Long-term health status as measured by EQ-5D among patients with metastatic breast cancer: comparison of first-line oral S-1 and taxane therapies in the randomized phase III SELECT BC trial.

PURPOSE: The goal of chemotherapy for metastatic breast cancer (MBC) is to prolong survival and maintain health-related quality of life. This study aimed to evaluate long-term health status of patients with MBC who participated in the phase III randomized SELECT BC trial. METHODS: In the SELECT BC trial, patients were randomly allocated to the S-1 or taxane (paclitaxel or docetaxel) arm. Health status was assessed by EQ-5D at pre-treatment, 3 and 6 months after randomization, and every 6 months thereafter to the extent possible. Least square mean scores were assessed to compare EQ-5D index values between groups. Time to deterioration analysis was also performed by defining the minimally important difference of EQ-5D as 0.05 or 0.1. RESULTS: The number of patients for EQ-5D analysis was 175 and 208 in the taxane and S-1 arms, respectively. Least square mean EQ-5D index values up to 60 months were 0.741 (95 % CI [0.713-0.769]) in the taxane arm and 0.748 [0.722-0.775] in the S-1 arm. The EQ-5D index value during PFS up to 12 months in the S-1 was superior to the corresponding index value in the taxane (0.812 [0.789-0.834] vs. 0.772 [0.751-0.792], P = 0.009). Time to deterioration analysis also revealed that S-1 significantly delayed the deterioration of EQ-5D index value during the period before progression (P = 0.002 and 0.003). CONCLUSIONS: Our findings suggest that the EQ-5D index value was higher in patients treated with S-1 during first-line chemotherapy. Considering non-inferiority of S-1 in terms of OS, obtained quality-adjusted life years may be greater in the S-1 arm.

Randomized trial of preoperative docetaxel with or without capecitabine after 4 cycles of 5-fluorouracil­ epirubicin­cyclophosphamide (FEC) in early-stage breast cancer: exploratory analyses identify Ki67 as a predictive biomarker for response to neoadjuvant chemotherapy.

This randomized, multicenter study compared the efficacy of docetaxel with or without capecitabine following fluorouracil/epirubicin/cyclophosphamide (FEC) therapy in operable breast cancer and investigated the role of Ki67 as a predictive biomarker. Patients were randomized to 4 cycles of docetaxel/capecitabine (docetaxel: 75 mg/m2 on day 1; capecitabine: 1,650 mg/m2 on days 1­14 every 3 weeks) or docetaxel alone (75 mg/m2 on day 1 every 3 weeks) after completion of 4 cycles of FEC (5-fluorouracil 500 mg/m2, epirubicin 100 mg/m2 and cyclophosphamide 500 mg/m2 on day 1 every 3 weeks). The primary endpoint was the pathological complete response (pCR) rate. Predictive factor analysis was conducted using clinicopathological markers, including hormone receptors and Ki67 labeling index (Ki67LI). A total of 477 patients were randomized; the overall response in the docetaxel/capecitabine and docetaxel groups was 88.3 and 87.4 %, respectively. There were no significant differences in the pCR rate (docetaxel/capecitabine: 23 %; docetaxel: 24 %; p = 0.748), disease-free survival, or overall survival. However, patients with mid-range Ki67LI (10­20 %) showed a trend towards improved pCR rate with docetaxel/capecitabine compared to docetaxel alone. Furthermore, multivariate logistic regression analysis showed pre-treatment Ki67LI (odds ratio 1.031; 95 % CI 1.014­1.048; p = 0.0004) to be a significant predictor of pCR in this neoadjuvant treatment setting. Docetaxel/capecitabine (after 4 cycles of FEC) did not generate significant improvement in pCR compared to docetaxel alone. However, exploratory analyses suggested that assessment of pre-treatment Ki67LI may be a useful tool in the identification of responders to preoperative docetaxel/capecitabine in early-stage breast cancer.

Hypoxia-induced Jagged2 promotes breast cancer metastasis and self-renewal of cancer stem-like cells.

Notch signaling is often and aberrantly activated by hypoxia during tumor progression; however, the exact pathological role of hypoxia-induced Notch signaling in tumor metastasis is as yet poorly understood. In this study, we aimed to define the mechanism of Notch-ligand activation by hypoxia in both primary tumor and bone stromal cells in the metastatic niche and to clarify their roles in tumor progression. We have analyzed the expression profiles of various Notch ligands in 779 breast cancer patients in GEO database and found that the expression of Jagged2 among all five ligands is most significantly correlated with the overall- and metastasis-free survival of breast cancer patients. The results of our immunohistochemical (IHC) analysis for Jagged2 in 61 clinical samples also revealed that both Jagged2 and Notch signaling were strongly upregulated at the hypoxic invasive front. Activation of Jagged2 by hypoxia in tumor cells induced EMT and also promoted cell survival in vitro. Notably, a γ-secretase inhibitor significantly blocked Notch-mediated invasion and survival under hypoxia by promoting expression of E-cadherin and inhibiting Akt phosphorylation. Importantly, Jagged2 was also found to be upregulated in bone marrow stroma under hypoxia and promoted the growth of cancer stem-like cells by activating their Notch signaling. Therefore, hypoxia-induced Jagged2 activation in both tumor invasive front and normal bone stroma has a critical role in tumor progression and metastasis, and Jagged2 is considered to be a valuable prognostic marker and may serve as a novel therapeutic target for metastatic breast cancer.

Laparoscopy-assisted appendectomy through an umbilical port in children.

INTRODUCTION: We report surgical techniques for single-incision laparoscopy-assisted surgery (SILAS) in the treatment of pediatric acute appendicitis. METHODS: We performed SILAS in 15 cases of acute appendicitis between January and September of 2009. SILAS is a surgical method that involves making the incision at the umbilicus, inserting a wound retractor XS, suspending the abdominal wall with a hook, and appendectomy with the same procedures as conventional appendectomy. RESULTS: SILAS appendectomy was performed in all 15 cases with the exception of one case where one 3-mm port was added. Compared to open appendectomy, blood loss was significantly lower and postoperative hospitalization time was shorter, although there was no significant decrease in operative time, or postoperative fasting time. No postoperative complications, such as wound infection, intestinal obstruction, intra-abdominal abscess, or bleeding, were encountered. CONCLUSION: SILAS was safely performed and is superior to open appendectomy with regard to cosmetic outcome.

[Novel biodegradable materials for drug delivery systems (DDS)].

The authors provide three new and different types of fibrin gels (FCs) and a chitosan sheet (BC) using an ultraviolet (UV)-crosslinking method. They are 1) FC-UV, 2) gelatin entrapped FC; FC (Gp)-UV, 3) chitosan entrapped FC; FC (Cs)-UV and 4) BC-UV. Each material was loaded with aqueous cis-platinum (CDDP), and both the degradation of the drug carriers and the release profile of the CDDP were examined in vitro. The FCs, 1)-3), gradually degraded and dissolved within 10-12 days. The BC, 4), maintained its original weight for more than 30 days. Each FC showed a sustained release of CDDP for 10 days, while BC provided an initial bursting of the loaded drug. New materials 2) and 3) show great potential as drug carriers for DDS and further in vivo studies are now proceeding.

Adenomatous polyposis coli (APC) gene promoter hypermethylation in primary breast cancers.

Similar to findings in colorectal cancers, it has been suggested that disruption of the adenomatous polyposis coli (APC)/beta-catenin pathway may be involved in breast carcinogenesis. However, somatic mutations of APC and beta- catenin are infrequently reported in breast cancers, in contrast to findings in colorectal cancers. To further explore the role of the APC/beta-catenin pathway in breast carcinogenesis, we investigated the status of APC gene promoter methylation in primary breast cancers and in their non-cancerous breast tissue counterparts, as well as mutations of the APC and beta- catenin genes. Hypermethylation of the APC promoter CpG island was detected in 18 of 50 (36%) primary breast cancers and in none of 21 non-cancerous breast tissue samples, although no mutations of the APC and beta- catenin were found. No significant associations between APC promoter hypermethylation and patient age, lymph node metastasis, oestrogen and progesterone receptor status, size, stage or histological type of tumour were observed. These results indicate that APC promoter CpG island hypermethylation is a cancer-specific change and may be a more common mechanism of inactivation of this tumour suppressor gene in primary breast cancers than previously suspected.

Induction of antitumor immunity by vaccination of dendritic cells transfected with MUC1 RNA.

Dendritic cells (DC) are potent APCs. In this study, murine bone marrow-derived DC were transfected with RNA encoding the MUC1 Ag that is aberrantly overexpressed in human breast and other carcinomas. The MUC1 RNA-transfected DC exhibited cell surface expression of MUC1 and costimulatory molecules. After injection at the base of the tail, the transfected DC were detectable in inguinal lymph nodes by dual immunochemical staining. Vaccination of wild-type mice with MUC1 RNA-transfected DC induced anti-MUC1 immune responses against MUC1-positive MC38/MUC1, but not MUC1-negative, tumor cells. Mice immunized with the transfected DC were protected against challenge with MC38/MUC1 tumor cells. Furthermore, mice with established MC38/MUC1 tumors were eliminated after receiving the vaccination. CTLs isolated from mice immunized with the transfected DC exhibited specific cytolytic activity against MC38/MUC1 tumor cells. In contrast to these findings, there was little if any anti-MUC1 immunity induced with the transfected DC in MUC1 transgenic (MUC1.Tg) mice. However, coadministration of the transfected DC and IL-12 reversed the unresponsiveness to MUC1 Ag in MUC1.Tg mice and induced MUC1-specific immune responses. These findings demonstrate that vaccination of DC transfected with MUC1 RNA and IL-12 reverses tolerance to MUC1 and induces immunity against MUC1-positive tumors.

[A novel fibrin clot with a sustained release of cis-platinum (CDDP)].

The authors devised a novel fibrin clot (FC) using an ultra-violet (UV)-crosslinking method. CDDP was impregnated into FCs, and the release profiles of the CDDP were examined in vitro. The microstructures of the FCs were studied with scanning electron microscopy (SEM). The release of CDDP from the FC-UV-CDDP was maintained for 10 days, while that from the FC-CDDP showed initial bursting with a following plateau of CDDP concentrations. SEM of UV-crosslinked FCs revealed highly organized, close and homogeneous micropore structures. Native FCs and non-crosslinked FCs showed rough fibrin networks with entangling fibrin fibers. These microstructural differences may play important roles in the release profiles of CDDP. Our newly devised UV-crosslinked material is promising as a drug carrier for sustained release.

Activation of antitumor cytotoxic T lymphocytes by fusions of human dendritic cells and breast carcinoma cells.

We have reported that fusions of murine dendritic cells (DCs) and murine carcinoma cells reverse unresponsiveness to tumor-associated antigens and induce the rejection of established metastases. In the present study, fusions were generated with primary human breast carcinoma cells and autologous DCs. Fusion cells coexpressed tumor-associated antigens and DC-derived costimulatory molecules. The fusion cells also retained the functional potency of DCs and stimulated autologous T cell proliferation. Significantly, the results show that autologous T cells are primed by the fusion cells to induce MHC class I-dependent lysis of autologous breast tumor cells. These findings demonstrate that fusions of human breast cancer cells and DCs activate T cell responses against autologous tumors.

Reversal of tolerance to human MUC1 antigen in MUC1 transgenic mice immunized with fusions of dendritic and carcinoma cells.

Immunological unresponsiveness established by the elimination or anergy of self-reactive lymphocyte clones is of importance to immunization against tumor-associated antigens. In this study, we have investigated induction of immunity against the human MUC1 carcinoma-associated antigen in MUC1 transgenic mice unresponsive to MUC1 antigen. Immunization of adult MUC1 transgenic mice with irradiated MUC1-positive tumor cells was unsuccessful in reversing unresponsiveness to MUC1. By contrast, fusions of dendritic cells with MUC1-positive tumor cells induced cellular and humoral immunity against MUC1. Immunization with the dendritic cell fusions that express MUC1 resulted in the rejection of established metastases and no apparent autoimmunity against normal tissues. These findings demonstrate that unresponsiveness to the MUC1 tumor-associated antigen is reversible by immunization with heterokaryons of dendritic cells and MUC1-positive carcinoma cells.

Establishment and characterization of a new human extrahepatic bile duct carcinoma cell line (ICBD-1).

A new human extrahepatic bile duct carcinoma cell line (ICBD-1) was established from surgically resected tumor of a 71-year-old Japanese male patient. ICBD-1 cells proliferate in a layer with a population doubling time of 31.5 h and secrete tissue polypeptide antigen. ICBD-1 cells have a tetraploid pattern with a DNA index of 1.83 and chromosome counts showed equally distribution in a range from 65 to 69. IC50 values for ICBD-1 cells were 200 ng/ml for adriamycin, 400 ng/ml for mitomycin C, 2 microg/ml for cisplatin and 300 ng/ml for 5-fluorouracil. ICBD-1 cells were successfully transplanted to male nude mice, inducing progressive tumor growth. Histologically, nude mouse tumors were less differentiated than the original human tumor. Tumor cells showed alveolar structures with thin fibrous stroma, classified as poorly-differentiated adenocarcinoma. ICBD-1 is the fourth established cell line that originate from extrahepatic bile duct carcinoma and it will be applicable for the experimental studies of this disease.

Induction of antitumor activity by immunization with fusions of dendritic and carcinoma cells.

Dendritic cells (DCs) are potent antigen-presenting cells that prime naive cytotoxic T-cells (CTLs). In this study, we have fused DCs with MC38 carcinoma cells. The fusion cells were positive for major histocompatibility (MHC) class I and II, costimulating molecules and intercellular cell adhesion molecule-1 (ICAM-1). The results show that the fusion cells stimulate naive T cells in the primary mixed lymphocyte reaction (MLR) and induce MC38 tumor-specific CTLs in vivo. Antibody-mediated depletion experiments demonstrate that induction of CD4+ and CD8+ CTLs protects against challenge with tumor cells. We also show that immunization with the fusion cells induces rejection of established metastases. These findings represent the first demonstration that fusions of DCs and tumor cells can be used in the treatment of cancer.

Induction of antigen-specific antitumor immunity with adenovirus-transduced dendritic cells.

Transduction of dendritic cells (DC) can result in presentation of tumor-associated antigens and induction of immunity against undefined epitopes. The present studies demonstrate adenovirus (Ad)-mediated transduction of the beta-galactosidase gene in mouse DC. Similar transductions have been obtained with the gene encoding the DF3/MUC1 tumor-associated antigen. We show that the Ad-transduced DC are functional in primary allogeneic mixed lymphocyte reactions. Mice immunized with Ad-transduced DC develop cytotoxic T lymphocytes that are specific for the beta-galactosidase or DF3/MUC1 antigens. The results also demonstrate that Ad MUC1-transduced DC induce a specific response which inhibits the growth of DF3/MUC1-positive tumors. These findings support the usefulness of Ad-transduced DC for in vivo immunization against tumor-associated antigens.

Epithelial-cadherin gene is not mutated in ductal carcinomas of the breast.

We investigated mutations of the epithelial (E)-cadherin gene and loss of heterozygosity (LOH) at flanking loci using three microsatellite markers on the long arm of chromosome 16 in 25 ductal carcinomas of the breast. Expression of E-cadherin was also investigated immunohistochemically. No mutations were detected in exons 6 through 9 of the E-cadherin gene. LOH was observed more frequently (42%) at D16S402 (16q-ter) than at D16S421 (16q22.3-q23.1) (17%), which is located near the E-cadherin gene. Expression of E-cadherin was observed at the cell borders in 92% (11/12) of the tumors examined. The absence of mutations in the E-cadherin gene and its conserved expression suggest that inactivation of E-cadherin does not contribute significantly to the invasion or metastatic potential of ductal carcinomas of the breast. Furthermore, the high frequency of LOH at 16q-ter suggests the existence of another tumor suppressor gene which may play a crucial role in the genesis of ductal carcinomas of the breast.

The sequential accumulation of genetic alterations characteristic of the colorectal adenoma-carcinoma sequence does not occur between gastric adenoma and adenocarcinoma.

We screened 30 gastric adenomas and 72 gastric adenocarcinomas for four genetic alterations (mutations of the K-ras, APC, and p53 genes and loss of heterozygosity at the DCC genetic locus) which are known to occur during colorectal tumourigenesis. We used polymerase chain reaction (PCR) single-strand conformation polymorphism analysis to detect mutations. Loss of heterozygosity (LOH) at the DCC locus was ascertained directly by performing PCR on the variable number of tandem repeats within the gene. Mutations of the K-ras gene were not detected in any gastric adenoma or carcinoma. APC mutations were detected in 20 per cent (6/30) of the adenomas but in only 1.4 per cent (1/72) of the carcinomas. In contrast, the p53 gene was frequently mutated in carcinomas (35 per cent; 25/72), but not in adenomas. LOH at the DCC locus was a frequent occurrence in carcinomas (58 per cent; 11/19 informative cases) but was infrequent in adenomas (14 per cent; 1/7). Alterations of the p53 and DCC genes occurred frequently both in differentiated and in undifferentiated gastric carcinomas. The considerable differences in the incidences of genetic alterations between gastric adenoma and carcinoma indicate that the sequential development of gastric carcinoma from adenoma is uncommon in gastric carcinogenesis.

A point mutation, C to T, in exon 8 of the porphobilinogen deaminase gene in a Japanese family with acute intermittent porphyria.

Acute intermittent porphyria (AIP) is an autosomal dominant disease characterized by a deficiency of porphobilinogen deaminase (PBGD). To date, only two mutations have been reported in Japanese patients. We report here another mutation of the gene in a Japanese patient. Analysis of the PCR amplified DNA fragments of the gene by direct-sequencing method revealed the gene abnormality responsible for the disease. The mutation found was a point mutation, C to T, in exon 8 of the gene at position 346 of the housekeeping cDNA from the translation codon ATG. This mutation resulted in an Arg116 to Trp substitution. Four carriers in the family were successfully diagnosed by detecting the mutation using restriction analysis of PCR products.

Frequent loss of heterozygosity at the deleted in colorectal carcinoma gene locus and its association with histologic phenotypes in breast carcinoma.

Loss of heterozygosity (LOH) at the deleted in colorectal carcinoma gene (DCC), a tumour suppressor gene that encodes a protein with high homology to the neural cell adhesion molecule, was investigated in 42 surgical specimens of primary breast carcinoma. LOH was analysed in breast carcinoma by amplifying the DNA, spanning a variable number of tandem repeats site and a restriction fragment length polymorphism site within DCC, using the polymerase chain reaction (PCR). Cell sorting was used to enrich carcinoma cells. The expression of the DCC gene was also investigated using a reverse transcription-PCR method followed by Southern blot hybridization. LOH at the DCC locus was detected in 15 (51.7%) of 29 informative cases and 10 of 13 cases having DCC-LOH showed distinct reduction or loss of DCC expression. The DCC-LOH was closely associated with certain histological phenotypes: DCC-LOH was more frequent in scirrhous carcinomas than in solid-tubular ones (P < 0.05), and was also more frequent in carcinomas with infiltration into fat tissue over the mammary gland than in those without infiltration (P < 0.05). DCC-LOH was detected in invasive lobular carcinomas (2/2), but in none of the noninvasive ductal carcinomas (0/2). These observations suggest that malignant histological phenotypes are associated with DCC-LOH.

Mutations of the APC gene occur during early stages of gastric adenoma development.

Mutations of the adenomatous polyposis coli (APC) gene have recently been shown to play an important role in colorectal tumorigenesis. We investigated mutations of the APC gene in 30 gastric adenomas obtained endoscopically. Mutations of the APC gene were examined by polymerase chain reaction-single-strand conformation polymorphism analysis followed by sequencing of the polymerase chain reaction products. Mutations were detected in 20% (6 of 30) of gastric adenomas. In addition, deletion of the remaining allele that subsequently led to complete inactivation of the APC gene was confirmed in one-half (3 of 6) of the tumors with APC gene mutations. Sequencing analysis confirmed that the mutations resulted in truncation of the gene products or in an amino acid change. The incidences of mutations of the APC gene remained constant regardless of the size or degree of histological atypia. Our observations suggest that mutations of the APC gene, similarly to those in colorectal tumorigenesis, occur during the early stages of gastric adenoma development.

Improved detection of loss of heterozygosity at retinoblastoma gene locus in human breast carcinoma.

Loss of heterozygosity (LOH) at the retinoblastoma (Rb) gene locus was investigated in 33 breast carcinomas by polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis after tumor cell enrichment by cell sorting. The efficacy of cell sorting was evaluated by comparing the results of PCR-SSCP with and without cell sorting. Ten of 17 (59%) informative cases showed LOH at this locus by cell sorting combined with PCR-SSCP, although LOH was detectable in six (35%) cases without cell sorting. Flow cytometry and histologic examination revealed that this underestimation may occur when the tumor cell population is less than 50% in the specimens analyzed. It is concluded that LOH of the Rb gene occurs more frequently in human breast carcinoma than previously thought, and thus may contribute significantly to the development and/or progression of this tumor.