SMARCA4 är en gen som kodar för ett protein vid namn Brahma-related gene 1 (BRG1). BRG1 utgör en väsentlig katalytisk subenhet av SWI/SNF-komplexet, vilket är ett proteinkomplex utvecklat för kromatinremodellering. Att modulera kromatinstrukturen bidrar till att viktiga funktioner, såsom transkription och celldifferentiering, kan utföras. En mutation i SMARCA4-genen kan ge upphov till en lungtumör med benämningen Thoracic SMARCA4-deficient undifferentiated tumor (SMARCA4-UT), vilken kännetecknas av en förlust av uttrycket BRG1 i de maligna cellerna. Då tumören kan variera i morfologiskt utseende och i många fall påminna om andra tumörtyper, kan tillämpning av immunhistokemiska metoder vara till stor hjälp vid den medicinska diagnosticeringen. Syftet med denna studie var att vidareutveckla ett befintligt protokoll för att uppnå en optimal immunhistokemisk infärgning av SMARCA4 (BRG1), för att diagnostiskt kunna differentiera mellan olika lungtumörer. För detta ändamål testades flera olika infärgningsprotokoll och kontrollvävnader, samt två antikroppar av olika kloner från företagen Abcam och Santa Cruz. Infärgning med Abcam-antikroppen (klon EPNCIR111A) uppvisade intensiva och specifika resultat, medan Santa Cruz-antikroppen (klon G-7) gav upphov till negativa infärgningar, trots flera försök till optimering. I samråd med en patolog fastställdes det infärgningsprotokoll som gav bäst resultat, vilket inkluderade en förbehandling bestående av buffert CC1 (64 min, 95° C), antikroppsinkubation med Abcam-antikroppen (32 min, 36° C, spädning 1:100), detektion med ultraView Universal DAB Detection Kit, samt kontrastfärgning med hematoxylin (8 min) och blåning (4 min). Denna immunfärgning kommer att införas som ny metod och användas inom den kliniska verksamheten. Däremot kommer metoden att fortsätta prövas under en tid framöver, för att helt säkerställa dess pålitlighet och kunna valideras inför framtida bruk.

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Les sarcomes épithélioides sont caractérisés dans 85% des cas par une perte d'expression nucléaire de la protéine SMARCB1, codée par un gène suppresseur de tumeurs situés en 22q11 impliqué dans la génèse des tumeurs rhabdoides malignes. L'exploration par BAC-FISH (Bacterial Artificial Chromosome- Fluorescence In Situ Hybridization) d'une série de 40 sarcomes épithélioides a permis d'établir que cette perte d'expression était secondaire dans 85% des cas à des délétions homozygotes et a mis en évidence le premier cas de sarcome épithélioide associé à une délétion germinale de SMARCB1, altération jusqu'alors uniquement identifiée dans les tumeurs rhabdoides malignes. Nous avons par la suite testé le gène suppresseur de tumeurs SMARCA4 comme gène candidat impliqué dans les sarcomes épithélioides SMARCB1-conservés à partir d'une série rétrospective de 16 cas. SMARCA4 code la sous-unité ATPase du complexe BAF dont SMARCB1 représente une sous unité. Ce screening initial a permis d'identifier 6 cas de sarcomes SMARCA4-inactivés dont la localisation était exclusivement thoracique et dont les caractéristiques clinique et anatomopathologique stéréotypées ont permis le recrutement prospectif et rétrospectif de nouveaux cas. L'étude par RNA-sequencing d'une fraction de notre cohorte (n=13/19) a confirmé leur homogénéité transcriptomique et souligné leur parenté avec les tumeurs rhabdoides SMARCB1 et SMARCA4 déficientes. L'absence de mutation germinale fréquente (n=1/11) a fait proposer le terme de sarcome thoracique SMARCA4-déficient (SMARCA4-DTS) en proscrivant l'utilisation du qualificatif « rhabdoide ». La parenté transcriptomique de ces tumeurs laisse entrevoir des vulnérabilités thérapeutiques communes qui restent à identifier / Epithelioid sarcomas (ES) display loss of SMARCB1 nuclear expression in 85% of cases. SMARCB1 is encoded by a tumor suppressor gene located in 22q11 which was first linked to cancer in malignant rhabdoid tumors. While investigating a series of 40 epithelioid sarcomas with BAC-FISH (Bacterial Artificial Chromosome-Fluorescence In Situ Hybridization), we demonstrated that SMARCB1 loss in ES occurred through genomic deletions in 85% of cases. We were also able to highlight the first case of ES associated with a heterozygous SMARCB1 deletion in the germ line, which feature was previously thought to be restricted to malignant rhadboid tumors (MRT). We subsequently investigated a series of 16 SMARCB1-retained ES to identify its underlying culprit gene with a focus on the candidate tumor suppressor gene SMARCA4. SMARCA4 encodes one of the ATPase subunit of BAF complexes. Interestingly, SMARCB1 is also a core submit of these complexes which regulate chromatin remodeling. We were able to identify a set of 6 cases displaying SMARCA4 inactivation with this discovery cohort. The review of medical records highlighted these cases had similar presentation : all tumors presented with large compressive and aggressive mediastinopulmonary masses. We further recruited 13 cases based on these characteristics including 5 prospective cases. The characterization of their transcriptomes by RNA-sequencing (n=13/19) confirmed their remarkable homogeneity, all our samples clustering together with MRT. However our variant diverge from malignant rhabdoid tumors as it lacks SMARCA4 alteration in the germline (n=0/11) and displays complex polyploidy genetic profiles. We therefore called this new tumor variant “SMARCA4-deficient thoracic sarcoma” (SMARCA4-DTS). The transcriptomic vicinity of SMARCA4-DTS and MRT let foresee they share common therapeutic vulnerabilities

abstract: Small Cell Carcinoma of the Ovary Hypercalcemic Type (SCCOHT) is a rare and highly aggressive ovarian cancer that affects children and young women at a mean age of 24 years. Most SCCOHT patients are diagnosed at an advanced stage and do not respond to chemotherapy. As a result, more than 75% of patients succumb to their disease within 1-2 years. To provide insights into the biological, diagnostic, and therapeutic vulnerabilities of this deadly cancer, a comprehensive characterization of 22 SCCOHT cases and 2 SCCOHT cell lines using microarray and next-generation sequencing technologies was performed. Following histological examination, tumor DNA and RNA were extracted and used for array comparative genomic hybridization and gene expression microarray analyses. In agreement with previous reports, SCCOHT presented consistently diploid profiles with few copy number aberrations. Gene expression analysis showed SCCOHT tumors have a unique gene expression profile unlike that of most common epithelial ovarian carcinomas. Dysregulated cell cycle control, DNA repair, DNA damage-response, nucleosome assembly, neurogenesis and nervous system development were all characteristic of SCCOHT tumors. Sequencing of DNA from SCCOHT patients and cell lines revealed germline and somatic inactivating mutations in the SWI/SNF chromatin-remodeling gene SMARCA4 in 79% (19/24) of SCCOHT patients in addition to SMARCA4 protein loss in 84% (16/19) of SCCOHT tumors, but in only 0.4% (2/485) of other primary ovarian tumors. Ongoing studies are now focusing on identifying treatments for SCCOHT based on therapeutic vulnerabilities conferred by ubiquitous inactivating mutations in SMARCA4 in addition to gene and protein expression data. Our characterization of the molecular landscape of SCCOHT and the breakthrough identification of inactivating SMARCA4 mutations in almost all cases of SCCOHT offers the first significant insight into the molecular pathogenesis of this disease. The loss of SMARCA4 protein is a highly sensitive and specific marker of the disease, highlighting its potential role as a diagnostic marker, and offers the opportunity for genetic testing of family members at risk. Outstanding questions remain about the role of SMARCA4 loss in the biology, histogenesis, diagnosis, and treatment of SCCOHT. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2014