Katzmann, Jerry A.

Abstract Background: Monoclonal gammopathy of undetermined significance (MGUS) is defined by a monoclonal protein concentration in serum of 3 g/dL or less and a proportion of plasma cells in the bone marrow of 10% or less, absence of lytic bone lesions, anemia, hypercalcemia, and renal insufficiency related to the proliferation of the monoclonal plasma cells. Two major questions remain unanswered: What proportion of patients with MGUS in the population is unrecognized clinically? What is the likely duration of MGUS at the time of its discovery? Methods: We examined the serum of 21,463 (77%) of the 28,038 enumerated residents of Olmsted County, Minnesota, in a population-based screening study and found that MGUS was present in 1.3% of persons 50 years of age and 3.8% of those 70 years of age. The risk of progression of MGUS to multiple myeloma (MM), primary amyloidosis (AL), Waldenstrom’s macroglobulinemia (WM), or related plasma cell disorders was 1% per year. Results: By age 70, only 21% of Olmsted County residents with MGUS, as determined by the prevalence study, had been recognized by routine clinical practice (Fig. 1). This is probably higher in Olmsted County than elsewhere because serum protein electrophoresis is frequently ordered on Mayo Clinic patients. The incidence of MGUS, estimated from the prevalence data, indicates onset of the condition long before clinical recognition. The duration of MGUS prior to detection at age 70 is shown in Fig. 2. Thus, 30% of patients recognized to have MGUS at age 70 probably had MGUS onset before age 50 and 55% had a prior (unrecognized) MGUS already by age 60. The median duration of MGUS prior to its recognition on the basis of clinical practice is 11 years (median). Linear extrapolation of the incidence curve implies a first appearance of MGUS around 30 years of age. Figure 1 Figure 1 Figure 2 Figure 2

Abstract Background: Immunoglobulin free light chains (FLC) form the substrate for synthesis of amyloid fibrils in patients with AL amyloidosis. Development of FLC assay (Freelite) has allowed us to better assess the clonal cell burden in patients with AL amyloid. The relationship of the light chain types and their levels in serum to the clinical feature at presentation and the eventual outcome has not been systematically studied. Methods: We identified 730 patients with biopsy proven AL amyloidosis, who were seen at Mayo Clinic between January 1980 and July 2006, who were seen within 90 days of their diagnosis and in whom FLC levels were performed or had stored serum available for analysis. Cardiac biomarkers (cTnT, NT ProBNP) were also performed on stored serum for some of the patients as part of previous studies. Clinical data and follow up status are prospectively collected into the Dysproteinemia database, which was used for the study. Results: The median age of the study population was 63 years (range; 32–90) and 463 patients (63%) were male. The median estimated survival for the entire group was 37 months from diagnosis and the median follow up for the 263 patients (37%) alive at the time of the analysis was 27.8 months (range; 1–177 months). The plasma cell clone was lambda in 528 patients (72%) and kappa in 202 (28%) patients. The median absolute difference between the FLCs was 19.6 mg/dL for the entire group (range; 0.01–2478); for the kappa patients was 29.4 mg/dL (range; 0.01–1359) and for the lambda patients was 18.2 mg/dL (range; 0.03–2478). Kappa AL was associated with more involvement of the GI tract and liver as indicated by higher alkaline phosphatase (1.8 vs. 1.3 fold; P=0.03), higher total bilirubin (0.95 vs. 0.78 mg/dL, P = 0.04) and lower serum carotene (119 vs. 155 ug/dL; P = 0.0002). On the contrary, renal involvement was more in the lambda AL with the 24 hour urinary albumin higher in the lambda AL (2.4 gm vs. 1.4 gm; P = 0.0002) and lower serum albumin (2.77 vs. 2.97 mg/dL; P = 0.001). While there was no difference in the overall survival (OS) between the kappa and lambda AL, the median OS for those without a heavy chain was significantly shorter (12.6 months vs. 29.9 months; P= 0.01) compared to those with a heavy chain identified. There was also a correlation between high FLC difference and degree of organ involvement, especially cardiac. Among the 202 kappa AL patients, the median survival for those with a high FLC difference ( > 29.4 mg/dL) was 13 months vs. 48.8 months for those with low difference (P = 0.001). Similarly among the 528 patients with lambda AL, those with high difference (> 18.2 mg/dL) had a median OS of 9.3 months compared to 33 months for those with low difference (P < 0.0001) (Figure). The FLC difference was independent of the cTnT, NT Pro BNP, uric acid and fold-increase in alkaline phosphatase, all of which were significant in the multivariable analysis. Conclusions: The type of FLC impacts the spectrum of organ involvement, with gastrointestinal involvement observed more frequently with kappa, and renal involvement with lambda FLC. The FLC burden correlates with the degree of organ involvement, and is a significant predictor of overall survival in AL amyloidosis. Lambda FLC exerts similar effect as kappa FLCs, but at lower levels confirming the increased “amyloidogenicity” of lambda FLC. Finally, lack of a chaperone heavy chain is associated with a poorer outcome. Figure 1. Overall survival form diagnosis in patients with high vs. low FLC difference (using median values for cutoffs for kappa and lambda FLC) Figure 1. Overall survival form diagnosis in patients with high vs. low FLC difference (using median values for cutoffs for kappa and lambda FLC)

Abstract Background: Smoldering multiple myeloma (SMM) is an asymptomatic plasma cell proliferative disorder with a high risk of progression to symptomatic multiple myeloma. Identification of risk factors that predict progression of SMM to symptomatic MM could identify higher risk patients who might benefit from chemoprevention or more intensive surveillance. We hypothesized that increased monoclonal free kappa or lambda immunoglobulin light chains in smoldering myeloma (SMM), as detected by the serum free light chain (FLC) assay, indicates an increased the risk of progression to active myeloma. Methods: Of 276 pathologically confirmed SMM patients seen at the Mayo Clinic from 1970 to 1995, baseline serum samples obtained within 30 days of diagnosis were available in 273. Results: At a median follow-up of surviving patients of 12.4 years, transformation to active disease has occurred in 161 (59%) patients. An abnormal FLC ratio was present at baseline in 90% of patients. The best break-point for predicting risk of progression was a FLC ratio less than or equal to 0.125 or greater than or equal to 8 (hazard ratio, 2.3; 95% CI, 1.6–3.2) [Figure 1]. The extent of abnormality of FLC ratio was independent of SMM risk categories defined by number of plasma cells in the bone marrow and size of serum M-proteins (bone marrow plasma cells ≥ 10% and serum M protein ≥ 3 g/dL; bone marrow plasma cells ≥ 10% but serum M protein < 3 g/dL; and serum M protein ≥ 3 g/dL but bone marrow plasma cells < 10%). Incorporating the FLC ratio into the risk model, the division of patients into high-, intermediate-, and low-risk groups is 28, 42, and 30% with 5 year progression rates of 76, 51, and 25%, respectively [Figure 2]. Conclusions: The serum immunoglobulin FLC ratio is an important additional determinant of clinical outcome in patients with SMM. Figure Figure Figure Figure

AbstractBackground: The detection of monoclonal free light chains (FLCs) is an important diagnostic aid for a variety of monoclonal gammopathies and is especially important in light-chain diseases, such as light-chain myeloma, primary systemic amyloidosis, and light-chain-deposition disease. These diseases are more prevalent in the elderly, and assays to detect and quantify abnormal amounts of FLCs require reference intervals that include elderly donors.Methods: We used an automated immunoassay for FLCs and sera from a population 21–90 years of age. We used the calculated reference and diagnostic intervals to compare FLC results with those obtained by immunofixation (IFE) to detect low concentrations of monoclonal κ and λ FLCs in the sera of patients with monoclonal gammopathies.Results: Serum κ and λ FLCs increased with population age, with an apparent change for those >80 years. This trend was lost when the FLC concentration was normalized to cystatin C concentration. The ratio of κ FLC to λ FLC (FLC K/L) did not exhibit an age-dependent trend. The diagnostic interval for FLC K/L was 0.26–1.65. The 95% reference interval for κ FLC was 3.3–19.4 mg/L, and that for λ FLC was 5.7–26.3 mg/L. Detection and quantification of monoclonal FLCs by nephelometry were more sensitive than IFE in serum samples from patients with primary systemic amyloidosis and light-chain-deposition disease.Conclusions: Reference and diagnostic intervals for serum FLCs have been developed for use with a new, automated immunoassay that makes the detection and quantification of monoclonal FLCs easier and more sensitive than with current methods. The serum FLC assay complements IFE and allows quantification of FLCs in light-chain-disease patients who have no detectable serum or urine M-spike.

AbstractBackground: Patients with multiple myeloma often have Bence Jones proteins composed of free monoclonal light chains of the κ or λ type in their urine. Usually, these light chains exist as monomeric or dimeric forms, but rarely, larger molecules, such as tetramers, have been reported in the serum.Methods and Results: We report the presence of trimeric complexes of λ light chain dimers in a patient who was diagnosed with a free λ light chain multiple myeloma 2 years earlier and subsequently underwent a stem cell transplant. Recently, the patient presented with a large serum M-spike (23 g/L) by protein electrophoresis. The spike consisted of monoclonal λ light chains without a heavy chain. The urine contained only 8 mg of λ light chain in a 24-h specimen. Quantitative analysis of the serum and urinary free light chains (FLCs) indicated the probability of larger aggregates of FLCs. Size-exclusion chromatography, electrophoresis, analytical ultracentrifugation, and mass spectrometric studies of the serum revealed almost exclusively the presence of trimolecular aggregates of λ light chain dimers without other multimeric species.Conclusion: Monoclonal λ light chains may present as hexameric aggregates that cannot be cleared by renal excretion.

Abstract Detection of an M-protein (monoclonal immunoglobulin) has been used to diagnose and monitor multiple myeloma (MM). As therapies for MM have improved, more sensitive methods have been used to define response: immunofixation electrophoresis (IFX) of serum and urine, normalization of the serum immunoglobulin free light chain (FLC) ratio, and high sensitivity flow cytometry to detect clonal plasma bone marrow cells. It is hoped that these more sensitive approaches will differentiate those patients with minimal residual disease (MRD) versus no residual disease (NRD), that later which could mean a cure. Flow cytometry of plasma cells requires bone marrow aspiration, which is inconvenient and expensive and is potentially limited by sampling bias. More sensitive methods to differentiate MRD from NRD using serum would be advantageous. We have developed a sensitive test for the presence of the monoclonal antibody produced by the plasma cells which may serve as a substitute for invasive bone marrow biopsy. Briefly, patient serum is enriched for immunoglobulins (Ig) and the Ig light chains are decoupled from the heavy chain by reduction with DTT. The mass distribution of the light chains is resolved using a micro LC-ESI-Q-TOF mass spectrometry and the presence of the M-protein is detected as a spike in the mass distribution. In addition to the detection, the accurate mass measurement of the light chain serves as a unique individualized marker which can aid in detection in subsequent patient monitoring. We have termed this method: monoclonal-immunoglobulin-Rapid-Accurate-Mass-Measurement (miRAMM). By spiking monoclonal immunoglobulins into human serum, we have demonstrated that miRAMM is approximately 1000x more sensitive than SPEP at detecting M-proteins. Initial results on 21 patients in stringent complete response (sCR) demonstrated that 67% (n=14) had detectable malignant specific clones by miRAMM. Given these promising results we have extended the method to a larger series of myeloma patients for whom we had long term follow-up and serum samples prior to treatment and at 6-12 months status post stem cell treatment. The results of this data will be correlated to the clinical outcomes for these patients. Early results demonstrate the potential of miRAMM to be a more sensitive, cost effective approach to detect MRD compared with current methods. Disclosures Murray: Mayo Clinic: Patent Application filed Patents & Royalties. Barnidge:Mayo Clinic: Patent filed Patents & Royalties.

Abstract This study evaluated the contributing roles of flow cytometric immunophenotyping of blood and bone marrow and immunohistochemical paraffin section staining of bone marrow biopsies in the staging of B-cell malignant lymphoma. Flow immunophenotyping was performed on a marrow specimen in 175 cases; a corresponding blood specimen was also immunophenotyped in 135 of these cases. Morphologic marrow involvement by lymphoma was found in 59 cases; flow immunophenotyping identified 54 cases with a monoclonal B-cell process: morphology-positive/flow-positive (n = 49), morphology-positive/flow-negative (n = 10), morphology-negative/flow-positive (n = 5), and morphology-negative/flow-negative (n = 111). The 10 morphology-positive/flow-negative cases included 5 follicular and 5 large-cell lymphomas with minimal marrow involvement. All 5 morphology-negative/flow-positive cases were from patients with large-cell lymphomas and bulky clinical disease. Because the blood contained the same B-cell clone in 2 of 2 morphology-negative/flow-positive cases studied, blood contamination of marrow may account for these findings. Blood flow cytometric immunophenotyping studies were positive in 32 cases; 30 had marrow involvement by morphology and were from patients with follicular, mantle cell, lymphoplasmacytic, small lymphocytic, or marginal zone lymphomas. From our results, we conclude that (1) bone marrow flow cytometric immunophenotyping is not a cost-effective replacement for good morphologic evaluation in lymphoma staging and that (2) a positive peripheral blood flow cytometric immunophenotyping study when performed in low-grade lymphomas correlates with marrow involvement.

Abstract Background: Monoclonal gammopathy of undetermined significance (MGUS) affects 3% of persons age 50 yrs or greater. Reliable predictors of progression of MGUS to myeloma or related malignancy are needed. We hypothesized that the presence of monoclonal unbound (free) kappa or lambda immunoglobulin light chains in MGUS, as detected by an abnormal serum kappa/lambda free light chain (FLC) ratio would indicate clonal evolution in the neoplastic plasma cell and increase risk of progression to malignancy. Methods: Patients (pts) from Southeastern Minnesota with MGUS seen at the Mayo Clinic from Jan 1, 1960 through Dec 31, 1994 who had baseline serum samples collected and stored at the time MGUS was first recognized were studied. Free kappa and lambda chains were measured using the serum FLC assay (FREELITETM, The Binding Site Ltd). Results: Of 1384 pts with MGUS during the defined time period, baseline serum samples obtained within 30 days of diagnosis of MGUS were available in 1148 pts. At a median follow-up of 15 yrs, malignant progression has occurred in 87 (7.6%) pts: myeloma (53 pts), IgM lymphoma (17), primary amyloidosis (6), macroglobulinemia (6), chronic lymphocytic leukemia (3) and plasmacytoma (2). An abnormal kappa/lamda FLC ratio (kappa/lambda ratio <0.26 or >1.65) indicating presence of monoclonal FLC was detected in 379 (33%) pts. In a Cox proportional hazards model, the risk of progression in pts with an abnormal kappa/lamda FLC ratio was significantly higher (hazard ratio 3.5, 95% CI 2.3–5.5; p<0.001) compared to pts with a normal ratio. After adjusting for the size of the serum monoclonal (M) protein by multivariate analysis, the hazard ratio was only slightly reduced to 2.8, p<0.001. The risk of progression to myeloma or related malignancy at 10 years was 17% with an abnormal kappa/lambda FLC ratio versus 5% with a normal ratio; corresponding rates at 20 years were 35%, and 13%, respectively. MGUS pts with both an abnormal serum FLC ratio and high serum M protein had a risk of progression at 20 years of 46% (high-risk), versus 26% with one of two risk factors (intermediate-risk) and 7% with no risk factors (low-risk) (Table). The true life-time (28 year) risk of myeloma or related malignancy in the low-risk group was only 5.3% when other competing causes of death were taken into account. Conclusions: Presence of monoclonal FLC in the serum as detected by an abnormal kappa/lamda FLC ratio is a major independent risk factor for progression of MGUS to myeloma or a related malignancy. The new risk-stratification identifies a low-risk subset with a remarkably small life-time risk of progression, a finding of significant importance for the management of MGUS. Risk-stratification of MGUS using the size of the serum M spike and free light chain ratio Risk Group No. of patients Hazard ratio Absolute risk of progression at 20 years Low-risk (Serum M protein <1.5 gm/dL and normal FLC ratio [0.26–1.65]) 606 1 7% Intermediate-risk (Either risk factor present) 373 3.5 26% High-risk (Serum M protein >/=1.5 gm/dL and abnormal FLC ratio [<0.26 or >1.65]) 169 6.8 46%

Abstract Abstract 4876 Background Recent studies have found obesity to be associated with a 1.5- to 2-fold elevated risk of developing multiple myeloma. This is of particular interest given that elevated levels of the pro-inflammatory cytokine interleukin (IL)-6 have been found in obese persons, and, at the same time, IL-6 has well-known proliferative and anti-apoptotic effects on monoclonal plasma-cells. Also insulin-like growth factors (IGFs) have been proposed to play a role since obesity often causes insulin resistance, which in turn modulates the bioavailability of IGF-1 Similar to IL-6, prior studies have found IGF-1 to have both growth and survival effects on monoclonal plasma-cells. Based on these facts, we have speculated that obesity might increase the risk of the multiple myeloma precursor monoclonal gammopathy of undetermined significance (MGUS), or, alternatively, that obesity may increase the risk for transformation from MGUS to multiple myeloma. We conducted the first large screening study designed to assess the association between obesity and MGUS among almost 2,000 African-American and Caucasian women. Methods We included 1000 African-American and 996 Caucasian women (age 40-79, median 48 years) from the Southern Community Cohort Study to assess MGUS risk in relation to obesity. Per our sampling strategy, about 50% of the participants were obese. Medical record-abstracted weight and height (measured on the day of study enrollment) and self-reported values had very high concordance (Pearson correlation >0.95). Serum samples from all subjects were analyzed by electrophoresis performed on agarose gel; samples with a discrete or localized band were subjected to immunofixation. Using logistic regression models, we estimated odds ratios (ORs) as measures of risk. Results Among all study participants, 39 (3.9%) African-Americans and 21 (2.1%) Caucasians were found to have MGUS, yielding a 1.9-fold (95%CI 1.1-2.3; p=0.021) higher risk of MGUS among African-Americans (vs. Caucasians). On multivariate analysis, we found obesity (OR=1.8, 95%CI 1.03-3.1; p=0.039), African-American race (OR=1.8, 95%CI 1.04-3.1; p=0.037), and increasing age (quartiles: ≥55 vs. <43 years) (OR=2.5, 95%CI 1.1-5.7; p=0.028) to be independently associated with an excess risk of MGUS. Another interesting finding was that the distribution of the monoclonal immunoglobulin isotype usage among African-American and Caucasian women was significantly different (p=0.007). Their respective rates were: IgG in 79.5% and 71.3 %; IgA in 7.7% and 0%; IgM in 7.7% and 19%; biclonal in 5.1% and 4.7%; and triclonal in 0% and 4.7%. The distribution of serum light-chain types between the two races was also significantly different (P=0.003, chi-square test): kappa in 53.8% and 47.6%; lambda in 43.6% and 42.8%; biclonal 2.6% and 4.7%; and triclonal in 0% and 4.7%. Conclusions Our finding that MGUS is twice as common among obese (vs. non-obese) women, and independent of race, supports the hypothesis that obesity is etiologically linked to myelomagenesis and may have public health impact. The observed 2-fold excess of MGUS among African-Americans (vs. Caucasians) of similar socio-economic status, coupled with other recent studies supports a role for susceptibility genes as the cause for racial disparity in the prevalence of MGUS. Disclosures No relevant conflicts of interest to declare.

The quantitative assay for free light chains (FLC) is a recently introduced commercial product (FREELITETM, The Binding Site, Ltd.) that has been reported to be sensitive and specific for detecting and monitoring free light chain diseases such as primary systemic amyloidosis (AL), light chain deposition disease (LCDD), non-secretory multiple myeloma (NSMM), and LCMM (light chain multiple myeloma). We have prospectively evaluated the test performance in clinical practice. Results : In 2003, our Clinical Laboratory received samples for FLC assays from 1020 Mayo Clinic patients. The majority of these patients (88%) had plasma cell disorders (PCD). All 120 patients who did not have PCD had normal K/L FLC ratios. Among these, 52 had non-AL amyloidosis: localized amyloid (23), hereditary (16), senile (6), secondary (3), and amyloid of unknown type (4). The 68 other patients who did not have a PCD were being tested because of peripheral neuropathy, rule out AL, anemia, proteinuria, lymphoproliferative disease, and a number of other indications with small numbers of patients (n=13). Among the monoclonal gammopathy patients were 330 with MM, 269 AL, 115 MGUS, 72 SMM, 22 plasmacytomas, 20 NSMM, 9 macroglobulinemia, 7 LCDD, and a variety of other diagnoses with smaller numbers of patients. The sensitivity of the K/L FLC ratio was 100% in LCDD (7/7) and 70% in NSMM (14/20). The 6 NSMM patients with normal K/L FLC ratios had all been treated with SCT and 5 of the 6 had achieved hematologic remission by bone marrow plasma cells. Among the 110 AL patients who had not been previously treated and who had a FLC assay performed within 120 days of diagnosis, the sensitivity of the K/L FLC ratio was 92% compared to 71% for serum IFE and 84% for urine IFE. Using all 3 assays, there was 99.1% (109/110) sensitivity for detecting monoclonal light chain in AL. Conclusion : The performance of the FLC assay in this prospective analysis matches the results from published retrospective validation studies.