Circulating KRAS G12D but not G12V is associated with survival in metastatic pancreatic ductal adenocarcinoma.

While high circulating tumor DNA (ctDNA) levels are associated with poor survival for multiple cancers, variant-specific differences in the association of ctDNA levels and survival have not been examined. Here we investigate KRAS ctDNA (ctKRAS) variant-specific associations with overall and progression-free survival (OS/PFS) in first-line metastatic pancreatic ductal adenocarcinoma (mPDAC) for patients receiving chemoimmunotherapy ("PRINCE", NCT03214250), and an independent cohort receiving standard of care (SOC) chemotherapy. For PRINCE, higher baseline plasma levels are associated with worse OS for ctKRAS G12D (log-rank p = 0.0010) but not G12V (p = 0.7101), even with adjustment for clinical covariates. Early, on-therapy clearance of G12D (p = 0.0002), but not G12V (p = 0.4058), strongly associates with OS for PRINCE. Similar results are obtained for the SOC cohort, and for PFS in both cohorts. These results suggest ctKRAS G12D but not G12V as a promising prognostic biomarker for mPDAC and that G12D clearance could also serve as an early biomarker of response.

Inflammatory and tissue injury marker dynamics in pediatric acute respiratory distress syndrome.

BACKGROUNDThe molecular signature of pediatric acute respiratory distress syndrome (ARDS) is poorly described, and the degree to which hyperinflammation or specific tissue injury contributes to outcomes is unknown. Therefore, we profiled inflammation and tissue injury dynamics over the first 7 days of ARDS, and associated specific biomarkers with mortality, persistent ARDS, and persistent multiple organ dysfunction syndrome (MODS).METHODSIn a single-center prospective cohort of intubated pediatric patients with ARDS, we collected plasma on days 0, 3, and 7. Nineteen biomarkers reflecting inflammation, tissue injury, and damage-associated molecular patterns (DAMPs) were measured. We assessed the relationship between biomarkers and trajectories with mortality, persistent ARDS, or persistent MODS using multivariable mixed effect models.RESULTSIn 279 patients (64 [23%] nonsurvivors), hyperinflammatory cytokines, tissue injury markers, and DAMPs were higher in nonsurvivors. Survivors and nonsurvivors showed different biomarker trajectories. IL-1α, soluble tumor necrosis factor receptor 1, angiopoietin 2 (ANG2), and surfactant protein D increased in nonsurvivors, while DAMPs remained persistently elevated. ANG2 and procollagen type III N-terminal peptide were associated with persistent ARDS, whereas multiple cytokines, tissue injury markers, and DAMPs were associated with persistent MODS. Corticosteroid use did not impact the association of biomarker levels or trajectory with mortality.CONCLUSIONSPediatric ARDS survivors and nonsurvivors had distinct biomarker trajectories, with cytokines, endothelial and alveolar epithelial injury, and DAMPs elevated in nonsurvivors. Mortality markers overlapped with markers associated with persistent MODS, rather than persistent ARDS.FUNDINGNIH (K23HL-136688, R01-HL148054).

KRAS activation in gastric cancer stem-like cells promotes tumor angiogenesis and metastasis.

Our previous work showed that KRAS activation in gastric cancer cells leads to activation of an epithelial-to-mesenchymal transition (EMT) program and generation of cancer stem-like cells (CSCs). Here we analyze how this KRAS activation in gastric CSCs promotes tumor angiogenesis and metastasis. Gastric cancer CSCs were found to secrete pro-angiogenic factors such as vascular endothelial growth factor A (VEGF-A), and inhibition of KRAS markedly reduced secretion of these factors. In a genetically engineered mouse model, gastric tumorigenesis was markedly attenuated when both KRAS and VEGF-A signaling were blocked. In orthotropic implant and experimental metastasis models, silencing of KRAS and VEGF-A using shRNA in gastric CSCs abrogated primary tumor formation, lymph node metastasis, and lung metastasis far greater than individual silencing of KRAS or VEGF-A. Analysis of gastric cancer patient samples using RNA sequencing revealed a clear association between high expression of the gastric CSC marker CD44 and expression of both KRAS and VEGF-A, and high CD44 and VEGF-A expression predicted worse overall survival. In conclusion, KRAS activation in gastric CSCs enhances secretion of pro-angiogenic factors and promotes tumor progression and metastasis.

The Clinical Implications of Reversions in Patients with Advanced Pancreatic Cancer and Pathogenic Variants in BRCA1, BRCA2, or PALB2 after Progression on Rucaparib.

PURPOSE: PARP inhibitors (PARPi) provide an effective maintenance option for patients with BRCA- or PALB2-mutated pancreatic cancer. However, mechanisms of PARPi resistance and optimal post-PARPi therapeutic strategies are poorly characterized. EXPERIMENTAL DESIGN: We collected paired cell-free DNA samples and post-PARPi clinical data on 42 patients with advanced, platinum-sensitive pancreatic cancer who were treated with maintenance rucaparib on NCT03140670, of whom 32 developed progressive disease. RESULTS: Peripherally detected, acquired BRCA or PALB2 reversion variants were uncommon (5/30; 16.6%) in patients who progressed on rucaparib. Reversions were significantly associated with rapid resistance to PARPi treatment (median PFS, 3.7 vs. 12.5 months; P = 0.001) and poor overall survival (median OS, 6.2 vs. 23.0 months; P < 0.0001). All patients with reversions received rechallenge with platinum-based chemotherapy following PARPi progression and experienced faster progression on this therapy than those without reversion variants (real-world time-to-treatment discontinuation, 2.4 vs. 5.8 months; P = 0.004). Of the patients who progressed on PARPi and received further chemotherapy, the OS from initiation of second-line therapy was significantly lower in those with reversion variants than in those without (5.5 vs. 12.0 months, P = 0.002). Finally, high levels of tumor shedding were independently associated with poor outcomes in patients who received rucaparib. CONCLUSIONS: Acquired reversion variants were uncommon but detrimental in a population of patients with advanced BRCA- or PALB2-related pancreatic ductal adenocarcinoma who received maintenance rucaparib. Reversion variants led to rapid progression on PARPi, rapid failure of subsequent platinum-based treatment, and poor OS of patients. The identification of such variants in the blood may have both predictive and prognostic value. See related commentary by Tsang and Gallinger, p. 5005.

Multianalyte Prognostic Signature Including Circulating Tumor DNA and Circulating Tumor Cells in Patients With Advanced Pancreatic Adenocarcinoma.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is associated with a poor prognosis. Multianalyte signatures, including liquid biopsy and traditional clinical variables, have shown promise for improving prognostication in other solid tumors but have not yet been rigorously assessed for PDAC. MATERIALS AND METHODS: We performed a prospective cohort study of patients with newly diagnosed locally advanced pancreatic cancer (LAPC) or metastatic PDAC (mPDAC) who were planned to undergo systemic therapy. We collected peripheral blood before systemic therapy and assessed circulating tumor cells (CTCs), cell-free DNA concentration (cfDNA), and circulating tumor KRAS (ctKRAS)-variant allele fraction (VAF). Association of variables with overall survival (OS) was assessed in univariate and multivariate survival analysis, and comparisons were made between models containing liquid biopsy variables combined with traditional clinical prognostic variables versus models containing traditional clinical prognostic variables alone. RESULTS: One hundred four patients, 40 with LAPC and 64 with mPDAC, were enrolled. CTCs, cfDNA concentration, and ctKRAS VAF were all significantly higher in patients with mPDAC than patients with LAPC. ctKRAS VAF (cube root; 0.05 unit increments; hazard ratio, 1.11; 95% CI, 1.03 to 1.21; P = .01), and CTCs ≥ 1/mL (hazard ratio, 2.22; 95% CI, 1.34 to 3.69; P = .002) were significantly associated with worse OS in multivariate analysis while cfDNA concentration was not. A model selected by backward selection containing traditional clinical variables plus liquid biopsy variables had better discrimination of OS compared with a model containing traditional clinical variables alone (optimism-corrected Harrell's C-statistic 0.725 v 0.681). CONCLUSION: A multianalyte prognostic signature containing CTCs, ctKRAS, and cfDNA concentration outperformed a model containing traditional clinical variables alone suggesting that CTCs, ctKRAS, and cfDNA provide prognostic information complementary to traditional clinical variables in advanced PDAC.

THSB2 as a prognostic biomarker for patients diagnosed with metastatic pancreatic ductal adenocarcinoma.

Patients newly diagnosed with metastatic pancreatic ductal adenocarcinoma generally have poor survival, with heterogeneous rates of progression. Biomarkers that could predict progression and/or survival would help inform patients and providers as they make care decisions. In a previous retrospective study, we discovered that circulating thrombospondin-2 (THBS2) could, in combination with CA19-9, better distinguish patients with PDAC versus healthy controls. Here we evaluated whether THBS2 levels, previously not known to be prognostic, were associated with outcome in 68 patients at time of diagnosis of metastatic PDAC. Specifically, we interrogated the association of THBS2 level, alone or in combination with CA19-9, with progression by 90 days and/or survival to 180 days. The results indicate that elevated THBS2 levels alone, at the time of a metastatic PDAC diagnosis, can identify patients with a shorter time to death and thus help patients and providers when planning treatment.

Optimization of Sources of Circulating Cell-Free DNA Variability for Downstream Molecular Analysis.

Circulating cell-free DNA (ccfDNA) is used increasingly as a cancer biomarker for prognostication, as a correlate for tumor volume, or as input for downstream molecular analysis. Determining optimal blood processing and ccfDNA quantification are crucial for ccfDNA to serve as an accurate biomarker as it moves into the clinical realm. Whole blood was collected from 50 subjects, processed to plasma, and used immediately or frozen at -80°C. Plasma ccfDNA was extracted and concentration was assessed by real-time quantitative PCR (qPCR), fluorimetry, and droplet digital PCR (ddPCR). For the 24 plasma samples from metastatic pancreatic cancer patients, the variant allele fractions (VAF) of KRAS G12/13 pathogenic variants in circulating tumor DNA (ctDNA) were measured by ddPCR. Using a high-speed (16,000 × g) or slower-speed (4100 × g) second centrifugation step showed no difference in ccfDNA yield or ctDNA VAF. A two- versus three-spin centrifugation protocol also showed no difference in ccfDNA yield or ctDNA VAF. A higher yield was observed from fresh versus frozen plasma by qPCR and fluorimetry, whereas a higher yield was observed for frozen versus fresh plasma by ddPCR, however, no difference was observed in ctDNA VAF. Overall, our findings suggest factors to consider when implementing a ccfDNA extraction and quantification workflow in a research or clinical setting.

CD40 agonistic monoclonal antibody APX005M (sotigalimab) and chemotherapy, with or without nivolumab, for the treatment of metastatic pancreatic adenocarcinoma: an open-label, multicentre, phase 1b study.

BACKGROUND: Standard chemotherapy remains inadequate in metastatic pancreatic adenocarcinoma. Combining an agonistic CD40 monoclonal antibody with chemotherapy induces T-cell-dependent tumour regression in mice and improves survival. In this study, we aimed to evaluate the safety of combining APX005M (sotigalimab) with gemcitabine plus nab-paclitaxel, with and without nivolumab, in patients with pancreatic adenocarcinoma to establish the recommended phase 2 dose. METHODS: This non-randomised, open-label, multicentre, four-cohort, phase 1b study was done at seven academic hospitals in the USA. Eligible patients were adults aged 18 years and older with untreated metastatic pancreatic adenocarcinoma, Eastern Cooperative Oncology Group performance status score of 0-1, and measurable disease by Response Evaluation Criteria in Solid Tumors version 1.1. All patients were treated with 1000 mg/m2 intravenous gemcitabine and 125 mg/m2 intravenous nab-paclitaxel. Patients received 0·1 mg/kg intravenous APX005M in cohorts B1 and C1 and 0·3 mg/kg in cohorts B2 and C2. In cohorts C1 and C2, patients also received 240 mg intravenous nivolumab. Primary endpoints comprised incidence of adverse events in all patients who received at least one dose of any study drug, incidence of dose-limiting toxicities (DLTs) in all patients who had a DLT or received at least two doses of gemcitabine plus nab-paclitaxel and one dose of APX005M during cycle 1, and establishing the recommended phase 2 dose of intravenous APX005M. Objective response rate in the DLT-evaluable population was a key secondary endpoint. This trial (PRINCE, PICI0002) is registered with ClinicalTrials.gov, NCT03214250 and is ongoing. FINDINGS: Between Aug 22, 2017, and July 10, 2018, of 42 patients screened, 30 patients were enrolled and received at least one dose of any study drug; 24 were DLT-evaluable with median follow-up 17·8 months (IQR 16·0-19·4; cohort B1 22·0 months [21·4-22·7], cohort B2 18·2 months [17·0-18·9], cohort C1 17·9 months [14·3-19·7], cohort C2 15·9 months [12·7-16·1]). Two DLTs, both febrile neutropenia, were observed, occurring in one patient each for cohorts B2 (grade 3) and C1 (grade 4). The most common grade 3-4 treatment-related adverse events were lymphocyte count decreased (20 [67%]; five in B1, seven in B2, four in C1, four in C2), anaemia (11 [37%]; two in B1, four in B2, four in C1, one in C2), and neutrophil count decreased (nine [30%]; three in B1, three in B2, one in C1, two in C2). 14 (47%) of 30 patients (four each in B1, B2, C1; two in C2) had a treatment-related serious adverse event. The most common serious adverse event was pyrexia (six [20%] of 30; one in B2, three in C1, two in C2). There were two chemotherapy-related deaths due to adverse events: one sepsis in B1 and one septic shock in C1. The recommended phase 2 dose of APX005M was 0·3 mg/kg. Responses were observed in 14 (58%) of 24 DLT-evaluable patients (four each in B1, C1, C2; two in B2). INTERPRETATION: APX005M and gemcitabine plus nab-paclitaxel, with or without nivolumab, is tolerable in metastatic pancreatic adenocarcinoma and shows clinical activity. If confirmed in later phase trials, this treatment regimen could replace chemotherapy-only standard of care in this population. FUNDING: Parker Institute for Cancer Immunotherapy, Cancer Research Institute, and Bristol Myers Squibb.

A Multianalyte Panel Consisting of Extracellular Vesicle miRNAs and mRNAs, cfDNA, and CA19-9 Shows Utility for Diagnosis and Staging of Pancreatic Ductal Adenocarcinoma.

PURPOSE: To determine whether a multianalyte liquid biopsy can improve the detection and staging of pancreatic ductal adenocarcinoma (PDAC). EXPERIMENTAL DESIGN: We analyzed plasma from 204 subjects (71 healthy, 44 non-PDAC pancreatic disease, and 89 PDAC) for the following biomarkers: tumor-associated extracellular vesicle miRNA and mRNA isolated on a nanomagnetic platform that we developed and measured by next-generation sequencing or qPCR, circulating cell-free DNA (ccfDNA) concentration measured by qPCR, ccfDNA KRAS G12D/V/R mutations detected by droplet digital PCR, and CA19-9 measured by electrochemiluminescence immunoassay. We applied machine learning to training sets and subsequently evaluated model performance in independent, user-blinded test sets. RESULTS: To identify patients with PDAC versus those without, we generated a classification model using a training set of 47 subjects (20 PDAC and 27 noncancer). When applied to a blinded test set (N = 136), the model achieved an AUC of 0.95 and accuracy of 92%, superior to the best individual biomarker, CA19-9 (89%). We next used a cohort of 20 patients with PDAC to train our model for disease staging and applied it to a blinded test set of 25 patients clinically staged by imaging as metastasis-free, including 9 subsequently determined to have had occult metastasis. Our workflow achieved significantly higher accuracy for disease staging (84%) than imaging alone (accuracy = 64%; P < 0.05). CONCLUSIONS: Algorithmically combining blood-based biomarkers may improve PDAC diagnostic accuracy and preoperative identification of nonmetastatic patients best suited for surgery, although larger validation studies are necessary.

Imaging and histopathologic correlates of plasma cell-free DNA concentration and circulating tumor DNA in adult patients with newly diagnosed glioblastoma.

BACKGROUND: Plasma cell-free DNA (cfDNA) concentration is lower in glioblastoma (GBM) compared to other solid tumors, which can lead to low circulating tumor DNA (ctDNA) detection. In this study, we investigated the relationship between multimodality magnetic resonance imaging (MRI) and histopathologic features with plasma cfDNA concentration and ctDNA detection in patients with treatment-naive GBM. METHODS: We analyzed plasma cfDNA concentration, MRI scans, and tumor histopathology from 42 adult patients with newly diagnosed GBM. Linear regression analysis was used to examine the relationship of plasma cfDNA concentration before surgery to imaging and histopathologic characteristics. In a subset of patients, imaging and histopathologic metrics were also compared between patients with and without a detected tumor somatic mutation. RESULTS: Tumor volume with elevated (>1.5 times contralateral white matter) rate transfer constant (K ep, a surrogate of blood-brain barrier [BBB] permeability) was independently associated with plasma cfDNA concentration (P = .001). Histopathologic characteristics independently associated with plasma cfDNA concentration included CD68+ macrophage density (P = .01) and size of tumor vessels (P = .01). Patients with higher (grade ≥3) perivascular CD68+ macrophage density had lower volume transfer constant (K trans, P = .01) compared to those with lower perivascular CD68+ macrophage density. Detection of at least 1 somatic mutation in plasma cfDNA was associated with significantly lower perivascular CD68+ macrophages (P = .01). CONCLUSIONS: Metrics of BBB disruption and quantity and distribution of tumor-associated macrophages are associated with plasma cfDNA concentration and ctDNA detection in GBM patients. These findings represent an important step in understanding the factors that determine plasma cfDNA concentration and ctDNA detection.

Highly specific enrichment of rare nucleic acid fractions using Thermus thermophilus argonaute with applications in cancer diagnostics.

Detection of disease-associated, cell-free nucleic acids in body fluids enables early diagnostics, genotyping and personalized therapy, but is challenged by the low concentrations of clinically significant nucleic acids and their sequence homology with abundant wild-type nucleic acids. We describe a novel approach, dubbed NAVIGATER, for increasing the fractions of Nucleic Acids of clinical interest Via DNA-Guided Argonaute from Thermus thermophilus (TtAgo). TtAgo cleaves specifically guide-complementary DNA and RNA with single nucleotide precision, greatly increasing the fractions of rare alleles and, enhancing the sensitivity of downstream detection methods such as ddPCR, sequencing, and clamped enzymatic amplification. We demonstrated 60-fold enrichment of the cancer biomarker KRAS G12D and ∼100-fold increased sensitivity of Peptide Nucleic Acid (PNA) and Xenonucleic Acid (XNA) clamp PCR, enabling detection of low-frequency (<0.01%) mutant alleles (∼1 copy) in blood samples of pancreatic cancer patients. NAVIGATER surpasses Cas9-based assays (e.g. DASH, Depletion of Abundant Sequences by Hybridization), identifying more mutation-positive samples when combined with XNA-PCR. Moreover, TtAgo does not require targets to contain any specific protospacer-adjacent motifs (PAM); is a multi-turnover enzyme; cleaves ssDNA, dsDNA and RNA targets in a single assay; and operates at elevated temperatures, providing high selectivity and compatibility with polymerases.

Clinical Utility of Plasma Cell-Free DNA in Adult Patients with Newly Diagnosed Glioblastoma: A Pilot Prospective Study.

PURPOSE: The clinical utility of plasma cell-free DNA (cfDNA) has not been assessed prospectively in patients with glioblastoma (GBM). We aimed to determine the prognostic impact of plasma cfDNA in GBM, as well as its role as a surrogate of tumor burden and substrate for next-generation sequencing (NGS). EXPERIMENTAL DESIGN: We conducted a prospective cohort study of 42 patients with newly diagnosed GBM. Plasma cfDNA was quantified at baseline prior to initial tumor resection and longitudinally during chemoradiotherapy. Plasma cfDNA was assessed for its association with progression-free survival (PFS) and overall survival (OS), correlated with radiographic tumor burden, and subjected to a targeted NGS panel. RESULTS: Prior to initial surgery, GBM patients had higher plasma cfDNA concentration than age-matched healthy controls (mean 13.4 vs. 6.7 ng/mL, P < 0.001). Plasma cfDNA concentration was correlated with radiographic tumor burden on patients' first post-radiation magnetic resonance imaging scan (ρ = 0.77, P = 0.003) and tended to rise prior to or concurrently with radiographic tumor progression. Preoperative plasma cfDNA concentration above the mean (>13.4 ng/mL) was associated with inferior PFS (median 4.9 vs. 9.5 months, P = 0.038). Detection of ≥1 somatic mutation in plasma cfDNA occurred in 55% of patients and was associated with nonstatistically significant decreases in PFS (median 6.0 vs. 8.7 months, P = 0.093) and OS (median 5.5 vs. 9.2 months, P = 0.053). CONCLUSIONS: Plasma cfDNA may be an effective prognostic tool and surrogate of tumor burden in newly diagnosed GBM. Detection of somatic alterations in plasma is feasible when samples are obtained prior to initial surgical resection.

Oncogenic KRAS and p53 Loss Drive Gastric Tumorigenesis in Mice That Can Be Attenuated by E-Cadherin Expression.

Gastric adenocarcinoma is the third leading cause of cancer-related death worldwide, but no models exist to readily investigate distant metastases that are mainly responsible for mortality in this disease. Here we report the development of a genetically engineered mouse model of gastric adenocarcinoma tumorigenesis based on KrasG12D expression plus inactivation of E-cadherin (Cdh1) and p53 in the gastric parietal cell lineage. Intestinal and diffuse gastric tumors arise rapidly in this model that displays a median survival of 76 days. Tumors occur throughout the stomach, with metastases documented in lymph nodes, lung, and liver. Mice otherwise identical but retaining one wild-type Cdh1 allele exhibited longer survival with only 20% penetrance of invasive tumors and no apparent lung or liver metastases. Notably, increased RAS activity and downstream MAPK signaling was observed in stomachs only when E-cadherin was absent. This model offers a valuable tool to investigate gastric adenocarcinoma subtypes where RAS/MAPK pathway activation and E-cadherin attenuation are common. Cancer Res; 77(19); 5349-59. ©2017 AACR.

Tumor vessel normalization after aerobic exercise enhances chemotherapeutic efficacy.

Targeted therapies aimed at tumor vasculature are utilized in combination with chemotherapy to improve drug delivery and efficacy after tumor vascular normalization. Tumor vessels are highly disorganized with disrupted blood flow impeding drug delivery to cancer cells. Although pharmacologic anti-angiogenic therapy can remodel and normalize tumor vessels, there is a limited window of efficacy and these drugs are associated with severe side effects necessitating alternatives for vascular normalization. Recently, moderate aerobic exercise has been shown to induce vascular normalization in mouse models. Here, we provide a mechanistic explanation for the tumor vascular normalization induced by exercise. Shear stress, the mechanical stimuli exerted on endothelial cells by blood flow, modulates vascular integrity. Increasing vascular shear stress through aerobic exercise can alter and remodel blood vessels in normal tissues. Our data in mouse models indicate that activation of calcineurin-NFAT-TSP1 signaling in endothelial cells plays a critical role in exercise-induced shear stress mediated tumor vessel remodeling. We show that moderate aerobic exercise with chemotherapy caused a significantly greater decrease in tumor growth than chemotherapy alone through improved chemotherapy delivery after tumor vascular normalization. Our work suggests that the vascular normalizing effects of aerobic exercise can be an effective chemotherapy adjuvant.

Genetic and phenotypic analysis of flagellar assembly mutants in Chlamydomonas reinhardtii.

Conditional mutants for flagellar assembly (fla) provide a useful tool to study intraflagellar transport (IFT) at the molecular level, and provide a unique set of tools to analyze cilia. The analysis of IFT phenotypes of fla mutants at the permissive temperature by a quantitative image analysis approach identified four distinct phases of the IFT cycle and directly demonstrated structural and functional remodeling of IFT particles at both axonemal extremities. In addition, the genetic analysis of fla mutants reveal interesting interactions among genes involved in flagellar assembly that help to provide information about the structure and function of IFT particles and their motors. This chapter provides protocols to isolate, characterize, and identify conditional Chlamydomonas flagellar assembly mutants and their genes and to test genetic interactions among proteins encoded by these genes.