Incidence Of Atypical Chronic Lymphocytic Leukemia In 1819 Patients With B Chronic Lymphoproliferative Disorder

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1770-1770
Author(s):  
Edouard Cornet ◽  
Alice-Sophie Boucher ◽  
Véronique Salaun ◽  
Florence Truquet ◽  
Michele Malet ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Lymphoproliferative Disorder ◽  
Who Classification ◽  
Cell Lymphoma ◽  
Hematologic Malignancies ◽  
Lymphocytic Leukemia ◽  
Histological Evidence

Abstract Flow cytometry is the diagnostic tool of choice to study abnormal lymphoid population detected in peripheral blood by morphological analysis. The main diagnosed chronic lymphoproliferative disorder (CLPD) is chronic lymphocytic leukemia (CLL). In a significant number of cases, a B-CLPD non-CLL can be diagnosed. Further molecular and histological examinations are then compulsory to characterize such hematologic malignancies. The objective of this study was to determine the incidence of atypical CLL among all B-CLPD diagnosed by flow cytometry. We retrospectively studied the B-CLPD consecutively diagnosed at the hospital of Caen (Normandy, France) between 2000 and 2013. The diagnosis of B-CLPD was based on the detection by flow cytometry of circulating lymphoid abnormal B cells. Multiparametric flow cytometry included markers CD19, CD20, CD22, CD79b, CD5, CD10, CD23, CD43, FMC7, CD38 and light chains (kappa and lambda) of surface immunoglobulin. The diagnosis of CLL was based on the criteria defined by Hallek et al (Blood 2008). The non-CLL B-CLPD were then explored by molecular analyses driven by the phenotype of B-cells (overexpression of cyclin D1 in case of CD5+/CD10-/CD23- B-CLPD and BCL2-JH rearrangement in case of CD10+ B-CLPD). In addition, histological evidence was necessary to classify the B-CLPD non-CLL. 1819 B-CLPD were detected by flow cytometry. The distribution of B-CLPD was as follows: 1156 cases (64%) of CLL or immunophenotypic equivalent (leukemic phase of small lymphocytic lymphoma (SLL) and monoclonal B-cell lymphocytosis (MBL)), 297 cases (16%) of marginal zone lymphoma (MZL), 84 cases (5%) of mantle cell lymphoma (MCL), 39 cases (2%) of follicular lymphoma (FL), 26 cases (1%) of hairy cell leukemia (HCL), 13 cases (<1%) of diffuse large B-cell lymphoma (DLBCL), 9 cases (<1%) of Waldenstrom's macroglobulinemia (WM) and 3 cases (<1%) of B-cells prolymphocytic leukemia (B-PLL). 65 cases (4%) remained unclassified due to lack of histological and molecular data. 127 cases (7%) did not meet the diagnostic criteria of CLL established by Hallek et al but were classified as atypical CLL because of the detection of a clonal B-cell proliferation expressing CD5+ / CD23+ / CD43+ / CD10- / FMC7+ / CD79b+ with moderate or high intensity and light chain kappa or lambda with moderate or strong intensity (absence of molecular or histological argument of MZL or MCL was required). CD20 marker was highly expressed in 113 cases (89%) of atypical CLL. We particularly studied the 1532 cases (84%) of B-CLPD expressing CD5 (table). CD5+ CD23+ B-CLPD cases accounted for 1293 (84%) with 1153 cases of CLL, 13 cases of MCL and 127 cases of atypical CLL. CD5+ CD23- B-CLPD accounted for 239 cases (16%) with 72 cases of MCL, 158 cases of MZL, 4 cases of FL, 2 cases of WM, 2 cases of CD23- CLL and one case of B-PLL.CD5+ casesCD5+ CD23+ B-CLPDCD5+ CD23- B-CLPDTotalCLL115301153Atypical CLL1272129MCL137285MZL0158158FL044WM022B-PLL011Total12932391532 WHO classification of hematologic malignancies do not include atypical CLL as defined by a clonal proliferation of B-cells expressing CD5+, CD23+, cyclin D1- with no histological evidence of MZL or MCL, and which do not meet all the diagnostic criteria of CLL (Hallek et al, Blood 2008). This concept of atypical CLL, first described by Criel et al (BJH 1997), is particularly interesting, because such B-CLPD seems to have a different outcome as compared with CLL (Oscier et al, BJH 1997) and to have a different biological presentation with atypical morphology of CLL cells (Criel et al, BJH 1997), more frequent trisomy 12 (Matutes et al, BJH 1996) and a stronger intensity of CD20 (Ugo et al, Leuk Lymphoma 2006). Diagnosis of B-CLPD relies on a multidisciplinary approach combining morphological, immunophenotypic, molecular and histological analyses. Despite detailed information of these analyses, there are B-CLPD which remain unclassifiable according WHO classification, especially CD5 positive B-CLPD. The concept of atypical CLL seems to take all its meaning to help define such unclassifiable entities. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Utility of Quantitative Flow Cytometry Immunophenotypic Analysis of CD5 Expression in Small B-Cell Neoplasms

2014 ◽  
Vol 138 (7) ◽  
pp. 903-909 ◽  
Author(s):  
Pramoda Challagundla ◽  
Jeffrey L. Jorgensen ◽  
Rashmi Kanagal-Shamanna ◽  
Inga Gurevich ◽  
Diane M. Pierson ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Lymphoma ◽  
Lymphocytic Leukemia ◽  
Positive Events ◽  
Expression Levels ◽  
Mantle Cell ◽  
Homogeneous Pattern

Context.—The value of assessing CD5 expression in the differential diagnosis of small B-cell neoplasms is well established. Assessment is usually done qualitatively. Objectives.—To assess CD5 expression levels by quantitative flow cytometry immunophenotyping and to determine possible differences among various small B-cell neoplasms. Design.—We performed 4-color flow cytometry analysis on specimens of peripheral blood and bone marrow aspirate and quantified CD5 expression in various small B-cell lymphomas and leukemias. We also assessed CD5 levels in peripheral blood samples of healthy blood donors. Results.—Cases of chronic lymphocytic leukemia and mantle cell lymphoma had higher levels of CD5 compared with control B cells (P &lt; .001). Cases of marginal zone lymphoma and hairy cell leukemia had CD5 levels similar to control B cells (P = .35 and P = .14, respectively), whereas cases of follicular lymphoma and lymphoplasmacytic lymphoma had significantly lower CD5 levels than control B cells (P &lt; .001 and P = .04, respectively). In B-cell neoplasms, a high level of CD5 expression was correlated with a homogeneous pattern of positive events, whereas lower CD5 levels were correlated with heterogeneous patterns of positive events. Conclusions.—Using flow cytometric immunophenotypic analysis to quantify CD5 levels can aid in diagnosis. CD5 expression levels are higher in patients with chronic lymphocytic leukemia and mantle cell lymphoma, and expression is observed in a homogeneous pattern, as compared with other B-cell neoplasms that are either negative for CD5 or express CD5 at lower levels with a heterogeneous pattern. However, there is some overlap in CD5 expression levels between a subset of atypical chronic lymphocytic leukemia and marginal zone lymphoma cases.

Expression of NTB-A in Normal Tissue, Non-Hodgkin Lymphomas and Investigation of Its Potential Therapeutic Impact for Chronic Lymphocytic Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4741-4741
Author(s):  
Xiaoxian Zhao ◽  
Wouter Korver ◽  
Nichole Prescott ◽  
Arie Abo ◽  
Eric Hsi
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Progenitor Cells ◽  
Blood Cells ◽  
Cell Lymphoma ◽  
White Blood Cells ◽  
B Cell Lymphoma ◽  
Hematologic Malignancies ◽  
Normal Tissues

Abstract Introduction: Expressed on T, B, NK cells and neutrophils, NTB-A belongs to the signaling lymphocytic activation molecule (SLAM) family of immune receptors. It was reported recently that cross-linking of NTB-A induces T cell activation and Ly108 (the presumed mouse orthologue of NTB-A) controls the production of reactive oxygen species in neutrophils. To date, little is known about its expression at the protein level in normal tissues, or in hematologic malignancies. Methods: We have generated monoclonal antibodies (mAbs) to NTB-A and used tissue microarrays (TMAs) to screen normal tissues and lymphomas. Western blotting and flow cytometry were used for confirmation of selected entities. Complement dependent cytotoxicity (CDC) assays were performed using the CellTiter-Glo Luminescent Cell Viability Assay kit (Promega). Results: NTB-A was not detected in normal tissues including heart, liver, breast, kidney, brain, lung, uterus, small intestine, skin, prostate, pancreas, ovary, bladder, testis and stomach (5/5 negative cases for all above tissues) by IHC. Expression was observed in lymphocytes of normal tonsil and spleen. In lymphomas, we found NTB-A expression in diffuse large B-cell lymphoma (DLBL), follicular lymphoma, small lymphocytic lymphoma (SLL), mantle cell lymphoma and Burkitt lymphoma. Western blotting and flow cytometry of B-cell lymphoma cell lines DOHH2 (DLBL) and Raji (Burkitt) confirmed NTB-A expression in these lines. Myeloma cell lines were negative for expression. Flow cytometry of normal blood cells showed expression of NTB-A in B-cells, T-cells and NK cells (95.5 ± 6.7%, 62.6 ± 29.5% and 72.7 ± 26.5%, respectively) but not in CD66+ granulocytes. Furthermore, CD34+ progenitor cells from bone marrow were negative. Because of the expression in SLL, we focused on chronic lymphocytic leykemia (CLL) cells. All (15/15) patient samples were confirmed positive by flow cytometry. No significant differences in expression levels were observed comparing CLL B-cells vs normal B-cells (n = 10). However, anti-NTB-A antibodies were capable of inducing CDC in white blood cells from CLL patients (n=5) but not in normal white blood cells (n=5), in addition to CDC activity against Raji cells. Preliminary data also suggests that the CDC effect is greater in purified CLL B-cells than purified normal B-cells. Conclusions: These results show that NTB-A is expressed in normal lymphocytes but not in other solid tissues or in CD34+ progenitor cells. B cell malignancies, including Non-Hodgkin lymphoma cells appear to express NTB-A. In particular CLL patient cells express NTB-A, and an anti-NTB-A mouse mAb is capable of inducing CDC, suggesting NTB-A may be a potential immunotherapeutic target. Further studies examining the expression patterns in hematologic malignancies are ongoing.

scholarly journals Next-Generation IgVH Sequencing of Monoclonal B-Cell Lymphocytosis Reveals Clonal Heterogeneity, Ongoing Mutation, and Repertoire Differences Relative to Chronic Lymphocytic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1960-1960
Author(s):  
Mark Klinger ◽  
Malek Faham ◽  
Jianbiao Zheng ◽  
Kojo S.J. Elenitoba-Johnson ◽  
Sherrie L. Perkins ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Lymphocytic Leukemia ◽  
Cell Repertoire ◽  
Clonal Heterogeneity ◽  
Cell Counts ◽  
Lambda Expression ◽  
Antigen Selection

Abstract Background: Chronic lymphocytic leukemia (CLL) usually develops from asymptomatic monoclonal expansions of CD5 positive B-cells termed monoclonal B-cell lymphocytosis (MBL), present in the peripheral blood (PB) of approximately 5% of otherwise healthy older individuals. Although MBL only occasionally progresses to CLL, cases that do progress typically have higher MBL cell counts in the 1500-4000/µL range. Although antigen selection appears to play a central role in the development CLL, it is unclear whether this occurs at an early MBL stage or primarily during the progression of MBL to CLL. One prior study has reported clonal heterogeneity in MBL finding it in 4 of 6 low count MBL cases from familial CLL kindreds using a single cell PCR technique (Leukemia 2010,24:133-140). In this study, we assessed the VH repertoire and degree of clonal heterogeneity in sporadic MBL cases using next-generation sequencing (NGS) of the rearranged immunoglobulin heavy chain (IgH) locus. Methods: The 35 cases selected for sequencing represented residual, cryopreserved material from PB specimens submitted to ARUP for clinical phenotyping studies. All contained polytypic CD5 negative B-cells in addition to MBL/CLL phenotype cells, and had 2 or more vials for analysis. The majority (80%) had counts of MBL cells below 1000/µL (mean 294/, range 795-30 cells/µL). FACS purification of MBL cells (CD20+CD5+) and CD5 negative B-cells was performed on all samples. The IgH repertoire from the unsorted and two sorted populations was determined by NGS using the LymphoSIGHT method. Results: Five cases could not be analyzed due to insufficient numbers of MBL cells. Clonal VDJ rearrangements or clonotypes were identified in the remaining 30 based on their high frequency within the B-cell repertoire of the unsorted sample, and having a higher frequency in the sorted MBL cells relative to the sorted CD5 negative B-cells. Functional clonotypes were identified in 29 of these 30 cases. Interestingly, 5 cases had 2 functional unrelated clonotypes using different D and/or J segments that also employed different V segments. Of the 5 cases with 2 unrelated clonotypes, 3 had MBL cell counts below 1000/µL (32, 275, and 865) and 2 above (1640, 2600). Moreover, 1 of the clones in the case with 865 cells/µL represented only 25% of the MBL cells or 220 cells/µL, while 1 clone in the case with 2600 MBL cells/µL represented 18% of the MBL cells or 470 cells/µL. By flow cytometry, the CD5+ CD20+ cells in 2 of the cases with 2 functional clonotypes showed polytypic kappa/lambda expression (ratios near 1), 2 cases had uniform dim monotypic kappa expression, and 1 case showed 90% dim kappa and 10% dim lambda expression. The most frequently used VH segments were V4-34 in 6/34 or 18% of functional clonotypes, followed by V3-23 (11%), and V3-21 (9%). The V1-69 segment was used by only 1/34 (3%) functional clonotypes. The VH segments in 72% of cases with functional clonotypes were mutated (homology to germline < 98%), with 6 cases showing clear evidence of ongoing mutation by having 2 or more related clones. Conclusions: We demonstrate that MBL exhibits considerable clonal heterogeneity, with 2 distinct unrelated clones identified in 17% of 30 analyzed cases. Finding 2 distinct clones cannot be explained by a lack of allelic exclusion or the presence of 1 cell with 2 productive IgH rearrangements since each clone had different frequencies within the sorted MBL cell repertoire. This is further supported by finding the ratios of the two MBL clones in 2 cases being different in the unsorted compared to the MBL sorted cells. Clonal heterogeneity appears to occur at an early stage since the majority of clones (6/10) had cell counts below 500 cells/µL. We also found that clonal heterogeneity of MBL may not be detectable by flow cytometry or may appear as polytypic CD5+CD20+ B-cells. To our knowledge, this represents the first report of clonal heterogeneity in sporadic MBL. Our identification of infrequent use of V1-69 (1/34) supports prior studies indicating the VH repertoire of MBL is different than CLL which frequently employs V1-69. Finding evidence of ongoing VH mutation suggests antigen selection may occur in early MBL. Overall, our findings are consistent with recent observations (Cancer Cell 2011, 20;246-259) suggesting that hematopoietic stem cells from CLL patients can generate mono-or oligoclonal MBL phenotype cells that can then be selected through antigen binding for expansion. Disclosures Faham: Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

scholarly journals A Novel Spirocyclic Dimer (36-286) Targeting the NF-Kappa B Pathway Displays Potent Anti-Tumor Properties in Chronic Lymphocytic Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1186-1186
Author(s):  
Alexandria P Eiken ◽  
Audrey L Smith ◽  
Sarbjit Singh ◽  
Sandeep Rana ◽  
Sunandini Sharma ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Growth ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Protein Translation ◽  
Lymphocytic Leukemia ◽  
Rna Seq ◽  
Resistant Cells

Abstract Introduction: Chronic lymphocytic leukemia (CLL) is an incurable, heterogenetic disease dependent on B cell receptor (BCR) signaling with subsequent nuclear factor-kappa B (NF-κB) activation resulting in the evasion of apoptosis and enhanced malignant B cell growth. Targeted therapies such as ibrutinib (IBR; BTK inhibitor) and venetoclax (VEN; BCL2 antagonist) have revolutionized the management of CLL, however ~20% of patients relapse, signifying the urgent need for novel therapeutics for CLL patients especially those with refractory/relapse (ref/rel) disease. Additionally, various tumor microenvironment (TME) stimuli fuel CLL growth and contribute to drug resistance through the activation of numerous signaling pathways (BCR, CD40R, TLR, BAFFR) and consequential sustained NF-κB activation. Currently, there are no FDA approved drugs that effectively target the NF-κB protein family. Herein we introduce 36-286 (N3), a novel spirocyclic dimer which displays NF-κB inhibitory activity and elicits potent anti-leukemic properties. N3 is a dimer of a spirocyclic α-methylene-γ-butyrolactone analog that covalently binds to surface exposed cysteine residues on NF-κB proteins (IKKβ and P65) (Rana S et al, 2016). Our study aims to investigate N3's mode of action (MOA) and to establish its anti-leukemic effects in CLL including drug-resistant disease, thereby introducing a novel therapeutic option for rel/ref disease. Methods: Cell growth via MTS proliferation assay was determined following treatment with N3 (0.125 - 2 μM) in a panel of malignant B cell lines [CLL (HG3, MEC1, OSUCLL), diffuse large B cell lymphoma (Pfeiffer, RC, RIVA), mantle cell lymphoma (Jeko1)], and in patient derived CLL cells stimulated with CpG ODN 2006 (CpG; 3.2 μM). Viability testing of normal B cells isolated from healthy donors was conducted following N3 treatment. Anti-tumor properties of N3 (1 - 2 μM; 4h) in the HG3 and OSUCLL cell lines were further confirmed under conditions mimicking different TME stimuli such as α-IgM (10 μg/mL), CD40L (100 ng/mL), BAFF (50 ng/mL) or CpG (3.2 μM). Protein expression of oncogenic MYC, select NF-κB pathway proteins (IKKα, IKKβ, P65, IκBα, RelB) and the anti-apoptotic protein MCL1 was determined following treatment with N3 (0.25 - 2 μM; 4h) by immunoblot (IB). Next, we induced IBR resistance in HG3 cells by prolonged exposure to increasing IBR concentrations (~10-15 fold its IC 50 in parental cells). Cell proliferation via MTS was determined following treatment with N3 on these resistant cells. To gain insight on the potential MOA of N3 in CLL, we adapted a proteomics-based approach (TMT labeled mass spectrometry) and conducted RNA-seq in OSUCLL cells treated with N3 (1 - 2 μM) for up to 24 h. Subsequent pathway analysis was performed to identify the top factors modulated by N3. Results: N3 showed remarkable efficacy (IC 50 &lt; 0.6 μM) across all the malignant B cell lines evaluated while sparing normal B cells. In CpG stimulated primary CLL, N3 resulted in marked anti-leukemic effects (0.125 μM) comparable to IBR (1 μM). N3 induced cell apoptosis in CLL cell lines in a dose-dependent manner with marked PARP cleavage. Furthermore, our IB analyses of N3 treated CLL cell lines showed reduced levels of NF-κB pathway proteins, MYC and MCL1. Notably, N3 was effective in reducing levels of the above-mentioned proteins in the presence of the various TME stimuli. Strikingly, N3 maintained its cytotoxic effects in ibrutinib resistant HG3 cells. Studies to confirm N3's cytotoxicity in VEN resistant CLL cells are ongoing. Top ten pathways from both proteomics and RNA-seq analyses revealed an upregulation of the unfolded protein response (UPR) and inhibition of cap-dependent protein translation. IB analyses of select factors related to UPR (CHOP, XBP1, PERK, IRE1) and protein translation (eIF2α, 4E-BP1, PDCD4) in N3 treated CLL cells validated our omics' findings. Efforts to identify the proteome wide direct targets of N3 in CLL cells are currently underway. Conclusion: N3 is a novel pre-therapeutic lead that targets multiple survival and proliferation pathways through the inhibition of NF-κB activity and upregulation of UPR. We show that its highly cytotoxic in tumor B cells while sparing normal B cells. Moreover, N3 sustained its anti-tumor properties under different TME stimuli and in IBR resistant cells, indicating the potential use of this compound in rel/ref patients following evaluation in murine CLL models. Disclosures No relevant conflicts of interest to declare.

Analysis of PRAME Protein Expression in Normal Lymphoid Tissues and during B Ontogenesis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3924-3924
Author(s):  
Lorena L. de Figueiredo-Pontes ◽  
Fabio M. do Nascimento ◽  
Rodrigo S. de Abreu e Lima ◽  
Rodrigo Proto-Siqueira ◽  
Aglair B. Garcia ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Protein Expression ◽  
B Cell ◽  
Mononuclear Cells ◽  
Hematologic Malignancies ◽  
Lymphocytic Leukemia ◽  
Lymphoid Tissues ◽  
Healthy Donors ◽  
Chronic Lymphoproliferative Diseases

Abstract PRAME (Preferentially Expressed Antigen in Melanoma) gene was originally isolated in melanoma. A significant increase in the number of PRAME transcripts has been demonstrated in hematologic malignancies such as acute myeloid and lymphoid leukemias, multiple myeloma and chronic lymphoproliferative diseases. Furthermore, our group generated an anti-PRAME monoclonal antibody (MoAb) and by quantitative flow cytometry has demonstrated that PRAME protein was aberrantly expressed in Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma. However, the expression of this antigen in normal lymphoid tissues and during B cells ontogeneis has not been characterized. To address this question, PRAME protein expression was studied by flow cytometry in peripheral blood (PB, n=15) and bone marrow (BM, n=6) from healthy donors, lymphonodes (n=4) and spleen (n=4) from patients submitted to lymphonode excision or splenectomy for non malignant diseases. First, we determined in which hematopoietic lineage PRAME was expressed by concomitantly staining PB, BM, lymphonode and spleen mononuclear cells (MCs) with anti-PRAME and a panel of MoAbs specific to B(CD19)/ T(CD3)/ NK(CD16/56), monocytic(CD14) and granulocytic(CD33) markers. PRAME was detected exclusively in CD19+ cells. The median percenatge of PRAME positive cells was 5,31% (2,55–12,34%), 13,01% (8,47–38,15%), 12,79% (3,15–23,06%) and 17,5% (12,67–27,43%) in PB, BM, lymphonode and spleen MCs, respectively. Amongst CD19+ cells, we have observed that PRAME was expressed by 42,39% (16,16–75,72%), 16% (13–69,5%), 15,16% (5,49–41,20%) and 48,82%(12,67–58,89%) in PB, BM, lymphonode and spleen, respectively. To establish in which stage of B ontogenesis PRAME was expressed on, cell suspensions stained with anti-CD19 were submitted to positive magnetic separation and labeled with anti-PRAME, CD5, CD27, CD38, CD34, CD10 and IgD MoAbs. PRAME+/CD19+ cells were CD5−, CD27+, CD38+, CD34−, CD10− and IgD+, thus suggesting that PRAME is expressed by the memory B cell compartment of the normal lymphoid tissues. This study defines PRAME as a B cell antigen that may accompany the neoplastic clone proliferation of mature B cell neoplasms. Although PRAME is mainly an embryonic antigen, expressed by carcinomas of immature phenotype, it is expressed by mature B cells in normal and pathological lymphoid tissues. Our findings suggest that maturational events occurring at the germinal center of lymphoid follicles affects PRAME expression.

Concomitant Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma Discovered by Flow Cytometry

2020 ◽  
Vol 154 (Supplement_1) ◽  
pp. S127-S127
Author(s):  
K M Erickson ◽  
D Lynch
Keyword(s):  
Flow Cytometry ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Mantle Cell Lymphoma ◽  
Light Chain ◽  
Cell Lymphoma ◽  
Lymphocytic Leukemia ◽  
Cell Populations ◽  
Light Chain Restriction ◽  
Mantle Cell

Abstract Casestudy: Chronic lymphocytic leukemia (CLL) accounts for about 30% of all lymphoid neoplasms and is the most common adult blood cancer in the Western world. Mantle cell lymphoma (MCL) accounts for only about 6% of all B-cell lymphomas in Western countries. MCL and CLL are both CD5 positive B-cell lymphoproliferative disorders. It is necessary to distinguish these two entities as MCL is a more aggressive disease, and requires specific treatment. MCL and CLL can occur in one patient at the same time and is often termed a composite lymphoma. We present an 84-year-old female with a history of endometrial cancer who was found to have splenomegaly and lymphadenopathy. Flow cytometry was performed upon her peripheral blood specimen which demonstrated two distinct populations of abnormal light chain restricted B-cell populations. One population demonstrated kappa light chain restriction and was positive for CD45, CD19, CD20, CD5, CD38, FMC-7, and CD22, representing MCL. The other population showed dim lambda light chain restriction that was also positive for CD45, CD19, dim CD20, CD5, and CD23, representing CLL. FISH studies demonstrated t(11;14), and four common deletions or chromosome aneuploidy associated with CLL. These findings confirmed the dual populations of CLL and MCL. This is an interesting case because it is a very rare combination with only a few cases having been reported with two distinct cell populations in one patient at the same time.

scholarly journals CD200 Expression Improves Differential Diagnosis Between Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5637-5637 ◽  
Author(s):  
Martin Spacek ◽  
Josef Karban ◽  
Martin Radek ◽  
Eva Babunkova ◽  
Jan Kvasnicka ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Differential Diagnosis ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Mantle Cell Lymphoma ◽  
Scoring System ◽  
Cell Lymphoma ◽  
Flow Cytometer ◽  
Lymphocytic Leukemia ◽  
Mantle Cell

Abstract Background Chronic lymphocytic leukemia (CLL) in most patients is diagnosed with early stage disease identified incidentally on blood counts obtained for unrelated purposes. Immunophenotyping of peripheral blood (PB) is required for the diagnosis of CLL. A scoring system that helps in the differential diagnosis between CLL and other mature B-cell neoplasms (MBN) has been described twenty years ago (Matutes et al., Leukemia 1994; modified by Moreau et al., Am J Clin Pathol 1997). CLL/SLL typically demonstrates low-intensity staining for surface immunoglobulin, low or absent expression of CD22, CD79b and FMC7 and moderate to strong expression of CD5 and CD23. However, this phenotype is not entirely specific and some overlap in immunophenotype exists between CLL and non-CLL MBN. In particular, leukemic phase of CD5 positive mantle cell lymphoma (MCL) can be misdiagnosed as CLL. Recently, it has been shown that CD200 expression may help in differential diagnosis between CLL and other MBN. The present study aimed to prove CD200 usefulness in differentiating CLL from MCL on a series of consecutive patients and to investigate whether adding CD200 could improve the utility of Matutes scoring system, especially in atypical CLL. Methods Between January 2013 and March 2014, PB of consecutive patients with MBN was assessed in this study. Analysis was performed on a FACSCalibur flow cytometer (Becton Dickinson) and samples were stained with panels of 4-color combinations of antibodies using a standard whole-blood assay. PB specimens were incubated with antibodies purchased from eBioscience (CD200 APC, clone OX-104), Immunotech (CD23, CD79b, FMC7), BD Biosciences (CD5, CD19), and DAKO (sIg). At least 5,000 B-cells were immediately acquired on flow cytometer. Diagnosis of CLL was made according to National Cancer Institute-Working Group criteria. Furthermore, tissue biopsies of 62 (31%) CLL cases were available for histological review, including all cases of atypical CLL. Diagnosis of MCL was based on morphology and immunohistochemical detection of cyclin D1 in tissue biopsies and further confirmed by detection of t(11;14) by FISH in selected cases. Results Table 1 provides details of the patient characteristics. In our series, CD200 was present on neoplastic B-cells of all 200 CLL cases (100%), whereas only 4 cases (8.7%) of MCL showed dim positivity of CD200. The remaining 42 cases (91.3%) of MCL were negative for CD200 expression. The revised Matutes score was calculated to classify CLL cases. All 179 cases of typical CLL (defined by a score ≥ 4) presented moderate to strong expression of CD200 (Median fluorescence intensity - MFI: median = 161). CD200 was also positive in all 21 cases of atypical CLL (defined by a score < 4), but showed lower intensity (MFI: median 128) than that observed in typical CLL (P = 0.02). Application of the Matutes scoring system to MCL cases showed that three cases scored 3 (6.5%), two cases scored 4 (4.3%) and none scored 5. Of note, CD200 was absent in two cases scoring 3 and was only dimly expressed in the remaining MCL cases scoring 3 or 4. Thus, the differential expression of CD200 in CLL and MCL retained even in those cases with otherwise indeterminate immunophenotype, therefore being particularly helpful for the distinction of atypical CLL and MCL. Conclusions Flow cytometry is an essential tool for the diagnosis of CLL. However, a significant immunophenotypic overlapping occurs especially between CLL and MCL cells. In this study, we investigated the expression of recently identified marker CD200 in PB of consecutive CLL and MCL patients. We have confirmed previous reports that CD200 is consistently expressed in all typical CLL. Furthermore, CD200 was expressed by all immunophenotypically atypical CLL cases. On the contrary, in MCL patients CD200 showed only a dim positivity in four subjects and was absent in the remaining 42. The inclusion of CD200 in the MBN routine flow cytometry panels facilitates the differential diagnosis between CLL and MCL and has a great impact on accurate diagnosis in cases with immunophenotypic aberrancies. This work was supported by grant RVO VFN64165 and PRVOUK P27/LF1/1 Table 1 MCL (46 pts.) CLL (200 pts.) Age (median, range) 66.7; 47.8-82.4 67.6; 32.2-90.7 Sex (F/M) 19/27 74/126 WBC x109/L (median, range) 10; 2.1-285.4 21.9; 2.8-375.2 % neoplastic B-cells of WBC (median, range) 17.1; 1.3-90.5 54; 1.7-94.7 CD200 MFI (median, range) 2.16; 1-53.2 147.5; 20.6-637 Disclosures No relevant conflicts of interest to declare.

scholarly journals Expression of FAK and Its Involvement in the Progression of B-Cell Chronic Lymphocytic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3309-3309
Author(s):  
Cristina Gattazzo ◽  
Andrea Visentin ◽  
Alberto Pavan ◽  
Veronica Martini ◽  
Federica Frezzato ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
B Lymphocytes ◽  
Focal Adhesion ◽  
Poor Prognosis ◽  
Lymphocytic Leukemia ◽  
Bcr Signaling ◽  
Cell Chronic Lymphocytic Leukemia

Abstract INTRODUCTION B-cell chronic lymphocytic leukemia (B-CLL) is a disorder characterized by the accumulation of clonal CD5+ B lymphocytes, due to uncontrolled growth and resistance to apoptosis. Although the prognosis and clinical outcome has dramatically improved by recent innovative therapies, B-CLL still remains an incurable disease. Since signaling events downstream the BCR engagement are important for the progression of B cells, BCR signaling has been investigated in B-CLL in order to design new agents to specifically treat this disease. We demonstrated that Lyn, one of the first kinases involved in BCR signaling pathway, is overexpressed, constitutively active and anomalously distributed in malignant B cells, as compared to normal B lymphocytes. The Focal adhesion kinase (FAK), a non-receptor protein tyrosine kinase, is the primary enzyme involved in the engagement of integrins and assembly of Focal Adhesion. FAK is regulated primarily through tyrosine phosphorylation by Lyn after BCR engagement and was found to be overexpressed in many kinds of human cancers. However, a downmodulation of FAK expression and its association to poor prognosis have also been reported. The aim of this study was to investigate the role of FAK in CLL patients. METHODS Blood samples were collected from 5 controls and 50 B-CLL patients. Informed consent was obtained according to the Declaration of Helsinki. Untouched peripheral blood B cells were purified using the RosetteSep for human B cells isolation kit. The samples that were used had at least 95% of normal CD19+ or neoplastic CD5+/CD19+ cells, as assessed by flow-cytometry. Level of FAK protein was evaluated by Western blotting (Wb) and Flow Cytometry assay (FC). Levels of FAK were correlated to clinical parameters of patients. RESULTS We observed that FAK was downmodulated in 56% of analyzed patients with respect to healthy subjects (respectively, Wb: 0.28±0.25 vs 0.85±0.32, p<0.001; FC: 35%±29 vs 60%±16, p<0.05). We also identified that lower levels of FAK expression were related to the prognostic markers of poor outcome (the expression of ZAP70, CD38 and an unmutated-IGHV genes status, p<0.05) and to a shorter Treatment Free Survival (p<0.05). Moreover, patients (n=6) who had an indolent course and were responsive to the standard treatment, showed normal expression of this kinase already at diagnosis. In contrast, patients (n=6) with a more aggressive disease, had a lower expression of FAK, that was further downmodulated during the progression of disease, irrespective of how the patients were treated. CONCLUSIONS From the data presented in this report we propose that FAK downmodulation could be considered as a new marker of poor prognosis and as a putative predictor for high-risk subgroups of CLL, even in early-stage disease. Disclosures No relevant conflicts of interest to declare.

scholarly journals Evaluation of CD160 and CD200 Expression as Differentiating Markers between Chronic Lymphocytic Leukemia and Other Mature B-Cell Neoplasms

2020 ◽  
Author(s):  
Wafaa Ahmed El- Neanaey ◽  
Rania Shafik Swelem ◽  
Omar Mohamed Ghallab ◽  
Sara Mohamed Abu-Shelou
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Diagnostic Tool ◽  
Diagnostic Markers ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Double Negative ◽  
The Mean

Background: The present work aimed to investigate the expression of CD160/ CD200 in CLL and other mature B-cell neoplasms (MBN) and their use as an additional diagnostic tool for differentiating CLL from other MBN. Materials and Methods: Using flow cytometry, we detected the expression of CD160 &CD200 on B-cells from 30 CLL patients, 30 other MBN patients in addition to 20 controls. CDs160/200 measurements were determined as a percentage expression (≥20% was considered positive) and as a ratio of the mean fluorescence intensities (MFIR) of leukemic cells/controls and were considered positive when the ratios were ≥2 and 20, respectively. Results: 90% and 100% of the CLL group expressed CDs160/200 in comparison to 60% and 63.3% of other MBN (p=0.007, p<0.001), respectively. By MFIR, 96.7% and 50% of our CLL group expressed CDs160/200 in comparison to 76.7% and 30% of other MBN, respectively. CDs160/ 200 were not expressed on the controls. Positive co-expression of CD160 and CD200 was found in 90% of the CLL cases, 60% of HCL patients and only in 40% of B-NHL. However, double negative expression of both markers was found only in 24% of the B-NHL patients. Conclusion: CD160 with CD200 can be used as additional diagnostic markers to the available routine panel to differentiate between B-CLL and other non-specified B-NHL patients.

ICOS Is Induced by B Cell Receptor Signalling on Chronic Lymphocytic Leukemia B Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 969-969 ◽  
Author(s):  
Tetsuya Fukuda ◽  
Traci L. Toy ◽  
Laura Z. Rassenti ◽  
Kanti R. Rai ◽  
Thomas J. Kipps
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Progressive Disease ◽  
Leukemia Cell ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Activated T Cells

Abstract Patients with chronic lymphocytic leukemia (CLL) cells that express unmutated immunoglobulin (Ig) heavy chain variable region genes (IgVH genes) generally have a more aggressive clinical course than do patients with leukemia cells that express mutated IgVH. The reason(s) accounting for this are not known. Microarray gene expression analyses revealed that CLL cells that express unmutated IgVH could be distinguished from the leukemia cells that express mutated IgVH via the differential expression of a relatively small number of genes, one of which encodes the zeta-associated chain of 70kD (ZAP-70), which generally is expressed by CLL cells that express unmutated IgVH. Although the expression of ZAP-70 is associated with expression of unmutated IgVH in CLL, this association is not absolute. This was the case for a pair of monozygotic twins who both developed CLL at age 57. Although each of the twins had leukemia cells that expressed mutated IgVH, only one of the twins had leukemia cells that lacked expression of ZAP-70 protein and has indolent, non-progressive disease (Blood100: 4609–14, 2002). We performed microarray analysis using Affymetrix HG-U133A array on the isolated leukemia cells of each twin to define the genes that were differentially expressed between the two. In addition to ZAP-70, we found that the CLL cells of the twin with progressive disease also expressed the inducible co-stimulatory molecule (ICOS), a member of the CD28/CTLA-4 family of immune accessory co-stimulatory molecules that ordinarily only is expressed by activated T cells. Expression of ICOS protein by this leukemia B cell population, but not by the CLL B cells population of the other twin, was confirmed using fluorochrome-labeled anti-ICOS mAb and flow cytometry. We examined the CLL B cells from 58 additional patients for expression of ICOS by flow cytometry and found that 16 (28%) also expressed ICOS. We found that expression of ICOS was associated with expression of ZAP-70, as assessed via flow cytometry and immunoblot analyses. Whereas 14 of the 29 ZAP-70+ cases expressed ICOS, only 2 of the 29 ZAP-70-negative cases expressed this immune co-stimulatory molecule. Nevertheless, we found that nearly all of the 56 of the 58 cases expressed B7h, the ligand for ICOS. The two cases that did not express detectable B7h expressed ZAP-70 and were ICOS+. In preliminary studies, we found that treatment of ICOS-negative, ZAP-70+ CLL cells (n = 2) with goat anti-human Ig could induce expression of ICOS, suggesting that, as on T cells, this molecule also might be inducible in some cases of B cell CLL. Culture of ICOS+ CLL cells with an anti-B7h mAb capable of blocking ICOS-B7h interactions significantly enhanced ICOS surface expression, as assess by flow cytometry, suggesting that B7h may down-modulate ICOS through paracrine/autocrine receptor-ligand interactions. Because of this we evaluated for functional expression of ICOS on CLL B cells. We found that ligation of ICOS could induce enhanced signaling via the PI3K/Akt pathway in isolated CLL B cells, resulting in enhanced phosphorylation and activation of Akt. As such, we speculate that the expression of ICOS and its ligand in B cell CLL may enhance leukemia cell survival and/or proliferation, potentially contributing to the more aggressive disease observed in some patients with this disease.

Sign in / Sign up

Export Citation Format

Share Document