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

2. -M onoclonal B-cell Lymphocytosis (MBL) indicates the presence of <5x10 9 clonal 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: <5x10 9 clonal CD5 + B-cells/L in PB of otherwise healthy subjects, with or without lymphocytosis -Typical: CD5 +, CD20 lo, CD79 lo and sIg lo -Atypical: CD5 + and CD20 hi and/or CD79b hi and/or sIg hi - Non-CLL-like MBL: <5x10 9 clonal CD5- 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 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 WHO CLASSIFICATION OF B-CLPD

5. IMMUNOPHENOTYPIC PATTERNS OF DIFFERENT TYPES OF B-CLPD ( 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 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 >5x10 9 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. -Between 5%-7% and 12% of healthy adults, display small clones of CLL-like and 2%-2.5% of healthy adults have small clones of non-CLL-like B-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 MONOCLONAL B LYMPHOCYTOSIS (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. 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 MONOCLONAL B CELL LYMPHOCYTOSIS AND CHRONIC LYMPHOCYTIC LEUKEMIA

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. 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 10 9 /L - N. of PB lymphocytes: 2,1±0,7 x 10 9 /L - N. of PB B lymphocytes: 0,16±0,1 x10 9 /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% 639 healthy adults > 40 years: 75 non-treated B-CLPD patients: Healthy subjects and patients studied Material & Methods

16. Methods: Immunophenotypic screening for aberrant PB B-cells -8-color direct immunofluorescence technique: PBPOFITCPE PerCP/ Cy5.5 PECY7APCAF700 CD20CD45 CD8 Anti-sIg CD56 Anti-sIg  CD4CD19CD3CD38 CD20CD45cyBcl2CD23CD19CD10CD5CD38 --Anti-sIg Anti-sIg  CD19CD10CD5-

17. 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 Window of analysis to simultaneously select for CD19+ and/or CD20+ events (B-cells), for > 5x10 6 leukocytes CD20+ events (B-cells), for > 5x10 6 leukocytes CD20 Pacific blue CD19 PerCP-Cy5.5 CD20 Pacific blue FSC-A SSC-A

18. Frequency of PB B-cell clones in healthy adults Frequency of PB B-cell clones in healthy adults Found in 94/637 cases (14.8%) No differences in distribution per sex group: 13.4% m /15.9% f ; p>0.05 637 No. of individuals studied 150 % of cases showing PB B-cell clones 10% 300 11.7% 400 14.2% 500 14.4% 14.8% Negative PBPositive PB Age (Mean ± 1SD)61 ± 1369 ± 11 Differences with age: p<0.0001 Frequency along the recruitment for the study (18 months)

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

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

21. Nieto et al, Blood 2009 Frequency of clonal B cell populations in PB of healthy adults En 95/639 casos (14,8%) 5.7% 5.9% 18.5% 21.3% 26% 75% Mean age: 70±11 years for MBL (vs. 61±13 years for non-MBL; p<0.05)

22. PB clonal B-cells in healthy adults: size of the B-cell population PB clonal B-cells in healthy adults: size of the B-cell population % of PB leukocytes% of PB B-lymphocytesMedian[range]0.01%[0.0004%-14.8%]0.53%[0.02%-96%]  0.01% Clonal B-cells >0.01-0.1% Clonal B-cells >0.1-1% >1% 54% % of PB leucocytes 29% 8.5% P>0.05 Age group (years) % infiltración respecto al total de leucocitos P>0.05 40-4950-5960-6970-7980-89>90 % of PB leukocytes

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

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

25. - Direct stain-lyse-wash immunofluoresce technique: Panel of 8-color MAb combinations for: Panel of 8-color MAb combinations for: CD11c,CD22,CD24,CD25,CD27,CD34,CD43,CD49d,CD103,CD79b, CD11c,CD22,CD24,CD25,CD27,CD34,CD43,CD49d,CD103,CD79b, FMC7,IgM,CCR6,cyZAP70. FMC7,IgM,CCR6,cyZAP70. 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) Purification of aberrant B-cells: FACSAria (purity: ≥98%) Genetic analysis of purified B-cells by iFISH: Immunophenotypic characterization of aberrant B-cells Statistical methods: SPSS 15.0 Methods: Characterization of (purified) circulating PB B-cell clones poblaciones 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.

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 MONOCLONAL BICLONAL Immunophenotypic features of the B-cell clones Immunophenotypic features of the B-cell clones

27. Median CLL count 13q14 deletion Trisomy 1211q deletion17p deletion CLL (17)>5,000/uL 54% (178/238) 16% (53/325) 18% (58/235) 7% (23/325) Clinic (Mayo) (12,13) 275744% (56/126)18% (21/126)2% (2/126)3% (4/126) Clinic (Leeds) (8) 314158% (19/33)21% (7/33)6% (2/33)3% (1/33) Population (Leeds) (8) 939% (15/38)18% (4/22)0% (0/21)0% (0/10) Population (Salamanca) (6)* 0.536% (16/45)8% (4/45)0% (0/45) Rawstron et al, Cytometry B, 2010 CLL vs CLL-LIKE MBL: Genetic features of clonal B-lymphocytes

28. Source Median CLL cell count CLL-like B- cells (median %) Cases with <98% IGHV homology Predominant CLL cell IGHV gene Similar to CLL? CLL (25)>5,000>95% 534/927 (57.6%) 3-07, 1-69, 4-34, 3-23 - Clinic (Leeds) (8) 3141>95%18/20 (90%)3-07, 3-23, 4-34Yes Clinic (Mayo) (12,13) 2757>95%84/109 (77%) 3-07, 1-69, 4-34, 3-23 Yes Familial (Duke) (24) 2625%12/16 (75%)3-07, 4-34Yes Population (Leeds) (8) 980%18/20 (90%)3-07, 3-23, 4-34Yes Population (Italy) (4) 1.07%36/51 (70%)4-59/61No Population (Salamanca) (6) 0.50.4%2/7 (29%) No CLL- associated No CLL vs CLL-LIKE MBL: Biological features of clonal B-lymphocytes Rawstron et al, Cytometry B, 2010

29. 0 10 20 30 40 50 60 70 Percentage of cases Normal*13q-+12 17p-;11q- MBL (n=35) CLL (n=65) P<0,05 60% 25% 38% 58% 7% 16% Genetic alterationMBLCLL 13q-67%±30%67%±31% +1250%±13%76%±17% Other (17p-/11q-) - 65%±29% % of altered cells No alterations detected by FISH with probes for 13q-,+12, 17p-,11q- Cytogenetic patterns of clonal CLL tumor cells vs PB CLL-like B-cells from healthy adults

30. CLL-like MBLs with genetic abnormalities have higher absolute numbers of CLL-like B-cells Trisomy 12Del (13q) No genetic abnormalities Abs. N. CLL-like B cells (cells/  L) P=0.001 Almeida et al, Leukemia 2011 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)

31. Baseline 0,0 0,1 +1 year % of clonal B-cells from leucocytes 0,2 0,3 0,4 0,5 p=0,04 BASAL+1 year % of clonal B-cells From all B cells p=0,03 0 5 10 15 20 25 Baseline+1 year N. of clonal B cells (x10 3 /  L) 0 3 6 9 12 15 p=0,045 MBL: CLINICAL SIGNIFICANCE 0 2 4 6 8 10 12 % of clonal B cells from leucocytes Baseline +1 year p=0,03 0 20 40 60 80 100 % of clonal B cells From all B cells p=0,2 Baseline +1 year 0 200 400 600 800 1,000 1,200 1,400 p=0,003 N. of clonal B cells (x10 3 /  L) Baseline +1 year NON-CLL-like MBL (11/13) CLL-like MBL (39/82) Nieto et al, Clinical Cytometry 2010

32. 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 molecular features 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. CONCLUSIONS

33. 100% 50% 0% 10.10.0151020405030 Whole series 10.10.0151020405030 40 - 59 years 10.10.0151020405030 60 - 69 years 100% 50% 0% 100% 50% 0% 100% 50% 0%  70 years PB Volume (mL) 10.10.011020405030 % of cases with a CLL-like clone 5 70% 100% 18% 46% 32% 88% 62% 36% 100% DETECTED FREQUENCY FREQUENCY OF CLL-like MBL IN HEALTHY ADULTS Almeida et al, Leukemia 2010

34. 100% 50% 0% 10.10.0151020405030 Whole series 10.10.0151020405030 40 - 59 years 10.10.0151020405030 60 - 69 years 100% 50% 0% 100% 50% 0% 100% 50% 0%  70 years PB Volume (mL) 10.10.011020405030 % of cases with a CLL-like clone 5 70% 100% 18% 46% 32% 88% 62% 36% 100% CALCULATED FREQUENCYDETECTED FREQUENCY FREQUENCY OF CLL-like MBL IN HEALTHY ADULTS Statistical predictive model (power regression analysis) Almeida et al, Leukemia 2010

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

36. Volume of PB analyzed: 1mL Volume of PB analyzed: 50mL % of CLL-like cells: 0.002% of the whole B-cell population Immunophenotypic identification of CLL-like clonal B- cells in healthy adults

37. Case N. Age (years) Sex N. of CLL- like B-cell populations* sIg light chain restriction Ratio  % of CLL-like B-cells from WBC (x10 -3 ) % of CLL-like B cells from all PB B-cells N. of CLL-like B cells /mL Monoclonal cases Case #178MOne NA0.8%0.041%0.066 Case #273MOne  NA0.73%0.05%0.036 Case #377MOne  NA1.3%0.08%0.112 Case #488MOne  NA0.14%0.013%0.009 Multiclonal cases Case #577FTwo  20 0.28% 0.014% 0.002% 0.001% 0.0018 0.0009 Case #672FTwo  0.72 0.08% 0.11% 0.006% 0.008% 0.007 0.009 Case #782FTwo  0.18 0.18% 0.95% 0.02% 0.01% 0.009 0.004 Case #873MTwo  1.8 0.49% 0.27% 0.016% 0.009% 0.032 0.018 Case #987MND NA<0.0006%<1.1x10 -6 <1x10 -6 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) Characteristics of CLL-like B-cells identified in 50 mL of PB from 9 healthy subjects older than 70 years Almeida et al, Leukemia 2011

38. B-CLPD: COMMON ABERRANT PHENOTYPES Diagnostic group Aberrant phenotype % of cases typCLL/SLLCD22 -/+d CD5 + 97% CD22 -/+d CD23 + 98% PLLsIg +++ 60% HCLCD11c +++ 69% CD19 +++ 69% CD103 +++ 92% FSC/SSC +++ 85% LPLCD22 -/+d CD10 - 67% FLBCL2+++CD10 + 85% MCLCD22 -/+d CD5 + 100% Sanchez et al, Leukemia, 2002

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

40. 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

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

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

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

44. Memory B-cells Somatic Hypermutation/IgH Switch Naïve B-cells Immature B-cells BMPB MATURATION-ASSOCIATED NORMAL PB B-CELL SUBSETSPlasmablasts/ Plasma cells

45. CD38:AF700 LOGICAL CD27:APC LOGICAL CD10:PE-Cy7 LOGICAL Immature Naïve Memory Plasmablasts PercentageAbsolute count (cells/  L )Immature 5.4%  3.7% (1.8%-4.5%) 9  9 (3-11) Naive 64%  12% (57%-73%) 101  57 (61-130) Memory 31%  12% (21%-40%) 52  39 (26-65) Plasmablasts/ Plasma cells 2.5%  1.4% (0.8%-2.3%) 3.0  6.8 (1.0-3.1) Distribution of B-cell maturation subsets in peripheral blood from normal individuals (N=600) MemoryPlasmablasts/Plasma cells sIgA + (21%  9%) sIgG + (23%  10%) sIgMD + (52%  15%) sIgG + (13%  11%) sIgM + (18%  12%) sIg neg (14%  12%) sIgD + (5%  5%) sIgA + (49%  18%) Caraux A Haematologica 2010, Perez-Andres M Clinical Cytometry 2010

46. CD27 EXPRESSION BY NORMAL PB CD5+ B CELLS

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

48. 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 AKNOwLEDGEMENTS 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 ) University Hospital of Salamanca MB Muñoz Criado (Microbiology) A Balanzategui (Hematology) M González Díaz (Hematology) MB Vidriales (Hematology) Regional Health Care Department of Castilla y León AT Vega Alonso (Epidemiology) Universidad Federal de Río de Janeiro (Brazil) CE Pedreira (Mathematics)

49. THANK YOU