bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from Phase I Study of Eribulin Mesylate Administered Once Every 21 Days in Patients with Advanced Solid Tumors
Authors: Tan Antoinette R. , Rubin Eric H. , Walton Diana C. , Shuster Dale E. , Wong Y. Nancy , Fang Fang , Ashworth Simon , Rosen Lee S.
Published: April 2023

Abstract
Purpose: To evaluate the maximum tolerated dose (MTD), dose-limiting toxicities (DLT), and pharmacokinetics of eribulin mesylate (E7389), a halichondrin B analogue, administered every 21 days in patients with advanced solid tumors.

Experimental Design: Eribulin mesylate was given as a 1-hour infusion every 21 days at doses of 0.25, 0.5, 1, 2, 2.8, and 4 mg/m2. The MTD was identified using an accelerated titration design. The pharmacokinetics of eribulin were evaluated in the plasma and urine with the first dose.

Results: Twenty-one patients were enrolled. At 4 mg/m2, three patients experienced a DLT of febrile neutropenia on day 7. The dose level was reduced to 2.8 mg/m2 where two of three patients experienced dose-limiting febrile neutropenia. Six additional patients were enrolled at 2 mg/m2 (seven patients in total received this dose) and one of these patients experienced a neutropenic DLT. The MTD of eribulin mesylate was therefore 2 mg/m2. Nonhematologic toxicities included alopecia, fatigue, anorexia, and nausea. Pharmacokinetic analysis showed linear kinetics for eribulin over the dose range studied and a terminal half-life of 2 days. The plasma-concentration-time profile exhibited a rapid distribution phase followed by a slow elimination phase. Drug clearance was nonrenal. One patient with non–small cell lung cancer achieved an unconfirmed partial response and 12 patients had stable disease.

Conclusions: Eribulin mesylate administered as a 1-hour infusion every 21 days has a manageable toxicity profile at 2 mg/m2, with further dose escalation limited by neutropenia.
Data from Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Reverses Mesenchymal to Epithelial Phenotype and Inhibits Metastasis in Inflammatory Breast Cancer
Authors: Zhang Dongwei , LaFortune Tiffany A. , Krishnamurthy Savitri , Esteva Francisco J. , Cristofanilli Massimo , Liu Ping , Lucci Anthony , Singh Balraj , Hung Mien-Chie , Hortobagyi Gabriel N. , Ueno Naoto T.
Published: April 2023

Abstract
Purpose: Inflammatory breast cancer (IBC) is a rare but aggressive type of advanced breast cancer. Epidermal growth factor receptor (EGFR) expression is an independent poor prognostic factor in IBC. The purpose of this study was to determine the effect on IBC tumorigenicity and metastasis of blocking the EGFR pathway.

Experimental Design: IBC cell lines, which express high level of EGFR, were treated with EGFR small interfering RNA and with the EGFR tyrosine kinase inhibitor erlotinib. The role of EGFR in IBC cell proliferation, motility, invasiveness, and change of the expression levels of epithelial-mesenchymal transition markers was examined. The role of extracellular signal–regulated kinase (ERK)-1/2 in erlotinib activity was also studied. The activity of erlotinib in tumor growth and metastasis was examined in an orthotopic xenograft model of IBC.

Results: Erlotinib inhibited proliferation and anchorage-independent growth of IBC cells, and this inhibition was ERK dependent. Erlotinib inhibited cell motility and invasiveness and reversed the mesenchymal phenotype of IBC cells to epithelial phenotype in three-dimensional culture. Erlotinib dramatically inhibited IBC tumor growth in a xenograft model. Interestingly, erlotinib inhibited spontaneous lung metastasis, even at a low dose that had no significant effect on primary tumor growth. These erlotinib-treated tumors were converted to epithelial phenotype from mesenchymal phenotype.

Conclusions: The EGFR pathway is involved in tumor growth and metastasis of IBC. Targeting EGFR through the ERK pathway may represent an effective therapeutic approach to suppress tumorigenicity and prevent metastasis in EGFR-expressing IBC. (Clin Cancer Res 2009;15(21):6639–48)
Data from Clinical Benefit of Allogeneic Melanoma Cell Lysate–Pulsed Autologous Dendritic Cell Vaccine in MAGE-Positive Colorectal Cancer Patients
Authors: Toh Han Chong , Wang Who-Whong , Chia Whay Kuang , Kvistborg Pia , Sun Li , Teo Kelly , Phoon Yee Peng , Soe Yatanar , Tan Sze Huey , Hee Siew Wan , Foo Kian Fong , Ong Simon , Koo Wen Hsin , Zocca Mai-Britt , Claesson Mogens H.
Published: April 2023

Abstract
Purpose: We evaluated the clinical benefit of an allogeneic melanoma cell lysate (MCL)–pulsed autologous dendritic cell (DC) vaccine in advanced colorectal cancer patients expressing at least one of six MAGE-A antigens overexpressed by the cell line source of the lysate.

Experimental Design: DCs were cultured from peripheral blood mononuclear cells (PBMC), pulsed with the allogeneic MCL, and matured using cytokines that achieved high CD83- and CCR7-expressing DCs. Each patient received up to 10 intradermal vaccinations (3-5 × 106 cells per dose) at biweekly intervals.

Results: Twenty patients received a total of 161 vaccinations. Treatment was well tolerated and quality of life measurements did not vary much across time. One patient experienced partial response [5%; 95% confidence interval (CI), 1-24%] and seven achieved stable disease (35%; 95% CI, 18-57%), one of whom also achieved late tumor regression, yielding a clinical benefit response rate of 40% (95% CI, 22-61%). Although overall median progression-free survival was 2.4 months (95% CI, 1.9-4.1 months), five patients (25%) experienced prolonged progression-free survival (>6 months), two of whom (10%) remain progression-free for >27 and >37 months, respectively. This result is particularly meaningful as all patients had progressive disease before treatment. Overall, DC vaccination was associated with a serial decline in regulatory T cells. Using an antibody array, we characterized plasma protein profiles in responding patients that may correlate with vaccine activity and report a prevaccination protein signature distinguishing responders from nonresponders.

Conclusion: This phase II vaccine study using mature, MCL-pulsed DCs has shown promising results and warrants further evaluation in a prospective randomized setting. (Clin Cancer Res 2009;15(24):7726–36)
Data from Validation of SAG/RBX2/ROC2 E3 Ubiquitin Ligase as an Anticancer and Radiosensitizing Target
Authors: Jia Lijun , Yang Jie , Hao Xinbao , Zheng Min , He Hongbin , Xiong Xiufang , Xu Liang , Sun Yi
Published: April 2023

Abstract
Purpose: Sensitive to apoptosis gene (SAG; also known as RBX2 or ROC2) was originally cloned as a redox-inducible antioxidant protein and was later characterized as a RING component of SCF E3 ubiquitin ligases. SAG overexpression inhibits apoptosis induced by many stimuli both in vitro and in vivo. SAG mRNA was overexpressed in human lung tumor tissues with a correlation to poor patient survival. To investigate whether SAG serves as an anticancer target, we determined the effect of SAG silencing on cell proliferation, survival, and radiosensitivity.

Experimental Design: SAG protein expression in human tumors was evaluated by immunohistochemical staining using tumor tissue arrays. SAG expression in cancer cells was knocked down by siRNA silencing. The anticancer effects of SAG silencing were evaluated by in vitro assays for cell growth and survival and by an in vivo orthotopic xenograft tumor model. Radiosensitization by SAG silencing of human cancer cells was determined by clonogenic survival assay. Apoptosis induction was evaluated by fluorescence-activated cell sorting analysis, caspase-3 activation assay, and Western blotting of apoptosis-associated proteins.

Results: SAG was overexpressed in multiple human tumor tissues compared with their normal counterparts. SAG silencing selectively inhibited cancer cell proliferation, suppressed in vivo tumor growth, and sensitized radiation-resistant cancer cells to radiation. Mechanistically, SAG silencing induced apoptosis with accumulation of NOXA, whereas SAG overexpression reduced NOXA levels and shortened NOXA protein half-life.

Conclusions: The findings showed that SAG E3 ubiquitin ligase plays an essential role in cancer cell proliferation and tumor growth and may serve as a promising anticancer and radiosensitizing target. Clin Cancer Res; 16(3); 814–24
Data from Mutation-Specific Antibodies for the Detection of EGFR Mutations in Non–Small-Cell Lung Cancer
Authors: Yu Jian , Kane Susan , Wu Jiong , Benedettini Elisa , Li Daiqiang , Reeves Cynthia , Innocenti Gregory , Wetzel Randy , Crosby Katherine , Becker Alison , Ferrante Michelle , Cheung Wan Cheung , Hong Xiqiang , Chirieac Lucian R. , Sholl Lynette M. , Haack Herbert , Smith Bradley L. , Polakiewicz Roberto D. , Tan Yi , Gu Ting-Lei , Loda Massimo , Zhou Xinmin , Comb Michael J.
Published: April 2023

Abstract
Purpose: Activating mutations within the tyrosine kinase domain of epidermal growth factor receptor (EGFR) are found in approximately 10% to 20% of non–small-cell lung cancer (NSCLC) patients and are associated with response to EGFR inhibitors. The most common NSCLC-associated EGFR mutations are deletions in exon 19 and L858R mutation in exon 21, together accounting for 90% of EGFR mutations. To develop a simple, sensitive, and reliable clinical assay for the identification of EGFR mutations in NSCLC patients, we generated mutation-specific rabbit monoclonal antibodies against each of these two most common EGFR mutations and aimed to evaluate the detection of EGFR mutations in NSCLC patients by immunohistochemistry.

Experimental Design: We tested mutation-specific antibodies by Western blot, immunofluorescence, and immunohistochemistry. In addition, we stained 40 EGFR genotyped NSCLC tumor samples by immunohistochemistry with these antibodies. Finally, with a panel of four antibodies, we screened a large set of NSCLC patient samples with unknown genotype and confirmed the immunohistochemistry results by DNA sequencing.

Results: These two antibodies specifically detect the corresponding mutant form of EGFR by Western blotting, immunofluorescence, and immunohistochemistry. Screening a panel of 340 paraffin-embedded NSCLC tumor samples with these antibodies showed that the sensitivity of the immunohistochemistry assay is 92%, with a specificity of 99% as compared with direct and mass spectrometry–based DNA sequencing.

Conclusions: This simple assay for detection of EGFR mutations in diagnostic human tissues provides a rapid, sensitive, specific, and cost-effective method to identify lung cancer patients responsive to EGFR-based therapies.
Data from Dominant Effect of Antiangiogenesis in Combination Therapy Involving Cyclophosphamide and Axitinib
Authors: Ma Jie , Waxman David J.
Published: April 2023

Abstract
Purpose: Antiangiogenic drug treatment inhibits tumor growth by decreasing blood supply, which can also reduce the delivery of other therapeutic agents. Presently, we investigated the effect of the vascular endothelial growth factor receptor tyrosine kinase inhibitor axitinib (AG-013736) on tumor vascular patency and chemotherapeutic drug uptake. Furthermore, the effect of axitinib on the antitumor activity of combination treatments with cyclophosphamide was examined.

Experimental Design: Prostate cancer PC-3 xenografts were used to evaluate the effect of axitinib treatment on tumor vascular morphology, fluorescent dye perfusion, hypoxia, and uptake of 4-hydroxycyclophosphamide, the active metabolite of the chemotherapeutic prodrug cyclophosphamide. Sequential or simultaneous schedules for axitinib and cyclophosphamide administration were evaluated in both PC-3 tumors and 9L gliosarcoma xenograft models.

Results: Axitinib monotherapy induced sustained growth stasis in PC-3 tumors in association with extensive apoptotic cell death. A substantial decrease in tumor vascular patency was observed, exemplified by a near complete loss of Hoechst 33342 perfusion and the absence of pimonidazole staining in the increasingly hypoxic tumors. Antitumor activity was significantly enhanced in both PC-3 and 9L tumors treated using an optimized schedule of sequential, intermittent axitinib-cyclophosphamide combination therapy despite a 40% to 70% decrease in tumor tissue uptake of 4-hydroxycyclophosphamide.

Conclusions: In axitinib-cyclophosphamide combination therapy, enhanced anticancer activity can be achieved when the reduced tumor cell exposure to the cancer chemotherapeutic agent is compensated by antiangiogenesis-induced tumor cell starvation. This intrinsic antitumor effect was particularly evident in PC-3 tumor xenografts, where tumor blood flow deprivation dominates the overall therapeutic response.
Data from A Cohort Study of Cyclin D1 Expression and Prognosis in 602 Colon Cancer Cases
Authors: Ogino Shuji , Nosho Katsuhiko , Irahara Natsumi , Kure Shoko , Shima Kaori , Baba Yoshifumi , Toyoda Saori , Chen Li , Giovannucci Edward L. , Meyerhardt Jeffrey A. , Fuchs Charles S.
Published: April 2023

Abstract
Purpose: Cyclin D1 and cyclin-dependent kinases (CDK) are commonly activated in colorectal cancer. The activity of cyclin D1 can be blocked by CDK inhibitors, including p27 (CDKN1B) and p21 (CDKN1A, which is induced by p53). However, prognostic significance of tumoral cyclin D1 remains uncertain, and no previous study has considered potential confounding effect of p53, p21, p27, and related molecular events [microsatellite instability (MSI), CpG island methylator phenotype, and LINE-1 hypomethylation].

Experimental Design: Among 602 colon cancer patients (stage I-IV) in two prospective cohort studies, cyclin D1 overexpression was detected in 330 (55%) tumors by immunohistochemistry. Cox proportional hazard models computed hazard ratios (HR) of colon cancer–specific and overall mortalities, adjusted for patient characteristics and tumoral molecular features, including p53, p21, p27, cyclooxygenase-2, fatty acid synthase, LINE-1 methylation, CpG island methylator phenotype, MSI, BMI, KRAS, and BRAF.

Results: Cyclin D1 overexpression was associated with a low cancer-specific mortality in Kaplan-Meier analysis (P = 0.006), and in both univariate Cox regression [unadjusted HR, 0.64; 95% confidence interval (CI), 0.47-0.88; P = 0.0063] and multivariate analyses (adjusted HR, 0.57; 95% CI, 0.39-0.84; P = 0.0048). Similar findings were observed for an overall mortality (adjusted HR, 0.74; 95% CI, 0.57-0.98; P = 0.036). Notably, the effect of cyclin D1 on survival might differ by MSI status (Pinteraction = 0.008). Compared with tumors that were both cyclin D1–negative and MSI-low/microsatellite stable, the presence of either cyclin D1 or MSI-high or both seemed to confer better clinical outcome (adjusted HR point estimates, 0.10-0.65).

Conclusions: Cyclin D1 overexpression is associated with longer survival in colon cancer.
Data from Snail Promotes CXCR2 LigandDependent Tumor Progression in NonSmall Cell Lung Carcinoma
Authors: Yanagawa Jane , Walser Tonya C. , Zhu Li X. , Hong Longsheng , Fishbein Michael C. , Mah Vei , Chia David , Goodglick Lee , Elashoff David A. , Luo Jie , Magyar Clara E. , Dohadwala Mariam , Lee Jay M. , St. John Maie A. , Strieter Robert M. , Sharma Sherven , Dubinett Steven M.
Published: April 2023

Abstract
Purpose: As a transcriptional repressor of E-cadherin, Snail has predominantly been associated with epithelial-mesenchymal transition, invasion, and metastasis. However, other important Snail-dependent malignant phenotypes have not been fully explored. Here, we investigate the contributions of Snail to the progression of nonsmall cell lung cancer (NSCLC).

Experimental Design: Immunohistochemistry was done to quantify and localize Snail in human lung cancer tissues, and tissue microarray analysis was used to correlate these findings with survival. NSCLC cell lines gene-modified to stably overexpress Snail were evaluated in vivo in two severe combined immunodeficiency murine tumor models. Differential gene expression between Snail-overexpressing and control cell lines was evaluated using gene expression microarray analysis.

Results: Snail is upregulated in human NSCLC tissue, and high levels of Snail expression correlate with decreased survival (P < 0.026). In a heterotopic model, mice bearing Snail-overexpressing tumors developed increased primary tumor burden (P = 0.008). In an orthotopic model, mice bearing Snail-overexpressing tumors also showed a trend toward increased metastases. In addition, Snail overexpression led to increased angiogenesis in primary tumors as measured by MECA-32 (P < 0.05) positivity and CXCL8 (P = 0.002) and CXCL5 (P = 0.0003) concentrations in tumor homogenates. Demonstrating the importance of these proangiogenic chemokines, the Snail-mediated increase in tumor burden was abrogated with CXCR2 blockade. Gene expression analysis also revealed Snail-associated differential gene expression with the potential to affect angiogenesis and diverse aspects of lung cancer progression.

Conclusion: Snail upregulation plays a role in human NSCLC by promoting tumor progression mediated by CXCR2 ligands. (Clin Cancer Res 2009;15(22):68209)
Data from Release of HMGB1 in Response to Proapoptotic Glioma Killing Strategies: Efficacy and Neurotoxicity
Authors: Candolfi Marianela , Yagiz Kader , Foulad David , Alzadeh Gabrielle E. , Tesarfreund Matthew , Muhammad A.K.M. Ghulam , Puntel Mariana , Kroeger Kurt M. , Liu Chunyan , Lee Sharon , Curtin James F. , King Gwendalyn D. , Lerner Jonathan , Sato Katsuaki , Mineharu Yohei , Xiong Weidong , Lowenstein Pedro R. , Castro Maria G.
Published: April 2023

Abstract
Purpose: In preparation for a phase I clinical trial using a combined cytotoxic/immunotherapeutic strategy with adenoviruses (Ad) expressing Flt3L (Ad-Flt3L) and thymidine kinase (Ad-TK) to treat glioblastoma (GBM), we tested the hypothesis that Ad-TK+GCV would be the optimal tumor-killing agent in relation to efficacy and safety when compared with other proapoptotic approaches.

Experimental Design: The efficacy and neurotoxicity of Ad-TK+GCV was compared with Ads encoding the proapoptotic cytokines [tumor necrosis factor-α, tumor necrosis factor–related apoptosis-inducing factor (TRAIL), and Fas ligand (FasL)], alone or in combination with Ad-Flt3L. In rats bearing small GBMs (day 4), only Ad-TK+GCV or Ad-FasL improved survival.

Results: In rats bearing large GBMs (day 9), the combination of Ad-Flt3L with Ad-FasL did not improve survival over FasL alone, whereas Ad-Flt3L combined with Ad-TK+GCV led to 70% long-term survival. Expression of FasL and TRAIL caused severe neuropathology, which was not encountered when we used Ad-TK+/−Ad-Flt3L. In vitro, all treatments elicited release of high mobility group box 1 protein (HMGB1) from dying tumor cells. In vivo, the highest levels of circulating HMGB1 were observed after treatment with Ad-TK+GCV+Ad-Flt3L; HMGB1 was necessary for the therapeutic efficacy of AdTK+GCV+Ad-Flt3L because its blockade with glycyrrhizin completely blocked tumor regression. We also showed the killing efficacy of Ad-TK+GCV in human GBM cell lines and GBM primary cultures, which also elicited release of HMGB1.

Conclusions: Our results indicate that Ad-TK+GCV+Ad-Flt3L exhibit the highest efficacy and safety profile among the several proapoptotic approaches tested. The results reported further support the implementation of this combined approach in a phase I clinical trial for GBM.
Data from PLK1 Is Transcriptionally Activated by NF-κB during Cell Detachment and Enhances Anoikis Resistance through Inhibiting β-Catenin Degradation in Esophageal Squamous Cell Carcinoma
Authors: Lin De-Chen , Zhang Yu , Pan Qin-Jing , Yang Hai , Shi Zhi-Zhou , Xie Zhi-Hui , Wang Bo-Shi , Hao Jia-Jie , Zhang Tong-Tong , Xu Xin , Zhan Qi-Min , Wang Ming-Rong
Published: April 2023

Abstract
Purpose: To investigate the molecular mechanisms through which polo-like kinase-1 (PLK1) takes part in anoikis resistance of esophageal squamous cell carcinoma (ESCC) cells.

Experimental Design: The role of PLK1 in cell anoikis resistance was examined by ectopic gene expression and siRNA-mediated knockdown. Glutathione S-transferase pull-down and co-immunoprecipitation assays were utilized to investigate PLK1-interacting proteins. Electrophoretic mobility shift assay, chromatin immunoprecipitation, and reporter gene assays were carried out to identify the transcription factors responsible for PLK1 expression during anoikis resistance.

Results: We found that detachment of ESCC cells triggers the upregulation of PLK1. Elevated PLK1 expression contributes to protection against anoikis in cancer cells through the regulation of β-catenin expression. Moreover, we showed that, through direct binding to the PLK1 promoter, the NF-κB subunit RelA transcriptionally activates PLK1, which inhibits the ubiquitination and degradation of β-catenin. Inhibition of the NF-κB pathway restores the sensitivity of cancer cells to anoikis by downregulating PLK1/β-catenin expression. In addition, RelA gene amplification and protein overexpression was significantly correlated with PLK1 expression in ESCC tissues.

Conclusions: Our findings suggest that upregulation of PLK1 triggered by cell detachment is regulated by RelA at the transcriptional level. PLK1 protects esophageal carcinoma cells from anoikis through modulation of β-catenin protein levels by inhibiting their degradation. Taken together, this study reveals critical mechanisms involved in the role of RelA/PLK1/β-catenin in anoikis resistance of ESCC cells. Clin Cancer Res; 17(13); 4285–95. ©2011 AACR.
Data from Aldehyde Dehydrogenase 1–Positive Cancer Stem Cells Mediate Metastasis and Poor Clinical Outcome in Inflammatory Breast Cancer
Authors: Charafe-Jauffret Emmanuelle , Ginestier Christophe , Iovino Flora , Tarpin Carole , Diebel Mark , Esterni Benjamin , Houvenaeghel Gilles , Extra Jean-Marc , Bertucci François , Jacquemier Jocelyne , Xerri Luc , Dontu Gabriela , Stassi Giorgio , Xiao Yi , Barsky Sanford H. , Birnbaum Daniel , Viens Patrice , Wicha Max S.
Published: April 2023

Abstract
Purpose: To examine the role of cancer stem cells (CSC) in mediating metastasis in inflammatory breast cancer (IBC) and the association of these cells with patient outcome in this aggressive type of breast cancer.

Experimental Design: CSCs were isolated from SUM149 and MARY-X, an IBC cell line and primary xenograft, by virtue of increased aldehyde dehydrogenase (ALDH) activity as assessed by the ALDEFLUOR assay. Invasion and metastasis of CSC populations were assessed by in vitro and mouse xenograft assays. Expression of ALDH1 was determined on a retrospective series of 109 IBC patients and this was correlated with histoclinical data. All statistical tests were two sided. Log-rank tests using Kaplan-Meier analysis were used to determine the correlation of ALDH1 expression with development of metastasis and patient outcome.

Results: Both in vitro and xenograft assays showed that invasion and metastasis in IBC are mediated by a cellular component that displays ALDH activity. Furthermore, expression of ALDH1 in IBC was an independent predictive factor for early metastasis and decreased survival in this patient population.

Conclusions: These results suggest that the metastatic, aggressive behavior of IBC may be mediated by a CSC component that displays ALDH enzymatic activity. ALDH1 expression represents the first independent prognostic marker to predict metastasis and poor patient outcome in IBC. The results illustrate how stem cell research can translate into clinical practice in the IBC field. Clin Cancer Res; 16(1); 45–55
Data from Predictors of Resistance to Preoperative Trastuzumab and Vinorelbine for HER2-Positive Early Breast Cancer
Authors: Harris Lyndsay N. , You Fanglei , Schnitt Stuart J. , Witkiewicz Agnes , Lu Xin , Sgroi Dennis , Ryan Paula D. , Come Steven E. , Burstein Harold J. , Lesnikoski Beth-Ann , Kamma Madhavi , Friedman Paula N. , Gelman Rebecca , Iglehart J. Dirk , Winer Eric P.
Published: April 2023

Abstract
Purpose: To assess pathologic complete response (pCR), clinical response, feasibility, safety, and potential predictors of response to preoperative trastuzumab plus vinorelbine in patients with operable, human epidermal growth factor receptor 2 (HER2)–positive breast cancer.

Experimental Design: Forty-eight patients received preoperative trastuzumab and vinorelbine weekly for 12 weeks. Single and multigene biomarker studies were done in an attempt to identify predictors of response.

Results: Eight of 40 (20%) patients achieved pCR (95% confidence interval, 9-36%). Of 9 additional patients recruited for protocol-defined toxicity analysis, 8 were evaluable; 42 of 48 (88%) patients had clinical response (16 patients, clinical complete response; 26 patients, clinical partial response). T1 tumors more frequently exhibited clinical complete response (P = 0.05) and showed a trend to exhibit pCR (P = 0.07). Five (13%) patients experienced grade 1 cardiac dysfunction during preoperative treatment. Neither HER2 nor estrogen receptor status changed significantly after exposure to trastuzumab and vinorelbine. RNA profiling identified three top-level clusters by unsupervised analysis. Tumors with extremes of response [pCR (n = 3) versus nonresponse (n = 3)] fell into separate groups by hierarchical clustering. No predictive genes were identified in pCR tumors. Nonresponding tumors were more likely to be T4 stage (P = 0.02) and express basal markers (P < 0.00001), growth factors, and growth factor receptors. Insulin-like growth factor-I receptor membrane expression was associated with a lower response rate (50% versus 97%; P = 0.001).

Conclusions: Preoperative trastuzumab plus vinorelbine is active and well tolerated in patients with HER2-positive, operable, stage II/III breast cancer. HER2-overexpressing tumors with a basal-like phenotype, or with expression of insulin-like growth factor-I receptor and other proteins involved in growth factor pathways, are more likely to be resistant to this regimen.
Data from Identification of Nicotinamide N-Methyltransferase as a Novel Serum Tumor Marker for Colorectal Cancer
Authors: Roeßler Markus , Rollinger Wolfgang , Palme Stefan , Hagmann Marie-Luise , Berndt Peter , Engel Alfred M. , Schneidinger Bernd , Pfeffer Michael , Andres Herbert , Karl Johann , Bodenmüller Heinz , Rüschoff Josef , Henkel Thomas , Rohr Gerhard , Rossol Siegbert , Rösch Wolfgang , Langen Hanno , Zolg Werner , Tacke Michael
Published: April 2023

Abstract
Purpose: The goal of this study was to identify and validate novel serum markers of human colorectal cancer as potential candidates for noninvasive detection of early colorectal neoplasm.

Experimental Design: Employing two-dimensional gel electrophoresis and mass spectrometry, we analyzed 16 matched colorectal cancer and adjacent normal tissue samples. Proteins found to be elevated in cancer tissue were further validated by generating antibodies which were used for immunoblotting of tissue samples and for the development of highly sensitive immunoassays for assessment of serum samples.

Results: In total, 735 different proteins were identified in colon tissue. Strong elevation in colorectal cancer for five proteins was confirmed by immunoblot analysis: transforming growth factor-β induced protein ig-h3 (βIG-H3), nicotinamide N-methyltransferase (NNMT), nucleoside diphosphate kinase A (nm23-H1), purine nucleoside phosphorylase (PNPH), and mannose-6-phosphate receptor binding protein 1 (M6P1). Elevated levels of NNMT, which is not predicted to be secreted but is known as a cytoplasmic protein, were found in serum from patients with colorectal cancer. Employing a receiver-operating characteristic curve based on the measurement of 109 patients with colorectal cancer and 317 healthy controls, we obtained an area under the curve of 0.84 for NNMT, which was superior to the established tumor marker carcinoembryogenic antigen with an area under the curve of 0.78.

Conclusions: It is proposed that NNMT serum levels may have significance in the early detection and in the management of patients with colorectal cancer.
Data from The Cytoskeleton Regulatory Protein hMena (ENAH) Is Overexpressed in Human Benign Breast Lesions with High Risk of Transformation and Human Epidermal Growth Factor Receptor-2–Positive/Hormonal Receptor–Negative Tumors
Authors: Di Modugno Francesca , Mottolese Marcella , Di Benedetto Anna , Conidi Andrea , Novelli Flavia , Perracchio Letizia , Venturo Irene , Botti Claudio , Jager Elke , Santoni Angela , Natali Pier Giorgio , Nisticò Paola
Published: April 2023

Abstract
Purpose: hMena (ENAH), a cytoskeleton regulatory protein involved in the regulation of cell motility and adhesion, is overexpressed in breast cancer. The aim of this study was to define at what stage of breast carcinogenesis hMena is overexpressed and to correlate hMena overexpression with established prognostic factors in breast cancer, focusing on human epidermal growth factor receptor-2 (HER-2).

Experimental Design: hMena expression was assessed immunohistochemically in a prospective cohort of cases (n = 360) encompassing a highly representative spectrum of benign breast diseases associated with different risk of transformation, in situ, invasive, and metastatic tumors. Correlations with conventional pathologic and prognostic variables, such as proliferation index, hormonal receptor status, and HER-2 overexpression, were also evaluated. In vitro experiments were done to study the effect of neuregulin-1 and Herceptin treatments on hMena expression.

Results: hMena protein is undetectable in normal breast and is weakly expressed in a small percentage of low-risk benign diseases (9%), but displays a progressive and significant increase of positivity in benign lesions at higher risk of transformation (slightly increased risk 43%; moderate increased risk 67%), in in situ (72%), invasive (93%), and metastatic breast cancer (91%). A significant direct correlation with tumor size (P = 0.04), proliferation index (P < 0.0001), and HER-2 overexpression (P < 0.0001) and an inverse relationship with estrogen (P = 0.036) and progesterone receptors (P = 0.001) are found in invasive carcinomas. In vitro experiments show that neuregulin-1 up-regulates, whereas Herceptin down-regulates, hMena expression.

Conclusions: Our data provide new insights into the relevance of actin-binding proteins in human breast carcinogenesis and indicate hMena overexpression as a surrogate indicator in breast disease management.
Data from Hepatocyte Growth Factor Reduces Susceptibility to an Irreversible Epidermal Growth Factor Receptor Inhibitor in EGFR-T790M Mutant Lung Cancer
Authors: Yamada Tadaaki , Matsumoto Kunio , Wang Wei , Li Qi , Nishioka Yasuhiko , Sekido Yoshitaka , Sone Saburo , Yano Seiji
Published: April 2023

Abstract
Purpose: The secondary T790M mutation in epidermal growth factor receptor (EGFR) is the most frequent cause of acquired resistance to the reversible EGFR tyrosine kinase inhibitors (EGFR-TKI), gefitinib and erlotinib, in lung cancer. Irreversible EGFR-TKIs are expected to overcome the reversible EGFR-TKI resistance of lung cancer harboring T790M mutation in EGFR. However, it is clear that resistance may also develop to this class of inhibitors. We showed previously that hepatocyte growth factor (HGF) induced gefitinib resistance of lung cancer harboring EGFR-activating mutations. Here, we investigated whether HGF induced resistance to the irreversible EGFR-TKI, CL-387,785, in lung cancer cells (H1975) harboring both L858R activating mutation and T790M secondary mutation in EGFR.

Experimental Design: CL-387,785 sensitivity and signal transduction in H1975 cells were examined in the presence or absence of HGF or HGF-producing fibroblasts with or without HGF-MET inhibitors.

Results: HGF reduced susceptibility to CL-387,785 in H1975 cells. Western blotting and small interfering RNA analyses indicated that HGF-induced hyposensitivity was mediated by the MET/phosphoinositide 3-kinase/Akt signaling pathway independent of EGFR, ErbB2, ErbB3, and ErbB4. Hyposensitivity of H1975 cells to CL-387,785 was also induced by coculture with high-level HGF-producing lung fibroblasts. The hyposensitivity was abrogated by treatment with anti-HGF neutralizing antibody, HGF antagonist NK4, or MET-TKI.

Conclusions: We showed HGF-mediated hyposensitivity as a novel mechanism of resistance to irreversible EGFR-TKIs. It will be clinically valuable to investigate the involvement of HGF-MET–mediated signaling in de novo and acquired resistance to irreversible EGFR-TKIs in lung cancer harboring T790M mutation in EGFR. Clin Cancer Res; 16(1); 174–83
Data from ShRNA-Targeted MAP4K4 Inhibits Hepatocellular Carcinoma Growth
Authors: Liu An-Wen , Cai Jing , Zhao Xiang-Li , Jiang Ting-Hui , He Tian-Feng , Fu Hua-Qun , Zhu Ming-Hua , Zhang Shu‐Hui
Published: April 2023

Abstract
Purpose: Mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) is overexpressed in many types of cancer. Herein, we aimed to investigate its expression pattern, clinical significance, and biological function in hepatocellular carcinoma (HCC).

Experimental Design: MAP4K4 expression was examined in 20 fresh HCCs and corresponding nontumor liver tissues. Immunohistochemistry for MAP4K4 was performed on additional 400 HCCs, of which 305 (76%) were positive for hepatitis B surface antigens. The clinical significance of MAP4K4 expression was analyzed. MAP4K4 downregulation was performed in HCC cell lines HepG2 and Hep3B with high abundance of MAP4K4, and the effects of MAP4K4 silencing on cell proliferation in vitro and tumor growth in vivo were evaluated. Quantitative real-time PCR arrays were employed to identify the MAP4K4-regulated signaling pathways.

Results: MAP4K4 was aberrantly overexpressed in HCCs relative to adjacent nontumor liver tissues. This overexpression was significantly associated with larger tumor size, increased histologic grade, advanced tumor stage, and intrahepatic metastasis, as well as worse overall survival and higher early recurrence rate. Knockdown of the MAP4K4 expression reduced cell proliferation, blocked cell cycle at S phase, and increased apoptosis. The antitumor effects of MAP4K4 silencing were also observed in vivo, manifested as retarded tumor xenograft growth. Furthermore, multiple tumor progression–related signaling pathways including JNK, NFκB, and toll-like receptors were repressed by MAP4K4 downregulation.

Conclusions: MAP4K4 overexpression is an independent predictor of poor prognosis of HCC patients, and inhibition of its expression might be of therapeutic significance. Clin Cancer Res; 17(4); 1–11. ©2010 AACR.
Data from The Biological Role of the Low-Affinity p75 Neurotrophin Receptor in Esophageal Squamous Cell Carcinoma
Authors: Okumura Tomoyuki , Tsunoda Shigeru , Mori Yukiko , Ito Tetsuo , Kikuchi Keiji , Wang Timothy Cragin , Yasumoto Shigeru , Shimada Yutaka
Published: April 2023

Abstract
In this study, we investigated the clinicopathologic significance of the low-affinity p75 neurotrophin receptor (p75NTR; which is expressed in the stem/progenitor cell fraction of normal esophageal epithelial cells) in 187 resected esophageal squamous cell carcinoma (ESCC) specimens and found that ∼50% of ESCC expressed p75NTR. Our investigation using ESCC cell lines showed that p75NTR was intensely expressed in the cells with high colony-forming capacity but they were sensitive to cell death on inhibition of p75NTR expression with transient transfection of small interfering RNA (siRNA). These findings suggest that p75NTR is necessary for survival and maintenance of ESCC tumors, providing us with a potential target for novel therapies.

Purpose: p75NTR is expressed in a stem/progenitor cell fraction of human normal esophageal epithelial cells. In this study, we investigated the expression and biological role of p75NTR in ESCC.

Experimental Design: The expression of p75NTR in 187 resected ESCC specimens was immunohistochemically investigated. The expression of p75NTR in 30 ESCC cell lines (KYSEs) was assessed by reverse transcription-PCR, immunocytochemistry, and flow cytometry. The p75NTR-bright and p75NTR-dim/negative cells were isolated from KYSE150 by magnetic beads and colony formation was investigated. The role of p75NTR in KYSEs was assessed by transient transfection of siRNA.

Results: p75NTR was expressed in 92 of 187 (49.2%) tumors. In well-differentiated tumors, positive staining was apparent in the first one to two layers from infiltrative margin of the tumors where most of the cells were actively proliferating. In moderately differentiated tumors, p75NTR was expressed in wider range from the margin of the tumors whereas p75NTR was diffusely distributed in poorly differentiated tumors. p75NTR was expressed in all examined KYSEs and the mean proportion of the p75NTR-bright fraction was 30.1%. The size of p75NTR-positive colonies was larger than that of p75NTR-negative colonies derived from KYSE150 (P < 0.0001). The purified p75NTR-bright cells formed p75NTR-positive large colonies more frequently than the p75NTR-dim/negative cells (P < 0.0001). Down-regulation of p75NTR expression by siRNA resulted in marked growth inhibition with induction of apoptosis.

Conclusions: Our findings suggest that p75NTR is necessary for survival and maintenance of ESCC tumors, providing us with a potential target for novel therapies.
Data from Whole-Genome Allelotyping Identified Distinct Loss-of-Heterozygosity Patterns in Mucinous Ovarian and Appendiceal Carcinomas
Authors: Feltmate Colleen M. , Lee Kenneth R. , Johnson Michael , Schorge John O. , Wong Kwong-kwok , Hao Ke , Welch William R. , Bell Debra A. , Berkowitz Ross S. , Mok Samuel C.
Published: April 2023

Abstract
Purpose: Mucinous adenocarcinoma of the ovary is one of the common histologic types of ovarian cancer. Its pathogenesis is largely unknown. In addition, the differential diagnosis of metastatic mucinous carcinomas to the ovaries, particularly those originating from the appendix, remains challenging. The purpose of this study is to identify molecular biomarkers for mucinous ovarian adenocarcinoma and compare them with those of appendiceal origin.

Experimental Design: Genome-wide loss-of-heterozygosity (LOH) analysis was done on DNA isolated from 28 microdissected primary mucinous ovarian carcinomas and five appendiceal adenocarcinomas. Markers from high-loss regions were selected for further analysis on a total of 32 ovarian and 14 appendiceal cancers.

Results: High levels of LOH rates (>40%) were detected on chromosome arms 9p, 17p, and 21q in mucinous ovarian carcinoma cases. The frequency of allelic loss was similar between high-grade and low-grade mucinous ovarian carcinoma cases but was significantly higher in ovarian versus appendiceal cases. In addition, LOH rates on five chromosomal loci were statistically different between ovarian and appendiceal carcinomas.

Conclusion: A high frequency of LOH can be found in mucinous ovarian adenocarcinomas independent of grade. Despite histologic similarities between mucinous ovarian carcinomas and metastatic appendiceal carcinomas, they have distinct LOH profiles, which may be used for distinguishing the two diseases.
Data from Expression of Adipose Differentiation-Related Protein: A Predictor of Cancer-Specific Survival in Clear Cell Renal Carcinoma
Authors: Yao Masahiro , Huang Ying , Shioi Koichi , Hattori Keiko , Murakami Takayuki , Nakaigawa Noboru , Kishida Takeshi , Nagashima Yoji , Kubota Yoshinobu
Published: April 2023

Abstract
Purpose: We recently found that adipose differentiation-related protein (ADFP) is a potential diagnostic and prognostic biomarker for clear cell subtype renal cell carcinoma (RCC). To further evaluate the correlation between ADFP expression levels and clinicopathologic characteristics and patient outcome, we retrospectively examined patients with clear cell RCC.

Experimental Design: A series of 432 consecutive patients with sporadic clear cell RCC who underwent nephrectomy between March 1986 and June 2004 were enrolled in the study. ADFP expression levels in the primary tumors and in 18 metastases were measured by real-time quantitative PCR. The clinicopathologic and prognostic data were collected, as well as the von Hippel-Lindau disease (VHL) gene alteration status in selected cases.

Results:ADFP expression was apparently high in cases without a symptomatic presentation, as well as in cases of low-stage, low-grade, or VHL alteration–positive clear cell RCC, whereas it was down-regulated in undifferentiated tumors with a spindle/pleomorphic component or metastatic lesions. Univariate analyses showed that high ADFP expression was associated with better cancer-specific survival and cancer-free survival. Further Cox multivariate analyses combined with the split-sample validation method showed that ADFP expression still remains an independent predictor for cancer-specific survival in all tumor stages and in advanced metastatic cases, whereas the predictive value of ADFP expression for cancer recurrence is rather weak.

Conclusions: The ADFP expression may represent the tumor differentiation status, and the detection of the expression levels provides useful prognostic information for cancer-specific survival in patients with clear cell RCC.
Data from WDR19 Expression is Increased in Prostate Cancer Compared with Normal Cells, but Low-Intensity Expression in Cancers is Associated with Shorter Time to Biochemical Failures and Local Recurrence
Authors: Lin Biaoyang , Utleg Angelita G. , Gravdal Karsten , White James T. , Halvorsen Ole J. , Lu Wei , True Lawrence D. , Vessella Robert , Lange Paul H. , Nelson Peter S. , Hood Leroy , Kalland Karl-Henning , Akslen Lars A.
Published: April 2023

Abstract
Purpose: Prostate cancer is the third leading cause of cancer death in the United States, following lung and colorectal cancer. We previously identified WDR19 as a prostate-specific, androgen-regulated gene. Here, we evaluate its utility as a prostate cancer tissue marker for diagnosis and prognostic evaluation.

Experimental Design: Real-time quantitative PCR was done on a panel of prostate tissue isolated by laser capture microdissection. After generating antibodies against WDR19, tissue microarrays (TMA) were employed to compare WDR19 expression between normal, benign prostatic hyperplasia, and prostate cancer tissue.

Results: Using microarrays and real-time quantitative PCR, we showed that WDR19 mRNA expression was increased in cancer. We further showed that WDR19 protein is localized to cytoplasmic subcellular granules and is expressed exclusively in prostate epithelia. Large-scale immunohistochemical staining using TMAs reveals a significant percentage of increase in intensely staining tissue cores in cancer tissue when compared with normal or benign prostatic hyperplastic tissue. Based on the analysis of a separate TMA for which clinical follow-up information was available, low-intensity WDR19 staining was significantly associated with decreased time to biochemical failure (P = 0.006) and with decreased time to locoregional recurrence (P = 0.050).

Conclusions: WDR19 should be added to the list of prostate cancer tissue markers. The continued expansion of a multiple-marker panel will conceivably increase the sensitivity and specificity of prostate cancer diagnosis and prognosis.
Data from A Novel Set of DNA Methylation Markers in Urine Sediments for Sensitive/Specific Detection of Bladder Cancer
Authors: Yu Jian , Zhu Tongyu , Wang Zhirou , Zhang Hongyu , Qian Ziliang , Xu Huili , Gao Baomei , Wang Wei , Gu Lianping , Meng Jun , Wang Jina , Feng Xu , Li Yixue , Yao Xuebiao , Zhu Jingde
Published: April 2023

Abstract
Purpose: This study aims to provide a better set of DNA methylation markers in urine sediments for sensitive and specific detection of bladder cancer.

Experimental Design: Fifty-nine tumor-associated genes were profiled in three bladder cancer cell lines, a small cohort of cancer biopsies and urine sediments by methylation-specific PCR. Twenty-one candidate genes were then profiled in urine sediments from 132 bladder cancer patients (8 cases for stage 0a; 68 cases for stage I; 50 cases for stage II; 4 cases for stages III; and 2 cases for stage IV), 23 age-matched patients with noncancerous urinary lesions, 6 neurologic diseases, and 7 healthy volunteers.

Results: Despite six incidences of four genes reported in 3 of 23 noncancerous urinary lesion patients analyzed, cancer-specific hypermethylation in urine sediments were reported for 15 genes (P < 0.05). Methylation assessment of an 11-gene set (SALL3, CFTR, ABCC6, HPR1, RASSF1A, MT1A, RUNX3, ITGA4, BCL2, ALX4, MYOD1, DRM, CDH13, BMP3B, CCNA1, RPRM, MINT1, and BRCA1) confirmed the existing diagnosis of 121 among 132 bladder cancer cases (sensitivity, 91.7%) with 87% accuracy. Significantly, more than 75% of stage 0a and 88% of stage I disease were detected, indicating its value in the early diagnosis of bladder cancer. Interestingly, the cluster of reported methylation markers used in the U.S. bladder cancers is distinctly different from that identified in this study, suggesting a possible epigenetic disparity between the American and Chinese cases.

Conclusions: Methylation profiling of an 11-gene set in urine sediments provides a sensitive and specific detection of bladder cancer.
Data from Frequent Inactivation of RAMP2, EFEMP1 and Dutt1 in Lung Cancer by Promoter Hypermethylation
Authors: Yue Wen , Dacic Sanja , Sun Quanhong , Landreneau Rodney , Guo Mingzhou , Zhou Wei , Siegfried Jill M. , Yu Jian , Zhang Lin
Published: April 2023

Abstract
Purpose: The goal of this study is to identify novel genes frequently silenced by promoter hypermethylation in lung cancer.

Experimental Designs: Bioinformatic analysis was done to identify candidate genes significantly down-regulated in lung cancer. The effects of DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine on the expression of the candidate genes were determined. Methylated CpG sites in the promoters of the candidate genes were identified using bisulfite DNA sequencing. Methylation-specific PCR was developed and used to analyze DNA methylation in cell lines and clinical specimen. Pathologic and functional analyses were done to study the role of one candidate gene, receptor activity-modifying protein 2 (RAMP2), in suppressing lung cancer cell growth.

Results: Among 54 candidate genes down-regulated in lung cancer, 31 were found to contain CpG islands in their promoters. Six of these 31 genes could be reactivated by 5-aza-2′-deoxycytidine in at least four of six lung cancer cell lines analyzed. Promoter hypermethylation of RAMP2, epidermal growth factor–containing fibulin-like extracellular matrix protein 1, and deleted in U Twenty Twenty cells was detected in 36% to 77% of 22 lung cancer cell lines and in 38% to 50% of 32 primary lung tumors, whereas hypermethylathion of these genes was rarely found in the matched normal samples. The methylation frequencies of these genes in lung cancer were similar to those of commonly used methylation markers, such as RAS association domain family protein 1A, p16, and methylguanine-DNA methyltransferase. Immunohistochemistry showed that RAMP2 was down-regulated in a majority of lung tumors, and RAMP2 down-regulation was correlated with high tumor grade. Ectopic expression of RAMP2 inhibited lung cancer cell growth and caused apoptotic cell death. Knockdown of RAMP2 by RNA interference stimulated cell proliferation.

Conclusions: Studying the newly identified genes may provide new insight into lung tumorigenesis. These genes might be useful as molecular markers of lung cancer.