Signet ring cell differentiation in mucinous colorectal carcinoma.

Approximately 10% of all colorectal carcinomas are mucinous carcinomas, characterized by extracellular mucin. Occasionally, mucin accumulates intracellularly in these tumours, causing signet ring cell differentiation. We hypothesized that signet ring cells arise from a separate genetic pathway. In this study the molecular background of signet ring cell differentiation was investigated by analysing genetic changes, changes in the expression of adhesion molecules, and mucin content. Furthermore, its clinical relevance was addressed. Cell lines of colorectal tumours with non-mucinous (AC), mucinous (MC), and signet ring cell phenotype (MCSRC) were used for Multiplex Ligation-dependent Probe Amplification to detect deletions and amplifications in specific oncogenes and tumour suppressor genes. Furthermore, the expression of E-cadherin, beta-catenin, ITF (intestinal trefoil factor), and MUC2 in signet ring cells was studied by immunohistochemistry, immunofluorescence, and mRNA in situ hybridization. Results were validated using a large cohort of rectal carcinomas from which clinicopathological data were available. Specific amplifications and deletions in cell lines of AC, MC, and MCSRC were detected. Bcl-2 was amplified in MCSRC and MC cell lines, but not in AC cell lines. Bcl-2 FISH analysis confirmed this in patient material. Signet ring cells had decreased expression of adhesion molecules (E-cadherin, beta-catenin) and were strongly positive for ITF and MUC2, two peptides which are normally only produced by goblet cells. RNA in situ hybridization confirmed the production of ITF. Mucinous carcinomas with signet ring cell differentiation presented at a higher T stage than adenocarcinomas and mucinous carcinomas (16% pT4 versus 3-5%, p<0.001) and were more frequently node positive (77% vs 39-44%; p<0.001). Prognosis was significantly worse. In conclusion, the presence of signet ring cells in carcinomas with mucinous differentiation correlates with increased T-stage and poor prognosis. These cells, characterized by ITF and MUC2 production, show disruption of the E-cadherin/beta-catenin complex, as well as amplification of Bcl-2.

Characteristic chromosomal aberrations in sporadic cerebellar hemangioblastomas revealed by comparative genomic hybridization.

Hemangioblastomas (HBs) of the central nervous system are benign tumors and occur as sporadic (sp) tumors (75%) or as a manifestation of the von Hippel-Lindau (VHL) disease (25%). VHL-disease is an autosomal dominant disorder characterized by HBs of the central nervous system and retina, renal cell carcinoma (RCC), phaeochromocytoma (PHEO), islet tumors of the pancreas, and endolympatic sac tumors as well as cysts and cystadenoma in the kidney, pancreas and epididymis. In VHL patients a large spectrum of germline mutations in the VHL gene has been detected. In spHBs VHL alleles are reported to be inactivated in up to 50% of the tumors. To our knowledge the involvement of other genes in spHBs has not been investigated. To elucidate the oncogenesis of spHBs, we performed CGH on 10 spHBs to screen for chromosomal imbalances throughout the entire tumor genome. Aberrations most frequently detected are losses of chromosomes 3 (70%), 6 (50%), 9 (30%), and 18q (30%) and a gain of chromosome 19 (30%). Based on these frequencies and the co-occurrence of these aberrations in the analyzed tumors we hypothesize that loss of chromosome 3 (harboring the VHL gene) is an early event in the oncogenesis of spHBs, followed by loss of 6, and then losses of chromosomes 9, 18q and gain of chromosome 19. Comparison of the chromosomal imbalances in spHBs to those previously reported in RCCs and PHEOs reveals that the pathway of spHBs shows similarities to both the RCCs and PHEOs.

The relationship between genetic aberrations as detected by comparative genomic hybridization and vascularization in glioblastoma xenografts.

Angiogenesis is of vital importance for the growth of solid tumors and constitutes a target for anti-cancer therapy. Glioblastomas (GBMs) are histologically characterized by striking microvascular proliferation. The identification of the mechanism of angiogenesis is of major importance for the further development of anti-angiogenic therapy. Tumor angiogenesis might be the result of a combination of local tissue conditions (especially hypoxia) and specific genetic alterations acquired during oncogenesis. In order to investigate the relationship between genetic aberrations and tumor angiogenesis in GBM xenograft lines, the genetic alterations were examined by Comparative Genomic Hybridization (CGH). Two vascular phenotypes of GBM xenografts could be identified: a well vascularized and a poorly vascularized type. In this model, the poorly vascularized type had a larger number of genetic alterations. However, there was no unequivocal correlation between angiogenesis, growth rate and patterns of genetic alterations as detected by CGH.

Subtyping of oligo-astrocytic tumours by comparative genomic hybridization.

Oligo-astrocytic tumours (OAs) histologically show both oligodendroglial and astrocytic differentiation. Unequivocal criteria for delineation of OAs from pure oligodendroglial (Os) and astrocytic (As) tumours and for grading of OAs are lacking. Molecular genetic analysis may allow for a better characterization of OAs and thereby guide prognostic and therapeutic decisions. Comparative genomic hybridization (CGH) was performed on 39 gliomas with variable phenotypic expression of histological features characteristic of both astrocytic and oligodendroglial differentiation. The results show that OAs are genetically more heterogeneous than Os. In addition to the "-1p/-19q" and "+7/-10" subtypes that have been previously recognized, two additional genetic subtypes, "intermediate" and "other", were identified in the present study. "Intermediate" OAs likely represent progression from "-1p/-19q" tumours. The "other" subtype appears to represent an additional, heretofore unrecognized, genetic pathway(s). Application of rigorously "strict" histopathological criteria, as opposed to "relaxed" criteria, for the selection of oligo-astrocytic tumours resulted in a higher percentage of "-1p/-19q" tumours, but some "-1p/-19q" tumours might be missed. The results suggest that molecular genetic analysis is a useful and valid additional tool for the classification of gliomas, particularly for the significant subset of tumours in which subjective histopathological criteria are insufficient for an unequivocal distinction between Os, As, and mixed OAs.

Molecular analysis of a familial case of renal cell cancer and a t(3;6)(q12;q15).

We identified a novel familial case of clear-cell renal cancer and a t(3;6)(q12;q15). Subsequent cytogenetic and molecular analyses showed the presence of several abnormalities within tumour samples obtained from different patients. Loss of the der(3) chromosome was noted in some, but not all, of the samples. A concomitant VHL gene mutation was found in one of the samples. In addition, cytogenetic and molecular evidence for heterogeneity was obtained through analysis of several biopsy samples from one of the tumours. Based on these results and those reported in the literature, we conclude that loss of der(3) and subsequent VHL gene mutation may represent critical steps in the development of renal cell cancers in persons carrying the chromosome 3 translocation. Moreover, preliminary data suggest that other (epi)genetic changes may be related to tumour initiation.

Hypoxia in a human intracerebral glioma model.

OBJECT: The development of hypoxia in human gliomas is closely related to functional vasculature and the presence of hypoxia has important biological and therapeutic consequences. Assessment of hypoxia is necessary to understand its role in treatment response and to evaluate treatment strategies to improve tumor oxygenation. In this study, the authors report findings of their analysis of the degree of hypoxia in relation to other vascular parameters in a human intracerebral glioma xenograft. METHODS: In sections of tumor, hypoxic regions were identified immunohistochemically by using the hypoxic marker pimonidazole. The S-phase marker bromodeoxyuridine was used to detect cell proliferation, and the perfusion marker Hoechst 33342 was used to delineate perfused vessels. Vascular structures were stained with an endothelial marker. Hypoxic tumor regions were clearly present in this human intracerebral glioma model. Hypoxic areas were usually found in nonperfused regions, whereas tumor cell proliferation was especially marked in perfused tumor areas. Furthermore, by using in situ hybridization the authors identified infiltrating tumor cells in the normal brain. This feature is often observed in gliomas in patients. CONCLUSIONS: This model is a representative human glioma model that provides the researcher with the opportunity to analyze the relationship between the degree of hypoxia and vascular parameters, as well as to examine the effects of treatments aimed at modification of the oxygenation status of a tumor.

PTEN mutation analysis in two genetic subtypes of high-grade oligodendroglial tumors. PTEN is only occasionally mutated in one of the two genetic subtypes.

We recently identified two genetic subtypes of high-grade oligodendroglial tumors (HG-OT): 1p-/19q- HG-OT are characterized by a loss of chromosome 1p32-36 (del(1)(p32-p36) and/or a del(19)(q13. 3); whereas +7/-10 HG-OT harbor a gain of chromosome 7 (+7) and/or a -10 without a loss of 1p32-36 and 19q13.3. Because a -10 and a +7 are most frequently detected in glioblastomas (GBM), the genotype of +7/-10 HG-OT suggests that these tumors are GBM with a prominent oligodendroglial phenotype rather than anaplastic oligodendrogliomas. PTEN is a tumor suppressor gene, located at 10q23.3, which is involved in tumor progression of GBM and other neoplasms. In this study, we screened for PTEN mutations in six low-grade oligodendroglial tumors (LG-OT), five 1p-/19q- HG-OT, seven +7/-10 HG-OT, and nine xenografted GBM. PTEN mutations were detected in none of the LG-OT and 1p-/19q- HG-OT, once in +7/-10 HG-OT, and frequently in GBM. As one of the +7/-10 HG-OT harbored a PTEN mutation, this demonstrates that PTEN can be involved in the oncogenesis of this genetic subtype of HG-OT. The lower frequency of PTEN mutations in +7/-10 HG-OT compared to GBM suggests that these tumors are of a distinct tumor type rather than GBM. Published by Elsevier Science Inc.

Frequent loss of 9p21 (p16(INK4A)) and other genomic imbalances in human malignant fibrous histiocytoma.

To search for new recurrent genetic aberrations in malignant fibrous histiocytoma (MFH), a combination of conventional cytogenetic, comparative genomic hybridization (CGH), and Southern blot analyses was applied to a series of 34 tumors. Cytogenetic analysis revealed the presence of multiple structural and numerical aberrations, including marker chromosomes, telomeric associations, double minutes, and ring chromosomes. The most frequent genomic imbalances in this series of neoplasms as detected by CGH were gains of 1q21-q22 (69%), 17q23-qter (41%), and 20q (66%), and losses of 9p21-pter (55%), 10q (48%), 11q23-qter (55%), and 13q10-q31 (55%). Southern blot analyses with p16(INK4A) (CDKN2A; 9p21) and RB1 (13q14) probes provided clear indications for frequent deletions of these tumor suppressor genes, and as such, substantiated the CGH results. Additionally, examination of the TP53 and MDM2 genes showed frequent loss and amplification, respectively. These data indicate that genes involved in the RB1- and TP53-associated cell cycle regulatory pathways may play prominent roles in the development of human MFH.

Genetic reflection of glioblastoma biopsy material in xenografts: characterization of 11 glioblastoma xenograft lines by comparative genomic hybridization.

OBJECT: Human tumors implanted as subcutaneous xenografts in nude mice are widely used for the study of tumor biology and therapy. Validation of these models requires knowledge of the genetic makeup of the xenografts. The aim of this study was to establish whether chromosomal imbalances in 11 xenograft lines derived from human glioblastomas multiforme (x-GBMs) are similar to those found in GBM biopsy samples. The authors also studied genetic stability during serial passaging of three xenograft lines. METHODS: Chromosomal imbalances in x-GBMs were detected using comparative genomic hybridization (CGH). The authors compared the CGH results in x-GBMs with those in the original GBMs (o-GBMs) that were used to establish three of the xenograft lines and with the GBM biopsy results reported in the literature (1-GBMs). In three xenograft lines two different passages were analyzed. CONCLUSIONS: The results show that the chromosomal imbalances in x-GBMs are similar to those in o-GBMs and 1-GBMs, indicating that the GBM xenograft lines used were valid models from a genetic point of view. The CGH analysis of two different passages of three xenograft lines indicates that x-GBMs (like 1-GBMs) show intratumoral genetic heterogeneity and do not acquire chromosomal imbalances as a result of serial passaging.

Comparative genomic hybridization of medulloblastomas and clinical relevance: eleven new cases and a review of the literature.

Medulloblastomas are highly malignant primitive neuroectodermal tumors of the cerebellum that display a wide variety of histopathological patterns. However, these patterns do not provide an accurate prediction of clinical-biological behavior and no satisfactory morphological grading system has ever been presented. Genetic alterations may provide additional diagnostic information and allow clinically relevant subgrouping of primitive neuroectodermal tumors. We examined 10 medulloblastomas and one medulloblastoma cell line. One amplification site on chromosome 8q24 was detected in the cell line corresponding to the known amplification of the c-myc gene in this cell line. The gain of 2p21-24 in two tumors was shown to represent amplification of the N-myc gene by Southern blot hybridization and fluorescence in situ hybridization. The data show that the isochromosome 17 can be recognized using comparative genomic hybridization (CGH) by the typical combination of loss of 17p combined with gain of 17q. No specific pattern of genetic alterations could be linked to the clinical behavior of the tumors. We have compared our results with previous CGH studies on medulloblastomas.

Isolation and characterization of glioblastoma-associated homozygously deleted DNA fragments from chromosomal region 9p21 suggests involvement of multiple tumour suppressor genes.

Representational difference analysis (RDA) of a human glioblastoma xenograft resulted in the isolation of five tumour-associated homozygously deleted DNA fragments, all originating from chromosome 9, region p21. Subsequent analysis of a series of ten glioblastomas using the newly isolated RDA fragments in conjunction with a series of known 9p21 DNA markers revealed homozygous deletions in nine of the ten (90 per cent) tumours. These deletions encompass the p15/p16 complex and two additional putative tumour suppressor loci. The RDA fragments correspond to the latter two loci. Taken together, these results suggest the involvement of multiple tumour suppressor genes from the 9p21 region in glioblastoma tumourigenesis. The novel RDA fragments will be instrumental in the isolation of the relevant genes.

Identification of subgroups of high-grade oligodendroglial tumors by comparative genomic hybridization.

In contrast to astrocytic tumors, approximately two thirds of anaplastic oligodendrogliomas are reported to be chemosensitive. Relatively little is known about the genetic aberrations in oligodendroglial tumors (OTs). In order to elucidate oligodendroglial oncogenesis and to find specific genetic aberrations that may have prognostic and therapeutic implications, we performed comparative genomic hybridization (CGH) to detect chromosomal copy number changes in 17 low-grade OTs (LG-OTs) and 12 high-grade OTs (HG-OTs) lacking a prominent astrocytic component. Loss of chromosome 1p (79%) and 19q (76%) were most frequently detected by CGH, all LG-OTs and 50% of the HG-OTs contained -1p (including 1p36-32), -19q (including 19q13.3), or both, and the rest of the HG-OTs showed +7, -10, or both. Since losses of 1p36-32 and 19q13.3 were mutually exclusive with +7 or -10, the HG-OTs could be divided in -1p/-19q and +7/-10 tumors. While the -1p/-19q tumors can be considered as pure anaplastic oligodendrogliomas, the +7/-10 tumors may rather be glioblastomas with prominent oligodendroglial differentiation. We conclude that CGH is a powerful tool to assist in the identification of 2 major subgroups of HG-OTs with prognostic and possibly therapeutic relevance.

Personality variables involved in chronic prostatitis.

Psychologic factors have been considered to play an important role in the etiology of chronic prostatitis. Earlier studies are often based on a psychoanalytical perspective and seldomly used quantitative approaches. In the present study quantitative tests are used to investigate personality variables which are suggested in the literature as underlying chronic prostatitis. A group of 50 chronic prostatitis patients was compared with a group of 50 patients seen for a vasectomy. Psychologic measures were taken by means of a personality inventory (NVM, Dutch short form of the MMPI), a symptom checklist (SLC-90), and a depression inventory (IDD). Results showed statistically significant differences between the groups, with the chronic prostatitis patients scoring consistently higher on the measures than vasectomy patients. However, these differences in scores were not of a great magnitude and minor compared with differences in scores from psychiatric patients. Discriminant analysis suggested somatization and depression to be the key variables to distinguish chronic prostatitis patients from vasectomy patients. Overall, it seemed unfounded to label chronic prostatitis patients "neurotic" or "psychopathologic," and it was impossible to conclude that there are personality variables that specifically identify the chronic prostatitis patients.