Banu, Matei A.

Abstract While the allure of targeted therapies in oncology has been their high degree of specificity and potency for key tumor drivers, they have been disappointing in glioblastoma (GBM), even for drugs that are blood brain barrier permeable and CNS retained. This point is highlighted by the experience with mitotic spindle inhibitors, drugs which block the G2M transition and induce mitotic catastrophe—a phenotype characterized by cell enlargement and polyploidy that leads to apoptotic cell death. We have shown that one of these, a potent inhibitor of the mitotic kinesin Kif11 (ispinesib), is highly active against GBM tumor initiating cells and prolongs survival in murine models of this disease. However, tumors eventually progress, reflecting the development of drug resistance. Although ispinesib resistant GBM cells develop mitotic catastrophe, they become highly resistant to the apoptosis that typically follows and continue to proliferate. We find that this apoptosis resistance requires phosphorylation of the transcription factor STAT3 at two residues—Y705 and S727. Phosphorylation of Y705, mediated by SRC kinase, translocates STAT3 to the nucleus where it induces transcription of anti-apoptotic proteins. Phosphorylation at S727, mediated by EGFR, translocates STAT3 to the mitochondria where it blocks release of cytochrome c—the penultimate effector in apoptosis. Simultaneously inhibiting both SRC and EGFR with FDA-approved, CNS permeant inhibitors reverses this resistance and significantly prolongs survival in ispinesib-treated GBM-bearing mice. Furthermore, we find that resistance to several other mitotic inhibitors also utilizes this STAT3-driven mechanism and can likewise be reversed with combined EGFR and SRC inhibition. Thus, our work demonstrates how a promising therapeutic approach, which has been disappointing in GBM, can in fact be rendered effective by anticipating and prospectively treating ab initio the mechanism that drives treatment resistance.

Abstract Introduction Distinguishing between tumor and treatment effect in post-treatment glioma, although crucial for clinical management, is difficult because contrast-enhancing regions are mixtures of recurrent tumor and reactive tissue, and definitive histopathological criteria do not exist. This study disentangles the marked intra-tumoral heterogeneity in the treatment-recurrent setting by developing an unsupervised framework to algorithmically categorize intraoperative MRI-localized biopsies into three clinically-relevant tissue clusters based on joint analysis of RNA sequencing and histopathological data. Methods A retrospective cohort of 84 MRI-localized biopsies from 37 patients with post-treatment recurrent glioblastoma underwent mRNA extraction and quantification via PLATEseq protocol. For 48 of 84 biopsies, a neighboring piece of tissue underwent quantitative histopathology based on labeling index (LI) for SOX2, CD68, NeuN, Ki67, and H&E. Correlation between LIs and gene expression for these 48 samples was performed. Genes significantly correlated (p<0.05) with ≥1 marker were used for hierarchical clustering of correlation matrix, identifying three mutually-exclusive tissue-specific gene sets. These sets were then used to perform ssGSEA to categorize each of 84 biopsies into one of three tissue types. Results Correlation analysis identified 7779 genes significantly correlated with ≥1 histopathological marker. Clustering revealed three gene sets associated with specific markers: SetA-3688 genes associated with SOX2/Ki67/H&E; SetB-2418 genes associated with CD68; SetC-1673 genes associated with NeuN. ssGSEA using these sets categorized each biopsy into one of three tissue types: 27 biopsies enriched in SetA, 28 in SetB, and 29 in SetC. Conclusions Using MRI-localized biopsies with both RNAseq and histopathological data, this algorithmic approach allows development of three orthogonal tissue-specific gene sets that can be applied to characterize the heterogeneity in post-treatment recurrent glioma: SetA: genes correlated with SOX2/Ki67/H&E, representing recurrent tumor; SetB: genes correlated with CD68 (microglia) representing reactive tissue consistent with treatment effect; SetC: genes correlated with NeuN (neurons), representing infiltrated brain.

OBJECTIVEThe long-term effects of surgical fusion on the growing subaxial cervical spine are largely unknown. Recent cross-sectional studies have demonstrated that there is continued growth of the cervical spine through the teenage years. The purpose of this multicenter study was to determine the effects of rigid instrumentation and fusion on the growing subaxial cervical spine by investigating vertical growth, cervical alignment, cervical curvature, and adjacent-segment instability over time.METHODSA total of 15 centers participated in this multi-institutional retrospective study. Cases involving children less than 16 years of age who underwent rigid instrumentation and fusion of the subaxial cervical spine (C-2 and T-1 inclusive) with at least 1 year of clinical and radiographic follow-up were investigated. Charts were reviewed for clinical data. Postoperative and most recent radiographs, CT, and MR images were used to measure vertical growth and assess alignment and stability.RESULTSEighty-one patients were included in the study, with a mean follow-up of 33 months. Ninety-five percent of patients had complete clinical resolution or significant improvement in symptoms. Postoperative cervical kyphosis was seen in only 4 patients (5%), and none developed a swan-neck deformity, unintended adjacent-level fusion, or instability. Of patients with at least 2 years of follow-up, 62% demonstrated growth across the fusion construct. On average, vertical growth was 79% (4-level constructs), 83% (3-level constructs), or 100% (2-level constructs) of expected growth. When comparing the group with continued vertical growth to the one without growth, there were no statistically significant differences in terms of age, sex, underlying etiology, surgical approach, or number of levels fused.CONCLUSIONSContinued vertical growth of the subaxial spine occurs in nearly two-thirds of children after rigid instrumentation and fusion of the subaxial spine. Failure of continued vertical growth is not associated with the patient’s age, sex, underlying etiology, number of levels fused, or surgical approach. Further studies are needed to understand this dichotomy and determine the long-term biomechanical effects of surgery on the growing pediatric cervical spine.