Department of Haematology University Hospital. University of Salamanca, Spain

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1 Immunophenotyping of Plasma Cells: Implications on management Department of Haematology University Hospital Cancer Research Centre J.F. San Miguel University of Salamanca, Spain

2 Faculty disclosure information Nothing to disclose

3 BACKGROUND IMMUNOPHENOTYPING - Acute Leukemias & Lymphoproliferative disorders: Mandatory for diagnosis & monitoring - Multiple Myeloma: Restricted to research Differential diagnosis of unusual cases

4 IMMUNOPHENOTYPING of PLASMA CELL Discrimination between normal vs. clonal PC - Differential diagnosis i of MGUS & MM - Risk of Transformation of MGUS & Smoldering MM - MRD investigation

5 Immunophenotype of normal and myelomatous PC Normal BM Multiple Myeloma Antigenic % cases Antigenic % cases pattern pattern CD19 heterogeneous ( 80% +ve cells) CD19 homogeneous 100% 93% (-ve) Gate CD 38 -> CD38 -> CD45 homogeneous (+strong) CD45 homogeneous 94% 83% (-ve) CD45 -> CD45 -> CD56 heterogeneous 100% CD56 homogeneous ( 10% +weak) (+strong) 57% Gate CD 38 -> CD38 -> Mateo et al. J Clin Oncol; 2008;26:2737

6 Immunophenotype of normal and myelomatous PC Antigenic pattern Normal BM % cases Antigenic pattern Multiple Myeloma % cases CD117 homogeneous ( -ve) CD % homogeneous 30% (+strong) Gate CD38 -> CD38 -> CD28 (-ve/ weak) 70% CD28 homogeneous 30% 25% (+strong) Gate CD38 -> CD38 -> CD33 (-ve/ weak) 80% 20% CD33 homogeneous (+strong) 20% Gate CD38 -> CD38 -> Mateo et al. J Clin Oncol; 2008;26:2737

7 Distinction between myelomatous & normal PC * Selected CD38 high plasma cells n-pc CD19 MM-PC CD56 CD45 *92% of MM patients Mateo G, et al. In: Methods in molecular medicine. Vol 113. Multiple myeloma: methods and protocols. New Jersey:Humana Press Inc.; 2005: 5-24.

8 Protocol for immunophenotypic investigation Panel of MoAb Double Acquisition (quadruple combinations) FITC CD38 CD138 CD20 PE PE/Cy5 CD56 CD28 CD117 CD19 CD33 CD138 APC CD45 CD38 CD38 FITC: fluorescein isotiocianate; PE: phycoerytrine; PE/Cy5: PE/cyanine5; APC: allo-phyco-cyanine 1 er er STEP Total BM cellularity CD38 FITC -> Identify the phenotypic aberrancy characteristic of malignant PC PATIENT-SPECIFIC PROBE In <10% of cases other combination were added to clarify the clonal nature of the PC: cl/ck/cd38/*cd nd STEP Live Gate on selected fraction* Gate CD38 FITC -> * 3,000 gated PC from 10 6 acquired leukocytes

9 Parameters analyzed Total cellularity PC compartment % myelomatous PC %MM-PC % normal PC %N-PC San Miguel et al Blood; 2002; 98: Proportion of N-PC referred to the total-pc %N-PC/ total-pc

10 IMMUNOPHENOTYPING of PLASMA CELL Discrimination between normal vs. clonal PC - Differential diagnosis of MGUS & MM - Risk of Transformation of Smoldering MM & MGUS - MRD investigation

11 Differential diagnosis between MM and MGUS Clonal MM Poly-Clonal MGUS versus Only 10% of MM patients showed > 5% poly-pc >5% poly-pc: 82% MGUS* The most powerful single criteria for differential diagnosis 1. Ocqueteau M, Am J Pathol 1998, 152: Updated with 1218 patients (811 MM & 407 MGUS)

12 IMMUNOPHENOTYPING of PLASMA CELL Discrimination between normal vs. clonal PC - Differential diagnosis of MGUS & MM - Risk of Transformation of MGUS & Smoldering MM - MRD investigation

13 MGUS: Flow Cytometry Results in 407 patients* %TotalPCinBM* 1.0 ( ) % of apc / BMPC compartment 73 (0-100) <95%aPC/BMPC 334 (82%) > 95% apc / BMPC 73 (18%) * Median (range) *Morphology: % BMPC: 4% (1-10%) Pérez-Persona et al; Blood. 2007: 110;

14 MGUS (403 patients)* Impact of % apc/bmpc by FC on PFS 1,0 0,8 p= years >95% apc/bmpc n= 73 (16 progressions) % of Pro ogression 0,6 0,4 24% Median 107 months 4% <95% apc/bmpc n= 330 (13 progressions) 0,2 0,0 Median Not reached Months Follow-up (months).52 (>24) Progression (%). 8% Pérez-Persona et al; Blood. 2007: 110;

15 MGUS (253 patients) Multivariate Analysis: apc/bmpc & DNA aneuploidy 1,0 0,8 p= % 5 years >95% apc/bmpc+ Aneuploidy n= 15 (9 progressions) % Progress sion 0,6 0,4 0,2 >95% apc/bmpc or Aneuploidy 11% n= 114 (15 progressions) 2% No adverse factors n= 124 (3 progressions) 0, Months Follow-up (months) 52 (>24) Progression (%).10% Pérez-Persona et al; Blood. 2007: 110;

16 IMMUNOPHENOTYPING of PLASMA CELL Discrimination between normal vs. clonal PC - Differential diagnosis of MGUS & MM - Risk of Transformation of MGUS & Smoldering MM - MRD investigation

17 Risk of Transformation of Smoldering MM into active MM or Amyloidosis Cumulative probability of progression: 73% at 15 years - 10% / year during the.. 1st 5 years - 3% / year during the.. next 5 years - 1% / year during the.. last 10 years Overal Survival : 60% at 5 years ; 34 % at 10 years Risk factor for progression: - MC (>4 g /dl); Heavy chain ( IgA); % PC in BM (>20%) - Diffuse pattern of BM infiltration. - Urinary light chain (+ and lambda), - Reduction of univolved Igs (one or two). Kyle NEJM 2007; 356:

18 Smoldering MM: Flow Cytometry Results in 93 patients* %TotalPCinBM* 2.9 ( ) % of apc / BMPC compartment 97 (35-100) < 95% apc / BMPC 37 (40%) >95%aPC/BMPC 56 (60%) * Median (range) * Morphology: % PC 14% (4-55%) Pérez-Persona et al; Blood. 2007: 110;

19 Smoldering Multiple Myeloma Impact of % apc/bmpc by FC on PFS 1,0 p= years % of Progr ression 0,8 0,6 0,4 63% 8% Median 40 months >95% apc/bmpc n= 56 (36 progressions) <95% apc/bmpc n= 37 (4 progressions) 0,2 0,0 Median Not reached Months Follow-up (months).56 (24-107) Progression (%). 42% Pérez-Persona et al; Blood. 2007: 110;

20 Multivariate analysis for PFS p HR % a PC /BMPC Immunoparesis Pérez-Persona et al; Blood. 2007: 110;

21 Impact of prognostic index on PFS Immunoparesis >95 95% apc/bmpc Score (n) (n=32 32) +/- -/+ 1(n= (n=27) + + 2(n= (n=27) Pérez-Persona et al; Blood. 2007: 110;

22 Impact of prognostic index* on TTP in Smoldering MM (based on apc/bmpc by FC & Immunoparesis) 10 1,0 5 years p= ,8 82% Median 23 months rogression % Time to p 0,6 0,4 0,2 42% Median 73 months 8% No adverse factors n= 32 (3 progr.) >95% apc/bmpc or paresis n= 27 (12 progr.) >95% apc/bmpc + paresis n= 27 (22 progr.) Median not reached 0, Months Pérez-Persona et al; Blood. 2007: 110;

23 Symptomatic Multiple Myeloma Clonal 10% of MM patients showed > 5% poly-pc Paiva et al Unpublished data

24 Symptomatic myeloma patients baseline characteristics according to the number of N-PC/BMPC (811 patients) 5% N-PC/BMPC (N=723) >5% N-PC/BMPC (N=88) P ISS stage I 33% 48%.02 β 2 M; >3.5 mg/l 46% 33%.01 Hemoglobin; <100 g/l 44% 20% <.001 Albumin; <3.5 g/dl 50% 38%.05 M-component; 3 g/dl 75% 44% <.001 Immunoparesis 84% 42%.003 PC by optimal microscope >30% 45% 17% <.001 High-risk cytogenetics 26% 3%.006 Any t(4;14), t(14;16) or del(17p)

25 > 5% N-PC/BMPC (n=88) 5% N-PC/BMPC (n=723) Symptomatic Multiple Myeloma: Survival according to the presence or absence of >5% N-PC/BMPC in bone marrow at diagnosis (N=811) PFS OS 100 p< p= Median: Not reached Median: 39 months Median: 51 months Median: 73 months Months Months

26 IMMUNOPHENOTYPING of PLASMA CELL Discrimination between normal vs. clonal PC - Differential diagnosis of MGUS & MM - Risk of Transformation of MGUS & Smoldering MM - MRD investigation

27 BACKGROUND High-dose chemotherapy and Novel Drugs Complete remission (CR): 25%-75% Relapse-free survival (RFS) at 5 year: 40%-70% However, patients with MM ultimately relapse MINIMAL RESIDUAL DISEASE (MRD) persistence of residual malignant cells

28 Impact on Survival of MRD by Immnunophenotyping in BM obtained 3 months after ASCT in CR patients (negative Immunofixation) (n=147) PFS OS % % 30% Median: 71 months Median: 37 months % Medians: Not reached 0 p< MRD negative (n=94) MRD positive (n=53) 0 p= y 5y Months Months Paiva et al; Blood. 2008; 112:

29 Multivariate Analysis PFS OS p risk p risk MRD+ at day High Risk Cytog* ns Age >60y. ns IFx+ at day +100 ns ns *t(4;14), t(4;16), del (17p) Paiva et al; Blood. 2008; 112:

30 CONCLUSIONS Immunophenotypic studies have clinical value: - Differential diagnosis MGUS/MM - Risk of transformation in MGUS & SMM - Investigation of MRD (immunophenotypic remission)

31 Grupo Español de Mieloma (GEM) Hospitales Clínico de Barcelona 12 Octubre (Madrid) Clínico de Salamanca Clínico de San Carlos (Madrid) Hospital de Badalona Clínico de Asturias Fr. Peset (Valencia) Universitario de Canarias Rio Ortega (Valladolid) Cínico de Zaragoza Hospital General de Jerez Ramón y Cajal (Madrid) Morales Meseguer (Murcia) La Fe (Valencia) C.U. de Navarra Galdakao (Vizcaya) Clínico de Valladolid Sant Pau (Barcelona) Arnau Vilanova (Lérida) Universitario de Santiago General Universitario de Valencia Universitario de Getafe (Madrid) Insular de las Palmas H. de La Princesa (Madrid) Severo Ochoa (Madrid) Juan XIII (Tarragona) Toledo Gandía (Valencia) Vall D Hebrón (Barcelona) San Jorge (Huesca) Verge de la Cinta (Tortosa) Alarcos (Ciudad Real) Mataró (Madrid) Juán Canalejo (Coruña) Ferrol Hospitales General lde Segovia Cruces (Bilbao) St. Coloma de Gramanet (Barcelona) Gregorio Marañon (Madrid) Carlos Haya (Málaga) H. Tauli (Gerona) Huesca Palencia Alcira (Valencia) H. Del Mar (Barcelona) Mahón (Baleares) Clínico de Málaga Xeral Cies (Vigo) Plasencia Cáceres Algeciras Ávila Jaén S. Pau i Sta Tecla (Tarragona) General de Guadalajara Sagunto (Valencia) Son Dureta (Mallorca) Cuenca Alicante SUS M. Valdecilla (Santander) Albacete H. Del Bierzo Fundación Jiménez Díaz (Madrid) Elda (Alicante) V. Del Rosel (Cartagena) Castellón Mutua Tarrasa Consorcio Tarrasa C. Corachán (Barcelona)

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