Application of urine immunofixation electrophoresis in prognostic evaluation of hematopoietic stem cell transplantation in patients with myeloma

Objectives: To investigate the value of urine immunofixation electrophoresis in prognostic evaluation of hematopoietic stem cell transplantation in patients with myeloma. Methods: Thirty-four patients with multiple myeloma admitted to Affiliated Hospital of Hebei University from November 2013 to December 2014 were included as research subjects. All patients received hematopoietic stem cell transplantation and were followed up for five years. Outcomes were evaluated according to the recovery status: complete response (CR), very good partial response (VGPR), partial response (PR), stable disease (SD), and progression disease (PD). In addition, the overall response rate (CR+VGPR) of patients was observed and their urine immunoglobulin status was measured by immunofixation electrophoresis. The Kaplan-Meier method was utilized to plot the survival curve, and the Log-rank method was adopted to analyze the relationship between CR+VGPR and PR and hematopoietic stem cell transplantation (HSCT) survival in patients with myeloma. Results: The basic clinical type of immunofixation electrophoresis was as follows: 19 cases (55.88%) of IgG, 7 cases (20.59%) of IgA, 6 cases (17.65%) of IgM, and 2 cases (5.88%) of light chain type. Outcomes: 13 cases (38.24%) of CR, 12 cases (35.29%) of VGPR, 9 cases (26.47%) of PR, and 25 cases (73.53%) of the overall response rate (CR+VGPR). Compared with IgG, CR, VGPR and PR of IgA, IgM and light chain had statistically significant differences in outcome (p<0.05), and CR+VGPR of patients with IgG was higher than that of patients with IgA, IgM and light chain type (p<0.05). Two of the 34 patients were lost to follow-up. The log-rank analysis showed that the survival rate of patients with CR+VGPR was higher than that of patients with PR (p<0.05). Patients with IgA, IgM, and light chain type had an increased number of prognostic death compared with those with IgG (p<0.05). Conclusion: Patients with IgG type myeloma are superior to those with IgA, IgM and light chain type in terms of the prognosis of hematopoietic stem cell transplantation, which has a certain clinical reference value. doi: https://doi.org/10.12669/pjms.38.1.4425 How to cite this:Zhu S, Yang C, Li W, Lin M. Application of urine immunofixation electrophoresis in prognostic evaluation of hematopoietic stem cell transplantation in patients with myeloma. Pak J Med Sci. 2022;38(1):315-319. doi: https://doi.org/10.12669/pjms.38.1.4425 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Relapse with BCR-ABL1 Elevation in Chronic Myeloid Leukemia after Progression to Multiple Myeloma from Monoclonal Gammopathy of Undetermined Significance with a Persistent KMT2D Mutation

Chronic myeloid leukemia (CML) and monoclonal gammopathy of undetermined significance (MGUS) are two different hematologic malignancies, the former arising from the myeloid cell lineage, and the latter arising from plasma cells. The concurrent diagnosis of CML and MGUS progression to multiple myeloma (MM) in one patient is an extremely rare event. A 59-year-old male was diagnosed with CML and MGUS with no discomfort in August 2012. Bone marrow (BM) aspiration suggested chronic myelogenous leukemia in chronic phase and perhaps myeloproliferative with 6.5% mature plasma cells (Figure 1A). FISH analysis detected that the BCR-ABL1 expression was 130%. And Next-generation sequencing (NGS) of BM showed: ASXL1 , KMT2D , SPEN , BRINP3 , ANKRD26 , PLCG1 , CUX1 were mutated (Figure 2I). The patient started oral imatinib 400 mg per day and achieved a complete cytogenetic response at 3 months. In September 2019, his IgG levels were 2,790 mg/dl (Figure 2J and serum immunofixation electrophoresis revealed monoclonal (M) protein of IgG-Lambda type (Figure 1E). BM aspiration revealed 9.5% plasma cell infiltration, including 6% mature plasma cells and 3.5% proplasmacyte (Figure 1C and 2H). Flow cytometry in BM showed 6.3% plasmacytoma and abnormal cell expressing CD38+CD138+CD56+CD117+clambda+ (Figure 1F). BM biopsy showed hematopoietic hyperplasia with abnormal growth of immature cells (Figure 1B). Fluorescent in situ hybridization (FISH) was negative. Mutations of KMT2D, SPEN, BRINP3, ANKRD26, PLCG1, CUX1, and ZMYM3 still existed(Figure 2I). In January 2020, examination of a new BM aspiration revealed that mature plasma cells were 3% and plasmablast and proplasmacyte were 4.5% (Figure 2H). In February 2020, he stopped IM therapy with undetectable BCR-ABL1 copies because he met the requirement of stopping TKI therapy . In March 2020, IgG levels were 3520 mg/dl and serum immunofixation electrophoresis still revealed monoclonal (M) protein of IgG-Lambda type. His BM aspiration demonstrated 13.5% plasma cells in April 2020 (Figure 2B and 2H). Flow cytometry in BM showed 6.44% (Figure 2F). BM biopsy showed extremely increased proliferation with abnormal growth of abnormal cells (Figure 2A). FISH demonstrated the presence of t(4;14)(p16;q32)(IGH/FGFR3) , 13q14 deletion（RB-1） and 13q14.3 （D13S319） (Figure 2C, 2D and 2E). The patient was diagnosed as MM (IgGλ type, D-S stage IA; ISS stage II) . BCR-ABL1 copies were still not detected at this point (Figure 2G). The patient continued his follow-up treatment of MM without chemotherapy.However, in June 2020, he was considered to have a molecular relapse with 0.2013% BCR-ABL1 copies in the peripheral blood (Figure 2G). NGS showed that the variant allele fractions of KMT2D, SPEN, BRINP3, ANKRD26, PLCG1, CUX1, and ZMYM3 mutations were similar to former . He restarted 400 mg daily IM therapy and BCR-ABL1 copies were undetectable againafter one month therapy (Figure 2G). BM aspiration revealed that the percentage of plasma cells increased to 25.5% in August 2020 (Figure 2H). Then the patient was started on treatment for ISS stage II standard risk myeloma with ID regimen: ixazomib 4 mg on days 1, 8 , 15 and dexamethasone 20 mg on days 1, 8, 15 , 22 in 28-day cycles. After 6 cycles , the patient got VGPR. BM aspiration demonstrated 13% plasma cells (Figure 2H). And he continued to receive myeloma treatment and imatinib . BCR-ABL1 were &lt;MR4.5 (Figure 2G). Our research indicated that KMT2D mutation may make MGUS progress to MM with NK cells functional defects and then promote the recurrence of BCR-ABL1. Co-existence of these two diseases is rare, therefore, additional investigations are warranted. Acknowledgment:The research was supported by the Public Technology Application Research Program of Zhejiang, China (LGF21H080003), the Key Project of Jinhua Science and Technology Plan, China (2020XG-29 and 2020-3-011), the Academician Workstation of the Fourth Affiliated Hospital of the Zhejiang University School of Medicine (2019-2024), the Key Medical Discipline of Yiwu, China (Hematology, 2018-2020) and the Key Medical Discipline of Jinhua, China (Hematology, 2019-2021). Correspondence to: Dr Jian Huang, Department of Hematology, The Fourth Affiliated Hospital of Zhejiang University School of Medicine. N1 Shangcheng Road. Yiwu, Zhejiang, Peoples R China. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

The Prognostic Utility of Serial MASS-FIX in Multiple Myeloma

Background: MASS-FIX is a novel method for detection, characterization and quantification of serum and urine monoclonal proteins in multiple myeloma (MM) which uses immunoglobulin enrichment coupled with matrix-assisted laser desorption ionization time-of-flight mass-spectrometry (MALDI-TOF). MASS-FIX has been endorsed by the International Myeloma Working Group (IMWG) as an alternative to immunofixation electrophoresis for diagnosis and disease monitoring in MM and has replaced immunofixation electrophoresis in our institution. This technique has demonstrated superior sensitivity and specificity compared to conventional gel-based methods, but its prognostic value is still unknown. We designed this study to evaluate whether serum M protein monitoring by MASS-FIX has prognostic significance in predicting disease progression in patients with MM. Methods: In the first part of the study, we included patients with MM who had a documented negative MASSFIX after first-line treatment and had serial MASS-FIX data available (n=187). We then compared the time to next treatment (TTNT) between patients who became positive by MASS-FIX and those who remained negative by last follow up. In the second part, we included patients who were positive by MASS-FIX within 90 days from diagnosis and had available serial MASS-FIX data (n=203). We compared TTNT between patients who became negative by MASS-FIX and those who remained positive by last follow up. TTNT was defined as the time of initiation of the first-line treatment to the time of initiation of second-line treatment or last follow up. Results: Part 1: We included 155 patients diagnosed with MM between January 2013 and December 2019 who had a documented negative MASSFIX with first-line treatment and had serial MASSFIX data available. After a median follow up of 3.1 (95%CI: 2.6-3.5) years, 44 (28%) patients became MASSFIX positive, while 111 (72%) still had negative MASS-FIX. The median time from treatment start to negative MASSFIX was 18.0 (IQR: 6.4-33.5) months. The median TTNT was 4.1 (95%CI: 3.2 - NR) years in patients who became MASSFIX positive and not reached (NR) (95%CI: NR-NR) in patients who were MASS-FIX negative by last follow up (P&lt;0.001). The 2-year OS was 97% and 100% in the 2 groups, respectively (P=0.27). Part 2: We included 203 patients who had positive MASSFIX within 90 days from diagnosis and available serial MASSFIX data. The median follow-up was 1.5 (95%CI: 1.5-1.7) years. By last follow up, 39 (19%) patients became MASSFIX negative while 164 (81%) still had positive MASSFIX. The median TTNT was NR (95%CI: 2.0-NR) years in patients who developed negative MASSFIX and 2.3 (95%CI: 1.9 - NR) years in patients who were still MASS-FIX positive by last follow up (P=0.03). The 2-year OS was 97% and 94% in the 2 groups, respectively (P=0.84) Conclusion: The utilization of MASS-FIX in disease monitoring of MM patients provides prognostic information; among patients who become MASS-FIX negative after first line treatment, an earlier conversion to positive MASS-FIX predicts disease progression. Similarly, persistent MASS-FIX positivity after treatment initiation predicts earlier disease progression. Larger studies and longer follow up are needed to evaluate whether the MASS-FIX is associated with overall survival. Disclosures Murray: Mayo Clinic: Other: Has received patents for the Mass-Fix technology which has been licensed to the Binding Site with potential royalties.. Dispenzieri: Sorrento Therapeutics: Consultancy; Oncopeptides: Consultancy; Pfizer: Research Funding; Alnylam: Research Funding; Takeda: Research Funding; Janssen: Consultancy, Research Funding. Kapoor: Karyopharm: Consultancy; Cellectar: Consultancy; BeiGene: Consultancy; Pharmacyclics: Consultancy; Sanofi: Consultancy; Amgen: Research Funding; Ichnos Sciences: Research Funding; Regeneron Pharmaceuticals: Research Funding; Glaxo SmithKline: Research Funding; Karyopharm: Research Funding; Sanofi: Research Funding; Takeda: Research Funding; AbbVie: Research Funding. Gertz: Akcea Therapeutics, Alnylam Pharmaceuticals Inc, Prothena: Consultancy; Akcea Therapeutics, Ambry Genetics, Amgen Inc, Celgene Corporation, Janssen Biotech Inc, Karyopharm Therapeutics, Pfizer Inc (to Institution), Sanofi Genzyme: Honoraria; Ionis Pharmaceuticals: Other: Advisory Board; Aurora Biopharma: Other: Stock option; AbbVie Inc, Celgene Corporation: Other: Data Safetly & Monitoring. Dingli: Sanofi: Consultancy; GSK: Consultancy; Novartis: Research Funding; Apellis: Consultancy; Janssen: Consultancy; Alexion: Consultancy. Kumar: Amgen: Consultancy, Research Funding; Bluebird Bio: Consultancy; Antengene: Consultancy, Honoraria; BMS: Consultancy, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Beigene: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche-Genentech: Consultancy, Research Funding; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Carsgen: Research Funding; Merck: Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Consultancy; Novartis: Research Funding; Tenebio: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding.

Study of prognostic factors in multiple myeloma

Background: Multiple myeloma (MM) is cancer of the plasma cell characterized by interpatient heterogeneity, in most of the cases it is incurable. It is the second most common hematological malignancy. Overall survival of patients has significantly increased recently. Lot of research is going on for prognostic factors, which can predict disease and response to therapy. During the past decades, biomarkers: M protein and β2 microglobulin have shaped the knowledge about MM. This study was undertaken to evaluate the role of prognostic factors in newly diagnosed and few follow up patients of MM before, during and after the treatment in Indian population.Methods: We analyzed 177 samples (90 MM patients and 87 healthy control) for creatinine, calcium, phosphorus, LDH, total protein, albumin, globulin, β2M, Immunoglobulines (Igs), IgG, IgA, IgM, Kappa light chain and Lambda light chains, serum protein electrophoresis (SPE) and immunofixation electrophoresis (IFE).Results: The result of our study are significant at p<0.0001 for creatinine, β2M, LDH, albumin, globulin and immunoglobulins whereas for calcium and phosphorus results are not significant at p<0.005. We observed that the levels of β2m, creatinine, LDH and M band concentration declined during the treatment (chemotherapy).Conclusions: We conclude that these results will help the clinicians to tailor-made the chemotherapy doses for betterment of patients and improve survival rate in MM patients.

Lambda monoclonal free light chain abnormalities detected by a serum immunofixation electrophoresis assay are underrepresented by quantitative serum free light chain results

Protein and immunofixation (IFIX) electrophoresis are used to diagnose and monitor monoclonal gammopathies. While IFIX detects clonal production of intact immunoglobulins and free light chains (FLC), the latter can also be quantified using a serum free light chain (SFLC) assay, in which polyclonal antisera detects epitopes specific for free kappa (KFLC) or lambda light chains (LFLC). An abnormal KFLC: LFLC ratio (KLR) serves as a surrogate for clonality. While the SFLC assay is highly sensitive, normal LFLC (&lt;2.63mg/dL) and KLR results (&gt;0.26 & &lt;1.65) were found in samples with distinct lambda monoclonal free light chains visualized by IFIX (X-LMFLC). To investigate this discordance, contemporaneous SFLC or KLR values were evaluated for their ability to accurately classify monoclonal FLCs identified by IFIX. We performed a retrospective analysis of serum and urine IFIX (Sebia Hydrasys) and SFLC (Freelite®, Binding Site) results from our institution between July 2010 through December 2020, using R 4.0.2 and Tidyverse packages. From among 9,594 encounters in which a single monoclonal component was initially identified by IFIX, 157 X-LMFLC and 131 X-KMFLC samples were analyzed. Elevated LFLC with normal KFLC was identified in 105/157 X-LMFLC samples (67%), while both LFLC and KFLC were elevated in 42/157 samples (27%). Concordance between X-KMFLC and KFLC was markedly higher, where 122/131 samples (93%) displayed elevated kappa FLC (&gt;1.94mg/dL) with normal LFLC, and only 7/131 X-KMFLC samples (5%) possessed both elevated KFLC and LFLC. The use of KLR to identify pathogenic monoclonal free light chains improved lambda concordance to 85%; however, 19/157 (12%) of X-LMFLC samples still exhibited normal KLR. High concordance of 98% was again observed for X-KMFLC with abnormal KLR. When samples were segregated according to normal or impaired renal function (eGFR &gt; or ≤60mL/min/1.73m², respectively), this disparate identification of X-LMFLC and X-KMFLC by the SFLC assay persisted, suggesting that renal dysfunction (as measured by eGFR) does not underlie this phenomenon. Lastly, we corroborated the above findings in a larger sample population by examining patients with urine Bence Jones FLC identified by IFIX who had free or intact monoclonal components in serum (N=724), grouped by lambda or kappa light chain involvement. The cause(s) of the discrepant performance by the Freelite® SFLC assay, relative to the Sebia Hydrasys IFIX assay, for identifying lambda FLC components is currently unclear. Possible contributory factors include assay reference range cutoffs, other patient disease parameters, and differences in assay-specific polyclonal antisera. Future analyses of these factors will help to further characterize SFLC assay performance and elucidate how interpretation of composite serum FLC test results can be improved to better guide patient management.

Immunoglobulin D Multiple Myeloma with a “Hidden” Lambda Light Chain

Introduction/Objective In rare cases, the conventional immunofixation gel electrophoresis technique fails to detect the light chain of an M-protein. We report a case of immunoglobulin (Ig) D multiple myeloma with a hidden lambda (λ) light chain. Methods/Case Report Capillary electrophoresis (CE) (Sebia CAPILLARYS 2) was used to detect and quantify M- proteins in serum specimens. Immunosubtraction (IS) on the CAPILLARYS 2 systems was used to identify the classes of M-proteins. Conventional gel immunofixation electrophoresis (IFE) was performed, using monospecific antisera for IgD, IgE, kappa (κ) or λ in the Sebia HYDRASYS system, and IgG, IgA, IgM, κ or λ in the Helena SPIFE3000 system. Beta-mercaptoethanol (BME) with Fluidil were used as reduction agents. Results (if a Case Study enter NA) Results of serum CE showed two abnormal peaks in beta 2 and gamma regions, suspected to be positive for M-proteins. IS results showed subtraction for λ light chain only in both peaks, suggesting two monoclonal λ light chains. In contrary, no monoclonal λ light chain was detected in gamma region by IFE (Sebia). Epitope masking in the folded monoclonal protein was suspected to cause the “hidden λ light chain” and was further investigated by two laboratory approaches. IFE performed on the Helena SPIFE3000 system found two λ bands in beta 2 and gamma regions, which was consistent with the results from IS. The treatment of BME with Fluidil helped unmasking the hidden epitope and revealed the λ band in gamma region on IFE (Sebia). Conclusion The medical laboratories should be aware of the described scenario. The failure to detect light chains of certain intact M-proteins is most likely due to the structurally inaccessibility of light chains. It is recommended that treatment with reduction agents or use of an alternative methodology or IS might be helpful for investigating suspected heavy chain only cases, due to the limitation of conventional methodology.

The efficiency of hypogammaglobulinemia reflex immunofixation electrophoresis in identifying monoclonal proteins

Introduction/Objective Hypogammaglobulinemia can be a common occurrence in disorders with monoclonal gammopathies. Because hypogammaglobulinemia may mask a monoclonal protein on serum protein electrophoresis (sPEP), its presence in the absence of a discernible M-spike is often the basis of reflexive testing by immunofixation electrophoresis (IFE). At our Institution, reflex IFE has historically been performed in cases where the gamma fraction on sPEP is &lt;0.6 g/dL. The aim of this study was to test the predictive performance of hypogammaglobulinemia in identifying abnormal bands on IFE in newly screened patients. Methods/Case Report All patients that underwent sPEP testing from November 2020 to May 2021 at our Institution were identified. Among them, patients with gamma fraction &lt;0.6 g/dL and no previous sPEP testing were included for analysis. Reflex IFE results were reviewed for identification of abnormal bands. Results (if a Case Study enter NA) Out of a total number of 1,374 patients tested for sPEP in the study period, 72 had serum gamma fraction &lt;0.6 g/dL (5.2%). Among them, 36 patients had no previous sPEP testing, and their reflex IFE were reviewed. In 38.8% of the cases, the IFE showed one or more abnormal (monoclonal) bands. When considering a new threshold for hypogammaglobulinemia IFE reflex of &lt;0.4 g/dL, the diagnostic yield for finding abnormal bands increased to 62.5%. Moreover, a percentage reduction of 64.2% was observed in the number of reflex IFE performed. Conclusion Although these data must be confirmed using a larger sample population, a lower threshold for hypogammaglobulinemia may be proposed at our Institution to reduce labor and costs and to improve efficiency of monoclonal protein detection.

Monitoring the M-protein of multiple myeloma patients treated with a combination of monoclonal antibodies: the laboratory solution to eliminate interference

Objectives The therapeutic monoclonal antibody (t-mAb) daratumumab, used to treat multiple myeloma (MM) patients, interferes with routine, electrophoretic based M-protein diagnostics. Electrophoretic response assessment becomes increasingly difficult when multiple t-mAbs are combined for use in a single patient. This is the first study to address the analytical challenges of M-protein monitoring when multiple t-mAbs are combined. Methods In this proof-of-principle study we evaluate two different methods to monitor M-protein responses in three MM patients, who receive both daratumumab and nivolumab. The double hydrashift assay aims to resolve t-mAb interference on immunofixation. The MS-MRD (mass spectrometry minimal residual disease) assay measures clonotypic peptides to quantitate both M-protein and t-mAb concentrations. Results After exposure to daratumumab and nivolumab, both t-mAbs become visible on immunofixation electrophoresis (IFE) as two IgG-kappa bands that migrate close to each other at the cathodal end of the γ-region. In case the M-protein co-migrates with these t-mAbs, the observed interference was completely abolished with the double IFE hydrashift assay. In all three patients the MS-MRD assay was also able to distinguish the M-protein from the t-mAbs. Additional advantage of the MS-MRD assay is that this multiplex assay is more sensitive and allows quantitative M-protein-, daratumumab- and nivolumab-monitoring. Conclusions Daratumumab and nivolumab interfere with electrophoretic M-protein diagnostics. However, the M-protein can be distinguished from both t-mAbs by use of a double hydrashift assay. The MS-MRD assay provides an alternative method that allows sensitive and simultaneous quantitative monitoring of both the M-protein and t-mAbs.

In vitro evaluation of potential interference of lokivetmab with protein electrophoresis and immunofixation

Objective In humans, misdiagnoses of monoclonal gammopathy after use of therapeutic monoclonal antibodies has been documented. This triggers concerns for similar misdiagnoses in animals treated with monoclonal antibodies. The aim of this study was to evaluate if lokivetmab interferes with serum protein electrophoresis and immunofixation electrophoresis in dogs. Material and methods Residual sera from 25 client-owned, healthy blood donor dogs from 2 veterinary hospitals in Germany were used. The residual sera were analysed with serum protein electrophoresis and immunofixation electrophoresis before and after being spiked with lokivetmab at a concentration of 10 µg/ml (corresponding to the mean peak serum concentration after a subcutaneous injection of 2 mg/kg lokivetmab). Results No monoclonal gammopathy was observed on serum protein electrophoresis and all proteins had a normal distribution pattern without any pathologic bands on immunofixation electrophoresis. The absolute γ-globulin values of spiked samples, however, were significantly higher than in the native sera although they remained within the reference interval. No other globulin fractions were significantly different. Conclusion and clinical relevance This study suggests that lokivetmab at a dose of 2 mg/kg is not detected as a monoclonal peak on serum protein electrophoresis or immunofixation electrophoresis, and thus is unlikely to lead to a misdiagnosis of other diseases that are characterised by monoclonal gammopathies.