The Impact Of The Tumor Immune Microenvironment And The Overall Immunity Condition On Prognosis Of NSCLC

【Objective】To evaluate whether the levels, locations and functional status of Tlymphocyte subgroups and cytokines in NSCLC tumor microenvironmentcorrelate with NSCLC recurrence and metastasis. In addition, T lymphocytesubgroups and cytokines in NSCLC peripheral blood were tested to determine thedifferences of immune response between tumor microenvironment and the globalbody. The immune situations at different time points after chemotherapy and theprognosis of NSCLC patients were analyzed. We also analyzed the impact ofdifferent chemotherapy strategies on the immune system of NSCLC patient andtheir complex interactions on patients' outcome. These data provide technical andtheoretical basis for improved anti-tumor strategies of chemotherapy incombination with immununotherapy.【Methods】 Part one: The archival samples were derived from107NSCLCpatients with radical surgical resection with curative intent between1998and2007and pathologically diagnosed and confirmed. Only those cases with tissuepreservation and complete following up were selected. Samples were tested byimmunohistochemistry (IHC) with following primary antibodies: CD3, CD4, CD8,CD28, Foxp3, CTLA-4,COX-2,TGF-β1,IL-2,IL-6,IL-10,IL-12R,HIF-1α. These targets in tumor microenvironment were analyzed to reveal theirrelationship with NSCLC recurrence and metastasis. Part two: Peripheral bloodsamples from84patients were tested by flowcytometry with the following targets:CD3+, CD4+,CD8+, CD28-, CD28+, CD4+CD25+, HLADR+, HLADR-. RT-PCR was applied to test the mRNAlevel in peripheral blood for the followingtargets: FOXp3,CTLA-4,COX-2,TGF-β,IL-2,IL-6,IL-10,IL-12,HIF-1α. The results represent the global situation of the NSCLC patients'immunesystem. Part three: Those with stage IIIb-IV whose tumor was unresectable wererandomly distributed into two different trial groups, the docetaxol group and thepemetrexed group. Flowcytometry was used to examine T lymphocyte subsets atdifferent time points. The effect of different chemotherapies on immune systemat different time points was evaluated to explore the best possible combination ofchemotherapy and immunotherapy.【Results】The immunohistochemical result of the NSCLC specimen showed thatthe Foxp3+Treg and IL-2level significantly correlate with progress free survivaltime and overall survival positively or negatively, respectively. The combinationof Foxp3+Treg and IL-2expression pattern iss an independent prognostic factorin spite of TNM system for both progress free survival time and overall survival.The level of CD3+lymphocyte significantly correlates positively with overallsurvival. In addition, CD3+,CD4+and CD8+cells mainly located in the stromainstead of tumor nests in the immune microenvironment. The level of CD28negatively correlateswith IL-6, IL-10, TGF-β1, as well as IL-10and IL-2. Thelevel of Foxp3+Treg positively correlates with TGF-β1, IL-10, CTLA-4. Thepositive correlations were also found between TGF-β1and IL-10and betweenCOX2and TGF-β1or IL-10. There is a negative correlation between IL-10andIL-2.Flow cytometry analysis revealed that the levels of CD3+, CD4+,CD4+/CD8+, CD3+HLADR-, CD4+HLADR-, CD8+HLADR-in the PBMC(peripheral blood mononuclear cells) of NSCLC patients were significantly lowerthan those of controls, while the CD4+CD25+Treg, CD3+HLADR+,CD4+HLADR+, CD8+HLADR+levels of NSCLC patients were higher. Thesubgroup analysis revealed that the CD3+,CD3+CD4+,CD3+HLADR-andCD4+HLADR-levels in the normal CEA group were higher than those in the abnormal CEA group. Patients with tumor size＞3cm showed lower levels ofCD3+HLADR+, CD4+HLADR+, CD8+HLADR+and higher level ofCD3+HLADR-. RT-PCR result showed lower expression of IL-2, IL-12andhigher expression of IL-10, COX2,TGF-β1and HIF-1a in the patient group.We also analyzed the change of the immune function after chemotherapy.The ratio of CD4+/CD8+and the levels of CD3+and CD3+CD4+increasedsignificantly on the4th and7th-10th day post-chemotherapy, and returned tonormal levels on the21th day. The CD3+level increased significantly in bothtreatment groups (pemetrexed and docetaxol) on all time points, while theCD3+CD4+, CD4+/CD8+levels significantly increased and the CD3+CD8+,CD8+CD28-levels significantly decreased on the4th day in Pemetrexed group.The CD3+CD4+levels increased significantly on the4th and7th-10th day and theCD8+CD28-levels decreased on the4th day in PR group. RT-PCR test showed atrend of decreased IL-6,IL-10,TGF-β1mRNA levels and increased IL-2mRNAlevel in the peripheral blood. However, the results were not statisticallysignificant.【Conclusion】The tumor microenvironment in NSCLC patients is immunesuppressive. The CD3+cells, Foxp3+Treg cells and IL-2level were closelyrelated to the prognosis of NSCLC patients. CD3related with OS while IL-2andFoxp3+Treg related to both PFS and OS. The combination of IL-2andFoxp3+Treg is an independent prognostic predictor in NSCLC independent ofTNM system. The test of the CD3, IL-2and Foxp3levels in the microenviromentsof NSCLC may help to identify the high-risk patient and lead them to moreeffective and individual care.The T lymphocyte subgroups in the PBMC of NSCLC patients were out ofbalance with enhanced Th1cytokine expression. Higher levels of COX2, TGF-β1, and HIF-1a were observed in the peripheral blood of NSCLC patientscomparing to those in the control group. These results suggest theimmunosuppressive condition of the NSCLC patients. The subgroup analysis showed more serious immune suppression in patients with higher CEA level andbigger tumor size. The chemotherapy may help to modulate the immune functionagainst cancer to some degree, while the problems remain in the selection ofchemotherapy regimen, the dose controlling and the way of combined treatment.