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Keywords:

  • 8p11-12 amplification;
  • array CGH;
  • familial breast cancer

Abstract

  1. Top of page
  2. Abstract
  3. Acknowledgements
  4. References
  5. Supporting Information

Amplification of 8p11-12 has been recurrently reported in sporadic breast cancer. These studies define a complex molecular structure with a set of minimal amplified regions, and different putative oncogenes that show a strong correlation between amplification and over-expression such as ZNF703/FLJ14299, SPFH2/C8orf2, BRF2 and RAB11FIP. However, none of these studies were carried out on familial breast malignancies. We have studied the incidence, molecular features and clinical value of this amplification in familial breast tumors associated with BRCA1, BRCA2 and non-BRCA1/2 gene mutations. We detected 9 out of 80 familial tumors with this amplicon by chromosomal comparative genomic hybridization. Next, we used a high-resolution comparative genomic hybridization array covering the 8p11-12 region to characterize this chromosomal region. This approach allowed us to define 2 cores of common amplification that largely overlap with those reported in sporadic tumors. Our findings confirm the molecular complexity of this chromosomal region and indicate that this genomic event is a common alteration in breast cancer, present not only in sporadic but also in familial tumors. Finally, we found correlation between the 8p11-12 amplification and proliferation (Ki-67) and cyclin E expression, which further proves in familial tumors the poor prognosis association previously reported in sporadic breast cancer. © 2006 Wiley-Liss, Inc.

The short arm of chromosome 8 is frequently altered in solid and hematological human tumors.1, 2, 3, 4 Amplification at 8p11-12 has been reported in 10–15% of sporadic breast cancer,3, 5, 6 although according to recent studies, using high-resolution array comparative genomic hybridization (aCGH) techniques, this frequency may be as high as 25%.7, 8

Recently, 3 different groups have characterized the 8p11-12 amplification in sporadic breast tumors and breast cancer cell lines using aCGH, to define the region and describe relevant genes that map in this chromosomal location.7, 8, 9 Gelsi-Boyer et al.9 defined 4 minimal amplicons in the 8p11-12 amplified region (Fig. 1). The most telomeric core of amplification (A1) spans 1.27 Mb and comprises the entire minimal region previously described by Garcia et al.8; this minimal region of common amplification is 1 Mb long and contains several candidate oncogenes (Fig. 1). This narrow region was similarly reported by a third group that suggests breakpoints and complex chromosomal rearrangements within the NRG1 locus (31.38–32.70 Mb) as likely mechanisms involved in the amplification.7 The second amplicon (A2), centromeric to A1, has a length of around 800 Kb and contains among other genes FGFR1. This gene has been previously proposed as candidate oncogene but its role as target of the 8p12 amplification remains to be established.3, 4, 5, 11, 12, 13, 14, 15, 16 Two more amplicons, A3 and A4, centromeric to the previous ones and with lengths of 1.25 Mb and 460 Kb, respectively, were also described. Candidate oncogenes at each of these amplicons are listed in Figure 1.

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Figure 1. Array-CGH profile across the 8p11-12 region. The 9 samples are represented in columns: 3 BRCA1 (1–3), 3 BRCA2 (4–6) and 3 BRCAX (7–9). Each row represents a clone on the array. Clones have been ordered by genome position according to the NCBI Build 35 from the most distal ones (top) to the most centromeric ones (bottom). The midpoint position (Mb) of each clone is indicated beside the clone name, as well as the genes mapping in the region. Colors represent discrete values according to the aCGH ratio: red indicates loss (ratios <0.8), black implies no change (0.8–1.2) and a green scale represents distinct levels of gains. Light green represents simple gain (1.2–1.5), midtone green is high-level gain (1.5–1.75) and dark green represents BAC amplification (>1.75). Grey cells correspond to data rejected after quality tests for signal intensity and replicate reproducibility. Genes in bold font present a good correlation between amplification and expression levels according to previous studies. Left colored thick bars represent the 4 cores of amplification described by Gelsi-Boyer et al. (A1, A2, A3, A4).9 The orange bar defines the 1-Mb minimal amplicon reported by us in a previous study.8 The blue box represents the cluster of breakpoints proximal to the UNC5D gene reported by Gelsi-Boyer et al. (BPC2).9, 10 Finally, the further right colored striped bars represent the region of loss distal to breakpoints (red box), the area where breaks mainly occurred (discontinuous grey boxes) and the 2 regions of gain/amplification (green boxes) we describe.

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Two regions of recurrent breakpoints in 8p have also been described in breast cancer: a telomeric cluster of breakpoints (BPC1) associated with rearrangements at the NRG1 locus (31.38–32.70 Mb),9, 10, 17 and a centromeric cluster of breakpoints (BPC2) proximal to the NRG1 and UNC5D genes8, 9, 10 (Fig. 1). The telomeric region of 8p distal to these breakpoints is frequently deleted as a result of these breaks.8, 9, 10 This pattern of molecular complexity has been recently reported by us, not only in breast cancer cell lines but also in colon and pancreatic cancer cell lines.10

All these data suggest that the 8p11-12 region is prone to present breaks and complex rearrangements, and could therefore participate in oncogenesis via inactivation of one or several potential tumor suppressor genes, and/or via amplification and over-expression of candidate oncogenes.

We have previously reported 9 cases (11.25%) showing 8p11-12 amplification in a set of 80 familial breast tumors (including 26 BRCA1, 18 BRCA2 and 36 non-BRCA1/2 tumors), using chromosomal comparative genomic hybridization (cCGH).18 Because familial tumors present different genetic and immunohistochemical (IHC) profiles among themselves and also with respect to sporadic tumors, we decided to investigate the features of this amplicon at the molecular level, to establish whether it is a common event in all breast cancer types.

Patients included in our previous study18 belonged to families with at least 3 women affected with breast/ovarian cancer and at least 1 of them diagnosed before 50 years, or to families with female breast/ovarian cancer and at least with 1 case of male breast cancer. All cases were studied for mutations in BRCA1 and BRCA2 genes and for large rearrangements alterations using standard procedures.19 Those that were negative for this mutational screening were considered as non-BRCA1/2 patients. We extracted DNA from paraffin-embedded tissue from the 9 familial breast tumor samples in our collection (3 BRCA1, 3 BRCA2 and 3 non-BRCA1/2) that displayed amplification at the 8p11-12 region. We used a xylene treatment and sodium thiocyanate incubation before proteinase K digestion and phenol chloroform extraction. We then hybridized these cases onto a new version of a previously used in-house BAC-array.8, 10, 17 Briefly, this platform comprised BACs ∼10 Mb apart across the whole genome; 1.5 Mb apart over chromosome 8; and at near-tiling-path density over 8p11-12. Interestingly, its resolution at 8p11-12 was further improved by adding 30 more clones to fill in existing gaps at the region. A total number of 91 BACs (versus 61 in the previous set) spans over 9.5 Mb at 8p11-12 between positions 31.03 Mb (RP11-473A17) and 43.38 Mb (CTD-2115H11). Name, position, size and accession number for these clones are available in Supplementary Table I. DNA labeling and hybridization as well as image acquisition and data analysis were done as previously published.8 Clinical and IHC data of the 80 cases studied by cCGH were collected from earlier studies by our group.20, 21

Table I. Correlations Between 8p11-12 Amplification and Immunohistochemical and Clinical Features
 No amplification, n (%)8p11-12 amplification,1n (%)p*
  • 1

    Cases whose 8p11-12 amplification was defined by cCGH and studied in the present work.

  • *

    p-value defined by Pearson's χ2 test. NS, non significant.

Age (years)
 <4425 (45.5)5 (55.6)NS
 ≥4430 (54.5)4 (44.4)
SBR grade
 117 (28.3)1 (11.1)NS
 217 (28.3)2 (22.2)
 326 (43.3)6 (66.7)
Estrogen receptor
 <1024 (35.8)4 (44.4)NS
 ≥1043 (64.2)5 (55.6)
Progesterone receptor
 <1032 (47.8)5 (55.6)NS
 ≥1035 (52.2)4 (44.4)
p53
 <2551 (77.3)7 (77.8)NS
 ≥2515 (22.7)2 (22.2)
Ki-67
 <2049 (74.2)3 (33.3)0.013
 ≥2017 (25.8)6 (66.7)
Cyclin D1
 <2532 (51.6)4 (50.0)NS
 ≥2530 (48.4)4 (50.0)
Cyclin D3
 <1034 (56.7)5 (71.4)NS
 ≥1026 (43.3)2 (28.6)
Cyclin E
 <1046 (74.2)3 (37.5)0.033
 ≥1016 (25.8)5 (62.5)
Cyclin A
 <1023 (37.7)1 (12.5)NS
 ≥1038 (62.3)7 (87.5)
Cyclin B1
 <1041 (68.3)3 (42.9)NS
 ≥1019 (31.7)4 (57.1)
p16
 <5022 (40.0)3 (37.5)NS
 ≥5033 (60.0)5 (62.5)
p21
 <1035 (57.4)2 (25.0)NS
 ≥1026 (42.6)6 (75.0)
p27
 <5026 (42.6)4 (50.0)NS
 ≥5035 (57.4)4 (50.0)

The genomic characterization of the 8p11-12 region in the 9 cases with amplification by means of hybridization on the high-resolution aCGH platform is summarized in Figure 1. The majority of the cases did not show any rearrangements at the previously described cluster of breakpoints BPC1, which has been associated with the NRG1 locus. In contrast, most of breakages occurred roughly between BACs CTD-2017E4 (36.01 Mb) and RP11-26K8 (37.18 Mb). Genomic losses telomeric to the breakpoints and gain/amplification centromeric to the breakpoints were observed, reproducing the genomic imbalances already defined at this chromosomal region.8, 9 This region of breakpoints overlaps with the previously described cluster of breakpoints centromeric to the UNC5D gene, BPC2 (Fig. 1).9, 10 These findings highlight this area as prone to develop breaks in different types of tumors, including familial breast cancer. Regarding amplified regions, all cases showed distinct levels of gains that were grouped in 2 main subregions (Fig. 1). The first one has a length of 2.13 Mb, and comprises the proximal half of A1, the whole of A2, and a small region of around 100 Kb between A2 and A3. This genomic area contains genes such as ZNF703/FLJ14299, SPFH2/C8orf2, PROSC, DDHD2, WHSC1L1 and FGFR1, all of them previously reported as genes of interest for further functional analysis because of their amplification-over-expression correlation.7, 8, 9 It is important to note that this chromosomal region includes the minimal 1 Mb amplicon already defined by us,8 supporting the candidate role of the genes located there. The second subregion encompasses the A4 core of amplification, has a length of 1.41 Mb, shows a lower level of gain and contains other genes already described as relevant such as GOLGA7, MYST3 and AP3M2.1, 9 Interestingly, the majority of cases in our sample set did not present amplification at the A3 region. This region was amplified in only 2 cases (cases 2 and 6) that presented large and continuous amplifications from about positions 35 and 36 Mb up to the centromere (Fig. 1). No differences in the pattern and distribution of the amplifications were observed among the 3 types of familial breast tumors (BRCA1, BRCA2 and non-BRCA1/2).

Amplification of 8p11-12 has been associated with high proliferation (high histological grade and Ki-67 expression)9 and an adverse effect on survival in breast cancer.7, 9 To evaluate these clinical associations in our series, we compared different IHC markers and clinical variables between the 9 tumors with this amplification and those cases without this genomic event (68 samples) (Table I). We found that tumors with 8p11-12 amplification had significantly higher Ki-67 and cyclin E expression (p = 0.013 and 0.033, respectively). Moreover, we observed a trend to present high grade, high expression of other cell cycle markers (such as cyclin A or B1) and an early age of onset in tumors with the amplification. These differences were not statistically significant, probably due to the low number of cases (Table I). These clinical and IHC features have been largely associated with tumor progression, proliferation and poor patient prognosis.22, 23, 24, 25, 26, 27 Therefore our data indicate that this amplified region would present clinical prognostic value in familial breast cancer, as it has been shown for sporadic breast tumors. Interestingly, the region A2 described as the amplification core associated with the most aggressive tumor behavior9 was amplified in all of our cases.

In summary, to our knowledge, this is the first time that the 8p11-12 amplification has been reported and analyzed in detail in familial breast cancer. We have defined 2 common regions of amplification that greatly overlap with the minimal regions of amplifications previously described in sporadic tumors. We also found a cluster of breakpoints centromeric to the UNC5D gene, similarly to what has been reported for sporadic neoplasms. Therefore our findings in a selected group of familial tumors confirm the molecular complexity of the 8p11-12 chromosomal region and suggest that these alterations, and probably some gene(s) mapping in these regions, are common in breast cancer pathogenesis, independent of the tumor type. Finally, we have found that the presence of this amplification is associated with high proliferation (Ki67) and cyclin E expression, which further supports the clinical value of this aberrations found in sporadic breast cancer.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Acknowledgements
  4. References
  5. Supporting Information

We thank the Spanish National Tumor Bank Network, the Familial Cancer Unit, the Immunohistological Unit and the Molecular Cytogenetics Group of the Spanish National Cancer Center for their technical support.

References

  1. Top of page
  2. Abstract
  3. Acknowledgements
  4. References
  5. Supporting Information
  • 1
    Borrow J, Stanton VP,Jr, Andresen JM, Becher R, Behm FG, Chaganti RS, Civin CI, Disteche C, Dube I, Frischauf AM, Horsman D, Mitelman F et al. The translocation t(8;16)(p11;p13) of acute myeloid leukaemia fuses a putative acetyltransferase to the CREB-binding protein. Nat Genet 1996; 14: 3341.
  • 2
    Emi M, Fujiwara Y, Nakajima T, Tsuchiya E, Tsuda H, Hirohashi S, Maeda Y, Tsuruta K, Miyaki M, Nakamura Y. Frequent loss of heterozygosity for loci on chromosome 8p in hepatocellular carcinoma, colorectal cancer, and lung cancer. Cancer Res 1992; 52: 536872.
  • 3
    Theillet C, Adelaide J, Louason G, Bonnet-Dorion F, Jacquemier J, Adnane J, Longy M, Katsaros D, Sismondi P, Gaudray P, Birnbaum D,. FGFRI and PLAT genes and DNA amplification at 8p12 in breast and ovarian cancers. Genes Chromosomes Cancer 1993; 7: 21926.
  • 4
    Chaffanet M, Popovici C, Leroux D, Jacrot M, Adelaide J, Dastugue N, Gregoire MJ, Hagemeijer A, Lafage-Pochitaloff M, Birnbaum D, Pebusque MJ. t(6;8), t(8;9) and t(8;13) translocations associated with stem cell myeloproliferative disorders have close or identical breakpoints in chromosome region 8p11–12. Oncogene 1998; 16: 9459.
  • 5
    Adelaide J, Chaffanet M, Imbert A, Allione F, Geneix J, Popovici C, van Alewijk D, Trapman J, Zeillinger R, Borresen-Dale AL, Lidereau R, Birnbaum D et al. Chromosome region 8p11-p21: refined mapping and molecular alterations in breast cancer. Genes Chromosomes Cancer 1998; 22: 18699.
  • 6
    Courjal F, Theillet C. Comparative genomic hybridization analysis of breast tumors with predetermined profiles of DNA amplification. Cancer Res 1997; 57: 436877.
  • 7
    Prentice LM, Shadeo A, Lestou VS, Miller MA, deLeeuw RJ, Makretsov N, Turbin D, Brown LA, Macpherson N, Yorida E, Cheang MC, Bentley J et al. NRG1 gene rearrangements in clinical breast cancer: identification of an adjacent novel amplicon associated with poor prognosis. Oncogene 2005; 24: 72819.
  • 8
    Garcia MJ, Pole JC, Chin SF, Teschendorff A, Naderi A, Ozdag H, Vias M, Kranjac T, Subkhankulova T, Paish C, Ellis I, Brenton JD, Edwards PA, Caldas C. A1 Mb minimal amplicon at 8p11-12 in breast cancer identifies new candidate oncogenes. Oncogene 2005; 24: 523545.
  • 9
    Gelsi-Boyer V, Orsetti B, Cervera N, Finetti P, Sircoulomb F, Rouge C, Lasorsa L, Letessier A, Ginestier C, Monville F, Esteyries S, Adelaide J et al. Comprehensive profiling of 8p11–12 amplification in breast cancer. Mol Cancer Res 2005; 3: 65567.
  • 10
    Pole JC, Courtay-Cahen C, Garcia MJ, Blood KA, Cooke SL, Alsop AE, Tse DM, Caldas C, Edwards PA. High-resolution analysis of chromosome rearrangements on 8p in breast, colon and pancreatic cancer reveals a complex pattern of loss, gain and translocation. Oncogene 2006; 25: 5693706.
  • 11
    Freeman KW, Welm BE, Gangula RD, Rosen JM, Ittmann M, Greenberg NM, Spencer DM. Inducible prostate intraepithelial neoplasia with reversible hyperplasia in conditional FGFR1-expressing mice. Cancer Res 2003; 63: 825663.
  • 12
    Freeman KW, Gangula RD, Welm BE, Ozen M, Foster BA, Rosen JM, Ittmann M, Greenberg NM, Spencer DM. Conditional activation of fibroblast growth factor receptor (FGFR) 1, but not FGFR2, in prostate cancer cells leads to increased osteopontin induction, extracellular signal-regulated kinase activation, and in vivo proliferation. Cancer Res 2003; 63: 623743.
  • 13
    Ugolini F, Adelaide J, Charafe-Jauffret E, Nguyen C, Jacquemier J, Jordan B, Birnbaum D, Pebusque MJ. Differential expression assay of chromosome arm 8p genes identifies Frizzled-related (FRP1/FRZB) and Fibroblast Growth Factor Receptor 1 (FGFR1) as candidate breast cancer genes. Oncogene 1999; 18: 190310.
  • 14
    Yang ZQ, Albertson D, Ethier SP. Genomic organization of the 8p11-p12 amplicon in three breast cancer cell lines. Cancer Genet Cytogenet 2004; 155: 5762.
  • 15
    Xian W, Schwertfeger KL, Vargo-Gogola T, Rosen JM. Pleiotropic effects of FGFR1 on cell proliferation, survival, and migration in a 3D mammary epithelial cell model. J Cell Biol 2005; 171: 66373.
  • 16
    Ray ME, Yang ZQ, Albertson D, Kleer CG, Washburn JG, Macoska JA, Ethier SP. Genomic and expression analysis of the 8p11–12 amplicon in human breast cancer cell lines. Cancer Res 2004; 64: 407.
  • 17
    Huang HE, Chin SF, Ginestier C, Bardou VJ, Adelaide J, Iyer NG, Garcia MJ, Pole JC, Callagy GM, Hewitt SM, Gullick WJ, Jacquemier J et al. A recurrent chromosome breakpoint in breast cancer at the NRG1/neuregulin 1/heregulin gene. Cancer Res 2004; 64: 68404.
  • 18
    Melchor L, Alvarez S, Honrado E, Palacios J, Barroso A, Diez O, Osorio A, Benitez J. The accumulation of specific amplifications characterizes two different genomic pathways of evolution of familial breast tumors. Clin Cancer Res 2005; 11: 857784.
  • 19
    Diez O, Osorio A, Duran M, Martinez-Ferrandis JI, de la Hoya M, Salazar R, Vega A, Campos B, Rodriguez-Lopez R, Velasco E, Chaves J, Diaz-Rubio E et al. Analysis of BRCA1 and BRCA2 genes in Spanish breast/ovarian cancer patients: a high proportion of mutations unique to Spain and evidence of founder effects. Hum Mutat 2003; 22: 30112.
  • 20
    Palacios J, Honrado E, Osorio A, Cazorla A, Sarrio D, Barroso A, Rodriguez S, Cigudosa JC, Diez O, Alonso C, Lerma E, Sanchez L et al. Immunohistochemical characteristics defined by tissue microarray of hereditary breast cancer not attributable to BRCA1 or BRCA2 mutations: differences from breast carcinomas arising in BRCA1 and BRCA2 mutation carriers. Clin Cancer Res 2003; 9: 360614.
  • 21
    Palacios J, Honrado E, Osorio A, Cazorla A, Sarrio D, Barroso A, Rodriguez S, Cigudosa JC, Diez O, Alonso C, Lerma E, Dopazo J et al. Phenotypic characterization of BRCA1 and BRCA2 tumors based in a tissue microarray study with 37 immunohistochemical markers. Breast Cancer Res Treat 2005; 90: 514.
  • 22
    Simpson PT, Reis-Filho JS, Gale T, Lakhani SR. Molecular evolution of breast cancer. J Pathol 2005; 205: 24854.
  • 23
    Foulkes WD, Brunet JS, Stefansson IM, Straume O, Chappuis PO, Begin LR, Hamel N, Goffin JR, Wong N, Trudel M, Kapusta L, Porter P et al. The prognostic implication of the basal-like (cyclin E high/p27 low/p53+/glomeruloid-microvascular-proliferation+) phenotype of BRCA1-related breast cancer. Cancer Res 2004; 64: 8305.
  • 24
    Chappuis PO, Donato E, Goffin JR, Wong N, Begin LR, Kapusta LR, Brunet JS, Porter P, Foulkes WD. Cyclin E expression in breast cancer: predicting germline BRCA1 mutations, prognosis and response to treatment. Ann Oncol 2005; 16: 73542.
  • 25
    Pavelic ZP, Pavelic L, Lower EE, Gapany M, Gapany S, Barker EA, Preisler HD. c-myc, c-erbB-2, and Ki-67 expression in normal breast tissue and in invasive and noninvasive breast carcinoma. Cancer Res 1992; 52: 2597602.
  • 26
    Kuhling H, Alm P, Olsson H, Ferno M, Baldetorp B, Parwaresch R, Rudolph P. Expression of cyclins E, A, and B, and prognosis in lymph node-negative breast cancer. J Pathol 2003; 199: 42431.
  • 27
    Potemski P, Kusinska R, Watala C, Pluciennik E, Bednarek AK, Kordek R. Cyclin E expression in breast cancer correlates with negative steroid receptor status, HER2 expression, tumor grade and proliferation. J Exp Clin Cancer Res 2006; 25: 5964.

Supporting Information

  1. Top of page
  2. Abstract
  3. Acknowledgements
  4. References
  5. Supporting Information

This article contains supplementary material available via the Internet at http://www.interscience.wiley.com/jpages/0020-7136/suppmat

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