Multidisciplinary Management of Isocitrate Dehydrogenase-Mutated Gliomas in a Contemporary Molecularly Defined Era.

Mutations in isocitrate dehydrogenase (IDH) genes, an early step in the ontogeny of lower-grade gliomas, induce global epigenetic changes characterized by a hypermethylation phenotype and are critical to tumor classification, treatment decision making, and estimation of patient prognosis. The introduction of IDH inhibitors to block the oncogenic neomorphic function of the mutated protein has resulted in new therapeutic options for these patients. To appreciate the implications of these recent IDH inhibitor results, it is important to juxtapose historical outcomes with chemoradiotherapy. Herein, we rationally evaluate recent IDH inhibitor data within historical precedents to guide contemporary decisions regarding the role of observation, maximal safe resection, adjuvant therapies, and the import of patient and tumor variables. The biological underpinnings of the IDH pathway and the mechanisms, impact, and limitations of IDH inhibitors, the actual magnitude of tumor regression and patient benefit, and emergence of resistance pathways are presented to guide future trial development. Management in the current, molecularly defined era will require careful patient selection and risk factor assessment, followed by an open dialog about the results of studies such as INDIGO, as well as mature data from legacy trials, and a discussion about risk-versus-benefit for the choice of treatment, with multidisciplinary decision making as an absolute prerequisite.

Proteomic Analysis of Spatial Heterogeneity Identifies HMGB2 as Putative Biomarker of Tumor Progression in Adult-Type Diffuse Astrocytomas.

Although grading is defined by the highest histological grade observed in a glioma, most high-grade gliomas retain areas with histology reminiscent of their low-grade counterparts. We sought to achieve the following: (i) identify proteins and molecular pathways involved in glioma evolution; and (ii) validate the high mobility group protein B2 (HMGB2) as a key player in tumor progression and as a prognostic/predictive biomarker for diffuse astrocytomas. We performed liquid chromatography tandem mass spectrometry (LC-MS/MS) in multiple areas of adult-type astrocytomas and validated our finding in multiplatform-omics studies and high-throughput IHC analysis. LC-MS/MSdetected proteomic signatures characterizing glioma evolution towards higher grades associated with, but not completely dependent, on IDH status. Spatial heterogeneity of diffuse astrocytomas was associated with dysregulation of specific molecular pathways, and HMGB2 was identified as a putative driver of tumor progression, and an early marker of worse overall survival in grades 2 and 3 diffuse gliomas, at least in part regulated by DNA methylation. In grade 4 astrocytomas, HMGB2 expression was strongly associated with proliferative activity and microvascular proliferation. Grounded in proteomic findings, our results showed that HMGB2 expression assessed by IHC detected early signs of tumor progression in grades 2 and 3 astrocytomas, as well as identified GBMs that had a better response to the standard chemoradiation with temozolomide.

A Review of Hodgkin Lymphoma in the Era of Checkpoint Inhibitors.

Hodgkin lymphoma (HL) is a hematopoietic malignancy of B-cells that has a bimodal distribution with respect to age and incidence. With the introduction of doxorubicin (Adriamycin), bleomycin, vinblastine, and dacarbazine (ABVD) and radiation combined, the prognosis of HL has significantly improved, with five-year survival rates approaching 95%. While HL has become highly curable, the side effect profiles of ABVD are dire and warrant continuous review. Because HL is often diagnosed in populations in their 20s-30s, patients are forced to undergo fertility preservation procedures as well as deal with other long-term side effects of chemotherapy (including doxorubicin dose-dependent cardiotoxicity and bleomycin-induced lung toxicity). The opportunity cost of the treatment in the short term and vulnerability to treatment-induced malignancies decades later dramatically affect the quality of life of HL patients. New therapies have developed over the past several decades with respect to immunotherapies, particularly programmed death protein 1 inhibitors (e.g., nivolumab and pembrolizumab). Studies have shown checkpoint inhibitors to be effective in treating HL with an objective response rate of 69% for relapsed/refractory classical HL for nivolumab use. Checkpoint inhibitors will continue to help maintain the high five-year survival rate for HL and hopefully have a more favorable side effect profile in the short term, as well as later in the patient's life. This article seeks to summarize treatment options for HL while comparing outcomes and side effect profiles with the addition of checkpoint inhibitors.

TRIB1 confers therapeutic resistance in GBM cells by activating the ERK and Akt pathways.

GBM (Glioblastoma) is the most lethal CNS (Central nervous system) tumor in adults, which inevitably develops resistance to standard treatments leading to recurrence and mortality. TRIB1 is a serine/threonine pseudokinase which functions as a scaffold platform that initiates degradation of its substrates like C/EBPα through the ubiquitin proteasome system and also activates MEK and Akt signaling. We found that increased TRIB1 gene expression associated with worse overall survival of GBM patients across multiple cohorts. Importantly, overexpression of TRIB1 decreased RT/TMZ (radiation therapy/temozolomide)-induced apoptosis in patient derived GBM cell lines in vitro. TRIB1 directly bound to MEK and Akt and increased ERK and Akt phosphorylation/activation. We also found that TRIB1 protein expression was maximal during G2/M transition of cell cycle in GBM cells. Furthermore, TRIB1 bound directly to HDAC1 and p53. Importantly, mice bearing TRIB1 overexpressing tumors had worse overall survival. Collectively, these data suggest that TRIB1 induces resistance of GBM cells to RT/TMZ treatments by activating the cell proliferation and survival pathways thus providing an opportunity for developing new targeted therapeutics.

Haploinsufficiency of NFKBIA reshapes the epigenome antipodal to the IDH mutation and imparts disease fate in diffuse gliomas.

Genetic alterations help predict the clinical behavior of diffuse gliomas, but some variability remains uncorrelated. Here, we demonstrate that haploinsufficient deletions of chromatin-bound tumor suppressor NFKB inhibitor alpha (NFKBIA) display distinct patterns of occurrence in relation to other genetic markers and are disproportionately present at recurrence. NFKBIA haploinsufficiency is associated with unfavorable patient outcomes, independent of genetic and clinicopathologic predictors. NFKBIA deletions reshape the DNA and histone methylome antipodal to the IDH mutation and induce a transcriptome landscape partly reminiscent of H3K27M mutant pediatric gliomas. In IDH mutant gliomas, NFKBIA deletions are common in tumors with a clinical course similar to that of IDH wild-type tumors. An externally validated nomogram model for estimating individual patient survival in IDH mutant gliomas confirms that NFKBIA deletions predict comparatively brief survival. Thus, NFKBIA haploinsufficiency aligns with distinct epigenome changes, portends a poor prognosis, and should be incorporated into models predicting the disease fate of diffuse gliomas.

Comparison of Gonadal Toxicity of Single-Fraction Ultra-High Dose Rate and Conventional Radiation in Mice.

Purpose: Increasing evidence suggests that ultra-high-dose-rate (UHDR) radiation could result in similar tumor control as conventional (CONV) radiation therapy (RT) while reducing toxicity to surrounding healthy tissues. Considering that radiation toxicity to gonadal tissues can cause hormone disturbances and infertility in young patients with cancer, the purpose of this study was to assess the possible role of UHDR-RT in reducing toxicity to healthy gonads in mice compared with CONV-RT. Methods and Materials: Radiation was delivered to the abdomen or pelvis of female (8 or 16 Gy) and male (5 Gy) C57BL/6J mice, respectively, at conventional (∼0.4 Gy/s) or ultrahigh (>100 Gy/s) dose rates using an IntraOp Mobetron linear accelerator. Organ weights along with histopathology and immunostaining of irradiated gonads were used to compare toxicity between radiation modalities. Results: CONV-RT and UHDR-RT induced a similar decrease in uterine weights at both studied doses (∼50% of controls), which indicated similarly reduced ovarian follicular activity. Histologically, ovaries of CONV- and UHDR-irradiated mice exhibited a comparable lack of follicles. Weights of CONV- and UHDR-irradiated testes were reduced to ∼30% of controls, and the percentage of degenerate seminiferous tubules was also similar between radiation modalities (∼80% above controls). Pairwise comparisons of all quantitative data indicated statistical significance between irradiated (CONV or UHDR) and control groups (from P ≤ .01 to P ≤ .0001) but not between radiation modalities. Conclusions: The data presented here suggest that the short-term effects of UHDR-RT on the mouse gonads are comparable to those of CONV-RT.

Long-Term Report of a Comprehensive Molecular and Genomic Analysis in NRG Oncology/RTOG 0424: A Phase II Study of Radiation and Temozolomide in High-Risk Grade II Glioma.

PURPOSE: This study sought to determine the prognostic significance of the WHO-defined glioma molecular subgroups along with additional alterations, including MGMT promoter methylation and mutations in ATRX, CIC, FUBP1, TERT, and TP53, in NRG/RTOG 0424 using long-term follow-up data. METHODS: Mutations were determined using an Ion Torrent sequencing panel. 1p/19q co-deletion and MGMT promoter methylation were determined by Affymetrix OncoScan and Illumina 450K arrays. Progression-free survival (PFS) and overall survival (OS) were estimated using the Kaplan-Meier method and tested using the log-rank test. Hazard ratios were calculated using the Cox proportional hazard model. Multivariable analyses (MVAs) included patient pretreatment characteristics. RESULTS: We obtained complete molecular data to categorize 80/129 eligible patients within the WHO subgroups. Of these, 26 (32.5%) were IDHmutant/co-deleted, 28 (35%) were IDHmutant/non-co-deleted, and 26 (32.5%) were IDHwild-type. Upon single-marker MVA, both IDHmutant subgroups were associated with significantly better OS and PFS (P values < .001), compared with the IDHwild-type subgroup. MGMT promoter methylation was obtained on 76 patients, where 58 (76%) were methylated and 18 (24%) were unmethylated. Single-marker MVAs demonstrated that MGMT promoter methylation was statistically significant for OS (P value < .001) and PFS (P value = .003). In a multimarker MVA, one WHO subgroup comparison (IDHmutant/co-deleted v IDHwild-type) was significant for OS (P value = .045), whereas MGMT methylation did not retain significance. CONCLUSION: This study reports the long-term prognostic effect of the WHO molecular subgroups, MGMT promoter methylation, and other mutations in NRG/RTOG 0424. These results demonstrate that the WHO molecular classification and MGMT both serve as strong prognostic indicators, but that MGMT does not appear to add statistically significant prognostic value to the WHO subgrouping, above and beyond IDH and 1p/19q status.

A Novel miR-146a-POU3F2/SMARCA5 Pathway Regulates Stemness and Therapeutic Response in Glioblastoma.

Rapid tumor growth, widespread brain-invasion, and therapeutic resistance critically contribute to glioblastoma (GBM) recurrence and dismal patient outcomes. Although GBM stem cells (GSC) are shown to play key roles in these processes, the molecular pathways governing the GSC phenotype (GBM-stemness) remain poorly defined. Here, we show that epigenetic silencing of miR-146a significantly correlated with worse patient outcome and importantly, miR-146a level was significantly lower in recurrent tumors compared with primary ones. Further, miR-146a overexpression significantly inhibited the proliferation and invasion of GBM patient-derived primary cells and increased their response to temozolomide (TMZ), both in vitro and in vivo. Mechanistically, miR-146a directly silenced POU3F2 and SMARCA5, two transcription factors that mutually regulated each other, significantly compromising GBM-stemness and increasing TMZ response. Collectively, our data show that miR-146a-POU3F2/SMARCA5 pathway plays a critical role in suppressing GBM-stemness and increasing TMZ-response, suggesting that POU3F2 and SMARCA5 may serve as novel therapeutic targets in GBM. IMPLICATIONS: miR-146a predicts favorable prognosis and the miR-146a-POU3F2/SMARCA5 pathway is important for the suppression of stemness in GBM.

Pediatric Gliosarcoma With and Without Neurofibromatosis Type 1: A Whole-exome Comparison of 2 Patients.

Gliosarcoma is rare among pediatric patients and among individuals with Neurofibromatosis Type 1 (NF1). Here we compare 2 pediatric gliosarcoma patients, one of whom has NF1. We performed whole-exome sequencing, methylation, and copy number analysis on tumor and blood for both patients. Whole-exome sequencing showed higher mutational burden in the tumor of the patient without NF1. Copy number analysis showed differences in chromosomal losses/gains between the tumors. Neither tumor showed O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation. The NF1 patient survived without progression while the other expired. This is the first reported case of gliosarcoma in a child with NF1.

Comprehensive Genomic Analysis in NRG Oncology/RTOG 9802: A Phase III Trial of Radiation Versus Radiation Plus Procarbazine, Lomustine (CCNU), and Vincristine in High-Risk Low-Grade Glioma.

PURPOSE: NRG Oncology/RTOG 9802 (ClinicalTrials.gov Identifier: NCT00003375) is a practice-changing study for patients with WHO low-grade glioma (LGG, grade II), as it was the first to demonstrate a survival benefit of adjuvant chemoradiotherapy over radiotherapy. This post hoc study sought to determine the prognostic and predictive impact of the WHO-defined molecular subgroups and corresponding molecular alterations within NRG Oncology/RTOG 9802. METHODS: IDH1/2 mutations were determined by immunohistochemistry and/or deep sequencing. A custom Ion AmpliSeq panel was used for mutation analysis. 1p/19q codeletion and MGMT promoter methylation were determined by copy-number arrays and/or Illumina 450K array, respectively. Progression-free survival (PFS) and overall survival (OS) were estimated using the Kaplan-Meier method. Hazard ratios (HRs) were calculated using the Cox proportional hazard model and tested using the log-rank test. Multivariable analyses (MVAs) were performed incorporating treatment and common prognostic factors as covariates. RESULTS: Of the eligible patients successfully profiled for the WHO-defined molecular groups (n = 106/251), 26 (24%) were IDH-wild type, 43 (41%) were IDH-mutant/non-codeleted, and 37(35%) were IDH-mutant/codeleted. MVAs demonstrated that WHO subgroup was a significant predictor of PFS after adjustment for clinical variables and treatment. Notably, treatment with postradiation chemotherapy (PCV; procarbazine, lomustine (CCNU), and vincristine) was associated with longer PFS (HR, 0.32; P = .003; HR, 0.13; P < .001) and OS (HR, 0.38; P = .013; HR, 0.21; P = .029) in the IDH-mutant/non-codeleted and IDH-mutant/codeleted subgroups, respectively. In contrast, no significant difference in either PFS or OS was observed with the addition of PCV in the IDH-wild-type subgroup. CONCLUSION: This study is the first to report the predictive value of the WHO-defined diagnostic classification in a set of uniformly treated patients with LGG in a clinical trial. Importantly, this post hoc analysis supports the notion that patients with IDH-mutant high-risk LGG regardless of codeletion status receive benefit from the addition of PCV.

Single Nucleotide Polymorphisms in ß-Carotene Oxygenase 1 are Associated with Plasma Lycopene Responses to a Tomato-Soy Juice Intervention in Men with Prostate Cancer.

BACKGROUND: Human plasma and tissue lycopene concentrations are heterogeneous even when consuming controlled amounts of tomato or lycopene. OBJECTIVES: Our objective is to determine whether single nucleotide polymorphisms (SNPs) in or near known or putative carotenoid metabolism genes [ß-carotene 15,15' monooxygenase 1 (BCO1), scavenger receptor class B type 1 (SCARB1), ATP-binding cassette transporter subfamily A member 1 (ABCA1), microsomal triglyceride transfer protein (MTTP), apolipoprotein B-48, elongation of very long chain fatty acids protein 2 (ELOVL2), and ATP-binding cassette subfamily B member 1 (ABCB1), and an intergenic superoxide dismutase 2, mitochondrial-associated SNP] are predictive of plasma lycopene responses to steady state tomato juice consumption. METHODS: Secondary linear regression analyses of data from a dose-escalation study of prostate cancer patients [n = 47; mean ± SEM age: 60 ± 1 y; BMI (in kg/m2): 32 ± 1] consuming 0, 1, or 2 cans of tomato-soy juice/d (163 mL/can; 20.6 mg lycopene 1.2 mg ß-carotene/can) for 24 ± 0.7 d before prostatectomy were conducted to explore 11 SNP genotype effects on the change in plasma lycopene and plasma and prostate tissue concentrations of lycopene, ß-carotene, phytoene, and phytofluene. RESULTS: Two BCO1 SNP genotypes were significant predictors of the change in plasma lycopene, with SNP effects differing in magnitude and direction, depending on the level of juice intake (rs12934922 × diet group P = 0.02; rs6564851 × diet group P = 0.046). Further analyses suggested that plasma ß-carotene changes were predicted by BCO1 rs12934922 (P < 0.01), prostate lycopene by trending interaction and main effects of BCO1 SNPs (rs12934922 × diet group P = 0.09; rs12934922 P = 0.02; rs6564851 P = 0.053), and prostate ß-carotene by BCO1 SNP interaction and main effects (rs12934922 × diet group P = 0.01; rs12934922 P < 0.01; rs7501331 P = 0.02). CONCLUSIONS: In conclusion, SNPs in BCO1 and other genes may modulate human plasma and prostate tissue responses to dietary lycopene intake and warrant validation in larger, human controlled feeding intervention and cohort studies. Genetic variants related to carotenoid metabolism may partially explain heterogeneous human blood and tissue responses and may be critical covariates for population studies and clinical trials. This trial was registered at clinicaltrials.gov as NCT01009736.

miR-4516 predicts poor prognosis and functions as a novel oncogene via targeting PTPN14 in human glioblastoma.

Glioblastomas (GBMs) are the most aggressive primary brain tumors, with an average survival of less than 15 months. Therefore, there is a critical need to develop novel therapeutic strategies for GBM. This study aimed to assess the prognostic value of miR-4516 and investigate its oncogenic functions and the underlying cellular and molecular mechanisms in GBM. To determine the correlation between miR-4516 expression and overall survival of patients with GBM, total RNAs were isolated from 268 FFPE tumor samples, miR expression was assayed (simultaneously) using the nCounter human miRNA v3a assay followed by univariable and multivariable survival analyses. Further, in vitro and in vivo studies were conducted to define the role of miR-4516 in GBM tumorigenesis and the underlying molecular mechanisms. Upon multivariable analysis, miR-4516 was correlated with poor prognosis in GBM patients (HR = 1.49, 95%CI: 1.12-1.99, P = 0.01). Interestingly, the significance of miR-4516 was retained including MGMT methylation status. Overexpression of miR-4516 significantly enhanced cell proliferation and invasion of GBM cells both in vitro and in vivo. While conducting downstream targeting studies, we found that the tumor-promoting function of miR-4516, in part, was mediated by direct targeting of PTPN14 (protein tyrosine phosphatase, non-receptor type 14) which, in turn, regulated the Hippo pathway in GBM. Taken together, our data suggest that miR-4516 represents an independent negative prognostic factor in GBM patients and acts as a novel oncogene in GBM, which regulates the PTPN14/Hippo pathway. Thus, this newly identified miR-4516 may serve as a new potential therapeutic target for GBM treatment.

MicroRNA Biomarkers for Patients With Muscle-Invasive Bladder Cancer Undergoing Selective Bladder-Sparing Trimodality Treatment.

PURPOSE: Trimodality therapy with maximal transurethral resection of bladder tumor and definitive chemoradiation reserving cystectomy for salvage of local recurrence is an accepted treatment alternative to upfront cystectomy for selected patients with muscle-invasive bladder cancer. There is a need for molecular biomarkers to predict which patients will respond to bladder preservation therapy. METHODS AND MATERIALS: We sought to identify biomarkers with the ability to predict response to chemoradiation and survival after selective bladder preservation therapy in a cohort of 40 patients using a microRNA profiling approach. In vitro experiments were performed using transitional cell carcinoma lines CRL1749, HTB5, and HTB4. RESULTS: We identified a panel of microRNAs associated with overall survival in our bladder preservation cohort and in the TCGA cohort. We also identified several microRNAs, including miR-23a and miR-27a, microRNAs of the miR-23a cluster, to be suggestively associated with complete response to chemoradiation therapy. The microRNAs were significantly associated with overall survival in The Cancer Genome Atlas cohort. In vitro studies suggest that the functional roles of miR-23a and miR-27a involve targeting the SFRP1 protein, a negative regulator of the Wnt signaling pathway. The upregulation of ß-catenin in the Wnt signaling pathway mediated proliferation, migration, invasion, and sensitivity to radiation and cisplatin treatment in bladder cancer cells. CONCLUSIONS: Our results indicate that miR-23a and miR-27a act as oncomirs, and once independently validated, they may help appropriately triage selected bladder cancer patients to individualize treatment.

Association of MGMT Promoter Methylation Status With Survival Outcomes in Patients With High-Risk Glioma Treated With Radiotherapy and Temozolomide: An Analysis From the NRG Oncology/RTOG 0424 Trial.

Importance: The initial report of NRG Oncology/Radiation Therapy Oncology Group (RTOG) 0424 demonstrated a 3-year overall survival benefit with the addition of temozolomide to radiotherapy compared with a historical control. However, an important end point of the trial-evaluation of the association between O6-methylgaunine-DNA-methyltransferase (MGMT) promoter methylation and survival outcomes-was not previously reported. Objective: To examine the proportion of patients in NRG Oncology/RTOG 0424 with MGMT promoter methylation and its association with survival outcomes. Design, Setting, and Participants: Specimens collected were analyzed after trial completion to determine MGMT promoter methylation and IDH1/2 status and the association between MGMT status and survival outcomes. A model derived from logistic regression (MGMT-STP27) was used to calculate MGMT promoter methylation status. Univariate and multivariable analyses were performed using the Cox proportional hazards regression model to determine the association of MGMT status with survival outcomes. Patient pretreatment characteristics were included as covariates in multivariable analyses. Main Outcomes and Measures: Progression-free survival (PFS) and overall survival (OS). Results: Of all 129 eligible patients in NRG Oncology/RTOG 0424, 75 (58.1%) had MGMT status available (median age, 48 years; age range, 20-76 years; 42 [56.0%] male): 57 (76.0%) methylated and 18 (24.0%) unmethylated. A total of 13 unmethylated patients (72.2%) had astrocytoma as opposed to oligoastrocytoma or oligodendroglioma, whereas 23 methylated patients (40.4%) had astrocytoma. On univariate analyses, an unmethylated MGMT promoter was significantly associated with worse OS (hazard ratio [HR], 3.52; 95% CI, 1.64-7.56; P < .001) and PFS (HR, 3.06; 95% CI, 1.55-6.04; P < .001). The statistical significances were maintained in multimarker multivariable analyses, including IDH1/2 status for both OS (HR, 2.70; 95% CI, 1.02-7.14; P = .045) and PFS (HR, 2.74; 95% CI, 1.19-6.33; P = .02). Conclusions and Relevance: In this study, MGMT promoter methylation was an independent prognostic biomarker of high-risk, low-grade glioma treated with temozolomide and radiotherapy. This is the first study, to our knowledge, to validate the prognostic importance of MGMT promoter methylation in patients with grade II glioma treated with combined radiotherapy and temozolomide and highlights its potential prognostic value beyond IDH1/2 mutation status. Trial Registration: ClinicalTrials.gov Identifier: NCT00114140.

Oncogenic transgelin-2 is differentially regulated in isocitrate dehydrogenase wild-type vs. mutant gliomas.

The presence of an isocitrate dehydrogenase (IDH1/2) mutation in gliomas is associated with favorable outcomes compared to gliomas without the mutation (IDH1/2 wild-type, WT). The underlying biological mechanisms accounting for improved clinical outcomes in IDH1/2 mutant gliomas remain poorly understood, but may, in part, be due to the glioma CpG island methylator phenotype (G-CIMP) and epigenetic silencing of genes. We performed profiling of IDH1/2 WT versus IDH1/2 mutant Grade II and III gliomas and identified transgelin-2 (TAGLN2), an oncogene and actin-polymerizing protein, to be expressed at significantly higher levels in IDH1/2 WT gliomas compared to IDH1/2 mutant gliomas. This differential expression of TAGLN2 was primarily due to promoter hypermethylation in IDH1/2 mutant gliomas, suggesting involvement of TAGLN2 in the G-CIMP. Our results also suggest that TAGLN2 may be involved in progression due to higher expression in glioblastomas compared to IDH1/2 WT gliomas of lower grades. Furthermore, our results suggest that TAGLN2 functions as an oncogene by contributing to proliferation and invasion when overexpressed in IDH1/2 WT glioma cells. Taken together, this study demonstrates a possible link between increased TAGLN2 expression, invasion and poor patient outcomes in IDH1/2 WT gliomas and identifies TAGLN2 as a potential novel therapeutic target for IDH1/2 WT gliomas.

The Role of miRNAs in Angiogenesis, Invasion and Metabolism and Their Therapeutic Implications in Gliomas.

MicroRNAs (miRNAs) are small, non-coding, endogenous RNA molecules that function in gene silencing by post-transcriptional regulation of gene expression. The dysregulation of miRNA plays a pivotal role in cancer tumorigenesis, including the development and progression of gliomas. Their small size, stability and ability to target multiple oncogenes have simultaneously distinguished miRNAs as attractive candidates for biomarkers and novel therapeutic targets for glioma patients. In this review, we summarize the most frequently cited miRNAs known to contribute to gliomagenesis and progression by regulating the defining hallmarks of gliomas, including angiogenesis, invasion, and cell metabolism. We also discuss their promising potential as prognostic and predictive biomarkers and novel therapeutic targets, in addition to the challenges that must be overcome before their translation from bench to bedside.

A novel miRNA-based predictive model for biochemical failure following post-prostatectomy salvage radiation therapy.

PURPOSE: To develop a microRNA (miRNA)-based predictive model for prostate cancer patients of 1) time to biochemical recurrence after radical prostatectomy and 2) biochemical recurrence after salvage radiation therapy following documented biochemical disease progression post-radical prostatectomy. METHODS: Forty three patients who had undergone salvage radiation therapy following biochemical failure after radical prostatectomy with greater than 4 years of follow-up data were identified. Formalin-fixed, paraffin-embedded tissue blocks were collected for all patients and total RNA was isolated from 1mm cores enriched for tumor (>70%). Eight hundred miRNAs were analyzed simultaneously using the nCounter human miRNA v2 assay (NanoString Technologies; Seattle, WA). Univariate and multivariate Cox proportion hazards regression models as well as receiver operating characteristics were used to identify statistically significant miRNAs that were predictive of biochemical recurrence. RESULTS: Eighty eight miRNAs were identified to be significantly (p<0.05) associated with biochemical failure post-prostatectomy by multivariate analysis and clustered into two groups that correlated with early (≤ 36 months) versus late recurrence (>36 months). Nine miRNAs were identified to be significantly (p<0.05) associated by multivariate analysis with biochemical failure after salvage radiation therapy. A new predictive model for biochemical recurrence after salvage radiation therapy was developed; this model consisted of miR-4516 and miR-601 together with, Gleason score, and lymph node status. The area under the ROC curve (AUC) was improved to 0.83 compared to that of 0.66 for Gleason score and lymph node status alone. CONCLUSION: miRNA signatures can distinguish patients who fail soon after radical prostatectomy versus late failures, giving insight into which patients may need adjuvant therapy. Notably, two novel miRNAs (miR-4516 and miR-601) were identified that significantly improve prediction of biochemical failure post-salvage radiation therapy compared to clinico-histopathological factors, supporting the use of miRNAs within clinically used predictive models. Both findings warrant further validation studies.

Allele-specific imbalance mapping identifies HDAC9 as a candidate gene for cutaneous squamous cell carcinoma.

More than 3.5 million nonmelanoma skin cancers were treated in 2006; of these 700,000 were cutaneous squamous cell carcinomas (cSCCs). Despite clear environmental causes for cSCC, studies also suggest genetic risk factors. A cSCC susceptibility locus, Skts5, was identified on mouse chromosome 12 by linkage analysis. The orthologous locus to Skts5 in humans maps to 7p21 and 7q31. These loci show copy number increases in ∼10% of cSCC tumors. Here, we show that an additional 15-22% of tumors exhibit copy-neutral loss of heterozygosity. Furthermore, our previous data identified microsatellite markers on 7p21 and 7q31 that demonstrate preferential allelic imbalance (PAI) in cSCC tumors. On the basis of these results, we hypothesized that the human orthologous locus to Skts5 would house a gene important in human cSCC development and that tumors would demonstrate allele-specific somatic alterations. To test this hypothesis, we performed quantitative genotyping of 108 single nucleotide polymorphisms (SNPs) mapping to candidate genes at human SKTS5 in paired normal and tumor DNAs. Nine SNPs in HDAC9 (rs801540, rs1178108, rs1178112, rs1726610, rs10243618, rs11764116, rs1178355, rs10269422 and rs12540872) showed PAI in tumors. These data suggest that HDAC9 variants may be selected for during cSCC tumorigenesis.

Differential expression of miR-1, a putative tumor suppressing microRNA, in cancer resistant and cancer susceptible mice.

Mus spretus mice are highly resistant to several types of cancer compared to Mus musculus mice. To determine whether differences in microRNA (miRNA) expression account for some of the differences in observed skin cancer susceptibility between the strains, we performed miRNA expression profiling of skin RNA for over 300 miRNAs. Five miRNAs, miR-1, miR-124a-3, miR-133a, miR-134, miR-206, were differentially expressed by array and/or qPCR. miR-1 was previously shown to have tumor suppressing abilities in multiple tumor types. We found miR-1 expression to be lower in mouse cutaneous squamous cell carcinomas (cSCCs) compared to normal skin. Based on the literature and our expression data, we performed detailed studies on predicted miR-1 targets and evaluated the effect of miR-1 expression on two murine cSCC cell lines, A5 and B9. Following transfection of miR-1, we found decreased mRNA expression of three validated miR-1 targets, Met, Twf1 and Ets1 and one novel target Bag4. Decreased expression of Ets1 was confirmed by Western analysis and by 3' reporter luciferase assays containing wildtype and mutated Ets1 3'UTR. We evaluated the effect of miR-1 on multiple tumor phenotypes including apoptosis, proliferation, cell cycle and migration. In A5 cells, expression of miR-1 led to decreased proliferation compared to a control miR. miR-1 expression also led to increased apoptosis at later time points (72 and 96 h) and to a decrease in cells in S-phase. In summary, we identified five miRNAs with differential expression between cancer resistant and cancer susceptible mice and found that miR-1, a candidate tumor suppressor, has targets with defined roles in tumorigenesis.

The impact of 3'UTR variants on differential expression of candidate cancer susceptibility genes.

Variants in regulatory regions are predicted to play an important role in disease susceptibility of common diseases. Polymorphisms mapping to microRNA (miRNA) binding sites have been shown to disrupt the ability of miRNAs to target genes resulting in differential mRNA and protein expression. Skin tumor susceptibility 5 (Skts5) was identified as a locus conferring susceptibility to chemically-induced skin cancer in NIH/Ola by SPRET/Outbred F1 backcrosses. To determine if polymorphisms between the strains which mapped to putative miRNA binding sites in the 3' untranslated region (3'UTR) of genes at Skts5 influenced expression, we conducted a systematic evaluation of 3'UTRs of candidate genes across this locus. Nine genes had polymorphisms in their 3'UTRs which fit the linkage data and eight of these contained polymorphisms suspected to interfere with or introduce miRNA binding. 3'UTRs of six genes, Bcap29, Dgkb, Hbp1, Pik3cg, Twistnb, and Tspan13 differentially affected luciferase expression, but did not appear to be differentially regulated by the evaluated miRNAs predicted to bind to only one of the two isoforms. 3'UTRs from four additional genes chosen from the locus that fit less stringent criteria were evaluated. Ifrd1 and Etv1 showed differences and contained polymorphisms predicted to disrupt or create miRNA binding sites but showed no difference in regulation by the miRNAs tested. In summary, multiple 3'UTRs with putative functional variants between susceptible and resistant strains of mice influenced differential expression independent of predicted miRNA binding.