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Introduction International guidelines recommend BRAF mutational status assessment in treatment-naive advanced non-squamous non-small cell lung carcinoma (NSCLC) patients since the presence of a BRAFV600 mutation enables specific BRAF inhibitor treatment. For this purpose, the mutational status needs to be obtained in 10 working days. Herein, we prospectively evaluated the feasibility of systematic assessment of the BRAF status using immunohistochemistry (IHC) in a single institution (LPCE, Nice) at baseline for NSCLC diagnosed. Methods 1317 NSCLC were evaluated using BRAF IHC from 2011 to 2019. Initially the BRAF status was prospectively assessed using NGS and/or pyrosequencing in 618 consecutively diagnosed NSCLC patients from 2012 to 2016; BRAFV600E and BRAF nonV600E mutated tumors detected in this cohort were retrospectively evaluated using BRAF IHC. Secondarily, 699 biopsies of NSCLC were prospectively analyzed between 2017 and 2019 using BRAF IHC. BRAF IHC positive tumors were tested using a rapid BRAF specific PCR based assay. Results Initially, 21/618 (3%) of tumors (15 early and 6 late stage tumors) were BRAFV600E mutated according to the results of NGS and/or pyrosequencing. BRAF IHC was positive in 21/21 of these cases and negative in 51/51 (100 %) BRAF non V600E mutated cases. In the prospective BRAF IHC tested cohort of patients, 24/699 (3%) tumors (13 early and 11 late stage tumors) were positive with VE1 IHC. The BRAF PCR assay was positive in 20/24 (83 %) of these cases. Conclusion BRAFV600E IHC screening of treatment-naive NSCLC patients is a rapid, specific and very sensitive method which can lead in advanced stage positive NSCLC tumors to a BRAF inhibitor treatment. This test can be routinely integrated into mandatory predictive biomarker ‘testing of NSCLC. According to the organization of patient care and the physician’s request, this practice can be proposed as an alternative to NGS-based tissue biopsy made at baseline.

The evaluation of an increasing number of diagnostic and predictive markers is playing a central role in precision thoracic oncology. Multiplex immunohistochemistry (mIHC), alongside next-generation sequencing, is ideally situated for this purpose and maximizes tumor tissue preservation for molecular analyses that use increasingly large panels. However, the standardization and validation of mIHC that supports routine clinical laboratory processes are mandatory. After a previous proof-of-concept study, we now (i) optimized two automated four-plex assays on a commercially available IHC autostainer for use in daily practices worldwide and (ii) evaluated the repeatability and concordance of the assessment of the cell density.Two four-plex mIHC assays [i) TTF1, p40, PD-L1, CD8; and, ii) ALK, ROS1, BRAFV600E, NTRK] were optimized on the BenchMark ULTRA autostainer (Ventana Medical Systems, Inc.), as determined in comparison to conventional IHC chromogenic assays. Intra-site repeatability was evaluated on serial tumor sections from non-small cell lung carcinomas (NSCLC). The concordance was assessed by linear fit to plots of the percentage staining evaluated on tumor sections from 89 NSCLC patients.Following optimization, an average concordance for a staining rate of 95.4% was achieved between conventional IHC and mIHC across all selected markers. Assessment of intra-site repeatability showed strong concordance for all these markers (average, ROur optimized mIHC assay gave a sensitive and repeatable assessment of two panels of eight diagnostic and predictive biomarkers for NSCLC. The availability of standardized protocols to determine these biomarkers on a widely available IHC platform will expand the number of pathology laboratories able to determine the eligibility of patients with NSCLC for targeted treatment or immunotherapy in a reliable and concordant manner, thus providing a unique sample-sparing tool to characterize limited tissue samples in thoracic oncology.

Due to increased demand for testing, as well as restricted supply chain resources, testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to face many hurdles. Pooling several samples has been proposed as an alternative approach to address these issues. We investigated the feasibility of pooling nasopharyngeal swab (NPS) or saliva samples for SARS-CoV-2 testing with a commercial assay (Idylla SARS-CoV-2 test; Biocartis). We evaluated the 10-pool and 20-pool approaches for 149 subjects, with 30 positive samples and 119 negative samples. The 10-pool approach had sensitivity of 78.95% (95% confidence interval [CI], 54.43% to 93.95%) and specificity of 100% (95% CI, 71.51% to 100%), whereas the 20-pool approach had sensitivity of 55.56% (95% CI, 21.20% to 86.30%) and specificity of 100% (95% CI, 25% to 100%). No significant difference was observed between the results obtained with pooled NPS and saliva samples. Given the rapidity, full automation, and practical advantages of the Idylla SARS-CoV-2 assay, pooling of 10 samples has the potential to significantly increase testing capacity for both NPS and saliva samples, with good sensitivity. IMPORTANCE To control outbreaks of coronavirus disease 2019 (COVID-19) and to avoid reagent shortages, testing strategies must be adapted and maintained for the foreseeable future. We analyzed the feasibility of pooling NPS and saliva samples for SARS-CoV-2 testing with the Idylla SARS-CoV-2 test, and we found that sensitivity was dependent on the pool size. The SARS-CoV-2 testing capacity with both NPS and saliva samples could be significantly expanded by pooling 10 samples; however, pooling 20 samples resulted in lower sensitivity.

Background Recent advances obtained with immune checkpoint inhibitors (ICIs) targeting the programmed cell death-1 (PD-1) protein have significantly improved the outcome of patients with metastatic melanoma. The PD-L1 expression in tumour cells as detected by immunohistochemistry is a predictive biomarker in some solid tumours, but appears insufficient as prognostic or predictive factor of response to ICIs in metastatic melanomas. Objectives We investigated whether the presence and the features of pretreatment CD8+ tumour-infiltrating T lymphocytes (TILs) could be a complementary prognostic or predictive biomarker in patients with metastatic melanoma. Methods In this retrospective study, we evaluated the association of PD-L1 expression ≥5% of tumour cells combined with TIL features (CD8, CD28, Ki67) with the overall survival (OS) among 51 patients treated with ICIs and 54 patients treated with other treatment options (non-ICIs). Results PD-L1 positivity was observed in 33% and 39% of primary melanomas and matched metastases, respectively, with, however, poor concordance between the primary and the matched metastatic site (κ = 0.283). No significant association was noted between PD-L1 expression and CD8+ TIL profile analysed as single markers and OS or response to immunotherapy. Instead, their combined analysis in primary melanoma samples showed that the PD-L1-/CD8+ status was significantly associated with prolonged OS in the whole population (P = 0.04) and in the subgroup treated with non-ICIs (P = 0.009). Conversely, the PD-L1+/CD8+ status was a good prognostic factor in patients treated with ICIs (P = 0.022), whereas was significantly associated with poor prognosis in patients treated with non-ICIs (P = 0.014). While the expression of CD28 was not related to outcome, the Ki67 expression was significantly associated with poor OS in the subgroup CD8+ TIL+/PD-L1- (P = 0.02). Conclusions The pretreatment combination of PD-L1 expression with the level of CD8+ TILs could better assess OS and predict therapeutic response of patients with metastatic melanoma treated by either immunotherapy or other treatment regimens.

The outcome of patients with cutaneous melanoma has been strongly modified by recent advances obtained with Immune Checkpoint Inhibitors (ICIs). However, despite this breakthrough, durable response to ICIs is limited to a subset of patients. We investigated whether the expression of TRF2, which preserves telomere integrity, and have an effect on tumor immunosurveillance notably by directly recruiting and activating myeloid-derived suppressor cells (MDSCs), could be a prognostic biomarker in patients with relapsed or metastatic melanoma based on different treatment regimens. We evaluated retrospectively the association of TRF2 expressed in melanoma cells in combination with intratumoral CD33+ CD15+ CD14- MDSCs, as detected by immunohistochemistry and quantified by digital analysis, to clinicopathological features and overall survival (OS) among 48 patients treated with ICIs and 77 patients treated with other treatment options. The densities/mm2 of TRF2+ cells (P=.003) and CD33+ cells (P=.004) were individually significantly related to poor OS. In addition, only the combined expression of CD33+/CD15+/CD14- cells/mm2 was significantly correlated to poor OS (P=.017) in the whole study population as well as in patients treated by ICIs (P=.023). There was no significant difference in OS when analyzing the other markers individually or in combination according to the treatment regimen. The pre-treatment assessment of TRF2 expression and CD33+ cells/mm2 along with the density of CD33+/CD15+/CD14- cells/mm2 could assess OS and better predict clinical response of patients with melanoma treated by ICIs.

In only a few months after its inception, the COVID-19 pandemic lead to the death of hundreds of thousands of patients and to the infection of millions of people on most continents, mostly in the United States and in Europe. During this crisis, it was demonstrated that a better understanding of the pathogenicity, virulence, and contagiousness of SARS-CoV-2, all of which were initially underestimated, was urgently needed. The development of diagnostic tests to identify SARS-CoV-2 or to detect anti-SARS-CoV2 antibodies in blood, of vaccines, and of preventive and curative treatments has been relying on intense activity of scientists in academia and industry. It is noteworthy that these scientists depend on the use of high-quality biological samples taken from positive COVID-19 patients in a manner that preserves their integrity. Given this unique and emergent situation, it was necessary to urgently establish biological collections clinically annotated for immediate development of clinical and translational research projects focusing on COVID-19 biological aspects. It is in this very specific context that biobanks must rapidly adapt their infrastructure and/or operational capacity to fulfill new critical needs. We report the establishment of a biobank dedicated to the collection of blood-derived products (plasma, serum, and leukocytes) from COVID-19 patients hospitalized in the Nice Pasteur Hospital (Nice, France).

Introduction Tumor mutational burden (TMB) has been proposed as a novel predictive biomarker for the stratification of patients with NSCLC undergoing immune checkpoint inhibitor (ICI) treatment. The assessment of TMB has recently been established using large targeted sequencing panels, and numerous studies are ongoing to harmonize TMB assessment. "Correlation" or the coefficient of determination has generally been used to evaluate the association between different panels. We hypothesized that these metrics might overestimate the comparability, especially for lower TMB values. Methods A total of 30 samples from patients with NSCLC undergoing ICI treatment were consecutively sequenced using the following three large, targeted sequencing panels: FoundationOne, Oncomine TML, and QiaSeq TMB. The TMB values were compared in the whole patient population and in a subset of patients in which the TMB assessed by FoundationOne was between 5 and 25 mutations/Mb. Prediction of durable clinical benefit (>6 mo with no progression) was assessed using receiver operator characteristics, and optimal cutoff values were calculated using the Youden J statistic. Results Correlation between the three targeted sequencing panels was strong in the whole patient population (R2 > 0.79) but was dramatically reduced in the subset of patients with TMB of 5 to 25 mutations/Mb. The agreement assessed using the Bland-Altman method was also very low. All panels were able to predict durable clinical benefit in the TMB-high population. Conclusions Assessment of TMB using the three targeted sequencing panels was possible and predictive of response to ICI treatment, but correlation was an inappropriate measurement to assess the association between the respective panels.

International audience; Background - The introduction of liquid biopsy using PCR-based assays into routine practice has had a strong impact on the treatment of EGFR-mutated lung adenocarcinoma and is now commonly used for routine testing of EGFR mutations in certain clinical settings. To assess whether the claimed benefits of PCR-based assays hold true in daily practice at a multicenter clinical institution, we assessed how treatment decisions are affected by PCR-based assays for the analysis of EGFR mutations from plasma samples in a centralized laboratory (LPCE, Nice, France). Patients and methods - A total of 345 samples were analyzed using the US Food and Drug Administration-approved Cobas EGFR Mutation Test v2 and 103 using the Therascreen EGFR Plasma RGQ PCR Kit over 3 years (395 samples from 324 patients). Eleven plasma samples were validated independently using Cobas at 3 institutions, and 130 samples were analyzed using Stilla digital PCR. Clinical data were collected for 175 (54%) of 324 patients. Results - Cobas was superior to the Therascreen assay and demonstrated 100% reproducibility. Digital PCR showed only 48%, 83%, and 58% concordance with Cobas for exon 19 deletions, L858R mutations, and T790M mutations, respectively. Liquid biopsies helped inform and change treatment when resistance occurred and enabled the detection of EGFR mutations in patients when biopsy tissue results were unavailable. Conclusion - PCR-based assays are a fast and convenient test, allowing the detection of primary and secondary EGFR mutations from plasma. Cobas proved to be a reliable test, whereas digital PCR produced too many inconclusive results to be currently recommended as a principal testing device.

Introduction The detection of a ROS1 rearrangement in advanced and metastatic lung adenocarcinoma (LUAD) led to a targeted treatment with tyrosine kinase inhibitors with favorable progression-free survival and overall survival of the patients. Thus, it is mandatory to screen for the ROS1 rearrangement in all these patients. ROS1 rearrangements can be detected using break-apart fluorescence in situ hybridization (FISH), which is the gold standard; however, ROS1 immunohistochemistry (IHC) can be used as a screening test because it is widely available, easy and rapid to perform, and cost-effective. Methods We evaluated the diagnostic accuracy and interpathologist agreement of two anti-ROS1 IHC clones, SP384 (Ventana, Tucson, Arizona) and D4D6 (Cell Signaling, Danvers, Massachusetts), in a training cohort of 51 positive ROS1 FISH LUAD cases, and then in a large validation cohort of 714 consecutive cases of LUAD from six routine molecular pathology platforms. Results In the two cohorts, the SP384 and D4D6 clones show variable sensitivity and specificity rates on the basis of two cutoff points greater than or equal to 1+ (all % tumor cells) and greater than or equal to 2+ (>30% stained tumor cells). In the validation cohort, the D4D6 yielded the best accuracy for the presence of a ROS1 rearrangement by FISH. Interpathologist agreement was moderate to good (interclass correlation 0.722–0.874) for the D4D6 clone and good to excellent (interclass correlation: 0.830–0.956) for the SP384 clone. Conclusions ROS1 IHC is an effective screening tool for the presence of ROS1 rearrangements. However, users must be acutely aware of the variable diagnostic performance of different anti-ROS1 antibodies before implementation into routine clinical practice.

In this study, we performed microRNA (miRNA) expression profiling on a large series of sporadic and hereditary forms of medullary thyroid carcinomas (MTC). More than 60 miRNAs were significantly deregulated in tumor vs adjacent non-tumor tissues, partially overlapping with results of previous studies. We focused our attention on the strongest up-regulated miRNA in MTC samples, miR-375, the deregulation of which has been previously observed in a variety of human malignancies including MTC. We identified miR-375 targets by combining gene expression signatures from human MTC (TT) and normal follicular (Nthy-ori 3-1) cell lines transfected with an antagomiR-375 inhibitor or a miR-375 mimic, respectively, and from an in silico analysis of thyroid cell lines of Cancer Cell Line Encyclopedia datasets. This approach identified SEC23A as a bona fide miR-375 target, which we validated by immunoblotting and immunohistochemistry of non-tumor and pathological thyroid tissue. Furthermore, we observed that miR-375 overexpression was associated with decreased cell proliferation and synergistically increased sensitivity to vandetanib, the clinically relevant treatment of metastatic MTC. We found that miR-375 increased PARP cleavage and decreased AKT phosphorylation, affecting both cell proliferation and viability. We confirmed these results through SEC23A direct silencing in combination with vandetanib, highlighting the importance of SEC23A in the miR-375-associated increased sensitivity to vandetanib. Since the combination of increased expression of miR-375 and decreased expression of SEC23A point to sensitivity to vandetanib, we question if the expression levels of miR-375 and SEC23A should be evaluated as an indicator of eligibility for treatment of MTC patients with vandetanib.

Histopathology is the fundamental tool of pathology used for more than a century to establish the final diagnosis of lung cancer. In addition, the phenotypic data contained in the histological images reflects the overall effect of molecular alterations on the behavior of cancer cells and provides a practical visual reading of the aggressiveness of the disease. However, the human evaluation of the histological images is sometimes subjective and may lack reproducibility. Therefore, computational analysis of histological imaging using so-called "artificial intelligence" (AI) approaches has recently received considerable attention to improve this diagnostic accuracy. Thus, computational analysis of lung cancer images has recently been evaluated for the optimization of histological or cytological classification, prognostic prediction or genomic profile of patients with lung cancer. This rapidly growing field constantly demonstrates great power in the field of computing medical imaging by producing highly accurate detection, segmentation or recognition tasks. However, there are still several challenges or issues to be addressed in order to successfully succeed the actual transfer into clinical routine. The objective of this review is to emphasize recent applications of AI in pulmonary cancer pathology, but also to clarify the advantages and limitations of this approach, as well as the perspectives to be implemented for a potential transfer into clinical routine.

Pembrolizumab monotherapy has been approved for the first- and second-line treatment of patients with PD-L1-expressing advanced non-small cell lung cancer (NSCLC). Testing for PD-L1 expression with the PD-L1 immunohistochemistry (IHC) 22C3 companion diagnostic assay, which gives a tumor proportion score (TPS), has been validated on tumor tissue. We developed an optimized laboratory-developed test (LDT) that uses the 22C3 antibody (Ab) concentrate on a widely available IHC autostainer for biopsy and cytology specimens. The PD-L1 TPS was evaluated with 120 paired whole-tumor tissue sections and biopsy samples and with 70 paired biopsy and cytology samples (bronchial washes, n = 40; pleural effusions, n = 30). The 22C3 Ab concentrate-based LDT showed a high concordance rate between biopsy (~100%) and cytology (~95%) specimens when compared to PD-L1 IHC expression determined using the PD-L1 IHC 22C3 companion assay at both TPS cut points (≥1%, ≥50%). The optimized LDT presented here, using the 22C3 Ab concentrate to determine the PD-L1 expression in both tumor tissue and in cytology specimens, will expand the ability of laboratories worldwide to assess the eligibility of patients with NSCLC for treatment with pembrolizumab monotherapy in a reliable and reproducible manner.

Background High expression of programmed death ligand-1 (PD-L1) on tumor cells (TC) and/or on tumor-infiltrating immune cells (IC) is associated with a high response rate in patients with advanced nonsmall-cell lung cancer (NSCLC) treated with PD-L1 inhibitors. The use of a PD-L1 immunohistochemical (IHC) test in determining the responsiveness to immunotherapy has raised the question of the reliability and reproducibility of its evaluation in lung biopsies compared with corresponding resected surgical specimens. Patients and methods PD-L1 expression in TC and IC was assessed in 160 patients with operable NSCLC on both whole surgical tissue sections and matched lung biopsies, by using a highly sensitive SP142 IHC assay. The specimens were scored as TC 0–3 and IC 0–3 based on increasing PD-L1 expression. Results PD-L1 expression was frequently discordant between surgical resected and matched biopsy specimens (the overall discordance rate=48%; 95% confidence interval 4.64–13.24) and κ value was equal to 0.218 (poor agreement). In all cases, the biopsy specimens underestimated the PD-L1 status observed on the whole tissue sample. PD-L1-positive IC tumors were more common than PD-L1-positive TC tumors on resected specimens. The discrepancies were mainly related to the lack of a PD-L1-positive IC component in matched biopsies. Conclusions Our results indicate relatively poor association of the PD-L1 expression in TC and IC between lung biopsies and corresponding resected tumors. Although these results need to be further validated in larger cohorts, they indicate that the daily routine evaluation of the PD-L1 expression in diagnostic biopsies can be misleading in defining the sensitivity to treatment with PD-L1 targeted therapy.

Background Mortality in early stage, resectable lung cancer is sufficiently high to warrant consideration of post-surgical treatment. Novel markers to stratify resectable lung cancer patients may help with the selection of treatment to improve outcome. Methods Primary tumour tissue from 485 patients, surgically treated for stage I–II lung adenocarcinoma, was analysed for the RNA expression of 31 cell cycle progression (CCP) genes by quantitative polymerase chain reaction (PCR). The expression average, the CCP score, was combined with pathological stage into a prognostic score (PS). Cox proportional hazards regression assessed prediction of 5-year lung cancer mortality above clinical variables. The PS threshold was tested for risk discrimination by the Mantel–Cox log-rank test. Results The CCP score added significant information above clinical markers (all patients, P = 0.0029; stage I patients, P = 0.013). The prognostic score was a superior predictor of outcome compared to pathological stage alone (PS, P = 0.00084; stage, P = 0.24). Five-year lung cancer mortality was significantly different between the low-risk (90%, 95% confidence interval (CI) 81–95%), and high-risk groups (65%, 95% CI 57–72%), P = 4.2 × 10–6). Conclusions The CCP score is an independent prognostic marker in early stage lung adenocarcinoma. The prognostic score provides superior risk estimates than stage alone. The threefold higher risk in the high-risk group defines a subset of patients that should consider therapeutic choices to improve outcome.

KRAS mutations are detected in over one third of lung adenocarcinomas, most frequently in Caucasian and smoker patients. The impact of KRAS mutations on lung adenocarcinoma prognosis is currently subject to debate, as is their impact on the response to chemotherapy and EGFR tyrosine kinase inhibitors. The different methods for KRAS status assessment, based on histological and cytological samples or biological fluids, offer varying sensitivities. Since no treatments are available in clinical routine for KRAS-mutated lung cancer patients, one of the current major challenges in thoracic oncology is developing new dedicated strategic therapies. Different molecules can be developed that act on a post-transcriptional KRAS protein level, blocking its cytoplasmic membrane recruitment. The efficacy of these molecules' targeting of the different signaling pathways activated by the KRAS mutation (such as the MEK and BRAF pathways) is related to the particular KRAS mutation subtype. New therapeutic strategies are currently focused on certain genes linked with KRAS inducing a synthetic lethal interaction. The purpose of this work is to provide an overview of i) the recent epidemiological and molecular findings concerning KRASmutated lung adenocarcinoma, ii) the prognostic impact of KRAS mutations, in particular during response to treatment, iii) the available methods for detecting this mutation, and iv) the current molecules under development for new therapeutic strategies and the clinical trials targeting this genomic alteration.

Interest in biomarkers in the field of thoracic oncology is focused on the search for new robust tests for diagnosis (in particular for screening), prognosis and theragnosis. These biomarkers can be detected in tissues and/or cells, but also in biological fluids, mainly the blood. In this context, there is growing interest in the detection of circulating tumor cells (CTCs) in the blood of lung cancer patients since CTC identification, enumeration and characterization may have a direct impact on diagnosis, prognosis and theragnosis in the daily clinical practice. Many direct and indirect methods have been developed to detect and characterize CTCs in lung cancer patients. However, these different approaches still hold limitations and many of them have demonstrated unequal sensitivity and specificity. Indeed, these methods hold advantages but also certain disadvantages. Therefore, despite the promises, it is currently difficult and premature to apply this methodology to the routine care of lung cancer patients. This situation is the consequence of the analysis of the methodological approaches for the detection and characterization of CTCs and of the results published to date. Finally, the advent of targeted cancer therapies in thoracic oncology has stimulated considerable interest in non-invasive detection of genomic alterations in tumors over time through the analysis of CTCs, an approach that may help clinicians to optimize therapeutic strategies for lung cancer patients. We describe here the main methods for CTC detection, the advantages and limitations of these different approaches and the potential usefulness and value of CTC characterization in the field of thoracic oncology.

Background: Previous studies indicate that endothelial injury, as demonstrated by the presence of circulating endothelial cells (CECs), may predict clinical outcome in cancer patients. In addition, soluble CD146 (sCD146) may reflect activation of angiogenesis. However, no study has investigated their combined clinical value in patients undergoing resection for non-small cell lung cancer (NSCLC). Methods: Data were collected from preoperative blood samples from 74 patients who underwent resection for NSCLC. Circulating endothelial cells were defined, using the CellSearch Assay, as CD146+CD105+CD45−DAPI+. In parallel, sCD146 was quantified using an ELISA immunoassay. These experiments were also performed on a group of 20 patients with small-cell lung cancer, 60 healthy individuals and 23 patients with chronic obstructive pulmonary disease. Results: The CEC count and the plasma level of sCD146 were significantly higher in NSCLC patients than in the sub-groups of controls (P

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.

International audience; Most lipomas are characterized by translocations involving the HMGA2 gene in 12q14.3. These rearrangements lead to the fusion of HMGA2 with an ectopic sequence from the translocation chromosome partner. Only five fusion partners of HMGA2 have been identified in lipomas so far. The identification of novel fusion partners of HMGA2 is important not only for diagnosis in soft tissue tumors but also because these genes might have an oncogenic role in other tumors. We observed that t(1;12)(p32;q14) was the second most frequent translocation in our series of lipomas after t(3;12)(q28;q14.3). We detected overexpression of HMGA2 mRNA and protein in all t(1;12)(p32;q14) lipomas. We used a fluorescence in situ hybridization-based positional cloning strategy to characterize the 1p32 breakpoint. In 11 cases, we identified PPAP2B, a member of the lipid phosphate phosphatases family as the 1p32 target gene. Reverse transcription-polymerase chain reaction analysis followed by nucleotide sequencing of the fusion transcript indicated that HMGA2 3' untranslated region (3'UTR) fused with exon 6 of PPAP2B in one case. In other t(1;12) cases, the breakpoint was extragenic, located in the 3'region flanking PPAP2B 3'UTR. Moreover, in one case showing a t(1;6)(p32;p21) we observed a rearrangement of PPAP2B and HMGA1, which suggests that HMGA1 might also be a fusion partner for PPAP2B. Our results also revealed that adipocytic differentiation of human mesenchymal stem cells derived from adipose tissue was associated with a significant decrease in PPAP2B mRNA expression suggesting that PPAP2B might play a role in adipogenesis. © 2013 Wiley Periodicals, Inc.

The objective of this study was to evaluate the potential detection of circulating tumor cells (CTCs) using the CellSearch (CS) Assay™ in patients with locally advanced head and neck squamous cell carcinoma (HNSCC) and then to identify the clinical factors predictive of the presence of CTCs. The presence and number of CTCs were determined using the CS system before treatment, and in 10 healthy individuals (control group). The CS system was able to successfully identify the presence of CTCs in 8 of 49 patients (16 %) before therapy. No CTC was found in the control group. CTCs were detected before therapy in 1 of 19 patients (5 %) with N0 tumor and in 7 of 30 patients (23 %) with N1-2c tumor (p = 0.12; Fisher's exact test). CTCs were identified in a relatively low proportion of patients with locally advanced HNSCC.

The assays for the assessment of the PD-L1 status by immunohistochemistry are available in clinical studies in thoracic oncology to predict response to immunotherapies targeting the PD-1/PD-L1 pathway. With the arrival of this new class of molecules in second line and very soon in first line of treatment for patients with advanced or metastatic non-small cell lung cancer, these tests will certainly be required in routine once these new drugs will be granted marketing authorization. The rapid introduction of these "companion" or "complementary" tests seems essential to select patients to benefit from these effective but also expensive and sometimes toxic therapies. Although challenged by some oncologists (as some patients not expressing PD-L1 may sometimes respond to PD-1/PD-L1 blockade), the anti-PD-L1 immunohistochemically approach seems inevitable in 2017. This new activity developed in the pathology laboratories raises several questions: which anti-PD-L1 clone should be used? On which device? What threshold of positivity should be considered? Should PD-L1 expression be assessed on tumor cells as well as on the immune cells? What controls should be used? Comparative studies are underway or have been already implemented in order to answer some of these questions. This review addresses the different evaluation criteria for immunohistochemistry using the main anti-PD-L1 antibodies used to date as well the recently published studies using these antibodies in thoracic oncology.

// Marius Ilie 1, 2, * , Edith Szafer-Glusman 3, * , Veronique Hofman 1, 2, 4 , Elodie Long-Mira 1, 2 , Rebecca Suttmann 3 , Walter Darbonne 3 , Catherine Butori 1 , Salome Lalvee 1 , Julien Fayada 4 , Eric Selva 4 , Wei Yu 3 , Charles-Hugo Marquette 5 , David S. Shames 3 , Elizabeth Punnoose 3 , Paul Hofman 1, 2, 4 1 Laboratory of Clinical and Experimental Pathology and Liquid Biopsy Laboratory, Pasteur Hospital, University Hospital Federation OncoAge, Universite Cote d’Azur, Nice, France 2 Institute for Research on Cancer and Ageing, Nice (IRCAN), INSERM U1081/UMR CNRS 7284, Team 3, Antoine Lacassagne Cancer Center, Nice, France 3 Department of Oncology Biomarker Development and Oncology Clinical Development, Genentech, Inc, South San Francisco, California, USA 4 Nice Hospital-Related Biobank (BB 0025-00033), Pasteur Hospital, Nice, France 5 Pneumology Department, Pasteur Hospital, Nice, France * These authors contributed equally to this work Correspondence to: Paul Hofman, email: hofman.p@chu-nice.fr Keywords: MET protein expression, circulating tumor cells, NSCLC, immunocytochemistry, targeted therapy Received: June 20, 2016 Accepted: January 28, 2017 Published: February 15, 2017 ABSTRACT Given the difficulty in obtaining adequate tissue in NSCLC, we investigated the utility of circulating tumor cells (CTCs) for MET status assessment in NSCLC patients. We used two platforms for CTC capture, and assessed MET expression in CTCs and matched-bronchial biopsies in patients with advanced-stage III/IV lung adenocarcinoma. Baseline peripheral blood was collected from 256 advanced-stage III/IV NSCLC patients from Genentech clinical trials, and from 106 patients with advanced-stage III/IV lung adenocarcinoma treated at the Department of Pneumology, Pasteur Hospital, Nice. CTCs were enriched using CellSearch (Genentech), or ISET technologies (Pasteur Hospital). MET expression was evaluated by immunofluorescence on CellSearch, and by immunocytochemistry on ISET-enriched CTCs and on matched FFPE tissue sections (Pasteur Hospital). CTCs were detected in 83 of 256 (32%) patients evaluated on CellSearch, with 30 samples (12%) exhibiting ≥ 5 CTCs/7.5 ml blood. On ISET, CTC were observed in 80 of 106 patients (75%), and 79 patients (75%) exhibited ≥ 5 CTCs/4 ml blood. MET expression on ISET CTCs was positive in 72% of cases, and the MET expression on matched-patient tissue was positive in 65% patients using the Onartuzumab IHC scoring algorithm (93% concordance). Quantification of MET expression using H-scores showed strong correlation between MET expression in tissue and CTCs (Spearman correlation, 0.93). MET status in CTCs isolated on ISET filters from blood samples of advanced-stage NSCLC patients correlated strongly with MET status in tumor tissue, illustrating the potential for using CTCs as a non-invasive, real-time biopsy to determine MET status of patients entering clinical trials.

Circulating tumors cells (CTCs) can be detected in the blood of metastatic melanoma patients (MMPs) both as isolated circulating tumor cells (iCTCs) and circulating tumor microemboli (CTMs), but their clinical significance remains unknown. The aim of this work was to evaluate the prognostic impact in metastatic cutaneous melanoma of CTMs and iCTCs identified by a cytomorphological approach using the isolation by size of tumor cell (ISET) method. We characterized the phenotype of CTCs using anti‐PS100, anti‐SOX10, anti‐CD10, and anti‐TRF2 antibodies. 128 MMPs and 37 control healthy individuals with benign nevi were included in this study. Results were compared to the follow‐up of patients. 109/128 (85%) MMPs showed CTCs, 44/128 (34%) with 2 to 6 CTMs and 65/128 (51%) with 4 to 9 iCTCs. PS100 expression was homogeneous in iCTCs and heterogeneous in CTMs. SOX10, CD10, and TRF2 were mainly expressed in CTMs. None of the control subjects demonstrated circulating malignant tumor cells. Overall survival was significantly decreased in patients with CTMs, independently of the therapeutic strategies. In conclusion, the presence of CTMs is an independent predictor of shorter survival from the time of diagnosis of MMPs.

PD-1/PD-L1 inhibitors demonstrated durable clinical responses in patients with lung squamous cell carcinoma. However, the expression pattern of PD-L1 and the presence of CD8

Background A subgroup of anaplastic lymphoma kinase (ALK)-rearranged lung tumours can respond to ALK inhibitors. Until now, the ALK status in circulating tumour cells (CTCs) isolated from patients with lung cancer has not been characterised. We assessed the ALK status in CTCs detected in patients with lung cancer and correlated the results to the ALK status defined in the corresponding tumour tissue. Patients and methods A total of 87 patients with lung adenocarcinoma showing CTCs isolated using the isolation by size of epithelial tumour cell method were screened for their ALK status both in tumour samples and in CTCs. ALK break-apart fluorescence in situ hybridisation (FISH) and immunoreactivity analyses using an anti-ALK antibody (5A4 clone) were carried out on CTCs and compared with the results obtained in the corresponding tissue specimens. Results A total of five patients showed ALK-gene rearrangement and strong ALK protein expression in CTCs and in the corresponding tumour samples. Both ALK-FISH and ALK immunoreactivity analyses show negative results in CTCs and corresponding tumour samples for 82 patients. Conclusions We demonstrated that the ALK status can be determined in CTCs isolated from patients with lung cancer by immunocytochemistry and FISH analyses. These results favour non-invasive, ALK-gene status pre-screening on a routine basis on CTCs isolated from patients with lung cancer and open new avenues for real-time monitoring for adapted targeted therapy.

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.

Clear cell meningiomas (CCM) are rare tumors of the nervous system that usually occur in young patients and display high recurrence rates and potentially aggressive behavior. In this report, we describe a primary CCM of the orbit in an 84-year-old man with a previous history of a clear cell carcinoma of the kidney. Histologically, the tumor demonstrated a sheet-like proliferation of clear polygonal cells. Differential diagnosis includes metastasis of clear cell carcinomas. Immunohistochemistry, by showing that tumor cells expressed vimentin, epithelial membrane antigen, and progesterone antigens, and cytogenetic analysis, by identifying a monosomy 22, confirmed the diagnosis of CCM.

Rosai–Dorfman disease (RDD) involves the gastrointestinal tract only in exceptional cases, and this very unusual site of presentation can confuse the pathologist. We present a case of RDD manifesting as an intestinal occlusion caused by colonic diverticulitis. The patient was a 79-year-old man with myelodysplasia, who presented with fever, abdominal pain, and constipation. Colonoscopy revealed sigmoiditis and diverticulosis. Microscopic study of the sigmoid colon surgical specimen showed the histological and immunological features of RDD. No human DNA of herpesvirus types 6 and 8 (HHV6/HHV8), Epstein–Barr virus (EBV), and cytomegalovirus (CMV) was detected in tissue by polymerase chain reaction. Electron microscopic study revealed no microbes or viral particles. Widespread nodal and extranodal RDD occurred, and the patient died 2 y after initial surgery. The etiology of RDD is still under debate. We discuss the association of RDD with hematological disorders.

Background Knowledge of the BRAFV600E status is mandatory in metastatic melanoma patients (MMP). Molecular biology is currently the gold standard method for status assessment. Objectives We assessed and compared the specificity, sensibility, cost-effectiveness and turnaround time (TAT) of immunohistochemistry (IHC) and molecular biology for detection of the BRAFV600E mutation in 188 MMP. Methods IHC, with the VE1 antibody, and pyrosequencing analysis were performed with formalin fixed paraffin embedded tumour samples. Results The BRAFV600E mutation was detected by pyrosequencing in 91/188 (48%) patients. IHC was strongly positive (3+) in all of these 91 cases. IHC was strongly positive in 9/188 (5%) cases in which the molecular testing failed due to non-amplifiable DNA. Weak or moderate staining was noted in 10/188 (5%) cases in which the molecular biology identified BRAF wild-type tumours. The ratio of the global cost for IHC/molecular biology testing was 1 : 2.2. The average TAT was 48 h vs. 96 h, for IHC vs. molecular biology testing, respectively. Conclusions This study showed that VE1 IHC should be a substitute for molecular biology in the initial assessment of the BRAFV600E status in MPP. This methodology needs to be set up in pathology laboratories in accordance with quality control/quality assurance accreditation procedures. Under these strict conditions the question is to know if BRAFV600E-IHC can serve not only as a prescreening tool, but also as a stand-alone test (at least in cases displaying an unequivocally staining pattern) as well as an alternative predictive test for samples for which the molecular biology failed.

Immunotherapy targeting the PD-L1/PD-1 axis has shown recently some promising results in metastatic lung cancer patients. This treatment seems to be more effective when a high expression of PD-L1 is detected by immunohistochemistry in bronchial biopsies. In this regard, this targeted therapy will be proposed soon in metastatic lung cancer patients. This immunotherapy could be dependent to the immunohistochemical (IHC) assessment of PD-L1 expression, thus considered as a companion diagnostic test. This near perspective poses challenges with regard to the positivity threshold value for PD-L1 expression before therapy administration, the positive cellular compartment (tumour cells and/or immune cells), the percentage of positive cells and the clone which is used. A couple of patients have a good response to treatment targeting the PD-1/PD-L1 axis despite the absence or a weak PD-L1 expression. However the assessment of PD-L1 expression by immunohistochemistry will be the only mandatory approach before therapeutic strategies targeting the PD1/PD-L1 axis for lung cancer patients. In this review, the main challenges of using PD-L1 immunohistochemistry as a potential companion diagnostic tool for metastatic lung cancer patient immunotherapy will be discussed.

The aim of accreditation of a pathology laboratory is to control and optimize, in a permanent manner, good professional practice in clinical and molecular pathology, as defined by internationally established standards. Accreditation of a pathology laboratory is a key element in fine in increasing recognition of the quality of the analyses performed by a laboratory and in improving the care it provides to patients. One of the accreditation standards applied to clinical chemistry and pathology laboratories in the European Union is the ISO 15189 norm. Continued functioning of a pathology laboratory might in time be determined by whether or not it has succeeded the accreditation process. Necessary requirements for accreditation, according to the ISO 15189 norm, include an operational quality management system and continuous control of the methods used for diagnostic purposes. Given these goals, one would expect that all pathologists would agree on the positive effects of accreditation. Yet, some of the requirements stipulated in the accreditation standards, coming from the bodies that accredit pathology laboratories, and certain normative issues are perceived as arduous and sometimes not adapted to or even useless in daily pathology practice. The aim of this review is to elaborate why it is necessary to obtain accreditation but also why certain requirements for accreditation might be experienced as inappropriate.

Background It can be useful to assess the NRAS mutation status in patients with metastatic melanoma because NRAS -activating mutations confer resistance to RAF inhibitors, and NRAS- mutated patients appear to be sensitive to mitogen-activated protein kinase (MEK) inhibitors. Objective We aimed to assess the diagnostic accuracy of an immunohistochemistry (IHC) approach using a novel anti-NRAS (Q61R) monoclonal antibody on formalin-fixed paraffin-embedded tissue samples from patients with metastatic melanoma. Methods We conducted a retrospective multicenter cohort study on 170 patients with metastatic melanoma. The automated IHC assay was performed using the SP174 clone, and compared with results of the molecular testing. Results Evaluation of a test cohort with knowledge of the mutation status established a specific IHC pattern for the mutation. In the independent blinded analysis of the remaining cases, the anti-NRAS (Q61R) antibody accurately identified all NRAS Q61R-mutated tumors, and demonstrated 100% sensitivity and specificity. Limitations Limitations include retrospective design and lack of multicenter interobserver reproducibility. Conclusion The NRAS (Q61R) IHC assay is reliable and specific for the evaluation of the Q61R mutation status in metastatic melanoma and may be an alternative to molecular biology in evaluation of metastatic melanoma in routine practice.

The concept of personalized or stratified medicine in thoracic oncology have led to the development of companion diagnostic testing in the laboratories in order to detect genomic alterations which can be targeted by therapeutic molecules. The use of these companion tests has to be associated with an optimized quality control with the aim of getting solid results before treatment administration to the patients. The great majority of these tests is based on molecular biology approach. However, since the commercial availability of different antibodies targeting genomic alterations which can be used in formalin fixed paraffin sections, an alternative method to the molecular approach is the immunohistochemistry (IHC). Some of these antibodies are or will be probably soon used in a daily routine practice (such as anti-ALK or anti-MET antibodies). Other antibodies have currently a more restricted use in thoracic oncology (such as anti-BRAF V600E, anti-ROS1 and mutation-specific anti-EGFR antibodies). In this review, we aim to detail the advantages and the limits of IHC method in thoracic oncology field for personalized medicine, in particular comparatively to the molecular biology technology. Moreover, we discuss the opportunity to provide accredited IHC tests in the context of stratified medicine for lung cancer patients.

One of the earliest events in epithelial carcinogenesis is the dissolution of tight junctions and cell polarity signals that are essential for normal epithelial barrier function. Here, we report that EFA6B, a guanine nucleotide exchange factor for the Ras superfamily protein Arf6 that helps assemble and stabilize tight junction, is required to maintain apico-basal cell polarity and mesenchymal phenotypes in mammary epithelial cells. In organotypic three-dimensional cell cultures, endogenous levels of EFA6B were critical to determine epithelial–mesenchymal status. EFA6B downregulation correlated with a mesenchymal phenotype and ectopic expression of EFA6B hampered TGFβ-induced epithelial-to-mesenchymal transition (EMT). Transcriptomic and immunohistochemical analyses of human breast tumors revealed that the reduced expression of EFA6B was associated with loss of tight junction components and with increased signatures of EMT, cancer stemness, and poor prognosis. Accordingly, tumors with low levels of EFA6B were enriched in the aggressive triple-negative and claudin-low breast cancer subtypes. Our results identify EFA6B as a novel antagonist in breast cancer and they point to its regulatory and signaling pathways as rational therapeutic targets in aggressive forms of this disease. Cancer Res; 74(19); 5493–506. ©2014 AACR.

With the ongoing need to improve therapy for non–small cell lung cancer (NSCLC) there has been increasing interest in developing reliable preclinical models to test novel therapeutics. Patient-derived tumor xenografts (PDX) are considered to be interesting candidates. However, the establishment of such model systems requires highly specialized research facilities and introduces logistic challenges. We aimed to establish an extensive well-characterized panel of NSCLC xenograft models in the context of a long-distance research network after careful control of the preanalytical steps. One hundred fresh surgically resected NSCLC specimens were shipped in survival medium at room temperature from a hospital-integrated biobank to animal facilities. Within 24 h post-surgery, tumor fragments were subcutaneously xenografted into immunodeficient mice. PDX characterization was performed by histopathological, immunohistochemical, aCGH and next-generation sequencing approaches. For this model system, the tumor take rate was 35%, with higher rates for squamous carcinoma (60%) than for adenocarcinoma (13%). Patients for whom PDX tumors were obtained had a significantly shorter disease-free survival (DFS) compared to patients for whom no PDX tumors (P = 0.039) were obtained. We established a large panel of PDX NSCLC models with a high frequency of mutations (29%) in EGFR, KRAS, NRAS, MEK1, BRAF, PTEN, and PI3KCA genes and with gene amplification (20%) of c-MET and FGFR1. This new patient-derived NSCLC xenograft collection, established regardless of the considerable time required and the distance between the clinic and the animal facilities, recapitulated the histopathology and molecular diversity of NSCLC and provides stable and reliable preclinical models for human lung cancer research.

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.

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.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.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.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.

International audience; Chronic obstructive pulmonary disease (COPD) is a risk factor for lung cancer. Migration of circulating tumor cells (CTCs) into the blood stream is an early event that occurs during carcinogenesis. We aimed to examine the presence of CTCs in complement to CT-scan in COPD patients without clinically detectable lung cancer as a first step to identify a new marker for early lung cancer diagnosis. The presence of CTCs was examined by an ISET filtration-enrichment technique, for 245 subjects without cancer, including 168 (68.6%) COPD patients, and 77 subjects without COPD (31.4%), including 42 control smokers and 35 non-smoking healthy individuals. CTCs were identified by cytomorphological analysis and characterized by studying their expression of epithelial and mesenchymal markers. COPD patients were monitored annually by low-dose spiral CT. CTCs were detected in 3% of COPD patients (5 out of 168 patients). The annual surveillance of the CTC-positive COPD patients by CT-scan screening detected lung nodules 1 to 4 years after CTC detection, leading to prompt surgical resection and histopathological diagnosis of early-stage lung cancer. Follow-up of the 5 patients by CT-scan and ISET 12 month after surgery showed no tumor recurrence. CTCs detected in COPD patients had a heterogeneous expression of epithelial and mesenchymal markers, which was similar to the corresponding lung tumor phenotype. No CTCs were detected in control smoking and non-smoking healthy individuals. CTCs can be detected in patients with COPD without clinically detectable lung cancer. Monitoring ''sentinel'' CTC-positive COPD patients may allow early diagnosis of lung cancer.

International audience; The number of biobanks, in particular hospital-integrated tumor biobanks (HITB), is increasing all around the world. This is the consequence of an increase in the need for human biological resources for scientific projects and more specifically, for translational and clinical research. The robustness and reproducibility of the results obtained depend greatly on the quality of the biospecimens and the associated clinical data. They also depend on the number of patients studied and on the expertise of the biobank that supplied the biospecimens. The quality of a research biobank is undoubtedly reflected in the number and overall quality of the research projects conducted with biospecimens provided by the biobank. Since the quality of a research project can be measured from the impact factor of resulting publications, this also provides some indication of the quality of a research biobank. It is necessary for the biobank community to define "surrogate" quality indicators, and to establish systems of evaluation in relation to current and future resource requirements. These indicators will help in the realistic assessment of biobanks by institutions and funding bodies, and they will help biobanks demonstrate their value, raise their quality standards, and compete for funding. Given that biobanks are expensive structures to maintain, funding issues are particularly important, especially in the current economic climate. Use of performance indicators may also contribute to the development of a biobank impact factor or "bioresource research impact factor" (BRIF). Here we review four major categories of indicators that appear to be useful for the evaluation of a(m) HITB (quality, activity, scientific productivity, and "visibility"). In addition, we propose a scoring system to measure the chosen indicators.

The development of molecular analyses for thyroid pathologies is on going. These analyses provide new diagnostic tools with the aim of accurately distinguishing malignant and benign thyroid tumors. They are particularly useful as most of them can be done preoperatively on thyroid fine-needle aspiration biopsy samples. Furthermore, molecular biomarkers may play a promising role since they are able to predict the prognosis of patients with thyroid tumors. Moreover, identification of molecular markers as well as a better understanding of thyroid carcinogenesis will help develop innovative targeted therapies, particularly in patients with metastatic iodo-resistant thyroid carcinoma. To date, four types of somatic genetic alterations are known to hold potential interest for the diagnosis and/or prognosis of follicular cell-derived thyroid carcinomas: BRAF and RAS mutations, and RET/PTC and PAX8/PPARγ rearrangements. Other recent molecular biomarkers have been investigated in thyroid oncology, in particular different microRNA signatures. This review describes the different aspects of ancillary methods, including those bassed on molecular biology, that are of current interest for the diagnosis, prognosis and treatment of follicular cell-derived thyroid carcinomas.

The quick emerging of the several targeted therapies and the concept of personnalized medicine underlie the necessity to develop and to well organize a molecular biology (or molecular pathology) unit of high quality, dedicated to clinical care, in order to look for tissular and cellular theragnosis biomarkers. This new and sudden area of activity for a clinical pathologist is strongly linked to the knowledge of a new medical speciality in health care institutions. Thus, the molecular pathology (or molecular biology made from cellular or tissular samples) can nicely be implemented in a clinical pathology laboratory. This new mission for a pathologist has to be done in respect with a great quality assurance which should allow obtaining in a short-term an ISO 15189 accreditation to keep going to perform this activity. The present work aims to describe the main steps to be set up in the order to get an ISO 15189 accreditation in molecular pathology. The different chapters of this norm will not be described in their exhaustivity, but in their large lines. Finally, we will describe the potential difficulties and pitfalls to be avoided before getting this accreditation.

The advent of targeted therapies and personalized medicine in oncology has led in France to the settlement and organisation of a network of hospital molecular genetic platforms under the impetus of the National Cancer Institute (INCa). These platforms are, according to the concerned sites, integrated or not in pathology laboratories. The development of molecular biology methods, the choice of the procedures, the establishment of sample workflow, the quality control and the selection of the genomic alterations to be detected on each platform, have been left to the discretion of the different laboratories. Based on calls for project made by the INCa, hospital molecular genetic platforms were able to adapt their activity according to the assigned budgets. While the presence of some genomic alterations (i.e. KRAS gene mutations in metastatic colon adenocarcinoma or EGFR gene mutations in lung adenocarcinomas), may lead to administration of targeted therapies under the Marketing Authorization Application (MAA), others are associated with therapeutic clinical trials. However, increasing number of MAA for new molecules targeting genomic alterations is likely in the near future. In this context, it is necessary to quickly adapt the organisation of work of the hospital pathology laboratories performing molecular biology tests in order to meet the growing demand of oncologists in the field of targeted therapies. The purpose of this article is to describe the different steps of the settlement of a molecular genetic platform in an academic pathology laboratory (LPCE, CHU de Nice) and to show the experience of this laboratory specifically oriented on the support of the morphological and molecular diagnosis of lung cancer, thyroid cancer and malignant melanoma.

The biobanking area is highly complex, and its complexity is increasing along with its growth and demand. Due to the advancements in genetic research, stem cell research and regenerative medicine, biobanking has become ever more important and plays a key role in biomedical research. The robustness and the reproducibility of research results depend greatly on the quality and on the number of the samples used, and thus on the expertise of biobanks having supplied these samples. Undoubtedly, the recognition of a research biobank depends on the impact of the research projects conducted with samples obtained from tumour bank(s), but also on many other criteria. It thus seems important to determine a number of indicators within a biobank to estimate objective criteria for the performance of these structures. These indicators can allow to make some strategic decisions knowing that biobanks are expensive structures to maintain in the present hospital context. The use of these indicators could also contribute to the elaboration of an "biobank impact factor of" or so called "bioresource research impact factor" (BRIF). We describe here four major categories of indicators (quality, activity, scientific production, visibility), which seem to be useful for the evaluation of a biobank by making a proposition of allocation of coefficients for the various considered items.

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.