Tomoko Sekiya

Penetrance of functioning and nonfunctioning pancreatic neuroendocrine tumors in multiple endocrine neoplasia type 1 in the second decade of life.

CONTEXT Data are scarce on the penetrance of multiple endocrine neoplasia type 1 (MEN1)-related nonfunctioning pancreatic neuroendocrine tumors (NF-PETs) and insulinomas in young MEN1 patients. A potential positive correlation between tumor size and malignancy (2-3 cm, 18%; >3 cm, 43%) has greatly influenced the management of MEN1 adults with NF-PETs. OBJECTIVE The aim of the study was to estimate the penetrance of NF-PETs, insulinomas, and gastrinomas in young MEN1 carriers. DESIGN The data were obtained from a screening program (1996-2012) involving 113 MEN1 patients in a tertiary academic reference center. PATIENTS Nineteen MEN1 patients (aged 12-20 y; 16 patients aged 15-20 y and 3 patients aged 12-14 y) were screened for NF-PETs, insulinomas, and gastrinomas. METHODS Magnetic resonance imaging/computed tomography and endoscopic ultrasound (EUS) were performed on 10 MEN1 carriers, magnetic resonance imaging/computed tomography was performed on five patients, and four other patients underwent an EUS. RESULTS The overall penetrance of PETs during the second decade of life was 42% (8 of 19). All eight PET patients had NF-PETs, and half of those tumors were multicentric. One-fifth of the screened patients (21%; 4 of 19) harbored at least one large tumor (>2.0 cm). Insulinoma was detected in two NF-PET patients (11%) at the initial screening; gastrinoma was not present in any cases. Six of the 11 (54%) screened patients aged 15-20 years who underwent an EUS had NF-PETs. Potential false-positive EUS results were excluded based on EUS-guided biopsy results, the reproducibility of the NF-PET findings, or the observation of increased tumor size during follow-up. Distal pancreatectomy and the nodule enucleation of pancreatic head tumors were conducted on three patients with large tumors (>2.0 cm; T2N0M0) that were classified as grade 1 neuroendocrine tumors (Ki-67<2%). CONCLUSIONS Our data demonstrated high penetrance of NF-PETs in 15- to 20-year-old MEN1 patients. The high percentage of the patients presenting consensus criteria for surgery for NF-PET alone or NF-PET/insulinoma suggests a potential benefit for the periodic surveillance of these tumors in this age group.

Penetrance and clinical features of pheochromocytoma in a six-generation family carrying a germline TMEM127 mutation.

CONTEXT The phenotype of familial pheochromocytoma (PHEO) associated with germline TMEM127 mutations (TMEM127-related PHEO) has not been clearly defined. OBJECTIVE This study aimed to investigate the penetrance, full phenotypic spectrum and effectiveness of clinical/genetic screening in TMEM127-related PHEO. DESIGN, SETTING, AND PARTICIPANTS Clinical and genetic screening, and genetic counseling were offered to 151 individuals from a six-generation family carrying a TMEM127 germline mutation in a referral center. INTERVENTION AND MAIN OUTCOME MEASURES TMEM127 genetic testing was offered to at-risk relatives and clinical surveillance for pheochromocytoma was performed in mutation-positive carriers. RESULTS Forty seven individuals carried the c.410-2A>C TMEM127 mutation. Clinical data were obtained from 34 TMEM127-mutation carriers followed up for 8.7 ± 8.1 years (range, 1-20 y). Pheochromocytoma was diagnosed in 11 carriers (32%) at a median age of 43 years. In nine patients, symptoms started at 29 years (range, 10-55 y) and two cases were asymptomatic. Tumors were multicentric in five (45%) and bilateral in five (45%) patients. Six patients (54%) had at least one adrenomedullary nodule less than 10 mm. No paragangliomas, distant metastases, or other manifestations were detected. Cumulative penetrance of pheochromocytoma was 0% at 0-20 years, 3% at 21-30 years, 15% at 31-40 years, 24% at 41-50 years, and 32% at 51-65 years. The youngest case was diagnosed at 22 years and the earliest symptoms were reported at age 10. CONCLUSIONS Tumor multicentricity, nodular adrenomedullary hyperplasia, and the occurrence of symptoms more than a decade earlier than the age at diagnosis are novel findings in TMEM127-related PHEO. The high penetrance of pheochromocytoma in this condition validates the benefits of genetic testing of at-risk relatives. We thus recommend that TMEM127 genetic testing should be offered to at-risk individuals at age 22 years and mutation carriers should undergo clinical surveillance annually.

Comprehensive assessment of the disputed RET Y791F variant shows no association with medullary thyroid carcinoma susceptibility

Accurate interpretation of germline mutations of the rearranged during transfection (RET) proto-oncogene is vital for the proper recommendation of preventive thyroidectomy in medullary thyroid carcinoma (MTC)-prone carriers. To gain information regarding the most disputed variant of RET, ATA-A Y791F, we sequenced blood DNA samples from a cohort of 2904 cancer-free elderly individuals (1261 via Sanger sequencing and 1643 via whole-exome/genome sequencing). We also accessed the exome sequences of an additional 8069 individuals from non-cancer-related laboratories and public databanks as well as genetic results from the Catalogue of Somatic Mutations in Cancer (COSMIC) project. The mean allelic frequency observed in the controls was 0.0031, with higher occurrences in Central European populations (0.006/0.008). The prevalence of RET Y791F in the control databases was extremely high compared with the 40 known RET pathogenic mutations (P=0.00003), while no somatic occurrence has been reported in tumours. In this study, we report new, unrelated Brazilian individuals with germline RET Y791F-only: two tumour-free elderly controls; two individuals with sporadic MTC whose Y791F-carrying relatives did not show any evidence of tumours; and a 74-year-old phaeochromocytoma patient without MTC. Furthermore, we showed that the co-occurrence of Y791F with the strong RET C634Y mutation explains the aggressive MTC phenotypes observed in a large affected family that was initially reported as Y791F-only. Our literature review revealed that limited analyses have led to the misclassification of RET Y791F as a probable pathogenic variant and, consequently, to the occurrence of unnecessary thyroidectomies. The current study will have a substantial clinical influence, as it reveals, in a comprehensive manner, that RET Y791F only shows no association with MTC susceptibility.

p27 variant and corticotropinoma susceptibility: a genetic and in vitro study

Germline mutations in p27(kip1) are associated with increased susceptibility to multiple endocrine neoplasias (MEN) both in rats and humans; however, the potential role of common polymorphisms of this gene in endocrine tumor susceptibility and tumorigenesis remains mostly unrecognized. To assess the risk associated with polymorphism rs2066827 (p27-V109G), we genotyped a large cohort of Brazilian patients with sporadic endocrine tumors (pituitary adenomas, n=252; pheochromocytomas, n=125; medullary thyroid carcinoma, n=51; and parathyroid adenomas, n=19) and 885 population-matched healthy controls and determined the odds ratios and 95% CIs. Significant associations were found for the group of patients with pituitary adenomas (P=0.01), particularly for those with ACTH-secreting pituitary adenomas (P=0.005). In contrast, no association was found with GH-secreting pituitary tumors alone or with the sporadic counterpart of MEN2-component neoplasias. Our in vitro analyses revealed increased colony formation and cell growth rate for an AtT20 corticotropin mouse cell line overexpressing the p27-V109G variant compared with cells transfected with the WT p27. However, the genotypic effects in genetic and in vitro approaches were divergent. In accordance with our genetic data showing specificity for ACTH-secreting pituitary tissues, the overexpression of p27-V109G in a GH3 somatotropin rat cell line resulted in no difference compared with the WT. Pituitary tumors are one of the major clinical components of syndromes associated with the p27 pathogenic mutations MENX and MEN4. Our genetic and in vitro data indicate that the common polymorphism rs2066827 may play a role in corticotropinoma susceptibility and tumorigenesis through a molecular mechanism not fully understood thus far.

Association between the p27 rs2066827 variant and tumor multiplicity in patients harboring MEN1 germline mutations

OBJECTIVE To date, no evidence of robust genotype-phenotype correlation or disease modifiers for multiple endocrine neoplasia type 1 (MEN1) syndrome has been described, leaving the highly variable clinical presentation of patients unaccounted for. DESIGN As the CDKN1B (p27) gene causes MEN4 syndrome and it is transcriptionally regulated by the product of the MEN1 gene (menin), we sought to analyze whether p27 influences the phenotype of MEN1-mutated patients. The cohort consisted of 100 patients carrying germline MEN1 gene mutations and 855 population-matched control individuals. METHODS Genotyping of the coding p27 c.326T>G (V109G) variant was performed by sequencing and restriction site digestion, and the genotypes were associated with clinical parameters by calculating odds ratios (ORs) and their 95% CIs using logistic regression. RESULTS There were significant differences in p27 V109G allele frequencies between controls and MEN1-mutated patients (OR=2.55, P=0.019, CI=1.013-5.76). Among patients who are ≥30 years old carrying truncating MEN1 mutations, the T allele was strongly associated with susceptibility to tumors in multiple glands (three to four glands affected vs one to two glands affected; OR=18.33; P=0.002, CI=2.88-16.41). This finding remained significant after the Bonferronis multiple testing correction, indicating a robust association. No correlations were observed with the development of MEN1-related tumors such as hyperparathyroidism, pituitary adenomas, and enteropancreatic and adrenocortical tumors. CONCLUSIONS Our study suggests that the p27 tumor suppressor gene acts as a disease modifier for the MEN1 syndrome associated with MEN1 germline mutations. If confirmed in independent patient cohorts, this finding could facilitate the management of this clinically complex disease.

Polymorphisms in the p27kip-1 and prohibitin genes denote novel genes associated with melanoma risk in Brazil, a high ultraviolet index region.

Ultraviolet (UV) radiation is a major environmental risk factor to the development of cutaneous melanoma as it induces pyrimidine dimers in DNA. Genes that exert their function by arresting the cell cycle are critical to avoid carcinogenic mutations, allowing the processing of DNA repair systems. This study was carried out to evaluate the role of polymorphisms in cell cycle genes such as TP53, p27kip-1, CDKN2A, prohibitin, and GADD153 in melanoma risk as well as their influence on known risk factors in a high UV index region. A hospital-based case–control study was carried out in Brazil to evaluate the contribution of polymorphisms in cell cycle genes toward melanoma risk. The study comprised 202 melanoma patients and 210 controls. The polymorphisms analyzed were TP53 Arg72Pro, p27kip-1 Val109Gly, GADD153 Phe10Phe (rs697221), CDKN2A 3′UTR C540G, and prohibitin 3′UTR C1703T. As regards, p27kip-1 Val109Gly, both heterozygous and homozygous Gly genotypes were shown to be protective genotypes on calculating both crude and adjusted odds ratios (ORs) for age, sex, and educational level [OR 0.37; 95% confidence interval (CI) 0.16–0.87; P<0.05]. Similarly, the prohibitin TT genotype increased melanoma risk in the crude and adjusted analyses (OR 2.40; 95% CI 1.10–5.26; P<0.05). The p27kip-1 Gly protective genotype decreased the risk for melanoma in a stratified analysis of the known risk factors such as hair and eye color, sunburns, pigmented lesions, and European ancestry. The prohibitin TT genotype increased the risk of melanoma by such host factors. Our results showed for the first time that polymorphisms in p27kip-1 Val109Gly and in prohibitin 3′UTR C1703T genotypes modulate the risk to melanoma in a high UV index region.

Narrowing the gap of personalized medicine in emerging countries: the case of multiple endocrine neoplasias in Brazil

The finished version of the human genome sequence was completed in 2003, and this event initiated a revolution in medical practice, which is usually referred to as the age of genomic or personalized medicine. Genomic medicine aims to be predictive, personalized, preventive, and also participative (4Ps). It offers a new approach to several pathological conditions, although its impact so far has been more evident in mendelian diseases. This article briefly reviews the potential advantages of this approach, and also some issues that may arise in the attempt to apply the accumulated knowledge from genomic medicine to clinical practice in emerging countries. The advantages of applying genomic medicine into clinical practice are obvious, enabling prediction, prevention, and early diagnosis and treatment of several genetic disorders. However, there are also some issues, such as those related to: (a) the need for approval of a law equivalent to the Genetic Information Nondiscrimination Act, which was approved in 2008 in the USA; (b) the need for private and public funding for genetics and genomics; (c) the need for development of innovative healthcare systems that may substantially cut costs (e.g. costs of periodic medical follow-up); (d) the need for new graduate and postgraduate curricula in which genomic medicine is emphasized; and (e) the need to adequately inform the population and possible consumers of genetic testing, with reference to the basic aspects of genomic medicine.

RET haplotype, not linked to the C620R activating mutation, associated with Hirschsprung disease in a novel MEN2 family

Hirschsprung disease is a congenital form of aganglionic megacolon that results from cristopathy. Hirschsprung disease usually occurs as a sporadic disease, although it may be associated with several inherited conditions, such as multiple endocrine neoplasia type 2. The rearranged during transfection (RET) proto-oncogene is the major susceptibility gene for Hirschsprung disease, and germline mutations in RET have been reported in up to 50% of the inherited forms of Hirschsprung disease and in 15–20% of sporadic cases of Hirschsprung disease. The prevalence of Hirschsprung disease in multiple endocrine neoplasia type 2 cases was recently determined to be 7.5% and the co-occurrence of Hirschsprung disease and multiple endocrine neoplasia type 2 has been reported in at least 22 families so far. It was initially thought that Hirschsprung disease could be due to disturbances in apoptosis or due to a tendency of the mutated RET receptor to be retained in the Golgi apparatus. Presently, there is strong evidence favoring the hypothesis that specific inactivating haplotypes play a key role in the fetal development of congenital megacolon/Hirschsprung disease. In the present study, we report the genetic findings in a novel family with multiple endocrine neoplasia type 2: a specific RET haplotype was documented in patients with Hirschsprung disease associated with medullary thyroid carcinoma, but it was absent in patients with only medullary thyroid carcinoma. Despite the limited number of cases, the present data favor the hypothesis that specific haplotypes not linked to RET germline mutations are the genetic causes of Hirschsprung disease.

Assessing the emerging oncogene protein kinase C epsilon as a candidate gene in families with Carney complex‐2

Carney complex (CNC) is a rare multiple endocrine neoplasia syndrome characterized by the co-occurrence of several types of skin tumours and pigmented lesions, endocrine neoplasms, schwannomas and myxomas (MIM *188830). Carney complex, type 1 (CNC-1), is now known to be caused by inactivating mutations in the PRKAR1A tumour suppressor gene – a key molecule from the cAMP signalling pathway. The gene locus for most of the remaining families with CNC, type 2 (CNC-2), has been suggested to be on the short arm of chromosome 2 (2p16-21) with a maximum 2-point lod score of 5Æ97 obtained in eleven North American kindreds. However, the gene responsible for the disease has still not been identified. The observed retention of heterozygosity in tumours from CNC-2 patients suggests possible involvement of an unknown proto-oncogene in this region rather than a tumour suppressor gene. In the current study, we analysed the coding regions of the emerging oncogene protein kinase C epsilon (Gene ID, NCBI: 5581), located at 2p16-21 region, as a possible candidate gene for CNC-2. Protein kinases C (PKCs) are serine/threonine kinases involved in key cellular signalling and functions, such as proliferation, migration, motility, differentiation, apoptosis and chemo-resistance. PKCs are classified according to their activation: PKCs activated by Caand/or diacylglycerol (DAG) and phorbol esters (PE) are called classical or conventional PKCs (a, bI bII, c); those activated by DAG/PE and calcium-independent are called novel PKCs (d, e, g, and h), while those unresponsive to Ca, DAG and PE are called atypical PKCs (i, f and PKMf). In the early 1990s, it was shown that in vitro overexpression of PKCe in NIH-3T3 fibroblasts leads to accelerated cell growth and that 100% of mice injected with PKCe overexpressing cells develop neoplasms. PKCe overexpression in colonic epithelial cells leads to their metastatic transformation, thus supporting its oncogenic role. As these first indications of PKCe having oncogenic potential, the status of PKCe has been comprehensively investigated in different tumours. It has been shown that PKCe is overexpressed and acts as an important player in bladder, brain, breast, head and neck, lung and prostate tumours. In addition to the initially reported PKCe involvement in apoptosis and cellular growth promotion, PKCe overexpression was later associated with processes involved in metastasis such as cell motility and invasion. MDA-MB231 breast cancer cells show elevated PKCe expression and PKCe levels in clinical breast cancers appear to be a useful predictive biomarker of aggressiveness and malignancy. PKCe was also reported as a prognostic biomarker for aggressiveness of oral cancer. Importantly, RNA interference or dominant negative PKCe inhibition in breast cancer cells, head and neck squamous cell carcinoma, and lung carcinoma, reduces the tumour growth rate and aggressiveness, indicating PKCe as a promising molecular target for anticancer therapy. Recently, it has been demonstrated that PKCe has several partners in signal transduction and transcription activation and that PKCe-mediated activation of Stat3 may contribute to the malignant phenotype of several tumours. Further, PKCe expresses its oncogenic activity by interacting with the ras pathway at the level of Raf-1 activation. Few reports have investigated PKCs in endocrine tumours. It has been shown that normal pituitary cells as well as GH3 cells express several PKC isoforms and that activation of PKCe was able to induce a proliferation of lactotroph cells through the ERK1/2 pathway. Another study has detected high expression of PKCalpha in invasive pituitary tumours harbouring a PKCalpha point mutation (p.D294G), which was not identified in benign pituitary tumours (MIM*176960). The same PKCalpha mutation was also detected in a subpopulation of thyroid follicular adenomas and carcinomas. In the present study, we sequenced the 15 coding exons and the flanking intronic junctions of the PKCe gene in six CNC indexcases in which germline inactivations in the PRKAR1A have previously been excluded. No pathogenic change was observed in the studied cohort. Several known polymorphic variants: intron 5 (c.694-32G>A; rs4953294), intron 7 (c.966+18G>Crs41308186), intron 9 (rs10188306; c.1064-33A>G) and exon 11 (c.1572C>T, p.H524H; rs17034455) (reference sequence NM_005400.2) were seen, all of them in heterozygous conditions, thus possibly excluding whole gene deletion. This is the first sequence analysis of PKCe in CNC-2 patients. Our data, showing only wild type PKCe coding sequence, suggest that PKCe is not likely to be the gene responsible for CNC-2 and further searches aiming to elucidate CNC molecular background are needed.