Shigefumi Yoshino

Th2 subset dominance among peripheral blood T lymphocytes in patients with digestive cancers.

BACKGROUND Two types of helper T cells (Th), which are categorized as Th1 and Th2 on the basis of cytokine production, have been reported. Th1 cells produce interleukin (IL)-2 and interferon (IFN)-gamma, while Th2 cells secrete IL-4, IL-6, and IL-10. We assessed the intracellular cytokine profiles of CD3/CD4 positive lymphocytes (CD4+ T-cells) in peripheral blood in patients with digestive cancers. METHODS Peripheral blood samples were collected from 50 patients with digestive cancers and 35 healthy volunteers. The proportions of CD4+ T-cells producing intracellular cytokines were determined using flow cytometry. RESULTS The percentages (mean +/- SD) of CD4+ T-cells producing IL-4, IL-6, and IL-10 in the cancer group (73.9% +/- 13.0%, 73.0% +/- 16.6%, and 58.0% +/- 21.0%, respectively) were significantly higher than in the healthy group (37.4% +/- 12.4%, 37.8% +/- 13.5%, and 34.0% +/- 14.1%, respectively; P <0.01). Proportions of CD4+ T-cells producing IL-4, IL-6, and IL-10 in 10 patients undergoing curative resection had decreased significantly 1 month after surgery (P <0.01). No significant difference was noted between groups in the percentages of CD4+ T-cells producing IFN-gamma. CONCLUSIONS Th2-dominant status develops in cancer patients. Such lymphocyte evaluations could find applications in diagnosis and therapeutic monitoring of cancer patients.

Anticachectic effects of Coptidis rhizoma, an anti-inflammatory herb, on esophageal cancer cells that produce interleukin 6

Herbs as alternative cancer therapies have attracted a great deal of recent attention due to their low toxicity and costs. In this study, the antitumor activity and anticachectic effect of Coptidis rhizoma, an anti-inflammatory herb, were investigated in nude mice carrying a human esophageal cancer cell line YES-2, which constitutively secretes interleukin-6 (IL-6) and induces cachexia when injected into these mice. In this study, in vivo growth of YES-2 cells was not affected by an oral supplement containing the extract powder of C. rhizoma at a final concentration of 1% (CR supplement). However, in comparison with normal diet, CR supplement significantly attenuated weight loss of tumor-bearing mice without a change in food or water intake. Tumor IL-6 levels were significantly lower in mice treated with CR supplement than in control mice (P<0.001). Serum IL-6 was detectable in four (50%) of eight control mice; IL-6 was not detected in mice treated with CR supplement. We also confirmed that berberine (8-32 microM), a major component of C. rhizoma, dose-dependently inhibited secretion of IL-6 by YES-2 cells in vitro. Moreover, reverse transcription-PCR assay showed that treatment of YES-2 cells with berberine (8-32 microM) for 24 h reduced IL-6 mRNA expression. Our results suggest that C. rhizoma may have an anticachectic effect on esophageal cancer and an effect is associated with the ability of berberine to down-regulate tumor IL-6 production.

Sequential paclitaxel followed by tegafur and uracil (UFT) or S-1 versus UFT or S-1 monotherapy as adjuvant chemotherapy for T4a/b gastric cancer (SAMIT): a phase 3 factorial randomised controlled trial

BACKGROUND The prognosis for locally advanced gastric cancer is poor despite advances in adjuvant chemotherapy. We did the Stomach cancer Adjuvant Multi-Institutional group Trial (SAMIT) to assess the superiority of sequential treatment (paclitaxel then tegafur and uracil [UFT] or paclitaxel then S-1) compared with monotherapy (UFT or S-1) and also the non-inferiority of UFT compared with S-1. METHODS We did this randomised phase 3 trial with a two-by-two factorial design at 230 hospitals in Japan. We enrolled patients aged 20-80 years with T4a or T4b gastric cancer, who had had D2 dissection and a ECOG performance score of 0-1. Patients were randomly assigned to one of four treatment groups with minimisation for tumour size, lymph node metastasis, and study site. Patients received UFT only (267 mg/m(2) per day), S-1 only (80 mg/m(2) per day) for 14 days, with a 7-day rest period or three courses of intermittent weekly paclitaxel (80 mg/m(2)) followed by either UFT, or S-1. Treatment lasted 48 weeks in monotherapy groups and 49 weeks in the sequential treatment groups. The primary endpoint was disease-free survival assessed by intention to treat. We assessed whether UFT was non-inferior to S-1 with a non-inferiority margin of 1·33. This trial was registered at UMIN Clinical Trials Registry, number C000000082. FINDINGS We randomly assigned 1495 patients between Aug 3, 2004, and Sept 29, 2009. 374 patients were assigned to receive UFT alone, 374 to receive S-1 alone, 374 to received paclitaxel then UFT, and 373 to receive paclitaxel then S-1. We included 1433 patients in the primary analysis after at least 3 years of follow-up (359, 364, 355, and 355 in each group respectively). Protocol treatment was completed by 215 (60%) patients in the UFT group, 224 (62%) in the S-1 group, 242 (68%) in the paclitaxel then UFT group, and 250 (70%) in the paclitaxel then S-1 group. 3-year disease-free survival for monotherapy was 54·0% (95% CI 50·2-57·6) and that of sequential treatment was 57·2% (53·4-60·8; hazard ratio [HR] 0·92, 95% CI 0·80-1·07, p=0·273). 3-year disease-free survival for the UFT group was 53·0% (95% CI 49·2-56·6) and that of the S-1 group was 58·2% (54·4-61·8; HR 0·81, 95% CI 0·70-0·93, p=0·0048; pnon-inferiority=0·151). The most common grade 3-4 haematological adverse event was neutropenia (41 [11%] of 359 patients in the UFT group, 48 [13%] of 363 in the S-1 group, 46 [13%] of 355 in the paclitaxel then UFT group, and 83 [23%] of 356 in the paclitaxel then S-1 group). The most common grade 3-4 non-haematological adverse event was anorexia (21 [6%], 24 [7%], seven [2%], and 18 [5%], respectively). INTERPRETATION Sequential treatment did not improve disease-free survival, and UFT was not non-inferior to S-1 (and S-1 was superior to UFT), therefore S-1 monotherapy should remain the standard treatment for locally advanced gastric cancer in Japan. FUNDING Epidemiological and Clinical Research Information Network.

Detection of amplified oncogenes by genome DNA microarrays in human primary esophageal squamous cell carcinoma: comparison with conventional comparative genomic hybridization analysis

Oncogene amplification in 20 surgically resected esophageal squamous cell carcinomas (ESCC) was examined with DNA microarrays that detected 57 oncogenes and two reference DNA. Alterations in DNA copy numbers detected by microarrays were compared to those obtained by conventional comparative genomic hybridization (CGH). Amplification of eight oncogenes (CCND1, FGF3/FGF4, EMS1, SAS, ERBB2, PDGFRA, MYC, and BCL2) was detected by DNA microarrays in 9 of 20 tumors. Although ERBB2 was 23.2 times higher than the control level in one case, the average magnitude of gene amplification was approximately two to four times that of the control level. EMS1, CCND1, and FGF3/FGF4, which are all located on 11q13, were amplified in 7, 5, and 4 of 20 ESCC, respectively, and they were coamplified in 3 tumors. A comparison of genome DNA microarrays and CGH data revealed that although most amplified oncogenes were included in chromosomal regions for which DNA copy number gains were detected by conventional CGH, not all amplified genes on microarrays showed concomitant DNA copy number gains on CGH. In conclusion, microarrays of oncogenes are useful for the comprehensive identification of amplified oncogenes and for analysis of areas of specific amplification within chromosomal regions with DNA copy number increases detected by CGH analysis.

Anticachectic effects of the natural herb Coptidis rhizoma and berberine on mice bearing colon 26/clone 20 adenocarcinoma

We previously showed that the natural herb Coptidis rhizoma has an anticachectic effect in nude mice bearing human esophageal cancer cells. We further investigated this phenomenon by examining the anticachectic effect of C. rhizoma in syngeneic mice bearing colon 26/clone 20 carcinoma cells, which cause IL‐6–related cachexia after cell injection. We evaluated nutritional parameters such as serum glucose level and wasting of adipose tissue and muscle in tumor‐bearing and non‐tumor‐bearing mice treated with C. rhizoma (CR) supplement or a normal diet. IL‐6 levels in those mice were quantified by ELISA and real‐time RT‐PCR. CR supplementation significantly attenuated weight loss in tumor‐bearing mice without changing food intake or tumor growth. Furthermore, these mice maintained good nutritional status. IL‐6 mRNA levels in tumors and spleens and IL‐6 protein levels in tumors and sera were significantly lower in tumor‐bearing mice treated with CR supplement than in those treated with a normal diet. CR supplementation did not affect food intake, body weight, nutritional parameters and IL‐6 levels in non‐tumor‐bearing mice. An in vitro study showed that C. rhizoma and its major component, berberine, inhibited IL‐1–induced IL‐6 mRNA expression in a dose‐dependent manner in colon 26/clone 20 cells. Our results showed that C. rhizoma exerts an anticachectic effect on colon 26/clone 20–transplanted mice and that its effect is associated with tumor IL‐6 production. We also suggest that its effect might be due to berberine.

Expression of estrogen receptor-α and -β mRNAs in human gastric cancer

Abstract To clarify the roles of estrogen receptors (ERs) in gastric cancers, we evaluated expression of ER-α and ER-β mRNAs in 41 pairs of tumorous and non-tumorous tissues of gastric cancer patients and in six gastric cancer cell lines by reverse transcription-polymerase chain reaction method. ER-α and ER-β mRNAs were detected in 21 (51%) and 30 (73%) of 41 tumors and in 15 (37%) and 36 (88%) of 41 corresponding normal tissues, respectively. There were no statistically significant associations between expression of ER-α and/or ER-β mRNAs in tumors and clinicopathologic factors. Between the tumorous and normal tissues, expression of ER-α and ER-β mRNAs were changed in 20 (49%) and unchanged in 21 (51%) of the 41 cases. The incidences of lymph node metastasis and liver metastasis were significantly higher in changed cases than in unchanged cases ( P =0.031 and P =0.021, respectively). We confirmed that ER-α and ER-β mRNA were expressed in 2 and 6 of the six gastric cancer cell lines, respectively. Together with this finding, our results indicate that ER-β mRNAs are preferentially expressed in gastric cancers. Our data also suggest that altered expression of ER-α and ER-β mRNAs in tumors compared with corresponding normal gastric tissues is related to increased metastatic potential in gastric cancers. Further studies are needed to clarify the role of ERs in gastric cancers.

A phase I clinical trial of vaccination with KIF20A-derived peptide in combination with gemcitabine for patients with advanced pancreatic cancer.

KIF20A (RAB6KIFL) belongs to the kinesin superfamily of motor proteins, which play critical roles in the trafficking of molecules and organelles during the growth of pancreatic cancer. Immunotherapy using a previously identified epitope peptide for KIF20A is expected to improve clinical outcomes. A phase I clinical trial combining KIF20A-derived peptide with gemcitabine (GEM) was therefore conducted among patients with advanced pancreatic cancer who had received prior therapy such as chemotherapy and/or radiotherapy. GEM was administered at a dose of 1000 mg/m2 on days 1, 8, and 15 in a 28-day cycle. The KIF20A-derived peptide was injected subcutaneously on a weekly basis in a dose-escalation manner (doses of 0.5, 1, and 3 mg/body; 3 patients/cohort). Safety and immunologic parameters were assessed. No severe adverse effects of grade 3 or higher related to KIF20A-derived peptide were observed. Of the 9 patients who completed at least one course of treatment, interferon-&ggr; (IFN-&ggr;)-producing cells were induced in 4 of 9 patients (P2, P3, P6, and P7), and IFN-&ggr;-producing cells were increased in 4 of the 9 patients (P1, P5, P8, and P9). Four of the 9 patients achieved stable disease. The disease control rate was 44%. The median survival time after first vaccination was 173 days and 1-year survival rate was 11.1%. IFN-&ggr;-producing cells were induced by the KIF20A-derived peptide vaccine at a high rate, even in combination with GEM. These results suggest that this combination therapy will be feasible and promising for the treatment of advanced pancreatic cancer.

Natural killer activity and serum immunosuppressive acidic protein levels in esophageal and gastric cancers

The natural killer (NK) activity of peripheral blood mononuclear cells and serum immunosuppressive acidic protein (IAP) levels were examined in patients with esophageal or gastric cancer, before and after surgery. Patients with stage IV esophageal or stage IV gastric cancer had significantly lower NK activity (39.5±14.8% and 37±11.6%, respectively), and also higher serum IAP levels (778±264 μg/mL and 633±156 μg/mL, respectively), than the corresponding control values (50±5.6% and 375±26 μg/mL, respectively). Patients with esophageal or gastric cancer who underwent curative resection had high NK activity (54.8±11.6% and 54.8±8.0%, respectively), and low IAP levels (471±116 μg/mL and 490±42 μg/mL, respectively), compared with those who underwent non-curative resection. Patients who underwent non-curative resection had lower NK activity and higher serum IAP levels than those who underwent curative resection, even 1 month after surgery. Mononuclear cells in the regional lymph nodes and tumor specimens showed significantly lower NK activity than those in the peripheral blood and spleen. Thus, NK activity and the IAP level reflected the immunocompetence, clinical course, and surgical curability of those patients. NK cells appeared not to have any significant antitumor activity in the regional lymph nodes or in the tumor itself, although they were still active in the peripheral blood.

Phase I study of irinotecan and doxifluridine for metastatic colorectal cancer focusing on the UGT1A1*28 polymorphism

(Cancer Sci 2010; 101: 722–727)

Successful immunogene therapy using colon cancer cells (colon 26) transfected with plasmid vector containing mature interleukin-18 cDNA and the Igκ leader sequence

IL-18 is a novel cytokine that induces interferon (IFN)-γ secretion and plays an important role in antitumor immunity. In the present study, we constructed plasmid vectors encoding the murine mature IL-18 cDNA linked with the Igκ leader sequence and the pro-IL-18 cDNA to estimate the efficacy of the mature IL-18 vector and to evaluate IL-18–producing tumor cells as a tumor vaccine. Colon 26 cells were transfected with the abovementioned vectors or with vector alone (mock). Reverse transcription-polymerase chain reaction analysis showed increased expression of murine IL-18 cDNA in both mature IL-18 and pro-IL-18 transfectants in comparison to that in mock transfected cells. The ability of the culture supernatants of mature IL-18 transfectants to induce IFN-γ secretion was extremely high (40–140 pg/106 cells) in comparison to that of pro-IL-18 transfectants (4–18 pg/106 cells). When injected into syngeneic BALB/c mice, the growth of mature IL-18 transfectants, but not pro-IL-18 transfectants, was significantly less than that in mock transfected cells (P<.01, by ANOVA and analysis of covariance). In addition, injection of colon 26 or Meth-A cells into mice immunized with a mature IL-18 transfectant revealed acquired immunity. Depletion of natural killer cells did not affect the growth of transfectants. However, the growth inhibitory effects were partially abrogated following treatment with anti-CD4+ and anti-CD8+ antibodies. These data suggest that the rejection of mature IL-18/colon 26 cells was mediated through T-cell activation. Gene therapy using mature IL-18 transfectants containing a plasmid vector and the Igκ leader sequence may be a useful tumor vaccine. Cancer Gene Therapy (2001) 8, 9–16