Association between decreased WWOX protein expression and thyroid cancer development.

CONTEXT: Chromosomal fragile sites are often related to cancer development. The WW domain-containing oxidoreductase gene (WWOX) spans the second most common chromosomal fragile site (FRA16D) and encodes an important proapoptotic protein. OBJECTIVE: To verify our hypothesis that underexpression of WWOX could contribute to malignant transformation of the thyroid cells. METHOD: We compared WWOX expression among follicular adenomas (FAs) and differentiated thyroid carcinomas [follicular thyroid carcinomas (FTCs) and papillary thyroid carcinomas (PTCs)] in 53 thyroid tumors resected from patients submitted to total thyroidectomy. DESIGN: Multiple fields of tumor areas of FAs, FTCs, and PTCs as well as normal thyroid tissue were stained with WWOX antiserum, and classified by the extent of staining (percentage of cells staining) and staining intensity. MAIN OUTCOME: PTCs showed a significantly decreased expression of WWOX when compared to FAs and FTCs. Further, using a unique model of comparison in patients in whom FAs and PTCs were concomitantly present, we detected the same result (i.e., no expression in PTCs). CONCLUSION: We conclude that WWOX underexpression is an important step that might increase the vulnerability to the carcinogenesis process in PTCs.

High frequency of loss of heterozygosity in imprinted, compared with nonimprinted, genomic regions in follicular thyroid carcinomas and atypical adenomas.

CONTEXT: Many mammalian genes that are imprinted regulate cell growth, differentiation, and apoptosis. Because imprinting silences one of the two alleles, resulting in functional haploinsufficiency, we hypothesized that loss of heterozygosity (LOH) at an imprinted locus may result in the deletion of the only functional copy of an imprinted tumor suppressor gene. OBJECTIVE: The goal of this study was to specifically address this hypothesis that in thyroid neoplasias loss of imprinted loci becomes enriched during oncogenesis. DESIGN: In total, thyroid tissue was obtained from 72 patients with thyroid neoplasias comprising 34 follicular thyroid carcinomas (FTCs) and 38 follicular adenomas. We performed PCR-based LOH analysis of DNA from paired normal-tumor samples using 18 markers mapped to imprinted regions (IR) and 13 markers in nonimprinted regions (NIR). RESULTS: Overall LOH frequencies for the IR markers were 26% for the adenomas and 38% for the carcinomas. In the NIR, the overall LOH frequency was 23 and 26% for adenomas and FTCs, respectively. The difference in LOH frequencies between IR and NIR was statistically significant only for the carcinomas (P = 0.001), although there was a similar trend for the atypical adenomas (ATY, P = 0.06). CONCLUSIONS: Our observations suggest that IRs are more prone to genomic instability in FTCs. The fact that the ATY trended toward differential IR/NIR LOH, similar to FTC, may suggest that loss of IR might be instrumental in the adenoma-carcinoma sequence in thyroid carcinogenesis and that ATY could be an important intermediate in this pathway.

Papillary thyroid carcinoma with brain metastases: an unusual 10-year-survival case.

BACKGROUND: Papillary thyroid carcinoma (PTC) is the most common endocrine malignancy with indolent clinical course and good prognosis. Brain metastases are extremely rare and the average survival time after diagnosis has been reported to be around 12 months. SUMMARY: We here report a 69-year-old patient who was admitted to the emergency room in January 2000 with progressive dizziness, headache, and vomiting. Five years before admission the patient underwent partial thyroidectomy for goiter. On admission, a diagnostic evaluation that included brain magnetic resonance imaging showed multiple brain lesions, and a stereotactic biopsy demonstrated a metastatic carcinoma from primary PTC, with the neoplastic cells staining for thyroglobulin. Total thyroidectomy was then performed, which showed colloid goiter and a PTC metastasis on a cervical lymph node. The patient received 200 mCi of radioactive iodine ((131)I) with suppressive therapy with l-thyroxine thereafter. Subsequently, serial whole-body scanning and magnetic resonance imaging showed multiple brain metastases and the patient received further (131)I treatment, with a total dose of 1.2 Ci in a 10-year span. She also underwent partial surgical resection of brain metastases because complete resection was not feasible. Thereafter, the patient was subjected to whole-brain body radiotherapy with a dose of 44 Gy, followed by two brain gamma knife radiosurgeries (15 Gy each). To date, biochemical tests are within the normal range and the patient remains asymptomatic. CONCLUSIONS: To our knowledge, this is the first report of a 10-year-survival case of brain metastases from PTC, despite this being a bad prognostic factor. A combined approach of surgical excision, (131)I, whole-brain radiotherapy, and gamma knife radiosurgery was successful to treat metastases derived from primary tumor.

HRPT2-related familial isolated hyperparathyroidism: could molecular studies direct the surgical approach?

It is still debatable which is the best management to familial forms of hyperparathyroidism. Conservative, minimally invasive or aggressive surgical approaches have been proposed from different groups around the world. Our objective was to study the gene mutation, expression of HRPT2 and the clinical outcome after 32 years of follow-up in one Brazilian kindred with familial isolated hyperparathyroidism (FIHP). Clinical and biochemical data, direct sequencing of the HRPT2 gene, analysis of parafibromin expression using RT-PCR, and immunohistochemistry were done. A nonsense mutation was found in exon 1 (c.96G>A)(p.Trp32X) in all affected members studied. Using RT-PCR, mRNA transcription was altered with complete absence of both transcripts in tumor tissue. Immunohistochemical analysis of tumors showed loss of parafibromin immunoreactivity. In this kindred there was a high prevalence of recurrence (75%), or persistence after less than subtotal parathyroidectomy that led us to consider a more aggressive surgical approach should be discussed among the affected family members, once surgical criteria was met. We concluded that it is necessary to individualize the surgical approach for HRPT2-related hyperparathyroidism until we can gather a better phenotype-genotype correlation in larger series, to best define their treatment.

Familial hyperparathyroidism: surgical outcome after 30 years of follow-up in three families with germline HRPT2 mutations.

BACKGROUND: Familial forms of hyperparathyroidism are responsible for approximately 10% of the cases of primary hyperparathyroidism, and their management is different from the sporadic forms. Our objective was to study the gene sequence and expression of HRPT2 and clinical outcome regarding recurrence or persistence rates in three Brazilian kindreds with familial hyperparathyroidism after up to 30 years of follow-up. METHODS: Clinical and biochemical data, direct sequencing of germline DNA of the HRPT2 gene, and analysis of parafibromin expression (HRPT2 gene product) using RT-PCR and immunohistochemistry of resected parathyroid neoplasms were performed. RESULTS: Affected members of kindred A were found to carry a novel, germline, nonsense mutation in exon 1 (c.96G>A; W32X) of HRPT2. Six of seven patients who have undergone less than total parathyroidectomy recurred after up to 30 years of follow-up. An unrelated affected patient from kindred B had a germline mutation in exon 7 (c.686delGAGT), and the disease recurred with several pulmonary metastases after 5 years follow-up. The affected member of kindred C also had a previously described mutation in exon 7 (c.679delAG) and the disease recurred after 10 years of follow-up. All parathyroid neoplasms from these families had diffuse loss of expression by immunohistochemistry. CONCLUSIONS: An unacceptable recurrence/persistence rate (80%) associated with increasingly difficult re-operations and risk of parathyroid carcinoma in the setting of germline mutations of HRPT2 gene with familial hyperparathyroidism suggest that a more aggressive operative approach should be undertaken in these patients. Parafibromin immunohistochemistry may serve as a cost-effective screen for HRPT2-related aggressive parathyroid disease.

Distinct expression profiles for PTEN transcript and its splice variants in Cowden syndrome and Bannayan-Riley-Ruvalcaba syndrome.

Cowden syndrome (CS) and Bannayan-Riley-Ruvalcaba syndrome (BRRS) are autosomal dominant hamartoma syndromes. Germline PTEN mutations have been associated with 85% of CS cases and 65% of BRRS cases and also with other disorders, which are collectively referred to as the "PTEN hamartoma tumor syndrome." The human PTEN gene has been previously found to express two naturally occurring splice variants (SVs). Recently, we identified eight novel naturally occurring PTEN SVs that result in different downstream signaling effects: SV3a, SV3b, SV3c (inclusion of various lengths of intron 3 3' of exon 3), SV5a, SV5b, SV5c, SV5d (inclusion of various lengths of intron 5 3' of exon 5), and SV Delta Ex6 (deletion of exon 6). We therefore sought to characterize the relative expression of 5', middle, and 3' full-length PTEN mRNA (FL-PTEN) and also of these eight PTEN SVs in 85 (65 female and 20 male) patients with CS/BRRS (with or without PTEN mutations) compared with 27 controls, using a SYBR green quantitative polymerase chain reaction method. Significantly reduced FL-PTEN levels were found in the probands, compared with those of controls (P < .01). Apart from FL-PTEN, SV3a is the most consistently relatively underexpressed in patients compared with controls. The patients showed relative underexpression of SV3a and SV3b and overexpression of SV5b (P = .005, P = .02, and P = .04, respectively). Indeed, there appears to be an SV expressional genotype-phenotype correlation in which the SV expressional profiles are distinct among CS, CS-like, and BRRS. The reduced FL-PTEN transcript expression, associated with differential expression of PTEN SVs, regardless of PTEN mutation status, supports the concept that modulation of PTEN inactivation may also occur at the transcription level influencing the specific phenotypes seen in these syndromes.

HRPT2-related familial isolated hyperparathyroidism: could molecular studies direct the surgical approach? / Formas familiares de hiperparatireoidismo relacionadas a mutações no gene HRPT2: estudos moleculares poderiam direcionar procedimentos cirúrgicos?

It is still debatable which is the best management to familial forms of hyperparathyroidism. Conservative, minimally invasive or aggressive surgical approaches have been proposed from different groups around the world. Our objective was to study the gene mutation, expression of HRPT2 and the clinical outcome after 32 years of follow-up in one Brazilian kindred with familial isolated hyperparathyroidism (FIHP). Clinical and biochemical data, direct sequencing of the HRPT2 gene, analysis of parafibromin expression using RT-PCR, and immunohistochemistry were done. A nonsense mutation was found in exon 1 (c.96G>A)(p.Trp32X) in all affected members studied. Using RT-PCR, mRNA transcription was altered with complete absence of both transcripts in tumor tissue. Immunohistochemical analysis of tumors showed loss of parafibromin immunoreactivity. In this kindred there was a high prevalence of recurrence (75 percent), or persistence after less than subtotal parathyroidectomy that led us to consider a more aggressive surgical approach should be discussed among the affected family members, once surgical criteria was met. We concluded that it is necessary to individualize the surgical approach for HRPT2-related hyperparathyroidism until we can gather a better phenotype-genotype correlation in larger series, to best define their treatment.

A melhor conduta nas formas familiares de hiperparatireoidismo relacionadas a mutações no gene HRPT2 ainda é controvertida. Cirurgias conservadoras, minimamente invasivas ou mais agressivas já foram propostas por diferentes grupos. Objetivamos estudar a seqüência e a expressão do gene HRPT2, além do desfecho clínico, após seguimento de até 32 anos de uma família brasileira com hiperparatireodismo familiar isolado (FIHP). Utilizamos dados clínicos e bioquímicos, seqüenciamento direto do HRPT2 além de análise da expressão da parafibromina através da RT-PCR e imunohistoquímica. Foi identificada mutação nonsense no éxon 1 (c.96G>A)(p.Trp32X) em todos os membros afetados que foram estudados. A análise do mRNA transcrito, através da RT-PCR, demonstrou ausência do transcrito no tecido tumoral. A imunohistoquímica também evidenciou ausência da parafibromina. Nessa família houve alta (75 por cento) prevalência de recorrência ou persistência da doença após paratireoidectomia parcial o que nos levou a considerar fundamental discutir uma abordagem cirúrgica mais agressiva com os outros familiares portadores da mutação caso critérios de indicação cirúrgica sejam atingidos. Dessa maneira, até que estudos mais amplos estabeleçam uma correlação genótipo-fenótipo no hiperparatireoidismo familiar relacionado a mutações no HRPT2, a abordagem cirúrgica deverá ser individualizada.

No evidence for oncogenic mutations in guanine nucleotilde-binding proteins of endocrine tumors

In recent years, the application of DNA technology has led to significant advances in the elucidation of the somatic defects which can occur in several tumors, including oncogene expression, allelic loss and inappropriate gene transcription and translation. Normal cell growth is regulated by many proto-oncogenes encoding proteins and specific mutations can convert these genes in oncogenes, leading to abnormal protein products that are responsible for the growth of malignant cells. Mutations that inhibit GTPase activity of the a subunit of the stimulatory G protein (Gsa) have been demonstrated in approximately a thrid of GH-secreting tumors, in 10 percent of functionless pituitary tumors, and also in corticotropinomas although with far less frequency. These mutations -gsp mutations - stabilize the Gsa in the active state (GTP-bound state), resulting in the permanent activation of adenylyl cyclase, leading to tumorigenesis. In addition, mutations in the a subunit of the inhibitory GTP-binding protein gene (Gi2a), or gip mutations, have been found in a subset of adrenocortical and ovarian tumors. In the present work, using the polymerase chain reaction and denaturing gradient gel electrophoresis, we investigated the existence of gsp and gip mutations in twenty three different endocrine tumors.