Aligning tumor mutational burden (TMB) quantification across diagnostic platforms: phase II of the Friends of Cancer Research TMB Harmonization Project.

BACKGROUND: Tumor mutational burden (TMB) measurements aid in identifying patients who are likely to benefit from immunotherapy; however, there is empirical variability across panel assays and factors contributing to this variability have not been comprehensively investigated. Identifying sources of variability can help facilitate comparability across different panel assays, which may aid in broader adoption of panel assays and development of clinical applications. MATERIALS AND METHODS: Twenty-nine tumor samples and 10 human-derived cell lines were processed and distributed to 16 laboratories; each used their own bioinformatics pipelines to calculate TMB and compare to whole exome results. Additionally, theoretical positive percent agreement (PPA) and negative percent agreement (NPA) of TMB were estimated. The impact of filtering pathogenic and germline variants on TMB estimates was assessed. Calibration curves specific to each panel assay were developed to facilitate translation of panel TMB values to whole exome sequencing (WES) TMB values. RESULTS: Panel sizes >667 Kb are necessary to maintain adequate PPA and NPA for calling TMB high versus TMB low across the range of cut-offs used in practice. Failure to filter out pathogenic variants when estimating panel TMB resulted in overestimating TMB relative to WES for all assays. Filtering out potential germline variants at >0% population minor allele frequency resulted in the strongest correlation to WES TMB. Application of a calibration approach derived from The Cancer Genome Atlas data, tailored to each panel assay, reduced the spread of panel TMB values around the WES TMB as reflected in lower root mean squared error (RMSE) for 26/29 (90%) of the clinical samples. CONCLUSIONS: Estimation of TMB varies across different panels, with panel size, gene content, and bioinformatics pipelines contributing to empirical variability. Statistical calibration can achieve more consistent results across panels and allows for comparison of TMB values across various panel assays. To promote reproducibility and comparability across assays, a software tool was developed and made publicly available.

Ado-trastuzumab emtansine (T-DM1) in patients with HER2-amplified tumors excluding breast and gastric/gastroesophageal junction (GEJ) adenocarcinomas: results from the NCI-MATCH trial (EAY131) subprotocol Q.

BACKGROUND: The National Cancer Institute-Molecular Analysis for Therapy Choice (NCI-MATCH) is a national precision medicine study incorporating centralized genomic testing to direct refractory cancer patients to molecularly targeted treatment subprotocols. This treatment subprotocol was designed to screen for potential signals of efficacy of ado-trastuzumab emtansine (T-DM1) in HER2-amplified histologies other than breast and gastroesophageal tumors. METHODS: Eligible patients had HER2 amplification at a copy number (CN) >7 based on targeted next-generation sequencing (NGS) with a custom Oncomine AmpliSeq™ (ThermoFisher Scientific) panel. Patients with prior trastuzumab, pertuzumab or T-DM1 treatment were excluded. Patients received T-DM1 at 3.6 mg/kg i.v. every 3 weeks until toxicity or disease progression. Tumor assessments occurred every three cycles. The primary end point was centrally assessed objective response rate (ORR). Exploratory end points included correlating response with HER2 CN by NGS. The impact of co-occurring genomic alterations and PTEN loss by immunohistochemistry were also assessed. RESULTS: Thirty-eight patients were enrolled and 36 included in efficacy analysis. Median prior therapies in the metastatic setting was 3 (range 0-9; unknown in one patient). Median HER2 CN was 17 (range 7-139). Partial responses were observed in two (5.6%) patients: one mucoepidermoid carcinoma of parotid gland and one parotid gland squamous cell cancer. Seventeen patients (47%) had stable disease including 8/10 (80%) with ovarian and uterine carcinomas, with median duration of 4.6 months. The 6-month progression-free survival rate was 23.6% [90% confidence interval 14.2% to 39.2%]. Common toxicities included fatigue, anemia, fever and thrombocytopenia with no new safety signals. There was a trend for tumor shrinkage with higher levels of gene CN as determined by the NGS assay. CONCLUSION: T-DM1 was well tolerated. While this subprotocol did not meet the primary end point for ORR in this heavily pre-treated diverse patient population, clinical activity was seen in salivary gland tumors warranting further study in this tumor type in dedicated trials.

Influence of fluorescence on screening decisions for oral mucosal lesions in community dental practices.

BACKGROUND: Quality of oral screening examinations is dependent upon the experience of the clinician and can vary widely. Deciding when a patient needs to be referred is a critical and difficult decision for general practice clinicians. A device to aid in this decision would be beneficial. The objective of this study was to to examine the utility of direct fluorescence visualization (FV) by dental practitioners as an aid in decision-making during screening for cancer and other oral lesions. METHODS: Dentists were trained to use a stepwise protocol for evaluation of the oral mucosa: medical history, head, neck and oral exam, and fluorescent visualization exam. They were asked to use clinical features to categorize lesions as low (LR), intermediate (IR), or high (HR) risk and then to determine FV status of these lesions. Clinicians made the decision of which lesions to reassess in 3 weeks and based on this reassessment, to refer forward. RESULTS: Of 2404 patients screened over 11 months, 357 initially had lesions with 325 (15%) identified as LR, 16 (4.5%) IR, and 16 (4.5%) HR. Lesions assessed initially as IR and HR had a 2.7-fold increased risk of FV loss persisting to the reassessment appointment versus the LR lesions. The most predictive model for lesion persistence included both FV status and lesion risk assessment. CONCLUSION: A protocol for screening (assess risk, reassess, and refer) is recommended for the screening of abnormal intraoral lesions. Integrating FV into a process of assessing and reassessing lesions significantly improved this model.

Blow fly Lucilia sericata nuclease digests DNA associated with wound slough/eschar and with Pseudomonas aeruginosa biofilm.

In chronic wounds, it may be clinically important to remove extracellular bacterial and patient DNA as its presence may impede wound healing and promote bacterial survival in biofilm, in which extracellular DNA forms part of the biofilm architecture. As medicinal maggots, larvae of Lucilia sericata Meigen (Diptera: Calliphoridae) have been shown to efficiently debride wounds it became of interest to investigate their excretions/secretions (ES) for the presence of a deoxyribonuclease (DNAse) activity. Excretions/secretions products were shown to contain a DNAse, with magnesium, sodium and calcium metal ion dependency, and a native molecular mass following affinity purification of approximately 45 kDa. The affinity purified DNAse degraded genomic bacterial DNA per se, DNA from the slough/eschar of a venous leg ulcer, and extracellular bacterial DNA in biofilms pre-formed from a clinical isolate of Pseudomonas aeruginosa. The latter finding highlights an important attribute of the DNAse, given the frequency of P. aeruginosa infection in non-healing wounds and the fact that P. aeruginosa virulence factors can be toxic to maggots. Maggot DNAse is thus a competent enzyme derived from a rational source, with the potential to assist in clinical wound debridement by removing extracellular DNA from tissue and biofilm, and promoting tissue viability, while liberating proteinaceous slough/eschar for debridement by the suite of proteinases secreted by L. sericata.

Crizotinib in patients with tumors harboring ALK or ROS1 rearrangements in the NCI-MATCH trial.

The NCI-MATCH was designed to characterize the efficacy of targeted therapies in histology-agnostic driver mutation-positive malignancies. Sub-protocols F and G were developed to evaluate the role of crizotinib in rare tumors that harbored either ALK or ROS1 rearrangements. Patients with malignancies that progressed following at least one prior systemic therapy were accrued to the NCI-MATCH for molecular profiling, and those with actionable ALK or ROS1 rearrangements were offered participation in sub-protocols F or G, respectively. There were five patients who enrolled on Arm F (ALK) and four patients on Arm G (ROS1). Few grade 3 or 4 toxicities were noted, including liver test abnormalities, and acute kidney injury. For sub-protocol F (ALK), the response rate was 50% (90% CI 9.8-90.2%) with one complete response among the 4 eligible patients. The median PFS was 3.8 months, and median OS was 4.3 months. For sub-protocol G (ROS1) the response rate was 25% (90% CI 1.3-75.1%). The median PFS was 4.3 months, and median OS 6.2 months. Data from 3 commercial vendors showed that the prevalence of ALK and ROS1 rearrangements in histologies other than non-small cell lung cancer and lymphoma was rare (0.1% and 0.4% respectively). We observed responses to crizotinib which met the primary endpoint for ALK fusions, albeit in a small number of patients. Despite the limited accrual, some of the patients with these oncogenic fusions can respond to crizotinib which may have a therapeutic role in this setting.

Confounding experimental considerations in nanogenotoxicology.

The development of novel nanomaterials with unique physico-chemical properties is increasing at a rapid rate, with potential applications across a broad range of manufacturing industries and consumer products. Nanomaterial safety is therefore becoming an increasingly contentious issue that has intensified over the past 4 years, and in response, a steady stream of studies focusing on nanotoxicology are emerging. However, it is becoming increasingly evident that nanomaterials cannot be treated in the same manner as chemical compounds with regards to their safety assessment, as their unique physico-chemical properties are also responsible for unexpected interactions with experimental components that generate misleading data-sets. In this report, we focus on nanomaterial interactions with colorimetric and fluorometric dyes, components of cell culture growth medium and genotoxicity assay components, and the resultant consequences on test systems are demonstrated. Thus, highlighting some of the potential confounding factors that need to be considered in order to ensure that in vitro genotoxicity assays report true biological impacts in response to nanomaterial exposure.

Tritium and carbon-14 distributions in seawater from under the ross ice shelf project ice hole.

The tritium and carbon-14 activities of seawater samples collected from 22 to 200 meters below the ice at the Ross Ice Shelf Project ice hole are reported. The tritium results show that the waters below the ice have exchanged with Ross Sea water since the advent of nuclear testing. The carbon-14 results indicate that waters in the upper layer exchange in time periods of less than 6 years. Measurements of these isotopes in seawater under the Ross Ice Shelf in McMurdo Sound show that this water has a different history.

The use of prophylaxis against HIV after sexual exposure in pregnancy to reduce the risk of vertical transmission.

We present a case demonstrating the use of HIV prophylaxis post-sexual exposure in pregnancy. This has not been presented before but has certainly been discussed.

Airway epithelial CFTR mRNA expression in cystic fibrosis patients after repetitive administration of a recombinant adenovirus.

We sought to evaluate the ability of an E1(-), E3(-) adenovirus (Ad) vector (Ad(GV)CFTR.10) to transfer the normal human cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to the airway epithelium of individuals with cystic fibrosis (CF). We administered Ad(GV)CFTR.10 at doses of 3 x 10(6) to 2 x 10(9) plaque-forming units over 9 months by endobronchial spray to 7 pairs of individuals with CF. Each 3-month cycle, we measured vector-derived versus endogenous CFTR mRNA in airway epithelial cells prior to therapy, as well as 3 and 30 days after therapy. The data demonstrate that (a) this strategy appears to be safe; (b) after the first administration, vector-derived CFTR cDNA expression in the CF airway epithelium is dose-dependent, with greater than 5% endogenous CFTR mRNA levels at the higher vector doses; (c) expression is transient, lasting less than 30 days; (d) expression can be achieved with a second administration, but only at intermediate doses, and no expression is observed with the third administration; and (e) the progressive lack of expression with repetitive administration does not closely correlate with induction of systemic anti-Ad neutralizing antibodies. The major advantage of an Ad vector is that it can deliver sufficient levels of CFTR cDNA to the airway epithelium so that CFTR expression protects the lungs from the respiratory manifestations of CF. However, this impressive level of expression is linked to the challenging fact that expression is limited in time. Although this can be initially overcome by repetitive administration, unknown mechanisms eventually limit this strategy, and further repetitive administration does not lead to repetitive expression.

Audit of hepatitis C antibody requests made by a genito-urinary department.

Requests for hepatitis C antibodies (HCV Ab) made by the Oxford Department of Genito-urinary (GU) Medicine were audited against national guidelines to determine how the service performed and to develop locally applicable standards. This resulted in more effective screening and cost savings.

Effects of early angiotensin-converting enzyme inhibition on cardiac gene expression after acute myocardial infarction.

BACKGROUND: ACE inhibition after myocardial infarction (MI) has been shown to have beneficial effects on cardiac anatomy and function. The purpose of this study was to examine the effects of ACE inhibition on cardiac gene expression after MI. METHODS AND RESULTS: Rats were randomized to receive captopril or no treatment 1 day after MI. Eight weeks later, cardiac function and hemodynamics were measured by use of indwelling catheters and perivascular flow probes. Myocardial gene expression was assessed with DNA microarrays and real-time reverse transcription-polymerase chain reaction. The ratios of heart and left ventricular weights to body weight were significantly increased by MI and normalized by captopril. Cardiac index and stroke volume index were lower in the untreated MI group than in sham controls but were normal in the MI+captopril group. Thirty-seven genes were found to be differentially expressed between the untreated MI group and sham controls; 31 were induced and 6 repressed. Captopril partially or completely inhibited changes in 10 of the genes. The 37 genes clustered into 11 functional groups, and 6 had >/=1 genes whose expression was modified by ACE inhibition. CONCLUSIONS: ACE inhibition after MI inhibits cardiac hypertrophy, preserves cardiac function, and attenuates changes in myocardial gene expression. Gene expression profiling reveals, however, that some elements of the pathophysiology may be unaffected by the treatment and be targets for new therapies.

Glucocorticoid induction of the adipocyte clone 5 gene requires high cell density.

We have previously reported the identification of a glucocorticoid and cell-density inducible gene (clone 5) that appears to play a regulatory role in adipocyte differentiation. In the current studies we have more carefully investigated the interplay between cell growth or differentiation and the glucocorticoid responsiveness of clone 5 RNA. We find that inducibility by steroid hormone is independent of the differentiated state but surprisingly, occurs only when cells are at high cell density. In subconfluent TA1 cells clone 5 RNA is refractory to induction whereas well characterized glucocorticoid-inducible promoters such as the those from mouse mammary tumor virus and alpha-1-acid glycoprotein are induced under all conditions. These results point to the necessity of ancillary factors, absent in low density cell culture, that are required for glucocorticoid responsiveness of the clone 5 gene.

CCAAT/enhancer binding protein expression is rapidly extinguished in TA1 adipocyte cells treated with tumor necrosis factor.

Tumor necrosis factor (TNF) has been shown to have diverse effects on a wide variety of cell types. In the mouse adipogenic TA1 cell line, TNF completely abolishes differentiation and reverts fully differentiated fat cells into fibroblasts. This block in differentiation and its reversal is due to the rapid reduction in the expression of adipose-specific genes. This study reports that the transcription factor, CCAAT/enhancer binding protein (C/EBP), previously reported to promote the differentiation of 3T3-L1 adipocytes, is expressed in TA1 cells. During their growth in culture, the levels of C/EBP, as evidenced by its cellular levels of specific mRNA, protein, and DNA binding activity, increase dramatically when cells reach confluence and proceed to differentiate. Addition of TNF to cultured preadipocytes or fully differentiated adipocytes rapidly reduces C/EBP levels and is accompanied by the decrease in expression of adipose-specific genes. C/EBP binding sites occur in several adipose-specific genes, and here it is demonstrated that its presence in a novel adipose-specific gene, Clone 47, also referred to as FSP27, may be responsible for the strong down-regulation of the expression of the Clone 47 (FSP27) promoter-linked chloramphenicol acetyl transferase gene by TNF. This study proposes that the loss of C/EBP in response to TNF treatment may in part explain the loss of the adipocyte differentiated state.

Atomic force microscopy and scanning tunnelling microscopy: refining techniques for studying biomolecules.

The scanning tunnelling microscope and the atomic force microscope offer the prospect of real-time, nanometre-scale imaging of biomolecules and biosurfaces under physiological environments. Much effort is therefore being made to establish these techniques as routine biophysical tools. The considerable recent progress that has been made in biotechnological applications is reviewed, highlighting specific examples of the applications of this new and exciting method of analysis.

A novel organic solvent-based coupling method for the preparation of covalently immobilized proteins on gold.

A novel organic solvent-based coupling method has been developed for the covalent immobilization of biological material to gold surfaces. The method employs the polar organic solvent anhydrous 2,2,2-trifluoroethanol as the reaction medium and involves dissolution of the protein (catalase) in the solvent allowing protein coupling to proceed under basic conditions in a dry organic environment. The advantage of this method is that protein attachment is favored over hydrolysis of the coupling reagent. We have shown qualitatively and quantitatively that following attachment to the gold surface a significant proportion of the enzyme catalase remains catalytically active (at least 20-31%).

Scanning tunnelling microscopy studies of beta-amyloid fibril structure and assembly.

Alzheimer's disease is in part characterised by the deposit of beta-amyloid peptide in the form of fibrils in the brain. In this study, the scanning tunnelling microscope (STM) has been used to provide high resolution images of synthetic fibril structure and formation as a function of time. Short fibrils are observed following brief peptide incubation times. At longer incubation periods ribbon like filaments were observed. These results suggest that beta-amyloid self-assembly is an ordered process, with a correlation between time of incubation and length of beta-amyloid filament growth.

Observation of DNA-polymer condensate formation in real time at a molecular level.

Dynamic real time assembly of toroidal and rod-like DNA condensates has been visualised using atomic force microscopy. Imaging has been conducted in an aqueous environment allowing the visualisation of hydrated, pegylated-polymer DNA condensates undergoing dynamic structural movement and conformational change. A major hurdle in the field of gene delivery is cellular transfection and the subsequent transfer of condensed genetic material to the cell nucleus. An increased understanding of the process of DNA condensation will aid the development and optimisation of gene delivery vectors.

In situ observation of streptavidin-biotin binding on an immunoassay well surface using an atomic force microscope.

Polystyrene microtitre wells are commonly used as supports for the enzyme-linked immunosorbent assay (ELISA) method of biomolecular detection, which is employed in the routine diagnosis of a variety of medical conditions. We have used an atomic force microscope (AFM) to directly monitor specific molecular interactions between individual streptavidin and biotin molecules on such wells. This was achieved by functionalising an AFM probe with biotin and monitoring the adhesive forces between the probe and a streptavidin coated immunoassay well. The results demonstrate that the AFM may be employed as an analytical tool to study the interactions between biomolecules involved in immunoassay systems.