Genetic Aberrations in Hypodiploid Breast Cancer
1998; Elsevier BV; Volume: 153; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)65560-5
ISSN1525-2191
AutoresMinna M. Tanner, Ritva Karhu, Nina N. Nupponen, Åke Borg, Bo Baldetorp, Tanja Pejović, Mårten Fernö, D. Killander, Jorma Isola,
Tópico(s)Cancer Genomics and Diagnostics
ResumoThe evolution of somatic genetic aberrations in breast cancer has remained poorly understood. The most common chromosomal abnormality is hyperdiploidy, which is thought to arise via a transient hypodiploid state. However, hypodiploidy persists in 1 to 2% of breast tumors, which are characterized by a poor prognosis. We studied the genetic aberrations in 15 flow cytometrically hypodiploid breast cancers by comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH). Surprisingly, numerous copy number gains were detected in addition to the copy number losses. The number of gains per tumor was 4.3 ± 3.2 and that of losses was 4.5 ± 3.3 (mean ± SD), which is similar to that previously observed in hyperdiploid breast cancers. Gains at chromosomes or chromosomal regions at 11q13, 1q, 19, and 16p and losses of 2q, 4, 6q, 9p, 13, and 18 were most commonly observed. Compared with unselected breast carcinomas, hypodiploid tumors showed certain differences. Loss of chromosome 4 (53%) and gain of 11q13 (60%) were significantly more common in hypodiploid tumors. The gain at 11q13 was found by FISH to harbor amplification of the Cyclin D1 oncogene, which is therefore three to four times more common in hypodiploid than in unselected breast cancers (15 to 20%). Structural chromosomal aberrations (such as Cyclin D1 amplification) were present both in diploid and hypodiploid tumor cell populations, as assessed by FISH and CGH after flow cytometric sorting. Together these results indicate that hypodiploid tumors form a distinct genetic entity of invasive breast cancer, although they probably share a common genetic evolution pathway where structural chromosomal aberrations precede gross DNA ploidy changes. The evolution of somatic genetic aberrations in breast cancer has remained poorly understood. The most common chromosomal abnormality is hyperdiploidy, which is thought to arise via a transient hypodiploid state. However, hypodiploidy persists in 1 to 2% of breast tumors, which are characterized by a poor prognosis. We studied the genetic aberrations in 15 flow cytometrically hypodiploid breast cancers by comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH). Surprisingly, numerous copy number gains were detected in addition to the copy number losses. The number of gains per tumor was 4.3 ± 3.2 and that of losses was 4.5 ± 3.3 (mean ± SD), which is similar to that previously observed in hyperdiploid breast cancers. Gains at chromosomes or chromosomal regions at 11q13, 1q, 19, and 16p and losses of 2q, 4, 6q, 9p, 13, and 18 were most commonly observed. Compared with unselected breast carcinomas, hypodiploid tumors showed certain differences. Loss of chromosome 4 (53%) and gain of 11q13 (60%) were significantly more common in hypodiploid tumors. The gain at 11q13 was found by FISH to harbor amplification of the Cyclin D1 oncogene, which is therefore three to four times more common in hypodiploid than in unselected breast cancers (15 to 20%). Structural chromosomal aberrations (such as Cyclin D1 amplification) were present both in diploid and hypodiploid tumor cell populations, as assessed by FISH and CGH after flow cytometric sorting. Together these results indicate that hypodiploid tumors form a distinct genetic entity of invasive breast cancer, although they probably share a common genetic evolution pathway where structural chromosomal aberrations precede gross DNA ploidy changes. Over the past decade a substantial amount of evidence has been collected to indicate that the development of malignant tumors requires the sequential accumulation of somatic genetic aberrations.1Devilee P Cornelisse C Somatic genetic changes in human breast cancer.Biochim Biophys Acta. 1994; 1198: 113-130PubMed Google Scholar, 2Bieche I Lidereau R Genetic alterations in breast cancer.Genes Chromosomes Cancer. 1995; 14: 227-251Crossref PubMed Scopus (283) Google Scholar These aberrations range from single nucleotide point mutations and structural chromosomal changes to gross change in chromosome copy number, called aneuploidy.1Devilee P Cornelisse C Somatic genetic changes in human breast cancer.Biochim Biophys Acta. 1994; 1198: 113-130PubMed Google Scholar, 2Bieche I Lidereau R Genetic alterations in breast cancer.Genes Chromosomes Cancer. 1995; 14: 227-251Crossref PubMed Scopus (283) Google Scholar Approximately 60 to 80% of breast cancers show clear evidence of an aneuploid DNA content by image and flow cytometry (FCM).3Fernö M Baldetorp B Borg Å Olsson H Sigurdsson H Killander D Flow cytometric DNA index and S-phase fraction in breast cancer in relation to other prognostic variables and to clinical outcome.Acta Oncol. 1992; 31: 157-165Crossref PubMed Scopus (61) Google Scholar, 4Kallioniemi OP Blanco G Alavaikko M Hietanen T Mattila J Lauslahti K Koivula T Tumour DNA ploidy as an independent prognostic factor in breast cancer.Br J Cancer. 1987; 56: 637-642Crossref PubMed Scopus (165) Google Scholar, 5Beerman H Kluin PM Hermans J van de Velde CJ Cornelisse CJ Prognostic significance of DNA-ploidy in a series of 690 primary breast cancer patients.Int J Cancer. 1990; 15: 34-39Crossref Scopus (113) Google Scholar Usually a single aneuploid stemline is observed by FCM, most frequently showing a hypotetraploid DNA content (DNA index, 1.7 to 1.8).1Devilee P Cornelisse C Somatic genetic changes in human breast cancer.Biochim Biophys Acta. 1994; 1198: 113-130PubMed Google Scholar, 3Fernö M Baldetorp B Borg Å Olsson H Sigurdsson H Killander D Flow cytometric DNA index and S-phase fraction in breast cancer in relation to other prognostic variables and to clinical outcome.Acta Oncol. 1992; 31: 157-165Crossref PubMed Scopus (61) Google Scholar Near-diploid (DNA index, 1.1 to 1.4), triploid (DNA index, 1.4 to 1.6), tetraploid (DNA index, 1.9 to 2.1), and hypertetraploid tumors (DNA index, >2.1) each constitute 5 to 15% of aneuploid breast cancers.1Devilee P Cornelisse C Somatic genetic changes in human breast cancer.Biochim Biophys Acta. 1994; 1198: 113-130PubMed Google Scholar, 3Fernö M Baldetorp B Borg Å Olsson H Sigurdsson H Killander D Flow cytometric DNA index and S-phase fraction in breast cancer in relation to other prognostic variables and to clinical outcome.Acta Oncol. 1992; 31: 157-165Crossref PubMed Scopus (61) Google Scholar Multiple DNA aneuploid stemlines have been observed in 5 to 10% of cases.1Devilee P Cornelisse C Somatic genetic changes in human breast cancer.Biochim Biophys Acta. 1994; 1198: 113-130PubMed Google Scholar, 3Fernö M Baldetorp B Borg Å Olsson H Sigurdsson H Killander D Flow cytometric DNA index and S-phase fraction in breast cancer in relation to other prognostic variables and to clinical outcome.Acta Oncol. 1992; 31: 157-165Crossref PubMed Scopus (61) Google Scholar An infrequent (incidence, 1 to 2%) type of nondiploid DNA content is hypodiploidy, defined as a cellular DNA content less than in normal cells (DNA index, 10%) for either type of tumor population are shown. Hypodiploid breast tumors show distinct pattern of genetic aberrations compared with unselected tumors. The frequency of loss of chromosome 4 and amplification of 11q13 (11q) are statistically significant.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Comparison of hypodiploid tumors with 55 unselected invasive ductal breast cancers (Tirkkonen et al15Tirkkonen M Tanner M Karhu R Kallioniemi A Isola J Kallioniemi OP Molecular cytogenetics of primary breast cancer by CGH.Genes Chromosomes & Cancer. 1998; 21: 177-184Crossref PubMed Scopus (325) Google Scholar) revealed a partly distinct pattern of genetic aberrations in hypodiploid tumors (Figure 3). Gain at 11q13 (60%; 95% confidence interval (CI), 38 to 88%) was more common in hypodiploid than in unselected tumors (18%; 95% CI, 9 to 31%; Figure 3A). Similarly, loss of entire chromosome 4 (53%; 95% CI 27 to 79%) was particularly characteristic for hypodiploid tumors compared with unselected tumors (frequency, 11%; 95% CI, 4 to 22%; Figure 3B). In other aberrations, especially for copy number gains, a rather similar frequency of aberrations was found.Validation of CGH FindingsSeven tumors were analyzed twice by CGH in two different laboratories with virtually identical findings (second analyses were done by T. Pejovic at the University of Yale). Furthermore, copy numbers for chromosomes 1, 2, 3, 4, 8, 9, 10, 11, 16, 17, 18, and X were established independently by FISH with centromere-specific probes. Among the eight tumors tested, 75% concordance (18 of 24 loci confirmed in eight tumors) was found between CGH and FISH. Representative examples are shown in Figure 4.Figure 4Examples of two-color FISH in hypodiploid tumors. Hybridized signals are visualized either in green or red fluorescence, and nuclei are counterstained with DAPI (blue). A tumor sample (I) shows loss of one copy of chromosomes 2 (green) and 18 (red) and a tumor (J) shows loss of one copy of chromosome 4 (green), whereas chromosome X (red) has retained both copies. DAPI (blue) was used for counterstaining of the nuclei. The corresponding CGH copy number profiles of chromosomes 2, 4, 18, and X are shown in below the FISH images.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Amplification of Cyclin D1 Oncogene in Hypodiploid TumorsCGH revealed a regional copy number gain at 11q13 in 9 of 15 cases (60%; Table 3 and Figure 5A). To ascertain whether the gain was due to the well characterized Cyclin D1 gene amplification, imprint touch preparations were analyzed by two-color FISH. A PAC probe for Cyclin D1 was hybridized together with a centromere probe to chromosome 11 (Figure 5B). An increased copy number indicatingCyclin D1 amplification was found in seven cases, all except one showing a regional gain at 11q13 by CGH (Table 3). In one case (H), CGH showed no gain at 11q13, although amplification of Cyclin D1 was found in a small subpopulation of cells by FISH. Thus, the overall frequency of Cyclin D1 amplification was 67% (10 of 15).Table 3Summary of CGH at 11q13 and FISH of Cyclin D1 Oncogene in 15 Hypodiploid Breast CancersCaseCGH at 11q13Cyclin D1 copy number by FISHChromosome 11 centromere copy number by FISHLevel of amplification (cyclin D1/11cen ratio)ANormalNTNTNTCNormal22No amplificationDNormal1–22No amplificationGNormalNTNTNTINormal22No amplificationBGain*Found in cells with both diploid and h
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