We have demonstrated the feasibility of using SNPs and high-density oligonucleotide arrays in genome-wide screening for allelic imbalance in human tumors. The SNP array used here yielded ~150 informative loci per patient, comparable with the number of STRs used in current genome-wide LOH screens. For example, in 17 different genome-wide allelotype studies conducted over the past 5 years, the average number of STRs used was 120, and the study using the largest number of loci for LOH analysis included 280 STR polymorphisms (Field et al. 1995
; Hahn et al. 1995
; Takeuchi et al. 1995
; Califano et al. 1996
; Johns et al. 1996
; Tamura et al. 1996
; Baccichet et al. 1997
; Boige et al. 1997
; Gleeson et al. 1997
; Kawanishi et al. 1997
; Mori et al. 1997
; Chambon-Pautas et al. 1998
; Hatta et al. 1998
; Piao et al. 1998
; Shih et al. 1998
; Mao et al. 1999
; Yustein et al. 1999
). However, for prognostic and diagnostic utility, genome-wide analysis will require a greater number of SNP markers that are more evenly distributed throughout the genome. In addition, because of the lower average heterozygosity rate of SNPs (0.33) compared with STRs, approximately three times the number of SNPs are required for an equivalent resolution (Kruglyak 1997
). Higher density SNP arrays should greatly increase the ability to detect small regions of chromosomal changes and will provide more information regarding the boundaries of loss regions. In addition, more markers increase confidence in a detected event: If multiple adjacent SNPs all show a consistent change, the confidence in the call is much higher than if it is based on only a single SNP. It is clearly feasible to increase the density of SNP markers as SNPs are abundant in the human genome and SNP discovery and mapping is rapidly advancing (Wang et al. 1998
; Cargill et al. 1999
; Halushka et al. 1999
). Because the array-based readout is parallel and scalable, larger numbers of markers can be assayed simultaneously without significant increases in time or labor.
SNP arrays have many advantages for LOH detection compared with traditional techniques. The PCR products containing SNP loci are typically smaller and more readily amplified in parallel than with STRs, and may be better for amplifying DNA from formalin-fixed or compromised tissues. Also, the amount of cellular DNA required to interrogate a SNP on an array is significantly less than that required for standard STR analysis, providing an opportunity to evaluate limited clinical samples.
Surgically removed tumor tissues often contain some normal cells that can interfere with the detection of changes in tumor cells. Therefore, it is important to be able to detect chromosomal changes in heterogeneous samples in which the tumor cells may represent only a portion of the sampled cell population. We simulated a heterogeneous cell population by preparing a mixture of purified aneuploid DNA with normal control DNA from the same patient. With the SNP arrays, we were able to detect chromosomal changes in heterogeneous samples, and changes can be clearly and reproducibly identified in samples with a background of up to 50% normal DNA (). As described previously, high sample purity is required to distinguish true LOH from other types of allelic imbalance because of the confounding effects of normal cell contamination (Barrett et al. 1996
; Boige et al. 1997
; Paulson et al. 1999
). Our mixing experiments reinforce the importance of working with purified samples to distinguish between true LOH and other mechanisms of allelic imbalance.
At present the SNP-based method cannot distinguish between loss and gain of alleles. With higher density SNP arrays, it may be possible to use signal intensity differences between tumor and normal samples to indicate chromosomal loss or gain. In a recent study, 3360 mapped cDNAs were used in a microarray hybridization assay (Pollack et al. 1999
). This technique provides an approach for the detection of DNA copy number changes, which is complementary to a SNP-based method that detects changes in allelic representation.
The identification and mapping of additional SNP markers is rapidly advancing, and array-based methods provide a scalable approach to the simultaneous genotyping of thousands of markers in parallel. The availability of more markers and higher capacity array designs will allow efficient, genome-wide, high-resolution searches for chromosomal changes associated with tumor initiation and progression. The patterns of chromosomal alterations may be useful for diagnostic purposes and to follow disease progression and guide patient care.