免疫微环境与肝细胞癌复发转移及“免疫微环境分子预测模型”的建立与验证
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摘要
肝细胞癌(Hepatocellular carcinoma,HCC,简称肝癌)是最常见的恶性肿瘤之一,位居全球恶性肿瘤发病率的第6位、死因的第3位,在我国为恶性肿瘤第2位死因。尽管近几十年以来肝癌临床和基础研究均取得了长足的进步,但总体而言,肝癌的预后并无显著改善,5年生存率不足5%。手术治疗仍是目前肝癌最有效的方法,但远期疗效欠满意:即使是根治性切除5年复发转移率仍高达60～70%。因此,术后转移复发已成为阻碍肝癌病人长期生存的关键和瓶颈,临床上迫切需要探索肝癌转移复发的分子机制、预测患者预后、寻找有效的干预靶点并设计干预治疗的新方法。
传统的肝癌研究注重肿瘤细胞自身,试图从癌细胞本身基因与表型改变来解释肿瘤。研究表明,作为与肿瘤密不可分的局部微环境对肿瘤演进起着不容忽视的重要作用。由于恶性细胞的遗传学和表观遗传学上的异质性、不稳定性,以及由此获得的逃避外界压力的克隆选择性生长和适应能力,一切忽视甚至是破坏机体免疫系统的抗肿瘤治疗最终将遭遇治疗抵抗和恶性细胞优势克隆选择性生长。公认为肿瘤的“第七大标志性特征(the Seventh Hallmarker)”的免疫逃逸在肿瘤防治中已处于不可或缺的地位。更有发现,肿瘤局部微环境免疫学因素是一个优于TNM分期的、迄今为止最准确的独立预后指标;肿瘤学家和免疫学家们逐渐达成共识:癌症是一种免疫和微环境疾病。当前,肿瘤治疗的热点和突破口已确定为两方面:一是针对与恶性细胞共生共栖的肿瘤微环境,一是利用机体自身免疫系统。肿瘤微环境之所以日益受到重视,就是在传统研究的不足基础上,看到了癌与宿主互动的极其重要性。基因组学、组织微阵列以及其他高通量研究技术的兴起与日益完善,为肿瘤微环境研究提供了技术与理论保证,既可对以往建立在现象观察基础上的假说进行验证,又可在大规模、无偏倚观察基础上提出新的理论与设想,从而大大推动研究的精确性与进程,成为目前肿瘤学研究的一个方兴未艾的热点,受到了广泛关注。
作为免疫特惠器官,肝脏具有独特的免疫系统并参与机体局部及整体水平免疫调节。有理由相信,免疫微环境在肝癌发生发展和侵袭转移中发挥了异常重要的作用。本研究首先从肝癌微环境免疫活性细胞的层面和角度,利用组织微阵列和免疫组化技术,原位、在体和系统地探索了局部微环境树突状细胞和T淋巴细胞的类型(亚型)、数目、部位与功能状态在肝癌复发转移中的作用;接着,利用高通量、高灵敏度和精确度的荧光实时定量PCR微阵列芯片研究了肝癌微环境过继免疫(Adaptive immunity)为主的免疫效应、免疫抑制(Immunosuppression)和炎症(Inflammation)相关的标志性基因的表达谱及其与肿瘤复发转移的关系,并在此基础之上建立了与肝癌复发转移相关的“免疫微环境分子预测模型”;最后,在另一个随机、独立的肝癌患者队列中证明了该“免疫微环境分子预测模型”的稳定性、有效性和通用性,并在细胞水平进行了与分子水平遥相呼应的验证。
第一部分微环境免疫活性细胞与肝细胞癌复发转移关系的研究
本研究旨在从细胞水平探索肝癌局部微环境树突状细胞和T淋巴细胞的类型(亚型)、数目、部位与功能状态在肝癌复发转移中的作用,为进一步深入进行免疫微环境与肝癌复发转移的高通量、系统性研究奠定基础。
本研究中,我们随机选取了两组分别于两个不同时间段(1991年1月—1992年12月,123例,为队列A)(1997年2月—1999年12月,302例,为队列B)在复旦大学附属中山医院行根治性手术切除的肝细胞癌患者。在队列A中,利用常规组织切片,借助HE染色、普通及双重免疫组化染色,研究了癌内及癌旁微环境S100+树突状细胞、CD3+总T细胞、CD8+效应性T细胞和CD45R0+记忆T细胞形态、分布、数目及其与癌转移复发的关系;在队列B中,借助高通量的组织微阵列及免疫组化染色,研究了癌内及癌旁微环境Foxp3+调节性T细胞、Granzyme B+活化的细胞毒性细胞、CD3+T细胞、CD4+T细胞、CDS+T细胞的分布、数目与肿瘤复发转移的关系。
结果发现:(1)HE染色下癌内淋巴细胞主要是区域性地分布在癌间质中,部分成片状或巢状,偶见生发中心,HE染色下癌与癌周的淋巴细胞数目分级与无瘤生存无明显相关(P=0.054,0.071)。树突状细胞形态上主要分为两种:一种近圆形、椭圆形,基本无突触;另一种为不规则形,较多的树突状突起,体积较大,常在淋巴细胞成簇区处密集。双重染色可见其突触与肿瘤细胞接触,跟淋巴细胞相连。(2)多因素生存分析发现,癌内树突状细胞与无瘤生存显著正相关(P=0.005);而癌周树突状细胞与无瘤生存无显著相关性(P=0.329);同样,癌内CD45R0+、CD8+T细胞数目与无瘤生存亦直接正相关(P=0.003、P=0.037)。联合树突状细胞与T细胞亚群进行分析,两者同时多者预后更好(P＜0.001)。(3)单因素分析显示,癌内Foxp3+调节性T细胞与无瘤生存及总生存均显著负相关(P=0.015,P=0.006),而Granzyme B+细胞毒性细胞仅与总生存显著正相关(P=0.026)。两者联合分组经多因素分析证实,癌内微环境中调节性T细胞与细胞毒性细胞的比例关系为无瘤生存(P=0.008)及总生存(P＜0.0001)的一个独立预测指标,预测能力甚至优于目前临床广泛应用的TNM分期和癌栓(最小的P值和最大的危险度HR)。(4)微环境调节性T细胞(Foxp3+)数目与肝癌血管侵犯及包膜不完整等高侵袭性表型密切相关:有血管侵犯和/或肿瘤包膜不完整者微环境调节性T细胞更多。
以上结果表明,作为构成免疫微环境的重要组成部分,肝癌局部免疫活性细胞是一个复杂的多细胞群体,它们之间及其与肿瘤细胞之间的相互作用,在肝癌复发转移中起了十分重要作用。局部免疫反应越强,越不利于肿瘤生长侵袭,反之,则越有利于肿瘤侵袭转移。通过综合评价免疫细胞类型、数量、分布以及功能状态可以准确地把握肝癌患者局部免疫状态,也提示了更深入地研究免疫微环境与肝癌的相互作用、探索肝癌侵袭转移和免疫逃逸机制的理论意义和临床指导价值。
第二部分免疫微环境与肝细胞癌复发转移的PCR微阵列研究及“免疫微环境分子预测模型”的建立
本研究旨在从分子水平,借助于高通量和高精确度的二代功能分类基因芯片(实时荧光定量PCR微阵列芯片)全面、系统地探索了免疫微环境各种组成成分在肝癌复发转移中的作用;并在此基础之上建立了来源于肿瘤微环境和机体免疫特征的肝癌分子预测模型。
本研究中,我们首先进行了PCR微阵列芯片实验平台的优化;通过广泛细致的检索文献,最终确立了27个免疫效应/过继免疫、免疫抑制和炎症相关的经典的、代表性和标志性基因为本研究的目的基因;借助于我们特别定制的PCR微阵列芯片检测了随机选取的122例肝癌患者手术切除冰冻保存的肿瘤标本中上述基因的表达情况,通过Cluster和Treeview软件对基因表达进行了回顾性的非监督聚类分析(Unsupervised Hierarchical Cluster)和相关性矩阵分析(Correlation Matrix),及聚类结果的生存分析,并进一步利用国际上通用的复发评分系统(Recurrence Score)建立了肝细胞癌复发转移相关的前瞻性预测模型。
结果发现:(1)实验优化结果显示,联合TBP和HPRT作为内参照在肝癌研究中既实用又有效。(2)总体25个免疫/炎症相关的标志性基因(排除极低表达或异常基因2个)的非监督聚类分析可初步将患者区分为无瘤生存时间显著差异的两组(P=0.046,HR=0.54,95%CI=0.29-0.99),然而却未能通过多因素验证(P=0.27),两组患者临床病理特征亦未见显著性差异。(3)相关性矩阵分析显示,免疫效应/过继免疫相关基因(GNLY、GZMB、TRAV10、CD3Z、TBX21、IFNG、GATA3、IRF1)和免疫抑制/炎症相关基因(两类:[IL8、MMP7、PGS2、IL1B、TGFB1、HIF1A、MMP9]和[ARG1、NOS2A、IL23A、VEGF、NEMO])各自紧密聚集成独立的类别。(4)单独免疫效应/过继免疫相关基因的非监督聚类分析可将患者区分为无瘤生存(P=0.00035,HR=4.07,95%CI=1.88-8.78)和总生存(P=0.010,HR=2.18,95%CI=1.20-3.97)均显著差异的两组,多因素分析亦验证了无瘤生存的显著性差异(P=0.017);其中,免疫效应/过继免疫相关基因低表达的B组,肿瘤复发、患者死亡、有癌栓、较大肿瘤、晚期TNM和CLIP的病例数要显著多于基因高表达的A组患者。(5)12个炎症/免疫抑制相关基因联合进行聚类分析可将患者被区分为无瘤生存(P=0.023,HR=0.50,95%CI=0.27-0.91)和总生存(P=0.0030,HR=0.45,95%CI=0.26-0.76)显著不同的两组,进一步多因素验证,结果均未见显著的统计学意义(P=0.26;P=0.13)。(6)25个基因逐个一一进行多因素生存分析时仅免疫效应/过继免疫相关基因显示显著的统计学意义,以这8个基因多因素分析的危险系数乘以基因表达值的积的总和而建立的肝癌复发评分预测模型,可将患者显著区分为高危和低危复发转移组(单因素P=0.00011;多因素P=0.0012),该模型的ROC曲线下面积(AUC=0.738)仅次于TNM分期(AUC=0.769),优于肿瘤血管侵犯(AUC=0.715)、数目、大小、包膜等临床病理指标。
以上结果表明,免疫效应/过继免疫微环境在肝癌复发转移中的具有核心地位,微环境过继免疫反应越强,肿瘤复发转移风险越小,肿瘤恶性生物学表型越轻微,反之,则复发转移风险越大,恶性程度越高;免疫抑制/炎症相关微环境亦在肝癌复发转移中起着一定的辅助和促进作用。基于免疫微环境建立肝癌复发转移分子预测模型是完全可行和有效的,该模型的稳定性、预测能力和通用性需要进一步检验。
第三部分“免疫微环境分子预测模型”的分子和细胞水平验证
本研究旨在通过另一个随机、独立的肝细胞癌患者队列来检验和评价我们在前一个患者队列中已经建立的“免疫微环境分子预测模型”的稳定性、有效性和通用性,并同时联合这两个队列在细胞水平进行与分子水平遥相呼应的验证。
第二部分中,我们利用免疫效应/过继免疫相关基因(GNLY、GZMB、TRAV10、CD3Z、TBX21、IFNG、GATA3、IRF1)建立了“免疫微环境分子预测模型”,本研究中,我们设计了针对这八个基因的特异性real time PCR引物,在随机选取的112例肝细胞癌患者手术切除冰冻保存的肿瘤标本利用real time PCR检测了这八个基因的表达,将其应用于已经建立的预测模型中,通过生存分析以检验该模型的稳定性、有效性和通用性;最后,联合这两个队列的患者构建了组织微阵列,用免疫组化方法检测了癌内及癌旁微环境免疫效应性/抑制性/炎症细胞(CD8+、Granzyme B+、CD57+、CD45R0+、CD68+、Foxp3+和αSMA+)数目及分布,即利用相同的患者在细胞水平再次一一对应地检验免疫微环境对肝癌复发转移的预测效能。
结果发现:(1)应用“免疫微环境分子预测模型”,可以将患者区分为高危和低危复发转移的两组(单因素和多因素P=0.020),高危组的复发转移风险是低危组的2.13倍以上(HR=2.13)。(2)无论采用细胞计数的中位数、最佳P值法作为分界点依据,还是原始的连续性数值进行分析,癌内微环境免疫效应性细胞(CD8+、Granzyme B+、CD57+、CD45R0+)、癌内(Foxp3+)和癌旁(CD68+、αSMA+)免疫抑制性/炎症细胞与患者复发转移均显著相关;联合两种细胞分析显著性更加明显。
以上结果表明,利用肿瘤微环境和免疫特征建立的肝癌复发转移分子预测模型是值得推广的、强有力的前瞻性预测手段,进一步提示了过继免疫微环境在肝癌复发转移中的核心地位;免疫微环境在细胞水平和分子水平具有相似的功能状态和预测能力。
结论
1.肝癌微环境树突状细胞、记忆T细胞和效应性T细胞与根治性切除术后的复发转移密切相关;微环境调节性T细胞和细胞毒性T细胞的比例关系更是肝癌术后复发转移和总生存独立的预测指标;从细胞水平表明了免疫微环境在肝癌发生发展中的重要作用。
2.实时荧光定量PCR常用的内参照基因(GAPDH、ACTB、B2M)在肝癌发生发展中并非稳定表达,联合应用两个内参照基因(TBP和HPRT)既方便实用又可以达到有效的内参作用。
3.以GNLY、GZMB、TRAV10、CD3Z、TBX21、IFNG、GATA3、IRF1高表达为特征的免疫效应/过继免疫微环境在肝癌复发转移中的处于核心地位,微环境过继免疫反应越强,肿瘤复发转移风险越小,肿瘤恶性生物学表型越轻微,反之,则复发转移风险越大,恶性程度越高;免疫抑制/炎症相关微环境亦在肝癌复发转移中起着一定的辅助和促进作用。
4.基于免疫微环境建立肝癌复发转移分子预测模型是完全可行和有效的,该模型的具有相当的稳定性、较强的预测能力和通用性,值得进一步推广应用。
创新点
1.首次发现和报道了肝癌微环境中树突状细胞和记忆T细胞与术后复发转移的密切关系,可以作为肝癌复发转移新的预测指标。
2.首次发现和报道了肝癌微环境调节性T细胞和细胞毒性T细胞比例关系是术后复发转移和总生存的独立预测指标,并显示了对筛选高危复发患者和指导术后治疗的临床应用价值。
3.首次发现和报道了real time PCR常用的内参照基因在乙肝相关肝癌中的差异表达,并提出联合应用TBP和HPRT作为肝癌研究用内参照的实用性和有效性。
4.通过对肝癌免疫/炎症微环境原位、在体和高通量、系统性研究,证明了过继免疫微环境在肝癌复发转移中的核心地位和决定性作用,以及免疫抑制/炎症微环境对肝癌复发转移的辅助和促进作用。
5.利用8个过继免疫相关基因建立了“免疫微环境分子预测模型”,并在验证组中证实了该模型的价值,这是肿瘤研究中首次建立的基于微环境和免疫特征的复发转移预测模型(显著区别于既往的以肿瘤细胞为基础的预测模型)。
潜在应用价值
1.肝癌微环境免疫活性细胞的类型(亚型)、数目、分布和功能状态是术后复发转移新的独立预测指标,可用于临床筛选高危复发患者和指导术后治疗。
2.“免疫微环境分子预测模型”准确、简便、实用,可以制备成临床检测用试剂盒,用于肝癌患者复发转移的前瞻性预测并指导个体化治疗的实施;同时,该模型以微环境非肿瘤细胞为基础,可以在其他肿瘤中推广应用。
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 levels
In 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.
Conclusions
1.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 study
1.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 project
1.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.
传统的肝癌研究注重肿瘤细胞自身,试图从癌细胞本身基因与表型改变来解释肿瘤。研究表明,作为与肿瘤密不可分的局部微环境对肿瘤演进起着不容忽视的重要作用。由于恶性细胞的遗传学和表观遗传学上的异质性、不稳定性,以及由此获得的逃避外界压力的克隆选择性生长和适应能力,一切忽视甚至是破坏机体免疫系统的抗肿瘤治疗最终将遭遇治疗抵抗和恶性细胞优势克隆选择性生长。公认为肿瘤的“第七大标志性特征(the Seventh Hallmarker)”的免疫逃逸在肿瘤防治中已处于不可或缺的地位。更有发现,肿瘤局部微环境免疫学因素是一个优于TNM分期的、迄今为止最准确的独立预后指标;肿瘤学家和免疫学家们逐渐达成共识:癌症是一种免疫和微环境疾病。当前,肿瘤治疗的热点和突破口已确定为两方面:一是针对与恶性细胞共生共栖的肿瘤微环境,一是利用机体自身免疫系统。肿瘤微环境之所以日益受到重视,就是在传统研究的不足基础上,看到了癌与宿主互动的极其重要性。基因组学、组织微阵列以及其他高通量研究技术的兴起与日益完善,为肿瘤微环境研究提供了技术与理论保证,既可对以往建立在现象观察基础上的假说进行验证,又可在大规模、无偏倚观察基础上提出新的理论与设想,从而大大推动研究的精确性与进程,成为目前肿瘤学研究的一个方兴未艾的热点,受到了广泛关注。
作为免疫特惠器官,肝脏具有独特的免疫系统并参与机体局部及整体水平免疫调节。有理由相信,免疫微环境在肝癌发生发展和侵袭转移中发挥了异常重要的作用。本研究首先从肝癌微环境免疫活性细胞的层面和角度,利用组织微阵列和免疫组化技术,原位、在体和系统地探索了局部微环境树突状细胞和T淋巴细胞的类型(亚型)、数目、部位与功能状态在肝癌复发转移中的作用;接着,利用高通量、高灵敏度和精确度的荧光实时定量PCR微阵列芯片研究了肝癌微环境过继免疫(Adaptive immunity)为主的免疫效应、免疫抑制(Immunosuppression)和炎症(Inflammation)相关的标志性基因的表达谱及其与肿瘤复发转移的关系,并在此基础之上建立了与肝癌复发转移相关的“免疫微环境分子预测模型”;最后,在另一个随机、独立的肝癌患者队列中证明了该“免疫微环境分子预测模型”的稳定性、有效性和通用性,并在细胞水平进行了与分子水平遥相呼应的验证。
第一部分微环境免疫活性细胞与肝细胞癌复发转移关系的研究
本研究旨在从细胞水平探索肝癌局部微环境树突状细胞和T淋巴细胞的类型(亚型)、数目、部位与功能状态在肝癌复发转移中的作用,为进一步深入进行免疫微环境与肝癌复发转移的高通量、系统性研究奠定基础。
本研究中,我们随机选取了两组分别于两个不同时间段(1991年1月—1992年12月,123例,为队列A)(1997年2月—1999年12月,302例,为队列B)在复旦大学附属中山医院行根治性手术切除的肝细胞癌患者。在队列A中,利用常规组织切片,借助HE染色、普通及双重免疫组化染色,研究了癌内及癌旁微环境S100+树突状细胞、CD3+总T细胞、CD8+效应性T细胞和CD45R0+记忆T细胞形态、分布、数目及其与癌转移复发的关系;在队列B中,借助高通量的组织微阵列及免疫组化染色,研究了癌内及癌旁微环境Foxp3+调节性T细胞、Granzyme B+活化的细胞毒性细胞、CD3+T细胞、CD4+T细胞、CDS+T细胞的分布、数目与肿瘤复发转移的关系。
结果发现:(1)HE染色下癌内淋巴细胞主要是区域性地分布在癌间质中,部分成片状或巢状,偶见生发中心,HE染色下癌与癌周的淋巴细胞数目分级与无瘤生存无明显相关(P=0.054,0.071)。树突状细胞形态上主要分为两种:一种近圆形、椭圆形,基本无突触;另一种为不规则形,较多的树突状突起,体积较大,常在淋巴细胞成簇区处密集。双重染色可见其突触与肿瘤细胞接触,跟淋巴细胞相连。(2)多因素生存分析发现,癌内树突状细胞与无瘤生存显著正相关(P=0.005);而癌周树突状细胞与无瘤生存无显著相关性(P=0.329);同样,癌内CD45R0+、CD8+T细胞数目与无瘤生存亦直接正相关(P=0.003、P=0.037)。联合树突状细胞与T细胞亚群进行分析,两者同时多者预后更好(P＜0.001)。(3)单因素分析显示,癌内Foxp3+调节性T细胞与无瘤生存及总生存均显著负相关(P=0.015,P=0.006),而Granzyme B+细胞毒性细胞仅与总生存显著正相关(P=0.026)。两者联合分组经多因素分析证实,癌内微环境中调节性T细胞与细胞毒性细胞的比例关系为无瘤生存(P=0.008)及总生存(P＜0.0001)的一个独立预测指标,预测能力甚至优于目前临床广泛应用的TNM分期和癌栓(最小的P值和最大的危险度HR)。(4)微环境调节性T细胞(Foxp3+)数目与肝癌血管侵犯及包膜不完整等高侵袭性表型密切相关:有血管侵犯和/或肿瘤包膜不完整者微环境调节性T细胞更多。
以上结果表明,作为构成免疫微环境的重要组成部分,肝癌局部免疫活性细胞是一个复杂的多细胞群体,它们之间及其与肿瘤细胞之间的相互作用,在肝癌复发转移中起了十分重要作用。局部免疫反应越强,越不利于肿瘤生长侵袭,反之,则越有利于肿瘤侵袭转移。通过综合评价免疫细胞类型、数量、分布以及功能状态可以准确地把握肝癌患者局部免疫状态,也提示了更深入地研究免疫微环境与肝癌的相互作用、探索肝癌侵袭转移和免疫逃逸机制的理论意义和临床指导价值。
第二部分免疫微环境与肝细胞癌复发转移的PCR微阵列研究及“免疫微环境分子预测模型”的建立
本研究旨在从分子水平,借助于高通量和高精确度的二代功能分类基因芯片(实时荧光定量PCR微阵列芯片)全面、系统地探索了免疫微环境各种组成成分在肝癌复发转移中的作用;并在此基础之上建立了来源于肿瘤微环境和机体免疫特征的肝癌分子预测模型。
本研究中,我们首先进行了PCR微阵列芯片实验平台的优化;通过广泛细致的检索文献,最终确立了27个免疫效应/过继免疫、免疫抑制和炎症相关的经典的、代表性和标志性基因为本研究的目的基因;借助于我们特别定制的PCR微阵列芯片检测了随机选取的122例肝癌患者手术切除冰冻保存的肿瘤标本中上述基因的表达情况,通过Cluster和Treeview软件对基因表达进行了回顾性的非监督聚类分析(Unsupervised Hierarchical Cluster)和相关性矩阵分析(Correlation Matrix),及聚类结果的生存分析,并进一步利用国际上通用的复发评分系统(Recurrence Score)建立了肝细胞癌复发转移相关的前瞻性预测模型。
结果发现:(1)实验优化结果显示,联合TBP和HPRT作为内参照在肝癌研究中既实用又有效。(2)总体25个免疫/炎症相关的标志性基因(排除极低表达或异常基因2个)的非监督聚类分析可初步将患者区分为无瘤生存时间显著差异的两组(P=0.046,HR=0.54,95%CI=0.29-0.99),然而却未能通过多因素验证(P=0.27),两组患者临床病理特征亦未见显著性差异。(3)相关性矩阵分析显示,免疫效应/过继免疫相关基因(GNLY、GZMB、TRAV10、CD3Z、TBX21、IFNG、GATA3、IRF1)和免疫抑制/炎症相关基因(两类:[IL8、MMP7、PGS2、IL1B、TGFB1、HIF1A、MMP9]和[ARG1、NOS2A、IL23A、VEGF、NEMO])各自紧密聚集成独立的类别。(4)单独免疫效应/过继免疫相关基因的非监督聚类分析可将患者区分为无瘤生存(P=0.00035,HR=4.07,95%CI=1.88-8.78)和总生存(P=0.010,HR=2.18,95%CI=1.20-3.97)均显著差异的两组,多因素分析亦验证了无瘤生存的显著性差异(P=0.017);其中,免疫效应/过继免疫相关基因低表达的B组,肿瘤复发、患者死亡、有癌栓、较大肿瘤、晚期TNM和CLIP的病例数要显著多于基因高表达的A组患者。(5)12个炎症/免疫抑制相关基因联合进行聚类分析可将患者被区分为无瘤生存(P=0.023,HR=0.50,95%CI=0.27-0.91)和总生存(P=0.0030,HR=0.45,95%CI=0.26-0.76)显著不同的两组,进一步多因素验证,结果均未见显著的统计学意义(P=0.26;P=0.13)。(6)25个基因逐个一一进行多因素生存分析时仅免疫效应/过继免疫相关基因显示显著的统计学意义,以这8个基因多因素分析的危险系数乘以基因表达值的积的总和而建立的肝癌复发评分预测模型,可将患者显著区分为高危和低危复发转移组(单因素P=0.00011;多因素P=0.0012),该模型的ROC曲线下面积(AUC=0.738)仅次于TNM分期(AUC=0.769),优于肿瘤血管侵犯(AUC=0.715)、数目、大小、包膜等临床病理指标。
以上结果表明,免疫效应/过继免疫微环境在肝癌复发转移中的具有核心地位,微环境过继免疫反应越强,肿瘤复发转移风险越小,肿瘤恶性生物学表型越轻微,反之,则复发转移风险越大,恶性程度越高;免疫抑制/炎症相关微环境亦在肝癌复发转移中起着一定的辅助和促进作用。基于免疫微环境建立肝癌复发转移分子预测模型是完全可行和有效的,该模型的稳定性、预测能力和通用性需要进一步检验。
第三部分“免疫微环境分子预测模型”的分子和细胞水平验证
本研究旨在通过另一个随机、独立的肝细胞癌患者队列来检验和评价我们在前一个患者队列中已经建立的“免疫微环境分子预测模型”的稳定性、有效性和通用性,并同时联合这两个队列在细胞水平进行与分子水平遥相呼应的验证。
第二部分中,我们利用免疫效应/过继免疫相关基因(GNLY、GZMB、TRAV10、CD3Z、TBX21、IFNG、GATA3、IRF1)建立了“免疫微环境分子预测模型”,本研究中,我们设计了针对这八个基因的特异性real time PCR引物,在随机选取的112例肝细胞癌患者手术切除冰冻保存的肿瘤标本利用real time PCR检测了这八个基因的表达,将其应用于已经建立的预测模型中,通过生存分析以检验该模型的稳定性、有效性和通用性;最后,联合这两个队列的患者构建了组织微阵列,用免疫组化方法检测了癌内及癌旁微环境免疫效应性/抑制性/炎症细胞(CD8+、Granzyme B+、CD57+、CD45R0+、CD68+、Foxp3+和αSMA+)数目及分布,即利用相同的患者在细胞水平再次一一对应地检验免疫微环境对肝癌复发转移的预测效能。
结果发现:(1)应用“免疫微环境分子预测模型”,可以将患者区分为高危和低危复发转移的两组(单因素和多因素P=0.020),高危组的复发转移风险是低危组的2.13倍以上(HR=2.13)。(2)无论采用细胞计数的中位数、最佳P值法作为分界点依据,还是原始的连续性数值进行分析,癌内微环境免疫效应性细胞(CD8+、Granzyme B+、CD57+、CD45R0+)、癌内(Foxp3+)和癌旁(CD68+、αSMA+)免疫抑制性/炎症细胞与患者复发转移均显著相关;联合两种细胞分析显著性更加明显。
以上结果表明,利用肿瘤微环境和免疫特征建立的肝癌复发转移分子预测模型是值得推广的、强有力的前瞻性预测手段,进一步提示了过继免疫微环境在肝癌复发转移中的核心地位;免疫微环境在细胞水平和分子水平具有相似的功能状态和预测能力。
结论
1.肝癌微环境树突状细胞、记忆T细胞和效应性T细胞与根治性切除术后的复发转移密切相关;微环境调节性T细胞和细胞毒性T细胞的比例关系更是肝癌术后复发转移和总生存独立的预测指标;从细胞水平表明了免疫微环境在肝癌发生发展中的重要作用。
2.实时荧光定量PCR常用的内参照基因(GAPDH、ACTB、B2M)在肝癌发生发展中并非稳定表达,联合应用两个内参照基因(TBP和HPRT)既方便实用又可以达到有效的内参作用。
3.以GNLY、GZMB、TRAV10、CD3Z、TBX21、IFNG、GATA3、IRF1高表达为特征的免疫效应/过继免疫微环境在肝癌复发转移中的处于核心地位,微环境过继免疫反应越强,肿瘤复发转移风险越小,肿瘤恶性生物学表型越轻微,反之,则复发转移风险越大,恶性程度越高;免疫抑制/炎症相关微环境亦在肝癌复发转移中起着一定的辅助和促进作用。
4.基于免疫微环境建立肝癌复发转移分子预测模型是完全可行和有效的,该模型的具有相当的稳定性、较强的预测能力和通用性,值得进一步推广应用。
创新点
1.首次发现和报道了肝癌微环境中树突状细胞和记忆T细胞与术后复发转移的密切关系,可以作为肝癌复发转移新的预测指标。
2.首次发现和报道了肝癌微环境调节性T细胞和细胞毒性T细胞比例关系是术后复发转移和总生存的独立预测指标,并显示了对筛选高危复发患者和指导术后治疗的临床应用价值。
3.首次发现和报道了real time PCR常用的内参照基因在乙肝相关肝癌中的差异表达,并提出联合应用TBP和HPRT作为肝癌研究用内参照的实用性和有效性。
4.通过对肝癌免疫/炎症微环境原位、在体和高通量、系统性研究,证明了过继免疫微环境在肝癌复发转移中的核心地位和决定性作用,以及免疫抑制/炎症微环境对肝癌复发转移的辅助和促进作用。
5.利用8个过继免疫相关基因建立了“免疫微环境分子预测模型”,并在验证组中证实了该模型的价值,这是肿瘤研究中首次建立的基于微环境和免疫特征的复发转移预测模型(显著区别于既往的以肿瘤细胞为基础的预测模型)。
潜在应用价值
1.肝癌微环境免疫活性细胞的类型(亚型)、数目、分布和功能状态是术后复发转移新的独立预测指标,可用于临床筛选高危复发患者和指导术后治疗。
2.“免疫微环境分子预测模型”准确、简便、实用,可以制备成临床检测用试剂盒,用于肝癌患者复发转移的前瞻性预测并指导个体化治疗的实施;同时,该模型以微环境非肿瘤细胞为基础,可以在其他肿瘤中推广应用。
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 levels
In 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.
Conclusions
1.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 study
1.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 project
1.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.
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