Yasushi Cho

Concurrent infiltration by CD8+ T cells and CD4+ T cells is a favourable prognostic factor in non-small-cell lung carcinoma.

The purpose of this study was to clarify the relationship between the number of tumour-infiltrating T lymphocytes and the clinicopathological features and clinical outcome in patients with non-small-cell lung cancer (NSCLC). Tissue specimens from 109 patients who underwent surgical resection for NSCLC were immunohistochemically analysed for CD4 and CD8 expression. Patients were classified into two groups according to whether their tumours exhibited a ‘high’ or ‘low’ level of CD8+ or CD4+ lymphocyte infiltration. Although the level of infiltration by CD8+ T cells alone had no prognostic significance, the survival rate for patients with both ‘high’ CD8+ and ‘high’ CD4+ T-cell infiltration was significantly higher than that for the other groups (log-rank test, P=0.006). Multivariate analysis indicated that concomitant high CD8+ and high CD4+ T-cell infiltration was an independent favourable prognostic factor (P=0.0092). In conclusion, the presence of high levels of both CD8+ T cells and CD4+ T cells is a significant indicator of a better prognosis for patients with NSCLC, and cooperation between these cell populations may allow a significantly more potent antitumour response than either population alone.

CD8+ tumor-infiltrating lymphocytes together with CD4+ tumor-infiltrating lymphocytes and dendritic cells improve the prognosis of patients with pancreatic adenocarcinoma.

Objective Recent studies have demonstrated the importance of tumor immunity for a cancer patients prognosis. In some types of cancer, it has been shown through immunohistochemical analysis that the existence of CD8+ tumor-infiltrating lymphocytes (TILs) is a crucial factor in determining prognosis. In an experimental model, CD4+ lymphocytes together with CD8+ lymphocytes contributed significantly to tumor immunity. Methods Specimens were taken from 80 surgically resected pancreatic adenocarcinomas between 1992 and 1999. Immunohistochemical staining of CD4, CD8, and S100 protein was performed, and the levels of these proteins were determined by microscopic analysis. The percentages of patients in the CD4(+) and CD8(+) groups were 59% (47/80) and 25% (16/80), respectively. When separated into 4 groups, CD4/8(+/+), CD4/8(+/−), CD4/8(−/+) and CD4/8(−/−), the overall survival rate was significantly higher in CD4/8(+/+) patients (13 cases) compared with those in all other groups combined (67 cases; P = 0.0098). CD4/8(+/+) status was negatively correlated with tumor depth and TNM stage. Multivariate analyses showed that CD4/8(+/+) status was an independent favorable prognostic factor. The number of tumor-infiltrating S100 protein positive cells was also significantly higher in the CD4/8(+/+) group than in others (P = 0.0084). Conclusions In pancreatic adenocarcinoma, the presence of CD4+ TILs together with CD8+ TILs serves as a good indicator of the patients outcome after surgical treatment.

Overexpression of hypoxia-inducible-factor 1alpha(HIF-1alpha) in oesophageal squamous cell carcinoma correlates with lymph node metastasis and pathologic stage.

The purpose of this study is to investigate the clinical and histopathologic significance of hypoxia-inducible-factor 1α (HIF-1α) expression in oesophageal squamous cell carcinoma. One hundred and thirty surgically resected specimens of OSCC were immunohistochemically assessed for HIF-1α expression with monoclonal antibody. High HIF-1α immunostaining was detected in 40 specimens. The percentage of high HIF-1α expression cases increased with tumour stage according to pTNM system. High HIF-1α expression correlated with pTNM stage, depth of tumour invasion, lymph node metastasis, distant metastasis, lymphatic invasion and positive surgical margin. The overall survival rate was worse in patients with high HIF-1α pattern than in patients with low-expression pattern. Univariate analyses identified high HIF-1α positivity, depth of tumour invasion, lymph node metastasis, distant metastasis, lymphatic invasion, and a positive surgical margin as risk factors. Multivariate analyses indicated that depth of tumour invasion, lymph node metastasis and positive surgical margin, but not HIF-1α, were independent prognostic factors. Survival in patients with a high HIF-1α expression was significantly worse than in those with low expression in patient treated with adjuvant therapy.

Clinical significance of immune cell infiltration within gallbladder cancer

To investigate the pathophysiological significance of infiltrating antitumour immune cells, we evaluated the quantity of immune cell intratumoral infiltration in 110 surgically resected gallbladder specimens by immunohistochemistry. We examined 45 cases of gallbladder cancer and 65 cases of benign gallbladder diseases for CD4+ T cells, CD8+ T cells, natural killer cells (NKCs), and dendritic cells (DCs). High levels of CD4+ T cell, CD8+ T cell, NKC, and DC infiltration were recognised in 51.1% (23 out of 45), 37.8% (17 out of 45), 33.3% (15 out of 45), and 48.9% (22 out of 45) of cancer specimens, respectively. High numbers of infiltrating CD4+ and CD8+ T cells correlated with decreasing tumour invasion, and high numbers of infiltrating DCs correlated with decreasing lymph-node tumour metastasis. Furthermore, increased infiltration of CD4+ and CD8+ T cells and DCs exhibited a significant correlation with prolonged survival. NKC infiltration, however, did not correlate with any of the clinicopathological factors examined. Additionally, high levels of infiltration were not identified in specimens from benign diseases, consistent with the cancer-specific activity of CD4+ and CD8+ T cells and DCs. In this study, we demonstrate that CD4+ and CD8+ tumour-infiltrating lymphocyte and DCs, but not NKCs, are important factors in the accurate prognosis of survival after surgical removal of gallbladder adenocarcinoma.

HLA class I antigen expression is associated with a favorable prognosis in early stage non-small cell lung cancer

Human leukocyte antigen (HLA) class I displays a repertoire of endogenously processed peptides to CD8+ T lymphocytes. The present study assessed correlations between HLA class I expression, clinicopathologic factors, and tumor‐infiltrating immune cells in human non‐small cell lung cancers (NSCLC). Expression of HLA class I was assessed in 161 resected primary NSCLC by immunohistochemistry using EMR8–5, a novel monoclonal anti‐pan HLA class I heavy chain antibody. Expression of HLA class I was classified into three categories: strongly positive, weakly positive, or negative. Tumor‐infiltrating CD8+ lymphocytes and CD56+ natural killer cells within cancer nests and stroma were also counted. Expression of HLA class I was strongly positive in 50 tumors, weakly positive in 57 tumors, and negative in 54 tumors. Down‐regulation of HLA class I was significantly correlated with male sex, history of smoking, non‐adenocarcinoma histology, and moderate‐/low‐grade differentiation. The density of cancer nest‐infiltrating CD8+ cells in HLA class I‐negative tumors was significantly decreased compared to that in HLA class I strongly positive tumors (P < 0.01). Kaplan–Meier analysis revealed a significant favorable influence on overall survival for patients displaying tumors with strongly positive expression of HLA class I (P < 0.01). Multivariate analysis revealed down‐regulation of HLA class I as an independent factor of poor prognosis in pathological stage I patients, but not in late‐stage patients. These results suggest that down‐regulation of HLA class I expression in NSCLC is a marker of poor prognosis, and this may play a critical role in immune surveillance of patients with NSCLC. (Cancer Sci 2007; 98: 1424–1430)

Fusion of HIV-1 Tat protein transduction domain to poly-lysine as a new DNA delivery tool

Effective gene therapy depends on the efficient transfer of therapeutic genes to target cells. None of the current technologies, however, satisfy all of the requirements necessary for gene therapy, because the plasma and nuclear membranes of mammalian cells are tight barriers against gene transfer using synthetic delivery systems. The protein transduction domain (PTD) of human immunodeficiency virus type 1 (HIV-1) Tat protein greatly facilitates protein transfer via membrane destabilisation. We synthesised polylysine peptides containing Tat PTD (TAT-pK), or other sequences, and investigated their potential as agents for gene transfer. The synthesised polypeptide TAT-pK retains DNA binding function and mediates delivery of a reporter gene to cultured cells. RGD motif binds with low affinity to alpha integrins which induce cell activation. Two control polypeptides, GGG-pK and RGD-pK, were synthesised and tested, but their gene transfer abilities were weaker than those of TAT-pK. TAT-pK-mediated gene transfer was enhanced in the presence of chloroquine or ammonium chloride, to a greater extent than that of cationic lipid-mediated gene transfer in most cancer cell lines tested. These data suggest that TAT-pK may be a potent candidate delivery vehicle that promotes gene transfer, dependent on the endocytic pathway. We conclude that the TAT-pK/DNA complex is useful as a minimal unit to package therapeutic genes and to transduce them into mammalian cells.

Metastases in Mediastinal and Hilar Lymph Nodes in Patients With Non-Small Cell Lung Cancer : Quantitative Assessment With Diffusion-Weighted Magnetic Resonance Imaging and Apparent Diffusion Coefficient

Objective: To evaluate diffusion-weighted magnetic resonance (DW-MR) imaging for detection of metastases in lymph nodes by using quantitative analysis. Methods: Seventy patients with non-small cell lung cancer were examined with DW and short inversion time inversion recovery (STIR) turbo-spin-echo MR imaging. Apparent diffusion coefficient of each lung cancer and lymph node was calculated from DW-MR images. Difference of the apparent diffusion coefficient in a lung cancer and a lymph node was calculated (D1). From STIR turbo-spin-echo MR images, ratios of signal intensity in a lymph node to that in a 0.9% saline phantom was calculated (lymph node-saline ratio [LSR1]). For quantitative analysis, the threshold value for a positive test was determined on a per node basis and tested for ability to enable a correct diagnosis on a per patient basis. Results of quantitative analyses of DW- and STIR-MR images were compared on a per patient basis with McNemar testing. Results: Mean D1 in the lymph node group with metastases was lower than that in the group without metastases (P < 0.001). When an D1 of 0.24 × 10−3 mm2/s was used as the positive test threshold, sensitivity, specificity, and accuracy were 69.2%, 100%, and 94.0%, respectively, on a per patient basis. There was no significant difference (P > 0.05) between quantitative analyses of DW-MR images and STIR-MR images. Conclusions: Quantitative analysis of DW-MR images enables differentiation of lymph nodes with metastasis from those without.

Infiltrating regulatory T cell numbers is not a factor to predict patient's survival in oesophageal squamous cell carcinoma

CD4/8 status has been previously reported to be a critical factor in the prognosis of oesophageal squamous cell carcinoma (OSCC). In the current study, we investigated the effect of regulatory T cells (Treg; Foxp3+ lymphocytes) on the status of CD4+ and CD8+ T cells in 122 patients with OSCC. Immunohistochemical analysis of Treg was performed using an antibody against Foxp3. The survival rate for low Foxp3 patients was significantly lower than for high Foxp3 patients (P=0.0028 by log-rank test), but Foxp3 status did not significantly correlate with prognosis in CD4/8(+/+) patients or CD4/8(+/−) or (−/+) patients (P=0.5185 and 0.8479, respectively, by log-rank test). We also found that Foxp3 status correlated with CD4/8 status (P=0.0002 by χ2 test) and that the variance of CD8/CD4 ratio in patients with low Foxp3 was larger than in patients with high Foxp3 (P<0.0001 by F-test). Thus, the results do not support the idea that Treg suppress anti-tumour immunity in patients with OSCC. Rather, the CD8/CD4 ratio and CD4/8 status appear to be critical factors in anti-tumour immunity. Furthermore, Treg numbers correlate with both the CD8/CD4 ratio and the CD4/8 status, suggesting that Treg number is not a factor to predict patients survival in OSCC.

DARPP-32 expression arises after a phase of dysplasia in oesophageal squamous cell carcinoma

This is the first report to correlate DARPP-32 immunoreactivity (dopamine and cAMP-regulated phosphoprotein, Mr 32u2009000) to clinicopathological status in human cancer. DARPP-32 is recognised as a neuronal protein. A recent study demonstrated that DARPP-32, and a truncated isoform t-DARPP, are overexpressed in gastric carcinoma during the process of carcinogenesis. The biological function of DARPP-32, however, is still unclear. The purpose of this study was to clarify the roles of DARPP-32 and t-DARPP in oesophageal squamous cell carcinoma (OSCC). Initially, we investigated DARPP-32 and t-DARPP expression in OSCC cell lines by Reverse transcription–polymerase chain reaction and Western blot. DARPP-32 expression was observed in four out of seven (57.1%) cell lines, but t-DARPP expression was not observed in any cell lines. In oesophageal tissue sample, DARPP-32 expression was observed in four out of seven (57.1%) tumour tissues, while t-DARPP was not observed in any tissues. Subsequently, DARPP expression was assessed by immunohistochemistry, using a polyclonal antibody, in tissue sections from 122 patients with primary OSCC. DARPP immunoreactivity was not observed in any normal oesophageal mucous membranes. On the other hand, positive DARPP immunostaining was detected in 37 patients (30.3%) and correlated inversely with pathologic stage (P=0.0284), pT (P=0.0438), pN (P=0.0303) and tumour size (P=0.012). The overall survival rate was worse in patients with DARPP-negative tumours than in patients with DARPP-positive tumours (P=0.0453). Interestingly, DARPP expression was observed in only one out of 45 cases of dysplasia. These observations suggest that DARPP-32 (rather than t-DARPP) expression arises after a phase of dysplasia in OSCC, and that tumours expressing DARPP-32 progress less rapidly than DARPP-32-negative tumours.

Association of CD8+ T cell infiltration in oesophageal carcinoma lesions with human leucocyte antigen (HLA) class I antigen expression and survival

Oesophageal cancer is one of the most aggressive tumours with a poor prognosis. However, little is known about the immune response in the tumour microenvironment. To investigate the role of immunosurveillance in the clinical course of oesophageal squamous cell carcinoma, 98 formalin‐fixed, paraffin‐embedded primary tumours were analysed using immunohistochemical methods for human leucocyte antigen (HLA) class I heavy chain and β2‐microglobulin expression and for CD4‐, CD8‐ and CD57‐positive cell infiltration. HLA class I expression of tumour cells was correlated positively with infiltration of CD8+ T cells into the cancer nest, but not with the clinical course of disease. However, CD8+ and CD4+ T cell infiltration was correlated with prognosis. These results suggest that tumour antigen‐specific cellular immune response plays a role in the clinical course of the disease and that HLA class I antigen expressed on tumour cells contribute to this association most probably by mediating the interactions between tumour cells and CD8+ T cells.