Prevalence and clinical association of gene mutations through multiplex mutation testing in patients with NSCLC: results from the ETOP Lungscape Project.

Background: Reported prevalence of driver gene mutations in non-small-cell lung cancer (NSCLC) is highly variable and clinical correlations are emerging. Using NSCLC biomaterial and clinical data from the European Thoracic Oncology Platform Lungscape iBiobank, we explore the epidemiology of mutations and association to clinicopathologic features and patient outcome (relapse-free survival, time-to-relapse, overall survival). Methods: Clinically annotated, resected stage I-III NSCLC FFPE tissue was assessed for gene mutation using a microfluidics-based multiplex PCR platform. Mutant-allele detection sensitivity is >1% for most of the ∼150 (13 genes) mutations covered in the multiplex test. Results: Multiplex testing has been carried out in 2063 (76.2%) of the 2709 Lungscape cases (median follow-up 4.8 years). FFPE samples mostly date from 2005 to 2008, yet recently extracted DNA quality and quantity was generally good. Average DNA yield/case was 2.63 µg; 38 cases (1.4%) failed QC and were excluded from study; 95.1% of included cases allowed the complete panel of mutations to be tested. Most common were KRAS, MET, EGFR and PIK3CA mutations with overall prevalence of 23.0%, 6.8%, 5.4% and 4.9%, respectively. KRAS and EGFR mutations were significantly more frequent in adenocarcinomas: PIK3CA in squamous cell carcinomas. MET mutation prevalence did not differ between histology groups. EGFR mutations were found predominantly in never smokers; KRAS in current/former smokers. For all the above mutations, there was no difference in outcome between mutated and non-mutated cases. Conclusion: Archival FFPE NSCLC material is adequate for multiplex mutation analysis. In this large, predominantly European, clinically annotated stage I-III NSCLC cohort, none of the mutations characterized showed prognostic significance.

Mechanisms of motor protein reversal.

Members of the kinesin superfamily of microtubule-based motors and the myosin superfamily of actin-based motors that move 'backwards' have been identified. As the core catalytic domains of myosins and kinesins are similar in structure, this raises the intriguing questions of how direction reversal is accomplished and whether kinesins and myosins share mechanisms for switching their motors into reverse.

Unconventional myosins.

The unconventional myosins form a large and diverse group of molecular motors. The number of known unconventional myosins is increasing rapidly and in the past year alone two new classes have been identified. Substantial progress has been made towards characterizing the properties and functions of these motor proteins, which have been hypothesized to play fundamental roles in processes such as cell locomotion, phagocytosis and vesicle transport.

Inactivation of the WASF3 gene in prostate cancer cells leads to suppression of tumorigenicity and metastases.

BACKGROUND: The WASF3 protein is involved in cell movement and invasion, and to investigate its role in prostate cancer progression we studied the phenotypic effects of knockdown in primary tumors and cell lines. METHODS: ShRNA was used to knockdown WASF3 function in prostate cell lines. Cell motility (scratch wound assay), anchorage independent growth and in vivo tumorigenicity and metastasis were then compared between knockdown and wild-type cells. RESULTS: Increased levels of expression were seen in high-grade human prostate cancer and in the PC3 and DU145 cell lines. Inactivation of WASF3 using shRNAs reduced cell motility and invasion in these cells and reduced anchorage independent growth in vitro. The loss of motility was accompanied by an associated increase in stress fiber formation and focal adhesions. When injected subcutaneously into severe combined immunodeficiency (SCID) mice, tumor formation was significantly reduced for PC3 and DU145 cells with WASF3 knockdown and in vivo metastasis assays using tail vain injection showed a significant reduction for PC3 and DU145 cells. The loss of the invasion phenotype was accompanied by down-regulation of matrix metalloproteinase 9. CONCLUSIONS: Overall, these observations demonstrate a critical role for WASF3 in the progression of prostate cancer and identify a potential target to control tumorigenicity and metastasis.

A new direction for myosin.

Members of the myosin superfamily of actin-based motor proteins were previously thought to move only towards the barbed end of the actin filament. In an extraordinary reversal of this dogma, an abundant and widespread unconventional myosin known as myosin VI has recently been shown to move towards the pointed end of the actin filament - the opposite direction of all other characterized myosins. This discovery raises novel and intriguing questions about the molecular mechanisms of reversal and the biological roles of this 'backwards' myosin.

Microtubules remodel actomyosin networks in Xenopus egg extracts via two mechanisms of F-actin transport.

Interactions between microtubules and filamentous actin (F-actin) are crucial for many cellular processes, including cell locomotion and cytokinesis, but are poorly understood. To define the basic principles governing microtubule/F-actin interactions, we used dual-wavelength digital fluorescence and fluorescent speckle microscopy to analyze microtubules and F-actin labeled with spectrally distinct fluorophores in interphase Xenopus egg extracts. In the absence of microtubules, networks of F-actin bundles zippered together or exhibited serpentine gliding along the coverslip. When microtubules were nucleated from Xenopus sperm centrosomes, they were released and translocated away from the aster center. In the presence of microtubules, F-actin exhibited two distinct, microtubule-dependent motilities: rapid ( approximately 250-300 nm/s) jerking and slow ( approximately 50 nm/s), straight gliding. Microtubules remodeled the F-actin network, as F-actin jerking caused centrifugal clearing of F-actin from around aster centers. F-actin jerking occurred when F-actin bound to motile microtubules powered by cytoplasmic dynein. F-actin straight gliding occurred when F-actin bundles translocated along the microtubule lattice. These interactions required Xenopus cytosolic factors. Localization of myosin-II to F-actin suggested it may power F-actin zippering, while localization of myosin-V on microtubules suggested it could mediate interactions between microtubules and F-actin. We examine current models for cytokinesis and cell motility in light of these findings.

Primary structure and cellular localization of chicken brain myosin-V (p190), an unconventional myosin with calmodulin light chains.

Recent biochemical studies of p190, a calmodulin (CM)-binding protein purified from vertebrate brain, have demonstrated that this protein, purified as a complex with bound CM, shares a number of properties with myosins (Espindola, F. S., E. M. Espreafico, M. V. Coelho, A. R. Martins, F. R. C. Costa, M. S. Mooseker, and R. E. Larson. 1992. J. Cell Biol. 118:359-368). To determine whether or not p190 was a member of the myosin family of proteins, a set of overlapping cDNAs encoding the full-length protein sequence of chicken brain p190 was isolated and sequenced. Verification that the deduced primary structure was that of p190 was demonstrated through microsequence analysis of a cyanogen bromide peptide generated from chick brain p190. The deduced primary structure of chicken brain p190 revealed that this 1,830-amino acid (aa) 212,509-D) protein is a member of a novel structural class of unconventional myosins that includes the gene products encoded by the dilute locus of mouse and the MYO2 gene of Saccharomyces cerevisiae. We have named the p190-CM complex "myosin-V" based on the results of a detailed sequence comparison of the head domains of 29 myosin heavy chains (hc), which has revealed that this myosin, based on head structure, is the fifth of six distinct structural classes of myosin to be described thus far. Like the presumed products of the mouse dilute and yeast MYO2 genes, the head domain of chicken myosin-V hc (aa 1-764) is linked to a "neck" domain (aa 765-909) consisting of six tandem repeats of an approximately 23-aa "IQ-motif." All known myosins contain at least one such motif at their head-tail junctions; these IQ-motifs may function as calmodulin or light chain binding sites. The tail domain of chicken myosin-V consists of an initial 511 aa predicted to form several segments of coiled-coil alpha helix followed by a terminal 410-aa globular domain (aa, 1,421-1,830). Interestingly, a portion of the tail domain (aa, 1,094-1,830) shares 58% amino acid sequence identity with a 723-aa protein from mouse brain reported to be a glutamic acid decarboxylase. The neck region of chicken myosin-V, which contains the IQ-motifs, was demonstrated to contain the binding sites for CM by analyzing CM binding to bacterially expressed fusion proteins containing the head, neck, and tail domains. Immunolocalization of myosin-V in brain and in cultured cells revealed an unusual distribution for this myosin in both neurons and nonneuronal cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Myosin-I nomenclature.

We suggest that the vertebrate myosin-I field adopt a common nomenclature system based on the names adopted by the Human Genome Organization (HUGO). At present, the myosin-I nomenclature is very confusing; not only are several systems in use, but several different genes have been given the same name. Despite their faults, we believe that the names adopted by the HUGO nomenclature group for genome annotation are the best compromise, and we recommend universal adoption.

GEOSAT Altimeter Observations of Kelvin Waves and the 1986-87 El Nino.

Two years of GEOSAT altimeter observations are used to investigate the response of sea level to anomalous westerly wind bursts in the tropical Pacific Ocean before and during the 1986-87 El Niño. Sea level time series along the equator show examples of both positive and negative anomalies of 10-centimeter amplitude and 2- to 4-week time scale propagating across the Pacific with phase speeds of 2.4 to 2.8 meters per second, suggesting downwelling and upwelling Kelvin waves, respectively. A comparison of island wind observations with sea level indicates one instance (May 1986) in which a positive sea level anomaly can be related to westerly winds caused by a cross-equatorial cyclone pair in the western Pacific. This episode was followed by additional wind bursts later in the year, and finally by sustained westerlies in the western Pacific during November-December 1986, at the height of El Niño. The GEOSAT observations reveal the sea level response to these meteorological events and provide a synoptic description of the El Niño oceanographic phenomenon.

Function of myosin-V in filopodial extension of neuronal growth cones.

The molecular mechanisms underlying directed motility of growth cones have not been determined. The role of myosin-V, an unconventional myosin, in growth cone dynamics was examined by chromophore-assisted laser inactivation (CALI). CALI of purified chick brain myosin-V absorbed onto nitrocellulose-coated cover slips inhibited the ability of myosin-V to translocate actin filaments. CALI of myosin-V in growth cones of chick dorsal root ganglion neurons resulted in rapid filopodial retraction. The rate of filopodial extension was significantly decreased, whereas the rate of filopodial retraction was not affected, which suggests a specific role for myosin-V in filopodial extension.

Bmf: a proapoptotic BH3-only protein regulated by interaction with the myosin V actin motor complex, activated by anoikis.

Bcl-2 family members bearing only the BH3 domain are essential inducers of apoptosis. We identified a BH3-only protein, Bmf, and show that its BH3 domain is required both for binding to prosurvival Bcl-2 proteins and for triggering apoptosis. In healthy cells, Bmf is sequestered to myosin V motors by association with dynein light chain 2. Certain damage signals, such as loss of cell attachment (anoikis), unleash Bmf, allowing it to translocate and bind prosurvival Bcl-2 proteins. Thus, at least two mammalian BH3-only proteins, Bmf and Bim, function to sense intracellular damage by their localization to distinct cytoskeletal structures.

A randomised controlled feasibility trial of alcohol consumption and the ability to appropriately use a firearm.

OBJECTIVE: To show the feasibility of using a controlled trial to investigate the effect of alcohol on firearm use. METHODS: Randomised, blinded, placebo-controlled trial in the Firearm Usage and Safety Experiments (FUSE) Lab. Treatment subjects (male, 21-40-year-old, non-habitual drinkers, with no professional firearms training) received alcohol; control subjects received placebo alcohol. The AIS PRISim Firearm Simulator, including real pistols retrofitted to discharge compressed air cartridges that simulate firearm recoil and sound, was used to measure firearm performance. Accuracy and speed for target shooting, reaction time scenarios, and scenarios requiring judgement about when to use a gun were measured. RESULTS: 12 subjects enrolled in the trial, completing 160 training scenarios. All subjects in the alcohol arm reached target alcohol level. 33% of placebo subjects reported alcohol consumption. Mechanical malfunction of the simulator occurred in 9 of 160 (5.6%) scenarios. Intoxicated subjects were less accurate, slower to fire in reaction time scenarios, and quicker to fire in scenarios requiring judgement relative to controls. CONCLUSIONS: The feasibility of a randomised, controlled trial exploring the relationship between alcohol consumption and firearm use was shown. The hypothesis that alcohol consumption worsens accuracy and retards judgement about when to use a gun should be tested. Larger trials could inform policies regarding firearm use while intoxicated.

A millennial myosin census.

The past decade has seen a remarkable explosion in our knowledge of the size and diversity of the myosin superfamily. Since these actin-based motors are candidates to provide the molecular basis for many cellular movements, it is essential that motility researchers be aware of the complete set of myosins in a given organism. The availability of cDNA and/or draft genomic sequences from humans, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Dictyostelium discoideum has allowed us to tentatively define and compare the sets of myosin genes in these organisms. This analysis has also led to the identification of several putative myosin genes that may be of general interest. In humans, for example, we find a total of 40 known or predicted myosin genes including two new myosins-I, three new class II (conventional) myosins, a second member of the class III/ninaC myosins, a gene similar to the class XV deafness myosin, and a novel myosin sharing at most 33% identity with other members of the superfamily. These myosins are in addition to the recently discovered class XVI myosin with N-terminal ankyrin repeats and two human genes with similarity to the class XVIII PDZ-myosin from mouse. We briefly describe these newly recognized myosins and extend our previous phylogenetic analysis of the myosin superfamily to include a comparison of the complete or nearly complete inventories of myosin genes from several experimentally important organisms.

FGFR3 and p53 protein expressions in patients with pTa and pT1 urothelial bladder cancer.

AIMS: This study is designed to evaluate the expression and prognostic value of FGFR3 protein expression in patients with pTa/pT1 tumours and to determine the significance of the combinations of FGFR3 and p53 protein expressions in bladder pathogenesis. MATERIALS AND METHODS: A tissue microarray (TMA) of 107 pTa, and 147 pT1 tumours was constructed. The TMA sections were immunostained with FGFR3 and p53 monoclonal antibodies. RESULTS: There were significant associations between loss of FGFR3 and tumour stage (p<0.001) and grade (p<0.001) and between p53 overexpression and tumour stage and grade (p<0.001 and p<0.001, respectively). There was no association between FGFR3 and p53 proteins (p=0.107). In addition, tumours with FGFR3+/p53- phenotype have slower recurrence rate than other (FGFR3+/p53+, FGFR3-/p53- and FGFR3-/p53+). CONCLUSION: 1-FGFR3 expression is significantly associated with two important prognostic factors; stage and grade. 2-FGFR3 protein expression is not an independent predictive factor for pTa/pT1 tumour recurrence and progression. 3-Tumours with FGFR3+/p53- phenotype seem to have a distinctive pathway in bladder tumorigenesis.

Raf1, Aurora-A/STK15 and E-cadherin biomarkers expression in patients with pTa/pT1 urothelial bladder carcinoma; a retrospective TMA study of 246 patients with long-term follow-up.

AIMS: The study was designed to evaluate Raf1, Aurora-A/STK15 and E-cadherin (E-CD) protein expression and their prognostic value in patients with pTa/pT1 tumours. MATERIALS AND METHODS: A tissue microarray of 105 pTa, and 141 pT1 tumours was constructed and sections were immunostained with these three antibodies. RESULTS: There were significant associations between Raf1 overexpression and tumour grade (p = 0.03), between Aurora-A overexpression/alterations of E-CD and tumour grade and stage (p < 0.001 and p < 0.001). In multiple Cox regression analysis, moderate/strong expression of E-CD seemed to be an independent factor in predicting slower tumour progression (p = 0.003) and Aurora-A overexpression (p = 0.022) displays an independent value in predicting tumour recurrences. CONCLUSION: Evaluation of E-CD and Aurora-A expressions in tissue of patients with pTa/pT1 UC have been proven to be useful in predicting tumours behavior and Raf1 protein expression seemed to have no potential use in this regard.

Effect of transdermal nicotine delivery as an adjunct to low-intervention smoking cessation therapy. A randomized, placebo-controlled, double-blind study.

To assess the smoking cessation efficacy of transdermal nicotine patches as an adjunct to low-intervention therapy, we conducted a double-blind, placebo-controlled trial in 158 smokers. Participants were randomly assigned to one of the following three study regimens that required daily application of two 15-cm2 patches: (1) 24-hour nicotine delivery, (2) nicotine delivery during wakeful hours only, and (3) placebo. The impact of the three regimens on smoking cessation rates and tobacco withdrawal symptoms was examined. During the last 2 weeks of the trial, 39% of the 24-hour nicotine regimen delivery group, 35% of those on wakeful hour nicotine regimens, and 13.5% of the placebo treatment group achieved abstinence. Self-reported quit rates for the two nicotine patch-wearing regimens, as compared with that for the placebo group, continued to be significantly higher at 6 months. Moreover, compared with placebo, the transdermal nicotine patches significantly reduced tobacco withdrawal symptoms during the first few weeks of quitting. The differences in quit rates and tobacco withdrawal symptoms between the two active groups were not statistically significant. The patches were well tolerated both topically and systemically. We concluded that transdermal nicotine, when used as an adjunct to low-intervention therapy, significantly reduced tobacco withdrawal symptoms and enhanced smoking cessation rates.

Photofrin photodynamic therapy for treatment of AIDS-related cutaneous Kaposi's sarcoma.

OBJECTIVE: Kaposi's sarcoma, the most common malignancy in AIDS patients, often presents with painful cutaneous lesions that are difficult to treat effectively despite a wide variety of therapeutic approaches. We used photodynamic therapy in an attempt to provide effective palliative treatment for this disease. METHODS: Photodynamic therapy utilizes the activation by light of a photosensitizing drug that preferentially accumulates in tumor tissue such as Kaposi's sarcoma. We enrolled 25 patients who received 1.0 mg/kg of Photofrin 48 h before exposure to 100-400 J/cm2 of 630 nm light. RESULTS: Of the 348 lesions treated, 289 were evaluable: 32.5% had complete clinical response, 63.3% had partial clinical response and 4.2% were clinical failures. There was a strong correlation between response and light dose: 54% of lesions achieved a complete clinical response at optimum light dose (> 250 J/cm2). There was no correlation of response with CD4 cell count nor was there a change in CD4 cell count post-treatment. At 400 J/cm2 full field scabbing and necrosis occurred in 90% of the treated fields. Thus, the maximum tolerated dose was determined to be 300 J/cm2. At light doses of 250 J/cm2 and below the toxicities were limited to erythema and edema in the treatment field. Forty-three biopsies were taken 0.5 h to 4 months post-treatment. These showed little change in the B and T cell infiltrates identified. Kaposi's sarcoma cells disappeared post-treatment in certain lesions. CONCLUSION: Photofrin is effective palliative treatment for HIV-associated Kaposi's sarcoma.

Inhaled cellulosic and plastic fibers found in human lung tissue.

We report the results of studies undertaken to determine whether inhaled plant (i.e., cellulosic; e.g., cotton) and plastic (e.g., polyester) fibers are present in human lungs and, if so, whether inhaled fibers are also present in human lung cancers. Specimens of lung cancer of different histological types and adjacent nonneoplastic lung tissue were obtained from patients undergoing a lung resection for removal of a tumor. With the protection of a laminar flow hood and safeguards to prevent contamination by extraneous fibers, fresh, nonfixed, and nonstained samples of lung tissue were compressed between two glass microscope slides. Specimens in these dual slide chambers were examined with a microscope configured to permit viewing with white light, fluorescent light, polarizing light, and phase-contrast illumination. Near-term fetal bovine lungs and nonlung human tumors were used as controls. In contrast to the observations of these control tissues, morphologically heterogeneous fibers were seen repetitively in freshly excised human lung tissue using polarized light. Inhaled fibers were present in 83% of nonneoplastic lung specimens (n = 67/81) and in 97% of malignant lung specimens (n = 32/33). Thus, of the 114 human lung specimens examined, fibers were observed in 99 (87%). Examination of histopathology slides of lung tissue with polarized light confirmed the presence of inhaled cellulosic and plastic fibers. Of 160 surgical histopathology lung tissue slides, 17 were selected for critical examination; of these, fibers were identified in 13 slides. The inhalation of mineral (e.g., asbestos) fibers has been described by many investigators; we believe, however, that this is the first report of inhaled nonmineral (e.g., plant and plastic) fibers. These bioresistant and biopersistent cellulosic and plastic fibers are candidate agents contributing to the risk of lung cancer.

New needle and syringe use, and use of needle exchange programmes by street recruited injection drug users in 1993.

BACKGROUND: Needle exchange programmes (NEP) provide injection drug users (IDU) with sterile injection equipment and receive used needles in exchange. In this paper we describe the use of new syringes and NEP by IDU and characteristics associated with using NEP in 1993. METHODS: Street-recruited IDU were interviewed in five US locations: Atlanta, Philadelphia, Chicago, New York City, and Los Angeles (LA) county. RESULTS: Most (75-95%) reported it was easy to get a new syringe and for their last injection, 45-77% used a new syringe and 2-18% used a syringe previously used by another IDU. Use of NEP ranged from 8% to 16% in Chicago, Philadelphia, and LA County. In LA County not having injected 'speedball' in the last 30 days, last injection with a new syringe, and reporting it was very easy to get a new syringe were associated with NEP use. In Philadelphia, NEP use was associated with 'speedball' injection in the last 30 days, and in Chicago, not injecting with 'speedball' and injecting with cocaine were associated with NEP use. CONCLUSIONS: In 1993, most street-recruited IDU in Chicago, Philadelphia, and LA County had not used NEP. Factors associated with NEP use were not consistent across sites. Dispersion of NEP and removal of legal barriers restricting access to sterile syringes may be more important in increasing the use of sterile syringes and NEP than client characteristics.

Subpopulations of lymphocytes in maternal peripheral blood during pregnancy.

The fetus can be considered an allograft with up to one-half of its MHC antigens being potentially recognized by the mother as foreign. This study compares expression of OKT3, OKT4, OKT8, Kappa, Lambda and Ia antigens on lymphocytes in the peripheral blood of normal non-pregnant women, normal pregnant women, patients who are chronic spontaneous aborters and pregnant insulin-dependent diabetic women. Monoclonal antibodies and cytofluorometric analyses were used for these determinations. There were no significant differences (P = 0.01) between these groups for T-cell markers. A statistically significant (P = 0.001) increased ratio of cells bearing surface immunoglobulin to those expressing Ia antigen (K&L/Ia) was observed between normal non-pregnant controls and women with a history of chronic spontaneous abortion. It is concluded that T-lymphocytes in the peripheral blood do not demonstrate a phenotypic abnormality that would account for the non-rejection of the fetal allograft; however, women with chronic spontaneous abortion may have abnormal B-cell differentiation or T-cell activation that mediates chronic spontaneous abortion.