Guidelines for May-Grünwald-Giemsa staining in haematology and non-gynaecological cytopathology: recommendations of the French Society of Clinical Cytology (SFCC) and of the French Association for Quality Assurance in Anatomic and Cytologic Pathology (AFAQAP).

OBJECTIVE: Since the guidelines of the International Committee for Standardisation in Haematology (ICSH) in 1984 and those of the European Committee for External Quality Assessment Programmes in Laboratory Medicine (EQALM) in 2004, no leading organisation has published technical recommendations for the preparation of air-dried cytological specimens using May-Grünwald-Giemsa (MGG) staining. DATA SOURCES: Literature data were retrieved using reference books, baseline-published studies, articles extracted from PubMed/Medline and Google Scholar, and online-available industry datasheets. RATIONALE: The present review addresses all pre-analytical issues concerning the use of Romanowsky's stains (including MGG) in haematology and non-gynaecological cytopathology. It aims at serving as actualised, best practice recommendations for the proper handling of air-dried cytological specimens. It, therefore, appears complementary to the staining criteria of the non-gynaecological diagnostic cytology handbook edited by the United Kingdom National External Quality Assessment Service (UK-NEQAS) in February 2015.

Cytomorphology of ovarian clear cell carcinomas in peritoneal effusions.

OBJECTIVE: To investigate and describe the cytomorphology of malignant effusions from ovarian clear cell carcinomas (OCCC). METHODS: Five cases of malignant peritoneal effusions from OCCC histologically confirmed were analysed and compared. RESULTS: Among the malignant peritoneal effusions exhibiting clear cell features, a characteristic feature of OCCC was the presence of large deposits of a hyaline matrix. This matrix may be typically arranged either in 'raspberry bodies' or 'globule-like' structures. Other rare neoplasms composed of clear cells must be considered in the differential diagnosis such as yolk sac tumour of the ovary, clear cell subtype of endometrial carcinoma and, less frequently, malignant peritoneal mesothelioma as well as metastatic renal cell carcinoma. CONCLUSIONS: Ovarian clear cell carcinomas have distinct morphological features that are helpful in making a cytological diagnosis of this entity. The role of cytological examination in ovarian neoplasms is of paramount importance, as stated by The International Federation of Gynecology and Obstetrics (FIGO) recommendations.

Vimentin and Ki67 expression in circulating tumour cells derived from castrate-resistant prostate cancer.

BACKGROUND: High circulating tumor cell (CTC) counts are associated with poor prognosis in advanced prostate cancer, and recently CTC number was suggested to be a surrogate for survival in metastatic castrate-resistant prostate cancer (mCRPC). Ki67 and vimentin are well-characterised markers of tumour cell proliferation and the epithelial-mesenchymal transition (EMT), respectively. Here we asked if the expression of vimentin and Ki67 in CTCs offered prognostic or predictive information in mCRPC. METHODS: In two separate patient cohorts, anti-vimentin or anti-Ki67 antibodies were added to the free channel in the CellSearch® system for analysis of peripheral blood samples. For each cohort, association of CTC number with clinical characteristics were assessed using Fisher's exact, Mann-Whitney and chi-squared tests. Kaplan-Meier method and log-rank tests were used to analyse overall survival (OS) of vimentin-expressing and Ki67-expressing CTC patient cohorts. RESULTS: In this retrospective analysis, CTC vimentin expression was analysed in 142 blood samples from 93 patients, and CTC Ki67 expression was analysed in 90 blood samples from 51 patients. In the vimentin cohort, 80/93 (86 %) of baseline samples from patients were CTC-positive overall (≥1 total CTC per 7.5 mls blood), and 30/93 (32.3 %) vimentin CTC-positive (≥1 vimentin-positive CTC per 7.5 mls blood). 41/51 (80.4 %) of baseline samples from patients in the Ki67 cohort were CTC-positive overall, and 23/51 (45.1 %) Ki67 CTC-positive (≥1 Ki67-positive CTC per 7.5 mls blood). There was no significant difference in baseline PSA in patients with vimentin-positive CTC at baseline versus those with no vimentin-positive CTC at baseline (p = 0.33). A significant reduction in OS was shown in patients with vimentin-positive CTC compared to those without vimentin-positive CTC (median 305 days vs 453 days, p = 0.0293). There was no significant difference in baseline PSA in patients with Ki67-positive CTC at baseline versus those without Ki67-positive CTC (p = 0.228), but OS was significantly reduced in the Ki67-positive CTC group (median 512 days vs 751 days, p = 0.0091). No changes in relative proportion of vimentin- or Ki67-positive CTCs were observed in post-treatment samples compared to baseline. CONCLUSIONS: Analysis of vimentin and Ki67 expression can straightforwardly be assessed in CTCs from patients with mCRPC. Poorer survival outcomes were observed in vimentin- and Ki67-positive CTC patients. TRANSLATIONAL STUDY PROTOCOLS: CEC-CTC (IDRCB2008-AOO585-50) and Petrus ( NCT01786031 ).

Seeking the driver in tumours with apparent normal molecular profile on comparative genomic hybridization and targeted gene panel sequencing: what is the added value of whole exome sequencing?

BACKGROUND: Molecular tumour profiling technologies have become increasingly important in the era of precision medicine, but their routine use is limited by their accessibility, cost, and tumour material availability. It is therefore crucial to assess their relative added value to optimize the sequence and combination of such technologies. PATIENTS AND METHODS: Within the MOSCATO-01 trial, we investigated the added value of whole exome sequencing (WES) in patients that did not present any molecular abnormality on array comparative genomic hybridization (aCGH) and targeted gene panel sequencing (TGPS) using cancer specific panels. The pathogenicity potential and actionability of mutations detected on WES was assessed. RESULTS: Among 420 patients enrolled between December 2011 and December 2013, 283 (67%) patients were analysed for both TGPS and aCGH. The tumour sample of 25 (8.8%) of them presented a flat (or low-dynamic) aCGH profile and no pathogenic mutation on TGPS. We selected the first eligible 10 samples-corresponding to a heterogeneous cohort of different tumour types-to perform WES. This allowed identifying eight mutations of interest in two patients: FGFR3, PDGFRB, and CREBBP missense single-nucleotide variants (SNVs) in an urothelial carcinoma; FGFR2, FBXW7, TP53, and MLH1 missense SNVs as well as an ATM frameshift mutation in a squamous cell carcinoma of the tongue. The FGFR3 alteration had been previously described as an actionable activating mutation and might have resulted in treatment by an FGFR inhibitor. CREBBP and ATM alterations might also have suggested a therapeutic orientation towards epigenetic modifiers and ataxia-telangectasia and Rad3-related inhibitors, respectively. CONCLUSION: The therapeutic added value of performing WES on tumour samples that do not harbour any genetic abnormality on TGPS and aCGH might be limited and variable according to the histotype. Alternative techniques, including RNASeq and methylome analysis, might be more informative in selected cases.

Antitumor activity in advanced cancer patients with thymic malignancies enrolled in early clinical drug development programs (Phase I trials) at Gustave Roussy.

OBJECTIVES: Thymic epithelial neoplasms (TENs) represent a rare entity with poor prognosis and limited systemic treatment options. The aim of this study was to assess the clinical benefit, the efficacy and toxicities of agents for patients with TEN enrolled in Phase I trials. MATERIALS AND METHODS: We reviewed retrospectively patients with advanced TEN enrolled in Phase I trials at Gustave Roussy (DITEP) between 1994 and 2012. Efficacy was assessed using RECIST version 1.1. RESULTS: Twenty-two treated patients were enrolled (15 with thymic carcinoma, 7 with thymoma). The median number of prior systemic therapies was 2 (0-8). The median age was 50 years (range 23-72), and 4 females were treated. Treatments received encompassed mTOR inhibitor (mTORi) in 4 of patients, antiangiogenic agents (AA) in 11 patients, and other targeted therapies in 7 patients. 18% had grade 3-4 toxicity, 85% all grade toxicity and no toxic death was reported. One patient experienced a complete response (CR) and 3 a partial response (PR); 16 patients had stable disease (median 6.6 months; range 1.0-30.7) and 2 had a progressive disease. The median overall survival was 54.5 months (95% CI 25-75.50). The median progression free survival (PFS) was 6.6 months (95% CI 1.35-11.59). Median PFS was 11.6 months for mTORi, 6.9 for AA, and 6.6 for other targeted therapies. CONCLUSION: Phase I trials appear as a sound therapeutic option in TENs pts progressing after standard treatments. Use of AA and mTORi seem to yield a good clinical response and warrant further investigation.

Guidelines for the Cytopathologic Diagnosis of Epithelioid and Mixed-Type Malignant Mesothelioma: a secondary publication.

OBJECTIVE: To provide practical guidelines for the cytopathologic diagnosis of malignant mesothelioma. DATA SOURCES: Cytopathologists with an interest in the field involved in the International Mesothelioma Interest Group (IMIG) and the International Academy of Cytology (IAC) contributed to this update. Reference material includes peer-reviewed publications and textbooks. RATIONALE: This article is the result of discussions during and after the IMIG 2012 conference in Boston, followed by thorough discussions during the 2013 IAC meeting in Paris. Additional contributions have been obtained from cytopathologists and scientists who could not attend these meetings, with final discussions and input during the IMIG 2014 conference in Cape Town.

High level of chromosomal instability in circulating tumor cells of ROS1-rearranged non-small-cell lung cancer.

BACKGROUND: Genetic aberrations affecting the c-ros oncogene 1 (ROS1) tyrosine kinase gene have been reported in a small subset of patients with non-small-cell lung cancer (NSCLC). We evaluated whether ROS1-chromosomal rearrangements could be detected in circulating tumor cells (CTCs) and examined tumor heterogeneity of CTCs and tumor biopsies in ROS1-rearranged NSCLC patients. PATIENTS AND METHODS: Using isolation by size of epithelial tumor cells (ISET) filtration and filter-adapted-fluorescence in situ hybridization (FA-FISH), ROS1 rearrangement was examined in CTCs from four ROS1-rearranged patients treated with the ROS1-inhibitor, crizotinib, and four ROS1-negative patients. ROS1-gene alterations observed in CTCs at baseline from ROS1-rearranged patients were compared with those present in tumor biopsies and in CTCs during crizotinib treatment. Numerical chromosomal instability (CIN) of CTCs was assessed by DNA content quantification and chromosome enumeration. RESULTS: ROS1 rearrangement was detected in the CTCs of all four patients with ROS1 rearrangement previously confirmed by tumor biopsy. In ROS1-rearranged patients, median number of ROS1-rearranged CTCs at baseline was 34.5 per 3 ml blood (range, 24-55). In ROS1-negative patients, median background hybridization of ROS1-rearranged CTCs was 7.5 per 3 ml blood (range, 7-11). Tumor heterogeneity, assessed by ROS1 copy number, was significantly higher in baseline CTCs compared with paired tumor biopsies in the three patients experiencing PR or SD (P < 0.0001). Copy number in ROS1-rearranged CTCs increased significantly in two patients who progressed during crizotinib treatment (P < 0.02). CTCs from ROS1-rearranged patients had a high DNA content and gain of chromosomes, indicating high levels of aneuploidy and numerical CIN. CONCLUSION: We provide the first proof-of-concept that CTCs can be used for noninvasive and sensitive detection of ROS1 rearrangement in NSCLC patients. CTCs from ROS1-rearranged patients show considerable heterogeneity of ROS1-gene abnormalities and elevated numerical CIN, a potential mechanism to escape ROS1-inhibitor therapy in ROS1-rearranged NSCLC tumors.

Targeting WNT1-inducible signaling pathway protein 2 alters human breast cancer cell susceptibility to specific lysis through regulation of KLF-4 and miR-7 expression.

The molecular basis for the resistance of tumor cells to cell-mediated cytotoxicity remains poorly understood and thus poses a major challenge for cancer immunotherapy. The present study was designed to determine whether the WNT1-inducible signaling pathway protein 2 (WISP2, also referred to as CCN5), a key regulator of tumor cell plasticity, interferes with tumor susceptibility to cytotoxic T-lymphocyte (CTL)-mediated lysis. We found that silencing WISP2 signaling in human breast adenocarcinoma MCF7 cells impairs CTL-mediated cell killing by a mechanism involving stem cell marker Kruppel-like factor-4 (KLF-4) induction and microRNA-7 (miR-7) downregulation. Inhibition of transforming growth factor beta (TGF-ß) signaling using the A83-01 inhibitor in MCF7-shWISP2 cells resulted in a significant reversal of the epithelial-to-mesenchymal-transitioned (EMT) phenotype, the expression of KLF-4 and a partial recovery of target susceptibility to CTLs. More importantly, we showed that silencing KLF-4 was accompanied by a reduction in MCF7-shWISP2 resistance to CTLs. Using human breast cancer tissues, we demonstrated the coexpression of KLF-4 with EMT markers and TGF-ß pathway signaling components. More importantly, we found that KLF-4 expression was accompanied by miR-7 inhibition, which is partly responsible for impairing CTL-mediated lysis. Thus, our data indicate that WISP2 has a role in regulating tumor cell susceptibility through EMT by inducing the TGF-ß signaling pathway, KLF-4 expression and miR-7 inhibition. These studies indicate for the first time that WISP2 acts as an activator of CTL-induced killing and suggests that the loss of its function promotes evasion of immunosurveillance and the ensuing progression of the tumor.

Breast elastography: the technical process and its applications.

Breast elastography is being increasingly used to better characterize breast lesions. Published studies have shown that it improved specificity of B mode ultrasound. Two elastography modes are available: free-hand elastography and shear wave elastography. Free-hand elastography is obtained by a mechanic wave induced by the ultrasound probe, deforming the target, either by small movements induced by breathe. An elastogram is obtained and displayed either as a colour map or a size ratio or elasticity ratio measurement. The second mode is shear wave elastography; two methods are available: Shear Wave Elastography (SWE) and ARFI mode (Acoustic Radiation Force Impulse). Shear wave elastography is less operator-dependent than free-hand elastography mode and provides a quantitative approach. A value of over 80kPa (SWE) or velocity results of over 2m/s (ARFI) are considered as suspicious. False negatives may occur in soft breast cancers (mucinous carcinoma, carcinoma with an inflammatory stroma, etc.) and false positives may be seen with poorly deformable benign lesions such as old fibrous adenomas. In practical use, elastography is a useful complementary tool for undetermined breast lesions categorized as BI-RADS 4a or BI-RADS 3, or for cystic lesions but cannot avoid fine needle aspiration or core biopsy if ultrasound features are clearly suspicious.

Pure and mixed mucinous carcinoma of the breast: fine needle aspiration cytology findings and review of the literature.

OBJECTIVE: Mucinous (colloid) breast carcinoma accounts for 1-6% of all breast cancer. It comprises pure mucinous tumours and mixed infiltrating ductal carcinomas with a mucinous component. As this latter mixed form has a worse prognosis than pure colloid carcinoma, making this diagnosis on fine needle aspiration cytology (FNAC) might influence the choice of treatment. METHODS: We report a consecutive series of 22 cases consisting of 17 mixed and five pure mucinous carcinomas diagnosed by cytology and verified on histopathology. Patients underwent FNAC at the one-stop clinic of our institution during a 7-year period of time. Cytological findings were evaluated by a semi-quantitative method and included percentage of smear surface occupied by mucin, shape of cell groupings, size and outline of tumour nuclei as well as presence or absence of nucleolus. RESULTS: Three of five pure mucinous carcinomas displayed at least two of the following features: abundant mucin, small nuclei and/or regular nuclear outlines. Sparse mucin, large nuclei, irregular nuclear outlines or the presence of nucleoli were found in 7 out of 17 mixed mucinous carcinomas but not in pure tumours. CONCLUSION: Cytopathological identification of patients with pure mucinous carcinomas may be performed in a limited number of cases.

Breast elasticity: principles, technique, results: an update and overview of commercially available software.

Breast ultrasound elasticity evaluation has become a routine tool in addition to diagnostic ultrasound during the last five years. Two elasticity evaluation modes are currently available: free-hand elastography and shear-wave elastography (SWE). Most of the commercially available elastography scanners have specific procedures which must be understood by the users. Free-hand elastography usually displays qualitative imaging such as an elastogram, but most of the companies now use it to quantify the relative stiffness between a lesion and the surrounding breast tissue. SWE is a new mode theoretically independent of the sonographer which displays more quantitative information, and can be useful for characterizing breast lesions. Recent studies on elastography suggest that elasticity imaging can increase B-mode accuracy and specificity in differentiating benign and malignant breast lesions. This functional imaging mode could help reduce the number of biopsies performed for benign breast lesions. This review gives a detailed description of the main commercially available systems and the results of current applications in the evaluation of breast elasticity.

Multinational study of oestrogen and progesterone receptor immunocytochemistry on breast carcinoma fine needle aspirates.

OBJECTIVES: To collect data on the variability of immunocytochemical (ICC) procedures used to detect oestrogen/progesterone receptors (ER/PR) on cytological material; to test the reproducibility of results; and to identify the crucial points in the ICC procedures that affect the result. METHODS: Ten laboratories from eight countries participated in a two-part study. In the first part, one of the participants (the coordinator) prepared and distributed cytospins from a fine needle aspirate of a primary breast carcinoma. Laboratories performed ICC staining for ER/PR according to their own methods on the test slides and in-house positive controls. Slides were returned to the coordinator together with information on the preparation of positive control slides and the ICC methodology used. In the second part, obligatory methods of fixation and antigen retrieval were specified. Evaluation of results included grading the number of positive cells, staining intensity, background staining, cytoplasmic staining, sample condition and cellularity. Participants evaluated their own results, which were subsequently evaluated by the coordinator. RESULTS: There was great variability in the preparation of slides for in-house controls and ICC methodology. The outcome of ICC staining of in-house control slides was excellent in two laboratories, adequate in three, sub-optimal in four and inadequate in one. Only six obtained a positive reaction on the test slides and not all were of a high quality. Results of the second run were greatly improved in terms of cellularity of in-house positive control slides, and scores for the percentage of stained cells and staining intensity of control and test slides. Cytospins and monolayer (ThinPrep(®)) preparations were superior to direct smears; methods of fixation and antigen retrieval were the key points in the staining process. CONCLUSIONS: Our experience points to the need for guidelines for hormonal receptor determination and external quality control on cytological material, in order for cytological methods to be used in routine clinical practice with a suitable degree of confidence.

Genetic variability and integration of Merkel cell polyomavirus in Merkel cell carcinoma.

Merkel cell polyomavirus (MCPyV) is associated to Merkel cell carcinoma (MCC). We studied 113 MCC tumoral skin lesions originating from 97 patients. MCPyV detection was higher in fresh-frozen (FF) biopsies (94%) than in formalin-fixed paraffin-embedded biopsies (39-47%). Mean viral load in FF tumor was of 7.5 copies per cell with a very wide range (0.01-95.4). Nineteen complete sequences of LTAg were obtained, mainly from FF biopsies when the viral load was high. Seventeen showed stop codons, all localized downstream of the pRb protein binding domain. Sequence comparison and phylogenetic analysis showed that all sequences clustered in the large C clade of MCPyV strains. MCPyV integration was demonstrated in 19 out of 27 FF MCC DNA biopsies without evidence of specific host cellular genome integration site. In 13/19 cases, the viral junction was located within the second exon of the LTAg, after the pRB binding domain.

[Radiobiology and breast cancer molecular classification: present or future?]. / Implications radiobiologiques de la classification moléculaire des cancers du sein : présent ou avenir?

Breast cancer treatment guidelines are based on usual prognostic factors such as size, histological grade, axillar lymph node involvement, expression of hormonal receptors. The intrinsic molecular classification is giving additional information over clinical and pathological features. Predictive models for systemic relapse have been established and are currently under clinical investigation to determine precisely when chemotherapy is needed. This review will look after the implications of this classification in terms of radiobiology: on one hand, we will look if this classification helps for loco-regional relapse prediction and on the other hand, if it is able to change the radiotherapy schedule within the molecular classification.

Morphological analysis of circulating tumour cells in patients undergoing surgery for non-small cell lung carcinoma using the isolation by size of epithelial tumour cell (ISET) method.

BACKGROUND AND OBJECTIVE: Recurrence rates after surgery for non-small cell lung cancer (NSCLC) range from 25 to 50% and 5-year survival is only 60-70%. Because no biomarkers are predictive of recurrence or the onset of metastasis, pathological TNM (pTNM) staging is currently the best prognostic factor. Consequently, the preoperative detection of circulating tumour cells (CTCs) might be useful in tailoring therapy. The aim of this study was to characterize morphologically any circulating non-haematological cells (CNHCs) in patients undergoing surgery for NSCLC using the isolation by size of epithelial tumour cell (ISET) method. METHODS: Of 299 blood samples tested, 250 were from patients with resectable NSCLC and 59 from healthy controls. The presence of CNHCs was assessed blindly and independently by 10 cytopathologists on May-Grünwald-Giemsa stained filters and the cells classified into three groups: (i) malignant cells, (ii) uncertain malignant cells, and (iii) benign cells. We assessed interobserver agreement using Kappa (κ) analysis as the measure of agreement. RESULTS: A total of 123 out of 250 (49%) patients showed CNHCs corresponding to malignant, uncertain malignant and benign cells, in 102/250 (41%), 15/250 (6%) and 6/250 (2%) cases, respectively. No CNHCs were detected in the blood of healthy subjects. Interobserver diagnostic variability was absent for CNHCs, low for malignant cells and limited for uncertain malignant and benign cells. CONCLUSION: Identification of CTCs in resectable NSCLC patients, using ISET technology and according to cytopathological criteria of malignancy, appears to be a new and promising field of cytopathology with potential relevance to lung oncology.

The Bethesda terminology for reporting thyroid cytopathology: from theory to practice in Europe.

OBJECTIVES: A 2007 conference held at the National Cancer Institute, Bethesda, Md., USA, proposed a new terminology for classifying the results of thyroid fine-needle aspiration (FNA) - The Bethesda System for Reporting Thyroid Cytology (TBSRTC). The need to standardize thyroid FNA terminology was emphasized during the 35th European Congress of Cytology in 2009. An interobserver review study to assess the new terminology for analyzing the results of thyroid FNA was organized by the scientific committee of the European Federation of Cytology Societies. STUDY DESIGN: Four experts in thyroid FNA examined and classified 116 FNAs according to the 6 levels of TBSRTC which are: nondiagnostic (ND); benign; atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS); follicular neoplasm/suspicious for a follicular neoplasm (FN/SFN), with those of Hürthle cell type reported as follicular neoplasm, Hürthle cell type/suspicious for a follicular neoplasm, Hürthle cell type (FNHCT/SFNHCT); suspicious (SUS), and malignant. RESULTS: The total consensus was 62.1%; the cytopathologists disagreed on 44 cases, including 8 cases of AUS/FLUS and 18 of FN/SFN; 59% of the cases had no consensus. They agreed on 73 and 80% of the cases classified as benign and malignant, respectively, and on 58.3% of the SUS cases. The percentage of no consensus for each expert was between 32 and 39%. CONCLUSIONS: Disagreement regarding the use of TBSRTC terminology for classifying the results of thyroid FNA mainly occurred in the most-often criticized categories of AUS/FLUS and FN/SFN.

Detection of circulating tumour cells with a hybrid (epithelial/mesenchymal) phenotype in patients with metastatic non-small cell lung cancer.

BACKGROUND: Circulating tumour cells (CTC) have a crucial role in metastasis formation and can consistently provide information on patient prognosis. Epithelial-mesenchymal transition (EMT) is considered as an essential process in the metastatic cascade, but there is currently very few data demonstrating directly the existence of the EMT process in CTCs. METHODS: CTCs were enriched by blood filtration using ISET (isolation by size of epithelial tumour cells), triply labelled with fluorescent anti-vimentin, anti-pan-keratin antibodies and SYTOX orange nuclear dye, and examined by confocal microscopy in six patients with metastatic non-small cell lung cancer (NSCLC). In parallel, CTCs were morphocytologically identified by an experienced cytopathologist. RESULTS: Isolated or clusters of dual CTCs strongly co-expressing vimentin and keratin were evidenced in all patients (range 5-88/5 ml). CTCs expressing only vimentin were detected in three patients, but were less frequent (range 3-15/5 ml). No CTC expressing only keratin was detected. CONCLUSION: We showed for the first time the existence of hybrid CTCs with an epithelial/mesenchymal phenotype in patients with NSCLC. Their characterisation should provide further insight on the significance of EMT in CTCs and on the mechanism of metastasis in patients with NSCLC.

A direct comparison of CellSearch and ISET for circulating tumour-cell detection in patients with metastatic carcinomas.

BACKGROUND: Circulating tumour cells (CTCs) can provide information on patient prognosis and treatment efficacy. However, there is no universal method to detect CTC currently available. Here, we compared the performance of two CTC detection systems based on the expression of the EpCAM antigen (CellSearch assay) or on cell size (ISET assay). METHODS: Circulating tumour cells were enumerated in 60 patients with metastatic carcinomas of breast, prostate and lung origins using CellSearch according to the manufacturer's protocol and ISET by studying cytomorphology and immunolabelling with anti-cytokeratin or lineage-specific antibodies. RESULTS: Concordant results were obtained in 55% (11 out of 20) of the patients with breast cancer, in 60% (12 out of 20) of the patients with prostate cancer and in only 20% (4 out of 20) of lung cancer patients. CONCLUSION: Our results highlight important discrepancies between the numbers of CTC enumerated by both techniques. These differences depend mostly on the tumour type. These results suggest that technologies limiting CTC capture to EpCAM-positive cells, may present important limitations, especially in patients with metastatic lung carcinoma.