Jaco Kraan

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.

CSF flow cytometry greatly improves diagnostic accuracy in CNS hematologic malignancies

Objective: To assess the diagnostic accuracy of flow cytometric immunophenotyping in comparison with classic cytomorphology for diagnosing CNS localizations of hematologic malignancies, and to evaluate the implications of CSF pleocytosis and protein content in this context. Methods: We reviewed the results of diagnostic evaluations of all CSF samples analyzed for localization of a hematologic malignancy between 2001 and 2004 at our center. Results: A total of 1,054 samples from 219 patients were available for analysis. Sixty patients had a CSF localization diagnosed by positive flow cytometry, cytomorphology, or both. The first sample was positive by flow cytometry in 44 (73%) patients, by cytomorphology in 19 (32%). Four first samples were positive by cytomorphology but negative by flow cytometry. Patients with positive cytomorphology had more frequent clinical symptomatology (95% vs 58%) and CSF pleocytosis (84% vs 25%), and tended to a poorer progression-free survival than patients with positive flow cytometry only. OR for CNS localization in case of CSF pleocytosis was 10.1 (95% CI 4.9 to 20.8); OR for CNS localization in case of elevated protein content was 2.9 (95% CI 1.5 to 5.4). Nevertheless, 26 of 137 (19%) patients with normal cell count and protein concentration had a CNS localization. Conclusions: The diagnostic value of flow cytometry is more than twice that of cytomorphology. However, cytomorphologic examination of the CSF has additional diagnostic and possibly prognostic value, and should still be performed in conjunction with flow cytometry.

Efficacy of Cabazitaxel in Castration-resistant Prostate Cancer Is Independent of the Presence of AR-V7 in Circulating Tumor Cells.

BACKGROUND Androgen receptor splice variant 7 (AR-V7) in circulating tumor cells (CTCs) from patients with metastatic castration-resistant prostate cancer (mCRPC) was recently demonstrated to be associated with resistance to abiraterone and enzalutamide. Cabazitaxel might, however, remain effective in AR-V7-positive patients. OBJECTIVE To investigate the association between AR-V7 expression in CTCs and resistance to cabazitaxel. DESIGN, SETTING, AND PARTICIPANTS We selected patients with mCRPC from the multicenter, randomized, phase 2, randomized, open-label, multicenter study in mCRPC on the pharmacodynamic effects of budesonide on cabazitaxel (Jevtana) (CABARESC). Before the start of the first and third cabazitaxel cycle, CTCs were enumerated using the CellSearch System. In patients with ≥10 CTCs in 7.5 ml blood at baseline, the expression of AR-V7 was assessed by quantitative polymerase chain reaction. OUTCOME MEASURES AND STATISTICAL ANALYSIS The primary end point was the association between the AR-V7 status and the CTC response rate (decrease to fewer than five CTCs in 7.5 ml blood during treatment). Secondary end points were the prostate-specific antigen (PSA) response rate (RR) and overall survival (OS). Analyses were performed using chi-square and log-rank tests. RESULTS AND LIMITATIONS AR-V7 was detected in 16 of 29 patients (55%) with ≥10 CTCs and was more frequently found in abiraterone pretreated patients (5 of 5 [100%] treated vs 7 of 20 [35%] untreated; p=0.009). We found no differences in CTC and PSA RRs. The presence of AR-V7 in CTCs was not associated with progression-free survival (hazard ratio [HR]: 0.8; 95% confidence interval [CI], 0.4-1.8) or overall survival (HR 1.6; 95% CI, 0.6-4.4). CONCLUSIONS The response to cabazitaxel seems to be independent of the AR-V7 status of CTCs from mCRPC patients. Consequently, cabazitaxel might be a valid treatment option for patients with AR-V7-positive CTCs. PATIENT SUMMARY Tools are needed to select specific treatments for specific patients at specific times. The presence of the gene AR-V7 in CTCs has been associated with resistance to anti-androgen receptor treatments. We investigated whether this holds true for cabazitaxel, but we found cabazitaxel to be effective independent of the presence of AR-V7.

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.

External quality assurance of circulating tumor cell enumeration using the CellSearch(®) system: a feasibility study.

Circulating tumor cells (CTCs) are cells that have detached from solid tumors and entered the blood. CTCs can be detected, among others, by semi‐automated immunomagnetic enrichment and image cytometry using CellSearch® (Veridex, Raritan, NJ). We studied the feasibility of external quality assurance (EQA) of the entire CellSearch procedure from blood draw to interpretation of results in multiple laboratories.

Circulating endothelial cells in oncology: pitfalls and promises

Adequate blood supply is a prerequisite in the pathogenesis of solid malignancies. As a result, depriving a tumour from its oxygen and nutrients, either by preventing the formation of new vessels, or by disrupting vessels already present in the tumour, appears to be an effective treatment modality in oncology. Given the mechanism by which these agents exert their anti-tumour activity together with the crucial role of tumour vasculature in the pathogenesis of tumours, there is a great need for markers properly reflecting its impact. Circulating endothelial cells (CEC), which are thought to derive from damaged vasculature, may be such a marker. Appropriate enumeration of these cells appears to be a technical challenge. Nevertheless, first studies using validated CEC assays have shown that CEC numbers in patients with advanced malignancies are elevated compared to healthy controls making CEC a potential tool for among other establishing prognosis and therapy-induced effects. In this review, we will address the possible clinical applications of CEC detection in oncology, as well as the pitfalls encountered in this process.

Reduction of intra‐ and interlaboratory variation in CD34+ stem cell enumeration using stable test material, standard protocols and targeted training

The European Working Group on Clinical Cell Analysis (EWGCCA) has, in preparation for a multicentre peripheral blood stem cell clinical trial, developed a single‐platform flow cytometric protocol for the enumeration of CD34+ stem cells. Using this protocol, stabilized blood and targeted training, the EWGCCA have attempted to standardize CD34+ stem cell enumeration across 24 clinical sites. Results were directly compared with participants in the UK National External Quality Assessment Scheme (NEQAS) for CD34+ Stem Cell Quantification that analysed the same specimens using non‐standardized methods. Two bead‐counting systems, Flow‐Count and TruCount, were also evaluated by the EWGCCA participants during trials 2 and 3. Using Flow‐Count, the intralaboratory coefficient of variation (CV) was ≤ 5% in 39% of the laboratories (trial 1), increasing to 65% by trial 3. Interlaboratory variation was reduced from 23.3% (trial 1) to 10.8% in trial 3. In trial 2, 70% of laboratories achieved an intralaboratory CV ≤ 5% using TruCount, increasing to 74% for trial 3; the interlaboratory CV was reduced from 23.4% to 9.5%. Comparative analysis of the EWGCCA and the UK NEQAS cohorts revealed that EWGCCA laboratories, using the standardized approach, had lower interlaboratory variation. Thus, the use of a common standardized protocol and targeted training significantly reduced intra‐ and interlaboratory CD34+ cell count variation.

Flow cytometric characterization of cerebrospinal fluid cells

Flow cytometry facilitates the detection of a large spectrum of cellular characteristics on a per cell basis, determination of absolute cell numbers and detection of rare events with high sensitivity and specificity. White blood cell (WBC) counts in cerebrospinal fluid (CSF) are important for the diagnosis of many neurological disorders. WBC counting and differential can be performed by microscopy, hematology analyzers, or flow cytometry. Flow cytometry of CSF is increasingly being considered as the method of choice in patients suspected of leptomeningeal localization of hematological malignancies. Additionally, in several neuroinflammatory diseases such as multiple sclerosis and paraneoplastic neurological syndromes, flow cytometry is commonly performed to obtain insight into the immunopathogenesis of these diseases. Technically, the low cellularity of CSF samples, combined with the rapidly declining WBC viability, makes CSF flow cytometry challenging. Comparison of flow cytometry with microscopic and molecular techniques shows that each technique has its own advantages and is ideally combined. We expect that increasing the number of flow cytometric parameters that can be simultaneously studied within one sample, will further refine the information on CSF cell subsets in low‐cellular CSF samples and enable to define cell populations more accurately.

Central memory CD4+ T cells dominate the normal cerebrospinal fluid†

To use cerebrospinal fluid (CSF) immune phenotyping as a diagnostic and research tool, we have set out to establish reference values of white blood cell (WBC) subsets in CSF.

Cells meeting our immunophenotypic criteria of endothelial cells are large platelets

Circulating endothelial cells (CEC) are shed from damaged vasculature, making them a rational choice to serve as surrogate marker for vascular damage. Currently, various techniques and CEC definitions are in use, and their standardization and validation is needed. A flow cytometric single platform assay defining CEC as forward light scatter (FSC)low‐to‐intermedate, sideward light scatter (SSC)low, CD45−, CD31++ and CD146+ is a promising approach to enumerate CEC because of its simplicity (Mancuso et al., Blood 2001;97:3658–3661). Here, we set out to confirm the endothelial nature of these cells.