Real Time Quantitative RTPCR

For the detection of leukemia-associated transcripts by RQ-PCR, RNA is initially converted to cDNA using RT protocols identical to those involved in conventional RT-PCR (reviewed in Reference 30). However, in contrast to the latter technique, in which two rounds of PCR amplification are routinely performed, RQ-PCR approaches involve only a single round of PCR. The basis of this technology is the measurement of the number of amplicons generated with each PCR cycle in the exponential phase of the reaction, detected as a proportionate rise in fluorescence intensity. PCR products may be detected through use of the fluorescent dye SYBR Green I, which binds to the minor groove of double-stranded DNA, or through specific "hybridization" or "hydrolysis" probes. SYBR Green I is not favored for

MRD detection, given its lack of sensitivity or specificity in comparison to the use of specific probes, which are significantly more expensive.

A key advantage of RQ-PCR approaches is the capacity to measure transcript levels of the fusion gene of interest, relative to the expression of endogenous control transcripts. This enables precise evaluation of kinetics of molecular response to treatment, documentation of rising leukemia-associated transcripts prior to frank relapse, and identification of poor-quality samples or problems with the RT step, which could give rise to false-negative results had conventional endpoint assays been used. Changes in leukemic target transcript level normalized to that of the endogenous control gene may be calculated on the basis of differences in Ct (cycle threshold) values (so-called AACT method), providing the efficiencies of the PCR reactions are comparable. Alternatively, normalized MRD data may be reported in terms of absolute copy numbers derived from respective plasmid standard curves run in parallel (reviewed in References 29 and 30).

For reliable performance of RQ-PCR, testing of diagnostic material is critical to define breakpoint location, particularly for rearrangements in which variable breakpoints occur at the genomic level (e.g., bcr 1, 2, and 3 for PML-RARA and types A, D, and E, which account for the majority of cases with the CBFB-MYH11 fusion), since RQ-

Figure 30-7. Detection of FLT3 mutations in AML. ITDs affect the juxtamembrane region of the FLT3 receptor, while point mutations (e.g., D835/I836) involve the kinase loop. Both classes of mutation (MUT) lead to constitutive activation of the receptor, activating a number of potential downstream targets. Presence of FLT3ITD is revealed by the amplification of a larger-sized band than that associated with wild-type (WT) receptor following PCR of genomic or complementary DNA (see lanes 4-8, upper right panel). In some cases, more than one aberrant band may be observed (e.g., lane 5); this may be

PCR assays are transcript specific. Furthermore, availability of diagnostic material is important so that normalized fusion gene expression levels can be related to the pre-treatment level. Extensive collaborative studies have been undertaken by the Europe Against Cancer (EAC) Group to optimize and standardize the methods for detection of leukemia-associated fusion transcripts and to define suitable endogenous control transcripts.29

Mutation Screening

There are a variety of methods available for detection of mutations, for example, involving genes encoding tyrosine kinases (e.g., KIT) or transcription factors (e.g., CEPBA). For small genes, PCR and direct sequencing may provide the most suitable approach. However, for larger genes, or in situations where large numbers of samples are to be tested, initial screening may be conveniently undertaken by methods such as single-strand conformation polymorphism (SSCP) analysis, chemical cleavage of mismatch, or denaturing high-performance liquid chromatography (DHPLC), with sequencing restricted solely to cases with a suspected mutation. Length mutations such as FLT3 ITDs are readily detected by PCR using genomic or complementary DNA as a template (see Figure 30-7). For muta-

due to biallelic FLT3 mutations in the leukemic clone, with the WT band being due to residual normal marrow elements. However, in some instances it may be due to leukemic subclones harboring different FLT3 mutations. Tyrosine kinase domain (TKD) mutations may be detected by PCR and restriction enzyme RE digest, with presence of a mutation leading to loss of the EcoR V restriction site. DDW, water control. (Figure kindly prepared by Rosemary Gale, University College London.)

IG-like domain

Juxtamembrane Transmembrane Juxtamembrane

Kinase 1 Kinase insert

Kinase 2 C-terminus

Internal tandem duplication

PCR + RE digest

TKD mutations D835/1836

Agarose gel electrophoresis 1 2 3 4 5 6 7

Internal tandem duplication

Agarose gel electrophoresis 1 2 3 4 5 6 7

Ladder WT

AML patients

Ladder WT

AML patients

EcoR V

tions that target a particular region of the gene, a specific assay may be used involving PCR combined with a restriction enzyme digest, for example, to detect FLT3 activation loop mutations (e.g., D835), or PCR using allele-specific primers, for example, for detection of JAK2 mutation in myeloproliferative disorders (see chapter 35). Rapid screening for presence of an underlying NPM1 mutation may be undertaken by immunohistochemistry using monoclonal antibodies against NPM that are in routine use for diagnosis of NPM-ALK-associated lymphoma, with presence of mutation being indicated by delocalization of NPM to the cytoplasm.12

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