bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from Epigenetic Silencing of MicroRNA-34b/c Plays an Important Role in the Pathogenesis of Malignant Pleural Mesothelioma
Authors: Kubo Takafumi , Toyooka Shinichi , Tsukuda Kazunori , Sakaguchi Masakiyo , Fukazawa Takuya , Soh Junichi , Asano Hiroaki , Ueno Tsuyoshi , Muraoka Takayuki , Yamamoto Hiromasa , Nasu Yasutomo , Kishimoto Takumi , Pass Harvey I. , Matsui Hideki , Huh Nam-ho , Miyoshi Shinichiro
Published: April 2023

Abstract
Purpose: Malignant pleural mesothelioma (MPM) is an aggressive tumor with a dismal prognosis. Unlike other malignancies, TP53 mutations are rare in MPM. Recent studies have showed that altered expression of microRNA (miRNA) is observed in human malignant tumors. In this study, we investigated the alterations of miR-34s, a direct transcriptional target of TP53, and the role of miR-34s on the pathogenesis of MPM.

Experimental Design: Aberrant methylation and expression of miR-34s were examined in MPM cell lines and tumors. miR-34b/c was transfected to MPM cells to estimate the protein expression, cell proliferation, invasion, and cell cycle.

Results: Aberrant methylation was present in 2 (33.3%) of 6 MPM cell lines and 13 (27.7%) of 47 tumors in miR-34a and in all 6 MPM cell lines (100%) and 40 (85.1%) of 47 tumors in miR-34b/c. Expression of miR-34a and 34b/c in all methylated cell lines was reduced and restored with 5-aza-2′-deoxycytidine treatment. Because epigenetic silencing was the major event in miR-34b/c, we investigated the functional role of miR-34b/c in MPM. miR-34b/c–transfected MPM cells with physiologic miR-34b/c expression exhibited antiproliferation with G1 cell cycle arrest and suppression of migration, invasion, and motility. The forced overexpression of miR-34b/c, but not p53, showed a significant antitumor effect with the induction of apoptosis in MPM cells.

Conclusions: We show that the epigenetic silencing of miR-34b/c by methylation is a crucial alteration and plays an important role in the tumorigenesis of MPM, suggesting potential therapeutic options for MPM. Clin Cancer Res; 17(15); 4965–74. ©2011 AACR.
Data from Inhibition of the Notch-Hey1 Axis Blocks Embryonal Rhabdomyosarcoma Tumorigenesis
Authors: Belyea Brian C. , Naini Sarasija , Bentley Rex C. , Linardic Corinne M.
Published: April 2023

Abstract
Purpose: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and remains refractory to combined-modality therapy in patients with high risk disease. In skeletal myogenesis, Notch signaling prevents muscle differentiation and promotes proliferation of satellite cell progeny. Given its physiologic role in myogenesis and oncogenic role in other human cancers, we hypothesized that aberrant Notch signaling may contribute to RMS tumorigenesis and present novel therapeutic opportunities.

Experimental Design: Human RMS cell lines and tumors were evaluated by immunoblot, IHC, and RT-PCR to measure Notch ligand, receptor, and target gene expression. Manipulation of Notch signaling was accomplished using genetic and pharmacologic approaches. In vitro cell growth, proliferation, and differentiation were assessed using colorimetric MTT and BrdU assays, and biochemical/morphologic changes after incubation in differentiation-promoting media, respectively. In vivo tumorigenesis was assessed using xenograft formation in SCID/beige mice.

Results: Notch signaling is upregulated in human RMS cell lines and tumors compared with primary skeletal muscle, especially in the embryonal (eRMS) subtype. Inhibition of Notch signaling using Notch1 RNAi or γ-secretase inhibitors reduced eRMS cell proliferation in vitro. Hey1 RNAi phenocopied Notch1 loss and permitted modest myogenic differentiation, while overexpression of an activated Notch moiety, ICN1, promoted eRMS cell proliferation and rescued pharmacologic inhibition. Finally, Notch inhibition using RNAi or γ-secretase inhibitors blocked tumorigenesis in vivo.

Conclusions: Aberrant Notch-Hey1 signaling contributes to eRMS by impeding differentiation and promoting proliferation. The efficacy of Notch pathway inhibition in vivo supports the development of Notch-Hey1 axis inhibitors in the treatment of eRMS. Clin Cancer Res; 17(23); 7324–36. ©2011 AACR.
Data from Phase I/II and Pharmacodynamic Study of Dovitinib (TKI258), an Inhibitor of Fibroblast Growth Factor Receptors and VEGF Receptors, in Patients with Advanced Melanoma
Authors: Kim Kevin B. , Chesney Jason , Robinson Douglas , Gardner Humphrey , Shi Michael M. , Kirkwood John M.
Published: April 2023

Abstract
Purpose: Dovitinib (TKI258) is an orally available inhibitor of fibroblast growth factor (FGF), VEGF, and platelet-derived growth factor receptors. This phase I/II dose–escalation study was conducted to evaluate the safety, pharmacodynamics, and preliminary efficacy of dovitinib in the treatment of advanced melanoma.

Experimental Design: Patients with advanced melanoma resistant or refractory to standard therapies or for whom no standard therapy was available were enrolled. Dovitinib was administered at doses ranging from 200 to 500 mg/d.

Results: Forty-seven patients were enrolled. The most frequently reported adverse events were fatigue (77%; grade ≥3, 28%), diarrhea (77%; grade ≥3, 11%), and nausea (77%; grade ≥3, 9%). Six dose-limiting toxicities were observed in the 400-mg and 500-mg dose cohorts, which consisted of grade 3 nausea, fatigue, and diarrhea and grade 4 fatigue events. The maximum tolerated dose was 400 mg/d. The best tumor response was stable disease, which was observed in 12 patients. Increases in plasma FGF23, VEGF, and placental growth factor and decreases in soluble VEGF receptor 2 were noted during the first cycle of treatment, consistent with FGF receptor (FGFR) and VEGF receptor (VEGFR) inhibition. Dynamic contrast-enhanced MRI analysis showed a dose-dependent decrease in tumor blood flow and vascular permeability with dovitinib therapy. A decrease in FGFR phosphorylation was observed in paired tumor biopsy samples from a patient treated with dovitinib at a dose of 400 mg/d.

Conclusions: At a dose of 400 mg/d, dovitinib showed an acceptable safety profile and limited clinical benefit and inhibited FGFR and VEGFR. Clin Cancer Res; 17(23); 7451–61. ©2011 AACR.
Data from Functional Analysis of the ATM-p53-p21 Pathway in the LRF CLL4 Trial: Blockade at the Level of p21 Is Associated with Short Response Duration
Authors: Lin Ke , Adamson Janet , Johnson Gillian G. , Carter Anthony , Oates Melanie , Wade Rachel , Richards Sue , Gonzalez David , Matutes Estella , Dearden Claire , Oscier David G. , Catovsky Daniel , Pettitt Andrew R.
Published: April 2023

Abstract
Purpose: This study sought to establish whether functional analysis of the ATM-p53-p21 pathway adds to the information provided by currently available prognostic factors in patients with chronic lymphocytic leukemia (CLL) requiring frontline chemotherapy.

Experimental Design: Cryopreserved blood mononuclear cells from 278 patients entering the LRF CLL4 trial comparing chlorambucil, fludarabine, and fludarabine plus cyclophosphamide were analyzed for ATM-p53-p21 pathway defects using an ex vivo functional assay that uses ionizing radiation to activate ATM and flow cytometry to measure upregulation of p53 and p21 proteins. Clinical endpoints were compared between groups of patients defined by their pathway status.

Results: ATM-p53-p21 pathway defects of four different types (A, B, C, and D) were identified in 194 of 278 (70%) samples. The type A defect (high constitutive p53 expression combined with impaired p21 upregulation) and the type C defect (impaired p21 upregulation despite an intact p53 response) were each associated with short progression-free survival. The type A defect was associated with chemoresistance, whereas the type C defect was associated with early relapse. As expected, the type A defect was strongly associated with TP53 deletion/mutation. In contrast, the type C defect was not associated with any of the other prognostic factors examined, including TP53/ATM deletion, TP53 mutation, and IGHV mutational status. Detection of the type C defect added to the prognostic information provided by TP53/ATM deletion, TP53 mutation, and IGHV status.

Conclusion: Our findings implicate blockade of the ATM-p53-p21 pathway at the level of p21 as a hitherto unrecognized determinant of early disease recurrence following successful cytoreduction. Clin Cancer Res; 18(15); 4191–200. ©2012 AACR.
Data from Rebiopsy of Lung Cancer Patients with Acquired Resistance to EGFR Inhibitors and Enhanced Detection of the T790M Mutation Using a Locked Nucleic Acid-Based Assay
Authors: Arcila Maria E. , Oxnard Geoffrey R. , Nafa Khedoudja , Riely Gregory J. , Solomon Stephen B. , Zakowski Maureen F. , Kris Mark G. , Pao William , Miller Vincent A. , Ladanyi Marc
Published: April 2023

Abstract
Background: The epidermal growth factor receptor (EGFR) mutation T790M is reported in approximately 50% of lung cancers with acquired resistance to EGFR inhibitors and is a potential prognostic and predictive biomarker. Its assessment can be challenging due to limited tissue availability and underdetection at low mutant allele levels. Here, we sought to determine the feasibility of tumor rebiopsy and to more accurately assess the prevalence of the T790M using a highly sensitive locked nucleic acid (LNA) PCR/sequencing assay. MET amplification was also analyzed.

Methods: Patients with acquired resistance were rebiopsied and samples were studied for sensitizing EGFR mutations. Positive cases were evaluated for T790M using standard PCR-based methods and a subset were re-evaluated with an LNA-PCR/sequencing method with an analytical sensitivity of approximately 0.1%. MET amplification was assessed by FISH.

Results: Of 121 patients undergoing tissue sampling, 104 (86%) were successfully analyzed for sensitizing EGFR mutations. Most failures were related to low tumor content. All patients (61/61) with matched pretreatment and resistance specimens showed concordance for the original sensitizing EGFR mutation. Standard T790M mutation analysis on 99 patients detected 51(51%) mutants. Retesting of 30 negative patients by the LNA-based method detected 11 additional mutants for an estimated prevalence of 68%. MET was amplified in 11% of cases (4/37).

Conclusions: The re-biopsy of lung cancer patients with acquired resistance is feasible and provides sufficient material for mutation analysis in most patients. Using high sensitivity methods, the T790M is detected in up to 68% of these patients. Clin Cancer Res; 17(5); 1169–80. ©2011 AACR.
Data from Suppression of Skeletal Muscle Turnover in Cancer Cachexia: Evidence from the Transcriptome in Sequential Human Muscle Biopsies
Authors: Gallagher Iain J. , Stephens Nathan A. , MacDonald Alisdair J. , Skipworth Richard J.E. , Husi Holger , Greig Carolyn A. , Ross James A. , Timmons James A. , Fearon Kenneth C.H.
Published: April 2023

Abstract
Purpose: The mechanisms underlying muscle wasting in patients with cancer remain poorly understood, and consequently there remains an unmet clinical need for new biomarkers and treatment strategies.

Experimental Design: Microarrays were used to examine the transcriptome in single biopsies from healthy controls (n = 6) and in paired biopsies [pre-resection baseline (weight-loss 7%) and 8 month post-resection follow-up (disease-free/weight-stable for previous 2 months)] from quadriceps muscle of patients with upper gastrointestinal cancer (UGIC; n = 12).

Results: Before surgery, 1,868 genes were regulated compared with follow-up (false discovery rate, 6%). Ontology analysis showed that regulated genes belonged to both anabolic and catabolic biologic processes with overwhelming downregulation in baseline samples. No literature-derived genes from preclinical cancer cachexia models showed higher expression in baseline muscle. Comparison with healthy control muscle (n = 6) revealed that despite differences in the transcriptome at baseline (941 genes regulated), the muscle of patients at follow-up was similar to control muscle (2 genes regulated). Physical activity (step count per day) did not differ between the baseline and follow-up periods (P = 0.9), indicating that gene expression differences reflected the removal of the cancer rather than altered physical activity levels. Comparative gene expression analysis using exercise training signatures supported this interpretation.

Conclusions: Metabolic and protein turnover–related pathways are suppressed in weight-losing patients with UGIC whereas removal of the cancer appears to facilitate a return to a healthy state, independent of changes in the level of physical activity. Clin Cancer Res; 18(10); 2817–27. ©2012 AACR.
Data from Characterization of a Cleavage Stimulation Factor, 3′ pre-RNA, Subunit 2, 64 kDa (CSTF2) as a Therapeutic Target for Lung Cancer
Authors: Aragaki Masato , Takahashi Koji , Akiyama Hirohiko , Tsuchiya Eiju , Kondo Satoshi , Nakamura Yusuke , Daigo Yataro
Published: April 2023

Abstract
Purpose: This study aims to discover novel biomarkers and therapeutic targets for lung cancers.

Experimental Design: We screened for genes showing elevated expression in the majority of lung cancers by genome-wide gene expression profile analysis of 120 lung cancers obtained by cDNA microarray representing 27,648 genes or expressed sequence tags. In this process, we detected a gene encoding cleavage stimulation factor, 3′ pre-RNA, subunit 2, 64 kDa (CSTF2) as a candidate. Immunohistochemical staining using tissue microarray consisting of 327 lung cancers was applied to examine the expression of CSTF2 protein and its prognostic value. A role of CSTF2 in cancer cell growth was examined by siRNA experiments.

Results: Northern blot and immunohistochemical analyses detected the expression of CSTF2 only in testis among 16 normal tissues. Immunohistochemical analysis using tissue microarray showed an association of strong CSTF2 expression with poor prognosis of patients with non–small cell lung cancer (P = 0.0079), and multivariate analysis showed that CSTF2 positivity is an independent prognostic factor. In addition, suppression of CSTF2 expression by siRNAs suppressed lung cancer cell growth, whereas exogenous expression of CSTF2 promoted growth and invasion of mammalian cells.

Conclusions: CSTF2 is likely to play an important role in lung carcinogenesis and be a prognostic biomarker in the clinic. Clin Cancer Res; 17(18); 5889–900. ©2011 AACR.
Data from Merkel Cell Polyomavirus-Specific CD8+ and CD4+ T-cell Responses Identified in Merkel Cell Carcinomas and Blood
Authors: Iyer Jayasri G. , Afanasiev Olga K. , McClurkan Christopher , Paulson Kelly , Nagase Kotaro , Jing Lichen , Marshak Joshua O. , Dong Lichun , Carter Joseph , Lai Ivy , Farrar Erik , Byrd David , Galloway Denise , Yee Cassian , Koelle David M. , Nghiem Paul
Published: April 2023

Abstract
Purpose: Merkel cell polyomavirus (MCPyV) is prevalent in the general population, integrates into most Merkel cell carcinomas (MCC), and encodes oncoproteins required for MCC tumor growth. We sought to characterize T-cell responses directed against viral proteins that drive this cancer as a step toward immunotherapy.

Experimental Design: Intracellular cytokine cytometry, IFN-γ enzyme-linked immunospot (ELISPOT) assay, and a novel HLA-A*2402–restricted MCPyV tetramer were used to identify and characterize T-cell responses against MCPyV oncoproteins in tumors and blood of MCC patients and control subjects.

Results: We isolated virus-reactive CD8 or CD4 T cells from MCPyV-positive MCC tumors (2 of 6) but not from virus-negative tumors (0 of 4). MCPyV-specific T-cell responses were also detected in the blood of MCC patients (14 of 27) and control subjects (5 of 13). These T cells recognized a broad range of peptides derived from capsid proteins (2 epitopes) and oncoproteins (24 epitopes). HLA-A*2402–restricted MCPyV oncoprotein processing and presentation by mammalian cells led to CD8-mediated cytotoxicity. Virus-specific CD8 T cells were markedly enriched among tumor infiltrating lymphocytes as compared with blood, implying intact T-cell trafficking into the tumor. Although tetramer-positive CD8 T cells were detected in the blood of 2 of 5 HLA-matched MCC patients, these cells failed to produce IFN-γ when challenged ex vivo with peptide.

Conclusions: Our findings suggest that MCC tumors often develop despite the presence of T cells specific for MCPyV T-Ag oncoproteins. The identified epitopes may be candidates for peptide-specific vaccines and tumor- or virus-specific adoptive immunotherapies to overcome immune evasion mechanisms in MCC patients. Clin Cancer Res; 17(21); 6671–80. ©2011 AACR.
Data from Expression of FoxM1 Is Required for the Proliferation of Medulloblastoma Cells and Indicates Worse Survival of Patients
Authors: Priller Markus , Pöschl Julia , Abrão Leticia , von Bueren André O. , Cho Yoon-Jae , Rutkowski Stefan , Kretzschmar Hans A. , Schüller Ulrich
Published: April 2023

Abstract
Purpose: The transcription factor Forkhead box M1 (FoxM1) is a key regulator of cell-cycle progression. It is involved in the development of multiple organs, and we have previously reported on its important role for the mitotic entry of cerebellar granule neuron precursors. Constitutive expression of FoxM1 is required for the growth of multiple cancer types. This study aimed to determine its role in medulloblastoma, the most frequent malignant brain tumor in childhood that can derive from cerebellar granule neuron precursors.

Experimental Design: We evaluated the expression of FoxM1 together with its prognostic value in two independent series of human medulloblastoma samples using immunohistochemistry (n = 43) and gene expression arrays (n = 193). The functional impact of FoxM1 expression was characterized by knockdown experiments in four human medulloblastoma cell lines, and the thiazole antibiotic siomycin A was tested to downregulate FoxM1 and inhibit tumor cell growth.

Results: FoxM1 was highly expressed in all subtypes of medulloblastoma. Importantly, expression levels of FoxM1 significantly correlated with unfavorable clinical outcome in univariate analysis (P = 0.0005), and FoxM1 was identified as an independent prognostic marker by multivariate analysis (P = 0.037). Knockdown of FoxM1 in medulloblastoma cell lines resulted in a significant decrease of cell viability which was caused by a failure in mitotic spindle formation and caspase-dependent mitotic catastrophe. Siomycin A significantly inhibited the expression of FoxM1 and the growth of medulloblastoma cells.

Conclusions: FoxM1 may be used as an additional prognostic marker and may represent a potential novel target to treat patients suffering from medulloblastoma. Clin Cancer Res; 17(21); 6791–801. ©2011 AACR.
Data from DNA Methylation Profiling Defines Clinically Relevant Biological Subsets of Non–Small Cell Lung Cancer
Authors: Walter Kim , Holcomb Thomas , Januario Tom , Du Pan , Evangelista Marie , Kartha Nithya , Iniguez Leonardo , Soriano Robert , Huw Ling , Stern Howard , Modrusan Zora , Seshagiri Somasekar , Hampton Garret M. , Amler Lukas C. , Bourgon Richard , Yauch Robert L. , Shames David S.
Published: April 2023

Abstract
Purpose: Non–small cell lung cancers (NSCLC) comprise multiple distinct biologic groups with different prognoses. For example, patients with epithelial-like tumors have a better prognosis and exhibit greater sensitivity to inhibitors of the epidermal growth factor receptor (EGFR) pathway than patients with mesenchymal-like tumors. Here, we test the hypothesis that epithelial-like NSCLCs can be distinguished from mesenchymal-like NSCLCs on the basis of global DNA methylation patterns.

Experimental Design: To determine whether phenotypic subsets of NSCLCs can be defined on the basis of their DNA methylation patterns, we combined microfluidics-based gene expression analysis and genome-wide methylation profiling. We derived robust classifiers for both gene expression and methylation in cell lines and tested these classifiers in surgically resected NSCLC tumors. We validate our approach using quantitative reverse transcriptase PCR and methylation-specific PCR in formalin-fixed biopsies from patients with NSCLC who went on to fail front-line chemotherapy.

Results: We show that patterns of methylation divide NSCLCs into epithelial-like and mesenchymal-like subsets as defined by gene expression and that these signatures are similarly correlated in NSCLC cell lines and tumors. We identify multiple differentially methylated regions, including one in ERBB2 and one in ZEB2, whose methylation status is strongly associated with an epithelial phenotype in NSCLC cell lines, surgically resected tumors, and formalin-fixed biopsies from patients with NSCLC who went on to fail front-line chemotherapy.

Conclusions: Our data show that patterns of DNA methylation can divide NSCLCs into two phenotypically distinct subtypes of tumors and provide proof of principle that differences in DNA methylation can be used as a platform for predictive biomarker discovery and development. Clin Cancer Res; 18(8); 2360–73. ©2012 AACR.
Data from Expansion of a CD8+PD-1+ Replicative Senescence Phenotype in Early Stage CLL Patients Is Associated with Inverted CD4:CD8 Ratios and Disease Progression
Authors: Nunes Claudia , Wong Ryan , Mason Malcolm , Fegan Chris , Man Stephen , Pepper Chris
Published: April 2023

Abstract
Purpose: Patients with chronic lymphocytic leukemia (CLL) display immune deficiency that is most obvious in advanced stage disease. Here we investigated whether this immune dysfunction plays a pathologic role in the progression of early stage disease patients.

Experimental Design: We carried out eight-color immunophenotyping analysis in a cohort of 110 untreated early stage CLL patients and 22 age-matched healthy donors and correlated our findings with clinical outcome data.

Results: We found a significant reduction in naive CD4+ and CD8+ T cells in CLL patients. Only the CD4+ subset showed significantly increased effector memory cells (TEM and TEMRA) in the whole cohort (P = 0.004 and P = 0.04, respectively). However, patients with inverted CD4:CD8 ratios (52 of 110) showed preferential expansion of the CD8 compartment, with a skewing of CD8+ TEMRA (P = 0.03) coupled with increased percentage of CD57+CD28−CD27− T cells (P = 0.008) and PD-1 positivity (P = 0.027), consistent with a replicative senescence phenotype. Furthermore, inverted CD4:CD8 ratios were associated with shorter lymphocyte doubling time (P = 0.03), shorter time to first treatment (P = 0.03), and reduced progression-free survival (P = 0.005).

Conclusions: Our data show that the emergence of CD8+PD-1+ replicative senescence phenotype in early stage CLL patients is associated with more aggressive clinical disease. Importantly, these findings were independent of tumor cell prognostic markers and could not be accounted for by patient age, changes in regulatory T-cell frequency, or cytomegalovirus serostatus. Clin Cancer Res; 18(3); 678–87. ©2011 AACR.
Data from Role of Apollon in Human Melanoma Resistance to Antitumor Agents That Activate the Intrinsic or the Extrinsic Apoptosis Pathways
Authors: Tassi Elena , Zanon Marina , Vegetti Claudia , Molla Alessandra , Bersani Ilaria , Perotti Valentina , Pennati Marzia , Zaffaroni Nadia , Milella Michele , Ferrone Soldano , Carlo-Stella Carmelo , Gianni Alessandro M. , Mortarini Roberta , Anichini Andrea
Published: April 2023

Abstract
Purpose: To assess the role of Apollon in melanoma resistance to intrinsic and extrinsic pathways of apoptosis and to identify strategies to reduce its expression.

Experimental Design: Apollon expression was assessed in melanoma cells in vitro and in vivo. Apollon modulation and melanoma apoptosis were evaluated by Western blot and/or flow cytometry in response to cytotoxic drugs, mitogen-activated protein/extracellular signal–regulated kinase (MEK)-, BRAFV600E-, and mTOR-specific inhibitors, TRAIL and anti-HLA class II monoclonal antibodies (mAb). Mitochondrial depolarization, caspase activation, apoptosis assays, and gene expression profiling were used to test effects of Apollon silencing, by siRNA, on melanoma response to antitumor agents.

Results: Apollon was constitutively expressed by melanoma cells, in vitro and in vivo, and at higher levels than in benign melanocytic lesions. Melanoma apoptosis correlated significantly with Apollon protein downmodulation in response to cytotoxic drugs, MEK, or BRAFV600E-specific inhibitors. Combinatorial treatment with MEK and mTOR inhibitors and HLA class II ligation, by a specific mAb, promoted Apollon downmodulation and enhanced melanoma apoptosis. Apollon downmodulation induced by antitumor agents was caspase independent, but proteasome dependent. Knockdown of Apollon, by siRNA, triggered apoptosis and/or significantly enhanced melanoma cell death in response to cytotoxic drugs, MEK- and BRAFV600E-specific inhibitors, and soluble or membrane-bound TRAIL. Apollon silencing promoted mitochondrial depolarization and caspase-2, caspase-8, caspase-9, and caspase-3 activation in response to different antitumor agents and altered the profile of genes modulated by MEK or BRAFV600E-specific inhibitors.

Conclusions: Targeting of Apollon may significantly improve melanoma cell death in response to antitumor agents that trigger the intrinsic or the extrinsic apoptosis pathways. Clin Cancer Res; 18(12); 3316–27. ©2012 AACR.
Data from MAPKAP Kinase 2 Overexpression Influences Prognosis in Gastrointestinal Stromal Tumors and Associates with Copy Number Variations on Chromosome 1 and Expression of p38 MAP Kinase and ETV1
Authors: Birner Peter , Beer Andrea , Vinatzer Ursula , Stary Susanne , Höftberger Romana , Nirtl Nadine , Wrba Fritz , Streubel Berthold , Schoppmann Sebastian F.
Published: April 2023

Abstract
Purpose: ETV1 has been proposed to be activated by KIT mutations in gastrointestinal stromal tumors (GIST). The aim of the study was to evaluate the clinical role of ETV1 and associated proteins in GIST.

Experimental Design: Expressions of ETV1, MAPKAP kinase 2 (MAPKAPK2), phosphorylated p38 MAP kinase (pp38), phosphorylated MSK1 (pMSK1), phosphorylated RSK1, COP1, and KIT protein were determined immunohistochemically in 139 GISTs. Sequence analysis of KIT, PDGFRA, and MAPKAPK2 and FISHs of ETV1 as well as chromosomes 1 and 7 were done.

Results: Prominent ETV1 expression was seen in 50% of GISTs, but no correlation with clinical outcome was found. Correlation of ETV1 expression and KIT mutation was seen in 60% of cases. MAPKAPK2 overexpression (n = 62/44.6%) correlated with pp38 expression (P = 0.021, χ2 test) and alterations of chromosome 1 (n = 17, P = 0.024, χ2 test). In one of 20 sequenced cases with high MAKAPK2 expression, a putative damaging MAPKAPK2 gene mutation was found. All relapsing GISTs with very low/low risk according to Fletcher showed high MAPKAPK2 and KIT expression. MAPKAPK2 overexpression was an independent prognostic factor for disease-free survival (P = 0.006, Cox regression).

Conclusion: ETV1 is not universally overexpressed in GIST and seems to also be induced by pathways other than KIT mutation. Nevertheless, its clinical relevance is low. Overexpression of ETV1 inhibitor MAPKAPK2 is associated with shorter survival in GIST, indicating a clinically relevant role of this gene not reported previously. Patients with low-risk GISTs showing MAPKAPK2 overexpression might profit from early adjuvant tyrosine kinase inhibitor therapy. Clin Cancer Res; 18(7); 1879–87. ©2012 AACR.
Data from The HSP90 Inhibitor XL888 Overcomes BRAF Inhibitor Resistance Mediated through Diverse Mechanisms
Authors: Paraiso Kim H.T. , Haarberg H. Eirik , Wood Elizabeth , Rebecca Vito W. , Chen Y. Ann , Xiang Yun , Ribas Antoni , Lo Roger S. , Weber Jeffrey S. , Sondak Vernon K. , John Jobin K. , Sarnaik Amod A. , Koomen John M. , Smalley Keiran S.M.
Published: April 2023

Abstract
Purpose: The clinical use of BRAF inhibitors is being hampered by the acquisition of drug resistance. This study shows the potential therapeutic use of the HSP90 inhibitor (XL888) in six different models of vemurafenib resistance.

Experimental Design: The ability of XL888 to inhibit growth and to induce apoptosis and tumor regression of vemurafenib-resistant melanoma cell lines was shown in vitro and in vivo. A novel mass spectrometry–based pharmacodynamic assay was developed to measure intratumoral HSP70 levels following HSP90 inhibition in melanoma cell lines, xenografts, and melanoma biopsies. Mechanistic studies were carried out to determine the mechanism of XL888-induced apoptosis.

Results: XL888 potently inhibited cell growth, induced apoptosis, and prevented the growth of vemurafenib-resistant melanoma cell lines in 3-dimensional cell culture, long-term colony formation assays, and human melanoma mouse xenografts. The reversal of the resistance phenotype was associated with the degradation of PDGFRβ, COT, IGFR1, CRAF, ARAF, S6, cyclin D1, and AKT, which in turn led to the nuclear accumulation of FOXO3a, an increase in BIM (Bcl-2 interacting mediator of cell death) expression, and the downregulation of Mcl-1. In most resistance models, XL888 treatment increased BIM expression, decreased Mcl-1 expression, and induced apoptosis more effectively than dual mitogen-activated protein–extracellular signal–regulated kinase/phosphoinositide 3-kinase (MEK/PI3K) inhibition.

Conclusions: HSP90 inhibition may be a highly effective strategy at managing the diverse array of resistance mechanisms being reported to BRAF inhibitors and appears to be more effective at restoring BIM expression and downregulating Mcl-1 expression than combined MEK/PI3K inhibitor therapy. Clin Cancer Res; 18(9); 2502–14. ©2012 AACR.
Data from Comprehensive Mapping of p53 Pathway Alterations Reveals an Apparent Role for Both SNP309 and MDM2 Amplification in Sarcomagenesis
Authors: Ito Moriko , Barys Louise , O'Reilly Terence , Young Sophie , Gorbatcheva Bella , Monahan John , Zumstein-Mecker Sabine , Choong Peter F. , Dickinson Ian , Crowe Philip , Hemmings Christine , Desai Jayesh , Thomas David M. , Lisztwan Joanna
Published: April 2023

Abstract
Purpose: Reactivation of p53 tumor suppressor activity in diseases such as soft-tissue sarcoma is considered an attractive means of targeted therapy. By systematically assessing alterations affecting the p53 pathway, we aimed to (a) classify sarcoma subtypes, (b) define a potential role in malignancy, and (c) identify potential patient biomarkers in this heterogeneous disease.

Experimental Design: We have mapped mutational events in a panel of 192 benign or malignant bone and soft-tissue sarcomas. Analyses included TP53 and CDKN2A mutational and SNP status, MDM2 and MDM4 amplification and MDM2 SNP309 status.

Results: We found an inverse relationship between MDM2 amplification and TP53 mutations, with a predominantly wild-type CDKN2A background. A high rate of point mutations in TP53 was observed uniquely in leiomyosarcoma, osteosarcoma, and MFH. Both MDM2 and MDM4 were also amplified in a subtype-specific manner, which was frequently seen as a coamplification event. We have also analyzed the risk allele frequencies for MDM2 SNP309, and show that the G allele was strongly associated with both liposarcomas and MDM2 amplification.

Conclusions: Our data emphasize the critical role of p53 inactivation in sarcomagenesis, whereby different pathway alterations may be related to the heterogeneity of the disease. Moreover, we observed a strong association of malignancy with TP53 mutation, or MDM2 amplification and the presence of a G allele in SNP309, especially in lipoma versus liposarcoma. We propose, therefore, that MDM2 markers along with TP53 sequencing should be considered as patient biomarkers in clinical trials of sarcomas using MDM2 antagonists. Clin Cancer Res; 17(3); 416–26. ©2010 AACR.
Data from Relationship between Quantitative GRB7 RNA Expression and Recurrence after Adjuvant Anthracycline Chemotherapy in Triple-Negative Breast Cancer
Authors: Sparano Joseph A. , Goldstein Lori J. , Childs Barrett H. , Shak Steven , Brassard Diana , Badve Sunil , Baehner Frederick L. , Bugarini Roberto , Rowley Steve , Perez Edith A. , Shulman Lawrence N. , Martino Silvana , Davidson Nancy E. , Kenny Paraic A. , Sledge George W. , Gray Robert
Published: April 2023

Abstract
Purpose: To conduct an exploratory analysis of the relationship between gene expression and recurrence in patients with operable triple-negative breast cancer (TNBC) treated with adjuvant doxorubicin-containing chemotherapy.

Experimental Design: RNA was extracted from archived tumor samples derived from 246 patients with stage I-III TNBC treated with adjuvant doxorubicin-containing chemotherapy, and was analyzed by quantitative reverse transcriptase PCR for a panel of 374 genes. The relationship between gene expression and recurrence was evaluated using weighted Cox proportional hazards model score tests.

Results: Growth factor receptor bound protein 7 (GRB7) was the only gene for which higher expression was significantly associated with increased recurrence in TNBC (Korn's adjusted P value = 0.04). In a Cox proportional hazards model adjusted for clinicopathologic features, higher GRB7 expression was associated with an increased recurrence risk (HR = 2.31; P = 0.04 using the median as the split). The 5-year recurrence rates were 10.5% [95% confidence intervals (CI), 7.8–14.1] in the low and 20.4% (95% CI, 16.5–25.0) in the high GRB7 groups. External validation in other datasets indicated that GRB7 expression was not prognostic in two adjuvant trials including variable systemic therapy, but in two other trials showed that high GBR7 expression was associated with resistance to neoadjuvant doxorubicin and taxane therapy.

Conclusions:GRB7 was associated with an increased risk of recurrence in TNBC, suggesting that GRB7 or GRB7-dependent pathways may serve as potential biomarkers for therapeutic targets. Therapeutic targeting of one or more factors identified which function as interaction nodes or effectors should also be considered. Clin Cancer Res; 17(22); 7194–203. ©2011 AACR.
Data from Membranous Expression of Ectodomain Isoforms of the Epidermal Growth Factor Receptor Predicts Outcome after Chemoradiotherapy of Lymph Node–Negative Cervical Cancer
Authors: Halle Cathinka , Lando Malin , Svendsrud Debbie Hege , Clancy Trevor , Holden Marit , Sundfør Kolbein , Kristensen Gunnar B. , Holm Ruth , Lyng Heidi
Published: April 2023

Abstract
Purpose: We compared the prognostic significance of ectodomain isoforms of the epidermal growth factor receptor (EGFR), which lack the tyrosine kinase (TK) domain, with that of the full-length receptor and its autophosphorylation status in cervical cancers treated with conventional chemoradiotherapy.

Experimental Design: Expression of EGFR isoforms was assessed by immunohistochemistry in a prospectively collected cohort of 178 patients with squamous cell cervical carcinoma, and their detection was confirmed with Western blotting and reverse transcriptase PCR. A proximity ligation immunohistochemistry assay was used to assess EGFR-specific autophosphorylation. Pathways associated with the expression of ectodomain isoforms were studied by gene expression analysis with Illumina beadarrays in 110 patients and validated in an independent cohort of 41 patients.

Results: Membranous expression of ectodomain isoforms alone, without the coexpression of the full-length receptor, showed correlations to poor clinical outcome that were highly significant for lymph node–negative patients (locoregional control, P = 0.0002; progression-free survival, P < 0.0001; disease-specific survival, P = 0.005 in the log-rank test) and independent of clinical variables. The ectodomain isoforms were primarily 60-kD products of alternative EGFR transcripts. Their membranous expression correlated with transcriptional regulation of oncogenic pathways including activation of MYC and MAX, which was significantly associated with poor outcome. This aggressive phenotype of ectodomain EGFR expressing tumors was confirmed in the independent cohort. Neither total nor full-length EGFR protein level, or autophosphorylation status, showed prognostic significance.

Conclusion: Membranous expression of ectodomain EGFR isoforms, and not TK activation, predicts poor outcome after chemoradiotherapy for patients with lymph node–negative cervical cancer. Clin Cancer Res; 17(16); 5501–12. ©2011 AACR.
Data from Extent and Patterns of MGMT Promoter Methylation in Glioblastoma- and Respective Glioblastoma-Derived Spheres
Authors: Sciuscio Davide , Diserens Annie-Claire , van Dommelen Kristof , Martinet Danielle , Jones Greg , Janzer Robert-Charles , Pollo Claudio , Hamou Marie-France , Kaina Bernd , Stupp Roger , Levivier Marc , Hegi Monika E.
Published: April 2023

Abstract
Purpose: Quantitative methylation-specific tests suggest that not all cells in a glioblastoma with detectable promoter methylation of the O6-methylguanine DNA methyltransferase (MGMT) gene carry a methylated MGMT allele. This observation may indicate cell subpopulations with distinct MGMT status, raising the question of the clinically relevant cutoff of MGMT methylation therapy. Epigenetic silencing of the MGMT gene by promoter methylation blunts repair of O6-methyl guanine and has been shown to be a predictive factor for benefit from alkylating agent therapy in glioblastoma.

Experimental Design: Ten paired samples of glioblastoma and respective glioblastoma-derived spheres (GS), cultured under stem cell conditions, were analyzed for the degree and pattern of MGMT promoter methylation by methylation-specific clone sequencing, MGMT gene dosage, chromatin status, and respective effects on MGMT expression and MGMT activity.

Results: In glioblastoma, MGMT-methylated alleles ranged from 10% to 90%. In contrast, methylated alleles were highly enriched (100% of clones) in respective GS, even when 2 MGMT alleles were present, with 1 exception (<50%). The CpG methylation patterns were characteristic for each glioblastoma exhibiting 25% to 90% methylated CpGs of 28 sites interrogated. Furthermore, MGMT promoter methylation was associated with a nonpermissive chromatin status in accordance with very low MGMT transcript levels and undetectable MGMT activity.

Conclusions: In MGMT-methylated glioblastoma, MGMT promoter methylation is highly enriched in GS that supposedly comprise glioma-initiating cells. Thus, even a low percentage of MGMT methylation measured in a glioblastoma sample may be relevant and predict benefit from an alkylating agent therapy. Clin Cancer Res; 17(2); 255–66. ©2010 AACR.
Data from An Antibody–Drug Conjugate Targeting the Endothelin B Receptor for the Treatment of Melanoma
Authors: Asundi Jyoti , Reed Chae , Arca Jennifer , McCutcheon Krista , Ferrando Ronald , Clark Suzanna , Luis Elizabeth , Tien Janet , Firestein Ron , Polakis Paul
Published: April 2023

Abstract
Purpose: To identify and evaluate targets amenable to antibody therapy in melanoma.

Experimental Design: We searched for mRNA transcripts coding for cell-surface proteins with expression patterns similar to that of the melanoma oncogene MITF. One such candidate, the endothelin B receptor (EDNBR), was first analyzed for a functional contribution to tumor growth by conditional induction of shRNA. Second, antibodies were raised to the receptor, conjugated with monomethyl auristatin E, and tested for efficacy against melanoma tumor models generated from cell lines.

Results: Conditional knockdown of the receptor in tumor xenograft models resulted in only a modest impact on tumor growth. A monoclonal antibody reactive with the N-terminal tail of EDNBR was found to internalize rapidly into melanoma cells. When conjugated with monomethyl auristatin E, the antibody–drug conjugate (ADC) showed remarkable efficacy against human melanoma cell lines and xenograft tumor models that was commensurate with levels of receptor expression. Comparative immunohistochemistry revealed a range of EDNBR expression across a panel of human melanomas, with the majority expressing levels equivalent to or greater than that in the models responsive to the ADC.

Conclusion: An ADC targeting the EDNBR is highly efficacious in preclinical models of melanoma. Clin Cancer Res; 17(5); 965–75. ©2011 AACR.
Data from IDH Mutation and Neuroglial Developmental Features Define Clinically Distinct Subclasses of Lower Grade Diffuse Astrocytic Glioma
Authors: Gorovets Daniel , Kannan Kasthuri , Shen Ronglai , Kastenhuber Edward R. , Islamdoust Nasrin , Campos Carl , Pentsova Elena , Heguy Adriana , Jhanwar Suresh C. , Mellinghoff Ingo K. , Chan Timothy A. , Huse Jason T.
Published: April 2023

Abstract
Purpose: Diffuse gliomas represent the most prevalent class of primary brain tumor. Despite significant recent advances in the understanding of glioblastoma [World Health Organization (WHO) IV], its most malignant subtype, lower grade (WHO II and III) glioma variants remain comparatively understudied, especially in light of their notable clinical heterogeneity. Accordingly, we sought to identify and characterize clinically relevant molecular subclasses of lower grade diffuse astrocytic gliomas.

Experimental Design: We conducted multidimensional molecular profiling, including global transcriptional analysis, on 101 lower grade diffuse astrocytic gliomas collected at our own institution and validated our findings using publically available gene expression and copy number data from large independent patient cohorts.

Results: We found that IDH mutational status delineated molecularly and clinically distinct glioma subsets, with IDH mutant (IDH mt) tumors exhibiting TP53 mutations, platelet—derived growth factor receptor (PDGFR)A overexpression, and prolonged survival, and IDH wild-type (IDH wt) tumors exhibiting EGFR amplification, PTEN loss, and unfavorable disease outcome. Furthermore, global expression profiling revealed three robust molecular subclasses within lower grade diffuse astrocytic gliomas, two of which were predominantly IDH mt and one almost entirely IDH wt. IDH mt subclasses were distinguished from each other on the basis of TP53 mutations, DNA copy number abnormalities, and links to distinct stages of neurogenesis in the subventricular zone. This latter finding implicates discrete pools of neuroglial progenitors as cells of origin for the different subclasses of IDH mt tumors.

Conclusion: We have elucidated molecularly distinct subclasses of lower grade diffuse astrocytic glioma that dictate clinical behavior and show fundamental associations with both IDH mutational status and neuroglial developmental stage. Clin Cancer Res; 18(9); 2490–501. ©2012 AACR.
Data from Cytokeratin-19 and Mammaglobin Gene Expression in Circulating Tumor Cells from Metastatic Breast Cancer Patients Enrolled in North Central Cancer Treatment Group Trials, N0234/336/436/437
Authors: Reinholz Monica M. , Kitzmann Kathleen A. , Tenner Kathleen , Hillman David , Dueck Amylou C. , Hobday Timothy J. , Northfelt Donald W. , Moreno-Aspitia Alvaro , Roy Vivek , LaPlant Betsy , Allred Jake B. , Stella Philip J. , Lingle Wilma L. , Perez Edith A.
Published: April 2023

Abstract
Purpose: To investigate the associations between baseline and posttreatment circulating tumor cell (CTC) gene expression and outcome of patients enrolled in four North Central Cancer Treatment Group metastatic breast cancer (MBC) trials in which specimens were shipped (at 4°C) from community-based sites to a reference laboratory (Mayo Clinic, Rochester, MN).

Experimental Design: Blood was collected at treating sites from MBC patients before (baseline), during, and at the end of treatment with erlotinib + gemcitabine (N0234), sorafenib (N0336), irinotecan + cetuximab (N0436), or paclitaxel-poliglumex + capecitabine (N0437). CTCs from 10 mL of EDTA blood were enriched with CD45 depletion, 24 to 30 hours postblood collection. Reverse transcription/quantitative PCR was used to determine cytokeratin-19 (CK19) and mammaglobin (MGB1) mRNA levels in CTCs from up to 13 (N0234), 16 (N0336), 18 (N0436), and 39 (N0437) patients. The gene expressions were normalized to β2-microglobulin and calibrated to healthy blood using the 2–ΔΔCq algorithm; positivity was defined as 2 or more.

Results:CK19+mRNA cells were detected in 56% to 75% and MGB1+mRNA cells in 23% to 38% of 86 patients at baseline. CK19+mRNA cells were detected in 30% to 67% and MGB1+mRNA cells in 14% to 64% of 110 postbaseline serial samples. The presence of baseline CK19+mRNA cells (P = 0.01) but not MGB1+mRNA cells (P = 0.14) was significantly associated with shorter overall survival. A decrease in MGB1+mRNA levels (baseline-week 8) seemed to be associated with clinical response (P = 0.05).

Conclusions: CTC gene expression analysis conducted by a reference laboratory is feasible when blood is collected from treating sites and processed 24 to 30 hours postcollection. The presence of baseline CK19+mRNA CTCs was associated with poor prognosis; a decrease in MGB1+mRNA CTCs may help predict response to therapy of MBC patients. Clin Cancer Res; 17(22); 7183–93. ©2011 AACR.
Data from Connexin 47 Mutations Increase Risk for Secondary Lymphedema Following Breast Cancer Treatment
Authors: Finegold David N. , Baty Catherine J. , Knickelbein Kelly Z. , Perschke Shelley , Noon Sarah E. , Campbell Diana , Karlsson Jenny M. , Huang Diana , Kimak Mark A. , Lawrence Elizabeth C. , Feingold Eleanor , Meriney Stephen D. , Brufsky Adam M. , Ferrell Robert E.
Published: April 2023

Abstract
Purpose: Secondary lymphedema is a frequent complication of breast cancer associated with surgery, chemotherapy, or radiation following breast cancer treatment. The potential contribution of genetic susceptibility to risk of developing secondary lymphedema following surgical trauma, radiation, and other tissue insults has not been studied.

Experimental Design: To determine whether women with breast cancer and secondary lymphedema had mutations in candidate lymphedema genes, we undertook a case–control study of 188 women diagnosed with breast cancer recruited from the University of Pittsburgh Breast Cancer Program (http://www.upmccancercenter.com/breast/index.cfm) between 2000 and 2010.

Candidate lymphedema genes, GJC2 (encoding connexin 47 [Cx47]), FOXC2, HGF, MET, and FLT4 (encoding VEGFR3), were sequenced for mutation. Bioinformatics analysis and in vitro functional assays were used to confirm significance of novel mutations.

Results: Cx47 mutations were identified in individuals having secondary lymphedema following breast cancer treatment but not in breast cancer controls or normal women without breast cancer. These novel mutations are dysfunctional as assessed through in vitro assays and bioinformatics analysis and provide evidence that altered gap junction function leads to lymphedema.

Conclusions: Our findings challenge the view that secondary lymphedema is solely due to mechanical trauma and support the hypothesis that genetic susceptibility is an important risk factor for secondary lymphedema. A priori recognition of genetic risk (i) raises the potential for early detection and intervention for a high-risk group and (ii) allows the possibility of altering surgical approach and/or chemo- and radiation therapy, or direct medical treatment of secondary lymphedema with novel connexin-modifying drugs. Clin Cancer Res; 18(8); 2382–90. ©2012 AACR.