Chromosomal gains and losses in uveal melanomas detected by comparative genomic hybridization

Eleven uveal melanomas were analyzed using comparative genomic hybridization (CGH). The most abundant genetic changes were loss of chromosome 3, overrepresentation of 6p, loss of 6q, and multiplication of 8q. The smallest overrepresented regions on 6p and 8q were 6pter-->p21 and 8q24-->qter, respectively. Several additional gains and losses of chromosome segments were repeatedly observed, the most frequent one being loss of 9p (three cases). Monosomy 3 appeared to be a marker for ciliary body involvement. CGH data were compared with the results of chromosome banding. Some alterations, e.g., gains of 6p and losses of 6q, were observed with higher frequencies after CGH, while others, e.g., 9p deletions, were detected only by CGH. The data suggest some similarities of cytogenetic alterations between cutaneous and uveal melanoma. In particular, the 9p deletions are of interest due to recent reports about the location of a putative tumor-suppressor gene for cutaneous malignant melanoma in this region.     

Genetic changes in intraductal breast cancer detected by comparative genomic hybridization

Ductal carcinoma in situ (DCIS) is considered a direct precursor of invasive ductal breast cancer (IDC). We combined tissue microdissection and comparative genomic hybridization to identify genetic changes in five DCIS lesions with no invasion and in two that were adjacent to IDC. Extensive genetic changes characterized pure DCIS cases with gains of 1q, 6q, 8q, and Xq as well as losses of 17p and chromosome 22 being most often involved. Except for the Xq gain, these changes are also common to IDC. Separate analysis of DCIS and IDC components in the same tumor revealed an almost identical pattern of genetic changes in one case, whereas substantial differences were found in another. We conclude that many of the common genetic changes in IDC may take place before development of invasive growth. However, a simple linear progression model may not always account for the DCIS-IDC transition.     

Initiating genetic events in small renal neoplasms detected by comparative genomic hybridization

PURPOSE: To identify the genetic alterations associated with renal adenomas. MATERIALS AND METHODS: We analyzed 37 renal adenomas obtained at autopsy (23 papillary and 14 non-papillary) by comparative genomic hybridization. RESULTS: In papillary tumors, the median number of gains and losses of genetic material per tumor was 2.0 and 1.0, respectively. Papillary tumors were characterized predominantly by gains of genetic material on chromosomes 7 (57%), 17 (35%), 16 (26%), 12 (26%), 3 (22%), 20 (22%) and loss of a sex chromosome (83%). In 6 papillary tumors less than or equal to 5 mm. in diameter, gain of chromosome 7 occurred in 4 specimens. Initiating events for papillary renal adenomas include gain of chromosome 7 and loss of a sex chromosome. In non-papillary tumors, the median number of gains and losses of genetic material per tumor was 1.0 and 1.0, respectively. Non-papillary tumors were characterized by loss of genetic material on chromosome 3p (50%), loss of a sex chromosome (36%) and a gain of chromosome 5 (43%). The initiating event for non-papillary renal adenomas is the loss of chromosome 3p. CONCLUSIONS: Renal adenomas demonstrate similar genetic alterations as clinically detected renal cell carcinomas. Their clinically indolent course may, in part, be a result of the lower number of genetic alterations per tumor than their clinically detected counterparts. Renal adenomas are thus small carcinomas which have not yet acquired the necessary genetic alterations leading to tumor progression.     

Unbalanced chromosomal aberrations in neuroendocrine lung tumors as detected by comparative genomic hybridization

Typical and atypical carcinoids (TC, ATC) and small (SCLC) and large cell neuroendocrine carcinomas (LCNEC) constitute the spectrum of neuroendocrine lung tumors. Chromosomal aberrations have not been studied in LCNEC and only rarely in carcinoids. Only SCLCs have been investigated frequently for chromosomal aberrations. We compared three typical and four atypical carcinoids, one atypical carcinoid/SCLC mixed type, three SCLC, and three LCNEC for chromosomal gains and losses using comparative genomic hybridization. Typical carcinoids showed either no changes or only few chromosomal gains. Atypical carcinoids appeared genetically heterogeneous: One case had no aberrations, and three cases had few aberrations; two of them showed a deletion of 11q. SCLC and LCNEC were characterized by many gains and losses, especially similar changes of 3p, 5q, 5p, and 13q. Although ATC resemble LCNEC morphologically, there were no similarities at the genetic level. We have found a reciprocal relationship of prognosis and the amount of aberrations. TCs and ATCs with few chromosomal changes have the best prognosis, whereas SCLCs and LCNECs were generally characterized by a great amount of aberrations and worst prognosis. There was no unbalanced aberration common in all types of neuroendocrine tumors of the lung.     

Chromosomal abnormalities in glioblastoma multiforme tumors and glioma cell lines detected by comparative genomic hybridization

Comparative genomic hybridization (CGH) is a recent molecular cytogenetic method that detects and localizes gains or losses in DNA copy number across the entire tumor genome. We used CGH to examine 9 glioma cell lines and 20 primary and 10 recurrent glioblastoma tumors. More than 25% of the primary tumors had gains on chromosome 7; they also had frequent losses on 9p, 10, 13 and Y. The losses on chromosome 13 included several interstitial deletions, with a common area of loss of 13q21. The recurrent tumors not only had gains on chromosome 7 and losses on 9p, 10, 13 and Y but also frequent losses on 6 and 14. One recurrent tumor had a deletion of 10q22-26. Cell lines showed gains of 5p, 7 and Xp; frequent amplifications at 8q22-24.2, 7q21-32 and 3q26.2-29 and frequent losses on 4, 10, 13, 14 and Y. Because primary and recurrent tumors and cell lines showed abnormalities of DNA copy number on chromosomes 7, 10, 13 and Y, these regions may play a fundamental role in tumor initiation and/or progression. The propensity for losses on chromosomes 6 and 14 to occur in recurrent tumors suggests that these aberrations play a role in tumor recurrence, the development of resistance to therapy or both. Analysis of common areas of loss and gain in these tumors and cell lines provides a basis for future attempts to more finely map these genetic changes.     

Detection of human papillomavirus DNA in CaSki and HeLa cells by fluorescent in situ hybridization. Analysis by flow cytometry and confocal laser scanning microscopy

CaSki and HeLa cell lines, isolated from human uterine carcinomas and containing integrated human papillomavirus (HPV) DNA type 16 and 18, respectively were used to evaluate the sensitivity of HPV-DNA detection on suspended cells by fluorescent in situ hybridization using flow cytometry and on corresponding cell deposits using confocal laser scanning microscopy (CLSM). HPV DNAs were detected in cell suspensions with biotinylated DNA probes and revealed with a three-step technique: a rabbit antibiotin antibody, a biotinylated goat anti-rabbit antibody and a streptavidin-fluorescein isothiocyanate complex. By flow cytometry, HPV DNA was detectable only in CaSki cells which contained about 600 copies of HPV DNA per cell. In HeLa cells, with only 20-50 copies of HPV DNA, flow cytometry could not detect HPV DNA, whereas CLSM permitted visualization of fluorescent labelling of HPV DNA hybrids. Furthermore, CLSM showed good preservation of cellular morphology and the nucleus was clearly recognizable after fluorescent in situ hybridization and counterstaining with propidium iodide. Moreover, this examination confirmed that the fluorescent foci were specifically confined to the cell nuclei.     

Multiple genetic aberrations including evidence of chromosome 11q13 rearrangement detected in pituitary adenomas by comparative genomic hybridization

OBJECT: This study was conducted to determine whether comparative genomic hybridization (CGH) is a more sensitive method for detecting genetic aberrations than other tests currently in use. METHODS: The authors used CGH to examine 40 primary and 13 recurrent adenomas obtained from 52 patients for loss and gain of genetic material. Copy number aberrations (CNAs) were detected in 25 (48%) of the 52 patients studied. The chromosomes affected were, in order of decreasing frequency, 11, 7, X, 1, 8, 13, 5, 14, 2, 6, 9, 10, 12, 3, 18, 21, 4, 16, 15, 19, 22, and Y. Endocrinologically active adenomas were more likely to contain (p = 0.009) and had a greater number (p = 0.003) of CNAs. Of 26 adenomas with CNAs, 18 showed multiple aberrations involving entire chromosomes or chromosome arms. The most frequent CNA involving a chromosome subregion, which was present in four (8%) of 53 adenomas, was the loss of all chromosome 11 material except for a preserved common segment containing 11q13. Immunoperoxidase staining did not detect cyclin D1 expression in those four cases, making cyclin D1 an unlikely target of this rearrangement. CONCLUSIONS: These findings indicate that genetic abnormalities are present in pituitary adenomas at a higher rate than previously reported, are associated with endocrinological activity, and often involve several chromosomes. Rearrangement at 11q13 may inactivate a tumor suppressor gene or activate an oncogene that is important in the initiation or progression of sporadic pituitary adenomas.     

Prenatal diagnosis of a (X;X) translocation by fluorescence in situ hybridization and laser scanning image cytometry

A de novo structural abnormality of one X chromosome was prenatally detected in a female fetus. This chromosomal abnormality has been analyzed by conventional cytogenetic methods, fluorescence in situ hybridization, and laser scanning image cytometry. The association of these techniques has demonstrated that this anomaly corresponds to a (X;X) translocation. Analysis of hybridization signals by laser scanning image cytometry allowed to localize that the breakpoints were at the X-centromeric region and Xp11.3, respectively. These results show the usefulness of image analysis and fluorescence in situ hybridization for a rapid characterization of de novo structural chromosome anomalies in prenatal diagnosis.     

Interphase cytogenetics and comparative genomic hybridization of human epithelial cancers and precursor lesions

The accuracy of cytogenetic analyses of human solid cancers has improved enormously over the past decade by the introduction and refinement of DNA in situ hybridization (ISH) techniques. This methodology can be applied to cells in the interphase state, thereby making it an excellent tool for the delineation of chromosomal aberrations in solid tumors. The use of non-isotopic ISH to intact and disaggregated cancer specimens will be discussed, as well as comparative genomic hybridization (CGH) with tumor-derived DNAs. In this review we will focus on hybridocytochemical interphase approaches for the detection of chromosomal changes in frequently occurring human epithelial malignancies, e.g., breast, lung, and prostate carcinomas. We will further discuss the use of ISH procedures for the genetic analysis of precursor conditions leading to invasive carcinomas. Knowledge concerning these precancerous conditions is increasing, and its importance in cancer prevention has been recognized. Interphase cytogenetics by ISH, as well as CGH, with DNAs derived from microdissected, precancerous, dysplastic tissue areas will increase our understanding of these lesions, both at the investigative and diagnostic levels.     

Synopsis of unbalanced chromosome aberrations in neuroblastoma by comparative genomic hybridization

A case of synchronous squamous cell carcinomas in the soft palate, larynx and esophagus is reported, along with findings of molecular-pathological analysis. A biopsy sample from the aryngeal carcinoma revealed well differentiated squamous cell carcinoma harboring two point mutations at codons 144 and 148 of the p53 gene but not at codon 299, and more than 50% of the cancer cells showed accumulation of p53 protein immunohistochemically. The esophageal tumor, which was moderately differentiated squamous cell carcinoma, showed immunoreactivity for p53 within the nuclei of 25-50% of cancer cells with a missense mutation at codon 299 but not at codon 144 or 148. This cancer also showed immunoreactivity for transforming growth factor alpha. On the other hand, the poorly differentiated squamous cell carcinoma in the soft palate showed negative immunoreactivity for p53 and no point mutation in exons 5 to 8 of the gene. These results suggest that the three synchronous squamous cell carcinomas arose as independent events.     

Genetic imbalances in 67 synovial sarcomas evaluated by comparative genomic hybridization

The present study investigated the role of the postinjection interval in determining the functional consequences of acute ethanol administration in the CNS. Regional cerebral blood flow (RCBF) was determined by the [14C]iodoantipyrine method in 33 brain structures of ethanol-naive Sprague-Dawley rats. In the first experiment, changes in RCBF were assessed 5 and 15 min after a 0.8 g/kg (i.p.) dose of ethanol or water. Five minutes after treatment, rates of RCBF were increased in the motor cortex, agranular insular cortex, and the olfactory tubercle compared to water controls. No significant differences compared to control were found at the 15-min time point, despite the continued presence of ethanol in the blood. Experiment 2 tested whether blood ethanol level was the sole determinant of this response to ethanol by comparing animals with the same blood ethanol level at the 5- and 15-min time points. Greater rates of RCBF were found at 5 min postinjection compared to 15 min, in the motor cortex, agranular insular cortex, caudate/putamen, cerebellum, and the lateral septum. These data demonstrate that the rates of cerebral blood flow are increased in regionally discrete portions of the rat brain shortly after ethanol administration. Furthermore, blood ethanol level is not the exclusive factor governing this functional response.     

Mapping of chromosomal imbalances in pancreatic carcinoma by comparative genomic hybridization

To identify recurrent chromosomal imbalances in pancreatic adenocarcinoma, 27 tumors were analyzed by using comparative genomic hybridization. In 23 cases chromosomal imbalances were found. Gains of chromosomal material were much more frequent than losses. The most common overrepresentations were observed on chromosomes 16p (eight cases), 20q (seven cases), 22q (six cases), and 17q (five cases) and under-representations on a subregion of chromosome 9p (eight cases). Distinct high-level amplifications were found on 1p32-p34, 6q24, 7q22, 12p13, and 22q. These data provide evidence for a number of new cytogenetically defined recurrent aberrations which are characteristic of pancreatic carcinoma. The overrepresented or underrepresented chromosomal regions represent candidate regions for potential oncogenes and tumor suppressor genes, respectively, possibly involved in pancreatic tumorigenesis.     

Comparative genomic hybridization: a comparison with molecular and cytogenetic analysis

A comparative analysis of the differentiation pattern, the proliferative behaviour, and the level of apoptosis between human benign and malignant neoplasms of smooth-muscle (SM) tissue is lacking. The clinical, histopathological, immunochemical, and immunocytochemical features of leiomyomas (LM) and leiomyosarcomas (LMS) were investigated by a panel of monoclonal antibodies specific for some differentiation markers of SM tissue (SM myosin and alpha-actin, desmin, and SM22) and for markers of non-muscle tissue (vimentin and non-muscle myosin). Proliferating normal and neoplastic cells were identified by proliferating-cell nuclear antigen (PCNA)/Ki67 immunostainings and the apoptotic cells were revealed by means of the terminal-deoxynucleotidyltransferase-mediated dUTP nick-end labelling technique. Gel electrophoresis and Western blotting, performed with anti-(SM1/SM2 myosin isoform) antibody, indicated quantitative differences between LMS and LM, which mirrored higher positive to negative nuclear ratios for PCNA, Ki67 and apoptosis in malignant as opposed to benign neoplasms. With LM, however, a similar SM1 to SM2 ratio could be associated with different proliferation levels. Uterine, gastric and intestinal LMS displayed specific patterns of SM1/SM2 and/or non-muscle myosin expression that were not paralleled by different levels of proliferation/apoptosis. While the level of PCNA/Ki67 correlated with the level of apoptosis in normal SM tissues and LM, that of LMS did not. In vivo at the cellular level, LM and uterine LMS displayed a near-uniform SM tissue differentiation, whereas the other LMS displayed a lesser or a heterogeneous immunoreactivity. In vitro, cultured LMS cells showed a limited and peculiar expression of SM myosin. In conclusion, there is no reciprocal relationship between degree of differentiation and the level of proliferation, as exemplified by the finding that the less differentiated intestinal LMS displays the lowest proliferative behaviour and that the relatively more differentiated gastric LMS/metastasis is more proliferative.     

Comparison of benign and malignant follicular thyroid tumours by comparative genomic hybridization

DNA copy number changes were compared in 29 histologically benign follicular adenomas, of which five were atypical, and 13 follicular carcinomas of the thyroid by comparative genomic hybridization. DNA copy number changes were frequent in adenomas (14 out of 29, 48%). Most changes were gains, and they always involved a gain of the entire chromosome 7 (10 out of 29, 34%); other common gains involved chromosomes 5 (28%), 9 (10%), 12 (24%), 14 (21%), 17 (17%), 18 (14%) and X (17%). Losses were found only in four (14%) adenomas. Two of the five atypical adenomas had DNA copy number losses, and none had gains. Unlike adenomas, gains were rare and losses were frequent in carcinomas. A loss of chromosome 22 or 22q was particularly common in carcinomas (6 out of 13, 46%), whereas a loss of chromosome 22 was found in only two (7%) adenomas, one of which was atypical (P = 0.002). A loss of 1p was also frequent in carcinomas (31%), but gains of chromosomes 5, 7, 12, 14 or X that were common in adenomas were not found. Loss of chromosome 22 or 22q was present in six of the eight widely invasive follicular carcinomas, but in only one of the five minimally invasive carcinomas. We conclude that large DNA copy number changes are common in thyroid adenomas. These changes are strikingly different from those found in follicular carcinomas consisting of few losses and frequent gains, especially those of chromosome 7. A loss of chromosome 22 is common in widely invasive follicular carcinoma.     

Genetic analysis of ovarian germ cell tumors by comparative genomic hybridization

Ovarian germ cell tumors (OGCTs) show a heterogeneity that is not seen in their testicular counterparts and include benign mature cystic teratomas, intermediate immature teratomas, malignant germ cell tumors [GCTs (dysgerminomas, endodermal sinus tumors, and mixed GCTs)], and GCTs arising in dysgenetic gonads of 46,XY individuals. Comparative genomic hybridization was used to analyze 27 OGCTs for regions of relative gain or loss. The analysis of 21 malignant OGCTs (12 dysgerminomas, 6 endodermal sinus tumors, and 3 mixed GCTs) demonstrated genetic alterations similar to those reported in adult testicular GCTs. The most common regions gained include chromosomes 12p (16 of 21 tumors), 21 (10 of 21 tumors), 8 (8 of 21 tumors), and 1q (6 of 21 tumors). The most common region lost was chromosome 13. Regions of high-level gain were identified at 12p11-12 and 4q11. The profile of gains and losses was similar in the different histological subtypes within this category. One tumor presented in a 46,XY patient; this tumor was diploid and showed a gain of 12p. Immature teratomas (six cases) showed only one case with an abnormality, which was a gain of chromosome 14. We conclude that malignant OGCTs are genetically similar to those found in the adult testis; however, immature teratomas show no consistent gains or losses and are therefore different from those presenting in the adult testis. A review of the literature suggests that genetic abnormalities in this group may herald a worse prognosis. Lastly, OGCTs in dysgenetic gonads arise in a diploid rather than a tetraploid cell line, yet they also show a gain of 12p.     

DNA copy number amplifications in human neoplasms: review of comparative genomic hybridization studies

This review summarizes reports of recurrent DNA sequence copy number amplifications in human neoplasms detected by comparative genomic hybridization. Some of the chromosomal areas with recurrent DNA copy number amplifications (amplicons) of 1p22-p31, 1p32-p36, 1q, 2p13-p16, 2p23-p25, 2q31-q33, 3q, 5p, 6p12-pter, 7p12-p13, 7q11.2, 7q21-q22, 8p11-p12, 8q, 11q13-q14, 12p, 12q13-q21, 13q14, 13q22-qter, 14q13-q21, 15q24-qter, 17p11.2-p12, 17q12-q21, 17q22-qter, 18q, 19p13.2-pter, 19cen-q13.3, 20p11.2-p12, 20q, Xp11.2-p21, and Xp11-q13 and genes therein are presented in more detail. The paper with more than 150 references and two tables can be accessed from our web site http://www.helsinki.fi/lglvwww/CMG.html. The data will be updated biannually until the year 2001.     

Characterization of nonrandom chromosomal gains and losses in multiple myeloma by comparative genomic hybridization

Clonal chromosomal changes in multiple myeloma (MM) and related disorders are not well defined, mainly due to the low in vivo and in vitro mitotic index of plasma cells. This difficulty can be overcome by using comparative genomic hybridization (CGH), a DNA-based technique that gives information about chromosomal copy number changes in tumors. We have performed CGH on 25 cases of MM, 4 cases of monoclonal gammopathy of uncertain significance, and 1 case of Waldenstrom's macroglobulinemia. G-banding analysis of the same group of patients demonstrated clonal chromosomal changes in only 13 (43%), whereas by CGH, the number of cases with clonal chromosomal gains and losses increased to 21 (70%). The most common recurrent changes detected by CGH were gain of chromosome 19 or 19p and complete or partial deletions of chromosome 13. +19, an anomaly that has so far not been detected as primary or recurrent change by G-banding analysis of these tumors, was noted in 2 cases as a unique change. Other recurrent changes included gains of 9q, 11q, 12q, 15q, 17q, and 22q and losses of 6q and 16q. We have been able to narrow the commonly deleted regions on 6q and 13q to bands 6q21 and 13q14-21. Gain of 11q and deletion of 13q, which have previously been associated with poor outcome, can thus be detected by CGH, allowing the use of this technique for prognostic evaluation of patients, without relying on the success of conventional cytogenetic analysis.     

Mapping of chromosomal gains and losses in primitive neuroectodermal tumors by comparative genomic hybridization

Pediatric fungal pulmonary infections are being seen with increasing frequency. The dimorphic fungi Histoplasma capsulatum. Blastomyces dermatitidis, Coccidioides immitis, and Cryptococcus neoformans frequently cause infections that are asymptomatic. However, patients may suffer pneumonia and disseminated disease. Diagnosis can be made definitively by isolation of the causative organism, but serology or skin testing is often necessary when this is not successful. Severe or life threatening infections are treated with amphotericin B. Recently, new oral azole antifungals are being used more frequently for mild to moderate disease with good success.     

Tumor cytogenetics revisited: comparative genomic hybridization and spectral karyotyping

Fluorescence in situ hybridization techniques allow the visualization and localization of DNA target sequences on the chromosomal and cellular level and have evolved as exceedingly valuable tools in basic chromosome research and cytogenetic diagnostics. Recent advances in molecular cytogenetic approaches, namely comparative genomic hybridization and spectral karyotyping, now allow tumor genomes to be surveyed for chromosomal aberrations in a single experiment and permit identification of tumor-specific chromosomal aberrations with unprecedented accuracy. Comparative genomic hybridization utilizes the hybridization of differentially labeled tumor and reference DNA to generate a map of DNA copy number changes in tumor genomes. Comparative genomic hybridization is an ideal tool for analyzing chromosomal imbalances in archived tumor material and for examining possible correlations between these findings and tumor phenotypes. Spectral karyotyping is based on the simultaneous hybridization of differentially labeled chromosome painting probes (24 in human), followed by spectral imaging that allows the unique display of all human (and other species) chromosomes in different colors. Spectral karyotyping greatly facilitates the characterization of numerical and structural chromosomal aberrations, therefore improving karyotype analysis considerably. We review these new molecular cytogenetic concepts, describe applications of comparative genomic hybridization and spectral karyotyping for the visualization of chromosomal aberrations as they relate to human malignancies and animal models thereof, and provide evidence that fluorescence in situ hybridization has developed as a robust and reliable technique which justifies its translation to cytogenetic diagnostics.