Gerald B W Wertheim1, Catherine Smith2, Marlise Luskin3, Alison Rager3, Maria E Figueroa4, Martin Carroll5, Stephen R Master6. 1. Department of Pathology, Children's Hospital of Philadelphia; Department of Pathology and Laboratory Medicine. 2. Department of Pathology, Children's Hospital of Philadelphia; 3. Division of Hematology and Oncology, and. 4. Department of Pathology, University of Michigan Medical School, Ann Arbor, MI. 5. Division of Hematology and Oncology, and Abramson Research Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA; 6. Department of Pathology and Laboratory Medicine, srmaster@mail.med.upenn.edu.
Abstract
BACKGROUND: Epigenetic dysregulation involving alterations in DNA methylation is a hallmark of various types of cancer, including acute myeloid leukemia (AML). Although specific cancer types and clinical aggressiveness of tumors can be determined by DNA methylation status, the assessment of DNA methylation at multiple loci is not routinely performed in the clinical laboratory. METHODS: We recently described a novel microsphere-based assay for multiplex evaluation of DNA methylation. In the current study, we validated and used an improved assay [termed expedited microsphere HpaII small fragment Enrichment by Ligation-mediated PCR (xMELP)] that can be performed with appropriate clinical turnaround time. RESULTS: Using the xMELP assay in conjunction with a new 17-locus random forest classifier that has been trained using 344 AML samples, we were able to segregate an independent cohort of 70 primary AML patients into methylation-determined subgroups with significantly distinct mortality risk (P = 0.009). We also evaluated precision, QC parameters, and preanalytic variables of the xMELP assay and determined the sensitivity of the random forest classifier score to failure at 1 or more loci. CONCLUSIONS: Our results demonstrate that xMELP performance is suitable for implementation in the clinical laboratory and predicts AML outcome in an independent patient cohort.
BACKGROUND: Epigenetic dysregulation involving alterations in DNA methylation is a hallmark of various types of cancer, including acute myeloid leukemia (AML). Although specific cancer types and clinicalaggressiveness of tumors can be determined by DNA methylation status, the assessment of DNA methylation at multiple loci is not routinely performed in the clinical laboratory. METHODS: We recently described a novel microsphere-based assay for multiplex evaluation of DNA methylation. In the current study, we validated and used an improved assay [termed expedited microsphere HpaII small fragment Enrichment by Ligation-mediated PCR (xMELP)] that can be performed with appropriate clinical turnaround time. RESULTS: Using the xMELP assay in conjunction with a new 17-locus random forest classifier that has been trained using 344 AML samples, we were able to segregate an independent cohort of 70 primary AMLpatients into methylation-determined subgroups with significantly distinct mortality risk (P = 0.009). We also evaluated precision, QC parameters, and preanalytic variables of the xMELP assay and determined the sensitivity of the random forest classifier score to failure at 1 or more loci. CONCLUSIONS: Our results demonstrate that xMELP performance is suitable for implementation in the clinical laboratory and predicts AML outcome in an independent patient cohort.
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