WO2019217486A1 - Methods and compositions for detecting myeloma - Google Patents

Abstract

The present technology relates to compositions and methods for detecting at least one multiple myeloma-related mutation in a patient. The methods disclosed herein eliminate the need for secondary diagnostic assays such as fluorescent in situ hybridization (FISH).

Description

METHODS AND COMPOSITIONS FOR DETECTING MYELOMA

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 62/668,520, filed May 8, 2018, the contents of which are incorporated herein by reference in their entirety.

TECHNICAL FIELD

[0002] The present technology relates to compositions and methods for detecting at least one multiple myeloma-related mutation in a patient. The methods disclosed herein eliminate the need for secondary diagnostic assays such as FISH.

STATEMENT OF GOVERNMENT SUPPORT

[0003] This invention was made with government support under CA008748, AI073736, AI095692, and AR068118, awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

[0004] The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.

[0005] Multiple myeloma (MM) is a hematological neoplasm that arises from

transformation and clonal proliferation of plasma cells (Corre J el al, Blood 125: 1870-1876 (2015)). Almost every case of MM is characterized by gross chromosomal rearrangements that take the form of either hyperdiploidy or translocations predominantly involving the immunoglobulin locus, including the immunoglobulin heavy locus (IGH) (Fonseca R et al, Leukemia 23: 2210-2221 (2009)).

[0006] Molecular studies are not routinely performed in myeloma outside of

investigational trials (Bolli et al, Blood Cancer J 6: e467 (2016)). Myeloma is a disease driven by an intricate and heterogeneous interplay of genetic events and these data have thus far failed to provide a unifying view of its pathogenesis and clinical behavior (Bolli et al, Blood Cancer J 6: e467 (2016)). Moreover, the increasing availability of new drugs that target recurrent genetic lesions has created a need for updated risk stratification and a rational approach to the use of new agents (Chang-Yew Leow et al, Blood Cancer J 3: el05 (2013); Bolli et al, Blood Cancer J 6: e467 (2016)). While translocations and copy number alterations can be detected with high sensitivity using whole genome sequencing, such methods are both complex and costly.

SUMMARY

[0007] In one aspect, the present disclosure provides a bait library comprising one or more baits comprising a sequence of any of SEQ ID NOs: 159-6088. The one or more baits may comprise a barcode and/or an adapter sequence. Additionally or alternatively, in some embodiments, the one or more baits comprise a detectable label. Additionally or

alternatively, in some embodiments, the one or more baits are configured to bind one or more of: (i) an IGH locus present in a multiple myeloma sample, (ii) at least one chromosome 14 translocation breakpoint present in a multiple myeloma sample, (iii) a single nucleotide variation (SNV) present in a multiple myeloma sample, (iv) a copy number aberration (CNA) present in a multiple myeloma sample, (v) a translocation present in a multiple myeloma sample, and/or (vi) an insertion/deletion (indel) present in a multiple myeloma sample.

[0008] In any of the embodiments disclosed herein, the one or more baits are configured to bind to one or more introns of one or more genes selected from the group consisting of ANP32E, APC, ATM, ATR, BIRC2, BIRC3, CDKN2A, CDKN2C, CKS1B, CSNK1A1, CYLD, DIS3, FAF1, FAM46C, FAT3, HIST1H1E, IRF4, KDM6A, KRAS, MAP3K14, MAX, MYC, NF1, NFKBI, NFKBIA, PRDM1, PTEN, PTPRD, RBI, ROB02, SETD2, SP140, TNFAIP3, TP53, TRAF2, TRAF3, WWOX, and XBP1.

[0009] Additionally or alternatively, in some embodiments, the one or more baits are configured to bind to one or more exons of one or more genes selected from the group consisting ofACTGI, ADCY5, AK2, APBB1IP, APC, ARID2, ATM, ATP13A4, ATR, AVEN, BCE2, BIRC2, BIRC3, BRAF, BRCA2, BTG1, CCNDI, CD274,CD38, CDC27, CDKN1B, CDKN2A, CDKN2C, CRBN, CSNK1A1, CUL4B, CXCR4, CYLD, DIS3, DEX6, DNAHU, DNAH5, DNAH9, DUSP2, EGRI, EPHA3, FAM46C, FAT1, FAT3, FAT4, FBRS, FBXW7, FCRL5, FGFR3, FLG, FSIP2, GPRC5D, HIST 1 HI B, HIST 1 HI C, HIST 1 HIE, HIST1H3G, ID HI, IDH2, IKBKB, IKZFJ IKZF3, IRF4, JAM2, KDM1A, KDM6A, KEHE6, KMT2D, KRAS, ETB, MAF, MAP3K1, MAX, MYC, MYD88, NCKAP5, NF1, NFKB2, NFKBIA, NR3d, NRAS, PABPC1, PCLO, PDCD1, PIK3CA, PIM1, PPM1D, PRDM1, PRDM6, PRKD2, PSMB5, PSMB8, PSMB9, PSMC2, PSMC3, PSMC4, PSMC5, PSMC6, PSMDI, PSMD12, PSMD13, PSMD2, PSMD6, PSMD7, PTEN, RTRNP, PTPRD, RAG2, RASA2, RBI, RHOT1, RIPKI, ROBOl, R0B02, SETD2, SF3BJ SLAMF7, SP140, TNFRSF17, TP53, TRAF2, TRAF3, WHSC1 (NSD2), XBP1, XPOl, and ZFHX4.

[0010] Additionally or alternatively, in certain embodiments, the one or more baits are configured to bind to one or more exons of a gene selected from the group consisting of ABCF1, ACTG1, ARID1A, ARID2, ATM, ATRX, BRAF, C80RF34, CCNDI, CDKN1B, CDKN2C, CREBBP, CYFD, DIS3, DNMT3A, DUSP2, EGR1, EP300, FAM46C, FGFR3, FUBP1, HIST 1 HIE, HUWE1, IDH1, IDH2, IRF4, KDM5C, KDM6A, KFHF6, KMT2B, KMT2C, KRAS, FTB, MAF, MAFB, MAMF2, MAN2C1, MAX, NCORI, NF1, NFKB2, NFKBIA, NRAS, PIK3CA, PRDM1, PRKD2, RTRNP, RASA2, RBI, RFTNJ SAM HD J SETD2, SF3BI, SP140, TET2, TGDS, TP53, TRAF2, TRAF3, UBR5, XBP1, ZFP36F1, and ZNF292. In some embodiments, the one or more baits are configured to bind to one or more exons of a gene selected from the group consisting of IDH1, DIS3, and TP53.

[0011] In one aspect, the present disclosure provides a method for detecting at least one multiple myeloma-related mutation in a patient in need thereof, comprising (i) contacting a nucleic acid sample obtained from the patient with any embodiment of the bait library disclosed herein, and (ii) detecting at least one multiple myeloma-related mutation in the sample via massively parallel DNA sequencing. The nucleic acid sample may comprise DNA, RNA or any combination thereof. In certain embodiments, the nucleic acid sample is contacted with the bait library via solution phase hybridization. In some embodiments, the patient is suspected of, or diagnosed as having multiple myeloma. Additionally or alternatively, in some embodiments, the nucleic acid sample is derived from a biopsy sample obtained from the patient. In certain embodiments, the nucleic acid sample is derived from a blood or bone marrow sample.

[0012] Additionally or alternatively, in some embodiments of the methods disclosed herein, detecting at least one multiple myeloma-related mutation in the sample comprises detecting a translocation ( e.g ., chromosome 14 translocation), a single nucleotide variant (SNV), a copy number aberration (CNA), and/or an insertion/deletion (indel).

[0013] Additionally or alternatively, in some embodiments of the methods of the present technology, massively parallel DNA sequencing is performed using one or more of pyrosequencing, a reversible dye terminator chemistry, an oligonucleotide ligation chemistry, proton detection, or phospholinked fluorescent nucleotide chemistry. [0014] In one aspect, the present disclosure provides a method for selecting a patient suspected of, or diagnosed as having multiple myeloma for treatment with a therapeutic agent comprising (i) contacting a nucleic acid sample obtained from the patient with any embodiment of the bait library disclosed herein, (ii) detecting at least one mutation in one or more genomic drivers associated with multiple myeloma, wherein the one or more genomic drivers are selected from the group consisting of IDH1, DIS3, TP53, NRAS, KRAS, or BRAF, and (iii) administering a therapeutic agent to the patient. In some embodiments, the one or more genomic drivers comprise mutations that are associated with poor survival outcomes.

In some embodiments of the method, a TP53 mutation is not detected in the sample, and the therapeutic agent is an immunomodulatory drug (IMiD), a proteasome inhibitor (PI), melphalan, vincristine, cyclophosphamide, etoposide, doxorubicin, bendamustine, thalidomide, lenalidomide, pomalidimodide, an histone deacetylase (HD AC) inhibitor, autologous hematopoietic stem cell transplant (auto-HCT), or any combination thereof. In other embodiments of the method, a TP53 mutation is detected in the sample, and the therapeutic agent is nutlin, RITA (Reactivation of p53 and Induction of Tumor Cell

Apoptosis), PRIMA-l (p53 reactivation and induction of massive apoptosis), rocaglate, CMLD010509, a USP7 inhibitor, a Weel inhibitor, a PARP-l inhibitor, or any combination thereof.

[0015] Additionally or alternatively, in some embodiments of the method, a KRAS mutation is detected in the sample, and the therapeutic agent is a Syk inhibitor, a Ron inhibitor, an integrin beta6 inhibitor, a MEK1 inhibitor, a MEK2 inhibitor, a MAPK pathway inhibitor, a CDK4 inhibitor, a CDK6 inhibitor, a miR-29b inhibitor, a NF-kB inhibitor, a TNFAIP3 inhibitor, a KRAS inhibitor, an EGFR inhibitor, an immune checkpoint inhibitor, a Galectin-3 inhibitor, or any combination thereof.

[0016] Additionally or alternatively, in some embodiments of the method, a NRAS mutation is detected in the sample, and the therapeutic agent is a MEK1 inhibitor, a MEK2 inhibitor, a MAPK pathway inhibitor, a CDK4 inhibitor, a CDK6 inhibitor, a

farnesyltransferase inhibitor, a PI3K inhibitor, an AKT inhibitor, a MET inhibitor, a

VEGFR2 inhibitor, a ERK inhibitor, a BRAF inhibitor, a KRAS inhibitor, a heat shock protein-90 (HSP90) inhibitor, an immune checkpoint inhibitor, or any combination thereof.

[0017] Additionally or alternatively, in certain embodiments of the method, a BRAF mutation is detected in the sample, and the therapeutic agent is a BRAF inhibitor, a MEK1 inhibitor, a MEK2 inhibitor, a MAPK pathway inhibitor, a CDK4 inhibitor, a CDK6 inhibitor, a EGFR inhibitor, an ALK inhibitor, a ROS1 inhibitor, an immune checkpoint inhibitor, a programmed cell death protein 1 (PDCD1) inhibitor, or any combination thereof.

[0018] Additionally or alternatively, in certain embodiments of the method, a IDH1 mutation is detected in the sample, and the therapeutic agent is an IDH1 inhibitor, an IDH2 inhibitor, a DNA damaging agent, a DNA repair inhibitor, a poly(ADP-ribose) polymerase (PARP) inhibitor, a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, a BCL-2 inhibitor, a BH3 mimetic, a multikinase inhibitor, an electron transport chain inhibitor, a biguanide, a cytochrome c oxidase inhibitor, an immune checkpoint inhibitor, or any combination thereof.

[0019] Additionally or alternatively, in some embodiments, a DIS3 mutation is detected in the sample, and the therapeutic agent is a DIS3 inhibitor, a DotLl inhibitor, or any combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 shows a schematic representation of the DNA custom capture assay for targeted Next-Generation Sequencing (NGS) for myeloma-related genes. Figure 1 describes the steps of DNA fragmentation, linker ligation, hybridization to capture probes, affinity purification, and sequencing (adapted from NimbleGen Sequence Capture Microarrays and Services Genomic Enrichment for Targeted High-Throughput Sequencing, Roche

NimbleGen, Inc., (2008)).

[0021] Figure 2 shows the immunoglobulin heavy locus (IGH), depicting various exemplary regions where the bait probes of the present technology are configured to bind (adapted from HGNC:5477).

[0022] Figure 3 shows a table of gene targets with corresponding Ensembl IDs (SEQ ID NOs: 1-158), which comprise exons and/or introns to which the bait probes of the present technology are configured to bind.

[0023] Figure 4 shows the concordance of aberrations by assay, for individual patients.

[0024] Figure 5 shows the concordance and discordance for the detection of multiple myeloma genomic aberrations using conventional methods versus the multiple myeloma- specific targeted NGS assay described herein.

[0025] Figure 6A shows the distribution of TP53 mutations among patients with mono- allelic and bi-allelic TP53 inactivation, respectively. [0026] Figure 6B shows the distribution of genomic alterations in all patients with TP53 aberrations (including those involved in bi-allelic inactivation) along with other co-occurring aberrations)

[0027] Figure 7 shows the progression- free survival in myeloma patients with bi-allelic TP53 inactivation, mono-allelic TP53 inactivation, and without TP53 inactivation.

[0028] Figure 8A shows the number of reads sequenced and duplicates per sample.

[0029] Figure 8B shows the proportion of PCR duplicates per sample.

[0030] Figure 9 shows mean target coverage across normal samples and tumors sequenced and across each bait type.

[0031] Figure 10A shows the distribution of coverage across the exons of different genes covered.

[0032] Figure 10B shows the coverage across the different types of targets captured by the the multiple myeloma-specific targeted NGS assay described herein.

[0033] Figure 10C shows the percentage of bases achieving at least 2x, lOx, 30x, 50x, and lOOx coverage.

[0034] Figure 11 shows the non-synonymous mutations identified in 156 patient samples.

[0035] Figure 12 shows a circle plot displaying the t(8;l4) rearrangements identified in the the Multiple Myeloma Research Foundation’s (MMRF) MMRF CoMMpass database.

The IGH locus is shown using a solid bar near top of the figure.

[0036] Figure 13A shows that FISH displays complex signal patterns with IGH/MYC fusions in 99% of cells.

[0037] Figure 13B shows the characteristic chromosome aberrations in one myeloma patient with a gain of lq, 5p, 6p, 8q (including MYC), 7, 9, 11, 15, 18, and deletion of Xp,

4q, 6q,-l3, and 17r (TP53)/l7q, and CN-LOH of 5q, l4q

[0038] Figure 13C shows the terminal gain of 8q with breakpoints at the MYC locus.

[0039] Figure 13D shows the gain of l4q, CN-LOH, terminal deletion including IGH.

[0040] Figure 14 shows the comparison of detection of IGH translocations by the multiple myeloma-specific targeted NGS assay described herein vs. FISH. DETAILED DESCRIPTION

[0041] Genetic assessment of cancer-related genes includes analyzing IGH

translocations, copy number variations, somatic mutations, SNPs/ SNVs, gene deletions, and/or gene amplifications. Currently, in the standard of care setting, conventional chromosome analysis, multiple myeloma targeted fluorescence in situ hybridization (FISH) panels, and single nucleotide polymorphism (SNP) microarrays are used to detect chromosome translocations and gains and losses in multiple myeloma.

[0042] Molecular profiling of tumors is becoming increasingly important in the management and treatment of advanced cancer. The present disclosure provides methods for detecting myeloma in a patient with high specificity and sensitivity. These methods are based on detecting alterations in target nucleic acid sequences corresponding to a specific set of multiple myeloma-related genes using a sensitive and comprehensive nucleic acid capture assay for targeted Next-Generation Sequencing (NGS). The bait libraries of the present technology are useful in methods for detecting at least one multiple myeloma-related mutation in a sample, and eliminate the need for secondary standard of care diagnostic assays such as FISH or SNP.

Definitions

[0043] As used herein, the term“about” in reference to a number is generally taken to include numbers that fall within a range of l%-5% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context.

[0044] As used herein, the terms“amplify” or“amplification” with respect to nucleic acid sequences, refer to methods that increase the representation of a population of nucleic acid sequences in a sample. Nucleic acid amplification methods, such as PCR, isothermal methods, rolling circle methods, etc., are well known to the skilled artisan. Copies of a particular nucleic acid sequence generated in vitro in an amplification reaction are called “amplicons” or“amplification products”.

[0045] As used herein, the term“actionable genetic alterations” refers to mutations that are associated with (1) treatment with an FDA approved drug, (2) a guideline supported drug treatment, (3) a guideline indication of sensitivity or resistance to a particular treatment, (4) ongoing clinical trials, (5) clinical data supporting an indication of resistance or sensitivity to drug treatment, (6) pre-clinical data showing strong evidence of resistance or sensitivity to a targeted treatment, or (6) a prognostic implication that may guide a physician’s treatment decisions.

[0046] As used herein, the term“genomic driver” a mutation that is required for the cancer development. All known genomic drivers can be classified into one of twelve pathways representing three core cellular processes: cell fate, cell survival, and genome maintenance. Cancer evolves from normal cells via successive genomic driver mutations that activate of oncogenes and/or inactivate of tumor suppressor genes. On an average, alterations of three to five genomic drivers is required to generate a cancer. During this process, a number of“passenger” mutations, arise, which are irrelevant to cancer development and accumulate through DNA replication.

[0047] The term“adapter” refers to a short, chemically synthesized, nucleic acid sequence which can be used to ligate to the end of a nucleic acid sequence in order to facilitate attachment to another molecule. The adapter can be single- stranded or double- stranded. An adapter can incorporate a short (typically less than 50 base pairs) sequence useful for PCR amplification or sequencing.

[0048] As used herein, an“alteration” of a gene or gene product ( e.g ., a marker gene or gene product) refers to the presence of a mutation or mutations within the gene or gene product, e.g., a mutation, which affects the quantity or activity of the gene or gene product, as compared to the normal or wild-type gene. The genetic alteration can result in changes in the quantity, structure, and/or activity of the gene or gene product in a cancer tissue or cancer cell, as compared to its quantity, structure, and/or activity, in a normal or healthy tissue or cell (e.g., a control). For example, an alteration which is associated with cancer, or predictive of responsiveness to anti-cancer therapeutics, can have an altered nucleotide sequence (e.g., a mutation), amino acid sequence, chromosomal translocation, intra-chromosomal inversion, copy number, expression level, protein level, protein activity, in a cancer tissue or cancer cell, as compared to a normal, healthy tissue or cell. Exemplary mutations include, but are not limited to, point mutations (e.g., silent, missense, or nonsense), deletions, insertions, inversions, linking mutations, duplications, translocations, inter- and intra-chromosomal rearrangements. Mutations can be present in the coding or non-coding region of the gene. In certain embodiments, the alterations are associated with a phenotype, e.g., a cancerous phenotype (e.g., one or more of cancer risk, cancer progression, cancer treatment or resistance to cancer treatment). In one embodiment, the alteration is associated with one or more of: a genetic risk factor for cancer, a positive treatment response predictor, a negative treatment response predictor, a positive prognostic factor, a negative prognostic factor, or a diagnostic factor.

[0049] “Bait”, as used herein, is a type of hybrid capture reagent that retrieves target nucleic acid sequences for sequencing. A bait can be a nucleic acid molecule, e.g., a DNA or RNA molecule, which can hybridize to (e.g., be complementary to), and thereby allow capture of a target nucleic acid. In one embodiment, a bait is an RNA molecule (e.g., a naturally-occurring or modified RNA molecule); a DNA molecule (e.g., a naturally-occurring or modified DNA molecule), or a combination thereof. In other embodiments, a bait includes a binding entity, e.g., an affinity tag, that allows capture and separation, e.g., by binding to a binding entity, of a hybrid formed by a bait and a nucleic acid hybridized to the bait. In one embodiment, a bait is suitable for solution phase hybridization.

[0050] As used herein,“bait set” refers to one or a plurality of bait molecules.

[0051] The terms“cancer” or“tumor” are used interchangeably and refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells are often in the form of a tumor, but such cells can exist alone within an animal, or can be a non-tumorigenic cancer cell. As used herein, the term“cancer” includes premalignant, as well as malignant cancers.

[0052] The terms“complementary” or“complementarity” as used herein with reference to polynucleotides (i.e., a sequence of nucleotides such as an oligonucleotide or a target nucleic acid) refer to the base-pairing rules. The complement of a nucleic acid sequence as used herein refers to an oligonucleotide which, when aligned with the nucleic acid sequence such that the 5' end of one sequence is paired with the 3’ end of the other, is in“antiparallel association.” For example, the sequence“5'-A-G-T-3”’ is complementary to the sequence “3’-T-C-A-5.” Certain bases not commonly found in naturally-occurring nucleic acids may be included in the nucleic acids described herein. These include, for example, inosine, 7- deazaguanine, Locked Nucleic Acids (LNA), and Peptide Nucleic Acids (PNA).

Complementarity need not be perfect; stable duplexes may contain mismatched base pairs, degenerative, or unmatched bases. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, base composition and sequence of the

oligonucleotide, ionic strength and incidence of mismatched base pairs. A complement sequence can also be an RNA sequence complementary to the DNA sequence or its complement sequence, and can also be a cDNA.

[0053] As used herein, a "control" is an alternative sample used in an experiment for comparison purpose. A control can be "positive" or "negative." A“control nucleic acid sample” or“reference nucleic acid sample” as used herein, refers to nucleic acid molecules from a control or reference sample. In certain embodiments, the reference or control nucleic acid sample is a wild type or a non-mutated DNA or RNA sequence. In certain

embodiments, the reference nucleic acid sample is purified or isolated ( e.g ., it is removed from its natural state). In other embodiments, the reference nucleic acid sample is from a non-tumor sample, e.g., a blood control sample, a normal adjacent tumor (NAT) sample, or any other non-cancerous sample from the same or a different subject.

[0054] “Detecting” as used herein refers to determining the presence of a mutation or alteration in a nucleic acid of interest in a sample. Detection does not require the method to provide 100% sensitivity.

[0055] “Gene” as used herein refers to a DNA sequence that comprises regulatory and coding sequences necessary for the production of an RNA, which may have a non-coding function (e.g., a ribosomal or transfer RNA) or which may include a polypeptide or a polypeptide precursor. The RNA or polypeptide may be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or function is retained. Although a sequence of the nucleic acids may be shown in the form of DNA, a person of ordinary skill in the art recognizes that the corresponding RNA sequence will have a similar sequence with the thymine being replaced by uracil, i.e., "T" is replaced with "U."

[0056] The term“hybridize” as used herein refers to a process where two substantially complementary nucleic acid strands (at least about 65% complementary over a stretch of at least 14 to 25 nucleotides, at least about 75%, or at least about 90% complementary) anneal to each other under appropriately stringent conditions to form a duplex or heteroduplex through formation of hydrogen bonds between complementary base pairs. Hybridizations are typically and preferably conducted with probe-length nucleic acid molecules, preferably 15- 100 nucleotides in length, more preferably 18-50 nucleotides in length. Nucleic acid hybridization techniques are well known in the art. See, e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, N.Y. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is influenced by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, and the thermal melting point (T_m) of the formed hybrid. Those skilled in the art understand how to estimate and adjust the stringency of hybridization conditions such that sequences having at least a desired level of complementarity will stably hybridize, while those having lower complementarity will not. For examples of hybridization conditions and parameters, see, e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition,

Cold Spring Harbor Press, Plainview, N.Y.; Ausubel, F. M. et al. 1994, Current Protocols in Molecular Biology, John Wiley & Sons, Secaucus, N.J. In some embodiments, specific hybridization occurs under stringent hybridization conditions. An oligonucleotide or polynucleotide (e.g., a probe or a primer) that is specific for a target nucleic acid will “hybridize” to the target nucleic acid under suitable conditions.

[0057] As used herein, the terms“individual”,“patient”, or“subject” are used

interchangeably and refer to an individual organism, a vertebrate, a mammal, or a human. In a preferred embodiment, the individual, patient or subject is a human.

[0058] As used herein, the term“library” refers to a collection of nucleic acid sequences, e.g., a collection of nucleic acids derived from whole genomic, subgenomic fragments, cDNA, cDNA fragments, RNA, RNA fragments, or a combination thereof. In one embodiment, a portion or all of the library nucleic acid sequences comprises an adapter sequence. The adapter sequence can be located at one or both ends. The adapter sequence can be useful, e.g., for a sequencing method (e.g., an NGS method), for amplification, for reverse transcription, or for cloning into a vector.

[0059] The library can comprise a collection of nucleic acid sequences, e.g., a target nucleic acid sequence (e.g., a tumor nucleic acid sequence), a reference nucleic acid sequence, or a combination thereof). In some embodiments, the nucleic acid sequences of the library can be derived from a single subject. In other embodiments, a library can comprise nucleic acid sequences from more than one subject (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30 or more subjects). In some embodiments, two or more libraries from different subjects can be combined to form a library having nucleic acid sequences from more than one subject. In one embodiment, the subject is human having, or at risk of having, a cancer or tumor.

[0060] A“library nucleic acid sequence” refers to a nucleic acid molecule, e.g., a DNA, RNA, or a combination thereof, that is a member of a library. Typically, a library nucleic acid sequence is a DNA molecule, e.g., genomic DNA or cDNA. In some embodiments, a library nucleic acid sequence is fragmented, e.g., sheared or enzymatically prepared, genomic DNA. In certain embodiments, the library nucleic acid sequences comprise sequence from a subject and sequence not derived from the subject, e.g., adapter sequence, a primer sequence, or other sequences that allow for identification, e.g.,“barcode” sequences.

[0061] “Massively parallel sequencing,”“Next-generation sequencing,” or“NGS” as used herein, refer to any sequencing method that determines the nucleotide sequence of either individual nucleic acid molecules (e.g., in single molecule sequencing) or clonally expanded proxies for individual nucleic acid molecules in a high throughput parallel fashion (e.g., greater than 10³, 10⁴, l0⁵or more molecules are sequenced simultaneously). In one embodiment, the relative abundance of the nucleic acid species in the library can be estimated by counting the relative number of occurrences of their cognate sequences in the data generated by the sequencing experiment. Next generation sequencing methods are known in the art, and are described, e.g., in Metzker, M. Nature Biotechnology Reviews 11:31-46 (2010).

[0062] As used herein,“oligonucleotide” refers to a molecule that has a sequence of nucleic acid bases on a backbone comprised mainly of identical monomer units at defined intervals. The bases are arranged on the backbone in such a way that they can bind with a nucleic acid having a sequence of bases that are complementary to the bases of the

oligonucleotide. The most common oligonucleotides have a backbone of sugar phosphate units. A distinction may be made between oligodeoxyribonucleotides that do not have a hydroxyl group at the 2' position and oligoribonucleotides that have a hydroxyl group at the 2' position. Oligonucleotides may also include derivatives, in which the hydrogen of the hydroxyl group is replaced with organic groups, e.g., an allyl group. Oligonucleotides of the method which function as primers or probes are generally at least about 10-15 nucleotides long and more preferably at least about 15 to 25 nucleotides long, although shorter or longer oligonucleotides may be used in the method. The exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. The

oligonucleotide may be generated in any manner, including, for example, chemical synthesis, DNA replication, restriction endonuclease digestion of plasmids or phage DNA, reverse transcription, PCR, or a combination thereof. The oligonucleotide may be modified e.g., by addition of a methyl group, a biotin or digoxigenin moiety, a fluorescent tag or by using radioactive nucleotides. [0063] As used herein, the term“primer” refers to an oligonucleotide, which is capable of acting as a point of initiation of nucleic acid sequence synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a target nucleic acid strand is induced, i.e., in the presence of different nucleotide triphosphates and a polymerase in an appropriate buffer (“buffer” includes pH, ionic strength, cofactors etc.) and at a suitable temperature. One or more of the nucleotides of the primer can be modified for instance by addition of a methyl group, a biotin or digoxigenin moiety, a fluorescent tag or by using radioactive nucleotides. A primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being substantially complementary to the strand. The term primer as used herein includes all forms of primers that may be synthesized including peptide nucleic acid primers, locked nucleic acid primers, phosphorothioate modified primers, labeled primers, and the like. The term “forward primer” as used herein means a primer that anneals to the anti-sense strand of dsDNA. A“reverse primer” anneals to the sense-strand of dsDNA.

[0064] As used herein,“primer pair” refers to a forward and reverse primer pair (i.e., a left and right primer pair) that can be used together to amplify a given region of a nucleic acid of interest.

[0065] As used herein, a“sample” refers to a substance that is being assayed for the presence of a mutation in a nucleic acid of interest. Processing methods to release or otherwise make available a nucleic acid for detection are well known in the art and may include steps of nucleic acid manipulation. A biological sample may be a body fluid or a tissue sample. Biological samples may include tissues, cells, protein or membrane extracts of cells, and biological fluids ( e.g ., ascites fluid or cerebrospinal fluid (CSF)) isolated from a subject, as well as tissues, cells and fluids present within a subject. Biological samples of the present technology include, but are not limited to, samples taken from breast tissue, renal tissue, the uterine cervix, the endometrium, the head or neck, the gallbladder, parotid tissue, the prostate, the brain, the pituitary gland, kidney tissue, muscle, the esophagus, the stomach, the small intestine, the colon, the liver, the spleen, the pancreas, thyroid tissue, heart tissue, lung tissue, the bladder, adipose tissue, lymph node tissue, the uterus, ovarian tissue, adrenal tissue, testis tissue, the tonsils, thymus, blood, hair, buccal, skin, serum, plasma, CSF, semen, prostate fluid, seminal fluid, urine, feces, sweat, saliva, sputum, mucus, bone marrow, lymph, and tears. Biological samples can also be obtained from biopsies of internal organs or from cancers. Biological samples can be obtained from subjects for diagnosis or research or can be obtained from non-diseased individuals, as controls or for basic research. Samples may be obtained by standard methods including, e.g., venous puncture and surgical biopsy. In some cases, a biological sample may consist of or comprise blood, plasma, sera, urine, feces, epidermal sample, vaginal sample, skin sample, cheek swab, sperm, amniotic fluid, cultured cells, bone marrow sample, tumor biopsies, aspirate and/or chorionic villi, cultured cells, and the like. In certain embodiments, the biological sample is a blood or bone marrow sample. Fresh, fixed or frozen tissues may also be used. In one embodiment, the sample is preserved as a frozen sample or as formaldehyde- or paraformaldehyde-fixed paraffin-embedded (FFPE) tissue preparation. For example, the sample can be embedded in a matrix, e.g., an FFPE block or a frozen sample. Whole blood samples of about 0.5 to 5 ml collected with EDTA, ACD or heparin as anti-coagulant are suitable.

[0066] The term“sensitivity,” as used herein in reference to the methods of the present technology, is a measure of the ability of a method to detect a preselected sequence variant in a heterogeneous population of sequences. A method has a sensitivity of S % for variants of F % if, given a sample in which the preselected sequence variant is present as at least F % of the sequences in the sample, the method can detect the preselected sequence at a preselected confidence of C %, S % of the time. By way of example, a method has a sensitivity of 90% for variants of 5% if, given a sample in which the preselected variant sequence is present as at least 5% of the sequences in the sample, the method can detect the preselected sequence at a preselected confidence of 99%, 9 out of 10 times (F=5%; C=99%; S=90%).

[0067] The term“specific” as used herein in reference to an oligonucleotide primer means that the nucleotide sequence of the primer has at least 12 bases of sequence identity with a portion of the nucleic acid to be amplified when the oligonucleotide and the nucleic acid are aligned. An oligonucleotide primer that is specific for a nucleic acid is one that, under the stringent hybridization or washing conditions, is capable of hybridizing to the target of interest and not substantially hybridizing to nucleic acids which are not of interest. Higher levels of sequence identity are preferred and include at least 75%, at least 80%, at least 85%, at least 90%, at least 95% and more preferably at least 98% sequence identity.

[0068] “Specificity,” as used herein, is a measure of the ability of a method to distinguish a truly occurring preselected sequence variant from sequencing artifacts or other closely related sequences. It is the ability to avoid false positive detections. False positive detections can arise from errors introduced into the sequence of interest during sample preparation, sequencing error, or inadvertent sequencing of closely related sequences like pseudo-genes or members of a gene family. A method has a specificity of X % if, when applied to a sample set of Ni_otai sequences, in which XT,_UC sequences are truly variant and X_{Not t}ru_e are not truly variant, the method selects at least X % of the not truly variant as not variant. E.g., a method has a specificity of 90% if, when applied to a sample set of 1,000 sequences, in which 500 sequences are truly variant and 500 are not truly variant, the method selects 90% of the 500 not truly variant sequences as not variant. Exemplary specificities include 90, 95, 98, and 99%.

[0069] The term“stringent hybridization conditions” as used herein refers to

hybridization conditions at least as stringent as the following: hybridization in 50% formamide, 5xSSC, 50 mM NathPCE, pH 6.8, 0.5% SDS, 0.1 mg/mL sonicated salmon sperm DNA, and 5x Denhart's solution at 42° C overnight; washing with 2x SSC, 0.1% SDS at 45° C; and washing with 0.2x SSC, 0.1% SDS at 45° C. In another example, stringent hybridization conditions should not allow for hybridization of two nucleic acids which differ over a stretch of 20 contiguous nucleotides by more than two bases.

[0070] As used herein, the terms“target sequence” and“target nucleic acid sequence” refer to a specific nucleic acid sequence to be detected and/or quantified in the sample to be analyzed.

[0071] “Treating” or“treatment” as used herein covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, i.e., arresting its development; (ii) relieving a disease or disorder, i.e., causing regression of the disorder; (iii) slowing progression of the disorder; and/or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder. In some embodiments, treatment means that the symptoms associated with the disease are, e.g., alleviated, reduced, cured, or placed in a state of remission.

NGS Platforms

[0072] In some embodiments, high throughput, massively parallel sequencing employs sequencing-by- synthesis with reversible dye terminators. In other embodiments, sequencing is performed via sequencing-by-ligation. In yet other embodiments, sequencing is single molecule sequencing. Examples of Next Generation Sequencing techniques include, but are not limited to pyrosequencing, Reversible dye-terminator sequencing, SOLiD sequencing,

Ion semiconductor sequencing, Helioscope single molecule sequencing etc. [0073] The Ion Torrent™ (Life Technologies, Carlsbad, CA) amplicon sequencing system employs a flow-based approach that detects pH changes caused by the release of hydrogen ions during incorporation of unmodified nucleotides in DNA replication. For use with this system, a sequencing library is initially produced by generating DNA fragments flanked by sequencing adapters. In some embodiments, these fragments can be clonally amplified on particles by emulsion PCR. The particles with the amplified template are then placed in a silicon semiconductor sequencing chip. During replication, the chip is flooded with one nucleotide after another, and if a nucleotide complements the DNA molecule in a particular microwell of the chip, then it will be incorporated. A proton is naturally released when a nucleotide is incorporated by the polymerase in the DNA molecule, resulting in a detectable local change of pH. The pH of the solution then changes in that well and is detected by the ion sensor. If homopolymer repeats are present in the template sequence, multiple nucleotides will be incorporated in a single cycle. This leads to a corresponding number of released hydrogens and a proportionally higher electronic signal.

[0074] The 454TM GS FLX™ sequencing system (Roche, Germany), employs a light- based detection methodology in a large-scale parallel pyro sequencing system.

Pyro sequencing uses DNA polymerization, adding one nucleotide species at a time and detecting and quantifying the number of nucleotides added to a given location through the light emitted by the release of attached pyrophosphates. For use with the 454™ system, adapter-ligated DNA fragments are fixed to small DNA-capture beads in a water-in-oil emulsion and amplified by PCR (emulsion PCR). Each DNA-bound bead is placed into a well on a picotiter plate and sequencing reagents are delivered across the wells of the plate. The four DNA nucleotides are added sequentially in a fixed order across the picotiter plate device during a sequencing run. During the nucleotide flow, millions of copies of DNA bound to each of the beads are sequenced in parallel. When a nucleotide complementary to the template strand is added to a well, the nucleotide is incorporated onto the existing DNA strand, generating a light signal that is recorded by a CCD camera in the instrument.

[0075] Sequencing technology based on reversible dye-terminators: DNA molecules are first attached to primers on a slide and amplified so that local clonal colonies are formed.

Four types of reversible terminator bases (RT-bases) are added, and non- incorporated nucleotides are washed away. Unlike pyrosequencing, the DNA can only be extended one nucleotide at a time. A camera takes images of the fluorescently labeled nucleotides, then the dye along with the terminal 3' blocker is chemically removed from the DNA, allowing the next cycle.

[0076] Helicos's single-molecule sequencing uses DNA fragments with added polyA tail adapters, which are attached to the flow cell surface. At each cycle, DNA polymerase and a single species of fluorescently labeled nucleotide are added, resulting in template-dependent extension of the surface-immobilized primer-template duplexes. The reads are performed by the Helioscope sequencer. After acquisition of images tiling the full array, chemical cleavage and release of the fluorescent label permits the subsequent cycle of extension and imaging.

[0077] Sequencing by synthesis (SBS), like the "old style" dye-termination

electrophoretic sequencing, relies on incorporation of nucleotides by a DNA polymerase to determine the base sequence. A DNA library with affixed adapters is denatured into single strands and grafted to a flow cell, followed by bridge amplification to form a high-density array of spots onto a glass chip. Reversible terminator methods use reversible versions of dye-terminators, adding one nucleotide at a time, detecting fluorescence at each position by repeated removal of the blocking group to allow polymerization of another nucleotide. The signal of nucleotide incorporation can vary with fluorescently labeled nucleotides, phosphate- driven light reactions and hydrogen ion sensing having all been used. Examples of SBS platforms include Illumina GA and HiSeq 2000. The MiSeq® personal sequencing system (Illumina, Inc.) also employs sequencing by synthesis with reversible terminator chemistry.

[0078] In contrast to the sequencing by synthesis method, the sequencing by ligation method uses a DNA ligase to determine the target sequence. This sequencing method relies on enzymatic ligation of oligonucleotides that are adjacent through local complementarity on a template DNA strand. This technology employs a partition of all possible oligonucleotides of a fixed length, labeled according to the sequenced position. Oligonucleotides are annealed and ligated and the preferential ligation by DNA ligase for matching sequences results in a dinucleotide encoded color space signal at that position (through the release of a fluorescently labeled probe that corresponds to a known nucleotide at a known position along the oligo). This method is primarily used by Life Technologies’ SOLiD™ sequencers. Before sequencing, the DNA is amplified by emulsion PCR. The resulting beads, each containing only copies of the same DNA molecule, are deposited on a solid planar substrate.

[0079] SMRT™ sequencing is based on the sequencing by synthesis approach. The DNA is synthesized in zero-mode wave-guides (ZMWs)-small well-like containers with the capturing tools located at the bottom of the well. The sequencing is performed with use of unmodified polymerase (attached to the ZMW bottom) and fluorescently labeled nucleotides flowing freely in the solution. The wells are constructed in a way that only the fluorescence occurring at the bottom of the well is detected. The fluorescent label is detached from the nucleotide at its incorporation into the DNA strand, leaving an unmodified DNA strand.

Bait Compositions of the Present Technology

[0080] In one aspect, the present disclosure provides a bait library comprising one or more baits comprising a sequence of any of SEQ ID NOs: 159-6088. Exemplary bait sequences of the present disclosure are disclosed below:

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[0081] In some embodiments, the bait sequences are configured to bind the entire IGH locus. Additionally or alternatively, in some embodiments, the bait sequences are configured to bind to (i) chromosome 14 translocation breakpoints found in multiple myeloma, (ii) single nucleotide polymorphisms (1 per 3 Mb) found in multiple myeloma, and/or (iii)

hyperdiploidy and other copy number aberrations (CNAs) found in multiple myeloma. [0082] The bait library may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75, at least 100, at least 125, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 1000, at least 1100, at least 1250, at least 1500, at least 1750, at least 2000, at least 2250, at least 2500, at least 2750, at least 3000, at least 3500, at least 4000, at least 4500, at least 5000, or at least 5500 unique baits.

[0083] In some embodiments, the bait library is configured to bind an IGH locus. In some embodiments, the bait library is configured to bind at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175 or at least 200 chromosome 14 translocation breakpoints present in a multiple myeloma sample.

[0084] Additionally or alternatively, in some embodiments, the bait library is configured to bind single nucleotide variations (SNVs) present in a multiple myeloma sample. In some embodiments, the bait library is configured to bind at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75, at least 100, at least 125, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 1000, at least 1100, at least 1250, at least 1500, at least 1750, at least 2000, at least 2250, at least 2500, at least 2750, at least 3000, at least 3500, at least 4000, at least 4500, at least 5000, or at least 5500 single nucleotide variations (SNVs) present in a multiple myeloma sample.

[0085] Additionally or alternatively, in some embodiments, the bait library is configured to bind copy number aberrations (CNAs) present in a multiple myeloma sample. In some embodiments, the bait library is configured to bind at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175 or at least 200 copy number aberrations (CNAs) present in a multiple myeloma sample.

[0086] Additionally or alternatively, in some embodiments, the bait library is configured to bind insertions/deletions (indels) present in a multiple myeloma sample. In some embodiments, the bait library is configured to bind at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75, at least 100, at least 125, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 1000, at least 1100, at least 1250, at least 1500, at least 1750, at least 2000, at least 2250, at least 2500, at least 2750, at least 3000, at least 3500, at least 4000, at least 4500, at least 5000, or at least 5500 insertions/deletions (indels) present in a multiple myeloma sample.

[0087] In some embodiments, the bait sequences of the present technology are configured to bind to one or more introns of one or more of the following plurality of genes: ANP32E, APC, ATM, ATR, BIRC2, BIRC3, CDKN2A, CDKN2C, CKS1B, CSNK1A1, CYLD, DIS3, FAF1, FAM46C, FAT3, HIST1H1E, IRF4, KDM6A, KRAS, MAP3K14, MAX, MYC, NF1, NFKB1, NFKBIA, PRDM1, PTEN, PTPRD, RBI, R0B02, SETD2, SP140, TNFAIP3, TP53, TRAF2, TRAF3, WWOX, and XBP1. See Figure 3 for corresponding SEQ ID NOs.: 121-158.

[0088] In some embodiments, the bait sequences of the present technology are configured to bind to one or more exons of one or more of the following plurality of genes: ACTG1, ADCY5, AK2, APB B1IP, APC, ARID2, ATM, ATP13A4, ATR, AVEN, BCE2, BIRC2, BIRC3, BRAF, BRCA2, BTG1, CCND1, CD274,CD38, CDC27, CDKN1B, CDKN2A, CDKN2C, CRBN, CSNK1A1, CUL4B, CXCR4, CYLD, DIS3, DEX6, DNAH11, DNAH5, DNAH9, DUSP2, EGR1, EPHA3, FAM46C, FAT1, FAT3, FAT4, FBRS, FBXW7, FCRL5, FGFR3, FEG, FSIP2, GPRC5D, HIST1H1B, HIST1H1C, HIST1H1E, HIST1H3G, IDH1, IDH2, IKBKB, IKZF1 , IKZF3, IRF4, JAM2, KDM1A, KDM6A, KEHE6, KMT2D, KRAS, LTB, MAF, MAP3K1, MAX, MYC, MYD88, NCKAP5, NF1, NFKB2, NFKBIA, NR3C1, NRAS, PABPC1, PCFO, PDCD1, PIK3CA, PIM1, PPM ID, PRDM1, PRDM6, PRKD2, PSMB5, PSMB8, PSMB9, PSMC2, PSMC3, PSMC4, PSMC5, PSMC6, PSMD1, PSMD12, PSMD13, PSMD2, PSMD6, PSMD7, PTEN, PTPN11, PTPRD, RAG2, RASA2, RBI, RHOT1, RIPK1, ROBOl, ROB02, SETD2, SF3B1, SFAMF7, SP140, TNFRSF17, TP53, TRAF2, TRAF3, WHSC1 ( NSD2 ), XBP1, XPOl, and ZFHX4. See Figure 3 for corresponding SEQ ID NOs.: 1-120.

[0089] In some embodiments, the bait sequences of the present technology are configured to bind to one or more mutant exons of genomic drivers identified in multiple myeloma patients. Exemplary genomic drivers include, but are not limited to, ABCF1, ACTG1,

ARID 1 A, ARID2, ATM, ATRX, BRAF, C80RF34, CCND1, CDKN1B, CDKN2C, CREBBP, CYFD, DIS3, DNMT3A, DUSP2, EGR1, EP300, FAM46C, FGFR3, FUBP1, HIST1H1E, HUWE1, IDH1, IDH2, IRF4, KDM5C, KDM6A, KFHF6, KMT2B, KMT2C, KRAS, LTB, MAF, MAFB, MAML2, MAN2C1, MAX, NCOR1, NF1, NFKB2, NFKBIA, NRAS, P1K3CA, PRDM1, PRKD2, PTPN11, RASA2, RBI, RFTN1, SAMHD1, SETD2, SF3B1, SP140, TET2, TGDS, TP53, TRAF2, TRAF3, UBR5, XBP1, ZFP36L1, and ZNF292. In some embodiments, the bait sequences of the present technology are configured to bind to one or more genomic drivers comprising mutations associated with poor survival outcomes. Exemplary genomic drivers comprising mutations associated with poor survival outcomes include, but are not limited to IDH1, DIS3, and TP53.

[0090] In some embodiments, the bait sequences of the present technology comprise a barcode sequence and/or an adapter sequence. DNA barcoding is a method that uses a short nucleic acid tag to identify the source of a nucleic acid sample. In combination with next generation sequencing methods, it may be possible to determine the identity of a sample with regard to its origin through detection of DNA sequence that is specific to a subject.

Additionally, it is possible that a unique, artificial DNA sequence not derived directly from a subject could be conjugated to sample derived from a subject prior to combination with other tumor samples with differing origins, and that this unique artificial DNA barcode could be read later by DNA sequence analysis in order to identify the origin of a sample under investigation. Additionally or alternatively, in some embodiments, the bait sequences of the present technology comprise an affinity tag ( e.g ., biotin).

[0091] In some embodiments, the bait sequences of the present technology comprise one or more detectable labels selected from fluorescent molecules or fluorochromes (such as sold by Invitrogen, e.g., see, The Handbook— A Guide to Fluorescent Bait sequences and Labeling Technologies, Invitrogen Detection Technologies, Molecular Bait sequences, Eugene, Oreg, or disclosed in U.S. Pat. No. 5,866,366 to Nazarenko et al, such as 4-acetamido-4'- isothiocyanatostilbene-2,2'disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2'-aminoethyl)aminonaphthalene-l- sulfonic acid (EDANS), 4- amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4- anilino-l-naphthyl)maleimide, anthranilamide, Brilliant Yellow, coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4- trifluoromethylcouluarin (Coumaran 151); cyanosine; 4',6-diaminidino-2- phenylindole (DAPI); 5',5"-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7- diethylamino- 3-(4'-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'- diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'- disulfonic acid; 5-[dimethylamino]naphthalene-l-sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl- 4'- isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2- yl)aminofluorescein (DTAF), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), and QFITC (XRITC); 2', 7'- difluoro fluorescein (OREGON GREEN®); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4- methylumbelliferone; ortho cresolphthalein; nitro tyro sine; pararosaniline; Phenol Red; B- phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1 -pyrene butyrate; Reactive Red 4 (Cibacron.RTM.

Brilliant Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6- carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, rhodamine green, sulforhodamine B, sulforhodamine 101 and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); N,N,N',N'-tetramethyl-6- carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives, thiol-reactive europium chelates which emit at approximately 617 nm (Heyduk and Heyduk, Analyt. Biochem. 248:216- 27, 1997; J. Biol. Chem. 274:3315-22, 1999), as well as GFP, LissamineTM, diethylaminocoumarin, fluorescein chlorotriazinyl, naphthofluorescein, 4,7- dichlororhodamine and xanthene (as described in U.S. Pat. No. 5,800,996 to Lee el al.) and derivatives thereof. Other fluorophores known to those skilled in the art can also be used, for example those available from Invitrogen Detection Technologies, Molecular Bait sequences (Eugene, Oreg.) and including the ALEXA FLUOR™ series of dyes (for example, as described in U.S. Pat. Nos. 5,696,157, 6,130,101 and 6, 716,979), the BODIPY series of dyes (dipyrrometheneboron difluoride dyes, for example as described in U.S. Pat. Nos. 4,774,339, 5,187,288, 5,248,782, 5,274,113, 5,338,854, 5,451,663 and 5,433,896), Cascade Blue (an amine reactive derivative of the sulfonated pyrene described in U.S. Pat. No. 5,132,432) and Marina Blue (U.S. Pat. No. 5,830,912), a fluorescent nanoparticle, such as a semiconductor nanocrystal, e.g., a QUANTUM DOTTM (obtained, for example, from QuantumDot Corp, Invitrogen Nanocrystal Technologies, Eugene, Oreg.; see also, U.S. Pat. Nos. 6,815,064, 6,682,596 and 6,649,138). The semiconductor nanocrystals described in e.g., U.S. Pat. No. 6,602,671, Bruchez et. al. (1998) Science 281:2013- 6, Chan et al. (1998) Science 281:2016- 8, and U.S. Pat. No. 6,274,323, U.S. Pat. Nos. 6,927,069; 6,914,256; 6,855,202; 6,709,929; 6,689,338; 6,500,622; 6,306,736; 6,225,198; 6,207,392; 6,114,038; 6,048,616; 5,990,479; 5,690,807; 5,571,018; 5,505,928; 5,262,357 and in U.S. Patent Publication No.

2003/0165951 as well as PCT Publication No. 99/26299 (published May 27, 1999), radioisotopes (such as 3H), metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+, and liposomes, enzymes, for example horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, b- galactosidase, b - glucuronidase or b lactamase, enzyme in combination with a chromogen, fluorogenic or luminogenic compound that generates a detectable signal, for example, those sold by

Invitrogen Corporation, Eugene Oreg.). Examples of suitable chromogenic compounds include diaminobenzidine (DAB), 4-nitrophenylpho spate (pNPP), fast red,

bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), BCIP/NBT, fast red,

AP Orange, AP blue, tetramethylbenzidine (TMB), 2,2'-azino-di-[3-ethylbenzothiazoline sulphonate](ABTS), o- dianisidine, 4-chloronaphthol (4-CN), nitrophenyl-.beta.-D- galactopyranoside (ONPG), o- phenylenediamine (OPD), 5-bromo-4-chloro-3-indolyl-.beta.- galactopyranoside (X-Gal), methylumbelliferyl-.beta.-D-galactopyranoside (MU-Gal), p- nitrophenyl-. alpha. -D- galactopyranoside (PNP), 5-bromo-4-chloro-3-indolyl-.beta.-D- glucuronide (X-Gluc), 3-amino- 9-ethyl carbazol (AEC), fuchsin, iodonitrotetrazolium (INT), tetrazolium blue and tetrazolium violet, among others.

Methods for Detecting Myeloma

[0092] Disclosed herein are methods that are based, at least in part, on multiple myeloma- specific targeted NGS assays. The baits used in the assays disclosed herein, are designed to cover the entire IgH locus. The advantages of the assays disclosed herein include efficient detection of genome-wide single-nucleotide polymorphisms for hyperdiploidy, and other copy number alterations (CNAs), IGH translocations, gene point mutations (SNVs or SNPs) and/or small insertions/deletions (indels). The assays disclosed herein are less labor intensive, and can be performed with low concentration nucleic acid samples.

[0093] In the current standard of care setting, conventional chromosome analysis, multiple myeloma targeted fluorescence in situ hybridization (FISH) panels, and single nucleotide polymorphism (SNP) microarrays are used to detect chromosome translocations and gains and losses in multiple myeloma. Conventional chromosome analysis is labor intensive, has low genomic resolution, and due to low mitotic activity and low percentage of plasma cells in bone marrow is often inadequate despite stimulating culture conditions.

Multiple myeloma targeted FISH panels, by default, are inherently limited to the targets of the selected probes. SNP microarrays frequently fail in multiple myeloma patients with less than 20% plasma cell infiltration of the bone marrow. Furthermore, none of the routine chromosomal platforms can detect point mutations or small insertions/deletions (indels). Importantly, these technologies require relatively large amounts of bone marrow sample material which, in turn, limits laboratories in their ability to perform additional parallel comprehensive genomic analysis (such as, somatic mutational characterization and V(D)J profiling for minimal residual disease characterization/tracking). The assays disclosed herein are less labor intensive, can be performed with limited quantities of nucleic acids, and can efficiently detect gene point mutations or small insertions/deletions (indels).

[0094] Capture based next generation sequencing (NGS) panels, such as FoundationOne CDx have proven their utility in identifying genomic alterations. However, none of the currently available capture based NGS panels reliably identify CNAs (e.g. gains and/or losses of chromosomes or large sections thereof) and none of them have been designed to capture structural rearrangements (e.g. IGH translocations) which are frequent in multiple myeloma. Therefore, the use of these available capture based NGS panels cannot replace the current standard of care assays.

[0095] Provided herein is a multiple myeloma-specific hybridization based custom sequencing panel designed to identify all rearrangements involving the IGH locus, arm level and focal copy number aberrations, as well as frequently mutated genes in the disease. The targeted NGS assays disclosed herein capture mutational status in multiple myeloma in a single assay, and therefore, can replace current standard of care assays. The data using 156 primary multiple myeloma bone marrow samples disclosed herein show that the assays disclosed herein are superior to the standard of care FISH and SNP microarrays with respect to the sensitivity, specificity, and reproducibility of detection of CNAs and IGH

translocations. The results disclosed herein reveal an extremely high concordance (sensitivity >98% and specificity >99%) between the methods disclosed herein and conventional FISH/SNP microarrays in detection of IGH translocations and CNAs.

[0096] In one aspect, the present disclosure provides a method for detecting at least one myeloma-related mutation in a patient in need thereof, comprising (i) contacting a nucleic acid sample obtained from the patient with any embodiment of the bait library disclosed herein, and (ii) detecting at least one myeloma-related mutation in the sample via massively parallel DNA sequencing. The nucleic acid sample may comprise DNA, RNA or any combination thereof. In certain embodiments, the nucleic acid sample is contacted with the bait library via solution phase hybridization. In some embodiments, the patient is suspected of, or diagnosed as having multiple myeloma. Additionally or alternatively, in some embodiments, the nucleic acid sample is derived from a biopsy sample obtained from the patient. In certain embodiments, the nucleic acid sample is derived from a blood or bone marrow sample.

[0097] In some embodiments of the methods disclosed herein, CDl38⁺ cells are isolated from bone marrow aspirates collected from patients. The samples may be collected through a bone marrow aspiration and then purified through magnetic bead sorting. Alternatively, CDl38⁺ cells may be enriched via flow cytometry. DNA from the enriched CDl38⁺ cells may be extracted using Qiagen DNA or DNA/RNA (Germantown, MD) extraction kits.

After extraction, gel electrophoresis and Qubit Picogreen Assay (Salt Lake City, UT) measurements may be carried out to assess the quality and quantity of the DNA. In some embodiments, about 250 ng of DNA per sample is used for sequencing.

[0098] After library preparation, all samples with sufficient DNA quality and quantity are sequenced with Next-Generation Sequencing (Illumina (San Diego, CA) HiSeq) using the bait probes of the present technology. See Figure 1.

[0099] In some embodiments, the bait probes of the present technology are configured to bind to one or more regions of the IGH locus (as depicted in Figure 2) that comprise a chromosomal alteration selected from the group consisting of t(l 1: 14), t( 14: 16), t(l4:20), t(4: 14), t(6: 14), t(8: 14), and 1.14.

[00100] Additionally or alternatively, in some embodiments, the bait probes of the present technology are configured to bind to one or more genomic regions that comprise a chromosomal alteration selected from the group consisting of dellp, amplq, dell2pl3.3l, dell3, dell6q, and dell7pl3.

[00101] Additionally or alternatively, in some embodiments, the bait probes of the present technology are configured to detect hyperdiploidy ( e.g ., gain of 3, 5, 7, 9, 11, 15, 19 and 21) and copy number single nucleotide polymorphisms (SNPs) evenly spaced across the genome ( i.e ., over a distance of 3 Mbp).

[00102] Additionally or alternatively, in some embodiments, the bait probes of the present technology are configured to bind to one or more introns of one or more of the following plurality of genes: ANP32E, APC, ATM, ATR, BIRC2, BIRC3, CDKN2A, CDKN2C, CKS1B, CSNK1A1, CYLD, DIS3, FAF1, FAM46C, FAT3, HIST1H1E, IRF4, KDM6A, KRAS, MAP3K14, MAX, MYC, NF1, NFKB1, NFKBIA, PRDM1, PTEN, PTPRD, RBI, ROB02, SETD2, SP140, TNFAIP3, TP53, TRAF2, TRAF3, WWOX, and XBP1. See Figure 3 for corresponding SEQ ID NOs.: 121-158.

[00103] Additionally or alternatively, in some embodiments, the bait probes of the present technology are configured to bind to one or more exons of one or more of the following plurality of genes: ACTG1, ADCY5, AK2, APBB1IP, APC, ARID2, ATM, ATP13A4, ATR, AVEN, BCF2, BIRC2, BIRC3, BRAF, BRCA2, BTG1, CCND1, CD274,CD38, CDC27, CDKN1B, CDKN2A, CDKN2C, CRBN, CSNK1A1, CUF4B, CXCR4, CYFD, DIS3, DFX6, DNAH11, DNAH5, DNAH9, DUSP2, EGR1, EPHA3, FAM46C, FAT1, FAT3, FAT4, FBRS, FBXW7, FCRF5, FGFR3, FFG, FSIP2, GPRC5D, HIST1H1B, HIST1H1C, HIST1H1E, HIST1H3G, IDH1, IDH2, IKBKB, IKZF1, IKZF3, IRF4, JAM2, KDM1A, KDM6A, KFHF6, KMT2D, KRAS, FTB, MAF, MAP3K1, MAX, MYC, MYD88, NCKAP5, NF1, NFKB2, NFKBIA, NR3C1, NRAS, PABPC1, PCFO, PDCD1, PIK3CA, PIM1, PPM1D, PRDM1, PRDM6, PRKD2, PSMB5, PSMB8, PSMB9, PSMC2, PSMC3, PSMC4, PSMC5, PSMC6, PSMD1, PSMD12, PSMD13, PSMD2, PSMD6, PSMD7, PTEN, PTPN11, PTPRD, RAG2, RASA2, RBI, RHOT1, RIPK1, ROBOl, R0B02, SETD2, SF3B1, SFAMF7, SP140,

TNFRSF17, TP53, TRAF2, TRAF3, WHSC1 ( NSD2 ), XBP1, XPOl, and ZFHX4. See Figure 3 for corresponding SEQ ID NOs.: 1-120.

[00104] Additionally or alternatively, in some embodiments, the bait probes of the present technology are configured to bind genomic regions that contain one or more of the following SNPs: rsl05250l, rsl0936599, rsl2374648, rsl26l4346, rs2237892, rs2285803, rs2383208, rs2839629, rs35767, rs4273077, rs4487645, rs562l9066, rs603965, rs6746082, rs72773978, rs7944584, and rs877529.

[00105] Additionally or alternatively, in some embodiments, the bait probes of the present technology are capable of detecting one or more of the following genetic alterations: IGH translocations, copy number variations, somatic mutations, SNPs, gene deletions, and/or gene amplifications.

[00106] Additionally or alternatively, in some embodiments, the bait probes of the present technology can be used to detect the presence of mutations or chromosomal alterations in a disease selected from the group consisting of monoclonal gammopathy of undermined significance, smoldering multiple myeloma, multiple myeloma (newly diagnosed as well as relapse patients), AL amyloidosis, multiple myeloma, plasmacytoma, localized myeloma and extramedullary myeloma. [00107] Samples may be read at a depth of 600x and mutations may be analyzed and called either with or without access to matched normal samples. In some embodiments, the tumor samples may be compared to matched normal samples. In other embodiments, the tumor samples may be compared to unmatched normal samples that have been run using the sequencing panel described herein. The genomic data may be analyzed using one or more of the following bioinformatic pipelines: BWA-mem, cnv-kit, Pindel, FACETS, and Caveman. These include tools for alignment, copy number variants, indels, breakpoints, and point mutations. Results can then be annotated using single nucleotide variant and somatic mutation databases such as ExAC, lOOOg, and COSMIC as well as the mutations from the published myeloma sequencing studies. Post-processing and variant annotation may be performed as described in Papaemmanuil el al, NEJM (2016) but for myeloma specific variants.

[00108] The genetic events may then be manually evaluated for final annotation. Findings can then be validated and compared to existing datasets of mutations in myeloma as well as COSMIC and other databases of somatic mutations. The genomic data can then be correlated to patient characteristics and outcome information. Biostatistical analyses including regression models and more may be performed. Results obtained using methods of the present technology may be compared to FISH results obtained for the same samples.

[00109] The methods of the present technology may include the use of one or more control samples that lack multiple myeloma-related mutations. In some embodiments, the one or more control samples are unmatched ( e.g ., are not derived from the patient samples). In some embodiments, control samples are combined into a pooled reference for CNA inference.

[00110] In some embodiments, the at least one bait sequence captures a part or the entire IGH locus. In some embodiments, the method detects insertions/deletions (indels) in multiple myeloma and other hematological malignancies (oncogenes, tumor suppressor genes, and members of pathways deemed actionable by targeted therapies). In some embodiments, the method detects the chromosome 14 translocation breakpoints, as well as genome wide single nucleotide polymorphisms (1 per 3 Mb) to assess hyperdip loidy and other copy number aberrations (CNAs).

[00111] In some embodiments, the at least one multiple myeloma-related mutation is identified using one or more computer algorithms. In some embodiments, the one or more computer algorithms are selected from the group consisting of CNVkit, Brass, Delly, CaVEMan, Strelka2, Mutect2, and Velvet de novo assembler. In some embodiments, the at least one multiple myeloma-related mutation is a copy number aberrations (CNA), a single nucleotide variant (SNV) or an insertion/deletion (indel). In some embodiments, the CNVKit algorithm is used to identify a CNA. Additionally or alternatively, in some embodiments, CaVEMan, Strelka2 or Mutect2 algorithms are used to identify a SNV. In some

embodiments, Pindel, Strelka2 or Mutect2 algorithms are used to identify an indel. In some embodiments, nucleotide sequences are filtered for known variation and artifacts using the methods disclosed herein.

[00112] In some embodiments, the methods featured in the present technology are used in a multiplex, multi-gene assay format, e.g., assays that incorporate multiple signals from a large number of diverse genetic alterations in a large number of genes.

Methods of Treatment Selection

[00113] The assays disclosed herein capture mutations relevant to clinical outcomes in multiple myeloma. As shown in Figures 11 and 13, among patients with 17r deletions captured by SNP microarrays, through its capture of mutations, identified high-risk multiple myeloma patients carrying both 17r deletions and TP53 mutations (i.e. double-hit bi-allelic TP 53 inactivation subtype). These results demonstrate a strong evidence of direct clinical utility for the assay in multiple myeloma patients.

[00114] Accordingly, in one aspect, the present disclosure provides a method for selecting a patient suspected of, or diagnosed as having multiple myeloma for treatment with a therapeutic agent comprising (i) contacting a nucleic acid sample obtained from the patient with any embodiment of the bait library disclosed herein, (ii) detecting at least one mutation in one or more genomic drivers associated with multiple myeloma, and (iii) administering a therapeutic agent to the patient. In some embodiments, the one or more genomic drivers are selected from the group consisting of ABCF1, ACTG1, ARID1A, ARID2, ATM, ATRX, BRAF, C80RF34, CCND1, CDKN1B, CDKN2C, CREBBP, CYLD, DIS3, DNMT3A, DUSP2, EGR1, EP300, FAM46C, FGFR3, FUBP1, HIST 1 HIE, HUWE1, IDH1, IDH2, IRF4, KDM5C, KDM6A, KLHL6, KMT2B, KMT2C, KRAS, LTB, MAF, MAFB, MAML2, MAN2C1, MAX, NCOR1, NF1, NFKB2, NFKBIA, NRAS, PIK3CA, PRDM1, PRKD2, PTPN11, RASA2, RBI, RFTN1, SAMHD1, SETD2, SF3B1, SP140, TET2, TGDS, TP53, TRAF2, TRAF3, UBR5, XBP1, ZFP36L1, and ZNF292. In some embodiments, the at least one mutation in the one or more genomic drivers are associated with poor survival outcomes. Examples of such mutations include, but are not limited to mutations in IDH1, DIS3, TP53, NRAS, KRAS, and/or BRAF.

[00115] In some embodiments, a TP53 mutation is not detected, and the therapeutic agent is an immunomodulatory drug (IMiD), a proteasome inhibitor (PI), melphalan, vincristine, cyclophosphamide, etoposide, doxorubicin, bendamustine, thalidomide, lenalidomide, pomalidimodide, an histone deacetylase (HD AC) inhibitor, an autologous hematopoietic stem cell transplant (auto-HCT) or any combination thereof. In other embodiments of the method, a TP 53 mutation is detected in the sample, and the therapeutic agent is nutlin, RITA

(Reactivation of p53 and Induction of Tumor Cell Apoptosis), PRIMA-l (p53 reactivation and induction of massive apoptosis), rocaglate, CMLD010509, a USP7 inhibitor, a Weel inhibitor, a PARP-l inhibitor, or any combination thereof.

[00116] Additionally or alternatively, in some embodiments of the method, a KRAS mutation is detected in the sample, and the therapeutic agent is a Syk inhibitor, a Ron inhibitor, an integrin beta6 inhibitor, a MEK1 inhibitor, a MEK2 inhibitor, a MAPK pathway inhibitor, a CDK4 inhibitor, a CDK6 inhibitor, a miR-29b inhibitor, a NF-kB inhibitor, a TNFAIP3 inhibitor, a KRAS inhibitor, an EGFR inhibitor, an immune checkpoint inhibitor, a Galectin-3 inhibitor, or any combination thereof.

[00117] Additionally or alternatively, in some embodiments of the method, a NRAS mutation is detected in the sample, and the therapeutic agent is a MEK1 inhibitor, a MEK2 inhibitor, a MAPK pathway inhibitor, a CDK4 inhibitor, a CDK6 inhibitor, a

farnesyltransferase inhibitor, a PI3K inhibitor, an AKT inhibitor, a MET inhibitor, a

VEGFR2 inhibitor, a ERK inhibitor, a BRAF inhibitor, a KRAS inhibitor, a heat shock protein-90 (HSP90) inhibitor, an immune checkpoint inhibitor, or any combination thereof.

[00118] Additionally or alternatively, in certain embodiments of the method, a BRAF mutation is detected in the sample, and the therapeutic agent is a BRAF inhibitor, a MEK1 inhibitor, a MEK2 inhibitor, a MAPK pathway inhibitor, a CDK4 inhibitor, a CDK6 inhibitor, a EGFR inhibitor, an ALK inhibitor, a ROS1 inhibitor, an immune checkpoint inhibitor, a programmed cell death protein 1 (PDCD1) inhibitor, or any combination thereof.

[00119] Additionally or alternatively, in certain embodiments of the method, a IDH1 mutation is detected in the sample, and the therapeutic agent is an IDH1 inhibitor, an IDH2 inhibitor, a DNA damaging agent, a DNA repair inhibitor, a poly(ADP-ribose) polymerase (PARP) inhibitor, a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, a BCL-2 inhibitor, a BH3 mimetic, a multikinase inhibitor, an electron transport chain inhibitor, a biguanide, a cytochrome c oxidase inhibitor, an immune checkpoint inhibitor, or any combination thereof.

[00120] Additionally or alternatively, in some embodiments, a DIS3 mutation is detected in the sample, and the therapeutic agent is a DIS3 inhibitor, a DotLl inhibitor, or any combination thereof.

[00121] In any of the embodiments disclosed herein, the therapeutic agent is compatible with the detected mutation.

EXAMPLES

[00122] The present technology is further illustrated by the following Examples, which should not be construed as limiting in any way.

Example 1: Detection ofIGH Translocations and Copy Number Alterations in Multiple Myeloma Samples Using the Methods of the Present Technology

[00123] To interrogate genomic aberrations in primary samples from patients with multiple myeloma, a targeted next generation sequencing (NGS) panel was developed. The mutation detection rates obtained using the NGS panel were compared to those obtained via FISH.

[00124] Methods. In the NGS assay of the present technology, baits were designed to capture (a) the entire IGH locus where the vast majority of the chromosome 14 breakpoints occur, (b) genome wide single nucleotide polymorphisms (SNPs) for hyperdiploidy and other copy number alterations (CNAs), as well as exons of 120 frequently mutated genes in multiple myeloma.

[00125] To validate the capture of IGH translocations and CNAs using the methods of the present technology, 46 samples from 22 patients with multiple myeloma as well as bone marrow samples from 16 healthy individuals were analyzed. All samples were sequenced using 126 bp paired end reads using Illumina HiSeq with a mean target depth of ~600x. All patient samples contained a high percentage of plasma cells. After sequencing, CNAs and translocations were identified using validated bioinformatic algorithms such as CNVkit,

Brass and Delly. [00126] Table 1 shows a summary of the IGH translocations and CNAs detected using the NGS methods of the present technology and conventional FISH.

[00127] Results. The NGS methods of the present technology were either comparable or in many instances more effective in detecting IGH translocations and CNAs compared to FISH. A higher number of t(4; 14) and t( 11 ; 14) and equally many t(l4;l6) IGH

translocations were detected using the NGS panel compared to FISH (Table 1).

Hyperdiploidy was detected in 14% and 23% with FISH and the NGS panel, respectively. A greater number of lq gains and l3/l3q deletions were detected using the NGS panel while the number of 17r deletions detected were similar to those detected by FISH. The NGS method of the present technology was able to detect additional CNAs such as 6q deletion, 8p deletion, l6q gain, and trisomy 8 that were not detected by FISH.

[00128] In 4 multiple myeloma patients enrolled on a study protocol allowing for several parallel bone marrow/extramedullary disease biopsies in the same patient, the same IGH translocations and CNAs were detected across all sites of extramedullary disease except in two samples where a 6q deletion and 8p deletion were not detected in one sample each. This inconsistency is likely attributable to clonal heterogeneity, i.e., the CNA was not present in the individual sample.

[00129] These results demonstrate that the sensitivity for detecting IGH translocations and CNAs in primary samples from patients with plasma cell myeloma were higher using the NGS methods disclosed herein as compared to FISH. Furthermore, additional CNAs were detected using the NGS methods of the present technology that were not detected by FISH.

[00130] Accordingly, the methods of the present technology are useful for detecting IGH translocations and chromosomal gains/losses in patients suffering from multiple myeloma. Example 2: Materials and Methods

[00131] Custom capture next generation sequencing panel. The test is based on a multiplex custom capture NGS assay designed to capture the most common and relevant genomic aberrations in multiple myeloma. To capture IGH (l4q32) rearrangements, includes the canonical IGH locus. CNAs are assessed through genome wide representation of SNPs, one in every 3Mb, to enable detection of arm level copy number events. These SNPs are tiled at a higher density to capture focal events that are recurrent in multiple myeloma.

Additionally, includes 120 genes that were selected on the basis of (a) genes frequently mutated in multiple myeloma from earlier reports, (b) genes that are involved in important signaling pathways in multiple myeloma ( e.g ., the NFKB pathway), (c) treatment targets and candidate genes for drug resistance in multiple myeloma (e.g., CRBN, IKZF1, and IKZF3), and (d) exons in candidate genes where SNPs were associated with an increased

susceptibility of developing multiple myeloma. The target space including all these regions came to a total of 2.06 Mb. The final bait design was created using Nimblegen SeqCap online tool using permissive setting.

[00132] Patient cohort : Details of the patient cort used in this study are shown in the Table below. All subjects were diagnosed as having Specimen Source multiple myeloma (MM). Bone marrow samples of the patients were used for subsequent studies. 159 primary multiple myeloma bone marrow aspirate samples obtained at diagnosis or relapse were included.

Three samples, which failed sample quality control (QC) or had low purity, were excluded from the study leaving 156 patients in our study cohort. All specimens, which were fresh bone marrow aspirates, were assessed for standard of care purposes by FISH and SNP microarrays. Plasma cells were enriched by CD138 positive selection through magnetic bead sorting in all samples and were divided for FISH and SNP microarray testing. DNA was extracted using commercial Quiagen DNA extraction kits and used for SNP microarray and testing. In addition, 16 unmatched normal bone marrow samples from healthy donors were used as control to filter sequencing and chemistry specific artifacts and germline variation.

[00133] Data generation and processing: After DNA extraction, barcoded sequence libraries (New England Biolabs, Kapa Biosystems, Wilmington, MA, USA) were subjected to exon capture by hybridization (Nimblegen SeqCap, Madison, WI, USA). 100 to 200 ng of genomic DNA (gDNA) was used as input for library construction. DNA was subsequently sequenced on an Illumina HiSeq 4000 to generate paired-end lOl-bp reads. [00134] Structural variations were called using the breakpoint via assembly (BRASS) system, an in-house algorithm, which first groups discordant read pairs that span the same breakpoint, and then performs local assembly within the vicinity to reconstruct and determine the exact position of the breakpoint to nucleotide precision using the Velvet de novo assembler. Zehir el al., Nat Med. 23(6):703-l3 (2017). Additionally, an orthogonal pipeline using Delly was used to identify structural rearrangements. Rausch et al., Bioinformatics 28(l8):i333-i9 (2012). The resulting calls retained after the described filters were manually curated. The CNVKit algorithm was used to identify somatic CNAs. The 16 unmatched control samples were combined into a pooled reference for CNA inference.

[00135] Single nucleotide variants (SNVs) were called using the CaVEMan, Strelka2 and Mutect2 algorithms, and indels were called using the Pindel, Strelka2 and Mutect2 algorithms. Jones et al., Current protocols in bioinformatics 56(1): 15.10.1-15.10.18 (2016); Kim et al., Nat Methods l5(8):59l-594 (2018); Cibulskis et al., Nature biotechnology 3l(3):2l3 (2013); and Raine et al., Current protocols in bioinformatics 52(1): 15.7.1-7.2 (2015). All SNVs and indels that passed by at least one caller were included for downstream analysis and variant annotation was done based on Ensembl v74 using VAGrENT. Menzies et al., Current protocols in bioinformatics 52(1): 15.8.1-11 (2015). Subsequently, filtering for known variation and artifacts were applied and all calls retained were manually curated.

[00136] FISH and SNP microarray: FISH panels for multiple myeloma were used for t(4; 14), t(6; 14), t(8; 14), t( 11 ; 14), t(l4;l6), t(l4;20), and also for del(lp), gain of lq, del(l3q) and del(l7p) (from Abbott Molecular, Des Plaines, IL, and Metasystems, Newton, MA). All FISH testing performed followed standard of care protocol; a total of 100 cells, if available, were analyzed. The cut-off values for a positive result was >10% for IGH translocations.

[00137] SNP microarrays with 2.67 million probes including 750 thousand common and rare SNP probes (Cytoscan, Affymetrix, Santa Clara, CA) were performed following manufacturers protocol and data analysis was performed using the Affymetrix ChAS 3 software. All samples were manually reviewed for any genomic imbalances, assisted with the cancer gene lists from Cancer Genomics Consortium, and from the MSKCC IMPACT™ heme gene list. Copy neutral loss of heterozygosity (CN-LOH) was reported if the size is at least 10 Mb at a terminal region or 20 Mb for an interstitial CN-LOH. For CNAs comparison in this study, only commonly implicated multiple myeloma aberrations, namely, lp, lq, 3p, 3q, 6q, 8p, 9q, l lq, 12r, l3q, l4q, l6q, 17r were included. [00138] Statistical methods: For comparisons of conventional FISH/SNP microarrays versus descriptive statistics were used. All statistical analysis was performed using R 3.4.3. Kaplan-Meier curves were used to assess survival for patients with no del 17p/77^J53, mono- allelic TP53 inactivation versus bi- allelic TP53 inactivation (Walker et al, Leukemia 33(1): 159-70 (2019)). See Figures 6-7.

[00139] FISH and SNP-array analysis: The FISH analysis targeted high risk genetic markers, namely; loss of lp32.3 (CDKN2C), gain of 1 q21.3 (CKS1B) (Metasystem, Newton, MA), t(4 ; 14)/IGH-FGFR3 fusion, Del(l3q) (D13S319 and LAMP1), IGH break-apart and del(l7p)/TP53 (Abbott Molecular, Des Plaines, IL). If t(4; 14) was negative and FISH with IGH break-apart showed an IGH translocation, reflex FISH tests was performed for t( 11 ; 14)/IGH-CCND 1 fusion, t(14;16)/IGH-MAF fusion, t(14;20)/IGH-MAFB fusion, and more recently for t(6; 14) and t(8;14)/IGH-MYC fusion. All FISH tests followed a standard protocol, and about 100 cells were analyzed, if available. The cut-off value for a positive result was greater than 10% for IGH translocations.

[00140] Genomic microarray tests used SNP array (Affymetrix Cytoscan) with 2.67 million probes including 750 thousand common and rare SNP probes. 200ng gDNA was used for the analysis, following the manufacturers protocol. The data analysis was performed using ChAS 3 software and Nexus copy number software (Biodiscovery, El Segundo, CA). All samples were manually reviewed for any genomic imbalances, assisted with the cancer gene lists from Cancer Gene Census, and from MSKCC IMPACT-HEME gene list. Futreal et al., Nature reviews cancer 4(3): 177 (2004); and He et al., Blood 127(24):3004-3014 (2016). Copy Neutral Loss of Heterozygosity (CN-LOH) was reported if the size is at least 10Mb at a terminal region or 20Mb for an interstitial one.

[00141] DNA extraction, amplification and target enrichment: Solution phase

hybridization-based exon capture and massively parallel DNA sequencing was used to capture all of the recurrent genomic aberrations described herein. Barcoded sequence libraries (New England Biolabs, Kapa Biosystems, Wilmington, MA, USA) were subjected to exon capture by hybridization (Nimblegen SeqCap, Madison, WI, USA). 100 to 200 ng of gDNA was used as input for library construction. Libraries were pooled at equimolar concentrations (100 ng per library) and input to a single exon-capture reaction as previously described. Cheng et al., J Mol Diagn 17(3):251-264 (2015). To prevent off-target hybridization, a pool of blocker oligonucleotides complementary to the full sequences of all barcoded adaptors was spiked in to a final total concentration of 10 pmol/L. DNA was subsequently sequenced on an Illumina HiSeq 4000 to generate paired-end 101 or 126 bp reads.

[00142] Alignment and somatic mutation calling: Sequence data were de-multiplexed using bcl2fastq and sample-wise paired-end fastqs were generated. Short insert paired-end reads were aligned to the GRCh37 reference human genome with 1000 genomes decoy contigs using BWA-mem. Li, arXiv: 1303.3997 (2013). Structural rearrangements were called using Delly (Rausch et al., Bioinformatics 28( 18):i333-i9 (2012)) and a modified version of BRASS using an unmatched control. Copy Number Aberrations were called using CNVKit in each tumor taking as background the pool of 16 constitutional samples. Talevich et al., PLoS computational biology l2(4):el004873 (2016). Single Nucleotide Variants (SNV) were called using CaVEMan, Strelka2 and Mutect2 and Indels were called using Pindel, Strelka2 and Mutect2. All calls that were passed by at least one caller were included for downstream filtering and subsequent analysis.

[00143] Substitutions, Small insertions and deletions: Single base substitutions were called using CaVEMan, Strelka2 and Mutect2. CaVEMan compares sequence data from each tumor sample albeit with an unmatched non-cancerous sample and calculates a mutation probability at each genomic locus. An un-matched constitutional sample is used when a matched sample is unavailable.

[00144] To improve specificity, a number of post-processing filters were applied to CaVEMan and these are elucidated below. All“PASS” calls by Strelka2 and Mutect2 were retained for downstream filtering.

[00145] Post-call filtering of SNV s identified by CaVEMan

1. At least a third of the alleles containing the mutant must have base quality >= 25.

2. If mutant allele coverage >= lOx there must be a mutant allele of at least base quality 20 in the middle 3rd of a read. If mutant allele coverage is < lOx a mutant allele of at least base quality 20 in the first 2/3 of a read is acceptable.

3. The mutation position is marked by <3 reads in any sample in the unmatched normal panel.

4. The mutant allele proportion must be >5 times than that in the matched normal sample (or it is zero in the matched normal). 5. If the mean base quality is <20 then less than 96% of mutations carrying reads are in one direction.

6. Mutations within simple repeats, centromeric repeats, regions of excessive depth and low mapping quality were excluded.

[00146] Small somatic indels were additionally identified using a modified version of Pindel, Strelka2 and Mutect2. To improve specificity of Indels called by Pindel, a number of post-processing filters were applied and described below.

[00147] Post-call filtering of Indels identified by Pindel

1) For regions with sequencing depth <200x mutant variant must be present in at least 8% of total reads.

2) For regions with sequencing depth >=200x mutant variant must be present in at least 4% of total reads.

3) The region with the variant should have <= 9 small (<4 nucleotides) repeats.

4) The variant is not seen in any reads in the matched normal sample or the unmatched normal panel.

5) The number of Pindel calls in the tumor sample is greater than 4 and either:

a. The number of mutant reads mapped by BWA in the tumor sample is greater than 0 or

b. The number of mutant reads mapped by BWA in the tumor sample is equal to 0 but there are no repeats in the variant region and there are reads mapped by Pindel in the tumor sample on both the positive and negative strand.

6) Pindel‘SUM-MS’ score (sum of the mapping scores of the reads used as anchors)

>=150

[00148] All SNVs and Indels that were passed by at least one caller were included for downstream analysis and variant annotation was done based on Ensembl v74 using

VAGrENT¹³. Subsequent, filtering for known variation and artifacts were applied as described below. All calls retained after filtering were manually curated.

[00149] Filtering of all SNVs and Indels : Calls retained after applying the above filters were additionally annotated with variants from the Interim Analysis of 9 exomes from the Multiple Myeloma Research Foundation’s (MMRF) MMRF CoMMpass database. Lonial et al., Am Soc Hematology , 2014; Bolli et al, Blood Cancer J 6: e467 (2016) and Lohr et al., Cancer Cell 25(l):9l- 101 (2014). Calls were annotated if present at the exact genomic position with the exact mutation of if present in close proximity of a mutation (+-9 bp). [00150] For all filtered SNV and Indels identified additional filters were applied and calls were filtered if:

• Present in the IGH locus

• Present in a gene not in the panel.

• Variant is annotated as a synonymous change.

• Minor Allele Frequency (MAF) > 3% in Exac (Version 0.3)

• Filter calls with > 0.5% MAF in Exac (Version 0.3) or 1000 Genomes.

• Present in an unmatched normal sequenced unless present in COSMIC (v8l) at the same position or occurring a the very least altering the same amino acid.

• Filter missense mutations between 0.0025-0.005, unless these are present in TP53 or BRCA1/2 and not present in COSMIC.

• Variants are present with reads only on 1 strand.

• Variant Allele Frequency (VAF) of variants is less than 3%.

• Variants have less than 5 supporting reads.

• Target depth of coverage is less than lOOx

[00151] For both substitutions and indels, variants that may have failed post processing filtering criteria but mapped to recurrent oncogenic mutations in COSMIC¹⁷ were retained for manual curation.

[00152] Structural rearrangements. Given the smaller fragment insert sizes in targeted capture, the 101 bp paired-end reads were trimmed to 50 bp from the 3’ end of the read for better discovery of in structural rearrangements. Alignment on the trimmed reads was performed as described herein and structural rearrangements were detected by an in house algorithm, BRASS, which first groups discordant read pairs that span the same breakpoint and then using the Velvet de novo assembler, performs local assembly within the vicinity to reconstruct and determine the exact position of the breakpoint to nucleotide precision. All calls having support of less than 5 reads were excluded. Additionally, translocations in which either of the break-points is not involved with the IGH locus were excluded for downstream analysis.

[00153] Additionally, an orthogonal pipeline using Delly (Version: 0.7.6) was used to identify structural rearrangements. Delly was run on each tumor sample using an unmatched control sample and only somatic calls were retained. All calls identified with ± 50 bp in the unmatched normal were filtered. Additionally, only those calls having at least 1 spanning read and 1 junction read, or at least 4 spanning reads were retained. The translocations having either of the break-points involving with the IGH locus were retained, and deletions, inversions and duplications were retained if neither breakpoint involved the IGH locus.

[00154] All calls from Delly and BRASS were further filtered for false positives using average MAPQ, CIGAR Match length and number of reads supporting the SV. MAPQ filter excludes SVs having average mapping quality of the reads supporting and SV with less than a value of 30. CIGAR Match length is the average match length in the CIGAR string of all the reads supporting the SV. A threshold of <60 was used to filter calls. Lastly, supporting reads is the number of reads supporting the SV. The threshold used for support is at least 30 reads.

[00155] Additionally, BRASS and Delly were run on the 16 constitutional samples sequenced using and the post-call filters as described were applied. All SVs identified in the tumor samples having a break-point detected within ± 50 bp of the SVs in the constitutional samples were excluded.

[00156] The resulting calls retained after the described filters were manually curated.

[00157] Copy number aberrations. For samples with unmatched sample, CNVKit was used to identify somatic copy number aberrations in the data. To negate sample specific biases in CNV analysis, all 16 control samples were combined into a pooled reference. Each tumor sample was then compared with the pooled reference to identify somatic Copy Number Aberrations (CNA) in each sample. CNVKit corrected for biases in regional coverage and GC content, according to the given reference before calculating the log-ratios between the built pooled reference and tumor. Subsequently, Circular Binary Segmentation (CBS) algorithm is applied to obtain the log2 segment means.

[00158] An arm level aberration was defined if the absolute value of log2 segment mean > 0.1 and if length of the segment covers at least 10% of the length of the entire arm. Similarly, a focal aberration was defined if absolute value of log2 segment mean > 0.1 and if length of the segment covers at least 10%.

[00159] Genomic coverage metrics. As shown in Figure 8, a median of 38 million paired end reads were obtained per sample across the entire cohort of 159 tumor and 16 nomals. However, as shown in Figure 9, an overall median target coverage of 65 lx per sample was obtained resulting from a formidable proportion (median=3l.l%) of reads aligning to off- target regions. After marking duplicates, a median of 29.9% duplicates were observed per sample with values ranging from 16.7% to 37.4%. [00160] Next, the coverage across the different regions of target space captured across the constitutional bone marrow samples was examined. This analysis was performed using constitutional samples alone to prevent for coverage biases caused due to rearrangements and copy number aberrations in the tumor samples. Overall, a median coverage of 8l5.6x across the constitutional samples was obtained. As shown in Figures 10A-10B, median coverage of 750.2x across the coding sequences, 618. lx across finger-printing SNPs, 5l0.6x for the SNPs in the intronic regions and 9l4.6x across the IGH locus was obtained.

[00161] Overall, the percentage of target space with no coverage was well below 1% overall (0.1%) and across the different types of regions with the exception of tiled intronic SNPs (median=l8.3%, Ql=l8.2%, Q3=l8.4) and IGH locus (median=3.4%, Ql=3%, Q3=3.7%). The low inter-quartile range of lack of coverage in the intronic SNPs suggests that the same set of SNPs are not being captured by the protocol potentially due to low sequence specificity in these regions.

[00162] As shown in Figure 10C, across the target space, median of 99% and 98% had atleast lOx and lOOx coverage respectively. The same for other captured regions namely exons, fingerprinting SNPs, and IGH locus was atleast 99% and 97% respectively (Figure 10B). The same for Intronic SNPs is 81% and 75% respectively consistent with lack of coverage in 18% of this targeted space.

[00163] The variation of coverage across each of the genes for which exons were captured was analyzed. The median coverage across these genes is 695 (IQR=327.5) while the same for previously reported significantly mutated myeloma genes is 7l2.6x. Lohr J el al, Cancer Cell 25(l):9l- 101 (2014).

Example 3: Sequencing Coverage for the NGS Methods of the Present Technology

[00164] The bait sequences of the present disclosure are represented by SEQ ID NOs: 159- 6088. As shown in Figures 8-9, sequencing generated a median of 38 million paired-end reads per sample resulting in a median target coverage of 65lxper sample. The difference between the median paired-end reads per sample and the median target coverage was attributable to a formidable proportion (median=3 l.l%) of reads aligning to off-target regions. The coverage across the IGH locus, exonic regions, genome-wide copy number SNPs and finger printing SNPs were homogenous across every probe type. Example 4: Comparison FISH/SNP microarray versus the NGS Methods of the Present Technology

[00165] Figure 14 and the Table below show the comparison of detection of IGH translocations by the multiple myeloma-specific targeted NGS assay described herein vs. FISH. As shown in the Table below, among all samples that were subjected to the NGS sequencing disclosed herein ( e.g ., myTYPE) or FISH, a total of 82 IGH translocations were identified. As shown in the table below and Figure 14, the NGS sequencing assays disclosed herein were more sensitive compared to FISH assays with respect to detecting IGH translocations.

[00166] Across the 153 samples that were tested by both and FISH panels, a total of 78 IGH translocations were detected. Of these, as shown in Figures 4-5, 77 and 76

rearrangements were detected by FISH and myTYPE, respectively.

[00167] The concordance of those identified by both assays was 96% (N=75), while 3 of the translocations were uniquely identified by FISH (N=2) and (N=l). The overall sensitivity and specificity for were 97% and 100%, respectively, using FISH as the reference. All 3 translocations uniquely identified by either of the assays were t(8; 14). In two patients, two translocations were detected by myTYPE, including one with t(4; 14) and t(8;l4), and the other with t( 11 ; 14) and t(8;l4). For all 28 IGH translocations detected by only re-test the samples by FISH was aimed to confirm the results: six patients did not have any remaining cell pellets for FISH tests, however, clinical FISH tests revealed IGH translocations in two patients, indirectly confirming the presence of the relevant IGH translocations. As shown in Figures 13A-13D, in two other patients, the t(8; 14), detected by FISH tests with high percentage, were not confirmed by myTYPE, indicative of variable break-points at MYC, which may be outside of primer coverage (See Figure 12). In addition, one patient with an t(8; 14) identified by was not confirmed by FISH in multiple repeated samples.

[00168] These results demonstrate that the library bait compositions of the present technology are useful in methods for detecting at least one multiple myeloma-related mutation in a sample, and eliminate the need for secondary standard of care diagnostic assays such as FISH or SNP.

Example 5: Comparison FISH/SNP Microarray Versus myTYPE: Copy Number Alterations

[00169] The comparison of CNAs between and array results was focused on the commonly implicated regions in multiple myeloma. Overall, 696 aberrations were identified across all 156 samples by either or SNP microarray with an overall sensitivity and specificity of 97% and 97%, respectively. Of the 658 aberrations identified by SNP microarrays, 97% (N=637) were called by myTYPE. In the main analysis, lesions of prognostic relevance in multiple myeloma, namely 17r, lq+, l3q-, and lp- were investigated and found that 179/182 aberrations identified by SNP microarrays were called by with a high sensitivity of 98%. Importantly, uniquely identified two 17r deletions, one lp gain and l3q deletions each. One aberration, each in lq+, 13r-, and lp-, were uniquely identified by SNP microarrays.

Table 2: Common multiple myeloma genomic aberrations and their detection rates by myTYPE and conventional methods, respectively.

[00170] These results in Table 2 demonstrate that the methods of present disclosure exhibit higher sensitivity as well as specificity compared to the conventional methods.

[00171] The Table below shows the comparison of CNAs between and SNP Arrays. As shown in the Table below and Table 2, among the recurring aberrations in multiple myeloma, namely 17r-, lq-, lp+,l3q-, l lq+, 12r-, l4q-, l6q-, 3p+, 3q+, 6q-, 8p-, 9q+, t(l l;l4), t(l4;l6), t(l4;20), t(4; 14), t(6; 14) and t(8; 14), 97% (75/77) of the translocations and 98% (570/602) of the CNA identified by FISH/SNP microarrays were also identified by myTYPE.

[00172] These results demonstrate that the library bait compositions of the present technology are useful in methods for detecting at least one multiple myeloma-related mutation in a sample, and eliminate the need for secondary standard of care diagnostic assays such as FISH or SNP.

Example 6: Mutations. Small Insertions and Deletions: Detection by myTYPE

[00173] At least one non- synonymous mutation was identified in 132 of 156 (85%) samples. In these 132 samples, a total of 362 non-synonymous single nucleotide variants (SNVs) and 51 indels were detected by (median = 3/sample). As shown in Figure 11, 21% of the samples harbored a KRAS mutation, and 18% and 13% of the samples had NRAS and TP53 mutations, respectively. Of clinical significance, 4% (N=6) harbored a BRAF V600E mutation and 3 samples harbored an IDH1 mutation including one sample with the hotspot R132C mutation.

[00174] Clinically important, among 25 patients with 17r deletions detected by SNP microarrays, found that 8% (N=l2) also harbored a TP53 mutation, causing a bi-allelic TP53 inactivation. As shown in Figure 13, significantly (p< 0.0001) poorer progression- free survival was found for multiple myeloma patients with bi-allelic TP53 inactivation.

[00175] Thes results disclosed herein represent the first large-scale head-to-head comparison of standard of care targeted FISH panel and SNP microarray versus a targeted NGS assay based on bone marrow specimens from a well-defined cohort of multiple myeloma patients. Recurring chromosomal abnormalities and the genomic landscape in multiple myeloma include various IGH translocations, copy number alterations (CNAs), and somatic mutations. For example, two broad main categories of genomic abnormalities have been described: hyperdiploidy (defined as gains of odd numbered chromosomes) and immunoglobulin heavy chain {IGH) translocations (including t(4; 14), t(6; 14), t( 11 ; 14), t(l4;l6), t(l4;20), and t(8;l4)). In addition, deletions (del) and losses of certain regions, such as, del(l3q), del(l7p), del(lq), as well as and gain of lp, were also frequently observed. In the most recent version of the International Staging System (R-ISS) for multiple myeloma, dcl( 17p)/7/^J53, t(4; 14), and t(l4;l6) are defined as high-risk aberrations. The assays disclosed herein can replace standard of care assays, including FISH and SNP microarrays.

[00176] Using the multiple myeloma-specific targeted NGS assay of the present technology, an extremely high concordance (sensitivity >98% and specificity >99% for myTYPE) was found between and conventional FISH/SNP microarrays in detection of IGH translocations and CNAs relevant to multiple myeloma. The clinical implications of these results are important in that they show that current standard of care bone marrow prognostic assays for multiple myeloma ( i.e . conventional cytogenetics, disease targeted FISH panels, and SNP microarrays) can be replaced by the novel single-assay myTYPE.

[00177] In addition to profiling of IGH translocations and CNAs, the multiple myeloma- specific targeted NGS assay of the present technology captures mutations of all relevant multiple myeloma genes and reveals critical information indicative of adverse clinical outcomes. Specifically, of 25 patients with deletion 17r captured by standard of care SNP microarrays, revealed TP53 mutations in 12 (48%) patients, resulting in bi-allelic TP53 inactivation. As shown in Figure 13, the patients with bi-allelic TP53 inactivation had a significantly reduced progression- free survival. The ability to capture all mutations relevant to clinical outcomes in multiple myeloma - in addition to the capture of all recurrent IGH translocations and all CNAs relevant to multiple myeloma - further supports the clinical strengths of as an optimal assay for patient care.

[00178] In accord with current multiple myeloma guidelines, conventional chromosome analysis and targeted FISH panels are typically used around the world to detect IGH translocations and recurring chromosome gains and losses. Manier et al., Nat Rev Clin Oncol 14(2): 100- 13 (2017). To improve the capture rate of chromosomal gains and losses, SNP microarrays have replaced conventional chromosome analysis at some laboratories.

However, none of the conventional clinical assays can detect mutations or small indels which may be clinically significant in terms of prognosis and treatment responses in multiple myeloma.

[00179] Due to the fact that current assays require relatively large amounts of bone marrow sample material, it limits laboratories in their ability to perform additional parallel comprehensive genomic analysis including mutational characterization. Furthermore, none of the currently available capture based NGS panels (such as FoundationOne CDx) are capable of capturing structural rearrangements {i.e. IGH translocations) which are frequently occurring in multiple myeloma, thus requiring the current standard of care assays ( i.e ., conventional chromosome analysis, multiple myeloma targeted FISH panels, and SNP microarrays) to be performed in parallel. In contrast to available capture based NGS panels, a single assay with full coverage of the IGH locus was designed allowing capture of all multiple myeloma-specific IGH translocations, CNAs, and all mutations relevant in multiple myeloma.

[00180] As discussed above, the multiple myeloma-specific targeted NGS assay of the present technology accurately and efficiently detected IGH translocations and CNAs.

Furthermore, regarding mutation calling, the prevalence of KRAS and NRAS as well as the overall frequency of somatic mutations was found to be in full accord with the literature. Bolli et al., Leukemia doi: l0.l038/leu.20l7.344 (2017); Walker et al., Journal of clinical oncology : official journal of the American Society of Clinical Oncology 33(33):39l 1-20 (2015); and Lohr et al., Cancer Cell 25(l):9l-l0l (2014). Thus, the multiple myeloma- specific targeted NGS assay of the present technology precisely captures comprehensive cytogenomic abnormalities beyond FISH and SNP-arrays and shows that it is possible to replace current standard of care prognostic bone marrow assays for multiple myeloma patients {i.e., conventional chromosome analysis, targeted FISH panels, and SNP

microarrays) with a single test.

[00181] High -risk multiple myeloma is a relative terminology that is subject to change as modern effective therapies continue to evolve. Per the International Myeloma Working Group consensus criteria, IGH translocations t(4; 14) and t( 14; 16) and 17r deletions are defined as high-risk multiple myeloma and lq gains have been included in earlier versions of high risk definitions. Manier et al., Nat Rev Clin Oncol 14(2): 100- 13 (2017). As illustrated in the current study, an advantage of assay is the integrated capture of CNAs and mutations co-occurring in the same genes, allowing for the assessment of bi-allelic events involving different types of aberrations. Indeed, of 25 patients with deletion of 17r, detected TP53 mutations in 12 patients, resulting in bi-allelic p53 inactivation. As shown in Figure 13, the multiple myeloma patients with bi-allelic TP53 inactivation have a poor progression- free survival. As therapies get better and patient benefit from modern effective combination therapies, it is expected that the difference in biology will become be less important for the majority of multiple myeloma patients. However, patients with short progression-free survival and overall survival after having received modern combination therapy are likely reflections of disease biologies which require fundamentally different therapies. [00182] As shown throughout this study, there is high concordance between conventional assays (targeted FISH panels and SNP microarrays) and the multiple myeloma- specific targeted NGS assay of the present technology when it comes to detection of IGH

translocations and CNAs. In this context, it should be emphasized that the analysis of panel sequencing data and the identification of these abnormalities is complex. For example, in an unmatched- normal with a multiple myeloma sample analysis, the heterozygous SNPs cannot be confirmed in the normal, which makes detection of LOH events difficult because inference is based on Variant Allele Frequency (VAF) of SNPs in tumor alone. Furthermore, for capturing of IGH translocations, the assay was designed to capture the IGH locus between 14:105994256 and 14:107288051. As shown in Figure 12, in the Multiple Myeloma Research Foundation’s CoMMpass study, the breakpoints for 26% of the t(8; 14)

translocations occurred outside this region and hence were not captured. This is most likely the case in our comparison study where two t(8; 14) detected by FISH were not confirmed by tests.

[00183] In conclusion, this large head-to-head comparison of targeted FISH panels and SNP microarrays versus the targeted NGS assay revealed extremely high concordance with regard to detection of all relevant IGH translocations and CNAs relevant in multiple myeloma. Additionally, the multiple myeloma-specific targeted NGS assay of the present technology is designed to capture all relevant mutations in multiple myeloma. Among 25 multiple myeloma patients with deletion of 17r captured by conventional SNP microarrays, the multiple myeloma-specific targeted NGS assay of the present technology detected TP53 mutations in 12 patients (48%), resulting in bi-allelic p53 inactivation. As shown in Figure 13, multiple myeloma patients with bi-allelic TP53 inactivation had a significantly reduced progression- free survival. Overall, these results show that has great potential for clinical applications with its advantages in detecting all IGH translocations, CNAs, and gene mutations that are relevant in multiple myeloma - all within a single assay.

EQUIVALENTS

[00184] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as were apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, were apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present technology is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[00185] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[00186] As were understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as“up to,”

“at least,”“greater than,”“less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as were understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

[00187] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

[00188] Other embodiments are set forth within the following claims.

Claims

1. A bait library comprising one or more baits comprising a sequence of any of SEQ ID NOs: 159-6088.

2. The bait library of claim 1, wherein the one or more baits comprise a barcode and/or an adapter sequence.

3. The bait library of claim 1 or claim 2, wherein the one or more baits comprise a detectable label.

4. The bait library of any one of claims 1-3, wherein the one or more baits are configured to bind an IGH locus.

5. The bait library of any one of claims 1-4, wherein the one or more baits are configured to bind at least one chromosome 14 translocation breakpoint present in a multiple myeloma sample.

6. The bait library of any one of claims 1-5, wherein the one or more baits are configured to bind a single nucleotide variation (SNV) present in a multiple myeloma sample.

7. The bait library of any one of claims 1-6, wherein the one or more baits are configured to bind a copy number aberration (CNA) present in a multiple myeloma sample.

8. The bait library of any one of claims 1-7, wherein the one or more baits are configured to bind a translocation present in a multiple myeloma sample.

9. The bait library of any one of claims 1-8, wherein the one or more baits are configured to bind an insertion/deletion (indel) present in a multiple myeloma sample.

10. The bait library of any one of claims 1-9, wherein the one or more baits are configured to bind to one or more introns of one or more genes selected from the group consisting of ANP32E, APC, ATM, ATR, BIRC2, BIRC3, CDKN2A, CDKN2C, CKS1B, CSNK1A1, CYLD, DIS3, FAF1, FAM46C, FAT3, HIST1H1E, IRF4, KDM6A, KRAS,

MAP3K14, MAX, MYC, NF1, NFKBI, NFKBIA, PRDM1, PTEN, PTPRD, RBI, R0B02, SETD2, SP140, TNFAIP3, TP53, TRAF2, TRAF3, WWOX, and XBPL

11. The bait library of any one of claims 1-10, wherein the one or more baits are configured to bind to one or more exons of one or more genes selected from the group consisting ofACTGI, ADCY5, AK2, APBB1IP, APC, ARID2, ATM, ATP13A4, ATR, AVEN, BCL2, BIRC2, BIRC3, BRAF, BRCA2, BTG1, CCNDI, CD274,CD38, CDC27, CDKN1B, CDKN2A, CDKN2C, CRBN, CSNK1A1, CUL4B, CXCR4, CYLD, DIS3, DLX6, DNAHU, DNAH5, DNAH9, DUSP2, EGRI, EPHA3, FAM46C, FAT1, FAT3, FAT4, FBRS, FBXW7, FCRL5, FGFR3, FEG, FSIP2, GPRC5D, HIST 1 HI B, HIST 1 HI C, HIST 1 HIE, HIST1H3G,

ID HI, IDH2, IKBKB, IKZFJ IKZF3, IRF4, JAM2, KDM1A, KDM6A, KEHE6, KMT2D, KRAS, ETB, MAF, MAP3K1, MAX, MYC, MYD88, NCKAP5, NF1, NFKB2, NFKBIA,

NR3C1, NRAS, PABPC1, PCLO, PDCD1, PIK3CA, PIM1, PPM1D, PRDM1, PRDM6, PRKD2, PSMB5, PSMB8, PSMB9, PSMC2, PSMC3, PSMC4, PSMC5, PSMC6, PSMOI, PSMD12, PSMD13, PSMD2, PSMD6, PSMD7, PTEN, RTRNP, PTPRD, RAG2, RASA2, RBI, RHOT1, RIPKl, ROBOl, R0B02, SETD2, SE3BI, SEAMF7, SP140, TNFRSF17,

TP53, TRAF2, TRAF3, WHSC1 (NSD2), XBP1, XPOl, and ZFHX4.

12. The bait library of any one of claims 1-11, wherein the one or more baits are configured to bind to one or more exons of a gene selected from the group consisting of ABCF1, ACTG1, ARID1A, ARID2, ATM, ATRX, BRAF, C80RF34, CCNDI, CDKN1B, CDKN2C, CREBBP, CYLD, DIS3, DNMT3A, DUSP2, EGRI, EP300, FAM46C, FGFR3, FUBP1, HIST 1 HIE, HUWE1, IDH1, IDH2, IRF4, KDM5C, KDM6A, KEHE6, KMT2B, KMT2C, KRAS, ETB, MAF, MAFB, MAME2, MAN2CI, MAX, NCORI, NEI, NFKB2, NFKBIA, NRAS, PIK3CA, PRDM1, PRKD2, RTRNP, RASA2, RBI, RETNI, SAM HD I, SETD2, SE3BI, SP140, TET2, TGDS, TP53, TRAF2, TRAF3, UBR5, XBP1, ZFP36L1, and ZNF292.

13. The bait library of any one of claims 1-12, wherein the one or more baits are configured to bind to one or more exons of a gene selected from the group consisting of IDH1, DIS3, and TP53.

14. A method for detecting at least one multiple myeloma-related mutation in a patient in need thereof, the method comprising

(i) contacting a nucleic acid sample obtained from the patient with the bait library of any of claims 1-13, and

(ii) detecting at least one multiple myeloma-related mutation in the sample via massively parallel DNA sequencing.

15. The method of claim 14, wherein the nucleic acid sample is derived from a biopsy sample obtained from the patient.

16. The method of claim 14 or claim 15, wherein the nucleic acid sample is a blood or bone marrow sample.

17. The method of any one of claims 14-16, wherein the patient is suspected of, or diagnosed as having multiple myeloma.

18. The method of any one of claims 14-17, wherein the nucleic acid sample comprises DNA, RNA or any combination thereof.

19. The method of any one of claims 14-18, wherein the nucleic acid sample is contacted with the bait library via solution phase hybridization.

20. The method any one of claims 14-19, wherein detecting at least one multiple myeloma-related mutation in the sample comprises detecting a chromosome 14 translocation.

21. The method of any one of claims 14-20, wherein detecting at least one multiple myeloma-related mutation in the sample comprises detecting a single nucleotide variant (SNV).

22. The method of any one of claims 14-21, wherein detecting at least one multiple myeloma-related mutation in the sample comprises detecting a copy number aberration (CNA).

23. The method of any one of claims 14-22, wherein detecting at least one multiple myeloma-related mutation in the sample comprises detecting a translocation.

24. The method of any one of claims 14-23, wherein detecting at least one multiple myeloma-related mutation in the sample comprises detecting an insertion/deletion (indel).

25. The method of any one of claims 14-24, wherein the massively parallel DNA sequencing is performed using one or more of pyrosequencing, reversible dye terminator chemistry, oligonucleotide ligation chemistry, proton detection, or phospholinked fluorescent nucleotide chemistry.

26. A method for selecting a patient suspected of, or diagnosed as having multiple myeloma for treatment with a therapeutic agent comprising

(i) contacting a nucleic acid sample obtained from the patient with the bait library of any of claims 1-13,

(ii) detecting at least one mutation in one or more genomic drivers associated with multiple myeloma, wherein the one or more genomic drivers are selected from the group consisting of IDH1, DIS3, TP53, NRAS, KRAS, or BRAF, and

(iii) administering a therapeutic agent to the patient.

27. The method of claim 26, wherein the one or more genomic drivers comprise mutations that are associated with poor survival outcomes.

28. The method of claim 26 or claim 27, wherein a TP 53 mutation is not detected in the sample, and the therapeutic agent is an immunomodulatory drug (IMiD), a proteasome inhibitor (PI), melphalan, vincristine, cyclophosphamide, etoposide, doxorubicin,

bendamu stine, thalidomide, lenalidomide, pomalidimodide, an histone deacetylase (HD AC) inhibitor, autologous hematopoietic stem cell transplant (auto-HCT), or any combination thereof.

29. The method of claim 26 or 27, wherein a TP53 mutation is detected in the sample, and the therapeutic agent is nutlin, RITA (Reactivation of p53 and Induction of Tumor Cell Apoptosis), PRIMA-l (p53 reactivation and induction of massive apoptosis), rocaglate, CMLD010509, a USP7 inhibitor, a Weel inhibitor, a PARP-l inhibitor, or any combination thereof.

30. The method of any one of claims 26-29, wherein a KRAS mutation is detected in the sample, and the therapeutic agent is a Syk inhibitor, a Ron inhibitor, an integrin beta6 inhibitor, a MEK1 inhibitor, a MEK2 inhibitor, a MAPK pathway inhibitor, a CDK4 inhibitor, a CDK6 inhibitor, a miR-29b inhibitor, a NF-kB inhibitor, a TNFAIP3 inhibitor, a KRAS inhibitor, an EGFR inhibitor, an immune checkpoint inhibitor, a Galectin-3 inhibitor, or any combination thereof.

31. The method of any one of claims 26-30, wherein a NRAS mutation is detected in the sample, and the therapeutic agent is a MEK1 inhibitor, a MEK2 inhibitor, a MAPK pathway inhibitor, a CDK4 inhibitor, a CDK6 inhibitor, a farnesyltransferase inhibitor, a PI3K inhibitor, an AKT inhibitor, a MET inhibitor, a VEGFR2 inhibitor, a ERK inhibitor, a BRAF inhibitor, a KRAS inhibitor, a heat shock protein-90 (HSP90) inhibitor, an immune checkpoint inhibitor, or any combination thereof.

32. The method of any one of claims 26-31, wherein a BRAF mutation is detected in the sample, and the therapeutic agent is a BRAF inhibitor, a MEK1 inhibitor, a MEK2 inhibitor, a MAPK pathway inhibitor, a CDK4 inhibitor, a CDK6 inhibitor, a EGFR inhibitor, an ALK inhibitor, a ROS1 inhibitor, an immune checkpoint inhibitor, a programmed cell death protein 1 (PDCD1) inhibitor, or any combination thereof.

33. The method of any one of claims 26-32, wherein an IDH1 mutation is detected in the sample, and the therapeutic agent is an IDH1 inhibitor, an IDH2 inhibitor, a DNA damaging agent, a DNA repair inhibitor, a poly(ADP-ribose) polymerase (PARP) inhibitor, a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, a BCL-2 inhibitor, a BH3 mimetic, a multikinase inhibitor, an electron transport chain inhibitor, a biguanide, a cytochrome c oxidase inhibitor, an immune checkpoint inhibitor, or any combination thereof.

34. The method of any one of claims 26-33, wherein a DIS3 mutation is detected in the sample, and the therapeutic agent is a DIS3 inhibitor, a DotLl inhibitor, or any combination thereof.