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.

Introduction: The purpose of this study was to study the efficacy of subsequent treatment lines for metastatic breast cancer (MBC), as well as the association between radiologic objective response rate (ORR) and overall survival (OS). Methods: In this retrospective study, consecutive patients treated for MBC in two centers in Greece from January 1, 1992, to December 31, 2016, were identified and clinicopathologic data regarding tumor characteristics and administered treatments were collected. The efficacy per treatment line in terms of ORR, progression-free survival (PFS) and OS, as well as the prognostic value of ORR at first line were investigated. Results: A total of 977 patients with MBC were identified; 950 received any treatment. At first line, ORR was 43.5%, PFS 11.4 months (95% CI 10.4–12.4), and median OS 52.4 months (95% CI 47.7–57.1). Lower ORR and shorter PFS were observed with each subsequent line. Median OS was significantly longer for patients that had an objective response at first line, 61.9 months (95% CI 51.1–69.7) for responders versus 41.3 months (95% CI 44.1–63.3) for nonresponders (p < 0.001). In multivariable analysis, failure to achieve an objective response was an independent predictor of poor survival (hazard ratio 1.70, 95% CI 1.34–2.15, p < 0.001). Conclusion: Late treatment lines for MBC seem to have limited efficacy, while response to first-line therapy is associated with long-term survival. The latter should be considered in the treatment strategy of patients with MBC.

The objective of this study is the determination of the various stages of deformation of brecciated marbles, under uniaxial compression, and the application of regression techniques, to investigate the relations of these stages, with physical, dynamical, and mechanical properties. Therefore, a total number of fifteen specimens, prepared from samples obtained from various locations from the southern part of the East Attica Prefecture, Greece. The laboratory program involved the determination of the uniaxial compressive strength (UCS), the crack initiation (Cci) and crack damage stress (Ccd) levels, the static elastic modulus (Es), as well as the basic physical and dynamical properties, such as the dry density (γd), the effective porosity (neff) and the ultrasonic velocities of both primary (VP) and secondary (VS) waves. Results obtained from the mechanical tests reveal that the onset of stable (Cci) and unstable (Ccd) crack growth varies between 0.19–0.38 and 0.66–0.92 of the UCS values, respectively. Applying simple nonlinear regression models, it was found that crack initiation and crack damage stresses, decrease exponentially with the increase of effective porosity, while increase exponentially with the increase of static elastic modulus, dynamic elastic modulus (Ed), and the primary wave velocity.

The National Archaeological Museum in Athens is the largest archaeological museum in Greece, and one of the most important museums in the world, devoted to Ancient Greek art and history. Among other exhibits, it owns a large collection of gemstones some of which were used during prehistoric times (Mycenaean period) as seals. Their shape varies from round to oval, flattish or cylindrical, and they are delicately engraved as intaglios with a variety of depictions (lions, bulls, man, etc). They show a wide range of colours from reddish, brown, to purplish and blue. The six sealstones described here come from the Chamber Tombs of Mycenae (15-14 cent. BC). Museum exhibit labels recognize them as varieties of quartz such as jasper (NAM 3138), sardonyx (NAM 2316 & 2865), agate (NAM 4928), amazonite (NAM 2863) and gold-mounted carnelian (NAM 6489). Raman Spectroscopic analysis has been carried out with a new MRM (Mobile Raman Microscope) using a Kaiser Holoprobe with a NIR 785nm laser. The Raman spectra acquired from 1s-60s measurements confirmed that, in all six sealstones, quartz was the major mineral species clearly identified by its characteristic peaks. The second most important phase was moganite, a little-known polymorph of quartz. The amber-coloured sealstone (NAM 6489) was confirmed as carnelian, whereas the blue-green amazonite-coloured sealstone (NAM 2863) was not detected as amazonite. Small amounts of haematite were detected in the NAM 2865 & NAM 3138.

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 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.

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.

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).

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.

Background: To investigate the clinical relevance of CK-19mRNA-positive circulating tumour cells (CTCs) detected before the initiation of front-line treatment in patients with metastatic breast cancer (MBC). Methods: The presence of CTCs was detected in 298 patients with MBC using a real-time PCR (RT-PCR assay. In 44 patients, the detection of CTCs was evaluated by both the CellSearch and the RT-PCR assay. Interaction with known prognostic factors and association of CTCs with clinical outcome were investigated. Results: There was a strong correlation between the detection of CTCs by both assays. CK-19mRNA-positive CTCs were detected in 201 (67%) patients and their detection was independent of various patients’ clinico-pathological characteristics. The median progression-free survival (PFS; 9.2 vs 11.9 months (mo), P =0.003) and the overall survival (OS; 29.7 vs 38.9 mo, P =0.016) were significantly shorter in patients with detectable CK-19mRNA-positive CTCs compared with patients without detectable CTCs. Multivariate analysis demonstrated that oestrogen receptor status, performance status and detection of CTCs were emerged as independent prognostic factors associated with decreased PFS and OS. Conclusion: The detection of CK-19mRNA-positive CTCs in patients with MBC before front-line therapy could define a subgroup of patients with dismal clinical outcome.

BACKGROUND:Since the detection of circulating tumor cells (CTCs) which express HER2 is an adverse prognostic factor in early breast cancer patients, we investigated the effect of trastuzumab on patients' clinical outcome. PATIENTS AND METHODS:Seventy five women with HER2 (-) breast cancer and detectable CK19 mRNA-positive CTCs before and after adjuvant chemotherapy, were randomized to receive either trastuzumab (n=36) or observation (n=39). CK19 mRNA-positive CTCs were detected by RT-PCR and double stained CK(+)/HER2(+) cells by immunofluorescence. The primary endpoint was the 3-year disease-free survival rate. RESULTS:Fifty-one (89%) of the 57 analyzed patients had HER2-expressing CTCs. After trastuzumab administration, 27 of 36 (75%) women became CK19 mRNA-negative compared to seven of 39 (17.9%) in the observation arm (p=0.001). After a median follow up time of 67.2 months, four (11%) and 15 (38%) relapses were observed in the trastuzumab and observation arm, respectively (p=0.008); subgroup analysis indicated that this effect was mainly confined to women with >3 involved axillary lymph nodes (p=0.004). The median DFS was also significantly higher for the trastuzumab-treated patients (p=0.008). CONCLUSION:Administration of trastuzumab can eliminate chemotherapy-resistant CK19 mRNA-positive CTCs, reduce the risk of disease recurrence and prolong the DFS.

Abstract Background: Previous studies have shown that the molecular detection of CK-19 mRNA in peripheral blood and the mitotic index of primary tumor have prognostic value in early breast cancer. The aim of the present 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 mitotic activity index (MAI) classified either as <5/10, 6–10/10 and >10/10 or <10/10 and >10/10 mitoses/hpf using a standardized protocol as previously reported (Baak JP et al, Breast Cancer Res Treat 2009, 115:241–254). Peripheral blood was also collected before the start and after the end of adjuvant chemotherapy for detection of CK-19 mRNA-positive cells by RT-PCR as previously described (Stathopoulou et al, Clin Cancer Res 2003, 9:5145–5151). Results: After a median follow-up of 118 months, 75 (33.6%) patients have experienced disease relapse and 56 (25.1%) have died of breast cancer. MAI was strongly associated with disease-free survival (DFS) and overall survival (OS) (p<0.001 for both DFS and OS). Detection of CK19 mRNA-positive cells in the peripheral blood before but not after adjuvant chemotherapy was marginally associated with worse DFS (p=0.055) and OS (p=0.059). There was no correlation between MAI and CK19 mRNA detection before chemotherapy. Cox regression analysis revealed that both MAI and CK19 mRNA-positive cell detection before adjuvant chemotherapy were independent variables associated with decreased DFS (p<0.001 and p=0.038, respectively) and OS (p<0.001 and p=0.029, respectively). The interaction test showed no significant association between MAI and detection of CK19 mRNA-positive cells. Conclusion: MAI of the primary tumor and detection CK-19 mRNA-positive cells in the blood before the start of adjuvant chemotherapy in women with early breast cancer are two independent prognostic factors associated with clinical outcome. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-01-16.

BACKGROUND:The detection of cytokeratin-19 (CK-19) mRNA-positive circulating tumor cells (CTC) before and/or after adjuvant chemotherapy in patients with operable breast cancer is associated with poor clinical outcome. Reliable prognostic markers for late disease relapse are not available. In this study we investigated the value of CTC detection during the first five years of follow-up in predicting late disease relapse.METHODS:Blood was analyzed from 312 women with operable breast cancer who had not experienced disease relapse during the first two years of follow-up. A real-time reverse transcriptase polymerase chain reaction (RT-PCR) for CK-19 mRNA was used to detect CTC three months after the completion of adjuvant chemotherapy and every six months thereafter for a follow-up period of five years.RESULTS:Eighty patients (25.6% of the study population) remained CTC free throughout the five-year period. A change in CTC status was observed in 133 patients (42.6%); 64 patients (20.5%) with initially CK-19 mRNA-positive CTC during the first 24 months turned CTC-negative afterwards while 69 (22.1%) who were initially CTC-negative became CTC-positive. Ninety-nine patients (31.7%) remained persistently CK-19 mRNA-positive. After a median follow-up period of 107 months (range: 38 to 161 months), the persistently CTC-positive patients with either hormonal receptor positive or negative tumors, had a higher risk of late-disease relapse compared to the persistently CTC-negative patients (36.4% versus 11.2%, P <0.001). Multivariate analysis revealed that persistently CTC-positive patients also had a shorter disease-free (P = 0.001) and overall survival (P = 0.001).CONCLUSIONS:Persistent detection of CK-19 mRNA-positive CTC during the first five years of follow-up is associated with an increased risk of late relapse and death in patients with operable breast cancer and indicates the presence of chemo-and hormonotherapy-resistant residual disease. This prognostic evaluation may be useful when deciding on subsequent adjuvant systemic therapy.

Abstract Purpose: To evaluate the clinical relevance of circulating CEACAM5mRNA-positive cells in patients with operable colorectal cancer (CRC). Methods: Peripheral blood was obtained from 265 patients with operable CRC before the initiation of adjuvant systemic therapy from 96 normal donors and RNA prepared from the Lovo and ARH-77 CRC and leukemic cell lines, respectively, was used as positive and negative controls. The detection of CEACAM5mRNA-positive cells was done using a real-time PCR assay. The association with known prognostic factors and the effect of CEACAM5mRNA-positive cells on patients' prognosis was investigated. Results: The analytical detection limit of the method was found to correspond to 0.7 Lovo cell equivalence/5 μg RNA, with a sensitivity of 1 tumor cell/105 normal cells and a specificity of 97%. Ninety-eight (37%) patients had detectable circulating CEACAM5mRNA-positive cells. Detection of CEACAM5mRNA-positive cells was significantly associated with higher relapse rate (P < 0.001), decreased disease-free survival (DFS; P < 0.001), higher death rate (P = 0.017), and decreased median overall survival (P = 0.025). Multivariate analysis revealed that the detection of circulating CEACAM5mRNA-positive cells was an independent prognostic factor for decreased DFS [HR = 3.4; 95% CI: 2.0–5.9; P < 0.001]. Conclusions: Detection of peripheral blood CEACAM5mRNA-positive cells is an adverse prognostic factor correlated with poor clinical outcome in patients with operable CRC. Clin Cancer Res; 17(1); 165–73. ©2010 AACR.

10553 Background: To investigate the clinical relevance of CK-19mRNA-positive CTCs detected before the initiation of front-line treatment in patients with metastatic breast cancer (MBC).METHODSA total of 300 patients with MBC (training set: 200 patients; validation set: 100 patients) at first diagnosis were enrolled. The presence of CK-19 mRNA-positive CTCs was assessed by real-time reverse transcriptase polymerase chain reaction before any systemic treatment. Interaction with known prognostic factors and association of CTCs with clinical outcome were investigated.RESULTSCK-19 mRNA-positive cells were detected in 139 (69.5%) and 62 (62%) patients of the training and validation set, respectively. There was no association between the detection of CK-19 mRNA-positive CTCs and other clinico-pathological parameters, the tumor burden or the achievement of objective response in either set. The median PFS was significantly lower in patients with detectable CK-19 mRNA-positive CTCs compared to patients without detectable CTCs both in the training (9.0mo vs 12.0mo, respectively; p=0.018) and the validation (10.0mo vs 12.5mo, respectively; p=0.010) set. Similarly, the median OS was significantly higher in patients without detectable CK-19 mRNA-positive CTCs compared to patients with detectable CTCs both sets (training set: 41.4mo vs 29.9mo, respectively, p=0.014; validation set: 46.0mo vs 29.9mo, respectively, p=0.022). Multivariate analysis including all patients (n=300) demonstrated that ER status (HR=1.483; 95% CI: 1.140-1.928) and detection of CTCs (HR=1.440; 95% CI: 1.440-1.913) were independently associated with a decreased PFS and OS (ER status: HR=1.510, 95% CI: 1.085-2.113; detection of CTCs: HR=1.656, 95% CI: 1.148-2.390).CONCLUSIONSThese data indicate that the detection of CK-19 mRNA-positive CTCs in patients with MBC at first diagnosis defines a subgroup of patients with dismal clinical outcome.

Abstract Background: Different methods are available for the detection of CTCs in pts with BC, however, the variable performance of these assays, the heterogeneity of CTCs, and the possible treatment-induced alteration of the markers evaluated, imply that no ideal method currently exists. We compared the efficiency of two methods to detect CTCs in pts with BC. Patients and Methods: Blood was obtained from 200 pts with early and 164 with metastatic BC before the start of adjuvant or first-line chemotherapy, respectively. Different aliquots of the same sample were evaluated by RT-PCR for CK-19 mRNA and by the CellSearch System. Blood samples were available after the end of adjuvant or first-line therapy in 99 and 93 pts, respectively. CTCs in 23 and 7.5 ml of blood were enumerated by CellSearch, in adjuvant and metastatic pts, respectively. Cut-off values of ≥1 and ≥2 CTCs/23 ml and ≥2 and ≥5 CTCs/7.5 ml were used. Twenty ml of blood were obtained for mRNA extraction in all RT-PCR experiments. Results: In early BC, 18.0% of samples were positive prior to therapy by RT-PCR and 37.0% (CTC≥1) and 16.5% (CTC≥2) by CellSearch. No significant correlation was shown for the detection of CTCs between methods. Overall (positive and negative) concordance was 62% for CTC≥1 and 73.5% for CTC≥2 (Chi-Square, p=0.161 and p=0.307). Post-chemotherapy, the positivity rate was 33.6% (CTC≥1) and 18.8% (CTC≥2) by CellSearch and 11.6% by RT-PCR (Spearman, R=-0.031, p=0.761). Overall agreement was 60.6% (CTC≥1) and 71.4% (CTC≥2) (Chi-Square, p=0.771 and p=0.708). In the metastatic setting, 37.8% of the samples were positive by RT-PCR, and 50% (CTC≥2) or 32.3% (CTC≥5), by CellSearch (Spearman, R=0.373, p=0.0001). Overall agreement was 70.0% for CTC≥5 (Chi-Square, p=0.0001). Post-chemotherapy, 21% were positive by RT-PCR and 13.7% and 8.4% by CellSearch for CTC≥2 and ≥5, respectively (Spearman, R=0.194, p=0.063). Overall concordance for CTC≥5 was 79.6% (Chi-Square, p=0.017). Agreement was also observed for CTC≥2, pre-and post-chemotherapy (Chi-Square, p=0.0001 and p=0.010). The concordance rates in the adjuvant and metastatic settings are shown in Table 1. Conclusions: A high concordance between the two methods was detected in metastatic but not in early disease. Patient follow-up will determine the clinical relevance of each individual assay or their combination in the assessment of patient prognosis. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-07-21.