Expression of hypoxia-inducible factor 1 alpha and its downstream targets in fibroepithelial tumors of the breast

INTRODUCTION: Hypoxia-inducible factor 1 (HIF-1) alpha and its downstream targets carbonic anhydrase IX (CAIX) and vascular endothelial growth factor (VEGF) are key factors in the survival of proliferating tumor cells in a hypoxic microenvironment. We studied the expression and prognostic relevance of HIF-1α and its downstream targets in phyllodes tumors and fibroadenomas of the breast. METHODS: The expression of HIF-1α, CAIX, VEGF and p53 was investigated by immunohistochemistry in a group of 37 primary phyllodes tumors and 30 fibroadenomas with known clinical follow-up. The tumor microvasculature was visualized by immunohistochemistry for CD31. Proliferation was assessed by Ki67 immunostaining and mitotic counts. Being biphasic tumors, immunoquantification was performed in the stroma and epithelium. RESULTS: Only two fibroadenomas displayed low-level stromal HIF-1α reactivity in the absence of CAIX expression. Stromal HIF-1α expression was positively correlated with phyllodes tumor grade (P = 0.001), with proliferation as measured by Ki67 expression (P < 0.001) and number of mitoses (P < 0.001), with p53 accumulation (P = 0.003), and with global (P = 0.015) and hot-spot (P = 0.031) microvessel counts, but not with CAIX expression. Interestingly, concerted CAIX and HIF-1α expression was frequently found in morphologically normal epithelium of phyllodes tumors. The distance from the epithelium to the nearest microvessels was higher in phyllodes tumors as compared with in fibroadenomas. Microvessel counts as such did not differ between fibroadenomas and phyllodes tumors, however. High expression of VEGF was regularly found in both tumors, with only a positive relation between stromal VEGF and grade in phyllodes tumors (P = 0.016). Stromal HIF-1α overexpression in phyllodes tumors was predictive of disease-free survival (P = 0.032). CONCLUSION: These results indicate that HIF-1α expression is associated with diminished disease-free survival and may play an important role in stromal progression of breast phyllodes tumors. In view of the absence of stromal CAIX expression in phyllodes tumors, stromal upregulation of HIF-1α most probably arises from hypoxia-independent pathways, with p53 inactivation as one possible cause. In contrast, coexpression of HIF-1α and CAIX in the epithelium in phyllodes tumors points to epithelial hypoxia, most probably caused by relatively distant blood vessels. On the other hand, HIF-1α and CAIX seem to be of minor relevance in breast fibroadenomas

Pathogenesis and progression of fibroepithelial breast tumors

Fibroadenoma and phyllodes tumor are fibroepithelial breast tumors. These tumors are biphasic, i.e. they are composed of stroma and epithelium. The behavior of fibroadenomas is benign, whereas phyllodes tumors can recur and even metastasize. Classification criteria for both tumors show considerable overlap, though. Further, morphological observations have suggested that fibroadenoma may progress to phyllodes tumor. The aim of this thesis can be summarized as a study of tumorigenesis and progression in fibroepithelial tumors, i.e. progression in fibroadenomas and progression in grade of phyllodes tumors. By a comprehensive study of its histology, we identified several regions of phylloid-like stromal progression in fibroadenomas. We subjected these areas to clonality analysis and demonstrated that they are monoclonal. Further, monoclonal epithelial progression to carcinoma in situ was found in fibroadenomas as well. We therefore demonstrated that fibroadenoma may show both epithelial (to carcinoma) and stromal (to phyllodes tumor) progression. The molecular mechanisms underlying development of fibroepithelial tumors are largely unknown. We showed that, in phyllodes tumors, stromal progression is driven by cell cycle deregulation and EGFR overexpression. In addition, stromal p53 expression turned out to be the strongest single prognosticator in phyllodes tumors. In the epithelial component of phyllodes tumors and in fibroadenomas these carcinogenic changes were absent. Deregulation of the cell cycle machinery may lead to loss of genetic integrity. We obtained copy number profiles of fibroepithelial tumors and demonstrated genomic instability in phyllodes tumors, but not in fibroadenomas. Although not related to grade, it seems that genomic instability plays a role in development of phyllodes tumors. Little information is available on the cytokines involved in neovascularization of fibroepithelial tumors. We found that HIF-1α expression, a pivotal angiogenic factor, correlates with tumor grade and predicts prognosis in phyllodes tumors. Surprisingly, phyllodes tumor epithelium showed HIF-1α expression as well, which most likely reflects relatively distant microvasculature causing mild hypoxia. Although similar quantities of microvessels are found in fibroadenomas as compared to phyllodes tumors, HIF-1α does not seem to play a role here. Finally, we compared gene expression profiles of fibroadenomas and phyllodes tumors. We found many novel genes which may contribute to genesis and progression of fibroepithelial tumors. As compared to fibroadenomas, phyllodes tumors showed altered expression of factors involved in transcription, cell adhesion, apoptosis, Wnt signalling, cellular integrity and extracellular matrix degradation. CTAG1, a gene of unknown function, showed a nearly 50-fold upregulation in phyllodes tumors which suggests that it may aid diagnostics. In this thesis we demonstrated that benign fibroadenoma may progress to the unpredictable phyllodes tumor. Further, we studied several major carcinogenic phenomena and found important roles for cell cycle deregulation, EGFR overexpression, angiogenic factors and genomic instability in phyllodes tumor genesis and progression. Although fibroadenoma and phyllodes tumor share morphological similarities, both tumors are different molecular entities. This thesis contains no data supportive of an active role of the epithelium in fibroepithelial tumors genesis., as had been suggested previously by some authors

Progressive Deregulation of the Cell Cycle With Higher Tumor Grade in the Stroma of Breast Phyllodes Tumors

We studied cell cycle–regulating proteins in phyllodes tumor pathogenesis by immunohistochemical analysis for Ki-67, cyclin A, cyclin D1, retinoblastoma protein (pRb), p53, p16INK4A, bcl-2, and p21waf1 in the epithelium and stroma of 40 primary (benign, 21; borderline, 8; malignant, 11) and 7 recurrent tumors of different grades. In most cases, the epithelium showed no altered expression of cell cycle regulators. Stromal overexpression of p16INK4A, p53, cyclin A, pRb, and p21waf1 correlated significantly with tumor grade. The number of altered proteins in stroma increased with higher grade and was accompanied by increased proliferation. Stromal cyclin A expression was the best separating marker between tumor grades. Correlations existed between stromal overexpression of p16INK4A and p21waf1, p16INK4A and p53, and p53 and pRb. No immunostaining differences were detected between primary tumors and recurrences. Four or more altered proteins and p53 expression in the stromal component were independent negative prognosticators for diseasefree survival. The stromal component of mammary phyllodes tumors displays an increasing level of cell cycle deregulation with higher tumor grade; the epithelial compartment mostly remains inconspicuous. Several combinations of aberrantly expressed cell cycle proteins seem important in the stromal progression of phyllodes tumors. The number of stromal cell cycle aberrations and stromal p53 expression might predict clinical behavior

Multiple fibroadenomas harbouring carcinoma in situ in a woman with a family history of breast/ovarian cancer

A 46 year old woman with a family history of breast and ovarian cancer presented with multiple fibroadenomas in both breasts. From three fibroadenomas removed from the left breast carcinoma in situ (CIS) had developed. One fibroadenoma gave rise to ductal CIS, whereas the other two harboured lobular CIS. This is the first report of three fibroadenomas simultaneously giving rise to CIS. In addition, synchronous fibroadenomas harbouring different types of CIS from one fibroadenoma to the other have never been described. Direct sequencing revealed a mutation (5075G-->A) in the BRCA1 gene, but retention of BRCA1 immunohistochemical staining and no loss of heterozygosity at the BRCA1 locus by polymerase chain reaction made a pathogenic mutation in BRCA1 unlikely. Furthermore, in this family no cosegregation of breast cancer with this BRCA1 mutation was seen. Indeed, this mutation is now regarded as a polymorphism. This case stresses the need for histological evaluation of all breast masses in women with a strong positive family history for breast and/or ovarian cancer

Analysis of the progression of fibroepithelial tumours of the breast by PCR-based clonality assay

Fibroadenoma and phyllodes tumour of the breast are both fibroepithelial tumours. Although progression to epithelial malignancy has been described, the behaviour of most fibroadenomas is benign. Phyllodes tumours, on the other hand, can display locally destructive growth and can even metastasize. A relationship between the two tumours has been suggested in the literature. This study investigated the clonality of both the stroma and the epithelium of these fibroepithelial tumours and attempted to construct a model in which fibroadenoma can progress in both an epithelial and a stromal direction. Fibroadenomas (n=25) and phyllodes tumours (n=12) were selected for analysis. Tissue was microdissected and analysed for clonality using a polymerase chain reaction (PCR)-based assay targeted at an X-linked polymorphic marker, the human androgen receptor gene (HUMARA). Nineteen fibroadenomas and nine phyllodes tumours could be analysed. Normal-appearing epithelium, hyperplastic epithelium, and stroma removed from fibroadenomas were polyclonal. As expected, carcinoma in situ (CIS) removed from four fibroadenomas was monoclonal. Three areas of apparent stromal expansion within fibroadenoma were monoclonal, suggesting stromal progression. Mostly, the stroma of phyllodes tumours was monoclonal and the epithelium polyclonal. In two cases, however, the epithelium seemed to be monoclonal, whereas in three other cases the stromal component was polyclonal. These findings indicate that fibroadenoma can progress in an epithelial direction to CIS and in a stromal direction to phyllodes tumour

Disease-free survival according to hypoxia-inducible factor 1 alpha (HIF-1α) status

<p><b>Copyright information:</b></p><p>Taken from "Expression of hypoxia-inducible factor 1 alpha and its downstream targets in fibroepithelial tumors of the breast"</p><p>Breast Cancer Research 2005;7(5):R808-R818.</p><p>Published online 5 Aug 2005</p><p>PMCID:PMC1242154.</p><p>Copyright © 2005 Kuijper et al, licensee BioMed Central Ltd.</p> The Kaplan–Meier survival curve illustrating disease-free survival for patients with breast phyllodes tumors with high expression (≥1%) versus low expression

Egfr amplification specific gene expression in phyllodes tumours of the breast, Cell. Oncol

Abstract. Background: Recently, we were able to show that amplifications of the epidermal growth factor receptor (egfr) gene and the overexpression of EGFR were associated with the initiation and progression of phyllodes tumours. Methods: In order to gain further insights into regulation mechanisms associated with egfr amplifications and EGFR expression in phyllodes tumours, we performed global gene expression analysis (Affymetrix A133.2) on a series of 10 phyllodes tumours, of these three with and seven without amplifications of an important regulatory repeat in intron 1 of egfr (CA-SSR I). The results were verified and extended by means of immunohistochemistry using the tissue microarray method on an extensively characterized series of 58 phyllodes tumours with antibodies against caveolin-1, eps15, EGF, TGF-α, pErk, pAkt and mdm2. Results: We were able to show that the presence of egfr CA-SSR I amplifications in phyllodes tumours was associated with 230 differentially expressed genes. Caveolin-1 and eps15, involved in EGFR turnover and signalling, were regulated differentially on the RNA and protein level proportionally to egfr gene dosage. Further immunohistochemical analysis revealed that the expression of caveolin-1 and eps15 were also significantly correlated with the expression of pAkt (p &lt; 0.05), pERK (p &lt; 0.05), mdm2 (p &lt; 0.01) and EGF (p &lt; 0.001 for caveolin-1). Eps15 and pERK were further associated with tumour grade (p &lt; 0.01 and p &lt; 0.001, respectively). Conclusion: Our results show that amplifications within regulatory sequences of egfr are associated with the expression of eps15 and caveolin-1, indicating an increased turnover of EGFR. The interplay between EGFR and caveolin-1, eps15, pAkt, mdm2 and pERK therefore seems to present a major molecular pathway in carcinogenesis and progression of breast phyllodes tumours

Genomic Profiling by Array Comparative Genomic Hybridization Reveals Novel DNA Copy Number Changes in Breast Phyllodes Tumours

Breast phyllodes tumour (PT) is a rare fibroepithelial tumour. The genetic alterations contributing to its tumorigenesis are largely unknown. To identify genomic regions involved in pathogenesis and progression of PTs we obtained genome-wide copy number profiles by array comparative genomic hybridization (CGH)