2698 Vol. 5, 2698–2703, October 1999 Clinical Cancer Research Advances in Brief Association between Keratin and Vimentin Expression, Malignant Phenotype, and Survival in Postmenopausal Breast Cancer Patients1 Patricia A. Thomas,2 Dawn A. Kirschmann, tumors. These latter two groups demonstrated similar James R. Cerhan, Robert Folberg, Kaplan-Meier survival curves; the former group (keratin Elisabeth A. Seftor, Thomas A. Sellers, and and vimentin in approximately similar amounts) demon- 3 strated a poorer survival, with a hazard ratio of 2.1 (95% Mary J. C. Hendrix confidence interval, 0.5–9.6). These data suggest that rela- University of Iowa Cancer Center [P. A. T., D. A. K., J. R. C., R. F., tive keratin and vimentin IF expression is more indicative of E. A. S., M. J. C. H.], Departments of Pathology [P. A. T., R. F.], prognosis and tumor phenotype than either IF marker de- Ophthalmology [R. F.], Anatomy and Cell Biology [D. A. K., E. A. S., M. J. C. H.], Preventive Medicine and Environmental Health tected independently. [J. R. C.], The University of Iowa, Iowa City, Iowa 52242-1109 and Health Sciences Research [T. A. S.], Mayo Clinic, Rochester, Introduction Minnesota 55905 IF4 proteins are best known for their cell type specificity and their static structural role as components of the vertebrate Abstract cell cytoskeleton. There is growing evidence for IF involvement Pathology observational reports and experimental data in a variety of dynamic cellular functions, including intercellular suggest that keratin and vimentin intermediate filament (IF) and cell-to-extracellular matrix signal transduction, cellular mo- coexpression in breast cancer confers a more aggressive tility, and tumor cell invasiveness (1–5). “interconverted” phenotype, expressing both epithelial and Infidelity of IF expression, i.e., expression of the mesen- mesenchymal markers. In this study, we extended previous chymal marker vimentin by an epithelial cell, or expression of observations by measuring the expression of keratin and more than one type of IF (coexpression) has been observed in vimentin, in relation to other selected biomarkers of disease vitro in certain tumor cell lines and in cellular and tissue progression, in postmenopausal women with breast cancer. samples of certain human malignancies (6–13). Recently, an in Using immunohistochemical analysis of 54 archival, forma- vitro model of keratin and vimentin coexpression in breast lin-fixed, paraffin-embedded invasive breast cancers from a cancer cells (developed as stable transfectants) has revealed that well-defined cohort, we examined relative IF (keratin and these keratin/vimentin IF-positive cells display increased prolif- vimentin) expression in a semiquantitative fashion and com- eration rates, invasive potential, clonogenicity, and tumorige- pared these results with other biological markers and sur- nicity when compared with keratin-positive/vimentin-negative vival. By univariate analysis, we found that vimentin expres- controls displaying low invasive potential (12). Furthermore, the sion was inversely associated with keratin expression alone keratin/vimentin-positive, transfected breast cancer cells dem- (P 5 0.0089) and directly related to histological grade (P 5 onstrated networks of well-formed IFs, which appear as inter- 0.017), nuclear grade (P 5 0.027), Ki67 growth fraction (P 5 woven keratin and vimentin filamentous networks. The in situ 0.024), and epidermal growth factor receptor immuno- appearance of vimentin immunoreactivity in human breast can- staining (P 5 0.019). The relative expression of keratin and cer cells, which normally express only the epithelial IF marker vimentin in approximately similar amounts characterized keratin, has been reported in up to 61% of human breast cancers tumors with the poorest prognosis, as compared with kera- (10, 13–19). The immunohistochemical detection of vimentin tin-high/vimentin-negative or keratin-low/vimentin-positive expression in invasive breast cancer has also been associated with biomarkers of poor prognosis and adverse clinical out- comes (14–19). In contrast, increased expression of certain keratins has been associated with a favorable outcome (20). Received 7/9/99; accepted 8/24/99. In this study, we postulated that expression of both keratin The costs of publication of this article were defrayed in part by the and vimentin IFs is associated with a more aggressive pheno- payment of page charges. This article must therefore be hereby marked type in human breast cancers, compared with tumors expressing advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. either IF alone. Furthermore, we propose that the relationship or 1 This work was supported by NIH/National Cancer Institute Minor- relative amounts of these two IF proteins are more important ity Supplement 3R01CA59702-06S1 (to P. A. T.), CA59702 (to than the mere aberrant expression of the mesenchymal marker M. J. C. H.), CA39712 (to T. A. S.), and the Search to Prevent Blindness vimentin. We therefore undertook a retrospective study using Senior Scientific Investigator Award (to R. F.). immunohistochemical analysis of keratin and vimentin IF pro- 2 Present address: Department of Pathology and Laboratory Medicine, Kansas University Medical Center, 3901 Rainbow Boulevard, Kansas City, Kansas 66160-7281. 3 To whom requests for reprints should be addressed, at Department of Anatomy and Cell Biology, 1–100 BSB, The University of Iowa, College of Medicine, 51 Newton Road, Iowa City, IA 52242-1109. Fax: 4 The abbreviations used are: IF, intermediate filament; FNAB, fine- (319) 335-7770; E-mail: [email protected]. needle aspiration biopsy. Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 1999 American Association for Cancer Research. Clinical Cancer Research 2699 teins in invasive breast cancers from a well-defined patient Table 1 Semiquantitative immunohistochemical analysis criteria cohort of postmenopausal women and assessed the relative None vs. expression of these IF protein markers in tumor tissue sections. some staining ,10% vs. $10% ,50% vs. $50% Vimentin expression was also compared with other known Vimentin Estrogen receptor Keratin prognostic markers for disease progression. We identified sta- p53 Progesterone receptor tistically significant associations between vimentin expression Cathepsin c-erbB2 and histological grade, nuclear grade, Ki67, epithelial growth Epithelial growth factor receptor factor receptor, and keratin expression. In addition, poorer sur- Histological sections were examined at 340 to identify areas of 3 vival of breast cancer patients was observed when tumor cells maximum tumor staining. At 400, 200 cells were analyzed for stain- ing, and the percentage of positive cells was recorded. This procedure expressed both keratin and vimentin IFs in approximately equal was repeated, and the two percentages were averaged. On the basis of ratios. The data derived from this study may provide important the known biological significance of the markers and sample size translational significance to the utility of using vimentin:keratin considerations, immunostaining was a priori categorized into the above ratios in the diagnosis of breast pathology, including fine-needle three groups for data analysis. aspirates. Materials and Methods graphic images were recorded using an Olympus AH-2 photomicroscope and a Kodographic digital color printer. Patients and Breast Cancer Tissues. Diagnostic breast Semiquantitative Immunohistochemical Scoring. cancer tissues from patients participating in the Iowa Women’s Each histological section was examined at 340 to identify areas Health Study were obtained from the University of Iowa Hos- of maximum tumor staining. At 3400, 200 cells were analyzed pitals and Clinics and the Iowa Methodist Hospital. Breast (in the areas of maximum tumor staining), and the percentage of cancer was diagnosed from 1986 to 1993, and follow-up through positive cells was recorded. This procedure was repeated, and the Iowa Cancer Registry was complete through December 31, the average of the two percentages was recorded. These aver- 1996. None of the patient population was lost to follow-up in aged values were originally stratified into four scoring groups: this study. Cause of death was determined from the death (a) no immunopositive cells identified; (b) ,10% positive tu- certificate (ICD 9 code), and the primary outcome variable was mor cells; (c) 10–50% positive tumor cells; and (d) .50% time-to-death (all causes). Death attributable to breast cancer positive tumor cells. On the basis of known biological signifi- was also evaluated. cance of the markers analyzed and sample size considerations, Paraffin-embedded tissue blocks of any histological sub- immunostaining was a priori further subcategorized into three type of invasive breast cancer, original H&E-stained slides, and groups for data analysis: (a) no staining versus some staining; surgical reports were collectively requested from University of (b) ,10% staining versus $10% staining; and (c) ,50% versus Iowa Hospitals and Clinics and Iowa Methodist Hospital. Cases $50% staining (Table 1). were excluded if H&E-stained slides were not available for Histological Grade and Nuclear Grade. Histological review, tissue blocks were not available, no invasive tumor grade was determined using the histological criteria described remained in the block, or if insufficient material was present for previously by Elston and Ellis (21). Nuclear grade