Gene expression analysis revealed downregulation of complement receptor 1 in clonal B cells in cold agglutinin disease.

Cold agglutinin disease (CAD) is a rare B-cell lymphoproliferative disorder of the bone marrow, manifested by autoimmune hemolytic anemia caused by binding of monoclonal IgM autoantibodies to the I antigen. Underlying genetic changes have previously been reported, but their impact on gene expression profile has been unknown. Here, we define differentially expressed genes in CAD B cells. To unravel downstream alteration in cellular pathways, gene expression by RNA sequencing was undertaken. Clonal B-cell samples from 12 CAD patients and IgM-expressing memory B cells from 4 healthy individuals were analyzed. Differential expression analysis and filtering resulted in 93 genes with significant differential expression. Top upregulated genes included SLC4A1, SPTA1, YBX3, TESC, HBD, AHSP, TRAF1, HBA2, RHAG, CA1, SPTB, IL10, UBASH3B, ALAS2, HBA1, CRYM, RGCC, KANK2, and IGHV4-34. They were upregulated at least 8-fold, while complement receptor 1 (CR1/CD35) was downregulated 11-fold in clonal CAD B cells compared to control B cells. Flow cytometry analyses further confirmed reduced CR1 (CD35) protein expression by clonal CAD IgM+ B cells compared to IgM+ memory B cells in controls. CR1 (CD35) is an important negative regulator of B-cell activation and differentiation. Therefore, reduced CR1 (CD35) expression may increase activation, proliferation, and antibody production in CAD-associated clonal B cells.

Cold agglutinin disease revisited: a multinational, observational study of 232 patients.

We retrospectively studied 232 patients with cold agglutinin disease (CAD) at 24 centers in 5 countries. In Norway and a northern region of Italy, the study was close to being population-based. For the first time, we demonstrate fourfold differences between cold and warmer climates regarding prevalence (20 vs 5 cases/million) and incidence (1.9 vs 0.48 cases/million per year). Mean baseline hemoglobin level was 9.3 g/dL, but 27% had hemoglobin <8 g/dL. Identification of typical features of CAD-associated lymphoproliferative disorder in the bone marrow was greatly increased by centralized biopsy assessment. CAD seems to be associated with a slightly increased risk of venous thrombosis. This work includes a follow-up study of therapies, focusing on the long-term outcomes of the rituximab plus bendamustine and rituximab plus fludarabine regimens. Rituximab plus bendamustine therapy resulted in responses in 35 (78%) of 45 patients; 24 (53%) achieved complete response. Interestingly, these rates were still higher than observed in the original (2017) prospective trial, and we also found a shift toward deeper responses with time. This is explained by the prolonged time to response seen in many patients, probably related to long-lived plasma cells. In patients responding to rituximab-bendamustine, median response duration was not reached after 88 months, and estimated 5-year sustained remission was 77%. The regimen appeared safe regarding late-occurring malignancies. Rituximab plus fludarabine therapy seems to carry a higher risk of long-term adverse effects.

Cold agglutinin disease: where do we stand, and where are we going?

Primary cold agglutinin disease (CAD) is characterized by a very indolent bone marrow clonal B-cell lymphoproliferative disorder that initiates an autoimmune hemolytic anemia. The clonal B cells produce a monoclonal autoantibody termed cold agglutinin, most often of the immunoglobulin (Ig) Mκ class. After binding to its antigen, the IgM initiates a complement classical pathway-driven erythrocyte destruction, predominantly mediated by opsonization with complement protein C3b and extravascular hemolysis in the liver. We review the molecular biology, histopathology, clinical features, and diagnostic procedures in CAD. Some patients are only slightly anemic and do not require treatment, but moderate or severe anemia frequently occurs, and the disease burden has been underestimated. CAD should not be treated with corticosteroids. Several B-cell-directed treatment options are available, and complement-directed approaches are being rapidly developed. Current and possible future therapies are reviewed.

Bendamustine plus rituximab for chronic cold agglutinin disease: results of a Nordic prospective multicenter trial.

Primary chronic cold agglutinin disease (CAD) is a well-defined clinicopathologic entity in which a bone marrow clonal B-cell lymphoproliferation results in autoimmune hemolytic anemia and cold-induced circulatory symptoms. Rituximab monotherapy and fludarabine-rituximab in combination are documented treatment options. In a prospective, nonrandomized multicenter trial, 45 eligible patients received rituximab 375 mg/m2 day 1 and bendamustine 90 mg/m2 days 1 and 2 for 4 cycles at a 28-day interval. Thirty-two patients (71%) responded; 18 (40%) achieved complete response (CR) and 14 (31%) partial response (PR). Among 14 patients previously treated with rituximab or fludarabine-rituximab, 7 (50%) responded to bendamustine-rituximab (3 CR and 4 PR). Hemoglobin levels increased by a median of 4.4 g/dL in the complete responders, 3.9 g/dL in those achieving PR, and 3.7 g/dL in the whole cohort. The 10th percentile of response duration was not reached after 32 months. Grade 3-4 neutropenia occurred in 15 patients (33%), but only 5 (11%) experienced infection with or without neutropenia. Thirteen patients (29%) had their dose of bendamustine reduced. In conclusion, bendamustine-rituximab combination therapy is highly efficient, sufficiently safe, and may be considered in first line for patients with CAD requiring therapy. The trial was registered at www.clinicaltrials.gov as #NCT02689986.

Folliculotropic Mycosis Fungoides with Skewed T-cell Receptor CDR3 Motif: Suggestive of Lipid-antigen Selection?

Folliculotropic mycosis fungoides (FMF), a variant of mycosis fungoides (MF) with distinct clinical features, is characterized by infiltration of malignant T cells in hair follicles. This raises the hypothesis that antigens in the hair follicle may contribute to the pathogenesis of FMF. T-cell receptor ß gene (TRB) sequences as well as dendritic cell subsets in patients with FMF (n = 21) and control patients with MF (n = 20) were studied to explore this hypothesis. A recurrent usage of the TRB junctional genes TRBJ2-1 and TRBJ2-7 was found in patients with FMF compared with those with MF. These genes contribute to an amino acid motif in the complementarity-determining region 3 (CDR3) of the T-cell receptor. This motif was previously found in T cells stimulated by lipids bound to CD1 on antigen-presenting cells. Additional immunohistochemical analysis revealed abundant CD1c- and CD1a- expressing dendritic cells in FMF. The combined findings support a role for lipid-antigen stimulation in FMF.

Comprehensive genome-wide transcription factor analysis reveals that a combination of high affinity and low affinity DNA binding is needed for human gene regulation.

BACKGROUND: High-throughput in vivo protein-DNA interaction experiments are currently widely used in gene regulation studies. Hitherto, comprehensive data analysis remains a challenge and for that reason most computational methods only consider the top few hundred or thousand strongest protein binding sites whereas weak protein binding sites are completely ignored. RESULTS: A new biophysical model of protein-DNA interactions, BayesPI2+, was developed to address the above-mentioned challenges. BayesPI2+ can be run in either a serial computation model or a parallel ensemble learning framework. BayesPI2+ allowed us to analyze all binding sites of the transcription factors, including weak binding that cannot be analyzed by other models. It is evaluated in both synthetic and real in vivo protein-DNA binding experiments. Analysing ESR1 and SPIB in breast carcinoma and activated B cell-like diffuse large B-cell lymphoma cell lines, respectively, revealed that the concerted binding to high and low affinity sites correlates best with gene expression. CONCLUSIONS: BayesPI2+ allows us to analyze transcription factor binding on a larger scale than hitherto achieved. By this analysis, we were able to demonstrate that genes are regulated by concerted binding to high and low affinity binding sites. The program and output results are publicly available at: http://folk.uio.no/junbaiw/BayesPI2Plus.

Multiple distinct T-cell clones in folliculotropic mycosis fungoides.

Multiple distinct T-cell clones have been demonstrated in a subset of mycosis fungoides (MF), but have so far not been documented in folliculotropic MF, a clinical and histological variant of MF. We analyzed T-cell receptor (TCR) gene rearrangements in 8 patients with folliculotropic MF with multiple biopsies (range, 2-5) taken during the course of disease. Two patients had disease stage IA-IIA, 5 stage IIB-IVA, whereas data were not available for 1 patient. TCR ß and γ gene rearrangements were analyzed according to the BIOMED-2 PCR protocol. Multiple clonal TCR gene rearrangements indicating more than 1 T-cell clone were found in 5 patients. Although the number of patients is small, the finding of multiple distinct T-cell clones in 5 out of 8 patients suggests that chronic T-cell stimulation contributes to the development of folliculotropic MF.

The impact of individual clinical features in cold agglutinin disease: hemolytic versus non-hemolytic symptoms.

INTRODUCTION: During the last decades, the pathogenesis of cold agglutinin disease (CAD) has been well elucidated and shown to be complex. Several documented or investigational therapies have been made available. This development has resulted in major therapeutic advances, but also in challenges in choice of therapy. AREAS COVERED: In this review, we address each step in pathogenesis: bone marrow clonal lymphoproliferation, composition and effects of monoclonal cold agglutinin, non-complement mediated erythrocyte agglutination, complement-dependent hemolysis, and other effects of complement activation. We also discuss the heterogeneous clinical features and their relation to specific steps in pathogenesis, in particular with respect to the impact of complement involvement. CAD can be classified into three clinical phenotypes with consequences for established treatments as well as development of new therapies. Some promising future treatment approaches - beyond chemoimmunotherapy and complement inhibition - are reviewed. EXPERT OPINION: The patient's individual clinical profile regarding complement involvement and hemolytic versus non-hemolytic features is important for the choice of treatment. Further development of treatment approaches is encouraged, and some candidate drugs are promising irrespective of clinical phenotype. Patients with CAD requiring therapy should be considered for inclusion in clinical trials.

The Histopathology of Cold Agglutinin Disease-Associated B-Cell Lymphoproliferative Disease.

OBJECTIVES: Primary cold agglutinin disease is a type of autoimmune hemolytic anemia caused by circulating antibodies against I antigen, a carbohydrate expressed on most cells, including red blood cells. The underlying disease has been characterized in recent years as a distinct B-cell lymphoproliferative disease of the bone marrow, occurring mostly in the elderly. The disease has been now been included as a separate entity in the most recent classifications of mature B-cell neoplasms. METHODS: A review of the characteristics of cold agglutinin disease is provided, with an emphasis on the pathology features. RESULTS: A detailed description of the histopathology, immunophenotype, and genetics of cold agglutinin disease is provided and compared to other B-cell lymphoproliferative diseases in the bone marrow with similar features. CONCLUSIONS: Recognition of the pathology features of cold agglutinin disease allows to distinguish it from other diseases, especially lymphoplasmacytic lymphoma and marginal zone lymphoma.

T-cell/histiocyte-rich large B-cell lymphoma shows transcriptional features suggestive of a tolerogenic host immune response.

BACKGROUND: Gene expression profiling has successfully identified the prognostic significance of the host response in lymphomas. The aggressive T-cell/histiocyte-rich large B-cell lymphoma and the indolent nodular lymphocyte-predominant Hodgkin's lymphoma are both characterized by a paucity of tumor cells embedded in an overwhelming background. The tumor cells of both lymphomas share several characteristics, while the cellular composition of their microenvironment is clearly different. DESIGN AND METHODS: We collected 33 cases of T-cell/histiocyte-rich large B-cell lymphoma and 56 cases of nodular lymphocyte-predominant Hodgkin's lymphoma and performed microarray gene expression profiling on ten cases of each lymphoma, to obtain a better understanding of the lymphoma host response. By quantitative reverse transcriptase polymerase chain reaction we verified that these 20 selected cases were representative of the entire population of T-cell/histiocyte-rich large B-cell and nodular lymphocyte-predominant Hodgkin's lymphomas. RESULTS: We observed that the microenvironment in nodular lymphocyte-predominant Hodgkin's lymphoma is molecularly very similar to a lymph node characterized by follicular hyperplasia, while the microenvironment in T-cell/histiocyte-rich large B-cell lymphoma is clearly different. The T-cell/histiocyte-rich large B-cell lymphoma signature is hallmarked by up-regulation of CCL8, interferon-gamma, indoleamine 2,3 dioxygenase, VSIG4 and Toll-like receptors. These features may be responsible for the recruitment and activation of T cells, macrophages and dendritic cells, characterizing the stromal component of this lymphoma, and may point towards innate immunity and a tumor tolerogenic immune response in T-cell/histiocyte-rich large B-cell lymphoma. CONCLUSIONS: The gene expression profile of T-cell/histiocyte-rich large B-cell lymphoma, in comparison with that of nodular lymphocyte-predominant Hodgkin's lymphoma, shows features suggestive of a distinct tolerogenic host immune response that may play a key role in the aggressive behavior of this lymphoma, and that may serve as a potential target for future therapy.

Polish Translation and Validation of the Tinnitus Handicap Inventory and the Tinnitus Functional Index.

Objective: The need for validated measures enabling clinicians to classify tinnitus patients according to the severity of tinnitus and screen the progress of therapies in our country led us to translate into Polish and to validate two tinnitus questionnaires, namely the Tinnitus Handicap Inventory (THI) and the Tinnitus Functional Index (TFI). Design: The original English versions of the questionnaires were translated into Polish and translated back to English by three independent translators. These versions were then finalized by the authors into a Polish THI (THI-Pl) and a Polish TFI (TFI-Pl). Participants from three laryngological centers in Poland anonymously answered the THI-Pl (N = 98) and the TFI-Pl (N = 108) in addition to the Polish versions of the Center for Epidemiologic Studies Depression Scale as a measure of self-perceived level of depression, and the Satisfaction With Life Scale to assess self-perceived quality of life. Both were used to determine discriminant validity. Two Visual Analog Scales were used to measure tinnitus annoyance and tinnitus loudness in order to determine convergent validity. Results: Similar to the original version of the THI, the THI-Pl showed a high internal consistency (Cronbach's α = 0.93). The exploratory factor analysis revealed that the questionnaire has a three-factorial structure that does not correspond to the original division for functional, catastrophic, and emotional subscales. Convergent and discriminant validities were confirmed. The TFI-Pl showed high internal consistency (Cronbach's α = 0.96) with the reliability ranging from 0.82 to 0.95 for its different subscales. Factor analysis confirmed an eight-factorial structure with factors assigning all items to appropriate subscales reported in the original version of the questionnaire. Discriminant and convergent validities were also confirmed for the TFI-Pl. Conclusion: We translated and validated the Polish versions of the THI and the TFI to make them suitable for clinical use in Poland.

PU.1 protein expression has a positive linear association with protein expression of germinal centre B cell genes including BCL-6, CD10, CD20 and CD22: identification of PU.1 putative binding sites in the BCL-6 promotor.

The transcription factor PU.1 has been shown to be crucial for the early stages of B cell development but its function at later stages of B cell development is less well known. We observed previously that PU.1 is expressed uniformly throughout the mature pre-plasma cell B cell population, the only exception being a subpopulation of germinal centre (GC) cells which showed exceptionally high expression of PU.1. This suggested that PU.1 may also have a role in GC B cell biology. To test this hypothesis and to screen for possible genes regulated by PU.1, we first evaluated semi-quantitatively the possible co-expression of PU.1 with proteins known to be upregulated or downregulated during GC B cell development. Normal lymphoid tissues and 255 B cell non-Hodgkin lymphomas of putative GC B cell origin were evaluated. PU.1 expression was positively associated with CD10 (p < 0.0001), CD20 (p = 0.043), CD22 (p = 0.005), CD79a (p = 0.024) and Bcl-6 (p < 0.0001) and negatively associated with cytoplasmic immunoglobulin light-chain expression (p = 0.036) in diffuse large B cell lymphoma. Identical or nearly identical associations were found in follicular lymphoma. Since CD20 is known to be partly regulated by PU.1 and putative PU.1-binding sites have been described in the regulatory regions of the CD22, CD79a and CD10 genes, we looked for putative PU.1 binding sites in the BCL6 promotor. Four such putative PU.1 binding sites were identified. Further analysis by gel-shift electromobility essay showed that PU.1 protein binds to three of the four putative binding sites in the BCL6 promotor. PU.1 and Bcl-6 were also found to be upregulated in centroblasts in the normal GC, but jointly downregulated in a subpopulation of centrocytes. Our findings support the contention that PU.1 may also have an important role in GC B cell development.

Splenic marginal zone lymphoma with villous lymphocytes shows on-going immunoglobulin gene mutations.

Splenic marginal zone lymphoma (also splenic lymphoma with villous lymphocytes) is a B-cell non-Hodgkin's lymphoma with a characteristic morphology and phenotype. We studied the pattern of somatic hypermutation of the rearranged immunoglobulin heavy chain genes on 23 cases and have correlated these data with survival as well as immunophenotypic and genetic characteristics of the cases. Two-thirds of the cases show immunoglobulin gene mutations, half of which show evidence of antigen selection, whereas one-third of the cases show no significant mutations. On-going mutation, a feature characteristic of follicular lymphoma, was demonstrated in all six cases randomly selected for this analysis, including one case with a low number of mutations (<2%). No statistical significant correlation was found between immunoglobulin mutation status and clinical, immunophenotypic, or genetic characteristics. Our results demonstrate that on-going somatic hypermutation is a prominent feature of splenic marginal zone lymphoma with circulating villous lymphocytes. On-going somatic hypermutation has previously been demonstrated in extra-nodal and nodal marginal zone lymphoma. Our results indicate that marginal zone lymphomas at different anatomical localizations may derive from a similar B-cell subset.