bluuebunny/crossref_metadata_2025 huggingface dataset:
β1,6-Branched Oligosaccharides Are Increased in Lymph Node Metastases and Predict Poor Outcome in Breast Carcinoma
Authors: Handerson Tamara , Camp Robert , Harigopal Malini , Rimm David , Pawelek John
Published: September 2005

Purpose: This study was designed to provide a comprehensive assessment on the role of β1,6-branched oligosaccharides in the metastasis and outcome of breast carcinoma. Generation of these structures on N-glycans is initiated by β1,6-N-acetylglucosaminyltransferase V and used by both myeloid cells and cancer cells in systemic migration.

Experimental Design: Tissue microarrays of >700 tumors (>400 patients; 30-year follow-up data) were stained through lectin histochemistry with leukocytic phytohemagglutinin (LPHA), a selective marker for β1,6-branched oligosaccharides. Node-negative and node-positive primary tumors and patient-matched lymph node metastases were scored by blinded observers.

Results: Metastases stained at significantly greater intensities than did the patient-matched primary tumors (P < 0.0001), demonstrating for the first time that the abundance of β1,6-branched oligosaccharides was directly associated with breast carcinoma nodal metastasis. Multivariate analyses revealed that β1,6-branched oligosaccharides in primary tumors were a predictor of poor outcome, most notably in node-negative tumors, where an LPHA staining score of 3+ gave a risk factor of 3.3, independent of tumor size, nuclear grade, or patient age (P = 0.007).

Conclusions: The data firmly establish a role for β1,6-N-acetylglucosaminyltransferase V activity and β1,6-branched oligosaccharides in breast carcinoma metastasis, and reemphasize the involvement, although poorly understood, of aberrant glycosylation in tumor progression.
Cell Death Independent of Caspases: A Review
Authors: Bröker Linda E. , Kruyt Frank A.E. , Giaccone Giuseppe
Published: September 2005

Patterns of cell death have been divided into apoptosis, which is actively executed by specific proteases, the caspases, and accidental necrosis. However, there is now accumulating evidence indicating that cell death can occur in a programmed fashion but in complete absence and independent of caspase activation. Alternative models of programmed cell death (PCD) have therefore been proposed, including autophagy, paraptosis, mitotic catastrophe, and the descriptive model of apoptosis-like and necrosis-like PCD. Caspase-independent cell death pathways are important safeguard mechanisms to protect the organism against unwanted and potential harmful cells when caspase-mediated routes fail but can also be triggered in response to cytotoxic agents or other death stimuli. As in apoptosis, the mitochondrion can play a key role but also other organelles such as lysosomes and the endoplasmic reticulum have an important function in the release and activation of death factors such as cathepsins, calpains, and other proteases. Here we review the various models of PCD and their death pathways at molecular and organelle level and discuss the relevance of the growing knowledge of caspase-independent cell death pathways for cancer.
Activation of the RAS Pathway Is Predictive for a Chemosensitive Phenotype of Acute Myelogenous Leukemia Blasts
Authors: Illmer Thomas , Thiede Christian , Fredersdorf Anke , Stadler Sylvia , Neubauer Andreas , Ehninger Gerhard , Schaich Markus
Published: September 2005

Purpose: Activation of the RAS pathway plays a major role in cancer cells. In acute myeloid leukemia (AML), mutations of the RAS genes cause an intrinsic activation of this pathway. Until now, clinical studies could not find clear association of RAS mutations with the clinical outcome after AML therapy. This could be due to alternative initiating events for activation of the RAS pathway like constitutive tyrosine kinase activation or mutations in Ras-regulating genes.

Experimental Design: In total, 191 AML patients (126 as training population and 65 as test population) were studied for Ras activity with a glutathione S-transferase pull-down assay using Raf binding of activated Ras.

Results: AML samples showed a wide range of Ras activity values, which was in contrast to normal bone marrow donors who showed no or very limited Ras activity. Using a Ras binding score based on semiquantitative Western blotting, we defined patients with strong Ras activity and compared Ras activity with RAS mutation. Surprisingly, only a minority of RAS mutated AML samples (22.2%) showed strong Ras activity, whereas 25 patients presented strong Ras activity in the absence of RAS mutations. Clinical outcome did not show differences according to RAS mutations. In contrast, Ras activity predicted for a high response rate (P < 0.05) and proved to be an independent factor for overall survival rate (P < 0.05) in younger AML patients receiving high-dose 1-β-d-arabinofuranosylcytosine as induction therapy.

Conclusion: The data highlight the role for alternative pathways of Ras activation without RAS mutations. Intrinsically activated Ras seems to increase sensitivity of the AML blast to high-dose 1-β-d-arabinofuranosylcytosine therapy.
Markers of Adenocarcinoma Characteristic of the Site of Origin: Development of a Diagnostic Algorithm
Authors: Dennis Jayne L. , Hvidsten Torgeir R. , Wit Ernst C. , Komorowski Jan , Bell Alexandra K. , Downie Ian , Mooney Jacqueline , Verbeke Caroline , Bellamy Christopher , Keith W. Nicol , Oien Karin A.
Published: September 2005

Purpose: Patients with metastatic adenocarcinoma of unknown origin are a common clinical problem. Knowledge of the primary site is important for their management, but histologically, such tumors appear similar. Better diagnostic markers are needed to enable the assignment of metastases to likely sites of origin on pathologic samples.

Experimental Design: Expression profiling of 27 candidate markers was done using tissue microarrays and immunohistochemistry. In the first (training) round, we studied 352 primary adenocarcinomas, from seven main sites (breast, colon, lung, ovary, pancreas, prostate and stomach) and their differential diagnoses. Data were analyzed in Microsoft Access and the Rosetta system, and used to develop a classification scheme. In the second (validation) round, we studied 100 primary adenocarcinomas and 30 paired metastases.

Results: In the first round, we generated expression profiles for all 27 candidate markers in each of the seven main primary sites. Data analysis led to a simplified diagnostic panel and decision tree containing 10 markers only: CA125, CDX2, cytokeratins 7 and 20, estrogen receptor, gross cystic disease fluid protein 15, lysozyme, mesothelin, prostate-specific antigen, and thyroid transcription factor 1. Applying the panel and tree to the original data provided correct classification in 88%. The 10 markers and diagnostic algorithm were then tested in a second, independent, set of primary and metastatic tumors and again 88% were correctly classified.

Conclusions: This classification scheme should enable better prediction on biopsy material of the primary site in patients with metastatic adenocarcinoma of unknown origin, leading to improved management and therapy.
Enhanced Natural Killer Cell Binding and Activation by Low-Fucose IgG1 Antibody Results in Potent Antibody-Dependent Cellular Cytotoxicity Induction at Lower Antigen Density
Authors: Niwa Rinpei , Sakurada Mikiko , Kobayashi Yukari , Uehara Aya , Matsushima Kouji , Ueda Ryuzo , Nakamura Kazuyasu , Shitara Kenya
Published: September 2005

Purpose: Recent studies have revealed that fucose removal from the oligosaccharides of human IgG1 antibodies results in a significant enhancement of antibody-dependent cellular cytotoxicity (ADCC) via improved IgG1 binding to FcγRIIIa. In this report, we investigated the relationship between enhanced ADCC and antigen density on target cells using IgG1 antibodies with reduced fucose.

Experimental Design: Using EL4 cell-derived transfectants with differential expression levels of exogenous human CC chemokine receptor 4 or human CD20 as target cells, ADCC of fucose variants of chimeric IgG1 antibodies specific for these antigens were measured. We further investigated IgG1 binding to natural killer (NK) cells and NK cell activation during ADCC induction to elucidate the mechanism by which low-fucose IgG1 induces ADCC upon target cells with low antigen expression.

Results: Low-fucose IgG1s showed potent ADCC at low antigen densities at which their corresponding high-fucose counterparts could not induce measurable ADCC. The quantitative analysis revealed that fucose depletion could reduce the antigen amount on target cells required for constant degrees of ADCC induction by 10-fold for CC chemokine receptor 4 and 3-fold for CD20. IgG1 binding to NK cells was increased by ligating IgG1 with clustered antigen, especially for low-fucose IgG1. Up-regulation of an activation marker, CD69, on NK cells, particularly the CD56dim subset, in the presence of both the antibody and target cells was much greater for the low-fucose antibodies.

Conclusions: Our data showed that fucose removal from IgG1 could reduce the antigen amount required for ADCC induction via efficient recruitment and activation of NK cells.
Inhibition of Glioblastoma Angiogenesis and Invasion by Combined Treatments Directed Against Vascular Endothelial Growth Factor Receptor-2, Epidermal Growth Factor Receptor, and Vascular Endothelial-Cadherin
Authors: Lamszus Katrin , Brockmann Marc A. , Eckerich Carmen , Bohlen Peter , May Chad , Mangold Ulrich , Fillbrandt Regina , Westphal Manfred
Published: September 2005

Purpose: Inhibition of angiogenesis can influence tumor cell invasion and metastasis. We previously showed that blockade of vascular endothelial growth factor receptor-2 (VEGFR-2) with the monoclonal antibody DC101 inhibited intracerebral glioblastoma growth but caused increased tumor cell invasion along the preexistent vasculature. In the present study, we attempted to inhibit glioma cell invasion using a monoclonal antibody against the epidermal growth factor receptor (EGFR), which in the context of human glioblastomas, has been implicated in tumor cell invasion. In addition, we analyzed whether blockade of vascular endothelial (VE)-cadherin as a different antiangiogenic target could also inhibit glioblastoma angiogenesis and growth.

Experimental Designs: Nude mice who received intracerebral glioblastoma xenografts were treated using monoclonal antibodies against VEGFR-2 (DC101), EGFR (C225), and VE-cadherin (E4G10) either alone or in different combinations.

Results: Increased tumor cell invasion provoked by DC101 monotherapy was inhibited by 50% to 66% by combined treatment with C225 and DC101. C225 inhibited glioblastoma cell migration in vitro, but had no effect on the volume of the main tumor mass or on tumor cell proliferation or apoptosis in vivo, either alone or in combination with DC101. The anti-VE-cadherin monoclonal antibody E4G10 was a weaker inhibitor of tumor angiogenesis and growth than DC101, and also caused a weaker increase in tumor cell invasion.

Conclusions: Inhibition of angiogenesis achieved by blocking either VEGFR-2 or VE-cadherin can cause increased glioma cell invasion in an orthotopic model. Increased tumor cell invasion induced by potent inhibition of angiogenesis with DC101 could be inhibited by simultaneous blockade of EGFR.
High Relapse Rate in Patients with MALT Lymphoma Warrants Lifelong Follow-up
Authors: Raderer Markus , Streubel Berthold , Woehrer Stefan , Puespoek Andreas , Jaeger Ulrich , Formanek Michael , Chott Andreas
Published: September 2005

Background: B-cell lymphoma of the mucosa-associated lymphoid tissue (MALT lymphoma) is thought to be an indolent disease, with a good prognosis following various forms of treatment. Little, however, is known about the rate and pattern of relapse following successful treatment.

Patients and Methods: We have analyzed time to and pattern of relapse in patients with MALT lymphoma, along with investigation of t(11;18)(q21;q21), t(1;14)(p22;q32), and t(14;18)(q32;q21) involving IGH/MALT1, trisomy 3, and trisomy 18. Eighty-six patients achieving complete remission (CR) after initial therapy with sufficient follow-up data were available. Primary site of disease was the stomach (n = 36), salivary gland (n = 19), ocular adnexa/orbit (n = 12), lung (n = 8), thyroid (n = 5), breast (n = 3), liver (n = 2), and skin (n = 1).

Results: Thirty-two patients (37%) relapsed between 14 and 307 months (median 47 months) after initial CR. Ten relapses were local, whereas the remaining patients relapsed in a distant organ. Eight of 36 gastric versus 24 of 50 nongastric MALT lymphomas (P = 0.02) relapsed. Five patients had a second recurrence 26 to 56 months after a second CR. Relapse rates were not related to forms of initial treatment. Chromosomal aberrations were detected in 14 of 28 (50%) relapsing patients, and chromosomal alterations were identical at diagnosis and relapse. No significant association of any of the genetic changes investigated with relapse was found. Interestingly, patients with t(11;18)(q21;q21) had a significantly longer median time to relapse (76 months) than patients without this translocation (29 months; P = 0.012).

Conclusions: In view of the late relapses seen in our series, lifelong observation of all patients treated for MALT lymphoma seems to be required.
Factors That Limit the Effectiveness of Herpes Simplex Virus Type 1 for Treatment of Oral Cancer in Mice
Authors: Shillitoe Edward J. , Pellenz Christopher
Published: September 2005

Although the growth of experimental oral cancers can be inhibited by infection with the herpes simplex virus type 1 (HSV-1), the effect is incomplete. To define factors that might limit the effectiveness of the virus, we examined the roles of the innate immune system and the replication status of the tumor cells. AT-84 tumors were induced in strains of mice that had specific immune defects and were treated with the virus. Explanted tumors and tumor cells in culture were also infected. No differences in viral replication or in the effect of virus on the tumor were seen between mice with a lack of T or B lymphocytes, natural killer cells, phagocytic spleen cells, or complement. The virus did not replicate significantly more in tumors that were maintained as explants. Immediately after recovery of cells from a tumor the proportion of cells in the S phase was around 18%, and replication of virus in those cells was very limited. After 3 weeks in culture, the proportion in S had increased to 50% and both the recovery of virus from the cells and the toxic effect of the virus on the cells had increased significantly.

The innate immune system thus seemed to have a minimal effect on replication of HSV-1 when used as an oncolytic virus for oral cancers in mice. Instead, the fraction of cells in the S phase was important. Because human oral cancers, like mouse tumors, have a low fraction of cells in the S phase, it is likely that the in vivo use of HSV-1 as cancer therapy will be limited by the replication of the virus.
Int6 Expression Can Predict Survival in Early-Stage Non–Small Cell Lung Cancer Patients
Authors: Buttitta Fiamma , Martella Carla , Barassi Fabio , Felicioni Lara , Salvatore Simona , Rosini Sandra , D'Antuono Tommaso , Chella Antonio , Mucilli Felice , Sacco Rocco , Mezzetti Andrea , Cuccurullo Franco , Callahan Robert , Marchetti Antonio
Published: September 2005

Purpose: The Int6 gene was originally identified as a common insertion site for the mouse mammary tumor virus in virally induced mouse mammary tumors. Recent studies indicate that Int6 is a multifaceted protein involved in the regulation of protein translation and degradation through binding with three complexes: the eukaryotic translation initiation factor 3, the proteasome regulatory lid, and the constitutive photomorphogenesis 9 signalosome. This study aimed to investigate the prognostic role of Int6 in a large series of stage I non–small cell lung cancers (NSCLC) patients with long-term follow-up.

Experimental Design: We determined the methylation status of Int6 DNA by methylation-specific PCR and the steady-state levels of Int6 RNA by quantitative real-time reverse transcription-PCR in 101 NSCLCs and matched normal lung tissues.

Results: In 27% of the tumors, Int6 RNA levels were reduced relative to normal tissue. In 85% of the tumors with reduced Int6 expression, the transcription promoter and first exon were hypermethylated, whereas only 4% of the tumors with elevated Int6 RNA levels were hypermethylated (P < 0.000001). Low levels of Int6 RNA were found a significant predictor of overall and disease-free survival (P = 0.0004 and P = 0.0020, respectively). A multivariate analysis confirmed that low Int6 expression was the only independent factor to predict poor prognosis, for both overall (P = 0.0006) and disease-free (P = 0.024) survival.

Conclusions: Our results suggest that Int6 expression, evaluated by quantitative real-time PCR, may represent a new prognostic factor in patients with stage I NSCLC.
NHS76/PEP2, a Fully Human Vasopermeability-Enhancing Agent to Increase The Uptake and Efficacy of Cancer Chemotherapy
Authors: Khawli Leslie A. , Hu Peisheng , Epstein Alan L.
Published: September 2005

Purpose: Previously, we have shown that the attachment of interleukin 2 (IL-2) to a tumor-targeting antibody can produce a 4-fold enhancement in the uptake of antibodies and drugs in tumors. More recently, we discovered that a 37-amino-acid linear sequence of IL-2 designated vasopermeability-enhancing peptide (PEP), contained the vasopermeability activity of IL-2, and could be used after linkage to tumor-targeting antibodies to produce the same enhancement of drugs and antibodies in tumors. We now describe the generation of a fully human antibody fusion protein, designated NHS76/PEP2, which can be used in patients to enhance the therapeutic potential of chemotherapy.

Methods: NHS76/PEP2 was expressed in NS0 cells using the glutamine synthetase gene amplification system. To show its clinical potential as a pretreatment to chemotherapy, NHS76/PEP2 was given i.v. 2 hours before the injection of suboptimal doses of etoposide, doxorubicin, Taxol, Taxotere, 5-fluorouracil, or vinblastine in mice bearing established solid tumors. Results were recorded by measuring tumor volumes thrice per week.

Results: Compared with drug treatment alone, NHS76/PEP2 pretreatment substantially improved the effectiveness of chemotherapeutic agents in solid tumor models. Tumor suppression was most pronounced in those groups of mice bearing tumors known to be sensitive to the specific drug under study. However, in certain instances, tumors previously known to be resistant to specific single chemotherapeutic agents were shown to respond by the addition of NHS76/PEP2 pretreatment.

Conclusions: NHS76/PEP2 seems an excellent candidate to improve the value of standard chemotherapy drug treatment by virtue of its ability to increase the uptake of drugs in solid tumors selectively.
Phase I Pharmacokinetic-Pharmacodynamic Study of 17-(Allylamino)-17-Demethoxygeldanamycin (17AAG, NSC 330507), a Novel Inhibitor of Heat Shock Protein 90, in Patients with Refractory Advanced Cancers
Authors: Ramanathan Ramesh K. , Trump Donald L. , Eiseman Julie L. , Belani Chandra P. , Agarwala Sanjiv S. , Zuhowski Eleanor G. , Lan Jing , Potter Douglas M. , Ivy S. Percy , Ramalingam Sakkaraiappan , Brufsky Adam M. , Wong Michael K.K. , Tutchko Susan , Egorin Merrill J.
Published: September 2005

Purpose: 17-(Allylamino)-17-demethoxygeldanamycin (17AAG), a benzoquinone antibiotic, down-regulates oncoproteins by binding specifically to heat shock protein 90 (HSP90). We did a phase I study of 17AAG to establish the dose-limiting toxicity and maximum tolerated dose and to characterize 17AAG pharmacokinetics and pharmacodynamics.

Experimental Design: Escalating doses of 17AAG were given i.v. over 1 or 2 hours on a weekly × 3 schedule every 4 weeks to cohorts of three to six patients. Plasma pharmacokinetics of 17AAG and 17-(amino)-17-demethoxygeldanamycin (17AG) were assessed by high-performance liquid chromatography. Expression of HSP70 and HSP90 in peripheral blood mononuclear cells was measured by Western blot.

Results: Forty-five patients were enrolled to 11 dose levels between 10 and 395 mg/m2. The maximum tolerated dose was 295 mg/m2. Dose-limiting toxicity occurred in both patients (grade 3 pancreatitis and grade 3 fatigue) treated with 395 mg/m2. Common drug-related toxicities (grade 1 and 2) were fatigue, anorexia, diarrhea, nausea, and vomiting. Reversible elevations of liver enzymes occurred in 29.5% of patients. Hematologic toxicity was minimal. No objective responses were observed. 17AAG pharmacokinetics was linear. Peak plasma concentration and area under the curve of 17AG, the active major metabolite of 17AAG, increased with 17AAG dose, but the relationships were more variable than with 17AAG. 17AAG and 17AG in plasma were >90% protein bound. There were no consistent changes in peripheral blood mononuclear cell HSP90 or HSP70 content.

Conclusions: 17AAG doses between 10 and 295 mg/m2 are well tolerated. 17AAG pharmacokinetics is linear. Peripheral blood mononuclear cell HSP90 and HSP70 are uninformative pharmacodynamic markers. The dose recommended for future studies is 295 mg/m2 weekly × 3, repeated every 4 weeks.
Resistance of Tumor Interstitial Pressure to the Penetration of Intraperitoneally Delivered Antibodies into Metastatic Ovarian Tumors
Authors: Flessner Michael F. , Choi Jaewah , Credit Kimberly , Deverkadra Ravi , Henderson Karla
Published: September 2005

Purpose: Despite evidence that regional chemotherapy improves the treatment of metastatic peritoneal ovarian carcinoma, monoclonal antibodies have not shown significant success in i.p. delivery. The present study was designed to address the hypothesis that convective penetration of macromolecular antineoplastic agents depends on a positive pressure difference between the i.p. therapeutic solution and the tumor.

Experimental Design: Nude rats with human ovarian xenografts implanted in the abdominal wall were used in experiments to facilitate in vivo measurement of tumor pressure and the treatment of the tumor with i.p. solutions at high pressures. Penetration of 125I-labeled trastuzumab was measured with quantitative autoradiography.

Results: Tumor pressure profiles showed peak pressures of 32 mm Hg with mean pressures (± SD, mm Hg) in 12 SKOV3 tumors of 9.7 ± 8.3 and in 15 OVCAR3 tumors of 12.5 ± 7.0. I.p. therapeutic dwells at 6 to 8 mm Hg (maximum feasible pressure) showed significantly less penetration of trastuzumab than in adjacent normal muscle. To establish a driving force for convection into the tumor, various maneuvers were attempted to reduce tumor pressure, including treatment with taxanes or prostaglandin E1, elimination of tumor circulation, and removal of the tumor capsule. Tumor decapsulation decreased the pressure to zero but did not enhance the penetration of antibody. Binding to specific trastuzumab receptors on each tumor was shown to be not a significant barrier to antibody penetration.

Conclusions: The results only partially support our hypothesis and imply that the microenvironment of the tumor is in itself a major barrier to delivery of charged macromolecules.
A Neutralizing Anti–Fibroblast Growth Factor 8 Monoclonal Antibody Shows Potent Antitumor Activity against Androgen-Dependent Mouse Mammary Tumors In vivo
Authors: Shimada Naoki , Ishii Toshihiko , Imada Teruyoshi , Takaba Katsumi , Sasaki Yuka , Maruyama-Takahashi Kumiko , Maekawa-Tokuda Yoshimi , Kusaka Hideaki , Akinaga Shiro , Tanaka Akira , Shitara Kenya
Published: September 2005

Purpose: Fibroblast growth factor 8b (FGF8b) has been implicated in oncogenesis of sex hormone–related malignancies. A murine monoclonal anti-FGF8 antibody, KM1334, has been raised against a FGF8b-derived peptide and shown to neutralize FGF8b activity in an androgen-dependent mouse mammary cell line (SC-3) in vitro growth. The purpose of this study was to evaluate KM1334 as a therapeutic agent for FGF8-dependent cancer.

Experimental Design: Specificity and neutralizing activity of KM1334 were examined in vitro. In vivo therapeutic studies were done in nude mice bearing SC-3 tumors s.c.

Results: KM1334 recognized FGF8b and FGF8f specifically out of four human FGF8 isoforms and showed little binding to other members of FGF family. Neutralizing activity of KM1334 was confirmed by both blocking of FGF8b binding to its three receptors (FGFR2IIIc, FGFR3IIIc, and FGFR4) and FGF8b-induced phosphorylation of FGFR substrate 2α and extracellular signal-regulated kinase 1/2 in SC-3 cells. The in vitro inhibitory effect could be extended to in vivo tumor models, where KM1334 caused rapid regression of established SC-3 tumors in nude mice. This rapid regression of tumors after KM1334 treatment was explained by two independent mechanisms: (a) decreased DNA synthesis, as evidenced by a decrease in uptake of 5-bromo-2′-deoxyuridine, and (b) induction of apoptosis as shown by the terminal deoxynucleotidyl transferase–mediated nick end labeling assay.

Conclusions: KM1334 possesses strong blocking activity in vitro and antitumor activity in vivo and therefore may be an effective therapeutic candidate for the treatment of cancers that are dependent on FGF8b signaling for growth and survival.
In vitro Induction of Myeloid Leukemia–Specific CD4 and CD8 T Cells by CD40 Ligand – Activated B Cells Gene Modified to Express Primary Granule Proteins
Authors: Fujiwara Hiroshi , Melenhorst J. Joseph , El Ouriaghli Frank , Kajigaya Sachiko , Grube Matthias , Sconocchia Giuseppe , Rezvani Katayoun , Price David A. , Hensel Nancy F. , Douek Daniel C. , Barrett A. John
Published: September 2005

The primary granule proteins (PGP) of myeloid cells are a source of multiple antigens with immunotherapeutic potential for myeloid leukemias. Therefore, we developed a method to induce T-cell responses to PGP protein sequences. We found that gene-transfected antigen-presenting cells efficiently expand functionally competent PGP-specific CD4 and CD8 T cells. The system was optimized using T-cell responses to autologous CD40-activated B cells (CD40-B) transfected with a cytomegalovirus pp65-encoding expression vector. To generate leukemia-specific T cells, expression vectors encoding the PGP proteinase 3 (PR3), human neutrophil elastase, and cathepsin-G were transfected into CD40-B cells to stimulate postallogeneic stem cell transplantation T cells from five patients with myeloid and three with lymphoid leukemias. T-cell responses to PGP proteinase 3 and human neutrophil elastase were observed in CD8+ and CD4+ T cells only in patients with myeloid leukemias. T-cell responses against cathepsin-G occurred in both myeloid and lymphoblastic leukemias. T cells from a patient with chronic myelogenous leukemia (CML) and from a posttransplant CML patient, expanded against PGP, produced IFN-γ or were cytotoxic to the patient's CML cells, demonstrating specific antileukemic efficacy. This study emphasizes the clinical potential of PGP for expansion and adoptive transfer of polyclonal leukemia antigen-specific T cells to treat leukemia.
Farnesyltransferase Inhibitor, ABT-100, Is a Potent Liver Cancer Chemopreventive Agent
Authors: Carloni Vinicio , Vizzutti Francesco , Pantaleo Pietro
Published: September 2005

Purpose: Treatment of hepatocellular carcinoma raised on cirrhotic liver represents a major endeavor because surgery and chemotherapeutic management fail to improve the clinical course of the disease. Chemoprevention could represent an important means to inhibit the process of hepatocarcinogenesis. Farnesyltransferase inhibitors are a class of drugs blocking the growth of tumor cells with minimal toxicity towards normal cells.

Experimental Design: In the present study, we investigated the effects of a novel farnesyltransferase inhibitor, ABT-100, on human liver cancer cell lines, HepG2 and Huh7, and on an animal model of hepatocarcinogenesis.

Results: ABT-100 inhibited HepG2 and Huh7 cell growth as well as the invading ability of Huh7 on Matrigel. In HepG2 and Huh7 cells, ABT-100 inhibited growth factor–stimulated phosphoinositide 3-kinase and Akt/protein kinase B activity. Furthermore, ABT-100 inhibited Akt-dependent p27Kip1 phosphorylation and this event was associated with increased levels of p27Kip1 in the nucleus and reduced activity of the cyclin-dependent kinase 2. Moreover, ABT-100 treatment resulted in a significant reduction in tumor incidence and multiplicity.

Conclusions: Taken together, these findings identify a mechanism of ABT-100 function and show the efficacy of ABT-100 as a chemopreventive agent of hepatocellular carcinoma.
Neutral Endopeptidase 24.11/CD10 Suppresses Progressive Potential in Ovarian Carcinoma In vitro and In vivo
Authors: Kajiyama Hiroaki , Shibata Kiyosumi , Terauchi Mikio , Morita Takanori , Ino Kazuhiko , Mizutani Shigehiko , Kikkawa Fumitaka
Published: September 2005

Recently, numerous studies have shown that endothelin-1 (ET-1) is expressed in ovarian carcinoma and that ET-1 selectively acts as an autocrine or paracrine growth factor through the endothelin A receptor (ETAR), and is involved in cell proliferation, invasiveness, neovascularization, and prevention of apoptosis. Neutral endopeptidase 24.11 (NEP) is a cell surface aminopeptidase with a ubiquitous expression and is capable of degrading a number of bioactive peptides including ET-1. Our previous report showed that stromal NEP expression in ovarian carcinoma was down-regulated as the histologic grade advanced. Here, we confirmed that NEP was expressed in tumor cells as well as stromal tissues in ovarian carcinoma, and investigated the functions of NEP in this carcinoma. We showed that there was a significant decrease in cell proliferation and invasiveness with a reduction in the concentration of ET-1 in the conditioned medium on the NEP overexpression of NEP in ovarian carcinoma cells. In addition, the overexpression of NEP enhanced susceptibility to paclitaxel, resulting in an increased occurrence of apoptotic morphologic change. Furthermore, tumorigenesis was reduced in vivo with the overexpression of NEP, down-regulation of both matrix metalloproteinase-2, and vascular endothelial growth factor expression. This evidence suggests that NEP functionally suppresses the progression of ovarian carcinoma and further study of this enzyme may reveal an effective way to target ET-1 for the treatment of this carcinoma.
Plasma Pharmacokinetics, Oral Bioavailability, and Interspecies Scaling of the DNA Methyltransferase Inhibitor, Zebularine
Authors: Holleran Julianne L. , Parise Robert A. , Joseph Erin , Eiseman Julie L. , Covey Joseph M. , Glaze Elizabeth R. , Lyubimov Alexander V. , Chen Ya-Fei , D'Argenio David Z. , Egorin Merrill J.
Published: September 2005

Purpose: Zebularine is a DNA methyltransferase inhibitor proposed for clinical evaluation.

Experimental Design: We developed a liquid chromatography/mass spectrometry assay and did i.v. and oral studies in mice, rats, and rhesus monkeys.

Results: In mice, plasma zebularine concentrations declined with terminal half-lives (t1/2) of 40 and 91 minutes after 100 mg/kg i.v. and 1,000 mg/kg given orally, respectively. Zebularine plasma concentration versus time curves (area under the curve) after 100 mg/kg i.v. and 1,000 mg/kg given orally were 7,323 and 4,935 μg/mL min, respectively, corresponding to a total body clearance (CLtb) of 13.65 mL/min/kg, apparent total body clearance (CLapp) of 203 mL/min/kg, and oral bioavailability of 6.7%. In rats, plasma zebularine concentrations declined with t1/2 of 363, 110, and 126 minutes after 50 mg/kg i.v., 250 mg/kg given orally, and 500 mg/kg given orally, respectively. Zebularine areas under the curve after 50 mg/kg i.v., 250 mg/kg given orally, and 500 mg/kg given orally were 12,526, 1,969, and 7,612 μg/mL min, respectively, corresponding to a CLtb of 3.99 mL/min/kg for 50 mg/kg i.v. and CLapp of 127 and 66 mL/min/kg for 250 and 500 mg/kg given orally, respectively. Bioavailabilities of 3.1% and 6.1% were calculated for the 250 and 500 mg/kg oral doses, respectively. In monkeys, zebularine t1/2 was 70 and 150 minutes, CLtb was 3.55 and 10.85 mL/min/kg after i.v. administration, and CLapp was 886 and 39,572 mL/min/kg after oral administration of 500 and 1,000 mg/kg, respectively. Zebularine oral bioavailability was <1% in monkeys. Interspecies scaling produced the following relationship: CLtb = 6.46(weight0.9).

Conclusions: Zebularine has limited oral bioavailability. Interspecies scaling projects a CLtb of 296 mL/min in humans.
Drg1 Expression in 131 Colorectal Liver Metastases: Correlation with Clinical Variables and Patient Outcomes
Authors: Shah Manish A. , Kemeny Nancy , Hummer Amanda , Drobnjak Marija , Motwani Monica , Cordon-Cardo Carlos , Gonen Mithat , Schwartz Gary K.
Published: September 2005

Purpose: The differentiation-related gene-1 (Drg1) is a recently identified gene down-regulated in malignancy and a putative suppressor of colorectal cancer metastases. Its expression is associated with improved survival in patients with prostate or breast cancer. Drg1 expression is also associated with resistance to irinotecan therapy in preclinical colorectal cancer models. The clinical evaluation of Drg1 in colorectal cancer has been limited. We performed this study to evaluate the role of Drg1 in a large cohort of patients with metastatic colorectal cancer who were irinotecan naïve.

Experimental Design: We examined Drg1 expression by immunohistochemistry in 131 patients with metastatic colorectal cancer enrolled in a clinical trial of adjuvant fluorouracil-based therapy from 1991 to 1995. We correlated expression of Drg1 to numerous clinical and tumor related variables and to patient outcomes, including a subset of patients who recurred and received irinotecan-based therapy.

Results: Drg1 expression was identified in all metastatic tissue samples. There was a trend for unilobar metastases with high Drg1 expression (P = 0.07) and a suggestion of improved 2-year survival (82.4% versus 69.6%, P = 0.148). High Drg1 expression suggested irinotecan resistance (P = 0.07).

Conclusions: In colorectal cancer, Drg1 expression may be associated with a less aggressive, indolent colorectal cancer. High Drg1 may also be associated with relative resistance to irinotecan. The role of Drg1 in malignancy continues to be defined.
Promoter Hypermethylation Profile of Ovarian Epithelial Neoplasms
Authors: Makarla Prakash B. , Saboorian M. Hossein , Ashfaq Raheela , Toyooka Kiyomi O. , Toyooka Shinichi , Minna John D. , Gazdar Adi F. , Schorge John O.
Published: September 2005

Purpose: Ovarian carcinomas are believed to arise de novo from surface epithelium, but the actual molecular pathogenesis is unknown. The aim of this study was to compare the promoter hypermethylation profiles of ovarian epithelial neoplasms to better understand the role of epigenetic silencing in carcinogenesis.

Experimental Design: We analyzed the DNA promoter methylation status of eight tumor suppressor and cancer-related genes (p16, RARβ, E-cadherin,H-cadherin, APC, GSTP1, MGMT, RASSF1A) in 23 benign cystadenomas, 23 low malignant potential (LMP) tumors, and 23 invasive carcinomas by methylation-specific PCR.

Results: Benign cystadenomas exhibited promoter hypermethylation in only two genes, p16 (13%) and E-cadherin (13%). LMP tumors also showed p16 (22%) and E-cadherin (17%) methylation, in addition to RARβ (9%) and H-cadherin (4%). All eight genes were hypermethylated in invasive cancers at a frequency of 9% to 30%. The mean methylation index was highest in invasive tumors [0.20 versus 0.065 (LMP) and 0.033 (cystadenomas); P = 0.001]. Promoter methylation of at least one gene was most commonly observed among invasive cancers [78% versus 44% (LMP; P = 0.03) and 26% (cystadenomas; P = 0.0009)]. Three genes exhibited higher methylation frequencies in invasive tumors: RASSF1A (30% versus 0%; P = 0.0002), H-cadherin (22% versus 2%; P = 0.013), and APC (22% versus 0%; P = 0.003).

Conclusions: Promoter hypermethylation is a frequent epigenetic event that occurs most commonly in invasive epithelial ovarian carcinomas. The profile of aberrant methylation suggests that an accumulation of events at specific genes may trigger malignant transformation of some benign cystadenomas and LMP tumors.
Distinct Methylation Patterns of Benign and Malignant Liver Tumors Revealed by Quantitative Methylation Profiling
Authors: Lehmann Ulrich , Berg-Ribbe Ina , Wingen Luzie U. , Brakensiek Kai , Becker Thomas , Klempnauer Jürgen , Schlegelberger Brigitte , Kreipe Hans , Flemming Peer
Published: September 2005

Purpose: A comparative quantitative methylation profiling of hepatocellular carcinoma and the most frequent benign liver tumor, hepatocellular adenoma, was set up for the identification of tumor-specific methylation patterns.

Experimental Design: The quantitative methylation levels of nine genes (RASSF1A, cyclinD2, p16INK4a, DAP-K, APC, RIZ-1, HIN-1, GSTπ1, SOCS-1) were analyzed in hepatocellular carcinoma and adjacent normal tissue (n = 41), hepatocellular adenoma and adjacent normal tissue (n = 26), focal nodular hyperplasia (n = 10), and unrelated normal liver tissue (n = 28). Accumulated methylation data were analyzed using various statistical algorithms, including hierarchical clustering, to detect tumor-specific methylation patterns.

Results: Cluster analysis revealed that hepatocellular adenoma displays a methylation profile much more similar to that found in normal liver tissue and focal nodular hyperplasia than to that found in hepatocellular carcinoma. Many characteristic differences were not detected when using mere qualitative methylation assays. The cyclinD2 gene was identified as a new and frequent target for aberrant hypermethylation in hepatocellular carcinoma (68%). In the control group of 28 liver specimens from healthy donors, a clear correlation between age of patient and frequency and level of aberrant methylation was seen, which could not be detected in the group of hepatocellular carcinoma specimens.

Conclusions: Methylation profiling can clearly contribute to the unequivocal classification of suspicious lesions, but only if done in a quantitative manner applying cell type and gene-specific thresholds. In hepatocellular carcinoma, the altered methylation patterns accompanying malignant transformation override the age-dependent increase in gene methylation.
Circulating Tumor Cells in Breast Cancer: Correlation to Bone Marrow Micrometastases, Heterogeneous Response to Systemic Therapy and Low Proliferative Activity
Authors: Müller Volkmar , Stahmann Nicole , Riethdorf Sabine , Rau Thomas , Zabel Tanja , Goetz Alexander , Jänicke Fritz , Pantel Klaus
Published: September 2005

Purpose: The incidence and biological characteristics of circulating tumor cells in the blood of patients with breast cancer were examined and subgroups were evaluated in the context of systemic treatment and the presence of disseminated tumor cells in bone marrow.

Experimental Design: Circulating tumor cells were isolated from the peripheral blood of patients with breast cancer using a gradient system designed for the enrichment of circulating tumor cells (OncoQuick). Circulating tumor cells were identified with the anti-cytokeratin antibody, A45-B/B3. In subsets of patients, expression of the proliferation-associated Ki-67 antigen in circulating tumor cells and the concomitant presence of micrometastases in bone marrow were examined.

Results: In patients with primary breast cancer (stage M0), circulating tumor cells were detected in 5 of 60 patients (8.3%) after surgery and before initiation of adjuvant chemotherapy; a positive correlation to the presence of disseminated tumor cells in bone marrow was observed (P = 0.030, n = 53). During the course of adjuvant chemotherapy, repeated analysis of 20 M0 patients revealed the occurrence of circulating tumor cells in 7 of 16 patients that were initially negative. Patients with metastatic disease (stage M1) showed circulating tumor cells in 25 of 63 cases (39.7%, P < 0.0001 as compared with M0 patients), and a positive finding was correlated with elevated concentrations of the serum tumor marker CA15.3 (P = 0.0093). Performing repeated analysis in a subgroup of 25 M1 patients, circulating tumor cells were found more frequently in patients with progressive disease than in patients with stable disease or remission (87.5% versus 43.8% of patients with circulating tumor cells, respectively; P = 0.047). Independent of the disease-stage, none of the 47 patients examined for the proliferative status of their circulating tumor cells showed coexpression of Ki-67.

Conclusions: Circulating tumor cells seem to be nonproliferating cells that persist during chemotherapy. Circulating tumor cell detection is linked to disease progression and elevated tumor marker concentrations in patients with metastatic breast cancer.
Detection and Staging of Preinvasive Lesions and Occult Lung Cancer in the Central Airways with 18F-Fluorodeoxyglucose Positron Emission Tomography: A Pilot Study
Authors: Pasic Arifa , Brokx Hes A. , Comans Emile F. , Herder Gerarda J. , Risse Elle K. , Hoekstra Otto S. , Postmus Pieter E. , Sutedja Tom G.
Published: September 2005

Purpose: To evaluate the role of 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) in radiologically occult preinvasive lesions and lung cancer in the central airways.

Experimental Design: Twenty-two patients with 24 preinvasive lesions and early squamous cell cancer (SCC) being occult on high-resolution computed tomography were studied. All lesions were diagnosed based on histology sampled using autofluorescence bronchoscopy. FDG-PET findings were correlated with WHO histologic classification. FDG-PET was considered true-positive when the final diagnosis was SCC and true-negative when the lesions were classified as severe dysplasia or less.

Results: FDG-PET was true-positive in 8 of 11 and true-negative in 11 of 13 cases corresponding with a sensitivity of 73% [95% confidence interval (CI), 0.43-0.91] and specificity of 85% (95% CI, 0.57-0.97). Positive and negative predictive values were 80% (95% CI, 0.48-0.96) and 79% (95% CI, 0.52-0.93), respectively.

Conclusions: Our very preliminary data suggest that FDG-PET might be useful for the evaluation of early central airway lesions, being positive in most SCC and negative in cases of severe dysplasia. Validation in a larger multicenter study is needed.
Systemic Administration of an Attenuated, Tumor-TargetingSalmonella typhimuriumto Dogs with Spontaneous Neoplasia: Phase I Evaluation
Authors: Thamm Douglas H. , Kurzman Ilene D. , King Ivan , Li Zujin , Sznol Mario , Dubielzig Richard R. , Vail David M. , MacEwen E. Gregory
Published: September 2005

Purpose: Genetically modified bacteria are a potentially powerful anticancer therapy due to their tumor targeting capacity, inherent antitumor activity, and ability to serve as efficient vectors for gene delivery. This study sought to characterize the acute and short-term toxicities and tumor colonization rates of a genetically modified Salmonella typhimurium (VNP20009) in dogs with spontaneous tumors, in the context of a phase I dose escalation trial.

Experimental Design: Forty-one pet dogs with a variety of malignant tumors received weekly or biweekly i.v. infusions of VNP20009, at doses ranging from 1.5 × 105 to 1 × 108 cfu/kg. Vital signs and clinicopathologic variables were monitored regularly. Incisional biopsies were obtained before and 1 week following the first infusion for histopathology and bacterial culture.

Results: The nominal maximum tolerated dose was 3 × 107 cfu/kg, with refractory fever and vomiting being the dose-limiting toxicities. One treatment-related acute death occurred. Bacteria were cultured from tumor tissue in 42% of cases. Thirty-five patients were evaluable for antitumor response. Major antitumor responses were seen in 15% (4 complete response and 2 partial response), and disease stabilization for at least 6 weeks in 10%.

Conclusions: Administration of VNP20009 at doses with acceptable toxicity results in detectable bacterial colonization of tumor tissue and significant antitumor activity in tumor-bearing dogs.
Decreased T-Cell Receptor Excision Circles in Cutaneous T-Cell Lymphoma
Authors: Yamanaka Kei-ichi , Yawalkar Nikhil , Jones David A. , Hurwitz Daniel , Ferenczi Katalin , Eapen Sara , Kupper Thomas S.
Published: September 2005

Purpose: The T cell repertoire in patients with advanced cutaneous T cell lymphoma (CTCL) is significantly contracted despite the presence of relatively normal absolute numbers of T cells. We propose that many normal T cells were being lost in patients with CTCL, with the remaining normal T cells expanding clonally to fill the T cell compartment. T-cell receptor excision circles (TREC) form as a result of the initial gene rearrangement in naïve T cells. Although they are stable, they do not replicate and are subsequently diluted with the expansion of a population of T cells. Their concentration is therefore a measure of unexpanded naïve T cells relative to T cells that have undergone expansion.

Experimental Design: We analyzed TRECs from unfractionated peripheral blood T cells from 108 CTCL patients by quantitative PCR. In patients with obvious peripheral blood involvement, we also analyzed TRECs from clonal and nonclonal T cells.

Results: We found a decrease in the number of TRECs in peripheral blood of patients with CTCL at all stages of disease, and this decrease was proportional to the loss of complexity of the T cell repertoire as measured by complementarity-determining region 3 spectratyping. In patients with leukemic CTCL and a numerically expanded clone, we also found a significantly lower-than-expected number of TRECs in the nonclonal normal T cells.

Conclusions: We hypothesize that the nonmalignant T cells have proliferated to fill the empty T cell repertoire space left by the loss of other T cells, leading to diminished TRECs and loss of T-cell receptor diversity.
Tumor-Associated Alterations in Caspase-14 Expression in Epithelial Malignancies
Authors: Krajewska Maryla , Kim Hoguen , Shin Eunah , Kennedy Susan , Duffy Michael J. , Wong Yick F. , Marr David , Mikolajczyk Jowita , Shabaik Ahmed , Meinhold-Heerlein Ivo , Huang Xianshu , Banares Steven , Hedayat Hirad , Reed John C. , Krajewski Stan
Published: September 2005

Purpose: Caspase-14 is unique among caspase family proteases in that its proteolytic processing has been principally associated with epithelial cell differentiation rather than apoptosis or inflammation. We investigated caspase-14 expression in several types of human epithelial malignancy by immunohistochemistry, correlating results with stage, histologic grade, and patient survival.

Experimental Design: Tumor-associated alterations in caspase-14 expression were observed for cervical, ovarian, breast, gastric, and colon cancers.

Results: In cervical (n = 445), ovarian (n = 91), and colon (n = 106) specimens, expression of caspase-14 was significantly reduced in cancers compared with normal epithelium. Decreases in caspase-14 immunopositivity correlated with the histologic progression of cervical cancer (P < 0.0001, ANOVA). In localized gastric cancers, caspase-14 immunostaining was significantly lower in poorly differentiated tumors compared with well-differentiated tumors (P = 0.02, Pearson's χ2 analysis). Lower caspase-14 expression was associated with advanced clinical stage in ovarian cancer (P = 0.04, ANOVA) and with shorter overall survival among ovarian cancer patients with serous tumors (n = 62) in both univariate (P = 0.005) and multivariate (P = 0.03) analysis. Lower caspase-14 expression correlated with shorter overall survival among patients with T3N0M0 stage gastric cancers (n = 94; P = 0.006, log-rank test). In contrast to cervical, ovarian, and colon cancers, caspase-14 expression was increased in ductal carcinoma in situ and invasive cancers compared with normal mammary epithelium (P = 0.001, t test).

Conclusions: The findings reveal tumor-specific alterations in caspase-14 expression and suggest that differences in its expression may define subsets of epithelial cancers with distinct clinical behaviors.