Kenji Shibuta

The expression of tumor-rejection antigen “MAGE” genes in human gastric carcinoma

BACKGROUND & AIMS The genes MAGE-1 and MAGE-3 both encode melanoma peptide antigens recognized by major histocompatibility complex-restricted cytotoxic T lymphocytes. The antigens may be a target for immunotherapy. There is, however, little information on the expression of these genes in gastric carcinomas. Therefore, the expression of MAGE genes in gastric carcinomas was evaluated. METHODS The expression of MAGE-1, MAGE-2, and MAGE-3 genes in tumors and corresponding normal tissue specimens was studied using a reverse-transcription polymerase chain reaction. The results were analyzed according to clinicopathologic factors of the tumor. RESULTS In the 68 gastric carcinomas studied, MAGE-1, MAGE-2, and MAGE-3 messenger RNA were detected in 41%, 31%, and 38%, respectively. Fifty percent of the gastric carcinomas expressed at least one of the MAGE genes. Messenger RNA for the three MAGE proteins was not detected in normal gastric tissue. MAGE gene expression in gastric carcinomas was not associated with a significant clincopathology of the tumor. However, gene expression was lower in mucinous carcinomas (3 of 10). CONCLUSIONS MAGE-1, MAGE-2, and MAGE-3 are expressed in a high percentage of gastric carcinomas. These tumor rejection antigens may provide tumor-specific targets for immunotherapy.

Regional Expression of CXCL12/CXCR4 in Liver and Hepatocellular Carcinoma and Cell-cycle Variation during in vitro Differentiation

The CXCL12/CXCR4 system may be important in carcinoma. Expression of the a‐chemokine SDF‐lα (stromal cell derived factor‐lα)/CXCL12 mRNA is reduced in many carcinomas, yet its tissue protein expression may guide metastasis. Here we first compare the mRNA and protein expression of CXCL12 and its receptor CXCR4 in human liver, hepatocellular carcinoma, and malignant cell lines, and then assess cell cycle variation in CXCR4 expression. CXCR4 mRNA was present in most normal human tissues and malignant cell lines; it was only marginally reduced in hepatomas, while CXCL12 was markedly reduced, P<0.0001. Immuno‐histochemical staining of adjacent non‐malignant liver showed regional CXCR4 cytoplasmic and cell‐surface staining, limited to those hepatocytes around the central vein, a distribution resembling that of CXCL12. CXCL12 protein was not present in hepatocellular carcinoma cells in vivo, nor was cytoplasmic CXCR4 staining; nuclear CXCR4 protein expression in some malignant hepatocytes and CXCR4 staining of capillary endothelial cells around tumor cells were noted. In some malignant cell lines that had no CXCL12 on northern blots CXCL12 was weakly detectable by RT‐PCR or protein staining in the cytoplasm of a few cells. With a view to future manipulation of CXCL12/CXCR4 expression and growth we noted that in HT‐29 cells CXCR4 protein expression was less on confluent than on non‐confluent cells and varied during the cell cycle. Higher expression was associated most closely with the percentage of cells in the S‐phase and inversely with the percentage of cells in the G1‐phase. Treatment of HT‐29 cells with butyrate reduced CXCR4 cell surface expression and reduced the percentage of cells in S‐phase. In summary, CXCL12 protein expression parallels its mRNA, being markedly reduced in malignant cell lines and hepatomas; in liver, the regional distributions of CXCL12 and cytoplasmic CXCR4 are similar; finally, in HT‐29, CXCR4 expression correlates with the S‐phase of the cell cycle and is reduced during butyrate‐induced differentiation.

Loss of chemokine SDF-1alpha-mediated CXCR4 signalling and receptor internalization in human hepatoma cell line HepG2.

Expression of the chemokine stromal cell-derived factor-1alpha (SDF-1alpha) is absent from many carcinomas, including hepatomas. We note an early signalling defect in the hepatocellular carcinoma (HCC) cell line HepG2 that expresses the CXCR4 receptor and binds biotin-labelled SDF, but fails to stimulate downstream signalling events after engagement with SDF. In HepG2, the SDF/CXCR4 interaction did not result in calcium influx, phosphorylation and internalization of CXCR4, nor in a rapid phosphorylation of p44/42 MAP kinase. There were no CXCR4 mutations in the second chemokine binding loop or C terminal phosphorylation and internalization domains. The downstream signalling machinery in HepG2 appears to be intact since transfection of wild-type CXCR4 restored functional responsiveness. We conclude that HepG2 is unresponsive to SDF stimulation because of a defect located after receptor binding but before the activation of the signalling cascade. A hypothetical blocking molecule could hinder receptor internalization or CXCR4 signalling.

RELAXATION OF INSULIN-LIKE GROWTH FACTOR 2 GENE IMPRINTING IN ESOPHAGEAL CANCER

Paternal allele‐specific expression is identified for the insulin‐like growth factor 2 (IGF2) gene. Relaxation or loss of IGF2 imprinting, however, has been reported in several neoplasms. We studied the expression of IGF2 mRNA in 35 squamous cancers of the esophagus and searched for the presence or absence of relaxation of IGF2 imprinting. In 28 (80%) cases, IGF2 mRNA was overexpressed in the tumor tissues (T) compared to the normal tissues (N). The patients whose tumor invaded the adventitia showed a higher T/N ratio than those whose tumor was restricted to the musculi propria layer. Heterozygosity was determined by using the Apa 1 polymorphism in exon 9. Thirteen of 35 cases showed heterozygosity. In these 13 cases, a similar analysis was performed on cDNA obtained by reverse transcriptase‐polymerase chain reaction. Consequently, 7 cases disclosed relaxation of IGF2 imprinting in the tumor tissue. The cases of esophageal cancer with relaxation of IGF2 imprinting showed a higher T/N ratio and deeper invasion than those without relaxation. The results suggest that overexpression of IGF2 mRNA plays an important role in esophageal cancer and, in certain cases, is associated with relaxation of IGF2 imprinting.

Rapid intraoperative visualization of breast lesions with γ-glutamyl hydroxymethyl rhodamine green

We previously developed γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG) as a tool to detect viable cancer cells, based on the fact that the enzyme γ-glutamyltranspeptidase (GGT) is overexpressed on membranes of various cancer cells, but is not expressed in normal tissue. Cleavage of the probe by GGT generates green fluorescence. Here, we examined the feasibility of clinical application of gGlu-HMRG during breast-conserving surgery. We found that fluorescence derived from cleavage of gGlu-HMRG allowed easy discrimination of breast tumors, even those smaller than 1 mm in size, from normal mammary gland tissues, with 92% sensitivity and 94% specificity, within only 5 min after application. We believe this rapid, low-cost method represents a breakthrough in intraoperative margin assessment during breast-conserving surgery.

Efficacy of goserelin plus anastrozole in premenopausal women with advanced or recurrent breast cancer refractory to an LH-RH analogue with tamoxifen: Results of the JMTO BC08-01 phase II trial

The aim of the present study was to assess the efficacy and tolerability of a luteinizing hormone-releasing hormone (LH-RH) analogue plus an aromatase inhibitor following failure to respond to standard LH-RH analogue plus tamoxifen (TAM) in premenopausal patients. Premenopausal women with estrogen receptor (ER)-positive and/or progesterone-receptor positive, advanced or recurrent breast cancer refractory to an LH-RH analogue plus TAM received goserelin (GOS) in conjunction with anastrozole (ANA). The primary endpoint was the objective response rate (ORR). Secondary endpoints included progression-free survival (PFS), overall survival (OS), clinical benefit rate (CBR) and safety. Between September 2008 and November 2010, 37 patients were enrolled. Thirty-five patients (94.6%) had ER-positive tumors, and 36 (97.3%) had human epidermal growth factor receptor (HER) 2-negative tumors. Thirty-six (97.3%) had measurable lesions and 1 (2.7%) had only bone metastasis. The ORR was 18.9% [95% confidence interval (CI), 8.0–35.2%], the CBR was 62.2% (95% CI, 44.8–77.5%) and the median PFS was 7.3 months. Eight patients had adverse drug reactions but none resulted in discontinuation of treatment. GOS plus ANA is a safe effective treatment for premenopausal women with hormone receptor-positive, recurrent or advanced breast cancer. The treatment may become viable treatment in the future, particularly when TAM is ineffective or contraindicated. Further studies and discussion are warranted.

L‐myc Restriction Fragment Length Polymorphism in Japanese Patients with Esophageal Cancer

L‐myc polymorphism is a representative genetic trait related to an individuals susceptibility to several cancers. However, there have been no reports concerning the association between esophageal cancer and L‐myc polymorphism. To analyze the distribution of polymorphism in Japanese patients with esophageal cancer, a molecular genotyping method using a polymerase chain reaction‐based restriction fragment length polymorphism (PCR‐RFLP) was used. Based on an analysis of 65 Japanese patients with esophageal cancer and 107 healthy control subjects, a significant difference was observed in either the distribution of genotypes (P=0.012) or of allele frequencies between the two groups (P 0.004). The relative risk of esophageal cancer for genotypes including the shorter allele was 2.9 compared to the longer allele homozygote. Furthermore, the patients with S‐allele had a tendency for poor prognosis among those with three genotypes. A significant difference between the distribution of genotypes and the incidence of lymph node metastasis was found based on the clinicopathological features of the cancers. These results suggest that L‐myc polymorphism may be implicated as a genetic trait affecting an individuals susceptibility to esophageal cancer, at least among Japanese patients.

The relevance of intrinsic subtype to clinicopathological features and prognosis in 4,266 Japanese women with breast cancer

BackgroundEstrogen receptor (ER), progesterone receptor (PgR), and HER2 expression status in breast cancer function as prognostic and predictive factors that enable individualized treatment. Intrinsic subtype classification has also been performed based on these and other biological and prognostic characteristics. However, clinical analysis of such subtypes in a large number of Japanese breast cancer patients has not yet been reported.MethodsBetween January 2003 and December 2007, 4,266 patients with primary breast cancer were registered. Four subtypes based on immunohistochemically evaluated ER/PgR/HER2 status, clinicopathological features, and prognosis were analyzed retrospectively.ResultsThe following subtype distribution was observed: luminal A type (ER+ and/or PgR+, HER2−), 3,046 cases (71%); luminal B type (ER+ and/or PgR+, HER2+), 321 cases (8%); HER2 type (ER−, PgR−, HER2+), 398 cases (9%); and triple negative (TN) type (ER−, PgR−, HER2−), 501 cases (12%). The HER2+ subtypes (luminal B and HER2 types) had a significantly higher incidence of lymph node metastasis and lymphatic permeation, while the hormone receptor negative subtypes (HER2 and TN types) showed a significantly higher nuclear grade. Overall, patients with HER2-type and TN-type disease had a significantly poorer prognosis than other subtypes.ConclusionIntrinsic breast cancer subtypes are associated with clinicopathological features and prognosis in Japanese women. Long-term clinical observation of the relationship between each subtype and therapies used should provide useful information for selecting appropriately tailored treatments.

Genetic polymorphism of N-acetyltransferase 2 in patients with esophageal cancer

OBJECTIVE:N-Acetylation polymorphism is a representative genetic trait related to an individuals susceptibility to several cancers. However, there remains a controversy and no consensus concerning whether there is a true association between esophageal cancer and N-acetylation polymorphism.METHODS:To analyze the distribution of N-acetyltransferase 2 polymorphism in Japanese patients with esophageal squamous cell cancer, a molecular genotyping method using a polymerase chain reaction-based restriction fragment length polymorphism was used.RESULTS:Based on an analysis of 71 Japanese patients with esophageal squamous cell cancer and 329 healthy control subjects, the distribution of the slow acetylator phenotype was significantly higher in esophageal cancer patients than in the controls (19.7% and 9.4%, respectively, p = 0.040). The odds ratio of esophageal cancer for the slow phenotype was 2.55 (95% CI = 1.15–5.65, p = 0.023) compared with the rapid type. Furthermore, a significant difference between the distribution of acetylator phenotype and the incidence of lymph node metastasis and lymphatic involvement was found based on the clinicopathological features of these cancers. Esophageal cancer patients with a higher smoking exposure history tended to have the rapid acetylator phenotype.CONCLUSION:These results suggest that N-acetylation polymorphism may be implicated as a genetic trait affecting an individuals susceptibility and biological behavior of esophageal squamous cell cancer.

Rapid diagnosis of lymph node metastasis in breast cancer using a new fluorescent method with γ-glutamyl hydroxymethyl rhodamine green.

Sentinel lymph node biopsy is performed as a standard procedure in breast cancer surgery, and the development of quick and simple methods to detect metastatic lesions is in high demand. Here, we validated a new fluorescent method using γ-glutamyl hydroxymethyl rhodamine green to diagnose metastatic lymph nodes in breast cancer. One hundred and forty-nine lymph nodes from 38 breast cancer patients were evaluated in this study. Comparison of fluorescent and pathological images showed that this fluorescent method was successful for visualizing breast cancer cells in lymph nodes. This method had a sufficiently high sensitivity (97%), specificity (79%) and negative predictive value (99%) to render it useful for an intraoperative diagnosis of cancer. These preliminary findings suggest that this novel method is useful for distinguishing non-cancerous specimens from those in need of careful examination and could help save time and cost for surgeons and pathologists.