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Molecular diagnostic assays are best performed on nucleic acid extracted from BM samples. When marrow is not available, fusion genes and mutations

can generally be reliably identified using blood (par-ticularly if blasts predominate). Ideally DNA and RNA should be extracted and viable cells stored; however, where cell numbers are limiting RNA extraction should be prioritized, as this is suitable for molecular screening for fusion genes and leukemia-associated mutations. Ideally diagnostic material should be stored for future research, along with constitutional DNA extracted from buccal swabs or other non-leukemic b

a

c

d

Figure 8.5.AML with inv(16)(p13.1q22); CBFB-MYH11.

a. Bone marrow morphology showing myeloblasts, a monoblast, granulocytic differentiation and abnormal baso-eosinophil precursors.

b. Flow cytometry showing three sub-populations of blast cells based on varying intensity of CD13 and CD45 antigen expression.

c. Flow cytometry showing the subpopulation of blast cells with strongest CD45 positivity to express CD14 monocyte-associated antigen (monoblasts).

d. Metaphase FISH for CBFB using a breakapart probe showing the normal red/green fusion signal and a rearranged CBFB (separated red and green signals) on the abnormal chromosome 16.

tissue, subject to appropriate ethical approval and informed patient consent.

Molecular screening for chimeric fusion genes

PML-RARA fusion gene: When APL is suspected molecular analysis for the PML-RARA fusion gene should be performed. This should be done irrespective of patient age to identify those likely to benefit from molecularly targeted therapies (ATRA and ATO) and to establish thePML breakpoint location necessary for subsequent MRD detection [45].

RUNX1-RUNX1T1 and CBFB-MYH11 fusion genes:

Molecular screening for presence of these fusion genes is also relevant in younger non-APL patients who are suitable for intensive treatment approaches including allogeneic transplantation. This is important as the overt cytogenetic lesion is not detected in ~10% of cases with CBF leukemia due to cryptic, simple variant or more complex rearrangements or cytogenetic fail-ures (reviewed in [1]). Cases that are molecularly pos-itive in the absence of the associated cytogenetic lesion should be subject to confirmation by reverse transcrip-tase polymerase chain reaction (RT-PCR) performed b

a

d c

Figure 8.6.Acute monoblastic leukemia with NPM1 exon 12 mutation.

a. Bone marrow aspirate morphology showing monoblasts.

b. Bone marrow trephine showing a monomorphic blast cell infiltrate of intermediate sized cells (hematoxylin and eosin (H&E)).

c. CD15 positivity of the myeloblasts in the bone marrow trephine (immunoperoxidase stain).

d. Cytoplasmic localization of NPM (immuno-alkaline phosphatase staining) of the bone marrow trephine biopsy.

e. Melt curve analysis for NPM1 exon 12 mutations showing normal wildtype single peaks (red). Three AML samples with NPM1 mutations (other colors) have different melt profiles corresponding to the presence of mutant and wildtype alleles.

f. High resolution capillary gel electrophoresis for NPM1 exon 12 mutations. PCR amplification of DNA containing the NPM1 exon 12 mutation yields a PCR product (*) that is four base pairs longer than the wildtype allele. (Red peaks are size markers).

in an independent laboratory and FISH. These analy-ses respectively serve to exclude the possibility that the result was spurious due to PCR contamination or was unlikely to be of clinical relevance, should the fusion gene have arisen in a minor clone. However, it is important to appreciate that small insertion events can potentially be missed by FISH when using large

probes, hence molecular screening for CBF leukemia is best undertaken by RT-PCR, for which standardized optimized protocols have been published by the BIOMED-1 group [49]. Patients with CBF leukemia identified on molecular screening are assumed to be biologically similar to those with the overt cytogenetic lesion. Recognition of such cases is clinically relevant, f

*

e

Figure 8.6.(cont.)

since they represent a group of patients who may be spared routine use of allogeneic transplant in first remission and can benefit from MRD monitoring by real-time quantitative polymerase chain reaction (RQ-PCR). Routine provision of samples for molecular diagnostics in the CBF leukemias is important to establish breakpoint location (variable for CBFB-MYH11) and to provide a baseline fusion transcript level for subsequent MRD assessment.

BCR-ABL1 fusion: Although rare in AML (~1%), molecular screening for the BCR-ABL1 fusion may also be worth including in the diagnostic panel. This will identify those patients with a very poor prognosis, who could potentially benefit from molecularly tar-geted therapy with imatinib and other tyrosine kinase inhibitors.

FIP1L1-PDGFRA fusion: In AML with a marked eosi-nophilic component and negative for CBFB-MYH11 there may be merit in screening for the FIP1L1-PDGFRA fusion using PCR or FISH-based approaches, given the sensitivity of this disease entity to imatinib [50].

Other fusion genes: Beyond identifying patients who can potentially be subject to MRD monitoring by RQ-PCR, more extensive screening for a wider range of AML fusion genes is of limited clinical utility and is currently not recommended [46].

Mutation screening

The diagnostic work-up for AML patients lacking APL or CBF leukemia subtypes and who are candi-dates for intensive treatment, should be routinely screened for mutations that may impact upon man-agement and clinical outcome. A number of studies have highlighted the complex interplay of mutations affecting NPM1, CEBPA and FLT3 on clinical out-come. Such information is increasingly being used to direct use of allogeneic transplantation infirst remis-sion (reviewed in [32]).

FLT3 mutations: For detection of FLT3-ITD muta-tions it is important to use a methodology that allows quantification of the mutant allele (e.g. Genescan) [20], since higher mutant ratios have been associated with poorer prognosis [19–21]. The prognostic signif-icance of TKD mutations remains uncertain and based

Table 8.4.Immunophenotyping panel for the diagnosis of acute myeloid leukemia (AML) and mixed phenotype acute leukemia.

Diagnosis of AML^a

Precursor stage CD34, CD38, CD117, CD133, HLA-DR Granulocytic

markers

CD13, CD15, CD16, CD33, CD65, cytoplasmic myeloperoxidase (cMPO)

Monocytic markers

Non-specific esterase (NSE), CD11c, CD14, CD64, lysozyme, CD4, CD11b, CD36, NG2 homologue^b Megakaryocytic

markers

CD41 (glycoprotein IIb/IIIa), CD61 (glycoprotein IIIa), CD42 (glycoprotein 1b)

Erythroid marker CD235a (glycophorin A) Diagnosis of mixed phenotype acute leukemia^c

Myeloid lineage MPO or evidence of monocytic differentiation (at least two of the following: NSE, CD11c, CD14, CD64, lysozyme) B-lineage CD19 (strong) with at least one of the following: CD79a, cCD22, CD10, or CD19 (weak) with at least two of the

following: CD79a, cCD22, CD10 T-lineage cCD3, or surface CD3

Adapted from European LeukemiaNet AML Guidelines [46].

aFor the diagnosis of AML, the table provides a list of selected markers rather than a mandatory marker panel.

bMost cases with 11q23 abnormalities express the NG2 homolog (encoded by CSPG4) reacting with the monoclonal antibody 7.1.

cRequirements for assigning more than one lineage to a single blast population adopted from the WHO classification (see Table 8.2). Note that the requirement for assigning myeloid lineage in mixed phenotype acute leukemia is more stringent than for establishing a diagnosis of AML.

Note also that mixed phenotype acute leukemia can be diagnosed if there are separate populations of lymphoid and myeloid blasts.

on current evidence there appears to be little merit in screening for this mutation outside clinical trials eval-uating FLT3 inhibitor therapy.

NPM1 exon 12 mutations: Molecular screening for NPM1 exon 12 mutations is most appropriately restricted to AML cases lacking any of the balanced chromosomal abnormalities recognized in the WHO classification. A number of methods are available to detect mutations in theNPM1 gene [51]. Presence of an NPM1 mutation to some extent ameliorates the adverse prognosis associated withFLT3-ITD, while NPM1-pos-itive/FLT3-ITD-negative AML has a particularly favor-able prognosis (Figure 8.7). NPM1 mutations have been shown to provide highly sensitive and reliable targets for MRD detection [47,52]. There is preliminary evidence to suggest, albeit based on analysis of small sample sizes, that patients with mutant NPM1 may benefit from ATRA therapy [53].

CEBPA mutations: Protocols have been established for mutation screening of CEBPA [26,29–31]. However, this is more challenging than analysis of FLT3 and

NPM1, given that mutations are widely distributed and the gene is highly GC-rich. Routine screening strat-egy may be influenced by the observation that favorable disease outcome correlates with presence of biallelic CEBPA mutations in the absence of FLT3-ITD [30–32]. Accordingly, CEBPA mutation screening could reasonably be restricted to cases that are:

1. FLT3-ITD-negative.

2. NPM1 mutation-negative.

3. Do not have balanced cytogenetic abnormalities.

4. Do not have adverse risk cytogenetics (defined below).

Other mutations: In situations in which clinical fea-tures suggest that AML has arisen on the background of a predisposing genetic disorder (e.g. Down syn-drome, Fanconi anemia), input should be sought from specialist laboratories for additional molecular characterization to help inform patient management.