Circulating Tumor Cell Clusters in Patients with Metastatic Breast Cancer: a SWOG S0500 Translational Medicine Study.

PURPOSE: Metastasis requires malignant cell circulation from the primary to a distant tissue. Elevated levels of circulating tumor cells (CTC) portend a poor prognosis in breast and other cancers. Recent studies have suggested that CTC clusters may be a factor in the metastatic process. We conducted a prospective retrospective study of the SWOG0500 clinical trial to test whether CTC clusters are associated with poorer prognosis. EXPERIMENTAL DESIGN: CTC CellSearch galleries from SWOG0500 trial were reread using prespecified criteria for CTC clusters, doublets, and enumeration. Survival analysis methods include Kaplan-Meier plots and log-rank tests. RESULTS: Patients were classified into three prognostic subgroups based on baseline CTC/7.5 mL whole blood (WB): Arm A: <5CTC; Arm B/C: ≥5CTC and then B (<5CTC) and C (≥5CTC)/7.5 mL WB at first follow-up. At baseline, 19% of patients had CTC doublets or clusters, which were more likely in Arm B/C versus Arm A (38% vs. 1.4%; P < 0.0001). Furthermore, doublets or clusters were significantly more common in patients who were ultimately assigned to Arm C versus B (54% vs. 25%; P < 0.0001). In Arm C, doublets and clusters were associated with worse overall survival than only doublets, clusters, or no doublets nor clusters at baseline (P = 0.008) and first follow-up (P = 0.010). When compared with enumeration alone, doublets, clusters, or both were not prognostic in patients who had 5-19 or ≥20 CTC/7.5 mL WB. CONCLUSIONS: In patients with metastatic breast cancer starting first-line chemotherapy, mortality is independent of the presence of CTC clusters, but rather depends on the number of CTC/7.5 mL WB.

Enumeration and characterization of circulating multiple myeloma cells in patients with plasma cell disorders.

We have developed an automated assay to enumerate and characterize circulating multiple myeloma cells (CMMC) from peripheral blood of patients with plasma cell disorders. CMMC show expression of genes characteristic of myeloma and fluorescence in situ hybridisation results on CMMC correlated well with bone marrow results. We enumerated CMMC from over 1000 patient samples including separate cohorts of newly diagnosed multiple myeloma and high/intermediate risk smouldering multiple myeloma (SMM) with clinical follow-up data. In newly diagnosed myeloma patient samples, CMMC counts correlated with other clinical measures of disease burden, including the percentage of bone marrow plasma cells, serum M protein, and International Staging System stage. CMMC counts decreased significantly from baseline when a remission was achieved due to treatment (P < 0·001). Patients with CMMC counts ≥100 at remission showed reduced survival relative to patients with CMMC counts <100. Patients with undetectable CMMC in remission showed further overall survival benefits. In the SMM cohort, there was a trend toward higher CMMC in patients with higher-risk myeloma precursor states. Significantly higher CMMC counts were observed between intermediate/high risk SMM patients that progressed versus those without progression (P = 0·031). CMMC allow a non-invasive means of monitoring tumour biology and may have use as a prognostic test for patients with plasma cell disorders.

Prognostic and predictive value of circulating tumor cells and CXCR4 expression as biomarkers for a CXCR4 peptide antagonist in combination with carboplatin-etoposide in small cell lung cancer: exploratory analysis of a phase II study.

Background Circulating tumor cells (CTCs) and chemokine (C-X-C motif) receptor 4 (CXCR4) expression in CTCs and tumor tissue were evaluated as prognostic or predictive markers of CXCR4 peptide antagonist LY2510924 plus carboplatin-etoposide (CE) versus CE in extensive-stage disease small cell lung cancer (ED-SCLC). Methods This exploratory analysis of a phase II study evaluated CXCR4 expression in baseline tumor tissue and peripheral blood CTCs and in post-treatment CTCs. Optimum cutoff values were determined for CTC counts and CXCR4 expression in tumors and CTCs as predictors of survival outcome. Kaplan-Meier estimates and hazard ratios were used to determine biomarker prognostic and predictive values. Results There was weak positive correlation at baseline between CXCR4 expression in tumor tissue and CTCs. Optimum cutoff values were H-score ≥ 210 for CXCR4+ tumor, ≥7% CTCs with CXCR4 expression (CXCR4+ CTCs), and ≥6 CTCs/7.5 mL blood. Baseline H-score for CXCR4+ tumor was not prognostic of progression-free survival (PFS) or overall survival (OS). Baseline CXCR4+ CTCs ≥7% was prognostic of shorter PFS. CTCs ≥6 at baseline and cycle 2, day 1 were prognostic of shorter PFS and OS. None of the biomarkers at their respective optimum cutoffs was predictive of treatment response of LY2510924 plus CE versus CE. Conclusions In patients with ED-SCLC, baseline CXCR4 expression in tumor tissue was not prognostic of survival or predictive of LY2510924 treatment response. Baseline CXCR4+ CTCs ≥7% was prognostic of shorter PFS. CTC count ≥6 at baseline and after 1 cycle of treatment were prognostic of shorter PFS and OS.

Liquid biopsy-based clinical research in early breast cancer: The EORTC 90091-10093 Treat CTC trial.

There is increasing evidence that breast cancer evolves over time under the selection pressure of systemic treatment. Today, treatment decisions in early breast cancer are based on primary tumour characteristics without considering the disease evolution. Chemoresistant micrometastatic disease is poorly characterised and thus it is not used in current clinical practice as a tool to personalise treatment approaches. The detection of chemoresistant circulating tumour cells (CTCs) has been shown to be associated with worse prognosis in early breast cancer. The ongoing Treat CTC trial is the first international, liquid biopsy-based trial evaluating the concept of targeting chemoresistant minimal residual disease: detection of CTCs following adjuvant chemotherapy (adjuvant cohort) or neoadjuvant chemotherapy in patients who did not achieve pathological complete response (neoadjuvant cohort). This article presents the rational and design of this trial and the results of the pilot phase after 350 patients have been screened and provides insights that might provide information for future trials using the liquid biopsy approach as a tool towards precision medicine (NCT01548677).

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.

Monitoring apoptosis and Bcl-2 on circulating tumor cells in patients with metastatic breast cancer.

BACKGROUND: Enumeration of circulating tumor cells (CTC) from whole blood permits monitoring of patients with breast carcinoma. Analysis of apoptosis & Bcl-2 expression in CTC might add additional prognostic and predictive information. We estimated the degree of these markers in CTC from patients being treated for metastatic breast cancer. METHODS: Eighty-three evaluable patients initiating a new therapy for metastatic breast cancer were enrolled. Whole blood was collected at baseline, at one of three short term time windows (24, 48, or 72 h) after initiating treatment, and at first follow-up (3-5 weeks). CTC were isolated, enumerated, and expression of M30 and Bcl2 was determined using the CellSearch(®) System. RESULTS: At baseline, window, and 3-5 weeks post-treatment, 41/80 (51%), 40/80 (50%) and 21/75 (28%) patients had ≥5 CTC, respectively. At baseline, the proportion of CTC-apoptosis (M30) was inversely correlated with CTC number, and modestly inversely correlated with CTC-Bcl-2. As expected, higher CTC levels at baseline or first follow-up were associated with worse prognosis. Surprisingly, in patients with elevated CTC, higher levels of CTC-apoptosis were associated with worse prognosis, while higher CTC-Bcl-2 levels correlated with better outcomes. CONCLUSIONS: CTC apoptosis and expression of Bcl-2 can be analytically determined in patients with metastatic breast cancer and may have biological and clinical implications. Characterization of CTC for these and other markers could further increase the utility of CTC monitoring patients in clinical investigations of new anti-neoplastic agents.

Monitoring serial changes in circulating human breast cancer cells in murine xenograft models.

Circulating tumor cells (CTC) are emerging as a powerful prognostic and predictive biomarker in several types of cancer, including breast, colon, and prostate. Studies of CTC in metastasis and further development of CTC as a biomarker in cancer have been limited by the inability to repetitively monitor CTC in mouse models of cancer. We have validated a method to enumerate CTC in blood samples obtained from living mice using a modified version of an in vitro diagnostic system for quantifying CTC in patients. Different routes of blood collection were tested to identify a method to reproducibly recover CTC from tumor-bearing mice without interference from contaminating normal murine epithelial cells. CTC are present in blood samples from mice bearing orthotopic xenografts of several different breast cancer cell lines and primary breast cancer cells from patient biopsies. We also show that this technology can be used for serial monitoring of CTC in mouse xenograft models of human breast cancer. These results establish a new method for studying CTC in mouse models of epithelial cancer, providing the foundation for studies of molecular regulation of CTC in cancer and CTC as biomarker for therapeutic efficacy.

Potential applications for circulating tumor cells expressing the insulin-like growth factor-I receptor.

PURPOSE: To detect insulin-like growth factor-IR (IGF-IR) on circulating tumor cells (CTC) as a biomarker in the clinical development of a monoclonal human antibody, CP-751,871, targeting IGF-IR. EXPERIMENTAL DESIGN: An automated sample preparation and analysis system for enumerating CTCs (CellTracks) was adapted for detecting IGF-IR-positive CTCs with a diagnostic antibody targeting a different IGF-IR epitope to CP-751,871. This assay was used in three phase I trials of CP-751,871 as a single agent or with chemotherapy and was validated using cell lines and blood samples from healthy volunteers and patients with metastatic carcinoma. RESULTS: There was no interference between the analytic and therapeutic antibodies. Eighty patients were enrolled on phase I studies of CP-751,871, with 47 (59%) patients having CTCs detected during the study. Before treatment, 26 patients (33%) had CTCs, with 23 having detectable IGF-IR-positive CTCs. CP-751,871 alone, and CP-751,871 with cytotoxic chemotherapy, decreased CTCs and IGF-IR-positive CTCs; these increased toward the end of the 21-day cycle in some patients, falling again with retreatment. CTCs were commonest in advanced hormone refractory prostate cancer (11 of 20). Detectable IGF-IR expression on CTCs before treatment with CP-751,871 and docetaxel was associated with a higher frequency of prostate-specific antigen decline by >50% (6 of 10 versus 2 of 8 patients). A relationship was observed between sustained decreases in CTC counts and prostate-specific antigen declines by >50%. CONCLUSIONS: IGF-IR expression is detectable by immunofluorescence on CTCs. These data support the further evaluation of CTCs in pharmacodynamic studies and patient selection, particularly in advanced prostate cancer.

Circulating tumor cells versus imaging--predicting overall survival in metastatic breast cancer.

PURPOSE: The presence of >or=5 circulating tumor cells (CTC) in 7.5 mL blood from patients with measurable metastatic breast cancer before and/or after initiation of therapy is associated with shorter progression-free and overall survival. In this report, we compared the use of CTCs to radiology for prediction of overall survival. EXPERIMENTAL DESIGN: One hundred thirty-eight metastatic breast cancer patients had imaging studies done before and a median of 10 weeks after the initiation of therapy. All scans were centrally reviewed by two independent radiologists using WHO criteria to determine radiologic response. CTC counts were determined approximately 4 weeks after initiation of therapy. Specimens were analyzed at one of seven laboratories and reviewed by a central laboratory. RESULTS: Interreader variability for radiologic responses and CTC counts were 15.2% and 0.7%, respectively. The median overall survival of 13 (9%) patients with radiologic nonprogression and >or=5 CTCs was significantly shorter than that of the 83 (60%) patients with radiologic nonprogression and <5 CTCs (15.3 versus 26.9 months; P=0.0389). The median overall survival of the 20 (14%) patients with radiologic progression and <5 CTCs was significantly longer than the 22 (16%) patients with >or=5 CTCs that showed progression by radiology (19.9 versus 6.4 months; P=0.0039). CONCLUSIONS: Assessment of CTCs is an earlier, more reproducible indication of disease status than current imaging methods. CTCs may be a superior surrogate end point, as they are highly reproducible and correlate better with overall survival than do changes determined by traditional radiology.

Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with nonmalignant diseases.

PURPOSE: The purpose of this study was to determine the accuracy, precision, and linearity of the CellSearch system and evaluate the number of circulating tumor cells (CTCs) per 7.5 mL of blood in healthy subjects, patients with nonmalignant diseases, and patients with a variety of metastatic carcinomas. EXPERIMENTAL DESIGN: The CellSearch system was used to enumerate CTCs in 7.5 mL of blood. Blood samples spiked with cells from tumor cell lines were used to establish analytical accuracy, reproducibility, and linearity. Prevalence of CTCs was determined in blood from 199 patients with nonmalignant diseases, 964 patients with metastatic carcinomas, and 145 healthy donors. RESULTS: Enumeration of spiked tumor cells was linear over the range of 5 to 1,142 cells, with an average recovery of >/=85% at each spike level. Only 1 of the 344 (0.3%) healthy and nonmalignant disease subjects had >/=2 CTCs per 7.5 mL of blood. In 2,183 blood samples from 964 metastatic carcinoma patients, CTCs ranged from 0 to 23,618 CTCs per 7.5 mL (mean, 60 +/- 693 CTCs per 7.5 mL), and 36% (781 of 2,183) of the specimens had >/=2 CTCs. Detection of >/=2 CTCs occurred at the following rates: 57% (107 of 188) of prostate cancers, 37% (489 of 1,316) of breast cancers, 37% (20 of 53) of ovarian cancers, 30% (99 of 333) of colorectal cancers, 20% (34 of 168) of lung cancers, and 26% (32 of 125) of other cancers. CONCLUSIONS: The CellSearch system can be standardized across multiple laboratories and may be used to determine the clinical utility of CTCs. CTCs are extremely rare in healthy subjects and patients with nonmalignant diseases but present in various metastatic carcinomas with a wide range of frequencies.

Apoptosis of circulating tumor cells in prostate cancer patients.

BACKGROUND: The prescence of circulating tumor cells (CTCs) in the peripheral blood of cancer patients and their frequency has been correlated with disease status. METHODS: In this study, CTCs were characterized by flow cytometry and fluorescence microscopy after immunomagnetic enrichment from 7.5-ml blood samples collected from patients with prostate cancer in evacuated blood-draw tubes that contained an anticoagulant and a preservative. Events were classified as tumor cell candidates if they expressed cytokeratin, lacked CD45, and stained with the nucleic acid dye 4,6-diamidino-2-phenylindole. RESULTS: In the blood of prostate cancer patients, only few of these events were intact cells. Other CTC events appeared as damaged cells or cell fragments by microscopy. By flow cytometry, these events stained variably with 4,6-diamidino-2-phenylindole and frequently expressed the apoptosis-induced, caspase-cleaved cytokeratin 18. Similar patterns of cell disintegration were observed when cells of the prostate line LNCaP were exposed to paclitaxel before spiking the cells into normal blood samples. CONCLUSIONS: The different observed stages of tumor cell degradation or apoptosis varied greatly between patients and were not found in blood of normal donors. Enumeration of CTCs and identification of CTCs undergoing apoptosis may provide relevant information to evaluate the response to therapy in cancer patients.