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.

The pathogenesis of sporadic endocrine pancreatic tumors (EPTs) is still primarily unknown. Comparative genomic hybridization studies revealed loss of 10q in a significant number (nine of 31) of EPTs. The tumor suppressor gene PTEN lies on 10q23, and so, is a candidate to play some role in EPT pathogenesis. Germline PTEN mutations are found in Cowden and Bannayan-Riley-Ruvalcaba syndromes, whereas somatic mutations and deletions are found in a variety of sporadic cancers. The mutation and expression status of PTEN in EPTs has not yet been examined. Mutation analysis of the entire coding region of PTEN including splice sites was performed in 33 tumors, revealing one tumor with somatic L182F (exon 6). Loss of heterozygosity of the 10q23 region was detected in eight of 15 informative malignant (53%) and in none of seven benign EPTs. PTEN expression was assessed in 24 available EPTs by immunohistochemistry using a monoclonal anti-PTEN antibody. Of these 24, 23 tumors showed strong immunoreactivity for PTEN. Only the EPTs with PTEN mutation lacked PTEN protein expression. Although normal islet cells always exhibited predominantly nuclear PTEN immunostaining, 19 of 23 EPTs had a predominantly cytoplasmic PTEN expression pattern. Exocrine pancreatic tissue was PTEN-negative throughout. PTEN mutation is a rare event in malignant EPTs and PTEN protein is expressed in most (23 of 24) EPTs. Thus, intragenic mutation or another means of physical loss of PTEN is rarely involved in the pathogenesis of EPTs. Instead, either an impaired transport system of PTEN to the nucleus or some other means of differential compartmentalization could account for impaired PTEN function. Loss of heterozygosity of the 10q23 region is a frequent event in malignant EPTs and might suggest several hypotheses: a different tumor suppressor gene in the vicinity of PTEN might be principally involved in EPT formation; alternatively, 10q loss, including PTEN, seems to be associated with malignant transformation, but the first step toward neoplasia might involve altered subcellular localization of PTEN.

The molecular pathogenesis as well as histogenesis of endocrine pancreatic tumors (EPTs) is not well understood , and the clinical behavior of EPTs is difficult to predict using current morphological criteria. Thus, more accurate markers of risk and better understanding of tumor initiation and progression are needed to allow a precise classification of EPTs. We have studied 44 benign and malignant EPTs by comparative genomic hybridization to correlate the overall number of genetic alterations with clinical and histopathological parameters and to identify chromosomal regions which might harbor genes involved in EPT pathogenesis and progression. Aberrations were found in 36 EPTs , and chromosomal losses (mean, 5.3) were slightly more frequent than gains (mean, 4.6). The most frequent losses involved Y (45% of male EPTs) , 6q (39%) , 11q (36%) , 3p , 3q , 11p (each 30%) , 6p (27%) , and 10q and Xq (each 25%) , whereas most common gains included 7q (43%) , 17q (41%), 5q and 14q (each 32%) , 7p , 9q , 17p , 20q (each 27%), and 12q and Xp (each 25%). A correlation was found between the total number of genetic changes per tumor and both tumor size and disease stage. In particular , losses of 3p and 6 and gains of 14q and Xq were found to be associated with metastatic disease. Furthermore , characteristic patterns of genetic changes were found in the various EPT subtypes , eg , 6q loss in malignant insulinomas , indicating that these groups might evolve along genetically different pathways. The highlighted genetic aberrations , including the newly found involvement of 6q losses and sex chromosome alterations , should stimulate the further analysis of these chromosomal regions, which may lead to the discovery of novel genes important in the tumorigenesis and evolution of EPTs. (Am J Pathol 1999, 155:1787-1794)

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.

Despite several loss of heterozygosity studies, a comprehensive genomic survey of pheochromocytomas is still lacking. To identify DNA copy number changes which might be important in tumor development and progression and which may have diagnostic utility, we evaluated genetic aberrations in 29 sporadic adrenal and extra-adrenal pheochromocytomas (19 clinically benign tumors and 10 malignant lesions). Comparative genomic hybridization was performed using directly fluorochrome-conjugated DNA extracted from frozen (16) and paraffin-embedded (13) tumor tissues. The most frequently observed changes were losses of chromosomes 1p11-p32 (86%), 3q (52%), 6q (34%), 3p, 17p (31% each), 11q (28%), and gains of chromosomes 9q (38%) and 17q (31%). No amplification was identified and no difference between adrenal and extra-adrenal tumors was detected. Progression to malignant tumors was strongly associated with deletions of chromosome 6q (60% versus 21% in clinically benign lesions, P = 0.0368) and 17p (50% versus 21%). Fluorescence in situ hybridization confirmed the comparative genomic hybridization data of chromosomes 1p, 3q, and 6q, and revealed aneuploidy in some tumors. Our results suggest that the development of pheochromocytomas is associated with specific genomic aberrations, such as losses of 1p, 3q, and 6q and gains of 9q and 17q. In particular, tumor suppressor genes on chromosomes 1p and 3q may be involved in early tumorigenesis, and deletions of chromosomes 6q and 17p in progression to malignancy.

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Neuroendocrine tumors (carcinoids) are a heterogeneous group of neoplasms arising from the diffuse neuroendocrine system. Genetic changes underlying their tumorigenesis are primarily unknown. We used comparative genomic hybridization to screen 32 well-differentiated neuroendocrine tumors (21 gastrointestinal and 11 bronchial) and three associated metastases for genomic alterations. There were striking differences of genomic imbalances between the two subgroups of neuroendocrine tumors. Losses of chromosome 18q and 18p were shown in eight (38%) and seven (33%), respectively, out of 21 gastrointestinal tumors and in none of the 11 bronchial tumors. Conversely, deletions of 11q occurred in four of 11 (36%) bronchial tumors but only in one gastrointestinal tumor. These comparative genomic hybridization findings were confirmed by interphase cytogenetics. Our data indicate that neuroendocrine tumors of the two subgroups develop via different molecular pathways. Inactivation of one or several tumor suppressor genes on chromosome 18 may be important for the biological behavior of gastrointestinal tumors, whereas gene inactivation on 11q seems to be associated with tumor development of the bronchi.

IGFII and MIB1 are helpful immunohistochemical markers to predict malignancy in adrenocortical neoplasms. These markers can be used in addition to clinical, gross and morphological features to establish a diagnosis in difficult cases.