Prognostic value and response to chemotherapy of immunohistochemical phenotypes of 141 operable breast cancer patients included in phase III trials of adjuvant therapy

Rafael Trujillo ¹, E Gallego ³, A Márquez ², N Ribelles ², D Perez ¹, C Quero ², D Olmos ¹, L Vicioso ³, A Rueda ¹, E Alba ¹

1. Hospital Costa del Sol, Medical Oncology Department, MARBELLA, SPAIN

2. Hospital Universitario Virgen de la Victoria, Medical Oncology Department, MALAGA, SPAIN

3. Hospital Universitario Virgen de la Victoria, Pathology Department, MALAGA, SPAIN

Abstract:

Background: Gene expression arrays and immunohistochemical phenotypes studies classified breast cancer in three distinct subtypes: basal, HER2/neu and luminal that are associated with different clinical outcomes.

Methods: In 141 patients with operable breast cancer included in several phase III adjuvant therapy trials, immunohistochemical staining was performed. A basal phenotype was defined by negative estrogen and progesterone receptor and positive cytokeratin 5/6 or EGFR immunoreactivity. HER2/neu phenotype as positive c-erb B2 by HercepTest™ and luminal phenotype by positive estrogen receptors, progesterone receptors and CK 7/8 and negative HER-2 were defined respectively. Survival curves were calculated by the Kaplan-Meier method. The differences between survivals were estimated using the log rank test.

Results: The median follow-up period was 52 months (range 1-103 months). During this period, 13.8% patients died from breast cancer and 27.7% patients relapsed. At the time of the primary diagnosis 10.4% of the patients had lymph node negative disease, 50.8% patients received standard chemotherapy and 7.7% Trastuzumab. Positivity for luminal phenotype was 65.2%, basal phenotype 9.9%, HER-2 phenotype 8.5% and 16.3% were null/no expression. Median disease free survival for basal phenotype was 24 months, but for luminal phenotype and HER-2 phenotypes has not been reached. DFS at 5 years were basal phenotype: 19%, luminal phenotype: 63% and HER-2: 56%. Detectable basal phenotype was highly significantly associated with a worse DFS compared with the presence of a non basal phenotype (p=0.0001). Multivariate analyses estimated that the prognostic effect of basal phenotype was independent of lymph node, stage and tumor size (HR 0.12; 95% CI 0.05-0.2; p<0.05). In the group of patients who received standard-based adjuvant chemotherapy, disease free survival was found to be significantly shorter in the basal phenotype (p<0.05).

Conclusions: We found that expression of basal phenotype was associated with poor prognostic in the context of randomized phase III trials. Standard adjuvant chemotherapy seems to be less effective in these tumors and new therapeutic approaches are indicated.

Correspondence to: Dr Rafael Trujillo. Hospital Costa del Sol, Medical Oncology Department, Autovía A-7 Km 187. 29603 Marbella , SPAIN.

Keywords: Breast cancer, genetics; Microarrays; Chemotherapy, adjuvant ; Breast cancer, prognostic factors

Introduction

Breast cancer is one of the most common malignancies in women (1). Currently, the most important prognostic factors are nodal status, tumor size, status of hormone receptor, and histological grade. Nevertheless numerous other clinicopathologic factors and novel molecular markers have been investigated to improve the prediction of clinical outcome (2). Individual prognostic factors provide limited information on the biology of the disease because of the heterogeneity of breast cancers as well as the interrelationship among prognostic markers.

Molecular classification based on gene expression patterns from complementary DNA (cDNA) microarrays has recently been applied for breast cancer (3,36,39,40,41)showing to be a powerful tool to predict tumors behavior. Studies focusing on breast cancer have been published showing that gene expression profiling distinguishes groups of patients representing specific tumor subtypes and/or prognosis (4,35,36,37,39,40,41). It has been shown that using the gene expression profile of breast cancer, prognosis can be more accurately predicted than by clinical variables only (5,35,36,37,39,40,41),and different subtypes of invasive breast carcinomas including luminal/estrogen receptor (ER)+, normal breast–like, HER2/neu+, and basal-like subtypes can be distinguished (6,36,37,39,40,41). Among these, the basal-like and HER2/neu+ subtypes are associated with poorer clinical outcomes than other types in Western countries.

Tumors expressing basal epithelium cytokeratins (CK5/14/17) constitute a subgroup distinct from basal cytokeratin-negative luminal epithelium-like tumors. Basal cytokeratin-positive tumors are characterized by hormone receptor negativity, a high tumor grade, and a high proliferation activity (7). Gene expression microarray studies have also distinguished the "basal-like" tumors from the other subtypes of tumors, and have associated the basal-like group with adverse patient survival (8). Different immunohistochemical markers have been used by different investigators to identify basal-like differentiation, but there is no a universally agreed-upon criterion or set of markers that define this subtype of breast cancer. Nielsen et al. (8) developed a panel for identifying basal-like breast cancers based on a comparison of immunohistochemical profiles. Using their definition, basal-like carcinomas are negative for estrogen receptors and HER2, in addition to being positive for either cytokeratin 5/6 or epidermal growth factor receptor (EGFR). Others have suggested that expression of high molecular weight cytokeratins alone (including CK5, CK14, and CK17) could be used (11). By immunohistochemistry, the expression of CK5/14/17 can be detected in 10% of breast tumors (9). The overwhelming majority of breast cancers are characterized by the expression of CK8/18 only, the predominant cytokeratins of the glandular epithelium lining the lumen of the breast ducts (10). Therefore, in most immunohistochemical studies, the classification of breast cancers into "basal phenotype" and "luminal phenotype" subgroups refers to the presence or absence of CK5/14/17 in the tumor cells, respectively (11), because CK8/18 can be detected in all sporadic CK5/14-positive breast tumors. It has been suggested that, until these criteria are developed, staining for estrogen receptors, progesterone receptors, and HER2 would correctly classify the majority of basal-like breast cancers (46). Currently, three large molecular subgroups have been proposed: HER2-overexpressing/Estrogen receptor (ER)-negative tumors, basal-like and luminal-like breast cancers.

In addition, the study of these molecular subtypes of breast cancer might offer some predictive information about treatment efficacy. Studies have investigated the predictive value of HER2 status for the efficacy of anthracyclines (12,13,14). In two out of four studies, Her2 expression was found to be predictive for the benefit of anthracyclines in terms of disease-free survival (13,14).Several studies have reported a good correlation between the molecular subclassification (determined by DNA microarray) and the efficacy of postoperative and preoperative anthracycline-based chemotherapy (12,13,14,38). Recently, Conforti et al (42), have published that estrogen receptors expression was a strong and independent predictive biomarker for the benefit of adjuvant chemotherapy. In this study, the authors also found that the P value for interaction between HER2 expression and benefit of chemotherapy was 0.37, and concluded that HER2 did not add predictive information to that provided by the estrogen receptors status. Furthermore, while the molecular subclassification was predictive for the efficacy of chemotherapy (test for interaction, P = 0.01), its determination did not add significant information to the predictive value provided by the estrogen receptors status (P = 0.32).

In the present study, in a well-characterized and followed series of primary breast cancer that entered in randomized phase III trials, we determined the immunohistochemically expression pattern in 141 breast tumors from clinical adjuvant therapy randomized trials. The results were correlated with the biological and clinicopathologic tumor characteristics and patient survival.

Patients and methods

Patients

One hundred and forty one cases of primary operable invasive breast carcinoma from patients that entered in randomized phase III trials were analyzed. Patients have been followed up at 3-month intervals initially, then 6-monthly, then annually. At the time of resection all tumours are managed in a standard fashion, incised and fixed immediately.

Tissue array and immunostaining

A basal phenotype was defined by negative estrogen and progesterone receptor and positive cytokeratin 5/6 or EGFR immunoreactivity. HER2/neu phenotype as positive c-erb B2 by HercepTest™ and luminal phenotype by positive estrogen receptors, progesterone receptors and CK 7/8 and negative HER-2 were defined respectively.

Immunohistochemical staining was performed on 3μm sections of paraffin blocks containing tissue-arrays of tumour tissue. To construct these blocks, two representative areas from the tumor were selected from a hematoxylin-eosin stained section of a donor block. Core cylinders (0.4 mm diameter) were punched from each of them and deposited into two separate recipient paraffin blocks. The sections were microwaved in citrate buffer, pH 6 for 20 min, to epitope retrieval. Immunohistochemical test was carried out in an autoimmunostainer (Tech Mate Horizon, Dako, Copenhagen, Denmark), using the Dako Real EnVision method to detect the antibody.