Monitoring MRD in Multiple Myeloma in Serum by Mass Spectrometry

Background

Multiple myeloma (MM) is a severe plasma cell cancer caused by malignant proliferation and accumulation of clonal plasma cells in the bone marrow. It is the second most common hematological malignancy. The malignant plasma cells produce either intact but abnormal immunoglobulins or free light kappa or lambda chains - called M-protein. These secreted abnormal proteins have, for decades, been used as biomarkers to monitor MM, assess response to treatment, and to diagnose relapse of the disease. The M-protein is measured by serum protein electrophoresis and protein immunofixation, and the free light chains are measured by immunoassays. International consensus criteria for treatment response assessment are based on the degree of suppression of the M-protein, involved free light chain and the number of clonal plasma cells in the bone marrow. The best defined response, stringent complete response (sCR) is defined as disappearance of M-protein in serum by immunofixation, normalized kappa/lambda ratio in serum, and absence of clonal plasma cells in bone marrow biopsy by immunohistochemistry or flowcytometry.

Novel treatments have considerably improved observed responses and survival, and deep suppression of the clonal cells is the key factor to improve survival. More sensitive analyses to assess residual disease have been established, and the International Myeloma Working Group (IMWG) has defined the concept "minimal residual disease" (MRD) negativity as the absence of clonal plasma cells in the bone marrow with a minimum sensitivity of 1 in 100,000 nucleated cells. "Sustained MRD-negativity" defines MRD-negativity confirmed minimum 1 year apart. Moreover, "imaging plus MRD negativity" further requires a normal bone marrow tracer uptake by FDG PET/CT. Imaging is included as MM residual disease may be patchy or heterogeneously located in the body.

The impact of achieving MRD negativity at 1 in 100,000 or lower has consistently been reported to be so important that MRD negativity rate is proposed as primary objective in clinical trials and is under consideration by FDA as an accepted surrogate marker for long-term survival in the drug approval process. To achieve and maintain MRD negativity will therefore become the new goal for treating patients with MM. Further, serial MRD analyses will enable guidance during continued or maintenance therapy

Two methods have been established for MRD analysis, next-generation flowcytometry (NGF), e.g., the EuroFlow MRD assay, and next-generation sequencing (NGS), e.g., the ClonoSeq assay. Both methods are highly sensitive; 1 in 100,000 as standard goal, and currently reaching for 1 in 1,000,000. However, both methods require bone marrow sampling, which particularly for serial analysis is inconvenient and painful for the patients.

Blood-based MRD analyses of high sensitivity would be desirable for the patients and would allow more frequent sampling and monitoring. However, methods based on circulating cell-free tumor DNA have been found to have rather low sensitivity compared to bone marrow analysis. Mass spectrometry (MS) based analysis of patient-specific M-protein is another potential candidate for sensitive and specific monitoring of patients in deep remission. The literature proposes two distinct methods: a clonotypic peptide method and an intact protein method. These methods are under development in some centers, e.g. at the Mayo Clinic, USA, but are not fully standardized, and no commercial assays have been approved for MRD monitoring.

The investigators will assess and validate the sensitivity of a MS-based MRD analysis of blood in patients with MM in remission. The investigators will compare the sensitivity of MS-based analysis with Euro-flow MRD NGF analysis and NGS LymphoTrack analysis, both performed on bone marrow aspirates. Establishing a blood-based MRD analysis will potentially not only be more convenient, it may, in some patients, also be more sensitive. The blood reflects disease status in the whole body, whereas bone marrow sampling specifically examines what is present at this particular site. In addition, MRD in MM may be patchily located in the skeleton, highlighting the relevance of including FDG PET/CT in assessments.

Design

The investigators will prospectively examine and monitor a cohort of 40 patients in sCR. The study will include patients that have been in sCR for more than six months; this to minimize the risk that low-level circulating M-protein, due to a long half-live, will still be present in some patients in otherwise cellular MRD negativity. Furthermore, the investigators will perform retrospective analyses based on an established biobank where possible.

Samples from bone marrow and/or CD138+ isolated plasma cells from diagnosis must be present to identify the patient-specific Ig heavy chain and Ig light chain re-arrangement for NGS and MS assay establishment.

At base-line (time of inclusion), after 12 and 24 months, all subjects are examined using blood for MS, bone marrow for NGS-MRD and NGF-MRD, and FDG-PET/CT.

Every three months, blood is sampled for MS. If MS turns from negative to positive, all base-line assessments will be repeated. If quantitative MS doubles, e.g. from 5 ng/ml to 10 ng/ml, all base-line assessment will be repeated.

The sensitivity of the MS-based analysis will be compared with NGF-MRD, NGS-MRD and FDG-PET/CT findings. Moreover, the study will compare the sensitivity and prognostic value to predict biochemical and clinical progression of all four methods.

At 24 months, all subjects will be end of study, but will be followed for time to progression.

Experimental methods

Mass spectrometry For intact protein measurements, sample preparation methods for the purification and intact protein measurements of lambda and kappa light chains and corresponding Ig heavy chains is already under developpment. Moreover, strategies for the identification of clonotypic amino acid sequences of the M-protein and kappa/lambda proteins will be done either through de novo peptide sequencing by proteolytic M-protein cleavage, using multiple proteases combined with LC-MSMS analysis and bioinformatics, or through NGS sequencing of clonal Ig rearrangements. Clonotypic peptide sequences will then be used as surrogate markers for the M-protein levels in serum. Specifically, disease-specific immunoglobulins will be isolated from patient samples using affinity enrichment strategies virtually as previously described. For the de novo sequencing strategy, isolated immunoglobulins will be proteolytically cleaved by several proteases including, but not exclusively, trypsin, Lysozyme C, and proteinase K. Proteolytic digests will be analyzed by LC-MSMS using data-dependent acquisition. Resulting tandem mass spectra with amino acid sequence information will be de novo sequenced for the identification of clone-specific amino acid sequences using bioinformatics tools.

For intact protein measurements, the isolated disease-specific immunoglobulins will be analyzed using a novel multidimensional separation approach based on reversed-phase LC combined with FAIMS (Field asymmetric ion mobility spectrometry) and MS detection by high resolution Orbitrap MS. Samples will be analyzed using both MS-methods for increased sensitivity and selectivity.

Identified clone specific immunoglobulins and derived clone specific peptides will then be used for disease monitoring during treatment and relapse.

Patient samples for method/assay development and equipment/instrumentations are already available at OUH for method comparison with gold standard method. Serum and plasma cell samples along with clinical data are already available for assay/method development. For clinical studies, patient samples collected at baseline (time of diagnosis), remission and relapse are also available for the present project.

Next generation sequencing of clonal rearrangements Clonal B cell rearrangements will be identified by NGS sequencing on the in-house Illumina MiSeq sequencing platform using the established LymphoTrack kit (Invivoscribe) for IgH and IgL kappa rearrangements and in-house developed assay for IgL lambda rearrangements. The method identifies plasma cell clones in CD138+ cells at diagnosis by the unique sequences of their antibody immunoglobulin repertoire, where each cell expansion will display its own genomic fingerprint. Clonal plasma cell populations will be quantified to a sensitivity of 10^-5 to 10^-6 for MRD assessment. These methods are established in the study lab which have a long and increasing experience in supporting clinical trials with NGS-MRD analyses.

Flowcytometry MM MRD assessment by flow cytometry using the standardized approach for sample processing and data analysis developed by the EuroFlow Consortium achieve high sensitivity (10^-5) by processing a large number of cells (10 x 10^6). EuroFlow standard operating protocol (SOP) for Bulk lysis, staining and Instrument set-up for BD FACS Lyric™ is used and data analyzed by experienced chief physicians using the Infinicyte™ Software program allowing identification of both normal and abnormal plasma cells in the sample.

FDG PET/CT Standardized FDG PET/CT scans will be performed at the Department of Nuclear Medicine, OUH.

Statistics The study will include 40 patients in sCR defined by IMWG criteria. Sample cohort size estimation is based on power calculations using a dichotomous endpoint of MRD negativity or positivity between two methods (80% power, significance threshold of 0.05, comparing proportions). Using estimated parameters of 2/3 flow-MRD negative patients in sCR, with 3/4 of these obtaining sustained MRD negativity throughout follow-up (i.e., 50% MRD+/-), 70% MRD positive rate by mass spectrometry, and three evaluations for each patient, the study will include 40 patients in sCR defined by IMWG criteria (120 samplings for blinded, unpaired assessment).