Donna Butcher

National Institutes of Health, 베서스다, Maryland, United States

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Publications (19)111.82 Total impact

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    ABSTRACT: Epithelial-mesenchymal transition (EMT) is a dynamic process whereby epithelial cells acquire mesenchymal properties. Despite the clinical significance of the acquired mesenchymal properties for metastasis and drug resistance, histopathological evidence of transitional cells in patient samples is lacking and EMT remains an unproven clinical hypothesis. We previously developed and validated a multiplex immunofluorescence assay (IFA) that quantifies the levels of EMT biomarkers (E-Cadherin, Vimentin, β-catenin) in snap-frozen, formalin-fixed, paraffin-embedded (FFPE) tumor tissue (Navas et al, AACR 2013). Building upon that method, we now report a precise, quantitative and unbiased IFA method of tissue analysis (EMT-IFA) using Definiens® software to quantify co-expression of the epithelial marker E-cadherin (E) and mesenchymal marker Vimentin (V) at the cellular level in FFPE clinical biopsies. FFPE human tumor xenografts and cell lines serve as calibrators and reference materials for establishing initial image acquisition parameters for segmented tumor regions of interest. Flanking H&E sections of the clinical biopsies are initially annotated by an anatomic pathologist to identify regions of tumor tissue, non-tumor related areas (including normal or stromal tissues), inflammatory and/or necrotic regions, and freeze artifacts. Adjacent sections are used for the EMT-IFA, and further tumor segmentation from stroma is applied to extracted regions of interests (fields) using the β-catenin layer. Within segmented tumor regions, E-cadherin (E) and Vimentin (V) sub-cellular pixel areas are measured using predetermined thresholds for each biomarker and reported as Log10 (V:E). High resolution images confirmed the co-localization of plasma membrane E-cadherin and cytoplasmic Vimentin to individual transitional tumor cells, and the co-localization of E and V within individual tumor cells of the segmented regions of interest was key for distinguishing EMT from tumor heterogeneity in which adjacent tumor cells can be exclusively E+ or V+. Quantitative analysis of a clinical biopsy series of various histologies revealed all possible phenotypes: epithelial (E+V- colorectal carcinoma), mesenchymal (E-V+ sarcomas), heterogeneous mixtures of E+V- and E-V+ subpopulations, and EMT. Importantly, using the EMT-IFA, we discovered that pharmacological targeting of VEGFR/FGFR/PDGFR signaling with the multi-kinase inhibitor pazopanib stimulated EMT in a preclinical xenograft model of gastric carcinoma (MKN45), as revealed by the significant increase in the V:E ratio (P = 0.0023) coupled with an increase in the EMT phenotype (co-localized V+E+ at the individual cell level) (P = 0.0070) in biopsy specimens. Thus, the EMT-IFA has potential value for investigating EMT in the clinic and its ramifications for drug response and other clinical endpoints. Funded by NCI Contract No. HHSN261200800001E.
    No preview · Article · Nov 2015 · Molecular Cancer Therapeutics
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    ABSTRACT: We previously reported the generation of rabbit monoclonal antibodies to twelve EMT (epithelial-to-mesenchymal transition) transcription factors and cancer stem cell (CSC) markers for the development of pharmacodynamic assays to inform clinical trials of new anticancer therapies (Pfister et al., AACR 2013). Here we demonstrate the functional utility of some of these reagents in detecting HGF-induced changes in EMT and CSC biology in a xenograft tumor model. Initial antibody characterization was performed in vitro and a subset [including SNAIL, SLUG, SOX9, Goosecoid (GSC), NANOG and CD133] was selected for further testing of functional utility in FFPE tissues by quantitative multiplex IFA. The antibodies were applied to xenograft tissues derived from the non-small cell lung cancer tumor line, NCI-H596, implanted in hHGFscid/scid, hHGFki/scid or hHGFki/ki mice to examine HGF-induced changes in EMT factors, CSC markers, as well as pY1235-MET expression in vivo. H596 tumors grown in either hHGFki/scid or hHGFki/ki mice exhibited enhanced EMT particularly in tumor microenvironments adjacent to mouse stroma containing the HGF knockin gene, compared to those in hHGFscid/scid mice. By quantitative immunofluorescence, H596 tumors showed increased Vimentin:E-cadherin ratio when grown in hHGFki/scid (P<0.0006) or hHGFki/ki (P<0.022) vs. HGFscid/scid mice. Moreover, significant increases in nuclear pY1235-MET, measured by%NAP (percentage nuclear area positive), were observed in H596 tumors in hHGFki/scid (P<0.0041) or hHGFki/ki (P<0.0058) vs. hHGFscid/scid. We detected varying levels of EMT/CSC marker expression, including CD44, CD133, ALDH, and GSC in the membrane or cytoplasm of noninvasive regions of H596 tumors in hHGFscid/scid mice. Sox9 was also co-expressed with GSC in some tumor cells but was predominantly in the nuclei. In tissues collected from hHGFki/scid or hHGFki/ki mice, Slug, Snail and Sox9 expression were increased in transitioning tissue regions, adjacent to HGF-containing stroma, coincident with diminished E-Cadherin expression and enhanced Vimentin expression. While Slug expression was predominantly cytoplasmic in invading tumor fronts, the expression was mutually exclusive with CD133 within non-invasive regions. Snail and Sox9 showed enhanced nuclear expression in tumor cells undergoing EMT. These markers are currently being investigated in additional tumor samples from human TNBC or CRC xenograft models treated with various drug combinations. To our knowledge, this is the first demonstration of EMT and changes in cancer stem cell biology in NSCLC induced by an HGF-knockin stromal microenvironment. Funded by NCI Contract No. HHSN261200800001E.
    No preview · Article · Sep 2015

  • No preview · Article · Aug 2015 · Cancer Research
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    ABSTRACT: We have previously reported the development of ELISA assays for intact MET, pY1235-MET, and pY1356-MET in tumor lysates (Srivastava et al, ASCO 2011). Here we report the development of a multiplex quantitative immunofluorescence assay (qIFA) for simultaneous detection of pY1235-MET and total MET in formalin-fixed paraffin-embedded (FFPE) tissues. We utilized a rabbit monoclonal antibody specific to pY1235-MET (with no detectable crossreactivity to pY1234-MET) to develop a highly specific and sensitive IFA assay with total MET (MET4 Mab; from G. Vande Woude, Van Andel Research Institute). Specificity of the IFA was demonstrated by HGF stimulation of A549 cells showing increased intensity of pY1235-MET which was completely inhibited by crizotinib but not by the non-MET specific multi-kinase inhibitor sorafenib, in vitro. The pY1235-MET epitope is stable in FFPE, and IFA detection was enhanced by EDTA (vs. Citrate) for antigen retrieval. Quantitative assessment of pY1235-MET was determined in xenograft models that demonstrate very high IFA expression of pY1235-MET and total MET via gene amplification (GTL-16 and SNU-5), lower expression in HGF-induced paracrine or autocrine cell lines (A549 and HT29), and no detectable staining for pY1235-MET in negative controls (SNU-1 and MDA-MB-231). We have demonstrated assay fitness for purpose using SNU-5 FFPE xenograft tumor samples from animals treated with increasing doses of crizotinib in vivo. Quantitative measurement by Definiens analysis of pY1235-MET and total MET in tissue regions of interest (ROI) showed 50% and 95% pY1235-MET inhibition with 12.5 mg/kg and 25 mg/kg crizotinib, respectively. There were no significant changes in total MET by IFA. A high correlation (R=0.899) was observed between % pY1235-MET/total MET ratio measured by IFA levels (expressed as the marker area/# nuclei for pY1235-MET and total MET per ROI) and % pY1235-MET/total MET ratio determined by ELISA levels (pM/ug protein). This quantitative MET IFA assay is currently being applied in conjunction with a previously developed EMT IFA assay (Navas et al, AACR 2013) for focal tissue analysis of pY1235-MET and total MET IFA expression as well as changes in epithelial to mesenchymal transition (EMT) in gastric tumor xenograft tissues treated with the VEGF inhibitor pazopanib, the MET inhibitor tivantinib (ARQ197), or the drug combination in vivo. These studies will determine whether pY1235-MET or total MET is induced by anti-VEGF inhibitors via increased EMT transition, and whether this effect can be reversed by combination with tivantinib. Funded by NCI Contract No. HHSN261200800001E
    No preview · Article · May 2014 · Proceedings of the American Association for Cancer Research
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    ABSTRACT: Background: Wnt/ß-catenin signaling and cadherin-mediated cell adhesion are two processes that promote cell proliferation and epithelial-mesenchymal transition (EMT) of carcinomas. Therapeutic inhibitors of Wnt-induced tumor activation prevent stabilization and nuclear translocation of β-catenin and putatively lead to inhibition of tumor metastasis by upregulation of E-Cadherin and downregulation of N-Cadherin and Vimentin. Methods: A multiplex quantitative immunofluorescence (qIFA) assay was developed to assess changes in levels of EMT biomarkers (E-Cadherin, N-Cadherin, Vimentin/Pancytokeratin) as well as monitor intracellular localization of β-catenin in tumor tissues. A panel of highly specific antibodies against EMT targets, directly-conjugated to various fluorophores, were optimized for multiplex staining of FFPE tissues using colorectal (CRC) and triple-negative breast (TNBC) cancer cell lines and xenograft tissues as tumor controls for target expression. Rat kidney was used as positive control for endogenous biomarker expression in tissues. Image acquisition was performed with Confocal and Epifluorescence microscopy. Image segmentation and quantitative multiplex analysis were performed using NIS Elements and Definiens Tissue Studio software to obtain total mean integrated signal per biomarker within a tissue region of interest. Results: Fitness for purpose evaluation of the biomarkers was performed in vitro with several cell lines and compounds to demonstrate nuclear translocation of β-catenin as well as EMT-mediated changes in E- to N-Cadherin ratio. A431 squamous epithelial cell lines treated with Lysophosphatidic acid (LPA) induced Adherens Junction (AJ) dissociation of membrane E-Cadherin and β-catenin while co-treatment with specific inhibitors to glycogen synthase kinase-3β (GSK-3β) induced a 5-fold increase in the nuclear to cytoplasmic mean intensity ratio of β-catenin after 24h. TGFβ-treatment of the NMuMG mouse epithelial cell line induced a significant decrease in E- to N-Cadherin membrane intensity ratio after 48 and 72h in vitro. Studies with human TNBC or APC-mutated CRC mouse xenograft models using HDAC or Src/Abl kinase inhibitors to compare pre- and post-treatment effects on EMT biomarker staining will be presented. Conclusion: We have developed a quantitative imaging assay that integrates multiplex changes in EMT biomarkers to assay pharmacodynamic activities of potential targeted agents currently in development at the National Cancer Institute (NCI). This assay could also be applied for retrospective analysis of clinical samples to document drug-induced changes in tumor phenotypes. Funded by NCI Contract No. HHSN261200800001E.
    No preview · Article · Aug 2013 · Cancer Research

  • No preview · Article · Aug 2013 · Cancer Research
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    ABSTRACT: Increased serum levels of IL-15 are reported in type 1 diabetes (T1D). Here we report elevated serum soluble IL-15Rα levels in human T1D. To investigate the role of IL-15/IL-15Rα in the pathogenesis of T1D, we generated double transgenic mice with pancreatic β-cell expression of IL-15 and IL-15Rα. The mice developed hyperglycemia, marked mononuclear cell infiltration, β-cell destruction, and anti-insulin autoantibodies that mimic early human T1D. The diabetes in this model was reversed by inhibiting IL-15 signaling with anti-IL2/IL15Rβ (anti-CD122), which blocks IL-15 transpresentation. Furthermore, the diabetes could be reversed by administration of the Janus kinase 2/3 inhibitor tofacitinib, which blocks IL-15 signaling. In an alternative diabetes model, nonobese diabetic mice, IL15/IL-15Rα expression was increased in islet cells in the prediabetic stage, and inhibition of IL-15 signaling with anti-CD122 at the prediabetic stage delayed diabetes development. In support of the view that these observations reflect the conditions in humans, we demonstrated pancreatic islet expression of both IL-15 and IL-15Rα in human T1D. Taken together our data suggest that disordered IL-15 and IL-15Rα may be involved in T1D pathogenesis and the IL-15/IL15Rα system and its signaling pathway may be rational therapeutic targets for early T1D.
    Preview · Article · Jul 2013 · Proceedings of the National Academy of Sciences
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    ABSTRACT: Reactive oxygen species generated by NADPH oxidase 5 (Nox5) have been implicated in physiological and pathophysiological signaling pathways, including cancer development and progression. However, because immunological tools are lacking, knowledge of the role of Nox5 in tumor biology has been limited; the expression of Nox5 protein across tumors and normal tissues is essentially unknown. Here, we report the characterization and use of a mouse monoclonal antibody against a recombinant Nox5 protein (600-746) for expression profiling of Nox5 in human tumors by tissue microarray analysis. Using our novel antibody, we also report the detection of endogenous Nox5 protein in human UACC-257 melanoma cells. Immunofluorescence, confocal microscopy, and immunohistochemical techniques were employed to demonstrate Nox5 localization throughout UACC-257 cells, with perinuclear enhancement. Tissue microarray analysis revealed, for the first time, substantial Nox5 overexpression in several human cancers including those of prostate, breast, colon, lung, brain, and ovary as well as in malignant melanoma and non-Hodgkin lymphoma; expression in most non-malignant tissues was negative to weak. This validated mouse monoclonal antibody will promote further exploration of the functional significance of Nox5 in human pathophysiology, including tumor cell growth and proliferation.
    No preview · Article · Jul 2013 · Free Radical Biology and Medicine
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    ABSTRACT: Pancreatitis is associated with release of proinflammatory cytokines and reactive oxygen species and plays an important role in the development of pancreatic cancer. We recently demonstrated that dual oxidase (Duox)2, an NADPH oxidase essential for reactive oxygen species-related, gastrointestinal host defense, is regulated by IFN-γ-mediated Stat1 binding to the Duox2 promoter in pancreatic tumor lines. Because LPS enhances the development and invasiveness of pancreatic cancer in vivo following TLR4-related activation of NF-κB, we examined whether LPS, alone or combined with IFN-γ, regulated Duox2. We found that upregulation of TLR4 by IFN-γ in BxPC-3 and CFPAC-1 pancreatic cancer cells was augmented by LPS, resulting in activation of NF-κB, accumulation of NF-κB (p65) in the nucleus, and increased binding of p65 to the Duox2 promoter. TLR4 silencing with small interfering RNAs, as well as two independent NF-κB inhibitors, attenuated LPS- and IFN-γ-mediated Duox2 upregulation in BxPC-3 cells. Induction of Duox2 expression by IFN-γ and LPS may result from IFN-γ-related activation of Stat1 acting in concert with NF-κB-related upregulation of Duox2. Sustained extracellular accumulation of H(2)O(2) generated by exposure to both LPS and IFN-γ was responsible for an ∼50% decrease in BxPC-3 cell proliferation associated with a G(1) cell cycle block, apoptosis, and DNA damage. We also demonstrated upregulation of Duox expression in vivo in pancreatic cancer xenografts and in patients with chronic pancreatitis. These results suggest that inflammatory cytokines can interact to produce a Duox-dependent pro-oxidant milieu that could increase the pathologic potential of pancreatic inflammation and pancreatic cancer cells.
    Preview · Article · Jan 2013 · The Journal of Immunology
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    ABSTRACT: Despite high cure rates for pediatric B-lineage acute lymphoblastic leukemia (B-ALL), short-term and long-term toxicities and chemoresistance are shortcomings of standard chemotherapy. Immunotherapy and chemoimmunotherapy based on monoclonal antibodies (mAbs) that target cell surface antigens with restricted expression in pediatric B-ALL may offer the potential to reduce toxicities and prevent or overcome chemoresistance. The receptor tyrosine kinase ROR1 has emerged as a candidate for mAb targeting in select B-cell malignancies. Using flow cytometry, Western blotting, immunohistochemistry, and confocal immunofluorescence microscopy, we analyzed the cell surface expression of ROR1 across major pediatric ALL subtypes represented by 14 cell lines and 56 primary blasts at diagnosis or relapse as well as in normal adult and pediatric tissues. Cell surface ROR1 expression was found in 45% of pediatric ALL patients, all of which were B-ALL, and was not limited to any particular genotype. All cell lines and primary blasts with E2A-PBX1 translocation and a portion of patients with other high risk genotypes, such as MLL rearrangement, expressed cell surface ROR1. Importantly, cell surface ROR1 expression was found in many of the pediatric B-ALL patients with multiply relapsed and refractory disease and normal karyotype or low risk cytogenetics, such as hyperdiploidy. Notably, cell surface ROR1 was virtually absent in normal adult and pediatric tissues. Collectively, this study suggests that ROR1 merits preclinical and clinical investigations as a novel target for mAb-based therapies in pediatric B-ALL. We propose cell surface expression of ROR1 detected by flow cytometry as primary inclusion criterion for pediatric B-ALL patients in future clinical trials of ROR1-targeted therapies.
    Full-text · Article · Dec 2012 · PLoS ONE
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    ABSTRACT: Over 50% of patients with colorectal cancer (CRC) will progress and/or develop metastases. Biomarkers capable of predicting progression, risk stratification and therapeutic benefit are needed. Cancer stem cells are thought to be responsible for tumor initiation, dissemination and treatment failure. Therefore, we hypothesized that CRC cancer stem cell markers (CRCSC) will identify a group of patients at high risk for progression. Paraffin-embedded tissue cores of normal (n=8), and histopathologically well-defined primary (n= 30) and metastatic (n=10) CRC were arrayed in duplicate on tissue microarrays (TMAs). Expression profiles of non-CD133 CRCSC (CD29, CD44, ALDH1A1, ALDH1B1, EpCam, and CD166) were detected by immunohistochemistry and the association with clinicopathological data and patient outcomes was determined using standard statistical methodology. An independent pathologist, blinded to the clinical data scored the samples. Scoring included percent positive cells (0 to 4, 0 = <10%, 1 = 10 - 24%, 2 = 25 - 49%, 3 = 50 - 74%, 4 = 75 - 100%), and the intensity of positively stained cells (0 to 4; 0 = no staining, 1 = diminutive intensity, 2 = low intensity, 3 = intermediate intensity, 4 = high intensity). The pathologic score represents the sum of these two values, reported in this paper as a combined IHC staining score (CSS). Of 30 patients 7 were AJCC stage IIA, 10 stage IIIB, 7 stage IIIC and 6 stage IV. Median follow-up was 113 months. DFI was 17 months. Median overall survival (OS) was not reached. Stage-specific OS was: II - not reached; III - not reached; IV - 11 months. In a univariate analysis, poor OS was associated with loss of CD29 expression; median OS, 32 months vs. not reached for CSS 3-7 vs. >7.5, respectively; p=0.052 comparing entire curves, after adjustment. In a Cox model analysis, loss of CD29 exhibited a trend toward association with survival (p=0.098) after adjusting for the effect of stage (p=0.0076). Greater expression of ALDH1A1 was associated with increasing stage (p=0.042 over stages 2, 3b, 3c, and 4) while loss of CD29 expression exhibited a trend toward being associated with stages 3 and 4 (p=0.08). Compared to normal colon tissue, primary tumors were associated with increased expression of ALDH1B1 (p=0.008). ALD1H1B1 expression level differed according to whether the tumor was moderately or poorly differentiated, well differentiated, or mucinous; the highest expression levels were associated with moderately or poorly differentiated tumors (p=0.011). Lymph node metastases were associated with a trend toward decreased expression of EpCAM (p = 0.06) when comparing 0 vs. 1 vs. 2+ positive lymph nodes, as was CD29 (p = 0.08) when comparing 0 vs. any positive lymph nodes. Compared to normal colon tissue metastatic colon cancers from different patients were associated with increased ALDH1B1 expression (p=0.001) whereas CD29 expression was higher in normal colonic tissue (p=0.014). CD29 may be associated with survival as well as clinical stage and number of lymph nodes. ALDH1B1 expression was associated with differentiation as well as type of tissue evaluated. ALDH1A1 was associated with clinical stage, and decreased EpCAM expression was found in patients with advanced lymph node stage. CRCSCs may be useful biomarkers to risk stratify, and estimate outcomes in CRC. Larger prospective studies are required to validate the current findings.
    Full-text · Article · Jun 2012 · Journal of Cancer

  • No preview · Article · Jul 2011 · Cancer Research
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    ABSTRACT: In vitro growth of alveolar soft part sarcoma (ASPS) and establishment of an ASPS cell line, ASPS-1, are described in this study. Using a recently developed xenograft model of ASPS derived from a lymph node metastasis, organoid nests consisting of 15 to 25 ASPS cells were isolated from ASPS xenograft tumors by capture on 70 μm filters and plated in vitro. After attachment to the substratum, these nests deposited small aggregates of ASPS cells. These cells grew slowly and were expanded over a period of 3 years and have maintained characteristics consistent with those of both the original ASPS tumor from the patient and the xenograft tumor including (1) presence of the alveolar soft part locus-transcription factor E3 type 1 fusion transcript and nuclear expression of the alveolar soft part locus-transcription factor E3 type 1 fusion protein; (2) maintenance of the t(X;17)(p11;q25) translocation characteristic of ASPS; and (3) expression of upregulated ASPS transcripts involved in angiogenesis (ANGPTL2, HIF-1-α, MDK, c-MET, VEGF, and TIMP-2), cell proliferation (PRL, PCSK1), metastasis (ADAM9), as well as the transcription factor BHLHB3 and the muscle-specific transcripts TRIM63 and ITGβ1BP3. This ASPS cell line forms colonies in soft agar and retains the ability to produce highly vascularized ASPS tumors in NOD.SCID/NCr mice. Immunohistochemistry of selected ASPS markers on these tumors indicated similarity to those of the original patient tumor as well as to the xenografted ASPS tumor. We anticipate that this ASPS cell line will accelerate investigations into the biology of ASPS including identification of new therapeutic approaches for treatment of this slow growing soft tissue sarcoma.
    No preview · Article · Jul 2011 · Journal of Pediatric Hematology/Oncology

  • No preview · Article · Jan 2011 · Cancer Research

  • No preview · Article · May 2010 · Gastroenterology
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    ABSTRACT: Cancer cells undergo significant changes in carbohydrate expression, and these alterations can be useful as biomarkers and therapeutic targets. In this study, we investigated the expression of carbohydrate antigens containing a terminal GalNAcalpha1-3Gal or GalNAcalpha1-6Gal on human cervix and cervical carcinoma. Monoclonal antibodies to each of these carbohydrates were generated by immunizing rabbits with the corresponding antigen conjugated to KLH followed by hybridoma production. Antibodies were screened and evaluated using a combination of carbohydrate microarray profiling, ELISA, dot blot and immunohistochemical staining to verify specificity. Antibody 132-3 was found to selectively recognize GalNAcalpha1-3Gal with little cross-reactivity to other structurally similar antigens such as GalNAcalpha1-6Gal, blood group A, Forssman antigen and the Tn antigen on both solution assays and human tissue. Although GalNAcalpha1-6Gal expression was not detected, GalNAcalpha1-3Gal expression was found on 55% of squamous cell carcinomas. Expression in normal tissue was observed but was restricted to the suprabasal epithelial layer. Importantly, we found expression of the antigen on cervical cancer had a statistically significant correlation with the 5-year survival rate of the patients (48 vs. 85% for antigen negative vs. positive, p = 0.017). Expression of GalNAcalpha1-3Gal did not correlate with other clinical factors including tumor stage, size and lymph node metastasis, indicating the antigen is a new, independent biomarker for the prognosis of cervical cancer.
    Preview · Article · Jan 2010 · International Journal of Cancer
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    ABSTRACT: In vivo growth of alveolar soft part sarcoma (ASPS) was achieved using subcutaneous xenografts in sex-matched nonobese diabetic severe combined immunodeficiency mice. One tumor, currently at passage 6, has been maintained in vivo for 32 months and has maintained characteristics consistent with those of the original ASPS tumor including (1) tumor histology and staining with periodic acid Schiff/diastase, (2) the presence of the ASPL-TFE3 type 1 fusion transcript, (3) nuclear staining with antibodies to the ASPL-TFE3 type 1 fusion protein, (4) maintenance of the t(X;17)(p11;q25) translocation characteristic of ASPS, (5) stable expression of signature ASPS gene transcripts and finally, the development and maintenance of a functional vascular network, a hallmark of ASPS. The ASPS xenograft tumor vasculature encompassing nests of ASPS cells is highly reactive to antibodies against the endothelial antigen CD34 and is readily accessible to intravenously administered fluorescein isothiocyanate-dextran. The therapeutic vulnerability of this tumor model to antiangiogenic therapy, targeting vascular endothelial growth factor and hypoxia-inducible factor-1 alpha, was examined using bevacizumab and topotecan alone and in combination. Together, the 2 drugs produced a 70% growth delay accompanied by a 0.7 net log cell kill that was superior to the antitumor effect produced by either drug alone. In summary, this study describes a preclinical in vivo model for ASPS which will facilitate investigation into the biology of this slow growing soft tissue sarcoma and demonstrates the feasibility of using an antiangiogenic approach in the treatment of ASPS.
    Full-text · Article · Sep 2009 · Journal of Pediatric Hematology/Oncology
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    Qian Li · Miriam R Anver · Donna O Butcher · Jeffrey C Gildersleeve
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    ABSTRACT: The tumor-associated Tn antigen has been investigated extensively as a biomarker and therapeutic target. Cancer vaccines containing the Tn antigen as a single tumor antigen or as a component of a polyvalent vaccine have progressed into phase I and II clinical trials. One major focus of Tn-based vaccines is the treatment of prostate cancer patients. Although expression of the antigen on prostate tumors is a critical prerequisite, previous reports investigating Tn expression in prostate tumors have produced conflicting results. Using a combination of immunohistochemistry and carbohydrate microarray profiling, we show that only 4% to 26% of prostate tumors express the Tn antigen. Based on our results, the majority of prostate cancer patients do not express the appropriate antigen. Therefore, efforts to preselect the subset of prostate cancer patients with Tn-positive tumors or apply Tn vaccines to other cancers with higher rates of antigen expression could significantly improve clinical response rates. Because conflicting information on carbohydrate expression is a general problem for the field, the approach described in this article of analyzing antigen expression with multiple antibodies and using carbohydrate microarray profiles to interpret the results will be useful for the development of other carbohydrate-based cancer vaccines and diagnostics.
    Preview · Article · May 2009 · Molecular Cancer Therapeutics
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    ABSTRACT: Alveolar soft-part sarcoma (ASPS) is an extremely rare, highly vascular soft tissue sarcoma affecting predominantly adolescents and young adults. In an attempt to gain insight into the pathobiology of this enigmatic tumor, we performed the first genome-wide gene expression profiling study. For seven patients with confirmed primary or metastatic ASPS, RNA samples were isolated immediately following surgery, reverse transcribed to cDNA and each sample hybridized to duplicate high-density human U133 plus 2.0 microarrays. Array data was then analyzed relative to arrays hybridized to universal RNA to generate an unbiased transcriptome. Subsequent gene ontology analysis was used to identify transcripts with therapeutic or diagnostic potential. A subset of the most interesting genes was then validated using quantitative RT-PCR and immunohistochemistry. Analysis of patient array data versus universal RNA identified elevated expression of transcripts related to angiogenesis (ANGPTL2, HIF-1 alpha, MDK, c-MET, VEGF, TIMP-2), cell proliferation (PRL, IGFBP1, NTSR2, PCSK1), metastasis (ADAM9, ECM1, POSTN) and steroid biosynthesis (CYP17A1 and STS). A number of muscle-restricted transcripts (ITGB1BP3/MIBP, MYF5, MYF6 and TRIM63) were also identified, strengthening the case for a muscle cell progenitor as the origin of disease. Transcript differentials were validated using real-time PCR and subsequent immunohistochemical analysis confirmed protein expression for several of the most interesting changes (MDK, c-MET, VEGF, POSTN, CYP17A1, ITGB1BP3/MIBP and TRIM63). Results from this first comprehensive study of ASPS gene expression identifies several targets involved in angiogenesis, metastasis and myogenic differentiation. These efforts represent the first step towards defining the cellular origin, pathogenesis and effective treatment strategies for this atypical malignancy.
    Preview · Article · Feb 2009 · BMC Cancer

Publication Stats

194 Citations
111.82 Total Impact Points

Institutions

  • 2013
    • National Institutes of Health
      • Laboratory of Pathology
      베서스다, Maryland, United States
    • National Cancer Institute (USA)
      Maryland, United States
  • 2009-2013
    • NCI-Frederick
      Фредерик, Maryland, United States
  • 2012
    • Leidos Biomedical Research
      Фредерик, Maryland, United States
  • 2010
    • SAIC
      Maryland, United States