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Applied Immunohistochemistry & Molecular Morphology:
doi: 10.1097/PAI.0b013e3182311d82
Research Articles

CD73 Expression as a Potential Marker of Good Prognosis in Breast Carcinoma

Supernat, Anna MSc*; Markiewicz, Aleksandra MSc*,†; Wełnicka-Jaśkiewicz, Marzena MD, PhD; Seroczyńska, Barbara PhD§; Skokowski, Jarosław MD, PhD§; Sejda, Aleksandra MD; Szade, Jolanta MD; Czapiewski, Piotr MD; Biernat, Wojciech MD, PhD; Żaczek, Anna PhD*

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*Department of Medical Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdańsk and Medical University of Gdańsk

Department of Oncology and Radiotherapy

Department of Pathomorphology, Medical University of Gdańsk

§Central Tissue Bank and Genetic Specimen, Gdańsk

Postgraduate 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.

Reprints: Anna Żaczek, PhD, Laboratory of Cell Biology, Department of Medical Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdańsk and Medical University of Gdańsk, Dębinki 1, 80-211 Gdańsk, Poland (e-mail: azaczek@gumed.edu.pl).

Received June 28, 2011

Accepted August 3, 2011

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Abstract

Ecto-5′-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. However, the actual role of CD73 in cancerogenesis remains unclear and requires further analysis.

Ecto-5′-nucleotidase (CD73, e-5NT) is a membrane-bound 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, α,ß-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 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.

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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 paraffin-embedded 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%).

Table 1
Table 1
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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 Gdańsk.

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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 (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 µm-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 heat-induced 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 positive cells. Positive CD73 status was considered as the score higher then median value, which equaled 100 among all samples.

Figure 1
Figure 1
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A mouse monoclonal antibody against Estrogen Receptor alpha (ER α, clone 1D5, Dako) and a mouse monoclonal antibody against Progesterone Receptor (PgR, clone 636, Dako) were used, according to the manufacturer’s instructions, at a dilution of 1:50 and antigen retrieval was performed at neutral pH by water-bath heating at 90°C for 30 minutes. Envision Dako system was used for visualization. Evaluation of the immunohistochemical nuclear staining was performed based on Allred score.13 HER2 receptor status was determined with the use of Hercept Test Kits (DAKO) according to the manufacturer’s guidelines.

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Statistical Analysis

All statistical analyses were performed using the STATISTICA software, version 9. Survival curves were generated by the Kaplan-Meier method. The endpoints 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 Kaplan-Meier 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 P≤0.10 were entered into a stepwise multivariate model to identify those with independent prognostic information. P values <0.05 were assumed in all analyses to indicate statistical significance. The study was performed in accordance with the REMARK criteria.14

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RESULTS

Positive CD73 status (defined as score ≥100) occurred in 74% of the cases. No correlation was found between CD73 expression and grading, tumor size, lymph node status, histologic type, ER, or progesterone receptor status.

CD73 expression strongly correlated with DFS (P=0.00167, Fig. 2) and OS (P=0.00819, Fig. 3). Recurrence occurred in 8 of 101 patients (7.9%) with CD73 expression and in 10 of 35 patients (28.6%) without CD73 expression. Univariate analysis (Table 2) showed that T stage and CD73 expression correlated with DFS [hazard ratio (HR)=2.19; 95% confidence interval (CI), 1.41-3.42; P=0.0005 and HR=0.26; 95% CI, 0.1-0.66; P=0.004, respectively] and OS (HR=1.9; 95% CI, 1.05-3.5; P=0.03 and HR=0.24; 95% CI, 0.07-0.85; P=0.027, respectively). In the multivariate regression analysis (Table 3), again T stage and CD73 expression were independent prognostic factors for DFS (HR=1.89, 95% CI, 1.2-2.95, P=0.005 and HR=0.34, 95% CI, 0.13-0.93, P=0.003, respectively). The same trend was observed for OS; however, statistical significance was not achieved.

Figure 2
Figure 2
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Figure 3
Figure 3
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Table 2
Table 2
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Table 3
Table 3
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DISCUSSION

Paradigm of adenosine promoting tumor progression2,3,7 and cancer cell lines with CD73 expression possessing higher invasiveness and aggressiveness4,7 is commonly accepted in molecular oncology. The aforementioned rationale, however, is based on in vitro experiments. There are no studies, which would investigate the importance of CD73 expression in actual tissue samples, collected from a representative and appropriately large cohort of patients. Therefore, this study aimed at elucidation of the clinical significance of CD73 expression in breast cancer.

Our results showing correlation of positive CD73 staining with longer DFS and OS, clearly demonstrate that CD73 expression may in fact be the indicator of good prognosis. To our best knowledge, there is no solid data proving direct correlation of CD73 expression with poor prognosis. Contradictory findings may be the consequence of the vast majority of experiments performed on cancer cell lines, not actual patient tissues. Results from traditional 2-dimensional cell cultures might not be easily transferrable to the results from in vivo studies, as the lack of 3-dimensional tissue architecture can greatly alter expression of genes, proteins, and thus the phenotype and cell behavior.15,16 In the discussed topic, the only study which investigated tumor breast cancer tissues in the context of CD73 expression was that of Leth-Larsen et al.17 Although the study showed that intense ecto-5-NT IHC staining was more common for patients with relapse, distant and/or lymph node metastases, the experiment has been performed on a small number of samples, with no survival analyses conducted.17

A number of studies show that activated adenosine receptors do not necessarily have to stimulate cell proliferation, they can also inhibit it.9–11 Furthermore, CD73 expression is known to vary highly among cell types, even within the mammary gland.7,18 Another fact supporting our result is the positive correlation between CD73 expression in stromal fibroblasts and ER tumor status.18 As the latter is the indicator of good prognosis, the data is in congruence. Furthermore, tumor stroma tends to be CD73-positive, whereas cancer cells seem to lose this expression.19 Thus, CD73 expression might be characteristic for healthy cells, suggesting that the more advanced the cancer, the lesser the CD73 expression.

Studies mentioned in this article might suggest the existence of an alternative mechanism of adenosine production, not related to CD73 activity. Furthermore, strong contradictory results between in vivo and in vitro studies might indicate the presence of some cell line culture-related factor, which changes the expression of CD73 in examined cell lines. In contrast, the limitation of this study was a small number of events.

To conclude, elevated CD73 expression in breast cancer may predict a good prognosis. The actual role of CD73 in cancerogenesis remains unclear and requires further analyses.

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REFERENCES

1. Zimmermann H. 5′-Nucleotidase: molecular structure and functional aspects. Biochem J. 1992;285(Pt 2):345–365

2. Wang L, Zhou X, Zhou T, et al. Ecto-5′-nucleotidase promotes invasion, migration and adhesion of human breast cancer cells. J Cancer Res Clin Oncol. 2008;134:365–372

3. Spychala J, Lazarowski E, Ostapkowicz A, et al. Role of estrogen receptor in the regulation of ecto-5′-nucleotidase and adenosine in breast cancer. Clin Cancer Res. 2004;10:708–717

4. Sadej R, Spychala J, Skladanowski AC. Expression of ecto-5′-nucleotidase (eN, CD73) in cell lines from various stages of human melanoma. Melanoma Res. 2006;16:213–222

5. Bavaresco L, Bernardi A, Braganhol E, et al. The role of ecto-5′-nucleotidase/CD73 in glioma cell line proliferation. Mol Cell Biochem. 2008;319:61–68

6. Stella J, Bavaresco L, Braganhol E, et al. Differential ectonucleotidase expression in human bladder cancer cell lines. Urol Oncol. 2010;28:260–267

7. Zhou X, Zhi X, Zhou P, et al. Effects of ecto-5′-nucleotidase on human breast cancer cell growth in vitro and in vivo. Oncol Rep. 2007;17:1341–1346

8. Spychala J. Tumor-promoting functions of adenosine. Pharmacol Ther. 2000;87:161–173

9. Kulkarni JS, Przywara DA, Wakade TD, et al. Adenosine induces apoptosis by inhibiting mRNA and protein synthesis in chick embryonic sympathetic neurons. Neurosci Lett. 1998;248:187–190

10. Saitoh M, Nagai K, Nakagawa K, et al. Adenosine induces apoptosis in the human gastric cancer cells via an intrinsic pathway relevant to activation of AMP-activated protein kinase. Biochem Pharmacol. 2004;67:2005–2011

11. El-Darahali A, Fawcett H, Mader JS, et al. Adenosine-induced apoptosis in EL-4 thymoma cells is caspase-independent and mediated through a non-classical adenosine receptor. Exp Mol Pathol. 2005;79:249–258

12. Tavassoli FA, Devilee PWHO Classification of tumours. In: Tavassoli FA, Devilee P, eds. Pathology and Genetics. Tumor of the Breast and Female Genital Organs. 2003 IARC press, Lyon

13. Allred DC, Harvey JM, Berardo M, et al. Prognostic and predictive factors in breast cancer by immunohistochemical analysis. Mod Pathol. 1998;11:155–168

14. McShane LM, Altman DG, Sauerbrei W, et al. REporting recommendations for tumour MARKer prognostic studies (REMARK). Br J Cancer. 2005;93:387–391

15. Pampaloni F, Reynaud EG, Stelzer EH. The third dimension bridges the gap between cell culture and live tissue. Nat Rev Mol Cell Biol. 2007;8:839–845

16. Bissell MJ, Rizki A, Mian IS. Tissue architecture: the ultimate regulator of breast epithelial function. Curr Opin Cell Biol. 2003;15:753–762

17. Leth-Larsen R, Lund R, Hansen HV, et al. Metastasis-related plasma membrane proteins of human breast cancer cells identified by comparative quantitative mass spectrometry. Mol Cell Proteomics. 2009;8:1436–1449

18. Krüger KH, Thompson LF, Kaufmann M, et al. Expression of ecto-5′-nucleotidase (CD73) in normal mammary gland and in breast carcinoma. Br J Cancer. 1991;63:114–118

19. Canbolat O, Durak I, Cetin R, et al. Activities of adenosine deaminase, 5′-nucleotidase, guanase, and cytidine deaminase enzymes in cancerous and non-cancerous human breast tissues. Breast Cancer Res Treat. 1996;37:189–193

Cited By:

This article has been cited 2 time(s).

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Expression of epithelial to mesenchymal transition-related markers in lymph node metastases as a surrogate for primary tumor metastatic potential in breast cancer
Markiewicz, A; Ahrends, T; Welnicka-Jaskiewicz, M; Seroczynska, B; Skokowski, J; Jaskiewicz, J; Szade, J; Biernat, W; Zaczek, AJ
Journal of Translational Medicine, 10(): -.
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Acta Biochimica Polonica
Dual, enzymatic and non-enzymatic, function of ecto-5 '-nucleotidase (eN, CD73) in migration and invasion of A375 melanoma cells
Sadej, R; Skladanowski, AC
Acta Biochimica Polonica, 59(4): 647-652.

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Keywords:

CD73; prognostic marker; immunohistochemistry; tissue microarrays

© 2012 Lippincott Williams & Wilkins, Inc.

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