Clonal B-cell Population Research Articles

Chronic Lymphocytic Leukemia: 2025 Update on the Epidemiology, Pathogenesis, Diagnosis, and Therapy.

Chronic lymphocytic leukemia (CLL) is the most frequent type of leukemia. It typically occurs in older patients and has a highly variable clinical course. Leukemic transformation is initiated by specific genomic alterations that interfere with the regulation of proliferation and apoptosis in clonal B-cells. The diagnosis is established by blood counts, blood smears, and immunophenotyping of circulating B-lymphocytes, which identify a clonal B-cell population carrying the CD5 antigen as well as typical B-cell markers. Two clinical staging systems, Rai and Binet, provide prognostic information by using the results of physical examination and blood counts. Various biological and genetic markers provide additional prognostic information. Deletions of the short arm of chromosome 17 (del(17p)) and/or mutations of the TP53 gene predict a shorter time to progression with most targeted therapies. The CLL international prognostic index (CLL-IPI) integrates genetic, biological, and clinical variables to identify distinct risk groups of patients with CLL. The CLL-IPI retains its significance in the era of targeted agents, but the overall prognosis of CLL patients with high-risk stages has improved. Only patients with active or symptomatic disease or with advanced Binet or Rai stages require therapy. When treatment is indicated, several therapeutic options exist: combinations of the BCL2 inhibitor venetoclax with obinutuzumab, or venetoclax with ibrutinib, or monotherapy with one of the inhibitors of Bruton tyrosine kinase (BTK). At relapse, the initial treatment may be repeated if the treatment-free interval exceeds 3 years. If the leukemia relapses earlier, therapy should be changed using an alternative regimen. Combinations of targeted agents now provide efficient therapies with a fixed duration that generate deep and durable remissions. These fixed-duration therapies have gained territory in the management of CLL, as they are cost-effective, avoid the emergence of resistance, and offer treatment free time to the patient. The cure rate of these novel combination regimens is unknown. Moreover, the optimal sequencing of targeted therapies remains to be determined. A medical challenge is to treat patients who are double-refractory to both BTK and BCL2 inhibitors. These patients need to be treated within experimental protocols using novel drugs.

Clonal Bronchopulmonary B-Cell Proliferations of Indeterminate Malignant Potential in Patients with Common Variable Immune Deficiency (CVID) associated Granulomatous Lymphocytic Interstitial Lung Disease (GLILD)

Abstract Introduction/Objective Pulmonary extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) is often challenging to distinguish from reactive nodular lymphoid hyperplasia, and assessing B-cell clonality in such cases is crucial in making the distinction. However, patients with common variable immune deficiency (CVID) can harbor clonal or oligoclonal lymphoid populations in their tissues, particularly in association with granulomatous lymphocytic interstitial lung disease (GLILD), making the distinction from MALT lymphoma extremely challenging. We report six cases of GLILD with clonal or oligoclonal B cell infiltrates in lung wedge resection specimens and describe their clinicopathologic characteristics, management, and outcomes. Methods/Case Report A retrospective review was performed to collect lung wedge resection specimens from patients with CVID showing atypical B-cell infiltrates associated with GLILD between 01/2015 and 08/2023. The clinicopathologic findings, management, and response to treatment were studied. Results (if a Case Study enter NA) A total of six GLILD cases were identified with variable degrees of architectural and cytologic lymphoid atypia. Clonality of the lymphoid infiltrate was assessed by kappa/lambda in situ hybridization staining in all six, flow cytometry in 3/6, and IGH gene rearrangement in 4/6 cases. The infiltrate showed frank lambda restriction in 1/6, lambda predominance in 1/6, polytypic expression in 1/6, and no significant plasmacytic component in 3/6 cases. Flow cytometry detected atypical light chain skewed B-cell populations in 3/3 of cases. IGH was positive for clonality in 3/4 and oligoclonal in 1/4 cases. Four patients received therapeutic intervention for GLILD, including one with Rituximab monotherapy, two with Rituximab+Azathioprine, and one with Rituximab+Mycophenolate leading to marked symptomatic and radiologic improvement in all four patients. The remaining two patients were observed to have stable disease. Conclusion Patients with CVID can harbor clonal B-cell populations due to their underlying immune dysfunction. Studies have shown that these can be transient and may not always progress to overt lymphoma. In the setting of GLILD, detecting these populations makes the distinction from MALT lymphoma very challenging. Given that the first- line therapy for both GLILD and MALT is anti-CD20 therapy, the malignant potential of these clones remains uncertain and can only be determined with long-term clinical follow-up.

Splenic marginal zone lymphoma with prolymphocytic transformation and cyclin D1 expression in the absence of CCND1 rearrangement.

Splenic marginal zone lymphoma (SMZL) is one of the most common B-cell lymphomas that affect the spleen. We report a case with splenomegaly and lymphocytosis that showed a clonal B-cell population lacking CD5 and CD10 expression. Notably, the atypical lymphoid cells showed prolymphocytoid morphology and expressed cyclin D1. Fluorescence in-situ hybridization was negative for CCND1/IgH rearrangement. The prolymphocytoid morphology and cyclin D1 expression present a diagnostic pitfall. The clinical presentation, morphology, immunophenotype, and molecular genetic findings are most consistent with a diagnosis of SMZL with prolymphocytic transformation and cyclin D1 expression. Here, we present this case along with a review of the literature, and summarize the clinicopathological characteristics of SMZL with prolymphocytic transformation.

The immune response behind peptide vaccination in diffuse midline glioma.

A first-in-human trial demonstrated that a vaccine targeting the histone mutation H3K27M can induce an immune response, in a mutation-specific manner, in patients with diffuse midline glioma. In a recent study by Boschert etal., the same group now dissects the functional immune response triggered after effective vaccination of one of the patients, who has been in remission for over 3 years. The H3K27M peptide vaccine, named H3-vac, induces a CD4+ T-cell-specific immune response in this patient and expands the repertoire of polyclonal H3K27M-specific T-cell receptors. A clonal H3K27M-reactive B-cell population was also detected in the patient's cerebrospinal fluid. Importantly, the immune response is induced across various human leukocyte antigen alleleotypes, indicating the potential efficacy of the vaccine in diverse populations. By exploring in detail the immune response linked to this patient's long-term survival, the authors prove peptide vaccinations as a viable therapeutic approach. This paves the way for personalised therapies harnessing immunogenic T- and B-cell responses against different tumour types.

Abstract 4961: Large-scale CyTOF data modeling of leukemia patient cohorts

Abstract Introduction: Cytometry by Time-of-Flight (CyTOF) is a single-cell proteomic assay that uses antibody-lanthanide metal conjugates to tag surface or intracellular proteins, quantified via mass spectrometry. Compared to flow cytometry, CyTOF significantly reduces overlapping signals and increases the maximum protein features from 8 to 50. CyTOF data is commonly analyzed with the ‘gating’ workflow, which is the iterative process of plotting cells by two protein features to select discrete cell populations. While CyTOF can comprehensively profile nuanced cell types, gating does not scale well in the number of features. Biases in gating often arise from the forced discretization of cell populations, subjectivity of gate boundaries, and intrinsic sample-sample variation. Contemporary challenges in CyTOF data analysis can be attributed to data volume and gating bias, which can lead to oversight of disease-related cell populations while hindering reproducibility. Methods: We developed a computational platform that enables the processing and analysis of large-scale CyTOF data. Our workflow includes modular components for data harmonization, normalization, transformation, batch correction, quality control, subsampling, and dimensionality reduction via UMAP of input sample files in Flow Cytometry Standard (FCS) format. Further, additional analytical modules are currently being integrated to deploy large-scale machine learning models for cell type identification. We use the results of these models as input for multi-scale analysis, where we aim to link single-cell CyTOF data with clinical outcomes. We test our workflow on previously published datasets and apply it to new leukemia patient cohorts of blood and bone marrow samples and a variety of marker panels. Results: We tested our workflow on previously published datasets of 4.7 M peripheral blood mononuclear cells from healthy donors (N= 20) and T cells (N = 8). We observed that samples mixed well upon integration and separated into major cell types like monocytes, CD4+/CD8+ T-cells, naïve/mature B-cells, and natural killer T-cells. We then applied it to a bone marrow cohort of healthy donors (N = 10), where we observe good mixing of samples but less clear separation of cell types. Finally, we applied our workflow to 9.3 M cells from 51 chronic lymphocytic leukemia (CLL) patients and 4 healthy donors, using a lymphocytic marker panel. Here, we observed a clear resolution of a major clonal B-cell population and immune T-cells. We are in the process of applying it to Acute Myeloid Leukemia (AML) patient data with myeloid and immune marker panels. Conclusions: We developed a computational platform that facilitates the simultaneous analysis of large-scale CyTOF data, both in the number of samples and cells per sample, a variety of marker panels, while minimizing gating biases common in traditional gating analysis. Citation Format: Garth Kong, Tania Vu, Evan Lind, Olga H. Nikolova. Large-scale CyTOF data modeling of leukemia patient cohorts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4961.

Clonal Relationship and Mutation Analysis in Lymphoplasmacytic Lymphoma/Waldenström Macroglobulinemia Associated With Diffuse Large B-cell Lymphoma

Patients with lymphoplasmacytic lymphoma/Waldenström macroglobulinemia (LPL/WM) occasionally develop diffuse large B-cell lymphoma (DLBCL). This mostly results from LPL/WM transformation, although clonally unrelated DLBCL can also arise. LPL/WM is characterized by activating MYD88 L265P (>95%) and CXCR4 mutations (~30%), but the genetic drivers of transformation remain to be identified. Here, in thirteen LPL/WM patients who developed DLBCL, the clonal relationship of LPL and DLBCL together with mutations contributing to transformation were investigated. In 2 LPL/WM patients (15%), high-throughput sequencing of immunoglobulin gene rearrangements showed evidence of >1 clonal B-cell population in LPL tissue biopsies. In the majority of LPL/WM patients, DLBCL presentations were clonally related to the dominant clone in LPL, providing evidence of transformation. However, in 3 patients (23%), DLBCL was clonally unrelated to the major malignant B-cell clone in LPL, of which 2 patients developed de novo DLBCL. In this study cohort, LPL displayed MYD88 L265P mutation in 8 out of eleven patients analyzed (73%), while CXCR4 mutations were observed in 6 cases (55%). MYD88 WT LPL biopsies present in 3 patients (27%) were characterized by CD79B and TNFAIP3 mutations. Upon transformation, DLBCL acquired novel mutations targeting BTG1, BTG2, CD79B, CARD11, TP53, and PIM1. Together, we demonstrate variable clonal B-cell dynamics in LPL/WM patients developing DLBCL, and the occurrence of clonally unrelated DLBCL in about one-quarter of LPL/WM patients. Moreover, we identified commonly mutated genes upon DLBCL transformation, which together with preserved mutations already present in LPL characterize the mutational landscape of DLBCL occurrences in LPL/WM patients.

Update on diagnosis and treatment of Chronic Lymphocytic Leukemia (CLL)

commonest leukemia found in adults western countries, affects more than 200000 people and is associated with approximately 4410 death in the USA annually. CLL is characterized by a progressive proliferation and accumulation of mature yet functionally incompetent lymphocytes. The disease typically occurs in elderly patients and has a highly variable clinical course. Leukemic transformation is initiated by specific genomic alterations that impair apoptosis of clonal B-cells. Diagnosis: The diagnosis of chronic lymphocytic leukemia (CLL) requires the presence of more than or equal to 5 × 109/L B lymphocytes (5000/μL) in the peripheral blood for the duration of at least 3 months. Immunophenotyping of circulating B-lymphocytes, which identify a clonal B-cell population carrying the CD5 antigen, as well as typical B-cell markers are confirmatory test. Prognosis: The two similar clinical staging systems, Binet and Rai stages, create prognostic information by using results of physical examination and blood counts. Various molecular biology markers also have prognostic value. Mutations of the TP53 gene and x chromosome 17 chromosome (del [17p) predict resistance to chemoimmunotherapy and a shorter time to progression, with most targeted therapies. A comprehensive, international prognostic score (CLL-IPI) integrates genetic, biological and clinical variables to identify distinct risk groups of CLL patients. Therapy: Early-stage disease of CLL patients are followed up regularly, only patients with active or symptomatic disease or with advanced stages of Binet or Rai require treatment. When treatment is indicated, most patients with CLL have several options: a combination of obinutuzumab and venetoclax, ibrutinib monotherapy, or immunochemotherapy. In patients under 65 years of age who are physically fit (particularly if they have a mutated IGVH gene), immunochemotherapy with fludarabine, cyclophosphamide, and rituximab remains the standard of care because of its potential therapeutic potential. In case of relapse, the initial treatment can be repeated if the treatment-free period exceeds 3 years. And in case of early recurrence of the disease, treatment should be switched to an alternative regime. Patients with del(17p) or TP53 mutations are another high-risk group and should be treated with targeted drugs. Allogeneic SCT can be considered in patients with TP53 or del(17p) mutations or in patients on inhibitor therapy.

Primary EBV Lymphoproliferative Disease Responsive to Therapy in a Patient with CTLA4-Haploinsufficiency

Epstein-Barr virus lymphoproliferative disease (EBV-LPD) occurs most often in patients with acquired immunosuppression. However, several inborn errors of immunity have been associated with primary EBV-LPD, including pathogenic variants in SH2D1A, ITK, MAGT1, CORO1A, CD27, CTPS1, RASGRP1, and ZAP70 (PMID29942301,34239742). Here, we present a patient with primary EBV-LPD in the setting of CTLA-4 haploinsufficiency. JC is a 25yo male with Evans syndrome, hypogammaglobulinemia, and central nervous system vasculitis who presented to the NIH for evaluation of EBV-LPD. After a complicated course of legionella pneumonia at age 23, he developed recurrent fevers, respiratory distress, hypoxia, and night sweats responsive to dexamethasone. Chest CT revealed innumerable bilateral pulmonary nodules and masses with mediastinal hilar lymphadenopathy and scattered ground glass opacities. Transbronchial biopsy demonstrated EBV-positive B-cell LPD with extensive necrosis and clonal gene rearrangements. He responded well to 1 cycle of rituximab. Two years later, he again had fever, dyspnea on exertion, and hypoxia requiring glucocorticoids. Chest CT revealed ground glass opacities of the lungs, lung nodules/masses, and consolidation. Lung biopsy showed T and B-cell infiltrates, EBV+ B-cells by immunohistochemistry, and a clonal B-cell population (Figure 1). His course was also complicated by concurrent Pneumocystis Jirovecii (PJP) infection. He received 1 cycle of rituximab, trimethoprim-sulfamethoxazole, and sirolimus for treatment of his EBV-LPD, PJP, and underlying CTLA-4 haploinsufficiency respectively with symptomatic and radiologic response (Figure 2).Download : Download high-res image (261KB)Download : Download full-size imageFigure 1. Histological features of needle core biopsy from the lung – (A) 10x and (B) 20× magnification H&E stain shows dense lympho-histiocytic infiltrate. (C) Lympho-histiocytic infiltrate is predominantly CD3+ T-cells. (D) Scattered CD79+ B-cells are also identified in the infiltrate. (E) 10x magnification (F) 20X magnification shows numerous EBV-encoded RNA (EBER) positive cells, mostly small in size. (D&F) EBER+ cells are in a similar distribution to CD79+ B cells.Download : Download high-res image (87KB)Download : Download full-size imageFigure 2. EBV viral load and absolute B-cell count over the course of therapy with rituximab and sirolimus. Dates of medication initiation (sirolimus) or infusion (rituximab) are listed about the graph. B) Radiologic improvement on therapy. May 9,2022 shows lung nodules, lung masses, and ground glass opacities prior to starting therapy. July 19, 2022 shows some improvement in lung nodules and masses after 1 dose of rituximab and initiation of sirolimus. September 27, 2022 shows marked improvement in lung nodules and masses and near resolution of ground glass opacities on continuous sirolimus therapy and 1 month after completion of rituximab. Figure 1. Histological features of needle core biopsy from the lung – (A) 10x and (B) 20× magnification H&E stain shows dense lympho-histiocytic infiltrate. (C) Lympho-histiocytic infiltrate is predominantly CD3+ T-cells. (D) Scattered CD79+ B-cells are also identified in the infiltrate. (E) 10x magnification (F) 20X magnification shows numerous EBV-encoded RNA (EBER) positive cells, mostly small in size. (D&F) EBER+ cells are in a similar distribution to CD79+ B cells. Figure 2. EBV viral load and absolute B-cell count over the course of therapy with rituximab and sirolimus. Dates of medication initiation (sirolimus) or infusion (rituximab) are listed about the graph. B) Radiologic improvement on therapy. May 9,2022 shows lung nodules, lung masses, and ground glass opacities prior to starting therapy. July 19, 2022 shows some improvement in lung nodules and masses after 1 dose of rituximab and initiation of sirolimus. September 27, 2022 shows marked improvement in lung nodules and masses and near resolution of ground glass opacities on continuous sirolimus therapy and 1 month after completion of rituximab. NIAID whole exome sequencing (WES) revealed 2 compound heterozygous variants in CTLA-4 in trans: a novel pathogenic variant c.373G > A, p.Gly125Arg, and a non-pathogenic c.326G > A, p.Gly109Glu, shared with his mother(allele frequency of 0.000367 popmaxv.3.1.1 and Cadd score 17). Other variants that may contribute to EBV-LPD are under investigation. EBV-LPD in CTLA-4 haploinsufficiency is rare, previously reported in only 2 pediatric cases (PMID34329649). Additionally, WES revealed compound heterozygous variants in CTLA-4; however, only 1 variant was proven pathogenic. While this patient initially demonstrated excellent response to first line therapy, long-term prognosis is guarded. Abatacept was not included in this patient’s treatment because of an association with progressive LPD in patients with and without CTLA-4 haploinsufficiency. Immune reconstitution with bone marrow transplantation will likely be required for permanent disease control.

Abstract 5222: Genetic variants associated with progression from monoclonal B-cell lymphocytosis (MBL) to chronic lymphocytic leukemia (CLL)

Abstract Low count (LC) MBL is characterized by a circulating population of clonal B-cells and is a precursor state to CLL. Most individuals with LC MBL do not progress to CLL and never come to clinical attention. An active area of research is to identify factors that distinguish those individuals with MBL who progress from those who do not. Currently, 41 single nucleotide polymorphisms (SNPs) are known to be associated with CLL risk, and a portion of these SNPs have also been found to be associated with MBL risk. We hypothesize that these CLL-susceptibility SNPs associated with both MBL and CLL risk are SNPs associated with developing a B-cell clone; whereas the remaining SNPs found not to be associated with MBL risk are associated with progression of the B-cell clone (i.e., progression from MBL to CLL). To test this hypothesis, we evaluated the association of these 41 SNPs with risk of progression to CLL using a case-control study of 1,911 CLL cases and 1,579 individuals with MBL from Mayo Clinic. Individuals with MBL were identified through screening using 8-color flow cytometry assay. Individuals with LC MBL (defined by having a clonal B-cell population with the percent clonal B-cells out of total B-cell count <85%) who had an immunophenotype consistent with CLL were included as controls. CLL cases were obtained through the Mayo Clinic hematology practice. DNA was genotyped from whole blood. We analyzed the 41 CLL susceptibility SNPs with risk of progression to CLL using logistic regression and estimated odds ratio (OR) and 95% confidence interval (CI), adjusting for sex and age. We evaluated the CLL polygenic risk score (CLL-PRS), comprised of a weighted average of the 41 SNPs, with risk of CLL. Out of the 41 established CLL susceptibility SNPs, 15 were associated with CLL progression from MBL to CLL (all P<0.05). Nine of these 15 significant SNPs were not previously found to be associated with risk of MBL. The most significant SNPs were rs9880772 (OR=1.23, CI: 1.11-1.36, P=6.1 × 10−5), rs888096 (OR=1.25, CI: 1.12-1.38, P=3.0 × 10−5), and rs305065 (OR=1.28, CI: 1.14-1.43, P=1.5 × 10−5). One SNP (rs77551289) had a suggestive association (OR=1.21, P=0.069). The remaining 25 SNPs had no evidence of association with progression (ORs ranging between 0.9-1.1 and all P>0.1). The CLL-PRS was also associated with CLL risk (continuous OR = 1.25, CI: 1.15 - 1.35, P = 6.7 × 10−8). In our study accounting for the presence of B-cell clonal population, we identified 15 out of 41 CLL-susceptibility SNPs to be significantly associated with progression from LC MBL to CLL. The remaining 25 non-significant CLL-SNPs were not associated with progression and may instead be associated with development or initiation of the B-cell clone. These findings suggest that the established CLL susceptibility SNPs may play differing role (i.e., development or progression of the clone) in the etiology of CLL. Citation Format: Raphael Mwangi, Geffen Kleinstern, Sara J. Achenbach, Dennis Robinson, Aaron D. Norman, Kari G. Rabe, Janet E. Olson, Neil E. Kay, Rosalie G. Waller, Nicholas J. Boddicker, James R. Cerhan, Esteban Braggio, Sameer A. Parikh, Curtis A. Hanson, Celine M. Vachon, Tait Shanafelt, Susan L. Slager. Genetic variants associated with progression from monoclonal B-cell lymphocytosis (MBL) to chronic lymphocytic leukemia (CLL). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5222.

Unique Presentation of Bing-Neel Syndrome With Co-existing Chronic Lymphocytic Leukemia.

Unique Presentation of Bing-Neel Syndrome With Co-existing Chronic Lymphocytic Leukemia.

Monoclonal B-cell lymphocytosis in the bone marrow: revisiting the criteria for chronic lymphocytic leukemia/small lymphocytic lymphoma

Monoclonal B-cell lymphocytosis is a clonal B-cell population in the peripheral blood (PB) of <5x10ˆ9/L without extramedullary (EM) disease, often with a chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) phenotype. The degree of bone marrow (BM) involvement is not currently a part of the diagnostic criteria for MBL or CLL/SLL, but CLL-type MBLs in BM can be seen in patients lacking PB lymphocytosis. Data are limited on the outcome of such cases. We assessed the clinicopathologic characteristics of isolated BM CLL-type MBL in patients who did not meet criteria for CLL/SLL. We evaluated BMs from 2006 to 2018 with CLL-like clonal B-cell populations in patients with a PB absolute lymphocyte count or monoclonal B-cell count of <5×109/L and without definite evidence of EM disease. We investigated the extent and pattern of marrow involvement, PB counts, flow cytometric data, genetics, concurrent hematopoietic diseases, and outcomes including progression and treatment. Thirty cases with BM MBL but <5x10E9/L PB monoclonal B cells and no EM disease were identified. Thirteen of 30 had additional hematopoietic neoplasms. The mean patient age was 74.1 years (median: 77 years, range: 43-91 years). No patients had lymphadenopathy (LAD) or splenomegaly by physical examination. By imaging, nine of 18 had LAD (8/9<1.5cm) and four of 18 had splenomegaly but with other attributable etiologies. Mean PB absolute lymphocyte count (ALC) was 1.8×10E9/L (range: 0.5-5.0×10E9/L). Twenty-four of 30 (80%) had low-level (<20%) BM involvement by MBL, and among these, none with available follow-up data progressed to diagnostic CLL/SLL. Six of 30 (20%) had >20% marrow involvement by MBL. Four of 6 were treated for CLL/SLL due to cytopenias, despite not meeting diagnostic criteria, and all 4 were CD38 or ZAP70 positive and had cytogenetic abnormalities, including trisomy 12. One of 6 developed overt CLL/SLL 3 years later and had cytogenetic abnormalities at the time of MBL diagnosis. One of 6 was monitored without treatment but had no cytogenetic abnormalities.Isolated BM CLL-type MBL represents a diagnostic gray area, and this study highlights the range of clinical outcomes. All cases with <20% BM involvement did not require CLL-specific treatment or progress to CLL/SLL. In the 4 cases where treatment was initiated due to cytopenias, patients had ≥20% BM involvement, CD38 or ZAP70 expression, and cytogenetic abnormalities but lacked a PB ALC of ≥5x10E9/L or LAD ≥1.5cm, suggesting that not all patients with clinically significant disease will meet criteria for CLL/SLL. The results also show that concurrent hematopoietic disorders can complicate the diagnosis, as the disease course or treatment may result in leukopenia, precluding PB absolute lymphocytosis. Though larger studies are needed, the degree of BM involvement, in conjunction with flow cytometric prognostic markers, and cytogenetic abnormalities may be a useful addition to the current diagnostic criteria for CLL/SLL which only considers a PB numerical cutoff and EM involvement.

Primary esophageal diffuse large B-cell lymphoma.

A 76-year-old man presented with dysphagia, epigastric pain and weight loss for the last two months. Heavy sweating was also presented. Past medical conditions included type 2 diabetes. He had no evidence of any immunosupressive disease including HIV infection. Physical examination only revealed low-grade fever. Laboratory data showed leukocytosis. Gastroscopy evidenced a complete esophageal stenosis starting at 30 cm, with a severely friable mucosa of malignant appearance. The results of biopsies were insufficient for diagnosis of malignancy. Computed tomography demonstrated a 10-cm irregular tumor located in the distal and middle thirds of the esophagus, which resulted in narrowing of the lumen. Involving tracheal carina, bronchus, and descending aorta were observed. Perforation signs were also seen. Distant metastases were not found. Empirical treatment with piperacillin/tazobactan was started. A surgical gastrostomy to allows nutritional support was performed. Two other gastroscopies were performed resulting in an inconclusive diagnosis. Finally, flow cytometry performed in samples obtained by endobronchial ultrasound-guided biopsy evidenced prominent clonal B-cell populations consistent with extranodal diffuse large B-cell lymphoma exhibing CD10 expression. A treatment with Rituximab in combination with cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) was started. Primary esophageal diffuse large B-cell lymphoma (DLBCL), a variant of non-Hodgkin's lymphoma, accounts for less than 1% of all cases of gastrointestinal lymphomas.

CD5-Negative, CD10-Negative Low-Grade B-Cell Lymphoproliferative Disorders of the Spleen.

CD5-negative, CD10-negative low-grade B-cell lymphoproliferative disorders (CD5-CD10-LPD) of the spleen comprise a fascinating group of indolent, neoplastic, mature B-cell proliferations that are essential to accurately identify but can be difficult to diagnose. They comprise the majority of B-cell LPDs primary to the spleen, commonly presenting with splenomegaly and co-involvement of peripheral blood and bone marrow, but with little to no involvement of lymph nodes. Splenic marginal zone lymphoma is one of the prototypical, best studied, and most frequently encountered CD5-CD10-LPD of the spleen and typically involves white pulp. In contrast, hairy cell leukemia, another well-studied CD5-CD10-LPD of the spleen, involves red pulp, as do the two less common entities comprising so-called splenic B-cell lymphoma/leukemia unclassifiable: splenic diffuse red pulp small B-cell lymphoma and hairy cell leukemia variant. Although not always encountered in the spleen, lymphoplasmacytic lymphoma, a B-cell lymphoproliferative disorder consisting of a dual population of both clonal B-cells and plasma cells and the frequent presence of the MYD88 L265P mutation, is another CD5-CD10-LPD that can be seen in the spleen. Distinction of these different entities is possible through careful evaluation of morphologic, immunophenotypic, cytogenetic, and molecular features, as well as peripheral blood and bone marrow specimens. A firm understanding of this group of low-grade B-cell lymphoproliferative disorders is necessary for accurate diagnosis leading to optimal patient management.

Chronic lymphocytic leukemia: 2022 update on diagnostic and therapeutic procedures

Chronic lymphocytic leukemia (CLL) is one of the most frequent types of leukemia. It typically occurs in elderly patients and has a highly variable clinical course. Leukemic transformation is initiated by specific genomic alterations that interfere with the regulation of proliferation and of apoptosis in clonal B-cells. The diagnosis is established by blood counts, blood smears, and immunophenotyping of circulating B-lymphocytes, which identify a clonal B-cell population carrying the CD5 antigen as well as typical B-cell markers. The clinical staging systems provide prognostic information by using the results of physical examination and blood counts. Various biological and genetic markers provide additional prognostic information. Deletions of the short arm of chromosome 17 (del[17p]) and/or mutations of the TP53 gene predict resistance to chemoimmunotherapy and a shorter time to progression with most targeted therapies. The CLL international prognostic index integrates genetic, biological, and clinical variables to identify distinct risk groups of patients with CLL. Only patients with active or symptomatic disease or with advanced Binet or Rai stages require therapy. When treatment is indicated, several therapeutic options exist: a combination of the B-cell lymphoma 2 (BCL2) inhibitor venetoclax with obinutuzumab, monotherapy with inhibitors of Bruton tyrosine kinase (BTK) such as ibrutinib and acalabrutinib, or chemoimmunotherapy. At relapse, the initial treatment may be repeated, if the treatment-free interval exceeds 3 years. If the disease relapses earlier, therapy should be changed using an alternative regimen. Patients with a del(17p) or TP53 mutation are usually resistant to chemotherapy and should, therefore, be treated with targeted agents. Combinations of targeted agents are now being investigated to create efficient, potentially curative therapies of CLL with fixed duration. One of the most relevant questions currently addressed in clinical trials is the comparison of monotherapies with BTK inhibitors with fixed duration combination therapies. Moreover, the optimal sequencing of targeted therapies remains to be determined. Alternative therapies are needed for patients with BTK and BCL2 inhibitor double-refractory disease.

Prevalence and Overall Survival of Low Count Monoclonal B-Cell Lymphocytosis (LC-MBL): A Screening Study of 8,297 Individuals from the Mayo Clinic Biobank

Prevalence and Overall Survival of Low Count Monoclonal B-Cell Lymphocytosis (LC-MBL): A Screening Study of 8,297 Individuals from the Mayo Clinic Biobank

Prevalence of Allelic Variants and Clonality of IGHV1-69 Expressing B-Cells in Patients with Different Severity of COVID 19 Disease

Introduction: Since the first months of the COVID-19 pandemic, efforts have been made to understand the importance of broadly neutralising natural antibodies in determining the response to SARS-CoV-2. Previous studies have shown that allelic variants of the IGHV1-69 gene play a dominant role in protective natural antibody responses to several other viral pathogens, including influenza virus, hepatitis C virus, human immunodeficiency virus and, most notably, the SARS-CoV-2-related viruses SARS-CoV and MERS-CoV. These allelic variants are commonly known as 51p1-related and differ from the other IGHV1-69 alleles (known as hv1263-related) in the presence of a Phe54 residue in the CDR2 region. Importantly, crystallographic studies have shown that the Phe54 residue is critical for the binding of IGHV1-69 antibodies to the SARS-CoV and MERS-CoV spike proteins. In this study, we evaluated the prevalence of 51p1 and hv1263 alleles and the clonality of 51p1- and hv1263-expressing B cells in a large cohort of healthy individuals and COVID-19 patients and correlated the findings with the severity of the disease.Мaterials and methods: A total of 419 samples were included in the study, of which 78 asymptomatic/mildly symptomatic individuals, 200 hospitalized patients with severe disease, 94 critically ill patients and 47 healthy donors. Peripheral blood was collected 8-20 days after the onset of symptoms and total cellular RNA was extracted from whole blood using an automated procedure. Аllelle-specific Ig-gene fingerprinting of IgM heavy chain transcripts was used to simultaneously analyse the clonality of the IgM+ B-cell population and the clonality of the 51p1- and hv1263-expressing B cell populations. The significance of the differences in the prevalence of clonal B-cell populations between healthy donors and patients and between patients with different severity of the disease was calculated with the Chi-Square test.Results: Analysis of the clonality of the IgM+ B-cell population showed a polyclonal pattern in most of the investigated healthy individuals (33/47, 70%) but in only 20% of all SARS-CoV-2 infected individuals (75/372, p<0.001). A significant difference was also observed between mildly affected and severely/critically ill patients [31/78 (39.7%) vs. 44/294 (15%), respectively) (p<0.001)], but not between severely and critically ill patients [28/200 (14,%) vs. 16/94 (17,1%), (p=n.s.)]. No 51p1 transcripts were detected in 74/372 (19.9%) of SARS-CoV-2 infected individuals and in 14/47 (29,8%) of the control group (p>0,01), while hv1263 transcripts were not detected in 155/289 (53,6%) and in 27/47 (68,6%) tasted patients and controls, respectively (p>0,05). We did not find a statistically significant difference in the prevalence of 51p1 and hv1263 alleles between patients with different disease severity. However, a significantly higher number of patients displayed clonal expansions of 51p1- or hv1263-expressing B cells (219/372(58.9%) and 118/244 (48,4%), respectively in comparison to healthy donors [5/47(10.6%) and 7/47(14.9%), respectively]. There was no statistically significant difference between mildly affected and severely/critically ill patients in the clonallity status of 51p1- 38/61 (62,3%) and 182/237 (76,7%) respectively or between hv1263- expressing B cells in the same two groups of patients [20/25 (80%) and 98/109 (89,9%), p>0.05].Conclusions: Our results show that SARS-CoV-2 infection stimulates clonal expansions of IGHV1-69 -expressing B-cells, but this is independent of the severity of the disease. In addition, no difference in the prevalence of IGHV1-69 alleles was observed between patients at different stages of the disease, indicating that natural neutralizing antibodies encoded by this gene are not an important determinant of COVID-19 severity and progression.DisclosuresNo relevant conflicts of interest to declare.

Low Mutational Burden of Extra Nodal Marginal Zone Lymphoma of Mucosa-Associated Lymphoid Tissue in Patients with Primary Sjogren's Syndrome

Abstract Background: Primary Sjogren's syndrome (pSS) is an autoimmune disease characterized by chronic inflammation of the exocrine glands. Patients with pSS are at increased risk of developing extra nodal marginal zone lymphoma of the mucosa-associated lymphoid tissue (MALT) type, predominantly in the salivary glands. The presence of cryoglobulinemia, low C4 levels and salivary gland enlargement, are important risk factors for lymphoma development in pSS. Although lymphoma associated mutations have been described in circulating B-cells of patients with pSS associated cryoglobulinemia, it is unknown which somatic aberrations underlie the development of pSS-associated MALT lymphomas. The aim of the current study was to define the genomic landscape of pSS salivary gland MALT lymphomas. Identification of specific recurrent mutations or copy number aberrations and defining the affected pathways would help to understand the mechanisms by which intra-epithelial B-cells undergo malignant transformation. Methods: Whole exome sequencing was performed on 14 fresh frozen and 3 paraffin embedded MALT tissue samples of pSS patients at the University Medical Center Groningen (UMCG). Matched peripheral white blood cell samples were available for 12 patients and were used as germline controls. Fluorescence in situ hybridization was performed for the detection of MALT1 translocations. Results: Presence of a clonal B-cell population, indicative of a MALT lymphoma was confirmed by IgH PCR (BIOMED-2) for all patients. Whole exome sequencing resulted in a median target coverage of 130x, ranging from 84-163. More than 90% of the target regions had a coverage of &amp;gt;50x. In total we identified 232 somatic mutations in 184 genes. Three cases had a relatively high mutational load (&amp;gt;25 / case), while the median number of mutations in the remaining 14 cases was 7. Across the 17 cases there were 18 recurrently mutated genes. PRKD1, associated with malignant epithelial tumor of the salivary glands, was the most frequently mutated gene (six cases). Besides PRKD1, five additional recurrently mutated genes are also involved in epithelial surface and/or extracellular matrix (MAMDC4, COL14A1, CAMSAP3, TMEM2 and MUC4). Five genes, all with mutations in two cases, were shown to be associated with lymphomagenesis (ID3, TBL1XR1, PAX5, IGLL5, APC). A total of 18 copy number alterations (CNAs) were detected in 8 of the 14 evaluable cases, with gains of part of chromosomes 2 and 19, and loss of chromosome 6 each being detected in two cases. With respect to outcome, only 2 cases with high mutational load relapsed outside of the salivary glands, suggesting a more advanced stage of lymphoma. Conclusion: The low mutational load and lack of a clear lymphoma related gene signature suggests that localized pSS MALT lymphomas are genetically stable and most likely depend on the inflammatory state of the micro-environment. Only those cases characterized by higher mutational load showed a relapse-remitting disease, as is typical for indolent lymphoma. These observations could be translated to the clinical setting and potentially have value as biomarker for identification of patients with a more aggressive disease. Confirmation in larger cohorts are required to confirm this potential association. This study also paves way for treatment strategies targeting the micro-environment. Disclosures No relevant conflicts of interest to declare.

Single Institutional Experience on Orbital Inflammatory Pseudotumor: Diagnostic and Management Challenge

Aims: Orbital inflammatory pseudotumor is considered a non-neoplastic inflammatory process. The finding of clonality of B or T-cell receptors in cases pathologically diagnosed as orbital inflammatory pseudotumor has unknown clinicopathologic significance. We sought to investigate potential B and T-cell clonality and concomitant diseases in cases pathologically diagnosed as orbital inflammatory pseudotumor.Methods: Cases diagnosed as orbital inflammatory pseudotumor at our institution were retrospectively analyzed. Hematoxylin and eosin-stained slides, immunohistochemically stained slides and polymerase chain reactions on cell block material for the investigation of clonality of B and T-cell receptors were evaluated, to confirm the diagnosis and investigate the prevalence of concomitant diseases.Results: A total of 13 cases showing characteristic histopathologic features of orbital inflammatory pseudotumor were identified. CD138, IgG, and IgG4 showed varying numbers of plasma cells in each case, with 5 cases (5/13, 38%) exhibiting relative increase in the presence of IgG4 plasma cells. However, no cases showed diagnostic findings of IgG4-related disease (IgG4-RD). polymerase chain reactions analysis showed clonal B-cell populations in 2 cases (2/13, 15%). No cases showed anaplastic lymphoma kinase expression by immunohistochemistry. There were no clinical reports of progression to lymphoma or development of systemic IgG4-RD in any of the patients (average follow-up of 300 days), with 38% of patients showing systemic autoimmune conditions.Conclusion: A small but significant percentage of typical orbital inflammatory pseudotumor on histology showed B-cell clonality on polymerase chain reactions analysis of B-cell receptors, or features suggestive, but not diagnostic of IgG4-RD. Close follow-up of these patients to identify development of lymphoma, systemic IgG4-RD, or other rheumatologic conditions may be clinically warranted.

Intrathecal Rituximab for Treatment of Leptomeningeal NonHodgkin’s Lymphoma: A Case Report

Introduction: Standard therapy for central nervous system (CNS) Non-Hodgkin’s lymphoma (NHL) is still to be determined and varies in clinical practice. Leptomeningeal metastasis (LM) is more common as compared to parenchymal disease in CNS NHL. We present a case of Leptomeningeal NHL which was found to be resistant to treatment with intrathecal (IT) Methotrexate, Cytarabine and whole brain radiation therapy (WBRT). Our patient achieved a complete remission only after treatment with IT Rituximab. Case Presentation: 74-year-old male presented to us with cough with purulent sputum. Upon thorough investigation including biopsy of axillary lymphadenopathy and a staging MRI, the patient was found to have low grade I/III B cell follicular lymphoma with LM. The patient was refractory to treatment with intrathecally administered Methotrexate, intrathecal (IT) Cytarabine and WBRT. After administration of IT Rituximab, patient showed clinical improvement and subsequent cerebrospinal fluid (CSF) cytology revealed complete elimination of clonal B-cell population. Conclusions: In LM where cure is generally not expected, administration of IT Rituximab showed a favorable efficacy and safety profile. Prospective trials are needed to establish the role of IT Rituximab as a standard of care for treatment of LM involvement in B-cell lymphomas.

Myelodysplasia and Bone Marrow Manifestations of Somatic UBA1 Mutated Autoinflammatory Disease

Somatic mutations in UBA1 in hematopoietic stem cells and myeloid cells have recently been described and are associated with adult-onset severe autoinflammatory diseases including relapsing polychondritis, Sweet syndrome, polyarteritis nodosa, and giant cell arteritis. This newly defined syndrome is named VEXAS (vacuoles, E1, X-linked, autoinflammatory, somatic). We performed clinicopathologic, cytogenetic, flow cytometric and molecular bone marrow assessment of 15 patients diagnosed with VEXAS and peripheral cytopenias. All patients were male, with a median age of 64y (IQR 45-80) and all had somatic missense mutations affecting the p.Met41 residue in UBA1 with variant allele frequencies (VAF) over 20% from peripheral blood or bone marrow samples and with lineage restriction to mature myeloid cells. Germline mutations at this residue are not reported in any public databases. Peripheral blood findings included macrocytic anemia in all patients (100%), thrombocytopenia in 9/15, and neutropenia in 1/15. All bone marrow aspirates (15/15; 100%) showed prominent vacuolization of both myeloid and erythroid precursors with few vacuoles noted in mature cells. Of the 9 patients tested for serum copper levels all were normal or borderline low. Marrow was hypercellular with granulocytic and megakaryocytic hyperplasia in 12/15 (80%). Definitive WHO criteria for MDS was met in 5/15 (40%) with 3 cases of MDS-MLD, and 2 of MDS-SLD. The remaining cases were suspicious for MDS with low or borderline levels of dyspoiesis that did not exceed greater than 10% of cells in respective lineages meeting criteria for clonal cytopenia of undetermined significance (CCUS). Of the MDS cases, dysplasia was seen in megakaryocytes (5/5), myeloid (2/5) and erythroid (1/5) precursors. Cytogenetic abnormalities were detected in 2/5 MDS patients involving t(3;12)(q21;q13) in one case, and del (5q)/del (13q) in another case. Next generation sequencing analyses were performed in 9/15 patients and showed mutations in DNMT3A in 2/5 MDS cases (VAF 43% and 44%), CSF1R in 1/5 MDS (VAF 3.1%), GNA11 in 1/5 MDS (VAF3.3%) and EZH2 mutation (VAF 22%) in one of the patients without overt MDS. Clonal plasma cell or B-cell populations were diagnosed in 6/15 patients with 3 plasma cell dyscrasias, and 2 cases of monoclonal B-cell lymphocytosis. The most common abnormalities detected by bone marrow flow cytometry analysis were severely reduced or absent B-cell precursors, inverted CD4:CD8 ratios and abnormal expression of CD56 on monocytes. In summary, hematologic disorders were identified in 10/15 VEXAS patients, including both myeloid and lymphoid clonal disease comprising MDS, plasma cell dyscrasias, and monoclonal B cell lymphocytosis. Based on the presence of UBA1 somatic mutations in hematopoietic cells of all VEXAS patients, the patients without evidence of overt neoplasia in this study met criteria for CCUS. Importantly, all patients had characteristic vacuoles in myeloid and erythroid precursors in the marrow without evidence of copper deficiency. These findings suggest that vacuolization of hematopoietic precursors in marrow of cytopenic patients, particularly in male patients and in the setting of systemic inflammation and normal copper levels, should prompt evaluation for somatic UBA1 mutations. In addition to severe autoinflammatory disease manifestations, VEXAS patients appear to confer increased risk for development of both myeloid and lymphoid/plasma cell neoplasia and require surveillance for disease progression. Figure Disclosures Young: Novartis: Research Funding.