Methylation of MMP2 and MMP9 in patients with localized and generalized forms of Ewing's sarcoma.

e23503 Background: MMPs play a critical role in tumor growth and progression, metastatic development, angiogenesis, and tumor invasion. Epigenetic regulation of MMP2 and MMP9 levels is often disrupted in cancer and is considered as potential biomarkers. In this study, we tried to assess the methylation of the MMP2 and MMP9 genes in patients with localized and generalized forms of Ewing's sarcoma (ES). Methods: As a material used DNA from FFPE of primary tumor of 20 patients with localized forms ES and 20 patients with generalized forms ES. After bisulfite conversion of total DNA and PCR, the level of CpG methylation was assessed by sequencing on a Genetic Analyzer Applied Biosystems 3500. Results: The analysis showed the presence of hypomethylation of the MMP2 in the group with generalized forms (100%) 1.25 times more often than in the group with localized forms (80%), and hypermethylation was observed only in the group with localized forms–20% of cases (χ2 = 4.234, p = 0.040). On the other hand, hypermethylation was 1.9 times more common in the group with localized forms of ES (90%) (χ2 = 8.313, p = 0.004). Differences in the methylation status of the MMP2 and MMP9 indicate that the presence of hypomethylation at two loci increases the risk of developing generalized ES by 22 times (OR = 22.2, p < 0.05; CI95% 2.460-20.769). Conclusions: Specific methylation of the matrix metalloproteinases genes MMP2 and MMP9 identified in the groups of localized and generalized Ewing's sarcoma. At the same time, the group with generalized forms of ES was characterized by gene hypomethylation.

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A DNA methylation-based classifier for accurate molecular diagnosis of bone sarcomas.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.11034 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. 11034-11034

Author(s):

Shengyang Wu ◽

Benjamin Thomas Cooper ◽

Fang Bu ◽

Christopher Bowman ◽

Keith Killian ◽

...

Keyword(s):

Dna Methylation ◽

Synovial Sarcoma ◽

Ewing’S Sarcoma ◽

Ewing's Sarcoma ◽

Clinical Sample ◽

Methylation Status ◽

Accurate Diagnosis ◽

Cpg Sites ◽

Bone Sarcomas ◽

Methylation Profiling

11034 Background: Bone sarcomas present a unique diagnostic challenge because of the considerable morphologic overlap between different entities. The choice of optimal treatment, however, is dependent upon accurate diagnosis. Genome-wide DNA methylation profiling has emerged as a new approach to aid in the diagnosis of brain tumors, with diagnostic accuracy exceeding standard histopathology. In this work we developed and validated a methylation based classifier to differentiate between osteosarcoma, Ewing’s sarcoma, and synovial sarcoma. Methods: DNA methylation status of 482,421 CpG sites in 15 osteosarcoma, 10 Ewing’s sarcoma, and 11 synovial sarcoma samples were measured using the Illumina HumanMethylation450 array. From this training set of 36 samples we developed a random forest classifier using the 400 most differentially methylated CpG sites (FDR q value < 0.001). This classifier was then validated on 10 synovial sarcoma samples from TCGA, 86 osteosarcoma samples from TARGET-OS, and 15 Ewing’s sarcoma from a recently published series (Huertas-Martinez et al., Cancer Letters 2016). Results: Methylation profiling revealed three distinct molecular clusters, each enriched with a single sarcoma subtype. Within the validation cohorts, all samples from TCGA were correctly classified as synovial sarcoma (10/10, sensitivity and specificity 100%). All but one sample from TARGET-OS were classified as osteosarcoma (85/86, sensitivity 98%, specificity 100%) and all but one sample from the Ewing’s sarcoma series was classified as Ewing’s sarcoma (14/15, sensitivity 93%, specificity 100%). The single misclassified osteosarcoma sample was classified as Ewing’s sarcoma, and was later determined to be a misdiagnosed Ewing’s sarcoma based on RNA-Seq demonstrating high EWRS1 and ETV1 expression. An additional clinical sample that was misdiagnosed as a synovial sarcoma by initial histolopathology, was accurately recognized as osteosarcoma by the methylation classifier. Conclusions: Osteosarcoma, Ewing’s sarcoma and synovial sarcoma have distinct epigenetic profiles. Our validated methylation-based classifier can be used to provide an accurate diagnosis when histological and standard techniques are inconclusive.

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