Tetsuya So

Haplotype loss of HLA class I antigen as an escape mechanism from immune attack in lung cancer.

One of tumor escape mechanisms from the hosts immunosurveillance system (i.e., a haplotype loss of HLA class I antigens) has been detected in various tumor cells. We hypothesize that the majority of tumor cells with normal HLA class I expression were attacked and eradicated by CTLs, and only a minority with an abnormal expression of HLA class I antigens could escape the hosts immunosurveillance system. Using HLA class I-transfected tumor variants as stimulators in A904L lung cancer cell line, which has a haplotype loss of HLA class I antigens, both the transfected HLA-A26 and HLA-B39-restricted CTL lines were induced from autologous lymphocytes. However, only one HLA-B39-restricted CTL clone (CTL G3b) was established, and it was then used to identify the antigen. SGT1B [suppressor of G2 allele of SKP1 (SGT1), suppressor of kinetochore protein (SKP1)] was identified as the antigen recognized by CTL G3b. Further experiments using 13 subclones from a primary culture of A904L were found to confirm our above-mentioned hypothesis. Tumor cells with a normal HLA class I expression may thus be killed by CTL at an early stage of carcinogenesis, and only tumor cells with a haplotype loss of HLA class I antigens can escape an immune attack and develop into clinical cancer.

Clinical significance of cancer/testis antigens expression in patients with non-small cell lung cancer

Cancer/testis antigens (CT antigens) are thought to be suitable targets for antigen-specific immunotherapy, because of the cancer-specific expression except for the testis among various normal tissues and no-expression of HLA class I in the testis. In the present study, the expressions of CT antigens (MAGE-A3, MAGE-A4, NY-ESO-1 and KK-LC-1) in non-small cell lung cancer (NSCLC) were analyzed by RT-PCR. The subjects were 239 patients with NSCLC who underwent surgery from 2001 to 2005 in our department. The expression rates of MAGE-A3, MAGE-A4, NY-ESO-1 and KK-LC-1 were 23.8%, 20.1%, 10.5% and 32.6% in patients with NSCLC, respectively. MAGE-A4 was expressed more frequently in male (25.3%) than in female (10.6%) (p<0.01). The positive proportion of MAGE-A4 was higher in stages II-IV (30.6%) than in stage I (12.8%) (p<0.01). Both of MAGE-A3 and MAGE-A4 were expressed more frequently in squamous cell carcinoma than in adenocarcinoma (p<0.01). Such tendency was not observed among NY-ESO-1 and KK-LC-1 expression. KK-LC-1 was expressed in 32.1% of patients with adenocarcinoma and in 36.5% of patients with squamous cell carcinoma. Patients with positive MAGE-A4 expression showed significantly poorer overall survival than those without MAGE-A4 expression (p=0.013), and such effect on survival was also observed, when the analysis was limited to patients at stage I (p=0.0037). Expression of MAGE-A3, NY-ESO-1 or KK-LC-1 did not affect survival of patients with NSCLC significantly, however, expression of at least one of such CT antigens negatively affect survival of patients with NSCLC (p=0.045).

Simultaneous Cellular and Humoral Immune Response against Mutated p53 in a Patient with Lung Cancer

We recently identified several Ags recognized by tumor-infiltrating B lymphocyte-derived Ab using SCID mice and a xenografted non-small cell lung cancer system. One of these identified Ags was mutated p53 with a point mutation resulting in the alteration of codon 158 from Arg to Leu. The aim of this study was to ascertain whether cellular immunity against mutated p53 exists in the same patient together with humoral immunity. Two different nona peptides (mutated p53150 and p53155 peptides), including a mutated amino acid derived from p53, were synthesized according to the binding motif of HLA class I of the established cancer cell line A904L from the patient. Mediastinal lymph node lymphocytes of the patient were stimulated weekly with the peptides. The mutated p53155 peptide-stimulated lymphocytes showed specific cytotoxicity against both autologous EBV-transformed B cells pulsed with mutated p53155 peptide and A904L. The mutated p53155 peptide-specific CTL clone in an HLA-Cw*0702 restriction was established and analyzed for its TCR usage. Clonotypic PCR using CDR3-specific primers was applied to the tumor tissue containing the tumor-infiltrating lymphocytes. The specific amplification of PCR was found in the tumor tissue. These results demonstrated that not only B lymphocytes producing specific Ab against the p53 protein, but also CTL against mutated p53, expressed in autologous lung cancer cells exist in the tumor tissue. This approach may allow us to better understand the mechanisms of T and B cell immunity against the same tumor Ag in cancer patients.

Association between lymphangiogenesis-/micrometastasis- and adhesion-related molecules in resected stage I NSCLC

BACKGROUND The purpose of this study was to clarify the role and clinical significance of lymphangiogenesis/micrometastases and adhesion molecules in resected stage I non-small cell lung cancer (NSCLC). METHODS Immunohistochemical (IHC) staining was used to analyze the protein expression of vascular endothelial growth factor-C (VEGF-C), VEGF, E-cadherin, alpha-catenin, beta-catenin, and gamma-catenin in paraffin-embedded tumor samples from 117 well-characterized stage I NSCLC patients and to compare the protein expression, clinical variables and survival outcome. As a micrometastatic parameter in lymph nodes (LNs), cytokeratin (CK) staining was performed. RESULTS The positive expression of VEGF-C and VEGF were detected in 54 (48.7%) and 86 (73.5%), respectively. We identified micrometastatic tumor cells in pathological N0 LNs in 34 (29.1%) of 117 patients. E-cadherin, alpha-catenin, beta-catenin, and gamma-catenin were identified in 70 (59.8%), 41 (35.0%), 83 (70.9%), and 61 (52.1%) specimens, respectively. The VEGF-C expression was found more frequently in squamous cell carcinoma (SQ) and in the tumors with negative expression of beta-catenin than counter features. The VEGF expression was found more frequently in the tumors with a negative expression of E-cadherin. Micrometastasis was found more frequently in a pathological T2 status and in the tumors with a negative expression of alpha-catenin. Beta-catenin and gamma-catenin expressions were found less and more frequently in SQ, respectively. A univariate and multivariate survival analysis demonstrated that old age, pathological T2 status, and micrometastasis were independently associated with an increased risk of poor survival in the patients who underwent a surgical resection of stage I NSCLC. CONCLUSIONS Complicated relationships exist between lymphangiogenesis/micrometastases and adhesion molecules with a specific histology. The detection of lymph nodal micrometastasis by CK may therefore be a useful marker for predicting a poor prognosis in patients who undergo a complete resection of stage I NSCLC.

Time Trends of Surgical Outcome in Patients with Non-small Cell Lung Cancer

Purpose: A surgical resection is a potentially curative treatment for non-small cell lung cancer (NSCLC). This study investigated the time trends of the surgical outcome in patients with NSCLC. Methods: This study clinicopathologically evaluated 1487 patients who had undergone a resection for NSCLC between 1979 and 2008 during the five periods of 1979–1988, 1989–1993, 1994–1998, 1999–2003, and 2004–2008. Results: The number of patients who underwent a resection during the five respective periods increased: 167, 261, 248, 382, and 429. The percentage of pathologic stage IA lung cancers was 16.2, 21.5, 23.0, 38.5, and 52.0% in each period, respectively, and it has risen rapidly in recent years. The percentage of adenocarcinoma has also progressively increased during each period: 49.1, 52.1, 54.7, 62.8, and 69.7%, respectively. The diameter of the tumors resected during each period was 36, 37, 38, 33, and 26 mm, respectively, showing that the tumor tended to be diagnosed at an increasingly smaller size. The postoperative 5-year survival rates during the five periods improved markedly: 34.1, 44.0, 44.9, 65.4, and 76.5%, respectively. Patients with pathologic stage IA lung cancer also exhibited increasingly higher 5-year survival rates during the five periods: 70.0, 71.2, 80.4, 89.2, and 88.7%, respectively. Conclusion: The prognosis of NSCLC patients has remarkably improved in recent years. The increase in the number of patients with adenocarcinoma in the early stage is thought to have strongly contributed to the favorable results. Thus, early diagnosis remains a key factor for improving the survival of lung cancer patients after surgical treatment.

Identification of a New Cancer/Germline Gene, KK-LC-1, Encoding an Antigen Recognized by Autologous CTL Induced on Human Lung Adenocarcinoma

The purpose of our present study is to identify a tumor-specific antigen capable of inducing a specific cellular immune response in lung cancer patients. We established a lung adenocarcinoma cell line, designated as F1121L, and induced tumor-specific CTL clone H1 from regional lymph node lymphocytes of patient F1121. CTL clone H1 lysed autologous tumor cells in an HLA-B*1507-restricted manner, but not autologous EBV-B, phytohemagglutinin-blast cells, and K562. The CTL clone also recognized allogeneic HLA-B*1501- or 1507-positive lung cancer cell lines in the HLA-restricted manner. Using the CTL clone, we identified an antigen-coding gene by cDNA expression cloning technique. The gene consisted of 556 bp, including an open reading frame consisted of 113 amino acids, designated as Kita-kyushu lung cancer antigen 1 (KK-LC-1). A 9-mer peptide (KK-LC-1(76-84); RQKRILVNL) was identified as an epitope peptide. The genomic DNA of this antigen was located in chromosome Xq22. A reverse transcription-PCR analysis revealed that the mRNA of this gene was only expressed in the testis among normal tissues. It was expressed in 9 of 18 (50%) allogeneic non-small-cell lung cancer cell lines and in 40 of 100 (40%) non-small-cell lung cancer tissues. We thus identified a new tumor antigen-coding gene categorized as a cancer/germline gene by an autologous lung cancer and CTL system. The new cancer/germline gene was located in Xq22, which is apparently different from the locations of previously reported cancer/germline genes.

Limited pulmonary resection for peripheral small-sized adenocarcinoma of the lung

BACKGROUND It was recently reported that a limited pulmonary resection (segmentectomy or wedge resection) was not inferior to a lobectomy in the management of peripheral small-sized adenocarcinoma (tumor ≦ 20 mm) of the lung. METHODS We retrospectively analyzed patients undergoing a lobectomy (n = 114) and a limited resection (n = 35) for peripheral small-sized adenocarcinoma of the lung during a 7-year period from April 2001 to March 2008. Our criteria for the limited resection of lung cancer were as follows: (1) adenocarcinoma of 10 mm or less in diameter and (2) adenocarcinoma of 11-20 mm in diameter, in which the ratio of the ground glass opacity is 50% or more, without pleural indentation on computed tomography. Additionally, the frozen sections of the tumors were intraoperatively diagnosed as Noguchi type A or B. The survival and clinical outcomes were analyzed. RESULTS The 5-year survival rates of the lobectomy group and limited resection groups were 89.2% and 100%, respectively. No recurrence was seen in the limited resection group. CONCLUSIONS Our results suggest that our criteria for limited resection were adequate for the management of small-sized adenocarcinoma of the lung.

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.

Gender Difference as a Prognostic Factor in Patients Undergoing Resection of Non-Small Cell Lung Cancer

PurposeWe studied the effects of gender difference on the incidence of lung cancer and its mortality rate, which is a subject of much discussion.MethodsWe examined gender difference in the clinical features of 491 men and 222 women who underwent resection of primary non-small cell lung cancer (NSCLC) between 1994 and 2004.ResultsThe histological types of cancer were adenocarcinoma in 249 (51%) of the men and 182 (82%) of the women, and squamous cell carcinoma in 182 (37%) of the men and 27 (12%) of the women. The incidence of adenocarcinoma was significantly higher in the women. The proportion of stage IA disease was significantly higher in the women than in the men (45% vs 29%, respectively). The 5-year overall survival rates were 50% in the men and 63% in the women. In a multivariate analysis, gender difference was an independent prognostic factor; however, when death as a result of unrelated disease was excluded, there was no significant difference in prognosis.ConclusionAlthough the higher incidences of adenocarcinoma and stage IA cancer contributed to the good results of surgery in women, the low incidence of death attributed to diseases other than lung cancer was a major reason for their better prognosis.

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.