Dutch National Round Robin Trial on Plasma-Derived Circulating Cell-Free DNA Extraction Methods Routinely Used in Clinical Pathology for Molecular Tumor Profiling

BACKGROUND: Efficient recovery of circulating tumor DNA (ctDNA) depends on the quantity and quality of circulating cell-free DNA (ccfDNA). Here, we evaluated whether various ccfDNA extraction methods routinely applied in Dutch laboratories affect ccfDNA yield, ccfDNA integrity, and mutant ctDNA detection, using identical lung cancer patient-derived plasma samples. METHODS: Aliquots of 4 high-volume diagnostic leukapheresis plasma samples and one artificial reference plasma sample with predetermined tumor-derived mutations were distributed among 14 Dutch laboratories. Extractions of ccfDNA were performed according to local routine standard operating procedures and were analyzed at a central reference laboratory for mutant detection and assessment of ccfDNA quantity and integrity. RESULTS: Mutant molecule levels in extracted ccfDNA samples varied considerably between laboratories, but there was no indication of consistent above or below average performance. Compared to silica membrane-based methods, samples extracted with magnetic beads-based kits revealed an overall lower total ccfDNA yield (-29%; P < 0.0001) and recovered fewer mutant molecules (-41%; P < 0.01). The variant allelic frequency and sample integrity were similar. In samples with a higher-than-average total ccfDNA yield, an augmented recovery of mutant molecules was observed. CONCLUSIONS: In the Netherlands, we encountered diversity in preanalytical workflows with potential consequences on mutant ctDNA detection in clinical practice. Silica membrane-based methodologies resulted in the highest total ccfDNA yield and are therefore preferred to detect low copy numbers of relevant mutations. Harmonization of the extraction workflow for accurate quantification and sensitive detection is required to prevent introduction of technical divergence in the preanalytical phase and reduce interlaboratory discrepancies

The origin of tumor DNA in urine of urogenital cancer patients: Local shedding and transrenal excretion

In urogenital cancers, urine as a liquid biopsy for non-invasive cancer detection holds great promise for future clinical application. Their anatomical position allows for the local shedding of tumor DNA, but recent data indicate that tumor DNA in urine might also result from transrenal excretion. This study aims to assess the origin of tumor-associated DNA in the urine of 5 bladder and 25 cervical cancer patients. Besides natural voided urine, paired urine samples were collected in which contact with the local tumor was circumvented to bypass local shedding. The latter concerned nephrostomy urine in bladder cancer patients, and catheter urine in cervical cancer patients. Methylation levels of GHSR, SST, and ZIC1 were determined using paired bladder tumor tissues and cervical scrapes as a reference. Urinary methylation levels were compared to natural voided urine of matched controls. To support methylation results, mutation analysis was performed in urine and tissue samples of bladder cancer patients. Increased methylation levels were not only found in natural voided urine from bladder and cervical cancer patients, but also in the corresponding nephrostomy and catheter urine. DNA mutations detected in bladder tumor tissues were also detectable in all paired natural voided urine as well as in a subset of nephrostomy urine. These results provide the first evidence that the suitability of urine as a liquid biopsy for urogenital cancers relies both on the local shedding of tumor cells and cell fragments, as well as the transrenal excretion of tumor DNA into the urine

Circulating tumor DNA analysis of EGFR-mutant non-small cell lung cancer patients receiving osimertinib following previous tyrosine kinase inhibitor treatment

Objectives: Circulating tumor (ct)DNA analysis is rapidly gaining acceptance as a diagnostic tool to guide clinical management of advanced non-small cell lung cancer (NSCLC). Clinically-actionable EGFR mutations can be detected in ctDNA before or after first-line EGFR-Tyrosine Kinase Inhibitor (TKI) treatment, but data are limited for patients with a complex treatment history. This study aimed to explore the feasibility of ctDNA testing in a clinical setting of NSCLC patients receiving osimertinib as a second or third line EGFR-TKI. Materials and Methods: Twenty EGFR T790M-positive NSCLC patients, who had received osimertinib as a second or third line EGFR-TKI and had donated blood samples while attending routine follow-up consultations between April and November 2016, were retrospectively selected to test plasma cfDNA for tumor-guided EGFR mutations. We used EGFR mutations previously identified in tumor-tissue to retrospectively test plasma ctDNA from 20 patients who had received osimertinib as a second or third line EGFR-TKI. Both EGFR-TKI sensitising and T790 M resistance mutations were analysed by droplet digital PCR (ddPCR) in plasma taken alongside routine consultations and ctDNA detection was correlated with response under osimertinib. Follow-up solid-tissue biopsies were obtained after disease progression. Results: CtDNA was detected under osimertinib treatment in four out of the eight patients (50 %) who showed no response, two out of the seven (29 %) who showed an initial response and none of the five patients (0 %) who showed an ongoing response. The fraction of EGFR-mutant ctDNA in plasma tended to be higher in non-responders (0.1–68 %), compared to the initial responders (0.2–1.1 %). Blood samples were donated up to 34, 27 and 49 weeks after the start of osimertinib for the non-, initial and ongoing responders, respectively. Conclusions: These findings support a potential role for ctDNA analysis in response monitoring of NSCLC patients with a complex EGFR-TKI treatment history. The weak trend between ctDNA detection and disease progression warrants larger studies to further investigate potential clinical utility

Focal 18F-FDG uptake predicts progression of pre-invasive squamous bronchial lesions to invasive cancers

Introduction: Pre-invasive squamous lesions of the central airways can progress into invasive lung cancers. Identifying these high-risk patients could enable detection of invasive lung cancers at an early stage. In this study, we investigated the value of 18F-fluorodeoxyglucose ( 18F-FDG) positron emission tomography (PET) scans in predicting progression in patients with pre-invasive squamous endobronchial lesions. Methods: In this retrospective study, patients with pre-invasive endobronchial lesions, who underwent an 18F-FDG PET scan at the VU University Medical Center Amsterdam, between January 2000 and December 2016, were included. Autofluorescence bronchoscopy (AFB) was used for tissue sampling and was repeated every 3 months. The minimum and median follow-up was 3 and 46.5 months. Study endpoints were the occurrence of biopsy proven invasive carcinoma, time-to-progression and overall survival (OS). Results: A total number of 40 of 225 patients met the inclusion criteria of which 17 (42.5%) patients had a positive baseline 18F-FDG PET scan. A total of 13 of 17 (76.5%) developed invasive lung carcinoma during follow-up, with a median time to progression of 5.0 months (range, 3.0–25.0). In 23 (57.5%) patients with a negative 18F-FDG PET scan at baseline, 6 (26%) developed lung cancer, with a median time to progression of 34.0 months (range, 14.0–42.0 months, p < 0.002). With a median OS of 56.0 months (range, 9.0–60.0 months) versus 49.0 months (range, 6.0–60.0 months) (p = 0.876) for the  18F-FDG PET positive and negative groups, respectively. Conclusions: Patients with pre-invasive endobronchial squamous lesions and a positive baseline 18F-FDG PET scan were at high-risk for developing lung carcinoma, highlighting that this patient group requires early radical treatment