bluuebunny/crossref_metadata_2025 huggingface dataset:
Is a Lymph Node Detected by the Dye-Guided Method a True Sentinel Node in Gastric Cancer?
Authors: Osaka Hirohisa , Yashiro Masakazu , Sawada Tetsuji , Katsuragi Kei , Hirakawa Kosei
Published: September 2005

Purpose: A sentinel node is defined as the initial lymph node, to which cancer cells metastasize from a primary tumor. Recently, sentinel node navigation surgery has been done using the dye-guided method. However, no study has shown that a lymph node detected by the dye-guided method is the true sentinel node from the viewpoint of micrometastasis. Micrometastases of lymph nodes, in which no metastasis was found by H&E staining, were examined to establish whether a lymph node detected by the dye-guided method is the true sentinel node.

Experimental Design: Isosulfan blue was injected endoscopically as the dye-guided method at a submucosal lesion of early gastric cancer. Total 345 lymph nodes, including 150 blue-dyed lymph nodes and 195 nondyed lymph nodes were collected from 57 patients and each was quartered. Two quarters were examined histologically by H&E staining and cytokeratin staining. The other specimens were used for quantitative reverse transcription-PCR of CEA and CK20 mRNAs.

Results: Lymph node disease was not found in any of 345 lymph nodes from the 57 patients by routine H&E staining. By contrast, either CEA or CK20 mRNA expression was detected in 21 of 345 lymph nodes obtained from the 10 (18%) of 57 patients by quantitative reverse transcription-PCR. Eight of the 21 micrometastasis-positive lymph nodes were confirmed to be positive for cytokeratin staining. Although micrometastasis of nondyed lymph nodes was found in three cases, these were included in the 10 cases with micrometastasis of blue-dyed nodes, such that there was no patient who only had micrometastasis in nondyed nodes. Six of 10 cases were micrometastasis-positive in a single node; all six were blue-dyed nodes.

Conclusion: A lymph node detected by the dye-guided method should be a true sentinel node to which cancer cells metastasize initially.
Using Protein Expressions to Predict Survival in Clear Cell Renal Carcinoma
Authors: Kim Hyung L. , Seligson David , Liu Xueli , Janzen Nicolette , Bui Matthew H. T. , Yu Hong , Shi Tao , Figlin Robert A. , Horvath Steve , Belldegrun Arie S.
Published: September 2005

Purpose: An accurate system for predicting survival for patients with solid tumors will allow for better patient selection for both established and novel therapies. We propose a staging system for clear cell variants of renal cell carcinoma (RCC) that includes molecular predictors and standard clinical predictors such as tumor-node-metastasis (TNM) stage, histological grade, and performance status (PS).

Experimental Design: A custom tissue array was constructed using clear cell RCC from 318 patients, representing all stages of localized and metastatic RCC, and immunohistochemically stained for molecular markers Ki67, p53, gelsolin, CA9, CA12, PTEN, EpCAM, and vimentin. We present a strategy for evaluating individual candidate markers for prognostic information and integrating informative markers into a multivariate prognostic system.

Results: The overall median follow-up and the median follow-up for surviving patients were 28 and 55 months, respectively. A prognostic model based primarily on molecular markers included metastasis status, p53, CA9, gelsolin, and vimentin as predictors and had high discriminatory power: its statistically validated concordance index (C-index) was found to be 0.75. A prognostic model based on a combination of clinical and molecular predictors included metastasis status, T stage, Eastern Cooperative Oncology Group PS, p53, CA9, and vimentin as predictors and had a C-index of 0.79, which was significantly higher (P < 0.05) than that of prognostic models based on grade alone (C = 0.65), TNM stage alone (C = 0.73), or the University of California Los Angeles integrated staging system (C = 0.76).

Conclusions: Protein expressions obtained using widely available technology can complement standard clinical predictors such as TNM stage, histological grade, and PS.
Enhancement of Xenograft Tumor Radiosensitivity by the Histone Deacetylase Inhibitor MS-275 and Correlation with Histone Hyperacetylation
Authors: Camphausen Kevin , Scott Tamalee , Sproull Mary , Tofilon Philip J.
Published: September 2005

Purpose: Histone deacetylase (HDAC) inhibitors are undergoing clinical evaluation in cancer therapy. Because HDAC modulation has been shown to enhance the radiosensitivity of tumor cells in vitro, we investigated the effects of the HDAC inhibitor MS-275 on the radioresponse of DU145 prostate carcinoma xenografts.

Experimental Design: As an indicator of HDAC inhibition in vivo, the histone acetylation status in tumor lysates was determined after two, four, and six injections of MS-275 delivered at 12-hour intervals, as well as 24 and 48 hours after the last injection. Tumor growth delay studies were then performed using this DU-145 xenograft model with radiation administered to leg tumors after the fourth dose of MS-275, which corresponded to the time of maximum histone hyperacetylation.

Results: An increase in histone hyperacetylation was detected in each tumor after two injections of MS-275 with a maximum hyperacetylation occurring after four to six injections. In tumor growth delay studies, the combination of MS-275 and radiation resulted in a greater than additive inhibition of tumor growth as compared with the individual modalities. As alternative sources for an indicator of drug radiosensitizing activity, histone hyperacetylation was determined in a series of normal tissues, including lymphocytes. Each of the normal tissues also had a maximal histone hyperacetylation after four to six injections of MS-275.

Conclusions: These studies show that MS-275 enhances the radiosensitivity of DU145 xenografts and suggest that histone hyperacetylation status can serve as a useful marker for drug radiosensitizing activity.
T Cell–Dependent Antibody Responses against Aberrantly Expressed Cyclin B1 Protein in Patients with Cancer and Premalignant Disease
Authors: Suzuki Hiroyuki , Graziano Daniel F. , McKolanis John , Finn Olivera J.
Published: September 2005

Purpose: Cyclin B1–derived peptides were shown by us to be targets of tumor-specific CD8+ T cells in patients with breast and head and neck cancer. We obtained further evidence of cyclin B1 immunogenicity and its potential to serve as a tumor-specific antigen by analyzing its ability to elicit T cell–dependent humoral immune responses in vivo in patients with different types of tumors.

Experimental Design: Recombinant cyclin B1 protein from two different sources was purified and used as antigen in ELISA assays to test sera from patients with breast, pancreatic, colon, and lung cancer for the presence of anti-cyclin B1 antibody. We also analyzed patients with benign lung disease, premalignant disease, and a known history of heavy smoking.

Results and Conclusions: Cyclin B1 elicits helper T cell–dependent antibody responses in vivo. Tumors with higher level of cyclin B1 expression elicit higher anti-cyclin B1 antibody levels. Antibodies in patients with breast and colon cancer are primarily of the IgG isotype whereas patients with pancreatic and lung cancer have in addition anti-cyclin B1 IgA. Cyclin B1–specific IgG was also detected in long-term smokers and in patients with preneoplastic lung disease. Immune responses to aberrantly expressed cyclin B1 in tumors and premalignant lesions should be further explored as diagnostic and prognostic markers, in addition to their immunotherapeutic potential.
Truncating
BRCA1
Mutations Are Uncommon in a Cohort of Hereditary Prostate Cancer Families with Evidence of Linkage to 17q Markers
Authors: Zuhlke Kimberly A. , Madeoy Jennifer J. , Beebe-Dimmer Jennifer , White Kirsten A. , Griffin Angela , Lange Ethan M. , Gruber Stephen B. , Ostrander Elaine A. , Cooney Kathleen A.
Published: September 2005

Purpose: A genome-wide scan of 175 hereditary prostate cancer families from the University of Michigan Prostate Cancer Genetics Project provided evidence of prostate cancer linkage to 17q markers near the BRCA1 gene. To examine the possibility that germ-line BRCA1 mutations were associated with hereditary prostate cancer, individuals from 93 families with evidence of linkage to chromosome 17q were screened for germ-line BRCA1 mutations.

Experimental Design: One individual from each of the 93 families, the majority with three or more cases of prostate cancer, were screened for BRCA1 mutations with denaturing high-performance liquid chromatography (HPLC). Fragments exhibiting denaturing HPLC variant patterns were additionally analyzed by direct sequencing.

Results: Sixty-five of the individuals selected for sequencing from 65 unrelated families were determined to have wild-type BRCA1 sequence by denaturing HPLC. One individual from a family with both prostate and ovarian cancer was found to have a truncating BRCA1 mutation (3829delT). An additional 27 germ-line variants were identified, including 15 missense variants.

Conclusions: These sequencing results suggest that BRCA1 truncating mutations do not account for the linkage evidence on chromosome 17 observed in University of Michigan Prostate Cancer Genetics Project families. A recently completed combined genome scan has also detected linkage to 17q22, and studies are ongoing to identify the relevant prostate cancer susceptibility gene in this region.
Aberrant Promoter Hypermethylation of Multiple Genes in Gallbladder Carcinoma and Chronic Cholecystitis
Authors: Takahashi Takao , Shivapurkar Narayan , Riquelme Erick , Shigematsu Hisayuki , Reddy Jyotsna , Suzuki Makoto , Miyajima Kuniharu , Zhou Xian , Bekele B. Nebiyou , Gazdar Adi F. , Wistuba Ignacio I.
Published: September 2005

Purpose: Aberrant methylation of 5′ gene promoter regions is an epigenetic phenomenon that is a major mechanism for silencing of tumor suppressor genes in many cancer types. There is limited information about the molecular changes involved in the pathogenesis of gallbladder carcinoma (GBC), including methylation status.

Experimental Design: We investigated the aberrant promoter methylation profile of 24 known or suspected tumor suppressor genes in 50 GBCs and compared those results with the findings in 25 chronic cholecystitis (CC) specimens without cancer. The methylation-specific polymerase chain reaction and combined restriction analysis methods were used to detect methylation, and the results were confirmed by sequencing of cloned polymerase chain reaction products.

Results: In GBC, gene methylation frequencies varied from 0% to 80%. Ten genes demonstrated relatively high frequencies of aberrant methylation: SHP1 (80%), 3-OST-2 (72%), CDH13 (44%), P15INK4B (44%), CDH1 (38%), RUNX3 (32%), APC (30%), RIZ1 (26%), P16INK4A (24%), and HPP1 (20%). Eight genes (P73, RARβ2, SOCS-1, DAPK, DcR2, DcR1, HIN1, and CHFR) showed low frequencies (2–14%) of methylation, and no methylation of the remaining six genes (TIMP-3, P57, RASSF1A, CRBP1, SYK, and NORE1) was detected. In CC, methylation was detected for seven genes: SHP1 (88%), P15INK4B (28%), 3-OST-2 (12%), CDH1 (12%), CDH13 (8%), DcR2 (4%), and P16INK4A (4%). Significantly higher frequencies of methylation in GBC compared with CC were detected for eight genes (3-OST-2, CDH13, CDH1, RUNX3, APC, RIZ1, P16INK4A, and HPP1). Of those, four genes showed frequent methylation (>30%) in GBCs. The mean methylation index, an expression of the amount of methylated genes by case, was significantly higher in GBC (0.196 ± 0.013) compared with CC (0.065 ± 0.008; P < 0.001).

Conclusions: Our study constitutes the most comprehensive methylation profile report available in GBC and demonstrates that this neoplasm has a distinct pattern of abnormal gene methylation. Whereas gallbladders from healthy individual were not available, our finding of methylation in CC cases without cancer suggests that this phenomenon represents an early event in the pathogenesis of GBC.
Tumor Suppressor Gene Promoter Hypermethylation in Serum of Breast Cancer Patients
Authors: Dulaimi Essel , Hillinck Jeanne , de Caceres Inmaculada Ibanez , Al-Saleem Tahseen , Cairns Paul
Published: September 2005

Purpose: Breast cancer is the most common malignancy in American women and the second leading cause of death from cancer. The genetic and epigenetic alterations that initiate and drive cancer can be used as targets for detection of neoplasia in bodily fluids. Tumor cell-specific aberrant promoter hypermethylation can be detected in nipple aspirate and ductal lavage from breast cancer patients. In this study, we examine serum, a more readily accessible bodily fluid known to contain neoplastic DNA from individuals with cancer, for methylation-based detection of breast neoplasia.

Experimental Design: We examined the promoter methylation status of three normally unmethylated biologically significant cancer genes, RAS association domain family protein 1A (RASSF1A), adenomatous polyposis coli (APC), and death-associated protein kinase (DAP-kinase), by sensitive methylation-specific PCR in 34 breast tumor and paired preoperative serum DNA. The 34 patients comprised 7 ductal carcinoma in situ (CIS), 3 lobular CIS, 5 stage I and 15 stage II to IV invasive ductal carcinomas, and 4 invasive lobular carcinomas. Normal and benign tissue and serum control DNA were also examined to determine the specificity of hypermethylation.

Results: Hypermethylation of one or more genes was found in 32 of 34 (94%) breast tumor DNA. APC was hypermethylated in 15 of 34 (47%), RASSF1A in 22 of 34 (65%), and DAP-kinase in 17 of 34 (50%) tumors. Twenty-six (76%) of the corresponding serum DNA were positive for promoter hypermethylation, including ductal CIS, lobular CIS, stage I disease, and lobular carcinoma patients. No hypermethylation of APC, RASSF1A, or DAP-kinase was observed in serum DNA from normal healthy women and patients with inflammatory breast disease or nonneoplastic breast tissue specimens. A gene unmethylated in the tumor DNA was always found to be unmethylated in the matched serum DNA (100% specificity).

Conclusions: Tumor cell specific promoter hypermethylation of APC, RASSF1A, and DAP-kinase is present in ductal CIS, lobular CIS, and all grades and stages of invasive breast cancer. Hypermethylation can be detected by methylation-specific PCR analysis in serum DNA from patients with preinvasive and early-stage breast cancer amenable to cure. If confirmed in additional studies, hypermethylation-based screening of serum, a readily accessible bodily fluid, may enhance early detection of breast cancer.
[18F]Fluoro-2-deoxy-d-glucose Positron Emission Tomography Detects Gastric Carcinoma in an Early Stage in an Asymptomatic E-Cadherin Mutation Carrier
Authors: van Kouwen Mariette C. A. , Drenth Joost P. H. , Oyen Wim J. G. , de Bruin Joyce H. F. M. , Ligtenberg Marjolijn J. , Bonenkamp J. J. (Han) , van Krieken J. Han J. M. , Nagengast Fokko M.
Published: September 2005

Purpose: Autosomal dominant hereditary diffuse gastric cancer (HDGC) is caused by germ-line E-cadherin (CDH1) gene mutations. Early detection of cancer in carriers is difficult because HDGC escapes endoscopic detection. We hypothesized that the glucose metabolism is enhanced in HDGC and that this can be detected with [18F]fluoro-2-deoxy-d-glucose positron emission tomography (FDG-PET).

Experimental Design and Results: An asymptomatic twenty-eight year-old female was seen at our outpatient clinic because of a request for screening on HDGC. Her father and younger sister died of diffuse gastric cancer, at the ages of 52 and 27, respectively. Mutational analysis of the CDH1 gene in this patient demonstrated a novel heterozygous splice-site mutation in exon 8 (1135delACGGTAATinsTTAGA). Upper gastrointestinal endoscopies revealed no macroscopic abnormalities, but one of the 40 random biopsy specimens showed well-differentiated signet-cell carcinoma. A FDG-PET scan demonstrated two spots of FDG accumulation, one located in the proximal part of the stomach and the second in the region of the pylorus. A total gastrectomy was performed and microscopic examination showed focal localization of intramucosal adenocarcinoma of the signet-cell type in the cardiac and antrum area. Most notably, the localization of the FDG accumulation matched the localization of the carcinoma.

Conclusions: We present an asymptomatic patient from a HDGC family carrying a novel CDH1 mutation in whom FDG-PET scanning facilitated early detection of HDGC. This calls for further investigation of the role of FDG-PET scan as a screening modality in HDGC.
Expression of Hypoxia-related Tissue Factors Correlates with Diminished Survival of Adjuvantly Treated Patients with Chromosome 1p Aberrant Oligodendroglial Neoplasms and Therapeutic Implications
Authors: Birner Peter , Preusser Matthias , Gelpi Ellen , Berger Johannes , Gatterbauer Brigitte , Ambros Inge M. , Ambros Peter F. , Acker Till , Plate Karl H. , Harris Adrian L. , Hainfellner Johannes A.
Published: September 2005

Purpose: Oligodendroglial neoplasms with chromosome 1p deletion are chemosensitive, and stratified adjuvant therapies have been proposed on the basis of 1p status. In this study, we evaluated expression of hypoxia-related factors and its influence on survival in oligodendroglial brain tumors with chromosome 1p aberrations.

Experimental Design: Forty-four primary and 16 recurrent oligodendroglial neoplasms with 1p aberrations (deletion or imbalance) were investigated immunohistochemically for expression of hypoxia-inducible factor 1α and carbonic anhydrase-9. We used in situ hybridization to investigate expression of vascular endothelial growth factor-mRNA. We defined as “low hypoxia score” expression of no or only one marker and as “high hypoxia score” expression of two or three markers. The predominant vascular patterns of tumors were defined as classic or bizarre vascular formations, based on anti-CD34–immunostaining.

Results: High hypoxia score was evident in 16 of 44 (36.4%) primary tumor specimens and in 14 of 16 (87.5%) recurrent tumors (P = 0.001). High hypoxia score was associated with the presence of bizarre vascular proliferations and WHO grade III. In the subgroup of patients who received adjuvant therapy, univariate analysis showed significantly shorter survival of patients with high hypoxia score (n = 27; P = 0.0145). For all of the primary tumors, hypoxia score was an independent prognostic factor (P = 0.045).

Conclusions: A fraction of oligodendroglial neoplasms with 1p aberrations shows evidence of tissue hypoxia, which significantly influences survival of patients receiving adjuvant therapy. Evaluation of tissue hypoxia could become useful for recruitment of patients for individualized therapy strategies, e.g., selection of patients with hypoxic tumors for hyperbaric oxygenation preceding radiotherapy.
Ferritin Contributes to Melanoma Progression by Modulating Cell Growth and Sensitivity to Oxidative Stress
Authors: Baldi Alfonso , Lombardi Daniela , Russo Patrizia , Palescandolo Emanuele , De Luca Antonio , Santini Daniele , Baldi Feliciano , Rossiello Luigi , Dell'Anna Maria Lucia , Mastrofrancesco Arianna , Maresca Vittoria , Flori Enrica , Natali Pier Giorgio , Picardo Mauro , Paggi Marco G.
Published: September 2005

Purpose: Employing an in vitro model system of human melanoma progression, we previously reported ferritin light chain (L-ferritin) gene overexpression in the metastatic phenotype. Here, we attempted to characterize the role of ferritin in the biology of human melanoma and in the progression of this disease.

Experimental Design: Starting from the LM human metastatic melanoma cell line, we engineered cell clones in which L-ferritin gene expression was down-regulated by the stable expression of a specific antisense construct. These cells were then assayed for their growth capabilities, chemoinvasive properties, and sensitivity to oxidative stress. Additionally, ferritin protein content in primary and metastatic human melanomas was determined by immunohistochemistry.

Results: Artificial L-ferritin down-regulation in the LM cells strongly inhibited proliferation and chemoinvasion in vitro and cell growth in vivo. In addition, L-ferritin down-regulated cells displayed enhanced sensitivity to oxidative stress and to apoptosis. Concurrently, immunohistochemical analysis of a human melanoma tissue array revealed that ferritin expression level in metastatic lesions was significantly higher (P < 0.0001) than in primary melanomas. Furthermore, ferritin expression was constantly up-regulated in autologous lymph node melanoma metastases when compared with the respective primary tumors in a cohort of 11 patients.

Conclusions: These data suggest that high ferritin expression can enhance cell growth and improve resistance to oxidative stress in metastatic melanoma cells by interfering with their cellular antioxidant system. The potential significance of these findings deserves to be validated in a clinical setting.
Generation of Potent Antitumor CTL from Patients with Multiple Myeloma Directed against HM1.24
Authors: Rew Steven B. , Peggs Karl , Sanjuan Irene , Pizzey Arnold R. , Koishihara Yasuo , Kawai Shigeto , Kosaka Masaaki , Ozaki Shuji , Chain Benjamin , Yong Kwee L.
Published: September 2005

Purpose: The purpose of this work was to test the suitability of the HM1.24 antigen as a CTL target for immunotherapy of patients with multiple myeloma.

Experimental Design: Antigen-specific T cells were generated from patients with multiple myeloma using stimulation with protein-pulsed dendritic cells and tested in ELISPOT and CTL assays.

Results: HM1.24-primed T cells responded selectively to HM1.24-loaded autologous peripheral blood mononuclear cells (PBMC) in an IFN-γ ELISPOT assay (median, 342; range, 198-495 IFN-γ–producing cells/105 cf. unloaded PBMC median, 98; range, 7-137; P < 0.05, n = 5) and also to autologous malignant plasma cells (MPC; median, 227; range, 153-335; P < 0.05 when compared with the response to allogeneic MPC median, 57; range, 22-158; n = 5). HM1.24-primed T cells lysed autologous MPC (at 20:1 E/T ratio: median, 48% specific killing; range, 23-88%; at 10:1 E/T ratio: median, 43%; range, 15-80%; n = 12) but not allogeneic MPC. Lysis of autologous MPC was inhibited by anti–MHC class I but not anti–MHC class II antibodies and was blocked by Concanamycin A. Lysis of autologous MPC was blocked by competition with autologous HM1.24-transfected dendritic cells (10:1 ratio with autologous MPC). Unmanipulated, or control plasmid–transfected dendritic cells had no effect on lysis of autologous MPC.

Conclusion: Our results indicate that HM1.24 is a promising target for immunotherapy of multiple myeloma.
Characterization of Mutator Phenotype in Familial Colorectal Cancer Patients Not Fulfilling Amsterdam Criteria
Authors: Kim Jin C. , Lee Kang H. , Ka In H. , Koo Kum H. , Roh Seon A. , Kim Hee C. , Yu Chang S. , Kim Tae W. , Chang Heung M. , Gong Gyeong Y. , Kim Jung S.
Published: September 2005

Purpose: Although the mutator phenotype, including genetic and epigenetic alterations of the mismatch repair (MMR) system, seems to be pronounced in familial colorectal cancer, there have been few integrative studies comprising the entire mutator pathway. This study was done to identify the entire mutator pathway determining risk factors in patients with familial colorectal cancer not fulfilling the Amsterdam criteria.

Experimental Design: We consecutively recruited 134 colorectal cancer patients with a family history of accompanying cancers. Patients with hereditary nonpolyposis colorectal cancer meeting the Amsterdam criteria, familial adenomatous polyposis, or those receiving preoperative radiotherapy were excluded. Mutator phenotype was assessed by assaying microsatellite instability (MSI) at 24 markers, hMLH1-promoter methylation, mutations at MMR genes (hMLH1, hMSH2, hMSH6, and hPMS2), and immune staining of MMR proteins (hMLH1, hMSH2, hMSH6, hPMS1, and hPMS2).

Results: Of the 208 cancers in first-degree and/or second-degree relatives of patients, colorectal and gastric cancers (81%) were most common. Of the 134 proband colorectal cancers, 23 (17%) were MSI in high level, and 32 (24%) were MSI in low level. MMR alterations, including known polymorphism and splicing substitution, were identified in eight patients (6%). Twenty-eight tumors with mutator phenotype were further identified by hMLH1-promoter methylation and/or loss of MMR protein expression. In 51 tumors (38%), mutator phenotype was associated with right-sided colon cancer (P < 0.001) and younger age at onset (P = 0.032), but the number of patients with a mutator phenotype did not differ with respect to inheritance patterns of accompanying cancers, either successive or horizontal transmission (P = 0.815). Familial impact value, which differentially associated the degree of relatives with all accompanying cancers, effectively discriminated MSI in high level from microsatellite stable/MSI in low level tumors.

Conclusion: Familial colorectal cancer may be associated with multiple occurrences of colorectal or accompanying cancers inherited by dominant or recessive transmission. MMR gene mutations, however, are less associated with mutator phenotype in familial colorectal cancer.
Effect of Human Papillomavirus-16 Infection on CD8+ T-Cell Recognition of a Wild-Type Sequence p53264–272 Peptide in Patients with Squamous Cell Carcinoma of the Head and Neck
Authors: Sirianni Nicky , Ha Patrick K. , Oelke Mattias , Califano Joseph , Gooding William , Westra William , Whiteside Theresa L. , Koch Wayne M. , Schneck Jonathan P. , DeLeo Albert , Ferris Robert L.
Published: September 2005

Purpose: Wild-type sequence (wt) p53 peptides are attractive candidates for broadly applicable cancer vaccines, currently considered primarily for patients whose tumors overexpress p53. Circumstances exist, however, where increased p53 degradation may result in appreciable presentation of p53-derived peptides, despite low p53 expression. Squamous cell carcinoma of the head and neck is associated with oncogenic human papillomavirus (HPV) subtypes, which inactivate p53 through proteasomal degradation. The criterion of p53 overexpression would exclude these individuals from wt p53-based immunotherapy.

Experimental Design: We tested the correlation of HPV infection with enhanced antigenicity of the p53 protein and postulated that removal of HPV-16+ tumors with enhanced p53264-272 peptide presentation might lead to a drop in T cells specific for this peptide in vivo. Circulating frequencies of T cells specific for the HLA A*0201:p53264-272 complex were measured ex vivo using dimeric HLA:peptide complexes in 15 head and neck cancer patients before and 6 months after tumor excision.

Results: CD8+ T-cell recognition of HLA A*0201restricted wt p53264-272 peptide presented by HPV-16− squamous cell carcinoma of the head and neck lines was enhanced by HPV-16 E6 expression, sometimes exceeding that of a naturally transformed, HPV-16+ wt p53 expressing squamous cell carcinoma of the head and neck cell line. In patients with HPV-16− tumors, the frequency of wt p53264–272–specific T cells remained largely unchanged after tumor removal. However, a significant decline in frequency of anti-p53264–272 T cells was observed postoperatively in HPV-16+ patients (P < 0.005).

Conclusions: Recognition of HPV-associated squamous cell carcinoma of the head and neck appears associated with levels of wt p53-specific T cells and inversely with p53 expression. p53 peptides may be useful tumor antigens for squamous cell carcinoma of the head and neck immunotherapy in addition to viral gene products.
Expression of the Tumor Suppressor Gene ARHI in Epithelial Ovarian Cancer Is Associated with Increased Expression of p21WAF1/CIP1 and Prolonged Progression-Free Survival
Authors: Rosen Daniel G. , Wang Lin , Jain Ajay N. , Lu Karen H. , Luo Robert Z. , Yu Yinhua , Liu Jinsong , Bast Robert C.
Published: September 2005

Purpose: ARHI, an imprinted putative tumor suppressor gene, is expressed in normal ovarian epithelial cells, but its expression is down-regulated or lost in most ovarian cancer cell lines. Reexpression of ARHI in cancer cells induces p21WAF1/CIP1, down-regulates cyclin D1 promoter activity and inhibits growth in cell culture and in heterografts. To determine the relevance of these observations to clinical cancer, we have now measured ARHI expression in normal, benign and malignant ovarian tissues using immunohistochemistry and in situ hybridization.

Experimental Design: Paraffin embedded tissues from 7 normal ovaries, 22 cystadenomas and 42 borderline lesions were analyzed using standard immunoperoxidase and in situ hybridization techniques to assess ARHI expression. In addition, immunohistochemistry against ARHI was performed on a tissue microarray containing 441 consecutive cases of ovarian carcinoma.

Results: Strong ARHI expression was found in normal ovarian surface epithelial cells, cysts and follicles using immunohistochemistry and in situ hybridization. Reduced ARHI expression was observed in tumors of low malignant potential as well as in invasive cancers. ARHI expression was down-regulated in 63% of invasive ovarian cancer specimens and could not be detected in 47%. When immunohistochemistry and in situ hybridization were compared, ARHI protein expression could be down-regulated in the presence of ARHI mRNA. ARHI expression was correlated with expression of p21WAF1/CIP1 (P = 0.0074) but not with cyclin D1 and associated with prolonged disease free survival (P = 0.001). On multivariate analysis, ARHI expression, grade and stage were independent prognostic factors. ARHI expression did not correlate with overall survival.

Conclusions: Persistence of ARHI expression in epithelial ovarian cancers correlated with prolonged disease free survival and expression of the cyclin dependent kinase inhibitor p21WAF1/CIP1.
A Predictive Model of Human Myelotoxicity Using Five Camptothecin Derivatives and the
In vitro
Colony-Forming Unit Granulocyte/Macrophage Assay
Authors: Masubuchi Noriko , May Richard D. , Atsumi Ryo
Published: September 2005

Purpose: Many promising anticancer drugs are limited by myelosuppression. It is difficult to evaluate human myelotoxicity before a Phase I study because of the susceptibility of humans and animals to hematotoxicity. The purpose of this study was to establish a reliable method to predict the human maximum tolerated dose (MTD) of five camptothecin derivatives: SN-38, DX-8951f, topotecan, 9-aminocamptothecin, and camptothecin.

Experimental Design: The myelotoxicity of SN-38 and DX-8951f were evaluated on bone marrow from mice, dogs, and humans using a 14-day colony-forming unit, granulocyte-macrophage (CFU-GM) assay to determine the 50%, 75%, and 90% inhibitory concentration values (IC50, IC75, and IC90, respectively).

Results: Species differences in myelotoxicity were observed for SN-38 and DX-8951f. Using human and murine IC90s for myelotoxicity of these compounds and other camptothecin compounds (topotecan, 9-aminocamptothecin, and camptothecin), in vivo toxicological data, and pharmacokinetic parameters (data referred to in the literature), human MTDs were predicted retrospectively. The mechanism-based prediction model that is proposed uses the in vitro camptothecin assay and in vivo parameters on the basis of free fraction of area under the concentration-curve at the MTD (r2 = 0.887) and suggests that the human MTDs were well predicted for the five camptothecin derivatives by this model rather than by other models.

Conclusion: The human MTDs of the camptothecin drugs were successfully predicted using the mechanism-based prediction model. The application of this model for in vitro hematotoxicology could play an important role for the development of new anticancer agents.
Centrosome Hyperamplification Predicts Progression and Tumor Recurrence in Bladder Cancer
Authors: Yamamoto Yoshiaki , Matsuyama Hideyasu , Furuya Tomoko , Oga Atsunori , Yoshihiro Satoru , Okuda Masaru , Kawauchi Shigeto , Sasaki Kohsuke , Naito Katsusuke
Published: September 2005

Purpose: Recent studies have reported that centrosome hyperamplification (CH) is closely related to chromosomal instability in bladder cancer. In this study, we investigated whether CH could be used as a prognostic biomarker for patients with bladder cancer.

Experimental Design: CH was evaluated by immunohistochemistry in 50 bladder cancers (≤pT1: 43; ≥pT2: 7). In addition, numerical aberrations of chromosomes 7, 9, and 17 and gain of 20q13, on which the Aurora-A gene is located, were evaluated by fluorescence in situ hybridization, and DNA ploidy was assessed. Preliminary experiments on eight bladder cancer cell lines found that six had over 5% of CH cells associated with a gain of 20q13 and overexpression of Aurora-A; therefore, CH-positive cases (CH+) were defined as those having over 5% of cells with ≥3 centrosomes per cell.

Results: CH+, 20q13 gain, chromosomal instability, and DNA aneuploidy were detected in 30 (60%), 18 (36%), 22 (44%), and 19 (38%) patients, respectively. There were significant differences in tumor number, grade, recurrence, and progression between the CH+ and CH− groups. The later had significantly higher recurrence-free and progression-free survivals than the former (P = 0.0028 and P = 0.0070, respectively, log-rank test). Multivariate analysis revealed that CH+ was the strongest predictor for tumor recurrence in nonmuscle invasive (pTa and pT1) bladder cancer (hazard ratio, 1.882; 95% confidence interval, 1.161–3.325; P = 0.0094).

Conclusions: Detection of CH may provide crucial prognostic information about tumor recurrence in bladder cancer.
Herceptin Down-Regulates HER-2/neu and Vascular Endothelial Growth Factor Expression and Enhances Taxol-Induced Cytotoxicity of Human Ewing's Sarcoma Cells In vitro and In vivo
Authors: Guan Hui , Jia Shu-Fang , Zhou Zhichao , Stewart John , Kleinerman Eugenie S.
Published: September 2005

We have previously shown that high levels of HER-2/neu protein were overexpressed in human Ewing's sarcoma cells (TC71, SK-ES1) relative to normal human osteoblasts. The purpose of this study was to determine whether herceptin alone or in combination with chemotherapeutic agents could inhibit the growth of Ewing's sarcoma in vitro and in vivo. Western blot analysis showed that the protein levels of HER-2/neu were decreased following herceptin treatment. Cell growth was also inhibited by herceptin in a dose-dependent manner with an IC50 of 4 mg/mL in TC71 and SK-ES1 cell line, whereas human immunoglobin had no effect. Northern blot and ELISA showed the RNA expression and protein levels of vascular endothelial growth factor were also inhibited by herceptin treatment with no alteration in HIF-1α protein and topoisomerase IIα expression. Furthermore, Ewing's sarcoma tumor growth was significantly delayed by 100 mg/kg herceptin treatment in our Ewing's sarcoma xenograft mouse model. Combining taxol with herceptin resulted in additive cytotoxicity, whereas herceptin-etoposide, doxorubicin, and 9-nitrocamptothecin combinations did not. Taxol-herceptin enhanced growth inhibition in TC71 cells in vitro compared with either agent alone. Ewing's sarcoma growth was also delayed in vivo and mean tumor size was significantly lower in mice treated with herceptin plus taxol than in those receiving taxol or herceptin alone. These data suggest that herceptin in combination with taxol may be a therapeutic option in the treatment of Ewing's sarcoma.
Induction of Endothelial Nitric Oxide Synthase Expression by Melanoma Sensitizes Endothelial Cells to Tumor Necrosis Factor-Driven Cytotoxicity
Authors: Mocellin Simone , Provenzano Maurizio , Rossi Carlo Riccardo , Pilati Pierluigi , Scalerta Romano , Lise Mario , Nitti Donato
Published: September 2005

Purpose: The cascade of molecular events leading to tumor necrosis factor (TNF)-mediated tumor regression is still incompletely elucidated. We investigated the role of endothelial nitric oxide synthase in determining the tumor-selective activity of TNF.

Experimental Design: Using quantitative real-time PCR, endothelial nitric oxide synthase gene levels were measured in melanoma metastases of the skin and normal skin biopsies obtained from 12 patients before undergoing TNF-based therapy. In vitro, the ability of melanoma cells supernatant to affect endothelial nitric oxide synthase transcription by endothelial cells and the influence of nitric oxide synthase inhibition on TNF cytotoxicity toward endothelial cells was evaluated.

Results: Endothelial nitric oxide synthase transcript abundance resulted significantly greater in tumor samples rather than in normal skin samples and in patients showing complete response to TNF-based treatment rather than in those showing partial/minimal response. In vitro, melanoma cells’ supernatant induced endothelial nitric oxide synthase gene expression by endothelial cells. Nitric oxide synthase inhibition slowed endothelial cells proliferation and, if induced before TNF administration, decreased the cytokine-mediated cytotoxicity on endothelial cells.

Conclusions: Taken together, these findings support the hypothesis that high expression of endothelial nitric oxide synthase in the tumor microenvironment might increase or be a marker for endothelial cells sensitivity to TNF. These observations may have important prognostic and/or therapeutic implications in the clinical setting.
Combined Epidermal Growth Factor Receptor Targeting with the Tyrosine Kinase Inhibitor Gefitinib (ZD1839) and the Monoclonal Antibody Cetuximab (IMC-C225)
Authors: Matar Pablo , Rojo Federico , Cassia Raúl , Moreno-Bueno Gema , Di Cosimo Serena , Tabernero José , Guzmán Marta , Rodriguez Sonia , Arribas Joaquín , Palacios José , Baselga José
Published: September 2005

Purpose: The epidermal growth factor receptor (EGFR) is abnormally activated in cancer and two classes of anti-EGFR agents, monoclonal antibodies and low-molecular-weight tyrosine kinase inhibitors, have shown antitumor activity in patients. Because these two classes of antireceptor agents target the EGFR at different sites, we decided to explore whether the combined administration of gefitinib, a tyrosine kinase inhibitor, and cetuximab, a monoclonal antibody, had superior antitumor activity than either agent given alone.

Experimental Design: We studied the effects of the combination of gefitinib and cetuximab in a panel of human cancer cell lines and in an EGFR-dependent human tumor xenograft model (A431). The effects of these two agents on EGFR signaling, proliferation, apoptosis, and vascularization were evaluated. In addition, we analyzed, with cDNA arrays, changes in gene expression profiles induced by both agents.

Results: The combined treatment with gefitinib and cetuximab resulted in a synergistic effect on cell proliferation and in superior inhibition of EGFR-dependent signaling and induction of apoptosis. In a series of in vivo experiments, single-agent gefitinib or cetuximab resulted in transient complete tumor remission only at the highest doses. In contrast, suboptimal doses of gefitinib and cetuximab given together resulted in a complete and permanent regression of large tumors. In the combination-treated tumors, there was a superior inhibition of EGFR, mitogen-activated protein kinase, and Akt phosphorylation, as well as greater inhibition of cell proliferation and vascularization and enhanced apoptosis. Using cDNA arrays, we found 59 genes that were coregulated and 45 genes differentially regulated, including genes related to cell proliferation and differentiation, transcription, DNA synthesis and repair, angiogenesis, signaling molecules, cytoskeleton organization, and tumor invasion and metastasis.

Conclusions: Our findings suggest both shared and complementary mechanisms of action with gefitinib and cetuximab and support combined EGFR targeting as a clinically exploitable strategy.
Recognition of Adult T-Cell Leukemia/Lymphoma Cells by CD4+ Helper T Lymphocytes Specific for Human T-Cell Leukemia Virus Type I Envelope Protein
Authors: Kobayashi Hiroya , Nagato Toshihiro , Yanai Mitsuru , Oikawa Kensuke , Sato Keisuke , Kimura Shoji , Tateno Masatoshi , Omiya Ryusuke , Celis Esteban
Published: September 2005

Purpose: Human T-cell leukemia virus type I (HTLV-I) can cause an adult T-cell leukemia/lymphoma (ATLL). Because ATLL is a life-threatening lymphoproliferative disorder and is resistant to chemotherapy, the establishment and enhancement of T-cell immunity to HTLV-I through the development of therapeutic vaccines could be of value. Thus, the identification of HTLV-I epitopes for both CD8+ and CD4+ T cells should facilitate the development of effective vaccines. Although numerous HTLV-I epitopes for CTLs have been identified, few epitopes recognized by CD4+ helper T cells against this virus have been described.

Experimental Design: Synthetic peptides prepared from several regions of the HTLV-I envelope (Env) sequence that were predicted to serve as helper T-cell epitopes were prepared with use of computer-based algorithms and tested for their capacity to trigger in vitro helper T-cell responses using lymphocytes from normal volunteers.

Results: The results show that the HTLV-I–Env317–331, and HTLV-I–Env384–398-reactive helper T lymphocytes restricted by HLA-DQw6 and HLA-DR15, respectively, could recognize intact HTLV-I+ T-cell lymphoma cells and, as a consequence, secrete lymphokines. In addition, HTLV-I Env196–210-reactive helper T lymphocytes restricted by HLA-DR9 were able to directly kill HTLV-I+ lymphoma cells and recognize naturally processed antigen derived from killed HTLV-I+ lymphoma cells, which was presented to the helper T cells by autologous antigen-presenting cells.

Conclusions: The present findings hold relevance for the design and optimization of T-cell epitope-based immunotherapy against HTLV-I–induced diseases such as ATLL.
Combination Therapy Enhances the Inhibition of Tumor Growth with the Fully Human Anti–Type 1 Insulin-Like Growth Factor Receptor Monoclonal Antibody CP-751,871
Authors: Cohen Bruce D. , Baker Deborah A. , Soderstrom Catherine , Tkalcevic George , Rossi Ann Marie , Miller Penny E. , Tengowski Mark W. , Wang Faye , Gualberto Antonio , Beebe Jean S. , Moyer James D.
Published: September 2005

Purpose: The insulin-like growth factor (IGF) signaling pathway is implicated in cellular mitogenesis, angiogenesis, tumor cell survival, and tumorigenesis. Inhibition of this pathway results in decreased cell growth, inhibition of tumor formation in animal models, and increased apoptosis in cells treated with cytotoxic chemotherapy. We generated and characterized a human monoclonal antibody that targeted the IGF receptor.

Experimental Design: By use of XenoMouse technology, we generated CP-751,871, a fully human IgG2 antibody with high affinity (Kd = 1.5 nmol/L) for human IGF-1R and evaluated its biological, pharmacologic, and antitumor properties.

Results: This antibody blocks binding of IGF-1 to its receptor (IC50 1.8 nmol/L), IGF-1-induced receptor autophosphorylation (IC50 0.42 nmol/L) and induced the down-regulation of IGF-1R in vitro and in tumor xenografts. The extent of IGF-1R down-regulation in vivo was proportional to CP-751,871 concentrations in the serum of tumor-bearing mice. Pharmacokinetic profiles in cynomolgus monkeys indicated a close to linear increase of exposure following i.v. dosing of antibody in the range of 3 to 100 mg/kg. CP-751,871 showed significant antitumor activity both as a single agent and in combination with Adriamycin, 5-fluorouracil, or tamoxifen in multiple tumor models. A biomarker assay was developed to establish the relationship between circulating antibody concentrations and down-regulation of IGF-1R in peripheral blood cells. The concentration of CP-751,871 required to down-regulate 50% of IGF-1R on peripheral blood cells was 0.3 nmol/L.

Conclusion: These data suggest that inhibition of the IGF cascade by use of this monoclonal antibody may be of clinical benefit in the treatment of human cancers.
Thrombopoietic Factors in Chronic Bone Marrow Failure States: The Platelet Problem Revisited
Authors: Kurzrock Razelle
Published: September 2005

Thrombocytopenia is a serious clinical problem in several different clinical settings. In chronic bone marrow failure states, which include aplastic anemia, myelodysplastic syndrome, and graft failure, the prolonged nature of thrombocytopenia often leads to alloimunization after repeated platelet transfusions, the consequence of which is a platelet-refractory state and enhanced risk of bleeding. Despite the introduction of several thrombopoietic factors into clinical trials, an effective way to alleviate thrombocytopenia has been elusive, and the problem in chronic bone marrow failure states has remained poorly addressed by clinical investigations. Even so, several studies by our group and others suggest that a subset of patients suffering from chronic bone marrow failure can respond to appropriate growth factor therapy. The temporal pace of response appears, however, to be much slower than that observed after administering growth factors which act on neutrophils. On the other hand, durable responses can be secured in some patients given thrombopoietic factors for long periods of time. Herein, we provide an overview of the clinical research investigations of thrombopoietic factors in chronic bone marrow failure, and the emerging insights these studies provide for understanding the process of thrombopoiesis and its therapy in this setting.
Vascular Endothelial Growth Factor-C Expression and Invasive Phenotype in Ovarian Carcinomas
Authors: Ueda Masatsugu , Hung Yao-Ching , Terai Yoshito , Kanda Koji , Kanemura Masanori , Futakuchi Hikari , Yamaguchi Hiroyuki , Akise Daisuke , Yasuda Masayuki , Ueki Minoru
Published: September 2005

Purpose: To investigate the biological correlation between vascular endothelial growth factor (VEGF)-C expression and invasive phenotype in ovarian carcinomas.

Experimental Design: Gene and protein expression levels of VEGF-C in 10 ovarian carcinoma cell lines were correlated with invasive activity of the cells. The correlation between immunohistochemical expression of VEGF-C and tumor aggressiveness in 73 ovarian carcinomas was also examined with respect to clinicopathologic features and patient outcome.

Results: VEGF-C gene and protein expression differed remarkably among the cell lines, and there was a statistical correlation among VEGF-C expression, in vitro invasive activity, and matrix metalloproteinase-2 (MMP-2) gene expression and its activity. Anti-VEGF-C and anti-MMP-2 antibodies inhibited the invasive activity of tumor cells. VEGF-C expression in clinical tissue samples was well correlated with clinical stages, retroperitoneal lymph node metastasis, MMP-2 expression, angiogenesis, lymphangiogenesis, and low apoptotic index (AI). The patients whose tumors had strong VEGF-C expression and low AI underwent a poorer prognosis than did those with weak VEGF-C expression and high AI.

Conclusion: VEGF-C expression is closely related to invasive phenotype and affects the patient's survival in ovarian carcinomas.