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Flashcards in 18.06.14 AML Deck (49):
1

What is AML?

heterogeneous disease resulting from the clonal expansion of myeloid blasts in the peripheral blood (PB), bone marrow (BM) or other tissue.

2

What is the incidence and mean age of onset of adult AML?

Median age 65 at diagnosis, 2.5/100,000/yr, slight male predominance, AML accounts for 25% of acute leukaemias, ~55% cytogenetically abnormal

3

What is the incidence and mean age of onset of paediatric AML?

<15 years, 0.7/100,000/yr, AML accounts for 15~20% of all acute leukaemias, with peak incidence in first year decreasing to 4 years (median 2 years), ~

4

What proportion of adult and paediatric AML is cytogenetically abnormal?

Adult - 55% cases abnormal cyto

Paed - 78% cases abnormal cyto

5

What are the symptoms of AML?

What is the incidence and mean age of onset of adult AML?

6

What are the recurrent abnormalities seen in AML?

1. t(8;21)(q22;q22); (RUNX1T1-RUNX1)

2. inv(16)(p13.1q22) or t(16;16)(p13.1q22); CBFB (16q22)-MYH11 (16p13)

3. t(9;11)(p22;q23); MLLT3-MLL (now known as KMT2A by HUGO)

4. t(15;17)(q24;q21)

5. t(6;9)(p23;q34)

6. inv(3)(q21q26.2) or t(3;3)(q21;q26.2); RPN1 (3q21)-EVI1 (3q26)

7

What rearrangement is seen in acute promyelocytic leukaemia?

t(15;17)(q24;q21); PML-RARA

8

Describe features of t(8;21)(q22;q22).

(RUNX1T1-RUNX1)
Good prognosis when treated with high dose cytrabine
FAB M5
Auer rods
Predominantly in younger pateints
Secondary chromsomes (>70%) cases -X, -Y, del(9q)
Mutations of KRAS and NRAS seen in 30%
KIT mutations in 20-25%

9

Describe features with inv(16)(p13.1q22)

(CBFB)
Found in 5-8% of AML
Good prognosis when treated with high dose cytrabine
Fab class M4
Occurs in all age groups, predominantly younger patients
Easy to miss on karyotype
FISH and RT-PCR may be required
Secondary findings in 40%: +22 (10-15%), +8, del(7q), +21
KIT mutations in 30%
Mutations of KRAS or NRAS seen in 30%

10

What is the core binding factor?

RUNX1-CBFB proteins heterodimerise to create CBF that binds target genes via the RUNX1 transcriptional activation domain, which regulates normal blood cell differentiation, and cell survival.

The fusion proteins produced by the above rearrangements allow the CBF to bind to the target genes, but the transcriptional activation is lost via a dominant negative inhibition leading to arrest of differentiation and TP53 induction being inhibited resulting to increased cell survival.

11

Describe APL

t(15;17)(q24;q21)
(PML-RARA)
Good prognosis
5-8% of AML
FAB M3
Predominates in mid-life
Small number have cryptic rearrangements required RT-PCR
Sensitive to ATRA

12

Describe the aetiology and treatment of APL

- Expression of PML- RARα is associated with inhibition of differentiation and increased cell self-renewal.

- The PML-RARα protein (a dominant negative form of RARα) binds to DNA and represses transcription of retinoic acid target genes, like the normal RARα protein. However, PML-RARα doesn’t respond to the transcriptional signal induction of the genes, so the genes remain repressed.

- Additionally, the function of the PML protein is disrupted: PML blocks cell growth and proliferation and induces apoptosis. However, PML-RARα does not block proliferation or induce apoptosis.

As a result, excess promyelocytes accumulate in the bone marrow and normal white blood cells cannot form, leading to APL

- A symptom of APL is DIC (disseminated intravascular coagulation): widespread activation of the clotting cascade resulting in the formation of blood clots in the small blood vessels throughout the body that organ damage. It also consumes the clotting factors resulting in severe bleeding can occur from various sites.

- All-trans-retinoic acid (ATRA), a ligand for RARα, is effective therapy for APL especially when given in combination with conventional chemotherapy.

- ATRA binds the fusion protein with resultant dissociation of co-repressor complexes, engagement of co-activation complexes by the chimerical receptor and subsequent degradation of the fusion protein. Promyelocytes can then undergo normal hematopoietic differentiation and apoptose naturally with a minimum release of the clotting factors therefore reducing the risk of haemorrhage.

13

Describe AML with t(9;11)(p22;q23)

9-12% of paed cases, 2% of adult cases
FAB M%
<20% blasts requrie close monitoring for more definitive evidence of AML
t(9;11) most common MLL
Secondary abn +8 (20%)
Prognosis Intermediate (better than other MLL translocations)

14

Describe AML with t(6;9)(p23;q34); DEK-NUP21

Found in 0.7-1.8% of AML. Occurs in both adults and children.
FAB class: all except M3, M7
Sole abnormality in most cases

Secondary abns: FLT3-ITD mutations occur in 69% of paediatric cases and 78% of adult cases, some cases have a complex karyotype.

FLT3-TKD mutations are uncommon in this subgroup.

Cases with <20% blasts must be monitored for more definite evidence of AML.

Generally a poor prognosis.

15

Describe AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2); RPN1 (3q21)-EVI1 (3q26)

Found in 1-2% of AML. Mostly in adults. Associated with tri-lineage dysplasia.

- A variety of abnormalities of 3q occur in myeloid malignancies, with the two named above, the most common & a distinct entity.

- RPN1: enhancer of EVI1 expression increased cell proliferation/ impaired differientiation

- Secondary chromosome abns (common): -7 most frequent (50%), -5q, complex karyotype. They may precede the development of the 3q26.2 abnormality.

Prognosis:

- Associated with aggressive disease and short survival.

16

Describe AML (megakaryoblastic) with t(1;22)(p13;q13); RBM15-MKL

Found in <1% of cases. FAB class: M7. It most commonly occurs in infants without Down syndrome, mainly in the first six months of life.

- Sole karyotypic abnormality in 75% cases

Secondary abns: high hyperdiploidy/ hypotriploid (chr 51-61)

Prognosis:

- Intermediate: Early reports suggested a poor prognosis, but more recent reports have found the patients respond well to intensive AML chemotherapy with long disease-free survival.

17

What is AML with myelodysplasia-related changes?

- An acute leukaemia with 20% or more blasts in BM or PB with morphological features of MDS or a prior history of MDS or MDS/MPN, or MDS-related cytogenetic abnormalities.

- 24-35% of all AML. Occurs mainly in the elderly, is rare in children.

- Chromosome abnormalities are similar to those found in MDS and often include gains/losses, most common: complex karyotypes, -5/del(5q) and -7,del(7q)

- Complex karyotype is defined as three or more unrelated abnormalities, none of which are included in the ‘AML with recurrent genetic abnormalities’ subgroup.

18

What are therapy-related myeloid neoplasms?

- Accounts for 10-20% of AML.

- Most patients have received alkylating agents and/or radiation as well as topoisomerase II inhibitors, so a division according to the type of therapy is usually not practical and is no longer recommended.

- 90% of patients with therapy-related neoplasms have cytogenetic abns identical to those observed in ‘AML with myelodysplasia-related features’ or in ‘AML with recurrent cytogenetic abnormalities’

- patients with therapy-related myeloid neoplasms have a significantly worse outcome than do their de novo counterparts with the same genetic abnormalities, suggesting that there are biologic di

19

What are the common abnormalities seen in myeloid sarcoma?

Also known as granulocytic sarcoma. A tumour mass consisting of myeloid blasts occurring at a site other than the bone marrow. Genetic abnormalities detected in ~55% cases include: -7, +8, MLL-rearrangements, t(8;21), inv(16) and many others. Clinical behaviour and response not affected by cytogenetics. Improved outcome with allogeneic or autologous BM transplantation.

20

What is Transient abnormal myelopoiesis (TAM)? Which gene is it associated with?

~10% of DS newborns present with TAM. Genetic alterations (GATA1 mutations) in blast cells can be similar to typical DS-associated AML. TAM spontaneously resolves over a period of several weeks to 3 months in 70-80% of cases. Remaining cases develop non-transient AML (Acute megakaryoblastic leukaemia) 1-3 years later.

21

Describe AML associated with Down syndrome?

- Children with DS have a 50-fold increased risk of developing AML in the first 5 years of life.

- Approximately 1-2% of children with DS develop AML, usually M7. Usually de novo, but sometimes preceded by TAM. Blast cells carry mutations of GATA1. AML in children with DS over 5 years old without GATA1 (located on X chromosome) mutations are regarded as conventional AML.

- Clinical outcome of GATA-1 mutated AML in young children with DS is associated with very favourable prognosis in comparison with AML in children without DS.

22

Which chromosomal rearrangements are associated with a favourable prognosis in paediatric AML?

t(8;21)(q22;q22)

inv(16)(p13q22)

23

Which chromosomal rearrangements are associated with a poor prognosis in paediatric AML?

5q abnormalities

-7

t(6;9)(p23;q34)

t(9;22)(q34;q11)

12p abnormalities

24

Which chromosomal rearrangements are associated with a favourable prognosis in adult AML?

t(8;21)(q22;q22)

inv(16)(p13q22)/t(16;16)(p13;q22)

t(15;17)(q24;q21)

25

Which chromosomal rearrangements are associated with a poor prognosis in adult AML?

abnormal 3q [excluding t(3;5)(q21~25;q31~35)]

inv(3)(q21q26)/t(3;3)(q21;q26)

add(5q), del(5q), -5,

-7, add(7q)/del(7q)

t(6;11)(q27;q23)

t(10;11)(p11~13;q23)

t(11q23) [excluding t(9;11)(p21~22;q23), (11;19)(q23;p13)]

t(9;22)(q34;q11)

-17/abn(17p)

Complex (≥4 unrelated abnormalities)2,3

26

What is the definition of a clone?

- gain of a chromosome or structural abnormality in 2 cells

- loss of a chromosome in 3 cells (however loss in only 2 cells can be reported if a structural abnormality is also present in both cells)

27

What is the significance of -Y and +15 detected in BM of elderly patients?

-Y and +15: both are seen in bone marrow cells of elderly patients with no haematological disease, but may also occur as markers of neoplastic myeloid clones

28

AML development is considered a multistep process requiring collaboration of at least two classes of mutation. Describe each.

Class I mutations: activate signal transduction pathways and confer proliferative advantage on haematopoietic cells.

Class II mutations: affect transcription factors and primarily serve to block haematopoietic differentiation.

29

What is the classic class I AML mutation?

fms-like tyrosine kinase 3 (FLT3, 13q12) is a receptor tyrosine kinase expressed on hematopoietic progenitors, mutated in approx a third of AML, including normal karyotype (CN-AML).

Expressed in normal myeloid and lymphoid hematopoietic progenitor cells (so overexpressed in AML blasts); important for cell differentiation, proliferation and survival. Expression lost as cells differentiate.

Majority of mutations are internal tandem duplications (ITDs) in exons 14 and 15 (75-80% of muts, in 30-40% CN-AML) leading to in-frame insertions within juxtamembrane region of the receptor. Causes loss of structure of autoinhibitory domain and ligand-independent activation of FLT3. - Poor prognosis adult and paed - survival rate 20-25% at 4 years

Tyrosine kinase domain mutations – codons Asp835 and Ile836 (5-10% of AML; 20-35% of FLT3 muts). Result in folding out of activation loop enabling ATP to access catalytic site resulting in constitutive kinase activation.
- Prognosis unclear, may depend on the presence of other mutations

30

Which cytogenetic abnormalities in AML is the FLT3-ITD most commonly seen with?

FLT3 mutations most frequent in AML with t(15;17), AML with t(6;9) and CN-AML; considered independent prognostic factor for survival in CN-AML.

Most affected AML patients have one type of FLT3 mutation, but some have both types. Particularly poor prognosis if wildtype FLT3 allele lost by deletion of 13q or monosomy 13.

FLT3 mutations can be acquired/lost at relapse or during disease progression; no use to monitor MRD.

31

What is the AML 17 trial?

LT3 mutations can be acquired/lost at relapse or during disease progression; no use to monitor MRD.

An objective of the AML 17 trail is to determine whether CEP-701 (FLT3 inhibitor therapy), given in sequence with standard chemo as 1st line can reduce risk of relapse and improve overall survival in patients with a FLT3 mutation.

32

Give examples of class I AML mutations.

PTPN11 (2-4%) - (12q24) encodes cytoplasmic tyrosine phosphatise, SHP-2; highly expressed in haemopoietic cells. Gain of function muts; role in pathogenesis and prognostic significance in AML unclear (also mutated in 50% Noonan’s patients).

RAS – NRAS (CN-AML and up to 40% CBF-AML) and KRAS (5-17% of CBF-AML); significance unknown. Increased incidence in inv(16)/t(16;16), inv(3)/t(3;3) and M4 FAB type, decreased incidence in t(15;17). Negative association between RAS and FLT3 mutations. Not good for predicting MRD.

cKIT - proto-oncogene (4q12) - encodes a member of type III tyrosine kinase family, important in survival, proliferation, differentiation and functional activation of hematopoietic progenitor cells. Gain-of-function mutations, mostly in core-binding factor AML (CBF-AML) and AML with +4 (sole abnormality). Associated with increased incidence of relapse and confers a poorer prognosis in AML with t(8;21), less clear for inv(16)/t(16;16). Studies suggest CBF-AML with c-KIT mutations should be treated as intermediate-risk, not favourable and considered for transplantation.

33

Give an example of a class II AML mutation.

RUNX1 - Runt-related transcription factor 1 (21q22.3); makes up alpha subunit of core binding factor and involved in normal hematopoiesis. Significant correlation with MLL-PTD and IDH mutations, inversely correlated with NPM1 and CEBPA mutations. Prognostic significance unclear – resistance to chemotherapy has been observed therefore ?poor

MLL (11q23) essential role in early development and haematopoeisis by acting as histone methyltransferase and transcriptional co-activator. Partial tandem duplication mutation in 5-10% CN-AML and in large proportion of AML cases with trisomy 11. Contributes to leukaemogenesis through DNA hypermethylation and epigenetic silencing of tumour suppressor genes. Negative impact on prognosis. Can be used for MRD monitoring.

34

What is the target for most class II AML mutations?

Multiple class II mutations target the core binding factor (CBF) in AML. CBF is a heterodimeric transcription factor comprised of the RUNX1 (also known as AML1) and CBFB subunits both of which are essential for haematopoietic development. CBF mutations are commonly gene fusions as a result of a translocation or inversion (cyto notes).

Evidence suggests the leukaemia-associated fusion proteins are dominant negative versions of CBF - inhibit CBF target genes by recruitment of co-repressor complexes including histone deacetylases.

35

Give some examples of genes in which unclassified mutations have been detected.

NPM2 (5q35.1)
WT1 (11p13)
TET2 (4q24)
IDH
DNMT3A (2p33)
TP53
ASX1
JAK2
CBL

36

Describe NPM1 and role in AML.

A third of AML cases, including approx 50% with normal karyotype, have heterozygous mutations in the carboxy-terminus of the nucleolar phosphoprotein, nucleophosmin (NPM1, 5q35.1) (provisional disease entity in WHO Classification 2008) Associated with GOOD prognosis.

37

What is the role of NPM1 and association with p53?

Leads to delocalisation of the protein to the cytoplasm.

Evidence that NPM1 mutations represent primary lesions in leukaemogenesis - generally mutually exclusive with balanced translocations and usually heterozygous.

Associated with common secondary chromosome abnormalities, including; -+8, +4, and del(9q).

NPM1 mutation and FLT3-ITD commonly co-exist in CN-AML, suggests may cooperate in leukaemia.

NPM1 thought to have relevant roles in cellular functions, including ribosome biogenesis, centrosome duplication, DNA repair and response to stress.

NPM1 also involved in functions of p53 and p19ARF. Wild-type NPM1 protects hematopoietic cells against p53-induced apoptosis under conditions of cellular stress; possible that failure of mutated NPM1 to protect cells may make them more sensitive to high-level genotoxic stress induced by chemotherapy – good prognosis.

Less frequent in paediatric AML.

Different mutations have been described – all frameshift and mostly involving a 4 base (TCTG) insertion into exon 12.

Associated with FLT3-ITD (40%), FLT3-TKD(10-15%) and IDH (~25%) mutations

38

What is the role of WT1 in AML?

WT1 - transcription factor implicated in regulation of apoptosis, proliferation and differentiation of hematopoietic progenitor cells; mutations in about 10% of AML patients. Initially considered exclusively as a tumour suppressor gene but has been found to also act as an oncogene. Significance of mutations uncertain.

39

What is the role of TET2 in AML?

TET2 (4q24); breakpoint associated with some AML translocations. Encodes enzyme with role as a candidate tumour suppressor gene for myeloid malignancies (mutated in MDS, MPN and AML). Also involved in myeloid differentiation and self-renewal of stem and progenitor cells. Loss-of function mutations - in most cases nonsense and frameshift resulting in inadequate production. Prognostic relevance controversial – some studies suggest adverse impact on certain AML sub-groups, another recent study reported no prognostic significance

40

What is the role of IDH in AML?

IDH - cytosolic IDH1 (older adults) and mitochondrial IDH2 (cases without IDH1) metabolic enzymes involved in cellular defence of oxidative damage. Gain of function mutations. Prognosis unclear – some studies showed no significance, others demonstrated a poor impact in certain AML sub-groups. Some functional overlap between IDH1/2 and TET2 mutations (IHD1/2 product inhibits TET2 function) - are mutually exclusive; patients with IDH1/2 and TET2 mutations show a similar epigenetic signature and global DNA hypermethylation. Some studies showed that IDH1 & 2 mutations are significantly associated with NPM1 mutations and predict a worse outcome for patients with mutated NPM1 without FLT3-ITD.

41

What is the role of DNMT3A in AML?

DNMT3A (2p23); methyl transferase involved in generating de novo DNA methylation. Important role in the epigenetic regulation of genes, loss of activity causes hypomethylation and uncontrolled expression. Loss of function mutations in ~14-18% of AML with a higher proportion in CN-AML. Associated with the presence of NPM1, FLT3 and IDH mutations. DNMT3A mutations are highly recurrent in patients with de novo AML with an intermediate-risk cytogenetic profile and are independently associated with poor outcome.

42

Why is RNA used in AML diagnosis?

RNA to identify specific gene fusions due to: a) SIZE: Gene fusions are too large to detect by PCR due to the presence of introns; b) STRUCTURE: There are multiple gene fusions due to alternative splicing; c) SENSITIVITY: Detects transcription of gene fusions, not just their presence. Complications of using RNA rather than DNA – RNA is more difficult to handle due to presence of RNases in the environment- requires aseptic techniques and mRNA transcripts are very unstable and have a short half-life.

43

What is the key objective of diagnosis work-up for AML patients?

In children and young adults, key objective of the diagnostic work up is to distinguish patients at different risk of relapse to guide the use of allogeneic transplantion.

Procedure-related mortality associated with allogeneic transplantation, only individuals at significant risk of relapse benefit.

In older adults (>60 yrs), major goal of pre-treatment assessment is to find patients who may benefit from intensive chemotherapy.

44

How is MRD performed in AML

Molecular markers such as defined gene fusions or certain mutations provide the basis for molecular based monitoring. Blood or bone marrow samples are received at regular intervals following diagnosis and the relative level of the mutation in comparison to a housekeeping gene (usually ABL) is determined by RQ-PCR.

Mointoring frequency depends on transcript required.

45

What alternative method to RQ-PCR can be used for disease monitoring?

flow - less sensitive but applicable to more AML cases

46

What are the different aspects of the transcript that relate to prognosis?

The transcript level at diagnosis e.g. study found pts have worsened survival if expressing high levels of AML1-ETO transcript as compared to those with low transcript levels

The extent of transcript reduction after the induction therapy e.g. it has been demonstrated that CBFB-MYH11 patients with a reduction of the transcript level of at least two logs have a significantly improved disease free survival.

The increase of the transcript level following achievement of complete remission. Any successive rises in transcript level may indicate impending clinical relapse before it happens and thus allow the opportunity for pre-emptive therapy. NB. Because of the sensitivity of RQ-PCR, low level positivity may be detected in patients even in long term remission.

47

What are the other applications of RQ-PCR in AML?

There have been a number of reports linking over-expression of particular genes and prognosis in AML, which in some instances have been shown to discriminate patients at differing risk of relapse within cytogenetic risk groups. T
1) EVI1 (ecotropic virus integration-1) gene located at 3q26, which is upregulated as a result of the inv(3)(q21q26) and t(3;3)(q21;q26) rearrangements.

A recent study lends support to routine screening by RQ-PCR for EVI1 expression, which can be associated with cytogenetically cryptic 3q26 abnormalities, to identify a subgroup of patients with a particularly poor prognosis.

The molecular basis of up-regulation of other genes that have been associated with poorer prognosis such as MN1, BAALC, FLT3 and ERG remains poorly understood.

48

What is the classic example of a targeted therapy in AML?

ATRA for APL t(15;17)(q24;q21)

49

Other than ATRA, what other examples of targeted therapies are there for AML?

Over the last decade, a variety of compounds have been developed in preclinical and clinical studies as potent inhibitors of FLT3. Many of the earlier agents under investigation, such as lestaurtinib, midostaurin, and sunitinib, were initially developed as inhibitors of other tyrosine kinases and as targeted therapies in a variety of malignancies. These compounds have been demonstrated to have some efficacy in clinical trials of AML, mainly manifesting as transient decreases in circulating blasts correlating with effective in vivo suppression of the FLT3 target. However, the pharmacokinetics of some compounds and the suboptimal specificity and potency of others have limited their therapeutic efficacy. In the last few years, newer, more potent and specific agents have been under investigation e.g. AC220. This agent has shown significant promise in early phases of clinical investigation, and is currently in more advanced clinical trials (Fathi et al, 2011).