Splenic Lymphoma With Villous Lymphocytes Research Articles

ROLE AND RELEVANCE OF THE EUROFLOW ANTIBODY PANEL IN IMMUNOPHENOTYPING OF CHRONIC LYMPHOPROLIFERATIVE DISORDERS

Detection of phenotypically aberrant and clonal mature lymphocytes is the diagnostic hallmark of chronic lymphoproliferative disorders (CLPD). B-CLPD is the commonest of all the CLPD. In this study we evaluated the role of CD200 and CD43 new markers that have been introduced in the Euroflow panel in the separation between chronic lymphocytic leukemia (CLL) and all other mature B cell malignancies .These markers were also correlated with the markers used in the Matutes score like FMC7 and Surface membrane Immunoglobulin. Patient samples between October 2017-February 2018, peripheral blood or bone marrow aspirates of patients with suspected B-cell lymphoproliferative disorders were subjected to evaluation by flow cytometry. After washing, samples were stained by antibodies targeting the antigens CD45, CD19, CD5, CD10, CD20, CD23, CD43, CD79b, CD200, FMC7, CD25,CD103 ,CD11c ,sIgM, kappa and lambda . Immunophenotyping was performed using a BD FACS Canto flow cytometer. There were a total of 108 cases of B CLPD that were analysed by flow cytometry . Mean age (SD ) was 65 years. There were 68 males and 40 female patients . There were 71 cases of typical CLL , 7 cases of atypical CLL , 3 cases of Mantle cell lymphoma (MCL) , 5 cases of Follicular lymphoma ( FCL ) , 8 cases of Splenic lymphoma with villous lymphocytes (SLVL) , 6 cases of Hairy cell leukemia ( HCL) , 6 cases of unclassifiable B-NHL and 2 cases of lymphoplasmacytic lymphoma. All our cases of typical CLL showed bright expression of CD200 .It was also brightly expressed in all our atypical cases of CLL . CD43 was brightly positive in all our cases of typical CLL . The expression of this marker along with CD200 negativity was helpful in diagnosing of MCL.There were diagnostic difficulties in differentiating atypical CLL from B-NHL unclassifiable. The lack or dim expression FMC7 expression in all these cases along with absent or dim expression of sIgM were informative. Their use along with the panel by Euroflow is suggested to provide an accurate diagnosis.

Fast Detection Of MYD88-L265P Mutation By PCR-RFLP In Chronic Lymphoproliferative Disorders

IntroductionWaldenstrom's macroglobulinemia (WΜ) is an incurable disorder characterized by bone marrow infiltration from neoplastic lymphoplasmacytic B-lymphocytes and monoclonal IgM production. Recent data suggest a possible causal relationship of MYD88-L265P mutation with the pathogenesis of the disease (Treon Sp et al. N Eng J Med 2012;367:826-33). Additional studies have conformed that MYD88-L265P is characteristic of WΜ, but it is also rarely present in other chronic lymphoproliferative disorders (LPDs) (Jiménez C et al. Leukemia 2013,doi: 10.1038/leu.2013.62; Varettoni M et al. Blood 2013;121:2522-8). The purpose of this study was to analyse the prevalence of the aforementioned mutation in patients with WM and other LPDs, using a fast and reliable polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) protocol. MethodsGenomic DNA was extracted from the bone marrow of 11 patients with WΜ, 12 patients with B-cell chronic lymphocytic leukemia (CLL), 8 with splenic lymphoma with villous lymphocytes (SLVL). Furthermore, consecutive samples of peripheral blood and bone marrow isolated CD19+ cells, derived from a patient with monoclonal IgM gammopathy of undetermined significance (MGUS-IgM), were also retrospectively analysed for the presence of MYD88-L265P defect. The detection of the mutation was performed by PCR amplification of the MYD88 exon 5 region, followed by RFLP analysis, since the presence of the mutation results in the generation of BsiEI restriction enzyme site. PCR-RFLP results were also confirmed by direct sequencing of purified CD19+cells. ResultsThe MYD88-L265P mutation was detected in 10 patients with WΜ (90.9%), in 1 patient with SLVL with markers of lymphoplasmatocytoid differentiation (12.5%) and was absent in all CLL patients. Interestingly, our PCR-RFLP protocol exhibited a greater sensitivity than direct sequencing, when applied to total bone marrow cells (12.5% vs 25%). Moreover, the patient with MGUS-IgM displayed the MYD88-L265P mutation in isolated CD19+cells of both bone marrow and peripheral blood in all consecutive samples and remains in a stable condition for the last 7 years. ConclusionPCR-RFLP is a rapid, sensitive and reliable technique for the detection of MYD88-L265P mutation in patients with WM and lymphomas with lymphoplasmatocytoid differentiation. Additionally, the presence of MYD88-L265P mutation in MGUS-IgM in the absence of disease progression for many years, suggests that this genetic defect alone is not sufficient to lead to overt neoplastic disease. Disclosures:No relevant conflicts of interest to declare.

A Hairy Cell Leukaemia Variant – A Rare Case Report

The aim of the article is to present a rare case of Hairy cell leukaemia variant (HCl-V) which is a distinct clinico-pathological entity with intermediate features between classical HCl (HCl-C) and B-cell prolymphocytic leukaemia. It is an uncommon disorder accounting for approximately 0.4% of chronic lymphoid malignancies and 10% of all HCl cases. A 58 year old woman presented with pain abdomen and loss of weight. On examination she had massive splenomegaly. Peripheral smear was reported as chronic lymphoproliferative disorder (? Hairy cell leukemia or splenic lymphoma with villous lymphocytes). On Bone marrow examination, differential diagnosis was given as splenic lymphoma with villous lymphocytes (SLVL) and prolymphocytic variant of Hairy cell leukemia. On flow cytometric analysis, these cells were positive for CD11c, CD19, CD20, and CD22. Based on the clinical, peripheral smear, bone marrow and flow cytometry findings, a diagnosis of hairy cell leukaemia variant was confirmed. The differential diagnosis should always include SLVL, HCL-C and Japanese variant HCL because they have different clinical and biological features, particularly regarding their response to purine analogue-based treatment or splenectomy.

Splenic lymphoma with villous lymphocytes (SLVL)

Review on Splenic lymphoma with villous lymphocytes (SLVL), with data on clinics, and the genes involved.

Splenic lymphoma with villous lymphocytes (SLVL)

Splenic lymphoma with villous lymphocytes (SLVL)

High Frequency of Marginal Zone Lymphomas Among Patients Coinfected with Human Immunodeficiency Virus and Hepatitis C Virus - ANRS CO16 LYMPHOVIR Cohort Study

AbstractAbstract 4156 Introduction:Human Immunodeficiency Virus (HIV) and hepatitis C virus (HCV) infections are both associated with an increased risk of B-cell non-Hodgkin lymphoma (B-NHL). They are known to be preferentially diffuse large B-cell lymphoma (DLBCL), and Burkitt lymphoma subtypes in HIV infection, while marginal zone lymphoma (MZL) and DLBCL represent the main subtypes in HCV infected patients. The absence of increased risk of B-NHL in HIV-HCV co-infected patients compared to HIV infected patients was reported in epidemiological studies, but the clinico-pathological characteristics of B-NHL in the setting of such co-infection particularly in the era of HAART remains unclear. Patients and methods:We present the data of adult patients with B-NHL and HIV-HCV co-infection from the French ANRS CO16 Lymphovir cohort. This multicentric prospective cohort includes HIV infected patients with lymphoma. Each patient is followed every 6 months during 5 years. At each follow-up, a blood sample is withdrawn allowing ancillary studies. Data collection concerns clinical presentation, treatment and evolution of B-NHL and HIV and HCV infections. Pathological and cytological materials are centralized in order to allow their review and a concerted analysis by a group of expert hematopathologists (MR, SP, DC), haematologists and immunologists. Results:Among the 49 patients with B-NHL included in the cohort, 6 were HIV-HCV coinfected, [5 men and 1 woman, median age 47 years (range 36–67)]. They were included between october 2007 and august 2009 in 6 French centres. Transmission risk was intravenous drug abuse (n=4) and heterosexual transmission (n=2). Median duration from HIV diagnosis to B-NHL was 11 years (range 1–20), and the median nadir CD4 T-cell count was 194/mm3 (range 151–746) (nadir not available for 2 patients). Only one patient had developed AIDS before the diagnosis of NHL. HCV genotypic distribution was: genotype 1 (n=3), 2 (n=1), 4 (n=1), and not available (n=1). Median HCV viral load was 6.0 log (range 5.4–7.1). One patient had compensated cirrhosis and none of the patients were tested for cryoglobulinemia. Immunological assays showed the presence of monoclonal IgM Kappa in 1 patient and no positive rheumatoid factor.At the diagnosis of B-NHL, 4 patients received HAART and had an undetectable HIV load. In contrast, none of the patients were treated for HCV infection. The median interval between B-NHL diagnosis and last follow-up is 17 months (range 0.1–32 months). The histological subtype distribution is 4 MZL -including 2 extranodal MZL of MALT, 1 splenic lymphoma with villous lymphocytes (SLVL) and 1 lymphoplasmacytic lymphoma- and 2 EBV-negative DLBCL. All patients have extranodal involvement, including digestive tract (n=3), liver (n=2), bone marrow (n=2) and spleen (n=1). Median CD4 T-cell count at B-NHL diagnosis was 582/mm3 (range 235–1322) in MZL patients and 274/mm3 (200 and 347) in DLBCL patients.Following diagnosis, all patients were treated with HAART, associated with peg-interferon plus ribavirin in 1 patient. The 2 patients with DLBCL received R-CHOP, associated with methotrexate in 1 case. One patient with a partial response exhibited a neuromeningeal relapse and was treated with COPADM and CYVE regimen, allowing a complete and sustained response. The other patient died of serious infection after 2 courses of chemotherapy. Among patients with MZL, one patient received R-CVP and one received chlorambucil, allowing a complete response in both patients. The patient with SLVL received HAART then peg-interferon plus ribavirin, allowing normalization of lymphocytes count (5.89 at baseline vs. 1.69 × 109/L at 12 weeks of anti-HCV therapy) with a correlation between HCV virological and hematological response. Patient with lymphoplasmacytic lymphoma died of cardiac ischemia before the initiation of chemotherapy. Conclusions:This study describes the resurgence of MZL in HIV-HCV co-infected patients with restored immunity under HAART. Such B-NHL, very rarely described in HIV patients, occurs mainly in patients with control of HIV without history of AIDS but with active HCV infection. Histological subtypes and localizations resemble more to that observed in HCV infected patients than in HIV infected patients. These findings suggest the role of chronic antigenic stimulation by HCV and/or HIV and question about the interest of HCV antiviral therapy on NHL in co-infected patients. Disclosures:No relevant conflicts of interest to declare.

De-Regulation of MicroRNA Expression Is Detected in Hairy Cell Leukemia.

Abstract 3462Poster Board III-350Hairy cell leukemia (HCL) is a chronic B cell lymphoproliferative disorder characterized by marked splenomegaly, pancytopenia and diffuse infiltration of the spleen, liver, and bone marrow by tumor cells exhibiting a hairy appearance. The normal cellular counterpart of HCL is still debated. A study based on genome-wide gene expression analysis suggested that HCL is derived from memory B cell (Basso K. et al., J Exp Med 199:59-68, 2004). Despite multiple efforts to dissect the complex biology of HCL, the pathogenesis of this disease remains largely obscure. Recently, a novel layer of biological complexity has been added by the discovery of microRNA (miRNA). To date, miRNAs have been implicated in development and cancer. We have previously shown that miRNAs are expressed in a stage- or transformation-specific fashion in B cells (Basso K. et al., Immunity 744-52, 2009), suggesting that they might play specific developmental and pathologic roles. Although miRNA expression signatures have been derived for several hematological malignancies, assessment of miRNA expression in HCL has not yet been reported. Here, we investigated the role of miRNAs in HCL pathogenesis and the possibility of using miRNA expression signatures as a diagnostic tool to distinguish HCL-like disorders, especially Splenic Lymphoma with Villous Lymphocytes (SLVL). We evaluated the expression of 470 human miRNAs in peripheral blood-derived CD19+ B cells from 8 HCL, 5 SLVL and 9 Chronic Lymphocytic Leukemia (CLL) patients by a commercial miRNA array platform (Agilent). For comparison, 6 each naïve, germinal center and memory B cells from healthy donors were also included in the analysis. Unsupervised hierarchical clustering of miRNA expression profiles generated from HCL, SLVL and CLL showed that HCL and CLL displayed homogenous phenotypes clearly distinguishable from each other. Conversely, SLVL did not form a single cluster likely due to their higher heterogeneity, which could not be dissected due to the limited number of cases. In order to identify miRNAs whose expression was specifically associated with HCL, supervised clustering was applied to the dataset including both the tumors and the normal B cells. The results showed that 6 miRNAs were specifically up-regulated in HCL compared to CLL, SLVL and normal B cells. To gain insights on the potential role of miRNAs in HCL pathogenesis, we applied four target prediction algorithms (MiRbase, TargetScan, RNA22, PicTar) to identify the candidate target genes of the HCL-specific miRNA signature. Pathway enrichment analysis was performed on the 269 predicted targets using the Database for Annotation, Visualization and Integrated Discovery (DAVID). Of interest, MAPK pathway appeared as the most significant one suggesting that miRNA de-regulated expression in HCL may negatively modulate MAPK signaling. Taken together, our data identified a HCL-specific miRNA signature which may be used as diagnostic tool and provided early insights toward the dissection of the role of miRNAs in HCL pathogenesis. DisclosuresFalini:Xenomics: Patents & Royalties.

Flow Cytometry Study of CD148, a Tyrosine Phosphatase Receptor, in 184 Chronic B Cell Malignancies, Reveals a High Expression in Mantle Cell and Marginal Zone Lymphomas.

Abstract 1923Poster Board I-946We recently characterized CD148 as a potential marker for mantle cell lymphoma using mass spectrometry analysis of cell-derived microvesicles in a restricted set of patients (ASH Annual Meeting Abstracts, Nov 2008; 112: 1766). CD148 is a plasma membrane receptor with phosphatase activity related to CD45, composed of an extracellular domain containing 8 fibronectin type II-like domains, a transmembrane region and a single intracellular phosphatase domain. Interestingly, it was recently shown that deletion of the phosphatase domain of CD148 in mice blocks B cell development at the transitional stage, with a dramatic increase of marginal zone B cells. BCR signalling events are also substantially altered in CD148/CD45 doubly deficient mice.In the present study, we analyzed the expression of CD148 using flow cytometry in a larger group of controls and patients with circulating pathologic B-cells, including 93 chronic lymphocytic leukemia (CLL), 46 small lymphocytic lymphomas with Matutes score inferior or equal to 3 (SLL), 35 MCL, all harboring (11;14) translocation and/or cyclin D1 overexpression, 5 marginal zone lymphomas (MZL), 5 splenic lymphoma with villous lymphocytes (SLVL), as well as 30 controls. Mean fluorescence intensity of direct CD148 staining with phycoerythrin conjugated 143−41 clone was used for expression comparison.CD148 MFI of the 30 control cells was weak and very homogeneous (mean = 168, SD = 31), as well as in the 93 CLL (mean = 199, SD = 84). SLL cases (n=46) were stained slightly higher (mean = 297, SD = 138) revealing a slight but significant difference with CLL (p<0.01). The results were strikingly different for the MCL group (n=35), in which the mean CD148 MFI was 635 (SD = 358) which is significantly different from CLL, SLL, and SLVL groups (p < 0.001), and from controls (p < 0.001), thus confirming our preliminary results. We observed also a high CD148 MFI in marginal zone lymphoma, that was not different from the values obtained in MCL but very significantly higher than the other groups studied (p<0.001). Staining of MZL is in accordance with the published expression of CD148 in normal marginal zone of human spleen and tonsils using immunochemistry, but normal mantle zone cells are not stained with anti-CD148 antibodies, suggesting an aberrant CD148 expression in this pathology.The realization of a Receiver operator characteristic test (ROC curve) between MCL and CLL/SLL showed that in our hands a CD148 MFI superior to 383 allows strongly to suspect MCL diagnosis, with 86% specificity (versus CLL and SLL) and a sensitivity of 90%. Then, a high expression of CD148 may lead to karyotype or cyclin D1 expression analysis for a potential MCL diagnosis.Low CD148 expressing MCL cases need to be more studied, as well as the higher expressing SLL and CLL, because they could represent particular subgroups. Also, CD148 expression needs to be further studied in the different chronic B cell proliferations, notably in MZL, in order to evaluate its usefulness in first line antibodies diagnostic panel. Disclosures:No relevant conflicts of interest to declare.

Splenic Marginal Zone B-Cell Lymphoma: Clinical Clustering of Immunoglobulin Heavy Chain Repertoires.

Splenic marginal zone B-cell lymphoma (SMZL) is a rare clinical and pathological entity recognized by the WHO classification. SMZL usually presents with isolated splenomegaly, bone marrow involvement, and leukemic picture. Nodal disease is generally rare at diagnosis. When lymphocytes with villous projections are found in peripheral blood, the disease is termed splenic lymphoma with villous lymphocytes (SLVL). High HCV-seroprevalence is frequently reported in SMZL. The disease commonly pursues an indolent course; however, about a third of pts follow a more aggressive course. SMZL is also heterogeneous with respect to the preferential usage of IgVH genes, as well as the percentage of mutation load. No previous studies correlated IgVH rearrangement features with the clinical characteristics of SMZL. The aim of the present study was to determine the tumor-related IgVH gene rearrangement and compare the gene usage and the mutation status with clinical features of 59 pts. Diagnosis was made according to the WHO classification and to the diagnostic criteria for SMZL (Matutes et al., Leukemia 2008). Paraffin sections from lesional tissues (14 spleens, and 59 bone marrow trephines) were available for all pts. Histology and blood smears were reviewed. Total RNA was extracted from PB (n=13) or BM (n=46) mononuclear cells. IgVH gene rearrangements were amplified using 6 family-specific VH leader primers coupled with a 3′ heavy chain joining (JH) primer or with a constant region of cμ chain primer. The sequence obtained from direct sequencing of the clonal PCR product was compared with germline in the IMGT database. Sixty VDJ rearrangements were amplified and 54 were functional. VH1 family was used in 19 rearrangements (32%), VH3 family in 33 (55%) and VH4 family in 8 (13%). The most frequent VH genes were VH1-02 (n=13), VH3-23 (n=15), VH3-30 (n=7) and VH4-34 (n=5) (67% of all sequences). VH was unmutated in 25%. DH segments were assignable in 56/60 rearrangements (all of the VH unmutated and 41/45 of VH mutated). The most frequent DH families were DH2 (n=9) and DH3 (n=21); the most frequent DH genes were DH3-03 (n=8), DH3-22 (n=6) and DH2-02 (n=6). Most rearrangements used JH4 family (n=21), JH5 (n=8), and JH6 (n=19), accounting for 80% of all rearrangements. JH4b (n=14), JH6b (n=10), and JH6c (n=8) were the most common JH genes. Median length of HCDR3 region was 17 aa (range 11–32). For antigen selection analysis P value was < 0.05 in 41% for CDRs and in 55% for FRs. No correlation was found among VH and DH families (p=0.1), among VH and JH families (p=0.09) and among DH and JH families (p=0.4). Forty-two % of pts were unmutated in VH1 family, 15% in VH3 family and 25% in VH4 family (p=0.09). Unmutated cases were 7% in VH3-23 group, 46% in VH1-02 group and 14% in VH3-30 group (p=0.03). For clinical correlations we considered only the 54 functional rearrangements. Villous lymphocytes >10% were detected in 53% of VH1 pts, in 57% of VH4 pts, and in 17% of VH3 pts (p=0.01). Villous lymphocytes >10% were detected in 21% of VH3-23 pts, in 50% of VH1-02 pts and in no VH3-30 pt (p=0.05). Liver involvement was present in 9% of VH3-23 pts, in no VH1-02 pt and in 50% of VH3-30 pts (p=0.009). HCV serology was positive in 7% of VH3-23 pts, 17% of VH1-02 pts and 50% of VH3-30 pts (p=0.04). Serum MC was detected in 50% of VH3-23 pts, in 9% of VH1-02 pts and in 17% of VH3-30 pts (p=0.06). BM was involved in 86% of VH3-23 pts, in 100% of VH1-02 pts and in 50% of VH3-30 pts (p=0.02). A sinusal localization was detected in 67% of VH3-23 pts, in 20% of VH1-02 and in 100% of VH3-30 (p=0.03). Unmutated status was significantly related to a higher percentage of BM infiltration (p=0.003). The proportion of intermediate and high risk patients according to the SMZL score (Arcaini et al. Blood 2006) was higher in the unmutated respect to the mutated group (69% vs 32%, p=0.05). After a median F-UP of 3 yrs, 10 pts died (7 of lymphoma, 3 of causes non related to lymphoma). The median OS was 12 yrs and the median PFS was 2 yrs. OS did not differ among VH families (p=0.9). Median PFS was 2 yrs for mutated pts and 1 yr for unmutated pts. We performed clustering of pts by applying a 2-means clustering algorithm, using as parameters nodal disease, villous lymphocytes >10%, HCV serology positive, BM involvement: in cluster 1 VH1 family pts accounted for 20%, VH3 family for 71%, VH4 family for 9%; in cluster 2 VH1 family pts accounted for 47%, VH3 family for 29% and VH4 family for 24% (p=0.01). In conclusion, VH rearrangement analysis in SMZL reveals a non-random preference for VH1-02, VH3-23 and VH3-30 genes, whose use differs according to distinctive clinical features at presentation.

Splenic lymphoma with villous lymphocytes

Splenic lymphoma with villous lymphocytes (SLVL) is a rare disorder that comprises less than 1% of lymphoid neoplasms. It is the leukemic counterpart of splenic marginal zone lymphoma (SMZL) and is characterized by splenomegaly, often with no lymphadenopathy, moderate lymphocytosis and villous lymphocytes on peripheral blood smear. Here, we report a case of SLVL in a 56-year-old male with very high leukocyte counts, massive splenomegaly and relatively few leukemic cells with subtle villous projections on the surface. This disorder is often confused with other chronic lymphoproliferative disorders, especially chronic lymphocytic leukemia (CLL) and hairy cell leukemia and should be differentiated from them. We are reporting this case to highlight the diagnostic pitfalls associated with this disorder.

Leukaemic Monocytoid Marginal Zone Lymphoma (LMMZL): A Rare Undescribed Lymphoproliferative Disorder with Unique Features.

We report five cases of clonal lymphocytosis with striking monocytoid features, immunophenotype and clinical features distinct from chronic lymphocytic leukaemia (CLL), splenic marginal zone lymphoma (MZL) also known as splenic lymphoma with villous lymphocytes (SLVL) and mantle cell lymphoma (MCL). We believe these cases to represent a unique leukaemic variant of monocytoid MZL. All cases presented with marked peripheral blood lymphocytosis, no smear cells and monocytoid morphology with voluminous sometimes vacuolated cytoplasm. Immunophenotyping confirmed clonal lymphocytosis with strong light chain restriction, positivity for CD19 and CD20, absent or weak expression of CD10, CD22, CD23 and FMC7 and variable but weak CD5 expression. Bone marrow trephine biopsies revealed marked hypercellularity with a non-nodular diffuse lymphoid infiltrate. Cytogenetic analysis in cases 1 and 5 was normal, and fluorescent in-situ hybridisation (FISH) for abnormalities associated with CLL (11q23, trisomy 12, 13q14 and 17p13) and MCL (t11,14) were negative. Case 1: A 70 year old male presented with symptoms of anaemia, generalised lymphadenopathy, splenomegaly and leucocytosis. He progressed through chlorambucil and prednisolone but responded to oral fludarabine. He has been successfully retreated with fludarabine on relapse and remains alive, more than six years post diagnosis. Case 2: A 79 year old female was admitted with abdominal pain and vomiting. Investigation revealed marked leucocytosis with grossly abnormal liver function tests. This patient's condition deteriorated rapidly and she died before intervention was possible, ten days post presentation. Case 3: An 87 year old female presented with an incidental finding of lymphocytosis and isolated splenomegaly. Six months following her presentation she developed anaemia, thrombocytopenia and progressive lymphocytosis. Her disease progressed through chlorambucil and cyclophosphamide but responded to fludarabine. She unfortunately died of a cerebrovascular accident, eighteen months after diagnosis. Case 4: A 72 year old female presented with B symptoms, axillary lymphadenopathy and massive splenomegaly. She developed progressive B symptoms and rapid doubling of her white cell count, necessitating initiation of treatment. Despite an initial partial response to chlorambucil and prednisolone, her disease continued to progress and was refractory to multiple lines of chemotherapy. She died six years after her initial presentation. Case 5: An 84 year old presented with mild anaemia and leucocytosis and had clinical splenomegaly. He was managed with supportive therapy only and died of unrelated causes.Summary: The clinical presentation of these cases mimics that of CLL. The morphology of the peripheral blood lymphocytes is, however, markedly different. Patients are typically elderly with a high white cell count, marked splenomegaly and heavy marrow infiltration. Marker analysis is distinct from typical CLL and extranodal features at presentation are atypical of SLVL and nodal MZL. FISH results in two cases were also atypical for CLL. The clinical behaviour is distinct with resistance to alkylating agents but sensitivity to purine analogues. We believe that these unique cases represent an as yet undescribed leukaemic variant of monocytoid MZL probably currently classed and treated as atypical CLL.

Splenic lymphoma with villous lymphocytes, mixed cryoglobulinemia and HCV infection: deciphering the role of HCV in B-cell lymphomagenesis

Chronic hepatitis C virus (HCV) infection is associated with mixed cryoglobulinemia (MC) which can be viewed as a low-grade non-malignant B-cell lymphoproliferation. HCV is also associated with overt B-cell lymphomas but the direct causal relationship has remained elusive. The finding that HCV-associated splenic lymphomas with villous lymphocytes (SLVL), a subset of splenic marginal zone lymphomas, is constantly associated with MC and responds to antiviral therapy, and furthermore that the viral load strongly correlates with the tumor burden, lends support to the hypothesis that HCV is associated with antigen-driven B-cell transformation in a mechanism reminiscent of Helicobacter pylori-associated gastric MALT lymphoma. Moreover, the finding that HCV-positive large B-cell lymphomas appear to be transformed from low-grade B-cell lymphomas and that cryoglobulinemia is an independent risk factor for lymphoma in HCV-infected patients add support to this hypothesis. However, HCV-associated antigen-driven lymphomagenesis may not be the sole mechanism by which the virus could induce lymphomas, and a direct transformation of B-cells may be at play in some cases. HCV is among the growing list of pathogens associated with the development of lymphomas. Antiviral therapy should be considered as first-line therapy in low-grade B-cell and possibly large-cell lymphomas associated with HCV, especially in the presence of MC.

Spleenic lymphoma villous lymphocytes: Case report and literature review

Splenic lymphoma with villous lymphocytes (SLVL) is a rare but recognized distinct disease entity among chronic B lymphoproliferative disorders. It is frequently misdiagnosed as chronic lymphocytic leukaemia, (CLL) Prolymphocytic leukaemia or Hairy Cell leukaemia. Few cases have been reported worldwide. The case records of a sixty year old Nigerian male with a splenic lymphoma and a review of the literature on the subject using MEDLINE, other internet sources and manual library search was utilized. RESULT A sixty-year-old male with splenic lymphoma with villous lymphocytes, seen at the Ahmadu Bello University Teaching Hospital Zaria is presented. However review of the blood film, bone marrow and Splenic aspirates showed absolute lymphocytosis, consisting of "villous" lymphocytes. He was commenced on an alkylating agent and a glucocorticoid, with partial remission in the first three months but was lost to follow up. Adequate morphologic evaluation is advocated particularly in the resource limited settings were Cytogenetics, immunohistochemistry and immunophenotyping are not available.

WNT6 mRNA Is Overexpressed in Variuos Non-Hodgkin's Lymphoma Cases with No Apparent Relation to Cyclin D1 Level.

A casual role for Wnts in the development of some human cancers seems to be established. Binding of Wnt to its receptor results in β-catenin stabilization and in consequence, translocation to the nucleus, where it activates the specific target gene's expression including CCND1 (cyclin D1). Abnormal levels of β-catenin may therefore contribute to neoplastic transformation by causing accumulation of cyclin D1. Overexpression of cyclin D1, as a result of the t(11;14)(q13;q32) translocation, which places cyclin D1 gene under the enhancer of immunoglobulin heavy chain is considered the hallmark of mantle cell lymphoma (MCL) but high levels of cyclin D1 have also been observed with various levels in some cases of non-MCL lymphoproliferative disorders not involving t(11;14), such as hairy cell leukemia (HCL), splenic lymphoma with villous lymphocytes (SLVL), prolymphocytic leukemia (PLL) and some atypical CLL cases. Little is known about potential Wnt signaling in mature lymphocytes or lymphoid malignancies. Recent finding, that the Wnt pathway regulates the proliferation and survival of normal B lymphocytes, together with WNT16 overexpression in leukemic cell lines and high expression of five other Wnts including WNT6 in CLL suggests that Wnt family members can contribute to tumorgenesis in the immune system. In the current study we evaluated the relationship between Wnts and cyclin D1 mRNA expression level in NHL patients. WNT6 and CCND1 mRNA expression level normalized to 18 sRNA was evaluated by RT-PCR on 200 biopsy samples taken from 150 lymphoma patients. In parallel, samples underwent pathological and flow cytometry examination. WNT6 mRNA was detected in all specimens tested. The 18 sRNA/WNT6 ratio ranged from 1 to 650. Most of the CLL specimens were among those with high WNT6 mRNA expression, but no apparent correlation between the level of WNT6 mRNA expression and cyclin D1 status was found so far.

In vitro Epstein-Barr virus-immortalized lymphoma cell line carrying t(9;14)(p13;q32) chromosome abnormality, derived from splenic lymphoma with villous lymphocytes

We herein describe splenic lymphoma with villous lymphocytes (SLVL) carrying t(9;14)(p13;q32). The t(9;14)(p13;q32) is a rare reciprocal chromosome translocation found in a subset of B-cell malignancies, mainly in low-grade non-Hodgkin's lymphomas. In t(9;14)(p13;q32), PAX-5 gene on 9p13 is involved with the immunoglobulin heavy-chain gene on 14q32. It has been thought that the deregulated expression of PAX-5 as a result of t(9;14)(p13;q32) may contribute to abnormal cell proliferation. Although continuous cell lines are invaluable tools for studying lymphomagenesis in the t(9;14)(p13;q32)-bearing lymphomas, establishment of such cell lines is extremely difficult since they are usually mature B-cell malignancies. In an attempt to transform the SLVL cells into a proliferating cell line, we examined the responses of the cells to infection by Epstein-Barr virus (EBV). SLVL cells were found to be susceptible to immortalization by EBV, resulting in a permanent cell line. The cell line, designated SL-15, possessed the t(9;14)(p13;q32). Genotype analysis and immunophenotype profiles confirmed that the cell line arose from the primary lymphoma cells. The cells had characteristic cytoplasmic villi. SL-15 cells has been growing over 2 years equivalent to 350-400 population doubling levels without proliferative crisis that is often observed in EBV-positive lymphoblastoid cell lines. Furthermore, SL-15 cells, when inoculated into nude mice, formed t(9;14)(p13;q32)-bearing tumors with cytoplasmic villi. The validated SLVL-derived cell line provide a useful model system to study molecular biology of t(9;14)(p13;q32)-bearing B-cell malignancies as well as lymphomagenesis of SLVL in vitro and in vivo.

Multiple M components in two patients with splenic lymphoma with villous lymphocytes

Splenic lymphoma with villous lymphocytes (SLVL) is a rare lymphoproliferative disorder characterized by the presence of typical lymphoid cells with villous projections and monoclonal immunoglobulin (M-Ig) in about 30% of patients. The simultaneous presence of more than one M-Ig in SLVL has not been reported. We present two patients with SLVL, each with three serum M components associated with the presence of rheumatoid factor (RF) and antiphospholipid antibodies (APLA) together with fatal thromboembolic events. Both patients presented with splenomegaly and typical bone marrow cytology with 30-50% infiltration of lymphoid cells that had the characteristics of villous lymphocytes. Immunohistochemistry of bone marrow histology showed CD20++, CD43-/+, CD5-, IgM+, lambda+ and kappa-. In serum, two M-IgM lambda components were combined with M-IgG lambda in case 1 and with M-IgA lambda in case 2. In both cases, M-IgM displayed RF as well as lupus anticoagulant activity and free monoclonal lambda (lambda) light chains were present. In addition to M-IgM, in case 1 M-IgG also behaved like an APLA. One patient was splenectomized. Both patients suffered thromboembolic complications and died 3 and 8 months after presentation with signs of massive pulmonary thromboembolism.

Phase III Study of Chlorambucil Versus Fludarabine as Initial Therapy for Waldenström's Macroglobulinemia and Related Disorders

The WM1 study is a prospective randomized open-label study that includes patients with previously untreated Waldenström's macroglobulinemia (WM), splenic lymphoma with villous lymphocytes (SLVL), and non–immunoglobulin (Ig) M lymphoplasmacytic lymphoma (LPL) who have an indication for treatment. At registration, patients are categorized as having WM, SLVL, or LPL, and these cohorts are to be analyzed separately. The aim of the study is to compare the efficacy of oral chlorambucil at a dose of 8 mg/m2 (6 mg/m2 for those > 75 years of age) for 10 days every 28 days to a maximum of 12 cycles with oral or intravenous (I.V.) fludarabine at a dose of 40 mg/m2 orally or 25 mg/m2 I.V. (30 mg/m2 orally or 20 mg/m2 I.V. for those > 75 years of age) for 5 days every 28 days to a maximum of 6 cycles. Primary endpoints are response to therapy and duration of response; secondary endpoints are improvement in hematologic parameters, toxicity of therapy, quality of life, and survival. To detect a difference in response rate of patients with WM of 15%, assuming that the overall response rates will be 50% to chlorambucil and 65% to fludarabine, with a power of 80%, requires the sample size of each group to be 183, indicating the need for collaboration among a number of national investigator groups. As of February 2005, accrual to the study stands at 143. Registration, randomization, and data collection are entirely Internet-based (www.waldenstroms.org), and the study is organized by an international collaboration, with a planned interim analysis and an external data monitoring committee.

Hairy Cell Leukemia Variant With Features of Intrasinusoidal Bone Marrow Involvement

Hairy cell leukemia variant (HCL-V) is a rare lymphoproliferative disorder. We report a case of HCL-V with an intrasinusoidal pattern of bone marrow involvement without interstitial or diffuse infiltration that is typical of HCL and its variant. The peripheral blood and bone marrow aspirates demonstrated abnormal lymphoid cells with cytoplasmic projections that were weakly positive for tartrate-resistant acid phosphatase cytochemical staining. Immunostaining of the bone marrow biopsy specimen showed that these cells were strongly positive for CD20, located within bone marrow sinusoids, and weakly positive for DBA44. By flow cytometry, these cells were positive for CD19, CD20, CD11c, and CD103, exhibited lambda light chain restriction, and were negative for CD25. The patient was initially diagnosed as having splenic lymphoma with villous lymphocytes (SLVL) or splenic marginal zone lymphoma (SMZL) (World Health Organization designation) and treated with fludarabine followed by splenectomy with simultaneous liver biopsy. The pathologic analysis of the spleen revealed infiltration of red pulp by the critical cells without white pulp involvement, which is characteristic of HCL and HCL-V but not of SLVL (SMZL). This case illustrates an atypical marrow presentation of HCL-V and emphasizes the need to correlate all clinical and pathologic data, including tissue biopsy, in reaching a diagnosis.

Splenic lymphoma with villous lymphocytes, associated with type II cryoglobulinemia and HCV infection: a new entity?

AbstractHepatitis C virus (HCV) has been associated with the development of B-cell non-Hodgkin lymphomas. We recently reported the regression of splenic lymphoma with villous lymphocytes (SLVL) in patients with HCV after antiviral treatment, demonstrating a direct role of HCV in lymphomagenesis. This study expands our previous results in 18 patients with chronic HCV and SLVL. Mixed cryoglobulinemia (MC) was present in all cases and was symptomatic in 13 (72%). All patients were treated with interferon alone or in association with ribavirin. Hematologic and virologic responses were correlated. Fourteen (78%) patients achieved a sustained complete hematologic response after clearance of HCV RNA. Two patients had a virologic partial response and achieved a complete hematologic response. Two virologic nonresponders achieved partial hematologic response. Regardless of the response, monoclonal immunoglobulin gene rearrangement persisted after treatment. This study underscores the role of HCV in the lymphomagenesis and the benefit of antiviral treatment for patients presenting with HCV-driven lymphoproliferations.

Importance of receptor density in alpha radioimmunotherapy in B cell malignancies: an in-vitro study

External beam radiotherapy and beta radioimmunotherapy (RIT) are effective treatments for lymphoid malignancies. The development of RIT with alpha emitters is attractive because of the high linear energy transfer (LET) and short path length, allowing higher tumour cell kill and lower toxicity to healthy tissues. To assess the binding of rituximab to samples of B cell chronic lymphocytic leukaemia (B-CLL) and splenic lymphoma with villous lymphocytes (SLVL), and to evaluate the induction of apoptosis by conventional therapies as well as with Bi conjugated to rituximab. 213Bi was eluted from a 225Ac generator and conjugated to CD20 antibody (rituximab) with CHX-A''-DTPA as chelator. Binding assays with 213Bi-rituximab were correlated to antibody binding capacity obtained by flow cytometry. Apoptosis was scored by flow cytometric analyses of the cells stained with annexin V-FITC and 7-amino-actinomycin D. Binding of 213Bi-rituximab was significantly lower for B-CLL compared to SLVL samples (12+/-3 and 42+/-10 213Bi atoms per cell, respectively, at 370 kBq.ml(-1)). The induction of apoptosis did not differ significantly between the two groups (B-CLL and SLVL) after external gamma irradiation or treatment with methylprednisolone and fludarabine (17+/-12% and 18+/-11%; 23+/-14% and 21+/-12%; 9+/-9% and 11+/-8%, respectively; all results expressed as percentages of all cells). Rituximab conjugated or not to 213Bi induced significantly more apoptosis in SLVL (42+/-19% and 42+/-17%) compared to B-CLL samples (27+/-12% and 6+/-8%). Binding assays confirm that SLVL samples present more CD20 antigens compared to B-CLL samples. Conventional therapies such as fludarabine, methylprednisolone or external gamma irradiation induce similar responses in the two populations but SLVL samples present higher sensitivity towards 213Bi-rituximab. These data are in favour of alpha-RIT in SLVL patients.