Surface Immunoglobulin Expression Research Articles

Delineating immune variation between adult and children COVID-19 cases and associations with disease severity

The SARS-CoV-2 pandemic has emphasized the need to explore how variations in the immune system relate to the severity of the disease. This study aimed to explore inter-individual variation in response to SARS-CoV-2 infection by comparing T cell, B cell, and innate cell immune subsets among primary infected children and adults (i.e., those who had never experienced SARS-CoV-2 infection nor received vaccination previously), with varying disease severity after infection. We also examined immune subset kinetics in convalescent individuals compared to those with persistent infection to identify possible markers of immune dysfunction. Distinct immune subset differences were observed between infected adults and children, as well as among adult cases with mild, moderate, and severe disease. IgM memory B cells were absent in moderate and severe cases whereas frequencies of B cells with a lack of surface immunoglobulin expression were significantly higher in severe cases. Interestingly, these immune subsets remained stable during recovery implying that these subsets could be associated with underlying baseline immune variation. Our results offer insights into the potential immune markers associated with severe COVID-19 and provide a foundation for future research in this area.

What Does Atypical Chronic Lymphocytic Leukemia Really Mean? A Retrospective Morphological and Immunophenotypic Study.

Atypical chronic lymphocytic leukemia (CLL) is still defined according to morphological criteria. However, deviance from the typical surface immunological profile suggests an atypical immunological-based CLL. A large cohort of patients with CLL was retrospectively evaluated aiming at assessing morphological (FAB criteria), immunophenotypical (two or more discordances from the typical profile), and clinical-biological features of atypical CLL. Compared to typical cases, morphologically atypical CLL showed a greater percentage of unmutated IgVH and CD38 positivity, and a higher expression of CD20. Immunophenotypically atypical CLL was characterized by more advanced clinical stages, higher expression of CD20, higher rate of FMC7, CD79b and CD49d positivity, and by an intermediate-high expression of membrane surface immunoglobulin, compared to typical cases. When patients were categorized based on immunophenotypic and morphologic concordance or discordance, no difference emerged. Finally, morphological features better discriminated patients' prognosis in terms of time-to-first treatment, while concordant atypical cases showed overall a worse prognosis. Discordant cases by immunophenotype and/or morphology did not identify specific prognostic groups. Whether-in the era of molecular markers used as prognostic indicators-it does make sense to focus on morphology and immunophenotype features in CLL is still matter of debate needing further research.

Localization of DC-SIGN Expressing Cells in Follicular Lymphoma

The high incidence of unusual mannose-rich glycans on the expressed surface immunoglobulins of follicular lymphoma (FL) suggests a pathogenic role for this modification. While lectin-mediated signaling has been proposed to provide trophic support to FL cells, the localization of the lectins has not been extensively characterized. Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN) is a mannose-selective lectin named for its expression on classic marrow-derived dendritic cells. In vitro, recombinant DC-SIGN induces signaling in primary FL cells. The nodules of Follicular lymphoma (nFL) are the neoplastic counterparts of normal germinal centers (GC). Both GC and nFL contain variable numbers of centrocytic and/or centroblastic B cells, T-cells, and follicular dendritic cells (FDCs), but macrophages are typically found only in GC. Although termed “follicular dendritic cells”, FDCs are thought to be derived from non-blood stem-cells, potentially pericytes. Therefore, nFL lack classic dendritic cells and macrophages so it is unclear what cells in FL could express DC-SIGN. Therefore, we systematically characterized FDC and DC- SIGN localization in sixteen FL and nine Follicular hyperplasia (FH) lymph nodes. Using flow cytometry of disaggregated FL nodes, we were not able to identify a specific cell population expressing DC-SIGN. However, using immunohistochemistry and Immunofluorescence methods, we identified nine cases of FH and five cases of FL (n=5/16) with DC-SIGN and FDC co-localization. Immunohistochemical stains of the FDCs using CD23 and CD21 showed that the follicular dendritic structures were disrupted in almost all the FL cases assessed showing either a peripheral crescentic staining or broken FDC meshwork within the follicle. We identified five cases of FL with an average of more than four well visualized and complete FDC structures staining per low power field (4X) while the remaining cases had an average of less than two complete FDC network staining. There was near complete absence of CD21 positive FDCs meshwork identified in 50% of the FL cases compared to only one case lacking CD23 positive FDCs structure staining. In contrast, FH cases (n=9) showed complete staining of the FDC meshwork within the follicles with both CD21/CD23. For FH, the average number of complete FDC staining with CD23 was 18 while an average of 10 complete FDC meshworks stained with CD21. Furthermore, immunofluorescence imaging analysis showed different pattern of localization of CD23 positive FDC with DC-SIGN with between the two conditions. In FH, DC-SIGN and CD23 colocalization was identified mainly in the light zone. For FL, we observed that DC-SIGN overlap with CD23 localized in the intra-follicular areas and co-localized with CD163 (M2 macrophages) in the inter-follicular zones. Confocal imaging confirmed colocalization of CD23 and DC-SIGN on FDCs with close association to MUM1-positive B cells in both GC and nFL. Additional analysis also showed that 80% (n=5) of the cases which had more than four complete FDC structures staining with CD23 co-localized with DC-SIGN in the intra-follicular areas and the cases with complete FDC network disruption did not. There was no significant relationship identified between the extent of FDC networks and the fraction of non-neoplastic B cells in the FL cases. In summary, our data show that DC-SIGN is expressed on FDCs in FL and FH even though FDCs are not the marrow-derived DCs for which DC-SIGN is named. Furthermore, localization of DC-SIGN to the follicle suggests a potential role for the microenvironment in the pathogenesis of FL and possible therapeutic target.

Detection of 13q deletion in patients with chronic lymphocytic leukemia and its correlation to hematopathological parameters

Background: Chronic lymphocytic leukemia(CLL) is a monoclonal malignancy characterized by an accumulation of small and mature looking B lymphocytes in the blood, bone marrow and other tissues, and is typically characterized by expression CD5+, CD23+, CD22 -, CD79b-, with weak expression of surface immunoglobulin (sIg). Two major clinical staging systems (Rai and Binet staging), that developed to estimate prognosis in CLL. However both these systems are unable to prospectively distinguish the rapidly developing patients from those appreciated to remain with a stable disease for decades. Several publications reported the prognostic important of 13q deletion in patients with chronic lymphocytic leukemia. 13q deletion is the most important cytogenetic abnormalities detected by Fluorescence In situ Hybridization in CLL patients and represent a good prognostic marker with 60% of patients alive after 5 years as compared with 27% for patients with a normal FISH analysis. Aims of the study: To investigate 13q deletion in patients with chronic lymphocytic leukemia by using FISH technique. To correlate the presence of 13q deletion with hematological and clinical prognostic markers including complete blood picture, absolute lymphocyte count, and modified Rai staging.

Burkitt leukaemia with B-cell precursor immunophenotype.

A 64-year-old male presented with malaise and B symptoms. An automated full blood count showed haemoglobin 124 g/L, white blood cells 40 × 109/L, platelet 51 × 109/L. Positron emission tomography/computed tomography indicated widespread activity above and below the diaphragm with bone marrow, peritoneal, pleural, hepatic, renal, small bowel and possible adrenal involvement. The blood film (Figure 1A, May-Grünwald-Giemsa stain ×100 objective) showed numerous large cells with basophilic cytoplasm and cytoplasmic vacuolation. By flow cytometry (Beckman Coulter Duraclone) (Figure 1E–H) these cells (red colour population) were CD45 low (Figure 1E). They were positive for surface CD19, CD10, HLADR, CD38 and cytoplasmic CD79a, terminal deoxynucleotidyl transferase (TdT) (Figure 1H); negative for surface CD34, CD20 (panel F), CD22, light chains (Figure 1G) and cytoplasmic CD3, myeloperoxidase (MPO). Bone marrow aspirate (Figure 1B, May-Grünwald-Giemsa stain x100 objective) was packed with medium to large-sized cells with basophilic cytoplasm, and frequent cytoplasmic vacuolation. No abnormalities were detected by reverse transcription-polymerase chain reaction screening for common leukaemic fusion genes using the Q30 leukaemia assay (QuanDX). Fluorescence in situ hybridization (FISH) analysis of the liquid sample detected immunoglobulin heavy chain/MYC translocation and gain of 1q. Next-generation sequencing (Archer VariantPlex) identified NRAS p.Gln61Arg variant (VAF 36%). Bone marrow histology (Figure 1C, haematoxylin and eosin stain x40 objective) revealed effacement by large cells with finely dispersed chromatin, with frequent mitoses and some with multiple small nucleoli. A classical ‘starry sky’ pattern was not seen. Immunohistochemical staining was strongly positive for CD45, CD20 (most positive) and TdT (Figure 1D, x40 objective); positive for paired box 5 (PAX5), CD19, CD79a, CD10, multiple myeloma 1; and negative for B-cell lymphoma 2 (BCL2), BCL6, CD5, cyclin D1, CD1a, CD99, CD117, CD34, MPO and light chains. Staining for Epstein-Barr virus ribonucleic acid was negative. The MIB1 proliferation factor was near 100%. FISH confirmed the MYC translocation but neither BCL6 nor BCL2 translocations were detected. A diagnosis of “Burkitt leukaemia (BL) with a B-cell precursor immunophenotyped”. The patient's medical comorbidities made him unfit for chemotherapy and he received palliative corticosteroid treatment. BL with precursor B cell immunophenotype is rare in adults. The WHO Classification of Tumours of Haematopoietic and Lymphoid Tissue (revised 4th edition) notes approximately 2% of paediatric cases of BL that “have a phenotype of precursor” B-cells, with an expression of TdT, and sometimes CD34, and absence of CD20 and surface immunoglobulin expression. The reason for this aberrant phenotype remains unknown [1]. A small study reported IG-MYC+ neoplasms with precursor B cell immunophenotype to resemble precursor B-cell acute lymphoblastic leukaemia/ lymphoblastic lymphoma rather than BL in genomic, epigenomic profiling, the mutational landscape, and the DNA methylation pattern, with frequent activation of the RAS pathway [2]. Further studies are needed for a better understanding of this rare subgroup of disease (see Figure 1). Ke Xu wrote the manuscript. Ke Xu, Anna Childerhouse and Rajeev Gupta critically revised the final version of the manuscript.

Atypical immunophenotype of chronic lymphocytic leukemia

Assessment of the immunophenotype plays a crucial role in the diagnostic process of chronic lymphocytic leukemia (CLL). The expression of CD5, CD19 and CD23 antigens with a concomitant reduction or lack of surface immunoglobulin expression as well as CD22 and CD79b antigens is the basic part of CLL diagnosis. A significant diagnostic challenge is atypical CLL with cells devoid of CD5 or CD23 antigens. The assessment of additional antigens in flow cytometry, especially the CD200 glycoprotein, may facilitate the process of differential diagnosis of atypical CLL from other B-cell lymphoproliferative neoplasms. The results of current studies analyzing the influence of atypical CLL on prognosis are inconclusive. The analysis of a large group of patients with atypical CLL is difficult because of the rare occurrence of CD5(–) or CD23(–) CLL and the misdiagnosis of this disease as other B-cell lymphoproliferative neoplasms. The following paper aims to show how important it is to include atypical CLL in the diagnostic process of this disease and to re-standardize the commonly used immunophenotypic scales for its diagnosis.

Novel Prognostic Markers in Previously Treated Chronic Lymphocytic Leukemia

Background: We need new biomarkers that manage CLL patients receiving novel chemotherapeutic agent treatments. In our earlier pilot study, we reported an association of inferior outcomes in those with higher IgM density, or those with IgG isotype expression, not explained by traditional markers such as unmutated IGHV or TP53 aberrations (Brander, et al., ASH abstract 118, 2011). The International Prognostic Index for CLL (CLL-IPI) was developed to provide CLL risk stratification, but this tool was largely studied in those treated conventionally (Lancet Oncol 17:779-790, 2016). Here we report studies of CLL cell IgM expression and outcomes in CLL patients treated with novel agents. We hypothesized that high IgM expression is associated with poor progression free survival (PFS) in patients treated with novel agents.Methods: We studied samples from 906 Duke University CLL patients. Fifty seven of 906 patients had received ibrutinib, acalabrutinib, and/or venetoclax. We classified CLL cell IgM expression surface isotype as low (<30%), moderate (31-60%), or high (>60%), and noted presence of class switching to IgG. We collected the following: social and geographical demographics, IGHV mutation status, CLL-IPI score, Rai staging, beta-2-microglobulin, FISH, cytogenetics, and treatment response per iwCLL definitions. PFS was defined as time from initiation of treatment with novel agents to progression of disease or death.Results: Of 57 CLL patients, 42 were alive at the time of followup. Median time for followup was 62 months for patients on BTK-inhibitors and 32 months for those on the Bcl-inhibitor venetoclax. Social demographics were similar across all treatment groups. Deaths were noted in each group (2/7 acalabrutinib, 13/45 ibrutinib, 6/13 venetoclax), with progression of disease as the leading cause. Many patients were not treatment naïve before starting novel agents. Specifically, 14/19 patients most recently on venetoclax had been exposed to a novel inhibitor. PFS did not correlate with surface IgM expression and outcomes for those treated with BTK inhibitors or venetoclax. Previously, approximately 25% of CLL patients with TP53 mut/del17p relapsed within 24 months of initiating traditional chemoimmunotherapeutics. Our TP53 mut/del17p group treated with BTK inhibitors had improved PFS (p=0.005) (Figure 1). Similarly, by univariate cox regression, we found statistically significant PFS in patients with high risk mutations treated with BTK inhibitors (p=0.042). There was no statistically significant correlation between CLL-IPI score and PFS, nor high IgM expression density and PFS for those treated with BTK inhibitors.Conclusions: We hypothesized that higher IgM expression density is associated with reduced PFS in patients treated with the novel agents. This was not identified in our study of 57 patients who had been treated with ibrutinib, acalabrutinib, or venetoclax. There was significantly shorter PFS for patients treated with BTK-inhibitors and TP53 mutand/or del17p compared to those without these genomic changes (p=0.005). This is consistent with prior studies. Historically, approximately 25% of CLL patients with TP53 mut/del17p relapsed within 24 months of initiating traditional chemoimmunotherapeutic agents. Our TP53 mut/del17p group treated with BTK inhibitors had improved PFS compared to the historical outcomes with a PFS of 66.5 months despite being a relapsed/refractory group. Our data supports prior studies identifying this as a high-risk subgroup of patients in need of investigational agent treatments (Catherwood MA, et al, J Clin Pathol, 72:343-346, 2019; and Gordon MJ, et al. Clin Cancer Res, In press, 2021). While the CLL-IPI score generally helps with prognostication of CLL patients, it was not significantly superior in our analyses in these patients. Other retrospective studies have also identified the limitations of the CLL-IPI score in the era of novel agents. Our work is limited by a small sample size of patients treated with the novel agents and by the retrospective nature of the study. With larger cohorts and longer followup, our future prospective studies will help clarify the role of surface immunoglobulin expression relative to CLL biology, and patient outcomes while receiving novel treatments. [Display omitted] DisclosuresBrander: BeiGene: Research Funding; Juno Therapeutics/Celgene/Bristol Myers Squibb: Research Funding; AstraZeneca: Research Funding; Verastem: Consultancy; Genentech: Consultancy, Research Funding; ArQule: Research Funding; Ascentage: Research Funding; DTRM: Research Funding; ArQule/Merck: Consultancy; LOXO: Research Funding; MEI Pharma: Research Funding; Novartis: Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; Pfizer: Consultancy, Other: Biosimilars outcomes research panel; TG Therapeutics: Consultancy, Research Funding; AbbVie: Consultancy, Other: informCLL registry steering committee, Research Funding; NCCN: Other: panel member.

Use of Flow Cytometry to Evaluate the Immunophenotypic Profile of Patients with Chronic Lymphocytic Leukemia from Rio Grande Do Norte State, Brazil

BACKGROUND: Chronic lymphocytic leukemia (B-CLL) is a clonal proliferation of mature B lymphocytes characterized by indolent clinical course. This clonality is characterized biologically by low expression of surface immunoglobulin (sIg) and restriction to a single immunoglobulin light chain associated with high expression of CD5 antigen and positivity to B cell antigen lymphocytes such as CD19, CD20 and CD23 and negativity to FMC7. The immunological profile and morphological analysis of lymphoid cells are the main means for the differential diagnosis of B-CLL from other chronic lymphoproliferative diseases. OBJECTIVE: The aim of this study was to evaluate the expression pattern of a variety of membrane antigens in leukemic cells originating from patients with B-CLL. METHODS: Peripheral blood samples from 80 patients with B-CLL were analyzed by multiparametric flow cytometry and routine hematologic exams, using a panel of monoclonal antibodies (MoAb): CD45/CD14, CD3/CD19/CD45, CD4/CD8/CD3, CD20/CD5/CD3, CD3/CD16-56/CD45, CD2/CD7, FMC7/CD23, CD103/CD22/CD20, HLADR/CD38, CD10/CD19, CD1a, CD11b in addition to IgM/gD, kappa and lambda immunoglobulin light chains to detect surface immunoglobulin and clonal restriction for immunoglobulin light chains. The hematological data were obtained using a hematological analyzer and cytomorphological analysis in blood film stained with Leishman's stain.RESULTS: The study sample consisted of 45 men and 35 women, ages ranging from 55 to 84 years (mean 65 years). Complete white blood cell count ranged from 10.0 to 42.0 x 109/L. (mean 50.0 x 109/L) and lymphocyte count was greater than 5.0 x 109/l in all cases. The neoplastic cells displayed B-CLL phenotype (CD5+/CD19+/CD20+/HLADR+/CD23+) in the vast majority of the cases, associated with failure to stain for T cell markers (CD1a, CD2, CD4, CD3, CD7, CD8), CD103, CD14 and FMC7. Leukemic cells of most patients also expressed low intensity of IgM and IgD, with clonal restricted kappa light chain in the majority of cases (59.7%). CONCLUSIONS: This investigation highlights the importance of immunophenotyping for correct diagnosis of chronic lymphoproliferative syndromes, and the MoAb panel used was sufficient for diagnostic confirmation of B-CLL. DisclosuresNo relevant conflicts of interest to declare.

A new score including CD43 and CD180: Increased diagnostic value for atypical chronic lymphocytic leukemia.

Moreau score has been used to differentiate chronic lymphocytic leukemia (CLL) from other mature B‐cell neoplasms. However, it showed limitations in Asian patients. Therefore, we conducted a new score system replacing CD5 and CD23 with CD43 and CD180 to evaluate its diagnostic value of CLL. 237 untreated samples diagnosed with mature B‐cell neoplasms were collected and were randomly divided into an exploratory and a validation cohort by a 2:1 ratio. The expression of CD5, CD19, CD20, CD23, CD43, CD79b, CD180, CD200, FMC7, and surface immunoglobulin (SmIg) were analyzed among all the samples. A proposed score was developed based on the logistic regression model. The sensitivity and specificity of the proposed score were calculated by ROC curves. CD43/CD180, CD200, FMC7, and CD79b were included in our new CLL score, which showed a sensitivity of 91.8% and a specificity of 83.1%. These results were confirmed in a validation cohort with a sensitivity of 90.5% (p = 0.808) and a specificity of 79.5% (p = 0.639). In CD5 negative or CD23 negative CLL group, the new CLL score displayed improved sensitivity of 79.4% compared to Moreau score and CLLflow score (41.2% and 47.1%, respectively). In atypical CLL group, the new CLL score showed improved sensitivity of 84.2% compared to Moreau score and CLLflow score (61.4% and 64.9%, respectively). This proposed atypical CLL score helped to offer an accurate differentiation of CLL from non‐CLL together with morphological and molecular methods, particularly in Chinese patients with atypical immunophenotype.

Waldenstrom's macroglobulinemia with peripheral neuropathy and favorable long-term treatment responses

Abstract Rationale: Waldenstrom's Macroglobulinemia (WM), is a rare hematological disorder with an indolent course that affects about 1 in 1,000,000 people over the age of 50. The two cases presented here highlight the favorable responses to targeted therapies and that such responses may show a longer progression-free survival than the standard regimens for WM (Dexamethasone + Rituximab + Cyclophosphamide and Bendamustine + Rituximab). Patient concerns: We report 2 Filipino patients with WM presenting with symptoms of peripheral nerve involvement characterized by numbness, dysesthesia and weakness. Diagnosis: Serum immunoelectrophoresis showed distinct bands in IgM and Kappa light chain channels, indicative of IgM kappa monoclonal gammopathy. Bone marrow aspirate flow cytometry showed a B-lymphoid population, bright expression of Kappa light chain surface immunoglobulin, all consistent with a B-cell neoplasm. Interventions: The first case received Dexamethasone + Rituximab + Cyclophosphamide. The second case was successfully treated with Rituximab + Bendamustine. Outcomes: The first case achieved complete remission for 9 years. However, in the 10th year of follow-up, there was recurrence of peripheral nerve symptomatology, which prompted a repeat course Rituximab monotherapy, with favorable response. The second case was successfully treated with Rituximab + Bendamustine over a 3-year course. Lessons: To the best of our knowledge, WM presenting with peripheral neuropathy with a long progression-free survival as described in these patients have not been reported before. The two cases highlight the potentially favorable responses to targeted therapies. Though the possibility of developing recurrence cannot be discounted, favorable outcomes with the use of the standard chemotherapies could still be expected.

Primary Mediastinal (Thymic) Large B-Cell Lymphoma: Fidelity of Diagnosis Using WHO Criteria

PurposeDiagnosing primary mediastinal (thymic) large B-cell lymphoma (PMBCL) is challenging because it is a clinicopathologic entity that shares characteristics with other lymphomas and lacks pathognomonic features. We sought to investigate the fidelity between a working diagnosis of PMBCL at our institution and the clinicopathologic criteria established within the 2017 World Health Organization (WHO) classification. Patients and MethodsMedical records and archived tissue of patients treated for stage I-II PMBCL from 1998 to 2018 were retrospectively reviewed for clinical and pathologic conformity with current WHO criteria. Disease was characterized as definitely PMBCL if all of the following were present: anterior mediastinal mass with or without lymph node involvement, no extranodal disease, B-cell antigen expression, Epstein-Barr virus negativity, and at least one supportive feature: female gender under age 40, bulky primary tumor, CD30 weakly positive, compartmentalizing alveolar fibrosis, lack of surface immunoglobulin expression, and MUM1 or CD23 positivity. Disease without supportive features or other pathologic findings more suggestive of other entities was characterized as equivocal for PMBCL. Lack of an anterior mediastinal mass, presence of distant lymph node involvement or extranodal disease, lack of B-cell antigen expression, or Epstein-Barr virus positivity were characterized as definitely not PMBCL. Clinical management and outcomes were also assessed. ResultsOf 63 patients treated for presumed stage I-II PMBCL, 58 (92%) met the criteria for PMBCL. The most common reason for a discordant diagnosis was lack of an anterior mediastinal mass (n = 3). Two additional patients were characterized as having disease equivocal for PMBCL. In retrospect, one patient most likely had a mediastinal gray zone lymphoma due to CD15 positivity and another diffuse large B cell, not otherwise specified, at pathologic review. Five-year progression-free and overall survival were 67% (95% confidence interval, 54-77) and 81% (95% confidence interval, 68-89), respectively, for all patients. ConclusionDespite the complexity of the clinicopathologic criteria of PMBCL, most patients (92%) who were treated for stage I-II PMBCL at our institution appear to have been accurately diagnosed.

Ia Antigen Expression by Human Malignant Lymphomas: Correlation With Conventional Lymphoid Markers

The Ia (p23,30) antigens are useful in determining the B- or T-cell origin of normal peripheral blood lymphocytes. However, exceptions to the preferential B-cell expression of Ia antigens exist, i.e., many plasma cells are Ia– and T cells are rarely Ia+. Therefore, we investigated Ia expression (assayed by direct immunofluorescence using heteroantisera) by cells isolated from 33 malignant lymphomas and 7 benign lymph nodes. This was compared with surface immunoglobulin (SIg) and sheep erythrocyte (SRBC) receptor (E rosette) expression. Results with normal lymph nodes were similar to those described for peripheral blood. Most lymph node B cells were Ia+SIg+. Occasional Ia+SIg– B cells and rare Ia+ T cells were present. Ia antigens were expressed in parallel with SIg in 15 of 19 B-cell lymphomas. In 4 B-cell lymphomas, substantial numbers of neoplastic Ia+SIg– cells were also present, suggesting the presence of neoplastic cells at varying stages of differentiation. Heterogeneity was also seen in those lymphomas associated with monoclonal proteins, as small numbers of Ia+ and Ia– plasma cells were present. Seven cases expressed the T-cell (la–SIg–E+) phenotype. The common acute lymphoblastic leukemia phenotype (Ia+SIg–E–) was rarely expressed and may be uncommon among lymphomas. Three lymphomas expressing the Ia+E+ phenotype were shown to be B-cell malignancies whose SIg had SRBC specificity. One Ia 4 T-cell lymphoproliferative disorder was found. Thus, Ia antigens are useful B-cell markers in most instances. Furthermore, their enumeration allows the demonstration of phenotype heterogeneity in B-cell malignancies, analogous to that described for T-cell malignancies.

Primary Mediastinal (Thymic) Large B-cell Lymphoma: The Working Diagnosis vs WHO Classification Criteria

Primary mediastinal (thymic) large B-cell lymphoma (PMBCL) is an uncommon but distinct entity in the WHO classification of hematologic malignancies. Accurately diagnosing PMBCL is challenging as it is a clinicopathological entity. There are no pathognomonic features. Many characteristics overlap with diffuse large B-cell lymphoma NOS, nodular sclerosis Hodgkin lymphoma, and so-called “grey-zone” lymphomas. Thus, it is likely that patients are occasionally misdiagnosed as having PMBCL. Accurate diagnosis is critical as it impacts systemic therapy selection and receipt of radiation therapy (RT). We sought to investigate the fidelity between a working diagnosis of PMBCL at our institution and the clinicopathological criteria established within the 2017 WHO classification. Patients treated for stage I-II PMBCL from 1998-2018 at our institution were retrospectively reviewed for clinical and pathological conformity with WHO criteria in collaboration with a hematopathologist who reviewed archival tissue. Patients were characterized as definitely having PMBCL if they had all of the following: an anterior mediastinal mass +/- regional lymph node involvement, no extranodal disease, B-cell antigen expression, and supportive feature(s)- female gender, young age, bulky primary tumor, CD30 weakly positive, compartmentalizing alveolar fibrosis, lack of surface immunoglobulin expression, and MUM1 or CD23 positivity. Patients without supportive features were characterized as equivocal. PMBCL was excluded if they had any of the following: no anterior mediastinal mass, distant lymph node involvement or extranodal disease, lack of B cell antigen expression, or EBV positivity. 63 patients were evaluated. 71% were female. Median age- 35 years (range, 20-73). Stage 1- 51%; stage II- 49%. Median mediastinal mass size- 10 cm (range, 3-20). 48% of patients received R-CHOP, 32% R-EPOCH, and 21% received other chemotherapy regimens; 79% received rituximab; 83% of patients received 6 chemotherapy cycles. 86% of patients had a post chemotherapy PET/gallium response assessment (78% CR; 22% PR). 64% of patients received RT with PR patients more likely to receive RT (83% vs 62%, p = 0.16). Median follow up was 6 years. Overall, 92% of patients met WHO criteria for PMBCL; 5% (n = 3) did not meet criteria, all due to a lack of an anterior mediastinal mass. 3% were equivocal- grey zone lymphoma (n = 1) and lack of supportive features (n = 1). 5-year PFS and OS was 67% (95% CI 54-77%) and 81% (95% CI 68-89%), respectively. Failure to receive rituximab was the only predictor associated with inferior PFS on univariate analysis, HR 2.79 (1.15, 6.78). Despite the complexity of the clinicopathological criteria of PMBCL, most (92%) patients who were treated for stage I-II PMBCL at our institution appear to have been accurately diagnosed. Treatment regimens varied significantly given changes in patterns of care over the time period. The importance of rituximab was confirmed.

Rapid Isolation of Rare Isotype-Switched Hybridoma Variants: Application to the Generation of IgG2a and IgG2b MAb to CD63, a Late Endosome and Exosome Marker.

CD63, a member of the tetraspanin superfamily, is used as a marker of late endosomes and lysosome-related organelles, as well as a marker of exosomes. Here, we selected rare isotype variants of TS63 by sorting hybridoma cells on the basis of their high expression of surface immunoglobulins of the IgG2a and IgG2b subclass. Pure populations of cells secreting IgG2a and IgG2b variants of TS63 (referred to as TS63a and TS63b) were obtained using two rounds of cell sorting and one limited dilution cloning step. We validate that these new TS63 variants are suitable for co-labeling with mAb of the IgG1 subclass directed to other molecules, using anti mouse subclass antibodies, and for the labeling of exosomes through direct binding to protein A-coated gold particles. These mAbs will be useful to study the intracellular localization of various proteins and facilitate electron microscopy analysis of CD63 localization.

Effect of binding immunoglobulin protein on induction of regulatory B cells with killer phenotype during inflammation and disease.

Immune responses result from different immune cells acting in synergy to successfully fight infections. This requires a high degree of regulation to prevent excessive production of inflammatory products leading to other disease forms. Regulatory B cells are classified based on surface immunoglobulin expression. These cells are reported to resolve inflammation during chronic or autoimmune diseases. However, during chronic inflammation, their frequencies have been shown to be affected, and they can be induced by exposure to extracellular binding immunoglobulin protein (BiP). This review focuses on the effects on immune cells by extracellular or secreted BiP during various chronic inflammatory responses. For example, cell stress associated with Mycobacterium tuberculosis infection leads to accumulation of unfolded proteins that subsequently activate BiP and its three signal transducers intracellularly. Furthermore, BiP can be translocated from the endoplasmic reticulum to the extracellular environment where it binds immune cells as an autoantigen and leads to functional changes.

Chimeric Antigen Receptor T Cells Redirected Towards Clonally Restricted Surface Immunoglobulin Efficiently Eradicate Mature B Lymphocyte-Derived Malignancies

Background: Indolent B cell non-Hodgkin lymphomas (B-NHL) are typically described as having smoldering clinical courses. Chemotherapy plus CD20-targeting antibodies have made remissions in this type of disease possible, with fit patients sometimes consolidated with autologous stem cell transplant. However, these lymphomas commonly progress or relapse inexorably, requiring further interventions. Some B-NHL subtypes, such as mantle cell lymphoma (MCL) or advanced follicular lymphoma (FL) have unpredictable and sometimes clinically aggressive courses which are refractory to even frontline treatments, and remain principally incurable.Adoptive chimeric antigen receptor T cells targeting CD19 (CD19.CAR-T) has shown therapeutic promise for these B-NHL subtypes. Targeting this antigen, however, does not distinguish between normal and malignant B cells and may cause B-cell aplasia and agammaglobulinemia when CD19.CAR-T persist long-term. FL and MCL, along with many diffuse large B cell lymphoma (DLBCL), express surface immunoglobulin (Ig) that is clonally restricted to either the kappa (Ig-κ) or lambda (Ig-l) light chains. We explored targeting the Ig-κ or Ig-l individually with CAR-T, which would possibly spare B lymphocytes expressing the reciprocal light chain, and consequently reduce the impairment of humoral immunity.Methods/Results: Previous studies showed modifying the hinge and transmembrane (TM) regions could result in amplified tumor cytotoxicity. We explored refining our own CAR-T approach by first altering the hinge and TM regions of the CAR molecule itself, using the CD8a hinge and TM regions in lieu of the currently used CD28 hinge and TM sections. The costimulatory endodomains used were either CD28 or 41BB, each combined with the CD3ζ chain. When compared to the CD28 hinge and TM containing CAR molecules, we demonstrated the CD8a-containing hinge and TM CAR molecules targeting Ig-κ (CAR.κ) or Ig-l (CAR.λ) had augmented cytotoxicity, as well as increased IFNg and IL-2 cytokine production, in vitro against B-NHL derived tumor cell lines expressing Ig-κ or Ig-l, respectively. Furthermore, both CAR.κ and CAR.λ demonstrated selective cytotoxicity of the appropriately expressed tumor-associated immunoglobulin, while sparing the tumor cells expressing the reciprocal surface Ig.We conducted in vivo experiments using a NOD-scid gamma (NSG) xenograft murine models. NSG mice were treated with CAR-T targeting CD19 (positive control), non-transduced cells (negative control), and depending on the Ig light chain target, either CAR.κ.CD28ζ and CAR.κ.41BBζ, or CAR.λ.CD28ζ and CAR.λ.41BBζ. We also established a humanized murine model that reconstituted human B lymphocytes in NSG mice post-irradiation using CD34+ umbilical cord blood cells, replicating a humanized humoral immune system replete with a full complement of humanized B cells. We then measured the selective ability of CAR.κ and CAR.λ cells to eliminate human B-lymphocytes expressing either the Ig-κ or Ig-l, respectively, while sparing the reciprocally expressed light chain-carrying B lymphocytes.We found that CAR.κ and CAR.λ showed respective high cytotoxic activity, similar to CD19.CAR, against the appropriate Ig-κ+ or Ig-l+ B-NHL tumor cell lines in vivo within the NSG xenograft murine model. We also demonstrated the selective elimination by the CAR.κ and CAR.λ of either the kappa or lambda light chain-expressing B lymphocytes, with sparing of the reciprocal light chain-expressing B lymphocytes, in the humanized murine model.Conclusion: Since κ/l expression is clonally restricted, and because mature B-lymphocyte derived malignancies are themselves clonally restricted, having a CAR-T that targets either light chain can potentially treat the full gamut of mature B-NHL subtypes. In addition, sparing the normal population of B lymphocytes would have negligible impact upon the patient's humoral immunity. Moreover, we showed equal efficacy of our CAR.κ.CD28 and CAR.λ.CD28 when compared with CD19.CAR. As such, adoptive transfer of CAR-T targeting the clonally restricted light chain can be a very feasible immunotherapy approach to treating advanced FL, MCL and DLBCL clinically, without entirely compromising humoral immunity. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

HETEROGENEITY OF CHILDHOOD B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA (EGIL SUBTYPE BIV)

Introduction. Surface immunoglobulin expression is a main immunophenotypic criteria of mature subtype of B-lineage acute lymphoblastic leukemia. Although the majority of such cases represents Burkitt leukemia/lymphoma, it was shown for several times that membrane IgM could be detected in the absence of other mature lymphomas signs. The aim of the study was to evaluate heterogeneity of childhood acute lymphoblastic leukemia (ALL) with surface IgM expression and to assess correspondence of BIV EGIL ALL subtype with Burkitt lymphoma (BL) bone marrow dissemination. Materials and methods . Immunophenotypic, cytomorfologic and genetic data of 54 BIV-ALL cases were analyzed. Results . Among the studied patients 39 had BL, while others belonged to B-cell precursor ALL (BCP-ALL). All BL patients and none of BCP-ALL patients carried C-MYC rearrangement while in BCP-ALL group in 8 cases and in any BL cases KMT2A rearrangements were found. None of BCP-ALL children had L3 morphology according to FAB classification. Conclusions. B-lineage ALL with surface IgM expression is rather heterogeneous group of cases including typical BL and rare cases of BCP-ALL even with KMT2A-rearrangements. Combination of all available diagnostic technologies will allow precise split of these two different disease and select the appropriate treatment scheme.

Early stages in the ontogeny of small B-cell lymphomas: genetics and microenvironment.

In this review, we focus on the mechanisms underlying lymphomagenesis in chronic lymphocytic leukaemia, follicular lymphoma, mantle cell lymphoma and splenic marginal zone lymphoma. The cells of origin of these small B-cell lymphomas are distinct, as are the characteristic chromosomal lesions and clinical courses. One shared feature is retention of expression of surface immunoglobulin. Analysis of this critical receptor reveals the point of differentiation reached by the cell of origin. Additionally, the sequence patterns of the immunoglobulin-variable domains can indicate a role for stimulants of the B-cell receptor before, during and after malignant transformation. The pathways driven via the B-cell receptor are now being targeted by specific kinase inhibitors with exciting clinical effects. To consider routes to pathogenesis, potentially offering earlier intervention, or to identify causative factors, genetic tools are being used to track pretransformation events and the early phases in lymphomagenesis. These methods are revealing that chromosomal changes are only one of the many steps involved, and that the influence of surrounding cells, probably multiple and variable according to tissue location, is required, both to establish tumours and to maintain growth and survival. Similarly, the influence of the tumour microenvironment may protect malignant cells from eradication by treatment, and the resulting minimal residual disease will eventually give rise to relapse. The common and different features of the four lymphomas will be summarized to show how normal B lymphocytes can be subverted to generate tumours, how these tumours evolve and how their weaknesses can be attacked by targeted therapies.

Expressions of immunoglobulin M and immunoglobulin D heavy chain in B-chronic lymphocytic leukemia and their correlation with CD38 expression

Background: B-chronic lymphocytic leukemia (CLL) is a monoclonal malignancy characterized by an accumulation of small mature-looking B lymphocytes in the blood, bone marrow, and other tissues. It is typically characterized by CD5+, CD23+, CD22−, and CD79b−, with weak expression of surface immunoglobulin (Ig). Objectives: The aim of the study was to assess the expression of IgM and IgD heavy chain by flow cytometry in untreated newly diagnosed B-cell CLL and correlate the heavy chain isotopes expression with CD38 expression. Materials And Methods: A prospective cross-sectional study was conducted on thirty patients with new diagnosis of B-CLL. The study was conducted at nursing home teaching laboratory in medical city hospital in Baghdad for the period from November 2016 to March 2017. Five milliliter of venous blood in ethylenediaminetetraacetic acid was collected from thirty patients for detection of expression of IgM, IgD, and CD38 by flow cytometry. The patients were randomly selected regarding the age, sex, and stage of the disease. Results: The mean age of all included patients was (61.73 ± 6.92) with more disease predominant in male than female. The most common presenting feature of the patients was lymphadenopathy found in 16 patients (43%). Regarding staging system, six (20%) patients were in with Binet Stage C and five patients (17%) were in modified Rai Stage 4. CD38 and IgM expression showed a significant relation to hemoglobin and platelets (P Conclusions: The current study showed that the expression of CD38 had a significant correlation with IgM expression as well as there was positive correlation with the stage of the disease which may point out to inferior prognosis for those patients with CLL.

Rituximab Preferentially Eliminates BCR Signaling Proficient Chronic Lymphocytic Leukemia B Cells In Vivo

The use of anti-CD20 antibody rituximab has significantly improved the outcome of patients with chronic lymphocytic leukemia (CLL). Rituximab has been shown to act through several mechanisms including antibody-dependent cell cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), direct induction of apoptosis, and sensitization to chemotherapy. However, the exact contribution of each of these mechanisms to the clinical efficacy of rituximab in vivo and the exact mechanism of its action remain unclear. Importantly, the levels of cell surface CD20 expression were shown to associate with the efficacy of rituximab.We and others have described that CLL cells that have recently exited the lymph node microenvironment to the peripheral blood express lower cell surface levels of the chemokine receptor CXCR4 and higher levels of the activation marker CD5 (Callisano et al., 2011). We observed that these CLL cells (CXCR4dimCD5bright) have also higher cell surface CD20 expression (~2-fold; P<0.0001) compared to CLL cells from the same patient circulating in the blood stream for a longer time (CXCR4brightCD5dim), and we showed that CD20 is up-regulated by stromal cell interactions through the binding of SDF-1α to CXCR4 (P<0.001). We further hypothesized that higher levels of CD20 on CXCR4dimCD5bright cells will make them the primary target for rituximab in vivo. To investigate this, we obtained peripheral blood samples from CLL patients (N=17) before rituximab administration (day 0) and 24 hours (day 1) after rituximab administration (375 mg/m2, single agent). This showed that rituximab primarily and nearly completely eliminates the CXCR4dimCD5bright subpopulation (8.3 % pre-rituximab vs. 2.1 % post-rituximab, P<0.0001), which harbors the highest levels of CD20 (P<0.005).It has been previously suggested that the CD20 plays a direct role in microenvironmental interactions and especially in B cell receptor signaling (BCR; Uchida et al., 2004). Therefore, we investigated the BCR signaling propensity of CXCR4dimCD5bright subpopulation. We have observed that CXCR4dimCD5bright CLL cells have higher surface immunoglobulin (Ig) expression than CXCR4brightCD5dim cells from the same patient (~2-fold, P<0.005). This was also coupled with higher responsiveness of CXCR4dimCD5bright cells to BCR crosslinking with anti-IgM as measured by calcium flux (P=0.005). Moreover, CXCR4dimCD5bright cells also had higher levels of CD19 (1.8-fold, P<0.0001), which is an important docking molecule in PI3K signaling, and its higher levels lower the threshold for BCR signaling activation.Altogether, this study shows that rituximab primarily and effectively eliminates, at least in the short term, the BCR signaling proficient CLL B cells. It is likely that one of the mechanisms of rituximab action is the indirect inhibition of BCR signaling by eliminating CLL cells with strongly activated BCR pathway. These observations might have important implications for combinatorial therapies with BCR signaling inhibitors such as ibrutinib or idelalisib.This work was supported by: the Ministry of Education, Youth and Sports of the Czech Republic under the project CEITEC 2020 (LQ1601); the European Union's Horizon 2020 research and innovation programme under grant agreement No. 692298; Czech Science Foundation (project No. 16-13334Y); the Ministry of Health of the Czech Republic, grant No. 16-29622A. All rights reserved. This work was financed from the SoMoPro II Programme (project No. 4SGA8684), co-financed by European Union and the South-Moravian Region. This publication reflects only the author's views and the Union is not liable for any use that may be made of the information contained therein. This work was supported by the Ministry of Health of the Czech Republic - conceptual development of research organization (FNBr, 65269705, Sup 3/16), MUNI/A/1028/2015, and G.P. is a city of Ostrava scholarship holder. contact: marek.mraz@email.cz DisclosuresNo relevant conflicts of interest to declare.