Capture probes and uses thereof

Abstract

To provide probes for detecting known and unknown fusion genes, related methods for detecting fusion genes, and uses and kits related thereto, in particular methods for diagnosing and monitoring a cancer.SOLUTION: Disclosed is a probe set comprising at least one probe comprising: a first portion of the probe complementary to a primary nucleotide portion of a target nucleic acid, and a second portion of the probe complementary to a secondary nucleotide portion of the target nucleic acid, where the probe overlaps a breakpoint between the primary and the secondary nucleotide portions of the target nucleic acid, and the length of the second portion of the probe represents about 1% to about 42% of a total probe length; and at least one further probe complementary only to the primary nucleotide portion of the target nucleic acid, where the further probe does not overlap the breakpoint between the primary and the secondary nucleotide portions of the target nucleic acid.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to the detection of fusion genes, particularly to the field of probes for detecting gene fusion events, methods for detecting fusion genes, related uses and kits. In particular, the present invention relates to methods for diagnosing and / or monitoring cancer.

Background Art

[0002] Fusion genes are important clinical biomarkers that serve as diagnostic guides, provide prognostic information, and support treatment decisions. Fusion genes are caused by chromosomal rearrangements such as translocations, inversions, and deletions, and may result in chimeric RNAs and proteins. Fusion genes usually contain two partners, a first fusion partner, which can be, for example, a kinase gene, and a second fusion partner.

[0003] Fusion genes can be detected by various molecular assays such as fluorescence in situ hybridization (FISH), reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), next-generation sequencing (NGS)-based techniques, etc.

[0004] FISH relies on nucleic acid probes containing fluorescent dye molecules that hybridize to specific nucleic acid sequences enabling visualization by fluorescence microscopy. In RT-PCR, specific primer sets designed to amplify by PCR targeting known fusion gene transcripts are used. In IHC, fusions are detected using antibodies that specifically bind to the fusion protein and detected microscopically. These techniques are widely used clinically but are characterized by low throughput and were limited to the detection of fusion events between known partners.

[0005] In contrast, NGS-based technologies can comprehensively and concurrently detect multiple gene fusion events in multiple patients. Several variations of this technology exist, each based on different library preparation methods. Multiplex PCR (mPCR) is a higher-throughput version of RT-PCR, using a pool of gene-specific primers to detect various fusion genes, which are then converted to an NGS library for sequencing. While this technique has the advantage of being relatively fast, it requires both fusion partners to be known. Anchor multiplex PCR (AMP), similar to mPCR but using a combination of gene-specific and general primers, partially overcomes this limitation, enabling the detection of novel fusion events for specific known fusion partners. However, both AMP and mPCR are subject to the inherent technical limitations of PCR, such as off-target primer binding and primer dimerization, which increase with increasing panel size (Heyer and Blackburn, 2020 Bioessays 42(7):e2000016).

[0006] Currently, alternatives to amplicon or PCR-based techniques are whole-genome sequencing (WGS) and RNA sequencing (RNA-seq). These techniques enable unbiased characterization of the complete genome or transcriptome (each), including known and novel fusions. However, due to the high cost of this type of genome-wide approach, these techniques have not yet been widely used in clinical applications. Hybridization capture-based NGS provides a cost-effective alternative in which WGS or RNA-seq libraries are subjected to a targeted enrichment step before sequencing. This step allows for the detection of both known and novel fusion events while limiting the regions that are sequenced (Hrdlickova et al., 2017, Wiley Interdiscip Rev RNA 2017, 8:e1364; Heyer and Blackburn, 2020, mentioned above).

[0007] Hybridization-based NGS is particularly interesting for fusion detection using RNA-seq libraries. In this technique, RNA molecules are converted to cDNA, then ligated to a sequencing adapter, amplified, and a whole transcriptome library is generated. These libraries are subjected to hybridization capture using probes conjugated to biochemical regions (usually biotin) designed to isolate the sequence of interest, followed by amplification and sequencing (Mercer et al., 2014, Nat Protoc. 9(5):989-1009; Heyer and Blackburn, 2020, mentioned above).

[0008] Therefore, a crucial step for successful hybridization capture is probe design. Various examples of probe design are known, and EP3647420A1 describes a probe design in which the probe hybridizes with a region derived from either gene A or B of a cDNA prepared from the transcript of a fusion gene containing a portion of gene A on the 5' side and a portion of gene B on the 3' side, which are linked to each other at a potential junction. Precise probe design ensures that only the target of interest is captured. In the detection of fusion genes, this is a particularly challenging task because probe design depends on the fusion partners and cleavage sites, which are not always known and vary greatly from medical application to medical application. Furthermore, a significant limitation in fusion probe design is how to position the probe to detect both novel and known fusions without capturing excessive reads from wild-type (WT) partner genes of diagnostic interest.

[0009] A probe suitable for detecting gene fusion events can be designed to be homologous to a known partner (hereinafter referred to as the first partner), thereby enabling the detection of almost all fusions containing that region of the partner (hereinafter referred to as a universal probe). Alternatively, the probe can be designed to overlap with both the first and second fusion partners, traversing the cleavage site, in order to enrich known fusions (hereinafter referred to as a specific probe). While universal probe design is the most common form of probe design, it has low specificity and may require deeper sequencing to detect low-expression fusions. Therefore, to ensure optimal detection of relevant known fusions, the approach is to also design probes specific to particular fusion / cleavage sites. However, this approach requires a careful design balance to optimize sequence reading.

[0010] Therefore, there is a need to improve probes and probe sets to simultaneously capture and detect known and novel fusion gene partners while limiting the capture of the counterpart of the WT (wild-type) second fusion partner. [Overview of the Initiative]

[0011] This invention is based on the finding that the probe design can be optimized to detect specific known fusions and minimize the capture of the WT's second fusion partner counterpart. This design reduces the capture of the WT counterpart of the second partner of a specific fusion, enabling fusion detection while minimizing the occurrence of sequence reads unrelated to fusion detection, thus leaving room to detect more fusions in parallel or to perform higher levels of patient multiplexing per NGS run.

[0012] The present invention relates in particular to the design of so-called low-overlap probes, including universal probes and specific probes. Probes are defined by the number of base pairs after the fusion breakpoint region that overlaps with the second partner, and all probes have a constant total length of 120 bp (i.e., 100% probe length). Universal probes (or 0 bp probes) do not overlap with the second fusion partner and cover only the first partner up to the end of the breakpoint. Specific probes start at the first fusion partner, overlap with the second fusion partner, and are up to 10 bp, 20 bp, 30 bp, 40 bp, or 50 bp beyond the fusion breakpoint (i.e., 9%, 17%, 25%, 33%, or 42% of the full-length probe) (Figure 1B). These low-overlap probes capture reads from the second partner resulting from the target fusion gene (Figures 2A, 3A vs. Figures 2B-D and 3B-D) while avoiding capture of reads originating from the WT counterpart.

[0013] In particular, the present invention further relates to mixtures of universal probes and specific low-overlap probes having overlaps of up to 10 bp, 20 bp, 30 bp, and 40 bp with respect to the second fusion partner. As shown in Examples 2 and 3, this mixture of overlapping probes can capture more fusion fragments than probes of 0 bp or 50 bp alone. Only by using a mixture of low-overlap probes is it possible to reduce the reading of the second partner of the WT compared to readings obtained using high-overlap probes.

[0014] In particular, the present invention further relates to mixtures of specific low-overlap probes having up to 10 bp, 20 bp, 30 bp, 40 bp, and 50 bp overlap with a universal probe and a second fusion partner, as well as mixtures of specific low-overlap probes having up to 50 bp overlap with a universal probe and a second fusion partner. These low-overlap probe mixtures capture the same or more fusion fragments as a 0 bp probe alone, but with some variation depending on the fusion being targeted, as shown in Example 4. However, only by using low-overlap probe mixtures is it possible to reduce the reading of the second partner of the WT compared to the reading obtained using a high-overlap probe.

[0015] In one embodiment, A first probe portion complementary to the first nucleotide portion of the target nucleic acid, and A second probe portion complementary to the second nucleotide portion of the target nucleic acid. A probe having at least one of the following: The probe overlaps with the break point between the first nucleotide portion and the second nucleotide portion of the target nucleic acid, The length of the second probe portion corresponds to approximately 1% to approximately 42% of the total length of the probe. The at least one probe, and At least one further probe complementary only to the first nucleotide portion of the target nucleic acid, wherein the further probe does not overlap with the break point between the first and second nucleotide portions of the target nucleic acid. A probe set comprising the following:

[0016] In one embodiment, A first probe portion complementary to the first nucleotide portion of the target nucleic acid, and A second probe portion complementary to the second nucleotide portion of the target nucleic acid. A probe having at least two such probes, The probe overlaps with the break point between the first nucleotide portion and the second nucleotide portion of the target nucleic acid, The length of the second probe portion corresponds to approximately 1% to approximately 42% of the total length of the probe, and The aforementioned probe has a second probe portion of a different length. A probe set comprising at least two of the aforementioned probes is provided.

[0017] In one embodiment, A first probe portion complementary to the first nucleotide portion of the target nucleic acid, and A second probe portion complementary to the second nucleotide portion of the target nucleic acid, A probe that includes, The probe overlaps with the break point between the first nucleotide portion and the second nucleotide portion of the target nucleic acid, The length of the second probe portion corresponds to approximately 1% to approximately 42% of the total length of the probe. A probe is provided.

[0018] In one embodiment, the present invention provides a probe or probe set in which the length of a second probe portion corresponds to about 1%, 9%, 17%, 25%, 33%, or 42% of the total length of the probe.

[0019] In one embodiment, the first probe portion is complementary to a first nucleotide portion of a target nucleic acid selected from a fusion gene or transcript encoding a first fusion partner, or an exon-skipping transcript encoding one exon, and the second probe portion is complementary to a second nucleotide portion of a target nucleic acid selected from a fusion gene or transcript encoding a second fusion partner, or an exon-skipping transcript encoding a second exon.

[0020] According to certain embodiments, the first nucleotide portion of the target nucleic acid is the first fusion partner which is a kinase molecule, and the second nucleotide portion of the target nucleic acid is the second fusion partner which is a non-kinase molecule.

[0021] According to certain embodiments, the probes of the present invention are suitable for capturing and detecting fusion genes, and thus DNA molecules. According to certain embodiments, the probes of the present invention are suitable for capturing and detecting transcripts of fusion genes and / or exon skipping transcripts, and thus RNA molecules.

[0022] According to certain embodiments, the probes of the present invention are suitable for capturing and detecting known or novel fusion gene partners.

[0023] According to certain embodiments, the probes of the present invention are suitable for capturing and detecting at least one fusion gene and / or exon skipping.

[0024] According to certain embodiments, the present invention provides a kit comprising at least one probe or probe set of the present invention.

[0025] According to certain embodiments, the present invention provides a composition comprising at least one probe or probe set of the present invention.

[0026] According to certain embodiments, the present invention provides at least one probe or probe set for use in target-enriched DNA sequencing or RNA sequencing.

[0027] According to certain embodiments, the present invention provides a method for detecting gene fusions and / or exon skipping in a sample, comprising the step of hybridizing at least one probe or probe set of the present invention with a complementary portion of a target nucleic acid.

[0028] In a particular embodiment, the present invention relates to a method for target DNA sequencing or RNA sequencing by target capture (i.e., hybridization capture-based target sequencing), which: a) A step of providing sample material containing nucleic acids; b) The process of preparing a nucleic acid sequencing library; c) A step of hybridizing at least one probe or probe set of the present invention to a target nucleic acid; d) A process for amplifying nucleic acids; e) the step of determining the sequence of nucleic acids; and f) A step of analyzing the nucleic acid sequence obtained in step e). This provides a method that includes this.

[0029] In a particular embodiment, the present invention provides a method for diagnosing or monitoring cancer, comprising the step of hybridizing at least one probe or probe set of the present invention with a complementary portion of a target nucleic acid present in a sample.

[0030] In a particular embodiment, the present invention provides a method for diagnosing or monitoring cancer, wherein the presence of cancer is indicated when the presence of oncogenic fusion genes and / or exon skipping is detected in a patient sample.

[0031] In a particular embodiment, the present invention provides the use of at least one probe or probe set in a method for detecting fusion genes and / or exon skipping, preferably fusion genes present in cancer. [Brief explanation of the drawing]

[0032] [Figure 1]Figure 1 shows example designs of universal probes (probes that do not overlap with the second fusion partner; referred to herein as 0 bp) and specific probes (probes that overlap with the second fusion partner), all of which have a constant total length of 120 bp. A) shows a high-overlap probe design in which the probe overlaps with the second fusion partner by up to 60 bp or 90 bp. Panel A) also shows a mixture of high-overlap probes (60 bp and 90 bp) and low-overlap probes (30 bp), as used in Examples 2 and 3. B) shows a low-overlap probe design of the present invention in which the probe overlaps with the second fusion partner by 10 bp, 20 bp, 30 bp, 40 bp, or 50 bp. [Figure 2] Figure 2 shows heatmaps of FGFR3-TACC3 fusion fragments starting at the fusion cleavage point (0 bp position) detected for the high-overlap probe (A) compared to those detected for the low-overlap probes (B-D) according to Example 2. B) 0 bp probe; C) 50 bp probe; and D) mixture of 0 bp, 10 bp, 20 bp, 30 bp, and 40 bp probes. The numbers on the left of plots B-D represent the number of unique molecules containing the cleavage point region supporting the fusion (considering 3 million fragment inputs), and the percentages on the right of the plots represent the percentage of unique molecules supporting the fusion compared to the total number of unique molecules in the region. [Figure 3] Figure 3 shows heatmaps of PAX8-PPARG fusion fragments starting at fusion breakpoints (0bp positions) detected for high-overlap probes (A) compared to those detected for low-overlap probes (B-D) according to Example 3. B) 0bp probe; C) 50bp probe; and D) mixture of 0bp, 10bp, 20bp, 30bp, and 40bp probes. The numbers on the left of plots B-D represent the number of unique molecules containing the breakpoint region supporting the fusion (considering 3 million fragment inputs), and the percentages on the right of the plots represent the percentage of unique molecules supporting the fusion compared to the total number of unique molecules in the region. [Figure 4]The bar plots show the number of fragments spanning the cleavage point (divided by 1000) captured by the low-overlap probe design (0 bp or 0+50 bp, or mixed (0 bp+10 bp+20 bp+30 bp+40 bp+50 bp)) for various fusions in clinical samples (S1, S2, S3) and the reference sample described in Example 4 (RS: Seraseq® FFPE Tumor Fusion RNA v2). Along with the number of detected fusion fragments, the number of fusion fragments under condition 0 bp, the magnification change of fusion fragments under condition 0+50 bp compared to 0 bp, and the magnification change of fusion fragments under the "mixed" condition compared to 0 bp are indicated above each bar. [Modes for carrying out the invention]

[0033] "Nucleic acid molecules" are selected from known nucleic acid molecules such as RNA (ribonucleic acid), DNA (deoxyribonucleic acid), LNA (locked nucleic acid), PNA (peptide nucleic acid), and cDNA (complementary DNA).

[0034] The terms “fusion gene,” “gene fusion,” “fusion nucleic acid molecule,” “hybrid gene,” “chimeric gene,” or “chimeric transcript” refer to a gene formed from two or more originally independent genes, including the whole or fragment of a first gene and the whole or fragment of a second gene and / or subsequent gene. Typically, a fusion gene includes (i) a first fusion partner or gene, often a kinase gene, and (ii) two partners of the second fusion partner or gene. Fusion genes result from chromosomal rearrangements such as translocations, inversions, and deletions, and may result in chimeric RNA and chimeric proteins. Fusion genes have been found in all major types of tumors in humans (i.e., benign, potentially malignant, or malignant (cancer)). Fusion genes are important clinical biomarkers that guide diagnosis, provide prognostic information, and support treatment decisions.

[0035] The term "breakpoint" refers to a nucleotide location in a chromosome or transcript where two distal genomic regions merge as a result of either genome rearrangement or splicing. Furthermore, the term "breakpoint" can refer to a point between the first and second nucleotide portions of a target nucleic acid (also called an exon junction), such as between the first and second fusion partners of a fusion gene or transcript, or between the first and second exons of an exon-skipping transcript.

[0036] Therefore, the first and second nucleotide portions of a target nucleic acid, such as the first and second fusion partners of a fusion gene or transcript, or the first and second exons of an exon-skipping transcript, are defined and separated by the cleavage site. It is understood that the second nucleotide portion of the target nucleic acid begins at the cleavage site. Often, the first nucleotide portion of the target nucleic acid can be defined as the 3' of the cleavage site and is sometimes called the 3' partner. Also, often the second nucleotide portion of the target nucleic acid may be at the 5' of the cleavage site and is sometimes called the 5' partner.

[0037] The terms “fusion” or “fusion molecule” encompass any fusion molecule, including genes, gene products (cDNA, mRNA, or polypeptides), and variants thereof, including the whole or fragments of a first fusion gene or partner, and the whole or fragments of a second fusion gene or partner.

[0038] The terms "known fusion," "known gene fusion," or "known fusion gene" refer to a fusion gene in which the whole or fragment of the first gene (the first fusion partner) and the whole or fragment of the second gene (the second fusion partner) are known. Ongoing efforts are being made to classify gene fusions and explain their roles in cancer, such as COSMIC: The Catalogue of Somatic Mutations in Cancer (Tate et al, 2019, COSMIC: the Catalogue Of Somatic Mutations In Cancer. Nucleic Acids Res. 47(D1):D941-D947; http: / / cancer.sanger.ac.uk / census), the Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer (https: / / mitelmandatabase.isb-cgc.org / ), and ChimerDB (Jang et al., 2020, ChimerDB 4.0: an updated and expanded database of fusion genes. Nucleic Acids Res. 48(D1):D817-D824; http: / / www.kobic.re.kr / chimerdb / ).

[0039] Conventional nomenclature for describing fusion genes is used herein, and includes, for example, the following: abbreviations (5'-3') for fusion genes such as FGFR3-TACC3, where FGFR3 is an abbreviation for Fibroblast Growth Factor Receptor 3 and is the first fusion gene, TACC3 is an abbreviation for Transforming acidic coiled-coil-containing protein 3 and is the second fusion gene, or in PAX8-PPARG, where PAX8 is an abbreviation for paired box gene 8 and is the second fusion gene, PPARG is an abbreviation for Peroxisome Proliferator Activated Receptor Gamma and is the first fusion gene, or, for example, abbreviations (5'-3') for fusion genes including the relevant transcripts and exons. For example, the FGFR3:BAIAP2L1(NM_000142:e17:NM_018842:e2) fusion includes a 5'FGFR3 transcript NM_00142 up to exon 17, fused with a 3' partner BAIAP2L1 transcript NM_018842 starting from exon 2.

[0040] The terms "unknown fusion," "novel fusion," "partner agnostic fusion," "unknown fusion gene," "novel fusion gene," or "partner agnostic gene fusion" refer to a novel fusion gene or a fusion gene whose second gene partner or isoform has not yet been identified.

[0041] The term "kinase" or "kinase molecule" refers to a group of enzymes that catalyze the transfer of phosphate groups from high-energy phosphate-containing molecules (such as ATP) to substrates. Examples of kinase enzymes include protein kinases (cyclin-dependent kinases (CDKs), mitogenic factor-activated protein kinases (MAPKs), etc.), lipid kinases (phosphatidylinositol kinases, sphingosine kinases (SKs), etc.), carbohydrate kinases (hexokinases, phosphofructokinases (PFKs), etc.), kinases that act on nucleotides (DNA and RNA), and kinases that act on other small molecules that are substrates of these kinases (e.g., creatine, phosphoglycerates, riboflavin, dihydroxyacetone, shikimic acid, etc.). The term "kinase gene" or "kinase transcript" refers to a group of genes or transcripts that encode kinase molecules.

[0042] The terms "non-kinase," "non-kinase gene," or "non-kinase transcript" refer to a group of genes or transcripts that do not encode kinase proteins.

[0043] The term "exon skipping" refers to a form of RNA splicing in which a cell removes one or more consecutive exons from messenger RNA. An "exon" is a region of a gene's nucleotide sequence that remains in the mature transcript's nucleotide sequence. Exon skipping is understood to occur due to deletion, mutation, or as a result of splicing regulation. Exons present in nucleic acid molecules that are adjacent to and contained in a skipped exon can be detected using the probes and principles disclosed herein.

[0044] The terms "RNA sequencing," "RNAseq," "RNA-seq," "transcriptome profiling," "RNA high-throughput sequencing," "large-scale parallel RNA sequencing," or "next-generation sequencing of cDNA" refer to sequencing techniques that use next-generation sequencing (NGS) to analyze the transcriptome of cells and reveal the presence and quantity of RNA in a biological sample at a specific point in time.

[0045] The term "targeted RNA sequencing" or "targeted RNA-seq" refers to RNA sequencing combined with specific target enrichment, which can be performed, for example, by targeted capture or amplicon-based approaches (amplicon sequencing). This method allows for the selection and sequencing of a specific transcript of interest (i.e., enrichment of the target RNA transcript or enrichment). Similarly, the term "targeted DNA sequencing" or "targeted DNA-seq" refers to DNA sequencing combined with specific target enrichment, which can be performed, for example, by targeted capture or amplicon-based approaches (amplicon sequencing).

[0046] The terms “targeted RNA-seq / DNA-seq by target capture,” “(RNA / DNA)CaptureSeq,” “hybridization capture (sequencing),” “target capture method,” or “hybridization capture-based targeted enrichment for NGS” refer to enrichment techniques that include a hybridization capture step. In this technique, RNA or DNA sequencing libraries are subjected to hybridization capture using probes specifically designed to isolate the target sequences and bound to biochemical parts (usually biotin). These targets are then amplified and sequenced in the enriched libraries.

[0047] The terms “probe,” “bait,” “(probe) nucleic acid molecule,” “capture probe,” or “(DNA / RNA) oligonucleotide (capture) probe” refer to a nucleic acid molecule that can hybridize to a target nucleic acid molecule. In the context of detecting fusion genes and exon skipping, a probe can hybridize or anneal to the nucleic acid molecule of a fusion gene / transcript and / or transcript resulting from exon skipping. If a probe hybridizes / anneals only to the nucleic acid molecule of a first fusion gene (partner), it enables the detection of any fusion involving that gene, and such a probe is referred to herein as a universal probe. If a probe hybridizes / anneals to the nucleic acid molecule of a first fusion gene (partner) and crosses the breakpoint to enter a second fusion gene (partner), it enables the detection of a known fusion gene, and such a probe is referred to herein as a specific probe.

[0048] The term "target nucleic acid" refers to a nucleic acid region within a gene or transcript that can be captured by the probe of the present invention; for example, a gene that may form a fusion gene or a transcript in which exon skipping may occur.

[0049] probe In one embodiment, A first probe portion complementary to the first nucleotide portion of the target nucleic acid, and A second probe portion complementary to the second nucleotide portion of the target nucleic acid. A probe that includes, The probe overlaps with the break point between the first nucleotide portion and the second nucleotide portion of the target nucleic acid, The length of the second probe portion corresponds to approximately 1% to approximately 42% of the total length of the probe. A probe is provided.

[0050] The fact that the length of the second probe portion corresponds to approximately 1% to 42% of the total probe length means that the second probe portion and the second portion of the target nucleic acid overlap by that percentage of the total probe length.

[0051] It is understood that the probe may contain a probe portion complementary to the first nucleotide portion of the target nucleic acid, and another probe portion complementary to the second nucleotide portion of the target nucleic acid.

[0052] Probes complementary to the first and second nucleotide portions of a target nucleic acid are specific probes, and it is understood that they enable the detection of, for example, known fusion genes or exon skipping.

[0053] In one embodiment, the target nucleic acid is a fusion gene or transcript, wherein the first nucleotide portion of the target nucleic acid is a fusion gene or transcript encoding a first fusion partner, and the second nucleotide portion of the target nucleic acid is a fusion gene or transcript encoding a second fusion partner.

[0054] In one embodiment, the target nucleic acid is an exon-skipping transcript, the first nucleotide portion of the target nucleic acid is an exon-skipping transcript encoding a first exon, and the second nucleotide portion of the target nucleic acid is an exon-skipping transcript encoding a second exon.

[0055] In one embodiment, the target nucleic acid is selected from DNA, RNA, and cDNA.

[0056] In one embodiment, A fusion gene or transcript encoding the first fusion partner, or Exon-skipping transcripts that encode a single exon A first probe portion complementary to the first nucleotide portion of the target nucleic acid, and A fusion gene or transcript encoding a second fusion partner, or Exon-skipping transcripts encoding the second exon A second probe portion that is complementary to the second nucleotide portion of the target nucleic acid. A probe that includes, The probe overlaps with the cleavage point between the first nucleotide portion and the second nucleotide portion of the target nucleic acid, and The length of the second probe portion corresponds to approximately 1% to approximately 42% of the total length of the probe. A probe is provided.

[0057] The nucleotide length of the second probe portion complementary to the second nucleotide portion of the target nucleic acid, expressed as a percentage of the total probe length, is not limited to, for example, 1% or more, 9% or more, 17% or more, 25% or more, 33% or more, about 42% or more, and about 42% or less; about 1%, 9% or less, 17% or less, 25% or less, 33% or less, and 42% or less; for example, 1%~42%, 1%~33%, 1%~25%, 1%~17%, 1%~9%, etc. Preferably, the length of the second probe portion, expressed as a percentage of the total probe length, is selected from about 1%, 9%, 17%, 25%, 33%, and 42%.

[0058] In one embodiment, A first probe portion complementary to the first nucleotide portion of the target nucleic acid, and A second probe portion complementary to the second nucleotide portion of the target nucleic acid. A probe that includes, The probe overlaps with the break point between the first nucleotide portion and the second nucleotide portion of the target nucleic acid, The second probe portion has a length of 1 to approximately 50 base pairs (bp), and the total length of the probe is approximately 120 bp. A probe is provided.

[0059] In one embodiment, A fusion gene or transcript encoding the first fusion partner, or Exon-skipping transcripts that encode a single exon A first probe portion complementary to the first nucleotide portion of the target nucleic acid, and A fusion gene or transcript encoding a second fusion partner, or Exon-skipping transcripts encoding the second exon A second probe portion that is complementary to the second nucleotide portion of the target nucleic acid. A probe that includes, The probe overlaps with the break point between the first nucleotide portion and the second nucleotide portion of the target nucleic acid, The second probe portion has a length of 1 to approximately 50 base pairs (bp), and the total length of the probe is approximately 120 bp. A probe is provided.

[0060] The nucleotide length of the second probe portion complementary to the second nucleotide portion of the target nucleic acid is not limited, but for example, 1 or more, 10 or more, 20 or more, 30 or more, 40 or more, about 50, and about 50 or less; 10 or less, 20 or less, 30 or less, 40 or less, and 50 or less; for example, 10 to 50, 10 to 40, 10 to 30, and 10 to 20. Preferably, the nucleotide length of the second probe portion complementary to the second nucleotide portion of the target nucleic acid is about 10, 20, 30, 40, and 50.

[0061] The total nucleotide length of the probe is not limited, but is, for example, 20 or more, 40 or more, 60 or more, 80 or more, 100 or more, 110 or more, or 115 or more, and 220 or less, 200 or less, 180 or less, 160 or less, 140 or less, 130 or less, or 125 or less; for example, 20-220, 60-180, 100-140, 110-130, 115-125, or 120. Preferably, the nucleotide length of the probe is about 120.

[0062] In one embodiment, a probe is provided that is complementary only to the first nucleotide portion of the target nucleic acid and does not overlap with the cleavage site between the first and second nucleotide portions of the target nucleic acid. In this embodiment, there is no second probe portion. In this embodiment, the second probe portion is 0 bp (0 bp probe). This probe is a universal probe and is understood to be capable of detecting unknown fusion genes.

[0063] The nucleotide length of the universal probe is not limited, but for example, it may be 20 or more, 40 or more, 60 or more, 80 or more, 100 or more, 110 or more, or 115 or more; or 220 or less, 200 or less, 180 or less, 160 or less, 140 or less, 130 or less, or 125 or less; for example, 20-220, 60-180, 100-140, 110-130, 115-125, or 120. Preferably, the nucleotide length of the probe is 120.

[0064] According to the present invention, a probe of the present invention comprising a probe complementary only to the first nucleotide portion of the target nucleic acid (a universal probe, or a 0 bp probe) and a second probe portion of length 1 to about 50 bp is referred to herein as a low-duplication probe. A probe comprising a second probe portion longer than 50 bp is referred herein as a high-duplication probe. The designs of the low-duplication probe and the high-duplication probe of the present invention are described in Example 1 and shown in Figure 1.

[0065] In one embodiment, A first probe moiety complementary to a fusion gene or transcript encoding a first fusion partner, wherein the first fusion partner is preferably a kinase molecule, and A second probe moiety complementary to a fusion gene or transcript encoding a second fusion partner, wherein the second fusion partner is preferably a non-kinase molecule. A probe that includes, The probe overlaps with the cutting point between the first fusion partner and the second fusion partner in the invention described herein. A probe is provided.

[0066] In one embodiment, a probe of the present invention is provided, comprising a first probe portion complementary to a fusion gene or transcript encoding a first fusion partner, wherein the first fusion partner is a kinase molecule. It is understood that such a kinase molecule may include any known kinase. Examples of kinases include, but are not limited to, protein kinases (CDK, MAPK, etc.), lipid kinases (phosphatidylinositol kinase, SK, etc.), carbohydrate kinases (hexokinase, PFK, etc.), kinases acting on nucleotides (DNA and RNA), and kinases acting on other small molecules. Preferably, the kinases are tyrosine kinases and serine / threonine kinases. Examples of specific kinases include, but are not limited to, ALK, RET, ROS, MET, BRAF, NTRK, etc.

[0067] In one embodiment, a probe of the present invention is provided, comprising a second probe portion complementary to a fusion gene or transcript encoding a second fusion partner, wherein the second fusion partner is a non-kinase molecule. It is understood that such a non-kinase molecule may include any known non-kinase molecule. Examples of non-kinases include, but are not limited to, EML4, CD74, ETV6, TACC3, LMNA, SLC34A2, and the like.

[0068] Table 1 shows examples of fusion genes that can be captured and detected by the probe of the present invention. [Table 1]

[0069] In one embodiment, the probe of the present invention is made from DNA or RNA, preferably from DNA.

[0070] In the description of probes, the terms "complementary" and "homonymous" are used interchangeably.

[0071] In one embodiment, a probe of the present invention is provided comprising any of the following nucleotide sequences: (a) a nucleotide sequence comprising at least 20, 40, 60, 80, 100, 110, 115, or 120 consecutive nucleotides complementary to the first and / or second nucleotide parity of a target nucleic acid; (b) a nucleotide sequence in which one or more nucleotides are added, deleted, and / or substituted in the nucleotide sequence of (a); (c) a nucleotide sequence having, for example, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity with nucleotide sequence (a); and (d) a nucleotide sequence having at least 20, 40, 60, 80, 100, 110, 115, or 120 consecutive nucleotides that hybridize under stringent conditions with the first and / or second nucleotide parity of a target nucleic acid.

[0072] In one embodiment, the probe of the present invention enriches (i.e., captures) a target gene fusion (DNA) or transcript (RNA).

[0073] In one embodiment, the probe of the present invention is suitable for capturing and detecting exon skipping and / or fusion genes.

[0074] In one embodiment, the probe of the present invention is suitable for capturing and detecting fusion genes containing two or more partners.

[0075] In another embodiment, the probe of the present invention is coupled to a detectable label such as biotin, magnetic beads, fluorophores, radioisotopes, or nanoparticles. It is understood that such a probe may be used in targeted enrichment methods.

[0076] In another embodiment, the probe of the present invention is associated with a microarray such as a DNA microarray, a cDNA microarray, or a SNP microarray. It is understood that such a probe may be used in hybrid capture reactions.

[0077] In another embodiment, the probe of the present invention has an array selected from sequence numbers 1 to 75.

[0078] In one embodiment, a universal probe selected from SEQ ID NOs: 4, 13, 19, and 20-26 is provided.

[0079] In one embodiment, a probe of the present invention is provided in which the length of the second probe portion is 10 bp, the total length of the probe is 120 bp, and the probe is selected from SEQ ID NOs: 6, SEQ ID NOs: 15, and SEQ ID NOs: 37-46.

[0080] In one embodiment, a probe is provided in which the length of the second probe portion is 20 bp, the total length of the probe is 120 bp, and the probe is selected from sequence numbers 7, 16, and 47-56.

[0081] In one embodiment, a probe of the present invention is provided in which the length of the second probe portion is 30 bp, the total length of the probe is 120 bp, and the probe is selected from SEQ ID NOs: 1, 8, 12, 17 and 57-66.

[0082] In one embodiment, a probe of the present invention is provided in which the length of the second probe portion is 40 bp, the total length of the probe is 120 bp, and the probe is selected from SEQ ID NOs: 9, SEQ ID NOs: 18, and SEQ ID NOs: 67-76.

[0083] In one embodiment, a probe of the present invention is provided in which the length of the second probe portion is 50 bp, the total length of the probe is 120 bp, and the probe is selected from SEQ ID NOs. 5, SEQ ID NOs. 14, and SEQ ID NOs. 27-36.

[0084] In one embodiment, a method for preparing the probe of the present invention is provided.

[0085] Standard methods for the chemical synthesis of DNA or RNA probes, such as those described in McBride LJ and Caruthers MH., 1983, Tetrahedron Letters 24(3):245-248, can be used.

[0086] In one embodiment, a method for preparing probes of the present invention is provided, comprising the synthesis of oligonucleotides on a microarray having a length in the range of about 60 to about 120 bases or about 80 to about 120 bases.

[0087] Prior to the enrichment step, the oligonucleotide pool (probe pool) is modified by a known process, such as the method described in Klocker et al., 2020, Chem. Soc. Rev.49, p. 8749-8773, to include detectable labels such as biotin, magnetic beads, fluorophores, radioisotopes, or nanoparticles.

[0088] It is understood that isolated probes can be used in liquid-phase methods, while probes on a microarray can be used in surface-phase methods.

[0089] probe set In another embodiment, a set (combination, mixture, or pool) of probes of the present invention is provided.

[0090] In one embodiment, a probe set is provided that includes at least two, at least three, at least four, at least five, or at least six probes of the present invention.

[0091] In one embodiment, a probe set is provided comprising at least 10, at least 20, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, or at least 500 probes of the present invention.

[0092] In one embodiment, the probe set of the present invention includes at least one universal probe of the present invention and at least one specific probe of the present invention.

[0093] In one embodiment, A first probe portion complementary to the first nucleotide portion of the target nucleic acid, and A second probe portion complementary to the second nucleotide portion of the target nucleic acid. A probe comprising at least one element, The probe overlaps with the break point between the first nucleotide portion and the second nucleotide portion of the target nucleic acid, The length of the second probe portion corresponds to approximately 1% to approximately 42% of the total length of the probe. The at least one probe, and At least one further probe complementary only to the first nucleotide portion of the target nucleic acid, wherein the further probe does not overlap with the break point between the first and second nucleotide portions of the target nucleic acid. A probe set comprising the following is provided.

[0094] In one embodiment, A first probe portion complementary to the first nucleotide portion of the target nucleic acid, and A second probe portion complementary to the second nucleotide portion of the target nucleic acid. A probe comprising at least one element, The probe overlaps with the break point between the first nucleotide portion and the second nucleotide portion of the target nucleic acid, The second probe portion has a length of 1 to approximately 50 base pairs (bp), and the total length of the probe is approximately 120 bp. The at least one probe, and At least one further probe complementary only to the first nucleotide portion of the target nucleic acid, wherein the further probe does not overlap with the break point between the first and second nucleotide portions of the target nucleic acid. A probe set comprising the following is provided.

[0095] The probe set of the present invention, comprising at least one universal probe and at least one specific probe, is understood to be suitable for capturing and detecting known and novel fusion gene partners.

[0096] In one embodiment, the probe set of the present invention includes at least two probes specific to the present invention.

[0097] In one embodiment, A first probe portion complementary to the first nucleotide portion of the target nucleic acid, and A second probe portion complementary to the second nucleotide portion of the target nucleic acid. A probe comprising at least two elements, The probe overlaps with the break point between the first nucleotide portion and the second nucleotide portion of the target nucleic acid, The length of the second probe portion corresponds to approximately 1% to approximately 42% of the total length of the probe. A probe set is provided, comprising at least two probes.

[0098] In one embodiment, A first probe portion complementary to the first nucleotide portion of the target nucleic acid, and A second probe portion complementary to the second nucleotide portion of the target nucleic acid. A probe comprising at least two elements, The probe overlaps with the break point between the first nucleotide portion and the second nucleotide portion of the target nucleic acid, The second probe portion has a length of 1 to approximately 50 base pairs (bp), and the total length of the probe is approximately 120 bp. A probe set is provided, comprising at least two probes.

[0099] A probe set comprising at least two specific probes of the present invention is understood to be suitable for capturing and detecting known fusion gene partners.

[0100] In another preferred embodiment, the probe set of the present invention includes at least two probes specific to the present invention that have different lengths of the second probe portion.

[0101] In one embodiment, the probe set of the present invention comprises probes that hybridize to different target nucleic acids, allowing multiple different target nucleic acids to be captured and detected with a single probe set.

[0102] In one embodiment, the probe set of the present invention includes probes that hybridize to the same first nucleotide portion of a target nucleic acid, such as a fusion gene or transcript encoding a first fusion partner, or an exon-skipping transcript encoding a single exon. An exemplary probe set includes both specific probes and / or universal probes that hybridize to the same first fusion gene, which is PPARG or FGFR3.

[0103] In one embodiment, the probe set of the present invention comprises at least two, at least three, at least four, at least five, at least ten, at least 20, at least 50, or at least 100 probes that hybridize with different first nucleotide portions of a target nucleic acid, such as a fusion gene or transcript encoding a first fusion partner, or an exon-skipping transcript encoding a single exon.

[0104] In one embodiment, the probe set of the present invention comprises at least one universal probe complementary only to the first nucleotide portion of the target nucleic acid, and at least one, at least two, at least three, at least four, or at least five probes (specific probes) whose second probe portion length corresponds to about 1% to about 42% of the total length of the probe, for example, about 1%, 9%, 17%, 25%, 33%, and / or 42%.

[0105] In one embodiment, the probe set of the present invention comprises at least one universal probe complementary only to the first nucleotide portion of the target nucleic acid, and at least one, at least two, at least three, at least four, or at least five probes (specific probes) whose second probe portion is about 1 to about 50 bp in length, for example, about 10 bp, 20 bp, 30 bp, 40 bp and / or 50 bp, and whose total length is about 120 bp.

[0106] In one embodiment, the probe set of the present invention includes at least one universal probe complementary only to the first nucleotide portion of the target nucleic acid, and at least one probe (specific probe) in which the length of the second probe portion corresponds to 42% of the total length of the probe.

[0107] In one embodiment, the probe set of the present invention includes at least one universal probe complementary only to the first nucleotide portion of the target nucleic acid, and at least one probe having a second probe portion length of 50 bp and a total probe length of about 120 bp.

[0108] In one embodiment, the probe set of the present invention includes at least one universal probe complementary only to the first nucleotide portion of the target nucleic acid, and at least five probes (specific probes) whose second probe portion length corresponds to about 1%, 9%, 17%, 25%, 33%, and / or 42% of the total length of the probe.

[0109] In one embodiment, the probe set of the present invention includes at least one universal probe complementary only to the first nucleotide portion of the target nucleic acid, and at least five probes (specific probes) in which the second probe portion is about 10 bp, 20 bp, 30 bp, 40 bp and / or 50 bp in length, and the total length of the probe is about 120 bp.

[0110] In one embodiment, the probe set of the present invention includes at least two probes (specific probes) in which the length of the second probe portion corresponds to about 1% to about 42% of the total length of the probe, for example, about 1%, 9%, 17%, 25%, 33%, and / or 42%.

[0111] In one embodiment, the probe set of the present invention includes at least two, at least three, at least four, or at least five probes (specific probes) whose second probe portion length corresponds to about 1% to about 42% of the total length of the probe, for example, about 1%, 9%, 17%, 25%, 33%, and / or 42%. Preferably, the probe set of the present invention includes at least five probes whose second probe portion length corresponds to about 1%, 9%, 17%, 25%, 33%, and / or 42% of the total length of the probe.

[0112] In one embodiment, the probe set of the present invention includes at least two probes (specific probes) in which the second probe portion is about 1 to about 50 bp in length, for example, about 10 bp, 20 bp, 30 bp, 40 bp and / or 50 bp, and the total length of the probe is about 120 bp.

[0113] In one embodiment, the probe set of the present invention includes at least two, at least three, at least four, or at least five probes (specific probes) whose second probe portion is about 1 to about 50 bp in length, for example, about 10 bp, 20 bp, 30 bp, 40 bp, and / or 50 bp, and whose total length is about 120 bp. Preferably, the probe set of the present invention includes at least five probes whose second probe portion is about 10 bp, 20 bp, 30 bp, 40 bp, and / or 50 bp in length, and whose total length is about 120 bp.

[0114] It is understood that the probe set of the present invention may be a composition or reaction mixture that optionally further comprises target nucleic acid molecules such as fusion nucleic acid molecules (fusion gene / transcript) and / or exon-skipping nucleic acid molecules (exon-skipping transcript).

[0115] It is understood that the probe set of the present invention may be a reaction of a mixture further containing target nucleic acid molecules derived from a patient sample.

[0116] The terms "probe set," "probe pool," or "probe pool" are interchangeable.

[0117] In one embodiment, a probe set of the present invention is provided, further comprising any probe known in the art.

[0118] Probe method and usage The probe of the present invention is designed to advantageously reduce the capture of the second fusion partner counterpart of the WT.

[0119] In a particular embodiment, the present invention provides at least one probe or probe set for detecting at least one fusion gene and / or exon skipping.

[0120] According to another specific embodiment, the present invention provides the use of at least one probe or probe set for detecting at least one fusion gene and / or exon skipping.

[0121] In a particular embodiment, the present invention provides at least one probe or probe set for detecting at least one fusion gene, wherein the fusion gene is known or unknown (i.e., a novel fusion or a partner unknown fusion).

[0122] In a particular embodiment, the present invention provides at least one probe or probe set for use in targeted enrichment DNA sequencing or RNA sequencing.

[0123] According to another specific embodiment, the present invention provides the use of at least one probe or probe set in a targeted enrichment DNA or RNA sequencing method.

[0124] According to a particular embodiment, the present invention provides at least one probe or probe set for use in next-generation sequencing (NGS)-based targeted enrichment DNA or RNA sequencing.

[0125] According to another specific embodiment, the present invention provides the use of at least one probe or probe set in a next-generation sequencing (NGS)-based targeted enrichment DNA or RNA sequencing method.

[0126] In a particular embodiment, the present invention provides at least one probe or probe set for use in methods such as microarrays, blotting, and immobilization of probes onto columns or beads for the purification of target DNA.

[0127] According to another specific embodiment, the present invention provides the use of at least one probe or probe set in methods such as microarrays, blotting, and immobilization of probes onto columns or beads for the purification of target DNA.

[0128] According to one embodiment, a method is provided for detecting gene fusion and / or exon skipping in a sample, comprising the step of hybridizing at least one probe or probe set of the present invention with a complementary portion of a target nucleic acid.

[0129] According to one embodiment, a method for targeting or RNA sequencing by target capture (i.e., hybridization capture sequencing) is provided: a) A step of providing sample material containing nucleic acids, b) The process of preparing a nucleic acid sequencing library; c) A step of hybridizing at least one probe or probe set with a target nucleic acid; d) A process for amplifying nucleic acids; e) the step of determining the sequence of nucleic acids; and f) A step of analyzing the nucleic acid sequence obtained in step e). A method including this is provided.

[0130] According to one embodiment, a method for determining a targeted RNA sequence by target capture, wherein step a) provides a sample material containing nucleic acid: a.1) A step of providing sample material containing nucleic acids; a.2) Step of arbitrarily selecting an mRNA transcript: a.3) A process to arbitrarily deplete rRNA (ribosomal RNA); a.4) Steps to fragment the mRNA transcript. A method including this is provided.

[0131] Steps a) and a.1) to a.4) can be carried out by any known method, such as polyA selection (a.2 and a.3), hybridization-based rRNA depletion (a.3), or chemical or enzymatic RNA fragmentation (a.4).

[0132] According to one embodiment, a method for sequencing targeted RNA by target capture, wherein step b) preparing a nucleic acid sequencing library: b.1) The process of converting sample RNA to cDNA (complementary DNA); b.2) A step of optionally converting double-stranded cDNA to blunt-ended DNA, followed by the addition of a non-template 3'dAMP(dA) nucleotide tail; b.3) A step of optionally ligating cDNA to a sequencing adapter; b.4) A step of optionally amplifying a cDNA adapter product to generate a whole transcriptome library. A method is provided that includes the following: Steps b) and b.1) to b.4) can be carried out by any known method such as reverse transcription, double-stranded cDNA synthesis (b1), end repair and A-tail formation (b2), enzymatic ligation (b3), PCR (b4). Step b.3 is required for the preparation of a nucleic acid sequencing library for NGS.

[0133] According to one embodiment, a method for targeting RNA sequencing by target capture, wherein step c) hybridizing at least one probe or probe set of the present invention with a target nucleic acid (i.e., enriching the sequence of interest): c.1) A step of preparing the individual or pooled cDNA libraries obtained in step b); c.2) A step of hybridizing at least one probe or probe set of the present invention with a target library; c.3) Washing of untargeted cDNA / library: c.4) Elution of targeted cDNA / library A method including this is provided.

[0134] Steps c) and c.1) to c.4) can be carried out by any known method, such as target hybridization capture.

[0135] In one embodiment, step c.2) is carried out by incubating individual or pooled cDNA libraries together with probes, preferably at 65°C.

[0136] In one embodiment, step c.2) is performed by incubating individual or pooled cDNA libraries with probes linked to biochemical moieties such as biotin, the probes annealing to the target sequence in the library, and the annealed product is then captured using a system that specifically selects the biochemical moieties to be used, such as magnetic beads bound to streptavidin.

[0137] In one embodiment, the probe is captured using a biotin-bound probe and a streptavidin-bound magnetic bead.

[0138] In step c.3), the probe annealed to the library is washed to remove nonspecifically bound sequences, the resulting product is amplified by PCR (step d), and then sequenced (step e).

[0139] In step c), the probe is hybridized with the target nucleic acid under conditions suitable for hybridization and washed under conditions that typically maintain hybridization between them, with nucleotide sequences being at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to each other.

[0140] Step d) The step of amplifying nucleic acids can be carried out by any known method, such as polymerase chain reaction (PCR).

[0141] Step e) The step of determining the nucleic acid sequence can be carried out by any known method, such as synthetic sequencing (Illumina), ion semiconductor sequencing (Ion Torrent), single-molecule real-time sequencing (SMRT) (Pacific Biosciences), or nanopore DNA / RNA sequencing (Oxford Nanopore Technologies).

[0142] The analysis in step f) can be carried out by any known method so as to quantify the number of fragments supporting the presence of the gene fusion event of interest.

[0143] Known and novel gene fusions are understood to be particularly oncogenic gene fusions.

[0144] According to one embodiment, a method for targeted DNA sequencing or RNA sequencing by target capture is provided, wherein the sample is obtained from a subject who is suffering from or suspected of suffering from a disease, preferably cancer.

[0145] According to one embodiment, a targeted DNA sequencing or RNA sequencing method by targeted capture is provided in which the detection of the presence of fusion genes and / or exon skipping in a sample indicates the presence of cancer.

[0146] composition According to another aspect of the present invention, a composition comprising at least one probe of the present invention is provided.

[0147] According to another aspect of the present invention, a composition comprising at least one probe set of the present invention is provided.

[0148] In certain embodiments, the compositions of the present invention are useful in the methods of the present invention, particularly in targeted DNA sequencing or RNA sequencing methods, and in methods for diagnosing or monitoring cancer.

[0149] According to a particular embodiment, the composition of the present invention is a reaction mixture, optionally further comprising a target nucleic acid molecule.

[0150] kit According to another aspect of the present invention, a kit is provided which includes at least one probe of the present invention and optionally instructions for use.

[0151] According to another aspect of the present invention, a kit is provided which includes at least one probe set of the present invention and optionally an instruction manual.

[0152] In certain embodiments, the kits of the present invention are useful in the methods of the present invention, particularly in targeted DNA sequencing or RNA sequencing methods, and in methods for diagnosing or monitoring cancer.

[0153] Methods of cancer diagnosis and monitoring In one embodiment, the expression of a fusion gene or exon skipping is detected in a patient sample, suggesting the presence of cancer.

[0154] In a particular embodiment, the present invention provides at least one probe or set of probes for use in a method for detecting fusion genes and / or exon skipping, preferably fusion genes and / or exon skipping present in cancer.

[0155] According to another specific embodiment, the present invention provides the use of at least one probe or set of probes in a method for detecting fusion genes and / or exon skipping, preferably fusion genes and / or exon skipping present in cancer.

[0156] According to one embodiment, a method is provided for diagnosing or monitoring cancer in a sample, comprising the step of hybridizing at least one probe or probe set of the present invention with a complementary portion of a target nucleic acid present in the cancer.

[0157] According to certain embodiments, methods for diagnosing or monitoring cancer are provided, including any method of the present invention as described herein.

[0158] According to another specific embodiment, a method for diagnosing or monitoring cancer: a) A step of providing sample material containing nucleic acids; b) The process of preparing a nucleic acid sequencing library; c) A step of hybridizing at least one probe or probe set of the present invention with a target nucleic acid; d) A process for amplifying nucleic acids; e) the step of determining the sequence of nucleic acids; and f) A step of analyzing the nucleic acid sequence obtained in step e). A method including this is provided.

[0159] According to one embodiment, a method for diagnosing or monitoring cancer is provided in which the presence of a fusion gene and / or exon skipping in a patient sample indicates the presence of cancer.

[0160] According to one embodiment, a method is provided for diagnosing or monitoring cancer, wherein a sample is obtained from a subject who has or is suspected of having a disease, preferably cancer.

[0161] According to one embodiment, a method is provided for diagnosing or monitoring cancer in which the detection of the presence of fusion genes and / or exon skipping in a patient sample indicates treatment of the patient.

[0162] In one embodiment, the sample is a patient sample and may be in the form of tissue, blood, saliva, or cytological specimen / preparation (FFPE, smear, etc.).

[0163] patient In one embodiment, the patient in the present invention is suffering from cancer.

[0164] In another specific embodiment, the patient in the present invention suffers from lung cancer, bile duct cancer, prostate cancer, pancreatic ductal adenocarcinoma, thyroid cancer, colorectal cancer, gastric cancer, glioblastoma, head and neck cancer, kidney cancer, endometrial cancer, etc.

[0165] In another specific embodiment, the patient in the present invention is suspected to have cancer.

[0166] In another specific embodiment, the patient in the present invention is receiving treatment for cancer.

[0167] References cited herein are incorporated herein by reference in their entirety. The present invention should not be limited in scope by the specific embodiments and drawings described herein, which are intended as single examples of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. The examples illustrating the present invention are not intended in any way to limit the scope of the invention. [Examples]

[0168] Example 1: Probe Design Fusion genes typically include a first partner (often a kinase enzyme) with relatively low expression levels in the WT (wild-type) gene, and a second partner, often a gene with higher WT expression levels. This means that, when the probe is designed to capture the WT counterpart of the second partner, there will be many reads consumed by the WT gene compared to the fusion gene. The following probe has been designed.

[0169] The following probes were designed: i) A universal probe, which does not overlap with the second fusion partner, i.e., referred to herein as a 0 bp probe, may have a constant length of 120 bp (Figure 1). This probe makes it possible to capture the novel fusion of the first fusion partner. ii) Specific probe, which is a probe that overlaps with the second fusion partner, i.e., the second probe portion is at least 1 bp. In a high-overlap probe design, the specific probe overlaps with the second fusion partner by more than approximately 50 bp, for example, up to 60 bp or 90 bp, and all probes are constant in total length of 120 bp (Figure 1A). In a low-overlap probe design (the present invention), the specific probe overlaps with the second fusion partner by up to 10 bp, 20 bp, 30 bp, 40 bp or 50 bp, and all probes are constant in total length of 120 bp (Figure 1B).

[0170] Example 2: Performance comparison of high-overlap probe design and low-overlap probe design in FGFR3-TACC3 fusion detection The performance of high-duplication and low-duplication probes was compared in the detection of FGFR3-TACC3 fusions. FGFR3 is the first fusion gene.

[0171] Materials and methods RNA sequencing libraries were prepared using 100 ng of RNA extracted from Seraseq® FFPE Tumor Fusion RNA v2 standard material. The individually barcoded whole transcriptome libraries were then captured using xGEN Lockdown IDT® probe-based capture technology with a custom panel. The probes were designed according to the present invention, including high-duplication probe mixtures (60 bp, 90 bp), low-duplication probe mixtures (30 bp) (Figure 2A), or low-duplication probes: 0 bp probes (Figure 2B), or low-duplication 50 bp probes (Figure 2C), or mixtures of 0 bp, 10 bp, 20 bp, 30 bp, and 40 bp low-duplication probes (Figure 2D), and including probes targeting the FGFR3-TACC3 fusion. In this fusion gene, FGFR3 is the first fusion gene and TACC3 is the second fusion gene. The captured libraries were then sequenced using an Illumina MiSeq instrument. Data were analyzed by mapping readouts to a homemade fusion-synthesized genome using a BWA-MEM aligner, generating a heatmap of fragment initiations associated with fusion breakpoints. The probe mixture included probes targeting different gene fusions; for clarity, only the probes targeting the FGFR3-TACC3 fusion are described, and the detection results for the FGFR3-TACC3 fusion are reported.

[0172] The following probes were used. a) A mixture of highly overlapping probes (60 bp and 90 bp) and a low-overlap probe (30 bp) (Figure 2A): [Table 2]

[0173] b) Universal probe (0 bp probe) (Figure 2B): [Table 3]

[0174] c) A specific probe that overlaps with the second fusion partner by 50 bp (Figure 2C): [Table 4]

[0175] d) Probe mixtures of a universal probe (0 bp probe) and specific probes that overlap with a second fusion partner by 10 bp, 20 bp, 30 bp, and 40 bp (Figure 2D): [Table 5]

[0176] Analysis of heatmaps of fragments starting from the fusion breakpoint of FGFR3-TACC3 fusions captured with highly overlapping probes (Figure 2A) showed that these probes captured a very large number of reads from the second partner in wild-type (WT) fusions. Approximately 60% of the fragments around the fusion may be attributable to the wild-type transcript of the second partner (TACC3). Adding a low-overlap probe (30 bp) to the mixture of highly overlapping probes did not reduce the reads from the second partner in WT fusions.

[0177] Analysis of heatmaps of fragments starting from fusion breakpoints of FGFR3-TACC3 fusions captured with low-duplication probes (Figures 2B, C, D) showed that these probes primarily captured second partner reads belonging to the target fusion. Advantageously, the probe mixture captured more fusion fragments than the 0bp and 50bp probes alone (i.e., probe mixture - 2108 fragment (Figure 2D); 0bp - 1101 fragment (Figure 2B); 50bp - 1302 fragment (Figure 2C)). The low-duplication probe mixture alone (Figure 2D) reduced the number of WT second partner reads compared to reads obtained using high-duplication probes (Figure 2A). Advantageously, this low-duplication probe mixture, including universal and specific low-duplication probes, enabled the detection of known and novel fusion gene partners.

[0178] These results indicate that low-overlap probes primarily capture reads of the second partner belonging to the target fusion, and advantageously, a mixture of low-overlap probes captures more fusion fragments than 0bp and 50bp probes alone. Only the mixture of low-overlap probes was able to reduce the reads of the second partner in the WT compared to reads obtained using high-overlap probes. Therefore, the design of fusion-specific probes for capturing known fusion / breakpoints was optimized to avoid capturing the WT's second partner counterpart.

[0179] Example 3: Performance comparison of high-overlap probe design and low-overlap probe design in PAX8-PPARG fusion detection The performance of high-duplication and low-duplication probes was compared in the detection of PAX8-PPARG fusions. PPARG is the first fusion gene.

[0180] Similar materials and methods as in Example 2 The probes were designed according to the present invention using a probe mixture comprising a high-duplication probe mixture (60 bp, 90 bp), a low-duplication probe (30 bp) (Figure 3A), or a low-duplication probe: 0 bp probe (Figure 3B), or a low-duplication 50 bp probe (Figure 3C), or a mixture of 0 bp, 10 bp, 20 bp, 30 bp, and 40 bp low-duplication probes (Figure 3D), and including a probe that targets the PAX8-PPARG fusion. In this fusion gene, PPARG is the first fusion gene and PAX8 is the second fusion gene. The probe mixture contains probes that target different gene fusions, and for clarity, only the probe that targets the PAX8-PPARG fusion is described, and the detection results of the PAX8-PPARG fusion are reported.

[0181] The following probes were used. a) A mixture of highly overlapping probes (60 bp and 90 bp) and low-overlap probes (30 bp) (Figure 3A): [Table 6]

[0182] b) Universal probe (0 bp probe) (Figure 3B): [Table 7]

[0183] c) A specific probe that overlaps with the second fusion partner by 50 bp (Figure 3C): [Table 8]

[0184] d) A mixture of a universal probe (0 bp probe) and specific probes that overlap with a second fusion partner by 10 bp, 20 bp, 30 bp, and 40 bp (Figure 3D): [Table 9]

[0185] Analysis of heatmaps of fragments starting from the fusion breakpoint of PAX8-PPARG fusions captured with highly overlapping probes (Figure 3A) showed that these probes captured a very large number of reads from the second partner in wild-type (WT) fusions. Approximately 39% of the fragments around the fusion may be attributable to the wild-type transcript of the second partner (PAX8). Adding a low-overlap probe (30 bp) to the mixture of highly overlapping probes did not reduce the reads from the second partner in WT fusions.

[0186] Analysis of heatmaps of fragments starting from fusion breakpoints in PAX8-PPARG fusions captured with low-duplication probes (Figures 3B, C, D) showed that these probes primarily captured second partner reads belonging to the target fusion. Advantageously, the probe mixture captured more fusion fragments than the 0bp and 50bp probes alone (i.e., probe mixture - 1827 fragments (Figure 3D); 0bp - 1032 fragments (Figure 3B); 50bp - 1351 fragments (Figure 3C)). The low-duplication probe mixture alone (Figure 3D) reduced the number of WT second partner reads compared to reads obtained using high-duplication probes (Figure 3A). Advantageously, this low-duplication probe mixture, including universal and specific low-duplication probes, enabled the detection of known and novel fusion gene partners.

[0187] These results indicate that low-overlap probes primarily capture reads of the second partner belonging to the target fusion, and advantageously, a mixture of low-overlap probes captures more fusion fragments than 0bp and 50bp probes alone. Only the mixture of low-overlap probes was able to reduce the reads of the second partner in the WT compared to reads obtained using high-overlap probes. Therefore, the design of fusion-specific probes for capturing known fusion / breakpoints was optimized to avoid capturing the WT's second partner counterpart.

[0188] Example 4: Capture of different fusions in clinical and reference samples using low-overlap probes We compared the number of fusion fragments captured with a low-overlap probe design for different fusions in clinical and reference samples.

[0189] Materials and methods RNA sequencing libraries were prepared using 100 ng of RNA extracted from clinical FFPE samples or reference samples (Seraseq® FFPE Tumor Fusion RNA v2 reference material). Individually barcoded whole transcriptome libraries were then captured using xGEN Lockdown IDT® probe-based capture technology with a custom panel. Probes were designed according to the present invention, using low-duplication probes (0 bp probe (0 bp)), or mixtures of low-duplication probes (mixture) including 0 bp, 10 bp, 20 bp, 30 bp, 40 bp, and 50 bp), or mixtures of 0 bp and 50 bp low-duplication probes (0+50 bp). The captured libraries were then sequenced using an Illumina MiSeq instrument. Data were analyzed by mapping readouts to a homemade fusion synthetic genome using a BWA-MEM aligner to generate heatmaps of fragment starts associated with fusion breakpoints. Fusions were detected in both clinical samples (solid tumors; samples 1 and 2 contained confirmed CD74-ROS1 fusions, and sample 3 contained confirmed EML4-ALK fusions (S1, S2, S3 respectively), and a reference material (RS; seracare) containing 14 different fusions and 2 exon skipping events. Various fusions were targeted, including EML4-ALK, KIF5B-RET, NCOA4-RET, SLC34A2-ROS1, TPM3-NTRK1, FGFR3-BAIAP2L1, PAX8-PPARG, FGFR3-TACC3, ETV6-NTRK3, LMNA-NTRK1, and SLC45A3-BRAF).

[0190] Along with the number of fusion fragments detected, the number of fusion fragments under condition 0 bp, the change in the fusion fragment ratio under condition 0+50 bp compared to 0 bp, and the change in the fusion fragment ratio under mixed conditions compared to 0 bp were reported (Figure 4).

[0191] The following probes were used. a) Universal probe (0bp probe): [Table 10] JPEG2022179442000011.jpg170166

[0192] b) A probe mixture consisting of a universal probe (0 bp probe) and a specific probe (0+50 bp) that overlaps with a second fusion partner by 50 bp: [Table 11] JPEG2022179442000013.jpg250153 JPEG2022179442000014.jpg250153 JPEG2022179442000015.jpg36164

[0193] c) Probe mixture (mixture) of a universal probe (0 bp probe) and specific probes that overlap with a second fusion partner by 10 bp, 20 bp, 30 bp, 40 bp, and 50 bp: [Table 12] JPEG2022179442000017.jpg250154 JPEG2022179442000018.jpg248152 JPEG2022179442000019.jpg249153 JPEG2022179442000020.jpg249153 JPEG2022179442000021.jpg248152 JPEG2022179442000022.jpg249154 JPEG2022179442000023.jpg249153 JPEG2022179442000024.jpg249153 JPEG2022179442000025.jpg36166

[0194] Note that the universal probes for FGFR3-TACC3(0bp) and FGFR3-BAIAP2L1(0bp) have the same sequence as sequence number 4. The universal probes for CD74-ROS1(0bp) and SLC34A2-ROS1(0bp) have the same sequence as sequence number 19.

[0195] These results indicate that, depending on the target fusion, the 0 bp probe, 0+50 bp probe, and a mixture of probes capture a similar or greater number of fusion fragments (Figure 4).

[0196] Therefore, the probe mixture / design of the present invention provides effective capture of fusion molecules and enriches desired specific fusions while optimizing readouts derived from mutant morphs compared to untargeted WT morphs of the gene.

Claims

1. a first probe portion complementary to a first nucleotide portion of the target nucleic acid; and a second probe portion complementary to a second nucleotide portion of the target nucleic acid At least one probe having the probe overlaps a cleavage point between a first nucleotide portion and a second nucleotide portion of the target nucleic acid; and The length of the second probe portion corresponds to 1% to 42% of the total length of the probe. the at least one probe, and A probe set comprising at least one additional probe complementary only to a first nucleotide portion of the target nucleic acid, said additional probe not overlapping a cleavage point between the first nucleotide portion and a second nucleotide portion of the target nucleic acid.

2. a first probe portion complementary to a first nucleotide portion of the target nucleic acid; and a second probe portion complementary to a second nucleotide portion of the target nucleic acid At least two probes having the probe overlaps a cleavage point between a first nucleotide portion and a second nucleotide portion of the target nucleic acid; and the length of the second probe portion corresponds to 1% to 42% of the total length of the probe; and The probe has a second probe portion of a different length. A probe set comprising the at least two probes.

3. the first nucleotide portion of the target nucleic acid is a fusion gene or transcript encoding a first fusion partner, or an exon-skipping transcript encoding one exon; and The probe set of claim 1 or 2, wherein the second probe portion complementary to the second nucleotide portion of the target nucleic acid is a fusion gene or transcript encoding a second fusion partner, or an exon-skipping transcript encoding a second exon.

4. 3. The probe set of claim 1 or 2, wherein the length of the second probe portion corresponds to 1%, 9%, 17%, 25%, 33% or 42% of the total length of the probe.

5. a fusion gene or transcript encoding a first fusion partner, or Exon-skipping transcripts encoding a single exon a first probe portion complementary to a first nucleotide portion of the target nucleic acid which is a fusion gene or transcript encoding a second fusion partner; or Exon-skipping transcripts encoding the second exon a second probe portion complementary to a second nucleotide portion of the target nucleic acid which is A probe comprising: the probe overlaps a cleavage point between a first nucleotide portion and a second nucleotide portion of the target nucleic acid; and The probe, wherein the second probe portion is 1-50 base pairs (bp) in length and the total length of the probe is 120 bp.

6. The probe of claim 5 , wherein the second probe portion has a length of 10 bp, 20 bp, 30 bp, 40 bp or 50 bp.

7. 7. The probe set of claim 3, or the probe of claim 5 or 6, wherein a first nucleotide portion of the target nucleic acid is a first fusion partner that is a kinase molecule and a second nucleotide portion of the target nucleic acid is a second fusion partner that is a non-kinase molecule.

8. A kit comprising a probe set described in claim 1 or 2 or at least one probe described in claim 5 or 6.

9. A composition comprising a probe set described in claim 1 or 2 or at least one probe described in claim 5 or 6.

10. A method for detecting fusion genes and / or exon skipping in a sample, the method comprising a step of hybridizing a probe set described in claim 1 or 2 or at least one probe described in claim 5 or 6 to a complementary portion of the target nucleic acid.

11. A method for detecting fusion genes and / or exon skipping according to claim 10, comprising: a) providing a sample material comprising nucleic acid; b) preparing a nucleic acid sequencing library; c) hybridizing the probe set according to claim 1 or 2 or at least one probe according to claim 5 or 6; d) amplifying the nucleic acid; e) determining the sequence of the nucleic acid; and f) analyzing the nucleic acid sequence obtained in step e); The method is targeted DNA or RNA sequencing with target capture comprising:

12. The method for detecting fusion genes and / or exon skipping according to claim 11, wherein the sample is obtained from a subject suffering from or suspected of suffering from a disease.

13. A method for diagnosing or monitoring cancer, comprising hybridizing a probe set described in claim 1 or 2 or at least one probe described in claim 5 or 6 with a complementary portion of a target nucleic acid present in a sample, wherein detection of the target nucleic acid, such as a target fusion gene and / or exon skipping, indicates the presence of cancer.

14. 7. Use of a probe set according to claim 1 or 2 or at least one probe according to claim 5 or 6 for detecting fusion genes and / or exon skipping.

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