bluuebunny/crossref_metadata_2025 huggingface dataset:
High-Level Expression of the Coxsackievirus and Adenovirus Receptor Messenger RNA in Osteosarcoma, Ewing’s Sarcoma, and Benign Neurogenic Tumors among Musculoskeletal Tumors
Authors: Gu Wenguang , Ogose Akira , Kawashima Hiroyuki , Ito Masayuki , Ito Tomoyuki , Matsuba Atsushi , Kitahara Hiroshi , Hotta Tetsuo , Tokunaga Kunihiko , Hatano Hiroshi , Morita Tetsuro , Urakawa Sayuri , Yoshizawa Tatsuya , Kawashima Hiroyuki , Kuwano Ryozo , Endo Naoto
Published: September 2005

Purpose: The sensitivity of human tumor tissues to infection with recombinant adenoviruses correlates with the expression of the coxsackievirus and adenovirus receptor (CAR). CAR has been shown to function as the primary receptor for adenoviruses and to play a critical role in adenovirus entry into host cells. It is important for clinical gene therapy to determine the expression level of CAR in tumor tissues.

Experimental Design: We analyzed the expression of CAR mRNA in 154 musculoskeletal tumor tissues from 154 patients and 10 normal mesenchymal tissues from 3 patients using reverse transcription-PCR and real-time quantitative PCR. An adenovirus infection assay was performed in two cell lines that were established from CAR-positive osteosarcoma tissue and CAR-negative malignant fibrous histiocytoma tissue.

Results: Ninety-nine of 154 tumors were detected as CAR positive by reverse transcription-PCR. We found that the expression levels of CAR mRNA varied markedly between different tumors as determined by real-time quantitative PCR. CAR mRNA was expressed at high levels in osteosarcoma, Ewing’s sarcoma, neurofibroma, and schwannoma; at intermediate levels in exostosis, giant cell tumor, liposarcoma, synovial sarcoma, malignant peripheral nerve sheath tumor, and hemangioma; and at low levels in alveolar soft part sarcoma and desmoid. Whereas the osteosarcoma cell line that expressed a high level of CAR mRNA, like its parent tumor, had a high efficiency of adenovirus infection, the malignant fibrous histiocytoma cell line with almost undetectable expression of CAR mRNA, like its parent tumor, had a low efficiency of infection.

Conclusions: Our data showed the great variations in CAR mRNA expression among human musculoskeletal tumors and mesenchymal tissues and implicated the potential usefulness of adenoviral vectors in gene therapy for osteosarcoma, Ewing’s sarcoma, neurofibroma, and schwannoma. Efficient transduction with adenovirus for gene therapy could be realized in appropriate, sensitive tumor types.
Prognostic Significance of Cyclooxygenase-2 in Oropharyngeal Squamous Cell Carcinoma
Authors: Chang Bryan W. , Kim David H. , Kowalski Diane P. , Burleson Joseph A. , Son Yung H. , Wilson Lynn D. , Haffty Bruce G.
Published: September 2005

Purpose: To determine the relative prognostic significance of cyclooxygenase (COX)-2 expression in patients with oropharyngeal squamous cell carcinoma (SCC).

Experimental Design: This retrospective cohort study included 82 patients with SCC referred to the Department of Therapeutic Radiology at Yale-New Haven Hospital (Connecticut) between 1980 and 1999 who were treated with primary external beam radiotherapy or gross total surgical resection and postoperative radiotherapy. A microarray of archival tumor tissue was constructed and stained with monoclonal antibodies directed against COX-2 and scored for intensity by a pathologist blinded to the clinical outcomes of the patients. COX-2 immunoreactivity and clinicopathological data were analyzed with respect to survival endpoints using bivariate and multivariate techniques.

Results: Frequency of COX-2 overexpression was 45%. In multivariate analysis, COX-2 positivity predicted poor 3-year survival (P = 0.02; odds ratio = 0.41; 95% confidence interval, 0.20–0.84). Increasing age was significantly associated with increased 3-year survival (P = 0.03; odds ratio = 1.04; 95% confidence interval, 1.004–1.09). Positive COX-2 status trended toward predicting decreased 3-year disease-free survival.

Conclusions: COX-2 was the most important predictor of poor survival in this patient cohort. In patients with oropharyngeal SCC treated with external-beam radiation therapy, overexpression of COX-2 may affect clinical outcome, and COX-2 may therefore prove valuable both as a prognostic factor and as a therapeutic target.
Reliable and Sensitive Identification of Occult Tumor Cells Using the Improved Rare Event Imaging System
Authors: Kraeft Stine-Kathrein , Ladanyi Andras , Galiger Kevin , Herlitz Anna , Sher Andrew C. , Bergsrud Danielle E. , Even Gaelle , Brunelle Stephanie , Harris Lyndsay , Salgia Ravi , Dahl Tom , Kesterson John , Chen Lan Bo
Published: September 2005

Purpose: The purpose of this study was to assess the feasibility of using rare event imaging system (REIS)-assisted analysis to detect occult tumor cells (OTCs) in peripheral blood (PB). The study also sought to determine whether REIS-assisted OTC detection presents a clinically viable alternative to manual microscopic detection to establish the true significance of OTC from solid epithelial tumors.

Experimental Design: We recently demonstrated proof of concept using a fluorescence-based automated microscope system, REIS, for OTC detection from the PB. For this study, the prototype of the system was adopted for high-throughput and high-content cellular analysis.

Results: The performance of the improved REIS was examined using normal blood (n = 10), normal blood added to cancer cells (n = 20), and blood samples obtained from cancer patients (n = 80). Data from the screening of 80 clinical slides from breast and lung cancer patients, by manual microscopy and by the REIS, revealed that as many as 14 of 35 positive slides (40%) were missed by manual screening but positively identified by REIS. In addition, REIS-assisted scanning reliably and reproducibly quantified the total number of cells analyzed in the assay and categorized positive cells based on their marker expression profile.

Conclusions: REIS-assisted analysis provides excellent sensitivity and reproducibility for OTC detection. This approach may enable an improved method for screening of PB samples and for obtaining novel information about disease staging and about risk evaluation in cancer patients.
c-Kit Expression in Patients with Uterine Leiomyosarcomas
Authors: Raspollini Maria Rosaria , Amunni Gianni , Villanucci Alessandro , Pinzani Pamela , Simi Lisa , Paglierani Milena , Taddei Gian Luigi
Published: September 2005

Purpose: Uterine leiomyosarcomas are rare tumors characterized by their resistance to chemotherapy and radiation treatment. Surgery is the primary method of treatment, but for patients with unresectable disease, alternate therapeutic options are clearly warranted. According to initial observations of c-KIT expression, correlation with a bad prognosis, and the successful therapeutic possibility of STI571 in gastrointestinal stromal tumors, the data have encouraged us to study c-KIT expression in these tumors.

Experimental Design: We analyzed the expression of c-KIT and genetic assessment of exon 11 of c-kit gene in 32 uterine leiomyosarcomas.

Results: In 17 cases (53.1%), we observed a c-KIT expression in tumor cells. Of the 17 patients with distinct c-KIT-positive immunoreactivity, eight had I or II stage disease and nine had III or IV stage disease. Molecular genetic analysis of exon 11, analyzed by direct DNA sequencing, was performed for all of the c-KIT-positive uterine leiomyosarcomas. No mutations were found.

Conclusion: The conventional chemotherapy in leiomyosarcomas appears to be ineffective for patients with metastatic or unresectable disease, and the management of these patients poses a special problem. In these women, new therapeutic strategies are warranted. The treatment with STI571 in leiomyosarcoma patients might be hypothesized, because uterine leiomyosarcomas also express c-KIT.
Clonal Isolation of Different Strains of Mouse Mammary Tumor Virus-Like DNA Sequences from Both the Breast Tumors and Non-Hodgkin’s Lymphomas of Individual Patients Diagnosed with Both Malignancies
Authors: Etkind Polly R. , Stewart Alexandre F. R. , Dorai Thambi , Purcell Daniel J. , Wiernik Peter H.
Published: September 2005

Purpose: In a previous study, we had detected the presence of mouse mammary tumor virus (MMTV)-like envelope (ENV) gene sequences in both the breast tumors and non-Hodgkin’s lymphoma tissue of two of our breast tumor patients who had been diagnosed simultaneously with both malignancies. The aim of this study was to determine if MMTV-like DNA sequences are present in the breast tumors and non-Hodgkin’s lymphomas of additional patients suffering from both malignancies and if so to characterize these sequences in detail.

Experimental Design: DNA was extracted from formalin-fixed, paraffin-embedded tissue sample blocks of breast tumors and non-Hodgkin’s lymphomas from patients suffering from both malignancies. A 250-bp region of the MMTV ENV gene and a 630-bp region of the MMTV long terminal repeat (LTR) open reading frame (ORF) that encodes the MMTV superantigen (sag) gene were amplified by PCR from the isolated DNA. Amplified products were analyzed by Southern blotting, cloned, and sequenced.

Results: MMTV-like ENV and LTR sequences were detected in both the breast tumors and non-Hodgkin’s lymphomas of 6 of 12 patients suffering from both malignancies. A novel mutant of the MMTV ENV gene was identified in these patients. Characterization of the MMTV-like LTR highly variable sag sequences revealed total or nearly total identity to three distinct MMTV proviruses from two different branches of the MMTV phylogenetic tree.

Conclusions: The presence of MMTV-like ENV and LTR sequences in both the breast tumors and non-Hodgkin’s lymphomas of 6 additional patients suggests a possible involvement of these sequences in these two malignancies. MMTV-like LTR sequence homology to different MMTV proviruses revealed the presence of more than one strain of MMTV-like sequences in each individual suggesting the possibility of multiple infections in these patients.
The Prognostic Value of Isolated Tumor Cells in Bone Marrow in Breast Cancer Patients
Authors: Naume Bjørn , Wiedswang Gro , Borgen Elin , Kvalheim Gunnar , Kåresen Rolf , Qvist Hanne , Janbu Jan , Harbitz Torstein , Nesland Jahn M.
Published: September 2005

Purpose/Experimental Design: Immunocytochemical detection of isolated tumor cells (ITCs) in the bone marrow (BM) is a prognostic factor in breast cancer. However, hematopoietic cells (HCs) can occasionally be stained by the techniques used. Morphological evaluation improves the specificity of ITC detection, but optimal separation of ITCs from false-positive HCs needs to be determined. Here, predetermined morphological categories of immunocytochemically (ICC)-positive cells in the BM and the number of detected ITCs were analyzed for association with clinical outcome in 817 early-stage breast cancer patients (median 49 months of follow-up). All ICC+ cells detected were categorized into one of the following groups: (a) tumor cell (TC); (b) uninterpretable cell (UIC); (c) probable HC; or (d) HC.

Results: Among the TC+ patients, 30.6% and 25.9% experienced systemic relapse (SR) and breast cancer death (BCD), respectively, as compared with 13.3% and 8.5% of patients without TCs in the BM (survival analyses: P < 0.001, log-rank). The SR and BCD rate was 19.7% and 15.8% for TC−/UIC+ patients versus 12.5% and 7.4% for TC−/UIC− patients. Survival analyses confirmed that the UIC+ group contained clinically significant cells (P = 0.018, log-rank). No difference in clinical outcome was observed, regardless of whether probable HCs or HCs were present. Analyzing the number of ITC+ cells, SR and BCD occurred in 12.4% and 7.4% of patients with 0 ITCs present, 21.3% and 18.5% of patients with 1 ITC present, 19.4% and 16.7% of patients with 2 ITCs present, and 42.5% and 32.5% of patients with ≥3 ITCs present.

Conclusions: Morphological categorization of ICC+ cells improves the clinical value of ITC detection in the BM. The presence of only one ITC reduces survival, and a greater number of ITCs further aggravates the prognosis.
A Randomized Phase II Trial of the Antiangiogenic Agent SU5416 in Hormone-Refractory Prostate Cancer
Authors: Stadler Walter M. , Cao Dingcai , Vogelzang Nicholas J. , Ryan Christopher W. , Hoving Kristin , Wright Russell , Karrison Theodore , Vokes Everett E.
Published: September 2005

Purpose: To assess the activity of the antiangiogenic agent and VEGFR2 inhibitor SU5416 in hormone-refractory prostate cancer.

Patients and Methods: Thirty-six chemotherapy naïve patients were randomized to treatment with SU5416 (145 mg/m2) and dexamethasone premedication or dexamethasone alone. Patients in the control arm could cross over to experimental therapy after progression. Prostate-specific antigen (PSA) was measured every 2 weeks, and radiological evaluation was performed every 8 weeks. In vitro assessment of SU5416 on PSA secretion was assessed in the LNCaP cell line. Baseline serum basic fibroblast growth factor and plasma vascular endothelial growth factor (VEGF) were explored as prognostic factors.

Results: VEGF receptor-2 expression is detectable in prostate cancer cell lines, and SU5416 inhibited in vitro PSA secretion. No effect of SU5416 on PSA secretion or time to progression is detectable in patients. VEGF and basic fibroblast growth factor were not prognostic. Headache and fatigue were the most common SU5416 toxicities, but hyperglycemia, hyponatremia, lymphopenia, infection, and adrenal suppression, all attributable to steroids and the required central line, were common.

Conclusion: No disease modifying effects of SU5416 were detectable in this small study. Modest toxicity, an inconvenient administration schedule, and availability of other VEGFR-targeted agents support the decision to halt further evaluation of SU5416 in prostate cancer.
Magnetic Resonance Imaging Measurements of the Response of Murine and Human Tumors to the Vascular-Targeting Agent ZD6126
Authors: Evelhoch Jeffrey L. , LoRusso Patricia M. , He Zhanquan , DelProposto Zachary , Polin Lisa , Corbett Thomas H. , Langmuir Peter , Wheeler Catherine , Stone Andrew , Leadbetter Joanna , Ryan Anderson J. , Blakey David C. , Waterton John C.
Published: September 2005

Purpose: ZD6126 is a novel vascular targeting agent currently undergoing clinical evaluation. It acts by destabilizing the microtubulin of fragile and proliferating neoendothelial cells in tumors. The drug leads to blood vessel congestion, the selective destruction of the vasculature, and extensive necrosis in experimental tumors. The aim of the study reported here was to assess the ability of dynamic contrast enhanced magnetic resonance imaging (MRI) to measure the antivascular effects of ZD6126 in tumors.

Experimental Design: The work was carried out in mice bearing C38 colon adenocarcinoma and in patients with advanced cancers. MRI was performed before and 6 h (human tumors) or 24 h (C38 tumors) after i.v. drug administration. Contrast agent (gadolinium diethylenetriaminepentaacetate) enhancement was characterized by the initial area under the gadolinium diethylenetriaminepentaacetate uptake versus time curve (IAUC). IAUC reflects blood flow, vascular permeability, and the fraction of interstitial space.

Results: The median IAUC was reduced in all C38 tumors after ZD6126 administration [by 6–48% at 50 mg/kg (n = 3)], 58–91% at 100 mg/kg (n = 4), and 11–93% at 200 mg/kg (n = 6). In contrast, the administration of vehicle only led to no consistent change in median IAUC (n = 4). The ZD6126-induced changes in median IAUC appeared to be dose dependent (P = 0.045). No ZD6126-induced changes were apparent in murine muscle. Similar effects were seen in preliminary data from human tumors (11 tumors studied, 9 patients). At doses of 80 mg/m2 and higher, the median IAUC post-ZD6126 treatment was reduced in all of the tumors studied (8 tumors, 6 patients) to 36–72% from the baseline value. There was a significant trend of increasing reductions with increasing exposure (P < 0.01). No drug-induced changes in human muscle or spleen IAUC were apparent. The reproducibility of the median IAUC parameter was investigated in patients. In 19 human tumors (measured in 19 patients) inter- and intratumor coefficients of variation were 64 and 18%.

Conclusions: The contrast enhanced-MRI measured median IAUC is a useful end point for quantifying ZD6126 antivascular effects in human tumors.
Hypoxia and Hypoxia-Inducible Factor-1 Target Genes in Central Nervous System Radiation Injury
Authors: Nordal Robert A. , Nagy Andras , Pintilie Melania , Wong C. Shun
Published: September 2005

Purpose: Microvascular permeability changes and loss of blood-brain barrier integrity are important features of central nervous system (CNS) radiation injury. Expression of vascular endothelial growth factor (VEGF), an important determinant of microvascular permeability, was examined to assess its role in CNS radiation damage. Because hypoxia mediates VEGF up-regulation through hypoxia-inducible factor-1α (HIF1α) induction, we studied the relationships of hypoxia, HIF1α expression, and expression of VEGF in this damage pathway.

Experimental Design: Expression of HIF1α, VEGF, and another hypoxia-responsive gene, glucose transporter-1, was assessed in the irradiated rat spinal cord using immunohistochemistry and in situ hybridization. Hypoxic areas were identified using the nitroimidazole 2-(2-nitro-1H-imidazole-l-yl)-N-(2,2,3,3,3,-pentafluoropropyl) acetamide. To determine the causal importance of VEGF expression in radiation myelopathy, we studied the response of transgenic mice with greater (VEGF-Ahi/+), reduced (VEGF-Alo/+), and wild-type VEGF activity to thoracolumbar irradiation.

Results: In rat spinal cord, the number of cells expressing HIF1α and VEGF increased rapidly from 16 to 20 weeks after radiation, before white matter necrosis and forelimb paralysis. A steep dose response was observed in expression of HIF1α and VEGF. HIF1α and VEGF expressing cells were identified as astrocytes. Hypoxia was present in regions where up-regulation of VEGF and glucose transporter-1 and increased permeability was observed. VEGF-Alo/+ mice had a longer latency to development of hindlimb weakness and paralysis compared with wild-type or VEGF-Ahi/+ mice.

Conclusions: VEGF expression appears to play an important role in CNS radiation injury. This focuses attention on VEGF and other genes induced in response to hypoxia as targets for therapy to reduce or prevent CNS radiation damage.
Generation of Carcinoembryonic Antigen (CEA)-Specific T-Cell Responses in HLA-A*0201 and HLA-A*2402 Late-Stage Colorectal Cancer Patients after Vaccination with Dendritic Cells Loaded with CEA Peptides
Authors: Liu Ko-Jiunn , Wang Chuan-Cheng , Chen Li-Tzong , Cheng Ann-Lii , Lin Dong-Tsamn , Wu Yu-Chen , Yu Wei-Lan , Hung Yi-Mei , Yang Hui-Yu , Juang Shin-Hun , Whang-Peng Jacqueline
Published: September 2005

Purpose: We intranodally immunized metastatic colorectal carcinoma patients, who had failed standard chemotherapy, with dendritic cells (DCs) pulsed with HLA-A*0201- or HLA-A*2402-restricted carcinoembryonic antigen (CEA) peptides to evaluate the safety of this treatment and the immune response against CEA peptides before and after the treatment.

Experimental Design: Six patients with the HLA-A*2402 genotype and 4 patients with the HLA-A*0201 genotype were enrolled. A single CEA peptide (YLSGANLNL) or two CEA peptides (QYSWFVNGTF and TYACFVSNL) were used for patients with the HLA-A*0201 or HLA-A*2402 genotype, respectively. Autologous DCs were generated by culturing adherent mononuclear cells with interleukin 4 and granulocyte macrophage colony-stimulating factor for 6 days. Maturation of DCs was then induced with tumor necrosis factor α for 40 h. Mature DCs were pulsed with appropriate CEA peptides for 2 h. After washing, 1 million peptide-pulsed DCs were injected into one inguinal lymph node under sonographic guidance. Each patient received four injections.

Results: No grade II/III toxicity or autoimmunity was observed. An increase in the number of CEA-specific T cells after DC vaccination could be detected in 7 of 10 (70%) patients. Two (20%) patients had stable disease for at least 12 weeks. One of these 2 patients experienced a transient decrease in CEA levels during the treatment period and also had the most significant T-cell response against the immunizing CEA peptides.

Conclusions: These results suggest that our vaccination procedure can generate or boost specific T-cell responses and may provide clinical benefit in certain cancer patients.
The dl1520 Virus Is Found Preferentially in Tumor Tissue after Direct Intratumoral Injection in Oral Carcinoma
Authors: Morley Stephen , MacDonald Gordon , Kirn David , Kaye Stan , Brown Robert , Soutar David
Published: September 2005

Purpose: dl1520 (also known as Onyx-015) is an E1B-deleted adenovirus designed to selectively lyse p53-deficient cancer cells. Clinical trials involving patients with recurrent squamous cell carcinoma of the head and neck have shown clinical efficacy, but no direct evidence as to the tumor or p53 selectivity of the virus was demonstrated. We wanted to determine whether dl1520 is selective for survival and replication within tumor tissue after direct injection and whether this is determined by p53 status of the tissues. We also wanted to ascertain whether the virus has any macroscopic effect on normal tissue.

Experimental Design: An open-label Phase II trial was devised in which a fixed dose of the virus was administered to 15 patients via a direct intertumoral injection before surgery for untreated oral squamous cell carcinoma. The agent was also delivered into an area of adjacent normal buccal mucosa. Specimens of the excised tumor and of biopsies of the injected normal tissue were assessed for viral presence and p53 status.

Results: We demonstrated that the virus replicates selectively in tumor as opposed to normal tissue after this direct injection. It was not possible to determine whether this selectivity was p53 related. It was found that dl1520 triggers an early rise in apoptosis levels in injected normal tissues. No adverse effects of viral injection were noted.

Conclusions: This is the first report of injection of dl1520 into previously untreated squamous cell cancer. The data support the concept that dl1520 is replication deficient in normal, compared with cancerous, tissues and has potential as a selective anticancer agent against tumor tissues.
Expression of the Forkhead Transcription Factor FOXP1 Is Associated with Estrogen Receptor α and Improved Survival in Primary Human Breast Carcinomas
Authors: Fox Stephen B. , Brown Philip , Han Cheng , Ashe Sally , Leek Russel D. , Harris Adrian L. , Banham Alison H.
Published: September 2005

Purpose: The FOXP1 protein belongs to a functionally diverse family of winged-helix or forkhead transcription factors that have diverse roles in cellular proliferation, differentiation, and neoplastic transformation. The FOXP1 gene, which maps to 3p14, shows common loss of heterozygosity in breast tumors and is a candidate tumor suppressor gene. However, its role in breast cancer is unknown.

Experimental Design: We have therefore investigated the pattern of FOXP1 expression in whole sections from normal (n = 16) and neoplastic (n = 90) breast tissues and correlated the level of expression in 283 invasive breast carcinomas on tissue microarrays with clinicopathological factors and survival. Because a relationship with estrogen receptor (ER) was identified, estrogen (17β-estradiol) regulation and ER/FOXP1 colocalization was also investigated.

Results: Expression of FOXP1 was significantly positively associated with ER (P = 0.03) and negatively with epidermal growth factor receptor (P = 0.01) but no association with age (P = 0.91), lymph node status (P = 0.94), size (P = 0.76), or grade (P = 0.22). In a multivariate analysis of survival, FOXP1 expression was associated with a significantly improved relapse-free (P = 0.03) and borderline overall (P = 0.09) survival. Unlike normal breast, there was common coexpression of FOXP1 and ER in cell lines and tumors, but no 17β-estradiol (10-9 m) regulation of FOXP1 in MCF-7 cells was demonstrated.

Conclusions: Our findings support a role for FOXP1 as a potential ER coregulator in human breast carcinoma and suggest that it may also independently regulate additional important pathways that control the progression of breast cancer.
Expression Pattern of the Novel Gene EG-1 in Cancer
Authors: Zhang Liping , Maul Raymond Scott , Rao JianYu , Apple Sophia , Seligson David , Sartippour Maryam , Rubio Rosalio , Brooks Mai N.
Published: September 2005

Purpose: We recently discovered a novel gene responsive to tumor-conditioned media: endothelial-derived gene 1 (EG-1). Its transcript has been shown to be present in epithelial cells, as well as in endothelial cells. In this study, we examined the levels of EG-1 protein expression in breast, colon, prostate, and lung cancers, which constitute the four most common solid malignancies in the United States.

Experimental Design: Polyclonal antibodies were generated that recognize the EG-1 peptide. These antibodies were used in immunoblot analysis, as well as immunohistochemistry of multiple human clinical specimens of cancer.

Results: In immunoblots of whole cell lysates, EG-1 antibodies revealed the presence of a 22-kDa peptide. Immunohistochemistry of breast, colon, and prostate specimens showed higher levels of EG-1 peptides in cancer tissues, in comparison with their benign counterparts. However, EG-1 expression was minimal in both benign and malignant lung tissues.

Conclusions: Here, we demonstrated that the expression of EG-1 is elevated in cancerous in comparison to benign epithelial cells, as seen in immunohistochemistry of human pathological specimens. These observations collectively support the hypothesis that the novel gene EG-1 is associated with the malignant phenotype of the common epithelial-derived cancers of the breast, colon, and prostate.
Chromatin Remodeling Factors and BRM/BRG1 Expression as Prognostic Indicators in Non-Small Cell Lung Cancer
Authors: Fukuoka Junya , Fujii Takeshi , Shih Joanna H. , Dracheva Tatiana , Meerzaman Daoud , Player Audrey , Hong Kyeong , Settnek Sharon , Gupta Ajay , Buetow Kenneth , Hewitt Stephen , Travis William D. , Jen Jin
Published: September 2005

We immunohistochemically examined 12 core proteins involved in the chromatin remodeling machinery using a tissue microarray composed of 150 lung adenocarcinoma (AD) and 150 squamous cell carcinoma (SCC) cases. Most of the proteins showed nuclear staining, whereas some also showed cytoplasmic or membranous staining. When the expression patterns of all tested antigens were considered, proteins with nuclear staining clustered into two major groups. Nuclear signals of BRM, Ini-1, retinoblastoma, mSin3A, HDAC1, and HAT1 clustered together, whereas nuclear signals of BRG1, BAF155, HDAC2, BAF170, and RbAP48 formed a second cluster. Additionally, two thirds of the cases on the lung tissue array had follow-up information, and survival analysis was performed for each of the tested proteins. Positive nuclear BRM (N-BRM) staining correlated with a favorable prognosis in SCC and AD patients with a 5 year-survival of 53.5% compared with 32.3% for those whose tumors were negative for N-BRM (P = 0.015). Furthermore, patients whose tumors stained positive for both N-BRM and nuclear BRG1 had a 5 year-survival of 72% compared with 33.6% (P = 0.013) for those whose tumors were positive for either or negative for both markers. In contrast, membranous BRM (M-BRM) staining correlated with a poorer prognosis in AD patients with a 5 year-survival of 16.7% compared with those without M-BRM staining (38.1%; P = 0.016). These results support the notion that BRM and BRG1 participate in two distinct chromosome remodeling complexes that are functionally complementary and that the nuclear presence of BRM, its coexpression with nuclear BRG1, and the altered cellular localization of BRM (M-BRM) are useful markers for non-small cell lung cancer prognosis.
Characterization of a New Humanized Anti-CD20 Monoclonal Antibody, IMMU-106, and Its Use in Combination with the Humanized Anti-CD22 Antibody, Epratuzumab, for the Therapy of Non-Hodgkin’s Lymphoma
Authors: Stein Rhona , Qu Zhengxing , Chen Susan , Rosario Adriane , Shi Victoria , Hayes Marianne , Horak Ivan D. , Hansen Hans J. , Goldenberg David M.
Published: September 2005

Purpose: A new humanized anti-CD20 monoclonal antibody (MAb), IMMU-106, was evaluated to elucidate its action as an antilymphoma therapeutic, as a single agent, and in combination with the anti-CD22 MAb, epratuzumab.

Experimental Design: Antiproliferative effects, apoptotic effects, and the ability of IMMU-106 to mediate complement-mediated cytotoxicity and antibody-dependent cellular cytotoxicity on a panel of non-Hodgkin’s lymphoma (NHL) cell lines were compared with the chimeric anti-CD20 MAb, rituximab, and evaluated in light of the various levels of antigen expression by the cell lines. In vivo therapy studies were performed in SCID mice bearing disseminated Raji lymphoma.

Results: The mechanisms of cytotoxicity of IMMU-106 were found to be similar to rituximab, and include direct apoptosis, antibody-dependent cellular cytotoxicity, and complement-mediated cytotoxicity. IMMU-106 was also found to be very similar to rituximab in terms of antigen-binding specificity, binding avidity, and dissociation constant. Treatment of Raji-bearing SCID mice with IMMU-106 yielded median survival increases of up to 4.2-fold compared with control mice. Survival in mice treated with IMMU-106 plus epratuzumab was compared with IMMU-106 treatment alone. Although the combined treatment did not improve median survival, an increased proportion of long-term survivors was observed. An enhanced antiproliferative effect was also observed in vitro in SU-DHL-6 cells when IMMU-106 was combined with epratuzumab. These findings are consistent with the up-regulation of CD22 expression observed after pretreatment of NHL cells in vitro with CD20 MAb (IMMU-106).

Conclusions: It is expected that in humans IMMU-106 should be at least as effective as rituximab and, due to its human framework construction, it may exhibit different pharmacokinetic, toxicity, and therapy profiles. In addition, it may be possible to enhance efficacy by combination therapy comprised of anti-CD20 and other B-cell lineage targeting MAbs, such as epratuzumab. The current results emphasize that in vitro as well as in vivo studies with many of the NHL cell lines were generally predictive of the known activity of anti-CD20 MAbs in NHL patients, as well as the enhanced efficacy of epratuzumab combined with rituximab observed in early clinical trials.
Induction of Cell Cycle Arrest and Apoptosis by the Proteasome Inhibitor PS-341 in Hodgkin Disease Cell Lines Is Independent of Inhibitor of Nuclear Factor-κB Mutations or Activation of the CD30, CD40, and RANK Receptors
Authors: Zheng Bei , Georgakis Georgios V. , Li Yang , Bharti Alok , McConkey David , Aggarwal Bharat B. , Younes Anas
Published: September 2005

Purpose: The malignant Hodgkin and Reed-Sternberg cells of Hodgkin disease (HD) are known to constitutively express high levels of activated nuclear factor κB (NF-κB), which plays an important role in their survival. The proteasome inhibitor PS-341 has been recently shown to modulate tumor cell proliferation and survival by inhibiting NF-κB and modulating critical cellular regulatory proteins, but its activity in cells carrying IκBα gene mutations has not been reported previously.

Experimental Design: The activity of PS-341 in four well-characterized, HD-derived cell lines. Cell proliferation and apoptosis were determined by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium (MTS) and Annexin-V binding methods, respectively. Cell cycle analysis was determined by flow cytometry. Intracellular protein levels were determined by Western blot.

Results: PS-341 demonstrated a strong antiproliferative activity, which was irrespective of the status of mutations in IκBα and even the presence of CD30, CD40, or RANK receptor activation. This effect was attributable to the induction of apoptosis and cell cycle arrest at the G2-M phase. PS-341 not only inhibited nuclear localization of NF-κB but also activated the caspase cascade, increased p21 and Bax levels, and decreased Bcl-2 levels. Furthermore, PS-341 enhanced the effect of gemcitabine chemotherapy and potentiated the effect of tumor necrosis factor-related apoptosis-inducing ligand/APO2L and two agonistic antibodies to tumor necrosis factor-related apoptosis-inducing ligand death receptors R1 and R2.

Conclusions: The in vitro activity of PS-341 against HD-derived cell lines suggests that PS-341 may have a therapeutic value for the treatment of HD.
Combined Targeting of Epidermal Growth Factor Receptor and MDM2 by Gefitinib and Antisense MDM2 Cooperatively Inhibit Hormone-Independent Prostate Cancer
Authors: Bianco Roberto , Caputo Roberta , Caputo Rosa , Damiano Vincenzo , De Placido Sabino , Ficorella Corrado , Agrawal Sudhir , Bianco A. Raffaele , Ciardiello Fortunato , Tortora Giampaolo
Published: September 2005

Purpose: The epidermal growth factor receptor (EGFR) may play a relevant role in the progression, hormone therapy resistance, and prognosis of prostate cancer patients. Also MDM2, a negative p53 regulator that interacts with retinoblastoma (Rb), E2F, p19arf and the ras-mitogen-activated protein kinase(MAPK) cascade plays an important role in prostate cancer progression and prognosis. On the basis of the EGFR and MDM2 role in integrating signaling pathways critical for prostate cancer progression, we investigated whether their selective combined blockade may have a cooperative antitumor effect in prostate cancer. For this purpose, we have used the EGFR tyrosine kinase inhibitor gefitinib (ZD1839, Iressa) and a second generation hybrid oligonucleotide antisense MDM2 (AS-MDM2), respectively.

Experimental Design: Gefitinib and AS-MDM2 were administered to hormone-refractory and hormone-dependent human prostate cancer cells in vitro and to mice bearing tumor xenografts, evaluating the effects on growth, apoptosis, and protein expression, in vitro and in vivo.

Results: We demonstrated that the combination of gefitinib and AS-MDM2 synergistically inhibits the growth of hormone-independent prostate cancer cells in vitro. This effect is accompanied by the inhibition of MDM2, phosphorylated Akt (pAkt), phosphorylated MAPK (pMAPK), and vascular endothelial growth factor (VEGF) expression and by Rb hypophosphorylation. The combination of the two agents in nude mice bearing the same hormone-independent tumors caused a potent cooperative antitumor effect. Tumor samples analysis confirmed the inhibition of MDM2, pAkt, pMAPK, VEGF, and basic fibroblast growth factor expression.

Conclusions: This study shows that EGFR and MDM2 play a critical role in the growth of prostate cancer, especially hormone-dependent, and that their combined blockade by gefitinib and AS-MDM2 causes a cooperative antitumor effect, supporting the clinical development of this therapeutic strategy.
Quantification of Colorectal Cancer Micrometastases in Lymph Nodes by Nested and Real-Time Reverse Transcriptase-PCR Analysis for Carcinoembryonic Antigen
Authors: Ho Samuel B. , Hyslop Ann , Albrecht Richard , Jacobson Amanda , Spencer Michael , Rothenberger David A. , Niehans Gloria A. , D’Cunha John , Kratzke Robert A.
Published: September 2005

Purpose: Reverse-transcriptase PCR (RT-PCR) assays for carcinoembryonic antigen (CEA) have been described to identify lymph node micrometastases. These assays are not quantitative and can be confounded by false-positive results. The purpose of this study was to determine whether quantification of CEA in lymph nodes could more readily identify clinically relevant groups.

Experimental Design: Specimens included 400 lymph nodes from 64 patients undergoing colon resections. Specimens were tested by immunohistochemistry and by RT-PCR using nested primers for CEA. Specimens from 59 patients that were positive by nested RT-PCR were further quantified by detection of CEA mRNA fluorescence increase at a threshold PCR cycle.

Results: CEA was detected by nested RT-PCR analysis in 4 of 34 (12%) nodes of nonneoplastic disease, 2 of 13 (15%) nodes from T1N0 patients, 32 of 81 (40%) nodes of T2N0 patients, 49 of 109 (45%) nodes from T3N0 patients, and 92 of 163 (56%) nodes from T1–4N1–2 patients. The overall presence of any RT-PCR–detectable CEA in nodes did not differentiate patient groups. Immunohistochemistry was positive in nodes from 7% of T3N0 patients and 100% of T1–3N1–2 patients. CEA quantification revealed that 0 of 7 patients with nonneoplastic disease and 2 of 17 (12%) patients with stage I T1–2N0 cancers had one or more lymph nodes with ≥1.0 × 102 CEA transcripts per sample. In contrast, 4 of 13 (31%) patients with stage II T3N0 cancer and 10 of 22 (45%) stage III patients with known metastases had lymph nodes with ≥1.0 × 102 CEA transcripts.

Conclusions: These data suggest that quantification of CEA levels in lymph nodes may more accurately identify patients at risk for cancer recurrence than does routine nested RT-PCR or immunohistochemistry.
Phosphorylation/Cytoplasmic Localization of p21Cip1/WAF1 Is Associated with HER2/
neu
Overexpression and Provides a Novel Combination Predictor for Poor Prognosis in Breast Cancer Patients
Authors: Xia Weiya , Chen Jin-Shing , Zhou Xian , Sun Pei-Rong , Lee Dung-Fang , Liao Yong , Zhou Binhua P. , Hung Mien-Chie
Published: September 2005

Purpose: The diversity of biological functions makes p21Cip1/WAF1 (p21) a controversial marker in predicting the prognosis of breast cancer patients. Recent laboratory studies revealed that the regulation of p21 function could be related to different subcellular localizations of p21 by Akt-induced phosphorylation at threonine 145 in HER2/neu-overexpressing breast cancer cells. The purpose of this study was to verify these findings in clinical settings.

Experimental Design: The expression status of the key biological markers in the HER2/neu-Akt-p21 pathway in 130 breast cancer specimens was evaluated by immunohistochemical staining and correlated with patients’ clinical parameters and survival. In addition, an antibody against phospho-p21 at threonine 145 [phospho-p21 (T145)] was also used for better validation of these findings.

Results: Cytoplasmic localization of p21 is highly correlated with overexpression of phospho-p21 (T145). Both cytoplasmic p21 and overexpression of phospho-p21 (T145) are associated with high expression of HER2/neu and phospho-Akt. Cytoplasmic localization of p21 and overexpression of phospho-p21 (T145), HER2/neu, and phospho-Akt are all associated with worse overall survival. Multivariate analysis of the Cox proportional hazard regression model revealed that cytoplasmic p21 and overexpression of HER2/neu are independently associated with increased risk of death. Combining these two factors stratified patients’ survival into four distinct groups, with a 5-year survival rate of 79% in low HER2/neu and negative/nuclear p21 patients, 60% in high HER2/neu and negative/nuclear p21 patients, 29% in low HER2/neu and cytoplasmic p21 patients, and 16% in high HER2/neu and cytoplasmic p21 patients.

Conclusions: The present study, in addition to supporting the mechanisms of p21 regulation derived from laboratory investigation, demonstrates the prognostic importance of phospho-p21 (T145) for the first time and also provides a novel combination of p21 and HER2/neu for better stratification of patients’ survival than any single clinicopathological or biological marker that may play important diagnostic and therapeutic roles for breast cancer patients.
Synergistic Induction of Oxidative Injury and Apoptosis in Human Multiple Myeloma Cells by the Proteasome Inhibitor Bortezomib and Histone Deacetylase Inhibitors
Authors: Pei Xin-Yan , Dai Yun , Grant Steven
Published: September 2005

Purpose: The purpose of this study was to examine interactions between the proteasome inhibitor bortezomib (Velcade) and the histone deacetylase (HDAC) inhibitors sodium butyrate and suberoylanilide hydroxamic acid in human multiple myeloma (MM) cells that are sensitive and resistant to conventional agents.

Experimental Design: MM cells were exposed to bortezomib for 6 h before the addition of HDAC inhibitors (total, 26 h), after which reactive oxygen species (ROS), mitochondrial dysfunction, signaling and cell cycle pathways, and apoptosis were monitored. The functional role of ROS generation was assessed using the free radical scavenger N-acetyl-l-cysteine.

Results: Preincubation with a subtoxic concentration of bortezomib markedly sensitized U266 and MM.1S cells to sodium butyrate- and suberoylanilide hydroxamic acid-induced mitochondrial dysfunction; caspase 9, 8, and 3 activation; and poly(ADP-ribose) polymerase degradation; resulting in synergistic apoptosis induction. These events were associated with nuclear factor κB inactivation, c-Jun NH2-terminal kinase activation, p53 induction, and caspase-dependent cleavage of p21CIP1, p27KIP1, and Bcl-2, as well as Mcl-1, X-linked inhibitor of apoptosis, and cyclin D1 down-regulation. The bortezomib/HDAC inhibitor regimen markedly induced ROS generation; moreover, apoptosis and c-Jun NH2-terminal kinase activation were attenuated by N-acetyl-l-cysteine. Dexamethasone- or doxorubicin-resistant MM cells failed to exhibit cross-resistance to the bortezomib/HDAC inhibitor regimen, nor did exogenous interleukin 6 or insulin-like growth factor I block apoptosis induced by this drug combination. Finally, bortezomib/HDAC inhibitors induced pronounced lethality in primary CD138+ bone marrow cells from MM patients, but not in the CD138− cell population.

Conclusions: Sequential exposure to bortezomib in conjunction with clinically relevant HDAC inhibitors potently induces mitochondrial dysfunction and apoptosis in human MM cells through a ROS-dependent mechanism, suggesting that a strategy combining these agents warrants further investigation in MM.
Evaluation of Nuclear Factor-κB, Urokinase-Type Plasminogen Activator, and HBx and Their Clinicopathological Significance in Hepatocellular Carcinoma
Authors: Chan Chun-Fung , Yau Tai-On , Jin Dong-Yan , Wong Chun-Ming , Fan Sheung-Tat , Ng Irene Oi-Lin
Published: September 2005

Purpose: Nuclear factor κB (NF-κB) signaling pathway is an important regulating pathway in human diseases and cancers. One of its downstream target genes is urokinase plasminogen activator (uPA), which is involved in cancer invasion and metastasis. The purpose of this study was to evaluate NF-κB activation, uPA up-regulation, and hepatitis B viral X protein (HBx) expression in human hepatocellular carcinoma (HCC) and to assess their clinicopathological significance.

Experimental Design: We evaluated NF-κB activation, expression of uPA, and presence of HBx in 32 human HCCs. Their clinicopathological significance was assessed by correlation with the clinicopathological features. Aberrant NF-κB signaling pathway and uPA up-regulation mediated by HBx were also analyzed in vitro.

Results: We found that NF-κB activation and uPA up-regulation were frequently (56% and 59%, respectively) observed in HCCs, and particularly in HBx-positive HCCs. NF-κB activation and uPA overexpression were closely associated with one another (P < 0.0001). Furthermore, both activation of NF-κB and up-regulation of uPA were significantly associated with a more aggressive tumor behavior in terms of venous invasion, direct liver invasion, and absence of tumor encapsulation. In vitro, NF-κB activation was induced by HBx transfection in HepG2 cells through inhibitor of nuclear factor-κB kinase β (IKKβ). HBx also up-regulated uPA and enhanced cell invasion synergistically with IKKβ.

Conclusions: The data indicate that NF-κB dysregulation and uPA overexpression may lead to a more aggressive tumor behavior in HCC. In addition, our data suggest that IKKβ plays a critical role in the HBx-activated NF-κB signaling pathway.
Pilot Trial Evaluating an 123I-Labeled 80-Kilodalton Engineered Anticarcinoembryonic Antigen Antibody Fragment (cT84.66 Minibody) in Patients with Colorectal Cancer
Authors: Wong Jeffrey Y. C. , Chu David Z. , Williams Lawrence E. , Yamauchi Dave M. , Ikle David N. , Kwok Cheuk S. , Liu An , Wilczynski Sharon , Colcher David , Yazaki Paul J. , Shively John E. , Wu Anna M. , Raubitschek Andrew A.
Published: September 2005

Purpose: The chimeric T84.66 (cT84.66) minibody is a novel engineered antibody construct (VL-linker-VH-CH3; 80 kDa) that demonstrates bivalent and high affinity (4 × 1010 m−1) binding to carcinoembryonic antigen (CEA). The variable regions (VL and VH) assemble to form the antigen-combining sites, and the protein forms dimers through self-association of the CH3 domains. In animal models, the minibody demonstrated high tumor uptake, approaching that of some intact antibodies, substantially faster clearance than intact chimeric T84.66, and superior tumor-to-blood ratios compared with the cT84.66 F(ab′)2 fragment, making it attractive for further evaluation as an imaging and therapy agent. The purpose of this pilot clinical study was to determine whether 123I-cT84.66 minibody demonstrated tumor targeting and was well tolerated as well as to begin to evaluate its biodistribution, pharmacokinetics, and immunogenicity in patients with colorectal cancer.

Experimental Design: Ten patients with biopsy-proven colorectal cancer (6 newly diagnosed, 1 pelvic recurrence, 3 limited metastatic disease) were entered on this study. Each received 5–10 mCi (1 mg) of 123I-labeled minibody i.v. followed by serial nuclear scans and blood and urine sampling over the next 48–72 h. Surgery was performed immediately after the last nuclear scan.

Results: Tumor imaging was observed with 123I-labeled minibody in seven of the eight patients who did not receive neoadjuvant therapy before surgery. Two patients received neoadjuvant radiation and chemotherapy, which significantly reduced tumor size before surgery and minibody infusion. At surgery, no tumor was detected in one patient and only a 2-mm focus was seen in the second patient. 123I-labeled minibody tumor targeting was not seen in either of these pretreated patients. Mean serum residence time of the minibody was 29.8 h (range, 10.9–65.4 h). No drug-related adverse reactions were observed. All 10 patients were evaluated for immune responses to the minibody, with no significant responses observed.

Conclusion: This pilot study represents one of the first clinical efforts to evaluate an engineered intermediate-molecular-mass radiolabeled antibody construct directed against CEA. cT84.66 minibody demonstrates tumor targeting to colorectal cancer and a faster clearance in comparison with intact antibodies, making it appropriate for further evaluation as an imaging and therapy agent. The mean residence time of the minibody in patients is longer than predicted from murine models. We therefore plan to further evaluate its biodistribution and pharmacokinetic properties with minibody labeled with a longer-lived radionuclide, such as 111In.