SNPs in PI3K-PTEN-mTOR and Brain Metastases in NSCLC—Letter
Authors: Abolfazl Avan, Mina Maftouh, Amir Avan, Carmelo Tibaldi, Paolo A. Zucali, Elisa Giovannetti
Journal: Clinical Cancer Research (2014)

We have read with interest the study by Li and colleagues (1) on single-nucleotide polymorphisms (SNP) predicting brain metastases in non–small cell lung cancer (NSCLC). Patients with the GT/GG genotype of AKT1-rs2498804, CT/TT of AKT1-rs2494732, and AG/AA of PIK3CA-rs2699887 had higher risk of brain metastasis. These data support the feasibility of studying germinal polymorphisms for risk stratification. However, several points should be discussed in more detail.First, the choice to focus on NSCLC is valuable, as treatment with prophylactic cranial irradiation is a matter of debate in this tumor. However, according to the “rule of tens,” in a multivariate analysis there should be a minimum of 10 events per predictor variable. As 16 SNPs were analyzed, a total of 160 patients would have to have brain metastases to prevent overfitting, because the results from an overfitted model are not generalizable to other populations. Another critical obstacle to the successful development of a genotype-based test is the high number of spurious associations (2). To avoid false-positive associations, a Bonferroni correction considering all the studied SNPs would require a P value of <0.05/16 = 0.003 for statistical significance.Emerging SNPs need to be validated in independent cohorts, and our dataset from stage IIIB/IV NSCLC (3) confirmed the association of AKT1-rs2498804, but not of PIK3CA:rs2699887, with brain metastases (Table 1). These conflicting data might be explained by several factors, such as ethnicity, sample size, different clinical settings, histotypes, stage, and treatment. However, Li and colleagues did not describe treatment details of their cohort, while all our patients received gefitinib. Similarly, although 50% of their patients were never-smoker, no information was provided on EGFR mutations or ALK translocations. Because almost half of ALK+ patients treated with crizotinib have central nervous system relapse (4), we wonder whether the same results would have been obtained after adjustment for EGFR/ALK genetic aberrations and targeted treatments. Ultimately, the role of AKT1-rs2498804 in clinical decision-making should be validated within prospective trials in homogeneous patient cohorts.In addition to pharmacogenetic trials, further research is needed to unravel the functional significance of these polymorphisms. Specifically, AKT1-rs2498804, located at 3′ untranslated region, may affect gene expression through changes in transcription factor–binding sites, microRNA target sequences, and/or splicing variants. Although this SNP has not been tested in experimental models (5), it could be evaluated at least in silico, using publicly available software such as PROMO (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3) to detect transcription factor–binding sites, MicroSNiper (http://cbdb.nimh.nih.gov/microsniper) for prediction of SNP effects on microRNA targets, and Human Splicing Finder (http://www.umd.be/HSF/) to study pre-mRNA splicing. In particular, additional studies should investigate whether differential expression levels of candidate microRNAs might explain the fact that this polymorphism was not associated with metastasis at sites other than the brain. Finally, direct evidence of the effects on protein expression could be obtained by immunohistochemistry of NSCLC primary and metastastic tissues.In conclusion, we thank Li and colleagues for their study, but we believe that additional parameters are essential to validate candidate SNPs beyond already available clinical factors for predicting brain metastases in NSCLC.See the Response, p. 3625No potential conflicts of interest were disclosed.This work was supported by grants from CCA foundation (to A. Avan and E. Giovannetti), Fondazione Humanitas (to E. Giovannetti and P.A. Zucali), the Netherlands Organization for Scientific Research “NWO-Veni grant” and AIRC/Start-Up (to E. Giovannetti).