Cassie Kline

Targeted next-generation sequencing of pediatric neuro-oncology patients improves diagnosis, identifies pathogenic germline mutations, and directs targeted therapy

Background Molecular profiling is revolutionizing cancer diagnostics and leading to personalized therapeutic approaches. Herein we describe our clinical experience performing targeted sequencing for 31 pediatric neuro-oncology patients. Methods We sequenced 510 cancer-associated genes from tumor and peripheral blood to identify germline and somatic mutations, structural variants, and copy number changes. Results Genomic profiling was performed on 31 patients with tumors including 11 high-grade gliomas, 8 medulloblastomas, 6 low-grade gliomas, 1 embryonal tumor with multilayered rosettes, 1 pineoblastoma, 1 uveal ganglioneuroma, 1 choroid plexus carcinoma, 1 chordoma, and 1 high-grade neuroepithelial tumor. In 25 cases (81%), results impacted patient management by: (i) clarifying diagnosis, (ii) identifying pathogenic germline mutations, or (iii) detecting potentially targetable alterations. The pathologic diagnosis was amended after genomic profiling for 6 patients (19%), including a high-grade glioma to pilocytic astrocytoma, medulloblastoma to pineoblastoma, ependymoma to high-grade glioma, and medulloblastoma to CNS high-grade neuroepithelial tumor with BCOR alteration. Multiple patients had pathogenic germline mutations, many of which were previously unsuspected. Potentially targetable alterations were identified in 19 patients (61%). Additionally, novel likely pathogenic alterations were identified in 3 cases: an in-frame RAF1 fusion in a BRAF wild-type pleomorphic xanthoastrocytoma, an inactivating ASXL1 mutation in a histone H3 wild-type diffuse pontine glioma, and an in-frame deletion within exon 2 of MAP2K1 in a low-grade astrocytic neoplasm. Conclusions Our experience demonstrates the significant impact of molecular profiling on diagnosis and treatment of pediatric brain tumors and confirms its feasibility for use at the time of diagnosis or recurrence.

IDH1 mutation can be present in diffuse astrocytomas and giant cell glioblastomas of young children under 10 years of age

Author(s): Ferris, Sean P; Goode, Benjamin; Joseph, Nancy M; Kline, Cassie N; Samuel, David; Gupta, Nalin; Bollen, Andrew; Perry, Arie; Mueller, Sabine; Solomon, David A

Perinatal factors associated with clinical presentation of osteosarcoma in children and adolescents

Osteosarcoma typically develops during puberty with tumors arising at sites of rapid bone growth, suggesting a role for growth‐regulating pathways in tumor etiology. Birthweight is one measure of perinatal growth that has been investigated as an osteosarcoma risk factor. Whether birthweight affects clinical features of osteosarcoma remains unexplored.

Inactivating MUTYH germline mutations in pediatric patients with high-grade midline gliomas

Two familial gastrointestinal neoplasia syndromes are linked with increased incidence of brain tumors. Hereditary nonpolyposis colorectal carcinoma (HNPCC), or Lynch syndrome, is an autosomal dominant disorder caused by inactivating germline mutations in mismatch repair genes MLH1, MSH2, MSH6, or PMS2 (OMIM 609310, 120435, 614350, and 614337). Patients with Lynch syndrome have markedly increased incidence of carcinoma of the colon, stomach, endometrium, and biliary system. A subset of Lynch syndrome patients also develop malignant gliomas and are said to have Turcot syndrome type 1. Familial adenomatous polyposis type 1 (FAP1) is an autosomal dominant syndrome caused by inactivating germline mutation in the APC gene (OMIM 175100). Patients with FAP1 invariably develop numerous gastrointestinal adenomas and carcinoma. A subset of patients with FAP1 also develop medulloblastomas and are said to have Turcot syndrome type 2. Here we report two pediatric patients with high-grade midline gliomas found to have inactivating germline mutations in the MUTYH gene on chromosome 1p34, which encodes a DNA repair enzyme involved in base excision repair. Biallelic inactivating MUTYH germline mutations, either homozygous or compound heterozygous, cause markedly increased incidence of gastrointestinal adenomas and carcinomas termed familial adenomatous polyposis type 2 (FAP2, OMIM 608456), but this syndrome has not been previously linked with brain tumors. – 6 Our two patients, however, strongly suggest that monoallelic germline MUTYH mutations may increase risk of malignant brain tumors. Patient #1 was a 6-year old boy who presented with lower extremity weakness. MR imaging revealed a heterogeneously enhancing intramedullary mass in the thoracic spinal cord (Fig. 1). Pathology from a subtotal resection demonstrated glioblastoma, WHO grade IV, with histone H3-K27M mutant protein expression. He received adjuvant radiation with concurrent temozolomide. However, 8 months after diagnosis he was found to have disseminated recurrence within the posterior fossa. Repeat irradiation was administered, followed with trials of temozolomide, lomustine, erlotinib, bevacizumab, and a PARP inhibitor. Unfortunately, he had progressive disease and died 18 months after diagnosis. Targeted next-generation sequencing of 510 cancer-associated genes was performed that identified a germline nonsense mutation in the MUTYH gene at codon 466, causing early truncation of the encoded protein. The tumor demonstrated retention of the remaining wild-type allele, but the possibility of epigenetic inactivation could not be excluded. Additionally, the tumor harbored somatic missense mutations in H3F3A, ACVR1, and PIK3CA, all known to be recurrently mutated in pediatric high-grade gliomas. Interestingly, activating ACVR1 mutations have been recurrently found in diffuse intrinsic pontine gliomas with histone H3 p.K27M mutations, but this is the first report of ACVR1 mutation in a pediatric glioma outside of the pons. Patient #2 initially presented at 5 years of age with emesis, headaches, and seizure. He was found to have an enhancing mass in the inferior vermis of the cerebellum. Pathology from gross total resection demonstrated medulloblastoma, WHO grade IV. He underwent irradiation followed by chemotherapy with vincristine, cisplatin, and lomustine. The patient was stable for 10 years until he presented with worsening ataxia, dysarthria, and diplopia. He was found to have an expansile nonenhancing lesion in the pons. Biopsy demonstrated high-grade infiltrative astrocytoma, at least WHO grade III. Targeted nextgeneration sequencing was performed that identified a germline mutation in MUTYH at the canonical splice acceptor of exon 11, predicted to disrupt appropriate splicing of the encoded mRNA. The tumor demonstrated loss of heterozygosity due to deletion of chromosome 1p eliminating the remaining wildtype MUTYH allele. The tumor also contained homozygous deletion of CDKN2A and 3 missense mutations in PDGFRA likely to be activating. Sanger sequencing over the MUTYH mutation in the patient’s prior medulloblastoma also revealed loss of heterozygosity in this tumor. The patient was treated with focal irradiation and nivolumab. Follow-up imaging 3 months later revealed improvement of the brainstem lesion but new onset of metastatic disease. Targeted therapy with dasatinib was attempted based on the genomic profiling but was discontinued due to anaphylactic shock. He is currently receiving lomustine and bevacizumab. These patients both exhibited inactivating MUTYH germline mutations known to cause a gastrointestinal polyposis syndrome (FAP2) that has not been previously reported in brain tumor patients. Neither patient had family history of gastrointestinal neoplasia, as expected given the lack of biallelic mutation. Inactivating germline mutations in 2 unrelated patients with high-grade gliomas and one with prior medulloblastoma strongly suggest that germline MUTYH mutations increase risk of malignant brain tumors. Of note, a recent study identified

The genetic landscape of anaplastic pleomorphic xanthoastrocytoma.

Pleomorphic xanthoastrocytoma (PXA) is an astrocytic neoplasm that is typically well circumscribed and can have a relatively favorable prognosis. Tumor progression to anaplastic PXA (WHO grade III), however, is associated with a more aggressive biologic behavior and worse prognosis. The factors that drive anaplastic progression are largely unknown. We performed comprehensive genomic profiling on a set of 23 PXAs from 19 patients, including 15 with anaplastic PXA. Four patients had tumor tissue from multiple recurrences, including two with anaplastic progression. We find that PXAs are genetically defined by the combination of CDKN2A biallelic inactivation and RAF alterations that were present in all 19 cases, most commonly as CDKN2A homozygous deletion and BRAF p.V600E mutation but also occasionally BRAF or RAF1 fusions or other rearrangements. The third most commonly altered gene in anaplastic PXA was TERT, with 47% (7/15) harboring TERT alterations, either gene amplification (n = 2) or promoter hotspot mutation (n = 5). In tumor pairs analyzed before and after anaplastic progression, two had increased copy number alterations and one had TERT promoter mutation at recurrence. Less commonly altered genes included TP53, BCOR, BCORL1, ARID1A, ATRX, PTEN, and BCL6. All PXA in this cohort were IDH and histone H3 wildtype, and did not contain alterations in EGFR. Genetic profiling performed on six regions from the same tumor identified intratumoral genomic heterogeneity, likely reflecting clonal evolution during tumor progression. Overall, anaplastic PXA is characterized by the combination of CDKN2A biallelic inactivation and oncogenic RAF kinase signaling as well as a relatively small number of additional genetic alterations, with the most common being TERT amplification or promoter mutation. These data define a distinct molecular profile for PXA and suggest additional genetic alterations, including TERT, may be associated with anaplastic progression.

Clinical trials in pediatric neuro-oncology: what is missing and how we can improve

Brain tumors are the most common solid tumor in childhood, yet outcomes vary dramatically. High-grade gliomas have dismal outcomes with poor survival. By contrast, low-grade gliomas, have high survival rates, but children suffer from morbidity of tumor burden and therapy-associated side effects. In this article, we discuss how current trial designs often miss the opportunity to include end points beyond tumor response and thus fail to offer complete assessments of therapeutic approaches. Quality of life, neurocognitive function and neurofunctional deficits need to be considered when assessing overall success of a therapy. Herein, we identify specific end points that should be included in the interpretation of clinical trial results and accordingly, offer a more comprehensive approach to treatment decision-making.

Abnormal Morphology of Select Cortical and Subcortical Regions in Neurofibromatosis Type 1

Purpose To evaluate whether patients with neurofibromatosis type 1 (NF1)-a multisystem neurodevelopmental disorder with myriad imaging manifestations, including focal transient myelin vacuolization within the deep gray nuclei, brainstem, and cerebellum-exhibit differences in cortical and subcortical structures, particularly in subcortical regions where these abnormalities manifest. Materials and Methods In this retrospective study, by using clinically obtained three-dimensional T1-weighted MR images and established image analysis methods, 10 intracranial volume-corrected subcortical and 34 cortical regions of interest (ROIs) were quantitatively assessed in 32 patients with NF1 and 245 age- and sex-matched healthy control subjects. By using linear models, ROI cortical thicknesses and volumes were compared between patients with NF1 and control subjects, as a function of age. With hierarchic cluster analysis and partial correlations, differences in the pattern of association between cortical and subcortical ROI volumes in patients with NF1 and control subjects were also evaluated. Results Patients with NF1 exhibited larger subcortical volumes and thicker cortices of select regions, particularly the hippocampi, amygdalae, cerebellar white matter, ventral diencephalon, thalami, and occipital cortices. For the thalami and pallida and 22 cortical ROIs in patients with NF1, a significant inverse association between volume and age was found, suggesting that volumes decrease with increasing age. Moreover, compared with those in control subjects, ROIs in patients with NF1 exhibited a distinct pattern of clustering and partial correlations. Discussion Neurofibromatosis type 1 is characterized by larger subcortical volumes and thicker cortices of select structures. Most apparent within the hippocampi, amygdalae, cerebellar white matter, ventral diencephalon, thalami and occipital cortices, these neurofibromatosis type 1-associated volumetric changes may, in part, be age dependent.

Chemotherapy of Pediatric Low-Grade Gliomas

Abstract Pediatric low-grade gliomas are a heterogeneous group of central nervous system malignancies. A number of histologic subtypes make up pediatric low-grade gliomas, but all fall into World Health Organization tumor grades I and II. These tumors are treated with surgical resection when feasible; however, anatomic location or involvement of critical structures can preclude resection. In these settings, chemotherapy may be considered. A wide variety of chemotherapy regimens have been employed over the years with many illustrating similar outcomes, and ultimate therapy choices being decided according to anticipated side effects and individual patient needs. In recent years and as we have made strides in identifying the molecular biology of these tumors, therapy options have become more targeted. Targeted agents are most commonly directed at BRAF or mTOR activating mutations within the MAPK and PI3K pathways. This chapter outlines the background of pediatric low-grade gliomas and the chemotherapy options used to treat them.

Early detection of recurrent medulloblastoma: the critical role of diffusion-weighted imaging

Background Imaging diagnosis of medulloblastoma recurrence relies heavily on identifying new contrast-enhancing lesions on surveillance imaging, with diffusion-weighted imaging (DWI) being used primarily for detection of complications. We propose that DWI is more sensitive in detecting distal and leptomeningeal recurrent medulloblastoma than T1-weighted postgadolinium imaging. Methods We identified 53 pediatric patients with medulloblastoma, 21 of whom developed definitive disease recurrence within the brain. MRI at diagnosis of recurrence and 6 months prior was evaluated for new lesions with reduced diffusion on DWI, contrast enhancement, size, and recurrence location. Results All recurrent medulloblastoma lesions demonstrated reduced diffusion. Apparent diffusion coefficient (ADC) measurements were statistically significantly lower (P = .00001) in recurrent lesions (mean=0.658, SD=0.072) as compared to contralateral normal region of interest (mean=0.923, SD=0.146). Sixteen patients (76.2%) with disease recurrence demonstrated contrast enhancement within the recurrent lesions. All 5 patients with nonenhancing recurrence demonstrated reduced diffusion, with a mean ADC of 0.695 ± 0.101 (normal=0.893 ± 0.100, P = .0027). While group 3 and group 4 molecular subtypes demonstrated distal recurrence more frequently, nonenhancing metastatic disease was found in all molecular subtypes. Conclusion Recurrent medulloblastoma lesions do not uniformly demonstrate contrast enhancement on MRI, but all demonstrate reduced diffusion. Our findings support that DWI is more sensitive than contrast enhancement for detection of medulloblastoma recurrence, particularly in cases of leptomeningeal nonenhancing disease and distal nonenhancing focal disease. As such, recurrent medulloblastoma can present as a reduced diffusion lesion in a patient with normal postgadolinium contrast MRI.

The genetic landscape of gliomas arising after therapeutic radiation

Radiotherapy improves survival for common childhood cancers such as medulloblastoma, leukemia, and germ cell tumors. Unfortunately, long-term survivors suffer sequelae that can include secondary neoplasia. Gliomas are common secondary neoplasms after cranial or craniospinal radiation, most often manifesting as high-grade astrocytomas with poor clinical outcomes. Here, we performed genetic profiling on a cohort of 12 gliomas arising after therapeutic radiation to determine their molecular pathogenesis and assess for differences in genomic signature compared to their spontaneous counterparts. We identified a high frequency of TP53 mutations, CDK4 amplification or CDKN2A homozygous deletion, and amplifications or rearrangements involving receptor tyrosine kinase and Ras–Raf–MAP kinase pathway genes including PDGFRA, MET, BRAF, and RRAS2. Notably, all tumors lacked alterations in IDH1, IDH2, H3F3A, HIST1H3B, HIST1H3C, TERT (including promoter region), and PTEN, which genetically define the major subtypes of diffuse gliomas in children and adults. All gliomas in this cohort had very low somatic mutation burden (less than three somatic single nucleotide variants or small indels per Mb). The ten high-grade gliomas demonstrated markedly aneuploid genomes, with significantly increased quantity of intrachromosomal copy number breakpoints and focal amplifications/homozygous deletions compared to spontaneous high-grade gliomas, likely as a result of DNA double-strand breaks induced by gamma radiation. Together, these findings demonstrate a distinct molecular pathogenesis of secondary gliomas arising after radiation therapy and identify a genomic signature that may aid in differentiating these tumors from their spontaneous counterparts.