Detection of Plasma Cell Disorders by Mass Spectrometry: A Comprehensive Review of 19,523 Cases.

OBJECTIVES: To verify the analytical performance of a new mass spectrometry-based method, termed MASS-FIX, when screening for plasma cell disorders in a routine clinical laboratory. PATIENTS AND METHODS: Results from 19,523 unique patients tested for an M-protein between July 24, 2018, and March 6, 2020, by a combination serum protein electrophoresis (SPEP) and MASS-FIX were examined for consistency with pretest implementation performance. MASS-FIX's ability to verify abnormal results from SPEP and free light chain measurements was then compared with that of immunofixation electrophoresis (IFE) using a separate cohort of 52,586 patients tested by SPEP/IFE during the same period. RESULTS: Overall, 62.4% of our cohort was negative for an M-protein. Importantly, 7.3% of all specimens had an M spike on SPEP (0.1 to 8.5 g/dL) and MASS-FIX detected an M-protein in all these samples. Of all samples, 30.3% had M-proteins that were detected by MASS-FIX but the SPEP finding was too small for quantification. Of the positive samples, 5.7% contained a therapeutic monoclonal antibody. Of the positive samples, 4.1% had an N-glycosylated light chain (biomarker of high-risk plasma cell disorders). MASS-FIX confirmed a higher percentage of SPEP abnormalities than IFE. MASS-FIX was slightly more sensitive than IFE when confirming an M-protein in samples with an abnormal free light chain ratio. MASS-FIX had a very low sample repeat rate (1.5%). MASS-FIX was highly automatable resulting in a higher number of samples/technologist/day than IFE (∼30% more). CONCLUSION: Overall, MASS-FIX was successful in maintaining validation characteristics. MASS-FIX was more sensitive in confirming SPEP abnormalities when compared with IFE. Ability to detect therapeutic monoclonal antibodies and glycosylated light chains was distinctly advantageous.

MALDI-TOF mass spectrometry can distinguish immunofixation bands of the same isotype as monoclonal or biclonal proteins.

BACKGROUND: Plasma cell disorders (PCDs) are typically characterized by excessive production of a single immunoglobulin, defined as a monoclonal protein (M-protein). Some patients have more than one identifiable M-protein, termed biclonal. Traditional immunofixation electrophoresis (IFE) cannot distinguish if two bands of the same isotype represent biclonal proteins or M-proteins with some other feature. A novel assay using immunoenrichment coupled to matrix-assisted laser desorption ionization time-of-flight mass-spectrometry (Mass-Fix) was applied to determine whether two bands of the same isotype represented (1) monomers and dimers of a single M-protein, (2) an M-protein plus a therapeutic monoclonal antibody (t-mAb), (3) an M-protein with light chain glycosylation, or (4) two distinct biclonal M-proteins. METHODS: Patient samples with two bands of the same isotype identified by IFE were enriched using nanobodies against IgG, IgA, IgM, or κ and λ light chains then analyzed by Mass-Fix. Light chain masses were used to differentiate IgGκ M-proteins from t-mAbs. Mass differences between peaks were calculated to identify N-glycosylation or matrix adducts. High-resolution mass spectrometry was used as a comparator method in a subset of samples. RESULTS: Eighty-one residual samples were collected. For IgA, 93% (n = 25) were identified as monoclonal. For IgG, 67% (n = 24) were monoclonal, and 33% (n = 12) were truly biclonal. Among the monoclonal IgGs, the second band represented a glycosylated form for 21% (n = 5), while 33% (n = 8) had masses consistent with a t-mAb. 44% (n = 8) of IgM samples were biclonal, and 56% (n = 10) were monoclonal, of which one was glycosylated. CONCLUSIONS: We demonstrate the utility of mass spectrometry in the characterization of multiple IFE bands of the same isotype. Improved reporting accuracy of M-proteins is useful for monitoring of patients with PCDs.

Treatment of AL Amyloidosis: Mayo Stratification of Myeloma and Risk-Adapted Therapy (mSMART) Consensus Statement 2020 Update.

Immunoglobulin light chain (AL) amyloidosis is a clonal plasma cell disorder leading to progressive and life-threatening organ failure. The heart and the kidneys are the most commonly involved organs, but almost any organ can be involved. Because of the nonspecific presentation, diagnosis delay is common, and many patients are diagnosed with advanced organ failure. In the era of effective therapies and improved outcomes for patients with AL amyloidosis, the importance of early recognition is further enhanced as the ability to reverse organ dysfunction is limited in those with a profound organ failure. As AL amyloidosis is an uncommon disorder and given patients' frailty and high early death rate, management of this complex condition is challenging. The treatment of AL amyloidosis is based on various anti-plasma cell therapies. These therapies are borrowed and customized from the treatment of multiple myeloma, a more common disorder. However, a growing number of phase 2/3 studies dedicated to the AL amyloidosis population are being performed, making treatment decisions more evidence-based. Supportive care is an integral part of management of AL amyloidosis because of the inherent organ dysfunction, limiting the delivery of effective therapy. This extensive review brings an updated summary on the management of AL amyloidosis, sectioned into the 3 pillars for survival improvement: early disease recognition, anti-plasma cell therapy, and supportive care.

MASS-FIX for the detection of monoclonal proteins and light chain N-glycosylation in routine clinical practice: a cross-sectional study of 6315 patients.

Immunoenrichment-based matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), termed MASS-FIX, offers several advantages over immunofixation for the detection and isotyping of serum monoclonal protein, including superior sensitivity and specificity, the ability to differentiate therapeutic monoclonal antibodies, and the rapid identification of light chain (LC) N-glycosylation. We identified 6315 patients with MASS-FIX performed at our institution since 2018. Of these, 4118 patients (65%) with a wide array of plasma cell disorders (PCD), including rare monoclonal gammopathies of clinical significance, had a positive MASS-FIX. Two-hundred twenty-one (5%) of the MASS-FIX positive patients had evidence of LC N-glycosylation, which was more commonly identified in IgM heavy chain isotype, kappa LC isotype, and in diagnoses of immunoglobulin light chain (AL) amyloidosis and cold agglutinin disease (CAD) compared to other PCD. This cross-sectional study describes the largest cohort of patients to undergo MASS-FIX in routine clinical practice. Our findings demonstrate the widespread utility of this assay, and confirm that LC N-glycosylation should prompt suspicion for AL amyloidosis and CAD in the appropriate clinical context.

Clinical Characteristics and Outcomes of Patients With Primary Plasma Cell Leukemia in the Era of Novel Agent Therapy.

OBJECTIVE: To evaluate the clinical outcomes of patients with primary plasma cell leukemia (pPCL) defined by 5% or greater clonal circulating plasma cells on peripheral blood smear and treated with novel agent induction therapies. PATIENTS AND METHODS: A cohort of 68 patients with pPCL diagnosed at the Mayo Clinic in Rochester, Minnesota, from January 1, 2000, to December 31, 2019, and treated with novel agent induction therapies was evaluated. RESULTS: The median follow-up was 46 (95% CI, 41 to 90) months. The median bone marrow plasma cell content was 85% (range, 10% to 100%) and median clonal circulaitng plasma cell percentage on the peripheral blood smear was 26% (range, 5% to 93%). There was a preponderance of t(11;14) primary cytogenetic abnormality in this cohort. The median time to next therapy (TTNT) and overall survival (OS) for all patients with pPCL patients in this cohort was 13 (95% CI, 9 to 17) and 23 (95% CI, 19 to 38) months, respectively. However, when stratified by cytogenetic risk, the median TTNT and OS were 16 and 51 months for standard risk vs 9 and 19 months for high risk (P=.01 for OS). CONCLUSION: Primary plasma cell leukemia remains an aggressive disease with poor prognosis despite novel agent-based therapies. Some patients have better than expected survival and this phenomenon may be influenced by the absence of high-risk cytogenetics. Newer treatment regimens are needed to improve the prognosis of this devastating disease.

The Impact of Socioeconomic Risk Factors on the Survival Outcomes of Patients With Newly Diagnosed Multiple Myeloma: A Cross-analysis of a Population-based Registry and a Tertiary Care Center.

BACKGROUND: Multiple myeloma (MM) is a heterogeneous clonal plasma cell disorder leading to differences in clinical outcomes such as overall survival (OS) among patients. We hypothesized that with expensive, novel therapeutic agents and paradigm shifts to maintain continuous therapy and improvement in OS, patients with MM are subject to the pressures of financial toxicity and the need for social support, which may be of prognostic importance. MATERIALS AND METHODS: In this study, we examined the records of 122,458 patients from the National Cancer Database (NCDB) to determine the significance of socioeconomic factors such as estimated annual household income and education level, which were based on the patient's ZIP Code and the United States Census Bureau's 5-year report from 2008 to 2012. These socioeconomic factors, in addition to marital status, were then assessed individually and as a cumulative socioeconomic score for prognostic significance in a cohort of 2543 patients treated at a tertiary care center utilizing known biologic risk factors, such as cytogenetic risk, International Staging System classification, and serum lactate dehydrogenase levels. RESULTS: Only marital status and estimated annual household income at diagnosis negatively impacted OS in a univariate analysis, but not in the context of a multivariable analysis incorporating known biologic risk factors. CONCLUSION: Future analyses in other academic and non-academic centers located in urban and rural regions are required to understand the socioeconomic drivers of OS disparity among patients with MM observed nationally.

Disease monitoring with quantitative serum IgA levels provides a more reliable response assessment in multiple myeloma patients.

Unlike IgG monoclonal proteins (MCPs), IgA MCP quantification is unreliable due to beta-migration of IgA MCPs on serum protein electrophoresis (SPEP). The utility of nephelometric quantitative IgA (qIgA) to monitor IgA multiple myeloma (MM) is unclear. We retrospectively studied disease response kinetics using qIgA versus MCPs by SPEP, and developed and validated novel qIgA disease assessment criteria in 491 IgA MM patients. The SPEP MCP nadir occurred a median of 41 (IQR 0-102) days before the qIgA. The median time to achieve a partial response (PR) was shorter using standard IMWG versus qIgA response criteria (32 vs 58 days, p < 0.001). Stratification by qIgA criteria, unlike IMWG criteria, led to clear separation of the progression-free survival curves of patients achieving a PR or very good PR. There was a consistent trend toward earlier detection of disease progression using qIgA versus IMWG progression criteria. In conclusion, monitoring IgA MM using MCP-based IMWG criteria may be falsely reassuring, given that MCP levels on SPEP decrease faster than qIgA levels. The qIgA response criteria more accurately stratify patients based on the progression risk and may detect disease progression earlier, which may lead to more consistent measurement of trial endpoints and improved patient outcomes.

Prognostic restaging after treatment initiation in patients with AL amyloidosis.

The utility of systemic light chain (AL) amyloidosis staging systems has been validated for newly diagnosed patients, but their role in restaging after treatment has not been explored. We designed this study to evaluate whether the currently used systems are of prognostic value at 3 and 6 months of starting first-line treatment, and whether stage migration from diagnosis impacts survival. This is a retrospective study including Mayo Clinic patients with AL amyloidosis diagnosed between 1 January 2006 and 30 June 2019; 536 and 204 patients had restaging data for at least 1 system at 3 and 6 months, respectively. Using modified Mayo 2004 staging at 3 months, median overall survival (OSs) were 11.8, 10.8, 4.6, and 1.1 years for stage I, II, IIIa, and IIIb, respectively. Using Mayo 2012, median OSs were 11.8, 9.0, 5.2, and 0.8 years for stage I, II, III, and IV, respectively. Using modified Mayo 2004 staging at 6 months, median OSs were not reached (NR), NR, 5.4, and 0.9 years for stage I, II, IIIa, and IIIb, respectively. Using Mayo 2012, OSs were NR, NR, 4.6, and 0.9 years for stage I, II, III, and IV, respectively. Worsening stage at 3 or 6 months was associated with worse survival than retaining baseline stage. In conclusion, the current staging systems can be used for restaging at 3 and 6 months from treatment initiation. Migration to higher stage predicts poor prognosis.

Comparison of the current renal staging, progression and response criteria to predict renal survival in AL amyloidosis using a Mayo cohort.

Three sets of criteria (International Society of Amyloidosis [ISA], Palladini and Kastritis) were independently developed for staging, progression and response criteria to predict renal survival in patients with AL amyloidosis. We evaluated these criteria using a cohort of 495 newly diagnosed AL amyloidosis patients with renal involvement using time to event competing risk analysis at baseline, 3, 6 and 12 months after treatment. Only Palladini and Kastritis had a staging system and both predicted a higher risk of end stage renal disease (ESRD) in the stage III vs stage I patients but only the Palladini model was predictive for stage II patients. At 3 months, risk of ESRD was significantly higher for Palladini and ISA renal progression (hazard ratio [HR] 2.8 [95% CI: 1.5-5.3, p = .001] and 2.5 [CI: 1.4-4.6, p = .004, respectively]), but renal response was not significantly protective; conversely, the risk of ESRD was not significantly higher for the Kastritis renal progression, but was significantly protective for the Kastritis renal responders (HR 0.38 [95% CI: 0.17-0.84], p = .017). Both progression and response with ISA, Palladini and Kastritis criteria were predictive of ESRD at 6 months and 12 months. While the Palladini staging criteria at baseline, and the ISA and Palladini criteria for progression at 3 months performed better than the Kastritis criteria at baseline and 3 months post-treatment, the Kastritis criteria performed better for response 3 months after treatment. All three sets of criteria performed well at and after 6 months post-treatment. These differences are important when choosing endpoints for clinical trials.

Characterization and prognostic implication of delayed complete response in AL amyloidosis.

INTRODUCTION: Little is known on continued response following completion of therapy in light chain (AL) amyloidosis. METHODS: We studied 373 AL amyloidosis patients who achieved complete response (CR) or very good partial response (VGPR) to first-line therapy. RESULTS: By end of therapy (EOT), 46% of patients achieved a CR and 54% a VGPR. With no further therapy, 17.5% of patients were upstaged from VGPR to CR (delayed CR), with a median of 9 months. Compared with CR and VGPR at EOT, patients with a delayed CR were characterized by higher proportion of t(11;14) and lower rate of trisomies. Autologous stem cell transplant was more frequent in the delayed CR group. Patients with a delayed CR were characterized by minimal residual disease negativity and organ response rates similar to patients with CR at EOT and higher than patients achieving VGPR at EOT. Patients with a delayed CR had a longer PFS/OS compared to patients with CR or VGPR by EOT (median PFS 149 vs 92 vs 52 months, P < .001; 10-year OS 87% vs 71% vs 56%, P < .001). CONCLUSIONS: This study characterizes delayed CR in AL amyloidosis, highlights its prognostic impact which is at least similar to those who achieved CR at EOT, and underlines another aspect of response monitoring.

Correlation between urine ACR and 24-h proteinuria in a real-world cohort of systemic AL amyloidosis patients.

A 24-h urine protein collection (24hUP), the gold standard for measuring albuminuria in systemic AL amyloidosis, is cumbersome and inaccurate. We retrospectively reviewed 575 patients with systemic AL amyloidosis to assess the correlation between a urine albumin to creatinine ratio (uACR) and the 24hUP. The uACR correlated strongly with 24hUP at diagnosis (Pearson's r = 0.87, 95% CI 0.83-0.90) and during the disease course (Pearson's r = 0.88, 95% CI 0.86-0.90). A uACR ≥300 mg/g estimated a 24hUP ≥ 500 mg with a sensitivity of 92% and specificity of 97% (area under the receiver operating curve = 0.938, 95% CI 0.919-0.957). A uACR cutoff of 3600 mg/g best predicted a 24hUP > 5000 g (sensitivity 93%, specificity 94%), and renal stage at diagnosis was strongly concordant using either 24hUP or uACR as the proteinuria measure (k = 0.823, 95% CI 0.728-0.919). In patients with serial urine collections, a > 30% decrease in uACR predicted a > 30% decrease in 24hUP with a sensitivity of 94%. In conclusion, the uACR is a reliable and convenient method for ruling out proteinuria >500 mg per day, prognosticating renal outcomes, and assessing renal response to therapy. Further studies are needed to validate the uACR cutoffs proposed in this study.

Characteristics of exceptional responders to autologous stem cell transplantation in multiple myeloma.

Autologous stem cell transplantation (ASCT) is an important treatment modality in multiple myeloma (MM). However, relapse following ASCT is considered almost inevitable. This study aimed to characterize exceptional responders to ASCT, defined as progression-free survival (PFS) >8 years in the absence of maintenance therapy. We retrospectively analyzed patients treated at Mayo Clinic between August 1, 1998 and January 3, 2006, and included those with symptomatic MM, treated with an ASCT within 12 months of diagnosis. We found that 46 (9%) of the 509 patients who underwent ASCT during the study period were exceptional responders. The median duration of follow-up from diagnosis was 16.2 (interquartile range 14.3-17.7) years. The best response to therapy was a complete response (CR) or better in 34 (74%) of patients, and less than a CR in 12 (26%) of patients. The median PFS was 13.8 (95% confidence interval 10.5-18.5) years, and at the time of the last hematology assessment, 24 of 46 (52%) patients remained in remission. In conclusion, we showed that a small subset of patients with MM attains durable disease control without maintenance therapy post ASCT. Pre-emptive identification of these patients may help prevent undue toxicities and costs of subsequent therapy.

Refining amyloid complete hematological response: Quantitative serum free light chains superior to ratio.

Response assessment in light chain (AL) amyloidosis is based on serum and urine monoclonal protein studies. Newly diagnosed patients (n = 373) who achieved very good partial response or complete response (CR) to first line therapy were assessed for the survival impact of each of the monoclonal protein studies. At end of therapy (EOT), negative serum/urine immunofixation (IFE) was achieved in 61% of patients, 72% achieved normal serum free light chain ratio (sFLCR), and the median involved free light chain (iFLC) and difference between involved to uninvolved light chain (dFLC) were 17 mg/L and 5 mg/L, respectively. Overall, 46% of patients achieved a CR at EOT. At EOT, iFLC ≤20 mg/L and dFLC ≤10 mg/L were additive in survival discrimination to negative serum/urine IFE and were independent predictors of overall survival. In contrast, normalization of sFLCR did not add survival discrimination to serum/urine IFE and was not independent predictor of survival. We propose a new definition for hematological CR to include serum/urine IFE negativity plus iFLC ≤20 mg/L or dFLC ≤10 mg/L, instead of the current definition of serum/urine IFE negativity and normal sFLCR. Complete response using dFLC ≤10 mg/L had the best performance in those with significant renal dysfunction and by light chain isotype, making it the preferred partner to IFE. Validation of these results in a multicenter cohort is warranted.

Correction: Bone marrow plasma cells 20% or greater discriminate presentation, response, and survival in AL amyloidosis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Metaphase cytogenetics and plasma cell proliferation index for risk stratification in newly diagnosed multiple myeloma.

Metaphase cytogenetic abnormalities, plasma cell proliferation index (PCPro), and gain 1q by fluorescence in situ hybridization (FISH) are associated with inferior survival in newly diagnosed multiple myeloma (MM) treated with novel agents; however, their role in risk stratification is unclear in the era of the revised International Staging System (R-ISS). The objective of this study was to determine if these predictors improve risk stratification in newly diagnosed MM when accounting for R-ISS and age. We studied a retrospective cohort of 483 patients with newly diagnosed MM treated with proteasome inhibitors and/or immunomodulators. On multivariable analysis, R-ISS, age, metaphase cytogenetic abnormalities (both in aggregate and for specific abnormalities), PCPro, and FISH gain 1q were associated with inferior progression-free (PFS) and overall survival (OS). We devised a risk scoring system based on hazard ratios from multivariable analyses and assigned patients to low-, intermediate-, and high-risk groups based on their cumulative scores. The addition of metaphase cytogenetic abnormalities, PCPro, and FISH gain 1q to a risk scoring system accounting for R-ISS and age did not improve risk discrimination of Kaplan-Meier estimates for PFS or OS. Moreover, they did not improve prognostic performance when evaluated by Uno's censoring-adjusted C-statistic. Lastly, we performed a paired analysis of metaphase cytogenetic and interphase FISH abnormalities, which revealed the former to be insensitive for the detection of prognostic chromosomal abnormalities. Ultimately, metaphase cytogenetics lack sensitivity for important chromosomal aberrations and, along with PCPro and FISH gain 1q, do not improve risk stratification in MM when accounting for R-ISS and age.

The role of bone marrow biopsy in patients with plasma cell disorders: should all patients with a monoclonal protein be biopsied?

We conducted a retrospective review of multiple myeloma (MM), smoldering multiple myeloma (SMM), and monoclonal gammopathy of undetermined significance (MGUS) patients seen at Mayo Clinic to determine whether a bone marrow biopsy (BM) is necessary in all patients diagnosed with a monoclonal protein. A total of 2254 MM, 397 SMM, and 5836 MGUS patients were included in the study. A total of 29 (1.3%) MM patients "without CRAB/FLC" were identified where BM or advanced imaging was critical for diagnosis, 8 (0.3% MM cohort) of whom were diagnosed with MM solely on BM findings (plasma cells > 60%). Without BM or advanced imaging none of these patients would be classified low-risk MGUS. A total of 314 (79%) MGUS-like SMM patients were identified where classification of SMM was based on BM findings. Without BM 97 would be classified as low/low-intermediate-risk MGUS and 151 intermediate or high-risk MGUS; 66 had missing information precluding classification. Only three (<1% SMM cohort) were low-risk MGUS without abnormalities in hemoglobin, calcium, and renal function. In patients presenting with low-risk MGUS and normal hemoglobin, calcium, and renal function, the risk of missing a diagnosis of SMM and MM by omitting BM is <1%. BM should be deferred in these patients in preference to clinical and laboratory monitoring.

Utilizing multiparametric flow cytometry in the diagnosis of patients with primary plasma cell leukemia.

The diagnosis of primary plasma cell leukemia (pPCL) has been made by quantifying circulating plasma cells (cPCs) morphologically on a peripheral blood (PB) smear. However, this technique is not sufficiently sensitive. Multiparametric flow cytometry (MFC) provides a readily available and highly sensitive method to identify and quantify cPCs that could complement PB smear assessment. However, an optimal quantitative cutoff for cPCs by MFC to identify pPCL has not been established. Thus, a total of 591 patients newly diagnosed multiple myeloma (NDMM) patients who had their PB samples evaluated morphologically by PB smear, and immunophenotypically by MFC prior to beginning therapy were evaluated. The presence of ≥200 cPCs/µL by MFC (N = 25 or 5% of the total population) was chosen to identify patients with ≥5% cPCs by PB smear with a specificity of 99% and a sensitivity of 77%. For patients with ≥200 cPCs/µL by MFC compared to the remainder of the cohort, the median Time to next therapy (TTNT) was 18 vs 30 months and the median OS was 38 vs 70 months respectively. Thus, MFC assessment of PB can be utilized in conjunction with the morphological assessment of a PB smear to aid in improving the identification of pPCL among NDMM patients.

Blood mass spectrometry detects residual disease better than standard techniques in light-chain amyloidosis.

In patients with immunoglobulin light-chain (AL) amyloidosis, depth of hematologic response correlates with both organ response and overall survival. Our group has demonstrated that screening with a matrix-assisted laser desorption/ionization-time-of-flight (TOF) mass spectrometry (MS) is a quick, sensitive, and accurate means to diagnose and monitor the serum of patients with plasma cell disorders. Microflow liquid chromatography coupled with electrospray ionization and quadrupole TOF MS adds further sensitivity. We identified 33 patients with AL amyloidosis who achieved amyloid complete hematologic response, who also had negative bone marrow by six-color flow cytometry, and who had paired serum samples to test by MS. These samples were subjected to blood MS. Four patients (12%) were found to have residual disease by these techniques. The presence of residual disease by MS was associated with a poorer time to progression (at 50 months 75% versus 13%, p = 0.003). MS of the blood out-performed serum and urine immunofixation, the serum immunoglobulin free light chain, and six-color flow cytometry of the bone marrow in detecting residual disease. Additional studies that include urine MS and next-generation techniques to detect clonal plasma cells in the bone marrow will further elucidate the full potential of this technique.