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1.  Histological Transformation and Progression in Follicular Lymphoma: A Clonal Evolution Study 
PLoS Medicine  2016;13(12):e1002197.
Background
Follicular lymphoma (FL) is an indolent, yet incurable B cell malignancy. A subset of patients experience an increased mortality rate driven by two distinct clinical end points: histological transformation and early progression after immunochemotherapy. The nature of tumor clonal dynamics leading to these clinical end points is poorly understood, and previously determined genetic alterations do not explain the majority of transformed cases or accurately predict early progressive disease. We contend that detailed knowledge of the expansion patterns of specific cell populations plus their associated mutations would provide insight into therapeutic strategies and disease biology over the time course of FL clinical histories.
Methods and Findings
Using a combination of whole genome sequencing, targeted deep sequencing, and digital droplet PCR on matched diagnostic and relapse specimens, we deciphered the constituent clonal populations in 15 transformation cases and 6 progression cases, and measured the change in clonal population abundance over time. We observed widely divergent patterns of clonal dynamics in transformed cases relative to progressed cases. Transformation specimens were generally composed of clones that were rare or absent in diagnostic specimens, consistent with dramatic clonal expansions that came to dominate the transformation specimens. This pattern was independent of time to transformation and treatment modality. By contrast, early progression specimens were composed of clones that were already present in the diagnostic specimens and exhibited only moderate clonal dynamics, even in the presence of immunochemotherapy. Analysis of somatic mutations impacting 94 genes was undertaken in an extension cohort consisting of 395 samples from 277 patients in order to decipher disrupted biology in the two clinical end points. We found 12 genes that were more commonly mutated in transformed samples than in the preceding FL tumors, including TP53, B2M, CCND3, GNA13, S1PR2, and P2RY8. Moreover, ten genes were more commonly mutated in diagnostic specimens of patients with early progression, including TP53, BTG1, MKI67, and XBP1.
Conclusions
Our results illuminate contrasting modes of evolution shaping the clinical histories of transformation and progression. They have implications for interpretation of evolutionary dynamics in the context of treatment-induced selective pressures, and indicate that transformation and progression will require different clinical management strategies.
Sohrab Shah and colleagues explore the evolutionary histories that shape clinical and transformation dynamics in follicular lymphoma
Author Summary
Why Was This Study Done?
Follicular lymphoma (FL) is a largely incurable malignancy in which early progression and transformation have consistently been linked to lymphoma-related mortality.
We contended that detailed characterization of clonal dynamics would reveal fundamental biological properties with implications for future patient management strategies relating to both transformation and progression.
We also sought to identify recurrent gene mutations associated with transformation and/or early progression in a large patient cohort.
What Did the Researchers Do and Find?
Using whole genome sequencing, deep allelic sampling by amplicon sequencing, and digital droplet PCR, we found dramatic clonal expansions in transformed disease, whereby dominant clones in transformation samples emerged from extremely low prevalence clones or from clones that were not detected in the diagnostic samples.
The dynamics of disease progression during treatment in the absence of transformation showed markedly different characteristics, with much of the clonal architecture preserved from diagnostic to relapse specimens.
Targeted capture-based sequencing in a large extension cohort then established genetic variants associated with transformation and early progression in the broader patient population.
What Do These Findings Mean?
Taken together, our findings illuminate previously undescribed patterns of clonal expansion underpinning FL clinical histories suggesting that contrasting management strategies will be necessary across the FL patient population.
We uncovered novel associations of gene mutations with early progression that could inform future prognostic assay development.
doi:10.1371/journal.pmed.1002197
PMCID: PMC5154502  PMID: 27959929
2.  Patterns of Immune Infiltration in Breast Cancer and Their Clinical Implications: A Gene-Expression-Based Retrospective Study 
PLoS Medicine  2016;13(12):e1002194.
Background
Immune infiltration of breast tumours is associated with clinical outcome. However, past work has not accounted for the diversity of functionally distinct cell types that make up the immune response. The aim of this study was to determine whether differences in the cellular composition of the immune infiltrate in breast tumours influence survival and treatment response, and whether these effects differ by molecular subtype.
Methods and Findings
We applied an established computational approach (CIBERSORT) to bulk gene expression profiles of almost 11,000 tumours to infer the proportions of 22 subsets of immune cells. We investigated associations between each cell type and survival and response to chemotherapy, modelling cellular proportions as quartiles. We found that tumours with little or no immune infiltration were associated with different survival patterns according to oestrogen receptor (ER) status. In ER-negative disease, tumours lacking immune infiltration were associated with the poorest prognosis, whereas in ER-positive disease, they were associated with intermediate prognosis. Of the cell subsets investigated, T regulatory cells and M0 and M2 macrophages emerged as the most strongly associated with poor outcome, regardless of ER status. Among ER-negative tumours, CD8+ T cells (hazard ratio [HR] = 0.89, 95% CI 0.80–0.98; p = 0.02) and activated memory T cells (HR 0.88, 95% CI 0.80–0.97; p = 0.01) were associated with favourable outcome. T follicular helper cells (odds ratio [OR] = 1.34, 95% CI 1.14–1.57; p < 0.001) and memory B cells (OR = 1.18, 95% CI 1.0–1.39; p = 0.04) were associated with pathological complete response to neoadjuvant chemotherapy in ER-negative disease, suggesting a role for humoral immunity in mediating response to cytotoxic therapy. Unsupervised clustering analysis using immune cell proportions revealed eight subgroups of tumours, largely defined by the balance between M0, M1, and M2 macrophages, with distinct survival patterns by ER status and associations with patient age at diagnosis. The main limitations of this study are the use of diverse platforms for measuring gene expression, including some not previously used with CIBERSORT, and the combined analysis of different forms of follow-up across studies.
Conclusions
Large differences in the cellular composition of the immune infiltrate in breast tumours appear to exist, and these differences are likely to be important determinants of both prognosis and response to treatment. In particular, macrophages emerge as a possible target for novel therapies. Detailed analysis of the cellular immune response in tumours has the potential to enhance clinical prediction and to identify candidates for immunotherapy.
To investigate tumor infiltration by different types of immune cells, H. Raza Ali and colleagues study gene expression profiles from large breast cancer datasets.
Author Summary
Why Was This Study Done?
Previous studies have shown that certain immune cells present in breast tumours are associated with risk of relapse.
Whether particular immune cell types are associated with a greater or lesser risk of relapse, however, and how these effects differ by breast cancer subtype, remains unclear.
What Did the Researchers Do and Find?
We conducted a large analysis of breast tumour gene expression profiles available in the public domain (10,988 cases) to derive estimates of the relative proportions of 22 subsets of immune cells, in order to investigate associations between the proportion of each cell type and disease relapse or response to chemotherapy.
We found that higher proportions of some immune cell types were associated with greater risk of relapse (or greater chemotherapy response), whereas others were associated with lesser risk, and that these associations were often different according to the oestrogen receptor (ER) status of the tumour.
In tumours lacking expression of ER, we found that the presence of CD8+ T cells and activated memory T cells was associated with a reduction in the risk of relapse, while tumours with high proportions of T follicular helper cells were more likely to respond to neoadjuvant chemotherapy.
In ER-positive tumours, the presence of M0 macrophages was associated with poor prognosis.
T regulatory cells were associated with poor prognosis in both ER-positive and ER-negative tumours.
What Do These Findings Mean?
These findings establish a complex relationship between the heterogeneity of intratumoural immune cells, tumour molecular subtype, and disease progression in breast cancer.
Treatments that aim to boost the immune response to tumours, i.e., immunotherapies, are effective in only a subset of patients, and our findings may help to identify this patient group and suggest targets for the development of new immunotherapies.
doi:10.1371/journal.pmed.1002194
PMCID: PMC5154505  PMID: 27959923
3.  Immunotherapy in the Precision Medicine Era: Melanoma and Beyond 
PLoS Medicine  2016;13(12):e1002196.
In a Perspective, Mack Su and David Fisher discuss the development of immunotherapies for treatment of melanoma and other cancer types.
doi:10.1371/journal.pmed.1002196
PMCID: PMC5154509  PMID: 27959922
4.  Predictors of Chemosensitivity in Triple Negative Breast Cancer: An Integrated Genomic Analysis 
PLoS Medicine  2016;13(12):e1002193.
Background
Triple negative breast cancer (TNBC) is a highly heterogeneous and aggressive disease, and although no effective targeted therapies are available to date, about one-third of patients with TNBC achieve pathologic complete response (pCR) from standard-of-care anthracycline/taxane (ACT) chemotherapy. The heterogeneity of these tumors, however, has hindered the discovery of effective biomarkers to identify such patients.
Methods and Findings
We performed whole exome sequencing on 29 TNBC cases from the MD Anderson Cancer Center (MDACC) selected because they had either pCR (n = 18) or extensive residual disease (n = 11) after neoadjuvant chemotherapy, with cases from The Cancer Genome Atlas (TCGA; n = 144) and METABRIC (n = 278) cohorts serving as validation cohorts. Our analysis revealed that mutations in the AR- and FOXA1-regulated networks, in which BRCA1 plays a key role, are associated with significantly higher sensitivity to ACT chemotherapy in the MDACC cohort (pCR rate of 94.1% compared to 16.6% in tumors without mutations in AR/FOXA1 pathway, adjusted p = 0.02) and significantly better survival outcome in the TCGA TNBC cohort (log-rank test, p = 0.05). Combined analysis of DNA sequencing, DNA methylation, and RNA sequencing identified tumors of a distinct BRCA-deficient (BRCA-D) TNBC subtype characterized by low levels of wild-type BRCA1/2 expression. Patients with functionally BRCA-D tumors had significantly better survival with standard-of-care chemotherapy than patients whose tumors were not BRCA-D (log-rank test, p = 0.021), and they had significantly higher mutation burden (p < 0.001) and presented clonal neoantigens that were associated with increased immune cell activity. A transcriptional signature of BRCA-D TNBC tumors was independently validated to be significantly associated with improved survival in the METABRIC dataset (log-rank test, p = 0.009). As a retrospective study, limitations include the small size and potential selection bias in the discovery cohort.
Conclusions
The comprehensive molecular analysis presented in this study directly links BRCA deficiency with increased clonal mutation burden and significantly enhanced chemosensitivity in TNBC and suggests that functional RNA-based BRCA deficiency needs to be further examined in TNBC.
Through molecular analyses of tissue from triple negative breast cancer patients, Christos Hatzis and colleagues report on a new tumor subtype with possible implications for treatment.
Author Summary
Why Was This Study Done?
Identifying chemosensitive triple negative breast cancers (TNBCs) could significantly impact the survival of patients with these difficult to treat cancers until novel targeted therapies become available.
We hypothesized that genomic somatic aberrations may provide important molecular clues about chemosensitivity in TNBC.
Our study used a carefully selected cohort of 29 uniformly treated TNBC patients who either achieved pathologic complete response (pCR) or had extensive residual disease after neoadjuvant anthracycline/taxane chemotherapy.
What Did the Researchers Do and Find?
We sequenced the coding genomic DNA of TNBC tumors and compared the somatic mutations found in the two groups at the two extremes of the chemosensitivity spectrum.
Our analysis revealed that, although mutations in single genes were not individually predictive, TNBC tumors bearing mutations in genes involved in the androgen receptor (AR) and FOXA1 pathways were much more sensitive to chemotherapy.
We also found that mutations that lowered the levels of functional BRCA1 or BRCA2 RNA were associated with significantly better survival outcomes; we derived a BRCA deficiency signature to define this new, highly chemosensitive subtype of TNBC.
BRCA-deficient TNBC tumors have a higher rate of clonal mutation burden, defined as more clonal tumors with a higher number of mutations per clone, and are also associated with a higher level of immune activation, which may explain their greater chemosensitivity.
What Do These Findings Mean?
Mutations in the AR/FOXA1 pathway provide a novel marker for identifying chemosensitive TNBC patients who may benefit from current standard-of-care chemotherapy regimens.
The newly defined RNA-based BRCA-deficient subtype includes up to 50% of the TNBC tumors that appear to be immune primed, and it would be of interest to investigate combinations of chemotherapy with immunotherapies, which could provide clinical benefit for these patients.
Although our study showed concordant results in three different datasets, our key findings need to be further validated in a larger, prospectively designed study with archival samples.
doi:10.1371/journal.pmed.1002193
PMCID: PMC5154510  PMID: 27959926
5.  Sequencing Strategies to Guide Decision Making in Cancer Treatment 
PLoS Medicine  2016;13(12):e1002189.
In a Perspective, James Topham and Marco Marra discuss progress in the use of genomic information to guide cancer treatment.
doi:10.1371/journal.pmed.1002189
PMCID: PMC5140043  PMID: 27923042
6.  Precision Cancer Diagnostics: Tracking Genomic Evolution in Clinical Trials 
PLoS Medicine  2016;13(12):e1002177.
In a Perspective, Francisco Beca and Andrew Beck discuss Charles Swanton and colleagues' accompanying Research Article on somatic mutations in patients with inflammatory breast cancer treated in a Phase II clinical trial.
doi:10.1371/journal.pmed.1002177
PMCID: PMC5140044  PMID: 27923047
7.  Somatic Genomics and Clinical Features of Lung Adenocarcinoma: A Retrospective Study 
PLoS Medicine  2016;13(12):e1002162.
Background
Lung adenocarcinoma (LUAD) is the most common histologic subtype of lung cancer and has a high risk of distant metastasis at every disease stage. We aimed to characterize the genomic landscape of LUAD and identify mutation signatures associated with tumor progression.
Methods and Findings
We performed an integrative genomic analysis, incorporating whole exome sequencing (WES), determination of DNA copy number and DNA methylation, and transcriptome sequencing for 101 LUAD samples from the Environment And Genetics in Lung cancer Etiology (EAGLE) study. We detected driver genes by testing whether the nonsynonymous mutation rate was significantly higher than the background mutation rate and replicated our findings in public datasets with 724 samples. We performed subclonality analysis for mutations based on mutant allele data and copy number alteration data. We also tested the association between mutation signatures and clinical outcomes, including distant metastasis, survival, and tumor grade. We identified and replicated two novel candidate driver genes, POU class 4 homeobox 2 (POU4F2) (mutated in 9 [8.9%] samples) and ZKSCAN1 (mutated in 6 [5.9%] samples), and characterized their major deleterious mutations. ZKSCAN1 was part of a mutually exclusive gene set that included the RTK/RAS/RAF pathway genes BRAF, EGFR, KRAS, MET, and NF1, indicating an important driver role for this gene. Moreover, we observed strong associations between methylation in specific genomic regions and somatic mutation patterns. In the tumor evolution analysis, four driver genes had a significantly lower fraction of subclonal mutations (FSM), including TP53 (p = 0.007), KEAP1 (p = 0.012), STK11 (p = 0.0076), and EGFR (p = 0.0078), suggesting a tumor initiation role for these genes. Subclonal mutations were significantly enriched in APOBEC-related signatures (p < 2.5×10−50). The total number of somatic mutations (p = 0.0039) and the fraction of transitions (p = 5.5×10−4) were associated with increased risk of distant metastasis. Our study’s limitations include a small number of LUAD patients for subgroup analyses and a single-sample design for investigation of subclonality.
Conclusions
These data provide a genomic characterization of LUAD pathogenesis and progression. The distinct clonal and subclonal mutation signatures suggest possible diverse carcinogenesis pathways for endogenous and exogenous exposures, and may serve as a foundation for more effective treatments for this lethal disease. LUAD’s high heterogeneity emphasizes the need to further study this tumor type and to associate genomic findings with clinical outcomes.
Maria Teresa Landi and colleagues report genomic tumor data for a cohort of patients with lung adenocarcinoma, focusing on implications for tumor initiation and distant metastasis.
Author Summary
Why Was This Study Done?
Lung adenocarcinoma (LUAD) is the most common histologic subtype of lung cancer and causes more than half a million deaths worldwide annually.
Genomic studies of LUAD can shed light on tumor initiation and progression and identify potential targets for treatment.
What Did the Researchers Do and Find?
We performed an integrative genomic analysis, incorporating whole exome sequencing (WES), DNA copy number and DNA methylation determination, and transcriptome sequencing in 101 LUAD samples. We replicated major findings using public genomic resources and combined all existing genomic data for an overall analysis of 825 LUAD samples.
We identified two novel driver genes and characterized the driver events and types of mutations that have a stronger role in tumor initiation versus tumor progression.
We found strong associations between DNA methylation and somatic mutation patterns.
The total number of somatic mutations and the fraction of C→T transitions were associated with increased risk of distant metastasis.
What Do These Findings Mean?
We characterized LUAD genomic architecture and linked major genomic features with clinical outcomes.
Tobacco smoking-related mutations appear to have a stronger role in tumor initiation, while mutations associated with endogenous processes are more prominent at a later stage of tumor development and are associated with tumor progression.
Our findings highlight the complexity and heterogeneity of LUAD. In addition to new driver genes, we found some tumors with no exonic mutations in known lung cancer driver genes. This suggests that there are further drivers (genetic or epigenetic) to be identified, and larger numbers of samples need to be studied to fully capture LUAD genomic characteristics.
doi:10.1371/journal.pmed.1002162
PMCID: PMC5140047  PMID: 27923066
8.  Expression of lncRNAs in Low-Grade Gliomas and Glioblastoma Multiforme: An In Silico Analysis 
PLoS Medicine  2016;13(12):e1002192.
Background
Each year, over 16,000 patients die from malignant brain cancer in the US. Long noncoding RNAs (lncRNAs) have recently been shown to play critical roles in regulating neurogenesis and brain tumor progression. To better understand the role of lncRNAs in brain cancer, we performed a global analysis to identify and characterize all annotated and novel lncRNAs in both grade II and III gliomas as well as grade IV glioblastomas (glioblastoma multiforme [GBM]).
Methods and Findings
We determined the expression of all lncRNAs in over 650 brain cancer and 70 normal brain tissue RNA sequencing datasets from The Cancer Genome Atlas (TCGA) and other publicly available datasets. We identified 611 induced and 677 repressed lncRNAs in glial tumors relative to normal brains. Hundreds of lncRNAs were specifically expressed in each of the three lower grade glioma (LGG) subtypes (IDH1/2 wt, IDH1/2 mut, and IDH1/2 mut 1p19q codeletion) and the four subtypes of GBMs (classical, mesenchymal, neural, and proneural). Overlap between the subtype-specific lncRNAs in GBMs and LGGs demonstrated similarities between mesenchymal GBMs and IDH1/2 wt LGGs, with 2-fold higher overlap than would be expected by random chance. Using a multivariate Cox regression survival model, we identified 584 and 282 lncRNAs that were associated with a poor and good prognosis, respectively, in GBM patients. We developed a survival algorithm for LGGs based on the expression of 64 lncRNAs that was associated with patient prognosis in a test set (hazard ratio [HR] = 2.168, 95% CI = 1.765–2.807, p < 0.001) and validation set (HR = 1.921, 95% CI = 1.333–2.767, p < 0.001) of patients from TCGA. The main limitations of this study are that further work is needed to investigate the clinical relevance of our findings, and that validation in an independent dataset is needed to determine the robustness of our survival algorithm.
Conclusions
This work identifies a panel of lncRNAs that appear to be prognostic in gliomas and provides a critical resource for future studies examining the role of lncRNAs in brain cancers.
In an in silico study, Anindya Dutta and colleagues study the expression of long noncoding RNAs in brain tumors and normal brain tissue and the possible implications for disease prognosis.
Author Summary
Why Was This Study Done?
Long noncoding RNAs (lncRNAs) have recently been shown to play a crucial role in normal physiology as well as various disease states; however, the role of lncRNAs in gliomas has not been well characterized.
This study was undertaken to determine to what extent lncRNAs are dysregulated in glial tumors, whether lncRNA expression can be used to assess patient prognosis, and to determine which of the thousands of newly discovered lncRNAs should be prioritized for mechanistic studies.
What Did the Researchers Do and Find?
We analyzed over 700 publicly available glioma, glioblastoma, and normal brain RNA sequencing datasets and identified hundreds of lncRNAs with altered expression in gliomas or glioblastomas relative to normal brain tissue.
The expression of many lncRNAs was found to be associated with a tumor’s mutational status as well as its molecular subtype.
Using lncRNA expression and Cox regression modeling, we developed a survival algorithm that was able to separate glioma patients into two distinct prognostic groups. Several lncRNAs were identified that also predicted different outcomes in glioblastomas.
What Do These Findings Mean?
Our analysis provides an important resource for studying lncRNAs in glial tumors by helping prioritize which lncRNAs to investigate for disease relevance.
An lncRNA panel could potentially be used in the future to help distinguish glioma patients with good versus poor prognosis.
doi:10.1371/journal.pmed.1002192
PMCID: PMC5140055  PMID: 27923049
9.  Clonal Evolutionary Analysis during HER2 Blockade in HER2-Positive Inflammatory Breast Cancer: A Phase II Open-Label Clinical Trial of Afatinib +/- Vinorelbine 
PLoS Medicine  2016;13(12):e1002136.
Background
Inflammatory breast cancer (IBC) is a rare, aggressive form of breast cancer associated with HER2 amplification, with high risk of metastasis and an estimated median survival of 2.9 y. We performed an open-label, single-arm phase II clinical trial (ClinicalTrials.gov NCT01325428) to investigate the efficacy and safety of afatinib, an irreversible ErbB family inhibitor, alone and in combination with vinorelbine in patients with HER2-positive IBC. This trial included prospectively planned exome analysis before and after afatinib monotherapy.
Methods and Findings
HER2-positive IBC patients received afatinib 40 mg daily until progression, and thereafter afatinib 40 mg daily and intravenous vinorelbine 25 mg/m2 weekly. The primary endpoint was clinical benefit; secondary endpoints were objective response (OR), duration of OR, and progression-free survival (PFS). Of 26 patients treated with afatinib monotherapy, clinical benefit was achieved in 9 patients (35%), 0 of 7 trastuzumab-treated patients and 9 of 19 trastuzumab-naïve patients. Following disease progression, 10 patients received afatinib plus vinorelbine, and clinical benefit was achieved in 2 of 4 trastuzumab-treated and 0 of 6 trastuzumab-naïve patients. All patients had treatment-related adverse events (AEs). Whole-exome sequencing of tumour biopsies taken before treatment and following disease progression on afatinib monotherapy was performed to assess the mutational landscape of IBC and evolutionary trajectories during therapy. Compared to a cohort of The Cancer Genome Atlas (TCGA) patients with HER2-positive non-IBC, HER2-positive IBC patients had significantly higher mutational and neoantigenic burden, more frequent gain-of-function TP53 mutations and a recurrent 11q13.5 amplification overlapping PAK1. Planned exploratory analysis revealed that trastuzumab-naïve patients with tumours harbouring somatic activation of PI3K/Akt signalling had significantly shorter PFS compared to those without (p = 0.03). High genomic concordance between biopsies taken before and following afatinib resistance was observed with stable clonal structures in non-responding tumours, and evidence of branched evolution in 8 of 9 tumours analysed. Recruitment to the trial was terminated early following the LUX-Breast 1 trial, which showed that afatinib combined with vinorelbine had similar PFS and OR rates to trastuzumab plus vinorelbine but shorter overall survival (OS), and was less tolerable. The main limitations of this study are that the results should be interpreted with caution given the relatively small patient cohort and the potential for tumour sampling bias between pre- and post-treatment tumour biopsies.
Conclusions
Afatinib, with or without vinorelbine, showed activity in trastuzumab-naïve HER2-positive IBC patients in a planned subgroup analysis. HER2-positive IBC is characterized by frequent TP53 gain-of-function mutations and a high mutational burden. The high mutational load associated with HER2-positive IBC suggests a potential role for checkpoint inhibitor therapy in this disease.
Trial Registration
ClinicalTrials.gov NCT01325428
In a phase 2 clinical trial in which patients received afatinib, plus vinorelbine on disease progression, Charles Swanton and colleagues study the mutational events in HER2-positive inflammatory breast cancers alongside early-stage clinical outcomes.
Author Summary
Why Was This Study Done?
Inflammatory breast cancer (IBC) is a rare and poorly understood form of breast cancer that grows and spreads very quickly. Fifty percent of IBC cases are HER2-positive.
Afatinib is an investigational drug that showed promise in early-stage trials in the setting of HER2-positive metastatic breast cancer.
Our study was designed to look at how effective and safe afatinib is in treating HER2-positive IBC patients, and to elucidate how afatinib treatment affects the tumours at the genomic level.
What Did the Researchers Do and Find?
We recruited 26 patients for this study and administered afatinib daily, and 10 patients went on to be treated with daily afatinib and weekly vinorelbine, a chemotherapy drug, upon disease progression.
Thirty-five percent (9 of 26) and 20% (2 of 10) of patients had clinical benefit from being treated with afatinib monotherapy and afatinib plus vinorelbine, respectively.
We sequenced tumour biopsies before and after afatinib treatment and found that IBC has a higher mutational load and more frequent mutations in the well-known cancer gene TP53, compared to non-IBC.
We did not identify any single gene or mutation that led to afatinib resistance, and biopsies before and after treatment were very similar genetically.
What Do These Findings Mean?
Afatinib appears to be clinically active in HER2-positive IBC, albeit in a relatively small patient cohort.
The high mutational load in IBC suggests that checkpoint inhibitors, a type of cancer immunotherapy, might potentially be an effective way of treating patients.
doi:10.1371/journal.pmed.1002136
PMCID: PMC5140058  PMID: 27923043
10.  Towards Equity in Health: Researchers Take Stock 
PLoS Medicine  2016;13(11):e1002186.
For the 2016 end-of-the-year editorial, the PLOS Medicine editors asked 7 global health leaders to discuss developments relevant to the equitable provision of medical care to all populations. The result is a collection of expert views on ethical trial design, research during outbreaks, high-burden infectious diseases, diversity in research and protection of migrants.
doi:10.1371/journal.pmed.1002186
PMCID: PMC5127492  PMID: 27898673
11.  The Dengue Vaccine Dilemma: Balancing the Individual and Population Risks and Benefits 
PLoS Medicine  2016;13(11):e1002182.
In a Perspective, Jacqueline Deeen discusses challenges in balancing the individual and population risks and benefits for CYD-TDV (Dengvaxia), the first available dengue vaccine.
doi:10.1371/journal.pmed.1002182
PMCID: PMC5127493  PMID: 27898675
12.  Lifestyle Advice Combined with Personalized Estimates of Genetic or Phenotypic Risk of Type 2 Diabetes, and Objectively Measured Physical Activity: A Randomized Controlled Trial 
PLoS Medicine  2016;13(11):e1002185.
Background
Information about genetic and phenotypic risk of type 2 diabetes is now widely available and is being incorporated into disease prevention programs. Whether such information motivates behavior change or has adverse effects is uncertain. We examined the effect of communicating an estimate of genetic or phenotypic risk of type 2 diabetes in a parallel group, open, randomized controlled trial.
Methods and Findings
We recruited 569 healthy middle-aged adults from the Fenland Study, an ongoing population-based, observational study in the east of England (Cambridgeshire, UK). We used a computer-generated random list to assign participants in blocks of six to receive either standard lifestyle advice alone (control group, n = 190) or in combination with a genetic (n = 189) or a phenotypic (n = 190) risk estimate for type 2 diabetes (intervention groups). After 8 wk, we measured the primary outcome, objectively measured physical activity (kJ/kg/day), and also measured several secondary outcomes (including self-reported diet, self-reported weight, worry, anxiety, and perceived risk). The study was powered to detect a between-group difference of 4.1 kJ/kg/d at follow-up. 557 (98%) participants completed the trial. There were no significant intervention effects on physical activity (difference in adjusted mean change from baseline: genetic risk group versus control group 0.85 kJ/kg/d (95% CI −2.07 to 3.77, p = 0.57); phenotypic risk group versus control group 1.32 (95% CI −1.61 to 4.25, p = 0.38); and genetic risk group versus phenotypic risk group −0.47 (95% CI −3.40 to 2.46, p = 0.75). No significant differences in self-reported diet, self-reported weight, worry, and anxiety were observed between trial groups. Estimates of perceived risk were significantly more accurate among those who received risk information than among those who did not. Key limitations include the recruitment of a sample that may not be representative of the UK population, use of self-reported secondary outcome measures, and a short follow-up period.
Conclusions
In this study, we did not observe short-term changes in behavior associated with the communication of an estimate of genetic or phenotypic risk of type 2 diabetes. We also did not observe changes in worry or anxiety in the study population. Additional research is needed to investigate the conditions under which risk information might enhance preventive strategies. (Current Controlled Trials ISRCTN09650496; Date applied: April 4, 2011; Date assigned: June 10, 2011).
Trial Registration
The trial is registered with Current Controlled Trials, ISRCTN09650496.
In a randomized controlled trial, Job Godino and colleagues study the effects of providing personalized information about genetic and phenotypic risk of type 2 diabetes as compared with standard lifestyle advice.
Author Summary
Why Was This Study Done?
Despite questions regarding their clinical validity and utility, genetic tests aimed at predicting risk of type 2 diabetes are now widely available.
Some researchers and direct-to-consumer genetic testing companies are optimistic that personalized genetic information will encourage people at risk to adopt healthier behavior than standard advice about lifestyle, but scientific evidence is needed.
What Did the Researchers Do and Find?
The researchers calculated the risk of developing type 2 diabetes for 569 healthy middle-aged adults.
The risk estimates were either based on a participant’s genetic makeup, as judged by presence of genetic information known to be associated with type 2 diabetes, or on phenotypic characteristics, such as sex, age, body mass index, etc.
Participants were randomly assigned to three groups to receive standard lifestyle advice, alone or in combination with their genetic risk estimate or phenotypic risk estimate. After 8 wk, participants’ physical activity, self-reported diet and weight, anxiety, worry, and beliefs about their risk were measured.
The researchers found that receipt of a genetic or phenotypic risk estimate did not affect participants’ physical activity or other relevant behaviors, in comparison with those receiving standard lifestyle advice. However, perception of risk became more accurate.
What Do These Findings Mean?
In this study, provision of personalized information about genetic or phenotypic risk of type 2 diabetes did not affect behavior when compared with standard lifestyle advice.
Provision of personalized information about risk of type 2 diabetes did not seem to cause anxiety.
More research is needed to understand the circumstances in which genetic risk information might motivate behavior change.
doi:10.1371/journal.pmed.1002185
PMCID: PMC5127499  PMID: 27898672
13.  A Core Outcome Set for the Benefits and Adverse Events of Bariatric and Metabolic Surgery: The BARIACT Project 
PLoS Medicine  2016;13(11):e1002187.
Background
Bariatric and metabolic surgery is used as a treatment for patients with severe and complex obesity. However, there is a need to improve outcome selection and reporting in bariatric surgery trials. A Core Outcome Set (COS), an agreed minimum set of outcomes reported in all studies of a specific condition, may achieve this. Here, we present the development of a COS for BARIAtric and metabolic surgery Clinical Trials—the BARIACT Study.
Methods and Findings
Outcomes identified from systematic reviews and patient interviews informed a questionnaire survey. Patients and health professionals were surveyed three times and asked to rate the importance of each item on a 1–9 scale. Delphi methods provided anonymised feedback to participants. Items not meeting predefined criteria were discarded between rounds. Remaining items were discussed at consensus meetings, held separately with patients and professionals, where the COS was agreed. Data sources identified 2,990 outcomes, which were used to develop a 130-item questionnaire. Round 1 response rates were moderate but subsequently improved to above 75% for other rounds. After rounds 2 and 3, 81 and 14 items were discarded, respectively, leaving 35 items for discussion at consensus meetings. The final COS included nine items: “weight,” “diabetes status,” “cardiovascular risk,” “overall quality of life (QOL),” “mortality,” “technical complications of the specific operation,” “any re-operation/re-intervention,” “dysphagia/regurgitation,” and “micronutrient status.” The main limitation of this study was that it was based in the United Kingdom only.
Conclusions
The COS is recommended to be used as a minimum in all trials of bariatric and metabolic surgery. Adoption of the COS will improve data synthesis and the value of research data. Future work will establish methods for the measurement of the outcomes in the COS.
Karen Coulman and colleagues present a core outcome set developed by patients and health professionals for BARIAtric and metabolic surgery Clinical Trials (BARIACT).
doi:10.1371/journal.pmed.1002187
PMCID: PMC5127500  PMID: 27898680
14.  Patient-Reported Barriers to Adherence to Antiretroviral Therapy: A Systematic Review and Meta-Analysis 
PLoS Medicine  2016;13(11):e1002183.
Background
Maintaining high levels of adherence to antiretroviral therapy (ART) is a challenge across settings and populations. Understanding the relative importance of different barriers to adherence will help inform the targeting of different interventions and future research priorities.
Methods and Findings
We searched MEDLINE via PubMed, Embase, Web of Science, and PsychINFO from 01 January 1997 to 31 March 2016 for studies reporting barriers to adherence to ART. We calculated pooled proportions of reported barriers to adherence per age group (adults, adolescents, and children). We included data from 125 studies that provided information about adherence barriers for 17,061 adults, 1,099 children, and 856 adolescents. We assessed differences according to geographical location and level of economic development. The most frequently reported individual barriers included forgetting (adults 41.4%, 95% CI 37.3%–45.4%; adolescents 63.1%, 95% CI 46.3%–80.0%; children/caregivers 29.2%, 95% CI 20.1%–38.4%), being away from home (adults 30.4%, 95% CI 25.5%–35.2%; adolescents 40.7%, 95% CI 25.7%–55.6%; children/caregivers 18.5%, 95% CI 10.3%–26.8%), and a change to daily routine (adults 28.0%, 95% CI 20.9%–35.0%; adolescents 32.4%, 95% CI 0%–75.0%; children/caregivers 26.3%, 95% CI 15.3%–37.4%). Depression was reported as a barrier to adherence by more than 15% of patients across all age categories (adults 15.5%, 95% CI 12.8%–18.3%; adolescents 25.7%, 95% CI 17.7%–33.6%; children 15.1%, 95% CI 3.9%–26.3%), while alcohol/substance misuse was commonly reported by adults (12.9%, 95% CI 9.7%–16.1%) and adolescents (28.8%, 95% CI 11.8%–45.8%). Secrecy/stigma was a commonly cited barrier to adherence, reported by more than 10% of adults and children across all regions (adults 13.6%, 95% CI 11.9%–15.3%; children/caregivers 22.3%, 95% CI 10.2%–34.5%). Among adults, feeling sick (15.9%, 95% CI 13.0%–18.8%) was a more commonly cited barrier to adherence than feeling well (9.3%, 95% CI 7.2%–11.4%). Health service–related barriers, including distance to clinic (adults 17.5%, 95% CI 13.0%–21.9%) and stock outs (adults 16.1%, 95% CI 11.7%–20.4%), were also frequently reported. Limitations of this review relate to the fact that included studies differed in approaches to assessing adherence barriers and included variable durations of follow up. Studies that report self-reported adherence will likely underestimate the frequency of non-adherence. For children, barriers were mainly reported by caregivers, which may not correspond to the most important barriers faced by children.
Conclusions
Patients on ART face multiple barriers to adherence, and no single intervention will be sufficient to ensure that high levels of adherence to treatment and virological suppression are sustained. For maximum efficacy, health providers should consider a more triaged approach that first identifies patients at risk of poor adherence and then seeks to establish the support that is needed to overcome the most important barriers to adherence.
In this systematic review and meta-analysis, Nathan Ford and colleagues assess the most frequently reported barriers by patients experiencing challenges adhering to antiretroviral therapy.
Author Summary
Why Was This Study Done?
Despite more than two decades of research on adherence to antiretroviral therapy (ART) and more than 17 million HIV-positive individuals on treatment, adherence to ART remains a major challenge.
This review aimed to assess the most frequently reported barriers to adherence by patients experiencing adherence challenges.
What Did the Researchers Do and Find?
Published data from 125 studies on patient-reported barriers to adherence were systematically reviewed and analyzed by age group.
The most frequently reported individual barriers across all age groups included forgetting, being away from home, and a change to daily routine. Depression was reported as a barrier to adherence by more than 15% of patients across all age categories, while alcohol/substance misuse was commonly reported as a barrier by adults and adolescents.
With respect to contextual barriers, secrecy/stigma was a commonly cited barrier to adherence, reported by more than 10% of patients across all regions.
Health service–related barriers were frequently reported, including distance to clinic and stock outs.
What Do These Findings Mean?
Evidence-based interventions exist that may address many of the most common patient-reported barriers to adherence.
However, no single intervention will be sufficient to ensure that high levels of adherence to treatment and virological suppression are sustained.
Health providers should consider a more triaged approach that first identifies patients at risk of poor adherence and then seeks to establish the support that is needed to overcome the most important barriers to adherence.
Several key health service improvements are also required to ensure that patients are able to consistently access ART.
doi:10.1371/journal.pmed.1002183
PMCID: PMC5127502  PMID: 27898679
15.  Leukocyte Telomere Length in Relation to 17 Biomarkers of Cardiovascular Disease Risk: A Cross-Sectional Study of US Adults 
PLoS Medicine  2016;13(11):e1002188.
Background
Leukocyte telomere length (LTL) is a putative biological marker of immune system age, and there are demonstrated associations between LTL and cardiovascular disease. This may be due in part to the relationship of LTL with other biomarkers associated with cardiovascular disease risk. However, the strength of associations between LTL and adiposity, metabolic, proinflammatory, and cardiovascular biomarkers has not been systematically evaluated in a United States nationally representative population.
Methods and Findings
We examined associations between LTL and 17 cardiovascular biomarkers, including lipoproteins, blood sugar, circulatory pressure, proinflammatory markers, kidney function, and adiposity measures, in adults ages 20 to 84 from the cross-sectional US nationally representative 1999–2002 National Health and Nutrition Examination Survey (NHANES) (n = 7,252), statistically adjusting for immune cell type distributions. We also examine whether these associations differed systematically by age, race/ethnicity, gender, education, and income. We found that a one unit difference in the following biomarkers were associated with kilobase pair differences in LTL: BMI -0.00478 (95% CI -0.00749–-0.00206), waist circumference -0.00211 (95% CI -0.00325–-0.000969), percentage of body fat -0.00516 (95% CI -0.00761–-0.0027), high density lipoprotein (HDL) cholesterol 0.00179 (95% CI 0.000571–0.00301), triglycerides -0.000285 (95% CI -0.000555–-0.0000158), pulse rate -0.00194 (95% CI -0.00317–-0.000705), C-reactive protein -0.0363 (95% CI 0.0601–-0.0124), cystatin C -0.0391 (95% CI -0.0772–-0.00107). When using clinical cut-points we additionally found associations between LTL and insulin resistance -0.0412 (95% CI -0.0685–-0.0139), systolic blood pressure 0.0455 (95% CI 0.00137–0.0897), and diastolic blood pressure -0.0674 (95% CI -0.126–-0.00889). These associations were 10%–15% greater without controlling for leukocyte cell types. There were very few differences in the associations by age, race/ethnicity, gender, education, or income. Our findings are relevant to the relationships between these cardiovascular biomarkers in the general population but not to cardiovascular disease as a clinical outcome.
Conclusions
LTL is most strongly associated with adiposity, but is also associated with biomarkers across several physiological systems. LTL may thus be a predictor of cardiovascular disease through its association with multiple risk factors that are physiologically correlated with risk for development of cardiovascular disease. Our results are consistent with LTL being a biomarker of cardiovascular aging through established physiological mechanisms.
David Rehkopf and colleagues analyze the association of leukocyte telomere length with cardiovascular disease risk biomarkers.
Author Summary
Why Was This Study Done?
Leukocyte telomere length (LTL) is a biomarker of white blood cell division and is strongly associated with age.
There are still mixed findings on how LTL is related to different types of mortality.
Observational evidence and quasi-experimental studies suggest that there is an association between shorter LTL and cardiovascular disease mortality.
It is unclear, however, whether or not LTL is completely independent of already known biological pathways of cardiovascular disease.
What Did the Researchers Do and Find?
We examined US nationally representative data on participants ages 25 to 84 that had LTL measured along with 17 other cardiovascular risk biomarkers.
We found associations between LTL and measures of adiposity (BMI, waist circumference, percentage of body fat), along with C-reactive protein, cystatin C, high-density lipoprotein (HDL) cholesterol, triglycerides, insulin resistance, systolic blood pressure, diastolic blood pressure, and pulse rate.
We did not find that there were a meaningful number of differences in these relationships by age, race/ethnicity, or socioeconomic position, suggesting that the associations we find are generally consistent across different population groups.
What Do These Findings Mean?
Our findings suggest that LTL is associated with cardiovascular risk factors across different physiological systems.
The study findings do not have any implications for whether LTL is a cause of cardiovascular disease.
doi:10.1371/journal.pmed.1002188
PMCID: PMC5127504  PMID: 27898678
16.  Will 10 Million People Die a Year due to Antimicrobial Resistance by 2050? 
PLoS Medicine  2016;13(11):e1002184.
Marlieke de Kraker and colleagues reflect on the need for better global estimates for the burden of antimicrobial resistance.
doi:10.1371/journal.pmed.1002184
PMCID: PMC5127510  PMID: 27898664
17.  Genetic Predisposition to an Impaired Metabolism of the Branched-Chain Amino Acids and Risk of Type 2 Diabetes: A Mendelian Randomisation Analysis 
PLoS Medicine  2016;13(11):e1002179.
Background
Higher circulating levels of the branched-chain amino acids (BCAAs; i.e., isoleucine, leucine, and valine) are strongly associated with higher type 2 diabetes risk, but it is not known whether this association is causal. We undertook large-scale human genetic analyses to address this question.
Methods and Findings
Genome-wide studies of BCAA levels in 16,596 individuals revealed five genomic regions associated at genome-wide levels of significance (p < 5 × 10−8). The strongest signal was 21 kb upstream of the PPM1K gene (beta in standard deviations [SDs] of leucine per allele = 0.08, p = 3.9 × 10−25), encoding an activator of the mitochondrial branched-chain alpha-ketoacid dehydrogenase (BCKD) responsible for the rate-limiting step in BCAA catabolism. In another analysis, in up to 47,877 cases of type 2 diabetes and 267,694 controls, a genetically predicted difference of 1 SD in amino acid level was associated with an odds ratio for type 2 diabetes of 1.44 (95% CI 1.26–1.65, p = 9.5 × 10−8) for isoleucine, 1.85 (95% CI 1.41–2.42, p = 7.3 × 10−6) for leucine, and 1.54 (95% CI 1.28–1.84, p = 4.2 × 10−6) for valine. Estimates were highly consistent with those from prospective observational studies of the association between BCAA levels and incident type 2 diabetes in a meta-analysis of 1,992 cases and 4,319 non-cases. Metabolome-wide association analyses of BCAA-raising alleles revealed high specificity to the BCAA pathway and an accumulation of metabolites upstream of branched-chain alpha-ketoacid oxidation, consistent with reduced BCKD activity. Limitations of this study are that, while the association of genetic variants appeared highly specific, the possibility of pleiotropic associations cannot be entirely excluded. Similar to other complex phenotypes, genetic scores used in the study captured a limited proportion of the heritability in BCAA levels. Therefore, it is possible that only some of the mechanisms that increase BCAA levels or affect BCAA metabolism are implicated in type 2 diabetes.
Conclusions
Evidence from this large-scale human genetic and metabolomic study is consistent with a causal role of BCAA metabolism in the aetiology of type 2 diabetes.
Claudia Langenberg and colleagues show that high circulating branched chain amino acids associate with future risk of type 2 diabetes.
Author Summary
Why Was This Study Done?
Higher circulating levels of isoleucine, leucine, and valine, i.e., the branched-chain amino acids (BCAAs), are strongly associated with the risk of future type 2 diabetes.
It is not known if this association reflects a causal relationship.
It is important to assess the aetiologic nature of this relationship. If it is causal, then intervening on BCAA levels or metabolism may reduce the risk of diabetes.
What Did the Researchers Do and Find?
We used a human genetics framework known as “Mendelian randomisation” to study this question. Mendelian randomisation postulates that if a biomarker is causally implicated in a disease, then genetic variants specifically associated with that biomarker should also be associated with the disease.
In a meta-analysis of 1,992 incident cases of type 2 diabetes and 4,319 non-cases, we found strong associations between higher levels of each of the BCAAs and a higher risk of type 2 diabetes.
In a genome-wide meta-analysis of 16,596 individuals, we identified five genomic regions where common genetic variants were associated with BCAA levels.
In a meta-analysis of genetic association studies including 47,877 cases of type 2 diabetes and 267,694 controls, a genetically predicted difference of one standard deviation in amino acid level was associated with an odds ratio of type 2 diabetes of 1.44 (95% confidence interval 1.26–1.65) for isoleucine, 1.85 (1.41–2.42) for leucine, and 1.54 (1.28–1.84) for valine.
What Do These Findings Mean?
Evidence from this large-scale human genetic and metabolomic study is consistent with a causal role of BCAA metabolism in the aetiology of type 2 diabetes.
Possible limitations of the study included the possibility of non-specific associations of the genetic variants included in the study (i.e., “pleiotropy”) and the relatively low proportion of heritability in BCAA levels explained by the identified genetic variants.
doi:10.1371/journal.pmed.1002179
PMCID: PMC5127513  PMID: 27898682
18.  The Long-Term Safety, Public Health Impact, and Cost-Effectiveness of Routine Vaccination with a Recombinant, Live-Attenuated Dengue Vaccine (Dengvaxia): A Model Comparison Study 
PLoS Medicine  2016;13(11):e1002181.
Background
Large Phase III trials across Asia and Latin America have recently demonstrated the efficacy of a recombinant, live-attenuated dengue vaccine (Dengvaxia) over the first 25 mo following vaccination. Subsequent data collected in the longer-term follow-up phase, however, have raised concerns about a potential increase in hospitalization risk of subsequent dengue infections, in particular among young, dengue-naïve vaccinees. We here report predictions from eight independent modelling groups on the long-term safety, public health impact, and cost-effectiveness of routine vaccination with Dengvaxia in a range of transmission settings, as characterised by seroprevalence levels among 9-y-olds (SP9). These predictions were conducted for the World Health Organization to inform their recommendations on optimal use of this vaccine.
Methods and Findings
The models adopted, with small variations, a parsimonious vaccine mode of action that was able to reproduce quantitative features of the observed trial data. The adopted mode of action assumed that vaccination, similarly to natural infection, induces transient, heterologous protection and, further, establishes a long-lasting immunogenic memory, which determines disease severity of subsequent infections. The default vaccination policy considered was routine vaccination of 9-y-old children in a three-dose schedule at 80% coverage. The outcomes examined were the impact of vaccination on infections, symptomatic dengue, hospitalised dengue, deaths, and cost-effectiveness over a 30-y postvaccination period. Case definitions were chosen in accordance with the Phase III trials.
All models predicted that in settings with moderate to high dengue endemicity (SP9 ≥ 50%), the default vaccination policy would reduce the burden of dengue disease for the population by 6%–25% (all simulations: –3%–34%) and in high-transmission settings (SP9 ≥ 70%) by 13%–25% (all simulations: 10%– 34%). These endemicity levels are representative of the participating sites in both Phase III trials. In contrast, in settings with low transmission intensity (SP9 ≤ 30%), the models predicted that vaccination could lead to a substantial increase in hospitalisation because of dengue. Modelling reduced vaccine coverage or the addition of catch-up campaigns showed that the impact of vaccination scaled approximately linearly with the number of people vaccinated. In assessing the optimal age of vaccination, we found that targeting older children could increase the net benefit of vaccination in settings with moderate transmission intensity (SP9 = 50%). Overall, vaccination was predicted to be potentially cost-effective in most endemic settings if priced competitively.
The results are based on the assumption that the vaccine acts similarly to natural infection. This assumption is consistent with the available trial results but cannot be directly validated in the absence of additional data. Furthermore, uncertainties remain regarding the level of protection provided against disease versus infection and the rate at which vaccine-induced protection declines.
Conclusions
Dengvaxia has the potential to reduce the burden of dengue disease in areas of moderate to high dengue endemicity. However, the potential risks of vaccination in areas with limited exposure to dengue as well as the local costs and benefits of routine vaccination are important considerations for the inclusion of Dengvaxia into existing immunisation programmes. These results were important inputs into WHO global policy for use of this licensed dengue vaccine.
Mark Jit and colleagues report findings from eight independent modelling groups on the long-term safety, public health impact, and cost-effectiveness of routine vaccination with a recombinant, live-attenuated dengue vaccine (Dengvaxia).
Author Summary
Why Was This Study Done?
Dengvaxia, the first vaccine against all dengue serotypes, has recently been licensed in several countries.
The World Health Organization initiated this study to inform its official position on the use of Dengvaxia.
What Did the Researchers Do and Find?
We used dynamical models with differences in how dengue epidemiology is reflected but common assumptions on vaccine effects, informed by trial observations, to predict the impact and cost-effectiveness of Dengvaxia in a variety of situations.
The results showed that Dengvaxia has the potential to reduce the number of hospitalisations because of dengue by 13%–25% and be cost effective in settings where dengue is common.
However, in settings with low dengue prevalence, vaccination may increase the incidence of severe illness.
What Do These Findings Mean?
WHO has now recommended countries consider the use of Dengvaxia in settings with high dengue endemicity.
Our results can guide countries on the general suitability of Dengvaxia introduction; however, local demographics, heterogeneities in endemicity, and health system costs will need to be taken into account.
doi:10.1371/journal.pmed.1002181
PMCID: PMC5127514  PMID: 27898668
19.  Minimally Invasive Autopsy: A New Paradigm for Understanding Global Health? 
PLoS Medicine  2016;13(11):e1002173.
Peter Byass reflects on the potential niche for minimally invasive autopsies in determining cause-of-death in low- and middle-income countries.
doi:10.1371/journal.pmed.1002173
PMCID: PMC5119692  PMID: 27875535
20.  Effectiveness of Seasonal Malaria Chemoprevention in Children under Ten Years of Age in Senegal: A Stepped-Wedge Cluster-Randomised Trial 
PLoS Medicine  2016;13(11):e1002175.
Background
Seasonal Malaria Chemoprevention (SMC) with sulfadoxine-pyrimethamine (SP) plus amodiaquine (AQ), given each month during the transmission season, is recommended for children living in areas of the Sahel where malaria transmission is highly seasonal. The recommendation for SMC is currently limited to children under five years of age, but, in many areas of seasonal transmission, the burden in older children may justify extending this age limit. This study was done to determine the effectiveness of SMC in Senegalese children up to ten years of age.
Methods and Findings
SMC was introduced into three districts over three years in central Senegal using a stepped-wedge cluster-randomised design. A census of the population was undertaken and a surveillance system was established to record all deaths and to record all cases of malaria seen at health facilities. A pharmacovigilance system was put in place to detect adverse drug reactions. Fifty-four health posts were randomised. Nine started implementation of SMC in 2008, 18 in 2009, and a further 18 in 2010, with 9 remaining as controls. In the first year of implementation, SMC was delivered to children aged 3–59 months; the age range was then extended for the latter two years of the study to include children up to 10 years of age. Cluster sample surveys at the end of each transmission season were done to measure coverage of SMC and the prevalence of parasitaemia and anaemia, to monitor molecular markers of drug resistance, and to measure insecticide-treated net (ITN) use. Entomological monitoring and assessment of costs of delivery in each health post and of community attitudes to SMC were also undertaken. About 780,000 treatments were administered over three years. Coverage exceeded 80% each month. Mortality, the primary endpoint, was similar in SMC and control areas (4.6 and 4.5 per 1000 respectively in children under 5 years and 1.3 and 1.2 per 1000 in children 5-9 years of age; the overall mortality rate ratio [SMC: no SMC] was 0.90, 95% CI 0.68–1.2, p = 0.496). A reduction of 60% (95% CI 54%–64%, p < 0.001) in the incidence of malaria cases confirmed by a rapid diagnostic test (RDT) and a reduction of 69% (95% CI 65%–72%, p < 0.001) in the number of treatments for malaria (confirmed and unconfirmed) was observed in children. In areas where SMC was implemented, incidence of confirmed malaria in adults and in children too old to receive SMC was reduced by 26% (95% CI 18%–33%, p < 0.001) and the total number of treatments for malaria (confirmed and unconfirmed) in these older age groups was reduced by 29% (95% CI 21%–35%, p < 0.001). One hundred and twenty-three children were admitted to hospital with a diagnosis of severe malaria, with 64 in control areas and 59 in SMC areas, showing a reduction in the incidence rate of severe disease of 45% (95% CI 5%–68%, p = 0.031). Estimates of the reduction in the prevalence of parasitaemia at the end of the transmission season in SMC areas were 68% (95% CI 35%–85%) p = 0.002 in 2008, 84% (95% CI 58%–94%, p < 0.001) in 2009, and 30% (95% CI -130%–79%, p = 0.56) in 2010. SMC was well tolerated with no serious adverse reactions attributable to SMC drugs. Vomiting was the most commonly reported mild adverse event but was reported in less than 1% of treatments. The average cost of delivery was US$0.50 per child per month, but varied widely depending on the size of the health post. Limitations included the low rate of mortality, which limited our ability to detect an effect on this endpoint.
Conclusions
SMC substantially reduced the incidence of outpatient cases of malaria and of severe malaria in children, but no difference in all-cause mortality was observed. Introduction of SMC was associated with an overall reduction in malaria incidence in untreated age groups. In many areas of Africa with seasonal malaria, there is a substantial burden in older children that could be prevented by SMC. SMC in older children is well tolerated and effective and can contribute to reducing malaria transmission.
Trial Registration
ClinicalTrials.gov NCT00712374
In a stepped-wedge cluster-randomized trial, Paul J. Milligan and colleagues determine the effectiveness of SMC in Senegalese children up to ten years of age.
Author Summary
Why was this study done?
SMC was originally intended for children under five years of age. In many areas of the sub-Sahel, there is a substantial burden of malaria in older children; this may justify extending the age range of SMC.
This study was done in central Senegal to evaluate the effectiveness of SMC when given to children up to ten years of age.
What did the researchers do and find?
SMC was introduced into the area served by 54 health posts by district health teams over three years using a randomised stepped-wedge design.
Malaria cases were detected at outpatient clinics and in hospitals, and deaths were recorded through a demographic surveillance system.
SMC reduced the incidence of malaria in children up to ten years of age by 60%. No reduction in all-cause child deaths was observed, but the incidence of severe malaria was reduced by 45%.
SMC administered to children up to ten years of age led to a reduction in malaria incidence in older age groups by 26%.
What do these findings mean?
SMC given to children up to ten years of age is highly effective in preventing malaria in children and can contribute to reducing overall malaria transmission.
SMC programmes have started in 11 countries. In many of these countries, there is a substantial burden of malaria in older children. The findings of this study indicate that a much greater impact could be achieved by extending the age range of SMC.
doi:10.1371/journal.pmed.1002175
PMCID: PMC5119693  PMID: 27875528
21.  Seasonal Malaria Chemoprevention: An Evolving Research Paradigm 
PLoS Medicine  2016;13(11):e1002176.
Robert W. Snow discusses the importance of empirical evidence, such as that provided in the trial published this week by Milligan and colleagues, in guiding malaria control in Africa.
doi:10.1371/journal.pmed.1002176
PMCID: PMC5119694  PMID: 27875534
22.  Validity of a Minimally Invasive Autopsy for Cause of Death Determination in Adults in Mozambique: An Observational Study 
PLoS Medicine  2016;13(11):e1002171.
Background
There is an urgent need to identify tools able to provide reliable information on the cause of death in low-income regions, since current methods (verbal autopsy, clinical records, and complete autopsies) are either inaccurate, not feasible, or poorly accepted. We aimed to compare the performance of a standardized minimally invasive autopsy (MIA) approach with that of the gold standard, the complete diagnostic autopsy (CDA), in a series of adults who died at Maputo Central Hospital in Mozambique.
Methods and Findings
In this observational study, coupled MIAs and CDAs were performed in 112 deceased patients. The MIA analyses were done blindly, without knowledge of the clinical data or the results of the CDA. We compared the MIA diagnosis with the CDA diagnosis of cause of death.
CDA diagnoses comprised infectious diseases (80; 71.4%), malignant tumors (16; 14.3%), and other diseases, including non-infectious cardiovascular, gastrointestinal, kidney, and lung diseases (16; 14.3%). A MIA diagnosis was obtained in 100/112 (89.2%) cases. The overall concordance between the MIA diagnosis and CDA diagnosis was 75.9% (85/112). The concordance was higher for infectious diseases and malignant tumors (63/80 [78.8%] and 13/16 [81.3%], respectively) than for other diseases (9/16; 56.2%). The specific microorganisms causing death were identified in the MIA in 62/74 (83.8%) of the infectious disease deaths with a recognized cause.
The main limitation of the analysis is that both the MIA and the CDA include some degree of expert subjective interpretation.
Conclusions
A simple MIA procedure can identify the cause of death in many adult deaths in Mozambique. This tool could have a major role in improving the understanding and surveillance of causes of death in areas where infectious diseases are a common cause of mortality.
Jaume Ordi and colleagues examine the validity of a standardized minimally invasive autopsy approach by comparison with that of the complete diagnostic autopsy in a series of adults who died at Maputo Central Hospital in Mozambique.
Author Summary
Why Was This Study Done?
While complete autopsies are considered the gold standard for the determination of cause of death, they are poorly accepted and difficult to perform in middle- and low-income countries. More feasible options, such as verbal autopsies and clinical records, are highly inaccurate.
Minimally invasive autopsy techniques have been proposed as a more acceptable alternative to complete autopsy, and the results of these techniques have been shown to be relatively accurate; however, current minimally invasive autopsy protocols generally involve high-tech imaging procedures not available in middle- and low-income settings.
Our study was designed to develop a simplified minimally invasive autopsy method that would be feasible in middle- and low-income countries, and to validate this method against the gold standard.
What Did the Researchers Do and Find?
The minimally invasive autopsy developed in this study consists of the collection of blood and cerebrospinal fluid samples and the collection of tissue samples from solid organs using biopsy needles, followed by histological and microbiological analyses.
Coupled minimally invasive and complete autopsies were performed in 112 deceased patients. The samples obtained by both techniques were evaluated independently.
Concordance between the putative diagnosis obtained with the minimally invasive autopsy and the gold standard diagnosis obtained by complete autopsy was 75.9%.
The agreement was particularly high for infectious diseases. Moreover, the specific microorganisms causing death were accurately identified in the minimally invasive autopsy samples.
What Do These Findings Mean?
Minimally invasive autopsy is a reliable tool that can improve our understanding of the causes of death in Mozambique, where infectious diseases are a common cause of mortality.
The use of this tool could improve health planning and priority setting for the most vulnerable populations in the world.
doi:10.1371/journal.pmed.1002171
PMCID: PMC5119723  PMID: 27875530
23.  Willingness to Know the Cause of Death and Hypothetical Acceptability of the Minimally Invasive Autopsy in Six Diverse African and Asian Settings: A Mixed Methods Socio-Behavioural Study 
PLoS Medicine  2016;13(11):e1002172.
Background
The minimally invasive autopsy (MIA) is being investigated as an alternative to complete diagnostic autopsies for cause of death (CoD) investigation. Before potential implementation of the MIA in settings where post-mortem procedures are unusual, a thorough assessment of its feasibility and acceptability is essential.
Methods and Findings
We conducted a socio-behavioural study at the community level to understand local attitudes and perceptions related to death and the hypothetical feasibility and acceptability of conducting MIAs in six distinct settings in Gabon, Kenya, Mali, Mozambique, and Pakistan. A total of 504 interviews (135 key informants, 175 health providers [including formal health professionals and traditional or informal health providers], and 194 relatives of deceased people) were conducted. The constructs “willingness to know the CoD” and “hypothetical acceptability of MIAs” were quantified and analysed using the framework analysis approach to compare the occurrence of themes related to acceptability across participants.
Overall, 75% (379/504) of the participants would be willing to know the CoD of a relative. The overall hypothetical acceptability of MIA on a relative was 73% (366/504). The idea of the MIA was acceptable because of its perceived simplicity and rapidity and particularly for not “mutilating” the body. Further, MIAs were believed to help prevent infectious diseases, address hereditary diseases, clarify the CoD, and avoid witchcraft accusations and conflicts within families. The main concerns regarding the procedure included the potential breach of confidentiality on the CoD, the misperception of organ removal, and the incompatibility with some religious beliefs. Formal health professionals were concerned about possible contradictions between the MIA findings and the clinical pre-mortem diagnoses. Acceptability of the MIA was equally high among Christian and Islamic communities. However, in the two predominantly Muslim countries, MIA acceptability was higher in Mali than in Pakistan.
While the results of the study are encouraging for the potential use of the MIA for CoD investigation in low-income settings, they remain hypothetical, with a need for confirmation with real-life MIA implementation and in populations beyond Health and Demographic Surveillance System areas.
Conclusions
This study showed a high level of interest in knowing the CoD of a relative and a high hypothetical acceptability of MIAs as a tool for CoD investigation across six distinct settings. These findings anticipate potential barriers and facilitators, both at the health facility and community level, essential for local tailoring of recommendations for future MIA implementation.
Using a mixed methods socio-behavioral approach, Maria Maixenchs and colleagues investigate the willingness to know the cause of death and acceptability of the Minimally Invasive Autopsy in Gabon, Kenya, Mali, Mozambique and Pakistan.
Author Summary
Why Was This Study Done?
Reliable data on the real causes of death in low- and middle-income countries are scarce. Without this information, it is difficult to focus on the real health problems of a population and to implement adequate health policies.
Minimally invasive autopsies are being studied as a new method to determine the cause of death.
What Did the Researchers Do and Find?
A total of 504 interviews were conducted in five different countries to investigate the willingness of individuals to know the cause of death of a relative and the hypothetical acceptability of minimally invasive autopsies.
Willingness to know the cause of death was high, as was the hypothetical acceptability of this innovative method to investigate cause of death.
What Do These Findings Mean?
These results are encouraging because they open new research lines for investigating the cause of death in areas where data are difficult to obtain and general information on mortality is necessary to design effective preventive policies.
doi:10.1371/journal.pmed.1002172
PMCID: PMC5119724  PMID: 27875532
24.  Educational Outreach with an Integrated Clinical Tool for Nurse-Led Non-communicable Chronic Disease Management in Primary Care in South Africa: A Pragmatic Cluster Randomised Controlled Trial 
PLoS Medicine  2016;13(11):e1002178.
Background
In many low-income countries, care for patients with non-communicable diseases (NCDs) and mental health conditions is provided by nurses. The benefits of nurse substitution and supplementation in NCD care in high-income settings are well recognised, but evidence from low- and middle-income countries is limited. Primary Care 101 (PC101) is a programme designed to support and expand nurses’ role in NCD care, comprising educational outreach to nurses and a clinical management tool with enhanced prescribing provisions. We evaluated the effect of the programme on primary care nurses’ capacity to manage NCDs.
Methods and Findings
In a cluster randomised controlled trial design, 38 public sector primary care clinics in the Western Cape Province, South Africa, were randomised. Nurses in the intervention clinics were trained to use the PC101 management tool during educational outreach sessions delivered by health department trainers and were authorised to prescribe an expanded range of drugs for several NCDs. Control clinics continued use of the Practical Approach to Lung Health and HIV/AIDS in South Africa (PALSA PLUS) management tool and usual training. Patients attending these clinics with one or more of hypertension (3,227), diabetes (1,842), chronic respiratory disease (1,157) or who screened positive for depression (2,466), totalling 4,393 patients, were enrolled between 28 March 2011 and 10 November 2011. Primary outcomes were treatment intensification in the hypertension, diabetes, and chronic respiratory disease cohorts, defined as the proportion of patients in whom treatment was escalated during follow-up over 14 mo, and case detection in the depression cohort. Primary outcome data were analysed for 2,110 (97%) intervention and 2,170 (97%) control group patients. Treatment intensification rates in intervention clinics were not superior to those in the control clinics (hypertension: 44% in the intervention group versus 40% in the control group, risk ratio [RR] 1.08 [95% CI 0.94 to 1.24; p = 0.252]; diabetes: 57% versus 50%, RR 1.10 [0.97 to 1.24; p = 0.126]; chronic respiratory disease: 14% versus 12%, RR 1.08 [0.75 to 1.55; p = 0.674]), nor was case detection of depression (18% versus 24%, RR 0.76 [0.53 to 1.10; p = 0.142]). No adverse effects of the nurses’ expanded scope of practice were observed. Limitations of the study include dependence on self-reported diagnoses for inclusion in the patient cohorts, limited data on uptake of PC101 by users, reliance on process outcomes, and insufficient resources to measure important health outcomes, such as HbA1c, at follow-up.
Conclusions
Educational outreach to primary care nurses to train them in the use of a management tool involving an expanded role in managing NCDs was feasible and safe but was not associated with treatment intensification or improved case detection for index diseases. This notwithstanding, the intervention, with adjustments to improve its effectiveness, has been adopted for implementation in primary care clinics throughout South Africa.
Trial Registration
The trial is registered with Current Controlled Trials (ISRCTN20283604)
In a cluster-randomized trial done in South Africa, Lara Fairall and co-workers investigate the effectiveness of a clinical management tool for non-communicable diseases in primary care.
Author Summary
Why Was This Study Done?
Non-communicable diseases (NCDs) are the leading cause of deaths worldwide, even in low- and middle-income countries (LMICs) that continue to battle to control communicable diseases like HIV and tuberculosis (TB).
Effective and affordable treatments prevent complications from NCDs like heart attacks and strokes, but access is limited by the variable availability and limited capacity of primary care health workers to detect and effectively manage these conditions. In many LMICs, non-physicians such as nurses provide primary care for NCDs.
Over the past 16 years, we have developed, evaluated, and refined integrated clinical management tools and training programmes that employ problem-based approaches to common symptoms like cough and priority health conditions including TB, HIV, asthma, and emphysema. We have shown them to be effective in improving the quality and outcomes of care for communicable diseases.
We have expanded this programme to include almost all NCDs and mental health. This study evaluated the impact, both benefits and harms, of introducing the expanded programme, called Primary Care 101 (PC101), in terms of the quality of primary care for four common chronic diseases: hypertension, diabetes, chronic respiratory disease, and depression.
What Did the Researchers Do and Find?
We compared the care offered to patients with one of these four chronic diseases in 18 clinics in which primary care health workers were trained in the use of PC101 with that in 18 clinics where nurses continued to use the predecessor tool, which focused on communicable diseases.
The trial had a pragmatic design, meaning it was conducted under usual conditions of health system operational constraints. Clinics in urban and rural areas serving people living in socio-economically deprived areas of South Africa were selected.
We enrolled 4,393 patients with one or more of the NCDs of interest and followed them up for 14 mo after introduction of PC101 at the intervention clinics. The primary outcome of interest was intensification of treatment (or diagnosis, in the case of depression) for the four NCDs, analysed separately.
The results confirmed very high rates of multimorbidity (patients having more than one condition at a time), under-diagnosis, under-treatment, and poor disease control.
Introducing PC101 did not result in intensification of treatment for the four NCDs, but neither was there evidence of harm from the nurses’ expanded scope of practice.
What Do These Findings Mean?
The trial confirmed that multimorbidity and poor detection and control of NCDs and depression are common in this setting. Interventions are necessary to limit the impact of these conditions on people’s health and quality of life.
PC101 offered a practical and acceptable tool to help expand the scope of practice of non-physician clinicians to include NCD care, but we were not able to show improvements in care, as we have previously done for communicable diseases.
The study illustrates the limitations of trials designed to study the effects of complex system interventions in real life, where even small changes across many endpoints, as seen in our study, may be useful to decision-makers under pressure to respond constructively to the rise of multimorbidity and NCDs.
PC101 has been adopted for country-wide implementation in primary care clinics in South Africa.
doi:10.1371/journal.pmed.1002178
PMCID: PMC5119726  PMID: 27875542
25.  Under-prescribing of Prevention Drugs and Primary Prevention of Stroke and Transient Ischaemic Attack in UK General Practice: A Retrospective Analysis 
PLoS Medicine  2016;13(11):e1002169.
Background
Stroke is a leading cause of death and disability; worldwide it is estimated that 16.9 million people have a first stroke each year. Lipid-lowering, anticoagulant, and antihypertensive drugs can prevent strokes, but may be underused.
Methods and Findings
We analysed anonymised electronic primary care records from a United Kingdom (UK) primary care database that covers approximately 6% of the UK population. Patients with first-ever stroke/transient ischaemic attack (TIA), ≥18 y, with diagnosis between 1 January 2009 and 31 December 2013, were included. Drugs were considered under-prescribed when lipid-lowering, anticoagulant, or antihypertensive drugs were clinically indicated but were not prescribed prior to the time of stroke or TIA. The proportions of strokes or TIAs with prevention drugs under-prescribed, when clinically indicated, were calculated.
In all, 29,043 stroke/TIA patients met the inclusion criteria; 17,680 had ≥1 prevention drug clinically indicated: 16,028 had lipid-lowering drugs indicated, 3,194 anticoagulant drugs, and 7,008 antihypertensive drugs. At least one prevention drug was not prescribed when clinically indicated in 54% (9,579/17,680) of stroke/TIA patients: 49% (7,836/16,028) were not prescribed lipid-lowering drugs, 52% (1,647/3,194) were not prescribed anticoagulant drugs, and 25% (1,740/7,008) were not prescribed antihypertensive drugs.
The limitations of our study are that our definition of under-prescribing of drugs for stroke/TIA prevention did not address patients’ adherence to medication or medication targets, such as blood pressure levels.
Conclusions
In our study, over half of people eligible for lipid-lowering, anticoagulant, or antihypertensive drugs were not prescribed them prior to first stroke/TIA. We estimate that approximately 12,000 first strokes could potentially be prevented annually in the UK through optimal prescribing of these drugs. Improving prescription of lipid-lowering, anticoagulant, and antihypertensive drugs is important to reduce the incidence and burden of stroke and TIA.
In a retrospective analysis of primary care data from the United Kingdom, Grace Turner and colleagues study the proportion of patients with a stroke or transient ischemic attack who had been prescribed the clinically indicated lipid lowering, antihypertensive or anticoagulant drugs.
Author Summary
Why Was This Study Done?
Atrial fibrillation, dyslipidaemia, and hypertension are risk factors for stroke and transient ischaemic attack (TIA). However, anticoagulant, lipid-lowering, and antihypertensive drugs have been found to reduce the incidence of stroke.
Prescribing of these drugs for primary stroke/TIA prevention may be suboptimal in general practice. The proportion of stroke/TIA patients not prescribed prevention drugs when anticoagulant, lipid-lowering, or antihypertensive drugs are clinically indicated is unclear.
What Did the Researchers Do and Find?
We conducted a retrospective analysis of the electronic primary care medical records of 29,043 stroke/TIA patients in the UK.
Half of the patients with clinical indications for anticoagulant drugs (52%; 1,647/3,194) or lipid-lowering drugs (49%; 7,836/16,028) were not prescribed these drugs prior to stroke/TIA. A quarter of the patients with clinical indications for antihypertensive drugs (25%; 1,740/7,008) were not prescribed these drugs prior to stroke/TIA.
There was no reduction in the proportion of stroke/TIA patients with prior missed opportunities for prevention with lipid-lowering or antihypertensive drugs between 2009 and 2013; however, prescribing of anticoagulant drugs improved during this period.
What Do These Findings Mean?
Improving prescribing of anticoagulant, lipid-lowering, and antihypertensive drugs in primary care could potentially prevent approximately 12,000 first strokes annually in the United Kingdom.
doi:10.1371/journal.pmed.1002169
PMCID: PMC5112771  PMID: 27846215

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