DNA amplifications at 20q13 and MDM2 define distinct subsets of evolved breast and ovarian tumours

DNA amplification seems to be particularly frequent in human breast tumours and has been associated with cancer evolution and aggressiveness. Recent data indicate that new events should be added to the list, such as the amplifications at chromosome 20q13 or the MDM2 gene. The present work aimed at determining the incidence and clinicopathological signification of these amplifications in a large series of breast and ovarian tumours. We tested 1371 breast and 179 ovarian tumours by Southern blotting and observed amplification of 20q13 in 5.4% breast and 2.8% ovarian carcinomas, whereas MDM2 was found amplified in 5.3% and 3.8% of breast and ovarian tumours respectively. MDM2 RNA expression levels were analysed in a subset of 57 breast tumours and overexpression was observed in 4/57 (7%) of the tumours. Elevated expression levels coincided with amplification of the gene. In breast cancer, 20q13 and MDM2 amplifications seem to define subsets of aggressive tumours. Indeed, 20q13 was correlated to axillary nodal involvement and occurred preferentially in younger patients (< 50 years). Furthermore, 20q13 correlated, as did MDM2 amplification, to aneuploidy. In parallel, we had also tested our tumour DNAs for amplification of CCND1, ERBB-2 and MYC, which made it possible to test for correlations with 20q13 or MDM2 amplifications. Whereas 20q13 showed a very strong correlation to CCND1 amplification, that of MDM2 was prevalent in MYC-amplified tumours. Interestingly, 20q13 and MDM2 amplifications showed some degree of correlation to each other, which may possibly be owing to the fact that both events occurred preferentially in aneuploid tumours. In ovarian cancer, no statistically significant correlation was observed. However, 20q13 amplification occurred preferentially in stage 3 tumours and MDM2 was correlated to ERBB-2 amplification. This may suggest that in ovarian tumours also, 20q13 and MDM2 amplifications occur in late or aggressive cancers.     

Cyclin DI amplification is not associated with reduced overall survival in primary breast cancer but may predict early relapse in patients with features of good prognosis

Amplification of chromosome 11q13 is frequently observed in human malignancies, including breast cancers. A candidate oncogene at this locus is the CCND1 gene, which encodes the cell cycle regulatory protein cyclin D1. Because published data on the relationship between 11q13 amplification and prognosis in breast cancer have been controversial, we investigated the clinical significance of CCND1 amplification and its association with established clinicopathological features of prognosis in 1014 primary breast cancer patients. Amplification of the CCND1 gene and the INT-2/FGF-3 gene, which also maps to 11q13, was 10% and 17%, respectively. There were no associations between CCND1 or INT-2 amplification and patient age, tumor size, tumor grade, axillary lymph node status, HER/neu amplification, MIB-1 monoclonal antibody to Ki67 antigen count, or p53 expression. CCND1 amplification was predominantly observed in hormone receptor-positive tumors; at a copy number >/=3, CCND1 amplification was significantly correlated with both estrogen receptor (ER; P = 0.036) and progesterone receptor (P = 0.012) positivity. After a median follow-up period of 66 months, CCND1 or INT-2 amplification was not associated with significant increases in relapse or death from breast cancer. However, in the node-negative and ER-positive subgroups, there was a trend for an increased relapse rate in patients with INT-2 or CCND1 amplification. Thus, in this study, assessment of CCND1 or INT-2 amplification at 11q13 by slot-blot hybridization was of little use in determining phenotype or disease outcome in the whole group of patients but had a potential role in identifying a subset of poor-prognosis patients within the node-negative or ER-positive, good-prognosis groups. Because the prevalence of CCND1 amplification is much lower than the reported prevalence of cyclin D1 overexpression, additional studies are required to determine the true prognostic significance of altered cyclin D1 expression in breast cancer.     

The structure and organization of the human NPAT gene

Ataxia telangiectasia (AT) is an autosomal recessive gene disorder, and ATM, a housekeeping gene, has been identified as the gene responsible for AT. Recently we found that another housekeeping gene, NPAT, is located upstream of ATM on human chromosome 11. The two housekeeping genes are transcribed in opposite directions and share a 0.5-kb 5' flanking sequence. The structure and organization of NPAT were determined by direct sequencing of cosmid clones carrying the gene and by application of the long and accurate (LA)-PCR method to amplify regions encompassing the exon/intron boundaries and all of the exons. The gene spans at least 44 kb and consists of 18 exons and 17 introns. It has been suggested that AT heterozygotes have an increased risk of developing cancer, especially breast cancer in women. Frequently, loss of heterozygosity at loci on 11q22-q24 has been observed in DNA isolated from tumors of the breast, uterine cervix, and colon, perhaps suggesting the location of a tumor suppressor gene in 11q22-q24. For investigation of the role of NPAT in AT and these tumors with allelic loss of 11q22-q24, appropriate primer sequences and PCR conditions for amplification of all the NPAT exons from genomic DNA were determined. We previously reported that no recombinations are found among Atm, Npat, and Acat1 (acetoacetyl-CoA thiolase) loci as determined by fine genetic linkage mapping of the mouse AT region. The results of the LA-PCR analysis using NPAT- and ACAT-specific primers and human genomic DNA allowed us to map ACAT 12 kb centromeric to NPAT.     

Distinct patterns of chromosomal alterations in high- and low-grade head and neck squamous cell carcinomas

Comparative genomic hybridization was performed on 30 primary head and neck squamous cell carcinomas. Fractional or entire DNA loss of chromosome 3p was a basic finding that occurred in 29 cases (97%). Additional DNA underrepresentations were observed in more than 50% of the cases on chromosomes 1p, 4, 5q, 6q, 8p, 9p, 11q, 13q, 18q, and 21q. Deletions on chromosomes 3p, 13q, and 17p were confirmed by loss of heterozygosity analysis. Entire or partial DNA copy number increases were identified for chromosome 3q in 26 cases (87%) with high-level amplifications at 3q24 and 3q27-qter. Overrepresentations were found in decreasing order of frequency at 11q13 (70%), 8q (57%), 19q (50%), 19p (47%), and 17q (47%). The use of comparative genomic hybridization superkaryograms of the group of well-differentiated carcinomas (G1) indicated that the deletions on chromosomes 3p and 9p along with the overrepresentation of 3q are associated with early tumor development. Accordingly, the undifferentiated tumors (G3) were characterized by additional deletions on chromosomes 4q, 8p, 11q, 13q, 18q, and 21q and overrepresentations on 1pter, 11q13, 19, and 22q, suggesting that these changes are preferentially associated with tumor progression.     

Independent amplification and frequent co-amplification of three nonsyntenic regions on the long arm of chromosome 20 in human breast cancer

DNA amplification at 20q13.2 is common in breast cancer, correlates with poor prognosis, and may reflect location of an important oncogene. Recently, other regions along 20q were also found to undergo amplification. Here, amplification levels and patterns of co-amplification were analyzed by interphase fluorescence in situ hybridization at 14 loci along 20q in 146 uncultured breast carcinomas and 14 cell lines. Three regions were independently amplified in uncultured tumors: RMC20C001 region at 20q13.2 (highly amplified in 9.6% of the cases), PTPN1 region 3 Mb proximal (6.2%), and AIB3 region at 20q11 (6.2%). Co-amplifications involving two or three of these regions were seen in 11 of the 19 highly amplified tumors. The results suggest that three distinct nonsyntenic regions along 20q may be important and that complex chromosomal rearrangements underlie their frequent co-amplification in breast cancer.     

Localization of putative tumor suppressor loci by genome-wide allelotyping in human pancreatic endocrine tumors

Only two tumor suppressor gene loci, one on 3p25 and the MEN1 gene on 11q13, have thus far been implicated in the pathogenesis of sporadic human pancreatic endocrine tumors (PETs). A genome-wide allelotyping study of 28 human PETs was undertaken to identify other potential tumor suppressor gene loci. In addition to those on chromosomes 3p and 11q, frequent allelic deletions were identified on 3q (32%), 11p (36%), 16p (36%), and 22q (29%). Finer deletion mapping studies localized the smallest regions of common deletion to 3q27, 11p13, and 16p12.3-13.11. Potential candidate genes at these loci include WT1 (11p13), TSC2 (16p13), and NF2 (22q12), but no known tumor suppressor gene localizes to 3q27. The mean fractional allelic loss among these human PETs is 0.126, and no correlation was observed between allelic loss and clinical parameters, including age, sex, hormonal subtype, and disease stage. These findings highlight novel locations of tumor suppressor gene loci that contribute to the pathogenesis of human PETs, and several of these on 3p, 3q, and 22q are syntenic with loci on mouse chromosomes 9 and 16 that are implicated in a murine transgenic model of PETs.     

Genetic alteration mapping on chromosome 7 in primary breast cancer

Alterations of chromosome 7 are among the most frequent cytogenetic abnormalities found in human breast carcinoma. We examined genetic changes on chromosome 7 in 113 primary human breast tumors, using both microsatellite and restriction fragment length polymorphism/variable number of tandem repeats polymorphism markers mapping to the long arm (15 markers) and the short arm (8 markers). Allelic imbalance at 1 or more loci was observed in 50 (44%) of 113 tumors on the long arm of chromosome 7 and in 41 (36%) tumors on the short arm. Genetic changes of one arm were significantly associated with alterations of the other arm. The 50 7q-altered tumor DNAs exclusively showed a loss of heterozygosity (LOH), 23 (46%) at all informative loci tested on 7q and 27 (54%) at some loci (interstitial and/or telomeric deletions on 7q). The pattern of LOH of these 27 tumors enabled us to identify 3 distinct consensus regions of deletions on 7q, only 1 of which (7q31 region) has already been described in breast cancer. Among the 41 7p-altered tumor DNAs, 32 had a gain and/or loss of the entire short arm of chromosome 7. Fourteen tumor DNAs showed an allelic gain, and 18 tumor DNAs showed a LOH at each locus on the short arm. The other 9 7p-altered tumors showing partial random alterations of chromosome 7p revealed no common altered regions. This is the first report of an association between alterations of DNA sequences on chromosome 7p and breast cancer. The results suggest that tumor suppressor genes are present on the long arm of chromosome 7 and are associated with breast tumorigenesis. Moreover, the frequent loss or gain of a whole copy of chromosome 7p suggests the involvement of a gene dosage effect of this chromosomal arm in the pathogenesis of breast cancer.     

Bronchial mucosal immunoreactivity of sensory neuropeptides in severe airway diseases

DNA copy number changes were investigated in 29 leiomyosarcomas by comparative genomic hybridization. The most frequent losses were detected in 10q (20 cases, 69%) and 13q (17 cases, 59%). The most frequent gains were detected in 17p (16 cases, 55%). The most frequent high-level amplifications were detected in 17p (7 cases, 24%) and 8q (6 cases, 21%). A total of 137 losses and 204 gains were detected. Small tumors (less than 5 cm in diameter) displayed fewer changes per sample (3 to 11; mean, 7) than the other tumors (4 to 22; mean, 13). There was an increase in the number of gains from small tumors (mean, 4) to very large tumors (>20 cm; mean, 10). However, the number of losses was similar in small, large, and very large tumors (mean, 4.5). Tumor size-related aberrations were observed. Gains in 16p were detected in all small tumors but were infrequent in large and very large tumors (27% and 11%, respectively). Similarly, gains and high-level amplifications in 17p were more common in small (80%) than in very large tumors (33%). Gains in 1q, 5p, 6q, and 8q were not seen in any of the small tumors but were detected in large and very large tumors. Gains in 6q and 8q occurred in 8 of 9 cases (89%) of very large tumors, 5 of them with a high-level amplification in 8q.     

Allelic imbalance study of 16q in human primary breast carcinomas using microsatellite markers

The high incidence of allelic imbalance on the long arm of chromosome 16 in breast cancer suggests its involvement in the development and progression of the tumor. Several loss of heterozygosity (LOH) studies have led to the assignment of commonly deleted regions on 16q where tumor suppressor genes may be located. The most recurrent LOH regions have been 16q22.1 and 16q22.4-qter. The aim of this study was to gain further insight into the occurrence of one or multiple "smallest regions of overlap" on 16q in a new series of breast carcinomas. Hence, a detailed allelic imbalance map was constructed for 46 sporadic breast carcinomas, using 11 polymorphic microsatellite markers located on chromosome 16. Allelic imbalance of one or more markers on 16q was shown by 30 of the 46 tumors (65%). Among these 30 carcinomas, LOH on the long arm of chromosome 16 was detected at all informative loci in 19 (41%); 13 of them showed allelic imbalance on the long but not on the short arm, with the occurrence of variable "breakpoints" in the pericentromeric region. The partial allelic imbalance in 11 tumors involved either the 16q22.1-qter LOH region or interstitial LOH regions. A commonly deleted region was found between D16S421 and D16S289 on 16q22.1 in 29 of the 30 tumors. The present data argue in favor of an important involvement of a tumor suppressor gene mapping to 16q22.1 in the genesis or progression of breast cancer.     

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.     

Carney complex, Peutz-Jeghers syndrome, Cowden disease, and Bannayan-Zonana syndrome share cutaneous and endocrine manifestations, but not genetic loci

Carney complex (CC), Peutz-Jeghers syndrome (PJS), Cowden disease (CD), and Bannayan-Zonana syndrome (BZS) share clinical features, such as mucocutaneous lentigines and multiple tumors (thyroid, breast, ovarian, and testicular neoplasms), and autosomal dominant inheritance. A genetic locus has been identified for CC on chromosome 2 (2p16), and the genes for PJS, CD, and BZS were recently identified; genetic heterogeneity appears likely in both CC and PJS. The genes for PJS and CD/BZS, STK11/LKB1 and PTEN, respectively, may act as tumor suppressors, because loss of heterozygosity (LOH) of the PJS and CD/BZS loci has been demonstrated in tumors excised from patients with these disorders. We studied 2 families with CC in whom the disease could not be shown to segregate with polymorphic markers from the 2p16 locus. Their members presented with lesions frequently seen in PJS and the other lentiginosis syndromes. We also tested 16 tumors and cell lines established from patients with CC for LOH involving the PJS and CD/BZS loci. DNA was extracted from peripheral blood, tumor cell lines, and tissues and subjected to PCR amplification with primers from microsatellite sequences flanking the STK11/LKB1 and PTEN genes on 19p13 and 10q23, respectively, and a putative PJS locus on 19q13. All loci were excluded as candidates in both families with LOD scores less than 2 and/or by haplotype analysis. LOH for these loci was not present in any of the tumors that were histologically identical to those seen in PJS. The overall rate of LOH for the PJS and CD/BZS loci in tumors from patients with CC was less than 10%. We conclude that despite substantial clinical overlap among CC, PJS, CD, and BZS, LOH for the STK11 and PTEN loci is an infrequent event in CC-related tumors. Linkage analysis excluded the PJS and CD/BZS loci on chromosomes 19 (19p13 and 19q13) and 10 (10q23) from harboring the gene defect(s) responsible for the phenotype in these 2 families.     

The prototypic Th2 autoimmunity induced by mercury is dependent on IFN-gamma and not Th1/Th2 imbalance

Chromosomal translocations leading to deregulation of specific oncogenes characterize approximately 50% of cases of diffuse large B-cell lymphomas (DLBL). To characterize additional genetic features that may be of value in delineating the clinical characteristics of DLBL, we studied a panel of 96 cases at diagnosis consecutively ascertained at the Memorial Sloan-Kettering Cancer Center (MSKCC) for incidence of gene amplification, a genetic abnormality previously shown to be associated with tumor progression and clinical outcome. A subset of 20 cases was subjected to comparative genomic hybridization (CGH) analysis, which identified nine sites of chromosomal amplification (1q21-23, 2p12-16, 8q24, 9q34, 12q12-14, 13q32, 16p12, 18q21-22, and 22q12). Candidate amplified genes mapped to these sites were selected for further analysis based on their known roles in lymphoid cell and lymphoma development, and/or history of amplification in tumors. Probes for six genes, which fulfilled these criteria, REL (2p12-16), MYC (8q24), BCL2 (18q21), GLI, CDK4, and MDM2 (12q13-14), were used in a quantitative Southern blotting analysis of the 96 DLBL DNAs. Each of these genes was amplified (four or more copies) with incidence ranging from 11% to 23%. This analysis is consistent with our previous finding that REL amplification is associated with extranodal presentation. In addition, BCL2 rearrangement and/or REL, MYC, BCL2, GLI, CDK4, and MDM2 amplification was associated with advanced stage disease. These data show, for the first time, that amplification of chromosomal regions and genes is a frequent phenomenon in DLBL and demonstrates their potential significance in lymphomagenesis.     

Karyotypic changes in phyllodes tumors of the breast

Cytogenetic analysis of short-term cultures of five phyllodes tumors of the breast-classified as benign (one tumor), borderline malignant (two tumors removed from the same breast in 1991 and 1993), and malignant (two tumors)--revealed clonal changes with simple structural abnormalities in the benign tumor, the borderline malignant tumors, and one malignant tumor in which benign areas and areas of borderline malignancy were also present. In contrast, the malignant tumor without admixed borderline malignant or benign areas had a complex karyotype. The karyotype of the benign phyllodes tumor was 46,XX,del(12)(p11p12)/46,XX,t(8;18)(p11;p11)/46,XX. The first borderline malignant phyllodes tumor had t(3;20)(p21;q13) as the sole abnormality. When the tumor recurred, this was no longer the only clone detected and the tumor karyotype was now 46,XX,t(3;20)(p21;q13)/46,XX,t(9;10)(p22;q22)/46,XX,t(1;8) (p34;q24)/46,XX,del(11)(q22-23)/46,XX. The malignant/borderline malignant/benign tumor had t(1;6)(p34;p22) as the sole clonal abnormality. Finally, the karyotype of the malignant phyllodes tumor which contained no benign or borderline malignant areas was 42,XX,der(1)t(1;4)(q21;q21),der(3)t(3;17)(q29;q21), -4,i(8)(q10), -10, -13,i(13)(q10),der(14)t(1;14)(q21;p11),der(14)t(4;14) (p12;p11), -17/80-90,idemx2, +del(1)(q12), +i(1)(p10), +dic(5;5)(p14;p14), +i(6)(p10), +del(7)(p11), +dup(7)(q11q36), +i(15)(q10),inc/46,XX. The findings indicate some cytogenetic similarities between benign/borderline malignant phyllodes tumors and fibroadenomas of the breast, presumably reflecting similar pathogenetic mechanisms in the two types of mixed-lineage tumors.     

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.     

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.     

Identification of a novel gene, MASL1, within an amplicon at 8p23.1 detected in malignant fibrous histiocytomas by comparative genomic hybridization

We used comparative genomic hybridization to study malignant fibrous histiocytomas (MFHs) from 19 patients to detect changes in the copy number of DNA sequences, along entire chromosomes. Together with losses and gains in various chromosomal regions, distinct high-level amplifications were found at six loci (4q12-21, 8p21-pter, 8q24.1-qter, 9q12-13, 12p11.2-pter, and 15q11.2-15), suggesting that those regions may contain unknown (proto) oncogenes. We focused on the 8p amplicon, where detailed characterization allowed us to determine that the minimal common amplified region lay between markers D8S1819 and D8S550 at 8p23.1. A novel gene designated MASL1 (MFH-amplified sequences with leucine-rich tandem repeats 1) was isolated from within this narrowly defined region. Expression of the MASL1 gene was enhanced significantly in MFH tumors bearing the 8p amplicon. The primary structure of its deduced product revealed an ATP/GTP-binding site, three leucine zipper domains, and a leucine-rich tandem repeat, all of which are important structural or functional elements for interactions among proteins related to the cell cycle. These features suggest that overexpression of MASL1 might well be oncogenic with respect to MFH.     

Amplification of c-myc, K-sam, and c-met in gastric cancers: detection by fluorescence in situ hybridization

Gene amplifications of c-myc, K-sam, and c-met were examined in cancer nuclei isolated from 154 primary gastric adenocarcinomas by fluorescence in situ hybridization (FISH) using cosmid probes for 8q24 (c-myc locus) and 7q31 (c-met), as well as a DNA probe for K-sam synthesized by PCR. The results were compared with those of Southern blot analysis. Dual-color FISH using gene locus and chromosome-specific probes detected gene amplifications of c-myc in 24 tumors (15.5%), c-met in 6 tumors (3.9%), and K-sam in 3 tumors (2.9%). The six tumors with c-myc amplification had also been found to have amplified c-erbB-2 in our previous study, and coamplification of c-myc and c-met was found in two other tumors. This technique also differentiated the amplified genes on the homogeneous staining region (HSR) and on double minute chromosomes (DMs) in metaphase spreads and interphase nuclei of cell lines established from poorly differentiated adenocarcinomas, KATO III, SNU 16, and HSC 39. Examination of FISH images of these cell lines suggested that the high-level amplifications of c-myc found in primary tumors occurred mainly on DM in four tumors and on HSR in one, and those of K-sam occured on DM in two tumors and on HSR in one. No high-level amplification of c-met was found. These high-level amplifications were also detected in formalin-fixed, paraffin-embedded tissues from primary gastric tumors and metastatic lymph nodes, in some of which heterogeneity of gene amplification was demonstrated within the same tumor. We conclude that FISH is an important tool for examining the proto-oncogene aberrations in intact cells in solid tumors.