Jenkins, Robert P.

Abstract BACKGROUND Tumor Treating Field (TTF) therapy exerts antineoplastic effects through application of alternating electrical fields that disrupt mitotic machinery and induce DNA damage and activation of cell cycle checkpoints. However, despite extensive utilization in clinical practice for the management of glioblastoma, molecular markers for TTF sensitivity have proven elusive. METHODS To assess impact of molecular alterations on TTF response, we evaluated our institutional cohort of IDH wild type glioblastomas (n=449) for whom chromosomal microarray data was available. RESULTS A total of 142 patients were found to have undergone TTF treatment (31.6%) with improved overall survival (OS) (p=0.0049, univariate analysis). We assessed TTF treatment responses when stratified by alterations in the MDM2/4-TP53 axis, as well as the CDK4/6-RB1 axis, given well-described roles for these pathways on cellular responses to DNA damage and mitotic stressors. On univariate analysis, MDM2/4 amplified tumors treated with TTFs demonstrated significantly improved OS compared to untreated tumors (p=0.0012), and while MDM2/4-unaltered, TP53 intact tumors showed OS benefit from TTFs (p=0.0192), TP53-altered tumors did not demonstrate such benefit (p=0.6591). We confirmed these findings with multivariate Cox regression after adjusting for age and sex, where TTF therapy improved OS in MDM2/4 amplified tumors (HR 0.3881, p=0.0062) and a signal towards improved OS was seen in TP53 intact tumors (HR 0.7552, p=0.0509) but no such benefit was seen in TP53-altered tumors (HR 1.0025, p=0.9916). TTF therapy appeared to demonstrate improved OS in CDK4/6 amplified tumors (p=0.0116) on univariate analysis, with continued signal towards this effect on multivariate Cox regression (HR 0.5975, p=0.0870). CONCLUSIONS TP53 intact status and MDM2/4 amplification may represent previously unappreciated molecular markers for sensitivity to TTF therapy in IDH wild type glioblastoma. Confirmatory work with multi-institutional collaborations and evaluation of other potential prognostic factors are currently underway.

Abstract BACKGROUND Chromosomal instability is a well-recognized hallmark of tumorigenesis. However, it is not known how markers for chromosomal instability, such as total distance of DNA altered or total copy number variations (CNV), affect survival, particularly in IDH wild type glioblastoma. Additionally, whether alterations in pathways that impact cellular responses to chromosomal instability, such as the TP53 or RB1 axis, influence survival, similarly remain poorly understood. METHODS To better characterize potential prognostic impact of markers of chromosomal instability on patient outcomes, we assessed the Mayo Clinic cohort of IDH wild type glioblastomas (n=449) for whom chromosomal microarray data was available. RESULTS We found that while total distance of DNA altered on microarray analysis was not associated with overall survival (OS) (p=0.4030, univariate analysis), the least altered tumors (stratified by quartiles) when assessed by numbers of CNVs demonstrated improved OS (p=0.0057, univariate analysis). We next evaluated alterations in the MDM2/4-TP53 signaling axis, as well as the CDKN2A/B-CDK4/6-RB1 axis, as both amplifications and losses impacting these pathways have been well characterized in adult diffuse gliomas. While TP53 status was not predictive of OS (p=0.1504, univariate analysis), amplification of MDM2 or MDM4 was predictive of decreased OS (p=0.0071, univariate analysis). We also found that tumors without alterations in the CDKN2A/B-CDK4/6-RB1 axis had improved OS when compared to altered tumors (p<0.0001, univariate analysis). Multivariate Cox regression analysis, adjusting for age and sex, confirmed prognostic impact of MDM2/4 amplification status (HR 1.4090, p=0.0370) and CNV status (HR 0.6798, p=0.00312) on OS. CONCLUSIONS CNV status and MDM2/4 amplification represent previously unappreciated molecular markers for prognostication in IDH wild type glioblastoma. Further work is underway to confirm our findings through multi-institutional collaborations and to account for additional potential prognostic factors.

Abstract Low-grade gliomas (LGG) are slowly growing brain cancers that ultimately undergo a malignant progression to lethal secondary glioblastomas with a dismal prognosis. Despite intensive research, very little progress has been made in our understanding of this transformation. Hence, outside of known alternations in well-characterized driver genes CDKN2A, PTEN and PDGFRA, genetic drivers that underlie LGG transformation are largely unknown. Contrary to other types of cancer, LGGs exhibit very few driver mutations (30-40 mutations) as they progress. Interestingly, LGGs also exhibit a highly prevalent and heterogenous pattern of copy-number-alterations (CNAs) suggesting that they might be driven by changes in gene dosages that result from widespread chromosomal instability observed within these tumors. Through analysis of the human Mayo Clinic and TCGA LGG datasets (~1500 patients), we have identified eleven highly recurrent CNAs affecting LGG patients, many of which correlate with patient survival. We hypothesized that these CNAs contain driver genes that confer a growth advantage resulting in the highly recurrent CNA pattern and prognostic associations. To investigate the tumor suppressive potential of the ~1800 genes located in recurrently deleted regions and the oncogenic potential of the ~1200 genes located in recurrently amplified regions, our lab developed a novel in vivo ‘CRISPR-Knock Out and Activation Linked Assay’ (CRISPR-KOALA). Stereotaxic delivery of arm-level CNA lentiviral gRNA libraries targeting the mouse orthologs of genes in CNAs into the brain of our animal model (rs557mut;IDHmut;P53KO;AtrxKO;Cas9GFP: Yanchus et. al Science 2022) resulted in rapid formation of tumors compared to animals injected with control libraries. Deconvoluting the screen revealed enrichment of immune modulators such as Them6 as well as a modulator of NMDA receptors (Grina) implicating immune system and glutamate dysregulation in LGG malignant transformation. Taken together, our study provides insights into mechanisms underlying LGG progression which may serve to inform intervention strategies for this lethal disease.