Decalcification can cause the failure of BRAF molecular analyses and anti‐BRAFV600E VE1 immunohistochemistry

International audienceBRAF mutation detection is worthwhile for the management of patients with some advanced cancers. The tumor samples are sometimes difficult to analyze using DNA-based molecular methods because of poor tumor DNA quality or quantity. Anti-BRAFV600E VE1 immunohistochemistry (IHC) has been proposed as a valuable ancillary tool to analyze some "molecularly challenging" tumor samples. In this technical study, we focused on its application in the field of decalcified tumor samples. We selected four patients with known BRAFV600E-mutated cancer (3 metastatic melanomas and 1 hairy cell leukemia) and paired non-decalcified/decalcified tumor samples. Molecular analyses failed in the four decalcified samples (3 bone metastases and 1 osteo-medullar biopsy) with non-contributive mutation status. Whereas non-decalcified tumor samples were all positive using anti-BRAFV600E VE1 IHC, the four decalcified samples were concluded negative. Because decalcified tumor samples are difficult to analyze from a molecular point of view, it is tempting to use IHC instead of DNA-based methods searching for BRAFV600E mutations in these samples. Nevertheless, the decalcification process may also cause false-negative results using VE1 IHC. Decalcified samples require specific and optimized IHC and molecular protocols and quality controls

About concomitant KRAS and other molecular alterations in non–small cell lung cancers

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Transformation d’un mélanocytome méningé en mélanome : étude clinique, histopathologique et cytogénétique

International audienceMeningeal melanocytic tumors are rare. We report an exceptional case of transformation of a meningeal melanocytoma in a malignant melanoma. The course of the disease extents from 61-years to 85-years and ends with the death of the patient. Besides histopathological and immunohistochemical data, we also report the array CGH study of the melanocytoma and melanoma components suggesting the malignant transformation from whole chromosome gains in the melanocytoma to additional segmental aberrations in the malignant melanoma. Beyond the rarity of this tumor subtype, this case report highlights the potential interest of molecular analyses for diagnostic and prognostic purposes in the field of meningeal melanocytic tumors

Avoiding non-contributive molecular results in cancer samples: proposal of a score-based approach for sample choice

International audienceMutational analyses have become crucial for therapeutic choices in patients with advanced lung cancer, colorectal cancer and melanoma. Short turnaround times for molecular analyses are necessary to match the patient's therapeutic management. Non-contributive molecular analyses may increase the delay in reaching a relevant mutational status. We attempted to identify criteria in samples associated with non-contributive molecular results to better anticipate them and select samples with contributive analyses. We compared several criteria such as cancer type, sample type, organ of origin and percentage of tumour cells between samples with non-contributive or contributive EGFR, KRAS, NRAS and BRAF mutation analyses. Among two sets of 3367 and 554 tumour samples analysed in 2015-2017 and 2018, respectively, 11.7% and 15.7% of sample analyses were non-contributive for at least one oncogene. Lung cancer and melanoma cancer subtypes [odds ratio (OR)=7.2], cytological (OR=1.8) or bone samples (OR=8.5) and a percentage of tumour cells ≤20% (OR=41.4) were significantly associated with non-contributive results. By combining these parameters in a scoring system, we were able to predict the contributive or non-contributive result of a molecular analysis with sensitivity and specificity higher than 80% in a validation set of samples. Predicting the contributive or non-contributive result of a molecular analysis is feasible in samples on the basis of simple features. A combination of these features could be used to better choose samples to analyse in order to reduce the rate of non-contributive molecular results and related treatment delays and costs in patients with advanced cancers

Non-secretory breast carcinomas lack NTRK rearrangements and TRK protein expression

International audienceAnti-TRK targeted therapies offer opportunities to treat patients with advanced NTRK1/2/3-rearranged cancers. Beyond NTRK-rearranged secretory breast carcinomas, little is known about NTRK rearrangements and the expression of TRK proteins in non-secretory breast carcinomas. We search for TRK proteins expressions using pan-TRK immunohistochemistry and NTRK1, NTRK2 and NTRK3 rearrangements using fluorescent in situ hybridization (FISH) tests in a set of tissue microarray included breast carcinomas. Only 1/339 invasive breast carcinomas, the only example of secretory subtype, was positive using pan-TRK immunohistochemistry and harboured a NTRK-rearrangement (NTRK1 positive FISH test). According to our results, druggable NTRK rearrangements and related-TRK proteins expression are not encountered in non-secretory breast carcinomas

Structural analysis of the complex between influenza B nucleoprotein and human importin-α

International audienceInfluenza viruses are negative strand RNA viruses that replicate in the nucleus of the cell. The viral nucleoprotein (NP) is the major component of the viral ribonucleoprotein. In this paper we show that the NP of influenza B has a long N-terminal tail of 70 residues with intrinsic flexibility. This tail contains the Nuclear Location Signal (NLS). The nuclear trafficking of the viral components mobilizes cellular import factors at different stages, making these host-pathogen interactions promising targets for new therapeutics. NP is imported into the nucleus by the importin-α/β pathway, through a direct interaction with importin-α isoforms. Here we provide a combined nuclear magnetic resonance and small-angle X-ray scattering (NMR/SAXS) analysis to describe the dynamics of the interaction between influenza B NP and the human importin-α. The NP of influenza B does not have a single NLS nor a bipartite NLS but our results suggest that the tail harbors several adjacent NLS sequences, located between residues 30 and 71

Variabilité de l’incidence des hospitalisations et de la mortalité entre les régions lors de la première vague de COVID-19 entre janvier et juin 2020 en France

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Cellules germinales primordiales aviaires au cœur des avancées de la conservation des ressources génétiques et de l’édition des génomes. L’exemple français

National audiencePrimordial germ cells (PGCs) are the germ line stem cells, precursors of gametes. In Aves, PGCs can be isolated due to their accessibility in the early embryo and in some species propagated in vitro. The progress made in the in vitro culture of chicken PGCs allows their prolonged maintenance and self-renewal in both sexes. This offers the possibility to genetically modify and/or cryopreserve them in sufficient numbers for their later use. Forexample, they could be transplanted into a recipient embryo, where they may develop into functional gametes and produce offspring. Due to the development of these biotechnological steps, chicken PGCs propagated invitro represent the reproductive cells of choice for the conservation and restoration of genetic diversity as well as the edition of avian genome. Being diploid cells, they ensure the conservation and restoration of the entire genome of animals of both sexes. In this context, we have developed at INRAE the reproductive biotechnology based on chicken PGCs and used it for the conservation and restoration of genetic resources of a local breed “la Poule Noire du Berry” as well as for the edition of BCMO1 gene. We succeeded for the first time in reviving a genotype in a single generation and in obtaining animals carrying the KO of BCMO1 gene. We also studied factors that could influence the integrity of PGCs during in vitro steps and their reproductive capacities.Les cellules germinales primordiales (PGCs pour « primordial germ cells ») sont des cellules souches de la lignée germinale, précurseurs des gamètes. Les PGCs aviaires peuvent être isolées grâce à leur accessibilité dans le jeune embryon et chez certaines espèces multipliées in vitro. Le progrès réalisé dans le système de culture des PGCs chez le poulet permet leur maintien et leur auto-renouvellement prolongés chez les deux sexes. Cela offre une possibilité de les modifier génétiquement et/ou les cryopréserver en nombre suffisant pour une utilisation ultérieure et notamment les transplanter dans un embryon receveur, où elles peuvent se développer en gamètes fonctionnels et donner une descendance. Grâce au développement de ces étapes, les PGCs de poulet cultivées invitro représentent les cellules reproductrices de choix pour la conservation et restauration des ressources génétiques ainsi que l’édition du génome aviaire. Etant des cellules diploïdes, elles assurent la conservation et la restauration du génome complet des animaux des deux sexes. Dans ce contexte, nous avons développé à INRAE la maîtrise de cette biotechnologie de reproduction basée sur les PGCs chez le poulet et l’avons appliquée à la conservation et à la restauration des ressources génétiques de la Poule Noire du Berry ainsi qu’à l’édition du gène BCMO1. Nous avons réussi pour la première fois à restaurer un génotype en une seule génération ainsi qu’obtenir les animaux porteurs du KO du gène BCMO1. Nous avons aussi étudié des facteurs qui pourraient lors des étapes in vitro impacter l’intégrité des PGCs et leur capacité de donner la descendance

High reproducibility is attainable in assessing histoprognostic parameters of pT1 colorectal cancer using routine histopathology slides and immunohistochemistry analyses

International audienceAssessment of the risk of lymph node invasion and tumour recurrence is critical to determine whether additional surgery is required in patients with endoscopically-removed pT1 colorectal cancer (CRC). A reproducible assessment of this risk of recurrence based on histopathological parameters is crucial for relevant therapeutic decisions. The inter-observer reproducibility of these parameters was the subject of our study. Two pathologists independently analysed 163 endoscopically-removed pT1 CRC recorded in a local digestive cancer registry database (Finistère, France). Using haematoxylin-eosin-saffron (HES) and immunohistochemistry slides, they evaluated several parameters related to the risk of tumour recurrence according to the international pT1 CRC-dedicated guidelines. Based on Kappa and intra-class correlation coefficients, good to very good inter-observer agreement was obtained by analysing vertical and lateral margins, submucosal invasion, tumour differentiation and lymphovascular invasion. The reproducibility of tumour budding quantification was only fair on the basis of HES slides but reached a very good agreement using cytokeratin immunohistochemistry. Dual colour cytokeratin and podoplanin immunohistochemistry also improved inter-observer agreement for the detection of lymphovascular invasion. All patients with loco-regional nodal metastases (7 of 101 who underwent complementary surgery) or distant metastases (3 patients) were diagnosed as having a high risk of recurrence and requiring an additional surgery by the two observers. Our study showed that good to very good inter-observer agreement is achievable in evaluating the pathological parameters of recurrence risk in endoscopically-removed pT1 CRC. In addition to HES slides, the detection of lymphovascular invasion and tumour budding can benefit with more reproducible immunohistochemical analyses

Histopathological factors help to predict lymph node metastases more efficiently than extra-nodal recurrences in submucosa invading pT1 colorectal cancer

International audienceThe therapeutic management of patients with endoscopic resection of colorectal cancer invading the submucosa (i.e. pT1 CRC) depends on the balance between the risk of cancer relapse and the risk of surgery-related morbidity and mortality. The aim of our study was to report on the histopathological risk factors predicting lymph node metastases and recurrences in an exhaustive case series comprising every pT1 CRC (of adenocarcinoma subtype only) diagnosed in Finistère (France) during 5-years. For 312 patients with at least 46 months follow-up included in the digestive cancers registry database, histopathological factors required for risk stratification in pT1 CRC were reviewed. Patients were treated by endoscopic resection only (51 cases), surgery only (138 cases), endoscopic resection followed by surgery (102 cases) or transanal resection (21 cases). Lymph node metastases were diagnosed in 19 patients whereas 15 patients had an extra-nodal recurrence (7 local recurrences only, 4 distant metastases only and 4 combining local and distant recurrences). Four patients with distant metastases died of their cancer. Poor tumor differentiation, vascular invasion and high grade tumor budding on HES slides were notably identified as strong risk-factors of lymph node metastases but the prediction of extra-nodal recurrences (local, distant and sometimes fatal) was less obvious, albeit it was more frequent in patients treated by transanal resection than with other treatment strategies. Beyond good performances in predicting lymph node metastases and guiding therapeutic decision in patients with pT1 CRC, our study points that extra-nodal recurrence of cancer is more difficult to predict and requires further investigations