Method for cancer prognosis

Abstract

The present invention relates to methods of prognosing and monitoring cancer using circulating cells and/or extracellular vesicles as indicators of the progression of plasma cell neoplasms in patients.

Claims

1. A method for the prognosis of a patient with a plasma cell neoplasm or suspected of having a plasma cell neoplasm, comprising isolating a sample comprising extracellular vesicles, or cells and extracellular vesicles, from said patient and determining the level of extracellular vesicles, or cells and extracellular vesicles, that are CD138.sup.− and P-glycoprotein.sup.+ (CD138.sup.−/P-gp.sup.+), wherein the determining step comprises using an immunoaffinity method, wherein antibodies are directed towards a cell surface and/or microparticle-associated proteins; and wherein the immunoaffinity methods comprise using affinity pull-downs, Western blot analysis, dot blot analysis, radio-immune assays, flow cytometry, fluorescence activated cell sorting (FACS) analysis, magnetic beads, ELISA, immunofluorescence and/or affinity chromatography.

2. The method according to claim 1, wherein the plasma cell neoplasm is myeloma or multiple myeloma.

3. The method according to claim 1, wherein the sample is a blood-derived sample, a plasma sample, or a platelet-free plasma sample.

4. The method according to claim 1, wherein the extracellular vesicles, or the cells and extracellular vesicles, are also CD41a.sup.−.

5. The method according to claim 1, wherein: when the sample comprises extracellular vesicles, or cells and extracellular vesicles, that are CD138.sup.−/P-gp.sup.+ at a level that is higher than a reference control indicative of a subject that does not have a plasma cell neoplasm or has a remissive plasma cell neoplasm, a prognosis that the neoplasm is stable, progressive or refractory is determined; when the sample comprises extracellular vesicles, or cells and extracellular vesicles, that are CD138.sup.−/P-gp.sup.+ at a level that is higher than a reference control indicative of a subject with a stable plasma cell neoplasm, a prognosis that the neoplasm is progressive or refractory is determined; or when the sample comprises extracellular vesicles, or cells and extracellular vesicles, that are CD138.sup.−/P-gp.sup.+ at a level that is higher than a reference control indicative of a subject with progressive plasma cell neoplasm, a prognosis that the neoplasm is refractory is determined.

6. The method according to claim 1, wherein the extracellular vesicles, or the cells and extracellular vesicles, are also CD34.sup.+.

7. The method according to claim 6, wherein: when the sample comprises extracellular vesicles, or cells and extracellular vesicles, that are CD138.sup.−/P-gp.sup.+/CD34.sup.+ at a level that is higher than a reference control indicative of a subject that does not have a plasma cell neoplasm or has a remissive plasma cell neoplasm, a prognosis that the neoplasm is stable, progressive or refractory is determined; when the sample comprises extracellular vesicles, or cells and extracellular vesicles, that are CD138.sup.−/P-gp.sup.+/CD34.sup.+ at a level that is higher than a reference control indicative of a subject with a stable plasma cell neoplasm, a prognosis that the neoplasm is progressive or refractory is determined; or when the sample comprises extracellular vesicles, or cells and extracellular vesicles, that are CD138.sup.−/P-gp.sup.+/CD34.sup.+ at a level that is higher than a reference control indicative of a subject with a progressive plasma cell neoplasm, a prognosis that the neoplasm is refractory is determined.

8. The method according to claim 1, wherein the extracellular vesicles, or the cells and extracellular vesicles, are also phosphatidylserine.sup.+ (PS.sup.+).

9. The method according to claim 8, wherein: when the sample comprises extracellular vesicles, or cells and extracellular vesicles that are CD138.sup.−/P-gp.sup.+/CD34.sup.+/PS.sup.+ at a level that is higher than a reference control indicative of a subject that does not have a plasma cell neoplasm or a remissive plasma cell neoplasm, a prognosis that the neoplasm is stable, progressive or refractory is determined; when the sample comprises extracellular vesicles, or cells and extracellular vesicles, that are CD138.sup.−/P-gp.sup.+/CD34.sup.+/PS.sup.+ at a level that is higher than a reference control indicative of a subject with a stable plasma cell neoplasm, a prognosis that the neoplasm is progressive or refractory is determined; or when the sample comprises extracellular vesicles, or cells and extracellular vesicles, that are CD138.sup.−/P-gp.sup.+/CD34.sup.+/PS.sup.+ at a level that is higher than a reference control indicative of a subject with a progressive plasma cell neoplasm, a prognosis that the neoplasm is refractory is determined.

10. A method for monitoring the progression of a patient with a plasma cell neoplasm or suspected of having a plasma cell neoplasm, comprising isolating a sample comprising extracellular vesicles, or cells and extracellular vesicles, from said patient at at least two time points and determining the change in the level of extracellular vesicles, or cells and extracellular vesicles, that are CD138.sup.−/P-gp.sup.+, wherein the determining step comprises using an immunoaffinity method, wherein antibodies are directed towards a cell surface and/or microparticle-associated proteins; and wherein the immunoaffinity methods comprise using affinity pull-downs, Western blot analysis, dot blot analysis, radio-immune assays, flow cytometry, fluorescence activated cell sorting (FACS) analysis, magnetic beads, ELISA, immunofluorescence and/or affinity chromatography.

11. The method according to claim 10, wherein: when the level of extracellular vesicles, or cells and/or and extracellular vesicles, that are CD138.sup.−/P-gp.sup.+ increase between the at least two time points, a prognosis that the severity of the neoplasm is increasing is determined; or when the level of extracellular vesicles, or cells and extracellular vesicles, that are CD138.sup.−/P-gp.sup.+ decrease between subsequent time points, a prognosis that the severity of the neoplasm is decreasing is determined.

12. The method according to claim 4, wherein the plasma cell neoplasm is myeloma or multiple myeloma.

13. The method according to claim 4, wherein the sample is selected from the group consisting of a blood-derived sample, a plasma sample, a platelet-free plasma sample.

14. The method according to claim 10, wherein the extracellular vesicles, or the cells and extracellular vesicles, are also CD41a.sup.−.

15. The method according to claim 4, wherein the extracellular vesicles, or the cells and extracellular vesicles, are also CD34.sup.+.

16. The method according to claim 4, wherein the extracellular vesicles, or the cells and extracellular vesicles, are also phosphatidylserine.sup.+ (PS.sup.+).

17. The method according to claim 1, wherein the sample comprises cells that: are produced in the bone marrow; are hematopoietic stem cells; originated from hematopoietic stem cells; are myeloid progenitor cells; originated from myeloid progenitor cells; are lymphoid progenitor cells; originated from lymphoid progenitor cells; originated from B lymphocyte cells; are plasma-like cells; are neoplastic cells; or are multiple myeloma cells.

18. The method according to claim 1, wherein the sample comprises extracellular vesicles that are derived from: cells produced in the bone marrow; cells that are hematopoietic stem cells; cells that originated from hematopoietic stem cells; cells that are myeloid progenitor cells; cells that originated from myeloid progenitor cells; cells that are lymphoid progenitor cells; cells that originated from lymphoid progenitor cells; cells that originated from B lymphocyte cells; plasma-like cells; neoplastic cells; or multiple myeloma cells.

19. The method according to claim 1, wherein the sample comprises extracellular vesicles that are exosomes, microparticles, oncosomes, large oncosomes or migrasomes.

20. The method according to claim 4, wherein the sample comprises extracellular vesicles that are derived from: cells produced in the bone marrow; cells that are hematopoietic stem cells; cells that originated from hematopoietic stem cells; cells that are myeloid progenitor cells; cells that originated from myeloid progenitor cells; cells that are lymphoid progenitor cells; cells that originated from lymphoid progenitor cells; cells that originated from B lymphocyte cells; plasma-like cells; neoplastic cells; or multiple myeloma cells.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings as follows.

(2) FIG. 1: P-gp.sup.+ microparticle increases in multiple myeloma. The P-gp.sup.+ microparticle counts in the total microparticle (CD41a.sup.−) population in multiple myeloma patients and healthy subjects were determined using TruCount™ beads (A) P-gp.sup.+ microparticle counts were significantly greater in the multiple myeloma patients (n=69) relative to healthy volunteers (n=25), p<0.01 (**). (B) P-gp.sup.+ microparticle counts were greater in patients in de novo (n=14) and progressive disease (PD, n=17) relative to healthy volunteers, (p<0.01 (**)). There was no significant difference in the P-gp.sup.+ microparticle count of healthy volunteers compared to patients in partial remission (PR, n=32) and complete remission (CR, n=13). P values were generated using Mann-Whitney U test and the data is represented as mean.

(3) FIG. 2: CD138.sup.+ microparticles do not significantly express P-gp. (A) The CD133.sup.+ P-gp.sup.+ microparticle count was elevated in multiple myeloma relative to healthy volunteers however was not significant in a CD133.sup.+ microparticle population. (B) Consequently, CD133.sup.+ P-gp.sup.+ microparticle count across de novo (n=14), PR (n=32), CR (n=13) and PD were elevated though not significant. (C) The CD138.sup.− P-gp.sup.+ microparticle count was significantly elevated in multiple myeloma patients relative to healthy volunteers (n=25) (p<0.01 (**)). (D) CD138.sup.− P-gp counts were significantly higher in de novo cohort (P=0.0002 (***)) and PD (p<0.05 (*)) however was not significant for CR, PR. P values were generated using Mann-Whitney U test and the data is represented as mean.

(4) FIG. 3: P-gp.sup.+ microparticles in a 56-year-old patient with aggressive disease during the course of treatment (patient 1) microparticles were isolated from the PFP of a 56-year-old multiple myeloma patient at diagnosis and during the course of treatment. The absolute P-gp.sup.+ microparticle counts (Y-axis) and time of microparticle sampling post diagnosis (X-axis) are shown. (A) (CyBorD, black dot; BorD, pink dot; VTD, blue dot lenalidomide/dexamethasone, red dot and D-PACE and melphalan, green dot) (B) CD41a.sup.− CD133.sup.+ microparticle profile of the patient 1 (C) Corresponding CD41a.sup.− CD133.sup.+ P-gp.sup.+ and (D) CD41a.sup.− CD133.sup.+ P-gp.sup.+ microparticle profiles of patient 1.

(5) FIG. 4: Isotype-matched control to define parameters for .sup.+/.sup.− staining patient 1 in partial remission and ‘dual positive’ population in May 2015. CD138.sup.+/.sup.− population was gated based on anti CD138.sup.−APC.sup.+/.sup.− staining for patient 1 in PR. Isotype-matched control was used to define gating parameters for positive and negative staining for CD138.sup.− P-gp.sup.+ CD34.sup.+ (4A, left panel) and CD138.sup.+/P-gp.sup.+/CD34.sup.+ (4A, right panel) for patient 1 in PR (B) Gates were defined for CD138.sup.− CD34.sup.+ annexin V.sup.+ (4B, left panel) and CD138.sup.+/CD34.sup.+ annexin V.sup.+ (4B, right panel) for patient 1 in PR. (C) microparticles were phenotyped patient 1 in partial remission status for the presence of ‘dual positive’ population based on CD138−/.sup.+ staining. We assessed the presence of P-gp, CD34 in CD138.sup.− (red events) and CD138.sup.+ (blue events) microparticle sub-sets by flow cytometry (4C, left and right panel respectively). (D) microparticles were also phenotyped for the levels of PS enrichment using annexin V in the in CD138.sup.− (4D, left panel, yellow events) and CD138.sup.+ subpopulation of microparticles (4D, right panel, orange events).

(6) FIG. 5: Elevated levels of ‘dual positive’ microparticles in patient 1 with aggressive disease. The presence of P-gp and CD34 in CD138.sup.− (red events) and CD138.sup.+ (blue events) microparticle subpopulations was established by flow cytometry in patient 1. (A) A sequential gating strategy using microparticle size gate (left panel) followed by gating for CD41a (middle panel) and CD138 (right panel) was applied to the total microparticle population (left panel). The CD41a.sup.− population was defined based on .sup.+/− staining for anti-CD41a.sup.− PE (middle panel). (B) The total population (CD41a.sup.−) was gated based on CD138−/.sup.+ staining (left panel, red events, right panel, blue events, respectively). Within this microparticle population, we phenotyped for CD138.sup.−/P-gp.sup.+/CD34.sup.+ (left panel, gate P1) population and CD138.sup.+ P-gp.sup.+ CD34.sup.+ sub-population (right panel, gate P4). (C) The CD138 microparticle subtypes (gate P1 & P4 of left and right panel respectively). The CD138 microparticle subtypes (gate P1 & P4 of left and right panel respectively) were gated and phenotyped for the presence PS exposure using annexin V (left panel, gate P11, yellow events) (right panel, gate P12, orange events) respectively.

(7) FIG. 6: Isotype-matched control to define parameters for .sup.+/− staining in patient 2 (PD) and 3 (stable). (A) CD138.sup.+/− population was gated based on anti-CD138.sup.−/APC .sup.+/− staining for patient 2 and 3. Isotype-matched control was used to define gating parameters for positive and negative staining for CD138.sup.−/P-gp.sup.+/CD34.sup.+ (2A, left panel) and CD138.sup.+/P-gp.sup.+/CD34.sup.+ (2A, right panel) for patient 2 (B) Gates were defined for CD138.sup.−/CD34.sup.+/annexin V.sup.+ (2B, left panel) and CD138.sup.+/CD34.sup.+/annexin V.sup.+ (2B, right panel) for patient 2 (C) Isotype-matched control was used to define gating parameters for positive and negative staining for CD138.sup.−/P-gp.sup.+/CD34.sup.+ (2C, left panel) and CD138.sup.+/P-gp.sup.+/CD34.sup.+ (2C, right panel) for patient 3 (D) Gates were defined for CD138.sup.−/CD34.sup.+/annexin V.sup.+ (2D, left panel) and CD138.sup.+/CD34.sup.+ annexin V.sup.+ (2D, right panel) for patient 3.

(8) FIG. 7: 66-year-old female patient in progressive disease (patient 2) and 63-year-old male patient (patient 3) in stable condition. The presence of P-gp and CD34 in CD138.sup.− (red events) and CD138.sup.+ (blue events) microparticle subpopulations was established by flow cytometry in patient 2 and 3. (A) The total population (CD41a.sup.−) was gated based on CD138−/.sup.+ staining (left panel, red events, right panel, blue events, respectively). Within this microparticle population, we phenotyped for CD138.sup.−/P-gp.sup.+/CD34.sup.+ (left panel, gate P1) population and CD138.sup.+/P-gp.sup.+/CD34.sup.+ sub-population (right panel, gate P4). (B) The CD138 microparticle subtypes (gate P1 & P4 of left and right panel respectively) were gated and phenotyped for the presence PS exposure using annexin V (left panel, gate P11, yellow events) (right panel, gate P12, orange events) respectively.

(9) FIG. 8: Isotype-matched control to define parameters for .sup.+/− staining in patient 4 (PR) and 5 (remission). (A) CD138.sup.+/− population was gated based on anti CD138− APC.sup.+/− staining for patient 4 and 5. Isotype-matched control was used to define gating parameters for positive and negative staining for CD138.sup.−/P-gp.sup.+/CD34.sup.+ (3A, left panel) and CD138.sup.+/P-gp.sup.+/CD34.sup.+ (3A, right panel) for patient 4 (B) Gates were defined for CD138.sup.−/CD34.sup.+/annexin V.sup.+ (3B, left panel) and CD138.sup.+/CD34.sup.+/annexin V.sup.+ (3B, right panel) for patient 4 (C) Isotype-matched control was used to define gating parameters for positive and negative staining for CD138.sup.−/P-gp.sup.+/CD34.sup.+ (3C, left panel) and CD138.sup.+/P-gp.sup.+/CD34.sup.+ (3C, right panel) for patient 5 (D) Gates were defined for CD138.sup.−/CD34.sup.+/annexin V.sup.+ (3D, left panel) and CD138.sup.+/CD34.sup.+/annexin V.sup.+ (3D, right panel) for patient 5.

(10) FIG. 9: 71-year-old male patient on partial remission (patient 4) and 62-year-old male patient in remission—long-term survivor (patient 5). The presence of P-gp and CD34 in CD138.sup.− (red events) and CD138.sup.+ (blue events) microparticle subpopulations was established by flow cytometry in patient 4 and 5. (A) The total population (CD41a.sup.−) was gated based on CD138−/.sup.+ staining (left panel, red events, right panel, blue events, respectively). Within this microparticle population, we phenotyped for CD138.sup.−/P-gp.sup.+/CD34.sup.+ (left panel, gate P1) population and CD138.sup.+/P-gp.sup.+/CD34.sup.+ sub-population (right panel, gate P4). (B) The CD138 microparticle subtypes (gate P1 & P4 of left and right panel respectively) were gated and phenotyped for the presence PS exposure using annexin V (left panel, gate P11, yellow events) (right panel, gate P12, orange events) respectively.

(11) FIG. 10: PS.sup.+ microparticle represents a more aggressive state in multiple myeloma. The annexin V.sup.+ microparticle counts in multiple myeloma patients and healthy subjects were compared using Trucount™ beads. (A) PS.sup.+ microparticle counts were significantly greater in multiple myeloma patients (n=74) relative to healthy volunteers (n=25) p<0.01 (**). (B) PS.sup.+ microparticle counts were greater in de novo (n=14), partial remission (n=31), and progressive disease (PD, n=18) relative to healthy volunteers (n=25). No significant difference in annexin V.sup.+ microparticle counts was observed between the CR (n=15) and healthy volunteers. P values were generated using Mann-Whitney U test and the data is represented as mean (P<0.01 (**), P<0.05 (*)).

(12) FIG. 11: PS.sup.+ and CD138 do not co-express in progressive disease. (A) PS microparticles in the CD133.sup.+ microparticle sub-set in multiple myeloma patients were significantly elevated in multiple myeloma patients compared to the healthy volunteers (p<0.01, (**)). (B) CD138.sup.+/PS.sup.+ microparticle levels in the de novo and PR cohort were significantly higher relative to healthy volunteers (p<0.01, (**)) while CR and PD had insignificant levels relative to healthy volunteers. (C) PS.sup.+ microparticles in the CD138.sup.− microparticle sub-set were significantly elevated in multiple myeloma patients relative to that for healthy volunteers. (D) CD138.sup.−/PS.sup.+ microparticles were significantly higher in de novo and PD cohorts relative to healthy volunteers. There was no significant difference in CR and PR cohorts relative to healthy volunteers. Mann-Whitney U test was conducted to generate P values and the data is represented as mean (P<0.01 (**)).

(13) FIG. 12: Table showing microparticle subtypes across different clinical states.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

(14) Preferred embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings.

Example 1

(15) Reagents & Antibodies

(16) Annexin V-V450 (BD Horizon™), anti-CD138-APC (clone M115), anti-CD41a-PE (clone HIP8), anti-P-gp-FITC (clone 17F9), anti-CD34-PE-Cy7 (clone 8G12 Y7), matched isotype controls BD™ CompBead anti-mouse-Ig k, Sphero™ Rainbow calibration particles and TruCount™ tubes were from BD Biosciences Australia. Latex beads of diameter 0.3 (cat. no. LB3) & 1.1 (cat. no. LB11) μm were purchased from Sigma-Aldrich, Australia.

(17) Study Design and Patients

(18) This study was approved by the Sydney Local Health District (SLHD)-Human Research Ethics Committee (HREC) of Concord Repatriation General Hospital (CRGH) [(HREC/11/CRGH/223-CH62/6/2011-150], Royal Prince Alfred Hospital (RPAH) HREC (SSA/12/RPAH/10) and the University of Technology Sydney (2012-004R). Blood samples were collected from multiple myeloma patients and healthy volunteers (>18 years of age) after informed consent at the CRGH and RPAH blood collection centres in accordance with the Declaration of Helsinki. The subjects were de-identified and assigned a code for accessing clinical information. Healthy volunteers were age-matched, non-cancer patients with normal hematology and devoid of any cytotoxic treatment or radiotherapy of any nature in the past 5 years. Pregnancy was also an exclusion criterion. In total, 25 normal subjects and 74 multiple myeloma subjects were assessed, which included treatment responsive (n=32, n=15 for partial remission and complete remission respectively), de novo (n=14) and relapsed (n=18) multiple myeloma patients. Patient responses were determined according to IMWG guidelines.

(19) Isolation and Flow Cytometric Detection of Microparticles.

(20) Platelet free plasma (PFP) was prepared as described previously. PFP was divided into 200 μl aliquots, which were subjected to direct immunolabeling or microparticle isolation by ultracentrifugation at 18,890×g, 4° C. for 30 min. The supernatant was removed and the microparticle pellet was immunolabeled for flow cytometry in technical triplicates for each patient microparticle count. Latex beads of 0.3 and 1.1 μm diameters were prepared and used according to the manufacturer's recommendation to define the microparticle gate and was applied to all samples during analysis (26). Flow cytometric analyses were conducted using LSRII flow cytometer/LSR Fortessa X20 and the CellQuest Pro, FACSDiva analysis software (BD Biosciences).

(21) Surface Phenotyping of Systemic Microparticles and Quantitation.

(22) Cell surface antibodies directed against CD138, CD41a, CD34, P-gp and phosphatidylserine were added to the microparticle pellet as previously described. Relevant isotype-matched and unstained controls were run in parallel. Platelet derived microparticles were excluded during the analysis using anti-CD41a-PE. microparticles were re-suspended in 500 μL PBS and quantitated using BD TruCount™ beads as previously described.

(23) Statistical Analysis

(24) Mann-Whitney (U) test was conducted for the non-parametric data using GraphPad Prism® version 7.0 for Mac (GraphPad, La Jolla, Calif., USA). The data presented as the mean and Mann-Whitney constant U. The results with a predictive value of (***) P<0002, (**) P<0.01 and (*) P<0.05 were considered significant.

(25) Results

(26) P-gp.sup.+ Microparticle Numbers are Elevated in De-Novo and Progressive Disease Multiple Myeloma Patients.

(27) It was observed that significantly greater numbers of P-gp.sup.+ microparticles were present in the total (CD41a.sup.−) microparticle population in multiple myeloma patients relative to healthy volunteers (FIG. 1A). The absolute number of P-gp.sup.+ microparticles from multiple myeloma patients was 5.1 fold greater number per μl relative to the healthy volunteers (U=605, p<0.01). Specifically, it was observed that there was a 5.67 (U=70, p<0.01) and 12.4 (U=104, p<0.01) fold increase in P-gp.sup.+ microparticles for de novo and progressive disease (PD), respectively, relative to the healthy volunteers (FIG. 1B). There was no significant difference between P-gp.sup.+ microparticles between healthy volunteers and patients in complete (CR) or partial remission (PR) (FIG. 1B).

(28) CD138.sup.+ Microparticles do not Express Significant Levels of P-gp on their Surface.

(29) P-gp expression on the CD133.sup.+ microparticle population in multiple myeloma patients was not significantly increased compared with the healthy volunteers (U=716, p=0.28) (FIG. 2A). There was no significant increase observed across de novo, CR, PR and PD subpopulations (FIG. 2B) respectively relative to healthy volunteers.

(30) In contrast, P-gp expression on the CD138.sup.− microparticles showed a significant 4.5 fold increase relative to healthy volunteers (U=553, p=0.009, FIG. 2C). The P-gp.sup.+/CD138.sup.− microparticle numbers were 3.6 fold higher in the de novo cohort (U=57, p=0.0003) and 12.2 fold in PD relative to the healthy volunteers (U=126, p=0.04). The absolute numbers were not significantly different in the CR (U=151, p=0.19) or PR (U=195, p=0.14) cohorts compared to the healthy volunteers. (FIG. 2D).

Example 2

(31) Five individual patients across all disease states were selected and their microparticles phoenotyped for the presence P-gp, CD34 and CD138. CD34 is a transmembrane protein belonging to the CD34 family of sialomucins and is an established haematopoietic stem cell marker. Although, not typically used in phenotyping plasma cells, CD34 is present on a minor subpopulation of multiple myeloma stem cell clones. The selected panel of individual patients included (a) aggressive disease, patient 1 (b) progressive disease, patient 2 (c) stable disease, patient 3 (d) partial remission, patient 4 and (e) a long-term survivor in remission, patient 5. We also phenotyped microparticles for the extent of phosphatidylserine (PS) using annexin V. PS is expressed preferentially on the surface of microparticles of cancer cells of ‘stem cell like’ origin. PS on microparticles has been recently shown to be required for interactions with vascular endothelial cells in neovascularisation and is associated with cancer progression.

(32) Case 1: 58-Year-Old Female Patient with Aggressive Disease.

(33) FIG. 3A-D demonstrates the serial P-gp.sup.+ microparticle count of a 58-year-old female patient (patient 1) who was diagnosed with IgG multiple myeloma in September 2013 with 86% plasmacytosis in the bone marrow aspirate. At diagnosis, the P-gp.sup.+ microparticle count was minimal. Induction therapy with cyclophosphamide, bortezomib, dexamethasone (CyBorD) commenced in September 2013 (FIG. 3 black dot) but in November 2013 cyclophosphamide was withdrawn due to severe anaemia (FIG. 3 pink dot). A bone marrow biopsy in December 2013 showed partial response with 46% plasmacytosis. During this time, the number of P-gp.sup.+ microparticle was increasing steadily which was consistent with the emergence of MDR. Thalidomide was added from January-April 2014 (FIG. 3, blue dot). A follow up biopsy showed reduced plasmacytosis of 23% in April 2014 (˜days 70-80). The paraprotein increased to 38.3 g/l (progressive disease) in June-July 2014 and the treatment regimen changed to lenalidomide/dexamethasone from July-October 2014 (˜100 days) (FIG. 3 red dot). Patient 1 relapsed with a right side posterior mass along the chest wall in early February 2015 (60% plasmacytosis, around 130 days) while the M-protein level was only 18 g/l at that point in time (data not shown). At this time, P-gp.sup.+ microparticles in PFP continued to significantly increase. Dexamethasone along with platinol, adriamycin, cyclophosphamide and etoposide (D-PACE) and melphalan added to treatment regimen at this point (FIG. 3 green dot) and patient 1 achieved partial remission (˜day 495). She had a successful autologous stem cell transplant in July 2015. However, she relapsed soon and became unreponsive to all therapy in November 2015 and passed away in December. FIG. 3B shows the profile of P-gp.sup.+/CD138.sup.+ microparticles in the same patient. Consistent with our previous findings, the levels of these microparticles correspond to disease burden and treatment outcome (FIG. 3B). In the context of counts, the CD138.sup.+ microparticle subtype was not the predominant P-gp.sup.+ population (FIG. 3C). Rather the predominant Pgp.sup.+ microparticle subtype was CD138.sup.− (FIG. 3D). Upon examining the microparticle profiles for annexin V.sup.+ sub populations in patient 1, a significant difference in the levels between the CD138.sup.+ and CD138.sup.− subtypes was not observed (data not shown).

(34) A blood sample was taken from patient 1 on 11 Feb. 2015 during progressive disease and prior to stem cell transplantation. This sample showed elevated numbers of CD34.sup.+ (496.81/μl) and P-gp.sup.+ (155.29/μl) total CD41a.sup.− microparticle events (Table 1) compared to that observed when the patient was in partial remission in May 2015 (7.33/μl and 6.31/μl for CD34.sup.+ and P-gp.sup.+, respectively, FIG. 4). Gating parameters were established to detect CD41a.sup.− events in the context of CD138 and are shown in FIG. 5A. The levels of CD34.sup.+ and P-gp.sup.+ microparticle events within CD138.sup.+ (red) and CD138.sup.− (blue) microparticle subtypes (FIGS. 4B and C) were compared. The predominant population which was P-gp.sup.+ and CD34.sup.+ was the CD138.sup.− microparticle subtype (referred to as the ‘dual positive’ population for simplicity) FIG. 5B, left panel, gate P1, 12.48/μl). Very little P-gp.sup.+/CD34.sup.+/CD138.sup.+ microparticles (FIG. 5B, right panel, gate P4, 0.30/μl) was detected. Additional microparticle sub-sets which were CD138.sup.−/P-gp.sup.+/CD34.sup.− (FIG. 4B, left panel, gate P3, 56.45/μl) and CD138.sup.+/P-gp.sup.−/CD34.sup.+ microparticle (FIG. 5B, right panel, gate P2, 28.5/μl) were also detected microparticles within the CD138.sup.+ population that were solely CD34.sup.+ and P-gp.sup.+ were not detected (FIG. 5B, right panel, gate P5 and P6).

(35) The CD138 microparticle subtypes were gated and phenotyped for the presence PS exposure using annexin V. The presence of annexin V.sup.+ microparticles (FIG. 5C, left panel, gate P11, 5/μl) within the CD138.sup.+/P-gp.sup.+/CD34.sup.− microparticle population was detected. In contrast, annexin V positive events on CD138.sup.+/P-gp.sup.+/CD34.sup.+ microparticles were not detected (FIG. 5C, right panel, gate P12, 0 events) (Table 1).

(36) In summary, this patient with an aggressive disease course demonstrated significantly elevated levels of P-gp on microparticles of ‘stem cell like’ origin (i.e. CD138.sup.−/P-gp.sup.+/CD34.sup.+). A small proportion of this population also was positive for PS.

(37) Case 2: 66-Year-Old Female Patient in Progressive Disease

(38) A 66-year-old female patient (patient 2) was diagnosed with kappa light chain myeloma in 2014. She was enrolled and treated in a clinical trial MLN9708 (cyclophosphamide/dexamethasone) from December 2014 until March 2015, which was stopped in February 2015 due to progressive disease with a rise in kappa light chains. At the time of sampling on 5 May 2015, she was on CyBorD therapy for her progressive disease. During this time it was observed that CD34.sup.+ (40.5/μl) and P-gp.sup.+ microparticle events (60/μl) within the total (CD41a.sup.−) microparticles (Isotype-matched control; FIG. 6) were present. Within this population, the presence of a CD138.sup.−/P-gp.sup.+/CD34.sup.+ population (FIG. 7A, left panel, gate P1, 4.6/μl) and a CD138.sup.+/P-gp.sup.+/CD34.sup.+ population was detected (FIG. 7A, right panel, gate P4, 0.5/μl). A sub-set of CD138.sup.−/P-gp.sup.+/CD34.sup.− microparticles (FIG. 7A, left panel, gate P3, 58.8/μl) and CD138.sup.+/P-gp.sup.−/CD34.sup.+ was also detected (FIG. 7A, right panel, gate P6, 3/μl).

(39) The CD138 microparticle subtypes were gated and phenotyped for the presence PS exposure using annexin V. A minimal presence of CD138.sup.−/P-gp.sup.+/CD34.sup.+ annexin V.sup.+ microparticles (FIG. 7B, left panel, gate P11, 1.1/μl) in this patient at this given point in time was detected. CD138.sup.+/P-gp.sup.+/CD34.sup.+ annexin V.sup.+ microparticle levels were also minimal (FIG. 7B, right panel, gate P12, 0.4/μl) (Table 1).

(40) In summary, compared to patient 1, this patient with progressive disease demonstrated lower levels of the ‘dual positive’ population and showed only minimal positivity with PS.

(41) Case 3: 63-Year Male Patient in Stable Condition

(42) A 63-year-old male in a stable disease state (patient 3) at the time of sampling was diagnosed with IgG kappa multiple myeloma 2011 (smoldering myeloma 2008, active myeloma July 2011). The induction therapy consisted of 6 cycles of cyclophosphamide, thalidomide and dexamethasone followed by autologous stem cell transplant on 30 Mar. 2012. The patient experienced severe peripheral neuropathy associated with thalidomide and an increase in serum paraprotein, which resulted in a treatment change to lenalidomide, and dexamethasone July 2012. At the time of sampling in May 2015, the patient was on lenalidomide and dexamethasone, zometa and aspirin.

(43) The patient presented with CD34.sup.+ (5.13/μl) and P-gp.sup.+ (6.3/μl) microparticles in total microparticles (CD41a.sup.−) at the time of sampling (Isotype-matched control; FIG. 6). Within this population, the presence of CD138.sup.−/P-gp.sup.+/CD34.sup.+ population (FIG. 7C, left panel, gate P1, 4.7/μl) and CD138.sup.+/P-gp.sup.+/CD34.sup.+ (FIG. 7C, right panel, gate P4, 0.2/μl) was detected. A sub-set of CD138.sup.−/P-gp.sup.+/CD34.sup.− (FIG. 7C, left panel, gate P3 23.13/μl) and CD138.sup.−/P-gp.sup.−/CD34.sup.+ (FIG. 7C, left panel, gate P2, 18.54/μl) was also found. A sub-set of CD138.sup.+/P-gp.sup.+/CD34.sup.+ (FIG. 7C, right panel, gate P5, 1.2/μl) CD138.sup.+/P-gp.sup.+/CD34.sup.− (FIG. 7C, right panel, gate P6, 1/μl) was also observed.

(44) The CD138 microparticle subtypes were gated and phenotyped for the presence PS exposure using annexin V. The presence of CD138.sup.−/P-gp.sup.+/CD34.sup.+ annexin V.sup.+ microparticles (FIG. 7D, left panel, gate P11, 1.6/μl) in this patient at this given point in time and CD138.sup.+/P-gp.sup.+/CD34.sup.+ annexin V.sup.+ microparticle (FIG. 7D, right panel, gate P12, 0.3/μl events) (Table 1) was observed.

(45) In summary, the ‘dual positive’ population was present in comparable levels in patient 3 relative to patient 2. The sub-set was also not significantly enriched with PS.

(46) Case 4: 71 Year Old Male Patient in Partial Remission

(47) A 71-year-old male (patient 4) was diagnosed on February 2014 following a biopsy of a right shoulder mass. He presented with widely disseminated skeletal disease with multiple lesions as evidenced by positron emission tomography scan. Induction therapy consisted of CyBorD treatment from April 2014. The patient achieved very good partial remission after 6 cycles and treatment was stopped at 6 cycles instead of 8 due to severe peripheral neuropathy resulting from bortezomib. The sample analyzed was taken on 12 Aug. 2014. It was observed that numbers of CD34.sup.+ (15.13/μl) and P-gp.sup.+ microparticle events (10/μl) within the total (CD41a.sup.−) microparticle population (Isotype-matched control; FIG. 8). Within this population, the presence of CD138.sup.−/P-gp.sup.+/CD34.sup.+ population (FIG. 9A, left panel, gate P1, 7.2/μl) and CD138.sup.+/P-gp.sup.+/CD34.sup.+ (FIG. 9A, right panel, gate P4, 0.5/μl) was detected, and a sub-set of CD138.sup.−/P-gp.sup.+/CD34.sup.− (FIG. 9A, right panel, gate P3, 36.53/μl) and CD138.sup.−/P-gp.sup.−/CD34.sup.+ (FIG. 9A, left panel, gate P2, 63.17/μl) was found in this sample. A very small sub-set of CD138.sup.+/P-gp.sup.−/CD34.sup.+ (FIG. 9A, right panel, gate P5, 4/μl) and CD138.sup.+/P-gp.sup.+/CD34.sup.− (FIG. 9A, right panel, gate P6, 2.2/μl) was also observed.

(48) The CD138 microparticle subtypes were gated and phenotyped for the presence PS exposure using annexin V. The presence of CD138.sup.−/P-gp.sup.+/CD34.sup.+ annexin V.sup.+ microparticles (FIG. 9B, left panel, gate P11, 2.5/μl) was detected and CD138.sup.+/P-gp.sup.+/CD34.sup.+ annexin V.sup.+ microparticle was negative (FIG. 9B, right panel, gate P12, 0 events) (Table 1).

(49) In summary, patient 4 demonstrated elevated albeit lower levels of the ‘dual positive’ population compared to that of the patient 1. The sub-set was enriched with PS however levels were lower than that detected for patient 1.

(50) Case 5: 62 Year Old Male Patient in Remission—Long-Term Survivor

(51) A 62-year-old male (patient 5) was diagnosed at 50 years of age with IgG kappa multiple myeloma with bone marrow biopsy showing 10-15% plasma cell infiltration. His induction regimen consisted of VAD (vincristine, adriamycin (doxorubicin) and dexamethasone). This was followed by an autologous stem cell transplant in 2007, after which he remained in an unmaintained complete remission for almost three years. He experienced a relapse in 2012 with rise in serum paraprotein albeit he had no other issues. He was given thalidomide and achieved very good partial response in early 2013 with bone marrow biopsy showing only 3% plasma cell infiltration and M-protein too low to quantitate. His M protein started to increase in late 2014 and reached 17 g/l in October 2014. The patient was subsequently enrolled and treated on a clinical trial (lenalidomide/dexamethasone plus or minus daratumumab) in December 2014. At the time of sampling the patient was responding very well and he eventually achieved stringent complete remission with ongoing chemotherapy. This patient is a long-term survivor (12 years) with successful therapeutic interventions over a long period.

(52) At the time of sampling on 5 May 2015, it was observed that numbers of CD34.sup.+ (5.13/μl) and P-gp.sup.+ microparticle events (6.3/μl) within the total (CD41a.sup.−) microparticle population (Isotype-matched control; FIG. 8). The presence of CD138.sup.−/P-gp.sup.+/CD34.sup.+ population (FIG. 9C, left panel, gate P1, 2.54/μl) and CD138.sup.+/P-gp.sup.+/CD34.sup.+ (FIG. 9C, right panel gate P4, 3.0/μl) was detected. We also found a sub set of CD138.sup.−/P-gp.sup.+/CD34.sup.− (FIG. 9C, left panel, gate P3, 52.83/μl) and CD138.sup.+/P-gp.sup.−/CD34.sup.+ (FIG. 9C, left panel, gate P2, 14.46/μl). A very small sub-set of CD138.sup.+/P-gp.sup.−/CD34.sup.+ (FIG. 9C, right panel, gate P5, 4.5/μl) and CD138.sup.+/P-gp.sup.+/CD34.sup.− (FIG. 9C, right panel, gate P6, 2.4/μl) was also observed.

(53) The CD138 microparticle subtypes were gated and phenotyped for the presence PS exposure using Annexin V. The presence of CD138.sup.−/P-gp.sup.+/CD34.sup.+ annexin V.sup.+ microparticles (FIG. 9D, left panel, gate P11, 0.5/μl) and CD138.sup.+/P-gp.sup.+/CD34.sup.+ annexin V.sup.+ microparticle (FIG. 9D, right panel, gate P12, 1.17/μl) (Table 1) was detected.

(54) In summary, this patient demonstrated significantly reduced levels of the ‘dual positive’ population compared to all other disease states. There were barely detectable PS exposure on this population of microparticles.

(55) Overall, it was observed that elevated levels of the CD138.sup.−/P-gp.sup.+/CD34.sup.+ microparticle sub-set in multiple myeloma patients with more severe forms of the disease.

(56) PS.sup.+ Microparticle Represents a More Aggressive State in Multiple Myeloma

(57) PS.sup.+ microparticle subpopulations within the total (CD41a.sup.−) microparticle population were significantly (5.6 fold) increased in multiple myeloma patients relative to the healthy volunteers (U=607, p=0.009) (FIG. 10 A). PS V.sup.+ microparticle counts were 3.1 fold elevated in the de novo cohort relative to healthy volunteers (U=80, p=004) while the partial remission cohort showed a 3.08 fold increase in annexin V.sup.+ microparticle counts (U=249, p=0.02) to that of healthy volunteers. The annexin V.sup.+ microparticle counts were 13.9 fold higher in the progressive disease cohort relative to healthy volunteers (U=136, p=0.02). We did not observe any significant difference in annexin V.sup.+ microparticle counts between the CR cohort and healthy volunteers (FIG. 10B).

(58) PS and CD138 do not Co-Express in Progressive Disease.

(59) FIG. 11A shows the CD138.sup.+ annexin V.sup.+ microparticle profile of multiple myeloma patients with respect to healthy volunteers. It was observed that a significant 2.4 fold greater PS exposure in CD138.sup.+ microparticle sub-set for multiple myeloma patients compared to healthy volunteers (U=452.5, p=0.006). In the cohort data, the de novo and PR cohorts showed a 3.1 (U=82.5, p=0.005) and 3.61 (U=96.5, p=002) fold increase, respectively in PS exposure to that of healthy volunteers whilst it was observed that no significant difference with CR or PD cohorts relative to healthy volunteers (FIG. 11B).

(60) FIG. 11C shows the CD138.sup.− annexin V.sup.+ microparticle profile of multiple myeloma patients. It was observed that a significant 4.3 fold increase in PS exposure in multiple myeloma patients compared to healthy volunteers in the CD138.sup.− sub-set of microparticles (U=570, P=0.009). In the cohort data, the de novo patients had a 7.73 fold higher PS exposure (U=77, p=0.003) on CD138.sup.− microparticles compared to healthy volunteers while patients with PD had a 6.9 fold higher PS exposure (U=94, p=0.003) in the CD138.sup.− sub-set (FIG. 11D) relative to healthy volunteers. A significant difference in PS exposure for the CR and PR cohorts with respect to healthy volunteers was not observed (FIG. 11D).

DISCUSSION

(61) The results show that multiple myeloma patients have higher P-gp.sup.+ events in the total CD41a.sup.− and CD138.sup.− microparticle population compared to healthy volunteers, specifically in the de novo and PD cohorts. P-gp.sup.+ events within the total microparticle population as well as within each microparticle subtype were shown to correspond to treatment response when levels were monitored in individual patients.

(62) While CD138 is a useful surrogate marker for plasma cells, the results showed that P-gp.sup.+ microparticle events in multiple myeloma patients were predominantly within the CD138.sup.− population. It was observed that significantly greater P-gp.sup.+ microparticle events in patients with progressive disease. Within the total CD41a.sup.− microparticle population we identified a number of different subtypes based on the presence of CD138 and P-gp.

(63) Patient 2 (PD) and patient 3 (stable) had almost identical ‘dual positive population’. Patient 3 was in a stable condition while patient 2 was already showing response after one cycle of bortezomib (as defined by a drop in light chain levels) at the time of sampling and correspondingly had less ‘dual population’ relative to patient's 1 and 4. Patient 5 had a barely detectable ‘dual positive’ microparticle population corresponding to the remission and long-term survivor status. This data clearly demonstrates an association between elevated levels of the ‘dual positive’ microparticle population and treatment unresponsiveness as well disease activity.

(64) PS is a ubiquitous marker of microparticles arising from loss of phospholipid asymmetry during microparticle biogenesis. It was observed that significantly elevated numbers of PS.sup.+ microparticles were in the total CD41a.sup.− microparticle population in multiple myeloma patients relative to healthy volunteers. Significantly higher PS.sup.+ events in the CD138.sup.− microparticle sub-set in multiple myeloma patients (specifically, in de novo and PD) were also observed.

(65) The results show that the presence of a ‘dual positive’ microparticle population (CD138.sup.+/CD34.sup.+/P-gp.sup.+) provides a marker of disease progression and treatment responsiveness within individual patients, specifically in aggressive disease. It appears that CD138.sup.+ cannot be considered a ‘static’ biomarker of multiple myeloma disease evolution. Whereas it plays an important role as a measure of tumor burden in responsive disease, its presence diminishes in an aggressive disease state.

Example 3

(66) Samples were collected over a range of time for four additional patients across all disease states and their microparticles phoenotyped retrospectively for the presence P-gp, CD34 and CD138 according to the methods described in Example 1. The patients had the following clinical profiles (as determined by conventional blood tests):

(67) MM34, Female

(68) The patient had progressive, refractory multiple myeloma, and progressed through cyclophosphamide, velcade and dexamethasone, before passing away on 4th April 2014.

(69) MM19, Male

(70) The patient was diagnosed as having a stable multiple myeloma on 3 Sep. 2013, and treated with Thalidomide 50 mg (alternating with 100 mg at night together with Prednisone 50 mg) for 7 days each month. The patient's multiple myeloma became progressive.

(71) MM79, Male

(72) Kappa light chain multiple myeloma diagnosed in April 2014, followed by treatment with VCAT induction followed by ASCT and ThaIVs Velcade maintenance. Responded well to treatment prior to partial relapse in October 2014.

(73) MM49, Male

(74) Diagnosed with IgA kappa myeloma in 2009, and responded well to treatment prior to partial relapse in May 2014. The results of the microparticle phenotyping are shown in Table 1 below.

(75) TABLE-US-00001 TABLE 1 Levels of CD138.sup.−/CD34.sup.+/P-gp.sup.+/CD41a.sup.− in patients CD41a.sup.− CD138.sup.− Diagnosed P-gp.sup.+ P-gp.sup.+ P-gp.sup.+ Gen- response CD34.sup.+ CD34.sup.+ CD34.sup.+ Patient der Time line state (μl) (μl) (μl) MM34 F 29 Nov. 2013 PD 122 775.32 238.44 13 Dec. 2013 PD 152.17 788.73 208.99 17 Jan. 2014 PD 77.57 312.01 69.32 20 Feb. 2014 PD 61.68 130.66 207.84 11 Mar. 2014 PD 98.80 485.65 150.64 MM19 M 30 Sep. 2013 Stable 38.17 177.54 43.40 4 Nov. 2013 PD 27.78 223.70 58.04 MM79 M 1 May 2014 PR 4.95 9.57 22.38 10 Jun. 2014 PR 6.99 45.85 37.7 176/14 PR 8.59 40.28 119.42 7 Jul. 2014 PR 10.83 145.51 74.65 MM49 M 3 Mar. 2014 PR 4.27 16.50 31.55 5 May 2014 PR 6.18 15.55 21.43 MM34 has consistently high levels of CD138−P-gp+CD34+ MPs, indicative of an aggressive/refractory cancer from which the patients does eventually die. MM19 has a high level of CD138−P-gp+CD34+ MPs on 30 Sep. 2013, despite being diagnosed as stable, after which the disease becomes progressive 4 Nov. 2013. MM79 has slowly increasing levels of CD138−P-gp+CD34+ MPs up until 7 Jul. 2014, after which point the patient was diagnosed with a partial relapse in October 2014. MM49 has an increased level of CD138−P-gp+CD34+ MPs at 5 May 2014, after which the patient was suspected of relapsing on 13 May 2014.