Performance characteristics of a polymerase chain reaction-based assay for the detection of EGFR mutations in plasma cell-free DNA from patients with non-small cell lung cancer using cell-free DNA collection tubes.

Survival rates in non-small cell lung cancer (NSCLC) are low. Detection of circulating tumor DNA in liquid biopsy (plasma) is increasingly used to identify targeted therapies for clinically actionable mutations, including EGFR mutations in NSCLC. The cobas® EGFR Mutation Test v2 (cobas EGFR test) is FDA-approved for EGFR mutation detection in tissue or liquid biopsy from NSCLC. Standard K2EDTA tubes require plasma separation from blood within 4 to 8 hours; however, Roche Cell-Free DNA (cfDNA) Collection Tubes (Roche cfDNA tube) enable whole blood stability for up to 7 days prior to plasma separation. This analysis assessed performance of Roche cfDNA tubes with the cobas EGFR test for the detection of EGFR mutations in plasma from healthy donors or patients with NSCLC. Overall, test performance was equally robust with either blood collection tube, eg, regarding limit of detection, linearity, and reproducibility, making Roche cfDNA tubes suitable for routine clinical laboratory use in this setting. Importantly, the Roche cfDNA tubes provided more flexibility for specimen handling versus K2EDTA tubes, eg, in terms of tube mixing, plasma separation, and sample stability, and do not require processing of blood within 8 hours thereby increasing the reach of plasma biopsies in NSCLC.

Sustained secretion of human growth hormone from TheraCyte devices encapsulated with PiggyBac-engineered retinal pigment epithelium cells.

Growth hormone (GH) deficiency is characterized by impaired growth and development, and is currently treated by repeated administration of recombinant human GH (hGH). Encapsulated cell therapy (ECT) may offer a less demanding treatment-strategy for long-term production and release of GH into circulation. We used PiggyBac-based (PB) transposon delivery for engineering retinal pigment epithelial cells (ARPE-19), and tested a series of viral and non-viral promoters as well as codon-optimization to enhance transgene expression. Engineered cells were loaded into TheraCyte macrocapsules and secretion was followed in vitro and in vivo. The cytomegalovirus (CMV) promoter supports strong and persistent transgene expression, and we achieved clonal cell lines secreting over 6 µg hGH/106 cells/day. Codon-optimization of the hGH gene did not improve secretion. ARPE-19 cells endured encapsulation in TheraCyte devices, and resulted in steady hormone release for at least 60 days in vitro. A short-term pilot experiment in immunodeficient SCID mice demonstrated low systemic levels of hGH from a single 40 µL capsule implanted subcutaneously. No significant increase in weight increase or systemic hGH was detected after 23 days in the GH-deficient lit/SCID mouse model using 4.5 µL capsules loaded with the highest secreting clone of ARPE-19 cells. Our results demonstrate that PB-mediated engineering of ARPE-19 is an efficient way to generate hormone secreting cell lines compatible with macroencapsulation, and our CMV-driven expression cassette allows for identification of clones with high level and long-term secretory activity without addition of insulator elements. Our results pave the way for further in vivo studies of encapsulated cell therapy.

Co-occurring MET Amplification Predicts Inferior Clinical Response to First-Line Erlotinib in Advanced Stage EGFR-Mutated NSCLC Patients.

BACKGROUND: Intrinsic resistance is a major obstacle in treatment of non-small cell lung cancer (NSCLC) patients with an activating mutation in the epidermal growth factor receptor (EGFR). We investigated co-occurring genetic alterations in circulating tumor DNA (ctDNA) as predictive markers of clinical response to first-line erlotinib. METHODS: Pretreatment plasma samples were collected from 76 patients with EGFR-mutated, advanced-stage NSCLC treated with first-line erlotinib. We isolated ctDNA from plasma for next-generation sequencing. RESULTS: Co-occurring oncogenic drivers were detected in 21% of pretreatment samples and correlated with decreased progression-free survival (PFS) (6.9 months vs 14.4 months; hazard ratio [HR], 2.088; 95% confidence interval [CI], 0.8119-5.370; P = .0355). Concurrent MET amplification was identified in 9 samples (12%), predicting inferior PFS (5.5 months vs 14.4 months; HR, 4.750; 95% CI, 0.5923-38.10; P = .0007) and overall survival (7.6 months vs 28.3 months; HR, 3.952; 95% CI, 0.8441-18.50; P = .0005). Co-occurring non-MET-amplification oncogenic alterations showed a tendency for shorter PFS (9.9 months vs 14.4 months; HR, 1.199; 95% CI, 0.3373-4.265; P = .7586). Clearing EGFR-mutated ctDNA during erlotinib treatment is a positive predictor of clinical outcomes. Among patients who cleared the EGFR mutation, 12% had a co-occurring oncogenic driver, with a tendency toward inferior PFS (8.7 months vs 16.1 months; HR, 1.703; 95% CI, 0.5347-5.424; P = .2508). CONCLUSION: Co-occurring MET amplification in pretreatment ctDNA samples predict inferior clinical response to first-line erlotinib in advanced-stage, EGFR-mutated NSCLC patients. Co-occurring oncogenic alterations were associated with inferior response and may be potential predictors of clinical outcome.

TERT promoter mutated circulating tumor DNA as a biomarker for prognosis in hepatocellular carcinoma.

BACKGROUND AND AIMS: Plasma circulating tumor DNA (ctDNA) with tumor-specific mutations is an attractive biomarker. The telomerase reverse transcriptase (TERT) C228T promoter mutation is the most prevalent tumor-associated mutation in hepatocellular carcinoma (HCC). We evaluated the presence and prognostic value of the TERT C228T mutation in plasma and tissue in a Danish HCC cohort. METHODS: We analyzed ctDNA from 95 HCC patients and 45 liver cirrhotic patients without HCC for the TERT mutation using droplet digital polymerase chain reaction. We also analyzed DNA from the corresponding primary tumor tissues in 34 HCC patients. RESULTS: The plasma TERT C228T mutation was detected in 42/95 HCC patients (44%) but in none of the non-HCC patients. The TERT mutation was detected in 23/34 tumor samples (68%). The TERT mutation was associated with increased mortality when detected in plasma (adjusted HR 2.16 (1.20-3.88), p = .010) but not in tumor tissue (adjusted HR 1.11 (0.35-3.56), p = .860). There was a positive correlation between the presence of the TERT mutation in plasma and an advanced TNM stage (p < .0001) and vascular invasion (p = .005). Analysis of the TERT mutation in plasma and tumor DNA from the same patient was concordant in 21/34 samples (62%; kappa value 0.31, p = .014). Non-concordance was associated with an early TNM stage. CONCLUSION: The plasma TERT mutation was detected in 44% of HCC patients and in none of non-HCC cirrhotic patients; and was associated with increased mortality. We propose the TERT C228T mutation in ctDNA as a promising HCC biomarker for prognosis.