bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of the HLA-Cw*0702-Restricted Tumor-Associated Antigen Recognized by a CTL Clone from a Lung Cancer Patient
Authors: Nagata Yoshika , Hanagiri Takeshi , Takenoyama Mitsuhiro , Fukuyama Takashi , Mizukami Makiko , So Tetsuya , Ichiki Yoshinobu , Sugaya Masakazu , Sugio Kenji , Yasumoto Kosei
Published: September 2005

Purpose: A large number of tumor-associated antigens have been used in vaccination trials for mainly melanomas. Our purpose of this study is to identify a novel tumor antigen useful for immunotherapy of lung cancer patients.

Experimental Design: Analysis of an autologous tumor-specific CTL clone F2a that was established from regional lymph node lymphocytes of a patient with lung cancer (A904) by a mixed lymphocyte-tumor cell culture.

Results: F2a recognized and killed autologous tumor cells (A904L), whereas it did not respond to autologous EBV-transformed B cells, phytohemagglutinin-blastoid T cells, and K562 cells. cDNA clone 31.2 was isolated by using cDNA expression cloning method as a gene encoding antigen. This gene was identical to the reported gene whose function was unknown. The antigen encoded by the cDNA was recognized by the CTL in a HLA-Cw*0702-restricted manner. Furthermore, a 9-mer peptide at positions 659 to 685 in cDNA clone 31.2 was identified as a novel epitope peptide. The CTL recognized some allogeneic cancer cell lines with HLA-Cw*0702 as well as some HLA-Cw*0702-negative cell lines when transfected with HLA-Cw*0702, thus indicating that the identified antigen was a cross-reactive antigen.

Conclusions: Although exact mechanism to process the encoded protein and present the antigen in the context of HLA class I remains to be elucidated, the CTL recognized some of tumor cells in the context of HLA-Cw*0702 but did not recognize a variety of normal cells and also autologous EBV-transformed B cells. These results indicated that the antigen identified in this study may therefore be a possible target of tumor-specific immunotherapy for lung cancer patients.
Cytochrome P450 1B1 Is Overexpressed and Regulated by Hypomethylation in Prostate Cancer
Authors: Tokizane Takashi , Shiina Hiroaki , Igawa Mikio , Enokida Hideki , Urakami Shinji , Kawakami Toshifumi , Ogishima Tatsuya , Okino Steven T. , Li Long-Cheng , Tanaka Yuichiro , Nonomura Norio , Okuyama Akihiko , Dahiya Rajvir
Published: September 2005

Purpose: Cytochrome P450 1B1 (CYP1B1), a dioxin inducible member of the CYP supergene family, is overexpressed in various human malignancies including prostate cancer. We hypothesized that promoter/enhancer CpG methylation contributes to the regulation of CYP1B1 expression in human prostate tissue.

Experimental Design: Expression and induction of the CYP1B1 gene in clinical prostate tissues and prostate cancer cell lines were investigated. The methylation status of the CYP1B1 gene was analyzed in 175 prostate cancer and 96 benign prostatic hyperplasia samples using methylation-specific PCR (MSP) and bisulfite-modified DNA sequencing. MSP primers covered dioxin response elements (DRE) and Sp1 sites that are important for the expression of CYP1B1.

Results: Expressions of CYP1B1 mRNA and protein were increased in prostate cancer. The aryl hydrocarbon receptor (AhR)/AhR nuclear translocator (ARNT) heterodimer complex activates gene transcription by binding to the DREs of CYP1B1. In prostate cancer cells, CYP1B1 mRNA was induced by 2,3,7,8-tetrachlorodigenzo-p-dioxin (TCDD) and/or demethylation agent (5-aza-2-deoxycytidine). There was no change in the expressions of AhR and ARNT. Methylation of promoter/enhancer regions was significantly higher in benign prostatic hyperplasia compared with prostate cancer. MSP-positive patients had significantly lower risk for prostate cancer as compared with MSP-negative patients. There was no correlation between CYP1B1 methylation status and clinicopathologic features.

Conclusions: CYP1B1 is overexpressed in prostate cancer and regulated by hypomethylation of its promoter/enhancer region. This is the first report about CYP1B1 regulation in human clinical prostate samples showing that hypomethylation of the CYP1B1 gene may play an important role in prostate cancer.
Pharmacokinetics of Dactinomycin in a Pediatric Patient Population: a United Kingdom Children's Cancer Study Group Study
Authors: Veal Gareth J. , Cole Michael , Errington Julie , Parry Annie , Hale Juliet , Pearson Andrew D.J. , Howe Karen , Chisholm Julia C. , Beane Carol , Brennan Bernadette , Waters Fiona , Glaser Adam , Hemsworth Sue , McDowell Heather , Wright Yvonne , Pritchard-Jones Kathy , Pinkerton Ross , Jenner Gail , Nicholson James , Elsworth Ann M. , Boddy Alan V. ,   UKCCSG Pharmacology Working Group
Published: September 2005

Purpose: Dactinomycin (actinomycin D) is an antitumor antibiotic used routinely to treat certain pediatric and adult cancers. Despite concerns over the incidence of toxicity, little is known about the pharmacology of dactinomycin. A study was done to investigate dactinomycin pharmacokinetics in children.

Experimental Design: Dactinomycin was administered to 31 patients by bolus i.v. infusion, at doses of 0.70 to 1.50 mg/m2. Plasma concentrations were determined by liquid chromatography-mass spectrometry up to 24 hours after drug administration and National Cancer Institute Common Toxicity Criteria was assessed.

Results: Pharmacokinetic data analysis suggested that a three-compartment model most accurately reflected dactinomycin pharmacokinetics. However, there was insufficient data available to fully characterize this model. A median peak plasma concentration (Cmax) of 25.1 ng/mL (range, 3.2-99.2 ng/mL) was observed at 15 minutes after administration. The median exposure (AUC0-6), determined in 16 patients with sampling to 6 hours, was 2.67 mg/L.min (range, 1.12-4.90 mg/L.min). After adjusting for body size, AUC0-6 and Cmax were positively related to dose (P = 0.03 and P = 0.04, respectively). Patients who experienced any level of Common Toxicity Criteria grade had a 1.46-fold higher AUC0-6, 95% confidence interval (1.02-2.09). AUC0-6 was higher in patients <40 kg, possibly indicating a greater toxicity risk.

Conclusions: Data presented suggest that dosing of dactinomycin based on surface area is not optimal, either in younger patients in whom the risk of toxicity is greater, or in older patients where doses are capped.
Increased Expression and Activity of CDC25C Phosphatase and an Alternatively Spliced Variant in Prostate Cancer
Authors: Ozen Mustafa , Ittmann Michael
Published: September 2005

Alterations in the control of cell cycle progression have been implicated in a wide variety of malignant neoplasms, including prostate cancer. CDC25 phosphatases belong to the tyrosine phosphatase family and play a critical role in regulating cell cycle progression by dephosphorylating cyclin-dependent kinases at inhibitory residues. CDC25C plays an important role in the G2-M transition by activating Cdc2/Cyclin B1 complexes. To determine whether CDC25C activity is altered in prostate cancer, we have examined the expression of CDC25C and an alternatively spliced variant in human prostate cancer samples and cell lines. CDC25C protein is up-regulated in prostate cancer in comparison with normal prostate tissue and is present almost exclusively in its active dephosphorylated form. Expression of a biologically active alternatively spliced CDC25C isoform is also increased in prostate cancer and expression of alternatively spliced CDC25C is correlated to occurrence of biochemical (prostate-specific antigen) recurrence. We have also developed a quantitative reverse transcriptase-PCR analysis of Ki-67 expression as a method of measuring proliferative activity in prostate cancer from RNA samples. Based on this analysis of Ki67 expression, some but not all of this increase in CDC25C and its alternatively spliced variants is correlated with increased proliferation in prostate cancer. This data suggests that CDC25C might play an important role in prostate cancer progression and could be used to monitor and predict the aggressiveness of this disease.
Prevention of Cancer Cachexia by a Novel Nuclear Factor κB Inhibitor in Prostate Cancer
Authors: Kuroda Kenji , Horiguchi Yutaka , Nakashima Jun , Kikuchi Eiji , Kanao Kent , Miyajima Akira , Ohigashi Takashi , Umezawa Kazuo , Murai Masaru
Published: September 2005

Purpose: To investigate the association between serum interleukin-6 (IL-6) and cachexia in patients with prostate cancer and the inhibitory effect of a new nuclear factor κB (NF-κB) inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), on IL-6 production and cachexia in an animal model of hormone-refractory prostate cancer.

Experimental Design: The association between serum IL-6 levels and variables of cachexia was evaluated in 98 patients with prostate cancer. The inhibitory effects of DHMEQ on IL-6 secretion and cachexia were investigated in in vitro and in vivo studies using JCA-1 cells derived from human prostate cancer.

Results: Serum IL-6 levels were significantly elevated and cachexia developed in JCA-1 tumor-bearing mice as well as in prostate cancer patients with progressive disease. IL-6 secretion was significantly inhibited in JCA-1 cells exposed to DHMEQ. Intraperitoneal administration of DHMEQ (8 mg/kg) to tumor-bearing mice produced a significant amelioration of the reduction in body weight, epididymal fat weight, gastrocnemius muscle weight, hematocrit, and serum levels of triglyceride and albumin when compared with administration of DMSO or no treatment. DHMEQ caused a significant decrease of serum IL-6 level in JCA-1 tumor-bearing mice (all P < 0.05).

Conclusions: These results suggested an association between serum IL-6 and cachexia in patients with prostate cancer and in JCA-1 tumor-bearing mice and that a new NF-κB inhibitor, DHMEQ, could prevent the development of cachexia in JCA-1 tumor-bearing mice presumably through the inhibition of IL-6 secretion. DHMEQ seems to show promise as a novel and unique anticachectic agent in hormone-refractory prostate cancer.
Activation of p53-Dependent Apoptosis by Acute Ablation of Glycogen Synthase Kinase-3β in Colorectal Cancer Cells
Authors: Ghosh Jagadish C. , Altieri Dario C.
Published: September 2005

Purpose: The restoration of checkpoint mechanisms may provide a rational anticancer approach, but the molecular circuitries of how this can be achieved therapeutically are poorly understood. A pivotal signaling network in colorectal cancer cells involves glycogen synthase kinase-3β (GSK3β), a multifunctional kinase whose role in tumor cell survival is not defined.

Experimental Design: We used molecular, genetic, and pharmacologic antagonists of GSK3β in p53+/+ or p53−/− colorectal cancer cells. We monitored kinase activity in immunoprecipitation, protein expression by immunoblotting, and cell death by multiparametric flow cytometry. A xenograft colorectal cancer model was used to study antitumor activity in vivo.

Results: Treatment of p53+/+ colorectal cancer cells with pharmacologic inhibitors of GSK3β resulted in sustained elevation of p53, with up-regulation of p21Waf1/Cip1 and loss of survivin levels. Molecular targeting of GSK3β by overexpression of a GSK3β dominant-negative mutant, or acutesilencing of GSK3β by RNA interference, reproduced the induction of transcriptionally active p53 in colorectal cancer cells. This pathway was recapitulated by deregulated Wnt/T-cell factor signaling, with elevation of the tumor suppressor p14ARF, and reduced expression of the p53 antagonist, MDM2. Rather than cell cycle arrest, GSK3β blockade resulted in p53-dependent apoptosis, which was contributed by acute loss of survivin and inhibition of colorectal cancer growth in mice.

Conclusions: Acute ablation of GSK3β in colorectal cancer cells activates p53-dependent apoptosis and antagonizes tumor growth. This pathway may be exploited for rational treatment of colorectal cancer patients retaining wild-type p53.
BRAF Mutation in Endometrial Carcinoma and Hyperplasia: Correlation with KRAS and p53 Mutations and Mismatch Repair Protein Expression
Authors: Feng Yu-Zhen , Shiozawa Tanri , Miyamoto Tsutomu , Kashima Hiroyasu , Kurai Miyuki , Suzuki Akihisa , Konishi Ikuo
Published: September 2005

Purpose: Although several gene abnormalities have been reported in endometrial carcinoma, the genetic alterations have not fully been elucidated. Recent studies have revealed frequent activating mutations of the gene for BRAF, an effector of Ras protein in the mitogen-activated protein kinase pathway, in several malignancies. However, the prevalence and significance of BRAF mutations in endometrial carcinoma remain unclear.

Experimental Design: We examined BRAF mutations in exons 11 and 15 in 97 cases of endometrial carcinoma (endometrioid type, 78; nonendometrioid type, 19), 9 cases of atypical endometrial hyperplasia, and 20 cases of normal endometrium by direct sequencing. In addition, mutations of KRAS and p53 and the immunohistochemical expression of hMLH1 and hMSH2 were also examined.

Results: Of the 97 carcinomas and 9 hyperplasias, 20 (21%) and 1 (11%) had BRAF mutations, most of them at previously unreported sites. Twenty samples of normal endometrium and 21 samples of normal endometrium obtained from sites adjacent to neoplastic lesions had no BRAF mutations. There was no apparent difference in the prevalence of BRAF mutation among stages, histologic subtypes, or grades. Mutations of KRAS and p53 were found in 18 (19%) and 22 (23%) cases, and 65 (67%) and 92 (95%) cases showed positive immunostaining for hMLH1 and hMSH2, respectively. BRAF mutation was more frequently found in hMLH1-negative cases (12 of 32, 41%) than in hMLH1-positive cases (7 of 65, 11%; P = 0.008), suggesting that it is associated with an abnormal mismatch repair function.

Conclusions: These findings suggest that mutations of the BRAF gene are partly involved in the malignant transformation of the endometrium.
Indoleamine 2,3-Dioxygenase Serves as a Marker of Poor Prognosis in Gene Expression Profiles of Serous Ovarian Cancer Cells
Authors: Okamoto Aikou , Nikaido Takashi , Ochiai Kazunori , Takakura Satoshi , Saito Misato , Aoki Yuko , Ishii Nobuya , Yanaihara Nozomu , Yamada Kyosuke , Takikawa Osamu , Kawaguchi Rie , Isonishi Seiji , Tanaka Tadao , Urashima Mitsuyoshi
Published: September 2005

Purpose: We aimed to find key molecules associated with chemoresistance in ovarian cancer using gene expression profiling as a screening tool.

Experimental Design: Using two newly established paclitaxel-resistant ovarian cancer cell lines from an original paclitaxel-sensitive cell line and four supersensitive and four refractory surgical ovarian cancer specimens from paclitaxel-based chemotherapy, molecules associated with chemoresistance were screened with gene expression profiling arrays containing 39,000 genes. We further analyzed 44 genes that showed significantly different expressions between paclitaxel-sensitive samples and paclitaxel-resistant samples with permutation tests, which were common in cell lines and patients' tumors.

Results: Eight of these genes showed reproducible results with real-time reverse transcription-PCR, of which indoleamine 2,3-dioxygenase gene expression was the most prominent and consistent. Moreover, by immunohistochemical analysis using a total of 24 serous-type ovarian cancer surgical specimens (stage III, n = 21; stage IV, n = 7), excluding samples used for GeneChip analysis, the Kaplan-Meier survival curve showed a clear relationship between indoleamine 2,3-dioxygenase staining patterns and overall survival (log-rank test, P = 0.0001). All patients classified as negative survived without relapse. The 50% survival of patients classified as sporadic, focal, and diffuse was 41, 17, and 11 months, respectively.

Conclusion: The indoleamine 2,3-dioxygenase screened with the GeneChip was positively associated with paclitaxel resistance and with impaired survival in patients with serous-type ovarian cancer.
The Chemopreventive Agent Resveratrol Stimulates Cyclic AMP–Dependent Chloride SecretionIn vitro
Authors: Blumenstein Irina , Keserü Benjamin , Wolter Freya , Stein Jürgen
Published: September 2005

Resveratrol and its analogs are promising cancer chemoprevention agents, currently under investigation in clinical trials. However, patients administered other plant polyphenols experienced severe diarrhea, likely due to an increase in intracellular cyclic AMP (cAMP). Resveratrol itself raises intracellular cAMP levels in breast cancer cells in vitro. Its future use as a cancer chemopreventive agent could therefore be compromised by its severe side effects. The aim of the study was (a) to define the influence of resveratrol on intestinal Cl− secretion and (b) to elucidate possible intracellular transduction pathways involved. Resveratrol caused a dose- and time-dependent increase in ΔIsc in T84 cells. The specificity of resveratrol was confirmed by using piceatannol 100 μmol/L, the hydroxylated resveratrol analog, which did not alter ΔIsc. A significant elevation of [cAMP]i by resveratrol was assessed in T84 cells. In mouse jejunum, resveratrol induced a time- and dose-dependent increase in ΔIsc as well. In bilateral Cl−-free medium, as well as after inhibition of protein kinase A, resveratrol-induced ΔIsc was reduced significantly. Preincubation of T84 cells with butyrate 2 mmol/L (24 and 48 hours) significantly inhibited resveratrol as well as forskolin-induced Cl− secretion. In summary, the main mechanism of action of resveratrol in intestinal epithelia is cAMP-induced chloride secretion which can be suppressed by butyrate. It can therefore be suggested that in cancer chemoprevention, both agents should be combined to reduce an undesired side effect such as diarrhea and to benefit from the known agonistic effect of both agents on differentiation of colon cancer cells.
Radiotherapy Sensitization by Tumor-Specific TRAIL Gene Targeting Improves Survival of Mice Bearing Human Non–Small Cell Lung Cancer
Authors: Zhang Xiaochun , Cheung Rex Min , Komaki Ritsuko , Fang Bingliang , Chang Joe Y.
Published: September 2005

Purpose: To sensitize non–small cell lung cancer (NSCLC) to radiotherapy by tumor-specific delivery of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) gene.

Experimental Design: The TRAIL was delivered to human NSCLC cell lines and normal human bronchial epithelial cells by the replication-defective adenoviral vector Ad/TRAIL-F/RGD using a tumor-specific human telomerase reverse transcriptase promoter. Cancer growth was studied using 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt and clonogenic assays. Activation of the apoptosis pathway was analyzed in a Western blot and sub-G1 DNA accumulation. A xenograft mouse lung cancer model was treated by intratumoral injections of Ad/TRAIL-F/RGD and local radiotherapy; the other groups received one of these treatments alone or a control agent. Apoptosis and TRAIL expression in tumors were also analyzed.

Results: Ad/TRAIL-F/RGD specifically targets human NSCLC cells without significant effect in normal human bronchial epithelial cells. The combination of Ad/TRAIL-F/RGD and radiotherapy significantly improved cell-killing effect in all NSCLC cell lines tested (P < 0.05). Expression of TRAIL showed a dose-dependent relationship with Ad/TRAIL-F/RGD, and radiation seemed to increase TRAIL expression. Activation of the apoptosis by TRAIL and radiation was shown by activation of caspase-9, caspase-8, caspase-3, and poly(ADP-ribose) polymerase and increased DNA sub-G1 accumulation. The combination of TRAIL and radiotherapy significantly increased apoptosis in vivo, inhibited tumor growth, and prolonged mean survival in mice bearing human NSCLC to 43.7 days compared with 23.7 days (TRAIL only) and 16.5 days (radiotherapy only; P < 0.05).

Conclusions: The combination of Ad/TRAIL-F/RGD and radiotherapy significantly improved therapeutic efficacy in suppressing NSCLC tumor growth and prolonging survival. Ad/TRAIL-F/RGD may improve the therapeutic ratio of radiotherapy in NSCLC.
Clinical Trial Design Issues in the Era of Targeted Therapies
Authors: Schiller Joan H.
Published: September 2005

Although our lack of effective therapies mandates the development of new, molecularly targeted therapies, a number of issues in the design of clinical trials and their end points remain to be considered.
Cyclooxygenase as a Target in Lung Cancer
Authors: Brown Joanne R. , DuBois Raymond N.
Published: September 2005

Preclinical studies suggest that cyclooxygenase (COX)-2 may be involved in the molecular pathogenesis of some types of lung cancer. Most of the available studies point to its involvement in non-small cell lung cancer. Survival of patients with non-small cell lung cancer expressing high levels of COX-2 is markedly reduced. Treatment of humans with the selective COX-2 inhibitor celecoxib augments the antitumor effects of chemotherapy in patients with non-small cell lung cancer. COX-2 has been shown to regulate some aspects of tumor-associated angiogenesis. Most of the results we have published point to effects on the regulation of vascular endothelial growth factor. However, prostaglandins derived from COX-2 affect other signaling pathways as well, such as the epidermal growth factor and its receptor. Others have recently shown that non-small cell lung cancer exhibits a COX-2 downstream enzyme expression pattern that is altered in lung tumor cells and tumor-supplying vessels. Therefore, COX-2 and prostaglandins may have a major impact on lung tumor progression and tumor-associated inflammation. Clinical trials currently underway are exploring the potential of targeting COX-2 in lung cancer.
Oblimersen Sodium (Genasense bcl-2 Antisense Oligonucleotide)
Authors: Herbst Roy S. , Frankel Stanley R.
Published: September 2005

Bcl-2 protein inhibits apoptosis and confers resistance to treatment with traditional cytotoxic chemotherapy, radiotherapy, and monoclonal antibodies. Oblimersen sodium is an antisense oligonucleotide compound designed to specifically bind to human bcl-2 mRNA, resulting in catalytic degradation of bcl-2 mRNA and subsequent decrease in bcl-2 protein translation. Both small cell and non-small cell lung cancer show baseline and inducible expression of bcl-2, which may contribute to resistance to therapy. Preclinical studies have shown that combining bcl-2 antisense with chemotherapy improves antitumor response, increases apoptosis of tumor cells, and increases survival. Preliminary data from a large international randomized trial in melanoma show a trend toward increased survival and significantly improved response rates and response duration when oblimersen is added to dacarbazine. Phase I studies in small cell lung cancer patients demonstrate that oblimersen can be combined with paclitaxel or carboplatin and etoposide. The combination of docetaxel and oblimersen has been shown to be feasible in Phase I studies and is currently undergoing evaluation in comparison with docetaxel alone as first-line salvage therapy in patients refractory or relapsed after one prior chemotherapy regimen. Enhancement of the efficacy of anticancer treatments with oblimersen bcl-2 antisense therapy represents a promising new apoptosis-modulating strategy.
Preclinical and Clinical Development of the Cyclin-Dependent Kinase Inhibitor Flavopiridol
Authors: Shapiro Geoffrey I.
Published: September 2005

Flavopiridol is the first potent inhibitor of cyclin-dependent kinases (cdks) to reach clinical trial. In the majority of solid tumor cell lines and xenografts, flavopiridol induces cell cycle arrest and tumor growth inhibition. This is reflected in clinical outcomes: across multiple Phase II trials there are subsets of patients with prolonged stable disease, although few responses have been observed. Flavopiridol displays sequence-dependent cytotoxic synergy with chemotherapy agents. These effects are most marked when chemotherapy precedes flavopiridol. In the case of DNA-damaging agents that impose S-phase delay, flavopiridol-mediated cdk inhibition disrupts the phosphorylation of E2F-1, leading to inappropriate persistence of its activity, inducing apoptotic pathways. This mechanism has been exploited in a Phase I trial of sequential gemcitabine and flavopiridol that has produced promising results. Flavopiridol is also synergistic with taxanes. Inhibition of cyclin B-cdk1 by flavopiridol accelerates exit from an abnormal mitosis associated with taxane-induced cell death and reduces the phosphorylation of survivin, preventing its stabilization and the cellular protection it affords after taxane exposure. The sequential combination of docetaxel and flavopiridol has been investigated in a Phase I trial in patients with advanced non-small cell lung cancer, and a randomized Phase II study is under way. Initial schedules of flavopiridol used prolonged continuous infusions that produced nanomolar levels of drug thought to be capable of achieving cdk inhibition based on results in tumor cell lines. Recently, it has been discovered that micromolar concentrations are likely to be more effective, and shorter infusions that achieve a higher Cmax have now been adopted. Loading followed by maintenance infusions are also under development, designed to achieve sustained micromolar drug levels. Clinical trials remain complicated by the absence of pharmacodynamic end points to confirm target inhibition.
Detection of Breast Cancer in Nipple Aspirate Fluid by CpG Island Hypermethylation
Authors: Krassenstein Rachel , Sauter Edward , Dulaimi Essel , Battagli Cristina , Ehya Hormoz , Klein-Szanto Andres , Cairns Paul
Published: September 2005

Purpose: New approaches to the early detection of breast cancer are urgently needed as there is more benefit to be realized from screening. Nipple aspiration is a noninvasive technique that yields fluid known to contain breast epithelial cells. Silencing of tumor suppressor genes such as p16INk4a, BRCA1, and hMLH1 have established hypermethylation as a common mechanism for tumor suppressor inactivation in human cancer and as a promising target for molecular detection.

Experimental Design: Using sensitive methylation-specific PCR, we searched for aberrant promoter hypermethylation in a panel of six normally unmethylated genes: glutathione S-transferase π 1 (GSTP1); retinoic acid receptor-β2 (RARβ2); p16INk4a; p14ARF; RAS association domain family protein 1A (RASSF1A); and death-associated protein kinase (DAP-kinase) in 22 matched specimens of tumor, normal tissue, and nipple aspirate fluid collected from breast cancer patients.

Results: Hypermethylation of one or more genes was found in all 22 tumor DNAs (100% diagnostic coverage) and identical gene hypermethylation detected in 18 of 22 (82%) matched aspirate fluid DNAs. In contrast, hypermethylation was absent in benign and normal breast tissue and nipple aspirate DNA from healthy women.

Conclusions: Promoter hypermethylation of important cancer genes is common in breast cancer and could be detected in matched aspirate DNAs from patients with ductal carcinoma in situ or stage I cancer. Promoter hypermethylation represents a promising marker, and larger studies may lead to its useful application in breast cancer diagnosis and management.
Immunomagnetic Enrichment, Genomic Characterization, and Prognostic Impact of Circulating Melanoma Cells
Authors: Ulmer Anja , Schmidt-Kittler Oleg , Fischer Jörg , Ellwanger Ulf , Rassner Gernot , Riethmüller Gert , Fierlbeck Gerhard , Klein Christoph A.
Published: September 2005

Purpose: The finding of melanoma cells in the peripheral blood, thus far mainly inferred from the PCR-based demonstration of tyrosinase mRNA, has been associated with metastatic melanoma. Neither the malignant nature nor the prognostic significance of circulating cells could be established. To address this question, we analyzed immunomagnetically isolated circulating melanoma cells for chromosomal aberrations and performed a clinical follow-up study of the enrolled patients.

Experimental Design: In a prospective study, blood samples were taken from 164 melanoma patients and 50 donors without malignant disease. Circulating melanoma cells were enriched by immunomagnetic cell sorting using a murine monoclonal antibody against the melanoma-associated chondroitin sulfate proteoglycan. To prove the malignant origin of the positive cells and to define their chromosomal aberrations, we analyzed the genomes of 15 individually isolated cells from seven patients by single-cell comparative genomic hybridization (SCOMP).

Results: Absolute and relative frequencies of circulating melanoma cells were associated with stage and with the presence or absence of detectable tumor. The detection of two or more cells correlated significantly with a reduced survival of patients with metastatic melanoma. All of the cells that were analyzed by SCOMP displayed multiple chromosomal changes and carried aberrations typical for melanoma.

Conclusions: Immunomagnetic enrichment enables isolation and genomic characterization of circulating melanoma cells. The prognostic impact on survival of metastatic patients apparently reflects the aggressiveness of an ongoing tumor spread. Direct genomic analysis of the enriched and isolated cells will help to clarify the molecular-genetic basis of the establishment of generalized melanoma.
Interlaboratory Comparison ofHER-2Oncogene Amplification as Detected by Chromogenic and Fluorescencein situHybridization
Authors: Isola Jorma , Tanner Minna , Forsyth Amanda , Cooke Timothy G. , Watters Amanda D. , Bartlett John M. S.
Published: September 2005

Purpose: Chromogenic in situ hybridization (CISH) is a new modification of the fluorescence in situ hybridization (FISH) technique for detection of oncogene amplification in archival tumor samples. In CISH, the oncogene probe is detected using a peroxidase reaction, allowing use of transmitted light microscopy. We compared detection of HER-2/neu amplification by CISH with a Food and Drug Administration-approved two-color FISH test in an interlaboratory setting.

Experimental Design: Formalin-fixed paraffin-embedded tumor samples from 197 breast cancers were analyzed for HER-2 amplification by CISH. Two-color FISH (PathVysion) CISH of 17 centromere was done if the observer considered it necessary to ascertain amplification status in tumors with borderline HER-2 CISH copy numbers.

Results: Paired CISH/FISH results were available from 192 (97%) of 197 cases, no clear difference in success rates of either method was observed. Centromere 17 CISH was considered necessary in seven tumors. CISH and two-color FISH results were concordant in 180 cases (93.8%). There were 92 and 88 tumors found HER-2 amplified and nonamplified, respectively, by both methods. Eight tumors were amplified by CISH but not by FISH, and four tumors exhibited the opposite condition (kappa coefficient 0.875). In 7 of 12 cases differences between the two methods could have related to a lack of CISH chromosome 17 information. The remaining cases were explained by difficult histology (ductal carcinoma in situ, poor representativity, dense lymphocytic infiltration, or intratumoral heterogeneity).

Conclusions: These results indicate that CISH could provide an accurate and practical alternative to FISH for clinical diagnosis of HER-2/neu oncogene amplification in archival formalin-fixed breast cancer samples.
Overexpression of RhoA, Rac1, and Cdc42 GTPases Is Associated with Progression in Testicular Cancer
Authors: Kamai Takao , Yamanishi Tomonori , Shirataki Hiromichi , Takagi Kentaro , Asami Hidekazu , Ito Yuji , Yoshida Ken-Ichiro
Published: September 2005

The Rho family of GTPases are involved in actin cytoskeleton organization and associated with carcinogenesis and progression of human cancers. We investigated the roles of Rho family GTPases, prototypes RhoA, Rac1, and Cdc42, and the major downstream targets of RhoA, ROCK-I, and ROCK-II in testicular cancer. We quantified protein expression in paired tumor and nontumor samples from surgical specimens from 57 consecutive patients with testicular germ cell tumors using Western blotting. Protein expression of RhoA, ROCK-I, ROCK-II, Rac1, and Cdc42 was significantly higher in tumor tissue than in nontumor tissue (P < 0.0001). Expression of protein for RhoA, ROCK-I, ROCK-II, Rac1, and Cdc42 was greater in tumors of higher stages than lower stages (P < 0.0001, P < 0.001, P < 0.001, P < 0.0001, P < 0.0001, respectively). Within stage II nonseminoma (31 patients), protein levels of RhoA, ROCK-I, ROCK-II, Rac1, and Cdc42 in the primary tumor were lower in the group of 24 patients with no evidence of disease after therapy compared with 7 patients with disease that was refractory/recurrent (P < 0.05). Rho family GTPases may be involved in the progression of testicular germ cell tumors.
Increased Expression and Secretion of Interleukin-6 in Patients with Barrett’s Esophagus
Authors: Dvorakova Katerina , Payne Claire M. , Ramsey Lois , Holubec Hana , Sampliner Richard , Dominguez Jessica , Dvorak Bohuslav , Bernstein Harris , Bernstein Carol , Prasad Anil , Fass Ronnie , Cui Haiyan , Garewal Harinder
Published: September 2005

Purpose: Barrett’s esophagus (BE) is a common premalignant lesion of the distal part of the esophagus that arises as a consequence of chronic duodenogastroesophageal reflux. Interleukin (IL)-6 is a pleiotropic cytokine that regulates immune defense mechanisms and hematopoiesis. In addition, IL-6 may also be involved in malignant transformation and tumor progression. IL-6 has been shown to inhibit apoptosis. The major aim of this study was to evaluate expression of IL-6 in BE at the protein and mRNA levels. In addition, we tested whether proteins that are associated with IL-6 signaling, phosphorylated signal transducer and activator of transcription 3 and two antiapoptotic proteins, Bcl-xL and Mcl-1, are also expressed in the same tissues.

Experimental Design: Biopsies of duodenum, BE, and squamous epithelium were evaluated by using a human cytokine protein array, ELISA, real-time PCR, and immunohistochemistry.

Results: Increased IL-6 levels were found to be secreted from BE tissue compared with duodenum or squamous epithelium from sites adjacent or 5 cm away from the BE lesion. IL-6 mRNA was also elevated in BE compared with duodenum or squamous epithelium in five of seven patients. Immunohistochemical studies confirmed IL-6 expression in intestinal glandular epithelium in BE tissue. Activated signal transducer and activator of transcription 3, Mcl-1, and Bcl-xL are present at higher levels in BE glands, with lower levels being found in duodenum or squamous epithelium

Conclusions: These data, taken together, suggest that elevated IL-6 levels in BE may contribute to the development of apoptosis resistance, thereby placing this epithelium at higher risk of developing malignancy.
Phase I/II Combined Chemoimmunotherapy with Carcinoembryonic Antigen–Derived HLA-A2–Restricted CAP-1 Peptide and Irinotecan, 5-Fluorouracil, and Leucovorin in Patients with Primary Metastatic Colorectal Cancer
Authors: Weihrauch Martin R. , Ansén Sascha , Jurkiewicz Elke , Geisen Caroline , Xia Zhinan , Anderson Karen S. , Gracien Edith , Schmidt Manuel , Wittig Burghardt , Diehl Volker , Wolf Juergen , Bohlen Heribert , Nadler Lee M.
Published: September 2005

Purpose: We conducted a phase I/II randomized trial to evaluate the clinical and immunologic effect of chemotherapy combined with vaccination in primary metastatic colorectal cancer patients with a carcinoembryonic antigen–derived peptide in the setting of adjuvants granulocyte macrophage colony-stimulating factor, CpG-containing DNA molecules (dSLIM), and dendritic cells.

Experimental Design: HLA-A2–positive patients with confirmed newly diagnosed metastatic colorectal cancer and elevated serum carcinoembryonic antigen (CEA) were randomized to receive three cycles of standard chemotherapy (irinotecan/high-dose 5-fluorouracil/leucovorin) and vaccinations with CEA-derived CAP-1 peptide admixed with different adjuvants [CAP-1/granulocyte macrophage colony-stimulating factor/interleukin-2 (IL-2), CAP-1/dSLIM/IL-2, and CAP-1/IL-2]. After completion of chemotherapy, patients received weekly vaccinations until progression of disease. Immune assessment was done at baseline and after three cycles of combined chemoimmunotherapy. HLA-A2 tetramers complexed with the peptides CAP-1, human T-cell lymphotrophic virus type I TAX, cytomegalovirus (CMV) pp65, and EBV BMLF-1 were used for phenotypic immune assessment. IFN-γ intracellular cytokine assays were done to evaluate CTL reactivity.

Results: Seventeen metastatic patients were recruited, of whom 12 completed three cycles. Therapy resulted in five complete response, one partial response, five stable disease, and six progressive disease. Six grade 1 local skin reactions and one mild systemic reaction to vaccination treatment were observed. Overall survival after a median observation time of 29 months was 17 months with a survival rate of 35% (6 of 17) at that time. Eight patients (47%) showed elevation of CAP-1–specific CTLs. Neither of the adjuvants provided superiority in eliciting CAP-1–specific immune responses. During three cycles of chemotherapy, EBV/CMV recall antigen–specific CD8+ cells decreased by an average 14%.

Conclusions: The presented chemoimmunotherapy is a feasible and safe combination therapy with clinical and immunologic efficacy. Despite concurrent chemotherapy, increases in CAP-1–specific T cells were observed in 47% of patients after vaccination.
Enhancement of In vitro and In vivo Tumor Cell Radiosensitivity by the DNA Methylation Inhibitor Zebularine
Authors: Dote Hideaki , Cerna David , Burgan William E. , Carter Donna J. , Cerra Michael A. , Hollingshead Melinda G. , Camphausen Kevin , Tofilon Philip J.
Published: September 2005

Aberrant DNA hypermethylation is a frequent finding in tumor cells, which has suggested that inhibition of DNA methylation may be an effective cancer treatment strategy. Because DNA methylation affects gene expression and chromatin structure, parameters considered to influence radioresponse, we investigated the effects of the DNA methylation inhibitor zebularine on the radiosensitivity of human tumor cells. Three human tumor cell lines were used in this study (MiaPaCa, DU145, and U251) and the methylation status of three genes frequently hypermethylated in tumor cells (RASSF1A, HIC-1, and 14-3-3σ) was determined as a function of zebularine exposure. Zebularine resulted in DNA demethylation in a time-dependent manner, with the maximum loss of methylation detected by 48 hours. Treatment of cells with zebularine for 48 hours also resulted in an increase in radiosensitivity with dose enhancement factors of >1.5. As a measure of radiation-induced DNA damage, γH2AX expression was determined. Whereas zebularine had no effect on radiation-induced γH2AX foci at 1 hour, the number of γH2AX foci per cell was significantly greater in the zebularine-treated cells at 24 hours after irradiation, suggesting the presence of unrepaired DNA damage. Zebularine administration to mice reactivated gene expression in U251 xenografts; irradiation of U251 tumors in mice treated with zebularine resulted in an increase in radiation-induced tumor growth delay. These results indicate that zebularine can enhance tumor cell radiosensitivity in vitro and in vivo and suggest that this effect may involve an inhibition of DNA repair.
Analysis of Cell Cycle Regulator Proteins in Encapsulated Thymomas
Authors: Baldi Alfonso , Ambrogi Vincenzo , Mineo Davide , Mellone Pasquale , Campioni Mara , Citro Gennaro , Mineo Tommaso Claudio
Published: September 2005

Purpose: Although survival of encapsulated thymomas is usually good, some patients present a higher incidence rate of recurrence and a shorter long-term survival. Abnormalities in the components of cell cycle checkpoints are extremely common among virtually all neoplasms. In this study, three components of the cell cycle machinery (i.e., p21, p27 and p53) were examined in a series of well-characterized encapsulated thymoma specimens to analyze coregulation and influence on recurrence and survival.

Experimental Design: Sixty-eight consecutive patients with thymoma were operated in our center from 1987 to 2000. Expression of p53, p21, and p27 was studied in specimens from 25 encapsulated thymomas using immunohistochemistry. Generic factors and gene expression influencing the probability of recurrence were studied. Positive expression was dichotomized defining positive when present in more than 5% of tumor cells. Mean follow up was 85.9 months; clinical data about recurrence were recorded.

Results: Univariate analysis suggests that positive p53 (P < 0.05), negative p21 (P = 0.01), and especially negative p27 expressions (P = 0.001) significantly correlate with poor prognosis for disease-free survival. Multivariate Cox regression analysis suggests that negative p27 immunohistology is the only significant variable for poor prognosis (P = 0.03; odds ratio, 0.08; 95% confidence interval, 0.01-0.88).

Conclusions: These results show that loss of control of cell cycle checkpoints is a common occurrence in thymomas and support the idea that functional cooperation between different cell cycle inhibitor proteins constitutes another level of regulation in cell growth control and tumor suppression.
Phase I Study of OSI-7904L, a Novel Liposomal Thymidylate Synthase Inhibitor in Patients with Refractory Solid Tumors
Authors: Beutel Gernot , Glen Hilary , Schöffski Patrick , Chick Jon , Gill Stan , Cassidy James , Twelves Chris
Published: September 2005

Purpose: OSI-7904L is a liposomal formulation of a potent noncompetitive thymidylate synthase inhibitor (TSI) that does not require polyglutamation for activity. This phase I study was done to establish the safety, tolerability, maximum tolerated dose, recommended dose, and pharmacokinetics of OSI-7904L in patients with advanced solid tumors refractory to standard therapy.

Design: OSI-7904L was given as a 30-minute i.v. infusion every 21 days to 31 patients at eight dose levels from 0.4 to 15.0 mg/m2, using three patients per dose level, up to 10 patients at the recommended dose. Baseline plasma homocysteine and 2′-deoxyuridine and genotype polymorphism were measured as potential predictors of biological activity.

Results: Minimal toxicity was reported up to 9.6 mg/m2, but dose-limiting toxicity was seen in both patients at 15 mg/m2 including stomatitis, fatigue, tachyarrhythmia, rash and hand-foot syndrome, diarrhea, and fatal neutropenic sepsis. Other toxicity such as nausea and vomiting was mild or moderate. This resulted in the investigation of an intermediate dose level of 12 mg/m2, identified as the recommended dose for phase II studies. Prolonged disease stabilization was reported in 11 of 31 heavily pretreated patients. Pharmacokinetic data indicate that this liposomal formulation alters the disposition properties of the parent drug resulting in a prolonged plasma residence time.

Conclusions: OSI-7904L given as a 30-minute i.v. infusion every 21 days is feasible and well tolerated at the recommended phase II dose of 12 mg/m2. The main toxicities are rash, pruritis, lethargy, stomatitis, and myelosuppression. Observed toxicities were predictable and characteristic for TSIs.
Clinical Evaluation of Dendritic Cell Vaccination for Patients with Recurrent Glioma: Results of a Clinical Phase I/II Trial
Authors: Yamanaka Ryuya , Homma Junpei , Yajima Naoki , Tsuchiya Naoto , Sano Masakazu , Kobayashi Tsutomu , Yoshida Seiichi , Abe Takashi , Narita Miwako , Takahashi Masuhiro , Tanaka Ryuichi
Published: September 2005

Purpose: To investigate the safety and the immunologic and clinical responses of dendritic cell therapy for patients with recurrent malignant glioma.

Experimental Design: Twenty-four patients with recurrent malignant glioma (6 grade 3 and 18 grade 4 patients) were evaluated in a phase I/II clinical study of dendritic cell therapy. All patients were resistant to the standard maximum therapy. The patient's peripheral blood dendritic cells were generated with granulocyte macrophage colony-stimulating factor, plus interleukin 4 with or without OK-432, and pulsed with an autologous tumor lysate. Dendritic cells were injected intradermally, or both intratumorally and intradermally every 3 weeks.

Results: The protocols were well tolerated with only local redness and swelling at the injection site in several cases. Clinical responses were as follows: 1 patient with partial response, 3 patients with minor response, 10 patients with stable disease, and 10 patients with progressive disease. The patients whose dendritic cells were matured with OK-432 had longer survival times than the dendritic cells from patients without OK-432 maturation. The patients with both intratumoral and intradermal administrations had a longer survival time than the patients with intradermal administration only. Increased ELISPOT and delayed-type hypersensitivity responses after vaccination could provide good laboratory markers to predict the clinical outcome of patients receiving dendritic cell vaccination. The overall survival of patients with grade 4 glioma was 480 days, which was significantly better than that in the control group.

Conclusions: This study showed the safety and clinical response of autologous tumor lysate-pulsed dendritic cell therapy for patients with malignant glioma. Dendritic cell therapy is recommended for further clinical studies in malignant glioma patients.
FGFR3 and Tp53 Mutations in T1G3 Transitional Bladder Carcinomas: Independent Distribution and Lack of Association with Prognosis
Authors: Hernández Silvia , López-Knowles Elena , Lloreta Josep , Kogevinas Manolis , Jaramillo Roberto , Amorós Alex , Tardón Adonina , García-Closas Reina , Serra Consol , Carrato Alfredo , Malats Núria , Real Francisco X.
Published: September 2005

FGFR3 and Tp53 mutations have been proposed as defining two alternative pathways in the pathogenesis of transitional bladder cancer. FGFR3 mutations are associated with low-grade tumors and a favorable prognosis. Tp53 alterations are associated with advanced tumors and, possibly, with a poor prognosis. We focus here on the subgroup of T1G3 superficial tumors because they are a major clinical challenge. Patients (n = 119) were identified from a prospective study of 1,356 cases. Mutations in FGFR3 (exons 7, 10, and 15) and Tp53 (exons 4-9) were analyzed using PCR and direct sequencing. All cases were followed for recurrence and death. Survival was analyzed using Kaplan-Meier curves and multivariable Cox regression. FGFR3 mutations were detected in 20 (16.8%) tumors; 100 mutations in Tp53 were found in tumors from 78 (65.5%) cases. Multiple alterations in Tp53 were present in 19 tumors (16%). Inactivating mutations were present in 58% of tumors. The combined mutation distribution (FGFR3/Tp53) was: wt/wt (34.5%), mut/wt (7.6%), wt/mut (48.7%), and mut/mut (9.2%), indicating that the presence of either mutation did not depend on the other (P value = 0.767). FGFR3 and Tp53 mutations were not associated with clinicopathologic characteristics of patients and did not predict, alone or in combination, recurrence or survival. Taking the risk of the wt/wt group as reference, the mutation-associated risks of cancer-specific mortality were: mut/wt 1.42 (0.15-13.75), wt/mut 0.67 (0.19-2.31), mut/mut 1.62 (0.27-9.59). These molecular features support the notion that T1G3 tumors are at the crossroads of the two main molecular pathways proposed for bladder cancer development and progression.