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Analysis of bone marrow for MDS-related aberrancies

According toInternational/ELN Flow Cytometry Working Group (IMDSFlow)

Anna PorwitLund, Sweden

Approach used at Hematopathology , Lund

• 1. new patients with cytopenia and <10% cells in blast region: start with Screening tube

• 2. if blast region>10%: full MDS panel

• 3. if ”Ogata score” >2: full MDS panel

• 4. patients where previous bm showed dysplasia : full MDS panel

• 5. patients with >20% cells in blast region : full AML panel

Screening tube

Fluorescence Ab clone Titre (ul)

FITC CD4 13B8.2 10

Kappa Polyclonal 10

PE CD8 B9.11 10

Lambda Polyclonal 10

ECD CD3 UCHT1 5

CD14 RMO52 3

PC5.5 CD33 D3HL60.251 3

PC7 CD20 B9E9 5

CD56 N901 10

APC CD34 581 5

A700 CD19 J3-119 3

A750 CD10 ALB1 5

PB CD5 BL1a 5

KO CD45 J.33 5

.

Rajab A, Porwit A. Screening bone marrow samples for abnormal lymphoid populations and myelodysplasia-related features with one 10-color 14-antibody screening tube. Cytometry B Clin Cytom. 2015;88(4):253-60.

Excludes abnormalB-cell population

Gives orientation inT-cell subsets

Ogata score = 3)

4-parameter screening

score

consists of:1. % CD34+ myeloid progenitor

cells among all nucleated cells

(<2%)

2.% CD34+ B cell precursors

among all CD34+ cells (>5%)

1.3. SSC of granulocytes

(ratio to lymphocytes >6)

4. CD45 expression of myeloid

progenitor cells

(ratio to lymphocytes 4-7.5)

Ogata et al., Blood 2006;108;1037-1044; Ogata et al., Haematologica 2009;94:1066-74; Della Porta MG, et al., Haematologica 2012;97:1209-17 Rajab & Porwit, Clin Cytometry, 2015;88(4):253-60Bardet et al. Haematologica, 2015 Apr;100(4):472-8

AML 1 AML 2 AML 3

FITC CD65 CD36 CD71

PE CD13 CD64 CD11c

ECD CD14 CD56 CD4

PC5.5 CD33 CD33 CD33

PC7 CD34 CD34 CD34

APC CD117 CD123CD2

APC_AlexaF700 CD7 CD19CD10

APC_AlexaF750 CD11b CD38CD235a

Pacific_BLUE CD16 HLA-DRCD15

Krome Orange CD45 CD45 CD45

Example of comprehensive 10 color

acute leukemia/MDS panel

Porwit A, Rajab A. Int J Lab Hematol. 2015 May;37 Suppl 1:133-43 Arnoulet C, Béné MC, et al Cytometry B Clin Cytom. 2010 Jan;78(1):4-10

https://www.leukemia-net.org/content/diagnostics/diagnostics/flow_cytometry_atlas/index_eng.html

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Recommended analysis Aberrancies

Percentage of cells in nucleated cell fraction Increased percentage

Expression of CD45 Lack of/decreased/increasedExpression of CD34 Lack of/decreased/increasedExpression of CD117 Homogenous under/overexpressionExpression of HLA-DR Lack of/increased expressionExpression of CD13 Lack of/decreased/increasedExpression of CD33 Lack of/decreased/increasedAsynchronous expression of CD11b Presence of mature markersAsynchronous expression CD15 Presence of mature markersExpression of CD5 Presence of lineage infidelity markersExpression of CD7 Presence of lineage infidelity markersExpression of CD19 Presence of lineage infidelity markersExpression of CD56 Presence of lineage infidelity markersExpression of CD10 Lack of/decreased/increasedAberrant SSC signal Altered

Immature myeloid and monocyticprogenitors

Example of aberrant precursors

Ratio: 7.3N 4-7.5

>2%

<5%

CD7+

CD56+

CD38 low

Visualization of all 10 markers by radar analysis

Blast region in low blast count MDS

Maturing neutrophils

Recommended analysis Aberrancy

Percentage of cells as ratio to lymphocytes

Decreased

SSC as ratio vs SSC of lymphocytes Decreased

Relationship of CD13 and CD11b Altered pattern

Relationship of CD13 and CD16 Altered pattern

Relationship of CD15 and CD10 Altered pattern; for example, lack of CD10 on mature neutrophils

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Examples of aberrant neutrophils

Ratio 4(n>6)CD56+

Aberrant pattern Low CD10

MonocytesRecommended analysis Aberrancy

Percentage of cells Decreased/increased

Distribution of maturation stages Shift towards immature

Relationship of HLA-DR and CD11b Altered pattern

Relationship of CD36 and CD14 Altered pattern

Expression of CD13 (Homogenous) under/overexpression

Expression of CD33 (Homogenous) under/overexpression

Expression of CD56 Presence of lineage infidelity marker

Asynchronous expression of CD34 Presence of immature marker

Examples of aberrancies in monocytes

Lack of monocytes

CD117+

CD2+CD56+

Erythroid compartment

Recommended analysis Aberrancy

Percentage of nucleated erythroid cells

Increased

Relationship CD71 and CD235a Altered pattern

Expression of CD71 Decreased

Expression of CD71 Increased CV

Expression of CD36 Decreased

Expression of CD36 Increased CV

Percentage of CD117-positive precursors

Increased

Expression of CD105 Altered expression

Expression of CD105 Percentage

Erythropoietic tube on non-lysed BM• 2.5 ml CD71-FITC,

• 2.5 ml CD13-PE,

• 5 ml CD117-ECD,

• 5 ml CD105-PE-Cy7,

• 5 ml CD36-PB,

• 2.5 ml CD45-KO

• DRAQ5 gating

Poster 031, Violidaki et al.

AnalysisNormal

bone marrow

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Radar pattern of erythroid maturation: see Poster 031 for details

Abnormal patterns in MDS

Ring sideroblastsLeft shift

Abnormal populationRight shift

Other

Relation of cell compartments

Percentage of mDCs in relation to total WBC low or absent

Percentage of pDCs in relation to total WBC low or absent

Percentage of basophils in relation to total WBC absent or increased

Percentage of eosinophils within neutrophil compartment absent or high

Progenitor B-cells

Enumeration as fraction of total CD34+ based on CD45/CD34/SSC in combination with CD10 or CD19

Decreased or absent

• A:

FCM analysis: NO MDS-related features

• B:

FCM analysis: some changes often seen in MDS

• C:

FCM analysis: consistent with MDS

How to report FCM findings?

Guidelines of the IMDSflow WG on FCM in MDS 2015

Loosdrecht AA van de, Westers TM. J Nat Compr Cancer Netw 2013;11:892-902

Westers TM, et al., Leukemia 2012;26:1730-41; Porwit A, et al., Leukemia 2014;28:1793-98

Diagnostic flow score

(Ogata et al.)

<2 <2 <2 <2 ≥2 ≥2 ≥2 ≥2

Dysplasia by FC

myeloid progenitors

- - + + - - + +

Dysplasia by FC

- Neutrophils (SSC or two or

more other aberrancies)

- Monocytes (CD56 or two or

more other aberrancies)

- Erythroid precursors (CD36

and/or CD71)

- + - + - + - +

Conclusion A A/B A/B C A/B B/C B/C C

Loosdrecht AA van de, Westers TM. J Natl Comp Canc Netw 2013;11:892-902;

Porwit A, Loosdrecht AA van de, et al., Leukemia 2014;28:1793-98

Integrated Flow Cytometric diagnostic approach

Scoring system score combined with FCSS (Wells)

parameters Case 1: Male 59 years old

• Previously healthy• Developed increasing fatigue

about 2 months before presentation

• 6 weeks before presentation GP found anemia

• Patient went for vacation to Barbados

• Felt even more fatigue after coming back

• No fever, night sweats or weight loss

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Status and Lab

• No lymphadenopathy or organomegaly

• No bruising or rash, no neurological deficit

• Hb 70g/L, MCV 115, reticulocytes 33x109/L

• WBC 24.4x109/L, no eosinophilia or basophilia

• Neutrophils 17.5x109/L, Monocytes 6.1x109/L

• Plt 148x109/L

• LDH and creatinine borderline

• Bone marrow biopsy with flow cytometry was performed

CD45 vs SSC

A. Blasts are not increased

B. Monocytes are increased

C. Granulopoiesis shows abnormal scatter

D. Lymphocytes are within normal limits

Which is NOT true?????

CD34+ cells >2%

CD7 expressionCD2 expressionNegative HLA-DRNo B-precursors

Aberrant features????

Monocytes are increasedand have abnormal expression of CD56 and HLA-DR

SSC Gr/ly= 3

Granulocytes are right shifted, have abnormal scatter and upregulated CD14 Blood morphology, blasts 1%

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Bone marrow smear, blasts 5% Bone marrow biopsy

CD34 CD117

CD61 Glyc C

Cytogenetics, FISH, molecular studies

• t(11;19)(q23;p13.1), MLL-ELL

• FISH confirmed MLL rearrangement

• JAK-2 mutation negative

• BCR-ABL1 negative

• 11q23 abnormalities

• leading to the MLL gene

• rearrangement are most

• common in AML

Diagnosis and Follow-up

• Chronic myelomonocytic leukemia (CMML-1)

• One month later blasts were 9%

• Due to cytogenetics this patient was at risk of rapid progression to AML

• Induction with FLAG-IDA

• Consolidation with 2 cycles of intensification protocol Dana-Farber

• Doing well after BMT with 10/10 matched unrelated donor

Reference

• Kakihana K, Kubo F, Wakabayashi S, Kurosu T, Miki T, Murakami N, Miura O. A novel variant form of MLL-ELL fusion transcript with t(11;19)(q23;p13.1) in chronic myelomonocytic leukemia transforming to acute myeloid leukemia. Cancer Genet Cytogenet. 2008 Jul 15;184(2):109-12.

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Case 2

• 52 year old male presented with pancytopenia, fatigue, bleeding gums

• Hb 107 g/L, MCV 99,• WBC 1,5x109/L, ANC

0.3x109/L, Plt 72x109/L• Smears 6-10% blasts in

various areas• Erythropoiesis 48% • Dysplasia

Screening tube on lysed sample

Highly discrepant numbers between differential on smearsand in screening tube on lysed sample

Screening tube: Ogata score

Ogata score 3

4.0

6.5

Flow cytometry on lysed BM sample

Flow cytometry on non-lysed sample Aberrant pattern of erythroid maturation

Erythropoiesis: 23%Early: 63%CD117+ blasts 7% erythroid 0.6%

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Case ctd IHC

CD34 and CD117 counted approx. 11-13%

P53 overexpressed

Cytogenetics/Molecular

• 41-44, X,-Y,-4,-5, der(7)t(?5;q31)p(1?5;q31),der(17)(t(7;17)(q31;p11),-18,-19,?der(20)t(5;20)(q31;p11),-22, +1-4mar [cp21]/46XY[4]

• Illumina Tru Sight 54 genes

• TP53 c747G>T Tier 1 57%

Azacytidine treatment started but progressed to AML within 3 months

• For FCM application for MDS diagnostics

– Follow International MDS Flow methodological recommendations

• For screening purposes

– Follow a mini-panel based on the so-called Ogata score

• For extended analysis: perform FCM in all cell compartments following ELN recommended antigen combinations

– Myeloid and lymphoid progenitor cells

– Maturing myelomonocytic cells

– Immature and mature erythroid cells

generate integrated Flow Score (A;B;C)

• Integrate Flow cytometry findings in the bone marrow report (morphology, cytogenetics, flow cytometry, molecular methods)

Summary of recommendations for FCM in Myelodysplastic syndromes

Malcovati L, et al., ELN guidelines 2013: Blood 2013;122:2943-64; Greenberg P, et al., J Nat Compr Netw

Canc 2013;11:838-74; Westers TM, et al., Leukemia 2012;26:1730-41; Van de Loosdrecht AA, Westers

TM. J Natl Comp Canc Netw 2013;11:892-902; Porwit A, et al., Leukemia 2014;28:1793-98

Publications• van de Loosdrecht AA, et al Standardization of flow cytometry in myelodysplastic syndromes:

report from the first European LeukemiaNet workingconference on flow cytometry in myelodysplastic syndromes. Haematologica. 2009 Aug;94(8):1124-34.

• Della Porta MG, et al. Multicenter validation of a reproducible flow cytometric score for the diagnosis of low-grade myelodysplastic syndromes: results of a European LeukemiaNET study. Haematologica. 2012 Aug;97(8):1209-17

• Westers TM,et al. Standardization of flow cytometry in myelodysplastic syndromes: a reportfrom an international consortium and the European LeukemiaNet WorkingGroup. Leukemia. 2012 Jul;26(7):1730-41.

• van de Loosdrecht AA, et al. Rationale for the Clinical Application of Flow Cytometry in Patients with Myelodysplastic Syndromes. Leuk Lymphoma. 2013 Mar;54(3):472-5.

• Porwit A, et al. Revisiting guidelines for integration of flow cytometry results in the WHO classification of myelodysplastic syndromes-proposal from the International/European LeukemiaNet Working Group for Flow Cytometry in MDS. Leukemia 2014 Jun 12;28:1793-8.

• Westers TM,et al; IMDSFlow Working Group. Immunophenotypic analysis of erythroid dysplasiain myelodysplastic syndromes. A report from the IMDSFlowworking group. Haematologica. 2017 Feb;102(2):308-319

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References• 1: Jafari K, Tierens A, Rajab A, Musani R, Schuh A, Porwit A. Visualization of cell

composition and maturation in the bone marrow using 10-color flow cytometry and radar plots. Cytometry B Clin Cytom. 2017 Mar 3 PubMed PMID: 28257592.

• 2: Porwit A. Is There a Role for Flow Cytometry in the Evaluation of Patients WithMyelodysplastic Syndromes? Curr Hematol Malig Rep. 2015 Sep;10(3):309-17.

• 3: Rajab A, Porwit A. Screening bone marrow samples for abnormal lymphoidpopulations and myelodysplasia-related features with one 10-color 14-antibody screening tube. Cytometry B Clin Cytom. 2015 Jul-Aug;88(4):253-60.

• 4: Porwit A, Rajab A. Flow cytometry immunophenotyping in integrateddiagnostics of patients with newly diagnosed cytopenia: one tube 10-color 14-antibody screening panel and 3-tube extensive panel for detection of MDS-related features. Int J Lab Hematol. 2015 May;37 Suppl 1:133-43.

• 5: Saft L, Björklund E, Berg E, Hellström-Lindberg E, Porwit A. Bone marrowdendritic cells are reduced in patients with high-risk myelodysplastic syndromes. Leuk Res. 2013 Mar;37(3):266-73.

• 6: Poster 031, Violidaki et al. ESCCA 2017