The Expressions Of CD44v2-CD44v10and CD44s Are Associated With The Survival Of Pancreatic Carcinoma Patients

BackgroundPancreatic carcinoma (PCa) is undoubtedly the most aggressive and the deadliestcancer. Early metastasis to regional lymph nodes or hematogenous spread to distant organsis largely responsible for the lowest5-year survival rate (less than5%). Numerousmolecμles have been described and extensively investigated for their potential roles in thetumorigenesis and progression of PCa; CD44is the most important of these molecμles.CD44is quite complicated. This molecμle is encoded by20exons and undergoesextensive alternative splicing to generate CD44s (CD44standard) and CD44v (CD44variants). CD44s consists of exons1-5and16-20and is called the constant form. Thevariable exons are typically numbered v1-v10(v1is not encoded in humans),corresponding to the genomic exons6-15, and are alternatively spliced and incorporatedinto the variable region either singly or in combination. This process has the potential togenerate thousands of different CD44isoforms, and as a resμlt, CD44has complex anddiverse functions.CD44was initially identified as a lymphocyte homing receptor and transmembraneglycoprotein commonly expressed in embryonic stem cells and in hematopoietic and cancerstem cells. Although CD44plays an important role in many cell processes, includinggrowth, differentiation and motility, there are discrepancies in the literature about the rolesof CD44v and CD44s in tumor progression. In certain cancers, CD44s and CD44v areconsidered to be tumor progression promoters, but in other cancers, CD44s and CD44v maybe involved in tumor suppression. These discrepancies may be the resμlt of differentmethods for detecting CD44(such as immunohistochemistry (IHC) or PCR) and identifyingdifferent CD44variants in different tumor types. The roles of CD44in PCa are stilldisputed. Tsukuda, H reported that CD44v6and CD44v2were expressed in the pancreatic juice of patients with pancreatic carcinoma, but the authors also reported CD44v expressionin normal cases. Rall, C. J. studied CD44expression in21clinical PCa tissues and reportedthe expression of CD44v6, CD44v8-9, CD44v8-10and CD44s, although only CD44v6maybe involved in tumor metastasis. Tomaszewska, R reported that CD44v6and CD44sexpression was positive in pancreatic carcinoma, and Gotoda, T. et al. reported that CD44v6and CD44v2may be usefμl markers for poor survival after studying the expression ofCD44v6, CD44v2and CD44s via IHC. However, all studies found no significantassociation between CD44s and tumor progression.In fact, the expression patterns of CD44in pancreatic carcinoma have not beensystematically investigated at the mRNA level. To investigate whether CD44expressionpatterns are related to pancreatic carcinoma metastasis and prognosis, we designed a primerspecific for each CD44variant (CD44v2-CD44v10and CD44s), detected the expressionpatterns of CD44in101clinical pancreatic carcinoma samples, and analyzed therelationship of those patterns with the clinical characteristics of pancreatic carcinoma.Methods:1. A total of101patients underwent surgery for pancreatic carcinoma at theDepartment of Hepatobiliary Surgery Institute, Southwest Hospital, Third Military MedicalUniversity, China, from January2008to January2010. All patients underwent curativeresection by pancreaticoduodenectomy or pylorus-preserving pancreaticoduodenectomywith lymph node dissection. None of the patients received neoadjuvant or adjuvantradio/chemotherapy. Correlations between the expressions of CD44v2-CD44v10, CD44sand clinical features were analyzed.2. The Aspc-1, Cfpac-1and Panc-1pancreatic carcinoma cell lines were purchasedfrom the Shanghai Biomedical Institute. All cell cμltures were supplemented with10%fetalbovine serum in the presence of100U/ml penicillin and100mg/ml streptomycin. All celllines were cμltured at37°C in a humidified atmosphere containing5%CO2. Their invasionabilities were also tested by transwell assay.3. Expressions of CD44v2-CD44v10and CD44s on fresh frozen specimens wereanalyzed by quantitative real-time PCR (qRT-PCR). The correlation between categoricalvariables was evaluated using the χ2test. Data were analyzed using Student’s t-test if theinvolved comparison groups followed normal distributions; otherwise, the nonparametric Mann-Whitney test was applied. Univariate analysis was conducted by the KM method (thelog-rank test). Mμltivariate analysis was performed using the stepwise Cox mμltivariateproportional hazard regression model (forward, likelihood ratio), and the values of variablesnot in the equation were picked from step1. P-values <0.05were considered to bestatistically significant.Results:1. The expression levels of CD44v2-CD44v10(CD44v), from highest to lowest, werein As, Cf and Pa. Interestingly, the expression levels of CD44s, from highest to lowest,were in Pa, Cf and As, which indicates that the pancreatic carcinoma cell lines expresseddifferent levels of CD44v and CD44s. besides, The As cell line showed the highest invasioncapacity; Cf, the median invasion capacity; and Pa, the lowest invasion capacity. Theseresμlts suggest that increased expression of CD44v or decreased expression of CD44s isassociated with the invasion potential of the PCa cell lines. Compared with peritumoraltissue, PCa showed both increased CD44v expression and decreased CD44s expression(CD44v5, v6, v9and v10and CD44s showed significant differences). Moreover, higherexpression of CD44v and lower expression of CD44s were found in metastatic samplescompared with non-metastatic samples (CD44v6and CD44s showed significantdifferences).2. In101clinical tissues, CD44v6+and CD44v9+were significantly correlated withlymph node metastasis (P=0.02), liver metastasis (P=0.049) and TNM stage (P=0.008andP=0.027, respectively), and CD44s-was significantly correlated with liver metastasis(P=0.049), differentiation (P=0.028) and TNM stage (P=0.028). However,CD44v2-CD44v5+, CD44v7+, CD44v8+and CD44v10+were not correlated with anyclinicopathological characteristics of pancreatic carcinoma.3. KM curve analysis showed that patients with CD44v6+and CD44v9+hadsignificantly low survival rates (P=0.01and P=0.047, respectively). In contrast, patientswith CD44s-had significantly low survival rates (P=0.032). Patients withCD44v2-CD44v5+, CD44v7+, CD44v8+and CD44v10+demonstrated no differences insurvival rate. Interestingly, pancreatic carcinoma patients with tumors that wereCD44v6+/CD44s-or CD44v9+/CD44s-had a significantly lower survival rate than didpatients with tumors expressing CD44v6+, CD44v9+or CD44s-alone. However, CD44v6+/CD44v9+did not further decrease the patients’survival rate.4. Univariate analysis revealed that lymph node metastasis (pN); vessel invasion (pV);hepatic metastases; TNM stage; and individual or co-expression of CD44v6+, CD44v9+and CD44s-were risk factors that significantly affected the survival of pancreaticcarcinoma patients. However, mμltivariate analysis showed that only CD44v6+/CD44s-andpN were independent risk factors affecting the OS of pancreatic carcinoma patients(P=0.003and P=0.002, respectively).Conclusions:We demonstrated that among all CD44variants, CD44v6+, CD44v9+and CD44s-were significantly associated with the metastasis and prognosis of pancreatic carcinoma.The different expression patterns of CD44v and CD44s may be usefμl markers forpredicting the prognosis of pancreatic carcinoma.