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 ≥ 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.

Does p53 codon 72 polymorphism have a prognostic value in carcinoma of the vulva and vagina?

Human papilloma virus (HPV) is considered to be responsible for a large part of vaginal and vulvar carcinomas, and the p53 codon 72 polymorphism has been implicated in susceptibility to cancer induced by this virus, but with contradicting results. In this study, we have investigated the prognostic value of the codon 72 polymorphism by real-time PCR (qPCR) in two cohorts of vaginal (n = 66) and vulvar (n = 123) carcinomas. In vaginal carcinoma, arginine homozygous patients were significantly associated with a higher primary cure rate (p = 0.023) but also associated with a higher recurrence rate (p = 0.073), significant at distant locations (p = 0.009). No significant differences were found in overall survival rate (p = 0.499) or cancer-specific survival rate (p = 0.222). A higher frequency of arginine homozygosity was noted in HPV-positive tumors (p = 0.190) in comparison with HPV-negative tumors. In vulvar carcinoma, the genotype homozygous for arginine was significantly associated with a larger tumor size at diagnosis in the entire cohort (p = 0.015) and a lower cancer-specific survival rate (p = 0.024) compared with heterozygous (arginine/proline) in HPV-negative tumors. Our results indicate that the relation between HPV and the p53 codon 72 polymorphism is complex and the significance and mechanisms responsible for this relationship need to be further elucidated.