Japanese Foundation For Cancer Research Research Articles

Prognostic Impact of Programmed Cell Death-1-Positive Cells on Follicular Lymphoma Patients Might Diminish in the Rituximab Era

Abstract 2645 BackgroundProgrammed cell death-1 (PD-1) is involved in one of the inhibitory pathways of the B7-cluster of differentiation (CD) 28 family; this pathway is known to be involved in the attenuation of T cell responses and promotion of T cell tolerance. PD-1 is known to negatively regulate T cell receptor-mediated proliferation and cytokine production, lead to alternation in the tumor microenvironment. Carreas et al. (J Clin Oncol. 2009; 27: 1470–1476) examined 100 follicular lymphoma (FL) patients and reported better prognosis in the group that had high levels of PD-1-positive cells. In contrast, in the study performed by Richendollar et al. (Human Pathol. 2011; 42: 552–557), which involved 91 FL patients, high levels of PD-1-positive cells were found to have a poor prognostic impact. Although these studies have shown that high levels of PD-1-positive cells in FL patients influence their prognosis, both studies included patients treated without rituximab, and the prognostic impact of PD-1 positivity in the rituximab-era (R-era) has not yet been elucidated. Materials and methodsWe retrospectively analyzed data for 91 FL patients uniformly treated by standard rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) therapy in 5 institutions between 2001 and 2009. The median age of the entire cohort was 58 years (range, 34–85 years), and 46 (51%) of the patients were men. We also collected and examined biopsy specimens for diagnosis with respect to PD-1 positivity. The PD-1-positive cells were counted using computer analysis at the Cancer Institute, Japanese Foundation for Cancer Research. ResultsThe FL grade on diagnosis was grade 1 for 34 (37%) patients, grade 2 for 41 (45%) patients, and grade 3 for 16 (18%) patients. The median positivity for PD-1 staining was 16.0% (range, 0.01–51.9%) and was significantly higher in the high beta-2 microglobulin (B2M; at least 3) group (P = 0.009); the men had a high tendency for PD-1 positivity (P = 0.08). After a median follow-up of 29.1 months, the 3-year progression-free survival (PFS) and overall survival (OS) were 61.1% and 88.6%, respectively. Stage 4 FL at diagnosis (P = 0.02) and bone marrow involvement (P = 0.05) resulted in worse PFS, and an Eastern Cooperative Oncology Group (ECOG) performance status of 2–4 (P = 0.04), high Follicular Lymphoma International Prognostic Index (FLIPI; P = 0.02), B symptoms (P = 0.04), and high B2M levels (P = 0.005) worsened OS. Multivariate analysis showed that age over 60 years (P = 0.04) and high B2M levels (P = 0.07) were prognostic factors for PFS. PD-1 positivity was not found to be a prognostic factor with respect to both PFS and OS. Because the addition of rituximab to therapy regimens has altered the clinical course and prognosis of FL, some new prognostic factors have been proposed, and the impact of known prognostic factors has been changing. Rituximab might also have changed the prognosis of FL patients with high levels of PD-1-positive cells. ConclusionHigh levels of PD-1-positive cells were not found to be a prognostic factor in this study, indicating that the prognostic impact of PD-1 positivity might be eliminated in the R-era. Disclosures:No relevant conflicts of interest to declare.

Mda-9/Syntenin Protein Positively Regulates the Activation of Akt Protein by Facilitating Integrin-linked Kinase Adaptor Function during Adhesion to Type I Collagen

The integrin-linked kinase (ILK)-PINCH1-α-parvin (IPP) complex functions as a signaling platform for integrins that modulates various cellular processes. ILK functions as a central adaptor for the assembly of IPP complex. We report here that mda-9/syntenin, a positive regulator of cancer metastasis, regulates the activation of Akt (also known as protein kinase B) by facilitating ILK adaptor function during adhesion to type I collagen (COL-I) in human breast cancer cells. COL-I stimulation induced the phosphorylation and plasma membrane translocation of Akt. Inhibition of mda-9/syntenin or expression of mutant ILK (E359K) significantly blocked the translocation of both ILK and Akt to the plasma membrane. mda-9/syntenin associated with ILK, and this association was increased at the plasma membrane by COL-I stimulation. Knockdown of mda-9/syntenin impaired COL-I-induced association of ILK with Akt and plasma membrane targeting of ILK-Akt complex. These results demonstrated that mda-9/syntenin regulates the activation of Akt by controlling the plasma membrane targeting of Akt via a mechanism that facilitates the association of Akt with ILK at the plasma membrane during adhesion to COL-I. On a striking note, inhibition of mda-9/syntenin impaired COL-I-induced plasma membrane translocation of the IPP complex and assembly of integrin β1-IPP signaling complexes. Thus, our study defines the role of mda-9/syntenin in ILK adaptor function and describes a new mechanism of mda-9/syntenin for regulation of cell migration.

Abstract 4149: Genetic analysis of circulating tumor cells using 3D immunostain and FISH

Abstract Background: Circulating tumor cells (CTCs) offer a non-invasive approach to characterize metastatic tumor cells. However, the low recovery rates of CTCs by methods performed in most clinical laboratory have limited their usefulness. Recent studies have suggested that discordance may exist between Her-2 status of a patient's CTC and that of primary breast tumor. To investigate Her-2 status in detail, we developed a new technique of the FISH analysis of CTCs. Most CTCs enrichment techniques are anti-EpCAM antibody based. However, some tumor cells express low or no EpCAM. So, we adopted the strategy independent of EpCAM antigenecity in order to reliably analyze genes even with very few CTCs. Material and methods: The method for Her-2 analysis that we established was based on three-dimensional multi-color imaging. Briefly, CTCs of tumor patients were concentrated by negative selection from peripheral blood by using antibodies against WBC, RBC, and platelets. The concentrated CTCs were labeled by fluorescent monoclonal antibodies against pan-cytokeratin and CD45. The specimens were then hybridized with FISH probes and were mounted in DAPI with antifade reagent. The preparations were screened for a series of Z-axis optical sections with a confocal microscope and 3D images were generated using Fluoview software. To inspect the availability of our method, we analyzed the peripheral blood or effusion specimens of patients with gastrointestinal tumors. This study was approved by the Institutional Review Board of Japanese Foundation for Cancer Research. Results: CTCs (Cytokeratin+/CD45-/DAPI+) were easily discriminated from remaining hematopoietic cells (CD45+). And these immunocytochemical staining had no crossover on FISH signal by the advantage of confocal imaging. In addition, three-dimensional imaging reconstruction enabled counting of DNA probe spots that differed in the Z plane but were at the same XY position in the cell. Recovery rates of tumor cells spiked into normal blood averaged 79%. By the analysis of the several cases of peripheral blood and the colon fiber biopsy derived from the same patients, it was suggested that the expression of EpCAM in CTCs was distinctly lower than it in the primary tumor site. Conclusions: Because our method does not depend on expression of EpCAM, even the CTCs that express little or no EpCAM can be identified. In addition, the three-dimensional imaging with high spacial/wavelength resolution made it possible to analyze Her-2 gene status of CTCs easily and accurately. Using this methods, we are currently working on the investigation of Her-2 state in CTCs derived from patients with gastrointestinal tumor and its relation of Her-2 status of primary tumor and metastases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4149. doi:10.1158/1538-7445.AM2011-4149

Identification of Chrysoplenetin from Vitex negundo as a Potential Cytotoxic Agent against PANC‐1 and a Panel of 39 Human Cancer Cell Lines (JFCR‐39)

Human pancreatic cancer is known to be the most deadly disease with the lowest 5-year survival rate and is resistant to well known conventional chemotherapeutic drugs in clinical use. Screening of medicinal plants from Myanmar utilizing antiausterity strategy led to the identification of Vitex negundo as one of the medicinal plants having potent preferential cytotoxic activity against PANC-1 human pancreatic cancer cells. Bioactivity-guided phytochemical investigation led to the isolation of chrysoplenetin (1) and chrysosplenol D (2) as the active constituents with a PC(50) value of 3.4 μg/mL and 4.6 μg/mL, respectively, against PANC-1 cells. Both these compounds induced apoptosis-like morphological changes in PANC-1 cells. Chrysoplenetin was further tested against a panel of 39 human cancer cell lines (JFCR-39) at the Japanese Foundation for Cancer Research, and 25 cell lines belonging to lung, breast, CNS, colon, melanoma, ovarian, prostate cancer and stomach cancer cell lines were found to be highly sensitive to chrysoplenetin at a submicromolar range. In the JFCR-39 panel, lung NCI-H522, ovarian OVCAR-3 and prostate PC-3 cells were found to be most sensitive with GI(50) of 0.12, 0.18 and 0.17 μm, respectively. The COMPARE analysis suggested that the molecular mode of action of chrysoplenetin was unique compared with the existing anticancer drugs.

Pelvic dimensions as a predictor of difficulty in laparoscopic surgery for rectal cancer

Laparoscopic surgery for rectal cancer is thought to be a technically more difficult operation than laparoscopic colectomy. Data from the Conventional versus Laparoscopic-Assisted Surgery In Colorectal Cancer (CLASICC) trial showed that circumferential resection margin positivity was greater in the laparoscopic group (12%) than in the open surgery group (6%) undergoing anterior resection, although this difference was not significant [1]. In contrast, many nonrandomized studies, including ours, have suggested that laparoscopic surgery for rectal cancer is safe and feasible with careful case selection and expertise [2, 3]. The recent randomized Comparison of Open versus laparoscopic surgery for mid and low REctal cancer After Neoadjuvant chemoradiotherapy (COREAN) trial showed that laparoscopic surgery after preoperative chemoradiotherapy for mid and low rectal cancer is safe and that the quality of oncologic resection is similar to that of open surgery [4]. Because the future may see increasing demand for laparoscopic surgery to treat rectal cancer, a better understanding of the factors associated with the difficulty of laparoscopic surgery is important for surgeons, especially those learning these operations for proper case selection. In the December issue of Surgical Endoscopy, Killeen et al. [5] examined the influence of pelvic dimensions measured by magnetic resonance imaging (MRI) on difficulties in laparoscopic surgery for rectal cancer. Operative time was used as a measurement of operative difficulty. Univariate analysis showed that a less acutely curved sacrum and a larger sagittal pelvic outlet were significantly correlated with longer operative time, especially for a pelvis with a narrow intertuberous distance. Although Killeen et al. [5] introduced our study as supportive of their findings, indeed, we came to different conclusions [6]. We showed by multivariate analysis that narrower pelvic outlet together with higher body mass index, shorter tumor distance from the anal verge, and advanced tumor depth was independently predictive of longer operative time in laparoscopic total mesorectal excision (TME) with intracorporeal rectal transection and double-stapling technique (DST) anastomosis. There may be some reasons for the different conclusions. First, the total number of patients analyzed in the study of Killeen et al. [5] was small (n = 25). Second, multivariate analysis including patientand tumor-related factors other than pelvic dimensions was not performed in their study. Third, they evaluated the total operative time as dependent variable, and patients who underwent either laparoscopic anterior resection or abdominoperineal resection were included. However, excluding the procedures outside the pelvis might be necessary for accurate analysis of the influence that pelvic dimensions has on difficulties in laparoscopic surgery for rectal cancer because pelvic dimensions influence the procedures more directly after the pelvic cavity is reached. We also have reported that narrow pelvic dimensions were not associated with overall postoperative morbidity and anastomotic leakage [6]. On the contrary, larger pelvic outlet was independently predictive of anastomotic leakage [6]. Considering these results, we concluded in our T. Akiyoshi (&) M. Ueno Gastroenterological Center, Department of Gastroenterological Surgery, Cancer Institute Hospital, Japanese Foundation for Cancer Research, 3-8-31 Ariake, Koto-ku, Tokyo 135-8550, Japan e-mail: takashi.akiyoshi@jfcr.or.jp

XELOX 療法における薬剤師外来の有用性

XELOX (capecitabine plus oxaliplatin) is a standard treatment for unresectable advanced colorectal cancer. Combining oral and intravenous administration, it is convenient method of treatment but full patient compliance regarding capecitabine is necessary to obtain efficacy. To help ensure good patient compliance for this drug, we established a pharmaceutical outpatient clinic at the Cancer Institute Hospital of the Japanese Foundation for Cancer Research (JFCR) where pharmacists interview patients prior to physicians' assessments. Medication compliance and adverse effects are investigated, and supportive care and prescription support provided.In this study, we evaluated the usefulness of the pharmaceutical outpatient clinic. We retrospectively investigated colorectal cancer patients who had received treatment with XELOX or XELOX + bevacizumab in our hospital. The end points included the number and content of inquiries concerning XELOX and the adoption rate of suggestions made regarding supportive care.In the results, inquiries concerning XELOX were made for 5.3% (14/260) of patients in a pharmaceutical intervention group (post-pharmaceutical outpatient clinic), in contrast to 16.8% (43/256)for a control group (no consultation in pharmaceutical outpatient clinic). The number of inquiries was significantly lower in the pharmaceutical intervention group than in the control group (p<0.001). In the pharmaceutical intervention group there were no inquiries about dosage. At 95.4%, the adoption rate for 592 prescription suggestions was very high. The results of our study indicate that the pharmaceutical outpatient clinic was useful for patients prescribed XELOX.

Cytochrome P450 1B1 Gene Polymorphisms as Predictors of Anticancer Drug Activity: Studies with In vitro Models

Cytochrome P450 1B1 (CYP1B1) is found in tumor tissue and is suspected to play a role in oncogenesis and drug resistance. CYP1B1 gene polymorphisms have been associated with the risk of developing lung and other cancers. They may be associated with tumor response to anticancer drugs. We have determined 4 frequent nonsynonymous gene polymorphisms of CYP1B1 in the human tumor cell lines panels of the National Cancer Institute (NCI) and the Japanese Foundation for Cancer Research (JFCR): rs10012 (R48G), rs1056827 (A119S), rs1056836 (L432V), and rs1800440 (N453S). Numerous anticancer drugs have been tested against these panels that offer the opportunity to detect associations between gene polymorphisms and drug sensitivity. CYP1B1 single nucleotide polymorphisms were in marked linkage disequilibrium. The L432V allelic variants were significantly associated with reduced sensitivity to DNA-interacting anticancer agents, alkylators, camptothecins, topoisomerase II inhibitors, and some antimetabolites. For instance, in the NCI panel, cell lines homozygous for the V432 allele were globally 2-fold resistant to alkylating agents (P = 5 × 10(-10)) and 4.5-fold to camptothecins (P = 6.6 × 10(-9)) than cell lines homozygous for the L432 allele. Similar features were exhibited by the JFCR panel. Cell lines homozygous for the V432 allele were globally less sensitive to DNA-interfering drugs than cell lines having at least 1 common allele. There was no significant association between mRNA expression of CYP1B1 and CYP1B1 genotype, and no significant association between CYP1B1 mRNA expression and drug cytotoxicity. These observations open the way to clinical studies exploring the role of CYP1B1 gene polymorphisms for predicting tumor sensitivity to chemotherapy.

Molecular Consequences of the ACVR1R206H Mutation of Fibrodysplasia Ossificans Progressiva

Fibrodysplasia ossificans progressiva (FOP), a rare genetic and catastrophic disorder characterized by progressive heterotopic ossification, is caused by a point mutation, c.617G>A; p.R206H, in the activin A receptor type 1 (ACVR1) gene, one of the bone morphogenetic protein type I receptors (BMPR-Is). Although altered BMP signaling has been suggested to explain the pathogenesis, the molecular consequences of this mutation are still elusive. Here we studied the impact of ACVR1 R206H mutation on BMP signaling and its downstream signaling cascades in murine myogenic C2C12 cells and HEK 293 cells. We found that ACVR1 was the most abundant of the BMPR-Is expressed in mesenchymal cells but its contribution to osteogenic BMP signal transduction was minor. The R206H mutant caused weak activation of the BMP signaling pathway, unlike the Q207D mutant, a strong and constitutively active form. The R206H mutant showed a decreased binding affinity for FKBP1A/FKBP12, a known safeguard molecule against the leakage of transforming growth factor (TGF)-beta or BMP signaling. The decreased binding affinity of FKBP1A to the mutant R206H ACVR1 resulted in leaky activation of the BMP signal, and moreover, it decreased steady-state R206H ACVR1 protein levels. Interestingly, while WT ACVR1 and FKBP1A were broadly distributed in plasma membrane and cytoplasm without BMP-2 stimulation and then localized in plasma membrane on BMP-2 stimulation, R206H ACVR1 and FKBP1A were mainly distributed in plasma membrane regardless of BMP-2 stimulation. The impaired binding to FKBP1A and an altered subcellular distribution by R206H ACVR1 mutation may result in mild activation of osteogenic BMP-signaling in extraskeletal sites such as muscle, which eventually lead to delayed and progressive ectopic bone formation in FOP patients.

Triterpene Glycosides from Curculigo orchioides and Their Cytotoxic Activity

Six new cycloartane glycosides (1-6) were isolated from the rhizomes of Curculigo orchioides. The structures of 1-6 were determined by spectroscopic analyses and the results of hydrolytic cleavage. Compounds 1-6, and their common aglycone (1a), were evaluated for cytotoxic activity against HL-60 human leukemia cells. Compounds 1 and 1a showed cytotoxic activity against HL-60 cells with IC(50) values of 9.0 and 1.8 microM, respectively. The cancer cell growth inhibition of 1a was also examined using a panel of 39 human cancer cell lines in the Japanese Foundation for Cancer Research.

Artificial Intelligence in Neuroscience and Systems Biology: Lessons Learnt, Open Problems, and the Road Ahead

1 Systems Biology Research Group, Centre for Molecular Biosciences, School of Biomedical Sciences, University of Ulster, Cromore Road, BT52 1SA, Coleraine, Northern Ireland 2 Systems Biology Department, Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, Japan 3Department of Complex Systems, Future University Hakodate, 116-2 Kamedanakano-cho, Hakodate, Hokkaido 041-8655, Japan 4School of Computing and Mathematics, Faculty of Computing and Engineering, University of Ulster, Shore Road, New-Townabbey, Co. Antrim, BT37 0QB, Northern Ireland 5Laboratory for Dynamics of Emergent Intelligence, RIKEN Brain Science Institute, Wako-shi, Saitama 351-0198, Japan

Factors involved in free flap thrombosis after reconstructive surgery for head and neck cancer

Objective Reconstructive surgery in which a free flap is used following extended resection for head and neck cancer has been popular for more than a quarter of a century. However, postoperative total flap necrosis has not been completely resolved, and few studies have analyzed the specific risk factors for this complication. Methods Over a three-year period from 2005 to 2007, 388 flaps were used for reconstructive surgery on 384 patients at Department of Head and Neck of the Cancer Institute Hospital of Japanese Foundation for Cancer Research, and the results for 22 flaps with thrombosis were analyzed. Results The only statistically significant risk factor for free flap thrombosis was the history of radical concurrent chemoradiotherapy. Conclusions It will be necessary to keep this observation in mind when performing salvage surgery following chemoradiotherapy, which is expected to be performed more often in the future.

Glaziovianin A, a new isoflavone, from the leaves of Ateleia glazioviana and its cytotoxic activity against human cancer cells

A new isoflavone, named glaziovianin A ( 1), was isolated from the leaves of Ateleia glazioviana (Legminosae) by means of a cytotoxicity-guided fractionation procedure against HL-60 leukemia cells. The chemical structure of 1 was determined by analysis of its extensive spectroscopic data. Glaziovianin A ( 1) displayed differential cytotoxicities in the Japanese Foundation for Cancer Research 39 cell line panel assay. The pattern of the differential cytotoxicities of 1 was found to correlate to that shown by TZT-1027, suggesting that 1 inhibited tubulin polymerization as an action mechanism. Although 1 had little influence on microtubule networks in interphase cells, 1-treated cells showed abnormal structures with unaligned chromosomes.

Survival Analysis for Gastric Cancer Using the Japanese Foundation Database Based on a Generalized Hazards Model Incorporating B-spline Functions

The Japanese Foundation for Cancer Research (JFCR) has provided a large database on gastric cancer for open use comprised of data on survival times after surgery and related covariates including prognostic factors. Data analysis based on the generalized hazards model incorporating the B-spline function (GHMBS) was performed using a dataset (JFCR dataset) composed of 9,631 cases within this database. A model selection method was adopted for clarifying the meaning of estimated parameters, because the GHMBS was comprised of the proportional hazards model (PHM) and accelerated failure time model (AFTM) as submodels. A preliminary simulation experiment to examine the performance of the model selection method based on the GHMBS was conducted under the condition that multiple covariates were considered, where one was the target covariate and the other was covariate for adjustment. After validation of the method by this simulation experiment, the method was applied to the JFCR dataset to estimate the period effect for prolonging survival time with an adjustment for the stage effect. The analysis revealed that the PHM was suitable for the period effect, while a mixture of the PHM and AFTM was for the stage effect and treatment for gastric cancer made steady progress from the 1950s through the 1990s.

Sesquiterpenoids and Flavonoids from the Aerial Parts of Tithonia diversifolia and Their Cytotoxic Activity

Cytotoxicity-guided fractionation of the 80% EtOH extract of Tithonia diversifolia has resulted in the isolation of twelve sesquiterpenoids (1-12), including three new ones (4, 10, 12), and three known flavonoids (13-15). The structures of the new compounds were determined by analysis of their spectroscopic data. The isolated compounds showed cytotoxic activity against HL-60 leukemia cells with IC(50) values ranging from 0.13 to 13.0 microM, when etoposide used as a positive control gave an IC(50) value of 0.43 microM. The cancer growth inhibitory property of 9, the main cytotoxic compound in T. diversifolia, was examined using a disease-oriented panel composed of 39 human cancer cell lines in the Japanese Foundation for Cancer Research.

High prevalence of HER-2/neu and p53 overexpression in inflammatory breast cancer

Inflammatory breast cancer (IBC) is one of the most aggressive forms of breast cancer. Although the survival of patients with IBC has been greatly improved by the use of combined treatment modalities, women with IBC still have lower survival rates. We have summarized a single-center experience involving IBC patients. Our objectives are to clarify molecular alterations of HER-2/neu and p53 in IBC and to investigate the prognostic factors. Between January 1990 and December 2000, 57 patients with IBC were referred to the Cancer Institute Hospital of the Japanese Foundation for Cancer Research. The incidence of IBC among primary breast cancers was 1.0% (57/5,757) in our hospital. Forty-six patients meeting Haagensen's criteria for inflammatory breast carcinoma were evaluated. All patients had biopsy-proven carcinomas but no distant metastases at referral. The median age at diagnosis for IBC was 51.8 (range, 28 to 70). All patients underwent a mastectomy. Chemotherapy was performed pre- or post-operatively. Three-year and 5-year survival rates were 56.5%, and 40.7%, respectively. Expressions of HER-2/neu and the p53 protein were determined retrospectively by immunohistochemical (IHC) staining of thin paraffin-embedded sections of primary tumors. Of 46 patients, 23 (50.0%) with tumors testing positive for HER-2/neu fared somewhat worse than those with negative tumors, but the differences were not significant for either overall survival (OS) or disease-free survival (DFS). Of 46 patients, 19 (41.3%) whose tumors were positive for p53 fared somewhat better than patients with negative tumors, with no significant differences in either OS or DFS. Patients presenting with less than ten pathologically involved axillary lymph nodes showed significantly better OS and DFS. Overexpression of HER-2/neu and the p53 protein were not significant prognostic factors in inflammatory breast cancer. However, the increased incidence of HER-2/neu and the poor outcome of IBC may be of clinical interest, suggesting the need for clinical trials of antibody therapy targeted to HER-2/neu. Moreover, a high prevalence of p53 may be useful in determining the specific use of chemotherapy.

Fibronectin Regulates Latent Transforming Growth Factor-β (TGFβ) by Controlling Matrix Assembly of Latent TGFβ-binding Protein-1

Fibronectin Regulates Latent Transforming Growth Factor-β (TGFβ) by Controlling Matrix Assembly of Latent TGFβ-binding Protein-1

Increase in the frequency of K‐ras codon 12 point mutation in colorectal carcinoma in elderly males in Japan: The 1990s compared with the 1960s

The incidence of colorectal carcinomas has been increasing over the last 50 years in Japan. In order to determine whether adenoma-carcinoma sequence (ACS) or de novo cancer development, generally considered to be two separate genetic pathways, might be responsible, K-ras codon 12 mutations, good markers for ACS, were examined in 59 and 84 cases of advanced colorectal cancer surgically resected in Cancer Institute Hospital of the Japanese Foundation for Cancer Research in 1960-1969 and in 1990-1999, respectively. There was no significant difference of K-ras codon 12 mutation between the 25.4% (15/59) in the 1960s and 36.9% (31/84) in the 1990s (P = 0.148), and the reference of distal colon cancer also showed no significant difference between 24.4% (11/45) and 36.4% (20/55). Yet elderly males showed a significant difference: 27.3% (6/22) in the 1960s and 59.3% (16/27) in the 1990s. The references of males, elderly patients (over 75 years old) and distal colon cancer in the 1990s were significantly more likely to demonstrate mutations than their counterparts in the 1960s. There was no variation with the tumor location. The results suggest that the ACS pathway might have primarily contributed to the increased incidence of colorectal cancer in elderly males in Japan.

Classification of ipsilateral breast tumor recurrence after breast-conserving therapy: New primary cancer allows a good prognosis

To classify and assess ipsilateral breast tumor recurrences (IBTR) after breast-conserving therapy. Between 1986 and 2001, 2,137 patients who had breast cancer underwent breast-conserving surgery with or without radiotherapy at the Cancer Institute Hospital of the Japanese Foundation for Cancer Research. Of these patients, 83 (3.9%) had an IBTR. We classified the IBTR as a new primary cancer (NP) if the primary tumor had completely negative margins at first operation by detailed pathological examination and if the IBTR had an intraductal component. All other IBTRs were judged true local recurrence (TR). Of the 83 patients, 42 patients were classified as TR (29 had no radiotherapy) and 41 as NP (40 had no radiotherapy). Mean time to disease recurrence was 37 months for TR (52% were within 2 years) versus 55 months for NP (19% were within 2 years) (p=0.031). Six patients (14%) with TR did not receive re-operation, and 67% received salvage mastectomy and 19% re-lumpectomy. All cases of NP were operable, 78% underwent salvage mastectomy and 22% underwent re-lumpectomy. Distant metastases were observed in 33% of patients with TR and 5% of patients with NP, and cause-specific death occurred in 6 cases with TR and in one with NP. The patients with NP had improved 5-year rates of overall survival (NP 91% vs. TR 76%, P=0.0627) and distant disease-free survival (NP 93% vs. TR 61%, P=0.0028). Patients with NP more often developed contralateral breast cancer (NP 37% vs. TR 12%, P=0.018) Patients with NP had better survival rates than those with TR. Distinguishing new primary breast carcinomas from local disease recurrences may have importance in therapeutic decisions and chemoprevention strategies.

Smad3 Induces Chondrogenesis through the Activation of SOX9 via CREB-binding Protein/p300 Recruitment

The transcriptional activation by SRY-type high mobility group box 9 (SOX9) and the transforming growth factor beta (TGF-beta) signals are necessary for chondrogenic differentiation. We have previously shown that CREB-binding protein (CBP/p300) act as an important SOX9 co-activator during chondrogenesis. In the present study, we investigated the relationship between TGF-beta-dependent Smad2/3 signaling pathways and the SOX9-CBP/p300 transcriptional complex at the early stage of chondrogenesis. Overexpressed Smad3 strongly induced the primary chondrogenesis of human mesenchymal stem cells. In addition, Smad3 enhanced the transcriptional activity of SOX9 and the expression of alpha1(II) collagen gene (COL2A1), and small interference RNA against Smad3 (si-Smad3) inhibited them. We observed that Smad2/3 associated with Sox9 in a TGF-beta-dependent manner and formed the transcriptional complexes with SOX9 on the enhancer region of COL2A1. Interestingly, the association between Sox9 and CBP/p300 was increased by Smad3 overexpression and was suppressed by si-Smad3. Our findings indicate that Smad3 has a stronger potential to stimulate the SOX9-dependent transcriptional activity by modulating the interaction between SOX9 and CBP/p300, rather than Smad2. This study suggests that the Smad3 pathway presents a key role for the SOX9-dependent transcriptional activation in primary chondrogenesis.

Type I Transforming Growth Factor β Receptor Binds to and Activates Phosphatidylinositol 3-Kinase

We have examined the interaction of transforming growth factor (TGF)beta receptors with phosphatidylinositol 3-(PI3) kinase in epithelial cells. In COS7 cells, treatment with TGFbeta increased PI3 kinase activity as measured by the ability of p85-associated immune complexes to phosphorylate inositides in vitro. Both type I and type II TGFbeta receptors (TbetaR) associated with p85, but the association of TbetaRII appeared to be constitutive. The interaction of TbetaRI with p85 was induced by treatment with TGFbeta. The receptor association with PI3 kinase was not direct as (35)S-labeled rabbit reticulocyte p85 did not couple with fusion proteins containing type I and type II receptors. A kinase-dead, dominant-negative mutant of TbetaRII blocked ligand-induced p85-TbetaRI association and PI3 kinase activity. In TbetaRI-null R1B cells, TGFbeta did not stimulate PI3 kinase activity. This stimulation was restored upon reconstitution of TbetaRI by transfection. In R1B and NMuMG epithelial cells, overexpression of a dominant active mutant form of TbetaRI markedly enhanced ligand-independent PI3 kinase activity, which was blocked by the addition of the TbetaRI kinase inhibitor LY580276, suggesting a causal link between TbetaRI function and PI3 kinase. Overexpressed Smad7 also prevented ligand-induced PI3 kinase activity. Taken together, these data suggest that 1) TGFbeta receptors can indirectly associate with p85, 2) both receptors are required for ligand-induced PI3 kinase activation, and 3) the activated TbetaRI serine-threonine kinase can potently induce PI3 kinase activity.