Comparative Genomic Hybridization Analysis of Astrocytomas: Prognostic and Diagnostic Implications.

Astrocytoma is comprised of a group of common intracranial neoplasms that are classified into four grades based on the World Health Organization histological criteria and patient survival. To date, histological grade, patient age, and clinical performance, as reflected in the Karnofsky score, are the most reliable prognostic predictors. Recently, there has been a significant effort to identify additional prognostic markers using objective molecular genetic techniques. We believe that the identification of such markers will characterize new chromosomal loci important in astrocytoma progression and aid clinical diagnosis and prognosis. To this end, our laboratory used comparative genomic hybridization to identify DNA sequence copy number changes in 102 astrocytomas. Novel losses of 19p loci were detected in low-grade pilocytic astrocytomas and losses of loci on 9p, 10, and 22 along with gains on 7, 19, and 20 were detected in a significant proportion of high-grade astrocytomas. The Cox proportional hazards statistical modeling showed that the presence of +7q and -10q comparative genomic hybridization alterations significantly increased a patient\u27s risk of dying, independent of histological grade. This investigation demonstrates the efficacy of comparative genomic hybridization for identifying tumor suppressor and oncogene loci in different astrocytic grades. The cumulative effect of these loci is an important consideration in their diagnostic and prognostic implications

Effect Of Methionine Oxidation And Deletion Of Amino-Terminal Residues On The Conformation Of Parathyroid Hormone. Circular Dichroism Studies.

Circular dichroism (CD) studies of parathyroid hormone (PTH), its oxidized forms, and some fragments of the hormone are described. The CD spectrum of native PTH (84 amino acids) and the active fragment, 1-34 PTH, suggests that most of the secondary structure resides in the amino-terminal segment of this hormone. Oxidation of the methionine residue at position 18 has a small impact on secondary structure, whereas oxidation of the methionine at position 8 produces substantial changes. Oxidation of both methionines produces secondary structure changes that are greater than the sum of those seen upon oxidation of the individual methionines. The CD spectrum for the 3-34 fragment of PTH is identical to that of the 1-34 fragment, and that of the 7-34 fragment is only slightly different. The spectra of the 13-34 and 19-34 fragments are markedly altered from that of the 1-34 peptide, and those of the 9-84 and 19-84 fragments of native PTH are significantly different from the intact hormone. Computer-assisted estimates of secondary structure content, and difference spectra, were utilized to evaluate the secondary structure content of the peptides. These results suggest that residues 6-12 are important in formation of helical secondary structure and that a reverse turn may be important for the folding of PTH into a conformation with high affinity for receptors. Residues 1 and 2 appear to make no contribution to the secondary structure and may be directly involved in activation of receptors

Biphasic Malignant Meningioma: A Comparative Genomic Hybridization Study.

To ascertain if a carcinoma-like component within a fibroblastic meningioma represented a metastatic carcinoma to a meningioma or malignant progression, we employed traditional immunohistochemical methods as well as comparative genomic hybridization (CGH) which compares chromosomal alterations. Vimentin and epithelial membrane antigen were strongly immunoreactive in both the fibroblastic and carcinoma-like components. The CGH profile in both components had similar chromosomal alterations, including losses of 1p, 14, 16p13--\u3ep10 and 22. However, the CGH profiles from the fibroblastic component showed losses of 4p, 10q23--\u3eq24 and 18, along with gains of 1q, 6q25--\u3eqter and 13q32--\u3eqter. The profile of the carcinoma-like component showed losses of chromosome 4, in addition to gains of 3p12--\u3eq13.11, 5q14.3--\u3eq23.2, 6pter--\u3ep23, and 13q14.2--\u3eqter. CGH analysis of a biphasic malignant meningioma confirmed that the disparate histologic components were genetically related and likely derivative from a common precursor, demonstrating genetic instability and clonal expansion. Furthermore, CGH showed that the histologically appearing low-grade fibroblastic component had not solely the characteristic alterations of a benign meningioma but had already progressed to an atypical meningioma

Morphologic and Molecular Genetic Aspects of Oligodendroglial Neoplasms.

Morphologic criteria for diagnosing oligodendrogliomas and for classifying them as well-differentiated (World Health Organization grade II) and anaplastic (World Health Organization grade III) are well recognized. Nevertheless, applying these guidelines to specific cases often reveals discrepancies among different observers. In addition, whether a given tumor also contains an astrocytic component may be debatable. Loss of heterozygosity studies have demonstrated that oligodendroglial neoplasms have a high incidence of loss of the 1p and 19q chromosomal arms. Although loss of heterozygosity for portions of 19q are sometimes seen in astrocytic neoplasms, these tumors seldom show complete loss of 19q accompanied by loss of 1p. Loss of 9p or homozygous deletion of the CDKN2 gene or both are associated with anaplastic oligodendrogliomas, whereas loss of 17p or TP53 gene mutations or both are frequent in astrocytomas, but rare in oligodendrogliomas. These observations suggest that molecular genetic parameters could provide an objective, reproducible framework for classifying oligodendroglial neoplasms

Molecular Pathogenesis of Malignant Gliomas.

De novo glioblastomas develop in older patients without prior clinical history of less malignant tumors. Progressive glioblastomas are common among younger patients and arise through progression from lower-grade astrocytomas. CDKN2A deletions, PTEN alterations, and EGFR amplification are more prevalent among de novo glioblastomas, whereas p53 mutations are more common among progressive glioblastomas. Loss of heterozygosity (LOH) for chromosome 10 is seen uniformly among both de novo and progressive high-grade astrocytomas. The inactivation of the PTEN gene is found in approximately 30% to 40% of astrocytomas with chromosome 10 loss, and LOH pattern in the remaining astrocytomas strongly supports the presence of another yet unidentified tumor suppressor gene telomeric to PTEN. More than 80% of oligodendrogliomas exhibit LOH for 1 p and 19q alleles. Oligoastrocytomas with 1p/19q LOH are related to oligodendrogliomas, and those with p53 mutations are related to astrocytomas

Application of Molecular Cytogenetic Techniques in a Case Study of Human Cutaneous Metastatic Melanoma.

Consistent structural chromosome rearrangements have rarely been identified in adult solid tumors. The introduction of advanced molecular cytogenetic techniques has provided new ways of analyzing highly complex karyotypes commonly encountered in these malignancies. This study describes a detailed molecular cytogenetic analysis of a sporadic human cutaneous melanoma biopsy, M92-047, using a combination of G-banding, fluorescence in situ hybridization (FISH), chromosome microdissection, and comparative genomic hybridization (CGH). G-banding revealed that this tumor was composed primarily of closely related near-diploid and near-tetraploid cell subpopulations containing several clonal numerical and structural chromosome alterations. Fluorescence in situ hybridization using whole chromosome painting probes and chromosome arm painting probes, was employed to verify the rearranged chromosomes; dic(1;4), der(8)t(1;8), and der(15)t(6;15), whereas marker chromosomes dic(8;1;16), der(12)t(9;12), and der(17)t(13;17) were discerned by chromosome microdissection and subsequent reverse in situ hybridization (rev ish) analysis. Comparative genomic hybridization illustrated DNA copy number changes in good agreement with the karyotypic analysis. Although this line exhibits recurrent alterations representative of melanoma, two unique breakpoints--1p13 and 8p21--were identified in two different rearranged chromosomes, suggesting potentially important regions for further dissection by molecular genetic techniques. This report demonstrates the advantages of combining multiple techniques in order to obtain a detailed description of cytogenetic changes in melanoma

The Gene For The APC-Binding Protein Beta-Catenin (CTNNB1) Maps To Chromosome 3p22, A Region Frequently Altered In Human Malignancies.

beta-Catenin is one of the E-cadherin associated proteins involved in the process of cellular adhesion. It has recently been shown to interact with the APC protein whose gene is known to be mutated in the germline of familial adenomatous polyposis patients. This interaction implies that beta-catenin is a potential regulator of the APC gene. The localization of the human beta-catenin gene (CTNNB1) to chromosome 3p22, by fluorescent in situ hybridization (FISH), has linked the gene to a region that is frequently altered in several human malignancies. The location of the gene and the protein interactions suggest the importance of beta-catenin in the etiology of various human cancers

Comparative Genetic Patterns of Glioblastoma Multiforme: Potential Diagnostic Tool for Tumor Classification.

Cytogenetic and molecular genetic studies of glioblastoma multiforme (GBM) have shown that the most frequent alterations are gains of chromosome 7, losses of 9p loci and chromosome 10, and gene amplification, primarily of the epidermal growth factor receptor (EGFR) gene. Although this profile is potentially useful in distinguishing GBM from other tumor types, the techniques used tend to be labor intensive, and some can detect only gains or losses of genetic loci. Comparative genomic hybridization (CGH) is a powerful technique capable of identifying both gains and losses of DNA sequences. The present study compares the CGH evaluation of 22 GBM with classic cytogenetics, loss of heterozygosity by allelotyping, and gene amplification by Southern blot analysis to determine the reliability of CGH in the genetic characterization of GBM. The CGH and karyotypic data were consistent in showing gain of chromosome 7 accompanied by a loss of chromosome 10 as the most frequent abnormality, followed by a loss of 9p in 17 of 22 GBM cases. Loss of heterozygosity of chromosomes 10 (19/22) and 9p (9/22) loci confirmed the underrepresentation by CGH. Genomic amplifications were observed by CGH in 5 of the 10 cases where gene amplification was detected by Southern blot analysis. The data show that CGH is equally reliable, compared with the more established genetic methods, for recognizing the prominent genetic alterations associated with GBM and support its use as a plausible adjunct to glioma classification

Multiple Features of Advanced Melanoma Recapitulated in Tumorigenic Variants of Early Stage (Radial Growth Phase) Human Melanoma Cell Lines: Evidence For a Dominant Phenotype.

The vast majority of primary human cutaneous melanomas undergo a slow and gradual progression from a clinically indolent, curable radial growth phase (RGP) to a malignant vertical growth phase. We sought to develop a way of isolating genetically related malignant variants from a benign RGP human melanoma, called WM35. The parent and variants were then used as a model system to examine to what extent the expression of clinically and biologically relevant phenotypic features characteristic of advanced melanomas are associated with (and thus perhaps causative of) such a malignant conversion. Such a model system could also be used as a means of eventually identifying genetic alterations and cellular changes involved in the malignant switch in melanoma progression. To develop such a model, we subjected WM35 cells to retroviral insertional mutagenesis, which was followed by selection for progressive growth of solid tumors in nude mice. Highly aggressive and phenotypically stable tumorigenic variants were derived which contained at least four integrated proviruses. In contrast to the parental WM35 cells, these cell lines expressed several phenotypic features characteristic of naturally derived, advanced-stage malignant melanoma cells. Thus, in addition to tumor-forming ability in nude mice, the variants were growth factor and anchorage independent, overexpressed the MUC18 adhesion molecule, and lost responsiveness to the growth-inhibitory effect of several cytokines, including interleukin 6, transforming growth factor beta, interleukin 1beta, and tumor necrosis factor-alpha. Tumorigenicity and multicytokine resistance were dominant traits since in somatic cell hybrids between the parental cells and a tumorigenic subline no suppressive effect of the former cell population was observed. These findings suggest that one or more dominantly acting genetic alterations might be involved in this progression of RGP melanoma cells. The identity of such alterations remains to be determined