Assessment of Different Circulating Tumor Cell Platforms for Uveal Melanoma: Potential Impact for Future Routine Clinical Practice.

Liquid biopsy and circulating tumor cell (CTC) screening has gained interest over the last two decades for detecting almost all solid malignancies. To date, the major limitation in terms of the applicability of CTC screening in daily clinical practice is the lack of reproducibility due to the high number of platforms available that use various technologies (e.g., label-dependent versus label-free detection). Only a few studies have compared different CTC platforms. The aim of this study was to compare the efficiency of four commercially available CTC platforms (Vortex (VTX-1), ClearCell FX, ISET, and Cellsearch) for the detection and identification of uveal melanoma cells (OMM 2.3 cell line). Tumor cells were seeded in RPMI medium and venous blood from healthy donors, and then processed similarly using these four platforms. Melan-A immunochemistry was performed to identify tumor cells, except when the Cellsearch device was used (automated identification). The mean overall recovery rates (with mean recovered cells) were 39.2% (19.92), 22.2% (11.31), 8.9% (4.85), and 1.1% (0.20) for the ISET, Vortex (VTX-1), ClearCell FX, and CellSearch platforms, respectively. Although paramount, the recovery rate is not sufficient to assess a CTC platform. Other parameters, such as the purpose for using a platform (diagnosis, genetics, drug sensitivity, or patient-derived xenograft models), reproducibility, purity, user-friendliness, cost-effectiveness, and ergonomics, should also be considered before they can be used in daily clinical practice and are discussed in this article.

Prospective evaluation of NGS-based liquid biopsy in untreated late stage non-squamous lung carcinoma in a single institution.

BACKGROUND: NGS from plasma samples in non-squamous cell lung carcinoma (NSCC) can aid in the detection of actionable genomic alterations. However, the absolute clinical value of NGS in liquid biopsy (LB) made at baseline is currently uncertain. We assessed the impact of plasma-based NGS using an in-house test and an outsourced test in comparison to a routine molecular pathology workflow. METHODS: Twenty-four advanced/metastatic treatment-naïve NSCC patients were prospectively included. NGS analyses were conducted both in-house using the Oncomine cfTNA Panel and in an external testing center using the Foundation Liquid assay. NGS analysis and/or specific molecular based assays were conducted in parallel on tissue or cytological samples. RESULTS: Both LB tests were well correlated. Tissue NGS results were obtained in 67% of patients and demonstrated good correlation with LB assays. Activating EGFR mutations were detected using LB tests in three patients. PD-L1 expression assessed in tissue sections enabled the initiation of pembrolizumab treatment in five patients. CONCLUSION: NGS from LB is feasible in routine clinical practice using an in-house or an outsourced test at baseline. However, the impact on therapy selection was limited in this small series of patients and LB was not able to replace tissue-based testing in our hands.

PD-L1 expression in basaloid squamous cell lung carcinoma: Relationship to PD-1+ and CD8+ tumor-infiltrating T cells and outcome.

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+ and PD-1+ tumor-infiltrating T cells in the basaloid variant of squamous cell carcinoma remain unknown. immunohistochemistry analysis of PD-L1 expression, with three recently validated monoclonal antibodies used in clinical trials (clones SP142, SP263, and 28-8), and detection of CD8+ and PD-1+ tumor-infiltrating T cells was performed on whole-tissue sections from 56 patients following surgery for basaloid squamous cell carcinoma. Data were correlated to clinicopathological parameters and outcome. Fair to poor concordance was observed between the SP142 vs SP263 clones, and SP142 vs 28-8 (κ range, 0.018-0.412), while the 28-8 and SP263 demonstrated a strong correlation in both the tumor cell and immune cell compartments (κ=0.883, and κ=0.721). Expression of PD-L1 correlated with a high content of CD8+ and PD-1+ tumor-infiltrating T cells when using SP142 (P=0.012; P=0.022), but not with SP263 or 28-8 (P=0.314; P=0.611). In the multivariate analysis, we found significantly better disease-free and overall survival rates for high PD-L1 expression with SP142, CD8+ and PD-1+ tumor-infiltrating T cells (P=0.003; P=0.007). No significant prognosis value was observed for SP263 and 28-8 clones, except a correlation between improved overall survival and SP263 in the univariate analysis (P=0.039), not confirmed in the multivariate model. In conclusion, we report that the expression of PD-L1 and the content of CD8+ and PD-1+ tumor-infiltrating T cells is an independent indicator of better outcome in basaloid squamous cell carcinoma patients, although the observed effect is dependent on the PD-L1 immunohistochemistry assay.

Shifting from Immunohistochemistry to Screen for ALK Rearrangements: Real-World Experience in a Large Single-Center Cohort of Patients with Non-Small-Cell Lung Cancer.

The identification of ALK fusions in advanced non-small-cell lung carcinoma (aNSCLC) is mandatory for targeted therapy. The current diagnostic approach employs an algorithm using ALK immunohistochemistry (IHC) screening, followed by confirmation through ALK FISH and/or next-generation sequencing (NGS). Challenges arise due to the infrequency of ALK fusions (3-7% of aNSCLC), the suboptimal specificity of ALK IHC and ALK FISH, and the growing molecular demands placed on small tissue samples, leading to interpretative, tissue availability, and time-related issues. This study investigates the effectiveness of RNA NGS as a reflex test for identifying ALK fusions in NSCLC, with the goal of replacing ALK IHC in the systematic screening process. The evaluation included 1246 NSCLC cases using paired techniques: ALK IHC, ALK FISH, and ALK NGS. ALK IHC identified 51 positive cases (4%), while RNA NGS detected ALK alterations in 59 cases (4.8%). Of the 59 ALK-positive cases identified via NGS, 53 (89.8%) were confirmed to be positive. This included 51 cases detected via both FISH and IHC, and 2 cases detected only via FISH, as they were completely negative according to IHC. The combined reporting time for ALK IHC and ALK FISH averaged 13 days, whereas ALK IHC and RNA NGS reports were obtained in an average of 4 days. These results emphasize the advantage of replacing systematic ALK IHC screening with RNA NGS reflex testing for a more comprehensive and accurate assessment of ALK status.

Reproducibility of c-Met Immunohistochemical Scoring (Clone SP44) for Non-Small Cell Lung Cancer Using Conventional Light Microscopy and Whole Slide Imaging.

Emerging therapies for non-small cell lung cancer targeting c-Met overexpression have recently demonstrated promising results. However, the evaluation of c-Met expression can be challenging. We aimed to study the inter and intraobserver reproducibility of c-Met expression evaluation. One hundred ten cases with non-small cell lung cancer (40 biopsies and 70 surgical specimens) were retrospectively selected in a single laboratory (LPCE) and evaluated for c-Met expression. Six pathologists (4 seniors and 2 juniors) evaluated the H-score and made a 3-tier classification of c-Met expression for all cases, using conventional light microscopy (CLM) and whole slide imaging (WSI). The interobserver reproducibility with CLM gave global Cohen Kappa coefficients (ƙ) ranging from 0.581 (95% CI: 0.364-0.771) to 0.763 (95% CI: 0.58-0.92) using the c-Met 3-tier classification and H-score, respectively. ƙ was higher for senior pathologists and biopsy samples. The interobserver reproducibility with WSI gave a global ƙ ranging from 0.543 (95% CI: 0.33-0.724) to 0.905 (95% CI: 0.618-1) using the c-Met H-score and 2-tier classification (≥25% 3+), respectively. ƙ for intraobserver reproducibility between CLM and WSI ranged from 0.713 to 0.898 for the c-Met H-score and from 0.600 to 0.779 for the c-Met 3-tier classification. We demonstrated a moderate to excellent interobserver agreement for c-Met expression with a substantial to excellent intraobserver agreement between CLM and WSI, thereby supporting the development of digital pathology. However, some factors (scoring method, type of tissue samples, and expertise level) affect reproducibility. Our findings highlight the importance of establishing a consensus definition and providing further training, particularly for inexperienced pathologists, for c-Met immunohistochemistry assessment in clinical practice.

[Accreditation of the activity of molecular pathology according to ISO 15189: key steps to follow and the main potential pitfalls]. / Accréditation de l'activité de pathologie moléculaire selon la norme ISO 15189. Principales étapes à respecter et principaux écueils possibles.

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 accreditation of a surgical pathology and somatic genetic laboratory (LPCE, CHU of Nice) according to the ISO 15189 norm: Sharing of experience]. / L'accréditation selon la norme ISO 15189 d'un laboratoire de pathologie et de génétique somatique (LPCE, CHU de Nice) : retour d'expérience.

Accreditation is going to be vital and unavoidable in the medium term for medical biology laboratories in France. This accreditation will certainly condition the authorization to conduct biological testing in the health care system. All the biological specialities are now affected by this procedure, including the somatic genetics. The anatomo-pathology, which is a medical speciality in France, may be also concerned by the accreditation. However, the nature and the practices of this specialty increase the complexity of this approach to be implemented according to the standard requested by the authorities, i.e. the ISO 15189 normative standard (standard on "specific requirements for quality and competence for medical biology analysis laboratories"). The present article recounts the experience of a hospital laboratory (LPCE, Nice University Hospital) composed of a surgical pathology and a somatic genetics unit: (1) in the accreditation process according to the ISO 15189 standard, (2) at the time of the audit made by the team of "COFRAC" evaluators, and, (3) in evaluating the strategy implemented following the audit.

[Setting up a department of molecular pathology in oncology in a pathology laboratory (LPCE, CHU de Nice)]. / Mise en place d'un secteur de pathologie moléculaire en oncologie au sein d'un laboratoire d'anatomie pathologique (LPCE, CHU de Nice).

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.

Need for a Dedicated Ophthalmic Malignancy Clinico-Biological Biobank: The Nice Ocular MAlignancy (NOMA) Biobank.

Ophthalmic malignancies include various rare neoplasms involving the conjunctiva, the uvea, or the periocular area. These tumors are characterized by their scarcity as well as their histological, and sometimes genetic, diversity. Uveal melanoma (UM) is the most common primary intraocular malignancy. UM raises three main challenges highlighting the specificity of ophthalmic malignancies. First, UM is a very rare malignancy with an estimated incidence of 6 cases per million inhabitants. Second, tissue biopsy is not routinely recommended due to the risk of extraocular dissemination. Third, UM is an aggressive cancer because it is estimated that about 50% of patients will experience metastatic spread without any curative treatment available at this stage. These challenges better explain the two main objectives in the creation of a dedicated UM biobank. First, collecting UM samples is essential due to tissue scarcity. Second, large-scale translational research programs based on stored human samples will help to better determine UM pathogenesis with the aim of identifying new biomarkers, allowing for early diagnosis and new targeted treatment modalities. Other periocular malignancies, such as conjunctival melanomas or orbital malignancies, also raise specific concerns. In this context, the number of biobanks worldwide dedicated to ocular malignancies is very limited. The aims of this article were (i) to describe the specific challenges raised by a dedicated ocular malignancy biobank, (ii) to report our experience in setting up such a biobank, and (iii) to discuss future perspectives in this field.

The Need to Set up a Biobank Dedicated to Lymphoid Malignancies: Experience of a Single Center (Laboratory of Clinical and Experimental Pathology, University Côte d'Azur, Nice, France).

Several therapies to improve the management of lymphoma are currently being investigated, necessitating the development of new biomarkers. However, this requires high-quality and clinically annotated biological material. Therefore, we established a lymphoma biobank including all available biological material (tissue specimens and matched biological resources) along with associated clinical data for lymphoma patients diagnosed, according to the WHO classification, between 2005 and 2022 in the Laboratory of Clinical and Experimental Pathology, Nice, France. We retrospectively included selected cases in a new collection at the Côte d'Azur Biobank, which contains 2150 samples from 363 cases (351 patients). The male/female ratio was 1.3, and the median age at diagnosis was 58 years. The most common lymphoma types were classical Hodgkin lymphoma, diffuse large B-cell lymphoma, and extra-nodal marginal zone lymphoma of MALT tissue. The main sites of lymphoma were the mediastinum, lymph node, Waldeyer's ring, and lung. The Côte d'Azur Biobank is ISO 9001 and ISO 20387 certified and aims to provide high quality and diverse biological material to support translational research projects into lymphoma. The clinico-pathological data generated by this collection should aid the development of new biomarkers to enhance the survival of patients with lymphoid malignancies.

Ultrafast Gene Fusion Assessment for Nonsquamous NSCLC.

Introduction: Gene fusion testing of ALK, ROS1, RET, NTRK, and MET exon 14 skipping mutations is guideline recommended in nonsquamous NSCLC (NS-NSCLC). Nevertheless, assessment is often hindered by the limited availability of tissue and prolonged next-generation sequencing (NGS) testing, which can protract the initiation of a targeted therapy. Therefore, the development of faster gene fusion assessment is critical for optimal clinical decision-making. Here, we compared two ultrafast gene fusion assays (UFGFAs) using NGS (Genexus, Oncomine Precision Assay, Thermo Fisher Scientific) and a multiplex reverse-transcriptase polymerase chain reaction (Idylla, GeneFusion Assay, Biocartis) approach at diagnosis in a retrospective series of 195 NS-NSCLC cases and five extrapulmonary tumors with a known NTRK fusion. Methods: A total of 195 NS-NSCLC cases (113 known gene fusions and 82 wild-type tumors) were included retrospectively. To validate the detection of a NTRK fusion, we added five NTRK-positive extrathoracic tumors. The diagnostic performance of the two UFGFAs and standard procedures was compared. Results: The accuracy was 92.3% and 93.1% for Idylla and Genexus, respectively. Both systems improved the sensitivity for detection by including a 5'-3' imbalance analysis. Although detection of ROS1, MET exon 14 skipping, and RET was excellent with both systems, ALK fusion detection was reduced with sensitivities of 87% and 88%, respectively. Idylla had a limited sensitivity of 67% for NTRK fusions, in which only an imbalance assessment was used. Conclusions: UFGFA using NGS and reverse-transcriptase polymerase chain reaction approaches had an equal level of detection of gene fusion but with some technique-specific limitations. Nevertheless, UFGFA detection in routine clinical care is feasible with both systems allowing faster initiation of therapy and a broad degree of screening.

Predictive clinical outcome of the intratumoral CD66b-positive neutrophil-to-CD8-positive T-cell ratio in patients with resectable nonsmall cell lung cancer.

BACKGROUND: The role of the interaction between tumor cells and inflammatory cells in nonsmall cell lung carcinoma (NSCLC) is unclear. In this study, the authors assessed the prognostic impact of intratumoral cluster of differentiation 66b (carcinoembryonic antigen-related cell adhesion molecule 8 [CD66b])-positive neutrophils and of the intratumoral CD66b-positive neutrophil-to-cluster of differentiation 8 (cell surface antigen T8 [CD8])-positive lymphocytes (the CD66b-positive neutrophil-to-CD8-positive lymphocyte ratio [iNTR]) in patients with resectable NSCLC. METHODS: Expression levels of CD66b and CD8 were evaluated by immunohistochemistry on tissue microarrays consisting of 632 NSCLC specimens from patients who underwent curative surgery. The relation between clinicopathologic variables and patient outcome was assessed. RESULTS: Intratumoral CD66b-positive neutrophils were elevated in 318 patients (50%). In univariate analysis, an increase in CD66b-positive cells was associated with a high cumulative incidence of relapse (CIR) (median CIR, 51 months for low CD66b-positive cell density; 36 months for high CD66b-positive cell density; P = .002) and trended toward worse overall survival (OS) (median OS, 57 months for low CD66b-positive cell density; 54 months for high CD66b-positive cell density; P = .088). The iNTR was elevated in 190 patients (30%). An increased iNTR was strongly associated with both a high CIR (median CIR: 43 months for an iNTR ≤1; 34 months for an iNTR >1; P < .0001) and poor OS (median OS: 60 months for an iNTR ≤1; 46 months for an iNTR >1; P < .0001). In multivariate analysis, independent prognostic factors for a higher CIR were high iNTR (hazard ratio [HR], 0.71; 95% confidence interval [CI], 0.56-0.90; P = .005) and tumor stage >I, (HR, 0.39; 95% CI, 0.30-0.52; P < .0001). Independent prognostic factors for worse OS were a high iNTR (HR, 0.70; 95% CI, 0.54-0.91; P = .007) and tumor stage >I (HR, 0.35; 95% CI, 0.26-0.47; P < .0001). CONCLUSIONS: The current results indicated that the iNTR is a novel, independent prognostic factor for a high rate of disease recurrence and poor OS in patients with resectable NSCLC.

[Setting up indicators in biobanking: why and how?]. / Mise en place d'indicateurs de suivi au sein d'une tumorothèque et/ou d'un centre de ressources biologiques : pourquoi et comment ?

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.

Comparison of Two Rapid Assays for the Detection of BRAF V600 Mutations in Metastatic Melanoma including Positive Sentinel Lymph Nodes.

Testing for the BRAF mutation is mandatory for the management of patients with locally advanced or metastatic melanoma. Molecular analysis based on DNA sequencing remains the gold-standard method for the screening of the different BRAF mutations. These methods must be rapid, sensitive, and specific enough to allow optimal therapeutic management in daily practice and also to include patients in clinical trials. Here, we compared the Idylla BRAF Mutation Test and the anti-BRAF V600E (clone VE1) immunohistochemistry (IHC) in 90 melanoma samples, with a focus on a challenging cohort of 32 positive sentinel lymph nodes. The BRAF status was assessed with both methods independently of the percentage of tumor cells. The concordance rate was calculated excluding both non-contributory analyses and BRAFV600K/R/M mutants due to the specific V600E-IHC test design. The incidence of the BRAFV600E mutation was 33% with both BRAF Idylla and BRAF IHC. The agreement rate was 91% (72/79). Although the agreement rate was high, we suggest that the use of IHC is more suitable for rapid BRAF testing on sentinel lymph node biopsies when associated with a low percentage and scattered tumor cells, which gave a high risk of non-contributory analysis and/or false negative results with the IdyllaTMBRAF Mutation Test.

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.

[The Nice CHU biobank experience to collect patients' informed consent for research context (2004-2009)]. / L'expérience de la tumorothèque du CHU de Nice pour le recueil des consentements éclairés dans le cadre de la recherche (2004-2009).

Over the last 10 years, significant financial support from the French National Institute of Cancer (INCa), the Ministry of Health (DGOS), and the Health and Research National Institute (Inserm) helped biobanks--of which tumour banks represent a prominent example of hospital-based infrastructures--to improve their operations, and in some instances to adopt the rules of Biological Ressource Centers as defined by OECD. Nowadays, the use of biological samples of human origin is strictly subordinated to regulations that integrate bioethical principles. However, in spite of the establishment of these regulations, requirement to obtain an authorisation and/or to register the biological collections with the Ministry of Research, many uncertainties persist. While French regulations mandate that samples can be used for research as long as patients did not oppose to such use, many biobank curators face practical and theoretical issues when establishing a Material Transfer Agreement with scientists, due to the lack of harmonization between national regulations--particularly due to a different perception of privacy and free will in anglo-american and other countries--and different demands on the side of private industry or editorial boards of scientific journals. The goal of this article is (1) to describe the procedure followed to collect patients' informed consent at the Biobank of CHU de Nice and (2) to assess the number of obtained consents in comparison to the number of collected samples between 01/09/2004 and 31/12/2009, the number of consents obtained before or after collecting the samples, and the number of patients' refusal to collect their biological resources. This balance-sheet is settled for the three major collections (thoracic, thyroid and head and neck tissues) from the Biobank of CHU de Nice. Results show that 88 % of consents were obtained during this period (82 % in a prospective manner and 6 % in a retrospective manner). Refusal was notified by writing in nine cases only. The percentage of consents varies slightly according to the collection involved and is stable from 2004 to 2009. Overall, our procedure is quite efficient at obtaining informed consents from a majority of patients for whom the tumour bank stores biological samples. This situation provides optimal conditions for the use of collected samples in the context of national and international research projects.

Targeted Assessment of the EGFR Status as Reflex Testing in Treatment-Naive Non-Squamous Cell Lung Carcinoma Patients: A Single Laboratory Experience (LPCE, Nice, France).

BACKGROUND: Assessment of actionable EGFR mutations is mandatory for treatment-naïve advanced or metastatic non-squamous lung carcinoma (NSLC), but the results need to be obtained in less than 10 working days. For rapid EGFR testing, an EGFR-specific polymerase chain reaction (PCR) assay is an alternative and simple approach compared to next generation sequencing (NGS). Here, we describe how a rapid EGFR-specific PCR assay can be implemented in a single laboratory center (LPCE, Nice, France) as reflex testing in treatment-naïve NSLC. METHODS: A total of 901 biopsies from NSLC with more than 10% of tumor cells were prospectively and consecutively evaluated for EGFR mutation status between November 2017 and December 2019 using the Idylla system (Biocartis NV, Mechelen, Belgium). NGS was performed for nonsmokers with NSLC wild type for EGFR, ALK, ROS1, and BRAF and with less than 50% PD-L1 positive cells using the Hotspot panel (Thermo Fisher Scientific, Waltham, MA, USA). RESULTS: Results were obtained from 889/901 (97%) biopsies with detection of EGFR mutations in 114/889 (13%) cases using the Idylla system. Among the 562 EGFR wild type tumors identified with Idylla, NGS detected one actionable and one nonactionable EGFR mutation. CONCLUSIONS: Rapid and targeted assessment of EGFR mutations in treatment-naïve NSLC can be implemented in routine clinical practice. However, it is mandatory to integrate this approach into a molecular algorithm that allows evaluation of potentially actionable genomic alterations other than EGFR mutations.

Real-world assessment of the BRAF status in non-squamous cell lung carcinoma using VE1 immunohistochemistry: A single laboratory experience (LPCE, Nice, France).

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-naïve 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.

Critical Assessment in Routine Clinical Practice of Liquid Biopsy for EGFR Status Testing in Non-Small-Cell Lung Cancer: A Single-Laboratory Experience (LPCE, Nice, France).

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.