bluuebunny/crossref_metadata_2025 huggingface dataset:
Prediction of Colorectal Neoplasia by Quantitative Methylation Analysis of Estrogen Receptor Gene in Nonneoplastic Epithelium from Patients with Ulcerative Colitis
Authors: Tominaga Keiichi , Fujii Shigehiko , Mukawa Kenichiroh , Fujita Mikio , Ichikawa Kazuhito , Tomita Shigeki , Imai Yasuo , Kanke Kazunari , Ono Yuko , Terano Akira , Hiraishi Hideyuki , Fujimori Takahiro
Published: March 2006

Purpose: The incidence of colorectal neoplasia has increased among patients with longstanding and extensive ulcerative colitis (UC). Therefore, surveillance colonoscopy has been widely recommended. However, there is controversy about the impact of cancer surveillance, and ways to improve its effectiveness are being sought. The estrogen receptor (ER) gene shows age-related methylation in the colorectal epithelium and is frequently methylated in colorectal neoplasia, suggesting that ER methylation occurs early in the process of colorectal tumorigenesis.

Experimental Design: To clarify whether methylation analysis of the ER gene in nonneoplastic epithelium can help predict an increased risk for UC-associated neoplasia, a total of 105 nonneoplastic colorectal epithelia from 18 patients with longstanding and extensive UC, including 8 patients with neoplasia and 10 patients without neoplasia, were analyzed. In all patients, multiple samples were taken from six regions of the colorectum. The combined bisulfite restriction analysis method was used to determine the methylation status of the ER gene.

Results: The mean methylation level of the ER gene was 25.4% in the nonneoplastic epithelia from UC patients with neoplasia, whereas it was only 4.0% in those without neoplasia (P < 0.001). The methylation level of the ER gene in UC patients with neoplasia was significantly higher than in UC patients without neoplasia throughout the colorectum except for the cecum. In UC patients with neoplasia, the mean ER methylation level in the distal colon (36.1%) was significantly higher than in the proximal colon (14.6%; P < 0.001).

Conclusions: These results suggest that the analysis of ER gene methylation in nonneoplastic colorectal epithelium could have the potential to be a useful adjunct for identifying individuals with longstanding and extensive UC who are at increased risk of neoplasia and contribute to more effective cancer surveillance.
High Serum Concentration of YKL-40 Is Associated with Short Survival in Patients with Acute Myeloid Leukemia
Authors: Bergmann Olav J. , Johansen Julia S. , Klausen Tobias W. , Mylin Anne K. , Kristensen Jørgen S. , Kjeldsen Eigil , Johnsen Hans E.
Published: March 2006

Purpose: YKL-40 is secreted by cancer cells, macrophages, and neutrophils. It may be a growth or differentiation factor, play a role in angiogenesis, or protect against apoptosis. High serum YKL-40 is associated with poor prognosis in solid carcinomas. The aim was to examine serum YKL-40 in patients with acute myeloid leukemia (AML).

Experimental Design: YKL-40 was measured by ELISA in serum from 77 patients recently diagnosed with AML before and during the first month of chemotherapy.

Results: Forty (52%) of the AML patients had elevated serum YKL-40 (compared with age-matched healthy subjects) and their survival was shorter than in patients with normal serum YKL-40 (median, 128 days; interquartile range, 18-629 days versus 386 days; interquartile range, 180-901; P = 0.018 Mann-Whitney test). Univariate analysis of serum YKL-40 (logarithmically transformed and treated as a continuous covariate) showed significant association with survival within the first month after start of chemotherapy [hazard ratio (HR), 1.7; 95% confidence interval (CI), 1.2-2.4; P = 0.002], first 12 months (HR, 1.6; 95% CI, 1.2-2.0; P = 0.0002), and overall survival (HR, 1.3; 95% CI, 1.1-1.6; P = 0.003). Multivariate Cox analysis showed that serum YKL-40 was an independent prognostic variable for survival (first month: HR, 1.7; P = 0.011; 12 months: HR, 1.6; P = 0.0002; overall survival: HR, 1.4; P = 0.002). High serum YKL-40 at start of chemotherapy was a risk factor for pneumonia within the first month, and serum YKL-40 increased (P = 0.002) at time of pneumonia and was unchanged in patients without infections.

Conclusions: Serum YKL-40 is a prognostic biomarker of survival in AML patients. Its role in AML and infections needs to be determined.
Pigment Epithelium–Derived Factor Gene Therapy Inhibits Human Pancreatic Cancer in Mice
Authors: Hase Ryunosuke , Miyamoto Masaki , Uehara Hirofumi , Kadoya Masatoshi , Ebihara Yuma , Murakami Yoshihiro , Takahashi Ryo , Mega Seiji , Li Li , Shichinohe Toshiaki , Kawarada Yo , Kondo Satoshi
Published: March 2006

Purpose: Pigment epithelium–derived factor (PEDF), which has recently been shown to be the most potent inhibitor of angiogenesis in the mammalian eye, is also expressed in the pancreas. Previously, we have screened the expression of PEDF by immunohistochemical analysis and showed that low expression of PEDF is associated with increased risk of hepatic metastasis and short survival. The purpose of this study was to investigate whether PEDF gene is a potent tumor suppressor and a potential candidate for cancer gene therapy.

Experimental Design: We investigated both in vitro and in vivo growth characteristics of human pancreatic adenocarcinoma cell lines that were stably transfected to overexpress human PEDF and therapeutic effects of lentivirus-based vectors expressing PEDF on tumor growth in murine s.c. tumor model.

Results: We discovered that cells secreted PEDF protein in the media and this exhibited strong inhibitory effects on proliferation and migration of human umbilical vein endothelial cells. The size of PEDF-overexpressing pancreatic adenocarcinoma tumors was significantly smaller than that of control tumors in s.c. tumor models. Moreover, the growth of PEDF-overexpressing pancreatic adenocarcinoma cells was significantly suppressed in comparison with control cells in peritoneal metastasis models. In gene transfer models, intratumoral injection of a lentivirus vector encoding PEDF (LV-PEDF) caused significant inhibition of tumor growth. The antitumor effect observed after treatment with LV-PEDF was associated with decreased microvessel density in tumors.

Conclusion: Our data suggest that PEDF may exert a biological effect on tumor angiogenesis and PEDF gene therapy may provide a new approach for treatment of pancreatic adenocarcinoma.
Prognostic and Predictive Effects of Immunohistochemical Factors in High-Risk Primary Breast Cancer Patients
Authors: Kröger Nicolaus , Milde-Langosch Karin , Riethdorf Sabine , Schmoor Claudia , Schumacher Martin , Zander Axel R. , Löning Thomas
Published: March 2006

Purpose: To analyze prognostic and predictive effects of immunohistochemical factors within a randomized study of high-dose versus standard-dose chemotherapy in high-risk breast cancer with >10 involved lymph nodes.

Experimental Design: Histopathologic specimens in 188 of 302 patients were analyzed for Ki-67, p16, maspin, Bcl-2, Her2/neu, and p53.

Results: In a univariate analysis after adjustment for therapy, tumor size, and estrogen receptor, Her2/neu positivity (P = 0.001) was a negative and Bcl2 positivity (P = 0.003) was a positive prognostic factor for event-free survival. In a multivariate analysis, Her2/neu positivity (hazard ratio, 3.68; 95% confidence interval, 2.01-6.73; P = 0.0001) had a negative influence on event-free survival, whereas p53 positivity (hazard ratio, 0.57; 95% confidence interval, 0.34-0.95; P = 0.03) and Bcl2 positivity (hazard ratio, 0.35; 95% confidence interval, 0.19-0.64; P = 0.0006) were associated with a better event-free survival. Analyzing the predictive effect of the immunohistochemical factors, an interaction between p53 and treatment could be shown (P = 0.005). The hazard ratio for high-dose chemotherapy versus standard chemotherapy is estimated as 2.3 (95% confidence interval, 0.67-7.92) in p53-negative patients and as 0.46 (95% confidence interval, 0.2-1.07) in p53-positive patients, which indicates a superiority of high-dose chemotherapy in p53-positive patients and an inferiority in p53-negative patients. No interactive effect could be shown for the other factors.

Conclusions: Her2/neu and Bcl-2 are prognostic but not predictive factors in patients with high-risk primary breast cancer; p53-positive patients might benefit more from high-dose chemotherapy than from standard chemotherapy, and p53-negative patients might benefit more from standard chemotherapy than from high-dose therapy.
Clear Cell Sarcoma of the Kidney: Up-regulation of Neural Markers with Activation of the Sonic Hedgehog and Akt Pathways
Authors: Cutcliffe Colleen , Kersey Donna , Huang Chiang-Ching , Zeng Yong , Walterhouse David , Perlman Elizabeth J. ,   for the Renal Tumor Committee of the Children's Oncology Group
Published: March 2006

Purpose and Experimental Design: Clear cell sarcoma of the kidney (CCSK), the second most common renal tumor in children, poses significant diagnostic challenges. No positive diagnostic markers are available, and the pathogenesis of CCSK remains an enigma. To address these challenges, the gene expression patterns of 14 CCSKs were compared with 15 Wilms tumors and 3 fetal kidney samples using oligonucleotide arrays.

Results: Using unsupervised methods, the gene expression profile of CCSK was distinctive: differentially expressed genes could largely be grouped into four categories: (a) a wide variety of neural markers, (b) members of the Sonic hedgehog pathway, (c) members of the phosphoinositide-3-kinase/Akt cell proliferation pathway, and (d) known therapeutic targets. Corresponding changes in critical proteins using Western blot and/or immunohistochemistry confirmed the up-regulation of these pathways and proteins. In particular, CD117 and epidermal growth factor receptor are up-regulated at the protein level in many CCSKs, providing potential therapeutic targets. One of the neural markers, nerve growth factor receptor, represents a promising diagnostic tool for CCSK.

Conclusions: This study suggests that CCSKs arise within a renal mesenchymal cell that shows a wide variety of neural markers. As such, it seems to be susceptible to genetic changes also seen in a variety of other neuroectodermal and neuronal tumors, including activation of Sonic hedgehog and phosphoinositide-3-kinase/Akt pathways. Involvement of these pathways in CCSKs implicates their widening role in tumorigenesis.
Molecular Context of theEGFRMutations: Evidence for the Activation of mTOR/S6K Signaling
Authors: Conde Esther , Angulo Barbara , Tang Moying , Morente Manuel , Torres-Lanzas Juan , Lopez-Encuentra Angel , Lopez-Rios Fernando , Sanchez-Cespedes Montserrat
Published: March 2006

Purpose: Activating somatic mutations in the epidermal growth factor receptor (EGFR) gene are present in a small subset of lung adenocarcinomas. These mutations cluster in specific regions and confer sensitivity to inhibitors of the tyrosine kinase activity of EGFR. To further determine the genetic and molecular characteristics of tumors carrying EGFR gene mutations, we investigated the EGFR gene status in lung adenocarcinomas and evaluated its association with specific characteristics of the patients and tumors, such as mutations at KRAS and p53, EGFR and ErbB2 gene amplification, levels of EGFR and HER2 proteins, and levels of downstream effectors of EGFR, such as phospho–extracellular signal-regulated kinase and phospho-S6 proteins.

Experimental Design: The mutational status of EGFR was determined by direct sequencing in 86 primary lung adenocarcinomas and 12 lung cancer cell lines, and was correlated with a number of variables relating to the tumor and patient. A tissue microarray containing 37 lung tumors was constructed to determine, by fluorescence in situ hybridization analysis, the number of copies of EGFR and ErbB2 genes and, by immunohistochemistry, the levels of EGFR, HER2, phospho-ERK, and phospho-S6 proteins.

Results: EGFR gene mutations were identified in 13% of the primary tumors. The type and clustering of the mutations were identical to those previously reported. Amplification of the EGFR occurred in 14% of the tumors and could arise in tumors with EGFR mutations. Interestingly, mTOR activation, as measured indirectly by augmented levels of phospho-S6 protein, was more frequent in tumors with gene alterations in either EGFR or KRAS (P = 0.00005; Fisher's exact test) than in their wild-type counterparts.

Conclusions: Our data agree with the accumulation of EGFR mutations in a subset of patients with lung cancer. Moreover, we report EGFR gene amplification in EGFR-mutant tumors and a positive correlation between EGFR or KRAS alterations and activation of mTOR signaling.
Inhibition of Vascular Endothelial Growth Factor (VEGF)-A Causes a Paradoxical Increase in Tumor Blood Flow and Up-Regulation of VEGF-D
Authors: Moffat Bradford A. , Chen Mark , Kariaapper Muhammed S.T. , Hamstra Daniel A. , Hall Daniel E. , Stojanovska Jadranka , Johnson Timothy D. , Blaivas Mila , Kumar Mahesh , Chenevert Thomas L. , Rehemtulla Alnawaz , Ross Brian D.
Published: March 2006

Purpose: Vascular endothelial growth factor (VEGF)-A is an important mediator of angiogenesis in almost all solid tumors. The aim of this study was to evaluate the effect of VEGF-A expression on tumor growth, perfusion, and chemotherapeutic efficacy in orthotopic 9L gliosarcomas.

Experimental Design: Stable 9L cell lines underexpressing and overexpressing VEGF-A were generated. Anatomic, susceptibility contrast, and continuous arterial spin-labeling magnetic resonance imaging were used to quantify the volume, blood volume, and blood flow of tumors orthotopically grown from these and wild-type 9L cells. Histologic, immunohistochemical, and quantitative reverse transcription-PCR analyses were also done on excised tumors. Finally, the effects of carmustine chemotherapy were also evaluated.

Results: Orthotopic tumors underexpressing VEGF-A had slower growth rates (increased median survival), greater blood flow, vessel density, and VEGF-D expression, but no statistical difference in blood volume and chemotherapeutic sensitivity, compared with tumors with wild-type levels of VEGF-A. Tumors overexpressing VEGF-A had faster growth rates, greater blood volume, vessel density, and blood flow but no statistical difference in VEGF-D expression and chemotherapeutic sensitivity compared with wild-type VEGF-A-expressing tumors.

Conclusion: Blood volume and blood flow are independent and different biomarkers of tumor perfusion. Therefore, both should be measured when characterizing the efficacy of antiangiogenic therapies. Underexpression of VEGF-A does not result in complete inhibition of angiogenesis. Moreover, these tumors have a different perfusion phenotype, suggesting that angiogenesis is mediated by an alternative pathway. The results indicate that VEGF-D is a plausible alternative mediator of this angiogenesis.
Comparison of Two Cancer Vaccines Targeting Tyrosinase: Plasmid DNA and Recombinant Alphavirus Replicon Particles
Authors: Goldberg Stacie M. , Bartido Shirley M. , Gardner Jason P. , Guevara-Patiño José A. , Montgomery Stephanie C. , Perales Miguel-Angel , Maughan Maureen F. , Dempsey JoAnn , Donovan Gerald P. , Olson William C. , Houghton Alan N. , Wolchok Jedd D.
Published: March 2006

Purpose: Immunization of mice with xenogeneic DNA encoding human tyrosinase-related proteins 1 and 2 breaks tolerance to these self-antigens and leads to tumor rejection. Viral vectors used alone or in heterologous DNA prime/viral boost combinations have shown improved responses to certain infectious diseases. The purpose of this study was to compare viral and plasmid DNA in combination vaccination strategies in the context of a tumor antigen.

Experimental Design: Using tyrosinase as a prototypical differentiation antigen, we determined the optimal regimen for immunization with plasmid DNA. Then, using propagation-incompetent alphavirus vectors (virus-like replicon particles, VRP) encoding tyrosinase, we tested different combinations of priming with DNA or VRP followed by boosting with VRP. We subsequently followed antibody production, T-cell response, and tumor rejection.

Results: T-cell responses to newly identified mouse tyrosinase epitopes were generated in mice immunized with plasmid DNA encoding human (xenogeneic) tyrosinase. In contrast, when VRP encoding either mouse or human tyrosinase were used as single agents, antibody and T-cell responses and a significant delay in tumor growth in vivo were observed. Similarly, a heterologous vaccine regimen using DNA prime and VRP boost showed a markedly stronger response than DNA vaccination alone.

Conclusions: Alphavirus replicon particle vectors encoding the melanoma antigen tyrosinase (self or xenogeneic) induce immune responses and tumor protection when administered either alone or in the heterologous DNA prime/VRP boost approaches that are superior to the use of plasmid DNA alone.
Correlations between Serum Monocyte Chemotactic Protein-1 Levels, Clinical Prognostic Factors, and HER-2/neu Vaccine-Related Immunity in Breast Cancer Patients
Authors: Dehqanzada Zia A. , Storrer Catherine E. , Hueman Matthew T. , Foley Rebecca J. , Harris Katie A. , Jama Yusuf H. , Kao Tzu-Cheg , Shriver Craig D. , Ponniah Sathibalan , Peoples George E.
Published: March 2006

Purpose: We studied serum monocyte chemotactic protein-1 (MCP-1) levels in breast cancer patients in relationship to their clinicopathologic variables and immune response to a /neu E75 vaccine.

Experimental Design: We measured MCP-1 levels in 32 /neu+ breast cancer patients before and after vaccination with a /neu E75 peptide + granulocyte macrophage colony-stimulating factor vaccine. Clinical prognostic variables were collected. Vaccine-specific immunologic responses were monitored.

Results: Serum MCP-1 levels >250 pg/mL (MCP-high) correlated with favorable prognostic variables. MCP-high patients compared with MCP-low (<250 pg/mL) patients showed statistically significant later onset of disease, earlier stage of disease, fewer nodal metastasis, and less chemotherapy. MCP-high patients had increased levels of preexisting immunity when compared with MCP-low patients (69% versus 21%; P = 0.02). However, MCP-low patients showed higher inducible levels of MCP-1 compared with MCP-high patients (median increase, 41% versus 0%; P = 0.001) after vaccination. Moreover, MCP-low patients with >50% increase in MCP-1 levels (response-high) had worse clinical prognostic variables compared with patients with <50% increase (response-low). Response-high patients had statistically significant more poorly differentiated tumors, later stage of disease, and higher percentage of large tumors. Patients with >30% postvaccination MCP-1 increase also showed significant increases in E75-specific CD8+ T-cells (0.05% versus 0.38%; P = 0.03) in response to vaccination.

Conclusions: High serum MCP-1 levels in breast cancer patients correlate with favorable prognostic variables and increased preexisting /neu immunity. E75 vaccination induces the largest MCP-1 response in patients with unfavorable clinicopathologic variables. Therefore, low serum MCP-1 levels may identify patients with worse prognosis and those most likely to benefit from this vaccination.
Expression of Interleukin-9 in Nasal Natural Killer/T-Cell Lymphoma Cell Lines and Patients
Authors: Nagato Toshihiro , Kobayashi Hiroya , Kishibe Kan , Takahara Miki , Ogino Takeshi , Ishii Hideyuki , Oikawa Kensuke , Aoki Naoko , Sato Keisuke , Kimura Shoji , Shimizu Norio , Tateno Masatoshi , Harabuchi Yasuaki
Published: March 2006

Purpose: Nasal natural killer (NK)/T-cell lymphoma is associated with EBV and has distinct clinical and histologic features. However, little is known about its genetic features. In this study, we examined the genes expressed by SNK-6 and SNT-8 cells, which were established from nasal NK/T-cell lymphomas, and found that interleukin (IL)-9 was specifically expressed in these two cell lines.

Experimental Design: cDNA array was used to examine the genes expressed by SNK-6 and SNT-8 cells. Expression of IL-9 and IL-9 receptor was investigated by reverse transcription-PCR, ELISA, and flow cytometry. Cell growth was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Immunohistologic staining and ELISA were used to examine IL-9 expression in biopsies and sera from patients, respectively.

Results: In cDNA array, expression of IL-9 mRNA was much higher in SNK-6 and SNT-8 cells than in NK-92 cells from non-nasal NK-cell lymphoma and peripheral blood mononuclear cells from healthy volunteers. Furthermore, IL-9 was specifically expressed by SNK-6 and SNT-8 cells but not by other NK-cell, NK-like T-cell, and T-cell lymphoma/leukemia cell lines. IL-9 receptor was also expressed on the surfaces of SNK-6 and SNT-8 cells. An IL-9-neutralizing antibody inhibited the growth of these two cell lines, whereas recombinant human IL-9 enhanced their growth. Most significantly, IL-9 was present in biopsies and sera from patients with this lymphoma.

Conclusions: These results suggest that IL-9 plays an important role in nasal NK/T-cell lymphoma possibly via an autocrine mechanism.
Reduced Expression ofCAMTA1Correlates with Adverse Outcome in Neuroblastoma Patients
Authors: Henrich Kai-Oliver , Fischer Matthias , Mertens Daniel , Benner Axel , Wiedemeyer Ruprecht , Brors Benedikt , Oberthuer André , Berthold Frank , Wei Jun Stephen , Khan Javed , Schwab Manfred , Westermann Frank
Published: March 2006

Purpose: A distal portion of 1p is frequently deleted in human neuroblastomas, and it is generally assumed that this region harbors at least one gene relevant for neuroblastoma development. A 1p36.3 commonly deleted region, bordered by D1S2731 and D1S214 has been defined. The present study surveys whether expression of genes mapping to this region is associated with tumor behavior.

Experimental Design: Candidate genes localized within the deleted region were identified by sequence data analysis. Their expression was assessed in a cohort of 49 primary neuroblastomas using cDNA microarray analysis. Gene expression patterns associated with known prognostic markers and patient outcome were further evaluated by quantitative real-time reverse transcription-PCR in a cohort of 102 neuroblastomas.

Results: The commonly deleted region spans 261 kb and encompasses two genes, FLJ10737 and CAMTA1. We found no evidence for an association of FLJ10737 expression with established prognostic variables or outcome. In contrast, low CAMTA1 expression characterized tumors with 1p deletion, MYCN amplification, and advanced tumor stages 3 and 4. Moreover, low CAMTA1 expression was significantly associated with poor outcome (P < 0.001). In multivariate analysis of event-free survival, the prognostic information of low CAMTA1 expression was independent of 1p status, MYCN status, tumor stage, and age of the patient at diagnosis (hazard ratio, 3.52; 95% confidence interval, 1.21-10.28; P = 0.02).

Conclusions: Our data suggest that assessment of CAMTA1 expression may improve the prognostic models for neuroblastoma and that it will be important to define the biological function of CAMTA1 in this disease.
Inclusion of Vasculature-Related Variables in the Dukes Staging System of Colon Cancer
Authors: Koukourakis Michael I. , Giatromanolaki Alexandra , Sivridis Efthimios , Gatter Kevin C. , Harris Adrian L. ,   for Tumour and Angiogenesis Research Group
Published: March 2006

Purpose: The Dukes stage is used to stratify colorectal cancer patients into groups of different prognosis and need of adjuvant radiotherapy and chemotherapy. However, ∼80% of patients with Dukes stage C colorectal cancer receive cytotoxic therapy without any expected benefit, for such patients would either not relapse without adjuvant therapy or they would inevitably do so because of tumor resistance to the available regimens. On the other hand, as 20% of Dukes stage B patients would relapse after surgery, adjuvant therapy could improve their survival. Improvement of the Dukes stage predictive accuracy is necessary to better assign patients for adjuvant therapies, especially nowadays when antiangiogenic agents are being incorporated in the clinical practice.

Patients and Methods: In this study, we examined the prognostic role of Dukes staging system in parallel with three vasculature-related variables (vascular invasion, tumor angiogenic activity, and vascular survival ability) in a series of 130 stage B/C patients with colorectal cancer treated with surgery alone (without adjuvant radiotherapy or chemotherapy).

Results: Inclusion of vasculature-related variables in the Dukes staging system significantly improved the prognostic categorization of patients, identifying subgroups of B-stage and C-stage patients with an up to 40% and 60% 5-year survival difference, respectively.

Conclusions: Preliminary results show that the prognostic value of Dukes staging system is significantly improved after taking into account vasculature-related variables, which may be useful in stratifying patients for adjuvant therapies, highlighting also subgroups that may benefit the most from antiangiogenic agents.
Adoptive transfer of allogeneic cytotoxic T lymphocytes equipped with a HLA-A2 restricted MART-1 T-cell receptor: a phase I trial in metastatic melanoma.
Authors: Duval Lone , Schmidt Henrik , Kaltoft Keld , Fode Kirsten , Jensen Jens Jorgen , Sorensen Steen Mellerup , Nishimura Michael I , von der Maase Hans
Published: March 2006

PURPOSE: We did a phase I dose-escalation trial to evaluate the feasibility and safety of intratumoral injections of C Cure 709, an allogeneic, continuous CTL cell line that, restricted by HLA-A2, recognizes MART-1-positive tumor cells through transduction with a T-cell receptor encoding gene. EXPERIMENTAL DESIGN: Cells were administered intratumorally in four dose levels ranging from 10(8) to 10(9) cells/d on days 1, 4, 7, 10, 14, and 28 of each treatment cycle to patients with metastatic melanoma. Main inclusion criteria were HLA-A2 tissue type, MART-1-positive tumor cells, and metastases suitable for ultrasound-guided injections. Patients were assessed for toxicity and response. Three to six patients were treated per dose level. Patients without progressive disease were offered up to three treatment cycles. RESULTS: Fifteen patients received a total of 24 treatment cycles with a total of 266 injections of C Cure 709. Toxicity was minor to moderate and most common injection site reactions were fever, fatigue, nausea/vomiting, and arthralgia/myalgia. Side effects disappeared in general within 24 hours. Toxicity was not dose dependent. One patient obtained a partial response, encompassing both metastases used and not used for intratumoral injections. Remaining patients did not achieve an overall response. In addition, we observed local regression of metastases used for injection in two patients and of metastases not used for injection in one patient. CONCLUSION: Intratumoral injections of C Cure 709 are feasible, safe, and capable of inducing tumor regression. Further investigation in a phase II setting is warranted.
Expression of EphA2 and Ephrin A-1 in Carcinoma of the Urinary Bladder
Authors: Abraham Shaji , Knapp Deborah W. , Cheng Liang , Snyder Paul W. , Mittal Suresh K. , Bangari Dinesh S. , Kinch Michael , Wu Lan , Dhariwal Jay , Mohammed Sulma I.
Published: March 2006

Purpose: The EphA2 receptor tyrosine kinase is believed to play a role in tumor growth and metastasis. The clinical significance of the expression of EphA2 was observed in breast, prostate, colon, skin, cervical, ovarian, and lung cancers. The purpose of this work was to determine the expression of EphA2 and its ligand, Ephrin A-1, and E-cadherin in carcinoma of the urinary bladder, and determine EphA2 as a new target for therapy in bladder cancer.

Experimental Design: EphA2 mRNA and protein expression was investigated by reverse transcription-PCR and Western blot, respectively, in bladder cancer cell lines. In addition, the expression of EphA2, Ephrin A-1, and E-cadherin in tissues from patients with different stages of urinary bladder cancer was determined by immunohistochemistry. Furthermore, the ability of Ephrin A-1 to inhibit growth of bladder cancer cells was also investigated using an adenoviral delivery system.

Results: Western blot analysis showed high EphA2 expression in TCCSUP, T24, and UMUC-3 cell lines. In tissues, the staining intensity of EphA2 was less in normal urothelium but increased greatly in advancing stages of urothelial carcinoma (P < 0.05). Similarly, the staining intensity of Ephrin A-1 was low in normal tissues and high in cancerous tissues, but it was similar across the various stages of urothelial carcinoma (Ta-T4). E-cadherin immunoreactivity decreased in urothelial cancer. Association of EphA2 and Ephrin A-1 expression was found to be significant between Ta stage and T1-T2 (P < 0.04) and Ta and T3-T4 stages (P < 0.0001). Adenovirus delivery of Ephrin A-1 inhibited proliferation of TCCSUP cells.

Conclusion: EphA2 may serve as a novel target for bladder cancer therapy.
ZAP-70 Expression in Normal Pro/Pre B Cells, Mature B Cells, and in B-Cell Acute Lymphoblastic Leukemia
Authors: Crespo Marta , Villamor Neus , Giné Eva , Muntañola Ana , Colomer Dolors , Marafioti Teresa , Jones Margaret , Camós Mireia , Campo Elias , Montserrat Emili , Bosch Francesc
Published: March 2006

Purpose: The ZAP-70 gene is normally expressed in T and natural killer cells, where it is required for the T-cell receptor (TCR) signaling. More recently, it has been described that ZAP-70 contributes to the B-cell development at early stages of B-cell differentiation in mice. The purpose was to investigate the presence of ZAP-70 in normal pro/pre B cells and mature B cells and in tumoral cells from B-acute lymphoblastic leukemias (B-ALL).

Experimental Design: ZAP-70 expression was ascertained by flow cytometry, immunofluorescence, Western blot, and quantitative reverse transcription-PCR. Analysis of ZAP-70 and other signaling proteins of the pre-TCR/TCR was done by Western blot.

Results: ZAP-70 was expressed in pro/pre B cells but not in normal mature B cells derived from bone marrow, peripheral blood, or tonsil. Among tumoral cells, ZAP-70 was expressed in 56% of B-ALLs with pro/pre B-cell phenotype and in 4 of 6 Burkitt/ALL lymphomas. In B-ALL cells, expression of CD38 protein correlated with ZAP-70 expression (P = 0.05). Mutational analysis of the ZAP-70 gene revealed the absence of mutations in cases lacking ZAP-70 expression. Moreover, other elements of the pre-TCR/TCR signaling pathway, like LAT and Lck, were also found in B-ALL cells.

Conclusions: Among normal B-cell subsets, ZAP-70 was found expressed in normal pro/pre B cells but not in a significant proportion of normal B cells with mature phenotype. Moreover, the presence of ZAP-70 in B-ALLs probably reflects their cellular origin. The lack of ZAP-70 expression in normal mature B cells suggests that its expression in mature-derived neoplasms with different cellular origin, such as Burkitt's lymphoma and chronic lymphocytic leukemia, might be due to an aberrant phenomenon.
Plasma Levels of Trefoil Factors are Increased in Patients with Advanced Prostate Cancer
Authors: Vestergaard Else Marie , Borre Michael , Poulsen Steen Seier , Nexø Ebba , Tørring Niels
Published: March 2006

Purpose: Through cDNA array analyses and immunohistochemistry on tissue microarrays, trefoil factor 3 (TFF3) was recently shown to be overexpressed in prostate cancer. The purpose of this study was to test the feasibility of using the levels of trefoil factors as a plasma marker for prostate cancer.

Experimental Design: In 79 patients with prostate cancer, 23 patients with benign prostatic hyperplasia, and 44 healthy individuals plasma TFF1, TFF2, and TFF3 were determined with ELISAs and compared with clinical stage and prostate-specific antigen (PSA) values. Plasma levels of TFF were compared with the immunohistochemical expression of TFF and chromogranin A in 30 prostate cancer tissue samples.

Results: Patients with advanced prostate cancer had significantly higher plasma concentrations of TFF1, TFF2, and TFF3 (P < 0.01) compared with patients with localized disease. Using a cutoff of 200 pmol/L, the sensitivity and specificity of plasma TFF3 in differentiating between patients with localized and advanced disease was 74% (59-85%) and 81% (66-91%). Plasma levels of TFF3 were highest in patients with bone metastases (P = 0.008). Patients with serum PSA >10 μg/L had significantly higher plasma TFF3 values than patients with serum PSA <10 μg/L (P = 0.03) and TFF3 levels were higher in patients with Gleason sums of ≥7 (P = 0.02). Expression of TFF1 and TFF3 determined by immunohistochemistry was increased in patients with prostate cancer but did not correlate with plasma trefoil factor values.

Conclusions: Plasma levels of trefoil factors are increased in patients with advanced prostate cancer. Prospective studies are needed to confirm the predictive utility of trefoil factors in prostate cancer.
Reduced Toxicity and Superior Therapeutic Activity of a Mitomycin C Lipid-Based Prodrug Incorporated in Pegylated Liposomes
Authors: Gabizon Alberto A. , Tzemach Dinah , Horowitz Aviva T. , Shmeeda Hilary , Yeh Jerry , Zalipsky Samuel
Published: March 2006

Purpose: A lipid-based prodrug of mitomycin C [MMC; 2,3-(distearoyloxy)propane-1-dithio-4′-benzyloxycarbonyl-MMC] was designed for liposome formulation. The purpose of this study was to examine the in vitro cytotoxicity, pharmacokinetics, in vivo toxicity, and in vivo antitumor activity of this new lipid-based prodrug formulated in polyethylene glycol–coated (pegylated) liposomes.

Experimental Design: MMC was released from the MMC lipid–based prodrug (MLP) by thiolytic-induced cleavage with a variety of thiol-containing reducing agents. MLP was incorporated with nearly 100% efficiency in cholesterol-free pegylated liposomes with hydrogenated phosphatidylcholine as the main component and a mean vesicle size of ∼90 nm. This formulation was used for in vitro and in vivo tests in rodents.

Results: In vitro, the cytotoxic activity of pegylated liposomal MLP (PL-MLP) was drastically reduced compared with free MMC. However, in the presence of reducing agents, such as cysteine or N-acetyl-cysteine, its activity increased to nearly comparable levels to those of free MMC. Intravenous administration of PL-MLP in rats resulted in a slow clearance indicating stable prodrug retention in liposomes and long circulation time kinetics, with a pharmacokinetic profile substantially different from that of free MMC. In vivo, PL-MLP was ∼3-fold less toxic than free MMC. The therapeutic index and absolute antitumor efficacy of PL-MLP were superior to that of free MMC in the three tumor models tested. In addition, PL-MLP was significantly more active than a formulation of doxorubicin in pegylated liposomes (DOXIL) in the M109R tumor model, a mouse tumor cell line with a multidrug-resistant phenotype.

Conclusions: Delivery of MLP in pegylated liposomes is a potential approach for effective treatment of multidrug-resistant tumors while significantly buffering the toxicity of MMC.
Targeting Heat Shock Proteins for Immunotherapy in Multiple Myeloma: Generation of Myeloma-Specific CTLs Using Dendritic Cells Pulsed with Tumor-Derived gp96
Authors: Qian Jianfei , Wang Siqing , Yang Jing , Xie Jin , Lin Pei , Freeman Muta E. , Yi Qing
Published: March 2006

Purpose: To develop effective immunotherapies for patients with multiple myeloma, it is important to use novel tumor antigens. Recent studies in solid tumors show that tumor-derived heat shock proteins (Hsp) can be used as immunogen; however, no such study has yet been reported in multiple myeloma.

Experimental Design: We examined whether myeloma-derived Hsp gp96 can be used as a myeloma antigen. Specific CTL lines were obtained after repeatedly stimulating T cells with autologous, HLA-A*0201+ dendritic cells pulsed with gp96 derived from HLA-A*0201+ human myeloma cell line (HMCL) U266 or primary myeloma cells.

Results: These T cells lysed not only gp96-pulsed dendritic cells, U266, and other HLA-A*0201+ HMCLs IM-9 and XG1 but also effectively killed HLA-A*0201+ primary myeloma cells from patients. No killing was observed against unpulsed dendritic cells, dendritic cells pulsed with control gp96, HLA-A*0201− HMCLs, and primary myeloma cells, or HLA-A*0201+ nonmyeloma cells. Cytotoxicity was mainly MHC class I/HLA-A*0201 restricted, suggesting that the CTLs recognized gp96-chaperoned peptides on HLA-A*0201 that were derived from shared myeloma antigens and that myeloma cells naturally present these peptides in the context of their surface MHC molecules. Upon antigen stimulation, these T cells secreted IFN-γ and tumor necrosis factor-α, indicating that they belong to type 1 T-cell subsets.

Conclusion: These results show that these T cells are potent CTLs that are able to effectively lyse myeloma cells but not normal blood cells and also suggest that Hsps from allogeneic tumor cells may be used as vaccines to immunize patients.
Homeodomain-Interacting Protein Kinase-2 Restrains Cytosolic Phospholipase A2–Dependent Prostaglandin E2 Generation in Human Colorectal Cancer Cells
Authors: D'Orazi Gabriella , Sciulli Maria Gina , Di Stefano Valeria , Riccioni Sabrina , Frattini Milo , Falcioni Rita , Bertario Lucio , Sacchi Ada , Patrignani Paola
Published: March 2006

Purpose: Homeodomain-interacting protein kinase-2 (HIPK2), a corepressor for homeodomain transcription factors, is a multifunctional kinase whose role in tumor cell survival is not completely clarified. We addressed whether HIPK2 restrains colon tumorigenesis by turning off cytosolic phospholipase A2 (cPLA2)-dependent prostaglandin E2 (PGE2) generation in the light of overwhelming evidence suggesting the contribution of this prostanoid in a variety of cancers.

Experimental Design: In the human colorectal cancer cell line, RKO, we studied the effect of RNA interference for HIPK2 (HIPK2i) on prostanoid biosynthesis, both in the absence and in the presence of the cPLA2 inhibitor arachidonyl trifluoromethyl ketone. We evaluated the role of HIPK2 in the cPLA2 gene regulation by reverse transcriptase-PCR, transcriptional activity, and chromatin immunoprecipitation analyses. The involvement of HIPK2 in tumorigenicity in vivo was studied by tumor growth of HIPK2i cells in nude mice. We compared the gene expression of HIPK2 and cPLA2 in human colorectal cancer specimens by reverse transcriptase-PCR.

Results: HIPK2 silencing was associated with rousing PGE2 biosynthesis that was profoundly suppressed by the cPLA2 inhibitor. HIPK2 overexpression, along with histone deacetylase-1, inhibited the cPLA2-luc promoter that is strongly acetylated in HIPK2i cells. The tumors derived from HIPK2i cells injected in nude mice showed noticeably increased growth compared with parental cells. HIPK2 mRNA levels were significantly higher in colorectal cancers of patients with familial adenomatous polyposis, which showed undetectable cPLA2 levels compared with sporadic colorectal cancer expressing cPLA2.

Conclusions: Our findings reveal the novel mechanism of HIPK2 to restrain progression of human colon tumorigenesis, at least in part, by turning off cPLA2-dependent PGE2 generation.
A multimarker model to predict outcome in tamoxifen-treated breast cancer patients.
Authors: Linke Steven P , Bremer Troy M , Herold Christopher D , Sauter Guido , Diamond Cornelius
Published: March 2006

PURPOSE: This study was designed to produce a model to predict outcome in tamoxifen-treated breast cancer patients based on clinicopathologic features and multiple molecular markers. EXPERIMENTAL DESIGN: This was a retrospective study of 324 stage I to III female breast cancer patients treated with tamoxifen for whom standard clinicopathologic data and tumor tissue microarrays were available. Nine molecular markers were studied by semiquantitative immunohistochemistry and/or fluorescence in situ hybridization. Cox proportional hazards analysis was used to determine the contributions of each variable to disease-specific and overall survival, and machine learning was used to produce a model to predict patient outcome. RESULTS: On a univariate basis, the following features were significantly associated with worse survival: high pathologic tumor or nodal class, histologic grade, epidermal growth factor receptor, ERBB2, MYC, or TP53; absent estrogen receptor (ER) or progesterone receptor; and low BCL2. CCND1 and CDKN1B did not reach statistical significance. On a multivariate basis, nodal class, ER, and MYC were statistically significant as independent factors for survival. However, the benefit of ER-positive status was moderated by BCL2, ERBB2, and progesterone receptor. BCL2 and TP53 also interacted as an independent risk factor. A kernel partial least squares polynomial model was developed with an area under the receiver operating characteristic curve of 0.90. CONCLUSIONS: Our data show the predictive value of BCL2, ERBB2, MYC, and TP53 in addition to the standard hormone receptors and clinicopathologic features, and they show the importance of conditional interpretation of certain molecular markers. Our multimarker predictive model performed significantly better than standard guidelines.
Phase I and Pharmacokinetic Study of Sequences of the Rebeccamycin Analogue NSC 655649 and Cisplatin in Patients with Advanced Solid Tumors
Authors: Ricart Alejandro D. , Hammond Lisa A. , Kuhn John G. , Takimoto Chris H. , Goetz Andrew , Forouzesh Bahram , Forero Leonardo , Ochoa-Bayona Jose L. , Berg Kristin , Tolcher Anthony W. , Rowinsky Eric K.
Published: March 2006

Purpose: To evaluate the feasibility of administering NSC 655649, a water-soluble rebeccamycin analogue that inhibits both topoisomerases I and II, in combination with cisplatin (CDDP) in adults with solid malignancies. Major toxicologic and pharmacologic differences between the two sequences of drug administration were also assessed.

Experimental Design: NSC 655649 was administered as a 60-minute i.v. infusion; CDDP was given i.v. before or after NSC 655649 on day 1. Each patient was treated with alternating drug sequences every 3 weeks; doses of each drug were escalated in separate cohorts of new patients. Sequential dose escalation of NSC 655649 or CDDP resulted in three dosage permutations of NSC 655649/CDDP: 440/50, 550/50, and 440/75 mg/m2. After the maximum tolerated dose level was determined, the feasibility of using granulocyte colony-stimulating factor to permit further dose escalation was explored.

Results: Twenty patients were treated with 70 courses of NSC 655649/CDDP. Myelosuppression was the principal toxicity. The incidence of severe neutropenia, often associated with severe thrombocytopenia, was unacceptably high in minimally pretreated patients at the NSC 655649/CDDP dose level of 550/50 mg/m2 without and with granulocyte colony-stimulating factor. Major pharmacokinetic interactions between NSC 655649 and CDDP were not apparent. No relevant sequence-dependent differences in toxicity or pharmacokinetic variables occurred. Three patients had partial responses.

Conclusions: NSC 655649 and CDDP were well tolerated by minimally pretreated subjects at 440 and 50 mg/m2, respectively. Neither pharmacokinetic interactions between the agents nor sequence-dependent toxicologic or pharmacokinetic effects were apparent. The tolerance and preliminary activity observed with this combination suggest that disease-directed evaluations of the regimen are warranted.
Effective cancer therapy with the alpha-particle emitter [211At]astatine in a mouse model of genetically modified sodium/iodide symporter-expressing tumors.
Authors: Petrich Thorsten , Quintanilla-Martinez Leticia , Korkmaz Zekiye , Samson Elenore , Helmeke Hans J√ºrgen , Meyer Geerd J√ºrgen , Knapp Wolfram H , P√∂tter Eyck
Published: March 2006

PURPOSE: The sodium/iodide symporter (NIS) gene is currently explored in several trials to eradicate experimental cancer with radiodine ((131)I) by its beta-emission. We recently characterized NIS-specific cellular uptake of an alternative halide, radioastatine ((211)At), which emits high-energy alpha-particles. The aim of this study was to investigate in vivo effects of the high linear energy transfer (LET) emitter (211)At on tumor growth and outcome in nude mice. EXPERIMENTAL DESIGN: We administered radioastatide in a fractionated therapy scheme to NMRI nude mice harboring rapidly growing solid tumors established from a papillary thyroid carcinoma cell line genetically modified to express NIS (K1-NIS). Animals were observed over 1 year. Tumor growth, body weight, blood counts, survival, and side effects were measured compared with control groups without therapy and/or lack of NIS expression. RESULTS: Within 3 months, radioastatide caused complete primary tumor eradication in all cases of K1-NIS tumor-bearing nude mice (n = 25) with no tumor recurrence during 1 year follow-up. Survival rates of the K1-NIS/(211)At group were 96% after 6 months and 60% after 1 year, in contrast to those of control groups (maximum survival 40 days). CONCLUSION: Our study indicates that (211)At represents a promising substrate for NIS-mediated therapy of various cancers either with endogenous or gene transfer-mediated NIS expression.
The role of interleukin 1 in growth and metastasis of human cancer xenografts.
Authors: Elaraj Dina M , Weinreich David M , Varghese Sheelu , Puhlmann Markus , Hewitt Stephen M , Carroll Nancy M , Feldman Elizabeth D , Turner Ewa M , Alexander H Richard
Published: March 2006

BACKGROUND: Interleukin 1 (IL-1) is a pluripotent cytokine that promotes angiogenesis, tumor growth, and metastasis in experimental models; its presence in some human cancers is associated with aggressive tumor biology. The purpose of these studies was to characterize the role of IL-1 in human cancers and determine if inhibition of IL-1 via its receptor antagonist, IL-1Ra, alters tumor growth and metastatic potential. METHODS: IL-1 mRNA or protein levels were determined in clinical tumor samples, cancer cell lines, and xenografts using quantitative reverse transcription-PCR or ELISA. Biological activity of tumor-derived IL-1 protein was shown via induction of permeability across endothelial cell monolayers. The effects of recombinant IL-1Ra on tumor lines in culture (cell proliferation and IL-8 secretion) and in xenograft models (tumor growth, metastatic potential, and intratumoral levels of IL-8 and VEGF) were characterized. The effects of IL-1Ra-mediated regression of xenograft growth on angiogenic proteins (IL-8 and VEGF) were evaluated in an IL-1-producing melanoma (SMEL) xenograft model. RESULTS: IL-1 mRNA was highly expressed in more than half of all tested metastatic human tumor specimens including non-small-cell lung carcinoma, colorectal adenocarcinoma, and melanoma tumor samples. Constitutive IL-1 mRNA expression was identified in several cancer cell lines; tumor supernatant from these cell lines produced a significant increase in endothelial cell monolayer permeability, a hallmark event in early angiogenesis, in an IL-1-dependent manner. Moreover, systemic recombinant IL-1Ra resulted in significant inhibition of xenograft growth and neovessel density of IL-1-producing, but not non-IL-1-producing, tumor cell lines. Subsequent analysis of SMEL, a melanoma cell line with constitutive IL-1 production, showed that neither exogenous IL-1 nor IL-1Ra altered tumor cell proliferation rates in vitro. Gene expression analyses of IL-1Ra-treated SMEL xenografts showed a >3-fold down-regulation of 100 genes compared with control including a marked down-regulation of IL-8 and VEGF. CONCLUSIONS: These data show that the IL-1 gene is frequently expressed in metastases from patients with several types of human cancers. IL-1Ra inhibits xenograft growth in IL-1-producing tumors but has no direct antiproliferative effects in vitro; decreased tumor levels of IL-8 and VEGF may be an early surrogate of IL-1Ra-mediated antitumor activity. IL-1Ra may have a role alone or with other agents in the treatment of human cancers.