Hypoxia-Inducible Factor-1AExpression Predicts a Poor Response to Primary ChemoendocrineTherapy and Disease-Free Survival in Primary Human Breast Cancer

Purpose: To investigate the relationship of hypoxia-inducible factor-1a (HIF-1a) tumor expression in predicting the response to epirubicin and disease-free survival (DFS) in patients with breast cancer enrolled in a single institution trial of primary anthracycline and tamoxifen therapy. Experimental Design: The expression of HIF-1awas assessed by immunohistochemistry in 187 patients withT2-4 N0-1breast cancer enrolled in a randomized trial comparing four cycles of single agent epirubicin versus epirubicin + tamoxifen as primary systemic treatment. All patients postoperatively received four cycles of the four weekly i.v. CMF regimen (cyclophosphamide, methotrexate, and 5-fluorouracil). Patients with estrogen receptor (ER)-positive primary tumors also underwent 5 years of treatment with adjuvant tamoxifen. Carbonic anhydrase IX (CAIX)was also scored as a marker of HIF activity. Results: Overall response to therapy progressively decreased with increasing tumor HIF-1a (P < 0.05), and HIF-1awas an independent predictor of response (P < 0.048). HIF-1aexpression was also associated with a significantly shorter DFS (P < 0.02) in all patients and in ER-positive but not in ER-negative patients. Furthermore, CAIX positivity conferred a significantly shorter DFS (P = 0.02) compared with CAIX-negative tumors in patients with HIF-1a-negative tumors. Conclusions: HIF-1a expression in patients with breast cancer is a marker of poor therapy response and outcome, especially in ER-positive patients. The combination of two hypoxia markers has greater utility than assessing just one, and patients with hypoxia markers in their tumors may be suitable for administration of drugs that reduce HIF-1aexpression and increase oxygen delivery to the tumor bed before starting neoadjuvant therapies. Tumor growth and metastasis is dependent on the generation of a neovasculature. However, newly formed vessels function poorly in supplying oxygen and nutrient requirements in many tumors. Hypoxia, the pathophysiologic consequence of the structurally and functionally disturbed microcirculation (1), is therefore a common feature in solid tumors. Tumors respond to cellular oxygen deprivation using the ubiquitous family of transcription factors known as hypoxia-inducible factors (HIF; ref. 2). Under normal oxygen tension, HIF-1a is hydroxylated by specific prolyl hydroxylases, leading to recognition and binding by the von Hippel-Lindau protein, and targeting for degradation through the proteasome. In conditions of hypoxia, molecular oxygen is not available for hydroxylase activity, which leads to HIF-1a protein stabilization and translocation to the nucleus where it binds to aryl hydrocarbon nuclear translocators resulting in the activation of several gene pathways involved in angiogenesis, glycolysis, erythropoiesis, and apoptosis (see ref. 3). Overexpression of HIF-1a protein has been identified in many tumor types (4), with high levels influencing the growth rate and metastatic potential of these cancers. In breast cancers, the frequency of HIF-1a-positive cells increases in parallel with increasing pathologic stage and is associated with a poor prognosis (5–7). Furthermore, upregulation of the hypoxia pathway by HIF has not only been shown to confer an aggressive phenotype, but also contributes to resistance to radiotherapy and chemotherapy. Both radiotherapy and chemotherapy improve patient survival, with response dependent on tumor and patient Imaging, Diagnosis, Prognosis Authors’Affiliations: Nuffield Department of Clinical Laboratory and Molecular Oncology Laboratories,Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom; Unita' di Patologia Mammaria, Breast Cancer Unit and Anatomia Patologica, Azienda Instituti Ospitalieri di Cremona, Cremona, Italy ; Oncologia Medica, Dipartimento di Scienze Cliniche e Biologiche, Universita' di Torino, Azienda Ospedaliera San Luigi di Orbassano, Orbassano, Italy; and Peter MacCallum Cancer Center, St. Andrews Place, East Melbourne,Victoria, Australia Received12/8/05; revised 3/28/06; accepted 5/5/06. Grant support: Associazione Patologia Oncologica Mammaria, Cremona, Italy; L’associazione Amici dell’Ospedale di Cremona; Consiglio Nazionale Ricerche, Rome, Italy; Cancer Research UK, United Kingdom; and the EU 6th Framework Grant Euroxy. The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section1734 solely to indicate this fact. Requests for reprints: Adrian L. Harris, Molecular Oncology Laboratories, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford, OX3 9DS, United Kingdom. Phone: 44-1865222420; Fax: 44-1865-222431; E-mail: aharris.lab@cancer.org.uk. F2006 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-05-2690 www.aacrjournals.org Clin Cancer Res 2006;12(15) August1, 2006 4562 characteristics. Predictive factors are used to forecast such response to a particular therapy (8). Each therapy should be evaluated independently in patient cohorts defined by the predictive factor (9). This can be optimally done in a prospective randomized clinical trial with primary chemotherapy being the optimal setting to study new biological markers in relation to the predictive information they provide. In addition, tumor biopsy specimens obtained in matched pair cases at diagnosis and definitive surgery provides valuable information on the interaction between biological markers and treatment. We have used an immunohistochemical approach to evaluate the putative hypoxia markers HIF-1a and carbonic anhydrase IX (CAIX) expression in a series of breast cancer specimens obtained before and after primary anthracycline and tamoxifen therapy. Our aims were (a) to test whether HIF-1a predicts response to treatment, (b) to assess whether HIF-1a predicts disease-free survival (DFS), and (c) whether using additional hypoxic markers helps define the hypoxic population. Patients andMethods Patients. Patients with T2-4 N0-1 breast cancer were recruited in a randomized trial comparing single agent epirubicin (EPI arm) versus epirubicin plus tamoxifen (EPI-TAM arm) as the primary systemic treatment (10). Patients were accrued from January 1997 to December 2001. The study was approved by the Institutional Investigations Committee. All patients gave written informed consent to the diagnostic procedures, the proposed treatment, and the biological evaluations. Two-hundred and eleven patients were enrolled, 105 were randomized to receive epirubicin alone, and 106 were randomized to receive epirubicin plus tamoxifen. On first presentation, an incision biopsy was done on each patient and a small tissue sample (0.5-0.8 cm) was removed. Chemotherapy was started within 2 days of diagnosis. Patients in the EPI arm received 60 mg/m of epirubicin (Farmorubicina, Pharmacia, Milan, Italy) by slow i.v. push on days 1 and 2; whereas patients on the EPI-TAM arm received 60 mg/m of epirubicin by slow i.v. push on days 1 and 2 and 30 mg of tamoxifen (Kessar, Pharmacia) daily. Epirubicin injections were repeated every 21 days for three or four cycles before definitive surgery, whereas tamoxifen was given continuously until definitive surgery. All patients postoperatively received four cycles of the CMF regimen [i.v. cyclophosphamide (600 mg/m), i.v. methotrexate (40 mg/m), and i.v. 5-fluorouracil (600 mg/m) on days 1 and 8, every 28 days; ref. 11]. Patients with estrogen receptor (ER)–positive primary tumor in both treatment arms received tamoxifen (20 mg, i.e., lower than the primary dose) starting after surgery, up to progression or for a maximum of 5 years. The median follow up of patients was 53 months (August 2004; range, 13-95). Treatment evaluation. Each month, the size of the primary tumor and the size of the axillary lymph nodes, when appreciable, were measured by the same clinician using a caliper. Response was assessed before definitive surgery by the clinical measurement of the changes in the product of the two largest diameters recorded in two successive evaluations. According to WHO criteria, tumor progression was defined as an increase of at least 25% in tumor size; stable disease was defined as an increase of <25%, or a reduction of <50%; partial response was defined as a tumor shrinkage >50%; and complete response was defined as the complete disappearance of all clinical signs of disease. Pathologic complete response was defined as the absence of neoplastic cells in the breast and in the axillary lymph nodes. Surgery was planned after full clinical reassessment. Quadrantectomy or modified radical mastectomy were done when indicated in association with full axillary node dissection. All patients subjected to quadrantectomy underwent irradiation of the residual breast (60 Gy delivered over 6 weeks). Histopathologic grade and immunohistochemistry. Tumor grade was evaluated using the Nottingham prognostic index (12). Immunohistochemical evaluation was done on paraffin-embedded tumor samples obtained at diagnosis and at definitive surgery. Bcl2, p53, ER, progesterone receptor (PgR), and Ki67 staining were done at the Pathology Unit of the Azienda Ospedaliera Istituti Ospitalieri of Cremona (Italy). The immunohistochemical method used in Cremona for routine markers is fully described elsewhere (13). Immunohistochemistry for HIF-1a and CAIX was done on 5 Am sections of tissue microarrays containing two 1-mm tumor cores taken from selected morphologically representative tumor regions of each paraffinembedded breast tumor from both the initial diagnostic incisional biopsy and from tumor remaining at definitive surgery. Quality control was assessed on each block by H&E staining. HIF-1a was detected using the ESEE 122 (IgG1 monoclonal antibody; dilution, 1:40) monoclonal antibody and CAIX