FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2894062761> ?p ?o ?g. }

• W2894062761 endingPage "2911" @default.
• W2894062761 startingPage "2902" @default.
• W2894062761 abstract "Patient-derived xenografts retain the genotype of the parent tumors more readily than tumor cells maintained in culture. The two previously reported clival chordoma xenografts were derived from recurrent tumors after radiation. To study the genetics of clival chordoma in the absence of prior radiation exposure we established a patient-derived xenograft at primary resection of a clival chordoma. Epicranial grafting of clival chordoma collected during surgery was performed. Tumor growth was established in a nonobese diabetic/severe combined immunodeficiency mouse and tumors have been passaged serially for seven generations. Physaliferous cell architecture was shown in the regenerated tumors, which stained positive for Brachyury, cytokeratin, and S100 protein. The tumors showed bone invasion. Single-nucleotide polymorphism analysis of the tumor xenograft was compared with the parental tumor. Copy number gain of the T gene (brachyury) and heterozygous loss of cyclin dependent kinase inhibitor 2A (CDKN2A) was observed. Heterozygous loss of the tumor-suppressor fragile histidine triad (FHIT) gene also was observed, although protein expression was preserved. Accumulation of copy number losses and gains as well as increased growth rate was observed over three generations. The patient-derived xenograft reproduces the phenotype of clival chordoma. This model can be used in the future to study chordoma biology and to assess novel treatments. Patient-derived xenografts retain the genotype of the parent tumors more readily than tumor cells maintained in culture. The two previously reported clival chordoma xenografts were derived from recurrent tumors after radiation. To study the genetics of clival chordoma in the absence of prior radiation exposure we established a patient-derived xenograft at primary resection of a clival chordoma. Epicranial grafting of clival chordoma collected during surgery was performed. Tumor growth was established in a nonobese diabetic/severe combined immunodeficiency mouse and tumors have been passaged serially for seven generations. Physaliferous cell architecture was shown in the regenerated tumors, which stained positive for Brachyury, cytokeratin, and S100 protein. The tumors showed bone invasion. Single-nucleotide polymorphism analysis of the tumor xenograft was compared with the parental tumor. Copy number gain of the T gene (brachyury) and heterozygous loss of cyclin dependent kinase inhibitor 2A (CDKN2A) was observed. Heterozygous loss of the tumor-suppressor fragile histidine triad (FHIT) gene also was observed, although protein expression was preserved. Accumulation of copy number losses and gains as well as increased growth rate was observed over three generations. The patient-derived xenograft reproduces the phenotype of clival chordoma. This model can be used in the future to study chordoma biology and to assess novel treatments. Chordoma is a very rare and slowly growing tumor arising in the ventral axial skeleton. Tumor cells express Brachyury, a transcription factor that is restricted topographically to nascent and migrating mesoderm and the notochord during development.1Herrmann B.G. Expression pattern of the Brachyury gene in whole-mount TWis/TWis mutant embryos.Development. 1991; 113: 913-917Crossref PubMed Google Scholar, 2Wilkinson D.G. Bhatt S. Herrmann B.G. Expression pattern of the mouse T gene and its role in mesoderm formation.Nature. 1990; 343: 657-659Crossref PubMed Scopus (687) Google Scholar Notochordal cell rests presently are hypothesized as precursors to chordoma.3Salisbury J.R. Deverell M.H. Cookson M.J. Whimster W.F. Three-dimensional reconstruction of human embryonic notochords: clue to the pathogenesis of chordoma.J Pathol. 1993; 171: 59-62Crossref PubMed Scopus (74) Google Scholar, 4Shen J. Li C.D. Yang H.L. Lu J. Zou T.M. Wang D.L. Deng M. Classic chordoma coexisting with benign notochordal cell rest demonstrating different immunohistological expression patterns of Brachyury and galectin-3.J Clin Neurosci. 2011; 18: 96-99Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 5Yamaguchi T. Suzuki S. Ishiiwa H. Ueda Y. Intraosseous benign notochordal cell tumours: overlooked precursors of classic chordomas?.Histopathology. 2004; 44: 597-602Crossref PubMed Scopus (102) Google Scholar Approximately a third of all patients with a diagnosis of chordoma have tumor located at the skull base.6McMaster M.L. Goldstein A.M. Bromley C.M. Ishibe N. Parry D.M. Chordoma: incidence and survival patterns in the United States, 1973-1995.Cancer Causes Control. 2001; 12: 1-11Crossref PubMed Scopus (698) Google Scholar These patients more commonly are younger in age and present most frequently with cranial nerve deficits.7Diaz R.J. Maggacis N. Zhang S. Cusimano M.D. Determinants of quality of life in patients with skull base chordoma.J Neurosurg. 2014; 120: 528-537Crossref PubMed Scopus (21) Google Scholar The median survival for patients with clival chordoma is 9.2 years.8Chambers K.J. Lin D.T. Meier J. Remenschneider A. Herr M. Gray S.T. Incidence and survival patterns of cranial chordoma in the United States.Laryngoscope. 2014; 124: 1097-1102Crossref PubMed Scopus (74) Google Scholar These tumors are exceptionally resistant to standard DNA-altering chemotherapy, limiting the effectiveness of adjuvant therapies subsequent to surgical resection.9Diaz R.J. Cusimano M.D. The biological basis for modern treatment of chordoma.J Neurooncol. 2011; 104: 411-422Crossref PubMed Scopus (31) Google Scholar To better understand the tumor response to specific adjuvant therapy, experimental models that allow the assessment of tumor cells in a living microenvironment are needed. Presently, four chordoma xenografts (two clival and two sacral) derived from primary tumor, without cell culture, have been reported in the literature (Table 1).10Davies J.M. Robinson A.E. Cowdrey C. Mummaneni P.V. Ducker G.S. Shokat K.M. Bollen A. Hann B. Phillips J.J. Generation of a patient-derived chordoma xenograft and characterization of the phosphoproteome in a recurrent chordoma.J Neurosurg. 2014; 120: 331-336Crossref PubMed Scopus (19) Google Scholar, 11Siu I.M. Salmasi V. Orr B.A. Zhao Q. Binder Z.A. Tran C. Ishii M. Riggins G.J. Hann C.L. Gallia G.L. Establishment and characterization of a primary human chordoma xenograft model.J Neurosurg. 2012; 116: 801-809Crossref PubMed Scopus (22) Google Scholar, 12Owen J.H. Komarck C.M. Wang A.C. Abuzeid W.M. Keep R.F. McKean E.L. Sullivan S. Fan X. Prince M.E.P. UM-Chor1: establishment and characterization of the first validated clival chordoma cell line.J Neurosurg. 2018; 128: 701-709Crossref PubMed Scopus (12) Google Scholar, 13Bozzi F. Manenti G. Conca E. Stacchiotti S. Messina A. Dagrada G. Gronchi A. Panizza P. Pierotti M.A. Tamborini E. Pilotti S. Development of transplantable human chordoma xenograft for preclinical assessment of novel therapeutic strategies.Neuro Oncol. 2014; 16: 72-80Crossref PubMed Scopus (8) Google Scholar, 14Trucco M.M. Awad O. Wilky B.A. Goldstein S.D. Huang R. Walker R.L. Shah P. Katuri V. Gul N. Zhu Y.J. McCarthy E.F. Paz-Priel I. Meltzer P.S. Austin C.P. Xia M. Loeb D.M. A novel chordoma xenograft allows in vivo drug testing and reveals the importance of NF-kappaB signaling in chordoma biology.PLoS One. 2013; 8: e79950Crossref PubMed Scopus (16) Google Scholar, 15Karikari I.O. Gilchrist C.L. Jing L. Alcorta D.A. Chen J. Richardson W.J. Gabr M.A. Bell R.D. Kelley M.J. Bagley C.A. Setton L.A. Molecular characterization of chordoma xenografts generated from a novel primary chordoma cell source and two chordoma cell lines.J Neurosurg Spine. 2014; 21: 386-393Crossref PubMed Scopus (15) Google Scholar, 16Hsu W. Mohyeldin A. Shah S.R. ap Rhys C.M. Johnson L.F. Sedora-Roman N.I. Kosztowski T.A. Awad O.A. McCarthy E.F. Loeb D.M. Wolinsky J.P. Gokaslan Z.L. Quinones-Hinojosa A. Generation of chordoma cell line JHC7 and the identification of Brachyury as a novel molecular target.J Neurosurg. 2011; 115: 760-769Crossref PubMed Scopus (86) Google Scholar, 17Presneau N. Shalaby A. Ye H. Pillay N. Halai D. Idowu B. Tirabosco R. Whitwell D. Jacques T.S. Kindblom L.G. Bruderlein S. Moller P. Leithner A. Liegl B. Amary F.M. Athanasou N.N. Hogendoorn P.C. Mertens F. Szuhai K. Flanagan A.M. Role of the transcription factor T (brachyury) in the pathogenesis of sporadic chordoma: a genetic and functional-based study.J Pathol. 2011; 223: 327-335Crossref PubMed Scopus (150) Google Scholar, 18Liu X. Nielsen G.P. Rosenberg A.E. Waterman P.R. Yang W. Choy E. Sassi S. Yang S. Harmon D.C. Yang C. Schwab J.H. Kobayashi E. Mankin H.J. Xavier R. Weissleder R. Duan Z. Hornicek F.J. Establishment and characterization of a novel chordoma cell line: CH22.J Orthop Res. 2012; 30: 1666-1673Crossref PubMed Scopus (32) Google Scholar, 19DeComas A.M. Penfornis P. Harris M.R. Meyer M.S. Pochampally R.R. Derivation and characterization of an extra-axial chordoma cell line (EACH-1) from a scapular tumor.J Bone Joint Surg Am. 2010; 92: 1231-1240Crossref PubMed Scopus (19) Google Scholar These tumors were established in the flanks of immunodeficient mice. The clival chordoma xenografts previously described have been derived from tumors with prior radiation exposure and therefore may harbor genetic aberrations that are not representative of the originating oncogenic mutations in the tumor. Therefore, we present the first clival chordoma primary xenograft derived from previously untreated tumor.Table 1Summary of Chordoma Xenograft ModelsStudy (year)NameSourceBrachyuryS100CKGenomicPresent studySMH5Clival PDX+ (nuc)NT+SNP∗SNP Affymetrix 6.0.Davies et al10Davies J.M. Robinson A.E. Cowdrey C. Mummaneni P.V. Ducker G.S. Shokat K.M. Bollen A. Hann B. Phillips J.J. Generation of a patient-derived chordoma xenograft and characterization of the phosphoproteome in a recurrent chordoma.J Neurosurg. 2014; 120: 331-336Crossref PubMed Scopus (19) Google Scholar (2014)SF8894Clival PDX+ (nuc)NT+NoneSiu et al11Siu I.M. Salmasi V. Orr B.A. Zhao Q. Binder Z.A. Tran C. Ishii M. Riggins G.J. Hann C.L. Gallia G.L. Establishment and characterization of a primary human chordoma xenograft model.J Neurosurg. 2012; 116: 801-809Crossref PubMed Scopus (22) Google Scholar (2012)JHH-2009-011Clival PDX+ (nuc)++SNP†SNP Illumina 600W (Illumina, San Diego, CA).Owen et al12Owen J.H. Komarck C.M. Wang A.C. Abuzeid W.M. Keep R.F. McKean E.L. Sullivan S. Fan X. Prince M.E.P. UM-Chor1: establishment and characterization of the first validated clival chordoma cell line.J Neurosurg. 2018; 128: 701-709Crossref PubMed Scopus (12) Google Scholar (2017)UM-Chor1Clival CLD+ (cyto)NTNTNone+ (nuc)Bozzi et al13Bozzi F. Manenti G. Conca E. Stacchiotti S. Messina A. Dagrada G. Gronchi A. Panizza P. Pierotti M.A. Tamborini E. Pilotti S. Development of transplantable human chordoma xenograft for preclinical assessment of novel therapeutic strategies.Neuro Oncol. 2014; 16: 72-80Crossref PubMed Scopus (8) Google Scholar (2014)4Sacral PDX+ (nuc)NTNTFISHTrucco et al14Trucco M.M. Awad O. Wilky B.A. Goldstein S.D. Huang R. Walker R.L. Shah P. Katuri V. Gul N. Zhu Y.J. McCarthy E.F. Paz-Priel I. Meltzer P.S. Austin C.P. Xia M. Loeb D.M. A novel chordoma xenograft allows in vivo drug testing and reveals the importance of NF-kappaB signaling in chordoma biology.PLoS One. 2013; 8: e79950Crossref PubMed Scopus (16) Google Scholar (2013)Sacral PDX+ (cyto)NTNTSNP‡HumanCNV370-Duo version 1.0 (Illumina).Karikari et al15Karikari I.O. Gilchrist C.L. Jing L. Alcorta D.A. Chen J. Richardson W.J. Gabr M.A. Bell R.D. Kelley M.J. Bagley C.A. Setton L.A. Molecular characterization of chordoma xenografts generated from a novel primary chordoma cell source and two chordoma cell lines.J Neurosurg Spine. 2014; 21: 386-393Crossref PubMed Scopus (15) Google Scholar (2014)DVC-4Sacral CLD+ (nuc)NTNTPCR at brachy locusHsu et al16Hsu W. Mohyeldin A. Shah S.R. ap Rhys C.M. Johnson L.F. Sedora-Roman N.I. Kosztowski T.A. Awad O.A. McCarthy E.F. Loeb D.M. Wolinsky J.P. Gokaslan Z.L. Quinones-Hinojosa A. Generation of chordoma cell line JHC7 and the identification of Brachyury as a novel molecular target.J Neurosurg. 2011; 115: 760-769Crossref PubMed Scopus (86) Google Scholar (2011)JHC7Sacral CLD+ (nuc)++KaryotypeG-bandingPresneau et al17Presneau N. Shalaby A. Ye H. Pillay N. Halai D. Idowu B. Tirabosco R. Whitwell D. Jacques T.S. Kindblom L.G. Bruderlein S. Moller P. Leithner A. Liegl B. Amary F.M. Athanasou N.N. Hogendoorn P.C. Mertens F. Szuhai K. Flanagan A.M. Role of the transcription factor T (brachyury) in the pathogenesis of sporadic chordoma: a genetic and functional-based study.J Pathol. 2011; 223: 327-335Crossref PubMed Scopus (150) Google Scholar (2011)U-CH1Sacral CLD+ (nuc)NTNTFISHLiu et al18Liu X. Nielsen G.P. Rosenberg A.E. Waterman P.R. Yang W. Choy E. Sassi S. Yang S. Harmon D.C. Yang C. Schwab J.H. Kobayashi E. Mankin H.J. Xavier R. Weissleder R. Duan Z. Hornicek F.J. Establishment and characterization of a novel chordoma cell line: CH22.J Orthop Res. 2012; 30: 1666-1673Crossref PubMed Scopus (32) Google Scholar (2012)CH22Sacral CLD+ (nuc)++NoneDeComas et al19DeComas A.M. Penfornis P. Harris M.R. Meyer M.S. Pochampally R.R. Derivation and characterization of an extra-axial chordoma cell line (EACH-1) from a scapular tumor.J Bone Joint Surg Am. 2010; 92: 1231-1240Crossref PubMed Scopus (19) Google Scholar (2010)EACH-1Scapular CLD+ (nuc)++KaryotypeCK, cytokeratin; CLD, cell-line–derived xenograft; cyto, cytoplasmic; FISH, fluorescence in situ hybridization; Genomic, type of genomic analysis conducted; NT, not tested; nuc, nuclear; PDX, patient-derived xenograft; SNP, single-nucleotide polymorphism; +, positive immunostaining.∗ SNP Affymetrix 6.0.† SNP Illumina 600W (Illumina, San Diego, CA).‡ HumanCNV370-Duo version 1.0 (Illumina). Open table in a new tab CK, cytokeratin; CLD, cell-line–derived xenograft; cyto, cytoplasmic; FISH, fluorescence in situ hybridization; Genomic, type of genomic analysis conducted; NT, not tested; nuc, nuclear; PDX, patient-derived xenograft; SNP, single-nucleotide polymorphism; +, positive immunostaining. The patient-derived chordoma xenograft has been characterized histopathologically and by molecular analysis to determine if phenotype and genotype are maintained in serial generations. We previously identified frequent loss of fragile histidine triad (FHIT) protein expression in chordoma and postulated a potential role in chordoma pathogenesis.20Diaz R.J. Guduk M. Romagnuolo R. Smith C.A. Northcott P. Shih D. Berisha F. Flanagan A. Munoz D.G. Cusimano M.D. Pamir M.N. Rutka J.T. High-resolution whole-genome analysis of skull base chordomas implicates FHIT loss in chordoma pathogenesis.Neoplasia. 2012; 14: 788-798Crossref PubMed Scopus (34) Google Scholar Therefore, FHIT expression and genetic aberration at the FHIT locus were investigated in this patient-derived chordoma xenograft. FHIT is a well-characterized tumor suppressor and a potential target for therapy.20Diaz R.J. Guduk M. Romagnuolo R. Smith C.A. Northcott P. Shih D. Berisha F. Flanagan A. Munoz D.G. Cusimano M.D. Pamir M.N. Rutka J.T. High-resolution whole-genome analysis of skull base chordomas implicates FHIT loss in chordoma pathogenesis.Neoplasia. 2012; 14: 788-798Crossref PubMed Scopus (34) Google Scholar Patient consent for use of tumor material for research purposes was obtained and tissue collection was conducted as per approval by the St. Michael's Hospital Institutional Review Board. The patient was a 68-year-old man who presented with a 1-year history of neck pain and tongue weakness. Preoperative magnetic resonance imaging and computed tomography scan of the skull base showed a central mass in the lower clivus, extending to the level of the C2 vertebra. No evidence of metastatic disease was found. Heterogenous contrast enhancement, T1-weighted hypointensity, T2-weighted hyperintensity, and bony erosion were observed (Figure 1). An extended image-guided endoscopic transnasal transphenoidal surgical resection was performed. After ensuring sufficient tumor tissue had been obtained for pathologic diagnosis, several pieces of tumor were wrapped in sterile gauze and placed in a partially filled specimen container containing saline to keep the tissue moist. The consulting pathologist confirmed the diagnosis of classic chordoma, not otherwise specified. The tumor showed integrase interactor 1 and p53 expression by immunohistochemistry. The patient had rapid progression of disease after surgical treatment. The specimen container was transported on ice. Under a biological containment hood the tumor sample was divided and a portion was frozen in liquid nitrogen and stored at −80°C. The remainder of the tissue was minced (≤1-mm fragments) and suspended in 750 μL of phosphate-buffered saline (PBS) and kept cold on ice. Two nonobese diabetic/severe combined immunodeficiency γ mice (The Jackson Laboratory, Bar Harbor, ME) were anesthetized by isoflurane gas and a small transverse incision (approximately 4 mm) was made over the posterior parietal skull and suboccipital musculature (n = 2) or over the flank (n = 1). The parietal bone was scraped with the scalpel to thin the outer cortex. The 250 μL of the minced tissue suspension was mixed with 250 μL of Matrigel (Becton Dickinson, Franklin Lakes, NJ) and then implanted by wide-bore pipette into the subcutaneous epicranial space above the posterior parietal bone and suboccipital musculature or into the subcutaneous space over the flank. The incisions were closed with absorbable suture and the mice recovered. Serial inspection of the wound and implantation site was performed on a daily basis for 1 week and then on a weekly basis. Mice received 5 mg/kg ketoprofen analgesic and a bolus of 0.5 mL 0.9% saline subcutaneously in the immediate period after surgery. Animal experiments were approved by the Hospital for Sick Children Animal Care Committee (AUP0204-H) and conducted in accordance with the Ontario Animals for Research Act and the Canadian Council for Animal Care guidelines. Palpable tumors were measured with a digital caliper to estimate the tumor volume (in mm3) using the following formula: tumor volume = length (mm) × width2 (mm2)/2. Serial transplantation of tumors was performed when the tumor exceeded 1.5 cm in maximum diameter. Mice were anesthetized using isoflurane gas and the tumor was excised and immediately placed into cold PBS for mincing. The tumor bulk was divided into four equal portions: one portion was frozen in liquid nitrogen, one portion was fixed in 4% formaldehyde or cryopreserved in liquid nitrogen isopentane for histologic analysis, and the remaining two portions were resuspended in 1 mL PBS with 1 mL Matrigel. From the PBS:Matrigel mix, 400 μL was implanted into the epicranial space above the posterior parietal bone and suboccipital musculature of anesthetized nonobese diabetic/severe combined immunodeficiency γ mice (n = 4) as per the procedure described in Initial Implantation Procedure. This process was continued for subsequent generations of tumor growth with two modifications. After P1, the implantation ratio was changed to one tumor to three mice for P2 to P4. From P5 and onward, the implantation ratio was one tumor to two mice in order to achieve faster tumor regeneration. The implantation volume was changed to a minced tissue suspension of 75 μL PBS and 75 μL Matrigel to allow for a smaller epicranial pocket. Tumor growth was monitored on a weekly basis and tumor volume measurements were initiated every 15 days when tumors reached a minimum of 0.2 mm in maximum diameter. Mice were euthanized for collection of tumor if any pain, severe disability, or tumor maximum diameter greater than 1.5 cm was reached in accordance with institutional humane animal use protocols. Upon death, the dorsal subcutaneous space was opened and inspected for invasion of the skull by tumor. Paraffin tissue sections (5 μm) were deparaffinized, rehydrated, and pretreated in citrate buffer, pH 6.0, for 15 minutes. Sections were blocked in 10% goat serum and incubated in primary antibodies for 1 hour at room temperature: Brachyury (1:100, 04-135; Millipore, Burlington, MA), FHIT (1:100, HPA018909; Sigma, St. Louis, MO), pan-cytokeratin (1:100, NBP2-29429; Novus Bio, Littleton, CO), Ki67 (1:50, CRM 325 A; Biocare Medical, Pacheco, CA), INI1 (1:50, 612110; BD Biosciences), and p53 (1:50, 554294; BD Biosciences). Flash-frozen OCT-embedded tumor tissue sections (8 μm) were blocked in 10% goat serum and incubated with anti-nuclei antibody clone 235-1 (1:100, MAB1812; Millipore)21Bissig-Choisat B. Wang L. Legras X. Saha P.K. Chen L. Bell P. Pankowicz F.P. Hill M.C. Barzi M. Kettlun Leyton C. Leung H.C. Kruse R.L. Himes R.W. Goss J.A. Wilson J.M. Chan L. Lagor W.R. Bissig K.D. Development and rescue of human familial hypercholesterolaemia in a xenograft mouse model.Nat Commun. 2015; 6: 7339Crossref PubMed Scopus (41) Google Scholar for 1 hour at room temperature. This antibody recognizes human nuclei, but not mouse or rat nuclei. After incubation with primary antibody and washing three times, biotinylated secondary antibody 1:100 (ABC kit; Vector Labs, Burlington, Ontario, Canada) was applied for 30 minutes at room temperature. Sections then were exposed to the avidin-biotin detection system for 40 minutes at room temperature. After three washes, signal was detected using diaminobenzidine, counterstained in hematoxylin, and mounted in Permount (Thermo Fisher Scientific, Waltham, MA). Micrographs were captured with an Infinity1 camera (Lumenera Corporation, Ottawa, Ontario, Canada) with Infinity1 software visualized through an Olympus BX43 light microscope (Olympus, Tokyo, Japan). The Ki67 index was calculated by counting the number of positive nuclei in 10 high-power fields (20× objective). Genomic DNA was isolated from tumor samples and snap-frozen in liquid nitrogen at the time of passaging. A proteinase K/SDS buffer was used to dissociate the tissue (10 mmol/L Tris-Cl [pH 8.0], 0.1 mol/L EDTA [pH 8.0], 0.5% (w/v) SDS, 20 mg/mL proteinase K) overnight at 55°C. Protein extraction was achieved with phenol-chloroform-isoamyl alcohol using phase-lock centrifuge gels (5-Prime, Gaithersburg, MD) and DNA was precipitated with 100% ethanol and 7.5 mol/L ammonium acetate. The pellet was washed with 75% ethanol and then dried. It was resuspended in Tris-ethanolamine (pH 8.0) buffer and quantified by NanoDrop (NanoDrop, Willmington, DE). Microarray processing was performed in The Centre for Applied Genomics, The Hospital for Sick Children. Briefly, genomic DNA was cleaved with Nsp and Sty nucleases, biotinylated, and hybridized to the Genome-Wide Human Single-Nucleotide Polymorphism (SNP) Array 6.0 (Affymetrix, Santa Clara, CA). Scanning of the microarray was performed on a Affymetrix GeneChip Scanner 3000. Copy number data were analyzed using Partek Genomic Suite 6.6 v6.15.1207 (Partek, St. Louis, MO). Samples were imported, background was normalized using Partek Defaults, and the copy number was calculated from allele-specific intensity. Genomic segmentation was performed using the following parameters: i) 150 minimum genomic markers were required; ii) P value threshold was 0.001; iii) signal-to-noise ratio was 0.3; and iv) diploid status was in the range of 1.7 and 2.3, losses had a copy number <1.7, and gains had a copy number >2.3. Copy number alteration counts were determined by summing the segmentation results <1.7 and >2.3. Data sets GSE25720 and GSE9023 were downloaded for comparative analysis from GEO at the NCBI website (https://www.ncbi.nlm.nih.gov/geo). GSE25720 samples were analyzed on the Genome-Wide Human SNP Array 6.0 and data analysis was performed as described in the previous paragraph. For GSE9023 samples, which were processed using the klingenkhh BAC 32K Full array, normalized log2 ratios were imported into Partek Genomic Suite to be visualized. Genomic segmentation was performed using the following parameters: i) 10 minimum genomic markers were required; ii) P value threshold was 0.001; iii) signal-to-noise ratio was 0.3; and iv) diploid status was in the range of −0.15 and 0.15, losses had a copy number <−0.15, and gains had a copy number >0.15. The RNAscope in situ hybridization (ACDBio, Newark, CA) assay was performed on formalin-fixed, paraffin-embedded sections from the originating patient-derived xenograft (PDX0) and third-generation (PDX3) tumors. Briefly, tissue sections were deparaffinized and treated with hydrogen peroxide, followed by RNA target retrieval and protease treatment. After the sections were prepared, a commercial probe against human T gene mRNA was used to hybridize to the target, followed by staining of the targets with RNAscope 2.5 HD Red Detection reagent (ACDBio). Slides were counterstained with hematoxylin and mounted using EcoMount (BioCare Medical, Pacheco, CA). Whole slide scans were acquired using an Aperio AT2 Scanner (Leica Biosystems, Wetzlar, Germany) at ×40 magnification. Images then were analyzed using Definiens Developer XD software version 2.6 (Definiens, Munich, Germany), using a custom-programed algorithm to count the number of spots per nucleus (indicating single RNA molecules) in the entire tumor region. Prism 7 (GraphPad Software, Inc., La Jolla, CA) was used for statistical analysis. All measures are reported as means ± SEM. Means for survival time, tumor diameter, and Ki67 index were compared by an unpaired Welch t-test with a P value <0.05 set as significant. RNAscope data were compared using negative binomial regression. Clival chordoma tissue obtained at the time of surgical resection (Figure 1) was implanted in a Matrigel (Corning, Corning, NY) slurry within the epicranial space over the posterior parietal bone and in the subcutaneous space of the flank of nonobese diabetic/severe combined immunodeficiency mice. After 4.5 months a palpable and visible tumor formed in the epicranial space in 2 of 2 mice (Figure 2). No tumor formed in the subcutaneous space at the flank of the third host mouse by 4.5 months. The tumor from the epicranial space from one of the mice was passaged serially and is now in the 7th generation (Table 2). The patient-derived xenograft has been made available to the research community through the Chordoma Foundation (https://www.chordomafoundation.org, last accessed September 4, 2017). On gross pathology, bony invasion was observed in each generation from PDX3 to PDX7; however, it was not apparent for every tumor within each generation (Table 2). Microscopic bony invasion consisted of nests of tumor cells surrounded by mesenchymal cells (Figure 2). A pattern of successive growth rate increase over successive generations was observed as the tumor to host transplantation ratio was changed from 1:4 to 1:2. The median time to the appearance of the first tumor measuring >0.2 mm in maximum diameter was 104 ± 12 days (n = 15) in PDX4 (1:3 transplant ratio) compared with 44 ± 11 days (n = 10) in PDX7 (1:2 transplant ratio) (P = 0.001). The estimated volumetric tumor growth rates showed a wide range of variability in the PDX4 compared with the PDX7 generation (Figure 3A). The Ki67 labeling index was significantly higher in the PDX7 generation (10% ± 2%) compared with parental tumor (3% ± 1%; P = 0.002) (Figure 3B). Along with the faster tumor growth a decrease in mouse survival was observed over generations, with mean survival times of 35 ± 3 weeks (n = 15) in PDX4 compared with 27 ± 1 week (n = 10) in PDX7 (P = 0.026).Table 2Tally of Tumors Formed in Each Generation after Serial Host-To-Host ImplantationGenerationMice implanted, nTumors formed, nTime to first measurable tumor >0.2 mm length, daysTumors with gross bony invasion, nPDX0221260PDX144890PDX2552240PDX399514PDX41715754PDX599305PDX61010306PDX71010157 Open table in a new tab Figure 3A: Tumor growth curves for individual mice over time since implantation of tumor xenograft. Growth in the mice in the fourth generation (PDX4) and seventh generation (PDX7) is compared showing earlier tumor formation in PDX7. B: Histologic sections of original tumor, PDX4, and PDX7 showing Ki67 immunohistochemistry. Graph quantifying the Ki67 index in original tumor, PDX4, and PDX7. The Ki67 index is higher in PDX7 tumor compared with original tumor. **P < 0.01. Scale bars = 50 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The xenograft tumor showed similar cytology as the original tumor with lobules of physaliferous cells (Figure 4). Nuclear Brachyury expression and cytokeratin expression was conserved over generations from original tumor to PDX3 (Figure 4). The original and PDX3 tumor did not express epithelial membrane antigen, as expected for a chordoma (Figure 4). The identity of the chordoma xenograft was confirmed by immunohistochemistry independently by Valasciences, Inc. (San Diego, CA). The physaliferous cells of the patient-derived xenograft expressed human FHIT in a cytoplasmic and perinuclear location, although cytoplasmic expression was prominent (Figure 5). FHIT expression was conserved over the first three generations. Absent anti-human FHIT immunolabeling of connective tissue components in the xenografts was observed, as would be expected owing to the antibody specificity (Figure 5). Concordantly, anti-human nucleus antibody staining of physaliferous cells, but not the connective tissue component of the xenograft tumors, was observed (Figure 5).Figure 5A: Histologic section of parental chordoma immunolabeled with anti-human FHIT antibody. B: Histologic section of third-generation xenograft" @default.
• W2894062761 created "2018-10-05" @default.
• W2894062761 creator A5003268046 @default.
• W2894062761 creator A5008966396 @default.
• W2894062761 creator A5011967500 @default.
• W2894062761 creator A5017136932 @default.
• W2894062761 creator A5029519500 @default.
• W2894062761 creator A5040249971 @default.
• W2894062761 creator A5046496883 @default.
• W2894062761 creator A5052443665 @default.
• W2894062761 creator A5053672556 @default.
• W2894062761 creator A5067215084 @default.
• W2894062761 creator A5083880051 @default.
• W2894062761 date "2018-12-01" @default.
• W2894062761 modified "2023-09-26" @default.
• W2894062761 title "Characterization of a Clival Chordoma Xenograft Model Reveals Tumor Genomic Instability" @default.
• W2894062761 cites W1491774226 @default.
• W2894062761 cites W1503533733 @default.
• W2894062761 cites W1530602351 @default.
• W2894062761 cites W1533601335 @default.
• W2894062761 cites W1563804318 @default.
• W2894062761 cites W1564861257 @default.
• W2894062761 cites W1566578393 @default.
• W2894062761 cites W1969418387 @default.
• W2894062761 cites W1974490995 @default.
• W2894062761 cites W2000611245 @default.
• W2894062761 cites W2004371430 @default.
• W2894062761 cites W2007419457 @default.
• W2894062761 cites W2027362881 @default.
• W2894062761 cites W2030998129 @default.
• W2894062761 cites W2046760010 @default.
• W2894062761 cites W2051311335 @default.
• W2894062761 cites W2053702504 @default.
• W2894062761 cites W2067708830 @default.
• W2894062761 cites W2069248616 @default.
• W2894062761 cites W2076283983 @default.
• W2894062761 cites W2096240841 @default.
• W2894062761 cites W2096585698 @default.
• W2894062761 cites W2102036561 @default.
• W2894062761 cites W2104952546 @default.
• W2894062761 cites W2126260685 @default.
• W2894062761 cites W2166253719 @default.
• W2894062761 cites W2166870017 @default.
• W2894062761 cites W2171019961 @default.
• W2894062761 cites W2264448154 @default.
• W2894062761 cites W2605860178 @default.
• W2894062761 cites W2741070606 @default.
• W2894062761 cites W2763732767 @default.
• W2894062761 doi "https://doi.org/10.1016/j.ajpath.2018.08.004" @default.
• W2894062761 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/30248342" @default.
• W2894062761 hasPublicationYear "2018" @default.
• W2894062761 type Work @default.
• W2894062761 sameAs 2894062761 @default.
• W2894062761 citedByCount "7" @default.
• W2894062761 countsByYear W28940627612019 @default.
• W2894062761 countsByYear W28940627612021 @default.
• W2894062761 countsByYear W28940627612022 @default.
• W2894062761 crossrefType "journal-article" @default.
• W2894062761 hasAuthorship W2894062761A5003268046 @default.
• W2894062761 hasAuthorship W2894062761A5008966396 @default.
• W2894062761 hasAuthorship W2894062761A5011967500 @default.
• W2894062761 hasAuthorship W2894062761A5017136932 @default.
• W2894062761 hasAuthorship W2894062761A5029519500 @default.
• W2894062761 hasAuthorship W2894062761A5040249971 @default.
• W2894062761 hasAuthorship W2894062761A5046496883 @default.
• W2894062761 hasAuthorship W2894062761A5052443665 @default.
• W2894062761 hasAuthorship W2894062761A5053672556 @default.
• W2894062761 hasAuthorship W2894062761A5067215084 @default.
• W2894062761 hasAuthorship W2894062761A5083880051 @default.
• W2894062761 hasBestOaLocation W28940627611 @default.
• W2894062761 hasConcept C142724271 @default.
• W2894062761 hasConcept C143425029 @default.
• W2894062761 hasConcept C178169997 @default.
• W2894062761 hasConcept C2781190729 @default.
• W2894062761 hasConcept C502942594 @default.
• W2894062761 hasConcept C54355233 @default.
• W2894062761 hasConcept C552990157 @default.
• W2894062761 hasConcept C71924100 @default.
• W2894062761 hasConcept C86803240 @default.
• W2894062761 hasConceptScore W2894062761C142724271 @default.
• W2894062761 hasConceptScore W2894062761C143425029 @default.
• W2894062761 hasConceptScore W2894062761C178169997 @default.
• W2894062761 hasConceptScore W2894062761C2781190729 @default.
• W2894062761 hasConceptScore W2894062761C502942594 @default.
• W2894062761 hasConceptScore W2894062761C54355233 @default.
• W2894062761 hasConceptScore W2894062761C552990157 @default.
• W2894062761 hasConceptScore W2894062761C71924100 @default.
• W2894062761 hasConceptScore W2894062761C86803240 @default.
• W2894062761 hasFunder F4320320879 @default.
• W2894062761 hasIssue "12" @default.
• W2894062761 hasLocation W28940627611 @default.
• W2894062761 hasLocation W28940627612 @default.
• W2894062761 hasOpenAccess W2894062761 @default.
• W2894062761 hasPrimaryLocation W28940627611 @default.
• W2894062761 hasRelatedWork W2012774801 @default.
• W2894062761 hasRelatedWork W2014862128 @default.
• W2894062761 hasRelatedWork W2020502949 @default.
• W2894062761 hasRelatedWork W2052570858 @default.

• next