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CANCER RESEARCH CENTER, UNIVERSITY & UNIVERSITY HOSPITAL of SALAMANCA (SPAIN)

MONOCLONAL B-CELL LYMPHOCYTOSIS. CANCER RESEARCH CENTER, UNIVERSITY & UNIVERSITY HOSPITAL of SALAMANCA (SPAIN) Multicolor Immunophenotyping: Standardization and Applications March 9-11, 2012 TMH, Mumbay (India).

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CANCER RESEARCH CENTER, UNIVERSITY & UNIVERSITY HOSPITAL of SALAMANCA (SPAIN)

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  1. MONOCLONAL B-CELL LYMPHOCYTOSIS CANCER RESEARCH CENTER, UNIVERSITY & UNIVERSITY HOSPITAL of SALAMANCA (SPAIN) Multicolor Immunophenotyping: Standardization and Applications March 9-11, 2012 TMH, Mumbay (India)

  2. Monoclonal B-cell Lymphocytosis(MBL) indicates the presence of <5x109clonal B-cell/L in PB of otherwise healthy subjects, with or without lymphocytosis MONOCLONAL B CELL LYMPHOCYTOSIS (MBL) (Marti et al, Br J Haematol 2005)

  3. CLL-like MBL: <5x109 clonalCD5+B-cells/L in PB of otherwise healthy subjects, with or without lymphocytosis Typical: CD5+, CD20lo, CD79lo and sIglo Atypical: CD5+ and CD20hi and/or CD79bhi and/or sIghi - Non-CLL-like MBL: <5x109 clonalCD5- B-cells/L in PB of otherwise healthy subjects, with or without lymphocytosis. CLASSIFICATION OF MONOCLONAL B CELL LYMPHOCYTOSIS (MBL) (Marti et al, Br J Haematol 2005)

  4. B CELL CHRONIC LYMPHOPROLIFERATIVE DISORDERS Heterogeneous group of diseases typically characterized by a monoclonal expansion of a mature-appearing neoplastic B-lymphocyte WHO CLASSIFICATION OF B-CLPD Mature/peripheral B cell chronic lymphoid leukemias: Chronic lymphocytic leukemia/Small B cell lymphocytic lymphoma Prolymphocytic leukemia Hairy cell leukemia Mature/pheripheral B-cell lymphomas: Lymphoplasmacytic lymphoma Splenic marginal zone lymphoma Extranodal marginal zone lymphoma (MALT-type) Nodal marginal zone lymphoma Follicular lymphoma Mantle cell lymphoma Diffuse large B-cell lymphoma Burkitt lymphoma Plasma cell neoplasias: Multiple myeloma/plasmacytoma

  5. IMMUNOPHENOTYPIC PATTERNS OF DIFFERENT TYPES OF B-CLPD (Orfao et al, In: “B-CLL”.Humana Press, 2004) sIg CD5 CD10 CD20 CD11c CD23 CD24 CD25 CD38 CD43 CD79b CD103 FMC7 B-CLL d + - d -/+ ++ + + -/+ + d - - PLL + -/+ - + -/+ -/+ + -/+ -/+ -/+ + - + HCL + - - ++ ++ - -/+ ++ - - + + + SMZL + -/+ - + + - + -/+ - - + -/+ + LPL + - - + - - + + -/+ - + - -/+ MCL + + - + -/+ - + -/+ - + + - -/+ FL + - + + -/+ -/d + -/+ + - + - + LDBCL + - - + -/+ - -/+ - + - + - + BL -/+ - + + - - + - ++ -/+ -/+ - +

  6. WHO: B-cell malignancies Immunophenotype CLL HCL

  7. WHO: B-cell malignancies Cytogenetics MCL BL Immunophenotype CLL HCL

  8. WHO: B-cell malignancies Histology & cytology DLBCL B-PLL Cytogenetics MCL BL Immunophenotype CLL HCL

  9. WHO: B-cell malignancies Histology & cytology DLBCL B-PLL Cytogenetics MCL BL Immunophenotype CLL HCL Clinic MALT

  10. - Chronic lymphocytic leukemia (CLL) is the most frequent subtype of leukemia in the Western World - Diagnosis of CLL requires the presence of >5x109 clonal B-cells/L in PB with a CLL-like immunophenotype even if there are no symptoms. MONOCLONAL B CELL LYMPHOCYTOSIS (MBL) vs CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) (Morton et al, Blood 2006; http://www.hmrn.org/)

  11. MONOCLONAL B LYMPHOCYTOSIS • Between 5%-7% and 12% of healthy adults, display small clones ofCLL-likeand 2%-2.5% of healthy adultshave small clones ofnon-CLL-likeB-cells in peripheral blood. - Recent studies show a transformation rate of MBL into CLL (frequently assymptomatic) of around 1% per year, most CLL patients being preceeded by MBL. Prediction of transformation of MBL into symptomatic remains a challenge (Rawstron et al, Blood 2002; Rawstron et al, NEJM 2008; Dagklis et al, Blood 2009, Nieto et al, Blood 2009, Almeida et al, Leukemia 2011) (Rawstron et al, NEJM 2008; Landgren et al, NEJM 2009)

  12. CLINICAL PROGRESSION OF A CLL-LIKE MBL CASE Rawstron et al, Cytometry B, 2010

  13. MONOCLONAL B CELL LYMPHOCYTOSIS AND CHRONIC LYMPHOCYTIC LEUKEMIA MBL cases presenting with or without lymphocytosis, is highly prevalent among the general population Increasing evidence suggests that this could represent a pre-leukemic condition, since CLL frequently develops in individuals with prior history of MBL

  14. AIM: • 1.- To determine the prevalence of CLL-like (and other B-CLPD) clonal PB B-cells in a groupd of healthy adults from the Salamanca healthcare area • 2.- To establish the immunophenotypic and genetic characteristics of each clonal B-cell population identified (vs neoplastic B-cells from CLL and other B-CLPD. • 3.- To investigate the potential clinical significance of the B-cell clones identified and its behaviour.

  15. Material & Methods Healthy subjects and patients studied 75 non-treated B-CLPD patients: 639 healthy adults > 40 years: • Randomly recruited (Health Care Area of Salamanca: • -Age:62±13years (range: 40-97) • -Sex: (m/f): 46% / 54% • -N. of PB leucocytes: 6,3±1,6 x 109/L • N. of PB lymphocytes: 2,1±0,7 x 109/L • N. of PB B lymphocytes: • 0,16±0,1 x109/L • CLL:65 • Transf. CLL/PLL:3 • MALT NHL:2 • FL:1 • MZL:1 • Unclassifiable: 3 • - Age: 67±13 years • (range: 35-91) • - Sex:(m/f): 57% / 43%

  16. Methods: Immunophenotypic screening for aberrant PB B-cells -8-color direct immunofluorescence technique:

  17. CD20 Pacific blue SSC-A CD20 Pacific blue FSC-A CD19 PerCP-Cy5.5 CD19 PerCP-Cy5.5 Methods: Immunophenotypic screening for aberrant PB B-cell populations -Data acquisition: FACSCanto II flow cytometer: Window of analysis to simultaneously select for CD19+ and/or CD20+ events (B-cells), for > 5x106 leukocytes

  18. Frequency of PB B-cell clones in healthy adults Found in 94/637 cases (14.8%) Frequency along the recruitment for the study (18 months) 500 300 400 150 637 No. of individuals studied 10% 11.7% 14.8% 14.4% 14.2% % of cases showing PB B-cell clones No differences in distribution per sex group: 13.4% m /15.9% f ; p>0.05 Differences with age: p<0.0001

  19. Immunophenotypic identification of PB B-cells with a CLL-like phenotype Normal B lymphocytes Clonal CLL-like B-cells *0.35% of all B-cells & O.O3% of all leucocytes

  20. Immunophenotypic identification of clonal B-cells in PB from healthy subjects 0.01% of leucocytes 0.7% of B-cells CLL-like phenotype CD23 PE SSC-A Kappa PE CD5 APC CD19 PerCP-Cy5.5 CD20 Pacific blue cyBCL2 FITC Lambda FITC 0.0015% of leucocytes 0.1% of B-cells CLL-like phenotype CD23 PE Kappa PE SSC-A CD5 APC CD19 PerCP-Cy5.5 CD20 Pacific blue cyBCL2 FITC Lambda FITC Non-CLL B-cell phenotype CD20 Pacific blue SSC-A Kappa PE CD23 PE CD19 PerCP-Cy5.5 CD19 PerCP-Cy5.5 cyBCL2 FITC Lambda FITC

  21. Frequency of clonal B cell populations in PB of healthy adults 100 90 80 70 60 50 Percentage of MBL cases 40 30 20 10 0 40 45 50 55 60 65 70 75 80 85 90 95 Age (years) En 95/639 casos (14,8%) Mean age: 70±11 years for MBL (vs. 61±13 years for non-MBL; p<0.05) 75% 26% 21.3% 18.5% 5.9% 5.7% Nieto et al, Blood 2009

  22. P>0.05 % infiltración respecto al total de leucocitos 40-49 50-59 60-69 70-79 80-89 >90 Age group (years) PB clonal B-cells in healthy adults: size of the B-cell population P>0.05 >1% Clonal B-cells • 0.01% Clonal B-cells >0.1-1% Clonal B-cells 8.5% 8.5% 54% % of PB leukocytes 29% >0.01-0.1% Clonal B-cells % of PB leucocytes

  23. The Salamanca Area within Spain Castilla y León (2.625.000 inhabitants) Salamanca (330.000 inhabitants)

  24. BICLONAL 21% (n=20) MONOCLONAL 79% (n=74) Frequency of biclonal cases among healthy individuals Immunophenotypic features of PB clonal B-cells from healthy subjects

  25. Methods: Characterization of (purified) circulating PB B-cell clones poblaciones Immunophenotypic characterization of aberrant B-cells - Direct stain-lyse-wash immunofluoresce technique: Panel of 8-color MAb combinations for: CD11c,CD22,CD24,CD25,CD27,CD34,CD43,CD49d,CD103,CD79b, FMC7,IgM,CCR6,cyZAP70. Purification of aberrant B-cells: FACSAria (purity:≥98%) Genetic analysis of purified B-cells by iFISH: CLL: Trisomy 12, del(13q14), del(11q22-23) (ATM & MLL genes), del(17p13) & del(6q21); FL: t(14;18) (LSI IgH/bcl2 dual color); MCL: t(11;14) (LSI IgH/CCND1 dual color); DLBCL & BL: probes for 3q27 (BCL6 gene), t(8;14) (q24;q32), t(2;8) & t(8;22) Sequencing of IGH e IGK o IGL genes: Repertoire of the V, D, J regions of the IGH gene & VJ of the IGK & IGL genes IGH gene mutational status. Statistical methods: SPSS 15.0

  26. B-CLL non-class NHL. MCL MALT MZSL Atypical B-CLL MZSL or HCLv MALT or MZSL B-CLL+B-CLL B-CLL + non-class. NHL MZSL + non-class NHL Non class. NHL + Non-class. NHL MZSL or MALT + CLL PB clonal B-cells from helthy subjects Immunophenotypic features of the B-cell clones BICLONAL MONOCLONAL

  27. CLL vs CLL-LIKE MBL: Genetic features of clonal B-lymphocytes Rawstron et al, Cytometry B, 2010

  28. CLL vs CLL-LIKE MBL: Biological features of clonal B-lymphocytes Rawstron et al, Cytometry B, 2010

  29. Cytogenetic patterns of clonal CLL tumor cells vs PB CLL-like B-cells from healthy adults P<0,05 70 60% 58% 60 MBL (n=35) CLL (n=65) 50 38% 40 Percentage of cases 25% 30 16% 16% 20 7% 10 0 17p-;11q- Normal* 13q- +12 % of altered cells • No alterations detected by FISH with probes for 13q-,+12, 17p-,11q-

  30. CLL-like MBLs with genetic abnormalities have higher absolute numbers of CLL-like B-cells P=0.001 Abs. N. CLL-like B cells (cells/mL) No genetic abnormalities Trisomy 12 Del (13q) 0/2 cases carrying trisomy 12 and only 2/12 cases with del(13q) showed less than 1 PB CLL-like B-cell/mL, while 11/21 cases without detectable genetic abnormalities were “low-count” MBL (<1 circulating CLL-like B-cell/mL) Almeida et al, Leukemia 2011

  31. 12 p=0,03 p=0,045 p=0,04 25 10 15 0,5 8 20 12 0,4 6 15 0,3 9 % of clonal B-cells From all B cells % of clonal B-cells from leucocytes N. of clonal B cells (x103/mL) 4 10 0,2 6 2 5 0,1 3 0 0 0,0 0 BASAL +1 year Baseline +1 year Baseline +1 year p=0,2 100 p=0,003 p=0,03 80 1,400 1,200 60 1,000 % of clonal B cells From all B cells % of clonal B cells from leucocytes 800 N. of clonal B cells (x103/mL) 40 600 400 20 200 0 0 Baseline +1 year +1 year Baseline Baseline +1 year MBL: CLINICAL SIGNIFICANCE CLL-like MBL (39/82) NON-CLL-like MBL (11/13) Nieto et al, ClinicalCytometry 2010

  32. CONCLUSIONS • A significant proportion (14.8%) of healthy adults aged >40 years display circulating B-cell clones frequently (13%) showing a CLL-like phenotype. • The frequency of such CLL-like (and other) circulating B-cell clones increase with age. • The higher frequency of circulating CLL-like clones could reflect the greater sensitivity of the screening approach used, half of the cases showing clonal B-cell numbers below previously reported threshold levels ( 0,01%). • The immunophenotypic, genetic and molecularfeatures of circulating B-cell clones overlap with those of clinically evident CLL and other B-CLPD. • In all tested cases circulating B-cell clones remain detectable after one year with minimal but significant changes (increase) in their distribution.

  33. 100% Whole series 100% 100% 70% 50% 0% 0.01 0.1 1 5 10 20 30 40 50 100% 40 - 59 years 46% 50% 32% % of cases with a CLL-like clone 18% 0% 0.01 0.1 1 5 10 20 30 40 50 100% 60 - 69 years 88% 50% 62% 36% 0% 0.01 0.1 1 5 10 20 30 40 50 100%  70 years 100% 100% 100% 50% 0% 5 0.01 0.1 1 10 20 30 40 50 PB Volume (mL) FREQUENCY OF CLL-like MBL IN HEALTHY ADULTS DETECTED FREQUENCY Almeida et al, Leukemia 2010

  34. FREQUENCY OF CLL-like MBL IN HEALTHY ADULTS DETECTED FREQUENCY CALCULATED FREQUENCY 100% Whole series 100% 100% 70% 50% 0% 0.01 0.1 1 5 10 20 30 40 50 100% 40 - 59 years 46% 50% 32% % of cases with a CLL-like clone 18% 0% 0.01 0.1 1 5 10 20 30 40 50 100% 60 - 69 years 88% 50% 62% 36% 0% 0.01 0.1 1 5 10 20 30 40 50 100%  70 years 100% 100% 100% 50% 0% 5 0.01 0.1 1 10 20 30 40 50 PB Volume (mL) Statistical predictive model (power regression analysis) Almeida et al, Leukemia 2010

  35. 1 mL 50 mL Kappa FITC CD5 APC CD5 APC CD20 Pacific blue CD20 Pacific blue Lambda PE Kappa FITC CD5 APC CD5 APC CD20 Pacific blue CD20 Pacific blue Lambda PE Immunophenotypic identification of CLL-like clonal B-cells in 50 mL from healthy adults Almeida et al, Leukemia 2011

  36. Characteristics of CLL-like B-cells identified in 50 mL of PB from 9 healthy subjects older than 70 years M: male; F: female; ND: Not detected. NA: Not applicable *CLL-like B-cells were identified as those cellular events expressing CD19+, CD5+, CD20+dim, CD79b+dim and surface immunoglobulin light chain Ig+dim. § Restricted to CLL-like B-cells (considered to be altered when ratio k/l >3.1 or <1:3) Almeida et al, Leukemia 2011

  37. B-CLPD: COMMON ABERRANT PHENOTYPES Diagnostic group Aberrant phenotype % of cases typCLL/SLL CD22-/+dCD5+ 97% CD22-/+dCD23+ 98% PLL sIg +++ 60% HCL CD11c+++ 69% CD19+++ 69% CD103+++ 92% FSC/SSC+++ 85% LPL CD22-/+dCD10- 67% FL BCL2+++CD10+ 85% MCL CD22-/+dCD5+ 100% Sanchez et al, Leukemia, 2002

  38. CUMULATIVE FREQUENCY OF CLL-LIKE MBL IN POPULATION- VS HOSPITAL-BASED COHORTS Rawstron et al, Cytometry B, 2010

  39. Conclusions • In healthy adults over 70 years, emergence of one or more CLL-like cell populations occurs whenever > 50mL PB are screened, suggesting that these clonal cells may more likely represent the normal counterpart of CLL cells, rather than a leukemic precursor

  40. ONCOGENIC EVENTS Genetic/environmental factors Genetic and Chromosomal instability Genetic instability Reactive process Malignant trasnformation Neoplastic trasnformation X ? (Wait and see vs early therapy) Wait and see Treatment

  41. . . . . . . . . . . . . . . . . . . . . MBL AND B-CLL: HYPOTHETICAL ONTOGENIC MODEL Identical primary mutation ? Identical IGH/IGL VDJ/VJ gene rearrangement Restricted expressiofn Ig (k or L) light chains Clonal expansion Evolution Secondary genetic alterations Subclones blocked at different maturation stages and clinical progression

  42. B CELL MATURATION PATHWAYS IN NORMAL HUMAN BONE MARROW (GATED ON CD19+) sIgM:FITC CD20:PB CD38:APC-H7 CD10:PECy7 sIgD:PE sIgM:FITC sIgD:PE CD10:PECy7 CD38:APC-H7 CD5:APC

  43. MATURATION-ASSOCIATED NORMAL PB B-CELL SUBSETS BM PB Immature B-cells Naïve B-cells Somatic Hypermutation/IgH Switch Memory B-cells Plasmablasts/ Plasma cells

  44. Immature CD10:PE-Cy7 LOGICAL Naïve Memory Plasmablasts CD38:AF700 LOGICAL Memory Plasmablasts/Plasma cells CD27:APC LOGICAL sIgneg (14% 12%) sIgA+ (21% 9%) sIgM+ (18% 12%) sIgMD+ (52% 15%) sIgD+ (5% 5%) sIgG+ (13% 11%) sIgA+ (49% 18%) sIgG+ (23% 10%) Distribution of B-cell maturation subsets in peripheral blood from normal individuals (N=600) Caraux A Haematologica 2010, Perez-Andres M Clinical Cytometry 2010

  45. CD27 EXPRESSION BY NORMAL PB CD5+ B CELLS

  46. Normal B-cell maturation stem cell Lymphoid tissue Bone marrow Memory B-cell Mature naive B-cell Germinal center B-cell Lymphoid progenitor MZL LPL ALL MCL Progenitor-B cell Plasma cell Pre-B cell DLBCL BL, FL Immature B-lymphocyte MM Peripheral blood CLL Lee et al.

  47. AKNOwLEDGEMENTS CIC, Department of Medicine & Cytometry Service (USAL) J Almeida J Ciudad A López Fernández A Nieto W Nieto A Rodríguez Caballero ML Sánchez-García C Teodosio Primary Health Care Area of Salamanca (SACYL) JA Romero Furones P Fernández Navarro Primary Health Care Group of Salamanca for the study of MBL (40 medical doctors from the primary health care area of Salamanca ) Regional Health Care Department of Castilla y León AT Vega Alonso (Epidemiology) University Hospital of Salamanca MB Muñoz Criado (Microbiology) A Balanzategui (Hematology) M González Díaz (Hematology) MB Vidriales (Hematology) Universidad Federal de Río de Janeiro (Brazil) CE Pedreira (Mathematics)

  48. THANK YOU

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