Comparative genomic hybridization reveals complex genetic changes in primary breast cancer tumors and their cell lines.

DNA copy number changes were characterized by comparative genomic hybridization (CGH) in 18 breast cancer cell lines. In 5 of these, the results were comparable with those from the primary tumors of which the cell lines were established. All of the cell lines showed extensive DNA copy number changes, with a mean of 16.3 +/- 1.1 aberrations per sample (range 7-26). All of the cell lines had a gain at 8q22-qter. Other common gains of DNA sequences occurred at 1q31-32 (89%), 20q12-q13.2 (83%), 8q13 (72%), 3q26.1-qter (67%), 17q21-qter (67%) 5p14 (61%), 6p22 (56%), and 22pter-qter (50%). High-level amplifications were observed in all cell lines; the most frequent minimal common regions were 8q24.1 (89%), 20q12 (61%), 1q41 (39%), and 20p11.2 (28%). Losses were observed less frequently than gains and the minimal common regions of the most frequent losses were Xq11-q12 (56%), Xp11.2-pter (50%), 13q21 (50%), 8p12-pter (44%), 4p13-p14 (39%), 6q15-q22 (39%), and 18q11.2-qter (33%). Although the cell lines showed more DNA copy number changes than the primary tumors, all aberrations, except one found in a primary tumor, were always present in the corresponding cell line. High-level amplifications found both in primary tumors and cell lines were at 1q, 8q, 17q, and 20q. The DNA copy number changes detected in these cell lines can be valuable in investigation of tumor progression in vitro and for a more detailed mapping and isolation of genes implicated in breast cancer.

A broad amplification pattern at 3q in squamous cell lung cancer--a fluorescence in situ hybridization study.

Frequent DNA copy number gain at 3q, with minimal overlapping area at 3q24-qter, has previously been reported in squamous cell carcinoma of the lung (SQCC), implicating the importance of genes at 3q in the tumorigenesis of SQCC. To further characterize the gain of DNA sequences at 3q, we performed interphase fluorescence in situ hybridization (FISH) analysis on 16 paraffin-embedded SQCC tumor samples that had previously been studied by comparative genomic hybridization (CGH). Eleven yeast artificial chromosome (YAC) probes located at 3q25-q27 and a chromosome 3-specific centromeric probe were used in the analysis. All SQCC tumors showed increase in DNA sequence copy number with 9-11 probes. In 5 tumors (31%) the number of centromeric signals varied from 3 to 5 and the YAC/centromeric signal ratio was 1.0, suggesting that the increase in DNA sequence copy number at 3q in these cases resulted from polysomy of chromosome 3. In 11 tumors (69%), the YAC/centromeric signal ratio varied between 1.5 and 4.7, indicating that the increase in DNA sequence copy number was due to intrachromosomal gain of DNA sequences at 3q. In each case, several YACs showed increased number of signals, demonstrating that the gained area was relatively large. Our findings therefore suggest that multiple genes located at 3q25-q27 are involved in the tumorigenesis of SQCC.

Deletions at 14q in malignant mesothelioma detected by microsatellite marker analysis.

Previous molecular cytogenetic studies by comparative genomic hybridization (CGH) on primary tumours of human malignant mesothelioma have revealed that loss of genetic material at chromosome 14q is one of the most frequently occurring aberrations. Here we further verify the frequency and pattern of deletions at 14q in mesothelioma. A high-resolution deletion mapping analysis of 23 microsatellite markers was performed on 18 primary mesothelioma tumours. Eight of these had previously been analysed by CGH. Loss of heterozygosity or allelic imbalance with at least one marker was detected in ten of 18 tumours (56%). Partial deletions of varying lengths were more common than loss of all informative markers, which occurred in only one tumour. The highest number of tumours with deletions at a specific marker was detected at 14q11.1-q12 with markers D14S283 (five tumours), D14S972 (seven tumours) and D14S64 (five tumours) and at 14q23-q24 with markers D14S258 (five tumours), D14S77 (five tumours) and D14S284 (six tumours). We conclude from these data that genomic deletions at 14q are more common than previously reported in mesothelioma. Furthermore, confirmation of previous CGH results was obtained in all tumours but one. This tumour showed deletions by allelotyping, but did not show any DNA copy number change at 14q by CGH. Although the number of tumours allelotyped was small and the deletion pattern was complex, 14q11.1-q12 and 14q23-q24 were found to be the most involved regions in deletions. These regions provide a good basis for further molecular analyses and may highlight chromosomal locations of tumour suppressor genes that could be important in the tumorigenesis of malignant mesothelioma.

DNA copy number losses in human neoplasms.

This review summarizes reports of recurrent DNA sequence copy number losses in human neoplasms detected by comparative genomic hybridization. Recurrent losses that affect each of the chromosome arms in 73 tumor types are tabulated from 169 reports. The tables are available online at http://www.amjpathol.org and http://www. helsinki.fi/ approximately lglvwww/CMG.html. The genes relevant to the lost regions are discussed for each of the chromosomes. The review is supplemented also by a list of known and putative tumor suppressor genes and DNA repair genes (see Table 1, online). Losses are found in all chromosome arms, but they seem to be relatively rare at 1q, 2p, 3q, 5p, 6p, 7p, 7q, 8q, 12p, and 20q. Losses and their minimal common overlapping areas that were present in a great proportion of the 73 tumor entities reported in Table 2 (see online) are (in descending order of frequency): 9p23-p24 (48%), 13q21 (47%), 6q16 (44%), 6q26-q27 (44%), 8p23 (37%), 18q22-q23 (37%), 17p12-p13 (34%), 1p36.1 (34%), 11q23 (33%), 1p22 (32%), 4q32-qter (31%), 14q22-q23 (25%), 10q23 (25%), 10q25-qter (25%),15q21 (23%), 16q22 (23%), 5q21 (23%), 3p12-p14 (22%), 22q12 (22%), Xp21 (21%), Xq21 (21%), and 10p12 (20%). The frequency of losses at chromosomes 7 and 20 was less than 10% in all tumors. The chromosomal regions in which the most frequent losses are found implicate locations of essential tumor suppressor genes and DNA repair genes that may be involved in the pathogenesis of several tumor types.

DNA gains in 3q occur frequently in squamous cell carcinoma of the lung, but not in adenocarcinoma.

We performed a comparative genomic hybridization study on 25 samples of adenocarcinoma and 19 samples of squamous cell carcinoma of the lung to detect recurrent changes in the genetic material. DNA copy number changes were found in 16 squamous cell carcinoma samples and 17 adenocarcinoma samples. The most common changes were gains of DNA sequences in 3q (43%), 1q (34%), 8q (32%), 5p, (30%), 7p (25%), and 12p (25%). Of the squamous cell carcinoma samples with DNA copy number changes, 94% (15/16) had a gain in 3q (minimal common region of overlap q24-qter), whereas only 24% (4/17) of the adenocarcinoma samples with DNA copy number changes showed a gain in 3q (q22-qter) (P < 0.001). Six high-level amplifications in 3q (q26.2-q26.3) were detected in the squamous cell carcinoma samples but none were observed in the adenocarcinoma samples. Our results suggest that amplification of genes in 3q may be important in the tumorigenesis of squamous cell carcinoma but not necessarily of adenocarcinoma.

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.

Comparison of DNA copy number changes in malignant mesothelioma, adenocarcinoma and large-cell anaplastic carcinoma of the lung.

The differential diagnosis of mesothelioma, primary adenocarcinomas and pleural metastases frequently causes problems. We have used the comparative genomic hybridization (CGH) technique on 34 malignant mesotheliomas and 30 primary lung carcinomas (adenocarcinoma, including bronchoalveolar carcinoma and large-cell anaplastic carcinoma) to compare their copy number changes and to evaluate the use of CGH to distinguish between these two types of tumour. In mesothelioma, gains of genetic material occurred as frequently as losses, whereas gains predominated over losses in carcinoma. In mesothelioma, the most frequent changes were losses in 4q, 6q and 14q and gains in 15q and 7p, whereas gains in 8q, 1q, 7p, 5p and 6p were the most common changes in carcinoma. Amplification of KRAS2 was detected in two adenocarcinomas by Southern blot analysis. CGH showed gains in 12p in the same tumours. Statistically significant differences between the two types of tumour were detected in chromosomes X, 1, 2p, 4, 8q, 10q, 12p, 14q, 15q and 18q. When comparing the frequency of gains and losses between mesothelioma and lung carcinoma using discriminant analysis, the sensitivity of CGH to differentiate mesotheliomas from lung carcinomas was 81% and the specificity 77%. The differences in DNA copy number changes between the two types of tumour suggest that they are genetically different tumour entities. Although CGH cannot be used as a definitive discriminatory method, we were able to distinguish between mesothelioma and lung carcinoma in a large proportion of the abnormal cases.

Comparative genomic hybridization study on pooled DNAs from tumors of one clinical-pathological entity.

Comparative genomic hybridization (CGH) was performed using DNAs pooled from numerous specimens from tumor categories studied case-by-case. The series of six DNA pools consisted of 28 diffuse centroblastic lymphomas (DCL), 28 gastrointestinal stromal tumors (GIST), 21 primary chondrosarcomas (CS), 17 samples from the Ewing family of tumors (ET), 14 liposarcomas (LS), and 14 mesotheliomas (MS). Losses and gains present in at least 50% of the individual specimens were always detected in the pooled DNAs. The loss of the whole p-arm of chromosome 1 was observed even when the affected proportion of individual specimens was only 25%. Gains were also detected at frequencies lower than 50%, but with a high-level amplification in one or more specimens. In conclusion, the present pooled DNA study revealed the following changes: DCL had a gain at 18q22-qter; GIST had losses at 14 and 22q12, and gains at 5p, 8q22-24, 17q22-qter, and 19q13; ET had gains at 1q and 8q13-qter; LS had gains at 1q21-25 and 12q; and MS had a loss at 9p22-pter. No changes were observed in the CS DNA pool. The results from individual specimens also stressed the importance of these chromosomal regions to the tumorigenesis in the corresponding malignancies. This pooled DNA approach can thus be used for fast screening of recurrent DNA copy number in a specific tumor entity.

Recurrent DNA copy number changes in 1q, 4q, 6q, 9p, 13q, 14q and 22q detected by comparative genomic hybridization in malignant mesothelioma.

Comparative genomic hybridization (CGH) analyses were performed on 27 human pleural mesothelioma tumour specimens, consisting of 18 frozen tumours and nine paraffin-embedded tumours, to screen for gains and losses of DNA sequences. Copy number changes were detected in 15 of the 27 specimens with a range from one to eight per specimen. On average, more losses than gains of genetic material were observed. The loss of DNA sequences occurred most commonly in the short arm of chromosome 9 (p21-pter), in 60% of the abnormal specimens. Other losses among the abnormal specimens were frequently detected in the long arms of chromosomes 4 (q31.1-qter, 20%), 6 (q22-q24, 33%), 13 (33%),14 (q24-qter, 33%) and 22 (q13, 20%). A gain in DNA sequences was found in the long arm of chromosome 1 (cen-qter) in 33% of the abnormal specimens. Our analysis is the first genome-wide screening for gains and losses of DNA sequences using comparative genomic hybridization in malignant pleural mesothelioma tumours. The recurrent DNA sequence changes detected in this study suggest that the corresponding chromosomal areas most probably contain genes important for the initiation and progression of mesothelioma.

Localization of a susceptibility locus for Peutz-Jeghers syndrome to 19p using comparative genomic hybridization and targeted linkage analysis.

Many human cancer susceptibility genes have been successfully mapped by genetic linkage studies. One that has so far eluded researchers is that for Peutz-Jeghers (P-J) syndrome, a condition characterized by intestinal hamartomatous polyposis and melanin spots of the lips, buccal mucosa and digits. A dramatically elevated risk of malignancy has also been documented. Gastrointestinal tumours as well as cancers of the breast, ovary, testis and uterine cervix appear to be overrepresented in families with this syndrome. The nature of hamartomatous polyps is equivicol. Hamartomas are usually considered histologically benign, but in the case of Peutz-Jeghers patients, there are reports of adenomatous and malignant changes in the polyps, and the possibility of a hamartoma-carcinoma sequence has been discussed. A search for a putative tumour suppressor locus was made using comparative genomic hybridization (CGH) of Peutz-Jeghers polyps, combined with loss of heterozygosity (LOH) study. Genetic linkage analysis in 12 families using markers from a deletion site demonstrated the presence of a high-penetrance locus in distal 19p with a multipoint lod score of 7.00 at marker D19S886 without evidence of genetic heterogeneity. The study demonstrates the power of CGH combined with LOH analysis in identifying putative tumour suppressor loci, and provides molecular evidence of malignant potential in hamartomas.

Gains and losses of DNA sequences in malignant mesothelioma by comparative genomic hybridization.

The molecular basis of malignant mesothelioma is poorly known. We examined genetic changes in 11 mesothelioma specimens by comparative genomic hybridization (CGH). Five DNA specimens originated from uncultured tumor tissues and six from cell lines established from the same patients. Findings from the classical karyotypic characterization of both primary tumors and cell lines have been reported previously. In the CGH analyses the most common genetic alterations in the 11 mesothelioma specimens were losses of chromosomal regions in 1p, 8p, 14q, and 22q and gains of 5p, 6p, 8q, 15q, 17q, and 20. The cell lines had on average a much higher total number of genetic changes than the uncultured tumor specimens. Clonal relationship between the cell lines and the uncultured tissue specimens could not usually be demonstrated even though they originated from the same patient. The observed differences may partly be due to high frequency of chromosomal rearrangements, which CGH cannot detect, partly due to contamination of tumor specimens with normal tissue, and partly due to genetic evolution in tumor cell lines.

Absence of trisomy 7 in nonneoplastic human ascitic and pleural fluid cells. An interphase cytogenetic study.

Trisomy 7 is a frequent aneuploid change in lymphomas, adenocarcinomas, and malignant mesenchymal and neurogenic tumors. Moreover, it has been observed in cultured and uncultured non-neoplastic cells from brain, kidney, liver, lung, and atherosclerotic plaques, among other tissues, opening debate on the role of this change in normal and neoplastic tissue. We used nonradioactive in situ hybridization (ISH) with a biotinylated chromosome 7-specific alpha-satellite DNA probe to seek an extra copy of chromosome 7 in ascitic and pleural fluid interphase cells from 26 donors. The donors comprised 24 patients with nonmalignant clinical history, one patient with non-Hodgkin's malignant lymphoma (positive control), and one patient with chronic myeloid leukemia (CML, negative control). The highest frequency of fluid cells with three hybridization signals in patients without neoplasia was 0.5%, in contrast to the frequency of 40.5% noted in the fluid cells of the patient with non-Hodgkin's malignant lymphoma. The results demonstrate that the frequency of trisomic cells in pleural as well as in ascitic fluid is very low, making possible use of the cells in ascitic or pleural fluids in identification of malignancy.

Prenatal sex determination by in situ hybridization on fetal nucleated cells in maternal whole venous blood.

Our aim was to evaluate whether the sex of a fetus could be determined in maternal whole venous blood by in situ hybridization without enrichment of fetal cells. This procedure is virtually without risks to the fetus or the mother. Blood samples were obtained from 59 women at different stages of pregnancy. Twenty preparations were discarded because they were technically unfit for in situ hybridization. Of the remaining 39 pregnant women, 18 had a male fetus, one had male twins, and 20 had a female fetus. Y-positive cells were detected in 12 of the 19 pregnancies with male fetuses and in two of the 20 pregnancies with a female fetus. The frequencies of cells with Y-signals ranged from 1 in 100,000 to 1 in 639. Our results show that fetal cells in maternal blood cannot be reliably used for prenatal diagnosis without prior enrichment of fetal cells.

ISH and PCR study with Y-specific probe/primers.

Our main aim was to evaluate whether maternal whole venous blood could be used for determination of fetal sex, when no enrichment of fetal cells was attempted and when "standard" interphase cytogenetics and PCR analysis were adopted. Altogether 39 pregnant women were studied by using ISH and 59 by using PCR. Out of the 59 pregnant women, 26 carried a male fetus and 33 a female fetus. By ISH, Y-positive cells were detected in 12 of 19 pregnancies with a male fetus and in two of the 20 pregnancies with a female fetus. The frequency of the fetal cells ranged from 1 in 639 to 1 in 100,000. By nested PCR with primers flanking a Y-specific repeat sequence, the positive band indicating a male fetus was found in one of the 26 pregnancies with a male fetus and in one of the 33 pregnancies with a female fetus. According to our results, fetal cells in maternal blood cannot be reliably used for prenatal diagnosis without enrichment of fetal cells.