Yoshiaki Ikuta

Identification of HLA-A2- or HLA-A24-Restricted CTL Epitopes Possibly Useful for Glypican-3-Specific Immunotherapy of Hepatocellular Carcinoma

Purpose and Experimental Design: We previously reported that glypican-3 (GPC3) was overexpressed, specifically in hepatocellular carcinoma (HCC) and melanoma in humans, and it was useful as a novel tumor marker. We also reported that the preimmunization of BALB/c mice with dendritic cells pulsed with the H-2Kd-restricted mouse GPC3298-306 (EYILSLEEL) peptide prevented the growth of tumor-expressing mouse GPC3. Because of similarities in the peptide binding motifs between H-2Kd and HLA-A24 (A*2402), the GPC3298-306 peptide therefore seemed to be useful for the immunotherapy of HLA-A24+ patients with HCC and melanoma. In this report, we investigated whether the GPC3298-306 peptide could induce GPC3-reactive CTLs from the peripheral blood mononuclear cells (PBMC) of HLA-A24 (A*2402)+ HCC patients. In addition, we used HLA-A2.1 (HHD) transgenic mice to identify the HLA-A2 (A*0201)–restricted GPC3 epitopes to expand the applications of GPC3-based immunotherapy to the HLA-A2+ HCC patients. Results: We found that the GPC3144-152 (FVGEFFTDV) peptide could induce peptide-reactive CTLs in HLA-A2.1 (HHD) transgenic mice without inducing autoimmunity. In five out of eight HLA-A2+ GPC3+ HCC patients, the GPC3144-152 peptide-reactive CTLs were generated from PBMCs by in vitro stimulation with the peptide and the GPC3298-306 peptide-reactive CTLs were also generated from PBMCs in four of six HLA-A24+ GPC3+ HCC patients. The inoculation of these CTLs reduced the human HCC tumor mass implanted into nonobese diabetic/severe combined immunodeficiency mice. Conclusion: Our study raises the possibility that these GPC3 peptides may therefore be applicable to cancer immunotherapy for a large number of HCC patients.

Identification of Glypican-3 as a Novel Tumor Marker for Melanoma

Purpose: We reported recently the novel tumor marker glypican-3 (GPC3) for hepatocellular carcinoma. In the present study, we investigated the expression of GPC3 in human melanoma cell lines and tissues and asked whether GPC3 could be a novel tumor marker for melanoma. Experimental Design: Expression of GPC3 mRNA and protein was investigated in human melanoma cell lines and tissues using reverse transcription-PCR and immunohistochemical analysis. Secreted GPC3 protein was quantified using ELISA in culture supernatants of melanoma cell lines and in sera from 91 patients with melanoma and 28 disease-free patients after surgical removal of primary melanoma. All of the subjects were Japanese nationals. Results: In >80% of melanoma and melanocytic nevus, there was evident expression of GPC3 mRNA and protein. Furthermore, GPC3 protein was evidenced in sera of 39.6% (36 of 91) of melanoma patients but not in sera from subjects with large congenital melanocytic nevus (0 of 5) and from healthy donors (0 of 60). Twenty-seven of 36 serum GPC3-positive patients were negative for both serum 5-S-cysteinyldopa and melanoma-inhibitory activity, well-known tumor markers for melanoma. The positive rate of serum GPC3 (39.6%) was significantly higher than that of 5-S-cysteinyldopa (26.7%) and of melanoma-inhibitory activity (20.9%). Surprisingly, we detected serum GPC3 even in patients with stage 0 in situ melanoma. The positive rate of serum GPC3 at stage 0, I, and II (44.4%, 40.0%, and 47.6%) was significantly higher than that of 5-S-cysteinyldopa (0.0%, 8.0%, and 10.0%). Also observed was the disappearance of GPC3 protein in sera from 11 patients after surgical removal of the melanoma. Conclusions: GPC3 is apparently a novel tumor marker useful for the diagnosis of melanoma, especially in early stages of the disorder.

Proliferation Potential-Related Protein, an Ideal Esophageal Cancer Antigen for Immunotherapy, Identified Using Complementary DNA Microarray Analysis

Purpose: To establish effective antitumor immunotherapy for esophageal cancer, we tried to identify an useful target antigen of esophageal cancer. Experimental Design: We did cDNA microarray analysis to find a novel candidate antigen, proliferation potential-related protein (PP-RP). We examined cytotoxicity against tumor cells in vitro and in vivo of CTLs specific to PP-RP established from esophageal cancer patients. Results: In 26 esophageal cancer tissues, an average of relative ratio of the expression of the PP-RP mRNA in cancer cells versus adjacent normal esophageal tissues was 396.2. Immunohistochemical analysis revealed that, in 20 of the 22 esophageal cancer tissues, PP-RP protein was strongly expressed only in the cancer cells and not so in normal esophageal epithelial cells. PP-RP protein contains 10 epitopes recognized by HLA-A24–restricted CTLs. These CTLs, generated from HLA-A24–positive esophageal cancer patients, had cytotoxic activity against cancer cell lines positive for both PP-RP and HLA-A24. Furthermore, adoptive transfer of the PP-RP–specific CTL line inhibited the growth of a human esophageal cancer cell line engrafted in nude mice. Conclusions: The expression of PP-RP in esophageal cancer cells was significantly higher than in normal cells, and the CTLs recognizing PP-RP killed tumor cells in vitro and also showed tumor rejection effects in a xenograft model. Therefore, PP-RP may prove to be an ideal tumor antigen useful for diagnosis and immunotherapy for patients with esophageal cancer. cDNA microarray analysis is a useful method to identify ideal tumor-associated antigens.

Highly sensitive detection of melanoma at an early stage based on the increased serum secreted protein acidic and rich in cysteine and glypican-3 levels

Purpose: There are no available tumor markers detecting primary melanoma at an early stage. The identification of such serum markers would be of significant benefit for an early diagnosis of melanoma. We recently identified glypican-3 (GPC3) as a novel tumor marker but could diagnose only 40% of melanomas. Thereby, we focused out attention on secreted protein acidic and rich in cysteine (SPARC) overexpressed in melanoma as another candidate for tumor marker. Experimental Design: Secreted SPARC protein was quantified using ELISA in the sera from 109 melanoma patients, five patients with large congenital melanocytic nevus, 61 age-matched healthy donors, and 13 disease-free patients after undergoing a surgical removal. We also quantified GPC3 and 5-S-cysteinyldopa in the same serum samples and compared these markers for their diagnostic value. Results: The serum SPARC concentrations in melanoma patients were greater than those in healthy donors (P = 0.001). When we fixed a cutoff value at the mean concentration plus 2 SD of the healthy donors, the serum SPARC was found to have increased in the sera of 36 of the 109 (33%) melanoma patients, whereas there were three (4.9%) false-positive cases of 61 healthy donors. Surprisingly, 19 of 36 patients showing increased SPARC levels were in stages 0 to II. The serum SPARC level decreased under the cutoff level in 10 of 13 patients after surgical removal. Using SPARC and GPC3 in combination thus enabled us to diagnose 47 of 75 (66.2%) melanoma patients at an early stage (0-II). Conclusions: SPARC or its combination with GPC3 is thus considered a potentially useful tumor marker, especially for melanoma at an early stage.

Is an estimation of physiologic ability and surgical stress able to predict operative morbidity after pancreaticoduodenectomy

BackgroundMortality rates after pancreaticoduodenectomy (PD) are below 4% in high volume centers, although morbidity rates still remain high. Therefore, it is important to clarify a predictor associated with operative morbidity after PD. The estimation of physiologic ability and surgical stress (E-PASS) score has been developed for comparative audit in general surgical patients.ObjectiveTo evaluate whether E-PASS scoring system could predict the occurrence of complications after PD.MethodsWe performed retrospective analysis of 69 patients (42.0% pancreatic cancer, 31.9% bile duct cancer, and others) who underwent PD using the E-PASS as a predictor of morbidity. Correlations between the incidence rates of postoperative complications and the preoperative risk score (PRS), surgical stress score (SSS) and comprehensive risk score (CRS) of the E-PASS scoring system were evaluated.ResultsOf the 69 patients 30 (43.5%) experienced a total of 54 postoperative complications. All E-PASS scores, especially PRS and CRS were significantly higher in the patients with postoperative complications than in the patients without complication. The complication rate gradually increased as the PRS, SSS and CRS score increased. Under receiver operating characteristic analysis, if a cut-off point of CRS was 0.75, sensitivity and specificity for the prediction of operative morbidity after PD was 80.0 and 79.5%, respectively. Neoadjuvant chemotherapy and intraoperative radiation therapy (IORT) did not influenced on operative morbidity after PD.ConclusionE-PASS scoring system is useful to evaluate for morbidity after PD. Neoadjuvant chemotherapy and IORT could be adapted without significant extra risk for surgical complication.

Multiple Antigen-targeted Immunotherapy With α-Galactosylceramide–loaded and Genetically Engineered Dendritic Cells Derived From Embryonic Stem Cells

Numerous tumor-associated antigens (TAA) have been identified and their use in immunotherapy is considered to be promising. For TAA-based immunotherapy to be broadly applied as standard anticancer medicine, methods for active immunization should be improved. In the present study, we demonstrated the efficacy of multiple TAA-targeted dendritic cell (DC) vaccines and also the additive effects of loading α-galactosylceramide to DC using mouse melanoma models. On the basis of previously established methods to generate DC from mouse embryonic stem cells (ES-DC), 4 kinds of genetically modified ES-DC, which expressed the melanoma-associated antigens, glypican-3, secreted protein acidic and rich in cysteine, tyrosinase-related protein-2, or gp100 were generated. Anticancer effects elicited by immunization with the ES-DC were assessed in preventive and also therapeutic settings in the models of peritoneal dissemination and spontaneous metastasis to lymph node and lung. The in vivo transfer of a mixture of 3 kinds of TAA-expressing ES-DC protected the recipient mice from melanoma cells more effectively than the transfer of ES-DC expressing single TAA, thus demonstrating the advantage of multiple as compared with single TAA-targeted immunotherapy. Loading ES-DC with α-galactosylceramide further enhanced the anticancer effects, suggesting that excellent synergic effects of TAA-specific cytotoxic T lymphocytes and natural killer T cells against metastatic melanoma can be achieved by using genetically modified ES-DC. With the aid of advancing technologies related to pluripotent stem cells, induced pluripotent stem cells, and ES cells, clinical application of DC highly potent in eliciting anticancer immunity will be realized in the near future.

Multiple gastrointestinal stromal tumors in neurofibromatosis type 1: Report of a case

This report presents a case of multiple gastrointestinal stromal tumors (GIST) with neurofibromatosis type 1 (NF1). A 68-year-old woman was admitted to the hospital because of a tumor close to the head of the pancreas. Imaging studies revealed submucosal tumors of the duodenum. The retroperitoneal tumor was diagnosed before surgery. Besides the main tumor in the duodenum, multiple small submucosal tumors were found in the duodenum and upper part of the jejunum during the operation. All of these tumors were resected. The histological diagnosis of all these tumors was GISTs. These tumors were immunohistochemically positive for KIT, but they demonstrated no mutation in c-kit exons 9, 11, 13, and 17, and platelet-derived growth factor receptor α exons 12 and 18. No recurrence occurred for a year after surgery.

Cancer cells spread through lymph vessels in the submucosal layer of the common bile duct in gallbladder carcinoma.

INTRODUCTION In the present study, we performed immunohistochemical staining with a lymphatic epithelium-specific marker, D2-40, to analyze the status of lymphatic spreading in the hepatoduodenal ligament in T2 gallbladder carcinoma (GC). METHODS One hundred and eighty-six paraffin-embedded specimens from 15 T2 GC patients were reviewed. RESULTS Lymph vessels lined with D2-40 were visualized in the submucosal layer of the common bile duct in all cases. In 3 of 15 patients, clusters of cancer cells were identified in the submucosal lymph vessels of the extrahepatic bile duct, and this lymphatic invasion of cancer cells failed to be detected with only conventional hematoxylin-eosin staining. The frequency of the invasion to the submucosal lymph vessels in T2 GC correlated with presence of microscopic invasion to hepatoduodenal ligament and perineural invasion. CONCLUSION There were lymph vessels in the submucosal layer of the common bile duct, and cancer cells can spread through these channels in addition to the large lymph vessels in subserosal layer around the extrahepatic bile duct in GC. The present results would support the concept of en bloc resection of the extrahepatic bile duct in curative resection for T2 GC.

Clinical significance of dihydropyrimidine dehydrogenase and thymidylate synthase expression in patients with pancreatic cancer

BackgroundLittle is known about the clinical significance of TS and DPD in pancreatic cancer. We aimed to evaluate TS and DPD expression levels in not only pancreatic cancer but also surrounding normal pancreatic tissues to assess the clinical implications of the expression of TS and DPD in this study.Patients and methodsPancreatic cancer and normal pancreatic tissues were obtained from 18 patients with pancreatic cancer who underwent pancreatic resection to measure TS and DPD activities. The TS and DPD activities were determined by enzyme-linked immunosorbent assay using non-fixed fresh-frozen specimens.ResultsPancreatic cancer tissues had significantly higher DPD and TS enzyme activities than surrounding normal tissue. Anaplastic ductal carcinoma had higher DPD and TS activities than the other histological types. Patients with high DPD in this study demonstrated poorer prognosis than those with low DPD. On the other hand, there was no statistically significant difference in survival between the high and the low TS groups.ConclusionsThe efficacy of 5-FU may be lower in pancreatic cancer tissue than in normal tissue because DPD activity is upregulated in pancreatic cancer tissue compared to normal pancreatic tissue. It is necessary to develop an effective 5-FU delivery system and/or 5-FU combined with an inhibitor for DPD that can be used when 5-FU must be administered to patients with pancreatic cancer. High DPD activity may be a prognostic factor in patients with pancreatic cancer.

Cutaneous metastases secondary to pancreatic cancer

AIM To evaluate prognoses after cutaneous metastases, derived from pancreatic cancer. METHODS We treated two patients with cutaneous metastases from pancreatic cancer. We reviewed 40 reported patients in addition to our cases and analyzed clinical features of cutaneous metastases from pancreatic cancer. RESULTS The median survival time (MST) was 5 mo after diagnoses of cutaneous metastases. The cumulative 2-year survival rate was 3.5%. The most frequent site of cutaneous metastases was the umbilicus. The MST of patients who were treated with chemotherapy or chemoradiotherapy (CRT) was 6.5 mo, which was statistically longer in comparison to patients without treatment. Prognoses of cutaneous metastases are similar to other metastatic sites from pancreatic cancer. Receiving chemotherapy or CRT was the only prognostic factor of cutaneous metastases from pancreatic cancer. CONCLUSION The prognoses of cutaneous metastases are similar to other metastatic pancreatic cancers. Receiving chemotherapy or CRT was the only prognostic factor of cutaneous metastases from pancreatic cancer.