bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased Intratumor Vα24-Positive Natural Killer T Cells: A Prognostic Factor for Primary Colorectal Carcinomas
Authors: Tachibana Tsuyoshi , Onodera Hisashi , Tsuruyama Tatsuaki , Mori Akira , Nagayama Satoshi , Hiai Hiroshi , Imamura Masayuki
Published: March 2006

Purpose: Human invariant natural killer T (NKT) cells are novel, distinct lymphocyte populations with a restricted T-cell receptor repertoire (Vα24-Vβ11). They play a pivotal role in immunoregulation and in antitumor activities. This study focused on Vα24+ NKT cells in colorectal carcinomas and their clinicopathologic significance.

Experimental Design: Vα24+ NKT-cell infiltration immunohistochemistry was studied in a total of 103 colorectal carcinoma cases. The degree of NKT-cell infiltration in tumors was evaluated as low (<7 NKT cells/5 HPF) or high (≥7 NKT cells/5 HPF). The correlation between the degree of infiltrated Vα24+ NKT cells and clinicopathologic variables was studied statistically.

Results: A small number of Vα24+ NKT cells were found in the normal colorectal mucosa (2.6 ± 3.7 cells/5 HPF); however, their number increased remarkably in colorectal carcinomas (15.2 ± 16.3 cells/5 HPF; P = 0.0003) and a majority showed phenotype of activation. Higher NKT-cell infiltration was more frequent in women than in men (P = 0.034) and correlated with fewer lymph node metastases (P = 0.042). Patients with high NKT-cell infiltration showed higher overall (P = 0.018) as well as disease-free (P = 0.0006) survival rates. Intratumor NKT-cell infiltration was an independent prognostic factor for the overall (P = 0.033) and disease-free (P = 0.0064) survival rates.

Conclusions: Increased infiltration of Vα24+ NKT cells was observed in colorectal carcinomas. Higher Vα24+ NKT-cell infiltration in colorectal carcinomas was an independent prognostic factor for favorable prognosis.
Silencing of Human Phosphatidylethanolamine-Binding Protein 4 Sensitizes Breast Cancer Cells to Tumor Necrosis Factor-α–Induced Apoptosis and Cell Growth Arrest
Authors: Wang Xiaojian , Li Nan , Li Hongzhe , Liu Bin , Qiu Jianming , Chen Taoyong , Cao Xuetao
Published: March 2006

Purpose: The current therapeutic approach is not so effective in breast cancer patients. Alternative treatment protocols aimed at different targets need to be explored. We recently reported a novel phosphatidylethanolamine-binding protein, human phosphatidylethanolamine-binding protein 4 (hPEBP4), as an antiapoptotic molecule. The finding led us to explore a promising approach for breast cancer therapy via silencing the expression of hPEBP4.

Experimental Design: hPEBP4 expression in clinical breast specimens was examined by Tissue Microarrays. RNA interference was used to silence hPEBP4 expression in MCF-7 breast carcinoma cells and the effects on cell proliferation, cell cycle progression, apoptosis, as well as underlying mechanisms, were investigated.

Results: hPEBP4 was found to be expressed in up to 50% of breast cancers but in only <4% of normal breast tissues. Silencing of hPEBP4 potentiated tumor necrosis factor-α (TNF-α)–induced apoptosis and cell cycle arrest in MCF-7 cells, which was due to the increased mitogen-activated protein kinase activation and the enhanced phosphatidylethanolamine externalization. Further investigation showed that silencing of hPEBP4 in MCF-7 cells promoted TNF-α-induced stability of p53, up-regulation of phospho-p53ser15, p21waf/cip, and Bax, and down-regulation of Bcl-2 and Bcl-xL, which were shown to depend on extracellular signal-regulated kinase 1/2 and c-jun NH2-terminal kinase activation by hPEBP4 silencing. Moreover, the increased proportion of cells in the G0-G1 phase of cell cycle was observed in hPEBP4-silenced MCF-7 cells on TNF-α treatment and the expression of cyclin A and cyclin E was down-regulated more significantly.

Conclusions: The antiapoptotic effect and the preferential expression pattern in breast cancer tissues make hPEBP4 a new target for breast cancer therapy. Silencing of hPEBP4 expression may be a promising approach for the treatment of breast carcinoma.
Vascular Endothelial Growth Factor Trap Combined with Paclitaxel Strikingly Inhibits Tumor and Ascites, Prolonging Survival in a Human Ovarian Cancer Model
Authors: Hu Limin , Hofmann Judith , Holash Jocelyn , Yancopoulos George D. , Sood Anil K. , Jaffe Robert B.
Published: March 2006

Ovarian cancer is characterized by i.p. carcinomatosis and massive ascites. Vascular endothelial growth factor (VEGF) plays a pivotal role in tumor angiogenesis and vascular leakage leading to ascites. We assessed the efficacy of a soluble decoy receptor (VEGF Trap) combined with paclitaxel, in a mouse model of human ovarian cancer. Tumor burden after VEGF Trap plus paclitaxel was reduced by ∼98% versus controls. No measurable ascites developed in the treated group. Morphologic studies showed that most residual tumor had degenerative changes. Diaphragmatic and hepatic tumors were not found in the VEGF Trap plus paclitaxel group in contrast to controls, indicating lack of metastasis. In vivo FITC-lectin tumor vessel imaging showed sparse, short, straight vessels in treated mice as compared to controls, in which vessels were numerous, irregular, tortuous, and leaky. In a survival study, all controls underwent euthanasia between 29 and 58 days after tumor cell inoculation (cachexia, extensive ascites, and tumor masses). In the VEGF Trap plus paclitaxel group, mice were ambulating and eating normally with no signs of disease for at least 81 days after tumor cell inoculation, and survival occurred for 129.9 ± 38.88 days with no further treatment. We conclude that combination therapy with VEGF Trap plus paclitaxel may provide a novel, long-lasting therapeutic strategy for treatment of patients with ovarian cancer associated with ascites.
Allogeneic Transplant with Reduced Intensity Conditioning Regimens may Overcome the Poor Prognosis of B-Cell Chronic Lymphocytic Leukemia with Unmutated Immunoglobulin Variable Heavy-Chain Gene and Chromosomal Abnormalities (11q− and 17p−)
Authors: Caballero Dolores , García-Marco Jose A. , Martino Rodrigo , Mateos Victoria , Ribera José M. , Sarrá José , León Angel , Sanz Guillermo , de la Serna Javier , Cabrera Rafael , González Marcos , Sierra Jorge , San Miguel Jesús
Published: March 2006

Purpose: To evaluate the efficacy of reduced intensity conditioning (RIC) allogeneic transplant in 30 patients with poor-prognosis chronic lymphocytic leukemia (CLL) and/or high-risk molecular/cytogenetic characteristics.

Experimental Design: Eighty-three percent of patients had active disease at the moment of transplant. That is, 14 of the 23 patients analyzed (60%) had unmutated immunoglobulin variable heavy-chain gene (IgVH) status; 8 of 25 patients (32%) had 11q−, with four of them also displaying unmutated IgVH; and six (24%) had 17p− (five were also unmutated).

Results: After a median follow-up of 47.3 months, all 22 patients alive are disease free; overall survival and event-free survival (EFS) at 6 years were 70% and 72%, respectively. According to molecular/cytogenetic characteristics, overall survival and EFS for unmutated CLL and/or with 11q− aberration (n = 13) were 90% and 92%, respectively, not significantly different to those with normal in situ hybridization, 13q− and +12, or mutated CLL (n = 7). All six patients with 17p deletion were transplanted with active disease, including three with refractory disease; all except one reached complete remission after the transplant and two are alive and disease free. Nonrelapse mortality (NRM) was 20%; more than two lines before transplant is an independent prognostic factor for NRM (P = 0,02), EFS (P = 0.02), and overall survival (P = 0.01). Patients older than 55 years have a higher risk of NRM (hazard ratio, 12.8; 95% confidence interval, 1.5-111). Minimal residual disease was monitored by multiparametric flow cytometry in 21 patients. Clearance of CD79/CD5/CD19/CD23 cells in bone marrow was achieved in 68% and 94% of the patients at days 100 and 360, respectively.

Conclusion: According to these results, RIC allogeneic transplant could overcome the adverse prognosis of patients with unmutated CLL as well as those with 11q− or 17p−.
Clinicopathologic and Biological Significance of Kallikrein 6 Overexpression in Human Gastric Cancer
Authors: Nagahara Hisashi , Mimori Koshi , Utsunomiya Tohru , Barnard Graham F. , Ohira Masaichi , Hirakawa Kosei , Mori Masaki
Published: March 2006

Purpose: Human kallikrein genes (KLK) have been reported to be involved in human malignancies and several KLKs are promising biomarkers of prostate, ovarian, testicular, and breast cancers. Herein, we investigated the clinicopathologic and biological significance of KLK6 gene expression in human gastric cancer.

Patients and Methods: Using real-time reverse transcription-PCR, we analyzed the KLK6 expression status with respect to various clinicopathologic variables in 66 patients with gastric cancer. In addition, we established a KLK6 stably suppressed gastric cancer cell line (MKN28) using small interfering RNA–mediated gene silencing, and investigated its effects on the cell proliferation rate, cell cycle, and invasiveness.

Results: The KLK6 gene expression in cancerous tissue (0.37 ± 0.53) was significantly (P < 0.000001) higher than that in noncancerous tissue (0.026 ± 0.060). Elevated KLK6 expression was significantly associated with lymphatic invasion (P = 0.03). Furthermore, patients with a high KLK6 expression had a significantly poorer survival rate than those with a low KLK6 expression (P = 0.03). Therefore, we showed that KLK6 gene silencing with KLK6 small interfering RNA effectively suppressed the cell proliferation rate (P = 0.002), cell population in the S phase (P < 0.01), and invasiveness (P < 0.01) in comparison to mock-transfected cells.

Conclusions: The KLK6 gene is markedly overexpressed in gastric cancer tissue and its expression status may be a powerful prognostic indicator for patients with gastric cancer. Our findings also suggest that KLK6 may possibly be a novel target for gastric cancer therapy by gene-silencing procedures.
Overexpression of Platelet-Derived Growth Factor Receptor α in Endothelial Cells of Hepatocellular Carcinoma Associated with High Metastatic Potential
Authors: Zhang Ti , Sun Hui-Chuan , Xu Yang , Zhang Ke-Zhi , Wang Lu , Qin Lun-Xiu , Wu Wei-Zhong , Liu Yin-Kun , Ye Sheng-Long , Tang Zhao-You
Published: March 2006

Purpose: Little information is available on the heterogeneity of the vascular endothelium in hepatocellular carcinoma. The aim of this study was to identify the differentially expressed genes in tumor endothelial cells from highly metastatic hepatocellular carcinoma.

Experimental Design: Magnetic beads conjugated with anti-CD31 antibody were used to isolate vascular endothelial cells from hepatocellular carcinoma xenografts with different metastatic potentials in nude mice. Gene expression profiles for different endothelial cells were compared by use of cDNA microarray. The up-regulated gene was confirmed by reverse transcription-PCR, real-time PCR, and immunohistochemistry.

Results: cDNA microarray analysis revealed differential expression patterns in seven genes consistently presented in endothelial cells isolated from hepatocellular carcinoma with different metastatic potentials. Overexpression of platelet-derived growth factor receptor α was found only in the endothelium of highly metastatic hepatocellular carcinoma, which was confirmed by reverse transcription-PCR, real-time PCR, and immunohistochemistry. Oral administration of STI571 (imatinib mesylate or Glivec), a protein tyrosine kinase inhibitor of platelet-derived growth factor receptor, combined with s.c. injection of IFN-α not only effectively reduced tumor weight (by 81.8%) and microvessel density (by 70.2%) but also inhibited lung metastasis (by 100%). Furthermore, immunohistochemical analysis of platelet-derived growth factor receptor α in human hepatocellular carcinoma tissues revealed its correlation with postoperative recurrence, especially in patients without microvessel invasion.

Conclusions: The gene expression of hepatocellular carcinoma vascular endothelium is different between tumors with different metastatic potential. Platelet-derived growth factor receptor α, which is overexpressed in endothelium of highly metastatic hepatocellular carcinoma, may serve as a biomarker for predicting metastasis and a therapeutic target for highly metastatic hepatocellular carcinoma.
Establishment of a Serum Tumor Marker for Preclinical Trials of Mouse Prostate Cancer Models
Authors: Huizen Isaac Van , Wu Guojun , Moussa Madeleine , Chin Joseph L. , Fenster Aaron , Lacefield James C. , Sakai Hideki , Greenberg Norman M. , Xuan Jim W.
Published: March 2006

Current prostate cancer research in both basic and preclinical trial studies employ genetically engineered mouse models. However, unlike in human prostate cancer patients, rodents have no counterpart of prostatic-specific antigen (PSA) for monitoring prostate cancer initiation and progression. In this study, we established a mouse serum tumor marker from a mouse homologue of human prostate secretory protein of 94 amino acids (PSP94). Immunohistochemistry studies on different histologic grades from both transgenic and knock-in mouse prostate cancer models showed the down-regulation of tissue PSP94 expression (P < 0.001), the same as for PSA and PSP94 in humans. The presence of mouse serum PSP94 was shown by affinity column and immunoprecipitation purification using a polyclonal mouse PSP94 antibody. A competitive ELISA protocol was established to quantify serum PSP94 levels with a sensitivity of 1 ng/mL. Quantified serum levels of mouse PSP94 ranged from 49.84 ng/mL in wild-type mice to 113.86, 400.45, and 930.90 ng/mL in mouse prostatic intraepithelial neoplasia with microinvasion, well differentiated, moderately differentiated, and poorly differentiated prostate cancer genetically engineered prostate cancer mice, respectively (P < 0.01, n = 68). This increase in serum PSP94 is also well correlated with age and tumor weight. Through longitudinal monitoring of serum PSP94 levels of castrated mice (androgen ablation therapy), we found a correlation between responsiveness/refractory prostate tissues and serum PSP94 levels. The utility of mouse serum PSP94 as a marker in hormone therapy was further confirmed by three-dimensional ultrasound imaging. The establishment of the first rodent prostate cancer serum biomarker will greatly facilitate both basic and preclinical research on human prostate cancer.
Kinetics of Tempol for Prevention of Xerostomia Following Head and Neck Irradiation in a Mouse Model
Authors: Cotrim Ana P. , Sowers Anastasia L. , Lodde Beatrijs M. , Vitolo Joseph M. , Kingman Albert , Russo Angelo , Mitchell James B. , Baum Bruce J.
Published: March 2006

Purpose: Radiotherapy is commonly used to treat the majority of patients with head and neck cancers. Salivary glands in the radiation field are dramatically affected by this procedure. The purpose of this study was to examine pharmacokinetic characteristics of the stable nitroxide 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (tempol) with respect to radioprotection of the salivary glands.

Experimental Design: To evaluate the effect of different doses and times of administration, the heads of C3H mice were exposed to a single irradiation dose of 15 Gy, with i.p. tempol injection. To analyze other routes of administration, we injected 275 mg/kg tempol by an i.m., i.v., or s.c. route, 10 minutes before irradiation. We also tested whether oral administration of tempol in a topical form (either in a mouthwash or gel) provided any salivary gland protection.

Results: Tempol treatment (137.5 or 275 mg/kg, i.p., 10 minutes before irradiation) significantly reduced irradiation-induced salivary hypofunction (∼50-60%). I.v. or s.c. administration of tempol also showed significant radioprotection, whereas i.m. administration proved to be ineffective. Topical use of tempol, either as a mouthwash or gel, also was radioprotective.

Conclusions: Our results strongly suggest that tempol is a promising candidate for clinical application to protect salivary glands in patients undergoing radiotherapy for head and neck cancers.
Elevated Expression of Angiogenin in Prostate Cancer and Its Precursors
Authors: Katona Terrence M. , Neubauer Blake Lee , Iversen Philip W. , Zhang Shaobo , Baldridge Lee Ann , Cheng Liang
Published: March 2006

Purpose: Angiogenin is a polypeptide involved in the formation and establishment of new blood vessels necessary for growth and metastasis of numerous malignant neoplasms, including prostatic adenocarcinoma. Antiangiogenin therapy inhibits the establishment, growth, and metastasis of prostatic adenocarcinoma in animal studies. In this study, we have investigated the expression of angiogenin in prostatic adenocarcinoma, high-grade prostatic intraepithelial neoplasia, and adjacent benign prostatic epithelium in a large cohort of prostatectomy specimens.

Methods: We have studied the expression of angiogenin by immunohistochemistry in prostatic adenocarcinoma, high-grade prostatic intraepithelial neoplasia, and adjacent benign prostatic tissue in 107 human total prostatectomy specimens.

Results: The percentage of cells staining positively for angiogenin in benign prostatic glandular epithelium (mean = 17%) was significantly less than for high-grade prostatic intraepithelial neoplasia (mean = 58%, P < 0.001) and prostatic adenocarcinoma (mean = 60%, P < 0.001). Compared with adjacent benign prostatic epithelium, the staining intensity was significantly greater in high-grade prostatic intraepithelial neoplasia (P < 0.001) and prostatic adenocarcinoma (P < 0.001). Furthermore, staining intensity has significantly stronger in prostatic adenocarcinoma versus high-grade prostatic intraepithelial neoplasia (P = 0.0023). However, there was no correlation of angiogenin expression with various clinical and pathologic variables examined, including age at surgery, Gleason scores, pathologic stage, tumor extent, angiolymphatic invasion, extraprostatic extension, seminal vesical invasion, lymph node metastasis, surgical margin status, presence of prostatic intraepithelial neoplasia, and perineural invasion.

Conclusion: Angiogenin expression in prostatic tissue increases as prostatic epithelial cells evolve from a benign to an invasive phenotype. The increasing expression of prostatic adenocarcinoma in the progression from benign prostate to high-grade prostatic intraepithelial neoplasia and ultimately to prostatic adenocarcinoma are consistent with previous studies showing the influential role that angiogenin plays in the growth, invasion, and metastasis of prostatic adenocarcinoma and many other malignant tumors.
Somatic Mutations of ERBB2 Kinase Domain in Gastric, Colorectal, and Breast Carcinomas
Authors: Lee Jong Woo , Soung Young Hwa , Seo Si Hyung , Kim Su Young , Park Cho Hyun , Wang Young Pil , Park Kyeongmee , Nam Suk Woo , Park Won Sang , Kim Sang Ho , Lee Jung Young , Yoo Nam Jin , Lee Sug Hyung
Published: March 2006

Purpose: Recent reports revealed that the kinase domain of the ERBB2 gene is somatically mutated in lung adenocarcinoma, suggesting the mutated ERBB2 gene as an oncogene in human cancers. However, because previous reports focused the mutational search of ERBB2 primarily on lung cancers, the data on ERBB2 mutations in other types of human cancers have been largely unknown.

Experimental Design: Here, we did a mutational analysis of the ERBB2 kinase domain by PCR single-strand conformational polymorphism assay in gastric, colorectal, and breast carcinoma tissues.

Results: We detected the ERBB2 kinase domain mutations in 9 of 180 gastric carcinomas (5.0%), in 3 of 104 colorectal carcinomas (2.9%), and in 4 of 94 breast carcinomas (4.3%). All of the detected ERBB2 mutations except for one in-frame deletion mutation were missense mutations. Of the 16 ERBB2 mutations detected, 4 affected Val777 in the exon 20 site, and 3 affected Leu755 in the exon 19 site. We simultaneously analyzed the somatic mutations of EGFR, K-RAS, PIK3CA, and BRAF genes in the 16 samples with ERBB2 mutations, and found that all of the 3 colorectal carcinoma samples with ERBB2 mutations harbored K-RAS mutations.

Conclusion: This study showed that in addition to lung adenocarcinomas, ERBB2 kinase domain mutation occurs in other common human cancers such as gastric, breast, and colorectal cancers, and suggested that alterations of ERBB2-mediated signaling pathway by ERBB2 mutations alone or together with K-RAS mutations may contribute to the development of human cancers.
Changes of Topoisomerase IIα Expression in Breast Tumors after Neoadjuvant Chemotherapy Predicts Relapse-Free Survival
Authors: Tinari Nicola , Lattanzio Rossano , Natoli Clara , Cianchetti Ettore , Angelucci Domenico , Ricevuto Enrico , Ficorella Corrado , Marchetti Paolo , Alberti Saverio , Piantelli Mauro , Iacobelli Stefano ,   on behalf of the Consorzio Interuniversitario Nazionale per la Bioncologia
Published: March 2006

Purpose: To assess the value of changes in the expression of topoisomerase IIα (TopoII) and the proto-oncogene erbB-2 (HER-2) as predictors of relapse-free survival in women with operable breast cancer treated with anthracycline-based neoadjuvant chemotherapy.

Patients and Methods: Seventy-seven patients with primary breast cancer who had undergone neoadjuvant anthracycline-based chemotherapy were included in the present study. TopoII and HER-2 were measured by immunohistochemistry in prechemotherapy and postchemotherapy (at the time of surgery) tumor specimens, and the value of their changes as predictors of relapse-free survival were evaluated by Kaplan-Meier and Cox proportional hazard regression analyses.

Results: Neoadjuvant chemotherapy resulted in a significant reduction in the percentage of cells expressing TopoII (P < 0.0001). No significant change was observed for HER-2. TopoII and HER-2 expression before chemotherapy predicted tumor response to treatment. Changes in TopoII expression after chemotherapy were strongly associated with a poor relapse-free survival (P < 0.0001) in a Cox multivariate analysis adjusted for other clinicopathologic prognostic factors.

Conclusion: Changes in TopoII expression after anthracycline-based neoadjuvant chemotherapy is an independent predictor of a poor relapse-free survival in patients with breast cancer. Tumor cells displaying an increased TopoII expression after treatment may be responsible for relapses, and may, therefore, define a group of patients with anthracycline-resistant breast cancer.
Methylation Assay for the Diagnosis of Lung Cancer on Bronchial Aspirates: A Cohort Study
Authors: Schmiemann Viola , Böcking Alfred , Kazimirek Marietta , Onofre Alexandre Sherlley Casimiro , Gabbert Helmut Erich , Kappes Rainer , Gerharz Claus Dieter , Grote Hans Juergen
Published: March 2006

Purpose: Recent studies have detected aberrant promoter methylation of adenomatous polyposis coli promoter 1 A (APC), cyclin-dependent kinase inhibitor-2A (p16INK4a), retinoic acid receptor β2, and RAS association domain family protein 1 (RASSF1A) in bronchial aspirates and suggested their use as biomarkers for lung cancer diagnostics. The purpose of this study was to validate these candidate marker genes in a retrospective cohort study.

Experimental Design: Bronchial aspirates collected from a cohort comprising 247 patients with suspected lung cancer were investigated retrospectively regarding aberrant promoter methylation using a quantitative methylation-specific real-time PCR (QMSP).

Results: Eighty-nine patients were diagnosed with primary lung cancer, 102 had benign lung disease, and 56 showed miscellaneous other conditions. A panel consisting of APC, p16INK4a, and RASSF1A emerged as useful combination. This panel detected aberrant methylation in bronchial aspirates of 22 of 35 (63%) and 21 of 44 (44%) centrally and peripherally located primary lung cancers, respectively. Bronchial aspirates also showed aberrant methylation in 5 of 7 (71%) patients with a recurrent lung cancer and in 8 of 30 (27%) cases without tumor recurrence. In contrast, only 1 of 102 patients with benign lung disease displayed a (false) positive test result. Rarely, aberrant methylation was found in patients with other malignancies (3 of 16). The QMSP assay correctly confirmed lung cancer in 8 of 12 (67%) cases with an ambiguous cytology. Moreover, it disclosed 9 of 26 (35%) of peripheral tumors lacking simultaneous cytologic or histologic diagnosis of malignancy.

Conclusions: Our findings suggest that the QMSP assay could be applied as a reflex test in cases of suspected lung cancer that defy a definite diagnosis by conventional methods. Thus, the assay could be a useful diagnostic adjunct especially regarding peripheral tumors.
Differing DNA Methylation Patterns and Gene Mutation Frequencies in Colorectal Carcinomas from Middle Eastern Countries
Authors: Chan Annie O. , Soliman Amr S. , Zhang Qing , Rashid Asif , Bedeir Ahmed , Houlihan P. Scott , Mokhtar Nadia , Al-Masri Nidal , Ozbek Ugur , Yaghan Rami , Kandilci Ayten , Omar Sherif , Kapran Yersu , Dizdaroglu Ferhunde , Bondy Melissa L. , Amos Christopher I. , Issa Jean-Pierre , Levin Bernard , Hamilton Stanley R.
Published: March 2006

Purpose: The epidemiology of colorectal carcinoma is well known to differ among countries but the molecular characteristics are usually assumed to be similar. International differences in molecular pathology have not been studied extensively but have implications for the management of patients in different countries and of immigrant patients.

Experimental Design: We evaluated the CpG island methylator phenotype pathway characterized by concordant methylation of gene promoters that often silences transcription of the genes, the microsatellite instability pathway, and K-ras and p53 gene status in 247 colorectal carcinomas from the three selected Middle Eastern countries of Egypt, Jordan, and Turkey.

Results: Colorectal carcinoma from Egypt had the lowest frequencies of methylation. In multinomial logistic regression analysis, Jordanian colorectal carcinoma more frequently had methylation involving the p16 tumor suppressor gene (odds ratio, 3.5; 95% confidence interval, 1.2-10.6; P = 0.023) and MINT31 locus (odds ratio, 2.3; 95% confidence interval, 1.0-5.1; P = 0.041). The K-ras proto-oncogene was more frequently mutated in colorectal carcinoma from Turkey (odds ratio, 2.9; 95% confidence interval, 1.2-6.7; P = 0.016), but p53 overexpression was more common in both Jordanian and Turkish colorectal carcinoma than in Egyptian cases (odds ratio, 2.5; 95% confidence interval, 1.2-5.5; P = 0.019; and odds ratio, 3.6; 95% confidence interval, 1.8-7.1; P = 0.0003, respectively). The findings in Turkish colorectal carcinoma were most similar to those reported for Western cases.

Conclusions: Colorectal carcinoma from Middle Eastern countries have differing gene methylation patterns and mutation frequencies that indicate dissimilar molecular pathogenesis, probably reflecting different environmental exposures. These molecular differences could affect prevention strategies, therapeutic efficacy, and transferability of clinical trial results.
Circulating Aminopeptidase N/CD13 Is an Independent Prognostic Factor in Patients with Non–Small Cell Lung Cancer
Authors: Murakami Haruyasu , Yokoyama Akihito , Kondo Keiichi , Nakanishi Shuhei , Kohno Nobuoki , Miyake Masayuki
Published: March 2006

Purpose: Aminopeptidase N, also known as CD13, has important roles in tumor metastasis and angiogenesis. Its expression in tumor tissue has been reported to be associated with poor prognosis. However, the clinical significance of circulating aminopeptidase N/CD13 in patients with solid tumors is unknown. We previously developed an aminopeptidase N/CD13–specific monoclonal antibody (mAb) MH8-11, which inhibits cell motility and angiogenesis in vitro. The aim of this study was to evaluate the clinical significance of circulating aminopeptidase N/CD13 protein detected by mAb MH8-11 in patients with non–small cell lung cancer (NSCLC).

Experimental Design: We used electrochemiluminescence immunoassay with mAb MH8-11 to determine circulating aminopeptidase N/CD13 levels in 90 healthy volunteers and 90 patients with NSCLC. Circulating aminopeptidase N/CD13 levels were measured in sera taken before treatment and evaluated for a relationship with clinical outcomes.

Results: A significant correlation was found between tumor progression and serum aminopeptidase N/CD13 concentrations (r = 0.23, P = 0.029). High serum aminopeptidase N/CD13 levels (n = 17) were associated with advanced stage (P = 0.004) or poor performance status (P = 0.001). The overall survival rate for patients with high serum aminopeptidase N/CD13 levels (n = 17) was significantly less than that of patients with low serum aminopeptidase N/CD13 levels (n = 73, P < 0.0001). In a multivariate survival analysis in patients with NSCLC, serum aminopeptidase N/CD13 levels had an independent influence on survival (relative risk, 4.1; 95% confidence interval, 1.9-8.8).

Conclusions: Our data suggest that a high level of circulating aminopeptidase N/CD13 at diagnosis is an independent prognostic factor in patients with NSCLC.
Whole-Genome Allelotyping Identified Distinct Loss-of-Heterozygosity Patterns in Mucinous Ovarian and Appendiceal Carcinomas
Authors: Feltmate Colleen M. , Lee Kenneth R. , Johnson Michael , Schorge John O. , Wong Kwong-kwok , Hao Ke , Welch William R. , Bell Debra A. , Berkowitz Ross S. , Mok Samuel C.
Published: March 2006

Purpose: Mucinous adenocarcinoma of the ovary is one of the common histologic types of ovarian cancer. Its pathogenesis is largely unknown. In addition, the differential diagnosis of metastatic mucinous carcinomas to the ovaries, particularly those originating from the appendix, remains challenging. The purpose of this study is to identify molecular biomarkers for mucinous ovarian adenocarcinoma and compare them with those of appendiceal origin.

Experimental Design: Genome-wide loss-of-heterozygosity (LOH) analysis was done on DNA isolated from 28 microdissected primary mucinous ovarian carcinomas and five appendiceal adenocarcinomas. Markers from high-loss regions were selected for further analysis on a total of 32 ovarian and 14 appendiceal cancers.

Results: High levels of LOH rates (>40%) were detected on chromosome arms 9p, 17p, and 21q in mucinous ovarian carcinoma cases. The frequency of allelic loss was similar between high-grade and low-grade mucinous ovarian carcinoma cases but was significantly higher in ovarian versus appendiceal cases. In addition, LOH rates on five chromosomal loci were statistically different between ovarian and appendiceal carcinomas.

Conclusion: A high frequency of LOH can be found in mucinous ovarian adenocarcinomas independent of grade. Despite histologic similarities between mucinous ovarian carcinomas and metastatic appendiceal carcinomas, they have distinct LOH profiles, which may be used for distinguishing the two diseases.
Increased Expression of the Polycomb Group Gene, EZH2, in Transitional Cell Carcinoma of the Bladder
Authors: Raman Jay D. , Mongan Nigel P. , Tickoo Satish K. , Boorjian Stephen A. , Scherr Douglas S. , Gudas Lorraine J.
Published: March 2006

Purpose: The Polycomb group gene, EZH2, functions as a transcriptional repressor involved in gene silencing. Amplification of EZH2 has been reported in several malignancies, including prostate, breast, and lymphoma. We evaluated EZH2 mRNA and protein expression in bladder specimens from patients and the EZH2 mRNA expression in five bladder cancer cell lines.

Experimental Design: EZH2 mRNA expression was assessed by reverse transcription-PCR (RT-PCR) in 38 bladder tissue specimens. We also evaluated 39 bladder cancer specimens for EZH2 protein expression using immunohistochemistry with affinity-purified antibodies to human EZH2. In addition, five human bladder cancer cell lines were analyzed by RT-PCR for EZH2 mRNA expression.

Results: Five of 14 (36%) nontumor bladder specimens versus 21 of 24 (88%) bladder tumors showed EZH2 mRNA expression (P = 0.003). All of the invasive tumors (10 of 10) had detectable EZH2 mRNA expression, compared with 11 of 14 (79%) superficial tumors. In addition, EZH2 mRNA expression was noted in 100% (16 of 16) of high-grade bladder tumors versus 50% (4 of 8) of low-grade tumors (P = 0.01). EZH2 protein expression, meanwhile, was increased in neoplastic tissue compared with nontumor urothelium (78% versus 69% of nuclei, P < 0.005). There were no differences in EZH2 protein levels between superficial and invasive tumors. High-grade tumors had increased EZH2 staining compared with normal urothelium (78% versus 68%, P < 0.005), whereas low-grade lesions did not. Four of five human bladder cancer cell lines expressed high levels of EZH2, whereas only low levels were detected in one cell line.

Conclusions: We report a significant increase in EZH2 expression in transitional cell carcinoma of the bladder compared with normal urothelium. These data suggest that similar to other human malignancies, increased EZH2 expression correlates with oncogenesis of the bladder.
Apurinic/Apyrimidinic Endonuclease Activity Is Associated with Response to Radiation and Chemotherapy in Medulloblastoma and Primitive Neuroectodermal Tumors
Authors: Bobola Michael S. , Finn Laura S. , Ellenbogen Richard G. , Geyer J. Russell , Berger Mitchel S. , Braga Justin M. , Meade Elizabeth H. , Gross Mary E. , Silber John R.
Published: March 2006

Purpose: Apurinic/apyrimidinic endonuclease (Ap endo) is a key DNA repair activity that confers resistance to radiation- and alkylator-induced cytotoxic abasic sites in human cells. We assayed apurinic/apyrimidinic endonuclease activity in medulloblastomas and primitive neuroectodermal tumors (PNET) to establish correlates with tumor and patient characteristics and with response to adjuvant radiation plus multiagent chemotherapy.

Experimental Design: Ap endo activity was assayed in 52 medulloblastomas and 10 PNETs from patients 0.4 to 21 years old. Ape1/Ref-1, the predominant human Ap endo activity, was measured in 42 medulloblastomas by immunostaining. Cox proportional hazards regression models were used to analyze the association of activity with time to tumor progression (TTP).

Results: Tumor Ap endo activity varied 180-fold and was significantly associated with age and gender. Tumor Ape1/Ref-1 was detected almost exclusively in nuclei. In a multivariate model, with Ap endo activity entered as a continuous variable, the hazard ratio for progression after adjuvant treatment in 46 medulloblastomas and four PNETs increased by a factor of 1.073 for every 0.01 unit increase in activity (P ≤ 0.001) and was independent of age and gender. Suppressing Ap endo activity in a human medulloblastoma cell line significantly increased sensitivity to 1,3-bis(2-chlororethyl)-1-nitrosourea and temozolomide, suggesting that the association of tumor activity with TTP reflected, at least in part, abasic site repair.

Conclusions: Our data (a) suggest that Ap endo activity promotes resistance to radiation plus chemotherapy in medulloblastomas/PNETs, (b) provide a potential marker of treatment outcome, and (c) suggest clinical use of Ap endo inhibitors to overcome resistance.
Oncolytic Herpes Simplex Virus Vector G47Δ in Combination with Androgen Ablation for the Treatment of Human Prostate Adenocarcinoma
Authors: Fukuhara Hiroshi , Martuza Robert L. , Rabkin Samuel D. , Ito Yoshikazu , Todo Tomoki
Published: March 2006

Purpose: The use of oncolytic herpes simplex virus type 1 is a promising stategy for cancer treatment. We constructed herpes simplex virus type 1 vector G47Δ by deleting the α47 gene and the promoter region of US11 from G207. We now report studies demonstrating the potential of G47Δ as a therapeutic modality for prostate cancer in combination with androgen ablation.

Experimental Design: The cytopathic activities of G47Δ at low multiplicities of infection was tested in human prostate cancer cell lines LNCaP, PC-3, and DU145 in vitro. Two androgen-dependent mouse s.c. tumor models, murine TRAMP and human HONDA, were used to investigate the in vivo efficacy of G47Δ in combination with androgen ablation.

Results: G47Δ at low multiplicities of infection showed more rapid tumor cell killing than G207 in LNCaP and DU145 in vitro and showed a 22-fold higher virus yield in a single-step growth experiment. In vivo, G47Δ treatment resulted in reduced tumor growth of established s.c. TRAMP and HONDA tumors and inhibited the growth of recurrent HONDA tumors that once regressed by androgen ablation therapy. In both TRAMP and HONDA tumor xenografts, the combination therapy of G47Δ with androgen ablation led to significantly enhanced inhibition of the tumor growth and prolonged survival.

Conclusions: These results suggest that oncolytic virus therapy with G47Δ can be usefully combined with androgen ablation therapy for the treatment of prostate cancer.
Expression of Multidrug Resistance–Associated Proteins Predicts Prognosis in Childhood and Adult Acute Lymphoblastic Leukemia
Authors: Plasschaert Sabine L.A. , de Bont Eveline S.J.M. , Boezen Marike , vander Kolk Dorina M. , Daenen Simon M.J.G. , Faber Klaas Nico , Kamps Willem A. , de Vries Elisabeth G.E. , Vellenga Edo
Published: March 2006

Purpose: Patients with acute lymphoblastic leukemia (ALL) are treated with a variety of chemotherapeutic drugs, which can be transported by six multidrug resistance–associated proteins (MRP). These MRPs have strongly overlapping functional activities. The aim of this study was to investigate the expression levels of MRP1 to MRP6 and study their effect on prognosis.

Experimental Design: The mRNA expression levels of MRP1 to MRP6 were analyzed by quantitative real-time PCR in leukemic blasts of 105 de novo ALL patients (adults, n = 49; children, n = 56) including 70% B-lineage and 30% T-lineage ALL patients.

Results: Adults showed a higher expressions of MRP1 (P = 0.008), MRP2 (P = 0.026), and MRP3 (P = 0.039) than children. Interestingly, this difference disappeared when patients were categorized based on clinical outcome. Relapsed patients showed a higher expression of all MRP genes, except MRP4. For the total group of ALL patients, the expressions of MRP1, MRP2, MRP3, MRP5, and MRP6 predicted relapse. Moreover, high expression of all MRP genes, except MRP4, was associated with a reduced relapse-free survival in children and adults (MRP1, P = 0.005; MRP2, P = 0.008; MRP3, P = 0.001; MRP5, P = 0.016; MRP6, P = 0.037).

Conclusions: The present study shows that a subset of ALL patients with high MRP expression has an unfavorable prognosis independently of age.
Relation of Erythropoietin and Erythropoietin Receptor Expression to Hypoxia and Anemia in Head and Neck Squamous Cell Carcinoma
Authors: Winter Stuart C. , Shah Ketan A. , Campo Leticia , Turley Helen , Leek Russell , Corbridge Rogan J. , Cox Graham J. , Harris Adrian L.
Published: March 2006

Purpose: The use of erythropoietin in head and neck squamous cell carcinoma (HNSCC) has been associated with poor survival. This study examines the protein and mRNA expression of erythropoietin and erythropoietin receptor in HNSCC and their relation to hypoxia, hemoglobin (Hb), and clinical outcome.

Experimental Design: The immunohistochemical expression of erythropoietin and erythropoietin receptor was assessed in 151 cases of HNSCC. Expression was compared with the hypoxia-dependent proteins hypoxia-inducible factor-1α (HIF-1α) and carbonic anhydrase-9 (CA-9) and correlated with clinical outcome. The mRNA expression of erythropoietin and erythropoietin receptor was measured in paired samples of HNSCC.

Results: Erythropoietin and erythropoietin receptor were expressed in 95% and 99% of tumors, respectively. Using a weighed expression score, there was a positive correlation between erythropoietin and erythropoietin receptor expression (r = 0.18, P = 0.03). HIF-1α (r = 0.38, P < 0.01) and CA-9 (r = 0.26, P = 0.002) correlated with erythropoietin expression, but there was no correlation with erythropoietin receptor. No correlation was found between Hb and erythropoietin (r = 0.07, P = 0.36) or erythropoietin receptor (r = −0.02, P = 0.8), and no survival difference between high and low erythropoietin or erythropoietin receptor expression (P = 0.59 and P = 0.98, respectively). The mRNA expression of erythropoietin (P = 0.03) but not erythropoietin receptor (P = 0.62) was significantly increased in 11 paired samples of HNSCC.

Conclusion: In vivo, the HIF pathway regulates erythropoietin at the mRNA level but not erythropoietin receptor expression in HNSCC. Anemia does not seem to influence the hypoxic microenvironment of tumors sufficiently to alter the expression of erythropoietin. The effects of exogenous erythropoietin may be acting via receptors expressed on tumor cells in vivo, or on vascular cells, which also express the pathway.
The insulin-like growth factor-I receptor inhibitor picropodophyllin causes tumor regression and attenuates mechanisms involved in invasion of uveal melanoma cells.
Authors: Girnita Ada , All-Ericsson Charlotta , Economou Mario A , Astr√∂m Kristina , Axelson Magnus , Seregard Stefan , Larsson Olle , Girnita Leonard
Published: March 2006

PURPOSE: Uveal melanoma has a high mortality rate due to a high incidence of metastasis (up to 50%), which preferentially occurs in the liver. Conventional chemotherapy, being the only therapeutic option today against metastatic uveal melanoma, has not proved to be effective. Therefore, new molecular targets important for malignant phenotype of uveal melanoma have to be found to design efficient pharmacologic agents. EXPERIMENTAL DESIGN: We previously reported data indicating that the insulin-like growth factor-1 receptor (IGF-IR) is a metastasis predictor as well as a therapeutic target for uveal melanoma. In the present study, we made use of the cyclolignan picropodophyllin (PPP), which is an inhibitor of the IGF-IR. RESULTS: We showed that PPP efficiently blocks growth and viability of uveal melanoma cells in cultures and causes tumor regression in xenografted mice. In addition, treatment with PPP inhibited several mechanisms involved in metastasis, including tumor cell adhesion to extracellular matrix proteins, activity and expression of matrix metalloproteinase 2, and cell migration as well as invasion through basement membranes and endothelial cell layers. Furthermore, PPP significantly delayed establishment of uveal melanoma tumors and drastically reduced the incidence of liver metastasis in mice. CONCLUSIONS: Our data suggest that IGF-IR is crucial for growth and survival as well as invasion and metastasis of uveal melanoma cells. Targeting this receptor may therefore comprise a strategy to treat ongoing disease (today incurable) as well as a strategy to prevent development of metastases in patients with primary disease.
Strategies for Delaying or TreatingIn vivoAcquired Resistance to Trastuzumab in Human Breast Cancer Xenografts
Authors: du Manoir Jeanne M. , Francia Giulio , Man Shan , Mossoba Miriam , Medin Jeffrey A. , Viloria-Petit Alicia , Hicklin Daniel J. , Emmenegger Urban , Kerbel Robert S.
Published: March 2006

Purpose: Acquired resistance to trastuzumab (Herceptin) is common in patients whose breast cancers show an initial response to the drug. The basis of this acquired resistance is unknown, hampering strategies to delay or treat such acquired resistance, due in part to the relative lack of appropriate in vivo tumorigenic models.

Experimental Design: We derived an erbB-2–positive variant called 231-H2N, obtained by gene transfection from the highly tumorigenic erbB-2/HER2–negative human breast cancer cell line, MDA-MB-231. Unlike MDA-MB-231, the 231-H2N variants was sensitive to trastuzumab both in vitro and especially in vivo, thus allowing selection of variant resistant to drug treatment in the latter situation after showing an initial response.

Results: The growth of established orthotopic tumors in severe combined immunodeficient mice was blocked for 1 month by trastuzumab, after which rapid growth resumed. These relapsing tumors were found to maintain resistance to trastuzumab, both in vitro and in vivo. We evaluated various therapeutic strategies for two purposes: (a) to delay such tumor relapses or (b) to treat acquired trastuzumab resistance once it has occurred. With respect to the former, a daily oral low-dose metronomic cyclophosphamide regimen was found to be particularly effective. With respect to the latter, an anti–epidermal growth factor receptor antibody (cetuximab) was effective as was the anti–vascular endothelial growth factor (anti-VEGF) antibody bevacizumab, which was likely related to elevated levels of VEGF detected in trastuzumab-resistant tumors.

Conclusions: Our results provide a possible additional rationale for combined biological therapy using drugs that target both erbB-2/HER2 and VEGF and also suggest the potential value of combining less toxic metronomic chemotherapy regimens not only with targeted antiangiogenic agents but also with other types of drug such as trastuzumab.
Role of Activating Fibroblast Growth Factor Receptor 3 Mutations in the Development of Bladder Tumors
Authors: Zieger Karsten , Dyrskjøt Lars , Wiuf Carsten , Jensen Jens L. , Andersen Claus L. , Jensen Klaus Møller-Ernst , Ørntoft Torben Falck
Published: March 2006

Purpose: Bladder tumors develop through different molecular pathways. Recent reports suggest activating mutations of the fibroblast growth factor receptor 3 (FGFR3) gene as marker for the “papillary” pathway with good prognosis, in contrast to the more malignant “carcinoma in situ” (CIS) pathway. The aim of this clinical follow-up study was to investigate the role of FGFR3 mutations in bladder cancer development in a longitudinal study.

Experimental Design: We selected 85 patients with superficial bladder tumors, stratified into early (stage Ta/grade 1-2, n = 35) and more advanced (either stage T1 or grade 3, n = 50) developmental stages. The patients were followed prospectively, and metachronous tumors were included. We did screening for FGFR3 and TP53 mutations by direct bidirectional sequencing and for genome-wide molecular changes with microarray technology.

Results: A total of 43 of 85 cases (51%) showed activating mutations of FGFR3. The mutations were associated with papillary tumors of early developmental stage. However, after stratifying for developmental stage, FGFR3-mutated tumors showed the same malignant potential as wild-type tumors. Tumors with concomitant CIS were generally FGFR3 wild type. They were characterized by different patterns of chromosomal changes and gene expression signatures compared with FGFR3-mutated tumors, indicating different molecular pathways.

Conclusions: FGFR3 mutations seem to have a central role in the early development of papillary bladder tumors. These tumors follow a common molecular pathway, which is different from tumors with concomitant CIS. FGFR3 mutations do not seem to play a role in bladder cancer progression.