Buried treasure

What determines whether colorectal cancer will recur after surgery? Duke’s system for histopathological staging has been the mainstay of colorectal cancer prognosis for many years. But patients with tumours of the same Duke’s stage can have radically different outcomes. Now, in the 19 January issue of The Lancet, Bert Vogelstein’s group describe a new molecular technique called ‘digital single-nucleotide polymorphism’ (SNP), which they use to see how allelic imbalance of chromosomes 8p and 18q within tumour cells relates to disease progression. These chromosomes have previously been shown to exhibit allelic imbalance in colorectal cancer, but their relevance to disease progression is not understood. The first step is to find an informative SNP on these chromosomes — one for which the patient is heterozygous — using DNA purified from normal tissue at the tumour margin. Once an informative marker has been found, purified DNA from microdissected tumour tissue is plated out robotically in 384-well plates at low concentrations, so that an average of one molecule containing the SNP lands in each well. After polymerase chain reaction (PCR) amplification, fluorescent probes that can distinguish the two alleles are used to count each allele one by one. Statistical algorithms are then used to determine whether there is an allelic imbalance in the sample. By this mechanism, the proportion of each allele in a patient’s DNA can be assessed. This technique is more accurate than traditional methods of measuring allelic imbalance, which use microsatellite markers, in part because the PCR products obtained are identical in size, eliminating any artefacts caused by preferential degradation of larger alleles. The authors studied 180 patients who had earlystage sporadic colorectal cancer with no sign of metastases. Digital SNP was used to test for imbalances of chromosomes 8p and 18q in surgically removed tumours. Tumour samples were divided into those with allelic imbalances in both chromosomes, those with imbalances in either (but not both) chromosome, and those that showed no imbalance at all on either chromosome. The five-year disease-free survival was remarkably different in the three groups: 100%, 74% and 58% in tumours with no allelic imbalance, imbalance in one chromosome, and imbalance in both chromosomes, respectively. These results were independent of other variables, including Duke’s stage. So, a higher allelic imbalance correlates with a lower probability of disease-free survival. How, then, does allelic imbalance affect prognosis? The authors propose three models. First, important tumour-suppressor genes on chromosomes 8p and 18q are lost. Second, allelic imbalance in these chromosomes might simply reflect instability within the cell as a whole. And third, if all colorectal cancers accumulate allelic imbalances at similar rates, allelic imbalance would simply represent a measure of how long the tumour has existed. Although the authors do not favour a particular model, previous studies have correlated allelic imbalances in at least nine chromosomes with prognosis in colorectal cancer, which indicates that aneuploidy in general, rather than loss of specific tumour suppressors, might drive tumour progression. Similar correlations have been made for other cancers. So perhaps we’ll eventually be able to use the same digital SNP-based assay to predict outcomes in every cancer patient. Sandra Clark