[A Patient with Multiple Colorectal Liver Metastases for Whom Partial Splenic Embolization Contributed Extremely to Maintain Multidisciplinary Treatment].

After approximately 2.5 years of chemotherapy at the referred hospital, a 69-year-old man with double colon cancer and unresectable liver metastases(H3)sought consultation. A total of 8 liver metastases were deemed resectable; however, the disease was progressive. He received 2 courses of mFOLFOX6 plus Bmab before hepatectomy. Seven weeks after starting chemotherapy, Grade 4 thrombocytopenia occurred, which required platelet transfusion. Ten weeks after, curative parenchymal- preserving hepatectomy was performed under platelet transfusion. Hematologic examination including bone marrow aspiration showed no significant abnormalities, including normal megakaryocyte formation. Therefore, the patient was diagnosed with thrombocytopenia due to sinusoidal obstruction syndrome associated with past chemotherapy including oxaliplatin. Partial splenic embolization(PSE)was performed 8 weeks after the first hepatectomy. The infarcted splenic ratio was 79.5%, and the infarcted splenic volume was 444.3 mL. Curative resection of the primary colorectal cancer and the 2nd hepatectomy for the newly developed recurrent liver lesions was successfully performed at 2 weeks and 19 weeks after PSE, respectively. Platelet transfusion was never required in the perioperative period of the 2 operations performed after the PSE. Forty-five months after the initial treatment, the patient is alive with no recurrent tumors and normal tumor marker levels.

[Collision Lung Cancer Consisting of Squamous Cell Carcinoma and Adenocarcinoma which Showed Identical Epidermal Growth Factor Receptor Mutation;Report of a Case].

We report a 79-year-old woman with collision cancer in the right middle lobe of the lung. She had a persistent abnormal shadow after treatment for pneumonia pointed out in right middle lung field on chest radiogram, and referred to our hospital. On examination, the chest computed tomography showed a pure-solid mass of 7.6 cm in diameter in right middle lobe of the lung which was thought to invade the superior pulmonary vein. She underwent a successful right pneumonecomty, and the postoperative course was uneventful. The tumor proved to be a collision cancer consisting of poor differentiated squamous cell carcinoma and invasive adenocarcinoma, lepidic predominanat by pathological examination. Epidermal growth factor receptor mutations (L858R) were found in both squamous cell carcinoma and adenocarcinoma of the tumor, possibly suggesting the same origin of both histological types.

HLA-A2-restricted glypican-3 peptide-specific CTL clones induced by peptide vaccine show high avidity and antigen-specific killing activity against tumor cells.

Glypican-3 (GPC3) is an onco-fetal antigen that is overexpressed in human hepatocellular carcinoma (HCC), and is only expressed in the placenta and embryonic liver among normal tissues. Previously, we identified an HLA-A2-restricted GPC3(144-152) (FVGEFFTDV) peptide that can induce GPC3-reactive CTLs without inducing autoimmunity in HLA-A2 transgenic mice. In this study, we carried out a phase I clinical trial of HLA-A2-restricted GPC3(144-152) peptide vaccine in 14 patients with advanced HCC. Immunological responses were analyzed by ex vivo γ-interferon enzyme-linked immunospot assay. The frequency of GPC3(144-152) peptide-specific CTLs after vaccination (mean, 96; range, 5-441) was significantly larger than that before vaccination (mean, 6.5; range, 0-43) (P < 0.01). An increase in the GPC3(144-152) peptide-specific CTL frequency was observed in 12 (86%) of 14 patients after vaccination. Additionally, there was a significant correlation between the maximum value of GPC3(144-152) peptide-specific CTLs after vaccination and the dose of the peptide injected (P = 0.0166, r = 0.665). Moreover, we established several GPC3(144-152) peptide-specific CTL clones from PBMCs of patients vaccinated with GPC3(144-152) peptide by single cell sorting using Dextramer and CD107a antibody. These CTL clones had high avidity (the recognition efficiency showing 50% cytotoxicity was 10(-10) or 10(-11) M) and could recognize HCC cell lines expressing GPC3 in an HLA-class I-restricted manner. These results suggest that GPC3(144-152) peptide vaccine can induce high avidity CTLs capable of killing HCC cells expressing GPC3. This trial was registered with University Hospital Medical Information Network number 000001395.

Detection of glypican-3-specific CTLs in chronic hepatitis and liver cirrhosis.

Glypican-3 (GPC3) is one of carcinoembryonic antigens known to be overexpressed in hepatocellular carcinoma (HCC). It has been suggested that GPC3 may be related to the development of HCC in a background of chronic hepatitis (CH) and liver cirrhosis (LC). Therefore, in an attempt to establish an early diagnostic marker of HCC, we quantified the number of GPC3-specific CTLs in the peripheral blood of CH and LC patients. We selected CH and LC patients who were HCV-RNA (+) or HBs antigen (+) within 6 months prior to the study and had no HCC nodules as detected by imaging. A total of 56 patients with CH and LC, and 45 patients with HLA-A24+ or HLA-A2+ were enrolled for this investigation. After isolation of mononuclear cells from each patient's peripheral blood specimens, we performed ELISPOT assay using HLA-A24- and HLA-A2-restricted GPC3 peptides. In the ELISPOT assay, GPC3-specific CTLs were detected in 10 of the 45 CH and LC cases (22%). In addition, the plasma titers of anti-GPC3 IgG were increased in the CH and LC patients as compared with those in healthy donors. GPC3-specific CTLs were found to be present not only in patients with HCC, but also in patients with CH and LC. This suggests the possibility of GPC3-specific CTLs serving as a marker for the early diagnosis of imaging-invisible HCC.

HLA-A2 and -A24-restricted glypican-3-derived peptide vaccine induces specific CTLs: preclinical study using mice.

We previously reported that glypican-3 (GPC3) is uniquely overexpressed in human hepatocellular carcinoma and melanoma and that it is an ideal tumor antigen for immunotherapy in mouse models. We recently identified both HLA-A24 (A*2402) and H-2Kd-restricted GPC3298-306 (EYILSLEEL) and HLA-A2 (A*0201)-restricted GPC3144-152 (FVGEFFTDV), both of which can induce GPC3-reactive cytotoxic T cells (CTLs). The present study was a preclinical study in a mouse model that was conducted in order to design an optimal schedule for clinical trial of GPC3-derived peptide vaccine. When BALB/c mice were intradermally vaccinated at the base of the tail with Kd-restricted GPC3298-306 peptide mixed with incomplete Freund's adjuvant (IFA), the peptide-specific CTLs were induced. But the peptide alone could not induce peptide-specific CD8+ T cells. Furthermore, proteomic analyses showed that IFA protected the peptide against degradation in the human serum. Peptide-reactive CTLs were induced by peptide vaccine in a dose-dependent manner. In addition, at least two vaccinations with a single dose >10 microg were needed for the induction of GPC3298-306-specific CTLs. But repeated vaccination with a lower dose of GPC3298-306 did not induce peptide-specific CTLs. Similarly, induction of an Ag-specific immune response by HLA-A2 GPC3144-152 depended on the dose administered. The results of this study suggested that IFA is one of the indispensable adjuvants for peptide-based immunotherapy, and that the immunological effect of peptide vaccines depends on the dose of peptide injected.

Embryonic stem cell-derived dendritic cells expressing glypican-3, a recently identified oncofetal antigen, induce protective immunity against highly metastatic mouse melanoma, B16-F10.

We have recently established a method to generate dendritic cells from mouse embryonic stem cells. By introducing exogenous genes into embryonic stem cells and subsequently inducing differentiation to dendritic cells (ES-DC), we can now readily generate transfectant ES-DC expressing the transgenes. A previous study revealed that the transfer of genetically modified ES-DC expressing a model antigen, ovalbumin, protected the recipient mice from a challenge with an ovalbumin-expressing tumor. In the present study, we examined the capacity of ES-DC expressing mouse homologue of human glypican-3, a recently identified oncofetal antigen expressed in human melanoma and hepatocellular carcinoma, to elicit protective immunity against glypican-3-expressing mouse tumors. CTLs specific to multiple glypican-3 epitopes were primed by the in vivo transfer of glypican-3-transfectant ES-DC (ES-DC-GPC3). The transfer of ES-DC-GPC3 protected the recipient mice from subsequent challenge with B16-F10 melanoma, naturally expressing glypican-3, and with glypican-3-transfectant MCA205 sarcoma. The treatment with ES-DC-GPC3 was also highly effective against i.v. injected B16-F10. No harmful side effects, such as autoimmunity, were observed for these treatments. The depletion experiments and immunohistochemical analyses suggest that both CD8+ and CD4+ T cells contributed to the observed antitumor effect. In conclusion, the usefulness of glypican-3 as a target antigen for antimelanoma immunotherapy was thus shown in the mouse model using the ES-DC system. Human dendritic cells expressing glypican-3 would be a promising means for therapy of melanoma and hepatocellular carcinoma.

Regression of intestinal adenomas by vaccination with heat shock protein 105-pulsed bone marrow-derived dendritic cells in Apc(Min/+) mice.

Heat shock protein (HSP) 105 is overexpressed in various cancers, but is expressed at low levels in many normal tissues, except for the testis. A vaccination with HSP105-pulsed bone marrow-derived dendritic cells (BM-DC) induced antitumor immunity without causing an autoimmune reaction in a mouse model. Because Apc(Min/+) mice develop multiple adenomas throughout the intestinal tract by 4 months of age, the mice provide a clinically relevant model of human intestinal tumor. In the present study, we investigated the efficacy of the HSP105-pulsed BM-DC vaccine on tumor regression in the Apc(Min/+) mouse. Western blot and immunohistochemical analyses revealed that the tumors of the Apc(Min/+) mice endogenously overexpressed HSP105. Immunization of the Apc(Min/+) mice with a HSP105-pulsed BM-DC vaccine at 6, 8, and 10 weeks of age significantly reduced the number of small-intestinal polyps accompanied by infiltration of both CD4(+) and CD8(+) T cells in the tumors. Cell depletion experiments proved that both CD4(+) and CD8(+) T cells play a critical role in the activation of antitumor immunity induced by these vaccinations. These findings indicate that the HSP105-pulsed BM-DC vaccine can provide potent immunotherapy for tumors that appear spontaneously as a result of the inactivation of a tumor suppressor gene, such as in the Apc(Min/+) mouse model.

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-2K(d)-restricted mouse GPC3(298-306) (EYILSLEEL) peptide prevented the growth of tumor-expressing mouse GPC3. Because of similarities in the peptide binding motifs between H-2K(d) and HLA-A24 (A*2402), the GPC3(298-306) peptide therefore seemed to be useful for the immunotherapy of HLA-A24+ patients with HCC and melanoma. In this report, we investigated whether the GPC3(298-306) 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 GPC3(144-152) (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 GPC3(144-152) peptide-reactive CTLs were generated from PBMCs by in vitro stimulation with the peptide and the GPC3(298-306) 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.

Phase I trial of a glypican-3-derived peptide vaccine for advanced hepatocellular carcinoma: immunologic evidence and potential for improving overall survival.

PURPOSE: The carcinoembryonic antigen glypican-3 (GPC3) is an ideal target of anticancer immunotherapy against hepatocellular carcinoma (HCC). In this nonrandomized, open-label, phase I clinical trial, we analyzed the safety and efficacy of GPC3 peptide vaccination in patients with advanced HCC. EXPERIMENTAL DESIGN: Thirty-three patients with advanced HCC underwent GPC3 peptide vaccination (intradermal injections on days 1, 15, and 29 with dose escalation). The primary endpoint was the safety of GPC3 peptide vaccination. The secondary endpoints were immune response, as measured by IFN-γ ELISPOT assay, and the clinical outcomes tumor response, time to tumor progression, and overall survival (OS). RESULTS: GPC3 vaccination was well-tolerated. One patient showed a partial response, and 19 patients showed stable disease 2 months after initiation of treatment. Four of the 19 patients with stable disease had tumor necrosis or regression that did not meet the criteria for a partial response. Levels of the tumor markers α-fetoprotein and/or des-γ-carboxy prothrombin temporarily decreased in nine patients. The GPC3 peptide vaccine induced a GPC3-specific CTL response in 30 patients. Furthermore, GPC3-specific CTL frequency after vaccination correlated with OS. OS was significantly longer in patients with high GPC3-specific CTL frequencies (N = 15) than in those with low frequencies (N = 18; P = 0.033). CONCLUSIONS: GPC3-derived peptide vaccination was well-tolerated, and measurable immune responses and antitumor efficacy were noted. This is the first study to show that peptide-specific CTL frequency can be a predictive marker of OS in patients with HCC receiving peptide vaccination.

Radiofrequency ablation for hepatocellular carcinoma induces glypican-3 peptide-specific cytotoxic T lymphocytes.

Glypican-3 (GPC3), a carcinoembryonic antigen, is an ideal target for anticancer immunotherapy against hepatocellular carcinoma (HCC). In this study, we attempted to compare the induction of the GPC3-specific T-cell-mediated immune response after locoregional therapies in HCC patients and tumor-bearing mice. Twenty-seven HCC patients treated with locoregional therapies, including radiofrequency ablation (RFA), surgical resection and transcatheter arterial chemo-embolization (TACE), were prospectively enrolled in this study. Additionally, we performed RFA experiments using a mouse model. GPC3-specific T-cell response was investigated pre-treatment and post-treatment by an interferon-γ enzyme-linked immunospot assay using peripheral blood mononuclear cells from HCC patients and lymph node cells from tumor-bearing mice. Circulating GPC3-specific cytotoxic T lymphocytes (CTLs) were increased in 5 of 9 patients after RFA and in 4 of 9 patients after TACE, but in only 1 of 9 patients after surgical resection. All 7 patients with GPC3-expressing HCCs exhibited an increase in GPC3-specific CTLs after RFA or TACE, whereas none of the 7 patients did after surgical resection. The number of increased GPC3-specific CTLs after RFA was significantly larger than that after surgical resection (P=0.023). Similarly, the frequency of GPC3-specific CTLs after RFA was significantly greater than that after surgical resection in the mouse model (P=0.049). We validated for the first time the stronger effect on the immune system brought by RFA compared with surgical resection for HCC patients and tumor-bearing mice. Combined treatment of RFA and immunotherapy is a reasonable strategy against HCC.

Immunotherapy with pluripotent stem cell-derived dendritic cells.

In vivo transfer of dendritic cells (DC) has proven efficient in the priming of T cells and is regarded as a powerful means of providing anti-cancer immunotherapy. Clinical trials of anti-cancer therapy with DC pulsed with peptide antigens have been carried out in many institutions, although dramatic therapeutic effect has not been observed in most of the trials. Negative regulation of the immune response by DC might be applicable to treatment of autoimmune diseases and transplantation medicine. Currently, the DC used for anti-cancer vaccine therapy are generated from the peripheral blood monocytes of the patients. However, there is a limitation in the number of available monocytes and the potential of monocytes to differentiate into DC varies depending on the individual blood donors. To resolve the issue of the cell source for DC therapy, several groups have developed methods to generate DC from pluripotent stem cells. This review introduces methods to generate functional DC from pluripotent stem cells of mouse and human.

Trichoblastic carcinoma arising from colostomy.

Trichoblastic carcinoma is a rare skin cancer originating from hair germ cells. We report a case of an 84-year-old man who presented with a tumor on the stoma of the descending colon, which was preoperatively diagnosed as colon cancer. He underwent colectomy with adjacent skin, and the tumor was diagnosed as trichoblastic carcinoma by postoperative pathological examination. We are not aware of any similar cases published in the English literature. Therefore, we report this case because it is quite a rare condition.

Pluripotent stem cells as source of dendritic cells for immune therapy.

Dendritic cells (DC) are the most potent antigen-presenting cells. In vivo transfer of antigen-bearing DC has proven efficient in priming T cell responses specific to the antigen. DC-based cellular vaccination is now regarded as a powerful means for immunotherapy, especially for anti-cancer immunotherapy. Clinical trials of therapy with DC pulsed with peptide antigens or genetically modified to present antigens are currently carried out in many institutions. In addition, antigen-specific negative regulation of immune response by DC is considered to be a promising approach for treatments of autoimmune diseases and also for regulation of allo-reactive immune response causing graft rejection and GVHD in transplantation medicine. DC for transfer therapy are now generated by in vitro differentiation of peripheral blood monocytes of the patients. However, there is a limitation in the number of available monocytes, and the DC-differentiation potential of monocytes varies depending on the blood donor. Embryonic stem (ES) cells possess both pluripotency and infinite propagation capacity. We consider ES cells to be an ideal source for DC to be used in immunotherapy. Several groups, including us, have developed methods to generate DC from ES cells. This review introduces the studies on generation, characterization, and genetic modification of DC derived from ES cells or induced pluripotent stem (iPS) cells. The issues to be resolved before clinical application of pluripotent stem cell-derived DC will also be discussed.

Acute afferent loop necrosis after Roux-en-Y cholangiojejunostomy.

Afferent loop necrosis after Roux-en-Y cholangiojejunostomy biliary reconstruction is rare. We present the case of a 36-year-old woman with acute necrotic afferent loop obstruction. The peripheral area of the Roux-en-Y limb, including the cholangiojejunostomy portion, was twisted just proximal to the cholangiojejunostomy. Cholangiojejunostomy was completely separated due to necrosis of the Roux-en-Y jejunum. In addition to the case report, we discuss features of cholangiojejunostomy that require special attention.

Multiple antigen-targeted immunotherapy with alpha-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 alpha-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 alpha-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.

Cancer prevention with semi-allogeneic ES cell-derived dendritic cells.

Dendritic cells (DC) genetically modified to present tumor-associated antigen are a promising means for anti-cancer immunotherapy. By introducing expression vectors into ES cells and subsequently inducing differentiation to DC (ES-DC), we can generate transfectant DC expressing the transgenes. In the future clinical application of this technology, the unavailability of human ES cells genetically identical to the patients will be a problem. However, in most cases, semi-allogeneic ES cells sharing some of HLA alleles with recipients are expected to be available. In the present study, we observed that model tumor antigen (OVA)-expressing mouse ES-DC transferred into semi-allogeneic mice potently primed OVA-reactive CTL and elicited a significant protection against challenge with OVA-expressing tumor. Genetic modification of ES-DC to overexpress SPI-6, the specific inhibitor of granzyme B, further enhanced their capacity to prime antigen-specific CTL in semi-allogeneic recipient mice. These results suggest the potential of ES-DC as a novel means for anti-cancer immunotherapy.

Therapeutic effect of alpha-galactosylceramide-loaded dendritic cells genetically engineered to express SLC/CCL21 along with tumor antigen against peritoneally disseminated tumor cells.

The close cooperation of both innate and acquired immunity is essential for the induction of truly effective antitumor immunity. We tested a strategy to enhance the cross-talk between NKT cells and conventional antigen-specific T cells with the use of alpha GalCer-loaded dendritic cells genetically engineered to express antigen plus chemokine, attracting both conventional T cells and NKT cells. DC genetically engineered to express a model antigen, OVA, along with SLC/CCL21 or monokine induced by IFN-gamma/CXCL9, had been generated using a method based on in vitro differentiation of DC from mouse ES cells. The ES-DC were loaded with alpha-GalCer and transferred to mice bearing MO4, an OVA-expressing melanoma, and their capacity to evoke antitumor immunity was evaluated. In vivo transfer of either OVA-expressing ES-DC, stimulating OVA-reactive T cells, or alpha-GalCer-loaded non-transfectant ES-DC, stimulating NKT cells, elicited a significant but limited degree of protection against the i.p. disseminated MO4. A more potent antitumor effect was observed when alpha-GalCer was loaded to ES-DC expressing OVA before in vivo transfer, and the effect was abrogated by the administration of anti-CD8, anti-NK1.1 or anti-asialo GM1 antibody. alpha-GalCer-loaded double transfectant ES-DC expressing SLC along with OVA induced the most potent antitumor immunity. Thus, alpha-GalCer-loaded ES-DC expressing tumor-associated antigen along with SLC can stimulate multiple subsets of effector cells to induce a potent therapeutic effect against peritoneally disseminated tumor cells. The present study suggests a novel way to use alpha-GalCer in immunotherapy for peritoneally

DNA vaccination of HSP105 leads to tumor rejection of colorectal cancer and melanoma in mice through activation of both CD4 T cells and CD8 T cells.

We report that HSP105, identified by serological identification of antigens by recombinant expression cloning (SEREX), is overexpressed in a variety of human cancers, including colorectal, pancreatic, thyroid, esophageal, and breast carcinoma, but is not expressed in normal tissues except for the testis. The amino acid sequences and expression patterns of HSP105 are very similar in humans and mice. In this study, we set up a preclinical study to investigate the usefulness of a DNA vaccine producing mouse HSP105 whole protein for cancer immunotherapy in vivo using BALB/c and C57BL/6 mice, Colon26, a syngeneic endogenously HSP105-expressing colorectal cancer cell line, and B16.F10, a melanoma cell line. The DNA vaccine was used to stimulate HSP105-specific T-cell responses. Fifty percent of mice immunized with the HSP105 DNA vaccine completely suppressed the growth of subcutaneous Colon26 or B16.F10 cells accompanied by massive infiltration of both CD4+ T cells and CD8+ T cells into tumors. In cell transfer or depletion experiments we proved that both CD4+ T cells and CD8+ T cells induced by these vaccines play critical roles in the activation of antitumor immunity. Evidence of autoimmune reactions was not present in surviving mice that had rejected tumor cell challenges. We found that HSP105 was highly immunogenic in mice and that the HSP105 DNA vaccination induced antitumor immunity without causing autoimmunity. Therefore, HSP105 is an ideal tumor antigen that could be useful for immunotherapy or the prevention of various human tumors that overexpress HSP105, including colorectal cancer and melanoma.

Glypican-3, overexpressed specifically in human hepatocellular carcinoma, is a novel tumor marker.

With the global pandemic of hepatitis B and C infections, the incidence of Hepatocellular carcinoma (HCC) is rapidly increasing world wide. We identified glypican-3 (GPC3), a novel oncofetal gene over-expressed specifically in human HCC, as based on data of cDNA microarrays. As GPC3 is a GPI-anchored membrane protein and could be secreted, we attempted to detect secreted GPC3 protein in sera from HCC patients using Western blotting and ELISA. GPC3 protein was positive in sera of 40.0% (16/40) of HCC patients, and negative in sera from subjects with liver cirrhosis (LC) (0/13), chronic hepatitis (CH) (0/34), and healthy donors (0/60). All subjects were Japanese. Although 12 of 40 HCC patients were negative for both alpha-fetoprotein (AFP) and PIVKA-II well known tumor markers of HCC, four of these were GPC3-positive in the sera. We also observed vanishing GPC3 protein in the sera of three patients after the surgical treatment for HCC. On the other hand, immunohistochemical analysis revealed that HCC expressed GPC3 protein in all 14 HCC patients tested. In conclusion, GPC3, as defined in this study was shown to be a useful tumor marker for cancer-diagnosis for large numbers of patients with HCC.