Immunoparesis defined by heavy/light chain pair suppression in smoldering multiple myeloma shows initial isotype specificity and involves other isotypes in advanced disease.

Smoldering multiple myeloma (SMM) is an asymptomatic and biologically heterogeneous plasma cell disorder, with a highly variable clinical course. Immunoparesis, defined by total immunoglobulin measurements, has been shown to be an independent risk factor for progression to symptomatic disease. The heavy/light chain (HLC) assay allows precise measurement of the polyclonal immunoglobulin of the same isotype, enabling the evaluation of isotype-matched immunoparesis (IMI). In this study, we prospectively characterized immunoparesis, as determined by HLC measurements, in 53 SMM patients. Severe IMI was present in 51% of patients, while severe IP of uninvolved isotypes (HLC IP) was present in 39%. Most of the patients with severe HLC IP presented with severe IMI, but not the other way around. Isotype specificity of immune suppression was suggested by lower relative values of isotype-matched HLC pairs, both for IgG and IgA SMM. Severe IMI was associated with other risk factors for progression while patients with severe IMI and severe HLC IP showed an even higher risk profile. Both severe IMI and severe IgM HLC IP showed a significantly shorter time to progression. Finally, gene expression analysis demonstrated differences in the bone marrow microenvironment between patients with IMI and IMI plus HLC IP, with an increased expression of genes associated with cytolytic cells. In conclusion, our data supports isotype specificity of early immunoglobulin suppression mechanisms. While suppression of both involved and uninvolved isotypes is associated with risk of progression, the later appears to develop with more advanced disease and could be mediated by different mechanisms.

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

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

Proteomic markers with prognostic impact on outcome of chronic lymphocytic leukemia patients under chemo-immunotherapy: results from the HOVON 109 study.

Despite recent identification of several prognostic markers, there is still a need for new prognostic parameters able to predict clinical outcome in chronic lymphocytic leukemia (CLL) patients. Here, we aimed to validate the prognostic ability of known (proteomic) markers measured pretreatment and to search for new proteomic markers that might be related to treatment response in CLL. To this end, baseline serum samples of 51 CLL patients treated with chemo-immunotherapy were analyzed for 360 proteomic markers, using Olink technology. Median event-free survival (EFS) was 23 months (range: 1.25-60.9). Patients with high levels of sCD23 (>11.27, p = 0.026), sCD27 (>11.03, p = 0.04), SPINT1 (>1.6, p = 0.001), and LY9 (>8.22, p = 0.0003) had a shorter EFS than those with marker levels below the median. The effect of sCD23 on EFS differed between immunoglobulin heavy chain variable gene-mutated and unmutated patients, with the shortest EFS for unmutated CLL patients with sCD23 levels above the median. Taken together, our results validate the prognostic impact of sCD23 and highlight SPINT1 and LY9 as possible promising markers for treatment response in CLL patients.

Mass Spectrometry-Based Method Targeting Ig Variable Regions for Assessment of Minimal Residual Disease in Multiple Myeloma.

Multiple myeloma is a systemic malignancy of monoclonal plasma cells that accounts for 10% of hematologic cancers. With development of highly effective therapies for multiple myeloma, minimal residual disease (MRD) assessment has emerged as an important end point for management decisions. Currently, serologic assays lack the sensitivity for MRD assessment, and invasive bone marrow sampling with flow cytometry or molecular methods has emerged as the gold standard. We report a sensitive and robust targeted mass spectrometry proteomics method to detect MRD in serum, without the need of invasive, sequential bone marrow aspirates. The method detects Ig-derived clonotypic tryptic peptides predicted by sequencing the clonal plasma cell Ig genes. A heavy isotope-labeled Ig internal standard is added to patient serum at a known concentration, the Ig is enriched in a light chain type specific manner, and proteins are digested and analyzed by targeted mass spectrometry. Peptides from the constant regions of the λ or κ light chains, Ig heavy chains, and clonotypic peptides unique to the patient monoclonal Igs are targeted. This technique is highly sensitive and specific for the patient-specific monoclonal Igs, even in samples negative by multiparametric flow cytometry. Our method can accurately and precisely detect monoclonal protein in serum of patients treated for myeloma and has broad implications for management of hematologic patients.

Differential levels of Alpha-1-inhibitor III, Immunoglobulin heavy chain variable region, and Hypertrophied skeletal muscle protein GTF3 in rat mammary tumorigenesis.

Early detection of breast cancer can be best facilitated by the development of precancerous markers. Serum proteins being the sensitive signatures, can be the ideal choice. We previously demonstrated the reduced levels of two serum proteins at a very early stage of tumorigenesis in a breast cancer model, developed in Wistar rats by 7,12-dimethylbenz[a]anthracene (DMBA) administration. Here we report the dysregulation of three more proteins in the serum collected at another early stage (15 weeks) of tumorigenesis in the same model. The proteins were identified (as Alpha-1-inhibitor III (Mug1), Immunoglobulin heavy chain variable region (IGHV), and Hypertrophied skeletal muscle protein GTF3) by MALDI-TOF MS after the screening and fingerprinting of serum samples by one-dimensional (1D) and two-dimensional (2D) electrophoresis respectively. Relative expression analysis of corresponding genes was also carried out, and the results were found as supporting the proteomic findings. In addition, the candidate proteins of the study and their corresponding ribonucleic acids (RNAs) were subjected to homology modelling and docking (using softwares like MODELLER, 3dRNA, Autodock4.0, and GROMACS etc), which revealed the binding sites for carcinogen (DMBA) and its nature of interaction with proteins and RNAs. Moreover, the network analysis by GeneMANIA unraveled the protein/gene functional network in which Mug1, IGHV, and GTF3 are involved. Based on the significant protein and gene expression alterations in early tumorigenesis, these proteins may prove very effective in search for biomarkers for the early detection of mammary cancer. Further, these proteins can also be tried as targets for chemotherapy.

Daratumumab plus CyBorD for patients with newly diagnosed AL amyloidosis: safety run-in results of ANDROMEDA.

Although no therapies are approved for light chain (AL) amyloidosis, cyclophosphamide, bortezomib, and dexamethasone (CyBorD) is considered standard of care. Based on outcomes of daratumumab in multiple myeloma (MM), the phase 3 ANDROMEDA study (NCT03201965) is evaluating daratumumab-CyBorD vs CyBorD in newly diagnosed AL amyloidosis. We report results of the 28-patient safety run-in. Patients received subcutaneous daratumumab (DARA SC) weekly in cycles 1 to 2, every 2 weeks in cycles 3 to 6, and every 4 weeks thereafter for up to 2 years. CyBorD was given weekly for 6 cycles. Patients had a median of 2 involved organs (kidney, 68%; cardiac, 61%). Patients received a median of 16 (range, 1-23) treatment cycles. Treatment-emergent adverse events were consistent with DARA SC in MM and CyBorD. Infusion-related reactions occurred in 1 patient (grade 1). No grade 5 treatment-emergent adverse events occurred; 5 patients died, including 3 after transplant. Overall hematologic response rate was 96%, with a complete hematologic response in 15 (54%) patients; at least partial response occurred in 20, 22, and 17 patients at 1, 3, and 6 months, respectively. Renal response occurred in 6 of 16, 7 of 15, and 10 of 15 patients, and cardiac response occurred in 6 of 16, 6 of 13, and 8 of 13 patients at 3, 6, and 12 months, respectively. Hepatic response occurred in 2 of 3 patients at 12 months. Daratumumab-CyBorD was well tolerated, with no new safety concerns versus the intravenous formulation, and demonstrated robust hematologic and organ responses. This trial was registered at www.clinicaltrials.gov as #NCT03201965.

Cardiac involvement in heavy and light chain amyloidosis: A case report and literature review.

INTRODUCTION: Heavy and light chain amyloidosis is an extremely rare condition. There are few reports referring to the clinical impact of cardiac involvement in heavy and light chain amyloidosis, and the significance of myocardial impairment has not yet been completely explained. PATIENT CONCERNS: A 66-year-old Japanese man was admitted to our hospital presenting with nephrotic syndrome and congestive heart failure. DIAGNOSIS: Kidney and endoscopic gastric mucosal biopsy demonstrated congophilic hyalinization in most of the glomeruli and surrounding vessel walls, which were highly positive for immunoglobulin A and lambda. Finally, the patient was diagnosed as an atypical multiple myeloma with systemic heavy and light chain amyloidosis. INTERVENTIONS: The patient was referred to hematology for further treatment and was moved to another hospital for the administration of chemotherapy using melphalan and dexamethasone. OUTCOMES: The patient was still alive after 15-month follow-up from the initial diagnosis. CONCLUSION: Initial screening and follow-up for cardiac involvement are important for heavy and light chain amyloidosis. Further investigation for the prognosis of heavy and light chain amyloidosis is required to improve the strategies of diagnosis and treatment options for patients with this disease.

A new risk model to predict time to first treatment in chronic lymphocytic leukemia based on heavy chain immunoparesis and summated free light chain.

BACKGROUND: Chronic lymphocytic leukemia (CLL) is frequently accompanied by immune dysregulation. AIMS: In this multicenter prospective study, we investigated whether heavy + light chains (HLC: IgGκ, IgGλ, IgAκ, IgAκ, IgMκ, IgMλ) and IgG subclasses (IgG1, IgG2, IgG3, and IgG4) could be used as novel prognostic markers of immunoparesis in 105 treatment-naïve patients with CLL. RESULTS: Heavy + light chains immunoparesis of ≥1, ≥2, and ≥3 isotypes was evident in 74 (70%), 58 (55%), and 36 (34%) patients, respectively. Severe HLC immunoparesis was identified in 40 (38%) patients. Of the IgG subclasses, IgG1 and IgG2 were most frequently suppressed, affecting 46 (44%) and 36 (34%) patients, respectively; 63 (60%) patients had low levels of at least one IgG subclass. In multivariate analysis, severe HLC immunoparesis (hazard ratio [HR]: 36.5; P = .010) and ΣFLC ≥ 70 mg/L (HR: 13.2; P = .004) were the only factors independently associated with time to first treatment (TTFT). A risk model including these variables identified patients with 0, 1, and 2 risk factors and significantly different TTFT (P < .001). Patients with two factors represented an ultra-high-risk group with a median TTFT of only 1.3 months. CONCLUSION: The above findings demonstrate the potential for the use of HLC immunoparesis, together with sFLC measurements, as future prognostic biomarkers in CLL.

Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life.

Antibodies and Fc-fusion antibody-like proteins have become successful biologics developed for cancer treatment, passive immunity against infection, addiction, and autoimmune diseases. In general these biopharmaceuticals can be used for blocking protein:protein interactions, crosslinking host receptors to induce signaling, recruiting effector cells to targets, and fixing complement. With the vast capability of antibodies to affect infectious and genetic diseases much effort has been placed on improving and tailoring antibodies for specific functions. While antibody:antigen engagement is critical for an efficacious antibody biologic, equally as important are the hinge and constant domains of the heavy chain. It is the hinge and constant domains of the antibody that engage host receptors or complement protein to mediate a myriad of effector functions and regulate antibody circulation. Molecular and structural studies have provided insight into how the hinge and constant domains from antibodies across different species, isotypes, subclasses, and alleles are recognized by host cell receptors and complement protein C1q. The molecular details of these interactions have led to manipulation of the sequences and glycosylation of hinge and constant domains to enhance or reduce antibody effector functions and circulating half-life. This review will describe the concepts being applied to optimize the hinge and crystallizable fragment of antibodies, and it will detail how these interactions can be tuned up or down to mediate a biological function that confers a desired disease outcome.

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 ß 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, ß-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 ß 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 ß region on SPE.

AL amyloidosis with non-amyloid forming monoclonal immunoglobulin deposition; a case mimicking AHL amyloidosis.

BACKGROUND: Immunoglobulin heavy-and-light-chain amyloidosis (AHL amyloidosis) is a newly established disease entity where both the immunoglobulin heavy-chain and light-chain compose amyloid fibrils. The immunoglobulins responsible for the amyloid fibrils are generally identified by immunostaining and/or laser microdissection-liquid chromatography-tandem mass spectrometry (LMD-LC-MS/MS). However, both techniques do not biochemically differentiate immunoglobulins that formed amyloid fibrils from non-responsible immunoglobulins. CASE PRESENTATION: We herein report a case of 67-year-old female patient with renal amyloidosis due to lymphoplasmacytic lymphoma secreting monoclonal immunoglobulin M (IgM)-kappa. Renal immunostaining monotypically positive for IgM-kappa and LMD-LC-MS/MS identification of mu heavy-chain and kappa light-chain were consistent with the diagnosis of AHL amyloidosis. In order to confirm that both the immunoglobulin heavy-chain and light-chain were forming amyloid fibrils, we performed LC-MS/MS of renal amyloid fibrils isolated by the traditional amyloid purification method. The additional LC-MS/MS identified kappa light-chain only without any heavy-chain component. These results were suggestive that amyloid fibrils were composed by kappa light-chain only and that the mu heavy-chain identified by immunostaining and LMD-LC-MS/MS was derived from the non-specific co-deposition of monoclonal IgM-kappa. CONCLUSION: The case was AL amyloidosis with non-amyloid forming monoclonal immunoglobulin deposition. While immunostaining and LMD-LC-MS/MS are irreplaceable techniques to classify amyloidosis, confident exclusion of the present condition should be required to diagnose AHL amyloidosis.

Prognostic utility of serum free light chain ratios and heavy-light chain ratios in multiple myeloma in three PETHEMA/GEM phase III clinical trials.

We investigated the prognostic impact and clinical utility of serum free light chains (sFLC) and serum heavy-light chains (sHLC) in patients with multiple myeloma treated according to the GEM2005MENOS65, GEM2005MAS65, and GEM2010MAS65 PETHEMA/GEM phase III clinical trials. Serum samples collected at diagnosis were retrospectively analyzed for sFLC (n = 623) and sHLC (n = 183). After induction or autologous transplantation, 309 and 89 samples respectively were available for sFLC and sHLC assays. At diagnosis, a highly abnormal (HA) sFLC ratio (sFLCr) (<0.03 or >32) was not associated with higher risk of progression. After therapy, persistence of involved-sFLC levels >100 mg/L implied worse survival (overall survival [OS], P = 0.03; progression-free survival [PFS], P = 0.007). Among patients that achieved a complete response, sFLCr normalization did not necessarily indicate a higher quality response. We conducted sHLC investigations for IgG and IgA MM. Absolute sHLC values were correlated with monoclonal protein levels measured with serum protein electrophoresis. At diagnosis, HA-sHLCrs (<0.29 or >73) showed a higher risk of progression (P = 0.006). Additionally, involved-sHLC levels >5 g/L after treatment were associated with shorter survival (OS, P = 0.001; PFS, P = 0.018). The HA-sHLCr could have prognostic value at diagnosis; absolute values of involved-sFLC >100 mg/L and involved-sHLC >5 g/L could have prognostic value after treatment.

Normal serum protein electrophoresis and mutated IGHV genes detect very slowly evolving chronic lymphocytic leukemia patients.

More than 35 years after the Binet classification, there is still a need for simple prognostic markers in chronic lymphocytic leukemia (CLL). Here, we studied the treatment-free survival (TFS) impact of normal serum protein electrophoresis (SPE) at diagnosis. One hundred twelve patients with CLL were analyzed. The main prognostic factors (Binet stage; lymphocytosis; IGHV mutation status; TP53, SF3B1, NOTCH1, and BIRC3 mutations; and cytogenetic abnormalities) were studied. The frequencies of IGHV mutation status, cytogenetic abnormalities, and TP53, SF3B1, NOTCH1, and BIRC3 mutations were not significantly different between normal and abnormal SPE. Normal SPE was associated with Binet stage A, nonprogressive disease for 6 months, lymphocytosis below 30 G/L, and the absence of the IGHV3-21 gene rearrangement which is associated with poor prognosis. The TFS of patients with normal SPE was significantly longer than that of patients with abnormal SPE (log-rank test: P = 0.0015, with 51% untreated patients at 5.6 years and a perfect plateau afterward vs. a median TFS at 2.64 years for abnormal SPE with no plateau). Multivariate analysis using two different Cox models and bootstrapping showed that normal SPE was an independent good prognostic marker for either Binet stage, lymphocytosis, or IGHV mutation status. TFS was further increased when both normal SPE and mutated IGHV were present (log-rank test: P = 0.008, median not reached, plateau at 5.6 years and 66% untreated patients). A comparison with other prognostic markers suggested that normal SPE could reflect slowly advancing CLL disease. Altogether, our results show that a combination of normal SPE and mutated IGHV genes defines a subgroup of patients with CLL who evolve very slowly and who might never need treatment.

Abnormal Heavy/Light Chain Ratio and Matched Pair Suppression Increase Residual Disease Detection Sensitivity in Patients With Multiple Myeloma With Deep Responses.

BACKGROUND: Heavy/light chain (HLC) assay can quantify involved as well as uninvolved immunoglobulin pairs and is used to detect monoclonal proteins. PATIENTS AND METHODS: We compared the sensitivity between HLC assay and serum protein electrophoresis, serum immunofixation electrophoresis (IFE), and free light chain (FLC) assay in patients with symptomatic multiple myeloma (n = 111) whose responses were stable disease or better. RESULTS: Among patients with negative IFE and normal FLC ratios, 84.4% (38 of 45) and 80% (36 of 45) exhibited normal HLC ratios and no pair suppression, respectively (13.3% [6 of 45], moderate pair suppression and 6.7% [3 of 45], severe pair suppression). The lower the monoclonal protein levels, the more the possibility that the patients had normal HLC ratios and no matched pair suppression (both P < .000001). HLC ratios or pair suppression combined with IFE results and FLC ratios were more sensitive for detecting monoclonal proteins than were IFE results and FLC ratios alone (P = .016 and .0039, respectively). A combination of all 4 methods (IFE, FLC, HLC, and pair suppression) was far more sensitive than were IFE findings plus FLC ratios alone (P = .00024). CONCLUSION: Abnormal HLC ratios and HLC-matched pair suppression can increase the sensitivity for detecting residual disease in patients with multiple myeloma with deep responses.

Heavy + light chain analysis to assign myeloma response is analogous to the IMWG response criteria.

Automated serum heavy + light chain (HLC) immunoassays can measure the intact immunoglobulins of each light chain type separately. We though to compare HLC assays with electrophoretic techniques in determining International Myeloma Working Group (IMWG) response criteria. 114 myeloma patients from 2 trials were included. HLC measurements were made utilizing archived sera and response assessments compared with those based on electrophoretic analysis at the time of the trials. Assessments at ∼90 days and maximal response were compared as was the power of the 2 techniques for predicting later responses, overall survival, and progression. The kappa statistic indicated good agreement between the 2 methods for determining IMWG response criteria, although HLC measurements might give better predictions of subsequent responses and frequently gave an earlier indication of change. HLC measurements could represent an alternative to electrophoretic techniques in determining IMWG response. Validation with a greater range of patient responses is needed for confirmation.

Cytogenetic Profiling of Myelomas, Association With Complete Blood Count: Study of 180 Patients.

OBJECTIVES: To analyze the most common primary and secondary cytogenetic events in myelomas using a probe panel designed in our laboratory, and to associate those events with hematological and biochemical findings. METHODS: Blood specimens from patients diagnosed with myeloma were processed to determine complete blood count and levels of albumin, creatinine, and beta-2 microglobulin. We evaluated bone-marrow specimens for plasma-cell percentage by light microscopy and for cytogenetic abnormalities by fluorescence in situ hybridization (FISH). The Mann-Whitney U test was used to compare hematological and biochemical parameters. RESULTS: We observed immunoglobulin heavy chain (IgH) gene translocations in 43.3% and t(4;14) in 21% of specimens; t(11;14) was observed in 7.7% of specimens. Gain of chromosomes was observed in 67.2% and loss observed in 16.6% of specimens. CONCLUSIONS: Gains of chromosomes were observed in two-thirds of patients with myeloma. The most common IgH translocation was t(4;14); del13/monosomy13 was the most common secondary cytogenetic abnormality. Partial or complete tetrasomies were associated with higher beta-2 microglobulin levels.