bluuebunny/crossref_metadata_2025 huggingface dataset:
Demethylation of Urokinase Promoter as a Prognostic Marker in Patients with Breast Carcinoma
Authors: Pakneshan Pouya , Têtu Bernard , Rabbani Shafaat A.
Published: September 2005

Purpose: Urokinase (uPA) is expressed in a number of highly invasive malignancies including breast cancer. Because production of uPA is associated with breast cancer progression and can serve as a useful prognostic marker, the purpose of this study was to examine the role of uPA promoter methylation as an indicator of uPA production in breast cancer patients.

Experimental Design: We examined the methylation status of the uPA promoter and the levels of uPA expression in normal human breast epithelial cells and several human breast cancer cells by bisulfite sequencing analysis and reverse transcription-PCR. We also analyzed the methylation status of the uPA promoter in surgical biopsy samples from patients with breast cancer of different grades, as determined by the Elston-Ellis histological grading system.

Results: Expression of uPA mRNA was only detected in the highly invasive estrogen receptor-negative breast cancer cell lines, where the promoter was completely demethylated. In normal and low invasive breast cancer cells, the uPA promoter was methylated, resulting in lack of uPA mRNA expression. Analysis of biopsy samples showed that demethylation of the uPA promoter is associated with malignant transformation. Reverse transcription-PCR analysis revealed that this demethylation of the uPA promoter is directly associated with induction of uPA mRNA expression, which is well known to be associated with poor prognosis in breast cancer patients.

Conclusions: This study indicated that uPA expression in breast cancer patients is under epigenetic control via methylation of its promoter. Determination of uPA promoter methylation can therefore serve as an early reliable indicator of uPA production in breast cancer patients.
A Comparison of thein Vitroandin VivoActivities of IgG and F(ab′)2 Fragments of a Mixture of Three Monoclonal Anti-Her-2 Antibodies
Authors: Spiridon Camelia I. , Guinn Sarah , Vitetta Ellen S.
Published: September 2005

Purpose: We have demonstrated previously that a mixture of three anti-Her-2 monoclonal antibodies (MAbs) that bind to different epitopes on the extracellular domain of Her-2 expressed on a human breast cancer cell line has more potent antitumor activity than the individual MAbs both in vitro and in xenografted severe combined immunodeficient mice. Because the activity of Herceptin is Fc dependent, we determined whether this would also be the case when a mixture of these three anti-Her-2 MAbs was used.

Experimental Design: IgG and highly purified F(ab′)2 fragments of the anti-Her-2 MAbs and Herceptin were prepared and evaluated for their ability to induce cell death, inhibit vascular endothelial growth factor secretion, and mediate antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity in vitro. They were also compared for their abilities to induce regression of large BT474 tumors in severe combined immunodeficient mice.

Results: All of the F(ab′)2 fragments were >95% pure and, as expected, did not mediate antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity in vitro. The in vitro antiproliferative and proapoptotic effects of the IgGs and F(ab′)2 fragments were similar. In contrast, the IgGs had significant antitumor activity in vivo, whereas their F(ab′)2 fragments were only marginally effective even at 5-fold higher doses to offset their shorter half-lives.

Conclusions: These results confirm the importance of the Fc portion of Herceptin for optimal in vivo activity and demonstrate that even a mixture of three anti-Her-2 MAbs that are highly effective at inducing cell death in vitro requires Fc-mediated effector function for optimal in vivo activity.
Cancer and Leukemia Group B 90206
Authors: Rini Brian I. , Halabi Susan , Taylor John , Small Eric J. , Schilsky Richard L. ,   for the Cancer Leukemia Group B
Published: September 2005

The majority of sporadic clear cell renal cell carcinoma (RCC) is characterized by loss of heterozygosity of the von Hippel-Lindau (VHL) tumor suppressor gene and somatic inactivation of the remaining VHL allele. The resulting VHL gene silencing leads to induction of hypoxia-regulated genes including vascular endothelial growth factor (VEGF). Thus, therapeutic inhibition of VEGF holds promise for treatment of this historically refractory malignancy. An antibody to VEGF (bevacizumab, Avastin) has demonstrated a significant prolongation of time to disease progression compared with placebo in patients with metastatic RCC. Interferon-α (IFN-α) is a standard initial cytokine therapy in RCC with a modest response rate and a survival advantage demonstrated in randomized trials. We hypothesized that the addition of anti-VEGF therapy to IFN-α would prolong survival in untreated metastatic RCC patients. A Phase III trial is now being conducted randomizing untreated, metastatic clear cell RCC patients to IFN-α alone or IFN-α plus Avastin.
Expression of Extracellular Matrix Metalloproteases Inducer on Micrometastatic and Primary Mammary Carcinoma Cells
Authors: Reimers Natalie , Zafrakas Kristine , Assmann Volker , Egen Cornelia , Riethdorf Lutz , Riethdorf Sabine , Berger Jürgen , Ebel Sebastian , Jänicke Fritz , Sauter Guido , Pantel Klaus
Published: September 2005

Purpose: EMMPRIN (extracellular matrix metalloprotease inducer) is a glycosylated member of the immunoglobulin superfamily known to stimulate the production of matrix metalloproteases (MMPs) 1, 2, and 3 and MT1-MMP in peritumoral fibroblasts. We here evaluated whether EMMPRIN expression is related to tumor progression in human breast cancer.

Experimental Design: An immunohistochemical study using high-density tissue microarrays (n = 2222 breast cancer samples) and EMMPRIN-specific antibodies HIM6 and MEM-M6/1 was performed, and staining results were statistically correlated with various clinicopathological parameters. To analyze the putative association between EMMPRIN expression and bone marrow (BM) micrometastasis, an additional set of 55 breast tumors from patients with or without micrometastatic cells as determined with anti-cytokeratin antibody A45-B/B3 were included in our study. Cytokeratin-positive cells in BM were costained with EMMPRIN-specific antibody 1G6.2.

Results: Positive EMMPRIN staining correlated significantly with various histopathological risk factors (higher tumor grade, increased tumor size, negative estrogen receptor status and progesterone receptor status, and higher mitotic index) as well as decreased tumor-specific survival (log-rank, P = 0.0027). In particular, in patients > 50 years (i.e., postmenopausal women), EMMPRIN expression was an independent prognosticator as shown by Cox regression analysis (relative risk = 1.7, 95% confidence interval 1.4–4.3, P = 0.036). An involvement of EMMPRIN in tumor progression was also supported by the fact that it was expressed on ∼90% of micrometastatic cells in BM.

Conclusions: EMMPRIN expression in primary tumor predicts an unfavorable prognosis in breast cancer, suggesting a crucial role of EMMPRIN in progression of human mammary carcinomas.
Targeting Vascular Endothelial Growth Factor for Relapsed and Refractory Adult Acute Myelogenous Leukemias
Authors: Karp Judith E. , Gojo Ivana , Pili Roberto , Gocke Christopher D. , Greer Jacqueline , Guo Chuanfa , Qian David , Morris Lawrence , Tidwell Michael , Chen Helen , Zwiebel James
Published: September 2005

Purpose: Vascular endothelial growth factor (VEGF) promotes acute myelogenous leukemia (AML) cell growth and survival and may contribute to drug resistance. bevacizumab, an anti-VEGF monoclonal antibody, exhibits clinical activity against diverse malignancies when administered with cytotoxic chemotherapy. We conducted a Phase II clinical trial of bevacizumab administered after chemotherapy to adults with refractory or relapsed AML, using a timed sequential therapy (TST) approach.

Experimental Design: bevacizumab 10 mg/kg was administered on day 8 after 1-β-d-arabinofuranosylcytosine 2 g/m2/72 h beginning day 1 and mitoxantrone 40 mg/m2 beginning day 4. In vivo laboratory correlates included AML cell VEGF receptor-1 (FLT-1) expression, marrow microvessel density, and free serum VEGF before and during TST with bevacizumab.

Results: Forty-eight adults received induction therapy. Myelosuppression occurred in all of the patients similar to other TST regimens. Toxicities were decreased ejection fraction (6%), cerebrovascular bleed (4%), and mortality of 15%. Overall response was 23 of 48 (48%), with complete response (CR) in 16 (33%). Eighteen (14 CR and 4 partial response) underwent one consolidation cycle and 5 (3 CR and 2 partial response) underwent allogeneic transplant. Median overall and disease-free survivals for CR patients were 16.2 months (64%, 1 year) and 7 months (35%, 1 year). Marrow blasts demonstrated FLT-1 staining before bevacizumab and marked decrease in microvessel density after bevacizumab. VEGF was detected in pretreatment serum in 67% of patients tested, increased by day 8 in 52%, and decreased in 93% (67% undetectable) 2 h after bevacizumab.

Conclusions: In this single arm study, cytotoxic chemotherapy followed by bevacizumab yields a favorable CR rate and duration in adults with AML that is resistant to traditional treatment approaches. The clearance of marrow blasts in some patients after bevacizumab suggests that VEGF neutralization might result directly in leukemic cell death. The potential biological and clinical activity of bevacizumab in AML warrants additional clinical and laboratory study.
Molecular Diagnosis of Surgical Margins and Local Recurrence in Head and Neck Cancer Patients
Authors: van Houten Viola M. M. , Leemans C. René , Kummer J. Alain , Dijkstra Janny , Kuik Dirk J. , van den Brekel Michiel W. M. , Snow Gordon B. , Brakenhoff Ruud H.
Published: September 2005

Purpose: Approximately 10–30% of surgically treated head and neck cancer patients develop local recurrences while the resection margins are histologically tumor free. These recurrences may arise from cancer cells left behind but not detected by the pathologist, or they may develop from precursor lesions adjacent to the tumor that were not completely resected. We have investigated whether TP53-mutated DNA in the surgical margins is suitable to identify patients with head and neck squamous cell carcinoma at risk for local and locoregional recurrence.

Experimental Design: In a prospective cohort study of 76 patients with histologically tumor-free margins, the presence of TP53-mutated DNA was determined in the surgical margins using the phage plaque assay and correlated to clinical outcome. Immunostaining of the molecular-positive margins for mutated p53 protein was used to identify whether unresected precursor lesions or residual tumor cells were left behind.

Results: The absence of TP53-mutated DNA in surgical margins was significantly associated with remaining free of local and locoregional recurrence (P = 0.027 and P = 0.028, respectively). Moreover, the presence of TP53-mutated DNA in the surgical margins was an independent prognosticator for locoregional recurrence (relative risk = 7.1; P = 0.021; 95% confidence interval, 0.9–56). In 20% of the cases, the presence of TP53-mutated DNA in the surgical margins was found to be related to the presence of tumor-related precursor lesions.

Conclusions: This study shows the value of TP53-mutated DNA as a molecular marker to predict locally recurrent head and neck squamous cell carcinoma. The observation that all patients who were negative for TP53-mutated DNA in the surgical margins remained free of local recurrence raises hope that molecular analysis of histologically tumor-free surgical margins can be exploited to decide on postoperative radiotherapy. Furthermore, our data provide evidence that local recurrences originate mainly from tumor cells left behind but also originate, in part, from unresected precursor lesions.
Phase I Study of Rituximab-CHOP Regimen in Combination with Granulocyte Colony-Stimulating Factor in Patients with Follicular Lymphoma
Authors: Niitsu Nozomi , Hayama Miyuki , Okamoto Masataka , Khori Mika , Higashihara Masaaki , Tamaru Jun-ichi , Hirano Masami
Published: September 2005

Purpose: Rituximab is an anti-CD20 monoclonal antibody, and it is used to treat B-cell lymphomas. Antibody-dependent cellular cytotoxicity (ADCC) is considered one of the mechanisms through which rituximab exerts its effects. Granulocyte colony-stimulating factor (G-CSF) enhances the cytotoxicity of neutrophils through ADCC, and it can be speculated that a combination of rituximab and G-CSF may augment the treatment efficacy of rituximab.

Experimental Design: We administered rituximab with CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) treatment with G-CSF to 15 patients with follicular lymphoma, and we investigated the safety and efficacy of this regimen. We investigated ADCC activity in neutrophils and the expression of cell surface antigens including Fcγ receptor type I [FcγRI (CD64)] on neutrophils to determine the optimal dose of G-CSF.

Results: Adverse reactions occurred in 14 of 15 patients and consisted mainly of grade 3/4 hematological toxicity. The response rate was 100%, with complete remission in 12 patients (80%) and partial remission in 3 patients (20%). At 14 months, the median length of the observation period, 2 of 12 patients had relapsed. G-CSF administration increased both FcγRI expression and ADCC activity. There were no significant differences in the levels of FcγRI expression or ADCC activity between the 2 μg/kg G-CSF and 5 μg/kg G-CSF groups, indicating that the optimal dose of G-CSF was 2 μg/kg.

Conclusions: We conclude that the combination of rituximab-CHOP and G-CSF is well tolerated. We plan to carry out a randomized trial to compare efficacy between rituximab-CHOP treatment and treatment with a combination of rituximab-CHOP and G-CSF.
Treatment of Pancreatic Cancer Cells with Dicumarol Induces Cytotoxicity and Oxidative Stress
Authors: Lewis Anne , Ough Matthew , Li Ling , Hinkhouse Marilyn M. , Ritchie Justine M. , Spitz Douglas R. , Cullen Joseph J.
Published: September 2005

Purpose: NAD(P)H:quinone oxidoreductase (NQO1) catalyzes the two-electron reduction of quinones to hydroquinones. This reaction is believed to prevent the one-electron reduction of quinones that would result in redox cycling with generation of superoxide (O2.-). We have recently demonstrated that inhibition of NQO1 with dicumarol increases intracellular O2.- production and inhibits the in vitro malignant phenotype of pancreatic cancer cells (J. Cullen et al., Cancer Res., 63: 5513–5520, 2003). We hypothesized that inhibition of NQO1 would increase cell killing, induce oxidative stress, and inhibit in vivo tumor growth.

Experimental Design and Results: In the human pancreatic cancer cell line MIA PaCa-2, dicumarol decreased cell viability, as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and decreased clonogenic survival. Dicumarol increased the percentage of apoptotic cells in a time-dependent and dose-dependent manner as measured by 3,3′-diaminobenzidine staining and flow cytometry, which was associated with cytochrome c release and poly(ADP-ribose) polymerase cleavage. Dicumarol also induced oxidative stress as evidenced by increased total glutathione and oxidized glutathione, as well as sensitizing to cell killing mediated by menadione. In established orthotopic pancreatic tumors in nude mice, intratumoral injections of dicumarol slowed tumor growth and extended survival.

Conclusions: Inhibition of NQO1 with dicumarol induces cell killing and oxidative stress in pancreatic cancer cells and speculate that dicumarol may prove to be useful in pancreatic cancer therapeutics.
Mechanisms of Inhibition of Tumor Angiogenesis and Vascular Tumor Growth by Epigallocatechin-3-Gallate
Authors: Fassina Gianfranco , Venè Roberta , Morini Monica , Minghelli Simona , Benelli Roberto , Noonan Douglas M. , Albini Adriana
Published: September 2005

Purpose: Green tea consumption has been linked to a reduced occurrence of some tumor types. Current data indicate that the principal mediator of this chemopreventive effect is epigallocatechin-3-gallate (EGCG), the most abundant polyphenol found in dried tea leaves. Here, we examined the effects of this compound on the two key cell populations typically involved in tumor growth: tumor cells and endothelial cells.

Experimental Design: The effects of green tea and EGCG were tested in a highly vascular Kaposi’s sarcoma (KS) tumor model and on endothelial cells in a panel of in vivo and in vitro assays.

Results: EGCG inhibited KS-IMM cell growth and endothelial cell growth, chemotaxis, and invasion over a range of doses; high concentrations also induced tumor cell apoptosis. EGCG inhibited the metalloprotease-mediated gelatinolytic activity produced by endothelial cell supernatants and the formation of new capillary-like structures in vitro. Green tea or purified EGCG when administered to mice in the drinking water inhibited angiogenesis in vivo in the Matrigel sponge model and restrained KS tumor growth. Histological analysis of the tumors were consistent with an anti-angiogenic activity of EGCG and green tea.

Conclusions: These data suggest that the green tea gallate or its derivatives may find use in the prevention and treatment of vascular tumors in a chemoprevention or adjuvant setting.
Distribution of 1-(2-Deoxy-2-fluoro-β-d-arabinofuranosyl) Uracil in Mice Bearing Colorectal Cancer Xenografts
Authors: Eiseman Julie L. , Brown-Proctor Clive , Kinahan Paul E. , Collins Jerry M. , Anderson Lawrence W. , Joseph Erin , Hamburger Deborah R. , Pan Su-shu , Mathis Chester A. , Egorin Merrill J. , Klecker Raymond W.
Published: September 2005

Purpose: In colorectal, breast, and head and neck cancers, response to 5-fluorouracil is associated with low expression of thymidylate synthase. In contrast, tumors with high expression of thymidylate synthase may be more sensitive to prodrugs such as 1-(2-deoxy-2-fluoro-β-d-arabinofuranosyl) uracil (FAU) that are activated by thymidylate synthase. These studies were designed to evaluate FAU as a potential therapeutic and diagnostic probe.

Experimental Design: [18F]-FAU and [3H]-FAU were synthesized with >97% radiochemical purity. [3H]-FAU or [18F]-FAU was administered intravenously to severe combined immunodeficient mice bearing either HT29 (low thymidylate synthase) or LS174T (high thymidylate synthase) human colon cancer xenografts. Four hours after [3H]-FAU dosing, tissue distribution of total radioactivity and incorporation of 1-(2-deoxy-2-fluoro-β-d-arabinofuranosyl) 5-methyluracil (FMAU), derived from thymidylate synthase activation of FAU, into tumor DNA was measured. Positron emission tomography (PET) images were obtained for 90 minutes after injection of [18F]-FAU. Thymidylate synthase activity was determined in vitro in tumors from untreated mice by [3H] release from [3H]dUMP. Each cell line was incubated in vitro with [3H]-FAU or [3H]-FMAU in the absence or presence of 5-fluoro-2′-deoxyuridine (FdUrd) and then was analyzed for incorporation of radiolabel into DNA.

Results: Thymidylate synthase enzymatic activity in LS174T xenografts was ∼3.5-fold higher than in HT29 xenografts, and incorporation of radioactivity derived from [3H]-FAU into LS174T DNA was ∼2-fold higher than into HT29 DNA. At 240 minutes, radioactivity derived from [3H]-FAU was ∼2-fold higher in tumors than in skeletal muscle. At times up to 90 minutes, PET imaging detected only small differences in uptake of [18F]-FAU between the tumor types. Fluorine-18 in skeletal muscle was higher than in tumor for the first 90 minutes and plateaued earlier, whereas [18F] in tumor continued to increase during the 90-minute imaging period. For both cell lines in vitro, FdUrd decreased the rate of incorporation of [3H]-FAU into DNA, whereas the incorporation of [3H]-FMAU was increased.

Conclusions: These results for FAU incorporation into DNA in vitro and in vivo further support clinical evaluation of FAU as a therapeutic agent in tumors with high concentrations of thymidylate synthase that are less likely to respond to 5-fluorouracil treatment. The high circulating concentrations of thymidine reported in mice may limit their utility in evaluating FAU as a PET probe.
A Phase I and Pharmacological Study with Imidazolium-
trans-
DMSO-imidazole-tetrachlororuthenate, a Novel Ruthenium Anticancer Agent
Authors: Rademaker-Lakhai Jeany M. , van den Bongard Desiree , Pluim Dick , Beijnen Jos H. , Schellens Jan H. M.
Published: September 2005

Purpose: NAMI-A {H2Im[trans-RuCl4(DMSO)HIm] or imidazolium-trans-DMSO-imidazole-tetrachlororuthenate} is a novel ruthenium-containing compound that has demonstrated antimetastatic activity in preclinical studies. This Phase I study was designed to determine the maximum-tolerated dose (MTD), profile of adverse events, and dose-limiting toxicity of NAMI-A in patients with solid tumors. Furthermore, the ruthenium pharmacokinetics (PK) after NAMI-A administration and preliminary antitumor activity were evaluated.

Patients and Methods: Adult patients with solid tumors received NAMI-A as an i.v. infusion over 3 h daily for 5 days every 3 weeks. PK of total and unbound ruthenium was determined during the first and second treatment using noncompartmental pharmacokinetic analysis. The total accumulation of ruthenium in WBCs was also quantified.

Results: Twenty-four patients were treated at 12 dose levels (2.4–500 mg/m2/day). At 400 mg/m2/day, blisters developed on the hands, fingers, and toes. At 500 mg/m2/day, blisters persisted from weeks to months and slowly regressed. Although no formal common toxicity criteria (CTC) grade 3 developed, painful blister formation was considered dose limiting. Because the first signs developed at 400 mg/m2/day, the advised dose for further testing of NAMI-A was determined to be 300 mg/m2/day on this schedule. PK analysis revealed a linear relationship between dose and area under the concentration-time curve (AUC) of total and unbound ruthenium (R2 = 0.75 and 0.96, respectively) over the whole dose range. Plasma clearance of total ruthenium was 0.17 ± 0.09 liter/h, and terminal half-life was 50 ± 19 h. The volume of distribution at steady state of total ruthenium was 10.1 ± 2.8 liters. The accumulation of ruthenium in WBC was not directly proportional to the increasing total exposure to ruthenium. One patient with pretreated and progressive nonsmall cell lung cancer had stable disease for 21 weeks.

Conclusion: NAMI-A can be administered safely as a 3-h i.v. infusion at a dose of 300 mg/m2/day for 5 days, every 3 weeks.
The Antimetastatic Role of Thrombomodulin Expression in Islet Cell-Derived Tumors and Its Diagnostic Value
Authors: Iino Satoshi , Abeyama Kazuhiro , Kawahara Ko-ichi , Yamakuchi Munekazu , Hashiguchi Teruto , Matsukita Sumika , Yonezawa Suguru , Taniguchi Shotaro , Nakata Masanori , Takao Sonshin , Aikou Takashi , Maruyama Ikuro
Published: September 2005

Islet cell tumors, endocrine neoplasm arising from pancreatic islets of Langerhans, are histologically difficult to diagnose as benign or malignant. Molecular markers are associated with the clinical characteristics that most of insulinoma are usually benign tumors, whereas other islet cell tumors are malignant but have not been identified. In this context, we newly found that an endothelial anticoagulant thrombomodulin was expressed in the normal islet β cells and insulinoma, but not of other islet components or noninsulinoma islet cell tumors. Clinically, all of the subjects (n = 15) of the insulinoma group showed no metastasis together with thrombomodulin expression in the lesions, whereas the other islet cell tumor groups showed a high incidence of metastasis (82%) and a low expression rate of thrombomodulin (6%). To examine the functional role of thrombomodulin, especially regarding the clinical characteristics of islet cell tumors, we tested the effect of exogenous thrombomodulin overexpression on cell adhesiveness and proliferation using MIN6 insulinoma cell line. In cell-based experiments, thrombomodulin overexpression reduced cell proliferation and enhanced Ca2+-independent cell aggregation, possibly through direct interaction with neural cell adhesion molecule. Taken together, these results are suggesting that thrombomodulin may act as antimetastatic molecule of insulinomas. In addition, thrombomodulin is a clinically useful molecular marker not only for identifying β-cell–origin islet cell tumors (i.e., insulinomas) but also for predicting disease prognosis of islet cell tumors.
Mcl-1 Is a Novel Therapeutic Target for Human Sarcoma
Authors: Thallinger Christiane , Wolschek Markus F. , Maierhofer Helmut , Skvara Hans , Pehamberger Hubert , Monia Brett P. , Jansen Burkhard , Wacheck Volker , Selzer Edgar
Published: September 2005

Purpose: Little is known about the role that Mcl-1, an antiapoptotic Bcl-2 family member, plays in solid tumor biology and susceptibility to anticancer therapy. We observed that the Mcl-1 protein is widely expressed in human sarcoma cell lines of different histological origin (n = 7). Because the expression of antiapoptotic Bcl-2 family proteins can significantly contribute to the chemoresistance of human malignancies, we used an antisense strategy to address this issue in sarcoma.

Experimental Design: SCID mice (n = 6/group) received s.c. injections of SW872 liposarcoma cells. After development of palpable tumors, mice were treated by s.c.-implanted miniosmotic pumps prefilled with saline or antisense or universal control oligonucleotides (20 mg/kg/day for 2 weeks). On days 2, 6, and 10, mice were treated with low-dose cyclophosphamide (35 mg/kg i.p) or saline control. During the experiments, tumor weight was assessed twice weekly by caliper measurements. On day 14, animals were sacrificed. Tumors were weighed and fixed in formalin for immunohistochemistry and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling analysis.

Results: Mcl-1 antisense oligonucleotides specifically reduced Mcl-1 protein expression but produced no reduction in tumor weight compared with saline-treated control animals. Cyclophosphamide monotreatment caused only modest tumor weight reduction compared with saline control. However, use of Mcl-1 antisense oligonucleotides combined with cyclophosphamide clearly enhanced tumor cell apoptosis and significantly reduced tumor weight by more than two-thirds compared with respective control treatments.

Conclusion: A combination of Mcl-1 antisense oligonucleotides with low-dose cyclophosphamide provides a synergistic antitumor effect and might qualify as a promising strategy to overcome chemoresistance in human sarcoma.
Alpha-Particle Emitting Atomic Generator (Actinium-225)-Labeled Trastuzumab (Herceptin) Targeting of Breast Cancer Spheroids
Authors: Ballangrud Åse M. , Yang Wei-Hong , Palm Stig , Enmon Richard , Borchardt Paul E. , Pellegrini Virginia A. , McDevitt Michael R. , Scheinberg David A. , Sgouros George
Published: September 2005

Purpose: The humanized monoclonal antibody, trastuzumab (Herceptin), directed against HER2/neu, has been effective in the treatment of breast cancer malignancies. However, clinical activity has depended on HER2/neu expression. Radiolabeled trastuzumab has been considered previously as a potential agent for radioimmunotherapy. The objective of this study was to investigate the efficacy of trastuzumab labeled with the α-particle emitting atomic generator, actinium-225 (225Ac), against breast cancer spheroids with different HER2/neu expression levels. 225Ac has a 10-day half-life and a decay scheme yielding four α-particles.

Experimental Design: The breast carcinoma cell lines MCF7, MDA-MB-361 (MDA), and BT-474 (BT) with relative HER2/neu expression (by flow cytometry) of 1:4:18 were used. Spheroids of these cell lines were incubated with different concentrations of 225Ac-trastuzumab, and spheroid growth was measured by light microscopy over a 50-day period.

Results: The activity concentration required to yield a 50% reduction in spheroid volume at day 35 was 18.1, 1.9, and 0.6 kBq/ml (490, 52, 14 nCi/ml) for MCF7, MDA, and BT spheroids, respectively. MCF7 spheroids continued growing but with a 20–30 day growth delay at 18.5 kBq/ml. MDA spheroid growth was delayed by 30–40 days at 3.7 kBq/ml; at 18.5 kBq/ml, 12 of 12 spheroids disaggregated after 70, days and cells remaining from each spheroid failed to form colonies within 2 weeks of being transferred to adherent dishes. Eight of 10 BT spheroids failed to regrow at 1.85 kBq/ml. All of the BT spheroids at activity concentrations 3.7 kBq/ml failed to regrow and to form colonies. The radiosensitivity of these three lines as spheroids was evaluated as the activity concentration required to reduce the treated to untreated spheroid volume ratio to 0.37, denoted DVR37. An external beam radiosensitivity of 2 Gy was found for spheroids of all three of the cell lines. After α-particle irradiation a DVR37 of 1.5, 3.0, and 2.0 kBq/ml was determined for MCF7, MDA, and BT, respectively.

Conclusion: These studies suggest that 225Ac-labeled trastuzumab may be a potent therapeutic agent against metastatic breast cancer cells exhibiting intermediate to high HER2/neu expression.
Adaptive Hypersensitivity to Estrogen
Authors: Santen Richard J. , Song Robert X. , Zhang Zhenguo , Yue Wei , Kumar Rakesh
Published: September 2005

Clinical observations demonstrate that women with breast cancer often respond to subsequent endocrine manipulation after resistance to initial hormonal therapy develops. As a mechanistic explanation for these findings, we hypothesized that human breast tumors can adapt in response to the pressure exerted by endocrine therapy with development of hypersensitivity to estradiol. To understand the signaling pathways responsible, we examined estrogenic stimulation of cell proliferation in a model system and provided in vitro and in vivo evidence that long-term deprivation of estradiol (LTED) causes adaptive hypersensitivity. Even though the estrogen receptor α (ERα) is markedly up-regulated in LTED cells, the enhanced responses to estradiol do not appear to involve mechanisms acting at the level of transcription of estrogen-regulated genes. We found that ERα co-opts a classical growth factor pathway and induces rapid nongenomic effects that are enhanced in LTED cells. Estradiol binds to cell membrane-associated ERs, physically associates with the adaptor protein Shc, and induces its phosphorylation. In turn, Shc binds Grb2 and Sos, which result in the rapid activation of mitogen-activated protein kinase. These nongenomic effects of estradiol produced biological effects, as evidenced by Elk-1 activation and by morphological changes in cell membranes. The mechanistic pathways involved in adaptive hypersensitivity suggest that inhibitors of the mitogen-activated protein kinase and phosphatidylinositol-3-OH kinase pathways might prevent the development of adaptive hypersensitivity and allow more prolonged efficacy of endocrine therapies.
Prevention Strategies with Aromatase Inhibitors
Authors: Goss Paul E. , Strasser-Weippl Kathrin
Published: September 2005

Aromatase (estrogen synthetase) inhibitors are superior to tamoxifen in terms of both efficacy and toxicity in the treatment of advanced breast cancer and also in the neoadjuvant setting. Recent results from the Arimidex, Tamoxifen, Alone or in Combination adjuvant trial showed a marked reduction in contralateral primary breast cancer with anastrozole, an apparent prevention effect. A similar effect was seen in the MA.17 adjuvant trial comparing letrozole with placebo after 5 years of adjuvant tamoxifen. This has accelerated interest in aromatase inhibitors as primary preventive therapy. Two studies being conducted by the National Cancer Institute of Canada’s Clinical Trials Group select women by virtue of mammographic breast density. The International Breast Cancer Intervention Study 2 trial randomizes women at elevated risk to anastrozole or placebo. Because of its steroidal structure, exemestane may be more effective than the nonsteroidal aromatase inhibitors and may protect bone and lipid metabolism from the effects of estrogen ablation. Elevated prostaglandin E2 levels from cyclooxygenase-2 induction by preinvasive and invasive breast lesions increase a number of tumor-promoting pathways, including aromatase, as well as angiogenetic, antiapoptotic, and others. Additive or synergistic effects between celecoxib, a cyclooxygenase-2 inhibitor, and exemestane have been demonstrated and have led to the National Cancer Institute of Canada’s Clinical Trials Group MAP.3 trial, which will randomize women at elevated risk to placebo or to exemestane with or without celecoxib. The efficacy and long-term toxicity data from the aromatase inhibitor prevention trials, and the identification of risk profiles from trial results, are awaited with interest.
ICAM-2Gene Therapy for Peritoneal Dissemination of Scirrhous Gastric Carcinoma
Authors: Tanaka Hiroaki , Yashiro Masakazu , Sunami Takeshi , Sakate Yoji , Kosaka Kinshi , Hirakawa Kosei
Published: September 2005

Purpose: Human scirrhous gastric carcinoma develops peritoneal dissemination with high frequency, and the prognosis of patients with peritoneal metastasis is poor. There have been few reports of an immunogene therapy for peritoneal dissemination. Intercellular adhesion molecule (ICAM)-2 is a second ligand of leukocyte function-associated antigen-1, which functions as a costimulatory molecule for effector cells. In the present study, we examined whether ICAM-2 transfection using adenovirus vector is effective gene therapy for peritoneal metastasis of gastric cancer.

Experimental Design: We constructed an adenovirus vector, AdICAM-2, that encodes the full-length human ICAM-2 gene under control of the cytomegalovirus promoter. This vector expresses high levels of ICAM-2 on the human gastric cancer cell line OCUM-2MD3, which has high peritoneal metastatic ability in nude mice. We investigated the antitumor effects of gene transfer of ICAM-2 using the adenovirus vector AdICAM-2 in vitro and in vivo.

Results: ICAM-2 expressed on OCUM-2MD3 cells by AdICAM-2 demonstrated significantly high adhesiveness to and cytotoxicity against peripheral blood mononuclear cells in vitro compared with the control adenovirus vector AdlacZ. Intratumoral injection of AdICAM-2 significantly inhibited the growth of s.c. tumor. Mice with peritoneal metastasis survived for a significantly longer time after AdICAM-2 injection, compared with injection of AdlacZ. Histopathological findings revealed that many natural killer cells infiltrated the peritoneal metastatic lesions after AdICAM-2 injection.

Conclusions: These findings suggest that transduction of ICAM-2 into cancer cells enhances the adhesion and activation of natural killer cells, resulting in a reduction of peritoneal metastasis. ICAM-2 transfection using adenovirus vector might be an effective form of gene therapy for peritoneal metastasis of gastric cancer.
Dysregulated Expression of Adamalysin-Thrombospondin Genes in Human Breast Carcinoma
Authors: Porter Sarah , Scott Stuart D. , Sassoon Elaine M. , Williams Mark R. , Jones J. Louise , Girling Anne C. , Ball Richard Y. , Edwards Dylan R.
Published: September 2005

The adamalysin-thrombospondin (ADAMTS) proteinases are a relatively newly described branch of the metzincin family that contain metalloproteinase, disintegrin, and thrombospondin motifs. They have been implicated in various cellular events, including cleavage of proteoglycans, extracellular matrix degradation, inhibition of angiogenesis, gonadal development, and organogenesis. However, in many cases, their normal physiological roles and their potential for dysregulation in malignancy remain to be established. The expression profile of ADAMTS1–20 in human breast carcinoma was undertaken by real-time PCR using RNA isolated from malignant tumors, nonneoplastic mammary tissue, and breast cancer cell lines to identify altered regulation that may have potential pathogenetic and prognostic significance. Our studies show that seven of the ADAMTS genes (ADAMTS1, 3, 5, 8, 9, 10, and 18) are consistently down-regulated in breast carcinomas with respect to nonneoplastic mammary tissue, irrespective of the heterogeneity of the samples and the tumor type or grade (Mann-Whitney U test, P < 0.0001 for each gene). Conversely, ADAMTS4, 6, 14, and 20 are consistently up-regulated in breast carcinomas (P = 0.005, P < 0.0001, P = 0.003, and P = 0.001, respectively). ADAMTS2, 7, 12, 13, 15, 16, 17, and 19 show no significant difference between the sample types. ADAMTS1, 2, 7, 8, 10, and 12 are expressed predominantly in stromal fibroblasts. ADAMTS3, 4, 5, 6, 9, and 13–20 inclusive are expressed predominantly in myoepithelial cells; all appear to be relatively poorly expressed in luminal epithelial cells. ADAMTS15 has emerged as being an independent predictor of survival, with RNA expression levels significantly lower (P = 0.007) in grade 3 breast carcinoma compared with grade 1 and 2 breast carcinoma.
Randomized Phase II Trial of Sequential Chemotherapy in Advanced Non-Small Cell Lung Cancer (SWOG 9806)
Authors: Edelman Martin J. , Clark Joseph I. , Chansky Kari , Albain Kathy , Bhoopalam Nirmala , Weiss Geoffrey R. , Giguere Jeffrey K. , Kelly Karen , Crowley John , Gandara David R.
Published: September 2005

Purpose: Improving chemotherapeutic efficacy in non-small cell lung cancer (NSCLC) will require the development of new drugs or new strategies to better use currently available agents. Sequential administration offers an opportunity to increase drug diversity while maintaining dose intensity. On the basis of the data indicating the activity of taxanes as second-line therapy and the lack of efficacy for more than three cycles of platinum-based therapy, this randomized Phase II study tested the concept of planned sequential chemotherapy in advanced NSCLC.

Experimental Design: Patients with selected stage IIIb (pleural effusion)/stage IV NSCLC, performance status of 0–1 and normal organ function were eligible. Therapy: arm 1, carboplatin (area under the curve = 5.5 mg/ml × min day 1) and gemcitabine (1000 mg/m2 days 1 and 8 every 21 days × 3) followed by paclitaxel (225 mg/m2 every 21 days × 3) or arm 2, cisplatin (100 mg/m2 day 1), vinorelbine (25 mg/m2 days 1 and 8 every 21 d × 3) followed by docetaxel (75–100 mg/m2 every 21 days × 3).

Results: Two-hundred four patients were accrued, of whom, 178 were eligible and evaluable. Eighty percent of patients were stage IV on arm 1 and 85% on arm 2. Response rates were 21 and 28% on arms 1 and 2, respectively. Median, 1-year and 2-year survivals were 9 months, 34 and 13%, and 9 months, 36 and 8%, on arms 1 and 2, respectively.

Conclusions: Sequential therapy, as used in this study, resulted in comparable efficacy to previous Southwest Oncology Group trials of two drug combinations in this population; however, it failed to meet criteria for further study.
Tumor Neoangiogenesis by CD31 and CD105 Expression Evaluation in Breast Carcinoma Tissue Microarrays
Authors: Charpin Colette , Dales Jean-Philippe , Garcia Stéphane , Carpentier Séverine , Djemli Amina , Andrac Lucile , Lavaut Marie-Noëlle , Allasia Claude , Bonnier Pascal
Published: September 2005

Purpose: The aim of the study was to evaluate CD31 and CD105 immunohistochemical expressions in tissue microarrays from 360 breast carcinomas.

Study design: Computerized (ACIS/Chromavision) assisted image analysis was performed to compare immunoreactions in tissue microarrays with those in current paraffin and frozen sections. We also aimed to determine the CD105 and CD31 prognostic significance and relevance in routine practice by correlating results of immunodetections with patients’ (n = 360) outcome (14.3-year follow-up).

Results: The results show (a) that in tissue microarrays, the CD31 and CD105 expression quantified by image analysis device did not correlate with the measurements assessed on routine paraffin sections; (b) that CD105 expression is endowed of a prognostic significance in paraffin sections in terms of overall survival (P < 0.01), whereas in contrast, CD31 on paraffin sections did not correlate with patients overall survival; (c) that semiquantitative analysis of CD105 expression correlated with the image analysis measurements in frozen sections (ρ = 0.671, P < 0.01) and paraffin (ρ = 0.824, P < 0.01) sections. However, paraffin sections were less immunostained than frozen ones.

Conclusions: It is concluded (a) that CD105 may be suitable in paraffin sections to evaluated neoangiogenesis; and (b) that tissue microarrays are not suitable substrates for neoangiogenesis evaluation as a prognostic indicator in breast carcinomas, in contrast to current tissue sections.
Antibody-Targeted Chemotherapy with the Calicheamicin Conjugate hu3S193-
N
-Acetyl γ Calicheamicin Dimethyl Hydrazide Targets Lewisy and Eliminates Lewisy-Positive Human Carcinoma Cells and Xenografts
Authors: Boghaert Erwin R. , Sridharan Latha , Armellino Douglas C. , Khandke Kiran M. , DiJoseph John F. , Kunz Arthur , Dougher Maureen M. , Jiang Fan , Kalyandrug Lyka B. , Hamann Philip R. , Frost Philip , Damle Nitin K.
Published: September 2005

Purpose: Linking a cytotoxic anticancer drug to an antibody that recognizes a tumor-associated antigen can improve the therapeutic index of the drug. We asked whether a conjugate of the cytotoxic antibiotic N-acetyl γ calicheamicin dimethyl hydrazide (CalichDMH) and an antibody recognizing Lewisy (Ley) antigen could eliminate carcinomas that express Ley. Because Ley is highly expressed on carcinomas of colon, breast, lung, ovary, and prostate, a CalichDMH conjugate targeting Ley could provide a treatment option for various cancers.

Experimental Design: The humanized anti-Ley antibody hu3S193 was conjugated to CalichDMH via the bifunctional AcBut linker. Selectivity and avidity of the conjugate (hu3S193-CalichDMH) for Ley-BSA or Ley+ cells was tested by BIAcore or flow cytometry. Cytotoxicity of hu3S193-CalichDMH was compared with toxicity of a control conjugate on monolayers of Ley+ and Ley− carcinoma cells. Inhibition of tumor growth by hu3S193-CalichDMH was assessed on three types of s.c. xenografts.

Results: Hu3S193-CalichDMH had similar selectivity as hu3S193. The conjugate had lower affinity for Ley-BSA but not for Ley+ cells. When tested on monolayers of human Ley+ carcinoma cells, hu3S193-CalichDMH was more cytotoxic than a control conjugate. This difference in efficacy was not noted on Ley− cells. Efficacy of hu3S193-CalichDMH depended on the expression of Ley and on the sensitivity of the cells to CalichDMH. In vivo, hu3S193-CalichDMH inhibited growth of xenografted human gastric (N87), colon (LOVO), and prostate carcinomas (LNCaP). When used against N87 xenografts, hu3S193-CalichDMH arrested tumor growth for at least 100 days.

Conclusion: Hu3S193-CalichDMH can specifically eliminate Ley+ tumors. These results support development of this conjugate for treatment of carcinomas.
Steroid Sulfatase and Estrogen Sulfotransferase in Human Endometrial Carcinoma
Authors: Utsunomiya Hiroki , Ito Kiyoshi , Suzuki Takashi , Kitamura Takako , Kaneko Chika , Nakata Taisuke , Niikura Hitoshi , Okamura Kunihiro , Yaegashi Nobuo , Sasano Hironobu
Published: September 2005

Purpose: Intratumoral metabolism and synthesis of estrogens are considered to play important roles in the pathogenesis and/or development of human endometrial carcinoma. Steroid sulfatase hydrolyzes biologically inactive estrogen sulfates to active estrogens, whereas estrogen sulfotransferase sulfonates estrogens to estrogen sulfates. However, the status of steroid sulfatase and/or estrogen sulfotransferase in human endometrial carcinoma has not been examined.

Experimental Design: We first examined the expression of steroid sulfatase and estrogen sulfotransferase in 6 normal endometrium and 76 endometrial carcinoma using immunohistochemistry to elucidate the possible involvement of steroid sulfatase and estrogen sulfotransferase. We then evaluated the enzymatic activity and the semiquantitative analysis of mRNA using reverse transcription-PCR in 21 endometrial carcinomas. We correlated these findings with various clinicopathological parameters including the expression of aromatase, 17β-hydroxysteroid dehydrogenase type 1 and type 2.

Results: Steroid sulfatase and estrogen sulfotransferase immunoreactivity was detected in 65 of 76 (86%) and 22 of 76 (29%) cases, respectively. Results of immunoreactivity for steroid sulfatase and estrogen sulfotransferase were significantly correlated with those of enzymatic activity and semiquantitative analysis of mRNA. No significant correlations were detected among the expression of the enzymes involved in intratumoral estrogen metabolism. There was a significant correlation between steroid sulfatase/estrogen sulfotransferase ratio and clinical outcomes of the patients. However, there were no significant differences between steroid sulfatase or estrogen sulfotransferase and estrogen receptor, progesterone receptor, Ki67, histologic grade, or clinical outcomes of the patients.

Conclusions: Results of our study demonstrated that increased steroid sulfatase and decreased estrogen sulfotransferase expression in human endometrial carcinomas may result in increased availability of biologically active estrogens and may be related to estrogen-dependent biological features of carcinoma.
Decreased Absolute Counts of T Lymphocyte Subsets and Their Relation to Disease in Squamous Cell Carcinoma of the Head and Neck
Authors: Kuss Iris , Hathaway Bridget , Ferris Robert L. , Gooding William , Whiteside Theresa L.
Published: September 2005

Purpose: Apoptosis of circulating CD8+ T cells seen in patients with squamous cell carcinoma of the head and neck [SCCHN (Hoffmann T, et al. Clin Cancer Res 2002;8:2553–62)] suggested a possibility of lymphocyte imbalance. Therefore, absolute numbers and percentages of lymphocyte subsets were examined in the peripheral blood of SCCHN patients and controls.

Experimental Design: Venous blood was obtained from 146 patients with SCCHN and 54 normal volunteers. Absolute numbers of CD3+, CD4+, and CD8+ T lymphocytes were determined using fluorobeads in a flow cytometry-based technique. Percentages of T lymphocyte subsets were also evaluated by flow cytometry. The patients were grouped at the time of blood draw [active versus no evidence of disease (NED), type of therapy administered, and the length of follow-up].

Results: Patients with SCCHN had significantly lower absolute numbers of CD3+ CD4+, and CD8+ T cells than normal controls. However, no differences in the percentages of T-cell subsets between patients and normal controls were observed. Patients with active disease had significantly lower CD3+ and CD4+ T-cell counts than those with NED. Patients who had NED after surgery and radiotherapy had the lowest T-cell counts among the NED cohort. Patients who had NED for >2 years did not recover their T-cell counts, and the T-cell imbalance was evident many years after curative surgery. The tumor-node-metastasis (TNM) stage or site of the disease was not related to the absolute T-cell count. Patients with recurrent disease at the time of blood draw tended to have the lowest CD4+ T-cell counts.

Conclusions: Patients with SCCHN have altered lymphocyte homeostasis, which persists for months or years after curative therapies.