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Infant high-grade gliomas appear clinically distinct from their counterparts in older children, indicating that histopathologic grading may not accurately reflect the biology of these tumors. We have collected 241 cases under 4 years of age, and carried out histologic review, methylation profiling, and custom panel, genome, or exome sequencing. After excluding tumors representing other established entities or subgroups, we identified 130 cases to be part of an “intrinsic” spectrum of disease specific to the infant population. These included those with targetable MAPK alterations, and a large proportion of remaining cases harboring gene fusions targeting ALK (n = 31), NTRK1/2/3 (n = 21), ROS1 (n = 9), and MET (n = 4) as their driving alterations, with evidence of efficacy of targeted agents in the clinic. These data strongly support the concept that infant gliomas require a change in diagnostic practice and management.Significance:Infant high-grade gliomas in the cerebral hemispheres comprise novel subgroups, with a prevalence of ALK, NTRK1/2/3, ROS1, or MET gene fusions. Kinase fusion–positive tumors have better outcome and respond to targeted therapy clinically. Other subgroups have poor outcome, with fusion-negative cases possibly representing an epigenetically driven pluripotent stem cell phenotype.See related video: https://vimeo.com/438254885See related commentary by Szulzewsky and Cimino, p. 904.This article is highlighted in the In This Issue feature, p. 890

The largest whole genome sequencing (WGS) endeavour involving cancer and rare diseases was initiated in the UK in 2015 and ran for 5 years. Despite its rarity, sarcoma ranked third overall among the number of patients' samples sent for sequencing. Herein, we recount the lessons learned by a specialist sarcoma centre that recruited close to 1000 patients to the project, so that we and others may learn from our experience. WGS data was generated from 597 patients, but samples from the remaining approximately 400 patients were not sequenced. This was largely accounted for by unsuitability due to extensive necrosis, secondary to neoadjuvant radiotherapy or chemotherapy, or being placed in formalin. The number of informative genomes produced was reduced further by a PCR amplification step. We showed that this loss of genomic data could be mitigated by sequencing whole genomes from needle core biopsies. Storage of resection specimens at 4 °C for up to 96 h overcame the challenge of freezing tissue out of hours including weekends. Removing access to formalin increased compliance to these storage arrangements. With over 70 different sarcoma subtypes described, WGS was a useful tool for refining diagnoses and identifying novel alterations. Genomes from 350 of the cohort of 597 patients were analysed in this study. Overall, diagnoses were modified for 3% of patients following review of the WGS findings. Continued refinement of the variant‐calling bioinformatic pipelines is required as not all alterations were identified when validated against histology and standard of care diagnostic tests. Further research is necessary to evaluate the impact of germline mutations in patients with sarcoma, and sarcomas with evidence of hypermutation. Despite 50% of the WGS exhibiting domain 1 alterations, the number of patients with sarcoma who were eligible for clinical trials remains small, highlighting the need to revaluate clinical trial design.

Summary Background Marked variation exists in the use of genomic data in tumour diagnosis, and optimal integration with conventional diagnostic technology remains uncertain despite several studies reporting improved diagnostic accuracy, selection for targeted treatments, and stratification for trials. Our aim was to assess the added value of molecular profiling in routine clinical practice and the impact on conventional and experimental treatments. Methods This population-based study assessed the diagnostic and clinical use of DNA methylation-based profiling in childhood CNS tumours using two large national cohorts in the UK. In the diagnostic cohort—which included routinely diagnosed CNS tumours between Sept 1, 2016, and Sept 1, 2018—we assessed how the methylation profile altered or refined diagnosis in routine clinical practice and estimated how this would affect standard patient management. For the archival cohort of diagnostically difficult cases, we established how many cases could be solved using modern standard pathology, how many could only be solved using the methylation profile, and how many remained unsolvable. Findings Of 484 patients younger than 20 years with CNS tumours, 306 had DNA methylation arrays requested by the neuropathologist and were included in the diagnostic cohort. Molecular profiling added a unique contribution to clinical diagnosis in 107 (35%; 95% CI 30–40) of 306 cases in routine diagnostic practice—providing additional molecular subtyping data in 99 cases, amended the final diagnosis in five cases, and making potentially significant predictions in three cases. We estimated that it could change conventional management in 11 (4%; 95% CI 2–6) of 306 patients. Among 195 historically difficult-to-diagnose tumours in the archival cohort, 99 (51%) could be diagnosed using standard methods, with the addition of methylation profiling solving a further 34 (17%) cases. The remaining 62 (32%) cases were unresolved despite specialist pathology and methylation profiling. Interpretation Together, these data provide estimates of the impact that could be expected from routine implementation of genomic profiling into clinical practice, and indicate limitations where additional techniques will be required. We conclude that DNA methylation arrays are a useful diagnostic adjunct for childhood CNS tumours. Funding The Brain Tumour Charity, Children with Cancer UK, Great Ormond Street Hospital Children's Charity, Olivia Hodson Cancer Fund, Cancer Research UK, and the National Institute of Health Research.

Infant high-grade gliomas appear clinically distinct from their counterparts in older children, indicating that histopathologic grading may not accurately reflect the biology of these tumors. We have collected 241 cases under 4 years of age, and carried out histologic review, methylation profiling, and custom panel, genome, or exome sequencing. After excluding tumors representing other established entities or subgroups, we identified 130 cases to be part of an “intrinsic” spectrum of disease specific to the infant population. These included those with targetable MAPK alterations, and a large proportion of remaining cases harboring gene fusions targeting ALK (n = 31), NTRK1/2/3 (n = 21), ROS1 (n = 9), and MET (n = 4) as their driving alterations, with evidence of efficacy of targeted agents in the clinic. These data strongly support the concept that infant gliomas require a change in diagnostic practice and management. Significance: Infant high-grade gliomas in the cerebral hemispheres comprise novel subgroups, with a prevalence of ALK, NTRK1/2/3, ROS1, or MET gene fusions. Kinase fusion–positive tumors have better outcome and respond to targeted therapy clinically. Other subgroups have poor outcome, with fusion-negative cases possibly representing an epigenetically driven pluripotent stem cell phenotype. See related video: https://vimeo.com/438254885 See related commentary by Szulzewsky and Cimino, p. 904. This article is highlighted in the In This Issue feature, p. 890

BACKGROUND: Paediatric High Grade Gliomas (HGG) have poor outcomes with conventional treatment. HGG in association with constitutional DNA mismatch repair deficiency (CMMRD) are hypermutated and have shown dramatic response to checkpoint inhibitors. Salvage following progression or failure to respond to check point inhibitors has rarely been reported. We describe a successful alternative therapeutic approach targeting the activated pathway (mTOR) in a hypermutated HGG. CASE SUMMARY: A 6-year-old girl presenting with seizures was diagnosed with left frontal lobe HGG with concurrent neck mass (Pilomatrixoma). Presence of synchronous tumours raised the possibility of cancer predisposition; the HGG was hypermutated with germline PMS2 mutation confirming diagnosis of CMMRD. Near total resection was undertaken followed by focal radiotherapy 54 Gy, with 1 cycle of concomitant CCNU. MRI post radiotherapy showed tumour progression. Anti-PDl inhibitor Nivolumab was commenced. CTLA-4 antibody, Ipilimumab was added after 4 cycles of Nivolumab due to poor response. Tumour response was seen, but dual therapy had to be discontinued due to toxicity. The tumour progressed following further single agent Nivolumab. In view of multiple mutations in the mTOR pathway (NF1, PIK3/PTEN, TSC1, TSC2), a mTOR inhibitor, Everolimus was commenced. There was 25% tumour reduction after 4 weeks treatment and further reduction after 6 months. Resection of residual tumour showed necrotic tissue only. There continues to be a sustained response to Everolimus for over 12 months. DISCUSSION: Approximately a third of CMMRD HGG respond to checkpoint inhibitors. For those that don’t, these hypermutated tumours offers the possibility of targeting specific molecular pathways. Response to Everolimus in HGG harbouring mTOR aberrations have been described. To our knowledge this is the first report of successful use of mTOR inhibitor in CMMRD HGG. CONCLUSION: Targeted molecular treatment for patients with CMMRD hypermutated brain tumours should be considered according to the mutated pathways.

Craniopharyngiomas are rare challenging tumours, with around 25% of cases recurring despite surgery and/or radiotherapy. Whilst transcriptomic and proteomic profiling of adamantinomatous craniopharyngioma (ACP) have revealed activation of the MAPK pathway and IL-6 mediated inflammation, relatively little is known about the biological processes involved in tumour recurrence. To address this, we have analysed a cohort of primary and relapsed ACP tumour accessed from tissue banks, local pathology departments and international collaborators. Serial samples of recurrent ACP (n=11), recurrent papillary craniopharyngioma (PCP) (n=4) and 3 non-recurrent ACP were analysed using β-catenin, pERK1/2 immunostaining, DNA methylation array and RNA sequencing. Differential expression and methylation analyses confirmed differences between ACP and PCP, with over representation of WNT pathway genes in ACP and the MAPK pathway genes in PCP. All of the primary and all except for one of the relapsed ACP tumours showed a pERK1/2 expression. Differences in the immune environment were also identified between ACP and PCP, with higher levels of some inflammatory mediators and CD14+ cell signatures in PCP compared with ACP. Whilst differential methylation and expression analysis revealed relatively stable methylomes and transcriptomes between serial samples of cases, segmental chromosomal alterations were identified in recurrence samples from five ACP cases (5/11,45%). One relapsed case showed histological and molecular signs of malignant transformation, including high ki67 and deletion of TP53. Surprisingly, this malignant tumour showed nuclear beta-catenin in all neoplastic epithelial cells and absence of pERK1/2 staining, despite the primary tumour showing the typical beta-catenin and pERK1/2 expression patterns. These results suggest that the molecular landscape of craniopharyngioma remains stable between recurrences in most cases, but, there is evidence of molecular evolution in a subset of cases. Activation of the MAPK pathway in the vast majority of ACP tumours supports the clinical evaluation of MAPK pathway inhibitors in ACP patients.

Summary Background Genotype–phenotype studies can identify subgroups of patients with specific clinical features or differing outcomes, which can help shape management. Objectives To characterize the frequency of different causative genotypes in congenital melanocytic naevi (CMN), and to investigate genotype–phenotype and genotype–outcome associations. Methods We conducted a large cohort study in which we undertook MC1R genotyping from blood, and high‐sensitivity genotyping of NRAS and BRAF hotspots in 156 naevus biopsies from 134 patients with CMN [male 40%; multiple CMN 76%; projected adult size (PAS) > 20 cm, 59%]. Results Mosaic NRAS mutations were detected in 68%, mutually exclusive with BRAF mutations in 7%, with double wild‐type in 25%. Two separate naevi were sequenced in five of seven patients with BRAF mutations, confirming clonality. Five of seven patients with BRAF mutations had a dramatic multinodular phenotype, with characteristic histology distinct from classical proliferative nodules. NRAS mutation was the commonest in all sizes of CMN, but was particularly common in naevi with PAS > 60 cm, implying more tolerance to that mutation early in embryogenesis. Facial features were less common in double wild‐type patients. Importantly, the incidence of congenital neurological disease, and apparently of melanoma, was not altered by genotype; no cases of melanoma were seen in BRAF‐mutant multiple CMN, however, this genotype is rare. Conclusions CMN of all sizes are most commonly caused by mutations in NRAS. BRAF is confirmed as a much rarer cause of multiple CMN, and appears to be commonly associated with a multinodular phenotype. Genotype in this cohort was not associated with differences in incidence of neurological disease in childhood. However, genotyping should be undertaken in suspected melanoma, for guidance of treatment. What's already known about this topic? Multiple congenital melanocytic naevi (CMN) have been shown to be caused by NRAS mosaic mutations in 70–80% of cases, by BRAF mosaicism in one case report and by inference in some previous cases.There has been debate about genotypic association with different sizes of CMN, and no data on genotype–outcome. What does this study add? NRAS mosaicism was found in 68%, BRAF in 7% and double wild‐type in 25% of cases of CMN. NRAS was the commonest mutation in all sizes of CMN, but was nearly universal in projected adult size > 60 cm. BRAF is often associated with a distinct multinodular clinical/histological phenotype.Adverse outcomes did not differ between genotypes on current numbers., https://doi.org/10.1111/bjd.18747 available online

In this study, we report three paediatric cases of Diffuse Glioneuronal Tumours with Oligodendroglioma-like features and Nuclear Clusters (DGONC).

Constitutional mismatch repair deficiency syndrome (CMMRD) is a rare cancer-predisposition syndrome associated with a high risk of developing a spectrum of malignancies in childhood and adolescence, including brain tumours. In this report, we present the case of an 8-year-old boy with acute headache, vomiting and an episode of unconsciousness in whom brain imaging revealed a high-grade glioma (HGG). The possibility of an underlying diagnosis of CMMRD was suspected radiologically on the basis of additional neuroimaging findings, specifically the presence of multiple supratentorial and infratentorial developmental venous anomalies (DVAs) and malformations of cortical development (MCD), namely, heterotopic grey matter. The tumour was debulked and confirmed to be a HGG on histopathology. The suspected diagnosis of CMMRD was confirmed on immunohistochemistry and genetic testing which revealed mutations in PMS2 and MSH6. The combination of a HGG, multiple DVAs and MCD in a paediatric or young adult patient should prompt the neuroradiologist to suggest an underlying diagnosis of CMMRD. A diagnosis of CMMRD has an important treatment and surveillance implications not only for the child but also the family in terms of genetic counselling.

Immune-therapy is an attractive alternative therapeutic approach for targeting central nervous system (CNS) tumors and the constituency of the Tumor Immune Microenvironment (TIME) likely to predict patient response. Here, we describe the TIME of >6000 primarily pediatric CNS tumors using a deconvolution approach (methylCIBERSORT). We produce and validate a custom reference signature defining 11 non-cancer cell types to estimate relative proportions of infiltration in a panCNS tumor cohort spanning 80 subtypes. We group patients into three broad immune clusters associated with CNS tumor types/subtypes. In cohorts of medulloblastomas (n = 2325), malignant rhabdoid tumors (n = 229) and pediatric high-grade gliomas (n = 401), we show significant associations with molecular subgroups/subtypes, mutations, and prognosis. We further identify tumor-specific immune clusters with phenotypic characteristics relevant to immunotherapy response (i.e. Cytolytic score, PDL1 expression). Our analysis provides an indication of the potential future therapeutic and prognostic possibilities of immuno-methylomic profiling in pediatric CNS tumor patients that may ultimately inform approach to immune-therapy., Here, using methylCIBERSORT, the authors characterize the tumour-immune microenvironment of paediatric central nervous system (CNS) tumours and its association with tumour type and prognosis. These findings suggest that immuno-methylomic profiling may inform immunotherapy approaches in paediatric patients with CNS tumour.

Immunoglobulin M (IgM) anti-myelin-associated glycoprotein (MAG) paraprotein-associated peripheral neuropathy (anti-MAG PN) is the most frequent type of paraprotein-associated neuropathy. It typically presents as a chronic demyelinating disorder with progressive ataxia, tremor and sensory disturbance.1 By definition, IgM paraproteinaemia and high-titre anti-MAG antibodies are present. Up to 50% of patients develop significant disability. Progressive disease-related disability is considered an indication to start treatment. However, there is no consensus on the optimal treatment approach and a high clinical need for effective therapies.1 IgM paraproteinaemia is the hallmark of Waldenstrom’s macroglobulinaemia (WM) and IgM monoclonal gammopathy of unknown significance (MGUS). WM is an indolent B-cell malignancy with lymphoplasmacytic differentiation typically localised in the bone marrow (BM), while IgM MGUS is considered a premalignant condition, defined as asymptomatic IgM paraproteinaemia with 90%) of patients with WM and approximately 50% of patients with IgM MGUS. The mutation is absent in healthy donors, multiple myeloma and non-IgM MGUS.3 MYD88 is an adaptor protein of the interleukin-1R and toll-like receptor signalling pathways that ultimately lead to activation of nuclear factor …

Background Central nervous system (CNS) tumours are the leading cause of childhood cancer deaths. There is little understanding of why tumours become untreatable but it is likely their biology changes over time and across space as they spread. One way to understand such advanced disease is to study the tumour biology at autopsy. We assembled a cohort of cases who had undergone autopsy for a childhood tumour. We assessed factors that could affect tumour biology in advanced disease and the feasibility of using archival post-mortem tissue with modern genomic technologies. Methods Cases were identified through BRAIN UK and local databases. Clinico-pathological data was analysed for local cases. Twenty-seven cases underwent DNA methylation array profiling and data was processed in R using the DKFZ CNS tumour classifier and Conumee package. Results We identified over 200 post-mortem paediatric CNS tumours across the UK. Among local cases (n=89), the median age was 3.8 years (range 0-31) and the median time between presentation and death was 4.5 months (range 0-144). At time of death, 46% of patients had not received cancer treatment compared to 54% who were receiving or had received treatment; 24% of patients were receiving palliative care. The cohort included a wide range of tumour types and origins. We had anticipated widespread dissemination in most cases but only in 68% of cases did the tumour infiltrate an area outside its origin. We could derive a confident diagnosis in 38% of cases using methylation data and in these, the molecular diagnosis matched the histology. Copy number changes could be interpreted in 81% of cases. Conclusions We established a diverse post-mortem paediatric CNS tumour cohort and demonstrated that both previously known and novel genomic aberrations could be identified within this tissue. Future work will investigate how advanced tumour biology changes in time and space.

Adamantinomatous craniopharyngiomas (aCPs) are complex epithelial neoplasms that arise from the progenitors of the pituitary gland. Although benign, these tumours can be locally aggressive invading vital neighbouring structures such as the hypothalamus, the cranial and optic nerves. Congenital forms of aCPs diagnosed during foetal development are very rare. The purpose of this article is to present with a histopathological and molecular characterisation of congenital craniopharyngioma. Here we report a case of in utero diagnosed aCP, detected at 21 weeks of gestation by ultrasound, visualised by MRI at 22 weeks and histologically diagnosed at 23 weeks. We provide with histopathological characterisation of rare form of congenital aCPs. Detailed examination of the tumour reveals the classical histological hallmarks of aCPs with the presence of stellate reticulum, palisading epithelium, wet keratin and calcification deposits. The tumour demonstrated complete absence of all pituitary hormones and the absence of the neuroendocrine marker, synaptophysin. Immunohistochemistry against β-catenin revealed occasional cells with nuclear-β-catenin localisation and the presence of pituitary progenitors positive for SOX9 and SOX2. Targeted Sanger sequencing revealed no genetic variants in oncogenes CTNNB1 and BRAF, previously associated with CP. In this article, we provide with in-depth molecular and histological characterisation of in utero aCP due to an unknown driving mutation that could represent a sub-cohort of congenital aCPs.

Intra-tumour heterogeneity is an important diagnostic, therapeutic and prognostic challenge. Its extent and mechanism in brain tumours is incompletely understood[1]. We describe a malignant tumour with unique pathological and genetic features. Most notably the tumour contained mutations in the SMARCB1 gene (typically associated with Atypical Teratoid/Rhabdoid Tumours[2]), the H3F3A gene (typically associated with high grade glioma in children[3]) and the BRAF gene. Furthermore, there was marked heterogeneity in mutation load between different parts of the tumour. This heterogeneity has implications both for the evolution of the tumour and for its diagnosis.

Here, we report a high incidence of PAX5 abnormalities observed in 32/68 (47%) of patients with genetically unclassified childhood precursor B-cell acute lymphoblastic leukaemia (pre-B ALL). Various deletions, gains, mutations and rearrangements of PAX5 comprised 45%, 12%, 29% and 14%, respectively, of the abnormalities found. 28% of patients showed more than one abnormality of the gene, implying bi-allelic impairment of PAX5. Novel PAX5-RHOXF2, PAX5-ELK3 and PAX5-CBFA2T2 rearrangements, which lead to aberrant expression of PAX5, were also identified. PAX5 rearrangements demonstrated a complex mechanism of formation including concurrent duplications/deletions of PAX5 and its partner genes. Finally, the splice variant c.1013-2A>G, seen in two patients with loss of one PAX5 allele, was confirmed to be germ-line in one patient and somatic in the other. PAX5 alterations were also found to be clinically associated with a higher white blood cell count (P = 0·015). These findings contribute to the knowledge of PAX5 alterations and their role in the pathogenesis of pre-B ALL.

The implementation of personalised medicine in childhood cancers has been limited by a lack of clinically validated multi-target sequencing approaches specific for paediatric solid tumours. In order to support innovative clinical trials in high-risk patients with unmet need, we have developed a clinically relevant targeted sequencing panel spanning 311 kb and comprising 78 genes involved in childhood cancers. A total of 132 samples were used for the validation of the panel, including Horizon Discovery cell blends (n=4), cell lines (n=15), formalin-fixed paraffin embedded (FFPE, n=83) and fresh frozen tissue (FF, n=30) patient samples. Cell blends containing known single nucleotide variants (SNVs, n=528) and small insertion-deletions (indels n=108) were used to define panel sensitivities of ≥98% for SNVs and ≥83% for indels [95% CI] and panel specificity of ≥98% [95% CI] for SNVs. FFPE samples performed comparably to FF samples (n=15 paired). Of 95 well-characterised genetic abnormalities in 33 clinical specimens and 13 cell lines (including SNVs, indels, amplifications, rearrangements and chromosome losses), 94 (98.9%) were detected by our approach. We have validated a robust and practical methodology to guide clinical management of children with solid tumours based on their molecular profiles. Our work demonstrates the value of targeted gene sequencing in the development of precision medicine strategies in paediatric oncology. We are enormously grateful to the Christopher's Smile charity (grant numbers CSM 002 and CSM 003)for their support and enthusiasm to provide children with more effective and less toxic targeted drugs through molecular profiling. We also thank the contribution of Children’s Cancer and Leukaemia Group (CCLG). Sí

Glioneuronal tumours are an important cause of treatment-resistant epilepsy. Subtypes of tumour are often poorly discriminated by histological features and may be difficult to diagnose due to a lack of robust diagnostic tools. This is illustrated by marked variability in the reported frequencies across different epilepsy surgical series. To address this, we used DNA methylation arrays and RNA sequencing to assay the methylation and expression profiles within a large cohort of glioneuronal tumours. By adopting a class discovery approach, we were able to identify two distinct groups of glioneuronal tumour, which only partially corresponded to the existing histological classification. Furthermore, by additional molecular analyses, we were able to identify pathogenic mutations in BRAF and FGFR1, specific to each group, in a high proportion of cases. Finally, by interrogating our expression data, we were able to show that each molecular group possessed expression phenotypes suggesting different cellular differentiation: astrocytic in one group and oligodendroglial in the second. Informed by this, we were able to identify CCND1, CSPG4, and PDGFRA as immunohistochemical targets which could distinguish between molecular groups. Our data suggest that the current histological classification of glioneuronal tumours does not adequately represent their underlying biology. Instead, we show that there are two molecular groups within glioneuronal tumours. The first of these displays astrocytic differentiation and is driven by BRAF mutations, while the second displays oligodendroglial differentiation and is driven by FGFR1 mutations. Electronic supplementary material The online version of this article (doi:10.1007/s00401-017-1773-z) contains supplementary material, which is available to authorized users.

Solid pseudopapillary neoplasm of the pancreas (SPNP) is a rare tumor with low malignant potential found in adolescent girls and young women. The pathogenesis of SPNP remains uncertain and its management is controversial. Genetic changes associated with SPNP have seldom been reported. We describe here the cytogenetic investigation of a case of SPNP in a 13-year-old girl whose tumor cells revealed two unrelated clones: one clone characterized by complex karyotypic changes, including breakpoints in two common fragile sites at chromosome 2, band q33, and chromosome 4, band q31, and the second clone defined by partial monosomy for chromosome X. Loss of heterozygosity for HRAS was also identified by array comparative genomic hybridization (a-CGH). These cumulative changes seem insufficient for activation of cell transformation, but could possibly play a role in priming the cell for future mutagenic events.

The t(12;21) is the commonest recurrent translocation in childhood acute lymphoblastic leukaemia (ALL), the presence of which has been suggested to be a good prognostic feature. We have studied 22 childhood cases of B-precursor ALL with this rearrangement, and have found no significant differences in event-free survival between these and a control group of patients with similar phenotypes. Using a variety of cytogenetic and molecular techniques, we have confirmed a strong association with co-expression of myeloid markers, frequent deletions of the short-arm of the untranslocated chromosome 12 homologue and duplication of the derivative chromosome 21. Intragenic deletion of the untranslocated ETV6 gene in 3/12 informative patients points to the likelihood of this gene being a target for deletion.

Congenital melanocytic nevi (CMN) can be associated with neurological abnormalities and an increased risk of melanoma. Mutations in NRAS, BRAF, and Tp53 have been described in individual CMN samples; however, their role in the pathogenesis of multiple CMN within the same subject and development of associated features has not been clear. We hypothesized that a single postzygotic mutation in NRAS could be responsible for multiple CMN in the same individual, as well as for melanocytic and nonmelanocytic central nervous system (CNS) lesions. From 15 patients, 55 samples with multiple CMN were sequenced after site-directed mutagenesis and enzymatic digestion of the wild-type allele. Oncogenic missense mutations in codon 61 of NRAS were found in affected neurological and cutaneous tissues of 12 out of 15 patients, but were absent from unaffected tissues and blood, consistent with NRAS mutation mosaicism. In 10 patients, the mutation was consistently c.181C>A, p.Q61K, and in 2 patients c.182A>G, p.Q61R. All 11 non-melanocytic and melanocytic CNS samples from 5 patients were mutation positive, despite NRAS rarely being reported as mutated in CNS tumors. Loss of heterozygosity was associated with the onset of melanoma in two cases, implying a multistep progression to malignancy. These results suggest that single postzygotic NRAS mutations are responsible for multiple CMN and associated neurological lesions in the majority of cases.

We report gene expression and other analyses to elucidate the molecular characteristics of acute lymphoblastic leukemia (ALL) in children with Down syndrome (DS). We find that by gene expression DS-ALL is a highly heterogeneous disease not definable as a unique entity. Nevertheless, 62% (33/53) of the DS-ALL samples analyzed were characterized by high expression of the type I cytokine receptor CRLF2 caused by either immunoglobulin heavy locus (IgH@) translocations or by interstitial deletions creating chimeric transcripts P2RY8-CRLF2. In 3 of these 33 patients, a novel activating somatic mutation, F232C in CRLF2, was identified. Consistent with our previous research, mutations in R683 of JAK2 were identified in 10 specimens (19% of the patients) and, interestingly, all 10 had high CRLF2 expression. Cytokine receptor-like factor 2 (CRLF2) and mutated Janus kinase 2 (Jak2) cooperated in conferring cytokine-independent growth to BaF3 pro-B cells. Intriguingly, the gene expression signature of DS-ALL is enriched with DNA damage and BCL6 responsive genes, suggesting the possibility of B-cell lymphocytic genomic instability. Thus, DS confers increased risk for genetically highly diverse ALLs with frequent overexpression of CRLF2, associated with activating mutations in the receptor itself or in JAK2. Our data also suggest that the majority of DS children with ALL may benefit from therapy blocking the CRLF2/JAK2 pathways.

Twenty-five cases of B-cell precursor acute lymphoblastic leukaemia (ALL) from Down syndrome (DS) patients were analyzed using array comparative genomic hybridization (aCGH) and compared with two other subgroups of non-DS patients with ALL; five cases with high-hyperdiploidy (HH) and nine cases with ETV6-RUNX1 positive clones. Seven cases of DS-acute megakaryoblastic leukaemia (AMKL) were also included, DS-ALL cases showed relatively stable karyotypes with cryptic losses and gains that most frequently involved chromosomes X, 1, 2, 9, 11, 16, and 17. The most consistent change involved a deletion in 2p, spanning region Chr2:88273220-91084234, which in some cases appeared to be homozygous. ALL from non-DS patients showed a similar overall karyotypic stability, although gains of chromosome 21 were infrequent in the ETV6-RUNX1 positive cases. The most consistent change in this group involved a 12p deletion, where Chr12:10383878-16017619 defined the common region of overlap. All HH-ALL karyotypes showed variable gains of chromosome 21. This overall analysis supports the suggestion that, although constitutional trisomy 21 predisposes to ALL/AMKL, the cytogenetic changes associated with DS-ALL in particular, are most similar to those found in non-DS ETV6-RUNX1 positive ALL. The HH-ALL group, however, undergoes distinct karyotypic evolution not dependent on chromosome translocation/deletion events.

INTRODUCTION: Characteristic patterns of the DNA methylation are key features in various tumours. A promising tool for DNA methylation studies is the Illumina Infinium 450k array that assesses the DNA methylation status of 450,000 individual CpG sites. An algorithm, developed in Heidelberg, is based on more than 2000 reference brain tumours and is able to distinguish brain tumour entities based on their methylation profile. The aim is to improve the diagnostic accuracy, important for paediatric tumours, and useful for entities with unusual morphology or clinical course. METHOD: We have set up a diagnostic pipeline and have performed a trial run on a set of morphologically well-characterized paediatric and adult brain tumours and of a set of complex, unresolved tumour cases. Unprocessed idat-files of Illumina Infinium Human Methylation 450 BeadChips are uploaded to the Heidelberg v9 classifier. This tool provides a molecular classification, based on the comparison to the reference set as well as a low resolution copy number plot from the array data for assessment of deletions, including 1p19q codeletion and amplifications, and provides reliable information on the MGMT promoter methylation status. RESULTS: We present the feasibility of setting up a 450k array platform in a diagnostic setting including validation steps and interpretation of the classifier results of rare entities and approaches to refining or revising histological diagnosis. CONCLUSION: The molecular classification by Heidelberg classifier is as yet a research tool, but it does show a great potential for improving diagnostic accuracy, risk stratification and a step closer to improved patient-tailored therapy

Abstract 11 Children with Down's Syndrome (DS) have an increased risk for acute lymphoblastic leukemia (ALL). We have previously identified somatic mutations in R683 of JAK2 in 20% of DS-ALLs (Bercovich et al, Lancet 2008;372:1484). However the cooperating cytokine receptor and other molecular features have been obscured. Here we report that gene expression analysis reveals that DS-ALL is a highly heterogeneous disease not definable as unique ALL subtype. However, 62% (33 of 53) of the DS-ALL samples analyzed were characterized by aberrant expression of the type I cytokine receptor CRLF2 caused by two types of genomic aberrations. CRLF2 encodes one chain of the heterodimeric receptor of the TSLP cytokine involved in inflammation and lymphoid development and has been recently reported to be involved in translocations to the IgH locus or micro-deletions in 3% of ALLs in children without DS. We have confirmed its cell surface expression on primary DS-ALL cells by Flow cytometry analysis. Consisting with a possible role of CRLF2 as a cytokine receptor, all specimens with the R683 mutated JAK2 had high CRLF2 expression. We further show that CRLF2 and R683 mutated JAK2 cooperate in conferring cytokine independent growth of pro-B cells. Furthermore in 3 of 23 DS-ALL patients with wt. JAK2 we identified a novel activating somatic mutation in CRLF2 replacing the juxta-membrane F232 with Cystein. Mutated CRLF2 but not wild type CRLF2 conferred cytokine independent growth of BaF3 cells associated with phosphorylation of STAT5. Aberrant expression of CRLF2 was also associated with a younger age at diagnosis and worse prognosis. Intriguingly, bioinformatic analysis revealed that gene expression of DS-ALLs is enriched with DNA damage and BCL6 responsive genes suggesting the possibility that DS is associated with B-cell lymphocytic genomic instability leading to the genomic aberrations activating CRLF2. Thus DS confers increased risk for genetically highly diverse ALLs with frequent somatic anomaly in CRLF2, whose higher expression further selects for the activating mutations in itself or in JAK2 (figure). Our data also suggests that the majority of DS children with ALL may benefit from therapy blocking the CRLF2/JAK2 pathway. Disclosures: No relevant conflicts of interest to declare.