Novel approaches in pharmacogenetic studies
During the past decades, several studies based on DNA/RNA microarrays have tried to develop predictive signatures to decide the most suitable chemotherapy approach for specific types or subtypes of tumors. Conflicting results and difficulties in replicating the data led to a limited impact on clinical practice, mainly due to: i) lack of sufficiently powered clinical trials; ii) use of sample collection and data analysis methods that are not standardized; iii) intrinsic intratumor heterogeneity.
However, in the last few years, technical advancements in the development of rapid whole-genome studies have initiated a revolution in the field that could potentially change the pharmacogenomic approach to perzonalized treatment in the future. These techniques are represented mainly by massively parallel or next‑generation sequencing (NGS) and GWASs.
During recent decades, thanks to the introduction of advanced systems that may generate millions of short DNA reads (36–150 base pairs), the landscape of DNA sequencing has evolved from f irst-generation sequencing, which was based on DNA sequencing using dideoxynucleotide termination chemistry, to second-generation sequencing or NGS [74]. Noncoding regions of DNA/RNA, in addition to exons, can be analyzed at very high resolution in a short time by NGS with the possibility of identifying any chromosomal rearrangement and variations in copy-number. Furthermore, thanks to the progression in the development of whole-genome amplification, an insight into intratumor heterogeneity has become feasible by performing genetic studies even at the singlecell level [75]. Hopefully, with the diffusion of NGS methodologies in the next few years, we will develop an increasing knowledge of tumor biology.
GWASs are genetic studies that allow the simultaneous evaluation of many polymorphisms, especially common copy-number variations and SNPs, in order to identify existing correlations among any genetic variant and tumor features. In particular, the application of these types of studies in pharmacogenomics aims to find out any possible correlation between polymorphisms and drug response or toxicity. Of note, GWASs allow the investigation of the entire genome independently from preliminary information on chromosomal loci involvement in drug effects. The massive amount of SNPs analyzed with GWASs and the difficulty represented by the definition of real correlations are the limitations of this method, increasing the risk of a high rate of false-positive data; a massive number of samples is necessary to obtain a sufficient statistical power. A recent study has been published by University of Texas MD Anderson researchers (TX, USA), reporting a genome-wide scan of 307,260 polymorphisms in 327 NSCLC patients treated with cisplatin or carboplatin [76]. In this analysis, the rs1878022 variant, which is located within an intron of the gene CMKLR1, has been strongly correlated with shorter survival. The findings of this study were also validated in two other independent cohorts of 315 patients enrolled at the Mayo Clinic (USA) and 420 cases recruited in the Spanish Lung Cancer Group PLATAX clinical trials. Interestingly, the use of this unbiased non-candidate gene-driven approach led to the identification as strong predictive survival factor of a polymorphic variant within an intronic region of a platinum-unrelated gene.
In order to validate these pharmacogenetic findings and use them in the clinical setting, future genomic analysis should use validated and standardized methodology. Moreover, future analysis should be performed on larger cohorts of patients and/or utilize the sharing of data through common websites in order
to create ‘virtual multicentric pharmacogenetic trials’.
Clinical relevance
During recent decades, a number of important genetic determinants have been identified in different tumor types, including NSCLC. The FDA has approved a number of genetic tests and some pharmacogenetic markers have been included in drug labels [102]. However, the application of pharmacogenetic testing in primary care is not as common as predicted. This leads to questions regarding the clinical relevance of pharmacogenetic analyses and on future studies that should successfully bridge the gap from research to clinical practice.
Unfortunately, the clinical relevance of most of the polymorphisms described in the current literature remains controversial because of the exiguous number of (heterogeneous) patients screened, the lack of prospective studies designed to identify specific correlations between gene alterations and drug response or disease prognosis, and the strong variability in analytical analysis, as well as lack of quality controls among different studies.
The development of prospective clinical trials, in which patient treatments selected on the basis of conventional criteria are directly compared with patients given specific treatments suggested by genetic characteristics, might overcome these controversies and reduce the reluctance to transfer the novel knowledge into the clinical routine. Another obstacle to the successful clinical application of the new findings is the lack of association with the main pharmacokinetic/pharmacodynamic parameters as well as functional genomic analysis of the underlying mechanisms of drug sensitivity/resistance. The recent discovery of miRNA has improved our knowledge regarding the control of gene expression, and in the future it may help to clarify the role of the signaling pathways involved in lung cancer oncogenesis. Hopefully, growing evidence relating to miRNA may provide a clue to solve individual drug-resistance problems [77].
In conclusion, despite the intriguing findings of several studies, small sample sizes together with interethnic differences and the retrospective nature of most studies make it difficult to draw any clear conclusions regarding the role of most pharmacogenetic biomarkers in determining the clinical outcome or toxicity in gefitinib and erlotinib treatment. Hopefully, the accurate planning of new prospective trials, the increased knowledge of key mechanisms affecting drug distribution/activity and the use of novel technologies, including genome-wide approaches, may provide essential tools to improve the prospects of pharmacogenetic research, as well as to optimize currently available treatments in selected NSCLC patients.
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