Bianca Mostert

Circulating tumor cells (CTCs): detection methods and their clinical relevance in breast cancer.

The enumeration of circulating tumor cells has long been regarded as an attractive diagnostic tool, as circulating tumor cells are thought to reflect aggressiveness of the tumor and may assist in therapeutic decisions in patients with solid malignancies. However, implementation of this assay into clinical routine has been cumbersome, as a validated test was not available until recently. Circulating tumor cells are rare events which can be detected specifically only by using a combination of surface and intracellular markers, and only recently a number of technical advances have made their reliable detection possible. Most of these new techniques rely on a combination of an enrichment and a detection step. This review addresses the assays that have been described so far in the literature, including the enrichment and detection steps and the markers used in these assays. We have focused on breast cancer as most clinical studies on CTC detection so far have been done in these patients.

mRNA and microRNA expression profiles in circulating tumor cells and primary tumors of metastatic breast cancer patients

Purpose: Molecular characterization of circulating tumor cells (CTC) holds great promise. Unfortunately, routinely isolated CTC fractions currently still contain contaminating leukocytes, which makes CTC-specific molecular characterization extremely challenging. In this study, we determined mRNA and microRNA (miRNA) expression of potentially CTC-specific genes that are considered to be clinically relevant in breast cancer. Experimental Design: CTCs were isolated with the epithelial cell adhesion molecule–based CellSearch Profile Kit. Selected genes were measured by real-time reverse transcriptase PCR in CTCs of 50 metastatic breast cancer patients collected before starting first-line systemic therapy in blood from 53 healthy blood donors (HBD) and in primary tumors of 8 of the patients. The molecular profiles were associated with CTC counts and clinical parameters and compared with the profiles generated from the corresponding primary tumors. Results: We identified 55 mRNAs and 10 miRNAs more abundantly expressed in samples from 32 patients with at least 5 CTCs in 7.5 mL of blood compared with samples from 9 patients without detectable CTCs and HBDs. Clustering analysis resulted in 4 different patient clusters characterized by 5 distinct gene clusters. Twice the number of patients from cluster 2 to 4 had developed both visceral and nonvisceral metastases. Comparing transcript levels in CTCs with those measured in corresponding primary tumors showed clinically relevant discrepancies in estrogen receptor and HER2 levels. Conclusions: Our study shows that molecular profiling of low numbers of CTCs in a high background of leukocytes is feasible and shows promise for further studies on the clinical relevance of molecular characterization of CTCs. Clin Cancer Res; 17(11); 3600–18. ©2011 AACR.

KRAS and BRAF mutation status in circulating colorectal tumor cells and their correlation with primary and metastatic tumor tissue

Although anti‐EGFR therapy has established efficacy in metastatic colorectal cancer, only 10‐20% of unselected patients respond. This is partly due to KRAS and BRAF mutations, which are currently assessed in the primary tumor. To improve patient selection, assessing mutation status in circulating tumor cells (CTCs), which possibly better represent metastases than the primary tumor, could be advantageous. We investigated the feasibility of KRAS and BRAF mutation detection in colorectal CTCs by comparing three sensitive methods and compared mutation status in matching primary tumor, liver metastasis and CTCs. CTCs were isolated from blood drawn from 49 patients before liver resection using CellSearch™. DNA and RNA was isolated from primary tumors, metastases and CTCs. Mutations were assessed by co‐amplification at lower denaturation temperature‐PCR (Transgenomic™), real‐time PCR (EntroGen™) and nested Allele‐Specific Blocker (ASB‐)PCR and confirmed by Sanger sequencing. In 43 of the 49 patients, tissue RNA and DNA was of sufficient quantity and quality. In these 43 patients, discordance between primary and metastatic tumor was 23% for KRAS and 7% for BRAF mutations. RNA and DNA from CTCs was available from 42 of the 43 patients, in which ASB‐PCR was able to detect the most mutations. Inconclusive results in patients with low CTC counts limited the interpretation of discrepancies between tissue and CTCs. Determination of KRAS and BRAF mutations in CTCs is challenging but feasible. Of the tested methods, nested ASB‐PCR, enabling detection of KRAS and BRAF mutations in patients with as little as two CTCs, seems to be superior.

Diagnostic applications of cell-free and circulating tumor cell-associated miRNAs in cancer patients

Recently, miRNA-expression profiling in primary tumors has yielded promising results. However, establishing miRNA expression in the circulation probably has advantages over determination in primary tumor tissue, further augmenting the potential applications of miRNA determination in oncology. Circulating tumor cells (CTCs) have rapidly developed as important prognostic and therapy-monitoring biomarkers in metastatic breast, colorectal and prostate cancer when enumerated, and their isolation enables subsequent analysis using various molecular applications, including miRNA-expression analysis. In addition to CTC-associated miRNAs, free circulating miRNAs have been identified in whole blood, plasma and serum. Determination of miRNAs in peripheral blood, either cell-free or CTC-associated, is expected to become important in oncology, especially when linked to and interpreted together with epithelial CTCs. In this article, we will discuss miRNA-expression profiling in primary tumors, depict the potential applications of measuring miRNA in the circulation and review the literature on cell-free circulating miRNAs, as well as offering some methodological and technical considerations on the measurement of circulating miRNAs.

CD49f-based selection of circulating tumor cells (CTCs) improves detection across breast cancer subtypes

Circulating tumor cells (CTCs) can be enumerated using CellSearch, but not all breast cancer subtypes, specifically those with epithelial-mesenchymal transition (EMT) characteristics, sufficiently express the enrichment (EpCAM) and selection (CK8/18/19) markers used in this method. While CD146 can detect EpCAM-negative CTCs, we here evaluated the value of various cytokeratins and CD49f to detect CK8/18/19-negative CTCs. The tested cytokeratins provided no substantial benefit, but adding CD49f to CK8/18/19 as a selection marker resulted in improved recovery of normal-like cell lines. Combined staining of CK8/18/19 and CD49f after CD146/EpCAM enrichment is likely to further improve CTC detection in breast cancer.

Circulating Tumor Cells and Sample Size: The More, the Better

TO THEEDITOR: With interest we read the article by Jiao et al, 1 who studied the presence of circulating tumor cells (CTCs) in blood of patients with colorectal liver metastases (CRLM) by automated immunomagnetic enrichment and image cytometry using the CellSearch system (Veridex, Raritan, NJ). They showed CTCs to be present in the hepatic macrocirculation in significantly higher numbersthanintheperipheralcirculation(median,187[range,0to500]v median 1 [range 0 to 6]). Despite the number of evaluated patients in this study being small, the low number of detected CTCs in the peripheral circulation may suggest that CTC enumeration and characterization plays no role in this specific patient population. Enumeration and in particular characterization of CTC holds great promise for patient management and research purposes. 2 Of severalassaysenablingCTCdetection(reviewedbyMostertetal 3 ),the CellSearch system has been approved by the US Food and Drug Administration for use in metastatic breast, prostate, and colorectal cancer.Accordingtothemanufacturer’sinstructions,CTCenumeration should be performed in 7.5 mL blood. In the first study of 196 patientswithmetastaticcolorectalcancerwhoseCTCsweremeasured with the CellSearch system. At least 2 CTCs per 7.5 mL blood were detected in 30% of patients, whereas only 17% had 5 CTCs per 7.5 mL blood. 4 In a subsequent publication by Cohen et al 5 investigating the prognostic role of CTCs in advanced colorectal cancer, patients with a CTC count above a threshold of 3 CTCs per 7.5 mL blood had a worse outcome after systemic therapy compared with patients with lower CTC counts. The 430 patients in this study received first-, second-, or third-line chemotherapy, of whom 26% had a CTC count3 CTC threshold, whereas 48% of the patients had1 CTC per 7.5 mL blood. In a third study of 451 patients with metastatic colorectal cancer, at least 3 CTCs per 7.5 mL blood were detected in 29% of the patients. 6 From these studies we can conclude that the number of detectable CTCs in patients with advanced colorectal cancer using the CellSearch System is low to even below detection limit, which is in contrast to other tumor types. In metastatic breast and prostate cancer, the percentage of patients with a CTC count of 5 CTCs per 7.5 mL was 66% and 49%, respectively. 7,8 Not surprisingly and in line with thefindings of Jiao et al, 1 in nonmetastastic colorectal cancer, the number of patients with detectable CTCs is even lower thaninadvanceddisease.Onestudyrevealed2CTCs/7.5mLintwo

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

IntroductionCirculating 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.MethodsCellSearch® 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.ResultsFor 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).ConclusionsThe 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.

Gene expression profiles in circulating tumor cells to predict prognosis in metastatic breast cancer patients

BACKGROUND A circulating tumor cell (CTC) count is an established prognostic factor in metastatic breast cancer (MBC). Besides enumeration, CTC characterization promises to improve outcome prediction and treatment guidance. Having shown the feasibility of quantifying clinically relevant mRNA transcripts in CTCs, we determined the prognostic value of CTC gene expression in MBC. PATIENTS AND METHODS CTCs were isolated and enumerated from blood of 197 MBC patients who were about to start first-line systemic therapy. Of these, 180 were assessable for quantification of mRNA expression by RT-qPCR in relation to time-to-treatment failure (TTF). A prognostic CTC gene profile was generated by leave-one-out cross validation in a 103 patient discovery set and validated in 77 patients. Additionally, all 180 patients were randomly divided into two equal sets to discover and validate a second prognostic profile. RESULTS CTC count predicted for TTF at baseline {≥5 versus <5 CTCs/7.5 ml blood, hazard ratio (HR) 2.92 [95% confidence interval (CI) 1.71-4.95] P < 0.0001}. A 16-gene CTC profile was generated in the first discovery set, which identified patients with death or TTF <9 months versus those with a better outcome. In multivariate analysis, the 16-gene profile was the only factor associated with TTF [HR 3.15 (95% CI 1.35-7.33) P 0.008]. Validation of this profile in the independent patient set pointed into the same direction, but was not statistically significant. A newly generated 8-gene profile showed similarly favorable test characteristics as the 16-gene profile, but did not significantly pass validation either. CONCLUSION A 16-gene CTC profile was identified, which provided prognostic value on top of CTC count in MBC patients. However, validation of this profile in an independent cohort, nor of a second profile, reached statistical significance, underscoring the need to further fine-tune the still promising approach of CTC characterization.

Improved Circulating Tumor Cell Detection by a Combined EpCAM and MCAM CellSearch Enrichment Approach in Patients with Breast Cancer Undergoing Neoadjuvant Chemotherapy

Circulating tumor cells (CTC) are detected by the CellSearch System in 20% to 25% of patients with primary breast cancer (pBC). To improve CTC detection, we investigated melanoma cell adhesion molecule (MCAM) as enrichment marker next to epithelial cell adhesion molecule (EpCAM) and tested the clinical relevance of MCAM-positive CTCs in patients with HER2-negative stage II/III pBC starting neoadjuvant chemotherapy (NAC) in the NEOZOTAC trial. Using the CellSearch System, EpCAM-positive and MCAM-positive CTCs were separately enriched from 7.5 mL blood, at baseline and after the first NAC cycle. Circulating endothelial cells (CEC) were measured using flow cytometry. Primary objective was to improve the CTC detection rate to ≥40% combining EpCAM/MCAM. Correlations of CTC and CEC counts and pathologic complete response (pCR) were also explored. At baseline, we detected EpCAM-positive and MCAM-positive CTCs in 12 of 68 (18%) and 8 of 68 (12%) patients, respectively. After one cycle, this was 7 of 44 (16%) and 7 of 44 (16%) patients, respectively. The detection rate improved from 18% at baseline and 16% after one cycle with EpCAM to 25% (P = 0.08) and 30% (P = 0.02), respectively, with EpCAM/MCAM. No patients with MCAM-positive CTCs versus 23% of patients without MCAM-positive CTCs at baseline achieved pCR (P = 0.13). EpCAM-positive CTCs and CEC counts were not correlated to pCR. Combined EpCAM/MCAM CellSearch enrichment thus increased the CTC detection rate in stage II/III pBC. We found no associations of CTC and CEC counts with pCR to NAC. The clinical relevance of MCAM-positive CTCs deserves further study. Mol Cancer Ther; 14(3); 821–7. ©2014 AACR.

mRNA expression profiles in circulating tumor cells of metastatic colorectal cancer patients

The molecular characterization of circulating tumor cells (CTCs) is a promising tool for the repeated and non‐invasive evaluation of predictive and prognostic factors. Challenges associated with CTC characterization using the only FDA approved method for CTC enumeration, the CellSearch technique, include the presence of an excess of leukocytes in CTC‐enriched blood fractions. Here we aimed to identify colorectal tumor‐specific gene expression levels in the blood of patients with and without detectable CTCs according to CellSearch criteria.