We interrogated >550,000 heritable variants in patients with advanced pancreatic cancer treated with chemotherapy in CALGB 80303. To our knowledge, this is the first GWAS in a cancer patient population in the context of a randomized, placebo-controlled, clinical trial. This preliminary study generates hypotheses on the role of the IL17F gene in the biology of advanced pancreatic cancer. If replicated, the IL17F SNPs might have prognostic significance.
encodes interleukin-17F, a cytokine with the ability to induce stromal cells to secrete pro-inflammatory cytokines. The most significant SNP in this study is rs763780 in IL17F
, a base substitution that alters the histidine to arginine at amino acid 161 (H161R). In vitro functional experiments demonstrated that, in contrast to the wild-type 161H interleukin-17F, the 161R variant form lacks the ability to activate the mitogen-activated protein kinase pathway, thereby restricting cytokine and chemokine production[15
]. Wild-type 161H interleukin-17F has also demonstrated a strong anti-angiogenesis effect by markedly inhibiting the angiogenesis of human endothelial cells and inducing them to produce interleukin-2, TGF-beta, and monocyte chemoattractant protein-1[16
]. A recent study has also shown the anti-angiogenetic and anti-tumor properties of wild-type interleukin-17F in vivo[17
]. With respect to these activities, the variant 161R form of interleukin-17F is a natural antagonist of the anti-angiogenic and pro-inflammatory effects of wild-type 161H interleukin-17F. For example, the 161R variant has been associated with protective effects in Asian patients with inflammatory and autoimmune conditions[18
]. The resulting pro-angiogenic effects of the variant 161R of interleukin-17F could be further magnified by the concomitant increased expression driven by the noncoding rs7771466 IL17F
variant. This variant is almost in complete linkage disequilibrium with the nonsynonymous rs763780, and its minor allele seems to increase interleukin-17F expression through splicing enhancement and introduction of an additional CDX1 transcription factor binding site. The putative synergistic effect of these IL17F
variants should be verified in experimental models of SNP functionality and angiogenesis.
As angiogenesis has been thought to play an important role in the growth and metastatic spread of pancreatic cancer[20
], we hypothesize that the angiogenesis potential of tumors of patients with the variant 161R interleukin-17F is higher than tumors with wild-type 161H interleukin-17F, conferring worse prognosis. However, other mechanisms related to the pro-inflammatory effects of interleukin-17F cannot be excluded.
rs763780 is the most important candidate SNP discovered by this study, due to 1) the genome-wide significance, 2) its already established molecular function, 3) the mechanistic hypothesis explaining the association with reduced OS, and 4) the suggestion that a trend could be detected in patients of African ancestry, despite the very small sample size. This study proposes that rs763780 in IL17F
might have a prognostic effect in advanced pancreatic cancer patients, also because stratification by treatment arm does not seem to negatively affect the association (Supplemental Material, Table 1
). Because gemcitabine is given in both arms, a true interaction between gemcitabine and IL17F
SNPs cannot be tested. Additionally, a review of the clinical characteristics of the patients heterozygous for IL17F
rs763780 did not show any obvious difference with respect to the characteristics of the overall population accrued into this study (data not shown).
In addition to IL17F
, this study proposes additional genes as putatively involved in determining differences in survival among patients with advanced pancreatic cancer. Among the SNPs listed in , rs11644322 in WWOX
demonstrated a gene-dosage effect, with median OS in the heterozygous patients (5.3 months, 4.3–6.9) that was intermediate between the other two genotype groups (3.3 months, 2.9–5.7, for the variant homozygotes; 7.1 months, 6.0–8.4, for the wild-type homozygotes; p 1.31×10−5
; ). WWOX
codes for the WW domain-containing oxidoreductase, a tumor suppressor in several tumors, including pancreatic cancer[21
SNPs showed the strongest linkage for prostate cancer susceptibility in a recent genome-wide scan[22
]. In multiple myeloma, loss of heterozygosity of 16q23, the location of WWOX
, was associated with adverse survival and reduced WWOX expression[23
]. Germline variants from a recent study mapped WWOX
as one of the genes associated with clinical staging in lung cancer[24
]. Contrary to the amino acid changing SNP in IL17F
, the molecular functions of the SNPs in WWOX
are not known at this time. Due to their association with reduced OS, the established tumor suppressor role of WWOX
, and their intronic location, we hypothesize that these SNPs might reduce the expression of WWOX, diminishing its tumor suppressor properties, and leading to worse prognosis.
A SNP (rs10883617) near BTRC
was associated with reduced OS, as homozygote patients had a reduced median OS (3.6 months, 2.7–6.8) compared to heterozygous patients (6.1 months, 4.9–7.3) and patients who were not carriers of this variant (6.9 months, 5.9–8.2; p 3.94×10−5
, ). BTRC
(beta-transducin repeat containing) encodes a protein involved in the ubiquitination processes that demonstrated an oncogenic activity in several cancers including pancreatic cancer[25
]. In most tumors, overexpression of BTRC results in the degradation of IKappaB, an inhibitor of the NFkappaB transcription factor, and thus the activation of NFkappaB and the uncontrolled cell proliferation in these tumors. The use of our functional results in lymphoblastoid cell lines treated with gemcitabine indicated that BTRC
rs10883617 is associated with increased IC50
and, hence, resistance to gemcitabine (r 0.21, p 0.008). As inhibiting and silencing NFkappaB have been shown to increase the sensitivity of pancreatic cancer cells to gemcitabine[28
], we hypothesize that BTRC
rs10883617 might have a predictive value in pancreatic cancer patients treated with gemcitabine, via a NFkappaB-mediated effect.
This study is limited by the large number of multiple comparisons typical of GWAS, increasing the chance of false positive associations. This limitation could be overcome by independent replication of the findings. Replication studies in cancer treatment outcome have intrinsic difficulties, as there may not be an existing trial (to be used for replication) with the same eligibility criteria and drug treatment of the trial used for discovery. In a relatively uncommon disease like pancreatic cancer, the access to a sufficiently powered replication set is particularly challenging. Ideally, validation of our top hits should be conducted in patients treated with gemcitabine and randomized to an experimental treatment, in order to ensure that the populations are comparable. A few published trials[30
] where patient DNA has been already collected may be considered for replication, due to similarity of treatment and/or disease, and randomized treatment. Additionally, the MAF of the variants in IL17F
is low in Caucasians (0.05 from HapMap), potentially limiting the ability to replicate this association in this population. However, the MAF of the variants in IL17F
is higher in Asians (0.13).
The results of this study are preliminary because of the limited sample size and the low MAF of the IL17F SNP. Due to the refractoriness to treatment of advanced pancreatic cancer and the lack of established markers of survival, the dissemination of these findings to the scientific community could facilitate their replication by others, even as we continue to conduct replication and validation studies. The association of IL17F variants with efficacy could be also tested in tumors other than pancreatic cancer. To support this, our data are now available in dbGaP, in accordance with NIH policy.