A Synthetic DNA Construct to Evaluate the Recovery Efficiency of Cell-Free DNA Extraction and Bisulfite Modification.

BACKGROUND: Despite improvements in the genetic and epigenetic analysis of cell-free DNA (cfDNA), there has been limited focus on assessing the preanalytical variables of recovery efficiency following cfDNA extraction and bisulfite modification. Quantification of recovery efficiency after these steps can facilitate quality assurance and improve reliability when comparing serial samples. METHODS: We developed an exogenous DNA Construct to Evaluate the Recovery Efficiency of cfDNA extraction and BISulfite modification (CEREBIS) after cfDNA extraction and/or subsequent bisulfite modification from plasma. The strategic placement of cytosine bases in the 180 bp CEREBIS enabled PCR amplification of the construct by a single primer set both after plasma DNA extraction and following subsequent bisulfite modification. RESULTS: Plasma samples derived from 8 organ transplant donors and 6 serial plasma samples derived from a liver transplant recipient were spiked with a known number of copies of CEREBIS. Recovery of CEREBIS after cfDNA extraction and bisulfite modification was quantified with high analytical accuracy by droplet digital PCR. The use of CEREBIS and quantification of its recovery was useful in identifying problematic extractions. Furthermore, its use was shown to be invaluable towards improving the reliability of the analysis of serial samples. CONCLUSIONS: CEREBIS can be used as a spike-in control to address the preanalytical variable of recovery efficiency both after cfDNA extraction from plasma and following bisulfite modification. Our approach can be readily implemented and its application may have significant benefits, especially in settings where longitudinal quantification of cfDNA for disease monitoring is necessary.

Cerebrospinal fluid liquid biopsy for detecting somatic mosaicism in brain.

Brain somatic mutations are an increasingly recognized cause of epilepsy, brain malformations and autism spectrum disorders and may be a hidden cause of other neurodevelopmental and neurodegenerative disorders. At present, brain mosaicism can be detected only in the rare situations of autopsy or brain biopsy. Liquid biopsy using cell-free DNA derived from cerebrospinal fluid has detected somatic mutations in malignant brain tumours. Here, we asked if cerebrospinal fluid liquid biopsy can be used to detect somatic mosaicism in non-malignant brain diseases. First, we reliably quantified cerebrospinal fluid cell-free DNA in 28 patients with focal epilepsy and 28 controls using droplet digital PCR. Then, in three patients we identified somatic mutations in cerebrospinal fluid: in one patient with subcortical band heterotopia the LIS1 p. Lys64* variant at 9.4% frequency; in a second patient with focal cortical dysplasia the TSC1 p. Phe581His*6 variant at 7.8% frequency; and in a third patient with ganglioglioma the BRAF p. Val600Glu variant at 3.2% frequency. To determine if cerebrospinal fluid cell-free DNA was brain-derived, whole-genome bisulphite sequencing was performed and brain-specific DNA methylation patterns were found to be significantly enriched (P = 0.03). Our proof of principle study shows that cerebrospinal fluid liquid biopsy is valuable in investigating mosaic neurological disorders where brain tissue is unavailable.

NTRK and ALK rearrangements in malignant pleural mesothelioma, pulmonary neuroendocrine tumours and non-small cell lung cancer.

OBJECTIVES: Gene rearrangements involving NTRK1, NTRK2, NTRK3, ROS1 and ALK have been identified in many types of cancer, including non-small cell lung cancer (NSCLC). Data in malignant pleural mesothelioma (MPM), lung neuroendocrine tumors (NETs) and small-cell lung cancer (SCLC) are lacking. Given the activity of NTRK, ROS-1 and ALK inhibitors in tumors harboring gene fusions, we sought to explore such rearrangements in these less common tumors in addition to NSCLC. METHODS: Archival tumor tissue from patients with MPM, lung NETs, SCLC and NSCLC were used to create tissue microarrays. Immunohistochemistry (IHC) was performed using a cocktail of antibodies against TRK, ROS1 and ALK. IHC positive samples underwent RNA sequencing using the ArcherDX FusionPlex CTL diagnostic assay. Clinical data were obtained through retrospective chart review. RESULTS: We performed IHC on 1116 samples: 335 MPMs, 522 NSCLCs, 105 SCLCs and 154 lung NETs. There were 23 IHC positive cases (2.1%) including eight MPMs (2.4%), eight NETs (5.2%), five SCLC (4.8%) and two NSCLC (0.4%). The following fusions were detected: one MPM with an NTRK ex10-TPM3 ex8, another MPM with an ALK ex20-EML4ex13, one lung intermediate-grade NET (atypical carcinoid) with an ALK ex20-EML4 ex6/intron6, and two NSCLCs with an ALK ex20-EML4 ex6/intron6 rearrangement. None of the patients received targeted treatment. CONCLUSIONS: To our knowledge, we report for the first time NTRK and ALK rearrangements in a small subset of MPM. An ALK rearrangement was also detected in lung intermediate-grade NET (or atypical carcinoid). Our data suggest that IHC could be a useful screening test in such patients to ensure that all therapeutic strategies including targeted therapy are utilized.

Publisher Correction: A reference collection of patient-derived cell line and xenograft models of proneural, classical and mesenchymal glioblastoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Standard dose osimertinib for erlotinib refractory T790M-negative EGFR-mutant non-small cell lung cancer with leptomeningeal disease.

BACKGROUND: Leptomeningeal spread in non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations who experience disease progression on TKIs portends a poor prognosis. Mutation profiling of tumour DNA in cerebrospinal fluid (CSF) samples can be used to determine the presence of the EGFR T790M resistance mutation, indicating that osimertinib, a CNS-penetrating 3rd generation TKI may be efficacious. METHODS: Eight patients on EGFR TKIs who progressed with cytology-proven leptomeningeal disease at our institution were studied. EGFR mutations were profiled in CSF using droplet digital PCR (ddPCR) and compared to matched plasma samples. Clinical characteristics and survival outcomes on subsequent therapies tailored to ddPCR analysis were reported. RESULTS: None of the four patients who developed leptomeningeal disease while receiving 1st generation EGFR TKIs developed the EGFR T790M mutation in CSF. One patient who did not have extra-cranial disease and was EGFR T790M-negative in both plasma and CSF was nevertheless treated with standard-dose osimertinib, and achieved a rapid and durable response lasting 9 months to date. Three patients developed leptomeningeal disease on osimertinib, with one patient developing the EGFR C797S mutation in a cis-allelic conformation with the EGFR T790M mutation in plasma. CONCLUSIONS: Standard-dose osimertinib resulted in a clinically meaningful response in a patient with EGFR T790M-negative 1st generation EGFR TKI refractory leptomeningeal disease. Next generation sequencing and ddPCR has a role at identifying the C797S mutation and its allelic conformation with the T790M mutation with clinical implications.

The Measurement of Donor-Specific Cell-Free DNA Identifies Recipients With Biopsy-Proven Acute Rejection Requiring Treatment After Liver Transplantation.

BACKGROUND: Assessment of donor-specific cell-free DNA (dscfDNA) in the recipient is emerging as a noninvasive biomarker of organ rejection after transplantation. We previously developed a digital polymerase chain reaction (PCR)-based approach that readily measures dscfDNA within clinically relevant turnaround times. Using this approach, we characterized the dynamics and evaluated the clinical utility of dscfDNA after liver transplantation (LT). METHODS: Deletion/insertion polymorphisms were used to distinguish donor-specific DNA from recipient-specific DNA. Posttransplant dscfDNA was measured in the plasma of the recipients. In the longitudinal cohort, dscfDNA was serially measured at days 3, 7, 14, 28, and 42 in 20 recipients. In the cross-sectional cohort, dscfDNA was measured in 4 clinically stable recipients (>1-y posttransplant) and 16 recipients (>1-mo posttransplant) who were undergoing liver biopsies. RESULTS: Recipients who underwent LT without complications demonstrated an exponential decline in dscfDNA. Median levels at days 3, 7, 14, 28, and 42 were 1936, 1015, 247, 90, and 66 copies/mL, respectively. dscfDNA was higher in recipients with treated biopsy-proven acute rejection (tBPAR) when compared to those without. The area under the receiver operator characteristic curve of dscfDNA was higher than that of routine liver function tests for tBPAR (dscfDNA: 98.8% with 95% confidence interval, 95.8%-100%; alanine aminotransferase: 85.7%; alkaline phosphatase: 66.4%; gamma-glutamyl transferase: 80.1%; and bilirubin: 35.4%). CONCLUSIONS: dscfDNA as measured by probe-free droplet digital PCR methodology was reflective of organ health after LT. Our findings demonstrate the potential utility of dscfDNA as a diagnostic tool of tBPAR.

A reference collection of patient-derived cell line and xenograft models of proneural, classical and mesenchymal glioblastoma.

Low-passage, serum-free cell lines cultured from patient tumour tissue are the gold-standard for preclinical studies and cellular investigations of glioblastoma (GBM) biology, yet entrenched, poorly-representative cell line models are still widely used, compromising the significance of much GBM research. We submit that greater adoption of these critical resources will be promoted by the provision of a suitably-sized, meaningfully-described reference collection along with appropriate tools for working with them. Consequently, we present a curated panel of 12 readily-usable, genetically-diverse, tumourigenic, patient-derived, low-passage, serum-free cell lines representing the spectrum of molecular subtypes of IDH-wildtype GBM along with their detailed phenotypic characterisation plus a bespoke set of lentiviral plasmids for bioluminescent/fluorescent labelling, gene expression and CRISPR/Cas9-mediated gene inactivation. The cell lines and all accompanying data are readily-accessible via a single website, Q-Cell (qimrberghofer.edu.au/q-cell/) and all plasmids are available from Addgene. These resources should prove valuable to investigators seeking readily-usable, well-characterised, clinically-relevant, gold-standard models of GBM.

Somatic GNAQ mutation in the forme fruste of Sturge-Weber syndrome.

OBJECTIVE: To determine whether the GNAQ R183Q mutation is present in the forme fruste cases of Sturge-Weber syndrome (SWS) to establish a definitive molecular diagnosis. METHODS: We used sensitive droplet digital PCR (ddPCR) to detect and quantify the GNAQ mutation in tissues from epilepsy surgery in 4 patients with leptomeningeal angiomatosis; none had ocular or cutaneous manifestations. RESULTS: Low levels of the GNAQ mutation were detected in the brain tissue of all 4 cases-ranging from 0.42% to 7.1% frequency-but not in blood-derived DNA. Molecular evaluation confirmed the diagnosis in 1 case in which the radiologic and pathologic data were equivocal. CONCLUSIONS: We detected the mutation at low levels, consistent with mosaicism in the brain or skin (1.0%-18.1%) of classic cases. Our data confirm that the forme fruste is part of the spectrum of SWS, with the same molecular mechanism as the classic disease and that ddPCR is helpful where conventional diagnosis is uncertain.

Sensitive quantitative detection of somatic mosaic mutation in "double cortex" syndrome.

Somatic mutation of the lissencephaly-1 gene is a cause of subcortical band heterotopia ("double cortex"). The severity of the phenotype depends on the level of mutation in brain tissue. Detecting and quantifying low-level somatic mosaic mutations is challenging. Here, we utilized droplet digital PCR, a sensitive method to detect low-level mutation. Droplet digital PCR was used in concert with classic genotyping techniques (SNaPshot assays and pyrosequencing) to detect and characterize the tissue mosaicism of a somatic mutation (LIS1 c.190A>T; p.K64X) in a patient with posterior bilateral SBH and refractory epilepsy. The high sensitivity of droplet digital PCR and the ability to target individual DNA molecules allowed us to detect the mutation at low level in the brain, despite the low quality of the DNA sample derived from formalin-fixed paraffin-embedded tissue. This low mutation frequency in the brain was consistent with the relatively subtle malformation resolved by magnetic resonance imaging. The presence of the mutation in other tissues from the patient permitted us to predict the timing of mutagenesis. This sensitive methodology will have utility for a variety of other brain malformation syndromes associated with epilepsy for which historical pathological specimens are available and specific somatic mosaic mutations are predicted.

GRIDSS: sensitive and specific genomic rearrangement detection using positional de Bruijn graph assembly.

The identification of genomic rearrangements with high sensitivity and specificity using massively parallel sequencing remains a major challenge, particularly in precision medicine and cancer research. Here, we describe a new method for detecting rearrangements, GRIDSS (Genome Rearrangement IDentification Software Suite). GRIDSS is a multithreaded structural variant (SV) caller that performs efficient genome-wide break-end assembly prior to variant calling using a novel positional de Bruijn graph-based assembler. By combining assembly, split read, and read pair evidence using a probabilistic scoring, GRIDSS achieves high sensitivity and specificity on simulated, cell line, and patient tumor data, recently winning SV subchallenge #5 of the ICGC-TCGA DREAM8.5 Somatic Mutation Calling Challenge. On human cell line data, GRIDSS halves the false discovery rate compared to other recent methods while matching or exceeding their sensitivity. GRIDSS identifies nontemplate sequence insertions, microhomologies, and large imperfect homologies, estimates a quality score for each breakpoint, stratifies calls into high or low confidence, and supports multisample analysis.

Interlaboratory Reproducibility of Droplet Digital Polymerase Chain Reaction Using a New DNA Reference Material Format.

Use of droplet digital PCR technology (ddPCR) is expanding rapidly in the diversity of applications and number of users around the world. Access to relatively simple and affordable commercial ddPCR technology has attracted wide interest in use of this technology as a molecular diagnostic tool. For ddPCR to effectively transition to a molecular diagnostic setting requires processes for method validation and verification and demonstration of reproducible instrument performance. In this study, we describe the development and characterization of a DNA reference material (NMI NA008 High GC reference material) comprising a challenging methylated GC-rich DNA template under a novel 96-well microplate format. A scalable process using high precision acoustic dispensing technology was validated to produce the DNA reference material with a certified reference value expressed in amount of DNA molecules per well. An interlaboratory study, conducted using blinded NA008 High GC reference material to assess reproducibility among seven independent laboratories demonstrated less than 4.5% reproducibility relative standard deviation. With the exclusion of one laboratory, laboratories had appropriate technical competency, fully functional instrumentation, and suitable reagents to perform accurate ddPCR based DNA quantification measurements at the time of the study. The study results confirmed that NA008 High GC reference material is fit for the purpose of being used for quality control of ddPCR systems, consumables, instrumentation, and workflow.

EGFR and KRAS mutations do not enrich for the activation of IL-6, JAK1 or phosphorylated STAT3 in resected lung adenocarcinoma.

Resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) against EGFR mutant lung adenocarcinoma develops after a median of nine to thirteen months. Upregulation of the interleukin-6 (IL-6)/Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway may be a potential source of resistance to EGFR TKIs. We undertook a detailed assessment of the IL-6/JAK1/phosphorylated STAT3 (pSTAT3) pathway in resected lung adenocarcinoma specimens, with special interest in whether the presence of an EGFR mutation enriched for pSTAT3 positivity. Tumours from 143 patients with resected lung adenocarcinoma were assessed. EGFR and KRAS mutation status were scanned for with high-resolution melting and confirmed by polymerase chain reaction. Immunohistochemisty (IHC) was performed for IL-6, gp130, JAK1 and pSTAT3. Two methods for assigning IHC positivity were assessed (the presence of any positivity, and the presence of positivity at an H score >40). We found statistically significant associations between IL-6, JAK1 and pSTAT3 measured by IHC, consistent with the activation of the pathway in clinical specimens. No relationship was demonstrated between members of this pathway and oncogenic mutations in EGFR or KRAS. However, a proportion of tumours with EGFR mutations showed staining for IL-6, JAK1 and pSTAT3. No correlations with clinicopathologic features or survival outcomes were found for IL-6, JAK1 or pSTAT3 staining. The presence of EGFR or KRAS mutations did not enrich for the activation of IL-6, JAK1 or pSTAT3. pSTAT3 may still play a role in resistance to EGFR TKIs in clinical practice.

De novo activating epidermal growth factor mutations (EGFR) in small-cell lung cancer.

In Australia, mutations in epidermal growth factor mutations (EGFR) occur in 15% of patients diagnosed with non-small-cell lung cancer and are found with higher frequency in female, non-smokers of Asian ethnicity. Activating mutations in the EGFR gene are rarely described in SCLC. We present two cases of de novo EGFR mutations in patients with SCLC detected in tissue and in plasma cell free DNA, both of whom were of Asian ethnicity and never-smokers. These two cases add to the growing body of evidence suggesting that screening for EGFR mutations in SCLC should be considered in patients with specific clinical features.

Combination Osimertinib and Gefitinib in C797S and T790M EGFR-Mutated Non-Small Cell Lung Cancer.

INTRODUCTION: Osimertinib, a third-generation EGFR tyrosine kinase inhibitor has demonstrated efficacy in tumors harboring the EGFR T790M resistance mutation. Inevitably, resistance to third-generation inhibitors results in disease progression, with the EGFR C797S mutation being one of several resistance pathways identified to date. On the basis of preclinical data, we report what is the first known case of a patient harboring the T790M and C797S mutations in trans treated with combination gefitinib and osimertinib. METHODS: On development of progressive disease after multiple therapies, the patient's plasma was sequenced using the Oncomine Lung cfDNA Assay (Thermo Fisher Scientific, Waltham, MA). Subsequent monitoring of circulating tumor DNA in plasma was performed by droplet digital polymerase chain reaction. RESULTS: Sequencing showed that the T790M and C797S mutations were in trans. Within 2 weeks of commencement of combination therapy, rapid clinical improvement occurred. Accompanying this, a rapid decline in the C797S mutation subclone in plasma was detected. However, the levels of the EGFR exon 19 deletion driver mutation and the T790M resistance mutation in the circulating tumor DNA continued to rise and the patient died from progressive disease 6 weeks after commencement of combination therapy. There were no adverse events seen with the combination therapy. CONCLUSION: This is, to the best of our knowledge, the first reported case of combination EGFR tyrosine kinase inhibitor therapy tailored to the allelic conformation of T790M and C797S mutation that resulted in brief clinical improvement without toxicity.

Reducing Artifactual EGFR T790M Mutations in DNA from Formalin-Fixed Paraffin-Embedded Tissue by Use of Thymine-DNA Glycosylase.

BACKGROUND: False-positive EGFR T790M mutations have been reported in formalin-fixed lung tumors, but the cause of the false positives has not been identified. The T790M mutation results from a C>T change at the cytosine of a CpG dinucleotide. The presence or absence of methylation at this cytosine has different consequences following deamination, resulting in a thymine or uracil, respectively, both of which however result in an artifactual change. Uracil-DNA glycosylase (UDG) can be used to eliminate DNA templates with uracil residues but is not active against artifactual thymines. We therefore investigated the use of thymine-DNA glycosylase (TDG) to reduce artifactual T790M mutations. METHODS: Formalin-fixed normal lung tissues and lung squamous cell carcinomas were tested to measure the frequency of false-positive EGFR mutations by use of droplet digital PCR before and after treatment with either UDG or TDG. Methylation at the cytosine at EGFR T790 was assessed by pyrosequencing and by analysis of public databases. RESULTS: Artifactual EGFR T790M mutations were detected in all of the archival formalin-fixed normal lung and lung squamous cell carcinomas at mutant allele frequencies of 1% or lower. The cytosine at EGFR T790 showed high levels of methylation in all lung cancer samples and normal tissues. Pretreatment of the formalin-fixed DNA with either UDG or TDG reduced the false EGFR T790M mutations, but a greater reduction was seen with the TDG treatment. CONCLUSIONS: Both U:G and T:G lesions in formalin-fixed tissue are sources of false-positive EGFR T790M mutations. This is the first report of the use of TDG to reduce sequence artifacts in formalin-fixed DNA and is applicable to the accurate detection of mutations arising at methylated cytosines.

Probe-Free Digital PCR Quantitative Methodology to Measure Donor-Specific Cell-Free DNA after Solid-Organ Transplantation.

BACKGROUND: Donor-specific cell-free DNA (dscfDNA) is increasingly being considered as a noninvasive biomarker to monitor graft health and diagnose graft rejection after solid-organ transplantation. However, current approaches used to measure dscfDNA can be costly and/or laborious. A probe-free droplet digital PCR (ddPCR) methodology using small deletion/insertion polymorphisms (DIPs) was developed to circumvent these limitations without compromising the quantification of dscfDNA. This method was called PHABRE-PCR (Primer to Hybridize across an Allelic BREakpoint-PCR). The strategic placement of one primer to hybridize across an allelic breakpoint ensured highly specific PCR amplification, which then enabled the absolute quantification of donor-specific alleles by probe-free ddPCR. METHODS: dscfDNA was serially measured in 3 liver transplant recipients. Donor and recipient genomic DNA was first genotyped against a panel of DIPs to identify donor-specific alleles. Alleles that differentiated donor-specific from recipient-specific DNA were then selected to quantify dscfDNA in the recipient plasma. RESULTS: Lack of amplification of nontargeted alleles confirmed that PHABRE-PCR was highly specific. In recipients who underwent transplantation, dscfDNA was increased at day 3, but decreased and plateaued at a low concentration by 2 weeks in the 2 recipients who did not develop any complications. In the third transplant recipient, a marked increase of dscfDNA coincided with an episode of graft rejection. CONCLUSIONS: PHABRE-PCR was able to quantify dscfDNA with high analytical specificity and sensitivity. The implementation of a DIP-based approach permits surveillance of dscfDNA as a potential measure of graft health after solid-organ transplantation.

Reduced abundance of the E3 ubiquitin ligase E6AP contributes to decreased expression of the INK4/ARF locus in non-small cell lung cancer.

The tumor suppressor p16INK4a, one protein encoded by the INK4/ARF locus, is frequently absent in multiple cancers, including non-small cell lung cancer (NSCLC). Whereas increased methylation of the encoding gene (CDKN2A) accounts for its loss in a third of patients, no molecular explanation exists for the remainder. We unraveled an alternative mechanism for the silencing of the INK4/ARF locus involving the E3 ubiquitin ligase and transcriptional cofactor E6AP (also known as UBE3A). We found that the expression of three tumor suppressor genes encoded in the INK4/ARF locus (p15INK4b, p16INK4a, and p19ARF) was decreased in E6AP-/- mouse embryo fibroblasts. E6AP induced the expression of the INK4/ARF locus at the transcriptional level by inhibiting CDC6 transcription, a gene encoding a key repressor of the locus. Luciferase assays revealed that E6AP inhibited CDC6 expression by reducing its E2F1-dependent transcription. Chromatin immunoprecipitation analysis indicated that E6AP reduced the amount of E2F1 at the CDC6 promoter. In a subset of NSCLC samples, an E6AP-low/CDC6-high/p16INK4a-low protein abundance profile correlated with low methylation of the gene encoding p16INK4a (CDKN2A) and poor patient prognosis. These findings define a previously unrecognized tumor-suppressive role for E6AP in NSCLC, reveal an alternative silencing mechanism of the INK4/ARF locus, and reveal E6AP as a potential prognostic marker in NSCLC.