ACLY alternative splicing correlates with cancer phenotypes.

ATP-citrate lyase (ACLY) links carbohydrate and lipid metabolism and provides nucleocytosolic acetyl-CoA 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 an intrinsically disordered region and includes at least one dynamically phosphorylated residue. 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 is increased in several cancers and correlated with poorer overall survival in a pan-cancer analysis, though not in individual tumor types. This 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.

Protein biomarkers and alternatively methylated cell-free DNA detect early stage pancreatic cancer.

OBJECTIVE: 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. DESIGN: 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. RESULTS: 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). CONCLUSION: A combined panel of circulating markers including proteins and methylated cfDNA increased discrimination compared with CA19-9 alone for early stage PDAC.

Plasma ctDNA Response Is an Early Marker of Treatment Effect in Advanced NSCLC.

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. MATERIALS AND METHODS: Patients with advanced EGFR-mutant NSCLC across three phase I NCI osimertinib combination trials were analyzed in this study, and an institutional cohort of patients with KRAS-, EGFR-, and BRAF-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 EGFR-mutant NSCLC treated with osimertinib. Across cohorts, we also studied ctDNA variability before treatment start. RESULTS: 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. CONCLUSION: 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.

Molecular mechanism of N-terminal acetylation by the ternary NatC complex.

Protein N-terminal acetylation is predominantly a ribosome-associated modification, with NatA-E serving as the major enzymes. NatC is the most unusual of these enzymes, containing one Naa30 catalytic subunit and two auxiliary subunits, Naa35 and Naa38; and substrate selectivity profile that overlaps with NatE. Here, we report the cryoelectron microscopy structure of S. pombe NatC with a NatE/C-type bisubstrate analog and inositol hexaphosphate (IP6), and associated biochemistry studies. We find that the presence of three subunits is a prerequisite for normal NatC acetylation activity in yeast and that IP6 binds tightly to NatC to stabilize the complex. We also describe the molecular basis for IP6-mediated NatC complex stabilization and the overlapping yet distinct substrate profiles of NatC and NatE.

Plasma IL-6 changes correlate to PD-1 inhibitor responses in NSCLC.

BACKGROUND: Blood-based biomarkers of anti-solid tumor immune checkpoint blockade (ICB) response are lacking. We hypothesized that changes in systemic cytokine levels with the initial doses of programmed cell death protein 1 (PD-1) pathway inhibitors would correlate with clinical responses. New ultrasensitive ELISA technology enables quantitation of plasma proteins in sub-picogram-per-milliliter concentrations. METHODS: We measured plasma cytokines by ultrasensitive single-molecule array assays in patients with non-small-cell lung carcinoma before and during treatment with anti-PD-1 therapy. Association with best overall response and progression-free survival (PFS) was assessed by Kruskall-Wallis test and Kaplan-Meier plots with log-rank test, respectively. RESULTS: A decrease in interleukin 6 (IL-6) levels was associated with improved PFS (n=47 patients, median PFS: 11 vs 4 months, HR 0.45 (95% CI 0.23 to 0.89), p=0.04). The extent of change in IL-6 differed between best overall response categories (p=0.01) and correlated with changes in C reactive protein levels. We also explored plasma cytokine levels in relation to immune-related adverse effects and observed some correlation. CONCLUSIONS: This study suggests the presence of a systemic, proteomic reflection of successful ICB outside the tumor microenvironment with plasma decreases in IL-6 and CRP.

Sensitivity of next-generation sequencing assays detecting oncogenic fusions in plasma cell-free DNA.

OBJECTIVES: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Salivary HPV DNA informs locoregional disease status in advanced HPV-associated oropharyngeal cancer.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Denaturation-Enhanced Droplet Digital PCR for Liquid Biopsies.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Liquid biopsy of fine-needle aspiration supernatant for lung cancer genotyping.

BACKGROUND: Tumor genotyping is transforming lung cancer care but requires adequate tumor tissue. Advances in minimally invasive biopsy techniques have increased access to difficult-to-access lesions, but often result in smaller samples. With the advent of highly sensitive DNA genotyping methods used for plasma analysis, we hypothesized that these same methods might allow genotyping of free DNA derived from fine needle aspiration supernatant (FNA-S). METHODS: We studied patients with known or suspected lung cancer undergoing fine needle aspirate (FNA). After spinning the sample for cellblock, the FNA-S (usually discarded) was saved for genotyping. Supernatant cell-free DNA (SN-cfDNA) was extracted and tested by both droplet digital PCR (EGFR, BRAF, KRAS mutations) and highly sensitive amplicon-based next-generation sequencing (NGS). RESULTS: 17 samples were studied, including 11 FNAs from patients with suspected lung cancer and 6 FNAs from patients with lung cancer and acquired drug resistance. Of 6 newly diagnosed adenocarcinomas, 4 had a driver mutations (1 EGFR, 2 KRAS, 1 HER2) found on tissue; all of these could be detected in SN-cfDNA. The EGFR driver mutation was detected in all 5 adenocarcinomas with acquired EGFR resistance and the EGFR T790 M in three cases, in agreement with cellblock. CONCLUSIONS: FNA-S is a rich source of fresh tumor DNA, potentially increasing the diagnostic yield from small FNAs. Through use of emerging techniques for highly sensitive genotyping, this widely available biospecimen has potential for facilitating rapid cancer genotyping at diagnosis and after drug resistance.

False-Positive Plasma Genotyping Due to Clonal Hematopoiesis.

Purpose: Plasma cell-free DNA (cfDNA) genotyping is increasingly used in cancer care, but assay accuracy has been debated. Because most cfDNA is derived from peripheral blood cells (PBC), we hypothesized that nonmalignant mutations harbored by hematopoietic cells (clonal hematopoiesis, CH) could be a cause of false-positive plasma genotyping.Experimental Design: We identified patients with advanced non-small cell lung cancer (NSCLC) with KRAS, JAK2, or TP53 mutations identified in cfDNA. With consent, PBC DNA was tested using droplet digital PCR (ddPCR) or next-generation sequencing (NGS) to test for CH-derived mutations.Results: We first studied plasma ddPCR results from 58 patients with EGFR-mutant NSCLC. Two had KRAS G12X detected in cfDNA, and both were present in PBC, including one where the KRAS mutation was detected serially for 20 months. We then studied 143 plasma NGS results from 122 patients with NSCLC and identified 5 JAK2 V617F mutations derived from PBC. In addition, 108 TP53 mutations were detected in cfDNA; for 33 of the TP53 mutations, PBC and tumor NGS were available for comparison, and 5 were present in PBC but absent in tumor, consistent with CH.Conclusions: We find that most JAK2 mutations, some TP53 mutations, and rare KRAS mutations detected in cfDNA are derived from CH not tumor. Clinicians ordering plasma genotyping must be prepared for the possibility that mutations detected in plasma, particularly in genes mutated in CH, may not represent true tumor genotype. Efforts to use plasma genotyping for cancer detection may need paired PBC genotyping so that CH-derived mutations are not misdiagnosed as occult malignancy. Clin Cancer Res; 24(18); 4437-43. ©2018 AACRSee related commentary by Bauml and Levy, p. 4352.