Our findings support the use of an unbiased sequencing approach to discover previously unsuspected, recurring mutations in a cancer genome. With improved sequencing techniques, we covered this genome more completely than the first one we sequenced (98% vs. 91% diploid coverage) and used fewer sequencing runs (16.5 vs. 98), resulting in a dramatically reduced cost of data generation. With better data quality and calling algorithms, we reduced the 96% false positive frequency of possible mutations for the first sequenced AML genome to a frequency of 47% of the high-confidence tier 1 and 2 mutations called in this genome. We predicted 1458 tumor-specific point mutations with high confidence; we tested 116 of these with validation sequencing and confirmed 61 of them (53%). Thus, this genome may contain approximately 750 somatic point mutations. We detected mutations in NRAS, NPMc, and IDH1 and a tier 2 mutation on chromosome 10 in more than one AML genome, suggesting that these mutations are not random and are probably important for the pathogenesis of this tumor.
We suggest that the 12 nonsynonymous mutations are the most likely to be relevant for pathogenesis, since they could potentially alter the function of expressed genes. Consistent with this idea and with the results of our previous study18
is the finding that all these mutations were retained in the dominant clone. Surprisingly, we found that virtually all the 52 tier 2 mutations were also present in nearly every tumor cell in the sample, suggesting that they are also a part of the same dominant clone. However, one cannot conclude that these mutations (or any of the tier 3 or 4 mutations) are relevant for pathogenesis simply because they are found at a high frequency in the dominant clone. It is more likely that most of these mutations are random, benign sequence changes that existed in the hematopoietic cell that was transformed (i.e., they were preexisting and carried along as benign “passengers,” irrelevant for pathogenesis). The finding that the percentage of mutations found in each tier closely approximated the total amount of DNA assayed in that tier supports this hypothesis. Collectively, these data suggest that the vast majority of the mutations that we detected in this genome are random, background mutations in the hematopoietic stem cell that was transformed.27
Functional validation will be required to prove which mutations are truly important.
The best test of the relevance of individual mutations for pathogenesis (in the absence of functional validation) is recurrence in other AML samples or other cancers. Of the 12 tier 1 mutations, 3 (occurring in NPM1, NRAS
, and IDH1
) were recurrent in patients with AML and therefore were likely to be important in the pathogenesis of this tumor. R132 mutations in the IDH1
gene had not previously been detected in the 45 patients with AML who were tested23
and are detected only rarely in tumor types other than malignant gliomas.22,24
R132H, C, and S mutations dramatically reduce the catalytic activity of the IDH1 enzyme; it has been suggested that IDH1 is a tumor suppressor that is inactivated by dominant mutations in R132.28
There are significant differences, however, between the IDH1
mutations found in gliomas and those in AML. We detected the R132C mutation in 8 of 16 patients with AML who carried an IDH1
mutation (50%). In contrast, the mutation was reported in only 7 of 161 patients with gliomas (4%, P<0.001 by Fisher’s exact test). The most common mutation in gliomas (R132H) was detected in 142 of 161 patients (88%) but in only 7 of 16 patients with AML (44%, P = 0.13). When the R132H mutation was overexpressed in a glioblastoma cell line, induction of messenger RNAs for several target genes of hypoxia-inducible factor 1α (HIF1α) was detected (GLUT1, VEGF
, and PGK1
However, in 13 patients with AML — 5 with R132H and 8 with R132C — there were no significant alterations in the expression of any of these genes (Fig. 3 in the Supplementary Appendix
Assuming that the number of point mutations in most AML genomes is similar to the number in the first 2 patients we studied (approximately 750), the likelihood that 2 of 188 patients will carry an identical mutation at the same position in the genome is extremely small (1.1×10−9). This suggests that the tier 2 somatic mutation at position 108,115,590 of chromosome 10 is unlikely to be a random event. It falls in a conserved region with regulatory potential, and its detection in a second patient with AML suggests that this region may contribute to pathogenesis through a novel mechanism that remains to be defined.
Although the potential of next-generation sequencing platforms for uncovering the genetic rules of cancer is great, the sequencing of thousands of additional cancer genomes will be required to fully unravel this complex and heterogeneous disease.29,30