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.

Predominant Mesangial IgM, C3 and λ Light Chain Depositions and Interstitial Nephritis in a Patient with Overlap Syndrome and Positivity for Anti-Mitochondrial M2 Antibody: A Case Report

Overlap syndrome refers to a group of conditions that have clinical features of more than one well-characterized rheumatic disease and meet the respective classification criteria. There are no typical renal histological findings in overlap syndrome. When patients with overlap syndrome develop renal dysfunction, various potential causes, including lupus nephritis (LN), renal crisis by systemic sclerosis, interstitial nephritis and so on, need to be distinguished. Here we report a 44-year-old woman with overlap syndrome involving systemic lupus erythematosus (SLE), diffuse cutaneous systemic scleroderma and Sjogren’s syndrome, who was also positive for anti-mitochondrial M2 antibody. She developed glomerular hematuria, proteinuria and increase in creatinine appeared gradually. Suspecting lupus nephritis, renal biopsy was performed. However, in the interstitium, mild infiltration of lymphocytes and plasma cells and very partial fibrosis were observed. Immunofluorescence (IF) microscopy revealed predominant mesangial IgM, C3 and λ light chain staining. Overall, LN was not diagnosed based on these findings. Renal dysfunction was normalized by glucocorticoid treatment for three months. This case suggests the importance of a renal diagnosis based on renal pathological findings, especially in a case of overlap syndrome including SLE.

MO158CLINICOPATHOLOGICAL FEATURES OF COEXISTENT LIGHT CHAIN CASE NEPHROPATHY AND LIGHT CHAIN DEPOSITION DISEASE IN PATIENTS WITH NEWLY DIAGNOSED MULTIPLE MYELOMA

Background and Aims Several patients with multiple myeloma suffered from more than one type of kidney disease simultaneously, of which the most common pattern is coexistent light chain cast nephropathy and light chain deposition disease (LCCN+LCDD). We investigated clinicopathological characteristics of LCCN+LCDD in comparison with pure LCCN and pure LCDD. Method Forty-seven percent of the renal biopsies revealed a monoclonal κ light chain restriction, ranging from 31% in the pure LCCN group to 90% in the pure LCDD group. Of note, more than half of the patients with LCCN+LCDD showed λ light chain isotype, which was significantly different from patients with pure LCDD (56% vs. 10%, p = 0.033). Compared with patients with pure LCDD, patients with LCCN+LCDD usually presented atypical features of LCDD with less nodular glomerulosclerosis (p = 0.003) and less diffuse deposit distribution (p = 0.027). Compared to patients with pure LCDD, patients with LCCN+LCDD had lower hemoglobin (126.5 g/L vs. 82.0 g/L, p = 0.008), higher incidence of AKI (20% vs. 89%, p = 0.003), lower percentage of urinary albumin excretion (%UAE) (68.9% vs. 5.1%, p = 0.003). There was no significant clinical difference between patients with LCCN+LCDD and patients with pure LCCN. Compared to patients with pure LCDD, patients with pure LCCN had lower hemoglobin (126.5 g/L vs. 87.0 g/L, p = 0.001), higher incidence of AKI (20% vs. 85%, p < 0.001), higher incidence of hemodialysis at diagnosis (10% vs. 65%, p = 0.003), higher serum creatinine (165.8 μmol/L vs. 627.0 μmol/L, p = 0.02) and lower incidence of hematuria (6/10 vs. 4/26, p = 0.035). Results Forty-seven percent of the renal biopsies revealed a monoclonal κ light chain restriction, ranging from 31% in the pure LCCN group to 90% in the pure LCDD group. Of note, more than half of the patients with LCCN+LCDD showed λ light chain isotype, which was significantly different from patients with pure LCDD (56% vs. 10%, p = 0.033). Compared with patients with pure LCDD, patients with LCCN+LCDD usually presented atypical features of LCDD with less nodular glomerulosclerosis (p = 0.003) and less diffuse deposit distribution (p = 0.027). Compared to patients with pure LCDD, patients with LCCN+LCDD had lower hemoglobin (126.5 g/L vs. 82.0 g/L, p = 0.008), higher incidence of AKI (20% vs. 89%, p = 0.003), lower percentage of urinary albumin excretion (%UAE) (68.9% vs. 5.1%, p = 0.003). There was no significant clinical difference between patients with LCCN+LCDD and patients with pure LCCN. Compared to patients with pure LCDD, patients with pure LCCN had lower hemoglobin (126.5 g/L vs. 87.0 g/L, p = 0.001), higher incidence of AKI (20% vs. 85%, p < 0.001), higher incidence of hemodialysis at diagnosis (10% vs. 65%, p = 0.003), higher serum creatinine (165.8 μmol/L vs. 627.0 μmol/L, p = 0.02) and lower incidence of hematuria (6/10 vs. 4/26, p = 0.035). Conclusion Pathologically, patients with LCCN+LCDD were more likely to have λ light chain isotype and presented atypical features of LCDD including less nodular glomerulosclerosis and less deposit distribution than patients with pure LCDD. In clinical characteristics, patients with LCCN+LCDD and patients with pure LCCN shared similar features. For patients with LCCD, especially those with λ restriction, nephrologists should carefully evaluate the kidney specimens to exclude the possibility of combined LCCN.

Computational investigation of the binding of a designed peptide to λ light chain amyloid fibril

Peptide P62 only binds to the canonical interface of the amyloid fibril. Lysine residues of P62 play an important role in the binding process by forming initial contacts with aspartic acids on the fibril surface.

Pyrrolidinedithiocarbamic Acid Ammonium Salt Inhibits Apop-tosis and Phenotypic Transformation of Co-Culture of Myeloma Cells and Renal Tubular Epithelial Cells by Reducing the Secretion of Light Chain Protein

Background: We investigate the effects of NFκB inhibitor pyrrolidinedithiocarbamic acid ammonium salt (PDTC) on the viability, apoptosis and cell phenotype of HK-2 cells in the co-culture system of myeloma cells in renal tubular epithelial cells. Methods: This study was performed in Qiqihar Medical University, Qiqihar, China from Jun 2018 to Jan 2019. RPMI-8226 cells and HK-2 cells were inoculated in the co-culture chamber and cultured to establish the co-culture system. An immunoturbidimetric assay was performed to detect κ light chain and λ light chain in RPMI-8226 cells. The effect of PDTC on the secretion of κ light chain and λ light chain of RPMI-8226 cells was detected by immunoturbidimetry and the ratio was calculated. Results: PDTC significantly increased the viability of HK-2 cells. PDTC reduced the apoptosis of renal tubular epithelial cells. After PDTC treatment, the expression of cell surface marker E-cadherin decreased, and the expression of α-SMA increased, which induced the renal interstitial fibrosis. The secretion of κ light chain and λ light chain of RPMI-8226 cells was significantly decreased after the addition of PDTC, but the ratio was not changed. Conclusion: PDTC can inhibit the cell activity, promote apoptosis, and reduce the secretion of secretion of κ light chain and λ light chain through inhibiting the NF-κB pathway activation of myeloma cell RPMI-8226.

PDTC inhibits apoptosis and phenotypic transformation of co-culture of myeloma cells and renal tubular epithelial cells by reducing the secretion of light chain protein

Background The human myeloma cell line RPMI-8226 is co-cultured with human proximal tubular epithelial cell HK-2, which is commonly used to simulate the renal environment in patients with multiple myeloma. This study aimed to investigate the effects of NFκB inhibitor PDTC on the viability, apoptosis and cell phenotype of HK-2 cells in the co-culture system of myeloma cells in renal tubular epithelial cells.Methods An in vivo environment was simulated through a cell co-culture system. RPMI-8226 cells and HK-2 cells were inoculated in the co-culture chamber and cultured for 24 h to establish the co-culture system. PDTC was used in the single culture group and the co-culture group, respectively. The activity of HK-2 cells and RPMI-8226 cells in each group was detected by MTT. An immunoturbidimetric assay was performed to assess the effect of PDTC on secretion of κ light chain and λ light chain in RPMI-8226 cells. Flow cytometry was used to detect apoptosis of HK-2 cells. Western blot was carried out to detect NF-κB activation in RPMI-8226 myeloma cells, as well as the expression levels of caspase-3, bcl-2, Bax, E-cadherin, and α-SMAin HK-2 cells. Caspse-3 assay kit was used to detect the activity of caspase-3. The effect of PDTC on the secretion of κ light chain and λ light chain of RPMI-8226 cells was detected by immunoturbidimetry and the ratio was calculated.Results PDTC had a potent inhibitory effect on proliferation of RPMI-8226 cells in a dose- and time-dependent manner. PDTC had no significant effect on the viability of HK-2 cells cultured alone, and the addition of PDTC to the co-culture system significantly increased the viability of HK-2 cells. PDTC did not significantly change the apoptosis of HK-2 cells cultured alone, but could reduce the apoptosis of renal tubular epithelial cells by regulating the activity of caspase3 and the ratio of bcl2/bax in HK-2 cells in the co-culture system. After PDTC treatment, the expression of cell surface marker E-cadherin decreased, and the expression of α-SMA increased, which induced the renal interstitial fibrosis. The secretion of κ light chain and λ light chain of RPMI-8226 cells was significantly decreased after the addition of PDTC, but the ratio was not changed.Conclusions PDTC can inhibit the cell activity, promote apoptosis, and reduce the secretion of secretion of κ light chain and λ light chain through inhibiting the NF-κB pathway activation of myeloma cell RPMI-8226, leading to increased activity of renal tubular epithelial cells HK-2 in the co-culture system, decreased apoptosis, and renal interstitial fibrosis.

Analysis of treatment efficacy in the GEM-CESAR trial for high-risk smoldering multiple myeloma patients: Comparison between the standard and IMWG MRD criteria and QIP-MS including FLC (QIP-FLC-MS).

8512 Background: The GEM-CESAR trial is a potentially curative strategy for high-risk smoldering multiple myeloma (HRsMM) patients (pts) in which the primary endpoint is the achievement of sustained minimal residual disease (MRD) negativity in the bone marrow (BM) by next generation flow (NGF). The value of BM MRD assessment in MM is proven, but alternative, non-invasive methods, accurately reflecting disease burden are needed. Methods: Pts received six 4-week cycles of KRd as induction (K:36mg/m2 twice weekly, R: lenalidomide 25mg po od days 1-21 and d:dexamethasone 40mg po weekly) followed by melphalan 200mg/m2, two further cycles of KRd as consolidation and up to 2 years of Rd (R:10mg/d, d:20mg/week). Efficacy was analyzed in parallel in BM samples by NGF and in serum by SPEP/IFE and QIP-MS/QIP-FLC-MS in 52 out of the 90 pts enrolled in the trial. Standard and MRD responses were carried out as per the IMWG guidelines. For QIP-MS serum immunoglobulins were purified using polyclonal antibodies (anti-IgG, -IgA, -IgM, -total κ and -total λ light chain, -free κ and -free λ light chain). Mass spectra were generated on a MALDI-TOF-MS system. Results: Overall response rate (ORR) was 98% post-induction, 98% post-ASCT, and 100% post-consolidation; 38.4%, 61.5% and 68.6% of pts reached ≥ complete response at each time-point and, among them, 23%, 44% and 55% achieved flow MRD-negativity. Using the combination of QIP-MS/QIP-FLC-MS, the percentage of pts without detectable disease at each timepoint lowered to 12%, 27% and 38% reflecting the higher sensitivity of the method. Against NGF, QIP-MS/QIP-FLC-MS provided negative predictive values of 67%, 92% and 89% (p = 0,0206; p < 0,001; p = 0,003) and identified disease in 95%, 97% and 92% of pts that were positive by NGF-MRD at each respective timepoint. Three pts from this cohort have progressed so far: two were NGF+/MS+ at the three timepoints whilst 1 remained NGF- but QIP-MS/FLC-MS+ throughout. Conclusions: The GEM-CESAR treatment strategy induces a high ORR in HRsMM pts, and the % of cases achieving flow-MRD negativity post-ASCT meets the primary endpoint of the trial. The combined use of QIP-MS and FLC-MS offers higher sensitivity relative to standard methods and may provide relevant information about the right timing for performing a BM aspirate/biopsy. Clinical trial information: NCT02415413 .