Supplementary Methods, Figures 1-5. Supplementary Figure 1. Outline of cohort selection. Supplementary Figure 2. Example of whole-genome single-nucleotide polymorphism (SNP)array profile and genome alteration print (GAP). Supplementary Figure 3. Pre-treatment serum CA125 levels. Supplementary Figure 4. Progression-free and overall survival in patients with SBT-EOC and EOC without adjacent SBT. Supplementary Figure 5. Progression-free and overall survival of patients with Grade 3 SBTEOC and Grade 3 EOC without adjacent SBT

Supplementary Highlighted Methods. This document contains the revised supplementary methods and results with changes highlighted

Supplementary Tables 1-7. Supplementary Table 1. SBT-EOC cases used for molecular analyses Supplementary Table 2. Mutations Screened on the Sequenom/OncoMap3 Platform Supplementary Table 3. Primer sequences for the indicated exons of TP53, ERBB2 and NRAS Supplementary Table 4. Clinico-pathologic characteristics of the subset of SBT-EOC used for molecular analysis compared to the entire SBT-EOC cohort Supplementary Table 5A. Somatic mutations found in borderline and invasive ovarian tumors Supplementary Table 5B. Genes in which somatic mutations were found according to grade and co-existing serous borderline regions Supplementary Table 6. Genes differentially expressed between paired borderline and invasive regions of SBTEOC (p<0.01, not corrected for multiple comparison). Supplementary Table 7. Clinico-pathological characteristics of SBTEOC cases used for progesterone receptor (PGR) immunostaining

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.

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 180bp 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.

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.

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.

Introduction: Over 90% of breast cancers occur sporadically with no obvious hereditary factors at play, therefore highlighting the need for biomarkers that can identify women who are predisposed to developing breast cancer. DNA methylation of the BRCA1 promoter region is detectable at low levels in the peripheral blood of some women. This phenomenon is referred to as constitutional BRCA1 methylation. Peripheral blood methylation of the BRCA1 gene has been identified as a predisposition factor to the development of BRCA1 methylated tumors. We performed a case-control study to assess peripheral blood BRCA1 methylation from healthy women, and from women with breast cancer. This study aimed to establish the frequency of peripheral blood methylation of the BRCA1 gene in women with and without breast cancer, to further our understanding of the role of constitutional BRCA1 methylation in breast cancer predisposition. Methods: Constitutional BRCA1 methylation was assessed in peripheral blood DNA of breast cancer patients and healthy women. Whole blood DNA was obtained from healthy women (n=327 controls) and women with breast cancer (n=300 cases) as part of the LifePool Project. Blood was collected on the day of mammography with BreastScreen Victoria. We used a probe-based droplet digital PCR (ddPCR) assay designed to quantify methylated and unmethylated alleles at the BRCA1 promoter region. Results: Previous research using less-sensitive methodologies has reported a BRCA1 methylation frequency of 2-4% in peripheral blood of healthy women. In this study, there was no significant difference in the observed BRCA1 methylation frequency between cases and controls (6.6% and 6.4% respectively). The discrepancy between our observed methylation frequencies and previously reported data can be attributed to the higher sensitivity of the ddPCR methodology. Nevertheless, the level of BRCA1 methylation in cases was significantly higher than the level of methylation observed in controls, consistent with previous studies. In addition, the frequency of BRCA1 methylation was higher in women diagnosed with breast cancer under the age of 50 compared to women diagnosed over 50 years old (average methylation frequency = 12.5% and 5.3% respectively). Conclusions: ddPCR methodology enabled accurate quantification of methylated BRCA1 alleles in peripheral blood DNA down to less than 0.1%. In this selected population of women undergoing mammography, there was no difference in the frequency of detectable BRCA1 methylation between cases and controls. Once age was considered, detectable BRCA1 methylation showed an association with age of onset. Tumours for the women with breast cancer will be retrieved and tested for BRCA1 methylation, to further examine the link between the methylation observed in peripheral blood DNA and development of a BRCA1-methylated tumor. Citation Format: Basant Ebaid, Hongdo Do, Jia Min Pang, Lisa Devereux, Alexander Dobrovic. A case-control study of constitutional BRCA1 methylation in a mammographically screened cohort [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 823.

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.

BackgroundLeptomeningeal 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.MethodsEight 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.ResultsNone 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.ConclusionsStandard-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.

e20558 Background: Gene rearrangements involving NTRK1, NTRK2, NTRK3, ROS1 and ALK result in oncogenic fusion proteins that have been identified in many tumor types, including lung, colorectal, salivary gland, sarcoma, papillary thyroid, glioblastoma, melanoma. Identification of these rare rearrangements enables potential therapeutic targeting with specific inhibitors. Given the limited data available in rare tumor types, we investigated a panel of fusion genes in mesothelioma, small cell lung cancer and carcinoid tumors. Methods: Tumor tissue samples from 335 patients with mesothelioma, 154 with carcinoid and 105 with small cell lung cancer were arrayed in triplicate onto tissue microarrays (TMAs). TMA slides were stained by immunohistochemistry(IHC) with antibodies against NTRK1,2,3 ROS1 and ALK. Positive samples by IHC underwent RNA sequencing using the ArcherDX FusionPlex CTL (Comprehensive Thyroid and Lung) diagnostic assay. Target-enriched libraries were sequenced on the Illumina Miseq NGS platform and analysed using the cloud-based Archer Analysis tool to call NTRK1,2,3, ROS1 or ALK fusion transcripts. Results: There were 15 positive cases via IHC, eight mesothelioma and 7 carcinoid. Of these, 1 mesothelioma patient with an NTRK ex10-TMP3 ex8 fusion, 1 mesothelioma patient with an ALK ex20-EML4ex13 and 1 atypical carcinoid patient with an ALK ex20-EML4 ex6/intron6 was found. Clinicopathological features of the NTRK positive mesothelioma patient demonstrated biphasic histology subtype, with documented asbestosis exposure. The ALK positive mesothelioma patient had sarcomatoid histology, whilst the carcinoid patient had resected stage 3A disease, but died after relapsing after 6 months with metastatic disease. Conclusions: This is the first study to demonstrate the existence of NTRK gene fusions in mesothelioma. ALK rearrangements were also detected in carcinoid and mesothelioma. In spite of the low incidence, IHC would be a useful screening test in such patients to ensure that all therapeutic strategies including targeted therapy are utilised.

To the Editor: Droplet digital PCR (ddPCR)1 is an emerging platform that is being increasingly adopted by clinical laboratories. The partitioning of a PCR reaction into discrete droplets enables the accurate detection and quantification of molecular targets. Single molecule analysis by this manner is accurate, cost-effective, and readily performed compared with next-generation sequencing and mass spectrometry. The typical ddPCR workflow comprises 4 key steps: ( a ) preparation of the PCR reaction, ( b ) droplet generation (DG), ( c ) endpoint PCR, and ( d ), droplet analysis. The process of DG is a critical step of the workflow because this step is considered to be most prone to droplet loss (1). It is thus important to recognize that a loss of droplets during DG will affect the number of droplets recoverable for droplet analysis in an optimized assay. Consequently, lower droplet numbers can lead to a loss of analytical sensitivity and may confound the evaluation of samples for molecular targets that are present in very low concentrations. To date, informative measures to evaluate DG performance within the ddPCR workflow remain unaddressed. The Levey–Jennings chart in conjunction with the “Westgard rules” is the staple of quality management in clinical chemistry measurement systems. Here, we outline an approach that uses the Levey–Jennings chart and a set of adapted Westgard rules to monitor DG performance in …

Our case demonstrated that the clinical efficacy of combination osimertinib with gefitinib was brief and paralleled a reduction in the C797S clone in plasma. However, the T790M and exon 19 deletion alleles continued to rise. As pointed out by Leone,1Leone A. C797S and T790M EGFR mutations in non-small-cell lung cancer: in trans or in separate clones?.J Thorac Oncol. 2018; 13: e21-e22Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar there are two possible situations in which the EGFR T790M and C797S mutations could exist on different DNA strands—occurring either within the same tumor cell or in different tumor cell clones. The case presented by Wang et al.2Wang Z. et al.Lung adenocarcinoma harboring EGFR T790M and in trans C797S responds to combination therapy of first- and third-generation EGFR TKIs and shifts allelic configuration at resistance.J Thorac Oncol. 2017; 12: 1723-1727Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar supports the existence of the C797S mutation occurring in trans within the same cell, as the allele frequencies appear to follow one another and the subsequent C797S was found to occur in cis on development of resistance. Our digital droplet polymerase chain reaction and deep sequencing results of plasma DNA do not provide a direct answer for this important question, but they suggest that the EGFR T790M and C797S mutations occurred in separate cellular clones. The level of the C797S mutation is expected to be similar to or lower than the level of the T790M mutation if both mutations are present in the same cells, and it would show either concomitant increase or decrease in response to the combination therapy. The levels of each EGFR mutation measured by digital droplet polymerase chain reaction from a series of plasma samples indicated that the T790M and C797S mutations were likely present in different tumor cells, a finding that as we suggested in our article, may be due to multiple clones with individual mutations and resultant differences in tyrosine kinase sensitivity.3Arulananda S. Do H. Musafer A. Mitchell P. Dobrovic A. John T. Combination osimertinib and gefitinib in C797S and T790M EGFR-mutated non–small cell lung cancer.J Thorac Oncol. 2017; 12: 1728-1732Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar In the blood collected before the combination therapy, the levels of the T790M and C797S mutations were detected at mutant allele frequencies of 2.8% and 4.5%, respectively. This indicates that the overall load of C797S-positive tumor was higher than that of T790M-positive tumor before the combination therapy. The observed clinical improvement without reduction in T790M or exon 19 deletion also supports this hypothesis of clonal heterogeneity rather than the mutation being present in the same cells. Definitive proof, however, would require single-cell analysis, which is not possible for this patient. C797S and T790M EGFR Mutations in Non–Small Cell Lung Cancer: In Trans or in Separate Clones?Journal of Thoracic OncologyVol. 13Issue 2PreviewI read with great interest the article “Combination Osimertinib and Gefitinib in C797S and T790M EGFR-Mutated Non–Small Cell Lung Cancer” by Arulananda et al. that was recently published in the Journal of Thoracic Oncology.1 Using up-to-date methodologies, the authors monitored the EGFR mutation status in circulating tumor DNA (ctDNA) in the plasma of a patient with progressive disease after multiple lines of therapy, including tyrosine kinase inhibitors (TKIs). They detected T790M and C797S resistance mutations on different DNA strands and postulated a trans configuration. Full-Text PDF Open Archive

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.

Introduction There is an increasing interest in the use of donor-specific cell-free DNA (dscfDNA) as a non-invasive biomarker of organ health after transplantation. We have developed a PCR-based approach that readily measures dscfDNA. Using this approach, we evaluated the utility of dscfDNA in two separate cohorts of recipients. Methods Deletion/insertion polymorphisms (DIP) were used to distinguish donor-specific DNA and recipient-specific DNA. Post-transplant dscfDNA was measured using a simple and novel probe-free droplet digital PCR (ddPCR) methodology. 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 quantified in 16 recipients (>3 months post-transplant) undergoing liver biopsies.\ Results DscfDNA levels were reflective of organ health after liver transplantation. In the recipients who underwent uncomplicated transplantation, dscfDNA markedly reduced at day 7 and remained at a low median level of 76.45 (IQR: 50.60-108.90) dscfDNA copies/mL from day 14 onwards. Furthermore, dscfDNA was consistently lower in recipients who did not have any evidence of rejection compared to those who developed biopsy-proven organ rejection (281.50 dscfDNA copies/mL vs 1,661.00 dscfDNA copies/mL respectively, p<0.05). The area under the receiver operator characteristic curve of dscfDNA was higher compared to routine serum liver biochemistry (dscfDNA: 0.818, ALT: 0.745, GGT: 0.627, ALP: 0.436). Conclusion In this study, we demonstrated a readily performed methodology to measure dscfDNA. We also highlighted the utility of dscfDNA as a promising non-invasive biomarker for the surveillance of organ health and the diagnosis of organ rejection after liver transplantation.

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.

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.

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.

Non-small cell lung cancer (NSCLC) spread to the central nervous system (CNS) is associated with universally poor outcomes. Diagnosis of leptomeningeal (LM) disease is particularly challenging, with radiological and cytological assessment of CSF often negative. Examination of cell-free DNA (cfDNA) is promising method for the assessment of tumor mutation status and disease monitoring. We examine its utility in the diagnosis and management of LM disease. Patients with EGFR mutated NSCLC with symptoms suggestive of LM disease underwent cerebrospinal fluid (CSF) sampling and ddPCR assessment. Matched plasma and/or tumor biopsy samples were obtained. Clinical data was collected retrospectively. Genomic DNA was extracted from 1 mL of CSF and 4 mL of plasma using the Qiagen QIAamp Circulating Nucleic Acid kit and T790M and L858R mutations were assessed by using the QX200 ddPCR assays. Survival was calculated from diagnosis and diagnosis of LM disease. Seven patients (pts) were included in the analysis. Most pts, 6/7 (86%) developed LM either during or after EGFR tyrosine kinase inhibitor (TKI) therapy. EGFR mutations were demonstrated in CSF in 6 (86%) pts, but only 2 (29%) demonstrated the same mutation profile in both CSF and plasma. Causes of discrepancy were between CSF and plasma/tissue were: two cases were CSF positive but had no extrathoracic disease to biopsy, 1 case was negative in the CSF for T790M but positive in plasma and on biopsy and one case was wild type in CSF but positive on plasma and biopsy for G719A+T790M mutation. Six pts (86%) received therapy after diagnosis of LM disease and OS from LM diagnosis ranged from 1.0 -15.8 months. ddPCR is able to detect cfDNA in CSF, where other diagnostic modalities may be negative, and demonstrates both initial and resistance mutations. Heterogeneous mutation status may guide therapy when LM disease remains sensitive to targeted therapy.