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• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6854—Immunoglobulins
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  • G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
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  • C12Q2600/00—Oligonucleotides characterized by their use
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  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
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  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/54—Determining the risk of relapse
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  • G01N2800/00—Detection or diagnosis of diseases
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  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/70—Mechanisms involved in disease identification
  • G01N2800/7023—(Hyper)proliferation
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  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
  • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids

Definitions

• the present invention relates to plasma cell dyscrasia detection.
• myelomas typically progress from asymptomatic precursor stages (monoclonal gammopathy of undetermined significance: MGUS) and smoldering multiple myeloma (SMM) to frank disease. Some exhibit rapid progression to MM, whilst others have lifelong indolent disease.
• MGUS monoclonal gammopathy of undetermined significance
• SMM multiple myeloma
• the heterogeneity and genomic complexity of the disease, and particularly intraclonal heterogeneity underpins the heterogeneous evolution of disease, responses to therapy as well as progression after "successful" treatment.
• Chr Chromosome
• a MYC translocations either alone, or in conjunction with Chrlq amplification identifies a poor prognostic subtype in hyperdiploid myeloma. While useful, cytogenetic approaches become problematic when two markers predicting opposing outcomes coexist in the same patient. They are also only of modest assistance in indicating appropriate therapeutic strategies and none of them provide predictive information.
• Standard blood-based biomarkers e.g., lactate dehydrogenase, albumin or ⁇ 2 microglobulin (prognostic markers) or serum free light chain (PLC) assays (for disease monitoring) while important in diagnosis and management, are affected by numerous factors including renal failure and other comorbidities or by the cytogenetic profile of an individual disease. They do not measure or encompass the biologic determinants of multiple myeloma,
• GEP70 70 genes, 30% located at the prognostic Chrl loci
• CTNI is a multigene centromere amplification-associated prognostic signature
• IF Ml 5 includes 15 genes linked to control of the ceil cycle (prognostic); HZDC (97 genes - linked to cell death) - prognostic; the PI signature (50 proliferation-associated genes) - prognostic; a signature derived from myeloma cell lines (HMCL - 248 genes - "high risk signature”);
• EMC92 is a 92-gene prognostic signature;
• CINGEC - a measure of chromosome instability (160 genes - prognostic) and a 17 gene set that may identify patients at risk of early relapse.
• the complex nature of cancer and therapeutic responsiveness comprises a series of "hallmarks", that include canonical pathways, e.g., RAS and NFKB pathway activation, as well as other features e.g., response to immunotherapy etc.
• canonical pathways e.g., RAS and NFKB pathway activation
• other features e.g., response to immunotherapy etc.
• the mutational landscape of newly diagnosed multiple myeloma is dominated by mutations in the RAS (43%) and NFKB (17%) pathways.
• RAS 43%) and NFKB (17%) pathways.
• the NETest has been developed for tumors with a neuroendocrine phenotype.
• This blood-based 51 -specific niRNA target assay does not require isolation of a specific population of target cells.
• Gene expression measurements in whole blood correlates with tissue levels and therefore provide direct information about the tumor, its pathophysiology and its state of evolution from stability to progression. This functions as a diagnostic tool and as a surrogate marker of neuroendocrine tumor behavior. Expression of all genes is prognostic; while a subset of genes, those involved in metabolism and the RAS/RAF pathway, predict the response to peptide receptor radiotherapy for this tumor type.
• a 32-gene expression tool for plasma cell dyscrasias like MGUS and myeloma can have high sensitivity and specificity (>95%) for the detection of a plasma cell dyscrasia and can differentiate minimal residual disease from progressive, active disease. In addition, it can detect patients who are no longer responding to a therapy. Patient clinical status (newly diagnosed, stable/remission or relapsed/refractory) can be predicted with an overall accuracy of >90%.
• One aspect of the present disclosure relates to a method for detecting a plasma cell dyscrasia in a subject in need thereof, comprising: (a) determining the expression level of at least 32 biomarkers from a test sample from the subject by contacting the test sample with a plurality of agents specific to detect the expression of the at least 32 biomarkers, wherein the at least 32 biomarkers comprise ASXL1, BHLHE40, BTG2, COP A, FBXW7, GNA13, IL8, JMJD1C, LARS 2, MALAT1, MBNL1, MCL1, NFKBIZ (2 splice variants), NR4A I (2 splice variants), PDE4B, P1AS2, PRKAAl (2 splice variants), SCYL2 (2 splice variants), SMARCD2, SP1 (2 splice variants), SRSF5, TAGAP, TANK, ILEA, TSC22D3, UBE2J1, and at least one housekeeping gene, (b) determining
• the method further comprises treating the subject identified as having a plasma cell dyscrasia with drug therapy.
• the first predetermined cutoff value is derived from a plurality of reference samples obtained from subjects free of a neoplastic disease.
• the reference sample can be blood, serum, plasma, or a non-neoplastic tissue.
• Another aspect of the present disclosure relates to a method for determining whether a plasma cell dyscrasia in a subject is stable or progressive, comprising: (a) determining the expression level of at least 32 biomarkers from a test sample from the subject by contacting the test sample with a plurality of agents specific to detect the expression of the at least 32 biomarkers, wherein the at least 32 biomarkers comprise -I S ⁇ 7 /.
• Another aspect of the present disclosure relates to a method for determining a risk of disease relapse in a subject having a plasma cell dyscrasia, comprising: (a) determining the expression level of at least 32 bioniarkers from a test sample from the subject after treatment by contacting the test sample with a plurality of agents specific to detect the expression of the at least 32 biomarkers, wherein the at least 32 bioniarkers comprise ASXL1, BHLHE40, BTG2, COP A, FBXW7, GNAJ3, IL8, . ⁇ /.
• Yet another aspect of the present disclosure relates to a method for determining a response by a subject having a plasma cell dyscrasia to a therapy, comprising: (a) determining a first expression level of at least 31 bioniarkers from a first test sample from the subject at a first time point by contacting the first test sample with a plurality of agents specific to detect the expression of the at least 31 biomarkers, wherein the at least 31 biomarkers comprise ASXLI, BHLHE40, BTG2, COP A, FBXW7, GNA13, IL8, JMJD1C, LARS2, MALAT1, MBNL1, MCI.
• the first time point is prior to the administration of the therapy to the subject. In some embodiments, the first time point is after the administration of the therapy to the subject. In some embodiments, the therapy comprises a targeted therapy (e.g., a proteasome inhibitor).
• a targeted therapy e.g., a proteasome inhibitor
• the plasma cell dyscrasia is MGUS or myeloma.
• the at least one housekeeping gene is selected from the group consisting of ALG9, SEPN, YWHAQ, VPS 37 A, PRRC2B, DOPEY2, NDUFB11, ND4, MRPL19, PSMC4, SF3A1, PllMl, ACTB, GAPD, GUSB, RPLP0, TFRC, MORI 41 /, 18S, PPL I. PGK1, RPL13A, B2M, YWHAl' , SDH A, HPRTl, TOX4, and TPTl.
• the method can have a specificity, sensitivity, and/or accuracy of at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments of any one of the above aspects, the method has a sensitivity of greater than 90%. In some embodiments of any one of the above aspects, the method has a specificity of greater than 90%.
• At least one of the at least 32 biomarkers is RNA, cDNA, or protein.
• the biomarker is R ' NA
• the RNA can be reverse transcribed to produce cDNA, and the produced cDNA expression level is detected.
• the expression level of the biomarker is detected by forming a complex between the biomarker and a labeled probe or primer.
• the biomarker is RNA or cDNA
• the RNA or cDNA can be detected by forming a complex between the RNA or cDNA and a labeled nucleic acid probe or primer.
• the biomarker is protein
• the protein can be detected by forming a complex between the protein and a labeled antibody.
• the label can be a fluorescent label .
• the test sample is blood, serum, plasma, or a neoplastic tissue.
• the reference sample is blood, serum, plasma, or a non-neoplastic tissue.
• the subject in need thereof is a subject diagnosed with a plasma cell dyscrasia, a subject having at least one plasma cell dyscrasia symptom, or a subject have a predisposition or familial history for developing a plasma cell dyscrasia.
• the subject is a human.
• the algorithm is XGB, RF, glmnet, cforest, CART, treebag, knn, nnet, SVM-radial, SVM-linear, NB, NNET, mlp, or logistic regression modelling.
• FIG. 1 is a graph showing interactive and functional analyses of the normalized 31 target transcripts (26 genes, 5 splice variants) markers of myeloma.
• Gene transcripts were significantly functionally linked at an interactome level consistent with a common pattern of regulatory activity.
• Two trancripts, MCL1 and COP A, were linked with Chromosome Iq, an area known to exhibit amplification in myeloma.
• the markers identified captured a series of biological processes including angiogenesis, apoptosis, immune responsiveness, phenotype definition, protein processing (secretion), proliferation, RNA processing and survival.
• a number of the transcripts encoded genes that were either potential drug targets or markers for drug target efficacy.
• FIGs. 2A-2B are graphs showing the receiver operator cuver analysis and metrics for MelanomX (normalized expression of the 26 genes and 5 splice variants) in Test Set I.
• FIG. 2B is a graph showing that the metrics for MelanomX as a diagnostic has sensitivity >95%, specificity 100%, PPV 100%, NPV 88.5%. The overall accuracy was 96%> in Test Set I.
• FIG. 3 A is a graph showing that MGUS patients / 1 8) exhibited significantly higher (39 ⁇ 9%, ? ⁇ 0.0001) scores than controls. Control levels were 12 ⁇ 8%.
• FIG. 3B is a graph showing that the AUC for differentiating MGUS from controls was 0 7 ⁇ 0.0 1 ( O.0001).
• FIGs. 4A-4B are graphs showing MyeiomX scores in different multiple myeloma subgroups.
• FIG. 4B is a graph showing that the AUC for differentiating stable from refractory disease was 0.97 ⁇ 0.03 i/ 0.0001 ).
• FIGs. 5A-5C are graphs showing MyeiomX scores in Test Set II.
• FIG. 5B is a graph showing that the AUC was 0.97 ⁇ 0.01 (/>- 0,0001 ) for differentiating between myeloma and controls.
• FIG. 5C is a graph showing that the metrics for MelanomX as a diagnostic (using 20 as a cut-off) has sensitivity 97,5%, specificity 93 ,6%, PPV 88.8%, NPV 98.6%. The overal l accuracy was 94% in Test Set II .
• FIG. 6 is a graph showing the effect of therapy on MelanomX. Therapy significantly ( 0.0001) decreased the score from 59 ⁇ 14 (pre-therapy) to 35 ⁇ I 2 in 40 patients. Ten patients al l with high scores (>40) relapsed at an early time point (within one year).
• FIGs. 8A-8B are a set of graphs showing MyeiomX score in 3 different myeloma cell lines.
• FIG. 8A identifies the cell lines demonstrate elevated expression - MyeiomX score ranging from 60 (MM-1R) to 86 (RPMI-8226).
• FIG. 8B identifies that spiking these ceils into blood from a subject that does not have a myeloma or other plasma cell dyscrasia, resulted in detectable gene expression and scores. A minimum of 1 ceil/ml of blood could be consistently identified .
• FIGs. 9A-9B are a set of graphs showing the MyelomX score in different FAC-sorted (CD 138+) and matched whole blood samples from 3 different multiple myeloma patients.
• FIG. 9A identifies the scores from FACS samples and whole bloods and identifies the scores are positive and identical.
• FIG. 9B identifies that gene expression in FACS samples compared to matched blood samples is highly concordant (correlation -0.90) consistent with the assay detecting circulating multiple myeloma cells.
• Plasma cell dyscrasias are a spectrum of progressively more severe monoclonal gammopathies in which a clone or multiple clones of pre-malignant or malignant plasma cells over-produce and secrete into the blood stream a myeloma protein, i.e., an abnormal monoclonal antibody or portion thereof.
• a plasma ceil dyscrasia can develop in different stages.
• the MGUS stage can be non-IgM MGUS, IgM MGUS, light chain MGUS, or monoclonal gammopathy of renal significance.
• the smoldering multiple myeloma (SMM) stage can be non-IgM SMM, smoldering Waldenstrom's macroglobulinemia, or light chain SMM.
• the malignant sgate can be solitary plasmacytoma, non- secretory multiple myeloma, plasma ceil myeloma with concomitant chronic lymphocytic leukemia/monoclonal B-Cell lymphocytosis, Waldenstrom's macroglobulinemia, multiple myeloma, light chain multiple myeloma, or plasma cell leukemia.
• the plasma cell dyscrasia is MGUS. In some embodiments, the plasma cell dyscrasia is myeloma.
• Symptoms can include, but are not limited to, bone pain, anemia, kidney failure, infection, and neurological symptoms (e.g., weakness, confusion, fatigue, headache, visual changes, retinopathy, radicular pain, loss of bowel, bladder control, or carpal tunnel syndrome).
• neurological symptoms e.g., weakness, confusion, fatigue, headache, visual changes, retinopathy, radicular pain, loss of bowel, bladder control, or carpal tunnel syndrome.
• myelomas can be diagnosed through a bood test or urine test.
• Myeloma cells produce M proteins and beta-2 microglobulin, which can be detected by a blood test. M proteins can also be detected by urine tests.
• Myelomas can be diagnosed through examination of the bone marrow. Specifically, a sample of bone marrow is removed, and the sample is examined for myeloma cells. Specialized tests, such as fluorescence in situ hybridization (FISH) can analyze myeloma ceils to understand their chromosome abnormalities. Tests are also done to measure the rate at which the myeloma cells are dividing. Imaging tests can also be performed to detect bone problems associated with multiple myeloma. Tests may include X-ray, MRI, CT or positron emission tomography (PET).
• FISH fluorescence in situ hybridization
• the present disclosure provides a MyelomX score that can be used for, inter alia, identifying active disease, providing an assessment of treatment responses, predicting risk of relapse, or identifying minimal residual in conjunction with standard clinical assessment and imaging.
• Measurements of circulating plasma cell dyscrasia transcripts - the MyelomX - can identify plasma cell dyscrasias, and decreases in the MyelomX score correlate with the efficacy of therapeutic interventions such as proteasome inhibitors and immunomodulators.
• Targeted gene expression profile of RNA can be isolated from the biological samples (e.g., peripheral blood) of patients with plasma cell dyscrasias. This expression profile can be evaluated in an algorithm and converted to an output (prediction).
• the present disclosure relates to a method for detecting a plasma cell dyscrasia in a subject in need thereof, comprising: (a) determining the expression level of at least 32 biomarkers from a test sample from the subject by contacting the test sample with a plurality of agents specific to detect the expression of the at least 32 biomarkers, wherein the at least 32 biomarkers comprise ,i.V. ⁇ 7.
• the provided methods are those that are able to classify or detect a plasma cell dyscrasia such as MGUS and myeloma.
• the provided methods can identify or classify a plasma cell dyscrasia in a human biological sample.
• the biological sample is a blood, serum, plasma, or a neoplastic tissue.
• the methods can provide such informati on with a specificity, sensitivity, and/or accuracy of at l east 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
• the agents can be any agents for detection of the biomarkers, and typically are isolated polynucleotides or isolated polypeptides or proteins, such as antibodies, for example, those that specifically hybridize to or bind to the at least 32 biomarkers.
• the biomarker can be RNA, cDNA, or protein. When the biomarker is RNA, the RNA can be reverse transcribed to produce cDNA (such as by RT-PCR), and the produced cDNA expression level is detected. The expression level of the biomarker can be detected by forming a complex between the biomarker and a labeled probe or primer.
• the RNA or cDNA can be detected by forming a complex between the RNA or cDNA and a labeled nucleic acid probe or primer.
• the complex between the RNA or cDNA and the labeled nucleic acid probe or primer can be a hybridization complex.
• the protein can be detected by forming a complex between the protein and a labeled antibody.
• the label can be any label, for example a fluorescent label, chemiluminescence label, radioactive label, etc.
• the protein level can be measured by methods including, but not limited to, immunoprecipitation, ELISA, Western blot analysis, or immunohistochemistry using an agent, e.g., an antibody, that specifically detects the protein encoded by the gene.
• the methods are performed by contacting the test sample with one of the provided agents, more typically with a plurality of the provided agents, for example, a set of polynucleotides that specifically bind to the at least 32 biomarkers.
• the set of polynucleotides includes DNA, RNA, cDNA, PNA, genomic DNA, or synthetic oligonucleotides.
• the methods include the step of isolating RNA from the test sample prior to detection, such as by RT-PCR, e.g., QPCR.
• detection of the biomarkers, such as expression levels thereof! includes detecting the presence, absence, or amount of RNA.
• the RNA is detected by PGR or by hybridization.
• the polynucleotides include sense and antisense primers, such as a pair of primers that is specific to each of the at least 32 biomarkers.
• the detection of the at least 32 biomarkers is carried out by PGR, typically quantitative or real-time PGR.
• detection is carried out by producing cDNA from the test sample by reverse transcription; then amplifying the cD A using the pairs of sense and antisense primers that specifically hybridize to the panel of at least 32 biomarkers, and detecting products of the amplification.
• the test sample can be any biological fluid obtained from the subject.
• the test sample is blood, serum, plasma, or a neoplastic tissue.
• the test sample is a blood sample,
• the first predetermined cutoff value can be derived from a plurality of reference samples obtained from subjects free of a neoplastic disease.
• the reference sample is blood, serum, plasma, or non-neoplastic tissue.
• the subject in need thereof can be a subject diagnosed with a plasma cell dyscrasia, a subject having at least one plasma cell dyscrasia symptom, or a subject have a predisposition or familial history for developing a plasma cell dyscrasia.
• the subject can be any mammal. Preferably, the subject is human.
• the terms "subject” and “patient” are used interchangeably herein.
• the method can further include determining a mathematically- derived expression level score of the at least 32 biomarkers in the test sample.
• This is the MyelomX score, which has a scale of 0 to 100.
• the MyelomX score is the product of a classifier built from a predictive classification algorithm, e.g., XGB, RF, glmnet, cforest, CART, treebag, knn, nnet, SVM-radial, SVM-linear, NB, mlp, or logistic regression modelling.
• the predictive classification algorithm used is XGB.
• the method can further include treating the subject identified as having a plasma cell dyscrasia with targeted therapy, biological therapy, chemotherapy, corticosteroids, stem cell transplantation, radiation therapy, or a combination thereof.
• Targeted therapy can include the use of proteasome inhibitors.
• the targeted therapy can include bortezomib and/or carfilzomib.
• Biological therapy can include immunomoduiators.
• the biological therapy can include thalidomide, lenalidomide, and/or pornalidomide.
• Chemotherapy can include any known chemotherapeutic drugs.
• Corticosteroids can be prednisone or dexamethasone.
• the present disclosure also provides a method for determining whether a plasma cell dyscrasia in a subject is stable or progressive, comprising: (a) determining the expression level of at least 32 biomarkers from a test sample from the subject by contacting the test sample with a plurality of agents specific to detect the expression of the at least 32 biomarkers, wherein the at least 32 biomarkers comprise ASXL1, BHLHE40, BTG2, COP A, FBXW7, GNA13, IL8, JMJD1C, LARS2, MALATl, MBNL1, MCL1, NFKBIZ (2 splice variants), NR4A1 (2 splice variants), PDE4B, P1AS2, PRKAA1 (2 splice variants), SCYL2 (2 splice variants), SMARCD2, SP1 (2 splice variants), SRSF5, TAGAP, TANK, TLE4, TSC22D3, UBE2J1, and at least one housekeeping gene, (b) normal
• the second predetermined cutoff value can be derived from a plurality of reference samples obtained from subjects whose plasma cell dyscrasias are demonstrating disease progression.
• the present disclosure also provides a method for determining a risk of disease relapse in a subject having a plasma ceil dyscrasia, comprising: (a) determining the expression level of at least 32 biomarkers from a test sample from the subject after treatment by contacting the test sample with a plurality of agents specific to detect the expression of the at least 32 biomarkers, wherein the at least 32 biomarkers comprise ASM.
• the third predetermined cutoff value can be derived from a plurality of reference samples obtained from subjects whose plasma cell dyscrasias are being adequately controlled by therapies.
• the present disclosure also provides a method for determining a response by a subject having a plasma ceil dyscrasia to a therapy, comprising: (a) determining a first expression level of at least 31 biomarkers from a first test sample from the subject at a first time point by contacting the first test sample with a plurality of agents specific to detect the expression of the at least 31 biomarkers, wherein the at least 31 biomarkers comprise ASXLl, BHLHE40, BTG2, COP A, FBXW7, GNA13, IL8, JMJD1C, LARS2, MALA Tl, MBNLl, MCLl, NFKBIZ (2 splice variants), NR4A1 (2 splice variants), PDE4B, P1AS2, PRKAAl (2 splice variants), SCYL2 (2 splice variants), SMARCD2, SP1 (2 splice variants), SRSF5, TAGAP, TANK, 11.1.4, TSC22D3,
• 6 second time point is after the first time point and after the administration of the therapy to the subject; (c) comparing the first expression level with the second expression level; and (d) producing a report, wherein the report identifies that the subject is responsive to the therapy when the second expression level is significantly decreased as compared to the first expression level.
• the methods can predict treatment responsiveness to, or determine whether a patient has become clinically stable following, or is responsive or non- responsive to, a plasma cell dyscrasia treatment, such as a drug therapy (for example, an immunotherapy or targeted therapy).
• a plasma cell dyscrasia treatment such as a drug therapy (for example, an immunotherapy or targeted therapy).
• the methods can do so with a specificity, sensitivity, and/or accuracy of at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
• the first and second test samples can be of the same type. In some embodiments, the first and second test samples can be of different types,
• the therapy can be a drug therapy.
• the drug therapy can be an immunotherapy, a targeted therapy, a chemotherapy, or a combination thereof.
• the therapy can be a radiation therapy.
• the first time point is prior to the administration of the therapy to the subject. In some embodiments, the first time point is after the administration of the therapy to the subject.
• the second time point can be a few days, a few weeks, or a few months after the first time point. For example, the second time point can be at least 1 day, at least 7 days, at least 14 days, at least 30 days, at least 60 days, or at least 90 days after the first time point.
• the second expression level is significantly decreased as compared to the first expression level when the second expression level is at least 10% less than the first expression level. In some embodiments, the second expression level is significantly decreased as compared to the first expression level when the second expression level is at least 20% less than the first expression level. In some embodiments, the second expression level is significantly decreased as compared to the first expression level when the second expression level is at least 25% less than the first expression level. In some embodiments, the second expression level is significantly decreased as compared to the first expression level when the second expression level is at least 30% less than the first expression level. In some embodiments, the second expression level is significantly decreased as compared to the first expression level when the second expression level is at least 40% less than the first expression level.
• the second expression level is significantly decreased as compared to the first expression level when the second expression level is at least 50% less than the first expression level. In some embodiments, the second expression level is significantly decreased as compared to the first expression level when the second expression level is at least 60% less than the first expression level. In some embodiments, the second expression level is significantly decreased as compared to the first expression level when the second expression level is at least 70% less than the first expression level. In some embodiments, the second expression level is significantly decreased as compared to the first expression level when the second expression level is at least 80% less than the first expression level. In some embodiments, the second expression level is significantly decreased as compared to the first expression level when the second expression level is at least 90% less than the first expression level.
• the method further comprises determining a third expression level of the at least 32 biomarkers from a third test sample from the subject at a third time point by contacting the third test sample with a plurality of agents specific to detect the expression of the at least 32 biomarkers, wherein the third time point is after the second time point.
• the method can further comprise creating a plot showing the trend of the expression level change.
• the present disclosure also provides an assay comprising: (a) determining the expression level of biomarkers consisting essentially of the following 32 biomarkers from a test sample from a patient diagnosed of a plasma cell dyscrasia or a subject suspected of having a plasma cell dyscrasia: ASXL1, BHLHE40, BTG2, COP A, FBXW7, (ISA 13, IL8, JMJDIC, LARS2, MALATl, MBNL1, MCL1, NFKBIZ (2 splice variants), NR4A1 (2 splice variants), PDE4B, PJAS2, PRKAA1 (2 splice variants), SCYL2 (2 splice variants), SMARCD2, SP1 (2 splice variants), SRSF5, TAGAP, TANK, TLE4, TSC22D3, UBE2J1, and at least one housekeeping gene; (b) normalizing the expression level of each of ASXL1, BHLHE40, BTG2, COP A.
• the present disclosure also provides an assay comprising: (a) determining the expression level of biomarkers consisting of the following 32 biomarkers from a test sample from patient diagnosed of a plasma ceil dyscrasia or a subject suspected of having a plasma cell dyscrasia: ASXLl, BHLHE40, BTG2, COP A, FBXW7, GNA13, 1L8, JMJDIC, LARS2, MALATl, MBNIJ, MCI , NFKBIZ (2 splice variants), NR4A 1 (2 splice variants), PDE4B, P1AS2, PRKAAI (2 splice variants), SCYL2 (2 splice variants), SMARCD2, SP1 (2 splice variants), SRSF5, TAGAP, TANK, TLE4, TSC22D3, UBE2J1, and at least one housekeeping gene; (b) normalizing the expression level of each oi ⁇ i V./.
• the at least one housekeeping gene is selected from the group consisting of ALG9, SEPN, ) H I I AO. VPS37A, PRRC2B, DOPEY2, NDlJFBll, ND4, MRPL19, PSMC4, SF3A1, PUMl, AC IB, GAPD, GUSB, RPLPO, TFRC, MORF4IJ, 18S, PPIA, PGK1, RPIJ3A, B2M, YWHAZ, SDHA, HPRT1, TOX4, and TPTI.
• the housekeeping gene is TPTI.
• two or more housekeeping genes can be used in normalizing the expression levels.
• the method can comprise: (1 ) normalizing the expression level of each of ,i.V. ⁇ /. /. BUI.111: 0, BTG2, COP A, FBXW7, GNA 13, IL8, JMJDIC, IARS2, MALA TI, MBNLI, MCLI, NFKBIZ (2 splice variants), NR4A1 (2 splice variants), PDE4B, P1AS2, PRKAA1 (2 splice variants), SCYL2 (2 splice variants), SM ARC D2.
• SP1 (2 splice variants), SRSF5, TAGAP, TANK, TLE4, TSC22D3, and UBE2JI to the expression level of a first housekeeping gene; (2) normalizing the expression level of each of ASXL1, BHLHE40, BTG2, COP A, FBXW7, GNA13, IL8, JMJDIC, LARS2, MALATI, MBNLI, MCLI, NFKBIZ (2 splice variants), NR4A1 (2 splice variants), PDE4B, P1AS2, PRKAA l (2 splice variants), SCYL2 (2 splice variants), SMARCD2, SP1 (2 splice variants), SRSF5, TAGAP, TANK, TLE4, TSC22D3, and UBE2JI to the expression level of a second housekeeping gene; and (3) averaging the first normalized expression level and the second normalized expression level to obtain an averaged normalized expression level.
• CTGGGCCCAC ACCCACACCA GCCTCCTTTC TGCCTGCCCT TCTACCTGAT CCCACCTTCA
• CTCTAGCT TCATGAACCC AGACAAGATC TCGGCTCCCT TGCTCATGCC CCAGAGACTC CCTTCTCCCT TGCCAGCTCA TCCGTCCGTC GACTCTTCTG TCTTGCTCCA AGCTCTGAAG CCAATCCCCC CTTTAA A CTT AGAAACCAAA GACTAAACTC TCTAGGGGAT CCTGCTGCTT TGCTTTCCTT CCTCGCTACT TCCTAAAAAG
• GAGGCACTCA CAGAGCACTA CAAACACCAC TGG ' i ' i ' iCCCG AAAAGCCGTC CAAGGGCTCC GGCTACCGCT GC ATTCGC A T CAACCACAAG ATGGACCCCA TCATCAGCAG GGTGGCCAGC CAGATCGGAC TCAGCCAGCC CCAGCTGCAC CAGCTGCTGC CCAGCGAGCT GACCCTGTGG GTGGACCCCT ATGAGGTGTC CTACCGCATT GGGGAGGACG GCTCCATCTG CGTCTTGTAC GAGGAGGCCC CACTGGCCGC CTCCTGTGGG CTCCTCACCT GCAAGAACCA AGTGCTGCTG GGCCGGAGCA GCCCCTCCAA GAACTACGTG ATGGCAGTCT CCAGGC CCTTCCGCCC CCCCTGGG CGCCGCCGTGACAACA GGCCACCACA TACCTCAACC TGGGGAACTG TATTTTTAAA TGAAGAGCTA TTTTAAA TGAAGAGCTA TTT
• CTAA A CTGCT GTGAAAATGA TAGAAAGCAA GTAGCTCCCT TATTCTGTTT TTGATTGCAG CCTTTTATCT TTTGCTAATT ATAGCAATAT TTATTGAGCA CCTGCCATGT GACTGTCACT GTTCTAGATA 1 1 1 1 ACATGT AATATACAGA TAAAAGAATA
• AAGTGTTCCC ACTAGAACTG ACCTAAGCCA CTGATTAATA TTTAATGAGG GAAGGTAGGG GAGAATCTAG CCATTTTATA ATGCCAGAAA TCTATATATG TTATCTGATG CCATTTTTCT GAAGTAGCCT CACATGTGGT CCCCCTGCAG TTCAGCAGTT AACAGATGAC TTTTTTAGTG TAATAAAATG TTTATCATCT ATG
• CTGAAAGAAG CAAAGTTCAG TTTCAGCAAC A A AC A A ACT l TGTTTGGGAA GCTATGGAGG AGGACTTTTA GATTTAGTGA AGATGGTAGG GTGGAAAGAC TTAATTTCCT TGTTGAGAAC AGGAAAGTGG CCAGTAGCCA GGCAAGTCAT AGAATTGATT ACCCGCCGAA TTCATTAATT TACTGTAGTG TTAAGAGAAG CACTAAGAAT GCCAGTGACC TGTGTAAAAG TTACAAGTAA TAGAACTATG ACTGTAAGCC TCAGTACTGT AC A AGGGAAG CTTTTCCTCT CTCTAATTAG CTTTCCCAGT ATACTTCTTA GAAAGTCCAA GTGTTCAGGA CTTTTATACC TGTTATACTT TGGCTTGGTT TCCATGATTC TTACTTTATT AGCCTAGTTT ATCACCAATA ATACTTGACG GAAGGCTCAG TAATTAGTTA TGAATATGGA TATCCTCAAT TCTTA
• GCCCATCTCA GAGCCATAAG GTCATCTTTG CTAGAGCTAT TTTTACCTAT GTATTTATCG TTCTTGATCA TAAGCCGCTT ATTTATATCA TGTATCTCTA AGGACCTAAA AGCACTTTAT GTAGTTTTTA ATTAATCTTA AGATCTGGTT ACGGTAACTA AAAAAGCCTG TCTGCCAAAT CCAGTGGAAA CAAGTGCATA GATGTGAATT GGTTTTTAGG GGCCCCACTT CCCAATTCAT TAGGTATGAC TGTGGAAATA CAGACAAGGA TCTTAGTTGA TATTTTGGGC TTGGGGCAGT GAGGGCTTAG GACACCCCAA GTGGTTTGGG AAAGGAGGAG GGGAGTGGTG GGTTTATAGG GGGAGGAGGA GGCAGGTGGT CTAAGTGCTG ACTGGCTACG TAGTTCGGGC AAATCCTCCA AAAGGGAAAG GGAGGATTTG CTTAGAAGGA TGGCTCCC AGTGACTACT TTTTGACT
• CAGGAAGATC TCGAGGGCCC TGGCTGAACT TCACCTTTTG GCTTTCTTGG CCTGATGCTG AACTCTCGAG GTTGAGCCCC ATATGGGGGT TGGCAGGCAG CAGAGAGGCC CCTTTCAAGG TGTTCGGGTA AAGAACTCAG TGAAGGAACT CCTGTTGCAC ATCCGAAGTC ATAAACAGAA GGCTTCTGGC CAAGCTGTGG ATGA ' l 1 1 ⁇ GACACAAGGT GTGAACATAG AACAGTTCAG
• CTTTTTCCCC CTCCCTTACT CTTCGTCCCC GGTCCCTCCC 84.1 CTCCCCACCC CTTTCCTTCT AGCTCCGACG TTTGCGGCCG
• CAGGAGCGGA ATCTGTCCCG AACCGGGTCT GTGAGGAACT CGCGAACTTG GATTAGGAAA TCCCGGAGCC CGGATCGACA AATCCCGGAA CCCGGAATTA AGATCGCCA A GTCCCGGATC GCGGAGCACA GAGCACGGAG TGGACTCGAC GCGGAGCCCG GAGTCCGGAT CGCGGCACCG CGGGACGGGA CGGAGCGATG TCGGGCCGAG GCGCGGGCGG GTTCCCGCTG CCCGCTAA
• CTAGGTCTCG TAGCCGATCC CGTTCCCGTA GTCGCAAATC TTACAGCCGG TCAAGAAGCAGGAGCAGGAG CCGGAGCCGG AGCAAGTCCC GTTCTGTTAG TAGGTCTCCC GTGCCTGAGA AGAGC C AGAA ACGTGGTTCT TCAAGTAGAT CTAAGTCTCC AGCATCTGTG GATCGCCAGA GGTCCCGGTC CCGATCAAGG TCCAGATCAG TTGACAGTGG CAATTAAACT GTAAATAACT TGCCCTGGGG GCCTTTTTTT AAAAAACAAA AACCACAAAA ATTCCCAAAC CATACTTGCT AAAAATTCTG GTAAGTATGT GCTTTTCTGT GGGGGTGGGA TTTGGAAGGG GGGTTGGGTT GGGCTGGATA TCTTTGTAGA TGTGGAC C AC CAAGGGGTTG TTGAAAACTA ATTGTATTAA ATGTCTI ' TIG ATAAGCCTTC
• CTCCCCGA GCGCCGCTCC GGCTGCACCG CGCTCGCTCC GAGTTTCAGG CTCGTGCTAA GCTAGCGCCG TCGTCGTCTC CCTTCAGTCG CCATCATGAT TATCTACCGG GACCTCATCA
• GGAAGTCTAA TTAAAATGCA CTCAAGAGAC TAACAGTCGC AGGCATGAAA TACAATACAG GTACATGGTT TTTTATTATG TGTGCATCTG CTTCAGTAAT AGGTGTGAAT TACTCATTTG GATCATTAGG AGTTTCAAAA TCTAGTTAAA TGACTAGATT TITGTTGATG TAAATTCTGT CATTCTGAAC TGCAGGGATT GTCAGTAACT TAACTGCAAA CTAAACTGGT GATA A TTATG GTAAAATTGC AAGACGAGCA ATAAATCTCA ACCAACTTGA GAGAACACTG ATAA
• VTS37A 011451 AGTGACGGCG GCGCGGGTGG TGGAGCGCTG GGCGGCCAGG
• TTCTTCCTCA ATTTATGTAA TGAAAATAAA ATTAATATAT CATCTAACAG TAGCACAAAA TTTGTAATAT GAAGTAAAGT ATG A AG A TAA TGAAGAAGTT GTTTTCTTTG TTGAAGCAGT TATATGGGTC TTTCTCAGTA TATTTCTCTT TTCTCTAAAA GTTTAAACTT ATTAAAAGAA TGTTATTTTT AACCTTTCAA AAAAAAAAAAA
• TCAGCCAGGG ACTTCCGTTG TCGTCAGCGG AAGCGGTGAC AGATCATCCC AGGCCACACA GAGGCCGGCT TGGTCACTAT GGAGGAGATA GGCATCTTGG TGGAGAAGGC TCAGGATGAG ATCCCAGCAC TGTCCGTGTC CCGGCCCCAG ACCGGCCTGT CCTTCCTGGG CCCTGAGCCT GAGGACCTGG AGGACCTGTA CAGCCGCTAC AAGGAGGAGG TGAAGCGAAT CCAAAGCATC CCGCTGGTCA TCGGACAATT TCTGGAGGCT GTGGATCAGA ATACAGCCAT CGTGGGCTCT ACCACAGGCT CCAACTATTA TGTGCGCATC CTGAGCACCA TCGATCGGGA GCTGCTCAAG
• CTTTCAGTTC TACCCACTAC TTAAGTACTT GTCATGTACT CTTAGAGGAG GCCAGTAATG ATCAGAACCA TTTTACTTTA AAATTAATAA TATTGTATTA GAGAATATAT TAAATGGTTA TATTGGGTTA TGTTAGGATA TATACTTGAA TGGAAATACA TGTACTATTA GCAATCATAT TTCATTTATC CCTGTAATTA GACAAGAAAG CATAATATAG CTCTACTCAT GGGTACACAT ACCAGTGTAT AAGATTTTTA GAAGTTTACT TTTTAAAAAT AAAAGCAAAA TGTAAGATCT TAAAAAAAAA AAAAAAAAAAAAA
• GGGAGTGGGT GGAGGCAGCC AGGGCTTACC TGTACACTGA CTTGAGACCA GTTGAATAAA AGTGCACACC TTAAAAATGA GGAAAAAAAA AAAAAAAAAA
• GACAAAACCT CCTCCTTTTC CAAGCGGCTG CCGAAGATGG 3 CGGAGGTGCA GGTCCTGGTG CTTGATGGTC GAGGCCATCT
• ATAAGTTAAC TTCCAATTTA CATACTCTGC TTAGAATTTG GGGGAAAATT TAGAAATATA ATTGACAGGA TTATTGGAAA TTTGTTATAA TGAATGAAAC ATTTTGTCAT ATAAGATTCA TATTTACTTC TTATACATTT GATAAAGTAA GGCATGGTTG TGGTTAATCT GGTTTATTTT TGTTCCACAA GTTAAATAAA TCATAAAACT TGATGTGTTA TCTCTTA
• GTATCCCCCC TCCCCCGCCA GCTCGACCCC GGTGTGGTGC 3 GCAGGCGCAG TCTGCGCAGG
• GACTGGCGGG ACTGCGCGGC GGCAACAGCA GACATGTCGG GGGTCCGGGG CCTGTCGCGG
• ATCTTTGTCA GCAGTTCCCT TTTAAATGCA AATCAATAAA TTCCCAAAAA TTTAAAAAAA AAAAAAAAAA AAAAA
• nucleic acid molecule As used herein, the terms “polynucleotide” and “nucleic acid molecule” are used interchangeably to mean a polymeric form of nucleotides of at least 10 bases or base pairs in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide, and is meant to include single and double stranded forms of DNA.
• a nucleic acid molecule or nucleic acid sequence that serves as a probe in a microarray analysis preferably comprises a chain of nucleotides, more preferably DNA and/or RNA.
• nucleic acid molecule or nucleic acid sequence comprises other kinds of nucleic acid structures such as for instance a DNA/RNA helix, peptide nucleic acid (PNA), locked nucleic acid (LNA) and/or a ribozyme.
• PNA peptide nucleic acid
• LNA locked nucleic acid
• nucleic acid molecule also encompasses a chain comprising non-natural nucleotides, modified nucleotides and/or non-nucleotide building blocks which exhibit the same function as natural nucleotides.
• hybridize As used herein, the terms “hybridize,” “hybridizing”, “hybridizes,” and the like, used in the context of polynucleotides, are meant to refer to conventional hybridization conditions, such as hybridization in 50% formamide/6XSSC/0.1 % SDS/100 ⁇ / ⁇ ! ssDNA, in which temperatures for hybridization are above 37 degrees centigrade and temperatures for washing in 0.1 XSSC/0.1% SDS are above 55 degrees C, and preferably to stringent hybridization conditions.
• normalization refers to the expression of a differential value in terms of a standard value to adjust for effects which arise from technical variation due to sample handling, sample preparation, and measurement methods rather than biological variation of biomarker concentration in a sample.
• the absolute value for the expression of the protein can be expressed in terms of an absolute value for the expression of a standard protein that is substantially constant in expression.
• normalizing the expression level of a gene to the expression level of a housekeeping gene means dividing the expression level of the gene by the expression level of the housekeeping gene.
• diagnosis also encompass the terms “prognosis” and “prognostics”, respectively, as well as the applications of such procedures over two or more time points to monitor the diagnosis and/or prognosis over time, and statistical modeling based thereupon.
• diagnosis includes: a. prediction (determining if a patient will likely develop aggressive disease (hyperproliferative/invasive)), b. prognosis (predicting whether a patient will likely have a better or worse outcome at a pre-selected time in the future), c. therapy selection, d. therapeutic drug monitoring, and e. relapse monitoring.
• providing refers to directly or indirectly obtaining the biological sample from a subject.
• providing may refer to the act of directly obtaining the biological sample from a subject (e.g., by a blood draw, tissue biopsy, lavage and the like).
• providing may refer to the act of indirectly obtaining the biological sample.
• providing may refer to the act of a laboratory receiving the sample from the party that directly obtained the sample, or to the act of obtaining the sample from an archive.
• “Accuracy” refers to the degree of conformity of a measured or calculated quantity (a test reported value) to its actual (or true) value.
• Clinical accuracy relates to the proportion of true outcomes (true positives (TP) or true negatives (TN) versus mi sclassified outcomes (false positives (FP) or false negatives (FN)), and may be stated as a sensitivity, specificity, positive predictive values (PPV) or negative predictive values (NPV), or as a likelihood, odds ratio, among other measures.
• biological sample refers to any sample of biological origin potentially containing one or more biomarkers.
• biological samples include tissue, organs, or bodily fluids such as whole blood, plasma, serum, tissue, lavage or any other specimen used for detection of disease,
• subject refers to a mammal, preferably a human.
• Treating” or “treatment” as used herein with regard to a condition may refer to preventing the condition, slowing the onset or rate of development of the condition, reducing the risk of developing the condition, preventing or delaying the development of symptoms associated with the condition, reducing or ending symptoms associated with the condition, generating a complete or partial regression of the condition, or some combination thereof.
• Biomarker levels may change due to treatment of the disease.
• the changes in biomarker levels may be measured by the present disclosure. Changes in biomarker levels may be used to monitor the progression of disease or therapy,
• a change may be an increase or decrease by 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100%*, or more, or any value in between 0% and 100%.
• the change may be 1-fold, 1.5- fold, 2-fold, 3-fold, 4-fold, 5-fold or more, or any values in between 1-fold and five-fold.
• the change may be statistically significant with a p value of 0.1, 0.05, 0.001, or 0.0001.
• stable disease refers to a diagnosis for the presence of a plasma cell dyscrasia, however the myeloma has been treated and remains in a stable condition, i.e. one that that is not progressive, as determined by imaging data and/or best clinical judgment.
• progressive disease refers to a diagnosis for the presence of a highly active state of a plasma cell dyscrasia, i.e. one has not been treated and is not stable or has been treated and has not responded to therapy, or has been treated and active disease remains, as determined by imaging data and/or best clinical judgment.
• the top performing algorithm (XGB - "gradient boosting") best predicted the training data.
• XGB produced probability scores that predicted the sample.
• Each probability score reflects the "certainty" of an algorithm that an unknown sample belongs to either "Stable Disease” or "Progressive” class.
• This sample would be considered a myeloma sample that exhibited progressive disease, given a score of 0.8. if the sample came from a patient with MRD or who was under treatment, the score would identify either they are exhibiting progressive disease (will relapse) or are failing the therapy.
• MyelomX scores >0.2 are considered indicative the sample is from a myeloma patient.
• the receiver operator cuver analysis and metrics are included in Figure 2B.
• the score exhibited an area under the curve (AUC) of 0.99.
• the metrics are: sensitivity >95%, specificity 100%, PPV 100%, NPV 88.5%.
• the overall accuracy is 96%.
• the tool can therefore differentiate between controls and aggressive and stable myeloma disease.
• test set 2 The test was evaluated in a second test set (test set 2) that included 155 healthy controls and 81 myeloma patients, the majority of whom exhibited stable disease including those with MRD.
• the mean MyelomX score in this myeloma group was 47 ⁇ 14 versus 12 ⁇ 8 in the control group ( Figure 5A).
• the receiver operator cuver analysis demonstrated the score exhibited an area under the curve (AUC) of 0.97 ( Figure SB) and the metrics were 89-99% (Figure 5C).
• EXAMPLE 5 Use of MyelomX score to diagnose myeloma, demonstrate MRD and define therapy response
• a confusion matrix identifying the accuracy of the MyelomX score in both data sets is included in Table 4.
• the score is 97% accurate for identifying active disease.
• MRD it is overall 75% accurate, but is 100% accurate for those who do not recur within one year.
• the score is 87% accurate for identifying responders and 97% for those who are failing therapy or are refractor ⁇ ' .
• Table 4 Confusion matrix showing classification accuracy of the XGB-model algorithm and score cut-offs for defining the dynamic state of myeloma disease in blood samples
• myeloma was the source for the blood-based gene expression assay by evaluating expression in different myeloma cell lines and in FAC-sorted multiple myeloma tumors form patients.
• centrosome index is a powerful prognostic marker in myeloma and identifies a cohort of patients that might benefit from aurora kinase inhibition. Blood. 2008; i l l : 1603-1609. Epub 2007 Nov 1615.
• Hose D, Reme T, Hielscher T, et al. Proliferation is a central independent prognostic factor and target for personalized and risk-adapted treatment in multiple myeloma.

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