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We reconstructed the natural history and temporal evolution of the most common childhood brain malignancy, medulloblastoma, by single-cell whole-genome sequencing (sc-WGS) of tumours representing its major molecular sub-classes and clinical risk groups. Favourable-risk disease sub-types assessed (MBWNT and infant desmoplastic/nodular MBSHH) typically comprised a single clone with no evidence of further evolution. In contrast, highest risk sub-classes (MYC-amplified MBGroup3 and TP53-mutated MBSHH) were most clonally diverse and displayed gradual evolutionary trajectories. Clinically adopted biomarkers (e.g. chromosome 6/17 aberrations; CTNNB1/TP53 mutations) were typically early-clonal/initiating events, exploitable as targets for early-disease detection; in analyses of spatially distinct tumour regions, a single biopsy was sufficient to assess their status. Importantly, sc-WGS revealed novel events which arise later and/or sub-clonally and more commonly display spatial diversity; their clinical significance and role in disease evolution post-diagnosis now require establishment. These findings reveal diverse modes of tumour initiation and evolution in the major medulloblastoma sub-classes, with pathogenic relevance and clinical potential.

Medulloblastoma is the most common malignant CNS tumor of childhood, affecting ~350 patients/year in the USA. In 2020, most children are cured of their disease, however, survivors are left with life-long late-effects as a consequence of intensive surgery, and application of chemo- and radio-therapy to the developing brain. The major contributor to improvements in patient survival has been the development of risk-stratified treatments derived from a better understanding of the prognostic value of disease biomarkers. The characterization and validation of these biomarkers has engendered a comprehensive understanding of the extensive heterogeneity that exists within the disease, which can occur both between and within tumors (inter- and intra-tumoral heterogeneity, respectively). In this review, we discuss inter-tumoral heterogeneity, describing the early characterization of clinical and histopathological disease heterogeneity, the more recent elucidation of molecular disease subgroups, and the potential for novel therapies based on specific molecular defects. We reflect on the limitations of current approaches when applied to a rare disease. We then review early investigations of intra-tumoral heterogeneity using FISH and immunohistochemical approaches, and focus on the application of next generation sequencing on bulk tumors to elucidate intra-tumoral heterogeneity. Finally, we critically appraise the applications of single-cell sequencing approaches and discuss their potential to drive next biological insights, and for routine clinical application.

Aims\ud \ud Biomarker‐driven therapies have not been developed for infant medulloblastoma (iMB). We sought to robustly sub‐classify iMB, and proffer strategies for personalized, risk‐adapted therapies.\ud \ud \ud \ud Methods\ud \ud We characterized the iMB molecular landscape, including second‐generation subtyping, and the associated retrospective clinical experience, using large independent discovery/validation cohorts (n = 387).\ud \ud \ud \ud Results\ud \ud iMBGrp3 (42%) and iMBSHH (40%) subgroups predominated. iMBGrp3 harboured second‐generation subtypes II/III/IV. Subtype II strongly associated with large‐cell/anaplastic pathology (LCA; 23%) and MYC amplification (19%), defining a very‐high‐risk group (0% 10yr overall survival (OS)), which progressed rapidly on all therapies; novel approaches are urgently required. Subtype VII (predominant within iMBGrp4) and subtype IV tumours were standard risk (80% OS) using upfront CSI‐based therapies; randomized‐controlled trials of upfront radiation‐sparing and/or second‐line radiotherapy should be considered. Seventy‐five per cent of iMBSHH showed DN/MBEN histopathology in discovery and validation cohorts (P < 0.0001); central pathology review determined diagnosis of histological variants to WHO standards. In multivariable models, non‐DN/MBEN pathology was associated significantly with worse outcomes within iMBSHH. iMBSHH harboured two distinct subtypes (iMBSHH‐I/II). Within the discriminated favourable‐risk iMBSHH DN/MBEN patient group, iMBSHH‐II had significantly better progression‐free survival than iMBSHH‐I, offering opportunities for risk‐adapted stratification of upfront therapies. Both iMBSHH‐I and iMBSHH‐II showed notable rescue rates (56% combined post‐relapse survival), further supporting delay of irradiation. Survival models and risk factors described were reproducible in independent cohorts, strongly supporting their further investigation and development.\ud \ud \ud \ud Conclusions\ud \ud Investigations of large, retrospective cohorts have enabled the comprehensive and robust characterization of molecular heterogeneity within iMB. Novel subtypes are clinically significant and subgroup‐dependent survival models highlight opportunities for biomarker‐directed therapies.

Medulloblastomas harbor clinically-significant intra-tumoral heterogeneity for key biomarkers (e.g. MYC/MYCN, β-catenin). Recent studies have characterized transcriptional heterogeneity at the single-cell level, however the underlying genomic copy number and mutational architecture remains to be resolved. We therefore sought to establish the intra-tumoural genomic heterogeneity of medulloblastoma at single-cell resolution. Copy number patterns were dissected by whole-genome sequencing in 1024 single cells isolated from multiple distinct tumour regions within 16 snap-frozen medulloblastomas, representing the major molecular subgroups (WNT, SHH, Group3, Group4) and genotypes (i.e. MYC amplification, TP53 mutation). Common copy number driver and subclonal events were identified, providing clear evidence of copy number evolution in medulloblastoma development. Moreover, subclonal whole-arm and focal copy number alterations covering important genomic loci (e.g. on chr10 of SHH patients) were detected in single tumour cells, yet undetectable at the bulk-tumor level. Spatial copy number heterogeneity was also common, with differences between clonal and subclonal events detected in distinct regions of individual tumours. Mutational analysis of the cells allowed dissection of spatial and clonal heterogeneity patterns for key medulloblastoma mutations (e.g. CTNNB1, TP53, SMARCA4, PTCH1) within our cohort. Integrated copy number and mutational analysis is underway to establish their inter-relationships and relative contributions to clonal evolution during tumourigenesis. In summary, single-cell analysis has enabled the resolution of common mutational and copy number drivers, alongside sub-clonal events and distinct patterns of clonal and spatial evolution, in medulloblastoma development. We anticipate these findings will provide a critical foundation for future improved biomarker selection, and the development of targeted therapies.

Key Points Question Can targeting tropomyosin receptor kinase with an existing topical kinase inhibitor, pegcantratinib, 0.5% (wt/wt), reduce cutaneous cylindroma tumor volume more than placebo? Findings In this phase 2 clinical trial that included 150 tumors from 15 patients with CYLD cutaneous syndrome, pegcantratinib-treated tumors did not achieve the primary outcome of response. Molecular analyses of biopsy material demonstrated drug penetration; however, drug concentrations achieved were inadequate to abrogate tropomyosin receptor kinase signaling in CYLD cutaneous syndrome tumors. Meaning These findings indicate that further studies should examine dose-escalation of pegcantratinib in these patients., Importance There are no medical interventions for the orphan disease CYLD cutaneous syndrome (CCS). Transcriptomic profiling of CCS skin tumors previously highlighted tropomyosin receptor kinases (TRKs) as candidate therapeutic targets. Objective To investigate if topical targeting of TRK with an existing topical TRK inhibitor, pegcantratinib, 0.5% (wt/wt), is safe and efficacious in CCS. Design, Setting, and Participants A phase 1b open-label safety study, followed by a phase 2a within-patient randomized (by tumor), double-blind, placebo-controlled trial (the Tropomyosin Receptor Antagonism in Cylindromatosis [TRAC] trial). The setting was a single-center trial based at a tertiary dermatogenetics referral center for CCS (Royal Victoria Infirmary, Newcastle, United Kingdom). Patients who had germline mutations in CYLD or who satisfied clinical diagnostic criteria for CCS were recruited between March 1, 2015, and July 1, 2016. Interventions In phase 1b, patients with CCS applied pegcantratinib for 4 weeks to a single skin tumor. In phase 2a, allocation of tumors was to either receive active treatment on the right side and placebo on the left side (arm A) or active treatment on the left side and placebo on the right side (arm B). Patients were eligible if they had 10 small skin tumors, with 5 matched lesions on each body side; patients were randomized to receive active treatment (pegcantratinib) to one body side and placebo to the other side once daily for 12 weeks. Main Outcomes and Measures The primary outcome measure was the number of tumors meeting the criteria for response in a prespecified critical number of pegcantratinib-treated tumors. Secondary clinical outcome measures included an assessment for safety of application, pain in early tumors, and compliance with the trial protocol. Results In phase 1b, 8 female patients with a median age of 60 years (age range, 41-80 years) were recruited and completed the study. None of the participants experienced any adverse treatment site reactions. Three patients reported reduced pain in treated tumors. In phase 2a (15 patients [13 female; median age, 51 years], with 150 tumors), 2 tumors treated with pegcantratinib achieved the primary outcome measure of response compared with 6 tumors treated with placebo. The primary prespecified number of responses was not met. The incidence of adverse events was low. Conclusions and Relevance In this study, pegcantratinib, 0.5% (wt/wt), applied once daily appeared to be well tolerated and to penetrate the tumor tissue; however, the low tumor drug concentrations demonstrated are likely to account for the lack of response. Dose-escalation studies to assess the maximal tolerated dose may be beneficial in future studies of CCS. Trial Registration isrctn.org Identifier: ISRCTN75715723, This phase 2 randomized clinical trial investigates if topical targeting of topical tropomyosin receptor kinase (TRK) with an inhibitor, pegcantratinib, is safe and efficacious in treating tumors in patients with CYLD cutaneous syndrome.

Transcriptomic profiling of skin disease using next generation sequencing allows for detailed information on aspects of RNA biology including gene expression, non-coding regulatory elements and gene splicing. The application of RNA sequencing to human skin disease and cancer is often hampered by degraded RNA. Here we describe a protocol that allows for consistently intact RNA to be extracted from snap frozen skin biopsy samples, which has been validated in a clinical trial setting. Human skin tumour punch biopsies (n=28) ranging from 4-6mm in diameter were obtained from 14 patients with an inherited skin tumour syndrome (CYLD cutaneous syndrome) and frozen in liquid nitrogen prior to being stored at -80°C. These samples were then subject to cyrostat sectioning, allowing for histological assessment, and were homogenised using a bead-based lysis platform. RNA extraction was performed using a silica column-based system. RNA concentration was measured using fluorescent quantitation and RNA integrity assessed using microfluidic gel electrophoresis. We also processed normal skin biopsies using the same protocol (n=10). The mean RNA integrity score of the tumour and normal samples was 9.5, and the quantity of RNA obtained from the small amounts of tissue used exceeded requirements for RNA-seq library generation. We propose that the method of RNA extraction suggested here allows for transcriptomic profiling from small pieces of human tissue without the need for PCR amplification during library preparation. This protocol could be utilised in healthy and diseased skin to improve mechanistic understanding in a range of human skin diseases.

Aim: Pegcantratinib is a mini-PEGylated K252a derivative, under clinical evaluation as an anticancer agent acting through inhibition of the tropomyosin receptor kinase. A method for quantifying pegcantratinib in skin tumor biopsies of patients was required to determine tumor drug penetration. Methods & results: A sensitive and PEGylated molecule specific HPLC–MS/MS method coupled with in-source collision-induced dissociation was developed. The method exhibited excellent precision (coefficient of variation ≤8.5%), accuracy in the range 95–102%, high and consistent recovery and no matrix effect. The assay was linear across a range of 1–500 ng/ml, with a limit of quantitation of 2.5 ng/ml. Conclusion: We have developed and validated a method for analyzing pegcantratinib in human tumor biopsies, with the approach successfully applied to clinical trial samples.

Tra2 proteins regulate pre-mRNA splicing in vertebrates and invertebrates, and are involved in important processes ranging from brain development in mice to sex determination in fruitflies. In structure Tra2 proteins contain two RS domains (domains enriched in arginine and serine residues) flanking a central RRM (RNA recognition motif). Understanding the mechanisms of how Tra2 proteins work to control splicing is one of the key requirements to understand their biology. In the present article, we review what is known about how Tra2 proteins regulate splicing decisions in mammals and fruitflies.

Sam68 and T-STAR are members of the STAR family of proteins that directly link signal transduction with post-transcriptional gene regulation. Sam68 controls the alternative splicing of many oncogenic proteins. T-STAR is a tissue-specific paralogue that regulates the alternative splicing of neuronal pre-mRNAs. STAR proteins differ from most splicing factors, in that they contain a single RNA-binding domain. Their specificity of RNA recognition is thought to arise from their property to homodimerize, but how dimerization influences their function remains unknown. Here, we establish at atomic resolution how T-STAR and Sam68 bind to RNA, revealing an unexpected mode of dimerization different from other members of the STAR family. We further demonstrate that this unique dimerization interface is crucial for their biological activity in splicing regulation, and suggest that the increased RNA affinity through dimer formation is a crucial parameter enabling these proteins to select their functional targets within the transcriptome., Sam68 and T-STAR are members of the STAR family of proteins, which regulate various aspects of RNA metabolism. Here, the authors reveal structural features required for alternative splicing regulation by these proteins.

Meiosis requires conserved transcriptional changes, but it is not known whether there is a corresponding set of RNA splicing switches. Here, we used RNAseq of mouse testis to identify changes associated with the progression from mitotic spermatogonia to meiotic spermatocytes. We identified ∼150 splicing switches, most of which affect conserved protein-coding exons. The expression of many key splicing regulators changed in the course of meiosis, including downregulation of polypyrimidine tract binding protein (PTBP1) and heterogeneous nuclear RNP A1, and upregulation of nPTB, Tra2β, muscleblind, CELF proteins, Sam68 and T-STAR. The sequences near the regulated exons were significantly enriched in target sites for PTB, Tra2β and STAR proteins. Reporter minigene experiments investigating representative exons in transfected cells showed that PTB binding sites were critical for splicing of a cassette exon in the Ralgps2 mRNA and a shift in alternative 5′ splice site usage in the Bptf mRNA. We speculate that nPTB might functionally replace PTBP1 during meiosis for some target exons, with changes in the expression of other splicing factors helping to establish meiotic splicing patterns. Our data suggest that there are substantial changes in the determinants and patterns of alternative splicing in the mitotic-to-meiotic transition of the germ cell cycle.

The RNA binding protein T-STAR was created following a gene triplication 520–610 million years ago, which also produced its two parologs Sam68 and SLM-1. Here we have created a T-STAR null mouse to identify the endogenous functions of this RNA binding protein. Mice null for T-STAR developed normally and were fertile, surprisingly, given the high expression of T-STAR in the testis and the brain, and the known infertility and pleiotropic defects of Sam68 null mice. Using a transcriptome-wide search for splicing targets in the adult brain, we identified T-STAR protein as a potent splicing repressor of the alternatively spliced segment 4 (AS4) exons from each of the Neurexin1-3 genes, and exon 23 of the Stxbp5l gene. T-STAR protein was most highly concentrated in forebrain-derived structures like the hippocampus, which also showed maximal Neurexin1-3 AS4 splicing repression. In the absence of endogenous T-STAR protein, Nrxn1-3 AS4 splicing repression dramatically decreased, despite physiological co-expression of Sam68. In transfected cells Neurexin3 AS4 alternative splicing was regulated by either T-STAR or Sam68 proteins. In contrast, Neurexin2 AS4 splicing was only regulated by T-STAR, through a UWAA-rich response element immediately downstream of the regulated exon conserved since the radiation of bony vertebrates. The AS4 exons in the Nrxn1 and Nrxn3 genes were also associated with distinct patterns of conserved UWAA repeats. Consistent with an ancient mechanism of splicing control, human T-STAR protein was able to repress splicing inclusion of the zebrafish Nrxn3 AS4 exon. Although Neurexin1-3 and Stxbp5l encode critical synaptic proteins, T-STAR null mice had no detectable spatial memory deficits, despite an almost complete absence of AS4 splicing repression in the hippocampus. Our work identifies T-STAR as an ancient and potent tissue-specific splicing regulator that uses a concentration-dependent mechanism to co-ordinately regulate regional splicing patterns of the Neurexin1-3 AS4 exons in the mouse brain., Author Summary Alternative splicing plays a key role in animal development and is largely controlled by the expression of RNA binding proteins. Most RNA binding proteins exist as families of sister proteins called paralogs, which result from gene amplification, including T-STAR, which is closely related to Sam68 and SLM-1. T-STAR, Sam68, and SLM-1 usually behave identically in splicing control in transfected cells. Here we report the physiological functions of T-STAR protein by knocking its parent gene out in the mouse. Surprisingly we observed no defects in germ cell maturation without T-STAR protein, an unexpected result given T-STAR protein is mainly expressed in the testis and its paralog Sam68 is essential for male fertility. Instead, we find T-STAR controls a panel of splicing targets that encode important synaptic proteins. T-STAR acts as a potent splicing repressor to establish regional splicing patterns of these target exons in the brain. Forebrain-derived structures like the hippocampus strongly express T-STAR protein to repress these target exons. Some T-STAR regulated splicing targets overlap with Sam68, but T-STAR also regulates its own distinct targets. Comparative genomic analyses are consistent with an ancient mechanism of splicing control by T-STAR that has been conserved since the radiation of bony vertebrates.

Background: Pegcantratinib is the first topically applied tropomyosin receptor kinase inhibitor allowing retention in the skin to exert its effects at nanomolar concentrations. Pegcantratinib is now under early clinical evaluation as an anti-tumour agent in patients with germline mutations in the tumour suppressor gene CYLD, which include multiple clinical presentations of rare, skin appendage tumours. No pharmacological treatment is currently available and patients face repeated and disfiguring surgery to control tumour burden. We report on the development and validation of an analytical method to quantify pegcantratinib in skin tumour biopsies of patients to determine tumour penetration. Methods: A sensitive and specific HPLC-MS/MS (API 4000) assay coupled with in-source CID was developed and validated according to EMA guidelines, for direct quantitative measurement of pegcantratinib in biopsies of human skin tumours. The sample preparation procedure required a 10 mg sample volume and involved protein precipitation with acetonitrile following tissue homogenization and addition of internal standard. Reversed-phase chromatography under gradient conditions (MP A - 1% formic acid; MP B - 1% formic acid in acetonitrile) was applied with separation on a Kinetex 2.6 μm C18 column (100 Å, 50 × 4.6 mm). Detection was obtained by SRM, following the transitions m/z 419.0 → 376.4. The method is rapid and selective, allowing good resolution of peaks in 2.4 min. Results: The assay was fully validated and the method exhibited good sensitivity, precision, and accuracy, with overall precision expressed as CV ≤8.5% and accuracy in the range 96-99%. Recovery was high (≥85%) and consistent with CV ≤13%. No matrix effect was observed in four independent matrix sources including 3 different tumours and normal skin, the calculated CV was 5%. The limit of quantitation was 2.5 ng/ml, with precision and accuracy of 6% and 99%, respectively. The assay developed was linear in the range 1.0-500 ng/ml, with mean precision Conclusion: We have successfully developed a LC-MS/MS method to measure the anti-tumour agent pegcantratinib in human skin tumour samples. Analysis of samples obtained from the Phase 1b/2a clinical trial is now underway. Acknowledgements: Work supported by the Department of Health and Wellcome Trust through the Health Innovation Challenge Fund and Cancer Research UK. Citation Format: Monique Zangarini, Neil Rajan, Marina Danilenko, Philip Berry, Gareth J. Veal. Development and validation of a LC-MS/MS method for the quantification of the tropomyosin receptor kinase (Trk) inhibitor pegcantratinib in human skin tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 333.