bluuebunny/crossref_metadata_2025 huggingface dataset:
The Expression of Functional Chemokine Receptor CXCR4 Is Associated with the Metastatic Potential of Human Nasopharyngeal Carcinoma
Authors: Hu Jinyue , Deng Xiyun , Bian Xiuwu , Li Guancheng , Tong Yongqing , Li Yuehui , Wang Qingliang , Xin Rong , He Xiaojuan , Zhou Guohua , Xie Pingli , Li Yanwen , Wang Ji Ming , Cao Ya
Published: September 2005

Purpose: Chemokine receptors are implicated in metastasis of several malignant tumors. This study was done to evaluate the contribution of chemokine receptors CXCR4 and CCR7 to metastasis of human nasopharyngeal carcinoma.

Experimental Design: Reverse transcription-PCR, immunohistochemistry, and flow cytometry were used to evaluate mRNA and protein expression of CXCR4 and CCR7 in nasopharyngeal carcinoma tumor tissues and cell lines. Chemotaxis assays were used to evaluate the function of CXCR4 in nasopharyngeal carcinoma cells. Antisense CXCR4 was used to inhibit receptor expression and to block metastasis of human nasopharyngeal carcinoma cells in vivo in athymic mice.

Results: CXCR4 protein was detected in tumor cells in 31 of 40 primary human nasopharyngeal carcinoma and in 13 of 15 lymph node metastases. CXCR4 transcripts were detected in eight CXCR4 protein–positive primary nasopharyngeal carcinoma tissues and seven nasopharyngeal carcinoma cell lines tested. On the other hand, the transcripts for CCR7 were detected only in four primary nasopharyngeal carcinoma tissues and in none of the nasopharyngeal carcinoma cell lines. In functional experiments, metastatic nasopharyngeal carcinoma cell lines that expressed high levels of CXCR4 were found to migrate in response to the CXCR4 ligand SDF-1α. Transfection of antisense CXCR4 in metastatic nasopharyngeal carcinoma cells inhibited the expression of CXCR4 and SDF-1α-induced cell migration in vitro and reduced the capacity of the tumor cells to form metastasis in the lungs and lymph nodes when injected in athymic mice.

Conclusion: The expression of functional CXCR4 but not CCR7 is correlated with the metastatic potential of human nasopharyngeal carcinoma cells. Therefore, CXCR4 may be considered as a potential target for the prevention of nasopharyngeal carcinoma metastasis.
Expression of HOXB2, a Retinoic Acid Signaling Target in Pancreatic Cancer and Pancreatic Intraepithelial Neoplasia
Authors: Segara Davendra , Biankin Andrew V. , Kench James G. , Langusch Catherine C. , Dawson Amanda C. , Skalicky David A. , Gotley David C. , Coleman Maxwell J. , Sutherland Robert L. , Henshall Susan M.
Published: September 2005

Purpose: Despite significant progress in understanding the molecular pathology of pancreatic cancer and its precursor lesion: pancreatic intraepithelial neoplasia (PanIN), there remain no molecules with proven clinical utility as prognostic or therapeutic markers. Here, we used oligonucleotide microarrays to interrogate mRNA expression of pancreatic cancer tissue and normal pancreas to identify novel molecular pathways dysregulated in the development and progression of pancreatic cancer.

Experimental Design: RNA was hybridized to Affymetrix Genechip HG-U133 oligonucleotide microarrays. A relational database integrating data from publicly available resources was created to identify candidate genes potentially relevant to pancreatic cancer. The protein expression of one candidate, homeobox B2 (HOXB2), in PanIN and pancreatic cancer was assessed using immunohistochemistry.

Results: We identified aberrant expression of several components of the retinoic acid (RA) signaling pathway (RARα, MUC4, Id-1, MMP9, uPAR, HB-EGF, HOXB6, and HOXB2), many of which are known to be aberrantly expressed in pancreatic cancer and PanIN. HOXB2, a downstream target of RA, was up-regulated 6.7-fold in pancreatic cancer compared with normal pancreas. Immunohistochemistry revealed ectopic expression of HOXB2 in 15% of early PanIN lesions and 48 of 128 (38%) pancreatic cancer specimens. Expression of HOXB2 was associated with nonresectable tumors and was an independent predictor of poor survival in resected tumors.

Conclusions: We identified aberrant expression of RA signaling components in pancreatic cancer, including HOXB2, which was expressed in a proportion of PanIN lesions. Ectopic expression of HOXB2 was associated with a poor prognosis for all patients with pancreatic cancer and was an independent predictor of survival in patients who underwent resection.
Characterization of CEBPA Mutations in Acute Myeloid Leukemia: Most Patients with CEBPA Mutations Have Biallelic Mutations and Show a Distinct Immunophenotype of the Leukemic Cells
Authors: Lin Liang-In , Chen Chien-Yuan , Lin Dong-Tsamn , Tsay Woei , Tang Jih-Luh , Yeh You-Chia , Shen Hwei-Ling , Su Fang-Hsien , Yao Ming , Huang Sheng-Yi , Tien Hwei-Fang
Published: September 2005

Purpose: The transcription factor CCAAT/enhancer binding protein α, encoded by the CEBPA, is crucial for the differentiation of immature granulocytes. Mutation of the CEBPA may play an important role in leukemogenesis and prognosis. We sought to characterize the CEBPA mutation in acute myeloid leukemia (AML) and to clarify if there is a distinct immunophenotype for leukemic cells with the mutation.

Experiment Design: One hundred and four patients with de novo AML were evaluated for the CEBPA mutation and immunophenotype of the leukemic cells.

Results: Twenty-two distinct mutations were identified in 16 (15%) of 104 AML patients. Fourteen patients had biallelic mutations, mostly involving both the NH2-terminal TAD1 region and the COOH-terminal basic leucine zipper domain (bZIP). The mutations in the bZIP region were always tandem duplications and were located at hot-spot regions for topoisomerase II sites. Sequential study of the CEBPA mutations showed that the mutations disappeared at complete remission but the same mutations reappeared at relapse. None of the patients developed novel mutations during the follow-up period. Patients with CEBPA mutations had significantly higher incidences of CD7 (73%), CD15 (100%), CD34 (93%), and HLA-DR (93%) expression on the leukemic cells.

Conclusion: These data revealed that most AML with CEBPA mutations were associated with an immunophenotype of HLA-DR+CD7+CD13+CD14−CD15+CD33+CD34+. The close relationship of CEBPA mutations with the leukemia status of the patients and the concordance of mutation in presenting and relapse samples implicate the CEBPA mutation as a potential marker for monitoring minimal residue disease.
The Antitumor Effects of Angelica sinensis on Malignant Brain Tumors In vitro and In vivo
Authors: Tsai Nu-Man , Lin Shinn-Zong , Lee Chau-Chin , Chen Shee-Ping , Su Hsuan-Chi , Chang Wen-Liang , Harn Horng-Jyh
Published: September 2005

Purpose: In this study, we have examined the antitumor effects of chloroform extract of Angelica sinensis (AS-C), a traditional Chinese medicine, on glioblastoma multiforme (GBM) brain tumors in vitro and in vivo.

Experimental Design: In vitro, GBM cells were treated with AS-C, and the cell proliferation, changes in distributions of cell cycle, and apoptosis were determined. In vivo, human DBTRG-05MG and rat RG2 GBM tumor cells were injected s.c. or i.c. and were treated with AS-C. Effects on tumor growth were determined by tumor volume, magnetic resonance imaging, survival, and histology analysis.

Results: The AS-C displays potency in suppressing growth of malignant brain tumor cells without cytotoxicity to fibroblasts. Growth suppression of malignant brain tumor cells by AS-C results from cell cycle arrest and apoptosis. AS-C can up-regulate expression of cdk inhibitors, including p21, to decrease phosphorylation of Rb proteins resulting in cell arrest at the G0-G1 phase for DBTRG-05MG and RG2 cells. The apoptosis-associated proteins are dramatically increased and activated in DBTRG-05MG cells and RG2 cells by AS-C but RG2 cells without p53 protein expression. In vitro results showed AS-C triggered both p53-dependent and p53-independent pathways for apoptosis. In in vivo studies, AS-C not only can suppress growths of malignant brain tumors of rat and human origin but also shrink the volumes of in situ GBM, significantly prolonging survivals.

Conclusions: The in vitro and in vivo anticancer effects of AS-C indicate that it has sufficient potential to warrant further investigation and development as a new anti–brain tumor agent.
Antiapoptotic Role of Growth Factors in the Myelodysplastic Syndromes: Concordance Between In vitro and In vivo Observations
Authors: Tehranchi Ramin , Fadeel Bengt , Schmidt-Mende Jan , Forsblom Ann-Mari , Emanuelsson Emma , Jadersten Martin , Christensson Birger , Hast Robert , Howe Robert B. , Samuelsson Jan , Zhivotovsky Boris , Hellström-Lindberg Eva
Published: September 2005

Purpose: Erythroid apoptosis in low-risk myelodysplastic syndrome (MDS) maybe mediated via mitochondrial release of cytochrome c and subsequent caspase activation. In the present study, we compared the in vitro and in vivo effects of proerythroid treatment with erythropoietin + granulocyte colony-stimulating factor (G-CSF) on myelodysplastic erythropoiesis regarding apoptosis and preferential growth of clones with cytogenetic abnormalities.

Experimental Design: We enrolled 15 refractory anemia (RA) and 11 refractory anemia with ringed sideroblasts (RARS), including 5q– aberration, monosomy 7, and trisomy 8, before initiation of treatment and followed nine patients after successful treatment. The effects of G-CSF and erythropoietin were assessed. The expression of G-CSF receptor (G-CSFR) was explored during erythroid maturation. The relative growth of erythroid progenitors with cytogenetic aberrations in presence of erythropoietin was investigated.

Results: Significant redistribution of cytochrome c was seen before treatment at all stages of erythroid differentiation. This release was blocked by G-CSF during the whole culture period and by erythropoietin during the latter phase. Both freshly isolated glycophorin A+ bone marrow cells and intermediate erythroblasts during cultivation retained their expression of G-CSFR. Cytochrome c release and caspase activation were significantly less pronounced in progenitors obtained from successfully treated nonanemic patients and showed no further response to G-CSF in vitro. Moreover, erythropoietin significantly promoted growth of cytogenetically normal cells from 5q– patients, whereas no such effect was observed on erythroblasts from monosomy 7 or trisomy 8 patients.

Conclusion: We conclude that growth factors such as erythropoietin and G-CSF can act both via inhibition of apoptosis of myelodysplastic erythroid precursors and via selection of cytogenetically normal progenitors.
Celecoxib Inhibits Prostate Cancer Growth: Evidence of a Cyclooxygenase-2-Independent Mechanism
Authors: Patel Manish I. , Subbaramaiah Kotha , Du Baoheng , Chang Mindy , Yang Peiying , Newman Robert A. , Cordon-Cardo Carlos , Thaler Howard T. , Dannenberg Andrew J.
Published: September 2005

Purpose: Selective cyclooxygenase-2 (COX-2) inhibitors may suppress carcinogenesis by both COX-2-dependent and COX-2-independent mechanisms. The primary purpose of this study was to evaluate whether celecoxib or rofecoxib, two widely used selective COX-2 inhibitors, possess COX-2-independent antitumor activity.

Experimental Design: PC3 and LNCaP human prostate cancer cell lines were used to investigate the growth inhibitory effects of selective COX-2 inhibitors in vitro. To complement these studies, we evaluated the effect of celecoxib on the growth of PC3 xenografts.

Results: COX-1 but not COX-2 was detected in PC3 and LNCaP cells. Clinically achievable concentrations (2.5-5.0 μmol/L) of celecoxib inhibited the growth of both cell lines in vitro, whereas rofecoxib had no effect over the same concentration range. Celecoxib inhibited cell growth by inducing a G1 cell cycle block and reducing DNA synthesis. Treatment with celecoxib also led to dose-dependent inhibition of PC3 xenograft growth without causing a reduction in intratumor prostaglandin E2. Inhibition of tumor growth occurred at concentrations (2.37-5.70 μmol/L) of celecoxib in plasma that were comparable with the concentrations required to inhibit cell growth in vitro. The highest dose of celecoxib led to a 52% reduction in tumor volume and an ∼50% decrease in both cell proliferation and microvessel density. Treatment with celecoxib caused a marked decrease in amounts of cyclin D1 both in vitro and in vivo.

Conclusions: Two clinically available selective COX-2 inhibitors possess different COX-2-independent anticancer properties. The anticancer activity of celecoxib may reflect COX-2-independent in addition to COX-2-dependent effects.
Expression of CXCR4 Predicts Poor Prognosis in Patients with Malignant Melanoma
Authors: Scala Stefania , Ottaiano Alessandro , Ascierto Paolo Antonio , Cavalli Manuela , Simeone Ester , Giuliano Paola , Napolitano Maria , Franco Renato , Botti Gerardo , Castello Giuseppe
Published: September 2005

Purpose: CXCR4 receptor and its unique ligand, the CXCL12 chemokine, have been recently implicated in cancer metastasis. Evidence about the role of CXCR4/CXCL12 axis has been reported in several cancers including melanoma. Our goal was to investigate if CXCR4 expression has a prognostic value in malignant melanoma.

Experimental Design: Immunohistochemical expression of CXCR4 was evaluated on 71 specimens of primary cutaneous melanoma with a Breslow tumor thickness of >1 mm after radical resection. Associations between baseline patient features and tumors were analyzed by χ2 test. The prognostic value of CXCR4 expression was evaluated by univariate and multivariate analyses adjusted by age, sex, Breslow tumor thickness, presence of ulceration, and sentinel lymph node metastases.

Results: CXCR4 expression was detected in 31 of 71 (43.6%) primary cutaneous melanomas. Membrane or cytoplasmic staining for CXCR4 protein was absent in 56% of the tumors. The positive cases were divided into three score classes according to their staining: low in 15 cases (21%), moderate in 10 (14%), and high in 6 (8%). After a median follow-up of 38 months, 26 patients progressed (16 of 26 expressed CXCR4) and 19 died (12 of 19 expressed CXCR4). The CXCR4 expression on tumor cells was correlated with an unfavorable prognosis with a median disease-free and overall survival of 22 and 35 months, respectively. The hazard ratios of relapse and death, compared with patients with CXCR4-negative tumors, were 2.5 (95% confidence interval, 1.2-6.1) and 3.1 (95% confidence interval, 1.1-7.2), respectively. Median time-to-event (progression and survival) was not reached in patients with CXCR4-negative tumors. In the multivariate analysis, CXCR4 expression, presence of ulceration, and sentinel lymph node status emerged as independent prognostic factors.

Conclusions: This article provides the first evidence that CXCR4 expression could be an independent and powerful prognostic marker in primary cutaneous malignant melanomas.
Metabotropic Glutamate Receptor 4 Expression in Colorectal Carcinoma and Its Prognostic Significance
Authors: Chang Hee Jin , Yoo Byong Chul , Lim Seok-Byung , Jeong Seung-Yong , Kim Woo Ho , Park Jae-Gahb
Published: September 2005

Purpose: Metabotropic glutamate receptors (mGluR) play a variety of roles in both neuronal and nonneuronal cells. Recently, we reported that mGluR4 mediates 5-fluorouracil resistance in a human colon cancer cell line. In this study, we evaluated the nonneural expression of mGluR4 and clarified the existence of mGluR4 in normal colon epithelium and colorectal carcinomas. We also investigated the association of mGluR4 expression levels with various clinicopathologic parameters.

Experimental Design: mGluR4 expression was investigated in 21 normal and 312 malignant tissues from various organs using immunohistochemistry. In addition, 241 cases of colorectal carcinomas were examined and correlations between mGluR4 expression and various clinicopathologic parameters were then statistically analyzed.

Results: Expression of mGluR4 was identified in the normal epithelia of the upper respiratory tract, gastrointestinal tracts, breast, uterine cervix, urinary bladder, and skin, whereas it was not detected in the thyroid, lung alveoli, liver, testis, or prostate. In the corresponding malignant tissues, mGluR4 expression was frequently identified in colorectal carcinoma (68%), followed by malignant melanoma, laryngeal carcinoma, and breast carcinomas. Expression of mGluR4 was detected in 131 (54%) of 241 colorectal carcinomas and 12 (5%) cases among them showed overexpression in their cytoplasms. Loss of mGluR4 expression was negatively associated with tumor differentiation (P = 0.028), whereas overexpression of mGluR4 was positively associated with recurrence (P = 0.034) and poor disease-free survival (P = 0.017) in multivariate analyses.

Conclusions: Our results suggest that mGluR4 signaling may play a role in colorectal carcinomas and that overexpression of mGluR4 is associated with poor prognosis.
Identification of HLA-DRB1*1501–Restricted T-cell Epitopes from Prostate-Specific Antigen
Authors: Klyushnenkova Elena N. , Link Jason , Oberle Warren T. , Kodak James , Rich Cathleen , Vandenbark Arthur A. , Alexander Richard B.
Published: September 2005

The development of immunotherapy for prostate cancer based on the induction of autoimmunity to prostate tissue is very attractive because prostate is not a vital organ beyond the reproductive years. CD4 T cells play an important role in the development of antitumor immune responses, yet the identification of naturally processed MHC Class II–restricted epitopes derived from prostate differentiation antigens has not been described. To facilitate the search for prostate-specific antigen (PSA)–derived MHC class II–restricted peptides, we immunized mice transgenic for HLA-DRB1*1501 with human PSA and showed a robust dose-dependent immune response to the antigen. Screening a library of overlapping 20-mer peptides that span the entire PSA sequence identified two 20-mer peptides, PSA171-190 and PSA221-240, which were responsible for this reactivity. Immunization of DR2b transgenic mice with these peptides induced specific responses to the peptide and whole PSA. Identified peptides were used to stimulate CD4 T cells from HLA-DRB1*1501+ patients with a rare condition, granulomatous prostatitis, and who seem to have a preexisting immune response directed against the prostate gland. We previously showed a linkage of granulomatous prostatitis to HLA-DRB1*1501, suggesting that this disease may have an autoimmune etiology. Peptide-specific CD4 T-cell lines were generated from the peripheral blood of these patients as well as one patient with prostate cancer. These lines also recognized whole, processed PSA in the context of HLA-DRB1*1501. This study will be instrumental in understanding the interaction between circulating self-reactive T cells, organ-specific autoimmunity, and antitumor immune response. The use of these peptides for the immunotherapy of prostate cancer is under investigation.
HA22 (R490A) Is a Recombinant Immunotoxin with Increased Antitumor Activity without an Increase in Animal Toxicity
Authors: Bang SookHee , Nagata Satoshi , Onda Masanori , Kreitman Robert J. , Pastan Ira
Published: September 2005

Purpose: RFB4 (dsFv)-PE38 (BL22) is a recombinant immunotoxin containing an anti-CD22 (Fv) fused to truncated Pseudomonas exotoxin A, which induces a high complete remission rate in patients with purine analogue–resistant hairy cell leukemia. HA22 is a mutant of BL22 with mutations in heavy-chain CDR3 resulting in increased cytotoxic activity. Our goal was to improve the activity of HA22.

Experimental Design: Arg490, which is located in the catalytic domain (III) of the immunotoxin HA22, was mutated to alanine. Purified immunotoxins were produced and tested for cytotoxic activity in cell culture and for antitumor activity and nonspecific toxicity in mice. ADP-ribosylation activity was also measured.

Results: HA22 (R490A) is ∼2-fold more cytotoxic than HA22 on several CD22-positive cell lines. When injected i.v., HA22 (R490A) has more potent antitumor activity than HA22 against CA46 tumors in mice. HA22 and HA22 (R490A) have similar LD50s (∼1.3 mg/kg) and similar plasma half-lives. The R490A mutation also improved the cytotoxicity of the antimesothelin recombinant immunotoxin SS1 (dsFv)-PE38 (SS1P). In vitro ADP-ribosylation assays show that HA22 R490A has increased activity. Increased cytotoxic activity is probably related to this increase in ADP-ribosylation activity.

Conclusion: Protein engineering can be used to increase the efficacy of recombinant immunotoxins. Because HA22 (R490A) has increased antitumor activity without increased animal toxicity, immunotoxins with this mutation are candidates for clinical development.
Phase 1 Clinical and Pharmacokinetics Evaluation of Oral CI-1033 in Patients with Refractory Cancer
Authors: Nemunaitis John , Eiseman Irene , Cunningham Casey , Senzer Neil , Williams Adrienne , Lenehan Peter F. , Olson Stephen C. , Bycott Paul , Schlicht Michael , Zentgraff Rebecca , Shin Dong M. , Zinner Ralph G.
Published: September 2005

Purpose: To determine the tolerability and pharmacokinetics of oral CI-1033, a pan-erbB tyrosine kinase inhibitor, administered over 14 consecutive days of a 21-day cycle.

Design: Phase 1, multicenter trial involving patients with solid tumors that are refractory to standard therapy. CI-1033 was administered initially at 300 mg/day to a minimum cohort of three patients. Dose escalation proceeded at ≤40% increments. Patients were evaluated for toxicity, pharmacokinetic profile, and evidence of response.

Results: Thirty-two patients entered the trial and were evaluable for safety assessment. Dose-limiting toxicity (diarrhea, rash, and/or anorexia) occurred at the 560 mg dose level; the maximum tolerated dose was 450 mg. No patients achieved objective responses and six patients achieved stable disease. Plasma CI-1033 concentrations increased with increasing dose. CI-1033 was not eliminated in urine to any appreciable extent.

Conclusions: CI-1033 is suitable for phase 2 testing at the 450 mg/day dose level when administered for 14 days in a 21-day cycle. The pharmacokinetic profile is consistent with biologically relevant plasma concentrations over the dosing interval.
The Antitumor and Antiangiogenic Activity of Vascular Endothelial Growth Factor Receptor Inhibition Is Potentiated by ErbB1 Blockade
Authors: Sini Patrizia , Wyder Lorenza , Schnell Christian , O'Reilly Terence , Littlewood Amanda , Brandt Ralph , Hynes Nancy E. , Wood Jeanette
Published: September 2005

Purpose: Receptor tyrosine kinases of the ErbB family play important roles in the control of tumor growth. Vascular endothelial growth factor (VEGF) stimulates endothelial cell proliferation, enhances vascular permeability, and plays an important role in tumor vascularization. We evaluated the effects of selective VEGF receptor (VEGFR; PTK787/ZK222584) and ErbB (PKI166 and ZD1839) inhibitors on tumor growth and angiogenesis and asked whether additional therapeutic benefit was conferred by combination treatment.

Experimental Design: The antitumor activity of each inhibitor alone or in combination was assessed in human cancer models in immunocompromised mice. ErbB receptor expression and activation of downstream signaling pathway was evaluated in both tumor and endothelial cells.

Results: Both ErbB inhibitors significantly enhanced the antitumor activity of PTK787/ZK222584. In vitro, ErbB1 inhibition blocked VEGF release by tumor cells and proliferation of both tumor and endothelial cells. In an in vitro angiogenesis assay, epidermal growth factor (EGF) stimulated the release of VEGF by smooth muscle cells resulting in increased angiogenesis, a response blocked by administration of PTK787/ZK222584. Under basal condition, both ZD1839 and PTK787/ZK222584 blocked sprouting, likely via inhibition of an autocrine ErbB1 loop and VEGFR signaling, respectively, in endothelial cells. In conditions of limiting VEGF, EGF plays an important role in endothelial cell proliferation, survival, and sprouting.

Conclusion: We have shown that activation of ErbB1 triggers a plethora of effects, including direct effects on tumor and endothelial cells and indirect effects mediated via induction of VEGF release. Simultaneous blockade of ErbB1 and VEGFR pathways results in a cooperative antitumor effect, indicating that this combination may represent a valid therapeutic strategy.
Up-Regulation of the Peripheral Benzodiazepine Receptor during Human Colorectal Carcinogenesis and Tumor Spread
Authors: Maaser Kerstin , Grabowski Patricia , Oezdem Yelda , Krahn Antje , Heine Bernhard , Stein Harald , Buhr Heinz , Zeitz Martin , Scherübl Hans
Published: September 2005

The peripheral benzodiazepine receptor (PBR) is overexpressed in a variety of cancers. In Unio Internationale Contra Cancrum (UICC) III colorectal cancers, a high level of PBR overexpression correlates with poor prognosis. However, little is known about the role of PBR in the development and progression of colorectal cancer. This study addresses the up-regulation of PBR during colorectal carcinogenesis and tumor spread. One hundred sixteen consecutive patients undergoing surgery for colorectal cancer with either regional (59 patients) or distant metastases (57 patients) were followed-up for 5 years or until death. Twenty-four of the 59 patients with initial UICC stage III cancers later developed distant metastases. PBR overexpression in tumor specimens was determined by immunohistochemistry. UICC stage III patients with colorectal primaries highly overexpressing PBR developed metastases significantly more often than patients with low PBR overexpression in their primary carcinoma. In 54 of the 116 patients adenomas and/or metastases and/or recurrences were available to be studied for PBR up-regulation during colorectal carcinogenesis and tumor spread. PBR was found to be overexpressed in 86% of early and late adenomas. Furthermore, 85% of primaries and of 86% of metastases displayed PBR overexpression. PBR overexpression was also detected at the mRNA level as revealed by real-time PCR. The extent of PBR protein overexpression was equivalent in colorectal adenomas and carcinomas but slightly increased in metastases. These data suggest a functional role of PBR during colorectal carcinogenesis and tumor spread. Thus, PBR qualifies as a target for innovative diagnostic and therapeutic approaches.
Minimal Residual Disease in Acute Myeloid Leukemia Is Predicted by an Apoptosis-Resistant Protein Profile at Diagnosis
Authors: van Stijn Amber , Feller Nicole , Kok Alice , van der Pol Marjoleine A. , Ossenkoppele Gert J. , Schuurhuis Gerrit J.
Published: September 2005

Purpose: Apoptosis is an important mechanism regulating survival of acute myeloid leukemia cells. The apoptosis-related protein profile at diagnosis is important for achieving complete remission thereby affecting survival variables such as disease-free survival (DFS) and overall survival (OS).

To investigate the role of the apoptosis protein profile in further response to therapy and outgrowth of disease.

Experimental Design: We studied whether Bcl-2, Bcl-xL, Mcl-1, Bax as well as the Bcl-2/Bax ratio and a combination of all (antiapoptosis index, AAI) are related to the frequency of malignant cells surviving the chemotherapy (i.e., minimal residual disease, MRD). MRD cells were identified by leukemia-associated aberrant phenotypes established at diagnosis by flow cytometry.

Results: We found that Bcl-2 (R = 0.55, P = 0.002), Bcl-2/Bax (R = 0.42, P = 0.02), and AAI (R = 0.47, P = 0.01) at diagnosis directly correlated with MRD after the first cycle of chemotherapy. In turn, MRD frequency after first cycle correlated with DFS (P = 0.04). Taken together, these results directly explain why Bcl-2/Bax and especially AAI (P = 0.007) at diagnosis correlate with DFS.

Conclusion: Our results show that apoptosis resistance plays an important role in the first stage of the therapy (i.e., to eliminate the bulk of malignant cells), in terms of achievement of complete remission and frequency of MRD after first cycle of therapy.
Growth Inhibitory and Antimetastatic Effect of Green Tea Polyphenols on Metastasis-Specific Mouse Mammary Carcinoma 4T1 Cells In vitro and In vivo Systems
Authors: Baliga Manjeshwar S. , Meleth Sreelatha , Katiyar Santosh K.
Published: September 2005

Purpose: Breast cancer is the second leading cause of cancer-related deaths among females. Dietary habits may have a role in breast cancer risk and prevention as well. Here, we examined the effect of green tea polyphenols (GTP) on growth and metastasis of highly metastatic mouse mammary carcinoma 4T1 cells in vitro and in vivo systems.

Experimental Design: 4T1 cells were treated with (−)-epigallocatechin-3-gallate (EGCG), and the effect was determined on cellular proliferation, induction of apoptosis, proapoptosis, and antiapoptotic proteins of Bcl-2 family, and caspase 3 and poly(ADP-ribose) polymerase activation following 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, flow cytometry, and Western blot analysis. Anticarcinogenic and antimetastatic effect of GTP in 4T1 cells was assessed in immunocompetent BALB/c mice.

Results: Treatment of 4T1 cells with EGCG resulted in inhibition of cell proliferation, induction of apoptosis in dose- and time-dependent manner. The increase in apoptosis was accompanied with decrease in the protein expression of Bcl-2 concomitantly increase in Bax, cytochrome c release, Apaf-1, and cleavage of caspase 3 and PARP proteins. Treatment of EGCG-rich GTP in drinking water to 4T1 cells bearing BALB/c mice resulted in reduction of tumor growth accompanied with increase in Bax/Bcl-2 ratio, reduction in proliferating cell nuclear antigen and activation of caspase 3 in tumors. Metastasis of tumor cells to lungs was inhibited and survival period of animals was increased after green tea treatment.

Conclusion: This study suggests that GTP have the ability to prevent the development of breast cancer and its metastasis; however, further in vivo studies are required to identify the molecular targets.
Mutation in the Tyrosine Kinase Domain of Epidermal Growth Factor Receptor Is a Predictive and Prognostic Factor for Gefitinib Treatment in Patients with Non–Small Cell Lung Cancer
Authors: Chou Teh-Ying , Chiu Chao-Hua , Li Ling-Hui , Hsiao Chun-Yen , Tzen Chin-Yuan , Chang Kuo-Ting , Chen Yuh-Min , Perng Reury-Perng , Tsai Shih-Feng , Tsai Chun-Ming
Published: September 2005

Purpose: Mutations in epidermal growth factor receptor (EGFR) can be used to predict the tumor response of patients receiving gefitinib for non–small cell lung cancer (NSCLC). We investigated the association between mutations in EGFR tyrosine kinase domain and tumor response and survival in gefitinib-treated NSCLC patients.

Experimental Design: EGFR mutations in exons 18 to 21 were analyzed by DNA sequencing of paraffin-embedded tumor tissues from gefitinib-treated NSCLC patients. The results were correlated with clinical variables.

Results: EGFR mutations were found in 61.1% (33 of 54) of cases; response rate and disease control rate were 56.8% and 68.5%, respectively. There was no significant difference in mutation rates between adenocarcinoma (29 of 43) and nonadenocarcinoma (4 of 11; P = 0.085). However, all four nonadenocarcinomas with EGFR mutations had no response to gefitinib. Presence of EGFR mutations was the only independent predictor for disease control (P = 0.003) and tumor response (P = 0.017) in multivariate analysis; positive predictive values were 87.9% and 70.8% and negative predictive values were 61.9% and 69.2%, respectively. In comparison with patients whose tumor was negative for EGFR mutations, patients with EGFR mutations had better progression-free survival (median, 7.6 versus 1.7 months; P = 0.011) and overall survival (median, 14.7 versus 4.7 months; P = 0.046).

Conclusions: Mutations in EGFR tyrosine kinase correlate with treatment response and survival in gefitinib-treated NSCLC patients and can be used as a predictive and prognostic factor. Thus, analysis of EGFR tyrosine kinase mutations in lung adenocarcinoma is of clinical significance, as it can permit the customization of treatment with EGFR tyrosine kinase inhibitors.
Carbonic Anhydrase IX Expression Predicts Outcome of Interleukin 2 Therapy for Renal Cancer
Authors: Atkins Michael , Regan Meredith , McDermott David , Mier James , Stanbridge Eric , Youmans Amanda , Febbo Philip , Upton Melissa , Lechpammer Mirna , Signoretti Sabina
Published: September 2005

Purpose: Renal cancer response to interleukin 2 (IL-2) therapy and patient survival has been correlated with tumor histology and carbonic anhydrase IX (CAIX) expression. In an effort to confirm and expand these observations, we examined CAIX expression in pathology specimens from renal cancer patients who had previously received IL-2 therapy.

Experimental Design: Paraffin-embedded tissue sections of renal cancer were immunostained with the MN-75 monoclonal antibody to CAIX and expression levels were correlated with histologic findings and clinical outcome.

Results: Tissue specimens were obtained from 66 patients; 27 of whom (41%) had responded to IL-2–based therapy. Fifty-eight specimens were assessed as clear cell, with 56, 33, and 4 having alveolar, granular, and papillary features, respectively. Twenty-four (36%), 31 (47%), and 11 (17%) were classified into good, intermediate, and poor prognosis groups according to the Upton pathology model. Forty-one specimens (62%) had high CAIX expression. Twenty-one of 27 (78%) responding patients had high CAIX expressing tumors compared with 20 of 39 (51%) nonresponders (odds ratio, 3.3; P = 0.04). Median survival was prolonged (P = 0.04) and survival >5 years was only seen in high CAIX expressers. In patients with intermediate pathologic prognosis, all nine responders had high CAIX expression versus 11 of 22 nonresponders. A resultant group with good pathologic prognosis alone or with intermediate pathologic prognosis and high CAIX contained 26 of 27 (96%) responders compared with 18 of 39 (46%) nonresponders (odds ratio, 30; P < 0.01) and exhibited longer median survival (P < 0.01).

Conclusions: CAIX expression seems to be an important predictor of outcome in renal cell carcinoma patients receiving IL-2–based therapy and may enhance prognostic information obtained from pathology specimens.
A Homozygous Mutation in MSH6 Causes Turcot Syndrome
Authors: Hegde Madhuri R. , Chong Belinda , Blazo Maria E. , Chin Lip Hon E. , Ward Patricia A. , Chintagumpala Murali M. , Kim John Y. , Plon Sharon E. , Richards C. Sue
Published: September 2005

Heterozygous mutations in one of the DNA mismatch repair genes cause hereditary nonpolyposis colorectal cancer (MIM114500). Turcot syndrome (MIM276300) has been described as the association of central nervous system malignant tumors and familial colorectal cancer and has been reported to be both a dominant and recessive disorder. Homozygous and compound heterozygous mutations in APC, MLH1, MSH2, and PMS2 genes have been reported in five families. Here we describe a nonconsanguineous Pakistani family, including a son with lymphoma and colorectal cancer diagnosed at ages 5 and 8, respectively, and an 8-year-old daughter with glioblastoma multiforme. Both children had features of neurofibromatosis type 1 including atypical café au lait spots and axillary freckling without a family history consistent with neurofibromatosis type 1, familial adenomatous polyposis, or hereditary nonpolyposis colorectal cancer. Mutational analysis was done for MLH1, MSH2, and MSH6 using denaturing high-performance liquid chromatography and sequencing of a blood sample from the daughter. A novel homozygous single base insertion mutation was identified (3634insT) resulting in a premature stop at codon 1,223 in exon 7 of the MSH6 gene. Both parents were found to be heterozygous for the 3634insT mutation. Microsatellite instability testing showed instability in the glioblastoma sample. We report here the first identification of a homozygous mutation in MSH6 in a family with childhood-onset brain tumor, lymphoma, colorectal cancer, and neurofibromatosis type 1 phenotype. Our findings support a role for MSH6 in Turcot syndrome and are consistent with an autosomal recessive mode of inheritance.
Somatic Mutations of EGFR Gene in Squamous Cell Carcinoma of the Head and Neck
Authors: Lee Jong Woo , Soung Young Hwa , Kim Su Young , Nam Hyo Kyung , Park Won Sang , Nam Suk Woo , Kim Min Sik , Sun Dong Il , Lee Youn Soo , Jang Ja June , Lee Jung Young , Yoo Nam Jin , Lee Sug Hyung
Published: September 2005

Purpose: Recently, the kinase domain mutations of epidermal growth factor receptor (EGFR) gene have been identified in non–small-cell lung cancer, and these mutations have been related to the clinical response to the tyrosine kinase inhibitor gefitinib. Gefitinib treatment has also shown clinical benefits in squamous cell carcinoma of the head and neck (SCCHN). The aim of this study was to explore the possibility that SCCHN harbored the EGFR mutations.

Experimental Design: In this study, we analyzed EGFR gene in 41 SCCHN for the detection of the somatic mutations by PCR-single-strand conformational polymorphism analysis.

Results: Overall, we detected three EGFR mutations (7.3%), and all of the mutations were the same in-frame deletion mutation in exon 19 (E746_A750del).

Conclusion: These data indicated that in addition to non–small-cell lung cancer, SCCHN harbors the EGFR gene mutations, and suggested the rationale for the clinical applicability of gefinitib to SCCHN patients.
Definition of an Immunogenic Region Within the Ovarian Tumor Antigen Stratum Corneum Chymotryptic Enzyme
Authors: Bondurant Kristina L. , Crew Mark D. , Santin Alessandro D. , O'Brien Timothy J. , Cannon Martin J.
Published: September 2005

Purpose: The serine protease stratum corneum chymotryptic enzyme (SCCE) is overexpressed by ovarian tumor cells, but is not expressed by normal tissues, suggesting that SCCE may be an attractive target for immunotherapy. In this study, we tested the hypothesis that dendritic cells loaded with SCCE peptides will induce ovarian tumor antigen–specific CD8+ CTL responses and antigen-specific CD4+ helper T cell responses.

Experimental Design: Computer algorithms were used to identify candidate HLA-A2.1-restricted CD8+ CTL epitopes and HLA-DR-binding CD4+ helper T cell epitopes within SCCE. CD8+ CTL stimulated with peptide-loaded dendritic cells were tested against targets expressing endogenous SCCE, including HLA-A2.1-matched ovarian tumor cells. Dendritic cells were also loaded with an extended SCCE peptide, SCCE 110-139, which encompassed a defined CD8+ CTL epitope and multiple candidate CD4+ T helper cell epitopes.

Results: CD8+ CTL specific for SCCE 123-131 lysed autologous macrophages infected with an SCCE-expressing recombinant adenovirus, and also lysed HLA-A2.1-matched, SCCE-expressing ovarian tumor cells. Dendritic cells loaded with SCCE 5-13 peptide stimulated an HLA-A2.1-restricted CD8+ CTL response, but with a reduced level of lysis against ovarian tumor cells. Dendritic cells loaded with SCCE 110-139 induced antigen-specific CD4+ T cell and CD8+ T cell responses. Although SCCE 110-139-loaded dendritic cells processed and presented the 123-131 epitope, the dominant CD8+ CTL response was directed against alternative epitopes within SCCE 110-139.

Conclusions: The 110-139 region of SCCE incorporates multiple CD8+ CTL and CD4+ helper T cell epitopes, and represents an attractive target antigen for immunotherapy of ovarian cancer.
p38MAPK-Dependent Sensitivity of Ewing's Sarcoma Family of Tumors to Fenretinide-Induced Cell Death
Authors: Myatt Stephen S. , Redfern Christopher P.F. , Burchill Susan A.
Published: September 2005

Purpose: There is an urgent need for new therapeutic strategies in Ewing's sarcoma family of tumors (ESFT). In this study, we have evaluated the effect of fenretinide [N-(4-hydroxyphenyl)retinamide] in ESFT models.

Experimental Design: The effect of fenretinide on viable cell number and apoptosis of ESFT cell lines and spheroids and growth of s.c. ESFT in nu/nu mice was investigated. The role of the stress-activated kinases p38MAPK and c-Jun NH2-terminal kinase in fenretinide-induced death was investigated by Western blot and inhibitor experiments. Accumulation of reactive oxygen species (ROS) and changes in mitochondrial transmembrane potential were investigated by flow cytometry.

Results: Fenretinide induced cell death in all ESFT cell lines examined in a dose- and time-dependent manner. ESFT cells were more sensitive to fenretinide than the neuroblastoma cell lines examined. Furthermore, fenretinide induced cell death in ESFT spheroids and delayed s.c. ESFT growth in mice. p38MAPK was activated within 15 minutes of fenretinide treatment and was dependent on ROS accumulation. Inhibition of p38MAPK activity partially rescued fenretinide-mediated cell death in ESFT but not in SH-SY5Y neuroblastoma cells. c-Jun NH2-terminal kinase was activated after 4 hours and was dependent on ROS accumulation but not on activation of p38MAPK. After 8 hours, fenretinide induced mitochondrial depolarization (Δψm) and release of cytochrome c into the cytoplasm in a ROS- and p38MAPK-dependent manner.

Conclusions: These data show that the high sensitivity of ESFT cells to fenretinide is dependent in part on the rapid and sustained activation of p38MAPK. The efficacy of fenretinide in preclinical models demands the evaluation of fenretinide as a potential therapeutic agent in ESFT.
In vivo Bioavailability and Pharmacokinetics of a c-MYC Antisense Phosphorodiamidate Morpholino Oligomer, AVI-4126, in Solid Tumors
Authors: Devi Gayathri R. , Beer Tomasz M. , Corless Christopher L. , Arora Vikram , Weller Doreen L. , Iversen Patrick L.
Published: September 2005

Phosphorodiamidate morpholino oligomers (PMO) inhibit targeted gene expression by preventing ribosomal assembly, thereby preventing mRNA translation. AVI-4126, a PMO targeted against c-MYC, has been extensively characterized in multiple cancer and other disease models and is currently in human clinical trials. A phase I clinical study was conducted to address the issue of PMO bioavailability in malignant tumors surgically excised from patients with adenocarcinoma of prostate and breast 1 day after i.v. administration of a single dose of 90 mg AVI-4126 PMO. The study objectives were to evaluate safety, to determine AVI-4126 concentration in tissue samples of the tumors, and to examine the distribution of AVI-4126 (margin versus tumor core). Significant concentrations of intact PMO similar to the animal models were detected in both human prostate and breast tumor tissues with increased distribution in the tumor core for the vascular breast tumors. No serious adverse events (graded according to National Cancer Institute Common Toxicity Criteria) were reported. Another phase I study was conducted in normal human volunteers to assess AVI-4126 plasma pharmacokinetics following single i.v. administration of 90 mg AVI-4126. Data from both human studies indicated similar plasma concentration-time profile. These studies show PMO bioavailability in tumor tissue and establish the feasibility of using PMO targeting specific genes in human cancer clinical trials.
Preclinical Evaluation of Gemcitabine Combination Regimens for Application in Acute Myeloid Leukemia
Authors: Shanks Ryan H. , Rizzieri David A. , Flowers James L. , Colvin O. Michael , Adams David J.
Published: September 2005

The DNA antimetabolite gemcitabine is an anticancer agent with shown preclinical and clinical utility and a low toxicity profile. In this study, we sought to identify and optimize drug partners for binary and tertiary combinations with gemcitabine for use in the treatment of acute myelogenous leukemia (AML). Drug interaction was assessed by growth inhibition assay with metabolic end points. The combination index method was used to evaluate combinations of gemcitabine with fludarabine, paclitaxel, chlorambucil, doxorubicin, mitoxantrone, and SN-38 in U937 human AML cells. A three-dimensional method was used to determine the effect of dose ratio and schedule on drug interaction. Mechanisms underlying interactions related to cell cycle effects and apoptosis were assessed by flow cytometric and caspase-3 and -7 assays, respectively. The most synergistic binary combination was gemcitabine + fludarabine. The most synergistic tertiary combination was gemcitabine + fludarabine + paclitaxel, where the interaction was sequence dependent with paclitaxel given before gemcitabine + fludarabine, producing a 2-fold increase in synergy. Cell cycle analysis did not reveal a significant G2-M arrest, suggesting that the synergistic effect of paclitaxel in this combination, which produced the greatest caspase activation, might be independent of microtubule stabilization. In contrast, the gemcitabine + fludarabine + mitoxantrone combination was synergistic and schedule independent. Moreover, few ratios of gemcitabine + fludarabine to mitoxantrone were antagonistic, which could be important for clinical translation. In conclusion, synergistic interactions with gemcitabine occurred with several drugs, the most promising being gemcitabine + fludarabine, gemcitabine + fludarabine + paclitaxel, and gemcitabine + fludarabine + mitoxantrone. These findings provided a rationale for clinical trials of gemcitabine + fludarabine and gemcitabine + mitoxantrone where responses were observed in heavily pretreated AML patients.
Up-Regulation of EphB4 in Mesothelioma and Its Biological Significance
Authors: Xia Guangbin , Kumar S. Ram , Masood Rizwan , Koss Michael , Templeman Claire , Quinn David , Zhu Sutao , Reddy Ramachandra , Krasnoperov Valery , Gill Parkash S.
Published: September 2005

Purpose: Mesothelioma is a rare malignancy that is incurable and carries a short survival despite surgery, radiation, or chemotherapy. This study was designed to identify novel targets for diagnostic, prognostic, and therapeutic approaches.

Experimental Design: The expression and functional significance of the receptor tyrosine kinase EphB4 was studied in vitro and in a murine model of mesothelioma.

Results: EphB4 was highly expressed in mesothelioma cell lines and primary tumor tissues but not in normal mesothelium. Knockdown of EphB4 using small interfering RNA and antisense oligodeoxynucleotide showed reduction in cell survival, migration, and invasion. EphB4 knockdown initiated a caspase-8-mediated apoptosis and down-regulation of the antiapoptotic protein bcl-xl. EphB4 knockdown also resulted in reduced phosphorylation of Akt and down-regulation of matrix metalloproteinase-2 transcription. In addition, murine tumor xenograft studies using EphB4 oligodeoxynucleotides showed a marked reduction in tumor growth accompanied by a specific decline in EphB4 protein levels, reduced cell division, apoptosis in tumor tissue, and decreased microvascular density.

Conclusions: EphB4 is expressed in mesothelioma, provides a survival advantage to tumor cells, and is therefore a potential novel therapeutic target.