Author ORCID Identifier
0000-0002-4339-8683
Date of Graduation
12-2023
Document Type
Thesis (MS)
Program Affiliation
Cancer Biology
Degree Name
Masters of Science (MS)
Advisor/Committee Chair
Andy Futreal, PhD
Committee Member
Courtney DiNardo, MD
Committee Member
Marina Konopleva, MD, PhD
Committee Member
Michael Andreeff, MD, PhD
Committee Member
Adi Diab, MD
Abstract
Measurable residual disease (MRD) detection is associated with cancer relapse. Leukemias with rearrangement of the lysine methyltransferase 2 A gene (KMT2Ar) have an adverse prognosis with more than 80 possible fusion partner genes (FPG) identified. With the advent of effective targeted therapies for KMT2Ar leukemia such as menin inhibitors, there is an unmet need for sensitive assays to detect these various fusions. Standard next-generation sequencing (NGS) methods cannot accurately detect genetic alterations at low frequencies because of inherent technical errors and the need for high sequencing depth. Blocker Displacement Amplification (BDA) technology enables selective detection of such rare alterations with NGS. Here, we demonstrate the effective detection of KMT2Ar and its various FPG with a novel NGS assay leveraging BDA. Primers were designed to flank any known exon junction of cDNA corresponding to transcripts of the gene of interest, while non-extensible oligonucleotides (blockers) span the wildtype (WT) exon junction, therefore suppressing amplification of WT while differentially amplifying fusion junctions. We tested this assay in cell line, establishing limits of detection and input and subsequently validated our findings in clinical samples from patients with KMT2Ar leukemia. In conclusion, FusionBDA is a novel, sensitive, and specific assay for KMT2Ar acute leukemia. This assay can agnostically detect KMT2Ar with various fusions at a predicted sensitivity of at least 0.005%, therefore allowing both MRD detection and target identification.
Keywords
Menin, KMT2A, MRD, Leukemia