Eradication of malignant cells by chemotherapy is a composite phenotype which depends not only on the somatically acquired characteristics of the malignant cells but also upon inherent patient characteristics. Childhood ALL has long served as a prototype for a malignancy that is curable with drugs. Early assessments of MRD strongly predict cure rates, and are used to modify therapy.3-9,48
Eradication of MRD is affected by genetic characteristics of the blasts (e.g. the presence of the Philadelphia chromosome or the TEL/AML1
translocation) and by host characteristics such as age.7,8
Using a candidate gene approach, a few germline genetic variations have been shown to affect the level of MRD,16,49
but this has not been previously assessed on a genome-wide level. Herein, we used an agnostic genome-wide interrogation to identify 102 germline genetic variations that affected the level of residual leukemia in two independent cohorts of patients, and found that a high proportion (63 of 102 SNPs or 61.7%) also affected early response, relapse risk, or antileukemic drug disposition.
One of the strongest signals from the genome-wide scan came from 5 SNPs located in and around the IL15
gene, a proliferation-stimulatory cytokine.50,51
IL15 can protect lymphoid tumors from glucocorticoid-induced apoptosis in vitro
expression in ALL blasts has been linked to both CNS involvement at diagnosis and an increased risk of CNS relapse.53
Both higher IL15
gene expression (P=0.0342 in St. Jude and P=0.003 in COG) and germline SNP genotypes were associated with an increased risk of positive MRD at the end of induction therapy (Supplemental Figure 4S
), and we found a trend (P=0.0701) towards a significant relationship between IL15
germline SNP genotypes and IL15
gene expression in ALL leukemic blasts. Several of the IL15
SNPs that predicted MRD have been associated with enhanced IL15
transcription/translation efficiency in vitro
Thus, it is plausible that germline genetic variation in IL15
plays a role in treatment response in childhood ALL via affecting IL15
’s function or quantity in ALL blasts, and the fact that IL15
SNPs were prominent from unbiased genome scans in two independently-treated cohorts points to its importance in determining ALL response, either as a prognostic marker or as a therapeutic target.
As genome-wide interrogations for pharmacogenetics are still in their infancy, there are no published whole-genome data linking polymorphisms with anticancer drug response. We had the opportunity to couple the findings from our genome-wide SNP interrogation for MRD with three relevant host pharmacokinetic phenotypes: systemic clearance of two antileukemic agents (etoposide and methotrexate) and intracellular disposition of the latter. Although 4-8 different antileukemic agents were used in these two cohorts, remarkably, 21 of the 102 MRD-predicting SNPs we identified were also significantly associated with disposition of these two antileukemic agents. Although many additional genetic variations would be expected to be specific for antileukemic drugs other than methotrexate and etoposide, and might therefore account for some of the remaining 81 MRD-predicting SNPs, several of the pathways involved in methotrexate disposition and etoposide disposition (http://www.pharmgkb.org
) are likely to be shared by other antileukemic agents. Particularly for etoposide, whose disposition involves cytochrome P4503A
metabolism and P-glycoprotein excretion, it is likely that there is overlap in the genetic determinants of its disposition with those affecting anthracyclines, glucocorticoids, and vincristine.34,37-41
The majority (83.3%) of the associations between SNP genotypes and drug disposition were pharmacologically intuitive, with genotypes that predicted increased drug exposure linked to lower levels of MRD. Together, these results suggest that more attention be given to details of drug administration and risk factors for rapid drug clearance, in addition to the considerable attention already placed upon better risk classification of ALL to tailor therapy intensity.
There was also a high proportion (21/102) of SNPs that were associated with not only MRD, but also with the risk of hematologic relapse in both cohorts. This high percentage is somewhat surprising in that the post-remission therapy (which would ultimately be expected to have a significant effect on relapse risk) differed substantially in the COG and St. Jude cohorts. This secondary analysis does lend credence to the hypothesis that we did identify true associations between SNP genotypes and poor response.
Like all risk features, genotypes that are informative for pharmacogenetic phenotypes are likely to be highly dependent upon therapy. For this reason, we purposefully chose two cohorts (St. Jude and COG) that had received somewhat different remission induction regimens, with slightly different time points for the primary phenotype (MRD), to identify polymorphisms more likely to have prognostic significance across multiple therapeutic regimens. The advantage of our bi-directional statistical approach is that the SNPs we identified may be more likely to have external validity for other patient groups; the disadvantage is that we might have missed SNPs more specific to the few elements of therapy that differed between the cohorts.
It is important to consider race, both from the standpoint of its possible effects on antileukemic drug efficacy55-57
and from its influence on germline SNP allele frequency.58
The influence of race on ALL cure rates may be due to differences among races in the delivery of care, patient compliance, frequencies of poor-prognosis ALL subtypes, or to differences in allele frequencies for germline polymorphisms.16
We found good agreement between self-declared race and that determined using ancestry-informative SNPs, and the 102 MRD-associated SNPs remained significant after adjusting for ancestry (Supplemental Table 2S
). Thus, population stratification was unlikely to have affected the SNP genotype/phenotype associations we discovered, consistent with other recent studies.59,60
The fact that SNP genotypes maintained their significance after adjusting for race, despite some cases of substantial differences in allele frequency by race (Supplemental Figure 5S
), suggests that inherent differences in ALL prognosis among racial groups are partly influenced by differences in allele frequencies among racial groups, which could in the future lead to “race neutral” (but genomically-based) individualization of therapy.
We acknowledge that despite the fact that these SNP genotypes were associated with MRD in two independent cohorts, there is a danger of false negative and false positive findings, especially when sample size is relatively small. However, phenotypes of interest in pharmacogenetic studies (e.g. CYP2C9/VKORC
) may have effect sizes that exceed those likely to be observed for multigenic common diseases (e.g. diabetes and arthritis),24
and therefore smaller sample size may suffice in the former. By identifying 102 SNPs based on evidence of association in two independent cohorts, and also by further validation of 62% of these SNPs (Supplemental Table 3S
) as associating with the related phenotypes of relapse, “super response” at days 8 or 19, and antileukemic drug pharmacokinetics, we have further decreased the chance for false discoveries. The SNPs we identified may be in linkage disequilibrium64
with the truly causative genetic variants that have not yet been interrogated directly by our genotyping platform (Supplemental Table 4S). Importantly, few of the 102 polymorphisms we identified have previously been suggested as candidates for affecting anticancer drug efficacy, and approximately half of the genomic variants are not annotated to genes at all, illustrating the need to further explore mechanisms by which germline genomic variation affects interindividual variability in antileukemic drug response.
Although the acquired genetic characteristics of tumor cells play a critical role in drug responsiveness, our results show that inherited genetic variation of the patient also affects effectiveness of anticancer therapy, and that genome-wide approaches can identify novel and yet plausible pharmacogenetic variation. Such variation may be factored into treatment decisions in the future, by placing additional emphasis on optimizing drug delivery to overcome host genetic variation, in addition to the current emphasis on tumor genetic variation.