Method for detecting oncogenic fusion genes and its applications

Abstract

This disclosure provides a method for detecting oncogenic fusion genes based on ddPCR, and its use in screening or monitoring for cancer treatment. Furthermore, this disclosure provides a method for detecting oncogenic fusion genes involved in the histone-lysine N-methyltransferase 2A (KMT2A) gene.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority under U.S. Provisional Patent Application No. 63 / 579,041, filed on 28 August 2023. The entire disclosure of the above application is incorporated herein by reference.

[0002] (Description of research and development funded by the federal government) Not applicable.

[0003] (References to be incorporated) A sequence listing constituting part of this disclosure includes a computer-readable format containing the nucleotide sequences of the present invention. The entire contents of the above sequence listing are incorporated herein by reference.

[0004] (Technical field) This disclosure relates in general to a method for detecting oncogenic fusion genes. [Background technology]

[0005] Acute myeloid leukemia (AML) is an invasive hematological cancer caused by a diverse but finite number of known oncogenic drivers. Detecting residual leukemia cells after treatment is essential for subsequent treatment selection and long-term prognosis prediction. Currently, methods for detecting measurable residual disease (MRD) after AML treatment include myelomorphological evaluation, multi-parameter flow cytometry (MPFC), and DNA sequencing. Myelomorphological evaluation can only detect leukemia cells, with a detection limit of 5%. MPFC is more sensitive, with a detection limit of 0.01–0.001%, but is difficult to interpret and not standardized across laboratories. DNA sequencing can identify leukemia cells through somatic mutation profiles, but the assay is costly and may be affected by clonal hematopoiesis in non-leukemic blood cells. In a subset of AML patients whose disease is driven by oncogenic fusion genes, these fusion genes themselves can serve as molecular markers and be utilized for highly sensitive detection of measurable residual disease.

[0006] Oncogenic fusion genes are currently used for disease monitoring in hematological malignancies such as chronic myeloid leukemia (CML). The BCR-ABL1 fusion gene is a typical driver mutation in CML. Reverse transcription quantitative polymerase chain reaction (RT-qPCR), by utilizing primers that cross fusion breakpoints, can identify the BCR-ABL1 fusion gene from mRNA transcripts with high sensitivity, at a detection limit of 1 in 100,000 cells. Therapeutic response is evaluated by a logarithmic decrease in the abundance of the transcript measured by RT-qPCR, with a decrease in the abundance of BCR-ABL1 being 10 -3 In this case, it is classified as a molecular genetic major response (MR3), and 10 -4.5 (MR4.5) to 10 -5In the case of (MR5), it is classified as a deep molecular genetic response, which is the detection limit in most available assays. With the increased efficacy of tyrosine kinase inhibitors, patients who are assessed as having cleared CML by RT-qPCR can discontinue treatment, and about half of them maintain long-term disease-free status. BCR-ABL1 is almost without exception associated with CML, but there are no similar specific oncogenic fusion genes associated with AML.

[0007] The most common translocations associated with de novo AML, including RUNX1-RUNX1T1, CBFB-MYH11, and PML-RARA, can be detected by qPCR-based assays. Droplet digital PCR (ddPCR) improves upon qPCR by separating each reaction into microfluidic droplets, enabling absolute quantification of nucleic acids in a sample. Improvements of ddPCR over qPCR assays include ease of implementation, improved detection limits, higher specificity, and absolute quantification (compared to relative quantification by standard curves in qPCR). ddPCR has already demonstrated its usefulness in detecting the BCR-ABL1 fusion gene associated with CML and the PML-RARA fusion gene associated with acute promyelocytic leukemia. This platform has the potential to improve the detection capability of rare AML caused by chromosomal translocations. However, implementing these techniques is challenging when many different partners are involved in the fusion gene. Such fusion genes involving multiple partners cannot be detected by a single assay.

[0008] Treatment-associated AML (t-AML) is a particularly aggressive, treatment-resistant subtype of AML that develops after chemotherapy or radiation exposure, typically following treatment for solid tumors. Previous studies have demonstrated that cytotoxic therapies selectively induce t-AML in hematopoietic stem cells and progenitor cells with pre-existing precancerous conditions. In other cases, the treatment itself triggers oncogenic initiations, such as the induction of double-strand DNA breaks. Topoisomerase II (TOP2) inhibitors, a type of chemotherapeutic agent, are specifically associated with oncogenic fusion genes involving the histone-lysine N-methyltransferase 2A (KMT2A) gene. While at least 80 KMT2A fusion gene partners are known, approximately 80% of fusion genes involve only five partners: AF9, AF6, AF4, ELL, and ENL. These fusion genes are potent drivers of leukemia. Mouse models have shown that introducing these fusion genes into healthy bone marrow progenitor cells induces aggressive AML as a specific event. Furthermore, childhood leukemia caused by the KMT2A fusion gene often occurs without any co-mutations. The development of t-AML in childhood cancer patients receiving high-dose TOP2 inhibitor therapy is a devastating consequence of primary cancer treatment and is almost without exception fatal. If KMT2A fusion genes generated in hematopoietic cells during treatment or remission can be detected with high sensitivity, it will be possible to identify patients at high risk of t-AML and classify them as patients requiring intervention. [Overview of the Initiative] [Means for solving the problem]

[0009] Various aspects of this disclosure include providing a method for detecting at least one oncogenic fusion gene in a subject.

[0010] In one embodiment, the present disclosure provides a method for monitoring the expression level of at least one oncogenic KMT2A fusion gene in a subject, wherein each oncogenic KMT2A fusion gene comprises a KMT2A fragment fused with a partner gene fragment at a fusion site. The method of the present disclosure comprises the steps of providing: a biological sample of the subject comprising a predetermined amount of RNA; at least one forward PCR primer targeting the KMT2A start codon of the KMT2A fragment upstream of the fusion site, and at least one reverse PCR primer targeting the partner gene start codon of the partner gene fragment downstream of the fusion site; at least one KMT2A probe comprising a first fluorescent reporter, each KMT2A probe configured to anneal to a KMT2A fragment between the KMT2A start codon and the fusion site; and at least one partner gene probe comprising a second fluorescent reporter, each partner gene probe configured to anneal to a partner gene fragment between the partner gene start codon and the fusion site. The method of the present disclosure further includes the steps of: extracting a predetermined amount of RNA from a biological sample and synthesizing a predetermined amount of cDNA from the predetermined amount of RNA; subjecting a mixture of the predetermined amount of cDNA, at least one forward PCR primer, at least one reverse PCR primer, at least one KMT2A probe, and at least one partner gene probe to droplet digital PCR (ddPCR) to obtain a plurality of fluorescence intensity pairs from a first fluorescent reporter and a second fluorescent reporter; and estimating the expression level of at least one oncogenic KMT2A fusion gene based on the plurality of fluorescence intensity pairs obtained by droplet digital PCR (ddPCR). In some embodiments, the first fluorescent reporter and the second fluorescent reporter are independently selected from fluorescein and hexachlorofluorescein, and the first fluorescent reporter and the second fluorescent reporter are different from each other.In some embodiments, at least one oncogenic KMT2A fusion gene is selected from KMT2A-AF9, KMT2A-AF4, KMT2A-AF6, KMT2A-ENL, KMT2A-ELL, at least one target-specific KMT2A fusion gene, and any combination thereof. In some embodiments, at least one oncogenic KMT2A fusion gene is one KMT2A fusion gene selected from the group consisting of KMT2A-AF9, KMT2A-AF4, KMT2A-AF6, KMT2A-ENL, and KMT2A-ELL. In some embodiments, at least one forward PCR primer targets the KMT2A start codon of a KMT2A fragment selected from KMT2A exon 7, KMT2A exon 9, and any combination thereof, and at least one reverse PCR primer targets the partner gene start codon of a partner gene fragment selected from AF9 exon 6, AF4 exon 5, AF6 exon 2, ENL exon 7, ELL exon 3, and any combination thereof. In some embodiments, at least one forward PCR primer includes a nucleotide sequence selected from SEQ ID NO: 1 targeting KMT2A exon 7, SEQ ID NO: 2 targeting KMT2A exon 9, and any combination thereof, and at least one reverse PCR primer includes a nucleotide sequence selected from SEQ ID NO: 7 targeting AF9 exon 6, SEQ ID NO: 5 targeting AF4 exon 5, SEQ ID NO: 9 targeting AF6 exon 2, SEQ ID NO: 13 targeting ENL exon 7, SEQ ID NO: 14 targeting ELL exon 3, and any combination thereof. In some embodiments, at least one KMT2A probe includes a nucleotide sequence selected from Sequence ID No. 16, which targets KMT2A exon 7, Sequence ID No. 22, which targets KMT2A exon 9, and any combination thereof.In some embodiments, at least one partner gene probe includes a nucleotide sequence selected from SEQ ID NO: 18 targeting AF9 exon 6, SEQ ID NO: 17 targeting AF4 exon 5, SEQ ID NO: 19 targeting AF6 exon 2, SEQ ID NO: 21 targeting ENL exon 7, SEQ ID NO: 23 targeting ELL exon 3, and any combination thereof. In some embodiments, the biological sample includes at least one of a peripheral blood sample, a bone marrow sample, a solid tumor sample, and any combination thereof. In some embodiments, the method of the Disclosure further includes the step of identifying at least one target-specific KMT2A fusion gene by nucleic acid sequencing of the target cancer, each target-specific KMT2A fusion gene comprising a KMT2A fragment fused with a target-specific partner gene fragment at a fusion site, at least one reverse PCR primer further comprising an additional reverse PCR primer targeting the target-specific partner gene start codon of the target-specific partner gene fragment downstream of the fusion site, and at least one partner gene probe further comprising at least one target-specific partner gene probe comprising a second fluorescent reporter, each target-specific partner gene probe configured to anneal to the target-specific partner gene fragment between the target-specific partner gene start codon and the fusion site. In some embodiments, the method of the Disclosure further includes the step of classifying the subject as having measurable residual disease if the expression level of at least one oncogenic KMT2A fusion gene, as measured by droplet digital PCR (ddPCR), exceeds a threshold of 0.001%.In some embodiments, the method of the present disclosure further includes the steps of providing a first biological sample and a second biological sample from a subject, the second biological sample being obtained after the subject has been treated; estimating a first expression level and a second expression level of at least one oncogenic KMT2A fusion gene based on the first and second biological samples; and estimating the efficacy of the treatment, the development of a treatment-related oncogenic KMT2A fusion gene, prognosis, and any combination thereof based on the change between the first and second expression levels of at least one oncogenic KMT2A fusion gene.

[0011] In another aspect, the present disclosure provides a method for individually monitoring the expression levels of at least one oncogenic KMT2A fusion gene in a subject, wherein each oncogenic KMT2A fusion gene comprises a KMT2A fragment fused with a partner gene fragment at a fusion site. The method of the present disclosure includes the steps of: providing an initial sequencing at nucleotide-level resolution for a leukemia of the subject; and identifying at least one oncogenic KMT2A fusion gene based on the initial sequencing. The method of the present disclosure further includes providing a biological sample of interest comprising a predetermined amount of RNA; at least one forward PCR primer targeting the KMT2A start codon of a KMT2A fragment upstream of a fusion site, and at least one reverse PCR primer targeting the partner gene start codon of a partner gene fragment downstream of the fusion site; at least one KMT2A probe comprising a first fluorescent reporter, each KMT2A probe configured to anneal to a KMT2A fragment between the KMT2A start codon and the fusion site; and at least one partner gene probe comprising a second fluorescent reporter, each partner gene probe configured to anneal to a partner gene fragment between the partner gene start codon and the fusion site. The method of the present disclosure further comprises the steps of: extracting a predetermined amount of RNA from a biological sample and synthesizing a predetermined amount of cDNA from the predetermined amount of RNA; subjecting a mixture of the predetermined amount of cDNA, at least one forward PCR primer, at least one reverse PCR primer, at least one KMT2A probe, and at least one partner gene probe to droplet digital PCR (ddPCR) to obtain a plurality of fluorescence intensity pairs from a first fluorescent reporter and a second fluorescent reporter; and estimating the expression level of at least one oncogenic KMT2A fusion gene based on the plurality of fluorescence intensity pairs obtained by droplet digital PCR (ddPCR).

[0012] In another aspect, the present disclosure provides an assay for monitoring the expression level of at least one oncogenic KMT2A fusion gene in a subject, wherein each oncogenic KMT2A fusion gene comprises a KMT2A fragment fused with a partner gene fragment at a fusion site. The assay of the present disclosure comprises at least one forward PCR primer targeting the KMT2A start codon of the KMT2A fragment upstream of the fusion site, and at least one reverse PCR primer targeting the partner gene start codon of the partner gene fragment downstream of the fusion site; 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 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 gene start codon and the fusion site. At least one forward PCR primer, at least one reverse PCR primer, at least one KMT2A probe, and at least one partner gene probe are combined with a predetermined amount of cDNA synthesized from a predetermined amount of RNA obtained from a target to form a mixture for processing in a droplet digital PCR (ddPCR) instrument. In some embodiments, the first and second fluorescent reporters are selected independently from fluorescein and hexachlorofluorescein, and the first and second fluorescent reporters are different from each other. In some embodiments, at least one oncogenic KMT2A fusion gene is selected from KMT2A-AF9, KMT2A-AF4, KMT2A-AF6, KMT2A-ENL, KMT2A-ELL, at least one target-specific KMT2A fusion gene, and any combination thereof. In some embodiments, at least one oncogenic KMT2A fusion gene is one KMT2A fusion gene selected from the group consisting of KMT2A-AF9, KMT2A-AF4, KMT2A-AF6, KMT2A-ENL, and KMT2A-ELL.In some embodiments, at least one forward PCR primer targets the KMT2A start codon of a KMT2A fragment selected from KMT2A exon 7, KMT2A exon 9, and any combination thereof, and at least one reverse PCR primer targets the partner gene start codon of a partner gene fragment selected from AF9 exon 6, AF4 exon 5, AF6 exon 2, ENL exon 7, ELL exon 3, and any combination thereof. In some embodiments, at least one forward PCR primer includes a nucleotide sequence selected from SEQ ID NO: 1 targeting KMT2A exon 7, SEQ ID NO: 2 targeting KMT2A exon 9, and any combination thereof, and at least one reverse PCR primer includes a nucleotide sequence selected from SEQ ID NO: 7 targeting AF9 exon 6, SEQ ID NO: 5 targeting AF4 exon 5, SEQ ID NO: 9 targeting AF6 exon 2, SEQ ID NO: 13 targeting ENL exon 7, SEQ ID NO: 14 targeting ELL exon 3, and any combination thereof. In some embodiments, at least one KMT2A probe includes a nucleotide sequence selected from SEQ ID NO: 16 targeting KMT2A exon 7, SEQ ID NO: 22 targeting KMT2A exon 9, and any combination thereof. In some embodiments, at least one partner gene probe includes a nucleotide sequence selected from SEQ ID NO: 18 targeting AF9 exon 6, SEQ ID NO: 17 targeting AF4 exon 5, SEQ ID NO: 19 targeting AF6 exon 2, SEQ ID NO: 21 targeting ENL exon 7, SEQ ID NO: 23 targeting ELL exon 3, and any combination thereof.

[0013] In another embodiment, the present disclosure provides a kit for monitoring the expression level of at least one oncogenic KMT2A fusion gene in a subject, wherein each oncogenic KMT2A fusion gene comprises a KMT2A fragment fused with a partner gene fragment at a fusion site. The kit of the present disclosure comprises at least one forward PCR primer targeting the KMT2A start codon of the KMT2A fragment upstream of the fusion site, and at least one reverse PCR primer targeting the partner gene start codon of the partner gene fragment downstream of the fusion site, 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 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 gene start codon and the fusion site. In some embodiments, the first and second fluorescent reporters are independently selected from fluorescein and hexachlorofluorescein, and the first and second fluorescent reporters are different from each other. In some embodiments, at least one oncogenic KMT2A fusion gene is selected from KMT2A-AF9, KMT2A-AF4, KMT2A-AF6, KMT2A-ENL, KMT2A-ELL, at least one target-specific KMT2A fusion gene, and any combination thereof. In some embodiments, at least one oncogenic KMT2A fusion gene is one KMT2A fusion gene selected from the group consisting of KMT2A-AF9, KMT2A-AF4, KMT2A-AF6, KMT2A-ENL, and KMT2A-ELL.In some embodiments, at least one forward PCR primer targets the KMT2A start codon of a KMT2A fragment selected from KMT2A exon 7, KMT2A exon 9, and any combination thereof, and at least one reverse PCR primer targets the partner gene start codon of a partner gene fragment selected from AF9 exon 6, AF4 exon 5, AF6 exon 2, ENL exon 7, ELL exon 3, and any combination thereof. In some embodiments, at least one forward PCR primer includes a nucleotide sequence selected from SEQ ID NO: 1 targeting KMT2A exon 7, SEQ ID NO: 2 targeting KMT2A exon 9, and any combination thereof, and at least one reverse PCR primer includes a nucleotide sequence selected from SEQ ID NO: 7 targeting AF9 exon 6, SEQ ID NO: 5 targeting AF4 exon 5, SEQ ID NO: 9 targeting AF6 exon 2, SEQ ID NO: 13 targeting ENL exon 7, SEQ ID NO: 14 targeting ELL exon 3, and any combination thereof. In some embodiments, at least one KMT2A probe includes a nucleotide sequence selected from SEQ ID NO: 16 targeting KMT2A exon 7, SEQ ID NO: 22 targeting KMT2A exon 9, and any combination thereof. In some embodiments, at least one partner gene probe includes a nucleotide sequence selected from SEQ ID NO: 18 targeting AF9 exon 6, SEQ ID NO: 17 targeting AF4 exon 5, SEQ ID NO: 19 targeting AF6 exon 2, SEQ ID NO: 21 targeting ENL exon 7, SEQ ID NO: 23 targeting ELL exon 3, and any combination thereof.

[0014] Further aspects and embodiments of methods for detecting at least one oncogenic fusion gene in a subject are disclosed in detail herein. [Brief explanation of the drawing]

[0015] Those skilled in the art will understand that the drawings described below are for illustrative purposes only. The drawings described below are not intended to limit the scope of this instruction in any way.

[0016] [Figure 1A] Figure 1A is a schematic diagram illustrating the design of primers and probes to enable detection of the KMT2A-AF4 fusion gene. The primers straddle the fusion cleavage site (KMT2A-AF4 shown). The nested fluorescently labeled probes recognize specific sequences in KMT2A (FAM) and its fusion partner AFF1-AF4 (HEX). [Figure 1B] Figure 1B is a representative graph of ddPCR output containing double-positive droplets marking KMT2A fusion gene transcripts. The ddPCR results include double-positive droplets containing cDNA with the oncogenic KMT2A fusion gene (+). Double-negative droplets do not contain wild-type KMT2AcDNA or DNA derived from KMT2A (-). [Figure 2A] Figure 2A is a graph showing the serial dilutions of 457 cell lines containing KMT2A fusion genes in OCI-AML3 cells (KMT2A458 wild-type) at six orders of magnitude concentrations. Oncogenic KMT2A fusion genes were detected using primer / probe pairs targeting 459 cell type-specific KMT2A fusion genes. [Figure 2B] Figure 2B is a graph summarizing the droplet digital PCR results of HEL cells edited with CRISPR / Cas9 using guides targeting the KMT2A and AF9 gene loci. HEL cells containing the KMT2A-AF9 fusion gene were detected on days 4, 14, and 21 (top panel). The abundance of the fusion transcript is shown in comparison to the abundance of the wild-type KMT2A transcript (bottom panel). [Figure 2C] Figure 2C shows the time-course quantification results of the KMT2A-AF9 fusion protein in the edited HEL cells shown in Figure 2B, normalized to the expression level of the wild-type KMT2A transcript. [Figure 3A]Figure 3A is a graph summarizing ddPCR results using pooled primer / probe pairs targeting the five most common KMT2A fusion genes (left side). The KMT2A fusion gene was detected in THP-1 cells (KMT2A-AF9) but not in Kasumi cells (wild-type KMT2A). For comparison, the expression level of wild-type KMT2A transcript is also shown. [Figure 3B] Figure 3B is a graph showing the expression level of KMT2A fusion gene transcripts from multiple cell lines known to harbor KMT2A fusion genes, normalized to the expression level of wild-type KMT2A transcript. [Figure 3C] Figure 3C is a graph showing the amount of KMT2A fusion gene transcripts from multiple patient samples known to harbor KMT2A fusion genes, normalized to the expression level of wild-type KMT2A transcript. [Figure 3D] Figure 3D is a graph of a serial dilution experiment spanning a 6-log concentration range using pooled primer / probe pairs targeting the five most common KMT2A fusion genes. [Figure 4] Figure 4 is a table showing primer and probe sequences for KMT2A fusion gene detection. All tested primers are listed. * indicates a primer shifted forward for the ddPCR assay. [Figure 5] Figure 5 is a table showing single guide RNA molecules designed for introducing KMT2A-AF9 rearrangement using CRISPR / Cas9. [Figure 6] Figure 6 is a set of UCSC Genome Browser mapping results (https: / / genome.ucsc.edu / ; last accessed 2 / 10 / 2023), primer / probe sequences, and cell line cDNA sequencing results for KMT2A fusion genes in cell lines from DepMap (https: / / depmap.org / portal / ; last accessed 12 / 17 / 2021). Gene annotations are provided by NCBI RefSeq and Ensembl (last access date: February 10, 2023). [Figure 7]Figure 7 is a series of graphs of a dilution series experiment performed to evaluate fusion-specific primer / probe pairs using qPCR. For comparison, a combination of primer / probe pairs targeting KMT2A wild-type transcripts is also shown. [Figure 8A] Figure 8A is a series of graphs summarizing ddPCR results of a dilution series in which KOPN8 (KMT2A-ENL) cell line harboring a known KMT2A fusion gene was diluted into OCI-AML3 cells (KMT2A wild-type). The KMT2A fusion gene was detected using a primer / probe pair designed to target a cell-type-specific KMT2A fusion gene. Dilutions from top to bottom are 50%, 5%, 0.5%, 0.05%, 0.005%, and 0.0005%. Horizontal and vertical lines indicate positive and negative cutoff values for fluorescence intensity. [Figure 8B] Figure 8B is a series of graphs summarizing ddPCR results of a dilution series in which MOLM13 (KMT2A-AF9) cell line harboring a known KMT2A fusion gene was diluted into OCI-AML3 cells (KMT2A wild-type). [Figure 8C] Figure 8C is a series of graphs summarizing ddPCR results of a dilution series in which MV4-11 KOPN8 (KMT2A-AF4) cell line harboring a known KMT2A fusion gene was diluted into OCI-AML3 cells (KMT2A wild-type). [Figure 8D] Figure 8D is a series of graphs summarizing ddPCR results of a dilution series in which OCI-AML2 (KMT2A-AF6) cell line harboring a known KMT2A fusion gene was diluted into OCI-AML3 cells (KMT2A wild-type). [Figure 8E] Figure 8E is a series of graphs summarizing ddPCR results of a dilution series in which THP-1 (KMT2A-AF9) cell line harboring a known KMT2A fusion gene was diluted into OCI-AML3 cells (KMT2A wild-type). [Figure 9A]Figure 9A is a series of graphs summarizing the ddPCR results for individual KMT2A fusion gene primer / probe pairs targeting KMT2A-AF9. To evaluate off-target amplification and probe binding, the tests were conducted in cell lines containing different KMT2A fusion genes. [Figure 9B] Figure 9B is a series of graphs summarizing the ddPCR results for individual KMT2A fusion gene primer / probe pairs targeting KMT2A-AF6. To evaluate off-target amplification and probe binding, the tests were conducted in cell lines containing different KMT2A fusion genes. [Figure 9C] Figure 9C is a series of graphs summarizing the ddPCR results for individual KMT2A fusion gene primer / probe pairs targeting KMT2A-AF4. To evaluate off-target amplification and probe binding, the tests were conducted in cell lines containing different KMT2A fusion genes. [Figure 9D] Figure 9D is a series of graphs summarizing the ddPCR results for individual KMT2A fusion gene primer / probe pairs targeting KMT2A-ELL. To evaluate off-target amplification and probe binding, the tests were conducted in cell lines containing different KMT2A fusion genes. [Figure 9E] Figure 9E is a series of graphs summarizing the ddPCR results for individual KMT2A fusion gene primer / probe pairs targeting KMT2A-ENL. To evaluate off-target amplification and probe binding, the tests were conducted in cell lines containing different KMT2A fusion genes. [Figure 10A] Figure 10A is a series of graphs summarizing the ddPCR results of pooled primer / probe pairs for THP-1 and OCI-AML2, cell lines containing the KMT2A fusion gene. The ddPCR results using pooled KMT2A fusion gene primers / probes (left) are shown in comparison with the KMT2A wild-type primer / probe pair (right). The KMT2A fusion gene was not detected in KMT2A wild-type cell lines (OCI-AML3, Kasumi, Jurkat). [Figure 10B]Figure 10B is a series of graphs summarizing the ddPCR results using pooled primer / probe pairs for cell lines MV4-11 and MOL13, which contain the KMT2A fusion gene. The ddPCR results using pooled KMT2A fusion gene primers / probes (left) are shown in comparison with those using KMT2A wild-type primer / probe pairs (right). [Figure 10C] Figure 10C is a series of graphs summarizing the ddPCR results using pooled primer / probe pairs for the cell line KOPN8, which contains the KMT2A fusion gene. The ddPCR results using pooled KMT2A fusion gene primers / probes (left) are shown in comparison with those using KMT2A wild-type primer / probe pairs (right). [Figure 10D] Figure 10D is a series of graphs summarizing the ddPCR results of pooled primer / probe pairs for cell lines OCI-AML3 and Kasumi, which do not possess the KMT2A fusion gene. The plots show the ddPCR results using pooled KMT2A fusion gene primers and probes (left) in comparison with the KMT2A wild-type primer / probe pair (right). The KMT2A fusion gene was not detected in KMT2A wild-type cell lines (OCI-AML3, Kasumi, Jurkat). [Figure 10E] Figure 10E is a series of graphs summarizing the ddPCR results of pooled primer / probe pairs for the Jurkat cell line, which does not possess the KMT2A fusion gene. The plots show the ddPCR results using pooled KMT2A fusion gene primers / probes (left) in comparison to the KMT2A wild-type primer / probe pair (right). The KMT2A fusion gene was not detected in KMT2A wild-type cell lines (OCI-AML3, Kasumi, Jurkat). [Figure 11A]Figure 11A is a series of graphs summarizing the ddPCR results measured using pooled primers / probes targeting the five most frequent KMT2A fusion genes in two AML patient samples with known KMT2A fusion genes. The plots show the ddPCR results using pooled KMT2A fusion gene primers / probes (left) compared to the results using a control pair of wild-type KMT2A primers / probes (right). [Figure 11B] Figure 11B is a series of graphs summarizing the ddPCR results measured using pooled primers / probes targeting the five most common KMT2A fusion genes in two additional AML patient samples with known KMT2A fusion genes. The plots show the ddPCR results using pooled KMT2A fusion gene primers / probes (left) compared to the results using a control KMT2A wild-type primer / probe pair (right). [Figure 11C] Figure 11C is a series of graphs summarizing the ddPCR results of AML patient samples without KMT2A fusion genes, tested using pooled primers / probes targeting the five most common KMT2A fusion genes. The plots show the ddPCR results using pooled KMT2A fusion gene primers / probes (left) compared to a control pair of KMT2A wild-type primers / probes (right). [Figure 11D] Figure 11D is a series of graphs summarizing the ddPCR results of additional AML patient samples without KMT2A fusion genes, tested using pooled primers / probes targeting the five most common KMT2A fusion genes. The plots show the ddPCR results using pooled KMT2A fusion gene primers / probes (left) compared to the KMT2A wild-type primer / probe pair as a control (right). [Figure 12A]Figure 12A is a series of graphs summarizing the ddPCR results for a pooled primer / probe dilution series when KOPN8 (KMT2A-ENL) cell lines containing known KMT2A fusion genes were diluted in OCI-AML3 cells (KMT2A wild-type). KMT2A fusion genes were detected using pooled primers / probes designed to target the five most common KMT2A fusion genes. [Figure 12B] Figure 12B is a series of graphs summarizing the ddPCR results for pooled primer / probe dilution series when MOLM13 (KMT2A-AF9) cell lines, which possess known KMT2A fusion genes, were diluted in OCI-AML3 cells (KMT2A wild-type). KMT2A fusion genes were detected using pooled primer / probes targeting the five most common KMT2A fusion genes. [Figure 12C] Figure 12C is a series of graphs summarizing the ddPCR results for pooled primer / probe dilution series when MV4-11 (KMT2A-AF4) cell lines, which possess known KMT2A fusion genes, were diluted in OCI-AML3 cells (KMT2A wild-type). KMT2A fusion genes were detected using pooled primer / probes targeting the five most common KMT2A fusion genes. [Figure 12D] Figure 12D is a series of graphs summarizing the ddPCR results for a pooled primer / probe dilution series when OCI-AML2 (KMT2A-AF6) cell lines containing known KMT2A fusion genes were diluted in OCI-AML3 cells (KMT2A wild-type). KMT2A fusion genes were detected using pooled primers / probes targeting the five most common KMT2A fusion genes. [Figure 12E] Figure 12E is a series of graphs summarizing the ddPCR results for pooled primer / probe dilution series when THP-1 (KMT2A-AF9) cell lines containing known KMT2A fusion genes were diluted in OCI-AML3 cells (KMT2A wild-type). KMT2A fusion genes were detected using pooled primer / probes targeting the five most common KMT2A fusion genes. [Figure 13] Figure 13 is a table summarizing the pooled primer / probe mixtures used to prepare the 10-fold concentrated master mix. The final master mix volume was 200 μL. In the final 20 μL ddPCR reaction, the stock primers and probes were 100 μL each, the primers were 1000 μL each, and the probes were 250 nM each. [Figure 14] Figure 14 is a table summarizing the ddPCR results of dilution series using fusion-specific primers / probes. It shows the concentrations of fusion transcripts and wild-type KMT2A transcripts in dilution series experiments performed by diluting cell lines containing known KMT2A fusion genes into OCI-AML3 cells. Fusions were measured using individual primer / probe pairs designed to detect the fusion. [Figure 15] Figure 15 is a table summarizing the ddPCR results for several patients with KMT2A fusion gene-driven leukemia. The amount of KMT2A fusion gene transcripts measured by ddPCR is compared with standard pathological assessments of leukemia burden and KMT2A fusion gene abundance (including the percentage of leukemic blasts by fluorescence insitu hybridization (FISH), cytogenetic assessment, whole-genome sequencing (WGS), morphological assessment, and flow cytometry). ND indicates not detected, and N / A indicates not applicable / not evaluated. [Figure 16] Figure 16 is a table summarizing the ddPCR results of the dilution series using pooled primers / probes. In the dilution series experiments using cell lines with known KMT2A fusion genes, the concentrations of the fusion transcript and the wild-type KMT2A transcript were measured after dilution in OCI-AML3 cells. To detect the panel of KMT2A fusion genes, the fusion was assayed using the pooled primer / probe pairs shown in Figure 13. [Modes for carrying out the invention]

[0017] In various embodiments, droplet digital PCR (ddPCR) assays for detecting oncogenic fusion genes are disclosed. In one exemplary embodiment, a ddPCR assay for detecting five of the most common KMT2A fusion genes, which account for the majority (approximately 80%) of oncogenic KMT2A fusion genes found in treatment-associated acute myeloid leukemia (t-AML), is disclosed. In various embodiments, the ddPCR assays of this disclosure can be used to improve disease detection and treatment decision-making in t-AML patients with KMT2A fusion genes, and to detect oncogenic fusion genes in precancerous conditions in patients at risk after chemotherapy exposure.

[0018] The assays of this disclosure build upon previous studies on the detection of multiple oncogenic fusion genes in Ewing sarcoma and improve the detection of various cancer types, including (but not limited to) fusion gene-driven fluid carcinomas. As described in the examples, the assays of this disclosure are benchmarked using known cell lines and primary patient samples containing the KMT2A fusion gene. The assays of this disclosure provide an inexpensive, rapid, easy-to-implement, highly sensitive, specific, and easily interpretable platform for the detection and quantification of the KMT2A fusion gene and other oncogenic fusion genes, improving the detection of measurable residual lesions in various patients, including (but not limited to) AML patients with the KMT2A fusion gene.

[0019] The method described herein uses droplet digital PCR (ddPCR) technology to identify oncogenic fusion genes, overcoming at least some of the limitations of existing oncogenic fusion gene detection methods such as RT-qPCR. Because quantification by ddPCR is absolute, using ddPCR as the detection modality eliminates the need for calibration using standard curves, which was used in conventional methods. While previous studies have reported qPCR and ddPCR assays for fusion gene detection in CML (BCR-ABL1), none have been reported for AML. In some embodiments, the assay described herein is suitable for detecting a variety of oncogenic fusion genes associated with various cancers, including (but not limited to) the oncogenic KMT2A fusion gene, one of the most common treatment-associated AML translocations.

[0020] Conventional oncogenic fusion gene assays have targeted a single oncogenic fusion gene. For example, the detection of the BCR-ABL1 fusion gene used in monitoring the progression of chronic myeloid leukemia (CML) falls into this category. Such assays were effective because BCR fuses only with ABL1. In contrast, KMT2A is known to fuse with up to 80 different binding partners, making KMT2A fusion genes difficult to target.

[0021] In some embodiments, the assays of the Disclosure are configured to detect KMT2A fusion genes involving multiple oncogenic fusion gene partners. In various embodiments, the assays of the Disclosure are configured to detect multiple different KMT2A fusion genes without being limited to the detection of a single oncogenic fusion gene. As a non-limiting example, Table 1 summarizes several oncogenic KMT2A fusion genes associated with AML / BALL leukemia types that can be detected using the assay methods of the Disclosure. In one embodiment, the assays of the Disclosure are configured to detect multiple KMT2A fusion gene partners covering 80% of the most commonly found KMT2A fusion genes in t-AML. Examples of KMT2A fusion genes include KMT2A-AF9(MLLT1), KMT2A-AF4(AFF1), KMT2A-AF6(AFDN), KMT2A-ENL(MLLT1), and KMT2A-ELL(ELL) fusion genes (their partner genes are shown in parentheses). The assay described herein provides a novel mechanism for detecting the above-mentioned fusion gene with high sensitivity in order to detect residual lesions.

[0022] [Table 1]

[0023] As described in the examples, a method for detecting KMT2A fusion genes by dual-color ddPCR according to one embodiment achieves highly sensitive detection across several logs of the dynamic range and can reliably exclude patient samples and cell lines that do not contain KMT2A fusion genes. The assay of this disclosure improves upon existing qPCR strategies by its ease of use, accurate quantification of transcripts, ease of multiplex analysis, and flexibility or reproducibility of modification or expansion of the target panel. Since quantification by ddPCR is absolute quantification, the assay of this disclosure does not require calibration with a standard curve. Previous efforts to develop digital quantification of KMT2A fusion genes have been limited by the polybinding nature of KMT2A fusion genes, which have more than 80 known KMT2A fusion gene partners.

[0024] In some embodiments, the assays of the present disclosure can be configured to target the five most common KMT2A fusion gene partners, which account for approximately 80% of KMT2A-reconstituted AML cases. As demonstrated in the examples, the detection limit of the assays of the present disclosure varies with the amount of input material, but in the dilution experiments described herein, cells with KMT2A fusion genes could be reliably identified even when their estimated abundance was as low as 10 out of a total of 2 million cells.

[0025] In various embodiments, the assays of the Disclosure are configured to detect at least one potential oncogenic fusion gene, without being limited to the detection of a single oncogenic fusion gene. In some embodiments, the assays of the Disclosure are configured to detect one oncogenic fusion gene. In such embodiments, at least a portion of all oncogenic fusion genes associated with a particular oncogenic type can be detected using multiple assays, each configured to detect a different oncogenic fusion gene associated with that oncogenic type. In other embodiments, the assays of the Disclosure are pooled assays configured to simultaneously detect two or more oncogenic fusion genes associated with a particular oncogenic type.

[0026] While not limited to any particular theory, increasing the number of detectable oncogenic fusion genes enhances the sensitivity of the assay. As a non-limiting example, the polybinding ability of KMT2A to translocate with at least 80 known fusion partners has so far hindered the development of highly sensitive RT-qPCR or ddPCR assays for detecting KMT2A fusion genes associated with t-AML. In one embodiment, the assay of this disclosure targets a subset of 80 potentially t-AML-related KMT2A fusion genes. In various embodiments, the subset of oncogenic fusion genes is selected considering the proportion of all known oncogenic fusion genes, for example, to represent at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of all known oncogenic fusion genes associated with t-AML or other cancers. In a non-limiting example, the assay of this disclosure targets five oncogenic KMT2A fusion genes, which account for at least 80% of all oncogenic KMT2A fusion genes known to be associated with t-AML. Even when limited to the most common KMT2A fusion genes, the assay of this disclosure can sensitively and specifically detect KMT2A fusion genes by ddPCR. Given the ease of development and validation of the assay of this disclosure, it is possible to add additional KMT2A fusion genes to the assay of this disclosure with minimal cost and effort.

[0027] In various embodiments, the assays of this disclosure utilize primer / probe pairs that target portions of DNA or RNA sequences located upstream and downstream of an oncogenic fusion gene detected by droplet digital PCR (ddPCR). The assays of this disclosure can be performed using any suitable ddPCR system, including but not limited to the QX200 droplet digital PCR system (Bio-Rad). The DNA or RNA sequences that crosslink the oncogenic fusion gene can be obtained from any suitable source, including but not limited to publicly available sequences. In some embodiments, the RNA sample is expressed on complementary DNA (cDNA) before performing ddPCR.

[0028] Each primer / probe pair comprises a primer that targets a portion of the cDNA sequence and a probe configured to anneal within the corresponding primer. In various embodiments, an upstream primer / probe pair is used that targets a portion of the cDNA sequence of the oncogenic fusion gene upstream of the break, and a downstream primer / probe pair is used that targets a portion of the cDNA sequence of the oncogenic fusion gene downstream of the break within the fusion partner region. In various embodiments, the upstream and downstream probes are labeled with different reporters to facilitate the detection of the oncogenic fusion gene.

[0029] In an exemplary embodiment, the assay of the present disclosure employs a dual-color ddPCR detection method using an upstream probe labeled with a first fluorescent reporter and a downstream probe labeled with a second fluorescent reporter. In various embodiments, the first and second fluorescent reporters are independently selected from fluorescein and hexachlorofluorescein.

[0030] Figure 1A shows the overall design approach of the oncogenic fusion gene detection method of this disclosure, applicable to ddPCR detection and quantification of KMT2A-AF4 fusion genes. As shown in Figure 1A, forward and reverse cDNA primers are provided to generate cDNA from an RNA sample containing the oncogenic fusion gene. As shown in Figure 1A, a forward PCR primer is used that targets the KMT2A start codon of the KMT2A fragment upstream of the fusion site, and at least one reverse PCR primer is used that targets the start codon of the partner gene (AF4) fragment downstream of the fusion site.

[0031] Referring again to Figure 1A, the upstream probe is configured to anneal to the KMT2A fragment between the KMT2A start codon and the fusion site, and the downstream probe is configured to anneal to the partner gene (AF4) fragment between the partner gene (AF4) start codon and the fusion site. As shown in Figure 1A, the first fluorescent reporter for the upstream probe is fluorescein (FAM), and the second fluorescent reporter for the downstream probe is hexachlorofluorescein (HEX).

[0032] Figure 1B shows typical output from ddPCR analysis using the dual-color probe described above. Each droplet is classified as either a double-positive droplet, corresponding to a droplet containing signals from both the first and second fluorescent reporters, or a double-negative droplet, corresponding to an empty droplet or a droplet containing wild-type (i.e., non-fused) KMT2A.

[0033] In various embodiments, the assays of this disclosure can be performed to detect and quantify one type of oncogenic KMT2A fusion gene as described above. In some embodiments, multiple assays can be used to individually detect and quantify multiple types of oncogenic KMT2A fusion genes, thereby quantifying the type and abundance of each oncogenic KMT2A fusion gene. In an assay configured to detect one type of oncogenic KMT2A fusion gene, the result of double-positive ddPCR (see Figure 1B) indicates the presence and abundance of one type of oncogenic KMT2A fusion gene. The result of double-negative ddPCR is associated with empty cells, cells containing wild-type KMT2A, and / or cells containing oncogenic KMT2A fusion genotypes other than the single fusion type designed for the assay.

[0034] In various other embodiments, the assay of the present disclosure can be performed to identify two or more oncogenic KMT2A fusion genes using a pooled primer / probe pair. In this embodiment, the assay of the present disclosure comprises at least one forward PCR primer targeting the KMT2A start codon of the KMT2A fragment upstream of the fusion site, and a plurality of reverse PCR primers. Each reverse PCR primer targets the partner gene start codon of each partner gene fragment downstream of the fusion site. In various embodiments, each reverse primer corresponds to a different partner gene in a single oncogenic KMT2A fusion genotype. Furthermore, in this embodiment, the assay of the present disclosure comprises at least one first probe as described above, and a plurality of second probes. Each second probe is configured to anneal to a single fusion-type partner gene fragment between the partner gene start codon and the fusion site. PCR of different fusion types is achieved by using a reverse primer that targets each partner protein of each fusion type, and a forward primer that is shared by all fusion type PCRs, since all fusion types share the upstream KMT2A.

[0035] In various embodiments, in pooled assays using multiple downstream primers and probes targeting different partner genes associated with different types of oncogenic KMT2A fusion genes, the output of ddPCR is similar to the representative output shown in Figure 1B. In pooled assays, all downstream probes targeting different partner proteins share the same type / color fluorescent reporter, so each double-positive result corresponds to one of the droplets containing any type of KMT2A fusion gene targeted by the assay. Therefore, the KMT2A fusion gene expression level obtained from the ddPCR result represents the pooled total of all types of KMT2A fusion genes targeted by the assay. In this pooled assay, double-negative results represent empty cells, cells containing wild-type KMT2A, and / or cells containing a different type of KMT2A fusion gene than the fusion targeted by the primer / probe pair of the assay.

[0036] In various embodiments, the design and methodology of the assays of the Disclosure can be readily adapted to include or exclude the detection of specific types of KMT2A fusion genes by including or excluding primer / probe pairs that target partner genes of specific types of KMT2A fusion genes. As a non-limiting example, the assays of the Disclosure can include primer / probe pairs that target a group of KMT2A fusion gene types that account for 80% of all KMT2A fusion genes observed in AML patients (see below for further details). As another non-limiting example, the assays of the Disclosure can be adapted to include patient-specific types in certain embodiments. For example, the assays of the Disclosure can be adapted to the KMT2A fusion gene types of individual patients by including primer / probe pairs that target partner genes of the fusion type identified by genome sequencing of the patient's leukemia or other cancer.

[0037] In various embodiments, forward and reverse PCR primers are configured to generate amplicons (unit replication sequences) containing the KMT2A fragment and a partner gene fragment adjacent to the fusion region. In various embodiments, forward and reverse PCT primers are configured to generate amplicons having lengths ranging from approximately 300 base pairs (bp) to approximately 500 bp. In various other embodiments, forward and reverse PCT primers are configured to generate amplicons having lengths ranging from approximately 300 bp to approximately 320 bp, approximately 310 bp to approximately 330 bp, approximately 320 bp to approximately 340 bp, approximately 330 bp to approximately 350 bp, approximately 340 bp to approximately 360 bp, approximately 350 bp to approximately 370 bp, approximately 350 bp to approximately 380 bp, approximately 370 bp to approximately 390 bp, approximately 380 bp to approximately 400 bp, and approximately 390 bp to approximately It is configured to generate amplicons having lengths in the range of 410bp, approximately 400bp to 420bp, approximately 410bp to 430bp, approximately 420bp to 440bp, approximately 430bp to 450bp, approximately 440bp to 460bp, approximately 450bp to 470bp, approximately 460bp to 480bp, approximately 470bp to 490bp, or approximately 480bp to 500bp.

[0038] In various other embodiments, the amplicon and probe are configured to provide preferred annealing conditions including an annealing temperature Tm of about 60°C. In various other embodiments, the amplicon and probe are configured to provide preferred annealing conditions including an annealing temperature Tm of about 52°C, about 53°C, about 54°C, about 55°C, about 56°C, about 57°C, about 58°C, about 59°C, about 60°C, about 61°C, about 62°C, about 63°C, or about 64°C.

[0039] Although the oncogenic fusion gene detection method described herein is described in relation to the analysis of RNA expression levels, the oncogenic fusion gene detection method described herein may be modified to analyze DNA expression patterns indicating oncogenic fusion genes.

[0040] In some embodiments, the assays of this disclosure detect oncogenic fusion genes by detecting RNA in a biological sample. RNA is typically less stable than DNA and requires fusion expression for detection, but RNA is well-suited for detecting oncogenic fusion genes in genes characterized by translocation breakpoint heterogeneity. As a non-limiting example, the KMT2A gene is characterized primarily by translocation breakpoint heterogeneity occurring between exon 7 and exon 11. Without limiting to any particular theory, assays that detect oncogenic fusion genes using DNA-based measurements require a very large number of primer / probe pairs to cover the same set of translocation breakpoints as the corresponding RNA-based assay.

[0041] In some embodiments, the RNA-based assays of this disclosure involve normalizing the measured fusion transcript abundance relative to housekeeping genes. While not limited to any particular theory, in RNA-based assays, the detection of RNA indicating an oncogenic fusion gene depends on the expression of the oncogene, and consequently, the transcript abundance does not necessarily correlate with the burden of leukemia or other cancers. In one embodiment of the assay of this disclosure, the abundance of a KMT2A fusion gene is compared to the expression of wild-type KMT2A, which should have similar gene regulation and expression levels. As shown in the examples of this specification, this normalization scheme was validated by cell line dilution experiments in which the proportion of cells containing the KMT2A fusion gene was estimated using the ratio of the abundance of the KMT2A fusion gene to the abundance of the wild-type KMT2A transcript. The estimated proportion of cells was consistent with the amounts in each serial dilution. While not limited to any particular theory, other ddPCR fusion assays also employ similar methods for normalizing fusion transcript abundance relative to housekeeping genes.

[0042] In various embodiments, the assays of this disclosure are suitable for use in detecting residual disease after treatment. In one embodiment, the assays of this disclosure are suitable for use in detecting AML with a KMT2A fusion gene and in the early detection of the KMT2A fusion gene in individuals at risk of developing KMT2A fusion gene-driven t-AML. In patients with KMT2A fusion gene-driven AML, the assays of this disclosure can enhance or replace standard methods for detecting residual disease during treatment, after hematopoietic stem cell transplantation, or during long-term follow-up. This is analogous to the dramatic improvement in CML treatment decision-making made possible by high-sensitivity qPCR-based BCR-ABL1 fusion gene detection. In one embodiment, the assays of this disclosure can be directly compared with MPFC and morphological data in the assessment of residual disease during AML treatment.

[0043] In various embodiments, the assays of this disclosure can be used to classify cancer patients as having measurable residual disease based on the expression levels of oncogenic fusion genes measured by ddPCR. In some embodiments, a cancer patient is classified as having measurable residual disease if the expression level of at least one oncogenic KMT2A fusion gene measured by ddPCR exceeds a threshold of 0.001%. In various other embodiments, the threshold for the expression level of at least one oncogenic KMT2A fusion gene measured by ddPCR may be 0.01%, 0.001%, 0.0001%, or 0.00001%.

[0044] In some aspects, patient classification can be used as information for treatment decisions by healthcare professionals. As a non-limiting example, healthcare professionals may recommend additional treatment to cancer patients classified as having measurable residual disease. Alternatively, they may recommend discontinuation of treatment and / or regular monitoring to patients not classified as having measurable residual disease.

[0045] In some embodiments, the assays of the present disclosure can be used to monitor patients after treatment or patients at risk of early recurrence, relapse, or new onset of cancer. In a non-limiting example, the assays of the present disclosure can be used as described above to assess whether a patient is classified as having measurable residual disease, i.e., whether treatment is required.

[0046] In some embodiments, the assays of the present disclosure can be incorporated into interventional clinical trials, and the detected oncogenic fusion genes can be used as biomarkers for treatment decisions. In other embodiments, the assays of the present disclosure can be used to identify precancerous KMT2A fusion genes in patients receiving TOP2 inhibitors and at risk of t-AML. While not limited to any particular theory, the risk of t-AML is considered to be highest in several cancers affecting children and adolescents, such as Ewing sarcoma, Hodgkin lymphoma, and neuroblastoma. In rare cases, treating t-AML is extremely difficult in populations that have already received large lifetime doses of chemotherapy. In other additional embodiments, the assays of the present disclosure can be used to identify individuals at risk and determine the need for intervention before fulminant disease develops.

[0047] The method for detecting oncogenic fusion genes described herein involves analyzing the expression of RNA or DNA from cells isolated from blood or bone marrow samples; however, in various embodiments, the method described herein may be modified to analyze RNA or DNA from cancer cells isolated from other samples, including but not limited to solid tumor samples, cell-free DNA from blood samples, and any other suitable samples containing cancer cells.

[0048] In various embodiments, the assays of this disclosure can be modified as described herein to detect any oncogenic fusion gene associated with any oncogene. A non-limiting list of oncogenic fusion gene products suitable for detection using the methods of this disclosure is provided in Table 1 below.

[0049] [Table 2-1]

[0050] [Table 2-2]

[0051] [Table 2-3]

[0052] [Table 2-4]

[0053] Molecular engineering

[0054] The following definitions and methods are provided to better define this disclosure and to guide those skilled in the art in the practice of this disclosure. Unless otherwise noted, terms should be understood in accordance with the conventional usage by those skilled in the art.

[0055] As used herein, the terms “heterogeneous DNA sequence,” “exogenous DNA fragment,” or “heterogeneous nucleic acid” refer, respectively, to sequences originating from a source different from that of a particular host cell, or, if originating from the same source as that of a particular host cell, to sequences modified from their original form. Therefore, heterogeneous genes in a host cell include genes that are endogenous to a particular host cell but have been modified, for example, by DNA shuffling or cloning. These terms also include multiple copies of naturally occurring DNA sequences that do not exist naturally. Thus, these terms refer to DNA fragments that are heterogeneous or heterologous to a cell, or homologous to a cell but located in a position not normally found within the host cell’s nucleic acid. Exogenous DNA fragments are expressed to produce exogenous polypeptides. A “homologous” DNA sequence is a DNA sequence that naturally binds to the host cell into which it is introduced.

[0056] Expression vectors, expression constructs, plasmids, or recombinant DNA constructs are generally understood to refer to nucleic acids produced by artificial intervention, including recombinant methods or direct chemosynthesis, using a set of specific nucleic acid elements that enable the transcription or translation of a particular nucleic acid in a host cell. An expression vector may be part of a plasmid, virus, or nucleic acid fragment. Generally, an expression vector may contain the nucleic acid to be transcribed, operably linked to a promoter.

[0057] A "promoter" is generally understood as a nucleic acid regulatory sequence that directs the transcription of a nucleic acid. Inducible promoters are generally understood as promoters that mediate the transcription of a gene that is operably linked in response to a specific stimulus. In the case of polymerase-type II promoters, a promoter may contain the necessary nucleic acid sequence near the transcription start site, such as a TATA element. Promoters may optionally contain distal enhancer or repressor elements, which can be located several thousand base pairs away from the transcription start site.

[0058] As used herein, “transcribeable nucleic acid molecule” refers to a nucleic acid molecule that can be transcribed into an RNA molecule. Methods are known for introducing constructs into cells so that the transcribed nucleic acid molecule is transcribed into a functional mRNA molecule (i.e., translated and expressed as a protein product). Alternatively, the construct may be constructed to express an antisense RNA molecule in order to inhibit the translation of a specific RNA molecule of interest. Conventional compositions and methods for constructing and using constructs and host cells to implement this disclosure are well known to those skilled in the art (see "Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, CP 1988. Methods in Enzymology 167, 747-754").

[0059] The "transcription start site" or "start site" is the position surrounding the first nucleotide, which is part of the transcription sequence, and is also defined as position + 1. All other sequences of the gene and their regulatory regions can be numbered relative to this site. Downstream sequences (i.e., further protein-coding sequences in the 3' direction) can be considered positive, and upstream sequences (most of the regulatory region in the 5' direction) can be considered negative.

[0060] "Operatively linked" or "functionally linked" preferably refers to the linkage of nucleic acid sequences on a single nucleic acid fragment such that the function of one is influenced by the function of the other. For example, if a regulatory DNA sequence is positioned to affect the expression of a coding DNA sequence (i.e., the coding sequence or functional RNA is under the transcriptional control of a promoter), the regulatory DNA sequence is said to be "operably linked" or "linked" to the DNA sequence encoding the RNA or polypeptide. The coding sequence may be operably linked to the regulatory sequence in a sense or antisense direction. The two nucleic acid molecules may be part of or adjacent to a single adjacent nucleic acid molecule. For example, if a promoter regulates or mediates the transcription of a gene of interest within a cell, the promoter is operably linked to the gene of interest.

[0061] A "construct" is generally understood to be a recombinant nucleic acid molecule such as a plasmid, cosmid, virus, autonomously replicating nucleic acid molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule, which originates from any source, is capable of genome integration or autonomous replication, and consists of one or more nucleic acid molecules functionally linked together.

[0062] The constructs of this disclosure may include a promoter operably ligated to a transcriptable nucleic acid molecule operably ligated to a 3' transcription termination nucleic acid molecule. In addition, the constructs may include, but are not limited to, additional regulatory nucleic acid molecules from, for example, the 3' untranslated region (3'UTR). Furthermore, the constructs may include, but are not limited to, the 5' untranslated region (5'UTR) of an mRNA nucleic acid molecule, which plays a crucial role in translation initiation and is a genetic component in the expression construct. These additional upstream and downstream regulatory nucleic acid molecules may be derived from natural or heterologous sources in relation to other elements present on the promoter construct.

[0063] The term "transformation" refers to the process of introducing nucleic acid fragments into the genome of a host cell to bring about genetically stable inheritance. Host cells containing transformed nucleic acid fragments are called "transgenic" cells, and organisms containing transgenic cells are called "transgenic organisms."

[0064] "Transformed," "transgenic," and "recombinant" refer to host cells or organisms, such as bacteria, cyanobacteria, animals, or plants, into which heterologous nucleic acid molecules have been introduced. Nucleic acid molecules are commonly known in the art and can be stably incorporated into genomes (see, for example, Sambrook 1989; Innis 1995; Gelfand 1995; Innis & Gelfand 1999). Known PCR methods, but not limited to, include methods using paired primers, nested primers, single-specific primers, degenerate primers, gene-specific primers, vector-specific primers, and partially mismatched primers. The term "untransformed" refers to normal cells that have not undergone the transformation process.

[0065] "Wild type" refers to viruses or organisms that exist in nature and have no known mutations.

[0066] The design, production, and testing of mutant nucleotides and their encoded polypeptides that possess the required percentage identity and retain the required activity of the expressed protein are within the scope of the art. For example, the directional evolution and rapid isolation of mutants can be carried out according to methods described in literature such as "Link et al. (2007) Nature Reviews 5(9), 680-688; Sanger et al. (1991) Gene 97(1), 119-123; Ghadessy et al. (2001) Proc Natl Acad Sci USA 98(8) 4552-4557," for example. Thus, a person skilled in the art can, for example, produce a large number of nucleotide and / or polypeptide mutants having at least 95-99% identity with respect to the reference sequences described herein and screen for the desired phenotype according to methods common in the art.

[0067] The nucleotide and / or amino acid sequence identity percentage (%) is understood as the proportion of nucleotide or amino acid residues in a candidate sequence that are identical to a reference sequence when two sequences are aligned. To determine the identity percentage, sequences are aligned and gaps are introduced if necessary to maximize the sequence identity percentage. Sequence alignment procedures for determining the identity percentage are well known to those skilled in the art. In many cases, commonly available computer software such as BLAST, BLAST2, ALIGN2, or Megalign (DNASTAR) software is used for sequence alignment. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm necessary to achieve the greatest alignment over the entire length of the sequences being compared. When sequences are aligned, the sequence identity percentage of a given sequence A to a given sequence B (which can also be rephrased as having a sequence identity percentage with respect to a given sequence B) can be calculated as follows: sequence identity percentage = X / Y100, where X is the number of residues scored as a perfect match by the alignment of A and B by the sequence alignment program or algorithm, and Y is the total number of residues in B. If the length of array A is not equal to the length of array B, the sequence identity percentage between A and B will not be equal to the sequence identity percentage between B and A.

[0068] Generally, conservative substitutions can be performed at any position as long as the required activity is maintained. So-called conservative exchanges, where the substituted amino acid has similar properties to the original amino acid, can be performed, such as substitution of Glu with Asp, Gln with Asn, Val with Ile, Leu with Ile, and Ser with Thr. Examples of amino acids with similar properties include aliphatic amino acids (e.g., glycine, alanine, valine, leucine, isoleucine), hydroxyl group or sulfur / selenium-containing amino acids (e.g., serine, cysteine, selenocysteine, threonine, methionine), cyclic amino acids (e.g., proline), aromatic amino acids (e.g., phenylalanine, tyrosine, tryptophan), basic amino acids (e.g., histidine, lysine, arginine), and acidic amino acids or their amides (e.g., aspartic acid, glutamic acid, asparagine, glutamine). Deletion, on the other hand, is the substitution of an amino acid by direct bonding. Deletion sites include the terminals of polypeptides and connections between individual protein domains. Insertions are the introduction of amino acids into a polypeptide chain, and direct linkages are formally substituted by one or more amino acids. Amino acid sequences can be modified with the help of computer simulation programs known in the art, which can generate polypeptides with improved activity or altered regulation, for example. Based on these artificially generated polypeptide sequences, corresponding nucleic acid molecules encoding such modified polypeptides can be synthesized in vitro using specific codon usage methods of the desired host cell.

[0069] "High-stringent hybridization conditions" are defined as hybridization at 65°C in 6XSSC buffer (i.e., 0.9 M sodium chloride and 0.09 M sodium citrate). Given these conditions, it is possible to determine whether a given set of sequences hybridizes by calculating the melting temperature (Tm) of the DNA double helix between the two sequences. If the melting temperature of a particular double helix is ​​lower than 65°C under the 6XSSC salt conditions, the two sequences will not hybridize. On the other hand, if the melting temperature is 65°C or higher under the same salt conditions, the two sequences will hybridize. Generally, the melting temperature of hybridized DNA:DNA sequence can be determined using the following formula:T m = 81.5°C + 16.6(log 10 [Na + ]) + 0.41 (fraction G / C content) - 0.63 (% formamide) - (600 / l). Furthermore, the T of DNA:DNA hybrids m For every 1% decrease in nucleotide identity, the temperature decreases by 1–1.5°C (see, for example, Sambrook and Russel, 2006).

[0070] Host cells can be transformed using various standard techniques known in the art (see, for example, "Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, CP 1988. Methods in Enzymology 167, 747-754"). Examples of such techniques, though not limited to these, include viral infection, calcium phosphate transfection, liposome-mediated transfection, microprojectile-mediated delivery, receptor-mediated uptake, cell fusion, and electroporation. Transfected cells can be selected and grown to provide recombinant host cells containing expression vectors stably integrated into the host cell genome.

[0071] [Table 3]

[0072] [Table 4]

[0073] [Table 5]

[0074] Exemplary nucleic acids that can be introduced into host cells include, for example, DNA sequences or genes from other species, or genes or sequences that originate from or exist in the same species but are incorporated into recipient cells by genetic engineering methods. The term “exogenous” is intended to refer to genes that are not normally present in the transformed cells, or genes that are perhaps simply absent in form, structure, etc., as seen in the transformed DNA fragment or gene, or genes that are normally present but are to be expressed in a manner different from their natural expression pattern, for example, to be overexpressed. Therefore, the term “exogenous” gene or DNA is intended to refer to the gene or DNA fragment introduced into recipient cells. Types of DNA included in exogenous DNA include DNA already present in the cell, DNA from another individual of the same species, DNA from a different organism, or DNA sequences containing antisense messages for genes, and DNA sequences encoding synthetic or modified versions of genes, as well as DNA generated from outside.

[0075] Host strains developed according to the approaches described herein can be evaluated by various means known in the art (see, for example, "Studier (2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253").

[0076] Methods for downregulating or silencing genes are known in the art. For example, expressed protein activity can be downregulated or eliminated using antisense oligonucleotides (ASOs), protein aptamers, nucleotide aptamers, and RNA interference (RNAi) (e.g., small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA)) (e.g., "Rinaldi and Wood (2017) Nature Reviews Neurology 14, describing ASO therapies; Fanning and Symonds (2006) Handb Exp Pharmacol. 173, 289-303G, describing hammerhead ribozymes and small hairpin RNA; Helene, et al. (1992) Ann. NY Acad. Sci. 660, 27-36; Maher (1992) Bioassays 14(12): 807-15, describing targeting deoxyribonucleotide sequences; Lee et al. (2006) Curr Opin Chem Biol. 10, Reynolds et al. (2004) Nature Biotechnology 22(3), 326 - 330, describing RNAi; Pushparaj and Melendez (2006) Clinical and Experimental Pharmacology and Physiology 33(5-6), 504-510, describing RNAi; Dillon et al. (2005) Annual Review of Physiology 67, 147-173, describing RNAi; see Dykxhoorn and Lieberman (2005) Annual Review of Medicine 56, 401-423, describing RNAi).RNAi molecules are commercially available from various sources (e.g., Ambion, TX; Sigma Aldrich, MO; Invitrogen). Several siRNA molecule design programs using various algorithms are known in the art (e.g., see Cenix algorithm, Ambion; BLOCK-iT(tm) RNAi Designer, Invitrogen; siRNA Whitehead Institute Design Tools, Bioinofrmatics & Research Computing). Characteristics that influence the definition of an optimal siRNA sequence include the G / C content at the siRNA terminus, the Tm of specific internal domains of the siRNA, the length of the siRNA, the position of the target sequence within the CDS (coding region), and the nucleotide content of the 3' overhang.

[0077] Genome editing

[0078] As described herein, oncogenic fusion genes can be generated in cell lines for use in the development and validation of the assays of this disclosure for detecting oncogenic fusion genes. In some embodiments, oncogenic fusion genes can be generated in cell lines using genome editing. Methods of genome editing are well known (see, for example, "Aldi 2018 Nature Communications 9(1911)"). Therefore, unless otherwise stated herein, the methods of this disclosure can be carried out in accordance with such methods.

[0079] For example, genome editing can include CRISPR / Cas9, CRISPR-Cpf1, TALEN, or ZNF. As one example, the CRISPR / CRISPR-related (Cas) system is a novel class of genome editing tools that target desired genomic sites in mammalian cells. A recently published type II CRISPR / Cas system uses the Cas9 nuclease to hybridize a 20-nucleotide DNA sequence to the NGG motif recognized by Cas9 (and therefore, (N) 20The NGG (Nigral Genome Graft) targets a genomic site by complexing with a synthetic guide RNA immediately preceding the NGG target DNA sequence. This results in a double-strand break three nucleotides upstream of the NGG motif. The double-strand break induces either non-homologous end joining or homology-directed repair. Non-homologous end joining is prone to errors and induces frameshift mutations that knock out an allele of a gene. Homologous-directed repair allows for the knock-in or correction of genomic mutations using an externally introduced double-stranded or single-stranded DNA repair template.

[0080] For example, the method of the present disclosure includes a method for modifying a target polynucleotide sequence within a cell, the method comprising the step of contacting the polynucleotide sequence with a Cas protein.

[0081] screening

[0082] In some embodiments, the oncogenic fusion gene detection method of this disclosure can be used as part of various screening methods. In these embodiments, changes in the expression of oncogenic fusion genes in cancer cells can be used to evaluate the efficacy of a candidate therapeutic composition or to characterize the biochemical pathways regulated by the candidate therapeutic composition.

[0083] The methods described herein are used to screen a variety of different candidate molecules (e.g., potentially therapeutic candidate molecules). Candidate substances to be screened according to the methods of this disclosure include, but are not limited to, tissue or cell fractions, nucleic acids, polypeptides, siRNA, antisense molecules, aptamers, ribozymes, triple helix compounds, antibodies, and small molecules (e.g., less than about 2000 mW, or less than about 1000 mW, or less than about 800 mW) of organic or inorganic molecules (e.g., salts, metals).

[0084] Candidate molecules encompass a variety of chemical classes, including organic molecules such as low-molecular-weight organic compounds with molecular weights between 50 daltons and approximately 2500 daltons. Candidate molecules may contain functional groups necessary for structural interactions with proteins, particularly hydrogen bonding, and generally include at least an amine group, carbonyl group, hydroxyl group, or carboxyl group, and usually at least two functional chemical groups. Candidate molecules may include cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups.

[0085] Candidate molecules may be compounds in a library database of compounds. Those skilled in the art will generally be familiar with numerous databases of commercially available compounds for screening (e.g., the ZINC database (UCSF) with 2.7 million compounds across 12 different molecular subsets; see "Irwin and Shoichet (2005) J Chem Inf Model 45, 177-182"). Those skilled in the art will also be familiar with various search engines for identifying commercially available sources and desirable compounds and compound classes for further testing (e.g., the ZINC database; eMolecules.com; electronic libraries of commercially available compounds provided by vendors (ChemBridge, Princeton BioMolecular, Ambinter SARL, Enamine, ASDI, Life Chemicals, etc.)).

[0086] Candidate molecules for screening according to the methods of this disclosure include both lead-like and drug-like compounds. Lead-like compounds are generally understood to have a relatively small scaffold-like structure (e.g., molecular weight of about 150 to about 350 kD) with relatively few features (e.g., less than 3 hydrogen donors and / or less than 6 hydrogen acceptors; hydrophobicity xlogP of about -2 to about 4) (see, e.g., Angewante (1999) Chemie Int. ed. Engl. 24, 3943-3948). In contrast, drug-like compounds are generally understood to have a relatively large scaffold (e.g., molecular weight of about 150 to about 500 kD) with relatively many features (e.g., less than 10 hydrogen acceptors and / or less than 8 rotatable bonds; hydrophobicity xlogP of less than 5) (see, e.g., Lipinski (2000) J. Pharm. Tox. Methods 44, 235-249). Initial screening can be performed using lead-like compounds.

[0087] When designing lead compounds from spatial orientation data, it is useful to understand whether a particular molecular structure is "drug-like." Such characterization can be based on a set of empirically recognized properties and can be derived by comparing the broad similarities with known drugs in the pharmacopoeia. A drug does not need to satisfy all or any of these characteristics, but if it is drug-like, the drug candidate is far more likely to be clinically successful.

[0088] Some of these “drug-like” characteristics are summarized in Lipinski’s Four Laws (commonly known as the “Rule of Five” due to the frequent use of the number 5). These laws generally relate to oral absorption and are used to predict the bioavailability of a compound when optimizing lead compounds, but they also serve as effective guidelines for constructing lead molecules in rational drug design efforts, as can be achieved by using the methods of this disclosure.

[0089] The four "rules of five" state that candidate drug-like compounds should have at least three of the following characteristics: (i) weight less than 500 daltons; (ii) logarithm P less than 5; (iii) 5 or fewer hydrogen bond donors (total of OH and NH groups); (iv) 10 or fewer hydrogen bond acceptors (total of N and O atoms). Additionally, drug-like molecules generally have a span (width) of approximately 8 Å to 15 Å.

[0090] kit

[0091] Furthermore, kits are also provided according to this disclosure. The kits of this disclosure may include the drug or composition of this disclosure and, in certain embodiments, instructions for administration. Such kits facilitate the implementation of the method for detecting oncogenic fusion genes of this disclosure. When provided as a kit, the different components of the composition of this disclosure may be packaged in separate containers and mixed immediately before use. The components include, but are not limited to, one or more upstream primer / probe pairs, one or more downstream primer / probe pairs, a solubilizer, and sterile packaging. The individual packaging of such components may, if desired, be presented in a pack or dispenser device containing one or more unit dosage forms comprising the composition of this disclosure. The pack may include, for example, metal or plastic foil such as a blister pack. In addition, the individual packaging of such components may, in certain cases, allow for long-term storage without loss of activity of the components.

[0092] The kit may also include reagents in separate containers, such as sterile water or saline solution added to separately packaged lyophilized active ingredients. For example, a sealed glass ampoule may contain the lyophilized ingredient, and another ampoule may contain sterile water or sterile saline solution, each packaged under a neutral, non-reactive gas such as nitrogen. The ampoules may be made of glass, organic polymers such as polycarbonate, polystyrene, ceramic, metal, or any other material commonly used to hold reagents. Other examples of suitable containers include bottles made from similar materials to ampoules, and cylinders with foil-lined interiors made of aluminum or alloy. Other containers include test tubes, vials, flasks, bottles, and syringes. Containers may have a sterile access port, such as a bottle with a stopper that can be pierced with a subcutaneous needle. Other containers may have two compartments separated by an easily removable membrane, where the ingredients are mixed when the membrane is removed. The removable membrane may be made of glass, plastic, rubber, etc.

[0093] In certain embodiments, the kit may be provided with instructions. These instructions may be printed on paper or other substrates, or provided on an electronically readable medium or as a video. The instructions may not be physically attached to the kit. In such cases, users may be directed to an internet website designated by the kit's manufacturer or distributor.

[0094] The control or reference samples described herein may be samples derived from healthy subjects. Instead of control or reference samples, reference values ​​previously obtained from healthy subjects or groups of healthy subjects may be used. Furthermore, control or reference samples may be samples containing known amounts of detectable compounds or additives.

[0095] The compositions and methods of this disclosure utilizing molecular biology protocols can conform to various standard techniques known in the art (e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, CP 1988. Methods in Enzymology 167, 747-754; Studier (2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).

[0096] The definitions and methods set forth herein are provided to better define this disclosure and to guide those skilled in the art in the practice of this disclosure. Unless otherwise noted, terms should be understood in accordance with the conventional usage by those skilled in the art.

[0097] In some embodiments, numbers representing quantities, properties, such as molecular weight or reaction conditions, used to describe and claim certain embodiments of this disclosure should be understood as being modified by the term “approximately.” In some embodiments, the term “approximately” is used to indicate that a value includes the mean standard deviation of the apparatus or method used to determine that value. In some embodiments, numerical parameters described herein and in the appended claims are approximations that can be modified depending on the desired properties to be obtained by a particular embodiment. In some embodiments, numerical parameters should be interpreted by applying common rounding techniques, taking into account the reported number of significant figures. Although the numerical ranges and parameters representing a wide range of some embodiments of this disclosure are approximations, the numerical values ​​shown in a particular example are reported as accurately as possible. The numerical values ​​shown in some embodiments of this disclosure may include certain errors that inevitably arise from the standard deviation observed in each of those test measurements. The description of value ranges herein is merely intended to serve as a simplified method for individually specifying individual values ​​that fall within that range. Unless otherwise specifically specified herein, individual values ​​are incorporated into the specification as if they were described individually herein. A discrete enumeration of values ​​is understood to include a range between each value.

[0098] In some embodiments, the terms “a,” “an,” and “the,” as well as similar terms used in the context describing a particular embodiment (in particular in the specific context of the claims below), can be interpreted as encompassing both singular and plural forms unless otherwise specified. In some embodiments, including the claims, the term “or” as used herein is used to mean “and / or” unless it is explicitly indicated that the choices are mutually exclusive or that the choices are mutually exclusive.

[0099] The terms “comprise,” “have,” and “include” are open-ended linking verbs. Any one or more variations or tenses of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes,” and “including,” are also open-ended. For example, any method that “comprises,” “has,” or “includes” one or more steps is not limited to having only those one or more steps, but may also include other steps not listed. Similarly, any composition or apparatus that “comprises,” “has,” or “includes” one or more features is not limited to having only those one or more features, but may also include other features not listed.

[0100] All methods described herein may be carried out in any suitable order, unless otherwise specified herein or unless the context clearly contradicts it. Any and all examples or exemplary language (e.g., "etc.") provided in reference to specific embodiments herein are intended solely to better illustrate this disclosure and do not impose limitations on the scope of the invention unless specifically stated in the claims. Nothing in this specification should be construed as indicating that any element not described in the claims is essential for carrying out this disclosure.

[0101] The grouping of alternative elements or embodiments of the present disclosure disclosed herein should not be construed as limitation. Each member of each group may be referred to and claimed individually or in any combination with other members of the group or other elements described herein. One or more members of a group may be included in or removed from a group for convenience or patentability reasons. If such inclusion or removal occurs, this specification shall be deemed to include the modified group and thus satisfy all descriptions of the Markush group used in the appended claims.

[0102] All publications, patents, patent applications, and other documents cited herein are incorporated herein by reference in whole for all purposes to the same extent as if each of them were specifically and individually cited. No reference to documents herein should be construed as an admission that they are prior art to this disclosure.

[0103] As this disclosure has been described in detail, it will be clear that modifications, variations, and equivalent embodiments are possible without departing from the scope of this disclosure as defined in the attached claims. Furthermore, it should be understood that all embodiments in this disclosure are provided as non-limiting embodiments.

[0104] Examples

[0105] The above-described non-limiting embodiments are provided for further illustration of the present disclosure. Those skilled in the art will understand that the techniques disclosed in these embodiments represent approaches that the inventors have found to work well in practicing the present disclosure and can therefore be considered examples of embodiments of the present disclosure. Furthermore, those skilled in the art will understand that various modifications can be made to specific embodiments of the present disclosure in light of the present disclosure, and that similar or comparable results can still be obtained without departing from the spirit and scope of the present disclosure.

[0106] Example 1: Development and validation of a droplet digital PCR assay for detecting oncogenic KMT2A fusion genes.

[0107] The following experiments were conducted to develop and validate a droplet digital PCR (ddPCR) assay for detecting and quantifying oncogenic KMT2A fusion genes.

[0108] While there are at least 80 known KMT2A fusion gene partners, approximately 80% of KMT2A fusion genes involve only five exome partners (AF9, AF6, AF4, ELL, ENL). Considering the diverse range of fusion partners, detecting these fusion genes using qPCR technology is challenging.

[0109] Using a different droplet PCR technique, droplet digital PCR (ddPCR), we developed an assay that provides detection of five of the most common KMT2A fusion genes, which account for the majority (approximately 80%) of all oncogenic KMT2A fusion genes found in treatment-related AML (t-AML) patients. The ddPCR assay was benchmarked using cell lines and primary patient samples containing KMT2A fusion genes. The assay described below provides an inexpensive, rapid, highly sensitive, and specific platform for KMT2A fusion gene detection, improving the detection of measurable residual disease (MRD) in AML patients with KMT2A fusion genes and enabling screening of patients at risk of developing t-AML after TOP2 inhibitor therapy.

[0110] Materials and methods

[0111] cell line

[0112] We designed, benchmarked, and validated ddPCR assays using human cell lines known to contain the KMT2A fusion gene. The cell lines used were THP-1 (KMT2A-AF9), MOLM-13 (KMT2A-AF9), MV4-11 (KMT2A-AF4), OCI-AML2 (KMT2A-AF6), and KOPN8 (KMT2A-ENL). We designed and tested the KMT2A-ELL reagent using t-AML patient samples containing the KMT2A-ELL fusion gene. Cell lines without the KMT2A fusion gene, K562, HEL, Kasumi, Jurkat, and OCI-AML3, were used as controls. In each ddPCR experiment, cell lines containing the KMT2A fusion gene and cell lines without the KMT2A fusion gene were used as controls. THP-1 cells and HEL cells were cultured with RPMI (ATCC30-2001), 10% thermo-inactivated fetal bovine serum (HI-FBS; Gibco), and 1% penicillin-streptomycin (P / S; Gibco). 0.05 mM β-mercaptoethanol (Sigma-Aldrich) was added to the THP-1 medium. MOLM-13, Jurkat, and KOPN8 cells were cultured with RPMI (Gibco), 10% HI-FBS, and 1% P / S. Kasumi cells were cultured with RPMI (Gibco), 20% HI-FBS, and 1% P / S. MV-4-11 cells were cultured with IMDM (Gibco), 10% HI-FBS, and 1% P / S. OCl-AML2 and OCI-AML3 cells were cultured with MemAlpha (Gibco), 20% HI-FBS, and 1% P / S.

[0113] Patient sample

[0114] Cryopreserved human t-AML patient specimens were stored at Washington University in St. Louis. All t-AML patients provided written informed consent for tissue repository storage and genomic sequencing in accordance with protocol #201011766 approved by the Institutional Review Board of Washington University in St. Louis. Anonymized control patient peripheral blood (PB) or bone marrow (BM) samples were obtained in accordance with a protocol approved by the Institutional Review Board for human subjects research of Washington University (WU 01-1014).

[0115] RNA Extraction and cDNA Synthesis

[0116] RNA was extracted from cell lines and patient specimens using the RNeasy Plus Mini Kit (Qiagen). For each experiment, a maximum of 1×10 6 cells were processed. RNA was extracted without modification in accordance with the manufacturer's recommendations. RNA was eluted from RNAeasy spin columns with 25 to 50 μL of RNase-free water. RNA concentration was quantified using a Qubit fluorometer (Thermo Fisher Scientific). Complementary DNA (cDNA) was synthesized using Superscript IV VILO (ThermoFisher Scientific). The synthesized cDNA molecules were stored at -20°C.

[0117] Droplet Digital PCR Primer / Probe Design

[0118] The genomic locations for translocations in each cell line containing the KMT2A fusion gene were identified from a cancer-dependent map (https: / / depmap.org / portal / ; last accessed 12 / 17 / 2021) and used to design primer sequences spanning the fusion (Figure 1(a), Figure 6, Figure 4). Primers and probes were designed using Primer3Plus. Multiple exon primers were designed for each translocation partner. Fusion-specific cDNA amplicons generated from cell lines containing the KMT2A fusion gene were sequenced by Sanger sequencing and mapped to the hg38 reference genome using BLAT to validate the fidelity of the primer pairs (Figure 6). Prime Time fluorescent probes (Figure 1(a), Figure 6, Figure 4) were designed to anneal within the fusion-specific primers to add sensitivity and specificity to the assay. For each fusion, one fluorescein (FAM)-labeled probe was designed to anneal to the KMT2A upstream of the fusion site, and one hexachlorofluorescein (HEX)-labeled probe was designed to anneal to the fusion partner downstream of the fusion site. The control primer / probe pair was designed to label wild-type KMT2A cDNA with the FAM-labeled probe and the HEX-labeled probe to anneal within the KMT2A exon.

[0119] • Reaction conditions for droplet digital PCR

[0120] Droplet digital PCR experiments were performed using a QX200 Droplet Digital PCR System (Bio-Rad). In each ddPCR reaction, the input cDNA was diluted so that less than 330 ng of cDNA was input per reaction. Each ddPCR reaction consisted of 10 μL of 2×ddPCR Supermix for dUTP-free probes, 1000 nM fusion-specific primers, 250 nM fusion-specific probes, cDNA (maximum 330 ng), and RNase / DNase-free water, totaling 20 μL. Droplets were generated using a QX200 Droplet Generator (Bio-Rad) according to the manufacturer's instructions. Droplet PCR amplification was performed under thermocycling conditions of 94°C for 10 seconds; 94°C for 30 seconds, 60°C for 1 minute for 40 cycles; 98°C for 10 minutes; and hold at 4°C. After amplification, imaging was performed using a QX200 droplet reader (Bio-Rad), and analysis was performed using the QuantaSoft Analysis Pro software package (Bio-Rad). Multiple negative controls (cell lines without known KMT2A fusion genes) and positive controls (cell lines with known KMT2A fusion genes) were used in each experiment. For each sample analyzed for the KMT2A fusion gene, individual aliquots were analyzed using primers and probes targeting the wild-type KMT2A locus spanning exon 7 to exon 9 to provide estimates of wild-type transcript abundance (Figure 4).

[0121] • Conditions for a pooled assay

[0122] To enable simultaneous detection of all five KMT2A fusion genes targeted by the assay, a pooled ddPCR assay was designed and benchmarked. In a single reaction mixture, forward primers / probes for KMT2A exon 7 and KMT2A exon 9 were combined with reverse primers / probes for AF9 exon 6, AF4 exon 5, AF6 exon 2, ENL exon 7, and ELL exon 3 (Figure 13). The pooled primer / probe mixture was concentrated 10-fold. Then, 20 μL of ddPCR reaction product was added to 2 μL of the pooled primer / probe mixture to achieve a final concentration of 1000 nM for each primer and 250 nM for each probe. Droplet generation, thermocycling conditions, droplet imaging, and droplet analysis were performed as described above.

[0123] Dilution series experiment

[0124] Cell lines containing the KMT2A fusion gene were serially diluted in OCI-AML3 cells (KMT2A wild-type). Cells were quantified using an automated cell counter (Nexcelom). Starting with a 50% mixture, 1 million cells containing the KMT2A fusion gene (e.g., THP-1) were mixed with 1 million OCI-AML3 cells. From this mixture, 200,000 cells were taken and added to 1.8 million OCI-AML3 cells to prepare a 5% mixture. Serial dilutions were repeated down to 0.0005%, at which point it was estimated that fewer than 10 cells containing the KMT2A fusion gene were present in the mixture. RNA was extracted and converted to cDNA as described above. The cDNA was diluted to 40 ng / μL and stored at -20°C. For 50%, 5%, 0.5%, and 0.05% dilutions, 80 ng of cDNA was used in a single ddPCR reaction well for fusion detection. At a 0.005% dilution, 320 ng of cDNA was used per well in four ddPCR reaction wells (1280 ng total). At a 0.0005% dilution, 320 ng of cDNA was used per well in eight ddPCR reaction wells (2560 ng total). At the 0.005% and 0.0005% dilutions, it was necessary to increase the amount of cDNA included in the reaction to analyze enough cells to detect the rare KMT2A fusion gene. These serial dilution experiments simulated the detection of rare leukemia cells with the KMT2A fusion gene in patients and established the detection limit of the assay.

[0125] Genetic editing of KMT2A fusion genes

[0126] The KMT2A fusion gene was generated using clustered regularly spaced short palindromic repeat (CRISPR) / Cas9 gene editing technology. The fusion breakpoint was located between exons 10 and 11 of KMT2A. Guide RNA (gRNA) sequences targeting the intronic regions of KMT2A and MLLT3 / AF9 were synthesized using the nucleotide sequences (Synthego) of SEQ ID NOs. 24 and 25, as listed in the table in Figure 5. A ribonucleoprotein (RNP) complex was formed by incubating the guide RNA (120 pmol) with Cas9 (Integrated DNA Technologies) at room temperature for 20 minutes. Nucleofection was performed using the Neon transfection system (Thermo Fisher Scientific). The RNP complex was mixed with 250,000 cells in 10 μL of buffer R. The cells were electroporated at 1700 V, 20 ms, and 1 pulse. After nucleofection, the cells were cultured in appropriate medium. RNA was extracted from these cells using the protocol described above and converted to cDNA. This cDNA was assayed using a combination of KMT2A exon 9 and AF9 primer / probe to detect oncogenic fusion genes.

[0127] result

[0128] Droplet Digital PCR Assay Design

[0129] Standard dual-color ddPCR assays for mutation detection use FAM-labeled and HEX-labeled probes that overlap different regions of interest by a single nucleotide or small indel. Competitive annealing of these probes at the locus provides specificity to distinguish between wild-type and mutant DNA molecules. This standard method for mutation detection is incompatible with fusion detection. To enable detection of low-frequency fusions, we developed a novel cDNA-based dual-color ddPCR assay that identifies each fusion by PCR primers that span across the fusion. The fluorescently labeled probes were then nested adjacent to the fusions (Figures 1 and 4). Generally, FAM-labeled probes are designed to anneal within KMT2A upstream of the fusion, and HEX-labeled probes are designed to anneal within the fusion partner downstream of the fusion.

[0130] Benchmarking of droplet digital PCR in cell lines

[0131] These primer / probe pairs were first benchmarked in dilution series experiments using bulk qPCR. Excellent performance was observed across the entire panel for both the KMT2A FAM probe and the fusion partner HEX probe (Figure 7). In each qPCR experiment, wild-type primer / probe pairs were also incorporated into separate reactions to estimate the abundance of wild-type KMT2A transcript for comparison and to ensure proper sample preparation and loading. After optimization, the primer / probe reagents were benchmarked on a ddPCR platform in dilution series experiments (Figures 8 and 14). Cell lines with known KMT2A fusion genes were serially diluted to OCI-AML3 cells (KMT2A wild-type) and analyzed using ddPCR. In this experiment, appropriate cell type-specific KMT2A fusion genes were detected within a dynamic range of 5–6 logs. The fractional abundance of KMT2A fusion gene transcripts was determined by calculating the concentration of KMT2A fusion gene transcripts and dividing by the total number of detected KMT2A (fusion and wild-type) transcripts, and was consistent with the expected abundance of KMT2A mutant cell lines across the entire dilution series (Figure 2A). Primer / probe pairs designed to target specific KMT2A fusion genes did not exhibit off-target activity when assaying cell lines with different KMT2A fusion genes that were not targets of the specific primer / pair (Figure 9).

[0132] To demonstrate the principle of KMT2A fusion gene detection in a setting mimicking patients receiving TOP2 inhibitor therapy, HEL cells containing wild-type KMT2A were edited with CRISPR / Cas9 and KMT2A fusion genes were introduced. The KMT2A fusion gene was detected 4 days after CRISPR / Cas9 treatment and persisted to days 14 and 21 of culture (Figure 2B). Interestingly, the fractional abundance ratio (ratio to wild-type transcript) of the KMT2A-AF9 fusion transcript remained stable throughout the experimental period (Figure 2C).

[0133] Development and validation of a pooled droplet digital PCR assay.

[0134] Using a single primer / probe pair to track leukemia clones is useful when the oncogenic KMT2A fusion gene is known. However, in detection settings where the KMT2A fusion gene is unknown, such as in screening for KMT2A fusion genes in patients receiving TOP2 inhibitors, all fusion genes need to be tested simultaneously. Since each reaction is limited by the availability of patient sample RNA, a pooled primer / probe strategy capable of detecting the five most common KMT2A fusion genes in the same reaction was designed (Figure 13, Methods). This pooled reagent detected the KMT2A fusion gene in all cell lines known to contain it, and did not detect evidence of the KMT2A fusion gene in cell lines known not to contain it (Figure 3A, Figure 10). The proportion of KMT2A transcripts derived from the KMT2A fusion gene was found to be 0.28–0.57 across all cell lines (Figure 3B).

[0135] Furthermore, this pooled reagent also detected the KMT2A fusion gene in AML patient samples known to contain the KMT2A fusion gene (Figure 11). The abundance of the KMT2A fusion gene transcript was calculated relative to the abundance of the wild-type KMT2A fusion gene and compared with an orthogonal metric of leukemia burden (Figure 15(c)). Four additional healthy human control samples without known KMT2A fusion genes were identified, but the pooled primer / probe assay did not detect any evidence of the KMT2A fusion gene (Figure 11).

[0136] Finally, cell line dilution series experiments were performed using pooled primer / probe reagents. Cell lines known to contain the KMT2A fusion gene were sequentially diluted in OCI-AML3 cells (KMT2A wild-type), and the KMT2A fusion gene was detected in each cell line dilution experiment using the same pooled primer / probe reagent (Figures 12 and 16). The ratio of the KMT2A fusion gene transcript to the KMT2A wild-type transcript was in agreement with the predicted dilution ratio over a range of 5 to 6 orders of magnitude (Figure 3D).

[0137] Consideration

[0138] The results of these experiments demonstrate a KMT2A fusion gene detection method using dual-color ddPCR. The assay of this disclosure was compared with conventional techniques using bulk qPCR. High sensitivity detection across a dynamic range of several logs was demonstrated. The assay of this disclosure was demonstrated to be specific and to reliably exclude patient samples and cell lines that do not contain the KMT2A fusion gene. The assay of this disclosure improves upon existing qPCR strategies by its ease of use, accurate quantification of transcripts, ease of multiplex analysis, and flexibility or reproducibility of modification or expansion of the target panel. Since quantification by ddPCR is absolute, the assay of this disclosure does not require calibration with a standard curve. Previous efforts to develop digital quantification of the KMT2A fusion gene have been limited by the polybinding nature of the KMT2A fusion gene, which has more than 80 known KMT2A fusion gene partners. The assay of this disclosure targets five of the most common KMT2A fusion gene partners, which account for approximately 80% of KMT2A rearrangement AML cases. The detection limit of the assay described herein varies depending on the amount of material introduced, but in dilution experiments, cells containing the KMT2A fusion gene could be reliably identified even when the estimated abundance of cells was only 10 out of a total of 2 million cells.

[0139] The assay described herein was able to detect KMT2A fusion genes with high sensitivity and specificity by ddPCR, even when limited to the most common fusion genes. Given the ease of development and validation of the assay described herein, it is possible to add additional KMT2A fusion genes to the assay described herein with minimal cost and effort. In the assay described herein, the abundance of KMT2A fusion genes is compared to the expression of wild-type KMT2A, which should have similar gene regulation. This standardization was supported by cell line dilution experiments in which the proportion of cells containing KMT2A fusion genes was estimated using the ratio of KMT2A fusion genes to wild-type KMT2A transcripts, and the abundance was consistent with that in each serial dilution.

Claims

1. A method for monitoring the expression level of at least one oncogenic KMT2A fusion gene in a subject, Each of the aforementioned oncogenic KMT2A fusion genes contains a KMT2A fragment fused with a partner gene fragment at the fusion site. This method is (a) (i) The biological sample of the subject containing a predetermined amount of RNA; (ii) at least one forward PCR primer targeting the KMT2A start codon of the KMT2A fragment upstream of the fusion site, and at least one reverse PCR primer targeting the 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, wherein each KMT2A probe is configured to anneal to the KMT2A fragment between the KMT2A start codon and the fusion site; and (iv) A partner gene probe comprising a second fluorescent reporter, wherein each partner gene probe is configured to anneal to the partner gene fragment between the partner gene start codon and the fusion site; the step of providing (b) The steps of extracting a predetermined amount of RNA from the biological sample and synthesizing a predetermined amount of cDNA from the predetermined amount of RNA, (c) A step of subjecting a mixture of a predetermined 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 fluorescence intensity pairs from the first fluorescent reporter and the second fluorescent reporter, (d) A method comprising the step of estimating the expression level of the at least one oncogenic KMT2A fusion gene based on the plurality of fluorescence intensity pairs obtained by droplet digital PCR (ddPCR).

2. The method according to claim 1, The first fluorescent reporter and the second fluorescent reporter are independently selected from fluorescein and hexachlorofluorescein, and The first fluorescent reporter and the second fluorescent reporter are different from each other.

3. A method according to claim 1 or 2, The method wherein the at least one oncogenic KMT2A fusion gene is selected from KMT2A-AF9, KMT2A-AF4, KMT2A-AF6, KMT2A-ENL, KMT2A-ELL, at least one target-specific KMT2A fusion gene, and any combination thereof.

4. A method according to any one of claims 1 to 3, The method wherein the at least one oncogenic KMT2A fusion gene is one KMT2A fusion gene selected from the group consisting of KMT2A-AF9, KMT2A-AF4, KMT2A-AF6, KMT2A-ENL, and KMT2A-ELL.

5. The method according to any one of claims 1 to 4, (a) The at least one forward PCR primer targets the KMT2A start codon of the KMT2A fragment selected from KMT2A exon 7, KMT2A exon 9, and any combination thereof, (b) A method wherein the at least one reverse PCR primer targets the partner gene start codon of the partner gene fragment selected from AF9 exon 6, AF4 exon 5, AF6 exon 2, ENL exon 7, ELL exon 3, and any combination thereof.

6. A method according to any one of claims 1 to 5, (a) The at least one forward PCR primer comprises a nucleotide sequence selected from Sequence ID No. 1 targeting KMT2A exon 7, Sequence ID No. 2 targeting KMT2A exon 9, and any combination thereof. (b) The at least one reverse PCR primer is (i) Sequence ID 7, which targets AF9 exon 6, (ii) Sequence ID No. 5, which targets AF4 exon 5, (iii) Sequence ID 9, which targets AF6 exon 2, (iv) Sequence ID 13, which targets ENL exon 7, (v) Sequence ID No. 14, which targets ELL exon 3, (vi) A method comprising any combination thereof, comprising a nucleotide sequence selected from.

7. A method according to any one of claims 1 to 6, The method wherein the at least one KMT2A probe comprises a nucleotide sequence selected from Sequence ID No. 16, which targets KMT2A exon 7, Sequence ID No. 22, which targets KMT2A exon 9, and any combination thereof.

8. A method according to any one of claims 1 to 7, The at least one partner gene probe is (a) Sequence ID No. 18, which targets AF9 exon 6, (b) Sequence ID No. 17, which targets AF4 exon 5 (c) Sequence ID No. 19, which targets AF6 exon 2. (d) Sequence ID No. 21, which targets ENL exon 7 (e) Sequence ID No. 23, which targets ELL exon 3, (f) A method comprising a nucleotide sequence selected from any combination thereof.

9. The method according to any one of claims 1 to 8, The method wherein the biological sample comprises at least one of a peripheral blood sample, a bone marrow sample, a solid tumor sample, and any combination thereof.

10. A method according to any one of claims 1 to 9, The method further includes the step of identifying at least one target-specific KMT2A fusion gene by nucleic acid sequencing of the target cancer, Each of the aforementioned target-specific KMT2A fusion genes includes a KMT2A fragment fused with a target-specific partner gene fragment at the fusion site. (a) The at least one reverse PCR primer further comprises an additional reverse PCR primer that targets the target-specific partner gene start codon of the target-specific partner gene fragment downstream of the fusion site, (b) A method wherein the at least one partner gene probe further comprises at least one target-specific partner gene probe including the second fluorescent reporter, each of the target-specific partner gene probes is configured to anneal to the target-specific partner gene fragment between the target-specific partner gene start codon and the fusion site.

11. A method according to any one of claims 1 to 10, A method further comprising the step of classifying a subject as having measurable residual disease if the expression level of at least one oncogenic KMT2A fusion gene, as measured by droplet digital PCR (ddPCR), exceeds a threshold of 0.001%.

12. A method according to any one of claims 1 to 11, (a) Providing a first biological sample and a second biological sample from the subject, wherein the second biological sample is obtained after the subject has been treated, (b) Estimating the first and second expression levels of the at least one oncogenic KMT2A fusion gene based on the first and second biological samples, (c) A method further comprising the step of estimating the effectiveness of the treatment, the occurrence of treatment-related oncogenic KMT2A fusion genes, prognosis, and any combination thereof, based on the change between the first and second expression levels of the at least one oncogenic KMT2A fusion gene.

13. A method for individually monitoring the expression level of at least one oncogenic KMT2A fusion gene in a subject, Each of the aforementioned oncogenic KMT2A fusion genes contains a KMT2A fragment fused with a partner gene fragment at the fusion site. This method is (a) A step of providing initial sequencing of the target leukemia at nucleotide level resolution, (b) The step of identifying the at least one oncogenic KMT2A fusion gene based on the initial sequencing, (c) (i) The biological sample of the subject containing a predetermined amount of RNA; (ii) at least one forward PCR primer targeting the KMT2A start codon of the KMT2A fragment upstream of the fusion site, and at least one reverse PCR primer targeting the partner gene start codon of the partner gene fragment downstream of the fusion site; (iii) A KMT2A probe comprising a first fluorescent reporter, wherein each KMT2A probe is configured to anneal to the KMT2A fragment between the KMT2A start codon and the fusion site, and (iv) A partner gene probe comprising a second fluorescent reporter, wherein each partner gene probe is configured to anneal to the partner gene fragment between the partner gene start codon and the fusion site; the step of providing (d) The steps of extracting a predetermined amount of RNA from the biological sample and synthesizing a predetermined amount of cDNA from the predetermined amount of RNA, (e) The step of subjecting the mixture of a predetermined 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 fluorescence intensity pairs from the first fluorescent reporter and the second fluorescent reporter, (f) A method comprising the step of estimating the expression level of the at least one oncogenic KMT2A fusion gene based on the plurality of fluorescence intensity pairs obtained by the droplet digital PCR (ddPCR).

14. An assay for monitoring the expression level of at least one oncogenic KMT2A fusion gene in a subject, Each of the aforementioned oncogenic KMT2A fusion genes contains a KMT2A fragment fused with a partner gene fragment at the fusion site. This assay is, (a) at least one forward PCR primer targeting the KMT2A start codon of the KMT2A fragment upstream of the fusion site, and at least one reverse PCR primer targeting the partner gene start codon of the partner gene fragment downstream of the fusion site, (b) A KMT2A probe comprising a first fluorescent reporter, wherein each KMT2A probe is configured to anneal to the KMT2A fragment between the KMT2A start codon and the fusion site, (c) A partner gene probe comprising a second fluorescent reporter, wherein each partner gene probe is configured to anneal to the partner gene fragment between the partner gene start codon and the fusion site, An assay comprising 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, which are combined with a predetermined amount of cDNA synthesized from a predetermined amount of RNA obtained from the subject to form a mixture for processing in a droplet digital PCR (ddPCR) instrument.

15. The assay according to claim 14, The first fluorescent reporter and the second fluorescent reporter are independently selected from fluorescein and hexachlorofluorescein, and The assay wherein the first fluorescent reporter and the second fluorescent reporter are different from each other.

16. The assay according to claim 14 or 15, The assay wherein the at least one oncogenic KMT2A fusion gene is selected from KMT2A-AF9, KMT2A-AF4, KMT2A-AF6, KMT2A-ENL, KMT2A-ELL, at least one target-specific KMT2A fusion gene, and any combination thereof.

17. An assay according to any one of claims 14 to 16, The assay is characterized in that the at least one oncogenic KMT2A fusion gene is one KMT2A fusion gene selected from the group consisting of KMT2A-AF9, KMT2A-AF4, KMT2A-AF6, KMT2A-ENL, and KMT2A-ELL.

18. An assay according to any one of claims 14 to 17, (a) The at least one forward PCR primer targets the KMT2A start codon of the KMT2A fragment selected from KMT2A exon 7, KMT2A exon 9, and any combination thereof, (b) an assay in which at least one reverse PCR primer targets the partner gene start codon of the partner gene fragment selected from AF9 exon 6, AF4 exon 5, AF6 exon 2, ENL exon 7, ELL exon 3, and any combination thereof.

19. The assay according to any one of claims 14 to 18, (a) The at least one forward PCR primer comprises a nucleotide sequence selected from Sequence ID No. 1 targeting KMT2A exon 7, Sequence ID No. 2 targeting KMT2A exon 9, and any combination thereof. (b) The at least one reverse PCR primer is (i) Sequence ID 7, which targets AF9 exon 6, (ii) Sequence ID No. 5, which targets AF4 exon 5, (iii) Sequence ID 9, which targets AF6 exon 2, (iv) Sequence ID 13, which targets ENL exon 7, (v) Sequence ID No. 14, which targets ELL exon 3, (vi) an assay comprising any combination thereof, comprising a nucleotide sequence selected from those.

20. An assay according to any one of claims 14 to 19, The assay comprising at least one KMT2A probe including a nucleotide sequence selected from Sequence ID No. 16, which targets KMT2A exon 7, Sequence ID No. 22, which targets KMT2A exon 9, and any combination thereof.

21. An assay according to any one of claims 14 to 20, The at least one partner gene probe is (a) Sequence ID No. 18, which targets AF9 exon 6, (b) Sequence ID No. 17, which targets AF4 exon 5 (c) Sequence ID No. 19, which targets AF6 exon 2. (d) Sequence ID No. 21, which targets ENL exon 7 (e) Sequence ID No. 23, which targets ELL exon 3, (f) an assay comprising any combination thereof, comprising a nucleotide sequence selected from them.

22. A kit for monitoring the expression level of at least one oncogenic KMT2A fusion gene in a subject, Each of the aforementioned oncogenic KMT2A fusion genes contains a KMT2A fragment fused with a partner gene fragment at the fusion site. This kit is, (a) at least one forward PCR primer targeting the KMT2A start codon of the KMT2A fragment upstream of the fusion site, and at least one reverse PCR primer targeting the partner gene start codon of the partner gene fragment downstream of the fusion site, (b) A KMT2A probe comprising a first fluorescent reporter, wherein each KMT2A probe is configured to anneal to the KMT2A fragment between the KMT2A start codon and the fusion site, (c) A kit comprising at least one partner gene probe including a second fluorescent reporter, each of which is configured to anneal to the partner gene fragment between the partner gene start codon and the fusion site.

23. The kit according to claim 22, The first fluorescent reporter and the second fluorescent reporter are independently selected from fluorescein and hexachlorofluorescein, and The first fluorescent reporter and the second fluorescent reporter are different kits.

24. A kit according to claim 22 or 23, The kit wherein the at least one oncogenic KMT2A fusion gene is selected from KMT2A-AF9, KMT2A-AF4, KMT2A-AF6, KMT2A-ENL, KMT2A-ELL, at least one target-specific KMT2A fusion gene, and any combination thereof.

25. A kit according to any one of claims 22 to 24, The kit is characterized in that the at least one oncogenic KMT2A fusion gene is one KMT2A fusion gene selected from the group consisting of KMT2A-AF9, KMT2A-AF4, KMT2A-AF6, KMT2A-ENL, and KMT2A-ELL.

26. A kit according to any one of claims 22 to 25, (a) The at least one forward PCR primer targets the KMT2A start codon of the KMT2A fragment selected from KMT2A exon 7, KMT2A exon 9, and any combination thereof, (b) A kit wherein at least one reverse PCR primer targets the partner gene start codon of the partner gene fragment selected from AF9 exon 6, AF4 exon 5, AF6 exon 2, ENL exon 7, ELL exon 3, and any combination thereof.

27. A kit according to any one of claims 22 to 26, (a) The at least one forward PCR primer comprises a nucleotide sequence selected from Sequence ID No. 1 targeting KMT2A exon 7, Sequence ID No. 2 targeting KMT2A exon 9, and any combination thereof. (b) The at least one reverse PCR primer is (i) Sequence ID 7, which targets AF9 exon 6, (ii) Sequence ID No. 5, which targets AF4 exon 5, (iii) Sequence ID 9, which targets AF6 exon 2, (iv) Sequence ID 13, which targets ENL exon 7, (v) Sequence ID No. 14, which targets ELL exon 3, (vi) A kit containing any combination of those, including a nucleotide sequence selected from them.

28. A kit according to any one of claims 22 to 27, The kit comprises at least one KMT2A probe, SEQ ID NO: 16, which targets KMT2A exon 7; SEQ ID NO: 22, which targets KMT2A exon 9; and any combination thereof.

29. A kit according to any one of claims 22 to 28, The at least one partner gene probe is (a) Sequence ID No. 18, which targets AF9 exon 6, (b) Sequence ID No. 17, which targets AF4 exon 5 (c) Sequence ID No. 19, which targets AF6 exon 2. (d) Sequence ID No. 21, which targets ENL exon 7 (e) Sequence ID No. 23, which targets ELL exon 3, (f) A kit containing any combination of those, including a nucleotide sequence selected from them.

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