Deborah J. Marsh

HRPT2 mutations are associated with malignancy in sporadic parathyroid tumours

Background: Hyperparathyroidism is a common endocrinopathy characterised by the formation of parathyroid tumours. In this study, we determine the role of the recently identified gene, HRPT2, in parathyroid tumorigenesis. Methods: Mutation analysis of HRPT2 was undertaken in 60 parathyroid tumours: five HPT-JT, three FIHP, three MEN 1, one MEN 2A, 25 sporadic adenomas, 17 hyperplastic glands, two lithium associated tumours, and four sporadic carcinomas. Loss of heterozygosity at 1q24-32 was performed on a subset of these tumours. Results:HRPT2 somatic mutations were detected in four of four sporadic parathyroid carcinoma samples, and germline mutations were found in five of five HPT-JT parathyroid tumours (two families) and two parathyroid tumours from one FIHP family. One HPT-JT tumour with germline mutation also harboured a somatic mutation. In total, seven novel and one previously reported mutation were identified. “Two-hits” (double mutations or one mutation and loss of heterozygosity at 1q24-32) affecting HRPT2 were found in two sporadic carcinomas, two HPT-JT-related and two FIHP related tumours. Conclusions: The results in this study support the role of HRPT2 as a tumour suppressor gene in sporadic parathyroid carcinoma, and provide further evidence for HRPT2 as the causative gene in HPT-JT, and a subset of FIHP. In light of the strong association between mutations of HRPT2 and sporadic parathyroid carcinoma demonstrated in this study, it is hypothesised that HRPT2 mutation is an early event that may lead to parathyroid malignancy and suggest intragenic mutation of HRPT2 as a marker of malignant potential in both familial and sporadic parathyroid tumours.

Differential Nuclear and Cytoplasmic Expression of PTEN in Normal Thyroid Tissue, and Benign and Malignant Epithelial Thyroid Tumors

Germline mutations in PTEN (MMAC1/TEP1) are found in patients with Cowden syndrome, a familial cancer syndrome which is characterized by a high risk of breast and thyroid neoplasia. Although somatic intragenic PTEN mutations have rarely been found in benign and malignant sporadic thyroid tumors, loss of heterozygosity (LOH) has been reported in up to one fourth of follicular thyroid adenomas (FAs) and carcinomas. In this study, we examined PTEN expression in 139 sporadic nonmedullary thyroid tumors (55 FA, 27 follicular thyroid carcinomas, 35 papillary thyroid carcinomas, and 22 undifferentiated thyroid carcinomas) using immunohistochemistry and correlated this to the results of LOH studies. Normal follicular thyroid cells showed a strong to moderate nuclear or nuclear membrane signal although the cytoplasmic staining was less strong. In FAs the neoplastic nuclei had less intense PTEN staining, although the cytoplasmic PTEN-staining intensity did not differ significantly from that observed in normal follicular cells. In thyroid carcinomas as a group, nuclear PTEN immunostaining was mostly weak in comparison with normal thyroid follicular cells and FAs. The cytoplasmic staining was more intense than the nuclear staining in 35 to 49% of carcinomas, depending on the histological type. Among 81 informative tumors assessed for LOH, there seemed to be an associative trend between decreased nuclear and cytoplasmic staining and 10q23 LOH (P = 0.003, P = 0.008, respectively). These data support a role for PTEN in the pathogenesis of follicular thyroid tumors.

Genotype-phenotype correlation in multiple endocrine neoplasia type 2: report of the International RET Mutation Consortium

Abstract. The International RET Mutation Consortium was first convened as part of the Fifth International Workshop on Multiple Endocrine Neoplasia, Stockholm, Sweden, in an attempt to analyse the relationship of RET mutation and disease phenotype in the autosomal dominantly inherited multiple endocrine neoplasia type 2 (MEN 2) syndromes. Out of 361 families studied, 41% had MEN 2A, 17.7% MEN 2B, 6.4% FMTC and the remaining subjects were unclassified. RET mutations were detected in 87.3% of families overall. Over 93% of MEN 2B families had the RET 918 ATG → ACG mutation, while the most frequent mutation detected in MEN 2A families was cysteine codon 634 (87% of all mutations).

Somatic mutations in the RET proto-oncogene in sporadic medullary thyroid carcinoma

OBJECTIVE We have determined the frequency of specific mutations in the RET proto‐oncogene in sporadic medullary thyroid carcinomas (MTCs) and correlated the presence or absence of a codon 918 mutation with the clinical characteristics of these tumours.

Loss of Nuclear Expression of Parafibromin Distinguishes Parathyroid Carcinomas and Hyperparathyroidism-Jaw Tumor (HPT-JT) Syndrome-related Adenomas From Sporadic Parathyroid Adenomas and Hyperplasias.

Parathyroid carcinoma is notoriously difficult to diagnose with confidence in borderline cases. Commonly there is a long lag time between diagnosis and clinical evidence of malignant behavior even in histopathologically straightforward lesions. There is therefore a need for a novel adjunctive marker to assist in the diagnosis of carcinoma. Parafibromin is the protein encoded by the putative tumor suppressor gene HRPT2. Mutations predicted to inactivate parafibromin were first detected in the germline of patients with hyperparathyroidism-jaw tumor (HPT-JT) syndrome. Subsequently, somatic mutations have been identified in the majority of sporadic carcinomas. We performed immunohistochemistry for parafibromin on 115 parathyroid tissues comprising 4 HPT-JT-related tumors (3 adenomas and 1 carcinoma), 11 sporadic parathyroid carcinomas, 79 sporadic adenomas, 3 multiple endocrine neoplasia 2A-related adenomas, 2 sporadic primary hyperplasias, 2 multiple endocrine neoplasia (MEN)-1–related hyperplasias, 6 secondary hyperplasias, 4 tertiary hyperplasias, and 4 normal parathyroid glands. There was complete absence of nuclear staining in 3 of 4 (75%) HPT-JT–related tumors and 8 of 11 (73%) sporadic parathyroid carcinomas and focal weak staining in 1 of 4 HPT-JT tumors and 2 of 11 sporadic parathyroid carcinomas. Only 1 parathyroid carcinoma exhibited diffuse strong nuclear expression of parafibromin. In contrast, 98 of 100 non-HPT-JT–related benign parathyroids showed diffuse strong nuclear positivity and 2 of 100 showed weak positive staining. We conclude that, in the correct clinical and pathologic context, complete absence of nuclear staining for parafibromin is diagnostic of parathyroid carcinoma or an HPT-JT–related tumor.

Over-representation of a germline RET sequence variant in patients with sporadic medullary thyroid carcinoma and somatic RET codon 918 mutation

The aetiology of sporadic medullary thyroid carcinoma is unknown. About 50% harbour a somatic mutation at codon 918 of RET (M918T). To investigate whether other RET sequence variants may be associated with or predispose to the development of sporadic medullary thyroid carcinoma, we analysed genomic DNA from the germline and corresponding tumour from 50 patients to identify RET sequence variants. In one patient, tumour DNA showed a novel somatic 12 bp in-frame deletion in exon 15. More interestingly, we found that the rare polymorphism at codon 836 (c.2439C>T; S836S) occurred at a significantly higher frequency than that in control individuals without sporadic medullary thyroid carcinoma (Fishers exact test, P=0.03). Further, among the nine evaluable cases with germline c.2439C/T, eight also had the somatic M918T mutation in MTC DNA which was more frequent than in patients with the more common c.2439C/C (89% vs 40%, respectively; Fishers exact test, P=0.01). These findings suggest that the rare sequence variant at codon 836 may somehow play a role in the genesis of sporadic medullary thyroid carcinoma.

Germline Inactivation of PTEN and Dysregulation of the Phosphoinositol-3-Kinase/Akt Pathway Cause Human Lhermitte-Duclos Disease in Adults

Lhermitte-Duclos disease (LDD), or dysplastic gangliocytoma of the cerebellum, is an unusual hamartomatous overgrowth disorder. LDD can be familial or, more commonly, sporadic. It has been only recently recognized that LDD may be associated with Cowden syndrome (CS). Over 80% of patients with CS carry germline mutations in PTEN. It remains unclear whether all cases of LDD, even without features of CS, are caused by germline PTEN mutation and whether somatic PTEN mutation occurs in sporadic LDD. We obtained paraffin-embedded LDD lesions from 18 unselected, unrelated patients and performed mutational analysis of PTEN. Overall, 15 (83%) of 18 samples were found to carry a PTEN mutation. All individuals with mutations were adult-onset patients, but the three without mutations were diagnosed at the ages of 1, 3, and 11 years. Germline DNA was available from six adult-onset cases, and all had germline PTEN mutations. Of these six, two had CS features, one did not have CS features, and three were of unknown CS status. Immunohistochemistry revealed that 75% of the LDD samples had complete or partial loss of PTEN expression accompanied by elevated phosphorylated Akt, specifically in the dysplastic gangliocytoma cells. These data suggest that the loss of PTEN function is sufficient to cause LDD. The high frequency and spectrum of germline PTEN mutations in patients ascertaining by LDD alone confirm that LDD is an important defining feature of CS. Individuals with LDD, even without apparent CS features, should be counseled as in CS.

Male breast cancer in Cowden syndrome patients with germline PTEN mutations.

Cowden syndrome (CS) (OMIM 158350) is a multiple hamartoma syndrome associated with germline mutations in thePTEN tumour suppressor gene. While CS is characterised most commonly by non-cancerous lesions (mucocutaneous trichilemmomas, acral and palmoplantar keratoses, and papillomatous papules), it is also associated with an increased susceptibility to breast cancer (in females) and thyroid cancer, as well as non-cancerous conditions of the breast and thyroid. Here we report two cases of male breast cancer occurring in patients with classical CS phenotypes and germline PTEN mutations. The first subject was diagnosed with CS indicated primarily by mucocutaneous papillomatosis, facial trichilemmomas, and macrocephaly with frontal bossing at the age of 31 years. He developed breast cancer at 41 years and subsequently died of the disease. A PTENmutation, c.802delG, was identified in this subject, yet none of his family members showed evidence of a CS phenotype, suggesting that thisPTEN mutation may be a de novo occurrence. The second subject had a CS phenotype including multiple trichilemmomas and thyroid adenoma, developed male breast cancer at 43 years, and died of the disease at 57 years. He was a carrier of aPTEN mutation c.347-351delACAAT that cosegregated with the CS phenotype in affected family members. These two cases of male breast cancer associated with germlinePTEN mutations and the CS phenotype suggest that CS may be associated with an increased risk of early onset male as well as female breast cancer.

Novel succinate dehydrogenase subunit B (SDHB) mutations in familial phaeochromocytomas and paragangliomas, but an absence of somatic SDHB mutations in sporadic phaeochromocytomas

Phaeochromocytomas arising in adrenal or extra-adrenal sites and paragangliomas of the head and neck, in particular of the carotid bodies, occur sporadically and also in a familial setting. In addition to mutations in RET and VHL in familial disease, germline mutations in SDHD and SDHB genes that encode subunits of mitochondrial complex II have also been associated with the development of familial phaeochromocytomas. To further investigate the role of SDHD and SDHB in the development of these tumours we determined the occurrence of germline SDHD and SDHB mutations in four patients with a family history of phaeochromocytoma with associated head and neck paraganglioma, one patient with a family history of phaeochromocytoma only and two patients with apparently sporadic extra-adrenal phaeochromocytoma, one of whom had early onset disease. Secondly, we investigated whether somatic SDHB mutations correlated with loss of heterozygosity at 1p36 in a subgroup of 11 sporadic and three MEN 2-associated RET-mutation-positive phaeochromocytomas. Novel SDHB mutations were identified in the probands from four families and two apparently sporadic cases (six of seven probands studied), including two missense mutations, a single nonsense and frameshift mutation, as well as two splice site mutations, one of which was shown to have partial penetrance resulting in ‘leaky’ splicing. Further, five intronic polymorphisms in SDHB were found. No SDHD mutations were identified. In addition, no somatic SDHB mutations were found in the remaining allele of the 11 sporadic adrenal phaeochromocytomas with allelic loss at 1p36 or the three MEN 2-associated RET-mutation-positive phaeochromocytomas. Therefore, we conclude that SDHB has a major role in the pathogenesis of familial phaeochromocytomas, but the possible role of SDHB in sporadic tumours showing allelic loss at 1p36 has yet to be ascertained.

Allelic imbalance, including deletion of PTEN/MMACI, at the cowden disease locus on 10q22-23, in hamartomas from patients with cowden syndrome and germline PTEN mutation

Cowden disease (CD) is a rare, autosomal dominant inherited cancer syndrome characterized by multiple benign and malignant lesions in a wide spectrum of tissues. While individuals with CD have an increased risk of breast and thyroid neoplasms, the primary features of CD are hamartomas. The gene for CD has been mapped by linkage analysis to a 6 cM region on the long arm of chromosome 10 at 10q22‐23. Loss of heterozygosity (LOH) studies of sporadic follicular thyroid adenomas and carcinomas, both component tumors of CD, have suggested that the putative susceptibility gene for CD is a tumor suppressor gene. Somatic missense and nonsense mutations have recently been identified in breast, prostate, and brain tumor cell lines in a gene encoding a dual specificity phosphatase, PTEN/MMAC1, mapped at 10q23.3. Furthermore, germline PTEN/MMAC1 mutations are associated with CD. In the present study, 20 hamartomas from 11 individuals belonging to ten unrelated families with CD have been examined for LOH of markers flanking and within PTEN/MMAC1. Eight of these ten families have germline PTEN/MMAC1 mutations. LOH involving microsatellite markers within the CD interval, and including PTEN/MMAC1, was identified in two fibroadenomas of the breast, a thyroid adenoma, and a pulmonary hamartoma belonging to 3 of 11 (27%) of these patients. The wild‐type allele was lost in these hamartomas. Semi‐quantitative PCR performed on RNA from hamartomas from three different tissues from a CD patient suggested substantial reduction of PTEN/MMAC1 RNA levels in all of these tissues. The LOH identified in samples from individuals with CD and the suggestion of allelic loss and reduced transcription in hamartomas from a CD patient provide evidence that PTEN/MMAC1 functions as a tumor suppressor in CD. Genes Chromosomes Cancer 21:61–69, 1998.