Detection of circulating tumor cells as a prognostic factor in patients undergoing radical surgery for non-small-cell lung carcinoma: comparison of the efficacy of the CellSearch Assay™ and the isolation by size of epithelial tumor cell method.

Comparison of the efficacy of different enrichment methods for detection of circulating tumor cells (CTCs) before radical surgery is lacking in non-small-cell lung carcinoma (NSCLC) patients. Detection and enumeration of CTCs in 210 consecutive patients undergoing radical surgery for NSCLC were evaluated with the CellSearch Assay™ (CS), using the CellSearch Epithelial Cell Kit, and by the isolation by size of epithelial tumor (ISET) method, using double immunolabeling with anti-cytokeratin and anti-vimentin antibodies. CTCs were detected in 144 of 210 (69%) patients using CS and/or ISET and in 104 of 210 (50%) and 82 of 210 (39%) patients using ISET and CS, respectively. Using ISET, 23 of 210 (11%) patients had vimentin-positive cells with cytological criteria of malignancy. Disease-free survival (DFS) was worse for patients with CTCs compared to patients without CTCs detected by CS alone (p < 0.0001; log rank = 30.59) or by ISET alone (p < 0.0001; log rank = 33.07). The presence of CTCs detected by both CS and ISET correlated even better with shorter DFS at a univariate (p < 0.0001; log rank = 42.15) and multivariate level (HR, 1.235; 95% CI, 1.056-1.482; p < 0.001). CS and ISET are complementary methods for detection of CTCs in preoperative radical surgery for NSCLC. CTC detection in resectable NSCLC patients using CS and/or ISET could be a prognostic biomarker of great interest and may open up new avenues into improved therapeutic strategies for lung carcinoma patients.

Overexpression of carbonic anhydrase XII in tissues from resectable non-small cell lung cancers is a biomarker of good prognosis.

The pattern of protein expression in tumors is under the influence of nutrient stress, hypoxia and low pH, which determines the survival of neoplastic cells and the development of tumors. Carbonic anhydrase XII (CAXII) is a transmembrane enzyme that catalyzes the reversible hydration of cell-generated carbon dioxide into protons and bicarbonate. Hypoxic conditions activate its transcription and translation and enhanced expression is often present in several types of tumors. The aim of our study was to assess the prognostic significance of CAXII tumor tissues expression in patients with NSCLC. Five hundred fifty-five tumors were immunostained for CAXII on tissue microarrays (TMA) and the results were correlated with clinicopathological parameters and outcome of patients. CAXII overexpression was present in 105/555 (19%) cases and was associated with tumors of lower grade (p = 0.015) and histological type (p < 0.001), being significantly higher in squamous cell carcinoma. High CAXII expression correlated with better overall and disease-specific survival of patients with resectable NSCLC in univariate (p < 0.001) and multivariate survival analyses (p < 0.001). In conclusion, this is the first study demonstrating that a high CAXII tumor tissue expression evaluated on TMAs is related to a better outcome in a large series of patients with resectable NSCLC.

BRAFV600E mutation analysis by immunohistochemistry in patients with thoracic metastases from colorectal cancer.

The BRAF(V600E) mutation confers worse prognosis to metastatic colorectal cancer (mCRC) patients. In addition, this mutation has a negative predictive value for response to treatment with monoclonal antibodies against EGFR in patients with KRAS wild-type (wt) mCRC. The utility of immunohistochemistry (IHC) as an alternative approach for detection of BRAF(V600E) in the thoracic metastases of sporadic mCRC patients has not been evaluated until now. The purpose of this study was to compare BRAF(V600E) IHC staining with molecular biology methods and to define the diagnostic value of the VE1 antibody for the detection of BRAF(V600E) in this population. BRAF mutations were analysed by two DNA sequencing methods (pyrosequencing and Sanger sequencing) in a Caucasian population of 310 sporadic mCRC with thoracic metastases patients expressing KRAS wt. Detection of the BRAF(V600E) mutation was performed in the corresponding tumours by IHC using the VE1 antibody and compared to results of the DNA-based assays. Thirty-nine out of 310 (13%) of tumours harboured a BRAF mutation, which corresponded to either a BRAF(V600E) in 34 of 310 (11%) cases or a non-BRAF(V600E) mutation in 5 of 310 (2%) cases. IHC with VE1 was strongly positive in 32 of 34 (88%) BRAF(V600E) mutated tumours and negative in non-BRAF(V600E) mutated tumours. IHC using the VE1 clone is a specific and sensitive method for the detection of BRAF(V600E) and may be either a complementary or an alternative method to molecular testing in mCRC patients.

Diagnostic value of immunohistochemistry for the detection of the BRAF(V600E) mutation in papillary thyroid carcinoma: comparative analysis with three DNA-based assays.

BACKGROUND: The aim of this study was to compare the detection of BRAF(V600E) by immunohistochemistry (IHC) using a mutation-specific antibody with molecular biology methods for evaluation of papillary thyroid carcinoma (PTC) patients. PATIENTS AND METHODS: This study concerned 198 consecutive conventional PTC patients, of which the majority were women (133/198; 67%), with a mean age of 56 years (range 19-79 years). BRAF mutation analysis was performed using DNA-based (direct sequencing, pyrosequencing, and SNaPshot) and IHC (VE1 antibody) methods. The sensitivity and specificity of IHC for BRAF(V600E) was compared with the molecular biology data. RESULTS: A BRAF mutational result was obtained in 194 cases. A BRAF(V600E) mutation was detected in 153/194 (79%) cases of PTC when using at least one molecular method, and in 151/194 (78%) cases with IHC. No false positive results were noted using IHC to detect the BRAF(V600E) mutation. Besides this mutation, other rare BRAF mutations (BRAF(V600K) and BRAF(K601E)), used as negative controls, were consistently negative with IHC. The sensitivity and specificity of IHC for the detection of this mutation were 98.7% and 100% respectively. The IHC test demonstrated excellent performance at a level equivalent to two DNA-based counterparts (pyrosequencing and SNaPshot). Failure to achieve a result was more frequent with the direct sequencing method than with the three other methods. CONCLUSION: IHC using the VE1 antibody is a specific and sensitive method for the detection of the BRAF(V600E) mutation in PTC. IHC may be an alternative to molecular biology approaches for the routine detection of this mutation in PTC patients.

Two panels of plasma microRNAs as non-invasive biomarkers for prediction of recurrence in resectable NSCLC.

The diagnosis of non-small cell lung carcinoma (NSCLC) at an early stage, as well as better prediction of outcome remains clinically challenging due to the lack of specific and robust non-invasive markers. The discovery of microRNAs (miRNAs), particularly those found in the bloodstream, has opened up new perspectives for tumor diagnosis and prognosis. The aim of our study was to determine whether expression profiles of specific miRNAs in plasma could accurately discriminate between NSCLC patients and controls, and whether they are able to predict the prognosis of resectable NSCLC patients. We therefore evaluated a series of seventeen NSCLC-related miRNAs by quantitative real-time (qRT)-PCR in plasma from 52 patients with I-IIIA stages NSCLC, 10 patients with chronic obstructive pulmonary disease (COPD) and 20-age, sex and smoking status-matched healthy individuals. We identified an eleven-plasma miRNA panel that could distinguish NSCLC patients from healthy subjects (AUC = 0.879). A six-plasma miRNA panel was able to discriminate between NSCLC patients and COPD patients (AUC = 0.944). Furthermore, we identified a three-miRNA plasma signature (high miR-155-5p, high miR-223-3p, and low miR-126-3p) that significantly associated with a higher risk for progression in adenocarcinoma patients. In addition, a three-miRNA plasma panel (high miR-20a-5p, low miR-152-3p, and low miR-199a-5p) significantly predicted survival of squamous cell carcinoma patients. In conclusion, we identified two plasma miRNA expression profiles that may be useful for predicting the outcome of patients with resectable NSCLC.

In papillary thyroid carcinoma, TIMP-1 expression correlates with BRAF (V600E) mutation status and together with hypoxia-related proteins predicts aggressive behavior.

BRAF (V600E) causes upregulation of tissue inhibitor of metalloproteinase-1 (TIMP-1), which promotes cell invasion in papillary thyroid carcinoma (PTC). Hypoxia-inducible factor-1α (HIF- α) is regulated by hypoxia and also by the BRAF-mediated signaling pathway in PTC. We assessed the association of expression of TIMP-1, HIF-1α, and hypoxia-inducible carbonic anhydrase IX (CAIX) and XII (CAXII) with clinical parameters in PTC. TPC-1/BRAF (WT) wild-type and BcPAP/BRAF (V600E) -mutated PTC cell lines were selected to study the effects of the BRAF (V600E) mutation and hypoxia on expression in vitro of TIMP-1, CAIX, and CAXII proteins by immunoblotting. Higher expression of all proteins was detected in BcPAP cells exposed to hypoxia. Tissue microarray immunohistochemistry analysis was performed to study protein expression in 114 BRAF-genotyped PTC samples. Expression data on tumor tissue were compared with clinicopathological variables. TIMP-1 expression had a sensitivity of 87 % and a specificity of 83 % in identifying a BRAF mutation (P < 0.001) and was associated with pT stage (P = 0.001), pN stage (P = 0.02), and multifocality (P = 0.03). HIF-1α expression correlated with pT stage (P = 0.05). CAIX expression was associated with pN stage (P = 0.02), and both CAIX (P = 0.004) and CAXII (P = 0.05) were strongly associated with vascular invasion. We conclude that TIMP-1 protein expression is a reliable surrogate marker for BRAF-mutated status in PTC. TIMP-1 and hypoxia-regulated proteins are promising as predictors of aggressiveness in PTC and warrant further investigation as new therapeutic targets for the treatment of highly aggressive forms of PTC.

Cytopathologic detection of circulating tumor cells using the isolation by size of epithelial tumor cell method: promises and pitfalls.

Detection of circulating tumor cells (CTCs) morphologically may be a promising new approach in clinical oncology. We tested the reliability of a cytomorphologic approach to identify CTCs: 808 blood samples from patients with benign and malignant diseases and healthy volunteers were examined using the isolation by size of epithelial tumor cell (ISET) method. Cells having nonhematologic features (so-called circulating nonhematologic cells [CNHCs]) were classified into 3 categories: CNHCs with malignant features, CNHCs with uncertain malignant features, and CNHCs with benign features. CNHCs were found in 11.1% and 48.9% of patients with nonmalignant and malignant pathologies, respectively (P < .001). CNHCs with malignant features were observed in 5.3% and in 43.1% of patients with nonmalignant and malignant pathologies, respectively. Cytopathologic identification of CTCs using the ISET method represents a promising field for cytopathologists. The possibility of false-positive diagnosis stresses the need for using ancillary methods to improve this approach.

Preoperative circulating tumor cell detection using the isolation by size of epithelial tumor cell method for patients with lung cancer is a new prognostic biomarker.

PURPOSE: Pathologic TNM staging is currently the best prognostic factor for non-small cell lung carcinoma (NSCLC). However, even in early-stage NSCLC, the recurrence rates after surgery range from 25% to 50%. The preoperative detection of circulating tumor cells (CTC) could be useful to tailor new therapeutic strategies in NSCLC. We assessed the presence of CTC in NSCLC patients undergoing surgery, using cytologic analyses, after their isolation by size of epithelial tumor cells (ISET method). The presence and the number of CTCs were considered and correlated with clinicopathologic parameters including patient follow-up. EXPERIMENTAL DESIGN: Of the 247 blood samples tested, 208 samples were from patients with resectable NSCLC and 39 from healthy subjects. The mean follow-up was 24 months. An image of detected cells with presumably nonhematologic features [initially defined as "circulating nonhematologic cells" (CNHC)] was recorded. The presence of CNHC was assessed blindly and independently by 10 cytopathologists, using cytologic criteria of malignancy on stained filters. The count of detected CNHCs was made for each filter. RESULTS: One hundred two of 208 (49%) patients showed CNHCs corresponding to CNHC with malignant cytopathologic features in 76 of 208 (36%) cases. CNHCs were not detected in the control group. A level of 50 or more CNHCs corresponding to the third quartile was associated with shorter overall and disease-free-survival, independently of disease staging, and with a high risk of recurrence and death in early-stage I + II-resectable NSCLC. CONCLUSION: A high percentage of NSCLC patients show preoperative detection of CNHC by the ISET method. The presence and level of 50 or more CNHCs are associated with worse survival of patients with resectable NSCLC.

Usefulness of tissue microarrays for assessment of protein expression, gene copy number and mutational status of EGFR in lung adenocarcinoma.

Specific inhibitors targeting the epidermal growth factor receptor (EGFR) can increase survival rates in certain lung adenocarcinoma patients with mutations in the EGFR gene. Although such EGFR-targeted therapies have been approved for use, there is no general consensus among surgical pathologists on how the EGFR status should be tested in lung adenocarcinoma tissues and whether the results of immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and mutational analysis by molecular methods correlate. We evaluated the EGFR status in 61 lung adenocarcinomas by IHC (using total and mutant-specific antibodies against EGFR), by FISH analysis on tissue microarrays (TMAs), and by direct sequencing. The results of each method were compared using χ² and κappa statistics. The sensitivity and negative predictive value estimating the presence of abnormal EGFR for each test was calculated. The results show that, with respect to expression patterns and clinicopathological parameters, the total and mutant-specific EGFR detected by immunohistochemistry and FISH analysis on TMAs are valid and are equivalent to conventional methods performed on whole-tissue sections. Abnormal EGFR was detected in 52.4% of patients by IHC, FISH, and sequencing. The best sensitivity (100%) and negative predictive value (100%) was determined by evaluating the EGFR status with all methods. Testing for molecular changes in EGFR using a single test is likely to underestimate the presence of EGFR abnormalities. Taken together, these results demonstrate the high potential of TMAs to test for the major mechanisms of EGFR activation in patients with lung adenocarcinoma.