The Relationship Of Tumor-infiltrating Lymphocytes With HCC Recurrence/Metastasis

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 and the chief cause of death due to cirrhosis in China. Despite tremendous achievements being made clinically 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 curative resection, the 5-year recurrence rate was as high as 60-70%.The higher rate was displayed in patients who accepted local therapy. Recurrence and metastasis after resection remains to be a major obstacle for more curative effect. For this reason, understanding the mechanisms that facilitate HCC cell invasion and metastasis, evaluation invasiveness and prognosis of HCC, preventing positively and searching for effective interventional target is of great importance for enhancing the curative effect of HCC.Recurrence and metastasis of HCC is dynamic process that involve multi-links, multi-stages and multi-gene. 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 malignant transformation. Along with progresses of gene and molecular biology technology, the scientists discovered that the tumor microenvironment relative tightly to tumor plays an important role to the tumor evolution. Between the microenvironment and the tumor cell, there are complex overlapping dialogues and mutual using relations. The result of mutually effects like this reflected, changed even reversed the tumor biology characteristic and has the important influence to the tumor apparent genetics, the stem cell, the immunity escape, and metastasis potentials,ect. As the insufficiency of the traditional researches, we realized the extremely importance of interactions between cancer and the host, then, we pay more attention to the tumor microenvironment day by day. This is further demonstrated by the findings in colorectal cancer that immune microenvironment-related parameters were the 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.As an immune-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 plays a critical role on hepatocarcinogenesis, metastatic invasion and dissemination.In our previous work of Gao,et al.,they analyzed 27 marks genes symboling or representing effectors immune response (mainly adaptive immunity),immunosuppression and inflammatory correlation gene comprehensively.Eight effectors immune response related genes(GNLY,GZMB,TRAV10,CD3Z,TBX21,IFNG,GATA3,IRF1) were discovered significantly correlated with recurrence and metastasis of HCC.Based on these findings,they established "immune microenvironment signature" which further confirmed the function of immune microenvironment in recurrence of HCC.Therefore,it is of vital significance to explore characteristics of immune cell in the microenvironment of HCC which to affect the tumor malignant phenotypes and the ability of metastasis for the diagnosis,stages,target tropism treatment,the prevention of recurrence and metastasis,and consequently enhancing the curative effect of HCC.In this study,we first evaluated the type,density,location and functional status of tumor-infiltrating lymphocytes.They were found associated with postoperative recurrence and metastasis in HCC(especially in the early HCC patients absence of tumor micro and/or macro vascular invasion).Immunohistochemical staining was applied on tissue microarray and routine sections,and pointed out the local immune condition is decided by the proportional relationship between immunosuppression cells and immunological effect cells.We validated and evaluated the efficiency of "immune microenvironment signature" in different cohorts of HCC patients(all patients,the early HCC patients absence of tumor micro and/or macro vascular invasion,BCLC stage A patients).In the end,we appraised the difference of each hypotype of tumor-infiltration lymphocytes between the high and low risk groups,indicating that the combination of"immune microenvironment signature" and intratumoral regular T lymphocytes is a better prognostic model for relapse and survival of HCC.We confirmed the vital significance of immune microenvironment in HCC prognosis from molecular and cellular perspectives,and confirmed the tumor microenvironment was a tendency process that has multi-levels,multiple perspectives and multi-links.PartⅠ.The relationship butween tumor-infiltrating lymphocytes and HCC recurrence/metastasisThis study aimed to investigate whether the type,density,location and functional status of tumor-infiltrating lymphocytes were associated with postoperative recurrence and metastasis in HCC and to find out whether the signature could predict recurrence/metastasis of HCC.In this study we enrolled a random cohort of 298 HCC patients who underwent curative resection in Zhongshan Hospital,Fudan University from Jan 2002 to Dec 2006.Immunohistochemical staingings CD57+NK cells,Foxp3+regular T cells,Granzyme B+ activity cytotoxic lymphocytes,CD45RO+memery T cells,CD8+cytotoxic T cells were applied on tissue microarrays,and their relationships with HCC recurrence/metastasis were also assessed.The results showed that(a) except that the intratumoral number of FOXP3+Treg was greater than the peritumoral number,other various immunological reactivity cells numbering peritumoral tissue was by far more than intratumoral.(b) the univariate analysis showed that,except intratumoral Foxp3+Treg is strongly inverse related to DFS and OS(P＜0.001),other various immunological reactivity cell(ICD57+, ICD45RO+,IGrB+,ICD8+) is positive associated to DFS and OS.In peritumor tissue, all cells were related to DFS,yet,only Foxp3+Treg and CD8+TILs statistically correlates to OS.(c) multivariate analysis discovered:that intratumoral Foxp3+Treg is strongly inverse correlation to DFS and OS(HR=2.312,P＜0.001,HR=2.267,P＜0.001,respectively).Intratumoral CD8+ and CD45RO+TILs significant related to DFS and OS(ICD8+:HR=0.499,P=0.005,HR=0.481,P=0.002;ICD45RO+: HR=0.436,P＜0.001,HR=0.634,P=0.038,respectively).CD8+TILs and CD45RO+TILs in peritumor tissue were related to DFS,CD8+TILs in peritumor tissue also were related to OS.In the clinical pathology characteristics,tumor differentiation,number,vascular invasion were significant inverse associated to DFS, and there are inverse correlation between OS and the blood serumγGT,liver cirrhosis,the tumor differentiation,vascular invasion.(d) both DFS and OS in the group(groupⅠ)with intratumoral high Tregs and low immunological effect cells were worse than the group(groupⅣ) with intratumoral low Tregs and high immunological effect cells,so the proportional relationship between intratumoral Foxp3+Treg and immunological effect cells were an independent predictor for both DFS and OS(P＜0.001).(e)all intratumoral TILs were related to tumor vascular invasion,high Tregs density were associated to presence of tumor vascular invasion,however,high immunological effect cells were associated to absence of tumor vascular invasion; Intratumoral Foxp3+Tregs and CD45RO+TILs were also closely related to tumor differentiation(more IFoxp3+Treg,worse tumor differentiation;more intratumoral CD45RO+TILs,higher tumor differentiation);the number of intratumoral CD45RO+ and CD8+TILs were inverse associated to tumor size;Intratumoral GrB+ TILs were inverse related to tumor encapsulation and tumor number.In addition,there was no correlation between peritumoral TILs and clinical pathology characteristics.These results suggested that as a pivotal component of tumor microenvironment and a complex family consisted of multi-functional subsets,tumor-infiltrating lymphocytes might have a key role on HCC recurrence and metastasis through their own cross-talk and interactions with tumor cells.In HCC local immune microenvironment,the balance of power between immunosuppression and immunological reactivity function decided the local immune condition,further affected even decided the tumor progress direction.Local immune microenvironment with powerful immunological effect could control the tumor effectively,while a local immune microenvironment in the state of immunosuppression promoted tumor progression.Only detailed analysis on type,density,location and functional status of TILs in local immune microenvironment,can relative precise quantative and qualitative impression on immune response in HCC tumor microenvironment be catched.A further investigation on the exact role of immunomicroenvrionment in HCC may substantially promote studies on HCC invasion,metastasis,and immune escape,as well as bear fundamental clinical significance.PartⅡ.Study on the relationship of tumor-infiltrating lymphocytes with early HCC recurrence/metastasisThis study aimed to investigate whether the type,dense,location and functional status of tumor-infiltrating lymphocytes were associated with postoperative recurrence and metastasis in early HCC patients absence of tumor micro and/or macro vascular invasion,and to find out the signature could predict recurrence/metastasis of this group of HCC patient,further to select the high risk group from whole early HCC patients.In this study we enrolled a cohort of 167 HCC patients who underwent curative resection in Zhongshan Hospital,Fudan University from Jan 2002 to Dec 2006.They were confirmed after pathology as absence of tumor micro and/or macro vascular invasion.We researched CD57+NK cells,Foxp3+regular T cells,Granzyme B+ activity cytotoxic lymphocytes,CD45RO+memery T cells,CD8+cytotoxic T cells in tumor microenvironment and their relationships with early HCC recurrence/metastasis using immunohistochemisty and tissue microarrays.The results showed that(a) except that the intratumoral number of FOXP3+Treg was greater than the peritumoral number,other various immunological reactivity cells numbering peritumoral tissue was by far more than intratumoral.(b) the univariate analysis showed that,the intratumoral Foxp3+Treg were strongly inverse related to DFS and OS(P=0.030,P=0.003,respectively),intratumoral CD45RO+TILs were positive associated to DFS and OS(P=0.010,P=0.012,respectively).Foxp3+Treg in peritumor tissue only statistically related to OS(P=0.032),GrB+TILs in peritumor tissue were positive related to DFS and OS(P=0.005,P=0.039,respectively).(c) multivariate analysis discovered:the intratumoral CD45RO+TILs were significant related to DFS and OS(P＜0.001,P=0.003,respectively),both intratumoral and peritumoral Foxp3+Tregs were inverse related to OS(P=0.034,P=0.033,respectively). In the clinical pathology characteristics,AFP was significant inverse related to DFS(P=0.002),and there were inverse association between OS and blood serumγGT, tumor differentiation(P=0.002,P=0.038,respectively).(d) both DFS and OS in the group(groupⅠ)with intratumoral high Tregs and low CD45RO+memory T cells were worse than the group(groupⅣ) with intratumoral low Tregs and high CD45RO+memory T cells,so the proportional relationship between intrtumoral Foxp3+Treg and CD45RO+memory T cells was an independent predictor for DFS and OS.(e) intratumoral,high Tregs density was associated to worse tumor differentiation and more tumor number,however,high intrtumoral CD45RO+TILs closely related to better tumor differentiation and early TNM stage,intratumoral GrB+ TILs were inverse related to tumor encapsulation.In addition,there was no correlation between peritumoral TILs and clinical pathology characteristics.These results suggested that immunological effect function displayed by CD45RO+ memory T cells had a superiority role in early HCC immune microenvironment,But immunosuppression played by Foxp3+ Tregs to be in the inferior position.It was confirmed that,in HCC local immune microenvironment,the power balance relationships between immunosuppression and immunological reactivity function decided the local immune condition,further affected even decided the tumor progress direction.Local immune microenvironment with powerful immunological effect could control the tumor effectively,while a local immune microenvironment in the state of immunosuppression promoted tumor progression. Unifying the first part of result,we also might conceive,along with the tumor growth progress,the tumor local immune balance was broken unceasingly,when to mid and late stage,the local immune condition has gradually formed immunosuppression which toke Foxp3+Tregs as representative for the leading.Only detailed analysis on type,density,location and fimctional status of TILs in local immune microenvironment,can relative precise quantative and qualitative impression on immune response in HCC tumor microenvironment be catched.After screening the high-risk group of early HCC patients,we could carry on intervenes early to enhance the survival rate of entire HCC patients.PartⅢ.The relationship between tumor-infiltrating lymphocytes and the "immune microenvironment signature"This study aimed to investigate the relationships of TILs in immune microenvironment with the "immune microenvironment signature" by cellular and molecular perspectives,and to discover the difference of recrudescent patients between who the model forecasted and actually recurrence,and to discuss whether the union of this forecast model and TILs could enhance ability of prediction.In the formerly works,Gao,et al.analyzed 27 marks genes symboling or representing effectors immune response(mainly adaptive immunity), immunosuppression and inflammatory correlation gene comprehensively.Eight effectors immune response related genes(GNLY,GZMB,TRAV10,CD3Z,TBX21,IFNG,GATA3,IRF1) were discovered significantly related to recurrence and metastasis of HCC.Based on this reasons,they had established "immune microenvironment signature",RS8=(-4.554×IFNG value) +(-4.820×TBX21 value)+ (-4.472×GATA3 value)+(-6.554×GZMB value) +(-6.934×IRF1 value) +(-4.810×GNLY value) +(-4.077×CD3Z value) +(-3.758×TRAV10 value).The value was higher,indicated the recrudescent risk of this patient to be bigger.This model had reflected that an effector immune response(mainly adaptive immunity) dominated microenvironment play a central role in HCC recurrence.In this study we enrolled a cohort of 298 HCC patients that used in first part of research.First,we detected the expression situation of eight effect immune response-related genes involved in the "immune microenvironment signature"(GNLY,GZMB,TRAV10,CD3Z,TBX21,IFNG,GATA3,IRF1),and calculated each patient's RS8 value according to the above model formula.We divided 298 example patients using the cutoff value earlier period research provided into the high recurrence risk group and the low danger group.Finally,we discovered the difference of recrudescent patients between who the model forecasted and actually recurrence,and discussed whether the union of this forecast model and TILs could enhance ability of prediction.The results showed that(a) K-M survival curve analysis:the "immune microenvironment signature" successfully and accurately classified three cohorts(all 298 example patients,167 early HCC patients,156 BCLC stage A patients) into high recurrence risk and the low danger group(P＜0.001,P=0.019,P=0.039,respectively) on DFS level.(b) intratumoral,there was remarkable correlation between CD57+, GRB+,CD45RO+,CD8+TILs and the RS8 model(P＜0.001,respectively),opposite, FOXP3+Treg and the RS8 had no statistical relevance.There were no TILs in peritrumor tissue related to the RS8 model.(c)The difference between recurrence actually patients and recurrence patients predicted according RS8 model was tightly related to number of intratumoral FOXP3+Treg.(d) the union of RS8 model and intratumoral FOXP3+Treg was better than alone RS8 model in forecasting the risk degree of DFS and OS,even superior than TNM and BCLC.These results revealed the "immune microenvironment signature" can predict recurrence of HCC satisfactory,but it has neglected the function of immunosuppression factor which in the local immune microenvironment displayed. The outstanding efficiency of the union of RS8 model and intratumoral FOXP3+Treg validated molecular and cellular these two different research levels was interaction. This phenomenon also explained the immunity condition of tumor local microenvironment was decided by the coordination between immunological reactivity function and immunosuppression function.Both restricted and affected each other. Therefore,it is of vital significance to explore characteristics of immune cell in the microenvironment of HCC which to affect the tumor malignant phenotypes and the ability of metastasis for the diagnosis,stages,target tropism treatment,the prevention of recurrence and metastasis,and consequently enhancing the curative effect of HCC.Conclusions1) Intratumoral regulatory T cell,memory T cell and cytotoxic T cell might serve as a predictive index for recurrence and metastasis of HCC.Intratumoral balance of immunosuppression cells(regulatory T cell) and immunological effect cells(NK cell,memory T cell,cytotoxic T cell) was a promising independent predictor for recurrence and survival in HCC.These might suggest a crucial role of immune microenvironment in HCC recurrence and progression on cellular level.2) intratumoral regulatory T cell and memory T cell were associated to recurrence and survival of early HCC(absence of tumor micro and/or macro vascular invasion),the proportional relationship between intratumoral regulatory T cell and memory T cell was a promising independent predictor for recurrence and survival in early HCC.3) The "immune microenvironment signature" was a reproducible and powerful strategy for prospective patient prediction..4) The combination of "immune microenvironment signature" and IFOXP3+Treg might serve as a better predictive index for recurrence and metastasis of HCC than model alone.The efficiency of this combination validated molecular and the cellular these two different research levels were interaction.Furthermore,it explained the immunity condition of tumor local microenvironment was decided by the coordination between immunological effect function and immunosuppression function.Both restricted and affected each other.The novelty of this study1) For the first time,we demonstrated and reported that intratumoral balance of regulatory and memory T cell was a promising independent predictor for recurrence and survival in early HCC(absence of tumor micro and/or macro vascular invasion),and demonstrated efficiency of screening the high-risk group of early HCC patients.2) Through to validate the "immune microenvironment signature" and study on the relationship between this model and tumor infiltration lymphocyte,we confirmed the vital role of immune microenvironment in recurrence and prognosis of HCC on two different levels of molecular and cellular.The potential application of this project1) The type,density,location and functional status of tumor microenvironment TILs can be serves 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 furore immunotherapies.2) The proportional relationship between intratumoral regulatory T cell and memory T cell is a promising independent predictor for recurrence and survival in early HCC(absence of tumor micro and/or macro vascular invasion).This finding may select the high-risk group in clinical early HCC,and further provide the target of prevention.3) The combination of"immune microenvironment signature" and regulatory T cell can enhance the efficiency of prediction in HCC relapse,moreover instructing the individualized treatment.