bluuebunny/crossref_metadata_2025 huggingface dataset:
Transforming Growth Factor β Receptor I Kinase Inhibitor Down-Regulates Cytokine Secretion and Multiple Myeloma Cell Growth in the Bone Marrow Microenvironment
Authors: Hayashi Toshiaki , Hideshima Teru , Nguyen Aaron N. , Munoz Olivier , Podar Klaus , Hamasaki Makoto , Ishitsuka Kenji , Yasui Hiroshi , Richardson Paul , Chakravarty Sarvajit , Murphy Alison , Chauhan Dharminder , Higgins Linda S. , Anderson Kenneth C.
Published: September 2005

Purpose: Transforming growth factors (TGFs) have pleiotropic biological effects on tumor cells and their environment. In multiple myeloma (MM), we have reported that bone marrow stromal cells (BMSCs) from MM patients produce more TGF-β1 than BMSCs from healthy donors, which in turn induces interleukin (IL)-6 secretion. We show here that the TGF-β receptor I kinase inhibitor SD-208 significantly decreases secretion of both IL-6 and vascular endothelial growth factor (VEGF) from BMSCs, as well as tumor cell growth triggered by MM cell adhesion to BMSCs.

Experimental Design: Cytokine production and MM cell proliferation triggered by TGF-β1 or adhesion to BMSCs were examined in the presence or absence of SD-208. Effects of SD-208 on TGF-β1–induced signaling pathways triggering IL-6 and VEGF transcription in BMSCs were also delineated.

Results: SD-208 significantly inhibits not only transcription but also secretion of both IL-6 and VEGF from BMSCs triggered by either TGF-β1 or adhesion of MM cells to BMSCs. Moreover, SD-208 decreased tumor cell growth triggered by MM cell adhesion to BMSCs. SD-208 works, at least in part, by blocking TGF-β1–triggered nuclear accumulation of Smad2/3 and hypoxia-inducible factor 1α, as well as related production of IL-6 and VEGF, respectively.

Conclusions: These studies indicate that SD-208 inhibits production of cytokines mediating MM cell growth, survival, drug resistance, and migration in the BM milieu, thereby providing the preclinical rationale for clinical evaluation of SD-208 to improve patient outcome in MM.
Epigenetic Inactivation of
ID4
in Colorectal Carcinomas Correlates with Poor Differentiation and Unfavorable Prognosis
Authors: Umetani Naoyuki , Takeuchi Hiroya , Fujimoto Akihide , Shinozaki Masaru , Bilchik Anton J. , Hoon Dave S. B.
Published: September 2005

Purpose: ID4 gene is a member of the inhibitor of DNA binding (ID) family proteins that inhibit DNA binding of basic helix-loop-helix transcription factors. The epigenetic inactivation of ID4 gene on colorectal cancer (CRC) development and its clinical significance was assessed.

Experimental Design: In CRC cell lines, ID4 methylation status of the promoter region was assessed by methylation-specific PCR and bisulfite sequencing. The mRNA expression level was assessed by quantitative real-time reverse transcription-PCR. The methylation status of 9 normal epithelia, 13 adenomas, 92 primary CRCs, and 26 liver metastases was assessed by methylation-specific PCR. ID4 protein expression was assessed by immunohistochemistry analysis of tissue specimen.

Results: CRC cell lines were shown to be hypermethylated, and mRNA expression was suppressed and could be restored by 5-aza–cytidine treatment. In clinical specimens from normal epithelia, adenomas, primary CRCs, and liver metastases, the frequency of ID4 hypermethylation was 0 of 9 (0%), 0 of 13 (0%), 49 of 92 (53%), and 19 of 26 (73%), respectively, with a significant elevation according to CRC pathological progression. Methylation status of primary CRCs significantly correlated with histopathological tumor grade (P = 0.028). Immunohistochemistry analysis showed ID4 expression of normal colon epithelia, adenomas, and unmethylated primary CRCs but not hypermethylated CRC specimens. Among 76 American Joint Committee on Cancer stage I to IV patients who had undergone curative surgical resection, overall survival was significantly poorer in patients with hypermethylated ID4 bearing tumors (P = 0.0066).

Conclusions: ID4 gene is a potential tumor suppressor gene for which methylation status may play an important role in the CRC progression.
Genetic and Expression Profiles of Squamous Cell Carcinoma of the Head and Neck Correlate with Cisplatin Sensitivity and Resistance in Cell Lines and Patients
Authors: Akervall Jan , Guo Xiang , Qian Chao-Nan , Schoumans Jacqueline , Leeser Brandon , Kort Eric , Cole Andrew , Resau James , Bradford Carol , Carey Thomas , Wennerberg Johan , Anderson Harald , Tennvall Jan , Teh Bin T.
Published: September 2005

Purpose: The choice of treatment for squamous cell carcinoma of the head and neck (SCCHN) is still primarily based on the tumor-node-metastasis classification. However, it is reasonable to believe that biological profiles of SCCHN may be independently associated with response to therapy. The aim of the present study was to examine genetic changes and gene expression profiles that might correlate with sensitivity to cisplatin [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay] in 10 SCCHN cell lines.

Experimental Design: Five cisplatin-sensitive and five cisplatin-resistant cell lines [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay] were studied by comparative genomic hybridization, spectral karyotyping, and cDNA microarray analysis (21,632 sequence-validated human cDNA; confirmation by reverse transcriptase-PCR for selected genes). For the MET proto-oncogene, which showed low expression in the chemosensitive cell lines, we did immunohistochemical staining on SCCHN of 29 patients who received induction chemotherapy.

Results: The five cisplatin-resistant cell lines showed significantly more genetic imbalances (regions of loss and amplification) and chromosomal abnormalities by comparative genomic hybridization and spectral karyotyping, respectively, than did the five cisplatin-sensitive cell lines. Microarray studies identified ∼60 genes that clearly distinguish between the two groups of cell lines. Some of these genes are known to be involved in tumor progression, metastasis, and drug resistance. We identified low expression of c-met (immunohistochemistry) as a predictive factor for complete response in nondiploid tumors (P = 0.026).

Conclusions: We conclude that cisplatin sensitivity and resistance are related to distinctive differences in the genetic and expression profiles in individual SCCHN tumor cell lines and in SCCHN patients. The genes we have identified may serve as potential targets for novel treatment strategies.
ING-1, a Monoclonal Antibody Targeting Ep-CAM in Patients with Advanced Adenocarcinomas
Authors: de Bono Johann S. , Tolcher Anthony W. , Forero Andre , Vanhove Gertrude F. A. , Takimoto Chris , Bauer Robert J. , Hammond Lisa A. , Patnaik Amita , White Mark L. , Shen Sui , Khazaeli Muhammad B. , Rowinsky Eric K. , LoBuglio Albert F.
Published: September 2005

Purpose: To determine the feasibility of administration, safety, toxicity, immunogenicity, pharmacokinetics, maximum tolerated dose, and biodistribution of ING-1, a high-affinity, Human-Engineered monoclonal antibody (heMAb) to the Mr 40,000 epithelial cell adhesion molecule Ep-CAM, in patients with advanced adenocarcinomas.

Experimental Design: ING-1 was initially administered to patients as a 1-hour intravenous infusion every 3 weeks. Toxicity and pharmacokinetic data led to the evaluation of a weekly schedule. The distribution of iodine-131 (131I)-labeled ING-1 was studied.

Results: Twenty-five patients received 82 courses of ING-1. Minimal toxicity was initially observed at the 0.03-, 0.10-, and 0.30-mg/kg dose levels. A patient dosed at 1.0 mg/kg developed acute pancreatitis with severe abdominal pain, nausea, and vomiting. A patient dosed at 0.3 mg/kg had an asymptomatic amylase and lipase elevation to 502 units/L and 1,627 units/L, respectively. Both patients made uncomplicated recoveries. No other dose-limiting toxicities were observed. Regardless of dose, the volume of distribution (mean ± SEM) was 46.6 ± 1.6 mL/kg. ING-1 clearance decreased with increasing dose. To minimize toxicity and increase dose intensity, we then administered ING-1 weekly. No significant toxicity was observed in 7 patients dosed at 0.1 mg/kg. Studies of 131I-labeled ING-1 biodistribution showed radiolocalization to colorectal and prostate cancers. A patient with colorectal cancer had an 80% decrement in the levels of carcinoembryonic antigen.

Conclusion: The recommended dose for ING-1 is 0.10 mg/kg by intravenous infusion weekly. The absence of severe toxicity at this dose, low immunogenicity, and preliminary evidence of ING-1 tumor localization and antitumor efficacy support the further clinical development of this antibody to treat Ep-CAM–positive malignant diseases.
Associations between Serum Testosterone Fall and Cognitive Function in Prostate Cancer Patients
Authors: Salminen Eeva K. , Portin Raija I. , Koskinen Aki , Helenius Hans , Nurmi Martti
Published: September 2005

Data on the association between cognition and testosterone levels in elderly men are inconclusive. Androgen deprivation therapy is commonly used in the treatment of prostate cancer with the aim of achieving castration levels of serum testosterone. The study group comprised 26 elderly men (mean age 65 years) with newly diagnosed prostate cancer. Cognitive testing was done at baseline and at 6 and 12 months on androgen deprivation therapy. Cognitive performances were evaluated using verbal, visuomotor, and memory tests as well as tests of processing speed and attention. Castration levels of testosterone were achieved in all patients by 6 months. Significant associations between cognitive performances and testosterone decline were documented: visuomotor slowing, slowed reaction times in some attentional domains including working memory and impaired hit rate in a vigilance test, impaired delayed recall and recognition speed of letters, but improvement in object recall. The results suggest selective associations between testosterone decline and cognition. Documentation of cognitive performance with changes in serum testosterone levels has substantial implications for informed patient support in prostate cancer.
Nuclear Factor-κB Nuclear Localization Is Predictive of Biochemical Recurrence in Patients with Positive Margin Prostate Cancer
Authors: Fradet Vincent , Lessard Laurent , Bégin Louis R. , Karakiewicz Pierre , Masson Anne-Marie Mes , Saad Fred
Published: September 2005

Purpose: Radical prostatectomy (RP) patients with positive surgical margins are at increased risk for recurrence, emphasizing the need for prognostic markers to stratify probable outcome for optimal patient management decisions. We tested the hypothesis that nuclear localization of nuclear factor (NF)-κB, a transcription factor involved in the regulation of cell growth, angiogenesis, invasion, and apoptosis, is associated with an increased risk of biochemical recurrence after RP.

Experimental Design: Analyses addressed data from 42 patients (age range, 52–72 years; mean age, 63.7 years) who exhibited positive surgical margins after RP. Immunohistochemical analysis of NF-κB (p65) was performed on the positive margin tissue. A nuclear staining cutoff of >5% was considered positive. The relation between nuclear NF-κB expression and biochemical recurrence (prostate-specific antigen >0.3 ng/mL and rising) after RP was tested in univariate and multivariate Cox regression models.

Results: Biochemical recurrence was recorded in 23 patients (54.8%; median follow-up, 3.2 years). Univariate Cox regression demonstrated a 4.9-fold (95% confidence interval, 1.5–16.7; P = 0.01) higher rate of recurrence in men with NF-κB > 5%. In the multivariate model, after controlling for primary (P = 0.004) and secondary (P = 0.7) Gleason patterns, lymph node (P = 0.06) and seminal vesicle invasion (P = 0.2), and preoperative prostate-specific antigen (P = 0.009), NF-κB > 5% was associated with a 6.2-fold higher risk of biochemical recurrence (95% confidence interval, 1.7–23.5; P = 0.007).

Conclusions: In univariate and multivariate analysis, NF-κB nuclear expression was strongly predictive of biochemical recurrence in patients with positive surgical margins after RP. We propose that nuclear NF-κB may serve as a useful independent molecular marker for stratifying patients at risk for recurrence.
Cyclin A as a Predictive Factor for Chemotherapy Response in Advanced Head and Neck Cancer
Authors: Rodriguez-Pinilla Maria , Rodriguez-Peralto Jose L. , Hitt Ricardo , Sanchez Jose J. , Ballestin Claudio , Diez Ana , Sanchez-Verde Lydia , Alameda Francesc , Sanchez-Cespedes Montserrat
Published: September 2005

Purpose: Overall survival of head and neck squamous cell cancer (HNSCC) patients has not improved despite advances in our understanding of the biology and molecular features of this disease. In particular, patients with advanced HNSCC have the poorest prognosis. To understand more about the contribution of cell cycle alterations to HNSCC development and their possible value in predicting prognosis and response to chemotherapy, we evaluated the levels of proteins involved in cell cycle control in patients diagnosed with advanced HNSCC.

Experimental Design: A tissue microarray was made with 122 HNSCC specimens obtained from biopsy material. Protein expression was evaluated by immunohistochemistry and correlated with clinical and pathological characteristics.

Results: Multiple alterations at various checkpoints of cell cycle progression were observed. Loss of P16 protein was less common in oropharyngeal tumors than at other HNSCC locations (P = 0.02). Evaluation of the simultaneous expression of different proteins highlighted direct correlations (P < 0.05) such as that of the cyclin-dependent kinases with their cyclin-partners, and the Ki-67 protein with cyclin-dependent kinases 1, cyclin A (CA) and cyclin B1. Median overall survival and time-to-progression were longer in patients with CA-expressing tumors (not reached versus 34.4 months, P = 0.02) and (47.3 versus 14.6 months, P = 0.006), respectively. Moreover, expression of CA in tumors predicted a better response to chemotherapy. Positive expression of cyclin E in tumors was also associated with an increased median time-to-progression (14.6 versus 25.8 months, P = 0.04). Finally, patients with cyclin D1-expressing tumors had shorter median overall survival (29.6 months versus not reached, P = 0.05) and shorter median time-to-progression (21.5 months versus not reached, P = 0.06). However, in a multivariate analysis a CA-negative–expressing tumor was the only independent poor prognostic factor in the entire cohort of HNSCC patients [odds ratio, 2.3; 95% confidence interval (CI) = 1.2–4.5; P = 0.01].

Conclusions: Our results provide detailed information on the molecular profile of cell cycle components in HNSCCs and identify CA-negative–expressing tumors as an independent marker of tumor progression and poor response to chemotherapy in patients diagnosed with advanced HNSCC.
7-Hydroxystaurosporine (UCN-01) Inhibition of Akt Thr308 but not Ser473 Phosphorylation
Authors: Kondapaka Sudhir B. , Zarnowski MaryJane , Yver Dena R. , Sausville Edward A. , Cushman Samuel W.
Published: September 2005

7-Hydroxystaurosporine (UCN-01) infused for 72 hours by continuous i.v. infusion induced insulin resistance during phase I clinical trials. To understand the mechanism for this observation, we examined the effect of UCN-01 on insulin-stimulated glucose transport activity with 3-O-methylglucose in isolated rat adipose cells. UCN-01 inhibits glucose transport activity in a dose-dependent manner at all insulin concentrations. At the clinically relevant concentration of 0.25 μmol/L UCN-01, glucose transport is inhibited 66, 29, and 26% at insulin concentrations of 10, 50, and 100,000 (100K) microunits/mL respectively, thus shifting the dose-response curve to the right. Increasing concentrations of UCN-01 up to 2.5 μmol/L progressively shift the insulin dose-response curve even further. As Akt is known to mediate in part action initiated at the insulin receptor, we also studied the effect of UCN-01 on Akt activation in whole-cell homogenates of these cells. Decreased glucose transport activity directly parallels decreased Akt Thr308 phosphorylation in both an insulin and UCN-01 dose-dependent manner, whereas Akt Ser473 phosphorylation is inhibited only at the lowest insulin concentration, and then, only modestly. UCN-01 also inhibits insulin-induced Thr308 but not Ser473 phosphorylation of Akt associated with the plasma membranes and low-density microsomes and inhibits translocation of GLUT4 from low-density microsomes to plasma membranes as expected from the glucose transport activity measurements. These data suggest that UCN-01 induces clinical insulin resistance by blocking Akt activation and subsequent GLUT4 translocation in response to insulin, and this effect appears to occur by inhibiting Thr308 phosphorylation even in the face of almost completely unaffected Ser473 phosphorylation.
Overexpression of Aurora-A Contributes to Malignant Development of Human Esophageal Squamous Cell Carcinoma
Authors: Tong Tong , Zhong Yali , Kong Jianping , Dong Lijia , Song Yongmei , Fu Ming , Liu Zhihua , Wang Mingrong , Guo Liping , Lu Shixin , Wu Ming , Zhan Qimin
Published: September 2005

Purpose: Aurora-A/STK15/BTAK, a centrosome-associated oncogenic protein, is implicated in the control of mitosis. Overexpression of Aurora-A has been shown to result in chromosomal aberration and genomic instability. Multiple lines of evidence indicate that Aurora-A induces cell malignant transformation. In the current study, we are interested in investigating the expression of Aurora-A in human esophageal squamous cell carcinoma (ESCC) and characterizing the association of Aurora-A with ESCCmalignant progression.

Experimental Design: Aurora-A protein expression was examined in 84 ESCC tissues and 81 paired normal adjacent tissues by either immunohistochemistry or Western blot analysis. In addition, a gene-knockdown small interfering RNA technique was used in ESCC cells to investigate whether Aurora-A contributes to the ability of a tumor to grow invasively.

Results: The amount of Aurora-A protein in ESCC was considerably higher than that in normal adjacent tissues. Overexpression of Aurora-A was observed in 57 of 84 (67.5%) ESCC samples. In contrast, <2% of normal adjacent tissue displayed high expression of Aurora-A. Interestingly, overexpression of Aurora-A seemed to correlate with the invasive malignancy of ESCC. Disruption of endogenous Aurora-A using small interfering RNA technique substantially suppressed cell migrating ability.

Conclusion: The findings presented in this report show that Aurora-A expression is elevated in human esophageal squamous cell carcinoma and is possibly associated with tumor invasion, indicating that overexpression of Aurora-A may contribute to ESCC occurrence and progression.
Immunohistochemical and Molecular Analysis of Human Melanomas for Expression of the Human Cancer-Testis Antigens NY-ESO-1 and LAGE-1
Authors: Vaughan Hilary A. , Svobodova Suzanne , MacGregor Duncan , Sturrock Sue , Jungbluth Achim A. , Browning Judy , Davis Ian D. , Parente Philip , Chen Yao-Tseng , Stockert Elisabeth , St. Clair Fiona , Old Lloyd J. , Cebon Jonathan
Published: September 2005

Purpose: NY-ESO-1 and LAGE-1 are homologous cancer-testis antigens, which are expressed in many different cancers. It is essential to type tumors accurately to assess patient suitability for clinical trials which target these. This study evaluates typing strategies used to distinguish these two homologous but distinct antigens and to characterize and quantitate expression of each in clinical samples.

Experimental Design: We typed 120 malignant melanomas for the expression of NY-ESO-1 and LAGE-1 with immunohistochemistry, reverse transcription-PCR (RT-PCR), and quantitative real-time (qRT-PCR), which was also used to explore the relationship between NY-ESO-1 and LAGE expression.

Results: The two monoclonal antibodies ES121 and E978 had very similar immunohistochemistry reactivities. Both were specific for NY-ESO-1 because neither bound to homologous LAGE-1 peptides despite 84% overall amino acid homology. Of 120 melanomas tested by immunohistochemistry, NY-ESO-1 was expressed in >50% of cells in 23 melanomas (19%), between 11 and 50% cells in 15 (12.5%), <11% cells in 16 (13.5%), and negative in 66 (55%). Although specific for both antigens, the PCR methods did not provide this information about microheterogeneity. Polymorphisms in the LAGE-1 gene resulted in false negative LAGE-1 typing by qRT-PCR by inhibiting binding of oligonucleotide primers, thereby showing the exquisite specificity of qRT-PCR as a typing method.

Conclusions: For NY-ESO-1 typing, immunohistochemistry compared favorably with the RT-PCR, with the added advantage of being able to characterize heterogeneity of antigen expression. Because neither mAb bound LAGE and because there was no coordinate expression LAGE and NY-ESO-1, separate typing for each is required.
Pharmacokinetic Model-Predicted Anticancer Drug Concentrations in Human Tumors
Authors: Gallo James M. , Vicini Paolo , Orlansky Amy , Li Shaolan , Zhou Feng , Ma Jianguo , Pulfer Sharon , Bookman Michel A. , Guo Ping
Published: September 2005

In an era when molecular and targeted anticancer therapeutics is a major focus and when understanding drug dynamics in tumor is critical, it seems advantageous to be able to relate drug concentrations in tumors to corresponding biological end points. To that end, a novel method, based on physiologically based hybrid pharmacokinetic models, is presented to predict human tumor drug concentrations. Such models consist of a forcing function, describing the plasma drug concentration-time profile, which is linked to a model describing drug disposition in tumors. The hybrid models are originally derived from preclinical data and then scaled to humans. Integral to the scale-up procedure is the ability to derive human forcing functions directly from clinical pharmacokinetic data. Three examples of this approach are presented based on preclinical investigations with carboplatin, topotecan, and temozolomide. Translation of these preclinical hybrid models to humans used a Monte Carlo simulation technique that accounted for intrasubject and intersubject variability. Different pharmacokinetic end points, such as the AUC tumor, were extracted from the simulated human tumor drug concentrations to show how the predicted drug concentrations might be used to select drug-dosing regimens. It is believed that this modeling strategy can be used as an aid in the drug development process by providing key insights into drug disposition in tumors and by offering a foundation to optimize drug regimen design.
Characterization and Multiparameter Analysis of Visual Adverse Events in Irofulven Single-Agent Phase I and II Trials
Authors: Raymond Eric , Kahatt Carmen , Rigolet Marie Hélène , Sutherland William , Lokiec François , Alexandre Jérôme , Tombal Bertrand , Elman Michael , Lee Michael S. , MacDonald John R. , Cullen Michael , Misset Jean-Louis , Cvitkovic Esteban
Published: September 2005

Purpose: Irofulven (6-hydroxymethylacylfulvene) is a novel agent, derived from illudin S, with potent apoptotic effects in preclinical models. In the Phase I trial evaluating intermittent weekly schedules, visual symptoms were dose limiting. The aim of this analysis was to better characterize the visual adverse events of irofulven and provide treatment guidelines.

Experimental Design: Clinical data from 277 patients entered in single-agent Phase I to II clinical trials who received irofulven on days 1 and 15 every 4 weeks; days 1, 8, and 15 every 4 weeks; or days 1 and 8 every 3 weeks were included in this multiparameter analysis.

Results: Overall, 74 patients (27%) experienced visual symptoms. The most frequently reported symptoms were flashing lights (12% of patients), blurred vision (9%), and photosensitivity (8%). Grade 3 toxicity was observed in 12 patients (4%). The incidence and severity of visual events were dose dependent, with no grade 3 visual events occurring at doses ≤0.50 mg/kg and grade 1 to 2 events in only 12% and 8% of patients, at doses of ≤0.50 mg/kg and ≤20 mg/m2, respectively. Grade 1 to 2 toxicity was reversible in most patients. Abnormal electroretinogram and abnormal visual fields were noted after irofulven treatment in 24 of 39 patients (62%) and 15 of 26 patients (58%), respectively. All but 1 patient who had electroretinogram assessment received doses >0.50 mg/kg. Clinical examination and visual field assessment were found to be better correlated with symptoms and appear to be more appropriate for surveillance of irofulven retinal symptoms than electroretinograms.

Conclusions: On the basis of retained antitumor activity and reversibility of grade 1 and 2 visual symptoms at lower doses, it appears that an irofulven dose of ≤0.50 mg/kg or ≤20 mg/m2, not to exceed 50 mg in a single dose, given as a 30-minute infusion on days 1 and 8 every 3 weeks or days 1 and 15 every 4 weeks minimizes the frequency and severity of visual symptoms.
Preradiotherapy Hemoglobin Level but not Microvessel Density Predicts Locoregional Control and Survival in Laryngeal Cancer Treated with Primary Radical Radiotherapy
Authors: Haugen Hedda , Magnusson Bengt , Svensson Marie , Mercke Claes
Published: September 2005

Purpose: To evaluate the roles of preradiotherapy hemoglobin level and microvessel density (MVD) as predictive factors for tumor control and survival in patients with laryngeal cancer treated with primary radiotherapy.

Experimental Design: Two hundred and fourteen patients with stage I-IV laryngeal cancer were included in the analysis. Patients were treated with once daily fractionated radiotherapy over 6.5 weeks or twice daily fractionated radiotherapy over 4.5 weeks up to total doses of 62 to 68 Gy. Preradiotherapy hemoglobin levels were obtained from patient journals, and pretreatment tumor biopsies were stained with CD34 antibody for the counting of microvessels. The prognostic implication of preradiotherapy hemoglobin level and MVD on tumor control and survival was tested.

Results: Five-year locoregional control probability was 88.9% for patients with preradiotherapy hemoglobin levels >137.5 g/L (median) and 64.4% for patients with preradiotherapy hemoglobin levels <137.5 g/L (P = 0.01). The corresponding figures for disease-free survival were 87.8 and 62.8% (P = 0.007), respectively, and for overall survival 58.1 and 40.3% (P < 0.001), respectively.

In multivariate analysis, tumor stage and preradiotherapy hemoglobin level were significant prognostic factors for locoregional control and disease-free survival, whereas tumor stage, preradiotherapy hemoglobin-level, gender, and age were significant prognostic factors for overall survival. No correlation was found between MVD and tumor control and survival.

Conclusion: Preradiotherapy hemoglobin level, but not MVD, predicts locoregional control and survival in patients with laryngeal cancer treated with radiotherapy.
Increased Levels of Interleukin-10 in Serum from Patients with Hepatocellular Carcinoma Correlate with Profound Numerical Deficiencies and Immature Phenotype of Circulating Dendritic Cell Subsets
Authors: Beckebaum Susanne , Zhang Xia , Chen Xiao , Yu Zhengya , Frilling Andrea , Dworacki Grzegorz , Grosse-Wilde Hans , Broelsch Christoph Erich , Gerken Guido , Cicinnati Vito R.
Published: September 2005

Increased levels of interleukin (IL)-10 have been described as a negative prognostic indicator for survival in patients with various types of cancer. IL-10 exerts tolerogenic and immunosuppressive effects on dendritic cells, which are crucial for the induction of an antitumor immune response. Blood dendritic cell antigen (BDCA)-2 and BDCA-4 are specifically expressed by CD123bright CD11c− plasmacytoid dendritic cells; whereas BDCA-1 and BDCA-3 define 2 distinct subsets of CD11c+ myeloid dendritic cells. In this study, the T-helper cell (Th)1/Th2 cytokine serum profile of 65 hepatocellular carcinoma patients was assessed. We found that serum levels of IL-10 were substantially increased in hepatocellular carcinoma patients as compared with controls. Peripheral blood mononuclear cells from healthy volunteers were exposed to recombinant human (rh)IL-10 in vitro to additionally characterize its impact on distinct blood dendritic cell subsets. A dramatic decrease of all myeloid dendritic cell (MDC) and plasmacytoid dendritic cell (PDC) subsets was detectable after 24 hours of continuous rhIL-10 exposure. Moreover, the expression of HLA-DR, CD80 and CD86, was significantly reduced on rhIL-10-treated dendritic cell subsets. Direct ex vivo flow cytometric analysis of various dendritic cell subpopulations in peripheral blood from hepatocellular carcinoma patients revealed an immature phenotype and a substantial reduction of circulating dendritic cells that was associated with increased IL-10 concentrations in serum and with tumor progression. These findings confirm a predominantly immunosuppressive role of IL-10 for circulating dendritic cells in patients with hepatocellular carcinoma and, thus, may indicate novel aspects of tumor immune evasion.
Inhibition of Proliferation and Induction of Apoptosis by 25-Hydroxyvitamin D3-3β-(2)-Bromoacetate, a Nontoxic and Vitamin D Receptor-Alkylating Analog of 25-Hydroxyvitamin D3 in Prostate Cancer Cells
Authors: Swamy Narasimha , Chen Tai C. , Peleg Sara , Dhawan Puneet , Christakos Sylvia , Stewart LaMonica V. , Weigel Nancy L. , Mehta Rajendra G. , Holick Michael F. , Ray Rahul
Published: September 2005

The 25-hydroxyvitamin D3 (25-OH-D3) is a nontoxic and low-affinity vitamin D receptor (VDR)-binding metabolic precursor of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. We hypothesized that covalent attachment of a 25-OH-D3 analog to the hormone-binding pocket of VDR might convert the latter into transcriptionally active holo-form, making 25-OH-D3 biologically active. Furthermore, it might be possible to translate the nontoxic nature of 25-OH-D3 into its analog. We showed earlier that 25-hydroxyvitamin D3-3-bromoacetate (25-OH-D3-3-BE) alkylated the hormone-binding pocket of VDR. In this communication we describe that 10−6 mol/L of 25-OH-D3-3-BE inhibited the growth of keratinocytes, LNCaP, and LAPC-4 androgen-sensitive and PC-3 and DU145 androgen-refractory prostate cancer cells, and PZ-HPV-7 immortalized normal prostate cells with similar or stronger efficacy as 1,25(OH)2D3. But its effect was strongest in LNCaP, PC-3, LAPC-4, and DU145 cells. Furthermore, 25-OH-D3-3-BE was toxic to these prostate cancer cells and caused these cells to undergo apoptosis as shown by DNA-fragmentation and caspase-activation assays. In a reporter assay with COS-7 cells, transfected with a 1α,25-dihydroxyvitamin D3-24-hydroxylase (24-OHase)-construct and VDR-expression vector, 25-OH-D3-3-BE induced 24-OHase promoter activity. In a “pull down assay” with PC-3 cells, 25-OH-D3-3-BE induced strong interaction between VDR and general transcription factors, retinoid X receptor, and GRIP-1. Collectively, these results strongly suggested that the cellular effects of 25-OH-D3-3-BE were manifested via 1,25(OH)2D3/VDR signaling pathway. A toxicity study in CD-1 mice showed that 166 μg/kg of 25-OH-D3-3-BE did not raise serum-calcium beyond vehicle control. Collectively, these results strongly suggested that 25-OH-D3-3-BE has a strong potential as a therapeutic agent for androgen-sensitive and androgen-refractory prostate cancer.
MAP2K4/MKK4 Expression in Pancreatic Cancer
Authors: Xin Wei , Yun Ki J. , Ricci Francesca , Zahurak Marianna , Qiu Wanglong , Su Gloria H. , Yeo Charles J. , Hruban Ralph H. , Kern Scott E. , Iacobuzio-Donahue Christine A.
Published: September 2005

MKK4 (MAP2K4/SEK1) is a member of the mitogen-activated protein kinase family, originally identified as a kinase involved in the stress-activated protein kinase pathway by directly phosphorylating c-Jun NH2-terminal kinase. MKK4 genetic inactivation has been observed in a subset of pancreatic carcinomas, implicating deregulation of the stress-activated protein kinase pathway in pancreatic carcinogenesis. We evaluated Mkk4 protein expression patterns by immunohistochemical labeling in a series of 60 resected primary infiltrating pancreatic adenocarcinomas (24 cases with known MKK4 genetic status), and 14 different tissue arrays representing the primary carcinoma and all of the gross metastases from 26 patients that died of metastatic pancreatic cancer. Among the surgically resected carcinomas, focal or diffuse-positive immunolabeling for Mkk4 protein was found in 52 of 60 cases (86.7%). Among the eight carcinomas with negative Mkk4 immunolabeling, three harbored a homozygous deletion or intragenic mutation of the MKK4 gene, in contrast to none of the 52 cases with positive Mkk4 immunolabeling (P < 0.01). Loss of Mkk4 immunolabeling showed a trend toward shorter survival, with Mkk4-positive carcinomas having half the risk of death than Mkk4-negative carcinomas (P = 0.09). Mkk4 immunolabeling patterns were also evaluated among unresectable primary and metastatic cancer tissues from autopsy specimens, indicating intact Mkk4 immunolabeling in 88.8% of the unresectable primary carcinomas as compared with 63.3% of distant metastases (P < 0.001). Our data indicate that the loss of Mkk4 protein expression in pancreatic carcinomas may be more frequent than suggested by the rates of genetic inactivation alone and that MKK4 loss may contribute to disease progression. The correlation of MKK4 genetic status with immunolabeling patterns validate this approach for the evaluation of MKK4 status in routine histologic sections and may provide useful information regarding patient prognosis.
Phase II Feasibility and Pharmacokinetic Study of Concurrent Administration of Trastuzumab and High-Dose Chemotherapy in Advanced HER2+ Breast Cancer
Authors: Nieto Yago , Vredenburgh James J. , Shpall Elizabeth J. , Bearman Scott I. , McSweeney Peter A. , Chao Nelson , Rizzieri David , Gasparetto Christina , Matthes Steve , Barón Anna E. , Jones Roy B.
Published: September 2005

Purpose: To evaluate the safety of concurrent treatment with trastuzumab and high-dose chemotherapy (HDC), using cyclophosphamide, cisplatin, and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), with autologous hematopoietic progenitor cells support, in patients with HER2+ advanced breast cancer.

Experimental Design: Patients with HER2-overexpressing high-risk primary breast cancer (HRPBC; defined as ≥4 involved nodes or inflammatory disease), or metastatic breast cancer (MBC) were eligible. Treatment consisted of a loading dose of trastuzumab at 4 mg/kg (day −5), HDC (days −5 to −2), autologous hematopoietic progenitor cells infusion on day 0, and weekly maintenance trastuzumab (2 mg/kg) from day +1 (minimum of 9 doses). Cardiac monitoring included serial left ventricular ejection fraction measurements before treatment and on days +20 and +65.

Results: Thirty-three patients were prospectively enrolled (13 HRPBC, 20 MBC). Toxicity seemed similar to that expected with this HDC regimen alone. Neutrophils and platelets engrafted promptly. There were no cases of grade 4 or 5 toxicity. One patient experienced symptomatic grade 3 acute cardiac failure on day −4, responsive to treatment. Trastuzumab did not alter the pharmacokinetics of HDC. Eleven of twelve MBC patients with measurable disease (nine of them refractory to previous chemotherapy) experienced an objective response (9 complete and 2 partial responses). At median follow-up of 34 (13–58) months, all HRPBC patients remain alive and free of disease; the MBC group has event-free survival and overall survival rates of 45 and 70%, respectively.

Conclusions: Incorporation of trastuzumab into HDC (cyclophosphamide, cisplatin, and BCNU) is feasible, with no apparent increased toxicity or pharmacokinetic interactions.
A Phase I Open Label Study of the Farnesyltransferase Inhibitor CP-609,754 in Patients with Advanced Malignant Tumors
Authors: Moulder Stacy L. , Mahany John J. , Lush Richard , Rocha-Lima Caio , Langevin Michael , Ferrante Karen J. , Bartkowski Lisa Michele , Kajiji Shama M. , Noe Dennis A. , Paillet Simone , Sullivan Daniel M.
Published: September 2005

Purpose: The purpose of this phase I clinical trial was to determine the maximum-tolerated dose and toxicity of CP-609,754 in patients with solid tumors refractory to standard therapies, to determine the cellular effects of CP-609,754 on its molecular target (farnesyltransferase), and to determine the recommended phase II dose (RP2D) of this agent.

Experimental Design: Consenting patients with adequate bone marrow, liver, and renal function were enrolled with an accelerated dose strategy with single-patient parallel cohorts in whom the drug was given orally either once or twice daily. Once a dose-limiting toxicity was encountered or two patients developed Common Toxicity Criteria ≥ grade 2 toxicities, a modified Fibonacci sequence was initiated. Blood samples were collected during cycle 1 for pharmacokinetic and pharmacodynamic analyses.

Results: A total of 68 cycles of CP-609,754 was administered to 21 patients enrolled in this study. The dose escalation was from 20 mg once daily to 640 mg twice per day, and at the highest dose level, one of six patients developed a dose-limiting toxicity of grade 3 neuropathy. The drug was otherwise well tolerated, and the maximum-tolerated dose was not reached because of the large number of tablets that would have been required for additional dose escalation. Pharmacokinetic analyses showed a proportional increase in exposure with dose, rapid oral absorption, and a half-life of ∼3 hours. Pharmacodynamic results predict a 95% maximal inhibition of peripheral blood mononuclear cell farnesyltransferase activity 2 hours postdose, on average, with a dose of 400 mg twice per day of CP-609,754.

Conclusions: On the basis of the safety findings and the pharmacokinetic and pharmacodynamic analyses, the RP2D of CP-609,754 is ≥640 mg twice per day.
Thymidylate Synthase 5′- and 3′-Untranslated Region Polymorphisms Associated with Risk and Progression of Squamous Cell Carcinoma of the Head and Neck
Authors: Zhang Zhengdong , Shi Qiuling , Sturgis Erich M. , Spitz Margaret R. , Hong Waun Ki , Wei Qingyi
Published: September 2005

Purpose: Folate deficiency and reduced DNA repair capacity are established risk factors for squamous cell carcinoma of the head and neck (SCCHN). We hypothesized that polymorphisms of the thymidylate synthase (TYMS) gene, which regulates a key enzyme in folate metabolism required for DNA synthesis and repair, are associated with SCCHN risk.

Experimental Design: In a hospital-based case-control study of 704 SCCHN cases and 1,085 controls, frequency matched by age, sex, and ethnicity, we genotyped the TSER (thymidylate synthase in the 5′-untranslated enhanced region) and TS3′UTR (thymidylate synthase in the 3′-untranslated region) polymorphisms.

Results: The TS3′UTR 0bp/0bp genotype was associated with a significantly decreased risk of SCCHN [adjusted odd ratio (OR) = 0.67, 95% confidence interval (CI) = 0.47–0.94] compared with the 6bp/6bp genotype, but the TSER polymorphism had no main effect on risk of SCCHN. When we evaluated the two polymorphisms together by the number of protective alleles (the TSER 3R and TS3′UTR 0bp alleles), we found that the combined genotypes with four protective alleles (the TSER 3R3R and TS3′UTR 0bp/0bp) was associated with significantly decreased SCCHN risk (OR = 0.60, 95% CI = 0.37–0.98). In addition, the TS3′UTR 0bp genotypes were associated in an allele dose-dependent manner with a decreased risk of overall stage IV oral cancer (OR = 0.84, 95% CI = 0.52–1.34 for the 6bp/0bp genotype and OR = 0.26, 95% CI = 0.08–0.87 for the 0bp/0bp genotype; Ptrend = 0.035).

Conclusion: The TSER and TS3′UTR polymorphisms are associated with SCCHN risk. The TSER 3R and TS3′UTR 0bp alleles seemed to jointly protect against SCCHN. In particular, the 0bp allele seemed to protect against oral cancer progression.
Detection of Methylated Apoptosis-Associated Genes in Urine Sediments of Bladder Cancer Patients
Authors: Friedrich Martin G. , Weisenberger Daniel J. , Cheng Jonathan C. , Chandrasoma Shahin , Siegmund Kimberly D. , Gonzalgo Mark L. , Toma Marieta I. , Huland Hartwig , Yoo Christine , Tsai Yvonne C. , Nichols Peter W. , Bochner Bernard H. , Jones Peter A. , Liang Gangning
Published: September 2005

Purpose: There is increasing evidence for a fundamental role for epigenetic silencing of apoptotic pathways in cancer. Changes in DNA methylation can be detected with a high degree of sensitivity, so we used the MethyLight assay to determine how methylation patterns of apoptosis-associated genes change during bladder carcinogenesis and whether DNA methylation could be detected in urine sediments.

Experimental Design: We analyzed the methylation status of the 5′ regions of 12 apoptosis-associated genes (ARF, FADD, TNFRSF21, BAX, LITAF, DAPK, TMS-1, BCL2, RASSF1A, TERT, TNFRSF25, and EDNRB) in 18 bladder cancer cell lines, 127 bladder cancer samples, and 37 samples of adjacent normal bladder mucosa using the quantitative MethyLight assay. We also analyzed the methylation status in urine sediments of 20 cancer-free volunteers and 37 bladder cancer patients.

Results: The 5′ regions of DAPK, BCL2, TERT, RASSFIA, and TNFRSF25 showed significant increases in methylation levels when compared with nonmalignant adjacent tissue (P ≤ 0.01). Methylation levels of BCL2 were significantly associated with tumor staging and grading (P ≤ 0.01), whereas methylation levels of RASSF1A and ARF were only associated with tumor stage (P ≤ 0.04), and TERT methylation and EDNRB methylation were predictors of tumor grade (P ≤ 0.02). To investigate clinical usefulness for noninvasive bladder cancer detection, we further analyzed the methylation status of the markers in urine samples of patients with bladder cancer. Methylation of DAPK, BCL2, and TERT in urine sediment DNA from bladder cancer patients was detected in the majority of samples (78%), whereas they were unmethylated in the urine sediment DNA from age-matched cancer-free individuals.

Conclusions: Our results indicate that methylation of the 5′ region of apoptosis-associated genes is a common finding in patients with bladder carcinoma. The ability to detect methylation not only in bladder tissue, but also in urine sediments, suggests that methylation markers are promising tools for noninvasive detection of bladder cancers. Our results also indicate that some methylation markers, such as those in regions of RASSF1A and TNFRSF25, might be of limited use for detection because they are also methylated in normal bladder tissues.
Antifolate Resistance in a HeLa Cell Line Associated With Impaired Transport Independent of the Reduced Folate Carrier
Authors: Zhao Rongbao , Chattopadhyay Shrikanta , Hanscom Marie , Goldman I. David
Published: September 2005

Prior studies from this laboratory documented the prevalence of methotrexate (MTX) transport activity with a low pH optimum in human solid tumor cell lines. In HeLa cells, this low pH activity has high affinity for pemetrexed [PMX (Alimta)] and is reduced folate carrier (RFC)-independent because it is not diminished in a RFC-null subline (R5). R5 cells also have residual transport activity, with high specificity for PMX, at neutral pH. In the current study, a R5 subline, R1, was selected under MTX selective pressure at a modest reduction in pH. There was markedly decreased MTX and PMX transport at both pH 5.5 and pH 7.4. When MTX was removed, there was a slow return of transport activity, and when MTX was added back, there was loss of transport at both pH values within 8 weeks. In R1 cells, there was a marked decrease in accumulation of PMX, MTX, and folic acid along with a decrease in growth inhibition by these and other antifolates that require a facilitative process to gain entry into cells. These data demonstrate that (i) RFC-independent transport in HeLa cells at low and neutral pH contributes to antifolate activity (in particular, to PMX activity) and can be diminished by antifolate selective pressure and (ii) the loss of these activities results in marked resistance to PMX, an agent for which there is little or no loss of activity when transport mediated by RFC is abolished. These observations suggest that transport activity in RFC-null HeLa R5 cells at neutral and low pH may reflect the same carrier-mediated process.
Immunocytochemical Identification of VPAC1, VPAC2, and PAC1 Receptors in Normal and Neoplastic Human Tissues with Subtype-Specific Antibodies
Authors: Schulz Solveig , Röcken Christoph , Mawrin Christian , Weise Wolfgang , Höllt Volker , Schulz Stefan
Published: September 2005

Human tumors frequently overexpress receptors for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP). However, none of the VIP/PACAP receptor proteins has been visualized individually in human tumors. Here, we developed and characterized a panel of antipeptide antibodies to the carboxyl-terminal regions of the VIP/PACAP receptor subtypes vasoactive intestinal peptide receptor (VPAC)1, VPAC2, and pituitary adenylate cyclase-activating peptide receptor (PAC)1. Specificity of the antisera was shown by the following: (1) detection of broad bands migrating at Mr 50,000 to 70,000 in Western blots of membranes from receptor-expressing tumors and receptor-transfected cells; (2) cell surface staining of VIP/PACAP receptor-transfected cells; (3) translocation of VIP/PACAP receptor immunostaining in transfected cells after agonist exposure; and (4) abolition of tissue immunostaining by preadsorbtion of the antibodies with their immunizing peptides. The distribution of VIP/PACAP receptors was investigated in 98 human tumors and their tissues of origin. VPAC1, VPAC2, and PAC1 receptors were clearly located at the plasma membrane of the tumor cells in a variety of human neoplasms. In the gastrointestinal tract, VPAC1 receptor immunoreactivity was abundant in the mucosa and myenteric neurons; VPAC2 receptor immunoreactivity was detected in neuroendocrine cells, blood vessels, and smooth muscle; and PAC1 receptor immunoreactivity was found in myenteric neurons. This is the first localization of all of the VIP/PACAP receptor subtypes in human formalin-fixed, paraffin-embedded tissues. VIP/PACAP receptor visualization with this simple and rapid immunohistochemical method will facilitate identification of tumors with a sufficient receptor overexpression for diagnostic or therapeutic intervention.