The Role Of Tumor Immune Microenvironment In Hepatocellular Carcinoma Recurrence/metastasis And The Establishment Of An "Immune Microenvironment Signature"

Hepatocellular carcinoma(HCC) is a malignancy of worldwide significance and has become increasingly important.HCC is currently the sixth most common solid tumor and the third leading cause of cancer-related death worldwide,as well as the second leading cause of death due to cancer in China.Despite tremendous achievements being made clinicaly and basically during past decades,the prognosis of HCC remains dismal:the overall 5-year survival of HCC is less than 5%.Although surgery achieves the best outcomes in well-selected candidates,the recurrence rate remains high after HCC resection.Even after small HCC resection,the 5-year recurrence rate after curative resection was as high as 60-70%.Recurrence and metastasis after resection remains a major obstacle for more curative effect.For this reason,understanding the mechanisms that facilitate HCC cell invasion and metastasis,evaluation invasiveness of HCC and searching for effective interventional target is of great importance.The vast majority of previous studies focused solely on malignant cells themselves,regarding tumors just as masses of autonomous cells and aiming at identifying the molecular and genetic changes associated with this malignant transformation.Recent research on tumor-host interactions collectively reveals that the bidirectional and dynamic interactions between the tumors and their microenvironments co-evolve during tumor initiation and progression.Therapeutic strategies that fail to harness the immune system will always be defeated by tumor resistance,due to the large "genomic space" that genetically plastic tumor cells can readily access to evolve resistance mechanisms.In addition,as proposed by Schreiber and colleagues,avoidance of immunosurveillance is recognized as the seventh hallmark of cancer,suggesting that permanent success of treatments for cancer might depend on using immunogenic chemotherapy to re-establish antitumor immune responses.This is further demonstrated by the findings that in colorectal cancer immune microenvironment-related parameters is strongest prognosticators up to now, even outperformed the most commonly used TNM staging system.Cancer immunologists with cancer biologists finally come to a consensus that in addition to the malignant cell itself,cancer is a disease of microenvironment and immunity.It is not surprise that therapeutic strategies targeting the co-evolving tumor microenvironment and awaking hosts' antitumor immune responses have gained a major interest and are capable of providing an all-out attack on cancer.The concept of tumor microenvironment pays particular importance on the tumor-host interactions,and hence is superior to these cancer cell-oriented studies. Technically,the introduction and development of high throughput analysis like genomic,protemics and tissue microarray greatly facilitate the emerging studies on tumor microenvironment.Based on these technological achievements and previous theoretical hypothesis,unprecedently large-scale and unbiased analyses could be conducted as well as precise and novel conclusions yielded.As an immuno- privileged organ,the liver has its own unique immune system, acting as a key immune regulator locally and systemically.It is hypothesized that local immune microenvironment has a critical role in hepatocarcinogenesis,metastatic invasion and dessimination.However,to date,systemic and comprehensive in vivo human studies are still lacking.To this end,in this study,we first evaluate whether the type,dense,location and functional status of tumor-infiltrating immunocompetent cells(dendritic cells and T cell subsets) are associated with postoperative recurrence and metastasis in HCC using immunohistochemical staining on tissue microarray and routine sections;then,we investigated the expression profiles of marker genes symboling or representing effector immune response(mainly adaptive immunity), immunosuppression and inflammation using real time RT-PCR arrays to correlate them with HCC recurrence and metastasis;finally,we prospectively construct a recurrence score model-"immune microenvironment signature" which has the power to accurately stratify patients into high vs.low recurrent risk,based on the expression profiles of 8 effector immune response-related genes;this recurrence score model is validated in an independent cohort of HCC patients and the tissue microarrays containing this two cohorts of patients yield similar immunohistochemical results regarding immune infiltration and tumor relapse.PartⅠ.Study on the relationship of tumor microenvironment immunocompetent cells with HCC recurrence/metastasis This study aims to investigate whether the type,dense,location and functional status of tumor-infiltrating immunocompetent cells(dendritic cells and T cell subsets) are associated with postoperative recurrence and metastasis in HCC.This preliminary study may also lay a theoretical as well as experimental foundation for further high throughput and comprehensive analysis on the role of immune microenvironment in HCC relapse and tumor aggressiveness.In this study we enrolled tow independent and random cohorts of HCC patients underwent curative resection in Zhongshan Hospital,Fudan University at distinct time intervals as study populations:cohort A,from Jan 1991 to Dec 1992,n=123;cohort B, from Feb 1997 to Dec 1999,n=302.In cohort A,using HE staining, immunohistochemical and double immunohistochemical staining on routine HCC tissue sections,we evaluated tumor-infiltrating S100+ dendritic cell,CD45RO memory T cell,CD8+ effector T cell and CD3+ total T cell both in cancer center and surrounding liver tissue and correlated with postoperative HCC recurrence.In cohort B,using immunohistochemisty and tissue microarrays,tumor microenvironment CD3+,CD4+,CD8+ and in particular,FOXP3+ as well as granzyme B+ T and their relationship with HCC recurrence/metastasis were assessed.The results showed that(a) The number grade of infiltrating immunocompetent cells in HCC nodules and pericancerous tissues under HE staining had no significant correlation with tumor-free survival time(P=0.054,0.071,respectively).DCs were mainly among tumor cells,encircling tumor cells with their pseudopodia and were in contact with T lymphocytes.A certain number of DCs in HCC nodules(≥25/10HPF) statistically correlated to tumor-free survival time(P=0.005),while a certain number of DCs in pericancerous tissues(≥28/10HPF) had no correlation with tumor-free survival time(P=0.329).The number of memory T cells,CD3+ T lymphocytes and CD8+ T lymphocytes in HCC nodules strongly correlated to tumor-free survival time (P=0.003,0.005,0.037,respectively).The tumor-free survival rate curves revealed that the more DCs or together with memory T cells/CD3+ T lymphocytes or that the more CD8+ T lymphocytes were detected in HCC nodules,the better the prognosis would be.(b) The presence of low intratumoral Tregs in combination with high intratumoral activated CD8+ cytotoxic cells(CTLs),a balance toward CTLs,was an independent prognostic factor for both improved DFS(P=0.001) and OS(P＜0.0001).Five-year OS and DFS rates were only 24.1%and 19.8%for the group with intratumoral high Tregs and low activated CTLs,compared with 64.0%and 59.4%for the group with intratumorai low Tregs and high activated CTLs,respectively.Either intratumoral Tregs alone(P=0.001) or intratumoral activated CTLs(P=0.001) alone is also an independent predictor for OS.In addition,high Tregs density was associated with both absence of tumor encapsulation(P=0.032) and presence of tumor vascular invasion(P=0.031).These results suggested that as a pivotal component of tumor microenvironment and a complex family consisted of multi-functional subsets,tumor-infiltrating immunocompetent cells may have a key role in HCC recurrence and metastasis through their own inner cross-talk and interactions with tumor cells.An immune microenvironment with potent anti-tumor activity can control the tumor effectively, while a microenvironment in the state of tolerance promotes tumor's progression. Only detailed analysis on type,density,location and functional status of local immune cells,can relative precise quantative and qualitative impression on immune response in HCC tumor microenvironment be catched.A further investigation on the exact role of immunomicroenvrionent in HCC may substantially promote studies on HCC invasion,metastasis and immune escape,as well as bear fundamental clinical significance.PartⅡ.PCR array analysis on the role of tumor immune microenvironment in HCC recurrence/metastasis and the construction of "immune microenvironment signature"In this study,to conduct a comprehensive analysis on relation of immune microenvironment with HCC recurrence/metastasis,we investigated the expression profiles of genes symboling or representing effector immune response(mainly adaptive immunity),immunosuppression and inflammation using real time RT-PCR arrays,the so-called "second generation focused cDNA microarray" with the feature of high throughput and high reproducibility.Also,we prospectively construct a recurrence score model-"immune microenvironment signature" which has the power to accurately stratify patients into high vs.low recurrent risk,based on the expression profiles of 8 effector immune response-related genes.Through comprehensive literature searching,we identified 27 marker genes symboling or representing effector immune response(mainly adaptive immunity), immunosuppression and inflammatory response as target genes and enrolled an independent as well as random cohort of surgical HCC patients from Zhongshan Hospital,Fudan University.After optimizing the PCR array platform,the expression profiles of these 27 genes were detected using our specifically customized PCR arrays. 2 genes of extremely low expression or unexpected expression were excluded for data analysis.The open source software Cluster and Treeview were used in conducting retrospective unsupervised hierarchical cluster and correlation matrix analysis, followed by survival analysis.Finally,an "immune microenvironment signature" was generated by the well-recognized recurrence score system for prospective patient stratification.The results showed that(a) Unsupervised hierarchical cluster with all the 25 genes classified patients into two groups with significantly different relapse-free survival(P=0.046,HR=0.54,95%CI=0.29-0.99),however,this significance failed in multivariate analysis(P=0.27) and no significant difference regarding clinicopathological features was detected between the two groups.(b) In correlation matrix,genes symboling effector immune response,immunosuppression and inflammatory response clearly formed independent clusters.(c) A hierarchical tree structure classifying the patients according to the expression levels of effector immune response related genes cluster revealed an inverse correlation between the expression of these genes and tumor recurrence(P=0.00035,HR=4.07,95%CI= 1.88-8.78) and survival(P=0.010,HR=2.18,95%CI=1.20-3.97),with the significance in recurrence further validated in multivariate test(P=0.017).The group with low expression of effector immune response related genes consisted of more patients with tumor recurrence or death,and tumors venous invasion,larger tumors, later TNM and CLIP stages as compared with the group with gene high expressed.(d) Unsupervised hierarchical cluster with all the 12 immunosuppression/inflammation -related genes([IL8,MMP7,PGS2,IL1B,TGFB1,HIF1A,MMP9]&[ARG1,NOS2A,IL23A,VEGF,NEMO]) classified patients into two groups with significantly different relapse(P=0.023,HR=0.50,95%CI=0.27-0.91) and survival (P=0.0030,HR=0.45,95%CI=0.26-0.76),however,significances disappeared in multivariate analysis(P=0.26;P=0.13).(e) Only the 8 effector immune response -related genes(GNLY,GZMB,TRAV10,CD3Z,TBX21,IFNG,GATA3,IRFI) remained significant when performing multivariate Cox regression analysis one by one and separately.A recurrence score model-"immune microenvironment signature" which has the power to accurately stratify patients into high vs.low recurrent risk, based on the expression profiles of 8 effector immune response-related genes was constructed(univariate P=0.00011,multivariate P=0.0020).In ROC curve evaluation,the area under the curve of the "immune microenvironment signature" (AUC=0.738) was just next to TNM stage(AUC=0.769) and superior to other clinicopathological parameters like venous invasion(AUC=0.715),tumor number, size and encapsulation.Collectively,these results demonstrated that an effector immune response (mainly adaptive immunity) dominated microenvironment play a central role in HCC recurrence.An effector immune response(mainly adaptive immunity) dominated microenvironment is associated with low tumor recurrence risk and limited aggressive behavior.The immunosuppression/inflammation-related microenvironment may function as accelerating HCC tumor recurrence.However,its reproducibility, predictive value and significance remain to be evaluated,an immune microenvironment based HCC recurrence predicting system is clinically feasible and rational.PartⅢ.Validation of the "immune microenvironment signature" on cellular and molecular levelsIn this study,we designed to validate the "immune microenvironment signature" using real time RT-PCR on another independent cohort of surgical HCC patients. Further,a tissue microarray containing both the original and validating cohorts was constructed for testifying on cellular levels.We designed and synthesized primer sets specifically detecting the 8 effector immune response-related genes involved in the "immune microenvironment signature"(GNLY,GZMB,TRAV10,CD3Z,TBX21,IFNG,GATA3,IRF1).We enrolled another independent and random cohort of 122 HCC patients received curative hepatectomy in Zhongshan Hospital,Fudan University.Real time RT-PCR using these 8 primer sets was performed on frozen specimens from this cohort of patients and gene expression level was applied to test the reproducibility,predictive power and significance of the signature.Then,a tissue microarray containing both the original and validating cohorts was constructed for further immunostaining of peritumoral and intratumoral effector immune/immunosuppression/inflammation -related cells(CD8+,Granzyme B+,CD57+,CD45RO+,CD68+,Foxp3+和αSMA+). The type,dense,location and functional status of these cells were used for patients' stratification and compared with immune gene expression level.The results showed that(a) the "immune microenvironment signature" successfully and accurately classified the validation cohort into high recurrence risk and low recurrence risk groups(P=0.020,0.047 for the uni- and multi- variate analyses).The high risk group is 2.1 times more likely to experience recurrence than the low risk group.(b) the intratumoral effector immune cells(CD8+,Granzyme B+,CD57+,CD45RO+) immunosuppression cell(Foxp3+) and peritumoral immnunosuppression/inflammation cells(CD68+,αSMA+) were all significantly associated with HCC recurrence/metastasis,whether patients were grouped using median cell number as cutoff,minimum P value cell number as cutoff or analyzed using cell number as continuous variable.In addition,combinations of two cell types were more significant than any single cell type analyses.Taken together,these results revealed the "immune microenvironment signature" is reproducible and powerful strategy for prospective patient prediction and further validated the concept that an effector immune response(mainly adaptive immunity) dominated microenvironment play a central role in HCC recurrence.Additionally,the predictive value of immune microenvironment is comparable molecularly and cellularly.Conclusions1.The number of intratumoral dendritic cells and memory T cell can serve as a predictive index for recurrence and metastasis of HCC.Regulatory T cells are associated with HCC invasiveness and intratumoral balance of regulatory and cytotoxic T cell is a promising independent predictor for recurrence and survival in HCC.These may suggest a crucial role of immune microenvironment in HCC recurrence and progression.2.The most commonly used housekeeping genes in real time RT-PCR like GAPDH and ATCB are heavily regulated during hepatocarcinogenesis and tumor progression. The combination of TBP and HPRT as internal controls is cost-effective and potent in HBV-related HCC analysis.3.An effector immune response(mainly adaptive immunity) microenvironment characterized by high expression of gene encoding GNLY,GZMB,TRAV10,CD3Z, TBX21,IFNG,GATA3 and IRF1 play a central role in HCC recurrence.An effector immune response(mainly adaptive immunity) dominated microenvironment is associated with low tumor recurrence risk and limited aggressive behavior.The immunosuppression/inflammation-related microenvironment may function as accelerating HCC tumor recurrence.4.The "immune microenvironment signature" is a reproducible and powerful strategy for prospective patient prediction,which is worth of further clinical testifying.The novelty of this study1.For the first time,we demonstrated and reported that the number of intratumoral dendritic cells and memory T cell can serve as a predictive index for recurrence and metastasis of HCC.Induction of antitumor immune response by activating the DCs may be a promising biological therapy and contribute to reduce postoperative recurrence/metastasis of HCC.2.For the first time,we demonstrated and reported that intratumoral balance of regulatory and cytotoxic T cell is a promising independent predictor for recurrence and survival in HCC.A combination of depletion of Tregs and concomitant stimulation of effector T cells may be an effective immunotherapy to reduce recurrence and prolong survival after surgery.3.For the first time,we demonstrated and reported that in HBV- related HCC the most commonly used housekeeping genes in real time RT-PCR like GAPDH and ATCB are heavily regulated during hepatocarcinogenesis and tumor progression.The combination of TBP and HPRT as internal controls is cost-effective and potent in HBV-related HCC analysis.4.Using high throughput genomic and in situ immunostaining analyses,we found that an effector immune response(mainly adaptive immunity) dominated microenvironment play a central role in HCC recurrence.The immunosuppression/inflammation-related microenvironment may function as accelerating HCC tumor recurrence.5.We constructed and validated an "immune microenvironment signature" featured by the expression of 8 effector immune response related genes.It is the first time that a predictive signature is derived from molecular features of tumor immune microenvironment in human cancer studies.The potential application of this project1.The type,density,location and functional status of tumor microenvironment immune cells can be served as novel and independent predictors of HCC recurrence and metastasis,which is beneficial in predicting which patients are at highest risk of recurrence,thus facilitating patient selection for more aggressive treatment,and identifying patients who may benefit by future immunotherapies.2.The "immune microenvironment signature" is powerful,reproducible and practically convenient,which could be prepared as clinically diagnostic regent in predicting HCC relapse.Moreover,this signature is tumor microenvironment derived, suggesting its potential of being universally used in other tumor types.