Liquid biopsy as an option for predictive testing and prognosis in patients with lung cancer

Background: The aim of this study was to investigate the clinical value of liquid biopsy as a primary source for variant analysis in lung cancer. In addition, we sought to characterize liquid biopsy variants and to correlate mutational load to clinical data. Methods: Circulating cell-free DNA was extracted from plasma from patients with lung cancer (n = 60) and controls with benign lung disease (n = 16). Variant analysis was performed using the AVENIO ctDNA Surveillance kit and the results were correlated to clinical and variant analysis data from tumor tissue or cytology retrieved from clinical routine diagnostics. Results: There were significantly more variants detected in lung cancer cases compared to controls (p = 0.011), but no difference between the histological subgroups of lung cancer was found (p = 0.465). Furthermore, significantly more variants were detected in patients with stage IIIb-IV disease compared to patients with stage I-IIIa (median 7 vs 4, p = 0.017). Plasma cfDNA mutational load was significantly associated with overall survival (p = 0.010). The association persisted when adjusted for stage and ECOG performance status (HR: 3.64, 95% CI 1.37-9.67, p = 0.009). Agreement between tumor and plasma samples significantly differed with stage; patients with stage IIIb-IV disease showed agreement in 88.2% of the cases with clinically relevant variants, compared to zero cases in stage I-IIIa (p = 0.004). Furthermore, one variant in EGFR, two in KRAS, and one in BRAF were detected in plasma but not in tumor samples. Conclusion: This study concludes that in the vast majority of advanced NSCLC patients a reliable variant analysis can be performed using liquid biopsy from plasma. Furthermore, we found that the number of variants in plasma is associated with prognosis, possibly indicating a strategy for closer follow up on this crucial patient group

Technical in-depth comparison of two massive parallel DNA-sequencing methods for formalin-fixed paraffin-embedded tissue from victims of sudden cardiac death

Sudden cardiac death (SCD) is a tragic and traumatic event. SCD is often associated with hereditary genetic disease and in such cases, sequencing of stored formalin fixed paraffin embedded (FFPE) tissue is often crucial in trying to find a causal genetic variant. This study was designed to compare two massive parallel sequencing assays for differences in sensitivity and precision regarding variants related to SCD in FFPE material. From eight cases of SCD where DNA from blood had been sequenced using HaloPlex, corresponding FFPE samples were collected six years later. DNA from FFPE samples were amplified using HaloPlex HS, sequenced on MiSeq, representing the first method, as well as amplified using modified Twist and sequenced on NextSeq, representing the second method. Molecular barcodes were included to distinguish artefacts from true variants. In both approaches, read coverage, uniformity and variant detection were compared using genomic DNA isolated from blood and corresponding FFPE tissue, respectively. In terms of coverage uniformity, Twist performed better than HaloPlex HS for FFPE samples. Despite higher overall coverage, amplicon-based HaloPlex technologies, both for blood and FFPE tissue, suffered from design and/or performance issues resulting in genes lacking complete coverage. Although Twist had considerably lower overall mean coverage, high uniformity resulted in equal or higher fraction of genes covered at &amp;gt;= 20X. By comparing variants found in the matched samples in a pre-defined cardiodiagnostic gene panel, HaloPlex HS for FFPE material resulted in high sensitivity, 98.0% (range 96.6-100%), and high precision, 99.9% (range 99.5-100%) for moderately fragmented samples, but suffered from reduced sensitivity (range 74.2-91.1%) in more severely fragmented samples due to lack of coverage. Twist had high sensitivity, 97.8% (range 96.8-98.7%) and high precision, 99.9% (range 99.3-100%) in all analyzed samples, including the severely fragmented samples.Funding Agencies|ALF funding Region Orebro County; Orebro County Council Research committee</p

Evaluation of Microsatellite instability score from GMS560 DNA panel

Microsatellite instability is characterised by gains or losses of nucleotides in short tandem repeat sequences, microsatellites, dispersed throughout the human genome. Microsatellite instability status is a molecular fingerprint for DNA mismatch repair deficiency. Clinical detection of microsatellite instability status is important for identifying inherited disease in patients with colorectal and endometrial cancer but has also a prognostic value for survival and prediction of treatment response. Lately, microsatellite instability has been used as a tumor agnostic biomarker that predicts response to immune checkpoint inhibitors. To identify microsatellite instability status clinically, PCR and immunohistochemistry have been the gold standard. On the contrary, next generation sequencing provide simultaneous accession of large number of microsatellite loci and can be combined with detection of several other biomarkers.  The national collaboration Genome Medicine Sweden have developed a solid tumour gene panel composed of 560 cancer associated genes with integrated microsatellite instability score. Our aim was to validate the microsatellite instability status based on microsatellite instability score from GMS560 DNA panel against the clinically used methods. Extracted DNA (100 ng) from formalin fixed paraffin embedded tissue sections with various tumour cell content &gt;10% were analysed. During target enrichment sequencing analysis, allelic distribution from 5000 microsatellite markers were calculated by MSIsensor Pro to generate an instability score.  The cohort consisted of microsatellite instable verified colorectal cancer samples (n=20), microsatellite stable solid tumour material (n=60). Preliminary results generated a microsatellite instability score for the colorectal cancer samples with a mean of 26.5 % (CI: 23.4-29.6, range: 16.9-32.3). Microsatellite stable tumour samples had a mean microsatellite instability score of 1.5 % (CI: 0.93-2.07, range: 1-4.45).  In conclusion, we found the microsatellite instability score from GMS560 DNA panel to be both diagnostically sensitive and specific for determining MSI status due to obvious separation in instability.

Evaluation of Microsatellite instability score from GMS560 DNA panel

Microsatellite instability is characterised by gains or losses of nucleotides in short tandem repeat sequences, microsatellites, dispersed throughout the human genome. Microsatellite instability status is a molecular fingerprint for DNA mismatch repair deficiency. Clinical detection of microsatellite instability status is important for identifying inherited disease in patients with colorectal and endometrial cancer but has also a prognostic value for survival and prediction of treatment response. Lately, microsatellite instability has been used as a tumor agnostic biomarker that predicts response to immune checkpoint inhibitors. To identify microsatellite instability status clinically, PCR and immunohistochemistry have been the gold standard. On the contrary, next generation sequencing provide simultaneous accession of large number of microsatellite loci and can be combined with detection of several other biomarkers.  The national collaboration Genome Medicine Sweden have developed a solid tumour gene panel composed of 560 cancer associated genes with integrated microsatellite instability score. Our aim was to validate the microsatellite instability status based on microsatellite instability score from GMS560 DNA panel against the clinically used methods. Extracted DNA (100 ng) from formalin fixed paraffin embedded tissue sections with various tumour cell content &gt;10% were analysed. During target enrichment sequencing analysis, allelic distribution from 5000 microsatellite markers were calculated by MSIsensor Pro to generate an instability score.  The cohort consisted of microsatellite instable verified colorectal cancer samples (n=20), microsatellite stable solid tumour material (n=60). Preliminary results generated a microsatellite instability score for the colorectal cancer samples with a mean of 26.5 % (CI: 23.4-29.6, range: 16.9-32.3). Microsatellite stable tumour samples had a mean microsatellite instability score of 1.5 % (CI: 0.93-2.07, range: 1-4.45).  In conclusion, we found the microsatellite instability score from GMS560 DNA panel to be both diagnostically sensitive and specific for determining MSI status due to obvious separation in instability.