Identification of a 7.1-mega base pairs minimal deletion at 14q31.1-32.11 in adenocarcinomas of the gastroesophageal junction.

In a recent evaluation by comparative genomic hybridization, we demonstrated chromosome 14q31-32.1 to be frequently deleted in adenocarcinomas of the gastroesophageal junction. This suggests the presence of a tumor suppressor gene in the deleted region. In the present study, we have performed a detailed loss of heterozygosity analysis in 34 gastroesophageal junction adenocarcinomas and 1 tumor-corresponding dysplastic Barrett's epithelium sample with 37 polymorphic microsatellite markers. Thirty-five markers are in the 14q24.3-32.33 region with a mean distance of 800 kilo base pairs. Of 34 tumor samples, 14 (41%) showed loss of 14q markers. We identified a minimal region of allelic loss of 7105440 base pairs between markers D14S1000 and D14S256 at cytogenetic location 14q31.1-32.11. Within this region, markers D14S1035, D14S55, D14S1037, D14S1022, D14S1052, D14S974, D14S73, D14S1033, D14S67, D14S68, and D14S1058 showed loss in all informative tumors with 14q loss. The region between markers D14S1000 and D14S256 contains 7 known genes. The identification of this minimal deletion and the data base information on the genes present in this region facilitate the search for the candidate tumor suppressor gene(s).

Clinical characteristics of pheochromocytoma patients with germline mutations in SDHD.

PURPOSE: We examined the value of SDHD mutation screening in patients presenting with apparently sporadic and familial pheochromocytoma for the identification of SDHD-related pheochromocytomas. PATIENTS AND METHODS: This retrospective study involved 126 patients with adrenal or extra-adrenal pheochromocytomas, including 24 patients with a family history of multiple endocrine neoplasia 2, von Hippel-Lindau disease, neurofibromatosis type 1, or paraganglioma (PGL). Conformation-dependent gel electrophoresis and sequence determination analysis of germline and tumor DNA were used to identify SDHD alterations. The clinical and molecular characteristics of sporadic and hereditary tumors were compared. We reviewed the literature and compared our results with those from previously published studies. RESULTS: Pathogenic germline SDHD mutations were identified in three patients: two (2.0%) of the 102 apparently sporadic pheochromocytoma patients and one patient with a family history of PGL. These patients presented with multifocal disease (two of three multifocal patients) or with a single adrenal tumor (one of 82 patients). In the literature, mutations are mostly found in patients

Increased sensitivity of B-cell clonality analysis in formalin-fixed and paraffin-embedded B-cell lymphoma samples using an enzyme blend with both 5'-->3' DNA polymerase and 3'-->5' exonuclease activity.

Polymerase chain reaction (PCR)-based detection of immunoglobulin heavy chain (IgH) gene rearrangement for determination of B-cell clonality needs to be simple but optimally sensitive. Efficient IgH PCR analysis can be hampered by sequence variability in the template DNA, despite of the use of degenerative primers. To improve sensitivity of the B-cell clonality analysis in formalin-fixed and paraffin-embedded (FFPE) tissues, we have performed framework three-area (FR3)/joining gene (JH) IgH PCR utilizing an enzyme blend (r Tth DNA Polymerase, XL) providing both 5'-->3' polymerase and 3'-->5' exonuclease activities. The DNA samples were extracted from FFPE biopsies of 43 mature B-cell lymphoma cases of so-called germinal center and post-germinal center origin, including 6 nodal follicular lymphomas (FL), 15 gastric mucosa-associated lymphoid tissue (MALT) lymphomas, and 22 gastric diffuse large B-cell lymphomas (DLBCL). Of the cases, 31 (17 DLBCL and 14 MALT lymphoma) represented small endoscopic biopsies. Serial dilutions of target DNA were applied to avoid inconsistent bands that may be seen when the input amount of template is too low, which can be the case when DNA is extracted from FFPE endoscopic gastric biopsies. Using conventional Taq polymerase, consistent monoclonal product was found in 53% (23/43) of the cases (FL: 67%; MALT lymphoma: 47%; DLBCL: 55%). The r Tth polymerase showed reproducible monoclonal pattern in 72% (31/43) of the cases (FL: 67%; MALT lymphoma: 73%; DLBCL: 73%); the sensitivity is compatible with one that can be detected with conventional FR3/JH PCR in fresh/frozen tissues. In conclusion, the r Tth DNA polymerase greatly improves sensitivity of FR3/JH PCR in FFPE biopsies of mature B-cell lymphomas, most probably by increasing the primer matches during PCR amplification.

Von Hippel-Lindau gene alterations in sporadic benign and malignant pheochromocytomas.

The Von Hippel-Lindau (VHL) gene product has a wide spectrum of tissue-specific functions, and specific germline mutations are associated with clinical phenotypes in VHL disease. In particular, missense mutations are correlated with the susceptibility to pheochromocytomas. An association between VHL aberrations and prognosis has been suggested in renal clear cell carcinoma but has not been studied in pheochromocytomas. We studied the frequency and spectrum of VHL alterations in apparently sporadic pheochromocytomas in relation to the clinical behavior in 72 patients, including 48 patients with clinically benign and 24 patients with malignant pheochromocytomas. Single-strand conformation polymorphism (SSCP) analysis followed by DNA sequencing, loss of heterozygosity analysis of the VHL locus and immunohistochemistry for VHL protein expression were used to investigate somatic VHL gene alterations. In 2 patients, 1 with a malignant tumor, germline mutations were identified in the stop codon. Tumor-specific intragenic VHL mutations and accompanying loss of heterozygosity were identified in 2 (4.3%) of 47 sporadic benign pheochromocytomas compared to 4 (17.4%) of 23 malignant tumors (p = 0.064). Only one of these mutations has been previously described, in a renal clear cell carcinoma. Expression of the VHL protein was observed in all pheochromocytomas. No distinction in the nature of VHL alterations between benign and malignant pheochromocytomas and no correlation with histopathologic or clinical features was observed. We report novel VHL mutations in sporadic pheochromocytomas, which are slightly correlated with malignancy. VHL mutations may have some impact on the malignant transformation of pheochromocytomas.

Frequent germ-line succinate dehydrogenase subunit D gene mutations in patients with apparently sporadic parasympathetic paraganglioma.

PURPOSE: Recently, familial paraganglioma (PGL) was shown to be caused bymutations in the gene encoding succinate dehydrogenase subunit D (SDHD). However, the prevalence of SDHD mutations in apparently sporadic PGL is unknown. We studied the frequency and spectrum of germ-line and somatic SDHD mutations in patients with parasympathetic PGL. EXPERIMENTAL DESIGH: We studied 57 unselected patients who developed parasympathetic PGLs (n = 105 tumors) and who were treated between 1987 and 1999 at the Erasmus MC (Rotterdam, the Netherlands). Thirty-eight (67%) of these patients (n = 51 tumors) lacked a family history of parasympathetic PGL. We used conformation-dependent gel electrophoresis and sequence determination analysis of germ-line and tumor DNA to identify SDHD mutations. We compared the clinical and molecular characteristics of sporadic and hereditary PGLs. RESULTS: Three different SDHD germ-line mutations were identified in 32 of the 57 (56%) patients. These included 19 of 19 (100%) patients with familial PGL and also 13 of 38 (34%) patients with apparently sporadic PGL. All three mutations were characterized as missense mutations (D92Y, L95P, and L139P) in highly conserved regions of the SDHD gene and were not observed in 200 control alleles. No somatic mutations were found. CONCLUSIONS: Germ-line mutations of the SDHD gene are present in a significant number of patients with apparently sporadic parasympathetic PGL. Somatic SDHD mutations do not play a significant role in the sporadic form of this tumor. Genetic testing for SDHD germ-line mutations should be considered for every patient presenting with this tumor, even if a personal or family history of PGL is absent, to allow appropriate clinical management.