help button home button Am J Pathol R & D Systems
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tanner, M. M.
Right arrow Articles by Isola, J. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tanner, M. M.
Right arrow Articles by Isola, J. J.
(American Journal of Pathology. 1998;153:191-199.)
© 1998 American Society for Investigative Pathology

Regular Articles

Genetic Aberrations in Hypodiploid Breast Cancer

Frequent Loss of Chromosome 4 and Amplification of CyclinD1 Oncogene

Minna M. Tanner* , Ritva A. Karhu* , Nina N. Nupponen* , Åke Borg{dagger} , Bo Baldetorp{dagger} , Tanja Pejovic{dagger} , Mårten Fernö{dagger} , Dick Killander{dagger} and Jorma J. Isola*

From the Laboratory of Cancer Genetics,* University and University Hospital of Tampere, Tampere, Finland, and the Department of Oncology,{dagger} University of Lund, Lund, Sweden


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
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.1,2 These aberrations range from single nucleotide point mutations and structural chromosomal changes to gross change in chromosome copy number, called aneuploidy.1,2 Approximately 60 to 80% of breast cancers show clear evidence of an aneuploid DNA content by image and flow cytometry (FCM).3-5 Usually a single aneuploid stemline is observed by FCM, most frequently showing a hypotetraploid DNA content (DNA index, 1.7 to 1.8).1,3 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.1,3 Multiple DNA aneuploid stemlines have been observed in 5 to 10% of cases.1,3 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, <0.95).3,6,7 The prognosis of patients with different types of aneuploid tumors have been studied extensively (reviewed in 1 and 2 ). In one study, hypodiploid aneuploidy was associated with the worst clinical outcome of all types of aneuploidy, even after adjustment of other prognostic factors.3

Apart from its prognostic clinical utility, DNA ploidy analysis may provide interesting global information on the genetic evolution of breast cancer. Because little information is available from premalignant (atypical hyperplasia) and preinvasive (ductal carcinoma in situ (DCIS)) breast lesions, the evolution of somatic genetic aberrations has been difficult to assess. Based on a flow cytometric and cytogenetic study,8 structural chromosomal aberrations and losses of entire chromosomes have been suggested to occur first during genetic evolution of breast tumors. These would lead to a transient hypodiploid cell clone, the DNA of which is then doubled by endoreduplication. This pathway would explain the most commonly observed hypotetraploid DNA content (DNA index of 1.7 to 1.9).1,8,9 Alternatively, it has been proposed that endoreduplication is an early event in the genetic cascade, which is followed by successive chromosomal changes that give additional growth advantage to the tumor cells.10 However, both theories have little direct empirical evidence, and the molecular pathogenesis of breast cancer has therefore remained poorly understood.

Comparative genetic hybridization (CGH) is a new molecular cytogenetic method that can detect unbalanced structural chromosomal aberrations anywhere in the genome in a single hybridization.11 So far, CGH has been used to characterize primary breast tumors,12-15 preinvasive intraductal cancers,16,17 and primary tumors and their metastases.18 Chromosomal aberrations by CGH have been correlated to DNA ploidy in two studies,14,15 both showing that aneuploid tumors contain a higher number of copy number aberrations than diploid tumors.14,15 We combined flow cytometric ploidy analyses with CGH and fluorescence in situ hybridization (FISH) to characterize genetic aberrations in hypodiploid breast cancer, an interesting tumor entity associated with genetic evolution and aggressive clinical course.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients and Tumor Material

Fifteen freshly frozen primary breast cancer samples were selected from a tumor bank of over 10,000 primary breast carcinomas at the Department of Oncology, University of Lund (Lund, Sweden). Air-dried imprint touch preparations were available from 10 of these tumors for FISH. The clinical features of the patients are shown in Table 1 . The mean age of patients was 58.8 years.


View this table:
[in this window]
[in a new window]
  		Table 1. Clinicopathological and DNA Flow Cytometric Characteristics of 15 Hypodiploid Cancers Studied by CGH and FISH

 
DNA Flow Cytometry

Freshly frozen samples (100 to 200 mg) were prepared, and flow cytometric (FCM) analysis was performed as previously described.19 Calculation of the DNA index was done after zero-point adjustment of the DNA histogram using the modal values of trout and chicken red blood cells. The mean channel numbers of all G0/G1 peaks were then used for the calculation of the DNA index with trout red blood cells as reference standard. A hypodiploid cell population was defined as a clearly detectable subpopulation (at least 15%) of cells having a DNA index less than 0.95.3 An example of a hypodiploid FCM histogram is shown in Figure 1 .



View larger version (21K):
[in this window]
[in a new window]
  		Figure 1. Flow cytometric DNA histogram demonstrating hypodiploidy in a breast cancer sample. A small DNA diploid G0/G1 stemline with a DNA index of 1.00 and a larger DNA hypodiploid stemline with a prominent G0/G1 peak with a DNA index of 0.93 (corresponding to 85% of all nuclei analyzed). The S-phase fraction (S) was 4.4%, indicating a low tumor proliferation rate. G2 refers to G2/M phase of the cell cycle for the hypodiploid stemline. C and T stand for chicken and trout red blood cells, which were used as internal controls.

 
Simultaneously with the DNA analysis of tumors B and E, nuclei from the two different G0/G1 peaks were separated by sorting into small glass vials. Electronic gates were set in the DNA histogram around the stemline peaks as sorting criteria. Phosphate-buffered saline was used as sheath fluid for sorting. The analysis rate was approximately 150 nuclei per second. The electronic controls for the sorting were set as follows: droplet frequency, 30 kHz; three droplets per sorted event; and coincidence check of five droplets, yielding an efficiency close to 100% and purity over 95%. Up to 100,000 sorted nuclei were collected for each peak and stored until DNA was extracted for CGH.

Comparative Genomic Hybridization

CGH was done according to a published protocol.20,21 Briefly, tumor DNA and normal female reference DNA were extracted using a standard protocol and labeled with fluorescein isothiocyanate (FITC)-dUTP and Texas-Red-dUTP (DuPont, Boston, MA) using standard nick translation. DNA from FCM-sorted nuclei was first amplified with universal primer polymerase chain reaction (DOP-PCR) to obtain a sufficient amount of starting material for CGH.22 Labeled DNAs (400 ng each) and 10 µg of unlabeled Cot-1 DNA (Gibco BRL, Gaithersburg, MD) were hybridized onto commercially available normal metaphase chromosomes (Vysis, Downers Grove, IL). The hybridizations were evaluated using a commercial digital image analysis systems (Vysis and SCILImage software package, TNO, Delft, The Netherlands).

DNA Probes for Fluorescence in Situ Hybridization

The following plasmid probes were used to detect {alpha}-satellite repeat sequences of each chromosome-specific pericentromeric region: pUC177 (for the centromere of chromosome 1), p{alpha}3.5 (chromosome 3), pJM128 (chromosome 8), pHuR98 (chromosome 9), pLC11A (chromosome 11), pA12H8 (chromosome 12), pHUR195 (chromosome 16), p17H8 (chromosome 17), p18R (chromosome 18), and pBAMX7 (chromosome X). In addition, commercial centromeric probes for chromosomes 2 (locus D2Z), 4 (D4Z1), and 10 (D10Z1) (Oncor, Gaithersburg, MD) were also used. A gene-specific P1 probe for the MYC oncogene (obtained from Resource for Molecular Cytogenetics, Berkeley, CA) at 8q24 and a PAC probe for the Cyclin D1 oncogene (at locus 11q13) were used to determine the gene copy numbers of these genes. The PAC probe for Cyclin D1 was obtained by screening a PAC library by PCR using specific primers for Cyclin D1.23 Probes were labeled either with biotin-14-dATP (Gibco BRL) or digoxigenin-11-dUTP (Boehringer Mannheim, Indianapolis, IN) using nick translation.

Fluorescence in Situ Hybridization

Imprint touch preparations were done by lightly pressing a semi-thawed frozen tumor onto cleaned and Vectabond-coated (Vector Laboratories, Burlingame, CA) microscope slides. After air drying, slides were fixed with 50%, 70%, and 100% Carnoy's solution (3:1 methanol/acetic acid, 10 minutes each). Samples were then fixed with 4% paraformaldehyde in PBS (5 minutes at 4°C), dehydrated in graded ethanols, air dried, and baked at 80°C for 30 minutes in a hybridization oven. Two-color FISH was carried out as previously described24 with minor modifications. Slides were denatured in a 70% formamide/2X SSC at 72°C to 74°C for 3 minutes followed by a proteinase K digestion (0.65 µg/ml, 5 minutes). Five nanograms of differentially labeled pericentromeric probes, 20 ng Cyclin D1 and MYC probes (when appropriate) and 10 µg human placental DNA were hybridized onto in a hybridization mixture described previously.24 After standard post-hybridization washes, bound probes were detected immunohistochemically with avidin-FITC (Vector Laboratories, Burlingame, CA) and anti-digoxigenin rhodamine (Boehringer Mannheim).24 Slides were counterstained with 0.2 µmol/L 4,6-diamino-2-phenylindole in an anti-fade solution (Vectashield, Vector Laboratories). Hybridization signals were scored using a Zeiss Axioplan 2 epifluorescence microscope equipped with dual band-pass fluorescence filter (Chromatechnology, Brattleboro, NV), which enables simultaneous detection of both fluorescein and Texas Red fluorescence. Hybridization signals from at least 300 nuclei were scored to assess the chromosome copy numbers and amplification status of Cyclin D1 and MYC. Digital images were taken with a Hamamatsu 9585 camera (Hamamatsu, Hamamatsu City, Japan) operated via ISIS image analysis software (MetaSystems, Altslussheim, Germany). Control hybridizations to normal fibroblasts were done to ascertain that the hybridization efficiencies of the test and reference probes were similar.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Overview of Copy Number Aberrations in 15 Hypodiploid Breast Cancers by CGH

Fourteen tumors contained copy number gains and thirteen contained losses by CGH (Table 2) . Gains and losses of entire chromosomes were detected in 8 and 12 tumors, respectively. Smaller subregional gains were detected in 14 tumors and losses in 11 tumors (Table 2) . The total number of gains per tumor was 4.3 ± 3.2 and that of losses was 4.5 ± 3.3 (mean ± SD). The most common gains were at 11q13 (9 of 15), 1q (7 of 15), 19 (7/15), and 16p (6/15), whereas the most frequent losses were entire chromosomes 4, 13, and 18 and subregional losses of 5p, 5q, 2q, 6q, and 9p (Figures 2 and 3) .


View this table:
[in this window]
[in a new window]
  		Table 2. Gains and Losses in 15 Hypodiploid Breast Cancers by CGH

 


View larger version (31K):
[in this window]
[in a new window]
  		Figure 2. Summary of the gains and losses in 15 hypodiploid breast cancers by CGH. Lines on the right side of the chromosome indicate copy number gains, and those on the left side indicate copy number losses. Thick lines indicate high-level amplification.

 


View larger version (38K):
[in this window]
[in a new window]
  		Figure 3. The comparison of the frequencies of the most common gains (A) and losses (B) in 15 hypodiploid (black columns) to 55 unselected (gray columns) breast cancers (Tirkkonen et al15). Only aberrations common (>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.

 
Comparison of hypodiploid tumors with 55 unselected invasive ductal breast cancers (Tirkkonen et al15 ) 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 Findings

Seven 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 .



View larger version (110K):
[in this window]
[in a new window]
  		Figure 4. Examples 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.

 
Amplification of Cyclin D1 Oncogene in Hypodiploid Tumors

CGH 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 indicating Cyclin 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).


View this table:
[in this window]
[in a new window]
  		Table 3. Summary of CGH at 11q13 and FISH of Cyclin D1 Oncogene in 15 Hypodiploid Breast Cancers

 


View larger version (78K):
[in this window]
[in a new window]
  		Figure 5. High-level amplification of Cyclin D1 oncogene at 11q13 in a hypodiploid breast tumor. A: CGH green-to-red fluorescence profile showing a copy number increase at 11q13. B: FISH of the same tumor demonstrating amplification of the Cyclin D1 gene (numberous green signals). A chromosome 11 centromere probe was as a reference red signals. DAPI blue was used as a counterstain.

 
Evidence that Subregional Copy Number Aberrations Precede Hypodiploidization

Because several copy number aberrations involving chromosome arms and smaller regions (such as gain at 11q13) were observed, we studied next whether chromosomal losses leading to hypodiploidy precede subregional aberrations or vice versa. For this, nuclear suspensions from two tumors (B and E) were flow cytometrically sorted into diploid and hypodiploid cell populations, which were then analyzed by CGH after amplifying the extracted genomic DNA by DOP-PCR. As illustrated in Figure 6 , genetic aberrations, such as gains of 11q13 and 16p, were present in cells with both diploid and hypodiploid DNA content. The hypodiploid tumor population showed additional losses of entire chromosomes (chromosome 8 in Figure 6 ).



View larger version (23K):
[in this window]
[in a new window]
  		Figure 6. Comparison of averaged CGH profiles from flow cytometrically sorted diploid and hypodiploid subpopulations of tumor B. Green-to-red fluorescence ratio profiles from chromosomes 8, 11, and 16 demonstrate that gain of 11q13 and 16p are present both in diploid and hypodiploid populations and that the latter shows an additional loss of the entire chromosome 8.

 
In addition to CGH performed from FCM-sorted cells, evidence that structural chromosomal aberrations precede hypodiploidization was also obtained by FISH. As illustrated in Figure 7 , amplification of Cyclin D1 oncogene was seen in cells with either one or two copies of chromosome 11. Similarly, FISH with probes to the MYC oncogene and chromosome 8 centromere revealed a mixture of cells with 2:1 and 4:2 ratios of MYC and centromere 8 probes, indicating that duplication of MYC has occurred before losing one copy of chromosome 8.



View larger version (72K):
[in this window]
[in a new window]
  		Figure 7. Evidence by FISH that subregional genetic aberrations are present in both diploid and hypodiploid tumor cell populations. A: Tumor C showing nuclei with a single copy of chromosome 8 (green) and a duplication of MYC oncogene (red) and cells with two copies of chromosome 8 and four copies of MYC. b: Tumor E showing amplification of Cyclin D1 gene (numberous green signals) in cells with a single copy and those with two copies of chromosome 11 (red signals).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present results provide for the first time a detailed genetic characterization of a subgroup of breast tumors defined as hypodiploid by DNA FCM. Our goal was to reveal the chromosomal losses that may be critical for development of the known aggressive phenotype.3 Surprisingly, we found numerous gains in 14 of 15 tumors studied. The gains included those of entire chromosomes (such as chromosome 19), chromosome arms (1q, 8q), and smaller regional copy number gains. Nevertheless, an estimation of a quantitative net effect of gains and losses on DNA content indicated predominance of losses in all but two tumors (data not shown). The estimation was done by measuring the size of each aberration in megabases and assuming losses and gains to be due to increase or decrease of one copy (2 to 3 for gains and 2 to 1 for losses). The overall loss of DNA material can be explained by the relatively small size of gains, which was overcompensated by losses of chromosome arms and entire chromosomes. Thus, the CGH results are quantitatively in agreement with FCM in hypodiploid breast tumors.

The hypodiploid tumors had on average 4.3 gains and 4.5 losses per tumor. These numbers are equal to those in unselected hyperdiploid breast carcinomas (4.8 and 4.3, respectively),15 and only slightly higher than in DNA diploid tumors (3.4 and 2.7).15 This similarity of the CGH aberration patterns in diploid and nondiploid breast cancers (Ref. 15 and current data) suggests that ploidy changes are secondary to the relative chromosomal copy number aberrations detected by CGH. This concept is supported by direct evidence from CGH analyses of FCM-sorted cell subpopulations from the same tumor showing that copy number aberrations (such as gain of 11q13 and 16p) were present already in diploid cells. Additional chromosome losses were detected in a hypodiploid subpopulation, indicating subsequent chromosome losses as a cause for hypodiploidy. These findings were confirmed by FISH, which showed Cyclin D1 amplification in cells monosomic or disomic for chromosome 11. Similarly, a structural aberration of chromosome 8 (MYC duplication) was present in another tumor before loss of the other homologue of chromosome 8. An isochromosome formation could also explain findings revealed by FISH, but CGH from the same case did not show 8p loss (Table 2) , which suggests that MYC duplication is due to subregional aberration, not isochromosome formation. In this study DOP-PCR CGH allowed comparison of two DNA FCM-defined subclones. These results, together with supporting evidence acquired by FISH, directly suggest that unbalanced structural chromosomal aberrations (gains and losses) occur earlier during breast carcinogenesis than aneuploidization. Both types of DNA alterations probably occur very early during carcinogenesis, as already the preinvasive breast carcinomas are often aneuploid1,5 and show chromosomal aberrations,16,17,25 loss of heterozygosity,26 and oncogene amplifications.27,28

Previously, allelotyping and FCM studies have hypothesized that the most common type of aneuploidy, the near tetraploidy (DNA index, 1.7 to 1.9), is a consequence of endoreduplication of a hypodiploid clone.1,8,9 These endoreduplicated cell clones would then gradually replace the original hypodiploid clone.1,8,9 Our results support the hypothesis of a common pathogenetic origin, because many gains typical of diploid and hyperdiploid cancers (eg, gains of 1q, 8q, and 16p) were common also in hypodiploid tumors. The reason why hypodiploid stemlines persist in 1 to 2% of breast tumors remains unknown.3 It is possible that the persisting hypodiploid clones have accumulated certain critical genetic changes and acquired a growth advantage, which is only marginally improved by polyploidization. Cells persisting in a hypodiploid state may be more susceptible to loss of tumor suppressor gene activity, because unmasking of a somatic recessive mutation would require loss of only one wild-type allele. Loss of several wild-type alleles is required for total tumor suppressor gene inactivation in tetraploid cells. A candidate for such a locus is chromosome 4, which was frequently lost in hypodiploid (53%) but rarely in unselected breast cancers (11%). Thus, chromosome 4 may harbor one or more critical tumor suppressor genes that may contribute to the maintenance of the hypodiploid state.

Hypodiploid tumors showed evidence for high-level gene amplification for only one locus, chromosome band 11q13. Nine of fifteen tumors (60%) showed this aberration by CGH, which led us to study this locus more specifically with FISH. Using a PAC probe we were able to demonstrate that the gain of 11q13 was due to amplification of the well characterized Cyclin D1 oncogene.29,30 Thus, the overall prevalence of Cyclin D1 amplification in hypodiploid tumors is approximately three to four times higher than in unselected breast tumors, where large studies have reported 15 to 20% occurrence.30-32 Cyclin D1 amplification is therefore a characteristic feature of hypodiploid breast cancers, similar to ERB-B2 amplification in intraductal comedo-type tumors.28 The Cyclin D1 oncogene amplification is known to be associated with positive hormone receptor status,30-32 and evidence for this was obtained also in the hypodiploid tumors. Seven of ten Cyclin D1-amplified tumors were hormone receptor positive, compared with two of five tumors with no Cyclin D1 amplification. Cyclin D1 amplification has also been associated with aggressive clinical outcome,31,32 similar to the hypodiploid DNA content.3 It is tempting to speculate that the poor prognosis of hypodiploid breast cancers may be explained in most cases by Cyclin D1 amplification. Using the information obtained from the present study, it is now possible to test more specifically the association of hypodiploidy and Cyclin D1 amplification with FCM and FISH.

In conclusion, the combination of CGH and FISH with DNA FCM turned out to be a powerful combination in characterizing genetic aberrations in hypodiploid cancers and defining the possible genetic evolution pathways. CGH revealed a partly distinct pattern of genetic aberrations in hypodiploid breast carcinomas compared with unselected ones. Loss of chromosome 4 and amplification of the Cyclin D1 oncogene were defined as characteristic aberrations without any previous knowledge of involvement of these aberrations in hypodiploid tumors. Thus, the present CGH and FISH study indicates that histologically indistinguishable ductal invasive breast cancers consist of several distinct entities (such as the hypodiploid tumors) that can be defined and characterized by modern molecular techniques. In the future, this molecular subclassification of breast cancers may benefit clinicians in selecting optimal therapy individually for each patient.


   Acknowledgements
 
We thank Mrs. Minna Ahlstedt-Soini, Maria Johansson, and Mrs. Ghita Fallenius for excellent technical assistance.


   Footnotes
 
Address reprint requests to Prof. Jorma Isola, University of Tampere, P.O. BOX 607 FIN-33101 Tampere, Finland. E-mail: bljois{at}uta.fi

Supported by the Finnish Cancer Foundation, the Scientific Foundation of Tampere University Hospital, the Swedish Cancer Society, the Mrs. Berta Kamprads Foundation, the Gunnar, Arvid, and Elisabeth Nilsson Foundation, the John and Augusta Persson Foundation, the Inga-Britt and Arne Lundberg Foundation, the Franke and Margareta Bergquist Foundation, the Anna-Lisa and Sven-Erik Lundgren Foundation, and the Hospital of Lund Foundation.

Accepted for publication April 10, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Devilee P, Cornelisse C: Somatic genetic changes in human breast cancer. Biochim Biophys Acta 1994, 1198:113-130[Medline]
  2. Bieche I, Lidereau R: Genetic alterations in breast cancer. Genes Chromosomes Cancer 1995, 14:227-251[Medline]
  3. Fernö 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, 31:157-165[Medline]
  4. Kallioniemi 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, 56:637-642[Medline]
  5. Beerman 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, 15:34-39
  6. Coulson PB, Thornthwaite JT, Woolley TW, Sugarbaker EV, Seckinger D: Prognostic indicators including DNA histogram type, receptor content, and staging related to human breast cancer patient survival. Cancer Res 1984, 44:4187-4196[Abstract/Free Full Text]
  7. Remvikos Y, Gerbault-Seurreau M, Vielh P, Zafrani B, Magdelenat H, Dutrillaux B: Relevance of DNA ploidy as a measure of genetic deviation: a comparison of flow cytometry and cytogenetics in 25 cases of human breast cancer. Cytometry 1988, 9:612-618[Medline]
  8. Dutrillaux B, Gerbault-Seureau M, Remvikos Y, Zafrani B, Prieur M: Breast cancer genetic evolution. I. Data from cytogenetics and DNA content. Breast Cancer Res Treat 1991, 19:245-255[Medline]
  9. Cornelisse CJ, Kuipers-Dijkshoorn N, van Vliet M, Hermans J, Devilee P: Fractional allelic imbalance in human breast cancer increases with tetraploidization and chromosome loss. Int J Cancer 1992, 50:544-548[Medline]
  10. Ewers SB, Långström E, Baldetorp B, Killander D: Flow-cytometric DNA analysis in primary breast carcinomas and clinicopathological correlations. Cytometry 1984, 5:408-419[Medline]
  11. Kallioniemi A, Kallioniemi OP, Sudar D, Rutovitz D, Gray JW, Waldman F, Pinkel D: Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors. Science 1992, 258:818-821[Abstract/Free Full Text]
  12. Kallioniemi A, Kallioniemi OP, Piper J, Tanner M, Stokke T, Chen L, Smith HS, Pinkel D, Gray JW, Waldman FM: Detection and mapping of amplified DNA sequences in breast cancer by comparative genomic hybridization. Proc Natl Acad Sci USA 1994, 91:2156-2160[Abstract/Free Full Text]
  13. Isola JJ, Kallioniemi OP, Chu LW, Fuqua SA, Hilsenbeck SG, Osborne CK, Waldman FM: Genetic aberrations detected by comparative genomic hybridization predict outcome in node-negative breast cancer. Am J Pathol 1995, 147:905-911[Abstract]
  14. Ried T, Just K, Holtgreve-Grez H, du Manoir S, Speicher M, Schröck E, Latnam C, Biegen H, Zetterberg A, Cremer T, Auer G: Comparative genomic hybridization of formalin-fixed paraffin-embedded breast tumors reveals different patterns of chromosomal gains and losses in fibroadenomas and diploid and aneuploid breast carcinomas. Cancer Res 1995, 55:5415-5423[Abstract/Free Full Text]
  15. Tirkkonen M, Tanner M, Karhu R, Kallioniemi A, Isola J, Kallioniemi OP: Molecular cytogenetics of primary breast cancer by CGH. Genes Chromosomes & Cancer 1998, 21:177-184[Medline]
  16. Kuukasjärvi T, Tanner M, Pennanen S, Karhu R, Kallioniemi OP, Isola J: Genetic aberrations in intraductal breast cancers by comparative genomic hybridization. Am J Pathol 1997, 150:1465-1471[Abstract]
  17. James A, Mitchell ELD, Menasce L, Varley JM: Comparative genomic hybridization of ductal carcinoma in situ of the breast: identification of regions of DNA amplification and deletion in common with invasive breast cancer. Oncogene 1997, 14:1059-1065[Medline]
  18. Kuukasjärvi T, Karhu R, Tanner M, Kähkönen M, Nupponen N, Pennanen S, Schaffer A, Kallioniemi A, Kallioniemi O, Isola J: Clonal heterogeneity and clonal evolution underlying development of asynchronous metastasis in human breast cancer. Cancer Res 1997, 57:1597-1604[Abstract/Free Full Text]
  19. Baldetorp B, Fernö M, Fallenius A, Fallenius-Vecchi G, Idvall I, Olsson H, Sigurdsson H, Åkerman M, Killander D: Image cytometric DNA analysis in human breast cancer analysis may add prognostic information in diploid cases with low S-phase fraction by flow cytometry. Cytometry 1992, 13:577-585[Medline]
  20. Kallioniemi OP, Kallioniemi A, Piper J, Isola J, Waldman FM, Gray JW, Pinkel D: Optimizing comparative genomic hybridization for analysis of DNA sequence copy number changes in solid tumors. Genes Chromosomes & Cancer 1994, 10:231-243[Medline]
  21. Karhu R, Kähkönen M, Kuukasjärvi T, Pennanen S, Tirkkonen M, Kallioniemi O: Quality control of CGH: impact of metaphase chromosomes and the dynamic range of hybridization. Cytometry 1997, 28:198-205[Medline]
  22. Kuukasjärvi T, Tanner M, Pennanen S, Karhu R, Visakorpi T, Isola J: Optimizing DOP-PCR for universal amplification of small DNA samples in comparative genomic hybridization. Genes Chromosomes & Cancer 1997, 18:94-101[Medline]
  23. Ioannou PA, Amemiya CT, Garnes J, Kroisel PM, Shizuya H, Chen C, Batzer MA, de Jong PJ: A new bacteriophage P1-derived vector for the propagation of large human DNA fragments. Nature Genet 1994, 6:84-89[Medline]
  24. Tanner MM, Tirkkonen M, Kallioniemi A, Collins C, Stokke T, Karhu R, Kowbel D, Shadravan F, Hintz M, Kuo WL, Waldman FM, Isola JJ, Gray JW, Kallioniemi O-P: Increased copy number at 20q13 in breast cancer: defining the critical region and exclusion of candidate genes. Cancer Res 1994, 54:4257-4260[Abstract/Free Full Text]
  25. Nielsen KV, Blichert-Toft M, Andersen J: Chromosome analysis of in situ breast cancer. Acta Oncol 1989, 28:919-922[Medline]
  26. Fujii H, Szumel R, Marsh C, Zhou W, Gabrielson E: Genetic progression, histological grade, and allelic loss in ductal carcinoma in situ of the breast. Cancer Res 1996, 56:5260-5265[Abstract/Free Full Text]
  27. Murphy DS, Hoare SF, Going JJ, Mallon EEA, George WD, Kaye SB, Brown R, Black DM, Keith WN: Characterization of extencive genetic alterations in ductal carcinoma in situ by fluorescence in situ hybridization and molecular analysis. J Natl Cancer Inst 1995, 87:1694-1704[Abstract/Free Full Text]
  28. Van deVijver M, Peterse JL, Mooi WJ, Wisman P, Lomans J, Dalesio O, Nusse R: Neu-protein overexpression in breast cancer: association with comedo-type ductal carcinoma in situ and limited prognostic value in stage II breast cancer. N Engl J Med 1988, 319:1239-1245[Abstract]
  29. Schuuring E: The involvement of the chromosome 11q13 region in human malignancies: cyclin D1 and EMS1 are two new candidate oncogenes. Gene 1995, 159:83-96[Medline]
  30. Dickson C, Fantl V, Gillett C, Brookes S, Bartek J, Smith R, Fisher C, Barnes D, Peters G: Amplification of chromosome band 11q13 and a role for cyclin D1 in human breast cancer. Cancer Lett 1995, 90:43-50[Medline]
  31. Borg Å, Sigurdsson H, Clark G, Fernö M, Fuqua S, Olsson H, Killander D, McGuire WL: Association of INT2/HST1 coamplification in primary breast cancer with hormone-dependent phenotype and poor prognosis. Br J Cancer 1991, 63:136-142[Medline]
  32. Berns EM, Foekens JA, vanStaveren IL, vanPutten WL, de Koning HY, Portengen H, Klijn JG: Oncogene amplification and prognosis in breast cancer: relationship with systemic treatment. Gene 1995, 159:11-18[Medline]



This article has been cited by other articles:


Home page
 J. Clin. Pathol.Home page
A E Pinto, L Roque, R Rodrigues, S Andre, and J Soares
Frequent 7q gains in flow cytometric multiploid/hypertetraploid breast carcinomas: a study of chromosome imbalances by comparative genomic hybridisation
J. Clin. Pathol., April 1, 2006; 59(4): 367 - 372.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
K. J. Reichenberger, R. D. Coletta, A. P. Schulte, M. Varella-Garcia, and H. L. Ford
Gene Amplification Is a Mechanism of Six1 Overexpression in Breast Cancer
Cancer Res., April 1, 2005; 65(7): 2668 - 2675.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
S. Staff, J. Isola, and M. Tanner
Haplo-insufficiency of BRCA1 in Sporadic Breast Cancer
Cancer Res., August 15, 2003; 63(16): 4978 - 4983.
[Abstract] [Full Text] [PDF]

Home page
 J. Nutr.Home page
G. L. Firestone and L. F. Bjeldanes
Indole-3-Carbinol and 3-3'-Diindolylmethane Antiproliferative Signaling Pathways Control Cell-Cycle Gene Transcription in Human Breast Cancer Cells by Regulating Promoter-Sp1 Transcription Factor Interactions
J. Nutr., July 1, 2003; 133(7): 2448S - 2455.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
G. P. Risbridger, J. F. Schmitt, and D. M. Robertson
Activins and Inhibins in Endocrine and Other Tumors
Endocr. Rev., December 1, 2001; 22(6): 836 - 858.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
K. Rennstam, B. Baldetorp, S. Kytölä, M. Tanner, and J. Isola
Chromosomal Rearrangements and Oncogene Amplification Precede Aneuploidization in the Genetic Evolution of Breast Cancer
Cancer Res., February 1, 2001; 61(3): 1214 - 1219.
[Abstract] [Full Text]

Home page
 Cancer Res.Home page
F. Forozan, E. H. Mahlamäki, O. Monni, Y. Chen, R. Veldman, Y. Jiang, G. C. Gooden, S. P. Ethier, A. Kallioniemi, and O.-P. Kallioniemi
Comparative Genomic Hybridization Analysis of 38 Breast Cancer Cell Lines: A Basis for Interpreting Complementary DNA Microarray Data
Cancer Res., August 1, 2000; 60(16): 4519 - 4525.
[Abstract] [Full Text]

Home page
 Am. J. Pathol.Home page
S. Knuutila, Y. Aalto, K. Autio, A.-M. Bjorkqvist, W.'e. El-Rifai, S. Hemmer, T. Huhta, E. Kettunen, S. Kiuru-Kuhlefelt, M. L. Larramendy, et al.
DNA Copy Number Losses in Human Neoplasms
Am. J. Pathol., September 1, 1999; 155(3): 683 - 694.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
E. J. Cram, B. D. Liu, L. F. Bjeldanes, and G. L. Firestone
Indole-3-carbinol Inhibits CDK6 Expression in Human MCF-7 Breast Cancer Cells by Disrupting Sp1 Transcription Factor Interactions with a Composite Element in the CDK6 Gene Promoter
J. Biol. Chem., June 15, 2001; 276(25): 22332 - 22340.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tanner, M. M.
Right arrow Articles by Isola, J. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tanner, M. M.
Right arrow Articles by Isola, J. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS