We report the results of a large integrated analysis of SNP array screening and gene expression profiling of the CLL genome. Significant advantages of our dataset include the use of an extremely high resolution platform and comparison to matched germline DNA, allowing clear determination of somatic events and filtering of previously undescribed germline CNVs. We find that CLL is quite genomically stable compared with most solid tumors, with a median of only 1 CNA per genome in stably untreated patients, often 13q deletion or trisomy 12. This estimate is lower than earlier studies without matched germline controls(4
), but similar to more recent studies that included matched germline analysis(5
). The genomic stability of indolent CLL is unsurprising given that many cases display a benign course with minimal progression for years.
We also found that an increasing number of somatic CNAs was predictive of short TTFT, as previously reported(8
). This finding was true in the entire cohort and in those lacking 17p and 11q deletions, suggesting that increasing CNAs is independently associated with short TTFT. Similarly, additional CNAs were the major predictor of short TTFT in the context of 13q deletion. The size of 13q deletion and whether it was mono- or bi-allelic have both been reported to have prognostic significance, but in this cohort neither feature was predictive. The most important predictor of TTFT was the presence of any additional somatic CNA defined by SNP array. Our data are therefore an extension of Doehner and colleagues’ original observation using FISH(1
) but our conclusions are based on a much higher resolution platform, and thus may allow more definitive prognostic prediction.
Although the number of CNAs has prognostic significance, it remains likely that specific recurrent CNAs may target genes important in CLL pathogenesis. We were therefore interested in identifying other genomic regions targeted recurrently in CLL and found three that were significantly associated with requiring therapy in our dataset: amplification of 3q26 focused on PIK3CA
, amplification of 8q24 focused on the known GWAS cancer risk region near MYC
, and 8p loss. All three broad chromosomal regions have been reported previously, but the targets of these broad events in CLL were not previously hypothesized. The very high resolution platform used here allowed us to identify very focal CNAs that suggest likely targets in two of these cases, PIK3CA
. In fact, a recent study by Beroukhim and colleagues that catalogued the CNAs observed in more than 3000 cancers found that amplifications do most commonly involve either the whole chromosome arm or are focal(2
), similar to what we observe in CLL. In the Beroukhim study, both PIK3CA
were targets of recurrent gains in cancer.
To date genomic alterations in PI3K have not been reported in CLL, although both amplifications and activating point mutations occur in solid tumors(21
). Here we report amplifications of the PIK3CA
locus in CLL, but we did not observe activating point mutations. A similar pattern of PIK3CA
amplification without mutation has been described in mantle cell lymphomas(37
), and amplifications in endometrial cancer show a distinct phenotype compared to somatic mutation, suggesting that amplification and somatic mutation may have distinct consequences(38
). The PI3 kinase pathway is constitutively(23
) and inducibly activated by multiple cell surface signals in CLL, including the B cell receptor pathway(39
). Some interest has focused on the question of which PI3 kinase catalytic isoform is most important in CLL, given that the delta isoform is highly expressed and the delta knockout mouse shows impairment in the B cell compartment(39
). Our data suggest that the delta isoform of p110 is most commonly in active complex with the p85 regulatory subunit in CLL, but that at least in several samples with alpha amplification, this balance shifts toward alpha. Our data suggest that PIK3CA
amplification may be one of many mechanisms contributing to PI3K activation in CLL. Currently a delta-specific PI3K inhibitor is showing marked clinical activity in CLL(43
); whether pan-PI3K or PI3K alpha inhibitors will have similar potency remains to be determined, as does the effect of PIK3CA
amplification on the activity of the delta inhibitor. Ultimately, prospective validation of the frequency of PIK3CA
amplification in CLL will be required to determine its importance in the disease.
A role for MYC
in the initiation or progression of CLL has been much less clear, although transgenic mice expressing MYC
together with BAFF
have recently been reported to develop a CLL-like disease(44
). This study also found that higher MYC
expression in CLL patient samples was associated with shorter TTFT(44
). Genomic analyses of Richter’s transformation, namely CLL that has transformed to a higher grade lymphoma, have identified MYC
amplification as a common event thought to be acquired at the time of transformation(45
). Here we report MYC
amplifications in CLL without transformation, through whole chromosome arm amplification or focal amplification of the 8q24 risk region near MYC
. We also identify rare somatic mutations in MYC
. These findings suggest multiple mechanisms of MYC
activation in CLL, albeit at low frequency. The 8q24 gains identified here while uncommon are associated with short TTFT, and therefore likely a poor prognosis.
We identify two CLLs with focal gains of the 8q24 risk region, in which a SNP (rs2456449) has been associated with germline risk of CLL(25
). Multiple studies have shown that this region can act as an enhancer for MYC
). These focal amplifications may therefore represent somatic amplification of a germline risk allele, as described previously in neuroblastoma(49
). If alleles identified by GWAS truly promote the risk of malignancy, additional similar instances will likely be identified over time.
Interestingly, gene expression analyses of our three CNA groups identified an overlapping set of CLLs with a shared expression pattern associated with induction of ES Polycomb gene sets, repression of ES MYC and proliferation gene sets, and induction of small specific modules of ES ‘core’ factors and targets, all of which have been previously associated with long-term self-renewing HSCs(32
). These findings raise the possibility of a role for histone methylation in CLL pathogenesis and an association with HSC biology, but further work will be required to validate this finding and determine its significance to CLL.
In summary, our comprehensive integrated analysis of CLL has characterized three recurrent CNAs associated with reduced TTFT. Two of these CNAs affect PIK3CA and MYC focally. These CNAs will require validation in uniformly treated prospective cohorts in order to better define their incidence and prognostic significance. These studies together with emerging sequencing data will hopefully define key molecular subgroups of CLL that will clarify prognosis and inform novel therapeutic avenues in the coming years.