Agnieszka Malecka

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-Hodgkins 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.

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-γ, 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.

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.

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. Copyright

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

BackgroundHigh-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.ResultsA 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.ConclusionsBayesPI2+ 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.

Immunoglobulin heavy and light chain gene features are correlated with primary cold agglutinin disease onset and activity

Immunoglobulin heavy chain ( IGH ) and light chain gene sequences of 27 patients with primary cold agglutinin disease (CAD) were studied to find features explaining the heterogeneity of clinical presentation and disease activity. CAD is a hemolytic anemia mediated by monoclonal IgM anti-I

Frequent somatic mutations of KMT2D (MLL2) and CARD11 genes in primary cold agglutinin disease

Additional Supporting Information may be found in the online version of this article: Fig S1. Survival data in 446* patients with primary myelofibrosis, stratified by absolute monocyte count and the Dynamic International Prognostic Scoring System (DIPSS)plus. Table SI. Multivariable analysis of overall survival in 291 patients with primary myelofibrosis with available information on absolute monocyte count, Dynamic International Prognostic Scoring System-plus risk status, driver mutational status and ASXL1/SRSF2 mutational status.

Monocytoid B cells: An Enigmatic B cell Subset Showing Evidence of Extrafollicular Immunoglobulin gene Somatic Hypermutation

Monocytoid B cells are IgM+, IgD−/+, CD27− B cells, localized in the perisinusoidal area of the lymph node. These cells are especially prominent in infections such as those caused by toxoplasma and HIV. The ontogeny of monocytoid B cells with respect to B cell maturation is incompletely known. We analysed clonal expansion, somatic hypermutation and expression of activation‐induced cytidine deaminase (AID) in monocytoid B cells. Sequence analysis of the rearranged immunoglobulin heavy chain genes amplified from microdissected monocytoid B cell zones with a high proportion of proliferating cells reveals the presence of multiple clones with low‐level ongoing mutations (mean frequency: 0.46 × 10−2 per bp). Mutation analysis of these ongoing mutations reveals strand bias, a preference of transitions over transversions as well as the occurrence of small deletions, as observed for somatically mutated immunoglobulin genes in the human germinal centre. Proliferation, ongoing mutation as well as expression of AID, combined, is evidence that monocytoid B cells acquire the mutations in the extrafollicular perisinusoidal area of the lymph node and pleads against a postgerminal centre B cell origin.

Multiple distinct T-cell clones in folliculotropic mycosis fungoides.

Abstract: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 &bgr; and &ggr; 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.

Primary diffuse large B-cell lymphoma associated with clonally-related monoclonal B lymphocytosis indicates a common precursor cell.

Bone marrow monoclonal small B-cell infiltration (MSBC) associated with monoclonal B lymphocytosis (MBL), is present at a higher frequency in patients with diffuse large B-cell lymphoma (DLBCL). We have prospectively collected blood and bone marrow (BM) samples in patients with primary DLBCL at