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.

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.

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.

Synthesis and characterisation of a degradable poly(lactic acid)-poly(ethylene glycol) copolymer with biotinylated end groups.

Poly(lactic acid)-poly(ethylene glycol)-biotin (PLA-PEG-biotin) is a degradable polymer with protein resistant properties that can undergo rapid surface engineering in aqueous media to create biomimetic surfaces. Surface engineering of this polymer is dependent on biomolecular interactions between the biotin end group and the protein avidin. Given the vigorous conditions of synthesis, it is essential that the manufacture of the polymer does not alter the biotin structure or its molecular recognition. Equally, it is important that the incorporation of biotin does not adversely affect the physicochemical properties of the polymer. (1)H NMR provides evidence of biotin attachment and structural integrity. (1)H NMR, gel permeation chromatography (GPC), and differential scanning calorimetry (DSC) analysis shows there is no significant effect on bulk properties induced by the biotin end group. Surface plasmon resonance (SPR) and fluorescent spectroscopy studies using the 2-(4'-hydroxyazobenzene) benzoic acid (HABA)/avidin complex show that the biotin moieties binding capabilities are not impaired by the synthesis.

Impact of steroids on recovery after uvulopalatopharyngoplasty.

OBJECTIVE: To determine whether perioperative systemic corticosteroid administration can reduce uvulopalatopharyngoplasty (UPPP) postoperative morbidities (e.g., pain, anorexia, sleep disturbance, mouth odor, and fatigue) or reduce narcotic analgesic usage. STUDY DESIGN: A prospective, double-blinded study with random assignment of treatment agent (placebo or corticosteroid). METHODS: From 1995 to 1998, a consecutive sample of 48 adults presenting for elective UPPP surgery alone or in combination with tonsillectomy or septoplasty, or both, were enrolled. Twenty-eight subjects completed the protocol and were equally distributed by random assignment to intramuscular (IM) and intravenous (IV) doses of placebo (saline) or corticosteroid (60 mg methylprednisolone IM and 12 mg dexamethasone IV). Acetaminophen with codeine analgesic was available to both groups as needed. Subjects recorded a diary of symptom severity scores over the first postoperative week relating to eight commonly reported morbidities (1-4 points) and the daily quantity of narcotic consumed. RESULTS: Statistical comparison (Wilcoxon's rank sum test) showed no significant differences between subjects treated with placebo or corticosteroid on postoperative day 1 or 7. Three subjects (21%) in each treatment group reported no postoperative use of narcotic analgesic. CONCLUSIONS: No statistically or clinically significant benefits were derived from perioperative systemic corticosteroid treatment in this sample of 28 adults treated with UPPP alone or in combination with tonsillectomy or septoplasty, or both. Some individuals tolerate post-UPPP discomfort without a narcotic analgesic.

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.

Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro.

In this article we present validation of a real-time RT-PCR method to quantitate mRNA expression levels of atrial natriuretic peptide and c-fos in an in vitro model of cardiac hypertrophy. This method requires minimal sample and no postreaction manipulation. In real-time RT-PCR a dual-labeled fluorescent probe is degraded concomitant with PCR amplification. Input target mRNA levels are correlated with the time (measured in PCR cycles) at which the reporter fluorescent emission increases beyond a threshold level. The use of an oligo(dt) magnetic bead protocol to harvest poly(A) mRNA from cultured cells in 96-well plates minimized DNA contamination. We show that the GAPDH gene chosen for normalization of the RNA load is truly invariant throughout the biological treatments examined. We discuss two methods of calculating fold increase: a standard curve method and the DeltaDelta Ct method. Real-time quantitative RT-PCR was used to determine the time course of c-fos induction and the effect of varying doses of four known hypertrophy agents on atrial naturitic factor messenger RNA expression in cultured cardiac muscle cells. Our results agree with published data obtained from Northern blot analysis.

Phenylephrine, endothelin, prostaglandin F2alpha' and leukemia inhibitory factor induce different cardiac hypertrophy phenotypes in vitro.

In these studies, we show that endothelin (ET), leukemia inhibitory factor (LIF), phenylephrine (PE), and prostaglandin F2alpha (PGF2alpha), which are all hypertrophic for neonatal rat cardiac myocytes in culture, induce distinct morphological, physiological, and genetic changes after a 48-h treatment. Transmission electron microscopy revealed differences in myofibril organization, with ET-treated cells containing the most mature-looking myofibrils and PGF2alpha- and LIF-treated cells the least. ET- and PE-treated cultures contained the same number of beating cells as control, but LIF and PGF2alpha treatment increased the number of beating cells 180%. Treatment with LIF, PE, and PGF2alpha increased the beat rate to 3.3 times that of control. After exposure to the beta-adrenergic agonist isoproterenol, the beat rate increased 50% for PGF2alpha' 54% for PE, 84% for LIF, and 125% for control. ET treatment did not increase the beat rate, nor did these cells respond to isoproterenol. ET, LIF, and PE increased the production of atrial natriuretic peptide (ANP) by three-fold and PGF2alpha by 18-fold over nontreated cells. Brain natriuretic peptide (BNP) was increased fourfold by ET and PE, 16-fold by LIF, and 29-fold by PGF2alpha. Interestingly, on a pmol/L basis, only LIF induced more BNP than ANP. Treatment with all agents led to a similar pattern of gene induction: increased expression of the embryonic genes for ANP and skeletal alpha-actin, and less than a twofold change in the constitutively expressed gene myosin light chain-2, with the exception that LIF did not induce skeletal alpha-actin. Each agent, however, induced ANP mRNA with a different time-course. We conclude that at least four distinct cardiac myocyte hypertrophy response programs can be induced in vitro. Further studies are necessary to determine whether these correlate to the different types of cardiac hypertrophy seen in vivo.

Effects of glycosylation on fragments of tumour associated human epithelial mucin MUC1.

The glycodecapeptide AcPAPGS(alpha GalNAc)T(alpha GalNAc)APPA and the C-terminal glycohexapeptide AcS(alpha GalNAc)T(alpha GalNAc)APPA have been synthesized by applying the N-terminal Fmoc group in combination with the heptyl ester cleavable by lipase-catalyzed hydrolysis at pH 7. The solution conformation of these MUC1-related synthetic glycopeptides and the control, non-glycosylated decapeptide AcPAPGSTAPPA have been investigated using NMR spectroscopy. The structural studies indicate that the glycohexapeptide has a folded structure in solution. For this molecule, unrestrained molecular dynamics has been used to confirm the presence of the observed solution through-space connections. The results indicate that the non-globular nature of MUC1 is due to both protein core sequence and the effect of carbohydrate.

Spatially controlled cell engineering on biodegradable polymer surfaces.

Controlling receptor-mediated interactions between cells and template surfaces is a central principle in many tissue engineering procedures (1-3). Biomaterial surfaces engineered to present cell adhesion ligands undergo integrin-mediated molecular interactions with cells (1, 4, 5), stimulating cell spreading, and differentiation (6-8). This provides a mechanism for mimicking natural cell-to-matrix interactions. Further sophistication in the control of cell interactions can be achieved by fabricating surfaces on which the spatial distribution of ligands is restricted to micron-scale pattern features (9-14). Patterning technology promises to facilitate spatially controlled tissue engineering with applications in the regeneration of highly organized tissues. These new applications require the formation of ligand patterns on biocompatible and biodegradable templates, which control tissue regeneration processes, before removal by metabolism. We have developed a method of generating micron-scale patterns of any biotinylated ligand on the surface of a biodegradable block copolymer, polylactide-poly(ethylene glycol). The technique achieves control of biomolecule deposition with nanometer precision. Spatial control over cell development has been observed when using these templates to culture bovine aortic endothelial cells and PC12 nerve cells. Furthermore, neurite extension on the biodegradable polymer surface is directed by pattern features composed of peptides containing the IKVAV sequence (15, 16), suggesting that directional control over nerve regeneration on biodegradable biomaterials can be achieved.

A novel method for real time quantitative RT-PCR.

A novel approach to quantitative reverse transcriptase polymerase chain reaction (QC RT-PCR) using real time detection and the 5' nuclease assay has been developed. Cystic fibrosis transmembrane transductance regulator (CFTR) target mRNA is reverse transcribed, amplified, detected, and quantitated in real time. A fluorogenic probe was designed to detect the CFTR amplicon. Relative increase in 6-carboxy-fluorescein reporter fluorescent emission is monitored during PCR amplification using an analytical thermal cycler. An internal control template containing the same primer sequences as the CFTR amplicon, but a different internal sequence, has been designed as a control. An internal control probe with a reporter fluorescent dye tetrachloro-6-carboxy-fluorescein was designed to hybridize to the internal control amplicon. The internal control template is placed in each reaction tube and is used for quantitative analysis of the CFTR mRNA. This method provides a convenient and high-throughput format for QC RT-PCR.

Cardiac fibroblasts produce leukemia inhibitory factor and endothelin, which combine to induce cardiac myocyte hypertrophy in vitro.

Cardiac fibroblasts in culture produce factor(s) that induce hypertrophy of neonatal rat ventricular myocytes in vitro. As in vivo, the myocyte hypertrophy response in culture is characterized by an increase in cell size and contractile protein content, and by the activation of embryonic genes, including the gene for atrial natriuretic peptide. The purpose of this study was to identify the factor(s) produced by fibroblasts that induce myocyte hypertrophy. The fibroblast hypertrophy activity was inhibited using a combination of the endothelin A receptor blocker BQ-123 and an antibody to leukemia inhibitory factor. The individual antagonists each caused a partial inhibition. The mRNAs for both leukemia inhibitory factor and endothelin were detected by RT-PCR analysis and the concentration of both proteins was determined to be approximately 200 pmol/L in the conditioned medium using immunoassays. Purified leukemia inhibitory factor and endothelin each induced distinctive morphological changes in the myocytes. Their combination generated a different morphology similar to that induced by fibroblast conditioned medium. Each factor also induced atrial natriuretic peptide production, but both were required for the myocytes to produce the levels measured after exposure to fibroblast conditioned medium. These results show that hypertrophy activity produced by cardiac fibroblasts in culture is a result of leukemia inhibitory factor and endothelin.

The Haemophilus influenzae sxy-1 mutation is in a newly identified gene essential for competence.

The sxy-1 mutation of Haemophilus influenzae causes a 100- to 1,000-fold increase in spontaneous natural competence. We have used mapping and sequencing to identify this mutation as a G-to-A transition in an open reading frame adjacent to the rec-1 locus. This mutation substitutes valine for isoleucine at amino acid 19 of the protein specified by this gene (now named sxy). A multicopy plasmid containing the wild-type sxy gene confers constitutive competence on wild-type cells. Cells carrying this plasmid exhibit, in all stages of growth, DNA uptake levels and transformation frequencies as high those normally seen only after full induction of competence by starvation; deletion of part of the sxy gene from the plasmid abolishes this effect. In contrast, a transposon insertion in sxy entirely prevents both DNA uptake and transformation, indicating that sxy encodes a function essential for competence. These findings suggest that sxy may act as a positive regulator of competence. However, because cells carrying the transposon-inactivated sxy::Tn allele grow slowly under conditions that do not induce competence, sxy may also have a role in noncompetent cells.