Katzmann, Jerry A.

Abstract Background: Patients with monoclonal gammopathies typically have a single clone making a unique type of monoclonal protein, one heavy chain and one light chain. In a small proportion of patients, more than one (typically two) types of monoclonal protein can be seen and differ with respect to the heavy chain or light chain or both. There is limited information on the clinical course of patients with more than one monoclonal protein identified in their serum or urine. Objectives: We report outcomes of patients with more than one monoclonal protein at our institution over the past thirty years. Patients and Methods: We queried the existing clinical database to identify all patients with at least two different monoclonal protein types identified on serum or urine studies during the course of their disease. Retrospective chart review was performed on 551 patients, who had more than one type of monoclonal protein on electrophoresis and/or immunofixation. Results: Among the entire study cohort (N=551), 461 pts (84%) had a biclonal pattern, 7 (1%) had a triclonal pattern and the remaining 83 (15%) had a monoclonal pattern at the time of initial positive study. The median age at the time of initial diagnosis was 69.7 years (range; 32-93); 359 (65%) were male. Among these, 390 were diagnosed as MGUS/BGUS, 18 with SMM, 36 with MM, 20 with WM, and rest with another lymphoproliferative disorders. The median duration between the initial detection of monoclonal protein and the emergence of the biclonal protein was 40 months for the 83 patients, who were monoclonal initially. The distributions of the dominant monoclonal proteins were GK (30%), GL (21%), MK (22%), ML (9%), AK (9%), and AL (8%). The distributions of the second monoclonal proteins were GK (27%), GL (24%), MK (16%), ML (15%), AK (10%), and AL (7%). Overall, 20 patients with a MGUS/BGUS progressed to smoldering or symptomatic myeloma, while 12 patients with an initial diagnosis of SMM progressed to MM. The median estimated follow up for the MGUS/BGUS group and those with SMM were 6.5 years (95% CI; 5, 7) and 9.8 (95% CI; 4, 13), respectively. This translates to 20 progressions over 3822 person years of follow up for the MGUS/BGUS patient group and 12 progressions over 131 person years of follow up for the SMM group. The rate of progression was ~1% per year for patients with MGUS/BGUS and the median estimated time to progression was 2.6 years for the SMM group. (Figure). In majority of patients, the dominant M spike increased with the disease progression. Conclusion: Patients with biclonal gammopathies appear to have a similar rate of progression compared to what has been historically described for MGUS/ SMM population. For the patients who progressed and received treatment, both M proteins typically responded to therapy, and during relapse, the original dominant M protein typically reemerged as the dominant M protein. Figure 1 Figure 1. Disclosures Kumar: Janssen: Consultancy, Research Funding; Array BioPharma: Consultancy, Research Funding; Sanofi-Aventis: Consultancy, Research Funding; Onyx Pharmaceuticals: Consultancy, Research Funding; Millennium: The Takeda Oncology Co.: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Skyline Diagnostics: Membership on an entity's Board of Directors or advisory committees.

Abstract Introduction Diffuse or polyclonal hypergammaglobulinemia has been associated with multiple medical conditions such as autoimmune diseases, infections, and chronic liver diseases. However, the possibility of differences in gamma globulin levels in different races and its association with environmental and socioeconomic factors is not well studied. Method A randomized sample of 917 adult males (50–74 years) in an urban community in Ghana underwent in-person interview and health examination. Serum from all persons was analyzed by electrophoresis performed on agarose gel. Those found to have a monoclonal protein (n=54) were excluded from further analysis. Gamma globulin levels were measured in the remaining 863 samples. Associations between selected characteristics and gamma globulin levels were assessed by Chi-square test and logistic regression models. Results The median gamma globulin level was 1.86 gm/dl. Seventy three percent of the study population had gamma globulin levels above the 1.6 gm/dl reference level and will be considered to have hypergammaglobulinemia. Surprisingly, 25% had gamma globulin levels above 2.17 gm/dl. In univariate analysis increased gamma globulin levels were associated with older age within the study population. Only 54% of the males aged 60 yrs or younger had gamma globulin levels above 1.6 gm/dl compared to 69% in those older than 60 yrs (p=0.007). Higher education level was associated with a low prevalence of hypergammaglobulinemia 51% compared with 74% in those with less than high school education (p <0.0001). Smoking was not associated (p=0.16) with gamma globulin levels. Although a higher proportion (71%) of people with history of alcohol use had hypergammaglobulinemia compared to the control (63%) this was not statistically significant (p=0.07). Self-reported history of genital warts, genital herpes and meningitis was associated with hypergammaglobulinemia, whereas syphilis, malaria, cholera, gonorrhea, hepatitis, measles and tuberculosis had no association with gamma globulin levels in univariate analysis. Conclusion Three quarters of this randomized sample of Ghanaian adult males has hypergammaglobulinemia based on current laboratory definitions. More work is needed to uncover underlying causes for the observed high gamma globulin levels in this population. Further evaluation of a younger population with less environmental influence may help explain whether this is due to genetic differences, environmental exposures, or a combination.

Abstract Background: The serum free light chain (FLC) ratio is a readily available laboratory test that has independent prognostic significance in monoclonal gammopathy of undetermined significance (MGUS) (Blood2005;106:812–17) and solitary plasmacytoma (Blood2006;108:1979–83). The purpose of this study was to determine if the FLC ratio will be of prognostic value in multiple myeloma (MM). Methods: We used a well characterized cohort of 1027 newly diagnosed MM patients seen at Mayo Clinic from January 1, 1985 to December 31, 1998 (Mayo Clin Proc2003;78:21–33) to ensure adequate follow up. Archived serum samples from blood drawn within 30 days of initial diagnosis were available on 790 of the 1027 patients. Quantitation of serum free κ and λ concentrations was performed on the archived sera by immunonephelometry, using specific antibodies on a BNII nephelometer. FLC ratio was calculated as κ /λ, i.e., free κ concentration divided by free λ, reference range 0.26–1.65. If the FLC ratio is >1.65, κ is considered to be the “involved” FLC and λ the “uninvolved” FLC, and vice versa if the ratio is less than 0.26. Incorporation of FLC into the International Staging System (ISS) was assessed in 576 of 790 patients (73%), in whom samples or data were available to estimate the ISS. Results: 790 patients (median age 66 years) were studied. The FLC ratio was outside the reference range in 95.1% of patients. The median involved κ and λ concentrations were 37.1 and 71.3 mg/dL, respectively. A cut-point κ /λ FLC ratio of <0.03 or >32 was chosen for further modeling analysis on the basis of its separation of the cohort into two roughly comparable parts. Overall survival was significantly inferior in patients with an FLC ratio <0.03 or >32 (n=479 patients) compared to those with an FLC ratio that was 0.03–32 (n=311 patients), with median survival of 30 versus 39 months, respectively, P<0.001 (hazard ratio 1.3, 95% CI 1.12–1.54). The FLC ratio (<0.03 or >32) retained independent significance after adjusting for the International Staging System (ISS) (P=0.03). The FLC ratio made the most significant contribution to predicting prognosis in patients with ISS Stage 2 (n=265 pts), separating this category of patients into those with 5 year survival rates of 20.5% (FLC ratio <0.03 or >32; n=152 pts) versus 35.2% (FLC ratio 0.03–32; n=113 pts), P=0.02. We studied the additional prognostic value of the serum FLC to the variables used in the ISS by defining FLC ratio <0.03 or >32, Sβ2M ≥3.5 g/L, and serum albumin <3.5 g/dL as three risk factors in a risk-stratification model. Patients with 0, 1, 2, or 3 risk factors had significantly different overall survival, with median overall survival times of 51, 39, 30 and 22 months, respectively, P<0.001. Conclusions: The serum FLC ratio at initial diagnosis is an important predictor of prognosis in myeloma. We show that the serum FLC ratio can be incorporated into the ISS, providing the greatest prognostic value in ISS Stage 2 patients, as well as providing a new risk-stratification model.

Abstract Abstract 1788 Poster Board I-814 Background Clinically available prognostic factors for progression of MGUS to myeloma include the size of the M-spike, heavy chain isotype, and free light chain (FLC) K/L ratio. The combined use of these variables provides approximately a 10-fold difference in risk for progression. The highest risk group, however, still has only a 27% twenty year risk of progression. Therefore, additional predictors of progression are needed. Suppression of uninvolved immunoglobulins has been an inconsistent predictor of progression. The novel Hevylite” assay measures concentrations of each isotype-specific immunoglobulin heavy light chain (HLC) pair (i.e., IgG kappa, IgG lambda, IgA kappa, IgA lambda) and enables us for the first time to determine if suppression of isotype-specific Ig of the opposite HLC is occurring and if it is predictive of progression of MGUS to myeloma. We studied the prognostic value of isotype specific HLC suppression of uninvolved immunoglobins as a predictor of progression in MGUS. Methods: Total immunoglobulins were quantitated by immunonephelometry, and suppression was defined as concentrations less than the lower limit of the reference range. HLC concentrations were quantitated with Hevylite reagents (The Binding Site, Ltd). The antibodies used in this investigational nephelometric assay span epitopes in the junctional areas between the HLC constant regions. Based on reference ranges for GK (407-1130 mg/dL), GL (188-583 mg/dL), AK (55-292 mg/dL), and AL (39-249 mg/dL) that we derived from 276 normal samples, we compared the proportion of patients who had isotype specific suppression of the opposite HLC pair among 30 patients with MGUS who progressed to myeloma versus 36 patients who had not progressed over a similar follow-up period. To confirm the relationship of isotype specific HLC pair suppression compared to suppression of uninvolved immunoglobulins of other heavy chain classes, we tested a second cohort of stored samples from 50 IgG and 14 IgA MGUS patients prior to the development of MM. Results In the initial samples from 36 IgG MGUS patients who had stable disease, 6% had suppression of uninvolved intact immunoglobulins of IgA and/or IgM, while 22% had isotype specific suppression of the opposite HLC pair (i.e., IgG lambda suppressed in IgG kappa MGUS, and vice versa). In contrast, in baseline samples from 30 IgG MGUS patients who eventually progressed to myeloma or related malignancy, 7% had suppression of IgA and/or IgM, while 40% had isotype specific suppression of the opposite IgG HLC pair. Thus, HLC pair suppression was more frequent than suppression of Igs from other heavy chain classes, and MGUS patients who eventually progressed had a 2-fold higher rate of isotype specific HLC pair suppression than stable MGUS patients. These findings were confirmed independently on a second cohort of patients who progressed to myeloma 5-10 years after blood collection. In samples from 50 IgG MGUS patients, 42% had suppression of Igs of other heavy chain classes, while 80% had suppression of the isotype specific HLC by the Hevylite assay. In the last samples prior to progression, the suppression of heavy chain classes increased to 60%, while isotype specific HLC suppression rose to 90%. Interestingly, the 14 IgA patients had equivalent suppression of gamma or mu heavy chain classes as compared to the HLC pairs, with 50% of the patients having suppression in the initial sample and 79% in the last sample prior to MM. Conclusion Our data suggest that suppression of the opposite IgG HLC pair is a predictor of patients destined for progression. Isotype specific HLC pair suppression of this nature is more frequent in these patients (more sensitive) than suppression of other heavy chain classes. We were not able to demonstrate a similar effect in IgA MGUS. Larger studies are underway to determine if HLC pair suppression is independent of M-spike quantitation, heavy chain isotype, and FLC ratio. Biologically, our findings suggest that clonal IgG plasma cells are able to suppress other IgG producing non-clonal plasma cells far more effectively than IgA or IgM secreting cells, and this occurs more frequently with cells that will eventually undergo malignant transformation. Disclosures Bradwell: The Binding Site Group Ltd: Equity Ownership.

Abstract Abstract 1803 Poster Board I-829 Background Plasma cell proliferative disorders are monitored by a variety of methods. Serum protein electrophoresis (SPEP) and/or urine PEP M-spike quantitation are commonly assessed in patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), and multiple myeloma (MM) to determine disease progression, response, or relapse. Serum immunoglobin (Ig) concentrations can be quantitated when the M-spike is large or if the migration is obscured within the SPEP beta fraction. Serum FLC quantitation provides a rapid indicator of response, will detect the rare occurrence of FLC escape, and will allow disease monitoring in the absence of a measurable serum or urine M-spike. The International Myeloma Working Group (IMWG) has recommended that the serum and urine M-spike should be used to monitor disease, and that FLC quantitation should be used only if there is no measurable disease by electrophoresis and if the monoclonal FLC concentration is greater than 10 mg/dL in the context of an abnormal FLC K/L ratio. We have studied serial samples in clinically stable patients in order to assess the total variability (analytic and biologic) of these assays and to confirm the IMWG recent recommendations. Methods Serial data from stable MGUS patients (n=35) were identified by the availability of all 3 serum test results (M-spike, Ig, FLC) in at least 4 serial samples that were obtained 9 months to 5 years apart and whose serum M-spikes varied by less than 25%. For MM (n=60) and SMM (n=48) the samples were within 9-15 months and serum M-spikes varied by less than 0.5 g/dL. Among the 60 MM, 48 SMM, and 35 MGUS patients, there were 23, 41, and 18 patients with measurable disease by serum M-spike (i.e. M-spike >1 g/dL); 19, 10, and 10 patients with an evaluable FLC (i.e. monoclonal FLC > 10 mg/dL and an abnormal FLC ratio); and 5, 5, and 1 patient with an evaluable urine (i.e. M-spike > 200mg/24 hr). The FLC data was analyzed as the involved FLC concentration (iFLC), the difference between the involved and uninvolved FLC concentration (dFLC), and the FLC K/L ratio (rFLC). The coefficients of variability (CV) were determined for each methodology in each patient sample set, and the average CVs were determined. Igs were quantitated by immunonephelometry using a Siemens BNII and Siemens reagent sets; kappa and lambda FLC were quantitated using a Siemens BNII and Freelite reagent sets from The Binding Site; M-spikes were quantitated using Helena SPIFE SPE and reagent sets. Results The CVs for the Ig quantitation, SPEP M-spike, FLC quantitation, and urine M-spike in each of the patient groups are listed in the table: Our laboratory's interassay analytic CV for replicate samples are 4-5% for Ig quantitation, 6-8% for SPEP M-spikes, 6-7% for FLC quantitation, and 5-7% for urine M-spikes. The analytic CVs of the methods are similar, but the total (analytic + biologic) CVs are very different. The samples have been selected by restricting the variability of serum M-spike values; when we apply the same criteria to the IgG quantitation, the IgG total CV comes closer to the serum M-spike CVs. The remaining differences, however, may be due to biologic variability contributed by polyclonal Ig. The total CV for iFLC is similar to the urine M-spike CV and suggests a previously unrecognized large biologic CV for serum FLC. The iFLC and dFLC CVs were comparable but were smaller than the rFLC CV. Conclusion The variability of the serum and urine M-spike, Ig, and FLC measurements confirm the IMWG recommendations for patient monitoring. If a patient has a measurable M-spike >1 g/dL, then the serum M-spike should be followed. If there is no measurable disease, then the iFLC can be monitored, provided that the rFLC is abnormal and the iFLC concentration is >10 mg/dL. Although the number of patients with evaluable urine M-spikes in this study is small, larger studies may confirm the utility of serum FLC compared to urine M-spike for monitoring patients with monoclonal gammopathies. Disclosures No relevant conflicts of interest to declare.

Abstract BACKGROUND Electrophoretic separation of serum and urine proteins has played a central role in diagnosing and monitoring plasma cell disorders. Despite limitations in resolution and analytical sensitivity, plus the necessity for adjunct methods, protein gel electrophoresis and immunofixation electrophoresis (IFE) remain front-line tests. METHODS We developed a MALDI mass spectrometry–based assay that was simple to perform, automatable, analytically sensitive, and applicable to analyzing the wide variety of monoclonal proteins (M-proteins) encountered clinically. This assay, called MASS-FIX, used the unique molecular mass signatures of the different Ig isotypes in combination with nanobody immunoenrichment to generate information-rich mass spectra from which M-proteins could be identified, isotyped, and quantified. The performance of MASS-FIX was compared to current gel-based electrophoresis assays. RESULTS MASS-FIX detected all M-proteins that were detectable by urine or serum protein electrophoresis. In serial dilution studies, MASS-FIX was more analytically sensitive than IFE. For patient samples, MASS-FIX provided the same primary isotype information for 98% of serum M-proteins (n = 152) and 95% of urine M-proteins (n = 55). MASS-FIX accurately quantified M-protein to &lt;1 g/dL, with reduced bias as compared to protein electrophoresis. Intraassay and interassay CVs were &lt;20% across all samples having M-protein concentrations &gt;0.045 g/dL, with the ability to detect M-proteins &lt;0.01 g/dL. In addition, MASS-FIX could simultaneously measure κ:λ light chain ratios for IgG, IgA, and IgM. Retrospective serial monitoring of patients with myeloma posttreatment demonstrated that MASS-FIX provided equivalent quantitative information to either protein electrophoresis or the Hevylite™ assay. CONCLUSIONS MASS-FIX can advance how plasma cell disorders are screened, diagnosed, and monitored.

Abstract BACKGROUND Current recommendations for screening for monoclonal gammopathies include serum protein electrophoresis (PEL), imunofixation electrophoresis (IFE), and free light chain (FLC) ratios to identify or rule out an M-protein. The aim of this study was to examine the feasibility of an assay based on immunoenrichment and MALDI-TOF-MS (MASS-SCREEN) to qualitatively screen for M-proteins. METHODS Serum from 556 patients previously screened for M-proteins by PEL and IFE were immunopurified using a κ/λ-specific nanobody bead mixture. Following purification, light chains (LC) were released from their heavy chains by reduction. MALDI-TOF analysis was performed and the mass-to-charge LC distributions were visually examined for the presence of an M-protein by both unblinded and blinded analysts. RESULTS In unblinded analysis, MASS-SCREEN detected 100% of the PEL-positive samples with an analytical sensitivity and specificity of 96% and 81% using IFE positivity as the standard. In a blinded analysis using 6 different laboratory personnel, consensus was reached in 92% of the samples. Overall analytical sensitivity and specificity were reduced to 92% and 80%, respectively. FLC ratios were found to be abnormal in 28% of MASS-SCREEN–negative samples, suggesting FLC measurements need to be considered in screening. CONCLUSIONS MASS-SCREEN could replace PEL in a panel that would include FLC measurements. Further studies and method development should be performed to validate the clinical sensitivity and specificity and to determine if this panel will suffice as a general screen for monoclonal proteins.