Critical considerations for communicating environmental DNA science.

The economic and methodological efficiencies of environmental DNA (eDNA) based survey approaches provide an unprecedented opportunity to assess and monitor aquatic environments. However, instances of inadequate communication from the scientific community about confidence levels, knowledge gaps, reliability, and appropriate parameters of eDNA-based methods have hindered their uptake in environmental monitoring programs and, in some cases, has created misperceptions or doubts in the management community. To help remedy this situation, scientists convened a session at the Second National Marine eDNA Workshop to discuss strategies for improving communications with managers. These include articulating the readiness of different eDNA applications, highlighting the strengths and limitations of eDNA tools for various applications or use cases, communicating uncertainties associated with specified uses transparently, and avoiding the exaggeration of exploratory and preliminary findings. Several key messages regarding implementation, limitations, and relationship to existing methods were prioritized. To be inclusive of the diverse managers, practitioners, and researchers, we and the other workshop participants propose the development of communication workflow plans, using RACI (Responsible, Accountable, Consulted, Informed) charts to clarify the roles of all pertinent individuals and parties and to minimize the chance for miscommunications. We also propose developing decision support tools such as Structured Decision-Making (SDM) to help balance the benefits of eDNA sampling with the inherent uncertainty, and developing an eDNA readiness scale to articulate the technological readiness of eDNA approaches for specific applications. These strategies will increase clarity and consistency regarding our understanding of the utility of eDNA-based methods, improve transparency, foster a common vision for confidently applying eDNA approaches, and enhance their benefit to the monitoring and assessment community.

Tourmaline: A containerized workflow for rapid and iterable amplicon sequence analysis using QIIME 2 and Snakemake.

BACKGROUND: Amplicon sequencing (metabarcoding) is a common method to survey diversity of environmental communities whereby a single genetic locus is amplified and sequenced from the DNA of whole or partial organisms, organismal traces (e.g., skin, mucus, feces), or microbes in an environmental sample. Several software packages exist for analyzing amplicon data, among which QIIME 2 has emerged as a popular option because of its broad functionality, plugin architecture, provenance tracking, and interactive visualizations. However, each new analysis requires the user to keep track of input and output file names, parameters, and commands; this lack of automation and standardization is inefficient and creates barriers to meta-analysis and sharing of results. FINDINGS: We developed Tourmaline, a Python-based workflow that implements QIIME 2 and is built using the Snakemake workflow management system. Starting from a configuration file that defines parameters and input files-a reference database, a sample metadata file, and a manifest or archive of FASTQ sequences-it uses QIIME 2 to run either the DADA2 or Deblur denoising algorithm; assigns taxonomy to the resulting representative sequences; performs analyses of taxonomic, alpha, and beta diversity; and generates an HTML report summarizing and linking to the output files. Features include support for multiple cores, automatic determination of trimming parameters using quality scores, representative sequence filtering (taxonomy, length, abundance, prevalence, or ID), support for multiple taxonomic classification and sequence alignment methods, outlier detection, and automated initialization of a new analysis using previous settings. The workflow runs natively on Linux and macOS or via a Docker container. We ran Tourmaline on a 16S ribosomal RNA amplicon data set from Lake Erie surface water, showing its utility for parameter optimization and the ability to easily view interactive visualizations through the HTML report, QIIME 2 viewer, and R- and Python-based Jupyter notebooks. CONCLUSION: Automated workflows like Tourmaline enable rapid analysis of environmental amplicon data, decreasing the time from data generation to actionable results. Tourmaline is available for download at github.com/aomlomics/tourmaline.

Dominance of Sulfurospirillum in Metagenomes Associated with the Methane Ice Worm (Sirsoe methanicola).

Sirsoe methanicola, commonly known as the methane ice worm, is the only macrofaunal species known to inhabit the Gulf of Mexico methane hydrates. Little is known about this elusive marine polychaete that can colonize rich carbon and energy reserves. Metagenomic analysis of gut contents and worm fragments predicted diverse metabolic capabilities with the ability to utilize a range of nitrogen, sulfur, and organic carbon compounds through microbial taxa affiliated with Campylobacterales, Desulfobacterales, Enterobacterales, SAR324, Alphaproteobacteria, and Mycoplasmatales. Entomoplasmatales and Chitinivibrionales were additionally identified from extracted full-length 16S rRNA sequences, and read analysis identified 196 bacterial families. Overall, the microbial community appeared dominated by uncultured Sulfurospirillum, a taxon previously considered free-living rather than host-associated. Metagenome-assembled genomes (MAGs) classified as uncultured Sulfurospirillum predicted thiosulfate disproportionation and the reduction of tetrathionate, sulfate, sulfide/polysulfide, and nitrate. Microbial amino acid and vitamin B12 biosynthesis genes were identified in multiple MAGs, suggesting nutritional value to the host. Reads assigned to aerobic or anaerobic methanotrophic taxa were rare. IMPORTANCE Methane hydrates represent vast reserves of natural gas with roles in global carbon cycling and climate change. This study provided the first analysis of metagenomes associated with Sirsoe methanicola, the only polychaete species known to colonize methane hydrates. Previously unrecognized participation of Sulfurospirillum in a gut microbiome is provided, and the role of sulfur compound redox reactions within this community is highlighted. The comparative biology of S. methanicola is of general interest given research into the adverse effects of sulfide production in human gut microbiomes. In addition, taxonomic assignments are provided for nearly 200 bacterial families, expanding our knowledge of microbiomes in the deep sea.

Influence of nutrient supply on plankton microbiome biodiversity and distribution in a coastal upwelling region.

The ecological and oceanographic processes that drive the response of pelagic ocean microbiomes to environmental changes remain poorly understood, particularly in coastal upwelling ecosystems. Here we show that seasonal and interannual variability in coastal upwelling predicts pelagic ocean microbiome diversity and community structure in the Southern California Current region. Ribosomal RNA gene sequencing, targeting prokaryotic and eukaryotic microbes, from samples collected seasonally during 2014-2020 indicate that nitracline depth is the most robust predictor of spatial microbial community structure and biodiversity in this region. Striking ecological changes occurred due to the transition from a warm anomaly during 2014-2016, characterized by intense stratification, to cooler conditions in 2017-2018, representative of more typical upwelling conditions, with photosynthetic eukaryotes, especially diatoms, changing most strongly. The regional slope of nitracline depth exerts strong control on the relative proportion of highly diverse offshore communities and low biodiversity, but highly productive nearshore communities.

Metagenomic and Metatranscriptomic Insights into Population Diversity of Microcystis Blooms: Spatial and Temporal Dynamics of mcy Genotypes, Including a Partial Operon That Can Be Abundant and Expressed.

Cyanobacterial harmful algal blooms (cyanoHABs) degrade freshwater ecosystems globally. Microcystis aeruginosa often dominates cyanoHABs and produces microcystin (MC), a class of hepatotoxins that poses threats to human and animal health. Microcystin toxicity is influenced by distinct structural elements across a diversity of related molecules encoded by variant mcy operons. However, the composition and distribution of mcy operon variants in natural blooms remain poorly understood. Here, we characterized the variant composition of mcy genes in western Lake Erie Microcystis blooms from 2014 and 2018. Sampling was conducted across several spatial and temporal scales, including different bloom phases within 2014, extensive spatial coverage on the same day (2018), and frequent, autonomous sampling over a 2-week period (2018). Mapping of metagenomic and metatranscriptomic sequences to reference sequences revealed three Microcystis mcy genotypes: complete (all genes present [mcyA-J]), partial (truncated mcyA, complete mcyBC, and missing mcyD-J), and absent (no mcy genes). We also detected two different variants of mcyB that may influence the production of microcystin congeners. The relative abundance of these genotypes was correlated with pH and nitrate concentrations. Metatranscriptomic analysis revealed that partial operons were, at times, the most abundant genotype and expressed in situ, suggesting the potential biosynthesis of truncated products. Quantification of genetic divergence between genotypes suggests that the observed strains are the result of preexisting heterogeneity rather than de novo mutation during the sampling period. Overall, our results show that natural Microcystis populations contain several cooccurring mcy genotypes that dynamically shift in abundance spatiotemporally via strain succession and likely influence the observed diversity of the produced congeners. IMPORTANCE Cyanobacteria are responsible for producing microcystins (MCs), a class of potent and structurally diverse toxins, in freshwater systems around the world. While microcystins have been studied for over 50 years, the diversity of their chemical forms and how this variation is encoded at the genetic level remain poorly understood, especially within natural populations of cyanobacterial harmful algal blooms (cyanoHABs). Here, we leverage community DNA and RNA sequences to track shifts in mcy genes responsible for producing microcystin, uncovering the relative abundance, expression, and variation of these genes. We studied this phenomenon in western Lake Erie, which suffers annually from cyanoHAB events, with impacts on drinking water, recreation, tourism, and commercial fishing.

The founding charter of the Omic Biodiversity Observation Network (Omic BON).

Omic BON is a thematic Biodiversity Observation Network under the Group on Earth Observations Biodiversity Observation Network (GEO BON), focused on coordinating the observation of biomolecules in organisms and the environment. Our founding partners include representatives from national, regional, and global observing systems; standards organizations; and data and sample management infrastructures. By coordinating observing strategies, methods, and data flows, Omic BON will facilitate the co-creation of a global omics meta-observatory to generate actionable knowledge. Here, we present key elements of Omic BON's founding charter and first activities.

Capturing marine microbiomes and environmental DNA: A field sampling guide.

The expanding interest in marine microbiome and eDNA sequence data has led to a demand for sample collection and preservation standard practices to enable comparative assessments of results across studies and facilitate meta-analyses. We support this effort by providing guidelines based on a review of published methods and field sampling experiences. The major components considered here are environmental and resource considerations, sample processing strategies, sample storage options, and eDNA extraction protocols. It is impossible to provide universal recommendations considering the wide range of eDNA applications; rather, we provide information to design fit-for-purpose protocols. To manage scope, the focus here is on sampling collection and preservation of prokaryotic and microeukaryotic eDNA. Even with a focused view, the practical utility of any approach depends on multiple factors, including habitat type, available resources, and experimental goals. We broadly recommend enacting rigorous decontamination protocols, pilot studies to guide the filtration volume needed to characterize the target(s) of interest and minimize PCR inhibitor collection, and prioritizing sample freezing over (only) the addition of preservation buffer. An annotated list of studies that test these parameters is included for more detailed investigation on specific steps. To illustrate an approach that demonstrates fit-for-purpose methodologies, we provide a protocol for eDNA sampling aboard an oceanographic vessel. These guidelines can aid the decision-making process for scientists interested in sampling and sequencing marine microbiomes and/or eDNA.

Improving metabarcoding taxonomic assignment: A case study of fishes in a large marine ecosystem.

DNA metabarcoding is an important tool for molecular ecology. However, its effectiveness hinges on the quality of reference sequence databases and classification parameters employed. Here we evaluate the performance of MiFish 12S taxonomic assignments using a case study of California Current Large Marine Ecosystem fishes to determine best practices for metabarcoding. Specifically, we use a taxonomy cross-validation by identity framework to compare classification performance between a global database comprised of all available sequences and a curated database that only includes sequences of fishes from the California Current Large Marine Ecosystem. We demonstrate that the regional database provides higher assignment accuracy than the comprehensive global database. We also document a tradeoff between accuracy and misclassification across a range of taxonomic cutoff scores, highlighting the importance of parameter selection for taxonomic classification. Furthermore, we compared assignment accuracy with and without the inclusion of additionally generated reference sequences. To this end, we sequenced tissue from 597 species using the MiFish 12S primers, adding 252 species to GenBank's existing 550 California Current Large Marine Ecosystem fish sequences. We then compared species and reads identified from seawater environmental DNA samples using global databases with and without our generated references, and the regional database. The addition of new references allowed for the identification of 16 additional native taxa representing 17.0% of total reads from eDNA samples, including species with vast ecological and economic value. Together these results demonstrate the importance of comprehensive and curated reference databases for effective metabarcoding and the need for locus-specific validation efforts.

A Phase 2 Study of Capmatinib in Patients With MET-Altered Lung Cancer Previously Treated With a MET Inhibitor.

INTRODUCTION: Capmatinib is approved for MET exon 14-altered NSCLC on the basis of activity in targeted therapy-naive patients. We conducted a phase 2 study to assess the efficacy of capmatinib in patients previously treated with a MET inhibitor. METHODS: Patients with advanced NSCLC harboring MET amplification or MET exon 14 skipping alterations received capmatinib 400 mg twice daily. The primary end point was the objective response rate. Secondary end points included progression-free survival, disease control rate (DCR), intracranial response rate, and overall survival. Circulating tumor DNA was analyzed to identify capmatinib resistance mechanisms. RESULTS: A total of 20 patients were enrolled between May 2016 and November 2019, including 15 patients with MET skipping alterations and five patients with MET amplification. All patients had received crizotinib; three had also received other MET-directed therapies. The median interval between crizotinib and capmatinib was 22 days (range: 4-374). Two patients (10%) achieved an objective response to capmatinib and 14 had stable disease, yielding a DCR of 80%. Among five patients who discontinued crizotinib for intolerance, the DCR was 83%, including two patients with the best tumor shrinkage of -25% and -28%. Intracranial DCR among four patients with measurable brain metastases was 100%, with no observed intracranial objective responses. Overall, the median progression-free survival and overall survival were 5.5 (95% confidence interval: 1.3-11.0) and 11.3 (95% confidence interval: 5.5-not reached) months, respectively. MET D1228 and Y1230 mutations and MAPK alterations were recurrently detected in postcrizotinib, precapmatinib plasma. New and persistent MET mutations and MAPK pathway alterations were detected in plasma at progression on capmatinib. CONCLUSIONS: Capmatinib has modest activity in crizotinib-pretreated MET-altered NSCLC, potentially owing to overlapping resistance mechanisms.

Coronavirus Disease 2019 Infection in a Patient Population with Lung Cancer: Incidence, Presentation, and Alternative Diagnostic Considerations.

INTRODUCTION: Lung cancer is associated with severe coronavirus disease 2019 (COVID-19) infections. Symptom overlap between COVID-19 and lung cancer may complicate diagnostic evaluation. We aimed to investigate the incidence, symptoms, differential diagnosis, and outcomes of COVID-19 in patients with lung cancer. METHODS: To determine an at-risk population for COVID-19, we retrospectively identified patients with lung cancer receiving longitudinal care within a single institution in the 12 months (April 1, 2019 to March 31, 2020) immediately preceding the COVID-19 pandemic, including an "active therapy population" treated within the last 60 days of this period. Among patients subsequently referred for COVID-19 testing, we compared symptoms, laboratory values, radiographic findings, and outcomes of positive versus negative patients. RESULTS: Between April 1, 2019 and March 31, 2020, a total of 696 patients received longitudinal care, including 406 (58%) in the active therapy population. Among 55 patients referred for COVID-19 testing, 24 (44%) were positive for COVID-19, representing a cumulative incidence of 3.4% (longitudinal population) and 1.5% (active therapy population). Compared with patients who were COVID-19 negative, those who were COVID-19 positive were more likely to have a supplemental oxygen requirement (11% versus 54%, p = 0.005) and to have typical COVID-19 pneumonia imaging findings (5 versus 56%, p = 0.001). Otherwise, there were no marked differences in presenting symptoms. Among patients who were COVID-19 negative, alternative etiologies included treatment-related toxicity (26%), atypical pneumonia (22%), and disease progression (22%). A total of 16 patients positive for COVID-19 (67%) required hospitalization, and seven (29%) died from COVID-related complications. CONCLUSIONS: COVID-19 was infrequent in this lung cancer population, but these patients experienced high rates of morbidity and mortality. Oncologists should maintain a low threshold for COVID-19 testing in patients with lung cancer presenting with acute symptoms.

Adenosine Signaling Is Prognostic for Cancer Outcome and Has Predictive Utility for Immunotherapeutic Response.

PURPOSE: There are several agents in early clinical trials targeting components of the adenosine pathway including A2AR and CD73. The identification of cancers with a significant adenosine drive is critical to understand the potential for these molecules. However, it is challenging to measure tumor adenosine levels at scale, thus novel, clinically tractable biomarkers are needed. EXPERIMENTAL DESIGN: We generated a gene expression signature for the adenosine signaling using regulatory networks derived from the literature and validated this in patients. We applied the signature to large cohorts of disease from The Cancer Genome Atlas (TCGA) and cohorts of immune checkpoint inhibitor-treated patients. RESULTS: The signature captures baseline adenosine levels in vivo (r 2 = 0.92, P = 0.018), is reduced after small-molecule inhibition of A2AR in mice (r 2 = -0.62, P = 0.001) and humans (reduction in 5 of 7 patients, 70%), and is abrogated after A2AR knockout. Analysis of TCGA confirms a negative association between adenosine and overall survival (OS, HR = 0.6, P < 2.2e-16) as well as progression-free survival (PFS, HR = 0.77, P = 0.0000006). Further, adenosine signaling is associated with reduced OS (HR = 0.47, P < 2.2e-16) and PFS (HR = 0.65, P = 0.0000002) in CD8+ T-cell-infiltrated tumors. Mutation of TGFß superfamily members is associated with enhanced adenosine signaling and worse OS (HR = 0.43, P < 2.2e-16). Finally, adenosine signaling is associated with reduced efficacy of anti-PD1 therapy in published cohorts (HR = 0.29, P = 0.00012). CONCLUSIONS: These data support the adenosine pathway as a mediator of a successful antitumor immune response, demonstrate the prognostic potential of the signature for immunotherapy, and inform patient selection strategies for adenosine pathway modulators currently in development.

Risk Factors for Negative Global Treatment Outcomes in Lumbar Spinal Stenosis Surgery: A Mixed Effects Model Analysis of Data from an International Spine Registry.

OBJECTIVE: To determine risk factors for negative global treatment outcomes as self-assessed by patients undergoing surgical treatment for lumbar spinal stenosis (LSS). METHODS: Patients from the Spine Tango registry undergoing first-time surgery for LSS were analyzed. The primary outcome was global treatment outcomes measured at the last available follow-up ≥3 months postoperatively using a single question rating how much the operation had helped the patient's back problem (negative = no change/operation made things worse). A 2-level logistic mixed effects model with the treating department as the random effect was used to assess factors associated with a negative outcome. RESULTS: A total of 4504 patients from 39 departments in 10 countries were included. Overall, 14.4% of patients reported a negative global treatment outcome after an average follow-up of 1.3 years. In patients with dominant leg pain, negative outcome was associated with higher baseline back pain; in those with dominant back pain, it was associated with higher baseline back pain, ASA (American Society of Anesthesiologists) ≥3, lower age, not having rigid stabilization, not having disc herniation, and the vertebral level of the most severely affected segment (L5/S1 vs. L3/4). Four departments had significantly higher odds of a negative outcome, whereas 1 department had significantly lower odds. Three out of the 4 negative effects were related to 2 departments from 1 country. CONCLUSIONS: LSS surgery fails to help at least 1 in 10 patients. High baseline back pain is the most important factor associated with a negative treatment outcome. Department-level and potentially country-level factors of unknown origin explained a nonnegligible variation in the treatment results.

Systematic review and meta-analysis of decay rates of waterborne mammalian viruses and coliphages in surface waters.

Surface waters are essential natural resources. They are also receiving waters for a variety of anthropogenic waste streams that carry a myriad of pollutants including pathogens. Watershed and fate and transport models can help inform the spatial and temporal extent of microbial pollution from point and non-point sources and thus provide useful information for managing surface waters. Viruses are particularly important water-related pathogens because they often have a low infectious dose, which means that ingestion of even a small volume of water containing a low concentration of virions has the potential to cause disease. We conducted a systematic review of the literature, following best practices, to gather decay rate constants (k) of mammalian waterborne viruses (enteroviruses, adenoviruses, noroviruses, astroviruses, rotaviruses, and hepatitis A viruses) and coliphages in raw surface waters to aid in the parameterization of virus fate and transport models. We identified 562 k values from the literature, with the largest number identified for enteroviruses and coliphages and the smallest for astrovirus, hepatitis A virus, and norovirus. Average k values for each virus varied from 0.07 to 0.9 per day, in order from smallest to largest: Norwalk virus (i.e., noroviruses) < Human astrovirus < Mastadenovirus (i.e., adenoviruses) < Hepatovirus A (i.e., hepatitis A viruses) < Rotavirus A < coliphages < Enterovirus. A meta-analysis investigated how k varied among viruses for experiments conducted with different virus serotypes or species at different temperatures, salinities, and sunlight exposures, and for experiments that enumerated viruses using different methodologies. Virus species or serotype did not affect k among decay experiments. k values were generally larger for experiments conducted at higher temperatures, in sunlight, and in estuarine waters, and enumerated using culture methods. k values were statistically different between virus types with Norwalk virus, Hepatovirus A, and Mastadenovirus having smaller k values than other viruses, controlling for experimental condition and enumeration method. While F+ coliphage k values were similar to those of Enterovirus, Human astrovirus, and Rotavirus A, they were different from those of the other mammalian viruses. This compilation of coliphage and mammalian virus k values provides essential information for researchers and risk assessors who model virus fate and transport in surface waters and identifies avenues for future research to fill knowledge gaps.

Sustained release and improved bioavailability in mice after subcutaneous administration of griseofulvin as nano- and microcrystals.

The objective of the study was to evaluate the pharmacokinetic profile after different subcutaneous (s.c.) administrations of nano- and microparticle suspensions of griseofulvin to mice. The solubility of the compound was determined as approximately 40 µM, at 37 °C, independent of particle size, stabilizer mixtures investigated and solvent used for measurement. The present in vivo studies demonstrated non-linear absorption kinetics (in peak concentration, Cmax) for griseofulvin up to 50 mg/kg after s.c. administration of nanocrystals and microsuspensions but linear increase in area under the curve (AUC) at all occasions investigated. Cmax was higher for smaller particles administered. Both investigated suspensions, at 10 and 50 mg/kg, showed significantly sustained plasma profiles compared to i.v. and p.o. administration. Administering 10 and 50 mg/kg of griseofulvin nanocrystals as 10 mL/kg, instead of 2.5 mL/kg, improved Cmax but AUC was unchanged. The present study showed that the bioavailability of griseofulvin, administered as nano- and microparticles, increased significantly after s.c. administration (60-100%) compared with p.o. dosing (17%). The drug is currently orally administered and clearly exposed to a significant first pass metabolism, i.e. an ideal candidate for an alternative administration route, like s.c. injection.

Red Sea SAR11 and Prochlorococcus Single-Cell Genomes Reflect Globally Distributed Pangenomes.

Evidence suggests many marine bacteria are cosmopolitan, with widespread but sparse strains poised to seed abundant populations under conducive growth conditions. However, studies supporting this "microbial seed bank" hypothesis have analyzed taxonomic marker genes rather than whole genomes/metagenomes, leaving open the possibility that disparate ocean regions harbor endemic gene content. The Red Sea is isolated geographically from the rest of the ocean and has a combination of high irradiance, high temperature, and high salinity that is unique among the oceans; we therefore asked whether it harbors endemic gene content. We sequenced and assembled single-cell genomes of 21 SAR11 (subclades Ia, Ib, Id, and II) and 5 Prochlorococcus (ecotype HLII) samples from the Red Sea and combined them with globally sourced reference genomes to cluster genes into ortholog groups (OGs). Ordination of OG composition could distinguish clades, including phylogenetically cryptic Prochlorococcus ecotypes LLII and LLIII. Compared with reference genomes, 1% of Prochlorococcus and 17% of SAR11 OGs were unique to the Red Sea genomes (RS-OGs). Most (83%) RS-OGs had no annotated function, but 65% of RS-OGs were expressed in diel Red Sea metatranscriptomes, suggesting they are functional. Searching Tara Oceans metagenomes, RS-OGs were as likely to be found as non-RS-OGs; nevertheless, Red Sea and other warm samples could be distinguished from cooler samples using the relative abundances of OGs. The results suggest that the prevalence of OGs in these surface ocean bacteria is largely cosmopolitan, with differences in population metagenomes manifested by differences in relative abundance rather than complete presence/absence of OGs.IMPORTANCE Studies have shown that as we sequence seawater from a selected environment deeper and deeper, we approach finding every bacterial taxon known for the ocean as a whole. However, such studies have focused on taxonomic marker genes rather than on whole genomes, raising the possibility that the lack of endemism results from the method of investigation. We took a geographically isolated water body, the Red Sea, and sequenced single cells from it. We compared those single-cell genomes to available genomes from around the ocean and to ocean-spanning metagenomes. We showed that gene ortholog groups found in Red Sea genomes but not in other genomes are nevertheless common across global ocean metagenomes. These results suggest that Baas Becking's hypothesis "everything is everywhere, but the environment selects" also applies to gene ortholog groups. This widely dispersed functional diversity may give oceanic microbial communities the functional capacity to respond rapidly to changing conditions.

Development of a quantification method for adenosine in tumors by LC-MS/MS with dansyl chloride derivatization.

Adenosine is known to be an important signaling molecule in many physiological processes and has recently been shown to be an important molecule in oncology. A fit for purpose method has been developed for the quantification of adenosine in murine tumor samples using pre-column derivatization and liquid chromatography-mass spectrometry (LC-MS/MS). To overcome adenosine quantification challenges, derivatization with dansyl chloride was employed. This derivatization technique, following protein precipitation and liquid-liquid extraction, improved the sensitivity and selectivity of adenosine in tumor samples through the reduction of endogenous interference and matrix effects. This method utilizes a mouse plasma calibration curve, qualified over a range of 0.019 µM-37 µM. The 15 min derivatization incubation time and 1 min chromatographic run time allow for higher throughput. The following established method overcomes challenges associated with the quantification of low molecular weight, polar, endogenous molecules, such as adenosine, using derivatization and LC-MS/MS. With the additional analysis of murine tumors, this method will contribute to the understanding of the impact adenosine plays in the tumor microenvironment and the bearing it has on targeted cancer therapies.

Impact of Age on Outcomes with Immunotherapy in Patients with Non-Small Cell Lung Cancer.

INTRODUCTION: Immunotherapy has revolutionized the treatment of NSCLC, but little is known about the activity of programmed cell death 1 and programmed death ligand 1 blockade across age groups. METHODS: We retrospectively evaluated patients with NSCLC who initiated programmed cell death 1 and programmed death ligand 1 inhibitors from January 2013 through July 2017. Medical records and radiographic imaging were reviewed to determine progression-free survival (PFS) and overall survival (OS). We also compared immunotherapy-related toxicities, steroid use, and hospitalizations by age. RESULTS: Of the 245 patients, 26.1% were younger than 60 years, 31.4% were age 60 to 69 years, 31.0% were age 70 to 79 years, and 11.4% were age 80 years or older. The median PFS times by age group were as follows: younger than 60 years, 1.81 months; age 60 to 69 years, 2.53 months; age 70 to 79 years, 3.75 months; and age 80 years or older, 1.64 months (log-rank p value = 0.055). The median OS times by age group were as follows: younger than 60 years, 13.01 months; age 60 to 69 years, 14.56 months; age 70 to 79 years, 12.92 months; and age 80 years or older, 3.62 months (log-rank p value = 0.011). Rates of immunotherapy-related toxicities, steroid use, and hospitalizations did not differ by age. CONCLUSIONS: Although the OS and PFS benefits of immunotherapy differ by age, the rates of toxicity are similar regardless of age.

Safety of Combined PD-1 Pathway Inhibition and Intracranial Radiation Therapy in Non-Small Cell Lung Cancer.

INTRODUCTION: Intracranial metastases are a common cause of morbidity and mortality in patients with advanced NSCLC, and are frequently managed with radiation therapy (RT). The safety of cranial RT in the setting of treatment with immune checkpoint inhibitors (ICIs) has not been established. METHODS: We identified patients with advanced NSCLC with brain metastases who received cranial RT and were treated with or without programmed cell death 1/programmed death ligand 1 inhibitors between August 2013 and September 2016. RT-related adverse events (AEs) were retrospectively evaluated and analyzed according to ICI treatment status, cranial RT type, and timing of RT with respect to ICI. RESULTS: Of 163 patients, 50 (31%) received ICIs, whereas 113 (69%) were ICI naive. Overall, 94 (58%), 28 (17%), and 101 (62%) patients received stereotactic radiosurgery, partial brain irradiation, and/or whole brain RT, respectively. Fifty percent of patients received more than one radiation course. We observed no significant difference in rates of all-grade AEs and grade 3 or higher AEs between the ICI-naive and ICI-treated patients across different cranial RT types (grade ≥3 AEs in 8% of ICI-naive patients versus in 9% of ICI-treated patients for stereotactic radiosurgery [p = 1.00] and in 8% of ICI-naive patients versus in 10% of ICI-treated patients for whole brain RT [p = 0.71]). Additionally, there was no difference in AE rates on the basis of timing of ICI administration with respect to RT. CONCLUSIONS: Treatment with an ICI and cranial RT was not associated with a significant increase in RT-related AEs, suggesting that use of programmed cell death 1/programmed death ligand 1 inhibitors in patients receiving cranial RT may have an acceptable safety profile. Nonetheless, additional studies are needed to validate this approach.