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.

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.

Decision making on detection and triage of oral mucosa lesions in community dental practices: screening decisions and referral.

UNLABELLED: Oral cancer is a substantial, often unrecognized issue globally, with close to 300 000 new cases reported annually. It is a management conundrum: a cancer site that is easily examined; yet more than 40% of oral cancers are diagnosed at a late stage when prognosis is poor and treatment can be devastating. Opportunistic screening within the dental office could lead to earlier diagnosis and intervention with improved survival. OBJECTIVE: To describe how clinicians make decisions about referral based on the risk classification of the lesion. METHODS: Eighteen dentists from 15 dental offices participated in a 1-day workshop on oral cancer screening. Participants then screened patients (medical history, conventional oral exam, fluorescent visualization examination) in-office for 11 months, triaging patients by apparent clinical risk: low risk (common benign conditions, geographic tongue, candidiasis, trauma), intermediate risk (lichenoid lesions) and high risk (white or red lesions or ulcers without apparent cause). Clinicians made the decision on which lesions to reassess in 3 weeks based on risk assessment and clinical judgment. Lesions of concern were seen by a community facilitator or referred to an oral medicine specialist. RESULTS: Of 2542 patients were screened, and 389 lesions were identified (15% of patients). 350 were determined to be low risk (90%), 19 intermediate risk (IR) (5%), and 20 high risk (HR) (5%). One hundred and sixty-six (43%) patients were recalled for 3-week reassessment: 90% of HR lesions, 63% of IR lesions (63%), and 39% of low-risk lesions. Compliance to recall was high (92% of cases). Reassessment eliminated the referral of 99/166 (60%) of lesions that had resolved. six lesions were biopsied with three low-grade dysplasias identified. CONCLUSIONS: Three key decision points were tested: risk assessment, need for reassessment, and need for referral. A 3-week reassessment appointment was invaluable to prevent the unnecessary referral due to confounders. There is a need for a well-defined triage pathway to facilitate oral cancer screening and a methodical and consistent approach to opportunistic screening in the dental office.

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.

Characterization of a ceramic ultrafiltration membrane in different operational states after its use in a heavy-metal ion removal process.

In the present study, the atomic force microscopy (AFM) technique has been used to characterize a Carbosep M5 ceramic membrane (MWCO=10kDa, TiO(2)-ZrO(2) active layer). This membrane was previously used in a polymer supported ultrafiltration (PSU) process to recover copper, using partially ethoxylated polyethylenimine as the water-soluble polymer. The membrane was characterized in four different operational states: new, new and cleaned, fouled in a PSU stage and cleaned after a PSU process. The influence of the membrane state on pore opening size distribution and roughness was studied, finding a 16% decrease in the former and a 20% increase in the latter due to foulant deposition upon the membrane active layer. Phase angle distribution was also analyzed to indicate the foulant spreading on the membrane surface. These phase angle measurements can be related to pore opening size and roughness, concluding that the cleaning procedure is not totally effective and that foulant presence on the membrane active layer is not remarkable. Finally, AFM was used to measure the influence of pH on adhesion forces between a silica probe and the membrane active layer. These results can be related to the flux evolution vs pH in PSU experiments, finding both lowest adhesion and highest flux at pH 6.

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.

AML with translocation t(8;16)(p11;p13) demonstrates unique cytomorphological, cytogenetic, molecular and prognostic features.

Balanced chromosomal rearrangements define distinct entities in acute myeloid leukemia (AML). Here, we present 13 AML cases with t(8;16)(p11;p13) with observed low incidence (13/6124 patients), but more frequent presentation in therapy-related AML than in de novo AML (7/438 versus 6/5686, P=0.00001). Prognosis was poor with median overall survival of 4.7 months. Cytomorphology was characterized by parallel positive myeloperoxidase and non-specific esterase staining, therefore, French-American-British (FAB)-classification was impossible and origin of the AML with t(8;16) from an early stem cell with myeloid and monoblastic potential is hypothesized. Erythrophagocytosis was observed in 7/13 cases. Using gene expression profiling on 407 cases, patients with t(8;16) were compared to AML FAB subtypes with normal karyotype. Principal component analyses demonstrated that AML with t(8;16) were distinct from FAB subtypes M1, M4, M5a/b. When further compared to AML showing balanced rearrangements, that is, current WHO categories t(15;17), t(8;21), inv(16) and t(11q23)/MLL, AML with t(8;16) cases were clustered close to t(11q23)/MLL sharing commonly expressed genes. Subsequently, a pairwise comparison discriminated AML with t(8;16) from AML with t(11q23)/MLL, thus defining a highly unique signature for AML with t(8;16). In conclusion, AML with t(8;16) demonstrates unique cytomorphological, cytogenetic, molecular and prognostic features and is a specific subtype of AML.

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.

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.

Stochastic elastohydrodynamics of a microcantilever oscillating near a wall.

We consider the thermally driven motion of a microcantilever in a fluid environment near a wall, a configuration characteristic of the atomic force microscope. A theoretical model is presented which accounts for hydrodynamic interactions between the cantilever and wall over a wide range of frequencies and which exploits the fluctuation-dissipation theorem to capture the Brownian dynamics of the coupled fluid-cantilever system. Model predictions are tested against experimental thermal spectra for a cantilever in air and water. The model shows how, in a liquid environment, the effects of non-delta-correlated Brownian forcing appear in the power spectrum, particularly at low frequencies. The model also predicts accurately changes in the spectrum in liquid arising through hydrodynamic wall effects, which we show are strongly mediated by the angle at which the cantilever is tilted relative to the wall.

Immune response in silico (IRIS): immune-specific genes identified from a compendium of microarray expression data.

Immune cell-specific expression is one indication of the importance of a gene's role in the immune response. We have compiled a compendium of microarray expression data for virtually all human genes from six key immune cell types and their activated and differentiated states. Immune Response In Silico (IRIS) is a collection of genes that have been selected for specific expression in immune cells. The expression pattern of IRIS genes recapitulates the phylogeny of immune cells in terms of the lineages of their differentiation. Gene Ontology assignments for IRIS genes reveal significant involvement in inflammation and immunity. Genes encoding CD antigens, cytokines, integrins and many other gene families playing key roles in the immune response are highly represented. IRIS also includes proteins of unknown function and expressed sequence tags that may not represent genes. The predicted cellular localization of IRIS proteins is evenly distributed between cell surface and intracellular compartments, indicating that immune specificity is important at many points in the signaling pathways of the immune response. IRIS provides a resource for further investigation into the function of the immune system and immune diseases.

Direct atomic force microscopy observations of monovalent ion induced binding of DNA to mica.

Multivalent ions in solution are known to mediate attraction between two like-charged molecules. Such attraction has proved useful in atomic force microscopy (AFM) where DNA may be immobilized to a mica surface facilitating direct imaging in liquid. Theories of DNA immobilization suggest that either 'salt bridging' or fluctuation in the positions of counter ions about both the mica surface and DNA backbone secure DNA to the mica substrate. Whilst both theoretical and experimental evidence suggest that immobilization is possible in the presence of divalent ions, very few studies identify that such immobilization is possible with monovalent ions. Here we present direct AFM evidence of DNA immobilized to mica in the presence of only monovalent ions. Our data depict E. coli plasmid pBR322 adsorbed onto the negatively charged mica both after short (10 min) and long (24 h) incubation periods. These data suggest the need to re-explore current theories of like-charge attraction to include the possibility of monovalent interactions. We suggest that this DNA immobilization strategy may offer the potential to image natural processes with limited immobilization forces and hence enable maximum conformational freedom of the immobilized biomolecule.

Electrostatic interactions observed when imaging proteins with the atomic force microscope.

The atomic force microscope (AFM) is now an established and valuable tool for the study of biological macromolecules in aqueous environments. In this paper we form a patterned boundary via the microcontact printing of individually isolated proteins, covalently attached to a solid support. We use this boundary to investigate electrostatic interactions that can occur between an AFM tip and a protein surface during imaging in solution. The observed height variations of the protein film are found to be a combination of not only structural considerations and thickness of the protein film, but also the repulsive contribution from electrostatic interactions between the AFM tip and the sample. These variations in measured heights of the protein surface can be described by Derjaguin, Landau, Verway, Overbeek (DLVO) theory. Our experimental results show that height measurements can be manipulated either negatively or positively by adjusting the pH and concentration of the electrolyte buffer that is utilised.

Non-invasive assessment of selective 5-HT(1B/1D)-receptor agonist-induced peripheral vascular effects in humans: comparison of different techniques.

OBJECTIVE: To compare the sensitivity of three non-invasive techniques for detecting serotonin (5-HT)(1B/1D)-receptor agonist-induced peripheral vascular effects in humans: the measurement of (1) systolic (SBP) and diastolic (DBP) blood pressures, (2) dorsal hand vein (DHV) diameter and (3) toe-arm systolic blood pressure gradient (DeltaSBP(toe-arm)). METHODS: A double-blind, placebo-controlled, three-way, cross-over study was performed in 12 healthy male volunteers. According to a randomly assigned allocation schedule, subjects were administered sumatriptan 3 mg, sumatriptan 6 mg or placebo subcutaneously. Measurements were performed at baseline, every 5 min for 30 min and at 40 min and 60 min after drug administration. SBP and DBP were recorded using a semi-automated oscillometric device. DHV diameter was measured using a linear variable differential transformer. DeltaSBP(toe-arm) was calculated after measuring toe and arm SBP with a strain-gauge technique. Sensitivity was evaluated with responsiveness statistics. RESULTS: Based on weighted mean and compared with placebo, sumatriptan 3 mg and 6 mg increased SBP by 3.3 mmHg ( P=0.023) and 6.4 mmHg ( P<0.001) and DBP by 5.0 mmHg ( P=0.006) and 7.5 mmHg ( P<0.001), respectively. Sumatriptan 3 mg and 6 mg decreased DHV diameter by 36% ( P=0.015) and 40% ( P=0.005), respectively. DeltaSBP(toe-arm) did not change. Peak changes were observed within 10-15 min after drug administration. The rank order of responsiveness was: BP > DHV diameter > DeltaSBP(toe-arm.) CONCLUSIONS: Clinically relevant doses of subcutaneous sumatriptan increased blood pressure and decreased DHV diameter without affecting DeltaSBP(toe-arm). The increase in blood pressure appeared to be dose dependent. Compared with DHV diameter and DeltaSBP(toe-arm), blood pressure measurement appeared to be the most sensitive technique for detecting selective 5-HT(1B/1D)-receptor agonist-induced peripheral vascular effects in humans.

Interactions of 3T3 fibroblasts and endothelial cells with defined pore features.

The colonization of biodegradable polymer scaffolds with cell populations has been established as the foundation for the engineering of a number of tissues, including cartilage, liver, and bone. Within these scaffolds, the cells encounter a porous environment in which they must migrate across the convoluted polymer surface to generate a homogenous cell distribution. Predicting the interactions between cells and pores is important if scaffold characteristics are to be optimized. Therefore, we investigated the behavior of two model cell types over a range of defined pore features. These pore features range from 5 to 90 microm in diameter and have been fabricated by photolithographic techniques. Quantitatively, the behavior of the cells is dependent on three factors: 1) percentage cell coverage of the surface; 2) pore size; and 3) cell type. Fibroblast cells displayed a co-operative pattern of cell spreading in which pores with diameters greater than the cell dimensions were bridged by groups of cells using their neighbors as supports. Endothelial cells were unable to use neighbors as support structures and failed to bridge pores greater than the cell diameter.

Single-nucleotide polymorphism alleles in the insulin receptor gene are associated with typical migraine.

We have identified a migraine locus on chromosome 19p13.3/2 using linkage and association analysis. We isolated 48 single-nucleotide polymorphisms within the locus, of which we genotyped 24 in a Caucasian population comprising 827 unrelated cases and 765 controls. Five single-nucleotide polymorphisms within the insulin receptor gene showed significant association with migraine. This association was independently replicated in a case-control population collected separately. We used experiments with insulin receptor RNA and protein to investigate functionality for the migraine-associated single-nucleotide polymorphisms. We suggest possible functions for the insulin receptor in migraine pathogenesis.