bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from Involvement of Mitochondrial DNA Sequence Variations and Respiratory Activity in Late Complications following Radiotherapy
Authors: Alsbeih Ghazi A. , Al-Harbi Najla M. , El-Sebaie Medhat M. , Al-Rajhi Nasser M. , Al-Hadyan Khaled S. , Abu-Amero Khaled K.
Published: April 2023

Abstract
Purpose: Mitochondria and ionizing radiation overlap in a number of features; for instance, both generate harmful reactive oxygen species, and that radiation can induce cell death through the intermediary of mitochondria. Because a number of genetic variations in nuclear genes are frequently associated with response to cancer treatment, the aim of this case-control study was to test the hypothesis that mitochondrial DNA (mtDNA) genetic variations can contribute to patient-to-patient variability in normal tissue response to radiotherapy.

Experimental Design: Thirty-two nasopharyngeal carcinomas patients treated with definitive radiotherapy were included. The grade (G) of s.c. and deep tissue fibrosis was scored according to the Radiation Therapy Oncology Group/European Organization for Research and Treatment of Cancer grading system. Coding and RNA mtDNA (between 611 and 15,978 bp) were sequenced, and genetic variations were scored. Mitochondrial respiratory activity was measured by resazurin reduction assay.

Results: Data showed a significantly (P = 0.003) higher number of nonsynonymous genetic variations in the radiosensitive (G2-G3; 16 patients) as compared with the control (G0-G1; 16 patients) groups. The nonsynonymous A10398G variation in the ND3 gene was significantly associated with fibrotic reaction (P = 0.01). The radiosensitive patients had a 7-fold (95% confidence interval, 1.16-51.65) higher risk of developing moderate to severe fibrosis (G2-G3) following radiotherapy. This was significantly correlated with lower mitochondrial respiratory activity (P = 0.001).

Conclusion: Mitochondria contribute to radiation sensitivity, and genetic variations can be associated with late reactions to radiotherapy. Predictive markers of radiosensitivity should take into account mtDNA genetic variations in addition to variations in nuclear genes. (Clin Cancer Res 2009;15(23):7352–60)
Data from Homozygous Deletion of MTAP Gene as a Poor Prognosticator in Gastrointestinal Stromal Tumors
Authors: Huang Hsuan-Ying , Li Shau-Hsuan , Yu Shih-Chen , Chou Fong-Fu , Tzeng Ching-Cherng , Hu Tsung-Hui , Uen Yih-Huei , Tian Yu-Fang , Wang Yu-Hui , Fang Fu-Min , Huang Wen-Wei , Wei Yu-Ching , Wu Jing-Mei , Li Chien-Feng
Published: April 2023

Abstract
Purpose: Chromosome 9 is frequently deleted in high-risk gastrointestinal stromal tumors (GISTs), whereas its specific tumor suppressor genes (TSGs) are less understood. We did an integrative study of MTAP gene at 9p21 to analyze its implication in GISTs.

Experimental Design: To search TSGs on chromosome 9, we used ultrahigh-resolution array comparative genomic hybridization to profile DNA copy number alterations of 22 GISTs, with special attention to MTAP gene. MTAP immunoexpression was assessable for 306 independent GISTs on tissue microarrays, with 146 cases analyzed for MTAP homozygous deletion, 181 for mutations of KIT and PDGFRA receptor tyrosine kinase genes, and 7 for MTAP hypermethylation.

Results: Array comparative genomic hybridization identified 11 candidate TSGs on 9p and six on 9q. MTAP and/or CDKN2A/CDKN2B at 9p21.3 were deleted in one intermediate-risk (11) and seven high-risk (70) GISTs with two cases homozygously codeleted at both loci. MTAP homozygous deletion, present in 25 of 146 cases, was highly associated with larger size and higher mitotic rate, Ki-67 index, and risk level (all P < 0.01) but not with receptor tyrosine kinase genotypes. Whereas MTAP homozygous deletion correlated with MTAP protein loss (P < 0.001), 7 of 30 GISTs without MTAP expression did not show homozygous deletion, including three MTAP-hypermethylated cases. MTAP homozygous deletion was univariately predictive of decreased disease-free survival (P < 0.0001) and remained multivariately independent (P = 0.0369, hazard ratio = 2.166), together with high-risk category (P < 0.0001), Ki-67 index >5 (P = 0.0106), and nongastric location (P = 0.0416).

Conclusions:MTAP homozygous deletion, the predominant mechanism to deplete protein expression, is present in 17 of GISTs. It correlates with important prognosticators and independently predicts worse outcomes, highlighting the role in disease progression. (Clin Cancer Res 2009;15(22):696372)
Data from Local Recurrence after Breast-Conserving Therapy in Relation to Gene Expression Patterns in a Large Series of Patients
Authors: Kreike Bas , Halfwerk Hans , Armstrong Nicola , Bult Peter , Foekens John A. , Veltkamp Sanne C. , Nuyten Dimitry S.A. , Bartelink Harry , van de Vijver Marc J.
Published: April 2023

Abstract
Purpose: The majority of patients with early-stage breast cancer are treated with breast-conserving therapy (BCT). Several clinical risk factors are associated with local recurrence (LR) after BCT but are unable to explain all instances of LR after BCT. Here, gene expression microarrays are used to identify novel risk factors for LR after BCT.

Experimental Design: Gene expression profiles of 56 primary invasive breast carcinomas from patients who developed a LR after BCT were compared with profiles of 109 tumors from patients who did not develop a LR after BCT. Both unsupervised and supervised methods of classification were used to separate patients into groups corresponding to disease outcome. In addition, for 15 patients, the gene expression profile in the recurrence was compared with that of the primary tumor.

Results: The two main clusters found by hierarchical cluster analysis of all 165 primary invasive breast carcinomas revealed no association with LR. Predefined gene sets (molecular subtypes and “chromosomal instability” signature) are associated with LR (P = 0.0002 and 0.003, respectively). Significant analysis of microarrays revealed an association between LR and cell proliferation, not captured by histologic grading. Class prediction analysis constructed a gene classifier, which was successfully validated, cross-platform, on an independent data set of 161 patients (log-rank P = 0.041). In multivariate analysis, young age was the only independent predictor of LR.

Conclusions: We have constructed and cross-platform validated a gene expression profile predictive for LR after BCT, which is characterized by genes involved in cell proliferation but not a surrogate for high histologic grade.
Data from PPM1D Is a Potential Therapeutic Target in Ovarian Clear Cell Carcinomas
Authors: Tan David S.P. , Lambros Maryou B.K. , Rayter Sydonia , Natrajan Rachael , Vatcheva Radost , Gao Qiong , Marchiò Caterina , Geyer Felipe C. , Savage Kay , Parry Suzanne , Fenwick Kerry , Tamber Narinder , Mackay Alan , Dexter Tim , Jameson Charles , McCluggage W. Glenn , Williams Alistair , Graham Ashley , Faratian Dana , El-Bahrawy Mona , Paige Adam J. , Gabra Hani , Gore Martin E. , Zvelebil Marketa , Lord Christopher J. , Kaye Stanley B. , Ashworth Alan , Reis-Filho Jorge S.
Published: April 2023

Abstract
Purpose: To identify therapeutic targets in ovarian clear cell carcinomas, a chemoresistant and aggressive type of ovarian cancer.

Experimental Design: Twelve ovarian clear cell carcinoma cell lines were subjected to tiling path microarray comparative genomic hybridization and genome-wide expression profiling analysis. Regions of high-level amplification were defined and genes whose expression levels were determined by copy number and correlated with gene amplification were identified. The effects of inhibition of PPM1D were assessed using short hairpin RNA constructs and a small-molecule inhibitor (CCT007093). The prevalence of PPM1D amplification and mRNA expression was determined using chromogenic in situ hybridization and quantitative real-time reverse transcription-PCR in a cohort of pure ovarian clear cell carcinomas and on an independent series of unselected epithelial ovarian cancers.

Results: Array-based comparative genomic hybridization analysis revealed regions of high-level amplification on 1q32, 1q42, 2q11, 3q24-q26, 5p15, 7p21-p22, 11q13.2-q13.4, 11q22, 17q21-q22, 17q23.2, 19q12-q13, and 20q13.2. Thirty-four genes mapping to these regions displayed expression levels that correlated with copy number gains/amplification. PPM1D had significantly higher levels of mRNA expression in ovarian clear cell carcinoma cell lines harboring gains/amplifications of 17q23.2. PPM1D inhibition revealed that PPM1D expression and phosphatase activity are selectively required for the survival of ovarian clear cell carcinoma cell lines with 17q23.2 amplification. PPM1D amplification was significantly associated with ovarian clear cell carcinoma histology (P = 0.0003) and found in 10% of primary ovarian clear cell carcinomas. PPM1D expression levels were significantly correlated with PPM1D gene amplification in primary ovarian clear cell carcinomas.

Conclusion: Our data provide strong circumstantial evidence that PPM1D is a potential therapeutic target for a subgroup of ovarian clear cell carcinomas.
Data from Resistance to Chemotherapy Is Associated with Fibroblast Growth Factor Receptor 4 Up-Regulation
Authors: Roidl Andreas , Berger Hans-Jürgen , Kumar Sushil , Bange Johannes , Knyazev Pjotr , Ullrich Axel
Published: April 2023

Abstract
Purpose: Establishment of antiapoptotic signaling pathways in tumor cells is a major cause for the failure of chemotherapy against cancer. To investigate the underlying mechanisms, we developed an experimental approach that is based on the genetic plasticity of cancer cells and the selection for cell survival on treatment with chemotherapeutic agents.

Experimental Design: Gene expression changes of surviving cell clones were analyzed by macroarrays. Involvement of fibroblast growth factor receptor 4 (FGFR4) in antiapoptotic pathways was elucidated by apoptosis assays, small interfering RNA experiments, and an antagonistic antibody.

Results: We show that FGFR4 gene expression is up-regulated in doxorubicin-treated, apoptosis-resistant cancer cell clones. Ectopic expression of FGFR4 in cancer cells led to reduced apoptosis sensitivity on treatment with doxorubicin or cyclophosphamide, whereas knockdown of endogenous FGFR4 expression in breast cancer cell lines had the opposite effect. FGFR4 overexpression resulted in Bcl-xl up-regulation at both mRNA and protein levels. Knockdown of FGFR4 expression by small interfering RNA caused a decrease in phospho-extracellular signal-regulated kinase 1/2 levels and reduced Bcl-xl expression. Moreover, an antagonistic FGFR4 antibody suppressed the resistance of cancer cells with endogenous FGFR4 expression against apoptosis-inducing chemotherapeutic agents.

Conclusion: Based on these findings, we propose an antiapoptotic signaling pathway that is initiated by FGFR4 and regulating the expression of Bcl-xl through the mitogen-activated protein kinase cascade. Our findings are exemplary for a novel strategy toward the elucidation of diverse signaling pathways that define antiapoptotic potential in cancer cells. These observations open new avenues toward the diagnosis of chemoresistant tumors and therapies targeting FGFR4-overexpressing cancers.
Data from Immunohistochemical Expression of Estrogen and Progesterone Receptors Identifies a Subset of NSCLCs and Correlates with EGFR Mutation
Authors: Raso Maria G. , Behrens Carmen , Herynk Matthew H. , Liu Suyu , Prudkin Ludmila , Ozburn Natalie C. , Woods Denise M. , Tang Ximing , Mehran Reza J. , Moran Cesar , Lee J. Jack , Wistuba Ignacio I.
Published: April 2023

Abstract
Purpose: To determine the frequency of estrogen receptor α and β and progesterone receptor protein immunohistochemical expression in a large set of non–small cell lung carcinoma (NSCLC) specimens and to compare our results with those for some of the same antibodies that have provided inconsistent results in previously published reports.

Experimental Design: Using multiple antibodies, we investigated the immunohistochemical expression of estrogen receptors α and β and progesterone receptor in 317 NSCLCs placed in tissue microarrays and correlated their expression with patients' clinicopathologic characteristics and in adenocarcinomas with EGFR mutation status.

Results: Estrogen receptors α and β were detected in the nucleus and cytoplasm of NSCLC cells; however, the frequency of expression (nucleus, 5-36% for α and 42-56% for β; cytoplasm: <1-42% for α and 20-98% for β) varied among the different antibodies tested. Progesterone receptor was expressed in the nuclei of malignant cells in 63% of the tumors. Estrogen receptor α nuclear expression significantly correlated with adenocarcinoma histology, female gender, and history of never smoking (P = 0.0048 to <0.0001). In NSCLC, higher cytoplasmic estrogen receptor α expression significantly correlated with worse recurrence-free survival (hazard ratio, 1.77; 95% confidence interval, 1.12, 2.82; P = 0.015) in multivariate analysis. In adenocarcinomas, estrogen receptor α expression correlated with EGFR mutation (P = 0.0029 to <0.0001). Estrogen receptor β and progesterone receptor but not estrogen receptor α expressed in the normal epithelium adjacent to lung adenocarcinomas.

Conclusions: Estrogen receptor α and β expression distinguishes a subset of NSCLC that has defined clinicopathologic and genetic features. In lung adenocarcinoma, estrogen receptor α expression correlates with EGFR mutations. (Clin Cancer Res 2009;15(17):5359–68)
Data from Oncolytic Adenoviral Mutants with E1B19K Gene Deletions Enhance Gemcitabine-induced Apoptosis in Pancreatic Carcinoma Cells and Anti-Tumor Efficacy In vivo
Authors: Leitner Stephan , Sweeney Katrina , Öberg Daniel , Davies Derek , Miranda Enrique , Lemoine Nick R. , Halldén Gunnel
Published: April 2023

Abstract
Purpose: Pancreatic adenocarcinoma is a rapidly progressive malignancy that is highly resistant to current chemotherapeutic modalities and almost uniformly fatal. We show that a novel targeting strategy combining oncolytic adenoviral mutants with the standard cytotoxic treatment, gemcitabine, can markedly improve the anticancer potency.

Experimental Design: Adenoviral mutants with the E1B19K gene deleted with and without E3B gene expression (AdΔE1B19K and dl337 mutants, respectively) were assessed for synergistic interactions in combination with gemcitabine. Cell viability, mechanism of cell death, and antitumor efficacy in vivo were determined in the pancreatic carcinoma cells PT45 and Suit2, normal human bronchial epithelial cells, and in PT45 xenografts.

Results: The ΔE1B19K-deleted mutants synergized with gemcitabine to selectively kill cultured pancreatic cancer cells and xenografts in vivo with no effect in normal cells. The corresponding wild-type virus (Ad5) stimulated drug-induced cell killing to a lesser degree. Gemcitabine blocked replication of all viruses despite the enhanced cell killing activity due to gemcitabine-induced delay in G1/S-cell cycle progression, with repression of cyclin E and cdc25A, which was not abrogated by viral E1A-expression. Synergistic cell death occurred through enhancement of gemcitabine-induced apoptosis in the presence of both AdΔE1B19K and dl337 mutants, shown by increased cell membrane fragmentation, caspase-3 activation, and mitochondrial dysfunction.

Conclusions: Our data suggest that oncolytic mutants lacking the antiapoptotic E1B19K gene can improve efficacy of DNA-damaging drugs such as gemcitabine through convergence on cellular apoptosis pathways. These findings imply that less toxic doses than currently practiced in the clinic could efficiently target pancreatic adenocarcinomas when combined with adenoviral mutants.
Data from A Mitochondrial Target Sequence Polymorphism in Manganese Superoxide Dismutase Predicts Inferior Survival in Breast Cancer Patients Treated with Cyclophosphamide
Authors: Glynn Sharon A. , Boersma Brenda J. , Howe Tiffany M. , Edvardsen Hege , Geisler Stephanie B. , Goodman Julie E. , Ridnour Lisa A. , Lønning Per E. , Børresen-Dale Anne-Lise , Naume Bjorn , Kristensen Vessela N. , Chanock Stephen J. , Wink David A. , Ambs Stefan
Published: April 2023

Abstract
Purpose: Manganese superoxide dismutase protects against oxidative damage and modulates the efficacy of chemotherapeutic drugs. A functional single-nucleotide polymorphism in codon 16 of SOD2 (rs4880), which encodes manganese superoxide dismutase, results in a substitution of valine by alanine (Val16Ala). We hypothesized that this single-nucleotide polymorphism affects breast cancer survival of patients receiving chemotherapy.

Experimental Design: Two patient populations from the United States (n = 248) and Norway (n = 340) were genotyped for Val16Ala. Kaplan-Meier survival and Cox proportional hazards regression analyses were used to examine the relationship between Val16Ala and disease-specific survival.

Results: Val16Ala was significantly associated with breast cancer outcome in both patient populations. Carriers of the Ala allele had inferior survival rates in the multivariate analysis [hazard ratio (HR), 2.44 and 95% confidence interval (95% CI), 1.11-5.37 in U.S. cohort; HR, 1.91 and 95% CI, 1.06-3.45 in Norway cohort for Ala/Ala versus Val/Val]. In an analysis of the combined cohorts, this association was significant for patients receiving adjuvant therapy (HR, 2.47; 95% CI, 1.46-4.19), but not for patients without it (HR, 1.47; 95% CI, 0.57-3.74). After further stratification by type of chemotherapy, the effect of the Ala allele was mostly restricted to cyclophosphamide-containing chemotherapy regimens (HR, 22.0; 95% CI, 5.22-92.9; Ala/Ala versus Val/Val).

Conclusion: The Val16Ala polymorphism affects survival of patients receiving cyclophosphamide-containing chemotherapy. The findings provide the first evidence pointing toward a mechanism for cyclophosphamide resistance in breast cancer patients.
Data from Microarray Analysis of Early Stage Serous Ovarian Cancers Shows Profiles Predictive of Favorable Outcome
Authors: Berchuck Andrew , Iversen Edwin S. , Luo Jingqin , Clarke Jennifer P. , Horne Hisani , Levine Douglas A. , Boyd Jeff , Alonso Miguel A. , Secord Angeles Alvarez , Bernardini Marcus Q. , Barnett Jason C. , Boren Todd , Murphy Susan K. , Dressman Holly K. , Marks Jeffrey R. , Lancaster Johnathan M.
Published: April 2023

Abstract
Purpose: Although few women with advanced serous ovarian cancer are cured, detection of the disease at an early stage is associated with a much higher likelihood of survival. We previously used gene expression array analysis to distinguish subsets of advanced cancers based on disease outcome. In the present study, we report on gene expression of early-stage cancers and validate our prognostic model for advanced-stage cancers.

Experimental Design: Frozen specimens from 39 stage I/II, 42 stage III/IV, and 20 low malignant potential cancers were obtained from four different sites. A linear discriminant model was used to predict survival based upon array data.

Results: We validated the late-stage survival model and show that three of the most differentially expressed genes continue to be predictive of outcome. Most early-stage cancers (38 of 39 invasive, 15 of 20 low malignant potential) were classified as long-term survivors (median probabilities 0.97 and 0.86). MAL, the most differentially expressed gene, was further validated at the protein level and found to be an independent predictor of poor survival in an unselected group of advanced serous cancers (P = 0.0004).

Conclusions: These data suggest that serous ovarian cancers detected at an early stage generally have a favorable underlying biology similar to advanced-stage cases that are long-term survivors. Conversely, most late-stage ovarian cancers seem to have a more virulent biology. This insight suggests that if screening approaches are to succeed it will be necessary to develop approaches that are able to detect these virulent cancers at an early stage.
Data from Cancer-Selective Targeting and Cytotoxicity by Liposomal-Coupled Lysosomal Saposin C Protein
Authors: Qi Xiaoyang , Chu Zhengtao , Mahller Yonatan Y. , Stringer Keith F. , Witte David P. , Cripe Timothy P.
Published: April 2023

Abstract
Purpose: Saposin C is a multifunctional protein known to activate lysosomal enzymes and induce membrane fusion in an acidic environment. Excessive accumulation of lipid-coupled saposin C in lysosomes is cytotoxic. Because neoplasms generate an acidic microenvironment, caused by leakage of lysosomal enzymes and hypoxia, we hypothesized that saposin C may be an effective anticancer agent. We investigated the antitumor efficacy and systemic biodistribution of nanovesicles comprised of saposin C coupled with dioleoylphosphatidylserine in preclinical cancer models.

Experimental Design: Neuroblastoma, malignant peripheral nerve sheath tumor and, breast cancer cells were treated with saposin C–dioleoylphosphatidylserine nanovesicles and assessed for cell viability, ceramide elevation, caspase activation, and apoptosis. Fluorescently labeled saposin C–dioleoylphosphatidylserine was i.v. injected to determine in vivo tumor-targeting specificity. Antitumor activity and toxicity profile of saposin C–dioleoylphosphatidylserine were evaluated in xenograft models.

Results: Saposin C–dioleoylphosphatidylserine nanovesicles, with a mean diameter of ∼190 nm, showed specific tumor-targeting activity shown through in vivo imaging. Following i.v. administration, saposin C–dioleoylphosphatidylserine nanovesicles preferentially accumulated in tumor vessels and cells in tumor-bearing mice. Saposin C–dioleoylphosphatidylserine induced apoptosis in multiple cancer cell types while sparing normal cells and tissues. The mechanism of saposin C–dioleoylphosphatidylserine induction of apoptosis was determined to be in part through elevation of intracellular ceramides, followed by caspase activation. In in vivo models, saposin C–dioleoylphosphatidylserine nanovesicles significantly inhibited growth of preclinical xenografts of neuroblastoma and malignant peripheral nerve sheath tumor. I.v. dosing of saposin C–dioleoylphosphatidylserine showed no toxic effects in nontumor tissues.

Conclusions: Saposin C–dioleoylphosphatidylserine nanovesicles offer promise as a novel, nontoxic, cancer-targeted, antitumor agent for treating a broad range of cancers. (Clin Cancer Res 2009;15(18):5840–51)
Data from Overcoming Cancer Immune Tolerance and Escape
Authors: Clifton Guy T. , Peoples George E.
Published: April 2023

Abstract
Although HER2/neu–targeted cancer vaccines have shown initial promise in the adjuvant setting, a therapeutic vaccine remains elusive due to the tumor escape mechanisms of established cancer. As described by Seavey et al. in this issue of CCR, a Listeria-delivered vaccine may help overcome immune tolerance, leading to an effective therapeutic vaccine.
Data from Multi-center, Placebo-controlled, Double-blind, Randomized Study of Oral Toceranib Phosphate (SU11654), a Receptor Tyrosine Kinase Inhibitor, for the Treatment of Dogs with Recurrent (Either Local or Distant) Mast Cell Tumor Following Surgical Excision
Authors: London Cheryl A. , Malpas Phyllis B. , Wood-Follis Stacey L. , Boucher Joseph F. , Rusk Anthony W. , Rosenberg Mona P. , Henry Carolyn J. , Mitchener Kathy L. , Klein Mary K. , Hintermeister John G. , Bergman Philip J. , Couto Guillermo C. , Mauldin Guy N. , Michels Gina M.
Published: April 2023

Abstract
Purpose: The purpose of this study was to determine the objective response rate (ORR) following treatment of canine mast cell tumors (MCT) with toceranib phosphate (Palladia, SU11654), a kinase inhibitor with both antitumor and antiangiogenic activity through inhibition of KIT, vascular endothelial growth factor receptor 2, and PDGFRβ. Secondary objectives were to determine biological response rate, time to tumor progression, duration of objective response, health-related quality of life, and safety of Palladia.

Experimental Design: Dogs were randomized to receive oral Palladia 3.25 mg/kg or placebo every other day for 6 weeks in the blinded phase. Thereafter, eligible dogs received open-label Palladia.

Results: The blinded phase ORR in Palladia-treated dogs (n = 86) was 37.2% (7 complete response, 25 partial response) versus 7.9% (5 partial response) in placebo-treated dogs (n = 63; P = 0.0004). Of 58 dogs that received Palladia following placebo-escape, 41.4% (8 complete response, 16 partial response) experienced objective response. The ORR for all 145 dogs receiving Palladia was 42.8% (21 complete response, 41 partial response); among the 62 responders, the median duration of objective response and time to tumor progression was 12.0 weeks and 18.1 weeks, respectively. Palladia-treated responders scored higher on health-related quality of life versus Palladia-treated nonresponders (P = 0.030). There was no significant difference in the number of dogs with grade 3/4 (of 4) adverse events; adverse events were generally manageable with dose modification and/or supportive care.

Conclusions: Palladia has biological activity against canine MCTs and can be administered on a continuous schedule without need for routine planned treatment breaks. This clinical trial further shows that spontaneous tumors in dogs are good models to evaluate therapeutic index of targeted therapeutics in a clinical setting.
Data from Molecular Profiling of Inflammatory Breast Cancer
Authors: Bièche Ivan , Lerebours Florence , Tozlu Sengül , Espie Marc , Marty Michel , Lidereau Rosette
Published: April 2023

Abstract
Purpose: Inflammatory breast cancer (IBC) is a rare but particularly aggressive form of primary breast cancer. The molecular mechanisms responsible for IBC are largely unknown.

Experimental Design: To obtain further insight into the molecular pathogenesis of IBC, we used real-time quantitative reverse transcription (RT)-PCR to quantify the mRNA expression of 538 selected genes in IBC relative to non-IBC.

Results: Twenty-seven (5.0%) of the 538 genes were significantly up-regulated in IBC compared with non-IBC. None were down-regulated. The 27 up-regulated genes mainly encoded transcription factors (JUN, EGR1, JUNB, FOS, FOSB, MYCN, and SNAIL1), growth factors (VEGF, DTR/HB-EGF, IGFBP7, IL6, ANGPT2, EREG, CCL3/MIP1A, and CCL5/RANTES) and growth factor receptors (TBXA2R, TNFRSF10A/TRAILR1, and ROBO2). We also identified a gene expression profile, based on MYCN, EREG, and SHH, which discriminated subgroups of IBC patients with good, intermediate, and poor outcome.

Conclusion: Our study has identified a limited number of signaling pathways that require inappropriate activation for IBC development. Some of the up-regulated genes identified here could offer useful diagnostic or prognostic markers and could form the basis of novel therapeutic strategies.
Data from Protein Kinase A Activation Confers Resistance to Trastuzumab in Human Breast Cancer Cell Lines
Authors: Gu Long , Lau Sean K. , Loera Sofia , Somlo George , Kane Susan E.
Published: April 2023

Abstract
Purpose: Trastuzumab is a monoclonal antibody targeted to the Her2 receptor and approved for treatment of Her2-positive breast cancer. Among patients who initially respond to trastuzumab therapy, resistance typically arises within 1 year. BT/HerR cells are trastuzumab-resistant variants of Her2-positive BT474 breast cancer cells. The salient feature of BT/HerR cells is failure to downregulate phosphoinositide 3-kinase/Akt signaling on trastuzumab binding. The current work addresses the mechanism of sustained signaling in BT/HerR cells, focusing on the protein kinase A (PKA) pathway.

Experimental Design: We performed microarray analysis on BT/HerR and BT474 cell lines to identify genes that were upregulated or downregulated in trastuzumab-resistant cells. Specific genes in the PKA pathway were quantified using reverse transcription-PCR and Western hybridization. Small interfering RNA transfection was used to determine the effects of gene knockdown on cellular response to trastuzumab. Electrophoretic mobility shift assays were used to measure cyclic AMP–responsive element binding activity under defined conditions. Immunohistochemistry was used to analyze protein expression in clinical samples.

Results: BT/HerR cells had elevated PKA signaling activity and several genes in the PKA regulatory network had altered expression in these cells. Downregulation of one such gene, the PKA-RIIα regulatory subunit, conferred partial trastuzumab resistance in Her2-positive BT474 and SK-Br-3 cell lines. Forskolin activation of PKA also produced significant protection against trastuzumab-mediated Akt dephosphorylation. In patient samples, PKA signaling appeared to be enhanced in residual disease remaining after trastuzumab-containing neoadjuvant therapy.

Conclusions: Activation of PKA signaling may be one mechanism contributing to trastuzumab resistance in Her2-positive breast cancer. We propose a molecular model by which PKA confers its effects. (Clin Cancer Res 2009;15(23):7196–206)
Data from Inhibition of Insulin-Like Growth Factor 1 Receptor by CP-751,871 Radiosensitizes Non–Small Cell Lung Cancer Cells
Authors: Iwasa Tsutomu , Okamoto Isamu , Suzuki Minoru , Hatashita Erina , Yamada Yuki , Fukuoka Masahiro , Ono Koji , Nakagawa Kazuhiko
Published: April 2023

Abstract
Purpose: Therapeutic strategies that target the insulin-like growth factor I receptor (IGF-1R) hold promise for a wide variety of cancers. We have now investigated the effect of CP-751,871, a fully human monoclonal antibody specific for IGF-IR, on the sensitivity of human non–small cell lung cancer (NSCLC) cell lines to radiation.

Experimental Design: The radiosensitizing effect of CP-751,871 was evaluated on the basis of cell death, clonogenic survival, and progression of tumor xenografts. Radiation-induced damage was evaluated by immunofluorescence analysis of the histone γ-H2AX and Rad51.

Results: A clonogenic survival assay revealed that CP-751,871 increased the sensitivity of NSCLC cells to radiation in vitro. CP-751,871 inhibited radiation-induced IGF-IR signaling, and potentiated the radiation-induced increases both in the number of apoptotic cells and in the activity of caspase-3. Immunofluorescence analysis of the histone γ-H2AX and Rad51 also showed that CP-751,871 inhibited the repair of radiation-induced DNA double-strand breaks. Finally, combination therapy with CP-751,871 and radiation delayed the growth of NSCLC tumor xenografts in nude mice to a greater extent than did either treatment modality alone.

Conclusions: These results show that CP-751,871 sensitizes NSCLC cells to radiation both in vitro and in vivo, and that this effect of CP-751,871 is likely attributable to the inhibition of DNA repair and enhancement of apoptosis that result from attenuation of IGF-IR signaling. Combined treatment with CP-751,871 and radiation thus warrants further investigation in clinical trials as a potential anticancer strategy. (Clin Cancer Res 2009;15(16):5117–25)
Data from Tumor Cells Circulate in the Peripheral Blood of All Major Carcinomas but not in Healthy Subjects or Patients With Nonmalignant Diseases
Authors: Allard W. Jeffrey , Matera Jeri , Miller M. Craig , Repollet Madeline , Connelly Mark C. , Rao Chandra , Tibbe Arjan G. J. , Uhr Jonathan W. , Terstappen Leon W. M. M.
Published: April 2023

Abstract
Purpose: The purpose of this study was to determine the accuracy, precision, and linearity of the CellSearch system and evaluate the number of circulating tumor cells (CTCs) per 7.5 mL of blood in healthy subjects, patients with nonmalignant diseases, and patients with a variety of metastatic carcinomas.

Experimental Design: The CellSearch system was used to enumerate CTCs in 7.5 mL of blood. Blood samples spiked with cells from tumor cell lines were used to establish analytical accuracy, reproducibility, and linearity. Prevalence of CTCs was determined in blood from 199 patients with nonmalignant diseases, 964 patients with metastatic carcinomas, and 145 healthy donors.

Results: Enumeration of spiked tumor cells was linear over the range of 5 to 1,142 cells, with an average recovery of ≥85% at each spike level. Only 1 of the 344 (0.3%) healthy and nonmalignant disease subjects had ≥2 CTCs per 7.5 mL of blood. In 2,183 blood samples from 964 metastatic carcinoma patients, CTCs ranged from 0 to 23,618 CTCs per 7.5 mL (mean, 60 ± 693 CTCs per 7.5 mL), and 36% (781 of 2,183) of the specimens had ≥2 CTCs. Detection of ≥2 CTCs occurred at the following rates: 57% (107 of 188) of prostate cancers, 37% (489 of 1,316) of breast cancers, 37% (20 of 53) of ovarian cancers, 30% (99 of 333) of colorectal cancers, 20% (34 of 168) of lung cancers, and 26% (32 of 125) of other cancers.

Conclusions: The CellSearch system can be standardized across multiple laboratories and may be used to determine the clinical utility of CTCs. CTCs are extremely rare in healthy subjects and patients with nonmalignant diseases but present in various metastatic carcinomas with a wide range of frequencies.
Data from Mitochondrial Mutations Contribute to HIF1α Accumulation via Increased Reactive Oxygen Species and Up-regulated Pyruvate Dehydrogenease Kinase 2 in Head and Neck Squamous Cell Carcinoma
Authors: Sun Wenyue , Zhou Shaoyu , Chang Steven S. , McFate Thomas , Verma Ajay , Califano Joseph A.
Published: April 2023

Abstract
Purpose: Mitochondrial mutations have been identified in head and neck squamous cell carcinoma (HNSCC), but the pathways by which phenotypic effects of these mutations are exerted remain unclear. Previously, we found that mitochondrial ND2 mutations in primary HNSCC increased reactive oxygen species (ROS) and conferred an aerobic, glycolytic phenotype with HIF1α accumulation and increased cell growth. The purpose of the present study was to examine the pathways relating these alterations.

Experimental Design: Mitochondrial mutant and wild-type ND2 constructs were transfected into oral keratinocyte immortal cell line OKF6 and head and neck cancer cell line JHU-O19 and established transfectants. The protein levels of HIF1α, pyruvate dehydrogenease (PDH), phosphorylated PDH, and pyruvate dehydrogenease kinase 2 (PDK2), together with ROS generation, were compared between the mutant and the wild type. Meanwhile, the effects of small molecule inhibitors targeting PDK2 and mitochondria-targeted catalase were evaluated on the ND2 mutant transfectants.

Results: We determined that ND2 mutant down-regulated PDH expression via up-regulated PDK2, with an increase in phosphorylated PDH. Inhibition of PDK2 with dichloroacetate decreased HIF1α accumulation and reduced cell growth. Extracellular treatment with hydrogen peroxide, a ROS mimic, increased PDK2 expression and HIF1α expression, and introduction of mitochondria-targeted catalase decreased mitochondrial mutation-mediated PDK2 and HIF1α expression and suppressed cell growth.

Conclusions: Our findings suggest that mitochondrial ND2 mutation contributes to HIF1α accumulation via increased ROS production, up-regulation of PDK2, attenuating PDH activity, thereby increasing pyruvate, resulting in HIF1α stabilization. This may provide insight into a potential mechanism, by which mitochondrial mutations contribute to HNSCC development.
Data from Reveromycin A Inhibits Osteolytic Bone Metastasis of Small-Cell Lung Cancer Cells, SBC-5, through an Antiosteoclastic Activity
Authors: Muguruma Hiroaki , Yano Seiji , Kakiuchi Soji , Uehara Hisanori , Kawatani Makoto , Osada Hiroyuki , Sone Saburo
Published: April 2023

Abstract
Purpose: The purpose of this study was to determine therapeutic effect of a novel antibiotic, reveromycin A, against osteolytic bone metastasis of human small cell lung cancer (SBC-5) cells.

Results: Reveromycin A induced apoptosis specifically in osteoclasts in vitro. Although reveromycin A did not inhibit SBC-5 cell proliferation, it suppressed the expression of parathyroid hormone–related peptide. Intravenous inoculation of SBC-5 cells in natural killer cell–depleted severe combined immunodeficient mice produced experimental metastases in multiple organs, including the bone. Daily administration of reveromycin A inhibited the bone metastasis, but not visceral metastasis, in a dose-dependent manner. Histologic analyses revealed that although treatment with reveromycin A did not affect the number of proliferating tumor cells, it decreased the number of osteoclasts and increased apoptotic cells in bone lesions.

Conclusions: These findings suggest that reveromycin A may inhibit osteolytic bone metastasis through suppression of osteoclast activity by directly inducing apoptosis and indirectly inhibiting tumor cell–derived parathyroid hormone–related peptide production. Therefore, reveromycin A may be a novel, potent therapeutic agent against osteolytic bone metastasis of lung cancer in humans.
Data from System-Level Analysis of Neuroblastoma Tumor–Initiating Cells Implicates AURKB as a Novel Drug Target for Neuroblastoma
Authors: Morozova Olena , Vojvodic Milijana , Grinshtein Natalie , Hansford Loen M. , Blakely Kim M. , Maslova Alexandra , Hirst Martin , Cezard Timothee , Morin Ryan D. , Moore Richard , Smith Kristen M. , Miller Freda , Taylor Paul , Thiessen Nina , Varhol Richard , Zhao Yongjun , Jones Steven , Moffat Jason , Kislinger Thomas , Moran Michael F. , Kaplan David R. , Marra Marco A.
Published: April 2023

Abstract
Purpose: Neuroblastoma (NB) is an aggressive tumor of the developing peripheral nervous system that remains difficult to cure in the advanced stages. The poor prognosis for high-risk NB patients is associated with common disease recurrences that fail to respond to available therapies. NB tumor-initiating cells (TICs), isolated from metastases and primary tumors, may escape treatment and contribute to tumor relapse. New therapies that target the TICs may therefore prevent or treat tumor recurrences.

Experimental Design: We undertook a system-level characterization of NB TICs to identify potential drug targets against recurrent NB. We used next-generation RNA sequencing and/or human exon arrays to profile the transcriptomes of 11 NB TIC lines from six NB patients, revealing genes that are highly expressed in the TICs compared with normal neural crest-like cells and unrelated cancer tissues. We used gel-free two-dimensional liquid chromatography coupled to shotgun tandem mass spectrometry to confirm the presence of proteins corresponding to the most abundant TIC-enriched transcripts, thereby providing validation to the gene expression result.

Results: Our study revealed that genes in the BRCA1 signaling pathway are frequently misexpressed in NB TICs and implicated Aurora B kinase as a potential drug target for NB therapy. Treatment with a selective AURKB inhibitor was cytotoxic to NB TICs but not to the normal neural crest-like cells.

Conclusion: This work provides the first high-resolution system-level analysis of the transcriptomes of 11 primary human NB TICs and identifies a set of candidate NB TIC-enriched transcripts for further development as therapeutic targets. Clin Cancer Res; 16(18); 4572–82. ©2010 AACR.
Data from Global Gene Expression Profile of Nasopharyngeal Carcinoma by Laser Capture Microdissection and Complementary DNA Microarrays
Authors: Sriuranpong Virote , Mutirangura Apiwat , Gillespie John W. , Patel Vyomesh , Amornphimoltham Panomwat , Molinolo Alfredo A. , Kerekhanjanarong Veerachai , Supanakorn Siripornchai , Supiyaphun Pakpoom , Rangdaeng Samreung , Voravud Narin , Gutkind J. Silvio
Published: April 2023

Abstract
A number of genetic and epigenetic changes underlying the development of nasopharyngeal carcinomas have recently been identified. However, there is still limited information on the nature of the genes and gene products whose aberrant expression and activity promote the malignant conversion of nasopharyngeal epithelium. Here, we have performed a genome-wide transcriptome analysis by probing cDNA microarrays with fluorescent-labeled amplified RNA derived from laser capture microdissected cells procured from normal nasopharyngeal epithelium and areas of metaplasia-dysplasia and carcinoma from EBV-associated nasopharyngeal carcinomas. This approach enabled the identification of genes differentially expressed in each cell population, as well as numerous genes whose expression can help explain the aggressive clinical nature of this tumor type. For example, genes indicating cell cycle aberrations (cyclin D2, cyclin B1, activator of S-phase kinase, and the cell cycle checkpoint kinase, CHK1) and invasive-metastatic potential (matrix metalloproteinase 11, v-Ral, and integrin β4) were highly expressed in tumor cells. In contrast, genes underexpressed in tumors included genes involved in apoptosis (B-cell CLL/lymphoma 6, secretory leukocyte protease inhibitor, and calpastatin), cell structure (keratin 7 and carcinoembryonic antigen-related cell adhesion molecule 6), and putative tumor suppressor genes (H-Ras-like suppressor 3, retinoic acid receptor responder 1, and growth arrested specific 8) among others. Gene expression patterns also suggested alterations in the Wnt/β-catenin and transforming growth factor β pathways in nasopharyngeal carcinoma. Thus, expression profiles indicate that aberrant expression of growth, survival, and invasion-promoting genes may contribute to the molecular pathogenesis of nasopharyngeal carcinoma. Ultimately, this approach may facilitate the identification of clinical useful markers of disease progression and novel potential therapeutic targets for nasopharyngeal carcinoma.
Data from A Proof-of-Principle Clinical Trial of Bexarotene in Patients with Non–Small Cell Lung Cancer
Authors: Dragnev Konstantin H. , Petty W. Jeffrey , Shah Sumit J. , Lewis Lionel D. , Black Candice C. , Memoli Vincent , Nugent William C. , Hermann Thomas , Negro-Vilar Andres , Rigas James R. , Dmitrovsky Ethan
Published: April 2023

Abstract
Purpose: Bexarotene is a rexinoid (selective retinoid X receptor agonist) that affects proliferation, differentiation, and apoptosis in preclinical studies. The relationship between bexarotene levels and biomarker changes in tumor tissues has not been previously studied.

Experimental Design: BEAS-2B human bronchial epithelial (HBE) cells, retinoid-resistant BEAS-2B-R1 cells, A427, H226, and H358 lung cancer cells were treated with bexarotene. Proliferation and biomarker expression were assessed. In a proof-of-principle clinical trial, bexarotene tumor tissue levels and intratumoral pharmacodynamic effects were assessed in patients with stages I to II non–small cell lung cancer. Bexarotene (300 mg/m2/day) was administered p.o. for 7 to 9 days before resection.

Results: Bexarotene-induced dosage-dependent repression of growth, cyclin D1, cyclin D3, total epidermal growth factor receptor (EGFR), and phospho-EGFR expression in BEAS-2B, BEAS-2B-R1, A427, and H358, but not H226 cells. Twelve patients were enrolled, and 10 were evaluable. Bexarotene treatment was well tolerated. There was nonlinear correlation between plasma and tumor bexarotene concentrations (r2 = 0.77). Biomarker changes in tumors were observed: repression of cyclin D1, total EGFR and proliferation in one case; repression of cyclin D3, total and phospho-EGFR in another. The cases with multiple biomarker changes had high tumor bexarotene (107-159 ng/g). A single biomarker change was detected in one case with low tumor bexarotene.

Conclusion: Bexarotene represses proliferation and biomarker expression in responsive, but not resistant HBE and lung cancer cells. Similar biomarker changes occur in lung tumors when therapeutic intratumoral bexarotene levels are achieved. This proof-of-principle trial approach is useful to uncover pharmacodynamic mechanisms in vivo and relate these to intratumoral pharmacokinetic effects.
Data from Loss and Reduction of Fus1 Protein Expression is a Frequent Phenomenon in the Pathogenesis of Lung Cancer
Authors: Prudkin Ludmila , Behrens Carmen , Liu Diane D. , Zhou Xian , Ozburn Natalie C. , Bekele B. Nebiyou , Minna John D. , Moran Cesar , Roth Jack A. , Ji Lin , Wistuba Ignacio I.
Published: April 2023

Abstract
Purpose:FUS1, a novel tumor-suppressor gene located in the chromosome 3p21.3 region, may play an important role in lung cancer development. Currently, FUS1-expressing nanoparticles have been developed for treating patients with lung cancer. However, the expression of Fus1 protein has not been examined in a large series of lung cancers and their sequential preneoplastic lesions.

Experimental Design: Using tissue microarrays, we examined Fus1 immunohistochemical expression in 281 non–small cell lung carcinoma (NSCLC) and 22 small cell lung carcinoma tissue specimens and correlated the findings with patients' clinicopathologic features. To investigate the expression of Fus1 in the early sequential pathogenesis of NSCLC, we studied Fus1 expression in 211 histologically normal and mildly abnormal bronchial epithelia, and 118 bronchial and alveolar preneoplastic lesions obtained from patients with lung cancer.

Results: Loss and reduction of expression was detected in 82% of NSCLCs and 100% of small cell lung carcinomas. In NSCLCs, loss of Fus1 immunohistochemical expression was associated with significantly worse overall survival. Bronchial squamous metaplastic and dysplastic lesions expressed significantly lower levels of Fus1 compared with normal (P = 0.014 and 0.047, respectively) and hyperplastic (P = 0.013 and 0.028, respectively) epithelia.

Conclusions: Our findings show a high frequency of Fus1 protein loss and reduction of expression in lung cancer, and suggests that this reduction may play an important role in the early pathogenesis of lung squamous cell carcinoma. These findings support the concept that FUS1 gene and Fus1 protein abnormalities could be used to develop new strategies for molecular cancer therapy for a significant subset of lung tumors.
Data from Enhanced In vitro and In vivo Cytotoxicity of Combined Reovirus and Radiotherapy
Authors: Twigger Katie , Vidal Laura , White Christine L. , De Bono Johann S. , Bhide Shreerang , Coffey Matt , Thompson Brad , Vile Richard G. , Heinemann Lucy , Pandha Hardev S. , Errington Fiona , Melcher Alan A. , Harrington Kevin J.
Published: April 2023

Abstract
Purpose: To test combination treatment schedules of reovirus and radiation in human and murine tumor cells in vitro and in vivo.

Experimental Design:In vitro cytotoxicity and cell cycle effects of reovirus given alone and combined with radiotherapy were assessed by colorimetric, tissue culture infectious dose 50, and fluorescence-activated cell sorting–based assays. Interactions between the agents were evaluated using combination index analysis. The effect of different schedules of reovirus and radiotherapy on viral replication and cytotoxicity was tested in vitro and the combination was assessed in three tumor models in vivo.

Results: Characterization of reovirus cytotoxicity in a panel of cell lines yielded a range of sensitivities. Combined reovirus and radiotherapy yielded statistically significantly increased cytotoxicity, particularly in cell lines with moderate susceptibility to reovirus alone. The enhanced cytotoxicity of the combination occurred independently of treatment sequence or schedule. Radiation did not affect viral replication and only reduced reoviral cytotoxicity after clinically irrelevant single doses (>50 Gy). Combination index analysis revealed synergy between radiation (3-10 Gy) and reovirus at multiplicities of infection between 0.001 and 1. Combination treatment significantly increased apoptosis in tumor cells relative to either single-agent treatment. In vivo studies using xenograft and syngeneic tumors showed enhanced activity of the combination relative to reovirus or radiation alone (P < 0.001).

Conclusions: Combining reovirus and radiotherapy synergistically enhances cytotoxicity in a variety of tumor cells in vitro and in vivo. These results offer strong support for translational clinical trials of reovirus plus radiotherapy that have been initiated in the clinic.