bluuebunny/crossref_metadata_2025 huggingface dataset:
Pak-1 Expression Increases with Progression of Colorectal Carcinomas to Metastasis
Authors: Carter Julia H. , Douglass Larry E. , Deddens James A. , Colligan Bruce M. , Bhatt Tejal R. , Pemberton Jackson O. , Konicek Susan , Hom Joanne , Marshall Mark , Graff Jeremy R.
Published: September 2005

Purpose: The p21-activated kinase-1 (Pak-1) promotes cell motility and invasiveness. Pak-1 is activated by the Rac, Rho, and Cdc42 small GTPases in response to a variety of stimuli including ras and phosphatidylinositol 3′-kinase/AKT pathway activation. Because Pak-1 plays a central role in regulating cell motility and invasiveness, we sought to determine whether Pak-1 may be involved in the malignant progression of colorectal carcinoma.

Experimental Design: Pak-1 expression was examined by immunohistochemistry in archived tissues from normal human colons, tubular and tubulovillous adenomas, invasive adenocarcinomas (stages I-III/IV), and lymph node metastases (184 total specimens from 38 patients). Specific cytoplasmic immunostaining was evaluated for overall intensity and uniformity to derive a combined histoscore (stain intensity × percentage of epithelium stained).

Results: Pak-1 expression was increased significantly with colorectal cancer progression from normal tissue to lymph node metastases (P < 0.0001). Furthermore, Pak-1 expression was increased significantly in adenomas, invasive carcinomas, and lymph node metastases compared with normal colon (P < 0.0001). Strikingly, Pak-1 expression was significantly higher in lymph node metastases than in invasive cancers, adenomas, or normal colon (P < 0.0001). Moreover, in patients with multiple lesions representing different stages of disease, Pak-1 expression was increased specifically in the most advanced lesions.

Conclusions: This study demonstrates that Pak-1 expression is increased significantly with malignant progression of human colorectal carcinoma. These data, along with numerous functional studies demonstrating a central role for Pak-1 activity in tumor invasiveness and motility, implicate Pak-1 as an exciting target for therapy of colorectal carcinoma.
Molecular Marker Profiles Predict Locoregional Control of Head and Neck Squamous Cell Carcinoma in a Randomized Trial of Continuous Hyperfractionated Accelerated Radiotherapy
Authors: Buffa Francesca M. , Bentzen Søren M. , Daley Frances M. , Dische Stanley , Saunders Michele I. , Richman Paul I. , Wilson George D.
Published: September 2005

Purpose: Identification of factors that assist prediction of tumor response to radiotherapy may aid in refining treatment strategies and improving outcome. Possible association of molecular marker expression profiles with locoregional control of head and neck squamous cell carcinoma was investigated in a randomized trial of conventional versus continuous hyperfractionated accelerated radiotherapy (CHART).

Experimental Design: Tumor material was obtained from 402 patients. Immunohistochemistry was used to assess Ki-67, CD31, p53, Bcl-2, and cyclin D1 expression. A hierarchical clustering algorithm with a Bayesian information criterion was used to group tumors with similar marker expression; resulting expression profiles were then compared in terms of their difference in outcome after CHART and conventionally fractionated radiotherapy.

Results: Molecular marker profile was an independent prognostic factor for locoregional control. This was confirmed in multivariate analysis, including clinical variables such as tumor and nodal status, primary site, histological grade, age, and gender (P < 0.001 and P = 0.006 for local and nodal relapse, respectively). In particular, Bcl-2-positive tumors responded significantly better than average in both arms of the trial. Tumors negative for p53- and Bcl-2, with high and randomly patterned Ki-67 expression, responded worse than average with no benefit from CHART. Tumors with similarly negative p53 and Bcl-2, but low Ki-67 staining, with an organized pattern, benefit significantly from CHART schedule.

Conclusions: This study demonstrates the potential of molecular profiles to predict radiotherapy response of head and neck squamous cell carcinoma and for treatment stratification. Distinct expression profiles correlate with three distinct clinical phenotypes, including good locoregional control, poor locoregional control, and an outcome strongly dependent upon fractionation schedule.
Prognostic Impact of ANX7-GTPase in Metastatic and HER2-Negative Breast Cancer Patients
Authors: Srivastava Meera , Bubendorf Lukas , Raffeld Mark , Bucher Christoph , Torhorst Jochen , Sauter Guido , Olsen Cara , Kallioniemi Olli P. , Eidelman Ofer , Pollard Harvey B.
Published: September 2005

Purpose: ANX7-GTPase located on chromosome 10q21 is significantly altered and associated with hormone-refractory metastatic prostate cancers. Therefore, we investigated whether levels of ANX7 correlate with breast cancer progression and survival

Experimental Design: A diagnostic tumor tissue microarray containing 525 human breast tissue specimens at different stages of the disease was assayed for ANX7 using immunocytochemical methods with ANX7 monoclonal antibody. A separate prognostic tumor tissue microarray containing 553 human breast tissue specimens annotated with clinicopathological parameters was assayed for ANX7, HER2, estrogen receptor, progesterone receptor, and p53 protein.

Results: We report here for the first time that the expression of ANX7-GTPase is significantly enhanced and associated with the presence of metastatic disease (P < 0.0001) in the 525 human breast tissue specimens analyzed. Furthermore, using a separate 553 case retrospective prognostic tumor tissue microarray, we found that increased ANX7 expression is also significantly associated with poor overall patient survival (P < 0.014). This is particularly true when restricted to patients in whom the BRE clinical grade is 2 (P < 0.001) or for whom there is a lack of HER2 expression (P < 0.002). Finally, Cox regression analysis shows that as the expression of ANX7 rises, the probability of survival decreases by more than 10-fold for those patients with HER2-negative tumors. These latter patients represented 66% of the population affected with breast cancer in this study.

Conclusions: High levels of ANX7 in tumor correlate strongly with poor survival of HER2-negative patients and the most aggressive forms of breast cancer. This is the first study to demonstrate that ANX7 antibody has the potential for development into an in vivo diagnostic and therapeutic tool. This simple and reliable immunohistochemical assay may therefore become an important biomarker for metastatic breast cancer diagnosis and management of HER2-negative breast tumor patients.
Antisense RNA Down-Regulation of bcl-2 Expression in DU145 Prostate Cancer Cells Does Not Diminish the Cytostatic Effects of G3139 (Oblimersen)
Authors: Raffo Anthony , Lai Johnathan C. , Stein C. A. , Miller Paul , Scaringe Steven , Khvorova Anastasia , Benimetskaya Luba
Published: September 2005

Purpose: Inhibition of the function of the bcl-2 protein has been postulated to sensitize cells to cytotoxic chemotherapy, and thus provides an attractive target for investigative therapies. G3139, a phosphorothioate antisense oligonucleotide targeted to the initiation codon region of the bcl-2 mRNA, is currently being evaluated in several Phase II and Phase III clinical trials. However, the mechanism of action of this molecule appears to depend on a combination of antisense plus nonantisense events. Indeed, the very idea that bcl-2 is a critical target is, at least in part, an extrapolation from experiments in which intracellular bcl-2 protein concentrations have been dramatically increased, yielding chemoresistant cells.

Experimental Design: In this work, we down-regulated the expression of bcl-2 protein by 80–90% by two different antisense RNA strategies (antisense RNA and small interfering RNA) in DU145 prostate cancer cells.

Results: Even after down-regulation of bcl-2 protein expression by either one of these strategies, the cellular phenotype induced by subsequent G3139 treatment (inhibition of cellular growth and the generation of reactive oxygen species) was essentially identical to that induced in mock-infected or wild-type DU145 cells in which bcl-2 protein expression had not been down-regulated previously.

Conclusions: These results strongly suggest that bcl-2 expression in DU145 cells is not strongly associated with the prolife phenotype and that the mechanism by which G3139 produces its cytostatic effects in this cell line is bcl-2 independent.
Identification of Altered Protein Expression and Post-Translational Modifications in Primary Colorectal Cancer by Using Agarose Two-Dimensional Gel Electrophoresis
Authors: Tomonaga Takeshi , Matsushita Kazuyuki , Yamaguchi Seiko , Oh-Ishi Masamichi , Kodera Yoshio , Maeda Tadakazu , Shimada Hideaki , Ochiai Takenori , Nomura Fumio
Published: September 2005

Purpose: Although numerous proteome studies have been performed recently to identify cancer-related changes in protein expression, only a limited display of relatively abundant proteins has been identified. The aim of this study is to identify novel proteins as potential tumor markers in primary colorectal cancer tissues using a high-resolution two-dimensional gel electrophoresis (2-DE).

Experimental Design: 2-DE using an agarose gel for isoelectric focusing was used to compare protein profiling of 10 colorectal cancer tissues and adjacent normal mucosa. Altered expression and post-translational modification of several proteins were examined using Western blot analysis and immunohistochemistry.

Results: Ninety-seven proteins of 107 spots (90.7%) that were differentially expressed between matched normal and tumor tissues were identified by mass spectrometry. Among them, 42 unique proteins (49 spots) significantly increased or decreased in the tumors. They include eukaryotic translation initiation factor 4H, inorganic pyrophosphatase, anterior gradient 2 homologue, aldolase A, and chloride intracellular channel 1, whose elevated expression in tumor tissues was confirmed by Western blot analysis and immunohistochemistry. Interestingly, only isoform 1 of two transcript variants of eukaryotic translation initiation factor 4H was greatly up-regulated in most of the tumor tissues. Moreover, post-translational modifications of the prolyl-4-hydroxylase β subunit and annexin A2 also were identified.

Conclusions: We identified several novel proteins with altered expression in primary colorectal cancer using agarose 2-DE. This method is a powerful technique with which to search for not only quantitative but also qualitative changes in a biological process of interest and may contribute to the deeper understanding of underlying mechanisms of human cancer.
Second Round Results of the Finnish Population-Based Prostate Cancer Screening Trial
Authors: Mäkinen Tuukka , Tammela Teuvo L. J. , Stenman Ulf-Håkan , Määttänen Liisa , Aro Jussi , Juusela Harri , Martikainen Paula , Hakama Matti , Auvinen Anssi
Published: September 2005

Purpose: Large randomized trials provide the only valid means of quantifying the benefits and drawbacks of prostate-specific antigen (PSA) screening, but the follow-up of ongoing studies is still too short to allow evaluation of mortality. We report here the intermediate indicators of screening efficacy from the second round of the Finnish trial.

Experimental Design: The Finnish trial, with ∼80,000 men in the target population, is the largest component in the European Randomized Study of Screening for Prostate Cancer. The first round was completed in 1996–1999. Each year 8,000 men 55–67 years of age were randomly assigned to the screening arm, and the rest formed the control arm. Men randomized to the screening arm in 1996 were reinvited 4 years later, in 2000, and PSA was determined.

Results: Of the eligible 6415 men, 4407 (69%) eventually participated in the second round of screening. Of the first-round participants, up to 84% (3833 of 4556) attended rescreening. A total of 461 screenees (10.5%) had PSA levels of ≥4 μg/liter. Altogether, 97 cancers were found, yielding an overall detection rate of 2.2% (97 of 4407). Seventy-nine cases were found among the 3833 second-time screenees (detection rate 2.1%) and 18 in those 574 men (3.1%) who had not participated previously. A PSA of ≥4 μg/liter, but negative biopsy in the first screening round was associated with an up to 9-fold risk of cancer in rescreening relative to those with lower PSA levels at baseline. Ninety-one (94%) of all of the detected cancers were clinically localized.

Conclusions: As surrogate measures of an effective screening program, both compliance as well as the overall and advanced prostate cancer detection rates remained acceptable. Men defined as screen-positive but with a negative confirmation of cancer at prevalence screen formed a high-risk group at rescreening.
Differential Methylation Status of Tumor-Associated Genes in Head and Neck Squamous Carcinoma
Authors: Maruya Shin-ichiro , Issa Jean-Pierre J. , Weber Randal S. , Rosenthal David I. , Haviland Joie C. , Lotan Reuben , El-Naggar Adel K.
Published: September 2005

Purpose: Promoter hypermethylation is one of the major mechanisms in the transcriptional inactivation of certain carcinoma-associated genes. Concurrent methylation analysis of multiple, functionally distinct genes may provide important information on their differential alterations and potential association in head and neck squamous carcinogenesis.

Experimental Design: Methylation-specific PCR analysis of the CpG islands of 8 cancer-related genes was performed on 19 cell lines and 32 primary head and neck squamous cell carcinoma (HNSC) specimens with matched histologically normal mucosa and 6 dysplastic lesions. The methylation status and histological features of the specimens were investigated.

Results: In histologically normal squamous mucosa, no to low-level methylation (0–22%) was noted in some specimens at all genes except RARβ2 (50%). Considerable variation in the incidence of methylation of these genes within and between cell lines and tumor specimens was noted. The highest incidences of methylation in the cell lines and primary tumors were noted in RARβ2 (53%), MGMT (37%), p16 (33%), and DAP-K (25%); low incidence of methylations were noted in E-cadherin (2%), p73 (2%) RASSF1A (10%), and p14 (20%) genes. The incidences of methylation of each gene were almost similar between the HNSC cell lines and primary cancer specimens, although methylation of RASSF1A was observed in cell line (26%), but not in dysplasia and primary tumor. RARβ, p16, and MGMT genes showed the highest incidences of methylation in premalignant and invasive carcinomas.

Conclusions: Methylation of p16, RARβ, and MGMT may constitute early events in HNSC tumorigenesis. The infrequent methylation at certain genes suggests a minimal role for this feature in their functional assessment in HNSC. The variability within and between cell lines and tumor specimens supports a heterogeneous and dynamic state of methylation in genes associated with HNSC tumorigenesis.
Rapid Identification of Dihydropyrimidine Dehydrogenase Deficiency by Using a Novel 2-13C-Uracil Breath Test
Authors: Mattison Lori K. , Ezzeldin Hany , Carpenter Mark , Modak Anil , Johnson Martin R. , Diasio Robert B.
Published: September 2005

Purpose: Dihydropyrimidine dehydrogenase (DPD)-deficient cancer patients have been shown to develop severe toxicity after administration of 5-fluorouracil. Routine determination of DPD activity is limited by time-consuming and labor-intensive methods. The purpose of this study was to develop a simple and rapid 2-13C-uracil breath test, which could be applied in most clinical settings to detect DPD-deficient cancer patients.

Experimental Design: Fifty-eight individuals (50 “normal,” 7 partially, and 1 profoundly DPD-deficient) ingested an aqueous solution of 2-13C-uracil (6 mg/kg). 13CO2 levels were determined in exhaled breath at various time intervals up to 180 min using IR spectroscopy (UBiT-IR300). DPD enzyme activity and DPYD genotype were determined by radioassay and denaturing high-performance liquid chromatography, respectively.

Results: The mean (±SE) Cmax, Tmax, δ over baseline values at 50 min (DOB50) and cumulative percentage of 13C dose recovered (PDR) for normal, partially, and profoundly DPD-deficient individuals were 186.4 ± 3.9, 117.1 ± 9.8, and 3.6 DOB; 52 ± 2, 100 ± 18.4, and 120 min; 174.1 ± 4.6, 89.6 ± 11.6, and 0.9 DOB50; and 53.8 ± 1.0, 36.9 ± 2.4, and <1 PDR, respectively. The differences between the normal and DPD-deficient individuals were highly significant (all Ps <0.001).

Conclusions: We demonstrated statistically significant differences in the 2-13C-uracil breath test indices (Cmax, Tmax, DOB50, and PDR) among healthy and DPD-deficient individuals. These data suggest that a single time-point determination (50 min) could rapidly identify DPD-deficient individuals with a less costly and time-consuming method that is applicable for most hospitals or physicians’ offices.
Differential Up-Regulation of Cytosolic and Membrane-Bound Heat Shock Protein 70 in Tumor Cells by Anti-Inflammatory Drugs
Authors: Gehrmann Mathias , Brunner Marion , Pfister Karin , Reichle Albrecht , Kremmer Elisabeth , Multhoff Gabriele
Published: September 2005

Purpose: Modulation of the heat shock protein (HSP) response affects sensitivity to therapeutic agents in cancer. Here, drugs with anti-inflammatory potential (cyclooxygenase 1/2 inhibitors) and peroxidase proliferator-activated receptor-γ agonists were analyzed for their capacity to affect Hsp70 expression in human cancer cells with a divergent Hsp70 membrane expression pattern.

Experimental Design: In dose kinetics, the nonlethal concentration of acetyl-salicyl acid, celecoxib, rofecoxib, and the insulin-sensitizer pioglitazone was identified for the human adenocarcinoma cell line CX−. With the exception of CLX, which was diluted in DMSO, all reagents were dissolved in water. After treatment with the different compounds at nontoxic concentrations for 6 h, followed by a 1-h recovery period, the cytosolic Hsp70 levels were measured in CX-2 and CX− tumor cells by Western blot analysis. Fold increase was calculated in relation to the housekeeping protein tubulin. Membrane-bound Hsp70 was analyzed by flow cytometry using a FITC-labeled Hsp70-specific monoclonal antibody. Untreated cells and cells incubated with equivalent amounts of the diluting agents served as controls. The immunological function was tested in granzyme B apoptosis assays, standard 51Cr release assays, and antibody blocking studies.

Results: Compared with aqua dest, the cytoplasmic amount of Hsp70 was equally enhanced in CX-2 and CX− cells by all compounds. An increase in membrane-bound Hsp70, detected selectively in CX− cells, corresponded to an enhanced sensitivity to granzyme B- and natural killer cell-mediated kill that was blockable by using a Hsp70-specific antibody.

Conclusions: Although increase in cytosolic Hsp70 levels conferred resistance to further stress, membrane-bound Hsp70 rendered tumor cells more sensitive to the immunological attack mediated by granzyme B and natural killer cells. Our data provide a biological rational for combining anti-inflammatory drugs with immunotherapy in cancer therapy.
Evidence That Transfer of Functional p53 Protein Results in Increased Apoptosis in Prostate Cancer
Authors: Pisters Louis L. , Pettaway Curtis A. , Troncoso Patricia , McDonnell Timothy J. , Stephens L. Clifton , Wood Christopher G. , Do Kim-Anh , Brisbay Shawn M. , Wang Xuemei , Hossan Elizabeth A. , Evans Robert B. , Soto Cindy , Jacobson Marc G. , Parker Karen , Merritt James A. , Steiner Mitchell S. , Logothetis Christopher J.
Published: September 2005

Purpose: INGN 201 (Ad-p53) is a replication-defective adenoviral vector that encodes a wild-type p53 gene driven by the cytomegalovirus promoter. INGN 201 has been shown to have antitumoral activity against human prostate cancer cell lines. This study was undertaken to determine the safety of INGN 201 in patients with locally advanced prostate cancer, to assess transgene expression, and to evaluate antitumoral activity.

Experimental Design: Our study included patients with clinical stage T3, T1c-T2a with Gleason score 8–10 disease, or T2a-T2b with Gleason score 7 disease and a prostate-specific antigen level >10 ng/ml. INGN 201 was administered by intraprostatic injection under ultrasonographic guidance. One course of INGN 201 was defined as three separate INGN 201 administrations 2 weeks apart. Biopsies at baseline and 24 h after the first administration were assessed for p53 protein by immunohistochemical staining and for apoptosis by terminal deoxynucleotidyl transferase-mediated nick end labeling assay.

Results: A total of 38 courses of INGN 201 gene therapy were administered to 30 patients, of whom 26 underwent radical prostatectomy. There were no grade 3 or 4 adverse events related to INGN 201 administration. Of the 11 patients with negative baseline immunostaining for p53 protein, 10 had positive p53 immunostaining after the first administration of INGN 201, and 8 had an increase in apoptotic cells by terminal deoxynucleotidyl transferase-mediated nick end labeling staining. All 26 of the patients who underwent radical prostatectomy had significant residual viable prostate cancer, and 12 have experienced biochemical failure (median follow-up, 42 months).

Conclusion: Intraprostatic INGN 201 gene therapy is safe and can reliably result in p53 protein production and apoptosis.
Alternative Splicing of the
Multidrug Resistance Protein 1/ATP Binding Cassette Transporter Subfamily
Gene in Ovarian Cancer Creates Functional Splice Variants and Is Associated with Increased Expression of the Splicing Factors PTB and SRp20
Authors: He Xiaolong , Ee P. L. Rachel , Coon John S. , Beck William T.
Published: September 2005

Purpose: Overexpression of multidrug resistance protein 1 (MRP1) confers resistance to a range of chemotherapeutic agents in cell lines and could be involved in clinical drug resistance of some tumor types also. We examined MRP1 expression in a small series of untreated human ovarian tumors and matched normal tissues.

Experimental Design: We analyzed ten pairs of snap-frozen ovarian tumor and matched normal total ovarian tissues from the same patients for expression of MRP1 by reverse transcription-PCR. Amplified PCR products were sequenced to reveal splicing events of MRP1. MRP1 splice variants were expressed as enhanced green fluorescent fusion proteins in HEK293T cells to demonstrate their localization in the cell and their activity in conferring resistance to doxorubicin. The expression of splicing factors PTB and SRp20 was examined by Western blot.

Results: MRP1 was expressed in all 10 of the pairs of specimens. Multiple MRP1 cDNA fragments of various sizes were amplified between exons 10 and 19. Of interest, more MRP1 cDNA fragments were detected in ovarian tumors than in matched normal tissues in 9 of 10 pairs. We identified 10 splicing forms between exons 10 and 19 of the MRP1 gene with exon skipping ranging from 1 to 7. Amplification of the entire coding region of MRP1 from 1 ovarian tumor revealed >20 splice variants. We found whole and partial exon skipping and partial intron inclusion in these splice variants. We expressed 3 of these MRP1 splice variants in HEK293T cells and found that they appeared to localize to the plasma membrane and were functional in conferring resistance to doxorubicin. In addition, we identified a few nucleotide variations in this gene. To understand the basis for increased splice variants in the tumors, we examined splicing factor expression in these tissues. Western blot analysis revealed that two splicing factors, PTB and SRp20, were overexpressed in most ovarian tumors compared with their matched normal ovarian tissues. Importantly, overexpression of both of these splicing factors was associated with the increased number of MRP1 splicing forms in the ovarian tissues.

Conclusion: The MRP1 gene undergoes alternative splicing at a higher frequency in ovarian tumors than in matched normal tissues. Some of these splice variants confer resistance to doxorubicin. Expression of splicing factors PTB and SRp20 is strongly associated with the alternative splicing of the MRP1 gene.
HER2-Targeted Therapy Reduces Incidence and Progression of Midlife Mammary Tumors in Female Murine Mammary Tumor Virus huHER2-Transgenic Mice
Authors: Finkle David , Quan Zhi Ricky , Asghari Vida , Kloss Jessica , Ghaboosi Nazli , Mai Elaine , Wong Wai Lee , Hollingshead Philip , Schwall Ralph , Koeppen Hartmut , Erickson Sharon
Published: September 2005

Purpose: This study examined the effectiveness of early and prolonged mu4D5 (the murine form of trastuzumab/Herceptin) treatment in transgenic mice that overexpress human HER2 (huHER2), under the murine mammary tumor virus promoter, as a model of huHER2-overexpressing breast cancer.

Experimental Design: Mice were randomly assigned to one of three treatment groups and received i.p. injections from 17 weeks of age until either 52 weeks of age or morbidity. Fourteen mice received 100 mg/kg mu4D5, 14 mice received 100 mg/kg antiherpes simplex virus glycoprotein D control antibody, and 11 mice received a diluent control.

Results: High levels of huHER2 expression were detectable in mammary glands of young virgin founder mice. Mammary adenocarcinomas were frequently found in female founders and progeny at an average age of 28 weeks, with some progressing to metastatic disease. The incidence of mammary tumors was significantly reduced, and tumor growth inhibition was observed in mice receiving mu4D5 compared with control mice. In addition, Harderian gland neoplasms, highly associated with overexpression of huHER2 in this transgenic line, were entirely absent in the mu4D5 treatment group, indicating down-regulation of huHER2 in vivo activity.

Conclusions: Early intervention with mu4D5 was of benefit in our transgenic mice at high risk for developing huHER2-overexpressing breast cancer. This study suggests a potential benefit of early treatment with Herceptin in HER2-positive primary breast cancer.
Identification and Characterization of a T-Helper Peptide from Carcinoembryonic Antigen
Authors: Ruiz Marta , Kobayashi Hiroya , Lasarte Juan José , Prieto Jesús , Borrás-Cuesta Francisco , Celis Esteban , Sarobe Pablo
Published: September 2005

Purpose: The purpose of this research was to identify promiscuous T-helper cell determinants (THd) from carcinoembryonic antigen (CEA) to be used to prime T-cell help for cancer therapy. CEA was selected because this antigen is expressed in an important variety of carcinomas.

Experimental Design: Potential promiscuous THd from CEA were predicted using available computer algorithms. Predicted peptides were synthesized and tested in binding experiments to different HLA-DR molecules. Binder peptides were then used to prime T-cell responses both in vitro and in vivo.

Results: Twenty 15-mer peptides from CEA were predicted to bind to different HLA-DR molecules. The promiscuous character of these peptides was demonstrated in binding experiments. Fifteen of 20 peptides tested were able to bind to HLA-DR4, but only CEA (625–639) was shown to be presented after processing of recombinant CEA. CEA (625–639) was also found to be presented by HLA-DR53. Moreover, immunization of HLA-DR4 transgenic mice with CEA (625–639) in conjunction with class I epitope OVA (257–264), induced a CTL response specific of OVA (257–264).

Conclusions: CEA (625–639) might be a relevant promiscuous THd peptide for cancer therapy.
Rapamycin-Induced Endothelial Cell Death and Tumor Vessel Thrombosis Potentiate Cytotoxic Therapy against Pancreatic Cancer
Authors: Bruns Christiane J. , Koehl Gudrun E. , Guba Markus , Yezhelyev Maksim , Steinbauer Markus , Seeliger Hendrik , Schwend Astrid , Hoehn Anna , Jauch Karl-Walter , Geissler Edward K.
Published: September 2005

Purpose: Despite current chemotherapies, pancreatic cancer remains an uncontrollable, rapidly progressive disease. Here, we tested an approach combining a recently described antiangiogenic drug, rapamycin, with standard gemcitabine cytotoxic therapy on human pancreatic tumor growth.

Experimental Design: Tumor growth was assessed in rapamycin and gemcitabine-treated nude mice orthotopically injected with metastatic L3.6pl human pancreatic cancer cells. H&E staining was performed on tumors, along with Ki67 staining for cell proliferation and immunohistochemical terminal deoxynucleotidyl transferase-mediated nick end labeling and CD31 analysis. Rapamycin-treated tumor vessels were also directly examined in dorsal skin-fold chambers for blood flow after thrombosis induction. Cell death in human umbilical vein endothelial cells was assessed by flow cytometry after annexin-V staining.

Results: Rapamycin therapy alone inhibited tumor growth and metastasis more than gemcitabine, with remarkable long-term tumor control when the drugs were combined. Mechanistically, H&E analysis revealed tumor vessel endothelium damage and thrombosis with rapamycin treatment. Indeed, dorsal skin-fold chamber analysis of rapamycin-treated tumors showed an increased susceptibility of tumor-specific vessels to thrombosis. Furthermore, terminal deoxynucleotidyl transferase-mediated nick end labeling/CD31 double staining of orthotopic tumors demonstrated apoptotic endothelial cells with rapamycin treatment, which also occurred with human umbilical vein endothelial cells in vitro. In contrast, gemcitabine was not antiangiogenic and, despite its known cytotoxicity, did not reduce proliferation in orthotopic tumors; nevertheless, rapamycin did reduce tumor proliferation.

Conclusions: Our data suggest a novel mechanism whereby rapamycin targets pancreatic tumor endothelium for destruction and thrombosis. We propose that rapamycin-based vascular targeting not only reduces tumor vascularization, it decreases the number of proliferating tumor cells to be destroyed by gemcitabine, thus introducing a new, clinically feasible strategy against pancreatic cancer.
Estrogen Receptor/Progesterone Receptor-Negative Breast Cancers of Young African-American Women Have a Higher Frequency of Methylation of Multiple Genes than Those of Caucasian Women1
Authors: Mehrotra Jyoti , Ganpat M. Michelle , Kanaan Yasmine , Fackler Mary Jo , McVeigh Megan , Lahti-Domenici Jaana , Polyak Kornelia , Argani Pedram , Naab Tammy , Garrett Elizabeth , Parmigiani Giovanni , Broome Carolyn , Sukumar Saraswati
Published: September 2005

Purpose: To provide a molecular rationale for negative prognostic factors more prevalent in African-American (AA) than Caucasian (Cau) women, we investigated the frequency of promoter hypermethylation in invasive ductal breast cancers in the two races.

Experimental Design: HIN-1, Twist, Cyclin D2, RAR-β, and RASSF1A were analyzed in DNA from 67 AA and 44 Cau invasive ductal breast cancers, stratified by age and estrogen receptor/progesterone receptor (ER/PR) status, by methylation-specific PCR. Hierarchical multiple logistic regression analysis was applied to determine estimated probabilities of methylation. Expression of HIN-1 mRNA was analyzed by in situ hybridization and quantitative reverse transcribed PCR.

Results: Significant differences between races were observed in the ER−/PR−, age < 50 subgroup; AA tumors had higher frequency of methylation (P < 0.001) in four of five genes as compared with Cau and also a higher prevalence (80 versus 0%; P < 0.005) of three or more methylated genes per tumor. No differences in gene methylation patterns were observed across the two races for ER+/PR+ tumors in all ages and ER−/PR− tumors in age > 50. ER+/PR+ status was associated with higher frequency of methylation in Cau tumors of all ages but only with the age > 50 subgroup in AA. Frequent Cyclin D2 methylation was significantly associated (P = 0.01) with shorter survival time.

Conclusion: ER−/PR−, age < 50 tumors in AA women, have a significantly higher frequency of hypermethylation than in those of Cau women. Comparative studies, such as these, could provide a molecular basis for differences in tumor progression and pathology seen in the two races.
Overexpression of Epithelial Cell Adhesion Molecule Antigen in Gallbladder Carcinoma Is an Independent Marker for Poor Survival
Authors: Varga Meri , Obrist Peter , Schneeberger Stephan , Mühlmann Gilbert , Felgel-Farnholz Christina , Fong Dominic , Zitt Mathias , Brunhuber Thomas , Schäfer Georg , Gastl Guenther , Spizzo Gilbert
Published: September 2005

Purpose: Gallbladder carcinoma is an aggressive type of cancer that is difficult to cure by conventional procedures. There thus is a need to identify novel molecular markers for the assessment of prognosis and as potential therapeutic targets. This retrospective study was designed to investigate the prognostic significance of epithelial cell adhesion molecule (Ep-CAM) overexpression in human gallbladder carcinoma.

Experimental Design: Ep-CAM expression was examined immunohistochemically on paraffin-embedded tissue specimens from 99 patients who underwent surgical treatment for gallbladder carcinoma in the period between August 1988 and May 1999.

Results: Ep-CAM overexpression was found in 63 (63.6%) of the tumor samples. Kaplan-Meier curves showed that Ep-CAM overexpression was significantly related to decreased overall survival (P < 0.01). Overall survival gradually worsened with increasing Ep-CAM scores. Notably, in the subgroup of pT1 tumors (n = 17), patients without Ep-CAM overexpression had a 5-year overall survival rate of 100% compared with 38% (P = 0.01) for patients with Ep-CAM-overexpressing tumors. By univariate analysis, no correlation was found with conventional clinicopathological parameters. Multivariate analysis, including Ep-CAM expression, pT stage, tumor grade, and resection margin involvement, showed that Ep-CAM overexpression was an independent prognostic marker in gallbladder carcinoma (P = 0.03; relative risk, 1.8).

Conclusions: These results demonstrate for the first time that Ep-CAM overexpression is an independent prognostic marker in gallbladder carcinoma and that its prognostic impact should be validated prospectively. Furthermore, the Ep-CAM antigen represents an attractive target for specific therapies with monoclonal antibodies or specific vaccines in patients with Ep-CAM-overexpressing gallbladder carcinoma.
CD95-Mediated Apoptosis Is Impaired at Receptor Level by Cellular FLICE-Inhibitory Protein (Long Form) in Wild-Type p53 Human Ovarian Carcinoma
Authors: Mezzanzanica Delia , Balladore Emanuela , Turatti Fabio , Luison Elena , Alberti Paola , Bagnoli Marina , Figini Mariangela , Mazzoni Alessandra , Raspagliesi Francesco , Oggionni Maria , Pilotti Silvana , Canevari Silvana
Published: September 2005

Purpose: Ovarian carcinoma is a highly lethal malignancy that often becomes resistant to chemotherapy. Alterations in apoptotic signals and p53 status contribute to drug resistance, and CD95-mediated apoptosis is also deficient in resistant cells. We analyzed the mechanism of resistance to CD95-mediated apoptosis in ovarian carcinoma cell lines differing in p53 status.

Experimental Design: CD95-mediated apoptosis was induced by agonistic anti-CD95 antibody, and the apoptotic cascade was monitored with biochemical and functional assays.

Results: CD95-mediated apoptosis was blocked in human ovarian cancer cells. In cell lines with wild-type p53, treatment with the protein synthesis inhibitor cycloheximide (CHX) together with anti-CD95 overcame the resistance, suggesting the presence of a labile inhibiting protein. Indeed, the labile protein cellular FLICE-inhibitory protein long form (c-FLIPL) was found to block caspase-8 recruitment to the death-inducing signaling complex (DISC), and sensitization of cells by CHX was due to c-FLIPL down-modulation at the DISC level. Down-regulation of c-FLIPL with antisense oligonucleotides increased CD95-mediated apoptosis as in cells sensitized by CHX, demonstrating the direct involvement of c-FLIPL in apoptosis resistance. Removal of c-FLIPL block at DISC level allowed full activation of the mitochondrial pathway and, eventually, apoptosis in wild-type p53 cells, whereas in cells with mutated p53, c-FLIPL involvement in CD95-mediated apoptosis resistance appeared to be irrelevant. Immunohistochemical analysis of an ovarian tumor tissue array revealed c-FLIPL expression in samples with no p53 accumulation (P = 0.034), and a significant (P = 0.037) inverse relationship between c-FLIPL and p53 expression levels was also observed in 27 epithelial ovarian cancer specimens with known p53 status.

Conclusion: The inhibitory protein c-FLIPL is involved in resistance to CD95-mediated apoptosis in ovarian carcinoma cells with wild-type p53.
Design of a pH-Sensitive Polymeric Carrier for Drug Release and Its Application in Cancer Therapy
Authors: Kamada Haruhiko , Tsutsumi Yasuo , Yoshioka Yasuo , Yamamoto Yoko , Kodaira Hiroshi , Tsunoda Shin-ichi , Okamoto Takayuki , Mukai Yohei , Shibata Hiroko , Nakagawa Shinsaku , Mayumi Tadanori
Published: September 2005

Purpose: In this study, to optimize the polymeric drug delivery system for cancer chemotherapy, we developed a new pH-sensitive polymeric carrier, poly(vinylpyrrolidone-co-dimethylmaleic anhydride) [PVD], that could gradually release native form of drugs with full activity, from the conjugates in response to changes in pH. We examined the usefulness of PVD as a polymeric drug carrier.

Experimental Design: PVD was radically synthesized with vinylpyrrolidone and 2,3-dimethylmaleic anhydride, which is known to be a pH-reversible amino-protecting reagent. Conjugates between PVD and other drugs, such as Adriamycin (ADR), were prepared under the slightly basic conditions (pH 8.5). The drug-release pattern and the antitumor activity of PVD were examined.

Results: At pH 8.5, the release of the drugs from the conjugate was not observed. In contrast, PVD could release fully active drugs in the native form in response to the change in pH near neutrality, and gradually released drugs at neutral pH (7.0) and slightly acidic pH (6.0). The drug-release pattern in serum was almost similar to that observed during these physiological conditions. The PVD-conjugated ADR showed superior antitumor activity against sarcoma-180 solid tumor in mice, and it had less toxic side effects than free ADR. This enhancement in the antitumor therapeutic window may be due to not only the improvement of plasma half-lives and tumor accumulation of ADR, but also its controlled and sustained release from the conjugates in vivo.

Conclusions: These results indicate that PVD is an effective polymeric carrier for optimizing cancer therapy.
Relevance of Different UGT1A1 Polymorphisms in Irinotecan-Induced Toxicity
Authors: Rouits Elisabeth , Boisdron-Celle Michèle , Dumont Agnès , Guérin Olivier , Morel Alain , Gamelin Erick
Published: September 2005

Purpose: We wanted to assess polymorphisms in the uridine diphosphoglucuronosyl transferase 1A1 (UGT 1A1) gene: the TATA box polymorphism and UGT 1A1 G71R and Y486D mutations in the coding sequence, the main mutations characterizing Gilbert’s syndrome, as predictors of severe toxic event occurrence after irinotecan (CPT-11) administration. Therefore, we set up a rapid, sensitive, and reliable technique in routine practice to detect before CPT-11 treatment, the at-risk patients.

Experimental Design: Seventy-five patients with advanced colorectal cancer and treated with CPT-11 and 5-fluorouracil, entered the study. We used the Pyrosequencing technology a real-time sequencing method, to detect the UGT 1A1 TATA box polymorphisms and mutations in the coding regions. Patients were also assessed for both biochemical and clinical evaluation and tolerance to treatment.

Results: No G71R and Y486D mutations were found in our population. Frequencies for UGT 1A1 TATA box polymorphisms were 41, 47, and 9% for wild-type 6/6, heterozygous 6/7, and Gilbert’s syndrome 7/7, respectively. Tolerance to treatment decreased with increased number of TA repeat with 71% of the patients in 7/7 group who experienced grade 3/4 toxicity.

Conclusions: The method we set up is suitable for the detection of UGT 1A1 polymorphism in routine practice before irinotecan treatment. It could help to detect the patients homozygous or heterozygous for Gilbert’s syndrome, at-risk of CPT 11-induced toxicity, and thus could help to individualize the dose to optimize efficacy and limit toxicity.
Prognostic Significance of
c-kit
Mutation in Localized Gastrointestinal Stromal Tumors
Authors: Kim Tae Won , Lee Hyoungnam , Kang Yoon-Koo , Choe Mi Sun , Ryu Min-Hee , Chang Heung Moon , Kim Jung Sun , Yook Jeong Hwan , Kim Byung Sik , Lee Jung Shin
Published: September 2005

Purpose: Constitutive mutational activation of c-kit has been found to be associated with the pathogenesis of gastrointestinal stromal tumors (GISTs). The prognostic significance of c-kit mutations, however, is still controversial.

Experimental Design: We examined 86 patients curatively resected for localized GIST. Genomic DNA was extracted from paraffin-embedded tumor tissues. Exons 9, 11, 13, and 17 of the c-kit gene were amplified by PCR and sequenced.

Results: Mutations in exon 11 were detected in 61 tumors, and mutations in exon 9 were observed in three tumors, whereas no mutations were detected in exons 13 or 17. The overall c-kit mutation frequency was 74%. Amino acid alterations in the 61 tumors with exon 11 mutations were deletion in 33 tumors, substitution in 20, both deletion and substitution in 4, insertion in 1, and duplication in 3. Histologically, tumors with c-kit mutations showed higher mitotic counts and higher cellularity. The 5-year relapse-free survival (RFS) in patients having GISTs with c-kit mutations was 21%, compared with 60% in those without c-kit mutations. Significantly higher RFS rates were observed in patients with tumors having mitotic counts < 5 mitoses/50 high power field, spindle-cell histology, tumor size < 5 cm, or gastric GISTs. Multivariate analyses indicated association of poorer RFS with a higher mitotic count [≥5 of 50 high power fields; odds ratio (OR) = 3.0], presence of c-kit mutations (OR = 5.6), and a larger tumor size (≥5 cm; OR = 4.2).

Conclusions: The presence of c-kit mutation, along with high mitotic count and larger tumor size, was an independent factor for poor prognosis in patients with localized GISTs.
Suppression of Constitutive and Tumor Necrosis Factor α-Induced Nuclear Factor (NF)-κB Activation and Induction of Apoptosis by Apigenin in Human Prostate Carcinoma PC-3 Cells: Correlation with Down-Regulation of NF-κB-Responsive Genes
Authors: Shukla Sanjeev , Gupta Sanjay
Published: September 2005

Purpose: Development of androgen independence and resistance to apoptosis in prostate cancer are often correlated with high levels of serum tumor necrosis factor (TNF)-α in these patients. The loss of sensitivity to TNF-α-induced apoptosis in androgen-insensitive prostate carcinoma cells is due in part to constitutive activation of Rel/nuclear factor (NF)-κB transcription factors that regulate several cell survival and antiapoptotic genes. Our previous studies have demonstrated growth inhibitory and apoptotic effects of apigenin, a common plant flavonoid, in a variety of human prostate carcinoma cells. Here we examined whether apigenin is effective in inhibiting NF-κB expression in androgen-insensitive human prostate carcinoma cells exhibiting high constitutive levels of NF-κB.

Experimental Design: Using androgen-insensitive human prostate carcinoma PC-3 cells, the effect of apigenin was assessed on NF-κB activation by electrophoretic mobility shift assay and reporter gene assay. Expression of NF-κB subunits p65 and p50, IκBα, p-IκBα, in-beads kinase assay and NF-κB-regulated genes were determined by Western blot analysis. Apoptosis was determined by annexin V/propidium iodide staining after fluorescence-activated cell-sorting analysis.

Results: Treatment of cells with 10–40-μm doses of apigenin inhibited DNA binding and reduced nuclear levels of the p65 and p50 subunits of NF-κB. Apigenin inhibited IκBα degradation and IκBα phosphorylation and significantly decreased IKKα kinase activity. Apigenin also inhibited TNF-α-induced activation of NF-κB via the IκBα pathway, thereby sensitizing the cells to TNF-α-induced apoptosis. The inhibition of NF-κB activation correlated with a decreased expression of NF-κB-dependent reporter gene and suppressed expression of NF-κB-regulated genes [specifically, Bcl2, cyclin D1, cyclooxygenase-2, matrix metalloproteinase 9, nitric oxide synthase-2 (NOS-2), and vascular endothelial growth factor].

Conclusions: Our results indicate that inhibition of NF-κB by apigenin may lead to prostate cancer suppression by transcriptional repression of NF-κB-responsive genes as well as selective sensitization of prostate carcinoma cells to TNF-α-induced apoptosis.
BRCA
Mutations and Risk of Prostate Cancer in Ashkenazi Jews
Authors: Kirchhoff Tomas , Kauff Noah D. , Mitra Nandita , Nafa Kedoudja , Huang Helen , Palmer Crystal , Gulati Tony , Wadsworth Eve , Donat Sheri , Robson Mark E. , Ellis Nathan A. , Offit Kenneth
Published: September 2005

Purpose: The Breast Cancer Linkage Consortium and other family-based ascertainments have suggested that male carriers of BRCA mutations are at increased risk of prostate cancer. Several series looking at the frequency of BRCA mutations in unselected patients with prostate cancer have not confirmed this finding. To clarify this issue, we conducted a large case-control study.

Experimental Design: Blood specimens from 251 unselected Ashkenazi men with prostate cancer were screened for the presence of one of the three common Ashkenazi founder mutations in BRCA1 and BRCA2. The incidence of founder mutations was compared with the incidence of founder mutations in 1472 male Ashkenazi volunteers without prostate cancer using logistic regression analysis after adjusting for age.

Results: Thirteen (5.2%) cases had a deleterious mutation in BRCA1 or BRCA2 compared with 28 (1.9%) controls. After adjusting for age, the presence of a BRCA1 or BRCA2 mutation was associated with the development of prostate cancer (odds ratio, 3.41; 95% confidence interval, 1.64–7.06; P = 0.001). When results were stratified by gene, BRCA2 mutation carriers demonstrated an increased risk of prostate cancer (odds ratio, 4.78; 95% confidence interval, 1.87–12.25; P = 0.001), whereas the risk in BRCA1 mutation carriers was not significantly increased.

Conclusions: BRCA2 mutations are more likely to be found in unselected individuals with prostate cancer than age-matched controls. These results support the hypothesis that deleterious mutations in BRCA2 are associated with an increased risk of prostate cancer.