Exhaled Breath Condensate (EBC) analysis of circulating tumour DNA (ctDNA) using a lung cancer specific UltraSEEK oncogene panel.

INTRODUCTION: Small diagnostic tissue samples can be inadequate in testing an expanding list of validated oncogenic driver alterations and fail to reflect intratumour heterogeneity (ITGH) in lung cancer. Liquid biopsies are non-invasive and may better reflect ITGH. Most liquid biopsies are performed in the context of circulating tumour DNA (ctDNA) in plasma but Exhaled Breath Condensate (EBC) shows promise as a lung-specific liquid biopsy. METHODS: In this prospective, proof-of-concept study we carried out targeted Next Generation Sequencing (NGS) on diagnostic tissue samples from 125 patients with lung cancer and compared results to plasma and EBC for 5 oncogenic driver mutations (EGFR, KRAS, PIK3CA, ERBB2, BRAF) using an ultrasensitive PCR technique (UltraSEEK™ Lung Panel on the MassARRAY® System, Agena Bioscience, San Diego, CA, USA). RESULTS: There was a significantly higher failure rate due to unamplifiable DNA in tissue NGS (57/125, 45.6%) compared to plasma (27/125, 21.6%, p < 0.001 and EBC (26/125,20.8%, p ≤ 0.001. Consequently, both plasma and EBC identified higher number of mutations compared to tissue NGS. Specifically, there were significantly higher numbers of mutations detected in EGFR, KRAS and PIK3CA in plasma (p = 9.82 × 10-3, p = 3.14 × 10-5, p = 1.95 × 10-3) and EBC (p = 2.18 × 10-3, p = 2.28 × 10-4,p = 0.016) compared to tissue NGS. There was considerable divergence in mutation profiles between plasma and EBC with 34/76 (44%) mutations detected in plasma and 37/74 (41.89%) in EBC unique to their respective liquid biopsy. CONCLUSIONS: The results suggest that EBC is effective in identifying clinically relevant alterations in patients with lung cancer using UltraSEEK™ and has a potential role as an adjunct to plasma testing.

Mindfulness Practice Is Associated With Subjective Wellbeing Homeostasis Resilience in People With Crohn's Disease but Not Ulcerative Colitis.

Objectives: People with Crohn's disease and ulcerative colitis (inflammatory bowel disease: IBD), commonly experience high levels of depressive symptoms and stress and low levels of subjective wellbeing (SWB). Mindfulness is increasingly considered an adjuvant IBD treatment. The relationships between depression, disease symptoms and mindfulness have not previously been considered within the theory of SWB homeostasis. This theory states that SWB is normally maintained by a homeostatic system around a setpoint range but can fail when psychological challenges dominate consciousness. This study explored the relationship among SWB and patient-reported psychological and IBD symptoms and investigated whether mindfulness practice is independently associated with SWB homeostatic resilience. Design: This cross-sectional study recruited participants through online IBD support groups. Methods: Participants (n = 739; 62% Crohn's disease) detailed symptoms of depression and stress, patient-reported disease symptoms, and regularity of mindfulness practice. Results: The sample had significantly lower SWB (hedges g = -0.98) than normative data. A logistic regression found mindfulness practice doubled the Crohn's disease participants' odds of reporting SWB within the normal homeostatic range, after controlling for psychological, physical, and demographic variables (OR 2.15, 95% CI: 1.27, 3.66). A one-point increase of patient-reported bowel symptoms reduced the participant's odds of reporting SWB in the normal homeostatic range by about a third (OR 0.66, 95% CI: 0.50, 0.85). However, the influence of mindfulness or disease symptoms on SWB was not observed for people with ulcerative colitis. Conclusion: These findings provide initial evidence for an association between mindfulness and SWB homeostatic resilience in a clinical population.

Low Subjective Wellbeing Is Associated with Psychological Distress in People Living with Inflammatory Bowel Disease.

BACKGROUND: Inflammatory bowel disease (IBD) is a common and debilitating disease of the gastrointestinal tract. Psychological distress is highly comorbid to IBD, especially during periods of active disease. However, a controversy exists on how to best manage its symptoms in the IBD population. AIMS: This study aimed to explore protective and risk factors of psychological distress in IBD. METHODS: A cross-sectional online survey was conducted via social media and online patient forums. Respondents (N = 235) filled out questionnaires on demographics, health characteristics and a range of psychological variables. Measures of pain, disease activity, comorbid functional symptom severity, social support, subjective wellbeing, sleep quality, fatigue, stress, age, BMI and gender were entered into the Classification and Regression Tree Analysis model. RESULTS: Overall, 87 participants (37%) reported distress. Self-reported stress significantly discriminated between cases of probable psychological distress. In those with high stress, patients with and without probable psychological distress were separated by subjective wellbeing. Among patients with low stress, fatigue was the primary discriminator. CONCLUSIONS: Monitoring patients for low subjective wellbeing and high stress in clinical settings could offer an opportunity to engage in early intervention to limit psychological distress development. Monitoring for fatigue in patients who seem otherwise psychologically well could offer preventative benefits.

Study Protocol for the COVID-19 Pandemic Adjustment Survey (CPAS): A Longitudinal Study of Australian Parents of a Child 0-18 Years.

BACKGROUND: The COVID-19 pandemic presents significant risks to the mental health and wellbeing of Australian families. Employment and economic uncertainty, chronic stress, anxiety, and social isolation are likely to have negative impacts on parent mental health, couple and family relationships, as well as child health and development. OBJECTIVE: This study aims to: (1) provide timely information on the mental health impacts of the emerging COVID-19 crisis in a close to representative sample of Australian parents and children (0-18 years), (2) identify adults and families most at risk of poor mental health outcomes, and (3) identify factors to target through clinical and public health intervention to reduce risk. Specifically, this study will investigate the extent to which the COVID-19 pandemic is associated with increased risk for parents' mental health, lower well-being, loneliness, and alcohol use; parent-parent and parent-child relationships (both verbal and physical); and child and adolescent mental health problems. METHODS: The study aims to recruit a close to representative sample of at least 2,000 adults aged 18 years and over living in Australia who are parents of a child 0-4 years (early childhood, N = 400), 5-12 years (primary school N = 800), and 13-18 years (secondary school, N = 800). The design will be a longitudinal cohort study using an online recruitment methodology. Participants will be invited to complete an online baseline self-report survey (20 min) followed by a series of shorter online surveys (10 min) scheduled every 2 weeks for the duration of the COVID-19 pandemic (i.e., estimated to be 14 surveys over 6 months). RESULTS: The study will employ post stratification weights to address differences between the final sample and the national population in geographic communities across Australia. Associations will be analyzed using multilevel modeling with time-variant and time-invariant predictors of change in trajectory over the testing period. CONCLUSIONS: This study will provide timely information on the mental health impacts of the COVID-19 crisis on parents and children in Australia; identify communities, parents, families, and children most at risk of poor outcomes; and identify potential factors to address in clinical and public health interventions to reduce risk.

Efficacy of a Smartphone App Intervention for Reducing Caregiver Stress: Randomized Controlled Trial.

BACKGROUND: Caregivers play a pivotal role in maintaining an economically viable health care system, yet they are characterized by low levels of psychological well-being and consistently report unmet needs for psychological support. Mobile app-based (mobile health [mHealth]) interventions present a novel approach to both reducing stress and improving well-being. OBJECTIVE: This study aims to evaluate the effectiveness of a self-guided mobile app-based psychological intervention for people providing care to family or friends with a physical or mental disability. METHODS: In a randomized, single-blind, controlled trial, 183 caregivers recruited through the web were randomly allocated to either an intervention (n=73) or active control (n=110) condition. The intervention app contained treatment modules combining daily self-monitoring with third-wave (mindfulness-based) cognitive-behavioral therapies, whereas the active control app contained only self-monitoring features. Both programs were completed over a 5-week period. It was hypothesized that intervention app exposure would be associated with decreases in depression, anxiety, and stress, and increases in well-being, self-esteem, optimism, primary and secondary control, and social support. Outcomes were assessed at baseline, postintervention, and 3-4 months postintervention. App quality was also assessed. RESULTS: In total, 25% (18/73) of the intervention participants were lost to follow-up at 3 months, and 30.9% (34/110) of the participants from the wait-list control group dropped out before the postintervention survey. The intervention group experienced reductions in stress (b=-2.07; P=.04) and depressive symptoms (b=-1.36; P=.05) from baseline to postintervention. These changes were further enhanced from postintervention to follow-up, with the intervention group continuing to report lower levels of depression (b=-1.82; P=.03) and higher levels of emotional well-being (b=6.13; P<.001), optimism (b=0.78; P=.007), self-esteem (b=-0.84; P=.005), support from family (b=2.15; P=.001), support from significant others (b=2.66; P<.001), and subjective well-being (b=4.82; P<.001). On average, participants completed 2.5 (SD 1.05) out of 5 treatment modules. The overall quality of the app was also rated highly, with a mean score of 3.94 out of a maximum score of 5 (SD 0.58). CONCLUSIONS: This study demonstrates that mHealth psychological interventions are an effective treatment option for caregivers experiencing high levels of stress. Recommendations for improving mHealth interventions for caregivers include offering flexibility and customization in the treatment design. TRIAL REGISTRATION: Australian New Zealand Clinical Trial Registry ACTRN12616000996460; https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=371170.

Identification and clinical impact of potentially actionable somatic oncogenic mutations in solid tumor samples.

BACKGROUND: An increasing number of anti-cancer therapeutic agents target specific mutant proteins that are expressed by many different tumor types. Successful use of these therapies is dependent on the presence or absence of somatic mutations within the patient's tumor that can confer clinical efficacy or drug resistance. METHODS: The aim of our study was to determine the type, frequency, overlap and functional proteomic effects of potentially targetable recurrent somatic hotspot mutations in 47 cancer-related genes in multiple disease sites that could be potential therapeutic targets using currently available agents or agents in clinical development. RESULTS: Using MassArray technology, of the 1300 patient tumors analysed 571 (43.9%) had at least one somatic mutation. Mutations were identified in 30 different genes. KRAS (16.5%), PIK3CA (13.6%) and BRAF (3.8%) were the most frequently mutated genes. Prostate (10.8%) had the lowest number of somatic mutations identified, while no mutations were identified in sarcoma. Ocular melanoma (90.6%), endometrial (72.4%) and colorectal (66.4%) tumors had the highest number of mutations. We noted high concordance between mutations in different parts of the tumor (94%) and matched primary and metastatic samples (90%). KRAS and BRAF mutations were mutually exclusive. Mutation co-occurrence involved mainly PIK3CA and PTPN11, and PTPN11 and APC. Reverse Phase Protein Array (RPPA) analysis demonstrated that PI3K and MAPK signalling pathways were more altered in tumors with mutations compared to wild type tumors. CONCLUSIONS: Hotspot mutational profiling is a sensitive, high-throughput approach for identifying mutations of clinical relevance to molecular based therapeutics for treatment of cancer, and could potentially be of use in identifying novel opportunities for genotype-driven clinical trials.

A Rasch Analysis of the Personal Well-Being Index in School Children.

The personal well-being index-school children (PWI-SC) is designed as a cross-cultural instrument to measure subjective well-being among high school-aged children. Several published cross-cultural studies have confirmed adequate psychometric performance in terms of reliability, validity, and measurement invariance. This study adds to this literature by applying the Rasch approach to estimate invariant comparison in a cross-cultural context, applied to both Australian and Portuguese high school students. Participants were an age- and gender-matched convenience sample of 1,040 adolescents (520 cases in each group, 51.54% male) who ranged in age from 12 to 18 years (M = 14.25 years, SD = 1.71 years). It is found that both Portuguese and Australian data fit the Rasch measurement model, with excellent levels of reliability at a country level. However, when all of the data were combined, a slight misfit was found. This was resolved by removing some issues with item thresholds in standard of living among the Australian data and splitting the data by country on health. This allowed both Australian and Portuguese cases to differ on the health item. We conclude that the PWI-SC is unidimensional, with some evidence of mild, but acceptable local dependency. This study further supports the cross-cultural validity of the PWI-SC and the use of this measure in the Australian and Portuguese context but also indicates a potential direction that development of the PWI-SC might proceed.

Stromal TRIM28-associated signaling pathway modulation within the colorectal cancer microenvironment.

BACKGROUND: Stromal gene expression patterns predict patient outcomes in colorectal cancer. TRIM28 is a transcriptional co-repressor that regulates an abundance of genes through the KRAB domain family of transcription factors. We have previously shown that stromal expression of TRIM28 is a marker of disease relapse and poor survival in colorectal cancer. Here, we perform differential epithelium-stroma proteomic network analyses to characterize signaling pathways associated with TRIM28 within the tumor microenvironment. METHODS: Reverse phase protein arrays were generated from laser capture micro-dissected carcinoma and stromal cells from fresh frozen colorectal cancer tissues. Phosphorylation and total protein levels were measured for 30 cancer-related signaling pathway endpoints. Strength and direction of associations between signaling endpoints were identified using Spearman's rank-order correlation analysis and compared to TRIM28 levels. Expression status of TRIM28 in tumor epithelium and stromal fibroblasts was assessed using IHC in formalin fixed tissue and the epithelium to stroma protein expression ratio method. RESULTS: We found distinct proteomic networks in the epithelial and stromal compartments which were linked to expression levels of TRIM28. Low levels of TRIM28 in tumor stroma (high epithelium: stroma ratio) were found in 10 out of 19 cases. Upon proteomic network analyses, these stromal high ratio cases revealed moderate signaling pathway similarity exemplified by 76 significant Spearman correlations (ρ ≥ 0.75, p ≤ 0.01). Furthermore, low levels of stromal TRIM28 correlated with elevated MDM2 levels in tumor epithelium (p = 0.01) and COX-2 levels in tumor stroma (p = 0.002). Low TRIM28 epithelium to stroma ratios were associated with elevated levels of caspases 3 and 7 in stroma (p = 0.041 and p = 0.036) and an increased signaling pathway similarity in stromal cells with 81 significant Spearman correlations (ρ ≥ 0.75, p ≤ 0.01). CONCLUSIONS: By dissecting TRIM28-associated pathways in stromal fibroblasts and epithelial tumor cells, we performed comprehensive proteomic analyses of molecular networks within the tumor microenvironment. We found modulation of several signaling pathways associated with TRIM28, which may be attributed to the pleiotropic properties of TRIM28 through its translational suppression of the family of KRAB domain transcription factors in tumor stromal compartments.

Testing an Incentive-Sensitisation Approach to Understanding Problem Slot-Machine Gambling Using an Online Slot-Machine Simulation.

This study aims to test the application of the incentive-sensitisation theory to slot-machine gambling behaviour. The theory posits that for problem gamblers (PGs), gambling strengthens the response of motivational pathways in the mid-brain to gambling cues, eliciting strong wanting, independent of liking. Non-problem gamblers (NPGs) experience weaker changes to motivational pathways so liking and wanting remain associated. Hence, it is predicted that wanting to gamble will be greater than liking for PGs but there will be no difference for NPGs; wanting will be greater for PGs than for NPGs; and, wanting but not liking will predict whether PGs continue gambling, whereas both will predict this for NPGs. During gambling on an online simulated slot-machine, 39 PGs and 87 NPGs rated 'liking' and 'wanting'. Participants played at least 3 blocks of 10-20 spins, and then had the option of playing up to 4 additional blocks; to continue playing they had to complete an effortful task, so that 'number of blocks played' acted as an additional indirect measure of wanting. Results supported hypotheses except on the indirect measure of wanting (the number of blocks played).

IHC-based subcellular quantification provides new insights into prognostic relevance of FLIP and procaspase-8 in non-small-cell lung cancer.

In this study, we developed an image analysis algorithm for quantification of two potential apoptotic biomarkers in non-small-cell lung cancer (NSCLC): FLIP and procaspase-8. Immunohistochemical expression of FLIP and procaspase-8 in 184 NSCLC tumors were assessed. Individual patient cores were segmented and classified as tumor and stroma using the Definiens Tissue Studio. Subsequently, chromogenic expression of each biomarker was measured separately in the nucleus and cytoplasm and reported as a quantitative histological score. The software package pROC was applied to define biomarker thresholds. Cox proportional hazards analysis was applied to generate hazard ratios (HRs) and associated 95% CI for survival. High cytoplasmic expression of tumoral (but not stromal) FLIP was associated with a 2.5-fold increased risk of death in lung adenocarcinoma patients, even when adjusted for known confounders (HR 2.47, 95% CI 1.14-5.35). Neither nuclear nor cytoplasmic tumoral procaspase-8 expression was associated with overall survival in lung adenocarcinoma patients; however, there was a significant trend (P for trend=0.03) for patients with adenocarcinomas with both high cytoplasmic FLIP and high cytoplasmic procaspase-8 to have a multiplicative increased risk of death. Notably, high stromal nuclear procaspase-8 expression was associated with a reduced risk of death in lung adenocarcinoma patients (adjusted HR 0.31, 95% CI 0.15-0.66). On further examination, the cells with high nuclear procaspase-8 were found to be of lymphoid origin, suggesting that the better prognosis of patients with tumors with high stromal nuclear procaspase-8 is related to immune infiltration, a known favorable prognostic factor. No significant associations were detected in analysis of lung squamous cell carcinoma patients. Our results suggest that cytoplasmic expression of FLIP in the tumor and nuclear expression of procaspase-8 in the stroma are prognostically relevant in non-small-cell adenocarcinomas but not in squamous cell carcinomas of the lung.

Somatic DNA Mutation Analysis.

Somatic mutations in patient tumor DNA samples can be readily detected based on mass spectrometry. The MassARRAY system is a high-throughput matrix-assisted laser desorption time-of-flight (MALDI) mass spectrometer for detection of nucleic acids. The technique is based on single-nucleotide base extension. A series of PCR assays amplify specific DNA regions of interest harboring mutations. A third primer is then introduced into the reaction which corresponds to the DNA template immediately in front of the mutation site. A final round of PCR is then performed using mass-modified nucleotides. These nucleotides are designed so that no additional bases can be added to the extension primer (terminating bases) after a single-base extension and are mass modified to exaggerate mass differences between nucleotides allowing easier identification by mass spectrometry.The sequences of the extension primer and possible extension products (wild type and mutations) are known; therefore, it is possible to calculate their mass. The mass spectrometer can identify the mass peaks for each assay and identify those with mutations (multiple peaks). The technique was originally designed to screen multiple single-nucleotide polymorphisms (SNPs) in a large number of specimens. A SNP in the coding region of DNA that alters the gene and subsequent protein expression is considered a mutation. Mutations often occur in genes whose protein product is in a key signaling pathway and/or drug target. Rationale treatment options can be designed based upon the presence or absence of these mutations. In this chapter, we describe the process for detection of somatic mutations in DNA extracted from formalin-fixed paraffin-embedded (FFPE) material.

Using dynamic factor analysis to provide insights into data reliability in experience sampling studies.

The past 2 decades have seen increasing use of experience sampling methods (ESMs) to gain insights into the daily experience of affective states (e.g., its variability, as well as antecedents and consequences of temporary shifts in affect). Much less attention has been given to methodological challenges, such as how to ensure reliability of test scores obtained using ESM. The present study demonstrates the use of dynamic factor analysis (DFA) to quantify reliability of test scores in ESM contexts, evaluates the potential impact of unreliable test scores, and seeks to identify characteristics of individuals that may account for their unreliable test scores. One hundred twenty-seven participants completed baseline measures (demographics and personality traits), followed by a 7-day ESM phase in which positive and negative state affect were measured up to 6 times per day. Analyses showed that although at the sample level, scores on these affect measures exhibited adequate levels of reliability, up to one third of participants failed to meet conventional standards of reliability. Where these low reliability estimates were not significantly associated with personality factors, they could-in some cases-be explained by model misspecification where a meaningful alternative structure was available. Despite these potential differences in factor structure across participants, subsequent modeling with and without these "unreliable" cases showed similar substantive results. Hence, the present findings suggest typical analyses based on ESM data may be robust to individual differences in data structure and/or quality. Ways to augment the DFA approach to better understand unreliable cases are discussed. (PsycINFO Database Record

Challenging the Cancer Molecular Stratification Dogma: Intratumoral Heterogeneity Undermines Consensus Molecular Subtypes and Potential Diagnostic Value in Colorectal Cancer.

PURPOSE: A number of independent gene expression profiling studies have identified transcriptional subtypes in colorectal cancer with potential diagnostic utility, culminating in publication of a colorectal cancer Consensus Molecular Subtype classification. The worst prognostic subtype has been defined by genes associated with stem-like biology. Recently, it has been shown that the majority of genes associated with this poor prognostic group are stromal derived. We investigated the potential for tumor misclassification into multiple diagnostic subgroups based on tumoral region sampled. EXPERIMENTAL DESIGN: We performed multiregion tissue RNA extraction/transcriptomic analysis using colorectal-specific arrays on invasive front, central tumor, and lymph node regions selected from tissue samples from 25 colorectal cancer patients. RESULTS: We identified a consensus 30-gene list, which represents the intratumoral heterogeneity within a cohort of primary colorectal cancer tumors. Using a series of online datasets, we showed that this gene list displays prognostic potential HR = 2.914 (confidence interval 0.9286-9.162) in stage II/III colorectal cancer patients, but in addition, we demonstrated that these genes are stromal derived, challenging the assumption that poor prognosis tumors with stem-like biology have undergone a widespread epithelial-mesenchymal transition. Most importantly, we showed that patients can be simultaneously classified into multiple diagnostically relevant subgroups based purely on the tumoral region analyzed. CONCLUSIONS: Gene expression profiles derived from the nonmalignant stromal region can influence assignment of colorectal cancer transcriptional subtypes, questioning the current molecular classification dogma and highlighting the need to consider pathology sampling region and degree of stromal infiltration when employing transcription-based classifiers to underpin clinical decision making in colorectal cancer. Clin Cancer Res; 22(16); 4095-104. ©2016 AACRSee related commentary by Morris and Kopetz, p. 3989.

Understanding Quality of Life in Medicine: A New Approach.

This article introduces the subjective side of quality of life as it has evolved within the discipline of psychology. Subjective well-being is also of special interest within medicine due to its links to pathology and the fact that it is managed by a homeostatic system. This form of management offers an explanation for the unusual properties of subjective well-being, including its normal positivity, stability, and nonlinear relationship to objective variables, such as physical health. Central to understanding is the proposition that subjective well-being mainly consists of a specific form of trait mood. This homeostatically protected mood has a genetic set point and it is the experience of this set-point mood that homeostasis is defending. The resources required to maintain normal homeostatic control are described. If these resources are inadequate to protect the experience of set-point mood, mood positivity falls, and there is a high probability of depression. In this article, the process of homeostasis is shown to assist understanding of intervention effectiveness within both psychology and medicine. This concerns matters of resilience, the nonlinear relationship between levels of subjective well-being, and the strength of challenging agents, and the important understanding that interventions designed to raise subjective well-being are critically dependent on its level at baseline. Key teaching points: The physiological process of homeostasis has a parallel in psychology in the homeostatic management of subjective well-being. Subjective well-being is a more globally informative construct than health-related quality of life. How people feel about themselves and their lives cannot be simply predicted through measures of health. When subjective well-being homeostasis is defeated, there is a high probability of depression.

THE HAPPINESS AND SUBJECTIVE WELL-BEING OF PEOPLE ON HAEMODIALYSIS.

BACKGROUND: Happiness is a construct that has been gaining more prominence in both social and health research. The measure of happiness, subjective well-being, has not been rigorously explored in the end-stage kidney disease (ESKD) population. OBJECTIVES: To measure the subjective well-being of people with ESKD on haemodialysis and to compare their subjective well-being with a general population cohort. DESIGN: A cross-sectional design measuring the subjective well-being of an Australian haemodialysis cohort compared with a non-dialysis age-matched cohort. PARTICIPANTS: The haemodialysis cohort (N = 172), recruited from eight dialysis centres, had a mean age of 64.04 years (SD = 14.82) and included 104 males (60.5%) and 66 (38.4%) females. The non-dialysis cohort (N = 200), randomly extracted from the 2012 Australian Unity Wellbeing Index, had a mean age of 63.97 (SD = 14.68) and included 101 males (50.5%) and 99 females (49.5%). MEASUREMENT: Subjective well-being was measured using the Personal Wellbeing Index. This seven-item measure rates satisfaction with life in seven domains: standard of living, health, achievements in life, relationships, safety, community and future security. RESULTS: The haemodialysis cohort reported lower general life satisfaction, life achievements, relationship and personal safety compared to the general population. There were no differences between the two groups for health, community and future security. Standard of living and satisfaction with life achievements carried higher importance in subjective well-being compared with health satisfaction CONCLUSION: Subjective well-being can be an important indicator of people's life quality to be considered by clinicians and nephrology researchers in future studies.

High-throughput oncogene mutation profiling shows demographic differences in BRAF mutation rates among melanoma patients.

Because of advances in targeted therapies, the clinical evaluation of cutaneous melanoma is increasingly based on a combination of traditional histopathology and molecular pathology. Therefore, it is necessary to expand our knowledge of the molecular events that accompany the development and progression of melanoma to optimize clinical management. The central objective of this study was to increase our knowledge of the mutational events that complement melanoma progression. High-throughput genotyping was adapted to query 159 known single nucleotide mutations in 33 cancer-related genes across two melanoma cohorts from Ireland (n=94) and Belgium (n=60). Results were correlated with various clinicopathological characteristics. A total of 23 mutations in 12 genes were identified, that is--BRAF, NRAS, MET, PHLPP2, PIK3R1, IDH1, KIT, STK11, CTNNB1, JAK2, ALK, and GNAS. Unexpectedly, we discovered significant differences in BRAF, MET, and PIK3R1 mutations between the cohorts. That is, cases from Ireland showed significantly lower (P<0.001) BRAF(V600E) mutation rates (19%) compared with the mutation frequency observed in Belgian patients (43%). Moreover, MET mutations were detected in 12% of Irish cases, whereas none of the Belgian patients harbored these mutations, and Irish patients significantly more often (P=0.027) had PIK3R1-mutant (33%) melanoma versus 17% of Belgian cases. The low incidence of BRAF(V600E)(-) mutant melanoma among Irish patients was confirmed in five independent Irish cohorts, and in total, only 165 of 689 (24%) Irish cases carried mutant BRAF(V600E). Together, our data show that melanoma-driving mutations vary by demographic area, which has important implications for the clinical management of this disease.

High CerS5 expression levels associate with reduced patient survival and transition from apoptotic to autophagy signalling pathways in colorectal cancer.

Ceramide synthase 5 is involved in the de novo synthesis of ceramide, a sphingolipid involved in cell death and proliferation. In this study, we investigated the role of ceramide synthase 5 in colorectal cancer by examining ceramide synthase 5 expression, clinico-pathological parameters and association with survival/death signalling pathways in cancer. Immunohistochemical analysis of CerS5 was performed on 102 colorectal cancer samples using tissue microarrays constructed from formalin-fixed and paraffin-embedded tissues. We found strong membranous ceramide synthase 5 staining in 57 of 102 (56%) colorectal cancers. A multivariate Cox regression analysis of ceramide synthase 5 expression adjusted for disease stage, differentiation and lymphovascular invasion revealed reduced 5-year overall survival (p = 0.001) and 5-year recurrence-free survival (p = 0.002), with hazard ratios of 4.712 and 4.322, respectively. The effect of ceramide synthase 5 expression on tumourigenic processes was further characterised by reverse phase protein array analysis. Reverse phase protein arrays were generated from laser capture microdissection-enriched carcinoma cells from 19 fresh-frozen colorectal cancer tissues. Measurements of phosphorylation and total levels of signalling proteins involved in apoptosis, autophagy and other cancer-related pathways revealed two distinct signalling networks; weak membranous ceramide synthase 5 intensity was associated with a proteomic network dominated by signalling proteins linked to apoptosis, whereas strong ceramide synthase 5 intensity was associated with a proteomic sub-network mostly composed of proteins linked to autophagy. In conclusion, high ceramide synthase 5 expression was found in colorectal cancer tissue and was associated with poorer patient outcomes. Our findings suggest that this may be mediated by a transition from apoptotic to autophagy signalling pathways in ceramide synthase 5 High expressing tumours, thus implicating ceramide synthase 5 in the progression of colorectal cancer.

The frequencies and clinical implications of mutations in 33 kinase-related genes in locally advanced rectal cancer: a pilot study.

BACKGROUND: Locally advanced rectal cancer (LARC: T3/4 and/or node-positive) is treated with preoperative/neoadjuvant chemoradiotherapy (CRT), but responses are not uniform. The phosphatidylinositol 3-kinase (PI3K), MAP kinase (MAPK), and related pathways are implicated in rectal cancer tumorigenesis. Here, we investigated the association between genetic mutations in these pathways and LARC clinical outcomes. METHODS: We genotyped 234 potentially clinically relevant nonsynonymous mutations in 33 PI3K and MAPK pathway-related genes, including PIK3CA, PIK3R1, AKT, STK11, KRAS, BRAF, MEK, CTNNB1, EGFR, MET, and NRAS, using the Sequenom platform. DNA samples were extracted from pretreatment LARC biopsy samples taken from 201 patients who were then treated with long-course neoadjuvant CRT followed by surgical resection. RESULTS: Sixty-two mutations were detected in 15 genes, with the highest frequencies occurring in KRAS (47 %), PIK3CA (14 %), STK11 (6.5 %), and CTNNB1 (6 %). Mutations were detected in BRAF, NRAS, AKT1, PIK3R1, EGFR, GNAS, MEK1, PDGFRA, ALK, and TNK2, but at frequencies of <5 %. As expected, a pathologic complete response (pCR) was associated with improved 5-year recurrence-free survival (RFS; hazard ratio, 0.074; 95 % CI 0.01-0.54; p = 0.001). Mutations in PI3K pathway-related genes (odds ratio, 5.146; 95 % CI 1.17-22.58; p = 0.030), but not MAPK pathway-related genes (p = 0.911), were associated with absence of pCR after neoadjuvant CRT. In contrast, in patients who did not achieve pCR, mutations in PI3K pathway-related genes were not associated with recurrence-free survival (p = 0.987). However, in these patients, codon 12 (G12D/G12 V/G12S) and 13 mutations in KRAS were associated with poor recurrence-free survival (hazard ratio, 1.579; 95 % confidence ratio, 1.00-2.48; p = 0.048). CONCLUSIONS: Mutations in kinase signaling pathways modulate treatment responsiveness and clinical outcomes in LARC and may constitute rational targets for novel therapies.