bluuebunny/crossref_metadata_2025 huggingface dataset:
Clinicopathologic Significance of the Mutations of theEpidermal Growth Factor ReceptorGene in Patients with Non–Small Cell Lung Cancer
Authors: Tomizawa Yoshio , Iijima Hironobu , Sunaga Noriaki , Sato Koji , Takise Atsushi , Otani Yoshimi , Tanaka Shigefumi , Suga Tatsuo , Saito Ryusei , Ishizuka Tamotsu , Dobashi Kunio , Minna John D. , Nakajima Takashi , Mori Masatomo
Published: March 2006

Purpose: It has been reported that the mutations of epidermal growth factor receptor (EGFR) are detected in lung cancers. Studies of EGFR mutations in large numbers of patients' tumors with clinical data including response to EGFR tyrosine kinase directed therapy are needed to develop a robust database for clinical use. The purpose of the present study is to gain further insights into the significance of EGFR mutation in non–small cell lung cancer (NSCLC).

Experimental Design: We investigated the clinicopathologic significance of tyrosine kinase domain (exons 18-21) EGFR mutations in 120 patients with primary NSCLC and the correlation between EGFR mutation and sensitivity to gefitinib in an additional 20 NSCLC patients treated with gefitinib. In addition, onocogenic KRAS mutations and RASSF1A promoter methylation were determined in the same samples.

Results: EGFR mutation was detected in 29 of 120 (24%) tumors. All of the 29 (40%) mutations occurred in 72 adenocarcinomas. EGFR mutation was significantly more frequent in females (47%) than males (12%, P < 0.0001), in younger patients (38%) than older patients (10%, P = 0.0005), in nonsmokers (47%) than smokers (13%, P < 0.0001), and in well-differentiated tumors (39%) than moderately and poorly differentiated tumors (7%, P < 0.0001). Mutation of the EGFR gene was preferentially observed in advanced disease. Furthermore, EGFR mutations were detected in 11 of 14 (79%) responders, whereas none of six (0%) nonresponders had the mutation (P = 0.0022).

Conclusions: These results in Japanese (East Asian) patients indicated that EGFR mutation plays an important role in pathogenesis of lung adenocarcinoma.
Ghrelin and a Novel Preproghrelin Isoform Are Highly Expressed in Prostate Cancer and Ghrelin Activates Mitogen-Activated Protein Kinase in Prostate Cancer
Authors: Yeh Anthony H. , Jeffery Penelope L. , Duncan Russell P. , Herington Adrian C. , Chopin Lisa K.
Published: March 2006

Purpose: There is evidence that the hormone ghrelin stimulates proliferation in the PC3 prostate cancer cell line although the underlying mechanism(s) remain to be determined. A novel, exon 3–deleted preproghrelin isoform has previously been detected in breast and prostate cancer cells; however, its characterization, expression, and potential function in prostate cancer tissues are unknown.

Experimental Design: Expression of ghrelin and exon 3–deleted preproghrelin was investigated in prostate cancer cell lines and tissues by reverse transcription-PCR and immunohistochemistry. Proliferation and apoptosis assays were done in the LNCaP prostate cancer cell line to determine if ghrelin stimulates proliferation and/or cell survival. Stimulation of mitogen-activated protein kinase (MAPK) pathway activation by ghrelin was determined in PC3 and LNCaP cells by immunoblotting with antibodies specific for phosphorylated MAPKs.

Results: Prostate cancer tissues display greater immunoreactivity for ghrelin and exon 3–deleted preproghrelin than normal prostate tissues, and prostate cancer cell lines secrete mature ghrelin into conditioned medium. Treatment with ghrelin (10 nmol/L), but not the unique COOH-terminal peptide derived from exon 3–deleted preproghrelin, stimulates proliferation in the LNCaP cells (45.0 ± 1.7% above control, P < 0.01) and rapidly activates the extracellular signal-regulated kinase-1/2 MAPK pathway in both PC3 and LNCaP cell lines. Ghrelin, however, does not protect prostate cancer cells from apoptosis induced by actinomycin D (1 μg/mL). The MAPK inhibitors PD98059 and U0126 blocked ghrelin-induced MAPK activation, as well as proliferation, in both cell lines.

Conclusions: These data suggest that these components of the ghrelin axis may have potential as novel biomarkers and/or adjunctive therapeutic targets for prostate cancer.
FTY720: A Promising Agent for Treatment of Metastatic Hepatocellular Carcinoma
Authors: Lee Terence K. , Man Kwan , Ho Joanna W. , Wang Xiang Hong , Poon Ronnie T.P. , Xu Yang , Ng Kevin T. , Chu Alan C. , Sun Chris K. , Ng Irene O. , Sun Hui Chuan , Tang Zao You , Xu Ray , Fan Sheung Tat
Published: March 2006

Purpose: Recurrence after resection and metastasis are common in hepatocellular carcinoma and are associated with poor prognosis. Therefore, effective treatment is urgently needed for improvement of patients' survival. Previously, we reported that FTY720 has an antimetastatic effect on hepatocellular carcinoma cell line through down-regulation of Rac signaling pathway. This study aims to investigate the in vivo antimetastatic potential of FTY720 in an orthotopic nude mice model using metastatic human hepatocellular carcinoma cell lines MHCC-97L (lower metastatic potential) and MHCC-97H (higher metastatic potential).

Experimental Design: The nude mice bearing liver tumors were randomized into a treatment group and a control group, each with 12 mice. FTY720 was administered at a dosage of 5 or 10 mg/kg via i.p. injection after 7 days of tumor inoculation. Thirty-five days later, the mice were sacrificed for record of intrahepatic and pulmonary metastases.

Results: After 35 days of FTY720 treatment at the dosages of 5 and 10 mg/kg, all 12 mice in the treatment group were alive and well. FTY720 at the dosages of 5 and 10 mg/kg significantly suppressed the tumor volume and intrahepatic and pulmonary metastases in the metastatic nude mice model. FTY720 suppressed intrahepatic and pulmonary metastases by inhibition of Rac expression, which at least in part down-regulated the vascular endothelial growth factor expression and CD34 staining in a dose-dependent manner.

Conclusion: FTY720 is a promising novel therapeutic drug for treatment of hepatocellular carcinoma metastasis.
Stable Suppression of Tumorigenicity by Pin1-Targeted RNA Interference in Prostate Cancer
Authors: Ryo Akihide , Uemura Hiroji , Ishiguro Hitoshi , Saitoh Tatsuya , Yamaguchi Akira , Perrem Kilian , Kubota Yoshinobu , Lu Kun Ping , Aoki Ichiro
Published: March 2006

Purpose: The peptidyl-prolyl isomrase Pin1 plays a catalytic role in oncogenesis in solid cancers, including prostate cancer. In the present study, we sought to determine the potential of Pin1-targeted gene silencing in inhibiting cellular growth and tumorigenicity in prostate cancer.

Experimental Design: A retrovirus-mediated RNA interference targeting Pin1 was expressed in PC3 and LNCaP cells, and cell growth and several transformed properties were investigated.

Results: The stable expression of Pin1-specific small interfering RNA constructs in PC3 and LNCaP cells significantly reduced cellular proliferation, colony formation, migration, and invasion but strongly enhanced the apoptotic response induced by serum depletion or treatment with anticancer agents. Furthermore, Pin1 depletion significantly suppressed tumorigenic potential in athymic mice, resulting in the inhibition of both tumor growth and angiogeneisis.

Conclusions: These results strongly suggest that Pin1 plays an important role not only in tumorigenesis but also in the maintenance of the transformed phenotype in prostate cancer cells. Hence, Pin1 may serve as a promising therapeutic target, particularly for recurrent prostate tumors.
Pharmacokinetic-Pharmacodynamic Relationships for the Heat Shock Protein 90 Molecular Chaperone Inhibitor 17-Allylamino, 17-Demethoxygeldanamycin in Human Ovarian Cancer Xenograft Models
Authors: Banerji Udai , Walton Michael , Raynaud Florence , Grimshaw Rachel , Kelland Lloyd , Valenti Melani , Judson Ian , Workman Paul
Published: March 2006

Purpose: To establish the pharmacokinetic and pharmacodynamic profile of the heat shock protein 90 (HSP90) inhibitor 17-allylamino, 17-demethoxygeldanamycin (17-AAG) in ovarian cancer xenograft models.

Experimental Design: The effects of 17-AAG on growth inhibition and the expression of pharmacodynamic biomarkers c-RAF-1, CDK4, and HSP70 were studied in human ovarian cancer cell lines A2780 and CH1. Corresponding experiments were conducted with established tumor xenografts. The variability and specificity of pharmacodynamic markers in human peripheral blood lymphocytes (PBL) were studied.

Results: The IC50 values of 17-AAG in A2780 and CH1 cells were 18.3 nmol/L (SD, 2.3) and 410.1 nmol/L (SD, 9.4), respectively. Pharmacodynamic changes indicative of HSP90 inhibition were demonstrable at greater than or equal the IC50 concentration in both cell lines. Xenograft experiments confirmed tumor growth inhibition in vivo. Peak concentrations of 17-AAG achieved in A2780 and CH1 tumors were 15.6 and 16.5 μmol/L, respectively, and there was no significant difference between day 1 and 11 pharmacokinetic profiles. Reversible changes in pharmacodynamic biomarkers were shown in tumor and murine PBLs in both xenograft models. Expression of pharmacodynamic markers varied between human PBLs from different human volunteers but not within the same individual. Pharmacodynamic biomarker changes consistent with HSP90 inhibition were shown in human PBLs exposed ex vivo to 17-AAG but not to selected cytotoxic drugs.

Conclusion: Pharmacokinetic-pharmacodynamic relationships were established for 17-AAG. This information formed the basis of a pharmacokinetic-pharmacodynamic-driven phase I trial.
Feasibility and Tolerability of Sequential Doxorubicin/Paclitaxel Followed by Cyclophosphamide, Methotrexate, and Fluorouracil and Its Effects on Tumor Response as Preoperative Therapy
Authors: Gianni Luca , Baselga José , Eiermann Wolfgang , Guillem Porta Vicente , Semiglazov Vladimir , Lluch Aña , Zambetti Milvia , Sabadell Dolores , Raab Günther , Llombart Cussac Antonio , Bozhok Alla , Martinez-Agulló Angel , Greco Marco , Byakhov Mikhail , Lopez Lopez Juan Josè , Mansutti Mauro , Valagussa Pinuccia , Bonadonna Gianni ,   European Cooperative Trial in Operable Breast Cancer Study Group
Published: March 2006

Purpose: The European Cooperative Trial in Operable breast cancer (ECTO) randomly tested whether efficacy of adjuvant doxorubicin followed by i.v. cyclophosphamide, methotrexate, and fluorouracil (CMF; doxorubicin → CMF, arm A) could be improved by adding paclitaxel (doxorubicin/paclitaxel → CMF) as adjuvant (arm B) or primary systemic therapy (PST, arm C). We report here feasibility, tolerability, locoregional antitumor activity, and breast conservation rate.

Methods: A total of 1,355 women entered the study. Feasibility and safety were compared in arm A versus arms B plus C. Surgical findings were compared in arms A plus B versus arm C.

Results: Grade 3 or 4 National Cancer Institute toxicities were low (<5%) in all arms. Neuropathy was more frequent in the paclitaxel-containing arms (grade 2, 20.5% versus 5.0%; grade 3, 1.3% versus 0.2%). At 31 months of follow-up, asymptomatic drop of left ventricular ejection fraction was similar in all arms, whereas symptomatic cardiotoxicity was recorded in three patients (0.5%) in A and in three patients (0.3%) in B plus C. PST induced clinical complete plus partial remission in 78%, with an in-breast pathologic complete response rate of 23% and an in-breast plus axilla pathologic complete response rate of 20%. In the multivariate analysis, only estrogen receptor (ER) status was significantly associated with pathologic complete response (odds ratio for ER negative, 5.77; 95% confidence interval, 3.49-9.52; P < 0.0001). PTS induced a significant axillary downstaging (P < 0.001), and breast sparing surgery was feasible in 65% versus 34% (P < 0.001).

Conclusions: Doxorubicin/paclitaxel → CMF is feasible, safe, and well tolerated. Given as PST, it is markedly active, allowing for breast-sparing surgery in a large fraction of patients.
Serum Markers in Patients with Resectable Pancreatic Adenocarcinoma: Macrophage Inhibitory Cytokine 1 versus CA19-9
Authors: Koopmann Jens , Rosenzweig C. Nicole White , Zhang Zhen , Canto Marcia I. , Brown David A. , Hunter Mark , Yeo Charles , Chan Daniel W. , Breit Samuel N. , Goggins Michael
Published: March 2006

Purpose: More accurate serum markers of pancreatic cancer could improve the early detection and prognosis of this deadly disease. We compared the diagnostic utility of a panel of candidate serum markers of pancreatic cancer.

Experimental Design: We collected preoperative serum from 50 patients with resectable pancreatic adenocarcinoma, as well as sera from 50 patients with chronic pancreatitis and 50 age/sex-matched healthy controls from our institution. Sera were analyzed for the following candidate markers of pancreatic cancer: CA19-9, macrophage inhibitory cytokine 1 (MIC-1), osteopontin, tissue inhibitor of metalloproteinase 1, and hepatocarcinoma-intestine-pancreas protein levels.

Results: By logistic regression analysis, MIC-1 and CA19-9 were significant independent predictors of diagnosis. Receiver operating characteristic curve analysis showed that MIC-1 was significantly better than CA19-9 in differentiating patients with pancreatic cancer from healthy controls (area under the curve is 0.99 and 0.78, respectively; P = 0.003), but not in distinguishing pancreatic cancer from chronic pancreatitis (area under the curve of 0.81 and 0.74, respectively; P = 0.63). Hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein, osteopontin, and tissue inhibitor of metalloproteinase 1 serum levels did not provide additional diagnostic power.

Conclusion: In the differentiation of patients with resectable pancreatic cancer from controls, serum MIC-1 outperforms other markers including CA19-9.
Overexpression of Stefin A in Human Esophageal Squamous Cell Carcinoma Cells Inhibits Tumor Cell Growth, Angiogenesis, Invasion, and Metastasis
Authors: Li Wendong , Ding Fang , Zhang Liyong , Liu Zhongmin , Wu Yu , Luo Aiping , Wu Min , Wang Mingrong , Zhan Qimin , Liu Zhihua
Published: March 2006

Purpose: Evidence is accumulating that an inverse correlation exists between stefin A level and malignant progression. The aim of this study is to investigate the role of stefin A in human esophageal squamous cell carcinoma cells and to evaluate the possibility of stefin A for cancer therapy.

Experimental Design: We stably transfected stefin A cDNA into human EC9706 or KYSE150 esophageal squamous cell carcinoma cells. Subsequently, we evaluated the effect of stefin A overexpression on cell growth, cathepsin B activity, cell motility and invasion, tumor growth, and metastasis. Immunoanalysis was done to assess the expression of factor VIII and to support the localization of stefin A and cathepsin B. We also evaluated the effect of CA074Me, a selective membrane-permeant cathepsin B inhibitor.

Results: Both transfection of stefin A and treatment with 10 μmol/L CA074Me significantly reduced cathepsin B activity and inhibited the Matrigel invasion. Combination of both further reduced cathepsin B activity and inhibited the Matrigel invasion. Overexpression of stefin A delayed the in vitro and in vivo growth of cells and significantly inhibited lung metastasis compared with 50% of lung metastasis in xenograft mice from EC9706 or empty vector cells. Transfection with stefin A showed a dramatic reduction of factor VIII staining in the tumors of xenograft mice.

Conclusions: Our data strongly indicate that stefin A plays an important role in the growth, angiogenesis, invasion, and metastasis of human esophageal squamous cell carcinoma cells and suggest that stefin A may be useful in cancer therapy.
Carboxylesterase Isoform 2 mRNA Expression in Peripheral Blood Mononuclear Cells Is a Predictive Marker of the Irinotecan to SN38 Activation Step in Colorectal Cancer Patients
Authors: Cecchin Erika , Corona Giuseppe , Masier Sara , Biason Paola , Cattarossi Giulio , Frustaci Sergio , Buonadonna Angela , Colussi Annamaria , Toffoli Giuseppe
Published: March 2006

Purpose: Irinotecan (CPT11) is a prodrug activated in humans mainly by carboxylesterase 2 (CES2) generating the SN38 metabolite responsible for the drug efficacy and toxicity. The interpatients variability in CPT11 activation step could cause unpredictable toxicity. To identify a predictive molecular marker for CPT11 activation in cancer patients, we investigated the CES2 mRNA expression in peripheral blood mononuclear cells (PBMC) and correlated it to CPT11 activation rate, toxic effects, and response.

Experimental Design: Forty-five colorectal cancer patients were treated with a CPT11-including regimen (FOLFIRI). CES2 mRNA expression in PBMC was quantified by reverse transcription-PCR in real time. Plasma concentrations of CPT11, SN38, and SN38-glucuronide were determined by high-performance liquid chromatography and the pharmacokinetic variables calculated adopting the noncompartmental model. Toxicity was evaluated by the National Cancer Institute Common Toxicity Criteria scale and response by the WHO criteria.

Results: A high interindividual variability in CES2 mRNA relative expression was observed (median, 1.45; range, 0.01-28.21). CES2 mRNA expression level was significantly associated with CPT11 activation ratio [(AUCSN38 + AUCSN38G)/AUCCPT11]. Patients with CES2 mRNA expression above the median cutoff value presented an activation ratio higher (median, 0.25; range, 0.15-0.42) than those with CES2 mRNA below the median (median, 0.20; range, 0.10-0.40; P = 0.013). This was associated with a nonsignificant trend of 1.34-fold increase of SN38 AUC in the group of patients with high CES2 mRNA expression (mean, 1.03 ± 0.62 versus 0.77 ± 0.32 μmol/L hour). Eight of 23 high CES2 mRNA–expressing patients (34.8%) developed grade 3 to 4 neutropenia or diarrhea compared with 2 of 22 (9.1%) in the low CES2-expressing group (P = 0.071).

Conclusion: Our data support a predictive power of CES2 mRNA expression in PBMC for the activation rate of CPT11.
Combined Abl Inhibitor Therapy for Minimizing Drug Resistance in Chronic Myeloid Leukemia: Src/Abl Inhibitors Are Compatible with Imatinib
Authors: O'Hare Thomas , Walters Denise K. , Stoffregen Eric P. , Sherbenou Daniel W. , Heinrich Michael C. , Deininger Michael W.N. , Druker Brian J.
Published: March 2006

Purpose: Chronic myeloid leukemia (CML) is effectively treated with imatinib. However, reactivation of Bcr-Abl via kinase domain mutations that reduce sensitivity to imatinib can cause relapse. As combination therapy is frequently used to prevent emergence of resistance, the combination of imatinib with an inhibitor of imatinib-resistant Bcr-Abl mutants (e.g., Src/Abl inhibitors AP23848 and BMS-354825) was investigated.

Experimental Design: To test this approach, cellular proliferation and Bcr-Abl tyrosine phosphorylation assays were done on Ba/F3 cells expressing wild-type (WT) Bcr-Abl and four common imatinib-resistant mutants (Y253F, E255K, T315I, and M351T). Colony-forming assays with primary CML cells were also done.

Results: Both Src/Abl inhibitors retained full inhibitory capacity when coadministered with imatinib at concentrations above typical clinical levels. For cells expressing WT Bcr-Abl or the marginally imatinib-resistant mutant M351T, inclusion of imatinib at therapeutic levels enhanced the effects of the Src/Abl inhibitors. By comparison, for the highly imatinib-resistant mutants Y253F and E255K, inclusion of imatinib at clinical levels resulted in only a slight enhancement beyond the effects of the Src/Abl inhibitors. None of the inhibitors affected Bcr-Abl T315I cells. Colony-forming assays with primary CML cells yielded analogous results.

Conclusions: Our results indicate that Src/Abl inhibitors are compatible with imatinib and suggest that combined Abl inhibitor therapy is a feasible treatment strategy for patients with CML.
Antitumor Efficacy of Capecitabine and Celecoxib in Irradiated and Lead-Shielded, Contralateral Human BxPC-3 Pancreatic Cancer Xenografts: Clinical Implications of Abscopal Effects
Authors: Blanquicett Carmelo , Saif M. Wasif , Buchsbaum Donald J. , Eloubeidi Mohamad , Vickers Selwyn M. , Chhieng David C. , Carpenter Mark D. , Sellers Jeffrey C. , Russo Suzanne , Diasio Robert B. , Johnson Martin R.
Published: March 2006

Purpose: X-ray therapy (XRT) remains one of the major modalities used to treat patients diagnosed with locally advanced pancreatic adenocarcinoma. However, the effect of XRT on metastatic tumors outside the field of irradiation (abscopal effect) remains largely unknown. In the current study, we examined the effect of XRT alone and in combination with capecitabine and/or celecoxib in both irradiated and lead-shielded contralateral BxPC-3 pancreatic cancer xenografts. This chemoradiation regimen was chosen based on our molecular analysis of pancreatic adenocarcinoma.

Experimental Design: Athymic mice were injected bilaterally with BxPC-3 cells and treatment was initiated 28 days postimplant. During XRT (2 Gy for 5 consecutive days, administered on days 0 and 24), one flank was irradiated whereas the rest of the body (including the contralateral tumor) was lead shielded. Capecitabine (350 mg/kg) was administered on days 0 to 13 and 24 to 37. Celecoxib was initiated in the diet at 100 ppm (equivalent to 20 mg/kg/d p.o.) and administered throughout the study.

Results: In irradiated xenografts, capecitabine and XRT showed synergistic anitiumor efficacy (P = 0.008), which was further improved with the addition of celecoxib (P < 0.001). In contralateral shielded xenografts, abscopal effects were observed. Whereas monotherapy with XRT showed significant reduction in tumor area in irradiated xenografts, growth was promoted by 23% (P < 0.001) in contralateral lead-shielded tumors in the same animals relative to untreated tumors. Interestingly, synergistic antiproliferative efficacy occurred in these contralateral tumors when capecitabine was administered (P < 0.001), despite being outside the irradiated field. The addition of celecoxib further inhibited tumor growth (P < 0.001). This trimodal combination most effectively stabilized disease in both shielded and irradiated tumors; however, tumor eradication was not observed. There were no significant changes in thymidine phosphorylase, dihydropyrimidine dehydrogenase, or cyclooxygenase-2 mRNA levels in irradiated or lead-shielded tumors, suggesting that efficacy cannot be predicted solely from these previously identified indicators of response. Immunohistochemistry examining the proliferation marker Ki-67 showed concordance with tumor response in both irradiated and contralateral shielded xenografts.

Conclusions: These results have implications in the rational design of treatment paradigms for pancreatic cancer where metastatic disease remains the primary cause of patient morbidity and abscopal effects in tumors outside the field of irradiation may affect tumor response.
Changes in Cytoskeletal Protein Composition Indicative of an Epithelial-Mesenchymal Transition in Human Micrometastatic and Primary Breast Carcinoma Cells
Authors: Willipinski-Stapelfeldt Birthe , Riethdorf Sabine , Assmann Volker , Woelfle Ute , Rau Thomas , Sauter Guido , Heukeshoven Jochen , Pantel Klaus
Published: March 2006

Purpose: The bone marrow is a frequent and clinically important homing site for early disseminated breast cancer cells. Here, we aimed to profile the protein expression of these cells using unique cell line models and to evaluate the prognostic relevance of candidate gene expression for breast cancer patients.

Experimental Design: To identify expression patterns characteristic for micrometastatic cells, three different cell lines (BC-K1, BC-P1, and BC-S1) established by SV40 immortalization of cancer cells isolated from the bone marrow of patients with breast cancer were compared with MCF-7 breast cancer and SV40 immortalized normal breast ductal cells (MTSV-1.7) using two-dimensional gel electrophoresis followed by MALDI-ToF analysis. The prognostic significance and clinicopathologic associations of selected differentially expressed proteins were evaluated using high-density breast cancer tissue microarrays.

Results: In contrast to MCF-7 and MTSV1-7 reference cell lines, all micrometastatic cancer cell lines displayed loss of epithelial cytokeratins (CK8, CK18, and CK19) and ectopic expression of vimentin commonly present in mesenchymal cells. Immunohistochemical analysis of 2,517 samples of breast cancer further showed that loss of cytokeratin and ectopic vimentin expression were significantly associated with a higher tumor grade, high mitotic index, and negative estrogen/progesterone-receptor status. Although in univariate analyses significantly related to clinical outcome, none of the cytokeratins analyzed were independently associated with either overall or cancer-specific survival.

Conclusions: Micrometastatic cancer cells exhibit marked changes in the expression pattern of cytoskeletal proteins indicative of an epithelial-mesenchymal transition. This phenotypical change could already be detected in primary tumors and is associated with the aggressive behavior of breast cancer cells in vivo.
Increased Expression of Vascular Endothelial Growth Factor C in Papillary Thyroid Carcinoma Correlates with Cervical Lymph Node Metastases
Authors: Yu Xiao-Min , Lo Chung-Yau , Chan Wai-Fan , Lam King-Yin , Leung Pauline , Luk John M.
Published: March 2006

Purpose: Despite recent studies showing that vascular endothelial growth factor C (VEGF-C) mRNA is up-regulated in papillary thyroid carcinoma (PTC), the role of VEGF-C in lymph node metastasis is still unclear. The aim of this study is to investigate the expression pattern of VEGF-C immunoreactive protein in PTC and its relationship with cervical lymph node metastasis.

Experimental Design: Tissue samples were obtained from 39 specimens of PTC (20 with and 19 without lymph node metastasis) as well as 20 benign thyroid nodules. Overexpression of the VEGF-C protein was evaluated by immunoblotting with specific anti-VEGF-C antibody in paired tumor and nontumor tissues from PTC. The data were compared with patients' clinicopathologic features and lymph node metastasis. Immunohistochemical staining was done on selected paraffin sections to determine cellular localization of VEGF-C and to assess flt-4 (or VEGFR-3)–positive vessel density in PTC lesions.

Results: Overexpression of VEGF-C was detected in 69% of the PTC and in 5% of the benign thyroid specimens. When comparing between the metastatic and nonmetastatic groups of PTC, a higher expression level of VEGF-C was detected in both the tumor (P = 0.004) and adjacent nontumor tissues (P = 0.011). Positive immunostaining for VEGF-C was confirmed in PTC tumor tissues and metastatic lymph nodes, which correlated with flt-4-positive vessel density in tumor and peritumor tissues. The increased expression of VEGF-C protein in PTC is associated with lymph node metastasis (P = 0.004) and lymphovascular permeation (P = 0.001) but is independent of other clinicopatholgic variables.

Conclusions: The VEGF-C immunoreactive protein is overexpressed in PTC lesions, which correlates with lymph node metastases. VEGF-C expression may play a role in lymphangiogenesis of PTC and further study is necessary to evaluate the clinical application of VEGF-C as a molecular marker for tumor metastases to cervical lymph nodes.
Magnetic Resonance Imaging Characteristics Predict Epidermal Growth Factor Receptor Amplification Status in Glioblastoma
Authors: Aghi Manish , Gaviani Paola , Henson John W. , Batchelor Tracy T. , Louis David N. , Barker Fred G.
Published: March 2006

Purpose: Two clinical-molecular glioblastoma subtypes have been described: “primary” glioblastomas arise de novo in older patients and often overexpress epidermal growth factor receptor (EGFR); “secondary” glioblastomas progress from lower-grade tumors in younger patients and commonly have TP53 mutations. EGFR overexpression correlates in experimental gliomas with increased angiogenesis, edema, and invasion. No radiographic predictors of molecular glioblastoma subtype are known.

Experimental Design: We retrospectively reviewed 75 glioblastomas, classified as TP53-mutated (n = 11), EGFR-amplified (n = 31), or neither (non-TP53/non-EGFR; n = 33). Four variables were derived from preoperative magnetic resonance imaging: (a) T2/T1, the ratio of T2-bright volume to enclosed T1-enhancing volume; (b) percentage of tumor volume that was necrosis; and (c and d) T1 and T2 border sharpness coefficients (BSC), the rates of change in grayscale intensity of adjacent 0.02-cm2 voxels traversing the anterior, posterior, and lateral borders on T1-enhanced and T2 images.

Results and Conclusions: Mean T2/T1 was 4.7 for EGFR-amplified glioblastomas, greater than that of TP53-mutated glioblastomas (2.3) or non-TP53/non-EGFR glioblastomas (2.6; P < 0.00005). All four tumors with T2/T1 > 7.2 were EGFR-amplified; 0 of 15 with T2/T1 < 4.7 underwent gross total resection. The mean T2 BSC of EGFR-amplified glioblastomas was 33.7, less sharp (P < 0.0000005) than TP53-mutated (72.2) and non-TP53/non-EGFR glioblastomas (81.2). All 15 glioblastomas with T2 BSC < 30.8 were EGFR-amplified. Percentage necrosis and T1 BSC did not differ between glioblastoma subtypes. The increased T2/T1 ratio and decreased T2 BSC in EGFR-overexpressing tumors are the first radiographic distinctions described between glioblastoma molecular subtypes. These findings may reflect increased angiogenesis, edema, and/or invasion in EGFR-overexpressing tumors.
Food and Drug Administration Drug Approval Summary: Temozolomide Plus Radiation Therapy for the Treatment of Newly Diagnosed Glioblastoma Multiforme
Authors: Cohen Martin H. , Johnson John R. , Pazdur Richard
Published: March 2006

On March 15, 2005, the U.S. Food and Drug Administration approved temozolomide (Temodar capsules, Schering-Plough Research Institute) for the treatment of adult patients with newly diagnosed glioblastoma multiforme concomitantly with radiotherapy and then as maintenance treatment. Five hundred seventy-three glioblastoma multiforme patients were randomized to receive either temozolomide + radiotherapy (n = 287) or radiotherapy alone (n = 286). Patients in the temozolomide + radiotherapy arm received concomitant temozolomide (75 mg/m2) once daily for the duration of radiation therapy (42-49 days). This was followed, 4 weeks later, by six cycles of temozolomide, 150 or 200 mg/m2 daily for 5 days, every 4 weeks. Patients in the control arm received radiotherapy only. In both arms, radiotherapy was delivered as 60 Gy/30 fractions to the tumor site with a 2 to 3 cm margin. Pneumocystis carinii pneumonia prophylaxis was required during temozolomide + radiotherapy treatment and was continued until recovery of lymphocytopenia (Common Toxicity Criteria grade <1). At disease progression, temozolomide salvage treatment was given to 161 of 282 patients (57%) in the radiotherapy alone arm, and to 62 of 277 patients (22%) in the temozolomide + radiotherapy arm. Patients receiving concomitant and maintenance temozolomide + radiotherapy had significantly improved overall survival. The hazard ratio was 0.63 (95% confidence interval, 0.52-0.75; log-rank, P < 0.0001). Median survival was 14.6 months (temozolomide + radiotherapy) versus 12.1 months (radiotherapy alone). Adverse events during temozolomide treatment included thrombocytopenia, nausea, vomiting, anorexia, constipation, alopecia, headache, fatigue, and convulsions.
Tumor Gene Expression and Prognosis in Breast Cancer Patients with 10 or More Positive Lymph Nodes
Authors: Cobleigh Melody A. , Tabesh Bita , Bitterman Pincas , Baker Joffre , Cronin Maureen , Liu Mei-Lan , Borchik Russell , Mosquera Juan-Miguel , Walker Michael G. , Shak Steven
Published: March 2006

Purpose: This study, along with two others, was done to develop the 21-gene Recurrence Score assay (Oncotype DX) that was validated in a subsequent independent study and is used to aid decision making about chemotherapy in estrogen receptor (ER)–positive, node-negative breast cancer patients.

Experimental Design: Patients with ≥10 nodes diagnosed from 1979 to 1999 were identified. RNA was extracted from paraffin blocks, and expression of 203 candidate genes was quantified using reverse transcription-PCR (RT-PCR).

Results: Seventy-eight patients were studied. As of August 2002, 77% of patients had distant recurrence or breast cancer death. Univariate Cox analysis of clinical and immunohistochemistry variables indicated that HER2/immunohistochemistry, number of involved nodes, progesterone receptor (PR)/immunohistochemistry (% cells), and ER/immunohistochemistry (% cells) were significantly associated with distant recurrence-free survival (DRFS). Univariate Cox analysis identified 22 genes associated with DRFS. Higher expression correlated with shorter DRFS for the HER2 adaptor GRB7 and the macrophage marker CD68. Higher expression correlated with longer DRFS for tumor protein p53-binding protein 2 (TP53BP2) and the ER axis genes PR and Bcl2. Multivariate methods, including stepwise variable selection and bootstrap resampling of the Cox proportional hazards regression model, identified several genes, including TP53BP2 and Bcl2, as significant predictors of DRFS.

Conclusion: Tumor gene expression profiles of archival tissues, some more than 20 years old, provide significant information about risk of distant recurrence even among patients with 10 or more nodes.
Combination Analysis of Activator Protein-1 Family Members, Sp1 and an Activator Protein-2α-Related Factor Binding to Different Regions of the Urokinase Receptor Gene in Resected Colorectal Cancers
Authors: Schewe Denis Martin , Biller Tobias , Maurer Gabriele , Asangani Irfan A. , Leupold Joerg H. , Lengyel Ernst R. , Post Stefan , Allgayer Heike
Published: March 2006

Purpose: Studies on the transactivation of genes via promoter elements have mostly been done on cell lines rather than resected tissues. This, however, is essential to address an in vivo or clinical relevance. We have previously shown tumor-specific binding of Sp1 and an activator protein (AP)-2–related factor to promoter region −152/−135 of the metastasis-related u-PAR gene in 60% of in vivo–resected cancer tissues. Cell lines have implicated an additional role, and potential synergism, of an AP-1 region (−190/−171) in u-PAR regulation. This study was done to (a) analyze AP-1 binding to this region in resected tumor and normal tissues, and define subgroups in which it is tumor-specific, and (b) to analyze transcription factor–binding patterns to both promoter motifs in resected tissues, supporting synergism, and draw first prognostic conclusions.

Experimental Design: In 103 patients with colorectal cancer, electrophoretic mobility shift assay/supershift analysis for u-PAR promoter region −190/−171 was done in tumors and normal tissues. In 71 patients, region −152/−135 was also analyzed. U-PAR protein was measured by ELISA.

Results: Tumor-specific AP-1 binding to region −190/−171 of the u-PAR promoter was found in 40% of patients. Subgroup analysis showed tumor-specific binding for c-Fos in 58%, for c-Jun in 50%, for JunD in 39%, and for Fra-1 in 4% of cases. AP-1 binding correlated significantly with u-PAR protein amounts in both normal and tumor tissues (P < 0.001), in contrast to a tumor-specific correlation with u-PAR of the AP-2/Sp1 region. In analyses for both promoter regions, 62% of cancers showed simultaneous binding for AP-1, AP-2, and Sp1, 11% for AP-1 and AP-2, 16% for AP-2 and Sp1, 4% for AP-2 only, 3% for AP-1 only, and 0% for Sp1 only. The binding of AP-1, AP-2, and Sp1 correlated significantly with each other (P < 0.001), the combination of AP-1 and AP-2 showing the highest correlation with u-PAR (P = 0.008). Preliminary survival analysis indicated a trend for poorer prognosis for binding of all three transcription factors.

Conclusion: This is the first study differentiating transcription factor–binding to two important u-PAR promoter regions in a large series of resected tumors and normal tissues. The AP-1 site seems to be a less tumor-specific regulator than the Sp1/AP-2 motif. Nevertheless, data corroborate the hypothesis of synergism between both elements in resected tumors.
Activated Epidermal Growth Factor Receptor–Stat-3 Signaling Promotes Tumor SurvivalIn vivoin Non–Small Cell Lung Cancer
Authors: Haura Eric B. , Zheng Zhong , Song Lanxi , Cantor Alan , Bepler Gerold
Published: March 2006

Purpose: Signal transducers and activators of transcription 3 (Stat3), a member of the STAT family of transcription factors, regulates multiple oncogenic pathways, including pathways regulating tumor cell survival. We evaluated Stat3 activation in early stage non–small cell lung cancers (NSCLC) and how this relates to upstream epidermal growth factor receptor (EGFR) activation, tumor apoptosis, and prognosis.

Experimental Design: High-density tissue microarrays using tissues from 176 surgically resected NSCLC were evaluated for expression of phosphorylated Stat3 (pStat3) and epidermal growth factor receptor (pEGFR) along with tumor apoptosis. Using NSCLC cell lines, we evaluated how pStat3 expression relates to EGFR mutations and sensitivity of cells to gefitinib.

Results: We identified nuclear pStat3 expression in 54% of tumors. pStat3 expression was correlated with smaller tumors (P < 0.0001) and with limited smoking history (P = 0.02). We identified a trend toward higher pStat3 expression in adenocarcinomas compared with other tumor histology (P = 0.09). No relationship was found between pStat3 and prognosis following surgical resection. Importantly, we found a strong positive correlation between pEGFR expression and pStat3 expression (P <0.0001) and an inverse correlation between pStat3 and apoptosis (P = 0.01) consistent with less apoptosis in tumors expressing high amounts of pStat3. Cell lines with mutant EGFR have increased levels of pStat3 compared with cell lines without mutant EGFR and this correlates with their sensitivity to gefitinib. Finally, antisense-mediated knockdown of Stat3 induces apoptosis in EGFR mutant lung cancer cells.

Conclusions: Early-stage NSCLC tumors have activated EGFR-Stat3 signaling with low apoptosis. Our findings suggest that pStat3 expression may be helpful in identifying patients appropriate for treatment with EGFR tyrosine kinase inhibitors.
Expression of Carbonic Anhydrase IX in Astrocytic Tumors Predicts Poor Prognosis
Authors: Haapasalo Joonas A. , Nordfors Kristiina M. , Hilvo Mika , Rantala Immo J. , Soini Ylermi , Parkkila Anna-Kaisa , Pastoreková Silvia , Pastorek Jaromir , Parkkila Seppo M. , Haapasalo Hannu K.
Published: March 2006

Purpose: Carbonic anhydrase IX (CA IX) is a hypoxia-inducible enzyme, which is associated with neoplastic growth. Ectopic CA IX expression has been observed in several tumors, whose normal counterparts do not express this enzyme. Normal human brain tissue shows only slight or no expression of CA IX.

Experimental Design: We describe CA IX expression in human diffusely infiltrating astrocytomas. The association of CA IX is evaluated with clinicopathologic and molecular factors including cell proliferation and apoptosis as well as the expression of p53 and epidermal growth factor receptor.

Results: CA IX immunopositivity was observed in 284 cases of 362 (78%) tumors. The positive areas were often located in close proximity to necrotic regions (P < 0.001). The CA IX immunoreactivity showed strong association with tumor malignancy grades (P < 0.0001). CA IX showed no association with p53 expression nor did it correlate with epidermal growth factor receptor–amplification, apoptosis, or cell proliferation. CA IX intensity had significant prognostic value in univariate (P=0.0011, log-rank test) and multivariate survival analysis (P = 0.038, Cox analysis).

Conclusions: CA IX expression is common in diffusely infiltrating high-grade astrocytomas. Our results suggest that CA IX is a useful biomarker for predicting poor prognosis of astrocytic tumors. It may also be a promising target molecule for the improvement of therapeutic interventions in astrocytomas.
Topotecan Combination Chemotherapy in Two New Rodent Models of Retinoblastoma
Authors: Laurie Nikia A. , Gray Jonathan K. , Zhang Jiakun , Leggas Mark , Relling Mary , Egorin Merrill , Stewart Clinton , Dyer Michael A.
Published: March 2006

Chemotherapy combined with laser therapy and cryotherapy has improved the ocular salvage rate for children with bilateral retinoblastoma. However, children with late-stage disease often experience recurrence shortly after treatment. To improve the vision salvage rate in advanced bilateral retinoblastoma, we have developed and characterized two new rodent models of retinoblastoma for screening chemotherapeutic drug combinations. The first model is an orthotopic xenograft model in which green fluorescent protein– or luciferase-labeled human retinoblastoma cells are injected into the eyes of newborn rats. The second model uses a replication-incompetent retrovirus (LIA-EE1A) encoding the E1A oncogene. Clonal, focal tumors arise from mouse retinal progenitor cells when LIA-EE1A is injected into the eyes of newborn p53−/− mice. Using these two models combined with pharmacokinetic studies and cell culture experiments, we have tested the efficacy of topotecan combined with carboplatin and of topotecan combined with vincristine for the treatment of retinoblastoma. The combination of topotecan and carboplatin most effectively halted retinoblastoma progression in our rodent models and was superior to the current triple drug therapy using vincristine, carboplatin, and etoposide. Vincristine had the lowest LC50 in culture but did not reduce tumor growth in our preclinical retinoblastoma models. Taken together, these data suggest that topotecan may be a suitable replacement for etoposide in combination chemotherapy for the treatment of retinoblastoma.
Morphologic Instability and Cancer Invasion
Authors: Cristini Vittorio , Frieboes Hermann B. , Gatenby Robert , Caserta Sergio , Ferrari Mauro , Sinek John
Published: March 2006

Purpose: A solid tumor embedded in host tissue is a three-dimensional arrangement of cells and extracellular matrix that acts as a sink of oxygen and cell nutrients, thus establishing diffusional gradients. This and variations in vascular density and blood flow typically produce intratumoral regions of hypoxia and acidosis, and may result in spatially heterogeneous cell proliferation and migration. Here, we formulate the hypothesis that through these mechanisms, microenvironmental substrate gradients may drive morphologic instability with separation of cell clusters from the tumor edge and infiltration into surrounding normal tissue.

Experimental Design: We used computer simulations and in vitro experiments.

Results: We provide evidence that morphologic instability could be suppressed in vivo by spatially homogeneous oxygen and nutrient supply because normoxic conditions act both by decreasing gradients and increasing cell adhesion and, therefore, the mechanical forces that maintain a well-defined tumor boundary. A properly working tumor microvasculature can help maintain compact noninfiltrating tumor morphologies by minimizing oxygen and nutrient gradients. In contrast, antiangiogenic therapy, by increasing microenvironmental heterogeneity, may promote morphologic instability, leading to invasive patterns even under conditions in which the overall tumor mass shrinks.

Conclusions: We conclude that therapeutic strategies focused solely on reduction of vascular density may paradoxically increase invasive behavior. This theoretical model accounts for the highly variable outcome of antiangiogenic therapy in multiple clinical trials. We propose that antiangiogenic strategies will be more consistently successful when aimed at “normalizing” the vasculature and when combined with therapies that increase cell adhesion so that morphologic instability is suppressed and compact, noninvasive tumor morphologies are enforced.
Carbonic Anhydrase IX Expression and Tumor Oxygenation Status Do Not Correlate at the Microregional Level in Locally Advanced Cancers of the Uterine Cervix
Authors: Mayer Arnulf , Höckel Michael , Vaupel Peter
Published: March 2006

Purpose: Carbonic anhydrase IX (CA IX) can be induced by hypoxia in vitro and shows an immunohistochemical expression pattern that is predominantly found in perinecrotic tumor areas and correlates with exogenous markers of hypoxia, such as pimonidazole. CA IX might therefore serve as an endogenous marker of tumor hypoxia, although comparisons of CA IX immunostaining with direct oxygenation measurements using pO2 microsensors have thus far yielded contradictory results.

Experimental Design: Because tumor heterogeneity may be among the factors responsible for the discrepancy between the two methods, CA IX expression in tissue samples originating from oxygen microelectrode tracks of locally advanced cervical cancers was assessed in this study. Seventy-seven biopsy specimens were analyzed immunohistochemically using an anti–CA IX rabbit polyclonal antibody and semiquantitative scoring.

Results: CA IX expression showed no correlation with the oxygenation variables median pO2 and hypoxic fraction 2.5, 5, or 10. Cases with higher International Federation of Gynecology and Obstetrics stages (IIb-IVa) exhibited stronger expression of CA IX (P = 0.035) and CA IX expression tended to be more prevalent in node-positive patients (P = 0.051).

Conclusions: These data indicate that CA IX cannot be recommended as a substitute for oxygen microelectrode measurements. That the expression of CA IX does not correlate with the oxygenation status may be due to the degree to which other factors, such as nutrient (e.g., glucose) deficiency or the action of oncogenic mutations, can modulate the in vivo expression of this protein, rendering a strict association with tumor hypoxia too unreliable for clinical use.
Mesenchymal Stem Cell Targeting of Microscopic Tumors and Tumor Stroma Development Monitored by Noninvasive In vivo Positron Emission Tomography Imaging
Authors: Hung Shih-Chieh , Deng Win-Ping , Yang Wen K. , Liu Ren-Shyan , Lee Chien-Chih , Su Tzu-Chi , Lin Rue-Jen , Yang Den-Mei , Chang Chi-Wei , Chen Wei-Hong , Wei Hon-Jian , Gelovani Juri G.
Published: March 2006

The aim of this study was to assess the efficacy human mesenchymal stem cells (hMSC) for targeting microscopic tumors and suicide gene or cytokine gene therapy. Immunodeficient mice were transplanted s.c. with human colon cancer cells of HT-29 Inv2 or CCS line, and 3 to 4 days later, i.v. with “tracer” hMSCs expressing herpes simplex virus type 1 thymidine kinase (HSV1-TK) and enhanced green fluorescent protein (EGFP) reporter genes. Subsequently, these tumors were examined for specificity and magnitude of HSV1-TK+, EGFP+ stem cell engraftment and proliferation in tumor stroma by in vivo positron emission tomography (PET) with 18F-labeled 9-(4-fluoro-3-hydroxymethylbutyl)-guanine ([18F]-FHBG). In vivo PET images of tumors growing for 4 weeks showed the presence of HSV1-TK+ tumor stroma with an average of 0.36 ± 0.24% ID/g [18F]-FHBG accumulation. In vivo imaging results were validated by in situ correlative histochemical, immunofluorescent, and cytometric analyses, which revealed EGFP expression in vWF+ and CD31+ endothelial cells of capillaries and larger blood vessels, in germinal layer of dermis and hair follicles proximal to the s.c. tumor site. These differentiated HSV1-TK+, GFP+ endothelial cells had limited proliferative capacity and a short life span of <2 weeks in tumor fragments transplanted into secondary hosts. We conclude that hMSCs can target microscopic tumors, subsequently proliferate and differentiate, and contribute to formation of a significant portion of tumor stroma. PET imaging should facilitate clinical translation of stem cell–based anticancer gene therapeutic approaches by providing the means for in vivo noninvasive whole-body monitoring of trafficking, tumor targeting, and proliferation of HSV1-tk-expressing “tracer” hMSCs in tumor stroma.