In most clinical applications, either total or mononuclear cells are used as a source of DNA. In general, SNP-A and CGH-A show a good concordance with metaphase cytogenetics for detection of previously known unbalanced chromosomal defects and may also allow for identification of lost chromosomal material, for example, metaphase-detected monosomy 17 may in fact represent a pure deletion of 17p alone as the 17q material may be translocated to other chromosomes and escape detection by traditional means (Jasek et al, 2008).
For clinical karyotyping platforms, increasing probe density to 250–500 K does not appear to result in a higher detection rate for microdeletions or UPD (Mohamedali et al, 2007); when Affymetrix 6.0 (Affymetrix, Santa Clara, CA, USA; over 900 K SNP probes) and 250 K arrays were compared, there was remarkable concordance in the diagnostic yield and additional defects were found using higher density arrays (Huh et al, 2008). Thus, very high-density arrays add significant cost but may provide only a marginal (if any) diagnostic gain. However, due to a more even/dense distribution of probes, they enable a more precise mapping of chromosomal defects and CNVs.
The diagnostic rules for clinical cytogenetic diagnosis and investigative applications of arrays may vary, but in both applications, the distinction of artefacts and germ line changes from somatic clonal lesions is of great importance. In constitutional CGH-A or SNP-A, analysis to validate aberrations by alternative approaches, such as FISH, is usually required. Confirmation of novel somatic chromosomal defects detected by array technologies may be important, but this requirement may depend on the size of the defect location. We proposed that if metaphase cytogenetics and SNP-A show a concordant result with regard to a defect, no further analysis of germ line lesions are needed for that particular site (Fig 3, lower panel). In addition, in the case of a non-informative metaphase cytogenetics examination, very large recurrent deletions or gains are unlikely to be constitutional. Should array karyotyping reveal microdeletions and gains not detectable by metaphase cytogenetics, the diagnostic algorithm needs to include comparison with databases of known CNVs and internal control samples; such changes can be excluded without a need for testing germ line DNA. Unfortunately, the available reference databases contain only limited sets of controls. It is likely that, with time, such resources will become available but in the meanwhile a laboratory-generated database of controls may be helpful. Some of the currently available software packages provide information about the degree of overlap with known CNVs but the frequency of each CNV in the general population is difficult to assess. Depending on the size of the control cohort, CNVs with a finite frequency can be identified and excluded. The remaining defects, if diagnostically significant (e.g. due to their location in important chromosomal areas), should be confirmed as somatic.
Results of clinical application
Recently, the application of array-based karyotyping technologies has been described for various haematological malignancies (Table II). These studies utilized arrays with increasing densities as they became available, including 10, 50, 250, 500 K and Affymetrix 6.0 arrays containing almost two million SNP and CNV probes as well as various densities of BAC or oligonucleotide CGH arrays. Target diseases included multiple myeloma (MM) (Gutierrez et al, 2004; Walker et al, 2006), chronic lymphocytic leukaemia (CLL) (Tyybakinoja et al, 2007a), acute lymphoblastic leukaemia (ALL) (Mullighan et al, 2007), acute myeloid leukaemia (AML) (Fitzgibbon et al, 2005; Raghavan et al, 2008; Tiu et al, 2008), MDS (Gondek et al, 2007a,b; Mohamedali et al, 2007) and myeloproliferative syndrome (MPD) (Gondek et al, 2007c) (Table II). Overall, it can be concluded that the addition of array-based karyotyping increases diagnostic yield when combined with routine metaphase cytogenetics (Huh et al, 2008). However, due to the established value of traditional karyotyping, its ability to resolve balanced translocations and a proportion of cases in which array-based approaches fail to identify the lesions detected by metaphase cytogenetics, both traditional and array-based karyotyping should be combined for a comprehensive analysis of the malignant genome rather than replace metaphase analysis with SNP-A or CGH-A-based cytogenetic technologies (Huh et al, 2008; Tiu et al, 2008).
In general, the results obtained in clinical applications so far demonstrate a larger complexity of chromosomal defects than judged by metaphase cytogenetics due the presence of previously cryptic lesions. SNP-A also resolved cases in which metaphase cytogenetics was unsuccessful and the overall detection rate of karyotypic aberrations was higher by array analysis. The most common newly identified defects included small deletions and gains of chromosomal material but in a minority of studies, a stringent distinction of somatic defects from CNVs has been provided. Of particular importance are recurrent lesions such as microdeletions involving specific genes present in patients sharing similar phenotypic features. Examples of such defects include microdeletions in 11q spanning CBL (Dunbar et al, 2008), 21q spanning RUNX1 (AML1) or microdeletions in chromosome 4q involving TET2 (Jankowska et al, 2009), among many others under intense investigations.
Acquired somatic UPD is a new type of chromosomal lesion frequently identified by SNP-A (Fig 2). Principally, the pathophysiology of UPD may include various mechanisms that are frequently associated and shared with deletions. Acquired somatic UPD may lead to the duplication of an activating somatic mutation or homozygosity for a disease-prone minor allele present in the germ line DNA. UPD can also result in increased or decreased gene expression due to duplication of a methylation pattern. A similar mechanism may operate in deletion with loss of the inactivated or unmethylated allele. In addition, deletion may result in haploinsufficiency or loss of the intact allele with the remaining allele deficient due to somatic polymorphism or an inactivating somatic mutation.
The first detection of recurrent acquired UPD in haematological disorders was in polycythaemia vera (PV) (Kralovics et al, 2002), which enabled the subsequent discovery of the JAK2 V617F mutation (Kralovics et al, 2005). However, systematic studies revealing a high frequency of acquired somatic UPD involving various chromosomes and precise mapping of regions affected were not performed until SNP-A became available. The utility of SNP-A arrays to detect UPD in AML was initially reported in a study of 60 AML patients using 10 K arrays (Raghavan et al, 2005). SNP-A also enabled the very efficient identification UPD9p (Gondek et al, 2007c; Yamamoto et al, 2007). By analogy to this lesion, UPD in other areas of the genome can indicate the presence of activating mutations in important genes, e.g. homozygous MPL mutations and UPD1p (Szpurka et al, 2009); homozygous mutations in CEBPA (UPD19q) (Fitzgibbon et al, 2005) or UPD13q and FLT3 mutations (Raghavan et al, 2005) (Table III). Recently, UPD11q was shown to harbour biallelic missense mutations in CBL (Dunbar et al, 2008). Similarly, inactivating mutations in a homozygous constellation were seen involving NF1 in patients with juvenile myelomon cytic leukaemia (JMML) with UPD17p (Flotho et al, 2008) and TP53 in AML patients with UPD17q cooperating with deletions of chromosomes 5 and 7 (Jasek et al, 2008) (Table II). The presence of homozygous mutations, such as in cases of AML with FLT3-ITD, may be associated with worse prognostic features as compared to heterozygous genotypes (Whitman et al, 2001).
Various studies have attempted to investigate the impact of CGH-A and SNP-A-identified lesions on various clinical outcomes (Mohamedali et al, 2007; Gondek et al, 2008; Starczynowski et al, 2008). Due to the complexity of karyograms and large number of areas involved, such analyses will require very large cohorts of patients. We have described how UPD7 shows a similarly poor prognosis as deletions of the corresponding regions. Similarly, UPD17q (Jasek et al, 2008) and UPD11q (Makishima et al, 2008) have poor prognosis and, if occurring in combination with otherwise known low risk defects, determine the overall prognosis. In a recent series of patients with AML who were serially tested, those who showed residual chromosomal abnormalities following remission induction had shortened relapse-free survival (unpublished observations). Similarly, serial testing also demonstrated that progression to more malignant phenotype is associated with appearance of new additional defects (unpublished observations).