Predicting sentinel node positivity in patients with melanoma: external validation of a risk-prediction calculator (the Melanoma Institute Australia nomogram) using a large European population-based patient cohort.

BACKGROUND: A nomogram to predict sentinel node (SN) positivity [the Melanoma Institute Australia (MIA) nomogram] was recently developed and externally validated using two large single-institution databases. However, there remains a need to further validate the nomogram's performance using population-based data. OBJECTIVES: To perform further validation of the nomogram using a European national patient cohort. METHODS: Patients with cutaneous melanoma who underwent SN biopsy in the Netherlands between 2000 and 2014 were included. Their data were obtained from the Dutch Pathology Registry. The predictive performance of the nomogram was assessed by discrimination (C-statistic) and calibration. Negative predictive values (NPVs) were calculated at various predicted probability cutoffs. RESULTS: Of the 3049 patients who met the eligibility criteria, 23% (691) were SN positive. Validation of the MIA nomogram (including the parameters Breslow thickness, ulceration, age, melanoma subtype and lymphovascular invasion) showed a good C-statistic of 0·69 (95% confidence interval 0·66-0·71) with excellent calibration (R2 = 0·985, P = 0·40). The NPV of 90·1%, found at a 10% predicted probability cutoff for having a positive SN biopsy, implied that by using the nomogram, a 16·3% reduction in the rate of performing an SN biopsy could be achieved with an error rate of 1·6%. Validation of the MIA nomogram considering mitotic rate as present or absent showed a C-statistic of 0·70 (95% confidence interval 0·68-0·74). CONCLUSIONS: This population-based validation study in European patients with melanoma confirmed the value of the MIA nomogram in predicting SN positivity. Its use will spare low-risk patients the inconvenience, cost and potential risks of SN biopsy while ensuring that high-risk patients are still identified.

Desmoplastic melanoma: a review of its pathology and clinical behaviour, and of management recommendations in published guidelines.

Desmoplastic melanomas are uncommon. Their behaviour differs from that of other melanoma subtypes; therefore, management guidelines for non-desmoplastic melanomas may not be applicable. This review sought to examine all available evidence relating to the behaviour and management of desmoplastic melanomas, based on review of all relevant English-language publications, and to critically assess the recommendations for their management in current published melanoma management guidelines. Compared with other melanoma subtypes, patients with 'pure' desmoplastic melanomas (where ≥90% of the invasive melanoma is of desmoplastic melanoma subtype) have much lower rates of sentinel node positivity and distant metastasis. Local recurrence rates are higher for desmoplastic melanomas, but resection margins wider than those recommended for non-desmoplastic melanomas have not been shown to be of benefit. Adjuvant radiotherapy reduces the risk of local recurrence when a satisfactory histological clearance (≥8 mm) cannot be achieved. Of 29 published melanoma management guidelines identified, only 11 specified management for desmoplastic melanomas, while seven simply stated that the feature should be reported. Desmoplastic melanoma is a unique melanoma subtype with biology that differs from that of other melanoma subtypes. It requires specific management strategies but few current guidelines address these.

Factors predisposing to wrong blood in tube incidents: a year's experience in the North East of England.

INTRODUCTION: Wrong blood in tube (WBIT) describes a transfusion sample collected from one patient but labelled with the identification details of a different patient. These incidents have the potential to result in catastrophic harm to patients. In 2011, the Serious Hazards of Transfusion (SHOT) organisation received 469 reports of WBIT across the UK. WHAT THIS STUDY ADDS: This was a prospective study of WBIT which collected information not only on the frequency of WBIT but also risk factors. METHOD: All hospitals in the North East region of England submitted details of known WBITs during a 12-month period starting from July 2011, including the time of day and location where samples were taken, the job title and competency of the sample taker, and how the WBIT was identified. Where possible, the sampler was interviewed to determine reasons for the WBIT. RESULTS: There were 48 WBITs, giving a corrected incidence of 1 : 2717 repeat transfusion samples. Doctors were responsible for 24 of 45 WBITs where the identity of the sampler was known. The rate as a proportion of samples was highest in medicine and paediatric specialties. The commonest risk factor for WBIT was labelling away from the bedside (44%). CONCLUSIONS: These findings support, and add to, the data collected by SHOT. If our figures are representative of the whole of the UK, then over 1160 WBITs will occur each year, justifying SHOT's concerns that WBITs are under reported. Interventions are needed to ensure labelling of transfusion samples is always carried out at the patient's side.

VEGF(121)b, a new member of the VEGF(xxx)b family of VEGF-A splice isoforms, inhibits neovascularisation and tumour growth in vivo.

BACKGROUND: The key mediator of new vessel formation in cancer and other diseases is VEGF-A. VEGF-A exists as alternatively spliced isoforms - the pro-angiogenic VEGF(xxx) family generated by exon 8 proximal splicing, and a sister family, termed VEGF(xxx)b, exemplified by VEGF(165)b, generated by distal splicing of exon 8. However, it is unknown whether this anti-angiogenic property of VEGF(165)b is a general property of the VEGF(xxx)b family of isoforms. METHODS: The mRNA and protein expression of VEGF(121)b was studied in human tissue. The effect of VEGF(121)b was analysed by saturation binding to VEGF receptors, endothelial migration, apoptosis, xenograft tumour growth, pre-retinal neovascularisation and imaging of biodistribution in tumour-bearing mice with radioactive VEGF(121)b. RESULTS: The existence of VEGF(121)b was confirmed in normal human tissues. VEGF(121)b binds both VEGF receptors with similar affinity as other VEGF isoforms, but inhibits endothelial cell migration and is cytoprotective to endothelial cells through VEGFR-2 activation. Administration of VEGF(121)b normalised retinal vasculature by reducing both angiogenesis and ischaemia. VEGF(121)b reduced the growth of xenografted human colon tumours in association with reduced microvascular density, and an intravenous bolus of VEGF(121)b is taken up into colon tumour xenografts. CONCLUSION: Here we identify a second member of the family, VEGF(121)b, with similar properties to those of VEGF(165)b, and underline the importance of the six amino acids of exon 8b in the anti-angiogenic activity of the VEGF(xxx)b isoforms.

VEGF 165 b, an antiangiogenic VEGF-A isoform, binds and inhibits bevacizumab treatment in experimental colorectal carcinoma: balance of pro- and antiangiogenic VEGF-A isoforms has implications for therapy.

Bevacizumab, an anti-vascular endothelial growth factor (VEGF-A) antibody, is used in metastatic colorectal carcinoma (CRC) treatment, but responses are unpredictable. Vascular endothelial growth factor is alternatively spliced to form proangiogenic VEGF(165) and antiangiogenic VEGF(165)b. Using isoform-specific enzyme-linked immunosorbent assay and quantitative polymerase chain reaction, we found that over 90% of the VEGF in normal colonic tissue was VEGF(xxx)b, but there was a variable upregulation of VEGF(xxx) and downregulation of VEGF(xxx)b in paired human CRC samples. Furthermore, cultured colonic adenoma cells expressed predominantly VEGF(xxx)b, whereas colonic carcinoma cells expressed predominantly VEGF(xxx). However, adenoma cells exposed to hypoxia switched their expression from predominantly VEGF(xxx)b to predominantly VEGF(xxx). VEGF(165)b overexpression in LS174t colon cancer cells inhibited colon carcinoma growth in mouse xenograft models. Western blotting and surface plasmon resonance showed that VEGF(165)b bound to bevacizumab with similar affinity as VEGF(165). However, although bevacizumab effectively inhibited the rapid growth of colon carcinomas expressing VEGF(165), it did not affect the slower growth of tumours from colonic carcinoma cells expressing VEGF(165)b. Both bevacizumab and anti-VEGF(165)b-specific antibodies were cytotoxic to colonic epithelial cells, but less so to colonic carcinoma cells. These results show that the balance of antiangiogenic to proangiogenic isoforms switches to a variable extent in CRC, regulates tumour growth rates and affects the sensitivity of tumours to bevacizumab by competitive binding. Together with the identification of an autocrine cytoprotective role for VEGF(165)b in colonic epithelial cells, these results indicate that bevacizumab treatment of human CRC may depend upon this balance of VEGF isoforms.

Expression of VEGF(xxx)b, the inhibitory isoforms of VEGF, in malignant melanoma.

Malignant melanoma is the most lethal of the skin cancers and the UK incidence is rising faster than that of any other cancer. Angiogenesis - the growth of new vessels from preexisting vasculature - is an absolute requirement for tumour survival and progression beyond a few hundred microns in diameter. We previously described a class of anti-angiogenic isoforms of VEGF, VEGF(xxx)b, that inhibit tumour growth in animal models, and are downregulated in some cancers, but have not been investigated in melanoma. To determine whether VEGF(xxx)b expression was altered in melanoma, PCR and immunohistochemistry of archived human tumour samples were used. In normal epidermis and in a proportion of melanoma samples, VEGF(xxx)b staining was seen. Some melanomas had much weaker staining. Subsequent examination revealed that expression was significantly reduced in primary melanoma samples (both horizontal and vertical growth phases) from patients who subsequently developed tumour metastasis compared with those who did not (analysis of variance (ANOVA) P<0.001 metastatic vs nonmetastatic), irrespective of tumour thickness, while the surrounding epidermis showed no difference in expression. Staining for total VEGF expression showed staining in metastatic and nonmetastatic melanomas, and normal epidermis. An absence of VEGF(xxx)b expression appears to predict metastatic spread in patients with primary melanoma. These results suggest that there is a switch in splicing as part of the metastatic process, from anti-angiogenic to pro-angiogenic VEGF isoforms. This may form part of a wider metastatic splicing phenotype.

Laparoscopic anterior resection and uterine manipulation: why make things difficult?

The uterus continues to be a source of difficulty in mobilization of the rectum. These difficulties are compounded in laparoscopic operations, particularly anterior resection, proctectomy and vaginal sacrocolpopexy. Techniques to overcome this have in general proved unsatisfactory. We have adopted a technique of using an extended Vesellum forceps from gynaecological practice and recommend its use by all. The technique is described.

Effect of a 915-MHz simulated mobile phone signal on cognitive function in man.

PURPOSE: To examine whether a simulated mobile telephone transmission at 915 MHz has an effect on cognitive function in man. MATERIALS AND METHODS: Thirty-six subjects in two groups were each given two training sessions and then three test sessions in a randomized three-way cross-over design. About 1 W mean power at 915 MHz from a quarter-wave antenna mounted on a physical copy of an analogue phone, as a sine wave, or modulated at 217 Hz with 12.5% duty cycle, or no power, was applied to the left squamous temple region of the subjects while they undertook a series of cognitive function tests lasting approximately 25-30 min. The second group was investigated for sleep, consumption of alcohol and beverages, and any other substances that might affect performance. RESULTS: In both groups, the only test affected was the choice reaction time and this showed as an increase in speed (a decrease in reaction time). There were no changes in word, number or picture recall, or in spatial memory. While an effect of visit-order was evident suggesting a learning effect of repeat tests, the design of the study allowed for this. Additionally, there was no systematic error introduced as a result of consumption of substances or sleep time. CONCLUSIONS: There was evidence of an increase in responsiveness, strongly in the analogue and less in the digital simulation, in choice reaction time. This could be associated with an effect on the angular gyrus that acts as an interface between the visual and speech centres and which lies directly under and on the same side as the antenna. Such an effect could be consistent with mild localized heating, or possibly a non-thermal response, which is nevertheless power-dependent.

Effect of cyclosporin on pancreatic events and development of diabetes in BB/Edinburgh rats.

The effect of cyclosporin administered from 30 to 100 days of age on pancreatic events and the development of insulin-dependent diabetes has been studied by serial pancreatic biopsy of individual diabetes-prone BB/Edinburgh rats. Cyclosporin completely prevented the development of diabetes up to 150 days of age and reduced the incidence to 50% of controls at 452 days of age. Islet cell surface antibodies paralleled the development of diabetes. Insulin autoantibodies were unrelated to diabetes and not affected by cyclosporin. Immunohistochemical analysis of pancreatic biopsies from untreated control diabetes-prone rats with monoclonal antibodies specific for rat MHC molecules and T- and B-lymphocyte and macrophage subsets showed that the first abnormality seen in rats that subsequently developed diabetes was hyperexpression of MHC class I molecules on vascular endothelium and islet cells. This was followed by accumulation of ED1+ macrophages at perivascular and periductal sites adjacent to noninfiltrated islets. Increased expression of MHC class II molecules on vascular endothelial cells was also noted. Most cells infiltrating the islets initially were also ED1+ macrophages, followed by increasing numbers of other activated effector cells including helper and cytotoxic-suppressor T lymphocytes and natural killer cells. Obliteration of insulin-containing cells was associated with regression of the infiltrate. Treatment with cyclosporin had no effect on pancreatic hyperexpression of MHC class I molecules but markedly inhibited accumulation of ED1+ cells at extraislet sites, the subsequent recruitment of immune effector cells, and islet infiltration. This resulted in a delay of the onset of diabetes in some rats and prevention of diabetes in others.

Interferon-gamma induces class II MHC antigens on RINm5F cells.

The ability of recombinant interferon-gamma (rIFN-gamma) to induce major histocompatibility complex (MHC) antigen expression in the rat insulinoma cell line RINm5F was investigated. The cells were stained with monoclonal antibodies specific for rat class I and class II MHC antigens. RINm5F cells endogenously expressed class I antigens; this was enhanced by rIFN-gamma. Class II antigens could not be detected on RINm5F cells, but both I-A and I-E were induced by rIFN-gamma.

Insulin autoantibodies, islet cell surface antibodies and the development of spontaneous diabetes in the BB/Edinburgh rat.

The presence of insulin autoantibodies (IAA) and islet cell surface antibodies (ICSA) was sought in two longitudinal studies, involving BB/Edinburgh rats of high (BB/E/H, n = 157) and low (BB/E/L, n = 61) susceptibility to diabetes development. Both studies were designed to correlate pancreatic morphology with cellular and humoral immunity. In Study I, groups of eight male and eight female non-diabetic rats of the BB/E/H line were killed at 15 day intervals from 30-105 days and plasma samples were obtained by cardiac puncture. In study II, 61 BB/E/H and 41 BB/E/L rats underwent pancreatic biopsy 1-3 times from 30 days of age until onset of diabetes or 150 days, plasma samples being taken from the tail vein at biopsy. Both studies revealed a higher prevalence for ICSA than IAA in BB/E rats. Whereas a highly significant association of ICSA with diabetes development was observed in study II (chi 2 = 8.30, P less than 0.005), IAA were associated with diabetes development only weakly (P less than 0.03, Mann-Witney U-rank test). No correlation between the presence of ICSA and IAA in individual rats was observed and IAA were not significantly associated with BB/E/H in preference to BB/E/L rats, although positive IAA values were significantly elevated in the former compared with the latter (P less than 0.01). These observations support the concept that IAA form part of a background of heightened autoimmunity against which frank diabetes develops in some animals.

Interferon-mediated enhancement of thyroid major histocompatibility complex antigen expression. A flow cytometric analysis.

Epithelial expression of class II antigens encoded by the major histocompatibility complex (MHC) has been proposed as a means by which autoimmune thyroid disease may be initiated and maintained. We studied a rat thyroid epithelial cell line (FRTL-5), which constitutively expresses class I (OX18) but not class II (OX6 or OX17) determinants to quantify in vitro MHC antigen induction using flow cytometry. Recombinant rat gamma interferon (rIFN-gamma) induced dose-dependent expression of OX6 (I-A) antigen at greater than 48 h (maximum 80-90% of cells in culture at 100 U/ml), which was abrogated by DB-1, a monoclonal antibody to rat IFN-gamma. OX17 antigen (I-E) was also induced (86%) and OX18 (class I) markedly increased under these conditions. Other thyroid-active agents including the calcium ionophore A23187, dibutyryl cyclic AMP, thyroid-stimulating autoantibodies from Graves' disease patients (LATS), and TSH, caused no I-A induction. Supernatants from spleen cells stimulated with plant lectins (concanavalin A or phytohaemagglutinin), but not lectin alone, evoked substantial class II induction, which was inhibited by DB-1. These findings suggest that IFN-gamma is the central mediator of thyroid epithelial class II expression. FRTL-5 provides a powerful model for the analysis of thyroid MHC class II dynamics and a potential means of analysing the role of epithelial class II in autoimmune pathogenesis.

Immunological responses of the BB rat colony in Edinburgh.

Several immunological responses of the spontaneously diabetic BB rat colony in Edinburgh designated (BB/E) have been studied. The proliferative responses to Con A and LPS, ability to make IL-2 and to show NK activity have been studied using diabetic and non-diabetic BB/E rats and normal Wistar rats. Our data suggest that the diabetic animals in the BB/E colony do not have marked deficiencies in any of these parameters. Lymphopenia and depressed T-cell responses do not appear to be a prerequisite for the development of diabetes in the BB/E colony.

Immunological defects in SJL mice.

SJL mice are shown to be defective in their ability to develop suppressor cells following stimulation with Con A, a polyclonal T-cell activator. They make a normal proliferative response to this mitogen. In addition to this suppressor T-cell defect, the SJL mouse (unlike most mouse strains) does not develop a spontaneous antibody response to bromelain-treated autologous red blood cells (BrMRBC) in vitro. Although the SJL makes a normal proliferative response to LPS, antibody-forming cells against bromelain-treated autologous red blood cells are not increased following LPS in vivo nor does it manifest an increased response to SRBC or TNP. This may signify the presence of a functional B-cell defect in these animals. DBA mice are also shown, in this report, to have small numbers of antibody-forming cells to bromelain-treated autologous red blood cells but to be capable of responding to LPS in vivo with an increase in SRBC and TNP antibody responses.

Cyclosporine affects the function of antigen-presenting cells.

The immunosuppressant drug cyclosporine (CsA) is known to affect T-cell function. We have studied the effect of CsA on the specific proliferative response of T-cell lines to antigen. In addition to blocking IL-2 release by specifically activated T-cell lines, CsA also affected the ability of irradiated spleen cells to present preprocessed antigen to T-cell lines. Irradiated spleen cells pulsed with antigen for 2 hr were able to stimulate a proliferative response in T-cell lines. Following a 2-hr pulse with CsA, antigen presentation by these irradiated spleen cells was reduced significantly, suggesting that CsA not only affects T cells, but also affects the function of antigen-presenting cells.

Autoreactive T-cell lines specific for mouse thyroglobulin.

Autoreactive T-cell specific for mouse thyroglobulin have been established and characterized. These Lyt 1+ T cells proliferated specifically in response to thyroglobulin presented by syngeneic irradiated spleen cells. The antigen-presenting cell requirements of these autoreactive T cells appeared to be the same as those for foreign antigen (PPD) reactive T cells. All lines tested required antigen-presenting cells compatible at the I-A subregion of the H-2 complex. Both T-cell types responded to antigen presented by peritoneal cells and splenic dendritic cells, but only gave optimal responses when whole spleen cells were used. The cross-reactivity patterns of responses to mouse, rat, pig and human thyroglobulins indicated that at least two different epitopes could be recognized by the autoreactive T cells. Furthermore, these epitopes appeared to be different from those recognized by the majority of serum autoantibodies to mouse thyroglobulin.