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As the p53 tumor suppressor is rarely mutated in conjunctival melanoma (CM), we investigated its activation as a potential therapeutic strategy. Preventing p53/Mdm2 interaction by Nutlin-3, the prototypical Mdm2 antagonist, or via direct siRNA Mdm2 depletion, increased p53 and inhibited viability in CM cell lines. The sensitivity to Nutlin-3 p53 reactivation with concomitant Mdm2 stabilization was higher than that achieved by siRNA, indicative of effects on alternative Mdm2 targets, identified as the cancer-protective IGF-1R. Nutlin-3 treatment increased the association between IGF-1R and β-arrestin1, the adaptor protein that brings Mdm2 to the IGF-1R, initiating receptor degradation in a ligand-dependent manner. Controlled expression of β-arrestin1 augmented inhibitory Nutlin-3 effects on CM survival through enhanced IGF-1R degradation. Yet, the effect of IGF-1R downregulation on cell proliferation is balanced by β-arrestin1-induced p53 inhibition. As mitomycin (MMC) is a well-established adjuvant treatment for CM, and it triggers p53 activation through genotoxic stress, we evaluated how these alternative p53-targeting strategies alter the cancer-relevant bioactivities of CM. In 2D and 3D in vitro models, Nutlin-3 or MMC alone, or in combination, reduces the overall cell tumor growth ~30%, with double treatment inhibition rate only marginally higher than single-drug regimens. However, histopathological evaluation of the 3D models revealed that Nutlin-3 was the most effective, causing necrotic areas inside spheroids and complete loss of nuclear staining for the proliferative marker Ki67. These findings were further validated in vivo; zebrafish xenografts demonstrate that Nutlin-3 alone has higher efficacy in restraining CM tumor cell growth and preventing metastasis. Combined, these results reveal that β-arrestin1 directs Mdm2 toward different substrates, thus balancing IGF-1R pro-tumorigenic and p53-tumor suppressive signals. This study defines a potent dual-hit strategy: simultaneous control of a tumor-promoter (IGF-1R) and tumor-suppressor (p53), which ultimately mitigates recurrent and metastatic potential, thus opening up targeted therapy to CM.

OBJECTIVES: To establish the utility of a novel in‐house method of CSF analysis using sedimentation cytology direct from the spinal needle for the detection of laboratory‐defined pleocytosis. MATERIALS AND METHODS: In dogs and cats undergoing routine CSF analysis for investigation of neurological signs, an additional preparation was made at the patient's side by inverting the spinal needle on a slide and sedimenting for at least 1 hour. Nucleated cellularity and differential counts were assessed and compared with “gold‐standard” analysis. Variability of cell counts between observers and within slides using the new method was evaluated to optimise the procedure. RESULTS: Using a ×50 objective, at least 10 fields and an average of more than five cells per field were considered appropriate guidelines to achieve correct classification of samples (normal or pleocytosis). The new method had high sensitivity (89%) and specificity (100%) for the detection of laboratory‐defined pleocytosis. Agreement on the type of pleocytosis was good. CLINICAL SIGNIFICANCE: Clinically useful information can be obtained from CSF samples in a patient‐side setting without additional equipment. This technique may be of benefit if little fluid is available or if logistical constraints limit the availability of rapid specialist results.

// Haruka Furuta 1 , Hidehito Yoshihara 1 , Toshiaki Fukushima 2, 3 , Yosuke Yoneyama 1, 6 , Akihiro Ito 4 , Claire Worrall 5 , Ada Girnita 5 , Leonard Girnita 5 , Minoru Yoshida 4 , Tomoichiro Asano 2 , Masayuki Komada 3 , Naoyuki Kataoka 1 , Kazuhiro Chida 1 , Fumihiko Hakuno 1 and Shin-Ichiro Takahashi 1 1 Departments of Animal Sciences and Applied Biological Chemistry, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo, Japan 2 Department of Medical Science, Graduate School of Medicine, Hiroshima University, Hiroshima, Japan 3 Cell Biology Center, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama, Japan 4 Chemical Genomics Research Group, RIKEN Center for Sustainable Resource Science, Saitama, Japan 5 Department of Oncology and Pathology, Cancer Center Karolinska, Karolinska Institute and Karolinska University Hospital, Stockholm, Sweden 6 Present address: Institute of Research, Tokyo Medical and Dental University, Tokyo, Japan Correspondence to: Shin-Ichiro Takahashi, email: atkshin@mail.ecc.u-tokyo.ac.jp Fumihiko Hakuno, email: ahakuno@mail.ecc.u-tokyo.ac.jp Keywords: ubiquitin; USP9X; IGF-I; IRS-2; prostate cancer Received: March 07, 2018 Accepted: July 21, 2018 Published: September 21, 2018 ABSTRACT Insulin-like growth factors (IGFs) have been shown to induce proliferation of many types of cells. Insulin receptor substrates (IRSs) are major targets of IGF-I receptor (IGF-IR) tyrosine kinase activated by IGFs, and are known to play important roles in the activation of downstream signaling pathways, such as the Erk1/2 pathway. Dysregulation of IGF signaling represents a central tumor promoting principle in human carcinogenesis. Prostate carcinoma is highly dependent on the IGF/IGF-IR/IRS axis. Here we identified the deubiquitinase, ubiquitin specific peptidase 9X (USP9X) as a novel binding partner of IRS-2. In a human prostate carcinoma cell line, small interfering RNA (siRNA)-mediated knockdown of USP9X reduced IGF-IR as well as IRS-2 protein levels and increased their ubiquitination. Knockdown of USP9X suppressed basal activation of the Erk1/2 pathway, which was significantly restored by exogenous expression of IRS-2 but not by IGF-IR, suggesting that the stabilization of IRS-2 by USP9X is critical for basal Erk1/2 activation. Finally, we measured anchorage-independent cell growth, a characteristic cancer feature, by soft-agar colony formation assay. Knockdown of USP9X significantly reduced anchorage-independent cell growth of prostate carcinoma cell line. Taken all together, our findings indicate that USP9X is required for the promotion of prostate cancer growth by maintaining the activation of the Erk1/2 pathway through IRS-2 stabilization.

Background IGF-1R, a member of the tyrosine receptor family, is well documented in various experimental models to be an important factor in cell transformation, tumor progression, protection from apoptosis and metastasis. In the absence of IGF-1R, most oncogenes are unable to induce malignant transformation, suggesting that some signaling pathways activated by IGF-1R are essential for transformation. We have recently demonstrated that β-arrestin1 (β-arr1), a key regulator of G Protein Coupled Receptor (GPCR), is involved in IGF-1R signaling. Upon ligand binding, β-arr1 brings Mdm2, an E3 ubiquitin ligase, to the IGF-1R resulting in receptor ubiquitination. β-arr1 also functions as a scaffold to activate the MAPK pathway like GPCR. This study aims to investigate whether the β-arr1 mediated signal of IGF-1R is necessary for malignant transformation. Methods Mouse embryonic fibroblasts (MEFs) and a bladder cancer cell line, T24 cells were used as target cells. β-arr1 knockdown was performed by siRNA transfection. Activation of MAPK and PI-3K pathways was analysed by western blot. Cell proliferation was evaluated by PrestoBlue assay. Cellular transformation was estimated by colony formation assay in soft agar. Results To investigate whether β-arr1 is important for transformation, we stably transfected WT MEF and β-arr1 deficient MEFs with some oncogenes such as HRasV12, PvMT, and vSrc. In the absence of β-arr1, HRasV12 was not able to induce malignant transformation. Similar sensitivity to absence of β-arrestin1 was not observed with PyMT or vSrc. β-arr1 regulates IGF-1-induced activation of the MAPK and PI-3K pathways, both pathways being less active in the absence of β-arr1. These data suggested that β-arr1 is involved in Ras-mediated malignant transformation. In T24 cells with the same mutation in HRas, β-arr1 knockdown attenuate IGF-1 induced MAPK and PI-3K pathways like MEFs. Consistent with less activation of the downstream, IGF-1 induced cell proliferation and colony formation are decreased in β-arr1 knockdown cells, indicating that β-arr1 is required for malignant phenotypes in T24 cells. Conclusions These data suggest that β-arr1-dependent signaling by the IGF-1R regulates mechanisms involved in malignant transformation and progression of cancer cells. Legal entity responsible for the study Karolinska institutes, Department of Oncology-Pathology, Leonard Girnita’s group. Funding Swedish Research Council, Swedish Cancer Society, The Swedish Childhood Cancer Foundation. Disclosure All authors have declared no conflicts of interest.

With very similar 3D structures, the widely expressed β-arrestin isoforms 1 and 2 play at times identical, distinct or even opposing roles in regulating various aspects of G protein-coupled receptors (GPCR) expression and signalling. Recent evidence recognizes the β-arrestin system as a key regulator of not only GPCRs, but also receptor tyrosine kinases, including the highly cancer relevant insulin-like growth factor type 1 receptor (IGF-1R). Binding of β-arrestin1 to IGF-1R leads to ligand-dependent degradation of the receptor and generates additional MAPK/ERK signalling, protecting cancer cells against anti-IGF-1R therapy. Because the interplay between β-arrestin isoforms governs the biological effects for most GPCRs, as yet unexplored for the IGF-1R, we sought to investigate specifically the regulatory roles of the β-arrestin2 isoform on expression and function of the IGF-1R. Results from controlled expression of either β-arrestin isoform demonstrate that β-arrestin2 acts in an opposite manner to β-arrestin1 by promoting degradation of an unstimulated IGF-1R, but protecting the receptor against agonist-induced degradation. Although both isoforms co-immunoprecipitate with IGF-1R, the ligand-occupied receptor has greater affinity for β-arrestin1; this association lasts longer, sustains MAPK/ERK signalling and mitigates p53 activation. Conversely, β-arrestin2 has greater affinity for the ligandunoccupied receptor; this interaction is transient, triggers receptor ubiquitination and degradation without signalling activation, and leads to a lack of responsiveness to IGF-1, cell cycle arrest and decreased viability of cancer cells. This study reveals contrasting abilities of IGF-1R to interact with each β-arrestin isoform, depending on the presence of the ligand and demonstrates the antagonism between the two β-arrestin isoforms in controlling IGF-1R expression and function, which could be developed into a practical anti-IGF-1R strategy for cancer therapy.

ObjectivesPhysician variation in the use of computed tomography (CT) is concerning due to the risks of ionizing radiation, cost, and downstream effects of unnecessary testing. The objectives of this study were to describe variation in CT-ordering rates among emergency physicians (EPs), to measure correlation between perceived and actual CT-ordering rates, to assess attitudes that influence decisions to order imaging tests, and to identify EP attitudes associated with higher CT utilization.MethodsThis study was a retrospective review of imaging and administrative billing records at two emergency department sites of a tertiary care adult teaching hospital. The study also included a cross-sectional survey of EPs at this hospital. We asked physicians about their perceived ordering behaviour, and what factors influenced their decision to order a CT. We examined correlations between perceived and actual CT-ordering rates. We adjusted ordering rates for shift distribution using a logistic regression model and identified outlier physicians whose ordering rate was significantly lower or higher than expected. We used multivariable regression analysis to determine which survey responses predicted higher CT utilization.ResultsDuring the study period, 59 EPs saw 45,854 patients, and ordered 6,609 CTs — a mean ordering rate of 14.4% (standard deviation (SD)=4.3%). The ordering rate for individual physicians ranged from 5.9% to 25.9%. Of the 59 EPs, 13 EPs were low-ordering outliers; 12 were high-ordering outliers. Forty-five EPs (76.3%) completed the survey. Mean perceived ordering rate was 12.6%, and was weakly correlated with actual ordering (r=0.19, p=0.21). 42 EPs (93.3%) believed they ordered “about the same” or “fewer” CTs than their peers. Of the 17 EPs in the two highest ordering quintiles, only 3 (18%) knew they were high orderers. In the multivariable analysis, higher ordering was associated with increasing strength of response to the following predictors: medico-legal risk (relative risk [RR]=1.18, 95% CI: 1.03–1.21), risk of contrast (RR=1.14, 95% CI: 1.07–1.22), what colleagues would do (RR=1.09, 95% CI: 0.99–1.19), risk of missing a diagnosis (RR=1.08, 95% CI: 0.98–1.21), and patient wishes (RR=1.07, 95% CI: 0.97–1.17).ConclusionsThere is large variation in CT ordering among EPs. Physicians’ self-reported ordering rate correlates poorly with actual ordering. High CT orderers were rarely aware that they ordered more than their colleagues. Higher rates of ordering were observed among physicians who reported increased concern with 1) risk of missing a diagnosis, 2) medico-legal risk, 3) risk of contrast, 4) patient wishes, and 5) what colleagues would do.

Melanoma tumors usually retain wild-type p53; however, its tumor-suppressor activity is functionally disabled, most commonly through an inactivating interaction with mouse double-minute 2 homolog (Mdm2), indicating p53 release from this complex as a potential therapeutic approach. P53 and the tumor-promoter insulin-like growth factor type 1 receptor (IGF-1R) compete as substrates for the E3 ubiquitin ligase Mdm2, making their relative abundance intricately linked. Hence we investigated the effects of pharmacological Mdm2 release from the Mdm2/p53 complex on the expression and function of the IGF-1R. Nutlin-3 treatment increased IGF-1R/Mdm2 association with enhanced IGF-1R ubiquitination and a dual functional outcome: Receptor downregulation and selective downstream signaling activation confined to the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway. This Nutlin-3 functional selectivity translated into IGF-1-mediated bioactivities with biphasic effects on the proliferative and metastatic phenotype: An early increase and late decrease in the number of proliferative and migratory cells, while the invasiveness was completely inhibited following Nutlin-3 treatment through an impaired IGF-1-mediated matrix metalloproteinases type 2 activation mechanism. Taken together, these experiments reveal the biased agonistic properties of Nutlin-3 for the mitogen-activated protein kinase pathway, mediated by Mdm2 through IGF-1R ubiquitination and provide fundamental insights into destabilizing p53/Mdm2/IGF-1R circuitry that could be developed for therapeutic gain.

Due to its ability to compensate for signals lost following therapeutic MAPKinhibition, insulin-like growth factor type 1 receptor (IGF-1R) co-targeting is a rational approach for melanoma treatment. However IGF-1R conformational changes associated with its inhibition can preferentially activate MAPK-pathway in a kinaseindependent manner, through a process known as biased signaling. We explored the impact of biased IGF-1R signaling, on response to MAPK inhibition in a panel of skin melanoma cell lines with differing MAPK and p53 mutation statuses. Specific siRNA towards IGF-1R down-regulates the receptor and all its signaling in a balanced manner, whilst IGF-1R targeting by small molecule Nutlin-3 parallels receptor degradation with a transient biased pERK1/2 activity, with both strategies synergizing with MEK1/2 inhibition. On the other hand, IGF-1R down-regulation by a targeted antibody (Figitumumab) induces a biased receptor conformation, preserved even when the receptor is exposed to the balanced natural ligand IGF-1. This process sustains MAPK activity and competes with the MEK1/2 inhibition. Our results indicate that IGF-1R down-regulation offers an approach to increase the sensitivity of melanoma cells to MAPK inhibition, and highlights that controlling biased signaling could provide greater specificity and precision required for multi-hit therapy.

Objectives: Computed tomography (CT) use is increasing in the emergency department (ED). Many physicians are concerned about exposing patients to radiation from CT scanning, but estimates of radiation doses vary. This study’s objective was to determine the radiation doses from CT scanning for common indications in a Canadian ED using modern multidetector CT scanners. Methods: We conducted a health records review of consecutive adult patients seen at two busy tertiary care EDs over a 2-month period who underwent CT scanning ordered by emergency physicians. Cases were identified by searching an imaging database. Data collected included patient age and sex, study indication, scanner model, body area, and reported dose-length product. Effective dose per scan was calculated from reported dose-length product. Data were collected on a standardized form, entered into an electronic database, and analyzed with descriptive statistics and 95% CIs. Results: During the study period, emergency physicians assessed 19,880 patients. Overall, 2,720 (13.7%) underwent CT scanning, and of these, 144 (5.3%) patients had more than one scan. Patients had a mean age of 59.0 years, and 45.3% were men. Mean doses for the most common indications were as follows: simple head, 2.9 mSv; cervical spine, 5.7 mSv; complex head, 9.3 mSv; CT pulmonary angiogram, 11.2 mSv; abdomen (nontraumatic abdominal pain), 15.4 mSv; and abdomen (renal colic), 9.8 mSv. Conclusions: Approximately one in seven ED patients had a CT scan. Emergency physicians should be aware of typical radiation doses for the studies they order and how the dose varies by protocol and indication.

Cell-surface receptors govern the critical information passage from outside to inside the cell and hence control important cellular decisions such as survival, growth, and differentiation. These receptors, structurally grouped into different families, utilize common intracellular signaling-proteins and pathways, yet promote divergent biological consequences. In rapid processing of extracellular signals to biological outcomes, posttranslational modifications offer a repertoire of protein processing options. Protein ubiquitination was originally identified as a signal for protein degradation through the proteasome system. It is now becoming increasingly recognized that both ubiquitin and ubiquitin-like proteins, all evolved from a common ubiquitin structural superfold, are used extensively by the cell and encompass signal tags for many different cellular fates. In this chapter we examine the current understanding of the ubiquitin regulation surrounding the insulin-like growth factor and insulin signaling systems, major members of the larger family of receptor tyrosine kinases (RTKs) and key regulators of fundamental physiological and pathological states.

Owing to its essential role in cancer, insulin-like growth factor type 1 receptor (IGF-1R)–targeted therapy is an exciting approach for cancer treatment. However, when translated into clinical trials, IGF-1R–specific antibodies did not fulfill expectations. Despite promising clinical responses in Ewing’s sarcoma (ES) phase I/II trials, phase III trials were discouraging, requiring bedside-to-bench translation and functional reevaluation of the drugs. The anti-IGF-1R antibody figitumumab (CP-751,871; CP) was designed as an antagonist to prevent ligand–receptor interaction but, as with all anti-IGF-1R antibodies, it induces agonist-like receptor down-regulation. We explored this paradox in a panel of ES cell lines and found their sensitivity to CP was unaffected by presence of IGF-1, countering a ligand blocking mechanism. CP induced IGF-1R/β-arrestin1 association with dual functional outcome: receptor ubiquitination and degradation and decrease in cell viability and β-arrestin1–dependent ERK signaling activation. Controlled β-arrestin1 suppression initially enhanced CP resistance. This effect was mitigated on further β-arrestin1 decrease, due to loss of CP-induced ERK activation. Confirming this, the ERK1/2 inhibitor U0126 increased sensitivity to CP. Combined, these results reveal the mechanism of CP-induced receptor down-regulation and characteristics that functionally qualify a prototypical antagonist as an IGF-1R–biased agonist: β-arrestin1 recruitment to IGF-1R as the underlying mechanism for ERK signaling activation and receptor down-regulation. We further confirmed the consequences of β-arrestin1 regulation on cell sensitivity to CP and demonstrated a therapeutic strategy to enhance response. Defining and suppressing such biased signaling represents a practical therapeutic strategy to enhance response to anti-IGF-1R therapies.

Introduction Breast cancer is increasingly a disease of the elderly, and combined with the NHS Breast Screening Extension means that more elderly patients are having mammography. Increasing age can make mammography more technically difficult. This is a technical note detailing the results of a local audit which may be of interest due to potential service implications. Method A retrospective audit of the first year of screening extension of The Shropshire Breast Screening Programme. Aims to collect data on patient demographics and describe the technical adaptations developed in Shropshire. Results Breast screening extension has increased by 2.5 times the number of women aged 70–74 screened, and doubled the overall numbers of women over 70 screened. Significantly more older patients are being screened to present technical challenges to a screening programme. Data was obtained from a month of screening showed that 29% of patients over 70 needed extra time for positioning. Reasons included 22% difficulty in obtaining adequate positioning and 15% needed a relative to aid with consent. Discussion In the Shropshire screening programme different technical adaptations have been developed and are key to ensuring adequate images. These include double appointments, two radiographers, thorough assessment, steeper angles, seated examinations, from-below imaging and pre-planning for subsequent screen. Conclusion Significantly more older women are having breast screening due to the increasing incidence of breast cancer and the Breast Screening Programme extension. Increasing age can significantly increase time taken for adequate imaging and present technical challenges. Development of technical adaptations to art of mammography is key to achieve adequate images.

Objective:The primary objective of this study was to compare the results of nurse-performed urinalysis (NPU) interpreted visually in the emergency department (ED) with laboratory-performed urinalysis (LPU) interpreted by reflectance photometry.Methods:This was a prospective observational study based on a convenience sample from my emergency practice. Emergency nurses, who were unaware of the study, performed usual dipstick analysis before sending the same urine sample to the laboratory for testing.Results:Of 140 urinalyses performed during the study period, 124 were suitable for analysis. When compared with the reference standard LPU, the NPU had an overall sensitivity of 100% (95% confidence interval [CI] 95%–100%) and a specificity of 49% (95% CI 33%–65%) for the presence of any 1 of blood, leukocyte esterase, nitrites, protein, glucose or ketones in the urine. Of 20 falsely positive NPUs, 18 were a result of the nurse recording 1 or more components as “trace” positive.Conclusion:Although NPU does not yield identical results to LPU, a negative LPU is expected when the initial NPU in the ED is negative.

The prime position of the insulin-like growth factor 1 receptor (IGF-1R), at the head of the principle mitogenic and anti-apoptotic signalling cascades, along with the resilience to transformation of IGF-1R deficient cells fuelled great excitement for its anti-cancer targeting. Yet its potential has not been fulfilled, as clinical trial results fell far short of expectations. Advancements in understanding of other receptors’ function have now begun to shed light on this incongruity, with the now apparent parallels highlighting the immaturity of our understanding of IGF-1R biology, with the model used for drug development now recognised as having been too simplistic. Gathering together the many advancements of the field of IGF-1R research over the past decade, alongside those in the GPCR field, advocates for a major paradigm shift in our appreciation of the subtle workings of this receptor. This review will emphasise the updating of the IGF-1R’s classification from an RTK, to an RTK/GPCR functional hybrid, which integrates both canonical kinase signalling with many functions characteristic of a GPCR. Recognition of the shortcomings of IGF-1R inhibitor drug development programs and the models used not only allows us to reignite the initial interest in the IGF-1R as an anti-cancer therapeutic target, but also points to the possibility of biased ligand therapeutics, which together may hold a very powerful key to unlocking the true potential of IGF-1R modulation.

β-Arrestins are multifunctional proteins that play central roles in G protein-coupled receptor (GPCR) trafficking and signaling. β-Arrestin1 is also recruited to the insulin-like growth factor-1 receptor (IGF-1R), a receptor tyrosine kinase (RTK), mediating receptor degradation and signaling. Because GPCR phosphorylation by GPCR-kinases (GRKs) governs interactions of the receptors with β-arrestins, we investigated the regulatory roles of the four widely expressed GRKs on IGF-1R signaling/degradation. By suppressing GRK expression with siRNA, we demonstrated that lowering GRK5/6 abolishes IGF1-mediated ERK and AKT activation, whereas GRK2 inhibition increases ERK activation and partially inhibits AKT signaling. Conversely, β-arrestin–mediated ERK signaling is enhanced by overexpression of GRK6 and diminished by GRK2. Similarly, we demonstrated opposing effects of GRK2 and -6 on IGF-1R degradation: GRK2 decreases whereas GRK6 enhances ligand-induced degradation. GRK2 and GRK6 coimmunoprecipitate with IGF-1R and increase IGF-1R serine phosphorylation, promoting β-arrestin1 association. Using immunoprecipitation, confocal microscopy, and FRET analysis, we demonstrated β-arrestin/IGF-1R association to be transient for GRK2 and stable for GRK6. Using bioinformatic studies we identified serines 1248 and 1291 as the major serine phosphorylation sites of the IGF-1R, and subsequent mutation analysis demonstrated clear effects on IGF-1R signaling and degradation, mirroring alterations by GRKs. Targeted mutation of S1248 recapitulates GRK2 modulation, whereas S1291 mutation resembles GRK6 effects on IGF-1R signaling/degradation, consistent with GRK isoform-specific serine phosphorylation. This study demonstrates distinct roles for GRK isoforms in IGF-1R signaling through β-arrestin binding with divergent functional outcomes.

Introduction Extra-levator abdomino-perineal resection (eLAP) has superseded standard abdomino-perineal excision (APER) for low rectal cancer. Reduction in rates of circumferential resection margin (CRM) involvement and tumour perforation offer improved oncological outcome. There is no consensus regarding management of the larger perineal defect that results. We report outcomes following primary closure following an eLAP. Method All eLAPs undertaken in a single centre over five years were identified. eLAP and primary closure is the local standard of care. A retrospective review recorded CRM involvement, primary wound healing and infection rates, the incidence of perineal herniae, and subsequent intervention if required. Results Fifty-one patients were identified, with a mean age of 70.2, and a male preponderance of 63%. Ten patients were classified ASA 3 (19.6%); the remainder were ASA 1–2. Thirty-two patients had a laparoscopic operation (63%), with a further 6 converted to open (12%). 14 patients had an open operation (27%). 38/51 patients (75%) had a clear CRM. There were no tumour perforations. EMVI was present in 13 patients (25%). Wound healing problems were identified in 12 patients (24%), with 6 requiring intervention (12%). The incidence of perineal hernia was 9/51 (18%) of which 5 (9.8%) were repaired surgically. Conclusion This series suggests that eLAP combined with primary closure of the perineum may offer the benefits of low rates of CRM involvement and tumour perforation yet minimal perineal wound related morbidity compared to published data. Disclosure of interest None Declared.

Cloned DNA markers which are closely linked to the gene defect causing cystic fibrosis have recently been described. These markers are sufficiently informative for carrier detection in 80% of families where there is a living cystic fibrosis child and unaffected sibs. The tightly linked DNA marker pJ3.11 was used in this study to identify carriers in six families and exclude carrier status in two subjects. Risk calculations for recessive diseases using linked DNA probes may be complex, but useful information for counselling can be obtained in this way.

Friedreich ataxia, a progressive neurodegenerative disorder, is an autosomal recessive disease with a carrier frequency of 1/110 in the United Kingdom. The pathophysiological basis for the disease is not known and the chromosomal location of the mutation remains unidentified. As part of an attempt to map the mutation using linked DNA markers, we demonstrate that the Friedreich ataxia gene is excluded from human chromosome 19. This study also demonstrates that the insulin receptor, which maps to chromosome 19 and may be associated with abnormal biochemical features in some patients, is not the basic defect.

The insulin-like growth factor type 1 receptor (IGF-1R) plays a key role in the development and progression of cancer; however, therapeutics targeting it have had disappointing results in the clinic. As a receptor tyrosine kinase (RTK), IGF-1R is traditionally described as an ON/OFF system, with ligand stabilizing the ON state and exclusive kinase-dependent signaling activation. Newly added to the traditional model, ubiquitin-mediated receptor downregulation and degradation was originally described as a response to ligand/receptor interaction and thus inseparable from kinase signaling activation. Yet, the classical model has proven over-simplified and insufficient to explain experimental evidence accumulated over the last decade, including kinase-independent signaling, unbalanced signaling, or dissociation between signaling and receptor downregulation. Based on the recent findings that IGF-1R "borrows" components of G-protein coupled receptor (GPCR) signaling, including β-arrestins and G-protein-related kinases, we discuss the emerging paradigm for the IGF-1R as a functional RTK/GPCR hybrid, which integrates the kinase signaling with the IGF-1R canonical GPCR characteristics. The contradictions to the classical IGF-1R signaling concept as well as the design of anti-IGF-1R therapeutics treatment are considered in the light of this paradigm shift and we advocate recognition of IGF-1R as a valid target for cancer treatment.

A COINCIDENCE technique for paper chromatography has recently been described1, in which a ‘shadowgram’ of a chromatogram is produced by placing the chromatogram over a photographic film and exposing the covered film to radiation of a wave-length which is absorbed by the ‘spots’ on the chromatogram. In this way a ‘shadowgram’ of purine or pyrimidine ‘spots’ may be produced by exposing the covered film to an ultra-violet source, or of amino-acid spots (after development with ninhydrin) by exposure to visible light.