Minimal methylation classifier (MIMIC): A novel method for derivation and rapid diagnostic detection of disease-associated DNA methylation signatures

Schwalbe, Ed C.; Hicks, Debbie; Rafiee, Gholamreza; Bashton, Matthew; Gohlke, Henning; Enshaei, Amir; Potluri, Sandeep; Matthiesen, Jessie; Mather, Michael; Taleongpong, Pim; Chaston, Ria; Silmon, Angela; Curtis, Amalia De; Lindsey, Janet C.; Crosier, Stephen; Smith, Amanda; Goschzik, Tobias; Doz, François; Rutkowski, Stefan; Lannering, Birgitta; Pietsch, Torsten; Bailey, Simon; Williamson, Daniel; Clifford, Steven C.

doi:10.1038/s41598-017-13644-1

Cited by 24 publications

(19 citation statements)

References 23 publications

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“…These results shed new light on a potential method for low-income countries based on a simple and feasible technique such as qPCR along with six probe/primer pairs plus reference genes with implementation of an approach recently described by Gómes and colleagues [3]. Their method fully discriminates between Group 3 and Group 4 based on the methylation status of 5 CpG’s, which is feasible for the real-time PCR platform through High Resolution Melting technology, and shall improve the molecular assignment [26].…”

Section: Discussionmentioning

confidence: 99%

“…The R language and environment for statistical computing and graphics was used for bioinformatic analysis. The ComplexHeatmap and circlize packages were used for Heatmap generation [5, 6] and the ggplot2package [26, 32] was used for graphics generation. Rtsne [14, 10] was used for the visualization of t-Distributed Stochastic Neighbor Embedding (t-SNE) and the NbClust and Factoextra packages [3, 11] were used to point out the best number of clusters and to visualize the results.…”

Section: Methodsmentioning

confidence: 99%

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A simplified approach using Taqman low-density array for medulloblastoma subgrouping

Cruzeiro

Salomão

Biagi

et al. 2019

acta neuropathol commun

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Next-generation sequencing platforms are routinely used for molecular assignment due to their high impact for risk stratification and prognosis in medulloblastomas. Yet, low and middle-income countries still lack an accurate cost-effective platform to perform this allocation. TaqMan Low Density array (TLDA) assay was performed using a set of 20 genes in 92 medulloblastoma samples. The same methodology was assessed in silico using microarray data for 763 medulloblastoma samples from the GSE85217 study, which performed MB classification by a robust integrative method (Transcriptional, Methylation and cytogenetic profile). Furthermore, we validated in 11 MBs samples our proposed method by Methylation Array 450 K to assess methylation profile along with 390 MB samples (GSE109381) and copy number variations. TLDA with only 20 genes accurately assigned MB samples into WNT, SHH, Group 3 and Group 4 using Pearson distance with the average-linkage algorithm and showed concordance with molecular assignment provided by Methylation Array 450 k. Similarly, we tested this simplified set of gene signatures in 763 MB samples and we were able to recapitulate molecular assignment with an accuracy of 99.1% (SHH), 94.29% (WNT), 92.36% (Group 3) and 95.40% (Group 4), against 97.31, 97.14, 88.89 and 97.24% (respectively) with the Ward.D2 algorithm. t-SNE analysis revealed a high level of concordance (k = 4) with minor overlapping features between Group 3 and Group 4. Finally, we condensed the number of genes to 6 without significantly losing accuracy in classifying samples into SHH, WNT and non-SHH/non-WNT subgroups. Additionally, we found a relatively high frequency of WNT subgroup in our cohort, which requires further epidemiological studies. TLDA is a rapid, simple and cost-effective assay for classifying MB in low/middle income countries. A simplified method using six genes and restricting the final stratification into SHH, WNT and non-SHH/non-WNT appears to be a very interesting approach for rapid clinical decision-making. Electronic supplementary material The online version of this article (10.1186/s40478-019-0681-y) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

A simplified approach using Taqman low-density array for medulloblastoma subgrouping

Cruzeiro

Salomão

Biagi

et al. 2019

acta neuropathol commun

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“…Because only material of mostly low quantity and quality was available, the HIT-SIOP PNET 4 samples were unsuitable for subgroup assessment using conventional approaches (DNA methylation array 19 or mRNA expression analysis by Nanostring 20 ); therefore, we analysed all samples using a mass spectrometry-minimal methylation classifier (MS-MIMIC) assay to assess their molecular subgroup. 16 For the validation cohort, samples were of sufficient quality and quantity to do Illumina 450k DNA methylation microarray (62 DNA samples were from frozen material and eight were from FFPE tissue) and consensus methylation subgroup was assigned as described previously. 6 …”

Section: Methodsmentioning

confidence: 99%

“…In this Article, we report comprehensive molecular and pathological characterisation of the HIT-SIOP PNET 4 cohort using novel technologies 16 , 17 developed and adapted for assessment of the remnant tumour material. This analysis, alongside an independent, demographically matched, standard-risk medulloblastoma validation cohort, enabled the discovery and validation of concerted whole chromosomal aberration signatures with prognostic value for patients with non-WNT/non-SHH medulloblastoma.…”

Section: Introductionmentioning

confidence: 99%

Prognostic effect of whole chromosomal aberration signatures in standard-risk, non-WNT/non-SHH medulloblastoma: a retrospective, molecular analysis of the HIT-SIOP PNET 4 trial

Goschzik

Schwalbe

Hicks

et al. 2018

The Lancet Oncology

105

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SummaryBackgroundMost children with medulloblastoma fall within the standard-risk clinical disease group defined by absence of high-risk features (metastatic disease, large-cell/anaplastic histology, and MYC amplification), which includes 50–60% of patients and has a 5-year event-free survival of 75–85%. Within standard-risk medulloblastoma, patients in the WNT subgroup are established as having a favourable prognosis; however, outcome prediction for the remaining majority of patients is imprecise. We sought to identify novel prognostic biomarkers to enable improved risk-adapted therapies.MethodsThe HIT-SIOP PNET 4 trial recruited 338 patients aged 4–21 years with medulloblastoma between Jan 1, 2001, and Dec 31, 2006, in 120 treatment institutions in seven European countries to investigate hyperfractionated radiotherapy versus standard radiotherapy. In this retrospective analysis, we assessed the remaining tumour samples from patients in the HIT-SIOP PNET 4 trial (n=136). We assessed the clinical behaviour of the molecularly defined WNT and SHH subgroups, and identified novel independent prognostic markers and models for standard-risk patients with non-WNT/non-SHH disease. Because of the scarcity and low quality of available genomic material, we used a mass spectrometry-minimal methylation classifier assay (MS-MIMIC) to assess methylation subgroup and a molecular inversion probe array to detect genome-wide copy number aberrations. Prognostic biomarkers and models identified were validated in an independent, demographically matched cohort (n=70) of medulloblastoma patients with non-WNT/non-SHH standard-risk disease treated with conventional therapies (maximal surgical resection followed by adjuvant craniospinal irradiation [all patients] and chemotherapy [65 of 70 patients], at UK Children's Cancer and Leukaemia Group and European Society for Paediatric Oncology (SIOPE) associated treatment centres between 1990 and 2014. These samples were analysed by Illumina 450k DNA methylation microarray. HIT-SIOP PNET 4 is registered with ClinicalTrials.gov, number NCT01351870.FindingsWe analysed methylation subgroup, genome-wide copy number aberrations, and mutational features in 136 assessable tumour samples from the HIT-SIOP PNET 4 cohort, representing 40% of the 338 patients in the trial cohort. This cohort of 136 samples consisted of 28 (21%) classified as WNT, 17 (13%) as SHH, and 91 (67%) as non-WNT/non-SHH (we considered Group3 and Group4 medulloblastoma together in our analysis because of their similar molecular and clinical features). Favourable outcomes for WNT tumours were confirmed in patients younger than 16 years, and all relapse events in SHH (four [24%] of 17) occurred in patients with TP53 mutation (TP53mut) or chromosome 17p loss. A novel whole chromosomal aberration signature associated with increased ploidy and multiple non-random whole chromosomal aberrations was identified in 38 (42%) of the 91 samples from patients with non-WNT/non-SHH medulloblastoma in the HIT-SIOP PNET 4 cohort. Biomarkers associate...

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“…DNA polymerase incorporates a single dideoxynucleotide triphosphate (ddNTP) that is complementary to the target base. Single base‐extended primer (i.e., SBE product) can be detected by various methods, including CE , MALDI‐TOF MS , surface enhanced Raman spectroscopy , and BeadChip array . However, all of these techniques are expensive, use high‐precision instruments, and require time‐consuming separation or cleanup/concentration of the SBE products.…”

Section: Introductionmentioning

confidence: 99%

Separation‐free single‐base extension assay with fluorescence resonance energy transfer for rapid and convenient determination of DNA methylation status at specific cytosine and guanine dinucleotide sites

Fujita

Hashimoto

2018

Electrophoresis

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A separation‐free single‐base extension (SBE) assay utilizing fluorescence resonance energy transfer (FRET) was developed for rapid and convenient interrogation of DNA methylation status at specific cytosine and guanine dinucleotide sites. In this assay, the SBE was performed in a tube using an allele‐specific oligonucleotide primer (i.e., extension primer) labeled with Cy3 as a FRET donor fluorophore at the 5′‐end, a nucleotide terminator (dideoxynucleotide triphosphate) labeled with Cy5 as a FRET acceptor, a PCR amplicon derived from bisulfite‐converted genomic DNA, and a DNA polymerase. A single base‐extended primer (i.e., SBE product) that was 5′‐Cy3‐ and 3′‐Cy5‐tagged was formed by incorporation of the Cy5‐labeled terminator into the 3′‐end of the extension primer, but only if the terminator added was complementary to the target nucleotide. The resulting SBE product brought the Cy3 donor and the Cy5 acceptor into close proximity. Illumination of the Cy3 donor resulted in successful FRET and excitation of the Cy5 acceptor, generating fluorescence emission from the acceptor. The capacity of the developed assay to discriminate as low as 10% methylation from a mixture of methylated and unmethylated DNA was demonstrated at multiple cytosine and guanine dinucleotide sites.

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Minimal methylation classifier (MIMIC): A novel method for derivation and rapid diagnostic detection of disease-associated DNA methylation signatures

Cited by 24 publications

References 23 publications

A simplified approach using Taqman low-density array for medulloblastoma subgrouping

A simplified approach using Taqman low-density array for medulloblastoma subgrouping

Prognostic effect of whole chromosomal aberration signatures in standard-risk, non-WNT/non-SHH medulloblastoma: a retrospective, molecular analysis of the HIT-SIOP PNET 4 trial

Separation‐free single‐base extension assay with fluorescence resonance energy transfer for rapid and convenient determination of DNA methylation status at specific cytosine and guanine dinucleotide sites

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