RESEARCH ARTICLE
CD73 Expression as a Potential Marker of Good Prognosis in Breast Carcinoma Anna Supernat, MSc,* Aleksandra Markiewicz, MSc,*w Marzena We!nicka-Jas´kiewicz, MD, PhD,z Barbara Seroczyn´ska, PhD,y Jaros!aw Skokowski, MD, PhD,y Aleksandra Sejda, MD,8 Jolanta Szade, MD,8 Piotr Czapiewski, MD,8 Wojciech Biernat, MD, PhD,8 and Anna Z˙aczek, PhD*
Abstract: Ecto-50 -nucleotidase (CD73) is a membrane-bound enzyme, which catalyzes the conversion of adenosine monophosphate to adenosine. CD73 has been postulated to play an important role in carcinogenesis, as adenosine promotes tumor progression and CD73-expressing cancer cell lines are more aggressive. However, other studies have shown that activated adenosine receptors may also inhibit cell proliferation. This study investigated the clinical significance of CD73 expression in breast cancer. The study group included 136 consecutive stage I-III breast cancer patients treated between 2001 and 2008 at 2 institutions. CD73 expression was examined by immunohistochemistry (IHC) on tissue microarrays, using antihuman mouse monoclonal antibody. Survival curves were generated by the Kaplan-Meier method and compared using the log-rank test. CD73 staining was expressed as the score calculated by multiplying the staining intensity (0 = negative, 1 = weak, 2 = intermediate, 3 = strong) and percentage of positive cells (0% to 100%). The median score among all samples was 100. Positive CD73 staining (defined as score equal or higher than 100) occurred in 74% of the cases. No correlation was found between CD73 expression and grading, tumor size, lymph node status, histologic type, estrogen receptor, or progesterone receptor status. Positive CD73 expression strongly correlated with longer disease-free survival (hazard ratio = 0.26; 95% confidence interval, 0.1-0.66; P = 0.0044) and overall survival (hazard ratio = 0.24; 95% confidence interval, 0.07-0.85; P = 0.027). Multivariate analysis for disease-free survival revealed correlation with tumor size and CD73 status. Elevated CD73 expression in breast cancer can predict a good prognosis.
Received for publication June 28, 2011; accepted August 3, 2011. From the *Department of Medical Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdan´sk and Medical University of Gdan´sk; zDepartment of Oncology and Radiotherapy; 8Department of Pathomorphology, Medical University of Gdan´sk; yCentral Tissue Bank and Genetic Specimen, Gdan´sk; and wPostgraduate School of Molecular Medicine, Warsaw, Poland. Supported by Grant from Ministry of Science and Higher Education: IP2010 050370. The authors declare no conflict of interest. ˙ aczek, PhD, Laboratory of Cell Biology, Department Reprints: Anna Z of Medical Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdan´sk and Medical University of Gdan´sk, D˛ebinki 1, 80-211 Gdan´sk, Poland (e-mail:
[email protected]). Copyright r 2011 by Lippincott Williams & Wilkins
Appl Immunohistochem Mol Morphol
However, the actual role of CD73 in cancerogenesis remains unclear and requires further analysis. Key Words: CD73, prognostic marker, immunohistochemistry, tissue microarrays (Appl Immunohistochem Mol Morphol 2012;20:103–107)
E
cto-50 -nucleotidase (CD73, e-5NT) is a membranebound enzyme, which catalyzes the conversion of adenosine monophosphate to adenosine.1 A number of findings suggest that CD73 might be of importance in various types of cancer.2–6 Adenosine has cytoprotective and immunosuppressive activity. It is essential for cell proliferation and has been found to accumulate in cancer cells at high concentrations. Adenosine is also known to stimulate angiogenesis and tumor growth.7,8 Nevertheless, there are studies on adenosine metabolism, which show that activated adenosine receptors may in fact inhibit cell proliferation.9–11 Numerous in vitro studies have investigated the role of CD73 in different human cancer cell lines. Experiments performed on T-47D breast cancer cell line transfected with pcDNA-NT5E plasmid (resulting in CD73 overexpression) showed that high levels of e-5NT promoted migration, invasion, and cell adhesion to extracellular matrix. Furthermore, a CD73 inhibitor, a,-methylene ADP (APCP), has been demonstrated to decrease the mobility of MB-MDA-231 breast cancer cell line that expressed CD73 at a high level.2 Analysis of e-5NT expression regulation in breast cancer cell lines differing in invasive and metastatic potential and estrogen receptor (ER) status showed that ER-negative cells—commonly considered more aggressive—were characterized by higher CD73 expression on both protein and mRNA levels.3 Studies conducted on cell lines derived from different stages of melanoma proved a positive correlation between e-5NT expression and the stage of the tumor from which cell line originated.4 Bavaresco et al5 have found that treatment with APCP resulted in a significant decrease of proliferation in human U138MG glioma cell line, whereas the addition of adenosine induced the cell growth, suggesting a vital role of CD73 in cell division. Studies on human bladder cancer cell lines, RT4 (grade 1) and
Volume 20, Number 2, March 2012
www.appliedimmunohist.com |
103
Supernat et al
Appl Immunohistochem Mol Morphol
T24 (grade 3), showed that both lines expressed CD73 mRNA.6 A number of studies show that activated adenosine receptors do not necessarily have to stimulate cell proliferation, they can also inhibit it.7,9–11 Kulkarni et al9 demonstrated that RNA and protein synthesis in neuronal cells was inhibited by adenosine. Furthermore, subsequent experiments performed on human gastric cancer cells proved that adenosine promoted apoptosis.10 Similar results were also obtained when studying adenosine role in EL-4 thymoma cells.11 Until now experiments concerning CD73 expression have primarily been performed in vitro. No studies examined the importance of CD73 expression in actual tissue samples, collected from a representative and appropriately large cohort of patients. Herby study aimed at investigation of CD73 expression and its significance in terms of becoming a potential molecular marker. This study demonstrates for the first time that elevated CD73 expression in breast cancer may in fact predict a good prognosis.
MATERIALS AND METHODS Patients and Tissue Specimens In this retrospective study, the group encompassed 136 consecutive breast cancer patients treated between 2001 and 2008 in Medical University of Gdansk and Regional Cancer Centre in Bydgoszcz. Inclusion criteria were stage I-III breast cancer and signed informed consent. The majority of patients (91%) underwent primary surgery followed by systemic treatment, radiotherapy, or both. Nine percent of patients were administered induction chemotherapy. Tumor samples were collected by surgical excision or excisional biopsy before any systemic treatment and were formalin-fixed paraffinembedded according to standard procedure. The mean age of the patients was 58.4 years (range, 27 to 86 y). Twenty-four percent of the patients were premenopausal and 76% postmenopausal (Table 1). Grading was determined according to Elston and Ellis, recommended by World Health Organization. TNM staging and classification were followed according to World Health Organization recommendations.12 Survival analysis was performed for all patients. After a median follow-up of 1.8 years (range, 0.1 to 3.5 y), 18 patients (13.2%) experienced recurrence of the disease and 10 died (7.4%). Patients were examined every 6 months after the inclusion into the study, with the last follow-up in May 2010. The study was accepted by the Ethics Committee of the Medical University of Gdan´sk.
Immunohistochemistry on Tissue Microarrays Tissue microarrays (TMAs) were constructed from formalin-fixed paraffin-embedded surgical resection tumor specimens and control samples. In brief, two 1.5 mm diameter cores from each tumor were obtained from the most representative areas using tissue-arraying instrument
104 | www.appliedimmunohist.com
Volume 20, Number 2, March 2012
TABLE 1. Patient Characteristics (N = 136) Variable
No. Cases (%)
Menopausal status Premenopausal Postmenopausal T stage T1 T2 T3 T4 Missing Data N stage N0 N1 N2 N3 Missing Data Grade G1 G2 G3 Histologic type Ductal Lobular Other ER Negative Positive Missing data PgR Negative Positive Missing data HER2 protein status Negative (0, 1+) Weakly positive (2+) Strongly positive ( 3+) Missing data
33 (24) 103 (76) 43 76 6 10 1
(32) (56) (4) (7) (1)
63 45 19 6 3
(46) (33) (14) (4) (2)
36 (26) 62 (46) 38 (28) 105 (77) 20 (15) 11 (8) 58 (43) 77 (57) 1 (1) 53 (39) 82 (60) 1 (1) 41+33 23 19 20
(30+24) (17) (14) (15)
ER indicates estrogen receptor; PgR, progesterone receptor.
(MTA-I, Beecher Instruments), and then reembedded in microarray blocks. Punches of normal breast tissue and tonsil samples were added to the “tumor array,” to introduce built-in internal controls to the system. Consecutive 4 mm-thick TMA sections were cut and placed on charged polylysine-coated slides (Superfrost Plus, BDH, Germany) for subsequent immunohistochemistry (IHC) analysis. CD73 expression was examined by IHC on TMA blocks using CD73 antihuman mouse monoclonal antibody (clone IE9, dilution 1:50, Santa Cruz Biotechnology) and Novolink Polymer Detection System (Novocastra) in accordance with the manufacturer’s guidelines, with antigen retrieval carried out by heatinduced epitope retrival at pH 6. Immunostaining was performed by 2 pathologists and scored following 4-step scale (0 = negative, 1 = weak, 2 = intermediate, 3 = strong). The percentage of stained cells in a quantitative manner (0% to 100%) was also determined. Figures 1A and B depict representative images of CD73 IHC staining. CD73 staining was expressed as the score calculated by multiplying the staining intensity and percentage of r
2011 Lippincott Williams & Wilkins
Appl Immunohistochem Mol Morphol
Volume 20, Number 2, March 2012
CD73 Expression as a Potential Prognostic Marker
FIGURE 2. Kaplan-Meier curves depicting disease-free survival (DFS) depending on CD73 expression status (F Cox test, P = 0.00167).
FIGURE 1. Representative images of negative (A) and positive (B) nuclear CD73 immunohistochemistry staining. In (B) positive nuclear staining of the breast neoplastic cells with negative reaction in the fibrovascular stroma can be seen.
for the study were disease-free survival (DFS) and overall survival (OS). DFS was defined as the time from tumor sample collection to an event or censoring. An event was defined as relapse (local or distant), second malignancy or death, whichever came first. A censoring was defined as lost to follow-up or alive without relapse at the end of follow-up. OS was defined as the time from sample collection to death or censoring. DFS and OS KaplanMeier curves for subgroups of patients were compared using the log-rank test. Cox proportional hazards regression analysis was used to identify the independent predictors of DFS. Univariate predictors significant with a value of Pr0.10 were entered into a stepwise multivariate model to identify those with independent prognostic information. P values
