. The detection of CK-19 mRNA-positive CTCs and DTCs was assessed by real-time PCR.Results:CK-19 mRNA-positive CTCs and DTCs were detected in 55.2 and 57.6% of patients before chemotherapy, respectively. After chemotherapy, CTCs and DTCs were identified in 44 (52.4%) and 43 (51.2%) of the 84 patients, respectively. There was a 93.9% (McNemar; P=0.344) and 72.6% (McNemar; P=0.999) concordance between blood and bone marrow samples before and after chemotherapy, respectively. The detection of CK-19 mRNA-positive CTCs or DTCs before chemotherapy was associated with decreased overall survival (P=0.024 and P=0.015, respectively). In addition, their simultaneous detection was also associated with an increased incidence of disease-related death and decreased overall survival (P=0.016).Conclusions:The detection of CK-19 mRNA-positive CTCs using reverse transcription-PCR (RT–PCR) both before and after chemotherapy is correlated with the detection of CK-19 mRNA-positive DTCs in patients with early-stage breast cancer. The determination of the CTC status by RT–PCR conveys clinically relevant information that is not inferior to DTC status and, owing to the ease of sampling, warrants further evaluation as a tool for monitoring minimal residual disease.

Molecular Detection and Prognostic Value of Circulating Cytokeratin-19 Messenger RNA–Positive and HER2 Messenger RNA–Positive Cells in the Peripheral Blood of Women with Early-Stage Breast Cancer

PURPOSE The aim of this study was to evaluate the clinical relevance of the simultaneous detection of cytokeratin (CK)-19 messenger RNA (mRNA)- and HER2 mRNA-positive cells in peripheral blood of women with early-stage breast cancer. PATIENTS AND METHODS CK-19 mRNA- and HER2 mRNA-positive cells were detected using a real-time and a nested reverse-transcriptase polymerase chain reaction assay, respectively, in a cohort of 185 women with early-stage breast cancer before the initiation of any adjuvant systemic treatment. Detection of CK-19 mRNA- and HER2 mRNA-positive cells in the peripheral blood was correlated with clinical outcome. RESULTS Overall, 63 of the 185 patients (34%) had detectable CK-19 mRNA-positive cells, and 33 (52.3%) also had detectable HER2 mRNA-positive cells. Patients with CK-19/HER2 mRNA-negative cells showed a trend toward longer disease-free survival (DFS) compared with patients with CK-19 mRNA-positive/HER2 mRNA-negative cells (P = .054) and had longer DFS than patients with CK-19/HER2 mRNA-positive cells (P < .001). Similarly, overall survival (OS) was higher in patients with CK-19/HER2 mRNA-negative cells compared with patients with CK-19 mRNA-positive/HER2 mRNA-negative cells (P = .039) or CK-19/HER2 mRNA-positive cells (P < .001). Patients with CK-19/HER2 mRNA-positive cells had shorter DFS but not OS compared with patients with CK-19 mRNA-positive/HER2 mRNA-negative cells. In multivariate analysis, the simultaneous detection of CK-19 mRNA- and HER2 mRNA-positive cells was independently associated with early relapse. CONCLUSION The simultaneous detection of CK-19 mRNA- and HER2 mRNA-positive cells in peripheral blood predicts poor clinical outcome for women with early-stage breast cancer.

Prognostic role of APC and RASSF1A promoter methylation status in cell free circulating DNA of operable gastric cancer patients

Gastric carcinogenesis is a multistep process including not only genetic mutations but also epigenetic alterations. The best known and more frequent epigenetic alteration is DNA methylation affecting tumor suppressor genes that may be involved in various carcinogenetic pathways. The aim of the present study was to investigate the methylation status of APC promoter 1A and RASSF1A promoter in cell free DNA of operable gastric cancer patients. Using methylation specific PCR, we examined the methylation status of APC promoter 1A and RASSF1A promoter in 73 blood samples obtained from patients with gastric cancer. APC and RASSF1A promoters were found to be methylated in 61 (83.6%) and 50 (68.5%) of the 73 gastric cancer samples examined, but in none of the healthy control samples (p < 0.001). A significant association between methylated RASSF1A promoter status and lymph node positivity was observed (p = 0.005). Additionally, a significant correlation between a methylated APC promoter and elevated CEA (p = 0.033) as well as CA-19.9 (p = 0.032) levels, was noticed. The Kaplan-Meier estimates of survival, significantly favored patients with a non-methylated APC promoter status (p = 0.008). No other significant correlations between APC and RASSF1A methylation status and different tumor variables examined was observed. Serum RASSF1A and APC promoter hypermethylation is a frequent epigenetic event in patients with early operable gastric cancer. The observed correlations between APC promoter methylation status and survival as well as between a hypermethylated RASSF1A promoter and nodal positivity may be indicative of a prognostic role for those genes in early operable gastric cancer. Additional studies, in a larger cohort of patients are required to further explore whether these findings could serve as potential molecular biomarkers of survival and/or response to specific treatments.

Differential effect of adjuvant taxane-based and taxane-free chemotherapy regimens on the CK-19 mRNA-positive circulating tumour cells in patients with early breast cancer

Background:To determine the effect of adjuvant taxane-free and taxane-based chemotherapy regimens on the elimination of circulating tumour cells (CTCs) in patients with early breast cancer.Methods:The presence of CK-19 mRNA-positive CTCs in the peripheral blood was evaluated before and after chemotherapy, using a real-time RT–PCR assay, in a historical comparison of two cohorts of women with stage I–III breast cancer treated with adjuvant taxane-free (N=211; FE75C or E75C) and taxane-based (N=334; T/E75C or T/E75) chemotherapy.Results:Taxane-based chemotherapy resulted in a higher incidence of CTCs’ elimination than taxane-free regimens since 49.7% (74 of 149) and 33.0% (29 of 88) of patients with detectable CTCs before chemotherapy, respectively, turned negative post-chemotherapy (P=0.015). Patients treated with taxane-free regimens had a significantly lower disease-free survival (DFS) (P=0.035) than patients treated with taxane-based regimens; this difference was observed in patients with but not without detectable CTCs before chemotherapy (P=0.018 and P=0.481, respectively). The incidence of deaths was significantly higher in the taxane-free cohort of patients with but not without detectable CTCs before chemotherapy compared with that of the taxane-based cohort (P=0.002). Multivariate analysis revealed that the chemotherapy regimen was significantly associated with prolonged DFS (HR: 2.00; 95% CI=1.20–3.34).Conclusion:Elimination of CK-19 mRNA-positive CTCs during adjuvant chemotherapy seems to be an efficacy indicator of treatment and is associated with a favourable clinical outcome of patients with detectable CTCs before chemotherapy.

Circulating tumor cells (CTCs) monitoring in HER2-negative early breast cancer: Implications for secondary anti-HER2 adjuvant treatment strategies

11502 Background: We have shown that the detection of peripheral blood CK19mRNA+ and HER2mRNA+ cells predicts poor outcome in early breast cancer and that trastuzumab can effectively eliminate these cells. Methods: We used real-time and nested RT-PCR for the detection of peripheral blood CK19mRNA+ and HER2mRNA+ cells, respectively, before and after adjuvant chemotherapy in 260 HER2-negative (HER2 0,1+ by immunohistochemistry) early breast cancer patients. Furthermore, we compared 3 prognosticators that classify a patient into poor vs good prognosis group: (A:CK19mRNA pre-chemotherapy alone; positive vs negative, B:CK19mRNA pre- and post-chemotherapy; at least once positive vs all negative, C:CK19mRNA and HER2mRNA pre- and post-chemotherapy; see Table) Results: Patient groups and their clinical outcome based on CTCs’ monitoring with CK19mRNA and HER2mRNA pre- and post- chemotherapy are depicted in Table. The median follow-up for patients that did not relapse was 52 months (range 10–106 months). The prognos...