FLC Assay Research Articles

Complete responses in AL amyloidosis are unequal: the impact of free light chain mass spectrometry in AL amyloidosis

AbstractAmyloidogenic serum free light chains (sFLCs) drive disease progression in AL amyloidosis. Matrix-assisted laser desorption/ionization time of flight mass spectrometry–based FLC assay (FLC-MS) has greater sensitivity than conventional sFLC assays allowing for the detection of serological residual disease. We report the utility of FLC-MS in a large series of patients with AL amyloidosis assessing the impact of FLC-MS negativity after treatment on overall survival (OS) and organ response rates. Serum samples were analyzed using FLC-MS at diagnosis and at 6 and 12 months after treatment. The impact of FLC-MS negativity over standard hematologic responses on survival and organ response was assessed. A total of 487 patients were included; 290 (59%) and 349 (71.5%) had cardiac and renal involvement, respectively. There was 100% concordance between the light chain (LC) fibril type and LC isotype identified by FLC-MS. At 6 and 12 months, 81 (16.6%) and 101 (20.7%) were FLC-MS negative. Of those achieving a conventional hematologic complete response (CR) at 6 and 12 months, 45 (27.7%) and 64 (39%) were FLC-MS negative. At 12 months, median OS for CR + FLC-MS negative was not reached vs 108 months in CR + FLC-MS positive (P = .024). At 12 months, 70% of patients with FLC-MS negativity (vs 50% FLC-MS positive) achieved a cardiac response (P = .015). In a multivariate analysis, FLC-MS negativity at 12 months was an independent predictor of better outcomes. FLC-MS can detect persistent monoclonal light chains in a significant proportion of patients in a conventional hematologic CR. FLC-MS assessment promises to be a new standard for response assessment in AL amyloidosis.

Evaluation of the siemens N Latex free light chain assay and comparison to Binding Site Freelite™ assay

Abstract Introduction/Objective The International Myeloma Working Group (IMWG) guidelines include serum free light chain (sFLC) level and κ/λ ratio as excellent indicators of clonality. The Binding Site Freelite ™ was the first FDA approved assay for quantitative measurement of sFLC, the assay was based on a mixture of polyclonal antibodies directed against a variety of FLC epitopes. The Siemens N-Latex assay employs a probe mixture of mouse monoclonal antibodies. Both assays can be run on nephelometers. We assessed the analytical performance of the N-Latex assays and compared it with the Freelite™ assays. Methods/Case Report Analytical accuracy, precision, reproducibility and linearity were evaluated according to the regulatory standards. Method comparison was performed with 220 clinical samples for statistic correlation and clinical concordance analysis. Results (if a Case Study enter NA) The N-Latex FLC κ and λ assays had coefficient variation of 1-5% with-in run and 3-8% between run precision. Accuracy was verified using assayed controls in the duration of 21 days. Within analytical measuring range, almost perfect linearity was achieved for both κ and λ FLC assays. In comparison study to Freelite™ with 220 clinical samples, good agreement in classification was observed for κ, λ and κ/λ ratio (Cohen’s κ 0.73, 0.82 and 0.87). Pearson correlation analysis showed correlation coefficient value r >0.90 for all the analytes. Conclusion The N-Latex FLC assay has good analytical performance, did not exhibit gross antigen excess and can be used in clinical practice. However, it showed markedly lower absolute values for κ/λ ratio compared with Freelite™. Our data demonstrated that although good clinical concordance between N-Latex and Freelite™ FLC assays was achieved, the absolute values from the two assay are not interchangeable. Further studies in modification of the assay-specific diagnostic (involved FLC/non-involved FLC) thresholds for smoldering multiple myeloma (SMM) and multiple myeloma (MM) are needed.

An evaluation of patient workflow in hematology clinics at a university hospital referred for the workup of monoclonal gammopathy of undetermined significance (MGUS).

207 Background: Monoclonal Gammopathy of Undetermined Significance (MGUS) is an asymptomatic premalignant condition that can be a precursor to multiple myeloma and lymphoproliferative disorders. There exists no best practice for the workup of MGUS, despite the number of patients diagnosed every year. We evaluated the workup of MGUS patients at a University Hospital Hematology clinic before and after implementation of an algorithm based on the Mayo Clinic Risk Stratification Model. Methods: This was a single-center IRB-approved retrospective study. Charts of 132 patients referred for MGUS were reviewed across two groups (A: June 2019 -May 2020 and B: June-Dec 2020). Use of the Mayo Clinic Risk Stratification Model for MGUS was implemented in May 2020. Data regarding initial work up, bone marrow studies and imaging were collected. Statistical analyses were performed using R software for computing (4.0.4). Results: Patient demographics and those who did not need further work up per the algorithm are summarized in table. All 86 new (100%) referrals had an initial CBC, creatinine, calcium, SPEP, and IFE while 7(8.1%) did not have an FLC assay. 43(50%) patients had a 24-hour urine protein electrophoresis. 65.1% (56/86) [group A: 26; group B: 30] met the criteria for no extended workup; 39.3% of these (22/56) underwent imaging while 7.1% (4/56) underwent bone marrow exam. After implementation of the algorithm, the number of patients who underwent imaging studies decreased from 50% (13/26) to 30% (9/30). Skeletal survey was the most ordered imaging modality (90.1%,20/22). Conclusions: Our study highlights the overutilization of imaging studies in low-risk MGUS patients. Approximately 50% of patients with MGUS are low risk with a lifetime risk of progression being less than 2%. In these patients avoiding extensive testing will minimize costs without adversely affecting clinical outcomes. We recommend a dedicated MGUS clinic to improve workup and monitoring of these patients. [Table: see text]

Normalization of the Serum Free Light Chain Ratio in Patients with Intact Immunoglobulin Multiple Myeloma Who Achieve Less Than Complete Response

BackgroundNormalization of the serum free light chain ratio (rFLC) following achievement of complete response (CR) denotes stringent CR in multiple myeloma (MM) and is associated with favorable prognosis. In contrast, the clinical significance of this phenomenon in patients not achieving CR has not been energetically studied. Only a few studies have reported better progression free survival (PFS) and overall survival (OS) in patients who achieved normalization of the rFLC at their best response than in those who did not. As monoclonal intact immunoglobulin (Ig) and monoclonal FLC are independent biomarkers for the neoplastic plasma cell burden in intact immunoglobulin MM (IIMM), it is not clear whether patients who failed to achieve CR but normalized the rFLC also had improved outcomes. Here, we retrospectively analyzed the prognostic significance of normalization of the rFLC in IIMM patients who did not achieve CR.Patients and methodsConsecutive patients with IIMM diagnosed and treated at the Kameda Medical Center from Jun 2007 to April 2017 were identified. IMWG response criteria were used to assess response. We excluded patients with negative immunofixation for monoclonal M-protein in the serum or urine at diagnosis and those who achieved CR. The FLC assay (FREELITE, The Binding Site, Birmingham, UK) was performed at least monthly as a part of routine clinical management. The following ranges were used for normal FLC values: κ FLC, 0.33-1.94 mg/dL; λ FLC, 0.57-2.63 mg/dL; and rFLC, 0.26-1.65.ResultsThere were 239 patients diagnosed with symptomatic MM, including 191 patients with IIMM and 48 patients with light chain MM (LCMM). Among 191 patients with IIMM, 78 achieved CR by chemotherapy; the remaining 113 patients were included in this analysis. No patient with LCMM achieving less than CR (n = 15) obtained normalization of rFLC during the whole treatment. The median observation period was 21.9 months (m) [range, 0.2-118.9 m] and the median age was 77.0 yr (range, 43.8-90.7 yr). Sixty-four of the 113 patients (56.6%) obtained a normal rFLC during the course of treatment. Patients who achieved a normal rFLC were significantly younger (median age, 75.3 vs 79.2 yr, P = 0.014), more frequently received autologous stem cell transplantation (15.6% vs 0%, P = 0.005), and had better response [very good partial response (VGPR) rate: 57.8% vs 22.4%, P < 0.001] than those who did not. No significant difference was seen between patients with and without normalization of the rFLC with regard to sex, heavy- or light-chain type, ISS category, revised ISS category, albumin, β2 microglobulin, or creatinine levels, hemoglobin concentration, lactate dehydrogenase abnormality, induction regimen, or administration of maintenance therapy. Patients with a normal rFLC had significantly longer PFS and OS than those without on univariate analysis (median PFS, 24.2 vs 6.6 m, P = 0.001; median OS, 59.6 vs 20.2 m, P < 0.001). Normalization of the rFLC retained its prognostic significance for both PFS and OS on multivariate analysis (both P < 0.001). We further examined the impact of achievement of a normal rFLC on PFS and OS in patients with a high or low baseline rFLC. Using receiver operating characteristic curve analysis, the optimal cutoff was 69.8; patients were divided into two groups based on the cutoff. As expected, patients with a higher baseline rFLC who achieved a normal rFLC had longer PFS and OS (median PFS, 29.7 vs 4.3 m, P < 0.001; median OS, 93.6 vs 15.8 m, P < 0.001), but those with a lower baseline rFLC did not (median PFS, 21.4 vs 7.1 m, P = 0.484; median OS, 53.9 vs 34.9 m, P = 0.120). In addition, patients with a lower baseline rFLC were more likely to achieve normal rFLC than those with a higher baseline rFLC, although the difference was not statistically significant (P = 0.074).ConclusionThis study demonstrated that achieving normalization of the rFLC during the course of treatment is robustly predictive for both PFS and OS in patients with IIMM who do not achieve CR. As monoclonal FLC is secreted independently of monoclonal intact Ig from neoplastic plasma cells, normalization of the rFLC also confers improved prognosis of patients with IIMM who had high levels of rFLC. These data indicate that serial measurement of FLC at all levels of response may be a valuable tool for accurate prognostication in IIMM even when achieving only VGPR or worse response to therapy. [Display omitted] DisclosuresKitadate:Fujimoto: Research Funding; Novartis: Research Funding; Kyowa Kirin: Research Funding; Otsuka: Research Funding; Toyama kagaku: Research Funding; Eisai: Research Funding; Asahi Kasei: Research Funding; Chugai: Research Funding; Pfizer: Research Funding.

Comparison of three different serum-free light-chain assays\u2014implications on diagnostic and therapeutic monitoring of multiple myeloma

The measurement of serum-free light chains (FLC) is standard of care in the diagnosis and management of multiple myeloma (MM). The revised international myeloma working group (IMWG) implemented the involved FLC/noninvolved FLC (iFLC/niFLC) ratio as a biomarker for MM requiring treatment. Recently, a new definition of high-risk smoldering MM (SMM) including iFLC/niFLC ratio was published. These recommendations were solely based on a single assay method (Freelite assay). Today, two additional assays, N Latex FLC and ELISA-based Sebia FLC, are available. Here, we report on a single-center-study comparing results of all three different assays for FLC correlation and its potential implications for diagnostic and clinical use. In total, 187 samples from 47 MM patients were examined, and determination of FLC was performed. Comparison analyses showed similar FLC results for Sebia FLC and N Latex FLC assay with markedly lower absolute values for κ/λ ratio compared with Freelite. Values of λ FLC exhibited high variability. The ratio of iFLC/niFLC showed significant discrepancies among these assays. Our data demonstrate that the three available assays may result in markedly discrepant results, and should not be used interchangeably to monitor patients. Furthermore, modifications of the assay-specific diagnostic (iFLC/niFLC) thresholds for SMM and MM are recommended.

Heavy/light chain assay in the monitoring of multiple myeloma

Serum protein (SPE) and immunofixation electrophoresis (IFE) have been extensively validated for the routine use of identifying, characterising and quantifying monoclonal proteins. However, accurate quantitation of IgA monoclonal proteins can be difficult when they migrate in to the β fraction, due to co-migration with transferrin and complement components. The heavy/light chain (HLC) immunoassay is an additional tool for measuring intact immunoglobulin monoclonal proteins. Therefore, we aimed to examine the clinical utility of the HLC assay for the disease monitoring of IgG and IgA multiple myeloma (MM) patients. A total of 177 samples from 30 MM patients (21 IgG and 9 IgA) were analysed retrospectively with median number of six follow up samples per patient (range 3-13). Serum free light chains (sFLC) and HLC were quantified using Freelite and Hevylite immunoassays. Details of M-protein concentration, β-globulin levels, total immunoglobulin levels and disease treatment response were obtained from the laboratory and patient information system. Passing-Bablok regression analysis was performed to compare (i) M-protein quantification with involved HLC (iHLC) and (ii) total immunoglobulin with summated HLC pairs for each immunoglobulin type (e.g., IgGκ+IgGλ). For 127 IgG MM samples, IgG iHLC levels showed a good correlation with SPE quantification (iHLC y=0.96x+4.9; r=0.917) and summated HLC showed a good correlation with total IgG concentration (summated HLC y=0.94x+5.74; r=0.91). In total, 95/127 (75%) IgG MM follow-up samples had an abnormal HLC ratio and 122/127 (96%) had a positive SPE, probably due to the lower sensitivity of HLC assay in detecting clonality in patients with IgG MM. Consistent with this, one patient assigned a very good partial response by International Myeloma Working Group criteria would be assigned a complete response based on HLC measurements. For 50 IgA MM samples, 42/50 (84%) had an abnormal HLC ratio. Conversely, 50/50 (100%) of M-proteins showed β fraction migration and were difficult to accurately quantify by SPE. Therefore, M-protein concentration and iHLC did not correlate as well in IgA MM (y=1.9x-8.4; r=0.8) compared to IgG MM. However, there was good correlation between total IgA and summated IgA HLC (IgAκ+IgAλ y=1.35x-0.33; r=0.95). Of the 8/50 (16%) IgA samples with a normal HLC ratio, 6/8 (75%) were consistent with the disease status being in complete remission. Interestingly, in one IgA MM patient, SPE and IFE were negative, but the serum FLC ratio and involved FLC were highly abnormal, consistent with the presence of light chain escape. Our data suggest HLC measurements could add value to the current disease monitoring of MM patients. In IgG MM patients, the M-protein level correlated well with HLC values. The HLC assay complements the serum FLC assay and is especially useful for monitoring of IgA MM patients who display M-proteins migrating in the β region on SPE.

QIP-MS: A specific, sensitive, accurate, and quantitative alternative to electrophoresis that can identify endogenous m-proteins and distinguish them from therapeutic monoclonal antibodies in patients being treated for multiple myeloma

Serum protein (SPE) and immunofixation electrophoresis (IFE) have been extensively validated for the routine use of identifying, characterising and quantifying monoclonal proteins. However, accurate quantitation of IgA monoclonal proteins can be difficult when they migrate in to the β fraction, due to co-migration with transferrin and complement components. The heavy/light chain (HLC) immunoassay is an additional tool for measuring intact immunoglobulin monoclonal proteins. Therefore, we aimed to examine the clinical utility of the HLC assay for the disease monitoring of IgG and IgA multiple myeloma (MM) patients.A total of 177 samples from 30 MM patients (21 IgG and 9 IgA) were analysed retrospectively with median number of six follow up samples per patient (range 3–13). Serum free light chains (sFLC) and HLC were quantified using Freelite and Hevylite immunoassays. Details of M-protein concentration, β-globulin levels, total immunoglobulin levels and disease treatment response were obtained from the laboratory and patient information system. Passing–Bablok regression analysis was performed to compare (i) M-protein quantification with involved HLC (iHLC) and (ii) total immunoglobulin with summated HLC pairs for each immunoglobulin type (e.g., IgGκ+IgGλ).For 127 IgG MM samples, IgG iHLC levels showed a good correlation with SPE quantification (iHLC y=0.96x+4.9; r=0.917) and summated HLC showed a good correlation with total IgG concentration (summated HLC y=0.94x+5.74; r=0.91). In total, 95/127 (75%) IgG MM follow-up samples had an abnormal HLC ratio and 122/127 (96%) had a positive SPE, probably due to the lower sensitivity of HLC assay in detecting clonality in patients with IgG MM. Consistent with this, one patient assigned a very good partial response by International Myeloma Working Group criteria would be assigned a complete response based on HLC measurements. For 50 IgA MM samples, 42/50 (84%) had an abnormal HLC ratio. Conversely, 50/50 (100%) of M-proteins showed β fraction migration and were difficult to accurately quantify by SPE. Therefore, M-protein concentration and iHLC did not correlate as well in IgA MM (y=1.9x–8.4; r=0.8) compared to IgG MM. However, there was good correlation between total IgA and summated IgA HLC (IgAκ+IgAλ y=1.35x–0.33; r=0.95). Of the 8/50 (16%) IgA samples with a normal HLC ratio, 6/8 (75%) were consistent with the disease status being in complete remission. Interestingly, in one IgA MM patient, SPE and IFE were negative, but the serum FLC ratio and involved FLC were highly abnormal, consistent with the presence of light chain escape.Our data suggest HLC measurements could add value to the current disease monitoring of MM patients. In IgG MM patients, the M-protein level correlated well with HLC values. The HLC assay complements the serum FLC assay and is especially useful for monitoring of IgA MM patients who display M-proteins migrating in the β region on SPE.

Evaluation of a new free light chain ELISA assay: bringing coherence with electrophoretic methods.

Serum free light chain (sFLC) measurements are increasingly important in the context of screening for monoclonal gammopathies, prognostic stratification, and monitoring of therapy responses. At the same time, analytical limitations have been reported with the currently available nephelometric and turbidimetric sFLC assays. We have evaluated a new quantitative sFLC ELISA for its suitability in routine clinical use. Reference ranges of the Sebia FLC assay were calculated from 208 controls. Assay interference, reproducibility, lot-to-lot variability, and linearity were assessed. Method comparison to the Freelite assay (Binding Site) was conducted by retrospective analysis of 501 patient sera. Reference ranges of the Sebia κ/λFLC-ratio were 0.37-1.44. We observed good sensitivity (1.5 mg/L) and linearity in both polyclonal and monoclonal sFLC samples and never experienced antigen excess. Sebia FLC reproducibility varied between 6.7% and 8.1% with good lot-to-lot consistency. Method comparison with Freelite showed the following correlations: κFLC R=0.94, λFLC R=0.92 and κ/λFLC-ratio R=0.96. The clinical concordance of the κ/λFLC-ratio of both methods was 94%. Significant quantitative differences were observed between both methods, mainly in sera with high FLC concentrations. The Sebia monoclonal FLC concentrations were coherent with those obtained by serum protein electrophoresis (SPE). Freelite monoclonal FLC concentrations were consistently higher, with a mean 12-fold overestimation compared to SPE. The Sebia FLC assay provides a novel platform for sensitive and accurate sFLC measurements. The Sebia FLC showed good clinical concordance with Freelite. Further studies are warranted to confirm the clinical value of this assay.

Comparison of the Freelite serum free light chain (SFLC) assay with serum and urine electrophoresis/immunofixation and the N Latex FLC assay

Few reports have compared available serum free light chain (SFLC) assays. Here, a retrospective audit of the Freelite SFLC assay compared results to electrophoresis (EP)/immunofixation (IFX) and the N Latex FLC assay.A total of 244 samples collected over 3.5 months were studied using the Freelite and N Latex FLC nephelometry assays. Results were compared with serum and/or urine EP/IFX. The precision and linearity of the N Latex FLC assay was examined.Detectable paraprotein by serum or urine EP/IFX was present in 94% of samples with kappa and 100% with lambda FLC restriction. The correlation between the assays was higher for kappa (rho = 0.97) than lambda (rho = 0.89) especially when lambda results were above the upper limit of normal (rho = 0.62). Agreement in the categorical diagnosis as measured by the Cohen's kappa statistic was good (0.70). The N Latex FLC assay displayed good precision and linearity. In discordant samples the Freelite and N Latex FLC assays had equivalent agreement with IFX.Traditional methods of EP/IFX detected paraproteins in the majority of cases. Correlation between the Freelite and N Latex FLC assay is better for kappa than lambda FLC. The two assays are not entirely equivalent. Care should be taken by interpreting physicians and laboratories considering switching assays.

Assessment of time from onset of first symptoms to the final diagnosis of multiple myeloma (MM) - possible risks and future solutions

Assessment of time from onset of first symptoms to the final diagnosis of multiple myeloma (MM) - possible risks and future solutions

IgD multiple myeloma: Clinical, biological features and prognostic value of the serum free light chain assay

IgD multiple myeloma (MM) is a rare subtype of myeloma, it affects less than 2% of patients with MM. To evaluate the clinical and prognostic attributes of serum free light chains (sFLCs) analysis, we examined 17 cases of IgD MM. From 1998 to 2012, we obtained 1250 monoclonal gammapathies including 590 multiple myeloma and 17 patients had IgD MM. With preponderance of men patients with a mean age at diagnosis of: 59±12years. Patients with IgD MM have a short survival (Median survival=9months). The presenting features included: bone pain (75%), lymphadenopathy (16%), hepatomegaly (25%), splenomegaly (8%), associated AL amyloidosis (6%), renal impairment function (82%), infections (47%), hypercalcemia (37%) and anemia (93%). Serum electrophoresis showed a subtle M-spike (Mean=13.22±10g/L) in all patients associated to a hypogammaglobulinemia. There was an over-representation of Lambda light chain (65%); high serum β2-microglobulin in 91% and Bence Jones proteinuria was identified in 71%. The median rate of sFLCs κ was 19.05mg/L and 296.75mg/L for sFLCs λ. sFLCR was abnormal in 93% of patients and it showed concordance between baseline sFLCR and the survival (P=0.034). The contribution of FLC assay is crucial for the prognosis of patients with IgD MM.

Retrospective audit of the freeliteTM serum free light chain (SFLC) assay: testing patterns, concordance with serum and urine electrophoresis/immunofixation and correlation with the N latex FLC assay

Aims A retrospective audit of the Freelite™ SFLC assay was conducted to compare concordance with electrophoresis (EPG)/immunofixation (IFX) and correlation with the N latex FLC assay. Methods 244 samples collected over 3.5 months were studied by nephelometry using the Freelite™ and N Latex FLC assays. Results were compared with serum (S) and/or urine (UEPG)/IFX. The precision and linearity of the N latex FLC assay were examined. Results 94% of samples with kappa restriction, and 100% with lambda restriction had detectable paraprotein by SEPG/IFX or UEPG/IFX. The correlation between the two assays was good for kappa (R 2 = 0.935) but not lambda (R 2 = 0.775) especially when lambda was above the ULN (R 2 = 0.406). Agreement in the categorical diagnosis of restricted SFLC was good (Cohen's kappa = 0.701). In discordant samples the Freelite™ assay displayed higher agreement with IFX (53% vs 47%). The N latex FLC assay displayed good precision and linearity. Discussion SFLC testing is popular despite traditional methods detecting paraproteins in most cases. Correlation between the Freelite™ and N latex FLC assays is better for kappa than lambda FLC. The two assays are not equivalent and the Freelite™ assay displays better agreement with IFX. Care should be taken by interpreting physicians and laboratories considering switching assays.

Phase II Trial Of Initial Safety and Toxicity Prior To The Phase III Trial Of Lenalidomide Versus Observation Alone In Patients With Asymptomatic High-Risk Smoldering Multiple Myeloma (E3A06): A Trial Coordinated By The Eastern Cooperative Oncology Group

The treatment and natural history of smoldering myeloma has been an area of intense preclinical and clinical study. Historical attempts to treat these patients have not demonstrated benefit, likely as a consequence of lack of attention to the vast heterogeneity contained within the diagnosis. More recent risk criteria from both US and Spanish investigators have identified a cohort of smoldering myeloma patients at high risk for progression in a short time, using different methodology. Recently the PETHEMA group published a randomized clinical trial testing lenalidomide/dexamethasone vs observation among high risk smoldering patients, and demonstrated a clear benefit in terms of progression free and overall survival favoring early intervention. However, the risk criteria utilized a highly sensitive method of flow cytometry, a test not routinely available in the US. It is not clear that the group of patients defined in the PETHEMA study share similar outcomes with those defined as high risk using the ECOG/Mayo criteria. A recent publication demonstrated only 25% overlap for the highest risk smoldering patients when using either the PETHEMA or the ECOG criteria. To further evaluate the potential benefit of early intervention in a high risk smoldering cohort, the ECOG myeloma group designed a phase II/III trial testing the safety and efficacy of single agent lenalidomide. We report here on the safety and efficacy of the phase II portion of the trial.The phase II group of patients received lenalidomide alone at a dose of 25 mg days 1-21 every 28 days. The primary endpoint for the safety study was the rate of any treatment-related grade 4-5 non-hematologic toxicity plus grade 3 non-hematologic toxicity that affects vital organ function (such as cardiac, hepatic, or thromboembolic events) observed within 6 cycles of treatment. Patients were to be diagnosed with high-risk smoldering multiple myeloma (SMM) within the past 12 months, as confirmed by both of the following: bone marrow plasmacytosis with >= 10% plasma cells or sheets of plasma cells and an abnormal serum free light chain ratio (<0.125 or >8.0) by serum FLC assay. Patients must have had measurable levels of monoclonal protein. Patients must have had no lytic lesions on skeletal surveys and no hypercalcemia (i.e. >= 11 mg/dL).Among the final phase II cohort (n=44 patients), 55% were female, and 43% were 65 years and older. Enrollment began in January 2011 and lasted 2 years with the last patient completing 6 cycles of treatment in August 2013. The median treatment duration of the entire cohort is 13.5 cycles (range 1-30 cycles). Mean percentage lenalidomide dose over the first 6 cycles was 94, 89, 82, 79, 76, 72. With a median follow up of 17 months, 2 patients had progression on therapy. Fifteen patients are off treatment for the following reasons: disease progression (n=2), AE/complication (n=5), death (n=2), patient withdrawal/refusal (n=5), other (n=1); 6 of the 15 patients ended treatment before completing 6 cycles. Of 44 patients assessed for toxicity, 11 patients [25.0%, 95% CI: (13.2%-40.3%)] experienced worst grade treatment-related non-hematologic toxicity of grade 3 or higher based on CTCAE v4, with neutropenia and fatigue being the most frequent. This includes 2 fatal deaths (MI and TE). Two patients [4.6%, 95% CI: (0.1%-15.5%)] experienced a serious adverse event as defined for the purposes of the phase II toxicity analysis that included treatment-related grade 4-5 non-hematologic toxicity or any grade 3 toxicity affecting vital organ function (such as cardiac, hepatic, or thromboembolic events) during the first 6 cycles of treatment. Twelve patients achieved partial response or better [33.3%, 95% CI: (15.0%-42.8%)] along with 25 patients reporting stable disease. Lenalidomide has shown promise in the treatment of high-risk smoldering myeloma and based on this analysis, accrual to the phase III portion is ongoing. In the context of the encouraging data from the PETHEMA group, completion of the phase III portion of this study is needed to validate their findings using a different criteria, and using a steroid sparing approach.This study was coordinated by the Eastern Cooperative Oncology Group (Robert L. Comis, M.D., Chair) and supported in part by Public Health Service Grants CA23318, CA66636, CA21115, CA13650, CA32102 and from the National Cancer Institute, National Institutes of Health and the Department of Health and Human Services. Disclosures:Lonial:Millennium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; BMS: Consultancy; Sanofi: Consultancy; Onyx: Consultancy.

Usage of HLC-Ratio, FLC-Ratio, Ife, PBMC Infiltration and Isotype Suppression At Best Response Reveals Isotype Suppression As Most Powerful Parameter for Identification of Multiple Myeloma Patients with Long Survival

AbstractAbstract 1817 Introduction:Identifying patients with optimal response and long survival is important for clinical guidance because patients with these features are likely not to need further therapy. The techniques applied for classifying these patients should be readily available, standardized, and not liable to subjective interpretation. Here we compare the clinical usability of Hevylite™ (HLC) assay, FLC assay, IFE (immunofixation electropheresis), PMPC (bone marrow plasma cell) infiltration and isotype suppression for identifying patients at their best response with long term survival. Methods and Patients:65 multiple myeloma patients (median age at maximum response 64, range 33–85; 42 IgG, 23 IgA) were enrolled following the minimum assignment of very good partial response using international myeloma working group guidelines. Patients had been enrolled into various clinical trials and been treated with different induction protocols (VAMP, VMCP+IFNa2b, Thal-Dex, MP, VMP). Median follow was 4.5 years, range 0.5–12 years. Heavy/light chain analysis was retrospectively performed for the monoclonal plasma cell immunoglobulin and its isotype matched pair (42 IgGκ / IgGλ and 23 IgAκ / IgAλ) using commercially available immunoassays (Hevylite™, The Binding Site, Birmingham, UK). Isotype matched immunoparesis was recorded if the patients immunoglobulin concentration was 33% below the bottom of the normal range (IgGk, IgGl, IgAk, IgAl), similarly immunoparesis was assessed if the patients total immunoglobulin levels were 33% below the normal range (IgG, IgA, IgM). Results were compared to bone marrow biopsy, serum free light chain (Freelite™, The Binding Site, Birmingham, UK) and standard immunoglobulin assays. Overall survival was estimated by the product limiting method of Kaplan Meier and survival compared using the log rank test, proportional hazards were assessed using the Cox proportional hazard model. Results:Comparison of patients at maximum response with and without IF-positivity, abnormal HLC ratios, abnormal FLC ratios or BMPC infiltration >5% did not reveal significantly different survival rates. Only patients with an abnormal HLC ratio showed a tendency for shorter survival (table 1). When all markers were combined a difference in the 5 year survival rate was noted (50% as compared to 100%), but due to the limited power, the statistical analysis revealed a tendency for reduced survival only.Discrimination of patients according to HLC pair suppression produced a highly significant difference in overall survival with a 5 year survival rate of 43% compared to 70% (figure 1). Median overall survival was 4.8 years vs. 8.5 years (HR, 2.5 CI: 1.1–5.54,p <0.02). In contrast, suppression of the non-involved isotype immunoglobulin (i.e. IgG concentrations in an IgA MM patient) was not found to be associated reduced overall survival (IgG or IgA, p=0.75, IgM, p=0.9)AssessmentnHazard Ratio (CI)P ValueAIFE611.23 (0.6–2.5)0.57B>5% BM infiltration32/610.69 (0.34–1.4)0.3CAbnormal FLC ratio36/651.49 (0.54–2.4)0.7DAbnormal HLC ratio27/651.65 (0.78–3.3)0.2EHLC chain pair suppression16/652.5 (1.15–5.54)<0.02A+B+C+D+E4/6124.4 (0.95–6116)0.064Discussion:These findings show the importance of immunoparesis at time of maximum response. This phenomenon is specific to the non-involved isotype matched immunoglobulin suggesting a preferential isotype specific inhibition mechanism not previously identified. Interestingly, among the other parameters only abnormal heavy light chain ratio showed a tendency for shorter survival. However, when all of the factors were considered together a small population (4/61, 7%) was identified as having an exceptionally long remission (OS, median: 5.9 years (2.0 – 10.2) Conclusion:The non-involved isotype matched pair suppression has previously been shown to be a prognostic factor in MGUS transformation and in predicting progression free survival in MM patients. Here for the first time we identify this marker as being the most important marker in predicting outcome at maximum response. [Display omitted] Disclosures:Carr-Smith:Binding Site: Employment. Hughes:Binding Site: Employment. Harding:Binding Site: Employment.

FLC Assay and Multiparameter Flowcytometry Based Clonal Plasma Cell Measurement Are Independent but Complementary in Assessing the Treatment Response in Multiple Myeloma.

Abstract 2969 Purpose:Although stringent complete response (sCR) defined by paraprotein negativity on immunofixation and serum free light chain (sFLC) ratio normalization are considered deeper responses in the IMWG criteria, recent report indicated that Multiparameter flow cytometry (MFC)-dased immunophenotypic response (IR) is a more relevant prognostic factor in MM patients. However, data on the prognostic impact of IR and sFLC ratio (sFLCκ/λ) normalization are still scarce. We investigated the prognostic impact of IR and sFLCκ/λ normalization in MM patients treated with novel agents. Patients and Methods:A total of 124 consecutive patients (M:F=68:56; median age, 71 yr) were treated by chemotherapy regimens containing at least one novel agent (thalidomide, bortezomib, lenalidomide)from April 2005 to May 2012. Treatment responses were assessed using the IMWG criteria, and the best response to treatment during the clinical course was assessed by simultaneous serum immunofixation, sFLC measurements, and MFC analysis of bone marrow (BM) plasma cells. Normalization of sFLCκ/λ was defined 2 consecutive normal sFLCκ/λ apart from at least 4 weeks. MFC-defined minimal residual disease (MRD) was evaluated by single-tube 6-color MFC, CD45-CD38 gating strategy, and combination CD19, CD56, and cytoplasmic κ-λ analysis. Clonal plasma cell (PC) negativity by MFC (MFC-negative) was defined as <10−4 neoplastic PCs in BM samples on MFC. Overall survival (OS) and progression-free survival (PFS) were analyzed by the Kaplan–Meier (K-M) method and differences between curves were calculated by two-sided log-rank test. Univariate analysis was used to assess the impacts of factors on sFLCκ/λ normalization and MFC negativity (age, Durie–Salmon stage, ISS stage, LDH, hemoglobin, serum albumin, serum creatinine, FISH at diagnosis). The Cox regression proportional hazard model (stepwise regression) was used to explore the independent effects of these variables on PFS and OS. Results:At a median follow-up of 25.8 months, 3- and 5-year OS of all patients were 61.0% and 42.4%, respectively. CR was obtained in 25% (31/124), very good partial response (VGPR) in 33.5% (41/124), partial response (PR) in 30.5% (38/124), and stable disease or less (SD) in 11% (14/124). Normal sFLCκ/λ was achieved in 81% of CR, 56% of VGPR, 13% of PR, and 0% of SD or less response of patients. K-M estimated 3- and 5-year OS were 100% in CR patients; these were significantly better than in VGPR (75.8% and 43.2%, respectively) and PR patients (63% and 26.7.%, respectively). There were no significant differences in 3- or 5-year OS between VGPR and PR patients. Normal sFLCκ/λ and MFC negativity were achieved in 25 (81%) and 18 (58%) of 31 CR patients, respectively. Among 25 CR patients with normal sFLCκ/λ (stringent CR), 15 (60%) were MFC-negative and 10 (40%) were MFC-positive; three of 6 CR patients (50%)without normal sFLCκ/λ were MFC-positive. Twenty-three of 41 VGPR patients (56%) obtained normal sFLCκ/λ, while only 5 (12%) became MFC-negative; all 5 MFC-negative patients also obtained normal sFLCκ/λ. Among 52 patients with less than PR, only 5 (9.6%) obtained normal sFLCκ/λ and none achieved MFC negativity. Patients with MFC-negative CR showed significantly better PFS than patients with MFC-positive CR (p<0.05). Although patients in stringent CR with MFC-negative showed slightly better PFS compared to patients in stringent CR with MFC-positive, difference between the curves were not significant. Within the group of VGPR, PFS and OS were significantly longer in normal sFLCκ/λ patients than abnormal sFLCκ/λ(P<0.001). Univariate analysis showed that hemoglobin 10.0 g/dl>, age >70 yr, and abnormal LDH had negative prognostic impacts on attaining normal sFLCκ/λ, but none of these factors remained significant on multivariate analysis. Cox analysis showed that sFLCκ/λ normalization was an independent prognostic factor for longer PFS and OS in patients with CR, VGPR and PR (P=0.001). Conclusions:This study confirmed that magnitude of CR and VGPR response defined by IMWG criteria was heterogeneous in terms of sFLCκ/λ normalization and MFC negativity. Although MFC and sFLC analysis frequently gave discrepant results among patients with CR and VGPR, both analyses appeared to give important complementary information for assessing the depth of CR and VGPR category. Disclosures:No relevant conflicts of interest to declare.

Phase II Trial of Initial Safety and Toxicity Prior to the Phase III Trial of Lenalidomide Versus Observation Alone in Patients with Asymptomatic High-Risk Smoldering Multiple Myeloma (E3A06): A Trial Coordinated by the Eastern Cooperative Oncology Group

AbstractAbstract 4079The treatment and natural history of asymptomatic or smoldering myeloma has been an area of intense preclinical and clinical study. Historical attempts to treat these patients have not demonstrated significant benefit, likely as a consequence of limited therapeutic options as well lack of attention to the vast heterogeneity contained within the diagnosis of smoldering myeloma. More recent risk criteria from both US and Spanish investigators have identified a cohort of smoldering myeloma patients at high risk for progression to myeloma in a short time. Recently the PETHEMA group has conducted a randomized clinical trial testing lenalidomide and dexamethasone vs observation among high risk smoldering patients and has demonstrated a clear benefit in terms of progression free survival and hints towards improvement in overall survival favoring early intervention. To further evaluate the potential benefit of early intervention in a high risk smoldering cohort, the ECOG myeloma group designed a phase II trial testing the safety and efficacy of single agent lenalidomide with the intent of broadening to a randomized phase III trial if toxicity was acceptable. We report here on the safety portion of the phase II trial.The phase II group of patients received lenalidomide at a dose of 25 mg days 1–21 every 28 days to evaluate the early toxicity and tolerance of this dosing. The dose of lenalidomide could be adjusted based on toxicity using a defined dose-modification guideline in the protocol. The primary endpoint for the safety study was the rate of any treatment-related grade 4–5 non-hematologic toxicity plus grade 3 non-hematologic toxicity that affects vital organ function (such as cardiac, hepatic, or thromboembolic events) observed within 6 cycles of treatment. The goal was to enroll 34 patients; if 9 or more patients experience toxicity as defined, then the study would not continue. In terms of eligibility, patients were to be diagnosed with asymptomatic high-risk smoldering multiple myeloma (SMM) within the past 12 months, as confirmed by both of the following: bone marrow plasmacytosis with >= 10% plasma cells or sheets of plasma cells and an abnormal serum free light chain ratio (<0.125 or >8.0) by serum FLC assay. Further, patients must have measurable levels of monoclonal protein (M-protein): >=1g/dL on serum protein electrophoresis or >=200 mg of monoclonal protein on a 24 hour urine protein electrophoresis. Patients must have no lytic lesions on skeletal surveys and no hypercalcemia.Among the 36 patients enrolled in the phase II study, 56% were female, and 44% were 65 years and older. Treatment and toxicity data at a minimum through cycle 6 is complete as of August 2, 2102. The last patient enrolled completed cycle 6 treatment on June 7, 2012. The median treatment duration of the entire cohort is 9 cycles (range 1–18 cycles), with 86% of patients completing at least 6 cycles of treatment. Ten patients are off treatment for the following reasons: disease progression (n=1), AE/complication (n=5), death (n=1) and patient withdrawal/refusal (n=3); 5 of the 10 patients ended treatment before completing 6 cycles. Of 36 patients assessed for toxicity, 8 patients [22.2%, 90% CI: (11.6%-36.5%)] experienced worst grade treatment-related non-hematologic toxicity of grade 3 or higher. Separately, 6 patients experienced unrelated non-hematologic toxicity of grade 3 or higher. Three patients [8.3%, 90% CI: (2.3%-20.2%)] experienced a serious adverse event as defined for the purposes of the phase II toxicity analysis (treatment-related grade 4–5 non-hematologic toxicity or grade 3 non-hematologic toxicity that affects vital organ function (such as cardiac, hepatic, or thromboembolic events). Based upon this analysis of the study, the phase II portion of the trial met the prespecified safety benchmark established to allow for phase III expansion, and accrual to the phase III portion of this study will begin. Efficacy data will be presented at the time of presentation. Disclosures:Lonial:Novartis: Consultancy; Millennium: Consultancy; Onyx: Consultancy; BMS: Consultancy; Celgene Corp: Consultancy; Merck: Consultancy. Off Label Use: Lenalidomide is not approved for treatment of smoldering MM. Fonseca:Medtronic: Consultancy; Otsuka: Consultancy; Celgene: Consultancy; Genzyme: Consultancy; BMS: Consultancy; Lilly: Consultancy; Onyx: Consultancy; Binding site: Consultancy; Millenium: Consultancy; AMGEN: Consultancy; Celgene : Research Funding; Onyx: Research Funding; prognostication of MM based on genetic categorization of the disease: Prognostication of MM based on genetic categorization of the disease, Prognostication of MM based on genetic categorization of the disease Patents & Royalties. Dhodapkar:Celgene: Research Funding; KHK: Research Funding.

Relationship between monoclonal gammopathy and cardiac amyloid type

Proper identification of cardiac amyloid type is essential for patient management, and has historically relied upon immunohistochemical- or immunofluorescence-based methods, often correlated with serum and urine protein electrophoresis (SPEP and UPEP) with immunofixation electrophoresis (IFE), and/or free light chain immunoassay (FLC). The recent implementation of mass spectrometry-based proteomic analysis for clinical amyloid typing allows us to determine the validity of these tests to predict amyloid type. Validity of SPEP/UPEP/IFE and FLC assays in cardiac amyloid prediction was examined. Retrospective analysis of two tertiary care populations (n=143, 2001-2010), of cardiac biopsy-proven amyloidosis, was performed. Amyloid of transthyretin (ATTR) type was found in 81 (57%) of 143 patients and immunoglobulin light chain amyloid was found in the remaining 62 (43%). SPEP/UPEP/IFE detected a monoclonal gammopathy in 76 individuals, 56 with AL and 20 with ATTR amyloid and was overall a poor predictor of AL amyloid in this patient population: specificity (75%; 95% CI, 65-83%) and positive predictive value (PPV 74%; 95% CI, 63-82%). The FLC assay detected an abnormal kappa/lambda ratio in 61 patients, 53 with AL and 8 with ATTR amyloid and was a better predictor of AL amyloid type in this patient population: specificity (90%, 95% CI, 82-95%) and PPV (87%, 95% CI, 76-93%). ATTR was the predominant amyloid type in this large cohort of endomyocardial biopsies characterized by mass spectrometry. Although FLC performs better than SPEP/UPEP/IFE, the performance of blood and urine studies for monoclonal proteins are not adequate to classify amyloid type. This large-scale retrospective analysis of cardiac amyloidosis shows that blood and urine monoclonal protein studies are not, by themselves, robust predictors of cardiac amyloid type in patients undergoing endomyocardial biopsy.

Further stratification of patients with multiple myeloma by International Staging System in combination with ratio of serum free κ to λ light chains

The serum free light chain (sFlc) levels were measured for 122 Chinese patients with newly diagnosed symptomatic multiple myeloma (NDSMM), and κ/λ ratios (rFlc) were calculated. The data were analyzed for the roles of sFlc and rFlc in the diagnosis and prognosis of MM. Abnormal sFlc and/or rFlc were detected in 99.2% of patients, demonstrating that the FLC assay is much more sensitive than the commonly used methods. Baseline sFlc and rFlc successfully predicted the overall survival (OS). The median OS was not reached (NR) versus 23 months for the low sFLC group (sFLC-κ 180 mg/L or sFLC-λ 592.5 mg/L) and high sFLC group (sFLC-κ ≥ 180 mg/L or sFLC-λ ≥ 592.5 mg/L) (p = 0.001), and NR versus 21 months for the low rFLC group (0.04 ≤ rFLC ≤ 25) and high rFLC group (p 0.001), respectively. Interestingly, the significant differences in OS between the low and high rFLC groups were not changed by bortezomib chemotherapy. In addition, patients were further stratified by three novel poor-prognosis factors (β2-microglobulin [β2-MG] 3.5 mg/L, albumin [ALB] 35 g/L, rFLC 25 or rFLC 0.04) that were developed from combination of the rFlc with the International Staging System (ISS): the low risk group (no factor), the low-intermediate risk group (one factor), the high-intermediate risk group (two factors) and the high risk group (three factors). The median OS for those groups was NR, NR, 24 months and 13 months, respectively (p 0.05). In conclusion, the sFLC assay was highly sensitive in the diagnosis of MM in Chinese patients. The prognostic potential of the ISS may be improved with the addition of rFLC.

Extended Use of Serum Free Light Chain as a Biomarker in Lymphoproliferative Disorders

Serum free light chain (sFLC) assays were shown to improve detection, management, and prognostication in plasma cell disorders. Recently, sFLC assays improved detection of M proteins when combined with standard methods of protein electrophoresis/immunofixation in patients with non-Hodgkin lymphoma/chronic lymphocytic leukemia (NHL/CLL). Incidence of abnormal sFLC ratio (sFLCr) varied from 0% to 36% and 29.7% to 59% in NHL and CLL, respectively. Increased sFLC levels or abnormal sFLCr predict shorter overall survival in early-stage CLL. Furthermore, abnormal sFLCr correlated with advanced disease stage and poorer outcome. In diffuse large B-cell lymphomas, increased sFLC was demonstrated as an independent, adverse prognostic factor for overall/event-free survival. Moreover, abnormal sFLCr can be a diagnostic tool in central nervous system lymphomas. Finally, the quantitative FLC assay has the potential to become a new, easily measured biomarker for predicting prognosis and enhanced detection in NHL/CLL. It may be used serially at follow-up evaluations to provide clues to relapse.

Serum Free Light Chain Ratio in Distinguishing Smoldering Multiple Myeloma From Active Multiple Myeloma,

Abstract 3948▪▪This icon denotes a clinically relevant abstract Background:Smoldering multiple myeloma (SMM) is an asymptomatic precursor disease of multiple myeloma, and is defined by excess bone marrow plasma cells and monoclonal protein without evidence of end-organ damage (hypercalcemia, renal insufficiency, anemia, or bone lesions [CRAB]). The identification of SMM patients with more aggressive underlying disease remains a challenge. We hypothesize that SMM is a clinical entity comprised of both premalignant, high-risk MGUS and early multiple myeloma in transition to malignant disease, which may be differentiated with the use of the serum FLC (FLC) ratio. Methods:This was a retrospective analysis of 586 patients with newly diagnosed SMM from 1970–2010 with available stored serum samples around the time of diagnosis to be utilized for quantification of FLC ratios. SMM was defined by the International Myeloma Working Group 2003 definition; serum M-protein ≥ 3 g/dL and/or ≥ 10% bone marrow plasma cells with no evidence of CRAB features. The immunoglobulin FLC assay (Binding Site, U.K.) was used for testing. The FLC ratio was calculated as κ/λ (reference range 0.26–1.65). The involved/uninvolved FLC ratio was recorded to simplify the reporting of data. Receiver Operating Characteristics (ROC) curves were created to assess the ability of the FLC ratio to discriminate patients who progressed to symptomatic multiple myeloma (MM) in the first 2 years or at any point during follow-up versus patients without evidence of progression. Patients with less than 24 months follow-up without progression were censored. The optimal diagnostic cut-point for FLC involved/uninvolved ratio to identify patients with progressive disease from the ROC curve was >88.6 (equivalent to <0.011 or >88.6). For ease of clinical application, the optimal value for involved/uninvolved FLC ratio was rounded to >100. Time to progression (TTP) from date of the initial FLC to active MM was calculated using Kaplan-Meier analysis and compared to patients with a high (>100) and low (<100) involved/uninvolved FLC ratio at time of SMM diagnosis. TTP within 24 months of the initial FLC was also calculated. Results:During the study period, 54% of patients progressed to active MM. On ROC analysis, a cut-point of >100 corresponded to a sensitivity of 25% (95% CI, 20.5–30.4) and specificity of 99.3% (97.3–99.9), with positive likelihood (+LR) ratio of 33.9 (38.1–41.0), negative likelihood ratio (−LR) of 0.75 (0.2–3.0), positive predictive value (PPV) of 97.6 (91.5–99.7) and negative predictive value of 53.0 (48.5–57.4). Using the ROC to assess progression to MM within 24 months (Figure 1), sensitivity was 29.6% (23.5–36.4), specificity 94.5% (91.7–96.5), +LR 5.36 (4.3–6.6), -LR 0.75 (0.5–1.1), PPV 85.8 (77.7–91.8), and NPV 54.3 (49.8–58.9). Median TTP to active MM in the FLC >100 group was 15 months (9–17) versus 52 months (44–60) in the FLC <100 group (p <.0001) [Figure 2]. In the FLC ratio >100 group, progression at 1 year was 47%, 76% at 2 years, and 90% at 3 years. Only 25% of the FLC <100 patients had progressed at 2 years. The most common progression event was bone disease (42%), followed by anemia (26%), renal impairment (23%), and hypercalcemia (5%). [Display omitted] Conclusion:Elevation of the FLC ratio >100 (or <0.01) is highly specific for the future development of active MM, with 76% of these patients developing end-organ damage requiring therapy within 2 years. Risk of transformation to MM in the FLC <100 group was similar to previously reported rates of 10% per year for the first 5 years. Development of an FLC ratio >100 is associated with increasing disease burden and in this study behaved in a malignant fashion rather than a precursor state. The FLC is a simple and useful predictor of progression to MM in SMM, and patients with FLC ratios of <0.01 or >100 within the first 2 years of SMM diagnosis should be monitored especially closely. Future studies are needed to determine optimum cutoffs for FLC ratio to where a change in definition of MM could be considered. Disclosures:No relevant conflicts of interest to declare.