ATM inhibition enhances the efficacy of radiation across distinct molecular subgroups of pediatric high-grade glioma.

BACKGROUND: Pediatric high-grade glioma (pHGG) is largely incurable and accounts for most brain tumor-related deaths in children. Radiation is a standard therapy, yet the benefit from this treatment modality is transient, and most children succumb to disease within 2 years. Recent large-scale genomic studies suggest that pHGG has alterations in DNA damage response (DDR) pathways that induce resistance to DNA damaging agents. The aim of this study was to evaluate the therapeutic potential and molecular consequences of combining radiation with selective DDR inhibition in pHGG. METHODS: We conducted an unbiased screen in pHGG cells that combined radiation with clinical candidates targeting the DDR and identified the ATM inhibitor AZD1390. Subsequently, we profiled AZD1390 + radiation in an extensive panel of early passage pHGG cell lines, mechanistically characterized response to the combination in vitro in sensitive and resistant cells and evaluated the combination in vivo using TP53 wild-type and TP53 mutant orthotopic xenografts. RESULTS: AZD1390 significantly potentiated radiation across molecular subgroups of pHGG by increasing mutagenic nonhomologous end joining and augmenting genomic instability. In contrast to previous reports, ATM inhibition significantly improved the efficacy of radiation in both TP53 wild-type and TP53 mutant isogenic cell lines and distinct orthotopic xenograft models. Furthermore, we identified a novel mechanism of resistance to AZD1390 + radiation that was marked by an attenuated ATM pathway response which dampened sensitivity to ATM inhibition and induced synthetic lethality with ATR inhibition. CONCLUSIONS: Our study supports the clinical evaluation of AZD1390 in combination with radiation in pediatric patients with HGG.

Model-based evaluation of image-guided fractionated whole-brain radiation therapy in pediatric diffuse intrinsic pontine glioma xenografts.

Radiation therapy (RT) is currently the standard treatment for diffuse intrinsic pontine glioma (DIPG), the most common cause of death in children with brain cancer. A pharmacodynamic model was developed to describe the radiation-induced tumor shrinkage and overall survival in mice bearing DIPG. CD1-nude mice were implanted in the brain cortex with luciferase-labeled patient-derived orthotopic xenografts of DIPG (SJDIPGx7 H3F3AWT / K27 M and SJDIPGx37 H3F3AK27M / K27M ). Mice were treated with image-guided whole-brain RT at 1 or 2 Gy/fraction 5-days-on 2-days-off for a cumulative dose of 20 or 54 Gy. Tumor progression was monitored with bioluminescent imaging (BLI). A mathematical model describing BLI and overall survival was developed with data from mice receiving 2 Gy/fraction and validated using data from mice receiving 1 Gy/fraction. BLI data were adequately fitted with a logistic tumor growth function and a signal distribution model with linear radiation-induced killing effect. A higher tumor growth rate in SJDIPGx37 versus SJDIPGx7 xenografts and a killing effect decreasing with higher tumor baseline (p < 0.0001) were identified. Cumulative radiation dose was suggested to inhibit the tumor growth rate according to a Hill function. Survival distribution was best described with a Weibull hazard function in which the hazard baseline was a continuous function of tumor BLI. Significant differences were further identified between DIPG cell lines and untreated versus treated mice. The model was adequately validated with mice receiving 1 Gy/fraction and will be useful in guiding future preclinical trials incorporating radiation and to support systemic combination therapies with RT.

Radiation dose response of neurologic symptoms during conformal radiotherapy for diffuse intrinsic pontine glioma.

PURPOSE: To estimate the rate and magnitude of neurologic symptom change during radiation therapy (RT) and impact of symptom change on survival outcomes in patients with diffuse intrinsic pontine glioma (DIPG). METHODS: From 2006 to 2014, 108 patients with newly diagnosed DIPG were treated with conventionally fractionated radiation therapy (RT) to 54 Gy (median) at our institution. The presence and severity of neurologic symptoms related to cranial neuropathy (CN) and cerebellar (CB) and long-tract (LT) signs was reviewed before and weekly during RT for each patient. The rate and magnitude of change for each symptom category was evaluated according to accumulated RT dose. The impact of clinical factors and radiation dose-volume parameters was determined using Cox proportional hazards models. RESULTS: Median dose to first sign of symptomatic improvement was 16.2 Gy (CN), 19.8 Gy (LT) and 21.6 Gy (CB). Most patients showed an improvement by 20 Gy. Larger uninvolved brainstem volume, alone or normalized to total brain (TB) or posterior fossa volume (PF), was associated with shorter time to LT sign improvement (P = 0.044, P = 0.033, and P = 0.05, respectively). Patients with any improvement in CN experienced significantly, yet modestly, prolonged progression-free survival (PFS) and overall survival (OS) (P = 0.002 and P = 0.008, respectively). Tumor volume, with or without normalization to TB or PF, was not significantly associated with PFS or OS. CONCLUSIONS: Low cumulative RT doses resulted in neurologic improvement in most patients with DIPG. The volume of brainstem spared by tumor influenced time to symptomatic improvement. Neurologic improvement during RT was associated with superior survival.

Preclinical Modeling of Image-Guided Craniospinal Irradiation for Very-High-Risk Medulloblastoma.

PURPOSE: Craniospinal irradiation (CSI) is a crucial component of treatment for medulloblastoma (MB), a brain tumor clinically stratified into prognostically distinct molecular subgroups. Preclinical models of clinically relevant CSI offer the potential to study radiation dose and volume effects in these subgroups and to identify subgroup-specific combination adjuvant therapies, particularly for very-high-risk MB in which treatments are often unsuccessful. METHODS AND MATERIALS: The commercially available Small Animal Radiation Research Platform equipped with a motorized variable collimator was used for image-guided CSI. Mice were implanted in brain cortices with patient-derived orthotopic xenografts (PDOXs) of very-high-risk Group 3 (G3) or Sonic Hedgehog (SHH) MB and were treated with fully fractionated CSI at 2 Gy/fraction for a cumulative 36 Gy. Radiation therapy dose response effects on tumor burden and overall survival were assessed. The pattern of treatment failure was determined using bioluminescence and then confirmed histologically. Acute toxicity was appraised by body weight measurements and blood work. RESULTS: We established an accurate and efficient preclinical protocol to administer CSI reproducibly to mice harboring MB. CSI improved the survival of mice bearing very-high-risk G3 or SHH MB PDOXs. However, radiation therapy dose responses across models suggested significant radio-responsiveness to conventionally fractionated CSI ≥20 Gy. CSI was well tolerated; mice had no significant changes in body weight, and acute leukopenia developed but resolved soon after therapy completion. CONCLUSIONS: Our protocol for preclinical CSI delivery was effective and well tolerated, and it can be readily integrated into preclinical pipelines for MB and other central nervous system-seeding tumors.

Establishing a Preclinical Multidisciplinary Board for Brain Tumors.

Purpose: Curing all children with brain tumors will require an understanding of how each subtype responds to conventional treatments and how best to combine existing and novel therapies. It is extremely challenging to acquire this knowledge in the clinic alone, especially among patients with rare tumors. Therefore, we developed a preclinical brain tumor platform to test combinations of conventional and novel therapies in a manner that closely recapitulates clinic trials.Experimental Design: A multidisciplinary team was established to design and conduct neurosurgical, fractionated radiotherapy and chemotherapy studies, alone or in combination, in accurate mouse models of supratentorial ependymoma (SEP) subtypes and choroid plexus carcinoma (CPC). Extensive drug repurposing screens, pharmacokinetic, pharmacodynamic, and efficacy studies were used to triage active compounds for combination preclinical trials with "standard-of-care" surgery and radiotherapy.Results: Mouse models displayed distinct patterns of response to surgery, irradiation, and chemotherapy that varied with tumor subtype. Repurposing screens identified 3-hour infusions of gemcitabine as a relatively nontoxic and efficacious treatment of SEP and CPC. Combination neurosurgery, fractionated irradiation, and gemcitabine proved significantly more effective than surgery and irradiation alone, curing one half of all animals with aggressive forms of SEP.Conclusions: We report a comprehensive preclinical trial platform to assess the therapeutic activity of conventional and novel treatments among rare brain tumor subtypes. It also enables the development of complex, combination treatment regimens that should deliver optimal trial designs for clinical testing. Postirradiation gemcitabine infusion should be tested as new treatments of SEP and CPC. Clin Cancer Res; 24(7); 1654-66. ©2018 AACR.

Bupivacaine and triamcinolone may be toxic to human chondrocytes: a pilot study.

BACKGROUND: Intraarticular injections of corticosteroids combined with local anesthetics are commonly used for management of chronic pain symptoms associated with degenerative joint diseases and after arthroscopic procedures. Several studies suggest chondrotoxicity of local anesthetics whereas others report chondroprotective and cytotoxic effects of corticosteroids on cartilage. Given the frequency of use of these agents, it is important to know whether they are in fact toxic. QUESTIONS/PURPOSES: We asked whether (1) bupivacaine and triamcinolone acetonide, alone and combined, were chondrotoxic to chondrocytes in culture; (2) buffering of the reagents diminished toxicity of the bupivacaine and triamcinolone; and (3) the presence of the superficial layer of articular cartilage protects against toxicity. MATERIALS AND METHODS: We obtained cartilage from three patients undergoing arthroplasty. To address triamcinolone acetonide, bupivacaine, and combinatorial toxicity to human chondrocytes, we set up monolayer chondrocyte cultures (n = 8 wells per condition). The question of buffering was addressed by performing the same assays as above, but the reagents were buffered. An MTT assay was used to assess chondrocyte survival in the monolayer. We harvested 21 articular plugs from each of three patients (total 63 plugs) and exposed them to the same reagents as above, including the buffered reagents. A Live/Dead assay was used to determine chondrocyte survival. RESULTS: Triamcinolone acetonide, bupivacaine, and their combination were toxic to human chondrocytes in the monolayer comparisons. The addition of buffering did not mitigate chondrocyte death. With the intact superficial layer in the plug group, bupivacaine was not toxic as compared with for the control group; all the other reagents (triamcinolone, combination bupivacaine/triamcinolone, buffered bupivacaine, buffered triamcinolone, and buffered combination) produced chondrotoxicity. CONCLUSIONS: Triamcinolone induced chondrotoxicity in the articular plug and monolayer culture, whereas bupivacaine induced chondrotoxicity only in monolayer culture. The combined used of triamcinolone and bupivacaine did not show additive chondrocyte death in any arm. Buffering of bupivacaine increased its chondrotoxicity. CLINICAL RELEVANCE: Although not necessarily reflecting in vivo conditions, our data suggest physicians should be cognizant of the potential in vitro chondrotoxicity of bupivacaine and triamcinolone when contemplating intraarticular administration.

In vitro effects of 3 common arthroscopic instruments on articular cartilage.

PURPOSE: The purpose of this study was to compare chondroplasty performed with an ExoJet high-pressure fluid-driven burr (Mitek, Norwood, MA), a mechanical shaver, and a bipolar radiofrequency (RF) wand on articular cartilage-covered condyles taken from sheep cadavers that were induced to have an osteoarthritic-like condition, and corresponding healthy control tissue. TYPE OF STUDY: Experimental designed animal cadaveric, biochemical, and histologic study. METHODS: Sheep condyles were used as a source of articular cartilage. Femurs were extracted approximately 1 hour postmortem and a transverse section of the condyles was made. Half of the samples were treated to induce an osteoarthritic-like condition. The condyles were then subjected to chondroplasty performed with the ExoJet high-pressure fluid-driven burr, a mechanical shaver, and a bipolar RF wand under sterile saline solution by an experienced orthopaedic surgeon. Twenty cross-sections from each condyle were examined by confocal microscopy to measure smoothness and depth of tissue damage to the articular cartilage caused by each of the 3 instruments. RESULTS: The ExoJet high-pressure fluid-driven burr and the bipolar RF wand left a smoother surface on the articular cartilage compared with the mechanical shaver. Additionally, the ExoJet fluid-burr caused slightly less tissue damage to the cartilage than the bipolar RF wand, both of which were less damaging than the shaver. CONCLUSIONS: Orthopaedists have multiple choices for surgical instruments used on cartilage. However, the effect on the integrity of the cartilage left remaining at the knee was previously unknown. Based on this study, a fluid-burr appears to leave the cartilage with a smaller zone of injury than does the RF wand or shaver. It also leaves the cartilage surface smoother than the shaver. During surgical procedures, minimizing cartilage breakdown and smooth remaining surfaces are desired because they minimize the vulnerable tissue to further destruction. A fluid burr leaves cartilage with less injury and with a smoother surface than do more traditional surgical instruments. CLINICAL RELEVANCE: This information should help surgeons in their selection of currently available surgical instruments and should aid engineers in the design of future instruments that function to modify articular cartilage.

Shaver, bipolar radiofrequency, and saline jet instruments for cutting meniscal tissue: a comparative experimental study on sheep menisci.

PURPOSE: The purpose of this study was to compare the quality of meniscal tissue cut with 3 different surgical instruments (traditional shavers, bipolar radiofrequency (RF) wands, and a high-pressure saline jet) and that of control menisci. TYPE OF STUDY: Experimental design, biochemical and histologic study. METHODS: Sixty samples of sheep menisci were separated into 4 groups. Three groups were shaved on the apical surface with the different instruments. The smoothness of the cut surfaces was evaluated visually by an orthopaedic surgeon and then scored by laser scanning cytometry and by line measurement analysis. The depth of tissue damage was measured by fluorescent cytochemistry. Means and standard deviations were calculated and comparative statistics used (P < .05). RESULTS: The edges cut by the saline jet and bipolar RF were significantly smoother when judged by the surgeon than those cut by traditional shaver. There was no significant difference between the saline jet and bipolar RF. There were no significant differences in smoothness when measured by laser scanning cytometry or by line measurement techniques. The control menisci had less depth of damage along the edge as measured by fluorescent cytochemistry than did any of the menisci cut with the instrument. The saline jet had significantly less depth of damage than did the shaver. No other significant differences existed between the instruments for depth of damage. CONCLUSIONS: The results of our investigation conclude that high-pressure saline instruments may cause less damage to residual meniscal tissue when compared with bipolar RF and shavers. Saline jets and bipolar RF also produce a smoother cut than shavers. CLINICAL RELEVANCE: Surgeons may want to consider the degree of residual damage to meniscal tissue from the application of various surgical instruments. Saline jets may be a superior cutting instrument than RF or shavers when considering depth of residual damage and smoothness of residual meniscal edges.