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DNA-FISH analysis of non-EGFR-amplified GBM CTCs.

Immunohistologic and DNA-FISH analysis of tumor cells and CTCs from a patient with metastatic GBM.

Methods and Supplementary Figure Legends

STEAM cocktail staining specifically identifies GBM cells.

Clinical association of CTC frequency

Unsupervised hierarchical clustering analysis of patient and xenograft single cell qRT-PCR expression data.

GBM8 and GBM24 growth in culture.

Tumor genotypes and CTC frequency

Supplementary Data from MAPK Pathway Genetic Alterations Are Associated with Prolonged Overall Survival in Low-Grade Serous Ovarian Carcinoma

Purpose:To characterize the somatic mutational landscape, investigate associations between genetic alterations and clinical outcomes, and determine the prevalence of pathogenic germline mutations in low-grade serous ovarian carcinomas (LGSC).Experimental Design:Patients with LGSC tumors who underwent panel-based sequencing of up to 505 genes were identified. Data on somatic and germline mutations; copy-number alterations; and clinicopathologic features, including age at diagnosis, platinum sensitivity, and overall survival (OS), were collected.Results:Following central pathology rereview, 119 patients with LGSC were identified for analysis. Of these, 110 (92%) had advanced-stage disease (stages III/IV). Somatic KRAS (33%), NRAS (11%), EIF1AX (10%), and BRAF (11%) alterations were the most common; MAPK pathway alterations were found in 60% (n = 71) of LGSCs. KRAS mutations were significantly associated with age at diagnosis more than 50 years (P = 0.02) and platinum-sensitive disease (P = 0.03). On multivariate analysis, MAPK pathway alterations (P = 0.02) and platinum sensitivity (P = 0.005) were significantly associated with improved OS. Seventy-nine patients (66%) underwent germline genetic testing; seven pathogenic germline mutations were identified: MUTYH (n = 2), BAP1 (n = 1), RB1 (n = 1), CHEK2 (n = 1), APC (n = 1), and FANCA (n = 1). There were no germline BRCA1/2 mutations. One germline MUTYH-associated LGSC harbored loss-of-heterozygosity at the MUTYH locus, and the patient with the germline BAP1 mutation also harbored a somatic BAP1 frameshift mutation.Conclusions:This study showed that MAPK pathway alterations in LGSC, including KRAS mutations, are independently associated with platinum sensitivity and prolonged survival. Germline data, which were limited, identified few pathogenic germline mutations in patients with LGSC.See related commentary by Veneziani and Oza, p. 4357

OBJECTIVE: To determine the effect of hyperthermic intraperitoneal chemotherapy (HIPEC) with carboplatin on the transcriptomic profiles of normal and ovarian cancer (OC) tissues. METHODS: Normal and tumor samples from four OCs were prospectively collected pre- and immediately post-HIPEC treatment and subjected to RNA-sequencing. Differential gene expression, gene ontology enrichment and pathway analyses were performed. Heat shock protein and immune-response protein expression was assessed using protein arrays and western blotting. RESULTS: RNA-sequencing revealed 4,231 and 322 genes significantly differentially expressed between pre- and post-treatment normal and OC tissues, respectively (both adjusted p-value

Purpose: To characterize the somatic mutational landscape, investigate associations between genetic alterations and clinical outcomes, and determine the prevalence of pathogenic germline mutations in low-grade serous ovarian carcinomas (LGSC). Experimental Design: Patients with LGSC tumors who underwent panel-based sequencing of up to 505 genes were identified. Data on somatic and germline mutations; copy-number alterations; and clinicopathologic features, including age at diagnosis, platinum sensitivity, and overall survival (OS), were collected. Results: Following central pathology rereview, 119 patients with LGSC were identified for analysis. Of these, 110 (92%) had advanced-stage disease (stages III/IV). Somatic KRAS (33%), NRAS (11%), EIF1AX (10%), and BRAF (11%) alterations were the most common; MAPK pathway alterations were found in 60% (n = 71) of LGSCs. KRAS mutations were significantly associated with age at diagnosis more than 50 years (P = 0.02) and platinum-sensitive disease (P = 0.03). On multivariate analysis, MAPK pathway alterations (P = 0.02) and platinum sensitivity (P = 0.005) were significantly associated with improved OS. Seventy-nine patients (66%) underwent germline genetic testing; seven pathogenic germline mutations were identified: MUTYH (n = 2), BAP1 (n = 1), RB1 (n = 1), CHEK2 (n = 1), APC (n = 1), and FANCA (n = 1). There were no germline BRCA1/2 mutations. One germline MUTYH-associated LGSC harbored loss-of-heterozygosity at the MUTYH locus, and the patient with the germline BAP1 mutation also harbored a somatic BAP1 frameshift mutation. Conclusions: This study showed that MAPK pathway alterations in LGSC, including KRAS mutations, are independently associated with platinum sensitivity and prolonged survival. Germline data, which were limited, identified few pathogenic germline mutations in patients with LGSC. See related commentary by Veneziani and Oza, p. 4357

PDF file - 102KB, Supplementary Figure S6. Sphere formation assay of MGG152 expressing N-MYC shRNA.

Supplementary Table 2. Relationship of NAMPT inhibitor sensitivity, MYC/MYCN amplification, and Myc/N-Myc protein expression status in glioblastoma tumorsphere lines

Supplementary Figures S1 and S2, new figures

''Temozolomide induced PARP activation deregulates NAD metabolism. Additive cytotoxic effect of TMZ and NAMPT inhibitors in IDH1 mutant cancers. NAD and cell viability effects with TMZ and NAMPT inhibitors in endogenous IDH1 mutant cancers. Combination treatment with TMZ and NAMPT inhibitor in IDH1 mutant and wild-type cells. Cell death mechanisms that underlie combined TMZ and NAMPT inhibitor treatment in IDH1 mutant cells. In vivo effects of combined TMZ and NAMPT inhibitor treatment for endogenous IDH1 mutant cancer.''

GSEA results comparing gene pathway expression differences between the 125 TCGA/TCIA gliomas differentiated by the T2-FLAIR mismatch sign. ES = Enrichment Score. NES = Normalized Enrichment Score. NOM = Nominal. FDR = False Discovery Rate. FWER = Family-wise error rate.

PDF file - 266KB, Supplementary Figure S5. Maintenance of genotype upon serial xenotransplantation.

'Temozolomide induced PARP activation deregulates NAD metabolism. Additive cytotoxic effect of TMZ and NAMPT inhibitors in IDH1 mutant cancers. NAD and cell viability effects with TMZ and NAMPT inhibitors in endogenous IDH1 mutant cancers. Combination treatment with TMZ and NAMPT inhibitor in IDH1 mutant and wild-type cells. Cell death mechanisms that underlie combined TMZ and NAMPT inhibitor treatment in IDH1 mutant cells. In vivo effects of combined TMZ and NAMPT inhibitor treatment for endogenous IDH1 mutant cancer.'

Supplementary Figure S1. Nampt inhibitor effects in glioblastoma tumorsphere cells. Supplementary Figure S2. Characterization of Nampt inhibitor effects in MYC/MYCN driven glioblastoma cells. Supplementary Figure S3. Characterization of NAD+-synthesis pathways in glioblastoma cells. Supplementary Figure S4. Nampt inhibitors induce apoptosis and disrupt intracellular metabolism pathways in MYC/MYCN amplified glioblastoma cells. Supplementary Figure S5. Fluorescent in situ hybridization (FISH) for MYC and MYCN in standard cancer cell lines. Supplementary Figure S6. Effect of Nampt inhibitors in Myc-driven standard cancer cell lines. Supplementary Figure S7. In vivo efficacy of Nampt inhibitor in MYC amplified H1975 xenografts. Supplementary Figure S8. NAMPT inhibitors were well tolerated in SCID mice bearing flank or orthotopic xenografts.

Supplementary Table 1 from Phase II Study of Iniparib with Concurrent Chemoradiation in Patients with Newly Diagnosed Glioblastoma

PDF file - 404KB, Supplementary Figure S2. IDH1-mutant glioma orthotopic xenografts recapitulate histopathological features of the respective patient tumors.

Summary of IHC of glioma subtypes

PDF file - 347KB, Supplementary Figure S3. Proliferation index of IDH1-mutant glioma orthotopic xenografts (MIB-1).

XLS file - 24KB, Supplementary Table S2. Primers used for sequencing.

Purpose: Measurements of objective response rates are critical to evaluate new glioma therapies. The hallmark metabolic alteration in gliomas with mutant isocitrate dehydrogenase (IDH) is the overproduction of oncometabolite 2-hydroxyglutarate (2HG), which plays a key role in malignant transformation. 2HG represents an ideal biomarker to probe treatment response in IDH-mutant glioma patients, and we hypothesized a decrease in 2HG levels would be measureable by in vivo magnetic resonance spectroscopy (MRS) as a result of antitumor therapy.Experimental Design: We report a prospective longitudinal imaging study performed in 25 IDH-mutant glioma patients receiving adjuvant radiation and chemotherapy. A newly developed 3D MRS imaging was used to noninvasively image 2HG. Paired Student t test was used to compare pre- and posttreatment tumor 2HG values. Test–retest measurements were performed to determine the threshold for 2HG functional spectroscopic maps (fSM). Univariate and multivariate regression were performed to correlate 2HG changes with Karnofsky performance score (KPS).Results: We found that mean 2HG (2HG/Cre) levels decreased significantly (median = 48.1%; 95% confidence interval = 27.3%–56.5%; P = 0.007) in the posttreatment scan. The volume of decreased 2HG correlates (R2 = 0.88, P = 0.002) with clinical status evaluated by KPS.Conclusions: We demonstrate that dynamic measurements of 2HG are feasible by 3D fSM, and the decrease of 2HG levels can monitor treatment response in patients with IDH-mutant gliomas. Our results indicate that quantitative in vivo 2HG imaging may be used for precision medicine and early response assessment in clinical trials of therapies targeting IDH-mutant gliomas. Clin Cancer Res; 22(7); 1632–41. ©2015 AACR.

PDF file - 791KB, Supplementary Figure S1. Additional mouse orthotopic xenografts generated from patient IDH1 R132H-mutant gliomas.

Supplementary document

Supplementary Table 1 Primers used for PCR amplification and sequencing Supplementary Table 2 Results of Multiplex PCR based MGH SNAPShot assay, Pyrosequencing and Sanger sequencing in YMG6 patient and xenograft cells Supplementary Table 3 Analysis of microsatellite instability in YMG6 parent and xenograft tumors Supplementary Table 4 Quantitative analysis for MGMT methylation status in YMG6 cells Supplementary Table 5 Multiplex PCR based MGH SnaPShot assay for oligodendroglial tumors Supplementary Table 6 Correlation of xenograft lethality and phosphorylation of PI3K pathway protein Supplementary Table 7 Phosphorylation in PI3K pathway in oligodendroglioma xenograft model Supplementary Table 8 Clinical characteristics in oligodendroglial tumor patients

Kaplan-Meier curves demonstrating progression free survival and overall survival associated with the LGGs from the TCGA/TCIA database. Blue curves represent IDHmut-Codel gliomas, dotted black curves represent IDHmut-Noncodel gliomas that were positive for the T2-FLAIR mismatch sign, solid black curves represent IDHmut-Noncodel gliomas that were negative for the T2-FLAIR mismatch sign, and red curves represent IDHwt gliomas.

Supplementary Figure S1-S7

This file contains 3 Tables and 9 Figures with additional results and details of analysis related to the main results presented in the manuscript. The complete list of figures and tables is provided below. Table 1S. Baseline metabolite levels relative to creatine. Table 2S. Demographic, genomic, molecular, histological, radiological and clinical data. Table 3S. Fractional changes for all imaging biomarkers. Table 4S. General Linear Model with change of KPS as dependent variable and grade, age and fractional volume of decreased 2HG/Cre as covariates. Figure 1S. 3D MRSI pulse sequence for 2HG imaging, detailed diagram. Figure 2S. Quantum mechanics simulations of 2HG spectral editing for MEGA-LASER, MEGA-PRESS and PRESS-97 sequences. Figure 3S. Simulations of 2HG editing for MEGA-LASER and PRESS-97 sequences in the presence of B1 field inhomogeneity. Figure 4S. Mitigation of subtraction artifacts caused by motion and scanner instability in J-difference MR spectroscopy using real-time prospective motion correction, shim update and reacquisition. Figure 5S. Metabolic maps before image interpolation. Figure 6S. Examples of OFF and difference (DIFF) MEGA-LASER spectra from 3D MRSI voxels located in the tumor and contralateral normal appearing white matter, pre- and post-treatment respectively. Figure 7S. Distributions of Δ2HG/Cre in the test-retest and post-pre longitudinal data sets. Figure 8S. Comparison of functional spectroscopic maps (fSM) and functional diffusion maps (fDM). Figure 9S. Relation between residual tumor measured by FLAIR hyperintensity volume and the SNR and CRLB of 2HG. Notes. Details related to data analysis in Figures 7S and 9S.

PDF file - 323KB, Supplementary Figure S4. Sequencing of the IDH1 R132 locus in IDH1-mutant glioma xenografts.

Purpose: Isocitrate dehydrogenase (IDH) gene mutations occur in low-grade and high-grade gliomas. We sought to identify the genetic basis of malignant phenotype heterogeneity in IDH-mutant gliomas.Methods: We prospectively implanted tumor specimens from 20 consecutive IDH1-mutant glioma resections into mouse brains and genotyped all resection specimens using a CLIA-certified molecular panel. Gliomas with cancer driver mutations were tested for sensitivity to targeted inhibitors in vitro. Associations between genomic alterations and outcomes were analyzed in patients.Results: By 10 months, 8 of 20 IDH1-mutant gliomas developed intracerebral xenografts. All xenografts maintained mutant IDH1 and high levels of 2-hydroxyglutarate on serial transplantation. All xenograft-producing gliomas harbored “lineage-defining” mutations in CIC (oligodendroglioma) or TP53 (astrocytoma), and 6 of 8 additionally had activating mutations in PIK3CA or amplification of PDGFRA, MET, or N-MYC. Only IDH1 and CIC/TP53 mutations were detected in non–xenograft-forming gliomas (P = 0.0007). Targeted inhibition of the additional alterations decreased proliferation in vitro. Moreover, we detected alterations in known cancer driver genes in 13.4% of IDH-mutant glioma patients, including PIK3CA, KRAS, AKT, or PTEN mutation or PDGFRA, MET, or N-MYC amplification. IDH/CIC mutant tumors were associated with PIK3CA/KRAS mutations whereas IDH/TP53 tumors correlated with PDGFRA/MET amplification. Presence of driver alterations at progression was associated with shorter subsequent progression-free survival (median 9.0 vs. 36.1 months; P = 0.0011).Conclusion: A subset of IDH-mutant gliomas with mutations in driver oncogenes has a more malignant phenotype in patients. Identification of these alterations may provide an opportunity for use of targeted therapies in these patients. Clin Cancer Res; 20(11); 2898–909. ©2014 AACR.

Purpose: Deregulated Myc drives an oncogenic metabolic state, including pseudohypoxic glycolysis, adapted for the constitutive production of biomolecular precursors to feed rapid tumor cell growth. In glioblastoma, Myc facilitates renewal of the tumor-initiating cell reservoir contributing to tumor maintenance. We investigated whether targeting the Myc-driven metabolic state could be a selectively toxic therapeutic strategy for glioblastoma.Experimental Design: The glycolytic dependency of Myc-driven glioblastoma was tested using 13C metabolic flux analysis, glucose-limiting culture assays, and glycolysis inhibitors, including inhibitors of the NAD+ salvage enzyme nicotinamide phosphoribosyl-transferase (NAMPT), in MYC and MYCN shRNA knockdown and lentivirus overexpression systems and in patient-derived glioblastoma tumorspheres with and without MYC/MYCN amplification. The in vivo efficacy of glycolyic inhibition was tested using NAMPT inhibitors in MYCN-amplified patient-derived glioblastoma orthotopic xenograft mouse models.Results: Enforced Myc overexpression increased glucose flux and expression of glycolytic enzymes in glioblastoma cells. Myc and N-Myc knockdown and Myc overexpression systems demonstrated that Myc activity determined sensitivity and resistance to inhibition of glycolysis. Small-molecule inhibitors of glycolysis, particularly NAMPT inhibitors, were selectively toxic to MYC/MYCN–amplified patient-derived glioblastoma tumorspheres. NAMPT inhibitors were potently cytotoxic, inducing apoptosis and significantly extended the survival of mice bearing MYCN-amplified patient-derived glioblastoma orthotopic xenografts.Conclusions: Myc activation in glioblastoma generates a dependency on glycolysis and an addiction to metabolites required for glycolysis. Glycolytic inhibition via NAMPT inhibition represents a novel metabolically targeted therapeutic strategy for MYC or MYCN-amplified glioblastoma and potentially other cancers genetically driven by Myc. Clin Cancer Res; 22(17); 4452–65. ©2016 AACR.

Supplementary Figure 1. A, Hematoxylin and eosin, Ki-67, and IDH1R132H staining in each specimen. B, Sanger sequencing, immunohistochemical analysis, pyrosequencing, and FISH to identify YMG6 tumors as oligodendroglial tumors. C, Multiplex Ligation-dependent Probe Amplification assay demonstrating chromosome 1p and 19q co-deletion in YMG6R3F (left) and YMG6R3T (right). Bars, 50 �m. Supplementary Figure 2. A, DNA fingerprinting showing matched DNA identification in YMG6R3F (upper), YMG6R3T (middle), and YMG6R3T xenograft (YMG6R3Tsc1, lower). Supplementary Figure 3. Sanger sequencing indicating MSH6Ala1293Ser (left, arrow) and STK11Pro281Leu (right, arrow) in YMG6I. B, Immunohistochemical analysis demonstrating MSH6 expression in YMG6I (left) and YMG6R4T (right). C, Immunohistochemical analysis demonstrating LKB1/STK11 expression in YMG6I (left) and YMG23 (right, positive control). D, Sanger sequencing indicating MSH6Gly409Glu (arrow) in YMG6R4T. E, F, Upper gastrointestinal endoscopy (E) and colonoscopy (F) demonstrating no abnormal lesion in YMG6 patient. G, FDG-PET indicating no abnormal uptake in YMG6 patient. Bars, 50 �m. Supplementary Figure 4. Microsatellite instability (MSI)-PCR indicating microsatellite stable (MSS) in YMG6R2 and YMG6R3T. MSI-H, microsatellite instability high. MSS, microsatellite stable. Supplementary Figure 5. Immunohistochemical analysis demonstrating ATRX expression and weak expression of p53 in YMG6R3T. Supplementary Figure 6. Pyrosequencing indicating PIK3CAE542K (arrows) in YMG6I and YMG6R3T. B, Immunohistochemical analysis demonstrating phospho-AKT, -4EBP1, and -S6K expression in YMG6I and YMG6R1. Supplementary Figure 7. A, Multiplex Ligation-dependent Probe Amplification assay indicating 1p and 19q co-deletion in YMG6R3Tsc1. B, Sanger sequencing showing mutation in IDH1 (arrow; c.395G>A, left) and TERT (arrow; c.-124C>T, right) of YMG6R4Tsc1. Supplementary Figure 8. A, Overview and microscopic view of hematoxylin and eosin (H&E, upper), Ki-67 (middle) and IDH1R132H (lower) in first (YMG6R3Tsc1, left), second (YMG6R3Tsc2, middle), and third (YMG6R3Tsc3, right) generation YMG6R3T xenograft. B, H&E (upper), Ki-67 (middle), and IDH1R132H (lower) in YMG6R4Tsc1 (left), YMG6R4Tsc2 (middle), and YMG6R4Tsc3 (right). C, Sanger sequencing showing mutation of IDH1 (arrows, c.395G>A, left) and TERT (arrows, c.-124C>T, right), and PIK3CA (arrows, c.1624G>A) in YMG6R3Tsc2 (upper), YMG6R4Tsc2 (middle), and YMG6R4Tsc3 (lower). D, Immunohistochemical analysis demonstrating strong expression of phospho-AKT (Ser473, left), phospho-4EBP1 (middle), and phospho-S6K (right) in YMG6R4Tsc3. Bars, 50 �m. Supplementary Figure 9. A, Upper, Sanger sequencing showing IDH1 mutation (arrow, c.395 G>A), TERT (arrow, c.-124C>T), and PIK3CA (arrow, c.3140A>G) in YMG23. Lower, MLPA demonstrating chromosome 1p/19q co-deletion and CDKN2A (chr.9p.21) loss in YMG23 patient tumor. B, Immunohistochemical analysis demonstrating negative expression of p16INK4a/CDKN2A in YMG23 (upper). YMG5 (CDKN2A intact) served as positive control. C, Sanger sequencing showing mutations of IDH1 (arrow, c.395G>A, R132H, left) and TERT (arrow, c.-124C>T) in YMG5. D, Sanger sequencing showing mutations of IDH1 (arrow, c.395G>A), TERT (arrow, c.-124C>T), and PIK3CA (arrow, c.3140A>G) in YMG23sc1. E, MLPA demonstrating chromosome 1p/19q co-deletion and CDKN2A loss (chr.9p.21) in YMG23sc1. F, immunohistochemical analysis demonstrating negative expression of p16INK4a/CDKN2A in YMG23sc1. Bars, 50 �m. Supplementary Figure 10. A, B, C, Sanger sequencing showing IDH1 mutation (arrows, c.395G>A, R132H, left) and TERT promoter mutation (c.-124C>T, C228T or c.-146C>T, C250T), and in YMG46 (A), YMG28 (B) and YMG53 (C). PIK3CA mutation (c.1406A>G, E542G, arrow, A, right) in YMG46. D, Contrast enhancing MRI showing non-enhanced tumor recurrence at right frontal lobe (left). H&E (upper) and IDH1R132H staining (lower) in YMG53 (AOD, middle). Overview of H&E staining indicating no xenograft formation in YMG53sc1 (right). Bars, 50 �m. Supplementary Figure 11. A, Overview and magnification of hematoxylin and eosin staining of second passaged YMG23 xenograft model (YMG23sc2). B, C, Immunohistochemical analysis demonstrating expression of IDHR132H (B, left), Ki-67 (B, right), phospho-AKT (C, left), phospho-4EBP1 (C, middle), and phospho-S6K (C, right) in YMG23sc2. D, Sanger sequencing showing mutations in IDH1 (arrow, c.395 G>A), TERT (arrow, c.-124C>T), and PIK3CA (arrow, c.3140A>G) in YMG23sc2. Bars, 50 �m. Supplementary Figure 12. (A-D) Contrast enhanced MRI of the indicated patients at pre-treatment (left) and post-chemotherapy with/without radiotherapy (right). Supplementary Figure 13. Kaplan Meier Curve indicating no survival difference (P = 0.9) of overall survival in PIK3CA/PIK3RI- mutant (blue) and -wild-type (red) oligodendroglial tumor patients. Supplementary Figure 14. A, Relative cell viability of YMG28 (left) and YMG46 (right) cells after 3-day treatment with FK866 combined with DMSO control (blue bars) or TMZ (200 �M, purple bars). *, P

Primary central nervous system lymphoma (PCNSL) is an isolated type of lymphoma of the central nervous system and has a dismal prognosis despite intensive chemotherapy. Recent genomic analyses have identified highly recurrent mutations of MYD88 and CD79B in immunocompetent PCNSL, whereas LMP1 activation is commonly observed in Epstein–Barr virus (EBV)-positive PCNSL. However, a lack of clinically representative preclinical models has hampered our understanding of the pathogenic mechanisms by which genetic aberrations drive PCNSL disease phenotypes. Here, we establish a panel of 12 orthotopic, patient-derived xenograft (PDX) models from both immunocompetent and EBV-positive PCNSL and secondary CNSL biopsy specimens. PDXs faithfully retained their phenotypic, metabolic, and genetic features, with 100% concordance of MYD88 and CD79B mutations present in PCNSL in immunocompetent patients. These models revealed a convergent functional dependency upon a deregulated RelA/p65-hexokinase 2 signaling axis, codriven by either mutated MYD88/CD79B or LMP1 with Pin1 overactivation in immunocompetent PCNSL and EBV-positive PCNSL, respectively. Notably, distinct molecular alterations used by immunocompetent and EBV-positive PCNSL converged to deregulate RelA/p65 expression and to drive glycolysis, which is critical for intracerebral tumor progression and FDG-PET imaging characteristics. Genetic and pharmacologic inhibition of this key signaling axis potently suppressed PCNSL growth in vitro and in vivo. These patient-derived models offer a platform for predicting clinical chemotherapeutics efficacy and provide critical insights into PCNSL pathogenic mechanisms, accelerating therapeutic discovery for this aggressive disease.Significance:A set of clinically relevant CNSL xenografts identifies a hyperactive RelA/p65-hexokinase 2 signaling axis as a driver of progression and potential therapeutic target for treatment and provides a foundational preclinical platform.

PDF file - 294KB, Supplementary Figure S7. PDGFRA and MET amplification sensitize IDH1-mutant glioma TICs to targeted inhibitors.

Supplementary Table S1-S12

Purpose:Oligodendroglioma has a relatively favorable prognosis, however, often undergoes malignant progression. We hypothesized that preclinical models of oligodendroglioma could facilitate identification of therapeutic targets in progressive oligodendroglioma. We established multiple oligodendroglioma xenografts to determine if the PI3K/AKT/mTOR signaling pathway drives tumor progression.Experimental Design:Two anatomically distinct tumor samples from a patient who developed progressive anaplastic oligodendroglioma (AOD) were collected for orthotopic transplantation in mice. We additionally implanted 13 tumors to investigate the relationship between PI3K/AKT/mTOR pathway alterations and oligodendroglioma xenograft formation. Pharmacologic vulnerabilities were tested in newly developed AOD models in vitro and in vivo.Results:A specimen from the tumor site that subsequently manifested rapid clinical progression contained a PIK3CA mutation E542K, and yielded propagating xenografts that retained the OD/AOD-defining genomic alterations (IDH1R132H and 1p/19q codeletion) and PIK3CAE542K, and displayed characteristic sensitivity to alkylating chemotherapeutic agents. In contrast, a xenograft did not engraft from the region that was clinically stable and had wild-type PIK3CA. In our panel of OD/AOD xenografts, the presence of activating mutations in the PI3K/AKT/mTOR pathway was consistently associated with xenograft establishment (6/6, 100%). OD/AOD that failed to generate xenografts did not have activating PI3K/AKT/mTOR alterations (0/9, P < 0.0001). Importantly, mutant PIK3CA oligodendroglioma xenografts were vulnerable to PI3K/AKT/mTOR pathway inhibitors in vitro and in vivo—evidence that mutant PIK3CA is a tumorigenic driver in oligodendroglioma.Conclusions:Activation of the PI3K/AKT/mTOR pathway is an oncogenic driver and is associated with xenograft formation in oligodendrogliomas. These findings have implications for therapeutic targeting of PI3K/AKT/mTOR pathway activation in progressive oligodendrogliomas.

Supplementary Figure Legends and Supplementary Methods

PDF file - 85KB, Supplementary Table S1. 2-hydroxyglutarate (2HG) measurements of mouse glioma orthotopic xenografts.

Purpose:Isocitrate dehydrogenase (IDH)-mutant glioma is a distinct glioma molecular subtype for which no effective molecularly directed therapy exists. Low-grade gliomas, which are 80%–90% IDH-mutant, have high RNA levels of the cell surface Notch ligand DLL3. We sought to determine DLL3 expression by IHC in glioma molecular subtypes and the potential efficacy of an anti-DLL3 antibody–drug conjugate (ADC), rovalpituzumab tesirine (Rova-T), in IDH-mutant glioma.Experimental Design:We evaluated DLL3 expression by RNA using TCGA data and by IHC in a discovery set of 63 gliomas and 20 nontumor brain tissues and a validation set of 62 known IDH wild-type and mutant gliomas using a monoclonal anti-DLL3 antibody. Genotype was determined using a DNA methylation array classifier or by sequencing. The effect of Rova-T on patient-derived endogenous IDH-mutant glioma tumorspheres was determined by cell viability assay.Results:Compared to IDH wild-type glioblastoma, IDH-mutant gliomas have significantly higher DLL3 RNA (P < 1 × 10−15) and protein by IHC (P = 0.0014 and P < 4.3 × 10−6 in the discovery and validation set, respectively). DLL3 immunostaining was intense and homogeneous in IDH-mutant gliomas, retained in all recurrent tumors, and detected in only 1 of 20 nontumor brains. Patient-derived IDH-mutant glioma tumorspheres overexpressed DLL3 and were potently sensitive to Rova-T in an antigen-dependent manner.Conclusions:DLL3 is selectively and homogeneously expressed in IDH-mutant gliomas and can be targeted with Rova-T in patient-derived IDH-mutant glioma tumorspheres. Our findings are potentially immediately translatable and have implications for therapeutic strategies that exploit cell surface tumor-associated antigens.

Purpose:Iniparib is a purported prodrug causing cell death through intracellular conversion to nitro radical ions. We assessed the efficacy and safety of iniparib with standard radiotherapy and temozolomide in patients with newly diagnosed glioblastoma (GBM).Patients and Methods:Adults meeting eligibility criteria were enrolled in this prospective, single-arm, open-label multi- institution phase II trial with median overall survival (mOS) compared with a historical control as the primary objective. A safety run-in component of radiotherapy + temozolomide + iniparib (n = 5) was followed by an efficacy study (n = 76) with the recommended phase II doses of iniparib (8.0 mg/kg i.v. twice/week with radiotherapy + daily temozolomide followed by 8.6 mg/kg i.v. twice/week with 5/28-day temozolomide).Results:The median age of the 81 evaluable participants was 58 years (63% male). Baseline KPS was ≥ 80% in 87% of participants. The mOS was 22 months [95% confidence interval (CI), 17–24] and the HR was 0.44 (95% CI, 0.35–0.55) per-person-year of follow-up. The 2- and 3-year survival rates were 38% and 25%, respectively. Treatment-related grade 3 adverse events (AEs) occurred in 27% of patients; 9 patients had AEs requiring drug discontinuation including infusion-related reaction, rash, gastritis, increased liver enzymes, and thrombocytopenia.Conclusions:Iniparib is well tolerated with radiotherapy and temozolomide in patients with newly diagnosed GBM at up to 17.2 mg/kg weekly. The primary objective of improved mOS compared with a historical control was met, indicating potential antitumor activity of iniparib in this setting. Dosing optimization (frequency and sequence) is needed prior to additional efficacy studies.

Purpose: Lower-grade gliomas (WHO grade II/III) have been classified into clinically relevant molecular subtypes based on IDH and 1p/19q mutation status. The purpose was to investigate whether T2/FLAIR MRI features could distinguish between lower-grade glioma molecular subtypes.Experimental Design: MRI scans from the TCGA/TCIA lower grade glioma database (n = 125) were evaluated by two independent neuroradiologists to assess (i) presence/absence of homogenous signal on T2WI; (ii) presence/absence of “T2–FLAIR mismatch” sign; (iii) sharp or indistinct lesion margins; and (iv) presence/absence of peritumoral edema. Metrics with moderate–substantial agreement underwent consensus review and were correlated with glioma molecular subtypes. Somatic mutation, DNA copy number, DNA methylation, gene expression, and protein array data from the TCGA lower-grade glioma database were analyzed for molecular–radiographic associations. A separate institutional cohort (n = 82) was analyzed to validate the T2–FLAIR mismatch sign.Results: Among TCGA/TCIA cases, interreader agreement was calculated for lesion homogeneity [κ = 0.234 (0.111–0.358)], T2–FLAIR mismatch sign [κ = 0.728 (0.538–0.918)], lesion margins [κ = 0.292 (0.135–0.449)], and peritumoral edema [κ = 0.173 (0.096–0.250)]. All 15 cases that were positive for the T2–FLAIR mismatch sign were IDH-mutant, 1p/19q non-codeleted tumors (P < 0.0001; PPV = 100%, NPV = 54%). Analysis of the validation cohort demonstrated substantial interreader agreement for the T2–FLAIR mismatch sign [κ = 0.747 (0.536–0.958)]; all 10 cases positive for the T2–FLAIR mismatch sign were IDH-mutant, 1p/19q non-codeleted tumors (P < 0.00001; PPV = 100%, NPV = 76%).Conclusions: Among lower-grade gliomas, T2–FLAIR mismatch sign represents a highly specific imaging biomarker for the IDH-mutant, 1p/19q non-codeleted molecular subtype. Clin Cancer Res; 23(20); 6078–85. ©2017 AACR.