The Expression And Clinical Significance Of CD39/CD73 Molecules In Tiunmn Gastric And Colorectal Carcinoma

BackgroundColorectal carcinoma(CRC) and gastric carcinoma(GC) are respectively the third and the fourth most common cancer diagnosed worldwide [1]. In spite of considerable improvements in high-quality screening, timely diagnosis and improved treatment, CRC and GC remain the fourth and the second leading cause of cancer-related deaths [1,2]. It should be noted that uncontrolled distant metastasis continue to be an important clinical concern for advanced patients with poor progression-free survival(PFS) and overall survival(OS) [3,4], and further investigation of prometastatic mechanisms and identification of molecular targets is a crucial issue faced in cancer research.The ectonucleotidases CD39 and CD73 are the rate-limiting component of an enzymatic cascade and the major sources of extracellular adenosine [5]; thus, representing a checkpoint in the conversion of proinflammatory adenosine triphosphate(ATP) into immunosuppressive adenosine [5,6]. Firstly, CD39 hydrolyzes extracellular ATP and adenosine diphosphate(ADP) into adenosine monophosphate(AMP) [6], and sequentially acts in concert with CD73, catalyzing the generation of immunosuppressive adenosine(ADO) that binds ADO receptors(A1、A2A、A2B、A3) and inhibits CD4+, CD8+ T cell and NK cell responses [7,8]. In addition to its immunoregulatory roles, the ectonucleotidase pathway contributes directly to the modulation of cancer cell growth, differentiation, invasion, migration, metastasis and tumor angiogenesis [5,9,10]. Recent lines of evidence suggest further evaluation of CD39 and CD73 expression and activity as potential prognostic markers in cancer patients, as well as the exploration of pharmacological modulation of these enzymes as innovative strategies to thwart neoplastic development and progression [5,11]. Intriguingly, several small molecule inhibitors and monoclonal antibodies developed against these enzymes show antitumor efficacy and a favorable tolerability profile in several murine tumor models [11,12], at least in part through inhibiting Treg-mediated immunosuppression and restoring T- and NK-cell immune responses [11,13]. Furthermore, recent study revealed that targeted blockade of CD73 can enhance the therapeutic activity of anti-PD-1 and anti-CTLA-4 mAbs and may thus potentiate therapeutic strategies targeting immune checkpoint inhibitors in general [14].As the novel tumor immune checkpoint(TIC), it is noteworthy that upregulation of CD39 and CD73 molecules in several human solid tumors are significantly assotiated with the tumor TNM stage, metastasis and prognosis [5,6,11,13,15,16]. Most importantly, CD73 expression levels were recently identified as potential predictive markers of benefit from cetuximab treatment in metastatic CRC(mCRC) [16]. The above evidence suggested the potential roles for these checkpoint molecules in promoting tumor growth and infiltration. However, there is few available data concerning CD39 and CD73 expression in huama CRC and GC, and the clinical significance of CD39 and CD73 is also less well investigated. In the current study, therefore, we explored the expression characteristics, clinical relevance and prognostic significance of CD39 and CD73 in huama CRC and GC.Part Ⅰ The expression and clinical significance of CD73molecule in human colorectal carcinomaObjective: The ectonucleotidase CD73 is the ratelimiting enzyme in the production of extracellular adenosine which potently inhibits host immune responses against cancer. This study investigated the expression level and prognostic significance of CD73 in human CRC.Methods: Fresh CRC samples and tissue microarray slides(TMA) containing 90 paired tumor samples and fresh tumor tissues(TNM I-IV) were used to examine the expression characteristics of CD73 by immunohistochemistry(IHC). The staining density of CD73 protein was assessed objectively and quantitatively using Aperio ImageScope software. Epithelial cells and stromal component were retrieved respectively from 5 paired tumor samples by laser capture microdissection(LCM), and the mRNA expression levels of CD73 were investigated by real time polymerase chain reaction(RT-PCR). Protein levels of CD73 in tumor tissues, tumor draining lymph nodes and liver metastases were also determined by Western blot(WB). Statistical analyses were used to associate levels of CD73 with tumor features and patient outcomes.Results: Our data demonstrated that CD73 staining strongly marked both malignant epithelial cells and stromal components where the protein and mRNA expression levels of CD73 were significantly increased compared with paracancerous controls. In addition to primary tumor tissues, CD73 was also abundantly expressed in tumor draining lymph nodes and liver metastases from mCRC patients. High CD73 expression in tumor cells can be used as an independent factor for predicting poor patients’ prognosis; however, patients with higher density of stromal CD73 were more likely to have favorable characteristics(early T and TNM stages) and overall survival. Notably, combined CD73 expression analysis in both tumoral and stromal compartments was more efficient to foretell patient’s outcome where patients with increased CD73 in tumor cells but decreased CD73 in stroma displayed a worst prognosis.Conclusion: The current study revealed CD73 expression was increased in both tumoral and stromal compartments. Although up-regulated CD73 expression in tumor cells correlates with a poor prognosis in patients with CRC, the combination of CD73 expression in malignant epithelial cells and tumor stroma may have a better prognostic value.Part Ⅱ The expression and clinical significance of CD39molecule in human colorectal carcinomaPurpose: The ectonucleotidase CD39 is pivotal in the conversion of immunostimulatory adenosine triphosphate(ATP) into immunosuppressive adenosine(ADO) within tumor tissues. This study investigated the expression level and prognostic significance of CD39 molecule in human CRC.Methods: Fresh CRC specimens(primary tumor tissues, tumor draining lymph nodes and liver metastases)and tissue microarray slides(TMA) containing 90 paired tumor samples were used to examine the expression characteristics of CD39 by IHC. The staining density of CD39 protein was assessed objectively and quantitatively using Aperio ImageScope software. Malignant and normal epithelial cells were retrieved respectively from 5 paired tumor samples by LCM, and the mRNA expression levels of CD39 were investigated by RT-PCR. Protein levels of CD39 in tumor tissues, tumor draining lymph nodes and liver metastases were also determined by Western blot. Statistical analyses were used to associate levels of CD39 with tumor features and patient outcomes.Results: Our data demonstrated that CD39 staining strongly marked malignant epithelial cells where the protein and mRNA expression levels of CD39 were significantly increased compared with paracancerous controls. In addition to primary tumor cells, CD39 was also abundantly expressed in tumor draining lymph nodes andliver metastases from patients with mCRC. Although patients with higher density oftumoral CD39 were more likely to have favorable characteristics(early TNM and Nstages) and overall survival(OS), the singular parameter cannot be used as anindependent factor for predicting patients’ prognosis. Furthermore, our data revealed thatthere was not a significant difference in tumoral expression levels between CD39 andCD73, but a positive correlation was observed. Intriguingly, combined analysis of CD39and CD73 expression was more efficient to foretell patient’s outcome where patients withincreased CD73 but decreased CD39 levels displayed a worst prognosis.Conclusion: The current study revealed that malignant epithelial cells of humanCRC strongly express CD39 molecule, the expression level of which was significantlyassociated with tumor TNM stage. Although high expression of CD39 in tumor cells iscorrelated with favorable clinical outcome, the combination of CD39 and CD73expression may have a better prognostic value. Part Ⅲ The expression and clinical significance of CD39/CD73molecules in human gastric carcinomaObjective: The purpose of this study was to determine the expression and clinicalrelevance of CD39 and CD73 molecules in human gastric carcinoma.Methods: The expression of CD39 and CD73 in freshly resected samples(n = 42,TNM I-III) containing primary tumor tissues, paired paracancerous specimens and tumordraining lymph nodes were examined by IHC. The staining density of CD39/CD73protein was assessed quantitatively using Aperio ImageScope software. The protein andmRNA expression levels of CD39 and CD73 were also investigated respectively by WBand RT-PCR. Statistical analyses were used to associate levels of CD39 and CD73 withtumor characteristics.Results: We found CD39 or CD73 expression was mainly expressed in thecytoplasm and membrane of malignant epithelial cells, whereas it was primarily restrictedto the lymphocytes or vascular endothelia cells in tumor stroma. Compared to theparacancerous normal tissues, CD39/CD73 mRNA expression levels were not significantdifference in tumor tissues, but the protein expression levels of CD39/CD73 wereobviously upregulated. Moreover, our data indicated that CD73 expression levels intumor cells were associated with tumor TNM stage, but CD39 expression levels were notsignificantly related with clinicopathologic characteristics.Conclusion: The current study revealed CD39 or CD73 expression was strongly express in malignant epithelial cells of human gastric carcinoma, where the protein expression levels of CD39/CD73 were significantly increased compared with paracancerous controls. CD73 expression levels in tumor cells were associated with tumor TNM stage, but the prognostic significances of CD73 and CD39 warrants further investigated.