bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from DNA Copy-Number Alterations Underlie Gene Expression Differences between Microsatellite Stable and Unstable Colorectal Cancers
Authors: Jorissen Robert N. , Lipton Lara , Gibbs Peter , Chapman Matthew , Desai Jayesh , Jones Ian T. , Yeatman Timothy J. , East Philip , Tomlinson Ian P.M. , Verspaget Hein W. , Aaltonen Lauri A. , Kruhøffer Mogens , Ørntoft Torben F. , Andersen Claus Lindbjerg , Sieber Oliver M.
Published: April 2023

Abstract
Purpose: About 15% of colorectal cancers harbor microsatellite instability (MSI). MSI-associated gene expression changes have been identified in colorectal cancers, but little overlap exists between signatures hindering an assessment of overall consistency. Little is known about the causes and downstream effects of differential gene expression.

Experimental Design: DNA microarray data on 89 MSI and 140 microsatellite-stable (MSS) colorectal cancers from this study and 58 MSI and 77 MSS cases from three published reports were randomly divided into test and training sets. MSI-associated gene expression changes were assessed for cross-study consistency using training samples and validated as MSI classifier using test samples. Differences in biological pathways were identified by functional category analysis. Causation of differential gene expression was investigated by comparison to DNA copy-number data.

Results: MSI-associated gene expression changes in colorectal cancers were found to be highly consistent across multiple studies of primary tumors and cancer cell lines from patients of different ethnicities (P < 0.001). Clustering based on consistent changes separated additional test cases by MSI status, and classification of individual samples predicted MSI status with a sensitivity of 96% and specificity of 85%. Genes associated with immune response were up-regulated in MSI cancers, whereas genes associated with cell-cell adhesion, ion binding, and regulation of metabolism were down-regulated. Differential gene expression was shown to reflect systematic differences in DNA copy-number aberrations between MSI and MSS tumors (P < 0.001).

Conclusions: Our results show cross-study consistency of MSI-associated gene expression changes in colorectal cancers. DNA copy-number alterations partly cause the differences in gene expression between MSI and MSS cancers.
Data from High-Throughput Screening Identifies Cardiac Glycosides as Potent Inhibitors of Human Tissue Kallikrein Expression: Implications for Cancer Therapies
Authors: Prassas Ioannis , Paliouras Miltiadis , Datti Alessandro , Diamandis Eleftherios P.
Published: April 2023

Abstract
Purpose: Human tissue kallikreins (KLK) comprise a subgroup of 15 homologous secreted serine proteases. Primarily known for their clinical use as cancer biomarkers (e.g., PSA), KLKs have recently been directly implicated in cancer-related processes, including invasion, angiogenesis, and tumor growth regulation. Therefore, the identification of compounds that would modulate expression of KLKs might be of considerable therapeutic value.

Experimental Design: A cell-based high-throughput screening (HTS) of three small molecule libraries (∼4,500 compounds) was undertaken; KLK expression in the breast cancer cell line MDA-MB-468 was assessed with sensitive ELISAs.

Results: The initial screening resulted in 66 “putative hits” that decreased KLK5 expression by at least 50% over control. Secondary screening and mini-dose-response assays resulted in 21 “validated hits.” These 21 compounds were clustered in only three distinct functional families and were further analyzed in vitro to determine their effectiveness (IC50s). Hits that failed to show dose-responsiveness or interfered with the viability of the cells were excluded. Multiple members of the cardiac glycoside family were found to be novel inhibitors of KLK expression, acting at low concentrations (10-50 nmol/L). Furthermore, members of the same family induced marked decreases in c-MYC and c-FOS expression, in a dose-dependent manner that correlated the KLK inhibition, suggesting a transcriptional mechanism of regulation of KLK expression.

Conclusions: We conclude that cardiac glycosides can dramatically suppress the transcription of KLKs and that these effects may be linked to proto-oncogene (c-myc/fos) expression. These findings may partially explain the recently realized antineoplastic actions of cardiac glycosides.
Data from Pharmacokinetic/Pharmacodynamic Modeling and Simulation of Neutropenia during Phase I Development of Liposome-Entrapped Paclitaxel
Authors: Fetterly Gerald J. , Grasela Thaddeus H. , Sherman Jeffrey W. , Dul Jeanne L. , Grahn Amy , Lecomte Diane , Fiedler-Kelly Jill , Damjanov Nevena , Fishman Mayer , Kane Michael P. , Rubin Eric H. , Tan Antoinette R.
Published: April 2023

Abstract
Purpose: To evaluate the maximum tolerated dose (MTD), dose-limiting toxicities (DLT), and pharmacokinetics of liposome-entrapped paclitaxel easy-to-use (LEP-ETU) and to characterize the relationship between LEP-ETU concentrations and the time course of neutropenia in cancer patients.

Experimental Design: LEP-ETU was administered to 88 patients and 63 were evaluable for pharmacokinetic/pharmacodynamic (PK/PD) analysis following 1.5- and 3-h infusions every 3 weeks (q3w; dose range, 135-375 mg/m2). MTD was identified using a 3 + 3, up-and-down dose-finding algorithm. PK/PD modeling was done to describe the temporal relationship between paclitaxel concentrations and neutrophil count. Simulations assessed the influence of dose and schedule on neutropenia severity to help guide dose selection.

Results: The MTD of LEP-ETU was identified as 325 mg/m2. DLTs occurring at 375 mg/m2 consisted of febrile neutropenia and neuropathy. The Cmax and area under the plasma concentration-time curve of LEP-ETU were less than proportional with increasing dose. The PK/PD model showed that LEP-ETU inhibition of neutrophil proliferation was 9.1% per 10 μg/mL of total paclitaxel concentration. The incidence of grade 4 neutropenia increased from 33% to 42% across the dose range of 275 to 325 mg/m2 q3w. For a dose of 110 mg/m2 given weekly, grade 4 neutropenia was estimated to be 16% compared with 42% for the same total dose administered q3w.

Conclusions: LEP-ETU can be administered safely at higher doses than Taxol. Modeling and simulation studies predict that 325 mg/m2 LEP-ETU q3w provides acceptable neutropenic events relative to those observed at 175 mg/m2 Taxol q3w. A 275 mg/m2 dose may offer an improved therapeutic index.
Data from Induction of Both CD8+ and CD4+ T-Cell–Mediated Responses in Colorectal Cancer Patients by Colon Antigen-1
Authors: Maccalli Cristina , Di Cristanziano Veronica , Fodale Valentina , Corsi Domenico , D'Agostino Giuseppina , Petrangeli Valentina , Laurenti Luca , Guida Sofia , Mazzocchi Arabella , Arienti Flavio , Perrone Maria P. , Castelli Chiara , Rivoltini Licia , Zagonel Vittorina , Tartaglia Marco , Parmiani Giorgio , Belardelli Filippo
Published: April 2023

Abstract
Purpose: Colon antigen-1 (COA-1) was recently identified as a novel antigen of colorectal cancer encoded by the UBXD5 gene. Here, we evaluated whether a specific T-cell-mediated response directed against this molecule can occur in colorectal cancer patients.

Experimental Design: Antigen- and tumor-specific immunologic responses of peripheral blood mononuclear cells (PBMC) stimulated in vitro with the MHC class II-associated immunogenic epitope of COA-1 (FSTFPPTLYQDDTLTLQAAG) were analyzed by IFN-γ ELISPOT assay.

Results: COA-1-specific and tumor-reactive T lymphocytes were isolated from all (n = 7) HLA-DRβ1*0402+ or *1301+ colorectal cancer patients with progressive disease (Dukes' C and D) but not in patients (n = 4) with early-stage tumor (Dukes' A and B) and in healthy donors (n = 5), suggesting that the immune response against this antigen is associated with the progression of colorectal cancer. COA-1- and tumor-specific T lymphocytes displayed a CD3+CD4+CD69+CD45RA+ phenotype, compatible with the activated effector-type T-cell subset, and most of them exerted cytotoxic activity against HLA-matched and COA-1+ tumor cells. COA-1-specific T cells could also be isolated by in vitro stimulation of peripheral blood mononuclear cells with autologous dendritic cells loaded with tumor lysate, suggesting that this antigen can generate a dominant immunologic response against colorectal cancer cells. Notably, we could identify also COA-1-derived epitopes binding to HLA-A*0201 molecules that elicited antigen- and tumor-specific CD8+ T-cell-mediated responses in colorectal cancer patients.

Conclusions: Both CD4+ and CD8+ T-cell responses against COA-1 can occur in colorectal cancer patients with metastatic disease, suggesting that this antigen is suitable for immunotherapeutic protocols of these patients.
Data from A Self-Quenched Galactosamine-Serum Albumin-RhodamineX Conjugate: A “Smart” Fluorescent Molecular Imaging Probe Synthesized with Clinically Applicable Material for Detecting Peritoneal Ovarian Cancer Metastases
Authors: Hama Yukihiro , Urano Yasuteru , Koyama Yoshinori , Gunn Andrew J. , Choyke Peter L. , Kobayashi Hisataka
Published: April 2023

Abstract
Purpose: Fluorophore activation after cellular internalization of a targeted fluorescently labeled conjugate is an effective molecular imaging strategy to increase target-to-background ratios. The d-galactose receptor on ovarian cancer cells has been used to target self-quenched avidin-rhodamineX conjugates in which the avidin component binds to d-galactose receptor and the rhodamines are optically activated by dequenching only after cellular internalization. As a nonimmunogenic alternative of avidin, galactosamine-conjugated serum albumin (GmSA) targets the d-galactose receptor with higher binding affinity and has more conjugation sites available for rhodamineX than avidin.

Experimental Design: GmSA was conjugated with 20 rhodamineX molecules (GmSA-20ROX) to create a self-quenching complex, which was compared with a conjugate consisting of GmSA and a single rhodamineX (GmSA-1ROX) in ex vivo chemical activation characteristics, intracellular activation, and in vivo molecular imaging for detecting peritoneal micrometastases of SHIN3 ovarian cancer.

Results: GmSA-20ROX was five times brighter than GmSA-1ROX when incubated with SHIN3 ovarian cancer cells for 3 h. Submillimeter SHIN3 ovarian cancer implants in the peritoneal cavity were clearly visualized in vivo with spectral fluorescence imaging due to the high tumor-to-background ratio. The sensitivity and specificity of GmSA-20ROX for implant detection were determined by colocalization of the rhodamineX emission with red fluorescent protein expressed constitutively in the SHIN3 tumor implants. Among 336 lesions, sensitivity and specificity were 99%/99%, respectively, for GmSA-20ROX, whereas the results for GmSA-1ROX were only 24%/100% (n = 388), respectively, for lesions ∼0.8 mm or greater in diameter.

Conclusion: Self-quenched GmSA-20ROX is more efficient than previous d-galactose–targeted fluorescent conjugates.
Data from Estrogen Receptor–Negative Breast Cancer: New Insights into Subclassification and Targeting
Authors: Zhao Jean J. , Silver Daniel P.
Published: April 2023

Abstract
Classification schemes for disease states can help identify distinctions with useful implications for therapy and prognosis. Speers and colleagues provide a new subclassification scheme for estrogen receptor-negative breast cancer based upon kinome-wide gene expression profiling with interesting findings that are potentially relevant for both treatment and clinical outcome. (Clin Cancer Res 2009;15(20):6309–10)
Data from Baseline Vascular Endothelial Growth Factor Concentration as a Potential Predictive Marker of Benefit from Vandetanib in Non–Small Cell Lung Cancer
Authors: Hanrahan Emer O. , Ryan Anderson J. , Mann Helen , Kennedy Sarah J. , Langmuir Peter , Natale Ronald B. , Herbst Roy S. , Johnson Bruce E. , Heymach John V.
Published: April 2023

Abstract
Purpose: Vandetanib [vascular endothelial growth factor (VEGF) receptor/epidermal growth factor receptor/RET inhibitor] has shown improvements in progression-free survival (PFS) in advanced non-small cell lung cancer in three randomized phase II studies: vandetanib versus gefitinib (study 3), docetaxel ± vandetanib (study 6), and carboplatin-paclitaxel and/or vandetanib (study 7). In study 7, vandetanib monotherapy was inferior to carboplatin-paclitaxel. We performed an exploratory retrospective analysis of the relationship between baseline circulating VEGF concentrations and PFS.

Experimental Design: Mean baseline VEGF levels were determined by ELISA from two baseline samples of plasma (163 of 168 patients, study 3; 65 of 127, study 6) or serum (144 of 181, study 7). High baseline VEGF values were above the immunoassay reference range for healthy subjects; low baseline VEGF values were within the range.

Results: Patients with low baseline VEGF had a lower risk of disease progression with vandetanib versus gefitinib [hazard ratio (HR), 0.55; 95% confidence interval (95% CI), 0.35-0.86; P = 0.01] or vandetanib 100 mg/d + docetaxel versus docetaxel (HR, 0.25; 95% CI, 0.09-0.68; P = 0.01). High VEGF patients had a similar risk of disease progression with vandetanib monotherapy versus gefitinib (HR, 1.03; 95% CI, 0.60-1.75; P = 0.92) or vandetanib 100 mg/d + docetaxel versus docetaxel (HR, 0.95; 95% CI, 0.25-3.61; P = 0.94). In study 7, low VEGF patients had a similar risk of disease progression with vandetanib monotherapy 300 mg/d versus carboplatin-paclitaxel (HR, 0.80; 95% CI, 0.41-1.56; P = 0.51); high VEGF patients progressed more quickly (HR, 1.60; 95% CI, 0.81-3.15; P = 0.17).

Conclusions: These analyses suggest that low baseline circulating VEGF may be predictive of PFS advantage in patients with advanced non-small cell lung cancer receiving vandetanib versus gefitinib or vandetanib + docetaxel versus docetaxel. Moreover, patients with low VEGF levels may have a similar outcome with either vandetanib monotherapy or carboplatin-paclitaxel.
Data from Multiplex Reverse Transcription-PCR Screening for EML4-ALK Fusion Transcripts
Authors: Takeuchi Kengo , Choi Young Lim , Soda Manabu , Inamura Kentaro , Togashi Yuki , Hatano Satoko , Enomoto Munehiro , Takada Shuji , Yamashita Yoshihiro , Satoh Yukitoshi , Okumura Sakae , Nakagawa Ken , Ishikawa Yuichi , Mano Hiroyuki
Published: April 2023

Abstract
Purpose: EML4-ALK is a fusion-type protein tyrosine kinase that is generated by inv(2)(p21p23) in the genome of non–small cell lung cancer (NSCLC). To allow sensitive detection of EML4-ALK fusion transcripts, we have now developed a multiplex reverse transcription-PCR (RT-PCR) system that captures all in-frame fusions between the two genes.

Experimental Design: Primers were designed to detect all possible in-frame fusions of EML4 to exon 20 of ALK, and a single-tube multiplex RT-PCR assay was done with total RNA from 656 solid tumors of the lung (n = 364) and 10 other organs.

Results: From consecutive lung adenocarcinoma cases (n = 253), we identified 11 specimens (4.35%) positive for fusion transcripts, 9 of which were positive for the previously identified variants 1, 2, and 3. The remaining two specimens harbored novel transcript isoforms in which exon 14 (variant 4) or exon 2 (variant 5) of EML4 was connected to exon 20 of ALK. No fusion transcripts were detected for other types of lung cancer (n = 111) or for tumors from 10 other organs (n = 292). Genomic rearrangements responsible for the fusion events in NSCLC cells were confirmed by genomic PCR analysis and fluorescence in situ hybridization. The novel isoforms of EML4-ALK manifested marked oncogenic activity, and they yielded a pattern of cytoplasmic staining with fine granular foci in immunohistochemical analysis of NSCLC specimens.

Conclusions: These data reinforce the importance of accurate diagnosis of EML4-ALK–positive tumors for the optimization of treatment strategies.
Data from Loss of Histone H4K20 Trimethylation Occurs in Preneoplasia and Influences Prognosis of Non–Small Cell Lung Cancer
Authors: Van Den Broeck Arnaud , Brambilla Elisabeth , Moro-Sibilot Denis , Lantuejoul Sylvie , Brambilla Christian , Eymin Beatrice , Khochbin Saadi , Gazzeri Sylvie
Published: April 2023

Abstract
Purpose: Epigenetic modifications of histone have crucial roles in the control of gene activity, nuclear architecture, and genomic stability. In this respect, they may contribute to the development and progression of cancer. We investigated whether epigenetic changes of histone H4 are involved in lung carcinogenesis.

Experimental Design: Epigenetic modifications of histone H4 were studied by immunohistochemistry in normal lung and 157 lung carcinoma using antibodies specifically recognizing the acetylated (Ac) lysines 5 (K5), K8, K12, K16, and trimethylated (me3) K20 residues of histone H4. Western blotting was used to validate the immunohistochemistry results. H4K20me3 was also studied in 17 preneoplastic lesions. Expression of the Suv4-20h1/2 trimethyltransferases was analyzed by quantitative reverse transcription-PCR in a subset of tumor samples.

Results: As compared with normal lung, cancer cells displayed an aberrant pattern of histone H4 modifications with hyperacetylation of H4K5/H4K8, hypoacetylation of H4K12/H4K16, and loss of H4K20 trimethylation. Alteration of H4K20 trimethylation was frequent in squamous cell carcinoma (67%) and was observed in early precursors lesions in which the level of H4K20me3 staining strongly decreased with disease progression. In adenocarcinoma, the down-regulation of H4K20me3 was less frequent (28%) but allowed the identification of a subgroup of stage I adenocarcinoma patients with reduced survival (P = 0.007). Loss of H4K20 trimethylation was associated with decreased expression of Suv4-20h2, a specific H4K20 trimethyltransferase involved in telomere length maintenance.

Conclusions: Our findings indicate an important role of histone H4 modifications in bronchial carcinogenesis and highlight H4K20me3 as a candidate biomarker for early detection of and therapeutic approaches to lung cancer.
Data from An Immune Response Enriched 72-Gene Prognostic Profile for Early-Stage Non–Small-Cell Lung Cancer
Authors: Roepman Paul , Jassem Jacek , Smit Egbert F. , Muley Thomas , Niklinski Jacek , van de Velde Tony , Witteveen Anke T. , Rzyman Witold , Floore Arno , Burgers Sjaak , Giaccone Giuseppe , Meister Michael , Dienemann Hendrik , Skrzypski Marcin , Kozlowski Miroslaw , Mooi Wolter J. , van Zandwijk Nico
Published: April 2023

Abstract
Purpose: Current staging methods are imprecise for predicting prognosis of early-stage non–small-cell lung cancer (NSCLC). We aimed to develop a gene expression profile for stage I and stage II NSCLC, allowing identification of patients with a high risk of disease recurrence within 2 to 3 years after initial diagnosis.

Experimental Design: We used whole-genome gene expression microarrays to analyze frozen tumor samples from 172 NSCLC patients (pT1-2, N0-1, M0) from five European institutions, who had undergone complete surgical resection. Median follow-up was 89 months (range, 1.2-389) and 64 patients developed a recurrence. A random two thirds of the samples were assigned as the training cohort with the remaining samples set aside for independent validation. Cox proportional hazards models were used to evaluate the association between expression levels of individual genes and patient recurrence-free survival. A nearest mean analysis was used to develop a gene-expression classifier for disease recurrence.

Results: We have developed a 72-gene expression prognostic NSCLC classifier. Based on the classifier score, patients were classified as either high or low risk of disease recurrence. Patients classified as low risk showed a significantly better recurrence-free survival both in the training set (P < 0.001; n = 103) and in the independent validation set (P < 0.01; n = 69). Genes in our prognostic signature were strongly enriched for genes associated with immune response.

Conclusions: Our 72-gene signature is closely associated with recurrence-free and overall survival in early-stage NSCLC patients and may become a tool for patient selection for adjuvant therapy.
Data from Nrf2 Enhances Cell Proliferation and Resistance to Anticancer Drugs in Human Lung Cancer
Authors: Homma Shinsuke , Ishii Yukio , Morishima Yuko , Yamadori Tadahiro , Matsuno Yosuke , Haraguchi Norihiro , Kikuchi Norihiro , Satoh Hiroaki , Sakamoto Tohru , Hizawa Nobuyuki , Itoh Ken , Yamamoto Masayuki
Published: April 2023

Abstract
Purpose: NF-E2-related factor 2 (Nrf2), a key transcription regulator for antioxidant and detoxification enzymes, is abundantly expressed in cancer cells. In this study, therefore, the role of Nrf2 in cancer cell proliferation and resistance to anticancer drugs was investigated.

Experimental Design: We used three human lung cancer cell lines with different degrees of Nrf2 activation: Nrf2 was highly activated in A549 cells, slightly activated in NCI-H292 cells, and not activated in LC-AI cells under unstimulated conditions.

Result: A549 cells showed higher resistance to cisplatin compared with NCI-H292 and LC-AI cells. The resistance to cisplatin was significantly inhibited in A549 but not in NCI-H292 or LC-AI cells by knockdown of Nrf2 with its specific small interfering RNA (Nrf2-siRNA). The cell proliferation was also most prominently inhibited in A549 cells by treatment with Nrf2-siRNA. In A549 cells, the expression of self-defense genes, such as antioxidant enzymes, phase II detoxifying enzymes, and drug efflux pumps, was significantly reduced by Nrf2-siRNA concomitant with a reduction of the cellular glutathione level. The degree of DNA crosslink and apoptosis after treatment with cisplatin was significantly elevated in A549 cells by Nrf2-siRNA. Knockdown of Nrf2 arrested the cell cycle at G1 phase with a reduction of the phosphorylated form of retinoblastoma protein in A549 and NCI-H292 cells but not in LC-AI cells.

Conclusion: These results indicate that the Nrf2 system is essential for both cancer cell proliferation and resistance to anticancer drugs. Thus, Nrf2 might be a potential target to enhance the effect of anticancer drugs.
Data from DNA-Dependent Protein Kinase Is a Therapeutic Target and an Indicator of Poor Prognosis in B-Cell Chronic Lymphocytic Leukemia
Authors: Willmore Elaine , Elliott Sarah L. , Mainou-Fowler Tryfonia , Summerfield Geoffrey P. , Jackson Graham H. , O'Neill Fran , Lowe Christopher , Carter Anthony , Harris Robert , Pettitt Andrew R. , Cano-Soumillac Celine , Griffin Roger J. , Cowell Ian G. , Austin Caroline A. , Durkacz Barbara W.
Published: April 2023

Abstract
Purpose: del(17p), del(11q), and associated p53 dysfunction predict for short survival and chemoresistance in B-cell chronic lymphocytic leukemia (CLL). DNA-dependent protein kinase (DNA-PK) is activated by DNA damage and mediates DNA double-strand break repair. We hypothesized that inhibiting DNA-PK would sensitize CLL cells to drug-induced DNA damage and that this approach could increase the therapeutic index of agents used to treat CLL.

Experimental Design: Fifty-four CLL cases were characterized for poor prognosis markers [del(17p), del(11q), CD38, and ZAP-70]. In selected cases, DNA-PK catalytic subunit (DNA-PKcs) expression and activity and p53 function were also measured. Ex vivo viability assays established sensitivity to fludarabine and chlorambucil and also tested the ability of a novel DNA-PK inhibitor (NU7441) to sensitize CLL cells to these drugs. The effects of NU7441 on fludarabine-induced DNA damage repair were also assessed (Comet assays and detection of γH2AX).

Results: DNA-PKcs levels correlated with DNA-PK activity and varied 50-fold between cases but were consistently higher in del(17p) (P = 0.01) and del(11q) cases. NU7441 sensitized CLL cells to chlorambucil and fludarabine, including cases with del(17p), del(11q), p53 dysfunction, or high levels of DNA-PKcs. NU7441 increased fludarabine-induced double-strand breaks and abrogated drug-induced autophosphorylation of DNA-PKcs at Ser2056. High DNA-PK levels predicted for reduced treatment-free interval.

Conclusions: These data validate the concept of targeting DNA-PKcs in poor risk CLL, and demonstrate a mechanistic rationale for use of a DNA-PK inhibitor. The novel observation that DNA-PKcs is overexpressed in del(17p) and del(11q) cases indicates that DNA-PK may contribute to disease progression in CLL.
Data from Angiogenic Switch of Angiopietins-Tie2 System and Its Prognostic Value in Bladder Cancer
Authors: Szarvas Tibor , Jäger Tobias , Tötsch Martin , Vom Dorp Frank , Kempkensteffen Carsten , Kovalszky Ilona , Romics Imre , Ergün Süleyman , Rübben Herbert
Published: April 2023

Abstract
Purpose: Vascular endothelial growth factor (VEGF), angiopoietins (Ang-1 and Ang-2), and their receptor Tie2 are critically involved in both normal and pathologic angiogenesis. The aim of this study was to explore the role of Ang-1, Ang-2, VEGF, and Tie2 in the development and progression of bladder cancer as well as to examine their prognostic value in this tumor type.

Experimental Design: Tumor samples of 113 bladder cancer patients, normal bladder epithelium of 5 noncancer patients, and two low-grade (UMUC3 and RT4) and two high-grade (J82 and T24) bladder cancer cell lines were analyzed by quantitative real-time PCR. The expression data were analyzed performing Wilcoxon rank-sum and Kaplan-Meier log-rank tests as well as univariate Cox analyses and Cox proportional hazards regression model.

Results: In tissues of noninvasive bladder tumors, Ang-1 expression was significantly lower (P < 0.001), whereas VEGF expression was significantly higher (P = 0.031) than in normal bladder tissue. These findings were also confirmed at the protein level by immunohistochemistry. In contrast, Tie2 and Ang-2 abundance in tumor did not differ significantly from that in normal bladder tissue. Multivariate analysis identified Ang-2 as a strong and independent predictor of tumor recurrence [hazard ratio (HR), 10.18; 95% confidence interval (95% CI), 2.69-38.49; P < 0.001] and Tie2 expression as an independent favorable prognostic factor for both metastasis (HR, 0.31; 95% CI, 0.11-0.89; P = 0.029) and disease-specific survival (HR, 0.25; 95% CI, 0.10-0.62; P = 0.003).

Conclusions: These data show the strongest change in expression of VEGF and Ang-1 in superficial bladder cancer in comparison with normal bladder epithelium and the invasive tumor stages. The prognostic significance of Ang-2 and Tie2 underlines the essential role of angiopoietins-Tie2 system in progression of bladder cancer.
Data from Epigenetic Inactivation of Galanin Receptor 1 in Head and Neck Cancer
Authors: Misawa Kiyoshi , Ueda Yo , Kanazawa Takeharu , Misawa Yuki , Jang Ilwhan , Brenner John Chadwick , Ogawa Tetsuya , Takebayashi Satoru , Grenman Reidar A. , Herman James G. , Mineta Hiroyuki , Carey Thomas E.
Published: April 2023

Abstract
Purpose: One copy of the galanin receptor 1 (GALR1) locus on 18q is often deleted and expression is absent in some head and neck squamous cell carcinoma (HNSCC) cell lines. To determine if loss of heterozygosity and hypermethylation might silence the GALR1 gene, promoter methylation status and gene expression were assessed in a large panel of HNSCC cell lines and tumors.

Experimental Design: Promoter methylation of GALR1 in 72 cell lines and 100 primary tumor samples was analyzed using methylation-specific PCR. GALR1 expression and methylation status were analyzed further by real-time PCR and bisulfite sequencing analysis.

Results: The GALR1 promoter was fully or partially methylated in 38 of 72 (52.7%) HNSCC cell lines but not in the majority 18 of 20 (90.0%) of nonmalignant lines. GALR1 methylation was also found in 38 of 100 (38%) primary tumor specimens. Methylation correlated with decreased GALR1 expression. In tumors, methylation was significantly correlated with increased tumor size (P = 0.0036), lymph node status (P = 0.0414), tumor stage (P = 0.0037), cyclin D1 expression (P = 0.0420), and p16 methylation (P = 0.0494) and survival (P = 0.045). Bisulfite sequencing of 36 CpG sites upstream of the transcription start site revealed that CpG methylation within transcription factor binding sites correlated with complete suppression of GALR1 mRNA. Treatment with trichostatin A and 5-azacytidine restored GALR1 expression. In UM-SCC-23 cells that have total silencing of GALR1, exogenous GALR1 expression and stimulation with galanin suppressed cell proliferation.

Conclusions: Frequent promoter hypermethylation, gene silencing, association with prognosis, and growth suppression after reexpression support the hypothesis that GALR1 is a tumor suppressor gene in HNSCC.
Data from Dendritic Cell Vaccination with Xenogenic Polypeptide Hormone Induces Tumor Rejection in Neuroendocrine Cancer
Authors: Papewalis Claudia , Wuttke Margret , Seissler Jochen , Meyer Yvonne , Kessler Caroline , Jacobs Benedikt , Ullrich Evelyn , Willenberg Holger S. , Schinner Sven , Baehring Thomas , Scherbaum Werner A. , Schott Matthias
Published: April 2023

Abstract
Purpose: No relevant breakthrough has yet been achieved in the identification of tumor antigens in many neuroendocrine cancer types that exist, such as malignant gastrinoma, insulinoma, or medullary thyroid carcinoma. The aim of this study was to proof the concept of dendritic cell immunization with a tumor cell-specific polypeptide hormone as a target molecule in a transgenic mouse model for medullary thyroid carcinoma (Ret/Cal mice).

Experimental Design: Ret/Cal mice were repeatedly immunized for up to 6 months with amino acid–modified (xenogenic) calcitonin-pulsed dendritic cells. Xenogenic calcitonin was chosen for immunization due to its higher immunogenicity as compared with murine calcitonin.

Results: Lymph nodes from control protein-immunized mice did not show any macroscopic abnormalities, whereas tumor peptide-treated mice revealed in general profoundly enlarged lymph nodes. In tetramer analysis of paratumorous lymph nodes, 1.9% to 3.1% of all infiltrating CD8+ T cells were specific for one of three tumor epitopes tested. Analysis of the activated IFN-γ-secreting component in splenic cells revealed an average of 2.8% tumor epitope-specific CD8+ cells. Immunohistochemistry revealed strong CD8+ tumor infiltration in calcitonin-vaccinated mice. In addition, these cells also showed strong in vitro lysis capacity at up to 63.3%. Most importantly, calcitonin-immunized mice revealed largely diminished tumor outgrowth (−74.3%) compared with control mice (P < 0.0001). Likewise, serum calcitonin levels in calcitonin-vaccinated Ret/Cal mice were lower than in the control group.

Conclusion: These results have a major effect, as they are the first to establish a role for xenogenic polypeptide hormones as target molecules for immunotherapy in endocrine malignancies.
Data from A Contemporary Prognostic Nomogram for Men with Hormone-Refractory Metastatic Prostate Cancer: A TAX327 Study Analysis
Authors: Armstrong Andrew J. , Garrett-Mayer Elizabeth S. , Yang Yi-Chun Ou , de Wit Ronald , Tannock Ian F. , Eisenberger Mario
Published: April 2023

Abstract
Purpose: To develop a prognostic model and nomogram using baseline clinical variables to predict death among men with metastatic hormone-refractory prostate cancer (HRPC).

Experimental Design: TAX327 was a clinical trial that randomized 1,006 men with metastatic HRPC to receive every three week or weekly docetaxel or mitoxantrone, each with prednisone. We developed a multivariate Cox model and nomogram to predict survival at 1, 2, and 5 years.

Results: Ten independent prognostic factors other than treatment group were identified in multivariate analysis: (a) presence of liver metastases [hazard ratio (HR), 1.66; P = 0.019], (b) number of metastatic sites (HR, 1.63 if ≥2 sites; P = 0.001), (c) clinically significant pain (HR, 1.48; P < 0.0001), (d) Karnofsky performance status (HR, 1.39 if ≤70; P = 0.016), (e) type of progression (HR, 1.37 for measurable disease progression and 1.29 for bone scan progression; P = 0.005 and 0.01, respectively), (f) pretreatment prostate-specific antigen (PSA) doubling time (HR, 1.19 if <55 days; P = 0.066), (g) PSA (HR, 1.17 per log rise; P < 0.0001), (h) tumor grade (HR, 1.18 for high grade; P = 0.069), (i) alkaline phosphatase (HR, 1.27 per log rise; P < 0.0001), and (j) hemoglobin (HR, 1.11 per unit decline; P = 0.004). A nomogram was developed based on this multivariate model and validated internally using bootstrap methods, with a concordance index of 0.69.

Conclusions: This multivariate model identified several new independent prognostic factors in men with metastatic HRPC, including PSA doubling time, and led to the successful development of a clinically applicable nomogram. External prospective validation may support the wider use of this prognostic baseline model for men with HRPC treated with chemotherapy.
Data from Second-Generation Anti–Carcinoembryonic Antigen Designer T Cells Resist Activation-Induced Cell Death, Proliferate on Tumor Contact, Secrete Cytokines, and Exhibit Superior Antitumor Activity In vivo: A Preclinical Evaluation
Authors: Emtage Peter C.R. , Lo Agnes S.Y. , Gomes Erica M. , Liu David L. , Gonzalo-Daganzo Rosa M. , Junghans Richard P.
Published: April 2023

Abstract
Purpose: This report describes the development and preclinical qualification tests of second-generation anti-carcinoembryonic (CEA) designer T cells for use in human trials.

Experimental Design: The progenitor first-generation immunoglobulin-T-cell receptor (IgTCR) that transmits Signal 1-only effectively mediated chimeric immune receptor (CIR)–directed cytotoxicity, but expressor T cells succumbed to activation-induced cell death (AICD). The second-generation CIR (termed “Tandem” for two signals) was designed to transmit TCR Signal 1 and CD28 Signal 2 to render T cells resistant to AICD and provide prolonged antitumor effect in vivo.

Results: A CIR was created that combines portions of CD28, TCRζ, and a single chain antibody domain (sFv) specific for CEA into a single molecule (IgCD28TCR). As designed, the gene-modified Tandem T cells exhibit the new property of being resistant to AICD, showing instead an accelerated proliferation on tumor contact. Tandem T cells are more potent than first generation in targeting and lysing CEA+ tumor. Tandem T cells secrete high levels of interleukin-2 and IFNγ on tumor contact that first-generation T cells lacked, but secretion was exhaustible, suggesting a need for interleukin-2 supplementation in therapy even for these second-generation agents. Finally, second-generation T cells were more effective in suppressing tumor in animal models.

Conclusion: An advanced generation of anti-CEA designer T cells is described with features that promise a more potent and enduring antitumor immune response in vivo. These preclinical data qualify the human use of this agent that is currently undergoing trial in patients with CEA+ cancers.
Data from New Strategies in Kidney Cancer: Therapeutic Advances through Understanding the Molecular Basis of Response and Resistance
Authors: Rini Brian I.
Published: April 2023

Abstract
The emergence of viable therapeutic strategies in metastatic renal cell carcinoma has invigorated translational and clinical research in this disease. Building upon the clinical activity observed with inhibition of vascular endothelial growth factor (VEGF) and mammalian target of rapamycin (mTOR) pathways, novel strategies are being investigated to extend existing clinical benefits. Preclinical study has identified potential molecular mechanisms of response and resistance, providing a rational basis for biomarker development as well as sequential and combination therapy strategies. Several treatment strategies have emerged that are in the early phases of clinical testing. Further clinical and translational research is needed to validate initial hypotheses and translate observations into novel treatment strategies. Clin Cancer Res; 16(5); 1348–54
Data from In vivo Diagnosis of Epidermal Growth Factor Receptor Expression using Molecular Imaging with a Cocktail of Optically Labeled Monoclonal Antibodies
Authors: Barrett Tristan , Koyama Yoshinori , Hama Yukihiro , Ravizzini Gregory , Shin In Soo , Jang Beom-Su , Paik Chang H. , Urano Yasuteru , Choyke Peter L. , Kobayashi Hisataka
Published: April 2023

Abstract
Purpose: Epidermal growth factor receptors (EGFR) play an important role in tumorigenesis and, therefore, have become targets for new molecular therapies. Here, we use a “cocktail” of optically labeled monoclonal antibodies directed against EGFR-1 (HER1) and EGFR-2 (HER2) to distinguish tumors by their cell surface expression profiles.

Experimental Design:In vivo imaging experiments were done in tumor-bearing mice following s.c. injection of A431 (overexpressing HER1), NIH3T3/HER2+ (overexpressing HER2), and Balb3T3/DsRed (non-expression control) cell lines. After tumor establishment, a cocktail of optically labeled antibodies: Cy5.5-labeled cetuximab (anti-HER1) and Cy7-labeled trastuzumab (anti-HER2) was i.v. injected. In vivo and ex vivo fluorescence imaging was done. For comparison with radionuclide imaging, experiments were undertaken using 111Indium-labeled antibodies. Additionally, a “blinded” diagnostic study was done for mice bearing one tumor type.

Results:In vivo spectral fluorescent molecular imaging of 14 mice with three tumor types clearly differentiated tumors using the cocktail of optically labeled antibodies both in vivo and ex vivo. Twenty-four hours after injection, A431 and NIH3T3/HER2+ tumors were detected distinctly by their peak on Cy5.5 and Cy7 spectral images, respectively; radionuclide imaging was unable to clearly distinguish tumors at this time point. In blinded single tumor experiments, investigators were able to correctly diagnose a total of 40 tumors.

Conclusion: An in vivo imaging technique using an antibody cocktail simultaneously differentiated two tumors expressing distinct EGFRs and enabled an accurate characterization of each subtype.
Data from CpG Oligodeoxynucleotides Alter Lymphocyte and Dendritic Cell Trafficking in Humans
Authors: Haining W. Nicholas , Davies Jeffrey , Kanzler Holger , Drury Linda , Brenn Thomas , Evans John , Angelosanto Jill , Rivoli Steven , Russell Kate , George Suzanne , Sims Paul , Neuberg Donna , Li Xiaochun , Kutok Jeffrey , Morgan Jeffrey , Wen Patrick , Demetri George , Coffman Robert L. , Nadler Lee M.
Published: April 2023

Abstract
Purpose: CpG oligodeoxynucleotides (CpG-ODN) are being investigated as cancer vaccine adjuvants because they mature plasmacytoid dendritic cells (PDC) into potent antigen-presenting cells. CpG-ODN also induce PDC to secrete chemokines that alter lymphocyte migration. Whether CpG-ODN TLR signals enhance antigen-specific immunity and/or trafficking in humans is unknown.

Experimental Design: We conducted a phase I study of CpG-ODN (1018 ISS) given as a vaccine adjuvant with granulocyte-macrophage colony-stimulating factor (GM-CSF) to induce T-cell immunity to a peptide vaccine from the tumor-associated antigen hTERT.

Results: The adjuvant effect was limited; only 1 of 16 patients showed a high-frequency hTERT-specific tetramer CD8+ T-cell response. However, CpG-ODN induced marked, transient peripheral blood lymphopenia. Biopsies showed dense lymphocytic infiltration at the vaccine site clustered around activated PDC. In vitro, CpG-ODN-treated PDC induced T-cell migration, showing that CpG-ODN stimulation of human PDC was sufficient to chemoattract T cells.

Conclusions: Our results show that (a) CpG-ODN with GM-CSF may not be an effective adjuvant strategy for hTERT peptide vaccines but (b) GM-CSF/CpG-ODN causes a PDC-mediated chemokine response that recruits T-cell migration to the peripheral tissues. These findings suggest a novel therapeutic role for targeted injections of CpG-ODN to direct lymphocyte migration to specific sites such as the tumor bed.
Data from Nuclear and Cytoplasmic Expression of ERβ1, ERβ2, and ERβ5 Identifies Distinct Prognostic Outcome for Breast Cancer Patients
Authors: Shaaban Abeer M. , Green Andrew R. , Karthik Suchita , Alizadeh Yalda , Hughes Thomas A. , Harkins Lynn , Ellis Ian O. , Robertson John F. , Paish Emma C. , Saunders Philippa T.K. , Groome Nigel P. , Speirs Valerie
Published: April 2023

Abstract
Purpose: Previous conflicting results about the prognostic significance of estrogen receptor (ER)-β in breast cancer may be explained by contribution of isoforms, of which five exist. Our aim was to elucidate the prognostic significance of ERβ1, ERβ2, and ERβ5 by immunohistochemistry in a large cohort of breast carcinomas with long-term follow-up.

Experimental Design: Tissue microarrays were stained with ERβ1, ERβ2, and ERβ5 antibodies and scored as percentage of positive tumor cells and using the Allred system. Nuclear and cytoplasmic staining was evaluated and correlated with histopathologic characteristics, overall survival (OS), and disease-free survival (DFS).

Results: Nuclear ERβ2 and ERβ5, but not ERβ1, significantly correlated with OS (P = 0.006, P = 0.039, and P = 0.099, respectively), and ERβ2 additionally with DFS (P = 0.013). ERβ2 also predicted response to endocrine therapy (P = 0.036); correlated positively with ERα, progesterone receptor, androgen receptor, and BRCA1; and correlated inversely with metastasis and vascular invasion. Tumors coexpressing ERβ2 and ERα had better OS and DFS. Cytoplasmic ERβ2 expression, alone or combined with nuclear staining, predicted significantly worse OS. Notably, patients with only cytoplasmic ERβ2 expression had significantly worse outcome (P = 0.0014).

Conclusions: This is the first study elucidating the prognostic role of ERβ1, ERβ2, and ERβ5 in a large breast cancer series. ERβ2 is a powerful prognostic indicator in breast cancer, but nuclear and cytoplasmic expression differentially affect outcome. Measuring these in clinical breast cancer could provide a more comprehensive picture of patient outcome, complementing ERα.
Data from Evaluation of Lapatinib and Topotecan Combination Therapy: Tissue Culture, Murine Xenograft, and Phase I Clinical Trial Data
Authors: Molina Julian R. , Kaufmann Scott H. , Reid Joel M. , Rubin Stephen D. , Gálvez-Peralta Marina , Friedman Robert , Flatten Karen S. , Koch Kevin M. , Gilmer Tona M. , Mullin Robert J. , Jewell Roxanne C. , Felten Sara J. , Mandrekar Sumithra , Adjei Alex A. , Erlichman Charles
Published: April 2023

Abstract
Purpose: Topotecan resistance can result from drug efflux by P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) as well as survival signals initiated by epidermal growth factor receptor family members. The present studies were done to determine the effect of combining topotecan and the dual epidermal growth factor receptor/HER2 inhibitor lapatinib in tissue culture, a murine xenograft model, and a phase I clinical trial.

Experimental Design: The effects of lapatinib on topotecan accumulation and cytotoxicity in vitro were examined in paired cell lines lacking or expressing Pgp or BCRP. Antiproliferative effects of the combination were assessed in mice bearing HER2+ BT474 breast cancer xenografts. Based on tolerability in this preclinical model, 37 patients with advanced-stage cancers received escalating doses of lapatinib and topotecan in a phase I trial.

Results: Lapatinib increased topotecan accumulation in BCRP- or Pgp-expressing cells in vitro, and the combination showed enhanced efficacy in HER2+ BT474 xenografts. In the phase I study, nausea, vomiting, diarrhea, and fatigue were dose limiting. The maximum tolerated doses were 1,250 mg/d lapatinib by mouth for 21 or 28 days with 3.2 mg/m2 topotecan i.v. on days 1, 8, and 15 of 28-day cycles. Pharmacokinetic analyses showed that combined drug administration resulted in decreased topotecan clearance consistent with transporter-mediated interactions. Seventeen (46%) patients had disease stabilization.

Conclusions: The lapatinib/topotecan combination is well tolerated and warrants further study.
Data from Silibinin Inhibits Established Prostate Tumor Growth, Progression, Invasion, and Metastasis and Suppresses Tumor Angiogenesis and Epithelial-Mesenchymal Transition in Transgenic Adenocarcinoma of the Mouse Prostate Model Mice
Authors: Singh Rana P. , Raina Komal , Sharma Girish , Agarwal Rajesh
Published: April 2023

Abstract
Purpose: The chronic nature of prostate cancer growth and progression leading to metastasis provides a large window for intervention. Herein, for the first time, we investigated the effect and associated mechanisms of silibinin phosphatidylcholine (silybin-phytosome) on established prostate tumors in transgenic adenocarcinoma of the mouse prostate (TRAMP) model.

Experimental Design: Twenty-week-old TRAMP male mice having palpable prostate tumor were fed with control or 0.5% and 1%, w/w, silybin-phytosome diets for 11 weeks and then sacrificed.

Results: Dietary silibinin inhibited the growth of prostate tumors (up to 60%, P < 0.001) and suppressed tumor progression from prostatic intraepithelial neoplasia to differentiated adenocarcinoma and poorly differentiated adenocarcinoma, with a complete absence of poorly differentiated adenocarcinoma at higher doses. It also inhibited the incidence of tumor invasion of seminal vesicle (up to 81%, P < 0.001) with complete absence of distant metastasis. Silibinin moderately inhibited tumor cell proliferation and induced apoptosis, but strongly suppressed tumor microvessel density (up to 60%, P < 0.001), vascular endothelial growth factor, and vascular endothelial growth factor receptor-2 expression. Antibody array analysis of plasma showed a decrease in the circulatory levels of vascular endothelial growth factor and basic fibroblast growth factor. Decreased levels of matrix metalloproteinases (MMP), snail-1, and vimentin, and an increased level of E-cadherin were also observed, indicating the anti–epithelial-mesenchymal transition effect of silibinin in tumors.

Conclusions: Overall, silibinin treatment of TRAMP mice bearing prostate tumor inhibited tumor growth, progression, local invasion, and distant metastasis involving suppression of tumor angiogenesis and epithelial-mesenchymal transition. These findings would have greater relevance for the ongoing phase II clinical trial with silibinin-phytosome in prostate cancer patients.
Data from Identification and Validation of a Novel Gene Signature Associated with the Recurrence of Human Hepatocellular Carcinoma
Authors: Wang Suk Mei , Ooi London Lucien P.J. , Hui Kam M.
Published: April 2023

Abstract
Purpose: To improve the clinical management of human hepatocellular carcinoma (HCC) by accurate identification, at diagnosis, of patients at risk of recurrence after primary treatment for HCC.

Experimental Design: Two clinicopathologic variables available at diagnosis, vascular invasion and cirrhosis, together with molecular profiling using Affymetrix human HG-U133A and HG-U133B oligonucleotide probe arrays, were used to identify recurrent HCC disease.

Results: HCC patients presented clinically at diagnosis with vascular invasion and cirrhosis showed a high rate (78-83%) of developing recurrent disease within 6 to 35 months. In comparison, most of the HCC patients (80-100%) without vascular invasion and cirrhosis remained disease-free. However, the risk of recurrent disease for HCC patients with either vascular invasion or cirrhosis could not be accurately ascertained. Using a pool of 23 HCC patients with either vascular invasion or cirrhosis as training set, a 57-gene signature was derived and could predict recurrent disease at diagnosis, with 84% (sensitivity 86%, specificity 82%) accuracy, for a totally independent test set of 25 HCC patients with either vascular invasion or cirrhosis. On further analysis, the disease-free rate was significantly different between patients that were predicted to recur or not to recur in the test group (P = 0.002).

Conclusion: We have presented data to show that by incorporating the status of vascular invasion and cirrhosis available at diagnosis for patients with HCC after partial curative hepatectomy and a novel 57-member gene signature, we could accurately stratify HCC patients with different risks of recurrence.