DIAGNOSIS OF MYELODYSPLASTIC SYNDROMES/NEOPLASMS

This presentation provides a thorough discussion of the diagnostic processes involved in Myelodysplastic Syndromes (MDS), a heterogeneous group of clonal hematopoietic disorders.

Key Topics Covered:
-Definition
-Diagnostic criteria of MDS
-Challenge in diagnosis
-Diagnostic Algorithm
-Cytopenia
-History & physical examination
-Blood tests for cytopenia
-Peripheral blood film features
-If no cause of cytopenia identified?
-Bone marrow - Aspirate
-WHO MDS defining morphology
-Bone marrow - Biopsy
-Value and impact of bone marrow histology & IHC in MDS
-Mimics of MDS
-Aplastic vs hypoplastic MDS
-PBF & BM- Summary
-Flow cytometry immunophenotyping
-Recurrent immunophenotypic abnormalities in MDS
-Flow cytometry features used in the diagnosis of putative MDS
-PNH clones by flow cytometry
-Cytogenetics
Prognostication- IPSS-R
-Molecular genetic testing
-Linking genotype to phenotype
-Inherited germ line predisposition
-VEXAS Syndrome
-IPSS-M Web calculator
-MDS classification
-Essential components on a newly diagnosed MDS

Audience
This presentation is intended for healthcare professionals, including hematologists, hemtopathologists, pathologists, and medical students, who seek to enhance their understanding of MDS diagnosis.

Objective
By the end of this presentation, attendees will have gained insight into the complexity of identifying MDS, allowing them to utilize their knowledge in clinical practice to enhance patient outcomes.

1. DIAGNOSIS OF MYELODYSPLASTIC SYNDROME Dr Durgadevi S. MBBS, MD (Path.), DNB (Path.), DM (Hematopathology) Associate Consultant Manipal Hospital (Old airport road), Bengaluru

2. Definition Myelodysplastic syndromes/neoplasms (MDS) are a heterogeneous group of hematologic neoplasms C haracterized by clonal hematopoiesis , cytopenias (i.e., anaemia, neutropenia, and/or thrombocytopenia), and dysplastic cellular morphology V ariable predilection for the development of acute myeloid leukaemia (AML) D isease of the elderly (median age - 75·7 years) C ommon in men (2:1) Swerdlow SH, et al. WHO classification of tumours of haematopoietic and lymphoid tissues. Lyon: IARC; 2017

3. Diagnostic criteria of MDS No single criterion is pathognomonic Combination of cytopenias and morphological bone marrow dysplasia Absolute prerequisite Unexplained cytopenia(s) WHO thresholds- Hb (F), <120 g/l; Hb (M), <130g/l; ANC <1·8 × 10 9 /l and platelets, <150 × 10 9 /l Higher values do not exclude the diagnosis if definitive morphological and/or cytogenetic abnormalities are present Morphologic dysplasia at least 10% of cells in at least 1 hematopoietic (erythroid, granulocytic, or megakaryocytic) lineage

4. Challenge in diagnosis The diagnosis of MDS can be challenging to distinguish from nonneoplastic myeloid proliferations myriad other causes of cytopenias presence of morphologic dysplasia (10%- subjective) the border between MDS and acute myeloid leukemia (AML)

5. ALGORITHM Killick, S. B.,et al. (2021). British Society for Haematology guidelines for the diagnosis and evaluation of prognosis of Adult Myelodysplastic Syndromes. British Journal of Haematology , 194 (2), 282-293.

6. Cytopenia Clinical suspicion ?MDS – anaemia > bicytopenia >pancytopenia Median duration in newly diagnosed MDS- 4 months More severe and persistent cytopenia(s) Mild cytopenia and/or cytopenia of relatively recent onset

7. History & physical examination

8. Blood tests for cytopenia Hasserjian RP, et al. Diagnosis and classification of myelodysplastic syndromes. Blood. 2023 Dec 28;142(26):2247-2257.

9. Peripheral blood film features Good quality smears and stains are essential Blast percentage 200 cell count

10. If no cause of cytopenia identified? H istory, physical examination, and screening blood tests for cytopenia No cause identified Bone marrow biopsy and aspirate Abnormal marrow morphology remains a major de fi ning feature of MDS C lassify and risk-stratify the disease

11. Bone marrow - Aspirate May– Grünwald –Giemsa (or equivalent)- stained smears 500 cell count or more nucleated cells, including 30 or more megakaryocytes Fresh specimens Processed within 2 hours Avoid excess of ethylenediamine tetra-acetic acid (EDTA) Stains should be well controlled and checked by examining non-MDS films Prussian Blue or Perls’ stain - to assess iron stores and to quantitate ring sideroblasts (RS) Mario Cazzola et.al, The genetic basis of myelodysplasia and its clinical relevance, Blood,Volume 122, Issue 25,2013

12. WHO MDS defining morphology

13. N uclear irregularities M icromegakaryocytes H ypogranularity , and/or nuclear hyposegmentation Mario Cazzola et.al, The genetic basis of myelodysplasia and its clinical relevance, Blood,Volume 122, Issue 25,2013

14. Bone marrow - Biopsy An adequate trephine biopsy (decalcified, paraffin or plastic-embedded) - essential Touch imprint smears can be made Dysplasia- harder to assess on biopsy Provides supportive information for diagnosis Cellularity Architectural disruption (disruption of erythroid islands; abnormal localisation of immature precursors) Fibrosis (with reticulin staining) Helpful for the diagnosis of hypocellular MDS and MDS/myeloproliferative neoplasms (MPN) overlap syndromes Mario Cazzola et.al, The genetic basis of myelodysplasia and its clinical relevance, Blood,Volume 122, Issue 25,2013

15. Bone marrow - Biopsy H ypercellular P aratrabecular localization of erythroid precursors P aratrabecular localization of megakaryocytes Mario Cazzola et.al, The genetic basis of myelodysplasia and its clinical relevance, Blood,Volume 122, Issue 25,2013

16. Bone marrow - Biopsy “Abnormal localization of immature precursors” (ALIP) (clusters of five or more  myeloblasts  and/or early immature myeloid cells in the interstitium ) IHC- CD34 immunostain ALIP Mario Cazzola et.al, The genetic basis of myelodysplasia and its clinical relevance, Blood,Volume 122, Issue 25,2013

17. Value and impact of bone marrow histology & IHC in MDS Valent P et al, Proposed minimal diagnostic criteria for myelodysplastic syndromes (MDS) and potential pre-MDS conditions. Oncotarget . 2017 Jul 5;8(43):73483-73500

18. Mimics of MDS Bejar R. Myelodysplastic Syndromes Diagnosis: What Is the Role of Molecular Testing? Curr Hematol Malig Rep. 2015 Sep;10(3):282-91

19. Aplastic vs hypoplastic MDS AA and hMDS may have overlapping pathogenetic mechanisms, but therapeutic approach and prognosis will differ Dyserythropoiesis can be prominent in AA and is not specific to hMDS hMDS Hypogranular neutrophils or pseudo– Pelger – Huet cells (>10%) Dysmegakaryopoiesis and marrow granulocytic dysplasia Presence of RS Fibrosis ALIP Increased blasts

20. PBF & BM- Summary

21. Flow cytometry immunophenotyping Many aberrancies of the progenitor cells, the myelomonocytic lineage and the erythroid lineage are described U seful in diagnosis, risk stratification and therapy guidance of MDS-patients Used by experts according to published guidelines the European Leukemia Net ( ELNet ) published guidelines Ogata-score, the RED-score, the erythroid parameters of the IMDSflow and the Integrated Flow score ( iFCs ) including the Flow Cytometric Scoring System (FCSS) of Wells et al Valent P., et al Proposed minimal diagnostic criteria for myelodysplastic syndromes (MDS) and potential pre-MDS conditions. Oncotarget . 2017; 8: 73483-73500.

22. Recurrent immunophenotypic abnormalities in MDS Duetz , C., & Westers, T. M. (2018). Clinical Implication of Multi-Parameter Flow Cytometry in Myelodysplastic Syndromes. Pathobiology , 86 (1), 14.

23. Flow cytometry immunophenotyping

24. Flow cytometry features used in the diagnosis of putative MDS

25. PNH clones by flow cytometry PNH clones- important implications for prognosis and treatment PNH clones can occur in both MDS and AA Detected by standard flow cytometric techniques Showing lack of glycophosphatidylinositol (GPI)-anchored proteins in the red cell, monocyte, and granulocyte compartments

26. PNH Samples analysis- within 24–48 h to ensure sensitivity PNH clones- reported in between 5.5% and 8% of patients with MDS and in 26.3% of patients with AA If present at >1%, follow-up testing should generally be performed every 6 months to determine clonal expansion Patients with PNH - benefit from treatment with eculizumab , an anticomplement C5 monoclonal antibody

27. ALGORITHM

28. Cytogenetics Karyotyping - the most widely used and unbiased method Assessing numerical and structural chromosomal abnormalities Present in 50% to 60% of patients Sample- bone marrow aspirate At least 20 metaphases should be analyzed Described according to the International System for Human Cytogenetic Nomenclature

29. Cytogenetics The most frequent single cytogenetic abnormalities - del(5q), monosomy 7, del(7q), trisomy 8, and del(20q) Swerdlow SH, et al. WHO classification of tumours of haematopoietic and lymphoid tissues. Lyon: IARC; 2017

30. Cytogenetics Interphase fluorescence in situ hybridization (FISH) is recommended- in repeated failure of standard G-banded karyotyping Chromosomal microarrays- to identify small, unbalanced abnormalities or cryptic copy number alterations, S ingle nucleotide polymorphism probes (SNP arrays) can detect loss of heterozygosity at key loci Provide a more precise, genome-wide analysis which is independent of metaphases

31. Prognostication- IPSS-R Schanz J, et al. New comprehensive cytogenetic scoring system for primary myelodysplastic syndromes (MDS) and oligoblastic acute myeloid leukemia after MDS derived from an international database merge. J Clin Oncol. 2012 Mar 10;30(8):820-9.

32. Molecular genetic testing Mutations in MDS More than 100 genes are recurrently mutated in MDS Somatic mutations- >90% in MDS cases 5 mutations- >10% of cases ( SF3B1, TET2, SRSF2, ASXL1, and DNMT3A ) 10-15 mutations- 5-10% of cases >90% of all bone marrow cells are clonal despite the blast count <5% Certain germline mutations (e.g . RUNX1, GATA2, DDX41 )- predisposition to MDS/AML

33. Molecular genetic testing Mutation driver genes in MDS RNA splicing DNA methylation Histone modification Transcription regulation DNA repair control Signaling Cohesin complex Mutational profiling – confer prognostic information and help predict response to therapy  Next Generation Sequencing (NGS) using targeted panels

34. Linking genotype to phenotype

35. Inherited germ line predisposition

36. Inherited germ line predisposition

37. VEXAS Syndrome Vacuolated myeloid and erythroid precursors on BM morphology E1 ubiquitin activating enzyme (coded for by UBA1 ) X chromosome Autoinflammation Somatic mutation

38. Molecular genetic testing Premalignant conditions and its characteristics

39. ( NEJM 2022 ) ( https://mds-risk-model.com ) IPSS-M Web calculator

40. MDS classification has evolved over time

41. Classification of MDS (WHO 2022)

42. WHO 2022 & ICC 2022 classification

43. WHO 2022 & ICC 2022 classification

44. ALGORITHM

45. Summary Given the heterogeneity of MDS the diagnosis of MDS is assessed by Bone marrow smears- morphology and special stain including iron stain Core biopsy- to assess cellularity Flow cytometric analysis to define the cellular immunophenotype Cytogenetics & molecular studies (NGS for myeloid neoplasm) used for risk stratification & prognostication

46. Essential components on a newly diagnosed MDS

47. Diagnosis of MDS THANK YOU