Long-Term Echocardiographic and Clinical Outcomes After Invasive and Non-Invasive Therapies for Sub-Massive and Massive Acute Pulmonary Embolism.

AIM: Acute pulmonary embolism (PE) is a significant cause of mortality in the hospital setting. The objective of this study was to outline the long-term outcomes after surgical and non-surgical management for patients with massive and submassive PE. METHODS: Population cohort observational study evaluating all patients who presented to three tertiary hospitals in the state of Western Australia with access to cardiothoracic services over 5 years (2013-2018). Reviewed notes of all patients as well as radiology, linked mortality data and all available echocardiography studies at the primary hospital. RESULTS: In total, 245 patients were identified, of which 41 received surgical management and 204 non-surgical management; demographic data was similar. Clinically, the surgical group had higher rates of shock requiring vasopressors, severe bradycardia, or cardiopulmonary resuscitation prior to intervention. The 28-day mortality was not statistically significantly different between the surgical embolectomy group (2/41 [4.2%]) and the non-surgical group (17/201 [8.3%]) (p=0.382). There was no difference in 12-month mortality, including when this was adjusted for vasopressors, right ventricular (RV) strain, troponin, and brain natriuretic peptide. In the massive PE sub-group, 28-day mortality was not significantly different: 2/29 (6.9%) surgical group vs 7/34 (20.2%) non-surgical group (p=0.064). Higher rates of severe RV impairment and dilatation were present in the surgical group. All patients with available echocardiography studies at outpatient follow-up returned to normal or mild RV impairment. CONCLUSION: Patients who presented with massive or submassive PE had similar outcomes whether treated with surgical or non-surgical management. Surgical embolectomy is a safe option in a cardiothoracic centre setting.

The effect of expressive writing on wound healing: Immunohistochemistry analysis of skin tissue two weeks after punch biopsy wounding.

OBJECTIVE: To investigate the effects of expressive writing and its timing (pre or post wounding) on re-epithelialisation and leucocyte subsets within healing tissue. We previously showed expressive writing pre-wounding improved re-epithelialisation. Here we investigate cellular processes in the wound. METHODS: In a 2(writing content) x 2(writing timing) randomized trial, 122 participants were randomized to perform either expressive or control writing, before or after a 4 mm punch biopsy wound. On day 14 post-wounding, participants had a 5 mm punch biopsy of the initial wound. Seven of 16 primary registered outcomes were analysed, including re-epithelialisation from two photographs of the 4 mm biopsy (previously reported). This paper reports immunohistochemistry analysis of five primary outcomes - Langerhans cells, immune cell activation (HLA and CD3+), and macrophages (CD68 and MPO) - in the 5 mm biopsies in a random sample of 96 participants. RESULTS: Participants who performed either writing task pre-wounding had greater Langerhans cell infiltration, than those who wrote post-wounding (F(1,85) = 7.86, p = .006, ηp2 = 0.08). Those who performed expressive writing also had greater Langerhans cell infiltration than those who performed control writing (F(1,85) = 4.00, p = .049, ηp2 = 0.04). There were no significant group or interaction effects on immune cell activation or macrophages. Healed wounds on day 10 had lower levels of macrophages (z = -1.96, p = .050), and CD3+ cells (z = -1.99, p = .046) than non-healed wounds. CONCLUSION: Langerhans cells in the healing skin are affected by the timing and topic of writing. More research is needed to further explore timing and corroborate these results. CLINICAL TRIALS REGISTRATION: Registered at https://www.anzctr.org.au/ (Trial ID: ACTRN12614000971639).

Clinical Validation of a Cell-Free DNA Gene Panel.

The use of liquid biopsies to identify driver mutations in patients with solid tumors holds great promise for performing targeted therapy selection, monitoring disease progression, and detecting treatment resistance mechanisms. We describe herein the development and clinical validation of a 28-gene cell-free DNA panel that targets the most common genetic alterations in solid tumors. Bioinformatic and variant filtering solutions were developed to improve test sensitivity and specificity. The panel and these tools were used to analyze commercially available controls, allowing establishment of a limit of detection allele fraction cutoff of 0.25%, with 100% (95% CI, 81.5%-100%) specificity and 89.8% (95% CI, 81.0%-94.9%) sensitivity. In addition, we analyzed a total of 163 blood samples from patients with metastatic cancer (n = 123) and demonstrated a >90% sensitivity for detecting previously identified expected mutations. Longitudinal monitoring of patients revealed a strong correlation of variant allele frequency changes and clinical outcome. Additional clinically relevant information included identification of resistance mutations in patients receiving targeted treatment and detection of complex patterns of mutational heterogeneity. Achieving lower limits of detection will require additional improvements to molecular barcoding; however, these data strongly support clinical implementation of cell-free DNA panels in advanced cancer patients.

Clinically Integrated Molecular Diagnostics in Adenoid Cystic Carcinoma.

BACKGROUND: Adenoid cystic carcinoma (ACC) is an aggressive salivary gland malignancy without effective systemic therapies. Delineation of molecular profiles in ACC has led to an increased number of biomarker-stratified clinical trials; however, the clinical utility and U.S.-centric financial sustainability of integrated next-generation sequencing (NGS) in routine practice has, to our knowledge, not been assessed. MATERIALS AND METHODS: In our practice, NGS genotyping was implemented at the discretion of the primary clinician. We combined NGS-based mutation and fusion detection, with MYB break-apart fluorescent in situ hybridization (FISH) and MYB immunohistochemistry. Utility was defined as the fraction of patients with tumors harboring alterations that are potentially amenable to targeted therapies. Financial sustainability was assessed using the fraction of global reimbursement. RESULTS: Among 181 consecutive ACC cases (2011-2018), prospective genotyping was performed in 11% (n = 20/181; n = 8 nonresectable). Testing identified 5/20 (25%) NOTCH1 aberrations, 6/20 (30%) MYB-NFIB fusions (all confirmed by FISH), and 2/20 (10%) MYBL1-NFIB fusions. Overall, these three alterations (MYB/MYBL1/NOTCH1) made up 65% of patients, and this subset had a more aggressive course with significantly shorter progression-free survival. In 75% (n = 6/8) of nonresectable patients, we detected potentially actionable alterations. Financial analysis of the global charges, including NGS codes, indicated 63% reimbursement, which is in line with national (U.S.-based) and international levels of reimbursement. CONCLUSION: Prospective routine clinical genotyping in ACC can identify clinically relevant subsets of patients and is approaching financial sustainability. Demonstrating clinical utility and financial sustainability in an orphan disease (ACC) requires a multiyear and multidimensional program. IMPLICATIONS FOR PRACTICE: Delineation of molecular profiles in adenoid cystic carcinoma (ACC) has been accomplished in the research setting; however, the ability to identify relevant patient subsets in clinical practice has not been assessed. This work presents an approach to perform integrated molecular genotyping of patients with ACC with nonresectable, recurrent, or systemic disease. It was determined that 75% of nonresectable patients harbor potentially actionable alterations and that 63% of charges are reimbursed. This report outlines that orphan diseases such as ACC require a multiyear, multidimensional program to demonstrate utility in clinical practice.

Expediting Comprehensive Molecular Analysis to Optimize Initial Treatment of Lung Cancer Patients With Minimal Smoking History.

INTRODUCTION: Lung cancer patients with tumors harboring actionable alterations can achieve very durable responses to first-line targeted therapy. However, identifying targetable alterations using next-generation sequencing (NGS) is a complex and time-intensive process. As actionable genetic alterations are enriched in lung cancers arising in patients with limited smoking history, we designed a workflow to expedite NGS testing for this group. METHODS: We developed a protocol to allow for next-day extraction of nucleic acids from frozen tissue. Specimens were designated as high priority during sequencing. We determined the interval between biopsy and NGS results to evaluate whether the workflow reduced the pre-analytical period and in-laboratory turnaround time and allowed for rapid initiation of genotype-matched therapy. RESULTS: Between January 2017 and May 2018, 21 patients participated in the expedited sequencing program. The median interval between biopsy and NGS results was 10.7 days. Six patients received results within 1 week of biopsy. Performing molecular analysis on frozen tissue and prioritizing sequencing and analysis of these specimens reduced the pre-analytical period from 3.5 to 1.3 days (p < 0.0001) and shortened in-laboratory turnaround time by 3 days (11.8 versus 8.4 business days, p < 0.0001). Ninety-three percent of patients with an actionable molecular alteration received first-line targeted therapy. The median time-to-initiation of treatment was 19.7 days from biopsy. CONCLUSIONS: Sequencing and analyzing nucleic acids from frozen tissue is a practical strategy for shortening the time to matched therapy. The significant advantage of upfront treatment with targeted therapies in subsets of lung cancer patients provides rationale for developing workflows that accelerate comprehensive molecular analysis.

A Nanopore Sequencing-Based Assay for Rapid Detection of Gene Fusions.

Structural chromosomal rearrangements leading to gene fusions are strong driver mutations in a variety of tumors. Identification of specific gene fusions can be essential for distinguishing benign from malignant conditions and for recognizing specific subtypes of neoplasms that can have different management and prognosis. Rapid identification of gene fusions is particularly critical for patients with acute leukemia who cannot wait more than a few days before initiating treatment and for whom treatment can be dramatically different depending on the leukemia subtype. We have developed an assay for rapid detection of oncogenic gene fusions (within 24 hours) that takes advantage of the long reads and real-time data generation of the Oxford Nanopore MinION sequencing system. By using a modification of the anchored multiplex PCR method for library construction, we confidently identified BCR-ABL1 fusion transcripts, with >100 reads within 15 minutes of sequencing. By using formalin-fixed, paraffin-embedded specimens routinely tested in our clinical molecular laboratory, fusions were successfully identified within 5 hours from acquisition of Illumina-ready libraries and 30 minutes of sequencing initiation, including cases diluted to a tumor fraction of 5%. In conclusion, we have developed a nanopore-based sequencing assay that can decrease turnaround time for detection of fusion oncogenes and may be a valid approach for laboratories with low specimen volume and for cases in need of rapid results.

Clinical Utility of Rapid EGFR Genotyping in Advanced Lung Cancer.

PURPOSE: Targeted therapy is the cornerstone of treatment of advanced EGFR-mutant non-small-cell lung cancer (NSCLC). Next-generation sequencing (NGS), the preferred method for genotyping, typically requires several weeks. Here, we assessed workflows designed to rapidly identify patients with actionable EGFR mutations and reduce time to initiation (TTI) of epidermal growth factor receptor (EGFR)-directed therapy. PATIENTS AND METHODS: We performed rapid testing for EGFR L858R mutations and exon 19 deletions on paraffin-embedded or frozen section biopsy specimens from newly diagnosed patients with metastatic NSCLC by using an EGFR-specific assay (rapid test). To determine clinical utility, we assessed concordance with NGS results, turnaround time, and TTI of EGFR therapy, and we evaluated reimbursement data. RESULTS: Between January 2015 and September 2017, we performed 243 rapid EGFR tests and identified EGFR mutations in 43 patients (18%). With NGS results as a reference, sensitivity and specificity of the rapid EGFR polymerase chain reaction assay were 98% and 100%, respectively. The median turnaround time for NGS was 14 days, compared with 7 days for rapid testing (P < .001). In the rapid group, 95% of patients received an EGFR inhibitor in the first-line setting. The median TTI of EGFR therapy was significantly shorter in the rapid cohort when compared with 121 historical cases (22 v 37 days; P = .01). Escalation of the initiative into an interdisciplinary ultra-rapid next-day frozen-section workflow for highly symptomatic patients (n = 8) resulted in a reduction in the median (± standard deviation) turnaround time to 1 ± 0.4 days and allowed several patients to initiate therapy within 1 week of biopsy. An extended 9-month clinical evaluation phase confirmed operational sustainability (turnaround times: ultra-rapid, 0.81 ± 0.4 days; rapid, 3 ± 1.5 days), and a 63% reimbursement rate indicated financial sustainability. CONCLUSION: Rapid genotyping facilitates earlier initiation of EGFR-directed therapies without compromising NGS workflows.

An examination of user costs in relation to smokers using a cessation service based in the UK.

BACKGROUND: Smoking cessation services provide support to smokers who desire to quit. Published studies to date have looked at the cost and benefit of service provision but typically focus on clinical trial data. Using routinely collected observational data, this study examined the costs involved in providing a service in terms of average health care expenditure per successful quit attempt in addition to population - level cost-effectiveness measures. METHODS: Data were analysed from Quit-51 smoking cessation service across five English regions between March 2013 and March 2016 (n = 9116). For each user, costs were estimated in relation to: (i) time spent with advisers; (ii) prescription of pharmacotherapy. The total costs compared against self-reported quit at 12 weeks, which represents the time period for which the service is offered. Cost per quit (CPQ), with 95% confidence interval (CI), was calculated by relating total expenditure to the number of quitters, firstly for the whole dataset and then by subgroups of key categorical variables, namely; gender, age group, the Fagerstrom test for nicotine dependence (FTND) and Index of Multiple Deprivation (IMD). Confidence intervals (CIs) for the mean estimates were derived using a non-parametric bootstrap procedure. Parameters derived from the calculation in relation to treatment were used to estimate potential long-term population outcomes under a scenario where the Quit 51 prescription was rolled out nationally. RESULTS: The overall mean CPQ for this sample as estimated at 12 weeks was £403.51 (95% CI = £393.36 to £413.76). The estimated CPQs at this time point were comparable for those aged 12-19 (£423.56, 95% CI = £369.45 to £492.60) and those aged 20-29 (£430.76, 95% CI = £395.95 to £470.56). Differences were also seen in relation to other subgroups considered. The treatment parameters translated to a projected increase of 1.5 quality-adjusted life years (QALYs) per 1000 smokers in the short-term and 23.4 QALYS per 1000 smokers based on a lifetime horizon. CONCLUSIONS: These figures throw light on service expenditure for each successful quit over the timeframe for which the service is offered in addition to highlighting variability in these costs across different subgroups of the user population.

Factors moderating the relative effectiveness of varenicline and nicotine replacement therapy in clients using smoking cessation services.

AIMS: To assess how far the greater effectiveness of varenicline over nicotine replacement therapy (NRT) is moderated by characteristics of the smokers or setting in clinical practice. DESIGN: We used observational data from 22 472 treatment episodes between 2013 and 2016 from smoking cessation services in England to assess whether differences between varenicline and NRT were moderated by a set of smoker and setting characteristics: these included level of social deprivation, age, gender, ethnic group, nicotine dependence and treatment context. From the above, 15 640 episodes were analysed in relation to 4-week quit and 14 273 episodes at 12 weeks. All two-way interactions involving pharmacotherapy were fitted in addition to the main effects and a parsimonious model identified using a backwards stepwise selection procedure. SETTING: England PARTICIPANTS: Clients of smoking cessation service (number of individuals in 4-week quit analysis = 15 640). MEASUREMENTS: Four-week carbon monoxide-validated (primary outcome) and 12-week self-reported (secondary outcome) quit success/failure. FINDINGS: At both follow-up points, varenicline was associated with higher success rates overall [P < 0.001 at both 4 and 12 weeks; adjusted odds ratio (OR) varenicline versus NRT = 1.82 (95% confidence interval (CI) = 1.61, 2.06) and 2.58 (95% CI = 2.26, 2.94) at 4 and 12 weeks, respectively]. At 12 weeks, the relative benefits of varenicline were found to be influenced by the setting in which advice was provided [P < 0.001 for interaction pharmacotherapy × setting; adjusted odds ratio for varenicline × pharmacy setting = 0.53 (95% CI = 0.42, 0.69) and for varenicline × general practice (GP) setting = 0.79 (95% CI = 0.64, 0.98) against a baseline of 1 for varenicline × community setting]. The same trends were evident at 4 weeks, but this did not translate to statistical significance. There was inconclusive evidence for moderating effects of other variables. CONCLUSIONS: Varenicline use was associated with higher smoking cessation rates than nicotine replacement therapy in routine clinical practice, irrespective of a wide range of smoker characteristics, but the difference was less in certain intervention settings, most notably pharmacy but also GP practice, compared with community setting.

Randomized clinical trial of expressive writing on wound healing following bariatric surgery.

OBJECTIVE: Writing emotionally about upsetting life events (expressive writing) has been shown to speed healing of punch-biopsy wounds compared to writing objectively about daily activities. We aimed to investigate whether a presurgical expressive writing intervention could improve surgical wound healing. METHOD: Seventy-six patients undergoing elective laparoscopic bariatric surgery were randomized either to write emotionally about traumatic life events (expressive writing) or to write objectively about how they spent their time (daily activities writing) for 20 min a day for 3 consecutive days beginning 2 weeks prior to surgery. A wound drain was inserted into a laparoscopic port site and wound fluid analyzed for proinflammatory cytokines collected over 24 hr postoperatively. Expanded polytetrafluoroethylene tubes were inserted into separate laparoscopic port sites during surgery and removed after 14 days. Tubes were analyzed for hydroxyproline deposition (the primary outcome), a major component of collagen and marker of healing. Fifty-four patients completed the study. RESULTS: Patients who wrote about daily activities had significantly more hydroxyproline than did expressive writing patients, t(34) = -2.43, p = .020, 95% confidence interval [-4.61, -0.41], and higher tumor necrosis factor-alpha, t(29) = -2.42, p = .022, 95% confidence interval [-0.42, -0.04]. Perceived stress significantly reduced in both groups after surgery. CONCLUSIONS: Expressive writing prior to bariatric surgery was not effective at increasing hydroxyproline at the wound site 14 days after surgery. However, writing about daily activities did predict such an increase. Future research needs to replicate these findings and investigate generalizability to other surgical groups. (PsycINFO Database Record

Analytical Validation of the Next-Generation Sequencing Assay for a Nationwide Signal-Finding Clinical Trial: Molecular Analysis for Therapy Choice Clinical Trial.

The National Cancer Institute-Molecular Analysis for Therapy Choice (NCI-MATCH) trial is a national signal-finding precision medicine study that relies on genomic assays to screen and enroll patients with relapsed or refractory cancer after standard treatments. We report the analytical validation processes for the next-generation sequencing (NGS) assay that was tailored for regulatory compliant use in the trial. The Oncomine Cancer Panel assay and the Personal Genome Machine were used in four networked laboratories accredited for the Clinical Laboratory Improvement Amendments. Using formalin-fixed paraffin-embedded clinical specimens and cell lines, we found that the assay achieved overall sensitivity of 96.98% for 265 known mutations and 99.99% specificity. High reproducibility in detecting all reportable variants was observed, with a 99.99% mean interoperator pairwise concordance across the four laboratories. The limit of detection for each variant type was 2.8% for single-nucleotide variants, 10.5% for insertion/deletions, 6.8% for large insertion/deletions (gap ≥4 bp), and four copies for gene amplification. The assay system from biopsy collection through reporting was tested and found to be fully fit for purpose. Our results indicate that the NCI-MATCH NGS assay met the criteria for the intended clinical use and that high reproducibility of a complex NGS assay is achievable across multiple clinical laboratories. Our validation approaches can serve as a template for development and validation of other NGS assays for precision medicine.

The effects of expressive writing before or after punch biopsy on wound healing.

OBJECTIVE: Recent studies have shown that written emotional disclosure (expressive writing) performed in the two weeks prior to wounding improves healing of punch biopsy wounds. In many clinical settings, it would be more practical for patients to perform this intervention after wounding. The aim of this study was to investigate whether expressive writing could speed the healing of punch biopsy wounds if writing was performed after wounds were made. METHODS: One hundred and twenty-two healthy participants aged between 18 and 55years were randomly allocated to one of four groups in a 2 (intervention) by 2 (timing) design. Participants performed either expressive writing or neutral writing, either before or after receiving a 4mm punch biopsy wound. Wounds were photographed on day 10 (primary endpoint) and day 14 after the biopsy to measure epithelisation. Participants also completed questionnaires on stress and affect two weeks prior to the biopsy, on the day of biopsy and two weeks after biopsy. RESULTS: There was a significant difference in healing at day 10 between groups, χ2(3, N=97)=8.84, p=0.032. A significantly greater proportion of participants who performed expressive writing before the biopsy had fully reepithelialised wounds on day 10 compared to participants who performed neutral writing either before or after wounding, with no other significant differences between groups. Amongst people who wrote expressively after wounding, those who finished writing over the first 6days were significantly more likely to be healed at 14days than those who finished writing later. There were significant differences in positive and negative affect over the healing period between the pre and post expressive writing groups. CONCLUSIONS: Expressive writing can improve healing if it is performed prior to wounding. Performing expressive writing after wounding may be able to improve healing depending on the timing of writing and wound assessment. Expressive writing causes affect to worsen followed by subsequent improvement and it is important to consider this in the timing of intervention delivery. Further research with patient groups is required to determine the clinical relevance of these findings.

Tumor Heterogeneity and Lesion-Specific Response to Targeted Therapy in Colorectal Cancer.

UNLABELLED: How genomic heterogeneity associated with acquired resistance to targeted agents affects response to subsequent therapy is unknown. We studied EGFR blockade in colorectal cancer to assess whether tissue and liquid biopsies can be integrated with radiologic imaging to monitor the impact of individual oncogenic alterations on lesion-specific responses. Biopsy of a patient's progressing liver metastasis following prolonged response to cetuximab revealed a MEK1(K57T) mutation as a novel mechanism of acquired resistance. This lesion regressed upon treatment with panitumumab and the MEK inhibitor trametinib. In circulating tumor DNA (ctDNA), mutant MEK1 levels declined with treatment, but a previously unrecognized KRAS(Q61H) mutation was also identified that increased despite therapy. This same KRAS mutation was later found in a separate nonresponding metastasis. In summary, parallel analyses of tumor biopsies and serial ctDNA monitoring show that lesion-specific radiographic responses to subsequent targeted therapies can be driven by distinct resistance mechanisms arising within separate tumor lesions in the same patient. SIGNIFICANCE: Molecular heterogeneity ensuing from acquired resistance drives lesion-specific responses to subsequent targeted therapies. Analysis of a single-lesion biopsy is inadequate to guide selection of subsequent targeted therapies. ctDNA profiles allow the detection of concomitant resistance mechanisms residing in separate metastases and assessment of the effect of therapies designed to overcome resistance.

Anchored multiplex PCR for targeted next-generation sequencing.

We describe a rapid target enrichment method for next-generation sequencing, termed anchored multiplex PCR (AMP), that is compatible with low nucleic acid input from formalin-fixed paraffin-embedded (FFPE) specimens. AMP is effective in detecting gene rearrangements (without prior knowledge of the fusion partners), single nucleotide variants, insertions, deletions and copy number changes. Validation of a gene rearrangement panel using 319 FFPE samples showed 100% sensitivity (95% confidence limit: 96.5-100%) and 100% specificity (95% confidence limit: 99.3-100%) compared with reference assays. On the basis of our experience with performing AMP on 986 clinical FFPE samples, we show its potential as both a robust clinical assay and a powerful discovery tool, which we used to identify new therapeutically important gene fusions: ARHGEF2-NTRK1 and CHTOP-NTRK1 in glioblastoma, MSN-ROS1, TRIM4-BRAF, VAMP2-NRG1, TPM3-NTRK1 and RUFY2-RET in lung cancer, FGFR2-CREB5 in cholangiocarcinoma and PPL-NTRK1 in thyroid carcinoma. AMP is a scalable and efficient next-generation sequencing target enrichment method for research and clinical applications.

Patient-derived models of acquired resistance can identify effective drug combinations for cancer.

Targeted cancer therapies have produced substantial clinical responses, but most tumors develop resistance to these drugs. Here, we describe a pharmacogenomic platform that facilitates rapid discovery of drug combinations that can overcome resistance. We established cell culture models derived from biopsy samples of lung cancer patients whose disease had progressed while on treatment with epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors and then subjected these cells to genetic analyses and a pharmacological screen. Multiple effective drug combinations were identified. For example, the combination of ALK and MAPK kinase (MEK) inhibitors was active in an ALK-positive resistant tumor that had developed a MAP2K1 activating mutation, and the combination of EGFR and fibroblast growth factor receptor (FGFR) inhibitors was active in an EGFR mutant resistant cancer with a mutation in FGFR3. Combined ALK and SRC (pp60c-src) inhibition was effective in several ALK-driven patient-derived models, a result not predicted by genetic analysis alone. With further refinements, this strategy could help direct therapeutic choices for individual patients.