Normal B-cell Subsets Research Articles

High-Sensitivity Analysis of Normal B-Cell Subsets in MBL and Untreated CLL Identifies Patients with Sub-Optimal Sars-Cov-2 Antibody Responses

Background: Infectious morbidity and mortality are an issue in early stage CLL and to a lesser extent in CLL-type MBL. Normal B-cell numbers are usually decreased but the extent of depletion varies and cannot be predicted from the total B-cell count. Aim: to determine if high-sensitivity analysis of residual normal B-cell subsets can identify individuals with CLL/MBL at increased risk of sub-optimal humoral response to infection/vaccination Methods:Patients: n=92 (55 male, 37 female), median age 75 years (range 46-93), of which 77 had a diagnosis of CLL/SLL with a median B-cell count of 9.6, range 0.7-59 x 10^9/L, and 15 had MBL with a B-cell count median 3.2, range 0.3-7.1 x 10^9/L (2 had fluctuating B-cell counts that did not persist above 5 x 10^9/L). Samples were collected for routine monitoring and additional analysis was performed on pseudonymised surplus material (REC 14/WS/0098). Full vaccination status is currently evaluable in 69/92. Flow cytometry: Analysis of normal B-cell subsets was performed using CD19 to identify B-cells, with CD20, CD5, CD79b, ROR1 & CD3 to exclude CLL & contaminating cells, and CD27, CD38, IgD and IgM to characterise normal B-cell subsets; 0.5-2million cells labelled cells were acquired using a Coulter Cytoflex LX. SARS-CoV-2 antibody response: IgG and IgM antibodies to the spike protein were detected in plasma separated from EDTA-anticoagulated blood using the Fortress diagnostics lateral flow immunoassay validated for the UK REACT-2 national immunosurveillance study. Results: IgG anti-SARS-Cov-2 antibody response was detected in 67/77 (87%) of people with untreated CLL (of which 38/67 also had IgM Ab) and in 14/15 (93%) of MBL (of which 11/14 also had detectable IgM Ab). The most recent vaccination was a median 5.1 months prior to assessment (range 1.3-11 months, 29% had received 2x BNT162b2, 30% 2x ChAdOx1, 22% 2x ChAdOx1 + 1x BNT162b2, 16% 3x BNT162b2 and 3% declined vaccination). Figure 1A shows the relationship between CLL-phenotype cell count and normal naive B-cell number for all patients. Total B-cell numbers, as well as naïve-B-cell and IgM+ memory B-cell numbers were significantly reduced in individuals with no IgG SARS-CoV-2 Ab response. The numbers of class-switched memory B-cell numbers were decreased but not significantly different (Figure 1B). There was no clear threshold in any B-cell subset that predicted a detectable SARS-CoV-2 antibody response in CLL/MBL, although 63/66 (95%) of people with a naïve B-cell count >=2/uL had a detectable IgG SARS-CoV-2 Ab response compared to 18/26 (69%) for those with <2/uL naïve B-cells. Of the 26 people with CLL/MBL and a naïve B-cell count <2/uL, none of the 18 with a detectable IgG SARS-CoV-2 Ab response have died on follow-up (median 9 months, range 5-12) while 3/8 with no IgG SARS-CoV-2 IgG Ab response have subsequently died at 2,3, and 6 months since assessment, all with COVID infection as a primary cause (CLL cell count at last assessment 0.7, 15, and 20 x 10^9/L respectively). Conclusions: SARS-CoV-2 antibody responses are impaired in a small proportion of people with untreated CLL or MBL who also have substantially depleted naïve/IgM memory B-cells. Measuring normal B-cell levels in people with CLL/MBL is technically challenging and may not be required if COVID vaccine response can be shown to be an effective surrogate for infectious risk in general. Alternative strategies such as the use of passive immunisation or early treatment of active Covid infection with neutralising antibodies or antivirals should be considered in CLL/MBL patient with poor response to vaccination. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

P673: DEPLETION AND RECOVERY OF NORMAL B-CELLS DURING AND AFTER TREATMENT WITH CHEMOIMMUNOTHERAPY, IBRUTINIB OR VENETOCLAX.

Background: Infectious complications are a major cause of morbidity and mortality in Chronic Lymphocytic Leukaemia (CLL). Therapeutic approaches that deplete CLL cells also affect normal B-cells. Optimal treatment would result in eradication of CLL cells and recovery of normal immune function. FLAIR (ISRCTN01844152) is a phase III trial for previously untreated CLL comparing ibrutinib plus rituximab (IR) with fludarabine, cyclophosphamide and rituximab (FCR) and subsequently amended to also compare ibrutinib plus venetoclax (I+V) and ibrutinib alone (I) with FCR. Measurable residual disease (MRD) and normal B-cell levels were assessed at multiple timepoints. Aims: To assess the depletion of normal B-cells during treatment and recovery after end of treatment. Methods: Participants aged under 75 years with <20% TP53-deleted cells were initially randomised to FCR or IR and subsequently to FCR, IR, I+V or I with the IR arm closed after randomisation of 771 participants to FCR/IR. FCR was given for 6 cycles, while treatment in the IR, I and I+V arms continued for up to 6 years except in participants attaining <0.01% MRD who continued treatment for the time taken to achieved MRD <0.01% and then stopped if MRD remained <0.01%. Month (M) 24 was earliest permitted stopping point. MRD flow cytometry was performed according to ERIC guidelines (panel: CD19/5/20/43/79/81+ROR1, acquisition of 0.5–2.2 million cells, BD Biosciences Lyric). Additional analysis of normal B-cell subsets was performed in a cohort of >500 patients (panel: CD19 to identify B-cells, CD20/5/79b+ROR1 and CD3 to exclude CLL & contaminating cells, with CD27/ 38/IgD/IgM to characterise normal B-cell subsets using a Coulter Cytoflex LX). Results: Normal B-cells were undetectable during FCR treatment and only rarely detectable until 12 months after last FCR cycle. Circulating normal B-cells were reduced in number or undetectable in participants receiving ibrutinib-containing regimens with greater depletion in the I+V and IR arms relative to I monotherapy. B-progenitors persist through FCR treatment but were depleted during I, I+R or I+V treatment. Normal B-cell levels at 24 and 36 months after randomisation, with time off-treatment if applicable, are shown in Figure 1. In the ibrutinib-containing arms (IR, I, and I+V), there was a trend towards fewer COVID-associated SAE at any time point for participants with detectable B-cells at 24M (4/181, 2.2%) compared to those with no detectable B-cells (14/344, 4.1%) and COVID-associated SAEs were not observed in FCR-treated participants who had recovered any level of normal B-cells by 24M (0/215). However, the data on COVID infections are limited and there was no apparent association between normal B-cell levels at 24M with the proportion of participants experiencing an infectious SAE overall. Assessment of normal B-cell subsets during ibrutinib-based treatment demonstrated a mix of naïve and memory B-cells. Serological response to COVID infection/vaccination in this cohort is currently being performed. Participants stopping I+V treatment at 24-30 months post-randomisation due to MRD eradication showed rapid recovery of normal naive B-cells within 6-12 months after end of treatment in the vast majority (>95%) of evaluable cases. Image:Summary/Conclusion: Normal circulating B-cells are depleted during treatment with rituximab but can persist at a low level during I, IR or I+V treatment. Most patients in remission after treatment with FCR or I+V show recovery of normal B-cells at 12 months of stopping treatment.

Loss of synergistic transcriptional feedback loops drives diverse B-cell cancers.

BackgroundThe most common B-cell cancers, chronic lymphocytic leukemia/lymphoma (CLL), follicular and diffuse large B-cell (FL, DLBCL) lymphomas, have distinct clinical courses, yet overlapping “cell-of-origin”. Dynamic changes to the epigenome are essential regulators of B-cell differentiation. Therefore, we reasoned that these distinct cancers may be driven by shared mechanisms of disruption in transcriptional circuitry.MethodsWe compared purified malignant B-cells from 52 patients with normal B-cell subsets (germinal center centrocytes and centroblasts, naïve and memory B-cells) from 36 donor tonsils using >325 high-resolution molecular profiling assays for histone modifications, open chromatin (ChIP-, FAIRE-seq), transcriptome (RNA-seq), transcription factor (TF) binding, and genome copy number (microarrays).FindingsFrom the resulting data, we identified gains in active chromatin in enhancers/super-enhancers that likely promote unchecked B-cell receptor signaling, including one we validated near the immunoglobulin superfamily receptors FCMR and PIGR. More striking and pervasive was the profound loss of key B-cell identity TFs, tumor suppressors and their super-enhancers, including EBF1, OCT2(POU2F2), and RUNX3. Using a novel approach to identify transcriptional feedback, we showed that these core transcriptional circuitries are self-regulating. Their selective gain and loss form a complex, iterative, and interactive process that likely curbs B-cell maturation and spurs proliferation.InterpretationOur study is the first to map the transcriptional circuitry of the most common blood cancers. We demonstrate that a critical subset of B-cell TFs and their cognate enhancers form self-regulatory transcriptional feedback loops whose disruption is a shared mechanism underlying these diverse subtypes of B-cell lymphoma.FundingNational Institute of Health, Siteman Cancer Center, Barnes-Jewish Hospital Foundation, Doris Duke Foundation.

Post-Transformation IGHV-IGHD-IGHJ Mutations in Chronic Lymphocytic Leukemia B Cells: Implications for Mutational Mechanisms and Impact on Clinical Course.

Analyses of IGHV gene mutations in chronic lymphocytic leukemia (CLL) have had a major impact on the prognostication and treatment of this disease. A hallmark of IGHV-mutation status is that it very rarely changes clonally over time. Nevertheless, targeted and deep DNA sequencing of IGHV-IGHD-IGHJ regions has revealed intraclonal heterogeneity. We used a DNA sequencing approach that achieves considerable depth and minimizes artefacts and amplification bias to identify IGHV-IGHD-IGHJ subclones in patients with prolonged temporal follow-up. Our findings extend previous studies, revealing intraclonal IGHV-IGHD-IGHJ diversification in almost all CLL clones. Also, they indicate that some subclones with additional IGHV-IGHD-IGHJ mutations can become a large fraction of the leukemic burden, reaching numerical criteria for monoclonal B-cell lymphocytosis. Notably, the occurrence and complexity of post-transformation IGHV-IGHD-IGHJ heterogeneity and the expansion of diversified subclones are similar among U-CLL and M-CLL patients. The molecular characteristics of the mutations present in the parental, clinically dominant CLL clone (CDC) differed from those developing post-transformation (post-CDC). Post-CDC mutations exhibit significantly lower fractions of mutations bearing signatures of activation induced deaminase (AID) and of error-prone repair by Polη, and most of the mutations were not ascribable to those enzymes. Additionally, post-CDC mutations displayed a lower percentage of nucleotide transitions compared with transversions that was also not like the action of AID. Finally, the post-CDC mutations led to significantly lower ratios of replacement to silent mutations in VH CDRs and higher ratios in VH FRs, distributions different from mutations found in normal B-cell subsets undergoing an AID-mediated process. Based on these findings, we propose that post-transformation mutations in CLL cells either reflect a dysfunctional standard somatic mutational process or point to the action of another mutational process not previously associated with IG V gene loci. If the former option is the case, post-CDC mutations could lead to a lesser dependence on antigen dependent BCR signaling and potentially a greater influence of off-target, non-IG genomic mutations. Alternatively, the latter activity could add a new stimulatory survival/growth advantage mediated by the BCR through structurally altered FRs, such as that occurring by superantigen binding and stimulation.

Loss of Synergistic Transcriptional Feedback Loops Drives Diverse B-Cell Cancers

Background: The most common B-cell cancers, chronic lymphocytic leukemia/lymphoma (CLL), follicular and diffuse large B-cell (FL, DLBCL) lymphomas, have distinct clinical courses, yet overlapping “cell-of-origin”. Dynamic changes to the epigenome are essential regulators of B-cell differentiation. Therefore, we reasoned that these distinct cancers may be driven by shared mechanisms of disruption in transcriptional circuitry. Methods: We compared purified malignant B-cells from 52 patients with normal B-cell subsets (germinal center centrocytes and centroblasts, naive and memory B-cells) from 36 donor tonsils using >325 high-resolution molecular profiling assays for histone modifications, open chromatin (ChIP-, FAIRE-seq), transcriptome (RNA-seq), transcription factor (TF) binding, and genome copy number (microarrays). Findings: From the resulting data, we identified gains in active chromatin in enhancers/super-enhancers that likely promote unchecked B-cell receptor signaling, including one we validated near the immunoglobulin superfamily receptors FCMR and PIGR. More striking and pervasive was the profound loss of key B-cell identity TFs, tumor suppressors and their super-enhancers, including EBF1, OCT2(POU2F2), and RUNX3. Using a novel approach to identify transcriptional feedback, we showed that these core transcriptional circuitries are self-regulating. Their selective gain and loss form a complex, iterative, and interactive process that likely curbs B-cell maturation and spurs proliferation. Interpretation: Our study is the first to map the transcriptional circuitry of the most common blood cancers. We demonstrate that a critical subset of B-cell TFs and their cognate enhancers form self-regulatory transcriptional feedback loops whose disruption is a shared mechanism underlying these diverse subtypes of B-cell lymphoma. Funding: National Institute of Health, Siteman Cancer Center, Barnes-Jewish Hospital Foundation, Doris Duke Foundation. Declaration of Interest: None to declare. Ethical Approval: De-identified peripheral blood, lymph node biopsies, and excised tonsils were obtained from FL, DLBCL, or CLL or healthy tonsillectomy patients (Table 1 and Fig. S1A) seen at Washington University School of Medicine who provided informed consent under IRB-approved protocols in concordance with the Declaration of Helsinki. Due to yield constraints, some samples were not subjected to all assays.

In This Issue

See article, p. 32.Inflammatory signaling perturbs hematopoietic stem cell (HSC) differentiation through a process termed emergency hematopoiesis. Whether an equivalent process has a role in regulating leukemia stem cells (LSC) is unclear. Here Xie and colleagues demonstrate that sphingosine-1-phosphate receptor 3 (S1RP3), a receptor for proinflammatory and bioactive lipid sphingosine-1-phosphate (S1P), drives myeloid differentiation in both HSC and LSC. S1RP3 expression is high in CD33+ myeloid cells and low in primitive CD34+ cells. When S1RP3 is upregulated, it promotes myeloid differentiation in synergy with proinflammatory signaling pathways in HSC and LSC. In addition, S1RP3 expression level could be utilized to stratify acute myeloid leukemia (AML) patients into more differentiated and more primitive cases. Moreover, pharmacologic disruption of S1P signaling by the S1P prodrug FTY720 reduces LSC frequency and decreases leukemia burden in vivo. The results suggest that S1RP3 could serve as both a biomarker and therapeutic target in human AML.See article, p. 54.Specific alterations in DNA methylation patterns are common in cancer cells across tissue origins and genetic drivers. In this study, Kretzmer and colleagues asked how early cancer-associated methylation patterns emerge during chronic lymphocytic leukemia (CLL) oncogenesis. They have profiled DNA methylomes of single cells and bulk samples across cell types representing key stages of oncogenesis, from normal germinal center B-cell subsets to preneoplastic monoclonal B-cell lymphocytosis (MBL) to serial CLL samples at diagnosis and throughout disease progression and therapy. The authors identify cancer-specific methylation patterns distinguishing normal B-cell subtypes from MBL and CLL. These methylation patterns are shared among all CLL clones in each patient case and are already present in nonmalignant MBL cell state. Persistence of the methylation pattern is particularly remarkable when considered on the backdrop of genetic and transcriptional heterogeneity. These findings shed light on possible CLL cell of origin and provide support to the concept that precancerous methylome may be a prerequisite for oncogenic transformation and maintenance of neoplastic state.See article, p. 70.Activated B-cell–like diffuse large B-cell lymphoma (ABC-DLBCL) is associated with poor prognosis when compared with germinal center B-cell–like (GCB)-DLBCL, and there is an urgent need to develop a preclinical animal model that closely models human ABC-DLBCL for the development of effective targeting therapies. MYD88 mutation and gain of BCL2 copy numbers are two of the most frequent genomic aberrations in ABC-DLBCL. Here Flümann and colleagues characterize the phenotypes of a Myd88/BCL2-driven (MBC) mouse lymphoma model and find that this model closely resembles the morphologic and gene expression signatures of human ABC-DLBCL. Moreover, MBC-derived lymphomas undergo apoptosis in response to venetoclax, revealing their druggable vulnerabilities against BCL2. Furthermore, combined treatment of venetoclax plus anti–PD-1 antibody RMP1-14 extends survival of MBC mice after they develop tumors far longer than either drug alone. These findings validate MBC mice as a tool to study ABC-DLBCL and demonstrate preclinical evidence that targeting both BCL2 and PD-1 could serve as a novel therapy for the treatment of ABC-DLBCL.See article, p. 92.PTEN is the second most frequently mutated tumor suppressor gene in cancer. In this study, Tottone and colleagues identified a distal regulatory element enhancing PTEN expression (PE) as a recurrent deletion in human T-ALL. PE displays enhancer-associated chromatin marks in hematopoietic lineage, but not solid tissues. In several human T-ALL cell lines, the PE region is occupied by multiple transcription factors including Notch, is bidirectionally transcribed, and engages in long-range interactions with the PTEN promoter. PE deletion in these cells results in decreased PTEN mRNA and decreased proliferation. PE is conserved across species, and its germline as well as conditional deletion in mice results in dose-dependent diminished PTEN expression in T cells, but not other lineages. Notch1-driven leukemia development is accelerated on PE-null mouse background, providing functional evidence of PE tumor-suppressive role consistent with its deletion profile in human T-ALL. Cancer-associated genetic inactivation of tumor suppressor genes is mostly documented within their promoter or protein-coding regions. This study reveals that distal enhancer deletion is another mechanism by which a classic and ubiquitous tumor suppressor gene can be inactivated in cancer. This work identifies previously uncharacterized PTEN enhancer activity in normal T cells and in T-ALL. It invites exploration of lineage-specific enhancer regulation of PTEN and other classic tumor suppressors in physiology and oncogenesis.In This Issue is written by Blood Cancer Discovery editorial staff. Readers are encouraged to consult the original articles for full details.

Identification of 2 DNA methylation subtypes of Waldenström macroglobulinemia with plasma and memory B-cell features

AbstractEpigenetic changes during B-cell differentiation generate distinct DNA methylation signatures specific for B-cell subsets, including memory B cells (MBCs) and plasma cells (PCs). Waldenström macroglobulinemia (WM) is a B-cell malignancy uniquely comprising a mixture of lymphocytic and plasmacytic phenotypes. Here, we integrated genome-wide DNA methylation, transcriptome, mutation, and phenotypic features of tumor cells from 35 MYD88-mutated WM patients in relation to normal plasma and B-cell subsets. Patients naturally segregate into 2 groups according to DNA methylation patterns, related to normal MBC and PC profiles, and reminiscent of other memory and PC-derived malignancies. Concurrent analysis of DNA methylation changes in normal and WM development captured tumor-specific events, highlighting a selective reprogramming of enhancer regions in MBC-like WM and repressed and heterochromatic regions in PC-like WM. MBC-like WM hypomethylation was enriched in motifs belonging to PU.1, TCF3, and OCT2 transcription factors and involved elevated MYD88/TLR pathway activity. PC-like WM displayed marked global hypomethylation and selective overexpression of histone genes. Finally, WM subtypes exhibited differential genetic, phenotypic, and clinical features. MBC-like WM harbored significantly more clonal CXCR4 mutations (P = .015), deletion 13q (P = .006), splenomegaly (P = .02), and thrombocytopenia (P = .004), whereas PC-like WM harbored more deletion 6q (P = .012), gain 6p (P = .033), had increased frequencies of IGHV3 genes (P = .002), CD38 expression (P = 4.1e-5), and plasmacytic differentiation features (P = .008). Together, our findings illustrate a novel approach to subclassify WM patients using DNA methylation and reveal divergent molecular signatures among WM patients.

Human BCR analysis of single-sorted, putative IgE+ memory B cells in food allergy

R. Jimenez-Saiz, Y. Ellenbogen and K. Bruton et al. developed a method to identify bona fide IgE+ MBCs in humans, demonstrated their extreme rarity in circulation and cautioned against the clinical utility of their assessment.

A multiple myeloma classification system that associates normal B-cell subset phenotypes with prognosis

AbstractDespite the recent progress in treatment of multiple myeloma (MM), it is still an incurable malignant disease, and we are therefore in need of new risk stratification tools that can help us to understand the disease and optimize therapy. Here we propose a new subtyping of myeloma plasma cells (PCs) from diagnostic samples, assigned by normal B-cell subset associated gene signatures (BAGS). For this purpose, we combined fluorescence-activated cell sorting and gene expression profiles from normal bone marrow (BM) Pre-BI, Pre-BII, immature, naïve, memory, and PC subsets to generate BAGS for assignment of normal BM subtypes in diagnostic samples. The impact of the subtypes was analyzed in 8 available data sets from 1772 patients' myeloma PC samples. The resulting tumor assignments in available clinical data sets exhibited similar BAGS subtype frequencies in 4 cohorts from de novo MM patients across 1296 individual cases. The BAGS subtypes were significantly associated with progression-free and overall survival in a meta-analysis of 916 patients from 3 prospective clinical trials. The major impact was observed within the Pre-BII and memory subtypes, which had a significantly inferior prognosis compared with other subtypes. A multiple Cox proportional hazard analysis documented that BAGS subtypes added significant, independent prognostic information to the translocations and cyclin D classification. BAGS subtype analysis of patient cases identified transcriptional differences, including a number of differentially spliced genes. We identified subtype differences in myeloma at diagnosis, with prognostic impact and predictive potential, supporting an acquired B-cell trait and phenotypic plasticity as a pathogenetic hallmark of MM.

Chronic Lymphocytic Leukemia B-Cell Normal Cellular Counterpart: Clues From a Functional Perspective.

Chronic lymphocytic leukemia (CLL) is characterized by the clonal expansion of small mature-looking CD19+ CD23+ CD5+ B-cells that accumulate in the blood, bone marrow, and lymphoid organs. To date, no consensus has been reached concerning the normal cellular counterpart of CLL B-cells and several B-cell types have been proposed. CLL B-cells have remarkable phenotypic and gene expression profile homogeneity. In recent years, the molecular and cellular biology of CLL has been enriched by seminal insights that are leading to a better understanding of the natural history of the disease. Immunophenotypic and molecular approaches (including immunoglobulin heavy-chain variable gene mutational status, transcriptional and epigenetic profiling) comparing the normal B-cell subset and CLL B-cells provide some new insights into the normal cellular counterpart. Functional characteristics (including activation requirements and propensity for plasma cell differentiation) of CLL B-cells have now been investigated for 50 years. B-cell subsets differ substantially in terms of their functional features. Analysis of shared functional characteristics may reveal similarities between normal B-cell subsets and CLL B-cells, allowing speculative assignment of a normal cellular counterpart for CLL B-cells. In this review, we summarize current data regarding peripheral B-cell differentiation and human B-cell subsets and suggest possibilities for a normal cellular counterpart based on the functional characteristics of CLL B-cells. However, a definitive normal cellular counterpart cannot be attributed on the basis of the available data. We discuss the functional characteristics required for a cell to be logically considered to be the normal counterpart of CLL B-cells.

Age-associated distribution of normal B-cell and plasma cell subsets in peripheral blood

Humoral immunocompetence develops stepwise throughout life and contributes to individual susceptibility to infection, immunodeficiency, autoimmunity, and neoplasia. Immunoglobulin heavy chain (IgH) isotype serum levels can partly explain such age-related differences, but their relationship with the IgH isotype distribution within memory B-cell (MBC) and plasma cell (PCs) compartments remains to be investigated. We studied the age-related distribution of MBCs and PCs expressing different IgH isotypes in addition to the immature/transitional and naive B-cell compartments. B-cell and PC subsets and plasma IgH isotype levels were studied in cord blood (n=19) and peripheral blood (n=215) from healthy donors aged 0 to 90years by using flow cytometry and nephelometry, respectively. IgH-switched MBCs expressing IgG1, IgG2, IgG3, IgA1, and IgA2 were already detected in cord blood and newborns at very low counts, whereas CD27+IgM++IgD+ MBCs only became detectable at 1 to 5months and remained stable until 2 to 4years, and IgD MBCs peaked at 2 to 4years, with both populations decreasing thereafter. MBCs expressing IgH isotypes of the second immunoglobulin heavy chain constant region (IGHC) gene block (IgG1, IgG3, and IgA1) peaked later during childhood (2-4years), whereas MBCs expressing third IGHC gene block immunoglobulin isotypes (IgG2, IgG4, and IgA2) reached maximum values during adulthood. PCs were already detected in newborns, increasing in number until 6 to 11months for IgM, IgG1, IgG2, IgG3, IgA1, and IgA2; until 2 to 4years for IgD; and until 5 to 9years for IgG4 and decreasing thereafter. For most IgH isotypes (except IgD and IgG4), maximum plasma levels were reached after PC and MBC counts peaked. PC counts reach maximum values early in life, followed by MBC counts and plasma IgH isotypes. Importantly, IgH isotypes from different IGHC gene blocks show different patterns, probably reflecting consecutive cycles of IgH isotype switch recombination through life.

Molecular Features of Germinal Cell Derived B-Cell Lymphomas Using miRNA Signatures

Background:B-cell development is a complex biological process that initiates in bone marrow and matures in lymph nodes. These hierarchical stages are tightly regulated by transcription factors and microRNAs (miRNAs). B-cells are prone to genetic alteration during these maturation stages due to DNA breaks required for B-cell receptor re-arrangement and additional changes that occur during the germinal center (GC)-reaction for class switch recombination and somatic hypermutation. The unwarranted genomic changes resulting from these processes can lead to B-cell malignancies with varied clinical and pathological characteristics. Of the lymphoma subtypes derived from mature B-cells, a subset of diffuse large B-cell lymphoma (DLBCL), primary mediastinal large cell lymphoma (PMBL), follicular lymphoma (FL) and Burkitt lymphoma (BL) are putatively derived from GC B-cells, with varied morphological, pathological and clinical characteristics. These tumor are characterized by distinct genetic abnormalities,[ t(14;18) in GCB-DLCBL, and FL or t(8;14)] in BL, and mutational profile. We postulate miRNA expression signatures, may also explain the wide-ranging clinico-pathological characteristics of these GC B- cell derived tumors.Methods:We performed a meta-analysis of miRNA profiles of DLBCL, BL, PBML, FL and EBV+DLBCL performed by us or others. (Iqbal 2015; 2012; Leich 2011; Andrade 2014). We also included lymphomas derived from naïve B-cells (MCL, SLL) and post GC B-cells (activated B cells like ABC-DLBCL), EBV (+) DLBCL, normal B-cell subsets and cell lines of distinct B-cell lineages. The majority of the cases have been characterized by gene expression profiling (GEP) with corresponding pathological clinical characteristics in above studies. The data was obtained using Taqman® human microRNA array (Applied Biosystems, CA) containing 380 miRNAs and analyzed using BRB-array Tools.ResultsIn our initial miRNA expression profile of 249 lymphomas including GCB-DLBCL (n=34), ABC-DLBCL(n= 29), PMBL(n= 9), BL(n= 33), FL (n= 32), EBV+DLBCL (n=8); MCL(n=30) and SLL(n=12)showed distinct hierarchical clusters enriched with GEP defined subgroup, with few interspersed cases from other subgroups suggesting that miRNAs can complement and delineate GEP defined molecular subgroups, an observation consistent with earlier findings (Iqbal 2015).When we examined miRNAs, that are specifically associated with GC B- derived lymphomas, we observed a subset of miRNA including miR-146a, miR-142-3p, miR-17, miR19b, and miR106a were present at higher levels (top 5% in abundance by CT values) in GC B lymphomas. Of these, somatic mutations in miR142 have been recently reported in GC derived lymphomas (Alyssa, Leukemia 2016). These miRNAs also represented the most abundant miRNAs in centroblast, suggesting lineage specificity of miRNA signature in these lymphomas. However, miR222 highly expressed in centroblast was not expressed at higher levels in GCB-DLBCL and BL. A number of miRNAs specifically abundant in distinct lymphomas included (BL: miR-20a, miR-16), (GCB-DLBCL: miR-223, miR-24) and (FL: miR222). MiR-150 was highly expressed in NHLs putatively derived from naive B-cells like MCL or SLL, but absent from GC-derived NHLs. Consistent with this, miR-150 was present in naive B-cells, but not in centroblast, suggesting that miR-150 may have discrete function during early B-cell differentiation and late maturation stages.Within GC derived lymphoma, we examined miRNA profiles in clinically aggressive (BL); indolent lymphomas (FL) and intermediate aggressive (GCB-DLBCL) and their association with normal B-cell subsets, and indeed a number of miRNAs (ex. miR-150) were associated with indolent lymphomas, whereas a subset of miRNAs was present in aggressive lymphomas (ex. miR-16), suggesting these miRNAs may contribute the distinct clinical characteristics. Though we have identified unique miRNA profiles in these GC derived NHLs, the target genes of these miRNAs remains to be identified.Conclusion:We identified microRNAs that were differentially expressed between GC-derived DLBCL and other lymphomas, which may help explain distinguishing features of these lymphomas. DisclosuresNo relevant conflicts of interest to declare.

Long Noncoding RNA Expression Profiling in Normal B-Cell Subsets and Hodgkin Lymphoma Reveals Hodgkin and Reed-Sternberg Cell–Specific Long Noncoding RNAs

Hodgkin lymphoma (HL) is a malignancy of germinal center (GC) B-cell origin. To explore the role of long noncoding RNAs (lncRNAs) in HL, we studied lncRNA expression patterns in normal B-cell subsets, HL cell lines, and tissues. Naive and memory B cells showed a highly similar lncRNA expression pattern, distinct from GC-B cells. Significant differential expression between HL and normal GC-B cells was observed for 475 lncRNA loci. For two validated lncRNAs, an enhanced expression was observed in HL, diffuse large B-cell lymphoma, and lymphoblastoid cell lines. For a third lncRNA, increased expression levels were observed in HL and part of Burkitt lymphoma cell lines. RNA fluorescence in situ hybridization on primary HL tissues revealed a tumor cell-specific expression pattern for all three lncRNAs. A potential cis-regulatory role was observed for 107 differentially expressed lncRNA-mRNA pairs localizing within a 60-kb region. Consistent with a cis-acting role, we showed a preferential nuclear localization for two selected candidates. Thus, we showed dynamic lncRNA expression changes during the transit of normal B cells through the GC reaction and widely deregulated lncRNA expression patterns in HL. Three lncRNAs showed a tumor cell-specific expression pattern in HL tissues and might therefore be of value as a biomarker.

DIFFERENTIAL EXPRESSION OF CD150/SLAMF1 IN NORMAL AND MALIGNANT B CELLS ON THE DIFFERENT STAGES OF MATURATION

Within B-cell lineage cell surface receptor CD150/SLAMF1 is broadly expressed starting from pre-B cells with upregulation toward plasma cells. However, expression of CD150 is rather limited on the surface of malignant B cells with the block of differentiation at the different stages of maturation. The aim of our work was to explore CD150 expression both on protein and mRNA levels with the emphasis on CD150 isoforms in malignant B-cell lines at the different stages of maturation in comparison with their normal B cell counterparts. Studies were performed on normal tonsillar B-cell subpopulations, B-lymphoblastoid cell lines, malignant B-cell lines of different origin, including pre-B acute lymphoblastic leukemia, Burkitt's lymphoma, Hodgkin's lymphoma, and multiple myeloma. Protein CD150 expression was assessed by western blot analysis and the expression level of CD150 isoforms was evaluated using qRT-PCR. Despite the similar CD150 expression both on mRNA and protein levels in normal B-cell subsets and B-lymphoblastoid cell lines, malignant B-cell lines demonstrated substantial heterogeneity in CD150 expression. Only Hodgkin's lymphoma cell lines, Burkitt's lymphoma cell lines BJAB and Raji, and also pre-B cell line BLIN-1 expressed CD150 protein. At the same time total CD150 and mCD150 mRNA was detected in all studied cell lines excluding pre-B cell line REH. The minor sCD150 isoform was found only in Hodgkin's lymphoma cell lines and Burkitt's lymphoma cell line Raji. The nCD150 isoform was broadly expressed in tested B cell lines with exception of REH and Daudi. Malignant B-cell lines at the different stages of maturation only partially resemble their normal counterparts by CD150 expression. In malignant B-cell lines, CD150 expression on mRNA level is much broader than on protein level. CD150 isoforms are differentially expressed in normal and malignant B cells with predominant expression of mCD150 isoform.

WNT5A: a motility-promoting factor in Hodgkin lymphoma.

Classical Hodgkin lymphoma (cHL) has a typical clinical manifestation, with dissemination involving functionally neighboring lymph nodes. The factors involved in the spread of lymphoma cells are poorly understood. Here we show that cHL cell lines migrate with higher rates compared with non-Hodgkin lymphoma cell lines. cHL cell migration, invasion and adhesion depend on autocrine WNT signaling as revealed by the inhibition of WNT secretion with the porcupine inhibitors Wnt-C59/IWP-2, but did not affect cell proliferation. While application of recombinant WNT5A or WNT5A overexpression stimulates HL cell migration, neither WNT10A, WNT10B nor WNT16 did so. Time-lapse studies revealed an amoeboid type of cell migration modulated by WNT5A. Reduced migration distances and velocity of cHL cells, as well as altered movement patterns, were observed using porcupine inhibitor or WNT5A antagonist. Knockdown of Frizzled5 and Dishevelled3 disrupted the WNT5A-mediated RHOA activation and cell migration. Overexpression of DVL3-K435M or inhibition of ROCK (Rho-associated protein kinase) by Y-27632/H1152P disrupted cHL cell migration. In addition to these mechanistic insights into the role of WNT5A in vitro, global gene expression data revealed an increased WNT5A expression in primary HL cells in comparison with normal B-cell subsets and other lymphomas. Furthermore, the activity of both porcupine and WNT5A in cHL cells had an impact on lymphoma development in the chick chorionallantoic membrane assay. Massive bleeding of these lymphomas was significantly reduced after inhibition of WNT secretion by Wnt-C59. Therefore, a model is proposed where WNT signaling has an important role in regulating tumor-promoting processes.

The Chemokine Receptor Profile As Distinctive Criterion Between Normal B-Cell Subsets and As Potential Discriminative Marker to Identify the Cell of Origin in Patients with Chronic Lymphocytic Leukemia and Richter Syndrome

Chemokine receptors are G-protein-coupled cell surface receptors, which dissociate upon activation by their ligands and cause downstream signaling. Several studies have revealed the crucial contribution of chemokine receptors and their ligands in normal B-cell differentiation and development of hematopoietic malignancies. The Richter syndrome (RS) represents the clinico-pathologic transformation of chronic lymphocytic leukaemia (CLL) to an aggressive lymphoma, most commonly diffuse large B-cell lymphoma (DLBCL). Due to the lack of knowledge on the chemokine receptor, we aimed to investigate their expression profile in patients with CLL and Richter syndrome. Therefore, we investigated the mRNA expression levels of 18 known chemokine receptors (CCR1-CCR9, CXCR1-CXCR7, XCR1, CX3CR1) by using semi-quantitative real-time PCR on seven samples of paired (CLL and transformed DLBCL) RS samples, additionally four CLL samples -all of them subsequently transformed into DLBCL-, and eight transformed DLBCL samples originating from CLL. Additionally, 30 samples of de-novo DLBCL, including 10 germinal center B-cell (GCB) lymphomas, 12 non-germinal center B-cell lymphomas (non-GCB), and 8 unclassified DLBCL were included. Four samples of naïve B-cells (CD5 neg), CD5+ naïve B-cells and CD27+ memory B-cells (n=12) served as non-neoplastic controls.No differences in the chemokine receptor profile were detected between CD5+ and negative naïve B-cells. When comparing CD27+ memory B-cells to naïve B-cells a significant lower expression level was found for CCR7 (7-fold), CXCR4 (4-fold), and CXCR5 (1.5 fold). CCR7 (5-fold) and CXCR4 (5-fold) were also lower expressed in CD27+ memory B-cells compared to CD5+ naïve B-cells.Five out of 18 chemokine receptors were differentially expressed comparing the distinct normal B-cell subsets with RS samples. Comparing CLL samples and RS samples to CD5+ naïve B-cells, CXCR4 (12-fold for CLLs and 10-fold for RS samples) and CXCR5 (2-fold for CLLs and 2.4-fold for RS samples) were lower expressed, whereas CXCR3 (10-fold for CLLs and 8.5-fold for the transformed samples) was higher expressed and CCR5 de-novo expressed. Compared to naïve B-cells, the same chemokine receptors were deregulated: CXCR4 (10-fold for CLLs and 8.5-fold for the RS samples) and CXCR5 (2-fold for CLLs and 2.4-fold for the transformed samples) were lower expressed, CXCR3 (45-fold for CLLs and 30-fold for the transformed samples) was higher expressed and CCR5 was de-novo expressed. Comparing CLL samples and transformed RS samples to CD27+ memory B-cells, CCR5 (5.1-fold for CLLs and 4.3-fold for the RS samples) and CCR7 (8.7-fold for CLLs and 10-fold for the transformed samples) were higher expressed in both malignancies.Only one chemokine receptor was found to be differentially expressed in our seven paired RS samples: CCR6 showed a trend of a higher expression (1.4-fold) in CLL components.Considering RS and GCB DLBCL, CCR1, CCR5, and CXCR6 were found to be significantly down-regulated in RS (at least 4-fold), in contrast to CCR7 and CXCR4, which showed higher expression levels in RS (6-fold). CCR1 and CCR5 were lower expressed comparing RS and non-GCB DLBCL (25-fold and 8-fold), whereas CCR7 again, together with CXCR7, was higher expressed (3- fold and 6-fold respectively).Our data indicate a difference in the chemokine receptor profile within normal B-cell subsets. These differences are also reflected in the different expression profile of low and high aggressive component of CLL/RS compared to the distinct B cell subtypes. Hence, in future these multiple deregulated CC and CXC receptors might serve as a further hint in identifying the cell of origin of different B-cell malignancies. DisclosuresNo relevant conflicts of interest to declare.

Subtyping of B-Cell Malignancies By B-Cell Subset Associated Gene Signatures (BAGS), Generated from Human Primary and Secondary Lymphoid Organs.

BackgroundToday's diagnostic tests for B-cell malignancies reflect the criteria of the updated WHO classification based on cellular biomarkers and clinicopathologic heterogeneity. To that end, we proposed a new biological subtyping of malignancies by B-cell subset associated gene signatures (BAGS), generated from the normal B-cell hierarchy in primary and secondary lymphoid organs. Our concept is that BAGS can be used for identification of the "cell of origin" subtyping in selected B-cell malignancies (1), as recently shown for diffuse large B-cell lymphoma (DLBCL) based on B-cell sorting of normal tonsil tissue (2), and currently investigated in Multiple myeloma (MM).Material and MethodsWe used multiparametric flow cytometry (MFC) phenotyping by a multicolor single tube Euro flow standard approach (CD3/CD10/CD19/CD20/CD27/CD34/CD38/CD45) to identify and sort distinct B-cell subset of 20,000-50,000 cells from normal sternal bone marrow for microarray analysis. Isolated mRNA from sorted subsets of PreB-I, PreB-II, immature (I), naïve (N), memory (M) and plasma cell (PC) was amplified and hybridized to Affymetrix Gene Chip Human Exon 1.0 ST array to generate gene expression profiles (GEP).Construction of the bone marrrow specific BAGS was based on median-centered probe sets from the GEP data using regularized multinomial regression with 5-6 discrete outcomes representing B-cell subtypes using the software R.The classification system was validated using online available GEP data from tumor biopsy samples of newly diagnosed MM patients. Each patient tumor GEP data underwent BAGS assignment according to the highest predicted probability score above 0.15 or was otherwise unclassified. To compensate for cohort-wise technical batch effects, each cohort was median centered and adjusted probe set-wise to have same variance as the normal bone marrow specific data.ResultsIn bone marrow from sternum splits, the CD45+ leukocyte population accounted for a median of 55% (range 39-63%) of total number of mononuclear cells aspirated and the B-cell compartment was enumerated as the CD45+/CD19+/CD3- compartment of median 14% (3-27%). Among these 0.7% (0.2-2.4%) were PreBI, and 28% (6-38%) were PreBII subsets. A median of 4% (2-6%) were I cells and 29% (16%-39%) were N cells. The post-germinal cells comprised a median of 5 % (2-10%) M subsets and 19 % (13-30%) PC subsets.Following FACS and GEP of these minor subsets we validated the quality of each subset by expression of the used CD genes and expected transcription factors (TF) as illustrated in Figure 1A-D and published for DLBCL (2).The resultant tumor assignments exhibited BAGS subtyping frequencies of patient cases in available cohorts of MM (N=1,126) as shown in Table 1 and in DLBCL (N= 1,139) as published (2). The percentage of patients in each subtype was not significantly different between data set of MM, as in DLBCL (2)ConclusionWe have validated datasets of distinct normal B-cell subsets and generated bone marrow and tonsil specific BAGS to be used for subtyping of multiple myeloma (2) and diffuse large B cell lymphomas (3), with impact on staging and prognosis.

Multiple Myeloma Subtyping That Associates Normal B-Cell Subset Phenotypes, do Correlate to Disease Stage and Prognosis - Indication of Reversible Phenotypic Plasticity as a Hallmark

e48 p1⁄40.0005). To further validate this signature, we generated 500 randomly computed re-sampling of the data sets. The three-miRNA signature was consistently significant with a p-value < 0.05 in more than 78% and < 0.10 in 86% of the 500 randomizations. In a multivariable Cox regression model, the circulating exosomal miRNA signature was an independent prognostic marker after adjusting for cytogenetics and ISS. In a receiver operating characteristic (ROC) analysis, a combination of this signature together with International Staging System (ISS) and cytogenetics had a better prognostic value than ISS and cytogenetics alone in the training set (2 years area under the ROC curve 0.64 [95% CI 0.560.72] vs. 0.60 [95% CI 0.52-0.69]) and the validation set (0.67 [0.59-0.75] vs. 0.58 [0.50-0.66]). Interpretation: This study demonstrates unprecedented evidence of the prognostic significance of exosomal miRNAs in patients with MM. We identified a threemiRNA signature in circulating exosomes that adds prognostic value to ISS and cytogenetic status and helps improve prognostic identification of newly diagnosed MM patients.

Global microRNA expression profiling uncovers molecular markers for classification and prognosis in aggressive B-cell lymphoma

AbstractWe studied the global microRNA (miRNA) expression in diffuse large B-cell lymphoma (DLBCL; n = 79), Burkitt lymphoma (BL; n = 36), primary mediastinal B-cell lymphoma (PMBL; n = 12), B-cell lines (n = 11), and normal subsets of naïve B cells, centroblasts (CBs), and peripheral blood B cells along with their corresponding gene expression profiles (GEPs). The normal B-cell subsets have well-defined miRNA signatures. The CB miRNA signature was significantly associated with germinal center B-cell (GCB)–DLBCL compared with activated B-cell (ABC)–DLBCL (P = .002). We identified a 27-miRNA signature that included v-myc avian myelomatosis viral oncogene homolog (MYC) targets and enabled the differentiation of BL from DLBCL, a distinction comparable with the “gold standard” GEP-defined diagnosis. Distinct miRNA signatures were identified for DLBCL subgroups, including GCB-DLBCL, activated B-cell (ABC)-DLBCL, and PMBL. Interestingly, most of the unclassifiable-DLBCL by GEP showed a strong similarity to the ABC-DLBCL by miRNA expression profiling. Consistent results for BL and DLBCL subgroup classification were observed in formalin-fixed, paraffin-embedded tissue, making such tests practical for clinical use. We also identified predictive miRNA biomarker signatures in DLBCL, including high expression of miR-155, which is significantly associated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) treatment failure. This finding was further supported by the observation that high expression of miR-155 sensitizes cells to v-akt murine thymoma viral oncogene homolog-1 inhibitors in vitro, suggesting a novel treatment option for resistant DLBCL.

IFI16 Expression Is Related to Selected Transcription Factors during B-Cell Differentiation.

The interferon-inducible DNA sensor IFI16 is involved in the modulation of cellular survival, proliferation, and differentiation. In the hematopoietic system, IFI16 is consistently expressed in the CD34+ stem cells and in peripheral blood lymphocytes; however, little is known regarding its regulation during maturation of B- and T-cells. We explored the role of IFI16 in normal B-cell subsets by analysing its expression and relationship with the major transcription factors involved in germinal center (GC) development and plasma-cell (PC) maturation. IFI16 mRNA was differentially expressed in B-cell subsets with significant decrease in IFI16 mRNA in GC and PCs with respect to naïve and memory subsets. IFI16 mRNA expression is inversely correlated with a few master regulators of B-cell differentiation such as BCL6, XBP1, POU2AF1, and BLIMP1. In contrast, IFI16 expression positively correlated with STAT3, REL, SPIB, RELA, RELB, IRF4, STAT5B, and STAT5A. ARACNE algorithm indicated a direct regulation of IFI16 by BCL6, STAT5B, and RELB, whereas the relationship between IFI16 and the other factors is modulated by intermediate factors. In addition, analysis of the CD40 signaling pathway showed that IFI16 gene expression directly correlated with NF-κB activation, indicating that IFI16 could be considered an upstream modulator of NF-κB in human B-cells.