Genetic Studies In Leukemia

Published Date: 02 Nov 2017

Abstract

The diagnosis of leukemia relies upon a multiparametric approach involving a number of different pathology disciplines. Molecular methods are increasingly employed to help refine diagnosis, establish prognosis and determine the most appropriate treatment, including rational therapies targeting the underlying genetic lesion. This review aims to highlight some of the molecular techniques commonly used in the diagnosis of leukemia using relevant examples. The focus is on procedures in current use and technologies showing promise in the research setting that are likely to enter clinical use in the near future. The list is not exhaustive, and this article concentrates on diagnosis of leukemia; techniques used to monitor response to therapy and molecular residual disease are mentioned but have not been covered extensively.

Genetic Studies in Leukemia

http://asimg.webmd.com/external/cleargif/1x1.gifIn many types of leukemia, genetic studies at diagnosis are considered to be crucially important, especially with regards to prognostication and therapeutic choice. In practice, a bone marrow or peripheral blood sample is referred to the diagnostic laboratory with a (provisional) diagnosis based on the initial morphologic and immunophenotypic findings. The techniques employed to define the genetic aberrations within the leukemia depend on a number of factors, including: the subtype of leukemia; clinical urgency; type, volume and age of the sample; relevance of the genetic marker; and also the available technology within the laboratory. An overview of these genetic techniques is provided in Table 1. The scope of this review is molecular methodologies, illustrated by selected clinical examples; therefore cytogenetic techniques will only be described in brief.

G-band Metaphase Chromosome Analysis ('Karyotyping')

Conventional cytogenetic analysis in the newly diagnosed leukemia patient relies on the presence of mitotically active dividing cells, which usually means that a bone marrow specimen must be cultured, although, if there are circulating leukemia cells then sometimes blood can be used as a surrogate. Cells are arrested in metaphase of the cell cycle, when the chromosomes are at their most condensed, and easily visible. The chromosomes in the 'metaphase spread' are processed so that staining with Giemsa (or Leishman) produces a characteristic banding pattern, allowing individual chromosomes to be distinguished, and numerical and structural abnormalities to be identified. A clonal abnormality is usually defined by the presence of at least two cells with the same aberration (three cells in the case of chromosome loss). Even when the aberration identified is not recurrent in leukemia, the finding of a clonal chromosomal defect can provide evidence for a malignant process.

Metaphase cytogenetic analysis of marrow is useful to detect translocations and related changes such as inversions and aneuplodies, which are common in AML, ALL and CML. Cytogenetic analysis provides a low-resolution whole-genome scan, and has the advantage of being able to detect balanced rearrangements, which are relatively common in leukemia. Its main drawbacks are limited resolution (typically 3–5 Mb), sensitivity (5–10%, depending on the number of cells analyzed, and assuming disease is present in the marrow) and the challenge of leukemia cells with a low mitotic index (for example in CLL). It is worth noting that gross chromosomal abnormalities can only be detected in a proportion of leukemia (e.g., in approximately 55% of AML). When metaphase cytogenetic analysis is applied to the study of hematological malignancies, many chromosomal defects will remain undetected.[3]

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Metaphase cytogenetic analysis is able to provide a definitive diagnosis upon detection of the pathognomonic rearrangements in CML (t(9;22)(q34;q11), the 'Philadelphia [Ph] chromosome', creating a BCR–ABL1 gene fusion), acute promyelocytic leukemia (t(15;17)(q24;q21) PML–RARA) and core-binding factor AML (either t(8;21)(q22;q22) RUNX1–RUNX1T1, or inv(16)(p13q22) or t(16;16)(p13;q22) CBFB–MYH11), even in the absence of other diagnostic features, such as a blast count >20% in AML.[1] Many other recurrent (and unique) abnormalities are found across all types of leukemia, and it can be anticipated that at least some of these will be considered pathognomonic of different subtypes of leukemia in the future.[4]

Chromosomal aberrations may also provide important prognostic information. In AML, cytogenetic findings are the most important parameter in establishing the prognosis, and three broad prognostic groups (good, intermediate and poor) are defined on the basis of the diagnostic karyotype, which can help to define those patients that may benefit from stem cell transplantation.[5] In CML, the finding of additional karyotypic abnormalities, namely a second Ph chromosome, trisomy 8, isochromosome 17q or trisomy 19, at presentation may have a negative impact on survival, and may signify that the leukemia has already progressed to accelerated phase or blast crisis.[6]

Fluorescence in situ Hybridization

FISH provides a useful adjunct to cytogenetic analysis, its main advantage being that it does not rely on dividing cells and can therefore be performed on cells in interphase. Blood can be used if marrow is unavailable, and results can be obtained more quickly because cells do not need to be cultured. FISH is also both more sensitive than conventional cytogenetic analysis (0.5–1%) and has higher resolution (100 kb, depending on the probe), although, depending on the type of probe, caution needs to be applied to avoid false-positive and -negative results due to signal colocalization or drop-out. Laboratories should establish cut-off values for each probe set to define unambiguous positive results.

FISH uses fluorescently labeled DNA probes to locate specific sequences of interest and can thus identify structural and numeric chromosomal changes including balanced rearrangements and microdeletions. A targeted approach using a limited number of probes is usually employed to look for the most common or clinically relevant aberrations within the subtype of leukemia, so, unlike karyotype analysis, the technique does not normally provide a genome-wide assessment. The exception is multiplex FISH (M-FISH), where paints identify individual metaphase chromosomes, which can be useful in resolving complex karyotypes or poor morphology metaphase spreads where chromosomes are difficult to identify by their G-banding pattern alone.

FISH is particularly useful in the diagnosis of leukemia with cryptic cytogenetic abnormalities. One striking example of this is the t(12;21)(p13;q22) translocation resulting in an ETV6–RUNX1 gene fusion, the most frequent translocation in pediatric B-lineage ALL, which as a result of the breakpoints being relatively close to the telomeres of the short arm of chromosome 12 and the long arm of chromosome 21 is cytogenetically 'invisible'. It is, however, vital to identify this aberration for risk-adapted therapeutic protocols, as it is associated with the good risk prognostic group.[7] The translocation can be detected using FISH or molecular techniques.

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Interphase FISH is able to identify genomic aberrations in approximately 80% of CLL cases using a disease-specific panel of probes,[8] and is particularly useful for identifying TP53 (17p13) and ATM (11q23) deletions, both being associated with poor prognosis. Interphase FISH analysis is generally considered to be superior to metaphase cytogenetic analysis in CLL owing to: the difficulties in persuading B cells to divide in culture (they divide slowly and need mitogen stimulation to produce enough metaphases for analysis); the fact that most clinically relevant aberrations in CLL are copy-number changes; the limited sensitivity of metaphase analysis such that small populations of abnormal cells may be missed; the fact that submicroscopic deletions of prognostic relevance will not be detected by conventional G-band cytogenetics; and the fact that interphase FISH is quicker than metaphase analysis and may be cheaper if limited panels of probes are used.

in vitro Molecular Techniques

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Molecular techniques that can be employed in the diagnosis of leukemia are many and varied, each with their own advantages and disadvantages. Detailed below are some of the more common techniques in routine clinical use, and some techniques that are currently used in the research setting but that are on the verge of transferring to clinical use. Selected examples of their use in leukemia diagnostics are mentioned.

Microarray-based Techniques

Whole-genome Scanning by Molecular Karyotyping: Array Comparative Genomic Hybridization

Comparative genomic hybridization (CGH) is used to compare the genetic material from a test individual, such as a leukemia patient, to that of a reference 'normal' individual (usually DNA pooled from several subjects), to identify the presence of copy-number changes in the test sample. Test and reference DNA are digested into small fragments and each labeled with a different fluorophore. In the past, the DNA was allowed to hybridize to normal metaphase spreads to identify copy-number changes with a resolution of 2–3 Mb via differential fluorophore binding, but to improve resolution metaphase spreads have now been replaced with microarrays. The DNA probes on the array can be bacterial artificial chromosomes (BACs), or more commonly, oligonucleotides. Resolution and sensitivity are determined by many factors including the length of the probes, the number of probes on the array (probe density), probe distribution, size of clonal population, DNA quality and software analysis algorithms. Deletions or insertions as small as 50 kb can be detected by array CGH (aCGH), which is a marked improvement over karyotyping. Whole-genome scanning arrays have allowed for the discovery of chromosomal aberrations in a much higher proportion of patients with leukemia, and it is hoped that identification of novel aberrations may lead to more precise prognostic schemes.[9] However, one distinct disadvantage of aCGH is that it is unable to detect balanced rearrangements, which are relatively common in leukemia.

Array design is critical and is determined by the application. CGH arrays normally have a high number of probes spaced evenly throughout the genome, with increased density of probes at regions of particular interest. For example, a 385K array has median probe spacing of 6 kb, but by targeting specific regions, breakpoints can be mapped within 5-kb intervals.[9] Arrays can also be designed to be targeted to only specific regions known to be associated with disease. An overview of the aCGH process is provided in Figure 1.

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Figure 1.

Overview of the array comparative genomic hybridization process. Fragmented test DNA and reference (control) DNA are labeled with different fluorophores and allowed to competitively hybridize to the microarray, consisting of oligonucleotides or bacterial artificial chromosome clones spotted onto a slide. If the test DNA contains a region of gain in copy number, it will be in excess and will preferentially bind to the array, resulting in increased test signal and decreased reference signal for the probes within the region of gain in copy number. If the test DNA has a decreased copy number, then reference DNA will be in excess for that region and there will be decreased test signal intensity (and increased reference signal intensity) for the affected probes.

CLL is ideally suited to analysis with copy-number arrays because the genetic lesions with known clinical relevance are chromosomal gains and losses. Sargent et al. created and validated a custom aCGH platform as a clinical assay for CLL genomic profiling, designed to interrogate all known CLL prognostic loci.[10] Their 60-mer, 44,000-probe oligonucleotide array with a 50-kb average spatial resolution was augmented with high-density probe tiling at loci that are frequently aberrant in CLL.

The results of these and other studies using array-based karyotyping to evaluate CLL have consistently reported high concordance with FISH panel results, with instances of nonconcordance explained by low tumor burden, the presence of small subclones or the relatively low resolution of the arrays used in the study.[11] aCGH profiling thus represents a feasible routine clinical test for CLL. Furthermore, Gunn et al. were able to identify clinically significant atypical 11q deletions (atypical because they did not include deletion of the ATM gene) in CLL with aCGH that may be missed by FISH panels used for prognostic stratification of the disease.[12]

aCGH has also been used as a research tool to study novel genomic imbalance in CLL; although recurrent chromosomal alterations occur, relatively few affected tumor suppressors and oncogenes have been implicated in the disease.[13] Using a BAC array, Gunn et al. found a high proportion of submicroscopic deletions, both monoallelic and biallelic, of chromosome 22q11.[14] They subsequently used a higher-resolution oligonucleotide-based array to show that the 22q11 deletions ranged in size from 0.34 Mb up to approximately 1 Mb, and demonstrated that genes in the minimally deleted region (including PRAME) had significantly reduced mRNA expression by reverse-transcription quantitative real-time PCR (RT-qPCR).

Whole-genome Scanning by Molecular Karyotyping: SNP Arrays

The resolution of microarray technology has been improved further still with the introduction of SNP arrays. In contrast to aCGH, SNP arrays do not rely on competitive binding of reference DNA, but genotype polymorphisms directly in test DNA, and the hybridization signal strength from individual probes allows for the estimation of gene copy number. Given the high density of SNPs that can be evaluated on a genome-wide level (>750,000), very small regions of copy-number alteration can be identified,[15] and such arrays have again proved popular in the study of CLL, using for example a 250K SNP array.[16] Here, 250K refers to the number of SNPs distributed across the genome (rather than their spacing), so a 250K array comprises 250,000 SNPs, whereas a 500K array has double the SNP density. Arrays with even higher numbers of probes are available, which can decrease the minimal detectable size of a deletion to approximately 25 kb.[17] However, whereas CGH arrays usually have probes evenly spaced across the genome, the distribution of probes on SNP arrays is dictated by the location of SNPs, so resolution within 'SNP deserts' can be relatively poor.[18] To increase resolution, arrays containing both SNPs and genomic probes have been created.[9] One such example is the Affymetrix CytoScan® HD, which contains 750,000 SNPs and 1.9 million nonpolymorphic probes, and whose coverage includes all 526 Sanger cancer genes, with an average marker spacing of 553 bp within the cancer genes (>25 markers per 100 kb).[102] This array is capable of identifying genomic breakpoints at the exonic level throughout the entire genome; however, as with any leukemia sample, the ability to resolve copy-number changes and loss of heterozygosity (LOH) depends on the level of mosaicism (proportion of leukemic cells versus normal cells) within the specimen.

Gunnarsson et al. used their 250K SNP array to analyze a cohort of newly diagnosed CLL patients and to detect clonal evolution in follow-up samples.[16] They identified copy-number aberrations in 90% of these CLL patients at diagnosis, with 70% carrying known recurrent alterations, including del(13q) (55%), trisomy 12 (10.5%), del(11q) (10%), and del(17p) (4%).[16] In addition, they identified a small number of patients with copy number neutral LOH (CN-LOH, also called acquired isodisomy or acquired uniparental disomy) on 13q, which is the loss of all or part of a chromosome, and a doubling of the remaining homologous genetic material to restore genomic balance (see next section for more detail). The ability to detect CN-LOH is a singular feature of SNP arrays, as this phenomenon is invisible by conventional cytogenetic analysis, FISH and most CGH arrays (SNPs have recently been introduced within some CGH arrays allowing CN-LOH detection [BlueGnome's Cytochip™ Cancer; Oxford Gene Technology's Cytosure™ ISCA UPD array]). SNP arrays are able to detect CN-LOH because in addition to measuring copy number, they also provide genotyping information, and can therefore detect diploid stretches of homozygosity within the genome. Acquired CN-LOH can also be detected using PCR-based molecular techniques; one of the first reports of CN-LOH in AML, of the long arm of chromosome 13 encompassing the FLT3 locus, was an incidental finding after using polymorphic microsatellite markers to study chimerism status post stem cell transplantation.[19]

Copy-number Neutral LOH Numerous studies have shown that CN-LOH is a frequent event in leukemia, particularly myeloid malignancy,[20] but one that went mostly unrecognized until SNP arrays were available for routine use. The utility of SNP arrays to detect CN-LOH in AML was initially reported in a study of 60 AML patients using 10K arrays.[21] CN-LOH is regularly identified in patients with a normal karyotype and no other clonal marker, and is particularly common in mixed myelodysplastic syndrome (MDS)/myeloproliferative neoplasm (MPN),[22] with abnormality rates of 48% in chronic myelomonocytic leukemia and 38% in MDS/MPN-unclassifiable cases,[23] although it should be noted that this study did not use paired constitutional DNA to validate somatically acquired changes so these values may be overestimates. Regions of CN-LOH in leukemia often encompass oncogenes or tumor suppressor genes, facilitating duplication of a mutation with concomitant loss of the wild-type allele, but without any genomic imbalance. Furthermore, studies of candidate genes within regions of recurrent CN-LOH identified by SNP arrays have led to the identification of novel mutated genes such as CBL (associated with CN-LOH 11q in myeloid malignancy[23]) and TET2 (linked to CN-LOH 4q in MDS and mixed MDS/MPN[24,25]). SNP arrays have also detected recurrent submicroscopic deletions, which again have enabled the identification of new cancer-related genes in the minimally deleted region, such as PAX5,IKZF1 and CDKN2A in pediatric ALL.[26–28]

Overall, array-based karyotyping increases diagnostic yield when combined with routine metaphase cytogenetics.[3] However, with both aCGH and SNP arrays, detection of unbalanced cytogenetic defects relies on a sufficient number of cells sharing a clonal abnormality, so in cases with a small clonal population, or multiple subclones, standard metaphase cytogenetics may still be the most appropriate technique to use in the investigation of leukemia despite its limited resolution and reliance on dividing cells. A further inherent challenge of array analysis lies in correctly distinguishing somatically acquired, cancer-specific lesions from patient-specific inherited copy-number variations or segments of homozygosity. Copy-number variations are surprisingly frequent and not highly recurrent, making paired studies with matched tumor and germline DNA samples critical for correctly ascertaining somatically acquired variants.[29]

Gene Expression Profiling

Microarrays can be used to study global gene expression, simultaneously measuring the transcriptional activity of thousands of genes. The application of microarrays for classification of leukemia subtypes has been demonstrated in numerous studies, including the multicenter Microarray Innovations in Leukemia (MILE) project involving 11 laboratories worldwide. The MILE study assessed the clinical utility of gene expression profiling (GEP) as a single test to subtype leukemias into conventional categories of myeloid and lymphoid malignancies based upon gene expression signatures associated with distinct clinical subtypes.[30] Over 3000 patients comprising 16 acute and chronic leukemia subclasses, MDS and a 'none of the target classes' control group were profiled on a custom 'AmpliChip Leukemia' and results compared to 'gold standard' diagnostic methods. The AmpliChip Leukemia was able to classify leukemia with a high level of accuracy, was robust, with a high degree of inter- and intra-laboratory correlation, and was able to generate results within 48 h.[30] The authors hope that the MILE study will pave the way to the standardized introduction of microarray technology in the diagnosis and treatment of leukemia, with potential applicability in developing countries that currently lack the expertise to perform more labor-intensive and sophisticated diagnostic approaches.

The first commercially available CE-marked chip for the classification of AML was launched in 2011 by Skyline Diagnostics. The AMLprofiler™ uses distinct gene expression profiles to identify patients with favorable cytogenetic reciprocal rearrangements and CEBPA bialleic mutations, based on the earlier work on class prediction by Valk et al..[31] For example, the presence of t(8;21)(q22;q22) is determined by 31 proprietary gene expression levels, with virtually 100% accuracy. The chip is unique in that it can also directly detect the common NPM1 gene mutation types A, B and D, as well as overexpression ofEVI1 and low expression of BAALC. In this way, a single method replaces three different technologies: cytogenetics, mutation analysis and expression analysis.[103]

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One disadvantage of GEP in the classification of AML is that prognostically significant abnormalities in signaling molecule genes such as FLT3 and RAS appear not to be readily predictable, perhaps on account of their less direct role in transcriptional modulation.[32]Outside class prediction, GEP has the potential to facilitate class discovery, referring to the identification of new subtypes of leukemia by grouping cases according to similar gene expression signatures (often referred to as clustering). Wouters et al. were able to identify a subgroup of patients with an expression profile resembling that of AML with bialleic CEBPA mutations despite these patients not carrying the respective mutations.[33] The subgroup was actually associated with silencing of the CEBPA gene, often due to promoter hypermethylation.[33] GEP also enables class comparison, identifying genes that are deregulated in certain subgroups, which may address biologic questions and facilitate the detection of new molecular targets for therapy.[32,34]

PCR-based Techniques

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The cornerstone of molecular diagnosis in leukemia is PCR. Manipulations of the reaction itself and downstream processing have resulted in a multitude of different techniques; some of the most relevant to the diagnosis of leukemia are described below.

Quantitative Techniques

Reverse-transcription PCR & Reverse Transcription Quantitative Real-time PCR

RNA is a prerequisite for the GEP techniques described above. RNA is also used as the template of choice when studying the molecular counterparts of chromosomal rearrangements (i.e., gene fusions) and for a few other selected applications within the genetics laboratory. This is not necessarily because the test relies on expression per se, but because RNA devoid of introns makes a more convenient template. Translocation breakpoints are usually patient specific within the DNA sequence, although they do have a tendency to cluster within particular regions (usually within certain introns, but occasionally within exons). By using RNA (or cDNA) as the template and locating primers just outside the breakpoint cluster regions, a common set of primers can be employed to detect the fusion gene counterparts of chromosomal translocations in the majority of patients, except those that have rare fusion subtypes with breakpoints outside the common regions (Figure 2). The disadvantage of using RNA is that it is labile; samples need to reach the laboratory quickly and ideally be extracted within 48–72 h to prevent the action of RNase enzymes that degrade RNA.[35]

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Figure 2.

The t(8;21)(q22;q22) translocation resulting in aRUNX1 – RUNX1T1 gene fusion. Breakpoints occur within intron 5 of RUNX1 and within intron 1 of RUNX1T1. To circumvent the need to design patient-specific primers near to the exact breakpoints in the genomic DNA sequence, cDNA (reverse transcribed from mRNA) can be amplified using a common pair of external primers (E) located within exon 4 of RUNX1, and exon 3 ofRUNX1T1. Increased sensitivity for minimal residual disease monitoring can be achieved by performing a second round of reverse-transcription PCR with primers positioned internally (I) to the first-round primers (nested reverse-transcription PCR). Cen: Centromere; Tel: Telomere. Courtesy of Jane Bryon, West Midlands Regional Genetics Laboratory (Birmingham, UK).

End-point reverse-transcription PCR (RT-PCR), where products are assessed after the completion of the amplification reaction, provides a nonquantitative (or if PCR is kept in the exponential phase, a semiquantitative) assessment of the presence or absence of the specific product of interest. This is a useful technique at leukemia presentation to confirm or exclude the presence of a gene fusion: it is quick; sensitive relative to FISH and cytogenetics; does not rely on dividing cells so can potentially be performed on blood samples; can detect cytogenetically and FISH cryptic rearrangements; and may be able to detect rare variants in addition to standard gene fusions depending on the location of the primers. We have been able to identify a total of 19 leukemia patients with rare variant BCR–ABL1,RUNX1–RUNX1T1, CBFB–MYH11 or PML–RARA transcripts using our RT-PCR primers that cannot be amplified using the standard reverse transcription quantitative real-time PCR (RT-qPCR) primers in routine use. These would have been missed if screening at presentation relied on RT-qPCR, with one variant CBFB–MYH11 fusion also being cytogenetically cryptic.[35]

Qualitative end-point RT-PCR can be used for minimal residual disease (MRD) monitoring,[36] but has been largely overtaken by quantitative techniques that allow estimation of disease levels, and in the case of negative results, give an indication of quality and sensitivity to avoid 'false negatives' due to suboptimal samples. RT-qPCR can be performed using various fluorescent chemistries (e.g., DNA binding dyes, TaqMan® hydrolysis probes [Roche], Lightcycler® dual hybridization probes [Roche], Molecular Beacons, Locked Nucleic Acid® probes) and on a range of platforms (such as ABI's 7500/7900/ViiA™ Real-Time PCR systems, Roche's LightCycler®, Bio-Rad's CFX96™ Real-Time PCR Detection System, Cepheid's SmartCycler® and Qiagen's Rotor-Gene® Q). The one thing they all have in common is that the reaction is monitored in real time, with fluorescently labeled products detected and measured in the exponential (also known as log-linear) phase. During this phase, the PCR product doubles after every cycle, assuming 100% reaction efficiency. A fractional PCR cycle early in this phase, when amplification can first be detected above background noise, is used for quantification, known variously as the threshold cycle, crossing point, take-off point or quantification cycle. This value is representative of the starting copy number in the original template; the greater the starting amount, the lower the PCR cycle at which fluorescence (amplification) can first be detected.

There are two main methods of quantification: absolute and relative. Absolute quantitation uses serially diluted standards of known concentrations to generate a standard curve from which the concentration of unknowns based on their quantification cycle values can be determined. With relative quantification, changes in sample gene expression are measured based on either an external standard or a reference sample, also known as a calibrator.[37] Pros and cons of the two methodologies and further detailed information on the design and theory behind RT-qPCR experiments can be found in the excellent publications by Wong and Medrano[37] and Bustin et al. [38]

Fluorescence-based real-time PCR is one of the most widely used methods of quantification because it has a high dynamic range, is very sensitive and specific, and requires no postamplification processing.[37,38] It is commonly used to measure response to therapy and monitor MRD in leukemia.[39] European standardized protocols for the measurement of fusion gene transcripts in acute leukemia and CML by RT-qPCR were published in 2003 and are still in widespread use today.[40] It is beyond the scope of this review to cover molecular monitoring of leukemia, but a wealth of information is available in the published literature on both methodologies and the clinical application of such monitoring.

Real-time PCR can also make use of DNA as a template. Several studies have shown that levels of MRD, measured at critical timepoints in both childhood and adult ALL, significantly correlate with clinical outcome.[41] There are a number of methods available, including identification and subsequent monitoring of clonal immunoglobulin and T-cell receptor gene rearrangements by PCR amplification of DNA, where patient-specific primers are designed complementary to the junctional sequences of the target. Again, European standardized assays[42,43] and consensus guidelines on interpretation of results[44] have been published. Other methods of monitoring MRD in ALL include RT-qPCR for fusion genes, as discussed above, and flow cytometry based on a leukemia-associated immunophenotype.[45]

Quantitative techniques based on real-time PCR have been available for leukemia diagnostics for a relatively long time; other innovative methods of molecular quantification are available, such as multiplex ligation probe amplification (MLPA®; MRC-Holland) and digital PCR, which are discussed below, and pyrosequencing, which is considered in the section on sequencing-based techniques.

Multiplex Ligation Probe Amplification

MLPA is a technique that was initially developed to identify copy-number changes, from whole-chromosome aneuploidy to single exon deletions and duplications,[46] that has since been further enhanced for methylation profiling and mutation detection. MLPA is based on the hybridization and subsequent ligation of two separate oligonucleotide probes specific to immediately adjacent target sequences. Ligation will only occur when both oligonucleotides anneal to the template. At either end of the ligated probe are universal primer sequences, which enable the probe (not the template) to be amplified in a multiplex PCR reaction, and a stuffer sequence of variable length, which facilitates discrimination of different probes based on size, allowing up to 50 targets to be multiplexed. Quantification of probe ligation products (via peak height or area following capillary electrophoresis; see later section titled 'Fragment analysis') provides a measure of the number of target sequences in the sample, and by comparing the peak pattern obtained to that of reference samples, indicates which sequences show aberrant copy numbers. Probe oligonucleotides that are not ligated only contain one primer sequence and as a consequence they cannot be amplified and will not generate a signal. An example of MLPA showing a deletion in NSD1 is shown in Figure 3. Commercial MLPA kits are available for CLL, MDS, intrachromosomal amplification of chromosome 21, ALL and 'hematological malignancies', the latter kit containing probes for several genes and chromosomal regions known to have a significant diagnostic or prognostic role in ALL, AML, MDS, CML and CLL, including 2p23 (ALK), 7p12 (IKZF1), 8q24 (MYC), 10q23 (PTEN), 11q23 (ATM), 12p13 (ETV6), 13q14 (RB1, MIR15A), 17p13 (TP53) and 21q22 (RUNX1) deletions and duplications.[104]

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Figure 3.

Multiplex ligation probe amplification (MLPA®) analysis of the NSD1 gene. The lighter bars (left peak within each doublet) are the normal reference sample with no copy-number change inNSD1. The darker bars (right peaks), representing the test sample, are approximately the same height as the reference sample for all exons except 19, 20 and 21 (highlighted by arrows), revealing a partial NSD1 deletion.

One undesirable feature of MLPA is that a SNP within the probe binding site can prevent hybridization of the probe to the target sequence, and can thus generate a false-positive (apparent deletion) result. This feature has been exploited by the designers of MLPA assays in order that MLPA can also be used for mutation detection by deliberately locating probes at the site of a base change. A kit is due to be launched for 'myeloproliferative neoplasms' that has been designed to detect the JAK2 mutations V617F, E543_D544del and N542_E543del, MPL W515K and W515L, IDH1 R132S and R132C, IDH2R140Q, IKZF1 and EZH2 deletions and FIP1L1–PDGFRA fusions. MLPA is thus one of very few techniques that allows detection of mutations and copy-number changes in a single reaction, and is superior to FISH in being a multiplex technique capable of detecting even single exon deletions or duplications.

Ligation-dependent PCR (LD-PCR), based on a subtle modification of the MLPA technique, has been developed for the quantification of point mutations within the ABL1 kinase domain (AKD) of BCR–ABL1.[47] Such mutations can interfere with binding of tyrosine kinase inhibitors to the BCR–ABL1 protein in patients with CML and Ph chromosome-positive ALL, a major cause of resistance to therapy. Over 100 different amino acids have been shown to be mutated to date, but only a small number of residues account for the majority of resistance-causing mutations. LD-PCR assays were developed for 18 of the most common mutations, including the T315I, which confers resistance to imatinib, dasatinib and nilotinib. LD-PCR has the advantage over some other detection techniques of being able to accurately quantify the proportion of mutant clones. Qualitative detection of an AKD mutation does not necessarily imply impending onset of clinically resistant disease, especially if the clone size is small, but demonstration of expansion of a mutant clone over time can provide evidence of clinical relevance.

Digital PCR

Digital PCR, by array or droplet technology, transforms the exponential, analog nature of PCR into a digital signal suitable for detecting predefined mutations present in a minor fraction of a cell population.[48] Single molecules of the target sequence are isolated by dilution and individually amplified by PCR in multiple individual parallel reactions, so that the resultant PCR products are homogeneous (i.e., completely mutant or completely wild-type). The homogeneity of these PCR products makes them easy to distinguish.

The limit of detection of digital PCR is defined by the number of individual reactions performed. Microfluidic sample handling systems, such as Fluidigm's BioMarkâ„¢ digital array, have been developed, which split one sample into thousands of individual reaction chambers that reside on an 'integrated fluidic circuit'. This miniaturization of the PCR, whereby reactions are performed in nanoliter volumes on the chip compared with milliliter volumes typically used in conventional PCR, provides a significant reagent cost saving. The miniaturized process allows performance of 39,960 individual PCR reactions per chip (48.770 Fluidigm Digital Array IFC[105]). Alternative and more sensitive approaches are offered by Bio-rad's Quantalifeâ„¢ and RainDance RainDropâ„¢ droplet digital PCR technologies. In the latter, millions of emulsified picoliter droplets are generated, which act as individual reaction chambers within the RainDrop Digital PCR System. By combining this technology with differentially labeled probes to mutant and wild-type, quantitative detection of low copy targets with sensitivities in the order of one in more than 1 million can be achieved, and multiplexing is also possible.[106,107]

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Digital PCR shows some promise for MRD monitoring in leukemia, and is adequately sensitive, successfully detecting BCR–ABL1 transcripts in samples with low-level MRD that were not detectable by conventional RT-qPCR.[49] It has also been used to identify and quantify the T315I AKD mutation in CML patients on the Fluidigm BioMark digital array, with a theoretical 1000-fold improvement in detection sensitivity versus conventional PCR, and was able to detect as few as three T315I-mutated molecules in a total background of 100,000 unmutated ABL1 molecules.[50] At diagnosis it may be applicable: for detection of tumor cells present at very low levels relative to a high background of normal DNA, such as KIT D816V-positive mast cells in systemic mastocytosis;[51,52] for detection of low-level subclones harboring key mutations; in the early stages of cancer development; or for detection of circulating tumor cells or cell-free DNA, particularly those derived from solid tumors, present in plasma. One potential example of the latter scenario may be confirmation of a CNS relapse of ALL, where the marrow tests negative.

Mutation Detection

Co-amplification at Lower Denaturation Temperature PCR

A major limitation of PCR-based methods is the inability to selectively amplify low levels of mutations in a wild-type background, as is frequently the case in leukemia. Co-amplification at lower denaturation temperature PCR (COLD-PCR) is an elegant technique for enriching minor populations, and mutation detection sensitivity can be improved by up to 100-fold by replacing regular PCR with COLD-PCR before sequencing or genotyping assays.[53] The cycling conditions facilitate cross-hybridization of mutant and wild-type alleles. COLD-PCR relies on identifying the critical temperature for denaturation of heteroduplexes, and because mutant alleles are in the minority, most mutant alleles will end up in a heteroduplex that has a lower melting temperature than fully matched wild-type or mutant homoduplexes. Heteroduplexes are then selectively denatured and amplified at critical temperature, whereas homoduplexes remain double-stranded and do not amplify efficiently. Li et al., who first described the technique, demonstrated vastly improved sensitivity of existing mutation detection methodologies and identified new mutations in TP53, EGFR and KRAS in tumor specimens simply by replacing regular PCR with COLD-PCR.[54]

Fragment Analysis

Fragment analysis discriminates PCR products based on size and is useful for the detection of mutations, such as insertions, deletions and duplications, and for identifying gene fusion subtypes. The process can be thought of as a more accurate and sensitive form of gel electrophoresis; PCR products are fluorescently labeled and, along with appropriate size standards, loaded onto a capillary filled with polymer and detected within an automated analyzer.

Two of the most common mutations found in AML can be detected in this way. FLT3 internal tandem duplications (FLT3-ITDs) are in-frame insertions of three to approximately 400 bp within the juxtamembrane domain of the FLT3 tyrosine kinase gene,[55] causing constitutive activation of the receptor. NPM1 gene mutations, which usually involve insertion of four bases in exon 12, cause a frame shift in the C terminus, disrupting the nucleolar-localization signal or generating a nuclear export motif, resulting in aberrant cytoplasmic accumulation of NPM.[56] Both types of mutation can therefore be detected by fragment analysis following a simple PCR using primers either side of the commonly duplicated/inserted region (Figure 4). Given that both mutations have prognostic relevance in AML, and it is recommended that both should be ascertained at presentation, many laboratories have combined the two tests into one assay.[57]

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Figure 4.

Fragment analysis for NPM1 insertion and FLT3- ITD mutations. NPM1 alleles on the left; wild-type allele 167 bp, mutant allele 171 bp representing the common type A 4-bp insertion. FLT3alleles on the right; wild-type FLT3 236 bp (cDNA) or 326 bp (genomic DNA), ITD allele 275 bp (cDNA) or 365 bp (genomic DNA), representing a 39-bp duplication. ITD: Internal tandem duplication. Courtesy of Kerry Wall, West Midlands Regional Genetics Laboratory (Birmingham, UK).

Allele-specific Oligonucleotide Analysis

Fragment analysis can be used in conjunction with allele-specific oligonucleotide analysis (ASO-PCR) in the detection of the JAK2 V617F mutation. This point mutation in the JAK2 gene, resulting in constitutive activation of the tyrosine kinase that it encodes, is found in a high proportion of MPN. The rationale behind the ASO assay is that a single nucleotide mismatch at the 3' end of a primer will prevent primer binding and extension. A mutant-specific primer is therefore included in the reaction, which will only bind to an allele that contains a thymine at nucleotide position 1849 in JAK2 (i.e., the V617F mutation), and will not bind to the wild-type sequence with a guanine at that position[58] (Figure 5). Wild-type and mutant products are discriminated by size.

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Figure 5.

Detection of the JAK2 V617F mutation. (A) JAK2 V617F allele-specific oligonucleotide assay. When only wild-type JAK2 is present, the F and R primers bind to the DNA, amplifying a 364-bp product, which can be detected via gel electrophoresis, or if one of the primers is fluorescently tagged, by fragment analysis. The M primer will only hybridize if the V617F single nucleotide substitution mutation (c.1849G>T, indicated by white diamond) is present, in which case a 203-bp amplicon is generated in addition to the 364-bp control fragment. (B) Fragment analysis trace showing a patient with the JAK2 V617F mutation using the ASO assay. F: Forward; M: Mutant; R: Reverse; V617F: Mutant allele; WT: Wild-type allele.

In 2011, a point mutation in the BRAF gene was reported to be present in all cases of hairy cell leukemia (HCL), and absent from other B-cell neoplasms.[59] HCL can be difficult to differentiate from HCL-like disorders such as splenic marginal zone lymphoma and HCL variant, and in addition, and in contrast to other chronic B-cell leukemias, HCL cells circulate at low percentages in the blood.[1] A genetic test is therefore very useful for accurate diagnosis. The BRAF V600E mutation, the disease-defining genetic event in HCL, is easily detected using ASO-PCR at diagnosis, and in HCL samples containing as few as 0.1% leukemic cells from patients after therapy in complete flow-cytometric remission.[60]

Amplification Refractory Mutation System

Amplification refractory mutation system (ARMS) is similar to ASO-PCR, exploiting the fact that oligonucleotide primers must be perfectly annealed at their 3' ends for PCR to take place. In ARMS, two primers are designed to anneal to the sequence/nucleotide of interest, one specific to the mutant sequence at the 3' end, and one specific to the wild-type sequence at the 3' end. In the detection ofJAK2 V617F, this enables homozygotes to be distinguished from heterozygotes, which may be clinically relevant.[61]

Restriction Enzyme Digestion/Restriction Fragment Length Polymorphism Analysis

Restriction enzyme digestion can also be combined with fragment analysis to detect specific point mutations, using fluorescently labeled primers for the PCR step. Following PCR, an enzyme is chosen that will cut mutant sequence but not wild-type, or vice versa, and the products are electophoresed to separate digested from intact amplicons. Care must be taken to avoid false-positive or -negative results caused by incomplete digestion. Again this method is commonly employed for the detection of JAK2V617F, where the mutation abolishes the BsaXI recognition site, which will therefore only cleave wild-type JAK2 sequence.[62]

Other Mutation Detection Techniques

Often a technique is required that will scan for multiple/unknown mutations in a gene, rather than a single specific mutation. Traditional techniques such as single-strand conformation polymorphism analysis, denaturing gradient gel electrophoresis and denaturing high-performance liquid chromatography analysis have been largely surpassed by high-resolution melt analysis (HRM) in many diagnostic laboratories. HRM is sensitive, inexpensive, rapid, simple to perform and downstream analysis is performed in a single tube immediately following PCR.

High-resolution Melt Analysis

HRM characterizes PCR products based on their denaturation (melting) behavior. Samples can be discriminated according to their sequence, length or methylation status (following bisulfite treatment), and the resolution is so good that even SNPs can be readily identified. The PCR is performed in the presence of an intercalating dye, such as LCGreen or EvaGreen, which binds to double-stranded DNA and fluoresces. Once cycling is complete, the PCR products are heated to approximately 95oC whilst being monitored on an HRM-enabled real-time PCR instrument. As the double-stranded product denatures, the fluorescent dye is released and the fluorescence profile changes. This is detected as a sequence-specific melt curve, and subtle differences in melting behavior from the normal wild-type product can be easily distinguished. Sometimes mutations produce characteristic profiles and are thus recognized without further analysis, but often a second technique, such as sequence analysis, will need to be performed to define the exact nature of the change.

HRM has been used to detect mutations in exon 12 of the JAK2 gene, which are associated with the MPN polycythemia vera. HRM was chosen by one group to replace the individual allele-specific PCR reactions that had been developed for some of the recognized exon 12 mutations, in the knowledge that additional mutations of exon 12 had since been identified and novel mutations may exist.[63] HRM proved to be a more generally applicable diagnostic assay, capable of detecting at least seven different types of exon 12 mutation, including duplications, with a sensitivity as low as 5% for some mutations.

An HRM assay for detection of BRAF V600E mutations in HCL has been described recently. This assay was able to detect the mutation with 100% specificity, when hairy cells were present at only 5–10% in a sample.[64]

MALDI-TOF

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MALDI-TOF is a form of mass spectrometry specially designed for the analysis of biomolecules, such as nucleic acids, that can fragment when ionized by more conventional ionization methods. A laser is fired at the matrix, which absorbs the laser energy and transfers protons to the analyte molecules, thus charging the analyte. The mass of the molecule is calculated with great accuracy, which in turn can enable the sequence composition of the nucleic acid to be determined based on the molecular weights of the individual bases, and can also reveal the methylation status of the molecule. MALDI-TOF has a sensitivity limit of approximately 5–10% for low-level mutation detection. This technique has been used to identify novel AML subgroups based on distinct DNA methylation patterns, and in turn to generate a methylation-based outcome predictor, supporting the use of genomic methylation markers for improved molecular classification and prognostication in adult AML.[65]

Sequencing Techniques

The gold-standard method of molecular analysis is to sequence the nucleic acid to determine the exact base composition and characterize changes at single nucleotide resolution. Sanger sequencing on capillary sequencers has been the dominant technology for two decades and was the workhorse of the human genome project. A major drawback, especially with regard to leukemia diagnostics and minor cell populations, is that it can only reliably identify mutations when the fraction of mutated alleles is greater than 10–20%, although COLD-PCR (described earlier) can be used to enhance this sensitivity. Sanger sequencing, being such a prevalent and familiar technique, will not be discussed further, but pyrosequencing and next-generation sequencing techniques are described in some detail below.

Pyrosequencing

In contrast to Sanger sequencing, which is based on dye terminator chemistry, pyrosequencing involves sequencing by synthesis, and relies on the ultimate detection of pyrophosphate release on nucleotide incorporation, which as a result of a chemical cascade is converted to light. Nucleotides are sequentially added to the reaction, and light is produced only when the nucleotide complements the first unpaired base of the template (Figure 6).

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Figure 6.

Pyrosequencing reaction: chemiluminescent cascade to detect incorporation of nucleotides according to the sequence of the template strand. dGTP: Deoxyguanosine triphosphate; PPi: Pyrophosphate.

Advantages of pyrosequencing include the quantitative measurement of alleles, which is not reliable with Sanger sequencing, simplicity and speed, as sequence data are available in real time without an additional post-PCR analysis procedure. Pyrosequencing is also mutation tolerant; unlike hybridization-based assays, pyrosequencing analysis generates a correct sequence regardless of the appearance of new, unexpected mutations.[108] One of the drawbacks is the maximum read length of only 50–60 bases, making it best suited to targeted analysis of regions of interest within genes, rather than sequencing of whole genes.

Pyrosequencing has been used for detecting mutations in some of the genes already mentioned, such as JAK2 V617F[62] and BRAF V600E,[66] and KIT D816V, where the quantitative nature of the pyrosequencing assay proved to be advantageous in aiding better disease classification.[67] It has also been developed for the detection of AKD mutations conferring tyrosine kinase inhibitor resistance in CML and ALL. Pyrosequencing is useful in this context because whilst being more sensitive than Sanger sequencing, it has the added advantage of quantifying mutant alleles, which may provide important clinical information on drug response.[68]

Next-generation Sequencing

Pyrosequencing is just one of a number of methodologies that have been further developed for extremely high-throughput sequencing, techniques known collectively as next-generation sequencing (NGS) or massively parallel sequencing. These technologies promise to transform molecular medicine, with the potential to facilitate the cost-effective sequencing of entire genomes in a matter of hours.

NGS holds great promise for the study of leukemia, and is already moving into clinical diagnostics; it has been suggested that this technology has the potential to eventually replace all other genetic analyses at diagnosis.[29] NGS has the ability to fully sequence thousands of genes in a single test and simultaneously detect deletions, insertions, base substitutions, copy-number alterations and translocations, including balanced rearrangements.

There are a number of different platforms for NGS on the market using different chemistries, each with their own advantages and disadvantages, and it is beyond the scope of this review to describe them in detail. The interested reader is directed to the excellent reviews by Shendure and Ji,[69] ten Bosch and Grody,[70] Metzker[71] and Su et al..[72] Natrajan et al. [73] and Ross et al. [74] have recently reviewed the application of NGS to cancer diagnosis and prognostication.

One of the first cancer genomes to be sequenced using NGS was that of an AML patient with an apparently normal karyotype.[75] The AML genome was sequenced to a depth of >30-fold coverage, and matched normal DNA from the patient's skin was used to exclude almost 98% of variants found from further study on the basis that they were considered to have been inherited. The AML genome contained ten nonsynonymous somatic mutations thought to be pathogenetically relevant; two were known recurrent mutations in AML (FLT3-ITD and NPM1 4-bp insertion), and eight were new mutations, all single base changes, as yet undescribed in AML yet present in virtually all tumor cells at presentation and relapse. Half of the affected genes were already associated with cancer pathogenesis, but not AML, and the other four somatic mutations occurred in genes not previously implicated in cancer. All eight genes are now the focus of further study. A further 500–1000 additional noncoding and nongenic somatic variants were identified in the AML genome, some of which may in time also prove to be of clinical significance. Whole-genome sequencing may thus be the means for discovering all of the mutations that are relevant for cancer pathogenesis.

The authors of the above study sequenced a second AML genome and investigated whether any of the variants identified were recurrent in additional AML tumors, which in the absence of functional validation is considered to be a good test of the relevance of individual mutations.[76] This time, 12 nonsynonymous mutations were identified as most likely to be relevant for pathogenesis, since they could potentially alter the function of expressed genes. Three of these mutations were found in some of the other AML samples, including mutations in NPM1 and NRAS (already associated with AML) and the glioblastoma-associated cancer gene IDH1, which encodes a metabolic enzyme. The IDH1 mutation was identified in 16% of samples from patients with cytogenetically normal AML, and has since been confirmed to be a new recurrent mutation with potential prognostic significance in AML,[77]demonstrating the potential of an unbiased sequencing approach to discover previously unsuspected recurring mutations in cancer. Recurrent acquired mutations in DNMT3A, a de novo DNA methyltransferase, have also been identified in AML using NGS.[78–80]

NGS has since been applied to the sequencing of eight relapsed AML genomes to ascertain the mutational spectrum associated with relapse.[81] This study revealed more novel, recurrently mutated genes in AML and also found two major clonal evolution patterns associated with AML relapse: either the founding clone in the primary tumor gains mutations and evolves into the relapse clone; or a subclone of the founding clone survives initial therapy, gains additional mutations and expands. The same group, using a similar study design, have also looked at the clonal evolution of secondary AML from antecedent MDS to identify the genetic changes underlying progression.[82] Other examples of NGS used to investigate the mutational spectrum of leukemia include those of Puente et al. [83] and Wang et al. [84] in CLL.

The development of NGS strategies as diagnostic tools will lead to some novel interpretational problems. Sequence variations that differ from reference sequences will need to be defined as either germline or somatically acquired; and then it must be determined whether the acquired change is likely to be a contributing oncogenic event (driver mutation) or a passenger event. For the former, parallel analysis of germline DNA may be required to determine the somatic changes – a reasonable option technically as NGS allows DNA barcoding of each sample. The latter is in part determined by known recurrent oncogenic mutations, but will increasingly require the development of functional assays, such as that designed for FLT3 mutations,[85] and bioinformatics resources to provide annotated information on the significance of each event. In addition, the expansion into analysis of multiple genes will increase the likelihood of detecting significant germline mutations in the gene involved with implications for predisposition to cancer which can extend to other family members who may also carry the mutation.

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NGS heralds a new era of molecular medicine and has great potential in the diagnostic setting for a heterogeneous disease such as AML, where it could facilitate the detection of all prognostically relevant mutations with a single test, which will in turn enable better classification of AML and a personalized approach to treatment. NGS is already being used to detect insertions, deletions and point mutations of CEBPA with 397-fold to 1194-fold depth of coverage.[86] An international consortium has assessed the robustness, precision and reproducibility of NGS for investigation of TET2, CBL and KRAS mutations in chronic myelomonocytic leukemia in the clinical laboratory setting.[87] The study was designed to test the utility of amplicon deep sequencing, where multiple genes or hotspot regions are sequenced in multiple patients in a massively parallel fashion. A median of 500-fold coverage per amplicon was achieved, and the authors concluded that the technique shows clinical applicability, with high concordance across multiple centers, and improved sensitivity compared with Sanger sequencing (1–2 vs 20%). While NGS makes sequencing of a limited repertoire of relevant genes at very high sensitivity possible, it can also be used to sequence the following: whole genomes, such as the examples of the AML genomes mentioned above; whole exomes, thus concentrating on the coding regions most likely to be of clinical relevance; or even whole transcriptomes. Exome sequencing is unable to detect most structural variants, such as chromosomal translocations with intronic breakpoints, so may be of limited utility in leukemia, whereas transcriptome sequencing can detect fusion transcripts produced by chromosomal rearrangements and also provides quantitative information about gene expression levels.[88]

Expert Commentary

Since the success of the Human Genome Mapping Project, knowledge of the underlying genetic alterations driving leukemia has expanded rapidly. A diverse range of genetic mutations has been identified, building on previous knowledge derived initially from cytogenetic analysis acting as a surrogate marker for gene mutations and rearrangements. In addition to gene fusions, amplification and deletion, we now have lists of genes with aberrant expression or mutations. Mutations can be activating or inactivating, by direct mutation or by epigenetic promotion or silencing. Mutations can be single base changes found at multiple sites across the majority of a gene (usually inactivating) or very specific mutations at a single codon (usually activating), and involve base changes, deletions, insertions and duplications. The molecular analysis of these events is being successfully delivered by a range of technical approaches – which continue to evolve – but the challenge remains of how to deliver ever-increasing numbers of relevant mutation tests on each patient at an affordable cost in a clinical timescale. Such growth of testing needs is unsustainable with the technologies commonly in use in most laboratories – hence we will need to look to more recent or new technologies to help us deliver testing on the scale likely to be required.

Each newly identified gene and/or mutation will need to be assessed for associations, prognostic significance and potential suitability to direct targeted therapies. The role of sample archives from clinical trials will be of great importance in allowing rapid retrospective studies on patient cohorts with defined treatments and outcome data.

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There is a trend led by the pharmaceutical industry towards targeted therapies with approved specific 'companion diagnostic' tests, but this is not the future. As illustrated by the discussions above, there are many technological approaches to achieving the same result, which is often a simple sequence change in the DNA, and a need to develop ever more informative and cheaper technologies to deliver these results. The technology used to deliver the companion diagnostic information should not be fixed because this will inhibit development of better or cheaper strategies. However, robust mechanisms for external quality assessment to validate a laboratory's ability to deliver the results will be required. The importance of quality control and standardization should not be underestimated; comparable assay results from all laboratories performing a test for the same analyte are critical for interpreting results and making clinical decisions. Where they exist, this can be accomplished through: adherence to 'best practice guidelines' and formulation of standard operating procedures; the use of quality-marked commercially available assays (in preference to 'in-house' methods); use of reference materials; and participation in external quality assurance schemes. Arguably the most important features for reassuring users that a clinical laboratory can produce reliable results are its accreditation and that its staff be registered with statutory bodies, wherever this is possible. Further information about standardization can be found in a previous edition of this journal in an article by Holden et al. [89]

Five-year View

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The potential of NGS is so great that in 5 years we can expect to see most molecular diagnosis in leukemia to be delivered through NGS technology, replacing today's multiple single-test approach with gene mutation panels providing multiplex, multigene, multipatient analysis (Patel et al. have recently published an example of how this 'integrated genetic profiling' approach could be applied for risk stratification in AML,[90] which is further discussed in the accompanying editorial[91]). Whole-exome or whole-genome sequencing will be viewed on a rapidly approaching horizon. Layered on top of sequence-based gene mutation analysis will be panels for gene fusions, expression of key genes and epigenetic markers. The costs of the technologies involved will have reduced so that such an approach is technically affordable. Such screening will produce genetic markers that will allow effective residual disease monitoring in all patients. The changing technologies will support the increased use of targeted therapies, the monitoring of responses to therapy and improved outcomes for patients.