Immunoglobulin Expression Levels as Biomarker for Proteasome Inhibitor Response

CROSS REFERENCE TO RELATED APPLICATIONS

[0001]This application is a division of U.S. application Ser. No. 14/910,668, filed Feb. 5, 2016, which is the U.S. national stage application of International Application No. PCT/US14/50333, filed Aug. 8, 2014, which claims the priority benefit of Provisional U.S. Patent Application No. 61/863,809, filed on Aug. 8, 2013, Provisional U.S. Patent Application No. 61/875,954, filed on Sep. 10, 2013, and Provisional U.S. Patent Application No. 62/005,904, filed on May 30, 2014, each of which is incorporated by reference in its entirety.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0002]Incorporated by reference in its entirety is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: 46,919,680 bytes ASCII (Text) file named “40058A_SeqListing.txt,” created on Aug. 8, 2014.”

TECHNICAL FIELD

[0003]This invention relates to the fields of molecular biology and cancer treatment. In some aspects, the invention relates to determining (e.g. predicting) a tumor's (e.g., a hematological tumor) sensitivity to treatment with a proteasome inhibitor. In some other aspects, the invention relates to methods useful for diagnosing, classifying, profiling, and treating cancer.

BACKGROUND

[0004]Multiple myeloma (MM) is an incurable malignancy that originates in the antibody-secreting bone marrow plasma cells. MM comprises approximately 10% of all hematologic malignancies. The progression of the tumor is well understood, and it can be diagnosed by the presence of multiple myeloma cells in the bone marrow and monitored by the amount of antibody secretion from the clonal population of plasma cells. With conventional treatment, median survival is approximately 3 to 4 years, but the clinical course is highly variable and difficult to predict. Several therapies for MM are now approved and many more are in development, promising improved outcomes for patients with this incurable cancer. With expanding treatment options, however, comes a pressing need to pair each patient with the most efficacious and safe treatment. With the narrow therapeutic index and the toxic potential of many available cancer therapies, such differential responses potentially contribute to patients undergoing unnecessary ineffective and even potentially harmful therapy regimens. If a designed therapy could be optimized to treat individual patients, such situations could be reduced or even eliminated. Furthermore, targeted designed therapy may provide more focused, successful patient therapy overall. Therefore, there is a need to better define patient-specific treatment strategies for the use of both standard and novel therapies.

[0005]Proteasome inhibition has emerged as an important strategy in cancer treatment, including in the treatment of multiple myeloma. By way of background, proteasomes are large, multienzyme complexes that play a key role in protein breakdown. The average human cell contains about 30,000 proteasomes, each of which contains several protein-digesting proteases. The proteasome mediates the proteasomal degradation pathway which is necessary to rid cells of excess and misfolded proteins. Proteasomal complexes help regulate a whole host of functions including transcription, viral infection, oncogenesis, cell cycle, stress response, ribosome biogenesis, abnormal protein catabolism, neural and muscular degeneration, antigen processing, DNA repair, and cellular differentiation. Proteasome activity is exquisitely controlled; when it becomes either overzealous (degrading more proteins than it should) or underachieving (neglecting to degrade certain proteins) disease can develop. Proteasome inhibitors (PIs), such as carfilzomib (marketed as Kyprolis® by Onyx Pharmaceuticals) and bortezomib (marketed as Velcade® by Millennium Pharmaceuticals), have become a standard therapy across all lines of MM therapy. Carfilzomib is a tetrapeptide epoxyketone, a selective proteasome inhibitor, and is approved for the treatment of patients with multiple myeloma who have already received at least two other treatments including bortezomib and an immunomodulatory agent (e.g., lenalidomide and/or thalidomide), and whose disease has progressed on their last therapy or within 60 days of their last therapy. Despite extensive study, the mechanism of selective tumor cell death following proteasome inhibition is poorly understood. Many patients have disease that does not respond to PIs, whereas others develop resistance, suggesting the need to better define patient-specific treatment strategies for the use of PI therapies.

SUMMARY

[0006]Provided herein are methods of treating a tumor in a subject. In exemplary embodiments, the method comprises (a) measuring the level of expression of (i) immunoglobulin (Ig), including gene or gene product, (ii) Fc gamma receptor 2B (FCGR2B), including gene or gene product, or (iii) both Ig and FCGR2B, in a sample obtained from the subject, wherein the sample comprises a cell from the tumor; and (b) administering to the subject an effective amount of a proteasome inhibitor, when the level of Ig expression and/or FCGR2B expression in the sample is greater than a reference level. When the level of Ig expression and/or FCGR2B expression in the sample is less than this reference level, then the patient is administered an alternative anti-tumor therapy that is not a proteasome inhibitor. In alternative or additional embodiments, the method comprises (a) measuring the level of expression of one or more genes or gene products listed in Table 4, in a sample obtained from the subject, wherein the sample comprises a cell from the tumor; and (b) administering to the subject an effective amount of a proteasome inhibitor, when (i) the level of expression of the one or more genes listed in Table 4 in the sample is greater than a reference level, and the change in gene expression level for the one or more genes or gene products is denoted in Table 4 as “up”, or (ii) the level of expression of the one or more genes or gene products listed in Table 4 in the sample is less than a reference level, and the change in gene expression level for the one or more genes or gene products is denoted in Table 4 as “down”, or (iii) both (i) and (ii).

[0007]In exemplary embodiments, the method comprises the step of administering to the subject an effective amount of a proteasome inhibitor. In exemplary aspects, the subject is a subject from which a sample was obtained and the level of expression of (i) immunoglobulin (Ig), (ii) Fc gamma receptor 2B (FCGR2B), or (iii) both Ig and FCGR2B, has been measured from the sample. In exemplary aspects, the proteasome inhibitor is administered, when the level of expression is greater than a reference level.

[0008]In alternative or additional aspects, the subject is a subject from which a sample was obtained and the level of expression of one or more genes or gene products listed in Table 4 has been measured from the sample. In exemplary aspects, the proteasome inhibitor is administered, when (i) the level of expression of the one or more genes or gene products listed in Table 4 in the sample is greater than a reference level, and the change in gene expression level for the one or more genes or gene products is denoted in Table 4 as “up”, or (ii) the level of expression of the one or more genes or gene products listed in Table 4 in the sample is less than a reference level, and the change in gene expression level for the one or more genes or gene products is denoted in Table 4 as “down”, or (iii) both (i) and (ii).

[0009]Also provided herein are methods of determining a treatment regimen for a subject with a tumor. In exemplary embodiments, the method comprises (a) measuring the level of expression of (i) immunoglobulin (Ig), (ii) Fc gamma receptor 2B (FCGR2B), or (iii) both Ig and FCGR2B, in a sample obtained from the subject, wherein the sample comprises a cell from the tumor, and (b) selecting a treatment regimen comprising administration of a proteasome inhibitor, when the level of Ig expression and/or FCGR2B expression in the sample is greater than a reference level. When the level of Ig expression and/or FCGR2B expression in the sample is less than this reference level, then the treatment regimen preferably includes administration of an alternative anti-tumor therapy that is not a proteasome inhibitor. In exemplary embodiments, the method comprises (a) measuring the level of expression of one or more genes or gene products listed in Table 4 in a sample obtained from the subject, wherein the sample comprises a cell from the tumor, and (b) selecting a treatment regimen comprising administration of a proteasome inhibitor, when (i) the level of expression of the one or more genes or gene products listed in Table 4 in the sample is greater than a reference level, and the change in gene expression level for the one or more genes or gene products is denoted in Table 4 as “up”, or (ii) the level of expression of the one or more genes or gene products listed in Table 4 in the sample is less than a reference level, and the change in gene expression level for the one or more genes or gene products is denoted in Table 4 as “down”, or (iii) both (i) and (ii). When (i) the level of expression of the one or more genes or gene products denoted in Table 4 as“up” is less than a reference level, or (ii) the level of expression of the one or more genes or gene products denoted in Table 4 as “down” is greater than a reference level, or both, then the treatment regimen preferably includes administration of an alternative anti-tumor therapy that is not a proteasome inhibitor.

[0010]Further provided herein are kits. In exemplary embodiments, the kit comprises one or more binding agents to an Ig gene or gene product, optionally an IgH, IgK or IgL gene segment or gene segment product, and a binding agent to FCGR2B gene or gene product. In exemplary embodiments, the kits comprises (i) one or more binding agents to an Ig gene or gene product, optionally an IgH, IgK or IgL gene segment or gene segment product, or a binding agent to FCGR2B gene or gene product and (ii) at least one binding agent to a gene or gene product listed in Table 4. In exemplary embodiments, the kit comprises at least a first binding agent and a second binding agent, wherein the first binding agent binds to a first gene or gene product encoded by a first gene listed in Table 4, wherein the second binding agent binds to a second gene or gene product encoded by a second gene listed in Table 4, wherein the first gene is different from the second gene.

[0011]Computer readable-storage media are furthermore provided herein. In exemplary embodiments, the computer readable storage medium is one having stored thereon a plurality of reference levels or ranges of reference levels, each reference level or range of reference levels corresponding to (i) an expression level of Ig or (ii) an expression level of FCGR2B, or (iii) an expression level of a gene listed in Table 4, or (iv) a combination thereof; and a data value that is an expression level of Ig and/or an expression level of FCGR2B and/or an expression level of a gene listed in Table 4, measured from a cell from a sample from a patient. In exemplary aspects, the the computer readable storage medium is one having stored thereon (I) a first set of data values and a second set of data values, wherein each data value of the first set is an expression level of Ig determined from a sample obtained from a responder and each data value of the second set is an expression level of Ig determined from a sample obtained from a non-responder; (II) a receiver operating characteristic (ROC) curve based on a first set of data values and a second set of data values, wherein each data value of the first set is an expression level of Ig determined from a sample obtained from a responder and each data value of the second set is an expression level of Ig determined from a sample obtained from a non-responder; or (III) a table listing (a) a plurality of cut-off points on the ROC curve of (II), (b) the % sensitivity associated with each cut-off point of (a), and (c) the % specificity associated with each cut-off point of (a).

[0012]In exemplary embodiments, the computer readable storage medium is one having stored thereon (I) a first set of data values and a second set of data values, wherein each data value of the first set is an expression level of FCGR2B determined from a sample obtained from a responder and each data value of the second set is an expression level of FCGR2B deteremined from a sample obtained from a non-responder; (II) a receiver operating characteristic (ROC) curve based on a first set of data values and a second set of data values, wherein each data value of the first set is an expression level of FCGR2B determined from a sample obtained from a responder and each data value of the second set is an expression level of FCGR2B deteremined from a sample obtained from a non-responder; or (III) a table listing (a) a plurality of cut-off points on the ROC curve of (II), (b) the % sensitivity associated with each cut-off point of (a), and (c) the % specificity associated with each cut-off point of (a).

[0013]In exemplary embodiments, the computer readable storage medium is one having stored thereon (I) a first set of data values and a second set of data values, wherein each data value of the first set is an expression level of a gene listed in Table 4 determined from a sample obtained from a responder and each data value of the second set is an expression level of a gene listed in Table 4 determined from a sample obtained from a non-responder; (II) a receiver operating characteristic (ROC) curve based on a first set of data values and a second set of data values, wherein each data value of the first set is an expression level of a gene listed in Table 4 determined from a sample obtained from a responder and each data value of the second set is an expression level of a gene listed in Table 4 determined from a sample obtained from a non-responder; or (III) a table listing (a) a plurality of cut-off points on the ROC curve of (II), (b) the % sensitivity associated with each cut-off point of (a), and (c) the % specificity associated with each cut-off point of (a). In exemplary aspects, the computer readable storage medium comprises two or more of the foregoing media.

[0014]In exemplary embodiments, the computer readable storage medium is one having stored thereon machine-readable instructions executable by a processor, comprising: (a) instructions for receiving a data value, a, relating to a test level of Ig expression from a sample obtained from a test subject; and (b) instructions for displaying an output relating to treating a patient for multiple myeloma with a proteasome inhibitor, when a is greater than β, a cutoff correlative with a % specificity of at least 50% and a % sensitivity of at least 50%, as determined by a receiver operating characteristic (ROC) curve.

[0015]In exemplary embodiments, the computer readable storage medium is one having stored thereon machine-readable instructions executable by a processor, comprising: (a) instructions for receiving a data value, a, relating to a test level of FCGR2B expression from a sample obtained from a test subject; and (b) instructions for displaying an output relating to treating a patient for multiple myeloma with a proteasome inhibitor, when α is greater than β, a cutoff correlative with a % specificity of at least 50% and a % sensitivity of at least 50%, as determined by a receiver operating characteristic (ROC) curve.

[0016]In exemplary embodiments, the computer readable storage medium is one having stored thereon machine-readable instructions executable by a processor, comprising: (a) instructions for receiving a data value, α, relating to a test level of expression of a gene or gene product listed in Table 4 from a sample obtained from a test subject, wherein the change in gene expression level for the gene is denoted in Table 4 as “up”; and (b) instructions for displaying an output relating to treating a patient for multiple myeloma with a proteasome inhibitor, when a is greater than β, a cutoff correlative with a % specificity of at least 50% and a % sensitivity of at least 50%, as determined by a receiver operating characteristic (ROC) curve.

[0017]In exemplary embodiments, the computer readable storage medium is one having stored thereon machine-readable instructions executable by a processor, comprising: (a) instructions for receiving a data value, α, relating to a test level of expression of a gene or gene product listed in Table 4 from a sample obtained from a test subject, wherein the change in gene expression level for the gene is denoted in Table 4 as “down”; and (b) instructions for displaying an output relating to treating a patient for multiple myeloma with a proteasome inhibitor, when a is less than β, a cutoff correlative with a % specificity of at least 50% and a % sensitivity of at least 50%, as determined by a receiver operating characteristic (ROC) curve.

[0018]The invention additionally provides systems comprising: a processor; a memory device coupled to the processor, and machine readable instructions stored on the memory device. In exemplary embodiments, the machine readable instructions, when executed by the processor, cause the processor to: (i.) receive a data value, α, relating to a test level of Ig expression from a sample obtained from a test subject; and (ii) display an output relating to treating a patient for multiple myeloma with a proteasome inhibitor, when a is greater than β, a cutoff correlative with a % specificity of at least 50% and a % sensitivity of at least 50%, as determined by a receiver operating characteristic (ROC) curve.

[0019]In exemplary embodiments, the machine readable instructions, when executed by the processor, cause the processor to: (i) receive a data value, α, relating to a test level of FCGR2B expression from a sample obtained from a test subject; and (ii) display an output relating to treating a patient for multiple myeloma with a proteasome inhibitor, when a is greater than β, a cutoff correlative with a % specificity of at least 50% and a % sensitivity of at least 50%, as determined by a receiver operating characteristic (ROC) curve.

[0020]In exemplary embodiments, the machine readable instructions, when executed by the processor, cause the processor to: (i) receive a data value, α, relating to a test level of expression of a gene or gene product listed in Table 4, wherein the change in gene expression level for the gene is denoted in Table 4 as “up”, from a sample obtained from a test subject; and (ii) display an output relating to treating a patient for multiple myeloma with a proteasome inhibitor, when a is greater than β, a cutoff correlative with a % specificity of at least 50% and a % sensitivity of at least 50%, as determined by a receiver operating characteristic (ROC) curve.

[0021]In exemplary embodiments, the machine readable instructions, when executed by the processor, cause the processor to: (i) receive a data value, α, relating to a test level of expression of a gene or gene product listed in Table 4, wherein the change in gene expression level for the gene is denoted in Table 4 as “down”, from a sample obtained from a test subject; and (ii) display an output relating to treating a patient for multiple myeloma with a proteasome inhibitor, when a is less than β, a cutoff correlative with a % specificity of at least 50% and a % sensitivity of at least 50%, as determined by a receiver operating characteristic (ROC) curve.

[0022]The invention further provides methods implemented by a processor in a computer. In exemplary embodiments, the method comprises the steps of: (a) receiving a data value, α, relating to a test level of Ig expression from a sample obtained from a test subject; and (b) displaying an output relating to treating a patient for multiple myeloma with a proteasome inhibitor, when α is greater than β, a cutoff correlative with a % specificity of at least 50% and a % sensitivity of at least 50%, as determined by a receiver operating characteristic (ROC) curve. In exemplary embodiments, the method comprises the steps of: (a) receiving a data value, α, relating to a test level of FCGR2B expression from a sample obtained from a test subject; and (b) displaying an output relating to treating a patient for multiple myeloma with a proteasome inhibitor, when a is greater than β, a cutoff correlative with a % specificity of at least 50% and a % sensitivity of at least 50%, as determined by a receiver operating characteristic (ROC) curve. In exemplary embodiments, the method comprises the steps of: (a) receiving a data value, α, relating to a test level of expression of a gene or gene product listed in Table 4 from a sample obtained from a test subject, wherein the change in gene expression level for the gene is denoted in Table 4 as “up”; and (b) displaying an output relating to treating a patient for multiple myeloma with a proteasome inhibitor, when a is greater than β, a cutoff correlative with a specificity of at least 50% and a % sensitivity of at least 50%, as determined by a receiver operating characteristic (ROC) curve. In exemplary embodiments, the method comprises the steps of: (a) receiving a data value, α, relating to a test level of expression of a gene or gene product listed in Table 4 from a sample obtained from a test subject, wherein the change in gene expression level for the gene is denoted in Table 4 as “down”; and (b) displaying an output relating to treating a patient for multiple myeloma with a proteasome inhibitor, when a is less than (3, a cutoff correlative with a % specificity of at least 50% and a % sensitivity of at least 50%, as determined by a receiver operating characteristic (ROC) curve.

[0023]Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024]FIGS. 1A and 1B are graphs demonstrating the association between IG expression and bortezomib response. IE, response not evaluable; PD, progressive disease; no change, NC; minimal response, MR; partial response, PR; complete response, CR.

[0025]FIGS. 2A and 2B are graphs demonstrating the lack of association between IG expression and dexamethasone response. IE, response not evaluable; PD, progressive disease; no change, NC; minimal response, MR; partial response, PR; complete response, CR.

[0026]FIGS. 3A and 3B are graphs demonstrating the association between IGH expression and carfilzomib response. PD, progressive disease; stable disease, SD; minimal response, MR; partial response, PR; very good partial response, VGPR.

[0027]FIG. 4 is a graph demonstrating the time-to-progression (TTP) for IGH-High and IGH-Low patients treated with carfilzomib.

[0028]FIG. 5 is graph demonstrating the expression level of IG genes from multiple myeloma cell line (U266) continuously exposed to either bortezomib (BTZ) or carfilzomib (CFZ) for 24 hours.

[0029]FIG. 6 is graphical representation of the transcriptional profiling data of tumors collected from patients subsequently treated with carfilzomib. Highlighted is a cluster of genes encoding proteins of the Ig structural superfamily, which includes FCGR2B

[0030]FIGS. 7A and 7B are graphs demonstrating the association between FCGR2b expression and carfilzomib response.

[0031]FIG. 8 is a is a graph demonstrating the time-to-progression (TTP) for IGH+FCGR2B positive an IGH+FCGR2G negative patients treated with carfilzomib.

[0032]FIG. 9 is a graph containing three receiver operating characteristic (ROC) curves, each curve plotting cutoff values corresponding to a % specificity and % sensitivity: IGH, FCGR2B, and the combination of IGH and FCGR2B.

[0033]FIG. 10 is a schematic of an exemplary embodiment 101 of a system 100 for determining a therapeutic regimen for a subject with a tumor.

[0034]FIG. 11A is a graph of the immunoglobulin expression (ng IgG protein per μg total cellular protein) by cells of Line A or Line B, as measured by ELISA. SN, supernatant of cell culture; L, cell lysate.

[0035]FIG. 11B is a graph of the viability of cells of Line A and Line B upon treatment with carfilzomib (CFZ). Viability is expressed as % viable cells relative to untreated control.

[0036]FIG. 12 is a graph of the amount of FCGR2B RNA as measured by qPCR. The FCGR2B RNA amount is expressed as normalized threshold cycle (Ct).

DETAILED DESCRIPTION

[0037]Methods of Treating a Tumor

[0038]The invention provides methods of treating a tumor in a subject. In exemplary embodiments, the method comprises (a) measuring the level of expression of (i) immunoglobulin (Ig), including gene or gene product, (ii) Fc gamma receptor 2B (FCGR2B), including gene or gene product or (iii) both Ig and FCGR2B, in a sample obtained from the subject, wherein the sample comprises a cell from the tumor; and (b) administering to the subject an effective amount of a proteasome inhibitor when the level of Ig and/or FCGR2B expression in the sample is greater than a reference level.

[0039]In alternative or additional embodiments, the method comprises (a) measuring the level of expression of one or more genes or gene products listed in Table 4, in a sample obtained from the subject, wherein the sample comprises a cell from the tumor; and (b) administering to the subject an effective amount of a proteasome inhibitor, when (i) the level of expression of the one or more genes or gene products listed in Table 4 in the sample is greater than a reference level, and the change in gene expression level for the one or more genes or gene products is denoted in Table 4 as “up”, or (ii) the level of expression of the one or more genes or gene products listed in Table 4 in the sample is less than a reference level, and the change in gene expression level for the one or more genes or gene products is denoted in Table 4 as “down”, or (iii) both (i) and (ii).

[0040]The invention additional provides methods of treating a tumor in a subject, wherein the method comprises the step of administering to the subject an effective amount of a proteasome inhibitor and the subject is a subject from which a sample was obtained and the level of expression of (i) immunoglobulin (Ig), (ii) Fc gamma receptor 2B (FCGR2B), or (iii) both Ig and FCGR2B, has been measured from the sample. In exemplary aspects, the proteasome inhibitor is administered, when the level of expression is greater than a reference level.

[0041]In alternative or additional embodiments, the method of treating a tumor in a subject comprises the step of administering to the subject an effective amount of a proteasome inhibitor and the subject is a subject from which a sample was obtained and the level of expression of one or more genes or gene products listed in Table 4 has been measured from the sample. In exemplary aspects, the proteasome inhibitor is administered, when (i) the level of expression of the one or more genes listed in Table 4 in the sample is greater than a reference level, and the change in gene expression level for the one or more genes or gene products is denoted in Table 4 as “up”, or (ii) the level of expression of the one or more genes or gene products listed in Table 4 in the sample is less than a reference level, and the change in gene expression level for the one or more genes or gene products is denoted in Table 4 as “down”, or (iii) both (i) and (ii).

[0042]Methods of Determining a Treatment Regimen for a Subject with a Tumor

[0043]Also provided herein are methods of determining a treatment regimen for a subject with a tumor. In exemplary embodiments, the method comprises (a) measuring the level of expression of (i) immunoglobulin (Ig), (ii) Fc gamma receptor 2B (FCGR2B), or (iii) both Ig and FCGR2B, in a sample obtained from the subject, wherein the sample comprises a cell from the tumor, and (b) selecting a treatment regimen comprising administration of a proteasome inhibitor, when the level of Ig expression and/or FCGR2B expression in the sample is greater than a reference level. In additional or alternative embodiments, the method of determining a treatment regimen for a subject with a tumor comprises (a) measuring the level of expression of one or more genes or gene products listed in Table 4 in a sample obtained from the subject, wherein the sample comprises a cell from the tumor, and (b) selecting a treatment regimen comprising administration of a proteasome inhibitor, when (i) the level of expression of the one or more genes listed in Table 4 in the sample is greater than a reference level, and the change in gene expression level for the one or more genes or gene products is denoted in Table 4 as “up”, or (ii) the level of expression of the one or more genes or gene products listed in Table 4 in the sample is less than a reference level, and the change in gene expression level for the one or more genes or gene products is denoted in Table 4 as “down”, or (iii) both (i) and (ii).

[0044]Measurement of Expression Levels

[0045]In the methods of the invention, gene expression level(s) or gene segment expression level(s) is/are measured in a sample obtained from the subject. In exemplary aspects, the method comprises measuring the level, concentration, or amount of RNA, e.g., mRNA, encoded by the gene or gene segments in the sample. Levels of RNA, e.g., mRNA, may be measured by any technique known in the art, including but not limited to northern blotting or quantitative PCR (qPCR), including methods such as reverse transcription qPCR, real time qPCR, and end-point qPCR. Alternatively, “tag based” technologies, such as Serial analysis of gene expression (SAGE) and RNA-Seq, may be carried out to provide a relative measure of the cellular concentration of different mRNAs. Exemplary methods of measuring levels of RNA (e.g., mRNA) are also described herein at Example 5.

[0046]In alternative or additional aspects, the method comprises measuring the level, concentration, or amount of the protein product encoded by the gene or gene segments in the sample. Suitable methods of determining expression levels of protein products are known in the art and include immunoassays (e.g., Western blotting, an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), and immunohistochemical assay. See, e.g., U.S. Patent Application Publication No. 2007/0212723 A1, Shang et al., Circulation Research 101: 1146-1154 (2007); and International Patent Application Publication Nos. WO/2012/094651 and WO/2010/129964. Exemplary methods of determining expression levels of protein products are also described herein at Example 6.

[0047]In alternative or additional aspects, the level of protein product is represented by a level of the protein product's biological activity, e.g., enzymatic activity. In exemplary aspects, the protein level is reflected by the levels of the substrate or product of the enzymatic reaction catalyzed by the protein product. Methods of assaying for the level of biological activity, e.g., enzymatic activity, are known in the art.

[0048]In some aspects, the level of the protein product is represented by the level of biological activity of a related protein, e.g., a protein which acts upstream or downstream of the protein product. For example, if the protein product is a phosphorylated protein in the active state, then, in some embodiments, the level of the protein product may be represented by the activity level of the kinase which phosphorylates the protein product. In other aspects, if the protein product is a transcription factor which activates expression of a gene, then, in some embodiments, the level of the protein product may be represented by the expression levels of the gene activated by the protein product.

[0049]In exemplary aspects, the expression level that is measured, may be normalized or calibrated to a level of a housekeeping gene. The housekeeping gene in some aspects is β-actin or GAPDH. In exemplary aspects, the housekeeping gene is any one of those set forth in the table below.

[0050]In exemplary aspects, the expression level that is measured, is not normalized or calibrated to a level of a housekeeping gene. In exemplary aspects, the expression level that is measured may represent an average expression level or a mean expression level based on more than one measurement of the expression level. In exemplary aspects, the measured expression level is an average or mean of several measurements of expression levels of the same sample. In exemplary aspects, the measured expression level is an average or mean of several measurements of expression levels of different samples containing the same components obtained from the same subject. In exemplary aspects, the measured expression level is quantile normalized, as is done in RNA Seq techniques.

[0051]Immunoglobulin (Ig)

[0052]In exemplary aspects, the sample obtained from the subject is measured for the expression level of immunoglobulins. Ig molecules comprise heavy chains and light chains, each of which have a constant region and a variable region. The genes encoding the segments of an Ig molecule are located at three loci within the human genome: the Ig heavy (IGH) locus, the Ig kappa (IGK) locus, and the Ig lambda (IGL) locus. The IGH locus is located on chromosome 14 (at ch. 14q32.33) and contains gene segments encoding Ig heavy chains. The IGK locus is located on chromosome 2 (at ch. 2p11.2) and contains gene segments encoding Ig light chains. The IGL locus is located on chromosome 22 (at ch. 22q11.2) and contains gene segments for Ig light chains. Each heavy chain and light chain gene contains multiple copies of different types of gene segments for the variable regions of the Ig molecule. For example, the immunoglobulin heavy chain region contains 44 Variable (V) gene segments[Matsuda et al., J Expmtal Med 188: 2151-2162 (1998)] plus 27 Diversity (D) gene segments and 6 Joining (J) gene segments. [Li et al., Blood 103: 4602-4609 (2004)]. Likewise, the light chain region possesses numerous V and J gene segments. However, light chain genes do not have D gene segments. DNA rearrangement causes one copy of each type of gene segment to be selected in any given lymphocyte, generating an enormous antibody repertoire; roughly 3×10¹¹ combinations are possible.

[0053]With regard to the inventive methods, when the sample obtained from the subject is measured for the expression level of immunoglobulins, the method may comprise measuring the expression level of any gene segment of the IGH locus, the IGK locus, or the IGL locus. In alternative or additional aspects, the method may comprise measuring the expression level of any IGH orphon gene segment which is not located at the IGH locus, any IGK orphon gene segment which is not located at the IGK locus, or any IGL orphon gene segment which is not located at the IGL locus. In exemplary aspects, the method comprises measuring the level of expression of one or more gene segments of the IGH locus, IGK locus, or IGL locus, or any IGH orphon gene segment, IGK orphon gene segment, or IGL orhon gene segment. In exemplary aspects, the method comprises measuring the level of expression of a combination of gene segments of at least two of the IGH locus, IGK locus, and IGL locus, or an orphon gene segment thereof. In exemplary aspects, the method comprises measuring the level of expression of a combination gene segments at each of the IGH locus, IGK locus, and IGL locus or at each of the IGH and IGK loci or at each of the IGH and IGL loci, or at each of the IGK and IGL loci. In alternative or additional aspects, the method comprises measuring the level of expression of a combination of IGH orphon gene segment(s), IGK orphon gene segment(s), and IGL orphon gene segment(s) or a combination of IGH orphon gene segments and IGK orphon gene segments or a combination of IGH orphon gene segments and IGL orphon gene segments or a combination of IGK orphon gene segments and IGL orphon gene segments.

[0054]In exemplary aspects, the method comprises measuring the level of expression of one or more gene segments at the IGH locus. In exemplary aspects, the one or more gene segments is selected from the group consisting of: IGHA1, IGHA2, IGHD, IGHD1-1, IGHD1-14, IGHD1-20, IGHD1-26, IGHD1-7, IGHD2-15, IGHD2-2, IGHD2-21, IGHD2-8, IGHD3-10, IGHD3-16, IGHD3-22, IGHD3-3, IGHD3-9, IGHD4-11, IGHD4-17, IGHD4-23, IGHD4-4, IGHD5-12, IGHD5-18, IGHD5-24, IGHD5-5, IGHD6-13, IGHD6-19, IGHD6-25, IGHD6-6, IGHD7-27, IGHE, IGHEP1, IGHEP2, IGHG1, IGHG2, IGHG3, IGHG4, IGHGP, IGHJ1, IGHJ1P, IGHJ2, IGHJ2P, IGHJ3, IGHJ3P, IGHJ4, IGHJ5, IGHJ6, IGHM, IGHMBP2, IGHV1-12, IGHV1-14, IGHV1-17, IGHV1-18, IGHV1-2, IGHV1-24, IGHV1-3, IGHV1-45, IGHV1-46, IGHV1-58, IGHV1-67, IGHV1-68, IGHV1-69, IGHV1-8, IGHV1OR21-1, IGHV2-10, IGHV2-26, IGHV2-5, IGHV2-70, IGHV2OR16-5, IGHV3-11, IGHV3-13, IGHV3-15, IGHV3-16, IGHV3-19, IGHV3-20, IGHV3-21, IGHV3-22, IGHV3-23, IGHV3-25, IGHV3-29, IGHV3-30, IGHV3-30-2, IGHV3-32, IGHV3-33, IGHV3-33-2, IGHV3-35, IGHV3-36, IGHV3-37, IGHV3-38, IGHV3-41, IGHV3-42, IGHV3-43, IGHV3-47, IGHV3-48, IGHV3-49, IGHV3-50, IGHV3-52, IGHV3-53, IGHV3-54, IGHV3-57, IGHV3-6, IGHV3-60, IGHV3-62, IGHV3-63, IGHV3-64, IGHV3-65, IGHV3-66, IGHV3-7, IGHV3-71, IGHV3-72, IGHV3-73, IGHV3-74, IGHV3-75, IGHV3-76, IGHV3-79, IGHV3-9, IGHV3OR16-8, IGHV4-28, IGHV4-31, IGHV4-34, IGHV4-39, IGHV4-4, IGHV4-55, IGHV4-59, IGHV4-61, IGHV4-80, IGHV5-51, IGHV5-78, IGHV6-1, IGHV7-27, IGHV7-34-1, IGHV7-40, IGHV7-56, IGHV7-81, IGHVII-1-1, IGHVII-15-1, IGHVII-20-1, IGHVII-22-1, IGHVII-26-2, IGHVII-28-1, IGHVII-30-1, IGHVII-31-1, IGHVII-33-1, IGHVII-40-1, IGHVII-43-1, IGHVII-44-2, IGHVII-46-1, IGHVII-49-1, IGHVII-51-2, IGHVII-53-1, IGHVII-60-1, IGHVII-62-1, IGHVII-65-1, IGHVII-67-1, IGHVII-74-1, IGHVII-78-1, IGHVIII-11-1, IGHVIII-13-1, IGHVIII-16-1, IGHVIII-2-1, IGHVIII-22-2, IGHVIII-25-1, IGHVIII-26-1, IGHVIII-38-1, IGHVIII-44, IGHVIII-47-1, IGHVIII-5-1, IGHVIII-51-1, IGHVIII-5-2, IGHVIII-67-2, IGHVIII-67-3, IGHVIII-67-4, IGHVIII-76-1, IGHVIII-82, and IGHVIV-44-1. In exemplary aspects, the one or more gene segments at the IgH locus comprises a sequence selected from the group consisting of SEQ ID NOs: 1-174. In exemplary aspects, the one or more gene segments at the IGH locus is one listed in the following table.

[0055]In exemplary aspects, the method comprises measuring the level of expression of one or more gene segments at the IGK locus. In exemplary aspects, the one or more gene segments is selected from the group consisting of: IGKC, IGKJ1, IGKJ2, IGKJ3, IGKJ4, IGKJ5, IGKV1-12, IGKV1-13, IGKV1-16, IGKV1-17, IGKV1-22, IGKV1-27, IGKV1-32, IGKV1-33, IGKV1-35, IGKV1-37, IGKV1-39, IGKV1-5, IGKV1-6, IGKV1-8, IGKV1-9, IGKV1D-12, IGKV1D-13, IGKV1D-16, IGKV1D-17, IGKV1D-22, IGKV1D-27, IGKV1D-32, IGKV1D-33, IGKV1D-35, IGKV1D-37, IGKV1D-39, IGKV1D-42, IGKV1D-43, IGKV1D-8, IGKV1OR22-1, IGKV2-10, IGKV2-14, IGKV2-18, IGKV2-19, IGKV2-23, IGKV2-24, IGKV2-26, IGKV2-28, IGKV2-29, IGKV2-30, IGKV2-36, IGKV2-38, IGKV2-4, IGKV2-40, IGKV2D-10, IGKV2D-14, IGKV2D-18, IGKV2D-19, IGKV2D-23, IGKV2D-24, IGKV2D-26, IGKV2D-28, IGKV2D-29, IGKV2D-30, IGKV2D-36, IGKV2D-38, IGKV2D-40, IGKV2OR22-3, IGKV2OR22-4, IGKV3-11, IGKV3-15, IGKV3-20, IGKV3-25, IGKV3-31, IGKV3-34, IGKV3-7, IGKV3D-11, IGKV3D-15, IGKV3D-20, IGKV3D-25, IGKV3D-31, IGKV3D-34, IGKV3D-7, IGKV3OR22-2, IGKV4-1, IGKV5-2, IGKV6-21, IGKV6D-21, IGKV6D-41, IGKV7-3. In exemplary aspects, the one or more gene segments at the IgK locus comprises a sequence selected from the group consisting of SEQ ID NOs: 175-260. In exemplary aspects, the one or more gene segments at the IGK locus is one listed in the following table.

[0056]In exemplary aspects, the method comprises measuring the level of expression of one or more gene segments at the IGL locus. In exemplary aspects, the one or more gene segments is selected from the group consisting of: IGLC1, IGLC2, IGLC3, IGLC4, IGLC5, IGLC6, IGLC7, IGLCOR22-1, IGLJ1, IGLJ2, IGLJ3, IGLJ4, IGLJ5, IGLJ6, IGLJ7, IGLL1, IGLL3, IGLON5, IGLV10-54, IGLV10-67, IGLV11-55, IGLV1-36, IGLV1-40, IGLV1-41, IGLV1-44, IGLV1-47, IGLV1-50, IGLV1-51, IGLV1-62, IGLV2-11, IGLV2-14, IGLV2-18, IGLV2-23, IGLV2-28, IGLV2-33, IGLV2-34, IGLV2-5, IGLV2-8, IGLV3-1, IGLV3-10, IGLV3-12, IGLV3-13, IGLV3-15, IGLV3-16, IGLV3-17, IGLV3-19, IGLV3-2, IGLV3-21, IGLV3-22, IGLV3-24, IGLV3-25, IGLV3-26, IGLV3-27, IGLV3-29, IGLV3-30, IGLV3-31, IGLV3-32, IGLV3-4, IGLV3-6, IGLV3-7, IGLV3-9, IGLV4-3, IGLV4-60, IGLV4-69, IGLV5-37, IGLV5-45, IGLV5-48, IGLV5-52, IGLV6-57, IGLV7-35, IGLV7-43, IGLV7-46, IGLV8-61, IGLV9-49, IGLVI-20, IGLVI-38, IGLVI-42, IGLVI-56, IGLVI-63, IGLVI-68, IGLVI-70, IGLVIV-53, IGLVIV-59, IGLVIV-64, IGLVIV-65, IGLVIV-66-1, IGLVV-58, IGLVV-66, IGLVVI-22-1, IGLVVI-25-1, and IGLVVII-41-1. In exemplary aspects, the one or more gene segments at the IgL locus comprises a sequence selected from the group consisting of SEQ ID NOs: 261-350. In exemplary aspects, the one or more gene segments at the IGL locus is one listed in the following table.

[0057]In exemplary aspects, the method comprises measuring the expression of one or more IGH, IGK, or IGL orphon gene segments, and optionally, the one or more gene segments is one listed in the following table.