Immunoglobulin Kappa Light Chain Research Articles

Dual T-cell constant β chain (TRBC)1 and TRBC2 staining for the identification of T-cell neoplasms by flow cytometry

The diagnosis of leukemic T-cell malignancies is often challenging, due to overlapping features with reactive T-cells and limitations of currently available T-cell clonality assays. Recently developed therapeutic antibodies specific for the mutually exclusive T-cell receptor constant β chain (TRBC)1 and TRBC2 isoforms provide a unique opportunity to assess for TRBC-restriction as a surrogate of clonality in the flow cytometric analysis of T-cell neoplasms. To demonstrate the diagnostic utility of this approach, we studied 164 clinical specimens with (60) or without (104) T-cell neoplasia, in addition to 39 blood samples from healthy donors. Dual TRBC1 and TRBC2 expression was studied within a comprehensive T-cell panel, in a fashion similar to the routine evaluation of kappa and lambda immunoglobulin light chains for the detection of clonal B-cells. Polytypic TRBC expression was demonstrated on total, CD4+ and CD8+ T-cells from all healthy donors; and by intracellular staining on benign T-cell precursors. All neoplastic T-cells were TRBC-restricted, except for 8 cases (13%) lacking TRBC expression. T-cell clones of uncertain significance were identified in 17 samples without T-cell malignancy (13%) and accounted for smaller subsets than neoplastic clones (median: 4.7 vs. 69% of lymphocytes, p < 0.0001). Single staining for TRBC1 produced spurious TRBC1-dim subsets in 24 clinical specimens (15%), all of which resolved with dual TRBC1/2 staining. Assessment of TRBC restriction by flow cytometry provides a rapid diagnostic method to detect clonal T-cells, and to accurately determine the targetable TRBC isoform expressed by T-cell malignancies.

Clinical specificity of two assays for immunoglobulin kappa and lambda free light chains.

Free light chain (FLC) assays and the ratio of κ/λ are recommended for diagnosis, prognosis and monitoring of plasma cell dyscrasias (PCD). Limited data exists on FLC clinical specificity in patients diagnosed with other conditions. We assessed the κ, λ, and κ/λ FLC ratio using the FreeLite assay and the Sebia FLC ELISA assay in 176 patients with clinical presentations of fatigue, anemia, polyclonal hypergammaglobulinemia, joint disorders, kidney disease and non PCD-cancers with no monoclonal protein observed on serum protein electrophoresis or MASS-FIX immunoglobulin isotyping. Manufacturer defined reference intervals (RI) and glomerular filtration rate (GFR) specific RI (renal RI) were utilized. For the κ/λ ratio, 68.7 % (121/176) of specimens on the FreeLite and 87.5 % (154/176) of specimens on the Sebia assay were within RI. For κ, 68.2 % (120/176) and 72.2 % (127/176) of results were outside RI for FreeLite and Sebia respectively. For λ, 37.5 % (66/176) and 84.1 % (148/176) of FreeLite and Sebia results were outside RI. With FreeLite and Sebia, patients with kidney disease (n=25) had the highest κ/λ ratios. 44 patients (25.0 %) had GFR <60 mL/min/BSA. When renal RI were applied, 13.6 % had a FLCr outside the renal RI with FreeLite, and 4.5 % with Sebia. In a cohort of patients with signs and symptoms suggestive of PCDs, but ultimately diagnosed with other conditions, Sebia FLC had improved clinical specificity relative to FreeLite, if one was using an abnormal κ/λ ratio as a surrogate for monoclonality.

Targeting Loss of the Tumor Suppressor TENT5C in Multiple Myeloma

Introduction: Multiple myeloma (MM) is a genetically complex and heterogeneous neoplasm in which multiple genomic events lead to tumor development and progression. Loss of TENT5C through mutation or deletion on chromosome 1p occurs in more than 20% of newly diagnosed MM patients and is associated with a poor progression free and overall survival. Although, many studies have identified TENT5C as a tumor suppressor gene in MM, the impact of gene loss on MM physiopathology remain to be elucidated. Here we sought to identify drugs which may specifically target TENT5C-null MM cells using targeted and high-throughput drug screens (HTS). Methods: We generated CRISPR/Cas9 knockout (ko) of TENT5C in wild type (wt) U266 and KMS-11 MM cell lines and over-expressed (oe) TENT5C in MM cell lines with biallelic inactivation (H929 -/- and LP-1 -/-). Modified cell lines were compared to their original counterparts for gene expression, proliferation, and cell cycle differences. Targeted and HTS were performed to identify synthetic lethal pathways related to loss of TENT5C in MM that will result in specific cell death. Results: In TENT5C ko cells the RNA poly(A) polymerase activity was decreased at two TENT5C top targeted substrates, immunoglobulin kappa light chain and signal sequence receptor. Consistent with TENT5C tumor suppressor function, cell proliferation analysis showed that TENT5C ko cells had a higher growth rate than TENT5C wt cells. Cell cycle analysis, after nocodazole synchronization, showed TENT5C-null cells had an increased G2/M population compared to TENT5C-positive cells (U266 TENT5C ko 19.86% vs. TENT5C wt 2.6%, p&amp;lt;0.001; H929 TENT5C -/- 8.22% vs. TENT5C oe 1.18%, p&amp;lt;0.001), indicating an increase in cell cycle progression in TENT5C-null cells. Given our prior data indicating an increase in splicing abnormalities and non-homologous end joining (NHEJ) activation in TENT5C -/- patient samples, we performed growth inhibition curves using appropriate drugs. The spliceosome inhibitors pladienolide B and isoginkgetin affected TENT5C ko cells at lower concentrations than TENT5C wt cells (U266 TENT5C ko GI 50=0.58 vs. TENT5C wt GI 50=1.43 nM, U266 TENT5C ko GI 50=7.03 vs. TENT5C wt GI 50=13.72 µM, p&amp;lt;0.001, respectively) as did NHEJ inhibitors STL127705 and SCR7 (U266 TENT5C ko GI 50=22.5 vs. TENT5C wt GI 50=62.55 µM, U266 TENT5C ko GI 50=41.93 vs. TENT5C wt GI 50=117.38 µM, p&amp;lt;0.01). These results were confirmed on the other modified cells to ensure TENT5C specificity. A HTS using the LOPAC1280 library also identified several hits specifically affecting TENT5C null cells. The hits were categorized according to their targets to identify pathway enrichment. Inhibitors targeting the cell cycle (roscovitine, apigenin, SP60012) and angiogenesis (amsacrine) were selected for validation and further investigation. The differential effect observed in the HTS data was confirmed with full dose kill-curves on the same cell line (U266) and validated in another TENT5C ko cell line (KMS-11) to ensure target specificity. For example, amsacrine, an angiogenesis inhibitor, exhibited differential activity onT ENT5C ko and TENT5C wt cells (U266 TENT5C ko GI 50=5.63 µM vs. U266 TENT5C wt GI 50=9.19, p&amp;lt;0.001; KMS-11 TENT5C ko GI 50=9.26 µM vs. KMS-11 TENT5C wt GI 50=14.38, p&amp;lt;0.001). Similarly, roscovitine, a CDK inhibitor, affected TENT5C ko cells at lower concentrations than TENT5C wt cells (U266 TENT5C ko GI 50=23.33 vs. U266 TENT5C wt GI 50=34.91 µM; KMS-11 TENT5C ko GI 50=13.47 vs. KMS-11 TENT5C wt GI 50=29.33 µM, p&amp;lt;0.001). Additionally, cell cycle analysis showed that roscovitine induced a G 0/G 1 arrest in a dose-response manner in U266 and KMS-11 cells. Interestingly, TENT5C ko cells were more sensitive to the CDK inhibitor, and the arrested KMS-11 G 0/G 1 populations increased from 3.35 in TENT5C wt to 6.46 % in TENT5C ko and from 15.51 in TENT5C wt to 21.15 % in TENT5C ko (p&amp;lt;0.05), after treatment with 12.5 and 25 µM of roscovitine, respectively. The same trend was also validated in U266 TENT5C ko cells to ensure target specificity. Conclusion: Our results indicate a tumor suppressor role of TENT5C in MM, with antiproliferative properties and a susceptibility to spliceosome and NHEJ inhibitors. The HTP screen identified other targets that are currently undergoing validation leading to the identification of the molecular mechanisms underlying this function.

The Effectiveness of Blinatumomab in Clearing Next-Generation Sequencing Measurable Residual Disease in Pediatric Patients with B-Cell Acute Lymphoblastic Leukemia

Introduction Blinatumomab is a bispecific T-cell engager antibody designed to redirect CD3+ T cells to bind to CD19+ target cells in relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). Measurable residual disease (MRD) is a significant prognostic factor for relapse and overall survival in B-ALL. Blinatumomab consolidation therapy has been shown to improve MRD remission (&amp;lt;10 -4), as detected by multiparameter flow cytometry (MFC) or reverse transcriptase-polymerase chain reaction (PCR), and survival outcomes in children with high-risk first relapsed B-ALL. The next-generation sequencing (NGS)-MRD is a highly sensitive technique that can detect very low levels of MRD (&amp;lt;10 -6) in patients who were considered “MRD negative” by less sensitive MFC or PCR technologies. Early achievement of NGS-MRD negativity (&amp;lt;10 -6) can help in identifying patients who have a very low risk of relapse. It has been suggested that blinatumomab treatment could help to attain deeper responses than that measured with a MRD sensitivity level of 10 -4. However, data on blinatumomab clearing MRD with high sensitivity of 10 -6 remain scarce. Hence, we evaluated the effectiveness of blinatumomab in eradicating extremely low level of MRD (&amp;lt;10 -6), as detected by NGS, in children with B-ALL. Methods Pediatric B-ALL patients who were MFC-MRD negative (&amp;lt;10 -4) but NGS-MRD positive (sensitivity of 10 -6) after chemotherapy followed by blinatumomab consolidation therapy between October 2021 and February 2023, were studied retrospectively. At diagnosis, NGS analysis was performed by high-throughput deep level sequencing using the Seq-MRD ® (ImmuQuad Biotech, Hangzhou, China) to detect clonal rearrangements. Genomic DNA was extracted from bone marrow samples using QIAamp DNA Mini kit (Qiagen, Hilden, Germany), amplified by quantitative PCR and sequenced using the Illumina® Novaseq PE150 (Illumina ® Novaseq, San Diego, USA) platform. The sequences of immunoglobulin heavy chain (IGH) variable (V), diversity (D), and joining (J) (IGH -VDJ), IGH-DJ, immunoglobulin kappa light chain (IGK), immunoglobulin kappa deleting element (IGKDE) and immunoglobulin light chain (IGL) were assessed for clonality with dominant index sequence(s). Results Overall, 19 patients were enrolled. The baseline patient characteristics are shown in Table 1. Median age at diagnosis was 4.4 years (range 0.3-10.6), with males accounting for 84% of patients. Eight patients (42%) had at least 1 unfavorable genetic abnormality (i.e., KMT2A rearrangement, E2A-PBX1). Seventeen (89%) patients had ≥2 Ig clonal rearrangement at diagnosis. In total, NGS detected 71 clonal rearrangements, including 27 IGH clonal rearrangements in 15 (79%) patients, 9 IGH-DJ in 6 (32%), 14 IGK in 8 (42%), 6 IGKDE in 9 (32%), and 12 IGL in 8 (42%) patients. All patients achieved hematological complete response (CR) with MFC-MRD &amp;lt;10 -4 after the induction or consolidation chemotherapy. However, all these patients were identified to be MRD positive by NGS, including NGS-MRD &amp;gt;10 -6 and positive but not quantifiable (NGS-MRD &amp;lt;10 -6). Though the patients were MFC-MRD negative, 19 IGH clones, 4 IGH-DJ clones, 8 IGK clones, 7 IGKDE clones and 3 IGL clones were still persisted. After blinatumomab, MRD negativity rate by MFC was 95% (18/19) and the NGS-MRD negativity rate was 68% at 10 -6 (13/19). The rate of MRD clearance after blinatumomab per NGS was 69% and 67% in CR1 and CR2 patients, respectively. The clearance rates of IGH, IGK, IGL, IGH-DJ, and IGKDE were 75%, 67%, 33%, 100%, and 83%, respectively (Table 2). Three NGS-MRD negative patients with available NGS-MRD at pre-, mid-, and post-treatment achieved MRD clearance rapidly after blinatumomab treatment that was maintained durably through day 28. Three of 6 NGS-MRD positive patients relapsed after blinatumomab with 3 being IGH positive and 2 being IGL positive. Conclusion To the best of our knowledge, for the first time it has been demonstrated that blinatumomab could further eradicate MRD in patients achieving MFC-MRD &amp;lt;10 -4. Advanced MRD detection using NGS and blinatumomab made deeper molecular remission in B-ALL patients achievable. Further large-scale studies are warranted to verify the prognostic value of NGS in detecting lower levels of MRD in ALL patients treated in the current immunotherapy era.

Immunophenotypic Profile of Chronic Lymphoproliferative Diseases in the State of Rio Grande Do Norte, Northeast, Brazil

INTRODUCTION: Chronic lymphoproliferative diseases (CLPD) are considered as a heterogeneous group of diseases characterized by monoclonal expansion and accumulation of apparently mature lymphocytes, which have a proliferative and/or survival advantage over normal lymphocytes in different organs such as bone marrow, peripheral blood and lymph nodes. This translates into the progressive accumulation of clonal cells and their products, causing peripheral blood and bone marrow lymphocytosis, in addition to lymphadenopathy, splenomegaly or other organomegaly. According to the World Health Organization, which reflects the international consensus and is based on pathological, genetic and clinical factors, lymphoproliferative neoplasms are classified according to the stage of maturation and the lineage where the neoplastic transformation occurs through flow cytometry (FC). OBJECTIVES: The aim of this study was to demonstrate the immunophenotypic diagnostic profiles of patients with CLPD in Rio Grande do Norte, Brazil by FC. METHODOLOGY: Were investigated 884 patients with CLPD of both sexes through CF with a panel of monoclonal antibodies directed to CLPD. In addition, other patient information, such as age, gender, and clinical and hematological data were also collected. RESULTS: The results showed that 89.7% of the cases were B Cell CLPD (B-CLPD) and 10.3% T and natural killer cells (T/NK-CLPD). The distribution of B-CLPD cases were as follows: 487 Chronic lymphocytic leukemia; 18 B-Cell Pro-lymphocytic leukemia; 25 Hairy cell leukemia; 6 variant Hairy cell leukemia; 6 Follicular lymphoma leukemized; 4 Splenic lymphoma with villous lymphocytes; 33 Mantle cell lymphoma; 4 Waldenstrom's macroglobulinemia; 69 Multiple myeloma; 14 Plasma cell leukemia; 11 Burkitt's lymphoma and 117 Leukemic Non-Hodgkin lymphoma (Table 1). The distribution of CLPD-T/NK was as follows: 12 Large granular lymphocytic leukemia; 14 T-Cell prolymphocytic leukemia; 18 Adult T-cell leukemia; 20 Sézary syndrome; and 24 Peripheral T-Cell lymphoma and 2 DLPC-NK (Table 1). DISCUSSION: The B-CLPD are the most commonly observed corresponding to more than 85% of all cases and are characterized by the presence of clonal B lymphocytes with expression of a single type of immunoglobulin light chain (kappa or lambda) in a proportion greater than 1/10, associated the strong positivity of Pam-B antigens such as CD19, CD22 and CD20 and the absence or low expression of Pam-T antigens. However, other antigens such as CD200, CD5, FMC7, CD79b and CD103, among others, may be present or absent, specifically characterizing these entities as Chronic lymphocytic leukemia (weak CD22+; CD200+, CD5+, FMC7-, CD79b-), Hairy cells leukemia (strong CD22+; FMC7+; CD79b+; CD5-; CD200-; CD103+; CD123+; CD11c+ and Follicular lymphoma (strongCD22+/CD20+, CD10+, CD200-, CD79B+, FMC7+). T/NK-CLPD are less frequent and are characterized by the expression of Pan-T antigens CD2, CD3, CD5, CD7, CD28 and TCL-1, with clonality evidenced by strong positivity of TCRα/β in most cases and increase of TCD4+ or TCD8+ lymphocytes in a ratio above 1:10 in most cases. In this group, large granular lymphocyte leukemia (LGLG) is a rare type of T/NK cell CLPD characterized by peripheral and medullary infiltration of large hypergranular lymphocytic cells, comprising about 2-5% of all cases, the most commonly seen being CD8+ T cells. CONCLUSION: The results presented demonstrate that immunophenotyping is a defining technique for clarifying the diagnosis of patients with CPLD, clonality was also successfully characterized in the investigated cases of CPLP of B ,T and NK lineages.

Computational designing of the ligands of Protein L affinity chromatography based on molecular docking and molecular dynamics simulations

Protein L is a multidomain protein from Peptostreptococcus magnus with binding affinity to kappa light chain of human immunoglobulin (Ig) which is used for the purification of antibody fragments by affinity chromatography. The advances in protein engineering and computational biology approaches lead to the development of engineered affinity ligands with improved properties including binding affinity. In this study, molecular dynamics simulations (MDs) and Osprey software were used to design single B domains of the Protein L with higher affinity to antibody fragments. The modified B domains were then polymerized to ligand with six B domains by homology modeling methods. The results showed that single B domain mutants of MB1 (Thr865Trp) and MB2 (Thr847Met-Thr865Trp) had higher binding affinity to Fab compared to the wild single B domain. Also, MDs and molecular docking results showed that the polymerized Proteins L including the wild and mutated six B domains (6B0, 6B1, and 6B2) were stable during MDs and the two mutants of 6B1 and 6B2 showed higher binding affinity to Fab relative to the wild type. Communicated by Ramaswamy H. Sarma

An armed anti-immunoglobulin light chain nanobody protects mice against influenza A and B infections.

The immune system eliminates pathogen intruders such as viruses and bacteria. To recruit immune effectors to virus-infected cells, we conjugated a small molecule, the influenza neuraminidase inhibitor zanamivir, to a nanobody that recognizes the kappa light chains of mouse immunoglobulins. This adduct was designed to achieve half-life extension of zanamivir through complex formation with the much-larger immunoglobulins in the circulation. The zanamivir moiety targets the adduct to virus-infected cells, whereas the anti-kappa component simultaneously delivers polyclonal immunoglobulins of indeterminate specificity and all isotypes. Activation of antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity promoted elimination of influenza neuraminidase-positive cells. A single dose of the conjugate protected mice against influenza A or B viruses and was effective even when given several days after infection with a lethal dose of virus. In the absence of circulating immunoglobulins, we observed no in vivo protection from the adduct. The type of conjugates described here may thus find application for both anti-influenza prophylaxis and therapy.

P339 LIGHT–CHAIN CARDIAC AMYLOIDOSIS WITH FULMINANT EVOLUTION, A NOT–SO–RARE CONDITION: THE IMPORTANCE OF EARLY DIAGNOSIS

Abstract Introduction Amyloid AL results from misfolding of immunoglobulin light chain (kappa or lambda) secreted by abnormal plasma cells such as in multiple myeloma. It is a rare, multisystemic disease with an estimated annual incidence rate of 6 to 10 cases per million inhabitans in Western countries. The prognosis of this hematologic neoplasm is guided by the extent of cardiac involvement; if untreated, survival rate is less than 6 months; with early initiation of chemotherapy treatment, median survival can range from 4 to 10 years, except for high–risk cases. Case report: 58–year–old patient presented to emergency department in May 2022 with the first syncopal episode and sphincter release. The patient had no cardiovascular risk factors. Echocardiografy showed markedly hypertrophic left ventricle with hyper–reflective walls with a granular–sparkling appearance. Electromyography revealed sensory–motor polyneuropathy, early–stage autonomic neuropathy and bilateral carpal tunnel syndrome. Implantation of a loop recorder was performed to investigate the cause of syncope. In suspected amyloidosis, the patient underwent electrophoresis, which showed no monoclonal peaks, serum and urine kappa and lambda light chains were negative. Bone scintigraphy showed no osteotropic radiopharmaceutical uptake in the heart. Cardiac magnetic resonance imaging (MRI) revealed biventricular hypertrophic phenocopy (concentric hypertrophy) with diffuse edema–like tissue alterations. The patient was asymptomatic. In August 2022, hematologic evaluation revealed a monoclonal band for lambda light chains in repeated immunofixation and bone marrow biopsy showed 9–10% monoclonal lambda plasma cells. The patient was referred to a specialized center for amyloidosis, where a biopsy of peri–umbilical fat was performed to confirm the diagnosis and initiate therapy. In October 2022, due to recurrent syncope, advanced atrioventricular block and extreme bradycardia at loop recorder, a dual–chamber pacemaker was implanted. A few days after discharge, the patient suffered cardiac arrest at home due to electromechanical dissociation and died, only 5 months after the first symptom. Conclusion AL amyloidosis is an aggressive hematological neoplasm, with cardiac involvement. Early diagnosis is crucial for timely initiation of therapy, which is the only means available to increase patient survival. It is important to maintain a high level of awareness and diagnostic suspicion for this condition.

Read the clonotype: Next-generation sequencing-based lymphocyte clonality analysis and perspectives for application in pathology.

Clonality assessment using the unique rearrangements of immunoglobulin (IG) and T-cell receptor (TR) genes in lymphocytes is a widely applied supplementary test for the diagnosis of B-cell and T-cell lymphoma. To enable a more sensitive detection and a more precise comparison of clones compared with conventional clonality analysis based on fragment analysis, the EuroClonality NGS Working Group developed and validated a next-generation sequencing (NGS)-based clonality assay for detection of the IG heavy and kappa light chain and TR gene rearrangements for formalin-fixed and paraffin-embedded tissues. We outline the features and advantages of NGS-based clonality detection and discuss potential applications for NGS-based clonality testing in pathology, including site specific lymphoproliferations, immunodeficiency and autoimmune disease and primary and relapsed lymphomas. Also, we briefly discuss the role of T-cell repertoire of reactive lymphocytic infiltrations in solid tumors and B-lymphoma.

A rare case of systemic AL-amyloidosis under the mask of myocardial infarction in the absence of myocardial hypertrophy: case report

Systemic amyloidosis is a group of diseases associated with extracellular deposition of fibrillar proteins, resulting in the loss of normal organ structure and function. AL-amyloidosis occurs when amyloid is deposited, consisting of full-length lambda or kappa immunoglobulin light chains, or fragments thereof. This article describes the complexity of timely diagnosis of systemic AL-amyloidosis with a predominant heart lesion in the absence of myocardial hypertrophy on the example of an elderly patient in whom the leading manifestation of the disease was heart failure with a preserved left ventricular ejection fraction.

A Single Next Generation Sequencing Assay for Detection of Driver Mutations, Translocations and Copy Number Alterations in Patients with Multiple Myeloma

Multiple myeloma (MM) is a plasma cell malignancy characterized by several genetic abnormalities that impact risk stratification, outcome, and response to therapy; with fluorescence in situ hybridization (FISH) considered the gold-standard assay to detect the presence of chromosomal translocations and copy-number variations (CNV). Here, we evaluate the performance of a single clinically actionable targeted sequencing (NGS) assay for detecting cytogenetic abnormalities in addition to recurrent driver mutations in MM. Patient-derived bone marrow aspirates were obtained and enriched for CD138+ plasma cells. Targeted sequencing was performed using a 2.3 Mb custom capture panel covering 139 genes and regions surrounding MYC, immunoglobulin heavy chain (IgH), and immunoglobulin light chain kappa (IgK) and lambda (IgL) loci. Chromosomal translocations were identified using an in-house algorithm designed specifically for targeted capture DNA sequencing data analyses. Structural variants were called based on discordant read pairs as well as junction split reads, solving the challenges from both the overlapping paired-end reads due to the longer read lengths (150 bp) and the mapping ambiguities in the Ig regions. CNV were assessed using both on and off target reads, using a 200 kb bin size for segmentation covering the entire genome. Clinical annotations and FISH panel reports were abstracted. We sequenced 264 MM patient samples, 94% of which had available FISH information. A median of two somatic mutated genes per sample (range 0-13) was found. The most commonly mutated genes per sample were: KRAS (35%), NRAS (25%), FAM46C (15%), DIS3 (13%), BRAF (13%), ATM (11%), and TP53 (11%). Comparing NGS with FISH, we found a perfect concordance for the identification of t(4;14), t(6;14), t(14;16), and t(14;20); a 100% specificity and 97% sensitivity for t(11;14); and sensitivity >90% and an average specificity of 85% for trisomies of chromosomes 3, 7, 9, 11, and 15. The NGS approach was also able to detect gain(1q) and mono(13) with a sensitivity and specificity >90%, and del(17p) with a 60% sensitivity and 100% specificity. The reduced sensitivity for del(17p) detection was mostly due to focal deletions and low tumor purity. Because the TP53 gene is included in the custom capture panel, we increased the sensitivity by determining six separate regions of coverage using on-target probes, which enabled the detection of focal del(17p), increasing the sensitivity to 78%. The NGS approach also allowed the characterization of 16 (6%) samples with unavailable FISH. Of the 27 samples with del(17p) detected by FISH, NGS was able to identify TP53 mutations (i.e., biallelic inactivation) in 14 samples (52%). Due to the existence of multiple and promiscuous chromosomal partners involved in IgH translocation, the identification of the chromosomal partners is limited to the clinical FISH probe sets; in fact, 19 study samples presented abnormal IgH without a known partner by FISH. Our NGS assay was able to identify these partners, confirming MYC-IGH translocations in seven (37%) of these cases. Additionally, we detected MYC translocations with immunoglobulin light chains: 14 (5%) cases with IGK, and eight (3%) with IGL. Although IgL translocations, particularly IgL-MYC translocations, have been recently associated to poor prognosis, they are not routinely evaluated in FISH assays. The risk stratification and clinical utility of the NGS assay was demonstrated by the shorter overall survival observed in samples classified as high risk (mSMART 3.0; hazard ratio [HR]= 2.81; CI:1.89-4.17; p<0.0001), which was also observed with risk assessment by FISH (HR = 2.48; CI:1.72-3.58; p<0.0001). Therefore, in addition to the detection of clinically relevant somatic mutations, the targeted NGS approach showed robust detection of recurrent MM specific IgH translocations and CNV. Furthermore, the detection of relevant abnormalities not routinely assessed in most clinical labs, such as TP53 bi-allelic inactivation, IgL and MYC rearrangements, supports the use of this small, targeted panel as a clinically relevant, single molecular assay, for the comprehensive profiling of MM.

Gene Expression Analysis Reveals Downregulation of Complement Receptor 1 in Cold Agglutinin Disease

Introduction Cold Agglutinin Disease (CAD) is a rare B-cell lymphoproliferative disorder of the bone marrow, manifested by autoimmune hemolytic anemia caused by binding of monoclonal IgM autoantibodies to I antigen. We have described the molecular background of CAD in the context of extensive clinical data, and found frequent somatic mutations in KMT2D and CARD11 genes and showed that CARD11 and CXCR4 gene mutations correlate with hemoglobin level. Moreover, we also have discovered trisomy 3 and 12 or 18 in CAD. Gene expression data has not been reported in CAD and here we present novel RNA-seq data. Differential expression analysis revealed that the complement receptor 1 (CR1) gene was one of the genes most significantly down-regulated in CAD. Flow cytometry analysis further confirmed reduced CR1 protein expression on clonal CAD B cells. CR1 is an important negative regulator of B cell differentiation towards immunoglobulin-producing plasma cells and may thus contribute to CAD pathogenesis. Materials for RNA sequencing We analyzed samples from 12 CAD patients collected for clinical trial (NTC02689986) and 4 healthy controls (HC). Clonal B cells from CAD patients and IGM+ memory B cells from HC, were procured using fluorescence-activated cell sorting of blood or bone marrow cells. IGM+ memory B-cells were used as controls in view of their shared CD19+/CD27+/IGM+ immunophenotype with clonal B cells in CAD. Flow cytometry validation of CR1 expression CR1 protein expression was assessed by flow cytometry of a blood or bone marrow samples obtained from 15 CAD patients and blood samples from 5 HC. For each sample, CR1 expression was compared between IGM+ memory B-cells expressing immunoglobulin kappa light chain (IGK+) and IGM+ memory B-cells expressing immunoglobulin lambda light chain (IGL+), respectively. For CAD patients, the former had previously been demonstrated to be clonal cells, the latter functioned as non-clonal control cells. ResultsGene expression by RNA-seq RNA-seq data analysis and filtering resulted in a selection of 93 the most differentially expressed genes. The top 20 of the most differentially expressed genes from that list are: SLC4A1, SPTA1, YBX3, TESC, HBD, AHSP, TRAF1, HBA2, RHAG, CR1, CA1, SPTB, IL10, UBASH3B, ALAS2, HBA1, CRYM, RGCC, KANK2, IGHV4-34. These genes are upregulated at least 8 times in clonal CAD B cells compared to HC, except for CR1 which is downregulated ~11 times. The p-value, and p-value adjusted for multiple testing, are below 3.0-12 and 2.7-9, respectively, indicating statistically significant differences. Results are illustrated in Figure 1. Validation of CR1 level by flow cytometry In CAD patients, CR1 expression in IGK+ B cells was significantly lower than in IGL+ B cells (Figure 2) as indicated by a median fluorescence intensity that was 10.8x lower in IGK+ than in IGL+ B-cells. HC samples showed similar levels of CR1 expression in IGK+ and IGL+ B cells, respectively. Samples from CAD patients usually showed a bimodal CR1 expression in IGK+ B cells, but not in IGL+ B cells. HC samples did not reveal heterogeneity of CR1 expression in IGM+ memory B cell populations. Discussion CR1 is the receptor for C3b and C4b complement peptides (Klickstein et al. 1988), and its main function is to inhibit the complement cascade (Fearon 1979) and regulate proliferation of B cells (Khera et al. 2009). CR1 blocks proliferation induced by B-cell receptor activation, inhibits the differentiation of B cells to plasmablasts, and the production of immunoglobulins (Kremlitzka et al. 2012). CR1 ligation decreases phosphorylation of key molecules of the BCR-induced signaling cascade (Kremlitzka et al. 2016). Reduced expression of CR1 on B cells has previously been observed in several autoimmune conditions (Prokopec et al. 2010; Kremlitzka et al. 2016). In conclusion, we demonstrated that CR1 expression is highly downregulated in clonal CAD B cells, both at the mRNA and the protein level. This may be an important finding in the context of CAD, because complement controls B and T cell responses, and regulates the adaptive immunity (Killick et al. 2018). Therefore, downregulation of CR1 in CAD might be responsible for increased proliferation, survival of autoreactive B cells and IgM autoantibody secretion. The demonstration of reduced CR1 expression by flow cytometry by IGK compared to IGL IGM+ memory B cells might also be exploited as a diagnostic test. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Diagnostic Accuracy of Vitreous Cytology in Patients with Vitreoretinal Lymphoma.

(1) Background: To determine the diagnostic value of vitreous cytology in patients with vitreoretinal lymphoma (VRL) and evaluate its diagnostic accuracy relative to that of other diagnostic tests. (2) Methods: In total, 38 eyes from 38 patients with VRL who underwent diagnostic vitrectomy and were followed up for at least 6 months were analyzed. The clinical manifestations and VRL diagnostic rates for all diagnostic tests were determined. (3) Results: The presence of vitreous cells/opacity was the most common ophthalmic finding (97.4%), followed by sub-retinal pigment epithelial infiltration (65.8%) and retinal hemorrhage (21.1%). The VRL diagnostic rates were 89.3% for interleukin (IL)-10 levels &gt; 50 pg/mL; 82.1% for IL-10/IL-6 ratios &gt; 1; 60.0% and 63.3% for immunoglobulin heavy chain and kappa light chain clonality assays, respectively; and 44.4% for vitreous cytology. The VRL diagnostic rate for vitreous cytology was significantly lower in the steroid pretreatment group than in the non-steroid pretreatment group (p = 0.007). (4) Conclusions: The VRL detection rate for vitreous cytology was lower than that for the other tests, especially in patients who received steroid pretreatment. These findings suggest that even if vitreous cytology findings are negative, other tests and characteristic fundus findings should be evaluated to confirm VRL diagnosis.

Role of immunoglobulin heavy and light chain gene rearrangement analysis in differentiating between benign and malignant bone marrow B-cell lymphoid aggregates

Lymphoid aggregates are found in a minority of bone marrow biopsy and aspirate specimens, and when present, the distinction between benign and malignant aggregates can represent a diagnostic challenge. Morphologic and immunophenotypic evaluation of the aggregates can aid in that distinction but in a few cases, detection of immunoglobulin heavy chain (IGH) and kappa light chain (IGK) gene rearrangements may be needed to rule in or out a malignant disease process. We studied the role of testing for IGH/IGK rearrangements in the distinction between benign and malignant B cell-predominant lymphoid aggregates. Only a few studies have addressed this issue and most lacked an adequate number of cases for establishing statistical significance. Our study retrospectively evaluated 120 bone marrow aspirate and biopsy specimens, 79 cases originally diagnosed with benign lymphoid aggregates [4,5], and 41 demonstrating a B-cell lymphoma with malignant aggregates. Immunohistochemical stains were performed on all cases in our study and flow cytometry results were available in the vast majority of cases (98%). All patients included in our study but 9 had at least 2 years of clinical follow-up information. Of the malignant lymphoma cases, IGH/IGK rearrangements were demonstrated by polymerase chain reactionin 60% of the cases. Moreover, clonal rearrangements were identified in 15% of the cases with benign aggregates. After at least 2 years of follow-up, only one case with a positive clonality study occurring in the setting of morphologically benign-appearing bone marrow lymphoid aggregates experienced a relapse of non-Hodgkin lymphoma. Molecular analysis of the IGH and IGK genes may have utility in confirming the presence of malignancy in bone marrow aspirates and biopsy specimens. False-negative results, however, are possible due to testing limitations and sampling issues. Moreover, patients with conditions such as autoimmune disorders or infectious diseases are shown to also develop clonal B-cell lymphoid aggregates. As a result, we recommend a thorough morphological examination, informative immunohistochemical staining, accurate flow cytometric analysis, and current IGH/IGK rearrangement testing when evaluating bone marrow specimens containing B cell-predominant lymphoid aggregates, with the knowledge that molecular clonality results should be carefully interpreted in the context of morphological and immunophenotypic findings to prevent misdiagnosis.

Diving in the big blue: Atypical Mott cells in rapidly fatal primary amyloidosis.

A 75-year-old man with no previous medical history presented at the emergency unit with asthenia, anorexia, weight loss, and progressively increasing dyspnea revealing acute heart failure (N-terminal pro b-type natriuretic peptide:16 667 ng/L; troponemia: 47 ng/L). Echocardiography showed a granular appearance of the myocardium and interventricular septal hypertrophy, suggestive of amyloidosis. Chemistry disclosed high levels of immunoglobulin kappa light chains at 1240 mg/L (lambda light chains 14 mg/L; κ/λ ratio 86) and a detectable but unquantifiable IgA κ peak. Myocardial biopsies pathology showed an endocardium thickened by fibrosis. Within the myocardium, the presence of interstitial fibrillary deposits stained by Congo red and birefringent in polarized light confirmed the diagnosis of cardiac amyloid light-chain (AL) amyloidosis. The search for other organ damage disclosed vegetative involvement with orthostatic hypotension, icteric cholestasis (bilirubin 51.8 mg/L), undocumented digestive involvement (diarrhea with evocative endoscopic lesions), and glomerulonephritis (urinary proteinuria 4 g/24 h with 3 g/24 h albumin and 1 g/24 h immunoglobulin light chains). A bone marrow aspirate displayed 17% of plasma cells. Interestingly, a particular pattern of huge atypical Mott cells was identified (Figure 1A,B). The latter had so many cytoplasmic inclusions that the cytoplasm, and the nucleus could not be discriminated. These atypical cells are however plasma cells with an endoplasmic reticulum loaded with immunoglobulins and should be integrated as such in cell counts. Classical plasma cells and typical Mott cells with a few Russel bodies were also present (Figure 1C) indicating a continuum in the shape of the diseased plasma cells. Treatment with cyclophosphamide–bortezomib–dexamethasone-daratumumab was initiated in emergency. Unfortunately, the patient died 3 days later, of multiple organ failure. No additional septic or toxic causes could be identified. Mott cells are activated plasma cells containing intracytoplasmic inclusions with a “bunch of grapes” appearance. These inclusions, called Russell bodies, are dilated cisternae of the endoplasmic reticulum containing condensed immunoglobulins. They were identified in 1901 by the surgeon F.W Mott in the brains of monkeys with trypanosomiasis. Mott cells have been described in various pathological conditions: reactive plasmacytosis, monoclonal gammopathies, high grade B-cell lymphomas, Wiskott-Aldrich syndrome, and von Recklinghausen neurofibromatosis. AL amyloidosis is caused by clonal expansion of plasma cells producing misfolded immunoglobulin light chains responsible for the deposition of insoluble fibrillar proteins in many organs. Those most frequently affected are the kidneys, heart, liver, and peripheral nervous system. AL amyloidosis is a rare disease, and its diagnosis is often delayed. Although associated with a poor prognosis, AL amyloidosis presents a heterogeneous clinical expression and evolution. To our knowledge, the presence of Mott cells has rarely been described in this pathology. KR, ME, and BLC wrote the manuscript. KR, ME, and BLC performed biological analyses. CS treated the patient. All authors critically reviewed the manuscript. The authors have no conflict of interest to disclose. The authors received no specific funding for this work. The research was conducted in accordance with the principles embodied in the Declaration of Helsinki and in accordance with local statutory requirements.

S2691 A Rare Presentation of Gastric Plastocytomas in a Patient With Multiple Myeloma

Introduction: Multiple myeloma is an uncommon hematological neoplasm. This malignancy involves unchecked plasma cell growth in the bone marrow, but in a subset of often more advanced cases, these cells may migrate to soft tissues causing extramedullary plasmacytoma. This is unusual, and, in the literature, few cases of extramedullary plasmacytomas involve the gastrointestinal tract. Within this system, the more common locations are the stomach, the liver, the large bowel, and more rarely, the small bowel. Case Description/Methods: A 59-year-old male with a medical history of chronic hepatitis C, and Stage III multiple myeloma status post four cycles of chemotherapy presented with altered mental status. Upon evaluation, he had significant electrolyte abnormalities, remarkable anemia, severe renal and liver dysfunction, and a positive hemoccult test. Our patient received intravenous fluid, and multiple blood products. He was then admitted to the intensive care unit for a high level of care. On the second day of admission, he developed new onset hematemesis, and the gastroenterology team was consulted. The patient was started on a pantoprazole drip. Urgent esophagogastroduodenoscopy showed several polypoid appearing masses with overlying ulceration in the gastric fundus (Figure a &b), and tissue biopsies were obtained. Biopsy result of the polypoid fundal mass showed a large cell neoplasm with plasmocytic differentiation. Gastric tissue sections showed an ulcerated lesion with submucosal, tightly packed atypical large cells with multinucleation, prominent nucleoli and relatively identifiable mitoses (Figurec). A panel of immunohistochemical stains with plasma cell markers were positive for kappa, Multiple Myloma-1 nuclear protein, and CD138 (Figure d-f). The overall findings were compatible with gastric involvement by a plasma cell neoplasm. Discussion: This case highlights a complicated, yet a uniquely rare presentation of extramedullary plasmacytoma in a patient diagnosed with multiple myeloma. Although the gastrointestinal tract is rarely involved in patients with multiple myeloma, it should be considered as the differential diagnosis in patients with gastrointestinal bleeding especially with a known concurrent systemic hematological malignancy. The presence of gastrointestinal involvement and the malignant phase of multiple myeloma are associated with a poor prognosis despite aggressive therapy, and it is therefore paramount to establish an accurate diagnosis early to avoid any delays in treatment.Figure 1.: (a,b) EGD shows polypoid EMP in the gastric fundus. (c) Infiltration of plasmablasts with highly atypical nuclei and prominent nucleoli (hematoxylin-eosin 20x). (d) Granular positivity for kappa immunoglobulin light chain. (e) Positive immunostaining for MUM-1. (f) Positive CD138 immunostaining highlights the plasma cells in the lamina propria of the gastric fundus EMP.

A clonality assay in canine B cell tumors targeting the immunoglobulin light chain lambda locus

Clonality assays for antigen receptor rearrangement have been used as adjunct examinations of lymphoproliferative diseases. These assays have been useful for differentiation between inflammation and clonal expansion of lymphocytes. Whereas the immunoglobulin heavy chain (IGH) and immunoglobulin light chain kappa (IGK) loci have been targeted in canine clonality assays previously, the immunoglobulin light chain lambda gene (IGL) locus has not yet been investigated. This study aimed to evaluate the usefulness of clonality assays in dogs using IGL. Canine diffuse large B cell lymphomas (DLBCL), cutaneous plasmacytomas, and pathologically diagnosed lymph nodes without lymphoma, were used in this study. Genomic DNA was extracted from formalin-fixed paraffin embedded sections. Sequences of IGLV and IGLJ were obtained from the ImMunoGeneTics database. Several primers against IGLVs and IGLJs were designed in the regions showing homology, by alignment of the gene segments. Products of polymerase chain reaction were analyzed on a capillary electrophoresis. In total, 20 of 23 cases of DLBCL showed clonality (87.0 %), whereas 8 of 30 cutaneous plasmacytomas were clonal (26.7 %). One of 23 lymph nodes without lymphoma showed clonality, thus the specificity was 95.7 %. These data indicate that the IGL locus could be a target for canine clonality assays and that the sensitivity of IGL-based clonality assays in cutaneous plasmacytomas was lower than that in DLBCL.

Cutaneous Ossifying Amyloidoma

Amyloidoma, otherwise known as tumoral amyloidosis, is a localized deposition of amyloid (AL-type or AA type) without systemic amyloidosis. It is the rarest form of tissue amyloid deposition, and up to 7% of amyloidomas develop systemic amyloidosis.Cutaneous AL-type amyloidoma is considered by many authors as an unusual variant of primary cutaneous marginal zone lymphoma. Although cutaneous amyloidoma can form calcifications, ossification is extremely unusual, with only 1 case previously published to date.We report the case of a 75-year-old woman with voluminous and strikingly ossifying AL-type amyloidoma in the left pretibial skin. Her medical history included excision of hepatic hydatidic cysts 25 years prior and diffuse large B-cell lymphoma of the left parotid gland 8 years prior treated with chemotherapy and radiotherapy with complete response. After the diagnosis of amyloidoma, an extension study with cervical, chest, abdominal, and pelvic TC was performed, with no additional lesions found. Serum and protein electrophoresis revealed elevations in kappa light chain and IgA immunoglobulin levels but did not reveal monoclonal bands. In situ hybridization for immunoglobulin light chains showed monotypic kappa expression in plasma cells infiltrating the amyloidoma.Extensive ossification in amyloidomas can make diagnosis difficult; therefore, we describe an interesting case of this histopathologically peculiar amyloidoma.

Clinical and pathologic features of primary membranous nephropathy in Turkey: a multicenter study by the Turkish Society of Nephrology Glomerular Diseases Working Group

Background We aimed to evaluate the features of primary membranous nephropathy (MNP) in Turkish people. Methods This is a retrospective analysis of patients with biopsy-proven primary MNP. We obtained the data collected between 2009 and 2019 in the primary glomerulonephritis registry of the Turkish Society of Nephrology Glomerular Diseases Study Group (TSN-GOLD). Patients with a secondary cause for MNP were excluded. Clinical, demographic, laboratory, and histopathological findings were analyzed. Results A total of 995 patients with primary MNP were included in the analyses. Males constituted the majority (58.8%). The mean age was 48.4 ± 13.9 years. The most common presentation was the presence of nephrotic syndrome (81.7%) and sub nephrotic proteinuria (10.3%). Microscopic hematuria was detected in one-third of patients. The median estimated glomerular filtration rate (eGFR) was 100.6 mL/min/1.73 m2 (IQR, 75.4–116.3), and median proteinuria was 6000 mg/d (IQR, 3656–9457). Serum C3 and C4 complement levels were decreased in 3.7 and 1.7% of patients, respectively. Twenty-four (2.4%) patients had glomerular crescents in their kidney biopsy samples. Basal membrane thickening was detected in 93.8% of cases under light microscopy. Mesangial proliferation and interstitial inflammation were evident in 32.8 and 55.9% of the patients, respectively. The most commonly detected depositions were IgG (93%), C3 complement (68.8%), and kappa and lambda immunoglobulin light chains (70%). Although renal functions were normal at presentation, vascular, interstitial, and glomerular findings were more prominent on biopsy in hypertensive patients. No significant effect of BMI on biopsy findings was observed. Conclusions Despite some atypical findings, the main features of primary MNP in Turkey were similar to the published literature. This is the largest MNP study to date conducted in Turkish people.

Multiple Myeloma Presenting with Multiple Thoracic Manifestations

Multiple myeloma is a malignant proliferation of plasma cells that affects mainly bone marrow but may also involve other organs as well. We report thoracic involvement in the form of left-sided pleural effusion, osseous lesions, bronchial infiltration, and mediastinal lymphadenopathy in a 61-year-old woman, non-smoker presented with chest pain, dyspnoea, cough and deterioration in general health over the preceding seven months. Immunoelectrophoresis and immunofixation showed raised kappa-light chain immunoglobulin G (IgG) in serum and pleural fluid. Bronchial and pleural biopsies documented myelomatous infiltration and bone marrow aspirate revealed extensive plasma cell infiltration. At eight months, following the fourth cycle of melphalan, endoxan and prednisone based chemotherapy, the patient died.