GENES, CHROMOSOMES & CANCER 53:991–998 (2014)
RESEARCH ARTICLES
Identification of an MLL4-GPS2 Fusion as an Oncogenic Driver of Undifferentiated Spindle Cell Sarcoma in a Child Elaine O’Meara,1 Deirdre Stack,1 Susan Phelan,1 Naomi McDonagh,1 Lorna Kelly,1 Raf Sciot,2 Maria Debiec-Rychter,3 Thomas Morris,4 Doug Cochrane,5 Poul Sorensen,6 and Maureen J. O’Sullivan1,7,8* 1
Cancer Genetics Program,The National Children’s Research Centre,Crumlin,Dublin12,Ireland Department of Pathology,K.U.Leuven,University Hospital Gasthuisberg,Leuven,Belgium 3 Department of Genetics,K.U.Leuven,University Hospital Gasthuisberg,Leuven,Belgium 4 Cytogenetics Laboratory,National Centre for Medical Genetics,Our Lady’s Children’s Hospital,Crumlin,Dublin12,Ireland 5 Division of Neurosurgery,British Columbia’s Children’s Hospital,Vancouver,V6H 3V4,Canada 6 Department of Molecular Oncology,British Columbia Cancer Center,Vancouver, V5Z1L3,Canada 7 Department of Pathology,Our Lady’s Children’s Hospital,Crumlin,Dublin12,Ireland 8 Department of Histopathology, School of Medicine,University of Dublin,Trinity College,Dublin 2,Ireland 2
Undifferentiated spindle cell sarcoma (UDS) is a poorly defined or understood entity, essentially a waste-basket for cases failing to fulfill criteria for better-established diagnoses based on combined histology, immunohistochemistry, and tumor genetic assays. We identified a novel chromosomal translocation t(17;19)(p13;q13) in a pediatric UDS and have characterized this alteration to show rearrangement of the MLL4 and GPS2 genes, resulting in an in-frame fusion gene MLL4GPS2, the expression of which promotes anchorage-independent growth. MLL4 was previously reported to be similarly rearranged in hepatocellular carcinomas, notably those positive for hepatitis B virus. Isolated reports of individual rearrangements of GPS2 in a prostate carcinoma cell line and in glioblastoma multiforme, each with different partner genes, recently emerged from high-throughput sequencing studies but have not been further evaluated for biological C 2014 Wiley Periodicals, Inc. V effect.
INTRODUCTION
Chromosomal translocations number among the major oncogenic events recognized in human cancer biology (Sandberg, 1983). In tumors with complex karyotypes that show multiple numerical and structural aberrations, it can be difficult to separate out the biologically important events from the “noise” of by-stander mutations that reflect chromosomal instability. Major progress, therefore, has occurred preferentially in tumors with “simple” karyotypes, harboring few or even solitary recurrent aberrations. Our index case was an undifferentiated spindle cell sarcoma (UDS), which arose as an intracranial, extra-axial mass in a 12-year-old girl. Karyotypic analysis revealed a balanced nonconstitutional t(17;19)(p13;q13) occurring as the solitary cytogenetic aberration. Following surgery, no additional treatment was offered and at relapse the identical tumor karyotype was observed. This consistent, solitary gross aberration prompted us to work the case up further, relating the findings also to a wider cohort of human tumors. C 2014 Wiley Periodicals, Inc. V
MATERIALS AND METHODS G-Banding and Fluorescence In Situ Hybridization
G-banded chromosome analysis was performed on disaggregated tumor biopsy cells arrested in metaphase after culture and reported according to the International System for Human Cytogenetic Nomenclature. Fluorescence in situ hybridization (FISH) was performed on air-dried touch preparations from the snap-frozen tumor, which were incubated in pepsin solution for 40–70 sec at 37 C prior to dehydration in a series of ethanol washes (70%–100%). Bacterial artificial chromosomes Additional Supporting Information may be found in the online version of this article. Supported by the Health Research Board of Ireland Clinician Scientist Award (CSA/2012/13) granted to MJO’S. *Correspondence to: Maureen J. O’Sullivan, Department of Pathology, Our Lady’s Children’s Hospital, Crumlin, Dublin 12, Ireland. E-mail: [email protected] Elaine O’Meara and Deirdre Stack contributed equally to this work. Received 26 May 2014; Accepted 24 July 2014 DOI 10.1002/gcc.22208 Published online 19 August 2014 in Wiley Online Library (wileyonlinelibrary.com).
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(BACs) were chosen from the UCSC Human Genome Browser to flank the chromosome breakpoints as observed from the G-banding pattern, and were obtained from the BACPAC resources center (Children’s Hospital Oakland Research Institute, Oakland, CA). Following sequential BAC FISH, which narrowed down the breakpoint region, fosmid FISH was performed using sequentially proximate fosmid probes, again chosen and obtained as per BAC probes. Commercial control probes included are listed in Supporting Information Table 1 along with the BACs and fosmids. The probe pairs were differentially labeled with red-dUTP and green-dUTP (Enzo Life Sciences, Ann Arbor, MI) using a Nick Translation Kit (Abbott Molecular, Des Plaines, IL). Appropriate fluorescently labeled BAC or commercial probe mixtures were placed on sample slides and cohybridized at 75 C for 4 min and then incubated at 37 C overnight in a Hybrite humidity chamber (Abbott Molecular Inc, Des Plaines, IL). The slides were washed in 0.43 SSC/0.3% NP-40 for 2 min at 72 C followed by 1 min in 23 SSC/0.3% NP-40 at room temperature. DAPI II was applied as a counterstain, and slides were coverslipped and viewed with an Olympus BX51 (Olympus, Center Valley, PA) fluorescence microscope with Cytovision software (Leica Biosystems, Milton Keynes, UK). Once the breakpoint region had been confirmed and validated as described below for each chromosome, a confirmatory FISH assay was used to evaluate a cohort of further tumors, including 21 snapfrozen UDSs collected from the Catholic University of Leuven, Belgium, (courtesy of Prof. Raf Sciot), and 26 hepatocellular carcinomas (HCCs) from the Pathology Department at Washington University of St. Louis, MO. The spindle cell sarcomas were all out of necessity from adult patients between 24- and 83-years-old derived from a variety of anatomic locations and with relatively more complex karyotypes than our index case (Supporting Information Table 3). The hepatitis B and C status of the HCC cases were unavailable due to anonymization. Rapid Amplification of cDNA Ends by Polymerase Chain Reaction (30 RACE PCR)
RACE Polymerase Chain Reaction (PCR) was performed using the 50 /30 Race Kit 2nd Generation (Roche, West Sussex, UK) as per the manufacturer’s protocol with total RNA extracted from snap-frozen tumor tissue from the index case using the RNeasy Kit (Qiagen, Manchester, UK). Genes, Chromosomes & Cancer DOI 10.1002/gcc
Briefly, first strand cDNA synthesis was performed using the Oligo-dT anchor primer provided and incubating the sample at 55 C for 60 min followed by 5 min at 85 C. PCR amplification of the cDNA was then performed using a gene-specific forward primer for MLL4 (RACE3 primer, Supporting Information Table 1) and a PCR anchor primer provided. Second round PCR was performed using a nested MLL4 primer (RACE4 primer, Supporting Information Table 1) and the PCR anchor primer. PCR products were visualized on 1.5% agarose gels using SYBR safe (Life Technologies, Paisley, UK). Bands of interest were then excised from the gel and purified using a Gel Extraction Kit (Qiagen, Manchester, UK) and the products were sequenced. Reverse-Transcriptase PCR (RT-PCR)
For reverse transcriptase (RT)-PCR confirmation of the fusion, total RNA was extracted from snap-frozen index tumor tissue using RNeasy kit (Qiagen, Manchester, UK). Reverse transcription was performed using Superscript III first strand synthesis system (Invitrogen, Paisley, UK). The primer set (Supporting Information Table 1) was designed to include an MLL4 forward and a GPS2 reverse primer. Amplification of the MLL4-GPS2 product was performed using HotStarTaq DNA polymerase (Qiagen, Manchester, UK). PCR products were sequenced by LGC Genomics GmBH (Berlin, Germany) on an Applied Biosystems 3730xl DNA analyser (Carlsbad, CA). Cell Culture Conditions
HEK293 and NIH3T3 cells with and without pcDNA3HA constructs were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Paisley, UK) containing 10% Fetal Bovine Serum (FBS) (Gibco, Paisley, UK) and 1% penicillin/ streptomycin solution (Sigma-Aldrich, Dorset, UK). Plasmid Construction
The sequence encoding GPS2 and MLL4-GPS2 was amplified by PCR and inserted into pcDNA3HA (Invitrogen, Paisley, UK) and phrGFPIIN (Stratagene, Santa Clara, CA) vectors. Briefly, cDNA was generated from spleen tissue RNA using Superscript III First Strand Synthesis System (Invitrogen, Paisley, UK). GPS2 was amplified from the cDNA using HotStarTaq (Qiagen, Manchester, UK) and GPS2 forward and reverse primers (Supporting Information
NOVEL T(17;19) IN UNDIFFERENTIATED SPINDLE CELL SARCOMA
Table 1) with engineered restriction sites for insertion into phrGFPIIN (phrGPS2For and phrGPS2Rev) and into pcDNA3HA (pcDNAGPS2For and pcDNAGPS2Rev). The amplicon was inserted into vector pCR2.1 using a TOPO TA cloning kit (Invitrogen, Paisley, UK). GPS2 was excised from the plasmid by restriction digest, and ligated into pcDNA3HA and phrGFPIIN. cDNA was generated from the index case of t(17;19) UDS as above, and MLL4-GPS2 fusion was amplified using MLL4 forward and GPS2 reverse primers (Supporting Information Table 1) for insertion into phrGFPIIN (phrMLL4For and phrGPS2Rev) and pcDNA3HA (pcDNAMLL4For and pcDNAGPS2Rev). The amplicon was inserted into pCR2.1, and subsequently pcDNA3HA and phrGFPIIN, as outlined above. All plasmid constructs were sequenced by LGC Genomics GmBH (Berlin, Germany) on an Applied Biosystems 3730xl DNA analyser (Carlsbad, CA). Transient Transfection
HEK293 and NIH3T3 cells were transiently transfected with empty phrGPFIIN, and phrGFPIIN containing GPS2 or MLL4-GPS2 using Lipofectamine 2000 (Life Technologies, Paisley, UK). Cells were imaged on an LSM 700 microscope (Zeiss, Cambridge, UK). Stable Transfection
HEK293 cells were transfected with pcDNA3HA empty vector, pcDNA3HA-GPS2, or pcDNA3HA-MLL4-GPS2 using TransIT-LT1 transfection reagent (Mirus, Madison, WI). To select cells stably expressing the HA-tagged proteins, the cells were treated with 800 mg/ml G418 (Sigma-Aldrich, Dorset, UK) for 3 weeks until control untransfected HEK293 cells were dead. Colony Formation in Soft Agar
Stable transfectants of HEK293 cells with pcDNA3HA, pcDNA3HA-GPS2, or pcDNA3HAMLL4-GPS2 were studied for their ability to grow without anchorage, in soft agar assays. A layer of 0.5% low gelling temperature agarose (SigmaAldrich, Dorset, UK) was allowed to solidify in 6well plates. This layer was overlaid with 0.375% agarose in DMEM (supplemented with FBS and Pen/Strep) containing 1 3 104 cells. Triplicate wells were seeded for each cell line. The cell/agarose was overlaid with DMEM medium containing FBS and Pen/Strep, and incubated for 15 days. Medium was changed on the plates every 4 days.
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At the end of the assay, the wells were stained with 0.05% crystal violet (Sigma-Aldrich, Dorset, UK) and the cell colonies counted. Western Blotting
Protein from HEK293 cells containing empty pcDNA3HA, pcDNA3HA-GPS2, or pcDNA3HAMLL4-GPS2 was harvested by direct lysis of cells in RIPA buffer (Sigma-Aldrich, Dorset, UK) containing protease and phosphatase inhibitors (Roche, West Sussex, UK). Protein concentration was determined by detergent compatible (DC) protein assay (Biorad, Hertfordshire, UK). The protein samples were separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels, and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Hertfordshire, UK). The membranes were blocked in 5% nonfat milk powder reconstituted in TBS-Tween (0.05%) for 1 hr at room temperature and antibody incubations were performed overnight at 4 C in 5% milk/TBS-Tween (0.05%). The antibodies used were HA-tag antibody (Abcam, Cambridge, UK) and anti-beta actin (Abcam, Cambridge, UK). The membranes were washed in TBS-Tween and subsequently incubated with HRP-conjugated anti-rabbit antibody (Abcam, Cambridge, UK) for 1 hr at RT. After washing, the membranes were incubated with Supersignal West Dura chemiluminescent substrate (Thermo Scientific, Loughborough, UK) and exposed to X-ray film (Thermo Scientific, Loughborough, UK). RESULTS
The patient underwent postoperative radiation therapy to a total dose of 54Gy over 30 fractions after second surgery with total resection. She remains disease-free 9 years post-therapy, albeit requiring a wheel-chair for mobilization. Morphologic examination of Hematoxylin and Eosin stained sections showed a variably cellular tumor with a rich supply of fine, curvilinear blood vessels, and composed of spindle cells with poorly defined cell borders and elongated bland-appearing nuclei (Fig. 1A), focal microcystic change (Fig. 1B) and localized condensation of cells arranged in a more obvious storiform pattern, within which area mitotic activity was maximal (Fig. 1C). Mitotic activity overall was
RESEARCH ARTICLES
Identification of an MLL4-GPS2 Fusion as an Oncogenic Driver of Undifferentiated Spindle Cell Sarcoma in a Child Elaine O’Meara,1 Deirdre Stack,1 Susan Phelan,1 Naomi McDonagh,1 Lorna Kelly,1 Raf Sciot,2 Maria Debiec-Rychter,3 Thomas Morris,4 Doug Cochrane,5 Poul Sorensen,6 and Maureen J. O’Sullivan1,7,8* 1
Cancer Genetics Program,The National Children’s Research Centre,Crumlin,Dublin12,Ireland Department of Pathology,K.U.Leuven,University Hospital Gasthuisberg,Leuven,Belgium 3 Department of Genetics,K.U.Leuven,University Hospital Gasthuisberg,Leuven,Belgium 4 Cytogenetics Laboratory,National Centre for Medical Genetics,Our Lady’s Children’s Hospital,Crumlin,Dublin12,Ireland 5 Division of Neurosurgery,British Columbia’s Children’s Hospital,Vancouver,V6H 3V4,Canada 6 Department of Molecular Oncology,British Columbia Cancer Center,Vancouver, V5Z1L3,Canada 7 Department of Pathology,Our Lady’s Children’s Hospital,Crumlin,Dublin12,Ireland 8 Department of Histopathology, School of Medicine,University of Dublin,Trinity College,Dublin 2,Ireland 2
Undifferentiated spindle cell sarcoma (UDS) is a poorly defined or understood entity, essentially a waste-basket for cases failing to fulfill criteria for better-established diagnoses based on combined histology, immunohistochemistry, and tumor genetic assays. We identified a novel chromosomal translocation t(17;19)(p13;q13) in a pediatric UDS and have characterized this alteration to show rearrangement of the MLL4 and GPS2 genes, resulting in an in-frame fusion gene MLL4GPS2, the expression of which promotes anchorage-independent growth. MLL4 was previously reported to be similarly rearranged in hepatocellular carcinomas, notably those positive for hepatitis B virus. Isolated reports of individual rearrangements of GPS2 in a prostate carcinoma cell line and in glioblastoma multiforme, each with different partner genes, recently emerged from high-throughput sequencing studies but have not been further evaluated for biological C 2014 Wiley Periodicals, Inc. V effect.
INTRODUCTION
Chromosomal translocations number among the major oncogenic events recognized in human cancer biology (Sandberg, 1983). In tumors with complex karyotypes that show multiple numerical and structural aberrations, it can be difficult to separate out the biologically important events from the “noise” of by-stander mutations that reflect chromosomal instability. Major progress, therefore, has occurred preferentially in tumors with “simple” karyotypes, harboring few or even solitary recurrent aberrations. Our index case was an undifferentiated spindle cell sarcoma (UDS), which arose as an intracranial, extra-axial mass in a 12-year-old girl. Karyotypic analysis revealed a balanced nonconstitutional t(17;19)(p13;q13) occurring as the solitary cytogenetic aberration. Following surgery, no additional treatment was offered and at relapse the identical tumor karyotype was observed. This consistent, solitary gross aberration prompted us to work the case up further, relating the findings also to a wider cohort of human tumors. C 2014 Wiley Periodicals, Inc. V
MATERIALS AND METHODS G-Banding and Fluorescence In Situ Hybridization
G-banded chromosome analysis was performed on disaggregated tumor biopsy cells arrested in metaphase after culture and reported according to the International System for Human Cytogenetic Nomenclature. Fluorescence in situ hybridization (FISH) was performed on air-dried touch preparations from the snap-frozen tumor, which were incubated in pepsin solution for 40–70 sec at 37 C prior to dehydration in a series of ethanol washes (70%–100%). Bacterial artificial chromosomes Additional Supporting Information may be found in the online version of this article. Supported by the Health Research Board of Ireland Clinician Scientist Award (CSA/2012/13) granted to MJO’S. *Correspondence to: Maureen J. O’Sullivan, Department of Pathology, Our Lady’s Children’s Hospital, Crumlin, Dublin 12, Ireland. E-mail: [email protected] Elaine O’Meara and Deirdre Stack contributed equally to this work. Received 26 May 2014; Accepted 24 July 2014 DOI 10.1002/gcc.22208 Published online 19 August 2014 in Wiley Online Library (wileyonlinelibrary.com).
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(BACs) were chosen from the UCSC Human Genome Browser to flank the chromosome breakpoints as observed from the G-banding pattern, and were obtained from the BACPAC resources center (Children’s Hospital Oakland Research Institute, Oakland, CA). Following sequential BAC FISH, which narrowed down the breakpoint region, fosmid FISH was performed using sequentially proximate fosmid probes, again chosen and obtained as per BAC probes. Commercial control probes included are listed in Supporting Information Table 1 along with the BACs and fosmids. The probe pairs were differentially labeled with red-dUTP and green-dUTP (Enzo Life Sciences, Ann Arbor, MI) using a Nick Translation Kit (Abbott Molecular, Des Plaines, IL). Appropriate fluorescently labeled BAC or commercial probe mixtures were placed on sample slides and cohybridized at 75 C for 4 min and then incubated at 37 C overnight in a Hybrite humidity chamber (Abbott Molecular Inc, Des Plaines, IL). The slides were washed in 0.43 SSC/0.3% NP-40 for 2 min at 72 C followed by 1 min in 23 SSC/0.3% NP-40 at room temperature. DAPI II was applied as a counterstain, and slides were coverslipped and viewed with an Olympus BX51 (Olympus, Center Valley, PA) fluorescence microscope with Cytovision software (Leica Biosystems, Milton Keynes, UK). Once the breakpoint region had been confirmed and validated as described below for each chromosome, a confirmatory FISH assay was used to evaluate a cohort of further tumors, including 21 snapfrozen UDSs collected from the Catholic University of Leuven, Belgium, (courtesy of Prof. Raf Sciot), and 26 hepatocellular carcinomas (HCCs) from the Pathology Department at Washington University of St. Louis, MO. The spindle cell sarcomas were all out of necessity from adult patients between 24- and 83-years-old derived from a variety of anatomic locations and with relatively more complex karyotypes than our index case (Supporting Information Table 3). The hepatitis B and C status of the HCC cases were unavailable due to anonymization. Rapid Amplification of cDNA Ends by Polymerase Chain Reaction (30 RACE PCR)
RACE Polymerase Chain Reaction (PCR) was performed using the 50 /30 Race Kit 2nd Generation (Roche, West Sussex, UK) as per the manufacturer’s protocol with total RNA extracted from snap-frozen tumor tissue from the index case using the RNeasy Kit (Qiagen, Manchester, UK). Genes, Chromosomes & Cancer DOI 10.1002/gcc
Briefly, first strand cDNA synthesis was performed using the Oligo-dT anchor primer provided and incubating the sample at 55 C for 60 min followed by 5 min at 85 C. PCR amplification of the cDNA was then performed using a gene-specific forward primer for MLL4 (RACE3 primer, Supporting Information Table 1) and a PCR anchor primer provided. Second round PCR was performed using a nested MLL4 primer (RACE4 primer, Supporting Information Table 1) and the PCR anchor primer. PCR products were visualized on 1.5% agarose gels using SYBR safe (Life Technologies, Paisley, UK). Bands of interest were then excised from the gel and purified using a Gel Extraction Kit (Qiagen, Manchester, UK) and the products were sequenced. Reverse-Transcriptase PCR (RT-PCR)
For reverse transcriptase (RT)-PCR confirmation of the fusion, total RNA was extracted from snap-frozen index tumor tissue using RNeasy kit (Qiagen, Manchester, UK). Reverse transcription was performed using Superscript III first strand synthesis system (Invitrogen, Paisley, UK). The primer set (Supporting Information Table 1) was designed to include an MLL4 forward and a GPS2 reverse primer. Amplification of the MLL4-GPS2 product was performed using HotStarTaq DNA polymerase (Qiagen, Manchester, UK). PCR products were sequenced by LGC Genomics GmBH (Berlin, Germany) on an Applied Biosystems 3730xl DNA analyser (Carlsbad, CA). Cell Culture Conditions
HEK293 and NIH3T3 cells with and without pcDNA3HA constructs were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Paisley, UK) containing 10% Fetal Bovine Serum (FBS) (Gibco, Paisley, UK) and 1% penicillin/ streptomycin solution (Sigma-Aldrich, Dorset, UK). Plasmid Construction
The sequence encoding GPS2 and MLL4-GPS2 was amplified by PCR and inserted into pcDNA3HA (Invitrogen, Paisley, UK) and phrGFPIIN (Stratagene, Santa Clara, CA) vectors. Briefly, cDNA was generated from spleen tissue RNA using Superscript III First Strand Synthesis System (Invitrogen, Paisley, UK). GPS2 was amplified from the cDNA using HotStarTaq (Qiagen, Manchester, UK) and GPS2 forward and reverse primers (Supporting Information
NOVEL T(17;19) IN UNDIFFERENTIATED SPINDLE CELL SARCOMA
Table 1) with engineered restriction sites for insertion into phrGFPIIN (phrGPS2For and phrGPS2Rev) and into pcDNA3HA (pcDNAGPS2For and pcDNAGPS2Rev). The amplicon was inserted into vector pCR2.1 using a TOPO TA cloning kit (Invitrogen, Paisley, UK). GPS2 was excised from the plasmid by restriction digest, and ligated into pcDNA3HA and phrGFPIIN. cDNA was generated from the index case of t(17;19) UDS as above, and MLL4-GPS2 fusion was amplified using MLL4 forward and GPS2 reverse primers (Supporting Information Table 1) for insertion into phrGFPIIN (phrMLL4For and phrGPS2Rev) and pcDNA3HA (pcDNAMLL4For and pcDNAGPS2Rev). The amplicon was inserted into pCR2.1, and subsequently pcDNA3HA and phrGFPIIN, as outlined above. All plasmid constructs were sequenced by LGC Genomics GmBH (Berlin, Germany) on an Applied Biosystems 3730xl DNA analyser (Carlsbad, CA). Transient Transfection
HEK293 and NIH3T3 cells were transiently transfected with empty phrGPFIIN, and phrGFPIIN containing GPS2 or MLL4-GPS2 using Lipofectamine 2000 (Life Technologies, Paisley, UK). Cells were imaged on an LSM 700 microscope (Zeiss, Cambridge, UK). Stable Transfection
HEK293 cells were transfected with pcDNA3HA empty vector, pcDNA3HA-GPS2, or pcDNA3HA-MLL4-GPS2 using TransIT-LT1 transfection reagent (Mirus, Madison, WI). To select cells stably expressing the HA-tagged proteins, the cells were treated with 800 mg/ml G418 (Sigma-Aldrich, Dorset, UK) for 3 weeks until control untransfected HEK293 cells were dead. Colony Formation in Soft Agar
Stable transfectants of HEK293 cells with pcDNA3HA, pcDNA3HA-GPS2, or pcDNA3HAMLL4-GPS2 were studied for their ability to grow without anchorage, in soft agar assays. A layer of 0.5% low gelling temperature agarose (SigmaAldrich, Dorset, UK) was allowed to solidify in 6well plates. This layer was overlaid with 0.375% agarose in DMEM (supplemented with FBS and Pen/Strep) containing 1 3 104 cells. Triplicate wells were seeded for each cell line. The cell/agarose was overlaid with DMEM medium containing FBS and Pen/Strep, and incubated for 15 days. Medium was changed on the plates every 4 days.
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At the end of the assay, the wells were stained with 0.05% crystal violet (Sigma-Aldrich, Dorset, UK) and the cell colonies counted. Western Blotting
Protein from HEK293 cells containing empty pcDNA3HA, pcDNA3HA-GPS2, or pcDNA3HAMLL4-GPS2 was harvested by direct lysis of cells in RIPA buffer (Sigma-Aldrich, Dorset, UK) containing protease and phosphatase inhibitors (Roche, West Sussex, UK). Protein concentration was determined by detergent compatible (DC) protein assay (Biorad, Hertfordshire, UK). The protein samples were separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels, and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Hertfordshire, UK). The membranes were blocked in 5% nonfat milk powder reconstituted in TBS-Tween (0.05%) for 1 hr at room temperature and antibody incubations were performed overnight at 4 C in 5% milk/TBS-Tween (0.05%). The antibodies used were HA-tag antibody (Abcam, Cambridge, UK) and anti-beta actin (Abcam, Cambridge, UK). The membranes were washed in TBS-Tween and subsequently incubated with HRP-conjugated anti-rabbit antibody (Abcam, Cambridge, UK) for 1 hr at RT. After washing, the membranes were incubated with Supersignal West Dura chemiluminescent substrate (Thermo Scientific, Loughborough, UK) and exposed to X-ray film (Thermo Scientific, Loughborough, UK). RESULTS
The patient underwent postoperative radiation therapy to a total dose of 54Gy over 30 fractions after second surgery with total resection. She remains disease-free 9 years post-therapy, albeit requiring a wheel-chair for mobilization. Morphologic examination of Hematoxylin and Eosin stained sections showed a variably cellular tumor with a rich supply of fine, curvilinear blood vessels, and composed of spindle cells with poorly defined cell borders and elongated bland-appearing nuclei (Fig. 1A), focal microcystic change (Fig. 1B) and localized condensation of cells arranged in a more obvious storiform pattern, within which area mitotic activity was maximal (Fig. 1C). Mitotic activity overall was
