Changes in chromatin accessibility are not concordant with transcriptional changes for single-factor perturbations.

A major goal in the field of transcriptional regulation is the mapping of changes in the binding of transcription factors to the resultant changes in gene expression. Recently, methods for measuring chromatin accessibility have enabled us to measure changes in accessibility across the genome, which are thought to correspond to transcription factor-binding events. In concert with RNA-sequencing, these data in principle enable such mappings; however, few studies have looked at their concordance over short-duration treatments with specific perturbations. Here, we used tandem, bulk ATAC-seq, and RNA-seq measurements from MCF-7 breast carcinoma cells to systematically evaluate the concordance between changes in accessibility and changes in expression in response to retinoic acid and TGF-ß. We found two classes of genes whose expression showed a significant change: those that showed some changes in the accessibility of nearby chromatin, and those that showed virtually no change despite strong changes in expression. The peaks associated with genes in the former group had lower baseline accessibility prior to exposure to signal. Focusing the analysis specifically on peaks with motifs for transcription factors associated with retinoic acid and TGF-ß signaling did not reduce the lack of correspondence. Analysis of paired chromatin accessibility and gene expression data from distinct paths along the hematopoietic differentiation trajectory showed a much stronger correspondence, suggesting that the multifactorial biological processes associated with differentiation may lead to changes in chromatin accessibility that reflect rather than driving altered transcriptional status. Together, these results show many gene expression changes can happen independently of changes in the accessibility of local chromatin in the context of a single-factor perturbation.

Evolved differences in energy metabolism and growth dictate the impacts of ocean acidification on abalone aquaculture.

Ocean acidification (OA) poses a major threat to marine ecosystems and shellfish aquaculture. A promising mitigation strategy is the identification and breeding of shellfish varieties exhibiting resilience to acidification stress. We experimentally compared the effects of OA on two populations of red abalone (Haliotis rufescens), a marine mollusc important to fisheries and global aquaculture. Results from our experiments simulating captive aquaculture conditions demonstrated that abalone sourced from a strong upwelling region were tolerant of ongoing OA, whereas a captive-raised population sourced from a region of weaker upwelling exhibited significant mortality and vulnerability to OA. This difference was linked to population-specific variation in the maternal provisioning of lipids to offspring, with a positive correlation between lipid concentrations and survival under OA. This relationship also persisted in experiments on second-generation animals, and larval lipid consumption rates varied among paternal crosses, which is consistent with the presence of genetic variation for physiological traits relevant for OA survival. Across experimental trials, growth rates differed among family lineages, and the highest mortality under OA occurred in the fastest growing crosses. Identifying traits that convey resilience to OA is critical to the continued success of abalone and other shellfish production, and these mitigation efforts should be incorporated into breeding programs for commercial and restoration aquaculture.

Proteomic changes associated with predator-induced morphological defences in oysters.

Inducible prey defences occur when organisms undergo plastic changes in phenotype to reduce predation risk. When predation pressure varies persistently over space or time, such as when predator and prey co-occur over only part of their biogeographic ranges, prey populations can become locally adapted in their inducible defences. In California estuaries, native Olympia oyster (Ostrea lurida) populations have evolved disparate phenotypic responses to an invasive predator, the Atlantic oyster drill (Urosalpinx cinerea). In this study, oysters from an estuary with drills, and oysters from an estuary without drills, were reared for two generations in a laboratory common garden, and subsequently exposed to cues from Atlantic drills. Comparative proteomics was then used to investigate molecular mechanisms underlying conserved and divergent aspects of their inducible defences. Both populations developed smaller, thicker, and harder shells after drill exposure, and these changes in shell phenotype were associated with upregulation of calcium transport proteins that could influence biomineralization. Inducible defences evolve in part because defended phenotypes incur fitness costs when predation risk is low. Immune proteins were downregulated by both oyster populations after exposure to drills, implying a trade-off between biomineralization and immune function. Following drill exposure, oysters from the population that co-occurs with drills grew smaller shells than oysters inhabiting the estuary not yet invaded by the predator. Variation in the response to drills between populations was associated with isoform-specific protein expression. This trend suggests that a stronger inducible defence response evolved in oysters that co-occur with drills through modification of an existing mechanism.

Coast-wide evidence of low pH amelioration by seagrass ecosystems.

Global-scale ocean acidification has spurred interest in the capacity of seagrass ecosystems to increase seawater pH within crucial shoreline habitats through photosynthetic activity. However, the dynamic variability of the coastal carbonate system has impeded generalization into whether seagrass aerobic metabolism ameliorates low pH on physiologically and ecologically relevant timescales. Here we present results of the most extensive study to date of pH modulation by seagrasses, spanning seven meadows (Zostera marina) and 1000 km of U.S. west coast over 6 years. Amelioration by seagrass ecosystems compared to non-vegetated areas occurred 65% of the time (mean increase 0.07 ± 0.008 SE). Events of continuous elevation in pH within seagrass ecosystems, indicating amelioration of low pH, were longer and of greater magnitude than opposing cases of reduced pH or exacerbation. Sustained elevations in pH of >0.1, comparable to a 30% decrease in [H+ ], were not restricted only to daylight hours but instead persisted for up to 21 days. Maximal pH elevations occurred in spring and summer during the seagrass growth season, with a tendency for stronger effects in higher latitude meadows. These results indicate that seagrass meadows can locally alleviate low pH conditions for extended periods of time with important implications for the conservation and management of coastal ecosystems.

Seagrass-driven changes in carbonate chemistry enhance oyster shell growth.

Quantifying the strength of non-trophic interactions exerted by foundation species is critical to understanding how natural communities respond to environmental stress. In the case of ocean acidification (OA), submerged marine macrophytes, such as seagrasses, may create local areas of elevated pH due to their capacity to sequester dissolved inorganic carbon through photosynthesis. However, although seagrasses may increase seawater pH during the day, they can also decrease pH at night due to respiration. Therefore, it remains unclear how consequences of such diel fluctuations may unfold for organisms vulnerable to OA. We established mesocosms containing different levels of seagrass biomass (Zostera marina) to create a gradient of carbonate chemistry conditions and explored consequences for growth of juvenile and adult oysters (Crassostrea gigas), a non-native species widely used in aquaculture that can co-occur, and is often grown, in proximity to seagrass beds. In particular, we investigated whether increased diel fluctuations in pH due to seagrass metabolism affected oyster growth. Seagrasses increased daytime pH up to 0.4 units but had little effect on nighttime pH (reductions less than 0.02 units). Thus, both the average pH and the amplitude of diel pH fluctuations increased with greater seagrass biomass. The highest seagrass biomass increased oyster shell growth rate (mm day-1) up to 40%. Oyster somatic tissue weight and oyster condition index exhibited a different pattern, peaking at intermediate levels of seagrass biomass. This work demonstrates the ability of seagrasses to facilitate oyster calcification and illustrates how non-trophic metabolic interactions can modulate effects of environmental change.

Differences in induced thermotolerance among populations of Olympia oysters.

An organism's ability to cope with thermal stress is an important predictor of survival in a changing climate. One way in which organisms may acclimatize to thermal stress in the short-term is through induced thermotolerance, whereby exposure to a sublethal heat shock enables the organism to subsequently survive what might otherwise be a lethal event. Whether induced thermotolerance is related to basal thermotolerance is not well understood for marine organisms. Furthermore, whether populations often differ in their capacity for induced thermotolerance is also unclear. Here, we tested for differences in basal thermotolerance and induced thermotolerance among six populations of Olympia oysters (Ostrea lurida) from three California estuaries. Oysters were raised under common-garden laboratory conditions for a generation and then exposed to two treatments (control or sublethal heat shock) followed by a spectrum of temperatures that bound the upper critical temperature in order to determine LT50 (temperature at which 50% of the population dies). All populations exhibited induced thermotolerance by increasing their LT50 to a similar maximum temperature when extreme thermal stress was preceded by a sublethal heat shock. However, populations differed in their basal thermotolerance and their plasticity in thermotolerance. Populations with the highest basal thermotolerance were least able to modify upper critical temperature, while the population with the lowest basal thermotolerance exhibited the greatest plasticity in the upper critical temperature. Our results highlight that populations with high basal thermotolerance may be most vulnerable to climate warming because they lack the plasticity required to adjust their upper thermal limits.

A computational approach to distinguish somatic vs. germline origin of genomic alterations from deep sequencing of cancer specimens without a matched normal.

A key constraint in genomic testing in oncology is that matched normal specimens are not commonly obtained in clinical practice. Thus, while well-characterized genomic alterations do not require normal tissue for interpretation, a significant number of alterations will be unknown in whether they are germline or somatic, in the absence of a matched normal control. We introduce SGZ (somatic-germline-zygosity), a computational method for predicting somatic vs. germline origin and homozygous vs. heterozygous or sub-clonal state of variants identified from deep massively parallel sequencing (MPS) of cancer specimens. The method does not require a patient matched normal control, enabling broad application in clinical research. SGZ predicts the somatic vs. germline status of each alteration identified by modeling the alteration's allele frequency (AF), taking into account the tumor content, tumor ploidy, and the local copy number. Accuracy of the prediction depends on the depth of sequencing and copy number model fit, which are achieved in our clinical assay by sequencing to high depth (>500x) using MPS, covering 394 cancer-related genes and over 3,500 genome-wide single nucleotide polymorphisms (SNPs). Calls are made using a statistic based on read depth and local variability of SNP AF. To validate the method, we first evaluated performance on samples from 30 lung and colon cancer patients, where we sequenced tumors and matched normal tissue. We examined predictions for 17 somatic hotspot mutations and 20 common germline SNPs in 20,182 clinical cancer specimens. To assess the impact of stromal admixture, we examined three cell lines, which were titrated with their matched normal to six levels (10-75%). Overall, predictions were made in 85% of cases, with 95-99% of variants predicted correctly, a significantly superior performance compared to a basic approach based on AF alone. We then applied the SGZ method to the COSMIC database of known somatic variants in cancer and found >50 that are in fact more likely to be germline.

Enrichment of Targetable Mutations in the Relapsed Neuroblastoma Genome.

Neuroblastoma is characterized by a relative paucity of recurrent somatic mutations at diagnosis. However, recent studies have shown that the mutational burden increases at relapse, likely as a result of clonal evolution of mutation-carrying cells during primary treatment. To inform the development of personalized therapies, we sought to further define the frequency of potentially actionable mutations in neuroblastoma, both at diagnosis and after chemotherapy. We performed a retrospective study to determine mutation frequency, the only inclusion criterion being availability of cancer gene panel sequencing data from Foundation Medicine. We analyzed 151 neuroblastoma tumor samples: 44 obtained at diagnosis, 42 at second look surgery or biopsy for stable disease after chemotherapy, and 59 at relapse (6 were obtained at unknown time points). Nine patients had multiple tumor biopsies. ALK was the most commonly mutated gene in this cohort, and we observed a higher frequency of suspected oncogenic ALK mutations in relapsed disease than at diagnosis. Patients with relapsed disease had, on average, a greater number of mutations reported to be recurrent in cancer, and a greater number of mutations in genes that are potentially targetable with available therapeutics. We also observed an enrichment of reported recurrent RAS/MAPK pathway mutations in tumors obtained after chemotherapy. Our data support recent evidence suggesting that neuroblastomas undergo substantial mutational evolution during therapy, and that relapsed disease is more likely to be driven by a targetable oncogenic pathway, highlighting that it is critical to base treatment decisions on the molecular profile of the tumor at the time of treatment. However, it will be necessary to conduct prospective clinical trials that match sequencing results to targeted therapeutic intervention to determine if cancer genomic profiling improves patient outcomes.

Transcriptomic responses to extreme low salinity among locally adapted populations of Olympia oyster (Ostrea lurida).

The Olympia oyster (Ostrea lurida) is a foundation species inhabiting estuaries along the North American west coast. In California estuaries, O. lurida is adapted to local salinity regimes and populations differ in low salinity tolerance. In this study, oysters from three California populations were reared for two generations in a laboratory common garden and subsequently exposed to low salinity seawater. Comparative transcriptomics was then used to understand species-level responses to hyposmotic stress and population-level mechanisms underlying divergent salinity tolerances. Gene expression patterns indicate Olympia oysters are sensitive to hyposmotic stress: All populations respond to low salinity by up-regulating transcripts indicative of protein unfolding, DNA damage and cell cycle arrest after sub-lethal exposure. Among O. lurida populations, transcriptomic profiles differed constitutively and in response to low salinity. Despite two generations in common-garden conditions, transcripts encoding apoptosis modulators were constitutively expressed at significantly different levels in the most tolerant population. Expression of cell death regulators may facilitate cell fate decisions when salinity declines. Following low salinity exposure, oysters from the more tolerant population expressed a small number of mRNAs at significantly higher levels than less tolerant populations. Proteins encoded by these transcripts regulate ciliary activity within the mantle cavity and may function to prolong valve closure and reduce mortality in low salinity seawater. Collectively, gene expression patterns suggest sub-lethal impacts of hyposmotic stress in Olympia oysters are considerable and that even oysters with greater low salinity tolerance may be vulnerable to future freshwater flooding events.

Genomic Alterations Observed in Colitis-Associated Cancers Are Distinct From Those Found in Sporadic Colorectal Cancers and Vary by Type of Inflammatory Bowel Disease.

BACKGROUND & AIMS: Patients with inflammatory bowel diseases, such as Crohn's disease (CD) and ulcerative colitis (UC), are at increased risk for small bowel or colorectal cancers (colitis-associated cancers [CACs]). We compared the spectrum of genomic alterations in CACs with those of sporadic colorectal cancers (CRCs) and investigated differences between CACs from patients with CD vs UC. METHODS: We studied tumor tissues from patients with CACs treated at Memorial Sloan Kettering Cancer Center or Weill Cornell Medical College from 2003 through 2015. We performed hybrid capture-based next-generation sequencing analysis of >300 cancer-related genes to comprehensively characterize genomic alterations. RESULTS: We performed genomic analyses of 47 CACs (from 29 patients with UC and 18 with CD; 43 primary tumors and 4 metastases). Primary tumors developed in the ileum (n = 2), right colon (n = 18), left colon (n = 6), and rectosigmoid or rectum (n = 21). We found genomic alterations in TP53, IDH1, and MYC to be significantly more frequent, and mutations in APC to be significantly less frequent, than those reported in sporadic CRCs by The Cancer Genome Atlas or Foundation Medicine. We identified genomic alterations that might be targeted by a therapeutic agent in 17 of 47 (36%) CACs. These included the mutation encoding IDH1 R132; amplification of FGFR1, FGFR2, and ERBB2; and mutations encoding BRAF V600E and an EML4-ALK fusion protein. Alterations in IDH1 and APC were significantly more common in CACs from patients with CD than UC. CONCLUSIONS: In an analysis of CACs from 47 patients, we found significant differences in the spectrum of genomic alterations in CACs compared with sporadic CRCs. We observed a high frequency of IDH1 R132 mutations in patients with CD but not UC, as well as a high frequency of MYC amplification in CACs. Many genetic alterations observed in CACs could serve as therapeutic targets.

Interactive effects of temperature, food and skeletal mineralogy mediate biological responses to ocean acidification in a widely distributed bryozoan.

Marine invertebrates with skeletons made of high-magnesium calcite may be especially susceptible to ocean acidification (OA) due to the elevated solubility of this form of calcium carbonate. However, skeletal composition can vary plastically within some species, and it is largely unknown how concurrent changes in multiple oceanographic parameters will interact to affect skeletal mineralogy, growth and vulnerability to future OA. We explored these interactive effects by culturing genetic clones of the bryozoan Jellyella tuberculata (formerly Membranipora tuberculata) under factorial combinations of dissolved carbon dioxide (CO2), temperature and food concentrations. High CO2 and cold temperature induced degeneration of zooids in colonies. However, colonies still maintained high growth efficiencies under these adverse conditions, indicating a compensatory trade-off whereby colonies degenerate more zooids under stress, redirecting energy to the growth and maintenance of new zooids. Low-food concentration and elevated temperatures also had interactive effects on skeletal mineralogy, resulting in skeletal calcite with higher concentrations of magnesium, which readily dissolved under high CO2 For taxa that weakly regulate skeletal magnesium concentration, skeletal dissolution may be a more widespread phenomenon than is currently documented and is a growing concern as oceans continue to warm and acidify.

Transcriptomic responses to seawater acidification among sea urchin populations inhabiting a natural pH mosaic.

Increasing awareness of spatial and temporal variation in ocean pH suggests some marine populations may be adapted to local pH regimes and will therefore respond differently to present-day pH variation and to long-term ocean acidification. In the Northeast Pacific Ocean, differences in the strength of coastal upwelling cause latitudinal variation in prevailing pH regimes that are hypothesized to promote local adaptation and unequal pH tolerance among resident populations. In this study, responses to experimental seawater acidification were compared among embryos and larvae from six populations of purple sea urchins (Strongylocentrotus purpuratus) inhabiting areas that differ in their frequency of low pH exposure and that prior research suggests are locally adapted to seawater pH. Transcriptomic analyses demonstrate urchin populations most frequently exposed to low pH seawater responded to experimental acidification by expressing genes within major ATP-producing pathways at greater levels than populations encountering low pH less often. Multiple genes within the tricarboxylic acid cycle, electron transport chain and fatty acid beta oxidation pathways were upregulated in urchin populations experiencing low pH conditions most frequently. These same metabolic pathways were significantly over-represented among genes both expressed in a population-specific manner and putatively under selection to enhance low pH tolerance. Collectively, these data suggest natural selection is acting on metabolic gene networks to redirect ATP toward maintaining acid-base homeostasis and enhance tolerance of seawater acidification. As a trade-off, marine populations more tolerant of low pH may have less energy to put towards other aspects of fitness and to respond to additional ocean change.

Timing of stressors alters interactive effects on a coastal foundation species.

The effects of climate-driven stressors on organismal performance and ecosystem functioning have been investigated across many systems; however, manipulative experiments generally apply stressors as constant and simultaneous treatments, rather than accurately reflecting temporal patterns in the natural environment. Here, we assessed the effects of temporal patterns of high aerial temperature and low salinity on survival of Olympia oysters (Ostrea lurida), a foundation species of conservation and restoration concern. As single stressors, low salinity (5 and 10 psu) and the highest air temperature (40°C) resulted in oyster mortality of 55.8, 11.3, and 23.5%, respectively. When applied on the same day, low salinity and high air temperature had synergistic negative effects that increased oyster mortality. This was true even for stressor levels that were relatively mild when applied alone (10 psu and 35°C). However, recovery times of two or four weeks between stressors eliminated the synergistic effects. Given that most natural systems threatened by climate change are subject to multiple stressors that vary in the timing of their occurrence, our results suggest that it is important to examine temporal variation of stressors in order to more accurately understand the possible biological responses to global change.

Plastic responses of bryozoans to ocean acidification.

Phenotypic plasticity has the potential to allow organisms to respond rapidly to global environmental change, but the range and effectiveness of these responses are poorly understood across taxa and growth strategies. Colonial organisms might be particularly resilient to environmental stressors, as organizational modularity and successive asexual generations can allow for distinctively flexible responses in the aggregate form. We performed laboratory experiments to examine the effects of increasing dissolved carbon dioxide (CO2) (i.e. ocean acidification) on the colonial bryozoan Celleporella cornuta sampled from two source populations within a coastal upwelling region of the northern California coast. Bryozoan colonies were remarkably plastic under these CO2 treatments. Colonies raised under high CO2 grew more quickly, investing less in reproduction and producing lighter skeletons when compared with genetically identical clones raised under current surface atmosphere CO2 values. Bryozoans held under high CO2 conditions also changed the Mg/Ca ratio of skeletal calcite and increased the expression of organic coverings in new growth, which may serve as protection against acidified water. We also observed strong differences between source populations in reproductive investment and organic covering reaction norms, consistent with adaptive responses to persistent spatial variation in local oceanographic conditions. Our results demonstrate that phenotypic plasticity and energetic trade-offs can mediate biological responses to global environmental change, and highlight the broad range of strategies available to colonial organisms.

Inducible defenses in Olympia oysters in response to an invasive predator.

The prey naiveté hypothesis suggests that native prey may be vulnerable to introduced predators because they have not evolved appropriate defenses. However, recent evidence suggests that native prey sometimes exhibit induced defenses to introduced predators, as a result of rapid evolution or other processes. We examined whether Olympia oysters (Ostrea lurida) display inducible defenses in the presence of an invasive predator, the Atlantic oyster drill (Urosalpinx cinerea), and whether these responses vary among oyster populations from estuaries with and without this predator. We spawned oysters from six populations distributed among three estuaries in northern California, USA, and raised their offspring through two generations under common conditions to minimize effects of environmental history. We exposed second-generation oysters to cue treatments: drills eating oysters, drills eating barnacles, or control seawater. Oysters from all populations grew smaller shells when exposed to drill cues, and grew thicker and harder shells when those drills were eating oysters. Oysters exposed to drills eating other oysters were subsequently preyed upon at a slower rate. Although all oyster populations exhibited inducible defenses, oysters from the estuary with the greatest exposure to drills grew the smallest shells suggesting that oyster populations have evolved adaptive differences in the strength of their responses to predators. Our findings add to a growing body of literature that suggests that marine prey may be less likely to exhibit naiveté in the face of invasive predators than prey in communities that are more isolated from native predators, such as many freshwater and terrestrial island ecosystems.

Interacting environmental mosaics drive geographic variation in mussel performance and predation vulnerability.

Although theory suggests geographic variation in species' performance is determined by multiple niche parameters, little consideration has been given to the spatial structure of interacting stressors that may shape local and regional vulnerability to global change. Here, we use spatially explicit mosaics of carbonate chemistry, food availability and temperature spanning 1280 km of coastline to test whether persistent, overlapping environmental mosaics mediate the growth and predation vulnerability of a critical foundation species, the mussel Mytilus californianus. We find growth was highest and predation vulnerability was lowest in dynamic environments with frequent exposure to low pH seawater and consistent food. In contrast, growth was lowest and predation vulnerability highest when exposure to low pH seawater was decoupled from high food availability, or in exceptionally warm locations. These results illustrate how interactions among multiple drivers can cause unexpected, yet persistent geographic mosaics of species performance, interactions and vulnerability to environmental change.

Comprehensive Genomic Profiling of Advanced Penile Carcinoma Suggests a High Frequency of Clinically Relevant Genomic Alterations.

BACKGROUND: Advanced penile squamous cell carcinoma (PSCC) is associated with poor survival due to the aggressiveness of the disease and lack of effective systemic therapies. Comprehensive genomic profiling (CGP) was performed to identify clinically relevant genomic alterations (CRGAs). MATERIALS AND METHODS: DNA was extracted from 40 µm of formalin-fixed, paraffin-embedded sections in patients with advanced PSCC. CGP was performed on hybridization-captured, adaptor ligation-based libraries to a mean coverage depth of 692× for 3,769 exons of 236 cancer-related genes plus 47 introns from 19 genes frequently rearranged in cancer. CRGAs were defined as genomic alterations (GAs) linked to targeted therapies on the market or under evaluation in mechanism-driven clinical trials. RESULTS: Twenty male patients with a median age of 60 years (range, 46-87 years) were assessed. Seventeen (85%) cases were stage IV and three cases (15%) were stage III. CGP revealed 109 GAs (5.45 per tumor), 44 of which were CRGAs (2.2 per tumor). At least one CRGA was detected in 19 (95%) cases, and the most common CRGAs were CDKN2A point mutations and homozygous deletion (40%), NOTCH1 point mutations and rearrangements (25%), PIK3CA point mutations and amplification (25%), EGFR amplification (20%), CCND1 amplification (20%), BRCA2 insertions/deletions (10%), RICTOR amplifications (10%), and FBXW7 point mutations (10%). CONCLUSION: CGP identified CRGAs in patients with advanced PSCC, including EGFR amplification and PIK3CA alterations, which can lead to the rational administration of targeted therapy and subsequent benefit for these patients. IMPLICATIONS FOR PRACTICE: Few treatment options exist for patients with advanced penile squamous cell carcinoma (PSCC). Outcomes are dismal with platinum-based chemotherapy, with median survival estimated at 1 year or less across multiple series. Biological studies of patients with PSCC to date have principally focused on human papillomavirus status, but few studies have elucidated molecular drivers of the disease. To this end, comprehensive genomic profiling was performed in a cohort of 20 patients with advanced PSCC. Findings of frequent mutations in CDKN2A, NOTCH1, PIK3CA, and EGFR (all in excess of 20%) point to potential therapeutic avenues. Trials of targeted therapies directed toward these mutations should be explored.

Comprehensive Genomic Profiling Identifies a Subset of Crizotinib-Responsive ALK-Rearranged Non-Small Cell Lung Cancer Not Detected by Fluorescence In Situ Hybridization.

INTRODUCTION: For patients with non-small cell lung cancer (NSCLC) to benefit from ALK inhibitors, sensitive and specific detection of ALK genomic rearrangements is needed. ALK break-apart fluorescence in situ hybridization (FISH) is the U.S. Food and Drug Administration approved and standard-of-care diagnostic assay, but identification of ALK rearrangements by other methods reported in NSCLC cases that tested negative for ALK rearrangements by FISH suggests a significant false-negative rate. We report here a large series of NSCLC cases assayed by hybrid-capture-based comprehensive genomic profiling (CGP) in the course of clinical care. MATERIALS AND METHODS: Hybrid-capture-based CGP using next-generation sequencing was performed in the course of clinical care of 1,070 patients with advanced lung cancer. Each tumor sample was evaluated for all classes of genomic alterations, including base-pair substitutions, insertions/deletions, copy number alterations and rearrangements, as well as fusions/rearrangements. RESULTS: A total of 47 patients (4.4%) were found to harbor ALK rearrangements, of whom 41 had an EML4-ALK fusion, and 6 had other fusion partners, including 3 previously unreported rearrangement events: EIF2AK-ALK, PPM1B-ALK, and PRKAR1A-ALK. Of 41 patients harboring ALK rearrangements, 31 had prior FISH testing results available. Of these, 20 were ALK FISH positive, and 11 (35%) were ALK FISH negative. Of the latter 11 patients, 9 received crizotinib based on the CGP results, and 7 achieved a response with median duration of 17 months. CONCLUSION: Comprehensive genomic profiling detected canonical ALK rearrangements and ALK rearrangements with noncanonical fusion partners in a subset of patients with NSCLC with previously negative ALK FISH results. In this series, such patients had durable responses to ALK inhibitors, comparable to historical response rates for ALK FISH-positive cases. IMPLICATIONS FOR PRACTICE: Comprehensive genomic profiling (CGP) that includes hybrid capture and specific baiting of intron 19 of ALK is a highly sensitive, alternative method for identification of drug-sensitive ALK fusions in patients with non-small cell lung cancer (NSCLC) who had previously tested negative using standard ALK fluorescence in situ hybridization (FISH) diagnostic assays. Given the proven benefit of treatment with crizotinib and second-generation ALK inhibitors in patients with ALK fusions, CGP should be considered in patients with NSCLC, including those who have tested negative for other alterations, including negative results using ALK FISH testing.

Ocean acidification alters the response of intertidal snails to a key sea star predator.

Organism-level effects of ocean acidification (OA) are well recognized. Less understood are OA's consequences for ecological species interactions. Here, we examine a behaviourally mediated predator-prey interaction within the rocky intertidal zone of the temperate eastern Pacific Ocean, using it as a model system to explore OA's capacity to impair invertebrate anti-predator behaviours more broadly. Our system involves the iconic sea star predator, Pisaster ochraceus, that elicits flee responses in numerous gastropod prey. We examine, in particular, the capacity for OA-associated reductions in pH to alter flight behaviours of the black turban snail, Tegula funebralis, an often-abundant and well-studied grazer in the system. We assess interactions between these species at 16 discrete levels of pH, quantifying the full functional response of Tegula under present and near-future OA conditions. Results demonstrate the disruption of snail anti-predator behaviours at low pH, with decreases in the time individuals spend in refuge locations. We also show that fluctuations in pH, including those typical of rock pools inhabited by snails, do not materially change outcomes, implying little capacity for episodically benign pH conditions to aid behavioural recovery. Together, these findings suggest a strong potential for OA to induce cascading community-level shifts within this long-studied ecosystem.

Historical baselines and the future of shell calcification for a foundation species in a changing ocean.

Seawater pH and the availability of carbonate ions are decreasing due to anthropogenic carbon dioxide emissions, posing challenges for calcifying marine species. Marine mussels are of particular concern given their role as foundation species worldwide. Here, we document shell growth and calcification patterns in Mytilus californianus, the California mussel, over millennial and decadal scales. By comparing shell thickness across the largest modern shells, the largest mussels collected in the 1960s-1970s and shells from two Native American midden sites (∼1000-2420 years BP), we found that modern shells are thinner overall, thinner per age category and thinner per unit length. Thus, the largest individuals of this species are calcifying less now than in the past. Comparisons of shell thickness in smaller individuals over the past 10-40 years, however, do not show significant shell thinning. Given our sampling strategy, these results are unlikely to simply reflect within-site variability or preservation effects. Review of environmental and biotic drivers known to affect shell calcification suggests declining ocean pH as a likely explanation for the observed shell thinning. Further future decreases in shell thickness could have significant negative impacts on M. californianus survival and, in turn, negatively impact the species-rich complex that occupies mussel beds.