1. Acute myeloid leukemia: morphology and beyond
Nancy Rosenthal, MD
Clinical Professor of Pathology

2. Objectives
Review clinical, morphologic, immunophenotypic, and cytogenetic abnormalities that allow us to classify AML
Introduce new cytogenetic and mutational abnormalities that may be leukemia defining in the future
Discuss upcoming updates to the WHO classification that will be utilized starting in 2016 (?)
No conflicts to disclose

3. Diagnosing and following adult patients with acute myeloid leukaemia in the genomic age
Standard and optional analyses and their contribution for diagnosing, prognosticating, and following adult patients with AML. Opt., Optional analyses; qPCR, Quantitative polymerase chain reaction; MRD, Minimal residual disease; (i)FISH, (Interphase) fluorescence in situ hybridization; AML, acute myelod leukaemia; APL, Acute promyelocytic leukaemia; aCGH, Array comparative genomic hybridization; LAIP, Leukaemia‐associated immunophenotype; WHO, World Health Organization.
© IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSION' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER.
British Journal of Haematology Volume 167, Issue 2, pages , 7 AUG 2014 DOI: /bjh

4. Diagnosing and following adult patients with acute myeloid leukaemia in the genomic age
Examples of output data from analyses in the diagnostic and clinical work‐up of AML patients. (A) May‐Grünwald‐Giemsa stained bone marrow smear with from a patient diagnosed with AML without maturation. Myeloblasts are characterized by prominent nucleoli, delicate nuclear chromatin, and irregular nucleoli. (B) Flow cytometry dot plots illustrating initial blast gating of side scatter low cells with dim expression of CD45 and subsequent identification of CD34 and CD117 positive blasts. (C) Standard G‐band karyotyping from a male patient with t(8;21) and an additional Y‐chromosome loss. (D) Quantification of minimal residual disease by quantitative polymerase reaction targeting the fusion transcript RUNX1‐RUNX1T1 in a t(8;21) positive patient. (E) Detection of mutated NPM1 type A by semiquantitative gel capillary electrophoresis. (F) Next generation sequencing flowgram reflecting the sequence of the strand of DNA. The intensity of the peaks in the flowgram is proportional to the number of nucleotides incorporated during the individual sequencing events. Icons denote analyses and points as detailed in Fig . AML, acute myeloid leukaemia; BM, bone marrow; WT, wild‐type.
© IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSION' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER.
British Journal of Haematology Volume 167, Issue 2, pages , 7 AUG 2014 DOI: /bjh

5. Is there still a role for morphology in the diagnosis of AML?
May allow establishment of a diagnosis quickly
Especially important in the diagnosis of acute promyelocytic leukemia so ATRA can be started
Leads to the ability to review all of hematopoiesis
Exclude relevant differential diagnoses

6. Do we need to do an aspirate and bone marrow biopsy?
Only in the setting of >20% blasts in the peripheral blood
Depends on how the patient will be treated
Does give information about cellularity, maturation patterns, stromal changes
Helpful baseline for comparison purposes during evaluation of treatment efficacy

7. Diagnosis of AML – WHO 2008 Algorithmic approach Previous therapy
Therapy related myeloid neoplasm
Recurrent cytogenetic abnormalities
t(9;22), t(8;21), inv16, etc.
AML with myelodysplasia related changes
AML
Not otherwise specified

8. AML with recurrent genetic abnormalities
Clues to the diagnoses can be obtained by evaluating morphology
Blasts may be less than 20%
Blasts (in all cases) should be counted the old fashion way, not based on flow cytometry

14. Clues from flow cytometry
t(8;21) – strong CD34, CD13+, weak CD33, CD19+, PAX5+, cytoplasmic CD79a+, weak TdT
t(9;22) – CD34 -, HLADR -, CD117+
Inv16 – myeloid and monocytic markers and CD2+

15. For a definitive diagnosis - need cytogenetic studies and/or FISH

16. Mutations in AML - 2008 Provisional entities Prognostic markers NPM1
CEPBA
Prognostic markers
FLT3
KIT

19. AML with recurrent cytogenetic abnormalities – new categories
New mutation subgroups are being proposed
AML with RUNX 1 mutation – older men, poor prognosis
AML with NPM1 – good prognosis
AML with CEBPADM – only changes prognosis (good) if present as a double mutant

20. Kaplan-Meier curves for overall survival stratified by (A) CEBPA–wild-type (WT) or CEBPA-mutant status, (B) CEBPA-WT, CEBPA-single, or CEBPA-double mutant status, (C) CEBPA-WT, single N-terminal (term), single C-term, or double N-term plus C-term mutant sta...
Kaplan-Meier curves for overall survival stratified by (A) CEBPA–wild-type (WT) or CEBPA-mutant status, (B) CEBPA-WT, CEBPA-single, or CEBPA-double mutant status, (C) CEBPA-WT, single N-terminal (term), single C-term, or double N-term plus C-term mutant status.
Claire L. Green et al. JCO 2010;28:
©2010 by American Society of Clinical Oncology

21. (A) Disease-free survival, (B) overall survival, and (C) event-free survival of patients younger than age 60 years with cytogenetically normal acute myeloid leukemia according to RUNX1 mutation status.
(A) Disease-free survival, (B) overall survival, and (C) event-free survival of patients younger than age 60 years with cytogenetically normal acute myeloid leukemia according to RUNX1 mutation status. RUNX1 mut, RUNX1 mutated; RUNX1 wt, RUNX1 wild type.
Jason H. Mendler et al. JCO 2012;30:
©2012 by American Society of Clinical Oncology

22. AML with myelodysplasia related changes
Previous MDS
Dyspoiesis – 50% of the cells in 2 cell lines
Cytogenetic abnormalities

23. AML with myelodysplasia-related changes

25. Survival data.
Survival data. Overall survival (A) and progression-free survival (B) for patients with AML-NOS and AML-MRC.
Olga K. Weinberg et al. Blood 2009;113:
©2009 by American Society of Hematology

26. Characteristics of acute myeloid leukemia with myelodysplasia‐related changes: A retrospective analysis in a cohort of Chinese patients
Overall survival (A) and disease‐free survival (B) for patients with AML‐MRC and AML‐NOS (P < 0.05). Overall survival (C) and disease‐free survival (D) for patients with MLD+ and MLD− AML (P > 0.05).
© IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSION' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER.
American Journal of Hematology Volume 89, Issue 9, pages , 19 JUN 2014 DOI: /ajh

27. Revised criteria for AML with MRC
Remove de novo cases with no MDS related cytogenetic abnormality if there is an NPM1 or CEBPAdm
Revise cytogenetic abnormalities
Del9q is an MDS related entity only in the absence of NPM1 mutations
NPM1 commonly associated with del9q and is likely not adverse in this setting

28. Case

35. Flow cytometry, cytogenetics and molecular studies
14% myeloid blasts positive for CD117, CD13, CD33, CD34, CD38, HLA-DR and partial expression of CD7.
42~44,XY,add(2)(p11.2), del3, del 5, del(6)(q21),7,add(8)(q24),add(9)(p2,2), add(13)(q22),17,der(19)add(19)(p13)add(19)(q13.1),ad d(22)(p11.2) +mar[cp19]/46,XY[1]
FLT3, NPM1 and CEBPA negative

36. Acute erythroid leukemia - 2008
2 subtypes recognized
Acute erythroid/myeloid leukemia
> 50% erythroid precursors
>20% of the non-erythroid cells are blasts
Acute erythroleukemia
First one is controversial as the diagnosis of acute leukemia can be made with a very small number of blasts.
Altered erythroid percentages can changed based on therapy, metabolic defects, use of erythropoietin

37. Comparison of 40 patients who had MDS with erythroid hyperplasia (MDS-E), 41 patients who had AML with myelodysplasia-related changes and erythroid hyperplasia (AML-MRC), and 124 patients with acute erythroid leukemia (AEL).
Comparison of 40 patients who had MDS with erythroid hyperplasia (MDS-E), 41 patients who had AML with myelodysplasia-related changes and erythroid hyperplasia (AML-MRC), and 124 patients with acute erythroid leukemia (AEL). The median OS values are 14, 8, and 10 months, respectively, which are not significantly different (P = .313).
Robert P. Hasserjian et al. Blood 2010;115:
©2010 by American Society of Hematology

38. Figure 1. Effects of the presence of erythroid predominance (EP) and diagnostic categories on overall survival.
Zuo Z, Medeiros LJ, Chen Z, Liu D, Bueso-Ramos CE, et al. (2012) Acute Myeloid Leukemia (AML) with Erythroid Predominance Exhibits Clinical and Molecular Characteristics that Differ from Other Types of AML. PLoS ONE 7(7): e doi: /journal.pone

39. Acute erythroleukemia
New update of WHO
>50% erythroid precursors and 5-19% blasts would be considered RAEB (actually myelodysplasia with excess blasts)
> 50% erythroid and >20% blasts would be acute myeloid leukemia (often AML with myelodysplasia related changes)
Pure erythroleukemia will remain in AML
Erythroleukemia will become an extremely rare disorder.

40. Case

44. Flow cytometry, cytogenetics and molecular studies
90% monocytic cells which express CD38, CD64, bright CD33, and partial CD36,HLA-DR, CD56, CD13 and CD14
Cytogenetics showed t(11;17)
FISH showed an MLL rearrangement but no RARA break-apart probe abnormality
NPM1, CEBPA and FLT3 were negative

45. Acute monocytic/monoblastic leukemia
Need a careful monocyte, promonocyte, monocyte count to make the diagnosis of AML
Presence of NPM1 mutation or 11q23 rearrangement is not leukemia defining
May herald rapid progression from CMML to AML is present and these patients should be watched closely

46. Promonocytes

48. Differentiation of types of monocytic cells can be problematic
Morphological evaluation of monocytes and their precursors
Haematologica 2009;94:994
Authors included Barbara Bain, Richard Brunning, etc.

49. Gray, occ granules, occ vacuoles Large 20-25 um
monoblast
Round/oval
Delicate/lace-like prominent nucleolus
Basophilic, rare azurophilic granules
Large um
promonocyte
Convoluted
indented
Variably basophilic
Variable azurophilic granules
Except for nuclear shape similar to monoblast
Immature monocyte
More condensed
Rare nucleolus
Less basophilic than blast but more than monocytes
Resemble monocytes but less mature and smaller
monocyte
Lobulated
Condensed
No nucleolus
Gray, occ granules, occ vacuoles
Large um

50. Monocyte immature monocyte promonocyte monoblast

51. Classification
90 digital images of monocytic cells circulated among 5 experts
All experts made the same identification on 25 of the cells, 4/5 made the same identification on 44 cells
Percentage of good concordance (4/5 experts) was 76%
For 21 cells the concordance rate was 3/5 or 2/5

52. 6 cells had only 2/5 concordance – were sent back for a 2nd reading
These cells were confirmed as immature monocytes
Highest concordance was for monoblasts and lowest was for immature monocytes
Proposed using their classification scheme and correlate with flow cytometric findings

53. Use of flow cytometry to detect abnormal monocytes
May lack CD13, CD33, or CD14
Express aberrant antigens such as CD56, CD19, CD7 or CD5
May be difficult to distinguish immature monocytes from promonocytes

54. My conclusion
Differentiating these cells is difficult for even those most expert in morphology
Making the diagnosis of acute monocytic leukemia based on counting promonocytes is problematic in some cases

55. Summary
The diagnosis of AML is ever changing and will continue to evolve based on increased understanding of molecular mechanisms.
Targeted therapy may further influence diagnostics in the future.
At the moment there is still a role for morphology !!!