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We report an isolated sulfite oxidase deficiency in the first child boy of a non-consanguineous Caucasian family. He's a compound heterozygote for the sulfite oxidase gene, presenting low cystine, undetectable homocysteine and normal uric acid blood concentrations and undetectable sulfite oxidase activity in his cultured fibroblasts. Both mutations are not reported yet. The clinical presentation was typical and severe, with generalized status epilepticus and premature death.

Summary Background Advances in molecular genetics of sarcoma have enabled the identification of type-specific aberrations. We aimed to assess the clinical effect of systematic implementation of molecular assays to improve sarcoma misdiagnosis. Methods In this multicentre, observational study, we recruited patients from 32 centres of the French Sarcoma Group/Reference Network in Pathology of Sarcomas. Eligibility criteria included: biopsy or surgical resection; suspicion of: dermatofibrosarcoma protuberans (cohort 1), dedifferentiated liposarcoma (cohort 2), Ewing's sarcoma family of tumours (cohort 3), synovial sarcoma (cohort 4), alveolar rhabdomyosarcoma (cohort 5), and myxoid or round cell liposarcoma (cohort 6); review by one sarcoma-expert pathologist; availability of frozen material (except for cohort 1 of patients with dermatofibrosarcoma protuberans because anti-CD34 immunohistochemistry is performed on paraffin-embedded tissue); and patient information. For each case, the pathologist made one primary diagnosis followed by up to two differential diagnoses, based on histological characteristics only. Each diagnosis was classified as certain, probable, or possible. For each case to determine the molecular classification, we did fluorescence in-situ hybridisation on paraffin-embedded samples. We also did comparative genomic hybridisation and quantitative PCR (cohort 2) or reverse transcriptase PCR (cohorts 3–6) on frozen and paraffin-embedded samples. We made a final diagnosis based on the molecular results. The clinical effect of diagnosis correction was assessed by a board of experts. Finding Between June 22, 2009, and Oct 30, 2012, 395 patients were enrolled in the study, of which 384 were eligible for inclusion. The diagnosis was eventually modified by molecular genetics for 53 patients: eight (16%) of 50 patients with dermatofibrosarcoma (cohort 1), seven (23%) of 30 patients with dedifferentiated liposarcoma (cohort 2), 13 (12%) of 112 with Ewing's sarcoma family of tumours (cohort 3), 16 (16%) of 97 patients with synovial sarcoma (cohort 4), seven (15%) of 46 patients with alveolar rhabdomyosarcoma (cohort 5), and two (4%) of 49 patients with myxoid or round cell liposarcoma (cohort 6), with an effect on primary management or prognosis assessment in 45 cases. Interpretation Molecular genetic testing should be mandatory for diagnostic accuracy of sarcoma and appropriate clinical management, even when histological diagnosis is made by pathologist experts in this field. Funding French National Cancer Institute and Nice University Hospital.

Resume Les progres dans le demantelement moleculaire des CBNPC ont conduit a des avancees significatives dans le traitement des tumeurs solides. Les alterations moleculaires qui peuvent etre ciblables par des therapeutiques ciblees correspondent soit a des mutations de genes (Epidermal Growth Factor Receptor [EGFR], KRAS, HER2, BRAF), soit a des rearrangements de genes (Anaplastic Lymphoma Kinase [ALK], ROS1, RET), impliquant qu'outils et methodes soient appropries pour les detecter. Cet article decrit les problematiques liees aux differents types de prelevements realises en vue d'une analyse moleculaire, les criteres de qualite/selection des echantillons tumoraux, les differentes techniques de biologie moleculaires utilisees pour la recherche des mutations, avec en perspective l'utilisation du sequencage de nouvelle generation (NGS), ainsi que l'interpretation des resultats.

Epidermal growth factor receptor (EGFR)–tyrosine kinase inhibitors have limited use as first-line treatment for mutated EGFR metastatic non–small cell lung cancer. The French National Cancer Institute has installed molecular genetics platforms implementing EGFR and KRAS testing. However, there is considerable uncertainty as to which detection methods should be applied for routine diagnosis. This study aimed to compare the EGFR and KRAS genotyping methods developed by the IFCT/ERMETIC2 network platforms in two blind panels: 25 samples of serial dilutions of cell line DNA (20 centers) and 74 FFPE lung tumor samples (10 centers). The best threshold of mutation detection on cell lines was obtained using allele-specific amplification-based technologies. Nonamplifiable tissue samples were significantly less common when using alternative testing versus direct sequencing [15%; 95% confidence interval (CI), 14%–16% versus 40%; 95% CI, 39%–42%; P

Sarcomas represent a complex and heterogeneous group of rare malignant tumors and their correct diagnosis is often difficult. Recent molecular biological techniques have been of great diagnostic use and there is a need to assess the cost of these procedures in routine clinical practice. Using prospective and observational data from eight molecular biology laboratories in France, we used "microcosting" method to assess the cost of molecular biological techniques in the diagnosis of five types of sarcoma. The mean cost of fluorescence in situ hybridization (FISH) was 318 € (273-393) per sample; mean reverse transcription polymerase chain reaction (RT-PCR) cost ranged from 300 € (229-481) per formalin-fixed, paraffin-embedded specimen to 258 € (213-339) per frozen specimen; mean quantitative polymerase chain reaction (Q-PCR) cost was 184 € (112-229) and mean CGH-array cost was 332 € (329-335). The cost of these recently implemented techniques varied according to the type of sarcoma; the method of tissue collection and local organizational factors including the level of local expertise and investment. The cost of molecular diagnostic techniques needs to be balanced against their respective performance.

Introduction Recent clinical success of epidermal growth factor (EGFR)-tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) have raised hopes that targeting other deregulated growth factor signaling, such as the hepatocyte growth factor/MET pathway, will lead to new therapeutic options for NSCLC. Furthermore, NSCLC present secondary EGFR-TKIs resistance related to exons 20 and 19 EGFR mutations or more recently to MET amplification. The aim of this study was to determine MET copy number related to EGFR copy number and K-Ras mutations in a targeted TKI naive NSCLC cohort. Methods We investigated 106 frozen tumors from surgically resected NSCLC patients. Genes copy number of MET and EGFR were assessed by quantitative relative real-time polymerase chain reaction and K-Ras mutations by sequencing. Results MET is amplified in 22 cases (21%) and deleted in nine cases (8.5%). EGFR is amplified in 31 cases (29%). K-Ras is mutated in 11 cases (10.5%). As observed for EGFR amplification, MET amplification is never associated with K-Ras mutation. MET amplification could be associated with EGFR amplification. MET amplification is not related to clinical and pathologic features. MET amplification and EGFR amplification showed a trend toward poor prognosis in adenocarcinomas. Conclusion In EGFR-TKIs naive NSCLC patients, MET amplification is a frequent event, which could be associated with EGFR amplification, but not with K-Ras mutation. MET amplification may identify a subset of NSCLC for new targeted therapy. It will also be important to evaluate MET copy number to properly interpret future clinical trials.

Contexte La recherche des mutations du gene EGFR est effectuee en routine sur les prelevements de cancers bronchiques non a petites cellules (CBNPC) non epidermoides. La cellularite tumorale de l’echantillon analyse (rapport en pourcentage correspondant au nombre total de cellules tumorales/nombre total de cellules, tous types confondus) est une donnee importante pour interpreter les resultats de biologie moleculaire en fonction de la sensibilite de la technique utilisee. Un pourcentage de cellules tumorales > 20 % est requis pour la recherche de mutation par sequencage direct, et > 10 % pour l’analyse de fragment. La tendance serait de ne pas tenter d’effectuer d’analyses moleculaires sur les echantillons n’atteignant pas ces seuils, et de demander la realisation d’un autre prelevement. Nous rapportons trois cas pour lesquels l’echantillon renfermait moins de 10 % de cellules tumorales, ce qui n’a pas empeche la mise en evidence d’une mutation d’ EGFR en raison d’une amplification genique d’ EGFR . Cas cliniques Un des cas correspondait a une biopsie d’opacite pulmonaire mettant en evidence un adenocarcinome (cellularite tumorale de 7 %), les deux autres a des culots d’inclusion en paraffine de liquides d’epanchement pleural metastatique d’adenocarcinomes pulmonaires (cellularite tumorale de 5 et 7 %). Une mutation de l’exon 19 d’ EGFR etait detectee dans les trois cas, en analyse de fragment et en sequencage direct, avec une intensite de la mutation evaluee a 90 % de cellules mutees dans l’echantillon par rapport au temoin positif. Une relecture anatomo-pathologique confirmait la faible cellularite tumorale et l’analyse moleculaire de nouveaux echantillons des memes prelevements confirmait les resultats initiaux, ce qui ecartait l’hypothese d’un faux-positif. Devant cette discordance, l’analyse du nombre de copies du gene EGFR etait realisee sur coupes en paraffine par technique SISH (Silver In Situ Hybridization, kit Ventana). Elle mettait en evidence une amplification du gene EGFR en clusters dans les trois cas. Discussion La presence de plus de 2 copies du gene EGFR peut influencer la sensibilite de detection des alleles mutes : augmentation de la sensibilite de detection quand l’amplification touche l’allele mute (comme dans nos trois cas), ou au contraire diminution de la sensibilite en cas d’amplification touchant l’allele sauvage. D’apres les donnees de la litterature, environ la moitie des CBNPC mutes EGFR presente une amplification genique associee, amplification touchant preferentiellement l’allele mute. Conclusion Nous montrons a travers trois cas de « petits prelevements » d’adenocarcinomes pulmonaires (une biopsie pulmonaire et deux culots d’inclusion de liquide d’epanchement pleural metastatique) qu’en cas d’amplification genique touchant l’allele mute du gene EGFR dans les cellules tumorales, une mutation peut etre mise en evidence malgre un faible pourcentage de cellules tumorales. Ces elements invitent a ne pas exclure d’emblee des prelevements dont la cellularite tumorale est inferieure au seuil de sensibilite usuel de la technique moleculaire utilisee.