Yoshika Nagata

Cytokine production of lung cancer cell lines: Correlation between their production and the inflammatory/immunological responses both in vivo and in vitro

Cytokines produced by tumor cells may have various effects on antitumor immune responses and tumor growth. In the present study, the cytokine production of 31 lung cancer cell lines was evaluated, while any correlation with the histological type, the induction of tumor‐specific cytotoxic T lymphocytes (CTL) in vitro, and angiogenesis and the infiltration of inflammatory cells in tumor tissues were also examined. Production of interleukin (IL)‐1α, IL‐1β, IL‐4, IL‐6, IL‐8, IL‐10, tumor necrosis factor (TNF)‐α, granulocyte macrophage colony stimulating factor (GM‐CSF), granulocyte colony stimulating factor, transforming growth factor (TGF)‐β and vascular endothelial growth factor (VEGF) in the culture supernatant was measured using enzyme‐linked immunosorbent assay. Each cytokine was produced in a substantial number of the tumor cell lines. In particular, IL‐6, IL‐8, TGF‐β and VEGF were produced in 18 (55%), 29 (94%), 31 (100%) and 28 (90%) of 31 cell lines, respectively. However, neither IL‐4 nor TNF‐α was produced at all by any tumor cell line. TGF‐β production was significantly higher in adenocarcinoma than in squamous cell carcinoma (P = 0.03). Immunohistochemical staining revealed the magnitude of macrophage infiltration, and angiogenesis in surgically resected tumor tissue specimens correlated well with GM‐CSF and IL‐8 production from the corresponding cell lines. Among six lung cancer cell lines, CTL were induced in the three lung cancer cell lines that produced a lower amount of TGF‐β (<100 pg/mL). These findings suggested that TGF‐β produced by tumor cells could inhibit the induction of CTL in vitro. The present results suggest that the production of various cytokines from tumor cells might exert various paracrine effects both in vivo and in vitro. (Cancer Sci 2007; 98: 1048–1054)

Preoperative CYFRA 21-1 and CEA as prognostic factors in patients with stage I non-small cell lung cancer

PURPOSE This study investigated the preoperative serum levels of CYFRA 21-1 and CEA as prognostic factors in patients with stage I non-small cell lung cancer. SUBJECTS This study evaluated 341 patients who had undergone a complete resection for stage I NSCLC between 2002 and 2008. RESULTS The patients included 193 males and 148 females. The mean age of the patients was 69.2 years (range: 19-88). The histological types included 264 adenocarcinomas, 56 squamous cell carcinomas, 11 large cell carcinomas, and 10 other types of carcinoma. A pneumonectomy was performed in 2 patients, a bilobectomy in 7, a lobectomy in 255, a segmentectomy in 46, and partial resection of the lung in 31 patients. The positive rates for CYFRA 21-1 in the adenocarcinoma and squamous cell carcinoma patients were 33.3% and 76.8%, respectively. The positive rates for CEA in adenocarcinoma and squamous cell carcinoma patients were 23.8% and 26.8%, respectively. The 5-year survival rate after surgery in the normal CYFRA 21-1 group and the high CYFRA 21-1 groups were 92.8% and 75.4%, respectively, in the patients with stage I NSCLC. There was a significant difference between the 2 groups (p<0.0001). The 5-year survival rate according to the serum level of CEA in the patients with stage I NSCLC were 88.3% for the normal group and 76.3% for the high group. In a multivariate analysis using the variables found to be significant prognostic factors in univariate analysis, a high CYFRA 21-1 level was found to be a significant independent prognostic factor (95% confidence interval 1.213-5.442, p=0.014). CONCLUSION A high preoperative CYFRA 21-1 level was a significant independent prognostic factor in patients with stage I NSCLC. The patients with a high CYFRA 21-1 level should carefully followed-up to rule out occult metastasis. Further clinical studies will be necessary to evaluate the efficacy of adjuvant therapy for the patients selected according to this criterion.

Clinical significance of the frequency of regulatory T cells in regional lymph node lymphocytes as a prognostic factor for non-small-cell lung cancer.

BACKGROUND Regulatory T cells (Tregs) are potent immunosuppressive cells that play a crucial role in tumor immune escape. The purpose of the present study was to evaluate the prognostic significance of the frequency of CD4+CD25+Foxp3+ Tregs in the regional lymph node lymphocytes (RLNL) and peripheral blood lymphocytes (PBL) in patients who underwent surgical resection of non-small cell lung cancer (NSCLC). METHODS The RLNL and PBL in 158 NSCLC patients who underwent complete surgical resection were collected at the time of surgery. The proportions of CD4+CD25+Foxp3+ cells in the RLNL and PBL were determined by flow cytometry. RESULTS The average proportions of Tregs in the RLNL and PBL were 1.28% and 0.76%, respectively. The proportion of Tregs in the RLNL was significantly higher than that in the PBL (p < 0.0001). The 5-year overall survival rates of the patients according to the proportion of Tregs in the RLNL were 84.4% and 63.5% in the lower and higher groups, respectively. A significant difference was observed in the survival rate between the higher and lower groups (p = 0.0056). Among the patients with stage I disease, the 5-year survival rate (91.4%) was significantly higher in patients with the lower proportion of Tregs in RLNL that in the higher group (72.1%) (p = 0.0147). CONCLUSIONS The higher proportion of Tregs in the RLNL was a significant unfavorable prognostic factor, even in patients with node-negative NSCLC. The information about the proportion of Tregs in the RLNL might improve the discriminatory power for assessing the risk of the recurrence of NSCLC.

Identification of the HLA-Cw*0702-Restricted Tumor-Associated Antigen Recognized by a CTL Clone from a Lung Cancer Patient

Purpose: A large number of tumor-associated antigens have been used in vaccination trials for mainly melanomas. Our purpose of this study is to identify a novel tumor antigen useful for immunotherapy of lung cancer patients. Experimental Design: Analysis of an autologous tumor-specific CTL clone F2a that was established from regional lymph node lymphocytes of a patient with lung cancer (A904) by a mixed lymphocyte-tumor cell culture. Results: F2a recognized and killed autologous tumor cells (A904L), whereas it did not respond to autologous EBV-transformed B cells, phytohemagglutinin-blastoid T cells, and K562 cells. cDNA clone 31.2 was isolated by using cDNA expression cloning method as a gene encoding antigen. This gene was identical to the reported gene whose function was unknown. The antigen encoded by the cDNA was recognized by the CTL in a HLA-Cw*0702-restricted manner. Furthermore, a 9-mer peptide at positions 659 to 685 in cDNA clone 31.2 was identified as a novel epitope peptide. The CTL recognized some allogeneic cancer cell lines with HLA-Cw*0702 as well as some HLA-Cw*0702-negative cell lines when transfected with HLA-Cw*0702, thus indicating that the identified antigen was a cross-reactive antigen. Conclusions: Although exact mechanism to process the encoded protein and present the antigen in the context of HLA class I remains to be elucidated, the CTL recognized some of tumor cells in the context of HLA-Cw*0702 but did not recognize a variety of normal cells and also autologous EBV-transformed B cells. These results indicated that the antigen identified in this study may therefore be a possible target of tumor-specific immunotherapy for lung cancer patients.

Clinical significance of HLA class I alleles on postoperative prognosis of lung cancer patients in Japan.

BACKGROUND The role of the HLA phenotype in cancer prognosis has been frequently discussed. We previously reported the correlation between HLA alleles and the postoperative prognosis of 204 patients with non-small cell lung cancer (NSCLC). The present study was based on 695 patients with NSCLC to confirm these correlations. METHODS We evaluated the medical records of 695 NSCLC patients who underwent surgical resection. The serological typing of HLA class I was performed using a microcytotoxicity test of lymphocytes or PCR-sequence-specific oligonucleotides (PCR-SSO), and the correlation between the HLA alleles and the clinicopathological features was analyzed. The survival curves were calculated, and then a comparison of the survival curves was carried out. RESULTS The HLA-A2 positive(A2(+)) group at stage I showed a more unfavorable prognosis than HLA-A2(-) group in overall survival. At stage II+III, the HLA-A24(+) group had a poorer prognosis than the HLA-A24(-) group, and the HLA-B52(+) group showed unfavorable prognosis. Multivariate analysis demonstrated that HLA-A2 at stage I and HLA-A24 at stage II+III were the independent factors that affected the survival period. CONCLUSIONS The expression of HLA-A2 was considered as one of the unfavorable prognostic factors in the NSCLC patients at stage I. HLA-A24(+) group showed a significant unfavorable prognosis at stage II+III. These results suggested that HLA-A2 and HLA-A24 could be the prognostic factors in patients with NSCLC according to the state of advancement of the disease.

Results of a surgical resection for patients with stage IV non--small-cell lung cancer.

PURPOSE This study retrospectively investigated the clinical significance of surgical treatment for stage IV non-small-cell lung cancer (NSCLC). SUBJECTS There were 36 patients who underwent surgical resection for stage IV NSCLC between 1999 and 2008. RESULTS The patients included 22 males and 14 females. All patients had either synchronous distant metastasis or pleural dissemination. The mean age of the patients was 65.8 years (range, 18 to 90 years). The histological types included 29 adenocarcinomas, 5 squamous-cell carcinomas and 2 large-cell carcinomas. The organs of metastasis were bone in 5 patients, brain in 4, adrenal gland in 4, axillary lymph nodes in 3, liver in 2, and 1 patient had a contralateral pulmonary metastasis. The number of metastases was one site in 13, two sites in 3, three sites in 1, and five sites in 2 patients. The patients with bone metastasis were treated with radiation, and the patients with brain metastasis underwent stereotaxic radiosurgery. The patients with either adrenal metastasis, axillary lymph node metastasis, or contralateral lung metastasis underwent surgical resection. Among the patients with distant metastasis, the 5-year survival rate was 30.1 %. There were 17 patients with pleural dissemination. The 5-year survival rate in these patients was 25.3%. The overall 5-year survival rate after surgery in the patients with stage IV disease was 26.8%. CONCLUSION Selected patients who can undergo surgical resection for the primary tumor and effective local therapy for metastatic lesions still have a chance to obtain long-term survival. Surgical treatment for NSCLC with oligometastatic disease can be considered as one arm of multidisciplinary treatment.

Identification of HLA‐A24 restricted shared antigen recognized by autologous cytotoxic T lymphocytes from a patient with large cell carcinoma of the lung

The aim of the present study was to elucidate the tumor‐specific cellular immunological responses occurring in a patient with large cell carcinoma of the lung who had no evidence of recurrence following surgical resections of both a primary lung lesion and a metastatic adrenal lesion. We analyzed an autologous tumor‐specific cytotoxic T lymphocytes (CTL clone F2b), which were HLA‐A*2402 restricted from regional lymph node lymphocytes. The F2b possessed T cell receptor (TCR) using the Vα5 and Vβ7 gene segment. The existence of precursor CTL (pCTL) against autologous tumor cells (A904L) was analyzed using CTL clone‐specific PCR. Lymphocytes with the same TCR as F2b were detected in the primary tumor tissue, regional lymph node and the peripheral blood collected from the patient 3 years after the operation. Using the F2b, we identified a cDNA clone encoding the tumor antigen using cDNA expression cloning method. The gene was found to encode splicing variant of the Tara gene. Finally, we identified the 9‐mer Ag peptide, using constructions of mini‐genes. The F2b recognized 3 out of 7 HLA‐A24 positive allogeneic tumor cell lines and in 1 out of 7 HLA‐A24 negative allogeneic tumor cell lines when transfected with HLA‐A24. This peptide is therefore considered to be potentially useful for performing specific immunotherapy in a significant proportion of lung cancer patients bearing HLA‐A24.

Serum Level of Osteopontin as a Prognostic Factor in Patients Who Underwent Surgical Resection for Non-Small-Cell Lung Cancer

BACKGROUND OPN is a multifunctional glycophosphoprotein originally described as a secreted protein from malignant epithelial cells. This study focused on the clinical significance of preoperative serum level of OPN in NSCLC patients who underwent a complete resection. PATIENTS AND METHODS The serum OPN level was assayed in 244 patients who underwent a complete resection of NSCLC by commercially available sandwich enzyme-linked immunosorbent assay kits. The patients were considered as a higher group, when the serum OPN levels exceeded 81.3 ng/mL. RESULTS The patients included 166 male and 78 female subjects. The histologic types included 172 adenocarcinomas, 49 squamous cell carcinomas, and 23 other types of carcinoma. The serum level of OPN in male patients (92.6 ng/mL) was significantly higher than that of female patients (76.9 ng/mL). The OPN level of squamous cell carcinoma was significantly higher than that of adenocarcinoma. The OPN level was significantly elevated in patients with the pleural invasion or microvascular invasion than those without the invasion. The 5-year survival rate after surgery in the lower OPN group (82.0%) was a significant favorable prognosis than that in the higher OPN group (63.7%) (P < .0001). The 5-year survival rates in the lower OPN group at stage I NSCLC (88.1%) was significantly better than that in the higher OPN group (80.5%) (P = .0321). CONCLUSION The preoperative serum OPN level was a useful predictor of an unfavorable prognosis, and it was found to be an independent prognostic determinant of outcome in patients who underwent surgery for NSCLC.

Correlation Between HLA Alleles and EGFR Mutation in Japanese Patients with Adenocarcinoma of the Lung

Introduction: The identification of activating mutations in the epidermal growth factor receptor (EGFR) gene is one of the most intriguing recent discoveries in the field of lung cancer research, and they are more commonly found in adenocarcinoma occurring in females, never/light smokers, and East Asian patients. Why such certain patients are susceptible to the development of EGFR-mutant tumors is currently unknown. Methods: This study evaluated the medical records of 437 patients with adenocarcinoma of the lung who underwent a surgical resection. The genetic status of the EGFR gene was investigated by polymerase chain reaction-based analyses. The serological typing of histocompatibility leukocyte antigen (HLA) class I was performed using a microcytotoxicity test of lymphocytes or polymerase chain reaction-sequence-specific oligonucleotides, and the correlation between the EGFR mutation and HLA alleles was analyzed. Results: An EGFR mutation was found more frequently in females and never/former smokers than their counterpart. In females, the incidences of EGFR mutation were 61.0% and 41.7% in HLA-A2 (+) and A2 (−) patients with adenocarcinoma of the lung, respectively (p = 0.008). The EGFR mutation was found more frequently in female patients with HLA-A2. However, no significant correlation was identified between the frequencies of other HLA alleles and EGFR mutations in the same patients group. Conclusions: EGFR mutations are associated with HLA-A2 in female patients with adenocarcinoma of the lung. Further research was needed to elucidate the other relevant factors in the histogenesis of lung cancer with an EGFR mutation.

Identification of tumor associated antigens recognized by IgG from tumor-infiltrating B cells of lung cancer: correlation between Ab titer of the patient's sera and the clinical course.

We previously demonstrated that TIB recognize tumor antigens and produce antibodies against them. In the present study, we identified three tumor antigens recognized by TIB in lung cancer and evaluated whether changes in the antibody titer against these antigens correlated with the patients clinical course. A lung cancer cell line, G603L, was established from a primary lung tumor of a patient, G603. Seven months later, adrenal metastasis was detected and surgically resected. The latter tumor was mildly infiltrated with B cells and xenotransplanted into SCID mice to obtain human IgG. A cDNA library was constructed from G603L and SEREX was carried out using TIB‐derived IgG. The seroreactive clones were sequenced and one of these antigens was revealed to be MAGE‐B2 whereas the others were novel antigens. In the immunomonitoring of the patients sera, high antibody titer against MAGE‐B2 was observed before operation and the titer decreased after resection of the primary tumor. It was elevated again at the time of adrenal metastasis, but then decreased after resection. The change in antibody titer against the second antigen was similar to MAGE‐B2, and the antibody titer against the third antigen was low before the primary operation but increased at the time of recurrence. Our results suggest that TIB recognized tumor antigens and the antibody titers against these antigens were changed along with the patients clinical course. Therefore, these antibodies could be used as tumor markers for the patient. (Cancer Sci 2005; 96: 882–888)