Distinct Molecular Variants Research Articles

Carvajal syndrome related to two distinct molecular variants in desmoplakin gene.

Carvajal syndrome related to two distinct molecular variants in desmoplakin gene.

Mass Spectrometry-Based Top-Down Proteomics in Nanomedicine: Proteoform-Specific Measurement of Protein Corona.

Conventional mass spectrometry (MS)-based bottom-up proteomics (BUP) analysis of the protein corona [i.e., an evolving layer of biomolecules, mostly proteins, formed on the surface of nanoparticles (NPs) during their interactions with biomolecular fluids] enabled the nanomedicine community to partly identify the biological identity of NPs. Such an approach, however, fails to pinpoint the specific proteoforms─distinct molecular variants of proteins in the protein corona. The proteoform-level information could potentially advance the prediction of the biological fate and pharmacokinetics of nanomedicines. Recognizing this limitation, this study pioneers a robust and reproducible MS-based top-down proteomics (TDP) technique for characterizing proteoforms in the protein corona. Our TDP approach has successfully identified about 900 proteoforms in the protein corona of polystyrene NPs, ranging from 2 to 70 kDa, revealing proteoforms of 48 protein biomarkers with combinations of post-translational modifications, signal peptide cleavages, and/or truncations─details that BUP could not fully discern. This advancement in MS-based TDP offers a more advanced approach to characterize NP protein coronas, deepening our understanding of NPs' biological identities. We, therefore, propose using both TDP and BUP strategies to obtain more comprehensive information about the protein corona, which, in turn, can further enhance the diagnostic and therapeutic efficacy of nanomedicine technologies.

Gynura japonica: A new host of Apple stem grooving virus and Chrysanthemum virus B in China.

Gynura japonica (Thunb.) Juel [Asteraceae; syn: G. segetum (Lour.) Merr] is an important perennial medicinal herb used in China for topical treatment of trauma injuries (Lin et al. 2003). It grows naturally in the southern provinces of China and is also sometimes cultivated. During 2018-2020, wild G. japonica plants exhibiting chlorotic spots and mosaic symptoms were observed in Zhejiang province, China. To identify the possible causal agents of the disease, a single symptomatic leaf sample was collected in August 2019 and sent to Zhejiang Academy of Agricultural Sciences (Hangzhou, China) for next generation sequencing (NGS). Total RNAs extracted with TRIzol (Invitrogen, Carlsbad, USA) were subjected to high throughput sequencing on the Illumina NovaSeq 6000 platform with PE150bp and data analysis was performed by CLC Genomic Workbench 11 with default parameters (QIAGEN, Hilden, Germany). A total of 37,314,080 paired-end reads were obtained, and 11,785 contigs (961 to 10,964 bp) were generated and compared with sequences in GenBank using BLASTn or BLASTx. Of the total of 12 viral-related contigs obtained, one with a length of 6,442 nt mapped to the genomic RNA of ASGV (MN495979), seven contigs with lengths ranging from 1,034 to 2,901 nt mapped to Chrysanthemum virus B (CVB), and four mapped to broad bean wilt virus 2 (BBWV2), a virus which is known to infect G. procumbens (Kwak et al. 2017). To further confirm the presence of ASGV and CVB, primers were designed and the complete nucleotide sequences of both viruses were amplified from the original NGS sample using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) according to the manufacturer's instructions (Tiosbio, Beijing, China). BLASTn analysis revealed that the complete 6,451 nt sequence of ASGV (GenBank accession No. MW259059) shared the highest identity (81.2%) with a Chinese isolate of ASGV from citrus (MN495979). The two isolates grouped with another Chinese isolate (from pear) in phylogenetic analysis. The predicted coat protein of the virus had the highest nt identity of 93.7% (96.2% amino acid sequence identity) with that of the Chinese ASGV isolate XY from apple (KX686100). The complete genomes of two distinct molecular variants of CVB (both 8,987 nt in length) were also obtained from this sample (GenBank accession Nos. MW269552, MW269553). They shared 86.8% nt identity with each other and had 81.1% and 82.1% identity to the only known complete sequence of CVB from chrysanthemum (AB245142). Ten additional wild G. japonica plants with mosaic symptoms were collected randomly during 2019-2020 from Hangzhou (n=6) and Ningbo (n=4) in Zhejiang province and tested by RT-PCR with specific primer pairs to detect BBWV2, ASGV and CVB. RT-PCR and subsequent sequencing revealed that these three viruses were present in all the samples tested, indicating that co-infection of G. japonica by ASGV, CVB and BBWV2 is common. CVB mainly infects chrysanthemum (Singh et al. 2012), while ASGV is known as a pathogen of various fruit trees especially in the family Rosaceae, although there are recent reports that it can also infect some plants in Gramineae, Asparagaceae and Nelumbonaceae (Bhardwaj et al. 2017; Chen et al. 2019; He et al. 2019). Our results provide the first report that Gynura is a natural host of CVB and ASGV. Further surveys and biological studies are underway to evaluate the importance of Gynura as a virus reservoir for epidemics among the various hosts.

P50.01 RELAY, Erlotinib Plus Ramucirumab in Untreated, EGFR-Mutated, Metastatic NSCLC: Outcomes by EGFR Exon 19-del Variants

EGFR Exon 19-del mutations consist of distinct molecular variants and represent a heterogeneous disease entity. Distinct Exon 19 variants confer a differential sensitivity to EGFR-TKIs as well as different patterns of resistance. This exploratory analysis aimed to determine the impact of Exon 19-del variants on clinical outcomes in the RELAY trial.

First report of grapevine rupestris vein feathering virus in grapevine in Australia.

Grapevine rupestris vein feathering virus (GRVFV; tentative genus Marafivirus; family Tymoviridae ) was first detected from a Greek grapevine (Vitis vinifera), with asteroid mosaic-like symptoms (El Beaino et al. 2001; Ghanem-Sabanadzovic et al. 2003) and was also infected with grapevine fleck virus. GRVFV has been detected in the United States, South Africa, Canada, Spain, China, New Zealand, Brazil, Germany, Korea, Slovakia, Hungary and Pakistan (Cho et al. 2018; Mahmood et al. 2019).Transmission vectors are currently unknown. In 2018, nine grapevine samples were collected between May to July in South Australia (SA) and Western Australia (WA) (Table S1), were analysed by high-throughput sequencing (HTS) to characterise grapevine viruses in Australian vineyards. Total RNA or double stranded RNA was extracted from grapevine canes using RNeasy 96 QIAcube HT kit (Qiagen) with MacKenzie buffer (MacKenzie et al. 1997) or using CF-11 (Balijja et al. 2008). Libraries were prepared using the NEBNext® Ultra II RNA library Prep Kit (NEB) or TruSeq® Stranded mRNA Prep kit (Illumina) with Ribo-Zero®gold plant kit for ribosomal depletion (Illumina, San Diego, CA). Libraries were sequenced using Illumina Miseq (SA) or Hiseq (WA) technology with 2x300 (SA) or 2x100 (WA) paired end reads which were trimmed using Trim Galore! (0.4.0) or BBmap (38.20), respectively. De novo assembly, using the SPAdes (version 3.12.0) genome assembler with default settings, resulted in twelve near full length GRVFV genomes (6713-6737nt), eight sequences from the WA samples and four from the SA samples. WA samples 171 and 178 and SA sample BV each had two distinct GRVFV molecular variants. Variants 171-1 and 171-2 (GenBank accessions MT084811, MT084812) from sample 171 shared 83.39% nucleotide (nt) identity. Variants 178-1 and 178-2 (MT084813, MT084814) from sample 178 shared 83.54% nt identity. Variants BV6799 and BV8822 (MN974274, MN974275) from sample BV shared 82.85% nt identity. Only one GRVFV sequence was obtained from all other samples. The genome of SA isolate LC1 (MN974273) was confirmed by RT-PCR amplification and Sanger sequencing of overlapping genome regions. Tissue from the infected LC1 isolate has been deposited into the Victorian plant pathogen reference collection (VPRI accession No. 43698). When the genomes of all Australian isolates were compared, they had 78.94% to 94.37 % nt identity with each other. The SA isolates LC1, BV8822, BV6799, and SEL-L (MN974276), and the WA isolates 172 (MT084807), 179 (MT084808), 180 (MT084809), and 182 (MT084810) were most closely related to the Swiss isolate CHASS (KY513702; 82.87% to 85.46% nt identity). The WA isolates 171-1, 171-2, 178-1 and 178-2 were most closely related to the New Zealand isolate Ch8021 (MF000325; 83.21% to 93.87%). Grapevine leafroll-associated virus 1 (GLRaV-1), GLRaV-3, GLRaV-4 (strain 6 and 9), grapevine virus A, grapevine rupestris stem pitting associated virus, grapevine yellow speckle viroid 1 and hop stunt viroid were also identified in the sequencing data. This is the first report of GRVFV in Australia. All WA samples were collected during dormancy and symptoms were not observed. Sample LC1 from SA had Shiraz disease, the other SA samples were asymptomatic, and none had asteroid mosaic-like symptoms. Further research is required to determine its distribution and association with disease in Australia.

Heterogeneity within the PF-EPN-B ependymoma subgroup.

Posterior fossa ependymoma comprise three distinct molecular variants, termed PF-EPN-A (PFA), PF-EPN-B (PFB), and PF-EPN-SE (subependymoma). Clinically, they are very disparate and PFB tumors are currently being considered for a trial of radiation avoidance. However, to move forward, unraveling the heterogeneity within PFB would be highly desirable. To discern the molecular heterogeneity within PFB, we performed an integrated analysis consisting of DNA methylation profiling, copy-number profiling, gene expression profiling, and clinical correlation across a cohort of 212 primary posterior fossa PFB tumors. Unsupervised spectral clustering and t-SNE analysis of genome-wide methylation data revealed five distinct subtypes of PFB tumors, termed PFB1-5, with distinct demographics, copy-number alterations, and gene expression profiles. All PFB subtypes were distinct from PFA and posterior fossa subependymomas. Of the five subtypes, PFB4 and PFB5 are more discrete, consisting of younger and older patients, respectively, with a strong female-gender enrichment in PFB5 (age: p = 0.011, gender: p = 0.04). Broad copy-number aberrations were common; however, many events such as chromosome 2 loss, 5 gain, and 17 loss were enriched in specific subtypes and 1q gain was enriched in PFB1. Late relapses were common across all five subtypes, but deaths were uncommon and present in only two subtypes (PFB1 and PFB3). Unlike the case in PFA ependymoma, 1q gain was not a robust marker of poor progression-free survival; however, chromosome 13q loss may represent a novel marker for risk stratification across the spectrum of PFB subtypes. Similar to PFA ependymoma, there exists a significant intertumoral heterogeneity within PFB, with distinct molecular subtypes identified. Even when accounting for this heterogeneity, extent of resection remains the strongest predictor of poor outcome. However, this biological heterogeneity must be accounted for in future preclinical modeling and personalized therapies.

BCL6/BCL2 and MYC Alterations Drawing the Gray Zone between Diffuse Large B-cell Lymphoma and Double, Triple Hit Lymphoma

Diffuse large B-cell lymphoma (DLBCL) Diffuse large B-cell lymphoma (DLBCL) is an aggressive type of non- Hodgkin lymphoma that develops from the B-cells in the lymphatic system. Under the microscope, large malignant lymphocytes are seen diffusely throughout the specimen. Diffuse Large B-Cell Lymphoma (DLBCL) accounts for 20– 30% of all lymphoid malignancies. This subtype forms distinct morphological, molecular, and phenotypic variants. Therefore, it is of a great value to classify diffuse large B-cell lymphoma according to gene expression profiling into germinal center B-cell subtype, activated B-cell subtype, and unclassified subtype. The outcomes vary between these subtypes, and the germinal center B-cell subtype has a better outcome with the current standard rituximab, cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone (R-CHOP) immunochemotherapy.

Nanog-driven cell-reprogramming and self-renewal maintenance in Ptch1+/\u2212 granule cell precursors after radiation injury

Medulloblastoma (MB) is the most common pediatric brain tumor, comprising four distinct molecular variants, one of which characterized by activation of the Sonic Hedgehog (SHH) pathway, driving 25–30% of sporadic MB. SHH-dependent MBs arise from granule cell precursors (GCPs), are fatal in 40–70% of cases and radioresistance strongly contributes to poor prognosis and tumor recurrence. Patched1 heterozygous (Ptch1+/−) mice, carrying a germ-line heterozygous inactivating mutation in the Ptch1 gene, the Shh receptor and negative regulator of the pathway, are uniquely susceptible to MB development after radiation damage in neonatal cerebellum. Here, we irradiated ex-vivo GCPs isolated from cerebella of neonatal WT and Ptch1+/− mice. Our results highlight a less differentiated status of Ptch1-mutated cells after irradiation, influencing DNA damage response. Increased expression levels of pluripotency genes Nanog, Oct4 and Sal4, together with greater clonogenic potential, clearly suggest that radiation induces expansion of the stem-like cell compartment through cell-reprogramming and self-renewal maintenance, and that this mechanism is strongly dependent on Nanog. These results contribute to clarify the molecular mechanisms that control radiation-induced Shh-mediated tumorigenesis and may suggest Nanog as a potential target to inhibit for adjuvant radiotherapy in treatment of SHH-dependent MB.

The current consensus on the clinical management of intracranial ependymoma and its distinct molecular variants

Multiple independent genomic profiling efforts have recently identified clinically and molecularly distinct subgroups of ependymoma arising from all three anatomic compartments of the central nervous system (supratentorial brain, posterior fossa, and spinal cord). These advances motivated a consensus meeting to discuss: (1) the utility of current histologic grading criteria, (2) the integration of molecular-based stratification schemes in future clinical trials for patients with ependymoma and (3) current therapy in the context of molecular subgroups. Discussion at the meeting generated a series of consensus statements and recommendations from the attendees, which comment on the prognostic evaluation and treatment decisions of patients with intracranial ependymoma (WHO Grade II/III) based on the knowledge of its molecular subgroups. The major consensus among attendees was reached that treatment decisions for ependymoma (outside of clinical trials) should not be based on grading (II vs III). Supratentorial and posterior fossa ependymomas are distinct diseases, although the impact on therapy is still evolving. Molecular subgrouping should be part of all clinical trials henceforth.

PDTB-08. Mir-449a, A POTENTIAL DIAGNOSTIC BIOMARKER FOR WNT GROUP OF MEDULLOBLASTOMA

Medulloblastoma (MB) is the most common malignant brain tumor in childhood. The five year disease-free survival rate is rather low. There is a consensus that MB can be divided into at least four clinically, transcriptionally, and genetically distinct molecular variants, being designated as wingless (WNT), sonic hedgehog (SHH), Group 3 and Group 4. It poses a great challenge to the design of therapeutic strategy for MB patients. Intensive clinical intervention, including high dose radiotherapy, is commonly used in treatment of high risk MB, most of which are considered to be Group 3 patients. But such intensive therapy should be avoided to protect neurologic function of patients in the lower risk WNT group. In present study, MB subgroup assignment in formalin-fixed paraffin embedded (FFPE) specimens from 45 Chinese patients were performed by Nanostring platform using 22 well-known signature genes. Based on comparative expression profiles of miRNA real-time PCR microarray in MB cells with and without treatment of demethylation reagent, as well as MSP assay, miR-449a was demonstrated to be significantly silenced by aberrant DNA methylation in tumor cells. Real-time PCR showed that expression level of miR-449a in WNT group was significantly different from other subgroups, although it was down-regulated in most of the MB samples. In conclusion, current study demonstrates for the first time the feasibility of using the Nanostring assay for subgrouping of medulloblastomas in Chinese patients. In addition, MiR-449a, a candidate tumor suppressor regulated by hypermethylation, is a novel potential diagnostic marker for WNT group of MBs.

Therapeutic Impact of Cytoreductive Surgery and Irradiation of Posterior Fossa Ependymoma in the Molecular Era: A Retrospective Multicohort Analysis.

Posterior fossa ependymoma comprises two distinct molecular variants termed EPN_PFA and EPN_PFB that have a distinct biology and natural history. The therapeutic value of cytoreductive surgery and radiation therapy for posterior fossa ependymoma after accounting for molecular subgroup is not known. Four independent nonoverlapping retrospective cohorts of posterior fossa ependymomas (n = 820) were profiled using genome-wide methylation arrays. Risk stratification models were designed based on known clinical and newly described molecular biomarkers identified by multivariable Cox proportional hazards analyses. Molecular subgroup is a powerful independent predictor of outcome even when accounting for age or treatment regimen. Incompletely resected EPN_PFA ependymomas have a dismal prognosis, with a 5-year progression-free survival ranging from 26.1% to 56.8% across all four cohorts. Although first-line (adjuvant) radiation is clearly beneficial for completely resected EPN_PFA, a substantial proportion of patients with EPN_PFB can be cured with surgery alone, and patients with relapsed EPN_PFB can often be treated successfully with delayed external-beam irradiation. The most impactful biomarker for posterior fossa ependymoma is molecular subgroup affiliation, independent of other demographic or treatment variables. However, both EPN_PFA and EPN_PFB still benefit from increased extent of resection, with the survival rates being particularly poor for subtotally resected EPN_PFA, even with adjuvant radiation therapy. Patients with EPN_PFB who undergo gross total resection are at lower risk for relapse and should be considered for inclusion in a randomized clinical trial of observation alone with radiation reserved for those who experience recurrence.

Mir-449a, a potential diagnostic biomarker for WNT group of medulloblastoma.

Medulloblastoma (MB) is the most common malignant brain tumor in childhood. The 5year disease-free survival rate is rather low. There is a consensus that MB can be divided into at least four clinically, transcriptionally, and genetically distinct molecular variants, being designated as wingless (WNT), sonic hedgehog (SHH), Group 3 and Group 4. It poses a great challenge to the design of therapeutic strategy for MB patients. Intensive clinical intervention, including high dose radiotherapy, is commonly used in treatment of high risk MB, most of which are considered to be Group 3 patients. But such intensive therapy should be avoided to protect neurologic function of patients in the lower risk WNT group. In present study, MB subgroup assignment in formalin-fixed paraffin embedded (FFPE) specimens from 45 Chinese patients were performed by Nanostring platform using 22 well-known signature genes. Based on comparative expression profiles of miRNA real-time PCR microarray in MB cells with and without treatment of demethylation reagent, as well as MSP assay, miR-449a was demonstrated to be significantly silenced by aberrant DNA methylation in tumor cells. Real-time PCR showed that expression level of miR-449a in WNT group was significantly different from other subgroups, although it was down-regulated in most of the MB samples. In conclusion, current study demonstrates for the first time the feasibility of using the Nanostring assay for subgrouping of MBs in Chinese patients. In addition, MiR-449a, a candidate tumor suppressor regulated by hypermethylation, is a novel potential diagnostic marker for WNT group of MBs.

Molecular subgroups of adult medulloblastoma: a long-term single-institution study.

Recent transcriptomic approaches have demonstrated that there are at least 4 distinct subgroups in medulloblastoma (MB); however, survival studies of molecular subgroups in adult MB have been inconclusive because of small sample sizes. The aim of this study is to investigate the molecular subgroups in adult MB and identify their clinical and prognostic implications in a large, single-institution cohort. We determined gene expression profiles for 13 primary adult MBs. Bioinformatics tools were used to establish distinct molecular subgroups based on the most informative genes in the dataset. Immunohistochemistry with subgroup-specific antibodies was then used for validation within an independent cohort of 201 formalin-fixed MB tumors, in conjunction with a systematic analysis of clinical and histological characteristics. Three distinct molecular variants of adult MB were identified: the SHH, WNT, and group 4 subgroups. Validation of these subgroups in the 201-tumor cohort by immunohistochemistry identified significant differences in subgroup-specific demographics, histology, and metastatic status. The SHH subgroup accounted for the majority of the tumors (62%), followed by the group 4 subgroup (28%) and the WNT subgroup (10%). Group 4 tumors had significantly worse progression-free and overall survival compared with tumors of the other molecular subtypes. We have identified 3 subgroups of adult MB, characterized by distinct expression profiles, clinical features, pathological features, and prognosis. Clinical variables incorporated with molecular subgroup are more significantly informative for predicting adult patient outcome.

Somatic mutations of DICER1 and KMT2D are frequent in intraocular medulloepitheliomas.

Intraocular medulloepithelioma (IO-MEPL) is an uncommon embryonal neuroepithelial neoplasm of the eye. Little is known about the cytogenetics, molecular biology, and pathogenesis of this tumor. In the present study we investigated the mutational landscape of 19 IO-MEPL using targeted next-generation sequencing. Routinely prepared paraffin-embedded samples were assessed with high-coverage genome sequencing on the Illumina NextSeq 500 platform using a customized gene panel set covering the coding region of 130 genes. This revealed several notable genomic alterations, including mutations of DICER1 (6 tumors) and KMT2D (also known as MLL2; 5 tumors)-which are frequently recurrent and mutually exclusive molecular events for IO-MEPL. Non-recurrent mutations in the cancer-associated genes BRCA2, BRCA1, NOTCH2, CDH1, and GSE1 were also identified. IO-MEPL samples harboring a DICER1 mutation disclosed few chromosomal alterations and formed a separate DNA methylation cluster, indicating potential differences in genetic and epigenetic events arising perhaps from the presence of this aberration in the tumor genome. The high proportion of recurrent somatic DICER1 and KMT2D mutations in this series of sporadic IO-MEPL points to their likely important roles in the molecular pathogenesis of these rare embryonal tumors, and perhaps suggests the existence of distinct molecular variants of IO-MEPL. Although the precise role of these recurrent mutations in the development of IO-MEPL, and their relationship to pro-oncogenic molecular mechanisms, have yet to be determined, unraveling their roles could eventually be exploited for nonsurgical therapies of these neoplasms.

Posterior fossa ependymoma: current insights.

Ependymoma is the third most common malignant tumor of the posterior fossa and is a major cause of neurological morbidity and mortality in children. Current treatments, particularly surgery and external beam irradiation result in relatively poor outcomes with significant neurological and cognitive sequelae from treatment. Historical approaches have considered all ependymomas as similar entities based on their morphological appearance. Recent advances in genomics and epigenetics have revealed, however, that ependymomas from different CNS locations represent distinct entities. Moreover, ependymoma of the posterior fossa, the most common location in children, is actually comprised of two distinct molecular variants. These two variants have marked differences in demographics, transcriptomes, structure, methylation patterns, and clinical outcomes. This allows for the development of new biology-based clinical risk stratification, which can both prioritize patients for de-escalation of therapy and identify those who will benefit from novel therapeutic strategies. Indeed, the identification of these two variants allows an opportunity for robust preclinical modeling for development of novel therapeutic strategies. Herein, we have summarized our current clinical approach to diagnosis and treatment of posterior fossa ependymoma, recent advances in understanding the biology of posterior fossa ependymoma and how these new insights can be translated into the clinic to form the basis of the next generation of clinical trials.

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.

Subgroup-specific structural variation across 1,000 medulloblastoma genomes

Medulloblastoma, the most common malignant paediatric brain tumour, is currently treated with nonspecific cytotoxic therapies including surgery, whole-brain radiation, and aggressive chemotherapy. As medulloblastoma exhibits marked intertumoural heterogeneity, with at least four distinct molecular variants, previous attempts to identify targets for therapy have been underpowered because of small samples sizes. Here we report somatic copy number aberrations (SCNAs) in 1,087 unique medulloblastomas. SCNAs are common in medulloblastoma, and are predominantly subgroup-enriched. The most common region of focal copy number gain is a tandem duplication of SNCAIP, a gene associated with Parkinson's disease, which is exquisitely restricted to Group 4α. Recurrent translocations of PVT1, including PVT1-MYC and PVT1-NDRG1, that arise through chromothripsis are restricted to Group 3. Numerous targetable SCNAs, including recurrent events targeting TGF-β signalling in Group 3, and NF-κB signalling in Group 4, suggest future avenues for rational, targeted therapy.

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.

FSTL5 Is a Biomarker of Medulloblastoma That Predicts Poor Prognosis

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Treatment regimens cure up to 75% of patients, yet leave many with cognitive and/or endocrine dysfunction. Based upon genomic and immunohistochemical analysis, four distinct molecular variants of MB have been identified: WNT (wingless), SHH (sonic hedgehog), group C, and group D. In contrast to WNT and SHH, the prognosis of non-WNT/non-SHH tumors is poor and the underlying genetic changes remain unknown. Hoping to identify new molecular biomarkers of non-WNT/non-SHH tumors that may aid in the stratification of high- versus low-risk patients, Remke and colleagues performed transcriptome and copy number analyses on primary MB. Their findings delineated genetic markers associated with the four MB subgroups and noted the association of group C and particularly group D tumors with advanced disease and poor prognosis. Interestingly, the gene for follistatin-like protein 5, FSTL5, was overexpressed in group C and some group D tumors and was significantly associated with reduced progression-free and overall survival across all disease variants. Little is known about the function of FSTL5, a member of the activin/follistatin system, and only a few reports link the gene family to cancer-related processes. Significantly, the current findings conclude that FSTL5 is a reliable independent prognostic marker for MB. Addition of FSTL5 to the four-class molecular staging system enhances prediction accuracy for non-WNT/non-SHH MB and will inform the development of more effective and less toxic treatment regimens for MB patients.Remke M, Hielscher T, Korshunov A, Northcott PA, Bender S, Kool M, et al. FSTL5 is a marker of poor prognosis in non-WNT/non-SHH medulloblastoma. J Clin Oncol. 2011 Sept 12. [Epub ahead of print]Note: Research Watch is written by Cancer Discovery Science Writers. Readers are encouraged to consult the original articles for full details.

Adult Medulloblastoma Comprises Three Major Molecular Variants

Medulloblastoma is a rare primary brain tumor in adults, whereas it constitutes the most common malignant brain tumor in children. Integrated genomics approaches revealed at least four distinct disease variants in children. The aim of this study was to investigate molecular subtypes and their prognostic implication in a large cohort of adult medulloblastomas as the biology in this age group remains poorly understood. We combined transcriptome and DNA copy number analyses for 28 adult medulloblastomas. Statistical and bioinformatic tools were applied to discover distinct molecular variants. Clinical and molecular characteristics of each biologic subtype were validated using immunohistochemistry on a tissue microarray derived from an independent patient cohort of adult medulloblastomas (n = 103). Gene expression profiles revealed three distinct molecular variants with stable subtype separation using the 300 most varying transcripts. Distinct demographics, genetics, transcriptome, and prognosis were noted for each subtype of adult medulloblastoma. Immunohistochemistry revealed aberrant activation of the sonic hedgehog (SHH) pathway in over half of adult medulloblastomas constituting a promising molecular therapeutic target. In contrast, subtype C tumors, which comprise a robust subtype in childhood medulloblastoma are only exceptionally seen in adult cohorts. Notably, adult subtype D and Wnt/wingless tumors were associated with worse prognosis than pediatric cohorts, whereas survival for SHH tumors was similar for both age groups. The transcriptome of adult medulloblastomas differs considerably from pediatric counterparts, both in terms of tumor biology and prognostic impact. Therefore, age-specific classification is required and must be adapted for use in clinical trials of adult medulloblastoma.