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.

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 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.

Prognostic significance of neuron-associated protein expression in non-muscle-invasive urothelial bladder cancer.

BACKGROUND: Numerous novel genes have been identified in urothelial bladder cancer (UBC); genes including ninjurin, synuclein and neuropilin seem to be associated with invasive tumours and aggressive behaviour. AIMS: To define the protein expression of these biomarkers and to reveal their prognostic value in a large series of superficial (pTa) and minimally invasive (pT1) cases of UBC with a long and adequate follow-up. METHODS: Tissue microarray was done on 183 paraffin-embedded tumour tissues (pTa 81, pT1 102). Statistical analysis was performed to define the association between each of these biomarkers, clinical data and tumour outcomes. RESULTS: There was a statistically significant association between synuclein expression and tumour stage (p = 0.029). Ninjurin expression was significantly associated with tumour progression in univariate analysis. Tumour grade seemed to have an independent value in predicting tumour recurrence and progression. CONCLUSION: Tumours with strong synuclein expressions are more likely to be more advanced tumours (pT1). Tumours expressing ninjurin tend to progress slower than those with no ninjurin expressions. Synuclein and neuropilin failed to show any value in predicting tumour behaviour.

Diagnostic utility of FLI-1 monoclonal antibody and dual-colour, break-apart probe fluorescence in situ (FISH) analysis in Ewing's sarcoma/primitive neuroectodermal tumour (EWS/PNET). A comparative study with CD99 and FLI-1 polyclonal antibodies.

AIMS: To compare the sensitivity and specificity of the recently commercially available FLI-1 monoclonal (FLI-1m) antibody with the currently used antibodies [CD99 and FLI-1 polyclonal (FLI-1p)] in the diagnosis of Ewing's sarcoma/primitive neuroectodermal tumour (EWS/PNET) and to determine the diagnostic value of the EWSR1 (22q12) dual-colour, break-apart rearrangement probe fluorescence in situ hybridization (FISH) technique. MATERIALS AND METHODS: Forty-three cases of well-documented EWS/PNET and 15 non-EWS/PNET cases were retrieved from the archival files. Immunohistochemistry (IHC) for FLI-1p, FLI-1m and FISH analysis was performed. RESULTS: The most sensitive and specific test panel for the diagnosis of EWS/PNET is the combination of CD99 and FLI-1p. FISH had a very high specificity (100%) but only a moderate sensitivity (50%). CONCLUSION: The combination of CD99 and FLI-1p is the method of choice for the diagnosis of EWS/PNET. EWRS1 (22q12) dual-colour, break-apart rearrangement probe FISH should be used as a confirmatory test in addition to CD99 and FLI1-p due to its high specificity.

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.

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.

The global burden of non-conflict related firearm mortality.

OBJECTIVE: Understanding global firearm mortality is hindered by data availability, quality, and comparability. This study assesses the adequacy of publicly available data, examines populations for whom firearm mortality data are not publicly available, and estimates the global burden of non-conflict related firearm mortality. DESIGN: The design is a secondary analysis of existing data. A dataset of countries, populations, economic development, and geographic regions was created, using United Nations 2000 world population data and World Bank classifications of economic development and global regions. Firearm mortality data were obtained from governmental vital statistics reported by the World Health Organization and published survey data. A qualitative review of literature informed estimates for the 15 most populous countries without firearm death data. For countries without data, estimates of firearm deaths were made using quartiles of observed rates and peer reviewed literature. MAIN OUTCOME MEASURES: Non-conflict related firearm deaths. RESULTS: Global non-conflict related firearm deaths were estimated to fall between 196,000 and 229,000, adjusted to the year 2000. 162,800 firearm deaths adjusted for the year 2000 came from countries reporting data and represent 35% of the world's 186 countries. Public data are not available for 122 of these 186 countries, representing more than three billion (54%) of the world's population, predominately in lower and lower middle income countries. Estimates of firearm death for those countries without data range from 33,200 to 66,200. CONCLUSIONS: This study provides evidence that the burden of firearm related mortality poses a substantial threat to local and global health.

CD117+ small cell lung cancer lacks the asp 816-->val point mutation in exon 17.

AIMS: To determine the frequency of point mutation in c-kit in CD117+ small cell lung cancer (SCLC). A significant proportion of SCLCs have been documented to be CD117+, thereby signifying they express the c-kit gene product. This finding suggests this tumour may be a potential target for tyrosine kinase inhibitor (TKI) agents directed at c-kit. A point mutation in exon 17 of the c-kit gene, however, can abrogate the binding of TKIs. This being the case, immunohistochemistry is necessary to identify potential candidates for treatment with TKIs, but DNA sequence analysis may need to be performed to determine if these tumours will respond. METHODS AND RESULTS: Tumour cells of 23 cases of SCLC showing immunoreactivity for CD117 were laser capture microdissected from archived formalin-fixed paraffin-embedded tissue and the DNA isolated. PCR on exon 17 of the c-kit gene was performed and the amplified product sequenced. No point mutations were detected. CONCLUSIONS: The absence of mutations in exon 17 of CD117+ SCLC suggests this tumour may respond to therapy with TKI.

Expression of the peripheral benzodiazepine receptor is decreased in skin cancers in comparison with normal skin.

BACKGROUND: The peripheral benzodiazepine receptor (PBR) is an 18-kDa protein receptor mainly found on the outer mitochondrial membrane of cells. The PBR plays a role in several cellular functions including haem synthesis, steroidogenesis, DNA synthesis, cell growth and differentiation, and apoptosis. PBR expression in normal skin correlates with proliferating, secretory and differentiated cellular structures. Increased or aberrant expression of PBR has been associated with aggressive behaviour in several tumour types including ovarian, colon and breast adenocarcinomas and glioblastoma. OBJECTIVES: To determine whether changes in normal PBR distribution would be useful as markers for skin cancers or possible target sites for therapies such as photodynamic therapy (PDT), we used immunohistochemistry to evaluate PBR expression and distribution in normal and photodamaged skin (actinic keratoses), skin cancers (in situ and invasive squamous cell carcinomas and superficial, nodular, morphoeiform and mixed pattern basal cell carcinomas) and several benign epithelial proliferations. METHODS: A rabbit polyclonal antibody to a synthetic peptide fragment of the PBR was developed and characterized by enzyme-linked immunosorbent assay and Western blot analysis. The antibody was used to stain formalin-fixed and paraffin-embedded tissue samples (n = 157) by a routine avidin-biotin immunohistochemical technique. Sections were evaluated for antibody localization, distribution (0-4+) and reaction intensity (negative to strong). RESULTS: Normal skin stained with a strong homogeneous positive reaction (3-4+) in the spinous and granular layers (with a gradient corresponding to increasing differentiation), the pilosebaceous units, eccrine gland ducts, endothelial cells and pilar muscle. In cutaneous neoplasms and other skin diseases, a heterogeneous pattern (0-4+) of PBR expression at lower intensity was seen depending on tumour type and degree of differentiation. PBR expression was greatest in well-differentiated tumours, synonymous with the PBR expression gradient seen in normal skin; and least in poorly differentiated and infiltrative tumour types. CONCLUSIONS: The haem biosynthetic pathway has been harnessed for PDT of skin carcinomas by application of exogenous aminolaevulinic acid to generate the endogenous photosensitizer protoporphyrin IX (PpIX). Owing to the role of PBR as a transporter of haem precursors in haem synthesis, PBR density and distribution in skin cancers could be a predictor of the capacity for PpIX production and subsequent response to PDT in skin cancers.

Photodynamic therapy for the treatment of extramammary Paget's disease.

BACKGROUND: Surgical and ablative treatment modalities for extramammary Paget's disease (EMPD) have high recurrence rates and can be associated with significant morbidity. OBJECTIVES: To evaluate photodynamic therapy (PDT) for the treatment of EMPD. METHODS: We conducted a retrospective review of notes and histology of five men with anogenital, groin and axillary EMPD treated with PDT at Roswell Park Cancer Institute between 20 April 1995 and 1 February 2001. RESULTS: Sixteen EMPD lesions were treated with topical aminolaevulinic acid (ALA)-PDT. Eleven of these lesions had failed previous Mohs micrographic surgery, excision or laser ablation. When evaluated 6 months after one treatment with ALA-PDT, eight of 16 (50%) sites achieved a complete clinical response (CR); six of eight CRs were in lesions that had failed prior conventional therapies. Three of the eight CRs (37.5%) recurred at 9, 10 and 10 months. One patient who was partially responsive to topical ALA-PDT subsequently received systemic Photofrin(R)-PDT, with a complete clinical and histological response at 1 year. Functional and cosmetic outcome was excellent in all patients. CONCLUSIONS: PDT is an effective treatment for EMPD. Recurrence rates are high with topical ALA-PDT, but comparable with standard therapies. Topical ALA-PDT causes little scarring and is preferred for superficial disease and mucosal surfaces. Systemic Photofrin(R)-PDT may be better suited for bulky disease. While further studies are indicated, PDT is well tolerated and appears to be a useful therapy for EMPD.

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.

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 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.

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.

The light chain composition of chicken brain myosin-Va: calmodulin, myosin-II essential light chains, and 8-kDa dynein light chain/PIN.

Class V myosins are a ubiquitously expressed family of actin-based molecular motors. Biochemical studies on myosin-Va from chick brain indicate that this myosin is a two-headed motor with multiple calmodulin light chains associated with the regulatory or neck domain of each heavy chain, a feature consistent with the regulatory effects of Ca(2+) on this myosin. In this study, the identity of three additional low molecular weight proteins of 23-,17-, and 10 kDa associated with myosin-Va is established. The 23- and 17-kDa subunits are both members of the myosin-II essential light chain gene family, encoded by the chicken L23 and L17 light chain genes, respectively. The 10-kDa subunit is a protein originally identified as a light chain (DLC8) of flagellar and axonemal dynein. The 10-kDa subunit is associated with the tail domain of myosin-Va.

Gastrointestinal stromal tumors: current diagnosis, biologic behavior, and management.

Gastrointestinal stromal tumors (GIST) are rare tumors of the gastrointestinal (GI) tract that arise from primitive mesenchymal cells. GISTs occur throughout the GI tract but are usually located in the stomach and small intestine. The majority of GISTs are immunohistochemically positive for c-kit protein (CD 117) and CD34. GISTs express a heterogeneous clinical course not easily predicted by standard pathological means. The most important prognostic factors are size > 5 cm, tumor necrosis, infiltration and metastasis to other sites, mitotic count > 1-5 per 10 high-powered fields, and most recently, mutation in the c-kit gene. Surgical resection remains the mainstay of treatment, as chemotherapy and radiation are ineffective. Long-term follow-up is imperative, as recurrence rates are high.