Jann N Sarkaria

Publications (38)252.18 Total impact

• Article: Efficacy of protracted temozolomide dosing is limited in MGMT unmethylated GBM xenograft models.

  Ling Cen, Brett L Carlson, Jenny L Pokorny, Ann C Mladek, Patrick T Grogan, Mark A Schroeder, Paul A Decker, S Keith Anderson, Caterina Giannini, Wenting Wu, Karla V Ballman, Gaspar J Kitange, Jann N Sarkaria
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  ABSTRACT: Background Temozolomide (TMZ) is important chemotherapy for glioblastoma multiforme (GBM), but the optimal dosing schedule is unclear.Methods The efficacies of different clinically relevant dosing regimens were compared in a panel of 7 primary GBM xenografts in an intracranial therapy evaluation model.ResultsProtracted TMZ therapy (TMZ daily M-F, 3 wk every 4) provided superior survival to a placebo-treated group in 1 of 4 O6-DNA methylguanine-methyltransferase (MGMT) promoter hypermethylated lines (GBM12) and none of the 3 MGMT unmethylated lines, while standard therapy (TMZ daily M-F, 1 wk every 4) provided superior survival to the placebo-treated group in 2 of 3 MGMT unmethylated lines (GBM14 and GBM43) and none of the methylated lines. In comparing GBM12, GBM14, and GBM43 intracranial specimens, both GBM14 and GBM43 mice treated with protracted TMZ had a significant elevation in MGMT levels compared with placebo. Similarly, high MGMT was found in a second model of acquired TMZ resistance in GBM14 flank xenografts, and resistance was reversed in vitro by treatment with the MGMT inhibitor O6-benzylguanine, demonstrating a mechanistic link between MGMT overexpression and TMZ resistance in this line. Additionally, in an analysis of gene expression data, comparison of parental and TMZ-resistant GBM14 demonstrated enrichment of functional ontologies for cell cycle control within the S, G2, and M phases of the cell cycle and DNA damage checkpoints.Conclusions Across the 7 tumor models studied, there was no consistent difference between protracted and standard TMZ regimens. The efficacy of protracted TMZ regimens may be limited in a subset of MGMT unmethylated tumors by induction of MGMT expression.
  Neuro-Oncology 03/2013; · 5.72 Impact Factor
• Article: Biopsy validation of 18F-DOPA PET and biodistribution in gliomas for neurosurgical planning and radiotherapy target delineation: results of a prospective pilot study.

  Deanna H Pafundi, Nadia N Laack, Ryan S Youland, Ian F Parney, Val J Lowe, Caterina Giannini, Brad J Kemp, Michael P Grams, Jonathan M Morris, Jason M Hoover, Leland S Hu, Jann N Sarkaria, Debra H Brinkmann
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  ABSTRACT: Background Delineation of glioma extent for surgical or radiotherapy planning is routinely based on MRI. There is increasing awareness that contrast enhancement on T1-weighted images (T1-CE) may not reflect the entire extent of disease. The amino acid tracer 18F-DOPA (3,4-dihydroxy-6-[18F] fluoro-l-phenylalanine) has a high tumor-to-background signal and high sensitivity for glioma imaging. This study compares 18F-DOPA PET against conventional MRI for neurosurgical biopsy targeting, resection planning, and radiotherapy target volume delineation.Methods Conventional MR and 18F-DOPA PET/CT images were acquired in 10 patients with suspected malignant brain tumors. One to 3 biopsy locations per patient were chosen in regions of concordant and discordant 18F-DOPA uptake and MR contrast enhancement. Histopathology was reviewed on 23 biopsies. 18F-DOPA PET was quantified using standardized uptake values (SUV) and tumor-to-normal hemispheric tissue (T/N) ratios.ResultsPathologic review confirmed glioma in 22 of 23 biopsy specimens. Thirteen of 16 high-grade biopsy specimens were obtained from regions of elevated 18F-DOPA uptake, while T1-CE was present in only 6 of those 16 samples. Optimal 18F-DOPA PET thresholds corresponding to high-grade disease based on histopathology were calculated as T/N > 2.0. In every patient, 18F-DOPA uptake regions with T/N > 2.0 extended beyond T1-CE up to a maximum of 3.5 cm. SUV was found to correlate with grade and cellularity.Conclusions18F-DOPA PET SUVmax may more accurately identify regions of higher-grade/higher-density disease in patients with astrocytomas and will have utility in guiding stereotactic biopsy selection. Using SUV-based thresholds to define high-grade portions of disease may be valuable in delineating radiotherapy boost volumes.
  Neuro-Oncology 03/2013; · 5.72 Impact Factor
• Article: Targeting SRC family kinases inhibits bevacizumab-induced glioma cell invasion.

  Deborah Huveldt, Laura J Lewis-Tuffin, Brett L Carlson, Mark A Schroeder, Fausto Rodriguez, Caterina Giannini, Evanthia Galanis, Jann N Sarkaria, Panos Z Anastasiadis
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  ABSTRACT: Anti-VEGF antibody therapy with bevacizumab provides significant clinical benefit in patients with recurrent glioblastoma multiforme (GBM). Unfortunately, progression on bevacizumab therapy is often associated with a diffuse disease recurrence pattern, which limits subsequent therapeutic options. Therefore, there is an urgent need to understand bevacizumab's influence on glioma biology and block it's actions towards cell invasion.To explore the mechanism(s) of GBM cell invasion we have examined a panel of serially transplanted human GBM lines grown either in short-term culture, as xenografts in mouse flank, or injected orthotopically in mouse brain. Using an orthotopic xenograft model that exhibits increased invasiveness upon bevacizumab treatment, we also tested the effect of dasatinib, a broad spectrum SFK inhibitor, on bevacizumab-induced invasion.We show that 1) activation of Src family kinases (SFKs) is common in GBM, 2) the relative invasiveness of 17 serially transplanted GBM xenografts correlates strongly with p120 catenin phosphorylation at Y228, a Src kinase site, and 3) SFK activation assessed immunohistochemically in orthotopic xenografts, as well as the phosphorylation of downstream substrates occurs specifically at the invasive tumor edge. Further, we show that SFK signaling is markedly elevated at the invasive tumor front upon bevacizumab administration, and that dasatinib treatment effectively blocked the increased invasion induced by bevacizumab.Our data are consistent with the hypothesis that the increased invasiveness associated with anti-VEGF therapy is due to increased SFK signaling, and support testing the combination of dasatinib with bevacizumab in the clinic.
  PLoS ONE 01/2013; 8(2):e56505. · 4.09 Impact Factor
• Article: The role of LAT1 in (18)F-DOPA uptake in malignant gliomas.

  Ryan S Youland, Gaspar J Kitange, Timothy E Peterson, Deanna H Pafundi, Judi A Ramiscal, Jenny L Pokorny, Caterina Giannini, Nadia N Laack, Ian F Parney, Val J Lowe, Debra H Brinkmann, Jann N Sarkaria
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  ABSTRACT: Positron emission tomography (PET) imaging with the amino acid tracer 6-(18)F-fluoro-L-3,4-dihydroxy-phenylalanine ((18)F-DOPA) may provide better spatial and functional information in human gliomas than CT or MRI alone. The L-type amino acid transporter 1 (LAT1) is responsible for membrane transport of large neutral amino acids in normal cells. This study assessed the relationship between LAT1 expression and (18)F-DOPA uptake in human astrocytomas. Endogenous LAT1 expression was measured in established glioblastoma (GBM) cell lines and primary GBM xenografts using Western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Uptake of (18)F-DOPA was approximated in vitro using (3)H-L-DOPA as an analog. Uptake of (3)H-L-DOPA was assessed in cells expressing LAT1 shRNA or LAT1 siRNA and compared to non-targeted (NT) control shRNA or siRNA sequences, respectively. To demonstrate the clinical relevance of these findings, LAT1 immunofluorescence staining was compared with corresponding regions of (18)F-DOPA PET uptake in patients with newly diagnosed astrocytomas. LAT1 mRNA and protein expression varies in GBM, and the extent of (3)H-L-DOPA uptake was positively correlated with endogenous LAT1 expression. Stable shRNA-mediated LAT1 knockdown in T98 and GBM28 reduced (3)H-L-DOPA uptake relative to NT shRNA by 57 (P < 0.0001) and 52 % (P < 0.001), respectively. Transient siRNA-mediated LAT1 knockdown in T98 reduced (3)H-L-DOPA uptake relative to NT siRNA up to 68 % (P < 0.01). In clinical samples, LAT1 expression positively correlated with (18)F-DOPA PET uptake (P = 0.04). Expression of LAT1 is strongly associated with (3)H-L-DOPA uptake in vitro and (18)F-DOPA uptake in patient biopsy samples. These results define LAT1 as a key determinant of (18)F-DOPA accumulation in GBM.
  Journal of Neuro-Oncology 10/2012; · 3.21 Impact Factor
• Source

  Available from: Aditi Nadkarni

  Article: ATM inhibitor KU-55933 increases the TMZ responsiveness of only inherently TMZ sensitive GBM cells

  Aditi Nadkarni, bullet Meena, Shrivastav @bullet, Ann C Mladek, Paul M Schwingler, Patrick T Grogan, bullet Junjie, Chen @bullet, Jann N Sarkaria
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  ABSTRACT: Ataxia telangiectasia mutated (ATM) kinase is critical in sensing and repairing DNA double-stranded breaks (DSBs) such as those induced by temozolomide (TMZ). ATM deficiency increases TMZ sensitivity, which suggests that ATM inhibitors may be effective TMZ sen-sitizing agents. In this study, the TMZ sensitizing effects of 2 ATM specific inhibitors were studied in established and xenograft-derived glioblastoma (GBM) lines that are inherently sensitive to TMZ and derivative TMZ-resistant lines. In parental U251 and U87 glioma lines, the addition of KU-55933 to TMZ significantly increased cell killing compared to TMZ alone [U251 survival: 0.004 ± 0.0015 vs. 0.08 ± 0.01 (p \\ 0.001), respectively, and U87 sur-vival: 0.02 ± 0.005 vs. 0.04 ± 0.002 (p \\ 0.001), respectively] and also elevated the fraction of cells arrested in G2/M [U251 G2/M fraction: 61.8 ± 1.1 % vs. 35 ± 0.8 % (p \\ 0.001), respectively, and U87 G2/M frac-tion 25 ± 0.2 % vs.18.6 ± 0.4 % (p \\ 0.001), respectively]. In contrast, KU-55933 did not sensitize the resistant lines to TMZ, and neither TMZ alone or combined with KU-55933 induced a G2/M arrest. While KU-55933 did not enhance TMZ induced Chk1/Chk2 activation, it increased TMZ-induced residual c-H2AX foci in the parental cells but not in the TMZ resistant cells. Similar sensitization was observed with either KU-55933 or CP-466722 combined with TMZ in GBM12 xenograft line but not in GBM12TMZ, which is resistant to TMZ due to MGMT overexpression. These find-ings are consistent with a model where ATM inhibition suppresses the repair of TMZ-induced DSBs in inherently TMZ-sensitive tumor lines, which suggests an ATM inhibitor potentially could be deployed with an improvement in the therapeutic window when combined with TMZ.
  Journal of Neuro-Oncology 10/2012; 110(1). · 3.21 Impact Factor
• Article: Molecular characterization of EGFR and EGFRvIII signaling networks in human glioblastoma tumor xenografts.

  Hannah Johnson, Amanda M Del Rosario, Bryan D Bryson, Mark A Schroeder, Jann N Sarkaria, Forest M White
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  ABSTRACT: Glioblastoma multiforme (GBM) is a malignant primary brain tumor with a mean survival of 15 months with the current standard of care. Genetic profiling efforts have identified the amplification, over-expression, and mutation of the wild-type (wt) epidermal growth factor receptor tyrosine kinase (EGFR) in approximately 50% of GBM patients. The genetic aberration of wtEGFR is frequently accompanied by the over-expression of a mutant EGFR known as EGFR variant III (EGFRvIII, de2-7EGFR, ΔEGFR), which is expressed in 30% of GBM tumors. The molecular mechanisms of tumorigenesis driven by EGFRvIII over-expression in human tumors have not been fully elucidated. To identify specific therapeutic targets for EGFRvIII driven tumors, it is important to gather a broad understanding of EGFRvIII specific signaling. Here, we have characterized signaling through the quantitative analysis of protein expression and tyrosine phosphorylation across a panel of glioblastoma tumor xenografts established from patient surgical specimens expressing wtEGFR or over-expressing wtEGFR (wtEGFR+) or EGFRvIII (EGFRvIII+). S100A10 (p11), major vault protein (MVP), guanylate-binding protein 1(GBP1) and carbonic anhydrase III (CAIII) were identified to have significantly increased expression in EGFRvIII expressing xenograft tumors relative to wtEGFR xenograft tumors. Increased expression of these 4 individual proteins was found to be correlated with poor survival in patients with GBM; the combination of these 4 proteins represents a prognostic signature for poor survival in gliomas. Integration of protein expression and phosphorylation data has uncovered significant heterogeneity among the various tumors and has highlighted several novel pathways, related to EGFR trafficking, activated in glioblastoma. The pathways and proteins identified in these tumor xenografts represent potential therapeutic targets for this disease.
  Molecular &amp Cellular Proteomics 09/2012; · 7.40 Impact Factor
• Article: p16-Cdk4-Rb axis controls sensitivity to a cyclin-dependent kinase inhibitor PD0332991 in glioblastoma xenograft cells.

  Ling Cen, Brett L Carlson, Mark A Schroeder, Jamie L Ostrem, Gaspar J Kitange, Ann C Mladek, Stephanie R Fink, Paul A Decker, Wenting Wu, Jung-Sik Kim, Todd Waldman, Robert B Jenkins, Jann N Sarkaria
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  ABSTRACT: Deregulation of the p16(INK4a)-Cdk4/6-Rb pathway is commonly detected in patients with glioblastoma multiforme (GBM) and is a rational therapeutic target. Here, we characterized the p16(INK4a)-Cdk4/6-Rb pathway in the Mayo panel of GBM xenografts, established from primary tissue samples from patients with GBM, and evaluated their response to PD0332991, a specific inhibitor of Cdk4/6. All GBM xenograft lines evaluated in this study had disruptions in the p16(INK4a)-Cdk4/6-Rb pathway. In vitro evaluation using short-term explant cultures from selected GBM xenograft lines showed that PD0332991 effectively arrested cell cycle in G1-phase and inhibited cell proliferation dose-dependently in lines deleted for CDKN2A/B-p16(INK4a) and either single-copy deletion of CDK4 (GBM22), high-level CDK6 amplification (GBM34), or deletion of CDKN2C/p18(INK4c) (GBM43). In contrast, 2 GBM lines with p16(INK4a) expression and either CDK4 amplification (GBM5) or RB mutation (GBM28) were completely resistant to PD0332991. Additional xenograft lines were screened, and GBM63 was identified to have p16(INK4a) expression and CDK4 amplification. Similar to the results with GBM5, GBM63 was resistant to PD0332991 treatment. In an orthotopic survival model, treatment of GBM6 xenografts (CDKN2A/B-deleted and CDK4 wild-type) with PD0332991 significantly suppressed tumor cell proliferation and prolonged survival. Collectively, these data support the concept that GBM tumors lacking p16(INK4a) expression and with nonamplified CDK4 and wild-type RB status may be more susceptible to Cdk4/6 inhibition using PD0332991.
  Neuro-Oncology 06/2012; 14(7):870-81. · 5.72 Impact Factor
• Article: Inhibition of histone deacetylation potentiates the evolution of acquired temozolomide resistance linked to MGMT upregulation in glioblastoma xenografts.

  Gaspar J Kitange, Ann C Mladek, Brett L Carlson, Mark A Schroeder, Jenny L Pokorny, Ling Cen, Paul A Decker, Wenting Wu, Gwen A Lomberk, Shiv K Gupta, Raul A Urrutia, Jann N Sarkaria
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  ABSTRACT: The therapeutic benefit of temozolomide in glioblastoma multiforme (GBM) is limited by resistance. The goal of this study was to elucidate mechanisms of temozolomide resistance in GBM. We developed an in vivo GBM model of temozolomide resistance and used paired parental and temozolomide-resistant tumors to define the mechanisms underlying the development of resistance and the influence of histone deacetylation (HDAC) inhibition. Analysis of paired parental and resistant lines showed upregulation of O6-methylguanine-DNA methyltransferase (MGMT) expression in 3 of the 5 resistant xenografts. While no significant change was detected in MGMT promoter methylation between parental and derivative-resistant samples, chromatin immunoprecipitation showed an association between MGMT upregulation and elevated acetylation of lysine 9 of histone H3 (H3K9-ac) and decreased dimethylation (H3K9-me2) in GBM12 and GBM14. In contrast, temozolomide resistance development in GBM22 was not linked to MGMT expression, and both parental and resistant lines had low H3K9-ac and high H3K9-me2 within the MGMT promoter. In the GBM12TMZ-resistant line, MGMT reexpression was accompanied by increased recruitment of SP1, C-JUN, NF-κB, and p300 within the MGMT promoter. Interestingly, combined treatment of GBM12 flank xenografts with temozolomide and the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) favored the evolution of temozolomide resistance by MGMT overexpression as compared with treatment with temozolomide alone. This study shows, for the first time, a unique mechanism of temozolomide resistance development driven by chromatin-mediated MGMT upregulation and highlights the potential for epigenetically directed therapies to influence the mechanisms of resistance development in GBM.
  Clinical Cancer Research 06/2012; 18(15):4070-9. · 7.74 Impact Factor
• Article: Alkylpurine-DNA-N-glycosylase confers resistance to temozolomide in xenograft models of glioblastoma multiforme and is associated with poor survival in patients.

  Sameer Agnihotri, Aaron S Gajadhar, Christian Ternamian, Thierry Gorlia, Kristin L Diefes, Paul S Mischel, Joanna Kelly, Gail McGown, Mary Thorncroft, Brett L Carlson, Jann N Sarkaria, Geoffrey P Margison, Kenneth Aldape, Cynthia Hawkins, Monika Hegi, Abhijit Guha
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  ABSTRACT: Glioblastoma multiforme (GBM) is the most common and lethal of all gliomas. The current standard of care includes surgery followed by concomitant radiation and chemotherapy with the DNA alkylating agent temozolomide (TMZ). O⁶-methylguanine-DNA methyltransferase (MGMT) repairs the most cytotoxic of lesions generated by TMZ, O⁶-methylguanine. Methylation of the MGMT promoter in GBM correlates with increased therapeutic sensitivity to alkylating agent therapy. However, several aspects of TMZ sensitivity are not explained by MGMT promoter methylation. Here, we investigated our hypothesis that the base excision repair enzyme alkylpurine-DNA-N-glycosylase (APNG), which repairs the cytotoxic lesions N³-methyladenine and N⁷-methylguanine, may contribute to TMZ resistance. Silencing of APNG in established and primary TMZ-resistant GBM cell lines endogenously expressing MGMT and APNG attenuated repair of TMZ-induced DNA damage and enhanced apoptosis. Reintroducing expression of APNG in TMZ-sensitive GBM lines conferred resistance to TMZ in vitro and in orthotopic xenograft mouse models. In addition, resistance was enhanced with coexpression of MGMT. Evaluation of APNG protein levels in several clinical datasets demonstrated that in patients, high nuclear APNG expression correlated with poorer overall survival compared with patients lacking APNG expression. Loss of APNG expression in a subset of patients was also associated with increased APNG promoter methylation. Collectively, our data demonstrate that APNG contributes to TMZ resistance in GBM and may be useful in the diagnosis and treatment of the disease.
  The Journal of clinical investigation 12/2011; 122(1):253-66. · 15.39 Impact Factor
• Article: North Central Cancer Treatment Group Phase I trial N057K of everolimus (RAD001) and temozolomide in combination with radiation therapy in patients with newly diagnosed glioblastoma multiforme.

  Jann N Sarkaria, Evanthia Galanis, Wenting Wu, Patrick J Peller, Caterina Giannini, Paul D Brown, Joon H Uhm, Steven McGraw, Kurt A Jaeckle, Jan C Buckner
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  ABSTRACT: The mammalian target of rapamycin (mTOR) functions within the PI3K/Akt signaling pathway as a critical modulator of cell survival. On the basis of promising preclinical data, the safety and tolerability of therapy with the mTOR inhibitor RAD001 in combination with radiation (RT) and temozolomide (TMZ) was evaluated in this Phase I study. All patients received weekly oral RAD001 in combination with standard chemoradiotherapy, followed by RAD001 in combination with standard adjuvant temozolomide. RAD001 was dose escalated in cohorts of 6 patients. Dose-limiting toxicities were defined during RAD001 combination therapy with TMZ/RT. Eighteen patients were enrolled, with a median follow-up of 8.4 months. Combined therapy was well tolerated at all dose levels, with 1 patient on each dose level experiencing a dose-limiting toxicity: Grade 3 fatigue, Grade 4 hematologic toxicity, and Grade 4 liver dysfunction. Throughout therapy, there were no Grade 5 events, 3 patients experienced Grade 4 toxicities, and 6 patients had Grade 3 toxicities attributable to treatment. On the basis of these results, the recommended Phase II dosage currently being tested is RAD001 70 mg/week in combination with standard chemoradiotherapy. Fluorodeoxyglucose (FDG) positron emission tomography scans also were obtained at baseline and after the second RAD001 dose before the initiation of TMZ/RT; the change in FDG uptake between scans was calculated for each patient. Fourteen patients had stable metabolic disease, and 4 patients had a partial metabolic response. RAD001 in combination with RT/TMZ and adjuvant TMZ was reasonably well tolerated. Changes in tumor metabolism can be detected by FDG positron emission tomography in a subset of patients within days of initiating RAD001 therapy.
  International journal of radiation oncology, biology, physics 10/2011; 81(2):468-75. · 4.59 Impact Factor
• Article: WWP2 is an E3 ubiquitin ligase for PTEN.

  Subbareddy Maddika, Sridhar Kavela, Neelam Rani, Vivek Reddy Palicharla, Jenny L Pokorny, Jann N Sarkaria, Junjie Chen
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  ABSTRACT: PTEN, a lipid phosphatase, is one of the most frequently mutated tumour suppressors in human cancer. Several recent studies have highlighted the importance of ubiquitylation in regulating PTEN tumour-suppressor function, but the enzymatic machinery required for PTEN ubiquitylation is not clear. In this study, by using a tandem affinity-purification approach, we have identified WWP2 (also known as atrophin-1-interacting protein 2, AIP-2) as a PTEN-interacting protein. WWP2 is an E3 ubiquitin ligase that belongs to the NEDD4-like protein family, which is involved in regulating transcription, embryonic stem-cell fate, cellular transport and T-cell activation processes. We show that WWP2 physically interacts with PTEN and mediates its degradation through a ubiquitylation-dependent pathway. Functionally, we show that WWP2 controls cellular apoptosis and is required for tumorigenicity of cells. Collectively, our results reveal a functional E3 ubiquitin ligase for PTEN that plays a vital role in tumour-cell survival.
  Nature Cell Biology 06/2011; 13(6):728-33. · 19.49 Impact Factor
• Article: PI3K/AKT pathway alterations are associated with clinically aggressive and histologically anaplastic subsets of pilocytic astrocytoma.

  Erika F Rodriguez, Bernd W Scheithauer, Caterina Giannini, Amanda Rynearson, Ling Cen, Bridget Hoesley, Heather Gilmer-Flynn, Jann N Sarkaria, Sarah Jenkins, Jin Long, Fausto J Rodriguez
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  ABSTRACT: Pilocytic astrocytomas (PA) are well-differentiated gliomas having a favorable prognosis when compared with other diffuse or infiltrative astrocytomas. Molecular genetic abnormalities and activation of signaling pathways associated with clinically aggressive PA and histologically anaplastic PA have not been adequately studied. We performed molecular genetic, gene expression, and immunohistochemical studies using three PA subsets, including conventional PA (n = 43), clinically aggressive/recurrent PA (n = 24), and histologically anaplastic PA (n = 25). A clinical diagnosis of NF1 was present in 28% of anaplastic PA. Molecular cytogenetic studies demonstrated heterozygous PTEN/10q and homozygous p16 deletions in 6/19 (32%) and 3/15 (20%) cases of anaplastic PA, respectively, but in neither of the two other groups. BRAF duplication was identified in 33% of sporadic anaplastic PA and 63% of cerebellar examples. BRAF (V600E) mutation was absent in four (of 4) sporadic cases lacking duplication. IDH1(R132H) immunohistochemistry was negative in 16 (of 16) cases. Neither PDGFRA nor EGFR amplifications were present. pERK staining levels were similar among the three PA subsets, but a stepwise increase in cytoplasmic pAKT and to a lesser extent pS6 immunoreactivity was noted by immunohistochemistry in aggressive PA groups. This was particularly true in histologically anaplastic PA when compared with conventional PA (p < 0.001 and p = 0.005, respectively). In addition, PTEN expression at the mRNA level was decreased in histologically anaplastic PA when compared to the other groups (p = 0.05). In summary, activation of the PI3K/AKT in addition to MAPK/ERK signaling pathways may underlie biological aggressiveness in PA. Specifically, it may mediate the increased proliferative activity observed in histologically anaplastic PA.
  Acta Neuropathologica 03/2011; 121(3):407-20. · 9.32 Impact Factor
• Article: Establishment, maintenance and in vitro and in vivo applications of primary human glioblastoma multiforme (GBM) xenograft models for translational biology studies and drug discovery.

  Brett L Carlson, Jenny L Pokorny, Mark A Schroeder, Jann N Sarkaria
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  ABSTRACT: Development of clinically relevant tumor model systems for glioblastoma multiforme (GBM) is important for advancement of basic and translational biology. One model that has gained wide acceptance in the neuro-oncology community is the primary xenograft model. This model entails the engraftment of patient tumor specimens into the flank of nude mice and subsequent serial passage of these tumors in the flank of mice. These tumors are then used to establish short-term explant cultures or intracranial xenografts. This unit describes detailed procedures for establishment, maintenance, and utilization of a primary GBM xenograft panel for the purpose of using them as tumor models for basic or translational studies.
  Current protocols in pharmacology 03/2011; Chapter 14:Unit 14.16.
• Article: A phase 2 study of temsirolimus (CCI-779) in patients with soft tissue sarcomas: a study of the Mayo phase 2 consortium (P2C).

  Scott Okuno, Howard Bailey, Michelle R Mahoney, Douglas Adkins, William Maples, Tom Fitch, David Ettinger, Charles Erlichman, Jann N Sarkaria
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  ABSTRACT: The primary goal of this trial was to evaluate the confirmed response rate of temsirolimus (CCI-779), a mammalian target of rapamycin in patients with advanced soft tissue sarcomas (STS). Patients ≥18 years with measurable advanced STS, no prior chemotherapy for metastatic disease (adjuvant and neoadjuvant chemotherapy allowed), adequate organ function, and performance status of ≤2 were eligible. After premedication with an antihistamine, CCI-779 was given intravenously at 25 mg over 30 minutes on Days 1, 8, 15, and 22, repeated every 4 weeks. The primary endpoint was confirmed response rate per Response Evaluation Criteria in Solid Tumors. Between June 2004 and November 2005, a total of 41 patients were enrolled and began treatment; 40 patients are evaluable for response and adverse events. The median age was 62 years (range, 28-72 years) with 56% women. Eighty percent had high-grade STS, and 22% had prior adjuvant chemotherapy. There were 2 patients (5%; 95% confidence interval [CI], 1-17) (undifferentiated fibrosarcoma and uterine leiomyosarcoma) who achieved a confirmed partial response lasting 3 and 17 months, respectively. Thirty-nine (95%) patients have progressed, with a median time to progression of 2.0 months (95% CI, 1.8-3.5). The median overall survival was 7.6 months (95% CI, 6.1-15.9). Forty-three percent experienced grade 3+ adverse events that were possibly related to therapy. Temsirolimus in this patient population of STS had limited clinical activity and had moderate toxicities.
  Cancer 02/2011; 117(15):3468-75. · 4.77 Impact Factor
• Article: Combination of temsirolimus (CCI-779) with chemoradiation in newly diagnosed glioblastoma multiforme (GBM) (NCCTG trial N027D) is associated with increased infectious risks.

  Jann N Sarkaria, Eva Galanis, Wenting Wu, Allan B Dietz, Timothy J Kaufmann, Michael P Gustafson, Paul D Brown, Joon H Uhm, Ravi D Rao, Laurence Doyle, Caterina Giannini, Kurt A Jaeckle, Jan C Buckner
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  ABSTRACT: The mammalian target of rapamycin (mTOR) functions within the phosphoinositide 3-kinase/Akt signaling pathway as a critical modulator of cell survival. The mTOR inhibitor temsirolimus (CCI-779) was combined with chemoradiotherapy in glioblastoma multiforme (GBM) patients in a dose-escalation phase I trial. The first 12 patients were treated with CCI-779 combined with radiation/temozolomide and adjuvant temozolomide. A second cohort of 13 patients was treated with concurrent CCI-779/radiation/temozolomide followed by adjuvant temozolomide monotherapy. Concomitant and adjuvant CCI-779 was associated with a high rate (3 of 12 patients) of grade 4/5 infections. By limiting CCI-779 treatment to the radiation/temozolomide phase and using antibiotic prophylaxis, the rate of infections was reduced, although 2 of 13 patients developed exacerbation of pre-existing fungal or viral infections. Dose-limiting toxicities were observed in 2 of 13 patients with this modified schedule. Weekly CCI-779 (50 mg/week) combined with radiation/temozolomide is the recommended phase II dose and schedule. The immune profile of patients in the second cohort was assessed before, during, and after CCI-779 therapy. There was robust suppression of helper and cytotoxic T cells, B cells, natural killer, cells and elevation of regulatory T cells during CCI-779/radiation/temozolomide therapy with recovery to baseline levels during adjuvant temozolomide of cytotoxic T cells, natural killer cells, and regulatory T cells. The increased infection rate observed with CCI-779 combined with chemoradiotherapy in GBM was reduced with antibiotic prophylaxis and by limiting the duration of CCI-779 therapy. The combined suppressive effects of CCI-779 and temozolomide therapy on discrete immune compartments likely contributed to the increased infectious risks observed.
  Clinical Cancer Research 10/2010; 16(22):5573-80. · 7.74 Impact Factor
• Article: Expression of CD74 in high grade gliomas: a potential role in temozolomide resistance.

  Gaspar J Kitange, Brett L Carlson, Mark A Schroeder, Paul A Decker, Bruce W Morlan, Wenting Wu, Karla V Ballman, Caterina Giannini, Jann N Sarkaria
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  ABSTRACT: Temozolomide (TMZ) is the most effective chemotherapeutic agent for glioblastoma (GBM). Resistance to this methylating agent is linked to DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). However, in recent studies MGMT status was not completely accurate as a predictor of TMZ response in GBM, suggesting other mechanisms of resistance. As part of an effort aimed at discovery of genes involved in TMZ resistance in GBM, the expression of CD74 was evaluated in GBM patient samples and the influence of CD74 on TMZ response was evaluated in GBM tumor models. Reverse transcription-polymerase-chain reaction (RT-PCR) demonstrated differential expression of CD74 mRNA among the GBM xenografts; 8 of 20 (40%) expressed CD74 mRNA. In a preliminary evaluation of whether CD74 expression might influence TMZ response, CD74 mRNA expression levels were inversely associated with in vivo TMZ resistance in 20 GBM xenograft lines (median survival 122 vs. 62.5 days; r = -0.48, P = 0.032). In follow up to this observation, CD74 shRNA knock down in U87 cells significantly suppressed in vitro proliferation and increased TMZ sensitivity as compared to a non-specific control shRNA. Consistent with an effect on proliferation and survival, silencing of CD74 by shRNA was associated with reduced Akt and Erk1/2 activation in response to stimulation by CD74 ligand macrophage-migration inhibition factor (MIF). Lastly, expression of CD74 protein was assessed in patient samples [nine anaplastic astrocytoma (AA), and 62 GBM] by immunohistochemistry, and appreciable expression was observed in 28% of samples. Collectively, these findings suggest that CD74 is expressed in a subset of high grade gliomas and may contribute to TMZ resistance.
  Journal of Neuro-Oncology 05/2010; 100(2):177-86. · 3.21 Impact Factor
• Article: Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1.

  Roel G W Verhaak, Katherine A Hoadley, Elizabeth Purdom, Victoria Wang, Yuan Qi, Matthew D Wilkerson, C Ryan Miller, Li Ding, Todd Golub, Jill P Mesirov, [......], Cameron Brennan, Ari Kahn, Paul T Spellman, Richard K Wilson, Terence P Speed, Joe W Gray, Matthew Meyerson, Gad Getz, Charles M Perou, D Neil Hayes
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  ABSTRACT: The Cancer Genome Atlas Network recently cataloged recurrent genomic abnormalities in glioblastoma multiforme (GBM). We describe a robust gene expression-based molecular classification of GBM into Proneural, Neural, Classical, and Mesenchymal subtypes and integrate multidimensional genomic data to establish patterns of somatic mutations and DNA copy number. Aberrations and gene expression of EGFR, NF1, and PDGFRA/IDH1 each define the Classical, Mesenchymal, and Proneural subtypes, respectively. Gene signatures of normal brain cell types show a strong relationship between subtypes and different neural lineages. Additionally, response to aggressive therapy differs by subtype, with the greatest benefit in the Classical subtype and no benefit in the Proneural subtype. We provide a framework that unifies transcriptomic and genomic dimensions for GBM molecular stratification with important implications for future studies.
  Cancer cell 01/2010; 17(1):98-110. · 25.29 Impact Factor
• Article: Phenotypic variations in NF1-associated low grade astrocytomas: possible role for increased mTOR activation in a subset.

  Mark Jentoft, Caterina Giannini, Ling Cen, Bernd W Scheithauer, Bridget Hoesley, Jann N Sarkaria, Patrice C Abell-Aleff, Erika F Rodriguez, Ying Li, Fausto J Rodriguez
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  ABSTRACT: Low grade astrocytomas are the most common CNS tumors in neurofibromatosis type 1(NF1) patients. While most are classic pilocytic astrocytomas (PA), some are difficult to classify, and have been termed "low grade astrocytoma subtype indeterminate" (LGSI). Some of these tumors exhibit peculiar morphologies, including plump cytoplasmic processes and macronucleoli. In the current study we performed electron microscopy, followed by gene expression, immunohistochemicai and western blot analyses in an effort to identify biological differences underlying phenotypic variation in NF1-associated low grade astrocytoma. Electron microscopy demonstrated intermediate filaments and frequent Rosenthal fiber material in both PA and LGSI. Dense core granules and/or aligned microtubules were present in the LGSI group (2 of 3 cases) and in the PA group (1 of 10 cases). Analysis of global gene expression data obtained using Affymetrix HG-U133 Plus2.0 chips (5 PA, 1 LGSI), and western blot analysis for phospho-S6 (1 LGSI, 2 PA) demonstrated a gene expression profile reflecting "neuronal differentiation" and increased phospho-S6 immunoreactivity consistent with mTOR activation in the LGSI compared with PA. These findings were confirmed by immunohistochemistry for neuronal markers, as well as combined phospho-S6/ phospho-p70S6K immunoreactivity in 4 (of 4) LGSI vs. 5 (of 13) NF1-associated PA (p=0.02), and 13 (of 39) sporadic PA. Phospho-ERK immunoreactivity was uniformly present in PA and LGSI groups, while BRAF duplication was absent by FISH in 8 NF1-associated low grade astrocytomas. In summary, differential expression of neuronal-related genes and increased mTOR activation may underlie phenotypic variations in NF1-associated low grade astrocytomas.
  International journal of clinical and experimental pathology 01/2010; 4(1):43-57. · 1.89 Impact Factor
• Article: Radiosensitizing effects of temozolomide observed in vivo only in a subset of O6-methylguanine-DNA methyltransferase methylated glioblastoma multiforme xenografts.

  Brett L Carlson, Patrick T Grogan, Ann C Mladek, Mark A Schroeder, Gaspar J Kitange, Paul A Decker, Caterina Giannini, Wenting Wu, Karla A Ballman, C David James, Jann N Sarkaria
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  ABSTRACT: Concurrent temozolomide (TMZ) and radiation therapy (RT) followed by adjuvant TMZ is standard treatment for patients with glioblastoma multiforme (GBM), although the relative contribution of concurrent versus adjuvant TMZ is unknown. In this study, the efficacy of TMZ/RT was tested with a panel of 20 primary GBM xenografts. Mice with intracranial xenografts were treated with TMZ, RT, TMZ/RT, or placebo. Survival ratio for a given treatment/line was defined as the ratio of median survival for treatment vs. placebo. The median survival ratio was significantly higher for O6-methylguanine-DNA methyltransferase (MGMT) methylated tumors versus unmethylated tumors following treatment with TMZ (median survival ratio, 3.6 vs. 1.5, respectively; p = 0.008) or TMZ/RT (5.7 vs. 2.3, respectively; p = 0.001) but not RT alone (1.7 vs. 1.6; p = 0.47). In an analysis of variance, MGMT methylation status and p53 mutation status were significantly associated with treatment response. When we analyzed the additional survival benefit conferred specifically by combined therapy, only a subset (5 of 11) of MGMT methylated tumors derived substantial additional benefit from combined therapy, while none of the MGMT unmethylated tumors did. Consistent with a true radiosensitizing effect of TMZ, sequential treatment in which RT (week 1) was followed by TMZ (week 2) proved significantly less effective than TMZ followed by RT or concurrent TMZ/RT (survival ratios of 4.0, 9.6 and 12.9, respectively; p < 0.0001). Concurrent treatment with TMZ and RT provides significant survival benefit only in a subset of MGMT methylated tumors and provides superior antitumor activity relative to sequential administration of RT and TMZ.
  International journal of radiation oncology, biology, physics 09/2009; 75(1):212-9. · 4.59 Impact Factor
• Article: Effective sensitization of temozolomide by ABT-888 is lost with development of temozolomide resistance in glioblastoma xenograft lines.

  Michelle J Clarke, Evan A Mulligan, Patrick T Grogan, Ann C Mladek, Brett L Carlson, Mark A Schroeder, Nicola J Curtin, Zhenkun Lou, Paul A Decker, Wenting Wu, E Ruth Plummer, Jann N Sarkaria
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  ABSTRACT: Resistance to temozolomide and radiotherapy is a major problem for patients with glioblastoma but may be overcome using the poly(ADP-ribose) polymerase inhibitor ABT-888. Using two primary glioblastoma xenografts, the efficacy of ABT-888 combined with radiotherapy and/or temozolomide was evaluated. Treatment with ABT-888 combined with temozolomide resulted in significant survival prolongation (GBM12: 55.1%, P = 0.005; GBM22: 54.4%, P = 0.043). ABT-888 had no effect with radiotherapy alone but significantly enhanced survival in GBM12 when combined with concurrent radiotherapy/temozolomide. With multicycle therapy, ABT-888 further extended the survival benefit of temozolomide in the inherently sensitive GBM12 and GBM22 xenograft lines. However, after in vivo selection for temozolomide resistance, the derivative GBM12TMZ and GBM22TMZ lines were no longer sensitized by ABT-888 in combination with temozolomide, and a similar lack of efficacy was observed in two other temozolomide-resistant tumor lines. Thus, the sensitizing effects of ABT-888 were limited to tumor lines that have not been previously exposed to temozolomide, and these results suggest that patients with newly diagnosed glioblastoma may be more likely to respond to combined temozolomide/poly(ADP-ribose) polymerase inhibitor therapy than patients with recurrent disease.
  Molecular Cancer Therapeutics 02/2009; 8(2):407-14. · 5.23 Impact Factor