Youland, Ryan S.

AbstractBackgroundGlioblastoma (GBM) represents an aggressive cancer type with a median survival of only 14 months. With fewer than 5% of patients surviving 5 years, comprehensive profiling of these rare patients could elucidate prognostic biomarkers that may confer better patient outcomes. We utilized multiple molecular approaches to characterize the largest patient cohort of isocitrate dehydrogenase (IDH)–wildtype GBM long-term survivors (LTS) to date.MethodsRetrospective analysis was performed on 49 archived formalin-fixed paraffin embedded tumor specimens from patients diagnosed with GBM at the Mayo Clinic between December 1995 and September 2013. These patient samples were subdivided into 2 groups based on survival (12 LTS, 37 short-term survivors [STS]) and subsequently examined by mutation sequencing, copy number analysis, methylation profiling, and gene expression.ResultsOf the 49 patients analyzed in this study, LTS were younger at diagnosis (P = 0.016), more likely to be female (P = 0.048), and MGMT promoter methylated (UniD, P = 0.01). IDH-wildtype STS and LTS demonstrated classic GBM mutations and copy number changes. Pathway analysis of differentially expressed genes showed LTS enrichment for sphingomyelin metabolism, which has been linked to decreased GBM growth, invasion, and angiogenesis. STS were enriched for DNA repair and cell cycle control networks.ConclusionsWhile our findings largely report remarkable similarity between these LTS and more typical STS, unique attributes were observed in regard to altered gene expression and pathway enrichment. These attributes may be valuable prognostic markers and are worth further examination. Importantly, this study also underscores the limitations of existing biomarkers and classification methods in predicting patient prognosis.

Abstract Background Despite advances in modern therapy, high-grade gliomas continue to portend a dismal prognosis and nearly all patients will experience relapse. Unfortunately, salvage options remain limited. In this study, we assessed outcomes for patients with recurrent gliomas treated with reirradiation. Methods We retrospectively identified 48 glioma patients treated with reirradiation between 2013 and 2016. All had radiographic or pathologic evidence of recurrence. Prognostic factors were abstracted from the electronic medical record. Results Initial surgery included biopsy in 15, subtotal resection in 21, and gross total resection in 12. Initial chemotherapy included temozolomide (TMZ) in 31, TMZ+dasatinib in 7, TMZ+vorinostat in 3, and procarbazine, lomustine, and vincristine in 2. The median dose of primary radiotherapy was 60 Gy delivered in 30 fractions. Median overall survival (OS) and progression-free survival (PFS) from initial diagnosis were 3.2 and 1.7 years, respectively. A total of 36 patients failed salvage bevacizumab before reirradiation. Salvage surgery was performed before reirradiation in 21 patients. Median time to reirradiation was 1.7 years. Median follow-up was 13.7 months from reirradiation. Concurrent systemic therapy was given in 33 patients (bevacizumab in 27, TMZ in 8, and lomustine in 2). Median PFS and OS after reirradiation were 3.2 and 6.3 months, respectively. Radionecrosis occurred in 4 patients and no radionecrosis was seen in patients receiving concurrent bevacizumab with reirradiation (0% vs 19%, P = .03). Conclusions Reirradiation may result in delayed tumor progression with acceptable toxicity. Prospective trials are needed to determine the impact of reirradiation on tumor progression and quality of life.

Abstract INTRODUCTION: The L-Type amino acid transporter 1 (LAT1) transports large neutral amino acids across the plasma membrane in a variety of cells. Positron emission tomography (PET) imaging with the amino acid tracer 6-18F-fluoro-L-dopa (18F-FDOPA) provides useful histological information in human gliomas in addition to providing better localizing information then CT or MRI alone. This study is the first to correlate LAT1 expression with uptake of the 18F-FDOPA analog, 3H-L-DOPA in human glioblastomas. METHODS: Endogenous expression of LAT1 was evaluated in two established glioblastoma multiforme (GBM) cell lines and three primary GBM xenografts using Western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Uptake of FDOPA was assessed in vitro using 3H-L-DOPA as an analog to 18F-FDOPA. Knockdown of LAT1 expression was achieved by lentiviral expression of anti-LAT1 shRNA in GBM lines with high LAT1 expression. The effect of LAT1 knockdown on 3H-L-DOPA uptake was then quantified. RESULTS: Results from Western blotting and qRT-PCR demonstrate that LAT1 is variably expressed in GBM. Levels of 3H-L-DOPA uptake were directly related to LAT1 expression (p<0.001) across the 5 lines. Knockdown of LAT1 was achieved in T98 cells (knockdown efficiency = 82.8%) and xenograft GBM 28 (knockdown efficiency = 73.7%) as confirmed by qRT-PCR and Western blotting. Knockdown of LAT1 reduced 3H-L-DOPA uptake by 57.2% (p<0.0001) in T98 and 52.1% in GBM 28 (p<0.001). CONCLUSIONS: This is the first report of 3H-L-DOPA uptake being driven by LAT1 in human GBM. Our findings suggest LAT1 as a transporter of 18F-FDOPA in GBM. Additional studies are underway to correlate LAT1 expression with PET 18F-FDOPA uptake in patients with GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4543. doi:1538-7445.AM2012-4543

Abstract Purpose/Background: The first-line chemotherapeutic temozolomide (TMZ) does not offer any therapeutic benefit in some cases of glioblastoma (GBM) and emergence of resistance leads to tumor progression in almost all patients. While the expression of O-6-methylguanine-DNA-methyltransferase (MGMT) correlates well with TMZ resistance, its expression varies among GBM tumors. The goal of this study is to identify additional genes with potential roles in TMZ resistance. Materials and Methods: Five primary GBM xenografts were generated from patient samples, and subsequent secondary models for resistance were developed by subjecting tumor-bearing mice to escalating doses of TMZ. Triplicate samples from parental and TMZ-resistant xenografts were analyzed for gene expression using the Illumina BeadChip microarray platform. The top 100 differentially expressed genes for each line were considered candidates. Genes were selected for qRT-PCR validation after ontological and pathway analysis. A lentivirus-mediated shRNA knockdown was performed to validate the role of several candidate genes in TMZ resistance in U87 and primary GBM12 cell lines. Knockdown was confirmed with qRT-PCR and western blots. TMZ responsiveness was assessed using CyQUANT and Neurosphere assays. Results: Upregulated MGMT expression was found in 3 of the 5 xenografts (GBM12TMZ, GBM14TMZ and GBM28TMZ) and was mechanistically linked with TMZ resistance in these lines using O6-benzylguanine to reverse the resistance phenotype. Differential expression of other DNA damage response genes included CCND2, CDKN1A, GADD45B, UBB as well as apoptosis regulators NFKB2 and BIRC3. In lines with unchanged MGMT expression (GBM22TMZ and GBM39TMZ), changes in anti-apoptosis genes NFKB2 and MAP3K1, transcription factor SRF and signal transduction gene PRKCDBP were validated by qRT-PCR. In GBM12TMZ, in addition to MGMT upregulation, expression of Ubiquitin B (UBB) was found to be the most significantly down-regulated gene both on microarray (Log2FC = −4.55, Fold change = 0.043) and qRT-PCR (Log2RQ =-4.04, RQ = 0.061). To test a possible role of UBB in TMZ resistance, knockdown was achieved in U87 (knockdown efficiency=91.2%) and in primary xenograft line GBM12 (knockdown efficiency=84.4%). The CyQUANT assay for U87UBB-KD and U87NT treated with escalating doses of TMZ revealed no significant difference in cell survival (10 M TMZ relative survival= 89.7% for U87UBB-KD and 79.2% for U87NT; p-value=0.53). Similarly, a neurosphere assay for GBM12UBB-KD and GBM12NT also yielded no significant difference in cell survival (10 M TMZ relative survival= 25.13% for GBM12UBB-KD and 23.35% for GBM12NT; p-value=0.83). Conclusions: We have used a TMZ resistance xenograft model to identify candidate TMZ resistance genes. The current data provides evidence on the role of MGMT in the development of acquired TMZ resistance while ruling out UBB as potential TMZ resistance gene in GBM. Additional studies are underway to assess the functional significance of other candidate genes, particularly those with differential expression in the setting of constant MGMT expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B68.