Preparation of Duplex Sequencing Libraries for Archival Paraffin-Embedded Tissue Samples Using Single-Strand-Specific Nuclease P1.

DNA from formalin-fixed paraffin-embedded (FFPE) tissues, which are frequently utilized in cancer research, is significantly affected by chemical degradation. It was suggested that approaches that are based on duplex sequencing can significantly improve the accuracy of mutation detection in FFPE-derived DNA. However, the original duplex sequencing method cannot be utilized for the analysis of formalin-fixed paraffin-embedded (FFPE) tissues, as FFPE DNA contains an excessive number of damaged bases, and these lesions are converted to false double-strand nucleotide substitutions during polymerase-driven DNA end repair process. To resolve this drawback, we replaced DNA polymerase by a single strand-specific nuclease P1. Nuclease P1 was shown to efficiently remove RNA from DNA preparations, to fragment the FFPE-derived DNA and to remove 5'/3'-overhangs. To assess the performance of duplex sequencing-based methods in FFPE-derived DNA, we constructed the Bottleneck Sequencing System (BotSeqS) libraries from five colorectal carcinomas (CRCs) using either DNA polymerase or nuclease P1. As expected, the number of identified mutations was approximately an order of magnitude higher in libraries prepared with DNA polymerase vs. nuclease P1 (626 ± 167/Mb vs. 75 ± 37/Mb, paired t-test p-value 0.003). Furthermore, the use of nuclease P1 but not polymerase-driven DNA end repair allowed a reliable discrimination between CRC tumors with and without hypermutator phenotypes. The utility of newly developed modification was validated in the collection of 17 CRCs and 5 adjacent normal tissues. Nuclease P1 can be recommended for the use in duplex sequencing library preparation from FFPE-derived DNA.

Changes in the concentration of EGFR-mutated plasma DNA in the first hours of targeted therapy allow the prediction of tumor response in patients with EGFR-driven lung cancer.

PURPOSE: This study aimed to analyze changes in the plasma concentration of EGFR-mutated circulating tumor DNA (ctDNA) occurring immediately after the start of therapy with EGFR tyrosine kinase inhibitors (TKIs). METHODS: Serial plasma samples were collected from 30 patients with EGFR-driven non-small cell lung cancer before intake of the first tablet and at 0.5, 1, 2, 3, 6, 12, 24, 36 and 48 h after the start of the therapy. The content of EGFR alleles (exon 19 deletions or L858R) in ctDNA was measured by ddPCR. RESULTS: ctDNA was detected at base-line in 25/30 (83%) subjects. Twelve (50%) out of 24 informative patients showed > 25% reduction of the ctDNA content at 48 h time point; all these patients demonstrated disease control after 4 and 8-12 weeks of therapy. The remaining 12 individuals showed either stable content of EGFR-mutated ctDNA (n = 5) or the elevation of ctDNA concentration (n = 7). 10 of 12 patients with elevated or stable ctDNA level achieved an objective response at 4 weeks, but only 5 of 10 evaluable patients still demonstrated disease control at 8-12 weeks (p = 0.032, when compared to the group with ctDNA decrease). The decline of the amount of circulating EGFR mutant copies at 48 h also correlated with longer progression-free survival (14.7 months vs. 8.5 months, p = 0.013). CONCLUSION: Comparison of concentration of EGFR-mutated ctDNA at base-line and at 48 h after the start of therapy is predictive for the duration of TKI efficacy.

Identification of recurrent pathogenic alleles using exome sequencing data: Proof-of-concept study of Russian subjects.

Whole exome sequencing (WES) is a powerful tool for the cataloguing of population-specific genetic diseases. Within this proof-of-concept study we evaluated whether analysis of a small number of individual exomes is capable of identifying recurrent pathogenic alleles. We considered 106 exomes of subjects of Russian origin and revealed 13 genetic variants, which occurred more than twice and fulfilled the criteria for pathogenicity. All these alleles turned out to be indeed recurrent, as revealed by the analysis of 1045 healthy Russian donors. Eight of these variants (NAGA c.973G>A, ACADM c.985A>C, MPO c.2031-2A>C, SLC3A1 c.1400T>C, LRP2 c.6160G>A, BCHE c.293A>G, MPO c.752T>C, FCN3 c.349delC) are non-Russian-specific, as their high prevalence was previously demonstrated in other European populations. The remaining five disease-associated alleles appear to be characteristic for subjects of Russian origin and include CLCN1 c.2680C>T (myotonia congenita), DHCR7 c.453G>A (Smith-Lemli-Opitz syndrome), NUP93 c.1162C>T (steroid-resistant nephrotic syndrome, type 12), SLC26A2 c.1957T>A (multiple epiphyseal dysplasia) and EIF3F c.694T>G (mental retardation). These recessive disease conditions may be of particular relevance for the Russian Federation and other countries with a significant Slavic population.

Content of circulating tumor DNA depends on the tumor type and the dynamics of tumor size, but is not influenced significantly by physical exercise, time of the day or recent meal.

PURPOSE: This study aimed to investigate factors, which influence the content of circulating tumor DNA (ctDNA). METHODS: 398 serial plasma samples were collected within 1-7 consecutive days from patients with EGFR-mutated lung cancer (n = 13), RAS/RAF-mutated colorectal cancer (n = 54) and BRAF-mutated melanoma (n = 17), who presented with measurable tumor disease. The amount of ctDNA was determined by ddPCR. RESULTS: Among 82 patients, who donated 2-6 serial plasma samples, 42 subjects were classified as ctDNA-positive; only 22% cases were mutation-positive across all consecutive tests, while 24/82 (29%) patients showed presence of mutated ctDNA in some but not all blood draws. Subjects with progressing tumors had higher probability of being detected ctDNA-positive as compared to patients, who responded to therapy or had stable disease (39/55 (71%) vs. 4/24 (17%); p = 0.0001). Our study failed to reveal the impact of the time of the day, recent meal or prior physical exercise on the results of ctDNA testing. CONCLUSIONS: Presence of ctDNA in plasma is particularly characteristic for patients, who experience clinical progression of tumor disease. Consecutive plasma tests may occasionally provide discordant data; thus, the repetition of analysis may be advised in certain cases in order to ensure the validity of negative ctDNA result.