Katsunori Imai

Identification of a Novel Tumor-Associated Antigen, Cadherin 3/P-Cadherin, as a Possible Target for Immunotherapy of Pancreatic, Gastric, and Colorectal Cancers

Purpose: To establish cancer immunotherapy, it is important to identify the tumor-associated antigens (TAA) that are strongly expressed in the tumor cells but not in the normal cells. In this study, to establish an effective anticancer immunotherapy, we tried to identify the useful TAA of pancreatic cancer. Experimental Design: Based on a previous genome-wide cDNA microarray analysis of pancreatic cancer, we focused on cadherin 3 (CDH3)/P-cadherin as a novel candidate TAA for anticancer immunotherapy. To identify the HLA-A2 (A*0201)–restricted CTL epitopes of CDH3, we used HLA-A2.1 (HHD) transgenic mice (Tgm). Furthermore, we examined the cytotoxicity against the tumor cells in vitro and in vivo of CTLs specific to CDH3 induced from HLA-A2–positive healthy donors and cancer patients. Results:CDH3 was overexpressed in the majority of pancreatic cancer and various other malignancies, including gastric and colorectal cancers, but not in their noncancerous counterparts or in many normal adult tissues. In the experiment using HLA-A2.1 Tgm, we found that the CDH3-4655-663 (FILPVLGAV) and CDH3-7757-765 (FIIENLKAA) peptides could induce HLA-A2–restricted CTLs in Tgm. In addition, peptides-reactive CTLs were successfully induced from peripheral blood mononuclear cells by in vitro stimulation with these two peptides in HLA-A2–positive healthy donors and cancer patients, and these CTLs exhibited cytotoxicity specific to cancer cells expressing both CDH3 and HLA-A2. Furthermore, the adoptive transfer of the CDH3-specific CTLs could inhibit the tumor growth of human cancer cells engrafted into nonobese diabetic/severe combined immunodeficiency mice. Conclusions: These results suggest that CDH3 is a novel TAA useful for immunotherapy against a broad spectrum of cancers, including pancreatic cancer.

HLA-A2-restricted CTL epitopes of a novel lung cancer-associated cancer testis antigen, cell division cycle associated 1, can induce tumor-reactive CTL

Toward the development of a novel cancer immunotherapy, we have previously identified several tumor‐associated antigens (TAAs) and the epitopes recognized by human histocompatibility leukocyte (HLA)‐A2/A24‐restricted cytotoxic T lymphocyte (CTL). In this study, we tried to identify a TAA of lung cancer (LC) and its HLA‐A2 restricted CTL epitopes to provide a target antigen useful for cancer immunotherapy of LC. We identified a novel cancer testis antigen, cell division cycle associated gene 1 (CDCA1), overexpressed in nonsmall cell LC using a cDNA microarray analysis. The expression levels of CDCA1 were also increased in the majority of small cell LC, cholangiocellular cancer, urinary bladder cancer and renal cell cancers. We used HLA‐A2.1 transgenic mice to identify the HLA‐A2 (A*0201)‐restricted CDCA1 epitopes recognized by mouse CTL, and we investigated whether these peptides could induce CDCA1‐reactive CTLs from the peripheral blood mononuclear cells (PBMCs) of HLA‐A2‐positive donors and a NSCLC patient. Consequently, we found that the CDCA165–73 (YMMPVNSEV) peptide and CDCA1351–359 (KLATAQFKI) peptide could induce peptide‐reactive CTLs in HLA‐A2.1 transgenic mice. In HLA‐A2+ donors, in vitro stimulation of PBMC with these peptides could induce peptide‐reactive CTLs which killed tumor cell lines endogenously expressing both HLA‐A2 and CDCA1. As a result, CDCA1 is a novel cancer‐testis antigen overexpressed in LC, cholangiocellular cancer, urinary bladder cancer and renal cell cancers, and CDCA1 may therefore be an ideal TAA useful for the diagnosis and immunotherapy of these cancers.

Identification of HLA-A2-restricted CTL epitopes of a novel tumour-associated antigen, KIF20A, overexpressed in pancreatic cancer

Background:Identification of tumour-associated antigens (TAAs) that induce cytotoxic T lymphocytes (CTLs) specific to cancer cells is critical for the development of anticancer immunotherapy. In this study, we aimed at identifying a novel TAA of pancreatic cancer for immunotherapy.Methods:On the basis of the genome-wide cDNA microarray analysis, we focused on KIF20A (also known as RAB6KIFL/MKlp2) as a candidate TAA in pancreatic cancer cells. The HLA-A2 (A*02:01)-restricted CTL epitopes of KIF20A were identified using HLA-A2 transgenic mice (Tgm) and the peptides were examined to check whether they could generate human CTLs exhibiting cytotoxic responses against KIF20A+, HLA-A2+ tumour cells in vitro.Results:KIF20A was overexpressed in pancreatic cancer and in some other malignancies, but not in their non-cancerous counterparts and many normal adult tissues. We found that KIF20A-2 (p12–20, LLSDDDVVV), KIF20A-8 (p809–817, CIAEQYHTV), and KIF20A-28 (p284–293, AQPDTAPLPV) peptides could induce HLA-A2-restricted CTLs in HLA-A2 Tgm without causing autoimmunity. Peptide-reactive human CTLs were generated from peripheral blood mononuclear cells of HLA-A2+ healthy donors by in vitro stimulation with the three peptides, and those CTLs successfully exhibited cytotoxic responses to cancer cells expressing both KIF20A and HLA-A2.Conclusion:KIF20A is a novel promising candidate for anticancer immunotherapeutic target for pancreatic cancers.

The forkhead box M1 transcription factor as a candidate of target for anti-cancer immunotherapy

The present study attempted to identify a target antigen for immunotherapy for cholangiocarcinoma. Forkhead box M1 (FOXM1) was selected as a candidate antigen based on the data of previous cDNA microarray analysis of clinical samples of cholangiocarcinoma. The level of FOXM1 mRNA was more than 4 times higher in cancer cells in comparison to adjacent normal epithelial cells, in all of 24 samples of cholangiocarcinoma tissues. An immunohistochemical analysis also detected FOXM1 protein in the cancer cells but not in the normal cells. Twenty‐three human FOXM1‐derived peptides predicted to bind to HLA‐A2 were analyzed to determine their ability to induce HLA‐A2‐restricted T cells in HLA‐A2 transgenic mice. FOXM1362‐370 (YLVPIQFPV), FOXM1373‐382 (SLVLQPSVKV), and FOXM1640‐649 (GLMDLSTTPL) peptides primed HLA‐A2‐restricted cytotoxic T lymphocytes (CTLs) in the HLA‐A2 transgenic mice. Human CTL lines reactive to these 3 peptides could also be established from HLA‐A2‐positive healthy donors and cancer patients. Natural processing of the 3 epitopes from FOXM1 protein was confirmed by specific killing of HLA‐A2‐positive FOXM1‐transfectants by peptide‐induced CTLs. FOXM1 is expressed in various types of cancers and it is also functionally involved in oncogenic transformation and the survival of cancer cells. Therefore, FOXM1 may be a suitable target for immunotherapy against various cancers including cholangiocarcinoma.

A novel tumor-associated antigen, cell division cycle 45-like can induce cytotoxic T-lymphocytes reactive to tumor cells

The present study attempted to identify a useful tumor‐associated antigen (TAA) for lung cancer immunotherapy and potential immunogenic peptides derived from the TAA. We focused on cell division cycle 45‐like (CDC45L), which has a critical role in the initiation and elongation steps of DNA replication, as a novel candidate TAA for immunotherapy based on a genome‐wide cDNA microarray analysis of lung cancer. The CDC45L was overexpressed in the majority of lung cancer tissues, but not in the adjacent non‐cancerous tissues or in many normal adult tissues. We examined the in vitro and in vivo anti‐tumor effects of cytotoxic T‐lymphocytes (CTL) specific to CDC45L‐derived peptides induced from HLA‐A24 (A*24:02)‐positive donors. We identified three CDC45L‐derived peptides that could reproducibly induce CDC45L‐specific and HLA‐A24‐restricted CTL from both healthy donors and lung cancer patients. The CTL could effectively lyse lung cancer cells that endogenously expressed both CDC45L and HLA‐A24. In addition, we found that CDC45L 556KFLDALISL564 was eminent in that it induced not only HLA‐A24 but also HLA‐A2 (A*02:01)‐restricted antigen specific CTL. Furthermore, the adoptive transfer of the CDC45L‐specific CTL inhibited the growth of human cancer cells engrafted into immunocompromised mice. These results suggest that these three CDC45L‐derived peptides are highly immunogenic epitopes and CDC45L is a novel TAA that might be a useful target for lung cancer immunotherapy. (Cancer Sci 2011; 102: 697–705)

Functional assessment versus conventional volumetric assessment in the prediction of operative outcomes after major hepatectomy

PURPOSE In this study we examined whether conventional future remnant liver volume (FR volume) or function (FR function) better predicted the operative outcome after major hepatectomy. METHODS Of 510 patients who underwent hepatectomy for various indications, 133 patients with major hepatectomy were enrolled in this study. FR volume and the corresponding FR function routinely were measured with a 99mTc-GSA scintigraphy single-proton emission computed tomography fusion system. FR function was defined as the future remnant liver uptake ratio of 99mTc-GSA per whole liver by single-proton emission computed tomography. FR volume or function in cases with insufficient FR volume or function required for major hepatectomy were defined as marginal. RESULTS Morbidity, liver dysfunction-related morbidity, and mortality after major hepatectomy occurred in 40 (30%), 10 (7.5%), and 8 (5.7%) patients, respectively. Thirty-two of the 133 patients were diagnosed as marginal using FR volume, but only 11 patients were diagnosed as marginal using FR function. These results indicated that 21 patients (16%) were switched to the safe group using functional assessment. Operative outcomes in patients with safe FR function (n = 122) were equivalent to those of patients with safe FR volume (n = 101), but patients with marginal FR function (n = 11) had substantially worse outcomes than patients with marginal FR volumes (n = 32). Logistic regression analysis identified marginal FR function, but not volume, as a risk factor for worse operative outcome after major hepatectomy. Portal vein embolization induced the substantially greater FR function compared with FR volume. Although liver volume equally corresponds to liver function under normal conditions, liver cirrhosis was greatly associated with the major discrepancy (more than 10%) in patients without portal vein embolization. CONCLUSION Functional assessment for future remnant liver identified patients who were eligible for curative hepatectomy despite a marginal status based on conventional volumetric assessment. Thus, marginal FR function is a better predictor of operative outcome after major hepatectomy than FR volume, especially in patients with nonhealthy liver.

Peptides derived from human insulin-like growth factor-II mRNA binding protein 3 can induce human leukocyte antigen-A2-restricted cytotoxic T lymphocytes reactive to cancer cells

Insulin‐like growth factor‐II mRNA binding protein 3 (IMP‐3) is an oncofetal protein expressed in various malignancies including lung cancer. This study aimed to identify immunogenic peptides derived from IMP‐3 that can induce tumor‐reactive and human leukocyte antigen (HLA)‐A2 (A*02:01)‐restricted cytotoxic T lymphocytes (CTL) for lung cancer immunotherapy. Forty human IMP‐3‐derived peptides predicted to bind to HLA‐A2 were analyzed to determine their capacity to induce HLA‐A2‐restricted T cells in HLA‐A2.1 (HHD) transgenic mice (Tgm). We found that three IMP‐3 peptides primed HLA‐A2‐restricted CTL in the HLA‐A2.1 Tgm. Among them, human CTL lines reactive to IMP‐3 515NLSSAEVVV523 were reproducibly established from HLA‐A2‐positive healthy donors and lung cancer patients. On the other hand, IMP‐3 199RLLVPTQFV207 reproducibly induced IMP‐3‐specific and HLA‐A2‐restricted CTL from healthy donors, but did not sensitize CTL in the HLA‐A2.1 Tgm. Importantly, these two IMP‐3 peptide‐specific CTL generated from healthy donors and cancer patients effectively killed the cancer cells naturally expressing both IMP‐3 and HLA‐A2. Cytotoxicity was significantly inhibited by anti‐HLA class I and anti‐HLA‐A2 monoclonal antibodies, but not by the anti‐HLA‐class II monoclonal antibody. In addition, natural processing of these two epitopes derived from the IMP‐3 protein was confirmed by specific killing of HLA‐A2‐positive IMP‐3‐transfectants but not the parental IMP‐negative cell line by peptide‐induced CTL. This suggests that these two IMP‐3‐derived peptides represent highly immunogenic CTL epitopes that may be attractive targets for lung cancer immunotherapy. (Cancer Sci 2011; 102: 71–80)

Identification of SPARC as a candidate target antigen for immunotherapy of various cancers

To establish efficient anticancer immunotherary, it is important to identify tumor‐associated antigens (TAAs) directing the immune system to attack cancer. A genome‐wide cDNA microarray analysis identified that secreted protein acidic and rich in cysteine (SPARC) gene is overexpressed in the gastric, pancreatic and colorectal cancer tissues but not in their noncancerous counterparts. This study attempted to identify HLA‐A24 (A*2402)‐restricted and SPARC‐derived CTL epitopes. We previously identified H‐2Kd‐restricted and SPARC‐derived CTL epitope peptides in BALB/c mice, of which H‐2Kd‐binding peptide motif is comparable with that of HLA‐A24 binding peptides. By using these peptides, we tried to induce HLA‐A24 (A*2402)‐restricted and SPARC‐reactive human CTLs and demonstrated an antitumor immune response. The SPARC‐A24‐1143–151 (DYIGPCKYI) and SPARC‐A24‐4225–234 (MYIFPVHWQF) peptides‐reactive CTLs were successfully induced from peripheral blood mononuclear cells by in vitro stimulation with these two peptides in HLA‐A24 (A*2402) positive healthy donors and cancer patients, and these CTLs exhibited cytotoxicity specific to cancer cells expressing both SPARC and HLA‐A24 (A*2402). Furthermore, the adoptive transfer of the SPARC‐specific CTLs could inhibit the tumor growth in nonobese diabetic/severe combined immunodeficient mice bearing human cancer cells expressing both HLA‐A24 (A*2402) and SPARC. These findings suggest that SPARC is a potentially useful target candidate for cancer immunotherapy.

Cystine/glutamic acid transporter is a novel marker for predicting poor survival in patients with hepatocellular carcinoma

Cystine/glutamic acid transporter (xCT) plays a role in tumor progression by regulating the redox status in several types of cancers. To demonstrate the importance of xCT expression for predicting the prognosis of hepatocellular carcinoma (HCC), we analyzed xCT gene expression in 130 paired HCC and non-cancerous tissues. xCT protein expression was confirmed using 7 HCC cell lines and samples from human subjects. xCT mRNA expression was detected in 34 (26%) tumor tissues. Expression of xCT was higher in HCC tissues compared to the corresponding normal tissues according to quantitative reverse transcriptase-polymerase chain reaction findings (P<0.0001). Patients in the group presenting with xCT mRNA expression showed poorer overall and disease-free survival than did those with an absence of xCT mRNA (P=0.0130 and 0.0416, respectively). xCT mRNA expression proved to be an independent factor for poor prognosis in a multivariate analysis of overall survival (hazard ratio, 1.68; 95% CI, 1.03-2.92). We observed xCT protein expression in both the HCC cell lines and in human tissue samples. In conclusion, the findings of the present study suggest that xCT is useful as a predictive marker for patient prognosis and that it may be a novel therapeutic target for HCC.

A case of adrenal rest tumor of the liver: Radiological imaging and immunohistochemical study of steroidogenic enzymes

A case of adrenal rest tumor in the liver of a 67‐year‐old female was reported. The tumor appeared on diagnostic imaging as a hypervascular and fat‐containing mass at the periphery of right hepatic lobe and required differential diagnosis from hepatocellular carcinoma (HCC). Pathological diagnosis of the intraoperative frozen section taken under laparoscopic liver biopsy could not exclude fat‐containing HCC. Therefore, she underwent partial resection of the right hepatic lobe. A yellowish‐brown mass, 17 × 14 × 11 mm in a diameter, was located in the subcapsular portion. Histologically, the mass presented features similar to those of the adrenal cortex and was composed of pale cells. Immunohistochemically, the mass expressed the adrenal 4‐binding protein and several enzymes involved in the synthesis of adrenocortical steroids. Based on these findings, the hepatic mass was finally diagnosed as an adrenal rest tumor of the liver.