Prognostic Significance Of Immunity Cytokines Level In The Peritumoral Tissues And Osteopontin In The Intratumoral Tissues Of Hepatocellular Carcinoma Patients After Curative Resection

Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies worldwide. It has an extremely poor prognosis, mainly attributed to the high frequency of intrahepatic metastatic recurrence. It is a challenge to identify patients who are at a greater risk for tumor recurrence after curative treatment for HCC, particularly in those with early stage disease who do not have significant vascular invasion, or regional lymph node or distant metastasis. Identification of molecular markers could provide supplemental and useful information for predicting clinical outcome in patients with a given stage of disease and improve the selection of patients for adjuvant therapies after resection. Studies of many kinds of human malignancies have shown that Th1-and Th2-like cytokines may play a prominent role in tumor progression and metastasis.In a recent study of gene expression profiling, we found that the liver tissues in patients with metastatic HCC had lower levels of pro-inflammatory Thl-like cytokines and remarkably higher levels of anti-inflammatory Th2-like cytokines in comparison to those without metastatic HCC. However, whether the protein expression levels of these Thl/Th2 cytokines can be used to predict the clinical outcome of HCC is not known. In our previous research, we found that IL-2 or IL-15 of immunity-associated genes signature should allow a sensitive and specific monitoring of survival in HCC. The accumulated evidence has clearly demonstrated that a dominant Th2-like cytokine profile and a decrease in Thl-like cytokines are associated with the metastatic phenotype. High IL-10 levels had a worse postresection outcome, and IL-10 may be a predictor marker of the of HCC patients after surgery.The research based on the micro-environment has achieved exciting results, but, as we know the metastasis and recurrence of HCC is closely related to not only microenvironment but also the characteristics of cancer cell itself. In our previous work, we collaborated with the National Cancer Insititute (NCI) of USA, Osteopontin (OPN) plays an important role in the development, invasion, and metastasis of malignancies. An OPN-neutralizing antibody efficiently blocked in vitro invasion and in vivo pulmonary metastasis of HCC cells. The current study was based on our previous findings and the concept that proteins affect cancer progression, thus serving as markers of disease and therapeutic targets. We examined the protein levels of 12 Thl/Th2 cytokines in noncancerous liver tissue samples from 192 patients (cohort A) with HCC using ELISA methods. Of the 12 tested cytokines, three (IL-2, IL-15, and IFN-y) were found to correlate with clinical outcome. IL-2 had the best prognostic performance in the training set, which was then further validated in the test set (n=64). The prognostic value of IL-2 was further confirmed in another independent cohort (cohort B) of 261 patients using in situ immunohistochemical staining. At the same time, we examined OPN expression in tumor tissue microarrays (TMA) containing 261cases (cohort B) in order to investigate the predictive value of peritumoral IL-2 and intratumoral OPN for the prognosis of HCC patients.Thl/Th2-like cytokine mRNA levels in noncancerous hepatic tissues from hepatocellular carcinoma (HCC) patients are associated with metastases and recurrence. The present study evaluated the prognostic values of Thl/Th2 cytokine in HCC patients.Two independent cohorts of 453 HCC patients were enrolled. Twelve Thl/Th2 cytokines in noncancerous hepatic tissues from cohort A (n=192) were quantified with enzyme-linked immunosorbent assays. This cohort was split into training and test sets. Prognostic cytokines were identified from the training set, validated with the testing set, and confirmed in cohort B (n=261) using in situ immunohistochemical staining.IL-2, IL-15 and IFN-γprotein levels in noncancerous liver tissues were significantly greater in HCC patients without than in those with tumor recurrence. On Cox regression stepwise variable selection analyses, IL-2 was the optimal independent predictor of outcome (HR for recurrence=0.4, P=0.001; HR for death=0.5, P=0.019), rather than IL-15. This was confirmed in the testing set and validated in cohort B (HR for recurrence=0.4; P<0.0001; HR for survival=0.6; P=0.005). Another finding was that IL-2 was mainly contributed by hepatocytes other than the lymphocytes in noncancerous liver parenchyma. HCC patients can be stratified into subgroups with different prognoses postoperatively based on noncancerous hepatic tissue IL-2 levels. IL-2 was the optimal predictor for both tumor recurrence and patient。The aim of this study was to assess the value of Thl/Th2-like cytokine for predicting tumor recurrence after curative resection in serum and peripheral blood mononuclear cell of hepatocellular carcinoma patients.Blood samples from HCC patients and liver cirrhosis patients were collected for cytokine quantitation. Because Thl/Th2-like cytokine concentrations were too low, we performed the cytometric bead array (CBA) assay, which utilizes the sensitivity of amplified fluorescence detection. The expression levels of cytokine mRNA in peripheral blood mononuclear cell was assessed by qRT-PCR.No significant difference in serum IL-2 (P=0.6907),IFN-γ(P=0.459),IL-4 (P=0.2044) and IL-10 (P=0.0837) level was found between the HCC patients (n=71), CG (n=9)and NG(n=13). Moreover, the serum IL-2, IFN-γ,IL-4 and IL-10level had no significant associations with OS or TTR in HCC patients using the median levels as cutoff. Similar results were also found in PBMC. The finding was that, unlike in liver tissues, serum IL-2 levels were not significantly correlated with tumor recurrence or survival of HCC patients.Osteopontin (OPN) plays an important role in the development, invasion, and metastasis of malignancies. It is generally classified as a pro-inflammatory cytokine because it modulates cell-mediated immunity by promoting the Thl response. In our present study, we found that peritumoral IL-2 was the optimal predictor for both tumor recurrence and overall survival time. The aim of this study was to explore the relationship of OPN and Thl/Th2 cytokines(IL-2, IFN-γ, IL-4 and IL-10)expression, then assess the value of OPN and IL-2 for predicting tumor recurrence after curative resection in hepatocellular carcinoma patients. Tissue microarrays of 261 (cohort B) HCC patients were used to detect the expressions of OPN and IL-2. Clinicopathologic data for these patients were evaluated. The prognostic significance was assessed using Kaplan-Meier survival estimates and log-rankt tests.No significant association was found between OPN and Thl/Th2 cytokines expression in cell lines. In a tissue microarray-immunohistochemical analysis, we found that patients with higher levels of intratumoral OPN and lower levels of peritumoral IL-2 had a significantly poorer prognosis than patients with lower OPN and higher IL-2 levels. The combination of OPN and IL-2 expression thus served as an effective prognosticator.These findings suggest that IL-2 alone or in combination with OPN may act as an independent indicator for HCC patients after curative resection.1. HCC patients can be stratified into subgroups with different prognoses postoperatively based on noncancerous hepatic tissue IL-2 levels. IL-2 was the optimal predictor for both tumor recurrence and patient.2. IL-2 was mainly contributed by hepatocytes other than the lymphocytes in noncancerous liver parenchyma.3. The third important finding was that, unlike in liver tissues, serum and PBMC immune/inflammatory levels were not significantly correlated with tumor recurrence or survival of HCC patients.4. Peritumoral IL-2 alone or in combination with intratumoral OPN may act as an independent indicator for HCC patients after curative resection.5. The metastasis and recurrence of HCC is closely related to not only microenvironment but also the characteristics of cancer cell itself. To prevent recurrence of HCC we must focus on them all. 1. We firstly demonstrated that HCC patients can be stratified into subgroups with different prognoses postoperatively based on noncancerous hepatic tissue IL-2 and IL-15 levels. IL-2 was the optimal predictor for both tumor recurrence and patient.2. For the first time, we found IL-2 was mainly contributed by hepatocytes other than the lymphocytes in noncancerous liver parenchyma.3. We firstly confirmed that peritumoral IL-2 alone or in combination with intratumoral OPN may act as an independent indicator for HCC patients after curative resection.Measuring the IL-2 protein level in noncancerous hepatic tissues could provide useful information for predicting tumor recurrence and prognosis in patients, even those with early stage HCC. This finding may potentially enable us to identify and select high-risk patients for effective adjuvant therapy. Regulation of the immune/inflammatory response may be a helpful strategy to control tumor relapse and further improve the treatment outcome of HCC patients. It has been applied for the patent (" Multiple inflammatory cytokines prediction kit for postoperative recurrence and metastasis of hepatocellular carcinoma"; Chinese patent applying number is 200810208150.6)...