Risk for molecular contamination of tissue samples evaluated for targeted anti-cancer therapy.

With the increasing usage of sensitive PCR technology for pharmacogenetics, cross contamination becomes a significant concern. Researchers employed techniques which basically include replacing laboratory equipment after each sample preparation; however, there are no recommended guidelines. In the present work we wanted to evaluate the risk of cross contamination during tissue processing using the routine precaution measures. Twenty-one surgical samples of lung adenocarcinoma were used, of which 7 contained EGFR exon 19 mutation, 7 contained EGFR exon 21 mutation (p.L858R) and 7 were EGFR wild-type. The samples were ordered by alternating the mutation group to maximize the potential for cross contamination and underwent tissue sectioning and de-paraffinization. The entire process was performed using the same tools. Following DNA extraction all samples underwent PCR amplification and were scrutinized for small fractions of EGFR mutation using deep sequencing with the Ion torrent PGM technology. Twenty samples yielded results. The fraction of mutated copies was 41 ± 23% (range 11-66) for the cases with known exon 19 mutation and 48±24% (range 0-65) for the cases with known exon 21 mutations. No in-frame exon 19 deletion mutations were identified in the wild-type (WT) and exon 21 groups. The fraction of EGFR exon 21 (codon 858) mutations was 0.018±0.014% (range 0-0.05%) in the WT and exon 19 groups, which was not statistically different than the background sequencing artifact noise for the same base-pair alteration (p = 0.21). Our results suggest that standard precautions are sufficient for molecular pathology diagnosis of surgical samples and are not associated with increased risk of cross contamination

Primers list.

<p>Primers list.</p

Study workflow.

<p>Study procedure was divided to three main stages: 1) Sequential cutting of the samples using the same knife 2) De-paraffinization using the same tools 3) DNA extraction and mutation analysis.</p

Mutation analysis using the Ion Torrent PGM.

<p>(A) A sample with a known <i>EGFR</i> exon 19 deletion mutation. (B) A sample with <i>EGFR</i> exon 21 mutation (p.L858R) is present (39% T > G transversion). (C) Sequencing result of <i>EGFR</i> exon 19 wild-type case with lack of in frame deletion. (D) A sample with wild-type <i>EGFR</i> exon 21 "hot-spot" position (100% T, no T >G transversion). Using the Integrative Genomics Viewer (IGV) software, we were able to determine the mutation and fraction of mutant copies.</p

Mutation characteristics of the samples.

<p>Mutation characteristics of the samples.</p

Comparison of EGFR exon 21 "hotspot" locus (c.2573T>G) mutation fraction and sequencing noise.

<p>There was no significant difference in the fraction of <i>EGFR</i> exon 21 (cDNA position 2573) "hotspot" mutation (T>G) from the same base alteration fraction in the other T residues in the sequence (noise). Statistical significance was assessed by Wilcoxon test.</p

Distribution of the different substitution allele frequencies in the non-”hot-spot” positions.

<p>As shown, allele frequencies higher than 0.3% were present in less than 5% of possible positions and we therefore set 0.3% allele frequency as the detection limit of our method.</p