Dendritic cell infiltration and prognosis of human hepatocellular carcinoma
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- Cai, X., Gao, Q., Qiu, S. et al. J Cancer Res Clin Oncol (2006) 132: 293. doi:10.1007/s00432-006-0075-y
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Aim: To elucidate the relationship between local immunocompetent cells and prognosis of human hepatocellular carcinoma (HCC) after resection. Methods: HE staining and immunohistochemical study were carried out on specimens from patients underwent surgical resection. Local immunocompetent cells, such as dendritic cells (DCs), memory T cells, CD3+ T lymphocytes and CD8+ T lymphocytes, were counted and their relationships with tumor-free survival rate were analyzed by grouping DCs with the T lymphocytes retrospectively. Results: 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. Conclusions: Marked infiltration of DCs in HCC nodules was closely related to the prognosis of HCC after surgical resection and can be served as a predictive index for recurrence and metastasis of HCC.
KeywordsHepatocellular carcinoma (HCC)Dendritic cells (DCs)T lymphocytesPrognosis
The tumor–host interaction that has become a focus of much attention in the field of tumor determines its prognosis (Liotta et al. 2001; Rosenberg et al. 2001; Dunn et al. 2002; Fidler 2003). The tumor gets blood supply and nutrition from circumferential stroma and induces immune escape by producing numerous kinds of factors. On the other hand, the host microenvironment can control the tumor to a certain extent or even eradicate it by modifying and restricting the tumor biomedically. It has been found in clinical and experimental studies that there is little correlation between the real anti-tumor responses in vivo and the detected immune responses in vitro in tumor-bearing hosts, which suggests little were known on the regulation of anti-tumor immunity in vivo, especially on the role of the tumor microenvironment (Srivastava et al. 2000; Howard et al. 2004; Feijoo et al. 2005). The immune microenvironment, as an important part of the tumor microenvironment, is gaining more and more attention (Ciavarra et al. 2004; Yu et al. 2005). Recently, NCI has initiated an “extraordinary funding opportunity” program to extend its signatures of the cancer cell to its microenvironment. 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. The liver, an immune-privileged organ (Starzl 2001), has a pivotal role in immune regulation locally and systemically with its special immune system and plays an important role in the occurrence and progression of hepatocellular carcinoma (HCC). In patients with HCC, poor immune function especially impotent immune defense response resulting from abnormal immune microenvironment is an important factor responsible for the immune escape and the metastatic and recurrence tendencies of HCC (Jewell 2005). It is of great significance to thoroughly research on immune regulation mechanism of liver and the way to improve local immunocompetent cells’ activities, especially their specific anti-tumor activity, in order to reveal the mechanism of the occurrence and progression of HCC. Consequently, elucidating the relationship between local immunocompetent cells in tumor sites and its prognosis is obviously an obligatory task, which would contribute to the understanding of local immune status, prediction of prognosis and selection of treatment modality in HCC.
Dendritic cells (DCs) are the most potent professional antigen-presenting cells, widely distributed in peripheral lymphatic tissues. DCs determine whether an immune response finally activates immune system or leads to immunological tolerance (Banchereau et al. 1998; Steinman 1999, 2000; Steinman et al. 2002). Although the significance of DCs on the immune regulation of liver is well established, DCs’ role in the occurrence and progression of HCC is still uncertain. In this study, general data, such as distribution, quantity and morphology of local DCs and T lymphocytes in tumor sites of HCC patients were detected, and then their relationships with the prognosis were analyzed. The results indicated that marked infiltration of DCs in HCC nodules was closely related to the prognosis of HCC and can be served as a predictive index for recurrence and metastasis of HCC.
Materials and methods
Clinicopathological data of the 123 patients with HCC
Mean ± SD
History of hepatitis
Serum albumin (g/l)
Tumor size (cm)
Tumor-free survival time (year)
Staining of immunocompetent cells by immunohistochemistry
Chromogens diaminobenzidine (DAB), hematoxylin and fuchsin, rabbit anti-human S-100 protein polyclonal antibody (1:600 dilution), mouse anti-human CD45RO, CD3, CD8 monoclonal antibodies (1:50 dilution) and goat anti-rabbit, goat anti-mouse EnVision secondary antibodies are purchased from Dako company (CA, USA). Alkaline phosphatase (AP)-goat anti-mouse secondary antibodies (1:100 dilution) are commercially available from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Samples were prepared for serial section (4 μm thick). Both conventional HE and immunohistochemical staining were employed. Two steps Envision protocol was applied to detect CD45RO+ T lymphocytes, CD3+ T lymphocytes, CD8+ T lymphocytes and S-100+ cells in HCC tissue samples. Paraffin sections were deparaffinized conventionally and then serially dipped in 3% H2O2, kept in microwave for antigen retrieval, incubated by normal calf serum. After incubation with primary antibodies and secondary antibodies serially, the sections were developed in DAB solution under microscope and counterstained with hematoxylin. The double immunohistochemical staining with double enzyme renovated by microwave protocol (Zhang 1998) was used to show the relationship between S-100+ DCs and CD45RO+ memory T lymphocytes or CD8+ T lymphocytes. Firstly, S-100+ DCs were stained using the same techniques described above. After DAB staining, sections were blocked and then antigen retrieval was carried out in microwave oven for 20 min. Secondly, sections were incubated with primary antibodies, mouse anti-human CD45RO or CD8 monoclonal antibodies both with a dilution of 1:50 at 37°C for 2 h followed by incubation with AP-conjugated secondary antibodies at 37°C for 30 min. Thirdly, sections were developed with fuchsin solution, counterstained with hematoxylin, treated with 37 mmol/l ammonia water and mounted with 50% glycerol.
Observation of local immunocompetent cells
Intratumoral and paratumoral T lymphocytes infiltration were detected by HE staining. Immunohistochemical staining displayed as follows: (1) both plasma and nuclei of S-100+ cells were dark brown with a large cell volume and a round or irregular shape. Cell membrane and plasma of CD45RO+, CD3+ or CD8+ T lymphocytes were brown with small cell volume. HCC nodules, pericancerous tissues and adjacent cirrhotic tissues were detected by comparing with HE staining sections. Ten high power (×400) fields were randomly selected to sum up and average cell numbers. (2) Both plasma and nuclei of S-100+ cells were dark brown while cell membrane and plasma of CD45RO+ or CD8+ T lymphocytes were purple under double immunohistochemical staining. The relationships between S-100+ cells and CD45RO+ or CD8+ T lymphocytes were documented.
Follow-up of patients
None of the patients had any adjuvant therapies before operation. Follow-up was finished until March 15, 2003. All patients were followed-up by monitoring serum AFP and ultrasonography every 2–6 months according to the postoperative time. For suspicious cases, computed tomography and/or magnetic resonance imaging were used to verify whether recurrence or not. Sixteen and thirty-four patients were given transcatheter artery chemoembolization treatment or immunotherapy after resection, respectively, while eight patients received both. All patients were divided into four groups by combination of the significant numbers of DCs and T cells in HCC nodules. Group I comprised of patients who had both the DCs and T cells infiltrations exceeding certain numbers; group II was made up of patients who had the DCs infiltration exceeding a certain number while the T cells infiltration did not; group III was classified as those patients who had a DCs infiltration and a T cells infiltration that was less than and exceeding a certain number, respectively; and group IV meant that patients who had both the DCs and T cells infiltrations under certain numbers.
SPSS 11.0 statistical package was employed. The numbers of local infiltrating immunocompetent cells were expressed as mean and median. One-way Kaplan–Meier survival analysis, multi-way Cox survival analysis and analysis of variance were employed to analyze the relationships between local immunocompetent cells and prognosis of HCC. Significance was accepted when P<0.05.
T lymphocytes in HCC nodules mainly distributed in the stroma of tumor, with sheet-like or nest-like areas formed partially, and germinal centers were detected occasionally. The grades were: (−) no or few infiltrating lymphocytes detected; (++) infiltrating lymphocytes that formed sheet-like or nest-like areas (i.e. >100/HPF); (+) infiltrating lymphocytes between the two grades described above detected. The ratio of (−):(+):(++) was 29:64:30. T lymphocytes infiltration in pericancerous tissues, one high power field by the tumor nodule, was graded the same as that in HCC nodules. The ratio of (−):(+):(++) was 24:66:33.
Immunohistochemistry study of DCs
There was 0–362/10HPF of DCs in HCC nodules. The mean and median were 24.75 and 14.00, respectively. S-100+ DCs were generally small and round in pericancerous tissues and were 0–181/10HPF. The mean and median were 28.17/10HPF and 22.00/10HPF, respectively. When adjacent cirrhotic liver tissues were observed, DCs were mainly detected in the stroma of the tumor, together with lymphocytes, and were not common among liver cells. DCs infiltrating adjacent cirrhotic liver tissues were generally round with few dendrites with a number of 0–51/10HPF (Fig. 1a). The mean and median numbers of DC infiltrating cirrhotic tissue were 13.34/10HPF and 10.00/10HPF, respectively. The relationship between the number of DCs and the clinical data (not including the tumor-free survival time) was analyzed without any significance (data not shown).
Immunohistochemistry study of T lymphocytes
Double immunohistochemical staining
Relationship between local immunocompetent cells and prognosis
One-way log-rank survival analysis was employed to analyze the clinical data which may correlate with prognosis of HCC one by one. Preoperative γ-GT, AKP, tumor size, TNM staging, Child–Pugh grade, portal vein tumor thrombus and the number of tumor nodules were found to contribute to the prognosis (P<0.05). Multi-way Cox survival analysis was employed to analyze the relationships between infiltrating immunocompetent cells in HCC nodules or pericancerous tissues and tumor-free survival time. 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). A certain number of DCs in HCC nodules (≥25/10HPF) was found to be correlated with tumor-free survival time (P=0.005). The more DCs were detected, the longer tumor-free survival time was. But a certain number of DCs in pericancerous tissues (≥28/10HPF) had no correlation with tumor-free survival time (P=0.329). The number of CD45+ 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).
Although the local immune responses cannot directly reflect the anti-tumor activity of immunocompetent cells in patients with tumor, it is of great significance in determining the progression, prognosis and treatment of the tumor (Kawata et al. 1992; Eisenthal et al. 2001; Yu et al. 2002; Dave et al. 2004). Due to the complexity of the immune microenvironment, there is still controversy in the anti-tumor immune response of its single component; however, the interaction among its components that might heavily contribute to anti-tumor immune responses is gaining attention (Cho et al. 2003). According to Nakano et al. (2001), in patients with renal cell carcinoma, just the tumor infiltrating CD8+ T lymphocytes did not indicate good prognosis, but the CD8+ T lymphocytes with high proliferative activity (i.e. antigen Ki-67 positive) did. In patients with squamous cell carcinoma of the esophagus, only the infiltration of both the CD4+ T lymphocytes and CD8+ T lymphocytes indicated good prognosis, and a combination of the two could serve as an independent prognostic factor (Cho et al. 2003). It is the same with researches on liver cancer. In patients with HCC, most of the local immunocompetent cells detected were lymphocytes, mostly T lymphocytes, and were phenotypically different from the circulating lymphocytes, which suggested the importance of T lymphocytes in the anti-tumor immunity of HCC (Wada et al. 1998; Norris et al. 1998; Doherty et al. 2000). Cases with more number of tumor infiltrating lymphocytes did not have the higher 5-year tumor-free survival rate compared to those with less infiltrating lymphocytes (Qin et al. 1997), which might attribute to immunosuppression of the lymphocytes (Unitt et al. 2005). The relationship between the number of tumor infiltrating DCs and clinicopathological features has been studied in esophageal (Ishigami et al. 2003), lung (Zeid et al. 1993), breast (Lespagnard et al. 1999), cervical (Bethwaite et al. 1996) and ovarian cancer (Eisenthal et al. 2001). Most of these studies demonstrated that the number of tumor infiltrating DCs positively correlate with the clinicopathological features including surgical outcome, and even could serve as an independent prognostic factor in some cancers (Bethwaite et al. 1996; Ishigami et al. 2003). However, in HCC, neither the tumor-infiltrating DCs alone nor the tumor-infiltrating lymphocytes alone, having any significant relationship with the postoperative recurrence-free time and survival rate, were reported. A marked infiltration of DCs together with lymphocytes in HCC tissue was closely related to the improved clinical prognosis and represented as an independent prognostic factor (Yin et al. 2002, 2003). However, in HCC cases, none of these studies have investigated the subtypes and their accurate number of local immunocompetent cells, as well as the direct evidence of interaction between DCs and lymphocytes. On the contrary, many factors, such as types, subtypes, functional state and interactions of local immune cells, must be analyzed in order to evaluate immune microenvironment in patients.
In this study, there was little correlation between the number grade of infiltrating lymphocytes and tumor-free survival rate by HE staining (P>0.05). Infiltration of immunocompetent cells is considered as an important kind of anti-tumor immunity. Using multi-way Cox survival analysis, we found that the number of tumor infiltrating memory T lymphocytes, CD3+ T lymphocytes and CD8+ T lymphocytes directly correlated to the tumor-free survival rate, which indicated the important role of T lymphocytes’ in anti-HCC immunity.
Hepatic DCs play an important role in the immune regulation of the liver. Absence of mature and activated CD83+ DCs in HCC nodules has been shown to implicate hepatocarcinogenesis (Chen et al. 2000). CD83+ DCs were observed predominantly in the cancer-invasive margin, which was a prognostic factor in patients with colorectal liver metastasis (Miyagawa et al. 2004). Also, it has been shown that DCs in the peripheral blood from patients with HCC were characterized by immature phenotype and with defective function: lower levels of HLA-DR, CD1a, CD80 and CD86; lower capacity to stimulate allogenic T cells; decreased amounts of induced interleukin-12 (Ninomiya et al. 1999; Wong et al. 2005). Using a number of different techniques, researchers have been able to prevent the growth of implanted tumors. The most promising of these techniques is based on the use of DCs, which are able to process and present antigens to activate naive T cells, and when loaded with tumor antigens, can stimulate a specific and durable anti-tumor response (O’Beirne et al. 2004). In our study, a larger number of DCs with large cell volume and irregular shape were detected mainly in HCC nodules, which was in proportions to the total number of tumor infiltrating DCs detected. DCs with small cell volume and round shape were also detected, indicating expression of S-100 protein in both mature and immature DCs. However, DCs detected in peritumoral cirrhotic and normal liver tissues were mainly immature DCs with small cell volume and round shape. We guess that these immature DCs, gathering in peritumoral sites, might consequently infiltrate into tumor nodules or perhaps were preparing for more effective tumor antigen processing.
Precursors of hepatic DCs are strong immune stimulators to memory T cells, and at the same time, DCs are indispensable to the maintenance of immunological memory due to its role in the maintenance of memory function of T lymphocytes (Ludewig et al. 1999). In our study, the number of the tumor infiltrating DCs significantly correlated with that of the memory T lymphocytes, as well as CD3+ T lymphocytes and CD8+ T lymphocytes. The more tumor infiltrating DCs were detected, the more tumor infiltrating memory T lymphocytes were and the higher the tumor-free survival rate was. Morphologically, DCs with more dendrites were often found where lymphocytes clustered. DCs were generally among tumor cells, encircling tumor cells with their pseudopodia, and some of its dendrites were in contact with tumor cells, others with lymphocytes. We also found that the more the number of DCs, alone or in combination with CD3+ or CD8+ T lymphocytes, the higher the tumor-free survival rate (P=0.000), which suggested the DCs’ connecting role between tumor cells and T lymphocytes, as well as their role in effective activation of cell-mediated anti-tumor immune response.
In summary, the tumor-free survival rate of the patients who had more intratumoral DCs was higher, irrespective of the number and subsets of infiltrating lymphocytes, which indicates that DCs play an important role in the activation of T lymphocytes and immune response and can be used as an important prognostic factor. Furthermore, we suggest that the number of intratumoral DCs can serve as a predictive index for recurrence and metastasis of HCC. Induction of anti-tumor immune response by activating the DCs may be a promising biological therapy in HCC and may contribute to reduce the postoperative recurrence and metastasis of HCC.
This work was supported by a grant from the National Natural Science Foundation of China (No. 30200268).