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.

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.

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.

Expression of thymidylate synthetase activity in Bacillus subtilis upon integration of a cloned gene from Escherichia coli.

The gene from Escherichia coli encoding thymidylate synthetase was cloned in the plasmid pBR322. The resulting chimeric plasmid, pER2, was effective in transforming both E. coli and Bacillus subtilis to thymine prototrophy. Uncloned linear E. coli chromosomal DNA was unable to transform thymine-requiring strains of B. subtilis to thymine independence. Linearization of the chimeric plasmid, pER2, with restriction enzymes markedly diminished its ability to transform B. subtilis auxotrophs. The Thy+ transformants derived from the transformation of B. subtilis with pER2 DNA did not contain detectable extrachromosomal DNA as demonstrated by Southern hybridization patterns and centrifugation in CsCl gradients of DNA isolated from B. subtilis colonies transformed with the chimeric plasmid. We conclude that the DNA from the chimeric plasmid was integrated into the chromosome of B. subtilis, demonstrating that extensive homology is not required for the integration of foreign DNA. This is the first reported case of a gene from a Gram-negative bacterium functioning in a Gram-positive organism.

Effect of site-specific endonuclease digestion on the thyP3 gene of bacteriophage phi 3T and the thyP11 gene of bacteriophage rho11.

phi 3T and rho11 are closely related bacteriophages of Bacillus subtilis which can "convent" thymine auxotrophs to thymine prototrophs upon infection or transfection. The effect of endonuclease digestion on the ability of both bacteriophage and prophage DNA from phi eT and rho11 to transform for thymine prototrophy was determined. All of the endonucleases tested: BamHI, Bg/II, BsuRI, EcoRI, HindII+ III, and HpaII reduced the efficiency of thyP transformation to an equal extent in prophage and bacteriophage DNA. Only HpaII completely abolished thyP transformation. The reduction in transformation with BamHI, Bg/II, BsuRI, EcoRI, and HpaII fragments is size related. The thyP transforming fragments generated by these endonucleases are potentially clonable.

Transformation of Bacillus subtilis and Escherichia coli by a hybrid plasmid pCD1.

The gene thyP3 from Bacillus subtilis bacteriophage phi 3T was cloned in the plasmid pMB9. The resulting chimeric plasmid, pCD1, is effective in transforming both Escherichia coli and Bacillus subtilis to thymine prototrophy. The activity of the thyP3 gene product, thymidylate synthetase, was assayed and found to be 9 times greater in a transformed strain of Escherichia coli than in a phi 3T lysogen of Bacillus subtilis. The physical location of restriction sites has been determined for two related plasmids pCD1 and pCD2. Hybridization studies clearly indicate that the plasmid gene responsible for Thy+ transformation is the gene from the bacteriophage phi 3T. The lack of restriction in this transformation process is consistent with our previous studies using bacterial DNA in heterospecific exchanges indicating that the nucleotide sequence surrounding the gene is the dominant factor in determining interspecific transformation.

Impact of serum lipids on long-term graft and patient survival after renal transplantation.

The results after primary cadaveric renal transplantation in 665 consecutive patients were reviewed with respect to posttransplant serum lipids. Data were available for 182 of 665 patients on serum total cholesterol and triglycerides at 1 year posttransplant. Hypercholesterolemia (cholesterol > 200 mg/dl) developed in 141 of 182 patients (77%) and hypertriglyceridemia developed in 73 of 166 patients (44%). At 1 year posttransplant, hypercholesterolemia and hypertriglyceridemia both correlated with age at transplant (P = 0.0001, P = 0.01). Hypercholesterolemia and hypertriglyceridemia were also correlated with obesity as determined by body mass index (kg/m2) (P = 0.006, P = 0.01). Hypertriglyceridemia at 1 year posttransplant correlated with pretransplant triglyceride level (P = 0.006), but hypercholesterolemia did not correlate with pretransplant cholesterol level (P = 0.53). Hyperlipidemia was not correlated with cyclosporine (CsA) or prednisone dose (mg/kg), CsA trough levels, number of rejection episodes, or serum creatinine at 1 year. Despite significant differences in serum cholesterol and triglycerides, actuarial graft and patient survival were similar between the normolipidemic and hyperlipidemic groups.

Obesity as a risk factor after combined pancreas/kidney transplantation.

The impact of pretransplant overweight/obesity was analyzed in a group of 268 consecutive primary pancreas renal transplant recipients. Obesity was defined as body mass index (BMI) greater than 27 kg/m2. BMI was available for 240 of the 268 patients. A total of 88% (212/240) of the patients had a BMI < or = 27 and 28/240 (12%) had BMI > 27. There were no significant differences in age, sex, or race between obese and nonobese patients. The incidence and degree of posttransplant hypertension, weight gain, increase in BMI, and hyperlipidemia did not differ on the basis of pretransplant BMI. Serum creatinine at one year posttransplant was slightly increased in obese patients, but the increase was not statistically significant. Cumulative prednisone dose (mg/kg) as well as cyclosporine (CsA) dose (mg/kg) at one year was not significantly different between obese and nonobese patients. However, there was a marginally significant negative correlation between BMI and one-year cumulative (mg/kg) prednisone dose (P = .06). Types and frequency of posttransplant complications were similar between obese and nonobese patients, although there was a slightly higher incidence of wound related complications in obese patients (11% vs. 6.8%) (P = NS). There was no difference in the frequency of acute rejection episodes in obese and nonobese patients. Actuarial patient survival was comparable between patients with BMI < or = 27 versus those with BMI > 27 (P = .10). However, actuarial graft survival, both pancreas and renal, were significantly decreased in patients with BMI > 27 (P = .029). The decrease in pancreas and kidney graft survival in obese patients could not be attributed to decreased "early" patient survival, increased incidence of perioperative or postoperative complications, differences in hypertension, acute rejection episodes, serum lipids, or immunosuppression dosage. The most common causes of graft loss were rejection and patient death in both obese and nonobese patients. After three years posttransplant, the decreased pancreas and renal graft survival in obese patients corresponded to decreased patient survival. The most common cause of patient death was cardiovascular complications in both obese and nonobese PKT recipients.

Multiple pulmonary arteriovenous fistulas demonstrated by dynamic radionuclide pulmonary perfusion scanning.

Dynamic radionuclide perfusion scintigraphy performed with 99mTc-labeled microspheres was instrumental in establishing the diagnosis of multiple, small-vessel, pulmonary arteriovenous fistulas in a 14-month-old patient with cyanosis. Computer analysis of the sequential distribution of tagged microspheres in the pulmonary parenchyma normally demonstrates a curve that rises rapidly to a plateau as the particles microembolize. In the case reported here, the pulmonary flow curve rose rapidly to a maximum and then fell within 2 sec to a plateau of less than 50% of the maximum count, indicating that a large proportion of the microsophers passed through the pulmonary circulation. Conventional pulmonary contrast angiography did not demonstrate any intracardiac shunting but did confirm the presence of multiple pulmonary arteriovenous fistulas.

A comparison of radiotracer and biochemical methods for the quantitation of experimental myocardial infarct weight: in vitro relationships.

Anterior myocardial infarction (MI) was produced in conscious dogs to evaluate the relationships among: a) cardiac technetium-99m stannous pyrophosphate (TcPPi) accretion, b) creatine phosphokinase (CPK) depletion, and c) postmortem MI weight, infarct structure, and histology. In vitro, there was a close relationship between measured MI weight and MI weight calculated by the TcPPi accretion (r = 0.96) or CPK depletion (r = 0.93) in representative "cross-sectional" MI samples. Cardiac TcPPi accretion and CPK depletion showed a curvilinear relationship over the spectrum of tissue samples. Adjacent to infarcts, there was marked TcPPi uptake and modest CPK depletion where histology suggested ischemia without infarction. Within infarcts, microscopically visible calcium was rare in this series, suggesting little intracellular calcium accumulation, insensitivity of the von Kossa staining technique, and/or other cellular mechanisms to account for Tc-PPi uptake in this conscious dog model without reperfusion.

Myocardial imaging with Cc-99m pyrophosphate in patients on adriamycin treatment for neoplasia.

Technetium-99m pyrophosphate was utilized for myocardial imaging in 15 patients on adriamycin treatment for neoplasia. We have noted abnormal accumulation of the pyrophosphate in several patients, particularly in those in whom the so-called poor-risk factors were operative, namely prior radiation, cyclophosphamide therapy, and ischemic heart disease.

The predictive value of myocardial radioisotope scanning in animals treated with doxorubicin.

Thirty-four New Zealand white rabbits were treated with doxorubicin and imaged weekly with Tc-99m pyrophosphate to define the value of abnormal myocardial images in predicting doxorubicin-induced cardiac toxicity. Increased myocardial uptake was detected in most animals on sustained treatment with doxorubicin. A greater proportion of the heart was involved with doxorubicin-related histologic changes in animals with strongly positive myocardial images than in treated animals with moderately positive or normal scans. The myocardial images returned to normal levels 2--6 wk after doxorubicin was discontinued. Five of seven rabbits that received doxorubicin after they had three moderately positive myocardial scans, died from congestive heart failure. Three rabbits whose doxorubicin was discontinued because of scan findings, survived for 6 wk or more before dying from renal failure. The three rabbits who received the highest total dose of doxorubicin died of renal failure without developing abnormal myocardial scans.

A study of irradiated bone. III. Scintigraphic and radiographic detection of radiation-induced osteosarcomas.

Within 1 yr after localized irradiation of a hind limb with single (1756 rads) or fractionated (4650 rads in 3 wk) x-ray doses, radiation-induced osteosarcomas were observed in four of nine single-dose rabbits and two of 11 fractionated-dose rabbits. Tumors were observed in the proximal tibia in five cases and the distal femur in one case. In terms of production of osteoid or osseous tissue, three tumors were well differentiated, one slightly differentiated, and two (spindle-cell tumors) undifferentiated. This report summarizes the Tc-99m pyrophosphate (TcPPi) imaging and autoradiographic, radiographic, and histologic studies of these osteosarcomas. The four differentiated osteosarcomas were detected 1--2.5 mo earlier by TcPPi imaging than by radiography, whereas the two undifferentiated tumors were suspected 2 wk or 3.5 mo earlier radiographically. Autoradiograms showed TcPPi localization in bone produced by differentiated osteosarcomas, and in regions of reactive bone resorption and formation peripheral to tumors. The results support a recommendation for combined radiographic and scintigraphic techniques for the early detection of osteosarcomas.

The interleukin-2 receptor and its expression in the acute leukaemias and lymphoproliferative disorders.

The expression of the interleukin-2 receptor (IL-2R) was investigated in a total of 117 cases of acute leukaemia and lymphoproliferative disorder. The results confirm reports that the interleukin-2 receptor can be expressed on cells of myeloid lineage and in cases of Hairy Cell Leukaemia. They extend knowledge of the expression of the IL-2R in cases of lymphoproliferative disorders.

Effects of inhibitors of 5-hydroxytryptamine uptake on plasma glucose and their interaction with 5-hydroxytryptophan in producing hypoglycaemia in mice.

The effects of various inhibitors of 5-HT uptake on plasma glucose have been studied in normal and monoamine oxidase inhibitor pretreated mice. Additionally their interaction with 5-hydroxytryptophan (5-HTP) in producing hypoglycaemia was studied. Clomipramine, fenfluramine, fluoxetine, ORG 6582 (dl-8-chloro-11-anti-amino-benzo-(b)-bicyclo[3.3.1]nona-3, 6 alpha(10 alpha)-diene hydrochloride), ORG 6997 (dl-4-exo-amino-8-chloro-benzo-(b)-bicyclo[3.3.1]nona-2-6 alpha(10 alpha)-diene hydrochloride), MK 212 and mazindol did not modify the plasma glucose in normal mice but produced hypoglycaemia in mice pretreated with either nialamide or pargyline. Dexamphetamine did not influence plasma glucose in either normal or monoamine oxidase inhibitor pretreated mice. Each of the above drugs except ORG 6997 but including dexamphetamine augmented the hypoglycaemic effect of 5-HTP in normal mice. These responses did not appear to be mediated by insulin since none of the drugs increased the plasma immunoreactive insulin concentration or augmented the hyperinsulinaemic effect of 5-HTP. Moreover, fenfluramine, fluoxetine and ORG 6582 did not augment the hypoglycaemic action of injected insulin although such an augmentation was produced by mazindol.

Stimulatory effect of forskolin on gastric acid secretion in the rat, in vitro and in vivo.

The effect of forskolin, a potent adenylate cyclase activator, was studied on rat gastric acid secretion in vitro and in vivo. In the isolated gastric mucosa, forskolin was 25 and 100 times more potent than histamine and dibutyryl cyclic AMP respectively, with an EC50 of 2 microM and a maximum effect at 8-10 microM. In the lumen-perfused anaesthetised rat, acid secretion in response to a series of bolus i.v. injections of forskolin failed to show dose dependency, despite dose-related hypotensive effects. Secretory responses to i.v. infusions of forskolin were dose-related and were associated with a rise in gastric mucosal blood flow despite marked hypotension. These results indicate that forskolin has a direct secretagogue effect on parietal cells which, under some circumstances may be modified by its other pharmacological actions.

The effects of levodopa on plasma glucose in two strains of rat.

The effects of levodopa on plasma glucose were examined in two strains of rats. In fasted Wistar rats levodopa produced a dose-dependent hyperglycaemic response which was augmented by pretreatment with nialamide. This response in nialamide-treated rats was prevented by pretreatment with phentolamine and converted to a hypoglycaemic response. Phentolamine increased the plasma concentration of immunoreactive insulin (IRI). In phentolamine-pretreated rats levodopa produced a further marked increase in the plasma IRI concentration. It is suggested that the prevention of levodopa hyperglycaemia by phentolamine is due to the marked increase in the plasma IRI concentration produced by pretreatment with phentolamine. Moreover the fall in the plasma glucose concentration produced by levodopa in phentolamine-pretreated rats is likely to be due, at least in part, to the additional increase in the plasma IRI concentration produced by levodopa under these conditions. In contrast, in fasted Sprague-Dawley rats pretreated with nialamide, levodopa produced either no effect on plasma glucose, or in larger doses, a marked hypoglycaemic effect followed by death. This hypoglycaemic effect was accompanied by a decrease in the plasma IRI concentration.

Risperidone in the treatment of two very young children with autism.

This report describes the use of risperidone in the treatment of two very young children with autistic disorder, a 29-month-old boy and a 23-month-old boy, respectively. These children presented with severe and persistent symptoms of aggression and irritability that had not responded to previous treatment. In both cases, risperidone significantly reduced aggression and improved social relatedness. One patient's treatment with risperidone was complicated by persistent tachycardia and QTc interval prolongation that was dose-related. Consideration should be given to the appropriate use of medication in the treatment of very young children with autism when other interventions do not prove helpful.

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.