•Next-generation sequencing (NGS) of cell-free DNA could expand access to precision medicine.•Technical differences between plasma NGS assays are poorly characterized.•We compare complex variants ...detection between two plasma NGS assays.•Using tumor as a reference, we identify biochemical and bioinformatic differences.
Plasma genotyping represents an opportunity for convenient detection of clinically actionable mutations in advanced cancer patients, such has been well-documented in non-small cell lung cancer (NSCLC). Oncogenic gene fusions are complex variants that may be more challenging to detect by next-generation sequencing (NGS) of plasma cell-free DNA (cfDNA). Rigorous evaluation of plasma NGS assays in the detection of fusions is needed to maximize clinical utility.
: Additional plasma was collected from patients with advanced NSCLC and ALK, ROS1, or RET gene fusions in tissue who had undergone clinical plasma NGS using Guardant360™(G360, Guardant Health). We then sequenced extracted cfDNA with a plasma NGS kit focused on known driver mutations in NSCLC (ctDx-Lung, Resolution Bioscience) with cloud-based bioinformatic analysis and blinded variant calling.
Of 16 patients assayed known to harbor anALK, ROS1, or RET in tumor, G360 detected fusions in 7 cases, ctDx-Lung detected fusions in 13 cases, and 3 cases were detected by neither. Of the 7 fusions detected by both assays, G360 reported lower mutant allelic fractions (AF). In cases missed by G360, tumor derived TP53 mutations were often detected confirming presence of tumor DNA. Raw sequencing data showed that inverted or out-of-frame variants were overrepresented in cases detected using ctDx-Lung but not by G360.
Focusing on complex, clinically actionable mutations using tumor as a reference standard allows for evaluation of technical differences in plasma NGS assays that may impact clinical performance. Noting the heterogeneity of fusion sequences observed in NSCLC, we hypothesize that differences in hybrid capture techniques and bioinformatic calling may be sources of variations in sensitivity among these assays.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
•Salivary HPV DNA levels align with local disease burden in HPV oropharyngeal cancer.•The rise and fall of salivary HPV DNA often predicts treatment response or failure.•Plasma HPV cell-free DNA more ...often correlates with distant disease burden.
Quantifying tumor DNA in tissue and circulating in blood permits high-quality molecular monitoring to detect and track cancer progression. Evaluating tumor DNA in both blood and saliva in human papillomavirus (HPV)-associated oropharyngeal cancer (OPC) could provide a non-invasive and clinically actionable method for real-time disease detection.
We previously validated an ultrasensitive droplet-digital (dd)PCR assay targeting the dominant high-risk HPV subtypes causally linked to OPC. Here we enrolled an observational cohort to evaluate the predictive and prognostic potential of paired plasma-salivary tumor DNA among 21 patients with advanced HPV+OPC.
In patients with recurrent, persistent locoregional (LR) disease, median baseline normalized salivary HPV DNA was 10.9 copies/ng total DNA, nearly 20x higher compared with those with distant disease only (p = 0.01). A cutoff of 5 copies/ng yielded 87% sensitivity and 67% specificity for accurately predicting LR disease. Total tumor burden among those with LR disease strongly correlated with salivary HPV DNA levels (R = 0.83, p = 0.02). The rise and fall of salivary HPV DNA predicted treatment failure and response, respectively, in all patients with LR disease, and predated imaging findings. Among paired salivary-plasma (cell-free) cfDNA samples, only higher plasma HPV cfDNA levels were associated with poor outcomes (p < 0.01), suggesting that each bodily fluid provides unique information about HPV disease status.
Salivary HPV DNA provides valuable information about tumor burden and predicts treatment response in advanced HPV+OPC. Paired blood-saliva samples could be used to monitor HPV DNA with broad applications to inform diagnosis, prognosis, and surveillance in HPV-associated diseases.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
ATP-citrate lyase (ACLY) links carbohydrate and lipid metabolism and provides nucleocytosolic acetyl-CoA necessary for protein acetylation. ACLY has two major splice isoforms: the full-length ...canonical “long” isoform and an uncharacterized “short” isoform in which exon 14 is spliced out. Exon 14 encodes 10 amino acids within a disordered region of the protein and includes at least one site that is dynamically phosphorylated. Both isoforms are expressed in healthy tissues to varying degrees. Analysis of human transcriptomic data revealed that the percent spliced in (PSI) of exon 14, i.e., the proportion of long isoform, is increased in several cancers and correlated with poorer overall survival in a pan-cancer analysis, though not in individual tumor types, which prompted us to explore potential biochemical and functional differences between ACLY isoforms. Here, we show that there are no discernible differences in enzymatic activity or stability between isoforms or phosphomutants of ACLY in vitro. Similarly, both isoforms and phosphomutants were able to rescue ACLY functions, including fatty acid synthesis and bulk histone acetylation, when re-expressed in Acly knockout cells. Deletion of Acly exon 14 in mice did not overtly impact development or metabolic physiology nor did it attenuate tumor burden in a genetic model of intestinal cancer. Notably, expression of epithelial splicing regulatory protein 1 (ESRP1) is highly correlated with ACLY PSI. We report that ACLY splicing is regulated by ESRP1. In turn, both ESRP1 expression and ACLY PSI are correlated with specific immune signatures in tumors. Despite these intriguing patterns of ACLY splicing in healthy and cancer tissues, functional differences between the isoforms remain elusive.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Pancreatic ductal adenocarcinoma (PDAC) is commonly diagnosed at an advanced stage. Liquid biopsy approaches may facilitate detection of early stage PDAC when curative treatments can be employed.
To ...assess circulating marker discrimination in training, testing and validation patient cohorts (total n=426 patients), plasma markers were measured among PDAC cases and patients with chronic pancreatitis, colorectal cancer (CRC), and healthy controls. Using CA19-9 as an anchor marker, measurements were made of two protein markers (TIMP1, LRG1) and cell-free DNA (cfDNA) pancreas-specific methylation at 9 loci encompassing 61 CpG sites.
Comparative methylome analysis identified nine loci that were differentially methylated in exocrine pancreas DNA. In the training set (n=124 patients), cfDNA methylation markers distinguished PDAC from healthy and CRC controls. In the testing set of 86 early stage PDAC and 86 matched healthy controls, CA19-9 had an area under the receiver operating characteristic curve (AUC) of 0.88 (95% CI 0.83 to 0.94), which was increased by adding TIMP1 (AUC 0.92; 95% CI 0.88 to 0.96; p=0.06), LRG1 (AUC 0.92; 95% CI 0.88 to 0.96; p=0.02) or exocrine pancreas-specific cfDNA methylation markers at nine loci (AUC 0.92; 95% CI 0.88 to 0.96; p=0.02). In the validation set of 40 early stage PDAC and 40 matched healthy controls, a combined panel including CA19-9, TIMP1 and a 9-loci cfDNA methylation panel had greater discrimination (AUC 0.86, 95% CI 0.77 to 0.95) than CA19-9 alone (AUC 0.82; 95% CI 0.72 to 0.92).
A combined panel of circulating markers including proteins and methylated cfDNA increased discrimination compared with CA19-9 alone for early stage PDAC.
Plasma circulating tumor DNA (ctDNA) analysis is routine for genotyping of advanced non-small-cell lung cancer (NSCLC); however, early response assessment using plasma ctDNA has yet to be well ...characterized.
Patients with advanced
-mutant NSCLC across three phase I NCI osimertinib combination trials were analyzed in this study, and an institutional cohort of patients with
,
, and
-mutant advanced NSCLC receiving systemic treatment was used for validation. Plasma was collected before treatment initiation and serially before each cycle of therapy, and key driver mutations in ctDNA were characterized by droplet digital polymerase chain reaction. Timing of plasma versus imaging response was compared in a separate cohort of patients with
-mutant NSCLC treated with osimertinib. Across cohorts, we also studied ctDNA variability before treatment start.
In the NCI cohort, 14/16 (87.5%) patients exhibited ≥ 90% decrease in mutation abundance by the first on-treatment timepoint (20-28 days from treatment start) with minimal subsequent change. Similarly, 47/56 (83.9%) patients with any decrease in the institutional cohort demonstrated ≥ 90% decrease in mutation abundance by the first follow-up draw (7-30 days from treatment start). All 16 patients in the imaging cohort with radiographic partial response showed best plasma response within one cycle, preceding best radiographic response by a median of 24 weeks (range: 3-147 weeks). Variability in ctDNA levels before treatment start was observed.
Plasma ctDNA response is an early phenomenon, with the majority of change detectable within the first cycle of therapy. These kinetics may offer an opportunity for early insight into treatment effect before standard imaging timepoints.
Abstract
Purpose: Human papillomavirus (HPV) is associated with the majority of oropharyngeal squamous cell carcinomas (OPSCC), with a rising incidence of OPSCC predominantly among middle-aged men. ...Tumor-derived HPV cell-free (cf)DNA can be detected and quantified in circulation with high sensitivity and specificity. Although cfDNA has limited clinical application in monitoring locoregional spread of these tumors, up to 10% of HPV+ OPSCC patients present or recur with distant, metastatic disease. The potential of HPV DNA monitoring in this setting remains largely unexplored.
Methods: We present a proof-of-concept prospective observational cohort of recurrent, metastatic (R/M) HPV+ OPSCC patients treated with standard systemic therapies. We utilized droplet digital (dd)PCR to identify and quantify HPV cfDNA (strains 16, 18, 31, 33 and 45) at multiple time points throughout treatment. We then compared HPV cfDNA concentration at various timepoints with clinical parameters such as disease burden and treatment response.
Results: Clinicopathologic data from 12 R/M patients revealed a predominantly male cohort (11/12, 92%) with a median age of 55 at diagnosis. Eight (67%) were on immunotherapy treatments and the other four on standard chemotherapy during the study period (2.5 months). Plasma HPV cfDNA was detected in 6/12 (50%) samples (range 0-4460 copies/mL) in at least one timepoint during the study. All patients with undetectable HPV cfDNA had evidence of stable, low burden disease or no measurable lesions (near-complete response) on their current therapy. HPV cfDNA levels strongly correlated with both disease burden (p < 0.01) and distant disease sites (p < 0.01). In three cases, HPV DNA levels declined prior to contrast enhanced imaging findings which later confirmed treatment response (up to 14 days prior to planned imaging).
Conclusion: Our results suggest that high sensitivity plasma HPV cfDNA levels can be an early indicator of treatment response. HPV cfDNA monitoring could significantly impact clinical decision-making and improve patient outcomes by informing treatment response or failure. Further studies are underway to validate these findings and determine how early cfDNA can detect metastatic disease.
Citation Format: Glenn J. Hanna, Julianna G. Supplee, Yanan Kuang, Kate Geary, Pasi A. Janne, Robert I. Haddad, Cloud P. Paweletz. Plasma HPV cell free DNA as an earlier predictor of treatment response in advanced oropharyngeal cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2581.
Abstract
Background: Non-invasive genotyping of cell-free DNA (cfDNA) is increasingly used in cancer care as tumor biopsies may be inadequate or unavailable. As clinical adaptation of liquid biopsies ...is driven in large part by commercial vendors that offer proprietary PCR or NGS-based tests, rigorous validation of these assays is essential to ensure maximum clinical benefit. Recent efforts to compare cfDNA diagnostics have not focused on clinically actionable mutations or used tumor to verify plasma results. The present study analyzes the concordance in reports of actionable gene fusions in NSCLC from two independent, commercial plasma NGS tests, compares the breakpoint characteristics between tissue and plasma, and highlights how differences in sequencing and bioinformatic analysis can be a source of false negatives.
Methods/Results: We studied a cohort of 169 NSCLC patients (pts) that had plasma analyzed by Guardant 360 between April 2016 and July 2018. In those with tumor positive ALK, ROS1, or RET fusions (n=16), banked cfDNA from a separate tube of plasma (69% collected within 2 weeks) underwent local testing and sequencing at DFCI using Resolution Bioscience's pre-production ctDx-Lung kit with remote bioinformatic variant calling performed by Resolution Bioscience; all parties involved were blinded to tumor genotype and Guardant results. Locked results were unblinded for post hoc analysis. The ctDx-lung kit detected 13 out 16 fusions (AF% range 80-0.3%), while Guardant360 detected 7 (AF% range 10-0.3%). Guardant360 tended to report lower AFs than ctDx-lung, though corroborating TP53 alterations were of similar AFs. Of the cases where both assays did not detect any fusions, no other shared SNVs were detected, possibly due to low shed of tumor DNA. Of the 13 cases where a fusion was detected in plasma by ctDx-lung, 4 rearrangements could be characterized as ‘non-productive’ due to opposite transcriptional orientations and comprised 50% (3/6) of the fusions not detected by Guardant360. Only one case detected by Guardant360 was ‘non-productive’. Of the 13 cases where a fusion was detected in plasma, 89% (8/9) of pts with productive fusions and 75% of pts (3/4) with ‘non-productive’ fusions responded to TKI therapies. Additional unblinding is ongoing to better understand false negative cases.
Conclusions: Here, we demonstrate that a rigorous approach to benchmarking plasma genotyping assays should 1) focus on actionable mutations, 2) use tumor as a gold standard for establishing true and false positives and negatives, and 3) include orthogonal validation to address assay design and bioinformatic analyses as sources of discordance. Our study further highlights the challenges of fusion detection and interpretation and the need for platform cross-comparisons to realize the potential of liquid biopsy to increase access to personalized cancer care.
Citation Format: Julianna G. Supplee, Marina S. Milan, Kristy T. Potts, Lynette M. Sholl, Tristan S. Shaffer, Lee P. Lim, Pasi A. Janne, Geoffrey R. Oxnard, Cloud P. Paweletz. Building an effective concordance study: Plasma Next Generation Sequencing (NGS) for oncogenic fusion detection in non-small cell lung carcinoma (NSCLC) 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 1374.
Abstract
Introduction: ATP-citrate lyase (ACLY) links carbohydrate and lipid metabolism as well as provides acetyl-CoA necessary for histone acetylation, linking cell nutrient status and epigenetic ...regulation. Several studies have shown that ACLY is upregulated in multiple cancer types. ACLY has two major splice isoforms: the full-length canonical “long” isoform and an uncharacterized “short” isoform where exon 14 is spliced out. RNAseq data from The Cancer Genome Atlas (TCGA) shows that the Percent Spliced In (PSI) of exon 14, i.e., the proportion of long isoform, is increased in several cancers compared to normal tissues. We therefore hypothesize that ACLY exon 14 inclusion can support tumor growth.
Experimental Procedures: Extensive statistical analysis was performed on TCGA data to explore the connection between ACLY PSI and clinical parameters including tumor type, stage, and survival. We also noted a serine within exon 14 that is dynamically phosphorylated according to multiple phosphoproteomics studies. We used an in vitro activity assay and differential scanning fluorimetry to biochemically characterize purified ACLY isoforms and phosphomutants. We also expressed these isoforms and mutants in cells lacking endogenous ACLY to evaluated rescue of known ACLY functions including proliferation, de novo lipogenesis, and histone acetylation. Since exon 14 is located near a putative nuclear localization sequence, we also used confocal microscopy to study subcellular localization of these constructs. Ultimately, we developed a new C57BL6/J mouse model with ACLY exon 14 deleted by CRISPR to study the role of exon 14 in metabolic physiology and tumor growth in vivo.
Results: In vitro assays showed no differences in activity and stability between ACLY isoforms and phosphomutants. Similarly, both isoforms and mutants were able to rescue known ACLY functions in cells, and deletion of exon 14 in mice does not impact normal metabolic physiology. Analysis of TCGA data offers compelling evidence that the long ACLY isoform is upregulated early in tumorigenesis and is associated with deadlier tumors and certain immune cell signatures, suggesting that exon 14 is particularly relevant in this disease context. Our mouse model was cross-bred to ApcMin/+ mice, a genetic model of colon cancer, and studies are underway to determine the impact of exon 14 on colon tumors and immune infiltration.
Conclusions: ACLY exon 14 does not have obvious roles in enzymatic activity and known ACLY functions. However, alternative splicing could impact pathophysiological phenotypes such as cancer or other yet undescribed functions of ACLY.
Citation Format: Julianna G. Supplee, Hayley C. Affronti, Richard Duan, Rebekah C. Brooks, Phuong Nguyen, Kollin Schultz, Ronen Marmorstein, Kathryn E. Wellen. Molecular and cancer-related roles of ATP-citrate lyase exon 14. abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3711.
Although interest in droplet-digital PCR technology (ddPCR) for cell-free circulating DNA (cfDNA) analysis is burgeoning, the technology is compromised by subsampling errors and the few clinical ...targets that can be analyzed from limited input DNA. The paucity of starting material acts as a "glass ceiling" in liquid biopsies because, irrespective how analytically sensitive ddPCR techniques are, detection limits cannot be improved past DNA input limitations.
We applied denaturation-enhanced ddPCR (dddPCR) using fragmented genomic DNA (gDNA) with defined mutations. We then tested dddPCR on cfDNA from volunteers and patients with cancer for commonly-used mutations. gDNA and cfDNA were tested with and without end repair before denaturation and digital PCR.
By applying complete denaturation of double-stranded DNA before ddPCR droplet formation the number of positive droplets increased. dddPCR using gDNA resulted in a 1.9-2.0-fold increase in data-positive droplets, whereas dddPCR applied on highly-fragmented cfDNA resulted in a 1.6-1.7-fold increase. End repair of cfDNA before denaturation enabled cfDNA to display a 1.9-2.0-fold increase in data-positive signals, similar to gDNA. Doubling of data-positive droplets doubled the number of potential ddPCR assays that could be conducted from a given DNA input and improved ddPCR precision for cfDNA mutation detection.
dddPCR is a simple and useful modification in ddPCR that enables extraction of more information from low-input clinical samples with minor change in protocols. It should be applicable to all ddPCR platforms for mutation detection and, potentially, for gene copy-number analysis in cancer and prenatal screening.
PDF
