METHOD OF DETECTING OR MONITORING MINIMAL RESIDUAL DISEASE IN A MONOCLONAL GAMMOPATHY PATIENT

Abstract

Method of Detecting or Monitoring Minimal Residual Disease The application describes a method of identifying minimal residual disease (MRD) in a monoclonal gammopathy patient, comprising detecting the presence or absence of a monoclonal free light chain (FLC) in a sample from the patient by mass spectrometry (MS).

Claims

1. A method of identifying minimal residual disease (MRD) in a monoclonal gammopathy patient, comprising detecting the presence or absence of a monoclonal free light chain (FLC clone) in a sample from the patient by mass spectrometry (MS).

2. A method according to claim 1, wherein the subject has had the monoclonal gammopathy treated prior to the screening and/or has been in remission from the monoclonal gammopathy.

3. A method according to claim 1, wherein if no FLC clone is detected then the subject is then monitored by detecting G, A, M, K, by MS.

4. A method according to claim 1, wherein if a FLC clone is detected then the clone is monitored by screening a further sample for a FLC clone by MS after a time interval.

5. A method according to claim 1, wherein if an FLC clone is detected then the clone is monitored by screening a still further sample for a FLC clone by MS after a time interval.

6. A method according to claim 1, wherein if no clone is detected then (a) either a further sample is tested after a time interval for a FLC clone by MS to see if a clone is detected or (b) a sample of bone marrow is tested for a FLC clone.

7. A method according to claim 6, wherein the subject may be further tested one or more times at intervals of time until no FLC clone is detected by MS.

8. A method according to claim 7, wherein if no clone is detected then a sample of bone marrow is tested for a clone.

9. A method according to claim 1, wherein MS is liquid chromatography MS or MALDI-TOF.

10. A method according to claim 1, wherein the sample is a sample of blood, serum, plasma, cerebrospinal fluid, or urine.

11. A method according to claim 1, wherein the monoclonal gammopathy is selected from multiple myeloma, LA amyloidosis plasmacytoma, Waldenström's macroglobulinaemia, B-cell non-Hodgkin lymphoma, and B-cell chronic lymphocytic leukaemia.

12. A method according to claim 1, wherein the subject is monitored by one or more additional techniques to monitor immunoglobulins in the sample prior to detecting a FLC clone by MS.

13. A method according to claim 12, wherein the presence of free light chain in the subject is monitored by nephelometry, turbidimetry or ELISA and FLC-MS screening is used once the presence of free light chains is observed to return to a predetermined normal level in the subject.

14. A method according to claim 12, wherein the immunoglobulins in the subject are detected by serum plasma electrophoresis (SPE) or immunofixation electrophoresis (IFE) and the FLC-MS screening is carried out if the (SPE) or IFE is identified not to be normal.

Description

[0047] The invention will now be described by way of example only with reference to the following examples:

[0048] FIG. 1 shows a flow diagram showing the typical steps in determining which assay to use to detect MRD.

[0049] FIG. 2 shows a patient (1) with a free Kappa monoclonal protein peak of m/z 11990 at presentation or baseline (a); reduced pre-ASCT (Autologous Stem Cell Transplant) (b); post-ASCT (c) and post-consolidation (d); FIG. 2 (e-h) shows QIP-MS of IgG, IgA, IgM, total kappa and total lambda at baseline (e), pre-ACST (f), post-ACST (g) and post-consolidation (h).

[0050] FIG. 3 shows a patient (2) with a free Kappa monoclonal protein peak of m/z 11672 at presentation or baseline (a); reduced pre-ASCT (Autologous Stem Cell Transplant) (b); post-ASCT (c) and post consolidation (d).

[0051] FIG. 4 shows a patient (3) with a free Kappa monoclonal protein peak of m/z 11552 at presentation or baseline (a); reduced pre-ASCT (Autologous Stem Cell Transplant) (b); post-ASCT (c); post-consolidation (d); FIG. 4 (d-f) shows QIP-MS of IgG, IgA, IgM, total kappa and total lambda at baseline (e), pre-ASCT (f), post-ACST (g), post consolidation(h).

[0052] FIG. 5 shows a patient (4) with a free lambda monoclonal peak of m/z 11445 at baseline, post-induction (b), 3 months post transplantation (c), FIG. 5 (d-f) shows QIP-MS of IgG, IgA, IgM, total kappa and total lambda at baseline (d), post induction (e), 3 months post-transplantation (f).

[0053] Typically the patient has been previously diagnosed with a monoclonal gammopathy such as multiple myeloma. The patient is then typically treated by one or more techniques known in the art, as discussed above. The patient may be monitored by nephelometry or turbidimetry or ELISA, to detect free light chains in the subject. Such free light assays include the assay sold under the tradename Freelite™ (The Binding Site Group Limited, Birmingham, England). The latter technique uses latex-enhanced nephelometry with anti-FLC antibodies. Once the amount of FLC is observed to return to a normal level for a normal healthy subject, then the blood, serum, plasma or urine from a patient may be screened for free light chains by mass spectrometry. Alternatively, SPEP/IFE may be used to also monitor FLC for other serum proteins. If the serum proteins appear to be abnormal, then FLCs may be detected by mass spectrometry, such as described in WO 2018/215768, incorporated herein in its entirety.

[0054] Once the FLC has been screened by mass spectrometry an FLC clone may be detected as a peak on the shoulder of the curve of different masses of polyclonal light chains in the background within the sample from the subject. If such a clone is identified, then this is typically monitored for FLC mass spectrometry, for example, every 0-3 months to see if the FLC clone is again present. If it is then further monitoring for FLC by mass spectrometry may be used. If no FLC clone is present after the first round of screening by mass spectrometry, then typically the subject is monitored by more conventional G, A, M, K, L mass spectrometry, such as reviewed by Murray and Willrich (Supra).

[0055] Where a further screen has taken place, then if no clone is present then alternatively further testing by mass spectrometry may occur after a period of typically 0-4, or 0-3 months. If an FLC clone is again present then retesting is then carried out. Alternatively, if no clone is present before or after that additional retesting, then a sample of bone marrow may be removed and tested as discussed above.

[0056] After the test, if MRD is detected then one or more further rounds of monitoring for FLC by mass spectrometry may occur.

[0057] Where a patient has been shown to be in remission and have been shown to be negative for MRD, they will be monitored for clonal FLC by mass spec to identify the occurrence or absence of relapse to MRD. Where a FLC clone is shown to be present by MS then this may act as a trigger for the treating clinician to carry out additional assessments.

EXAMPLES

Methods

[0058] The standard of care for multiple myeloma patients is to receive high-dose chemotherapy with autologous stem cell rescue—or autologous stem cell transplant (ASCT)—after completion of induction therapy. ASCT can provide significant reduction in disease, extending patient survival. Many patients will continue to receive maintenance or post-consolidation therapy post transplantation to reduce the risk of relapse. To demonstrate the detection and sensitivity of monoclonal paraproteins, throughout these phases using the mass spectrometry technology referred to in the application, longitudinal clinical samples were obtained from 4 patients undergoing diagnosis and treatment for Multiple Myeloma. Each sample was diluted in phosphate buffered saline+tween (PBST) to appropriate levels and immunocaptured using 7 different magnetic bead types; Free Kappa specificity and Free Lambda specificity or, total immunoglobulin (IgG specificity, IgA specificity, IgM specificity, Total Kappa specificity, Total Lambda specificity). The captured samples were washed sequentially (PBST and water) and then eluted using 5% acetic Acid and 20 mM tris(2-carboxyethyl) phosphine (TCEP). The eluted samples were co-spotted onto MALDI target plates alongside matrix (α-Cyano-4-hydroxycinnamic acid in acetonitrile and water spiked with trifluoroacetic acid) and dried to allow for MALDI analysis. The resulted mass spectra were acquired on a Bruker Microflex Biotyper MALDI Mass Spectrometer over the mass-range 5000-32000 m/z in the positive ion mode. The data is presented for the +2charge state ions of immunoglobulin light chains.

Results

[0059] Patient 1 (FIGS. 2a-2h). The presentation or baseline sample's mass spectra showed a clear monoclonal protein peak of m/z 11990 in the Free-kappa spectra. Indicating the presence of a Free kappa paraprotein. This peak reduced in intensity at the pre-ASCT stage and had disappeared post-ASCT and post-consolidation. No monoclonal peaks were seen in the Free-lambda spectra. Similarly, the same monoclonal peak was observed in the IgG and total kappa immunoglobulin spectra. Indicating the presence of an IgGK+free kappa paraprotein. This data shows that both free-light chain or total-light chain MALDI-TOF-MS can be employed successful to follow successful therapy over many months in this patient.

[0060] In this example the total immunoglobulin light chain assessment is seen to be more sensitive for the monoclonal protein, however, this observation may be due to the difference in clearance between the intact monoclonal protein and the Free light chains. Free light chains are relatively small molecules that are readily cleared by the kidney and so have a very short half-life (Kappa FLCs—2 hours; lambda FLCs 4-6 hours) and will therefore clear relatively quickly when there is no longer any production by an aberrant plasma cell clone. In contrast, Intact immunoglobulins are relatively large and are not readily cleared by the kidney, furthermore IgG immunoglobulins are recycled via the FcRN receptor and their half-life is very long (IgA and IgM 5-6 days; IgG ˜21 days).

[0061] Patient 2 (FIGS. 3a-3d). The baseline mass spectra showed a clear monoclonal peak of m/z 11672 in the Free-kappa spectra. Indicating the presence of a Free kappa paraprotein. This peak reduced in intensity in the pre-ASCT, post-ASCT and post-consolidation stage but did not entirely disappear. No monoclonal peaks were seen in the Free-lambda spectra. This shows that free-light chain in the absence of total-immunoglobulin MALDI-TOF-MS can be monitored by mass spectrometry to follow therapy over many months and years in a patient that has not completely responded to a therapy regime.

[0062] Patient 3 (FIGS. 4a-4h). The baseline mass spectra showed a clear monoclonal peak of m/z 11552 in the Free-kappa spectra. Indicating the presence of a Free kappa paraprotein. This peak diminished in intensity at the pre-ASCT stage and post-ASCT stage. The monoclonal FLC was not shown to be present in the sample from the post-consolidation stage of treatment. No monoclonal peaks were seen in the Free-lambda spectra. Correspondingly, the same monoclonal m/z peak was observed in the IgA and total Kappa spectra from baseline and pre-ASCT samples but had largely disappeared by the post-ASCT phase. This shows that both free-light chain or total-light chain MALDI-TOF-MS can be employed to follow successful therapy in this patient. The FLC assessment is shown to be more sensitive in this patient sample series and the assessment of FLCs avoids having to consider the impact of the longer half-life of intact immunoglobulins when using the result to interpret the sensitivity for the presence of an aberrant plasma cell producing a monoclonal protein.

[0063] Patient 4 (FIGS. 5a-5f). The baseline mass spectra showed a clear monoclonal peak of m/z 11445 in the Free-lambda spectra. Indicating the presence of a Free lambda paraprotein. This peak reduced in intensity at the post induction (therapy) stage and had virtually disappeared 3 months after transplant. No monoclonal peaks were seen in the Free-kappa spectra. The same monoclonal peak was observed in the IgA and total lambda baseline spectra, suggesting the presence of an IgAL and free lambda paraprotein, but this had largely disappeared by the post-induction phase. This indicates that both free-light lambda chain or total-light chain MALDI-TOF-MS can be employed successful to follow successful therapy in this lambda-patient. The FLC assessment is shown to be more sensitive in this patient sample series and the assessment of FLCs avoids having to consider the impact of the longer half-life of intact immunoglobulins when using the result to interpret the sensitivity for the presence of an aberrant plasma cell producing a monoclonal protein.

[0064] The data demonstrates the utility of the mass spectrometry method when monitoring for the presence or absence of an aberrant monoclonal protein producing plasma cell clone during longitudinal therapy. It indicates the additional sensitivity of FLCs over assessment of intact immunoglobulins. And demonstrates this is a useful approach to employ prior to applying more sensitive and potentially more invasive methods to assess for MRD.