Supplementary Figures S1-S2 from Prognostic Value of the Molecular Detection of Circulating Tumor Cells Using a Multimarker Reverse Transcription-PCR Assay for Cytokeratin 19, Mammaglobin A, and HER2 in Early Breast Cancer

Supplementary Figures S1-S3; Supplementary Tables S1-S2.

Background The detection of circulating tumour cells (CTC) is prognostic for disease recurrence in early breast cancer (BC). This study aims to investigate whether this prognostic effect persists or varies over time. Methods The study population consisted of prospectively included stage I–III BC patients. The presence of CK19 mRNA-positive CTC in the peripheral blood was evaluated before and after adjuvant chemotherapy, using a real-time RT–PCR assay. Longitudinal samples were collected for a subset of patients. Results Baseline CTC data were available from 1220 patients, while 1132 had both pre- and post-therapy data. After a median follow-up of 134.1 months, CTC positivity at baseline was associated with shorter overall survival (OS; HR adj = 1.72, 95% CI 1.34–2.21, p < 0.001). For disease-free survival, an interaction with time ( p = 0.045) was observed. CTC positivity predicted early (within 5 years; HR adj = 1.76, 95 % CI 1.33–2.32, p < 0.001) but not late recurrence (HR adj = 1.10, 95% CI 0.79–1.53, p = 0.577). Following adjuvant chemotherapy, more patients converted from CTC-positive to CTC-negative than vice versa ( p < 0.001). Ten-year OS was 68.6% for + /+ and 86.7% for −/− group ( p < 0.001). CTC status at follow-up predicted disease recurrence. Conclusion CTC detection pre- and post-adjuvant chemotherapy is prognostic for early relapse, supporting investigations for novel adjuvant therapeutic approaches.

Detection of CTCs represents a poor prognostic factor in patients with early and metastatic breast cancer (mBC) and treatment with everolimus–exemestane (E/E) is an established effective treatment in hormone receptor-positive/HER2-negative mBC patients. The effect of E/E on CTCs in mBC patients was prospectively investigated. CTCs from 50 pre-treated patients with mBC receiving E/E were analyzed using the CellSearch (CS) platform and triple immunofluorescence (IF) staining for cytokeratin, M30 and Ki67 expression to assess their proliferative and apoptotic status. CTCs (by CS) were detected in 64% of patients before treatment and E/E administration resulted in their decreased prevalence [(n = 18; 36%, p = 0.004) and (n = 7; 19.4%, p = 0.019) post-1st and post-3rd treatment cycle, respectively] whereas it was significantly increased at disease progression (PD: 61%) compared to post-1st and post-3rd cycle (p = 0.049 and p = 0.021, respectively). Ki67-positive CTCs were detected in 60%, 60%, 17% and 50% of patients before treatment, post-1st, post-3rd cycle and at PD, respectively, while the opposite was observed for M30-positive CTCs (0% at baseline, 10% after the 1st cycle, 50% after the 3rd cycle and 0% at PD). The detection of even ≥ 1 CTC/5 ml after one cycle was associated with decreased PFS (3.3 vs 9.0 months, p = 0.025) whereas the detection of even ≥ 2 CTCs at PD was associated with decreased OS (32.4 vs 19.5 months; p = 0.009). The combination of E/E resulted in early elimination of proliferating CTCs in mBC patients and this effect was associated with a favorable clinical outcome.

The aim of the study was to investigate the effect of 2 nd -line pazopanib on the different CTCs subpopulations in SCLC patients and evaluate the clinical relevance of their changes. Different CTCs subpopulations were evaluated before pazopanib initiation (n = 56 patients), after one-cycle (n = 35) and on disease progression (n = 45) by CellSearch and double immunofluorescence using anti-CKs and anti-Ki67, anti-M30 or anti-Vimentin antibodies. Before treatment, CTCs were detected in 50% of patients by CellSearch whereas 53.4%, 15.5% and 74.1% patients had CK + /Ki67 + , CK + /M30 + and CK + /Vim + CTCs, respectively. One pazopanib cycle significantly decreased the number of CTCs as detected by CellSearch ( p = 0.043) as well as the number of CK + /Ki67 + ( p < 0.001), CK + /M30 + ( p = 0 . 015) and CK + /Vim + ( p < 0 . 001) cells. On disease progression, both the incidence and CTC numbers were significantly increased (CellSearch, p = 0.027; CK + /Ki67 + , p < 0.001; CK +/ M30 + , p = 0.001 and CK + /Vim + , p < 0 . 001) . In multivariate analysis, the detection of CK + /Vim + CTCs after one treatment cycle (HR: 7.9, 95% CI: 2.9–21.8; p < 0.001) and CTCs number on disease progression, as assessed by CellSearch, (HR: 2.0, 95% CI: 1.0–6.0; p = 0.005) were emerged as independent factors associated with decreased OS. In conclusion, pazopanib can eliminate different CTC subpopulations in patients with relapsed SCLC. The analysis of CTCs could be used as a dynamic biomarker of treatment efficacy.

Background: We directly compared CTC detection rates and prognostic significance, using three different methods in patients with breast cancer (BC). Methods: Early (n=200) and metastatic (n=164) patients were evaluated before initiating adjuvant or first-line chemotherapy, using the CellSearchTM System, an RT-qPCR for CK-19 mRNA detection and by double immunofluorescence (IF) microscopy using A45-B/B3 and CD45 antibodies. Results: Using the CellSearchTM System, 37% and 16.5% of early BC patients were CTC-positive (at ≥1 and ≥2 CTCs/23 ml of blood), 18.0% by RT-qPCR and 16.9% by IF; no agreement was observed between methods. By the CellSearchTM 34.8% and 53.7% (at≥ 5 and ≥ 2 CTCs/7.5 ml) of metastatic patients were CTC-positive, 37.8% by RT-qPCR and 28.5% by IF. A significant agreement existed only between the CellSearchTM and RT-qPCR. In 60.8% of cases, differential EpCAM and CK-19 expression on CTCs by IF could explain the discrepancies between the CellSearchTM and RT-qPCR. CTC-positivity by either method was associated with decreased overall survival in metastatic patients. Conclusion: A significant concordance was observed between the CellSearchTM and RT-qPCR in metastatic but not in early BC. Discordant results could be explained in part by CTC heterogeneity. CTC detection by all methods evaluated had prognostic relevance in metastatic patients.

The aim of the study was to evaluate the phenotypic CTCs heterogeneity (TTF-1 + and/or CD56 + ) in SCLC patients and correlate it with the CellSearch. Peripheral blood was obtained from 108 consecutive patients. CTCs were detected by CellSearch and double-immunofluorescence using anti-CD45, anti-TTF-1 and anti-CD56 antibodies. Before chemotherapy TTF-1 + /CD45 − , CD56 + /CD45 − and TTF-1 + /CD56 + CTCs were detected in 66(61.1%), 55(50.9%) and 46(42.6%) patients, respectively; 60.2% of patients were CellSearch + . Among the 22 patients with 0 CTCs/7.5 ml on CellSearch, TTF-1 + /CD45 − , CD56 + /CD45 − and TTF-1 + /CD56 + CTCs were detected in 8(36.4%), 6(27.3) and 6(27.3%) patients, respectively; no CK + /EpCAM + or TTF1 + /EpCAM + CTCs were detected in these patients. One-chemotherapy cycle decreased both the number of positive patients ( p < 0.001) and their CTC number ( p < 0.001), irrespectively of their phenotype and the detection method. The incidence and number of the different CTC subpopulations on PD, was significantly increased at their baseline levels. Multivariate analysis revealed that the increased number of CTCs at baseline and on PD were significantly associated with decreased PFS ( p = 0.048) and OS ( p = 0.041), respectively. There is an important CTC heterogeneity in such patients according to the expression of TTF-1 and CD56 which could detect EpCAM − CTC subpopulations and, thus, undetectable by CellSearch. These CTC subpopulations are dynamically correlated with treatment efficacy and disease-progression.

The detection of circulating tumor cells (CTCs) is of prognostic significance in several tumor types. The present study evaluated the detection and the clinical relevance of CK19mRNA(+) CTCs in patients with advanced/metastatic non-small cell lung cancer before and after front-line chemotherapy.

The accumulation of Myeloid-derived Suppressor Cells (MDSCs) and the Circulating Tumor Cells (CTCs) have been proposed as negative prognostic biomarkers in several tumors, including SCLC. However, no studies have shown a correlation of the MDSCs with CTCs in SCLC patients. We aimed to investigate the clinical relevance of CTCs and MDSCs in progressing patients with advanced SCLC. Peripheral blood was obtained from 32 SCLC patients at the time of progression after 1st line chemotherapy and 31 healthy controls (HC). Immune cells were determined using flow cytometry and CTCs were detected using both the CellSearch System (CS) and immunofluorescence double staining of PBMCs with anti-TTF1 and/or anti-CD56 and anti-CD45 antibodies (IF). The median percentage of patients MDSCs at baseline was used to characterize MDSCs as high or low. For the CTCs detection using the CS and IF, the cut-off values were ≥5 and ≥1 CTCs, respectively. The percentage of naïve CD4+ T cells was decreased in patients vs NC at baseline. The whole population of MDSCs (CD33+Lin-HLA-DR-/low), as well as the granulocytic (G)-(CD15+CD14-CD33+CD11b+) and the monocytic (M)-MDSCs (CD14+CD15+CD33+CD11b+Lin-HLA-DR-/low) were significantly increased in patients (p < 0.0001). Only M-MDSCs levels were significantly correlated with TTF1+ (p <0.018) but not with CD56+ or TTF1+/CD56+ CTCs. Conversely, there was no correlation of the different MDSCs subtypes with the CTCs detected using the CS. Patients with high M-MDSCs levels had a higher absolute number of TTF-1+ CTCs compared to patients with low M-MDSC levels (Mean: 46 vs 2 cells; p = 0.02) whereas the frequency of M-MDSCs was higher in patients with TTF-1+ CTCs compared to those with TTF-1- CTCs (mean: 4.6% vs 1.7%; p = 0.01). We showed an increased frequency of M-MDSCs in the blood of patients with SCLC at relapse exhibiting a positive correlation with CTCs. These observations seem to indicate that some CTCs' subpopulations may be escape and proliferate in patients with active immunosuppressive mechanisms.

8562 Background: To investigate the effect of 2nd line PZN on the number and phenotype of CTCs in patients with SCLC. Methods: CTCs were detected in58 pts using the CelleSearch assay (CS) and double immunofluorescece (IF) staining with anti-CK, anti-CD56 and anti-TTF1 antibodies, before, after 1 cycle of PZN and at the time of disease progression (PD). Results: Using the CS and IF, CTCs were detected in 50% and 51.8% of pts, respectively. Patients with < 5 CTCs/7.5 ml of blood had a significantly higher probability to respond to PZN compared to patients with > 5 CTCs/7.5 ml of blood (p= 0.006). After 1 cycle of PZN, the decrease of the median number of CTCs was associated with the probability to achieve an objective response (p= 0.006). In 48.1% of the pts with < 5 CTCs/7.5ml of blood at the time of PD, CTCs could be detected using IF. The detection of > 5 CTCs/7.5 ml of blood at baseline was associated with a decreased PFS (p< 0.001) and OS (p= 0.001) and multivariate analysis revealed that the number of CTCs after 1 cycle of PZN was an independent factor associated with OS (p= 0.005). Conclusions: The enumeration and phenotypic characterization of CTCs in patients with SCLC treated with PZN are of clinical relevance and could be considered as a dynamic biomarker for the monitoring of treatment efficacy.

Abstract Introduction: The aim of our study was to evaluate the prognostic significance of our previously developed EpCAM independent RT-qPCR assay for CK-19 mRNA (Stathopoulou et al, Int J Cancer 2006) and validate its analytical and clinical performance in early breast cancer. Methods: Quality control on analytical sensitivity and specificity, linearity, intra- and inter-assay reproducibility, and stability of the external standard used for the preparation of the calibration curves was performed. Reproducibility of the assay between our labs was evaluated by analyzing 26 cDNAs. The prognostic significance of the assay in respect to DFI and OS was evaluated by analysing peripheral blood of 179 patients with stage I/II breast cancer postoperatively, before the administration of adjuvant chemotherapy. Results: The limit of detection (LOD) is 3 copies/reaction and limit of Quantitation (LOQ) 10 copies/reaction. The linear range of the calibration curve was 10-105copies/reaction and the intra- and inter-assay reproducibility are shown below: CK19 transcriptsReproducibility of the assay (n = 3)copies/reactionIntra-assay RSD (%)Inter-assay RSD (%)101.973.151020.932.241030.450.861040.122.581050.11.52 The inter-lab reproducibility of the assay was evaluated by analyzing 26 cDNA samples in both labs and there was a 100% concordance. During the follow up period (8 years) 32/179 (17.9%) patients relapsed and 18/179 (10%) patients died from the disease. 45/179 (25.1%) samples were found positive for CK-19 and 134/179 (74.9%) negative. In the group of CK-19 positive patients 15/45(33.3%) relapsed and 9/45(20.0%) died while in the group of CK-19 negative patients 17(12.7%) patients relapsed and 9(6.7%) died. Kaplan-Meier analysis showed that CK-19 mRNA positivity was significantly associated with DFI (P = 0.014) and OS (P = 0.051). Conclusions: This EpCAM independent assay can be used for a high-throughput detection of CTCs in peripheral blood and has prognostic significance in early breast cancer. Citation Format: Areti Strati, Athina Markou, Aliki Stathopoulou, Stella Apostolaki, Dimitris Mavroudis, Vasilis Georgoulias, Evi S. Lianidou. Analytical and clinical validation of an EpCAM-independent assay for CTC detection in peripheral blood of early breast cancer patients based on Cytokeratin-19 (CK-19) RT-qPCR. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 373. doi:10.1158/1538-7445.AM2015-373

Abstract Background: The utility of CTC enumeration in predicting patient (pt) outcome has been demonstrated in metastatic breast cancer (MBC) treated with chemotherapy or endocrine therapy. In this study we evaluated the clinical impact of CTC assessment in terms of both enumeration and characterization in breast cancer pts treated with exemestane plus everolimus. Patients and methods: Thirty-nine pts with hormone receptor (HR)-positive, HER2-negative MBC, received exemestane plus everolimus. CTC enumeration in peripheral blood (7.5 ml) was performed before treatment (n=39), post cycles 1 (n=39) and 3 (n=29), on disease re-evaluation and on relapse, whichever occurred first, using the CellSearch System. CTC characteristics were determined at the same time points by immunofluorescence (IF) analysis of PBMC cytospins (106 cells), triple stained with pancytokeratin (CK) antibody along with Ki67 and M30 as proliferation and apoptosis markers, respectively, using the Ariol System. Patients were assessed by CT scans and bone scan, every 3 months or as clinically indicated. Results: At the cut-off of ≥ 1 CTC, 25 of 39 (64%) pts had detectable CTCs at baseline, 12 (31%) of 39 post-1st and 10 (34.5%) of 29 post-3rd cycle. Ten (25.6%) pts remained CTC(+) and 12 (30.8%) CTC(-) both at baseline and post-1st cycle; 15 (38.5%) CTC(+) pts turned to CTC(-) and 2 (5%) CTC(-) turned to (+). CTC positivity after the first cycle was associated with shorter median progression-free survival (PFS) compared to CTC(-) status (3.9 vs 8 mo, p=0.031). Shorter PFS was also recorded for pts that remained CTC(+) at both time points compared to all other (p=0.02). At the cut-offs of ≥ 2 and ≥ 5 CTCs, 16 (41%) and 9 (23%) pts were CTC(+) at baseline, respectively; post-1st cycle, 7 (18%) and 4 (10%) pts were CTC(+) (at ≥ 2 and ≥ 5 CTCs, respectively). Post-3rd cycle the positivity rate was 17% for both cut-offs and these pts had significantly shorter PFS compared to CTC(-) pts (3.7 vs 8.7 months, p=0.048). Efficacy assessment revealed partial response in 3 (7.7%) pts, stable disease in 27 (69.23%) and progressive disease (PD) in 8 (20.5%); 1 pt was non-evaluable for response. Among pts determined CTC(+) post-1st cycle (cut-off ≥ 2 CTCs), 57% progressed compared to 13% of CTC(-) pts (p=0.02). In addition, at the post-3rd cycle evaluation, pts with PD had significantly higher CTC counts compared to non-progressors (mean ± SEM; 10 ± 5.78/pt vs 1.62±0.83/pt, p=0.027). By the use of IF 43%, 44% and 40% of CTC(+) pts had proliferative [Ki67(+)/M30(-)] CTCs at baseline, post -1st and -3rd cycles, respectively (cut-off ≥ 1 CTC); 67%, 50% and 50% of those pts, respectively, experienced PD. Apoptotic [Ki67(-)/M30(+)] CTCs were detected in 14%, 22% and 60% of CTC(+) pts at baseline, post -1st and -3rd cycles, respectively; none of the pts with apoptotic CTCs experienced PD. Conclusions: CTC enumeration and characterization in terms of proliferation and apoptosis during the course of treatment has significant predictive and prognostic implications in patients with MBC receiving the combination of exemestane plus everolimus. Citation Format: Sofia Agelaki, Dimitris Mavroudis, Maria Spiliotaki, Eleni Politaki, Maria A Papadaki, Stella Apostolaki, Christos Nikolaou, Vassilis Georgoulias. CTC enumeration and characterization has predictive and prognostic implications in patients with metastatic breast cancer treated with exemestane plus the mTOR inhibitor everolimus [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-01-13.

7573 Background: The diagnosis of SCLC is based on morphology and immunocytochemistry using cytokeratins and neuroendocrine markers. We aimed to evaluate the clinical significance of the detection of Circulating Tumor Cells (CTCs) in patients with SCLC using the CellSearch system (CS) and immunofluorescence (IF). Methods: Peripheral blood was obtained from 101 patients with SCLC before treatment, 68 patients after one cycle of 1st line chemotherapy (etoposide/platinum) and 52 patients at the time of disease progression. CTCs were detected by double immunofluorescent staining of one million PBMC’s on cytospins using anti-TTF1 and anti-CD56 antibodies, and by the CS using anti-CK antibody. Results: Prior to the initiation of 1st line treatment, 61% of patients had detectable CTCs by IF and 78% with the CS (range: 1-169 and 2-10000 CTCs, respectively). After 1 cycle of chemotherapy, the positivity of CTC detection decreased to 44% (range: 1-56 CTCs) and 45% (range: 1-4882 CTCs) using IF and CS, respectively; at the time of disease progression (PD) the detection of CTCs almost reached the baseline levels (60% and 74%; range: 2-186 and 1-11143 CTCs for IF and CS, respectively). The detection of CTCs at baseline, after 1 cycle of chemotherapy and on PD was significantly associated with decreased PFS (7.0 vs 8.5 months, p = 0.021; 5.6 vs 8.1 months, p = 0.018; 5.7 vs 7.6 months, p = 0.014, respectively) and OS (11.0 vs 26.0 months, p = 0.021; 8.9 vs 17.0 months, p = 0.009; 10.0 vs 17.0 months, p = 0.021, respectively) compared to the patients with undetectable CTCs. Multivariate analysis revealed that performance status, disease stage and the detection of CTCs after 1 cycle of chemotherapy and at the time of progression emerged as independent factors associated with reduced PFS and OS. Conclusions: Detection of CTCs using either immunofluorescence or the CellSearch is an adverse prognostic factor correlated with poor clinical outcome in patients with SCLC receiving 1st line chemotherapy.

11042 Background: The utility of CTCs in predicting patient (pt) outcome has been demonstrated in metastatic breast cancer treated with chemotherapy or endocrine therapy. In this trial we evaluated the prognostic impact of CTC assessment in pts treated with exemestane plus the targeted therapy everolimus. Methods: Forty pts with HER2-negative metastatic breast cancer received exemestane plus everolimus. Blood was drawn for CTC assessment by CellSearch System and immunofluorescence (IF) analysis (7.5 and 10 ml, respectively) on day 1 of cycles 1 (n=40), 2 (n=39), 4 (n=28), on each disease evaluation and on relapse, whichever occurred first. Cytospins (106cells) of peripheral blood mononuclear cells (PBMCs) from 31 of 40 pts were double stained with pancytokeratin (CK) along with Ki-67 antibodies. Results: Using CellSearch 25 (62.5%) pts had detectable CTCs (cut-off ≥ 1 CTC) at baseline, 12 (30.8%) post-1st and 9 (32.1%), post-third cycle. In 9 pts no CTCs were observed at any time point. CTCs ≥5 were identified in 10 (25%), 6 (15.4%) and 3 (10.7%) pts at baseline, post-firstand post-third cycles, respectively. Four (40%) pts with baseline counts of CTCs ≥5 turned to <5 post-first ycle. Total CTC counts declined from 332 at baseline (mean number/pt 8.3, range 0-103) to 90 (mean 2.3, range 0-34) and 46 (mean 1.64, range 0 - 16) post-first and -third cycles, respectively. Using IF, 7 (22.5%) pts had detectable CK(+) CTCs (cutoff ≥ 1 CTC) at baseline; 8 (25.8%) of 31 and 7 (28%) of 25 were CTC(+) post -first and -third cycles, respectively. Total CTC numbers were 197 at baseline (mean, 16.42, range, 0-192) and 58 (mean 1.87, range 0-34) and 336 (mean 13.44, range 0 - 290) post-first and -third cycle, respectively; the ratio of Ki-67(+)/CK(+) CTCs declined from 32% to 13.8% and 13.4%, post-first and -third cycle, respectively. Conclusions: Exemestane plus everolimus effectively decreases CTC counts and the population of Ki-67(+) CTCs in HER2-negative metastatic breast cancer. Correlations with patient characteristics and outcome will be presented.

We assessed the association of cytokeratin (CK)-19 mRNA in blood before and after adjuvant chemotherapy and the mitotic activity index (MAI) of primary tumors in 223 early-stage breast cancer patients. Our results indicate that MAI and detection of CK-19 mRNA-positive cells before adjuvant chemotherapy are independent prognostic variables associated with clinical outcome.Background: Previous studies showed that molecular detection of CK-19 mRNA in peripheral blood and the mitotic index of primary tumors have prognostic value in early breast cancer. The aim of this study was to assess the association between these variables. Patients and Methods: The primary tumors of 223 operable breast cancer patients (92 premenopausal and 131 postmenopausal) were evaluated for the MAI classified as either <= 5 per 10, 6 to 10 per 10 and > 10 per 10 or < 10 per 10 and > 10 per 10 mitoses per high power field using a standardized protocol previously reported. Peripheral blood was also collected before and after the end of adjuvant chemotherapy for detection of CK-19 mRNA-positive cells using reverse transcription polymerase chain reaction previously described. Results: After a median follow-up of 118 months, 75 patients (33.6%) experienced disease relapse and 56(25.1%) died of breast cancer. MAI was strongly associated with disease-free survival (DFS) and overall survival (OS) (P < .001 for DFS and OS together). Detecting CK-19 mRNA-positive cells in the peripheral blood before but not after adjuvant chemotherapy was associated with marginally worse DFS (P = .055) and OS (P = .059). Cox regression analysis revealed that MAI and CK-19 mRNA-positive cell detection before adjuvant chemotherapy were independent variables associated with decreased DFS (P < .001 and P = .038, respectively) and OS (P < .001 and P = .029, respectively). There was no significant interaction between MAI and detection of CK-19 mRNA-positive cells. Conclusion: MAI of the primary tumor and detection of CK-19 mRNA-positive cells in the blood before adjuvant chemotherapy in early breast cancer patients are 2 independent prognostic factors associated with clinical outcome.

Circulating tumor cells (CTCs) have been studied in breast cancer with the CellSearch® system. Given the low CTC counts in non-metastatic breast cancer, it is important to evaluate the inter-reader agreement.

To evaluate the effect of front-line chemotherapy on CK-19mRNA+ circulating tumor cells (CTCs) and their relevance in patients with metastatic breast cancer (MBC).

Introduction Circulating tumor cells (CTCs) could represent a non-invasive source of cancer cells used for longitudinal monitoring of the tumoral mutation status throughout the course of the disease. The aims of the present study were to investigate the detection of KRAS mutations in CTCs from patients with metastatic colorectal cancer (mCRC) and to compare their mutation status during treatment or disease progression with that of the corresponding primary tumors. Materials and Methods Identification of the seven most common KRAS mutations on codons 12 and 13 was performed by Peptide Nucleic Acid (PNA)-based qPCR method. The sensitivity of the assay was determined after isolation of KRAS mutant cancer cells spiked into healthy donors' blood, using the CellSearch Epithelial Cell kit. Consistent detection of KRAS mutations was achieved in samples containing at least 10 tumor cells/7.5 ml of blood. Results The clinical utility of the assay was assessed in 48 blood samples drawn from 31 patients with mCRC. All patients had PIK3CA and BRAF wild type primary tumors and 14 KRAS mutant tumors. CTCs were detected in 65% of specimens obtained from 74% of patients. KRAS mutation analysis in CTC-enriched specimens showed that 45% and 16.7% of patients with mutant and wild type primary tumors, respectively, had detectable mutations in their CTCs. Assessing KRAS mutations in serial blood samples revealed that individual patient's CTCs exhibited different mutational status of KRAS during treatment. Conclusions The current findings support the rationale for using the CTCs as a dynamic source of tumor cells which, by re-evaluating their KRAS mutation status, could predict, perhaps more accurately, the response of mCRC patients to targeted therapy.

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. 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. 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). 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.