Antonis Dagklis

The immunoglobulin gene repertoire of low-count chronic lymphocytic leukemia (CLL)-like monoclonal B lymphocytosis is different from CLL: diagnostic implications for clinical monitoring.

In the revised National Cancer Institute Working Group (NCI-WG)/International Workshop on Chronic Lymphocytic Leukemia (IWCLL) guidelines for CLL, CLL-like monoclonal B lymphocytosis (MBL) is defined as the presence of less than 5 x 10(9)/L B lymphocytes in the peripheral blood. However, the concentration of MBL in the blood is extremely variable. MBL in subjects with lymphocytosis require treatment at a rate of 1.1% per year and present immunoglobulin (IG) gene features and similar to good prognosis CLL. Little is known about low-count MBL cases, accidentally found in the general population. We analyzed IGHV-D-J rearrangements in 51 CLL-like MBL cases from healthy individuals, characterized by few clonal B cells. Seventy percent of the IGHV genes were mutated. The most frequent IGHV gene was IGHV4-59/61, rarely used in CLL, whereas the IGHV1-69 gene was lacking and the IGHV4-34 gene was infrequent. Only 2 of 51 (3.9%) MBL cases expressed a CLL-specific stereotyped HCDR3. Therefore, the IG gene repertoire in low-count MBL differs from both mutated and unmutated CLL, suggesting that the detection of MBL in an otherwise healthy subject is not always equivalent to a preleukemic state. Detailed IG analysis of individual MBL may help to identify cases that necessitate continuous clinical monitoring to anticipate disease progression.

General population low-count CLL-like MBL persists over time without clinical progression, although carrying the same cytogenetic abnormalities of CLL

Monoclonal B-cell lymphocytosis (MBL) is classified as chronic lymphocytic leukemia (CLL)-like, atypical CLL, and CD5(-) MBL. The number of B cells per microliter divides CLL-like MBL into MBL associated with lymphocytosis (usually detected in a clinical setting) and low-count MBL detected in the general population (usually identified during population screening). After a median follow-up of 34 months we reevaluated 76 low-count MBLs with 5-color flow cytometry: 90% of CLL-like MBL but only 44.4% atypical CLL and 66.7% CD5(-) MBL persisted over time. Population-screening CLL-like MBL had no relevant cell count change, and none developed an overt leukemia. In 50% of the cases FISH showed CLL-related chromosomal abnormalities, including monoallelic or biallelic 13q deletions (43.8%), trisomy 12 (1 case), and 17p deletions (2 cases). The analysis of the T-cell receptor β (TRBV) chains repertoire showed the presence of monoclonal T-cell clones, especially among CD4(high)CD8(low), CD8(high)CD4(low) T cells. TRBV2 and TRBV8 were the most frequently expressed genes. This study indicates that (1) the risk of progression into CLL for low-count population-screening CLL-like MBL is exceedingly rare and definitely lower than that of clinical MBL and (2) chromosomal abnormalities occur early in the natural history and are possibly associated with the appearance of the typical phenotype.

Over 30% of patients with splenic marginal zone lymphoma express the same immunoglobulin heavy variable gene: ontogenetic implications

We performed an immunogenetic analysis of 345 IGHV-IGHD-IGHJ rearrangements from 337 cases with primary splenic small B-cell lymphomas of marginal-zone origin. Three immunoglobulin (IG) heavy variable (IGHV) genes accounted for 45.8% of the cases (IGHV1-2, 24.9%; IGHV4-34, 12.8%; IGHV3-23, 8.1%). Particularly for the IGHV1-2 gene, strong biases were evident regarding utilization of different alleles, with 79/86 rearrangements (92%) using allele (*)04. Among cases more stringently classified as splenic marginal-zone lymphoma (SMZL) thanks to the availability of splenic histopathological specimens, the frequency of IGHV1-2(*)04 peaked at 31%. The IGHV1-2(*)04 rearrangements carried significantly longer complementarity-determining region-3 (CDR3) than all other cases and showed biased IGHD gene usage, leading to CDR3s with common motifs. The great majority of analyzed rearrangements (299/345, 86.7%) carried IGHV genes with some impact of somatic hypermutation, from minimal to pronounced. Noticeably, 75/79 (95%) IGHV1-2(*)04 rearrangements were mutated; however, they mostly (56/75 cases; 74.6%) carried few mutations (97-99.9% germline identity) of conservative nature and restricted distribution. These distinctive features of the IG receptors indicate selection by (super)antigenic element(s) in the pathogenesis of SMZL. Furthermore, they raise the possibility that certain SMZL subtypes could derive from progenitor populations adapted to particular antigenic challenges through selection of VH domain specificities, in particular the IGHV1-2(*)04 allele.

Immunogenetics shows that not all MBL are equal: the larger the clone, the more similar to CLL.

Chronic lymphocytic leukemia (CLL) -like monoclonal B-cell lymphocytosis (MBL) shares common immunophenotype and cytogenetic abnormalities with CLL, from which it is discriminated by a cutoff value of 5 × 10(9)/L circulating clonal B cells. However, the clonal size in MBL is extremely variable and allows discrimination of two distinct entities (high-count [HC] and low-count [LC]-MBL) based on a cutoff value of 0.5 × 10(9)/L clonal B cells. HC-MBL is associated with lymphocytosis and progresses to CLL requiring treatment at a rate of 1.1% per year, whereas LC-MBL is found in the general population only through high-sensitivity techniques and carries limited, if any, risk of progression. We performed an immunogenetic profiling of 333 cases with CLL-like MBL supplemented by detailed comparisons with CLL, focusing especially on CLL Rai stage 0 (CLL-0). LC- and HC-MBL had similar somatic hypermutation status, yet different IGHV gene repertoires and frequencies of B-cell receptor (BcR) stereotypy. In particular, stereotyped BcRs were infrequent in LC-MBL and were often not CLL specific. In contrast, HC-MBL exhibited clear immunogenetic similarities to CLL-0. These findings indicate that LC-MBL may not represent a true preleukemic condition, thus differing from HC-MBL/CLL-0 in which the identification of factors endowing malignant potential is strongly warranted.

HS1 has a central role in the trafficking and homing of leukemic B cells

The function of the intracellular protein hematopoietic cell-specific Lyn substrate-1 (HS1) in B lymphocytes is poorly defined. To investigate its role in migration, trafficking, and homing of leukemic B lymphocytes we have used B cells from HS1(-/-) mice, the HS1-silenced human chronic lymphocytic leukemia (CLL) MEC1 cell line and primary leukemic B cells from patients with CLL. We have used both in vitro and in vivo models and found that the lack of expression of HS1 causes several important functional effects. In vitro, we observed an impaired cytoskeletal remodeling that resulted in diminished cell migration, abnormal cell adhesion, and increased homotypic aggregation. In vivo, immunodeficient Rag2(-/-)γ(c)(-/-) mice injected with HS1-silenced CLL B cells showed a decreased organ infiltration with the notable exception of the bone marrow (BM). The leukemic-prone Eμ-TCL1 transgenic mice crossed with HS1-deficient mice were compared with Eμ-TCL1 mice and showed an earlier disease onset and a reduced survival. These findings show that HS1 is a central regulator of cytoskeleton remodeling that controls lymphocyte trafficking and homing and significantly influences the tissue invasion and infiltration in CLL.

Monoclonal B Cell Lymphocytosis in Hepatitis C Virus Infected Individuals

Monoclonal B cell lymphocytosis (MBL) is a preclinical condition characterized by an expansion of clonal B cells in the absence of B lymphocytosis (BALC < 5 × 109/L) in the peripheral blood, without clinical signs, suggestive of a lymphoproliferative disorder. B cell clonal expansions are also associated with hepatitis C virus (HCV) infection and they can evolve into lymphoproliferative disorders such as mixed cryoglobulinemia and non‐Hodgkin lymphomas (NHL). The relationship between MBL and HCV infection has not been established yet.

Monoclonal B-cell lymphocytosis in healthy blood donors: an unexpectedly common finding

Circulating monoclonal B cells may be detected in healthy adults, a condition called monoclonal B-cell lymphocytosis (MBL). MBL has also been identified in donated blood, but no systematic study of blood donors has been reported. Using sensitive and specific laboratory methods, we detected MBL in 149 (7.1%; 95% confidence interval, 6.0% to 8.3%) of 2098 unique donors ages 45 years or older in a Midwestern US regional blood center between 2010 and 2011. Most of the 149 donors had low-count MBL, including 99 chronic lymphocytic leukemia-like (66.4%), 22 atypical (14.8%), and 19 CD5(-) (12.8%) immunophenotypes. However, 5 donors (3.4%) had B-cell clonal counts above 500 cells per µL, including 3 with 1693 to 2887 cells per µL; the clone accounted for nearly all their circulating B cells. Four donors (2.7%) had 2 distinct MBL clones. Of 51 MBL samples in which immunoglobulin heavy chain (IGH)V-D-J genotypes could be determined, 71% and 29% used IGHV3- and IGHV4-family genes, respectively. Sequencing revealed 82% with somatic hypermutation, whereas 18% had >98% germ-line identity, including 5 with entirely germ-line sequences. In conclusion, MBL prevalence is much higher in blood donors than previously reported, and although uncommon, the presence of high-count MBL warrants further investigations to define the biological fate of the transfused cells in recipients.

Immunoglobulin gene repertoire in ocular adnexal lymphomas: hints on the nature of the antigenic stimulation

Evidence from certain geographical areas links lymphomas of the ocular adnexa marginal zone B-cell lymphomas (OAMZL) with Chlamydophila psittaci (Cp) infection, suggesting that lymphoma development is dependent upon chronic stimulation by persistent infections. Notwithstanding that, the actual immunopathogenetical mechanisms have not yet been elucidated. As in other B-cell lymphomas, insight into this issue, especially with regard to potential selecting ligands, could be provided by analysis of the immunoglobulin (IG) receptors of the malignant clones. To this end, we studied the molecular features of IGs in 44 patients with OAMZL (40% Cp-positive), identifying features suggestive of a pathogenic mechanism of autoreactivity. Herein, we show that lymphoma cells express a distinctive IG repertoire, with electropositive antigen (Ag)-binding sites, reminiscent of autoantibodies (auto-Abs) recognizing DNA. Additionally, five (11%) cases of OAMZL expressed IGs homologous with autoreactive Abs or IGs of patients with chronic lymphocytic leukemia, a disease known for the expression of autoreactive IGs by neoplastic cells. In contrast, no similarity with known anti-Chlamydophila Abs was found. Taken together, these results strongly indicate that OAMZL may originate from B cells selected for their capability to bind Ags and, in particular, auto-Ags. In OAMZL associated with Cp infection, the pathogen likely acts indirectly on the malignant B cells, promoting the development of an inflammatory milieu, where auto-Ags could be exposed and presented, driving proliferation and expansion of self-reactive B cells.

Bone marrow histopathology in the diagnostic evaluation of splenic marginal-zone and splenic diffuse red pulp small B-cell lymphoma: a reliable substitute for spleen histopathology?

Primary splenic small B-cell lymphomas mostly comprise the distinct entity of splenic marginal-zone lymphoma (SMZL) and the provisional category of splenic lymphoma/leukemia unclassifiable, mainly represented by the hairy cell leukemia variant and splenic diffuse red pulp small B-cell lymphoma (SDRL). Until recently, histopathologic examination of splenectomy specimens was considered mandatory for the diagnosis of SMZL. However, nowadays, mainly because of advances in chemoimmunotherapy, splenectomy is performed much less frequently. We evaluated the diagnostic efficacy of bone marrow biopsy (BMB) histopathology in the diagnostic approach toward SMZL and SDRL and tested whether it may serve as a substitute for spleen histopathology in the differential diagnosis between these 2 entities. To this end, we conducted a paired assessment of BMB and spleen diagnostic samples from 46 cases with a diagnosis of SMZL (n=32) or SDRL (n=14) based on spleen histopathology. We demonstrate that detailed immunohistopathologic BMB evaluation offers adequate evidence for the confirmation of these entities and their differential diagnosis from other small B-cell lymphoma histotypes. Notably, the immunophenotypical profile of SMZL and SDRL was identical in both BMB and spleen specimens for 21 evaluated markers. Paired assessment of BMB and spleen specimens did not identify discriminating patterns of BMB infiltration, cytology, and/or immunohistology between SMZL and SDRL. Accordingly, bone marrow histopathology contributes significantly in confirming the diagnosis of SMZL and SDRL. However, presently it is not possible to distinguish SMZL from SDRL on the basis of BMB evaluation alone; hence, histopathologic examination of the spleen remains the “gold standard” approach.

KAP1 regulates gene networks controlling mouse B lymphoid cell differentiation and function

Chromatin remodeling is fundamental for B-cell differentiation. In the present study, we explored the role of KAP1, the cofactor of KRAB-ZFP transcriptional repressors, in this process. B-lymphoid-specific Kap1-KO mice displayed reduced numbers of mature B cells, lower steady-state levels of Abs, and accelerated rates of decay of neutralizing Abs after viral immunization. Transcriptome analyses of Kap1-deleted B splenocytes revealed an up-regulation of PTEN, the enzymatic counteractor of PIK3 signaling, and of genes encoding DNA-damage response factors, cell-cycle regulators, and chemokine receptors. ChIP/seq studies established that KAP1 bound at or close to several of these genes and controlled chromatin status at their promoters. Genome wide, KAP1 binding sites lacked active B cell-specific enhancers and were enriched in repressive histone marks, further supporting a role for this molecule in gene silencing in vivo. Likely responsible for tethering KAP1 to at least some of these targets, a discrete subset of KRAB-ZFPs is enriched in B lymphocytes. Our results therefore reveal the role of KRAB/KAP1-mediated epigenetic regulation in B-cell development and homeostasis.