International study on inter-reader variability for circulating tumor cells in breast cancer

Introduction 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. Methods CellSearch® images (N = 272) of either CTCs or white blood cells or artifacts from 109 non-metastatic (M0) and 22 metastatic (M1) breast cancer patients from reported studies were sent to 22 readers from 15 academic laboratories and 8 readers from two Veridex laboratories. Each image was scored as No CTC vs CTC HER2- vs CTC HER2+. The 8 Veridex readers were summarized to a Veridex Consensus (VC) to compare each academic reader using % agreement and kappa (κ) statistics. Agreement was compared according to disease stage and CTC counts using the Wilcoxon signed rank test. Results For CTC definition (No CTC vs CTC), the median agreement between academic readers and VC was 92% (range 69 to 97%) with a median κ of 0.83 (range 0.37 to 0.93). Lower agreement was observed in images from M0 (median 91%, range 70 to 96%) compared to M1 (median 98%, range 64 to 100%) patients (P < 0.001) and from M0 and <3CTCs (median 87%, range 66 to 95%) compared to M0 and ≥3CTCs samples (median 95%, range 77 to 99%), (P < 0.001). For CTC HER2 expression (HER2- vs HER2+), the median agreement was 87% (range 51 to 95%) with a median κ of 0.74 (range 0.25 to 0.90). Conclusions The inter-reader agreement for CTC definition was high. Reduced agreement was observed in M0 patients with low CTC counts. Continuous training and independent image review are require

Gene expression profile of circulating tumor cells in breast cancer by RT-qPCR

<p>Abstract</p> <p>Background</p> <p>Circulating tumor cells (CTCs) have been associated with prognosis especially in breast cancer and have been proposed as a liquid biopsy for repeated follow up examinations. Molecular characterization of CTCs is difficult to address since they are very rare and the amount of available sample is very limited.</p> <p>Methods</p> <p>We quantified by RT-qPCR <it>CK-19, MAGE-A3, HER-2, TWIST1, hTERT α+β+</it>, and <it>mammaglobin </it>gene transcripts in immunomagnetically positively selected CTCs from 92 breast cancer patients, and 28 healthy individuals. We also compared our results with the CellSearch system in 33 of these patients with early breast cancer.</p> <p>Results</p> <p>RT-qPCR is highly sensitive and specific and can detect the expression of each individual gene at the one cell level. None of the genes tested was detected in the group of healthy donors. In 66 operable breast cancer patients, <it>CK-19 </it>was detected in 42.4%, <it>HER-2 </it>in 13.6%, <it>MAGE-A3 </it>in 21.2%, <it>hMAM </it>in 13.6%, <it>TWIST-1 </it>in 42.4%, and <it>hTERT α+β+ </it>in 10.2%. In 26 patients with verified metastasis, <it>CK-19 </it>was detected in 53.8%, <it>HER-2 </it>in 19.2%, <it>MAGE-A3 </it>in 15.4%, <it>hMAM </it>in 30.8%, <it>TWIST-1 </it>in 38.5% and <it>hTERT </it>α⁺β⁺in 19.2%. Our preliminary data on the comparison between RT-qPCR and CellSearch in 33 early breast cancer patients showed that RT-qPCR gives more positive results in respect to CellSearch.</p> <p>Conclusions</p> <p>Molecular characterization of CTCs has revealed a remarkable heterogeneity of gene expression between breast cancer patients. In a small percentage of patients, CTCs were positive for all six genes tested, while in some patients only one of these genes was expressed. The clinical significance of these findings in early breast cancer remains to be elucidated when the clinical outcome for these patients is known.</p

International study on inter-reader variability for circulating tumor cells in breast cancer

Introduction: 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.Methods: CellSearch® images (N = 272) of either CTCs or white blood cells or artifacts from 109 non-metastatic (M0) and 22 metastatic (M1) breast cancer patients from reported studies were sent to 22 readers from 15 academic laboratories and 8 readers from two Veridex laboratories. Each image was scored as No CTC vs CTC HER2- vs CTC HER2+. The 8 Veridex readers were summarized to a Veridex Consensus (VC) to compare each academic reader using % agreement and kappa (κ) statistics. Agreement was compared according to disease stage and CTC counts using the Wilcoxon signed rank test.Results: For CTC definition (No CTC vs CTC), the median agreement between academic readers and VC was 92% (range 69 to 97%) with a median κ of 0.83 (range 0.37 to 0.93). Lower agreement was observed in images from M0 (median 91%, range 70 to 96%) compared to M1 (median 98%, range 64 to 100%) patients (P < 0.001) and from M0 and <3CTCs (median 87%, range 66 to 95%) compared to M0 and ≥3CTCs samples (median 95%, range 77 to 99%), (P < 0.001). For CTC HER2 expression (HER2- vs HER2+), the median agreement was 87% (range 51 to 95%) with a median κ of 0.74 (range 0.25 to 0.90).Conclusions: The inter-reader agreement for CTC definition was high. Reduced agreement was observed in M0 patients with low CTC counts. Continuous training and independent image review are required

The clinical use of circulating tumor cells (CTCs) enumeration for staging of metastatic breast cancer (MBC): International expert consensus paper

BACKGROUND: The heterogeneity of metastatic breast cancer (MBC) necessitates novel biomarkers allowing stratification of patients for treatment selection and drug development. We propose to use the prognostic utility of circulating tumor cells (CTCs) for stratification of patients with stage IV disease. METHODS: In a retrospective, pooled analysis of individual patient data from 18 cohorts, including 2436 MBC patients, a CTC threshold of 5 cells per 7.5\u2009ml was used for stratification based on molecular subtypes, disease location, and prior treatments. Patients with 65 5 CTCs were classified as Stage IVaggressive, those with < 5 CTCs as Stage IVindolent. Survival was analyzed using Kaplan-Meier curves and the log rank test. RESULTS: For all patients, Stage IVindolent patients had longer median overall survival than those with Stage IVaggressive (36.3 months vs. 16.0 months, P\u2009<\u20090.0001) and similarly for de novo MBC patients (41.4 months Stage IVindolent vs. 18.7 months Stage IVaggressive, p\u2009<\u20090.0001). Moreover, patients with Stage IVindolent disease had significantly longer overall survival across all disease subtypes compared to the aggressive cohort: hormone receptor-positive (44 months vs. 17.3 months, P\u2009<\u20090.0001), HER2-positive (36.7 months vs. 20.4 months, P\u2009<\u20090.0001), and triple negative (23.8 months vs. 9.0 months, P\u2009<\u20090.0001). Similar results were obtained regardless of prior treatment or disease location. CONCLUSIONS: We confirm the identification of two subgroups of MBC, Stage IVindolent and Stage IVaggressive, independent of clinical and molecular variables. Thus, CTC count should be considered an important tool for staging of advanced disease and for disease stratification in prospective clinical trials

Articles Clinical validity of circulating tumour cells in patients with metastatic breast cancer: a pooled analysis of individual patient data

Summary Background We aimed to assess the clinical validity of circulating tumour cell (CTC) quantifi cation for prognostication of patients with metastatic breast cancer by undertaking a pooled analysis of individual patient data

Comparative study between molecular methods and immunofluorescence for the detection of circulating tumor cells in the blood of patients with breast cancer

Background: Several techniques have been developed for the detection of circulating tumor cells (CTCs) in patients with breast cancer. We designed this study to compare the CellSearch system, with real time reverse transcription quantitative polymerase chain reaction (RT-qPCR) and an immunofluorescent method for CTC detection in patients with breast cancer. Methods: Blood was obtained from patients with early and metastatic breast cancer before the initiation of adjuvant or first-line chemotherapy. Different aliquots of the same blood sample were evaluated for the presence of CTCs by the CellSearch System, the RT-qPCR and by double immunofluorescence (IF) microscopy.Results: In early breast cancer, using the CellSearch System, 37% and 16.5% of patients were CTC-positive (cut-offs ≥1 and ≥2 CTCs per 23 ml of blood), 18.0% by RT-qPCR and 16.9% by IF. In the metastatic setting, 34.8% of patients had ≥ 5 and 53.7% had ≥ 2 CTCs per 7.5 ml of blood, 37.8% were CTC-positive by RT-qPCR, and 28.5% by IF. In 60.8% of cases presenting discordant results by CellSearch and RT-qPCR, the differential expression of EpCAM (Epithelial cell adhesion molecule) and CK19 on CTCs, could explain the discrepancies observed. Conclusion: A significant concordance in CTC detection between the CellSearch and RT-qPCR was observed in metastatic setting. CTC detection by either method had prognostic relevance in metastatic patients. Discordant results could be explained in part by CTC heterogeneity.Εισαγωγή: Πολλές τεχνολογίες απομόνωσης και ανίχνευσης των κυκλοφορούντων καρκινικών κυττάρων (circulating tumor cells, CTCs ή ΚΚΚ) έχουν αναπτυχθεί τα τελευταία χρόνια με ραγδαίο ρυθμό. Παρ΄ όλα αυτά η ασυμφωνία μεταξύ των διαφορετικών μεθόδων είναι μεγάλη, γεγονός που δυσχεραίνει την εφαρμογή τους στην καθημερινή κλινική πράξη. Σε αυτή την μελέτη, σκοπός είναι να συγκρίνουμε τρεις μεθοδολογίες ανίχνευσης των κυττάρων στο αίμα ασθενών με μεταστατικό και πρώιμο καρκίνο μαστού: την ποσοτική αλυσιδωτή αντίδραση της πολυμεράσης πραγματικού χρόνου [Reverse transcription quantitative polymerase chain reaction (RT-qPCR)] για την ανίχνευση του mRNA της κυτταροκερατίνης 19 [Cytokeratin 19 (CK19)], τον ανοσοφθορισμό σε επιστρωμένες αντικειμενοφόρες πλάκες και το εγκεκριμένο από τον Αμερικάνικο Οργανισμό Τροφίμων και Φαρμάκων (Food and Drug Administration, FDA) σύστημα Cell Search (Menarini, Silicon Biosystems, Italy), καθώς και να εκτιμήσουμε την αποτελεσματικότητα της ανίχνευσης σε σχέση με την κλινική έκβαση των ασθενών. Μέθοδοι: 200 ασθενείς με πρώιμο καρκίνο μαστού και 164 με μεταστατικό καρκίνο μαστού ελέγχθησαν πριν την έναρξη συμπληρωματικής ή πρώτης γραμμής θεραπείας χρησιμοποιώντας το σύστημα Cell Search , την RT-qPCR για την ανίχνευση του CK-19 mRNA και την τεχνική του ανοσοφθορισμού για την ανίχνευση των CTCs.Αποτελέσματα: Με το Cell Search System, το ποσοστό των ασθενών με πρώιμη νόσο που είχαν CTCs (cut off ≥1 και ≥2 CTCs σε 23 ml αίματος) ήταν 37% και 16.5% αντίστοιχα, με RT-qPCR 18.0% και με ανοσοφθορισμό 16.9%. Μεταξύ των μεθόδων δεν υπήρξε συμφωνία. Όσον αφορά στους μεταστατικούς ασθενείς, με το Cell Search System, ανιχνεύτηκαν CTCs στο 34.8% και 53.7% των ασθενών αντίστοιχα (για ≥ 5 και ≥ 2 CTCs σε 7.5 ml αίματος), με RT-qPCR 37.8% και με ανοσοφθορισμό 28.5%. Μία στατιστικά σημαντική συμφωνία παρατηρήθηκε μεταξύ του Cell Search System και της RT-qPCR. Σε ποσοστό 60.8% των περιπτώσεων, διαφορετική έκφραση του EpCAM (Epithelial cell adhesion molecule) και της CK-19 στα CTCs με ανοσοφθορισμό θα μπορούσε να εξηγήσει εν μέρει τις διαφωνίες μεταξύ CellSearch System και RT-qPCR. Στην ομάδα των ασθενών με μεταστατική νόσο η ανίχνευση των CTCs με οποιαδήποτε από τις τρείς μεθόδους σχετίζονταν με μειωμένη συνολική επιβίωση. Συμπέρασμα: Μία στατιστικά σημαντική συμφωνία παρατηρήθηκε όσον αφορά την ανίχνευση των CTCs με την τεχνολογία Cell Search System και RT-qPCR στους μεταστατικούς ασθενείς και όχι στους ασθενείς με πρώιμη νόσο μαστού. Εξαιτίας της ετερογένειας των καρκινικών κυττάρων, κάποιες διαφορές μεταξύ των αποτελεσμάτων θα μπορούσαν να δικαιολογηθούν. Η ανίχνευση των CTCs με όλες τις μεθόδους αποδείχθηκε ότι είχε προγνωστική σημασία στις ασθενείς με μεταστατική νόσο