Precursor lesions in patients with multiple endocrine neoplasia type 1-associated duodenal gastrinomas.

BACKGROUND & AIMS: The identification of precursor lesions has a great impact on the understanding of tumorigenesis. Precursor lesions of endocrine tumors are known to occur in the setting of the MEN1 syndrome. The aim of this study was to test the hypothesis that MEN1-associated duodenal gastrinomas originate from diffuse preneoplastic gastrin cell changes. Precursor lesions may precede the development of duodenal gastrinomas because, in contrast to sporadic gastrinomas, these tumors are usually multiple. METHODS: The distribution of endocrine cells in the nontumorous duodenal tissue was analyzed qualitatively and quantitatively for 25 patients operated on for a duodenal gastrinoma. MEN1 status was assessed clinically and by polymerase chain reaction-based mutational analysis. RESULTS: Fourteen of 25 patients with gastrinoma had proliferative, hyperplastic lesions consisting of gastrin cells in the nontumorous duodenal mucosa, similar to the gastric enterochromaffin-like cell lesions observed in chronic atrophic gastritis. All patients with Zollinger-Ellison syndrome with proven MEN1 had such proliferative gastrin cell lesions, and all patients with Zollinger-Ellison syndrome without precursor lesions were MEN1 negative. CONCLUSIONS: Duodenal gastrinomas in MEN1, but not sporadic duodenal gastrinomas, are associated with proliferative gastrin cell changes within the nontumorous mucosa. It is likely that these lesions precede the development of MEN1-associated duodenal gastrinomas.

Abnormalities of proinsulin processing in functioning insulinomas: clinical implications.

OBJECTIVE: Abnormal proinsulin processing in insulinomas may result in secretory granules containing both insulin and proinsulin, a finding not encountered in healthy beta-cells. The aim of this study was to test whether such abnormalities in the proinsulin to insulin conversion have clinical implications in patients with hypoglycaemic disorders. DESIGN: Case-series. PATIENTS AND METHODS: Fifteen patients with histologically confirmed insulinoma and two patients with islet cell hyperplasia were included. The immunohistochemical distribution pattern of proinsulin within the tumour cells was classified as Golgi pattern (predominantly perinuclear immunolabelling) or diffuse pattern (immunolabelling in the periphery of the cells, indicating the presence of proinsulin in secretory granules). Data obtained from the 72-h fast and arterial calcium stimulation and hepatic venous sampling (ASVS) test were related to the morphological classification. RESULTS: Six insulinomas exhibited a diffuse proinsulin distribution pattern, while nine insulinomas and two islet cell hyperplasias disclosed a Golgi pattern. Median proinsulin concentrations at the termination of the fast tended to be higher in patients with the diffuse proinsulin distribution pattern than in patients with the Golgi pattern (86.9 vs. 18.8 pmol/l, P = 0.07). Higher insulin (P < 0.005) and proinsulin (P < 0.05) concentrations were significantly correlated with earlier occurrence of hypoglycaemia during the prolonged fast. During the ASVS test, tumours with the diffuse proinsulin distribution pattern exhibited a higher increase in both insulin (median, 37.3- vs. 10.5-fold, P < 0.05) and proinsulin (6.3- vs. 1.6 fold, P < 0.005) concentrations following calcium stimulation than the tumours with the Golgi pattern. CONCLUSIONS: Abnormalities in the proinsulin to insulin conversion in patients with insulinomas and islet cell hyperplasia correlate with impaired regulation of both insulin and proinsulin secretion during the prolonged fast as well as the ASVS test.

Absence of BRAF gene mutations differentiates spitz nevi from malignant melanoma.

BACKGROUND: Distinction of Spitz nevus from malignant melanoma is sometimes difficult on the basis of conventional histology. A high rate of BRAF gene mutations in malignant melanomas (66%) and nevi (82%) has recently been reported. MATERIALS AND METHODS: We screened a series of 20 Spitz nevi for BRAF mutations in exons 11 and 15 by denaturing gradient gel electrophoresis (DGGE). RESULTS: BRAF mutations could not be identified in Spitz nevi. CONCLUSION: Our results show that mutations within the BRAF gene are useful markers for the differential diagnosis between Spitz nevus and malignant melanoma.

Bmi1 is essential for cerebellar development and is overexpressed in human medulloblastomas.

Overexpression of the polycomb group gene Bmi1 promotes cell proliferation and induces leukaemia through repression of Cdkn2a (also known as ink4a/Arf) tumour suppressors. Conversely, loss of Bmi1 leads to haematological defects and severe progressive neurological abnormalities in which de-repression of the ink4a/Arf locus is critically implicated. Here, we show that Bmi1 is strongly expressed in proliferating cerebellar precursor cells in mice and humans. Using Bmi1-null mice we demonstrate a crucial role for Bmi1 in clonal expansion of granule cell precursors both in vivo and in vitro. Deregulated proliferation of these progenitor cells, by activation of the sonic hedgehog (Shh) pathway, leads to medulloblastoma development. We also demonstrate linked overexpression of BMI1 and patched (PTCH), suggestive of SHH pathway activation, in a substantial fraction of primary human medulloblastomas. Together with the rapid induction of Bmi1 expression on addition of Shh or on overexpression of the Shh target Gli1 in cerebellar granule cell cultures, these findings implicate BMI1 overexpression as an alternative or additive mechanism in the pathogenesis of medulloblastomas, and highlight a role for Bmi1-containing polycomb complexes in proliferation of cerebellar precursor cells.

CGH analysis shows genetic similarities and differences in atypical fibroxanthoma and undifferentiated high grade pleomorphic sarcoma.

BACKGROUND: Atypical fibroxanthoma (AFX) and undifferentiated high grade pleomorphic sarcoma (UpS) are histologically very similar, if not identical. However, they differ significantly in clinical outcome. MATERIALS AND METHODS: We used comparative genomic hybridization (CGH) to screen 24 AFX and 12 UpS for genomic alterations. RESULTS: DNA copy number changes were observed in 20/24 AFX and in all UpS. The most frequent alterations occurring with comparable frequency in both tumors were deletions on chromosomes 9p and 13q. We also detected statistically significant differences of genetic alterations between the two tumors concerning deletions on 1q, 3p, 5q, 11p, 11q, gains on 7q, 12q and high level gains on 5p and 11q. CONCLUSION: Despite their very similar histology, AFX and UpS show clear differences in their genetic alterations. This might contribute to the different biological behavior of the two tumors. On the other hand the similarities in genetic alterations on chromosomes 9p and 13q might suggest a common pathogenetic pathway.

A novel succinate dehydrogenase subunit B gene mutation, H132P, causes familial malignant sympathetic extraadrenal paragangliomas.

We report a family with malignant sympathetic paragangliomas (PGL) exhibiting a new type of germline mutation in the succinate dehydrogenase subunit B (SDHB) gene. Two affected brothers, presenting with symptoms at the ages of 25 and 52 yr, suffered from malignant abdominal extraadrenal sympathetic PGL. They died of their disease at ages 43 and 61 yr. Their mother had the same history of signs and symptoms, suggesting a catecholamine-producing tumor at the age of 55 yr. Analysis of the germline DNA from these three patients revealed a novel mutation in exon 4 (H132P) of the SDHB gene. This mutation was absent in 160 control chromosomes. Loss of heterozygosity analysis of the tumors showed a loss of one SDHB allele, and RT-PCR-based expression analysis confirmed the exclusive expression of the mutated allele in both tumors. A review of the published PGL families revealed malignant tumors in seven of 12 well-documented families with SDHB mutation-associated extraadrenal PGL. These findings, as well as findings of the family reported here, suggest a strong causal relationship of SDHB germline mutations with malignant extraadrenal abdominal PGL and imply the necessity of a close follow-up of affected individuals and family members.

DPC4/Smad4: no mutations, rare allelic imbalances, and retained protein expression in pancreatic endocrine tumors.

Several chromosomal loci involved in tumorigenesis of pancreatic endocrine tumors (PET) have been identified. To date, the only gene known to be frequently altered is the MEN1 gene. Recently, DPC4 mutations and homozygous deletions have been described in 5/9 (55%) non-functioning PET, thus representing the most frequent genetic aberration described in PET. However, these data are in discordance with comparative genomic hybridization (CGH) results that rarely show genetic losses on chromosome 18. They have also been challenged by immunohistochemical data. We performed a detailed combined DPC4 mutation and deletion analysis in 34 benign and malignant PET. Mutations of the conserved C-terminal exons were not found in all examined PET and allelic loss (LOH) was found to be rare (<6%) by combined microsatellite PCR and FISH analysis. In addition, DPC4 protein expression was retained in all PET that were examined by immunohistochemistry. Therefore, DPC4 inactivation by mutation or deletion appears to be very rare in PET, which confirms the current concept of unrelated mechanisms of tumorigenesis of endocrine versus exocrine pancreatic tumors.

Immunostaining for the tumour suppressor gene p16 product is a useful marker to differentiate melanoma metastasis from lymph-node nevus.

Upon the introduction of extensive sampling protocols of sentinel node biopsies, pathologists are increasingly confronted with small melanoma metastases. Using conventional histology, it proves sometimes difficult or impossible to differentiate small melanoma metastases from lymph-node nevi. Loss of the tumour suppressor gene p16 has been shown to be associated with tumour progression of melanoma. We investigated nevus and melanoma cells for the presence of the product of the gene p16, using immunohistochemistry. All nevus cells, independent of their location (nodal or skin) displayed an extensive nuclear and cytoplasmic staining for p16. In contrast, all cells of melanoma metastases, except one skin metastasis, lacked nuclear staining for p16. These findings indicate that p16 is a reliable marker to distinguish lymph-node nevi from melanoma metastasis.

ALG-2: a Ca2+ -binding modulator protein involved in cell proliferation and in cell death.

During the development of an organism, cell proliferation, differentiation and cell death are tightly balanced, and are controlled by a number of different regulators. Alterations in this balance are often observed in a variety of human diseases. The role of Ca(2+) as one of the key regulators of the cell is discussed with respect to a recently discovered Ca(2+)-binding protein, ALG-2, which is highly upregulated in cancerous tissues of different origins. The role of ALG-2 as a possible clinical marker and, molecularly, as a possible modulator at the interface between cell proliferation and cell death is discussed.

Absence of somatic SDHD mutations in sporadic neuroendocrine tumors and detection of two germline variants in paraganglioma patients.

Allelic loss of the long arm of chromosome 11 is frequent in neuroendocrine tumors (NET) of different organs. However, the MEN1 gene on 11q13 is mutated only in a subset of NET and allelic losses on 11q frequently extend to the telomere. In this genetic region lies the tumor suppressor gene SDHD which is associated with hereditary paragangliomas (PGL1). We sought to determine whether SDHD plays a role in the development of sporadic NET. By mutation and deletion analysis of SDHD we were unable to detect any SDHD mutation in 45 NET of the lung, gastrointestinal tract, pancreas or parathyroid. However, we found allelic deletions in 20 to 50% of all tumors but parathyroid adenomas. Furthermore, we found heterozygous germline variants in 2/8 paragangliomas. A first case of variant c.149 A>G (H50R) was found in a patient with an extra-adrenal pheochromocytoma, the other variant c.34 G>A (G12S) in a patient with a paratracheal paraganglioma, C-cell hyperplasia of the thyroid and hyperplasia of ACTH-producing cells of the pituitary gland. Both variants were absent in 93 controls. Our results demonstrate that somatic SDHD mutations are rare in sporadic NET. However, LOH alone could lead to a complete loss of function since SDHD is an imprinted gene. Furthermore, we describe two germline variants possibly causing hereditary paragangliomas.

Molecular Diagnosis of Multiple Endocrine Neoplasia (MEN) in Paraffin-Embedded Specimens.

In this article, we summarize our recent findings on rearranged during transfection (RE7) mutations in a series of 46 sporadic as well as multiple endocrine neoplasia (MEN) type 2- associated tumors and present results of our family screening efforts to identify MEN 2 and MEN 1 gene carriers. A nonisotopic polymerase chain reaction-based single-strand conformation polymorphism (PCR-SSCP) analysis and heteroduplex gel electrophoresis method was used to screen DNA extracted from archival specimens of 22 patients with MEN 2-associated and 24 patients with sporadic tumors for mutations in RETexons 1O, 11, 13, and 16. Point mutations were identified by nonisotopic cycle sequencing of PCR products using an automated DNA sequencer. We found six different missense germ line mutations at cysteine residues encoded by exons 10 and 11 in all patients with MEN 2A or familial medullary thyroid carcinoma (FMTC). The frequency of mutations at codon 634 was higher in patients with MEN 2A than with FMTC and a (63)Cys - Arg mutation was associated with parathyroid disease. A germline Met -* Thr point mutation at codon 918 of the RETtyrosine kinase domain encoded by exon 16 was identified in all MEN 2B patients. Nonpredicted inheritable medullary thyroid carcinomas (MTCs) were detected in two patients and a mosaic postzygotic mutation was found in one additional patient. Tumor-specific (somatic) Met - Thr point mutations at codon 918 were identified in 5 of 13 sporadic MTCs and 2 of 8 sporadic pheochromocytomas (PCCs). The remaining sporadic tumors lacked mutations in all four RET exons tested. In exon 13, a nucleic acid polymorphism (CTT/CTG; Leu) at codon 769 was identified, which is present in approx 40% of the examined population. Our study demonstrates that the molecular methods used are not only suitable to identify asymptomatic individuals at risk for MEN 2A, FMTC, and MEN 2B, but also to distinguish sporadic from inherited tumors using archival tissue specimens; and that more tumors than clinically expected are inheritable, indicating the need for genetic analysis of all MTC and PCC patients.