Acute Myeloid Leukaemia

Question 1

List the different categories and prognosis used in the molecular classification of AML

Answer 1

1) AML with recurrent cytogenetic abnormalities

2) AML with mutated NPM1
- Generally good risk, depends upon co-mutations
- FLT3 ITD + DNMT3A= poor risk
- NRAS + DNMT3A= good risk (unexpectedly!)

3) Chromatin-splicing group
- Chromatin: ASXL1, EZH2, BCOR, STAG2
- Splicing: SRSF2, SF3B1, U2AF1, ZRSF2
- Poor risk

4) AML with TP53, chromosomal aneuploidies and complex karyotype
- Includes isolated chromosomal arm losses
- Poor risk

5) IDH2 R172
- Good risk

6) AML; no driver mutations present
- Approximately 4% have no driver mutations on NGS

7) AML; drivers but not class defining
- DNMT3A, TET2, IDH1, FLT3, NRAS

Question 2

List the early granulocytic markers used in flow cytometry

Answer 2

CD117
CD13
CD33
Cytoplasmic MPO

Question 3

List the granulocytic maturation markers

Answer 3

CD15

CD65

Question 4

List the monocytic markers

Answer 4

CD4, CD14, CD11c, CD36, CD64, CD68, lysosome, nonspecific esterase

Question 5

List the megakaryocytic markers

Answer 5

CD41, CD42, CD61

Question 6

List the erythroid markers

Answer 6

CD235a (glycophorin A), haemoglobin A, E-cadherin

Question 7

What is the definition of acute myelomonocytic leukaemia?

Answer 7

Neutrophils and their precursors constitute ≥20% of bone marrow cells AND monocytes and their precursors constitute ≥20% of bone marrow cells.

Question 8

What is the definition of acute monocytic leukaemia?

Answer 8

AML in which ≥80% of the leukaemic cells are of the monocytic lineage (cells are mostly promonocytes and monocytes)

Question 9

What is the definition of acute monoblastic leukaemia?

Answer 9

AML in which ≥80% of the leukaemic cells are of monocytic lineage AND ≥80% of the leukaemic cells are monoblasts (cells are mostly monoblasts)

Question 10

What is the definition of pure erythroid leukaemia?

Answer 10

> 80% of the bone marrow cells are erythroid with ≥30% proerythroblasts.
Myeloid blasts should not be increased.

Question 11

What is the definition of acute megakaryoblastic leukaemia?

Answer 11

AML where ≥50% of blasts are of megakaryocyte lineage.

Question 12

What are the immunophenotypic features of acute erythroid leukaemia?

Answer 12

PAS usually positive
CD34 and HLA-DR often negative
CD117 often positive
Glycophorin A (CD235a), Haemoglobin A and the less lineage specific marker CD71a usually positive but can be negative in early, undifferentiated blasts
E-cadherin positive in most cases (present in immature erythroid cells)

Question 13

What are the immunophenotypic features of acute megakaryocytic leukaemia and what are some of the disease/ cytogenetic associations seen with this type of leukaemia?

Answer 13

PAS may be positive
CD45  negative.
CD34 and HLA-DR negative.
Variable expression of CD13 and CD33
≥1 megakaryocytic markers: CD41, CD61 or CD42b.

Genetic associations: AML in patients with Down syndrome, t(1;22), AML with inv(3) or t(3;3), AML with MRC

Question 14

What are the features of acute basophilic leukaemia?

Answer 14

• AML in which the primary differentiation is to basophils.
• Characteristic metachromic staining with toluidine blue.
• MPO negative
• PAS may be positive
• Acid phosphatase positive
• May express CD34 and HLA-DR.
• CD117 negative.
• Myeloid maturation markers CD13 and CD33 usually positive.
• Monocytic markers negative with the exception of CD11b.

Question 15

What is acute panmyelosis with fibrosis

Answer 15

Panmyeloid proliferation with blasts ≥20% and accompanying fibrosis of the bone marrow.

Characterised by abnormal, dysplastic megakaryocytes and an acute clinical illness with fevers and bone pain.

• MPO usually negative
• Express CD34 with ≥1 myeloid associated antigen: CD117, CD13, CD33.
• Complex karyotype and abnormalities in chromosome 5 and/ or 7 are common
• Poor prognosis

Question 16

What five mutations are associated with an especially poor prognosis in AML?

Answer 16

DNMT3A 
TP53
ASXL1
SRSF2
RUNX1

Question 17

Describe the features of AML with t(8;21)

Answer 17

• AML with maturation
• High intensity CD34 and MPO, HLA-DR+, CD13+
• CD33 usually relatively weak
• Aberrant expression of B cell antigens (CD19, CD79a, PAX5) common
• CD56 may be positive and is associated with KIT mutations
• > 70% will have additional mutations (loss of a sex chromosome del(9q) most common)
• KIT in 20-30%

Question 18

Describe the features of AML with inv(16)

Answer 18

• Acute myelomonocytic leukaemia
• Characteristic abnormal eosinophil population
• Complex immunophenotype with many different blast populations present.
• 40% have additional cytogenetic abnormalities; +22 (very specific for inv(16) and +8 most common
• KIT in 30-40%

Question 19

Describe the features of AML with t(15;17)

Answer 19

• Characterised by low or absent expression of HLA-DR and CD34.
• CD117+. Bright CD33 and heterogeneous CD13.
• Granulocyte differentiation markers CD15 and CD65 are negative or only weakly expressed.
• 10% express CD56 which may be associated with a poorer prognosis
• CD34 and CD2 expression can be seen in the microgranular variant.
• CD2 expression is associated with FLT3-ITD and may therefore be a poor prognostic marker
• CD2 and FLT3 expression associated with microgranular variant and bcr3 breakpoint.
• ~40% have secondary cytogenetic abnormalities: +8 most common.
• FLT3-ITD and FLT3-TKD seen in 30-40%.

Question 20

Describe the features of AML with t(9;11)

Answer 20

• AML with monocytic/ monoblastic features
• Seen in therapy related myeloid neoplasms (particularly those induced by topoisomerase inhibitors).
• Overexpression of MECOM in 40% of cases. Reported to be associated with a poorer prognosis.
• Gain of chromosome 8 is the most common additional chromosomal abnormality seen and usually MECOM negative
• Intermediate prognosis. Better than translocations with other 11q23 partner genes

Question 21

Describe the features of AML with t(6;9) DEK-NUP214

Answer 21

• AML that is often associated with basophilia and multilineage dysplasia
• Sole karyotypic abnormality in the vast majority of cases.
• FLT3-ITD mutations are common occurring in up to 78% of adult cases.
• Poor prognosis

Question 22

Describe the features of AML with inv(3)

Answer 22

• AML that results in deregulated MECOM and GATA2 expression.
• Platelet count is characteristically normal or elevated at diagnosis
• Trilineage dysplasia common but characteristic feature is dysplastic megakaryocytes.
• A subset may show megakaryocytic markers/ differentiation
• Aberrant expression of CD7
• Additional cytogenetic abnormalities are common with aberrations in chromosome 5 and 7 most commonly seen.
• RAS genes mutated in 98%
• Poor prognosis

Question 23

Describe the features of AML with t(1;22)

Answer 23

• Generally has megakaryocyte differentiation
• Sole karyotypic abnormality.
• High risk disease.

Question 24

Describe the features of AML with BCR-ABL

Answer 24

• De novo AML that shows no evidence of a preceding CML.
• Less basophilia than CML. Normal non-blast M:E.
• Aberrant expression of CD19, CD7 and TDT appears common.
• Most cases demonstrate the p210 fusion
• Most have additional cytogenetic abnormalities: -7, +8, complex karyotypes.
• Aggressive disease. Poor response to therapy.

Question 25

Describe the features of AML with NPM1

Answer 25

- 35% of adult AML and up to 64% with a normal karyotype. - +8 and del(9q) can be seen. - Strong association between myelomonocytic and acute monocytic leukaemia and NPM1 mutation - IHC showing aberrant cytoplasmic localisation of NPM1 can be used as a surrogate for molecular testing. - HLA-DR and CD34 negative with positive CD117, bright CD33 and variable CD13. - Secondary mutations are common: FLT3 and DNMT3A most common.  - Good prognosis.

Question 26

Describe the features of AML with biallelic CEBPA

Answer 26

- >70% have a normal karyotype. - +8 and del(9q) can be seen.  - GATA2 mutations are seen in approximately 39% of cases. - Good prognosis.

Question 27

Describe the features of AML with mutated RUNX1

Answer 27

- Higher frequency amongst leukaemia presenting with minimal differentiation (seen in up to 65%). - Cooperating mutations are common: ASXL1, KMT2A-PTD, FLT3-ITD, IDH-1, IDH-2.  - Poor prognosis.

Question 28

What are the diagnostic criteria for AML with MRC?

Answer 28

Diagnostic criteria: All 3 criteria must be met 1) ≥20% blood or bone marrow blasts 2) Any of the following: - History of MDS or MDS/MPN overlap - MDS related cytogenetic abnormality - Multilineage dysplasia (must be ≥50% in ≥2 cell lines) 3) Absence of both of the following - Prior cytotoxic or radiation therapy for an unrelated disease - Recurrent cytogenetic abnormality (i.e. the presence of molecular abnormalities does not exclude this diagnosis)

Question 29

What are some of the MDS related cytogenetic abnormalities?

Answer 29

- Loss of chromosome 7 or del(7q) - Del (5q) or t(5q) - Isochromosome 17q or t(17p) - Loss of chromosome 13 or del(13q) - del(11q) - del(12p) or t(12p) - Complex karyotype

Question 30

Discuss the genetic features of therapy associated myeloid neoplasms

Answer 30

- >90% of t-MN have an abnormal karyotype. - Unbalanced loss of genetic material more common after treatment with alkylating agents. Most commonly involves chromosome 5 or 7, complex karyotypes also common and mutations or loss of TP53 occur in 50% - Balanced chromosomal abnormalities more common following treatment with topoisomerase inhibitors. Frequently involve 11q23 (KMT2A) or 21q22 (RUNX1).

Question 31

How do therapy related myeloid neoplasms caused by exposure to alkylating agents differ from those caused by topoisomerase

Answer 31

- Longer duration between exposure and onset (5 to 10 years vs 1 to 5 years) - Risk increases with increased age (vs risk the same in all age groups) - MDS prior to developing AML (versus present with AML) - Unbalanced loss of genetic material involving Chr 5, 7 and 17p common (vs balanced translocations)

Question 32

What are the high risk chromosomal and molecular abnormalities as per ELN?

Answer 32

``` t(6;9) DEK-NUP214 t(v;11q23.3) KMT2A rearranged t(9;22) inv(3), t(3;3) -5, -5q, -7, -17, abn 17p Complex karyotype Monosomal karyotype Wild type NPM1 and FLT3-ITD high Mutated RUNX1 Mutated ASXL1 Mutated TP53 ```

Question 33

What is the definition of a monosomal karyotype in AML?

Answer 33

The term "monosomy" is used to describe the absence of one member of a pair of chromosomes. Monosomal karyotype is defined as the presence of at least 2 autosomal monosomies or a single autosomal monosomy associated with at least one structural abnormality.

Question 34

What are the clinical implications of positive MRD in AML?

Answer 34

Association between MRD and clinical outcome o MRD positivity associated with an increase risk of relapse, treatment failure, reduced overall survival and poorer outcomes following HSCT. o Rising transcript levels following treatment reliably predict clinical relapse. o Intervening when there is evidence of molecular relapse can prevent haematological relapse which may improve survival. o MRD status may be used to guide treatment decisions in those with low risk of intermediate (i.e HSCT)  

Question 35

What does MRD assessment aim to achieve?

Answer 35

To detect the smallest amount of leukaemic blasts that are capable of causing relapse and to discriminate these from healthy stem cells or leukaemic blasts that are not capable of initiating relapse.

Question 36

What is the difference between using PB and BM for MRD assessment?

Answer 36

There is a correlation between PB and BM MRD in some studies. With molecular methods the peripheral blood is usually 10-fold less sensitive than BM Paired blood and bone marrow samples are currently recommended for molecular assessment, BM for flow cytometry.

Question 37

What is an optimal target for MRD assessment in AML?

Answer 37

To be used as an MRD target the marker must: 1) Indicate that the stem cell can initiate relapse. - This excludes mutations that were present before leukaemia developed. - CHIP mutations: ASXL1, TET2 and DNMT3A. - Germline mutations: CEPBA and RUNX1. 2) Be reliably positive both at diagnosis and at relapse 3) Represent clonal rather than sub-clonal events

Question 38

What time points are recommended by the ELN for MRD monitoring in AML?

Answer 38

1) At diagnosis 2) After two cycles of induction chemotherapy 3) At the end of treatment 4) In those undergoing allogenic HSCT; MRD should be evaluated 28days prior to the start of the conditioning regime. 5) During the follow up phase, MRD should be analysed every three months for at least 2 years.

Question 39

What are the advantages and disadvantages to using flow cytometry as an MRD method in AML?

Answer 39

Advantages: - Applicable to nearly all AML cases (90%) - Can distinguish viable from dead cells - Short turnaround time   Disadvantages: - Operator dependent, high experience needed - Lower sensitivity and specificity than PCR - Difficult to standardise - Leukaemic phenotype can change over time

Question 40

What are the advantages and disadvantages to using qPCR methods as an MRD method in AML?

Answer 40

Advantages: - Standardisation efforts - Operator- independent - Short turnaround time - Widely available Disadvantages: - Restricted applicability to patients harbouring a specific target (30-50%) - Relative quantification (to the test sample/ standard curve)

Question 41

What immunohistochemical features are diagnostic of BPDCN?

Answer 41

CD123, CD4, CD56 The diagnosis of blastic plasmacytoid dendritic cell neoplasm (BPDCN) requires positivity of CD4 and/ or CD56 and at least two of the four plasmacytoid dendritic cell markers (CD123, CD303, TCL1, MXA), with negativity for CD3, CD20, myeloperoxidase, and lysozyme.