PIK3CA-CDKN2A clonal evolution in metastatic breast cancer and multiple points cell-free DNA analysis.

BACKGROUND: Daily experience tells us that breast cancer can be controlled using standard protocols up to the advent of a relapse. Now new frontiers in precision medicine like liquid biopsy of cell free DNA (cfDNA) give us the possibility to understand cancer evolution and pick up the key mutation on specific cancer driver gene. However, tight schedule of standardized protocol may impair the use of personalized experimental drugs in a timely therapeutic window. MAIN BODY: Here, using a combination of deep next generation sequencing and cfDNA liquid biopsy, we demonstrated that it is possible to monitor cancer relapse over time. We showed for the first time the exact correspondence from the increasing clonal expansion and clinical worsening of metastatic breast cancer. CONCLUSION: Thanks to liquid biopsy may be possible to introduce new experimental drugs in the correct therapeutic window which would lead in the near future to an effective treatment which otherwise remains challenging.

A DNA pool of FLT3-ITD positive DNA samples can be used efficiently for analytical evaluation of NGS-based FLT3-ITD quantitation - Testing several different ITD sequences and rates, simultaneously.

Internal tandem duplication (ITD) in the fms-like tyrosine kinase 3 (FLT3) gene is one of the most frequent genetic alteration in acute myeloid leukemia (AML), and it is associated with worse clinical outcome. Not only the presence but also the size, localization and the rate of this variant or the presence of multiple ITDs has prognostic information. The traditional PCR based diagnostic methods cannot provide information about all of these parameters in one assay, however the application of next generation sequencing (NGS) technique can be a reliable solution for this diagnostic problem. In order to evaluate the analytical properties of an NGS-based FLT3-ITD detection assay a quality control sample was prepared from DNA of AML patients containing 19 different FLT3-ITD variants identified by NGS. The higher the total read count was in a certain sample of the NGS run, the more ITD variant types could be detected. The maximal sensitivity of FLT3-ITD detection by NGS technique was as low as 0.007% FLT3-ITD/total allele rate, however, below 0.1% rate, the reproducibility of the quantitation was poor (CV > 25%). DNA pools with several FLT3-ITDs can be used efficiently for analytical evaluation of NGS-based FLT3-ITD quantitation testing several different ITD sequences and rates, simultaneously.

EGFR T790M detection rate in lung adenocarcinomas at baseline using droplet digital PCR and validation by ultra-deep next generation sequencing.

BACKGROUND: Routine testing of baseline EGFR T790M mutation may have important clinical impact but many discordant data have been reported regarding the diagnostic, prognostic and predictive role of this marker. In this study we aimed to assess T790M frequency in 164 untreated EGFR-mutated NSCLCs using methods with different sensitivity as well as to analyze the relationship between baseline T790M mutation status, patient's clinicopathologic features and tyrosine kinase inhibitors (TKI) treatment outcomes. METHODS: We compared the diagnostic performance, sensitivity and specificity of three methods, namely MALDI-TOF mass spectrometry (MS), Allele-Specific Real Time PCR (AS-PCR), droplet digital PCR (ddPCR). Ultra-deep next generation sequencing (NGS) validation of T790M-mutant NSCLCs was performed using SiRe® panel. RESULTS: Baseline T790M occurred in 17% of the tumors. Intermediately sensitive techniques such as MALDI-TOF MS (detection limit of T790M ≥5%) allow to detect T790M in 2% of cases exhibiting mutant-allele fractions ranging from 11.5% to 17%. Median overall survival (OS) in these patients was poor (7.3 months) and progression free survival (PFS) was of 3.3 months in patients treated with a 1st generation EGFR TKI. The remaining T790M-positive cases showed very low mutant-allele fractions ranging from 0.07% to 0.38% and required highly sensitive methods such as ddPCR and NGS to be identified. All these cases showed a concurrent sensitizing EGFR mutation (mainly exon 19 deletion), and clinicopathological features similar to those observed in EGFR mutant cancers. Median OS of these patients was 27 months while median PFS after TKI treatment was 20 months. CONCLUSIONS: Routine test of baseline EGFR T790M may have an important role in the prediction to EGFR TKI therapy response and should be performed using highly sensitive and quantitative methods, such as ddPCR and NGS, in order to reliably distinguish NSCLCs with high or very low T790M mutant-allele fraction.

Beyond the whole genome consensus: unravelling of PRRSV phylogenomics using next generation sequencing technologies.

The highly heterogeneous porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent responsible for an economically important pig disease with the characteristic symptoms of reproductive losses in breeding sows and respiratory illnesses in young piglets. The virus can be broadly divided into the European and North American-like genotype 1 and 2 respectively. In addition to this intra-strains variability, the impact of coexisting viral quasispecies on disease development has recently gained much attention; owing very much to the advent of the next-generation sequencing (NGS) technologies. Genomic data produced from the massive sequencing capacities of NGS have enabled the study of PRRSV at an unprecedented rate and details. Unlike conventional sequencing methods which require knowledge of conserved regions, NGS allows de novo assembly of the full viral genomes. Evolutionary variations gained from different genotypic strains provide valuable insights into functionally important regions of the virus. Together with the advancement of sophisticated bioinformatics tools, ultra-deep NGS technologies make the detection of low frequency co-evolving quasispecies possible. This short review gives an overview, including a proposed workflow, on the use of NGS to explore the genetic diversity of PRRSV at both macro- and micro-evolutionary levels.