Whole genome and transcriptome integrated analyses guide clinical care of pediatric poor prognosis cancers.

The role for routine whole genome and transcriptome analysis (WGTA) for poor prognosis pediatric cancers remains undetermined. Here, we characterize somatic mutations, structural rearrangements, copy number variants, gene expression, immuno-profiles and germline cancer predisposition variants in children and adolescents with relapsed, refractory or poor prognosis malignancies who underwent somatic WGTA and matched germline sequencing. Seventy-nine participants with a median age at enrollment of 8.8 y (range 6 months to 21.2 y) are included. Germline pathogenic/likely pathogenic variants are identified in 12% of participants, of which 60% were not known prior. Therapeutically actionable variants are identified by targeted gene report and whole genome in 32% and 62% of participants, respectively, and increase to 96% after integrating transcriptome analyses. Thirty-two molecularly informed therapies are pursued in 28 participants with 54% achieving a clinical benefit rate; objective response or stable disease ≥6 months. Integrated WGTA identifies therapeutically actionable variants in almost all tumors and are directly translatable to clinical care of children with poor prognosis cancers.

The impact of whole genome and transcriptome analysis (WGTA) on predictive biomarker discovery and diagnostic accuracy of advanced malignancies.

In this study, we evaluate the impact of whole genome and transcriptome analysis (WGTA) on predictive molecular profiling and histologic diagnosis in a cohort of advanced malignancies. WGTA was used to generate reports including molecular alterations and site/tissue of origin prediction. Two reviewers analyzed genomic reports, clinical history, and tumor pathology. We used National Comprehensive Cancer Network (NCCN) consensus guidelines, Food and Drug Administration (FDA) approvals, and provincially reimbursed treatments to define genomic biomarkers associated with approved targeted therapeutic options (TTOs). Tumor tissue/site of origin was reassessed for most cases using genomic analysis, including a machine learning algorithm (Supervised Cancer Origin Prediction Using Expression [SCOPE]) trained on The Cancer Genome Atlas data. WGTA was performed on 652 cases, including a range of primary tumor types/tumor sites and 15 malignant tumors of uncertain histogenesis (MTUH). At the time WGTA was performed, alterations associated with an approved TTO were identified in 39 (6%) cases; 3 of these were not identified through routine pathology workup. In seven (1%) cases, the pathology workup either failed, was not performed, or gave a different result from the WGTA. Approved TTOs identified by WGTA increased to 103 (16%) when applying 2021 guidelines. The histopathologic diagnosis was reviewed in 389 cases and agreed with the diagnostic consensus after WGTA in 94% of non-MTUH cases (n = 374). The remainder included situations where the morphologic diagnosis was changed based on WGTA and clinical data (0.5%), or where the WGTA was non-contributory (5%). The 15 MTUH were all diagnosed as specific tumor types by WGTA. Tumor board reviews including WGTA agreed with almost all initial predictive molecular profile and histopathologic diagnoses. WGTA was a powerful tool to assign site/tissue of origin in MTUH. Current efforts focus on improving therapeutic predictive power and decreasing cost to enhance use of WGTA data as a routine clinical test.

Post-mortem Examination of the Nervous System: Fresh versus Fixed.

BACKGROUND: Post-mortem examination of the nervous system is a complex task that culminates in "brain cutting". It relies on expertise in neuroanatomy, clinical neurosciences, neuroimaging and experience in order to recognise the most subtle abnormalities. Like any specialist examination in medicine, it warrants formal training, a standardised approach and optimal conditions. Revelations of aberrant tissue retention practices of a select few pathologists (e.g. Goudge, Liverpool and Alder Hey inquiries) and a motivated sociopolitical climate led some Canadian jurisdictions to impose broad restrictions on tissue retention. This raised concerns that nervous system examinations for diagnosis, education and research were at risk by limiting examinations to the fresh or incompletely fixed state. Professional experience indicates that cutting an unfixed or partly fixed brain is inferior. METHODS: To add objectivity and further insight we sought the expert opinion of a group of qualified specialists. Canadian neuropathologists were surveyed for their opinion on the relative merits of examining brains in the fresh or fully fixed state. RESULTS: A total of 14 out of 46 Canadian neuropathologists responded (30%). In the pervasive opinion of respondents, cutting and sampling a brain prior to full fixation leads to a loss of diagnostic accuracy, biosafety and academic deliverables. CONCLUSIONS: Brain cutting in the fresh state is significantly impaired along multiple dimensions of relevance to a pathologist's professional roles and obligations.

NTRK2 Fusion driven pediatric glioblastoma: Identification of oncogenic Drivers via integrative Genome and transcriptome profiling.

This is the first report of a NACC2-NTRK2 fusion in a histological glioblastoma. Oncogenomic analysis revealed this actionable fusion oncogene in a pediatric cerebellar glioblastoma, which would not have been identified through routine diagnostics, demonstrating the value of clinical genome profiling in cancer care.

Pmp22 super-enhancer deletion causes tomacula formation and conduction block in peripheral nerves.

Copy number variation of the peripheral nerve myelin gene Peripheral Myelin Protein 22 (PMP22) causes multiple forms of inherited peripheral neuropathy. The duplication of a 1.4 Mb segment surrounding this gene in chromosome 17p12 (c17p12) causes the most common form of Charcot-Marie-Tooth disease type 1A, whereas the reciprocal deletion of this gene causes a separate neuropathy termed hereditary neuropathy with liability to pressure palsies (HNPP). PMP22 is robustly induced in Schwann cells in early postnatal development, and several transcription factors and their cognate regulatory elements have been implicated in coordinating the gene's proper expression. We previously found that a distal super-enhancer domain was important for Pmp22 expression in vitro, with particular impact on a Schwann cell-specific alternative promoter. Here, we investigate the consequences of deleting this super-enhancer in vivo. We find that loss of the super-enhancer in mice reduces Pmp22 expression throughout development and into adulthood, with greater impact on the Schwann cell-specific promoter. Additionally, these mice display tomacula formed by excessive myelin folding, a pathological hallmark of HNPP, as have been previously observed in heterozygous Pmp22 mice as well as sural biopsies from patients with HNPP. Our findings demonstrate a mechanism by which smaller copy number variations, not including the Pmp22 gene, are sufficient to reduce gene expression and phenocopy a peripheral neuropathy caused by the HNPP-associated deletion encompassing PMP22.

Comparative RNA-Sequencing Analysis Benefits a Pediatric Patient With Relapsed Cancer.

Clinical detection of sequence and structural variants in known cancer genes points to viable treatment options for a minority of children with cancer.1 To increase the number of children who benefit from genomic profiling, gene expression information must be considered alongside mutations.2,3 Although high expression has been used to nominate drug targets for pediatric cancers,4,5 its utility has not been evaluated in a systematic way.6 We describe a child with a rare sarcoma that was profiled with whole-genome and RNA sequencing (RNA-Seq) techniques. Although the tumor did not harbor DNA mutations targetable by available therapies, incorporation of gene expression information derived from RNA-Seq analysis led to a therapy that produced a significant clinical response. We use this case to describe a framework for inclusion of gene expression into the clinical genomic evaluation of pediatric tumors.

Genomic analysis of diffuse intrinsic pontine gliomas identifies three molecular subgroups and recurrent activating ACVR1 mutations.

Diffuse intrinsic pontine glioma (DIPG) is a fatal brain cancer that arises in the brainstem of children, with no effective treatment and near 100% fatality. The failure of most therapies can be attributed to the delicate location of these tumors and to the selection of therapies on the basis of assumptions that DIPGs are molecularly similar to adult disease. Recent studies have unraveled the unique genetic makeup of this brain cancer, with nearly 80% found to harbor a p.Lys27Met histone H3.3 or p.Lys27Met histone H3.1 alteration. However, DIPGs are still thought of as one disease, with limited understanding of the genetic drivers of these tumors. To understand what drives DIPGs, we integrated whole-genome sequencing with methylation, expression and copy number profiling, discovering that DIPGs comprise three molecularly distinct subgroups (H3-K27M, silent and MYCN) and uncovering a new recurrent activating mutation affecting the activin receptor gene ACVR1 in 20% of DIPGs. Mutations in ACVR1 were constitutively activating, leading to SMAD phosphorylation and increased expression of the downstream activin signaling targets ID1 and ID2. Our results highlight distinct molecular subgroups and novel therapeutic targets for this incurable pediatric cancer.

Diffusion-weighted imaging and pathological correlation in pediatric medulloblastomas-"They are not always restricted!".

INTRODUCTION: Some investigators have suggested that medulloblastomas can be distinguished from other cerebellar neoplasms by demonstrating "restricted diffusion" on the Apparent Diffusion Coefficient (ADC) map obtained from diffusion-weighted imaging (DWI) sequences on magnetic resonance imaging. Previous authors have postulated that this observed restricted diffusion is a reflection of very high cell density. There has been a tendency to assert that pediatric medulloblastoma uniformly demonstrates restricted diffusion on DWI. However, our clinical observation has been that there are pediatric medulloblastomas that exhibit normal or even increased diffusion on DWI. The current study was undertaken primarily to determine whether restricted diffusion is uniformly present in pediatric medulloblastoma and secondly to look for pathological features that might distinguish medulloblastomas with and without restricted diffusion. METHODS: The DWI characteristics of pathologically confirmed medulloblastomas diagnosed at our institution were retrospectively reviewed. The ADC was obtained in two non-overlapping, solid, non-hemorrhagic, non-necrotic regions of tumor and averaged. An ADC below 1 × 10(-3) mm(2)/s was considered to represent restricted diffusion. A detailed pathologic review of each tumor was conducted. RESULTS: Ten cases of medulloblastoma were reviewed, of which two demonstrated average ADCs above 1 × 10(-3) mm(2)/s (1.223 and 1.169 × 10(-3) mm(2)/s, respectively), indicating no restricted diffusion. Pathologic review revealed that both of these non-restricting cases displayed a lack of reticulin deposition by light microscopy. CONCLUSION: DWI does not appear to be an entirely reliable means of distinguishing medulloblastomas from other cerebellar neoplasms. Histologically, restricted diffusion in medulloblastomas may be related to reticulin deposition.

Unusual clinical, laboratory, and muscle histopathological findings in a family with myotonic dystrophy type 2.

Myotonic dystrophy type 2 (DM2) is a multisystem degenerative disorder with distinctive clinical and electrophysiological features. Recently, genetic confirmation has become available with the identification of the molecular defect, an expansion of a CCTG repeat located in intron 1 of the zinc finger protein 9 (ZNF9) gene. We present two first-degree relatives with an athletic clinical phenotype, pathological evidence of subsarcolemmal vacuolation, and molecular genetic confirmation of DM2. When found in the proper clinical context, athleticism and pathological subsarcolemmal vacuoles should not dissuade the clinician from the possible diagnosis of DM2.