Investigating the feasibility of tumour molecular profiling in gastrointestinal malignancies in routine clinical practice.

Background: Targeted capture sequencing can potentially facilitate precision medicine, but the feasibility of this approach in gastrointestinal (GI) malignancies is unknown. Patients and methods: The FOrMAT (Feasibility of a Molecular Characterisation Approach to Treatment) study was a feasibility study enrolling patients with advanced GI malignancies from February 2014 to November 2015. Targeted capture sequencing (mainly using archival formalin-fixed paraffin-embedded diagnostic/resection samples) was carried out to detect mutations, copy number variations and translocations in up to 46 genes which had prognostic/predictive significance or were targets in current/upcoming clinical trials. Results: Of the 222 patients recruited, 215 patients (96.8%) had available tissue samples, 125 patients (56.3%) had ≥16 genes successfully sequenced and 136 patients (61.2%) had ≥1 genes successfully sequenced. Sample characteristics influenced the proportion of successfully sequenced samples, e.g. tumour type (colorectal 70.9%, biliary 52.6%, oesophagogastric 50.7%, pancreas 27.3%, P = 0.002), tumour cellularity (high versus low: 78.3% versus 13.3%, P ≤ 0.001), tumour content (high versus low: 78.6% versus 27.3%, P = 0.001) and type of sample (resection versus biopsy: 82.4% versus 47.6%, P ≤ 0.001). Currently, actionable alterations were detected in 90 (40.5%) of the 222 patients recruited (66% of the 136 patients sequenced) and 2 patients subsequently received a targeted therapy. The most frequently detected currently actionable alterations were mutations in KRAS, BRAF, TP53 and PIK3CA. For the 205 patients with archival samples, the median time to obtain sequencing results was 18.9 weeks, including a median of 4.9 weeks for sample retrieval and 5.1 weeks for sequencing. Conclusions: Targeted sequencing detected actionable alterations in formalin-fixed paraffin-embedded samples, but tissue characteristics are of critical importance in determining sequencing success. Routine molecular profiling of GI tumours outside of clinical trials is not an effective use of healthcare resources unless more targeted drugs become available. ClinicalTrials.gov identifier: NCT02112357.

Reprogramming axonal behavior by axon-specific viral transduction.

The treatment of axonal disorders, such as diseases associated with axonal injury and degeneration, is limited by the inability to directly target therapeutic protein expression to injured axons. Current gene therapy approaches rely on infection and transcription of viral genes in the cell body. Here, we describe an approach to target gene expression selectively to axons. Using a genetically engineered mouse containing epitope-labeled ribosomes, we find that neurons in adult animals contain ribosomes in distal axons. To use axonal ribosomes to alter local protein expression, we utilized a Sindbis virus containing an RNA genome that has been modified so that it can be directly used as a template for translation. Selective application of this virus to axons leads to local translation of heterologous proteins. Furthermore, we demonstrate that selective axonal protein expression can be used to modify axonal signaling in cultured neurons, enabling axons to grow over inhibitory substrates typically encountered following axonal injury. We also show that this viral approach also can be used to achieve heterologous expression in axons of living animals, indicating that this approach can be used to alter the axonal proteome in vivo. Together, these data identify a novel strategy to manipulate protein expression in axons, and provides a novel approach for using gene therapies for disorders of axonal function.

Microduplication of Xp11.23p11.3 with effects on cognition, behavior, and craniofacial development.

We report an ~1.3 Mb tandem duplication at Xp11.23p11.3 in an 11-year-old boy with pleasant personality, hyperactivity, learning and visual-spatial difficulties, relative microcephaly, long face, stellate iris pattern, and periorbital fullness. This clinical presentation is milder and distinct from that of patients with partially overlapping Xp11.22p11.23 duplications which have been described in males and females with intellectual disability, language delay, autistic behaviors, and seizures. The duplicated region harbors three known X-linked mental retardation genes: FTSJ1, ZNF81, and SYN1. Quantitative polymerase chain reaction from whole blood total RNA showed increased expression of three genes located in the duplicated region: EBP, WDR13, and ZNF81. Thus, over-expression of genes in the interval may contribute to the observed phenotype. Many of the features seen in this patient are present in individuals with Williams-Beuren syndrome (WBS). Interestingly, the SYN1 gene within the duplicated interval, as well as the STX1A gene, within the WBS critical region, co-localize to presynaptic active zones, and play important roles in neurotransmitter release.

Combination of flow cytometry and functional imaging for monitoring of residual disease in myeloma.

The iliac crest is the sampling site for minimal residual disease (MRD) monitoring in multiple myeloma (MM). However, the disease distribution is often heterogeneous, and imaging can be used to complement MRD detection at a single site. We have investigated patients in complete remission (CR) during first-line or salvage therapy for whom MRD flow cytometry and the two imaging modalities positron emission tomography (PET) and diffusion-weighted magnetic resonance imaging (DW-MRI) were performed at the onset of CR. Residual focal lesions (FLs), detectable in 24% of first-line patients, were associated with short progression-free survival (PFS), with DW-MRI detecting disease in more patients. In some patients, FLs were only PET positive, indicating that the two approaches are complementary. Combining MRD and imaging improved prediction of outcome, with double-negative and double-positive features defining groups with excellent and dismal PFS, respectively. FLs were a rare event (12%) in first-line MRD-negative CR patients. In contrast, patients achieving an MRD-negative CR during salvage therapy frequently had FLs (50%). Multi-region sequencing and imaging in an MRD-negative patient showed persistence of spatially separated clones. In conclusion, we show that DW-MRI is a promising tool for monitoring residual disease that complements PET and should be combined with MRD.

Prediction of outcome in newly diagnosed myeloma: a meta-analysis of the molecular profiles of 1905 trial patients.

Robust establishment of survival in multiple myeloma (MM) and its relationship to recurrent genetic aberrations is required as outcomes are variable despite apparent similar staging. We assayed copy number alterations (CNA) and translocations in 1036 patients from the NCRI Myeloma XI trial and linked these to overall survival (OS) and progression-free survival. Through a meta-anlysis of these data with data from MRC Myeloma IX trial, totalling 1905 newly diagnosed MM patients (NDMM), we confirm the association of t(4;14), t(14;16), t(14;20), del(17p) and gain(1q21) with poor prognosis with hazard ratios (HRs) for OS of 1.60 (P=4.77 × 10-7), 1.74 (P=0.0005), 1.90 (P=0.0089), 2.10 (P=8.86 × 10-14) and 1.68 (P=2.18 × 10-14), respectively. Patients with 'double-hit' defined by co-occurrence of at least two adverse lesions have an especially poor prognosis with HRs for OS of 2.67 (P=8.13 × 10-27) for all patients and 3.19 (P=1.23 × 10-18) for intensively treated patients. Using comprehensive CNA and translocation profiling in Myeloma XI we also demonstrate a strong association between t(4;14) and BIRC2/BIRC3 deletion (P=8.7 × 10-15), including homozygous deletion. Finally, we define distinct sub-groups of hyperdiploid MM, with either gain(1q21) and CCND2 overexpression (P<0.0001) or gain(11q25) and CCND1 overexpression (P<0.0001). Profiling multiple genetic lesions can identify MM patients likely to relapse early allowing stratification of treatment.

Overexpression of EZH2 in multiple myeloma is associated with poor prognosis and dysregulation of cell cycle control.

Myeloma is heterogeneous at the molecular level with subgroups of patients characterised by features of epigenetic dysregulation. Outcomes for myeloma patients have improved over the past few decades except for molecularly defined high-risk patients who continue to do badly. Novel therapeutic approaches are, therefore, required. A growing number of epigenetic inhibitors are now available including EZH2 inhibitors that are in early-stage clinical trials for treatment of haematological and other cancers with EZH2 mutations or in which overexpression has been correlated with poor outcomes. For the first time, we have identified and validated a robust and independent deleterious effect of high EZH2 expression on outcomes in myeloma patients. Using two chemically distinct small-molecule inhibitors, we demonstrate a reduction in myeloma cell proliferation with EZH2 inhibition, which leads to cell cycle arrest followed by apoptosis. This is mediated via upregulation of cyclin-dependent kinase inhibitors associated with removal of the inhibitory H3K27me3 mark at their gene loci. Our results suggest that EZH2 inhibition may be a potential therapeutic strategy for the treatment of myeloma and should be investigated in clinical studies.

Spatial genomic heterogeneity in multiple myeloma revealed by multi-region sequencing.

In multiple myeloma malignant plasma cells expand within the bone marrow. Since this site is well-perfused, a rapid dissemination of "fitter" clones may be anticipated. However, an imbalanced distribution of multiple myeloma is frequently observed in medical imaging. Here, we perform multi-region sequencing, including iliac crest and radiology-guided focal lesion specimens from 51 patients to gain insight into the spatial clonal architecture. We demonstrate spatial genomic heterogeneity in more than 75% of patients, including inactivation of CDKN2C and TP53, and mutations affecting mitogen-activated protein kinase genes. We show that the extent of spatial heterogeneity is positively associated with the size of biopsied focal lesions consistent with regional outgrowth of advanced clones. The results support a model for multiple myeloma progression with clonal sweeps in the early phase and regional evolution in advanced disease. We suggest that multi-region investigations are critical to understanding intra-patient heterogeneity and the evolutionary processes in multiple myeloma.In multiple myeloma, malignant cells expand within bone marrow. Here, the authors use multi-region sequencing in patient samples to analyse spatial clonal architecture and heterogeneity, providing novel insight into multiple myeloma progression and evolution.

Bi-allelic inactivation is more prevalent at relapse in multiple myeloma, identifying RB1 as an independent prognostic marker.

The purpose of this study is to identify prognostic markers and treatment targets using a clinically certified sequencing panel in multiple myeloma. We performed targeted sequencing of 578 individuals with plasma cell neoplasms using the FoundationOne Heme panel and identified clinically relevant abnormalities and novel prognostic markers. Mutational burden was associated with maf and proliferation gene expression groups, and a high-mutational burden was associated with a poor prognosis. We identified homozygous deletions that were present in multiple myeloma within key genes, including CDKN2C, RB1, TRAF3, BIRC3 and TP53, and that bi-allelic inactivation was significantly enriched at relapse. Alterations in CDKN2C, TP53, RB1 and the t(4;14) were associated with poor prognosis. Alterations in RB1 were predominantly homozygous deletions and were associated with relapse and a poor prognosis which was independent of other genetic markers, including t(4;14), after multivariate analysis. Bi-allelic inactivation of key tumor suppressor genes in myeloma was enriched at relapse, especially in RB1, CDKN2C and TP53 where they have prognostic significance.

Effects of adenosine on inositol 1,4,5-trisphosphate formation and intracellular calcium changes in formyl-Met-Leu-Phe-stimulated human neutrophils.

In the presence of adenosine, formyl-Met-Leu-Phe-stimulated human neutrophils show a greatly diminished production of superoxide anion. Analysis of changes in levels of intracellular calcium revealed that the immediate increase (occurring within seconds) in intracellular calcium following addition of formyl-Met-Leu-Phe is not affected by the presence of adenosine, although there are significantly lower intracellular calcium levels during the late phase (occurring 1-4 min after addition of formyl-Met-Leu-Phe). Consistent with these findings is the fact that adenosine does not affect the production of inositol 1,4,5-triphosphate in formyl-Met-Leu-Phe-stimulated neutrophils. These data suggest that the profound inhibitory effects of adenosine on superoxide responses in formyl-Met-Leu-Phe-stimulated neutrophils may be related to an action of adenosine occurring late in the sequence of events of signal transduction.

Absence of FMLP receptors on rat macrophages.

Although rat peritoneal neutrophils in the presence of cytochalasin B demonstrate superoxide (O2-) responses to the chemotactic peptide N'-formyl-met-leu-phe (FMLP), neither elicited rat peritoneal macrophages nor rat alveolar macrophages show an O2- response to FMLP (in the presence or absence of cytochalasin B), although a good O2- response to opsonized zymosan is demonstrated by both types of macrophages. Using Fura-2 loaded cells, peritoneal macrophages failed to show an increase in intracellular calcium after exposure to FMLP, f-nor-leu-phe, F-met-met-met, or F-norleu-leu-phe-norleu-lys. FMLP also failed to induce elevations in intracellular calcium in alveolar macrophages. In 3H-FMLP binding studies, the lack of responsiveness of peritoneal and alveolar macrophages was associated with the lack of FMLP receptors on these cell types, in striking contrast to the presence of functional receptors on rat neutrophils.

The use of flow cytometry to measure neutrophil function.

Neutrophils are important professional phagocytic cells that provide the host with a first line of defense against acute bacterial and fungal diseases and recurrent, severe or unusual infections are associated with inherited defects of neutrophil function. Furthermore, abundant evidence links inappropriate neutrophil-mediated tissue damage to the pathogenesis of conditions such as acute respiratory distress syndrome, septicemia with multiorgan failure, ischemia-reperfusion injury and rheumatoid arthritis. Flow cytometry has been increasingly used to evaluate the functional capabilities of neutrophils. In this review, we discuss the use of flow cytometry to assess neutrophil functional responses including calcium mobilization, F-actin assembly, adhesion, aggregation, degranulation, phagocytosis and reactive oxygen species (ROS) production. The use of flow cytometry to identify neutrophil priming is also discussed. The advantage of flow cytometry is that the majority of neutrophil functions can be measured using a small volume of whole blood that reduces artifactual changes in function caused by purification procedures. The advent of numerous new fluorochromes and multiparametric analysis allows the simultaneous measurement of several neutrophil functions in the same population of cells. Flow cytometric analysis provides a rapid screen for abnormalities of neutrophil function and reflects more accurately their behavior in vivo.

Prenatal diagnosis of renal anomalies.

Within the past 24 months, we have performed prenatal diagnostic studies in 4 pregnancies known to be at risk for well-described genetic syndrome involving renal abnormalities, ie, Meckel syndrome, Roberts syndrome, and bilateral renal agenesis. The diagnostic techniques utilized were ultrasonographic scanning (B-mode and grey scale), biochemical assays, and radiographic evaluation. The ultrasound finding common to the 3 affected cases was extreme oligohydramnios, which we considered indirect evidence that renal anomalies were present. The ultrasound scans of the fetuses affected with Meckel and Roberts syndrome demonstrated anechoic cystic spaces in the abdomen, representing the enlarged dysplastic cystic kidneys. An encephalocele was well demonstrated by B-mode scan in the fetus with Meckel syndrome. The absence of normal limbs in the Roberts syndrome was evident on serial grey scale scans of the fetus. Biochemical and radiographic studies provided results consistent with the suspected diagnoses. The importance of providing genetic counseling and prenatal diagnosis to families at risk is emphasized.

Prenatal diagnosis of bilateral renal agenesis.

Bilateral renal agenesis (BRA), or Potter's syndrome, is a rare genetic disorder in which agenesis of the kidneys is associated with pulmonary hypoplasia and characteristic facial features. Oligohydramnios or virtual absence of amniotic fluid is found in most pregnancies with BRA. Clinical observation and serial ultrasonography scans made it possible to diagnose BRA prenatally in a fetus at risk. Postmortem examination confirmed the diagnosis.

Effect of mechanical deformation on structure and function of polymorphonuclear leukocytes.

The present studies were designed to test the hypothesis that mechanical deformation of polymorphonuclear leukocytes (PMN) leads to functional changes that might influence their transit in the pulmonary capillaries. Human leukocytes were passed through 5- or 3-micron-pore polycarbonate filters under controlled conditions. Morphometric analysis showed that the majority of PMN were deformed and that this deformation persisted longer after filtration through 3-micron filters than through 5-micron filters (P < 0.05) but did not result in the cytoskeletal polarization characteristic of migrating cells. Flow cytometric studies of the filtered PMN showed that there was a transient increase in the cytosolic free Ca2+ concentration after both 3- and 5-micron filtration (P < 0.01) with an increase in F-actin content after 3-micron filtration (P < 0.05). Although L-selectin expression on PMN was not changed by either 5- or 3-micron filtration, CD18 and CD11b were increased by 3-micron filtration (P < 0.05). Priming of the PMN with N-formyl-methionyl-leucyl-phenylalanine (0.5 nM) before filtration resulted in an increase of CD11b by both 5 (P < 0.05)- and 3-micron (P < 0.01) filtration. Neither 5- nor 3-micron filtration induced hydrogen peroxide production. We conclude that mechanical deformation of PMN, similar to what occurs in the pulmonary microvessels, induces both structural and functional changes in the cells, which might influence their passage through the pulmonary capillary bed.

Mutagenicity of selected functionalized benz(c)acridines and a benz(a)phenazine in the Salmonella typhimurium/microsome assay.

Five functionalized benz(c)acridines - 5,6-dimethylbenz(c)acridine; 5,6,7-trimethylbenz(c)acridine; 7-chloro-5,6-dimethylbenz(c)acridine; 7-amino-5,6-dimethylbenz(c)acridine; 7-oxo-5,6-dimethylbenz(c)-acridine and 5,6-dimethylbenz(a)phenazine - were tested for mutagenic activity in the Ames Salmonella typhimurium assay. Compounds were initially screened by spot tests with 5 tester strains and both plate incorporation and pre-incubation assays were performed when the results of the tests were positive or weakly positive. All assays were done with and without S9 activation. 7-Amino-5, 6-dimethylbenz(c)acridine and 7-chloro-5, 6-dimethylbenz(c)acridine were found to be moderately mutagenic with the 3 frameshift strains TA1537, TA98, and TA97.

Mutagenicity of benzylic acetates, sulfates and bromides of polycyclic aromatic hydrocarbons.

Studies were performed to determine the direct mutagenicity of the acetates and some bromides and sulfates of hydroxymethyl polycyclic aromatic hydrocarbons in S. typhimurium strains TA98 and TA100. Benzylic acetates, bromides and sulfates were synthesized and characterized. The compounds tested were benzyl alcohol, 5-hydroxymethylchrysene, 1-hydroxymethylpyrene, 6-hydroxymethylbenzo[a]pyrene, 6-(2-hydroxyethyl)benzo[a]pyrene, 6-hydroxymethylanthanthrene, 9-hydroxymethylanthracene, 9-hydroxymethyl-10-methylanthracene, 7-hydroxymethylbenz[a]anthracene, 7-(2-hydroxyethyl)benz[a]anthracene, 12-hydroxymethylbenz[a]anthracene, 7-hydroxymethyl-12-methylbenz[a]anthracene, 12-hydroxymethyl-7-methylbenz[a]anthracene, 1-hydroxy-3-methylcholanthrene, 2-hydroxy-3-methylcholanthrene, 3-hydroxy-3, 4-dihydrocyclopental[cd]pyrene and 4-hydroxy-3, 4-dihydrocyclopental[cd]pyrene. The benzylic sulfate esters of 6-hydroxymethylbenzo[a]pyrene and 7-hydroxymethylbenz[a]anthracene were the most mutagenic compounds, whereas the aliphatic sulfate ester of 7-hydroxyethylbenz[a]anthracene did not cause an increase in mutations above background. All meso-anthracenic benzylic acetate esters were mutagenic in both strains with various degrees of activity, whereas the corresponding non-benzylic esters were inactive, as expected. Of the non-meso-benzylic acetate esters, only the 3-acetoxy-3, 4-dihydrocyclopenta[cd]pyrene was mutagenic. In the benzylic bromide series, only the eight mesoanthracenic were mutagenic, whereas benzyl bromide and 5-bromomethylchrysene were inactive. The aliphatic bromides, 6-(2-bromoethyl)benzo[a]pyrene and 7-(2-bromoethyl)benz[a]anthracene did not display significant activity. The potencies of the acetate esters more accurately reflect the mutagenicity because the rate of solvolysis did not compete with the reactivity of the esters with bacterial DNA. In the case of benzylic sulfates and bromides, the rate of solvolysis was very rapid and could have diminished the level of mutagenicity, depending on the assay conditions. These results demonstrate that meso-anthracenic benzylic acetates, sulfates and bromides are mutagenic, whereas benzylic acetate esters attached to other carbon atoms are inactive.

Microbial mutagenicity of selected hydrazines.

Selected hydrazines and related compounds were examined for their mutagenic activity in S. typhimurium strains TA1535 and TA1537. These in vitro assays were conducted with and without metabolic activation by Aroclor-induced rat-liver enzymes. Relatively high levels of mutagenicity were observed with phenylhydrazine X HCl, methylhydrazine, N'-acetyl-4-(hydroxymethyl)phenylhydrazine, and 4-(hydroxymethyl)benzenediazonium tetrafluoroborate, the stabilized salt of a carcinogenic metabolite of agaritine; only low levels of mutagenicity were observed with other compounds, although most are strong carcinogens. Several of the compounds were highly toxic to the bacteria, and detection of mutagenicity was enhanced by calculating the increase in mutagenic activity on the basis of the surviving fractions of bacteria.

Effects of adenosine on guinea pig pulmonary eosinophils.

Extracellular adenosine has pharmacological activity on a wide variety of cell types and may play an important role as an inflammatory modulator with both pro- and anti-inflammatory activities. These studies examine the effects of adenosine on guinea pig pulmonary eosinophils. Adenosine alone did not directly induce superoxide (O2-) production. Pretreatment with adenosine primed the O2- response of guinea pig pulmonary eosinophils following the addition of 1 or 10 microM platelet-activating factor (PAF). Priming was seen at adenosine concentrations greater than 1 microM and was maximal at 100 microM. At this maximal dose, adenosine priming increased the O2- response to 1 microM and 10 microM PAF by 86% and 51%, respectively. Priming by adenosine was not seen when ionomycin or phorbol myristate acid (PMA) were used as agonists. In fura-2 loaded eosinophils, the addition of 100 microM adenosine resulted in a small but significant rise in intracellular calcium of 54.4 +/- 9.2 nM above baseline. In contrast, similar adenosine concentrations had no effect on cytosolic calcium levels in guinea pig neutrophils. These data demonstrate a pro-inflammatory role for adenosine in elicited guinea pig pulmonary eosinophils.

Bladder freeze ulceration and sodium saccharin feeding in the rat: examination for urinary nitrosamines, mutagens and bacteria, and effects on hepatic microsomal enzymes.

We previously demonstrated that long-term feeding of sodium saccharin, a non-mutagen, induced bladder carcinomas when administered to F344 male rats with regenerative hyperplasia of the urothelium induced by the freeze-ulceration technique, even without prior chemical initiation (Cohen et al. Cancer Res. 1982, 42, 65). In the present study, we examined the urine of rats subjected to freeze ulceration of the bladder and then fed sodium saccharin at 5% in the diet to evaluate the possibility of a mutagen being generated as a result of ulceration and/or saccharin feeding. Urine was collected into a syringe by aspiration from the urinary bladder after ligating the urethra for 2 hr at intervals from day 0 to day 14 after ulceration. After ulceration and/or sodium saccharin feeding, the urine showed no bacterial contamination, no mutagenic activity in the standard Ames assay, no production of nitrosamines, and no nitrosating environment. In addition, no significant changes in activities of liver microsomal enzymes (i.e. cytochrome P-450, NADPH-cytochrome c reductase, aniline hydroxylase, or ethylmorphine N-demethylase) were observed in rats fed sodium saccharin for 1, 5 or 14 days. Thus, freeze ulceration, and the consequent regenerative hyperplasia of the epithelium, compared with sodium saccharin feeding do not involve the administration of an exogenous mutagenic substance or the generation of a detectable mutagen in the urine.