Persistent Polyclonal B-cell Lymphocytosis Research Articles

Patients with Persistent Polyclonal B-Cell Lymphocytosis Share the Symptomatic Criteria of Systemic Exertion Intolerance Disease.

Introduction: Persistent polyclonal B-cell lymphocytosis (PPBL) is a rare and still poorly understood entity, with 90% of cases occurring in female smokers. Patients often appear tired and in pain, but the clinical symptoms remain imprecise. The main risk is the development of lymphoma in some cases. To better understand the characteristics of the fatigue associated with PPBL and study its relationship with systemic exertion intolerance disease (SEID), we analyzed the symptoms in a cohort of patients with PPBL included in the French national registry. Material and methods: An anonymous questionnaire following the recommendations of the Institute of Medicine/National Academy of Medicine for screening of the new SEID criteria was created in French and mailed to 50 patients. Results: Thirty-nine (78%) contacted patients responded. The studied population was mainly constituted of women (90%) with an average age of 50 (18–59) years. Smoking was a constant factor in all patients. A total of 28/39 (72%) respondents met the SEID symptoms criteria. Severe chronic fatigue for more than 6 months was noted in 36/39 cases (92%). Unrefreshing sleep, post-exertional malaise, cognitive impairment, and orthostatic intolerance were described in 30/39 (77%), 32/39 (82%), 28/39 (72%), and 27/39 (69%) cases, respectively. Pain (arthralgia, myalgia, headache) was present in 26/39 (67%) cases. The most prominent SEID symptoms were fatigue, followed by post-exercise discomfort and cognitive difficulties. The most disabling symptom was non-restorative sleep, followed by pain. An inflammatory and/or autoimmune context was noted in 13 patients (33%), and these comorbidities could have favored the deterioration of the general condition. Three patients also presented with fibromyalgia. However, 3 patients did not mention any complaints. Conclusion: This survey indicated that patients with PPBL most often initially presented with disabling chronic fatigue, chronic pain, and other symptoms suggestive of SEID but requiring more studies to confirm it. Education of medical staff about the symptoms of PPBL should be encouraged to better assess this peculiar condition.

Des lymphocytes dystrophiques

AbstractA case of persistent polyclonal B-cell lymphocytosis (PPBL) is reported. The difficulty of carrying out the initial blood count is highlighted, in particular for assessing the leucocyte differential. Supplementary tests and follow-up in such patient.

Abnormal characteristic “round bottom flask” shape volume-based scattergram as a trigger to suspect persistent polyclonal B-cell lymphocytosis

Background and aimsThe diagnosis of persistent polyclonal B-cell lymphocytosis (PPBL) is often challenging because of the lack of features and the overlap with the peripheral expression of splenic marginal zone lymphomas (SMZL). To obtain new clues for PPBL detection and diagnosis, all data provided by the DxH 800 analyzer (including scatter and cell population data (CPD)) was exploited and combined using a machine learning (ML) approach. Materials and methodsA total 211 samples from 101 normal controls and 110 patients (PPBL and SMZL) were assessed. Age, gender, full blood count, CPD, scatter, flags and CellaVision differentials were also considered. A ML model was built for true classification purposes. ResultsPPBL and SMZL shared increased absolute lymphoid counts, atypical lymphoid flag presence and CPD values (8 out of 14). A typical “round-bottom-flask” shape scattergram was described for the first time for PPBL which was also present in 51.4% of SMZL cases. The developed ML model render a global classification accuracy of 93.4%, allowing the detection of all pathological cases, with mean misclassification rates of 12% among PPBL and SMZL. ConclusionOur ML model represents a new unbiased tool than can be widely applied in the laboratory as an aid for detection of PPBL.

SDHA gain-of-function engages inflammatory mitochondrial retrograde signaling via KEAP1-Nrf2.

Whether screening the metabolic activity of immune cells facilitates discovery of molecular pathology remains unknown. Here we prospectively screened the extracellular acidification rate as a measure of glycolysis and the oxygen consumption rate as a measure of mitochondrial respiration in B cells from patients with primary antibody deficiency. The highest oxygen consumption rate values were detected in three study participants with persistent polyclonal B cell lymphocytosis (PPBL). Exome sequencing identified germline mutations in SDHA, which encodes succinate dehydrogenase subunit A, in all three patients with PPBL. SDHA gain-of-function led to an accumulation of fumarate in PPBL B cells, which engaged the KEAP1-Nrf2 system to drive the transcription of genes encoding inflammatory cytokines. In a single patient trial, blocking the activity of the cytokine interleukin-6 in vivo prevented systemic inflammation and ameliorated clinical disease. Overall, our study has identified pathological mitochondrial retrograde signaling as a disease modifier in primary antibody deficiency.

Persistent polyclonal B-cell lymphocytosis (PPBL). Case report

Persistent polyclonal B-cell lymphocytosis (PPBL). Case report

Whole Genome Sequencing and RNA Sequencing of 27 Patients with Persistent Polyclonal B-Cell Lymphocytosis Reveals a High Mutation Frequency/Overexpression of Lymphoma Associated Genes: Really a Benign Disorder?

Background: Persistent polyclonal B-cell lymphocytosis (PPBL) is a rare disorder, occurs almost exclusively in smoking women and is characterized by a chronic polyclonal lymphocytosis with circulating binucleated lymphocytes, clonal cytogenetic abnormalities involving chromosome 3, and chromosomal instability. Outcome of PPBL patients is mostly benign, but subsequent malignancies (non-Hodgkin´s lymphomas and solid tumors) were described. Potential molecular factors leading to their development are yet unclear.Aims: Detailed molecular genetic characterization of PPBL by whole genome sequencing (WGS) and RNA sequencing (RNAseq) in comparison to the well-characterized lymphoid malignancy CLL.Patient cohorts and methods: The total cohort comprised 27 PPBL (3 male, 24 female) and 250 CLL cases (163 male, 87 female). WGS was performed for all patients: 150bp paired-end reads where generated on Illumina HiseqX and NovaSeq 6000 machines (Illumina, San Diego, CA). A mixture genomic DNA from multiple anonymous donors was used as normal controls. To remove potential germline variants, each variant was queried against the gnomAD database, variants with global population frequencies >1% where excluded. Final analysis was performed only on protein-altering and splice-site variants. For further analysis, a virtual panel of 355 lymphoid genes was selected. All reported p-values are two-sided and were considered significant at p<0.05. For gene expression analysis, estimated gene counts were normalized applying Trimmed mean of M-values (TMM) normalization method and the resulting log2 counts per million (CPMs) were used as a proxy of gene expression in each sample. Genes were kept if they were expressed (> 5 CPM) in at least 66% of the samples. Genes with FDR (false discovery rate) < 0.05 and an absolute logFC > 1.5 were considered differentially expressed (DE).Results: Median age was 46 years for PPBL patients (range: 23-67 years) and 67 years for CLL patients (range: 39-94 years). Mean number of mutations per patient was 18 for PPBL and 20 for CLL. For both entities, the majority of mutations were missense mutations (88% in PPBL vs. 81% in CLL), followed by splice-site mutations (7% vs. 10%), other mutation types were only rarely detected. In PPBL, 42 genes were found to be mutated at a frequency of >15%, including ATM (22%), CREBBP (19%), NCOR2 (19%), AHNAK2 (15%), JAK3 (15%), NOTCH2 (15%) and TRAF1 (15%), all of which have been associated with a variety of cancers. Moreover, ATM, NOTCH2 and TRAF1 mutations were described before to be associated with lymphomas. In PPBL patients, mutations in TRAF1 and ATM as well as mutations in TRAF1 and NOTCH2 were found to be mutually exclusive. For CLL patients, 29 genes showed a mutation frequency of >15%, comprising ATM (26%), KMT2D (23%), NOTCH1 (23%), LRP1B (19%), TP53 (16%) and CREBBP (15%). Comparison of the mutation frequencies between the two entities revealed several genes with significant differences: whereas mutations in CKAP5 (11% vs. 2%, p=0.022), DNMT3A (11% vs. 3%, p=0.033), MAP2 (19% vs. 4%, p=0.009), ROBO1 (15% vs. 4%, p=0.046) and TRAF1 (15% vs. 2%, p=0.006) were found to be more frequent in PPBL cases compared to CLL cases, KMT2D (4% vs. 23%, p=0.014), TDRD6 (0% vs. 14%, p=0.032) and TP53 (4% vs. 16%, p=0.048) mutations were more abundantly detected in CLL patients. Moreover, NOTCH1 was mutated more frequently in CLL cases (7% vs. 23%, p=0.082), whereas mutated NOTCH2 (known to be frequently mutated in splenic marginal zone lymphoma), was more abundant in PPBL patients (15% vs. 6%, p=0.116), although both correlations were not statistically significant. Gene expression analyses by RNAseq revealed 337 genes to be differentially expressed between the entities. 207 genes were upregulated in PPBL, including PTPRK, CXCR1, BCL11B, CEPBA, CCR4 and MYC, whereas 130 genes were found to be upregulated in CLL cases, comprising ID3, BCL2, FGF2 and FLT1.Conclusions: 1) WGS analysis identifies high frequencies of cancer/lymphoma-associated gene mutations in PPBL, including mutated ATM, NOTCH2 and TRAF1. 2) Five genes showed a higher mutation frequency compared to CLL including TRAF1,DNMT3A, CKAP5 and MAP2. 3) Lymphoma associated genes (BCL11B and MYC) were overexpressed in PPBL vs CLL. 4) Taken together our results question PPBL as a benign entity and identify molecular markers that might contribute to development of subsequent malignancies. DisclosuresStengel:MLL Munich Leukemia Laboratory: Employment. Höllein:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Walter:MLL Munich Leukemia Laboratory: Employment. Hutter:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Analysis of BRCA1 and BRCA2 Mutations and Gene Expression in Hematological Malignancies - Implications for Differential Involvement in Pathogenesis of Lymphoid Neoplasms

Background: BRCA1 and BCRA2 (breast cancer 1 and 2) are tumor suppressor genes involved in repair of DNA double-strand breaks. Loss of BRCA1/2 activity by (germline) mutations (mut) or downregulation (DR) of gene expression (GE) correlates with an increased risk of breast and ovarian cancer. Mut in or reduced GE of BRCA1/2 have also been implicated in some hematological malignancies, although data are so far scarce. Moreover, PARP1 inhibitors, known to induce synthetic lethality in tumors harboring BRCA1/2 mut were shown to affect FLT3(ITD)-positive AML (acute myeloid leukemia) cells.Aims: Investigation of BRCA1/2 mut and GE in 3340 patients with different hematological malignancies by whole genome sequencing (WGS) and RNA sequencing.Patient cohorts and methods: WGS was performed for 3340 patients with different hematological malignancies: 150bp paired-end sequences where generated on Illumina HiseqX and NovaSeq 6000 machines (Illumina, San Diego, CA). A mixture genomic DNA from multiple anonymous donors was used as normal controls. All reported p-values are two-sided and were considered significant at p<0.05. For GE analysis, estimated gene counts were normalized and the resulting log2 counts per million were used as a proxy of GE in each sample.Results: We identified 14/3340 patients diagnosed with a variety of hematological neoplasms with a pathogenic (according to ClinVar) BRCA1/2 mut (BRCA1: n=11; BRCA2: n=3; incidence: 0.42%). Analysis of the mut frequencies of 1067 cancer associated genes revealed RNF213 (6/14, 43%), ARID1B, CREBBP (5/14, 36%, respectively), JAK3, TET2, NUMA1, POLD1, ZFHX3 (4/14, 29%), ATM, KMT2D and POLE (3/14, 21%) as most frequently mutated genes in cases with BRCA1/2 mut. Three of them (ATM, POLD1, POLE) are known to be directly involved in DNA repair. Comparison of mut frequencies of these 11 genes in cases with/without BRCA1/2 mut revealed JAK3 and POLD1 to be significantly more abundant in BRCA1/2 mut (JAK3: 29% vs. 9%, p=0.028; POLD1: 29% vs. 8%, p=0.026). GE analyses of BRCA1/2 showed DR of BRCA1 and/or BRCA2 in several lymphoid malignancies: for BRCA1, the strongest reduction was detected in PPBL (persistent polyclonal B-cell lymphocytosis; normalized BRCA1 expression: 1.5; control: 5.2), CLL (chronic lymphocytic leukemia; 1.6), T-PLL (T-cell prolymphocytic leukemia; 1.7) and MZL (marginal zone lymphoma; 2.2). DR of BRCA2 was less pronounced (control: 6.3; PPBL: 3.5; HCL, hairy cell leukemia: 4.0; T-PLL: 4.1; MZL: 4.6). In myeloid malignancies reduction was less (see Figure). For further analysis, the 50 lymphoid cases with the lowest and highest BRCA1/2 GE (BRCA1/2 high/low) were selected: these included for BRCA1 low (median GE: 0.8) mainly CLL (33/50), T-PLL (4/50) and PPBL (4/50), whereas BRCA1 high (median: 5.4) comprised B-ALL (23/50), T-ALL (15/50) and Burkitt lymphoma (7/50). BRCA2 low (median: 3.4) included only 2/50 CLL cases, as mainly PPBL (14/50), T-PLL (10/50), multiple myeloma (9/50) and HCL (9/50) cases showed a low BRCA2 GE. BRCA2 high (median: 6.6) included T-ALL (21/50), B-ALL (16/50) and Burkitt lymphoma (4/50). Regarding molecular mut, all entities with a strong DR of BRCA1/2 show a high mut frequency of ATM (analyzed cohort: PPBL, 22%; MZL, 24%; CLL, 25%; HCL, 28%, T-PLL: 74%); BRCA1 (p=0.023) and BRCA2 (p<0.001) GE was significantly lower in ATM mut cases. Comparison of BRCA1/2 low/high revealed several significant differences in detected gene mut: for BRCA1 low vs. high, JAK3 was more frequently mutated in low (18% vs. 4%, p=0.05), whereas KAT6B (4% vs. 24%, p=0.008), BCL11B (0% vs. 18%, p=0.003) and PHF6 (0% vs. 18%, p=0.003) were more abundant in high. For BRCA2, results were similar (JAK3: 20% vs. 4%, p=0.028; BCL11B: 2% vs. 18%, p=0.025; PHF6: 0% vs. 24%, p<0.001), moreover BRAF (20% vs. 2%, p=0.008) and STAT5B (16% vs. 0%, p=0.006) were more frequently mutated in low.Conclusions: 1) The incidence of BRCA1/2 mut in patients with hematological malignancies is comparable to the frequency in a normal population. 2) Mut in JAK3 and POLD1 are significantly more frequent in cases with BRCA1/2 mut. 3) GE analyses revealed a strong DR of BRCA1 in CLL, T-PLL and MZL. 4) T-ALL and B-ALL were not found to show a BRCA1/2 DR. 5) DR correlates with a high mut frequency of ATM and JAK3, for BRCA2 also of BRAF and STAT5B. 6) Thus, not molecular mut in, but DR of BRCA1/2 in CLL, T-PLL and MZL could play a more important role in pathogenesis than so far expected. [Display omitted] DisclosuresStengel:MLL Munich Leukemia Laboratory: Employment. Walter:MLL Munich Leukemia Laboratory: Employment. Hutter:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Persistent polyclonal binucleated B-cell lymphocytosis and MECOM gene amplification.

BackgroundPersistent Polyclonal Binucleated B-cell Lymphocytosis (PPBL) is characterized by a chronic polyclonal B-cell lymphocytosis with binucleated lymphocytes and a polyclonal increase in serum immunoglobulin-M. Cytogenetic is characterized by the presence of a supernumerary isochromosome +i(3)(q10), premature chromosome condensation and chromosomal instability. Outcome of PPBL patients is mostly benign, but subsequent malignancies could occur. The aim of our study is to provide an update of clinical and cytogenetic characteristics of our large cohort of PPBL patients, to describe subsequent malignancies occurring during the follow-up, and to investigate the role of the long arm of chromosome 3 in PPBL.ResultsWe analyzed clinical, biological and cytogenetic characteristics (conventional cytogenetic analysis and fluorescent in situ hybridization) of 150 patients diagnosed with PPBL. We performed high-resolution SNP arrays in 10 PPBL patients, comparing CD19+ versus CD19− lymphoid cells. We describe the cytogenetic characteristics in 150 PPBL patients consisting in the presence of supernumerary isochromosome +i(3)(q10) (59 %) and chromosomal instability (55 %). In CD19+ B-cells, we observed recurrent copy number aberrations of 143 genes with 129 gains (90 %) on 3q and a common minimal amplified genomic region in the MECOM gene. After a median follow-up of 60 months, we observed the occurrence of 12 subsequent malignancies (12 %), 6 solid tumors and 6 Non-Hodgkin’s Lymphomas, and 6 monoclonal gammopathies of undetermined significance (MGUS), requiring a long-term clinical follow-up.ConclusionsOur clinical and cytogenetic observations lead us to hypothesize that isochromosome 3q, especially MECOM abnormality, could play a key role in PPBL.

Unique Versus Common: Disease-Biased Immunoglobulin Gene Repertoires Along with Public Antigen Receptor Stereotypes in Marginal Zone B-Cell Lymphoproliferations

B cells residing the marginal zone (MZ) provide a first line of defense against blood borne pathogens, producing the greater part of circulating natural antibodies conferring protection against infection. Dysregulated homeostasis and function of MZ B cells has been implicated in a wide range of B lymphoproliferations, encompassing the distinct MZ lymphomas recognized by the WHO Classification, the related provisional entities and even chronic lymphocytic leukemia (CLL), for which a MZ derivation has been proposed. Here, taking advantage of a large multi-institutional series, we aimed at obtaining insight into the ontogenetic relationship of MZ lymphoproliferations, related entities and CLL through cross-comparison of their B cell receptor immunoglobulin (BcR IG) gene repertoires. Our sequence dataset included 3660 unique IGHV-IGHD-IGHJ gene rearrangement sequences from our collaborative centers and/or public databases derived from: (1) MZ lymphomas: splenic (SMZL), n=379; nodal (NMZL), n=37; extranodal (ENMZL), n=95; (2) provisional entities of postulated MZ origin, including splenic diffuse red pulp lymphoma (SDRL, n=16) and clonal B cell lymphocytosis of MZ origin (CBL-MZ, n=60); (3) persistent polyclonal B cell lymphocytosis (PPBL), n=286 (from 2 cases); (4) MZ cells isolated from six spleen specimens free of neoplastic cells at histological inspection (non-malignant MZ), obtained at surgery for cancer, n=489; (5) autoimmune conditions, n=1243; (6) various types of normal B cells, n=1055. The most pronounced IG gene repertoire skewing was observed in SMZL with the IGHV1-2*04 gene accounting for 26% of cases. Restrictions, though less striking, were also identified in the other MZ lymphomas as well: (i) the IGHV4-34 gene predominated in NMZL (14.3%); and, (ii) the IGHV1-69 gene predominated in ENMZL (14.6%), albeit with significantly different distribution depending on the primary site of involvement, ranging from 38% in salivary ENMZL to 11% in gastric ENMZL to 4% in ocular adnexa ENMZL (p<0.01). The vast majority of MZL cases showed at least some impact of somatic hypermutation (SHM), with the proportion of cases lacking any SHM ranging from 0% in salivary ENMZ to only 13% in SMZL. Following established bioinformatics approaches, we searched for stereotyped BcR IG sequences i.e. IGHV-IGHD-IGHJ gene rearrangements with restricted antigen-binding site sequence motifs. For the purposes of this analysis, the present sequence dataset was cross-compared to a large dataset of 20451 IGHV-IGHD-IGHJ gene rearrangement sequences from CLL patients from the IMGT/CLL-DB. Overall, 6437 different clusters with stereotyped BcR IG sequences were identified in the merged dataset, including from only 2 to more than 350 sequences. Two categories of clusters with stereotyped BcR IG were identified: disease-specific (n=4813) and 'mixed' (n=1624) i.e. comprised of cases with different diagnosis. The great majority of clusters in the former category concerned exclusively CLL and corresponded to well-established CLL stereotyped subsets, while only a small minority concerned exclusively MZ lymphomas, all with a diagnosis of SMZL. Mixed clusters were relatively small in size, with only 4 populated by more than 10 cases; of these, 2 utilized the IGHV1-69 gene, while the remaining 2 utilized the IGHV3-7 and IGHV4-59 gene, respectively. They comprised rearrangements from various entities, including SMZL, ENMZL (gastric, salivary gland, ocular adnexa), CLL, hepatitis C virus-associated diffuse large B cell lymphoma (DLBCL), but also rheumatoid factors and non-malignant spleen MZ cells. Notably, shared (recurrent) amino acid changes introduced by SHM (i.e. the same amino acid replacement at the same position) were identified in each mixed cluster. In conclusion, we document different immunogenetic signatures for MZ lymphomas, with limited overlap both amongst the various distinct and provisional WHO entities but also versus CLL. These findings indicate distinct antigen exposure histories and/or different (micro)environments underlying the ontogeny of MZ lymphomas. That said, the existence of rare public stereotypes raises the intriguing possibility that common, pathogen-triggered, immune-mediated mechanisms, may result in diverse B lymphoproliferations due to targeting versatile progenitor B cells and/or operating in particular microenvironments. DisclosuresGhia:Janssen Pharmaceuticals: Research Funding.

Occurrence of Non-Hodgkin Lymphoma after the Diagnosis of Persistent Polyclonal B-cell Lymphocytosis

Persistent polyclonal B cell lymphocytosis (PPBL) is a rare indolent condition characterized by a polyclonal expansion of B cells associated with binucleated lymphocytes observable on blood smears. Though polyclonal, recurrent genetic abnormalities have been described in PPBL, supernumerary isochromosome 3q (+i(3)(q10)) being the most frequently described. We report here two clinical observations showing the occurrence of NHL after the diagnosis of PPBL. These observations lead us to recommend a closer follow-up of PPBL patients and raise the question of a relationship between PPBL and NHL.

Persistent polyclonal B-cell lymphocytosis: extensively proliferated CD27+IgM+IgD+ memory B cells with a distinctive immunophenotype

Persistent polyclonal B-cell lymphocytosis: extensively proliferated CD27+IgM+IgD+ memory B cells with a distinctive immunophenotype

Persistent Polyclonal B Cell Lymphocytosis B Cells Can Be Activated through CD40-CD154 Interaction.

Persistent polyclonal B cell lymphocytosis (PPBL) is a rare disorder, diagnosed primarily in adult female smokers and characterized by an expansion of CD19+CD27+IgM+ memory B cells, by the presence of binucleated lymphocytes, and by a moderate elevation of serum IgM. The clinical course is usually benign, but it is not known whether or not PPBL might be part of a process leading to the emergence of a malignant proliferative disorder. In this study we sought to investigate the functional response of B cells from patients with PPBL by use of an optimal memory B cell culture model based on the CD40-CD154 interaction. We found that the proliferation of PPBL B cells was almost as important as that of B cells from normal controls, resulting in high immunoglobulin secretion with in vitro isotypic switching. We conclude that the CD40-CD154 activation pathway is functional in the memory B cell population of PPBL patients, suggesting that the disorder may be due to either a dysfunction of other cells in the microenvironment or a possible defect in another B cell activation pathway.

Making “Sense” Of Persistent Polyclonal B Cell Lymphocytosis

Neuronal regulation of the hematopoietic stem cell niche includes elements of the sympathetic nervous system however, a role for sensory neurons has not been explored. While investigating sensory nerves in hematopoiesis, we unexpectedly found mice deficient in the neurotransmitter substance P (Tac-1-/-) to exhibit peripheral blood lymphocytosis that is persistent from 8 weeks to at least 35 weeks of age. Flow cytometry showed the increased population to be comprised of polyclonal B-lymphocytes. Hematopathology review demonstrated abnormal peripheral blood bi-nucleated lymphocytes and enlarged splenic marginal zones. Lymph nodes were not affected. To confirm that sensory nerve disruption and not hematopoietically derived substance P was the cause, wildtype C57BL/6 mice were chemically denervated and found to exhibit similar peripheral blood lymphocytosis. By immunohistochemistry, sensory nerves synapsed at splenic marginal zones, consistent with previous reports and providing a neuro-anatomical link for the observed lymphoid hyperplasia. A human correlate to this hematological phenotype featuring long-term peripheral polyclonal B cell lymphocytosis, bi-nucleated lymphocytes and expanded splenic marginal zones is Persistent Polyclonal B-cell Lymphocytosis (PPBL), a rare disorder affecting predominantly female chronic smokers. The pathoetiology of this disorder is unknown but its strong association with smoking supports a novel hypothesis where chronic smoking leads to respiratory tract sensory nerve desensitization with possibly more systemic effects, including on the spleen, thereby mimicking the substance P deficient environment of Tac-1-/- mice. Indeed, pulmonary function tests on chronic smokers have shown a sub-population of patients with hyporeactivity to respiratory sensory nerve stimulation, explained molecularly by increased activation thresholds. It will be interesting to determine if PPBL patients fall into this category. In Tac-1-/- mice, further characterization of the expanded B cell population, immunoglobulin profiles, VDJ utilization and effect of neurokinin-1 receptor modulators is proposed. These results support a neuroimmune regulatory network involving sensory nerves with potential relevance in both lymphoid homeostasis and lymphoproliferative disease. Disclosures:No relevant conflicts of interest to declare.

Morphologic features of binucleated lymphocytes to assess the diagnosis of persistent B-cell polyclonal lymphocytosis or other mature B-cell neoplasms

The observation of binucleated lymphocytes (BNLs) on a peripheral blood smear is essential to the diagnosis of persistent polyclonal B-cell lymphocytosis (PPBL). Only a few case reports have mentioned their presence in other contexts, mainly mature B-cell neoplasms such as chronic lymphocytic leukemia or reactive circumstances such as multiple sclerosis undergoing natalizumab treatment. We sought to investigate whether any particular morphologic features of BNL were more specific to PPBL than other diseases. We reviewed peripheral blood smears of a series of patients affected by PPBL or harboring BNLs whatever the diagnosis. We found that BNLs in PPBL were heterogeneous, but mostly medium-sized with a moderately abundant basophilic cytoplasm, sometimes vacuolated. The chromatin was mature in an asymmetrically bilobed nucleus showing one to two nucleoli. Though mainly observed in patients with PPBL, all these criteria remained non-specific. Conversely, the presence of either monocytoid or hyperbasophilic cells had clinical value to confirm PPBL. We conclude that a substantial percentage of BNLs possessing the morphologic features described and observed in a context of monocytoid and/or hyperbasophilic lymphocytes is predictive of the diagnosis of PPBL.

Morphologic features of the binucleated lymphocytes for the assesment of persistent B-cell polyclonal lymphocytosis

Observation of binucleated lymphocytes on a blood film is a cornerstone event to assess persistent polyclonal B-cell lymphocytosis diagnosis (PPBL). But seldom case reports mentioned their finding in other contexts, mainly B-cell lymphoproliferative neoplasms such chronic lymphocytic leukemia or in reactive circumstances as multiple sclerosis undergoing natalizumab treatment. We aimed to check if some particular morphologic features were more specificaly linked to PPBL, reviewing blood films of series of patients affected by PPBL or harbouring binucleated lymphocytes whatever the diagnosis was. We found that binucleated cells in PPBL were heterogeneous but mostly midde-sized cells, asymetricaly bilobed with nucleoli, and with a moderately basophilic cytoplasm. Though mainly observed in PPBL patients, all that criteria remained unspecific. Conversely, the morphologic features of the other lymphoid cells were of good clinical value to affirm PPBL, either monocytoid or hyperbasophilic cells being systematically found. We think that a substantial percentage of binucleated lymphocytes, harbouring morphologic features such as described, and in a context of monocytoid and/or hyperbasophilic lymphocytes is predictive of PPBL diagnosis.

Persistent Polyclonal B-cell Lymphocytosis With Splenomegaly

Persistent polyclonal B-cell lymphocytosis is a rare, benign lymphoproliferative disorder characterized by a stable, polyclonal CD19-positive CD5-negative lymphocytosis, the presence of binucleated lymphocytes in peripheral blood, and a polyclonal increase in serum immunoglobulin-M that may occasionally be accompanied by splenomegaly. Histopathologic diagnosis of these splenectomy specimens is difficult because of the massive spleen infiltration and the rarity of the descriptions of this condition. We describe the histopathologic findings from 2 splenectomy specimens. These included a partially preserved architecture with infiltration of the red pulp by small lymphocytes and partial replacement of the white pulp. Suggestions for identifying the disorder are made.

Histological and immunohistochemical features of the spleen in persistent polyclonal B-cell lymphocytosis closely mimic splenic B-cell lymphoma

Persistent polyclonal B-cell lymphocytosis (PPBL) is rare and intriguing hematological disorder predominantly reported in young to middle- aged smoking women. It is characterized by persistent moderate polyclonal B-cell lymphocytosis with circulating hallmark binucleated lymphocytes and elevated polyclonal serum IgM. Most patients have benign clinical course on long-term follow-up. Some pathologic features of PPBL may resemble malignant lymphoma, including morphology as well as frequent cytogenetic and molecular abnormalities. Significant symptomatic splenomegaly requiring splenectomy is very unusual for this disorder; therefore there is a lack of descriptions of the morphologic features of the spleen in the literature. We present here one of the first detailed descriptions of the morphologic and immunohistochemical features of the spleen from a young female with PPBL who developed massive splenomegaly during 6-year follow up. Splenectomy was performed for symptomatic relief and suspicion of malignant process. The morphological and immunohistochemical features of the spleen closely mimicked involvement by B-cell lymphoma, however there was no monotypic surface light chain restriction seen by flow cytometry and no clonal rearrangement of IgH gene was detected by molecular analysis. Evaluating a splenectomy sample in cases like this may present a diagnostic challenge to pathologists. Therefore, correlation with B cell clonality studies (by flow cytometry and molecular analysis), clinical findings and peripheral blood morphology searching for characteristic binucleated lymphocytes is essential to avoid misdiagnosing this benign process as B-cell lymphoma. We also present here a literature review on pathogenesis of PPBL.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/5329558967545656

Persistent polyclonal B-cell lymphocytosis

![Figure][1] A 45-year-old woman had asthenia, diffuse myalgias, and mild splenomegaly. Her history included smoking and no evidence of viral illness. There was no family history of lymphoid disorders. Laboratory tests showed lymphocytosis (6.1 × 109/L) that had been present for years

EDTA-dependent lymphoagglutination in persistent polyclonal B-cell lymphocytosis

A 38-year-old female smoker was referred for assessment of a persistent polyclonal B-cell lymphocytosis (PPBL). She complained of profound asthenia and arthralgia. There was no palpable enlargement of lymph nodes, liver or spleen. Laboratory investigations showed moderate isolated lymphocytosis (4AE9 · 10/l) with 3% binucleated cells, increased polyclonal immunoglobulin (Ig) M (8AE2 g/l), HLA-DR7 phenotype and the presence of an abnormal karyotype: 47,XY,+i(3)(q10)[4]/46,XY[29]. Peripheral blood immunophenotyping demonstrated a polyclonal increase in the B-cell pool with IgM, IgD, CD19, CD20, CD21, CD22, CD24, CD27, CD40, CD79b and FMC7 positivity and CD5, CD10, CD23, CD25, CD30, CD34, CD38, CD43, CD54, CD122, CD138 and CD154 negativity, in keeping with a memory B-cell origin. In addition, clumps of lymphocytes including binucleated cells were observed under the microscope (left), but were absent when a specimen was taken into citrate (right), suggesting an artifactual EDTA-dependent lymphoagglutination. PPBL occurs mainly in middle-aged female smokers, presenting with a moderate chronic lymphocytosis with atypical binucleated lymphocytes comprising 1–10% of the lymphocytes and increased polyclonal serum IgM. The B cells are polyclonal with kappa and lambda light-chain expression and no clonal rearrangement of the immunoglobulin heavy chain genes. The cytogenetic profile includes an additional isochromosome for the long arm of chromosome 3, genetic instability and premature chromosome condensation. About 3% of patients develop lymphoma. Lymphoagglutination is a rare EDTA-dependent phenomenon; the use of other anticoagulants (citrate or heparin) avoids this artefact in nearly all cases. It occurs most often in B-cell lymphoproliferative disorders, particularly marginal zone lymphoma. The occurrence in PPBL may be attributed to the likely origin of these cells from the splenic marginal zone.

Linfocitosis B policlonal persistente: estudio de 35 casos

Background and objectives Persistent polyclonal B-cell lymphocytosis (PPBL) is a rare entity, presenting especially in adult smoker women. It is characterized by an increase of serum IgM, DR7-HLA haplotype, cytogenetic abnormalities and multiple IgH/BCL-2 rearrangements. To date, it has not been elucidated whether this is a benign or premalignant disorder. We analyzed the PPBL characteristics with especial attention to its evolution. Patients and methods Thirty-five PPBL patients from 5 hospitals in Catalonia were retrospectively analyzed. A simultaneous morphologic review of the blood smears was performed by members of the GCCH in a 16 multiple-observer optic microscope. Clinical and biological data were also analyzed. Results PPBL presents in the majority of cases with persistent polyclonal B-cell lymphocytosis and affects primarily smoker women. The morphologic hallmark, in absence of viral infections, is the presence of activated lymphocytes with bilobulated and/or cleaved nuclei, and nuclear pockets in the ultrastructural study. Increased serum IgM, HLA-DR7 haplotype, chromosomal abnormalities such as i(3)(q10) and multiple IgH/BCL-2 rearrangements were detected. Thirty-four out of 35 patients are alive after a median follow up of 70.7 months. One patient died because of lung adenocarcinoma and another developed a follicular lymphoma without relation to PPBL. Conclusions PPBL has an asymptomatic and stable evolution, although it frequently presents genetic abnormalities. It remains unknown whether it is a premalignant entity, similar to monoclonal gammopathies of unknown significance. Hence, accurate cytologic diagnosis and follow-up are essential.