Development of adoptive immunotherapy with KK-LC-1-specific TCR-transduced γδT cells against lung cancer cells.

The present study analyzed the antitumor effect of γδT cells transduced with the TCR of cancer-specific CTLs to establish forceful cancer-specific adoptive immunotherapy. We cloned the TCRαß genes from CTLs showing HLA-B15 restricted recognition of Kita-Kyushu lung cancer antigen-1 (KK-LC-1), a cancer/germline gene antigen, identified in a lung adenocarcinoma case (F1121). The TCRαß and CD8 genes were transduced into γδT cells induced from PBLs of healthy volunteers stimulated with zoledronate and IL-2. The KK-LC-1-specific TCRαß-CD8 γδT cells showed cytotoxic activity against the KK-LC-1 positive lung cancer cell line F1121L and produced IFN-γ against F1121L and KK-LC-1 peptide-pulsed F1121 EBV-B cells. These responses were blocked by HLA class I and HLA-B/C antibodies. An in vivo assay using NOD/SCID mice with xenotransplantation of human lung cancer cells was performed, and the TCRαß-CD8 transduced γδT cells (TCRαß-CD8 γδT cells) were intravenously injected. Growth inhibition of KK-LC-1+ , HLA-B15+ lung cancer cells was confirmed in mice with injection of the TCRαß-CD8 γδT cells from 1 wk after xenotransplantation of cancer cells but not in those treated 2 wk after xenotransplantation. The resected specimens of the tumor, 2 wk after xenotransplantation, highly expressed FasL but not programmed death ligand-1 (PD-L1) by immunohistochemical staining. FasL highly expressed cancer cells xenotransplanted 2 wk ago were resistant to TCRαß-CD8 γδT cells injection. These results suggested that apoptosis of Fas-positive TCRαß-CD8 γδT cells may be induced by a Fas-mediated signal after interacting with FasL-positive cancer cells.

Prognostic implications of human leukocyte antigen class I expression in patients who underwent surgical resection for non-small-cell lung cancer.

BACKGROUND: The purpose of the present study was to clarify the prognostic significance of human leukocyte antigen (HLA) class I expression in patients with non-small-cell lung cancer who underwent complete surgical resection. PATIENTS AND METHODS: The expression of HLA class I molecules was evaluated in 403 resected NSCLC specimens using immunohistochemistry. The results were scored as the percentage of stained tumor cells and were categorized into three groups: 0%-24% (decreased), 25%-79% (heterogeneous), and 80% or more (normal). RESULTS: The expression of HLA class I was evaluated in 124 tumors in the normal expression group, 181 tumors in the heterogeneous expression group, and 98 tumors in the decreased expression group. The 5-year survival rate of all patients after surgery according to the HLA class I expression in the normal, heterogeneous, and decreased groups was 76.6%, 65.9%, and 76.1%, respectively. The prognosis was significantly better in the normal expression group than in the heterogeneous group. Normal HLA class I expression also correlated with favorable survival in patients with stage I disease. CONCLUSIONS: The normal expression of HLA class I was associated with a favorable prognosis compared with the heterogeneous expression group, but no significant difference was observed between the normal expression and decreased expression groups.

Clinical significance of human leukocyte antigen loss and melanoma-associated antigen 4 expression in smokers of non-small cell lung cancer patients.

BACKGROUND: Melanoma-associated antigen-A4 (MAGE-A4) is one of the candidates for a target of immunotherapy and is expressed in non-small cell lung cancer (NSCLC). However, tumors sometimes lose human leukocyte antigen (HLA) class I expression, and tumor-specific T cells cannot eliminate the tumor with loss of HLA. However, the relationship between MAGE-A4 expression and HLA loss has remained unclear. METHODS: Among 363 NSCLC patients who consecutively underwent curative surgery, 187 cases whose material could be analyzed were reviewed. The expression of HLA class I molecules was assessed by immunohistochemical staining. The expression of MAGE-A4 was analyzed by RT-PCR. RESULTS: Seventy-seven tumors expressed HLA normally; however, 110 tumors lost HLA. The proportion of patients with a smoking habit and expressing the MAGE-A4 gene in patients with HLA loss was higher than those with HLA expression (p = 0.04 and 0.028, respectively). Five-year overall survival (OS) rate in the patients expressing MAGE-A4 but with loss of HLA was 52.4 %, and OS was significantly poorer than their counterparts (74.0 %, p = 0.036). Multivariate analysis indicated that advanced stage or history of smoking and HLA loss was an independently poor prognostic predictor of OS in NSCLC (p < 0.01 and p = 0.04, respectively). CONCLUSION: HLA class I loss in NSCLC was related to smoking history and MAGE-A4 expression of tumors. HLA class I loss in smokers or patients with the MAGE-A4 gene was a prognostic factors in NSCLC.

Antitumor activity of human γδ T cells transducted with CD8 and with T-cell receptors of tumor-specific cytotoxic T lymphocytes.

The difficulty in the induction and preparation of a large number of autologous tumor-specific cytotoxic T lymphocytes (CTL) from individual patients is one of major problems in their application to adoptive immunotherapy. The present study tried to establish the useful antitumor effectors by using γδ T cells through tumor-specific TCRαß genes transduction, and evaluated the efficacy of their adoptive transfer in a non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice model. The TCRαß gene was cloned from the HLA-B15-restricted CTL clone specific of the Kita-Kyushu Lung Cancer antigen-1 (KK-LC-1). The cloned TCRαß as well as the CD8 gene were transduced into γδ T cells induced from peripheral blood lymphocytes (PBL). Cytotoxic T lymphocyte activity was examined using a standard 4 h (51) Cr release assay. Mice with a xenotransplanted tumor were treated with an injection of effector cells. Successful transduction of TCRαß was confirmed by the staining of KK-LC-1-specific tetramers. The γδ T cells transduced with TCRαß and CD8 showed CTL activity against the KK-LC-1-positive lung cancer cell line in a HLA B15-restricted manner. Adoptive transfer of the effector cells in a mice model resulted in marked growth suppression of KK-LC-1- and HLA-B15-positive xenotransplanted tumors. Co-transducing TCRαß and CD8 into γδ T cells yielded the same antigen-specific activity as an original CTL in vitro and in vivo. The TCRαß gene transduction into γδ T cells is a promising strategy for developing new adoptive immunotherapy.

Identification of ribosomal protein L19 as a novel tumor antigen recognized by autologous cytotoxic T lymphocytes in lung adenocarcinoma.

The purpose of the present study was to identify a novel tumor-specific antigen capable of inducing a specific cellular immune response in lung cancer patients. The co-culture of regional lymph node lymphocytes and the CD80-transfected autologous lung adenocarcinoma cell line H1224L resulted in a successful induction of bulk cytotoxic T lymphocytes (CTL). CTL clone L7/8 was established by the limiting dilution method from these bulk CTLs and lysed H1224L but not autologous Epstein-Barr virus-transformed B cells or K562. The CTL clone also recognized allogeneic lung cancer cell lines in an HLA-A*31012-restricted manner. Using the CTL clone, an antigen-coding gene was identified using the cDNA expression cloning technique, which encodes ribosomal protein L19 (RPL19). Finally, a 9 mer antigenic peptide was identified by means of construction of mini-genes. RPL19 was overexpressed in the lung cancer tissue from patient H1224. All of the normal tissues examined expressed lower levels of RPL19 mRNA than that of the lung cancer tissue. RPL19 was also found to be overexpressed in 12 of 30 (40%) non-small-cell lung cancer tissues by immunohistochemical staining. The expression level of RPL19 in tumor cell lines correlated positively with the production of interferon (IFN)-gammaby CTL clone L7/8 in response to such cell lines. In addition, the suppression of RPL19 expression by transfection with small interfering RNA resulted in the suppression of cyclinD1, D3 synthesis, and the growth inhibition of lung cancer cell lines overexpressing RPL19. Therefore, this growth suppression could be ascribed to the inhibition of the cell cycle. These results may indicate that RPL19 is a novel overexpressed antigen which may therefore be a useful candidate as a target for specific immunotherapy.

Identification of a lung cancer antigen evading CTL attack due to loss of human leukocyte antigen (HLA) class I expression.

The human lung cancer cell line, C831L, lost HLA class I expression due to a mutation of the ß2-microglobulin (ß2m) gene, and it may have been the result of immunoediting by CTL cytotoxicity. By restoration of HLA class I expression, we could identify the antigen that may be associated with HLA downregulation. Such an antigen might be a promising target of immunotherapy because it potentially may induce a sufficient immune response to eradicate cancer cells. The CTL clone could be established from lymph node lymphocytes in patient C831 by stimulation with wild-type ß2m-transduced C831L (C831L-wß2m). The CTL clone showed reactivity against C831L-wß2m in a HLA-B*0702-restricted manner, but not Parental-C831L or autologous normal cells. The cDNA expression cloning method was used to identify the antigen coding gene recognized by the CTL clone. The cDNA clone exhibited a homology with a part of the mRNA that codes for leucine rich repeat containing eight family member A (LRRC8A). A transfection analysis of minigenes indicated that the antigen peptide was derived from protein translated from the downstream of the registered open reading frame in LRRC8A mRNA. The antigenic 9-mer peptide (GPRESRPPA) was identified. The present methodology should be useful to find the crucial tumor antigens, which are potentially associated with loss of HLA expression. Furthermore, such an antigen may help in achieving a better understanding of the immunological escape mechanisms and it may also provide a favorable immune response in cancer immunotherapy.

Extraskeletal osteosarcoma of the pleura:report of a case.

Extraskeletal osteosarcoma is a rare malignant tumor occurring very rarely in the pleura. We herein report the case of 67-year-old man with asbestos exposure, who underwent biopsies of the large tumor from the chest wall, and diagnosed as a suspicious of fibrosarcoma. Surgical resection was done, and the pathological diagnosis was extraskeletal osteosarcoma arising from the pleura. The differential diagnosis is malignant pleural mesothelioma with osseous and cartilaginous which is also very rare and one of the histopathological subtypes with heterologous elements. Identification of epithelial components, labeling for cytokeratins in spindle cells and its' anatomical distribution may help to distinguish them. In the neoplasm arising from the parietal pleura, primary extraskeletal osteosarcoma of the pleura is very rare, but should be considered.

Cancer/testis antigen expression as a predictor for epidermal growth factor receptor mutation and prognosis in lung adenocarcinoma.

OBJECTIVES: Immune therapy targeting cancer/testis (CT) antigens improve the survival in several types of solid tumours. The expression of CT antigens is related to poor survival in non-small-cell lung cancer (NSCLC). The epidermal growth factor receptor (EGFR) mutation is the best predictive factor for the sensitivity to tyrosine kinase inhibitors in lung adenocarcinoma. The aim of this study was to elucidate the correlation between the expression of CT antigens and clinicopathological factors, including the EGFR mutation, and to analyse the prognosis in lung adenocarcinoma. METHODS: Data were collected from a total of 281 lung adenocarcinoma patients who underwent surgery. Among them, 125 cases, whose specimens were too small to extract sufficient DNA and/or RNA, and 2 cases with the coexistence of another histological lung cancer were excluded. A total of 154 patients were reviewed. The expression of CT antigens (melanoma-associated antigen gene [MAGE]-A4 and KK-LC-1) and the EGFR-activating mutation (L858R point mutation in exon 21 and inframe deletion in exon 19) was evaluated by using polymerase chain reaction amplification. RESULTS: The expression of MAGE-A4 and KK-LC-1 was detected in 14 (9%) and 54 patients (35%) with adenocarcinoma. The EGFR-activating mutation was found in 64 patients (42%). Univariate and multivariate analyses demonstrated that tumours expressing at least one CT antigen were associated with no EGFR mutation (odds ratio = 0.3; 95% confidence interval, 0.14-0.71; P < 0.01). A survival analysis was performed in 135 patients who underwent complete resection and the 5-year overall survival rate was 71.1% in those with any expression of CT antigens and 83.2% in those without expression of the genes (P < 0.04). CONCLUSION: Two different therapeutic targets, EGFR-activating mutation and CT antigen, have a negative relationship with each other.

Immunosuppressive effect of regulatory T lymphocytes in lung cancer, with special reference to their effects on the induction of autologous tumor-specific cytotoxic T lymphocytes.

It is not easy to induce cytotoxic T lymphocytes (CTLs) against cancer in in vitro culture. Regulatory T cells (Tregs) are considered to play a pivotal role in tumor immune escape. In this study, we analyzed the distribution of Tregs among tumor-infiltrating lymphocytes (TILs), regional lymph node lymphocytes (RLNLs) and peripheral blood lymphocytes (PBLs) in patients with lung cancer, and analyzed the effect of Tregs on the induction of CTLs in vitro. A total of 84 patients with non-small cell lung cancer underwent surgery between January 2003 and December 2004. The TILs, RLNLs and PBLs from these patients were subjected to a comparison analysis. The proportion of CD4(+)CD25(+)Foxp3(+) cells in these lymphocytes was determined by flow cytometry. The effects of Tregs on the induction of CTLs was analyzed by the depletion of Tregs in mixed lymphocyte-tumor cell culture (MLTC). The average proportions of Tregs in the TILs, RLNLs and PBLs were 10.4±9.5, 4.4±2.4 and 2.8±2.1%, respectively. The proportion of Tregs in the RLNLs was significantly higher than that in the PBLs (P<0.001); furthermore, TILs contained a larger number of Tregs than RLNLs (P=0.034). These Tregs substantially suppressed the induction of CTLs against autologous tumor cells. The depletion of Tregs in the MLTC resulted in the successful induction of CTLs. Tregs were found at a higher frequency in the TILs and RLNLs than in the PBLs in lung cancer patients. Since Tregs inhibited the induction of CTLs, the depletion of Tregs may represent a new therapeutic strategy for lung cancer patients.

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).