Circulating Tumor DNA as a Predictive Biomarker for Clinical Outcomes With Margetuximab and Pembrolizumab in Pretreated HER2-Positive Gastric/ Gastroesophageal Adenocarcinoma.

PURPOSE: To assess the ability of circulating tumor DNA (ctDNA)-based testing to identify patients with HER2 (encoded by ERBB2)-positive gastric/gastroesophageal adenocarcinoma (GEA) who progressed on or after trastuzumab-containing treatments were treated with combination therapy of anti-HER2 and anti-PD-1 agents. METHODS: ctDNA analysis was performed retrospectively using plasma samples collected at study entry from 86 patients participating in the phase 1/2 CP-MGAH22-05 study (NCT02689284). RESULTS: Objective response rate (ORR) was significantly higher in evaluable ERBB2 amplification-positive vs - negative patients based on ctDNA analysis at study entry (37% vs 6%, respectively; P = .00094). ORR was 23% across all patients who were evaluable for response. ERBB2 amplification was detected at study entry in 57% of patients (all HER2 positive at diagnosis), and detection was higher (88%) when HER2 status was determined by immunohistochemistry fewer than 6 months before study entry. ctDNA was detected in 98% (84/86) of patients tested at study entry. Codetected ERBB2-activating mutations were not associated with response. CONCLUSIONS: Current ERBB2 status may be more effective than archival status at predicting clinical benefit from margetuximab plus pembrolizumab therapy. ctDNA testing for ERBB2 status prior to treatment will spare patients from repeat tissue biopsies, which may be reserved for reflex testing when ctDNA is not detected.

Margetuximab plus pembrolizumab in patients with previously treated, HER2-positive gastro-oesophageal adenocarcinoma (CP-MGAH22-05): a single-arm, phase 1b-2 trial.

BACKGROUND: Margetuximab, a novel, investigational, Fc-engineered, anti-HER2 monoclonal antibody, is designed to more effectively potentiate innate immunity than trastuzumab. We aimed to evaluate the safety, tolerability, and antitumour activity of margetuximab plus pembrolizumab (an anti-PD-1 monoclonal antibody) in previously treated patients with HER2-positive gastro-oesophageal adenocarcinoma. METHODS: CP-MGAH22-05 was a single-arm, open-label, phase 1b-2 dose-escalation and cohort expansion study done at 11 academic centres in the USA and Canada and 15 centres in southeast Asia (Korea, Taiwan, and Singapore) that enrolled men and women aged 18 years or older with histologically proven, unresectable, locally advanced or metastatic, HER2-positive, PD-L1-unselected gastro-oesophageal adenocarcinoma, with an Eastern Cooperative Oncology Group performance status of 0 or 1, who had progressed after at least one previous line of therapy with trastuzumab plus chemotherapy in the locally advanced unresectable or metastatic setting. In the dose-escalation phase, nine patients were treated: three received margetuximab 10 mg/kg intravenously plus pembrolizumab 200 mg intravenously every 3 weeks and six received the recommended phase 2 dose of margetuximab 15 mg/kg plus pembrolizumab 200 mg intravenously every 3 weeks. An additional 86 patients were enrolled in the phase 2 cohort expansion and received the recommended phase 2 dose. The primary endpoints were safety and tolerability, assessed in the safety population (patients who received at least one dose of either margetuximab or pembrolizumab) and the objective response rate as assessed by the investigator according to both Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1, in the response-evaluable population (patients with measurable disease at baseline and who received the recommended phase 2 dose of margetuximab and pembrolizumab). This trial is registered with ClinicalTrials.gov, NCT02689284. Recruitment for the trial has completed and follow-up is ongoing. FINDINGS: Between Feb 11, 2016, and Oct 2, 2018, 95 patients were enrolled. Median follow-up was 19·9 months (IQR 10·7-23·1). The combination therapy showed acceptable safety and tolerability; there were no dose-limiting toxicities in the dose-escalation phase. The most common grade 3-4 treatment-related adverse events were anaemia (four [4%]) and infusion-related reactions (three [3%]). Serious treatment-related adverse events were reported in nine (9%) patients. No treatment-related deaths were reported. Objective responses were observed in 17 (18·48%; 95% CI 11·15-27·93) of 92 evaluable patients. INTERPRETATION: These findings serve as proof of concept of synergistic antitumour activity with the combination of an Fc-optimised anti-HER2 agent (margetuximab) along with anti-PD-1 checkpoint blockade (pembrolizumab). FUNDING: MacroGenics.

A systematic genome-wide analysis of zebrafish protein-coding gene function.

Since the publication of the human reference genome, the identities of specific genes associated with human diseases are being discovered at a rapid rate. A central problem is that the biological activity of these genes is often unclear. Detailed investigations in model vertebrate organisms, typically mice, have been essential for understanding the activities of many orthologues of these disease-associated genes. Although gene-targeting approaches and phenotype analysis have led to a detailed understanding of nearly 6,000 protein-coding genes, this number falls considerably short of the more than 22,000 mouse protein-coding genes. Similarly, in zebrafish genetics, one-by-one gene studies using positional cloning, insertional mutagenesis, antisense morpholino oligonucleotides, targeted re-sequencing, and zinc finger and TAL endonucleases have made substantial contributions to our understanding of the biological activity of vertebrate genes, but again the number of genes studied falls well short of the more than 26,000 zebrafish protein-coding genes. Importantly, for both mice and zebrafish, none of these strategies are particularly suited to the rapid generation of knockouts in thousands of genes and the assessment of their biological activity. Here we describe an active project that aims to identify and phenotype the disruptive mutations in every zebrafish protein-coding gene, using a well-annotated zebrafish reference genome sequence, high-throughput sequencing and efficient chemical mutagenesis. So far we have identified potentially disruptive mutations in more than 38% of all known zebrafish protein-coding genes. We have developed a multi-allelic phenotyping scheme to efficiently assess the effects of each allele during embryogenesis and have analysed the phenotypic consequences of over 1,000 alleles. All mutant alleles and data are available to the community and our phenotyping scheme is adaptable to phenotypic analysis beyond embryogenesis.

The zebrafish reference genome sequence and its relationship to the human genome.

Zebrafish have become a popular organism for the study of vertebrate gene function. The virtually transparent embryos of this species, and the ability to accelerate genetic studies by gene knockdown or overexpression, have led to the widespread use of zebrafish in the detailed investigation of vertebrate gene function and increasingly, the study of human genetic disease. However, for effective modelling of human genetic disease it is important to understand the extent to which zebrafish genes and gene structures are related to orthologous human genes. To examine this, we generated a high-quality sequence assembly of the zebrafish genome, made up of an overlapping set of completely sequenced large-insert clones that were ordered and oriented using a high-resolution high-density meiotic map. Detailed automatic and manual annotation provides evidence of more than 26,000 protein-coding genes, the largest gene set of any vertebrate so far sequenced. Comparison to the human reference genome shows that approximately 70% of human genes have at least one obvious zebrafish orthologue. In addition, the high quality of this genome assembly provides a clearer understanding of key genomic features such as a unique repeat content, a scarcity of pseudogenes, an enrichment of zebrafish-specific genes on chromosome 4 and chromosomal regions that influence sex determination.

Risk Factors for Venous Thromboembolism in Pediatric Trauma Patients and Validation of a Novel Scoring System: The Risk of Clots in Kids With Trauma Score.

OBJECTIVES: Identify risk factors for venous thromboembolism and develop venous thromboembolism risk assessment models for pediatric trauma patients. DESIGN: Single institution and national registry retrospective cohort studies. SETTING: John Hopkins level 1 adult and pediatric trauma center and National Trauma Data Bank. PATIENTS: Patients 21 years and younger hospitalized following traumatic injuries at John Hopkins (1987-2011). Patients 21 years and younger in the National Trauma Data Bank (2008-2010 and 2011-2012). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Clinical characteristics of Johns Hopkins patients with and without venous thromboembolism were compared, and multivariable logistic regression analysis was used to identify independent venous thromboembolism risk factors. Weighted risk assessment scoring systems were developed based on these and previously identified factors from National Trauma Data Bank patients (2008-2010); the scoring systems were validated in this cohort from Johns Hopkins and a cohort from the National Trauma Data Bank (2011-2012). Forty-nine of 17,366 pediatric trauma patients (0.28%) were diagnosed with venous thromboembolism after admission to our trauma center. After adjusting for potential confounders, venous thromboembolism was independently associated with older age, surgery, blood transfusion, higher Injury Severity Score, and lower Glasgow Coma Scale score. These and additional factors were identified in 402,329 pediatric patients from the National Trauma Data Bank from 2008 to 2010; independent risk factors from the logistic regression analysis of this National Trauma Data Bank cohort were selected and incorporated into weighted risk assessment scoring systems. Two models were developed and were cross-validated in two separate pediatric trauma cohorts: 1) 282,535 patients in the National Trauma Data Bank from 2011 to 2012 and 2) 17,366 patients from Johns Hopkins. The receiver operating curve using these models in the validation cohorts had area under the curves that ranged 90-94%. CONCLUSIONS: Venous thromboembolism is infrequent after trauma in pediatric patients. We developed weighted scoring systems to stratify pediatric trauma patients at risk for venous thromboembolism. These systems may have potential to guide risk-appropriate venous thromboembolism prophylaxis in children after trauma.

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.

Performance of the Abbott Architect Immuno-Chemiluminometric NT-proBNP Assay.

Background: We evaluated the performance of the Abbott N-terminal pro-brain natriuretic peptide (NT-proBNP) assay against the Roche NT-proBNP immunoassay across two sites. Methods: Precision, linearity, and sensitivity studies were performed. A combined method of comparison and regression analysis was performed between the Roche and Abbott assays using samples from both sites (n = 494). To verify biotin interference, lyophilised biotin powder was reconstituted and spiked into serum samples at two medical decision levels (final concentration 500/4250 ng/mL) and compared to controls. NT-proBNP was also measured in anonymised leftover sera (n = 388) in a cardio-renal healthy population and stratified into three age bands­<50 (n = 145), 50−75 (n = 183) and >75 (n = 60). Results: Between-run precision (CV%) for NT-proBNP was 4.17/4.50 (139.5/142.0 pg/mL), 3.83/2.17 (521.6/506.3), and 4.60/2.51 (5053/4973), respectively. The assay was linear from 0.7−41,501 pg/mL. The limit of blank/quantitation was 1.2/7.9 pg/mL. The assay showed no interference from biotin up to 4250 ng/mL. Passing−Bablok regression analysis showed excellent agreement between the two assays (r = 0.999, 95% CI 0.999 to 0.999, p < 0.0001). The Roche assay had a slightly persistent, negative bias across different levels of NT-proBNP. ESC age cut-offs for diagnosing acute heart failure are applicable for the Abbott assay, with the median NT-proBNP of subjects < 50 years old at 43.0 pg/mL (range 4.9−456 pg/mL), 50−75 years old at 95.1 pg/mL (range 10.5−1079 pg/mL), and >75 years old at 173.1 pg/mL (range 23.2−1948 pg/mL). Conclusions: The Abbott Architect NT-proBNP assay has good performance that agrees with the manufacturer's specifications. ESC/AHA recommended NT-proBNP age groups for acute heart failure diagnosis are applicable to this assay.

Successful Treatment with Ensartinib After Alectinib-induced Hyperbilirubinemia in ALK-Positive NSCLC.

BACKGROUND: Alectinib is approved for the treatment of advanced non-small-cell lung cancer (NSCLC) harboring ALK rearrangements. Although generally well tolerated, alectinib can cause serious or life-threatening side effects. CASE PRESENTATION: Here, we report a case of a patient with NSCLC with an EML4-ALK fusion and was treated with alectinib but who developed grade 4 hyperbilirubinemia after five months on therapy. Alectinib was discontinued, and an artificial liver support system (ALSS) was used with an impressive decline in bilirubin levels. After two months drug-free, the patient experienced disease progression. Ensartinib was initiated as second-line treatment with a best response of stable disease after three months of therapy with no evidence of hyperbilirubinemia. CONCLUSION: This is the first report of ensartinib treatment after alectinib-induced hyperbilirubinemia which was successfully relieved by ALSS treatment and targeted drug cessation.

Landscape and Clonal Dominance of Co-occurring Genomic Alterations in Non-Small-Cell Lung Cancer Harboring MET Exon 14 Skipping.

MET exon 14 skipping alterations (METex14) comprise a diverse set of actionable oncogene drivers in non-small-cell lung cancer (NSCLC). Recent studies have established the efficacy of tyrosine kinase inhibitors for this patient population. The landscape of co-occurring genetic alterations in METex14 NSCLC and their potential impact to therapeutic sensitivities has not yet been fully described. MATERIALS AND METHODS: METex14 NSCLC cases were collected from three cohorts: the VISION trial, and data sets from Guardant360 and GenePlus. Clinicopathologic characteristics and METex14 mutation sites were analyzed and compared across data sets. Co-occurring genetic alterations and the clonality relationships to METex14 were evaluated. RESULTS: Of 40,824 NSCLCs, 692 METex14 cases (1.7%) were identified, including 332 in Guardant360, 188 in VISION, and 172 in GenePlus. The demographics and mutation type and/or sites were similar in the Asian versus Western cohorts. MET amplification, which were found to be associated with sensitivity to MET kinase inhibitors, co-occurs in 7.6%-13.8% of cases, whereas kinase domain secondary mutation of MET co-occurs in 5%-6%. When co-occurring with METex14, EGFR mutations were often identified as the dominant clone (78%, 7 of 9), whereas when co-occurring, METex14 (39%, 7 of 18) and KRAS (44%, 8 of 18) had similar rates of clonal dominance. PIK3CA and PTEN mutations were almost always subclones (89%, 16 of 18) to METex14. Moreover, RET-CCDC6 fusion and EGFR mutation were detected following crizotinib treatment in two patients, suggesting novel mechanisms of resistance. CONCLUSION: METex14 mutations frequently co-occur with other potential driver oncogenes with differing patterns of clonal dominance observed among the drivers. This cellular context can provide insights into whether METex14 is acting as a primary oncogenic driver or resistance mechanism and help guide treatment choices.

Alpha-galactosidase A-Tat fusion enhances storage reduction in hearts and kidneys of Fabry mice.

The protein transduction domain from human immunodeficiency virus (HIV) Tat allows proteins to penetrate the cell membrane. Enhanced cellular uptake of therapeutic proteins could benefit a number of disorders. This is especially true for lysosomal storage disorders (LSDs) where enzyme replacement therapy (ERT) and gene therapy have been developed. We developed a novel recombinant lentiviral vector (LV) that engineers expression of alpha-galactosidase A (alpha-gal A)-Tat fusion protein for correction of Fabry disease, the second-most prevalent LSD with manifestations in the brain, kidney and heart. In vitro experiments confirmed mannose-6-phosphate independent uptake of the fusion factor. Next, concentrated therapeutic LV was injected into neonatal Fabry mice. Analysis of tissues at 26 wks demonstrated similar alpha-gal A enzyme activities but enhanced globotriaosylceramide (Gb3) reduction in hearts and kidneys compared with the alpha-gal A LV control. This strategy might advance not only gene therapy for Fabry disease and other LSDs, but also ERT, especially for cardiac Fabry disease.

Use of purified Clostridium difficile spores to facilitate evaluation of health care disinfection regimens.

Clostridium difficile is a major cause of antibiotic-associated diarrheal disease in many parts of the world. In recent years, distinct genetic variants of C. difficile that cause severe disease and persist within health care settings have emerged. Highly resistant and infectious C. difficile spores are proposed to be the main vectors of environmental persistence and host transmission, so methods to accurately monitor spores and their inactivation are urgently needed. Here we describe simple quantitative methods, based on purified C. difficile spores and a murine transmission model, for evaluating health care disinfection regimens. We demonstrate that disinfectants that contain strong oxidizing active ingredients, such as hydrogen peroxide, are very effective in inactivating pure spores and blocking spore-mediated transmission. Complete inactivation of 106 pure C. difficile spores on indicator strips, a six-log reduction, and a standard measure of stringent disinfection regimens require at least 5 min of exposure to hydrogen peroxide vapor (HPV; 400 ppm). In contrast, a 1-min treatment with HPV was required to disinfect an environment that was heavily contaminated with C. difficile spores (17 to 29 spores/cm²) and block host transmission. Thus, pure C. difficile spores facilitate practical methods for evaluating the efficacy of C. difficile spore disinfection regimens and bringing scientific acumen to C. difficile infection control.

Routine Plasma-Based Genotyping to Comprehensively Detect Germline, Somatic, and Reversion BRCA Mutations among Patients with Advanced Solid Tumors.

PURPOSE: PARP inhibitors (PARPi) are efficacious in multiple cancers harboring germline (and possibly somatic) BRCA1/2 mutations. Acquired reversions can restore BRCA1/2 function, causing resistance to PARPi and/or platinum-based chemotherapy. The optimal method of identifying patients with germline, somatic, and/or reversion mutations in BRCA1/2 has not been established. Next-generation sequencing (NGS) of cell-free DNA (cfDNA) provides a platform to identify these three types of BRCA1/2 mutations. EXPERIMENTAL DESIGN: Patients with advanced breast, ovarian, prostate, or pancreatic cancer were tested using a clinically validated 73-gene cfDNA assay that evaluates single-nucleotide variants and insertion-deletion mutations (indels) in BRCA1/2, and distinguishes somatic/reversion from germline mutations with high accuracy. RESULTS: Among 828 patients, one or more deleterious BRCA1/2 mutations were detected in 60 (7.2%) patients, including germline (n = 42) and somatic (n = 18) mutations. Common coexisting mutations included TP53 (61.6%), MYC (30%), PIK3CA (26.6%), BRAF (15%), and ESR1 (11.5%). Polyclonal reversion mutations (median, 5) were detected in 9 of 42 (21.4%) germline BRCA1/2-mutant patients, the majority (77.7%) of whom had prior PARPi exposure (median duration, 10 months). Serial cfDNA demonstrated emergence of reversion BRCA mutations under therapeutic pressure from initial PARPi exposure, which contributed to subsequent resistance to PARPi and platinum therapy. CONCLUSIONS: cfDNA NGS identified high rates of therapeutically relevant mutations without foreknowledge of germline or tissue-based testing results, including deleterious somatic BRCA1/2 mutations missed by germline testing and reversion mutations that can have important treatment implications. Further research is needed to confirm clinical utility of these findings to guide precision medicine approaches for patients with advanced malignancies.

Analysis of Cell-Free DNA from 32,989 Advanced Cancers Reveals Novel Co-occurring Activating RET Alterations and Oncogenic Signaling Pathway Aberrations.

PURPOSE: RET is an emerging oncogenic target showing promise in phase I/II clinical trials. An understudied aspect of RET-driven cancers is the extent to which co-occurring genomic alterations exist and how they may impact prognosis or therapeutic response. EXPERIMENTAL DESIGN: Somatic activating RET alterations were identified among 32,989 consecutive patients with metastatic solid tumors tested with a clinical cell-free circulating tumor DNA (cfDNA) assay. This comprehensive next-generation sequencing (NGS) assay evaluates single-nucleotide variants, and select indels, fusions, and copy number gains in 68-73 clinically relevant cancer genes. RESULTS: A total of 176 somatic activating RET alterations were detected in 170 patients (143 fusions and 33 missense mutations). Patients had non-small cell lung (NSCLC, n = 125), colorectal (n = 15), breast (n = 8), thyroid (n = 8), or other (n = 14) cancers. Alterations in other oncogenic signaling pathway genes were frequently identified in RET-positive samples and varied by specific RET fusion gene partner. RET fusions involving partners other than KIF5B were enriched for alterations in MAPK pathway genes and other bona fide oncogenic drivers of NSCLC, particularly EGFR. Molecular and clinical data revealed that these variants emerged later in the genomic evolution of the tumor as mechanisms of resistance to EGFR tyrosine kinase inhibitors. CONCLUSIONS: In the largest cancer cohort with somatic activating RET alterations, we describe novel co-occurrences of oncogenic signaling pathway aberrations. We find that KIF5B-RET fusions are highly specific for NSCLC. In our study, only non-KIF5B-RET fusions contributed to anti-EGFR therapy resistance. Knowledge of specific RET fusion gene partner may have clinical significance.

Geeks, Fandoms, and Social Engagement.

The Internet changed the way the global community interacts and communicates. This cultural shift allows like-minded individuals to connect and share ideas. It creates spaces for stigmatized communities to gather in a virtual presence. Youth now have greater options to explore identity, and to reach peers with niche interests. From a clinical perspective, it is helpful to understand the nature of communities our patients join on the Internet. This article focuses on the broad umbrella of "geek" culture, exploring a variety of interests such as cosplay, fanfiction, and gaming.

Reconciling icetexane biosynthetic connections with their chemical synthesis: total synthesis of (+/-)-5,6-dihydro-6alpha-hydroxysalviasperanol, (+/-)-brussonol, and (+/-)-abrotanone.

A unified strategy for the chemical synthesis of the icetexane diterpenoids brussonol and 5,6-dihydro-6alpha-hydroxysalviasperanol has led to a structural revision of the recently isolated natural products abrotandiol and abrotanone.

A variant by any name: quantifying annotation discordance across tools and clinical databases.

BACKGROUND: Clinical genomic testing is dependent on the robust identification and reporting of variant-level information in relation to disease. With the shift to high-throughput sequencing, a major challenge for clinical diagnostics is the cross-identification of variants called on their genomic position to resources that rely on transcript- or protein-based descriptions. METHODS: We evaluated the accuracy of three tools (SnpEff, Variant Effect Predictor, and Variation Reporter) that generate transcript and protein-based variant nomenclature from genomic coordinates according to guidelines by the Human Genome Variation Society (HGVS). Our evaluation was based on transcript-controlled comparisons to a manually curated set of 126 test variants of various types drawn from data sources, each with HGVS-compliant transcript and protein descriptors. We further evaluated the concordance between annotations generated by Snpeff and Variant Effect Predictor and those in major germline and cancer databases: ClinVar and COSMIC, respectively. RESULTS: We find that there is substantial discordance between the annotation tools and databases in the description of insertions and/or deletions. Using our ground truth set of variants, constructed specifically to identify challenging events, accuracy was between 80 and 90% for coding and 50 and 70% for protein changes for 114 to 126 variants. Exact concordance for SNV syntax was over 99.5% between ClinVar and Variant Effect Predictor and SnpEff, but less than 90% for non-SNV variants. For COSMIC, exact concordance for coding and protein SNVs was between 65 and 88% and less than 15% for insertions. Across the tools and datasets, there was a wide range of different but equivalent expressions describing protein variants. CONCLUSIONS: Our results reveal significant inconsistency in variant representation across tools and databases. While some of these syntax differences may be clear to a clinician, they can confound variant matching, an important step in variant classification. These results highlight the urgent need for the adoption and adherence to uniform standards in variant annotation, with consistent reporting on the genomic reference, to enable accurate and efficient data-driven clinical care.

Zebrafish models of cancer: progress and future challenges.

The need for scalable strategies to probe the biological consequences of candidate cancer genes has never been more pressing. The zebrafish, with its capacity for high-throughput transgenesis, in vivo imaging and chemical/genetic screening, has ideal features for undertaking this task. Unique biological insights from zebrafish have already led to the identification of novel oncogenic drivers and small molecules being used to treat the human cancer. This review summarizes the recent main findings and describes pertinent areas where the zebrafish can greatly contribute to our understanding of cancer biology and treatment.

The genetic heterogeneity and mutational burden of engineered melanomas in zebrafish models.

BACKGROUND: Melanoma is the most deadly form of skin cancer. Expression of oncogenic BRAF or NRAS, which are frequently mutated in human melanomas, promote the formation of nevi but are not sufficient for tumorigenesis. Even with germline mutated p53, these engineered melanomas present with variable onset and pathology, implicating additional somatic mutations in a multi-hit tumorigenic process. RESULTS: To decipher the genetics of these melanomas, we sequence the protein coding exons of 53 primary melanomas generated from several BRAF(V600E) or NRAS(Q61K) driven transgenic zebrafish lines. We find that engineered zebrafish melanomas show an overall low mutation burden, which has a strong, inverse association with the number of initiating germline drivers. Although tumors reveal distinct mutation spectrums, they show mostly C > T transitions without UV light exposure, and enrichment of mutations in melanogenesis, p53 and MAPK signaling. Importantly, a recurrent amplification occurring with pre-configured drivers BRAF(V600E) and p53-/- suggests a novel path of BRAF cooperativity through the protein kinase A pathway. CONCLUSION: This is the first analysis of a melanoma mutational landscape in the absence of UV light, where tumors manifest with remarkably low mutation burden and high heterogeneity. Genotype specific amplification of protein kinase A in cooperation with BRAF and p53 mutation suggests the involvement of melanogenesis in these tumors. This work is important for defining the spectrum of events in BRAF or NRAS driven melanoma in the absence of UV light, and for informed exploitation of models such as transgenic zebrafish to better understand mechanisms leading to human melanoma formation.

Psychopathology, families, and culture: autism.

Autism spectrum disorders (ASDs) are now considered to be the most common of the developmental disorders, although the effect of cultural influences on the diagnosis and treatment of ASDs has received limited attention. The existing literature on this topic suggests that both macro-level and microlevel cultural factors can affect the characterization, diagnosis, and treatment of ASDs. As a result, it is important for clinicians to consider cultural factors throughout the diagnostic, treatment planning, and intervention implementation processes. In this article, cultural influences on the prevalence of autism and the diagnostic and treatment processes are reviewed and synthesized through a consideration of the developmental context and through clinical practice suggestions.