Intracellular Patterns of Her-2/neu, ras, and Ploidy Abnormalities in Primary Human Breast Cancers Predict Postoperative Clinical Disease-Free Survival

Purpose: In an earlier study (S. E. Shackney et al., Cancer J. Sci. Am., 2: 106, 1996), the presence of aneuploidy, Her-2/neu overexpression, and ras overexpression in the same cells (triple-positive cells) was of prognostic significance (P < 0.015) in 91 patients with localized breast cancer (median follow up, 32 months). Here, we present results involving a larger group of patients with longer follow-up. Experimental Design: Fixed cell suspensions prepared from primary tumors of 189 patients with early breast cancer were studied prospectively by multiparameter flow cytometry. Correlated intracellular fluorescence-based measurements of cell DNA content and Her-2/neu and ras protein were obtained on each of >2000 cells in each tumor. Intracellular combinations of abnormalities in these measurements were correlated with subsequent patient disease-free survival (DFS). Median time on study was 54 months (range, 7–128 months). Results: DFS of patients with ≥5% triple-positive tumor cells was shorter than those who did not meet this criterion (P = 0.004). The difference remained statistically significant after accounting for nodal status, tumor size, and each of the component abnormalities (P = 0.006). Node-negative patients whose tumors had fewer than 2 abnormalities/cell had an especially favorable clinical course, with a 5-year DFS of 96% (lower confidence bound, 86%). Conclusions: Patterns of accumulated intracellular molecular abnormalities in cells of primary human breast cancers are predictive for subsequent DFS independently of the abnormalities themselves taken individually.

[1]  G. Landberg,et al.  Deregulation of cyclin E and D1 in breast cancer is associated with inactivation of the retinoblastoma protein , 1997, Oncogene.

[2]  S. Hirohashi,et al.  c-erbB-2 gene product directly associates with beta-catenin and plakoglobin. , 1995, Biochemical and biophysical research communications.

[3]  S. Hirohashi,et al.  Dominant negative inhibition of the association between beta-catenin and c-erbB-2 by N-terminally deleted beta-catenin suppresses the invasion and metastasis of cancer cells. , 1996, Oncogene.

[4]  M. Sliwkowski,et al.  Biologic effects of heregulin/neu differentiation factor on normal and malignant human breast and ovarian epithelial cells , 1999, Oncogene.

[5]  S. Parsons,et al.  Src family kinases and HER2 interactions in human breast cancer cell growth and survival , 2001, Oncogene.

[6]  P. Nowell.  The clonal evolution of tumor cell populations. , 1976, Science.

[7]  Isabelle Bedrosian,et al.  Cyclin E and survival in patients with breast cancer. , 2002, The New England journal of medicine.

[8]  V. Rotter,et al.  Cooperation between gene encoding p53 tumour antigen and ras in cellular transformation , 1984, Nature.

[9]  J. Schlom,et al.  Loss of a c-H-ras-1 allele and aggressive human primary breast carcinomas. , 1986, Cancer research.

[10]  N. Rosen,et al.  Degradation of HER2 by ansamycins induces growth arrest and apoptosis in cells with HER2 overexpression via a HER3, phosphatidylinositol 3'-kinase-AKT-dependent pathway. , 2002, Cancer research.

[11]  James M. Roberts,et al.  Expression of cell-cycle regulators p27Kip1 and cyclin E, alone and in combination, correlate with survival in young breast cancer patients , 1997, Nature Medicine.

[12]  T. Julian,et al.  Distinctive patterns of Her-2/neu, c-myc, and cyclin D1 gene amplification by fluorescence in situ hybridization in primary human breast cancers. , 2001, Cytometry.

[13]  R. Hartsock,et al.  Discrepancies between flow cytometric and cytogenetic studies in the detection of aneuploidy in human solid tumors. , 1990, Cytometry.

[14]  M. Ladanyi,et al.  Impact of polysomy 17 on HER-2/neu immunohistochemistry in breast carcinomas without HER-2/neu gene amplification. , 2003, The Journal of molecular diagnostics : JMD.

[15]  L. Gu,et al.  Intracellular coexpression of epidermal growth factor receptor, Her-2/neu, and p21ras in human breast cancers: evidence for the existence of distinctive patterns of genetic evolution that are common to tumors from different patients. , 1998, Clinical cancer research : an official journal of the American Association for Cancer Research.

[16]  G. Landberg,et al.  The cyclin D1 high and cyclin E high subgroups of breast cancer: separate pathways in tumorogenesis based on pattern of genetic aberrations and inactivation of the pRb node , 2002, Oncogene.

[17]  W. McGuire,et al.  Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. , 1987, Science.

[18]  Carlos Caldas,et al.  Cancer: The molecular outlook , 2002, Nature.

[19]  Robert A. Weinberg,et al.  Tumorigenic conversion of primary embryo fibroblasts requires at least two cooperating oncogenes , 1983, Nature.

[20]  N. Rosen,et al.  Inhibition of Hsp90 function by ansamycins causes retinoblastoma gene product-dependent G1 arrest. , 2000, Cancer research.

[21]  J F Silverman,et al.  Correlations among p53, Her-2/neu, and ras overexpression and aneuploidy by multiparameter flow cytometry in human breast cancer: evidence for a common phenotypic evolutionary pattern in infiltrating ductal carcinomas. , 2000, Clinical cancer research : an official journal of the American Association for Cancer Research.

[22]  R. Gelber,et al.  Meeting highlights: International Consensus Panel on the Treatment of Primary Breast Cancer. Seventh International Conference on Adjuvant Therapy of Primary Breast Cancer. , 2001, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[23]  V. Keshamouni,et al.  Temporal and quantitative regulation of mitogen-activated protein kinase (MAPK) modulates cell motility and invasion , 2001, Oncogene.

[24]  R. Tibshirani,et al.  Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[25]  E. Devilard,et al.  Gene expression profiles of poor-prognosis primary breast cancer correlate with survival. , 2002, Human molecular genetics.

[26]  H. Ruley,et al.  Rescue of cells from ras oncogene-induced growth arrest by a second, complementing, oncogene. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[27]  R. Hartsock,et al.  Sequential paraformaldehyde and methanol fixation for simultaneous flow cytometric analysis of DNA, cell surface proteins, and intracellular proteins. , 1992, Cytometry.

[28]  Christine Solbach,et al.  Identification of high risk breast-cancer patients by gene expression profiling , 2002, The Lancet.

[29]  M. Wigler,et al.  Signaling pathways in Ras-mediated tumorigenicity and metastasis. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[30]  S. Shackney,et al.  Model for the genetic evolution of human solid tumors. , 1989, Cancer research.

[31]  S E Shackney,et al.  The accumulation of multiple genetic abnormalities in individual tumor cells in human breast cancers: clinical prognostic implications. , 1996, The cancer journal from Scientific American.

[32]  Jan F. Silverman,et al.  Molecular Evolutionary Patterns in Breast Cancer , 2003, Advances in anatomic pathology.

[33]  R. Jove,et al.  Inhibition of pp60c-Src reduces Bcl-XL expression and reverses the transformed phenotype of cells overexpressing EGF and HER-2 receptors , 1999, Oncogene.

[34]  S. Regauer,et al.  Aromatase expression in low-grade endometrial stromal sarcomas: an immunohistochemical study , 2004, Modern Pathology.

[35]  T. Shankey,et al.  Common patterns of genetic evolution in human solid tumors. , 1997, Cytometry.

[36]  R. Gelber,et al.  Meeting highlights: International Consensus Panel on the Treatment of Primary Breast Cancer. , 1995, Journal of the National Cancer Institute.

[37]  D. Visscher,et al.  Detection of chromosome aneuploidy in breast lesions with fluorescence in situ hybridization: comparison of whole nuclei to thin tissue sections and correlation with flow cytometric DNA analysis. , 1995, Cytometry.

[38]  M. Aapro,et al.  Genetic alterations of c-myc, c-erbB-2, and c-Ha-ras protooncogenes and clinical associations in human breast carcinomas. , 1989, Cancer research.

[39]  J. Bartek,et al.  Cyclin D1 is dispensable for G1 control in retinoblastoma gene-deficient cells independently of cdk4 activity , 1995, Molecular and cellular biology.

[40]  W Godolphin,et al.  Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. , 1989, Science.

[41]  Yudong D. He,et al.  Gene expression profiling predicts clinical outcome of breast cancer , 2002, Nature.

[42]  D. Hayes,et al.  Adjuvant therapy for all patients with breast cancer? , 2001, Journal of the National Cancer Institute.

[43]  Å. Borg,et al.  The retinoblastoma gene in breast cancer: allele loss is not correlated with loss of gene protein expression. , 1992, Cancer research.

[44]  D. Farkas,et al.  Multiparameter analysis of human epithelial tumor cell lines by laser scanning cytometry. , 2000, Cytometry.

[45]  N. Hellyer,et al.  Heregulin-dependent Activation of Phosphoinositide 3-Kinase and Akt via the ErbB2/ErbB3 Co-receptor* , 2001, The Journal of Biological Chemistry.

[46]  L. Neckers,et al.  Geldanamycin abrogates ErbB2 association with proteasome-resistant beta-catenin in melanoma cells, increases beta-catenin-E-cadherin association, and decreases beta-catenin-sensitive transcription. , 2001, Cancer research.

[47]  I. Macdonald,et al.  Activated ras regulates the proliferation/apoptosis balance and early survival of developing micrometastases. , 2002, Cancer research.

[48]  C. Benz,et al.  Incidence of activating ras oncogene mutations associated with primary and metastatic human breast cancer. , 1989, Cancer research.