bluuebunny/crossref_metadata_2025 huggingface dataset:
In vitro
Stimulation with WT1 Peptide-Loaded Epstein-Barr Virus-Positive B Cells Elicits High Frequencies of WT1 Peptide-Specific T Cells with
In vitro
and
In vivo
Tumoricidal Activity
Authors: Doubrovina Ekaterina S. , Doubrovin Mikhail M. , Lee Sangyull , Shieh Jae-Hung , Heller Glen , Pamer Eric , O’Reilly Richard J.
Published: September 2005

The Wilms tumor protein (WT1) is overexpressed in most acute and chronic leukemias. To develop a practicable, clinically applicable approach for generation of WT1-specific T cells and to comparatively evaluate the immunogenicity of WT1 in normal individuals, we sensitized T cells from 13 HLA-A0201+ and 5 HLA-A2402+ donors with autologous EBV-transformed B cells or cytokine-activated monocytes, loaded with the HLA-A0201-binding WT1 peptides 126–134RMFPNAPYL or 187–195SLGEQQYSV or a newly identified HLA-A2402-binding WT1 peptide 301–310RVPGVAPTL. WT1-specific T cells were regularly generated from each donor. T cells sensitized with peptide-loaded EBV-transformed B cells generated higher numbers of WT1-specific T cells than peptide-loaded cytokine-activated monocytes. Contrary to expectations, the frequencies of WT1 peptide-specific T cells were equivalent to those generated against individual highly immunogenic HLA-A0201-binding EBV peptides. Each of these T-cell lines specifically killed WT1+ leukemias and solid tumors in an HLA-restricted manner but did not lyse autologous or HLA-matched normal CD34+ hematopoietic progenitor cells or reduce their yield of colony-forming unit-granulocyte-macrophage (CFU-GM), burst-forming unit erythroid (BFU-E), or mixed colonies (CFU-mix). Furthermore, WT1 peptide-specific T cells after adoptive transfer into nonobese diabetic-severe combined immunodeficient mice bearing subcutaneous xenografts of WT1+ and WT1− HLA-A0201+ leukemias preferentially accumulated in and induced regressions of WT1+ leukemias that expressed the restricting HLA allele. Such cells are clinically applicable and may prove useful for adoptive cell therapy of WT1+ malignant diseases in humans.
Therapeutic Synergy of Oral Taxane BMS-275183 and Cetuximab
versus
Human Tumor Xenografts
Authors: Rose William C. , Wild Robert
Published: September 2005

Purpose: Combination therapy consisting of an oral taxane, BMS-275183, and the anti-epidermal growth factor receptor monoclonal antibody, cetuximab, was assessed for enhanced therapeutic benefit in preclinical tumor models.

Experimental Design: Mice bearing human tumor xenografts, either L2987 lung or GEO colon carcinoma, were administered the aforementioned treatments singly or in combination regimens. Delays in tumor growth and tumor-free status were evaluated and combination treatments were assessed relative to optimal solo treatments.

Results: Combination therapies with the oral taxane plus cetuximab were tolerated and therapeutic synergistic outcomes obtained. The therapeutic enhancements were >1 log cell kill greater than the antitumor effect caused by either solo agent applied optimally. For example, at the maximum-tolerated dose of BMS-275183, 60 mg/kg/administration, given p.o. once every 3 days for a total of six administrations (q3d×6), 1.0 gross log cell kill was achieved in mice bearing well established (100 to 200 mg) s.c. implanted L2987 tumors. Cetuximab, at an optimal dose of 1 mg/mouse, given i.p. q3d×6, produced 1.3 log cell kill. When cetuximab, 1 mg/mouse, i.p., plus BMS-275183, 25 mg/kg/administration, p.o., were both given q3d×6, the result was 2.6 log cell kill with three of eight mice cured (P < 0.01). Similar efficacy benefits were obtained in the GEO tumor model.

Conclusions: The combination of oral taxane BMS-275183 plus cetuximab provided therapeutically synergistic antitumor activity in two different human tumor xenograft models. Clinical evaluation of this combination is recommended.
Methylenetetrahydrofolate Reductase Genotype Affects Risk of Relapse after Hematopoietic Cell Transplantation for Chronic Myelogenous Leukemia
Authors: Robien Kim , Ulrich Cornelia M. , Bigler Jeannette , Yasui Yutaka , Gooley Ted , Bruemmer Barbara , Potter John D. , Radich Jerald P.
Published: September 2005

Purpose: Methylenetetrahydrofolate reductase (MTHFR) directs intracellular folate toward homocysteine metabolism and away from nucleotide synthesis. Two common MTHFR polymorphisms, C677T and A1298C, are associated with reduced enzyme activity. We evaluated the association of these polymorphisms with risk of relapse and bcr-abl mRNA transcript detection among 336 Caucasian patients who underwent allogeneic hematopoietic cell transplantation for chronic myelogenous leukemia.

Experimental Design: Data on the transplant course and folate-related exposures were abstracted from medical records. MTHFR C677T and A1298C genotypes were determined using polymerase chain reaction/restriction fragment length polymorphism and TaqMan assays. Qualitative bcr-abl mRNA testing was conducted using a two-step reverse transcription-polymerase chain reaction assay. Cox regression analysis was used to assess the association between MTHFR genotypes and time to relapse and bcr-abl mRNA detection.

Results: A statistically significant decreased risk of relapse was observed in patients with the variant A1298C genotype [1298AC, hazard ratio (HR) = 0.48 and 95% confidence interval (CI) = 0.26–0.88; 1298CC, HR = 0.28 and 95% CI = 0.09–0.84; P-trend < 0.01). For the joint C677T/A1298C genotype, variant genotypes were associated with a decreased risk of relapse when compared with the wild-type 677CC/1298AA genotype. This risk was lowest for the 677CC/1298CC genotype (HR, 0.23; 95% CI, 0.08–0.72). MTHFR genotypes were not associated with bcr-abl transcript detection.

Conclusions: These findings suggest that individuals with the 677CC/1298AA genotype are at higher risk of relapse after hematopoietic cell transplantation and that the balance of intracellular folate metabolites available for nucleotide synthesis (regulated by the relative activity of the MTHFR enzyme) may affect the progression from bcr-abl positivity to clinical relapse.
Expression of Matrix Metalloproteinase (MMP)-2 and MMP-9 in Breast Cancer with a Special Reference to Activator Protein-2, HER2, and Prognosis
Authors: Pellikainen Johanna M. , Ropponen Kirsi M. , Kataja Vesa V. , Kellokoski Jari K. , Eskelinen Matti J. , Kosma Veli-Matti
Published: September 2005

Purpose: In the present study, we investigated the expression and prognostic value of matrix metalloproteinase (MMP)-2 and MMP-9 in breast cancer as well as their relation to transcription factor activator protein (AP)-2 and HER2 oncogene. The role of invasion and metastasis-promoting MMPs and their potential regulators, AP-2 and HER2, is currently still unclear in breast cancer.

Experimental Design: MMP-2 and MMP-9 expressions were analyzed immunohistochemically in a large prospective series of 421 breast cancer patients diagnosed and treated between 1990 and 1995 at Kuopio University Hospital (Kuopio, Finland). The relation of MMP-2 and MMP-9 expressions to AP-2, HER2, clinicopathological data, and survival was investigated.

Results: Both MMP-2 and MMP-9 were expressed in the cytoplasm of malignant and stromal cells. High expression of MMPs in carcinoma cells was related to small tumors (T1, stage I), whereas positive stromal expression of MMPs was associated with aggressive factors. High expression of MMP-2 and MMP-9 in carcinoma cells, but not in stromal cells, was related to high AP-2 expression. Positive stromal MMP-2 expression was associated with HER2 overexpression in the whole patient group and in the node-negative patient subgroup. Positive stromal MMP-9 expression was related to HER2 overexpression in estrogen receptor (ER)-positive disease. In the univariate survival analysis, positive stromal MMP-9 predicted shorter recurrence-free survival (RFS; P = 0.0389) and breast cancer-related survival (BCRS; P = 0.0081) in ER+ disease, especially in the subgroup of ER+ tumors of ≤2 cm in diameter (T1; P = 0.0031 for RFS, and P = 0.0089 for BCRS). High MMP-9 expression in cancer cells predicted longer RFS (P = 0.0351) in the whole patient group. In the multivariate analysis of the whole patient group, the independent predictors of shorter RFS were reduced MMP-9 expression in carcinoma cells (P = 0.0248), HER2 overexpression (P = 0.0001), and advanced-stage disease (P = 0.0002). Shorter BCRS was predicted by advanced-stage disease (P < 0.0001).

Conclusions: Expression of MMP-2 and MMP-9 in breast cancer seems to be partly related to expression of AP-2 and HER2. Positive stromal MMP-9 expression predicts poor survival in the hormone-responsive small tumors, whereas MMP-9 expression in carcinoma cells favors survival. Evaluation of MMP-9 expression seems to add valuable information on breast cancer prognosis.
Small Integrin Binding Ligand
N
-Linked Glycoprotein Gene Family Expression in Different Cancers
Authors: Fisher Larry W. , Jain Alka , Tayback Matt , Fedarko Neal S.
Published: September 2005

Purpose: Members of the small integrin binding ligand N-linked glycoprotein (SIBLING) gene family have the capacity to bind and modulate the activity of matrix metalloproteinases (MMPs). The expression levels of five SIBLING gene family members [bone sialoprotein (BSP), osteopontin (OPN), dentin matrix protein 1 (DMP1), matrix extracellular phosphoglycoprotein (MEPE), and dentin sialophosphoprotein (DSPP)] and certain MMPs were determined using a commercial cancer array.

Experimental Design: Cancer profiling arrays containing normalized cDNA from both tumor and corresponding normal tissues from 241 individual patients were used to screen for SIBLING and MMP expression in nine distinct cancer types.

Results: Significantly elevated expression levels were observed for BSP in cancer of the breast, colon, stomach, rectum, thyroid, and kidney; OPN in cancer of the breast, uterus, colon, ovary, lung, rectum, and thyroid; DMP1 in cancer of the breast, uterus, colon, and lung; and dentin sialophosphoprotein in breast and lung cancer. The degree of correlation between a SIBLING and its partner MMP was found to be significant within a given cancer type (e.g., BSP and MMP-2 in colon cancer, OPN and MMP-3 in ovarian cancer; DMP1 and MMP-9 in lung cancer). The expression levels of SIBLINGs were distinct within subtypes of cancer (e.g., breast ductal tumors compared with lobular tumors). In general, SIBLING expression increased with cancer stage for breast, colon, lung, and rectal cancer.

Conclusions: These results suggest SIBLINGs as potential markers of early disease progression in a number of different cancer types, some of which currently lack vigorous clinical markers.
Hypoxia Is Important in the Biology and Aggression of Human Glial Brain Tumors
Authors: Evans Sydney M. , Judy Kevin D. , Dunphy Isolde , Jenkins W. Timothy , Hwang Wei-Ting , Nelson Peter T. , Lustig Robert A. , Jenkins Kevin , Magarelli Deirdre P. , Hahn Stephen M. , Collins Ruth A. , Grady M. Sean , Koch Cameron J.
Published: September 2005

We investigated whether increasing levels of tissue hypoxia, measured by the binding of EF5 [2-(2-nitro-1-H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide] or by Eppendorf needle electrodes, were associated with tumor aggressiveness in patients with previously untreated glial brain tumors. We hypothesized that more extensive and severe hypoxia would be present in tumor cells from patients bearing more clinically aggressive tumors. Hypoxia was measured with the 2-nitroimidazole imaging agent EF5 in 18 patients with supratentorial glial neoplasms. In 12 patients, needle electrode measurements were made intraoperatively. Time to recurrence was used as an indicator of tumor aggression and was analyzed as a function of EF5 binding, electrode values and recursive partitioning analysis (RPA) classification. On the basis of EF5 binding, WHO grade 2 tumors were characterized by modest cellular hypoxia (pO2s ≈ 10%) and grade 3 tumors by modest-to-moderate hypoxia (pO2s ≈ 10%- 2.5%). Severe hypoxia (≈0.1% oxygen) was present in 5 of 12 grade 4 tumors. A correlation between more rapid tumor recurrence and hypoxia was demonstrated with EF5 binding, but this relationship was not predicted by Eppendorf measurements.
Preparation and Biological Evaluation of Copper-64–Labeled Tyr3-Octreotate Using a Cross-Bridged Macrocyclic Chelator
Authors: Sprague Jennifer E. , Peng Yijie , Sun Xiankai , Weisman Gary R. , Wong Edward H. , Achilefu Samuel , Anderson Carolyn J.
Published: September 2005

Purpose: Somatostatin receptors (SSTr) are expressed on many neuroendocrine tumors, and several radiotracers have been developed for imaging these types of tumors. For this reason, peptide analogues of somatostatin have been well characterized. Copper-64 (t1/2 = 12.7 hours), a positron emitter suitable for positron emission tomography (PET) imaging, was shown recently to have improved in vivo clearance properties when chelated by the cross-bridged tetraazamacrocycle 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo(6.6.2)hexadecane (CB-TE2A) compared with 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA).

Experimental Design: CB-TE2A and TETA were conjugated to the somatostatin analogue tyrosine-3-octreotate (Y3-TATE) for evaluation of CB-TE2A as a bifunctional chelator of 64Cu. The in vitro affinity of each compound for SSTr was determined using a homologous competitive binding assay. In vivo characteristics of both radiolabeled compounds were examined in biodistribution and microPET studies of AR42J tumor-bearing rats.

Results: Cu-CB-TE2A-Y3-TATE (Kd = 1.7 nmol/L) and Cu-TETA-Y3-TATE (Kd = 0.7 nmol/L) showed similar affinities for AR42J derived SSTr. In biodistribution studies, nonspecific uptake in blood and liver was lower for 64Cu-CB-TE2A-Y3-TATE. Differences increased with time such that, at 4 hours, blood uptake was 4.3-fold higher and liver uptake was 2.4-fold higher for 64Cu-TETA-Y3-TATE than for 64Cu-CB-TE2A-Y3-TATE. In addition, 4.4-times greater tumor uptake was detected with 64Cu-CB-TE2A-Y3-TATE than with 64Cu-TETA-Y3-TATE at 4 hours postinjection. MicroPET imaging yielded similar results.

Conclusions: CB-TE2A appears to be a superior in vivo bifunctional chelator of 64Cu for use in molecular imaging by PET or targeted radiotherapy due to both improved nontarget organ clearance and higher target organ uptake of 64Cu-CB-TE2A-Y3-TATE compared with 64Cu-TETA-Y3-TATE.
Results of a Multicenter Randomized Phase II Trial of Thalidomide and Prednisone Maintenance Therapy for Multiple Myeloma after Autologous Stem Cell Transplant
Authors: Stewart A. Keith , Chen Christine I. , Howson-Jan Kang , White Darrell , Roy Jean , Kovacs Michael J. , Shustik Chaim , Sadura Anna , Shepherd Lois , Ding Keyue , Meyer Ralph M. , Belch Andrew R.
Published: September 2005

We report a multicenter, randomized phase II trial conducted to assess the tolerability of combined thalidomide and prednisone maintenance in multiple myeloma. Eligibility required administration of melphalan (200 mg/m2) with blood stem cell support within 1 year of treatment onset and initiation of maintenance within 60 to 100 days after stem cell infusion. All patients received 50 mg of prednisone by mouth on alternate days and thalidomide at a starting dose of either 200 or 400 mg daily by mouth. The primary end point was the incidence of dropout or dose reduction due to treatment toxicity within 6 months. Sixty-seven patients were enrolled. Median follow-up is 36.8 months. The primary end point was reached by 31% of patients on the 200 mg of thalidomide arm and 64% of patients on the 400 mg of thalidomide arm. Allowing for dose reduction, 76% of patients assigned to the 200 mg of thalidomide arm and 41% of patients assigned to the 400 mg of thalidomide arm remained on any maintenance therapy 18 months after registration. Eighty-eight percent of all patients dose-reduced thalidomide and 72% of all patients dose-reduced prednisone within 2 years of beginning maintenance. The median progression-free survival post-transplant is 32.3 months, or 42.2 months from diagnosis. Only the 200 mg of thalidomide arm of this trial met our definition of a tolerable maintenance therapy, defined as no dose reductions or discontinuation due to toxicity in at least 65% of patients for a minimum of 6 months, thus establishing a dosing schedule for phase III trials.
Prognostic Significance of Tissue Transglutaminase in Drug Resistant and Metastatic Breast Cancer
Authors: Mehta Kapil , Fok Jansina , Miller Fred R. , Koul Dimpy , Sahin Aysegul A.
Published: September 2005

Purpose: Drug resistance and metastasis pose major impediments in the successful treatment of cancer. We previously reported that multidrug-resistant breast cancer cells exhibit high levels of tissue transglutaminase (TG2; EC 2.3.2.13). Because the drug-resistant and metastatic phenotypes are thought to share some common pathways, we sought to determine whether metastatic breast cancer cells express high levels of TG2.

Experimental Design: The metastatic breast cancer cell line MDA-MB-231 and the sublines derived from it were tested for TG2 expression. Similarly, several sublines derived from an immortal but normal breast epithelial cell line, MCF10A, representing various stages in breast cancer progression were studied for TG2 expression. The primary and nodal tumor samples from 30 patients with breast cancer were also studied for TG2 expression.

Results: The MDA-MB-231 cells expressed high basal levels of TG2. Two clones derived from this cell line, MDA231/cl.9 and MDA231/cl.16, showed a 10- to 15-fold difference in TG2 level. TG2-deficient MDA231/cl.9 cells exhibited higher sensitivity to doxorubicin and were less invasive than were the TG2-sufficient MDA231/cl.16 cells. The MCF10A-derived sublines had increased TG2 expression as they advanced from noninvasive to an invasive phenotype. Importantly, the metastatic lymph node tumors from patients with breast cancer showed significant higher levels of TG2 expression compared with the primary tumors from the same patients.

Conclusions: TG2 expression is up-regulated in drug-resistant and metastatic breast cancer cells, and it can serve as a valuable prognostic marker for these phenotypes.
Fas Ligand Expression in Metastatic Renal Cell Carcinoma During Interleukin-2 Based Immunotherapy
Authors: Donskov Frede , von der Maase Hans , Marcussen Niels , Hamilton-Dutoit Stephen , Madsen Hans Henrik Torp , Jensen Jens Jorgen , Hokland Marianne
Published: September 2005

Purpose: It has been hypothesized that tumor cells expressing Fas ligand (FasL) might be able to counterattack and neutralize tumor-infiltrating lymphocytes. We assessed the effect of FasL tumor counterattack on the clinical outcome of interleukin-2 (IL-2)–based immunotherapy in metastatic renal cell carcinoma.

Experimental Design: Tumor core needle biopsies were obtained before IL-2–based immunotherapy in 86 patients and repeated within the first cycle in 57 patients. Tumor cells expressing FasL and intratumoral lymphocyte subsets expressing CD4, CD8, CD56, and CD57 were analyzed by immunohistochemistry.

Results: At baseline, negative FasL staining in tumor cells was seen in 10 of 86 (12%) biopsies, whereas intense FasL staining was seen (a) in fewer than 10% of tumor cells in 26 (30%) biopsies; (b) in 11 to 50% of tumor cells in 25 (29%) biopsies; (c) in 51 to 90% of tumor cells in 18 (21%) biopsies; and (d) in >90% of tumor cells in 7 (8%) biopsies. On treatment, tumor FasL expression did not change from baseline levels. Moreover, tumor FasL expression was not correlated with objective response or survival whereas the absolute number of CD4+, CD8+, CD56+, and CD57+ cells per mm2 tumor tissue at baseline was significantly higher in responding patients compared with nonresponding patients (P = 0.01, P = 0.008, P = 0.015, and P < 0.001, respectively). During the first course of immunotherapy, the absolute number of CD4+, CD8+, and CD57+ cells per mm2 tumor tissue was significantly higher in responding patients compared with nonresponding patients (P = 0.034, P < 0.001, and P < 0.001, respectively). However, no correlation was observed between the number of intratumoral lymphocytes and tumor FasL expression level.

Conclusion: These observations do not support the hypothesis that FasL tumor “counterattack” has an effect on the clinical outcome in metastatic renal cell carcinoma during IL-2–based immunotherapy.
Potent and Specific Antitumor Efficacy of CMC-544, a CD22-Targeted Immunoconjugate of Calicheamicin, against Systemically Disseminated B-Cell Lymphoma
Authors: DiJoseph John F. , Goad Mary E. , Dougher Maureen M. , Boghaert Erwin R. , Kunz Arthur , Hamann Philip R. , Damle Nitin K.
Published: September 2005

Purpose: CMC-544 is a CD22-targeted immunoconjugate of calicheamicin and exerts a potent cytotoxic effect against CD22+ B-cell lymphoma. This study evaluated antitumor efficacy of CMC-544 against systemically disseminated B-cell lymphoma.

Experimental Design: Scid mice received i.v. injections of CD22+ Ramos B-cell lymphoma cells for their systemic dissemination. CMC-544, G5/44, CD33-targeted CMA-676 (control conjugate) or rituximab were given i.p. 3, 9, 15, or 21 days after B-cell lymphoma dissemination. Diseased mice were monitored daily for hind-limb paralysis and death. Histopathological examination of CMC-544-treated and vehicle-treated diseased mice was also performed.

Results: Mice with disseminated B-cell lymphoma developed hind-limb paralysis within 35 days. When given up to 15 days after B-cell lymphoma dissemination, CMC-544 extended survival of the diseased mice to >100 days, and these mice were considered cured. CMC-544 was efficacious when given during both the early initiation phase and the late established phase of the disease. A single dose of CMC-544 was effective in delaying the occurrence of hind-limb paralysis. In contrast, neither CMA-676 nor unconjugated G5/44 was effective. Rituximab was effective when given early in the disease process but not when the disease was established. Histopathological analysis revealed B-cell lymphoma infiltration in brain, spinal cord, bone marrow, and kidney in vehicle-treated but not in CMC-544–treated diseased mice. Consistent with its efficacy against the disseminated B-cell lymphoma, CMC-544 also caused regression of established Ramos B-cell lymphoma xenografts in scid mice.

Conclusions: CMC-544 confers strong therapeutic activity against systemic disseminated B-cell lymphoma and protects mice from hind-limb paralysis and death. These results support clinical evaluation of CMC-544 in the treatment of CD22+ lymphoid malignancies.
Hypermethylation of the GATA Genes in Lung Cancer
Authors: Guo Mingzhou , Akiyama Yoshimitsu , House Michael G. , Hooker Craig M. , Heath Elizabeth , Gabrielson Edward , Yang Stephen C. , Han Yu , Baylin Stephen B. , Herman James G. , Brock Malcolm V.
Published: September 2005

Purpose: In lung cancer, DNA hypermethylation is known to be a common event.

Experimental Design: Gene expression and methylation status of GATA-4, GATA-5, and GATA-6 were analyzed with cell lines and primary human lung cancers. Methylation profiles of primary lung cancers were analyzed and correlated with clinical as well as histopathological data.

Results: Complete methylation was detected by methylation-specific PCR for both GATA-4 and GATA-5 in four cell lines (H358, DMS-53, A549, and H1299), all of which had no expression by reverse transcription-PCR analysis. Demethylation with 5-aza-2′deoxycytidine restored expression in each case. GATA-6 was ubiquitously expressed in all of the six cell lines. GATA-4 bisulfite sequencing revealed complete methylation of the GATA-4 promoter in H358 cells, correlating well with its lack of expression at the mRNA level. Only a few CpG sites showed methylation by bisulfite sequencing within the GATA-4 promoter in a cell line that expressed the gene. In 63 cases of primary lung cancers, GATA-4 and GATA-5 promoter methylation was detected in (42 of 63) 67% and (26 of 63) 41%, respectively. GATA-6 remained unmethylated both in cell lines and primary tumors. Six autopsy specimens of normal lung tissue showed no aberrant promoter hypermethylation for the GATA genes. Correlation of concomitant GATA-4 and GATA-5 methylation with clinicopathological parameters only found a statistically significant increase in methylation frequency with increasing patient age (P < 0.001).

Conclusions: These epigenetic changes in the GATA genes in lung cancer are tumor-specific, relate to the loss of GATA gene expression, and occur increasingly in the elderly.
Differential Radiosensitization in DNA Mismatch Repair-Proficient and -Deficient Human Colon Cancer Xenografts with 5-Iodo-2-pyrimidinone-2′-deoxyribose
Authors: Seo Yuji , Yan Tao , Schupp Jane E. , Colussi Valdir , Taylor Kerri L. , Kinsella Timothy J.
Published: September 2005

Purpose: 5-Iodo-2-pyrimidinone-2′-deoxyribose (IPdR) is a pyrimidinone nucleoside prodrug of 5-iododeoxyuridine (IUdR) under investigation as an orally administered radiosensitizer. We previously reported that the mismatch repair (MMR) proteins (both hMSH2 and hMLH1) impact on the extent (percentage) of IUdR-DNA incorporation and subsequent in vitro IUdR-mediated radiosensitization in human tumor cell lines. In this study, we used oral IPdR to assess in vivo radiosensitization in MMR-proficient (MMR+) and -deficient (MMR−) human colon cancer xenografts.

Experimental Design: We tested whether oral IPdR treatment (1 g/kg/d for 14 days) can result in differential IUdR incorporation in tumor cell DNA and subsequent radiosensitization after a short course (every day for 4 days) of fractionated radiation therapy, by using athymic nude mice with an isogenic pair of human colon cancer xenografts, HCT116 (MMR−, hMLH1−) and HCT116/3-6 (MMR+, hMLH1+). A tumor regrowth assay was used to assess radiosensitization. Systemic toxicity was assessed by daily body weights and by percentage of IUdR-DNA incorporation in normal bone marrow and intestine.

Results: After a 14-day once-daily IPdR treatment by gastric gavage, significantly higher IUdR-DNA incorporation was found in HCT116 (MMR−) tumor xenografts compared with HCT116/3-6 (MMR+) tumor xenografts. Using a tumor regrowth assay after the 14-day drug treatment and a 4-day radiation therapy course (days 11–14 of IPdR), we found substantial radiosensitization in both HCT116 and HCT116/3-6 tumor xenografts. However, the sensitizer enhancement ratio (SER) was substantially higher in HCT116 (MMR−) tumor xenografts (1.48 at 2 Gy per fraction, 1.41 at 4 Gy per fraction), compared with HCT116/3-6 (MMR+) tumor xenografts (1.21 at 2 Gy per fraction, 1.20 at 4 Gy per fraction). No substantial systemic toxicity was found in the treatment groups.

Conclusions: These results suggest that IPdR-mediated radiosensitization can be an effective in vivo approach to treat “drug-resistant” MMR-deficient tumors as well as MMR-proficient tumors.
ZD6474, a Potent Inhibitor of Vascular Endothelial Growth Factor Signaling, Combined With Radiotherapy
Authors: Williams Kaye J. , Telfer Brian A. , Brave Sandra , Kendrew Jane , Whittaker Lynsey , Stratford Ian J. , Wedge Stephen R.
Published: September 2005

Purpose: Vascular endothelial growth factor (VEGF) plays a key role in tumor angiogenesis and acts as a radiation survival factor for endothelial cells. ZD6474 (N-(4-bromo-2-fluorophenyl)-6-methoxy-7-[(1-methylpiperidin-4-yl)methoxy]quinazolin-4-amine) is a potent VEGF receptor 2 (KDR) tyrosine kinase inhibitor (TKI) that has additional activity versus the epidermal growth factor receptor. This study was designed to determine the efficacy of combining ZD6474 and radiotherapy in vivo.

Experimental Design: The Calu-6 (non–small-cell lung cancer) tumor model was selected because it was found to be unresponsive to treatment with a selective epidermal growth factor receptor TKI but responds significantly to treatment with selective VEGF receptor TKIs. Tumor-bearing mice received either vehicle or ZD6474 (50 mg/kg, by mouth, once daily) for the duration of the experiment, with or without radiotherapy (3 × 2 Gy, days 1–3). Two combination schedules were examined: (a) ZD6474 given before each dose of radiation (concurrent schedule); and (b) ZD6474 given 30 minutes after the last dose of radiotherapy (sequential schedule).

Results: The growth delay induced using the concurrent schedule was greater than that induced by ZD6474 or radiation treatment alone (22 ± 1 versus 9 ± 1 and 17 ± 2 days, respectively; P = 0.03 versus radiation alone). When administered sequentially, the growth delay was markedly enhanced (36 ± 1 days; P < 0.001 versus radiation alone or the concurrent schedule). Intravenous administration of Hoechst 33342 showed a trend toward reduced tumor perfusion after ZD6474 treatment, and a pairwise comparison (versus control) was significant after three doses of ZD6474 (P = 0.05 by one-tailed t test). Thus, impaired reoxygenation between fractions in the concurrent protocol may be the causal basis for the schedule dependency of the radiopotentiation observed.

Conclusions: ZD6474 may be a successful adjuvant to clinical radiotherapy, and scheduling of the treatments could be important to ensure optimal efficacy.
Administration of CI-1033, an Irreversible Pan-erbB Tyrosine Kinase Inhibitor, Is Feasible on a 7-Day On, 7-Day Off Schedule
Authors: Calvo Emiliano , Tolcher Anthony W. , Hammond Lisa A. , Patnaik Amita , de Bono Johan S. , Eiseman Irene A. , Olson Stephen C. , Lenehan Peter F. , McCreery Heather , LoRusso Patricia , Rowinsky Eric K.
Published: September 2005

Purpose: To determine the maximum tolerated dose of administrating CI-1033, an oral 4-anilinoquinazoline that irreversibly inhibits the tyrosine kinase domain of all erbB subfamilies, on an intermittent schedule, and assess the interaction of CI-1033 with food on the pharmacokinetic behavior.

Experimental Design: Escalating doses of CI-1033 from a dose level of 300 mg/day for 7 days every other week were administered to patients with advanced solid malignancies. Plasma concentration-time data sets from all evaluable patients were used to develop a population pharmacokinetic model. Noncompartmental methods were used to independently assess the effect of a high-fat meal on CI-1033 absorption and bioavailability.

Results: Twenty-four patients were treated with 69 twenty-eight day courses. The incidence of unacceptable toxicity, principally diarrhea and skin rash, was observed at the 300 mg/day dose level. At the 250 mg/day level, toxicity was manageable, and protracted administration was feasible. A one-compartment linear model with first-order absorption and elimination adequately described the pharmacokinetic disposition. CL/F, apparent volume of distribution (Vd/F), and ka (mean ± relative SD) were 280 L/hour ± 33%, 684 L ± 20%, and 0.35 hour−1± 69%, respectively. Cmax values were achieved in 2 to 4 hours. Systemic CI-1033 exposure was largely unaffected by administration of a high-fat meal. At 250 mg, concentration values exceeded IC50 values required for prolonged pan-erbB tyrosine kinase inhibition in preclinical assays.

Conclusions: The recommended dose on this schedule is 250 mg/day. Its tolerability and the biological relevance of concentrations achieved at the maximal tolerated dose warrant consideration of disease-directed evaluations. This intermittent treatment schedule can be used without regard to meals.
Inhibition of Cyclooxygenase (COX)-2 Expression by Tet-Inducible COX-2 Antisense cDNA in Hormone-Refractory Prostate Cancer Significantly Slows Tumor Growth and Improves Efficacy of Chemotherapeutic Drugs
Authors: Dandekar Devendra S. , Lokeshwar Bal L.
Published: September 2005

Purpose: Overexpression of the proinflammatory enzyme cyclooxygenase (COX)-2 is associated with the progression of various malignancies; the role of COX-2 in prostate cancer is less clear. The significance of COX-2 in prostate cancer growth and response to chemotherapy was investigated in an androgen-refractory prostate cancer cell line using a Tet-inducible antisense COX-2 expression system.

Experimental Design: An antisense COX-2 cDNA construct under the control of a doxycycline-inducible promoter was transfected into a prostate cancer cell line, PC-3ML. Modulations of cell growth, apoptosis, and chemosensitivity in the presence or absence of doxycycline were analyzed. Tumor incidence, growth rate, and response to two cytotoxic drugs, COL-3 [chemically modified tetracycline-3-(6-demethyl-6-deoxy-4-dedimethylamino-tetracycline)] and Taxotere (docetaxel), were investigated in tumor xenografts. Apoptotic incidences and tumor microvessel density in tumors were determined by immunohistochemistry.

Results: Conditional suppression of COX-2 in PC-3ML caused reduced cell proliferation, decreased levels of phosphorylated AKT, G0-G1 arrest, and increased apoptosis and caspase-3 activity. Suppression of COX-2 increased Bax protein and decreased Bcl-xL protein in vitro. COX-2 antisense-expressing PC-3ML tumors showed a 57% growth delay compared with nontransfected or vector controls. Oral administration of COL-3 (40 mg/kg, oral gavage) or Taxotere (2.3 mg/kg, intraperitoneally; 3× per week) in tumor-bearing mice further slowed tumor growth (65% and ∼94%, respectively). Compared with the control group, the occurrence of apoptosis in antisense COX-2 tumors was eight times higher, and the tumor microvessel density was three times lower.

Conclusions: These results provide direct evidence that constitutive expression of COX-2 in prostate cancer has both angiogenic and cytoprotective functions. Suppression of tumor cell COX-2 is sufficient to enhance chemotherapy response in prostate cancer.
Enhanced Tumor Cell Radiosensitivity and Abrogation of G2 and S Phase Arrest by the Hsp90 Inhibitor 17-(Dimethylaminoethylamino)-17-demethoxygeldanamycin
Authors: Bull Elizabeth E. A. , Dote Hideaki , Brady Kristin J. , Burgan William E. , Carter Donna J. , Cerra Michael A. , Oswald Kelli A. , Hollingshead Melinda G. , Camphausen Kevin , Tofilon Philip J.
Published: September 2005

Purpose: Because of the potential for affecting multiple signaling pathways, inhibition of Hsp90 may provide a strategy for enhancing tumor cell radiosensitivity. Therefore, we have investigated the effects of the orally bioavailable Hsp90 inhibitor 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) on the radiosensitivity of human tumor cells in vitro and grown as tumor xenografts.

Experimental Design: The effect of 17-DMAG on the levels of three proteins (Raf-1, ErbB2, and Akt) previously implicated in the regulation of radiosensitivity was determined in three human solid tumor cell lines. A clonogenic assay was then used to evaluate cell survival after exposure to 17-DMAG followed by irradiation. For mechanistic insight, the G2- and S-phase checkpoints were evaluated in 17-DMAG–treated cells. Finally, the effect of in vivo administration of 17-DMAG in combination with radiation on the growth rate of xenograft tumors was determined.

Results: 17-DMAG exposure reduced the levels of the three radiosensitivity-associated proteins in a cell line-specific manner with ErbB2 being the most susceptible. Corresponding concentrations of 17-DMAG enhanced the radiosensitivity of each of the tumor cell lines. This sensitization seemed to be the result of a 17-DMAG–mediated abrogation of the G2- and S-phase cell cycle checkpoints. The oral administration of 17-DMAG to mice bearing tumor xenografts followed by irradiation resulted in a greater than additive increase in tumor growth delay.

Conclusions: These data indicate that 17-DMAG enhances the in vitro and in vivo radiosensitivity of human tumor cells. The mechanism responsible seems to involve the abrogation of radiation-induced G2- and S-phase arrest.
Therapeutic Efficacy of DTI-015 using Diffusion Magnetic Resonance Imaging as an Early Surrogate Marker
Authors: Hall Daniel E. , Moffat Bradford A. , Stojanovska Jadranka , Johnson Timothy D. , Li Zhuolin , Hamstra Daniel A. , Rehemtulla Alnawaz , Chenevert Thomas L. , Carter Julie , Pietronigro Dennis , Ross Brian D.
Published: September 2005

To investigate diffusion weighted magnetic resonance imaging as a quantitative surrogate marker for evaluating the therapy-induced cellular changes in an orthotopic experimental glioma model, tumors were treated with direct intratumoral administration of DTI-015, a solution of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in 100% EtOH. Intracerebral 9L tumors were induced in Fischer 344 rats, and three treatment groups were established: DTI-015, EtOH, and sham. Two groups of rats received intratumoral injection of either 67 mg/mL BCNU in EtOH or EtOH alone at 50% of the tumor volume up to a maximum of 30 μl under stereotactic guidance. Diffusion magnetic resonance images were acquired before treatment and after treatment at 1, 24, 48, and 72 hours and then 3 times per week thereafter. Tumor cell viability was examined using multislice diffusion weighted magnetic resonance imaging with diffusion weighted transverse magnetic resonance images and histogram plots of each tumor quantified over time. Control animals (EtOH- or sham-treated animals) showed mean apparent diffusion coefficients (ADCs) that remained essentially unchanged over the experimental time course. In contrast, rats treated with DTI-015 showed a significant increase in ADC relative to the pretreatment within 24 hours, which further increased over time, followed by a significant therapeutic response as evidenced by subsequent tumor volume shrinkage, development of a cystic region, and enhanced animal survival. Finally, not only were ADC measurements predictive of differences between treatment groups, but they also yielded spatial and temporal data regarding the efficacy of treatment within individual treated animals that could be used to guide subsequent therapy.
Targeting of HER2 Antigen for the Treatment of Disseminated Peritoneal Disease
Authors: Milenic Diane E. , Garmestani Kayhan , Brady Erik D. , Albert Paul S. , Ma Dangshe , Abdulla Alia , Brechbiel Martin W.
Published: September 2005

The studies reported herein demonstrate the efficacy of α-particle–targeted radiation therapy of peritoneal disease with Herceptin as the targeting vehicle. Using the CHX-A-DTPA linker, Herceptin was radiolabeled with indium-111 and bismuth-213 with high efficiency without compromising immunoreactivity. A pilot radioimmunotherapy study treating mice bearing 5-day LS-174T (i.p.) xenografts, a low but uniform HER2 expressing, human colon carcinoma, with a single dose of 213Bi-CHX-A”-Herceptin, proved disappointing. This defined the effect of tumor burden/size on tumor response to radioimmunotherapy with α-radiation. A more successful experiment with a lower tumor burden (3 days) in mice followed. A specific dose-response (P = 0.009) was observed, and although a maximum-tolerated dose was not determined, a dose of 500 to 750 μCi was selected as the operating dose for future experiments based on changes in animal weight. Median survival was increased from 20.5 days for the mock-treated mice to 43 and 59 days with 500 and 750 μCi, respectively. The therapeutic effectiveness of 213Bi-CHX-A”-Herceptin was also evaluated in a second animal model for peritoneal disease with a human pancreatic carcinoma (Shaw). The results of this study were not as dramatic as with the former model, and higher doses were required to obtain an increase in survival of the mice (P = 0.001).
Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Does Not Improve Paclitaxel Effect in an Orthotopic Mouse Model of Lung Cancer
Authors: Onn Amir , Isobe Takeshi , Wu Wenjuan , Itasaka Satoshi , Shintani Tomoaki , Shibuya Keiko , Kenji Yokoi , O’Reilly Michael S. , Fidler Isaiah J. , Herbst Roy S.
Published: September 2005

Purpose: The purpose is to evaluate whether inhibition of epidermal growth factor receptor (EGFR) activation by PKI166, an EGFR-tyrosine kinase inhibitor, affects growth of human lung cancer implanted orthotopically into the lungs of nude mice.

Experimental Design: Lungs of mice were injected with NCI-H358 human bronchioloalveolar cancer cells. In three experiments, groups of mice (n = 10 per group) were randomized 7 days after tumor implantation to receive one of the following treatments: i.p. paclitaxel 100 or 200 μg (4 or 8 mg/kg) once per week, oral PKI166 100 or 200 mg/kg three times per week, paclitaxel plus PKI166, or i.p. saline and oral PKI166-vehicle (control) for 5 weeks. Mice were killed 6.5 to 8 weeks after tumor implantation. The experiments were repeated with PC14PE6 human lung adenocarcinoma cells to assess effect on survival.

Results: Immunohistochemical analyses revealed the expression and phosphorylation of EGFR in the growing tumors. Treatment with PKI166 alone or in combination with paclitaxel diminished activation of EGFR on tumor cells, yet maximal therapeutic effect was observed in mice treated with paclitaxel alone. Activated mitogen-activated protein kinase and basic fibroblast growth factor expression were similar in all treatment groups. Survival in mice treated with the combination of paclitaxel and PKI166 was shorter than in those treated with paclitaxel alone.

Conclusions: Our results suggest that concurrent administration of EGFR-tyrosine kinase inhibitor and chemotherapy is equivalent and may indeed be inferior to chemotherapy alone, even if EGFR is functional and its phosphorylation effectively inhibited. Our data show that the interaction of EGFR-TKIs and chemotherapy is complex and suggest that other growth factors may activate the downstream signaling events.
High Frequency of Epidermal Growth Factor Receptor Mutations with Complex Patterns in Non–Small Cell Lung Cancers Related to Gefitinib Responsiveness in Taiwan
Authors: Huang Shiu-Feng , Liu Hui-Ping , Li Ling-Hui , Ku Yuan-Chieh , Fu Yu-Ning , Tsai Hsien-Yu , Chen Ya-Ting , Lin Yung-Feng , Chang Wen-Cheng , Kuo Han-Pin , Wu Yi-Cheng , Chen Yi-Rong , Tsai Shih-Feng
Published: September 2005

Purpose: Epidermal growth factor receptor (EGFR) mutations related to gefitinib responsiveness in non–small cell lung cancer have been found recently. Detection of EGFR mutations has become an important issue for therapeutic decision-making in non–small cell lung cancer.

Experimental Design: Mutational analysis of the kinase domain of EGFR coding sequence was done on 101 fresh frozen tumor tissues from patients without prior gefitinib treatment and 16 paraffin-embedded tumor tissues from patients treated with gefitinib. Detection of phosphorylated EGFR by immunoblot was also done on frozen tumor tissues.

Results: The 101 non–small cell lung cancer tumor specimens include 69 adenocarcinomas, 24 squamous cell carcinomas, and 8 other types of non–small cell lung cancers. Mutation(s) in the kinase domain (exon 18 to exon 21) of the EGFR gene were identified in 39 patients. All of the mutations occurred in adenocarcinoma, except one that was in an adenosquamous carcinoma. The mutation rate in adenocarcinoma was 55% (38 of 69). For the 16 patients treated with gefitinib, 7 of the 9 responders had EGFR mutations, and only 1 of the 7 nonresponders had mutations, which included a nonsense mutation. The mutations seem to be complex in that altogether 23 different mutations were observed, and 9 tumors carried 2 mutations.

Conclusions: Data from our study would predict a higher gefitinib response rate in lung adenocarcinoma patients in Chinese and, possibly, other East Asian populations. The tight association with adenocarcinoma and the high frequency of mutations raise the possibility that EGFR mutations play an important role in the tumorigenesis of adenocarcinoma of lung, especially in East Asians.
Relative Bcl-2 Independence of Drug-Induced Cytotoxicity and Resistance in 518A2 Melanoma Cells
Authors: Benimetskaya Luba , Lai Johnathan C. , Khvorova Anastasia , Wu Sijian , Hua Emily , Miller Paul , Zhang Li-Ming , Stein Cy A.
Published: September 2005

Purpose: Inhibition of the function of Bcl-2 protein has been postulated to sensitize cells to cytotoxic chemotherapy. G3139 (Genasense) is a phosphorothioate anti–Bcl-2 antisense oligonucleotide, but its mechanism of action is uncertain. The aim of the present work is to investigate inhibition of Bcl-2 expression in 518A2 melanoma cells, the cell line on which recent phase II and phase III clinical trials employing this agent were based.

Experimental Design: We down-regulated the expression of Bcl-2 protein by two different strategies in these cells: one employing G3139 and controls, and the other using a small interfering RNA approach. Cell viability after treatment with oligonucleotides or small interfering RNA and cytotoxic agents including gemcitibine, DDP, docetaxel, and thapsigargin was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. A 518A2 melanoma cell line stably overexpressing Bcl-2 protein was constructed and treated with either these cytotoxic agents or G3139.

Results: The cytotoxic effects of either G3139 or small interfering RNA treatment of 518A2 melanoma cells are Bcl-2 independent. In addition, in the Bcl-2–overexpressing cells, only a modest increment in chemoresistance was observed, and treatment with G3139 not only did not down-regulate Bcl-2 expression but produced essentially identical toxicity as was observed in the wild-type or mock-transfected cells.

Conclusions: Our results suggest that the mechanism whereby G3139 produces drug-induced cytotoxicity in the 518A2 melanoma line is not dependent on levels of Bcl-2. These findings emphasize the nonsequence specific effects of this phosphorothioate oligonucleotide and call into question the validity of Bcl-2 as a target in this cell line.
Down-Regulation of Intratumoral
Aromatase
Messenger RNA Levels by Docetaxel in Human Breast Cancers
Authors: Miyoshi Yasuo , Kim Seung Jin , Akazawa Kenji , Kamigaki Shunji , Ueda Satsuki , Yanagisawa Tetsu , Inoue Tomoo , Taguchi Tetsuya , Tamaki Yasuhiro , Noguchi Shinzaburo
Published: September 2005

Purpose: The reason why chemotherapy induces resistance to subsequent hormonal therapy remains to be clarified in postmenopausal breast cancers. We hypothesized that chemotherapy might down-regulate the intratumoral biosynthesis of estrogens. Thus, we have studied the influence of chemotherapy (docetaxel) on intratumoral aromatase mRNA expression because aromatase is a key enzyme for intratumoral biosynthesis of estrogens.

Experimental Design: The mRNA levels of aromatase and its inducers [tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and cyclooxygenase 2 (COX-2)] were determined by a real-time polymerase chain reaction assay in breast cancer tissues obtained before and after neoadjuvant chemotherapy with docetaxel (four cycles of 60 mg/m2 every 3 weeks) in 16 postmenopausal patients with estrogen receptor (ER)- and/or progesterone receptor (PR)-positive breast cancers. ER and PR levels in tumor tissues were also determined by enzyme immunoassay before and after chemotherapy.

Results: The intratumoral aromatase mRNA levels decreased significantly (P < 0.05) after chemotherapy from 0.84 ± 0.28 (mean ± SE) to 0.47 ± 0.28. The intratumoral TNF-α mRNA levels also decreased significantly (P < 0.05) after chemotherapy from 2.40 ± 0.52 to 0.95 ± 0.25. On the contrary, the intratumoral IL-6 and COX-2 mRNA levels showed a marginally significant increase (P = 0.07) and a significant increase (P < 0.05), respectively, after chemotherapy. PR levels showed a marginally significant decrease (P = 0.08) after chemotherapy, whereas ER levels were almost constant before and after chemotherapy.

Conclusions: Antitumor activity of docetaxel is mediated, at least in part, through a down-regulation of aromatase expression in tumor tissues, resulting in the suppression of intratumoral estradiol synthesis. Aromatase expression seems to be regulated mostly by TNF-α, but not IL-6 and COX-2.