摘要
肝细胞癌(Hepatocellular carcinoma, HCC)是世界上最常见、恶性程度最高的肿瘤之一,其病死率在全球所有癌症中占第3位。手术切除仍是目前肝癌获得长期生存的主要手段,但术后5年复发率高达60%以上(小肝癌也达40%-50%)。转移复发已成为进一步延长肝癌病人生存期、提高远期疗效的瓶颈问题,也是最终攻克肝癌的关键。在肝癌术后病人中区分高、低复发风险人群有相当的困难,特别是那些没有明显血管侵犯、局部淋巴结转移或远处转移的早期病人。鉴定相关的分子标签可以为肝癌患者预测临床结果提供额外的有益信息,并为术后甄别需要接受辅助治疗的病人提供帮助。以往大量研究指出,Thl和Th2类细胞因子在多种人类恶性肿瘤的进展中起重要作用。此前我们与美国国立卫生研究院(NIH)的合作研究构建了包含17个免疫细胞因子的预测模型预测肿瘤复发。本课题组在此基础上采用微流体低密度表达芯片技术(qRT-PCR),应用生物信息学机器学习方法对以上预测模型优化后发现癌旁IL-2、IL-15基因表达可以作为早期肝癌术后预后的独立预测因子。以上是基于基因转录水平的研究,然而Th1/Th2类细胞因子蛋白是否可以作为肝癌的临床预测指标尚不十分肯定。本课题组前期基于较小样本量的初步研究发现在蛋白水平IL-2和IL-15也与肝癌的复发相关。基于其他恶性肿瘤的研究也提示Th1型细胞因子可以减少肿瘤的转移复发,而Th2细胞因子表达增高则可增加术后复发率。特别是IL-10在许多恶性肿瘤中被认为是预后不良的分子标签。
基于肿瘤微环境的研究成果令人振奋,但是肝癌的转移复发除与微环境免疫状态密切相关外,更与肝癌细胞自身的转移特性密切相关。我们的前期研究发现调控肝癌转移潜能的基因改变在其原发瘤阶段即已存在,此为转移理论的重要进展。研究还发现骨桥蛋白Osteopontin, OPN)表达升高与肝癌转移密切相关。骨桥蛋白是一种分泌型的非胶原化富含唾液酸的磷酸化糖蛋白,OPN正常情况下参与体内许多生理过程。许多文献报道OPN表达增高与恶性肿瘤演进、侵袭转移密切相关。为此,本研究中我们在前期研究的基础上首先通过ELISA方法在一组包含192例样本的队列中检测12种Th1/Th2相关细胞因子,发现IL-2,IL-15和IFN-γ与肝癌复发相关,IL-2是最佳的预测预后的因子,并进一步扩大样本量通过高通量的组织芯片技术加以验证。同时利用组织芯片检测肝癌组织中OPN的水平,分析其与免疫相关因子联合应用在预测肝癌预后中的作用。
本研究拟从蛋白水平检测癌旁非癌肝组织中免疫相关因子的表达,研究其与肝癌复发的关系。
我们利用酶联免疫吸附反应(enzyme-linked immunosorbent assays ELISAs)定量检测192例(cohort A训练组n=128,测试组n=64)肝癌患者癌旁肝组织中12个Th1/Th2相关细胞因子。同时用组织芯片免疫组化技术在包含261例患者的独立阵列中(cohort B)进行验证,评价癌旁微环境中免疫相关细胞因子的表达,在组织中的分布情况及其与术后肿瘤复发之间的关系。
结果发现,无复发组肝癌患者中,癌旁肝组织中IL-2,IL-15和IFN-γ蛋白表达显著高于复发组。Cox逐步回归分析发现,仅有IL-2是效果最佳的独立预测因子(HR for recurrence=0.4, P=0.001; HR for death=0.5, P=0.019)。这一结果在队列A的测试组及队列B(HR for recurrence=0.4; P<0.0001; HR for survival=0.6; P=0.005)中都得到了验证。本研究中我们还发现,在癌旁肝组织中IL-2主要存在于肝细胞内,而非淋巴细胞。
癌旁肝组织中的IL-2,IL-15和IFN-γ蛋白水平与肝癌的复发有关,但IL-2是预测复发及总体生存的最佳的预测因子,可以基于癌旁肝组织中的IL-2水平将肝癌患者分为不同预后的亚群。本研究的另一结论是在癌旁肝组织中IL-2主要存在于肝细胞中,而非淋巴细胞中。
本研究拟从转录水平及蛋白水平研究外周血单个核细胞及血清中Th1/Th2平衡状况,探索其对肝癌预后的预测作用。
我们首先收集肝癌患者、乙型肝炎肝硬化无肝癌患者及健康对照组的血清,通过先进的流式微球捕获技术(Cytometric Bead Array, CBA)检测血清Th1/Th2类细胞因子。同时收集与之配对的外周血单个核细胞(PBMC),行qRT-PCR检测,了解机体全身(血清细胞因子蛋白水平及外周血单个核细胞基因转录水平)免疫状态改变及其与肝癌复发及预后的关系。
我们的研究发现肝癌患者、乙型肝炎肝硬化无肝癌组及正常人,血清IL-2(P=0.6907),IFN-γ(P=0.459)、IL-4(P=0.2044)和IL-10(P=0.0837)表达无显著差异。外周单个核细胞(PBMC),行qRT-PCR检测IL-2(P=0.2959),IFN-γ(P=0.8225)、IL-4(P=0.2968)和IL-10(P=0.3239)mRNA表达组间亦无明显差异。本研究中检测的4种细胞因子其表达水平与肝癌患者的总生存时间及至复发时间无显著相关性。
本研究尚未在血清或外周单个核细胞(PBMC)中发现可用于预测肝癌预后的免疫炎症相关因子。
我们前面的研究发现IL-2是预测复发及总体生存的最佳的预测因子。Osteopontin (OPN)在恶性肿瘤的发生发展中起重要作用,与多种恶性肿瘤的预后关系密切。有研究认为OPN可以起类似促炎因子作用可增强Th1类细胞因子的表达,我们通过western blot及细胞免疫荧光染色,分析不同转移潜能肝癌细胞株OPN表达情况,流式微球捕获技术分析细胞培养上清中的IL-2,IFNγ,IL-4和IL-10的表达量,了解OPN是否与Th1或Th2类细胞因子的表达存在关联。并检测肝癌癌旁正常肝组织中IL-2以及癌组织中OPN的表达,研究两者的相关性及其与肝癌复发的关系。我们采用配对标本组织芯片免疫组化技术在含有261例患者的队列(cohort B)进行研究,评价癌旁正常肝组织IL-2以及癌组织中OPN的表达,在组织中的分布情况以及两者的相关性及其与术后肿瘤复发及肿瘤侵袭能力指标之间的关系。分析OPN联合IL-2在肝癌复发中的预测作用。
结果,未发现OPN和炎症相关细胞因子表达有明显相关性,癌旁IL-2和癌内OPN是互不干扰的相对独立的预测肝癌预后的指标。癌旁IL-2与肝癌术后复发关系密切,IL-2高表达有较好的总体生存率及更长的至复发时间(TTR：P<0.0001,0S：P=0.004)。癌组织内OPN高表达的肝癌患者预后显著差于OPN低表达的患者(TTR：P=0.0007,0S：P=0.0214)。OPN联合IL-2表达可以充当肝癌预后的有效的预测因子(TTR：P<0.0001,0S：P=0.0039)。其预测效能优于两个指标单一使用时的预测效能,亦优于传统的临床病理指标。
1.癌旁微环境炎症免疫相关因子与肝癌术后复发及总体生存密切相关。癌旁肝组织中的IL-2表达水平与肝癌的复发预后有关,IL-2是预测复发及总体生存的最佳的预测因子。
2.在癌旁肝组织中IL-2主要存在于肝细胞中,而癌旁T淋巴细胞中IL-2很少存在。
3.本研究尚未在血清或外周单个核细胞(PBMC)中发现免疫炎症相关因子可用于预测肝癌的预后。但不能就此认为肝癌的预后与机体全身免疫状态无关。
4.癌旁IL-2和癌内OPN表达水平是预测肝癌术后生存及复发的独立的预测因子,癌旁肝组织中IL-2联合癌内OPN可提高肝癌术后复发的预测效能。
5.肝癌的转移复发是一个复杂的过程。无论“土壤(微环境)”还是“种子(癌细胞本身)”在肝癌的复发中都起着重要的作用,预防肝癌术后复发要从多靶点着手,多途径干预,既针对“土壤”又针对“种子”才有可能取得理想的效果。
1.首先报道癌旁肝组织中的IL-2和IL-15蛋白表达水平与肝癌的复发预后有关,IL-2是预测复发及总体生存的最佳的预测因子。
2.我们首次发现在癌旁肝组织中IL-2主要存在于肝细胞中,而非淋巴细胞中。并用连续切片定位染色方法加以验证。
3.本研究同时从癌旁微环境和肿瘤细胞本身着手,证实癌旁IL-2和癌内OPN水平是肝癌预后的独立预测指标,既针对“土壤(微环境)”又针对“种子(肿瘤细胞本身)”,为早期肝癌的复发寻找分子标签。
1.癌旁IL-2和癌内OPN水平能够提供预测肝癌病人生存和复发的有益信息,尤其针对早期病人效果更显著。这一发现有助于我们界定和选择高复发风险人群采取个体化措施预防复发。
2.本研究中的方法实施方便,便于在临床应用,一旦成熟临床极易推广。“基于炎症因子的肝癌患者术后转移复发多分子预测试剂盒”已经申请国家专利,申请号：200810208150.6。
3.调控癌旁微环境,调变肿瘤细胞侵袭特性,多方出击方能战胜肿瘤。本研究为临床上控制肿瘤复发,提高肝癌的远期疗效提供新的思路。
Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies worldwide. It has an extremely poor prognosis, mainly attributed to the high frequency of intrahepatic metastatic recurrence. It is a challenge to identify patients who are at a greater risk for tumor recurrence after curative treatment for HCC, particularly in those with early stage disease who do not have significant vascular invasion, or regional lymph node or distant metastasis. Identification of molecular markers could provide supplemental and useful information for predicting clinical outcome in patients with a given stage of disease and improve the selection of patients for adjuvant therapies after resection. Studies of many kinds of human malignancies have shown that Th1-and Th2-like cytokines may play a prominent role in tumor progression and metastasis.In a recent study of gene expression profiling, we found that the liver tissues in patients with metastatic HCC had lower levels of pro-inflammatory Thl-like cytokines and remarkably higher levels of anti-inflammatory Th2-like cytokines in comparison to those without metastatic HCC. However, whether the protein expression levels of these Thl/Th2 cytokines can be used to predict the clinical outcome of HCC is not known. In our previous research, we found that IL-2 or IL-15 of immunity-associated genes signature should allow a sensitive and specific monitoring of survival in HCC. The accumulated evidence has clearly demonstrated that a dominant Th2-like cytokine profile and a decrease in Thl-like cytokines are associated with the metastatic phenotype. High IL-10 levels had a worse postresection outcome, and IL-10 may be a predictor marker of the of HCC patients after surgery.
The research based on the micro-environment has achieved exciting results, but, as we know the metastasis and recurrence of HCC is closely related to not only microenvironment but also the characteristics of cancer cell itself. In our previous work, we collaborated with the National Cancer Insititute (NCI) of USA, Osteopontin (OPN) plays an important role in the development, invasion, and metastasis of malignancies. An OPN-neutralizing antibody efficiently blocked in vitro invasion and in vivo pulmonary metastasis of HCC cells. The current study was based on our previous findings and the concept that proteins affect cancer progression, thus serving as markers of disease and therapeutic targets. We examined the protein levels of 12 Thl/Th2 cytokines in noncancerous liver tissue samples from 192 patients (cohort A) with HCC using ELISA methods. Of the 12 tested cytokines, three (IL-2, IL-15, and IFN-y) were found to correlate with clinical outcome. IL-2 had the best prognostic performance in the training set, which was then further validated in the test set (n=64). The prognostic value of IL-2 was further confirmed in another independent cohort (cohort B) of 261 patients using in situ immunohistochemical staining. At the same time, we examined OPN expression in tumor tissue microarrays (TMA) containing 261cases (cohort B) in order to investigate the predictive value of peritumoral IL-2 and intratumoral OPN for the prognosis of HCC patients.
Thl/Th2-like cytokine mRNA levels in noncancerous hepatic tissues from hepatocellular carcinoma (HCC) patients are associated with metastases and recurrence. The present study evaluated the prognostic values of Thl/Th2 cytokine in HCC patients.
Two independent cohorts of 453 HCC patients were enrolled. Twelve Thl/Th2 cytokines in noncancerous hepatic tissues from cohort A (n=192) were quantified with enzyme-linked immunosorbent assays. This cohort was split into training and test sets. Prognostic cytokines were identified from the training set, validated with the testing set, and confirmed in cohort B (n=261) using in situ immunohistochemical staining.
IL-2, IL-15 and IFN-γprotein levels in noncancerous liver tissues were significantly greater in HCC patients without than in those with tumor recurrence. On Cox regression stepwise variable selection analyses, IL-2 was the optimal independent predictor of outcome (HR for recurrence=0.4, P=0.001; HR for death=0.5, P=0.019), rather than IL-15. This was confirmed in the testing set and validated in cohort B (HR for recurrence=0.4; P<0.0001; HR for survival=0.6; P=0.005). Another finding was that IL-2 was mainly contributed by hepatocytes other than the lymphocytes in noncancerous liver parenchyma. HCC patients can be stratified into subgroups with different prognoses postoperatively based on noncancerous hepatic tissue IL-2 levels. IL-2 was the optimal predictor for both tumor recurrence and patient。
The aim of this study was to assess the value of Thl/Th2-like cytokine for predicting tumor recurrence after curative resection in serum and peripheral blood mononuclear cell of hepatocellular carcinoma patients.
Blood samples from HCC patients and liver cirrhosis patients were collected for cytokine quantitation. Because Thl/Th2-like cytokine concentrations were too low, we performed the cytometric bead array (CBA) assay, which utilizes the sensitivity of amplified fluorescence detection. The expression levels of cytokine mRNA in peripheral blood mononuclear cell was assessed by qRT-PCR.
No significant difference in serum IL-2 (P=0.6907),IFN-γ(P=0.459)、IL-4 (P=0.2044) and IL-10 (P=0.0837) level was found between the HCC patients (n=71), CG (n=9)and NG(n=13). Moreover, the serum IL-2, IFN-γ、IL-4 and IL-10level had no significant associations with OS or TTR in HCC patients using the median levels as cutoff. Similar results were also found in PBMC. The finding was that, unlike in liver tissues, serum IL-2 levels were not significantly correlated with tumor recurrence or survival of HCC patients.
Osteopontin (OPN) plays an important role in the development, invasion, and metastasis of malignancies. It is generally classified as a pro-inflammatory cytokine because it modulates cell-mediated immunity by promoting the Thl response. In our present study, we found that peritumoral IL-2 was the optimal predictor for both tumor recurrence and overall survival time. The aim of this study was to explore the relationship of OPN and Thl/Th2 cytokines(IL-2, IFN-γ, IL-4 and IL-10)expression, then assess the value of OPN and IL-2 for predicting tumor recurrence after curative resection in hepatocellular carcinoma patients. Tissue microarrays of 261 (cohort B) HCC patients were used to detect the expressions of OPN and IL-2. Clinicopathologic data for these patients were evaluated. The prognostic significance was assessed using Kaplan-Meier survival estimates and log-rankt tests.
No significant association was found between OPN and Thl/Th2 cytokines expression in cell lines. In a tissue microarray-immunohistochemical analysis, we found that patients with higher levels of intratumoral OPN and lower levels of peritumoral IL-2 had a significantly poorer prognosis than patients with lower OPN and higher IL-2 levels. The combination of OPN and IL-2 expression thus served as an effective prognosticator.
These findings suggest that IL-2 alone or in combination with OPN may act as an independent indicator for HCC patients after curative resection.
1. HCC patients can be stratified into subgroups with different prognoses postoperatively based on noncancerous hepatic tissue IL-2 levels. IL-2 was the optimal predictor for both tumor recurrence and patient.
2. IL-2 was mainly contributed by hepatocytes other than the lymphocytes in noncancerous liver parenchyma.
3. The third important finding was that, unlike in liver tissues, serum and PBMC immune/inflammatory levels were not significantly correlated with tumor recurrence or survival of HCC patients.
4. Peritumoral IL-2 alone or in combination with intratumoral OPN may act as an independent indicator for HCC patients after curative resection.
5. The metastasis and recurrence of HCC is closely related to not only microenvironment but also the characteristics of cancer cell itself. To prevent recurrence of HCC we must focus on them all.
1. We firstly demonstrated that HCC patients can be stratified into subgroups with different prognoses postoperatively based on noncancerous hepatic tissue IL-2 and IL-15 levels. IL-2 was the optimal predictor for both tumor recurrence and patient.
2. For the first time, we found IL-2 was mainly contributed by hepatocytes other than the lymphocytes in noncancerous liver parenchyma.
3. We firstly confirmed that peritumoral IL-2 alone or in combination with intratumoral OPN may act as an independent indicator for HCC patients after curative resection.
Measuring the IL-2 protein level in noncancerous hepatic tissues could provide useful information for predicting tumor recurrence and prognosis in patients, even those with early stage HCC. This finding may potentially enable us to identify and select high-risk patients for effective adjuvant therapy. Regulation of the immune/inflammatory response may be a helpful strategy to control tumor relapse and further improve the treatment outcome of HCC patients. It has been applied for the patent (" Multiple inflammatory cytokines prediction kit for postoperative recurrence and metastasis of hepatocellular carcinoma"; Chinese patent applying number is 200810208150.6)
引文
1. Parkin DM, Bray F, Ferlay J, Pisani P. Estimating the world cancer burden: Globocan 2000. Int J Cancer,2001,94:153-156.
2. Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics,2002. CA Cancer J Clin 2005,55:74-108.
3.Izzo F, Cremona F, Ruffolo F. Outcome of 67 patients with hepatocellular cancer detected during screening of 1125 patients with chronic hepatitis. Ann Surg 1998, 227:513-518.
4.Zhou XD, Tang ZY, Yang BH. Experience of 1000 patients who underwent hepatectomy for small hepatocellular carcinoma. Cancer,2001;91:1479-1486.
5.Tang ZY, Yu YQ, Zhou XD. Hepatocellular carcinoma-Cause, treatment and metastasis. World J Gastroenterol,2001;7:445-454.
6.Li LD, Zhang SW, Lu FZ. Research on characteristics of mortality spectrum and type composition of malignant tumors in China. Chin J Oncol,1997;19:323-328.
7. Tang ZY, Ye SL, Liu YK, Qin LX, Sun HC, Ye QH, Wang L, Zhou J, Qiu SJ, Li Y, Ji XN, Liu H, Xia JL, Wu ZQ, Fan J, Ma ZC, Zhou XD, Lin ZY, Liu KD. A decade's studies on metastasis of hepatocellular carcinoma. J Cancer Res Clin Oncol,2004; 130:187-196.
8.Jemal A, Thomas A, Murray T, Thun M. Cancer statistics,2002. CA Cancer J Clin, 2002; 52:23-47.
9.Llovet JM, Burroughs A, Bruix J. Hepatocellular carcinoma. Lancet, 2003;362:1907-1917.
10.Axelrod R, Axelrod DE, Pienta KJ. Evolution of cooperation among tumor cells. Proc Natl Acad Sci U S A.2006; 103:1347-1349.
11. Bhowmick NA, Neilson EG, Moses HL. Stromal fibroblasts in cancer initiation and progression. Nature.2004; 432:332-337.
12. Bagley RG, Weber W, Rouleau C, Teicher BA. Pericytes and endothelial precursor cells:cellular interactions and contributions to malignancy. Cancer Res.2005; 65:9741-9750.
13.Overall CM, Kleifeld O. Tumour microenvironment-opinion:validating matrix metalloproteinases as drug targets and anti-targets for cancer therapy. Nat Rev Cancer. 2006; 6:227-239.
14.Muller AJ, Scherle PA. Targeting the mechanisms of tumoral immune tolerancewithsmall-molecule inhibitors.Nat Rev Cancer.2006; 6:613-625.
15.Zitvogel L, Tesniere A, Kroemer G. Cancer despite immunosurveillance: immunoselection and immunosubversion. Nat Rev Immunol.2006; 6:715-727.
16. Galon J, Costes A, Sanchez-Cabo F, Kirilovsky A, Mlecnik B, Lagorce-Pages C, Tosolini M, Camus M, Berger A, Wind P, Zinzindohoue F, Bruneval P, Cugnenc PH, Trajanoski Z, Fridman WH, Pages F. Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science.2006; 313: 1960-1964.
17. Gao Q, Qiu SJ, Fan J, Zhou J, Wang XY, Xiao YS, Xu Y, Li YW, Tang ZY. Intratumoral balance of regulatory and cytotoxic T cells is associated with prognosis of hepatocellular carcinoma after resection.[J] Clin Oncol.2007; Jun 20; 25(18):2586-2593.
18. Dranoff G. Cytokines in cancer pathogenesis and cancer therapy. Nat Rev Cancer. 2004; 4:11-22.
19.Zou W. Immunosuppressive networks in the tumour environment and their therapeutic relevance. Nat Rev Cancer.2005; 5:263-274.
20. Thomas GR, Chen Z, Leukinova E, Van Waes C, Wen J. Cytokines IL-1 alpha, IL-6, and GM-CSF constitutively secreted by oral squamous carcinoma induce down-regulation of CD80 costimulatory molecule expression:restoration by interferon gamma. Cancer Immunol Immunother.2004; 53:33-40.
21. Rhodus NL, Ho V, Miller CS, Myers S, Ondrey F. NF-kappaB dependent cytokine levels in saliva of patients with oral preneoplastic lesions and oral squamous cell carcinoma. Cancer Detect Prev.2005; 29:42-45.
22. Budhu A, Forgues M, Ye QH, Jia HL, He P, Zanetti KA, Kammula US, Chen Y, Qin LX, Tang ZY, Wang XW. Prediction of venous metastases, recurrence, and prognosis in hepatocellular carcinoma based on a unique immune response signature of the liver microenvironment. Cancer Cell.2006; 10:99-111.
23. Ye QH, Qin LX, Forgues M, He P, Kim JW, Peng AC, Simon R, Li Y, Robles AI, Chen Y, Ma ZC, Wu ZQ, Ye SL, Liu YK, Tang ZY, Wang XW. Predicting hepatitis B virus-positive metastatic hepatocellular carcinomas using gene expression profiling and supervised machine learning. Nat Med.2003; 9:416-23.
24.MacDougall JR, Matrisian LM. Contributions of tumor and stromal matrix metalloproteinases to tumor progression, invasion and metastasis. Cancer Metastasis Rev 1995; 14:351-362.
25.Mahabeleshwar GH, Kundu GC. Syk, a protein-tyrosine kinase, suppresses the cell motility and nuclear factor kappa B-mediated secretion of urokinase type plasminogen activator by inhibiting the phosphatidylinositol 3'-kinase activity in breast cancer cells. J Biol Chem 2003; 278:6209-6221.
26.Yeatman TJ, Chamers AF. Osteopontin and colon cancer progression. Clin Cancer Res 2003;20(1):85-90.
27.Tuck AB, Chambers AF. The role of osteopontin in breast cancer:clinical and experimental studies. J Mammary Gland Biol Neoplasia 2001;6(4):419-429.
28. Koopmann J, Fedarko NS, Jain A, Maitra A, Iacobuzio-Donahue C, Rahman A, Hruban RH, Yeo CJ, Goggins M. Evaluation of osteopontin as biomarker for pancreatic adenocarcinoma. Cancer Epidemiol Biomarkers Prev 2004;13(3):487-491.
29.Khodavirdi AC, Song Z, Yang S, Zhong C, Wang S, Wu H, Pritchard C, et al. Increased expression of osteopontin contributes to the progression of prostate cancer. Cancer Res 2006; 66:883-888.
30.Fedarko, NS. Elevated serum bone sialoprotein and osteopontin in colon, breast, prostate, and lung cancer. Clin. Cancer Res 2001;7(12),4060-4066.
31.Bramwell VH, Doig GS, Tuck AB, Wilson SM, Tonkin KS, Tomiak A, Perera F, Vandenberg TA, Chambers AF. Serial plasma osteopontin levels have prognostic value in metastatic breast cancer. Clin Cancer Res 2006;12(11 Pt1):3337-3343.
32.Llovet JM, Bruix J. Novel advancements in the management of hepatocellular carcinoma in 2008. J Hepatol.2008; 48 Suppl 1, S20-37. 紧邻原发灶。这部分研究我们采用总共包括453个病人的大样本,两个相对独立的研究队列,两种不同的研究方法(ELISA和IHC)得出相对一致的结论,提高了本研究的可靠性。且ELISA和IHC方法实施方便,便于在临床应用。总之,基于大样本量的研究,我们发现癌旁工L-2水平能够提供预测肝癌病人生存和复发的有益信息,对早期病人效果亦显著。这一研究结果告诉我们调节免疫炎症反应可能为控制肿瘤复发,提高肝癌的远期疗效提供有益的帮助。
1. Galon J, Costes A, Sanchez-Cabo F, Kirilovsky A, Mlecnik B, Lagorce-Pages C, Tosolini M, Camus M, Berger A, Wind P, Zinzindohoue F, Bruneval P, Cugnenc PH, Trajanoski Z, Fridman WH, Pages F.. Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science 2006; 313:1960-1964.
2. Toi M, Bando H, Ogawa T, Muta M, Hornig C, Weich HA.. Significance of vascular endothelial growth factor (VEGF)/soluble VEGF receptor-1 relationship in breast cancer. Int J Cancer 2002; 98:14-18.
3. Altman DG, Lausen B, Sauerbrei W, Schumacher M.. Dangers of using "optimal" cutpoints in the evaluation of prognostic factors. J Natl Cancer Inst 1994; 86:829-835.
4.Tang ZY, Ye SL, Liu YK, Qin LX, Sun HC, Ye QH, Wang L, Zhou J, Qiu SJ, Li Y, Ji XN, Liu H, Xia JL, Wu ZQ, Fan J, Ma ZC, Zhou XD, Lin ZY, Liu KD. A decade's studies on metastasis of hepatocellular carcinoma. J Cancer Res Clin Oncol.2004; 130:187-196.
5.Llovet JM. Updated treatment approach to hepatocellular carcinoma. J Gastroenterol.2005; 40:225-235.
6. Llovet JM, Chen Y, Wurmbach E, Roayaie S, Fiel MI, Schwartz M, Thung SN, Khitrov G, Zhang W, Villanueva A, Battiston C, Mazzaferro V, Bruix J, Waxman S, Friedman SL.. A molecular signature to discriminate dysplastic nodules from early hepatocellular carcinoma in HCV cirrhosis. Gastroenterology 2006; 131:1758-1767.
7. Mann CD, Neal CP, Garcea G, Manson MM, Dennison AR, Berry DP. Prognostic molecular markers in hepatocellular carcinoma:a systematic review. Eur J Cancer 2007; 43:979-992.
8. Budhu A, Jia HL, Forgues M, Liu CG, Goldstein D, Lam A, Zanetti KA, Ye QH, Qin LX, Croce CM, Tang ZY, Wang XW.. Identification of metastasis-related microRNAs in hepatocellular carcinoma. Hepatology 2008; 47:897-907.
9.Lee J S, Chu I S, Heo J, Calvisi DF, Sun Z, Roskams T.Classification and prediction of survival in 29 hepatocellular carcinoma by gene expression profiling. Hepatology 2004; 40:667-676.
10.Zitvogel L, Tesniere A, Kroemer G. Cancer despite immunosurveillance: immunoselection and immunosubversion. Nat Rev Immunol.2006; 6:715-727.
11. Dranoff G. Cytokines in cancer pathogenesis and cancer therapy. Nat Rev Cancer. 2004; 4:11-22.
12. Zou W. Immunosuppressive networks in the tumour environment and their therapeutic relevance. Nat Rev Cancer.2005; 5:263-274.
13. Gao Q, Qiu SJ, Fan J, Zhou J, Wang XY, Xiao YS, Xu Y, Li YW, Tang ZY. Intratumoral balance of regulatory and cytotoxic T cells is associated with prognosis of hepatocellular carcinoma after resection. [J] Clin Oncol.2007;Jun 20; 25(18):2586-2593.
14. Zhu XD, Zhang JB, Zhuang PY, Zhu HG, Zhang W, Xiong YQ, Wu WZ, Wang L, Tang ZY, Sun HC. High expression of macrophage colony-stimulating factor in peritumoral liver tissue is associated with poor survival after curative resection of hepatocellular carcinoma. J Clin Oncol.2008;26:2707-2716.
15. Budhu A, Forgues M, Ye QH, Jia HL, He P, Zanetti KA, Kammula US, Chen Y, Qin LX, Tang ZY, Wang XW. Prediction of venous metastases, recurrence, and prognosis in hepatocellular carcinoma based on a unique immune response signature of the liver microenvironment. Cancer Cell.2006; 10:99-111.
16. Sato E, Olson SH, Ahn J, Bundy B, Nishikawa H, Qian F, Jungbluth AA, Frosina D, Gnjatic S, Ambrosone C, Kepner J, Odunsi T, Ritter G, Lele S, Chen YT, Ohtani H, Old LJ, Odunsi K. Intraepithelial CD8+ tumor-infiltrating lymphocytes and a high CD8+/regulatory T cell ratio are associated with favorable prognosis in ovarian cancer. Proc Natl Acad Sci U S A. Proc Natl Acad Sci USA.2005;102:18538-18543.
17.Chang AE and Rosenberg SA. Overview of interleukin-2 as an immunotherapeutic agent. Semin Surg Oncol 1989; 5:385-390.
18. Rosenberg SA. Progress in human tumour immunology and immunotherapy. Nature.2001; 411:380-384.
19. Spiess PJ, Yang JC, and Rosenberg SA. In vivo antitumor activity of tumor-infiltrating lymphocytes expanded in recombinant interleukin-2. J Natl Cancer Inst.1987; 79:1067-1075.
20. Smyth MJ, Cretney E, Kershaw MH, et al. Cytokines in cancer immunity and immunotherapy. Immunol Rev 2004; 202:275-293.
21. McDermott DF, and Atkins MB. Application of IL-2 and other cytokines in renal cancer. Expert Opin Biol Ther 2004; 4:455-468.
22. Ikeguchi M, and Hirooka Y. Interleukin-2 gene expression is a new biological prognostic marker in hepatocellular carcinomas. Onkologie 2005; 28:255-9.
23. Young MR, Wright MA, Lozano Y, Matthews JP, Benefield J, Prechel MM.. Mechanisms of immune suppression in patients with head and neck cancer:influence on the immune infiltrate of the cancer. Int J Cancer 1996; 67:333-338.
24. Beckebaum S, Zhang X, Chen X, Yu Z, Frilling A, Dworacki G, Grosse-Wilde H, Broelsch CE, Gerken G, Cicinnati VR. Increased levels of interleukin-10 in serum from patients with hepatocellular carcinoma correlate with profound numerical deficiencies and immature phenotype of circulating dendritic cell subsets. Clin Cancer Res 2004; 10:7260-7269.
25. Akiba J, Yano H, Ogasawara S, Higaki K, Kojiro M. Expression and function of interleukin-8 in human hepatocellular carcinoma. Int J Oncol 2001; 18:257-264.
26.Tuck AB, Arsenault DM, O'Malley FP, Hota C, Ling MC, Wilson SM, Chambers AF. Osteopontin induces increased invasiveness and plasminogen activator expression of human mammary epithelial cells. Oncogene 1999; 18:4237-4246. 原因可能因为我们研究中的肝癌病人大多有乙型肝炎病史(90%强),此类病人的机体全身的免疫平衡状态不仅仅与肿瘤及其肝脏局部微环境有关,可能还会受到乙肝病毒感染复制情况,肝炎活动情况的影响[23-24]。因此对于具有乙肝背景的肝癌病人来讲,其免疫状态错踪复杂,在血液水平寻求准确预测预后的免疫相关的分子标签难度极大。这部分研究中我们还发现,尽管血清及外周血单个核细胞中IL-2,IFN-γ,IL-4和IL-10与肝癌的预后无显著相关性。但是我们发现IL-10血清水平与肝癌患者的总体生存有轻度相关,但无显著意义(P=0.0579),与以往研究者的报道不同,我们发现IL-10表达高的患者有相对稍好的总生存率,前面一部分组织芯片免疫组化染色我们也看到了相似的现象,而以往大量研究认为IL-10是肿瘤预后不良的指标。有学者认为IL-10是一种免疫负调节因子,但其可能具有双向调节效应,IL-10可以下调免疫抑制性分子如一氧化氮、前列腺素等发挥免疫刺激作用瞳副,但是这方面的证据还不十分充分。如前所述,肝癌病人大多有肝炎病史哺],此类肝癌病人的死亡原因除了死于肝癌,一部分也可以因为肝炎进展肝衰竭而死亡,我们认为可能因为IL-10作为重要的炎症抑制因子,抑制了乙型肝炎的进展,使其慢性化,减缓了肝细胞及肝功能损害,因而更有利于病人的生存[27],而非IL-10抑制了肝癌的进展延长病人的生存。
通过本研究我们认为肿瘤患者的系统免疫状态并不能真正代表局部免疫状态,对于具有乙型肝炎背景的肝癌患者来说甚至两者并无明确相关性。总之,由于细胞因子功能的多样性和其作用网络的复杂性,全面准确地掌握机体全身和肿瘤局部免疫状态的特点,并将其应用于肝癌的复发的预测预防任重而道远。
1.Umemura T, Ichijo T, Yoshizawa K, Tanaka E, Kiyosawa K..Epidemiology of hepatocellular carcinoma in Japan.J Gastroenterol.2009;44 Suppl 19:102-7.
2.Wu HC, Wang Q, Yang HI, Ahsan H, Tsai WY, Wang LY, Chen SY, Chen CJ, Santella RM. Aflatoxin B1 exposure, hepatitis B virus infection, and hepatocellular carcinoma in Taiwan.Cancer Epidemiol Biomarkers Prev.2009;18(3):846-53.
3.Yao F, Terrault N. Hepatitis C and hepatocellular carcinoma. Curr Treat Options Oncol.2001;2:473-483.
4. Koike K, Moriya K, Kimura S. Role of hepatitis C virus in the development of hepatocellular carcinoma:transgenic approach to viral hepatocarcinogenesis. J Gastroenterol Hepatol.2002; 17:394-400.
5.Yuen MF, Wong DK, Fung J, Ip P, But D, Hung I, Lau K, Yuen JC, Lai CL. HBsAg Seroclearance in chronic hepatitis B in Asian patients:replicative level and risk of hepatocellular carcinoma. Gastroenterology.2008; 135(4):1192-9.
[6].Chemin I, Zoulim F. Hepatitis B virus induced hepatocellular carcinoma. Cancer Lett.2009:13. [Epub ahead of print]
7. Mosmann TR, Cherwinski H, Bond MW, Giedlin MA, Coffman RL Two types of murine helper T cell clone. J Immunol.1986; 136:2348-2357.
8. Coussens LM, Werb Z. Inflammation and cancer. Nature.2002;420:860-867.
9.Enzler T, Gillessen S, Manis JP, Ferguson D, Fleming J, Alt FW, Mihm M, Dranoff G. Deficiencies of GM-CSF and interferon gamma link inflammation and cancer. J Exp Med.2003; 197:1213-1219.
10.Balkwill F, Mantovani A. Inflammation and cancer:back to irchow? Lancet.2001;357:539-545.
11. Parish CR. Cancer immunotherapy:the past, the present and the future. Immunol Cell Biol.2003; 81:106-113.
12. Gao Q, Qiu SJ, Fan J, Zhou J, Wang XY, Xiao YS, Xu Y, Li YW, Tang ZY. Intratumoral balance of regulatory and cytotoxic T cells is associated with prognosis of hepatocellular carcinoma after resection. [J] Clin Oncol.2007;Jun20; 25(18):2586-2593.
13. Dranoff G. Cytokines in cancer pathogenesis and cancer therapy. Nat Rev Cancer. 2004; 4:11-22.
14.Zou W.Immunosuppressive networks in the tumour environment and their therapeutic relevance. Nat Rev Cancer.2005; 5:263-274.
15.Yamamura M, Modlin RL, Ohmen JD, Moy RL.Local expression of anti-inflammatory cytokines in cancer [J]. J Clin Invest,1993;91:1005.
16.Kharkevitch DD, Seito D, Balch GC, Maeda T, Balch CM, Itoh K. Characterization of autologous tumor-specific T-helper2 cells in tumor-infiltrating lymphocytes from a patient with metastatic melanoma [J]. Int J Cancer,1994;58:317.
17.Tabata T, Hazama S, Yoshino S, Oka M. Th2 subset dominance among peripheral blood T lymphocytes in patients with digestivecancers [J]. Am J Surg,1999;177:203.
18. Zhu XD, Zhang JB, Zhuang PY, Zhu HG, Zhang W, Xiong YQ, Wu WZ, Wang L, Tang ZY, Sun HC.High expression of macrophage colony-stimulating factor in peritumoral liver tissue is associated with poor survival after curative resection of hepatocellular carcinoma.J Clin Oncol.2008;26:2707-2716.
19. Budhu A, Forgues M, Ye QH, Jia HL, He P, Zanetti KA, Kammula US, Chen Y, Qin LX, Tang ZY, Wang XW. Prediction of venous metastases, recurrence, and prognosis in hepatocellular carcinoma based on a unique immune response signature of the liver microenvironment. Cancer Cell.2006;10:99-111.
20.Llovet, JM.Updated treatment approach to hepatocellular carcinoma.J Gastroenterol.2005; 40,225-235.
21.Lun-Xiu Qin, Zhao-You Tang. Recent progress in predictive biomarkers for metastatic recurrence of human hepatocellular carcinoma:a review of the literature. J Cancer Res Clin Oncol.2004;130:497-513.
22. Morgan E, Varro R, Sepulveda H, Ember JA, Apgar J, Wilson J, Lowe L, Chen R, Shivraj L, Agadir A, Campos R, Ernst D, Gaur A. Cytometric bead array:a multiplexed assay platform with applications in various areas of biology. Clin Immunol.2004; 110:252-266.
23. Falasca K, Ucciferri C, Dalessandro M, Zingariello P, Mancino P, Petrarca C, Pizzigallo E, Conti P, Vecchiet J.. Cytokine patterns correlate with liver damage in patientswith chronic hepatitisB and C[J]. Ann Clin Lab Sci.2006;36 (2):144-150.
24. Ariyasu T, Tanaka T, Fujioka N, Yanai Y, Yamamoto Y, Yamauchi H, Ikegami H, Ikeda M, Kurimoto M. Effects of interferon-alpha subtypes on the TH1/TH2 balance in peripheral blood mononuclear cells from patientswith hepatitis virus infection associated liver disorders.[J]. In V itro Cell Dev B iol Anim.2005;41 (1-2): 50-56.
25.Dokka S, Shi X, Leonard S, Wang L, Castranova V, Rojanasakul Y. Interleukin-10-mediated inhibition of free radical generation in macrophages. Am J Physiol Lung Cell Mol Physiol.2001;Jun;280(6):L1196-202.
26.Anzola, M. Hepatocellular carcinoma:role of hepatitis B and hepatitis C viruses proteins in hepatocarcinogenesis. J. Viral Hepat.2004;11,383-393.
27. Tsai SL, Liaw YF, Chen MH, Huang CY, Kuo GC. Detection of type 2-like T-helper cells in hepatitis C virus infection:implication for hepatitis virus chronicity. Hepatology,1997,25:449-458. 细胞内MMP-2和uPA的表达增加促进肝癌的侵袭转移,又能通过激活MEK/ERK1/2信号转导途径参与肝癌细胞生长增殖的调节[23]。在肝癌中OPN促进肿瘤侵袭转移的作用肯定,而其促炎作用还不肯定。我们知道如果两个预测指标呈显著正相关那么联合使用其预测效能未必提高,基于这部分的研究我们认为OPN是反应肝癌细胞自身侵袭特性的重要分子[14],癌旁IL-2也是预测肝癌预后的重要分子标签,两者相互独立,联合使用可以作为预测肝癌预后的指标。
总之,通过这部分研究我们认为对于肝癌转移复发的预测和预防要从肿瘤本身及其周围微环境两方面着眼,多方面着手。同时兼顾“种子”和“土壤”才有可能取得良好的效果,单纯强调某一方面都会有失偏颇。
1.Llovet JM. Updated treatment approach to hepatocellular carcinoma. J Gastroenterol.2005;40:225-235.
2. Tang ZY, Ye SL, Liu YK, Qin LX, Sun HC, Ye QH, Wang L, Zhou J, Qiu SJ, Li Y, Ji XN, Liu H, Xia JL, Wu ZQ, Fan J, Ma ZC, Zhou XD, Lin ZY, Liu KD. A decade's studies on metastasis of hepatocellular carcinoma. J Cancer Res Clin Oncol.2004; 130:187-196.
3. Budhu A, Jia HL, Forgues M, Liu CG, Goldstein D, Lam A, Zanetti KA, Ye QH, Qin LX, Croce CM, Tang ZY, Wang XW. Identification of metastasis-related microRNAs in hepatocellular carcinoma. Hepatology 2008; 47:897-907.
4. Lee J S, Chu I S, Heo J, Calvisi DF, Sun Z, Roskams T. Classification and prediction of survival in 29 hepatocellular carcinoma by gene expression profiling. Hepatology 2004; 40:667-676.
5. Llovet JM, Chen Y, Wurmbach E, Roayaie S, Fiel MI, Schwartz M, Thung SN, Khitrov G, Zhang W, Villanueva A, Battiston C, Mazzaferro V, Bruix J, Waxman S, Friedman SL. A molecular signature to discriminate dysplastic nodules from early hepatocellular carcinoma in HCV cirrhosis. Gastroenterology 2006; 131:1758-1767.
6. Mann CD, Neal CP, Garcea G, Manson MM, Dennison AR, Berry DP.. Prognostic molecular markers in hepatocellular carcinoma:a systematic review. Eur J Cancer 2007; 43:979-992.
7. Budhu A, Forgues M, Ye QH, Jia HL, He P, Zanetti KA, Kammula US, Chen Y, Qin LX, Tang ZY, Wang XW. (2006) Prediction of venous metastases, recurrence, and prognosis in hepatocellular carcinoma based on a unique immune response signature of the liver microenvironment. Cancer Cell.10:99-111.
8. Sato E, Olson SH, Ahn J, Bundy B, Nishikawa H, Qian F, Jungbluth AA, Frosina D, Gnjatic S, Ambrosone C, Kepner J, Odunsi T, Ritter G, Lele S, Chen YT, Ohtani H, Old LJ, Odunsi K. (2005) Intraepithelial CD8+ tumor-infiltrating lymphocytes and a high CD8+/regulatory T cell ratio are associated with favorable prognosis in ovarian cancer. Proc Natl Acad Sci U S A. Proc Natl Acad Sci USA 102:18538-18543.
9.Zitvogel L, Tesniere A, Kroemer G. Cancer despite immunosurveillance: immunoselection and immunosubversion. Nat Rev Immunol.2006; 6:715-727.
10. Galon J, Costes A, Sanchez-Cabo F, Kirilovsky A, Mlecnik B, Lagorce-Pages C, Tosolini M, Camus M, Berger A, Wind P, Zinzindohoue F, Bruneval P, Cugnenc PH, Trajanoski Z, Fridman WH, Pages F. Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science.2006; 313:1960-1964.
11. Dranoff G. Cytokines in cancer pathogenesis and cancer therapy. Nat Rev Cancer. 2004; 4:11-22.
12.Zou W. Immunosuppressive networks in the tumour environment and their therapeutic relevance. Nat Rev Cancer.2005; 5:263-274.
13.Gao Q, Qiu SJ, Fan J, Zhou J, Wang XY, Xiao YS, Xu Y, Li YW, Tang ZY. Intratumoral balance of regulatory and cytotoxic T cells is associated with prognosis of hepatocellular carcinoma after resection. [J] Clin Oncol.2007 Jun 20; 25(18):2586-2593.
14. Ye QH, Qin LX, Forgues M, He P, Kim JW, Peng AC, Simon R, Li Y, Robles AI, Chen Y, Ma ZC, Wu ZQ, Ye SL, Liu YK, Tang ZY, Wang XW. Predicting hepatitis B virus-positive metastatic hepatocellular carcinomas using gene expression profiling and supervised machine learning. Nat Med 2003; 9:416-423.
15. Zhang H, Ye QH, Ren N, Zhao L, Wang YF, Wu X, Sun HC, Wang L, Zhang BH, Liu YK, Tang ZY, Qin LX.. The prognostic significance of preoperative plasma levels of osteopontin in patients with hepatocellular carcinoma. J Cancer Res Clin Oncol 2006; 132:709-717.
16. Sun J, Xu HM, Zhou HJ, Dong QZ, Zhao Y, Fu LY, Hei ZY, Ye QH, Ren N, Jia HL, Qin LX. The prognostic significance of preoperative plasma levels of osteopontin in patients with TNM stage-I of hepatocellular carcinoma. J Cancer Res Clin Oncol 2010; 136:1-7.
17.Weber GF, Zawaideh S, Hikita S, Kumar VA, Cantor H, Ashkar S. Phosphorylation-dependent interaction of osteopontin with its receptors regulates macrophage migration and activation. J Leukoc Biol.2002; 72(4):752-761.
18.Bruemmer D, Collins AR, Noh G, Wang W, Territo M, Arias-Magallona S, Fishbein MC, Blaschke F, Kintscher U, Graf K, Law RE, Hsueh WA. Angiotensin II-accelerated atherosclerosis and aneurysm formation is attenuated in osteopontindeficient mice. J Clin Invest.2003; 112(9):1318-1331.
19. Rollo EE, Laskin DL, Denhardt DT. Osteopontin inhibits nitric oxide production and cytotoxicity by activated RAW264.7 macrophages. J Leukoc Biol.1996; 60(3):397-404.
20.Heilmann K, Hoffmann U, Loddenkemper C, Sina C, Schreiber S. Hayford C, et al. Osteopontin as a two-sided mediator of intestinal inflammation. J Cell Mol Med 2008.;[Epub ahead of print]
21.Xanthou G, Alissafi T, Semitekolou M, Simoes DC, Economidou E, Gaga M, et al. Osteopontin has a crucial role in allergic airway disease through regulation of dendritic cell subsets. Nat Med 2007;13:570-8.
22.Weber GF, Cantor H. Differential roles of osteopontin/Eta-1 in early and late lpr disease. Clin Exp Immunol 2001;126:578-83.
23. Sun BS, Dong QZ, Ye QH, Sun HJ, Jia HL, Zhu XQ, Liu DY, Chen J, Xue Q, Zhou HJ, Ren N, Qin LX. Lentiviral-mediated miRNA against osteopontin suppresses tumor growth and metastasis of human hepatocellular carcinoma. Hepatology.2008; 48:1834-42.
1.我们发现癌旁炎症免疫相关的微环境中的IL-2和IL-15表达水平与肝癌的复发有关,IL-2是预测复发及总体生存的最佳的预测因子。可能需要多中心随机对照研究来进一步验证。
2.在癌旁肝组织中IL-2主要存在于肝细胞中,而非淋巴细胞中,癌旁T淋巴细胞中IL-2很少存在,免疫组化方法几乎不能检测。但是我们不能就此得出IL-2就是肝细胞产生而非淋巴细胞产生的结论。尚应该从基因转录水平进一步寻求证据。我们拟用激光显微切割技术,获取肝组织冰冻切片上的肝细胞,但是此仅为形态学的判断。肝脏细胞表面缺乏特定的表面标记物,所以仅靠形态学判断缺乏说服力。进一步研究的方法我们在探索中。
1.Tang ZY, Ye SL, Liu YK, Qin LX, Sun HC, Ye QH, Wang L, Zhou J, Qiu SJ, Li Y, Ji XN, Liu H, Xia JL, Wu ZQ, Fan J, Ma ZC, Zhou XD, Lin ZY, Liu KD. A decade's studies on metastasis of hepatocellular carcinoma. J Cancer Res Clin Oncol. 2004;130:187-196.
2.Llovet JM. Updated treatment approach to hepatocellular carcinoma. J Gastroenterol.2005;40:225-235.
3.Zitvogel L, Tesniere A, Kroemer G. Cancer despite immunosurveillance: immunoselection and immunosubversion. Nat Rev Immunol.2006; 6:715-727.
4. Galon J, Costes A, Sanchez-Cabo F, Kirilovsky A, Mlecnik B, Lagorce-Pages C, Tosolini M, Camus M, Berger A, Wind P, Zinzindohoue F, Bruneval P, Cugnenc PH, Trajanoski Z, Fridman WH, Pages F. Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science.2006;313: 1960-1964.
5. Gao Q, Qiu SJ, Fan J, Zhou J, Wang XY, Xiao YS, Xu Y, Li YW, Tang ZY. Intratumoral balance of regulatory and cytotoxic T cells is associated with prognosis of hepatocellular carcinoma after resection.[J] Clin Oncol.2007;25(18):2586-2593.
6. Dranoff G. Cytokines in cancer pathogenesis and cancer therapy. Nat Rev Cancer. 2004;4:11-22.
7.Zou W. Immunosuppressive networks in the tumour environment and their therapeutic relevance. Nat Rev Cancer.2005; 5:263-274.
8. Coussens LM, Werb Z. Inflammation and cancer. Nature.2002; 420:860-867.
9.Enzler T, Gillessen S, Manis JP, Ferguson D, Fleming J, Alt FW, Mihm M, Dranoff G. Deficiencies of GM-CSF and interferon gamma link inflammation and cancer. J Exp Med.2003; 197:1213-1219.
10. Balkwill F, Mantovani A. Inflammation and cancer:back to irchow? Lancet.2001; 357:539-545.
11. Parish CR. Cancer immunotherapy:the past, the present and the future. Immunol Cell Biol.2003;81:106-113.
12. Shibata M, Nezu T, Kanou H, Abe H, Takekawa M, Fukuzawa M. Decreased production of interleukin-12 and type 2 immune responses are marked in cachectic patients with colorectal and gastric cancer. J Clin Gastroenterol.2002;34:416-420.
13. Skinnider BF, Mak TW. The role of cytokines in classical Hodgkin lymphoma. Blood.2002;99:4283-4297.
14. Ito N, Nakamura H, Tanaka Y, Ohgi S. Lung carcinoma:analysis of T helper type 1 and 2 cells and T cytotoxic type 1 and 2 cells by intracellular cytokine detection with flow cytometry. Cancer.1999;85:2359-2367.
15. Kioi M, Takahashi S, Kawakami M, Kawakami K, Kreitman RJ, Puri RK. Expression and Targeting of Interleukin-4 Receptor for Primary and Advanced Ovarian Cancer Therapy. Cancer Res.2005;65:8388-8396.
16. Filella X, Alcover J, Zarco MA, Beardo P, Molina R, Ballesta AM. Analysis of type T1 and T2 cytokines in patients with prostate cancer. Prostate.2000;44:271-274.
17. de Vita F, Orditura M, Galizia G, Romano C, Lieto E, Iodice P, Tuccillo C, Catalano G. Serum interleukin-10 is an independent prognostic factor in advanced solid tumors. Oncol Rep.2000;7:357-361.
18.Sakamoto N, Lef J S, Kato K, Umezu K, Yoneda K, Wada T, Sato S, Koyanagi Y, Oyamada M. Significance of the Thl/Th2 Balance and Tcl/Tc2 Balance in Advanced Colon Cancer Patients. Biotherapy.2000; 14:17-19.
19. Sheu BC, Lin RH, Lien HC, Ho HN, Hsu SM, Huang SC. Predominant Th2/Tc2 Polarity of Tumor-Infiltrating Lymphocytes in Human Cervical Cancer. J Immunol.2001; 167:2972-2978.
20. Clerici M, Shearer GM, Clerici E. Cytokine Dysregulation in Invasive Cervical Carcinoma and Other Human Neoplasias:Time to Consider the TH1/TH2 Paradigm. J Natl Cancer Inst.1998;90:261-263.
21. Reed MJ. The discriminant-function test:a marker of Thl/Th2 cell cytokine secretion and breast tumour oestrogen synthesis. Mol Med Today.1995; 1:98-103.
22.Tabata T, Hazama S, Yoshino S, Oka M. Th2 subset dominance among peripheral blood T lymphocytes in patients with digestive cancers. Am J Surg.1999; 177: 203-208.
23.Kharkevitch DD, Seito D, Balch GC, Maeda T, Balch CM, Itoh K. Characterization of autologous tumor-specific T-helper 2 cells in tumor-infiltrating lymphocytes from a patient with metastatic melanoma. Int J Cancer.1994;58:317-323.
24.Cameron AJ, Ott BJ, Payne WS. The incidence of adenocarcinoma in columnar-lined (Barrett's) esophagus. N Engl J Med.1985;313:857-859.
25. Dahms BB, Rothstein FC. Barrett's esophagus in children:A consequence of chronic gastroesophageal reflux. Gastroenterology.1984; 86:318-323.
26. Collins RH Jr, Feldman M, Fordtran JS. Colon cancer, dysplasia and surveillance in patients with ulcerative colitis:A critical review. N Engl J Med.1987; 316:1654-1658.
27. Korelitz BI. Carcinoma of the intestinal tract in Crohn's disease:Results of a survey conducted by the National Foundation for Ileitis and Colitis. Am J Gastroenterol.1983;78:44-46.
28. Sipponen P, Kekki M, Haapakoski J, Ihamaki T, Siurala M. Gastric cancer risk in chronic atrophic gastritis:statistical calculations of cross-sectional data. Int J Cancer.1985;35:173-177.
29.Yao F, Terrault N. Hepatitis C and hepatocellular carcinoma. Curr Treat Options Oncol.2001;2:473-483.
30. Koike K, Moriya K, Kimura S. Role of hepatitis C virus in the development of hepatocellular carcinoma:transgenic approach to viral hepatocarcinogenesis. J Gastroenterol Hepatol.2002; 17:394-400.
31. Gottschalk S, Rooney CM, Heslop HE. Post-transplant lymphoproliferative disorders. Annu Rev Med.2005;56:29-44.
32. Young LS, Rickinson AB. Epstein-Barr virus:40 years on. Nat Rev Cancer.2004; 4:757-768.
33.Kiippers R. Mechanisms of B-cell lymphoma pathogenesis. Nat Rev Cancer.2005; 5:251-262.
34. Falcone M, Sarvetnick N. Cytokines that regulate autoimmune responses. Curr Opin Immunol.1999; 11:670-676.
35. Harris DP, Haynes L, Sayles PC, Duso DK, Eaton SM, Lepak NM, Johnson LL, Swain SL, Lund FE. Reciprocal regulation of polarized cytokine production by effector B and T cells. Nat Immunol.2000; 1:475-482.
36. Song le H, Binh VQ, Duy DN, Kun JF, Bock TC, Kremsner PG, Luty AJ. Serum cytokine profiles associated with clinical presentation in Vietnamese infected with hepatitis B virus. J Clin Virol.2003;28:93-103.
37. Kitaoka S, Shiota G, Kawasaki H. Serum levels of interleukin-10, interleukin-12 and soluble interleukin-2 receptor in chronic liver disease type C. Hepatogastroenterology.2003;50:1569-1574.
38. Michelson S, Miller BE, Glicksman AS, Leith JT. Tumor micro-ecology and competitive interactions. J Theor Biol.1987;128:223-246.
39. Liotta LA, Steeg PS, Stetler-Stevenson WG. Cancer metastasis and angiogenesis: an imbalance of positive and negative regulation. Cell.1991;64:327-336.
40. de Visser KE, Coussens LM. The Inflammatory Tumor Microenvironment and Its Impact on Cancer Development. Contrib Microbiol. Basel Karger.2006;13:118-137.
41. Bissell MJ, Radisky D. Putting tumors in context. Nat Rev Cancer.2001;1:46-54.
42. Hasebe T, Sasaki S, Imoto S, Ochiai A. Highly proliferative fibroblasts forming fibrotic focus govern metastasis of invasive ductal carcinoma of the breast. Mod Pathol.2001;14:325-337.
43. Sung SY, Hsieh CL, Wu D, Chung LW, Johnstone PA. Tumor Microenvironment Promotes Cancer Progression, Metastasis, and Therapeutic Resistance. Curr Probl Cancer.2007;31:36-100.
44. Kinzler KW, Vogelstein B. Life (and death) in a malignant tumour. Nature.1996; 379:19-20.
45. Kurose K, Hoshaw-Woodard S, Adeyinka A, Lemeshow S, Watson PH, Eng C. Genetic model of multi-step breast carcinogenesis involving the epithelium and stroma:clues to tumour-microenvironment interactions. Hum Mol Genet.2001;10:1907-1913.
46. Dumont N, Arteaga CL. The tumor microenvironment:a potential arbitrator of the tumor suppressive and promoting actions of TGF-beta Differentiation.2002;70:574-582.
47.Schindler M, Grabski S, Hoff E, Simon SM. Defective pH regulation of acidic compartments in human breast cancer cells (MCF-7) is normalized in adriamycin-resistant cells (MCF-7adr). Biochemistry.1996;35:2811-2817.
48. Loeffler DA, Juneau PL, Heppner GH. (Natural killer-cell activity under conditions reflective of tumor micro-environment. Int J Cancer.1991;48:895-899.
49. Graeber TG, Osmanian C, Jacks T, Housman DE, Koch CJ, Lowe SW, Giaccia AJ. ypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours. Nature.1996;379:88-91.
50. Liyanage UK, Moore TT, Joo HG, Tanaka Y, Herrmann V, Doherty G, Drebin JA, Strasberg SM, Eberlein TJ, Goedegebuure PS, Linehan DC. Prevalence of regulatory T cells is increased in peripheral blood and tumor microenvironment of patients with pancreas or breast adenocarcinoma. J. Immunol.2002; 169:2756-2761.
51. Viguier M, Lemaitre F, Verola O, Cho MS, Gorochov G, Dubertret L, Bachelez H, Kourilsky P, Ferradini L. Foxp3 expressing CD4+CD25(high) regulatory T cells are overrepresented in human metastatic melanoma lymph nodes and inhibit the function of infiltrating T cells. J Immunol.2004;173:1444-1453.
52. Shekhar MPV, Pauley R, Heppner G. Host microenvironment in breast cancer development Extracellular matrix-stromal cell contribution to neoplastic phenotype of epithelial cells in the breast. Breast Cancer Research.2003;5:130-135.
53. Pollard, JW. Tumor-educated macrophages promote tumor progression and metastasis. Nat. Rev. Cancer.2004;4:71-78.
54. Galon J, Costes A, Sanchez-Cabo F, Kirilovsky A, Mlecnik B, Lagorce-Pages C, Tosolini M, Camus M, Berger A, Wind P, Zinzindohoue F, Bruneval P, Cugnenc PH, Trajanoski Z, Fridman WH, Pages F. Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science.2006;313:1960-1964.
55. Sato E, Olson SH, Ahn J, Bundy B, Nishikawa H, Qian F, Jungbluth AA, Frosina D, Gnjatic S, Ambrosone C, Kepner J, Odunsi T, Ritter G, Lele S, Chen YT, Ohtani H, Old LJ, Odunsi K. Intraepithelial CD8+ tumor-infiltrating lymphocytes and a high CD8+/regulatory T cell ratio are associated with favorable prognosis in ovarian cancer. Proc Natl Acad Sci U S A. Proc Natl Acad Sci USA.2005; 102:18538-18543.
56. Zhu XD, Zhang JB, Zhuang PY, Zhu HG, Zhang W, Xiong YQ, Wu WZ, Wang L, Tang ZY, Sun HC. High expression of macrophage colony-stimulating factor in peritumoral liver tissue is associated with poor survival after curative resection of hepatocellular carcinoma. J Clin Oncol.2008;26:2707-2716.
57. Hao C, Parney IF, Roa WH, Turner J, Petruk KC, Ramsay DA. Cytokine and cytokine receptor mRNA expression in human glioblastomas:evidence of Th1, Th2 and Th3 cytokine dysregulation. Acta Neuropathol.2002; 103:171-178.
58. Chattopadhyay S, Chakraborty NG Continuous presence of Thl conditions is necessary for longer lasting tumor-speciWc CTL activity in stimulation cultures with PBL. Hum Immunol.2005;66:884-891.
59. Sato M, Goto S, Kaneko R, Ito M, Sato S, Takeuchi S. Impaired production of Thl cytokines and increased frequency of Th2 subsets in PBMC from advanced cancer patients. Anticancer Res.1998;18:3951-3955.
60. Rouleau M, Cottrez F, Bigler M, Antonenko S, Carballido J M, Zlotnik A, Roncarolo M G, Groux H. IL-10 transgenic mice present a defect in T cell development reminiscent of SCID patients. J. Immunol.1999; 163:1420-1427.
61. Sharma S, Stolina M, Lin Y, Gardner B, Miller PW, Kronenberg M, Dubinett SM. T cell-derived IL-10 promotes lung cancer growth by suppressing both T cell and APC function J Immunol.1999;163:5020-5028.
62. Kiessling R, Wasserman K, Horiguchi S, Kono K, Sjoberg J, Pisa P, Petersson M. Tumor-induced immune dysfunction. Cancer Immunol Immunother.1999;48:353-362.
63. Tisdale MJ. Biology of cachexia. J Natl Cancer Inst. (1997) 89:1763-1773.
64. Yang FC, Agematsu K, Nakazawa T, Mori T, Ito S, Kobata T, Morimoto C, Komiyama A. CD27/CD70 interaction directly induces natural killer cell killing activity. Immunology.1996;88:289-293.
65. Akiba J, Yano H, Ogasawara S, Higaki K, Kojiro M. Expression and function of interleukin-8 in human hepatocellular carcinoma. Int J Oncol.2001; 18:257-264.
66. Chau GY, Wu C W, Lui W Y, Chang TJ, Kao HL, Wu LH, King KL, Loong CC, Hsia CY, Chi CW. Serum Interleukin-10 But Not Interleukin-6 Is Related to Clinical Outcome in Patients With Resectable Hepatocellular Carcinoma. Annals of Surgery.2000; 231:552-558.
67. Budhu A, Forgues M, Ye QH, Jia HL, He P, Zanetti KA, Kammula US, Chen Y, Qin LX, Tang ZY, Wang XW. Prediction of venous metastases, recurrence, and prognosis in hepatocellular carcinoma based on a unique immune response signature of the liver microenvironment. Cancer Cell.2006; 10:99-111.
68. Lauerova L, Dusek L, Simickova M, Kocak I, Vagundova M, Zaloudik J, Kovarik J. Malignant melanoma associates with Thl/Th2 imbalance that coincides with disease progression and immunotherapy response. Neoplasma.2002;4 9:159-166.
69. Topp MS, Papadimitriou CA, Eitelbach F, Koenigsmann M, Oelmann E, Koehler B, Oberberg D, Reufi B, Stein H, Thiel E, et al. Recombinant human interleukin 4 has antiproliferative activity on human tumor cell lines derived from epithelial and nonepithelial histologies. Cancer Res.1995;55:2173-2176.
70. Kawakami M, Kawakami K, Stepensky VA, Maki RA, Robin H, Muller W, Husain SR, Puri RK. Interleukin 4 receptor on human lung cancer:a molecular target for cytotoxin therapy. Clin Cancer Res.2002;8:3503-3511.
71. Colombo MP, Trinchieri G. Interleukine-12 in antitumor immunity and immunotherapy. Cytokine and Growth Factor Rew.2002;13:155-168.
72. Wang L, Wu WZ, Sun HC, Wu XF, Qin LX, Liu YK, Liu KD, Tang ZY. Mechanism of interferon alpha on inhibition of metastasis and angiogenesis of hepatocellular carcinoma after curative resection in nude mice. J Gastrointest Surg.2003;7:587-594.
73. Oka H, Emori Y, Ohya O, Kobayashi N, Sasaki H, Tanaka Y, Hayashi Y, Nomoto K. An immunomodulatory arabinomannan extracted from Mycobacterium tuberculosis, Z-100, restores the balance of Thl/Th2 cell responses in tumor bearing mice. Immunol Lett.1999;70:109-117.
74. Wagner HJ, Bollard CM, Vigouroux S, Huls MH, Anderson R, Prentice HG, Brenner MK, Heslop HE, Rooney CM. A strategy for treatment of Epstein-Barr virus-positive Hodgkin's disease by targeting interleukin 12 to the tumor environment using tumor antigen-specific T cells. Cancer Gene Ther.2004;11:81-91.
75.Oya M, Asakura H, Mizuno R, Marumo K, Murai M. Repeated regression of pulmonary metastases from renal cell carcinoma after treatment using different interferon-alpha preparations. Biomed Res.2005;26:135-137. 76.Schadendorf D. Gene-based therapy of malignant melanoma. Semin Oncol.2002;29:503-512.
77. Loskog A, Dzojic H, Vikman S, Ninalga C, Essand M, Korsgren O, Totterman TH. Adenovirus CD40 ligand gene therapy counteracts immune escape mechanisms in the tumor Microenvironment. J Immunol.2004; 172:7200-7205.
78. Okinaga K, Iinuma H, Ogiwara T, Ogawa F. A phase I study of immunotherapy with tumor cell-dendritic cell fusion vaccine in patients with advanced or recurrent gastrointestinal cancer. Journal of Clinical Oncology.2004:2615.
79. Park HR, Jo SK, Jung U, Yee ST. Restoration of the immune functions in aged mice by supplementation with a new herbal composition, HemoHIM. Phytother Res.2008;22:36-42.
80. Li T, Tamada K, Abe K, Tada H, Onoe Y, Tatsugami K, Harada M, Kubo C, Nomoto K. The restoration of the antitumor T cell response from stress-induced suppression using a traditional Chinese herbal medicine Hochu-ekki-to (TJ-41:Bu-Zhong-Yi-Qi-Tang). Immunopharmacology.1999;43:11-21.
81. Decker D, Schondorf M, Bidlingmaier F, Hirner A, von Ruecker AA. Surgical stress induces a shift in the type-1/type-2 T-helper cell balance, suggesting down-regulation of cell-mediated and up-regulation of antibody-mediated immunity commensurate to the trauma Surgery.1996; 119:316-325.
82.Mack VE, McCarter MD, Naama HA, Calvano SE, Daly JM. Dominance of T-helper 2-type cytokines after severe injury. Arch Surg.1996;131:1303-1308.
83. Hiraki S, Ono S, Kinoshita M, Tsujimoto H, Seki S, Mochizuki H. Interleukin-18 restores immune suppression in patients with nonseptic surgery, but not with sepsis. Am J Surg.2007; 193:676-680.
84.van Sandick JW, Gisbertz SS, ten Berge IJ, Boermeester MA, van der Pouw Kraan TC, Out TA, Obertop H, van Lanschot JJ. Immune responses and prediction of major infection in patients undergoing transhiatal or transthoracic esophagectomy for cancer. Ann Surg.2003;237:35-43.
85. Kanaoka Y, Yagi T, Sadamori H, Matsukawa H, Matsuda H, Inagaki M, Ishikawa T, Saito S, Iwagaki H, Tanaka N. Analysis of host response to hepatectomy by simultaneous measurement of cytokines in the portal vein, caval vein and radial artery. J Int Med Res.2002;30:496-505.
86. Lekanne Deprez RH, Fijnvandraat AC, Ruijter JM, Moorman AF. Sensitivity and accuracy of quantitative real-time polymerase chain reaction using S YBR green I depends on cDNA synthesis conditions. Anal Biochem.2002;307:63-69.
87. Gygi SP, Rochon Y, Franza BR, Aebersold R. Correlation between protein and mRNA abundance in yeast. Mol Cell Biol.1999;19:1720-1730.
88. Le Naour F, Hohenkirk L, Grolleau A, Misek DE, Lescure P, Geiger JD, Hanash S, Beretta L. Profiling changes in gene expression during differentiation and maturation of monocyte-derived dendritic cells using both oligonucleotide microarrays and proteomics. J Biol Chem.2001;276:17920-17931.
89. Anderson L, Seilhamer J. A comparison of selected mRNA and protein abundances in human liver. Electrophoresis.1997;18:533-537.
90. Goto S, Sato M, Kaneko R, Itoh M, Sato S, Takeuchi S. Analysis of Thl and Th2 cytokine production by peripheral blood mononuclear cells as a parameter of immunological dysfunction in advanced cancer patients. Cancer Immunol Immunother.1999;48:435-442.
91. Lindner P, Fjalling M, Hafstrom L, Kierulff-Nielsen H, Mattsson J, Schersten T, Rizell M, Naredi P. Isolated hepatic perfusion with extracorporeal oxygenation using hyperthermia, tumour necrosis factor alpha and melphalan. Eur J Surg Oncol.1999;25:179-185.
92. Kreher CR, Dittrich MT, Guerkov R, Boehm BO, Tary-Lehmann M. CD4+ and CD8+ cells in cryopreserved human PBMC maintain full functionality in cytokine ELISPOT assays. J Immunol Methods.2003;278:79-93.
93. Samri A, Durier C, Urrutia A, Sanchez I, Gahery-Segard H, Imbart S, Sinet M, Tartour E, Aboulker JP, Autran B, Venet A; ANRS ELISpot Standardization Group. Evaluation of the interlaboratory concordance in quantification of human immunodeficiency virus-specific T cells with a gamma interferon enzyme-linked immunospot assay. Clin Vaccine Immunol.2006; 13:684-697.
94. Hricik DE, Rodriguez V, Riley J, Bryan K, Tary-Lehmann M, Greenspan N, Dejelo C, Schulak JA, Heeger PS. Enzyme linked immunosorbent spot (ELISPOT) assay for interferon-gamma independently predicts renal function in kidney transplant recipients. Am J Transplant.2003;3:878-884.
95. Nickel P, Presber F, Bold G, Biti D, Schonemann C, Tullius SG, Volk HD, Reinke P. Enzyme-linked immunosorbent spot assay for donor-reactive interferon-gamma-producing cells identifies T-cell presensitization and correlates with graft function at 6 and 12 months in renal-transplant recipients. Transplantation.2004;78:1640-1646.
96. Clay TM, Hobeika AC, Mosca PJ, Lyerly HK, Morse MA. Assays for monitoring cellular immune responses to active immunotherapy of cancer. Clin Cancer Res.2001;7:1127-1135.
97. Reome JB, Hylind JC, Dutton RW, Dobrzanski MJ. Type 1 and type 2 tumor infiltrating effector cell subpopulations in progressive breast cancer. Clin Immunol.2004;111:69-81.
98. Horiuchi Y, Hanazawa A, Nakajima Y, Nariai Y, Asanuma H, Kuwabara M, Yukawa M, Ito H. T-helper (Th)1/Th2 imbalance in the peripheral blood of dogs with malignant tumor. Microbiol Immunol.2007;51:1135-1138.
99. Morgan E, Varro R, Sepulveda H, Ember JA, Apgar J, Wilson J, Lowe L, Chen R, Shivraj L, Agadir A, Campos R, Ernst D, Gaur A. Cytometric bead array:a multiplexed assay platform with applications in various areas of biology. Clin Immunol.2004;110:252-266.
100. Cook EB, Stahl JL, Lowe L, Chen R, Morgan E, Wilson J, Varro R, Chan A, Graziano FM, Barney NP. Simultaneous measurement of six cytokines in a single sample of human tears using microparticle-based flow cytometry:allergics vs. non-allergics. J Immunol Methods.2001;254:109-118.
101. Wong CK, Lam CW, Wu AK, Ip WK, Lee NL, Chan IH, Lit LC, Hui DS, Chan MH, Chung SS, Sung JJ. Plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome. Clin Exp Immunol.2004;136:95-103.
102. Templin MF, Stoll D, Schrenk M, Traub PC, Vohringer CF, Joos TO. Protein microarray technology. Trends Biotechnol.2002;20:160-166.
103. Huang RP, Huang R, Fan Y, Lin Y. Simultaneous detection of multiple cytokines from conditioned media and patient's sera by an antibody-based protein array system. Anal Biochem.2001;294:55-62.
104. Kodadek T. Development of protein-detecting microarrays and related devices. Trends Biochem Sci.2002;27:59-63.
105. Lin Y, Huang R, Santanam N, Liu YG, Parthasarathy S, Huang RP. Profiling of human cytokines in healthy individuals with vitamin E supplementation by antibody array. Cancer Lett.2002; 187:17-24.
106. Kverka M,Burianova J, Zadnikova RL, Kocourkova I, Cinova J, Tuckova L,Hogenova HT. Cytokine Profiling in Human Colostrum and Milk by Protein Array. Clinical Chemistry.2007;53:955-962.
107. Chen XM, Jiang HL, Zhou LF, Pan XP, Hu ZR, Liu RH, Chen XM, Chen Z. Immunomodulatory function of orally administered thymosin alphal J Zhejiang Univ Sci B.2005;6:873-876.
108.Chia CS, Ban K, Ithnin H, Singh H, Krishnan R, Mokhtar S, Malihan N, Seow HF. Expression of interleukin-18, interferon and interleukin-10 in hepatocellular carcinoma. Immunol Lett.2002;84:163-172.