Red Pulp Small B-cell Lymphoma Research Articles

Understanding splenic B-cell lymphoma/leukaemia with prominent nucleoli: Diagnosis, underpinnings for disease classification and future directions.

The 5th edition of the WHO classification of haematolymphoid tumours (WHO-HAEM5) introduced a new category, splenic B-cell lymphoma/leukaemia with prominent nucleoli (SBLPN). The diagnostic entity B-cell prolymphocytic leukaemia (B-PLL) has been discontinued and the category of hairy cell leukaemia variant (HCLv) has been conceptually reframed. B-PLL and HCLv diagnoses were uncommon. Overlap existed between B-PLL and other indolent lymphomas like chronic lymphocytic leukaemia/small lymphocytic lymphoma (CLL/SLL). HCLv lacked consistent cytomorphological, immunophenotypic and genetic features. To address these issues, the WHO-HAEM5 classification has introduced SBLPN to serve as a temporary holding ground for entities that do not neatly fit into the existing classification. Cases previously classified as CD5-negative B-PLL and HCLv fall under the SBLPN category. Some splenic marginal zone lymphoma and splenic diffuse red pulp small B-cell lymphoma cases with higher number of medium or large nucleolated B cells would also be classified as SBLPN under the WHO-HAEM5. This review explores the rationale for discontinuing B-PLL and HCLv diagnoses. It then examines the concept of SBLPN, offers practical guidance for diagnosis and discusses future directions in classifying splenic B-cell lymphomas.

Splenic B Cell Lymphoma Innate Immune Cells: Detection of Red Versus White Pulp Lymphoma Differences in NK Cell Populations By High-Parameter Flow Cytometry

Introduction Splenic B-cell lymphomas, (SLs, total incidence ~1:100,000/year) comprise four distinct indolent B-cell non-Hodgkin lymphomas. Three predominantly involve the red pulp of the spleen (classical hairy cell leukemia (HCL), hairy cell leukemia variant (HCLv) and splenic diffuse red pulp small B-cell lymphoma (SDRPL)) and the fourth predominantly involves the white pulp (splenic marginal zone lymphoma (SMZL)). HCL is usually diagnosed by detection of an activation mutation in BRAF and responds well to treatment. We focused our study on the remaining three diseases, which have overlapping clinical presentations with only subtle differences in their morphology and phenotypes, but have considerable differences in prognosis, response to treatment and outcome. The white pulp lymphoma SMZL generally has good treatment responses and longer survival, whereas the red pulp SLs, HCLv and SDRPL are less responsive to treatments with shorter survival. Cells of the tumor microenvironment (TME) may have distinct characteristics between these three diseases. We hypothesized that TME innate immune cells from red pulp SLs (HCLv, and SDRPL) would be similar to each other and distinct from the white pulp SL, SMZL.In this study, we report that natural killer (NK) cells, which are important innate immune cells critical for immune surveillance, vary in proportions among red and white pulp SLs. Methods We studied splenectomy specimens from patients with SMZL (n=8), HCLv (n=8), SDRPL (n=7), and controls (healthy, post-trauma, n=8). Cryo-preserved bulk splenic suspension cell isolates from fresh (<4 hour) surgical specimens and intact formalin-fixed paraffin embedded (FFPE) tissue sections were used. Human specimen collection and usage was conducted with written informed consent after approval of the Institutional Research Subjects Review Board. FFPE tissue sections were studied by immunohistochemical (IHC) staining and splenic suspension cells were analyzed by high-parameter fluorescence flow cytometry using a Cytek Aurora spectral flow cytometer with a 25-color myeloid and a 28-color NK/B-cell panel. Results IHC characterization showed an extensive network of innate immune cells as reflected by CD163 + macrophages. Using high-parameter flow cytometry, we focused on innate immune cells in the non-B- non-T-cell (CD3 -CD19 -) population. The average proportions of this population were: NK cells 25-36%, monocyte/macrophages 20-34%, dendritic cells 5-11%, and granulocytes 2%. Among the patient groups, the median percentage of NK cells was greater for controls (34%) and SMZL (35%) as compared to red pulp lymphomas (HCLv (28%), SDRPL (24%)). Next, we examined NK subtypes: 1) cytotoxic NK cells (CD56 +CD16 +), 2) cytokine-secreting weakly cytotoxic NK cells (CD56 +CD16 -), and 3) undefined NK cells (CD56 -CD16 +) as a median percentage of total NK cells. The major subtype for control and SMZL was CD56 +CD16 - with values of 58 and 44%, respectively. This subtype comprises only 38%-40% of red pulp SLs. Proportionally the CD56 +CD16 + subtype was the largest for red pulp SLs at 52-57% as compared to 30% for control and 38% for SMZL. The remaining CD56 -CD16 + NK cell subtype was generally higher among red pulp SLs (HCLv (9%), SDRPL (12%)) as compared to control (6%) and SMZL (4%) patients. The increase in cytotoxic CD56 +CD16 + subtype among red pulp SLs was confirmed by measuring the percentage of granzymeB +perforin + cells among mature cytotoxic NK cells (CD56 +CD16 +/ CD57 +NKG2a -). SLs had higher proportions of this NK phenotype (78-86% compared to control 69%) and red pulp SLs had the most granzymeB +perforin + proportions with 82 and 86% for HCLv and SDRPL, respectively. Discussion In summary, spleens from patients with SMZL had similar percentages of total NK cells to controls, with lower percentages in red pulp SLs. All SLs had a higher percentage of cytotoxic NK cells than control, with the red pulp SLs having the most. These data suggest that monoclonal antibody treatments targeting SL antigen, such as anti-CD20, may be effective by activating cytotoxic NK cell to target lymphoma cells by antibody-dependent cellular cytotoxicity. Thus, uncovering the characteristics of the TME may aid the development of non-invasive diagnostic procedures and/or precision therapies.

Hairy Cell Leukemia with Severe Leukocytosis

Introduction Hairy cell leukemia (HCL) is a rare indolent mature B-cells neoplasm that typically manifests with pancytopenia and splenomegaly. Nevertheless, significant splenomegaly, high hairy cells count in the blood, elevated β 2 microglobulin, and increased LDH can predict resistance to purine analogs. The absence of BRAF V600E mutation in HCL is rare and is associated with poorer outcomes. Case Presentation A 68-year-old female patient presented with symptomatic massive splenomegaly, leukocytosis (89,000 /microliter), monocytopenia, and thrombocytopenia. β 2microgobulin level was 4.4 mcg/L. Lactate dehydrogenase was 521 U/L. Peripheral blood and bone marrow examinations revealed typical hairy cells morphology, and the cells were positive for CD19, CD20, CD103, CD25, CD11c, CD200 (dim), and surface-Kappa. CD38 was negative. BRAF V600E result was wild-type. IGHV was mutated (IGHV3-49). She received first-line therapy with a 5-day course of Cladribine (0.15 mg/kg/day) with initial resolution of leukocytosis, normalization of platelets, and a 50% reduction in spleen size. This was followed by consolidation with an 8-weekly course of Rituximab (375 mg/m 2). Soon after the 2 nd dose of Rituximab, leukocytosis recurred, and she was initiated on second-line therapy with Bendamustine (90 mg/m 2 on days 1 and 2) along with Rituximab (BR). She has not achieved any durable response following two cycles of BR. Unfortunately, she suffered a septic shock requiring admission to our intensive care unit. Upon routine mental status evaluation, brain MRI revealed innumerable supratentorial and infratentorial bilateral acute/subacute and old microhemorrhages (Figure 1). This prompted further evaluation of unexplained recurrent episodes of pulmonary hemorrhages while on mechanical ventilation that were found to be secondary to pulmonary end-arterial cavitating lesions suggestive of hairy cells infiltration (Figure 2 arrowheads). As she was clinically unstable, we attempted to control leukocytosis with corticosteroids, leukapheresis aggressively and started the patient on Ibrutinib, but she succumbed to her disease. Discussion Recently, the 5 th edition of the World Health Organization classification of hematolymphoid tumors highlighted the splenic B-cell lymphoma and leukemia family that encompasses hairy cell leukemia (HCL), splenic B-cell lymphoma/leukemia with prominent nucleoli (SBLPN), formerly known as hairy-cell leukemia variant, splenic diffuse red pulp small B-cell lymphoma (SDRPL) and splenic marginal zone lymphoma (SMZL). HCL is typically an indolent mature B-cell neoplasm with characteristic pathologic features that preferentially insinuate the bone marrow and the spleen causing impaired hematopoiesis and splenomegaly. Unlike other disorders within this group, HCL commonly harbors BRAF V600E somatic mutation in almost all cases. Rarely, patients with HCL can have extramedullary involvement by the malignant cells. Leukocytosis in HCL appears to increase the risk of developing extramedullary lesions that can present unique clinical challenges necessitating early and effective therapy. According to the literature, brain lesions in HCL were described in three other cases, and leukocytosis was a common denominator. Our patient with significant leukocytosis had brainand pulmonary lesions, and the latter was the cause of her demise due tounusually aggressive and refractory disease. Therefore, we hypothesize that leukocytosis in HCL can be “the tip of the iceberg”. The higher the leukocytosis, the higher the burden of extramedullary disease, as in our case, who had simultaneous lesions in the brain and lungs. Patients with HCL and leukocytosis may warrant initial diagnostic evaluation to ensure the absence of potentially sinister lesions, which can inform subsequent monitoring and management plans. Additionally, more novel therapies could be considered sooner in the treatment course to control the disease and these lesions. Finally, our patient did not harbor BRAF V600E mutation, which likely resulted in further worse outcomes and limited the usage of targeted therapies like BRAF inhibitors.

Indolent B-cell splenic lymphomas have T cells with an exhausted phenotype

Abstract T cell dysregulation has been observed in many tumors, but has not been well-characterized in rare B-cell splenic lymphomas. We began defining the T cell populations in the tumor microenvironments of four human indolent B cell splenic lymphomas: splenic marginal zone lymphoma (SMZL), hairy cell leukemia (HCL), hairy cell leukemia variant (HCLv), and splenic diffuse red pulp small B-cell lymphoma (SDRPL). After Institutional Research Subjects Review Board approval, we studied de-identified cryopreserved bulk splenic tumor suspension cell isolates from fresh (less than 4 h) splenectomy tissue and matched intact formalin-fixed paraffin embedded (FFPE) tissue sections from 17 splenic lymphoma patients and 4 trauma patients (controls). Initial immunohistochemical staining analysis of FFPE tissue sections suggests that the distribution of intratumoral T cells is markedly different among B cell splenic lymphomas. In HCL, residual T cell zones appear retained, albeit diminished, while HCLv and SDRPL have markedly reduced T cells throughout with non-distinct zonation, and SMZL has prominent peritumoral collections of T cells at the interface of the neoplastic white pulp. High-parameter (32-color) fluorescence spectral flow cytometry (Cytek Aurora) analysis suggests that splenic lymphomas exhibit CD4+ and CD8+ T cells with higher proportions of transitional memory, regulatory, and exhausted phenotypes than controls. T cell activation may be enhanced in HCLv. HCLv and SMZL may have decreased naïve T cells. Finally, splenic lymphomas had higher proportions of T cells with an exhausted phenotype compared to trauma samples. In summary, our data suggests T cells are dysregulated in B-cell splenic lymphomas. American Association of Immunologists, Hairy Cell Leukemia Foundation / Sass Foundation for Medical Research, and generous donations by Elizabeth Aaron.

Transformations of marginal zone lymphomas and lymphoplasmacytic lymphomas: Report from the 2021 SH/EAHP Workshop.

To summarize the conclusions of the 2021 Society for Hematopathology/European Association for Haematopathology workshop regarding transformations of marginal zone lymphoma (MZL) and lymphoplasmacytic lymphoma (LPL). Nineteen cases were submitted to this portion of the workshop. Additional studies were performed in cases with sufficient material. Cases included splenic MZL (n = 4), splenic diffuse red pulp small B-cell lymphoma (n = 2), nodal MZL (n = 4), extranodal MZL (n = 1), and LPL (n = 8). The most common transformation was to diffuse large B-cell lymphoma (DLBCL), but others included classic Hodgkin lymphoma, high-grade B-cell lymphomas with MYC and BCL6 rearrangements, plasmablastic lymphoma, and plasma cell leukemia. Two splenic MZLs with transformation to DLBCL contained t(14;19)(q32;q13.3) IGH::BCL3 rearrangements in both samples. Paired sequencing studies in 5 MZLs with transformation to clonally related DLBCL identified a variety of mutations and gene fusions at the time of transformation, including CARD11, IGH::MYC, NOTCH2, P2RY8, TBLX1X1, and IGH::CD274. Marginal zone lymphoma and LPL may undergo a variety of transformation events, most commonly to DLBCL, which is usually, although not always, directly clonally related to the underlying low-grade lymphoma. Multiparameter analysis including broad-based sequencing studies can assist in the diagnosis and classification of these uncommon cases.

Clinical features and prognosis of eight patients with splenic diffuse red pulp small B-cell lymphoma

Objective: To investigate the clinical characteristics, response, and prognosis of splenic diffuse red pulp small B-cell lymphoma (SDRPL) . Methods: Eight cases of SDRPL were diagnosed and treated at Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, between May 2017 and April 2022. Data on the clinical features, laboratory results, bone marrow and spleen biopsy results, response, and prognosis were collected and analyzed. Results: The median age at diagnosis was 54 (42-69) years. Splenomegaly and lymphocytosis were present in all cases, and PET/CT revealed normal to slightly elevated splenic FDG uptake. All cases were in stage Ⅳ, with spleen, peripheral blood, and bone marrow but no proximal lymph nodes involved. The cytoplasm of neoplastic villous cells was abundant, and splenic pathology showed that small homogenous lymphocytes permeated the splenic sinus and splenic cord, and the white pulp atrophied. Immunohistochemistry was not typical, and B-cell markers including CD19, CD20 and CD79α were positive. After a median follow up of 35.5 (4-60) months, 7 cases were alive after splenectomy with or without chemoimmunotherapy. The patient with CCND3 P284A and MYC S146L mutation developed to B-cell prolymphocytic leukemia (B-PLL) 1 month after splenectomy and died at 16 months of follow-up. Conclusion: A rare indolent B-cell lymphoma that primarily affects the elderly, SDRPL. Most patients achieved long-term survival, but the prognosis of patients who progress to B-PLL was poor.

Innate Immune Microenvironment of Indolent B Cell Splenic Lymphomas: Role in Diagnosis and Treatment

Indolent splenic B-cell lymphomas are rare diseases (incidence <1:100,000) that include splenic marginal zone lymphoma (SMZL), hairy cell leukemia (cHCL), hairy cell leukaemia variant (HCLv), and splenic diffuse red pulp small B-cell lymphoma (SDRPL). These splenic lymphomas can have overlapping clinical presentations with only subtle differences in their morphology and phenotypes. However, there are considerable differences in prognosis, response to treatment, and outcomes. Better understanding of the tumor microenvironment (TME) is essential to design expedient and accurate diagnostic methods and precision drug treatments for each type of splenic B-cell lymphoma. We hypothesized each splenic lymphoma would have a distinct TME with some overlap for the red pulp lymphomas (cHCL, HCLv, and SDRPL). Human specimen collection and usage was approved by the Institutional Research Subjects Review Board. We studied cryo-preserved bulk splenic tumor suspension cell isolates from fresh (<4 hour) splenectomy tissue and intact formalin-fixed paraffin embedded (FFPE) tissue sections from 17 splenic lymphoma patients and 4 trauma patients (controls). FFPE tissue sections were studied by immunohistochemical (IHC) staining and splenic suspension cells analyzed by high-parameter fluorescence flow cytometry with a 29-color myeloid panel using a Cytek Aurora spectral flow cytometer. Initial IHC characterization of the splenic TME illustrated a markedly different distribution of intratumoral T cells (CD3) and macrophages (Mθ) (CD163). In cHCL, residual T cell zones appear retained, albeit diminished, while HCLv and SDRPL have markedly reduced T cells throughout with non-distinct zonation, and SMZL has prominent peritumoral collections of T cells at the interface of the neoplastic white pulp. Tissue architecture, as evidenced by splenic macrophage distribution, was markedly different. cHCL and HCLv showed diffuse but distorted networks of interdigitating macrophages, while SDRPL retained a nearly normal sinusoidal distribution of abundant macrophages, and SMZL showed total exclusion of CD163-positive Mθ from areas of confluent neoplastic white pulp. Analysis of splenic suspension cells showed an average of 72% B cells in the lymphoma specimens, twice that of trauma spleens. T cells made up the greatest fraction of non-B cells (~63%) for both the diseased and control samples. We analyzed the non-T and non-B cells to examine the innate immune cells. Granulocytes comprised on average 4% with no difference between the splenic lymphomas or trauma (Fig.1A). Trauma spleen had 86% macrophage/monocytes (M/Mθ)s, with similar values for HCLv (80%) and cHCL (74%). The percentage of M/Mθ was lower in SDRPL (55%) and SMZL (48%). We then analyzed the percentage of M/Mθ subtypes. As shown in (Fig.1B), CD14+CD16- was the major subtype for trauma (58%) and HCLv (50%). Conversely for cHCL, CD14loCD16+ was predominant (60%) subtype. SDRPL and SMZL shared similar subtype profiles with equal proportions of CD14+CD16- and CD14loCD16+ (40%). We then measured the dendritic cells of the innate immune population. SDRPL (41%) and SMZL (48%) had higher percentages than trauma (12%), HCLv (16%), or cHCL (20%). Conventional dendritic cell 2 (cDC2) was the predominant subtype comprising 50-74% of the DCs. Average conventional dendritic cell 1 (cDC1) and plasmacytoid dendritic cells (pDCs) were 30% and 9%, respectively. pDC was lower in cHCL than the other lymphomas (1%). Our data show a similar M/Mθ component of the innate immune TME in the red pulp lymphoma SDRPL and white pulp lymphoma SMZL. SDRPL and SMZL also had higher proportions of dendritic cells compared to cHCL, HCLv, or trauma spleens. cDC2 was the predominant DC type in the spleen regardless of disease state. We are currently analyzing flow cytometry panels to access natural killer cell distribution and T cell functionality. We aim to better understand immune cell interactions in the TME and discover variations between the splenic B-cell lymphomas, resulting in improved diagnosis and therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Splenic Diffuse Red Pulp Small B-Cell Lymphoma Descriptors and Clinicopathologic Determinants of Clinical Outcomes: Analysis of a Pooled Database

Introduction: Splenic Diffuse Red Pulp Small B-Cell Lymphoma (SDRPL) is a rare clinical entity representing <1% of B-Cell Lymphomas. Previously identified as a variant of SMZL, the WHO has recognized it as a provisional entity since its 2008 classification updates. The diagnosis can be challenging as it shares multiple clinical and laboratory features with Splenic Marginal Zone Lymphoma (SMZL), Hairy Cell Leukemia (HCL) and HCL variant (HCL-v). It is characterized by diffuse involvement of the splenic red pulp by small monomorphic B-cells. Due to our limited understanding of this disease, we conducted this pooled database analysis of all cases of SDRPL to delineate key clinicopathological characteristics, prognostic indicators and treatment modalities that affect outcomes in this rare lymphoma subtype. Methods: In order to study the demographic characteristics, molecular and immunohistochemical signatures, therapeutic interventions, survival, and prognostic factors, we compiled a pooled database of 80 cases. Kaplan-Meier survival curves were constructed. Cox proportional hazards model and Log-rank tests were used to assess the influence of demographic and clinicopathologic factors on overall (OS) and disease-free survival (DFS) survival. Results: A total of 80 patients with confirmed SDRPL were identified. The median age was 66.5 years, with a peak incidence between 69 and 70. There was a male preponderance with M:F ratio of 1.7. Splenomegaly, hepatomegaly (HM), lymphadenopathy (LN), constitutional symptoms (CS), ECOG0-1, and bone marrow (BM) involvement were present in 91%, 33%, 21%, 44%, 95%, and 95%, respectively. The median splenic weight was 2450g. While splenic red pulp was solely involved in 69% of the cases, 31% had mixed red and white pulp involvement. Three patients had active HBV, one had EBV, 4 had skin involvement, and 1 transformed into B-PLL. SDRPL Immunophenotype consisted of: TRAP(37%), Annexin A1(5%), CD5(14%), CD11c(63%), CD19(97%), CD20&22(100%), CD25(11%), CD27(60%), CD79(100%), CD103(26%), Bcl2(80%), CyclinD1(10%). CyclinD3(55%), and BRAF(16%). The median OS and DFS of the whole group were not reached (mean 138 mo) and 86mo, respectively. Age > 60 adversely impacted DFS (84mo vs. NR, p=0.03). The same trend was noted for age and OS. Compared to no treatment, splenectomy alone and the addition of combination chemotherapy were superior, with a median DFS of 36, 105, and 100 months respectively (p=0.06). HM, BM involvement, WBC>20K, absolute lymphocyte count>5K, and platelets<100K tended to adversely impact DFS, while only BM involvement and platelets<100K tended to impact OS adversely. CD25+ status was detrimental to DFS (36 vs. 105mo, p=0.049) whereas CD103+ status was advantageous to DFS (NR vs. 42mo, p=0.06). When chemotherapy was used, purine analogues tended to have better DFS and OS (76 vs. 26mo, p=0.09) compared to CHOP-like regimens. Neither DFS nor OS was impacted by sex, CS, LN, splenic weight, or splenic white pulp involvement. Conclusions: This study presents an updated clinicopathologic data from a pooled cohort of patients with SDRPL. It identifies the clinical, immunohistochemical, and treatment modalities that are major determinants of DFS and OS in this rare and distinct B-cell lymphoma entity.

A Review on Splenic Diffuse Red Pulp Small B-Cell Lymphoma.

Splenic diffuse red pulp small B-cell lymphoma (SDRPL) is a rare disease, representing <1% of all non-Hodgkin lymphomas (NHL). The most common clinical manifestations include splenomegaly, lymphocytosis, and hemocytopenia. A diagnosis of SDRPL can be challenging, as it shares multiple clinical and laboratory features with splenic marginal zone lymphoma (SMZL), hairy cell leukemia (HCL), and HCL variant (HCL-v). Obtaining splenic tissue remains the gold standard for diagnosis. In the cases where splenic tissue is not available, diagnosis can be established by a review of peripheral blood and bone marrow studies. SDRPL is characterized by a diffuse involvement of the splenic red pulp by monomorphous small-to-medium sized mature B lymphocytes effacing the white pulp. The characteristic immunophenotype is positive for CD20, DBA.44 (20 to 90%), and IgG, and typically negative for CD5, CD10, CD23, cyclin D1, CD43, annexin A1, CD11c, CD25, CD123, and CD138. The Ki-67 proliferative index is characteristically low. Cyclin D3 is expressed in the majority of SDRPL in contrast with other types of small B-cell lymphomas, thus facilitating the recognition of this disease. There is no standard treatment regimen for SDRPL. Initial treatment options include splenectomy, rituximab monotherapy, or a combination of both. Chemoimmunotherapy should be considered in patients with advanced disease at baseline or progression.

CD5-Negative, CD10-Negative Low-Grade B-Cell Lymphoproliferative Disorders of the Spleen.

CD5-negative, CD10-negative low-grade B-cell lymphoproliferative disorders (CD5-CD10-LPD) of the spleen comprise a fascinating group of indolent, neoplastic, mature B-cell proliferations that are essential to accurately identify but can be difficult to diagnose. They comprise the majority of B-cell LPDs primary to the spleen, commonly presenting with splenomegaly and co-involvement of peripheral blood and bone marrow, but with little to no involvement of lymph nodes. Splenic marginal zone lymphoma is one of the prototypical, best studied, and most frequently encountered CD5-CD10-LPD of the spleen and typically involves white pulp. In contrast, hairy cell leukemia, another well-studied CD5-CD10-LPD of the spleen, involves red pulp, as do the two less common entities comprising so-called splenic B-cell lymphoma/leukemia unclassifiable: splenic diffuse red pulp small B-cell lymphoma and hairy cell leukemia variant. Although not always encountered in the spleen, lymphoplasmacytic lymphoma, a B-cell lymphoproliferative disorder consisting of a dual population of both clonal B-cells and plasma cells and the frequent presence of the MYD88 L265P mutation, is another CD5-CD10-LPD that can be seen in the spleen. Distinction of these different entities is possible through careful evaluation of morphologic, immunophenotypic, cytogenetic, and molecular features, as well as peripheral blood and bone marrow specimens. A firm understanding of this group of low-grade B-cell lymphoproliferative disorders is necessary for accurate diagnosis leading to optimal patient management.

VILLOUS LYMPHOCYTES IN BLOOD AND BONE MARROW IN SOME FORMS OF B-CELL LYMPHOID MALIGNANCIES

The cytological and immunocytochemical features of the lymphocytes with villous morphology in peripheral blood and bone marrow in some B-lymphoproliferative disorders were studied. The diagnosis of hairy cell leukemia, a hairy cell leukemia variant, splenic marginal zone lymphoma and splenic diffuse red pulp small B-cell lymphoma was ascertained in accordance with the new revision of the WHO classification (2016). The neoplastic cells of hairy cell leukemia were determined by the presence of high tartrate resistant acid phosphatase (TRAP) activity. Cell surface expression of CD19, CD20 and CD21 antigens was detected. Also, the expression of CD25, CD103 and CD200, and in some cases cyclin D1, was found out. CD5, CD10 and CD23 were not detected. The immunophenotype of cells in splenic marginal zone lymphoma with villous processes also corresponded to the mature B cells. The expression of CD19, CD20 and CD21 was observed in all cases, CD11c – in 50% of patients, CD25 or CD5 – in 10% of patients. In 80% of patients, the pathologic cells did not show TRAP activity. In the bone marrow and peripheral blood cells of patients with diffuse red pulp lymphoma, TRAP activity was not detected. An immunophenotype in the hairy cell leukemia variant was different from those of classic HCL (CD19+CD20+CD22+CD103+CD11c+CD5–CD10–CD23–). Characterized immunophenotypical markers, which have differential diagnostic values in several forms of lymphoid tumors of B cell origin, will be important for the choice of treatment methods and prognosis

Targeted next‐generation sequencing reveals molecular heterogeneity in non‐chronic lymphocytic leukemia clonal B‐cell lymphocytosis

Non-chronic lymphocytic leukemia (non-CLL) clonal B-cell lymphocytosis (CBL) encompasses a heterogeneous group of hematologic disorders that are still poorly understood. To shed light on their biological aspects, we retrospectively analyzed a highly selected series of 28 patients, who had a clonal B-cell population in the peripheral blood and in the bone marrow, without evidence of lymphoma. Extended targeted next-generation sequencing revealed wide molecular heterogeneity with MYD88 (14%), PDE4DIP (14%), BIRC3 (11%), CCND3 (11%), NOTCH1 (11%), and TNFAIP3 (11%) as the most mutated genes. Mutations of MYD88 were "nonclassic" in most cases. Although some genetic lesions were overlapping with indolent lymphomas, mainly splenic B-cell lymphomas of marginal zone origin and splenic diffuse red pulp small B-cell lymphoma, the genetic profile of our non-CLL CBL series seemed to suggest that various pathways could be involved in the pathogenesis of these disorders, not mirroring any specific lymphoma entity. These data better enlighten the molecular characteristics of non-CLL CBL; however, more efforts are needed in order to improve the diagnostic process, prognostication, and clinical management.

Clinicopathological analysis of splenic red pulp low-grade B-cell lymphoma.

Primary splenic low-grade B-cell lymphoma of the red pulp comprises hairy cell leukemia (HCL) and splenic B-cell lymphoma/leukemia, unclassifiable (SPLL-U). SPLL-U is a rare disease that includes subtypes of a hairy cell leukemia-variant (HCL-v), splenic diffuse red pulp small B-cell lymphoma (SDRPL) and other types that are known as narrow sense SPLL-U (SPLL-U-NS). Notably, limited information is available regarding the BRAF mutation (V600E) and cyclin D3 expression in subtypes of SPLL-U. Therefore, we performed a pathological analysis of the BRAF mutation (V600E) and characterized pathological features of SPLL-U. We reviewed the pathological findings of 12 SPLL-U cases. The 12 cases considered included two cases of HCL-v, six cases of SPLL-U-NS and four undetermined cases. The BRAF mutation (V600E) was detected in three cases, which were all SPLL-U-NS. Cases with the BRAF mutation (V600E) have increased levels of CD103 expression and decreased cyclin D3 and cyclin D1 expression compared with cases that lacked the BRAF mutation. These findings suggest that the BRAF mutation might play a significant role in SPLL-U. Therefore, the significance of the BRAF mutation should be evaluated via genomic or transcriptional analyses of a large cohort of SPLL-U patients.

Tumors of lymphoid and hematopoietic tissue of spleen: a clinicopathologic analysis of 53 cases

Objective: To study the clinicopathologic features, diagnosis and differential diagnosis of the tumors of lymphoidand hematopoietic tissue of the spleen(TLTS). Methods: Fifty-three cases of TLTS were selected from the pathologic files from Peking University People's Hospital from April 2002 to April 2017. According to WHO classification of tumors of hematopoietic and lymphoid tissues (2008) and its updated classification (2016), the cases were studied by microscopy, immunohistochemistry and in situ hybridization, combined with the bone marrow biopsy and clinical examination. Results: In 53 cases of TLTS, the male to female ratio was 3.4∶1.0; the mean age was 55.4 years (range 21-76 years), and all patients presented with variable degree of splenomegaly. Laboratory examination showed increased percentage of lymphocyte in peripheral blood in 22 cases, and elevated serum LDH level in 24 cases. Abnormal blood counts were seen in 26 cases pre-operatively, in which 22 cases showed complete or partial correction of these abnormalities post-operatively (84.6%, 22/26). The clinical symptoms included abdominal pain or distension, fatigue, fever, and weight loss, etc. Seventeen cases presented with lymphadenopathy of abdomen or other sites. Bone marrow biopsy was performed in 30 cases, and 19 cases were involved by tumor (63.3%). Of all 53 cases, 43 were diagnosed as primary splenic lymphoma (PSL), and the remaining 10 cases as secondary TLTS. According to Ann Arbor staging, 14 cases were stages Ⅰ or Ⅱ, 6 were stage Ⅲ and 28 were stage Ⅳ. By histopathologic classification, 43 cases of PSL were splenic B-cell marginal zone lymphoma (SMZL; 48.8%, 21/43), diffuse large B cell lymphoma (DLBCL; 23.3%, 10/43), splenic diffuse red pulp small B-cell lymphoma (11.6%, 5/43), mantle cell lymphoma (9.3%, 4/43), follicular lymphoma (4.7%, 2/43), and composite lymphoma (CL, DLBCL and classical Hodgkin lymphoma; 2.3%, 1/43). The remaining 10 cases were chronic lymphocytic leukaemia/small lymphocytic lymphoma (4 cases), hairy cell leukaemia (1 case), hepatosplenic T-cell lymphoma (HSTL; 5 cases), with lesions in other sites. Of the 53 cases of TLTS, 47 were B cell neoplasm (88.7%, 47/53), and the T cell neoplasms were all HSTL(5 cases, 9.4%, 5/53), 1 case was composite lymphoma. In 11 cases of TLTS, EBER in situ hybridization was performed and all cases were negative. Forty eight cases had follow-up data, and the median survival period was 17.0 months(range: 1-96 months). The survival of patients with SMZL and DLBCL were 25.7 and 18.6 months respectively. Thirteen patients died (27.1%, 13/48). The prognosis of those with elevated LDH level, high clinical stage, B symptoms and older than 60 years of age was worse. And the prognosis of DLBCL was worse than that of SMZL. There was no statistically significant difference between these factors and prognosis (P>0.05). Conclusions: Most TLTS cases present with splenomegaly and abnormal blood counts, and complete or partial remission of blood counts isseen after splenectomy. The most common pathologic types of TLTS are SMZL and DLBCL. Definite diagnosis of TLTS could be made by combining clinical features, histopathology, immunophenotype, genetics, bone marrow biopsy and laboratory examination.

Exome sequencing identifies recurrent BCOR alterations and the absence of KLF2, TNFAIP3 and MYD88 mutations in splenic diffuse red pulp small B-cell lymphoma.

Splenic diffuse red pulp lymphoma is an indolent small B-cell lymphoma recognized as a provisional entity in the World Health Organization 2008 classification. Its precise relationship to other related splenic B-cell lymphomas with frequent leukemic involvement or other lymphoproliferative disorders remains undetermined. We performed whole-exome sequencing to explore the genetic landscape of ten cases of splenic diffuse red pulp lymphoma using paired tumor and normal samples. A selection of 109 somatic mutations was then evaluated in a cohort including 42 samples of splenic diffuse red pulp lymphoma and compared to those identified in 46 samples of splenic marginal zone lymphoma and eight samples of hairy-cell leukemia. Recurrent mutations or losses in BCOR (the gene encoding the BCL6 corepressor) – frameshift (n=3), nonsense (n=2), splicing site (n=1), and copy number loss (n=4) – were identified in 10/42 samples of splenic diffuse red pulp lymphoma (24%), whereas only one frameshift mutation was identified in 46 cases of splenic marginal zone lymphoma (2%). Inversely, KLF2, TNFAIP3 and MYD88, common mutations in splenic marginal zone lymphoma, were rare (one KLF2 mutant in 42 samples; 2%) or absent (TNFAIP3 and MYD88) in splenic diffuse red pulp lymphoma. These findings define an original genetic profile of splenic diffuse red pulp lymphoma and suggest that the mechanisms of pathogenesis of this lymphoma are distinct from those of splenic marginal zone lymphoma and hairy-cell leukemia.

NOTCH1, TP53, and MAP2K1 Mutations in Splenic Diffuse Red Pulp Small B-cell Lymphoma Are Associated With Progressive Disease.

Splenic diffuse red pulp small B-cell lymphoma (SDRPL) is considered an indolent neoplasm and its pathogenesis is not well known. We investigated the molecular characteristics of 19 SDRPL patients, 5 of them with progressive disease. IGHV genes were mutated in 9/13 (69%). Cytogenetic and molecular studies identified complex karyotypes in 2 cases, and IGH rearrangements in 3, with PAX5 and potentially TCL1 as partners in each one of them. Copy number arrays showed aberrations in 69% of the tumors, including recurrent losses of 10q23, 14q31-q32, and 17p13 in 3, and 9p21 in 2 cases. Deletion of 7q31.3-q32.3 was present in only 1 case and no trisomies 3 or 18 were detected. NOTCH1 and MAP2K1 were mutated in 2 cases each, whereas BRAF, TP53, and SF3B1 were mutated each in single cases. No mutations were found in NOTCH2 or MYD88. Four of the 5 patients with aggressive disease had mutations in NOTCH1 (2 cases), TP53 (1 case), and MAP2K1 (1 case). The progression-free survival of patients with mutated genes was significantly shorter than in the unmutated (P=0.011). These findings show that SDRPL share some mutated genes but not chromosomal alterations, with other splenic lymphomas, that may confer a more aggressive behavior.

Pathomorphological Diagnosis of Splenic Diffuse Red Pulp Small B-Cell Lymphoma

Background. Unclassifiable splenic B-cell lymphoma/leukemia is a rare and poorly studied disorder introduced in the WHO classification of hematopoietic and lymphoid tissue malignancies for the first time in 2008. This type of lymphoma requires differential diagnosing between hairy cell leukemia-variant (HCL-V) and splenic diffuse red pulp small B-cell lymphoma (SDRPL). Aim. To develop criteria for diagnosis of SDRPL by comparison of bone marrow biopsies (BMB) and surgical specimens of the spleen. Methods. In the Department of Morbid Anatomy of the Hematology Research Center, preoperative BMBs and surgical specimens of the spleen (2013-2015) were compared in 71 patients (men/women ratio 1:2.6, age range 44-81, median age 58 years) using morphological and extended immunohistochemical studies. Sanger sequencing and PCR assay were carried out to analyze the mutational status of IgHV and to identify mutations in MAP2K1, NOTCH, BRAF. Results. SDRPL was diagnosed in 5 (7 %) of 71 patients. In 2 groups of patients (with normal and high WBC count), the morphological features of spleen tissue were similar to those of a neoplastic substrate of HCL-V. The immunohistochemical assay demonstrates monomorphic expression of CD20 and DBA.44 and heterogeneous expression of CD11c, TRAP, CD103, CD123 in all cases. In none of the 5 cases, expression of CD25, CD27, Cyclin D1, Annexin-1 was found. In bone marrow (unlike HCL and HCL-V), predominantly interstitial and intravascular scant CD20+ lymphoid infiltration (4 of 5 cases) was found without detectable nucleoli in nuclei of small lymphoid cells. In 1 case, there was a combined lymphoid infiltration: CD20+ microfocalinterstitial infiltration with an intravascular component. No persistent molecular mutations in the spleen tissue specimens were found. Conclusion. SDRPL is diagnosed in 7 % of splenic B-cell lymphomas. It is a rare disorder, whose verification requires an integrated approach taking into account clini cal and laboratory data, results of flow cytometry, cytological, morphological, extended IHC and molecular biological studies.

New insights in the management of patients with hairy cell leukemia.

Although hairy cell leukemia (HCL) was identified in 1958 by Bouroncle and colleagues, HCL remains in 2015 a mysterious disease. Accurate diagnosis of HCL relies on the recognition of hairy cells by morphology and flow cytometry in blood and/or bone marrow. However, there are cases difficult to diagnose, particularly in variants of HCL. Furthermore, some diseases such as splenic diffuse red pulp small B-cell lymphoma are very close to HCL and may be misdiagnosed. Major advances in the management of patients who have HCL have been made following the use of purine nucleoside analogs. However, new treatment options can be available in relapsed/refractory HCL: monoclonal antibody therapy, BRAF inhibitors, or immunotoxins. The presence of the BRAFV600E mutation was recently identified in most cases of HCL and its absence in variants of HCL and in other B-cell chronic lymphoproliferative disorders. The precise cellular origin of HCL remains elusive but BRAF mutations were detected in hematopoietic stem cells of patients with HCL. Assessment for minimal residual disease is important in clinical trials. Minimal residual disease detection can clearly predict inferior long-term outcomes or early relapses in patients with HCL. Recent reports have shown that inhibition of BRAF kinase by drugs such as vemurafenib is effective in relapsed/refractory HCL. Immunotoxins offer new opportunities even in patients without BRAF mutations. All these findings have major implications for diagnosis, monitoring, and treatment of HCL and variant forms of HCL.

Chronic B-cell lymphoproliferative disorders with hairy cells

The standardized blood smear examination is the first step in the diagnosis of a B-cell chronic lymphoproliferative disorder and can guide further investigations. In the laboratory, the identification of hairy cells on blood smear is a matter of daily practice. Hairy cell proliferations represent heterogeneous entities and their respective diagnoses can be difficult. If hairy cell leukemia (HCL) and splenic marginal zone lymphoma (SMZL) represent separate entities, the variant form of HCL (HCLv) and splenic diffuse red pulp small B-cell lymphoma (SDRPL) remain provisional entities in the 2008 WHO classification. We discuss the main clinical and biological characteristics of these four entities and appropriate means to characterize, identify and distinguish from each other; standardized blood smear examination, multiparameter flow cytometry analysis, analysis of the repertoire of immunoglobulins heavy chains genes and their mutational status (mutated or unmutated profile), molecular analyses: BRAF gene V600E mutation in HCL and MAP2K1 gene mutations in HCLv. We also discuss the main therapeutic aspects with emphasis on the new targeted drugs that enter into force in the therapeutic arsenal.

Splenic Diffuse Red Pulp Small B Cell Lymphoma: Transformation To Diffuse Large Cells B Lymphoma

ObjectiveReport immunophenotypic and genetic aspects and clinical course of a case of Splenic Diffuse Red Pulp Small B Cell Lymphoma with transformation to diffuse large cell. IntroductionUntil recently, only Hairy Cell Leukemia was recognized as B chronic lymphoproliferation with primary involvement of the splenic red pulp. The 2008 WHO classification included two new provisional entities: Splenic diffuse red pulp small-B cell lymphoma and Hairy Cell Leukemia variant. These entities are rare, encompassing less than 1% of B chronic lymphoproliferation, characteristically presenting as an indolent clinical course and good control with splenectomy. Case reportFemale, 29 years old, with non-replicative chronic hepatitis B, and splenomegaly detected 7 years before without evidence of portal hypertension or schistosomiasis. Two months before admission to our hospital, she developed increasing splenomegaly for 39cm, B symptoms and abdominal lymphadenopathy. CBC showed Hb: 8,1g/dl, platelet: 80.000/mm3, WBC: 12.000/mm3 with 85% of abnormal lymphoid cells with immunophenotyping CD45++/CD19+/CD20++/CD25+/CD23dim/CD200dim/FMC7+/CD11c-/CD103- and cykappa restriction. Complex karyotype in PB suggested lymphoproliferative disorder, with strutural abnormalities in 14q32, isocromosomes 8 and 17, deletions 6q and 7q and trisomy 18. FISH studies showed extra copy of MYC and p53 deletion. Bone marrow aspirate showed 7,6% of cells with same phenotype. CT and PET-CT showed splenomegaly of 39cm (SUV: 6.1), hepatomegaly (SUV: 3.2), abdominal lymph node conglomerate (SUV: 6.3), cervical and mediastinal lymphadenopathy with no increase in dimensions (SUV: 3,8). The patient was submitted to splenectomy and histological findings revelead mature lymphoid neoplasm, ranging from small to intermediate cells with few large cells, primary from spleen red pulp and diffusely infiltrating the sinusoidal spaces and secondly the white pulp (CD20+, DBA44+, CD5-, CD10-, CD23-, CD3-, cyclin D1- and Ki67: 40%). The cytogenetic analysis of the spleen showed similar complex karyotype as seen in PB. With the diagnosis of Splenic Diffuse Red Pulp Small B Cell Lymphoma with aggressive transformation to diffuse large cell, she was submitted to 8 cycles of R-CHOEP and is in complete remission, waiting for consolidation with high-dose chemotheraphy followed with peripheral stem cells rescue. DiscussionSplenic Diffuse Red Pulp Small B-cell Lymphoma was included as a provisional entity in WHO 2008 classification. The few cases reported show an indolent course with good response to splenectomy. In this case the diagnosis was suspect with clinical course, cytogenetic and flow cytometry PB studies and confirmed after splenectomy when the clinical course of disease changed with B symptoms, spread to peripheral blood with atypical cells and increased proliferative markers, such as LDH. This fact highlights the difficulty in establishing the diagnosis of this entity during the course of prolonged and indolent disease, manifested by splenomegaly oligosymptomatic often neglected by physicians and by the patients themselves. Disclosures:No relevant conflicts of interest to declare.