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Testicular germ cell tumor (TGCT) is one of the most heritable forms of cancer. In last years, many evidence suggested that constitutional genetic factors, mainly single nucleotide polymorphisms, can increase its risk. However, the possible contribution of copy number variations (CNVs) in TGCT susceptibility has not been substantially addressed. Indeed, an increasing number of studies have focused on the effect of CNVs on gene expression and on the role of these structural genetic variations as risk factors for different forms of cancer. E2F1 is a transcription factor that plays an important role in regulating cell growth, differentiation, apoptosis and response to DNA damage. Therefore, deficiency or overexpression of this protein might significantly influence fundamental biological processes involved in cancer development and progression, including TGCT. We analyzed E2F1 CNVs in 261 cases with TGCT and 165 controls. We found no CNVs in controls, but 17/261 (6.5%) cases showed duplications in E2F1. Blot analysis demonstrated higher E2F1 expression in testicular samples of TGCT cases with three copies of the gene. Furthermore, we observed higher phosphorylation of Akt and mTOR in samples with E2F1 duplication. Interestingly, normal, non-tumoral testicular tissue in patient with E2F1 duplication showed lower expression of E2F1 and lower AKT/mTOR phosphorylation with respect to adjacent tumor tissue. Furthermore, increased expression of E2F1 obtained in vitro in NTERA-2 testicular cell line induced increased AKT/mTOR phosphorylation. This study suggests for the first time an involvement of E2F1 CNVs in TGCT susceptibility and supports previous preliminary data on the importance of AKT/mTOR signaling pathway in this cancer.

A third of patients with paraganglial tumors, pheochromocytoma, and paraganglioma, carry germline mutations in one of the susceptibility genes, RET, VHL, NF1, SDHAF2, SDHA, SDHB, SDHC, SDHD, TMEM127, and MAX. Despite increasing importance, data for long-term prognosis are scarce in pediatric presentations. The European-American-Pheochromocytoma–Paraganglioma-Registry, with a total of 2001 patients with confirmed paraganglial tumors, was the platform for this study. Molecular genetic and phenotypic classification and assessment of gene-specific long-term outcome with second and/or malignant paraganglial tumors and life expectancy were performed in patients diagnosed at <18 years. Of 177 eligible registrants, 80% had mutations, 49% VHL, 15% SDHB, 10% SDHD, 4% NF1, and one patient each in RET, SDHA, and SDHC. A second primary paraganglial tumor developed in 38% with increasing frequency over time, reaching 50% at 30 years after initial diagnosis. Their prevalence was associated with hereditary disease (P=0.001), particularly in VHL and SDHD mutation carriers (VHL vs others, P=0.001 and SDHD vs others, P=0.042). A total of 16 (9%) patients with hereditary disease had malignant tumors, ten at initial diagnosis and another six during follow-up. The highest prevalence was associated with SDHB (SDHB vs others, P<0.001). Eight patients died (5%), all of whom had germline mutations. Mean life expectancy was 62 years with hereditary disease. Hereditary disease and the underlying germline mutation define the long-term prognosis of pediatric patients in terms of prevalence and time of second primaries, malignant transformation, and survival. Based on these data, gene-adjusted, specific surveillance guidelines can help effective preventive medicine.

Pheochromocytomas (PCCs) and paragangliomas (PGLs) are rare neuroendocrine neoplasias of neural crest origin that can be part of several inherited syndromes. Although their mRNA profiles are known to depend on genetic background, a number of questions related to tumor biology and clinical behavior remain unanswered. As microRNAs (miRNAs) are key players in the modulation of gene expression, their comprehensive analysis could resolve some of these issues. Through characterization of miRNA profiles in 69 frozen tumors with germline mutations in the genes SDHD, SDHB, VHL, RET, NF1, TMEM127, and MAX, we identified miRNA signatures specific to, as well as common among, the genetic groups of PCCs/PGLs. miRNA expression profiles were validated in an independent series of 30 composed of VHL-, SDHB-, SDHD-, and RET-related formalin-fixed paraffin-embedded PCC/PGL samples using quantitative real-time PCR. Upregulation of miR-210 in VHL- and SDHB-related PCCs/PGLs was verified, while miR-137 and miR-382 were confirmed as generally upregulated in PCCs/PGLs (except in MAX-related tumors). Also, we confirmed overexpression of miR-133b as VHL-specific miRNAs, miR-488 and miR-885-5p as RET-specific miRNAs, and miR-183 and miR-96 as SDHB-specific miRNAs. To determine the potential roles miRNAs play in PCC/PGL pathogenesis, we performed bioinformatic integration and pathway analysis using matched mRNA profiling data that indicated a common enrichment of pathways associated with neuronal and neuroendocrine-like differentiation. We demonstrated that miR-183 and/or miR-96 impede NGF-induced differentiation in PC12 cells. Finally, global proteomic analysis in SDHB and MAX tumors allowed us to determine that miRNA regulation occurs primarily through mRNA degradation in PCCs/PGLs, which partially confirmed our miRNA–mRNA integration results.

Although the authors of the present review have contributed to genetic discoveries in the field of pheochromocytoma research, we can legitimately ask whether these advances have led to improvements in the diagnosis and management of patients with pheochromocytoma. The answer to this question is an emphatic Yes! In the field of molecular genetics, the well-established axiom that familial (genetic) pheochromocytoma represents 10% of all cases has been overturned, with >35% of cases now attributable to germline disease-causing mutations. Furthermore, genetic pheochromocytoma can now be grouped into five different clinical presentation types in the context of the ten known susceptibility genes for pheochromocytoma-associated syndromes. We now have the tools to diagnose patients with genetic pheochromocytoma, identify germline mutation carriers and to offer gene-informed medical management including enhanced surveillance and prevention. Clinically, we now treat an entire family of tumors of the paraganglia, with the exact phenotype varying by specific gene. In terms of detection and classification, simultaneous advances in biochemical detection and imaging localization have taken place, and the histopathology of the paraganglioma tumor family has been revised by immunohistochemical-genetic classification by gene-specific antibody immunohistochemistry. Treatment options have also been substantially enriched by the application of minimally invasive and adrenal-sparing surgery. Finally and most importantly, it is now widely recognized that patients with genetic pheochromocytoma/paraganglioma syndromes should be treated in specialized centers dedicated to the diagnosis, treatment and surveillance of this rare neoplasm.

The precise diagnosis of thyroid neoplasias will guide surgical management. Primary thyroid paraganglioma has been rarely reported. Data on prevalence, immunohistochemistry (IHC), and molecular genetics in a systematic series of such patients are pending. We performed a multinational population-based study on thyroid paraganglioma and analyzed prevalence, IHC, and molecular genetics. Patients with thyroid paraganglioma were recruited from the European-American-Head-and-Neck-Paraganglioma-Registry. Demographic and clinical data were registered. Histopathology and IHC were re-investigated. All patients with thyroid paraganglioma underwent molecular genetic analyses of the SDHA, SDHB, SDHC, SDHD, SDHAF2, VHL, RET, TMEM127, and MAX genes. Analyses included Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA) for detection of large rearrangements. Of 947 registrants, eight candidates were initially identified. After immunohistochemical analyses of these eight subjects, 5 (0.5%) were confirmed to have thyroid paraganglioma. IHC was positive for chromogranin, synaptophysin, and S-100 and negative for calcitonin in all five thyroid paragangliomas, whereas the three excluded candidate tumors stained positive for pan-cytokeratin, a marker excluding endocrine tumors. Germline variants, probably representing mutations, were found in four of the five confirmed thyroid paraganglioma cases, two each in SDHA and SDHB, whereas the excluded cases had no mutations in the tested genes. Thyroid paraganglioma is a finite entity, which must be differentiated from medullary thyroid carcinoma, because medical, surgical, and genetic management for each is different. Notably, approximately 80% of thyroid paragangliomas are associated with germline variants, with implications for additional tumors and a potential risk for the family. As opposed to sporadic tumors, surgical management and extent of resection are different for heritable tumors, each guided by the precise gene involved.

Pancreatic neuroendocrine tumors (PanNETs) are rare in von Hippel–Lindau disease (VHL) but cause serious morbidity and mortality. Management guidelines for VHL-PanNETs continue to be based on limited evidence, and survival data to guide surgical management are lacking. We established the European-American-Asian-VHL-PanNET-Registry to assess data for risks for metastases, survival and long-term outcomes to provide best management recommendations. Of 2330 VHL patients, 273 had a total of 484 PanNETs. Median age at diagnosis of PanNET was 35 years (range 10–75). Fifty-five (20%) patients had metastatic PanNETs. Metastatic PanNETs were significantly larger (median size 5 vs 2 cm; P < 0.001) and tumor volume doubling time (TVDT) was faster (22 vs 126 months; P = 0.001). All metastatic tumors were ≥2.8 cm. Codons 161 and 167 were hotspots for VHL germline mutations with enhanced risk for metastatic PanNETs. Multivariate prediction modeling disclosed maximum tumor diameter and TVDT as significant predictors for metastatic disease (positive and negative predictive values of 51% and 100% for diameter cut-off ≥2.8 cm, 44% and 91% for TVDT cut-off of ≤24 months). In 117 of 273 patients, PanNETs >1.5 cm in diameter were operated. Ten-year survival was significantly longer in operated vs non-operated patients, in particular for PanNETs <2.8 cm vs ≥2.8 cm (94% vs 85% by 10 years; P = 0.020; 80% vs 50% at 10 years; P = 0.030). This study demonstrates that patients with PanNET approaching the cut-off diameter of 2.8 cm should be operated. Mutations in exon 3, especially of codons 161/167 are at enhanced risk for metastatic PanNETs. Survival is significantly longer in operated non-metastatic VHL-PanNETs.