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.

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.

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.

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.

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.

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.

Establishment of a Collection of Blood-Derived Products from COVID-19 Patients for Translational Research: Experience of the LPCE Biobank (Nice, France).

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).

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.

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.

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.

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.

Redefining malignant pleural mesothelioma types as a continuum uncovers immune-vascular interactions.

BACKGROUND: Malignant Pleural Mesothelioma (MPM) is an aggressive disease related to asbestos exposure, with no effective therapeutic options. METHODS: We undertook unsupervised analyses of RNA-sequencing data of 284 MPMs, with no assumption of discreteness. Using immunohistochemistry, we performed an orthogonal validation on a subset of 103 samples and a biological replication in an independent series of 77 samples. FINDINGS: A continuum of molecular profiles explained the prognosis of the disease better than any discrete model. The immune and vascular pathways were the major sources of molecular variation, with strong differences in the expression of immune checkpoints and pro-angiogenic genes; the extrema of this continuum had specific molecular profiles: a "hot" bad-prognosis profile, with high lymphocyte infiltration and high expression of immune checkpoints and pro-angiogenic genes; a "cold" bad-prognosis profile, with low lymphocyte infiltration and high expression of pro-angiogenic genes; and a "VEGFR2+/VISTA+" better-prognosis profile, with high expression of immune checkpoint VISTA and pro-angiogenic gene VEGFR2. We validated the gene expression levels at the protein level for a subset of five selected genes belonging to the immune and vascular pathways (CD8A, PDL1, VEGFR3, VEGFR2, and VISTA), in the validation series, and replicated the molecular profiles as well as their prognostic value in the replication series. INTERPRETATION: The prognosis of MPM is best explained by a continuous model, which extremes show specific expression patterns of genes involved in angiogenesis and immune response.

Monitoring BRAF and NRAS mutations with cell-free circulating tumor DNA from metastatic melanoma patients.

The mutation status of the BRAF and NRAS genes in tumor tissue is used to select patients with metastatic melanoma for targeted therapy. Cell-free circulating DNA (cfDNA) represents an accessible, non-invasive surrogate sample that could provide a snapshot of the BRAF and NRAS genotype in these patients. We investigated the feasibility of the Idylla™ assay for detection of BRAF and NRAS mutations in cfDNA of 19 patients with metastatic melanoma at baseline and during the course of treatment. The cfDNA genotype obtained with Idylla was compared to the results obtained with matched-tumor tissue and to clinical outcome. At baseline, 47% of patients harbored a BRAFV600 mutation in their cfDNA. Two months after targeted treatment the BRAFV600 mutant cfDNA was undetectable in all patients and 3 were disease-free. Moreover, 15% of patients harbored a NRAS mutation that was detected with plasma before treatment. The sensitivity and specificity were 80% and 89% for the BRAF status, and 79% and 100% for the NRAS status in pretreatment cfDNA compared to results obtained with a tissue test. Due to the small size of the population, no significant correlation was observed between the presence of BRAF or NRAS mutations in cfDNA and the metastatic tumor load or overall survival. In conclusion, this study demonstrated that evaluation with the Idylla system of the BRAF and NRAS mutation status in cfDNA may be a surrogate for determination of the BRAF and NRAS status in tumor tissue.

Establishing a Dedicated Lung Cancer Biobank at the University Center Hospital of Nice (France). Why and How?

Lung cancer is the major cause of death from cancer in the world and its incidence is increasing in women. Despite the progress made in developing immunotherapies and therapies targeting genomic alterations, improvement in the survival rate of advanced stages or metastatic patients remains low. Thus, urgent development of effective therapeutic molecules is needed. The discovery of novel therapeutic targets and their validation requires high quality biological material and associated clinical data. With this aim, we established a biobank dedicated to lung cancers. We describe here our strategy and the indicators used and, through an overall assessment, present the strengths, weaknesses, opportunities and associated risks of this biobank.

Ensuring the Safety and Security of Frozen Lung Cancer Tissue Collections through the Encapsulation of Dried DNA.

Collected specimens for research purposes may or may not be made available depending on their scarcity and/or on the project needs. Their protection against degradation or in the event of an incident is pivotal. Duplication and storage on a different site is the best way to assure their sustainability. The conservation of samples at room temperature (RT) by duplication can facilitate their protection. We describe a security system for the collection of non-small cell lung cancers (NSCLC) stored in the biobank of the Nice Hospital Center, France, by duplication and conservation of lyophilized (dried), encapsulated DNA kept at RT. Therefore, three frozen tissue collections from non-smoking, early stage and sarcomatoid carcinoma NSCLC patients were selected for this study. DNA was extracted, lyophilized and encapsulated at RT under anoxic conditions using the DNAshell technology. In total, 1974 samples from 987 patients were encapsulated. Six and two capsules from each sample were stored in the biobanks of the Nice and Grenoble (France) Hospitals, respectively. In conclusion, DNA maintained at RT allows for the conservation, duplication and durability of collections of interest stored in biobanks. This is a low-cost and safe technology that requires a limited amount of space and has a low environmental impact.

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.

Study of LAT1 Expression in Brain Metastases: Towards a Better Understanding of the Results of Positron Emission Tomography Using Amino Acid Tracers.

Positron emission tomography using radiolabeled amino acid (PET-AA) appears to be promising in distinguishing between recurrent tumour and radionecrosis in the follow-up of brain metastasis (BM). The amino acid transporter LAT1 and its cofactor CD98, which are involved in AA uptake, have never been investigated in BM. The aim of our study was to determine and compare the expression of LAT1 and CD98 in BM and in non-tumoral brain tissue (NT). The expression of LAT1 and CD98 were studied by immunohistochemistry in 67 BM, including 18 BM recurrences after radiotherapy, in 53 NT, and in 13 cases of patients with previously irradiated brain tumor and investigated by [18F] FDOPA-PET. LAT1 and CD98 expression were detected in 98.5% and 59.7% of BM respectively and were significantly associated with BM tissue as compared to NT (p<0.001). LAT1 expression in recurrent BM was significantly increased as compared to newly occurring BM. Ten cases investigated by [18F] FDOPA-PET corresponding to recurrent BM displayed significant [18F] FDOPA uptake and LAT1 overexpression whereas three cases corresponding to radionecrosis showed no or low uptake and LAT1 expression. LAT1 expression level and [18F] FDOPA uptake were significantly correlated. In conclusion, we hypothesized that BM may overexpress the AA transporter LAT1. We have shown that LAT1 overexpression was common in BM and was specific for BM as compared to healthy brain. These results could explain the specific BM uptake on PET-AA.

MicroRNA-375/SEC23A as biomarkers of the in vitro efficacy of vandetanib.

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.

High expression of TRF2, SOX10, and CD10 in circulating tumor microemboli detected in metastatic melanoma patients. A potential impact for the assessment of disease aggressiveness.

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.

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

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