Insights from the bone marrow: Characterization of monoclonal gammopathy of undetermined significance through flow cytometry analysis of plasma cells

Abstract

Introduction and aims: Multiple myeloma (MM) is a hematological malignancy, characterized by the proliferation and accumulation of malignant plasma cells in the bone marrow (BM), leading to overproduction of monoclonal protein (M-protein) and/or free light-chains (FLC). MM is consistently preceded by monoclonal gammopathy of undetermined significance (MGUS), an asymptomatic condition common among older adults (affecting around 3-5% of individuals aged 50 or older), progressing to MM and related disorders at a rate of approximately 1% per year. Although the overall risk of progression is relatively low, MGUS is a heterogeneous condition. Some individuals exhibit rapid malignant transformation, while others may follow an indolent clinical course. Consequently, accurate risk assessment and optimal clinical management represent key challenges in MGUS care. Multiparameter flow cytometry (MFC) provides immunophenotypic characterization at the single-cell level, enabling direct detection of plasma cell clonality based on aberrant antigen expression patterns. Although not routinely used in the diagnostic work-up of MGUS, MFC can complement traditional serological markers and potentially improve diagnostic accuracy and risk stratification. The clinical utility of MFC in this context is evaluated in this thesis, based on three studies. The first study aimed to develop a predictive model for assessing hemodilution in BM aspirates, which causes underestimation of plasma cell proportions. The second study aimed to refine the use of serum FLC ratio as a surrogate marker for plasma cell clonality in light-chain MGUS (LC-MGUS). The third study investigated the frequency and clinical significance of clonal plasma cell detection in IgA and IgG MGUS. Methods: All three studies were based on immunophenotypic analysis of BM samples from participants in the Iceland Screens, Treats, or Prevents Multiple Myeloma (iStopMM) population-based screening study. The EuroFlow next-generation flow (NGF) MM-minimal residual disease method was used to identify and quantify phenotypically aberrant (clonal) plasma cells and other cell populations in the BM. In the first study, first and second pull BM aspirated samples, used as references for optimal and suboptimal sample quality, were compared to identify markers of hemodilution. The most discriminatory populations were combined using linear discriminant analysis to develop the Bone Marrow Quality Index (BMQI). Validation included experimentally induced hemodilution. In the second study, the relationship between serum FLC ratio and clonal plasma cell detection by NGF was assessed to define an optimal FLC ratio cutoff. Longitudinal FLC dynamics and progression outcomes were used to evaluate the clinical relevance of the identified threshold. In the third study, NGF was used to assess clonal plasma cell detection in IgA and IgG MGUS. Clinical outcomes, including the rate of transient M-proteins and disease progression, were evaluated by clonal plasma cell detection status. Results: In study I, 351 BM samples from 219 individuals with plasma cell disorders were included. Second pull samples showed significantly reduced plasma cell proportions relative to paired first pull samples (median ratio 0.31; p<0.001). Nucleated red cells and myeloid precursors provided the most robust discrimination between first and second pull samples (area under the curve (AUC) values of 0.87 and 0.85), and were combined into the BMQI. BMQI scores correlated with plasma cell percentages and experimentally induced hemodilution. In study II, clonal plasma cells were detected in 53.6% of BM samples from LC-MGUS individuals and all those with more advanced disease. Serum FLC ratio strongly predicted clonal plasma cell presence, with an optimal cutoff of 3.15 (AUC=0.98; 96.7% sensitivity and 91.7% specificity). Individuals with FLC ratios between 1.65-3.15 showed stable FLC values and no progression, while those with ratios >3.15 exhibited overall increasing FLC ratio and significantly higher rates of progression to smoldering or active MM. In study III, clonal plasma cells were detected in 79.1% of MGUS individuals, and more frequently in IgA MGUS (93.3%) than IgG MGUS (71.2%; p<0.05). All MGUS cases without detectable clonal plasma cell population had either transient M-protein (52.6%) or a low-level stable IgG M-proteins (47.4%), and none progressed to more advanced disease over a median follow-up of five years. Conclusions: Altogether, MFC enables sensitive detection of clonal plasma cells and can improve diagnostic characterization in MGUS. The BMQI provides an objective, reproducible measure of BM sample quality and can inform interpretation of MFC-based plasma cell analysis. A refined FLC ratio cutoff of 3.15 improves diagnostic specificity in LC-MGUS and immunophenotypic confirmation of plasma cell clonality might be useful in borderline cases in this subgroup. Additionally, absence of clonal plasma cells identifies an MGUS subset with an indolent clinical course, characterized by transient or low-level IgG M-proteins, and no disease progression. Integrating standardized MFC methods, such as NGF, into MGUS evaluation may enhance diagnostic precision and inform individualized follow-up strategies.

Mergæxli er illkynja blóðsjúkdómur sem einkennist af stjórnlausri fjölgun og uppsöfnun einstofna plasmafrumna í beinmerg og leiðir til offramleiðslu á einstofna mótefni (M-prótein). Þeir sem greinast með mergæxli hafa áður verið með forstig þess, góðkynja einstofna mótefnahækkun (e. monoclonal gammopathy of undetermined significance; MGUS), sem er einkennalaust ástand þar sem M-prótein eða óeðlilegt hlutfall léttra keðja mælist í sermi. MGUS er algengt meðal eldra fólks (um 3-5% einstaklinga 50 ára og eldri), en aðeins hluti þeirra þróar með sér mergæxli eða skylda sjúkdóma á lífsleiðinni. Ein af helstu áskorunum í greiningu á MGUS felst í því að bera kennsl á þá sem eru í aukinni áhættu á illkynja sjúkdómsframvindu og aðgreina þá frá þeim sem hafa hægfara eða meinslausan gang. Markmið þessa doktorsverkefnis var að meta notagildi frumuflæðisjárrannsókna á plasmafrumum í beinmerg við greiningu á einstaklingum með MGUS. Slíkar rannsóknir veita ítarlegar upplýsingar um ónæmissvipgerð einstakra frumuhópa og með þeim er meðal annars hægt að meta, út frá afbrigðilegu tjáningarmynstri yfirborðssameinda, hvort plasmafrumur séu einstofna. Þrátt fyrir að slík greining sé ekki hluti af hefðbundnu greiningarferli MGUS, getur hún veitt frekari upplýsingar samhliða hefðbundnum blóðrannsóknum og mögulega leitt til nákvæmari greiningar og áhættumats.