Methods of detecting oncogenic fusions and uses thereof

A method for personalized monitoring an abundance of expression of at least one oncogenic KMT2A fusion in a patient, each KMT2A fusion comprising a KMT2A fragment fused at a fusion site to a partner gene fragment, the method comprising:

• a. providing an initial sequencing of the patient's leukemia at a nucleotide-level resolution;
• b. identifying at least one oncogenic KMT2A fusion based on the initial sequencing;
• c. providing:
• i. a biological sample from the patient, the sample comprising an amount of RNA;
• ii. at least one forward PCR primer targeting a KMT2A start codon of the KMT2A fragment upstream of the fusion site and at least one reverse PCR primer targeting a partner gene start codon of the partner gene fragment downstream of the fusion site;
• iii. at least one KMT2A probe comprising a first fluorescent reporter, each KMT2A probe configured to anneal to the KMT2A fragment between the KMT2A start codon and the fusion site;
• and
• iv. at least one partner gene probe comprising a second fluorescent reporter, each partner gene probe configured to anneal to the partner gene fragment between the partner start codon and the fusion site;
• d. extracting the amount of RNA from the biological sample and synthesizing an amount of cDNA from the amount of RNA;
• e. subjecting a mixture of the amount of cDNA, the at least one forward PCR primer, the at least one reverse PCR primer, the at least one KMT2A probe, and the at least one partner gene probe to droplet digital PCR (ddPCR) to obtain a plurality of paired fluorescence intensities from first and second reporters;
• and
• f. estimating the abundance of expression of the at least one oncogenic KMT2A fusion based on the plurality of paired fluorescence intensities obtained from the ddPCR.

An assay to monitor an abundance of expression of at least one oncogenic KMT2A fusion in a subject, each KMT2A fusion comprising a KMT2A fragment fused at a fusion site to a partner gene fragment, the assay comprising:

• a. at least one forward PCR primer targeting a KMT2A start codon of the KMT2A fragment upstream of the fusion site and at least one reverse PCR primer targeting a partner gene start codon of the partner gene fragment downstream of the fusion site;
• b. at least one KMT2A probe comprising a first fluorescent reporter, each KMT2A probe configured to anneal to the KMT2A fragment between the KMT2A start codon and the fusion site;
• and
• c. at least one partner gene probe comprising a second fluorescent reporter, each partner gene probe configured to anneal to the partner gene fragment between the partner start codon and the fusion site;
• wherein the at least one forward PCR primer, the at least one reverse PCR primer, the at least one KMT2A probe, and the at least one partner gene probe are combined with an amount of cDNA synthesized from an amount of RNA from the subject to form a mixture for processing in a droplet digital PCR (ddPCR) device.

A kit for monitoring an abundance of expression of at least one oncogenic KMT2A fusion in a subject, each KMT2A fusion comprising a KMT2A fragment fused at a fusion site to a partner gene fragment, the kit comprising:

• a. at least one forward PCR primer targeting a KMT2A start codon of the KMT2A fragment upstream of the fusion site and at least one reverse PCR primer targeting a partner gene start codon of the partner gene fragment downstream of the fusion site;
• b. at least one KMT2A probe comprising a first fluorescent reporter, each KMT2A probe configured to anneal to the KMT2A fragment between the KMT2A start codon and the fusion site;
• and
• c. at least one partner gene probe comprising a second fluorescent reporter, each partner gene probe configured to anneal to the partner gene fragment between the partner start codon and the fusion site.