Detection of chromosomal aberrations in seminomatous germ cell tumours using comparative genomic hybridization

Comparative genomic hybridization (CGH) was used to evaluate tissue specimens from 16 seminomas in order to elucidate the pathogenesis of germ cell tumours in males. A characteristic pattern of losses and gains within the entire genomes was detected in 94% of the seminomas by comparing the ratio profiles of the tumours with a standard of cytogenetically normal genomic DNA. Losses represented 43% of the total number of alterations often affecting chromosomes and chromosome arms 4, 5, 11, 13q, and 18q. Gains amounted to 57% and were often observed on 1q, 7, 8, 12, 14q, 15q, 21q, and 22q. Aberrations of 12p and 21q appeared most consistently. Results from CGH analysis displayed no relationship to the clinical stages of the malignancy. Some rare aberrations appeared, however, only in clinical stage II and in tumours showing relapse in the contralateral testis following orchiectomy, although the alterations were not present in all of the tumours in question. Losses of 16q13-21 and gains of 9q22.1-22.2 were demonstrated in both groups, while loss of 16p12 and gains of 6p21 and 6q23.3-24 were detected in the latter group as well. In conclusion, a specific pattern of chromosomal alterations was demonstrated in the seminomas by improved detection criteria, which increased specificity and sensitivity. The rare aberrations, which appeared only in tumours in improved detection criteria, which increased specificity and sensitivity. The rare aberrations, which appeared only in tumours in clinical stage II and relapsed tumours, may be linked to tumour progression, invasiveness, and bilateral disease.     

Recurrent gains of 1q, 8 and 12 in the Ewing family of tumours by comparative genomic hybridization

Comparative genomic hybridization (CGH) was used to detect copy number changes of DNA sequences in the Ewing family of tumours (ET). We analysed 20 samples from 17 patients. Fifteen tumours (75%) showed copy number changes. Gains of DNA sequences were much more frequent than losses, the majority of the gains affecting whole chromosomes or whole chromosome arms. Recurrent findings included copy number increases for chromosomes 8 (seven out of 20 samples; 35%), 1q (five samples; 25%) and 12 (five samples; 25%). The minimal common regions of these gains were the whole chromosomes 8 and 12, and 1q21-22. High-level amplifications affected 8q13-24, 1q and 1q21-22, each once. Southern blot analysis of the specimen with high-level amplification at 1q21-22 showed an amplification of FLG and SPRR3, both mapped to this region. All cases with a gain of chromosome 12 simultaneously showed a gain of chromosome 8. Comparison of CGH findings with cytogenetic analysis of the same tumours and previous cytogenetic reports of ET showed, in general, concordant results. In conclusion, our findings confirm that secondary changes, which may have prognostic significance in ET, are trisomy 8, trisomy 12 and a gain of DNA sequences in 1q.     

CGH (comparative genomic hybridization)]

Comparative Genomic Hybridization (CGH) is a powerful new method which allows genome-wide mapping of regions with DNA sequence copy number changes (both increases and decreases) in a single experiment without previous knowledge of the locations of the regions of abnormality. CGH is based on in situ hybridization of differentially labeled total genomic tumor DNA and normal DNA to normal human metaphase chromosomes. After hybridization copy number variations among the sequences in the tumor DNA are detected by measuring the tumor/normal fluorescence intensity ratio for each locus in the target chromosomes. Many previously unknown chromosomal regions with relative copy number changes have been detected in various tumors by CGH. Some changes have been identified as genetic markers associated with biological and clinico-pathological characteristics (i.e., histopathological grade, and clinical outcome). We review the published CGH articles and discuss briefly on current progress in CGH analysis to ovarian and uterine cervical cancer in our laboratory.     

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.     

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.     

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.     

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 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.     

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.     

Comparison of DNA copy numbers in original oral squamous cell carcinomas and corresponding cell lines by comparative genomic hybridization

We analyzed regional DNA copy numbers in 4 oral squamous cell carcinomas (SCCs) by using comparative genomic hybridization, and compared them with those in cell lines derived from the SCCs. In the original tumors, DNA copy number increases were observed on chromosomes 5p (4/4 cases), 8q (4/4), 20p (3/4), 3q (2/4), 5q (2/4), 7p (2/4), 7q (2/4), 11p (2/4), 11q (2/4) and 13q (2/ 4). Although most of these changes have been described previously for SCC tumors in the head and neck, the incidence of increases in 8q and 20p was much higher in the present study; this may be important in relation to cell line establishment, since 8q contains e-myc, which is involved in immortalization. No common chromosomal region with DNA copy number decreases was observed, except for 18q (2/4). When the original tumors and the cell lines were compared, their profiles were essentially similar with one exception. Further, there was no region that commonly changed in the cell lines, but not in the original tumors, suggesting that the DNA copy number changes observed in the cell lines mostly represent those of the original tumors.     

Telomerase activity in benign and malignant thyroid diseases

BACKGROUND: Telomerase, an enzyme associated with cellular immortality, is expressed by most malignant cells and is inactive in most normal somatic cells, with the excitation of proliferative stem cells, male germ cells, and activated lymphocytes. The measurement of telomerase activity in clinically obtained tissue samples may provide useful information as both a diagnostic and prognostic marker. In this study, we sought to determine whether telomerase activity might prove helpful in the assessment of benign and malignant thyroid tumors. METHODS: A modified, semiquantitative polymerase chain reaction-based telomeric repeat amplification protocol assay was used for detection of telomerase activity in 59 samples obtained at thyroidectomy, including 15 thyroid cancers, 22 benign thyroid diseases, and 22 adjacent normal thyroid tissues. RESULTS: Four of 13 differentiated thyroid carcinomas (30%) and 2 of 2 medullary carcinomas (100%) expressed telomerase activity. Unexpectedly, we also detected activity in 3 of 22 (14%) adjacent normal thyroid tissues and 6 of 22 (28%) benign thyroid diseases. Pathologic review of the telomerase-positive benign specimens revealed that many contained extensive lymphoid infiltrates with germinal centers (six of nine, 67%), as did two of four telomerase-positive papillary carcinomas. CONCLUSIONS: In contradistinction to other epithelial carcinomas, telomerase does not appear to be frequently reactivated in differentiated thyroid carcinomas.     

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.     

Immunohistological differentiation of benign thyroid follicular cell tumors from malignant ones: usefulness of anti-peroxidase and JT-95 antibodies

An immunohistological investigation using anti-thyroid peroxidase (PO) and JT-95 (JT) antibodies was conducted on surgical specimens of papillary carcinoma (n = 12), follicular carcinoma (n = 8), follicular adenoma with prominent papillary structure (n = 12), follicular adenoma (n = 8) and adenomatous goiter (n = 8). In benign lesions, follicle-forming and papillae-forming epithelia showed positive staining with anti-PO antibody. In 26 of 28 benign lesions, PO-positive areas covered 50% or more of the cut surface area of the lesions. However in carcinomas, PO-positive areas were non-existent (13/20) or only focal (7/20) and the papillae did not stain. The incidence of JT-positive cases was higher in papillary carcinomas (12/12) than in other lesions (13/36). JT-positive areas were much wider than PO-positive areas in papillary carcinomas, whereas in benign lesions, PO-positive areas were wider than or at least roughly equal to, JT-positive areas. Follicular carcinomas did not stain or stained only focally with these antibodies. In conclusion, these two antibodies seem useful in differentiating benign from malignant follicular cell tumors.     

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.     

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.     

Telomerase activity in benign and malignant human thyroid tissues

Telomerase is a specialized ribonucleoprotein polymerase that directs the synthesis of telomerase repeats at chromosome ends. Accumulating evidence has indicated that telomerase is stringently repressed in normal human somatic tissues but reactivated in cancers and immortal cells, suggesting that activation of telomerase activity plays a role in carcinogenesis and immortalization. In this work, the status of telomerase activity during the development of human thyroid cancer was determined using telomeric repeat amplification protocol (TRAP) in 14 nodular hyperplasia, 14 adenomas, 23 papillary carcinomas and 11 follicular carcinomas. Positive telomerase activity was detected in 2 of 14 nodular hyperplasias (14%), 4 of 14 adenomas (29%), 12 of 23 papillary carcinomas (52%) and 10 of 11 follicular carcinomas (91%). The cancers that are negative for telomerase activity are mostly in early stage (stage I or II). These results suggest that telomerase reactivation plays a role during the development of thyroid cancer.     

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.     

Intratumoral cytogenetic heterogeneity detected by comparative genomic hybridization and laser scanning cytometry in human gliomas

Although it is well-known that cancers show intratumoral phenotypic heterogeneity, genotypic studies have been scarce. Using comparative genomic hybridization and laser scanning cytometric analyses, we investigated intratumoral cytogenetic heterogeneity in 21 surgically removed gliomas including 11 glioblastomas (GBMs), 8 anaplastic astrocytomas (AAs) and 2 low-grade astrocytomas. Comparative genomic hybridization analysis revealed gain or amplification of 7p in 63%, gain of 7q in 73%, loss of 9p in 53%, loss of 10p in 47%, loss of 10q in 47%, loss of 13q in 53%, and loss of 22q in 37% of high-grade astrocytomas. Because these aberrations were region-independent within the same tumor, they did not contribute to intratumoral cytogenetic heterogeneity. Such heterogeneity was due to cytogenetic changes other than the above region-independent aberrations. Intratumoral cytogenetic heterogeneity was detected in 8 of 11 GBMs, 4 of 8 AAs, and none of the 2 low-grade astrocytomas. These observations suggest that cytogenetic changes at chromosomes 7, 9p, 10, 13, and 22 are primary events in high-grade astrocytomas and that subsequent cytogenetic changes involving increases in copy number provide intratumoral heterogeneity. DNA aneuploidy was detected by laser scanning cytometry in 5 of 11 GBMs and 1 of 8 AAs. All tumors with DNA aneuploidy exhibited intratumoral cytogenetic heterogeneity, and there was a significant correlation between DNA aneuploidy and intratumoral cytogenetic heterogeneity. These results support the notion that cytogenetic heterogeneity results from genetic instability within a tumor.