bluuebunny/crossref_metadata_2025 huggingface dataset:
Cycloxygenase-2 Inhibition Augments the Efficacy of a Cancer Vaccine
Authors: Haas Andrew R. , Sun Jing , Vachani Anil , Wallace Africa F. , Silverberg Michael , Kapoor Veena , Albelda Steven M.
Published: March 2006

Tumor-derived cyclooxygenase-2 (COX-2) and its product, prostaglandin E2, exert strong immunoinhibitory effects that block dendritic cell function and CD4+ and CD8+ T-cell proliferation and function. We have shown previously that the addition of an oral COX-2 inhibitor to immunogene therapy using IFN-β markedly augmented therapeutic efficacy in murine tumor models. In this study, we hypothesized that COX-2 inhibition might also augment an antitumor vaccination strategy. Mice bearing tumors derived from TC1 cells, a tumor line that expresses the human papillomavirus (HPV) E7 protein, were thus vaccinated with an adenoviral vector expressing HPV E7 protein (Ad.E7). This vaccine approach effectively generated E7-specific CD8+ cells and slowed the growth of small tumors but had little effect on large tumors. However, feeding mice with the COX-2 inhibitor, rofecoxib, restored the effectiveness of the vaccine against large tumors and prolonged survival. This effect was accompanied by a larger percentage of E7-specific CD8+ cells in the regional draining lymph nodes and a markedly increased number of tumor-infiltrating E7-specific CD8+ cells (as determined by flow cytometry) and total CD8+ T cells (as determined by immunohistochemical staining). Increased immunocyte trafficking was likely mediated by the generation of a Th1-type tumor microenvironment because COX-2 inhibition increased expression levels of mRNA for IFN-γ, interleukin-12, IP-10, and MIG while lowering the expression of vascular endothelial growth factor within tumors. This study shows that the effectiveness of a cancer vaccine can be significantly improved by adding COX-2 inhibition.
Combination Immunotherapy with Clinical-Scale Enriched Human γδ T cells, hu14.18 Antibody, and the Immunocytokine Fc-IL7 in Disseminated Neuroblastoma
Authors: Otto Mario , Barfield Raymond C. , Martin William J. , Iyengar Rekha , Leung Wing , Leimig Thasia , Chaleff Stanley , Gillies Stephen D. , Handgretinger Rupert
Published: March 2006

Purpose: To evaluate a combined cellular and humoral immunotherapy regimen in a mouse model of disseminated human neuroblastoma. We tested combinations of clinical-grade, isolated human γδ T cells with the humanized anti-GD2 antibody hu14.18 and a novel fusion cytokine, Fc-IL7.

Experimental Design: γδ T cells were large-scale enriched from leukapheresis product obtained from granulocyte colony-stimulating factor–mobilized donors. γδ T cell cytotoxicity was tested in a europium-TDA release assay. The effect of Fc-IL7 on γδ T-cell survival in vitro was assessed by flow cytometry. NOD.CB17-Prkdcscid/J mice received 1 × 106 NB-1691 neuroblastoma cells via the tail vein 5 to 6 days before therapy began. Treatment, for five consecutive weeks, consisted of injections of 1 × 106 γδ T cells weekly, 1 × 106 γδ T cells weekly, and 20 μg hu14.18 antibody four times per week, or 1 × 106 γδ T cells weekly with 20 μg hu14.18 antibody four times per week, and 20 μg Fc-IL7 once weekly.

Results: The natural cytotoxicity of γδ T cells to NB-1691 cells in vitro was dramatically enhanced by hu14.18 antibody. Fc-IL7 effectively kept cultured γδ T cells viable. Combination therapy with γδ T cells and hu14.18 antibody significantly enhanced survival (P = 0.001), as did treatment with γδ T cells, hu14.18 antibody, and Fc-IL7 (P = 0.005). Inclusion of Fc-IL7 offered an additional survival benefit (P = 0.04).

Conclusions: We have shown a new and promising immunotherapy regimen for neuroblastoma that requires clinical evaluation. Our approach might also serve as a therapeutic model for other malignancies.
Identification of a Novel Estrogen-Regulated Gene, EIG121, Induced by Hormone Replacement Therapy and Differentially Expressed in Type I and Type II Endometrial Cancer
Authors: Deng Lei , Broaddus Russell R. , McCampbell Adrienne , Shipley Gregory L. , Loose David S. , Stancel George M. , Pickar James H. , Davies Peter J.A.
Published: March 2006

Purpose: The identification of genes and pathways that are affected by estrogenization may shed light on the mechanisms of estrogen action. Here, we describe the expression pattern of a novel estrogen-induced gene, EIG121, in distinct types of endometrial cancer.

Experimental Design: EIG121 was identified by cDNA microarray analysis of endometrial RNA from women receiving either placebo or estrogen replacement therapy. The expression level of EIG121 was then measured by real-time quantitative reverse transcription-PCR in benign, hyperplastic, and malignant endometrial samples. A polyclonal antibody was used to detect EIG121 protein by immunohistochemistry.

Results: In postmenopausal endometrium, estrogen replacement therapy with Premarin and synthetic estrogen sulfate conjugates induced the expression of EIG121 2- and 3-fold, respectively. In premenopausal endometrium, the expression of EIG121 was higher in the estrogen-dominated proliferative phase than the secretory phase. In endometrial complex, hyperplasia, and endometrioid adenocarcinoma, neoplastic proliferations associated with estrogen excess, the expression of EIG121 was significantly elevated (on average 3.8-fold in hyperplasias and 21-fold in grade 1 tumors). Although the level of EIG121 mRNA in grade 3 endometrioid carcinoma was still 3.5-fold of that in benign endometrium, EIG121 expression tended to decline with increasing tumor grade and disease stage. Immunohistochemistry showed faint staining of normal endometrial epithelium, but intense staining of endometrioid tumors. In sharp contrast, EIG121 expression was significantly suppressed in both uterine papillary serous carcinoma and uterine malignant mixed mullerian tumor, two tumors not associated with estrogen exposure, to <5% of the level in benign endometrium.

Conclusions: Our results suggest that EIG121 is a good endometrial biomarker associated with a hyperestrogenic state and estrogen-related type I endometrial adenocarcinoma.
Inhibition of Heat Shock Protein 90 Function by 17-Allylamino-17-Demethoxy-Geldanamycin in Hodgkin's Lymphoma Cells Down-Regulates Akt Kinase, Dephosphorylates Extracellular Signal–Regulated Kinase, and Induces Cell Cycle Arrest and Cell Death
Authors: Georgakis Georgios V. , Li Yang , Rassidakis Georgios Z. , Martinez-Valdez Hector , Medeiros L. Jeffrey , Younes Anas
Published: March 2006

Purpose: Heat shock protein 90 (HSP90) is a chaperone for several client proteins involved in transcriptional regulation, signal transduction, and cell cycle control. HSP90 is abundantly expressed by a variety of tumor types and has been recently targeted for cancer therapy. The objective of this study was to determine the role of HSP90 in promoting growth and survival of Hodgkin's lymphoma and to determine the molecular consequences of inhibiting HSP90 function by the small-molecule 17-allylamino-17-demethoxy-geldanamycin (17-AAG) in Hodgkin's lymphoma.

Experimental Design: HSP90 expression in Hodgkin's lymphoma cell lines was determined by Western blot and in primary lymph node sections from patients with Hodgkin's lymphoma by immunohistochemistry. Cell viability was determined by the 3-(4,5-dimethyl-thiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Apoptosis and cell cycle fractions were determined by flow cytometry. Expression of intracellular proteins was determined by Western blot.

Results: HSP90 is overexpressed in primary and cultured Hodgkin's lymphoma cells. Inhibition of HSP90 function by 17-AAG showed a time- and dose-dependent growth inhibition of Hodgkin's lymphoma cell lines. 17-AAG induced cell cycle arrest and apoptosis, which were associated with a decrease in cyclin-dependent kinase (CDK) 4, CDK 6, and polo-like kinase 1 (PLK1), and induced apoptosis by caspase-dependent and caspase-independent mechanisms. Furthermore, 17-AAG depleted cellular contents of Akt, decreased extracellular signal–regulated kinase (ERK) phosphorylation, and reduced cellular FLICE-like inhibitory protein levels (FLIP), and thus enhanced the cytotoxic effect of doxorubicin and agonistic anti–tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) death receptor antibodies.

Conclusion: Inhibition of HSP90 function induces cell death and enhances the activity of chemotherapy and anti–tumor necrosis factor–related apoptosis-inducing ligand death receptor antibodies, suggesting that targeting HSP90 function might be of therapeutic value in Hodgkin's lymphoma.
Immunization Using Autologous Dendritic Cells Pulsed with the Melanoma-Associated Antigen gp100-Derived G280-9V Peptide Elicits CD8+ Immunity
Authors: Linette Gerald P. , Zhang Dongsheng , Hodi F. Stephen , Jonasch Eric P. , Longerich Simonne , Stowell Christopher P. , Webb Iain J. , Daley Heather , Soiffer Robert J. , Cheung Amy M. , Eapen Sara G. , Fee Sharon V. , Rubin Krista M. , Sober Arthur J. , Haluska Frank G.
Published: March 2006

Purpose: To determine the toxicity, maximal tolerated dose, and clinical and immunologic response to autologous dendritic cells pulsed with melanoma-associated antigen gp100-derived G280-9V peptide.

Patients and Methods: Twelve HLA-A*0201+ patients with advanced melanoma were administered dendritic cells pulsed with G280-9V peptide. Cohorts of three patients were administered 5 × 106, 15 × 106, and 50 × 106 cells i.v. every 3 weeks for six doses according to a dose escalation scheme. Three additional patients were treated at the highest dose. No additional cytokines or therapies were coadministered. The immunogenicity of G280-9V-pulsed dendritic cells was measured by IFN-γ ELISPOT assay, tetramer assay, and 51Cr release assay comparing prevaccination to postvaccination blood samples. Response to treatment was assessed by Response Evaluation Criteria in Solid Tumors.

Results: CD8+ immunity to the native G280 was observed in 8 (67%) patients as measured by ELISPOT and in 12 (100%) patients as measured by tetramer assay. Of the 9 patients tested, 9 (100%) had measurable high-avidity CTL activity as defined by lysis of allogeneic melanoma lines, which coexpress HLA-A*0201 and gp100. The median follow-up of the entire cohort is 43.8 months. Two (17%) partial responses were observed and 3 (25%) patients had stable disease. The median survival of the treated population was 37.6 months. At this time, three patients are alive, including one patient who continues to respond without additional treatment.

Conclusion: The high rate of immunization as measured by three independent assays and the occurrence of clinical regression support continued investigation of G280-9V peptide as a candidate epitope in melanoma vaccine formulations.
Phase I and Pharmacokinetic Study of Flavopiridol followed by 1-β-d-Arabinofuranosylcytosine and Mitoxantrone in Relapsed and Refractory Adult Acute Leukemias
Authors: Karp Judith E. , Passaniti Antonino , Gojo Ivana , Kaufmann Scott , Bible Keith , Garimella Tushar S. , Greer Jacqueline , Briel Janet , Smith B. Douglas , Gore Steven D. , Tidwell Michael L. , Ross Douglas D. , Wright John J. , Colevas A. Dimitrios , Bauer Kenneth S.
Published: March 2006

Purpose: The serine/threonine kinase inhibitor flavopiridol targets multiple cyclin-dependent kinases, induces checkpoint arrest, and interrupts transcriptional elongation. We designed a phase I clinical trial using a timed sequential therapy approach where flavopiridol was given for the dual purpose of initial cytoreduction and enhancing cell cycle progression of the remaining leukemia cell cohort followed by cycle-dependent drugs 1-β-d-arabinofuranosylcytosine (ara-C) and mitoxantrone.

Experimental Design: Flavopiridol was given by 1-hour infusion daily for 3 days beginning day 1 followed by 2 g/m2/72 h ara-C beginning day 6 and 40 mg/m2 mitoxantrone beginning day 9. In vivo correlates included pharmacokinetics, modulation of blast cycle regulators, and serum and marrow supernatant vascular endothelial growth factor levels.

Results: Of 34 adults receiving induction therapy, 16 (47%) evinced direct leukemia cytotoxicity with ≥50% drop in peripheral blast counts and tumor lysis in 9 (26%). Four (12%) died during therapy (two fungal infections and two sudden death). Dose-limiting toxicity occurred at 60 mg/m2/d with profound neutropenia >40 days duration, and maximal tolerated dose was 50 mg/m2/d. Overall response rate was 31% in 26 acute myelogenous leukemia and 12.5% in acute lymphoblastic leukemia. Pharmacokinetics showed that a linear two-compartment model with first-order elimination provided the best fit of the observed concentration versus time data. Flavopiridol down-regulated one or more target proteins in marrow blasts in vivo. Vascular endothelial growth factor was detected in sera and marrow supernatant pretreatment, and sera obtained on day 3 inhibited bovine aortic endothelial cell proliferation by a mean of 32% (range, 10-80%).

Conclusions: Our data suggest that flavopiridol is cytotoxic to leukemic cells and, when followed by ara-C and mitoxantrone, exerts biological and clinical effects in patients with relapsed and refractory acute leukemias. These findings warrant continuing development of flavopiridol at 50 mg/m2/d × 3 days in combination with cytotoxic and biological agents for acute leukemias.
Cyclooxygenase-2-Dependent Activation of Signal Transducer and Activator of Transcription 3 by Interleukin-6 in Non–Small Cell Lung Cancer
Authors: Dalwadi Harnisha , Krysan Kostyantyn , Heuze-Vourc'h Nathalie , Dohadwala Mariam , Elashoff David , Sharma Sherven , Cacalano Nicholas , Lichtenstein Alan , Dubinett Steven
Published: March 2006

Purpose: Cyclooxygenase-2 (COX-2), phosphorylated signal transducers and activators of transcription 3 (STAT3), and interleukin-6 (IL-6) are elevated in non–small cell lung cancer (NSCLC). These molecules affect numerous cellular pathways, including angiogenesis and apoptosis resistance, and, therefore, may act in concert in NSCLC.

Experimental Design: We examined IL-6 and phosphorylated STAT3 in COX-2-overexpressing [COX-2 sense-oriented (COX-2-S)] NSCLC cells and control cells. The effect of IL-6, STAT3, phosphatidylinositol 3-kinase, and mitogen-activated protein/extracellular signal-regulated kinase kinase on vascular endothelial growth factor (VEGF) production and apoptosis resistance was assessed in COX-2-overexpresing cells.

Results: We report that NSCLC cells overexpressing COX-2 (COX-2-S) have increased IL-6 and phosphorylated STAT3 expression compared with control cells. IL-6 induced expression of VEGF in NSCLC cells. Moreover, blocking IL-6, mitogen-activated protein/extracellular signal-regulated kinase kinase, or phosphatidylinositol 3-kinase decreased VEGF production in COX-2-S cells. The addition of IL-6 to NSCLC cells resulted in increased apoptosis resistance. Furthermore, the inhibition of STAT3 or IL-6 induced apoptosis and reduced survivin expression, a member of the inhibitor of apoptosis protein family in COX-2-S cells.

Conclusions: Overall, these findings suggest a novel pathway in which COX-2 activates STAT3 by inducing IL-6 expression. This pathway could contribute to tumor formation by promoting survivin-dependent apoptosis resistance and VEGF production. These findings provide a rationale for the future development of STAT3, IL-6, and/or COX-2-targeted therapies for the treatment of lung cancer.
A Novel Cancer Testis Antigen That Is Frequently Expressed in Pancreatic, Lung, and Endometrial Cancers
Authors: Okada Takaho , Akada Masanori , Fujita Tomonobu , Iwata Takashi , Goto Yasufumi , Kido Kenji , Okada Tsutomu , Matsuzaki Yuriko , Kobayashi Kouichi , Matsuno Seiki , Sunamura Makoto , Kawakami Yutaka
Published: March 2006

Purpose: To isolate cancer testis antigens that are expressed in pancreatic cancers and may be useful in clinical applications.

Experimental Design: To efficiently isolate cancer testis antigens, a testis cDNA library was immunoscreened (SEREX) with serum from a patient with pancreatic ductal adenocarcinoma. The expression of isolated antigens in various cancer cell lines and tissues was evaluated by reverse transcription-PCR and Northern blot analyses. The immunogenicity of the antigen in cancer patients was evaluated by detection of the IgG antibody in sera from patients with various cancers.

Results: Of the three clones isolated through screening of a total of 2 × 106 cDNA library clones, one clone (KU-CT-1) was found to be expressed in various cancers but only in testis among normal tissues, indicating that it was a novel cancer testis antigen. The KU-CT-1 gene is located on chromosome 10p12 and produces two splice variants, which encode proteins of 397 and 872 amino acids, respectively. KU-CT-1 was expressed in pancreatic cancer tissues (3 of 9, 33%), lung cancer tissues (9 of 24, 38%), and endometrial cancer tissues (7 of 11, 64%). Specific serum IgG antibodies were detected in 3 of 20 pancreatic cancer patients, 2 of 12 endometrial cancer patients, 1 of 18 colon cancer patients, and 1 of 10 prostate cancer patients but not detected in 30 healthy individuals.

Conclusions: KU-CT-1 is a new cancer testis antigen that is expressed in pancreatic, lung, and endometrial cancers and may be useful for diagnosis and immunotherapy for patients with various cancers.
Polyclonal Evolution of Multiple Secondary KIT Mutations in Gastrointestinal Stromal Tumors under Treatment with Imatinib Mesylate
Authors: Wardelmann Eva , Merkelbach-Bruse Sabine , Pauls Katharina , Thomas Nadja , Schildhaus Hans-Ulrich , Heinicke Thomas , Speidel Nicola , Pietsch Torsten , Buettner Reinhard , Pink Daniel , Reichardt Peter , Hohenberger Peter
Published: March 2006

Gastrointestinal stromal tumors (GIST) are characterized by a strong KIT receptor activation most often resulting from KIT mutations. In a smaller subgroup of tumors without KIT mutations, analogous activating mutations are found in the platelet-derived growth factor receptor α (PDGFRα) gene. Both PDGFRα and KIT receptors are targets of the tyrosine kinase inhibitor imatinib (Glivec) which has improved the treatment of advanced GISTs significantly. However, a subgroup of tumors show a secondary progress under therapy with imatinib after initial response. One possible mechanism of secondary resistance is the development of newly acquired KIT mutations. In the present study, we evaluated the frequency of such secondary KIT mutations in a series of GIST patients in which tumor tissue was resected under treatment. We examined one to seven different tumor areas in 32 cases (total of 104 samples) and found up to four newly acquired KIT mutations in 14 patients (43.8%). These were always located in exons encoding the first or second tyrosine kinase domain (exon 13, 14, or 17). Mutations were found only in a subset of samples analyzed from each case whereas others retained the wild-type sequence in the same region. There was never more than one new mutation in the same sample. Consistent with a secondary clonal evolution, the primary mutation was always detectable in all samples from each tumor. According to our results, the identification of newly acquired KIT mutations in addition to the primary mutation is dependent on the number of tissue samples analyzed and has high implications for further therapeutic strategies.
Botulinum toxin potentiates cancer radiotherapy and chemotherapy.
Authors: Ansiaux R√©ginald , Baudelet Christine , Cron Greg O , Segers J√©r√¥me , Dessy Chantal , Martinive Philippe , De Wever Julie , Verrax Julien , Wauthier Val√©rie , Beghein Nelson , Gr√©goire Vincent , Buc Calderon Pedro , Feron Olivier , Gallez Bernard
Published: March 2006

PURPOSE: Structural and functional abnormalities in the tumor vascular network are considered factors of resistance of solid tumors to cytotoxic treatments. To increase the efficacy of anticancer treatments, efforts must be made to find new strategies for transiently opening the tumor vascular bed to alleviate tumor hypoxia (source of resistance to radiotherapy) and improve the delivery of chemotherapeutic agents. We hypothesized that Botulinum neurotoxin type A (BoNT-A) could interfere with neurotransmitter release at the perivascular sympathetic varicosities, leading to inhibition of the neurogenic contractions of tumor vessels and therefore improving tumor perfusion and oxygenation. EXPERIMENTAL DESIGN: To test this hypothesis, BoNT-A was injected locally into mouse tumors (fibrosarcoma FSaII, hepatocarcinoma transplantable liver tumor), and electron paramagnetic resonance oximetry was used to monitor pO(2) in vivo repeatedly for 4 days. Additionally, contrast-enhanced magnetic resonance imaging was used to measure tumor perfusion in vivo. Finally, isolated arteries were mounted in wire myograph to monitor specifically the neurogenic tone developed by arterioles that were co-opted by the surrounding growing tumor cells. RESULTS: Using these tumor models, we showed that local administration of BoNT-A (two sites; dose, 29 units/kg) substantially increases tumor oxygenation and perfusion, leading to a substantial improvement in the tumor response to radiotherapy (20 Gy of 250-kV radiation) and chemotherapy (cyclophosphamide, 50 mg/kg). This observed therapeutic gain results from an opening of the tumor vascular bed by BoNT-A because we showed that BoNT-A could inhibit neurogenic tone in the tumor vasculature. CONCLUSIONS: The opening of the vascular bed induced by BoNT-A offers a way to significantly increase the response of tumors to radiotherapy and chemotherapy.
The Novel Poly(ADP-Ribose) Polymerase Inhibitor, AG14361, Sensitizes Cells to Topoisomerase I Poisons by Increasing the Persistence of DNA Strand Breaks
Authors: Smith Lisa M. , Willmore Elaine , Austin Caroline A. , Curtin Nicola J.
Published: March 2006

Poly(ADP-ribose) polymerase (PARP) inhibitors enhance DNA topoisomerase I (topo I) poison-induced cytotoxicity and antitumor activity in vitro and in vivo, but the mechanism has not been defined. We investigated the role of PARP-1 in the response to topo I poisons using PARP-1−/− and PARP-1+/+ mouse embryonic fibroblasts and the potent PARP-1 inhibitor, AG14361 (Ki < 5 nmol/L). PARP-1−/− mouse embryonic fibroblasts were 3-fold more sensitive to topotecan than PARP-1+/+ mouse embryonic fibroblasts (GI50, 21 and 65 nmol/L, respectively). AG14361 caused a >3-fold sensitization of PARP-1+/+ cells to topotecan compared with a <1.4-fold sensitization in PARP-1−/− cells. In human leukemia K562 cells, AG14361 caused a 2-fold sensitization to camptothecin-induced cytotoxicity. AG14361 did not affect the cellular activity of topo I as determined by measurement of cleavable complexes and topo I relaxation activity, showing that sensitization was not due to topo I activation. In contrast, repair of DNA following camptothecin removal, normally very rapid, was significantly retarded by AG14361, resulting in a 62% inhibition of repair 10 minutes after camptothecin removal. This led to a 20% increase in the net accumulation of camptothecin-induced DNA strand break levels in cells coexposed to AG14361 for 16 hours. We investigated the DNA repair mechanism involved using a panel of DNA repair–deficient Chinese hamster ovary cells. AG14361 significantly potentiated camptothecin-mediated cytotoxicity in all cells, except the base excision repair–deficient EM9 cells. Therefore, the most likely mechanism for the potentiation of topo I poison-mediated cytotoxicity by AG14361 is via PARP-1-dependent base excision repair.
Increased NBS1 Expression Is a Marker of Aggressive Head and Neck Cancer and Overexpression of NBS1 Contributes to Transformation
Authors: Yang Muh-Hwa , Chiang Wei-Chung , Chou Teh-Ying , Chang Shyue-Yih , Chen Po-Min , Teng Shu-Chun , Wu Kou-Juey
Published: March 2006

Purpose: Head and neck squamous cell carcinoma (HNSCC) represents the sixth most frequent type of cancer worldwide. However, the molecular genetic alterations underlying its malignant behavior and progression are little known. We showed previously that c-MYC directly activates the expression of the DNA double-strand break repair gene NBS1, and NBS1 overexpression contributes to transformation. Here, we investigate the role of NBS1 overexpression in HNSCC.

Experimental Design: Immunohistochemistry analysis of NBS1 expression was done in 81 locally advanced HNSCC patients. Real-time PCR and Western blot analysis were used to confirm immunohistochemistry results. Human hypopharyngeal cancer cell lines (FADU) with overexpressing NBS1 (FADUNBS) or inducible short interference RNA to repress endogenous NBS1 (FADUNBSi) were generated by stable transfection. Soft agar clonogenicity assay was used to determine the transformation activity. Western blot analysis and phosphatidylinositol 3-kinase (PI3K) assay were done to evaluate the signaling pathways that were involved.

Results: NBS1 overexpression was identified in 45% of advanced HNSCC patients. It was an independent marker of poor prognosis. NBS1 expression levels correlated with the transformation activity of FADU clones and also correlated with the phosphorylation levels of Akt and its downstream target mammalian target of rapamycin (mTOR). PI3K activity was increased in NBS1-overexpressing FADU clones. NBS1 overexpression also correlated with increased Akt phosphorylation levels in tumor samples.

Conclusions: Increased NBS1 expression is a significant prognostic marker of advanced HNSCC, and the underlying mechanism may involve the activation of the PI3K/Akt pathway.
Peritumoral Lymphatic Vessel Density and Vascular Endothelial Growth Factor C Expression in Early-Stage Squamous Cell Carcinoma of the Uterine Cervix
Authors: Gombos Zoltan , Xu Xiaowei , Chu Christina S. , Zhang Paul J. , Acs Geza
Published: March 2006

Purpose: Lymphatic invasion and nodal metastasis plays a major role in the spread of cervical cancer; however, little is known about the mechanisms whereby tumor cells enter the lymphatic system.

Experimental Design: We examined the intra- and peritumoral lymphatic vessel density (LVD) using D2-40 immunohistochemistry in 111 cervical squamous cell carcinomas and correlated them with vascular endothelial growth factor (VEGF)-C expression, clinicopathologic tumor features, and outcome.

Results: Compared with benign cervix, intratumoral and peritumoral LVD was significantly increased (P < 0.0001). Peritumoral LVD was significantly higher than intratumoral LVD (P = 0.009). High peritumoral, but not intratumoral, LVD showed significant correlation with high tumor stage, lymphatic invasion, and nodal metastasis. VEGF-C showed increased expression at the invasive edge compared with the center of tumors (P < 0.0001) and correlated with high peritumoral LVD, lymphatic invasion, and nodal metastasis. High peritumoral LVD and VEGF-C expression at the invasive edge of tumors were associated with poor overall and recurrence-free survival in univariate analysis. In multivariate analysis, peritumoral LVD was the only independent term predictive of overall survival.

Conclusions: Our findings suggest a potential role for VEGF-C in tumor-induced lymphangiogenesis represented by high peritumoral LVD, which may be one of the mechanisms leading to lymphatic invasion and metastatic spread. High peritumoral LVD may be an independent prognostic factor in early-stage cervical cancer.
Therapeutic Advantage of Pretargeted Radioimmunotherapy Using a Recombinant Bispecific Antibody in a Human Colon Cancer Xenograft
Authors: Karacay Habibe , Brard Pierre-Yves , Sharkey Robert M. , Chang Chien-Hsing , Rossi Edmund A. , McBride William J. , Ragland Dan R. , Horak Ivan D. , Goldenberg David M.
Published: March 2006

Purpose: To assess if pretargeting, using a combination of a recombinant bispecific antibody (bsMAb) that binds divalently to carcinoembryonic antigen (CEA) and monovalently to the hapten histamine-succinyl-glycine and a 90Y-peptide, improves therapeutic efficacy in a human colon cancer-nude mouse xenograft compared with control animals given 90Y-humanized anti-CEA immunoglobulin G (IgG).

Experimental Design: Clearance and biodistribution were monitored by whole-body readings and necropsy. Animals were monitored for 34 weeks with a determination of residual disease and renal pathology in survivors. Hematologic toxicity was assessed separately in non-tumor-bearing NIH Swiss mice.

Results: Hematologic toxicity was severe at doses of 100 to 200 μCi of 90Y-IgG, yet mild in the pretargeted animals given 500 or 700 μCi of the 90Y-peptide. Evidence of end-stage renal disease was found at 900 μCi of the pretargeted 90Y-peptide whereas animals given 700 μCi showed only mild renal pathology, similar to that seen in control animals given 90Y-IgG. Biodistribution data indicated that the average amount of tumor radioactivity by a 700-μCi dose of the pretargeted peptide over a 96-hour period was increased 2.5-fold (48 μCi/g) compared with 150 μCi of 90Y-IgG (18.9 μCi/g). At these doses, survival (i.e., time to progression to 2.5 cm3) was significantly improved (P < 0.04) compared with 90Y-IgG, with ablation of about one third of the tumors, whereas viable tumor was present in all of the 90Y-IgG–treated animals.

Conclusion: Pretargeting increases the amount of radioactivity delivered to colorectal tumors sufficiently to improve the therapeutic index and responses as compared with conventional radioimmunotherapy.
Estrogen Signaling Is Active in Cartilaginous Tumors: Implications for Antiestrogen Therapy as Treatment Option of Metastasized or Irresectable Chondrosarcoma
Authors: Cleton-Jansen Anne-Marie , van Beerendonk Hetty M. , Baelde Hans J. , Bovée Judith V.G.M. , Karperien Marcel , Hogendoorn Pancras C.W.
Published: March 2006

Purpose: Chondrosarcoma is a malignant cartilaginous matrix–producing tumor that can be lethal in 10% to 50% of the patients. Surgery is the only effective treatment known as these tumors are notorious refractory to all types of conventional chemotherapy or radiotherapy. To identify a target for therapy, we want to determine whether estrogen signaling is active in chondrosarcoma because estrogen is important in the regulation of longitudinal growth that is initiated by chondrocyte proliferation and differentiation in the epiphyseal growth plate of long bones.

Experimental Design: We studied protein expression of the estrogen receptor in 35 cartilaginous tumors as well as mRNA levels for the estrogen receptor and for aromatase, an enzyme for estrogen synthesis and another potential therapeutic target. Furthermore, the activity of aromatase was determined in vitro by the tritiated water release assay. Dose-response experiments with chondrosarcoma cultured cells were done with estrogen, androstenedione, and exemestane.

Results: All chondrosarcomas tested showed mRNA and nuclear protein expression of the estrogen receptor. Also, aromatase mRNA was detected. The aromatase activity assay showed a functional aromatase enzyme in primary chondrosarcoma cultures and in a cell line. Growth of chondrosarcoma cell cultures can be stimulated by adding estrogen or androstenedione, which can be inhibited by exemestane.

Conclusions: These results show, on the RNA, protein, and cell biological levels, that the ligand and the receptor are active in estrogen-mediated signal transduction. This observation implicates potential use of targeted drugs that interfere with estrogen signaling, such as those applied for treating breast cancer.
Tumor-Associated Antigen Recognized by the 22-1-1 Monoclonal Antibody Encourages Colorectal Cancer Progression under the Scanty CD8+ T Cells
Authors: Oshikiri Taro , Miyamoto Masaki , Morita Takayuki , Fujita Miyoshi , Miyasaka Yuji , Senmaru Naoto , Yamada Hidehisa , Takahashi Toshiyuki , Horita Shoichi , Kondo Satoshi
Published: March 2006

Purpose: The receptor-binding cancer antigen expressed on SiSo cells (RCAS1) is a novel tumor-associated antigen. Although evidence suggests that RCAS1 suppresses immunity by inducing tumor-infiltrating lymphocyte (TIL) apoptosis, RCAS1 function in humans is controversial. RCAS1 overexpression leads to the generation of the Tn glycan antigen (N-acetyl-d-galactosamine, GalNAc) recognized by the 22-1-1 monoclonal antibody. The objective of this study is to examine Tn glycan antigen function in colorectal cancer and to determine its relationship to CD8+ T cells and prognosis.

Experimental Design: Immunohistochemical analyses examined Tn expression in tumor cells and CD8 on TILs in 146 surgically resected colorectal cancer.

Results: Of 146 samples, 68 tumors (47%) were Tn+; 72 tumors (49%) were CD8+. Using Cox multivariate analysis and the Kaplan-Meier method, Tn and CD8 positivity were determined to be mutually independent prognostic factors (P = 0.0266 and 0.0210, respectively). Tn+ patients with CD8+ TILs exhibited better survival than Tn+/CD8− patients (P = 0.0129). For CD8− patients, Tn positivity was associated with decreased survival from that seen in Tn− patients (P = 0.0097), suggesting that Tn exerts a function independent of CD8+ T cells in tumor progression. In all patients and cases with synchronous liver metastases (n = 29), the Tn+/CD8− survival rate was significantly lower than that seen for other groups (P = 0.0001 and 0.0063, respectively). The average number of liver metastases in Tn+/CD8− cases also increased (mean, 8.2 tumors; P = 0.0032). Multivariate analysis identified Tn+/CD8− status and Dukes' staging as independent prognostic factors (P = 0.0016 and <0.0001, respectively).

Conclusions: Tn may encourage invasion and innidiation through a mechanism independent of CD8+ T cells. Thus, Tn+/CD8− status is a risk factor for multiple liver metastases development and an independent negative prognostic factor for colorectal cancer.
213Bi-[DOTA0, Tyr3]Octreotide Peptide Receptor Radionuclide Therapy of Pancreatic Tumors in a Preclinical Animal Model
Authors: Norenberg Jeffrey P. , Krenning Boudewijn J. , Konings Inge R.H.M. , Kusewitt Donna F. , Nayak Tapan K. , Anderson Tamara L. , de Jong Marion , Garmestani Kayhan , Brechbiel Martin W. , Kvols Larry K.
Published: March 2006

Purpose: The somatostatin analogue [DOTA0, Tyr3]octreotide (DOTATOC) has previously been labeled with low linear energy transfer (LET) β-emitters, such as 177Lu or 90Y, for tumor therapy. In this study, DOTATOC labeled with the high-LET α-emitter, 213Bi, was evaluated.

Experimental Design: The radiolabeling, stability, biodistribution, toxicity, safety, and therapeutic efficacy of 213Bi-DOTATOC (specific activity 7.4 MBq/μg) were investigated. Biodistribution studies to determine somatostatin receptor specificity were done in Lewis rats at 1 and 3 hours postinjection. Histopathology of various organs was used to evaluated toxicity and safety. Therapeutic efficacy of 4 to 22 MBq 213Bi-DOTATOC was determined in a rat pancreatic carcinoma model.

Results: Radiolabeling of the 213Bi-DOTATOC was achieved with radiochemical purity >95% and an incorporation yield ≥99.9%. Biodistribution data showed specific binding to somatostatin receptor–expressing tissues. Administration of free 213Bi, compared with 213Bi-DOTATOC, resulted in higher radioactivity accumulation at 3 hours postinjection in the kidneys [34.47 ± 1.40% injected dose/g (ID/g) tissue versus 11.15 ± 0.46%, P < 0.0001] and bone marrow (0.31 ± 0.01% ID/g versus 0.06 ± 0.02%, P < 0.0324). A significant decrease in tumor growth rate was observed in rats treated with >11 MBq of 213Bi-DOTATOC 10 days postinjection compared with controls (P < 0.025). Treatment with >20 MBq of 213Bi-DOTATOC showed significantly greater tumor reduction when compared with animals receiving <11 MBq (P < 0.02).

Conclusions: 213Bi-DOTATOC showed dose-related antitumor effects with minimal treatment-related organ toxicity. No acute or chronic hematologic toxicities were observed. Mild, acute nephrotoxicity was observed without evidence of chronic toxicity. 213Bi-DOTATOC is a promising therapeutic radiopharmaceutical for further evaluation.
Nitric oxide in breast cancer: induction of vascular endothelial growth factor-C and correlation with metastasis and poor prognosis.
Authors: Nakamura Yasushi , Yasuoka Hironao , Tsujimoto Masahiko , Yoshidome Katsuhide , Nakahara Masaaki , Nakao Kazuyasu , Nakamura Misa , Kakudo Kennichi
Published: March 2006

PURPOSE: Metastasis to regional lymph nodes through the lymphatic vessels is a common step in the progression of cancer. Recent evidence suggests that tumor production of vascular endothelial growth factor-C (VEGF-C) promotes lymphagiogenesis, which in turn promotes lymphatic metastasis. Nitric oxide (NO) may also increase metastatic ability in human cancers. EXPERIMENTAL DESIGN: Nitrite/nitrate levels and VEGF-C production were assessed in MDA-MB-231 breast cancer cells after induction and/or inhibition of NO synthesis. Formation of nitrotyrosine, a biomarker for peroxynitrate formation from NO in vivo, was analyzed in primary human breast carcinoma with long-term follow-up. The relationship between nitrotyrosine levels and lymph node status, VEGF-C immunoreactivity, and other established clinicopathologic variables, as well as prognosis, was analyzed. RESULTS: Production of nitrite/nitrate and VEGF-C in MDA-MB-231 cells was increased by treatment with the NO donor DETA NONOate. The NO synthase inhibitor N(G)-nitro-l-arginine methyl ester eliminated this increase. High-grade nitrotyrosine staining was observed in 57.5% (65 of 113) of the invasive breast carcinomas. Nitrotyrosine levels were significantly correlated with VEGF-C immunoreactivity and lymph node metastasis. Survival curves determined by the Kaplan-Meier method showed that high nitrotyrosine levels were associated with reduced disease-free and overall survival. In multivariate analysis, high nitrotyrosine levels emerged as a significant independent predictor for overall survival. CONCLUSIONS: Our data showed a role for NO in stimulating VEGF-C expression in vitro. Formation of its biomarker nitrotyrosine was also correlated with VEGF-C expression and lymph node metastasis. Furthermore, high nitrotyrosine levels may serve as a significant prognostic factor for long-term survival in breast cancer.
Vascular Endothelial Growth Factor Messenger RNA Expression Level Is Preserved in Liver Metastases Compared with Corresponding Primary Colorectal Cancer
Authors: Kuramochi Hidekazu , Hayashi Kazuhiko , Uchida Kazumi , Miyakura Satoru , Shimizu Daisuke , Vallböhmer Daniel , Park Seongjin , Danenberg Kathleen D. , Takasaki Ken , Danenberg Peter V.
Published: March 2006

Purpose: Increased vascular endothelial growth factor (VEGF) expression is associated with colorectal cancer liver metastases. It is reasonable to expect that measurement of VEGF in liver metastases would provide the best prediction of therapy benefit for VEGF-targeted drugs, such as bevacizumab (Avastin). In this study, we evaluated how VEGF mRNA level in primary colorectal cancer was related to that in corresponding liver metastases. Thirty-one pairs of primary colorectal cancer and corresponding liver metastases were analyzed.

Experimental Design: Formalin-fixed, paraffin-embedded tumor specimens were dissected by using laser-captured microdissection. RNA was extracted and cDNA was prepared by reverse transcription. Quantitation of VEGF and internal reference gene (β-actin) was done using real-time PCR (Taqman PCR).

Results: There was no difference between median VEGF mRNA levels of primary colorectal cancer and liver metastases (median value 3.79 versus 3.97: P = 0.989). On an individual basis, there was a significant correlation in VEGF mRNA expression between primary colorectal cancer and corresponding liver metastases (rs = 0.6627, P < 0.0001). In addition, the VEGF mRNA levels of the patients who had two or more liver metastatic tumors were significantly higher than those of the patient who had solitary liver metastatic tumor in both primary cancer (5.02 versus 3.34: P = 0.0483) and liver metastases (4.38 versus 3.25: P = 0.0358).

Conclusion: Good prediction of VEGF mRNA levels in liver metastases can be obtained by measuring those of primary colorectal cancer. The risk of multiple liver metastatic tumors might be predictable by measuring VEGF mRNA expression in primary colorectal cancer. Further study is required to confirm these preliminary results.
T-Cell Responses Directed against Multiple HLA-A*0201-Restricted Epitopes Derived from Wilms' Tumor 1 Protein in Patients with Leukemia and Healthy Donors: Identification, Quantification, and Characterization
Authors: Rezvani Katayoun , Brenchley Jason M. , Price David A. , Kilical Yasemin , Gostick Emma , Sewell Andrew K. , Li Jongming , Mielke Stephan , Douek Daniel C. , Barrett A. John
Published: March 2006

Purpose: Antigens derived from the Wilms' tumor (WT1) protein, which is overexpressed in leukemias, are attractive targets for immunotherapy. Four HLA-A*0201-restricted WT1-derived epitopes have been identified: WT37, WT126, WT187, and WT235. We determined the natural immunogenecity of these antigens in patients with hematologic malignancies and healthy donor.

Experimental Design: To detect very low frequencies of WT1-specific CD8+ T cells, we used quantitative reverse transcription-PCR to measure IFN-γ mRNA production by WT1 peptide–pulsed CD8+ T cells from 12 healthy donors, 8 patients with chronic myelogenous leukemia, 6 patients with acute myelogenous leukemia, and 8 patients with acute lymphoblastic leukemia.

Results: Responses were detected in 5 of 8 chronic myelogenous leukemia patients, 4 of 6 patients with acute myelogenous leukemia, and 7 of 12 healthy donors. No responses were detected in patients with acute lymphoblastic leukemia. The magnitude and extent of these CD8+ T-cell responses was greater in patients with myeloid leukemias than in healthy donors. Clonotypic analysis of WT1-specific CD8+ T cells directly ex vivo in one case showed that this naturally occurring population was oligoclonal. Using fluorescent peptide-MHC class I tetramers incorporating mutations in the α3 domain (D227K/T228A) that abrogate binding to the CD8 coreceptor, we were able to confirm the presence of high-avidity T-cell clones within the antigen-specific repertoire.

Conclusion: The natural occurrence of high-avidity WT1-specific CD8+ T cells in the periphery could facilitate vaccination strategies to expand immune responses against myeloid leukemias.
Epigenetic Inactivation of Wnt Inhibitory Factor-1 Plays an Important Role in Bladder Cancer through Aberrant Canonical Wnt/β-Catenin Signaling Pathway
Authors: Urakami Shinji , Shiina Hiroaki , Enokida Hideki , Kawakami Toshifumi , Tokizane Takashi , Ogishima Tatsuya , Tanaka Yuichiro , Li Long-Cheng , Ribeiro-Filho Leopoldo A. , Terashima Masaharu , Kikuno Nobuyuki , Adachi Hiroyuki , Yoneda Tatsuaki , Kishi Hirofumi , Shigeno Kazushi , Konety Badrinath R. , Igawa Mikio , Dahiya Rajvir
Published: March 2006

Purpose: Aberrant activation of the Wingless-type (Wnt) pathway plays a significant role in the pathogenesis of several human cancers. Wnt inhibitory factor-1 (Wif-1) was identified as one of the secreted antagonists that can bind Wnt protein. We hypothesize that Wif-1 plays an important role in bladder cancer pathogenesis.

Experimental Design: To test this hypothesis, epigenetic and genetic pathways involved in the Wif-1 gene modulation and expression of Wnt/β-catenin-related genes were analyzed in 4 bladder tumor cell lines and 54 bladder tumor and matched normal bladder mucosa.

Results: Wif-1 mRNA expression was significantly enhanced after 5-aza-2′-deoxycytidine treatment in bladder tumor cell lines. Wif-1 promoter methylation level was significantly higher and Wif-1 mRNA expression was significantly lower in bladder tumor samples than in bladder mucosa samples. In the total bladder tumor and bladder mucosa samples, an inverse correlation was found between promoter methylation and Wif-1 mRNA transcript levels. However, loss-of-heterozygosity at chromosome 12q14.3 close to the Wif-1 gene loci was a rare event (3.7%). Nuclear accumulation of β-catenin was significantly more frequent in bladder tumor than in bladder mucosa and inversely correlated with Wif-1 expression. In addition, known targets of the canonical Wnt/β-catenin signaling pathway, such as c-myc and cyclin D1, were up-regulated in bladder tumor compared with bladder mucosa, and this up-regulation was associated with reduced Wif-1 expression at both mRNA and protein levels. Furthermore, transfection of Wif-1 small interfering RNA into bladder tumor cells expressing Wif-1 mRNA transcripts had increased levels of c-myc and cyclin D1 and accelerated cell growth.

Conclusion: This is the first report showing that CpG hypermethylation of the Wif-1 promoter is a frequent event in bladder tumor and may contribute to pathogenesis of bladder cancer through aberrant canonical Wnt/β-catenin signaling pathway. The present study elucidates novel pathways that are involved in the pathogenesis of bladder cancer.
Chemokine C Receptor 7 Expression and Protection of Circulating CD8+ T Lymphocytes from Apoptosis
Authors: Kim Jeong-Whun , Ferris Robert L. , Whiteside Theresa L.
Published: March 2006

Chemokine C receptor 7 (CCR7) expression is important for lymphocyte homing to tissues. We hypothesized that CCR7 also plays a role in CD8+ T-cell protection from apoptosis. Its expression was determined on circulating T cells in patients with cancer and related to that of molecules responsible for lymphocyte susceptibility/resistance to apoptosis. Peripheral blood mononuclear cells were obtained from 36 patients with squamous cell carcinoma of the head and neck and 16 normal controls. Multicolor flow cytometry was used to evaluate CCR7, Fas, Bax, and Bcl-2 expression in CD8+ T cells. Annexin V binding to CD8+CCR7+ and CD8+CCR7− T-cell subsets was compared. Fewer CD8+CCR7+ T cells bound Annexin V than CD8+CCR7− T cells in normal control and patients (P < 0.0001). CCR7 expression correlated with higher Bcl-2 but lower Bax and Fas expression levels in CD8+ T cells in both normal control and patients (P < 0.0001). In patients, the CD8+CCR7+ subset was reduced relative to normal control (P = 0.008) and replaced with an excess of apoptosis-sensitive CD8+CCR7− T cells. To study CCR7 signaling, CD8+ T cells were stimulated with CCR7 ligands, chemokine C ligands 19 or 21. Ligand binding to CCR7 resulted in phosphorylation of Akt and increased Bcl-2 expression in CD8+CCR7+ T cells, suggesting that CCR7 protects effector T cells from apoptosis through the phosphatidylinositol 3-kinase/Akt pathway. The absence of CCR7 expression on the majority of CD8+ T cells in the peripheral circulation of patients with squamous cell carcinoma of the head and neck contributes to apoptosis and a rapid turnover of these effector cells.