A IMPORTANCIA ATUAL DA IMUNOFENOTIPAGEM NO DIAGNOSTICO

E CLASSIFICAÇAO DA SMD

HEMO 2016 Congreso Brasileiro de Hematologia, Hemoterapia y Terapia CelularFlorianopolis, 10 de Novembro de 2016

CANCER RESEARCH CENTER IBSAL-UNIVERSITY OF SALAMANCA/CSIC

Conflict of Interest DisclosureI hereby declare the following potential conflicts of interest concerning my presentation:• Membership on an Entity’s Advisory Committee: Janssen, Celgene.

MDS: CONTRIBUTION OF IMMUNOPHENOTYPING

WHO 2002: “The contribution of immunophenotyping to the diagnosis of MDS

remains limited”

WHO 2008: “Immunophenotyping of MDS may be of diagnostic/prognostic utility”*

WHO 2016: “Immunophenotyping of MDS is of diagnostic/prognostic utility”*

Diagnosis, classification and prognostication of MDS are largely based on (heterogeneous) clinical,

cytomorphological and cytogenetic features

* Immunophenotypic criteria for MDS: 3 phenotypic alterations in suspicious cases

Prerequisites:1.- Marked prolonged (≥6months) cytopenia (<110g Hb/L or

< 1500 neutrophils/µl or <100.000 platelets/µl) in >1 cell lineages2.- Other hematological and non-hematological diseases excluded

Additional diagnostic criteria:1.- Morphologic dysplasia in >10% erythroid and/or neutrophil

and/or megakaryocytic lineages or >15% ringed sideroblasts2.- Characteristic cytogenetic abnormalities3.- Blast cell number of between 5% and 19%

Diagnostic co-criteria:1.- Flow cytometry immunophenotypic abnormalities.2.- Molecular signs of clonality (Chromosome X inactivation tests) andmolecular tests (e.g. RAS mutations)3.- Marked and persistently reduced CFU-assays

Valent et al, Leuk Res, 2007

MDS: MINIMAL DIAGNOSTIC CRITERIA

MDS:Aberrant granulomonocytic phenotypes

Stetler-Stevenson, Blood, 2001

Diagnostic utility of FCM in MDS

No cases Sensitivity Specificity

Stetler-Stevenson et al, Blood 2001 65 78% N.R.

Wells et al, Blood 2003 115 55% 100%

Cherian et al, Cytometry B 2005 15 73% 90%

Malcovati et al, Leukemia 2005 103 87% 100%

Ogata et al, Blood 2006 27 59% 100%

Loosdrecht et al, Blood 2008 50 82% 100%

Scott et al, Blood 2008 152 60% N.R.

Matarraz et al, Leukemia 2008 50 100% 100%

Ogata et al, Haematologica 2009 81 89% 90%

Differences in: 1) target cell populations, 2) phenotypic criteria for MDS, 3) cohorts of patients & controls

MDS: FCM IMMUNOPHENOTYPIC VARIABLES

Cell populations of interest Phenotypic alterations

Hematopoietic precursors/myeloblastsAltered cell numbers

Maturing neutrophilsAsynchronous maturation

Monocytes and monocytic precursorsAberrant markers

Nucleated red blood cellsMarker overexpression

Other potentially informative cells: Marker underexpression- Platelets- Eosinophils Altered scatter- Basophils - granularity- Mast cells - size- Dendritic cells- Mature red cells

Modified from van der Loordescht, Leukemia 2012 *52 immunophenotypic variables/case

Flow Cytometry Immunophenotyping of CD34+ HPC in MDS vs Normal/ Reactive BM

SUBSET OF CD34+ HPC

IMMATURE

NEUTROPHIL

B-CELL

ERYTHROID

RA RCMD RAEB-1 RAEB-2

Matarraz et al, Leukemia, 2008

RAEB-1

MDS vs normal BM: myelomonocytic maturation

Normal BM

CD34+ HPC:

Neutrophil

Immature

Pre-B

Neutrophil

Immature

CyMPO-PE

CyMPO-PE HLADR-FITC

HLADR-FITC

CD11

7-PE

CD11

7-PE

Diagnostic utility of FCM in MDS: the Ogata “simplified” score

Ogata et al, Haematologica 2012

Diagnostic utility of FCM in MDS: the Ogata “simplified” score

Ogata et al, Haematologica 2012

Diagnostic utility of FCM in MDS: validation of the Ogata “simplified” score

Matarraz et al, Cytometry B 2010; Mathis et al, Leukemia 2013Eidenschink et al, Cytometry B 2015;

Cremers et al, Haematologica 2016; Westerns et al, Haematologica 2016

Diagnostic utility of FCM in MDS:evaluation of the erythroid lineage

Altered erythorid phenotypes:- lower CD71 expression,- greater CD71 CV- greater CD36 CV- greater/lower CD117 expression

Increased sensitivity and specificity

Limited reproducibility

CD71 MFI

Tran

sf-S

SC

Wells et al, Blood 2003; Scott et al Blood 2008

Prognostic impact of FCM in MDS: The Well et al FCSS

Prognostic impact of FCM in MDS: The Well et al FCSS

POTENTIAL CONTRIBUTION OF IMMUNOPHENOTYPING IN MDS

Diagnosis:Suspected MDS (normal vs MDS),

ICUS,Unexplained monocytosis

Valent at al, Leuk Res, 2007; van de Loosdrecht et al, Haematologica, 2009; Leukemia 2012 & Leuk Lymph 2013; Bene et al, Leukemia 2014

Refine prognostic scoring systems

Yes but, how?

Diagnostic subclassificationUni- vs multi-lineage MDS

MDS with other clonal disorders

Disease monitoring(follow-up of FCM alterations)

Untreated MDS, After therapy

Inconclusive cases

ALTERED NEUTROPHIL MATURATION IN MDS BM

Normal BM RCMD RAEBCompatible with MDS

ALL

CLONAL HEMATOPOIETIC STEM CELL DISORDERS

AMLSM

MPN

PNH

AA

MDS

“DE NOVO” AML: ABERRANT PHENOTYPES & CLONAL HEMATOPOIESIS (n=59)

Phenotype Polyclonal AML# Clonal AML*N=10 N=49

Normal phenotype 58% 2%

>2 altered lineages 17% 92%

N. of altered lineages 0.7 2.7

Total 10/59 (17%) 49/59 (83%)

Fernández et al, Leukemia 2013

# One ISM-AML case with KIT mutation restricted to mast cells* Two cases showed coexistence of t(8;21) & D816V KIT mutation in all cellular compartments analyzed

ALTERED HEMATOPOIESIS AT DIAGNOSIS IN MULTIPLE MYELOMA & MGUS BONE MAROW

N.of altered cases/ total cases (%)

Matarraz et al, Leukemia 2014

Altered phenotypes* 33/250 (13%)

Cytogenetic changes 15/32 (47%)or clonal HUMARA

- del(5q32), del(7q31), -Y, +8 7/22

- Clonal HUMARA 8/10

*13/25 MM died during the first year after therapy and only 2/25 reached CR

MYELOPROLIFERATIVE/MYELODYSPLASTICNEOPLASMS: ALTERED PHENOTYPES IN CMML

Subira et al Translat Res 2008

Monocyticcells

Maturing neutrophils

CD45 MFI

Tran

sfor

med

SS

C Eosinophils

CD34+ Precursors

Erythroid cells

CD

13 I

MF

CD11b MFI

CD

13 M

FI

CD71 MFI

Tran

sf-S

SC

IMMUNOPHENOTYPIC ALTERATIONS OF BM MYELOID CELLS IN CHRONIC MYELOMONOCYTIC LEUKEMIA

Maturing neutrophils & Monocytic cells

Erythroid precursors

10 10 10 10 100 1 2 3 4

12704.008CD56 PE ->

CD56-PE

T-S

SC

Monocytic cells

EUROFLOW DATABASES: WHAT IS THEIR UTILITY?

- To evaluate the performance of antibody panels.

- To prospectively evaluate new cases against reference groups.

- To identify the most informative markers

- To automatically identify, gate and name multiple cell populations present in a sample.

Neutrophil maturation vs normal BMOverlayed Normal vs cytopenic BM

NEUTROPHIL MATURATION DATA BASE: BM case vs a reference normal BM data base

Neutrophil maturation: reactive vs normal BM

Overlayed Normal vs reactive BM

Overlayed Normal vs MDS BM

+3SD

-3SD

+10SD

-10SD

Concluding remarks

- Multiple studies have shown that FCM can assist in the diagnosis andprognostication in MDS as well as in cases suspected of MDS and in pre-MDS conditions.

- Despite no marker-abnormality or aberrant phenotype is specific forMDS, such alterations might help in the differential diagnosis betweenreactive and MDS bone marrow. However, the final diagnosis of MDShas to be based on other criteria

- Despite all the above, no generally accepted consensus exists as regardsthe optimal SOPs to apply.

- Current efforts and undergoing multi-center projects have the aim tostandardize the approach and to facilitate its reproducible use in routinediagnostics.

THE CIC/USAL-IBSAL TEAM

MUITO OBRIGADO