23/10/2017. Analysis of bone marrow for MDSrelated. Approach used at Hematopathology, Lund

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1 Approach used at Hematopathology, Lund Analysis of bone marrow for MDSrelated aberrancies According to International/ELN Flow Cytometry Working Group (IMDSFlow) Anna Porwit Lund, Sweden 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 13B Kappa Polyclonal 10 PE CD8 B Lambda Polyclonal 10 ECD CD3 UCHT1 5 CD14 RMO52 3 PC5.5 CD33 D3HL PC7 CD20 B9E9 5 CD56 N APC CD Excludes abnormal B-cell population Gives orientation in T-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) A700 CD19 J 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): Ogata et al., Blood 2006;108; ; Ogata et al., Haematologica 2009;94: ; Della Porta MG, et al., Haematologica 2012;97: Rajab & Porwit, Clin Cytometry, 2015;88(4): Bardet et al. Haematologica, 2015 Apr;100(4):472-8 Example of comprehensive 10 color acute leukemia/mds panel 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 CD123 CD2 APC_AlexaF700 CD7 CD19 CD10 APC_AlexaF750 CD11b CD38 CD235a Pacific_BLUE CD16 HLA-DR Krome Orange CD45 CD45 CD15 CD45 Porwit A, Rajab A. Int J Lab Hematol May;37 Suppl 1: Arnoulet C, Béné MC, et al Cytometry B Clin Cytom Jan;78(1):4-10 1

2 Immature myeloid and monocytic progenitors Recommended analysis Percentage of cells in nucleated cell fraction Expression of CD45 Expression of CD34 Expression of CD117 Expression of HLA-DR Expression of CD13 Expression of CD33 Asynchronous expression of CD11b Asynchronous expression CD15 Expression of CD5 Expression of CD7 Expression of CD19 Expression of CD56 Expression of CD10 Aberrant SSC signal Aberrancies Increased percentage Homogenous under/overexpression Lack of/increased expression Presence of mature markers Presence of mature markers Presence of lineage infidelity markers Presence of lineage infidelity markers Presence of lineage infidelity markers Presence of lineage infidelity markers Altered Example of aberrant precursors Visualization of all 10 markers by radar analysis >2% CD7+ Ratio: 7.3 N <5% CD56+ CD38 low Maturing neutrophils Blast region in low blast count MDS Recommended analysis Percentage of cells as ratio to lymphocytes SSC as ratio vs SSC of lymphocytes Relationship of CD13 and CD11b Relationship of CD13 and CD16 Relationship of CD15 and CD10 Aberrancy Decreased Decreased ; for example, lack of CD10 on mature neutrophils 2

3 Examples of aberrant neutrophils Aberrant pattern CD56+ Low CD10 Ratio 4 (n>6) Recommended analysis Percentage of cells Distribution of maturation stages Monocytes Relationship of HLA-DR and CD11b Relationship of CD36 and CD14 Expression of CD13 Expression of CD33 Expression of CD56 Asynchronous expression of CD34 Aberrancy Decreased/increased Shift towards immature (Homogenous) under/overexpression (Homogenous) under/overexpression Presence of lineage infidelity marker Presence of immature marker Examples of aberrancies in monocytes Erythroid compartment CD2+ CD117+ CD56+ Lack of monocytes Recommended analysis Percentage of nucleated erythroid cells Relationship CD71 and CD235a Expression of CD71 Expression of CD71 Expression of CD36 Expression of CD36 Percentage of CD117-positive precursors Expression of CD105 Expression of CD105 Aberrancy Increased Decreased Increased CV Decreased Increased CV Increased Altered expression 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 Analysis Normal bone marrow DRAQ5 gating Poster 031, Violidaki et al. 3

4 Radar pattern of erythroid maturation: seeposter 031 for details Abnormal patterns in MDS Abnormal population Right shift Ring sideroblasts Left shift Progenitor B-cells Other Enumeration as fraction of total CD34+ based on CD45/CD34/SSC in combination with CD10 or CD19 Decreased or absent How to report FCM findings? Guidelines of the IMDSflow WG on FCM in MDS 2015 A: FCM analysis: NO MDS-related features B: FCM analysis: some changes often seen in MDS Relation of cell compartments Percentage of mdcs in relation to total WBC Percentage of pdcs in relation to total WBC Percentage of basophils in relation to total WBC low or absent low or absent absent or increased C: FCM analysis: consistent with MDS Percentage of eosinophils within neutrophil compartment absent or high Loosdrecht AA van de, Westers TM. J Nat Compr Cancer Netw 2013;11: Westers TM, et al., Leukemia 2012;26: ; Porwit A, et al., Leukemia 2014;28: Integrated Flow Cytometric diagnostic approach Scoring system score combined with FCSS (Wells) parameters Diagnostic flow score (Ogata et al.) 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) <2 <2 <2 < Conclusion A A/B A/B C A/B B/C B/C C 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 Loosdrecht AA van de, Westers TM. J Natl Comp Canc Netw 2013;11: ; Porwit A, Loosdrecht AA van de, et al., Leukemia 2014;28:

5 Status and Lab No lymphadenopathy or organomegaly No bruising or rash, no neurological deficit Hb 70g/L, MCV 115, reticulocytes 33x10 9 /L WBC 24.4x10 9 /L, no eosinophilia or basophilia Neutrophils 17.5x10 9 /L, Monocytes 6.1x10 9 /L Plt 148x10 9 /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% Monocytes are increased and have abnormal expression of CD56 and HLA-DR Aberrant features???? CD7 expression CD2 expression Negative HLA-DR No B-precursors Granulocytes are right shifted, have abnormal scatter and upregulated CD14 Blood morphology, blasts 1% SSC Gr/ly= 3 5

6 Bone marrow smear, blasts 5% Bone marrow biopsy Cytogenetics, FISH, molecular studies CD34 CD117 t(11;19)(q23;p13.1), MLL-ELL FISH confirmed MLL rearrangement JAK-2 mutation negative BCR-ABL1 negative CD61 Glyc C 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 Jul 15;184(2):

7 Case 2 52 year old male presented with pancytopenia, fatigue, bleeding gums Hb 107 g/l, MCV 99, WBC 1,5x10 9 /L, ANC 0.3x10 9 /L, Plt 72x10 9 /L Smears 6-10% blasts in various areas Erythropoiesis 48% Dysplasia Screening tube on lysed sample Highly discrepant numbers between differential on smears and in screening tube on lysed sample Screening tube: Ogata score Flow cytometry on lysed BM sample Ogata score Flow cytometry on non-lysed sample Aberrant pattern of erythroid maturation Erythropoiesis: 23% Early: 63% CD117+ blasts 7% erythroid 0.6% 7

8 Case ctd IHC CD34 and CD117 counted approx % 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 Summary of recommendations for FCM in Myelodysplastic syndromes 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) 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 Aug;94(8): 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 Aug;97(8): Westers TM,et al. Standardization of flow cytometry in myelodysplastic syndromes: a report from an international consortium and the European LeukemiaNet WorkingGroup. Leukemia Jul;26(7): van de Loosdrecht AA, et al. Rationale for the Clinical Application of Flow Cytometry in Patients with Myelodysplastic Syndromes. Leuk Lymphoma Mar;54(3): 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: Westers TM,et al; IMDSFlow Working Group. Immunophenotypic analysis of erythroid dysplasia in myelodysplastic syndromes. A reportfrom the IMDSFlowworking group. Haematologica Feb;102(2): Malcovati L, et al., ELN guidelines 2013: Blood 2013;122: ; Greenberg P, et al., J Nat Compr Netw Canc 2013;11:838-74; Westers TM, et al., Leukemia 2012;26: ; Van de Loosdrecht AA, Westers TM. J Natl Comp Canc Netw 2013;11: ; Porwit A, et al., Leukemia 2014;28:

9 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 Mar 3 PubMed PMID: : Porwit A. Is There a Role for Flow Cytometry in the Evaluation of Patients With Myelodysplastic Syndromes? Curr Hematol Malig Rep Sep;10(3): : 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 Jul-Aug;88(4): : Porwit A, Rajab A. Flow cytometry immunophenotyping in integrated diagnostics of patients with newly diagnosed cytopenia: one tube 10-color 14- antibody screening panel and 3-tube extensive panel for detection of MDSrelated features. Int J Lab Hematol May;37 Suppl 1: : Saft L, Björklund E, Berg E, Hellström-Lindberg E, Porwit A. Bone marrow dendritic cells are reduced in patients with high-risk myelodysplastic syndromes. Leuk Res Mar;37(3): : Poster 031, Violidaki et al. ESCCA