Loss of heterozygosity in 11q13-14 regions in gastric neuroendocrine tumors not associated with multiple endocrine neoplasia type 1 syndrome.

Loss of heterozygosity (LOH) at the MEN1 gene locus at 11q13 is commonly found in type II gastric carcinoid tumors, which are associated with multiple endocrine neoplasia type 1 (MEN-1). In contrast, information is scanty or absent for other types of gastric neuroendocrine tumors, represented by type I carcinoids (associated with chronic atrophic gastritis), type III (sporadic) carcinoids, and neuroendocrine carcinomas. Moreover, LOH analysis of the allelic region distal to the MEN1 gene, which is postulated to contain an additional tumor suppressor gene effective in MEN-1-associated and sporadic endocrine tumors, has never been performed. To clarify these issues, DNA extracted from archival tissue from 25 type I carcinoids, 4 type III carcinoids, and 2 neuroendocrine carcinomas was amplified by PCR, using primers for six polymorphic markers located on chromosome 11q13 (PYGM, D11S4946, and D11S913) and 11q14 (D11S916, D11S901, and D11S1365), for analysis of LOH. Allelic losses in the 11q13-14 region with at least two polymorphic markers were found in 12 of 25 (48%) type I carcinoids. When LOH was found in the 11q13 region, it was large and continuous and extended to the most telomeric marker investigated. In one tumor, retention of heterozygosity for markers in the MEN1 region and LOH for distal markers were observed. No LOH was found in three of four type III carcinoids. Large deletions in both the 11q13 and 11q14 regions were observed in both neuroendocrine carcinomas investigated. In conclusion, LOH in the 11q13-14 regions is frequently found in type I carcinoids and neuroendocrine carcinomas of the stomach, suggesting the involvement of the MEN1 gene and/or a more telomeric tumor suppressor gene in the pathogenesis of these non-MEN-1-associated neuroendocrine tumors. The low rate of LOH at 11q13-14 suggests the predominance of different genetic mechanisms in type III carcinoids, which also differ from other types of gastric carcinoids in the lack of a promoter role for gastrin.     

Deletion mapping of endocrine tumors localizes a second tumor suppressor gene on chromosome band 11q13

Multiple endocrine neoplasia type 1 syndrome (MEN1, MIM 131100), an autosomal dominant disease, is characterized by parathyroid hyperplasia, pancreatic endocrine tumors, and pituitary adenomas. These tumors also occur sporadically. Both the familial (MEN1) and the sporadic tumors reveal loss of heterozygosity (LOH) for chromosome band 11q13 sequences. Based on prior linkage and LOH analyses, the MEN1 gene was localized between PYGM and D11S460. Recently, the MEN1 gene (menin) has been cloned from sequences 30-kb distal to PYGM. We performed deletion mapping on 25 endocrine tumors (5 MEN1 and 20 sporadic) by using 21 polymorphic markers on chromosome band 11q13. Of these, two (137C7A, 137C7B) were derived from PYGM-containing BAC (bacterial artificial chromosome-137C7) sequences, one from INT2-containing cosmid sequences and the marker D11S4748, a (CA)₂₀ repeat marker that was developed by us. The LOH analysis shows that the markers close to the MEN1 (menin) gene were not deleted in three of the tumors. These tumors, however, showed LOH for distal markers. Thus, the data suggest the existence of a second tumor suppressor gene on chromosome band 11q13. Genes Chromosomes Cancer 22:130–137, 1998. © 1998 Wiley-Liss, Inc.     

Mutations and allelic deletions of the MEN1 gene are associated with a subset of sporadic endocrine pancreatic and neuroendocrine tumors and not restricted to foregut neoplasms

Endocrine pancreatic tumors (EPT) and neuroendocrine tumors (NET) occur sporadically and rarely in association with multiple endocrine neoplasia type 1 (MEN1). We analyzed the frequency of allelic deletions and mutations of the recently identified MEN1 gene in 53 sporadic tumors including 30 EPT and 23 NET (carcinoids) of different locations and types. Allelic deletion of the MEN1 locus was identified in 18/49 (36.7%) tumors (13/30, 43.3% in EPT and 5/19, 26.3% in NET) and mutations of the MEN1 gene were present in 8/52 (15.3%) tumors (4/30 (13.3%) EPT and 4/22 (18.1%) NET). The somatic mutations were clustered in the 5' region of the coding sequence and most frequently encompassed missense mutations. All tumors with mutations exhibited a loss of the other allele and a wild-type sequence of the MEN1 gene in nontumorous DNA. In one additional patient with a NET of the lung and no clinical signs or history of MEN1, a 5178-9G-->A splice donor site mutation in intron 4 was identified in both the tumor and blood DNA, indicating the presence of a thus far unknown MEN1 syndrome. In most tumor groups the frequency of allelic deletions at 11q13 was 2 to 3 times higher than the frequency of identified MEN1 gene mutations. Some tumor types, including rare forms of EPT and NET of the duodenum and small intestine, exhibited mutations more frequently than other types. Furthermore, somatic mutations were not restricted to foregut tumors but were also detectable in a midgut tumor (15.2% versus 16.6%). Our data indicate that somatic MEN1 gene mutations contribute to a subset of sporadic EPT and NET, including midgut tumors. Because the frequency of mutations varies significantly among the investigated tumor subgroups and allelic deletions are 2 to 3 times more frequently observed, factors other than MEN1 gene inactivation, including other tumor-suppressor genes on 11q13, may also be involved in the tumorigenesis of these neoplasms.     

Loss of heterozygosity on chromosome arm 11q in lung carcinoids

Neuroendocrine lung tumors such as typical carcinoid, atypical carcinoid, small-cell lung carcinoma, and large-cell neuroendocrine carcinoma represent a variable group with different biologic characteristics and unclear genetical relationships. We investigated the pattern of allelic loss on chromosome arm 11q in 20 sporadic carcinoid tumors of the lung using 10 microsatellite markers. Loss of heterozygosity was found in 13 of 20 tumors. In 5 of 9 typical carcinoids, 3 distinct regions of allelic loss were identified: 11q13.1 (D11S1883), 11q14.3-11q21 (D11S906), and 11q25 (D11S910). Atypical carcinoids showed loss of heterozygosity at 4 different regions: the first, most proximal region at 11q13 between markers PYGM and D11S937; the second at 11q14.3-11q21 (D11S906); and the third and fourth defined by markers D11S939 (11q23.2-23.3) and D11S910 (11q25). However, the region 11q13 harboring the MEN1 gene was more frequently affected in atypical carcinoids (7 of 11) than in typical carcinoids (2 of 9). The high rate of allelic losses within chromosomal region 11q13 in atypical carcinoids emphasizes the importance of this region for tumor development. We also recognized that more aggressive atypical carcinoids defined by high mitotic counts, vascular invasion, and/or organ metastasis are combined with increased allelic losses. HUM PATHOL 32:333-338.     

Different patterns of 11q allelic losses in digestive endocrine tumors

Most foregut digestive endocrine neoplasms may be associated with the multiple endocrine type 1 (MEN-1) syndrome. In contrast, midgut/hindgut carcinoids never show such association. To investigate the pathogenetic involvement of the MEN-1 gene and of putative additional oncosuppressor gene(s) distal to it, a comparative analysis of loss of heterozygosity (LOH) at chromosome 11q13 to 11qter was performed in 27 foregut (pancreatic endocrine tumors [PETs]), 23 midgut (ileal and appendiceal), and 3 hindgut (rectal) endocrine tumors. LOH at the MEN-1 gene locus at 11q13 was observed in 52% of the 23 sporadic and in all 4 MEN-1-associated PETs and was found to consistently and continuously span to the most distal marker investigated at 11qter. In contrast, only occasional, discontinuous, and mostly interstitial LOH for 11q markers was observed in ileal (midgut) carcinoids, whereas no LOH was found in all appendiceal (midgut) and rectal (hindgut) carcinoids. The consistent extension of LOH from the MEN-1 region to 11qter in sporadic PETs suggests a mechanism of gene inactivation via chromosomal breakage and complete loss of chromosome 11q; furthermore, these results expand beyond the 11q13 region the search for additional oncosuppressor gene(s) potentially involved in the genesis of these neoplasms. The low frequency, limited extension, and discontinuous distribution of 11q deletions in midgut/hindgut carcinoids suggest that MEN-1 gene is not involved in the pathogenesis of these tumors.     

Genetic alterations on 3p, 11q13, and 18q in nonfamilial and MEN 1-associated pancreatic endocrine tumors.

Pancreatic endocrine tumors occur sporadically and as part of the multiple endocrine neoplasia type 1 (MEN 1) and von Hippel-Lindau (VHL) syndromes. The MEN1 locus on 11q13 and a candidate tumor suppressor locus on 3p are known to be hemi- or homozygously mutated in a subset of these tumors. Chromosome arm 18q harbors the SMAD4/DPC4 tumor suppressor gene that is frequently deleted and inactivated in tumors of the exocrine pancreas. We have analyzed 22 nonfamilial and 16 MEN 1-associated pancreatic endocrine tumors for loss of heterozygosity (LOH) at 3p, 11q13, and 18q. LOH at 3p was revealed in 45% and 36% of tumors from 31 patients with nonfamilial and MEN 1-associated disease, respectively. The corresponding proportions for 11q13 were 55% and 91%, and for 18q 27% and 25%, respectively. A striking relation between LOH at 11q13 and 3p and a malignant phenotype was found for the nonfamilial tumors. None of the six benign tumors (all of them insulinomas) had allelic loss at 3p or 11q13, whereas 92% (P < 0.01) of the malignant tumors (including malignant insulinomas) had such deletions. Besides the 11q13 abnormality, more than half of the MEN 1-associated tumors had additional genetic lesions affecting 3p or 18q. LOH analysis of several tumors from two MEN 1 patients suggested different clonal origin of the lesions. Sequencing of the SMAD4/DPC4 gene did not identify mutations in coding regions or at exon/intron boundaries in tumors with LOH at 18q. The data indicate involvement of tumor suppressor genes on 3p and 18q, in addition to the MEN1 gene at 11q13, in the tumorigenesis of both nonfamilial and MEN 1-associated pancreatic endocrine tumors.     

Role of disease-causing genes in sporadic pancreatic endocrine tumors: MEN1 and VHL

Pancreatic endocrine tumors (PETs) occur in association with multiple endocrine neoplasia type 1 (MEN1) and von Hippel-Lindau (VHL) syndromes caused by germline alterations in MEN1 and VHL, respectively. It is thus expected that these genes will also be altered in a proportion of sporadic PETs. Indeed, MEN1 is altered in about 25% of nonfamilial PETs, although no mutations have been found in VHL. For all clinical subtypes, the frequency of allelic loss on chromosome arm 11q mirrors observed mutational frequencies, with the exception of nonfunctional tumors (NF-PETs), in which mutations have been reported in only 8% of cases. As allelic loss on 11q is the most frequent event found in these neoplasms, this low frequency is somewhat puzzling, particularly in light of the fact that most MEN1-associated PETs are nonfunctioning. To clarify the role of these genes in sporadic PETs, we analyzed 31 sporadic NF-PETs, nine insulinomas, and one VIPoma for alterations in MEN1 and VHL. As somatic mutations were observed in eight (26%) of the NF tumors and in one insulinoma, it would therefore appear unlikely that an additional tumor suppressor gene related to sporadic PET pathogenesis is located on 11q. One insulinoma also had a somatic mutation in VHL, and thus this gene may also be altered in these neoplasms, albeit in a small proportion of cases.     

MEN1 gene mutations in sporadic neuroendocrine tumors of foregut derivation

Foregut-derived neuroendocrine (NE) tumors occur sporadically or in association with multiple endocrine neoplasia type 1 (MEN1) syndrome. Thirty-nine sporadic NE tumors of foregut derivation (six thymic, 21 bronchial, three gastric, and nine pancreatic tumors) as well as two hindgut-derived rectal carcinoids for somatic MEN1 gene mutation were analyzed by direct sequencing analysis. Five tumors showed mutations: nonsense mutations (Q393X and R98X) in thymic and pancreatic NE tumors, respectively, a 4 b.p. deletion (357del4) in a gastric NE carcinoma, and missense mutations (D172Y and S178Y) in pancreatic NE tumors. No mutation was identified in pulmonary or rectal NE tumors. In a patient with a pancreatic NE tumor (D172Y), the corresponding germline DNA showed the same mutation, suggesting that sporadic MEN1 syndrome was masked in this case. Somatic MEN1 gene mutations and deletions may play a crucial role in the tumorigenesis of a subset of foregut-derived NE tumors. Sporadic MEN1 syndrome may occur as a sporadic NE tumor of the pancreas.     

Mutation analysis of the MEN1 gene in Israeli patients with MEN1 and familial isolated hyperprolactinemia

Multiple endocrine neoplasia type 1 (MEN-1) is characterized by hyperfunction and tumor formation of the parathyroids, anterior pituitary and endocrine pancreas. We carried out exon-specific, PCR-based DNA sequencing of the coding exons of the MEN1 gene in 8 Israeli MEN1 patients: 4 familial and 4 sporadic. We similarly analyzed Israeli families with a unique phenotype of isolated hyperprolactinemia (HPRL). Four mutations were detected in 4 MEN1 patients: C to T alteration at nucleotide 2608 resulting in R108X, and three intronic insertions/deletions (a 13 basepair (bp) deletion and a 1 bp insertion both in intron 1, and a 2 bp insertion in intron 3) leading to exonic frame shifts as they encompass the splice junctions. An additional patient exhibited a compound mutation: a G to T change at position 7614 resulting in E463X, and insertion/deletion of 9 bp at position 7622-7630 resulting in EAE466-468X. Haplotype analysis showed no segregation of phenotype with 11q13 markers in 4 familial HPRL, and no men 1 germline mutations were detected in three representative individuals, from 3 families. Our results confirm that men 1 gene germline mutations occur in the majority of patients with clinically diagnosed MEN1, and that familial HPRL is a genetically distinct disorder.     

Sporadic follicular thyroid tumors show loss of a 200-kb region in 11q13 without evidence for mutations in the MEN1 gene.

Loss of heterozygosity (LOH) in 11q13 where the tumor suppressor gene for multiple endocrine neoplasia type 1 (MEN 1) is located has been demonstrated in several tumor types, including follicular thyroid tumors, but whether the MEN1 gene is actually involved in their tumorigenesis is not known. In the present study, the involvement of the MEN1 gene in follicular thyroid tumors was investigated. By using 14 MEN1-linked microsatellite markers, LOH was demonstrated in 12 out of 60 follicular thyroid tumors: 2/18 adenomas, 4/15 atypical adenomas, 1/6 Hürthle cell adenomas, 1/9 carcinomas, 3/6 Hürthle cell carcinomas, and 1/6 anaplastic carcinomas. In the tumors with LOH, a single minimal region of overlapping deletions was mapped to the 200-kb interval between D11S4946 and D11S4939. Tumors that showed 11q13 LOH were screened for mutations of the MEN1 gene using single-strand conformation analysis. Abnormal shifts detected in seven tumors in two exons were sequenced, which revealed two different polymorphisms present in both tumor and constitutional DNA, but without somatic mutation. Taken together, these results suggest that in this region, a tumor suppressor gene other than MEN1 might be involved in the tumorigenesis of follicular thyroid tumors. Genes Chromosomes Cancer 26:35-39, 1999.     

Chromosome 11 monosomy in conjunction with a mutated SDHD initiation codon in nonfamilial paraganglioma cases

Paragangliomas of the head and neck region are a group of rare, usually benign, slow-growing tumors developing from paraganglionic chemoreceptors in most patients. Mutations in a subunit of the mitochondrial enzyme II complex (succinate dehydrogenase [SDHD]) were shown to be responsible for the formation of paragangliomas. In addition, loss of heterozygosity (LOH) on chromosome 11, mainly in 11q23 (PGL1), was observed recently. We analyzed DNA derived from tumor sections of three unrelated paraganglioma patients (one case with multiple paragangliomas, two cases with single tumors; all of them sporadic cases) for mutations in the SDHD gene by direct sequencing. Microsatellite-based LOH was performed, and events of chromosomal loss were validated by fluorescence in situ hybridization (FISH) on paraffin-embedded tumor and normal tissue by using centromeric satellite DNA. Sequence analysis revealed mutations in SDHD exon 1 in all patients, affecting the initiation codon (M1V). Another alteration was detected in exon 2 but was lacking in tumor DNA and therefore classified as polymorphism (H50R). LOH and FISH analyses demonstrated partial/total monosomy for chromosome 11 in the tumor samples tested. A common genetic mechanism appears to be the pathophysiologic basis for sporadic tumor development because the proposed two-hit model comprising both LOH and point mutation is manifest in our patients. Loss of chromosome 11 regions, including the deletion of PGL1 and PGL2 loci, may result in a more severe phenotype, as exemplified by the development of multiple tumors in one of the patients.     

Deletions of 11q22.3-q25 Are Associated with Atypical Lung Carcinoids and Poor Clinical Outcome

Carcinoids are slow-growing neuroendocrine tumors that, in the lung, can be subclassified as typical (TC) or atypical (AC). To identify genetic alterations that improve the prediction of prognosis, we investigated 34 carcinoid tumors of the lung (18 TCs, 15 ACs, and 1 unclassified) by using array comparative genomic hybridization (array CGH) on 3700 genomic bacterial artificial chromosome arrays (resolution ≤1 Mb). When comparing ACs with TCs, the data revealed: i) a significant difference in the average number of chromosome arms altered (9.6 versus 4.2, respectively; P = 0.036), with one subgroup of five ACs having more than 15 chromosome arms altered; ii) chromosomal changes in 30% of ACs or more with additions at 9q (≥1 Mb) and losses at 1p, 2q, 10q, and 11q; and iii) 11q deletions in 8 of 15 ACs versus 1 of 18 TCs (P = 0.004), which was confirmed via fluorescence in situ hybridization. The four critical regions of interest in 45% ACs or more comprised 11q14.1, 11q22.1-q22.3, 11q22.3-q23.2, and 11q24.2-q25, all telomeric of MEN1 at 11q13. Results were correlated with patient clinical data and long-term follow-up. Thus, there is a strong association of 11q22.3-q25 loss with poorer prognosis, alone or in combination with absence of 9q34.11 alterations (P = 0.0022 and P = 0.00026, respectively).Pulmonary carcinoids comprise a group of usually smoking-unrelated neuroendocrine tumors. Compared with poorly differentiated neuroendocrine tumors of the lung, ie, large-cell neuroendocrine carcinoma and small-cell lung cancer, carcinoids are well-differentiated and characterized by a low metastatic rate and a relatively favorable prognosis. On the basis of histopathologic features (number of mitoses and presence of necrosis), lung carcinoid tumors are classified as typical carcinoids (TCs) or atypical carcinoids (ACs), although classification is sometimes difficult and its reliability to predict disease outcome is variable.¹ Compared with TCs, in general, ACs more often exhibit malignant behavior and are associated with a lower 5-year survival rate (61% to 88% and 92% to 100%, respectively).² Metastases will develop in 4% to 64% of patients with carcinoids (TCs, 4% to 14%, and ACs, 35% to 64%), usually in regional lymph nodes but also at distant sites including liver, bone, brain, subcutaneous tissue, and breast.^2,3 Although most patients remain cancer-free within 5 years after surgery, there is no curative treatment available for metastatic disease.A few studies have reported clinical and molecular factors associated with higher risk of developing metastases or with poor disease outcome. Clinical factors with prognostic value include size 3.5 cm or larger, mitotic index, degree of differentiation, presence of necrosis, co-secretion of peptides, and metastasis.^4,5 Immunohistochemistry on TC samples revealed that a high Ki-67 labeling index or up-regulation of the anti-apoptotic proteins Bcl-2 and p53 were associated with metastatic disease and shorter survival time, whereas immunostaining for the adhesion molecule CD44 was associated with localized disease and lower mortality.⁶ An additional study of 121 pulmonary neuroendocrine tumors including 21 carcinoids demonstrated a shift to low Bax and high Bcl-2 expression in association with ACs, resulting in an unfavorable prognosis.⁷ The Rb pathway is more often modified in ACs than in TCs. P16 negativity was observed in 23% of ACs, compared with 9% of TCs, and absent staining for pRb in 21% of ACs and no TCs.⁸Genomic alterations contribute to carcinogenesis by changing the expression levels of critical oncogenes and tumor-suppressor genes. In lung carcinoid tumorigenesis, few p53 and no EGFR or KRAS gene mutations have been detected, although the percentage of lung carcinoids expressing EGFR is higher in TCs than in ACs.^9,10 Previous studies have primarily demonstrated multiple endocrine neoplasia type 1 (MEN1) gene mutations and/or chromosome 11q deletions.^11–17 MEN1 is a syndrome in which an inherited mutation in the MEN1 gene, located at 11q13, predisposes to formation of multiple neuroendocrine tumors. Although formation of bronchial carcinoid tumors has been observed in only 2% of patients with MEN1,¹⁸ functional inactivation of menin, the MEN1 gene product, has been implicated in the tumorigenesis of sporadic lung carcinoids. In these bronchial carcinoids not associated with MEN1 syndrome, the frequency of loss of heterozygosity at 11q (36%) is higher than the somatic MEN1 mutation rate (18%), pointing to the presence of other tumor-suppressor genes at this chromosome arm and/or involvement of epigenetic silencing mechanisms.¹²To improve the discrimination between pulmonary carcinoid tumors with a favorable or poor prognosis and to identify critical genetic events in lung carcinoid tumorigenesis, we investigated 34 reclassified bronchial carcinoids by using array-based comparative genomic hybridization (array CGH) with a resolution of ≤1 Mb (megabase). Fluorescence in situ hybridization (FISH) was used to determine chromosome copy numbers and to validate array CGH data. Furthermore, the array CGH data were correlated with available histopathologic data and long-term clinical follow-up.     

Interstitial deletion of 11q13 sequences in HeLa cells

Previous cytogenetic and molecular genetic studies have shown that the HeLa (cervical carcinoma) cell line D98/AH-2 contains two apparently normal copies of chromosome 11 and additional 11q13-25 material translocated onto a chromosome 3 marker. To determine the 11q13 breakpoint, we performed fluorescence in situ hybridization (FISH) using 18 different 11q13 specific BAC (bacterial artificial chromosome) and cosmid probes spanning a 5.6 Mb interval. Markers localized to the multiple endocrine neoplasia type 1 (MEN1) gene (menin) were also included in the analysis. The FISH study identified an interstitial deletion between markers D11S449 and GSTP1, an interval of 2.3 Mb, in the marker chromosome. This deletion did not include the MEN1 gene. Because point mutations and methylations can inactivate the MEN1 gene, single stranded conformational polymorphism (SSCP) and Northern and Western blot analyses were performed with MEN1 specific probes and antibody. SSCP did not reveal mutations of the MEN1 gene in HeLa or in seven other cervical cancer cell lines. Northern and Western blot studies revealed normal levels of expression of this gene in the cervical cancer cell lines as well as in HeLa cell derived tumorigenic hybrids. Because deletions of tumor suppressor genes often occur in cancer progression, we hypothesize that the inactivation of a tumor suppressor gene other than MEN1, localized to the 2.3 Mb interval on 11q13, might play a role in the abnormal growth behavior of HeLa cells in vitro or in vivo.     

Allelic losses on chromosome band 11q13 in aldosterone-producing adrenal tumors

We examined loss of heterozygosity (LOH) in 14 aldosterone-producing adrenal tumors, with six linearly ordered restriction fragment length polymorphism (RFLP) markers that map within a 12-cM region containing the MEN 1 locus on 11q13. Among 11 tumors that were informative for at least one marker, five showed LOH at one or more loci, and two distinct regions of deletion were identified. The proximal region overlapped with the location of the MEN 1 locus previously predicted by linkage analyses in MEN 1 families and the commonly deleted region in hyperparathyroid tumors. This suggests that one of the genes associated with development of aldosterone-producing adrenal tumors may coincide with the MEN 1 locus, and that a second gene, distal to the MEN 1 locus, may also play a role in the development of this type of tumor.     

Independent genetic events associated with the development of multiple parathyroid tumors in patients with primary hyperparathyroidism

Multiple parathyroid tumors, as opposed to hyperplasia, have been reported in a subset of patients with sporadic primary hyperparathyroidism (PHPT). It is not clear whether these multiple tumors are representative of a neoplastic process or whether they merely represent hyperplasia that has affected the parathyroid glands differentially and resulted in asynchronous growth. The molecular genetic techniques of comparative genomic hybridization (CGH), loss of heterozygosity (LOH), and MEN1 mutation analysis were performed on a series of five patients with multiglandular PHPT, each of which had two parathyroid tumors removed. Analysis of these multiple parathyroid tumors from patients with PHPT revealed that independent genetic events were associated with the development of a subset of these tumors. The DNA sequence copy number changes, identified by CGH analyses, either involved different chromosomal regions in the paired glands of a patient (two patients), or those regions implicated in one gland were not changed in a second gland from the same patient (two patients). Each of the three patients exhibiting LOH demonstrated different changes between the paired glands. Where LOH was detected in one gland from a patient, the other gland from the same patient either exhibited no allelic loss or the loss detected was in another region. Each of the three tumors exhibiting LOH at 11q13 was found to contain a somatic MEN1 mutation in the remaining allele, however these mutations were not present in the germline or in the paired gland from the same patient. Although it is possible that a separate series of genetic changes has arisen randomly in two separate glands within the same individual, it seems more likely that the development of these multiple tumors has arisen because of the involvement of other unknown factors. These factors may be genetic [such as the involvement of one or more germline mutations in an unknown low-penetrance gene(s), germline mosaicism or alterations in calcium-sensing receptor gene(s)], epigenetic, physiological, or environmental.     

Alterations of the SDHD gene locus in midgut carcinoids,Merkel cell carcinomas,pheochromocytomas, and abdominal paragangliomas

Several types of endocrine tumors show frequent somatic deletions of the distal part of chromosome arm 11q, where the tumor-suppressor gene SDHD (succinate-ubiquinone oxidoreductase subunit D), constitutionally mutated in paragangliomas of the head and neck, is located. In this study, we screened 18 midgut carcinoids, 7 Merkel cell carcinomas, 46 adrenal pheochromocytomas (37 sporadic and 9 familial), and 7 abdominal paragangliomas for loss of heterozygosity (LOH) and/or mutations at the SDHD gene locus. LOH was detected in 5 out of 8 (62%) informative midgut carcinoids, in 9 out of 30 (30%) sporadic pheochromocytomas, in none of the familial pheochromocytomas (0%), and in 1 out of 6 (17%) abdominal paragangliomas. No sequence variants were detected in the pheochromocytomas or paragangliomas. However, two constitutional putative missense mutations, H50R and G12S, were detected in two midgut carcinoids, which were both associated with LOH of the other allele. The same sequence variants were also detected in two Merkel cell carcinomas. In addition, the S68S polymorphism was found to coexist with the G12S sequence variant in both cases. In conclusion, we show that alterations of the SDHD gene seem to be involved in the tumorigenesis of both midgut carcinoids and Merkel cell carcinomas.     

Six novel MEN1 gene mutations in sporadic parathyroid tumors

We report nine mutations of the multiple endocrine neoplasia type 1 (MEN1) gene in sporadic parathyroid adenomas. Six of them have not previously been described: E60X, P32R, 261delA, 934+2T-->G, S443P, and 1593insC. The tissue samples were initially submitted to LOH analysis at 11q13 followed by SSCP screening of LOH-positive samples. Mutations were identified by direct sequencing and subcloning. Three (E60X, P32R, and 261delA) were in exon 2, one (934+2bp) in the splice junction of exon 5, one (S443P) in exon 9, and one (1593insC) in exon 10. The 3 mutations in exon 2 were associated with loss and/or creation of a restriction site. The corresponding germline sequence of the MEN1 gene was normal. Most mutations would likely result in a nonfunctional menin protein, and therefore in the loss of a tumor suppressor protein.     

Comparative genomic hybridization analysis of thymic neuroendocrine tumors.

Thymic neuroendocrine (carcinoid) tumors are a rare neoplasm of the anterior mediastinum. The tumors frequently exhibit a wide spectrum of histology and appear to follow a more aggressive behavior than their nonthymic counterparts. Given the differing clinicopathologic manifestations, thymic neuroendocrine tumors may also possess different cytogenetic abnormalities from those that occur in foregut carcinoid tumors. In this study, we employed comparative genomic hybridization to detect genomic instability in 10 sporadic thymic neuroendocrine tumors and one multiple endocrine neoplasia type 1 (MEN1)-associated case. Gross chromosomal imbalances were found in nine cases, including gains of chromosomal material on regions X, 8, 18 and 20p and losses on 3, 6, 9q, 13q and 11q. We did not observe deletion at locus 11q13 where the MEN1 gene is located. These findings were essentially dissimilar to those reported in sporadic and MEN1-associated foregut carcinoid tumors. Consequently, we consider that a distinctive cytogenetic mechanism is at work in the development of thymic neuroendocrine tumors, which is different from that of foregut carcinoid tumors.