A Longitudinal Evaluation of Euroflow and Combined Quantitative Immunoprecipitation (QIP) and Free Light Chain (FLC) Mass Spectometry (MS) in Functional High Risk Multiple Myeloma

Introduction: The achievement of minimal residual disease (MRD) negativity is being increasingly recognised as the optimal measure of therapeutic response for both newly diagnosed and relapsed and/or refractory multiple myeloma (MM) patients. Bone marrow (BM) evaluation with either Next Generation Sequencing (NGS) or Next Generation Flow-cytometry (NGF) affords a high level of sensitivity and the attainment of MRD negativity (< 1 in 10^-5 MM cells) with either approach is a powerful predictor of superior progression free survival (PFS). Both, however, are limited by the requirement for invasive bone marrow biopsy and the technical limitations imposed by variability in sample quality. Moreover, we and others have demonstrated the presence of significant spatial heterogeneity in MM that increases in the context of disease progression. Against this background we have evaluated a blood-based strategy for disease burden evaluation, Quantitative ImmunoPrecipitation (Mass Spectometry (QIP MS) and Free Light Chain Mass Spectometry (FLC MS) in a uniformly treated cohort of functional high-risk MM patients also undergoing sequential NGF (EuroFlow platform) MRD evaluation.

Methods: Newly diagnosed MM patients failing (<partial remission [PR] as best response) front-line bortezomib-based induction therapy were enrolled onto the Australasian Leukaemia and Lymphoma Group (ALLG) MM17 trial (ACTRN12615000934549) evaluating an intensive salvage approach utilising a combination of carfilzomib, thalidomide and dexamethasone (KTd) as re-induction (KTd x 6 cycles) and as post autologous stem cell transplantation (ASCT) consolidation (KTd x 2 cycles followed by Td x10 cycles). NGF MRD status was determined pre-ASCT, post-ASCT and post-KTd consolidation utilising the standardised 8-colour EuroFlow platform. Matched serum samples from the 3 time-points were evaluated in parallel with QIP and FLC MS. Briefly, polyclonal antibodies (anti-IgG, -IgA, -IgM, -total κ, -total λ, free κ and free λ) covalently attached to paramagnetic microparticles were incubated with serum, washed and treated to simultaneously elute and reduce patient immunoglobulins. Light chain mass spectra were generated on a MALDI-TOF-MS system. Concordance between NGF and MS was assessed via the derivation of Cohen's kappa values.

Results: Fifty patients were enrolled onto the ALLG MM17 trial. QIP and/or FLC MS identified the serum monoclonal paraprotein (PP) at baseline in all cases (100% sensitivity). Serum samples for MS with matched BM for NGF were available on 33 patients pre-ASCT, 32 post-ASCT and 26 post-KTd consolidation (91 matched samples in total). Sequential MS demonstrated serological complete remission (disappearance of MS baseline detectable monoclonal intact immunoglobulin [PP] and/or FLC) (CR_MS) in 11%, 47% and 53% of patients pre-ASCT, post-ASCT and post-KTd, respectively. NGF MRD negativity at the same time points was 39%, 52% and 71% (the latter equivalent to a 50% MRD negativity rate within the original n=50 intention-to-treat population). The Cohen's kappa values for the 3 time-points were 0.21, 0.18 and 0.35 indicating fair to moderate concordance with the best concordance at the post-KTd consolidation time-point and with a Cohen's kappa value for the entire cohort (n=91) of 0.30. The sequential MS demonstrated that 12 patients had discordant disappearance of baseline PP and free light chains (FLC) prior to achieving CR_MS. In 11 the FLC disappeared before the PP and in 1 the PP prior to the FLC. The former though to be due to either the FLC falling below the sensitivity of the technique following successful therapy or the presence of 2 sub-clones with differential drug sensitivity, whereas the latter was likely secondary to the persistence of a FLC expressing sub-clone. Post-KTd MS demonstrated good concordance with serological response (Cohen's kappa value = 0.61) but with 18% of patients demonstrating sCR/CR despite persisting MS detectable PP and/or FLC.

Conclusion: These preliminary data confirm the utility of QIP MS and FLC MS for the sequential monitoring of tumour burden in HR MM. Concordance with standard monitoring was good with MS detectable disease in some patients with serological sCR/CR consistent with the higher sensitivity of MS. Concordance with NGF was only fair to moderate mandating the future comparison of larger sample sets to better understand the relationship between the 2 methodologies.