Adenoid Cystic Carcinoma of the Vulva and Vagina: A Clinicopathologic, Immunohistochemical, and Molecular Characterization of Five Cases.

Adenoid cystic carcinoma (ACC) is a rare neoplasm most frequently observed in the salivary glands, that can occur in other organs, including the vulva and vagina. Oncogenic mechanisms involving MYB, NFIB, and MYB-NFIB rearrangements have been described, but evidence in the vulva and vagina remains scarce. Our aim is to report the clinicopathologic features, immunohistochemical, and molecular findings in a series of vulvar and vaginal ACCs. Five cases were included. Medical records and slides were reviewed. Formalin-fixed paraffin-embedded material was available in 4 cases, where additional immunohistochemical and molecular studies were carried out. Fluorescence in situ hybridization using MYB, MYBL1, and NFIB bacterial artificial chromosome-clones break-apart and MYB::NFIB BAC-clones fusion probes was performed. The patients' mean age at diagnosis was 52 years. Tumor size ranged from 0.5 to 5 cm. Microscopic examination revealed tubular, cribriform, and solid patterns. Perineural invasion was seen in 4 cases. Patients were treated with surgery, some with adjuvant radiation therapy. During follow-up (mean: 11 yr), 4 patients developed local recurrences. Recently, one of these patients developed pulmonary disease. Cam 5.2, CK5/6, CD117, and DOG-1 were positive in all 4 cases and S100 and calponin were positive in 3 cases. MYB rearrangement was present in 3 cases, including one with concurrent MYB amplification. There were no MYBL1 or NFIB rearrangements and no MYB::NFIB fusions. Our findings corroborate that the histologic, immunohistochemical, and oncogenic background is similar between ACCs of the lower female genital tract and ACCs elsewhere, although the canonical MYB::NFIB fusion seems to be a less common finding in this location.

Clinical and molecular characterization of parathyroid carcinoma in multiple endocrine neoplasia type 1.

Nineteen cases of parathyroid carcinoma in patients with multiple endocrine neoplasia type 1 have been reported in the literature, of which 11 carry an inactivating germline mutation in the MEN1 gene. Somatic genetic abnormalities in these parathyroid carcinomas have never been detected. In this paper, we aimed to describe the clinical and molecular characterization of a parathyroid carcinoma identified in a patient with MEN1. A 60-year-old man was diagnosed with primary hyperparathyroidism during the postoperative period of lung carcinoid surgery. Serum calcium and parathyroid hormone levels were 15.0 mg/dL (8.4-10.2) and 472 pg/mL (12-65), respectively. The patient underwent parathyroid surgery, and histological findings were consistent with parathyroid carcinoma. Analysis of the MEN1 gene by next-generation sequencing (NGS) identified a novel germline heterozygous nonsense pathogenic variant (c.978C>A; p.(Tyr326*)), predicted to encode a truncated protein. Genetic analysis of the parathyroid carcinoma revealed a c.307del, p.(Leu103Cysfs*16) frameshift truncating somatic MEN1 variant in the MEN1 gene, which is consistent with MEN1 tumor-suppressor role, confirming its involvement in parathyroid carcinoma etiology. Genetic analysis of CDC73, GCM2, TP53, RB1, AKT1, MTOR, PIK3CA and CCND1 genes in the parathyroid carcinoma DNA did not detect any somatic mutations. To our knowledge, this is the first report of a PC case presenting both germline (first-hit) and somatic (second-hit) inactivation of the MEN1 gene.

Target therapy for BRAF mutated anaplastic thyroid cancer: a clinical and molecular study.

OBJECTIVES: Anaplastic thyroid carcinoma (ATC) has a poor survival. The combination of Dabrafenib plus Trametinib (DT) had a significant impact in survival of BRAF p.V600E patients. However, durable responses may be compromised by resistance. We aim to present our experience with DT in BRAF positive ATC patients and compare the outcomes with usual therapy, and to study tumor molecular alterations in the DT group. METHODS: Patients treated between May 2018 and April 2022 in a tertiary referral center, assessed for BRAF status were included. Patients were divided in three groups: BRAF p.V600E treated with DT, BRAF wild type (WT) under multimodal therapy (MT), and BRAF WT under compassionate care (CC). Response was assessed monthly in the first 6 months and every 3 months afterwards, by RECIST 1.1. Overall survival (OS) and progression-free survival (PFS) were estimated with the Kaplan-Meier method and compared with the log-rank test. RESULTS: Twenty-seven ATC patients were included (DT = 9, MT = 8, and CC = 10). Median OS was 475 days for DT, 156 days for MT, and 39 days for CC (P < .001). At 12 months, only patients in the DT group were alive (71%). Median PFS was 270 days, in the DT group, compared with less than 32 days in BRAF WT (P < .001). No severe adverse events were reported. Molecular profiling showed that in one of the four clinical progressions, a pathogenic NRAS mutation was found. CONCLUSIONS: Our results show a significant real-world efficacy of Dabrafenib plus Trametinib in both survival and recurrence compared with standard treatment, with a good safety profile.

Malignant peripheral nerve sheath tumor mimicking an adnexal mass: a radio-pathologic correlation.

We report the case of a pelvic malignant peripheral nerve sheath tumor mimicking an adnexal mass. A 59-year-old postmenopausal woman presented with a 3-month history of diffuse abdominal bloating and urinary frequency. Laboratory tests revealed an increased CA 125. Radiologic evaluation depicted a large, heterogeneous solid mass located right to the uterus, pushing it to the left. After a multidisciplinary board discussion, the diagnosis of a right adnexal lesion was assumed, and the patient was referred to surgery. The final diagnosis was only achieved after pathology examination, which prove to be a malignant peripheral nerve sheath tumor. This paper highlights some clinical, radiologic and pathological features of malignant peripheral nerve sheath tumors, a rare entity that should be considered as a differential in patients presenting with pelvic tumors of uncertain origin.

Establishment and characterization of a novel ovarian high-grade serous carcinoma cell line-IPO43.

BACKGROUND: Epithelial ovarian cancer (EOC) is an aggressive and lethal malignancy and novel EOC cell lines with detailed characterization are needed, to provide researchers with diverse helpful resources to study EOC biological processes and cancer experimental therapies. METHODS: The IPO43 cell line was established from the ascitic fluid of a patient with a diagnosis of high-grade serous carcinoma (HGSC) of the ovary, previously treated with chemotherapy. Cell immortalization was achieved in 2D cell culture and growth obtained in 2D and 3D cell cultures. The characterization of immortalized cells was done by immunocytochemistry, flow cytometry, cell proliferation, chromosomal Comparative Genomic Hybridization (cCGH), STR profile and Next Generation Sequencing (NGS). RESULTS: Characterization studies confirmed that IPO43 cell line is of EOC origin and maintains morphological and molecular features of the primary tumor. cCGH analysis showed a complex profile with gains and losses of specific DNA regions in both primary ascitic fluid and cell line IPO43. The cell line was successfully grown in a 3D system which allows its future application in more complex assays than those performed in 2D models. IPO43 cell line is resistant to standard drug treatment in vitro. CONCLUSIONS: IPO43 is available for public research and we hope it can contribute to enrich the in vitro models addressing EOC heterogeneity, being useful to investigate EOC and to develop new therapeutic modalities.

Selumetinib for plexiform neurofibromas in neurofibromatosis type 1: a single-institution experience.

BACKGROUND: Plexiform neurofibromas (PN) are the most frequent tumors associated with Neurofibromatosis type 1 (NF-1). PN can cause significant complications, including pain, functional impairment, and disfigurement. There is no efficient medical treatment and, surgical resection of large PN is frequently infeasible. Selumetinib (AZD6244/ARRY-142886) is a mitogen-activated protein kinase enzyme (MEK1/2) inhibitor and works by targeting the MAPK pathway. It is an investigational treatment option for inoperable symptomatic PN associated with NF-1. Herein, we describe a single institutional experience with selumetinib for inoperable PN in NF-1. METHODS: Case series study of demographics, clinical, baseline characteristics, treatment effect, and follow-up of consecutive genetically confirmed NF1 patients with inoperable PN associated with significant or potential significant morbidity treated with selumetinib (April 2018 to April 2019). RESULTS: Nineteen patients were treated with selumetinib. Predominant target locations were head and neck (31.6%, 6/19), chest (26.3%, 5/19) and pelvis (21%, 4/19) and the most important comorbidities were disfigurement (47.4%, 9/19) and pain (26.3%, 5/19). The mean follow-up time was 223 days (range 35-420 days). All but one had sustained clinical improvement, mainly in the first 60-90 days of treatment. In one patient, the treatment was suspended after 168 days (lack of clear benefit and left ventricular ejection fraction drop). There were no adverse effects leading to treatment suspension. CONCLUSIONS: In the first observational study of selumetinib for NF-1 associated PN we showed that the drug was associated with clinical and radiological improvement. Our study also confirms the safety described in the clinical trials.

HDAC Overexpression in a NUT Midline Carcinoma of the Parotid Gland with Exceptional Survival: A Case Report.

NUT midline carcinoma (NMC) is a recently described entity with a predilection for young individuals, characterised by a rearrangement of NUT, most commonly with BRD4. It usually involves midline structures, with a minority of cases presenting outside the midline axis. Given its dismal prognosis, new molecularly targeted therapies (eg, HDAC inhibitors) are gaining ground, but the HDAC expression pattern remains unknown. We describe the exceptional evolution of a NMC arising in the parotid gland. A 34-year-old male presented with a rapidly growing 35 mm left-parotid mass. Parotidectomy and lymphadenectomy were performed. The tumour invaded the surrounding soft tissue and lay adjacent to the surgical margin. No lymph node metastases were identified. Histology revealed blue nests of undifferentiated cells merging with foci of necrosis and occasional abrupt foci of keratinising squamous epithelium. FISH analysis confirmed a rearrangement of NUT, but not of BRD4. A diagnosis of NMC was rendered. Currently, after adjuvant chemoradiotherapy and 47 months after diagnosis, the patient is alive and well. The tumour was found to have increased immunoexpression of HDAC2, 4 and 6 and phospho-HDAC4/5/7. This case emphasises the importance of considering NMC in the differential diagnosis of poorly differentiated carcinomas of the head and neck region in young adults, even away from midline structures. As molecular targets hold the promise of successful therapy for the vast majority of NMC patients, the knowledge of their HDAC expression patterns will probably be relevant.

Monocytes as Endothelial Progenitor Cells (EPCs), Another Brick in the Wall to Disentangle Tumor Angiogenesis.

Bone marrow contains endothelial progenitor cells (EPCs) that, upon pro-angiogenic stimuli, migrate and differentiate into endothelial cells (ECs) and contribute to re-endothelialization and neo-vascularization. There are currently no reliable markers to characterize EPCs, leading to their inaccurate identification. In the past, we showed that, in a panel of tumors, some cells on the vessel wall co-expressed CD14 (monocytic marker) and CD31 (EC marker), indicating a putative differentiation route of monocytes into ECs. Herein, we disclosed monocytes as potential EPCs, using in vitro and in vivo models, and also addressed the cancer context. Monocytes acquired the capacity to express ECs markers and were able to be incorporated into blood vessels, contributing to cancer progression, by being incorporated in tumor neo-vasculature. Reactive oxygen species (ROS) push monocytes to EC differentiation, and this phenotype is reverted by cysteine (a scavenger and precursor of glutathione), which indicates that angiogenesis is controlled by the interplay between the oxidative stress and the scavenging capacity of the tumor microenvironment.

Clinical insights gained by refining the 2016 WHO classification of diffuse gliomas with: EGFR amplification, TERT mutations, PTEN deletion and MGMT methylation.

BACKGROUND: Significant advances in the molecular profiling of gliomas, led the 2016 World Health Organization (WHO) Classification to include, for the first-time, molecular biomarkers in glioma diagnosis: IDH mutations and 1p/19q codeletion. Here, we evaluated the effect of this new classification in the stratification of gliomas previously diagnosed according to 2007 WHO classification. Then, we also analyzed the impact of TERT promoter mutations, PTEN deletion, EGFR amplification and MGMT promoter methylation in diagnosis, prognosis and response to therapy in glioma molecular subgroup. METHODS: A cohort of 444 adult gliomas was analyzed and reclassified according to the 2016 WHO. Mutational analysis of IDH1 and TERT promoter mutations was performed by Sanger sequencing. Statistical analysis was done using SPSS Statistics 21.0. RESULTS: The reclassification of this cohort using 2016 WHO criteria led to a decrease of the number of oligodendrogliomas (from 82 to 49) and an increase of astrocytomas (from 49 to 98), while glioblastomas (GBM) remained the same (n = 256). GBM was the most common diagnosis (57.7%), of which 55.2% were IDH-wildtype. 1p/19q codeleted gliomas were the subgroup associated with longer median overall survival (198 months), while GBM IDH-wildtype had the worst outcome (10 months). Interestingly, PTEN deletion had poor prognostic value in astrocytomas IDH-wildtype (p = 0.015), while in GBM IDH-wildtype was associated with better overall survival (p = 0.042) as well as MGMT promoter methylation (p = 0.009). EGFR amplification and TERT mutations had no impact in prognosis. Notably, EGFR amplification predicted a better response to radiotherapy (p = 0.011) and MGMT methylation to chemo-radiotherapy (p = 0.003). CONCLUSION: In this study we observed that the 2016 WHO classification improved the accuracy of diagnosis and prognosis of diffuse gliomas, although the available biomarkers are not enough. Therefore, we suggest MGMT promoter methylation should be added to glioma classification. Moreover, we found two genetic/clinical correlations that must be evaluated to understand their impact in the clinical setting: i) how is PTEN deletion a favorable prognostic factor in GBM IDH wildtype and an unfavorable prognostic factor in astrocytoma IDH wildtype and ii) how EGFR amplification is an independent and strong factor of response to radiotherapy.

Establishment and characterization of a new patient-derived anaplastic thyroid cancer cell line (C3948), obtained through fine-needle aspiration cytology.

PURPOSE: Anaplastic thyroid cancer (ATC) is among the most aggressive and unresectable tumors, presenting a bad prognosis. A better comprehension of the functional and molecular mechanisms behind the aggressiveness of this cancer, as well as new biomarkers for aggressiveness, prognosis, and response to therapy are required. However, owing to their irresectability, ATC tissue is not always accessible. Here we describe the establishment and characterization of a new patient-derived cell line, obtained from an unresectable ATC through fine-needle aspiration cytology (FNAC). METHODS: The morphology, expression of epithelial and thyroid markers, cytogenetic, mutational and gene expression profiles, doubling time, and drug-resistance profile of the new cell line, designated C3948, were investigated using several methodologies: immunostaining, karyotype analysis, comparative genomic hybridization (CGH), fluorescent in situ hybridization (FISH), next-generation sequencing (NGS), Sanger sequencing, gene expression microarrays, cell counting, and IC50 determination. RESULTS: Results indicate that C3948 cell line has a histological phenotype representative of original ATC cells and a completely aberrant karyotype with many chromosomal losses and gains; harbors mutated TP53, STK11, and DIS3L2 genes; presents a gene expression profile similar to C643 ATC commercial cell line, but with some unique alterations; has a doubling time similar to C643; and the IC50 profile for paclitaxel, doxorubicin, and cisplatin is similar to C643, although higher for cisplatin. CONCLUSIONS: These observations are consistent with a typical ATC cell profile, supporting C3948 cell line as a novel preclinical model, and FNAC as a useful approach to better study anaplastic thyroid cancer, including testing of new anticancer therapies.

Fine-needle aspiration features of extraskeletal myxoid chondrosarcoma: A study of cytological and molecular features.

BACKGROUND: Extraskeletal myxoid chondrosarcoma (EMC) is a tumor of uncertain differentiation. Few data are available regarding its cytomorphological features in fine-needle aspiration (FNA). Specific cytogenetic alterations involving the NR4A3 gene are found in EMC and can be identified in FNA samples. METHODS: We retrospectively reviewed 14 FNAs performed in 11 patients with an EMC; 10 FNAs were performed preoperatively on primary tumors; 2 were performed on recurrences and 2 were performed on metastasis. Cytological features were compared with histological findings. Immunohistochemistry (IHC) and molecular studies were performed both in FNA and histological specimens. RESULTS: A preoperative cytological diagnosis of EMC was rendered in eight FNAs performed in newly diagnosed tumors and in recurrent/metastatic cases. A descriptive diagnosis of a myxoid neoplasm was made in the remaining two cases. Smears were moderately hypercellular, composed of plasmocytoid to fusiform cells, with scant, pale cytoplasm, bland nuclei and inconspicuous nucleoli, dispersed as cords, strands and occasionally with a lace-like pattern, in an abundant chondromyxoid matrix. IHC performed showed focal positivity for S100 and NSE. Fluorescence in situ hybridization technique performed in three FNA specimens showed EWSR1 gene rearrangements in all, concomitant with NR4A3 gene rearrangement in one case. Histological specimens showed typical features of EMC and NR4A3 gene rearrangements were found in all cases tested. CONCLUSION: FNA cytology is a reliable method to perform a preoperative diagnosis of EMC regarding its cytomorphological and molecular features. Main differential diagnoses include myoepithelial tumors of soft tissue, myxoid liposarcoma and myxofibrosarcoma. Ancillary studies are helpful when coupled with cytomorphology evaluation.

NUT Carcinoma of the Salivary Glands: Clinicopathologic and Molecular Analysis of 3 Cases and a Survey of NUT Expression in Salivary Gland Carcinomas.

NUT carcinoma (NC) represents a rare subset of highly aggressive poorly differentiated carcinomas characterized by rearrangement of the NUT (aka NUTM1, nuclear protein in testis) gene, most commonly fused to BRD4. Originally described as a mediastinal/thymic malignancy, NC has been reported at a variety of anatomic regions including the upper and lower aerodigestive tract. To date, only 7 NC cases of probable salivary gland origin have been reported. We herein describe 3 new cases (all affecting the parotid gland) in 2 women (39- and 55-y old) and 1 man (35-y old). Histologic examination showed poorly differentiated neoplasms composed of poorly cohesive small-sized to medium-sized cells with variable squamoid cell component that was focal and abrupt. Immunohistochemistry showed uniform expression of p63 and distinctive punctate expression of the NUT antigen in the tumor cell nuclei. Review of the reported salivary gland NC cases (total, 10) showed a male:female ratio of 1.5:1 and an age range of 12 to 55 years (median, 29 y). Site of the primary tumor was the parotid (7), sublingual (2), and submandibular (1) glands. All presented as rapidly growing masses treated by surgery followed by adjuvant radiotherapy/chemotherapy. Initial nodal status was positive in 8/10. At last follow-up (1 to 24 mo; median, 5 mo), 7/10 patients died of disease at a median of 5.5 months (1 to 24 mo) and only 2 were disease free at 7 and 14 months. Of 9 cases with genetic data, the fusion partner was BRD4 (n=7), non-BRD4/3 (n=1), or undetermined (n=1). None of 306 carcinomas spanning the spectrum of salivary carcinoma types screened by NUT immunohistochemistry was positive. This is the first small series on salivary NC highlighting the importance to include this rare disease in the differential diagnosis of poorly differentiated salivary gland carcinomas and in cases of presumable poorly differentiated carcinoma of unknown origin.

Fine-needle aspiration of lipoblastoma: Cytological, molecular, and clinical features.

BACKGROUND: Lipoblastomas are rare, benign adipocytic tumors that present mostly during infancy. In about 70% of cases, these tumors carry abnormalities in chromosome 8, mainly leading to rearrangements of the PLAG1 gene. METHODS: We report a series of histologically proven lipoblastomas with previous fine-needle aspiration (FNA) cytology from 9 patients (n = 10 samples) and describe their clinical, cytological, and molecular features. RESULTS: Our cohort included 5 boys and 4 girls (median age, 2.5 years [range, 10 months to 13 years]) who presented with soft tissue masses in the thorax (n = 3), abdomen (n = 2), axilla (n = 2), and thigh (n = 2). In 1 patient, the FNA diagnosis was inconclusive due to hypocellularity, and in another patient a diagnosis of benign lipomatous tumor was made. In the remaining 8 samples (one of which confirmed relapse), a correct preoperative FNA diagnosis was rendered. Smears were hypo- to moderately cellular and contained fragments of mature adipose tissue with thin branching vessels admixed with some lipoblasts in a myxoid matrix. Spindle cells and naked oval nuclei with no atypia were observed in the background. Of the 4 patients tested for PLAG1 rearrangement using FISH probes, 3 harbored this alteration (1 was made on a FNA smear and 1 was made in a tumor imprint). All the patients are alive and well, except for 1 patient with a retroperitoneal tumor who, after an initial incomplete excision, died of local disease progression. CONCLUSION: FNA, especially if used together with molecular biology techniques (eg, PLAG1 FISH analysis), is a reliable and accurate diagnostic tool. Cancer Cytopathol 2017;125:934-9. © 2017 American Cancer Society.

Composição corporal e avaliação antropométrica de adultos / Body composition and anthropometric evaluation of adults / Composición corporal y evaluación antropométrica de adultos

Objetivo: avaliar o estado nutricional e composição corporal de adultos. Método: estudo transversal, descritivo, quantitativo, realizado em uma clínica escola, com 80 indivíduos de ambos os sexos. O estado nutricional foi avaliado por meio do índice de massa corporal e da circunferência da cintura. A composição corporal foi determinada por bioimpedância. Os dados foram analisados por meio do teste qui-quadrado e correlação de Pearson. A pesquisa foi aprovada pelo Comitê de ética em Pesquisa, CAAE nº 19987213.2.0000.5210. Resultados: observou-se que 51,25% dos participantes possuíam excesso de peso, que 45% apresentaram circunferência da cintura aumentada ou muito aumentada, e 74,68% tinham percentual de gordura aumentado. Conclusão: a proporção de adultos com excesso de peso e adiposidade foi elevada no grupo estudado. Isso mostra a necessidade ações de educação alimentar e de incentivo à prática de atividades físicas como medidas de promoção de saúde.(AU)

Objective: to evaluate the nutritional status and body composition of adults. Method: it is a cross-sectional, descriptive and quantitative study, performed at a school clinic with 80 individuals of both genders. The nutritional status was assessed by body mass index and waist circumference. Body composition was determined by bioimpedance. Data were analyzed using the chi-square test and Pearson's correlation. The study was approved by the Research Ethics Committee, CAAE Number 19987213.2.0000.5210. Results: it was found that 51.25% of the participants had overweight, 45% showed that their waist circumference was increased or very increased, and 74.68% had increased fat percentage. Conclusion: the proportion of adults with overweight and obesity was high in this group. This shows the necessity of actions for food education and to encourage physical activity as health promotion measures.(AU)

Objetivo: evaluar el estado nutricional y composición corporal de adultos. Método: estudio transversal, descriptivo, cuantitativo, realizado en una clínica escuela, con 80 individuos de ambos sexos. El estado nutricional fue evaluado por medio del índice de masa corporal y de la circunferencia de la cintura. La composición corporal fue determinada por bioimpedancia. Los datos fueron analizados por medio del test chicuadrado y correlación de Pearson. La investigación fue aprobada por el Comité de ética en Investigación, CAAE nº 19987213.2.0000.5210. Resultados: se observó que 51,25% de los participantes poseían exceso de peso, que 45% presentaron circunferencia de la cintura aumentada o muy aumentada, y 74,68% tenían porcentaje de gordura aumentada. Conclusión: la proporción de adultos con exceso de peso y adiposidad fue elevada en el grupo estudiado. Eso muestra la necesidad acciones de educación alimentar y de incentivo a la práctica de actividades físicas como medidas de promoción de salud.(AU)

Identification of the first germline HRPT2 whole-gene deletion in a patient with primary hyperparathyroidism.

OBJECTIVE: Germline mutations in the HRPT2 gene are associated with the hereditary hyperparathyroidism-jaw tumour syndrome (HPT-JT) and a subset of familial isolated hyperparathyroidism (FIHP). Somatic HRPT2 mutations are detected in sporadic parathyroid carcinomas and less frequently in cystic adenomas. The purpose of this study was to investigate the underlying HRPT2 defect in a young patient with symptomatic hyperparathyroidism due to an apparently sporadic parathyroid adenoma with cystic features. DESIGN AND METHODS: HRPT2 mutations in the patient's genomic and parathyroid tumour DNA were screened by PCR-based sequencing. Tumour loss of heterozygosity (LOH) at the HRPT2 locus was assessed with microsatellite markers. A large germline HRPT2 deletion was investigated by real-time quantitative PCR analysis (qPCR). Genomic DNA losses were also appraised by chromosomal comparative genomic hybridization (cCGH). RESULTS: No germline HRPT2 point mutation was detected by direct sequencing. A novel hemizygous HRPT2 somatic mutation (c.32delA) was identified in the tumour. Apparent constitutional homozygosity for HRPT2 flanking microsatellite markers, and absence of LOH at a distal marker, suggested a large germline deletion. Gene dose mapping by qPCR unveiled a de novo deletion of the whole HRPT2 gene and adjacent loci (<9·3 Mb in size). cCGH confirmed germline DNA loss involving the HRPT2 locus. CONCLUSIONS: We report the first large germline deletion of the HRPT2 gene, which was not detectable by conventional PCR-based sequencing methods. This finding emphasizes that qPCR should be implemented in HRPT2 molecular analysis, which may improve genetic assessment and clinical management of patients with FIHP and HPT-JT.

Familial non-medullary thyroid carcinoma (FNMTC): analysis of fPTC/PRN, NMTC1, MNG1 and TCO susceptibility loci and identification of somatic BRAF and RAS mutations.

Linkage analysis has identified four familial non-medullary thyroid carcinoma (FNMTC) susceptibility loci: fPTC/PRN (1p13.2-1q22), NMTC1 (2q21), MNG1 (14q32) and TCO (19p13.2). To date, there is no evidence for the involvement of genes from the RAS/RAF signalling pathway in FNMTC. The aim of our study was to evaluate the role of the four susceptibility loci, and RAS/RAF signalling pathway genes, in FNMTC. In total, 8 FNMTC families, and 27 thyroid lesions from family members (22 papillary thyroid carcinomas (PTCs): 11 classic, 10 of the follicular variant and 1 of the mixed variant; 4 follicular thyroid adenomas (FTAs) and 1 nodular goitre (NG)), were evaluated for the involvement of the four susceptibility regions, using linkage and loss of heterozygosity (LOH) analyses. BRAF and H-, N- and K-RAS mutations were also screened in the 27 lesions and patients. Linkage analysis in seven informative families showed no evidence for the involvement of any of the four candidate regions, supporting a genetic heterogeneity for FNMTC. Twenty tumours (74%), of which 18 were PTCs, showed no LOH at the four susceptibility loci. The remaining seven tumours (four PTCs, two FTAs and one NG) showed variable patterns of LOH. Fourteen tumours (52%) had somatic mutations: BRAF-V600E mutation was observed in 9 out of the 22 PTCs (41%); and H-RAS and N-RAS mutations were detected in 5 out of the 22 PTCs (23%). Our data suggest that the four candidate regions are not frequently involved in FNMTC and that the somatic activation of BRAF and RAS plays a role in FNMTC tumourigenesis.

PLAG1 gene alterations in salivary gland pleomorphic adenoma and carcinoma ex-pleomorphic adenoma: a combined study using chromosome banding, in situ hybridization and immunocytochemistry.

Pleomorphic adenoma is the most common benign tumor of the salivary glands. It has marked histological diversity with epithelial, myoepithelial and mesenchymal-type cells arranged in a variety of architectural and differentiation patterns. Pleomorphic adenoma gene 1 (PLAG1), shown to be consistently rearranged in pleomorphic adenomas, is activated by chromosomal translocations involving 8q12, the chromosome region that is most frequently affected in these tumors. In this study, we evaluated PLAG1 involvement in salivary gland tumorigenesis by determining the frequency of its alterations in a selected group of 20 salivary gland tumors: 16 pleomorphic adenomas and four carcinomas ex-pleomorphic adenoma, having in common the presence of karyotypic chromosome 8 deviations, either structural, with 8q12 rearrangements, or numerical, with gain of chromosome 8. PLAG1 status was analyzed using in situ hybridization techniques, on metaphase cells, by fluorescence detection and/or interphase cells in paraffin sections, by chromogenic detection. Except for one pleomorphic adenoma case (5%) that lacked PLAG1 involvement, 17 tumors (85%), (14 pleomorphic adenomas and three carcinomas ex-pleomorphic adenoma) showed intragenic rearrangements of PLAG1 and the remaining two cases (10%), (one pleomorphic adenoma and one carcinoma ex-pleomorphic adenoma), had chromosome trisomy 8 only. To further investigate the role of PLAG1 on pleomorphic adenomas tumorigenesis, as well as the putative morphogenesis mechanism, we attempted to identify the cell types (epithelial vs myoepithelial) carrying 8q12/PLAG1 abnormalities by a combined phenotypic/genotypic analysis in four cases (three pleomorphic adenoma and one carcinoma ex-pleomorphic adenoma) characterized by 8q12 translocations and PLAG1 rearrangement. In these cases, both cells populations carried PLAG1 rearrangements. This finding further supports the pluripotent single-cell theory, which postulates that the tumor-initiated, modified myoepithelial cell, evolves into the varied somatic cell phenotypes present in pleomorphic adenoma, and reinforces the role of PLAG1 on the tumorigenesis of benign and malignant pleomorphic adenoma.

A study of MECT1-MAML2 in mucoepidermoid carcinoma and Warthin's tumor of salivary glands.

The t(11;19)(q21;p13) chromosomal translocation has been described in two distinct types of salivary gland neoplasms: mucoepidermoid carcinoma (MEC) and Warthin's tumor (WT). Since this translocation has been recently shown to generate a MECT1-MAML2 fusion gene, we evaluated 10 primary MEC and seven primary WT to further define the molecular association of these two entities using cytogenetic, as well as in situ hybridization (ISH) and reverse transcriptase-polymerase chain reaction (RT-PCR) analyses directed against the fusion gene. A karyotype was established in all neoplasms except for two MEC cases. Of the eight karyotyped MECs, five showed the t(11;19)(q21;p13), two had a normal karyotype, and one case presented a -Y and +X. Three of the WT revealed a normal karyotype and four had several abnormalities which did not involve chromosomes 11 and 19. ISH analysis performed in cytogenetic suspension and/or in tumor paraffin sections demonstrated MAML2 rearrangement in 7 of 10 cases of MEC: all five cases with t(11;19), one case with normal karyotype, and one unkaryotyped case. RT-PCR analysis confirmed the expression of the MECT1-MAML2 gene in all MEC cases that were positive by ISH analysis. Neither the t(11;19) nor MECT1-MAML2 was detected in any case of WT, nor in control samples from polymorphous low-grade adenocarcinoma, acinic cell carcinoma, or normal parotid gland tissue. We have demonstrated that ISH and RT-PCR are sensitive methods for detecting MECT1-MAML2 in MEC. In contrast, we did not detect the t(11;19) nor MECT1-MAML2 expression in seven cases of WT.

Comparative genomic hybridization analysis of a pleuropulmonary blastoma.

Pleuropulmonary blastoma (PPB) is a rare, aggressive dysontogenetic tumor of childhood. We report the comparative genomic hybridization (CGH) study performed on a case of PPB in a 3-year-old-boy. The tumor was characterized by several chromosomal imbalances. Gains observed affected regions: 1q12-q23, 3q23-qter, 8pter-q24.1, 9p13-q21, 17p12-p11, 17q11-q22, 17q23-q25, 19pter-p11, and 19q11-q13.3. Whole chromosome gains were detected at 2 and 7. Loss of genetic material was found at regions: 6q13-qter, 10pter-p13, 10q22-qter, and 20p13. To our knowledge, there have been no CGH reports on PPB, but it is interesting to note that 1) the alterations found confirm previous cytogenetic reports describing gains of chromosomes 2 and 8 as recurrent abnormalities in this type of tumor, suggesting that a gene or genes of putative relevance in PPB pathogenesis are mapped at 8p11-p12, and 2) the CGH profile of this case is very similar to those observed in embryonal rhabdomyosarcomas, in which gains of 2 or 2q, 7 or 7q, and 8 or 8p and loss of 10q22-qter are consistently found. This finding supports the hypothesis that PPB may be tumorigenetically related with embryonal rhabdomyosarcoma.