bluuebunny/crossref_metadata_2025 huggingface dataset:
Phase I Study of Immunization with Dendritic Cells Modified with Fowlpox Encoding Carcinoembryonic Antigen and Costimulatory Molecules
Authors: Morse Michael A. , Clay Timothy M. , Hobeika Amy C. , Osada Takuya , Khan Shubi , Chui Stephen , Niedzwiecki Donna , Panicali Dennis , Schlom Jeffrey , Lyerly H. Kim
Published: September 2005

Purpose: To determine the safety and immunologic and clinical efficacy of a dendritic cell vaccine modified to hyperexpress costimulatory molecules and tumor antigen.

Experimental Design: In this phase I study, we administered one or two cycles of four triweekly s.c./intradermal injections of ex vivo generated dendritic cells modified with a recombinant fowlpox vector encoding carcinoembryonic antigen (CEA) and a triad of costimulatory molecules [rF-CEA(6D)-TRICOM]. Controls consisted of immature dendritic cells loaded with tetanus toxoid and a HLA A2–restricted peptide derived from cytomegalovirus pp65 protein.

Results: Fourteen patients (11 with colorectal cancer and 3 with non–small cell lung cancer) were enrolled and 12 completed at least one cycle of immunization. There were no grade 3/4 toxicities directly referable to the immunizations. One patient had a decrease in the CEA level from 46 to 6.8 and a minor regression in adenopathy that occurred several months after completion of the immunizations. Five other patients were stable through at least one cycle of immunization (3 months). Direct analysis of peripheral blood mononuclear cells using the ELISpot assay showed an increase in the frequency of CEA-specific T cells in 10 patients (range, 10-541 CEA-specific cells/105 peripheral blood mononuclear cells). There was a trend for a greater peak frequency of CEA-specific T cells among those with either a minor response or a stable disease following at least one cycle of therapy. A second cycle was not associated with higher T-cell frequencies. Cytokine flow cytometry showed CEA-specific immune response among both CD4+ and CD8+ T cells in all immune responders.

Conclusion: This immunization strategy is safe and activates potent CEA-specific immune responses.
Automated Quantitative Analysis of E-Cadherin Expression in Lymph Node Metastases Is Predictive of Survival in Invasive Ductal Breast Cancer
Authors: Harigopal Malini , Berger Aaron J. , Camp Robert L. , Rimm David L. , Kluger Harriet M.
Published: September 2005

Purpose: The tumor suppressor adhesion molecule E-cadherin is believed to have an anti-invasive role in breast cancer. Lymph node involvement is the best prognostic marker known, yet there is variability in outcome among node-positive patients. We investigated the relationship between E-cadherin expression in primary invasive ductal tumors and corresponding nodal metastases, and determined the prognostic value of E-cadherin expression in node-positive breast cancer.

Experimental Design: Membrane E-cadherin expression was studied by immunohistochemical staining of tissue microarrays with fluorescent-labeled antibodies. An objective method of automated quantitative analysis (AQUA) was used. AQUA uses cytokeratin to define pixels as breast cancer (tumor mask) within the array spot, and measures E-cadherin expression using a Cy5-conjugated antibody within the mask.

Results: We employed a tissue microarray containing 207 primary and matched nodal metastases suitable for AQUA analysis. There was no significant difference in mean staining intensity between the primary and nodal specimens (P = 0.8). A scattergram was generated which identified a subset of patients (25%) with high E-cadherin expression in nodal metastases, and this top quartile had improved survival (P = 0.028). On univariate analysis, increased E-cadherin expression in nodal metastases was strongly associated with improved survival (P = 0.007), whereas expression in primary tumors was not (P = 0.13). On multivariate analysis, nodal E-cadherin expression retained its independent association with survival, as did tumor size and HER2/neu status.

Conclusions: Strong E-cadherin expression in lymph node metastases was highly predictive of improved survival. This suggests that expression of adhesion molecules at metastatic sites portends less aggressive tumor behavior.
Promoter Methylation of DAL-1/4.1B Predicts Poor Prognosis in Non–Small Cell Lung Cancer
Authors: Kikuchi Shinji , Yamada Daisuke , Fukami Takeshi , Masuda Mari , Sakurai-Yageta Mika , Williams Yuko N. , Maruyama Tomoko , Asamura Hisao , Matsuno Yoshihiro , Onizuka Masataka , Murakami Yoshinori
Published: September 2005

Purpose: DAL-1/4.1B is an actin-binding protein originally identified as a molecule whose expression is down-regulated in lung adenocarcinoma. We have previously shown that a lung tumor suppressor, TSLC1, associates with DAL-1, suggesting that both proteins act in the same cascade. The purpose of this study is to understand the molecular mechanisms and clinical significance of DAL-1 inactivation in lung cancer.

Experimental Design: We studied aberration of the DAL-1 in 103 primary non–small cell lung cancers (NSCLC) and 18 lung cancer cells. Expression and allelic and methylation status of DAL-1 was examined by reverse transcription-PCR, microsatellite analysis, and bisulfite sequencing or bisulfite single-strand conformational polymorphism, respectively.

Results: Loss of DAL-1 expression was strongly correlated with promoter methylation in lung cancer cells, whereas DAL-1 expression was restored by a demethylating agent, 5-aza-2′-deoxycytidine. The DAL-1 promoter was methylated in 59 (57%) primary NSCLC tumors, 37% of which were associated with loss of heterozygosity around the DAL-1 on chromosomal region 18p11.3. In squamous cell carcinomas, DAL-1 methylation was observed in 9 of 10 tumors at stage I, whereas the incidence of methylation gradually increased in adenocarcinomas as they progressed [13 of 36 (36%), 4 of 12 (33%), 14 of 17 (82%), and 3 of 3 (100%) tumors at stages I, II, III, and IV, respectively; P = 0.0026]. Furthermore, in adenocarcinomas, disease-free survival and overall survival were significantly shorter in patients with tumors harboring the methylated DAL-1 (P = 0.0011 and P = 0.045, respectively).

Conclusions: DAL-1 methylation is involved in the development and progression of NSCLC and provides an indicator for poor prognosis.
A Haplotype Analysis of HER-2 Gene Polymorphisms: Association with Breast Cancer Risk, HER-2 Protein Expression in the Tumor, and Disease Recurrence in Korea
Authors: Han Wonshik , Kang Daehee , Lee Jong Eun , Park In Ae , Choi Ji-Yeob , Lee Kyung-Mu , Bae Ji Yeon , Kim Sook , Shin Eun-Soon , Lee Jeong Eon , Shin Hyuk-Jae , Kim Seok Won , Kim Sung-Won , Noh Dong-Young
Published: September 2005

Purpose: A single-nucleotide polymorphism (SNP) in codon 655 of HER-2 has been extensively studied with inconclusive results. The purpose of this study was to investigate the association between common variants of HER-2 and breast cancer risk, HER-2 expression, and survival using a haplotype-based stepwise approach.

Experimental Design: Twenty-nine SNPs listed in the National Center for Biotechnology Information database were screened to identify novel polymorphisms of HER-2 gene in 90 healthy Korean women. Six of 29 SNPs were polymorphic and had greater than 10% of minor allele frequencies. Using these six SNPs, linkage disequilibrium and haplotype patterns were characterized. We tested association between the haplotypes and breast cancer in a large case–control study (n = 1,039 cases and 995 controls). Six-hundred two breast cancer patients with follow-up at least 24 months were analyzed for outcome in relation to haplotype. Expression of HER-2 protein was determined by immunohistochemistry in 1,094 cases of invasive breast cancer.

Results: All six SNPs showed a strong linkage disequilibrium pattern and were considered to belong to one haplotype block. Two haplotype-tagging SNPs (I655V and P1170A) for three common haplotypes (>5%) were genotyped in cases and controls. The haplotypes and individual SNPs were not associated with breast cancer risk. In patients with at least one copy of haplotype I (the most common haplotype), HER-2 expression was 1.5 times higher (P = 0.009) and the prognosis was worse (P = 0.032) compared with patients without having that haplotype.

Conclusions: Our results suggest that the currently identified genetic polymorphisms of HER-2 are not associated with an increased risk of breast cancer in Korean women, whereas one haplotype does affect protein expression of the tumor and disease outcome.
Translational Crossroads for Biomarkers
Authors: Bast Robert C. , Lilja Hans , Urban Nicole , Rimm David L. , Fritsche Herbert , Gray Joe , Veltri Robert , Klee George , Allen Andrew , Kim Nam , Gutman Steven , Rubin Mark A. , Hruszkewycz Andrew
Published: September 2005

A group of investigators met at a Specialized Programs of Research Excellence Workshop to discuss key issues in the translation of biomarker discovery to the development of useful laboratory tests for cancer care. Development and approval of several new markers and technologies have provided informative examples that include more specific markers for prostate cancer, more sensitive tests for ovarian cancer, more objective analysis of tissue architecture and an earlier indication of response to treatment in breast cancer. Although there is no clear paradigm for biomarker development, several principles are clear. Marker development should be driven by clinical needs, including early cancer detection, accurate pretreatment staging, and prediction of response to treatment, as well as monitoring disease progression and response to therapy. Development of a national repository that uses carefully preserved, well-annotated tissue specimens will facilitate new marker development. Reference standards will be an essential component of this process. Both hospital-based and commercial laboratories can play a role in developing biomarkers from discovery to test validation. Partnering of academe and industry should occur throughout the process of biomarker development. The National Cancer Institute is in a unique position to bring together academe, industry, and the Food and Drug Administration to (a) define clinical needs for biomarkers by tumor type, (b) establish analytic and clinical paradigms for biomarker development, (c) discuss ways in which markers from different companies might be evaluated in combination, (d) establish computational methods to combine data from multiple biomarkers, (e) share information regarding promising markers developed in National Cancer Institute–supported programs, and (f) exchange data regarding new platforms and techniques that can accelerate marker development.
Gene Signatures of Progression and Metastasis in Renal Cell Cancer
Authors: Jones Jon , Otu Hasan , Spentzos Dimitrios , Kolia Shakirahmed , Inan Mehmet , Beecken Wolf D. , Fellbaum Christian , Gu Xuesong , Joseph Marie , Pantuck Allan J. , Jonas Dietger , Libermann Towia A.
Published: September 2005

Purpose: To address the progression, metastasis, and clinical heterogeneity of renal cell cancer (RCC).

Experimental Design: Transcriptional profiling with oligonucleotide microarrays (22,283 genes) was done on 49 RCC tumors, 20 non-RCC renal tumors, and 23 normal kidney samples. Samples were clustered based on gene expression profiles and specific gene sets for each renal tumor type were identified. Gene expression was correlated to disease progression and a metastasis gene signature was derived.

Results: Gene signatures were identified for each tumor type with 100% accuracy. Differentially expressed genes during early tumor formation and tumor progression to metastatic RCC were found. Subsets of these genes code for secreted proteins and membrane receptors and are both potential therapeutic or diagnostic targets. A gene pattern (“metastatic signature”) derived from primary tumor was very accurate in classifying tumors with and without metastases at the time of surgery. A previously described “global” metastatic signature derived by another group from various non-RCC tumors was validated in RCC.

Conclusion: Unlike previous studies, we describe highly accurate and externally validated gene signatures for RCC subtypes and other renal tumors. Interestingly, the gene expression of primary tumors provides us information about the metastatic status in the respective patients and has the potential, if prospectively validated, to enrich the armamentarium of diagnostic tests in RCC. We validated in RCC, for the first time, a previously described metastatic signature and further showed the feasibility of applying a gene signature across different microarray platforms. Transcriptional profiling allows a better appreciation of the molecular and clinical heterogeneity in RCC.
Mining the Tumor Phosphoproteome for Cancer Markers
Authors: Lim Yoon Pin
Published: September 2005

Despite decades of cancer research, mortality rates remain high largely due to the failure of early detection, poor understanding of the epidemiology of rational drug targets, and molecular etiology of human cancers. The discovery of disease markers promises to deliver some solutions to these formidable challenges. Gene and protein expression profiling through DNA microarray and proteomics have already made a tremendous effect in this area. However, protein/gene expression does not necessarily reflect protein activity, which is often regulated via post-translation modifications, of which phosphorylation is one of the most prominent. This is an important consideration because the activity of protein is a more relevant phenotype than its expression during pathogenesis. Tyrosine kinases represent a very important class of enzymes that are critical regulators of mitogenic and angiogenic signaling, hence attractive targets for anticancer drugs as exemplified by BCR-ABL and ErbB2. More than 50% of them are overexpressed or mutated resulting in a gain of function in various human cancers. In this review, we discuss the potential effect of phosphoproteins as cancer markers in cancer diagnosis and therapeutics. Phosphoproteomics strategies that might pave the way to high-throughput analysis for routine clinical applications are also described.
Green Tea Component, Catechin, Induces Apoptosis of Human Malignant B Cells via Production of Reactive Oxygen Species
Authors: Nakazato Tomonori , Ito Keisuke , Ikeda Yasuo , Kizaki Masahiro
Published: September 2005

Purpose: Green tea polyphenol, (−)-epigallocatechin-3-gallate, has been shown to inhibit cellular proliferation and induce apoptosis of various cancer cells. The aim of this study was to investigate the possibility of (−)-epigallocatechin-3-gallate as a novel therapeutic agent for the patients with B-cell malignancies including multiple myeloma.

Experimental Design: We investigated the effects of (−)-epigallocatechin-3-gallate on the induction of apoptosis in HS-sultan as well as myeloma cells in vitro and further examined the molecular mechanisms of (−)-epigallocatechin-3-gallate-induced apoptosis.

Results: (−)-Epigallocatechin-3-gallate rapidly induced apoptotic cell death in various malignant B-cell lines in a dose- and time-dependent manner. (−)-Epigallocatechin-3-gallate-induced apoptosis was in association with the loss of mitochondrial transmembrane potentials (Δψm); the release of cytochrome c, Smac/DIABLO, and AIF from mitochondria into the cytosol; and the activation of caspase-3 and caspase-9. Elevation of intracellular reactive oxygen species (ROS) production was also shown during (−)-epigallocatechin-3-gallate-induced apoptosis of HS-sultan and RPMI8226 cells as well as fresh myeloma cells. Antioxidant, catalase, and Mn superoxide dismutase significantly reduced ROS production and (−)-epigallocatechin-3-gallate-induced apoptosis, suggesting that ROS plays a key role in (−)-epigallocatechin-3-gallate-induced apoptosis in B cells. Furthermore, a combination with arsenic trioxide (As2O3) and (−)-epigallocatechin-3-gallate significantly enhanced induction of apoptosis compared with As2O3 alone via decreased intracellular reduced glutathione levels and increased production of ROS.

Conclusions: (−)-Epigallocatechin-3-gallate has potential as a novel therapeutic agent for patients with B-cell malignancies including multiple myeloma via induction of apoptosis mediated by modification of the redox system. In addition, (−)-epigallocatechin-3-gallate enhanced As2O3-induced apoptosis in human multiple myeloma cells.
Treatment of Relapsed Chronic Lymphocytic Leukemia by 72-Hour Continuous Infusion or 1-Hour Bolus Infusion of Flavopiridol: Results from Cancer and Leukemia Group B Study 19805
Authors: Byrd John C. , Peterson Bercedis L. , Gabrilove Janice , Odenike Olatoyosi M. , Grever Michael R. , Rai Kanti , Larson Richard A. ,   the Cancer and Leukemia Group B
Published: September 2005

Purpose: Flavopiridol has in vitro activity in chronic lymphocytic leukemia (CLL) and promotes apoptosis independent of p53 function or prior fludarabine exposure. We sought to determine if flavopiridol has activity in previously treated CLL using two schedules of administration.

Patients and Methods: Patients with previously treated CLL were enrolled in two sequentially done phase II studies. Patients in the first trial received flavopiridol (50 mg/m2/d) as a continuous infusion (CI) for 72 hours every 2 weeks. Patients in the second trial received flavopiridol 50 mg/m2 as a 1-hour bolus (IVB) daily for 3 days repeated every 3 weeks. Patients received up to 12 (CI cohort) or 8 (IVB cohort) cycles of therapy.

Results: Fifteen patients were enrolled in the 72-hour CI phase II trial; 6 (40%) had intermediate-risk (Rai stage I or II) and 9 (60%) had high-risk (Rai stage III and IV) stages. No responses were noted in this group; 27% had stable disease and 73% had progressive disease. Thirty-six patients were enrolled in the second IVB trial, with 13 (36%) having intermediate and 23 (64%) having high-risk disease. Four patients (11%) had partial responses, 19 (53%) had stable disease, and 13 (36%) had progressive disease. The progression-free survival for responders in the IVB trial was 3, 3, 9, and 19 months. The median progression-free survival was 2 months [95% confidence interval (95% CI), 1.8-3.8] for patients in the CI trial and 3 months (95% CI, 2.5-7.4) for the IVB trial. The median overall survival was 27 months (95% CI, 20-42) for the CI trial and 24 months (95% CI, 18-31) for the IVB trial. Toxicity was manageable and included mainly myelosuppression, infections, diarrhea, and fatigue.

Conclusions: Flavopiridol has modest, schedule-dependent clinical activity in relapsed CLL and warrants future investigation utilizing alternative schedules of administration.
Comparative Preclinical and Clinical Pharmacokinetics of a Cremophor-Free, Nanoparticle Albumin-Bound Paclitaxel (ABI-007) and Paclitaxel Formulated in Cremophor (Taxol)
Authors: Sparreboom Alex , Scripture Charity D. , Trieu Vuong , Williams Paul J. , De Tapas , Yang Andrew , Beals Bridget , Figg William D. , Hawkins Michael , Desai Neil
Published: September 2005

Purpose: To compare the preclinical and clinical pharmacokinetic properties of paclitaxel formulated as a Cremophor-free, albumin-bound nanoparticle (ABI-007) and formulated in Cremophor-ethanol (Taxol).

Experimental Design: ABI-007 and Taxol were given i.v. to Harlan Sprague-Dawley male rats to determine pharmacokinetic and drug disposition. Paclitaxel pharmacokinetic properties also were assessed in 27 patients with advanced solid tumors who were randomly assigned to treatment with ABI-007 (260 mg/m2, 30 minutes; n = 14) or Taxol (175 mg/m2, 3 hours; n = 13), with cycles repeated every 3 weeks.

Results: The volume of distribution at steady state and clearance for paclitaxel formulated as Cremophor-free nanoparticle ABI-007 were significantly greater than those for paclitaxel formulated with Cremophor (Taxol) in rats. Fecal excretion was the main elimination pathway with both formulations. Consistent with the preclinical data, paclitaxel clearance and volume of distribution were significantly higher for ABI-007 than for Taxol in humans [21.13 versus 14.76 L/h/m2 (P = 0.048) and 663.8 versus 433.4 L/m2 (P = 0.040), respectively].

Conclusions: Paclitaxel formulated as ABI-007 differs from paclitaxel formulated as Taxol, with a higher plasma clearance and a larger volume of distribution. This finding is consistent with the absence of paclitaxel-sequestering Cremophor micelles after administration of ABI-007. This unique property of ABI-007 could be important for its therapeutic effectiveness.
Humoral Immune Response to Mesothelin in Mesothelioma and Ovarian Cancer Patients
Authors: Ho Mitchell , Hassan Raffit , Zhang Jingli , Wang Qing-cheng , Onda Masanori , Bera Tapan , Pastan Ira
Published: September 2005

Purpose: Mesothelin is a glycosyl-phosphatidylinositol–anchored glycoprotein present on the cell surface. Mesothelin is a differentiation antigen that is highly expressed on mesothelioma, ovarian cancer, and pancreatic cancer. The existence of a spontaneous humoral immune response to mesothelin in humans has not been fully studied. Here we addressed the issue of whether mesothelin elicits a humoral immune response in patients with mesothelioma and ovarian cancer.

Experimental Design: Using an ELISA, we analyzed immunoglobulin G antibodies specific for mesothelin in sera from patients with mesothelioma and epithelial ovarian cancer. Tumor specimens were examined by immunohistochemistry for mesothelin protein expression.

Results: Elevated levels of mesothelin-specific antibodies were detected in the sera of 39.1% of patients with mesothelioma (27 of 69 patients) and 41.7% with epithelial ovarian cancer (10 of 24 patients) when compared with a normal control population (44 blood donors; P < 0.01 for both mesothelioma and ovarian cancer). We also found that 53% to 56% of patients with mesothelin immunostaining-positive mesothelioma and ovarian cancer had antibodies specific for mesothelin, whereas only 0% to 8% of patients with negative mesothelin immunostaining had detectable mesothelin-specific antibodies (χ2 test: P < 0.01 for mesothelioma and P = 0.025 for ovarian cancer).

Conclusions: Our findings indicate that mesothelin is a new tumor antigen in patients with mesothelioma and ovarian cancer and the immunogenicity of mesothelin is associated with its high expression on the tumor cells. Mesothelin represents an excellent target for immune-based therapies.
Phase I Trial of 131I-huA33 in Patients with Advanced Colorectal Carcinoma
Authors: Chong Geoffrey , Lee Fook Thean , Hopkins Wendie , Tebbutt Niall , Cebon Jonathan S. , Mountain Angela J. , Chappell Bridget , Papenfuss Anthony , Schleyer Paul , U Paul , Murphy Roger , Wirth Veronika , Smyth Fiona E. , Potasz Nicole , Poon Aurora , Davis Ian D. , Saunder Tim , O'Keefe Graeme J. , Burgess Antony W. , Hoffman Eric W. , Old Lloyd J. , Scott Andrew M.
Published: September 2005

Purpose: Humanized monoclonal antibody A33 (huA33) targets the A33 antigen which is expressed on 95% of colorectal cancers. A previous study has shown excellent tumor-targeting of iodine-131 labeled huA33 (131I-huA33). Therefore, we did a phase I dose escalation trial of 131I-huA33 radioimmunotherapy.

Experimental Designs: Fifteen patients with pretreated metastatic colorectal carcinoma each received two i.v. doses of 131I-huA33. The first was an outpatient trace-labeled “scout” dose for biodistribution assessment, followed by a second “therapy” dose. Three patients were treated at 20, 30, and 40 mCi/m2 dose levels, and six patients at 50 mCi/m2 to define the maximum tolerated dose.

Results: Hematologic toxicity was 131I dose-dependent, with one episode of grade 4 neutropenia and two episodes of grade 3 thrombocytopenia observed at 50 mCi/m2. The maximum tolerated dose was determined to be 40 mCi/m2. There were no acute infusion-related adverse events, and gastrointestinal toxicity was not observed despite uptake of 131I-huA33 in bowel. Seven patients developed pruritus or rash, which was not related to 131I dose. There was excellent tumor-targeting of 131I-huA33 shown in all patients. The serum T1/2β of 131I-huA33 was (mean ± SD) 135.2 ± 46.9 hours. The mean absorbed tumor dose was 6.49 ± 2.47 Gy/GBq. Four patients developed human anti-human antibodies. At restaging, 4 patients had stable disease, whereas 11 patients had progressive disease.

Conclusion: Radioimmunotherapy using 131I-huA33 shows promise in targeting colorectal tumors, and is deliverable at a maximum tolerated dose of 40 mCi/m2. Further studies of 131I-huA33 in combination with chemotherapy are planned.
Methylation Profiling of Archived Non–Small Cell Lung Cancer: A Promising Prognostic System
Authors: Safar A. Mazin , Spencer Horace , Su Xiaobo , Coffey Maureen , Cooney Craig A. , Ratnasinghe Luke D. , Hutchins Laura F. , Fan Chun-Yang
Published: September 2005

Purpose: Enhanced prognostication power is becoming more desirable in clinical oncology. In this study, we explored the prognostic potential of multigene hypermethylation profiling in non–small-cell lung cancer.

Experimental Design: We evaluated a panel of eight genes (p16, APC, ATM, hMLH1, MGMT, DAPK, ECAD, and RASSF1A) using methylation-specific PCR in 105 archived specimens of non–small-cell lung cancer representing all stages of the illness. We analyzed the effect of gene methylation status on outcome individually in a cumulative manner and in a combinatorial approach using recursive partitioning to identify methylation profiles, which affect overall survival.

Results: In this data set, tumors harboring promoter hypermethylation at two or more genes exhibit similar survival trends to others in the cohort. Using recursive partitioning, three genes (APC, ATM, and RASSF1A) emerged as determinants of prognostic groups. This designation retained its statistical significance even when disease stage and age were entered into a multivariate analysis. Using this approach, patients whose tumors were hypermethylated at APC and those hypermethylated at only ATM (not also at APC or RASSF1A) enjoyed substantially longer 1- and 2-year survival than patients in the remaining groups. In 32 adjacent histologically normal lung tissue specimens, we detected similar methylation abnormalities.

Conclusion: Assessment of promoter hypermethylation aberrations may facilitate prognostic profiling of lung tumors, but validation in independent data sets is needed to verify these profiles. This system uses material that is abundantly available with linked outcome data and can be used to generate reliable epigenetic determinants.
P53 Mutation Analysis of Colorectal Liver Metastases: Relation to Actual Survival, Angiogenic Status, and p53 Overexpression
Authors: de Jong Koert P. , Gouw Annette S.H. , Peeters Paul M.J.G. , Bulthuis Marian , Menkema Lorian , Porte Robert J. , Slooff Maarten J.H. , van Goor Harry , van den Berg Anke
Published: September 2005

Purpose: To correlate TP53 mutations with angiogenic status of the tumor and prognosis after liver surgery in patients with colorectal liver metastases and to correlate immunohistochemical staining of p53 protein with TP53 gene mutations.

Experimental Design: Tumors of 44 patients with surgically treated colorectal liver metastases were analyzed for (a) TP53 mutations using denaturing gradient gel electrophoresis followed by sequencing, (b) microvessel density using the hot spot overlap technique, (c) apoptotic rate in tumor cells and endothelial cells of tumor microvessels using double immunostaining for anti–cleaved caspase 3 and anti-CD34, and (d) expression of p53 protein using immunohistochemistry.

Results:TP53 mutations were detected in 36% of the metastases and occurred more frequently in liver metastases from left-sided colon tumors than from right-sided colon tumors (P = 0.04). In metastases with TP53 mutations, microvessel density was higher compared with tumors with wild-type p53. Endothelial cell apoptosis was not different in tumor microvessels from TP53-mutated versus nonmutated tumors. The 5-year actual survival was not influenced by TP53 mutational status, microvessel density, or endothelial cell apoptotic rate of the tumors. Based on immunohistochemical p53 overexpression, the positive and negative predictive values of TP53 mutations were 61% and 82%.

Conclusions: In patients with surgically treated colorectal liver metastases, TP53 mutations and angiogenic status did not influence prognosis. Immunohistochemistry is not a reliable technique for detecting TP53 mutations.
Dual Inhibition of mTOR and Estrogen Receptor SignalingIn vitroInduces Cell Death in Models of Breast Cancer
Authors: Boulay Anne , Rudloff Joelle , Ye Jingjing , Zumstein-Mecker Sabine , O'Reilly Terence , Evans Dean B. , Chen Shiuan , Lane Heidi A.
Published: September 2005

Purpose: RAD001 (everolimus), a mammalian target of rapamycin (mTOR) pathway inhibitor in phase II clinical trials in oncology, exerts potent antiproliferative/antitumor activities. Many breast cancers are dependent for proliferation on estrogens synthesized from androgens (i.e., androstenedione) by aromatase. Letrozole (Femara) is an aromatase inhibitor used for treatment of postmenopausal women with hormone-dependent breast cancers. The role of the mTOR pathway in estrogen-driven proliferation and effects of combining RAD001 and letrozole were examined in vitro in two breast cancer models.

Experimental Design: The role of the mTOR pathway in estrogen response was evaluated in aromatase-expressing MCF7/Aro breast cancer cells by immunoblotting. Effects of RAD001 and letrozole (alone and in combination) on the proliferation and survival of MCF7/Aro and T47D/Aro cells were evaluated using proliferation assays, flow cytometry, immunoblotting, and apoptosis analyses.

Results: Treatment of MCF7/Aro cells with estradiol or androstenedione caused modulation of the mTOR pathway, a phenomenon reversed by letrozole or RAD001. In MCF7/Aro and T47D/Aro cells, both agents inhibited androstenedione-induced proliferation; however, in combination, this was significantly augmented (P < 0.001, two-way ANOVA, synergy by isobologram analysis). Increased activity of the combination correlated with more profound effects on G1 progression and a significant decrease in cell viability (P < 0.01, two-way ANOVA) defined as apoptosis (P < 0.05, Friedman test). Increased cell death was particularly evident with optimal drug concentrations.

Conclusion: mTOR signaling is required for estrogen-induced breast tumor cell proliferation. Moreover, RAD001-letrozole combinations can act in a synergistic manner to inhibit proliferation and trigger apoptotic cell death. This combination holds promise for the treatment of hormone-dependent breast cancers.
Prediction of BRCA1 Status in Patients with Breast Cancer Using Estrogen Receptor and Basal Phenotype
Authors: Lakhani Sunil R. , Reis-Filho Jorge S. , Fulford Laura , Penault-Llorca Frederique , van der Vijver Marc , Parry Suzanne , Bishop Timothy , Benitez Javier , Rivas Carmen , Bignon Yves-Jean , Chang-Claude Jenny , Hamann Ute , Cornelisse Cees J. , Devilee Peter , Beckmann Matthias W. , Nestle-Krämling Carolin , Daly Peter A. , Haites Neva , Varley Jenny , Lalloo Fiona , Evans Gareth , Maugard Christine , Meijers-Heijboer Hanne , Klijn Jan G.M. , Olah Edith , Gusterson Barry A. , Pilotti Silvana , Radice Paolo , Scherneck Siegfried , Sobol Hagay , Jacquemier Jocelyne , Wagner Teresa , Peto Julian , Stratton Michael R. , McGuffog Lesley , Easton Douglas F. ,   the Breast Cancer Linkage Consortium
Published: September 2005

Purpose: To investigate the proportion of breast cancers arising in patients with germ line BRCA1 and BRCA2 mutations expressing basal markers and developing predictive tests for identification of high-risk patients.

Experimental Design: Histopathologic material from 182 tumors in BRCA1 mutation carriers, 63 BRCA2 carriers, and 109 controls, collected as part of the international Breast Cancer Linkage Consortium were immunohistochemically stained for CK14, CK5/6, CK17, epidermal growth factor receptor (EGFR), and osteonectin.

Results: All five basal markers were commoner in BRCA1 tumors than in control tumors (CK14: 61% versus 12%; CK5/6: 58% versus 7%; CK17: 53% versus 10%; osteonectin: 43% versus 19%; EGFR: 67% versus 21%; P < 0.0001 in each case). In a multivariate analysis, CK14, CK5/6, and estrogen receptor (ER) remained significant predictors of BRCA1 carrier status. In contrast, the frequency of basal markers in BRCA2 tumors did not differ significant from controls.

Conclusion: The use of cytokeratin staining in combination with ER and morphology provides a more accurate predictor of BRCA1 mutation status than previously available, that may be useful in selecting patients for BRCA1 mutation testing. The high percentage of BRCA1 cases positive for EGFR suggests that specific anti-tyrosine kinase therapy may be of potential benefit in these patients.
Enhanced Pharmacodynamic and Antitumor Properties of a Histone Deacetylase Inhibitor Encapsulated in Liposomes or ErbB2-Targeted Immunoliposomes
Authors: Drummond Daryl C. , Marx Corina , Guo Zexiong , Scott Gary , Noble Charles , Wang Donghui , Pallavicini Maria , Park John W. , Kirpotin Dmitri B. , Benz Christopher C.
Published: September 2005

ErbB2-overexpressing human cancers represent potentially sensitive targets for therapy by candidate histone deacetylase (HDAC) inhibitors as we have shown that HDAC inhibitors can selectively reduce ErbB2 expression by repressing the ErbB2 promoter and accelerating the decay of cytoplasmic ErbB2 transcripts. To extend these in vitro findings and enhance the in vivo pharmacodynamic properties of HDAC inhibitors, we stably encapsulated a potent hydroxamate-based HDAC inhibitor (LAQ824) within long-circulating liposomes (Ls-LAQ824) and immunoliposomes (ILs-LAQ824) bearing >10,000 LAQ824 molecules per nanovesicle. Liposomal LAQ824 exhibits prolonged in vivo stability and, unlike free LAQ824, circulates with a half-life of 10.8 hours following a single i.v. injection. Three weekly i.v. injections of 20 to 25 mg/kg Ls-LAQ824 in nude mice with ErbB2 overexpressing BT-474 breast tumor xenografts significantly impairs tumor growth, and administration of ErbB2-targeted ILs-LAQ824 may further improve this antitumor activity. Studies of tumor-bearing mice 24 hours after single treatment indicate that: (a) >10% of injected liposomal LAQ824 is still circulating (whereas free LAQ824 is undetectable in the blood after 15 minutes); and (b) tumor uptake of Ls-LAQ824 and ILs-LAQ824 is >3% injected drug per gram of tumor, producing levels of acetylated tumor histones that are 5- to 10-fold increased over those following free LAQ824 or saline treatments and resulting in concordantly reduced levels of tumor ErbB2 mRNA. These preclinical results support the clinical evaluation of HDAC inhibitors against ErbB2-overexpressing malignancies, and further indicate that encapsulation into targeted and nontargeted liposomes substantially improves the in vivo pharmacokinetics, tumor uptake, and antitumor properties of hydroxamate-based HDAC inhibitors.
Trastuzumab-Mediated Antibody-Dependent Cellular Cytotoxicity against Esophageal Squamous Cell Carcinoma
Authors: Mimura Kousaku , Kono Koji , Hanawa Mitsuhiko , Kanzaki Mirei , Nakao Atsuhito , Ooi Akishi , Fujii Hideki
Published: September 2005

Purpose: In the present study, we investigated the degree of protein expression and gene amplification of HER-2 in esophageal squamous cell carcinoma (SCC) cell lines and freshly isolated tumors, and trastuzumab-mediated biological activity, in particular antibody-dependent cellular cytotoxicity (ADCC) against HER-2–expressing esophageal SCC cell lines.

Experimental Design: Ten different SCC cell lines with various levels of HER-2 status evaluated by flow cytometry, immunocytochemistry (HercepTest), and fluorescence in situ hybridization were evaluated for ADCC, growth inhibitory, or apoptosis-inducing activities mediated by trastuzumab.

Results: Trastuzumab induced ADCC against HER-2–expressing esophageal SCC and the activities reflected the degree of HER-2 expression analyzed by flow cytometric analysis, but not by HercepTest nor fluorescence in situ hybridization analysis. Furthermore, trastuzumab-mediated ADCC against transforming growth factor-β–producing SCC was enhanced by the treatment with SB-431542, which is a selective inhibitor of the phosphorylation induced by transforming growth factor-β. There were very marginal effects of anti-proliferative or apoptosis-inducing activities mediated by trastuzumab for HER-2–expressing esophageal SCC.

Conclusion: HER-2–expressing esophageal SCC cells could be killed by trastuzumab-mediated ADCC and the activity reflected the degree of HER-2 expression detected by flow cytometry.
Antiangiogenic Properties of Gold Nanoparticles
Authors: Mukherjee Priyabrata , Bhattacharya Resham , Wang Ping , Wang Ling , Basu Sujit , Nagy Janice A. , Atala Anthony , Mukhopadhyay Debabrata , Soker Shay
Published: September 2005

Here, we report an intrinsic property of gold nanoparticles (nanogold): they can interact selectively with heparin-binding glycoproteins and inhibit their activity. Gold nanoparticles specifically bound vascular permeability factor/vascular endothelial growth factor (VPF/VEGF)-165 and basic fibroblast growth factor, two endothelial cell mitogens and mediators of angiogenesis resulting in inhibition of endothelial/fibroblast cell proliferation in vitro and VEGF-induced permeability as well as angiogenesis in vivo. In contrast, nanogold did not inhibit VEGF-121 or epidermal growth factor, two non–heparin-binding growth factors, mediated cell proliferation. Gold nanoparticles significantly inhibited VEGF receptor-2 phosphorylation, intracellular calcium release, and migration and RhoA activation in vitro. These results report for the first time a novel property of gold nanoparticles to bind heparin-binding proteins and thereby inhibit their subsequent signaling events.
Flavopiridol Sensitivity of Cancer Cells Isolated from Ascites and Pleural Fluids
Authors: Richard Christina , Matthews Donald , Duivenvoorden Wilhelmina , Yau Jonathan , Wright Paul S. , Th'ng John P.H.
Published: September 2005

Purpose: We examined the efficacy of flavopiridol, a cyclin-dependent kinase inhibitor that is undergoing clinical trials, on primary cancer cells isolated from the ascites or pleural fluids of patients with metastatic cancers.

Experimental Design: Metastasized cancer cells were isolated from the pleural fluids (n = 20) or ascites (n = 15) of patients, most of whom were refractory to chemotherapy. These primary cancer cells were used within 2 weeks of isolation without selecting for proliferative capacities. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide viability assay was used to characterize the response of these cancer cells to commonly used chemotherapeutic agents, and their response to flavopiridol was compared with rapidly dividing cultured cell lines.

Results: The primary cancer cells displayed phenotypes that were different from established cell lines; they had very low replication rates, dividing every 1 to 2 weeks, and underwent replicative senescence within five passages. These primary tumor cells retained their resistance to chemotherapeutic drugs exhibited by the respective patients but did not show cross-resistance to other agents. However, these cancer cells showed sensitivity to flavopiridol with an average LD50 of 50 nmol/L (range, 21.5-69 nmol/L), similar to the LD50 in established cell lines. Because senescent cells also showed similar sensitivity to flavopiridol, it suggests that the mechanism of action is not dependent on the activity of cyclin-dependent kinases that regulate the progression of the cell cycle.

Conclusion: Using cancer cells isolated from the ascites or pleural fluids, this study shows the potential of flavopiridol against cancer cells that have developed resistance to conventional chemotherapeutic agents.
Expression of Cell Cycle–Regulated Proteins pRB2/p130, p107, E2F4, p27, and pCNA in Salivary Gland Tumors: Prognostic and Diagnostic Implications
Authors: Russo Giuseppe , Zamparelli Alessandra , Howard Candace M. , Minimo Corrado , Bellan Cristiana , Carillo Giovanna , Califano Luigi , Leoncini Lorenzo , Giordano Antonio , Claudio Pier Paolo
Published: September 2005

The retinoblastoma family consists of the tumor suppressor nuclear phosphoprotein pRb/p105 and related proteins p107 and pRb2/p130. Recent immunohistochemical studies of the retinoblastoma family of proteins in lung and endometrial cancer and choroidal melanomas show a tight inverse correlation between the histologic grading in the most aggressive tumor types and pRb2/p130 expression. This led us to investigate the role of pRb2/p130 in salivary tumors. We studied the expression of pRb2/p130, p107, E2F4, p27, and PcNA by immunohistochemistry in a panel of 44 salivary gland tumors. We found a direct correlation between the cytoplasmic expression of pRb2/p130 and tumor grading and the presence of metastasis that was highly statistically significant (P < 0.001). Additionally, increased cytoplasmic pRb2/p130 expression was significantly correlated with a decreased probability of survival (P < 0.001). Interestingly, p107 nuclear expression showed a strong direct correlation when compared with the same variables. pRb2/p130 showed the highest percentage of undetectable nuclear levels in the specimens examined and the tightest inverse correlation (P < 0.0001) with both the histologic grading and pCNA expression in malignant salivary tumors. Additionally, E2F4 showed an identical localization pattern as to that of pRb2/p130. These data suggests an important role for pRb2/p130 in the pathogenesis and progression of certain salivary gland cancers.
Low-Molecular Weight Caldesmon as a Potential Serum Marker for Glioma
Authors: Zheng Ping-Pin , Hop Wim C. , Sillevis Smitt Peter A.E. , van den Bent Martin J. , Avezaat Cees J.J. , Luider Theodorus M. , Kros Johan M.
Published: September 2005

Purpose: Testing the feasibility of using the serum low-molecular weight caldesmon (l-CaD) level as a serum marker for the presence of glioma.

Experimental Design: Within a total of 230 serum samples, the l-CaD level was measured in healthy volunteers (30), patients with gliomas (57), nonglial intracranial tumors (107), and nontumor neurologic diseases (36) by ELISA. The specificity of the assay was monitored by combination of immunoprecipitation and immunoblotting.

Results: The serum level of l-CaD is significantly higher in the group of glioma patients as compared with any of the other groups (P < 0.001). The cutoff value of 45 yields optimal sensitivity and specificity of the assay (91% and 84%, respectively; area under the curve score = 0.91). The specificity of ELISA was confirmed by the immunoprecipitation/immunoblotting control experiments. There were no significant differences in serum l-CaD levels between patients with low- or high-grade gliomas.

Conclusions: The serum l-CaD level as determined by ELISA is a good discriminator between glioma patients versus patients with other intracranial tumors, other neurologic diseases, and healthy people. Prospective studies are required to test the contribution of the assay in making the diagnosis of glioma, or its feasibility for monitoring the tumor during treatment.
Analysis of KIT Mutations in Sporadic and Familial Gastrointestinal Stromal Tumors: Therapeutic Implications through Protein Modeling
Authors: Tarn Chi , Merkel Erin , Canutescu Adrian A. , Shen Wei , Skorobogatko Yuliya , Heslin Martin J. , Eisenberg Burton , Birbe Ruth , Patchefsky Arthur , Dunbrack Roland , Arnoletti J. Pablo , von Mehren Margaret , Godwin Andrew K.
Published: September 2005

Purpose: Gastrointestinal stromal tumors (GIST) are characterized by expressing a gain-of-function mutation in KIT, and to a lesser extent, PDGFR. Imatinib mesylate, a tyrosine kinase inhibitor, has activity against GISTs that contain oncogenic mutations of KIT. In this study, KIT and PDGFRα mutation status was analyzed and protein modeling approaches were used to assess the potential effect of KIT mutations in response to imatinib therapy.

Experimental Design: Genomic DNA was isolated from GIST tumors. Exons 9, 11, 13, and 17 of c-KIT and exons 12, 14, and 18 of PDGFRα were evaluated for oncogenic mutations. Protein modeling was used to assess mutations within the juxtamembrane region and the kinase domain of KIT.

Results: Mutations in KIT exons 9, 11, and 13 were identified in GISTs with the majority of changes involving the juxtamembrane region of KIT. Molecular modeling indicates that mutations in this region result in disruption of the KIT autoinhibited conformation, and lead to gain-of-function activation of the kinase. Furthermore, a novel germ-line mutation in KIT was identified that is associated with an autosomal dominant predisposition to the development of GIST.

Conclusions: We have used protein modeling and structural analyses to elucidate why patients with GIST tumors containing exon 11 mutations are the most responsive to imatinib mesylate treatment. Importantly, mutations detected in this exon and others displayed constitutive activation of KIT. Furthermore, we have found tumors that are both KIT and PDGFRα mutation negative, suggesting that additional, yet unidentified, abnormalities may contribute to GIST tumorigenesis.
Correlation between DNA Repair Capacity in Lymphocytes and Acute Side Effects to Skin during Radiotherapy in Nasopharyngeal Cancer Patients
Authors: Wang Wei-dong , Chen Zheng-tang , Li De-zhi , Cao Zheng-huai , Sun Shi-liang , Pu Ping , Chen Xiao-pin
Published: September 2005

Purpose: Repair of radiation-induced DNA damage plays a critical role for both the susceptibility of patients to side effects after radiotherapy and their subsequent cancer risk. The study objective was to evaluate whether DNA repair data determined in vitro are correlated with the occurrence of acute side effects during radiotherapy.

Experimental Design: Nasopharyngeal cancer patients receiving radiation therapy were recruited in a prospective epidemiologic study. As an indicator for clinical radiosensitivity, adverse reactions of the skin were recorded. Cryopreserved lymphocytes from 100 study participants were γ-irradiated with 5 Gy in vitro and analyzed using the alkaline comet assay. Reproducibility of the assay was determined by repeated analysis (n = 22) of cells from a healthy donor. A coefficient of variation of 0.24 was calculated.

Results: The various parameters determined to characterize the individual DNA repair capacity showed large differences between patients. Twenty-one patients were identified with considerably enhanced DNA damage induction, and 19 patients exhibited severely reduced DNA repair capacity after 15 and 30 minutes. Eight patients were considered as clinically radiosensitive, indicated by moist desquamation of the skin after a total radiation dose of 70 Gy.

Conclusions: Using the alkaline comet assay as described here, nasopharyngeal cancer patients were identified showing abnormal cellular radiation effects, but this repair deficiency corresponded only at a very limited extent to the acute radiation sensitivity of the skin.