Intratumoural evolutionary landscape of high-risk prostate cancer: the PROGENY study of genomic and immune parameters.

BACKGROUND: Intratumoural heterogeneity (ITH) is well recognised in prostate cancer (PC), but its role in high-risk disease is uncertain. A prospective, single-arm, translational study using targeted multiregion prostate biopsies was carried out to study genomic and T-cell ITH in clinically high-risk PC aiming to identify drivers and potential therapeutic strategies. PATIENTS AND METHODS: Forty-nine men with elevated prostate-specific antigen and multiparametric-magnetic resonance imaging detected PC underwent image-guided multiregion transperineal biopsy. Seventy-nine tumour regions from 25 patients with PC underwent sequencing, analysis of mutations, copy number and neoepitopes combined with tumour infiltrating T-cell subset quantification. RESULTS: We demonstrated extensive somatic nucleotide variation and somatic copy number alteration heterogeneity in high-risk PC. Overall, the mutational burden was low (0.93/Megabase), but two patients had hypermutation, with loss of mismatch repair (MMR) proteins, MSH2 and MSH6. Somatic copy number alteration burden was higher in patients with metastatic hormone-naive PC (mHNPC) than in those with high-risk localised PC (hrlPC), independent of Gleason grade. Mutations were rarely ubiquitous and mutational frequencies were similar for mHNPC and hrlPC patients. Enrichment of focal 3q26.2 and 3q21.3, regions containing putative metastasis drivers, was seen in mHNPC patients. We found evidence of parallel evolution with three separate clones containing activating mutations of ß-catenin in a single patient. We demonstrated extensive intratumoural and intertumoural T-cell heterogeneity and high inflammatory infiltrate in the MMR-deficient (MMRD) patients and the patient with parallel evolution of ß-catenin. Analysis of all patients with activating Wnt/ß-catenin mutations demonstrated a low CD8+/FOXP3+ ratio, a potential surrogate marker of immune evasion. CONCLUSIONS: The PROGENY (PROstate cancer GENomic heterogeneitY) study provides a diagnostic platform suitable for studying tumour ITH. Genetic aberrations in clinically high-risk PC are associated with altered patterns of immune infiltrate in tumours. Activating mutations of Wnt/ß-catenin signalling pathway or MMRD could be considered as potential biomarkers for immunomodulation therapies. CLINICAL TRIALS.GOV IDENTIFIER: NCT02022371.

Detection of ubiquitous and heterogeneous mutations in cell-free DNA from patients with early-stage non-small-cell lung cancer.

BACKGROUND: The aim of this pilot study was to assess whether both ubiquitous and heterogeneous somatic mutations could be detected in cell-free DNA (cfDNA) from patients with early-stage non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: Three stage I and one stage II primary NSCLC tumors were subjected to multiregion whole-exome sequencing (WES) and validated with AmpliSeq. A subset of ubiquitous and heterogeneous single-nucleotide variants (SNVs) were chosen. Multiplexed PCR using custom-designed primers, coupled with next-generation sequencing (mPCR-NGS), was used to detect these SNVs in both tumor DNA and cfDNA isolated from plasma obtained before surgical resection of the tumors. The limit of detection for each assay was determined using cfDNA from 48 presumed-normal healthy volunteers. RESULTS: Tumor DNA and plasma-derived cfDNA was successfully amplified and sequenced for 37/50 (74%) SNVs using the mPCR-NGS method. Twenty-five (68%) were ubiquitous and 12 (32%) were heterogeneous SNVs. Variant detection by mPCR-NGS and WES-AmpliSeq in tumor tissue was well correlated (R(2) = 0.8722, P < 0.0001). Sixteen (43%) out of 37 SNVs were detected in cfDNA. Twelve of these were ubiquitous SNVs with a variant allele frequency (VAF) range of 0.15-23.25%, and four of these were heterogeneous SNVs with a VAF range of 0.28-1.71%. There was a statistically significant linear relationship between the VAFs for tumor and cfDNA (R(2) = 0.5144; P = 0.0018). For all four patients, at least two variants were detected in plasma. The estimated number of copies of variant DNA present in each sample ranged from 5 to 524. The average number of variant copies required for detection (VCRD) was 3.16 (range: 0.2-7.6 copies). CONCLUSIONS: The mPCR-NGS method revealed intratumor heterogeneity in early-stage NSCLC tumors, and was able to detect both ubiquitous and heterogeneous SNVs in cfDNA. Further validation of mPCR-NGS in cfDNA is required to define its potential use in clinical practice.

Retrospective Derivation and Validation of an Automated Electronic Search Algorithm to Identify Post Operative Cardiovascular and Thromboembolic Complications.

BACKGROUND: With increasing numbers of hospitals adopting electronic medical records, electronic search algorithms for identifying postoperative complications can be invaluable tools to expedite data abstraction and clinical research to improve patient outcomes. OBJECTIVES: To derive and validate an electronic search algorithm to identify postoperative thromboembolic and cardiovascular complications such as deep venous thrombosis, pulmonary embolism, or myocardial infarction within 30 days of total hip or knee arthroplasty. METHODS: A total of 34 517 patients undergoing total hip or knee arthroplasty between January 1, 1996 and December 31, 2013 were identified. Using a derivation cohort of 418 patients, several iterations of a free-text electronic search were developed and refined for each complication. Subsequently, the automated search algorithm was validated on an independent cohort of 2 857 patients, and the sensitivity and specificities were compared to the results of manual chart review. RESULTS: In the final derivation subset, the automated search algorithm achieved a sensitivity of 91% and specificity of 85% for deep vein thrombosis, a sensitivity of 96% and specificity of 100% for pulmonary embolism, and a sensitivity of 100% and specificity of 95% for myocardial infarction. When applied to the validation cohort, the search algorithm achieved a sensitivity of 97% and specificity of 99% for deep vein thrombosis, a sensitivity of 97% and specificity of 100% for pulmonary embolism, and a sensitivity of 100% and specificity of 99% for myocardial infarction. CONCLUSIONS: The derivation and validation of an electronic search strategy can accelerate the data abstraction process for research, quality improvement, and enhancement of patient care, while maintaining superb reliability compared to manual review.

Is routine molecular screening for common alpha-thalassaemia deletions necessary as part of an antenatal screening programme?

Antenatal sickle and thalassaemia screening programmes are now established in most high prevalence areas in England. Although screening reliably detects beta-thalassaemia trait, in many cases, results state that alpha-thalassaemia trait cannot be excluded. The detection of couples at risk of a child with hydrops fetalis is one of the aims of the national programme. We, therefore, performed polymerase chain reaction (PCR) for the common alpha-thalassaemia gene deletions to assess the usefulness of this technique in routine screening practice. Between August 2001 and August 2002, of the 5092 women booked at the antenatal clinic, 425 were found to have a mean corpuscular haemoglobin (MCH) <27 pg in the absence of beta-thalassaemia trait; 189 (44.5%) had an MCH <25 pg. All 425 patients underwent PCR analysis for the common deletions: -SEA (South-East Asian), -MED (Mediterranean), -alpha(20.5), -FIL (Filipino), -alpha 3.7 and -alpha 4.2 genotypes. In total, 130 (31%) women were positive for alpha-thalassaemia deletion; 86 (24.7%) were heterozygous for -alpha 3.7, 19 (4.4%) were homozygous for -alpha 3.7, 12 (2.8%) were heterozygous for -alpha 4.2, 1 (0.2%) was homozygous for -alpha 4.2, 11 (2.6%) were heterozygous for -SEA and one (0.2%) was heterozygous for the -MED genotype. Although the detection rate for alpha(+)-thalassaemia was high, a strategy of selective screening using MCH <25 pg and ethnic group (SEA, Middle East or Eastern MED) would have identified all individuals heterozygous for alpha(0)-thalassaemia. Routine molecular screening for all forms of alpha-thalassaemia trait is unjustified in antenatal screening.

Gains on 9p are common genomic aberrations in idiopathic myelofibrosis: a comparative genomic hybridization study.

Ideopathic myelofibrosis (IMF) is a chronic myeloproliferative disorder resulting in bone marrow fibrosis as a consequence of growth factor release from clonal haematopoiesis. Conventional cytogenetic analysis identifies abnormalities in approximately a third of cases at diagnosis, although rarely uncovers unique, primary genetic events. We have used comparative genomic hybridization (CGH) to study 25 IMF cases and have compared the results with conventional cytogenetics. Metaphase cells were available for analysis in 13 cases, of which seven showed an abnormal karyotype. CGH chromosomal profiles showed imbalances in 21 of 25 cases. The most frequent aberrations were gains of 9p (12 cases), 2q (seven cases), 3p (seven cases), chromosome 4 (seven cases), 12q (seven cases), 13q (eight cases). The main losses were at 17q and occurred in six cases. The results for CGH and cytogenetics were matched for one case only. Investigation of IMF by CGH suggests that genomic aberrations are much more common than has been previously indicated by conventional cytogenetic analysis and occur in the majority of cases. Gains of 9p were the most frequent finding, occurring in 50% of patients and suggests that genes on 9p may play a crucial role in the pathogenesis of IMF.

Contemporary gene flow and the spatio-temporal genetic structure of subdivided newt populations (Triturus cristatus, T. marmoratus).

Gene flow and drift shape the distribution of neutral genetic diversity in metapopulations, but their local rates are difficult to quantify. To identify gene flow between demes as distinct from individual migration, we present a modified Bayesian method to genetically test for descendants between an immigrant and a resident in a nonmigratory life stage. Applied to a metapopulation of pond-breeding European newts (Triturus cristatus, T. marmoratus) in western France, the evidence for gene flow was usually asymmetric and, for demes of known census size (N), translated into maximally seven reproducing immigrants. Temporal sampling also enabled the joint estimation of the effective demic population size (Ne) and the immigration rate m (including nonreproductive individuals). Ne ranged between 4.1 and 19.3 individuals, Ne/N ranged between 0.05 and 0.65 and always decreased with N; m was estimated as 0.19-0.63, and was possibly biased upwards. We discuss how genotypic data can reveal fine-scale demographic processes with important microevolutionary implications.

Biomechanical properties of quadruple tendon and patellar tendon femoral fixation techniques.

Two femoral fixation techniques for quadruple hamstring tendon grafts were compared under cyclic loading with the patellar tendon: the rectangular inserted pin (TransFix) and biodegradable interference screw fixation of the quadruple tendon and titanium interference screw fixation of the middle third of the patellar tendon. Porcine specimens were mounted onto a tension load machine, and the tendon-fixation-femur-complex was tested for stiffness, displacement during 800 cycles of loading between 50 and 250 N and ultimate tension load. TransFix fixation showed the greatest stiffness at 183.6 N/mm ( P<0.05). The least displacement under cyclic loading was observed for the titanium interference screw followed by the TransFix and biodegradable interference screw ( P<0.01). The ultimate tension load was greatest for the TransFix fixation at 1303+/-282 N, followed by patellar tendon fixation with 763+/-103 N and the biodegradable interference screw fixation with 480+/-133 N ( P<0.001). To reduce initial elongation of the graft and displacement at the fixation site, preconditioning of both the tendon and tendon-fixation complex is especially important when using quadruple tendons. TransFix fixation provides better stability and greater stiffness and pull-out strength than the other techniques. This finding is of clinical relevance to surgeons of the anterior cruciate ligament.

Control elements of Dictyostelium discoideum prespore specific gene 3B.

Expression of the prespore-specific gene 3B in Dictyostelium discoideum Ax-2 cells is first detectable late in development with 3B mRNA levels peaking at 18 h (Corney et al., 1990). Sequence analysis of 3B cDNA and genomic clones revealed two exons, 319bp and 341bp long, separated by an 82bp intron, which encode a 219 residue protein with no significant similarity to any other reported gene product. Transcription starts at an A residue 45bp upstream from the translation initiation codon, preceded by a TATA-like sequence and an oligo-dT stretch. The 5' flanking sequence of the 3B gene is extremely A + T rich but contains five G/C rich stretches, each approximately 7bp long, which have strong sequence similarity to the G boxes found upstream of other developmentally regulated Dictyostelium genes. Analysis of both 3B promoter-CAT reporter gene and 3B promoter-lacZ reporter gene constructs showed that 908bp of 5' flanking sequence is sufficient to confer correct developmental and cell-type specific regulation. Sequential 5' deletion analysis revealed that positive elements lie upstream of position -304 and that negative element(s) lie between positions -264 and -241. Nevertheless, a 286bp promoter fragment containing only sequence located downstream of position -241 directed essentially correct reporter gene expression. Point mutation analysis identified cis-acting elements within this 'sufficient' promoter fragment which activate transcription (G box V and psp-AT type sequences). A short (56bp) region of the 3B promoter sequence containing both G box IV and the psp-AT type element binds two types of nuclear factor, one present in cells throughout development and a second that appears only in late development with a time course comparable to 3B gene induction.

Melatonin for treatment of sleep disorders in children with developmental disabilities.

This study explored the safety and efficacy of synthetic melatonin in the treatment of sleep problems in 20 children with developmental disabilities, in a randomized, double-blind, placebo-controlled 6-week trial of melatonin versus placebo. All but 2 children fell asleep more quickly when receiving melatonin than placebo. Overall, the greater the sleep latency (time to fall asleep) was at baseline or when receiving placebo, the more pronounced was the decrease in sleep latency with melatonin. The effect of melatonin on sleep latency was significant (P < .05). The duration of sleep while receiving melatonin was significantly greater than baseline (P < .007) but was not significantly different from placebo, and no difference in the number of awakenings was noted. No side effects were reported. Eleven of 18 parents (61%) correctly identified the weeks their child received melatonin. This study suggests that synthetic melatonin reduces sleep latency in children with developmental disabilities.

Identification of novel FLT-3 Asp835 mutations in adult acute myeloid leukaemia.

Genomic DNA from 97 cases of adult de novo acute myeloid leukaemia (AML) was screened using polymerase chain reaction (PCR) and conformation-sensitive gel electrophoresis (CSGE) for FLT3 exon 20 mutations. Initial sequencing of four cases, representing the spectrum of CSGE abnormalities, revealed changes affecting codon Asp835 in three cases and also an intron 20 A to G change. In order to identify all possible Asp835 alterations, as well as the frequency of the intronic change nucleotide 2541 + 57 A-->G, the patient PCR products were digested with EcoRV and NlaIII respectively. Seven cases (7.2%) possessed a mutation affecting Asp835; these were identified, following DNA sequencing, as Asp835Tyr (n = 5), Asp835His (n = 1) and Asp835del (n = 1). Alterations affecting Asp835 were not found in 80 normal control DNA samples. In contrast, the nucleotide 2541 + 57 A-->G change was shown to be a polymorphism, with an allelic frequency of 0.24 for the G and 0.76 for the A allele. This study reports, for the first time, point mutations in the human FLT3 gene that, because of their homology with other class III receptor tyrosine kinase mutations, probably result in constitutive activation of the receptor.

Simultaneous choroidal melanoma and rhegmatogenous retinal detachment.

A case is reported of a 30-year-old man who presented with the unusual combination of a unilateral inferonasal choroidal melanoma and an unassociated rhegmatogenous retinal detachment attributable to a large horseshoe tear in the superotemporal quadrant. Over 20 cases of simultaneous occurrence of these two phenomena have now been reported but this patient appears to be the youngest. The reports of this uncommon association are reviewed.

Is epidural analgesia associated with an improved outcome following open Nissen fundoplication?

Postoperative epidural analgesia is increasingly popular in paediatric practice, although evidence of its benefit is scarce. We performed a retrospective analysis of a series of 104 consecutive open Nissen fundoplications, to determine whether mode of analgesia, epidural (n=65) or opioid infusion (n=39), influenced certain outcome measures, including intensive care utilization, duration of hospital stay, morbidity and mortality. The two groups were similar in terms of demographic characteristics and associated pathologies. Overall, morbidity and mortality (2%) rates were low. Mean duration of hospital stay was significantly greater for the opioid group, compared to those receiving epidural analgesia (13 vs. 8 days, P < 0.05). The number of patients who remained in hospital for more than 7 days was also significantly greater in the opioid group. Accepting the limitations of a retrospective study, these data suggest that epidural analgesia might be associated with an improved outcome following Nissen fundoplication and this merits a prospective study.

Smoke gets in your eyes: smoking and visual impairment in New Zealand.

AIM: To estimate the burden of visual impairment attributable to smoking in New Zealand. METHODS: Review of Medline-indexed literature on the relationship between smoking and eye disease and use of relevant New Zealand morbidity and smoking prevalence data. RESULTS: The international literature indicates there is strong evidence that smoking is a major cause of eye disease and blindness--particularly for cataracts and age-related macular degeneration (AMD). Using the most relevant international risk estimates, we estimated that 1335 people who are registered blind in New Zealand have AMD attributable to current and past smoking (26.8% of all AMD cases in the 55 years plus age-group). It was also estimated that 31 of the registered cases of visual impairment due to cataract and 396 hospitalisations for cataract surgery per year, are attributable to smoking. While subject to various methodological limitations, these estimates are probably under-estimates of the true burden of eye disease attributable to smoking. CONCLUSIONS: Smoking is a major cause of untreatable visual impairment and also a significant reason for cataract surgery in New Zealand. There is a need for more intensive tobacco control activities in New Zealand.

FLT3 internal tandem duplication mutations in adult acute myeloid leukaemia define a high-risk group.

Genomic DNA from 106 cases of adult de novo acute myeloid leukaemia (AML) was screened by polymerase chain reaction (PCR) and gel electrophoresis for FLT3 internal tandem duplication (ITD) mutations within the juxtamembrane (JM) domain. FLT3 mutations were detected in 14 cases (13.2%) and occurred in FAB types M1 (4 out of 14 cases), M3 (1 out of 10 cases), M4 (5 out of 37 cases) and M5 (4 out of 11 cases). Sequence analysis of four cases with abnormal PCR electrophoretic patterns revealed in frame duplications in the region of exon 11 of between 27 and 111 base pairs. Three are predicted to result in the tandem duplication of adjacent amino acid residues and one to result in a tandem duplication plus insertion of a novel amino acid motif. Statistical analysis showed the FLT3 mutations to be a strong prognostic factor, with patients lacking the mutation surviving significantly longer from diagnosis (mean 29.1 months) than those with an ITD (mean 12.8 months; P = 0.0002). Thirteen of the 14 patients with FLT3 mutations died within 18 months of diagnosis. FLT3 mutations were of prognostic significance in good risk disease (P = 0.04), as well as in patients with standard risk disease (P = 0.0096). This study demonstrates that the FLT3 ITD mutation occurs in a significant percentage of adult AML cases and is an important adverse prognostic factor that appears independent of conventional karyotypic findings.

Results of liver transplantation for nodular regenerative hyperplasia.

Liver transplantation has been performed in individuals with a pretransplant clinical diagnosis of cirrhosis but with nodular regenerative hyperplasia histologically. The purpose of this report is to investigate the results of liver transplantation in patients proven to have nodular regenerative hyperplasia post-transplant. A retrospective review was undertaken of four patients who underwent liver transplantation with a histologic diagnosis of nodular regenerative hyperplasia. All were felt to be cirrhotic on clinical grounds. Final histology of the explanted liver was confirmed by a single pathologist. Their ages ranged from 39 to 54 years, and three of the four were male. Three had pretransplant needle liver biopsies, two percutaneous and one transjugular. All revealed nonspecific reactive changes. Ultrasound and MRI were interpreted as consistent with cirrhosis in four of four and three of four cases, respectively. Portal vein flow was hepatopedal in three and absent in one. Pretransplant clinical characteristics and frequency were as follows: bleeding varices two, clinical ascites three, encephalopathy three, and impaired hepatic synthetic function two. All four patients underwent successful liver transplantation. There were no episodes of acute rejection. All are alive and well with normal graft function 2 to 4 years post-transplant. We conclude the following. 1) Patients with clinical end-stage liver disease due to underlying nodular regenerative hyperplasia can successfully undergo transplantation. 2) Nodular regenerative hyperplasia can present with signs and symptoms of liver failure, is difficult to diagnose by needle biopsy, and can be difficult to discriminate clinically from cirrhosis. 3) Although each case must be individually evaluated transplantation may be the optimal therapy in patients presenting with complications of liver failure.

Preoperative evaluation of living renal donors: comparison of CT angiography and MR angiography.

PURPOSE: To compare computed tomographic (CT) angiography and magnetic resonance (MR) angiography for preoperative evaluation of living renal donors. MATERIALS AND METHODS: Thirty-five living renal donors underwent preoperative contrast material-enhanced CT angiography and gadolinium-enhanced MR angiography. Each study was interpreted by two independent radiologists blinded to all other studies and to interpretations provided by other reviewers. Eighteen kidneys had surgical correlation. RESULTS: CT demonstrated 33 supernumerary arteries in 19 patients, bilateral solitary arteries in 16 patients, and 18 proximal arterial branches in 16 patients. MR demonstrated 26 supernumerary arteries in 15 patients, bilateral solitary renal arteries in 20 patients, and 21 proximal arterial branches in 16 patients. Interobserver agreements for MR (kappa = 0. 74) and CT (kappa = 0.73) were similar to the agreement between MR and CT (kappa = 0.74). Among the kidneys chosen for nephrectomy, one small accessory artery and one proximal arterial branch were missed with CT and MR. Two of the accessory arteries suggested at CT were not found at nephrectomy. By averaging data for both modalities, supernumerary arteries were present in 49% of kidney donors and were bilateral in approximately 17%. Proximal arterial branches were present in 46% of kidney donors. CONCLUSION: Preoperative CT and MR angiography of the renal arteries in renal donors demonstrate substantial agreement. Interobserver disagreement in the interpretation of CT and MR angiograms is related to 1-2-mm-diameter vessels.