B-cell Chronic Lymphoproliferative Disorders Research Articles

Outcome of Hairy Cell Leukemia: A Single-center Study

Abstract: BACKGROUND: Hairy cell leukemia (HCL) is a rare chronic B-cell lymphoproliferative disorder. Modern therapy with purine analogs and immunotherapy can provide long-term remission, but the risk of recurrence remains about 40%–50%. The aim of this study was to evaluate the outcome of patients with HCL who received treatment in Nanakali Hospital. PATIENTS AND METHODS: A retrospective cross-sectional study was carried out on 50 patients of HCL diagnosed from 2004 to 2022 in Nanakali Hospital in Erbil City, Kurdistan Region, Iraq. Demographics, clinical presentation, treatment data, complications, response, recurrence, and survival data were collected from medical records. The results were presented with descriptive statistics. Variables were compared by Chi-square analysis. RESULTS: The mean age was 52.64 ± 12.37 years, and 84% were male. The most common presenting symptoms were splenomegaly (18%) and fatigue (14%). The majority (69.6%) received cladribine; the response rate was 73.9%, with a complete remission (67.4%). 47.8% had recurrent disease. The most common adverse effects were febrile neutropenia (58.7%) and Grade III and IV hematologic toxicity (41.3%). The results were significantly associated with ANC pretreatment (P = 0.019), comorbidity (P = 0.001), and treatment response (P = 0.004). Cladribine–rituximab combination resulted in complete remission (100%). Ten-year overall survival was 70%. CONCLUSIONS: The results were broadly consistent with literature reports, demonstrating the efficacy and safety of cladribine with/without rituximab as first-line therapy for HCL but with a 30% mortality of concern. Further studies should identify modifiable factors that affect poor prognosis in subgroups to guide improvements in risk management of HCL.

SLAM Family Receptors in B Cell Chronic Lymphoproliferative Disorders.

The signaling lymphocytic activation molecule (SLAM) receptor family (SLAMF) consists of nine glycoproteins that belong to the CD2 superfamily of immunoglobulin (Ig) domain-containing molecules. SLAMF receptors modulate the differentiation and activation of a wide range of immune cells. Individual SLAMF receptors are expressed on the surface of hematopoietic stem cells, hematopoietic progenitor cells, B cells, T cells, NK cells, NKT cells, monocytes, macrophages, dendritic cells, neutrophils, and platelets. The expression of SLAMF receptors was studied during normal B cell maturation. Several SLAMF receptors were also detected in cancer cell lines of B-lymphoid origin and in pathological B cells from patients with B cell chronic lymphoproliferative disorders (B-CLPD), the most frequent hematological malignancies in adults. This review summarizes current knowledge on the expression of SLAMF receptors and their adaptor proteins SAP and EAT-2 in B-CLPD. Several SLAMF receptors could be regarded as potential diagnostic and differential diagnostic markers, prognostic factors, and targets for the development of novel drugs for patients with B-CLPD.

Waldenström macroglobulinemia: a review of pathogenesis, current treatment, and future prospects.

Waldenström macroglobulinemia (WM) is a chronic B-cell lymphoproliferative disorder characterized by lymphoplasmacytic cell overgrowth in the bone marrow and increased secretion of IgM immunoglobulins into the serum. Patients with WM have a variety of clinical outcomes, including long-term survival but inevitable recurrence. Recent advances in disease knowledge, including molecular and genetic principles with the discovery of MYD88 and CXCR4 mutations, have rapidly increased patient-tolerable treatment options. WM patients may benefit from chemotherapy regimens that include rituximab-based regimens, alkylating drugs, proteasome inhibitors, monoclonal antibodies, and drugs targeting Bruton tyrosine kinase inhibitors. In light of these advancements, patients can now receive treatment customized to their specific clinical characteristics, focusing on enhancing the depth and durability of their response while limiting the adverse effects. Despite the rapidly developing therapeutic armament against WM, a lack of high-quality evidence from extensive phase 3 trials remains a significant challenge in the research. We believe clinical outcomes will keep improving when new medicines are introduced while preserving efficacy and minimizing toxicity.

High Frequency of Monoclonal B-cell Lymphocytosis with a High Prevalence of Biclonal Cases in the Colombian Population

The World Health Organization (WHO) defines monoclonal B-Cell Lymphocytosis (MBL) as the presence in Peripheral Blood (PB) of monoclonal populations of B-Lymphocytes (BL) of up to 5x109/L, with the phenotype of Chronic Lymphocytic Leukemia (CLL) typical, CLL atypical, or non-CLL, in the absence of B symptoms or clinical manifestations related to chronic B-Cell Lymphoproliferative Disorders (B-CLPD). It is classified as Low Count (LC) (<500 clonal BL/μL) and High Count (HC) (>500 clonal BL/μL) [1]. It has been described in up to 12% of the healthy adult population [1], a proportion that increases in relatives of patients with CLL/Small Lymphocytic Lymphoma (SLL) to around 15% [2]. It is usually a more frequent entity (28.5%) in people with Hepatitis C virus (HCV) [3] infection, and it tends to persist for a long time (90% of cases) with a progression rate of the MBL-HC to clinical manifest B-CLPD between 1-4% per year [1,2]. Currently, it continues to be an entity under study since its possible evolution to CLL or another B-CLPD is clearly unknown [4].

Evaluation of Diagnostic Usefulness of CD200 Expression in B-cell Chronic Lymphoproliferative Disorders.

Immunophenotyping by flow cytometry (FCM) is a useful diagnostic tool for the evaluation of mature B-cell neoplasms (MBN). Here, CD200 expression may play a significant role and improve the distinction between various MBNs, but any potential as a prognostic marker is yet to be established. The present prospective study was conducted on all the suspected cases of MBNs. Immunophenotyping was done using a BD FACS Canto FCM using a panel of 4 to 6 color combinations of monoclonal antibodies; CD45, CD34, CD5, CD19, CD20, CD22, CD23, CD79b, FMC7, CD10, CD38, ZAP70, CD200, IgG, IgM, CD25, CD103, CD2, CD3, CD11c as well as κ and λ light chains. CD200 expression was compared in different subgroups. Of the total of 130 cases included in the study, CD200 was positive in 118 cases (90%). CD200 was expressed in 100% of the cases of CLL(86 cases), atypical CLL(06 cases), HCL(14 cases), FL(02 cases), SMZL(04 cases), LPL (01 case), and low-grade NHL (05 cases), with the highest intensity of fluorescence in HCL followed by CLL. All the cases of MCL and PLL were exclusively negative for CD200. In conclusion, the results of the present study support inclusion of this marker in the flow cytometric panels for the differential diagnosis of MBNs.

A Flow Cytometry Panel for Differential Diagnosis of Mantle Cell Lymphoma from Atypical B-Chronic Lymphocytic Leukaemia

Differential diagnosis of chronic lymphoproliferative disorders (CLDs) has remained challenging due to the highly variable morphology features and immunophenotyping. Currently, the development of multiple-marker panel analyses by flow cytometry has opened a broad way for diagnosis of CLDs. We analyzed the peripheral blood and bone marrow samples of 131 patients with B-cell CLDs (including 91 chronic lymphocytic leukemia (CLL), 15 atypical CLL, 14 mantle cell lymphoma (MCL), and 11 CD5-/CD10-lymphoma patients) from April 2018 to April 2019, using a panel of specific markers by flow cytometry. Our results indicated that the expression pattern of CD22, CD23, FMC-7, and CD5 allowed us to accurately and differentially diagnose the B-CLL, MCL, and CD5-/CD10- lymphoma, while it was not capable of differentiating MCL from atypical CLL. We, however, found that the expression patterns of CD38 and immunoglobulin light chain differed significantly between atypical B-CLL and MCL. CD38 and lambda light chain were remarkably expressed in MCL patients (92.8% and 85%, respectively) compared to the atypical CLL (1.1% and 0% respectively), with the p value less than 0.001 for both markers. In contrast to MCL patients, all the patients with atypical CLL, expressed kappa light chain. The immunohistochemistry method used for cyclin D1 confirmed that the flow cytometry detection of kappa and lambda light chains could provide a new approach with high sensitivity (91%) and moderate specificity (50%) to distinguish MCL patients from atypical B-CLL. Expression of CD5, CD20 (bright), CD22, FMC-7, CD38, and lambda light chain with no expression of CD23 can accurately detect MCL and differentiate it from atypical B-CLL

Waldenstrom macroglobulinemia with 11q deletion: A rarely diagnosed entity with review of literature

Waldenstrom macroglobulinemia (WM) is a rare, chronic, and indolent B-cell lymphoproliferative disorder characterized by bone marrow infiltration by small lymphocytes, lymphoplasmacytoid cells, and plasma cells along with the presence of a detectable monoclonal immunoglobulin M. It represents 1%–2% of hematological malignancies with an overall incidence of 3–4 cases/million persons/year. Some deletions are associated with a more aggressive IgM gammopathy and have a high probability of symptomatic transformation. 6q deletion, the most common cytogenetic abnormality, which is present in 42% of cases whereas 11q deletion is rare in WM and is present in only 8% of cases. We are presenting a case of a 70-year-old male patient diagnosed as WM with 11q deletion.

IBCL-429 Prevalence of B-Cell Chronic Lymphoproliferative Disorders at a Large Tertiary Care Center in India

Chronic lymphoproliferative disorders (CLPD) are a heterogeneous group of diseases characterized by uncontrolled production of lymphocytes that cause monoclonal lymphocytosis, lymphadenopathy, bone marrow infiltration, and specific immunophenotypic profile. CLPDs are relatively uncommon in Asian populations, constituting around 2% of all leukemias. The frequency of CLPDs shows geographical variations owing to distinct genetic or environmental factors that play a role in the development of these diseases. To determine the frequency and distribution of various B-cell CLPDs (B-CLPDs) diagnosed at a large tertiary care center in north India. We conducted a retrospective analysis of all patients diagnosed with B-CLPD at the All India Institute of Medical Sciences, New Delhi, between the years 2015 and 2019 to determine the prevalence, frequency, and distribution of B-CLPD in our patient population. The diagnosis of B-CLPD was based on morphologic and immunophenotypic criteria proposed by the World Health Organization (WHO) in 2008. A total of 439 patients were diagnosed with B-CLPD at our center with a median age of 57 years, and male:female ratio was 2.5:1. Chronic lymphocytic leukemia (CLL)/small lymphocytic leukemia was the most common form, constituting 58.31% of all. Other B-CLPDs identified in our cohort were follicular lymphoma (FL) 13.43%, mantle cell lymphoma (MCL) 6.83%, marginal zone lymphoma 6.37%, lymphoplasmacytic lymphoma/Waldenström macroglobulinemia 2.51%, mucosa-associated lymphatic tissue lymphoma 2.05%, hairy cell leukemia 1.59%, prolymphocytic leukaemia 0.68% and rest 8.6% were B-CLPD unclassified. This is one of the largest studies from India. CLL is the most common B-CLPD followed by FL and MCL.

Poster: IBCL-429 Prevalence of B-Cell Chronic Lymphoproliferative Disorders at a Large Tertiary Care Center in India

Poster: IBCL-429 Prevalence of B-Cell Chronic Lymphoproliferative Disorders at a Large Tertiary Care Center in India

Hairy Cell Leukemia (HCL) and HCL Variant: Updates and Spotlights on Therapeutic Advances.

This article aims to bring an update on the recent discoveries in hairy cell leukemia (HCL), especially findings in pathophysiology and therapeutic advances. Major discoveries have been made in genetics and epigenetics of HCL. Moreover, the importance of several signaling pathways and tumor microenvironment has been recently highlighted. These findings led to the development of new targeted therapies which have shown interesting results in recent clinical trials. HCL is a chronic B-cell lymphoproliferative disorder. Most patients respond to purine nucleoside analogs (PNA) like cladribine or pentostatin. However, relapses are frequent and the disease often becomes less sensitive to chemotherapy. Recent discoveries in pathophysiology, like the presence of the V600E mutation of the B-raf proto-oncogene (BRAF) gene and the importance of the B-cell receptor (BCR) pathway, led to the development of new drugs for relapsed/refractory (R/R) HCL patients. The variant-type of HCL (HCL-V) is usually less sensitive to PNA. Chemo-immunotherapy using PNA and rituximab (R), BRAF, MEK, or Bruton Tyrosine Kinase (BTK) inhibitors may be used. Good results were recently published and achieved with moxetumomab pasudotox (Moxe), an anti-CD22 immunoconjugate. In this review, we will present an update on HCL and HCL-V, focusing on pathophysiology and recent therapeutic advances.

Research progress on epigenetics of small B-cell lymphoma.

Small B-cell lymphoma is the classification of B-cell chronic lymphoproliferative disorders that include chronic lymphocytic leukaemia/small lymphocytic lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma/Waldenstrom macroglobulinemia. The clinical presentation is somewhat heterogeneous, and its occurrence and development mechanisms are not yet precise and may involve epigenetic changes. Epigenetic alterations mainly include DNA methylation, histone modification, and non-coding RNA, which are essential for genetic detection, early diagnosis, and assessment of treatment resistance in small B-cell lymphoma. As chronic lymphocytic leukemia/small lymphocytic lymphoma has already been reported in the literature, this article focuses on small B-cell lymphomas such as follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, and Waldenstrom macroglobulinemia. It discusses recent developments in epigenetic research to diagnose and treat this group of lymphomas. This review provides new ideas for the treatment and prognosis assessment of small B-cell lymphoma by exploring the connection between small B-cell lymphoma and epigenetics.

The Contribution of CD148, CD180 and CD200 Combination in the Diagnosis of Chronic B-Cell Lymphoproliferative Disorders

Introduction: B-cell immunophenotype could be swiftly assessed by flow cytometry on blood samples or bone marrow aspirate specimens. It provides crucial information later refined with histologic, genetic and molecular features to assert accurate diagnosis of chronic B-cell lymphoproliferative disorders (B-CLPD). Besides Matutes score we identified additional useful markers, i.e. CD148 and CD180 to classify mantle cell lymphoma (MCL) and marginal zone lymphoma (MZL), respectively. Furthermore, CD200 is known to be highly expressed in chronic lymphoid leukemia (CLL) while absent in MCL.Hypothesis: The determination of CD148, CD180 and CD200 expression on B-cells by flow cytometry on blood samples and/or bone marrow aspirates could be a potent tool to accurately identify B-CLPD. We postulated the existence of the following specific expression patterns in B-CLPD: CD148 dim/CD180 dim/CD200 bright for CLL, CD148 dim/CD180 dim/CD200 dim for lymphoplasmocytic lymphoma (LPL), CD148 bright/CD180 dim/CD200 neg/dim for MCL and CD148 dim/CD180 bright/CD200 dim for MZL .Methods: In a prospective study we investigated the expression of CD148/CD180/CD200 on B-cells from 673 patients at the time of B-CLPD diagnosis in our hospital from 2014 to 2020. We analyzed 440 blood and 233 bone marrow aspirate specimens using a BD FACSCanto II flow cytometry instrument. Based solely on CD148/CD180/CD200 specific expression patterns we postulated a diagnosis of CLL, LPL, MCL or MZL. These postulated diagnoses were later confronted to the final diagnoses when all histologic, genetic and molecular features were finalized. Sensitivity, specificity, positive and negative predictive values of the expression profiles were determined. In addition, to investigate the relative importance of these three CD markers we then normalized their mean fluorescence intensities (MFI) and applied several supervised machine learning algorithms including Logistic Regression, Random Forest and Light Gradient Boosting Machine (LightGBM).Results: Out of the 673 clinical samples the CD148/CD180/CD200 expression patterns classified 212 specimens as CLL/SLL (30.8%), 160 as LPL (23.8%), 76 as MCL (11.28%) and 169 as MZL (25%). These diagnosis hypotheses were retrospectively compared to the final diagnoses based on all histologic, genetic and molecular features These diagnosis hypotheses of CLL, LPL, MCL and MZL were consistent with the final diagnosis in 583 out of the 617 corresponding cases (94%) with high positive and negative predictive values. The characteristics of the diagnosis accuracy are detailed in the table below. HCL and FL were not further investigated as their immunophenotype usually do not overlap with those of other B-CLPD.Seventeen out of 617 patients (17/617, 5.3%) did not displayed a clear CD148/CD180/CD200 pattern: 9 LPL, 4 CLL and 4 MZL. In sixteen patients (16/617, 5.0%) the diagnosis hypothesis based on this strategy was not confirmed after completion of the exploration including karyotype, MYD88 L265P mutational status, CCND1 overexpression and pathology explorations.We next investigated the relative importance of these 3 markers. We focused on MFI values of CD148, CD180 and CD200 and three categorical “positive or negative” markers (CD5, CD23, FMC7) that were assembled into a composite marker. After Cox-box normalization of CD148, CD180 and CD200 MFIs, a set of supervised machine learning algorithms including Logistic Regression, Random Forest and Light Gradient Boosting Machine (LightGBM) were applied to the cohort of CLL, LPL, MCL and MZL.We established that the highest diagnosis weights were obtained for CD200 in CLL, CD200 and CD148 in MCL (negatively and positively, respectively), CD180 in MZL. In LPL, CD148, CD180 and CD200 had the highest weights using LightGBM and Random Forest algorithms, while Logistic Regression determined that CD5 and CD23 had the highest (negative) weights.In conclusion, the determination of CD148/CD180/CD200 surface expression patterns by flow cytometry, along with morphology, allowed to assert an accurate diagnosis hypothesis in CLL, MCL, LPL and MZL with high positive and negative predictive values. Machine learning algorithms allowed to measure the relative importance of these markers, that could be of great help in case of discordant expression of the main diagnosis markers. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

IBCL-034: Hairy Cell Leukemia: Single-Center Experience

Context Hairy cell leukemia (HCL) is an uncommon, chronic B-cell lymphoproliferative disorder that represents approximately 2% of all leukemias. The lack of large national and international registries on HCL has limited the ability to identify clear clinico-epidemiological characteristics in this rare disease. Objective To evaluate the HCL patients in the Egyptian population with regard to epidemiological, pathological, immunophenotypic, and clinical data, as well as treatment modalities and outcomes. Patients This is a retrospective observational study in which 92 patients diagnosed with HCL were reviewed through electronic health records between January 1, 2008, and December 31, 2019. Results Median age of patients was 50 years. The prevalence of hepatitis C virus (HCV) infection was triple that of the general population and appeared to have no effect on overall survival (OS). Most of the patients had varying degrees of cytopenia, varying from pancytopenia (50%), bicytopenia (43.5%), thrombocytopenia (69.3%), leukopenia (53%), and anemia only (6.5%). Hairy cells in peripheral blood samples were observed in 18.5% of cases. Sixty-one patients received cladribine, five patients received interferon (INF-a), six patients had palliative splenectomies, five patients were subjected to watchful waiting, and 15 patients received supportive therapy. The presence of comorbidities, age > 60 years, fever, pancytopenia, hemoglobin level Conclusions HCL in Egypt is a rare NHL that has different clinicopathological features, especially in association with hepatitis C infection, which necessitates participation in international registries for HCL, e.g., Hairy Cell Leukemia Foundation, to investigate the most optimal treatment strategies for these neoplasms in the real-world population.

Preclinical Anti-Lymphoma Activity of the BCL-2 BH4 Domain Antagonist Bda-366 in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is an aggressive and incurable B-cell non-Hodgkin lymphoma. MCL is genetically characterized by t(11;14) translocation, which leads to cyclin D1 overexpression. Despite the introduction of high-dose chemotherapy followed by autologous stem cell transplantation and novel agents including the Bruton's tyrosine kinase inhibitor ibrutinib, the proteasome inhibitor bortezomib and the immunomodulator lenalidomide, MCL still remains an incurable disease. BCL-2 is often amplified and/or overexpressed in MCL; high-level copy number gains of the BCL2 locus at 18q21.3 are most frequent. The novel BCL-2BH4 domain antagonist BDA-366 converts anti-apoptotic BCL-2 into the pro-apoptotic molecule (Han et al., Cancer Cell 2015). Considering the promising clinical activity of the BH3 mimetics venetoclax (Davids et al., JCO 2017), we investigated anti-lymphoma effects of BDA-366 in MCL. A total of eight MCL cell lines (Z-138, JVM-2, Granta-519, MINO, JeKo-1, REC-1, MAVER-1 and NCEB-1) were used. Z-138, Granta-519, MINO and NCEB-1 have high-level DNA amplification of BCL-2 . BDA-366 induced dose- and time-dependent phosphatidylserine externalization in MCL cells, with 72-h ED50 (effective concentration inducing 50% cell killing as measured by annexin V positivity) values ranging from 9.3 to 240 nM. 72-h IC50 values (concentration at which cell growth is inhibited by 50%) were also determined, which ranged from 5 to 30 nM. BDA-366 showed smaller differences in sensitivity than venetoclax, raising the possibility that BDA-366 may exhibit broader anti-lymphoma activity against heterogeneous MCL backgrounds. Basal levels of BCL-2 (determined by flow cytometry) did not significantly affect MCL cell susceptibility (ED50) to BDA-366 (r = -0.08, P = 0.85). The presence of high-level DNA amplification of BCL-2 was not associated with BDA-366 sensitivity either. BDA-366 did not increase total BCL-2 levels but it actively promoted the exposure of pro-apoptotic BH3 domain of BCL-2, supporting its proposed mode of action. BDA-366 did not significantly affect cell cycle distribution; S and G2/M phase cells appeared to be susceptible to BDA-366-induced apoptosis. BDA-366 induced loss of mitochondrial membrane potential, caspase-3 cleavage and BAX activation, indicating mitochondrial apoptosis. p53 knockdown (>85% knockdown efficiencies, as determined by Western blotting) desensitized p53 wild-type cell lines Z-138 and JVM-2 to BDA-366 (ED50 values of 29 nM and 10 nM in Z-138; 303 nM and 108 nM in JVM-2), implying the interference of p53 with BCL-2 signaling. BDA-366 synergized with venetoclax to induce apoptosis in venetoclax-sensitive Z-138 (averaged combination index (aCI) = 0.25) and MINO cells (aCI = 0.14), indicating that the combination may abrogate BCL-2-mediated survival signaling in MCL. The combination effects of BDA-366 with doxorubicin or AraC were additive (aCI ~ 1.0). BDA-366 induced apoptosis in primary leukemia cells from patients with B-cell chronic lymphoproliferative disorders. BDA-366 appeared to take longer time to fully induce apoptosis than ABT-199, perhaps reflecting the distinct mode of action of BDA-366 that changes BCL-2 conformation. From a clinical viewpoint, the slower pharmacodynamics in inhibiting BCL-2 and inducing MCL cell death may reduce the risk of tumor lysis syndromes in patients receiving BDA-366. Collectively, BCL-2 BH4 inhibition may offer a novel therapeutic strategy for MCL. DisclosuresNo relevant conflicts of interest to declare.

Flowcytometry in Chronic B-Cell Lymphoproliferative Disorders: A Retrospective Study from a Tertiary Oncology Centre, Trivandrum, India

Introduction: Chronic B-Cell Lymphoproliferative Disorders (B- CLPD) are malignant neoplasms of B lymphocytes characterised by accumulation of mature B lymphocytes in the Bone Marrow (BM), peripheral blood, and lymphoid tissues. Multiparameter flowcytometry has become a powerful tool in diagnosing B-CLPD that identifies a clonal light-chain restricted population expressing B-cell markers. Aim: To study the morphologic and immunophenotypic profile of B-CLPD by flowcytometry and to determine the incidence of various subtypes. Materials and Methods: All consecutively diagnosed cases of chronic B lymphoproliferative disorders at Regional Cancer Centre, Trivandrum, Kerala, India from 1st December 2016 to 30th November 2018 were retrospectively analysed and studied. Diagnosis of CLPD was made based on peripheral smear and BM aspiration and immunophenotyping by flowcytometry. Flowcytometry was performed using BD FACS Verse flowcytometer. The results of immunophenotyping by flowcytometry were reviewed and analysed. Results: During the study period, 231 cases were diagnosed as Chronic Lymphoproliferative Disorder (CPLD), of which 209 cases were B-CLPDs. Male to female ratio (M:F) was 2:1. In the present study, incidence of Chronic Lymphocytic Leukaemia (CLL) was 138 cases (66.03%), Follicular Lymphoma (FL) was 14 cases (6.7%), Hairy Cell Leukaemia (HCL) was 11 cases 5.26% and Mantle Cell Lymphoma (MCL) was 9 cases, (4.31%). Prolymphocytic leukaemia and splenic Marginal Zone Lymphoma (MZL) constituted 4 cases (1.91%) each. Some cases of B-CLPDs had no definite diagnoses which were diagnosed as CD5 positive B-CLPD unclassified 13 cases (6.22%) and CD5 negative B-CLPD unclassified 16 cases (7.66%). Conclusion: Flowcytometry has helped in definite subtyping of B-CLPD in most cases. Most common subtype in the present study was CLL. CD5+/CD23+ is highly specific for diagnosing CLL. CD200 is revealed to be an excellent marker to distinguish CLL from MCL.

Cell Surface Proteins of B Cells and Plasmacells Are Differently Expressed in Waldenström’s Macroglobulinemia (WM) Patients Vs. Subjects with Monoclonal Gammopathy of Uncertain Significance (IgMMGUS) Vs. Healthy Donors

We performed gene expression profiling (GEP) of 36 patients with Waldenström's Macroglobulinemia (WM), 13 subjects with Monoclonal Gammopathy of Uncertain Significance (IgMMGUS), and 7 healthy subjects (CTRLs) by Affymetrix GeneChip Human Genome U133 Plus 2.0. GEP was performed on 36 WM bone marrow (BM) CD19+ cells vs. 10 IgMMGUS BM CD19+cells vs. 7 CTRLs BM CD19+ cells. We analyzed 32 WM BM CD138+ cells vs. 10 IgMMGUS BM CD138+ cells vs. 7 CTRLs BM CD138+ cells by microarray. Data was preprocessed and normalized using RMA and ComBat and analyzed using Statistical Analysis for Microarrays (SAM) on 3 groups and a false discovery rate threshold of 5%, followed by a pair-wise SAM test. We found 2038 and 29 unique genes in the comparison between WM, IgMMGUS and CRTLs in CD19+ and CD138+ cells, respectively. There were 16 differently expressed (DE) genes in common between the CD19+ and the CD138+ cells. Genes were grouped accordingly to the following patterns of differential expression: 1) WM>MGUS>CTRL, 2) WM<MGUS<CTRL, 3) WM>MGUS but MGUS<CTRL, 4) WM<MGUS but MGUS>CTRL (Tab.1). Functional enrichment analysis and annotation clustering (DAVID) were run on each pattern. We demonstrated gene expression changes between WM, IgMMGUS and CTRLs in genes encoding hematopoietic cell lineage markers. We showed thatIL2RA, CD1D,andCD44were over expressed in WM vs. IgMMGS, and WM vs. CTRLs.CD25expression characterizes Waldenström's clone (García-Sanz et. al. Best Pract Res Clin Haematol 2016). The over expression ofCD1Don WM B cells compared to IgMMGUS and CTRLs, suggests its possible role as a marker in WM as previously shown in B-cell chronic lymphoproliferative disorders (Kotsianidis et. al. Am J Clin Pathol 2011). We found thatCD44,encoding a glycoprotein over expressed in cancer stem cells, was up regulated in WM B cells compared to the same cell counterpart of IgMMGUS and CTRLs (Chen et. al. J Hematol Oncol 2018). Functional clustering identified several genes encoding cell adhesion molecules (CAM) which are expressed on the surface of B lymphocytes and are involved in the binding with other cells, immune response (T cells), and inflammation. Class I histocompatibility genesHLA-A, HLA-C, HLA-B, HLA-E,andHLA-Gwere over expressed in WM vs. IgMMGUS, and WM vs. CTRLs. Previous studies demonstrated the higher levels of HLA-Gs in WM and IgMMGUS compared to CTRL. Moreover, HLA-G could represent a marker of clonality since high levels of HLA-Gs were observed in both IgG and IgA MGUS and MM patients (Moreau et. al. EJH 2008). CNTNAP2andADAM23were down regulated in WM vs. CTRLs.CNTNAP2,encoding CASPR2 protein, is expressed in CNS and PNS and is involved in neurodevolpmental disorders and the autoimmune disease Sjogren's syndrome (SS) (Xiao Clin Rheumatol 2017). In addition, Caspr2 and ADAM23 take part in the peripheral neuropathy by controlling the activity of potassium channels in neurological autoimmune diseases (Saint-Martin et al. Eur J Neurosci 2018). We can speculate the role of both genes in WM patients with Sjogren's syndrome. VWF(von Willebrand factor),TFPI, andSERPINE1belonging to the complement and coagulation cascade were under expressed in WM vs. IgMMGUS. Notably, some authors have demonstrated that a low level of VWF was associated with high serum IgM levels and withCXCR4mutations in WM patients, and that response to treatment improves VWF in WM patients (Castillo et. al. BJH 2019). Genes encoding zinc finger proteins (ZNF) (n=120) were deregulated in the comparison between WM vs. IgMMGUS, and vs. CTRLS CD19+ cells in all patterns. We suggest the possible role of ZNF in the transcription regulation and cancer progression in the comparison between the 3 groups of subjects. Of note,ZNF804AandZNF215were upregulated in WM vs. IgMGUS and WM vs. CTRLs with high FC (Guerrera et. al. Haemat 2018). TJP1was under expressed in WM CD19+ and WM CD138+ cells. We noted very high FC in CD138+ cells: WM vs. IgMMGUS (FC=-2,65), and WM vs. CTRLs (FC=-6,18). HighTJP1expression in myeloma plasma cells was associated to a better and longer response to protease Inhibitors (PI) suggesting this gene as a biomarker to identify patients which can benefit from PI-based therapy (Zhang et. al. Cancer Cell 2016). Our findings demonstrate that B cells show different gene expression levels in membrane adhesion and cell lineage pathways in WM compared to IgMMGUS and CTRLs, suggesting the possible role of these genes in the progression of IgMMGUS to WM. Disclosures Tedeschi: AstraZeneca:Consultancy, Membership on an entity's Board of Directors or advisory committees;Janssen spa:Membership on an entity's Board of Directors or advisory committees, Speakers Bureau;AbbVie:Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau;BeiGene:Consultancy, Membership on an entity's Board of Directors or advisory committees;Department of Hematology Niguarda Hospital Milano:Current Employment;Sunesis:Consultancy.Montillo:F. Hoffmann-La Roche:Honoraria, Research Funding;Astra Zeneca:Honoraria;AbbVie:Honoraria, Speakers Bureau;Janssen:Honoraria, Speakers Bureau;Verastem:Honoraria;Gilead:Honoraria, Speakers Bureau.

Long-Term Treatment of Hairy Cell Leukemia Patients with Interferon: Clinical and Molecular Aspects

Long-Term Treatment of Hairy Cell Leukemia Patients with Interferon: Clinical and Molecular Aspects

Difference of genomic copy numbers alterations between hairy cell leukemia-variant and classical hairy cell leukemia: a pilot retrospective study in Chinese.

Background: Hairy cell leukemia (HCL) is a rare chronic B-cell lymphoproliferative disorder. It has two pathological subtypes: classical HCL (HCL-C) and HCL-variant (HCL-V). HCL-C and HCL-V are distinct in morphology and immunophenotype. Their differentiation is important for patient management and clinical outcome, with HCL-V responding poorly to conventional HCL treatments. Recently, whole genomic sequencing has been used to identify the difference between HCL-C and HCL-V and mutation of BRAFV600E has been proved to be a molecular hallmark of HCL-C. However, BRAF inhibitors were not effective in all HCL-C cases and HCL-V seems be lack of the high-frequency mutations. Therefore, it is necessary to compare the genomic changes between HCL-C and HCL-V by high-resolution studies, especially in Chinese population, the genomic alterations of HCL have rarely be reported.Methods: In this study, the clinical features of a total of 18 Chinese HCL patients were described. Single nucleotide polymorphism (SNP) array analysis was performed to evaluate the genomic copy number alterations (CNA) and copy neutral loss of heterozygosity (CN-LOH) on six HCL-Vs with CD25-/BRAFV600E- and four HCL-Cs with CD25+/BRAFV600E+.Results: A total of 24 CNAs including seven chromosomal gains and 17 chromosomal losses, and 22 CN-LOHs were revealed. Five of the six cases of HCL-V showed 15 CNAs including four cryptic chromosomal gains and 11 chromosomal losses. Overlapping regions involving micro-deletion of chromosome 2q13 and large chromosomal loss of 14q were showed in HCL-V. In HCL-C, a total of nine CNAs were revealed in three of the four cases including three chromosomal gains and six chromosomal losses. No overlapping area was observed among the CNVs. 15 CN-LOHs were showed in five of the six cases of HCL-V and seven CN-LOHs was demonstrated in all of the four HCL-Cs.Conclusions: Comparing to Westerners, a relatively higher proportion of HCL-V in all HCL is observed in this study. CNAs and CN-LOHs were common in both HCL-V and HCL-C but the CNAs were different in them. HCL-C was characterized with the higher ratio of large chromosomal changes but lacked of recurrent CNAs, while HCL-V was presented with the higher incidence of cryptic CNAs and recurrent CNAs involving tumor-associated genes. It is necessary to further investigate the association of the genes, such as NPHP1 and TRAF3 genes, and HCL-V in the future study.

Clonal B-Cell Lymphocytosis with Mariginal-Zone Features: Comparison with Overt Splenic Marginal-Zone Lymphomas in 77 Patients from a Monocentric Series

Diagnosis of non-CLL B Cell Chronic Lymphoproliferative Disorders (BCLPD), defined by the detection of peripheral blood (PB) B-Cell clone with flow cytometry (FC) Matutesscore≤3, is not an infrequent event in clinical daily practice. Only a portion of these cases may be actually classified according to the 2016 revision of WHO classification of lymphoid neoplasms, as in these cases a non-bone marrow (BM) tissue biopsy is rarely available due to "liquid-only disease" (BM, PB, splenomegaly) or difficult accessible sites. The two most likely diagnosis of BCLPD according to WHO 2016 are splenic marginal-zone lymphoma (SMZL) and monoclonal B Cell lymphocytosis (MBL) with non-CLL phenotype (CD19+, CD20+, SIg+, CD5-), sometimes reported as MZL-like, defined by monotypic clonal B lymphocytes (CBL) <5 x 109/l. A recent study pointed out a similar outcome regardless CBL value exceeding or not this arbitrary cut-off in strictly defined cases (absence of cytopenias, splenomegaly and adenopathy) of clonal B cell lymphocytosis with marginal-zone features (CBL-MZ) and confirmed the really indolent behaviour of this entity (Xochelli, Blood 2014). On these bases we decided to retrospectively collect clinical, immunophenotypic, molecular features and outcome of all consecutive patients (pts) with CD5- CBL-MZ owing to the Department of Hematology of University of Insubria (Varese, Italy) from 2011 to 2019 and to compare them with overt SMZL cases. Excluding 5 SMZL pts who were splenectomized, all cases were diagnosed by means of non-BM tissue biopsy, namely BM histology and PB/BM FC, FISH and molecular studies, when appropriate, and complete laboratory and imaging staging procedures (CT scan or US). The primary objective of the study was time to progression (TTP) for CBL-MZ group. Secondary objectives were overall survival (OS), progression-free survival (PFS) for treated pts and evaluation of early progression <24 months (Early POD) (Luminari, Blood 2019). Overall we collected complete data of 33 pts with CD5- CBL-MZ, and 44 with SMZL (Table 1). Focusing on CBL-MZ, FC immunophenotypic findings showed a B-Cell population with Matutes score 0 to 2 in all cases, uniform expression of B Cell antigens, namely CD19, CD20 strong, FMC7 (93%), SIg (intermediate/bright in 71%), while CD10 and CD5 were consistently negative. CD23 was expressed in 18%, CD38 in 9% and CD49b in 95%. BM sinusoidal infiltration pattern was detected in 50% of cases. TP53 mutation/deletion was detected in 4/16 pts (25%) while trisomy 12 and del13q in 1 pt each. Twenty pts presented with <5000/mmc CBL (MBL-MZ) and 13 with ≥5000/mmc CBL. With a median follow-up of 1.8 years, 6 pts (18%) progressed (4 in SMZL, 1 NMZL and 1 SLL; Fig 2) with a median TTP of 1.7 yrs (range 0.3- 12.3), while the remaining 27 remained completely stable. Among 4 pts treated, 2 received Rituximab-based regimen, both achieving CR and 2 with Chlorambucil both experiencing progression. Overall, 5 pts died (2 for progressive lymphoma), with an estimated 5-yrs of 80%. Concerning prognostic factors, after adjusting by competing risk method, at univariate analysis age >60 yrs, CBL >5000/mmc and albumin <3.5 g/dl, were associated to shorter TTP, while age >60 yrs, Hb <11 g/dl, ECOG PS ≥2 and albumin <3.5 g/dl resulted predictive of worse OS (Table 2). By applying multivariate Cox regression model, only age >60 yrs and CBL>5000/mmc retained prognostic significance for TTP (p<0.0001), while ECOG PS ≥2 and albumin <3.5 g/dl remained independently associated with inferior OS (p<0.0001). Considering the SMZL group, 34 pts underwent systemic treatment (rituximab-based in 32, including R-Bendamustine in 17). With a median follow-up time of 2.8 yrs, 9 pts progressed (4 of whom within 24 months from treatment initiation), with a median PFS 2.9 yrs, and 8 died (7 due to progression, including 3 DLBCL transformations) with an estimated 5-yrs OS of 79%. Notably, Early POD SMZL pts exhibited a significant increased risk of death (p<0.0001, fig.2), while in CBL-MZ did not (p=0.4). In conclusion, CBL-MZ confirmed to be associated with indolent course and preferential evolution toward SMZL, of which it can be viewed as an initial phase or a precursor entity. In contrast with previous findings, the WHO 16 CBL cut-off defining MBL (5 x 109/l), together with advanced age, retained prognostic relevance for TTP. Finally, we confirmed for the first time in an independent series of SMZL the highly prognostic impact of Early POD24. Disclosures Passamonti: Roche: Consultancy; Janssen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau.

French National Retrospective Cohort of Hairy-Cell Leukemias: Risk of Second Malignancies after 10 Years of Follow-up

Introduction: Hairy-cell leukemia (HCL) is a chronic B-cell lymphoproliferative disorder with a favorable outcome thanks to treatment with purine analogues (PNA) like cladribine and pentostatin. Here, we updated the French national retrospective cohort of HCL after 10 years of follow-up, in order to evaluate the risk of second cancers in these patients. Methods: Data were collected up to June 2018 through a questionnaire sent to the members of the Société Française d'Hématologie, and centralized in the cohort database. We described the second malignancies observed during the follow-up, distinguishing second 'solid' cancers from second hematological malignancies. Then, using a Fine and Gray model, we performed a multivariate analysis in order to identify second cancer risk factors. Finally, to evaluate the excess of cancers in our cohort in comparison with the French general population, we calculated the standardized incidence ratio (SIR). Results: 279 patients (pts) from 19 centers were included in our retrospective cohort. The median age was 59 years old (range 29-88). 21% had an infectious disease at diagnosis, 23% had a familial history of cancer and 11% a personal history of cancer before HCL diagnosis. The median number of lines of treatments was 1 (0-7). PNA (cladribine or pentostatin) were the first therapeutic choice in frontline (75% of pts) and at relapse (69%). With a median follow-up of 127 months (2-413), the median overall survival for the overall study population was 328 months (95% CI 299-357) and the median relapse-free survival (RFS) was 136 months (95% CI 109-163). Pts treated with cladribine or pentostatin in first line had a statistically significant better RFS than pts treated with 'other' treatments (log rank test, p < 0.001). The 10-year cumulative incidence of relapse was 39% (95% CI 33-46). Pts who received treatments other than PNA in first line had a higher risk of relapse (Gray's test, p < 0.001). For pts receiving PNA in first and second lines, there was no difference in outcomes between those who switched PNA and those who did not. In this cohort, we observed 68 second malignancies during the follow-up: 49 solid cancers (most prevalent: prostate and non-melanoma skin cancers) and 19 hematological malignancies (most prevalent: monoclonal gammopathy of undetermined significance (MGUS) and myelodysplastic syndromes (MDS)). The median onset of second cancer, second solid cancer and second hematological malignancy from HCL diagnosis was 81 months, 99 months and 78 months, respectively. The median age at diagnosis of cancer, solid cancer and hematological malignancy was 70, 69 and 77 years old, respectively. Considering death as a competing risk, the 10-year cumulative incidence of cancer, solid cancer and hematological malignancy was 15% (95% CI 11-19), 11% (95% CI 7.2-15), and 5.0% (95% CI 2.8-8.2), respectively. In multivariate analyses, IFN treatment was associated with a decreased risk for all cancers (Fine and Gray regression model, subdistribution Hazard Ratio (sdHR) 0.53 (95% CI 0.29-0.97); p = 0.038), a familial history of cancer was a risk factor for solid cancers (sdHR 2.12 (95% CI 1.15-3.91); p = 0.017), a personal history of cancer was a risk factor for hematological malignancies (sdHR 3.47 (95% CI 1.14-10.55); p = 0.028). Even after excluding non-melanoma skin cancers and MGUS, there was an excess of cancers (SIR = 2.22), solid cancers (SIR = 1.81) and hematological malignancies (SIR = 6.67). Conclusions: In this updated real-world retrospective cohort with a long follow-up and most pts treated with PNA, we highlighted the importance and the excess of second cancers in HCL patients, in particular hematological malignancies. Figure Disclosures Paillassa: Janssen: Other: Bibliography board with young hematologists. Thieblemont:Roche: Honoraria, Research Funding; Gilead: Honoraria; Novartis: Honoraria; Kyte: Honoraria; Janssen: Honoraria; Celgene: Honoraria; Cellectis: Membership on an entity's Board of Directors or advisory committees. Hermine:AB Science: Membership on an entity's Board of Directors or advisory committees. Feugier:janssen: Honoraria, Research Funding, Speakers Bureau; gilead: Honoraria, Research Funding, Speakers Bureau; roche: Honoraria, Research Funding, Speakers Bureau; abbvie: Honoraria, Research Funding, Speakers Bureau. Troussard:Innate Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche: Other: Research Support; Sysmex: Other: Research Support.