Resistance mechanisms to targeted therapy in BRAF-mutant melanoma - A mini review.

BACKGROUND: The introduction of targeted therapies for the treatment of BRAF-mutant melanomas have improved survival rates in a significant proportion of patients. Nonetheless, the emergence of resistance to treatment remains inevitable in most patients. SCOPE OF REVIEW: Here, we review known and emerging molecular mechanisms that underlay the development of resistance to MAPK inhibition in melanoma cells and the potential strategies to overcome these mechanisms. MAJOR CONCLUSIONS: Multiple genetic and non-genetic mechanisms contribute to treatment failure, commonly leading to the reactivation of the MAPK pathway. A variety of resistance mechanisms are enabled by the underlying heterogeneity and plasticity of melanoma cells. Moreover, it has become apparent that resistance to targeted therapy is underpinned by early functional adaptations involving the rewiring of cell states and metabolic pathways. GENERAL SIGNIFICANCE: The evidence presented suggest that the use of a combinatorial treatment approach would delay the emergence of resistance and improve patient outcomes.

Comparison of Circulating Tumour DNA and Extracellular Vesicle DNA by Low-Pass Whole-Genome Sequencing Reveals Molecular Drivers of Disease in a Breast Cancer Patient.

There is increasing recognition of circulating tumour DNA (ctDNA) as a non-invasive alternative to tumour tissue for the molecular characterisation and monitoring of disease. Recent evidence suggests that cancer-associated changes can also be detected in the DNA contained within extracellular vesicles (EVs). As yet, there has been limited investigation into the relationship between EV DNA and ctDNA, and no studies have examined the EV DNA of breast cancer patients. The aim of this study was to use low-pass whole-genome sequencing to identify copy number variants (CNVs) in serial samples of both ctDNA and EV DNA from a patient with breast cancer. Of the 52 CNVs identified in tumour DNA, 36 (69%) were detected in at least one ctDNA sample and 13 (25%) in at least one EV DNA sample. The number of detectable variants in ctDNA and EV DNA increased over the natural history of the patient's disease, which was associated with progression to cerebral metastases. This case study demonstrates that, while CNVs are detectable in patient EV DNA, ctDNA has greater sensitivity than EV DNA for serial monitoring of breast cancer.

Detection of BRAF splicing variants in plasma-derived cell-free nucleic acids and extracellular vesicles of melanoma patients failing targeted therapy therapies.

The analysis of plasma circulating tumour nucleic acids provides a non-invasive approach to assess disease burden and the genetic evolution of tumours in response to therapy. BRAF splicing variants are known to confer melanoma resistance to BRAF inhibitors. We developed a test to screen cell-free RNA (cfRNA) for the presence of BRAF splicing variants. Custom droplet digital PCR assays were designed for the detection of BRAF splicing variants p61, p55, p48 and p41 and then validated using RNA from cell lines carrying these variants. Evaluation of plasma from patients with reported objective response to BRAF/MEK inhibition followed by disease progression was revealed by increased circulating tumour DNA (ctDNA) in 24 of 38 cases at the time of relapse. Circulating BRAF splicing variants were detected in cfRNA from 3 of these 38 patients; two patients carried the BRAF p61 variant and one the p55 variant. In all three cases the presence of the splicing variant was apparent only at the time of progressive disease. BRAF p61 was also detectable in plasma of one of four patients with confirmed BRAF splicing variants in their progressing tumours. Isolation and analysis of RNA from extracellular vesicles (EV) from resistant cell lines and patient plasma demonstrated that BRAF splicing variants are associated with EVs. These findings indicate that in addition to plasma ctDNA, RNA carried by EVs can provide important tumour specific information.

Pain and psychiatric comorbidities among two groups of Iraq and Afghanistan era Veterans.

This study aimed to (1) identify the prevalence and severity of pain and psychiatric comorbidities among personnel who had been deployed during Operation Iraqi Freedom (OIF), Operation Enduring Freedom (OEF), and Operation New Dawn (OND) and (2) assess whether the Department of Veterans Affairs (VA) Polytrauma System of Care and an OIF/OEF/OND registry reflect real differences among patients. Participants (N = 359) were recruited from two VA hospitals. They completed a clinical interview, structured diagnostic interview, and self-report measures. Results indicated pain was the most common complaint, with 87 percent experiencing pain during the prior week and 56 percent reporting moderate or severe pain. Eighty percent of participants met criteria for at least one of seven assessed comorbid problems (moderate or severe pain, postconcussional disorder, posttraumatic stress disorder [PTSD], anxiety disorder, mood disorder, substance use disorder, psychosis), and 59 percent met criteria for two or more problems. PTSD and postconcussional disorder rarely occurred in the absence of pain or other comorbidities (0.3% and 0%, respectively). The Polytrauma group had more comorbid psychiatric conditions (χ(2) = 48.67, p < 0.05) and reported greater severity of symptoms (p < 0.05) than the Registry group. This study confirmed the high prevalence of pain and concurrent mental health problems among personnel returning from military deployment.

Comparative Genomics of Two Closely Related Wolbachia with Different Reproductive Effects on Hosts.

Wolbachia pipientis are obligate intracellular bacteria commonly found in many arthropods. They can induce various reproductive alterations in hosts, including cytoplasmic incompatibility, male-killing, feminization, and parthenogenetic development, and can provide host protection against some viruses and other pathogens. Wolbachia differ from many other primary endosymbionts in arthropods because they undergo frequent horizontal transmission between hosts and are well known for an abundance of mobile elements and relatively high recombination rates. Here, we compare the genomes of two closely related Wolbachia (with 0.57% genome-wide synonymous divergence) that differ in their reproductive effects on hosts. wVitA induces a sperm-egg incompatibility (also known as cytoplasmic incompatibility) in the parasitoid insect Nasonia vitripennis, whereas wUni causes parthenogenetic development in a different parasitoid, Muscidifurax uniraptor Although these bacteria are closely related, the genomic comparison reveals rampant rearrangements, protein truncations (particularly in proteins predicted to be secreted), and elevated substitution rates. These changes occur predominantly in the wUni lineage, and may be due in part to adaptations by wUni to a new host environment, or its phenotypic shift to parthenogenesis induction. However, we conclude that the approximately 8-fold elevated synonymous substitution rate in wUni is due to a either an elevated mutation rate or a greater number of generations per year in wUni, which occurs in semitropical host species. We identify a set of genes whose loss or pseudogenization in the wUni lineage implicates them in the phenotypic shift from cytoplasmic incompatibility to parthenogenesis induction. Finally, comparison of these closely related strains allows us to determine the fine-scale mutation patterns in Wolbachia Although Wolbachia are AT rich, mutation probabilities estimated from 4-fold degenerate sites are not AT biased, and predict an equilibrium AT content much less biased than observed (57-50% AT predicted vs. 76% current content at degenerate sites genome wide). The contrast suggests selection for increased AT content within Wolbachia genomes.

Validity of Center for Epidemiologic Studies Depression (CES-D) scale in a sample of Iraq and Afghanistan Veterans.

OBJECTIVES: Optimal depression screening necessitates measurement tools that are valid across varied populations and in the presence of comorbidities. METHODS: This study assessed the test properties of two versions of the Center for Epidemiologic Studies Depression scale against psychiatric diagnoses established by the Mini International Neuropsychiatric Interview among a clinical sample of US Veterans deployed during Operations Enduring Freedom, Iraqi Freedom, and New Dawn. Participants (N = 359) recruited from two Department of Veterans Affairs hospitals completed a clinical interview, structured diagnostic interview, and self-reported measures. RESULTS: Based on diagnostic interview and the Diagnostic and Statistical Manual of Mental Disorders 4th Edition criteria, 29.5% of the sample met diagnostic criteria for major depressive disorder and 26.5% met diagnostic criteria for post-traumatic stress disorder. Both Center for Epidemiologic Studies Depression-20 and Center for Epidemiologic Studies Depression-10 scales performed well and almost identically against the Mini International Neuropsychiatric Interview-major depressive disorder in identifying Veterans with major depressive disorder (Center for Epidemiologic Studies Depression-20 area under the Receiver Operating Characteristic curve 91%; Center for Epidemiologic Studies Depression-10 area under the ROC curve 90%). Overall, higher cut points for the Center for Epidemiologic Studies Depression scales performed better in correctly identifying true positives and true negatives for major depressive disorder (Center for Epidemiologic Studies Depression-20 cut point 18+ sensitivity 92% specificity 72%; Center for Epidemiologic Studies Depression-10 cut point 10+ sensitivity 92% specificity 69%). CONCLUSIONS: The specificity of the Center for Epidemiologic Studies Depression scales was poor among Veterans with co-occurring post-traumatic stress disorder (13% and 16%). Veterans with post-traumatic stress disorder who have a positive depression screen should have a more thorough assessment of mental health symptoms and comorbidities, rather than immediate diagnosis of and treatment for depression.

Post-deployment Multi-symptom Disorder rehabilitation: An integrated approach to rehabilitation.

BACKGROUND: Veterans and active duty service members returning from Operation New Dawn and those having returned from Operations Iraqi and Enduring Freedom frequently report the presence of overlapping, co-morbid symptom clusters consisting of chronic pain, mild cognitive complaints, and posttraumatic stress symptoms/disorder or mood disturbance. This presentation has been called Post-deployment Multi-symptom Disorder (PMD) and its implications not only impact various functional domains, but have also influenced a system/continuum of care to rise to meet the challenges of treating PMD. This continuum is based on innovation informed by evidence-based therapies, systemic limitations, and a focus on functional improvement rather than diagnostic classification. OBJECTIVE: The purpose of this paper is to describe the symptomatic, functional and systemic challenges inherent to PMD conceptualization and treatment. METHOD: The constituent clusters of PMD are defined and exemplified, its functional impact is illustrated, and a continuum of care at a large southeastern Veterans Affairs (VA) hospital offering an interdisciplinary approach to integrated rehabilitation is described. Three case examples are provided that that underscore the importance of vocation for improved behavioral health and quality of life. CONCLUSION: The case examples demonstrate how vocational rehabilitation services are an integral component of PMD treatment.}

Smoking behavior and motivation to quit among chronic pain patients initiating multidisciplinary pain treatment: a prospective study.

OBJECTIVE: The primary aim of this study was to assess smoking characteristics and cessation motivation prior to and after initiation of multidisciplinary chronic pain treatment. A secondary aim was to identify predictors of cessation motivation among smokers initiating treatment for chronic pain. DESIGN: We used a prospective, nonrandomized, repeated measures design. SETTING: The study was conducted in a multidisciplinary specialty pain treatment program at a veterans hospital. PATIENTS: Smokers (N = 90) referred to a multidisciplinary pain program for the treatment of chronic pain. MEASURES: Patients completed questionnaires assessing pain-related and smoking-related factors prior to (baseline) and 8 weeks post (follow-up) specialty pain treatment initiation. Primary outcome measures were the Contemplation Ladder and the Stages of Change (SOC) algorithm. RESULTS: At baseline, patients reported moderate levels of cessation motivation, and 69% were in the contemplation stage or higher on the SOC. Motivation to quit smoking was higher at follow-up compared with baseline on both continuous, t(89) = 2.11, P < 0.05, and stage-based, z = 3.69, P < 0.01, measures. At follow-up, participants reported greater interest in receiving cessation interventions, and 7.8% of patients had quit smoking. Pain-related predictors of motivation (e.g., pain intensity) were subsumed by more general predictors (e.g., nicotine dependence). CONCLUSIONS: Patients in this sample were more motivated to quit smoking a few weeks after, as compared with before initiating specialty pain treatment. Future research into pain-specific predictors of cessation motivation is warranted to inform the development of interventions that address pain patients' unique needs.

Access to the US Department of Veterans Affairs health system: self-reported barriers to care among returnees of Operations Enduring Freedom and Iraqi Freedom.

BACKGROUND: The U.S. Department of Veterans Affairs (VA) implemented the Polytrauma System of Care to meet the health care needs of military and veterans with multiple injuries returning from combat operations in Afghanistan and Iraq. Studies are needed to systematically assess barriers to use of comprehensive and exclusive VA healthcare services from the perspective of veterans with polytrauma and with other complex health outcomes following their service in Afghanistan and Iraq. These perspectives can inform policy with regard to the optimal delivery of care to returning veterans. METHODS: We studied combat veterans (n = 359) from two polytrauma rehabilitation centers using structured clinical interviews and qualitative open-ended questions, augmented with data collected from electronic health records. Our outcomes included several measures of exclusive utilization of VA care with our primary exposure as reported access barriers to care. RESULTS: Nearly two thirds of the veterans reported one or more barriers to their exclusive use of VA healthcare services. These barriers predicted differences in exclusive use of VA healthcare services. Experiencing any barriers doubled the returnees' odds of not using VA exclusively, the geographic distance to VA barrier resulted in a 7 fold increase in the returnees odds of not using VA, and reporting a wait time barrier doubled the returnee's odds of not using VA. There were no striking differences in access barriers for veterans with polytrauma compared to other returning veterans, suggesting the barriers may be uniform barriers that predict differences in using the VA exclusively for health care. CONCLUSIONS: This study provides an initial description of utilization of VA polytrauma rehabilitation and other medical care for veteran returnees from all military services who were involved in combat operations in Afghanistan or Iraq. Our findings indicate that these veterans reported important stigmatization and barriers to receiving services exclusively from the VA, including mutable health delivery system factors.

Conceptualizing and Treating Comorbid Chronic Pain and PTSD.

The purpose of this paper is to review the rationale for concurrent, evidence-based treatment of chronic pain and posttraumatic stress disorder (PTSD). To meet this end, we review pertinent definitions and extant theories related to the two conditions and their correlations with each other. We then synthesize theoretical components into a proposal of a comprehensive conceptual framework for understanding the relationship and clinical complexity of overlapping chronic pain and PTSD. We conclude with an example of an integrated treatment model designed specifically to address a fundamental factor associated with pain and PTSD: avoidance.

Chronic pain and traumatic brain injury in OEF/OIF service members and Veterans.

The co-occurrence of chronic pain and traumatic brain injury (TBI) are 2 of the most common concerns among the Operations Enduring Freedom and Iraqi Freedom population and present unique challenges for evaluation and treatment. Previous research suggests that almost half the cohort report clinically significant pain, while up to 1 in 4 experiences some form of TBI. There is limited information regarding how TBI affects the presence and course of pain, and how pain impacts TBI and its symptoms. The present paper provides an overview of the range and degree of TBIs as well as a brief summary of current knowledge regarding the interaction between chronic pain and TBI, particularly in light of the numerous variables impacting it. Information on ways to best assess for and treat pain in the TBI population, including in those with multiple system injuries or associated affective symptoms, is provided. In addition, several innovative approaches for addressing the needs of this complex cohort of patients are described, which may stimulate further research and clinical innovation for this important subgroup.

Opioid cessation and multidimensional outcomes after interdisciplinary chronic pain treatment.

OBJECTIVES: Although the efficacy of interdisciplinary treatment for chronic noncancer pain has been well-established in the literature, there is limited research examining interdisciplinary programs that require opioid cessation. As the long-term use of opioid analgesics remains controversial, further investigation is warranted. The aim of this study was to evaluate the associations between opioid cessation and subsequent multidomain treatment outcomes among veterans admitted to a pain rehabilitation program at a large Veterans Affairs tertiary care hospital in the southeastern United States. METHODS: A retrospective design examined the medical records of 705 consecutive admissions comparing those using opioids at admission with those who were not. Participants taking opioids agreed to taper off of these medications using a "pain cocktail" approach; otherwise patients received identical treatment. Outcome measures were administered at program admission and discharge. RESULTS: Repeated measures analyses were used to compare responses across time. Those who completed the program (n=600) demonstrated improvement in all outcome measures from admission to discharge, and the opioid group improved as much or more than the nonopioid group on all measures despite opioid cessation during treatment. DISCUSSION: Results indicated that both groups experienced significant improvement on outcome measures, and that opioid analgesic use at admission had no discernible impact on treatment outcome in this large sample of veterans with moderate to severe chronic pain syndrome. The clinical implications of these findings for long-term chronic pain treatment, in light of the risks associated with opioid analgesics, are discussed.

Complete bacteriophage transfer in a bacterial endosymbiont (Wolbachia) determined by targeted genome capture.

Bacteriophage flux can cause the majority of genetic diversity in free-living bacteria. This tenet of bacterial genome evolution generally does not extend to obligate intracellular bacteria owing to their reduced contact with other microbes and a predominance of gene deletion over gene transfer. However, recent studies suggest intracellular coinfections in the same host can facilitate exchange of mobile elements between obligate intracellular bacteria-a means by which these bacteria can partially mitigate the reductive forces of the intracellular lifestyle. To test whether bacteriophages transfer as single genes or larger regions between coinfections, we sequenced the genome of the obligate intracellular Wolbachia strain wVitB from the parasitic wasp Nasonia vitripennis and compared it against the prophage sequences of the divergent wVitA coinfection. We applied, for the first time, a targeted sequence capture array to specifically trap the symbiont's DNA from a heterogeneous mixture of eukaryotic, bacterial, and viral DNA. The tiled array successfully captured the genome with 98.3% efficiency. Examination of the genome sequence revealed the largest transfer of bacteriophage and flanking genes (52.2 kb) to date between two obligate intracellular coinfections. The mobile element transfer occurred in the recent evolutionary past based on the 99.9% average nucleotide identity of the phage sequences between the two strains. In addition to discovering an evolutionary recent and large-scale horizontal phage transfer between coinfecting obligate intracellular bacteria, we demonstrate that "targeted genome capture" can enrich target DNA to alleviate the problem of isolating symbiotic microbes that are difficult to culture or purify from the conglomerate of organisms inside eukaryotes.

Decoupling of host-symbiont-phage coadaptations following transfer between insect species.

Transferring endosymbiotic bacteria between different host species can perturb the coordinated regulation of the host and bacterial genomes. Here we use the most common maternally transmitted bacteria, Wolbachia pipientis, to test the consequences of host genetic background on infection densities and the processes underlying those changes in the parasitoid wasp genus Nasonia. Introgressing the genome of Nasonia giraulti into the infected cytoplasm of N. vitripennis causes a two-order-of-magnitude increase in bacterial loads in adults and a proliferation of the infection to somatic tissues. The host effect on W. pipientis distribution and densities is associated with a twofold decrease in densities of the temperate phage WO-B. Returning the bacteria from the new host species back to the resident host species restores the bacteria and phage to their native densities. To our knowledge, this is the first study to report a host-microbe genetic interaction that affects the densities of both W. pipientis and bacteriophage WO-B. The consequences of the increased bacterial density include a reduction in fecundity, an increase in levels of cytoplasmic incompatibility (CI), and unexpectedly, male-to-female transfer of the bacteria to uninfected females and an increased acceptance of densely infected females to interspecific mates. While paternal inheritance of the W. pipientis was not observed, the high incidence of male-to-female transfer in the introgressed background raises the possibility that paternal transmission could be more likely in hybrids where paternal leakage of other cytoplasmic elements is also known to occur. Taken together, these results establish a major change in W. pipientis densities and tissue tropism between closely related species and support a model in which phage WO, Wolbachia, and arthropods form a tripartite symbiotic association in which all three are integral to understanding the biology of this widespread endosymbiosis.

The mushroom habitat as an ecological arena for global exchange of Wolbachia.

Wolbachia infect a variety of arthropod and nematode hosts, but in arthropods, host phylogenetic relationships are usually poor predictors of strain similarity. This suggests that new infections are often established by horizontal transmission. To gain insight into the factors affecting the probability of horizontal transmission among host species, we ask how host phylogeny, geographical distribution and ecology affect patterns of Wolbachia strain similarity. We used multilocus sequence typing (MLST) to characterize Wolbachia strain similarity among dipteran hosts associated with fleshy mushrooms. Wolbachia Supergroup A was more common than Supergroup B in Diptera, and also more common in mycophagous than non-mycophagous Diptera. Within Supergroup A, host family within Diptera had no effect on strain similarity, and there was no tendency for Wolbachia strains from sympatric host species to be more similar to one another than to strains from hosts in different biogeographical realms. Supergroup A strains differed between mycophagous and non-mycophagous Diptera more than expected by chance, suggesting that ecological associations can facilitate horizontal transmission of Wolbachia within mycophagous fly communities. For Supergroup B, there were no significant associations between strain similarity and host phylogeny, biogeography, or ecology. We identified only two cases in which closely related hosts carried closely related Wolbachia strains, evidence that Wolbachia-host co-speciation or early introgression can occur but may not be a major contributor to overall strain diversity. Our results suggest that horizontal transmission of Wolbachia can be influenced by host ecology, thus leading to partial restriction of Wolbachia strains or strain groups to particular guilds of insects.

Designing an automated clinical decision support system to match clinical practice guidelines for opioid therapy for chronic pain.

BACKGROUND: Opioid prescribing for chronic pain is common and controversial, but recommended clinical practices are followed inconsistently in many clinical settings. Strategies for increasing adherence to clinical practice guideline recommendations are needed to increase effectiveness and reduce negative consequences of opioid prescribing in chronic pain patients. METHODS: Here we describe the process and outcomes of a project to operationalize the 2003 VA/DOD Clinical Practice Guideline for Opioid Therapy for Chronic Non-Cancer Pain into a computerized decision support system (DSS) to encourage good opioid prescribing practices during primary care visits. We based the DSS on the existing ATHENA-DSS. We used an iterative process of design, testing, and revision of the DSS by a diverse team including guideline authors, medical informatics experts, clinical content experts, and end-users to convert the written clinical practice guideline into a computable algorithm to generate patient-specific recommendations for care based upon existing information in the electronic medical record (EMR), and a set of clinical tools. RESULTS: The iterative revision process identified numerous and varied problems with the initially designed system despite diverse expert participation in the design process. The process of operationalizing the guideline identified areas in which the guideline was vague, left decisions to clinical judgment, or required clarification of detail to insure safe clinical implementation. The revisions led to workable solutions to problems, defined the limits of the DSS and its utility in clinical practice, improved integration into clinical workflow, and improved the clarity and accuracy of system recommendations and tools. CONCLUSIONS: Use of this iterative process led to development of a multifunctional DSS that met the approval of the clinical practice guideline authors, content experts, and clinicians involved in testing. The process and experiences described provide a model for development of other DSSs that translate written guidelines into actionable, real-time clinical recommendations.

Non-coding changes cause sex-specific wing size differences between closely related species of Nasonia.

The genetic basis of morphological differences among species is still poorly understood. We investigated the genetic basis of sex-specific differences in wing size between two closely related species of Nasonia by positional cloning a major male-specific locus, wing-size1 (ws1). Male wing size increases by 45% through cell size and cell number changes when the ws1 allele from N. giraulti is backcrossed into a N. vitripennis genetic background. A positional cloning approach was used to fine-scale map the ws1 locus to a 13.5 kilobase region. This region falls between prospero (a transcription factor involved in neurogenesis) and the master sex-determining gene doublesex. It contains the 5'-UTR and cis-regulatory domain of doublesex, and no coding sequence. Wing size reduction correlates with an increase in doublesex expression level that is specific to developing male wings. Our results indicate that non-coding changes are responsible for recent divergence in sex-specific morphology between two closely related species. We have not yet resolved whether wing size evolution at the ws1 locus is caused by regulatory alterations of dsx or prospero, or by another mechanism. This study demonstrates the feasibility of efficient positional cloning of quantitative trait loci (QTL) involved in a broad array of phenotypic differences among Nasonia species.

Functional and evolutionary insights from the genomes of three parasitoid Nasonia species.

We report here genome sequences and comparative analyses of three closely related parasitoid wasps: Nasonia vitripennis, N. giraulti, and N. longicornis. Parasitoids are important regulators of arthropod populations, including major agricultural pests and disease vectors, and Nasonia is an emerging genetic model, particularly for evolutionary and developmental genetics. Key findings include the identification of a functional DNA methylation tool kit; hymenopteran-specific genes including diverse venoms; lateral gene transfers among Pox viruses, Wolbachia, and Nasonia; and the rapid evolution of genes involved in nuclear-mitochondrial interactions that are implicated in speciation. Newly developed genome resources advance Nasonia for genetic research, accelerate mapping and cloning of quantitative trait loci, and will ultimately provide tools and knowledge for further increasing the utility of parasitoids as pest insect-control agents.