bluuebunny/crossref_metadata_2025 huggingface dataset:
Paired Toll-like Receptor Agonists Enhance Vaccine Therapy through Induction of Interleukin-12
Authors: Zheng Rongxiu , Cohen Peter A. , Paustian Christopher A. , Johnson Terrence D. , Lee Walter T. , Shu Suyu , Koski Gary K.
Published: June 2008

Minimal requirements for generating effective immunity include the delivery of antigenic (signal 1) and costimulatory (signal 2) signals to T lymphocytes. Recently, a class of third signals, often delivered by antigen-presenting dendritic cells, has been shown to greatly enhance immune responses, especially against tumors. Among signal 3 factors, interleukin (IL)-12 is particularly effective and can be conditionally induced by agonists of Toll-like transmembrane receptors (TLR). In this study, we assessed the therapeutic effect of adjuvant TLR agonist administration upon the capacity of dendritic cell (DC)-tumor electrofusion hybrids to eradicate established MCA205 sarcomas in syngeneic mice. Paired, but not solitary combinations of polyinosine:polycytadilic acid (P[I:C]; TLR3 agonist) and CpG DNA (ODN1826l; TLR9 agonist) stimulated IL-12 secretion from DCs in vitro and synergized with vaccination to achieve potent tumor rejection. Therapeutic effects, however, required coadministration of paired TLR agonists and DC-tumor fusion hybrids. The administration of TLR agonists alone or with fusion vaccine induced transient splenomegaly but without apparent toxicity. The therapeutic effects of this immunization regimen were significantly abrogated through the neutralization of IL-12p70, indicating that production of this third signal was essential to the observed tumor regression. These results show the profound functional consequences of TLR cooperativity and further highlight the critical role of IL-12 in antitumor immunity. [Cancer Res 2008;68(11):4045–9]
Wnt/β-Catenin Signaling Contributes to Activation of Normal and Tumorigenic Liver Progenitor Cells
Authors: Yang Wen , Yan He-Xin , Chen Lei , Liu Qiong , He Ya-Qin , Yu Le-Xing , Zhang Shu-Hui , Huang Dan-Dan , Tang Liang , Kong Xiao-Ni , Chen Chao , Liu Shu-Qin , Wu Meng-Chao , Wang Hong-Yang
Published: June 2008

Adult hepatic progenitor (oval) cells are facultative stem cells in liver, which participate in a range of human liver diseases, including hepatocellular carcinoma (HCC). However, the molecular pathways regulating the expansion and differentiation of these cells are poorly understood. We show that active Wnt/β-catenin signaling occurs preferentially within the oval cell population, and forced expression of constitutively active β-catenin mutant promotes expansion of the oval cell population in the regenerated liver. More importantly, we identify a subpopulation of less differentiated progenitor-like cells in HCC cell lines and primary HCC tissues, which are defined by expression of the hepatic progenitor marker OV6 and endowed with endogenously active Wnt/β-catenin signaling. These OV6+ HCC cells possess a greater ability to form tumor in vivo and show a substantial resistance to standard chemotherapy compared with OV6− tumor cells. The fraction of tumor cells expressing OV6 is enriched after Wnt pathway activation, whereas inhibition of β-catenin signaling leads to a decrease in the proportion of OV6+ cells. In addition, the chemoresistance of OV6+ HCC progenitor-like cells can be reversed by lentivirus-delivered stable expression of microRNA targeting β-catenin. These results highlight the importance of the Wnt/β-catenin pathway in activation and expansion of oval cells in normal rodent models and human HCCs. OV6+ tumor cells may represent the cellular population that confers HCC chemoresistance, and therapies targeted to the Wnt/β-catenin signaling may provide a specific method to disrupt this resistance mechanism to improve overall tumor control with chemotherapy. [Cancer Res 2008;68(11):4287–95]
Genome-Wide Copy Number Analysis in Esophageal Adenocarcinoma Using High-Density Single-Nucleotide Polymorphism Arrays
Authors: Nancarrow Derek J. , Handoko Herlina Y. , Smithers B. Mark , Gotley David C. , Drew Paul A. , Watson David I. , Clouston Andrew D. , Hayward Nicholas K. , Whiteman David C. ,   for the Australian Cancer Study and the Study of Digestive Health
Published: June 2008

We applied whole-genome single-nucleotide polymorphism arrays to define a comprehensive genetic profile of 23 esophageal adenocarcinoma (EAC) primary tumor biopsies based on loss of heterozygosity (LOH) and DNA copy number changes. Alterations were common, averaging 97 (range, 23–208) per tumor. LOH and gains averaged 33 (range, 3–83) and 31 (range, 11–73) per tumor, respectively. Copy neutral LOH events averaged 27 (range, 7–57) per EAC. We noted 126 homozygous deletions (HD) across the EAC panel (range, 0–11 in individual tumors). Frequent HDs within FHIT (17 of 23), WWOX (8 of 23), and DMD (6 of 23) suggest a role for common fragile sites or genomic instability in EAC etiology. HDs were also noted for known tumor suppressor genes (TSG), including CDKN2A, CDKN2B, SMAD4, and GALR1, and identified PDE4D and MGC48628 as potentially novel TSGs. All tumors showed LOH for most of chromosome 17p, suggesting that TSGs other than TP53 may be targeted. Frequent gains were noted around MYC (13 of 23), BCL9 (12 of 23), CTAGE1 (14 of 23), and ZNF217 (12 of 23). Thus, we have confirmed previous reports indicating frequent changes to FHIT, CDKN2A, TP53, and MYC in EAC and identified additional genes of interest. Meta-analysis of previous genome-wide EAC studies together with the data presented here highlighted consistent regions of gain on 8q, 18q, and 20q and multiple LOH regions on 4q, 5q, 17p, and 18q, suggesting that more than one gene may be targeted on each of these chromosome arms. The focal gains and deletions documented here are a step toward identifying the key genes involved in EAC development. [Cancer Res 2008;68(11):4163–72]
Twist Is Required for Thrombin-Induced Tumor Angiogenesis and Growth
Authors: Hu Liang , Roth Jennifer M. , Brooks Peter , Ibrahim Sherif , Karpatkin Simon
Published: June 2008

Twist, a master regulator of embryonic morphogenesis, induces functions that are also required for tumor invasion and metastasis. Because thrombin contributes to the malignant phenotype by up-regulating tumor metastasis, we examined its effect on Twist in five different tumor cell lines and two different endothelial cell lines. Thrombin up-regulated Twist mRNA and protein in all seven cell lines. Down-regulation of Twist in B16F10 tumor cell lines led to a ∼3-fold decrease in tumor growth on a chorioallantoic membrane assay and ∼2-fold decrease in syngeneic mice. Angiogenesis was decreased ∼45% and 36%, respectively. The effect of Twist on angiogenesis was further examined and compared with the effect of thrombin. In studies using a Twist-inducible plasmid, several identical vascular growth factors and receptors were up-regulated ∼2- to 3-fold in tumor cells as well as human umbilical vascular endothelial cells by both Twist as well as thrombin (vascular endothelial growth factor, KDR, Ang-2, matrix metalloproteinase 1, GRO-α, and CD31). Thrombin-induced endothelial cell chemotaxis and Matrigel endothelial cell tubule formation were similarly regulated by Twist. Thus, thrombin up-regulates Twist, which is required for thrombin-induced angiogenesis as measured by endothelial cell migration, Matrigel tubule formation, and tumor angiogenesis. [Cancer Res 2008;68(11):4296–302]
Epigenetic Inactivation of the Groucho Homologue Gene TLE1 in Hematologic Malignancies
Authors: Fraga Mario F. , Berdasco Maria , Ballestar Esteban , Ropero Santiago , Lopez-Nieva Pilar , Lopez-Serra Lidia , Martín-Subero José I. , Calasanz Maria J. , de Silanes Isabel Lopez , Setien Fernando , Casado Sara , Fernandez Agustin F. , Siebert Reiner , Stifani Stefano , Esteller Manel
Published: June 2008

An undifferentiated status and the epigenetic inactivation of tumor-suppressor genes are hallmarks of transformed cells. Promoter CpG island hypermethylation of differentiating genes, however, has rarely been reported. The Groucho homologue Transducin-like Enhancer of Split 1 (TLE1) is a multitasked transcriptional corepressor that acts through the acute myelogenous leukemia 1, Wnt, and Notch signaling pathways. We have found that TLE1 undergoes promoter CpG island hypermethylation–associated inactivation in hematologic malignancies, such as diffuse large B-cell lymphoma and AML. We also observed a mutual exclusivity of the epigenetic alteration of TLE1 and the cytogenetic alteration of AML1. TLE1 reintroduction in hypermethylated leukemia/lymphoma cells causes growth inhibition in colony assays and nude mice, whereas TLE1-short hairpin RNA depletion in unmethylated cells enhances tumor growth. We also show that these effects are mediated by TLE1 transcriptional repressor activity on its target genes, such as Cyclin D1, Colony-Stimulating Factor 1 receptor, and Hairy/Enhancer of Split 1. These data suggest that TLE1 epigenetic inactivation contributes to the development of hematologic malignancies by disrupting critical differentiation and growth-suppressing pathways. [Cancer Res 2008;68(11):4116–22]
Interaction of Tomato Lycopene and Ketosamine against Rat Prostate Tumorigenesis
Authors: Mossine Valeri V. , Chopra Pankaj , Mawhinney Thomas P.
Published: June 2008

Prior investigations on the beneficial effect of dietary processed tomato products and lycopene on prostate cancer risk suggested that lycopene may require the presence of other constituents to exert its chemopreventive potential. We investigated whether ketosamines, a group of carbohydrate derivatives present in dehydrated tomato products, may interact with lycopene against prostate tumorigenesis. One ketosamine, FruHis, strongly synergized with lycopene against proliferation of the highly metastatic rat prostate adenocarcinoma MAT-LyLu cell line in vitro. The FruHis/lycopene combination significantly inhibited in vivo tumor formation by MAT-LyLu cells in syngeneic Copenhagen rats. Energy-balanced diets, supplemented with tomato paste, tomato powder, or tomato paste plus FruHis, were fed to Wistar-Unilever rats (n = 20 per group) treated with N-nitroso-N-methylurea and testosterone to induce prostate carcinogenesis. Survival from carcinogenesis was lowest in the control group (median survival time, 40 weeks) and highest in the group fed the tomato paste/FruHis diet (51 weeks; P = 0.004, versus control). The proportions of dying rats with macroscopic prostate tumors in the control, tomato paste, tomato powder, and tomato paste/FruHis groups were 63% (12 of 19), 39% (5 of 13), 43% (6 of 14), and 18% (2 of 11), respectively. FruHis completely blocked DNA oxidative degradation at >250 μmol/L in vitro, whereas neither ascorbate nor phenolic antioxidants from tomato were effective protectors in this assay. FruHis, therefore, may exert tumor-preventive effect through its antioxidant activity and interaction with lycopene. [Cancer Res 2008;68(11):4384–91]
Epigenetic Silencing of MicroRNA-34b/c and B-Cell Translocation Gene 4 Is Associated with CpG Island Methylation in Colorectal Cancer
Authors: Toyota Minoru , Suzuki Hiromu , Sasaki Yasushi , Maruyama Reo , Imai Kohzoh , Shinomura Yasuhisa , Tokino Takashi
Published: June 2008

Altered expression of microRNA (miRNA) is strongly implicated in cancer, and recent studies have shown that, in cancer, expression of some miRNAs cells is silenced in association with CpG island hypermethylation. To identify epigenetically silenced miRNAs in colorectal cancer (CRC), we screened for miRNAs induced in CRC cells by 5-aza-2′-deoxycytidine (DAC) treatment or DNA methyltransferase knockout. We found that miRNA-34b (miR-34b) and miR-34c, two components of the p53 network, are epigenetically silenced in CRC; that this down-regulation of miR-34b/c is associated with hypermethylation of the neighboring CpG island; and that DAC treatment rapidly restores miR-34b/c expression. Methylation of the miR-34b/c CpG island was frequently observed in CRC cell lines (nine of nine, 100%) and in primary CRC tumors (101 of 111, 90%), but not in normal colonic mucosa. Transfection of precursor miR-34b or miR-34c into CRC cells induced dramatic changes in the gene expression profile, and there was significant overlap between the genes down-regulated by miR-34b/c and those down-regulated by DAC. We also found that the miR-34b/c CpG island is a bidirectional promoter which drives expression of both miR-34b/c and B-cell translocation gene 4 (BTG4); that methylation of the CpG island is also associated with transcriptional silencing of BTG4; and that ectopic expression of BTG4 suppresses colony formation by CRC cells. Our results suggest that miR-34b/c and BTG4 are novel tumor suppressors in CRC and that the miR-34b/c CpG island, which bidirectionally regulates miR-34b/c and BTG4, is a frequent target of epigenetic silencing in CRC. [Cancer Res 2008;68(11):4123–32]
Identification and Characterization of Ovarian Cancer-Initiating Cells from Primary Human Tumors
Authors: Zhang Shu , Balch Curt , Chan Michael W. , Lai Hung-Cheng , Matei Daniela , Schilder Jeanne M. , Yan Pearlly S. , Huang Tim H-M. , Nephew Kenneth P.
Published: June 2008

The objective of this study was to identify and characterize a self-renewing subpopulation of human ovarian tumor cells (ovarian cancer-initiating cells, OCICs) fully capable of serial propagation of their original tumor phenotype in animals. Ovarian serous adenocarcinomas were disaggregated and subjected to growth conditions selective for self-renewing, nonadherent spheroids previously shown to derive from tissue stem cells. To affirm the existence of OCICs, xenoengraftment of as few as 100 dissociated spheroid cells allowed full recapitulation of the original tumor (grade 2/grade 3 serous adenocarcinoma), whereas >105 unselected cells remained nontumorigenic. Stemness properties of OCICs (under stem cell–selective conditions) were further established by cell proliferation assays and reverse transcription–PCR, demonstrating enhanced chemoresistance to the ovarian cancer chemotherapeutics cisplatin or paclitaxel and up-regulation of stem cell markers (Bmi-1, stem cell factor, Notch-1, Nanog, nestin, ABCG2, and Oct-4) compared with parental tumor cells or OCICs under differentiating conditions. To identify an OCIC cell surface phenotype, spheroid immunostaining showed significant up-regulation of the hyaluronate receptor CD44 and stem cell factor receptor CD117 (c-kit), a tyrosine kinase oncoprotein. Similar to sphere-forming OCICs, injection of only 100 CD44+CD117+ cells could also serially propagate their original tumors, whereas 105 CD44−CD117− cells remained nontumorigenic. Based on these findings, we assert that epithelial ovarian cancers derive from a subpopulation of CD44+CD117+ cells, thus representing a possible therapeutic target for this devastating disease. [Cancer Res 2008;68(11):4311–20]
The Previously Undescribed ZKSCAN3 (ZNF306) Is a Novel “Driver” of Colorectal Cancer Progression
Authors: Yang Lin , Hamilton Stanley R. , Sood Anil , Kuwai Toshio , Ellis Lee , Sanguino Angela , Lopez-Berestein Gabriel , Boyd Douglas D.
Published: June 2008

A relatively new view of colorectal cancer is that its development/progression reflects the contribution of a large set of altered gene products in varying combinations, each providing a “fitness advantage.” In searching for novel contributing gene products using Unigene cluster data mining, we found overrepresentation of expressed sequence tags corresponding to a previously uncharacterized gene (ZKSCAN3) in colorectal tumors. ZKSCAN3 was pursued for several reasons: (a) its sequence similarity with bowl required for Drosophila hindgut development; (b) it lies in a chromosomal region (6p22.1) amplified in colorectal cancer; and (c) its coding sequence predicts tandem C2H2 zinc finger domains present in a class of proteins gaining attention for their role in oncogenesis/tumor progression. Reverse transcription-PCR confirmed overexpression in colorectal tumor tissue compared with adjacent nonmalignant mucosa due in part to gene amplification determined by Southern blotting. Further, immunohistochemistry with an antibody generated to the predicted protein sequence revealed higher ZKSCAN3 expression in invasive compared with noninvasive tumors. Intriguingly, the ZKSCAN3 protein was also expressed in tumors wild-type for genes (APC, p53, K-Ras) commonly targeted in colorectal cancer. ZKSCAN3 knockdown in two independent colon cancer cell lines impaired anchorage-independent growth and orthotopic tumor growth, whereas overexpression in a third cell line had the opposite effect and increased 5-fluorouracil resistance. Liposomal delivery of a ZKSCAN3-targeting small interfering RNA reduced tumorigenicity of orthotopic colon cancer. Thus, the hitherto uncharacterized ZKSCAN3 adds to an expanding set of encoded products contributing to the progression of colorectal cancer. [Cancer Res 2008;68(11):4321–30]
Molecular Interactions between Dying Tumor Cells and the Innate Immune System Determine the Efficacy of Conventional Anticancer Therapies
Authors: Apetoh Lionel , Tesniere Antoine , Ghiringhelli François , Kroemer Guido , Zitvogel Laurence
Published: June 2008

The efficacy of anticancer treatments is mostly assessed by their ability to directly inhibit the proliferation of tumor cells. Recently, we showed that tumor cell death triggered by chemotherapy or radiotherapy initiates an immunoadjuvant pathway that contributes to the success of cytotoxic treatments. The interaction of high mobility group box 1 protein (HMGB1) released from dying tumor cells with Toll-like receptor 4 (TLR4) on dendritic cells was required for the crosspresentation of tumor antigens and the promotion of tumor specific cytotoxic T-cell responses. Breast cancer patients harboring the loss-of-function Asp299Gly polymorphism of TLR4 relapsed earlier after receiving anthracycline-based chemotherapy. These data suggests that HMGB1- and TLR4-dependent immune responses elicited by conventional cancer treatment may increase the probability to achieve a durable therapeutic success. [Cancer Res 2008;68(11):4026–30]
The E2F3-Oncomir-1 Axis Is Activated in Wilms' Tumor
Authors: Kort Eric J. , Farber Leslie , Tretiakova Maria , Petillo David , Furge Kyle A. , Yang Ximing J. , Cornelius Albert , Teh Bin T.
Published: June 2008

Oncomir-1 is an oncogenic cluster of microRNAs (miRNA) located on chromosome 13. Previous in vitro studies showed that it is transcriptionally regulated by the transcription factor E2F3. In this report, we combine expression profiling of both mRNA and miRNAs in Wilms' tumor (WT) samples to provide the first evidence that the E2F3-Oncomir-1 axis, previously identified in cell culture, is deregulated in primary human tumors. Analysis of RNA expression signatures showed that an E2F3 gene signature was activated in all WT samples analyzed, in contrast to other kidney tumors. This finding was validated by immunohistochemistry on the protein level. Expression of E2F3 was lowest in early-stage tumors and highest in metastatic tissue. Expression profiling of miRNAs in WT showed that expression of each measured member of the Oncomir-1 family was highest in WT relative to other kidney tumor subtypes. Quantitative PCR confirmed that these miRNAs were overexpressed in WT relative to normal kidney tissue. These results suggest that the E2F3-Oncomir-1 axis is activated in WT. Our study also shows the utility of integrated genomics combining gene signature analysis with miRNA expression profiling to identify protein-miRNA interactions that are perturbed in disease states. [Cancer Res 2008;68(11):4034–8]
AlkB Homologue 2–Mediated Repair of Ethenoadenine Lesions in Mammalian DNA
Authors: Ringvoll Jeanette , Moen Marivi N. , Nordstrand Line M. , Meira Lisiane B. , Pang Bo , Bekkelund Anders , Dedon Peter C. , Bjelland Svein , Samson Leona D. , Falnes Pål Ø. , Klungland Arne
Published: June 2008

Endogenous formation of the mutagenic DNA adduct 1,N6-ethenoadenine (εA) originates from lipid peroxidation. Elevated levels of εA in cancer-prone tissues suggest a role for this adduct in the development of some cancers. The base excision repair pathway has been considered the principal repair system for εA lesions until recently, when it was shown that the Escherichia coli AlkB dioxygenase could directly reverse the damage. We report here kinetic analysis of the recombinant human AlkB homologue 2 (hABH2), which is able to repair εA lesions in DNA. Furthermore, cation exchange chromatography of nuclear extracts from wild-type and mABH2−/− mice indicates that mABH2 is the principal dioxygenase for εA repair in vivo. This is further substantiated by experiments showing that hABH2, but not hABH3, is able to complement the E. coli alkB mutant with respect to its defective repair of etheno adducts. We conclude that ABH2 is active in the direct reversal of εA lesions, and that ABH2, together with the alkyl-N-adenine-DNA glycosylase, which is the most effective enzyme for the repair of εA, comprise the cellular defense against εA lesions. [Cancer Res 2008;68(11):4142–9]
Focal Adhesion Kinase Expression in Human Neuroblastoma: Immunohistochemical and Real-time PCR Analyses
Authors: Beierle Elizabeth A. , Massoll Nicole A. , Hartwich Joseph , Kurenova Elena V. , Golubovskaya Vita M. , Cance William G. , McGrady Patrick , London Wendy B.
Published: June 2008

Purpose: The focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase important in signaling between cells and their extracellular matrix. Studies have shown that FAK expression is up-regulated in several human tumors and is related to tumor progression. We recently found an increase in p125FAK expression in human neuroblastoma cells lines and wished to determine its expression in human neuroblastoma specimens and evaluate for a possible correlation between p125FAK expression and known prognostic factors for neuroblastoma. We hypothesized that p125FAK expression would be up-regulated in advanced human neuroblastomas.

Experimental Design: Using immunohistochemical techniques with monoclonal antibody 4.47 specific for p125FAK expression, we analyzed 70 formalin-fixed, paraffin-embedded human neuroblastoma specimens for p125FAK staining. In addition, real-time PCR was used to determine the abundance of FAK mRNA in 17 matched human neuroblastoma mRNA specimens.

Results: FAK staining was present in 51 of the 70 tumor specimens (73%). Immunohistochemical staining of p125FAK in the ganglion-type tumor cells correlated with advanced International Neuroblastoma Staging System tumor stages and FAK mRNA abundance. In addition, p125FAK staining was significantly increased in stage IV tumors with amplification of the N-MYC oncogene.

Conclusions: These novel findings provide evidence that FAK is expressed by advanced-stage neuroblastoma and provide a rationale for targeting FAK in the treatment of this tumor.
Changes in Tumor Metabolism as Readout for Mammalian Target of Rapamycin Kinase Inhibition by Rapamycin in Glioblastoma
Authors: Wei Liu Hua , Su Helen , Hildebrandt Isabel J. , Phelps Michael E. , Czernin Johannes , Weber Wolfgang A.
Published: June 2008

Purpose: Inhibition of the protein kinase mammalian target of rapamycin (mTOR) is being evaluated for treatment of a variety of malignancies. However, the effects of mTOR inhibitors are cytostatic and standard size criteria do not reliably identify responding tumors. The aim of this study was to evaluate whether response to mTOR inhibition could be assessed by positron emission tomography (PET) imaging of tumor metabolism.

Experiment Design: Glucose, thymidine, and amino acid utilization of human glioma cell lines with varying degrees of sensitivity to mTOR inhibition were assessed by measuring in vitro uptake of [18F]fluorodeoxyglucose ([18F]FDG), [18F]fluorothymidine ([18F]FLT), and [3H]l-tyrosine before and after treatment with the mTOR inhibitor rapamycin. The tumor metabolic activity in vivo was monitored by small-animal PET of tumor-bearing mice. The mechanisms underlying changes in metabolic activity were analyzed by measuring expression and functional activity of enzymes and transporters involved in the uptake of the studied imaging probes.

Results: In sensitive cell lines, rapamycin decreased [18F]FDG and [18F]FLT uptake by up to 65% within 24 hours after the start of therapy. This was associated with inhibition of hexokinase and thymidine kinase 1. In contrast, [3H]l-tyrosine uptake was unaffected by rapamycin. The effects of rapamycin on glucose and thymidine metabolism could be imaged noninvasively by PET. In sensitive tumors, [18F]FDG and [18F]FLT uptake decreased within 48 hours by 56 ± 6% and 52 ± 8%, respectively, whereas there was no change in rapamycin-resistant tumors.

Conclusions: These encouraging preclinical data warrant clinical trials evaluating [18F]FDG and [18F]FLT-PET for monitoring treatment with mTOR inhibitors in patients.
Human High Molecular Weight–Melanoma-Associated Antigen: Utility for Detection of Metastatic Melanoma in Sentinel Lymph Nodes
Authors: Goto Yasufumi , Ferrone Soldano , Arigami Takaaki , Kitago Minoru , Tanemura Atsushi , Sunami Eiji , Nguyen Sandy L. , Turner Roderick R. , Morton Donald L. , Hoon Dave S.B.
Published: June 2008

Purpose: Detection of micrometastasis in melanoma-draining lymph nodes is important for staging and prognosis. Immunohistochemical staining (IHC) using S-100p-HMB-45–, and MART-1–specfic antibodies is used for detecting metastases in sentinel lymph nodes (SLN). However, improvement in IHC is needed for melanoma micrometastasis detection.

Experimental Design: Paraffin-embedded archival tissue (PEAT) specimens were obtained from 42 non-SLN macrometastases, 42 SLN metastases, and 16 tumor-negative SLNs of 100 melanoma patients who underwent SLN biopsy. PEAT specimens were assessed by IHC with high molecular weight−melanoma-associated antigen (HMW-MAA)–specific monoclonal antibodies (mAb) and with S-100p-, HMB-45–, and MART-1–specific antibodies. Quantitative real-time reverse-transcriptase PCR assay was used for HMW-MAA and MART-1 mRNA detection.

Results: Expression frequency and immunostaining intensity were higher for HMW-MAA than MART-1 in nodal macrometastases (P < 0.0001 and P < 0.0001, respectively) and micrometastases (P < 0.0001 and P = 0.004, respectively). All 52 (100%) macrometastases were positive with HMW-MAA–specific mAbs, whereas 43 (83%) were positive with MART-1–specific mAbs. In a comparison analysis, 23 of 23 (100%) micrometastases were HMW-MAA–positive, whereas 21 (91%) and 18 (78%) specimens were S-100p– and HMB-45–positive, respectively. Quantitative real-time reverse-transcriptase PCR analysis of 48 nodal metastases showed HMW-MAA mRNA detection in SLNs with metastases.

Conclusions: HMW-MAA is more sensitive and specific than MART-1, S-100p, and HMB-45 for IHC-based detection of SLN micrometastases. SLN PEAT–based detection specificity of melanoma micrometastases can be improved by IHC with HMW-MAA–specific mAbs.
Combination of Docetaxel and Recombinant Vaccine Enhances T-Cell Responses and Antitumor Activity: Effects of Docetaxel on Immune Enhancement
Authors: Garnett Charlie T. , Schlom Jeffrey , Hodge James W.
Published: June 2008

Purpose: Taxanes comprise some of the most widely used cancer chemotherapeutic agents. Members of this drug family, including docetaxel, are commonly used to treat breast, prostate, and lung cancers, among others. This study was designed to determine if this taxane has the ability to modulate components of the immune system independent of antitumor activity and to investigate the potential synergistic activities of the combination of docetaxel and vaccine therapy.

Experimental Design: We examined the in vivo effects of docetaxel on immune-cell subsets and on the function of CD4+, CD8+, and regulatory T-cell (Treg) populations in response to antigen-specific vaccination. We also examined the antitumor effects of the combination of docetaxel and vaccine in a preclinical model in which docetaxel has no observable effect on tumor growth.

Results: These studies show for the first time that (a) docetaxel modulates CD4+, CD8+, CD19+, natural killer cell, and Treg populations in non–tumor-bearing mice; (b) unlike cyclophosphamide, docetaxel does not inhibit the function of Tregs; (c) docetaxel enhances CD8+ but not CD4+ response to CD3 cross-linking; (d) docetaxel given after vaccination provides optimal enhancement of immune response to recombinant viral vaccines; (e) docetaxel combined with recombinant viral vaccine is superior to either agent alone at reducing tumor burden; and (f) docetaxel plus vaccine increases antigen-specific T-cell responses to antigen in the vaccine, as well as to cascade antigens derived from the tumor.

Conclusions: These findings suggest potential clinical benefit for the combined use of docetaxel and recombinant cancer vaccines.
Involvement of Hepcidin in the Anemia of Multiple Myeloma
Authors: Sharma Sanjai , Nemeth Elizabeta , Chen Yi-Hsiang , Goodnough Julia , Huston Alissa , Roodman G.D. , Ganz Tomas , Lichtenstein Alan
Published: June 2008

Purpose: Hepcidin is a liver-produced peptide implicated in the anemia of inflammation. Because interleukin (IL)-6 is a potent inducer of hepcidin expression and its levels are elevated in multiple myeloma, we studied the role of hepcidin in the anemia of multiple myeloma.

Experimental Design: Urinary hepcidin and serum levels of IL-6, ferritin, C-reactive protein, tumor necrosis factor-α, and IL-1β were studied in newly diagnosed myeloma patients. In vitro hepcidin induction assay was assessed by real-time PCR assay.

Results: Pretreatment urinary hepcidin levels in 44 patients with stage III multiple myeloma were 3-fold greater than normal controls. In the subset of multiple myeloma patients without renal insufficiency (n = 27), a marked inverse correlation was seen between hemoglobin at diagnosis and urinary hepcidin level (P = 0.014) strongly supporting a causal relationship between up-regulated hepcidin expression and anemia. The urinary hepcidin also significantly (P < 0.05) correlated with serum ferritin and C-reactive protein, whereas its correlation with serum IL-6 levels was of borderline significance (P = 0.06). Sera from 14 multiple myeloma patients, with known elevated urinary hepcidin, significantly induced hepcidin mRNA in the Hep3B cells, whereas normal sera had no effect. For 10 patients, the ability of anti-IL-6 and anti-IL-6 receptor antibodies to prevent the serum-induced hepcidin RNA was tested. In 6 of these patients, hepcidin induction was abrogated by the anti-IL-6 antibodies, but in the other 4 patients, the neutralizing antibodies had no effect.

Conclusions: These results indicate hepcidin is up-regulated in multiple myeloma patients by both IL-6-dependent and IL-6-independent mechanisms and may play a role in the anemia of multiple myeloma.
Identification of Genes Differentially Expressed in Benign versus Malignant Thyroid Tumors
Authors: Prasad Nijaguna B. , Somervell Helina , Tufano Ralph P. , Dackiw Alan P.B. , Marohn Michael R. , Califano Joseph A. , Wang Yongchun , Westra William H. , Clark Douglas P. , Umbricht Christopher B. , Libutti Steven K. , Zeiger Martha A.
Published: June 2008

Purpose: Although fine-needle aspiration biopsy is the most useful diagnostic tool in evaluating a thyroid nodule, preoperative diagnosis of thyroid nodules is frequently imprecise, with up to 30% of fine-needle aspiration biopsy cytology samples reported as “suspicious” or “indeterminate.” Therefore, other adjuncts, such as molecular-based diagnostic approaches are needed in the preoperative distinction of these lesions.

Experimental Design: In an attempt to identify diagnostic markers for the preoperative distinction of these lesions, we chose to study by microarray analysis the eight different thyroid tumor subtypes that can present a diagnostic challenge to the clinician.

Results: Our microarray-based analysis of 94 thyroid tumors identified 75 genes that are differentially expressed between benign and malignant tumor subtypes. Of these, 33 were overexpressed and 42 were underexpressed in malignant compared with benign thyroid tumors. Statistical analysis of these genes, using nearest-neighbor classification, showed a 73% sensitivity and 82% specificity in predicting malignancy. Real-time reverse transcription–PCR validation for 12 of these genes was confirmatory. Western blot and immunohistochemical analyses of one of the genes, high mobility group AT-hook 2, further validated the microarray and real-time reverse transcription–PCR data.

Conclusions: Our results suggest that these 12 genes could be useful in the development of a panel of markers to differentiate benign from malignant tumors and thus serve as an important first step in solving the clinical problem associated with suspicious thyroid lesions.
Retinoblastoma RB94 Enhances Radiation Treatment of Head and Neck Squamous Cell Carcinoma
Authors: Araki Koji , Ahmad Sidrah M. , Li Guoyan , Bray David A. , Saito Koichiro , Wang Daiyou , Wirtz Uwe , Sreedharan Sunil , O'Malley Bert W. , Li Daqing
Published: June 2008

Purpose: To assess whether adenovirus-mediated retinoblastoma 94 (Ad-RB94) transgene expression enhances efficacy of radiation therapy (XRT) of human head and neck squamous cell carcinoma (HNSCC).

Experimental Design: The HNSCC cell lines (JHU006 and JHU012) were treated in vitro and in a nude mouse xenograft model with Ad-RB94, Ad-DL312 control vector, or untreated as mock control. Cell viability and tumor growth were evaluated and combined RB94/XRT antitumor activity was analyzed by measuring DNA double-strand breaks, apoptosis-associated early DNA fragmentation, and levels of RB-regulated cell cycle progression E2F1 transcription factor.

Results: Ad-RB94/XRT resulted in significant HNSCC cell growth inhibition compared with XRT alone or Ad-RB94 alone in vitro and caused significant tumor regression compared with XRT alone and Ad-DL312/XRT in JHU006 and with XRT alone, Ad-DL312/XRT and Ad-RB94 alone in JHU012 in vivo. Neutral comet analysis revealed that DNA damage was significantly elevated in cells treated with Ad-RB94 alone and Ad-RB94/XRT. Tumors treated with Ad-RB94 alone showed a striking increase in early apoptosis DNA fragmentation, and DNA fragmentation was further enhanced with XRT. In addition, levels of E2F1 were up-regulated by Ad-RB94/XRT combination, whereas Ad-RB94 alone did not affect E2F1 levels and XRT alone led to down-regulation of E2F1.

Conclusions: A potent antitumor effect has been observed after Ad-RB94/XRT combination treatment in HNSCC xenograft tumors. Enhanced tumor regression correlated with increased apoptosis. Ad-RB94 treatment enhances the efficacy of XRT through tumor cell sensitization by arresting the cells at the radiation-sensitive G2-M cell cycle and via E2F1 up-regulation.
Methylated NEUROD1 Promoter is a Marker for Chemosensitivity in Breast Cancer
Authors: Fiegl Heidi , Jones Allison , Hauser-Kronberger Cornelia , Hutarew Georg , Reitsamer Roland , Jones Robin L. , Dowsett Mitch , Mueller-Holzner Elisabeth , Windbichler Gudrun , Daxenbichler Günter , Goebel Georg , Ensinger Christian , Jacobs Ian , Widschwendter Martin
Published: June 2008

Purpose: Chemotherapy can be an integral component of the adjuvant management strategy for women with early stage breast cancer. To date, no tool is available to predict or monitor the efficacy of these therapies. The aim of this proof-of-principle study was to assess whether NEUROD1 DNA methylation is able to predict the response to neoadjuvant and adjuvant chemotherapy.

Experimental Design: Recently, we showed that NEUROD1 DNA is differentially methylated in neoplastic versus nonneoplastic breast tissue samples. In this study, we used MethyLight and analyzed NEUROD1 methylation in (a) 74 breast cancer tissue samples, (b) two independent sets of pretreatment core biopsies of 23 (training set) and 21 (test set) neoadjuvantly treated breast cancer patients, and (c) pretherapeutic and posttherapeutic serum samples from 107 breast cancer patients treated with adjuvant chemotherapy.

Results: High-grade tumors showed higher NEUROD1 methylation levels. Estrogen receptor–negative breast cancers with high NEUROD1 methylation were 10.8-fold more likely to respond with a complete pathologic response following neoadjuvant chemotherapy. Patients with positive serum pretreatment NEUROD1 methylation, which persisted after chemotherapy, indicated poor relapse-free and overall survival in univariate and multivariate analyses (relative risk for relapse, 6.2; 95% confidence interval, 1.6-24; P = 0.008, and relative risk for death, 14; 95% confidence interval, 1.6-120; P = 0.02).

Conclusions: These data support the view that NEUROD1 methylation is a chemosensitivity marker in estrogen receptor–negative breast cancer.
Preclinical Evaluation of the α-Particle Generator Nuclide 225Ac for Somatostatin Receptor Radiotherapy of Neuroendocrine Tumors
Authors: Miederer Matthias , Henriksen Gjermund , Alke Andrea , Mossbrugger Ilona , Quintanilla-Martinez Leticia , Senekowitsch-Schmidtke Reingard , Essler Markus
Published: June 2008

Purpose: Peptide receptor radionuclide therapy (PRRT) using somatostatin analogues labeled with β-particle–emitting isotopes such as 90Y or 177Lu has been a promising treatment strategy for metastasized neuroendocrine tumors. Although remission can be accomplished in a high percentage of neuroendocrine tumors, some tumors do not respond to this treatment. α-Emitting isotopes—such as the 10-day half-life α-emitting generator nuclide Actinum-225 (225Ac)—are characterized by extremely high cytotoxic activity on the cellular level, and may be superior in the treatment of neuroendocrine tumors not responding to PRRT using β-emitting isotopes.

Experimental Design: Radiolabeling of 225Ac 1,4,7,10-tetra-azacylododecane N,N′,N′′,N′′′-J-tetraacetic acid-Tyr3-octreotide (DOTATOC) was done at pH 5 (60 minutes at 70°C) without further purification. Biodistribution in nude mice bearing AR42J rat pancreas neuroendocrine tumor xenografts were measured for up to 24 hours. Toxicity was tested by weight changes, retention variables (blood urea nitrogen and creatine), and histopathology in mice 7 months after treatment with 10 to 130 kBq (n = 4-5). Therapeutic efficacy was assessed by tumor weighing in animals treated 4 days after xenotransplantation and compared with 177Lu-DOTATOC as a reference.

Results: Activities up to 20 kBq had no significant toxic effects in mice. In contrast, activities higher than 30 kBq induced tubular necrosis. Biodistribution studies revealed that 225Ac-DOTATOC effectively accumulated in neuroendocrine xenograft tumors. 225Ac-DOTATOC activities were shown to be nontoxic (12-20 kBq), reduced the growth of neuroendocrine tumors, and showed improved efficacy compared with 177Lu-DOTATOC.

Conclusions: 225Ac might be suitable to improve PRRT in neuroendocrine tumors.
The Iroquois Homeobox Gene 5 Is Regulated by 1,25-Dihydroxyvitamin D3 in Human Prostate Cancer and Regulates Apoptosis and the Cell Cycle in LNCaP Prostate Cancer Cells
Authors: Myrthue Anne , Rademacher Brooks L.S. , Pittsenbarger Janet , Kutyba-Brooks Bozena , Gantner Marin , Qian David Z. , Beer Tomasz M.
Published: June 2008

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3], the most active metabolite of vitamin D3, has significant antitumor activity in a broad range of preclinical models of cancer. In this study, we show that the Iroquois homeobox gene 5 (Irx5) is down-regulated by 1,25(OH)2D3 in human prostate cancer samples from patients randomly assigned to receive weekly high-dose 1,25(OH)2D3 or placebo before radical prostatectomy. Down-regulation of Irx5 by 1,25(OH)2D3 was also shown in the human androgen-sensitive prostate cancer cell line LNCaP and in estrogen-sensitive MCF-7 breast cancer cells. Knockdown of Irx5 by RNA interference showed a significant reduction in LNCaP cell viability, which was accompanied by an increase in p21 protein expression, G2-M arrest, and an increase in apoptosis. The induced apoptosis was partially mediated by p53, and p53 protein expression was increased as a result of Irx5 knockdown. Cell survival was similarly reduced by Irx5 knockdown in the colon cancer cell line HCT 116 and in MCF-7 breast cancer cells, each being derived from clinical tumor types that seem to be inhibited by 1,25(OH)2D3. Overexpression of Irx5 led to a reduction of p21 and p53 expression. This is the first report that Irx5 is regulated by 1,25(OH)2D3 in humans and the first report to show that Irx5 is involved in the regulation of both the cell cycle and apoptosis in human prostate cancer cells. Irx5 may be a promising new therapeutic target in cancer treatment.
Adjuvant Radioimmunotherapy Trial with Iodine-131–Labeled Anti–Carcinoembryonic Antigen Monoclonal Antibody F6 F(ab′)2 after Resection of Liver Metastases from Colorectal Cancer
Authors: Ychou Marc , Azria David , Menkarios Cathy , Faurous Patrick , Quenet François , Saint-Aubert Bernard , Rouanet Philippe , Pèlegrin Monique , Bascoul-Mollevi Caroline , Guerreau Dominique , Saccavini Jean-Claude , Mach Jean-Pierre , Artus Jean-Claude , Pèlegrin André
Published: June 2008

Purpose: To evaluate the feasibility of radioimmunotherapy (RIT) with radiolabeled anti–carcinoembryonic antigen antibodies after complete resection of liver metastases (LM) from colorectal cancer.

Patients and Methods: Twenty-two patients planned for surgery of one to four LM received a preoperative diagnostic dose of a 131I-F(ab′)2–labeled anti-carcinoembryonic antigen monoclonal antibody F6 (8-10 mCi/5 mg). 131I-F(ab′)2 uptake was analyzed using direct radioactivity counting, and tumor-to-normal liver ratios were recorded. Ten patients with tumor-to-normal liver ratios of >5 and three others were treated with a therapeutic injection [180-200 mCi 131I/50 mg F(ab′)2] 30 to 64 days after surgery.

Results: Median 131I-F(ab′)2 immunoreactivity in patient serum remained at 91% of initial values for up to 96 hours after injection. The main and dose-limiting-toxicity was hematologic, with 92% and 85% grades 3 to 4 neutropenia and thrombocytopenia, respectively. Complete spontaneous recovery occurred in all patients. No human anti-mouse antibody response was observed after the diagnosis dose; however, 10 of the 13 treated patients developed human anti-mouse antibody ∼3 months later. Two treated patients presented extrahepatic metastases at the time of RIT (one bone and one abdominal node) and two relapsed within 3 months of RIT (one in the lung and the other in the liver). Two patients are still alive, and one of these is disease-free at 93 months after resection. At a median follow-up of 127 months, the median disease-free survival is 12 months and the median overall survival is 50 months.

Conclusion: RIT is feasible in an adjuvant setting after complete resection of LM from colorectal cancer and should be considered for future trials, possibly in combination with chemotherapy, because of the generally poor prognosis of these patients.
A Dose-Escalation Study of Recombinant Human Interleukin-18 Using Two Different Schedules of Administration in Patients with Cancer
Authors: Robertson Michael J. , Kirkwood John M. , Logan Theodore F. , Koch Kevin M. , Kathman Steven , Kirby Lyndon C. , Bell William N. , Thurmond Linda M. , Weisenbach Jill , Dar Mohammed M.
Published: June 2008

Purpose: Interleukin-18 (IL-18) is an immunostimulatory cytokine with antitumor activity in preclinical models. A phase I study of recombinant human IL-18 (rhIL-18) was done to determine the toxicity, pharmacokinetics, and biological activities of rhIL-18 administered at different doses in two different schedules to patients with advanced cancer.

Experimental Design: Cohorts of three to four patients were given escalating doses of rhIL-18 as a 2-h i.v. infusion either on 5 consecutive days repeated every 28 days (group A) or once a week (group B) for up to 6 months. Toxicities were graded using standard criteria. Blood samples were obtained for safety, pharmacokinetic, and pharmacodynamic measurements.

Results: Nineteen patients (10 melanoma and 9 renal cell cancer) were given rhIL-18 in doses of 100, 500, or 1,000 μg/kg (group A) or 100, 1,000, or 2,000 μg/kg (group B). Common side effects included chills, fever, headache, fatigue, and nausea. Common laboratory abnormalities included transient, asymptomatic grade 1 to 3 lymphopenia, grade 1 to 4 hyperglycemia, grade 1 to 2 anemia, neutropenia, hypoalbuminemia, liver enzyme elevations, and serum creatinine elevations. No dose-limiting toxicities were observed. Biological effects of rhIL-18 included transient lymphopenia and increased expression of activation antigens on lymphocytes. Increases in serum concentrations of IFN-γ, granulocyte macrophage colony-stimulating factor, and IL-18–binding protein were observed following dosing.

Conclusions: rhIL-18 can be given in biologically active doses by either weekly infusions or daily infusions for 5 days repeated every 28 days to patients with advanced cancer. Toxicity was generally mild to moderate, and a maximum tolerated dose of rhIL-18 by either schedule was not determined.
Disseminated Tumor Cells of Breast Cancer Patients: A Strong Prognostic Factor for Distant and Local Relapse
Authors: Bidard Francois-Clément , Vincent-Salomon Anne , Gomme Stéphanie , Nos Claude , de Rycke Yann , Thiery Jean Paul , Sigal-Zafrani Brigitte , Mignot Laurent , Sastre-Garau Xavier , Pierga Jean-Yves ,   for the Institut Curie Breast Cancer Study Group
Published: June 2008

Purpose: Clinical significance of disseminated tumor cells (DTC) in bone marrow of early breast cancer patients has been reported, but improvements in detection methods are needed.

Experimental Design: Bone marrow aspirates from 621 patients with stage I to III breast cancer were screened for cytokeratin-positive (CK+) cells. CK+ cells were categorized into DTC only if they had specific morphologic features of tumor cells. Bone marrow status and clinical and pathologic variables of the patients were correlated with clinical outcome after a median follow-up of 56 months.

Results: DTC and non-DTC CK+ cells were detected in 15% and 34% of patients, respectively, with no correlation with clinical and pathologic variables. On univariate analysis, DTC detection was associated with a poorer distant metastasis-free survival (DMFS; P = 0.0013) and overall survival (OS; P = 0.005). Moreover, DTC detection was also associated with local relapse-free survival (P = 0.0009). On multivariate analysis, DTC detection was an independent prognostic factor for DMFS, local relapse-free survival, and OS. There was no significant interaction between DTC detection and hormonal receptors status (P = 0.34). Non-DTC CK+ cells had no clinical significance.

Conclusion: DTC detection is a powerful prognostic marker for DMFS and OS in early breast cancer patients and can be individualized from irrelevant non-DTC CK+ cells by morphologic criteria. Biologically, despite high rates of systemic adjuvant therapy and locoregional irradiation in this series, DTC detection remains a prognostic factor of distant and, more strikingly, of local relapse, in favor of resistance to treatment of locally or distant disseminated cancer cells in DTC-positive patients.