Guiding the global evolution of cytogenetic testing for hematologic malignancies.

Cytogenetics has long represented a critical component in the clinical evaluation of hematologic malignancies. Chromosome banding studies provide a simultaneous snapshot of genome-wide copy number and structural variation, which have been shown to drive tumorigenesis, define diseases, and guide treatment. Technological innovations in sequencing have ushered in our present-day clinical genomics era. With recent publications highlighting novel sequencing technologies as alternatives to conventional cytogenetic approaches, we, an international consortium of laboratory geneticists, pathologists, and oncologists, describe herein the advantages and limitations of both conventional chromosome banding and novel sequencing technologies and share our considerations on crucial next steps to implement these novel technologies in the global clinical setting for a more accurate cytogenetic evaluation, which may provide improved diagnosis and treatment management. Considering the clinical, logistic, technical, and financial implications, we provide points to consider for the global evolution of cytogenetic testing.

Quantification of aneuploidy in targeted sequencing data using ASCETS.

SUMMARY: The expansion of targeted panel sequencing efforts has created opportunities for large-scale genomic analysis, but tools for copy-number quantification on panel data are lacking. We introduce ASCETS, a method for the efficient quantitation of arm and chromosome-level copy-number changes from targeted sequencing data. AVAILABILITY AND IMPLEMENTATION: ASCETS is implemented in R and is freely available to non-commercial users on GitHub: https://github.com/beroukhim-lab/ascets, along with detailed documentation. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Re-evaluating tumors of purported specialized prostatic stromal origin reveals molecular heterogeneity, including non-recurring gene fusions characteristic of uterine and soft tissue sarcoma subtypes.

Tumors of purported specialized prostatic stromal origin comprise prostatic stromal sarcomas (PSS) and stromal tumors of uncertain malignant potential (STUMP). Prior studies have described their clinicopathologic characteristics, but the molecular features remain incompletely understood. Moreover, these neoplasms are morphologically heterogeneous and the lack of specific adjunctive markers of prostatic stromal lineage make precise definition more difficult, leading some to question whether they represent a specific tumor type. In this study, we used next-generation DNA and RNA sequencing to profile 25 primary prostatic mesenchymal neoplasms of possible specialized prostatic stromal origin, including cases originally diagnosed as PSS (11) and STUMP (14). Morphologically, the series comprised 20 cases with solid architecture (11 PSS and 9 STUMP) and 5 cases with phyllodes-like growth pattern (all STUMP). Combined DNA and RNA sequencing results demonstrated that 19/22 (86%) cases that underwent successful sequencing (either DNA or RNA) harbored pathogenic somatic variants. Except for TP53 alterations (6 cases), ATRX mutations (2 cases), and a few copy number variants (-13q, -14q, -16q and +8/8p), the findings were largely nonrecurrent. Eight gene rearrangements were found, and 4 (NAB2-STAT6, JAZF1-SUZ12, TPM3-NTRK1 and BCOR-MAML3) were useful for reclassification of the cases as specific entities. The present study shows that mesenchymal neoplasms of the prostate are morphologically and molecularly heterogeneous and include neoplasms that harbor genetic aberrations seen in specific mesenchymal tumors arising in other anatomic sites, including soft tissue and the uterus. These data suggest that tumors of purported specialized prostatic stromal origin may perhaps not represent a single diagnostic entity or specific disease group and that alternative diagnoses should be carefully considered.

Correlation of methylthioadenosine phosphorylase (MTAP) protein expression with MTAP and CDKN2A copy number in malignant pleural mesothelioma.

AIMS: Methylthioadenosine phosphorylase (MTAP) immunohistochemical expression is a specific marker of CDKN2A deletion in malignant mesothelioma. However, the relationship of MTAP expression with MTAP copy number remains unexplored. METHODS AND RESULTS: Forty malignant pleural mesotheliomas were characterised by targeted next-generation sequencing (29), single-nucleotide polymorphism microarray (seven), or both (four). MTAP and CDKN2A copy numbers were correlated with MTAP expression. Twenty-seven (68%) tumours showed CDKN2A deletion (14 heterozygous; 13 homozygous), of which 20 (74%) showed MTAP codeletion (15 heterozygous; five homozygous). No tumours showed MTAP deletion without CDKN2A codeletion. Loss of MTAP expression was seen in 16 (40%) tumours, and was 75% sensitive and 95% specific for MTAP deletion, and 59% sensitive and 100% specific for CDKN2A deletion. Nine of 40 (23%) tumours showed heterogeneous MTAP staining, and the percentage of tumour cells with MTAP loss correlated with molecular detection of MTAP deletion. CONCLUSIONS: MTAP is frequently codeleted with CDKN2A in pleural mesothelioma. However, homozygous deletion of both genes occurs in a minority of tumours (5/40; 13%); CDKN2A deletion often co-occurs with heterozygous MTAP deletion or neutral MTAP copy number; and MTAP expression correlates inconsistently with heterozygous MTAP deletion. Correspondingly, MTAP immunohistochemistry is a highly specific but only moderately sensitive assay for CDKN2A deletion.

Characterization of molecular signatures of supratentorial ependymomas.

Ependymomas show poor correlation between World Health Organization grade and clinical outcome. A subgroup of supratentorial ependymomas are characterized by C11orf95-RELA fusions, presumed to be secondary to chromothripsis of chromosome 11, resulting in constitutive activation of the NF-κB signaling pathway and overexpression of cyclin D1, p65, and L1 cell adhesion molecule (L1CAM). These RELA-fused ependymomas are recognized as a separate, molecularly defined World Health Organization entity and might be associated with poor clinical outcome. In this study, we show that immunohistochemistry for NF-κB signaling components, such as L1CAM, p65, and cyclin D1, can help distinguish RELA-fused from non-RELA-fused supratentorial ependymomas. Furthermore, these three markers can reliably differentiate RELA-fused ependymomas from a variety of histologic mimics. Lastly, we report that RELA-fused ependymomas may be associated with different chromosomal copy number changes and molecular alterations compared to their non-RELA-fused counterparts, providing additional insight into the genetic pathogenesis of these tumors and potential targets for directed therapies.

Molecular characterization of localized pleural mesothelioma.

Localized pleural mesothelioma is a rare solitary circumscribed pleural tumor that is microscopically similar to diffuse malignant pleural mesothelioma. However, the molecular characteristics and nosologic relationship with its diffuse counterpart remain unknown. In a consecutive cohort of 1110 patients with pleural mesotheliomas diagnosed in 2005-2018, we identified six (0.5%) patients diagnosed with localized pleural mesotheliomas. We gathered clinical history, evaluated the histopathology, and in select cases performed karyotypic analysis and targeted next-generation sequencing. The cohort included three women and three men (median age 63; range 28-76), often presenting incidentally during radiologic evaluation for unrelated conditions. Neoadjuvant chemotherapy was administered in two patients. All tumors (median size 5.0 cm; range 2.7-13.5 cm) demonstrated gross circumscription (with microscopic invasion into lung, soft tissue, and/or rib in four cases), mesothelioma histology (four biphasic and two epithelioid types), and mesothelial immunophenotype. Of four patients with at least 6-month follow-up, three were alive (up to 8.9 years). Genomic characterization identified several subgroups: (1) BAP1 mutations with deletions of CDKN2A and NF2 in two tumors; (2) TRAF7 mutations in two tumors, including one harboring trisomies of chromosomes 3, 5, 7, and X; and (3) genomic near-haploidization, characterized by extensive loss of heterozygosity sparing chromosomes 5 and 7. Localized pleural mesotheliomas appear genetically heterogeneous and include BAP1-mutated, TRAF7-mutated, and near-haploid subgroups. While the BAP1-mutated subgroup is similar to diffuse malignant pleural mesotheliomas, the TRAF7-mutated subgroup overlaps genetically with adenomatoid tumors and well-differentiated papillary mesotheliomas, in which recurrent TRAF7 mutations have been described. Genomic near-haploidization, identified recently in a subset of diffuse malignant pleural mesotheliomas, suggests a novel mechanism in the pathogenesis of both localized pleural mesothelioma and diffuse malignant pleural mesothelioma. Our findings describe distinctive genetic features of localized pleural mesothelioma, with both similarities to and differences from diffuse malignant pleural mesothelioma.

Voltage-gated potassium channel EAG2 controls mitotic entry and tumor growth in medulloblastoma via regulating cell volume dynamics.

Medulloblastoma (MB) is the most common pediatric CNS malignancy. We identify EAG2 as an overexpressed potassium channel in MBs across different molecular and histological subgroups. EAG2 knockdown not only impairs MB cell growth in vitro, but also reduces tumor burden in vivo and enhances survival in xenograft studies. Mechanistically, we demonstrate that EAG2 protein is confined intracellularly during interphase but is enriched in the plasma membrane during late G2 phase and mitosis. Disruption of EAG2 expression results in G2 arrest and mitotic catastrophe associated with failure of premitotic cytoplasmic condensation. While the tumor suppression function of EAG2 knockdown is independent of p53 activation, DNA damage checkpoint activation, or changes in the AKT pathway, this defective cell volume control is specifically associated with hyperactivation of the p38 MAPK pathway. Inhibition of the p38 pathway significantly rescues the growth defect and G2 arrest. Strikingly, ectopic membrane expression of EAG2 in cells at interphase results in cell volume reduction and mitotic-like morphology. Our study establishes the functional significance of EAG2 in promoting MB tumor progression via regulating cell volume dynamics, the perturbation of which activates the tumor suppressor p38 MAPK pathway, and provides clinical relevance for targeting this ion channel in human MBs.

Technical laboratory standards for interpretation and reporting of acquired copy-number abnormalities and copy-neutral loss of heterozygosity in neoplastic disorders: a joint consensus recommendation from the American College of Medical Genetics and Genomics (ACMG) and the Cancer Genomics Consortium (CGC).

The detection of acquired copy-number abnormalities (CNAs) and copy-neutral loss of heterozygosity (CN-LOH) in neoplastic disorders by chromosomal microarray analysis (CMA) has significantly increased over the past few years with respect to both the number of laboratories utilizing this technology and the broader number of tumor types being assayed. This highlights the importance of standardizing the interpretation and reporting of acquired variants among laboratories. To address this need, a clinical laboratory-focused workgroup was established to draft recommendations for the interpretation and reporting of acquired CNAs and CN-LOH in neoplastic disorders. This project is a collaboration between the American College of Medical Genetics and Genomics (ACMG) and the Cancer Genomics Consortium (CGC). The recommendations put forth by the workgroup are based on literature review, empirical data, and expert consensus of the workgroup members. A four-tier evidence-based categorization system for acquired CNAs and CN-LOH was developed, which is based on the level of available evidence regarding their diagnostic, prognostic, and therapeutic relevance: tier 1, variants with strong clinical significance; tier 2, variants with some clinical significance; tier 3, clonal variants with no documented neoplastic disease association; and tier 4, benign or likely benign variants. These recommendations also provide a list of standardized definitions of terms used in the reporting of CMA findings, as well as a framework for the clinical reporting of acquired CNAs and CN-LOH, and recommendations for how to deal with suspected clinically significant germline variants.

Yield of additional genetic testing after chromosomal microarray for diagnosis of neurodevelopmental disability and congenital anomalies: a clinical practice resource of the American College of Medical Genetics and Genomics (ACMG).

PURPOSE: Chromosomal microarray (CMA) is recommended as the first-tier test in evaluation of individuals with neurodevelopmental disability and congenital anomalies. CMA may not detect balanced cytogenomic abnormalities or uniparental disomy (UPD), and deletion/duplications and regions of homozygosity may require additional testing to clarify the mechanism and inform accurate counseling. We conducted an evidence review to synthesize data regarding the benefit of additional testing after CMA to inform a genetic diagnosis. METHODS: The review was guided by key questions related to the detection of genomic events that may require additional testing. A PubMed search for original research articles, systematic reviews, and meta-analyses was evaluated from articles published between 1 January 1983 and 31 March 2017. Based on the key questions, articles were retrieved and data extracted in parallel with comparison of results and discussion to resolve discrepancies. Variables assessed included study design and outcomes. RESULTS: A narrative synthesis was created for each question to describe the occurrence of, and clinical significance of, additional diagnostic findings from subsequent testing performed after CMA. CONCLUSION: These findings may be used to assist the laboratory and clinician when making recommendations about additional testing after CMA, as it impacts clinical care, counseling, and diagnosis.

Somatic cell transfer of c-Myc and Bcl-2 induces large-cell anaplastic medulloblastomas in mice.

A highly aggressive subgroup of the pediatric brain tumor medulloblastoma is characterized by overexpression of the proto-oncogene c-Myc, which encodes a transcription factor that normally maintains neural progenitor cells in an undifferentiated, proliferating state during embryonic development. Myc-driven medulloblastomas typically show a large-cell anaplastic (LCA) histological pattern, in which tumor cells display large, round nuclei with prominent nucleoli. This subgroup of medulloblastoma is therapeutically challenging because it is associated with a high rate of metastatic dissemination, which is a powerful predictor of short patient survival times. Genetically engineered mouse models have revealed important insights into the pathogenesis of medulloblastoma and served as preclinical testing platforms for new therapies. Here we report a new mouse model of Myc-driven medulloblastoma, in which tumors arise in situ after retroviral transfer and expression of Myc in Nestin-expressing neural progenitor cells in the cerebella of newborn mice. Tumor induction required concomitant loss of Tp53 or overexpression of the antiapoptotic protein Bcl-2. Like Myc-driven medulloblastomas in humans, the tumors induced in mice by Myc + Bcl-2 and Myc - Tp53 showed LCA cytoarchitecture and a high rate of metastatic dissemination to the spine. The fact that Myc - Tp53 tumors arose only in Tp53(-/-) mice, coupled with the inefficient germline transmission of the Tp53-null allele, made retroviral transfer of Myc + Bcl-2 a more practical method for generating LCA medulloblastomas. The high rate of spinal metastasis (87% of brain tumor-bearing mice) will be an asset for testing new therapies that target the most lethal aspect of medulloblastoma.

FISHing in the dark: How the combination of FISH and conventional karyotyping improves the diagnostic yield in CpG-stimulated chronic lymphocytic leukemia.

Despite significant advances in molecular genetic approaches, fluorescence in situ hybridization (FISH) remains the gold standard for the diagnostic evaluation of genomic aberrations in patients with chronic lymphocytic leukemia (CLL). Efforts to improve the diagnostic utility of molecular cytogenetic testing have led to the expansion of the traditional 4-probe CLL FISH panel. Not only do these efforts increase the cost of testing, they remain hindered by the inherent limitations of FISH studies - namely the inability to evaluate genomic changes outside of the targeted loci. While array-based profiling and next generation sequencing (NGS) have critically expanded our understanding of the molecular pathogenesis of CLL, these methodologies are not routinely used by diagnostic laboratories to evaluate copy number changes or the mutational profile of this disease. Mitogenic stimulation of CLL specimens with CpG-oligonucleotide (CpG-ODN) has been identified as a reliable and reproducible means of obtaining a karyotype, facilitating a low-resolution genome-wide analysis. Across a cohort of 1255 CpG-ODN-stimulated CLL specimens, we describe the clinical utility associated with the combinatorial use of FISH and karyotyping. Our testing algorithm achieves a higher diagnostic yield (∼10%) through the detection of complex karyotypes, well-characterized chromosomal aberrations not covered by the traditional CLL FISH panel and through the detection of concurrent secondary malignancies. Moreover, the single cell nature of this approach permits the evaluation of emerging new clinical concepts including clonal dynamics and clonal evolution. This approach can be broadly applied by diagnostic laboratories to improve the utility of traditional and molecular cytogenetic studies of CLL. Am. J. Hematol. 91:978-983, 2016. © 2016 Wiley Periodicals, Inc.

Medulloblastoma subgroups remain stable across primary and metastatic compartments.

Medulloblastoma comprises four distinct molecular variants with distinct genetics, transcriptomes, and outcomes. Subgroup affiliation has been previously shown to remain stable at the time of recurrence, which likely reflects their distinct cells of origin. However, a therapeutically relevant question that remains unanswered is subgroup stability in the metastatic compartment. We assembled a cohort of 12-paired primary-metastatic tumors collected in the MAGIC consortium, and established their molecular subgroup affiliation by performing integrative gene expression and DNA methylation analysis. Frozen tissues were collected and profiled using Affymetrix gene expression arrays and Illumina methylation arrays. Class prediction and hierarchical clustering were performed using existing published datasets. Our molecular analysis, using consensus integrative genomic data, establishes the unequivocal maintenance of molecular subgroup affiliation in metastatic medulloblastoma. We further validated these findings by interrogating a non-overlapping cohort of 19 pairs of primary-metastatic tumors from the Burdenko Neurosurgical Institute using an orthogonal technique of immunohistochemical staining. This investigation represents the largest reported primary-metastatic paired cohort profiled to date and provides a unique opportunity to evaluate subgroup-specific molecular aberrations within the metastatic compartment. Our findings further support the hypothesis that medulloblastoma subgroups arise from distinct cells of origin, which are carried forward from ontogeny to oncology.

Integrated genomic analysis identifies the mitotic checkpoint kinase WEE1 as a novel therapeutic target in medulloblastoma.

BACKGROUND: Medulloblastoma is the most common type of malignant brain tumor that afflicts children. Although recent advances in chemotherapy and radiation have improved outcomes, high-risk patients do poorly with significant morbidity. METHODS: To identify new molecular targets, we performed an integrated genomic analysis using structural and functional methods. Gene expression profiling in 16 medulloblastoma patient samples and subsequent gene set enrichment analysis indicated that cell cycle-related kinases were associated with disease development. In addition a kinome-wide small interfering RNA (siRNA) screen was performed to identify kinases that, when inhibited, could prevent cell proliferation. The two genome-scale analyses were combined to identify key vulnerabilities in medulloblastoma. The inhibition of one of the identified targets was further investigated using RNAi and a small molecule inhibitor. RESULTS: Combining the two analyses revealed that mitosis-related kinases were critical determinants of medulloblastoma cell proliferation. RNA interference (RNAi)-mediated knockdown of WEE1 kinase and other mitotic kinases was sufficient to reduce medulloblastoma cell proliferation. These data prompted us to examine the effects of inhibiting WEE1 by RNAi and by a small molecule inhibitor of WEE1, MK-1775, in medulloblastoma cell lines. MK-1775 inhibited the growth of medulloblastoma cell lines, induced apoptosis and increased DNA damage at nanomolar concentrations. Further, MK-1775 was synergistic with cisplatin in reducing medulloblastoma cell proliferation and resulted in an associated increase in cell death. In vivo MK-1775 suppressed medulloblastoma tumor growth as a single agent. CONCLUSIONS: Taken together, these findings highlight mitotic kinases and, in particular, WEE1 as a rational therapeutic target for medulloblastoma.

Recurrence patterns across medulloblastoma subgroups: an integrated clinical and molecular analysis.

BACKGROUND: Recurrent medulloblastoma is a therapeutic challenge because it is almost always fatal. Studies have confirmed that medulloblastoma consists of at least four distinct subgroups. We sought to delineate subgroup-specific differences in medulloblastoma recurrence patterns. METHODS: We retrospectively identified a discovery cohort of all recurrent medulloblastomas at the Hospital for Sick Children (Toronto, ON, Canada) from 1994 to 2012 (cohort 1), and established molecular subgroups using a nanoString-based assay on formalin-fixed paraffin-embedded tissues or frozen tissue. The anatomical site of recurrence (local tumour bed or leptomeningeal metastasis), time to recurrence, and survival after recurrence were assessed in a subgroup-specific manner. Two independent, non-overlapping cohorts (cohort 2: samples from patients with recurrent medulloblastomas from 13 centres worldwide, obtained between 1991 and 2012; cohort 3: samples from patients with recurrent medulloblastoma obtained at the NN Burdenko Neurosurgical Institute [Moscow, Russia] between 1994 and 2011) were analysed to confirm and validate observations. When possible, molecular subgrouping was done on tissue obtained from both the initial surgery and at recurrence. RESULTS: Cohort 1 consisted of 30 patients with recurrent medulloblastomas; nine with local recurrences, and 21 with metastatic recurrences. Cohort 2 consisted of 77 patients and cohort 3 of 96 patients with recurrent medulloblastoma. Subgroup affiliation remained stable at recurrence in all 34 cases with available matched primary and recurrent pairs (five pairs from cohort 1 and 29 pairs from cohort 2 [15 SHH, five group 3, 14 group 4]). This finding was validated in 17 pairs from cohort 3. When analysed in a subgroup-specific manner, local recurrences in cohort 1 were more frequent in SHH tumours (eight of nine [89%]) and metastatic recurrences were more common in group 3 and group 4 tumours (17 of 20 [85%] with one WNT, p=0·0014, local vs metastatic recurrence, SHH vs group 3 vs group 4). The subgroup-specific location of recurrence was confirmed in cohort 2 (p=0·0013 for local vs metastatic recurrence, SHH vs group 3 vs group 4,), and cohort 3 (p<0·0001). Treatment with craniospinal irradiation at diagnosis was not significantly associated with the anatomical pattern of recurrence. Survival after recurrence was significantly longer in patients with group 4 tumours in cohort 1 (p=0·013) than with other subgroups, which was confirmed in cohort 2 (p=0·0075), but not cohort 3 (p=0·70). INTERPRETATION: Medulloblastoma does not change subgroup at the time of recurrence, reinforcing the stability of the four main medulloblastoma subgroups. Significant differences in the location and timing of recurrence across medulloblastoma subgroups have potential treatment ramifications. Specifically, intensified local (posterior fossa) therapy should be tested in the initial treatment of patients with SHH tumours. Refinement of therapy for patients with group 3 or group 4 tumours should focus on metastases.

Multisite Evaluation and Validation of Optical Genome Mapping for Prenatal Genetic Testing.

Prenatal diagnostic testing of amniotic fluid, chorionic villi, or more rarely, fetal cord blood is recommended following a positive or unreportable noninvasive cell-free fetal DNA test, abnormal maternal biochemical serum screen, abnormal ultrasound, or increased genetic risk for a cytogenomic abnormality based on family history. Although chromosomal microarray is recommended as the first-tier prenatal diagnostic test, in practice, multiple assays are often assessed in concert to achieve a final diagnostic result. The use of multiple methodologies is costly, time consuming, and labor intensive. Optical genome mapping (OGM) is an emerging technique with application for prenatal diagnosis because of its ability to detect and resolve, in a single assay, all classes of pathogenic cytogenomic aberrations. In an effort to characterize the potential of OGM as a novel alternative to traditional standard of care (SOC) testing of prenatal samples, OGM was performed on a total of 200 samples representing 123 unique cases, which were previously tested with SOC methods (92/123 = 74.7% cases tested with at least two SOCs). OGM demonstrated an overall accuracy of 99.6% when compared with SOC methods, a positive predictive value of 100%, and 100% reproducibility between sites, operators, and instruments. The standardized workflow, cost-effectiveness, and high-resolution cytogenomic analysis demonstrate the potential of OGM to serve as a first-tier test for prenatal diagnosis.

The RNA-binding protein Musashi1 affects medulloblastoma growth via a network of cancer-related genes and is an indicator of poor prognosis.

Musashi1 (Msi1) is a highly conserved RNA-binding protein that is required during the development of the nervous system. Msi1 has been characterized as a stem cell marker, controlling the balance between self-renewal and differentiation, and has also been implicated in tumorigenesis, being highly expressed in multiple tumor types. We analyzed Msi1 expression in a large cohort of medulloblastoma samples and found that Msi1 is highly expressed in tumor tissue compared with normal cerebellum. Notably, high Msi1 expression levels proved to be a sign of poor prognosis. Msi1 expression was determined to be particularly high in molecular subgroups 3 and 4 of medulloblastoma. We determined that Msi1 is required for tumorigenesis because inhibition of Msi1 expression by small-interfering RNAs reduced the growth of Daoy medulloblastoma cells in xenografts. To characterize the participation of Msi1 in medulloblastoma, we conducted different high-throughput analyses. Ribonucleoprotein immunoprecipitation followed by microarray analysis (RIP-chip) was used to identify mRNA species preferentially associated with Msi1 protein in Daoy cells. We also used cluster analysis to identify genes with similar or opposite expression patterns to Msi1 in our medulloblastoma cohort. A network study identified RAC1, CTGF, SDCBP, SRC, PRL, and SHC1 as major nodes of an Msi1-associated network. Our results suggest that Msi1 functions as a regulator of multiple processes in medulloblastoma formation and could become an important therapeutic target.

Aberrant patterns of H3K4 and H3K27 histone lysine methylation occur across subgroups in medulloblastoma.

Recent sequencing efforts have described the mutational landscape of the pediatric brain tumor medulloblastoma. Although MLL2 is among the most frequent somatic single nucleotide variants (SNV), the clinical and biological significance of these mutations remains uncharacterized. Through targeted re-sequencing, we identified mutations of MLL2 in 8 % (14/175) of MBs, the majority of which were loss of function. Notably, we also report mutations affecting the MLL2-binding partner KDM6A, in 4 % (7/175) of tumors. While MLL2 mutations were independent of age, gender, histological subtype, M-stage or molecular subgroup, KDM6A mutations were most commonly identified in Group 4 MBs, and were mutually exclusive with MLL2 mutations. Immunohistochemical staining for H3K4me3 and H3K27me3, the chromatin effectors of MLL2 and KDM6A activity, respectively, demonstrated alterations of the histone code in 24 % (53/220) of MBs across all subgroups. Correlating these MLL2- and KDM6A-driven histone marks with prognosis, we identified populations of MB with improved (K4+/K27-) and dismal (K4-/K27-) outcomes, observed primarily within Group 3 and 4 MBs. Group 3 and 4 MBs demonstrate somatic copy number aberrations, and transcriptional profiles that converge on modifiers of H3K27-methylation (EZH2, KDM6A, KDM6B), leading to silencing of PRC2-target genes. As PRC2-mediated aberrant methylation of H3K27 has recently been targeted for therapy in other diseases, it represents an actionable target for a substantial percentage of medulloblastoma patients with aggressive forms of the disease.

TERT promoter mutations are highly recurrent in SHH subgroup medulloblastoma.

Telomerase reverse transcriptase (TERT) promoter mutations were recently shown to drive telomerase activity in various cancer types, including medulloblastoma. However, the clinical and biological implications of TERT mutations in medulloblastoma have not been described. Hence, we sought to describe these mutations and their impact in a subgroup-specific manner. We analyzed the TERT promoter by direct sequencing and genotyping in 466 medulloblastomas. The mutational distributions were determined according to subgroup affiliation, demographics, and clinical, prognostic, and molecular features. Integrated genomics approaches were used to identify specific somatic copy number alterations in TERT promoter-mutated and wild-type tumors. Overall, TERT promoter mutations were identified in 21 % of medulloblastomas. Strikingly, the highest frequencies of TERT mutations were observed in SHH (83 %; 55/66) and WNT (31 %; 4/13) medulloblastomas derived from adult patients. Group 3 and Group 4 harbored this alteration in <5 % of cases and showed no association with increased patient age. The prognostic implications of these mutations were highly subgroup-specific. TERT mutations identified a subset with good and poor prognosis in SHH and Group 4 tumors, respectively. Monosomy 6 was mostly restricted to WNT tumors without TERT mutations. Hallmark SHH focal copy number aberrations and chromosome 10q deletion were mutually exclusive with TERT mutations within SHH tumors. TERT promoter mutations are the most common recurrent somatic point mutation in medulloblastoma, and are very highly enriched in adult SHH and WNT tumors. TERT mutations define a subset of SHH medulloblastoma with distinct demographics, cytogenetics, and outcomes.

Subgroup-specific alternative splicing in medulloblastoma.

Medulloblastoma comprises four distinct molecular variants: WNT, SHH, Group 3, and Group 4. We analyzed alternative splicing usage in 14 normal cerebellar samples and 103 medulloblastomas of known subgroup. Medulloblastoma samples have a statistically significant increase in alternative splicing as compared to normal fetal cerebella (2.3-times; P < 6.47E-8). Splicing patterns are distinct and specific between molecular subgroups. Unsupervised hierarchical clustering of alternative splicing events accurately assigns medulloblastomas to their correct subgroup. Subgroup-specific splicing and alternative promoter usage was most prevalent in Group 3 (19.4%) and SHH (16.2%) medulloblastomas, while observed less frequently in WNT (3.2%), and Group 4 (9.3%) tumors. Functional annotation of alternatively spliced genes reveals overrepresentation of genes important for neuronal development. Alternative splicing events in medulloblastoma may be regulated in part by the correlative expression of antisense transcripts, suggesting a possible mechanism affecting subgroup-specific alternative splicing. Our results identify additional candidate markers for medulloblastoma subgroup affiliation, further support the existence of distinct subgroups of the disease, and demonstrate an additional level of transcriptional heterogeneity between medulloblastoma subgroups.

Copy Number Analysis in Cancer Diagnostic Testing.

Accurate detection of copy number alterations (CNAs) has become increasingly important in clinical oncology for the purpose of diagnosis, prognostication, and disease management. Cytogenetic approaches for the detection of CNAs, including karyotype, fluorescence in situ hybridization (FISH), and chromosomal microarray, remain mainstays in clinical laboratories. Yet, with rapidly decreasing costs and improved accuracy of CNA detection using emerging technologies such as next-generation sequencing and optical genome mapping, we are approaching a new era of cytogenomics and molecular oncology. The aim of this review is to describe the benefits and limitations associated with the routine clinical application of available classic, emerging, and projected future technologies for the detection of CNAs in oncology.