bluuebunny/crossref_metadata_2025 huggingface dataset:
The
In vitro
and
In vivo
Effects of Re-Expressing Methylated
von Hippel-Lindau
Tumor Suppressor Gene in Clear Cell Renal Carcinoma with 5-Aza-2′-deoxycytidine
Authors: Alleman Wade G. , Tabios Ray L. , Chandramouli Gadisetti V. R. , Aprelikova Olga N. , Torres-Cabala Carlos , Mendoza Arnulfo , Rodgers Craig , Sopko Nikolai A. , Linehan W. Marston , Vasselli James R.
Published: September 2005

Purpose: Clear cell renal carcinoma (ccRCC) is strongly associated with loss of the von Hippel-Lindau (VHL) tumor suppressor gene. The VHL gene is functionally lost through hypermethylation in up to 19% of sporadic ccRCC cases. We theorized that re-expressing VHL silenced by methylation in ccRCC cells, using a hypo-methylating agent, may be an approach to treatment in patients with this type of cancer. We test the ability of two hypo-methylating agents to re-express VHL in cell culture and in mice bearing human ccRCC and evaluate the effects of re-expressed VHL in these models.

Experimental Design: Real-time reverse transcription-PCR was used to evaluate the ability of zebularine and 5-aza-2′-deoxycytidine (5-aza-dCyd) to re-express VHL in four ccRCC cell lines with documented VHL gene silencing through hypermethylation. We evaluated if the VHL re-expressed after hypo-methylating agent treatment could recreate similar phenotypic changes in ccRCC cells observed when the VHL gene is re-expressed via transfection in cell culture and in a xenograft mouse model. Finally we evaluate global gene expression changes occurring in our cells, using microarray analysis.

Results: 5-Aza-dCyd was able to re-express VHL in our cell lines both in culture and in xenografted murine tumors. Well described phenotypic changes of VHL expression including decreased invasiveness into Matrigel, and decreased vascular endothelial growth factor and glucose transporter-1 expression were observed in the treated lines. VHL methylated ccRCC xenografted tumors were significantly reduced in size in mice treated with 5-aza-dCyd. Mice bearing nonmethylated but VHL-mutated tumors showed no tumor shrinkage with 5-aza-dCyd treatment.

Conclusion: Hypo-methylating agents may be useful in the treatment of patients having ccRCC tumors consisting of cells with methylated VHL.
Phase II Clinical Trial of Interleukin-12 in Patients with Relapsed and Refractory Non-Hodgkin’s Lymphoma and Hodgkin’s Disease
Authors: Younes Anas , Pro Barbara , Robertson Michael J. , Flinn Ian W. , Romaguera Jorge E. , Hagemeister Fredrick , Dang Nam H. , Fiumara Paolo , Loyer Evelyne M. , Cabanillas Fernando F. , McLaughlin Peter W. , Rodriguez Maria Alma , Samaniego Felipe
Published: September 2005

Purpose: The purpose of this study was to evaluate the clinical activity and toxicity of recombinant human Interleukin (IL)-12 in patients with relapsed and refractory non-Hodgkin’s lymphoma (NHL) or Hodgkin’s disease (HD).

Experimental Design: Forty-two previously treated patients (32 patients with NHL and 10 patients with HD) were enrolled on the study. Patients were treated with either intravenous (n = 11) or subcutaneous (n = 31) administration of IL-12. The patients had received a median of three prior treatment regimens, and 16 patients had undergone prior autologous stem cell transplantation.

Results: All patients were assessable for toxicity, and 39 of 42 (93%) patients were assessable for response. Six of 29 (21%) patients with NHL had a partial or complete response, whereas none of the 10 patients with HD responded. Furthermore, 15 patients had stable disease that lasted for up to 54 months. Progression-free survival in patients with indolent NHL, aggressive NHL, and HD was 6, 2, and 2.5 months, respectively. Treatment was well tolerated, and the most common toxicity was flu-like symptoms. Reversible grade 3 hepatic toxicity was observed in three patients requiring dose reduction. IL-12 therapy increased the median number of peripheral blood CD8 T lymphocytes from 423/μl to 576/μl (P = 0.0019). Furthermore, IL-12 therapy decreased serum vascular endothelial growth factor and basic fibroblast growth factor concentrations in 37% of the patients.

Conclusions: The ability of recombinant human IL-12 therapy to increase the number of circulating CD8+ cells and induce clinical remissions in patients with relapsed NHL warrants further investigation of the drug.
TEL
Deletion Analysis Supports a Novel View of Relapse in Childhood Acute Lymphoblastic Leukemia
Authors: Zuna Jan , Ford Anthony M. , Peham Martina , Patel Naina , Saha Vaskar , Eckert Cornelia , Köchling Joachim , Panzer-Grümayer Renate , Trka Jan , Greaves Mel
Published: September 2005

Purpose: TEL (ETV6)-AML1 (RUNX1) chimeric gene fusions are frequent genetic abnormalities in childhood acute lymphoblastic leukemia (ALL). They often arise prenatally as early events or initiating events and are complemented by secondary postnatal genetic events of which deletion of the non-rearranged, second TEL allele is the most common. This consistent sequence of molecular pathogenesis facilitates an analysis of the clonal origins of relapse in this leukemia, which has some unusual clinical features.

Experimental Design: We compared the boundaries, by microsatellite mapping, of TEL deletions at relapse versus diagnosis in 15 informative patients. Moreover, we compared the relatedness of diagnostic and relapse clones using immunoglobulin and T-cell receptor genes rearrangements and clonotypic TEL-AML1 genomic fusion.

Results: Five patients retained the apparent same size TEL deletion, seven had larger deletions, and three had smaller deletions at relapse. In all of the cases evaluated, the clonal relatedness of diagnostic and relapse cells was confirmed by the retention of clonotypic TEL-AML1 genomic sequence and/or at least one identical immunoreceptor gene rearrangement.

Conclusions: These data provide further evidence that TEL deletions are secondary to TEL-AML1 fusions in ALL. They are compatible with the novel idea that in at least some cases of childhood ALL, remission occurs with persistence of a preleukemic “fetal” clone, and subsequent relapse reflects the emergence of a new subclone from this reservoir after an independent “second hit,” i.e., independent TEL deletion. To our knowledge, the study is the most extensive and comprehensive analysis of the relationship between diagnostic and relapse clones in childhood ALL presented thus far.
Hypermethylation in Histologically Distinct Classes of Breast Cancer
Authors: Bae Young Kyung , Brown Amy , Garrett Elizabeth , Bornman Daniel , Fackler Mary Jo , Sukumar Saraswati , Herman James G. , Gabrielson Edward
Published: September 2005

Purpose: A number of different genes are known to be inactivated by aberrant hypermethylation in breast cancer, but it is still unknown to what extent these epigenetic alterations differ according to specific breast cancer phenotypes. We sought to determine whether the extent of hypermethylation or defined profiles of gene hypermethylation are associated with biological characteristics of breast cancers.

Experimental Design: We evaluated methylation status of 12 different genes in a series of 109 invasive breast tumors, representing the ductal, lobular, and mucinous histologic subtypes using methylation-specific PCR. Frequencies of methylation were compared across the recognized histologic classes, and multivariate techniques (latent class analysis, factor analysis, recursive partitioning, and hierarchical clustering) were used to seek patterns of methylation for individual genes that distinguish recognized histologic types of breast cancer or define breast cancer phenotypes on a molecular level.

Results: All 109 cases studied have aberrant methylation of multiple genes (3 to 10 genes per case), demonstrating that gene hypermethylation is pervasive in breast cancer. Lobular cancers and mucinous cancers, which often have relatively low levels of chromosomal changes, have higher overall frequencies of hypermethylation than ductal cancers (49% in lobular and mucinous versus 40% in ductal), but there is a relatively unimodal distribution of methylation frequency for all three histologic types. Only one of the individual genes studied, BRCA1, has a variable frequency of methylation that is significantly dependent on histologic pattern of tumor growth, with a higher frequency of methylation in mucinous cancers than ductal or lobular cancers. Although some trends of histology-specific gene methylation were seen, methylation patterns could not definitively classify breast cancers according to histologic type.

Conclusions: Although a more comprehensive hypermethylation profile could potentially be useful for breast cancer classification and understanding the biology of this disease, it appears that the hypermethylation patterns across various forms of breast cancer are less distinct than those between breast cancer and cancers of different tissue origins. Furthermore, the relatively unimodal distribution of methylation frequency for all three histologic types does not support there being a distinct CpG island methylator phenotype for breast cancer.
A Phase I/II Study of LY900003, an Antisense Inhibitor of Protein Kinase C-α, in Combination with Cisplatin and Gemcitabine in Patients with Advanced Non–Small Cell Lung Cancer
Authors: Villalona-Calero Miguel A. , Ritch Paul , Figueroa Jose A. , Otterson Gregory A. , Belt Robert , Dow Edward , George Sebastian , Leonardo James , McCachren Spence , Miller G. Lance , Modiano Manuel , Valdivieso Manuel , Geary Richard , Oliver Jennifer W. , Holmlund Jon
Published: September 2005

Purpose: Protein kinase C-α has been implicated in malignant transformation and proliferation. Based on in vivo superadditive interaction between the protein kinase C-α antisense oligonucleotide LY900003 (Affinitak, ISIS 3521) and cisplatin, we designed this phase I/II trial of LY900003 with cisplatin/gemcitabine

Experimental Design: The safety of the combination, as well as potential pharmacokinetic interactions, was evaluated in the phase I portion of the trial. The phase II portion evaluated the antitumor activity of the combination in previously untreated patients with stage IIIB/IV non–small-cell lung cancer (NSCLC).

Results: Seven patients received 18 cycles of the combination during the phase I portion. Dose-limiting toxicity was only observed in one of six evaluable patients (grade 3 fatigue). However, due to a relatively high frequency of thrombocytopenia, cisplatin 80 (mg/m2) and gemcitabine (1,000 mg/m2) were recommended for the phase II portion. Antitumor activity was observed in two patients (one with NSCLC and one with pancreatic carcinoma), and prolonged stabilization was observed in two others. No pharmacokinetic interactions occurred. In the phase II portion, 55 NSCLC patients received the combination at two gemcitabine doses [1,000 mg/m2, n = 44 (original cohort); 1,250 mg/m2, n = 11 (expanded cohort)]. Fourteen of 39 evaluable patients in the original cohort had a response rate (1 complete response and 13 partial responses; response, 36%), whereas 2 of 9 evaluable patients in the expanded cohort experienced partial response (combined response rate, 33%). The median time to treatment failure was 3.9 months, whereas the median time response to progression for the 48 patients with evaluable response was 4.4 months (confidence interval, 3.5–5.5 months). Intent to treat median survival time was 8.9 months. Forty-eight percent of the patients experienced catheter-related events.

Conclusions: LY900003 can be administered safely in combination with cisplatin and gemcitabine and is associated with antitumor activity in patients with advanced NSCLC. Better characterization of subsets of patients most likely to benefit from this combination therapy is needed.
Proliferation Potential-Related Protein, an Ideal Esophageal Cancer Antigen for Immunotherapy, Identified Using Complementary DNA Microarray Analysis
Authors: Yoshitake Yoshihiro , Nakatsura Tetsuya , Monji Mikio , Senju Satoru , Matsuyoshi Hidetake , Tsukamoto Hirotake , Hosaka Seiji , Komori Hiroyuki , Fukuma Daiki , Ikuta Yoshiaki , Katagiri Toyomasa , Furukawa Yoichi , Ito Hiromi , Shinohara Masanori , Nakamura Yusuke , Nishimura Yasuharu
Published: September 2005

Purpose: To establish effective antitumor immunotherapy for esophageal cancer, we tried to identify an useful target antigen of esophageal cancer.

Experimental Design: We did cDNA microarray analysis to find a novel candidate antigen, proliferation potential-related protein (PP-RP). We examined cytotoxicity against tumor cells in vitro and in vivo of CTLs specific to PP-RP established from esophageal cancer patients.

Results: In 26 esophageal cancer tissues, an average of relative ratio of the expression of the PP-RP mRNA in cancer cells versus adjacent normal esophageal tissues was 396.2. Immunohistochemical analysis revealed that, in 20 of the 22 esophageal cancer tissues, PP-RP protein was strongly expressed only in the cancer cells and not so in normal esophageal epithelial cells. PP-RP protein contains 10 epitopes recognized by HLA-A24–restricted CTLs. These CTLs, generated from HLA-A24–positive esophageal cancer patients, had cytotoxic activity against cancer cell lines positive for both PP-RP and HLA-A24. Furthermore, adoptive transfer of the PP-RP–specific CTL line inhibited the growth of a human esophageal cancer cell line engrafted in nude mice.

Conclusions: The expression of PP-RP in esophageal cancer cells was significantly higher than in normal cells, and the CTLs recognizing PP-RP killed tumor cells in vitro and also showed tumor rejection effects in a xenograft model. Therefore, PP-RP may prove to be an ideal tumor antigen useful for diagnosis and immunotherapy for patients with esophageal cancer. cDNA microarray analysis is a useful method to identify ideal tumor-associated antigens.
Enhancement of the Antibody-Dependent Cellular Cytotoxicity of Low-Fucose IgG1 Is Independent of FcγRIIIa Functional Polymorphism
Authors: Niwa Rinpei , Hatanaka Shigeki , Shoji-Hosaka Emi , Sakurada Mikiko , Kobayashi Yukari , Uehara Aya , Yokoi Haruhiko , Nakamura Kazuyasu , Shitara Kenya
Published: September 2005

Purpose: The most common polymorphic variant of Fcγ receptor type IIIa (FcγRIIIa), FcγRIIIa-158F, has been associated with inferior clinical responses to anti-CD20 chimeric IgG1 rituximab compared with FcγRIIIa-158V. As we previously found that removal of fucose residues from the oligosaccharides of human IgG1 results in enhanced antibody-dependent cellular cytotoxicity, we compared the effects of the FcγRIIIa gene (FCGR3A) polymorphism on normal and low-fucose versions of rituximab on antibody-dependent cellular cytotoxicity.

Experimental Design: The polymorphism at position 158 of FcγRIIIa was determined for the peripheral blood mononuclear cells (PBMCs) of 20 healthy donors. The PBMCs were then used as effector cells to compare the antibody-dependent cellular cytotoxicity of rituximab and a low-fucose version, KM3065. The contributions of the different cell types within the PBMC to antibody-dependent cellular cytotoxicity were examined.

Results: We found KM3065-mediated antibody-dependent cellular cytotoxicity was increased 10 to 100-fold compared with rituximab for each of the 20 donors. In contrast to rituximab, KM3065 antibody-dependent cellular cytotoxicity enhancement was similar for both FCGR3A alleles and thus independent of genotype. In addition, antibody-dependent cellular cytotoxicity of both KM3065 and rituximab requires natural killer cells but not monocytes nor polymorphonuclear cells. The antibody-dependent cellular cytotoxicity (ADCC) of each of the 20 donors correlated with the natural killer cell numbers present in the PBMCs. Importantly, using KM3065, the ADCC mediated by effector cells bearing the lower affinity variant FcγRIIIa-158F was significantly increased compared with rituximab-mediated ADCC using effector cells bearing the higher affinity FcγRIIIa-158V receptors.

Conclusions: The use of low-fucose antibodies might improve the therapeutic effects of anti-CD20 therapy for all patients independent of FcγRIIIa phenotype beyond that currently seen with even the most responsive patients.
Prognostic Significance of Tissue Factor in Pancreatic Ductal Adenocarcinoma
Authors: Nitori Nobuhiro , Ino Yoshinori , Nakanishi Yukihiro , Yamada Tesshi , Honda Kazufumi , Yanagihara Kazuyoshi , Kosuge Tomoo , Kanai Yae , Kitajima Masaki , Hirohashi Setsuo
Published: September 2005

Tissue factor (TF) is a transmembrane glycoprotein that plays roles in the blood coagulation and intracellular signaling pathways, and has also been suggested to modulate the biological behavior of cancer cells. In order to examine the clinicopathologic significance of TF expression in pancreatic ductal adenocarcinoma, TF expression was determined by immunohistochemistry using a newly raised anti-TF monoclonal antibody in 113 patients who had undergone surgical resection of pancreatic ductal adenocarcinoma. According to the incidence of tumor cell immunopositivity, patients were divided into “negative TF” (0%), “weak TF” (<25%), or “high TF” (25% or more) groups, which accounted for 11.6% (n = 13), 44.2% (n = 50), and 44.2% (n = 50) of the total, respectively. Increased TF expression was correlated with the extent of the primary tumor (P = 0.0043), lymph node metastasis (P = 0.0043), lymphatic distant metastasis (P = 0.0039), advanced tumor-node-metastasis stage (P = 0.0002), and high tumor grade (P = 0.0164). Multivariate analysis using the Cox proportional hazards model showed that high TF expression was an independent negative predictor for survival (hazard ratio, 2.014; P = 0.0076). Moreover, patients with TF-negative tumors had a significantly better prognosis even if lymph node metastasis was present (P < 0.0001). We also showed that TF knockdown by RNA interference suppressed the invasiveness of a pancreatic adenocarcinoma cell line in vitro. These results indicate that TF expression may contribute to the aggressiveness of pancreatic ductal adenocarcinoma by stimulating tumor invasiveness, and that evaluation of the primary tumor for TF expression may identify patients with a poor prognosis.
Troglitazone Acts on Cellular pH and DNA Synthesis through a Peroxisome Proliferator-Activated Receptor γ-Independent Mechanism in Breast Cancer-Derived Cell Lines
Authors: Turturro Francesco , Friday Ellen , Fowler Rocky , Surie Diya , Welbourne Tomas
Published: September 2005

Purpose: The purpose of this study was to assess whether troglitazone (TRO) would induce cellular acidosis by inhibiting Na+/H+ exchanger (NHE) 1 in breast carcinoma-derived cell lines and, if so, whether cellular acidosis would be associated with a reduction in proliferation.

Experimental Design: Intracellular pH (pHi) and acid extrusion capacity after an exogenous acid load were assayed using (2, 7)-biscarboxyethyl-5(6)-carboxyfluorescein in MCF-7 and MDA-MB-231 cells treated with TRO. Radiolabeled thymidine incorporation was used to assess DNA synthesis. Peroxisome proliferator-activated receptor (PPAR) γ involvement was assessed using an antagonist and PPARγ−/− NIH3T3 cells.

Results: TRO induced a prompt (<4 minute) and severe cellular acidosis in both MCF-7 (7.54 ± 0.23 to 6.77 ± 0.06; P < 0.001) and MDA-MB-231 cells (7.38 ± 0.18 to 6.89 ± 0.25; P < 0.05) after 12 minutes, without increasing acid production. Acid extrusion as assessed by the response to an exogenous acid load (NH4Cl pulse) was markedly blunted (MDA-MB-231, P < 0.01) or eliminated (MCF-7, P < 0.001). Chronic exposure to TRO resulted in NHE1 activity reduction (P < 0.05) and a dose-dependent decrease in DNA synthesis (<75% inhibition at 100 μmol/L; P < 0.001 and P < 0.01 for MCF-7 and MDA-MB-231, respectively) associated with a decreased number of viable cells. TRO-mediated inhibition of proliferation was not reversed by the presence of the PPARγ inhibitor GW9662 and was demonstrable in PPARγ−/− NIH3T3 cells, consistent with a PPARγ-independent mechanism.

Conclusions: TRO induces marked cellular acidosis in MCF-7 and MDA-MD-231 cells. Sustained acidosis is consonant with decreased proliferation and growth that is not reversed by a PPARγ antagonist. Our results support a NHE-mediated action of TRO that exerts its effect independent of PPARγ.
Modulation of Pulmonary Leukotriene B4 Production by Cyclooxygenase-2 Inhibitors and Lipopolysaccharide
Authors: Mao Jenny T. , Tsu I-Hsien , Dubinett Steven M. , Adams Bradley , Sarafian Theodore , Baratelli Felicita , Roth Michael D. , Serio Kenneth J.
Published: September 2005

Purpose: Emerging data continue to link carcinogenesis to inflammatory events involving the eicosanoid metabolic pathways. We therefore evaluated the effects of cyclooxygenase (COX)-2 inhibition on leukotriene (LT) B4 synthesis in the lungs of active smokers, as part of a pilot lung cancer chemoprevention study with celecoxib (Celebrex), an oral COX-2 inhibitor.

Experimental Design: Bronchoalveolar lavage was performed before celecoxib treatment and after 1 month of celecoxib treatment to recover alveolar macrophages (AMs) and lining fluid for study. After harvest, AMs were immediately stimulated in vitro with the calcium ionophore A23187. AMs obtained from smokers before treatment and from ex-smoker control subjects were also cultured overnight with SC58236, a selective COX-2 inhibitor, with or without lipopolysaccharide stimulation.

Results: Treatment with oral celecoxib only modestly increased LTB4 levels in bronchoalveolar lavage, without increasing the mRNA transcription of 5-lipoxygenase (5-LOX) or 5-LOX-activating protein in AMs, whereas the acute calcium ionophore-stimulated LTB4 production from smokers’ AMs was markedly increased by 10.6-fold. In addition, smokers’ AMs were twice as responsive in producing LTB4 when exposed to lipopolysaccharide compared with ex-smokers’ AMs. Concomitant COX-2 inhibition with SC58236, however, did not significantly impact these changes, whereas the 5-LOX inhibitor Zileuton blocked the generation of LTB4 in a dose-responsive manner. Finally, cycloheximide increased the production of LTB4 under all conditions, suggesting a shunting phenomenon and/or the presence of pathway inhibitors.

Conclusions: Our findings suggest that whereas oral celecoxib is capable of modulating LTB4 production in the lung microenvironment, under physiologic conditions, this effect is probably not functionally significant.
Enhanced Sensitivity to the HER1/Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Erlotinib Hydrochloride in Chemotherapy-Resistant Tumor Cell Lines
Authors: Dai Qun , Ling Yi-He , Lia Marie , Zou Yi-Yu , Kroog Glenn , Iwata Kenneth K. , Perez-Soler Roman
Published: September 2005

Purpose: Erlotinib (Tarceva, OSI-774) is a potent and specific inhibitor of the HER1/epidermal growth factor receptor (EGFR) tyrosine kinase. In phase II clinical studies, oral erlotinib monotherapy has shown antitumor activity in patients with advanced non–small cell lung cancer, head and neck cancer, and ovarian cancer after the failure of standard chemotherapy. We hypothesized that some tumors treated with multiple cytotoxic therapies may become more dependent on the HER1/EGFR signaling pathways for survival.

Experimental Design: The growth-inhibitory effect of erlotinib was tested on 10 pairs of chemosensitive, parental, and chemoresistant tumor cell lines.

Results: Enhanced sensitivity to erlotinib was observed in the doxorubicin-resistant human breast cancer cell line MCF-7, paclitaxel-resistant human ovarian carcinoma cell line A2780, and cisplatin-resistant human cervical carcinoma cell line ME180. The IC50 values of erlotinib in the resistant cell lines were 2- to 20-fold lower than those in the corresponding parental cell lines. This enhanced sensitivity to erlotinib correlated with higher HER1/EGFR and phospho-HER1/EGFR expression when compared with the corresponding parental cell lines. Acquired resistance to cytotoxic agents was not associated with cross-resistance to erlotinib. AE-ME180/CDDP-resistant xenografts showed greater sensitivity to erlotinib than parental ME180 xenografts did.

Conclusions: Our findings suggest that acquired resistance to cytotoxic therapy in some tumors is associated with enhanced sensitivity to HER1/EGFR inhibitors, which correlates with increased HER1/EGFR expression. These data may explain some of the observed clinical activity of HER1/EGFR inhibitors in patients previously treated with multiple therapies. HER1/EGFR tyrosine kinase inhibitors may be more effective as second- or third-line treatment for certain patients with tumors that were previously treated with multiple chemotherapy regimens.
Heregulin-Induced Activation of HER2 and HER3 Increases Androgen Receptor Transactivation and CWR-R1 Human Recurrent Prostate Cancer Cell Growth
Authors: Gregory Christopher W. , Whang Young E. , McCall Wesley , Fei Xiaoyin , Liu Yuanbo , Ponguta Liliana A. , French Frank S. , Wilson Elizabeth M. , Earp H. Shelton
Published: September 2005

Purpose: The androgen receptor (AR) is a ligand-dependent transcription factor that mediates gene expression and growth of normal and malignant prostate cells. In prostate tumors that recur after androgen withdrawal, the AR is highly expressed and transcriptionally active in the absence of testicular androgens. In these “androgen-independent” tumors, alternative means of AR activation have been invoked, including regulation by growth factors and their receptors in prostate cancer recurrence.

Experimental Design and Results: In this report, we show that HER receptor tyrosine kinases 1 through 4 are expressed in the CWR-R1 recurrent prostate cancer cell line; their stimulation by epidermal growth factor (EGF) and heregulin activates downstream signaling, including mitogen-activated protein kinase and phosphatidylinositol-3 kinase and Akt pathways. We show that heregulin activates HER2 and HER3 and increases androgen-dependent AR transactivation of reporter genes in CWR-R1 cells. Tyrosine phosphorylation of HER2 and HER3, AR transactivation, and cell proliferation induced by heregulin were more potently inhibited by the EGFR/HER2 dual tyrosine kinase inhibitor GW572016 (lapatinib) than the EGFR-specific inhibitor ZD1839 (gefitinib). Basal proliferation in the absence of growth factors was also inhibited by GW572016 to a greater extent than ZD1839, suggesting that low level HER2/HER3 activation perhaps by an autocrine pathway contributes to the proliferation signal.

Conclusions: These data indicate that heregulin signaling through HER2 and HER3 increases AR transactivation and alters growth in a recurrent prostate cancer cell line. Therefore, inhibition of low-level HER2 signaling may be a potential novel therapeutic strategy in prostate cancer.
Elevated Expression of Wnt Antagonists Is a Common Event in Hepatoblastomas
Authors: Koch Arend , Waha Andreas , Hartmann Wolfgang , Hrychyk Aksana , Schüller Ulrich , Waha Anke , Wharton Keith A. , Fuchs Serge Y. , Schweinitz Dietrich von , Pietsch Torsten
Published: September 2005

Hepatoblastomas are the most frequent malignant liver tumors of childhood. A high frequency of activating β-catenin mutations in hepatoblastomas indicates that the Wnt signaling pathway plays an important role in the development of this embryonic neoplasm. Stabilization of β-catenin leads to an increased formation of nuclear β-catenin-T-cell factor complexes and altered expression of Wnt-inducible target genes. In this study, we analyzed the mRNA expression levels of nine Wnt genes, including c-JUN, c-MYC, CYCLIN D1, FRA-1, NKD-1, ITF-2, MMP-7, uPAR, and β-TRCP, by competitive reverse transcription-PCR. We analyzed 23 hepatoblastoma biopsies for which matching liver tissue was available, 6 hepatoblastoma cell lines, and 3 human fetal liver samples. β-TRCP and NKD-1 were highly expressed in all hepatoblastoma samples, independent of the β-catenin mutational status, in comparison with their nontumorous counterparts. β-TRCP mRNA overexpression was associated with accumulation of intracytoplasmic and nuclear β-TrCP protein. In human liver tumor cells without β-catenin mutations, Nkd-1 inhibited the Wnt-3a-activated Tcf-responsive-luciferase reporter activity, whereas Nkd-1 in hepatoblastomas with β-catenin mutations had no antagonistic effect. Our data emphasize the inhibitory effect of β-TrCP and Nkd-1 on the Wnt signaling pathway in a manner analogous to Conductin (AXIN2) and Dkk-1, inhibitors shown previously to be up-regulated in hepatoblastomas. Our findings indicate that overexpression of the Wnt antagonists Nkd-1 and β-TrCP reveals an activation of the Wnt signaling pathway as a common event in hepatoblastomas. We propose that Nkd-1 and β-TrCP may be used as possible diagnostic markers for the activated Wnt signaling pathway in hepatoblastomas.
Morphology of Tumor Cell Nuclei Is Significantly Related with Survival Time of Patients with Glioblastomas
Authors: Nafe Reinhold , Franz Kea , Schlote Wolfgang , Schneider Berthold
Published: September 2005

Purpose: To investigate whether histomorphology of tumor cell nuclei has a significant and independent relation to survival time of patients with glioblastomas.

Experimental Design: Seventy-two tumors from 72 patients were investigated by means of digital image analysis. Proliferating and nonproliferating nuclei were separately measured and parameters of nuclear size, shape, texture, and spatial relationships (topometric parameters) were detected. Survival analysis was done regarding morphometric data together with the patients' age, the amount of resection (total or subtotal), and the classification of the tumor as a “primary” (de novo) or “secondary” glioblastoma.

Results: The overall relation of all morphometric data to the time of survival was highly significant (Cox analysis, P < 0.0001). Apart from the extent of surgical resection, parameters of nuclear shape and topometric variables, such as the distance between two nuclei lying nearest to each other, showed an independent and significant relation to survival time. The patients' age had also a significant but comparably slight relation to survival time.

Conclusions: The morphology of tumor cell nuclei, as represented by morphometric data, shows a significant relation to survival time of patients with glioblastomas. This relation is statistically independent from the amount of surgical resection, from the patients' age and from the classification of the glioblastoma as being primary or secondary. The results support the view that histomorphometry of tumor cell nuclei is a valuable prognostic marker for patients with glioblastomas. We believe that such a marker ought to be incorporated into the formation of individual therapeutic decisions.
Prospective Study of Quantitation of Plasma DNA Levels in the Diagnosis of Malignant versus Benign Prostate Disease
Authors: Boddy Jane L. , Gal Shira , Malone Peter R. , Harris Adrian L. , Wainscoat Jim S.
Published: September 2005

Purpose: The aim of this study was to determine the potential of cell-free DNA levels as a diagnostic marker for prostate cancer, having first established the effect that blood sample processing has on this measurement.

Experimental Design: A total of 152 blood samples were collected prospectively from patients before their prostate biopsy and 25 from men in two distinct control groups. Blood was processed to yield three components: one-spin plasma (n = 68), two-spin plasma (n = 152), and serum (n = 56) samples.

Results: Having established the effect of sample preparation on the measured DNA level, the more reliable two-spin plasma sample was used to determine the relationship between DNA and the presence of prostate cancer. Those patients with cancer (n = 78) had a significantly higher level of DNA compared with the control groups (P < 0.0001 and P < 0.0001). However, DNA levels in patients with a benign biopsy (n = 74) were significantly higher than the 78 patients confirmed to have cancer (P = 0.02).

Conclusions: We conclude that the sample type used in the quantitation of cell-free DNA has an effect on the level reported. Elevated levels are present in the two-spin plasma samples of patients with prostate cancer compared with healthy controls but are not of diagnostic value during the management of prostate cancer.
Phase I Study of Combined Pegylated Liposomal Doxorubicin with Protracted Daily Topotecan for Ovarian Cancer
Authors: Mirchandani Deepu , Hochster Howard , Hamilton Anne , Liebes Leonard , Yee Herman , Curtin John P. , Lee Sang , Sorich Joan , Dellenbaugh Cornelia , Muggia Franco M.
Published: September 2005

Purpose: To determine the maximum tolerated dose and dose-limiting toxicity of Doxil with low-dose continuous infusion topotecan and subsequently with low-dose oral topotecan. Other specific aims were preliminary assessment of activity in advanced ovarian and tubal malignancies, pharmacokinetics of oral topotecan, and correlation of response with topoisomerase I and II expression in tumors.

Methods: Eligible patients had histopathologically documented advanced cancers beyond standard therapy, performance status <2, and adequate organ functions. Doxil (30-40 mg/m2 i.v.) was given on day 1, with topotecan either oral topotecan 0.4 mg/m2 bid for 14 days or continuous infusion topotecan (0.3-0.4 mg/m2/d) for 14 to 21 days, in 28-day cycles. Fifty-seven patients, 23 with epithelial ovarian or tubal cancers were enrolled. Plasma levels of lactone form of topotecan were determined on patients receiving oral topotecan.

Results: Grade 4 neutropenia and thrombocytopenia and grade 3 diarrhea were dose-limiting toxicities at the highest dose levels explored. Doxil (40 mg/m2/day 1) and continuous infusion topotecan at 0.4 mg/m2/days 1 to 14 could be safely given and is the recommended phase II dose. Oral topotecan was limited by low and erratic plasma topotecan levels and frequent gastrointestinal toxicity. Particularly long partial responses and stable disease were observed in patients with epithelial ovarian or tubal cancers. Clinical benefit (objective responses and stable diseases) correlated with elevated expression of both topoisomerases by immunohistochemistry in four of six epithelial ovarian or tubal cancer tumor samples.

Conclusion: Doxil with 14-day topotecan infusion is a well-tolerated regimen and suitable for study in platinum-resistant or refractory ovarian or tubal cancers. Frequent gastrointestinal toxicity and/or erratic absorption complicate treatment with a longer topotecan infusion or with oral topotecan, respectively, and these combinations are not recommended.
The Clinical Relevance of Steroid Hormone Receptor Corepressors
Authors: Kumar Rakesh , Gururaj Anupama E. , Vadlamudi Ratna K. , Rayala Suresh K.
Published: September 2005

Steroid hormone receptors are ligand-dependent transcription factors that control a variety of essential physiologic and developmental processes in humans. The functional activity of a steroid receptor is regulated not only by hormones but also by an array of regulatory proteins such as coactivators, corepressors, and chromatin modifiers. Contrary to an earlier notion that corepressors and coactivators exist in separate complexes, these molecules, which have apparently opposite functions, are increasingly being found in the same complex, which allows for efficient transcriptional control mechanisms. These control mechanisms are in turn regulated by an array of post-translational modifications under the influence of upstream and local signaling networks. Because the outcome of steroidal hormone receptor transcriptional complexes is measured in terms of the expression of target genes, any dysregulation of coregulator complexes perturbs normal homeostasis and could contribute to the development and maintenance of malignant phenotypes. Increasing evidence implicating steroid hormone receptors and their coregulators in various pathophysiologic conditions has elicited interest in their structure and biology. Further advances in this field of study should open up a unique window for novel targeted therapies for diseases such as cancer. Here we briefly review the clinical relevance of corepressors, with a particular focus on their role in the development of cancerous phenotypes.
Dedifferentiation Precedes Invasion in the Progression from Barrett's Metaplasia to Esophageal Adenocarcinoma
Authors: Helm James , Enkemann Steven A. , Coppola Domenico , Barthel James S. , Kelley Scott T. , Yeatman Timothy J.
Published: September 2005

Purpose: Adenocarcinoma arises in Barrett's esophagus by progression from metaplasia to cancer through grades of dysplasia. Our aim in this exploratory study was to characterize the broad changes in gene expression that underlie this histologic progression to cancer and assess the potential for using these gene expression changes as a marker predictive of malignant progression in Barrett's epithelium.

Experimental Design: Microarray analysis was used to obtain individual gene expression profiles from endoscopic biopsies of nine esophageal adenocarcinomas and the Barrett's epithelia from which three of the cancers had arisen. Pooled samples from the Barrett's epithelia of six patients without cancer or dysplasia served as a reference.

Results: Barrett's epithelia from which cancer had arisen differed from the reference Barrett's epithelia primarily by underexpression of genes, many of which function in governing cell differentiation. These changes in gene expression were found even in those specimens of Barrett's epithelia from which cancer had arisen that lacked dysplasia. Each cancer differed from the Barrett's epithelium from which it had arisen primarily by an overexpression of genes, many of which were associated with tissue remodeling and invasiveness. Cancers without identifiable Barrett's epithelium differed from cancers that had arisen from a Barrett's epithelium by having an even greater number of these overexpressed genes.

Conclusions: Histologic progression from Barrett's epithelium to cancer is associated with a gradient of increasing changes in gene expression characterized by an early loss of gene function governing differentiation that begins before histologic change; gain in function of genes related to remodeling and invasiveness follows later. This correlation of histologic progression with increasing changes in gene expression suggests that gene expression changes in biopsies taken from Barrett's epithelium potentially could serve as a marker for neoplastic progression that could be used to predict risk for developing cancer.
Nuclear Factor-κB Activation Correlates with Better Prognosis and Akt Activation in Human Gastric Cancer
Authors: Lee Byung Lan , Lee Hye Seung , Jung Jieun , Cho Sung Jin , Chung Hee-Yong , Kim Woo Ho , Jin Young-Woo , Kim Chong Soon , Nam Seon Young
Published: September 2005

Purpose: Because the biological significance of constitutive nuclear factor-κB (NF-κB) activation in human gastric cancer is unclear, we undertook this study to clarify the regulatory mechanism of NF-κB activation and its clinical significance.

Experimental Design: Immunohistochemistry for NF-κB/RelA was done on 290 human gastric carcinoma specimens placed on tissue array slides. The correlations between NF-κB activation and clinicopathologic features, prognosis, Akt activation, tumor suppressor gene expression, or Bcl-2 expression were analyzed. We also did luciferase reporter assay, Western blot analysis, and reverse transcription-PCR using the SNU-216 human gastric cancer cell line transduced with retroviral vectors containing constitutively active Akt or the NF-κB repressor mutant of IκBα.

Results: Nuclear expression of RelA was found in 18% of the gastric carcinomas and was higher in early-stage pathologic tumor-node-metastasis (P = 0.019). A negative correlation was observed between NF-κB activation and lymphatic invasion (P = 0.034) and a positive correlation between NF-κB activation and overall survival rate of gastric cancer patients (P = 0.0228). In addition, NF-κB activation was positively correlated with pAkt (P = 0.047), p16 (P = 0.004), adenomatous polyposis coli (P < 0.001), Smad4 (P = 0.002), and kangai 1 (P < 0.001) expression. An in vitro study showed that NF-κB activity in gastric cancer cells is controlled by and controls Akt.

Conclusions: NF-κB activation was frequently observed in early-stage gastric carcinoma and was significantly correlated with better prognosis and Akt activation. These findings suggest that NF-κB activation is a valuable prognostic variable in gastric carcinoma.
Pulsatile Administration of the Epidermal Growth Factor Receptor Inhibitor Gefitinib Is Significantly More Effective than Continuous Dosing for Sensitizing Tumors to Paclitaxel
Authors: Solit David B. , She Yuhong , Lobo Jose , Kris Mark G. , Scher Howard I. , Rosen Neal , Sirotnak Frank M.
Published: September 2005

Purpose: Gefitinib is an inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase. Continuous inhibition of EGFR signaling is thought necessary for optimal inhibition of tumor cell proliferation. We hypothesized that continuous gefitinib may antagonize the effects of cytotoxics that inhibit tumor cells in other phases of the cell cycle. Furthermore, we hypothesized that intermittent dosing would allow for dose escalation and greater inhibition of EGFR-dependent antiapoptotic pathways.

Experimental Design: To test these assertions, we compared combinations of paclitaxel and gefitinib using either intermittent or continuous dosing schedules in mice.

Results: We found that when used in combination with paclitaxel, pulsatile gefitinib was significantly superior to continuous dosing. When gefitinib was administered for one or two consecutive days before paclitaxel, much higher doses could be given safely. Two days of gefitinib treatment before paclitaxel was most effective, causing significantly greater mean tumor regression and a higher percentage of complete responses than other schedules.

Conclusions: The results suggest that the dose and schedule of an EGFR inhibitor required to effectively inhibit proliferation may differ from that required to stimulate apoptosis or to induce other effects. The former may require continuous EGFR inhibition to maintain cell cycle arrest, whereas sensitization to apoptosis may be optimally induced by profound but temporary inhibition of survival pathways. Our data suggest that the effects of receptor inhibition vary as a function of dose and schedule and that continuous administration of tyrosine kinase inhibitors may not be the best schedule with which to combine such agents with taxanes.
Proton Beam Therapy for Hepatocellular Carcinoma: A Retrospective Review of 162 Patients
Authors: Chiba Toshiya , Tokuuye Koichi , Matsuzaki Yasushi , Sugahara Shinji , Chuganji Yoshimichi , Kagei Kenji , Shoda Junichi , Hata Masaharu , Abei Masato , Igaki Hiroshi , Tanaka Naomi , Akine Yasuyuki
Published: September 2005

Purpose: We present results of patients with hepatocellular carcinoma (HCC) treated with proton beam therapy.

Experimental Design: We reviewed 162 patients having 192 HCCs treated from November 1985 to July 1998 by proton beam therapy with or without transarterial embolization and percutaneous ethanol injection. The patients in the present series were considered unsuitable for surgery for various reasons, including hepatic dysfunction, multiple tumors, recurrence after surgical resection, and concomitant illnesses. The median total dose of proton irradiation was 72 Gy in 16 fractions over 29 days.

Results: The overall survival rate for all of the 162 patients was 23.5% at 5 years. The local control rate at 5 years was 86.9% for all 192 tumors among the 162 patients. The degree of impairment of hepatic functions attributable to coexisting liver cirrhosis and the number of tumors in the liver significantly affected patient survival. For 50 patients having least impaired hepatic functions and a solitary tumor, the survival rate at 5 years was 53.5%. The patients had very few acute reactions to treatments and a few late sequelae during and after the treatments.

Conclusions: Proton beam therapy for patients with HCC is effective, safe, well tolerable, and repeatable. It is the useful treatment mode for either cure or palliation for patients with HCC irrespective of tumor size, tumor location in the liver, insufficient feeding of the tumor with arteries, presence of vascular invasion, impaired hepatic functions, and coexisting intercurrent diseases.
Induction of p53 Up-Regulated Modulator of Apoptosis Messenger RNA by Chemotherapeutic Treatment of Locally Advanced Breast Cancer
Authors: Middelburg Rutger , de Haas Richard R. , Dekker Henk , Kerkhoven Ron M. , Pohlmann Paula R. , Fuentes-Alburo Adolfo , Mohar Alejandro , Pinedo Herbert M. , Lankelma Jan
Published: September 2005

Purpose: In biopsies of patients with locally advanced breast cancer, we investigated the in vivo changes of the gene expression pattern induced by chemotherapy to find genes that are potentially responsible for the efficacy of the drug.

Experimental Design: Early cellular responses to chemotherapy-induced damage, both in vivo and in vitro, were investigated by analyzing chemotherapy-induced changes in gene expression profiles. Core biopsies were taken from nine patients with locally advanced breast cancer, before and at 6 hours after initiation of doxorubicin-based chemotherapy. Both samples were cohybridized on the same microarray containing 18,000 cDNA spots.

Results: The analysis revealed marked differences in gene expression profile between treated and untreated samples. The gene which was most frequently found to be differentially expressed was p53 up-regulated modulator of apoptosis (PUMA). This gene was up-regulated in eight of nine patients with an average factor of 1.80 (range, 1.36-2.73). In vitro MCF-7 breast cancer cells exposed to clinically achievable doxorubicin concentrations for 6 hours revealed marked induction of PUMA mRNA, as well.

Conclusions: This is the first report describing PUMA mRNA to be up-regulated as a response to chemotherapy in patients. Because PUMA is a known member of the family of BH3-only proapoptotic proteins, this finding suggests PUMA's potential importance for the response to anticancer drugs.
Prognostic Significance of Fascin Overexpression in Human Esophageal Squamous Cell Carcinoma
Authors: Hashimoto Yosuke , Ito Tetsuo , Inoue Harutaka , Okumura Tomoyuki , Tanaka Eiji , Tsunoda Shigeru , Higashiyama Motoshige , Watanabe Go , Imamura Masayuki , Shimada Yutaka
Published: September 2005

Purpose: Fascin, an actin bundling protein, induces membrane protrusions and increased cell motility in various transformed cells. The expression of fascin in epithelial neoplasms has been described only recently, and the role of fascin in esophageal squamous cell carcinoma (ESCC) is still unknown.

Experimental Design: Paraffin sections of 200 patients with ESCC were immunohistochemically investigated. The expression levels of fascin mRNA in 20 ESCC tissues were compared with that in corresponding normal esophageal epithelium by semiquantitative reverse transcription-PCR. We also examined fascin protein expression in 33 ESCC cell lines. The role of fascin in cell motility and invasiveness in ESCC cells was assessed by the vector-based small interfering RNA.

Results: In immunohistochemical study, the intensity of fascin expression was usually increased in the tumor compared with that in normal epithelium. Fascin overexpression was significantly associated with a poor prognosis (immunoreactive rate, P = 0.033; immunoreactive intensity, P = 0.031). The fascin immunoreactive rate was associated with extent of the tumor (P = 0.002) and lymph node metastasis (P = 0.003). Multivariate analysis showed that fascin expression intensity was an independent prognostic factor, but the immunoreactive rate was not.

In addition, up-regulation of fascin mRNA was found in 60% (12 of 20) of patients. In vitro study revealed that all 33 ESCC cell lines expressed fascin protein at a certain level. KYSE170, one of the fascin-overexpressed cells, decreased its motile and invasive properties after down-regulation of fascin expression.

Conclusion: Our findings suggest that fascin overexpression may play an important role in the progression of ESCC.