Targeted therapy for pediatric diffuse intrinsic pontine glioma: a single-center experience.

Background: Diffuse intrinsic pontine glioma (DIPG) is a fatal disease with a median overall survival (OS) of less than 12 months after diagnosis. Radiotherapy (RT) still remains the mainstay treatment. Several other therapeutic strategies have been attempted in the last years without a significant effect on OS. Although radiological imaging is the gold standard for DIPG diagnosis, the urgent need to improve the survival has led to the reconsideration of biopsy with the aim to better understand the molecular profile of DIPG and support personalized treatment. Methods: In this study, we present a single-center experience in treating DIPG patients at disease progression combining targeted therapies with standard of care. Biopsy was proposed to all patients at diagnosis or disease progression. First-line treatment included RT and nimotuzumab/vinorelbine or temozolomide. Immunohistochemistry-targeted research included study of mTOR/p-mTOR pathway and BRAFv600E. Molecular analyses included polymerase chain reaction, followed by Sanger sequences and/or next-generation sequencing. Results: Based on the molecular profile, targeted therapy was administered in 9 out of 25 patients, while the remaining 16 patients were treated with standard of care. Personalized treatment included inhibition of the PI3K/AKT/mTOR pathway (5/9), PI3K/AKT/mTOR pathway and BRAFv600E (1/9), ACVR1 (2/9) and PDGFRA (1/9); no severe side effects were reported during treatment. Response to treatment was evaluated according to Response Assessment in Pediatric Neuro-Oncology criteria, and the overall response rate within the cohort was 66%. Patients treated with targeted therapies were compared with the control cohort of 16 patients. Clinical and pathological characteristics of the two cohorts were homogeneous. Median OS in the personalized treatment and control cohort was 20.26 and 14.18 months, respectively (p = 0.032). In our experience, the treatment associated with the best OS was everolimus. Conclusion: Despite the small simple size of our study, our data suggest a prognostic advantage and a safe profile of targeted therapies in DIPG patients, and we strongly advocate to reconsider the role of biopsy for these patients.

Droplet digital PCR-based detection of circulating tumor DNA from pediatric high grade and diffuse midline glioma patients.

BACKGROUND: The use of liquid biopsy is of potential high importance for children with high grade (HGG) and diffuse midline gliomas (DMG), particularly where surgical procedures are limited, and invasive biopsy sampling not without risk. To date, however, the evidence that detection of cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA) could provide useful information for these patients has been limited, or contradictory. METHODS: We optimized droplet digital PCR (ddPCR) assays for the detection of common somatic mutations observed in pediatric HGG/DMG, and applied them to liquid biopsies from plasma, serum, cerebrospinal fluid (CSF), and cystic fluid collected from 32 patients. RESULTS: Although detectable in all biomaterial types, ctDNA presented at significantly higher levels in CSF compared to plasma and/or serum. When applied to a cohort of 127 plasma specimens from 41 patients collected from 2011 to 2018 as part of a randomized clinical trial in pediatric non-brainstem HGG/DMG, ctDNA profiling by ddPCR was of limited use due to the small volumes (mean = 0.49 mL) available. In anecdotal cases where sufficient material was available, cfDNA concentration correlated with disease progression in two examples each of poor response in H3F3A_K27M-mutant DMG, and longer survival times in hemispheric BRAF_V600E-mutant cases. CONCLUSION: Tumor-specific DNA alterations are more readily detected in CSF than plasma. Although we demonstrate the potential of the approach to assessing tumor burden, our results highlight the necessity for adequate sample collection and approach to improve detection if plasma samples are to be used.

ALK2 inhibitors display beneficial effects in preclinical models of ACVR1 mutant diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine glioma (DIPG) is a lethal childhood brainstem tumour, with a quarter of patients harbouring somatic mutations in ACVR1, encoding the serine/threonine kinase ALK2. Despite being an amenable drug target, little has been done to-date to systematically evaluate the role of ACVR1 in DIPG, nor to screen currently available inhibitors in patient-derived tumour models. Here we show the dependence of DIPG cells on the mutant receptor, and the preclinical efficacy of two distinct chemotypes of ALK2 inhibitor in vitro and in vivo. We demonstrate the pyrazolo[1,5-a]pyrimidine LDN-193189 and the pyridine LDN-214117 to be orally bioavailable and well-tolerated, with good brain penetration. Treatment of immunodeprived mice bearing orthotopic xenografts of H3.3K27M, ACVR1R206H mutant HSJD-DIPG-007 cells with 25 mg/kg LDN-193189 or LDN-214117 for 28 days extended survival compared with vehicle controls. Development of ALK2 inhibitors with improved potency, selectivity and advantageous pharmacokinetic properties may play an important role in therapy for DIPG patients.

Functional diversity and cooperativity between subclonal populations of pediatric glioblastoma and diffuse intrinsic pontine glioma cells.

The failure to develop effective therapies for pediatric glioblastoma (pGBM) and diffuse intrinsic pontine glioma (DIPG) is in part due to their intrinsic heterogeneity. We aimed to quantitatively assess the extent to which this was present in these tumors through subclonal genomic analyses and to determine whether distinct tumor subpopulations may interact to promote tumorigenesis by generating subclonal patient-derived models in vitro and in vivo. Analysis of 142 sequenced tumors revealed multiple tumor subclones, spatially and temporally coexisting in a stable manner as observed by multiple sampling strategies. We isolated genotypically and phenotypically distinct subpopulations that we propose cooperate to enhance tumorigenicity and resistance to therapy. Inactivating mutations in the H4K20 histone methyltransferase KMT5B (SUV420H1), present in <1% of cells, abrogate DNA repair and confer increased invasion and migration on neighboring cells, in vitro and in vivo, through chemokine signaling and modulation of integrins. These data indicate that even rare tumor subpopulations may exert profound effects on tumorigenesis as a whole and may represent a new avenue for therapeutic development. Unraveling the mechanisms of subclonal diversity and communication in pGBM and DIPG will be an important step toward overcoming barriers to effective treatments.

Integrated Molecular Meta-Analysis of 1,000 Pediatric High-Grade and Diffuse Intrinsic Pontine Glioma.

We collated data from 157 unpublished cases of pediatric high-grade glioma and diffuse intrinsic pontine glioma and 20 publicly available datasets in an integrated analysis of >1,000 cases. We identified co-segregating mutations in histone-mutant subgroups including loss of FBXW7 in H3.3G34R/V, TOP3A rearrangements in H3.3K27M, and BCOR mutations in H3.1K27M. Histone wild-type subgroups are refined by the presence of key oncogenic events or methylation profiles more closely resembling lower-grade tumors. Genomic aberrations increase with age, highlighting the infant population as biologically and clinically distinct. Uncommon pathway dysregulation is seen in small subsets of tumors, further defining the molecular diversity of the disease, opening up avenues for biological study and providing a basis for functionally defined future treatment stratification.

Myoblast-mediated gene therapy via encephalomyosynangiosis--a novel strategy for local delivery of gene products to the brain surface.

An encephalomyosynangiosis (EMS) is a temporal muscle graft that is placed onto the surface of the brain to serve as a source for collateral vessel growth for brain revascularization in patients with Moyamoya Disease (MMD). To facilitate an EMS in patients with occlusive cerebrovascular diseases other than MMD, the transfer of pro-angiogenic genes via transplantation of retrovirally transduced myoblasts into the temporal muscle may represent an innovative approach to augment collateralization. Thus, we tested whether retrovirally transfected myoblasts can spontaneously fuse with the non-ischemic and uninjured muscle tissue and if a reporter gene can be stably expressed within the temporal muscle of the EMS. Primary mouse myoblasts expressing a reporter gene were implanted into the temporal muscle prior to an EMS being performed on C57/BL6 mice. Three different implantation modalities were evaluated: (a) intramuscular injection, (b) application of a cell pellet and (c) a combination of both techniques. Myoblast implantation resulted in spontaneous fusion with the host muscle fibers and stable reporter gene expression at both the muscle/brain interface and within the non-ischemic and uninjured temporal muscle in all animals. The mean number of fused hybrid myofibers was 59±28 after injection, 37±30 after pellet application and 60±23 after a combination of both techniques. Regardless of the implantation modality, an abundant extracellular expression of the reporter gene was evident at the muscle/brain interface; in the case of myoblast delivery by injection, expression was also observed around the needle tract marking the implantation site. This method could be used in the future to deliver angiogenic growth factors to the muscle/brain interface in order to improve revascularization after an EMS.

Effects of sunitinib on tumor hemodynamics and delivery of chemotherapy.

Current clinical protocols favor a combination of antiangiogenic/antivascular compounds with classical chemotherapy. However, it remains unclear to what extent an antiangiogenic/antivascular therapy influences the delivery of chemotherapy. Therefore, the aim of the present study was to characterize the effects of the antiangiogenic tyrosine kinase inhibitor sunitinib on tumor microhemodynamics and delivery of chemotherapy. SF126 tumor cells were implanted subcutaneously into nude mice and were analyzed repeatedly by intravital microscopy. Treatment with sunitinib was initiated 7 days after implantation. To assess the effects of sunitinib on tumor vasculature and hemodynamics, we analyzed total and functional vessel densities, microvascular diameter, and microvascular blood flow rate. To study the delivery of chemotherapy, autofluorescent doxorubicin was systemically administered and its vascular delivery to the tumor tissue was quantified. Histological analysis included endothelial cell proliferation, pericyte coverage of tumor vessels, and tumor cell proliferation. Sunitinib significantly suppressed tumor growth by both antivascular and antiangiogenic effects. However, a number of tumor vessels escaped antiangiogenic therapy. Interestingly, in these surviving blood vessels sunitinib treatment resulted in an increased microvascular blood flow rate resulting in an improved delivery of chemotherapy via these blood vessels. Besides its potent antiangiogenic and antivascular efficacy, sunitinib treatment results in improved microhemodynamics and blood flow in tumor blood vessels that escape therapy leading to an improved vascular delivery of chemotherapy. These results provide the basis for a potential chemosensitizing effect of sunitinib.