Differences in sensitivity to tumor-specific CTLs between primary and metastatic esophageal cancer cell lines derived from the same patient.

PURPOSE: MHC antigens and adhesion molecules, such as the intracellular adhesion molecule (ICAM-I), play an important role in cellular immune response. We examined the expression patterns of these molecules in both primary and metastatic esophageal carcinoma cells from the same patient and evaluated the cellular immune responses against these cells. MATERIALS AND METHODS: In the esophageal cancer patient (H122), tumor cell lines were established from primary and subcutaneous metastatic lesions. We compared the expression of cell surface molecules on the metastatic tumor cell line (H122SC) with that on the primary tumor cell line (H122ESO) using flow cytometry. Moreover, we analyzed the differences in cellular immune responses against these cell lines, which expressed similar levels of the Tara antigen, using the Tara antigen-specific CTL clone. RESULTS: H122SC ICAM-1 expression was significantly lower in H122ESO, and the Tara antigen-specific CTL clone produced lower levels of TNF in response to H122SC than H122ESO. ICAM-1 transfection into the H122SC rendered these cells as sensitive to the CTL clone as the H122ESO. CONCLUSION: The metastatic tumor cells displayed lower regulated ICAM-1 expression levels and were less sensitive to specific CTLs. ICAM-1 downregulation may be one mechanism by which tumor cells escape immunologic surveillance.

Identification of a tumour associated antigen in lung cancer patients with asbestos exposure.

BACKGROUND: This study analysed the humoral immune response in asbestos exposed lung cancer patients to identify new surrogate markers of the carcinogenic risk in populations exposed to asbestos. METHODS AND RESULTS: A serological analysis identified five distinct antigens reactive with IgG derived from a lung cancer patient with high asbestos exposure. In one of the isolated antigens, quantitative RT-PCR indicated that annexin A2 (AnxA2) was overexpressed in lung cancer tissues and normal lung from patients with high asbestos exposure. Antibody against AnxA2 was detected in 9/15 (60%) of lung cancer patients with high asbestos exposure; however, in only 1/12 (8%) of lung cancer patients with low asbestos exposure. AnxA2 was also overexpressed in malignant mesothelioma cells, and the antibody was also positive in 8/15 (53%) of patients with malignant mesothelioma. CONCLUSION: The antibody titer against AnxA2 may be a potentially useful new diagnostic surrogate marker for asbestos-related lung cancer and malignant mesothelioma.

Mediastinal metastasis from a fibrolamellar hepatocellular carcinoma: Report of a case.

Fibrolamellar hepatocellular carcinoma (FL-HCC) is an uncommon clinicopathological variant of hepatocellular carcinoma (HCC). The etiology of FL-HCC is unknown, but FL-HCC is not associated with hepatic viruses or alcohol. Hepatocellular carcinoma usually occurs in cases of chronic hepatitis or cirrhosis, whereas FL-HCC predominantly occurs in a normal liver and in younger adults. Fibrolamellar HCC shows relatively slow growth, and late recurrence is common. Repeated resections for recurrences should be considered not only because there is a lack of other effective treatment options but also because FL-HCC shows a relatively better prognosis after a resection in comparison to common HCC. This report presents a case of a rare mediastinal metastasis from FL-HCC in a patient who had undergone a previous resection for retroperitoneal metastasis after the initial hepatic operation. This is the second report of the same case. This patient also had a mediastinal neurogenic tumor, and these mediastinal tumors were concurrently resected.

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).

Clinicopathological characteristics of invasive lobular carcinoma of the breast.

BACKGROUND: The clinical features of invasive lobular carcinoma (ILC) of the breast have remained unclear due to the rarity of such cases. This study investigated the clinical and pathological features of ILC. METHODS: The medical records of 413 patients with invasive breast cancer who underwent surgery in our department were reviewed. These cases included 13 patients with ILC (3.1%). RESULTS: The age of the patients ranged from 36 to 77 years old (mean, 56). The tumour size was evaluated as T1 in five patients, T2-4 in 8. The lymph node metastasis was diagnosed as negative in six patients, positive in six. In this patient population, 11 (85%) and nine (69%) patients were positive for oestrogen and progesterone receptors, respectively. The 5-year survival rate was 76.2%, which was not significantly different from other types of invasive breast cancer. Extensive intraperitoneal metastasis was observed in two of the three patients. Two patients had bilateral carcinoma and one patient had a multicentric lesion in the ipsilateral breast. CONCLUSION: Multicentric development of breast cancer and intraperitoneal metastasis were one of clinical characteristics of ILC. The prognosis of ILC was not significantly different from other types of invasive breast carcinoma.

Malignant mesothelioma-associated antigens recognized by tumor-infiltrating B cells and the clinical significance of the antibody titers.

Malignant pleural mesothelioma (MPM) is difficult to diagnose at an early stage. The present study attempted to obtain a tumor-specific antibody against MPM derived from tumor-infiltrating B lymphocytes in MPM by using a xenotransplanted severe combined immunodeficiency (SCID) mouse model, and to identify the antigens recognized by the antibodies. Among the antigen-antibody relationships, the clinical usefulness of antibody titers in the sera was evaluated from the viewpoint of diagnosis of MPM and monitoring of therapeutic effects. Tumor tissue specimens from two patients with MPM were engrafted subcutaneously in SCID mice and blood samples were obtained and pooled every 2 weeks after xenotransplantation until 14 weeks when the mice were killed. A cDNA library was constructed from the mRNA of a MPM cell line (K921MSO). Immunoscreening of the libraries was carried out by serological identification of antigens by a recombinant expression cloning method (SEREX) and four antigens were identified as MPM-associated antigens. Among them, antibody titers against two antigens, Gene-X and thrombospondin-2 (THBS-2), were analyzed by phage plaque assay as the first step. ELISA systems correlated with the phage plaque assay to detect antibody titers against the two antigens were constructed using 20-mer peptides of the antigen-coding genes. The cut-off value was decided by the average and standard deviation of normal healthy persons. Antibody against Gene-X was detected in 10 out of 18 (55.6%) mesothelioma patients and antibody against THBS-2 was detected in 16 out of 18 (88.9%) mesothelioma patients. No patients with lung cancer regardless of asbestos exposure exhibited positive antibody titer against the two antigens. Furthermore, the serum antibody titers decreased after surgical treatment of MPM and increased after recurrence of the disease. The titers of the antibodies against Gene-X and THBS-2 could be used as tumor markers for the diagnosis and follow up of patients with MPM.

Prospective phase II study of gefitinib in non-small cell lung cancer with epidermal growth factor receptor gene mutations.

BACKGROUND: This study prospectively assessed the efficacy of gefitinib and the survival benefit for non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations. METHOD: Patients with either recurrent disease after undergoing surgery or advanced NSCLC disease (IIIB or IV) which demonstrated EGFR mutations were eligible for this study. EGFR mutations in exons 19-21 were examined. The patients with EGFR mutations were enrolled in this study after obtaining their informed consent a second time, and they were thereafter treated with gefitinib. RESULTS: EGFR mutations were detected in 20 of 48 patients with NSCLC, and 19 patients were enrolled onto this study and treated with gefitinib. Seven patients had an exon 19 deletion, 10 had L858R, 1 had both, and 1 had an exon 19 deletion and G796A. The overall response rate was 63.2%, and the disease control rate was 89.5%. In patients with an exon 19 del and L858R, the response rates were 71.4% and 60.0%, respectively. The median progression-free survival time was 7.1 months, and the median survival time was 20.0 months. No life-threatening toxicity was observed. Four of five acquired resistant tumors showed an acquired T790M mutation. CONCLUSIONS: EGFR mutations in exons 19 or 21 are considered to be a good predictor of the efficacy of gefitinib.

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.

Significance of Smoking as a Postoperative Prognostic Factor in Patients with Non-small Cell Lung Cancer.

INTRODUCTION: In this study, we investigated the influence of smoking on the postoperative prognosis in patients with non-small cell lung cancer. METHODS: The subjects consisted of 770 patients who underwent a resection of lung cancer in our department between 1994 and 2005. We compared the clinico-pathological findings between the smoking and never-smoking groups. The pack-year index (PYI) was used as a smoking index. RESULTS: The smoking group consisted of 569 patients (74%), and the never-smoking group consisted of 201 patients (26%). The smokers were composed of 492 men and 77 women. Among the adenocarcinoma patients, there were 293 (61%) smokers and 185 (39%) never-smokers. The patients with squamous cell carcinoma included 204 (95%) smokers and 10 (5%) never-smokers. The proportion of patients with stage IA disease was significantly higher in the never-smokers than that of the smokers. The 5-year survival rate after surgery was 66% in the never-smoking group; however, the rates were 56% in patients with a PYI more than or equal to 20, and 55% in those with PYI more than 20. Seventy-nine (13.9%) patients in the smoking group and seven (3.5%) patients in the never-smoking group died of other diseases, with a significant difference (p < 0.01). Of these patients, 44 (56%) and 13 (16%) in the smoking group died of respiratory and cardiovascular disorders, respectively. In our series, excluding those who died of other diseases, there were no significant differences in the postoperative prognosis. CONCLUSIONS: In the smoking group, the prognosis was poorer than that in the never-smoking group. The higher proportion of early stage disease (stage IA) and female gender were major causes of the better prognosis of the never-smokers. Nevertheless, the high pulmonary/cardiovascular complication-related mortality was another cause of the poor prognosis of the smokers with lung cancer.

Surgical treatment for chest wall invasion due to the local recurrence of breast cancer.

OBJECTIVE: In this study, we investigated the surgical results for chest wall invasion of local recurrence of breast cancer. PATIENTS AND METHODS: We reviewed eight patients who underwent a chest wall resection for local recurrence of breast cancer in our department between 1986 and 2004. RESULTS: All of the patients had local recurrence without any distant metastasis. All of them had skin ulcers with blood oozing. The operation procedures were Bt + Ax + Ic + Mj + Mn (Halsted mastectomy) in four patients, Bt + Ax + Ic + Mn (Patey procedure) in two patients, Bt + Ax + Ic (muscle-preserving mastectomy) in one patient, and Bt + Ax (Auchincloss procedure) in one patient. The intervals from the primary operation ranged from 14 months to 20 years. The maximum and minimum areas of the chest wall defect were 18 x 16 cm and 4.5 x 3.5 cm, respectively. Reconstruction of the chest wall was performed using a flap of the rectus abdominis muscle with polypropylene (Marlex) mesh in four patients, a flap of the rectus abdominis muscle combined with sandwich prosthesis of polypropylene mesh and methylmethacrylate in one patient, a flap of latissimus dorsi muscle in one patient, polypropylene mesh with pectoralis major muscle in one patient, and by direct closure in one patient. A survival of more than 3 years was achieved in seven patients and only one patient died 1 year and 2 months after the chest wall resection. CONCLUSION: In patients with the chest wall recurrence of breast cancer without distant metastasis, a surgical resection of the chest wall may be effective both for relieving pain and for control of the local hemorrhage. Seven out of the eight patients survived more than 3 years, suggesting that this surgical treatment could facilitate home health care and maintain a good quality of life for patients with breast cancer.

Postoperative prognosis in patients with non-small cell lung cancer according to the method of initial detection.

INTRODUCTION: In this study, we investigated the difference in the surgical results of non-small cell lung cancer according to the method of initial detection. METHODS: We reviewed the medical records of 796 patients who underwent pulmonary resection for non-small cell lung cancer between 1994 and 2005. The subjects consisted of 171 patients whose cancer was detected by a medical checkup or mass health screening (group I), 316 patients who were under evaluation for other diseases or with symptoms related to other diseases (group II), and 309 patients with lung cancer-related symptoms (group III). The mean ages of the three groups were 63.2, 69.7, and 68.2 years old, respectively, with group I being significantly younger than the other groups. The proportion of women in the symptomatic group was significantly lower than that of men. RESULTS: Pathologic stage I lung cancer was found in 112 (65.5%), 209 (65.2%), and 110 (35.6%) patients in groups I, II, and III, respectively. In comparison with stage II-IV cancer, stage I cancer was diagnosed more frequently in group I. According to the histologic type, adenocarcinoma was found in 132 patients (77.2%) in group I. However, squamous cell carcinoma was detected in only 27 patients (15.8%) in group I. The overall 5-year survival rates were 71.9%, 60.2%, and 48.0% in groups I, II, and III, respectively. Groups I and II had significantly better prognoses than group III. CONCLUSION: Groups I and II had favorable prognoses, and the presence of symptoms related to lung cancer was a significantly unfavorable prognostic factor independent of all other factors.

Antitumor effect of antibody against a SEREX-defined antigen (UOEH-LC-1) on lung cancer xenotransplanted into severe combined immunodeficiency mice.

We previously reported the humoral immune response of tumor-infiltrating B lymphocytes in a lung cancer patient and 22 genes coding tumor-associated antigens identified using the serological identification of antigens by recombinant expression cloning method. In this study, we investigated one of these genes, designated University of Occupational and Environmental Health-Lung cancer antigen-1 (UOEH-LC-1), which has an extracellular domain. Quantitative reverse transcription-PCR revealed that UOEH-LC-1 was expressed ubiquitously in the normal tissues tested. However, it was overexpressed in 5 of 11 (45.5%) lung cancer cell lines and also in 9 of 15 (60%) lung cancer tissues compared with the paired normal lung tissues. A sequence analysis revealed that UOEH-LC-1 has a transmembrane domain. Flow cytometry analysis using a polyclonal antibody against UOEH-LC-1 revealed positive staining on lung cancer cell lines that were positive for expression of mRNA of UOEH-LC-1. Phage plaque assay showed the specific reactivity of anti-UOEH-LC-1 antibody against UOEH-LC-1 protein derived from the antigen encoding phage. By immunohistochemical staining with the anti-UOEH-LC-1 antibody, 7 of 28 (25.0%) lung cancer specimens showed positive staining on the cell surface. The administration of anti-UOEH-LC-1 antibody inhibited the growth of the UOEH-LC-1-positive tumors that were xenotransplanted into severe combined immunodeficiency mice. Complement-dependent cytotoxicity was one of the mechanisms to suppress the tumor growth. These results suggest that the antibody against UOEH-LC-1 therefore seems to have a promising therapeutic potential as a treatment for lung cancer.

Lack and restoration of sensitivity of lung cancer cells to cellular attack with special reference to expression of human leukocyte antigen class I and/or major histocompatibility complex class I chain related molecules A/B.

Both cytotoxic T lymphocytes (CTL) and natural killer (NK) cells may play major roles in the host defense against cancer. However, their relationship against the same tumor remains to be elucidated. Among 26 human lung cancer cell lines established in our laboratory, 10 (38%) exhibited human leukocyte antigen (HLA)-class I haplotype loss and three (12%) lost HLA-class I expression totally by flow cytometry analysis. The two cell lines (E522L and C831L) that lost their expression of HLA-class I in vitro and in vivo were applied for further evaluations. Genetic abnormalities of beta2-microglobulin gene were observed in both E522L (loss of mRNA) and C831L (point mutation). Transduction of the wild-type beta2-microglobulin gene rendered them positive for HLA-class I expression. The CTL were induced from autologous peripheral blood mononuclear cells or regional lymph node lymphocytes by stimulation with wild-type beta2-microglobulin transduced-E522L or -C831L, and they showed tumor-specific cytotoxicity against wild-type beta2-microglobulin-transductant, but not parental cells. In NK cell cytotoxicity, E522L showed high sensitivity to NK cells; however, C831L showed resistance despite loss of HLA-class I expression. E522L expressed MHC class I chain related molecules A/B, but C831L did not. The transduction of the MHC class I chain related molecule A gene from E522L rendered C831L positive for expression and sensitive to NK cell cytotoxicity. Reconstruction of HLA-class I and MHC class I chain related molecules A expression could abrogate evasion from cellular attack by CTL and NK cells, and it may lead to a breakthrough in the development of cancer immunotherapy.

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)-1alpha, IL-1beta, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor (TNF)-alpha, granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor, transforming growth factor (TGF)-beta 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-beta 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-alpha was produced at all by any tumor cell line. TGF-beta 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-beta (<100 pg/mL). These findings suggested that TGF-beta 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.

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 Valpha5 and Vbeta7 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.

Antigens recognized by IgG derived from tumor-infiltrating B lymphocytes in human lung cancer.

BACKGROUND: Lung cancer tissues are often infiltrated by B lymphocytes, but it is not clear whether these infiltrations represent tumor-specific immune response or a nonspecific reaction. MATERIALS AND METHODS: The serological analysis of recombinant cDNA expression libraries (SEREX) were previously modified using a severe combined immunodeficient (SCID) mice model engrafted with fresh human lung cancer. Here, a panel of antigens recognized by tumor-infiltrating B lymphocytes (TIB) in human lung cancer were characterized. RESULTS: The modified SEREX analysis identified 22 distinct antigens in a large cell carcinoma of the lung. Sequence analysis and real time-PCR analysis showed that 55% of isolated antigens were overexpressed in tumor tissues and 9% had mutation. CONCLUSION: The results of this study indicate that the humoral immune response of TIB in lung cancer patients can be detected in the xenotransplanted SCID mouse model and our modification shows high sensitivity and specificity for identification of tumor antigens.

Effect of IgG produced by tumor-infiltrating B lymphocytes on lung tumor growth.

BACKGROUND: Tumor-infiltrating B lymphocytes (TIB) are often observed in lung cancer. The role of TIB in tumor growth has not been well investigated. MATERIALS AND METHODS: Forty-four surgically-resected human lung cancer tissues were xenotransplanted into SCID mice. Their blood was collected and the volume of the transplanted tumors was measured regularly. The correlations between the IgG titer in the sera and the growth of the transplanted tumors according to the clinicopathological variables were examined. RESULTS: Human IgG production from TIB was observed in the all xenotransplanted mice. Twenty-seven out of the 44 tumors regressed gradually. The average serum human IgG level of the tumor regressors (n = 10) was significantly higher than that of the progressors (n = 9) in squamous cell carcinoma (p = 0.02), while there was no significant difference in the other histological groups. CONCLUSION: IgG produced by TIB might play a crucial role in preventing tumor growth in squamous cell carcinoma.

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.

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 patient's 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 sero-reactive clones were sequenced and one of these antigens was revealed to be MAGE-B2 whereas the others were novel antigens. In the immuno-monitoring of the patient's 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 patient's clinical course. Therefore, these antibodies could be used as tumor markers for the patient.

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.