bluuebunny/crossref_metadata_2025 huggingface dataset:
Phase I Study of MetXia-P450 Gene Therapy and Oral Cyclophosphamide for Patients with Advanced Breast Cancer or Melanoma
Authors: Braybrooke Jeremy P. , Slade Andrew , Deplanque Gael , Harrop Richard , Madhusudan Srinivasan , Forster Martin D. , Gibson Rachel , Makris Andreas , Talbot Denis C. , Steiner Jan , White Linda , Kan On , Naylor Stuart , Carroll Miles W. , Kingsman Sue M. , Harris Adrian L.
Published: September 2005

Purpose: MetXia-P450 is a novel recombinant retroviral vector that encodes the human cytochrome P450 type 2B6 gene (CYP2B6), Escherichia coli lacZ, and neomycin resistance marker genes. Cytochrome P450 enzymes are primarily expressed in the liver and convert the prodrug cyclophosphamide to an active phosphoramide mustard and acrolein. Gene-based delivery of CYP2B6 to the tumor site leads to local prodrug activation and higher concentrations of the active metabolites at the target site.

Experimental Design: MetXia-P450 was directly injected into metastatic cutaneous tumor nodules on days 1 and 2 and nodules biopsied on day 7. Oral cyclophosphamide (100 mg/m2) was administered between days 8 and 22. Subsequent cycles of oral cyclophosphamide were repeated for 2 of 4 weeks. Gene transfer levels in biopsy samples were measured by histologic and quantitative PCR analyses. Safety assessments were made using PCR for vector dissemination to the blood after injection and using PCR and serologic analyses to detect replicating virus. Secondary end points included clinical response, toxicity, and evaluation of antitumor immune responses by measurement of carcinoembryonic antigen and 5T4 antibodies.

Results: Twelve patients with breast cancer (n = 9) and melanoma (n = 3) received three dose levels of MetXia-P450 (∼8 × 105, ∼8 × 106, and ∼8 × 107 lacZ transferring units/mL). The product was safe and well tolerated. The lacZ transgene was detected in biopsy material by immunohistochemistry in 10 of 12 patients and integrated viral sequences by PCR in 3 of 6 patients. One (8%) patient with breast cancer had a partial response and received 7 months of oral cyclophosphamide. Four (33%) patients had stable disease for ≥3 months and the rest had progressive disease. Preliminary immunologic analyses were suggestive of an antitumor response in two patients (partial response in one patient and stable disease in one patient).

Conclusion: MetXia was safe and well tolerated. Gene transfer was detected at all dose levels, and the initial suggestion of an antitumor response indicates that MetXia-P450 should undergo further clinical assessment.
Pharmacokinetics and Tissue Disposition of Indole-3-carbinol and Its Acid Condensation Products after Oral Administration to Mice
Authors: Anderton Mark J. , Manson Margaret M. , Verschoyle Richard D. , Gescher Andreas , Lamb John H. , Farmer Peter B. , Steward William P. , Williams Marion L.
Published: September 2005

Indole-3-carbinol (I3C) and 3,3′-diindolylmethane (DIM) are promising cancer chemopreventive agents in rodent models, but there is a paucity of data on their pharmacokinetics and tissue disposition. The disposition of I3C and its acid condensation products, DIM, [2-(indol-3-ylmethyl)-indol-3-yl]indol-3-ylmethane (LTr1), indolo[3,2b]carbazole (ICZ) and 1-(3-hydroxymethyl)-indolyl-3-indolylmethane (HI-IM) was studied, after oral administration of I3C (250 mg/kg) to female CD-1 mice. Blood, liver, kidney, lung, heart, and brain were collected between 0.25 and 24 h after administration and the plasma and tissue concentrations of I3C and its derivatives determined by high-performance liquid chromotography. I3C was rapidly absorbed, distributed, and eliminated from plasma and tissues, falling below the limit of detection by 1 h. Highest concentrations of I3C were detected in the liver where levels were approximately 6-fold higher than those in the plasma. Levels of DIM, LTr1, and HI-IM were much lower, although they persisted in plasma and tissues for considerably longer. DIM and HI-IM were still present in the liver 24 h after I3C administration. Tissue levels of DIM and LTr1 were found to be in equilibrium with plasma at almost every time point measured. In addition to acid condensation products of I3C, a major oxidative metabolite (indole-3-carboxylic acid) and a minor oxidative metabolite (indole-3-carboxaldehyde) were detected in plasma of mice after oral administration of I3C. ICZ was also tentatively identified in the liver of these mice. This study shows for the first time that, after oral administration to mice, I3C, in addition to its acid condensation products, is absorbed from the gut and distributed systemically into a number of well-perfused tissues, thus allowing the possibility for some pharmacological activity of the parent compound in vivo.
Breast Cancer Chemoprevention Phase I Evaluation of Biomarker Modulation by Arzoxifene, a Third Generation Selective Estrogen Receptor Modulator
Authors: Fabian Carol J. , Kimler Bruce F. , Anderson Julie , Tawfik Ossama W. , Mayo Matthew S. , Burak William E. , O’Shaughnessy Joyce A. , Albain Kathy S. , Hyams David M. , Budd G. Thomas , Ganz Patricia A. , Sauter Edward R. , Beenken Samuel W. , Grizzle William E. , Fruehauf John P. , Arneson Dora W. , Bacus James W. , Lagios Michael D. , Johnson Karen A. , Browne Doris
Published: September 2005

Purpose: Arzoxifene, a new selective estrogen receptor modulator with strong breast antiestrogen activity and absence of uterine agonist activity, was explored as a potential chemoprevention agent. We performed a multi-institutional evaluation of arzoxifene in women with newly diagnosed ductal carcinoma in situ or T1/T2 invasive cancer.

Experimental Design: In a Phase IA trial, 50 pre- or postmenopausal women were randomized to 10, 20, or 50 mg of arzoxifene daily in the interval between biopsy and re-excision or were enrolled as no-treatment controls. In a Phase IB trial, 76 postmenopausal women were randomized to 20 mg of arzoxifene versus matched placebo. Serum specimens collected at entry and at re-excision were assayed for various hormones and growth factors. Tissue from biopsies (estrogen receptor + and/or progesterone receptor +) and re-excision specimens was evaluated immunohistochemically for proliferation (Ki-67 by MIB-1 and proliferating cell nuclear antigen) and other biomarkers.

Results: In both trials, increases in serum sex hormone binding globulin were noted, as were decreases in insulin-like growth factor (IGF)-I and the IGF-I:IGF binding protein-3 ratio (P < 0.007 versus control/placebo). For 45 evaluable women in Phase IA, decreases in proliferation indices were more prevalent for arzoxifene (particularly 20 mg) than for controls. For 58 evaluable women in Phase IB, a decrease in estrogen receptor expression for arzoxifene was observed compared with no change with placebo (P = 0.0068). However, decreases in proliferation indices for arzoxifene were not statistically different from placebo, perhaps due to a confounding effect of stopping hormone replacement therapy before entry.

Conclusion: Given the favorable side effect profile and the biomarker modulations reported here, arzoxifene remains a reasonable candidate for additional study as a breast cancer chemoprevention agent.
Leptin Interferes with the Effects of the Antiestrogen ICI 182,780 in MCF-7 Breast Cancer Cells
Authors: Garofalo Cecilia , Sisci Diego , Surmacz Eva
Published: September 2005

Purpose: Obesity is a risk factor for breast cancer development in postmenopausal women and correlates with shorter disease-free and overall survival in breast cancer patients, regardless of menopausal status. Adipose tissue is a major source of leptin, a cytokine regulating energy balance and controlling different processes in peripheral tissues, including breast cancer cell growth. Here, we investigated whether leptin can counteract antitumorigenic activities of the antiestrogen ICI 182,780 in breast cancer cells.

Experimental Design: Mitogenic response to leptin and the effects of leptin on ICI 182,780-dependent growth inhibition were studied in MCF-7 estrogen receptor α-positive breast cancer cells. The expression of leptin receptor and the activation of signaling pathways were studied by Western immunoblotting. The interference of leptin with ICI 182,780-induced estrogen receptor α degradation was probed by Western immunoblotting, fluorescence microscopy, and pulse-chase experiments. Leptin effects on estrogen receptor α–dependent transcription in the presence and absence of ICI 182,780 were studied by luciferase reporter assays and chromatin immunoprecipitation.

Results: MCF-7 cells were found to express the leptin receptor and respond to leptin with cell growth and activation the signal transducers and activators of transcription 3, extracellular signal-regulated kinase-1/2, and Akt/GSK3/pRb pathways. The exposure of cells to 10 nmol/L ICI 182,780 blocked cell proliferation, induced rapid estrogen receptor α degradation, inhibited nuclear estrogen receptor α expression, and reduced estrogen receptor α–dependent transcription from estrogen response element–containing promoters. All of these effects of ICI 182,780 were significantly attenuated by simultaneous treatment of cells with 100 ng/mL leptin.

Conclusions: Leptin interferes with the effects of ICI 182,780 on estrogen receptor α in breast cancer cells. Thus, high leptin levels in obese breast cancer patients might contribute to the development of antiestrogen resistance.
Asparaginase Pharmacokinetics After Intensive Polyethylene Glycol-Conjugated L-Asparaginase Therapy for Children with Relapsed Acute Lymphoblastic Leukemia
Authors: Hawkins Douglas S. , Park Julie R. , Thomson Blythe G. , Felgenhauer Judy L. , Holcenberg John S. , Panosyan Eduard H. , Avramis Vassilios I.
Published: September 2005

Purpose: Asparaginase therapy is an important component in the treatment of children with acute lymphoblastic leukemia. Polyethylene glycol-conjugated asparaginase (PEG-ASNase) has significant pharmacological advantages over native Escherichia coli asparaginase. We investigated the pharmacokinetics of PEG-ASNase, presence of antibodies to PEG-ASNase, and concentrations of asparagine in serum and cerebrospinal fluid (CSF) in combination chemotherapy for relapsed pediatric acute lymphoblastic leukemia.

Experimental Design: Twenty-eight pediatric patients with relapsed medullary (n = 16) and extramedullary (n = 11) acute lymphoblastic leukemia were enrolled at three pediatric institutions and had at least two serum and CSF samples obtained for analysis. Patients received induction therapy (including PEG-ASNase 2500 IU/m2 intramuscularly weekly on days 2, 9, 16, and 23) and intensification therapy (including PEG-ASNase 2500 IU/m2 intramuscularly once on day 7). Serum samples were obtained weekly during induction and intensification. CSF samples were obtained during therapeutic lumbar punctures during induction and intensification.

Results: Weekly PEG-ASNase therapy resulted in PEG-ASNase activity of >0.1 IU/ml in 91–100% of patients throughout induction. During intensification, PEG-ASNase on day 7 resulted in PEG-ASNase activity >0.1 IU/ml in 94% and 80% of patients on days 14 and 21, respectively. Serum and CSF asparagine depletion was observed and maintained during induction and intensification in the majority of samples. PEG-ASNase antibody was observed in only 3 patients.

Conclusions: Intensive PEG-ASNase therapy in the treatment of relapsed acute lymphoblastic leukemia reliably results in high-level serum PEG-ASNase activity, and asparagine depletion in serum and CSF is usually achieved. Incorporation of intensive PEG-ASNase in future trials for recurrent acute lymphoblastic leukemia is warranted.
Expression Profiling of T-Cell Lymphomas Differentiates Peripheral and Lymphoblastic Lymphomas and Defines Survival Related Genes
Authors: Martinez-Delgado Beatriz , Meléndez Barbara , Cuadros Marta , Alvarez Javier , Castrillo Jose Maria , Ruiz de la Parte Ana , Mollejo Manuela , Bellas Carmen , Diaz Ramon , Lombardía Luis , Al-Shahrour Fatima , Domínguez Orlando , Cascon Alberto , Robledo Mercedes , Rivas Carmen , Benitez Javier
Published: September 2005

Purpose: T-Cell lymphomas constitute heterogeneous and aggressive tumors in which pathogenic alterations remain largely unknown. Expression profiling has demonstrated to be a useful tool for molecular classification of tumors.

Experimental Design: Using DNA microarrays (CNIO-OncoChip) containing 6386 cancer-related genes, we established the expression profiling of T-cell lymphomas and compared them to normal lymphocytes and lymph nodes.

Results: We found significant differences between the peripheral and lymphoblastic T-cell lymphomas, which include a deregulation of nuclear factor-κB signaling pathway. We also identify differentially expressed genes between peripheral T-cell lymphoma tumors and normal T lymphocytes or reactive lymph nodes, which could represent candidate tumor markers of these lymphomas. Additionally, a close relationship between genes associated to survival and those that differentiate among the stages of disease and responses to therapy was found.

Conclusions: Our results reflect the value of gene expression profiling to gain insight about the molecular alterations involved in the pathogenesis of T-cell lymphomas.
Expression of Pro- and Antiapoptotic Proteins in Circulating CD8+ T Cells of Patients with Squamous Cell Carcinoma of the Head and Neck
Authors: Kim Jeong-Whun , Tsukishiro Takashi , Johnson Jonas T. , Whiteside Theresa L.
Published: September 2005

Objective: Apoptosis of T lymphocytes in the circulation of patients with squamous cell carcinoma of the head and neck (SCCHN) was shown to target effector CD8+ rather than CD4+ T cells. This study evaluates the contribution of pro- and antiapoptotic components of the mitochondria-dependent pathway to apoptosis of circulating CD8+ T cells in these patients.

Experimental Design: Blood samples were obtained from 77 patients with SCCHN and 51 normal control(s) (NC). Percentages of CD8+Annexin V+ (ANX+) and CD8+CD95+ cells, changes in mitochondrial membrane potential and levels of expression of Bcl-2, Bcl-XL, and Bax in CD8+ T lymphocytes were measured by quantitative flow cytometry.

Results: Elevated percentages (P < 0.001) of early apo-ptotic (CD8+ANX+ CD95+) T cells in the circulation distinguish SCCHN patients from NCs but not patients with no evidence of disease (NED) from those with active disease (AD). Circulating CD8+ but not CD4+ T cells in patients were found to contain higher levels of proapoptotic Bax and antiapoptotic Bcl-XL (P < 0.01) than NC cells. The Bax/Bcl-2 ratio was elevated in CD8+ T cells of patients relative to NCs (P < 0.01), and it correlated with the percentage of ANX+CD8+ T cells (P = 0.007). The Bax/Bcl-XL ratio discriminated AD from NED patients.

Conclusion: Apoptosis of circulating CD8+T cells is found in SCCHN patients with AD or NED. Up-regulated Bax and Bcl-XL expression, the elevated Bax/Bcl-2 ratio and its association with ANX binding implicate the mitochondrial pathway in death of CD8+ T cells of patients with SCCHN. Understanding of molecular mechanisms of T-cell death and survival is essential for the development of more effective biotherapies for SCCHN.
Comprehensive Characterization of
Annexin I
Alterations in Esophageal Squamous Cell Carcinoma
Authors: Hu Nan , Flaig Michael J. , Su Hua , Shou Jian-Zhong , Roth Mark J. , Li Wen-Jun , Wang Chaoyu , Goldstein Alisa M. , Li Guang , Emmert-Buck Michael R. , Taylor Philip R.
Published: September 2005

Purpose: The purpose is to characterize alterations of the annexin I gene, its mRNA, and protein expression in esophageal squamous cell carcinoma.

Experimental Design: Fifty-six cases of esophageal squamous cell carcinoma were analyzed using four microsatellite markers flanking the annexin I gene (9q11-q21) to identify loss of heterozygosity. In addition, we performed (a) single-strand conformation polymorphism and DNA sequencing along the entire promoter sequence and coding region to identify mutations, (b) real-time quantitative reverse transcription-PCR of RNA from frozen esophageal squamous cell carcinoma tissue (n = 37) and in situ hybridization (n = 5) on selected cases to assess mRNA expression, and (c) immunohistochemistry (n = 44) to evaluate protein expression. The prevalence of the allelic variants identified in the first 56 patients was refined in 80 additional esophageal squamous cell carcinoma patients and 232 healthy individuals.

Results: Forty-six of 56 (82%) esophageal squamous cell carcinoma patients showed loss of an allele at one or more of the four microsatellite markers; however, only one (silent) mutation was seen. Two intragenic variants were identified with high frequency of allelic loss (A58G, 64%; L109L, 69%). Thirty of 37 (81%) esophageal squamous cell carcinoma patients showed reduced annexin I mRNA expression, which was confirmed by in situ hybridization, whereas annexin I protein expression was reduced in 79% of poorly differentiated tumor cell foci but in only 5% of well-differentiated tumor foci, although allelic loss on chromosome 9 was found in both tumor grades.

Conclusions: Allelic loss of annexin I occurs frequently, whereas somatic mutations are rare, suggesting that annexin I is not inactivated in esophageal squamous cell carcinoma via a two-hit mechanism. A decrease in annexin I protein expression was confirmed, consistent with a quantitative decrease in mRNA expression, and appeared to be related to tumor cell differentiation. We conclude that annexin I is not the tumor suppressor gene corresponding to the high levels of loss of heterozygosity observed on chromosome 9 in esophageal squamous cell carcinoma; however, dysregulation of mRNA and protein levels is associated with this tumor type.
Sequential Immunization of Melanoma Patients with GD3 Ganglioside Vaccine and Anti-Idiotypic Monoclonal Antibody That Mimics GD3 Ganglioside
Authors: Chapman Paul B. , Wu Dianna , Ragupathi Govind , Lu Sharon , Williams Linda , Hwu Wen-Jen , Johnson Diantha , Livingston Philip O.
Published: September 2005

GD3 ganglioside is an attractive target for immunotherapy of melanoma because it is abundantly expressed on all melanomas but not expressed on most normal tissues. Although GD3 has proven to be one of the least immunogenic gangliosides, our recent studies showed that anti-GD3 antibodies can be induced in patients immunized either with GD3-lactone-KLH (GD3-L-KLH) plus QS-21 adjuvant or with BEC2 anti-idiotypic monoclonal antibody vaccine, which mimics GD3, plus Bacillus Calmette-Guérin. We compared the immunogenicity of these two vaccines and tested whether one vaccine could prime an antibody response to the other. This is the first clinical trial immunizing patients with both antigen and anti-idiotypic monoclonal antibody vaccine. Twenty-four melanoma patients were randomized to be immunized with either BEC2 followed by GD3-L-KLH or in the opposite order. Our prior study suggested that a 25-μg dose of BEC2 was more immunogenic than our standard dose of 2.5 mg and therefor was used in this trial. Overall, 10 of 24 patients (42%) developed anti-GD3 antibodies detectable by ELISA, five in each cohort. All antibody responses were in response to the GD3-L-KLH vaccine. We found no evidence of priming by either vaccine. Antibody responses did not correlate with survival outcomes. Cellular responses were detected by enzyme-linked immunospot against BEC2, Bacillus Calmette-Guérin, and KLH, but not against GD3. We confirmed that GD3-L-KLH vaccine induces anti-GD3 antibodies, but we were unable to confirm our previous finding that a 25-μg dose of BEC2 is immunogenic. Future multivalent ganglioside vaccines should include the GD3-L-KLH vaccine.
CXC Chemokine Receptor 3 and CC Chemokine Receptor 4 Expression in T-Cell and NK-Cell Lymphomas with Special Reference to Clinicopathological Significance for Peripheral T-Cell Lymphoma, Unspecified
Authors: Ishida Takashi , Inagaki Hiroshi , Utsunomiya Atae , Takatsuka Yoshifusa , Komatsu Hirokazu , Iida Shinsuke , Takeuchi Genji , Eimoto Tadaaki , Nakamura Shigeo , Ueda Ryuzo
Published: September 2005

We recently reported expression of the chemokine receptors CXC chemokine receptor 3 (CXCR3) and CC chemokine receptor 4 (CCR4) in adult T-cell leukemia/lymphoma and showed a preferential expression of CCR4 and its association with an unfavorable outcome. In the present study, we extend our adult T-cell leukemia/lymphoma study to other subtypes of T- and NK-cell lymphoma, to clarify whether a characteristic chemokine receptor expression pattern is obtained for each of the subtypes defined by the WHO classification. CXCR3 and CCR4 were rarely expressed in three well-defined subtypes, precursor T-lymphoblastic lymphoma, anaplastic lymphoma kinase-positive anaplastic large cell lymphoma, and extranodal NK/T-cell lymphoma. A CXCR3-dominant expression pattern was observed in angioimmunoblastic T-cell lymphoma, while a CCR4-dominant expression pattern was observed in mycosis fungoides in transformation and in anaplastic lymphoma kinase-negative anaplastic large cell lymphoma. CXCR3 and CCR4 were heterogeneously expressed in peripheral T-cell lymphomas, unspecified (PTCLU). We next focused on PTCLU and analyzed the clinical significance of the chemokine receptors and their association with FoxP3, a hallmark of immunoregulatory T (Treg) cells. Multivariate analysis showed that CCR4 expression was an independent and significant unfavorable prognostic factor (P < 0.001). A significant correlation was found between mRNA expression of CCR4 and FoxP3, suggesting a possible association of CCR4-positive tumors with Treg cells and thereby with an immunocompromised state. Chemokine receptors may be useful not only for further characterization of the T- and NK-cell lymphomas but also in predicting clinical outcomes for patients. We suggest that a specific therapy targeting the CCR4 molecule may be developed as an alternative treatment for patients with CCR4-positive tumors.
Tumor Cells Circulate in the Peripheral Blood of All Major Carcinomas but not in Healthy Subjects or Patients With Nonmalignant Diseases
Authors: Allard W. Jeffrey , Matera Jeri , Miller M. Craig , Repollet Madeline , Connelly Mark C. , Rao Chandra , Tibbe Arjan G. J. , Uhr Jonathan W. , Terstappen Leon W. M. M.
Published: September 2005

Purpose: The purpose of this study was to determine the accuracy, precision, and linearity of the CellSearch system and evaluate the number of circulating tumor cells (CTCs) per 7.5 mL of blood in healthy subjects, patients with nonmalignant diseases, and patients with a variety of metastatic carcinomas.

Experimental Design: The CellSearch system was used to enumerate CTCs in 7.5 mL of blood. Blood samples spiked with cells from tumor cell lines were used to establish analytical accuracy, reproducibility, and linearity. Prevalence of CTCs was determined in blood from 199 patients with nonmalignant diseases, 964 patients with metastatic carcinomas, and 145 healthy donors.

Results: Enumeration of spiked tumor cells was linear over the range of 5 to 1,142 cells, with an average recovery of ≥85% at each spike level. Only 1 of the 344 (0.3%) healthy and nonmalignant disease subjects had ≥2 CTCs per 7.5 mL of blood. In 2,183 blood samples from 964 metastatic carcinoma patients, CTCs ranged from 0 to 23,618 CTCs per 7.5 mL (mean, 60 ± 693 CTCs per 7.5 mL), and 36% (781 of 2,183) of the specimens had ≥2 CTCs. Detection of ≥2 CTCs occurred at the following rates: 57% (107 of 188) of prostate cancers, 37% (489 of 1,316) of breast cancers, 37% (20 of 53) of ovarian cancers, 30% (99 of 333) of colorectal cancers, 20% (34 of 168) of lung cancers, and 26% (32 of 125) of other cancers.

Conclusions: The CellSearch system can be standardized across multiple laboratories and may be used to determine the clinical utility of CTCs. CTCs are extremely rare in healthy subjects and patients with nonmalignant diseases but present in various metastatic carcinomas with a wide range of frequencies.
Estrogen Receptor β (ERβ) Level but Not Its ERβcx Variant Helps to Predict Tamoxifen Resistance in Breast Cancer
Authors: Esslimani-Sahla Majida , Simony-Lafontaine Joelle , Kramar Andrew , Lavaill Roselyne , Mollevi Caroline , Warner Margaret , Gustafsson Jan-Åke , Rochefort Henri
Published: September 2005

The antiestrogen tamoxifen, a major endocrine therapy of estrogen receptor (ER)-positive breast cancer, is nevertheless inefficient in 30 to 40% of cases for unknown reasons. We retrospectively studied 50 ER-positive primary breast carcinomas. All of the patients had received tamoxifen as the only adjuvant therapy. They were divided into two groups depending on whether they relapsed within 5 years (16 tamoxifen-resistant cases) or did not relapse within 5 years (34 tamoxifen-sensitive cases). The expression of total ERβ protein, and of ERβcx protein, was estimated anonymously in formalin-fixed, paraffin-embedded tumor sections, by using specific antibodies and quantifiying nuclear immunostaining with a computer image analyzer. All of the tumors were found to be HER-2/neu-negative by immunohistochemistry.

Univariate analysis showed that Scarff-Bloom-Richardsson grade modified by Elston (SBR grade; P < 0.001), tumor size (P = 0.042), and MIB-1 proliferation index (P = 0.02) were significantly higher in tamoxifen-resistant tumors. A low level of total ERβ, whether in percentage of positive cells or in quantitative immunocytochemical (QIC) score, was also associated with tamoxifen resistance (P = 0.004). ERβcx expression and lymph node status were similar between the two groups. The expression of ERβ in the total population was positively correlated with ERβcx (r = 0.63, P < 0.001), and was independent of the other parameters. In a multivariate analysis, ERβ expression was the most important variable (P = 0.001), followed by SBR grade (I+II versus III; P = 0.008), and MIB-1 (P = 0.016).

To conclude, tamoxifen resistance is associated with classical variables of aggressive tumors (high SBR grade, proliferation index, and tumor size) but not with node invasiveness. Low ERβ level is an additional independent marker, better than ERα level, to predict tamoxifen resistance.
Inhibition of the Type III Epidermal Growth Factor Receptor Variant Mutant Receptor by Dominant-Negative EGFR-CD533 Enhances Malignant Glioma Cell Radiosensitivity
Authors: Lammering Guido , Hewit Theodore H. , Holmes Mathew , Valerie Kristoffer , Hawkins William , Lin Peck-Sun , Mikkelsen Ross B. , Schmidt-Ullrich Rupert K.
Published: September 2005

Purpose: The commonly expressed variant epidermal growth factor receptor (EGFR), the type III EGFR variant (EGFRvIII), functions as an oncoprotein promoting neoplastic transformation and tumorigenicity. The role of EGFRvIII in cellular responses to genotoxic stress, such as ionizing radiation, is only minimally defined. Thus, we have investigated EGFRvIII as a potential modulator of cellular radiation responses and explored the feasibility of adenovirus (Ad)-mediated expression of dominant-negative EGFR-CD533 as a gene therapeutic approach for inhibiting EGFRvIII function in vitro and in vivo.

Experimental Design and Results: EGFR-CD533 and EGFRvIII were expressed in vitro and in vivo in malignant U-373 MG glioma cells through transduction with an Ad vector, Ad-EGFR-CD533 and Ad-EGFRvIII, respectively. In vivo studies defined the importance of EGFRvIII as a modulator of radiation responses, demonstrating a 2.6-fold activation of EGFRvIII in U-373 malignant glioma tumors. Concomitant expression of EGFR-CD533 inhibited the radiation-induced activation of EGFRvIII in vitro and completely abolished the enhanced clonogenic survival conferred by EGFRvIII. The ability of EGFR-CD533 to inhibit EGFRvIII function was further confirmed in vivo through complete inhibition of EGFRvIII-mediated increased tumorigenicity and radiation-induced activation of EGFRvIII. Growth delay assays with U-373 xenograft tumors demonstrated that the expression of EGFR-CD533 significantly enhanced radiosensitivity of tumor cells under conditions of intrinsic and Ad-mediated EGFRvIII expression.

Conclusions: We conclude that EGFRvIII confers significant radioresistance to tumor cells through enhanced cytoprotective responses, and we have demonstrated that dominant-negative EGFR-CD533 effectively inhibits EGFRvIII function. These data affirm the broad potential of EGFR-CD533 to radiosensitize human malignant glioma cells.
The Fibroblast Growth Factor Receptor-4 Arg388 Allele Is Associated with Prostate Cancer Initiation and Progression
Authors: Wang Jianghua , Stockton David W. , Ittmann Michael
Published: September 2005

Purpose: Increased expression of fibroblast growth factors that can activate the fibroblast growth factor receptor-4 (FGFR-4) occurs in a substantial fraction of human prostate cancers in vivo. A germline polymorphism of the FGFR-4 gene resulting in expression of arginine at codon 388 (Arg388) is associated with aggressive disease in patients with breast and colon cancer. We therefore sought to determine whether the FGFR-4 Arg388 allele was associated with prostate cancer incidence and/or the occurrence of aggressive disease.

Experimental Design: The FGFR-4 genotype of men undergoing radical prostatectomy and controls of the same race was determined and the genotype correlated with clinical and pathologic markers of disease aggressiveness. PNT1A cell lines expressing predominantly the FGFR-4 Arg388 or Gly388 allele were established, and cell migration and invasiveness of these cells were assessed by a wounding assay and by quantitative determination of invasion through Matrigel. Expression of urokinase-type plasminogen activator receptor was determined by quantitative RT-PCR and enzyme-linked immunoabsorption assay.

Results: Homozygosity for the FGFR-4 Arg388 allele is strongly associated with the occurrence of prostate cancer in white men. The presence of the FGFR-4 Arg388 allele is also correlated with the occurrence of pelvic lymph node metastasis and biochemical (prostate-specific antigen) recurrence. Expression of FGFR-4 Arg388 in immortalized prostatic epithelial cells results in increased cell motility and invasion through Matrigel and was associated with increased expression of urokinase-type plasminogen activator receptor.

Conclusion: The FGFR-4 Arg388 allele is associated with both an increased incidence and clinical aggressiveness of prostate cancer and results in changes in cellular motility and invasiveness in immortalized prostate epithelial cells consistent with the promotion of metastasis.
Nuclear Localization of Apoptosis Protease Activating Factor-1 Predicts Survival after Tumor Resection in Early-Stage Non–Small Cell Lung Cancer
Authors: Besse Benjamin , Candé Céline , Spano Jean-Philippe , Martin Antoine , Khayat David , Le Chevalier Thierry , Tursz Thomas , Sabatier Laure , Soria Jean-Charles , Kroemer Guido
Published: September 2005

The proapoptotic protein apoptosis protein activating factor-1 (Apaf-1), which is normally located in the cytoplasm, can translocate to the nucleus before non-small cell lung carcinoma (NSCLC) cells manifest signs of apoptosis such as mitochondrial damage, caspase activation, or chromatin condensation. This may indicate a stage of imminent apoptosis. Importantly, we found that 24% (15 of 62) of resected stage I NSCLC (T1N0M0 or T2N0M0), manifested a marked nuclear localization of Apaf-1 (Apaf-1Nuc), as compared with the mostly cytoplasmic localization of Apaf-1 found in the remaining tumors (Apaf-1Cyt). After a median follow-up of 6.31 years, the actuarial 5-year overall survival rates were 89% (56–98%) in the Apaf-1Nuc group and 54% (36–71%) in the Apaf-1Cyt group (P = 0.039). No correlation between the subcellular localization of Apaf-1 and that of p53 and Hsp70 could be established. Thus, the subcellular location of Apaf-1 (but not that of p53 or Hsp70) constitutes an accurate prognostic factor for overall survival in NSCLC.
Ionizing Radiation Causes a Dose-Dependent Release of Transforming Growth Factor αIn vitrofrom Irradiated Xenografts and during Palliative Treatment of Hormone-Refractory Prostate Carcinoma
Authors: Hagan Michael , Yacoub Adly , Dent Paul
Published: September 2005

Purpose: Characterize the radiation response for transforming growth factor (TGF) α shedding in vitro and in vivo. We also report the shedding of TGF α by patients undergoing irradiation for hormone-refractory prostate cancer.

Experimental Design: TGF α levels were determined by ELISA. DU145 xenografts were established on the flanks of athymic nu/nu mice. Expression of phospho-extracellular signal-regulated kinase (ERK)1/2 and phospho-epidermal growth factor receptor (EGFR) and the DNA repair proteins XRCC1 and ERCC1 were determined by Western analyses.

Results: Exposure to ionizing radiation results in a dose-dependent release of TGF α. Once released, TGF α stimulates EGFR-ERK1/2 signaling in unirradiated cells. Blockade of the EGFR with the tyrphostin AG1478 eliminates the up-regulation XRCC1 and ERCC1 by TGF α or irradiation. After irradiation, cells are refractory to additional transactivation of EGFR by additional irradiation for 8 to 12 hours. Irradiation during this refractory period does not increase the expression of XRCC1 or ERCC1. Ligand activation of EGFR is maintained during the refractory period. Irradiation of DU145 xenografts also results in the activation of ERK1/2, release of TGF α, and a similar refractory period. Ionizing irradiation also results in the release of TGF α for patients undergoing radiation therapy for hormone-refractory prostate cancer.

Conclusions: Irradiation results in a dose-dependent increase in TGF α capable of enhancing the growth of DU145 xenografts. TGF α is also shed following radiation therapy of patients treated for hormone-refractory prostate cancer. Radiation transactivation of the EGFR produces a radio-refractory period, which lasts for several hours. During this period, additional irradiation fails to induce XRCC1, ERCC1, or additional TGF α release.
Phase II Study of the Proteasome Inhibitor Bortezomib (PS-341) in Patients with Metastatic Neuroendocrine Tumors
Authors: Shah Manisha H. , Young Donn , Kindler Hedy L. , Webb Iain , Kleiber Barbara , Wright John , Grever Michael
Published: September 2005

Purpose: This phase II study was undertaken to assess objective response, toxicity, tumor marker response, and pharmacodynamics of bortezomib in patients with metastatic neuroendocrine (carcinoid and islet cell) tumors.

Experimental Design: A total of 16 patients with measurable metastatic carcinoid (n = 12) or islet cell (n = 4) tumors received i.v. bolus of single agent bortezomib at a dose of 1.5 mg/m2 on days 1, 4, 8, and 11 every 21 days. Tumor response was assessed at 12-week intervals using Response Evaluation Criteria in Solid Tumors (RECIST) criteria. All patients were chemotherapy naïve and had Eastern Cooperative Oncology Group performance status of 0 to 1.

Results: No patient achieved a partial or a complete remission. The patients received total of 264 doses of therapy with a median of 15 doses per patient. Grade 4 toxicities were not observed. The most common grade 3 adverse events included peripheral sensory neuropathy (37%), diarrhea (25%), vomiting (18%), and ileus (18%). Six of 10 patients who experienced grade 2 to 3 peripheral sensory neuropathy also had grade 2 to 3 dizziness (n = 2), orthostatic hypotension (n = 2), syncope (n = 1), ileus (n = 2), or abdominal cramps (n = 1). Changes in tumor marker levels did not correlate with tumor response. The mean percentage of 20S proteasome inhibition achieved in whole blood at 1 and 24 hours after bortezomib administration was 68 and 30%, respectively.

Conclusions: Despite achieving the surrogate biologic end point, single-agent bortezomib did not induce any objective responses in patients with metastatic carcinoid or islet cell tumors. Additional investigation is warranted to clarify the possible association of autonomic neuropathy with bortezomib.
B7-H1 Expression on Non-Small Cell Lung Cancer Cells and Its Relationship with Tumor-Infiltrating Lymphocytes and Their PD-1 Expression
Authors: Konishi Jun , Yamazaki Koichi , Azuma Miyuki , Kinoshita Ichiro , Dosaka-Akita Hirotoshi , Nishimura Masaharu
Published: September 2005

Purpose: B7-H1/PD-L1 (B7-H1) and B7-DC/PD-L2 (B7-DC) are ligands for the receptor PD-1, which is known to negatively regulate T-cell activation. In the present study, we investigated the expression of B7-H1 and B7-DC in tumor specimens of non-small cell lung cancer and their relationships with clinicopathological variables and postoperative survival. Furthermore, we examined the correlation between B7-H1 expression on tumor cells and the number of tumor-infiltrating lymphocytes (TILs) or PD-1 expression on TILs.

Experimental Design: The expression of B7-H1 and B7-DC in 52 surgically resected specimens of non-small cell lung cancer was evaluated immunohistochemically.

Results: Expression of B7-H1 and B7-DC was focally observed in all non-small cell lung cancer tumor specimens. No relationship was found between the expression of B7-H1 or B7-DC and clinicopathological variables or postoperative survival. However, in the same sections evaluated, significantly fewer TILs were identified in B7-H1-positive tumor regions than in B7-H1-negative tumor regions in a subset of five patients (P = 0.01). Moreover, the percentage of TILs expressing PD-1 was significantly lower in B7-H1-positive tumor regions than in B7-H1-negative tumor regions (P = 0.02).

Conclusions: The expression of B7-H1 on tumor cells in local areas reciprocally correlated with the number of TILs, and this may contribute to negative regulation in antitumor immune responses in non-small cell lung cancer.
Phase I Study of Tirapazamine Plus Cisplatin/Etoposide and Concurrent Thoracic Radiotherapy in Limited-Stage Small Cell Lung Cancer (S0004)
Authors: Le Quynh-Thu , McCoy Jason , Williamson Stephen , Ryu Janice , Gaspar Laurie E. , Edelman Martin J. , Dakhil Shaker R. , Sides Stanley D. , Crowley John J. , Gandara David R.
Published: September 2005

Purpose: To determine the feasibility and a recommended phase II dose of tirapazamine when combined with chemoradiotherapy in limited-stage small cell lung cancer (LSCLC).

Experimental Design: Concurrent chemoradiotherapy consisted of two cycles of cisplatin, etoposide, and once-daily radiation to 61 Gy. Tirapazamine (260 mg/m2) was given 1 h before cisplatin with planned dose escalation to 330 mg/m2 in the absence of dose-limiting toxicity, defined as ≥33% esophagitis (grade 3 or above). Consolidation therapy consisted of two cycles of tirapazamine (330 mg/m2), cisplatin, and etoposide. Complete responders received prophylactic cranial irradiation.

Results: Thirty patients were enrolled at the 260 mg/m2 tirapazamine dose. All had performance status of 0–1. By comparison with S9713, a predecessor Southwest Oncology Group study in LSCLC that used the same concurrent chemoradiotherapy without tirapazamine, the present trial showed a higher rate of grade 3–4 esophagitis (34% versus 22%), vomiting (34% versus 23%), and febrile neutropenia (7% versus 2%). The consolidation phase was relatively well tolerated, with grade 4 neutropenia in 44% and febrile neutropenia in 5% of patients. There were two treatment-related deaths: one from neutropenic fever and one from respiratory infection. The overall response rate was 80%, and the median survival was 22 months.

Conclusions: Protocol-defined dose-limiting toxicity was observed at the initial tirapazamine dose, precluding dose escalation. Compared with S9713, the addition of tirapazamine increased the incidence of vomiting, neutropenia, and febrile neutropenia, although the overall toxicity profile remained acceptable. In view of the observed favorable survival, further study of tirapazamine in LSCLC is warranted.
Irofulven Cytotoxicity Depends on Transcription-Coupled Nucleotide Excision Repair and Is Correlated with XPG Expression in Solid Tumor Cells
Authors: Koeppel Florence , Poindessous Virginie , Lazar Vladimir , Raymond Eric , Sarasin Alain , Larsen Annette K.
Published: September 2005

Background: Irofulven is a novel alkylating agent with promising clinical activity, particularly toward ovarian and hormone-refractory prostate cancers. To facilitate additional clinical development, we have aimed to identify biological markers associated with sensitivity to the compound.

Methods: Fibroblasts derived from patients with xeroderma pigmentosum or Cockayne’s syndrome along with a panel of 20 human cancer cell lines (eight different tumor types) were examined to establish the importance of nucleotide excision repair proteins in the sensitivity to irofulven.

Results: Human cells deficient in nucleotide excision repair are up to 30-fold more sensitive to the cytotoxic effects of irofulven compared with repair-proficient controls, clearly indicating that nucleotide excision repair plays a crucial role in the sensitivity to the drug. Interestingly, our results show that irofulven-induced lesions are recognized by transcription-coupled repair but not by global genome repair. Another unique feature is the pronounced sensitivity of XPD and XPB helicase-deficient cells to the drug. Comparison of the IC50 values for irofulven, cisplatin, and ecteinascidin 743 with the expression levels of ERCC1, XPD, and XPG genes in different solid tumor cell lines shows no correlation between the expression levels of any of the three nucleotide excision repair proteins and the sensitivity to ecteinascidin 743. In contrast, expression of the XPG endonuclease was correlated with the cytotoxicity for irofulven and, to a lesser degree, for cisplatin. Importantly, XPG expression was also correlated with cellular nucleotide excision repair activity.

Conclusions: Increasing evidence indicates that compromised nucleotide excision repair activity is frequent in several solid tumor types. The results presented here suggest that XPG expression in such tumors may be a useful marker to predict their sensitivity to irofulven.
Overexpression of Collagen XVIII Is Associated with Poor Outcome and Elevated Levels of Circulating Serum Endostatin in Non–Small Cell Lung Cancer
Authors: Iizasa Toshihiko , Chang Hao , Suzuki Makoto , Otsuji Mizuto , Yokoi Sana , Chiyo Masako , Motohashi Shinichiro , Yasufuku Kazuhiro , Sekine Yasuo , Iyoda Akira , Shibuya Kiyoshi , Hiroshima Kenzo , Fujisawa Takehiko
Published: September 2005

Purpose: The aim of this study was to determine whether collagen XVIII expression is correlated with circulating serum endostatin and whether this has any prognostic value in patients with non–small cell lung cancer (NSCLC).

Experimental Design: Serum endostatin levels were measured quantitatively by a competitive enzyme immunoassay, and collagen XVIII expression in tumor tissue was investigated with an immunohistochemical method in a series of 94 patients who underwent surgery for NSCLC.

Results: Sixty cases (63.8%) had positive immunohistochemical staining with anticollagen XVIII polyclonal antibodies, including strongly positive staining in 11 (11.7%) cases. The mean (± SD) serum endostatin level was 41.6 ± 34.4 ng/ml in the patient group and 16.3 ± 10.3 ng/ml in the control group (P < 0.0001). The 11 cases who were strongly collagen XVIII-positive had significantly higher serum endostatin levels than the cases who were negative or weakly positive (P = 0.0297). The 5-year survival rates of negative, weakly positive, and strongly positive patients were 77.8%, 56.9%, and 43.8%, respectively. The cases with strongly positive collagen XVIII expression had a significantly poorer outcome than cases with negative expression (P = 0.0027). A multivariate analysis with Cox proportional hazards model for disease-specific survival revealed that expression of collagen XVIII (strongly positive versus negative; weakly positive versus negative), tumor classification, and regional lymph node classification were independent prognostic factors.

Conclusions: Our results suggest that expression of collagen XVIII in tumor tissue is strongly associated with a poorer outcome in NSCLC and correlates with elevated levels of circulating serum endostatin.
Phase I and Pharmacokinetic Study of the Oral Fluoropyrimidine S-1 on a Once-Daily-for-28-Day Schedule in Patients with Advanced Malignancies
Authors: Chu Quincy Siu-Chung , Hammond Lisa A. , Schwartz Garry , Ochoa Leonel , Rha Sun-Young , Denis Louis , Molpus Kathleen , Roedig Brian , Letrent Stephen P. , Damle Bharat , DeCillis Arthur P. , Rowinsky Eric K.
Published: September 2005

Purpose: The oral fluoropyrimidine S-1, which consists of a mixture of a 5-fluorouracil (5-FU) prodrug (tegafur), a dihydropyrimidine dehydrogenase inhibitor [5-chloro-2,4-dihydroxypyrimidine (CDHP)], and an inhibitor of orotate phosphoribosyltransferase [potassium oxonate (oxonic acid)], was developed to increase the feasibility and therapeutic index of 5-FU administered orally. The principal objective of this study was to assess the feasibility of administering S-1 on a once-daily-for-28-day schedule every 5 weeks, determine the maximum tolerated dose, characterize the pharmacokinetics of S-1, and seek evidence of anticancer activity.

Experimental Design: Patients with advanced solid malignancies were treated with escalating doses of S-1 on a once-daily oral schedule for 28 days every 5 weeks. The maximum tolerated dose was defined as the highest dose in which fewer than two of the first six new patients experienced dose-limiting toxicity. The pharmacokinetic profiles of the tegafur, CDHP, and oxonic acid constituents were characterized.

Results: Twenty patients were treated with 72 courses of S-1 at three dose levels ranging from 50 to 70 mg/m2/day. Diarrhea, which was often associated with abdominal discomfort and cramping, was the principal dose-limiting toxicity of S-1 on this protracted schedule. Nausea, vomiting, mucositis, fatigue, and cutaneous effects were also observed but were rarely severe. Myelosuppression was modest and uncommon. A partial response and a 49% reduction in tumor size were observed in patients with fluoropyrimidine- and irinotecan-resistant colorectal carcinoma. The pharmacokinetic data suggested potent inhibition of 5-FU clearance by CHDP, with resultant 5-FU exposure at least 10-fold higher than that reported from equitoxic doses of tegafur modulated by uracil in the oral fluoropyrimidine UFT.

Conclusions: The recommended dose for Phase II studies of S-1 administered once daily for 28 consecutive days every 5 weeks is 50 mg/m2/day. The pharmacokinetic data indicate substantial modulation of 5-FU clearance by CDHP. Based on these pharmacokinetic data, the predictable toxicity profile of S-1, and the low incidence of severe adverse effects at the recommended Phase II dose, evaluations of S-1 on this schedule are warranted in malignancies that are sensitive to the fluoropyrimidines.