Decoding duality: Unraveling biclonal myeloma through flow cytometry and cytogenetics

Diagnostic workup

Following the initial clinical and imaging assessments, a comprehensive diagnostic workup was initiated to confirm the diagnosis of MM and characterize the plasma cell proliferation.

Bone marrow aspiration revealed normocellular bone marrow with 8% plasma cells, while the trephine biopsy showed 12% plasma cells. The presence of clonal bone marrow plasma cells at or above 10% is a key diagnostic criterion for MM.^[1,5]

Serum light chain assay results were particularly informative, showing high levels of both serum free Kappa light chains (150 mg/L, reference range: 3.3–19.4 mg/L) and free Lambda light chains (45 mg/L, reference range: 5.7–26.3 mg/L). The calculated free kappa/lambda ratio was 3.32 (reference range: 0.26–1.65), which is elevated. While the patient’s ratio was elevated, it did not meet this specific threshold, suggesting that isolated free light chain elevation might not always be sufficient for a definitive diagnosis of biclonality without further corroboration.

Despite the elevated free light chains, serum capillary electrophoresis and immunotyping revealed only a thin discrete band in the beta 2 region identified as IgA lambda. This finding was critical because it failed to detect the presence of two distinct monoclonal light chains, which was later confirmed by more advanced methods. This discrepancy highlighted a significant limitation of traditional protein-based diagnostics in this complex case.

MFC

Given the suggestive yet inconclusive findings from traditional methods, MFC was employed, proving to be pivotal for the definitive diagnosis. An ethylene diamine tetra acetic acid anticoagulated, first pull aspirate bone marrow sample was used for the assay, a recommended practice to ensure accurate plasma cell percentage detection. Cytoplasmic staining was performed for Kappa and Lambda light chains, while a surface stain-lyse-wash method was utilized for other antibodies.

The analysis was performed on a 10-multicolour Navios Ex Flow Cytometer (Beckman Coulter) using Kaluza software. The antibody panel utilized for the assay is detailed in Table 2, including markers such as Kappa, Lambda, cluster of differentiation (CD)19, CD27, CD56, CD138, CD45, CD38, CD117, CD20, CD81, and CD229.^[1] This panel aligns with European Myeloma Network (EMN) guidelines, which recommend CD38, CD138, and CD45 for plasma cell identification, and CD19, CD56 as minimal markers for detecting abnormal plasma cells. A more comprehensive panel, including CD20, CD117, CD28, and CD27, is preferred to increase the detection rate of abnormal plasma cells to over 95% of patients.^[4]

A sequential gating strategy was applied for plasma cell identification and analysis [Figure 2]. Singlets were identified from the FS Integral VS FS Peak graph, and viable cells were separated from debris by side scatter (SS) versus forward scatter (FS), and then by CD45 versus SS.

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Figure 2: Sequential gating strategy. (a) Isolation of mononuclear cells (MNC’s) using singlets to remove doublets followed by viability gating to remove debris followed by use of CD38 vs side scatter for MNC isolation, (b) Isolation of plasma cells (PC) by using different combinations of CD38/CD138 and CD45, (c) Downregulation of CD81 in abnormal PC (magenta and orange population) as compared to normal expression in normal PC (blue), (d) Isolation of different PC populations by CD19 vs CD56, and followed by light-chain clonality assessment, Blue (CD19+, CD56-, polyclonal - nPC), Magenta (CD19-, CD56-, lambda clonal, aPC), Orange (CD19-, CD56+, kappa clonal, aPC), (e) Downregulation of CD19 and CD27 in abnormal plasma cells (aPC - Orange) compared with normal plasma cells (nPC - Blue). (f) DNA ploidy analysis using FxCycle™ Violet, a fluorescent DNA-binding dye showing hypodiploid CD56+ abnormal plasma cells (Orange) and diploid CD56− abnormal plasma cells (Magenta).

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Further gating strategies adhered to EMN guidelines, with plasma cell detection performed on CD138 versus CD38, CD138 versus CD45, and CD38 versus CD45 positive cell gates. The EMN consensus recommends that the primary gate for identifying plasma cells should be based on CD38 versus CD138 expression to ensure comprehensive detection.^[4]

The final analysis by flow cytometry revealed 1.25% total plasma cells among all viable cells, with 76% of these identified as abnormal clonal plasma cells. Crucially, the analysis identified two distinct plasma cell populations. The predominant population constituted 62.8% of all plasma cells and exhibited lambda restriction. The smaller population, comprising 13.2% of all plasma cells, showed kappa restriction. The immunophenotypic profiles of these two distinct populations are detailed in Table 3. This direct cellular-level identification of two distinct clonal populations, each producing a different light chain, provided the definitive evidence of biclonal MM, a diagnosis that traditional protein-based methods had missed. The ability of flow cytometry to identify distinct cellular populations based on immunophenotypic aberrancies and light chain restriction, rather than solely on protein migration patterns, offers a superior diagnostic resolution. This fundamental shift from protein detection to cellular characterization is paramount for accurate diagnosis in complex plasma cell dyscrasias, especially when traditional methods are ambiguous or fail.

DNA ploidy analysis and fluorescent in situ hybridization (FISH)

Further characterization included DNA ploidy analysis, performed using FX violet dye, which revealed an index of 0.75 indicating a hypodiploid karyotype. This finding is significant for prognostic assessment, as hypodiploidy is generally associated with a more aggressive disease course.^[7]

FISH for myeloma was also performed. The results indicated the presence of deletion 13q14.2 (RB1/DELU) and gain of 1q21.22 (CKS1B). Importantly, FISH was negative for deletion 17p13 and IGH rearrangement. These specific cytogenetic abnormalities are crucial for determining the patient’s risk stratification and anticipating the disease’s clinical behavior and response to therapy.

The enigma of biclonal MM

Biclonal MM is a rare and complex entity within the spectrum of plasma cell dyscrasias, with only a limited number of cases reported in the medical literature. It is precisely defined by the simultaneous presence of two distinct monoclonal proteins, which arise from the proliferation of two separate monoclonal plasma cell clones.^[2,3]

The pathophysiology underlying biclonal MM can involve two primary mechanisms: the proliferation of two entirely independent plasma cell clones, each originating from a separate transforming event and producing an unrelated monoclonal immunoglobulin; or, alternatively, a single monoclonal clone undergoing clonal evolution, leading to the production of a second M-protein.^[2,3] While a single clone can undergo class switching between different immunoglobulin heavy chains during proliferation, the phenomenon of class switching between kappa and lambda light chains within a single clone is not known to occur unless significant clonal evolution has taken place.^[2,3]

The patient in this case presented with both IgA-κ and IgA-λ monoclonal gammopathies. This specific combination, involving distinct light chains, is particularly rare and carries profound implications. It strongly suggests the presence of two truly distinct plasma cell clones or a highly complex clonal evolution that resulted in two separate light chain-producing populations. This observation moves beyond a simple heavy chain class switch, pointing to a more intricate biological origin for the biclonal nature of the disease in this individual. The presence of M-protein with more than one immunoglobulin light chain restriction is a strong indicator of the existence of multiple neoplastic plasma cell clones.^[2,3]

Diagnostic superiority of flow cytometry

The diagnostic journey in this case vividly illustrates the limitations of traditional protein-based assays and the indispensable advantages of MFC in identifying biclonal MM.

Traditional methods, SPEP and IFE, are foundational for detecting monoclonal proteins. However, in this patient, despite elevated serum free kappa and lambda light chains, SPEP and immunofixation failed to identify two distinct monoclonal light chains, revealing only IgA lambda. This outcome is not uncommon, as electrophoretic methods can suffer from insufficient sensitivity to detect small quantities of monoclonal protein, or issues such as co-migration of different proteins, or subjective interpretation of bands, all of which can lead to inaccuracies or false-negative results.^[8] For instance, IgA molecules have a tendency to polymerize, which can result in multiple bands on SPEP, simulating biclonality when only a single monoclonal protein is present.^[8] These inherent limitations mean that traditional methods may not reliably distinguish true biclonality from artifacts or subtle presentations.

In stark contrast, MFC proved crucial for confirming the diagnosis. This technique offers superior sensitivity and specificity compared to traditional methods, with a typical sensitivity ranging from 10^-4 to 10^-5, and next-generation flow cytometry capable of reaching 10^-6.^[4,9] Flow cytometry provides a direct assessment of the plasma cell populations by analyzing their immunophenotypic features, thereby revealing the presence of two distinct clones. The ability to classify plasma cells as biclonal is greatly aided by the detection of antigenic aberrancies on these cells, emphasizing the necessity of a comprehensive antibody panel and an accurate gating strategy. Aberrant immunophenotypes, such as the absence of CD19 (normally positive on plasma cells) or the presence of CD56 (normally negative), are key indicators that distinguish neoplastic plasma cells from normal ones.^[4] The adherence to EMN guidelines for gating strategies, focusing on markers such as CD138, CD38, and CD45, ensures robust and reproducible identification of plasma cell populations. The definitive identification of two distinct abnormal plasma cell populations, each with different light chain restrictions, directly by flow cytometry, provided the conclusive evidence of biclonality that protein-based assays could not. This demonstrates that flow cytometry provides a cellular-level diagnosis, which is far more precise and less susceptible to protein-specific artifacts than SPEP/IFE, making it an indispensable tool for accurate detection of true biclonal MM.

Prognostic implications of genetic abnormalities

The genetic landscape of MM is a critical determinant of disease prognosis and response to therapy. The patient’s genetic analysis revealed several high-risk cytogenetic abnormalities that collectively point to a particularly aggressive disease biology [Table 4].

DNA ploidy analysis indicated a hypodiploid index, with an index of 0.75 in CD56+ abnormal clonal PC and diploid index, with 1.05 in CD56- abnormal clonal PC [Figure 2f]. Hypodiploidy, characterized by fewer chromosomes than usual in myeloma cells, is considered a more severe form of MM and is a major independent prognostic factor associated with significantly shorter overall survival (OS) compared to hyperdiploid karyotypes.^[6]

FISH further identified deletion 13q14.2 (RB1/DELU) and gain of 1q21.22 (CKS1B). Deletion 13q14.2 is a common secondary genetic abnormality in MM, strongly correlated with poor prognosis and increased drug resistance. Patients with this deletion typically exhibit a significantly lower response rate to conventional chemotherapy and a shorter OS.^[6] It is also often associated with higher serum beta-2 microglobulin levels and an increased percentage of bone marrow plasma cells.^[10]

Gain of 1q21.22 (CKS1B) is another frequent secondary genetic abnormality in MM, also associated with poor prognosis and increased risk of drug resistance, disease progression, and death. Patients with 1q21+ tend to present with a higher tumor burden, more extensive end-organ damage, and a greater incidence of co-occurring high-risk cytogenetic abnormalities.^[11] There is a known correlation between gain of 1q21–22 and hypodiploidy, as observed in this patient. Furthermore, the coexistence of deletion 13q and gain of 1q21 has been shown to significantly reduce progression-free survival and OS times.^[12]

The simultaneous presence of multiple high-risk cytogenetic abnormalities – deletion 13q14.2, gain 1q21.22, and hypodiploidy – in this patient is not merely an additive factor but likely represents a synergistic effect, indicating a particularly aggressive disease biology. This clustering of adverse genetic features suggests a highly challenging clinical course with an increased likelihood of drug resistance and rapid disease progression. Such a profile necessitates a more intensive and personalized therapeutic approach from the outset to mitigate the compounded negative impact on survival and treatment response.

Clinical significance and management considerations

The clinical significance of biclonality in MM remains a subject of ongoing debate. While some studies suggest that biclonal MM may follow a more aggressive course, others indicate that its clinical features might be similar to those of monoclonal gammopathy.^[2,3] However, the presence of BG in plasma cell dyscrasia is often considered a manifestation of clonal evolution, potentially serving as a marker of poor prognosis.^[2,3] The presence of two clones in this patient suggests a complex pathophysiology involving both clonal evolution and inherent heterogeneity within the malignant cell population.

Monitoring biclonal MM poses unique challenges. Traditional methods such as SPEP and urine protein electrophoresis (UPEP) may not be sensitive enough to track two distinct M-proteins, or they may be complicated by co-migration issues, making it difficult to accurately assess disease stability or progression.^[13] Newer assays, such as serum free light chain (Freelite^®) and Hevylite^® assays, offer improved sensitivity and can be used in conjunction with electrophoresis to track disease and differentiate it from therapeutic antibody interference, providing more objective quantification of monoclonal proteins.^[13]

Therapeutic strategies for biclonal MM may require adjustments due to the presence of multiple clones, necessitating consideration for therapies that can target both types of paraproteins. A significant therapeutic challenge arises from the observation that anti-MM therapies may elicit a differential response between distinct clones within the same patient.^[14] Studies have shown that while the primary MM clone may respond well to therapy, a co-existing benign monoclonal gammopathy of undetermined significance clone might respond less effectively or not at all.^[14] This means that a standard “onesize-fits-all” approach may be insufficient, potentially leading to selection pressure that favors the less responsive clone. Such a scenario could result in persistent disease from the less sensitive clone, contributing to relapse or refractory disease. This necessitates innovative monitoring strategies capable of tracking both clones independently and potentially tailored, multi-pronged therapeutic approaches designed to target the unique vulnerabilities of each clonal population.

Despite tremendous advancements, MM remains largely incurable, with most patients eventually experiencing relapse. This is often driven by induced drug resistance and the emergence of genetically heterogeneous sub-clones, a challenge that is amplified in biclonal MM due to its inherent clonal complexity.^[15] Current MM treatment paradigms typically involve combinations of medications, including quadruplet and triplet regimens, autologous stem cell transplantation (ASCT), and chimeric antigen receptor T-cell therapies for relapsed or refractory MM.^[15] For transplant-eligible patients, standard of care often includes induction therapy (e.g., Bortezomib + Lenalidomide + Dexamethasone [VRd] and Daratumumab + Bortezomib + Lenalidomide + Dexamethasone [DVRd]) followed by ASCT and subsequent maintenance therapy (e.g., Revlimid).^[6]

Future research directions

The complexities surrounding biclonal MM underscore the urgent need for further dedicated research. The precise etiology of BG is not yet fully understood,^[2,3,16] and a deeper understanding of its clinical behavior, specific genetic mutations, and long-term outcomes is essential. Future investigations should focus on the genetic study of these distinct clones to elucidate the underlying physiopathology of the biclonal peak.^[16]

Understanding clonal evolution and heterogeneity is paramount. The investigation of processes driving MM clonal evolution using next-generation sequencing techniques is particularly important, as the selection of drug-resistant subclones is a major contributor to treatment failure.^[17] A comprehensive understanding of clonal heterogeneity and evolution is critical for addressing the challenges of treatment failure and cancer relapse.^[17] Given the current lack of specific management guidelines for biclonal MM and the observed differential therapeutic responses between clones,^[14] future research must move beyond general MM studies. Dedicated investigations into biclonal MM, particularly focusing on single-cell genomics, are crucial to unravel the unique genetic landscapes and evolutionary patterns of each clone. This granular understanding is vital for identifying specific vulnerabilities of each clone, which can then inform the development of truly personalized and effective multi-targeted therapies.

While MFC has proven invaluable in diagnosing biclonal MM, continued optimization of diagnostic and monitoring tools is necessary, especially for highly sensitive minimal residual disease detection in these complex cases. Ultimately, the goal is to develop more effective therapeutic strategies, particularly for patients who become resistant to multiple drug classes and those presenting with highly heterogeneous myeloma cell populations.^[15]