Frequent MAGE mutations in human melanoma.

Abstract: Background: Cancer/testis (CT) genes are expressed only in the germ line and certain tumors and are most frequently located on the X-chromosome (the CT-X genes). Amongst the best studied CT-X genes are those encoding several MAGE protein families. The function of MAGE proteins is not well understood, but several have been shown to potentially influence the tumorigenic phenotype. Methodology/Principal Findings: We undertook a mutational analysis of coding regions of four CT-X MAGE genes, MAGEA1, MAGEA4, MAGEC1, MAGEC2 and the ubiquitously expressed MAGEE1 in human melanoma samples. We first examined cell lines established from tumors and matching blood samples from 27 melanoma patients. We found that melanoma cell lines from 37% of patients contained at least one mutated MAGE gene. The frequency of mutations in the coding regions of individual MAGE genes varied from 3.7% for MAGEA1 and MAGEA4 to 14.8% for MAGEC2. We also examined 111 fresh melanoma samples collected from 86 patients. In this case, samples from 32% of the patients exhibited mutations in one or more MAGE genes with the frequency of mutations in individual MAGE genes ranging from 6% in MAGEA1 to 16% in MAGEC1. Significance: These results demonstrate for the first time that the MAGE gene family is frequently mutated in melanoma.

Isolated congenital atrioventricular block diagnosed in utero: natural history and outcome.

BACKGROUND: Isolated congenital atrioventricular block (CAVB) diagnosed in utero is associated with a high morbidity and mortality. Prognosis is especially poor when heart rate drops below 55 beats per minute (bpm) and when fetal hydrops develops. We describe the natural history and outcome of 24 infants with isolated CAVB diagnosed in utero, review the literature, and assess the risk factors that could predict outcome. METHODS: This was a retrospective multicenter study of 24 patients with isolated CAVB diagnosed in utero. RESULTS: CAVB was detected at a mean gestational age (GA) of 24.7 +/- 5.1 weeks. Ten fetuses initially presented with complete heart block. Low heart rate or incomplete heart block was the first documentation of bradyarrhythmia in the other 14 fetuses. In 11 of them, CAVB developed during pregnancy after a median time of 3 (range 1-16) weeks. Fetal hydrops developed in 10 of 24 (42%) fetuses at a mean GA of 27.6 +/- 5.1 weeks. Hydropic fetuses showed lower heart rates during pregnancy (47 +/- 10 bpm) than non-hydropic fetuses (57 +/- 10 bpm). There were three intrauterine deaths; all were hydropic and female. Nine viable females and 12 males were born at a mean GA of 37.1 +/- 6.1 weeks with an average birth weight of 3097 +/- 852 g. Fifteen CAVB patients required pacemaker (PM) intervention, 10 of them immediately after birth. Dilated cardiomyopathy (DCM) developed in three infants of whom two died of congestive heart failure, shortly after the diagnosis was made; one is still alive. Mortality before or after birth was 21%, and was associated with heart rates below 50 bpm and development of fetal hydrops. Poor outcome, defined as death, PM implantation, or development of DCM, occurred in 83% of cases and was associated with heart rates below 60 bpm during pregnancy. CONCLUSIONS: Isolated CAVB diagnosed in utero is associated with high morbidity and mortality. Patients who develop fetal hydrops show lower heart rates during pregnancy than patients who do not. A fetal heart rate below 50 bpm and development of fetal hydrops is associated with increased mortality. Rates below 60 bpm are associated with PM requirement and/or DCM.

Prolonged survival with valproic acid use in the EORTC/NCIC temozolomide trial for glioblastoma.

Objective: This analysis was performed to assess whether antiepileptic drugs (AEDs) modulate the effectiveness of temozolomide radiochemotherapy in patients with newly diagnosed glioblastoma.Methods: The European Organization for Research and Treatment of Cancer (EORTC) 26981-22981/National Cancer Institute of Canada (NCIC) CE.3 clinical trial database of radiotherapy (RT) with or without temozolomide (TMZ) for newly diagnosed glioblastoma was examined to assess the impact of the interaction between AED use and chemoradiotherapy on survival. Data were adjusted for known prognostic factors.Results: When treatment began, 175 patients (30.5%) were AED-free, 277 (48.3%) were taking any enzyme-inducing AED (EIAED) and 135 (23.4%) were taking any non-EIAED. Patients receiving valproic acid (VPA) only had more grade 3/4 thrombopenia and leukopenia than patients without an AED or patients taking an EIAED only. The overall survival (OS) of patients who were receiving an AED at baseline vs not receiving any AED was similar. Patients receiving VPA alone (97 [16.9%]) appeared to derive more survival benefit from TMZ/RT (hazard ratio [HR] 0.39, 95% confidence interval [CI] 0.24-0.63) than patients receiving an EIAED only (252 [44%]) (HR 0.69, 95% CI 0.53-0.90) or patients not receiving any AED (HR 0.67, 95% CI 0.49-0.93). Conclusions: VPA may be preferred over an EIAED in patients with glioblastoma who require an AED during TMZ-based chemoradiotherapy. Future studies are needed to determine whether VPA increases TMZ bioavailability or acts as an inhibitor of histone deacetylases and thereby sensitizes for radiochemotherapy in vivo.

Predictors of the risk of fibrosis at 10 years after breast conserving therapy for early breast cancer: a study based on the EORTC Trial 22881-10882 'boost versus no boost'.

The EORTC 22881-10882 trial in 5178 conservatively treated early breast cancer patients showed that a 16 Gy boost dose significantly improved local control, but increased the risk of breast fibrosis. To investigate predictors for the long-term risk of fibrosis, Cox regression models of the time to moderate or severe fibrosis were developed on a random set of 1797 patients with and 1827 patients without a boost, and validated in the remaining set. The median follow-up was 10.7 years. The risk of fibrosis significantly increased (P<0.01) with increasing maximum whole breast irradiation (WBI) dose and with concomitant chemotherapy, but was independent of age. In the boost arm, the risk further increased (P<0.01) if patients had post-operative breast oedema or haematoma, but it decreased (P<0.01) if WBI was given with >6 MV photons. The c-index was around 0.62. Nomograms with these factors are proposed to forecast the long-term risk of moderate or severe fibrosis.

Familial evaluation in catecholaminergic polymorphic ventricular tachycardia: disease penetrance and expression in cardiac ryanodine receptor mutation-carrying relatives.

BACKGROUND: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia syndrome associated with mutations in the cardiac ryanodine receptor gene (Ryr2) in the majority of patients. Previous studies of CPVT patients mainly involved probands, so current insight into disease penetrance, expression, genotype-phenotype correlations, and arrhythmic event rates in relatives carrying the Ryr2 mutation is limited. METHODS AND RESULTS: One-hundred sixteen relatives carrying the Ryr2 mutation from 15 families who were identified by cascade screening of the Ryr2 mutation causing CPVT in the proband were clinically characterized, including 61 relatives from 1 family. Fifty-four of 108 antiarrhythmic drug-free relatives (50%) had a CPVT phenotype at the first cardiological examination, including 27 (25%) with nonsustained ventricular tachycardia. Relatives carrying a Ryr2 mutation in the C-terminal channel-forming domain showed an increased odds of nonsustained ventricular tachycardia (odds ratio, 4.1; 95% CI, 1.5-11.5; P=0.007, compared with N-terminal domain) compared with N-terminal domain. Sinus bradycardia was observed in 19% of relatives, whereas other supraventricular dysrhythmias were present in 16%. Ninety-eight (most actively treated) relatives (84%) were followed up for a median of 4.7 years (range, 0.3-19.0 years). During follow-up, 2 asymptomatic relatives experienced exercise-induced syncope. One relative was not being treated, whereas the other was noncompliant. None of the 116 relatives died of CPVT during a 6.7-year follow-up (range, 1.4-20.9 years). CONCLUSIONS: Relatives carrying an Ryr2 mutation show a marked phenotypic diversity. The vast majority do not have signs of supraventricular disease manifestations. Mutation location may be associated with severity of the phenotype. The arrhythmic event rate during follow-up was low.

Guidelines for the use and interpretation of assays for monitoring autophagy.

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

Low dose endoluminal photodynamic therapy improves murine T cell-mediated colitis.

BACKGROUND AND STUDY AIMS: Low dose photodynamic therapy (LDPDT) may modify the mucosal immune response and may thus provide a therapy for Crohn's disease. We evaluated the efficacy and safety of this technique in a murine T cell-mediated colitis model. METHODS: The safety of LDPDT was first tested in BALB/c mice. Naïve T cells were used to induce colitis in mice with severe combined immunodeficiency, which were followed up endoscopically, and a murine endoscopic index of colitis (MEIC) was developed. The efficacy of LDPDT (10 J/cm (2); delta-aminolevulinic acid, 15 mg/kg bodyweight) was then tested on mice with moderate colitis, while a disease control group received no treatment. The MEIC, weight, length, and histology of the colon, cytokine expression indices, number of mucosal CD4 (+) T cells, percentage of apoptotic CD4 (+) T cells, body weight, and systemic side effects were evaluated. RESULTS: LDPDT improved the MEIC ( P = 0.011) and the histological score ( P = 0.025), diminished the expression indices of the proinflammatory cytokines, interleukin-6 ( P = 0.042), interleukin-17 ( P = 0.029), and interferon-gamma ( P = 0.014), decreased the number of mucosal CD4 (+) T cells, and increased the percentage of apoptotic CD4 (+) T cells compared with the disease control group. No local or systemic side effects occurred. CONCLUSION: LDPDT improves murine T cell-mediated colitis, decreases the proinflammatory cytokines interleukin-6, interleukin-17, and interferon-gamma, and decreases the number of CD4 (+) T cells. No adverse events were observed. Therefore, this technique is now being evaluated in patients with inflammatory bowel disease.

Acvbp Versus Acvbp Plus Rituximab For Young Patients With Localized Low-Risk Diffuse Large B-Cell Lymphoma: A Study By The Groupe D'etude Des Lymphomes De L'adulte

Introduction: In a prior study, we demonstrated that ACVBP + consolidation was superior to 3 cycles of CHOP + radiotherapy in young patients (pts) with localized aggressive lymphoma (Reyes F et al. N Engl J Med 2005;352:1197). This randomized trial compared in these pts ACVBP vs. ACVBP + a short course of rituximab (R-ACVBP).Methods: untreated pts between 18 and 65y with stage I/II DLBCL and no adverse prognostic factors according to the aa-IPI were eligible. ACVBP consisted of 3 induction cycles given every 2 weeks: doxorubicin (75 mg/m2) day 1, cyclophosphamide (1.2g/m2) day 1, vindesine (2 mg/m2) day 1 and 5, bleomycin (10 mg) day 1 and 5, prednisone (60 mg/m2) day 1 to 5 followed by consolidation with metothrexate, ifosfamide, VP-16 and cytarabine. R-ACVBP consisted of the same regimen combined with 4 doses of rituximab (375 mg/m2) on day 1, 15, 29 and 43. Primary objective was EFS.Results: From 01/04 to 03/08, 223 pts were randomized, 113 in ACVBP and 110 in R-ACVBP arm. Characteristics were: median age 49y (18-65), stage I 63%, extranodal involvement 45%, bulky disease 4%. CR was 94% in ACVBP and 97% in ACVBP arm (ns). With a median follow-up of 43 months, the 3-y EFS was 82% (95% CI, 73% to 88%) in ACVBP and 93% (95% CI, 87% to 97%) in R-ACVBP group (P=0.0487). The 3-y PFS was 83% (95% CI, 74% to 89%) and 95% (95% CI, 89% to 98%) respectively (P=0.0205). OS did not significantly differ with a 3-y estimates of 97% (95% CI, 90% to 99%) for ACVBP and 98% (95% CI, 92% to 100%) for R-ACVBP (P=0.686). In multivariate analysis, a longer PFS was associated with R-ACVBP arm (P=0.0302) and lower b2-m level (P=0.0164). The same proportion of pts (27%) experienced at least 1 SAE in both groups. There were 4 deaths in each arm, with 1 treatment-related death in R-ACVBP (pneumocystis jiroveci pneumonia).Conclusion: the addition of only 4 doses of rituximab to ACVBP significantly improves EFS and PFS in younger pts with low-risk localized DLBCL.

Phase II study of imatinib in patients with recurrent gliomas of various histologies: a European Organisation for Research and Treatment of Cancer Brain Tumor Group Study.

PURPOSE: To evaluate the safety and the efficacy of imatinib in recurrent malignant gliomas. PATIENTS: AND METHODS: This was a single-arm, phase II study. Eligible patients had recurrent glioma after prior radiotherapy with an enhancing lesion on magnetic resonance imaging. Three different histologic groups were studied: glioblastomas (GBM), pure/mixed (anaplastic) oligodendrogliomas (OD), and low-grade or anaplastic astrocytomas (A). Imatinib was started at a dose of 600 mg/d with dose escalation to 800 mg in case of no toxicity; during the trial this dose was increased to 800 mg/d with escalation to 1,000 mg/d. Trial design was one-stage Fleming; both an objective response and 6 months of progression-free survival (PFS) were considered a successful outcome to treatment. RESULTS: A total of 112 patients (51 patients with GBM, 25 patients with A, and 36 patients with OD) were enrolled. Imatinib was in general well tolerated. The median number of cycles was 2.0 (range, 1 to 43 cycles). Five patients had an objective partial response, including three patients with GBM; all had 6 months of PFS. The 6-month PFS rate was 16% (95% CI, 8.0% to 34.0%) in GBM, 4.0% (95% CI, 0.3% to 15.0%) in OD, and 9% (95% CI, 2.0% to 25.0%) in A. The exposure to imatinib was significantly lower in patients using enzyme-inducing antiepileptic drugs. The presence of ABCG2 point mutations were not correlated with pharmacokinetic findings. No somatic activating mutations of KIT or platelet-derived growth factor receptor-A or -B were found. CONCLUSION: In the dose range of 600 to 1,000 mg/d, single-agent imatinib is well tolerated but has limited antitumor activity in patients with recurrent gliomas.

Mechanisms of Symptomatic Spinal Cord Ischemia After TEVAR: Insights From the European Registry of Endovascular Aortic Repair Complications (EuREC).

Abstract Purpose: To test the hypothesis that simultaneous closure of at least 2 independent vascular territories supplying the spinal cord and/or prolonged hypotension may be associated with symptomatic spinal cord ischemia (SCI) after thoracic endovascular aortic repair (TEVAR). Methods: A pattern matching algorithm was used to develop a risk model for symptomatic SCI using a prospective 63-patient single-center cohort to test the positive predictive value (PPV) of prolonged intraoperative hypotension and/or simultaneous closure of at least 2 of 4 the vascular territories supplying the spinal cord (left subclavian, intercostal, lumbar, and hypogastric arteries). This risk model was then applied to data extracted from the multicenter European Registry on Endovascular Aortic Repair Complications (EuREC). Between 2002 and 2010, the 19 centers participating in EuREC reported 38 (1.7%) cases of symptomatic spinal cord ischemia among the 2235 patients in the database. Results: In the single-center cohort, direct correlations were seen between the occurrence of symptomatic SCI and both prolonged intraoperative hypotension (PPV 1.00, 95% CI 0.22 to 1.00, p = 0.04) and simultaneous closure of at least 2 independent spinal cord vascular territories (PPV 0.67, 95% CI 0.24 to 0.91, p = 0.005). Previous closure of a single vascular territory was not associated with an increased risk of symptomatic spinal cord ischemia (PPV 0.07, 95% CI 0.01 to 0.16, p = 0.56). The combination of prolonged hypotension and simultaneous closure of at least 2 territories exhibited the strongest association (PPV 0.75, 95% CI 0.38 to 0.75, p<0.0001). Applying the model to the entire EuREC cohort found an almost perfect agreement between the predicted and observed risk factors (kappa 0.77, 95% CI 0.65 to 0.90). Conclusion: Extensive coverage of intercostal arteries alone by a thoracic stent-graft is not associated with symptomatic SCI; however, simultaneous closure of at least 2 vascular territories supplying the spinal cord is highly relevant, especially in combination with prolonged intraoperative hypotension. As such, these results further emphasize the need to preserve the left subclavian artery during TEVAR.

Sagopilone (ZK-EPO, ZK 219477) for recurrent glioblastoma. A phase II multicenter trial by the European Organisation for Research and Treatment of Cancer (EORTC) Brain Tumor Group.

Background: Sagopilone (ZK 219477), a lipophylic and synthetic analog of epothilone B, that crosses the blood-brain barrier has demonstrated preclinical activity in glioma models.Patients and methods: Patients with first recurrence/progression of glioblastoma were eligible for this early phase II and pharmacokinetic study exploring single-agent sagopilone (16 mg/m(2) over 3 h every 21 days). Primary end point was a composite of either tumor response or being alive and progression free at 6 months. Overall survival, toxicity and safety and pharmacokinetics were secondary end points.Results: Thirty-eight (evaluable 37) patients were included. Treatment was well tolerated, and neuropathy occurred in 46% patients [mild (grade 1) : 32%]. No objective responses were seen. The progression-free survival (PFS) rate at 6 months was 6.7% [95% confidence interval (CI) 1.3-18.7], the median PFS was just over 6 weeks, and the median overall survival was 7.6 months (95% CI 5.3-12.3), with a 1-year survival rate of 31.6% (95% CI 17.7-46.4). Maximum plasma concentrations were reached at the end of the 3-h infusion, with rapid declines within 30 min after termination.Conclusions: No evidence of relevant clinical antitumor activity against recurrent glioblastoma could be detected. Sagopilone was well tolerated, and moderate-to-severe peripheral neuropathy was observed in despite prolonged administration.

Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial.

BACKGROUND: In 2004, a randomised phase III trial by the European Organisation for Research and Treatment of Cancer (EORTC) and National Cancer Institute of Canada Clinical Trials Group (NCIC) reported improved median and 2-year survival for patients with glioblastoma treated with concomitant and adjuvant temozolomide and radiotherapy. We report the final results with a median follow-up of more than 5 years. METHODS: Adult patients with newly diagnosed glioblastoma were randomly assigned to receive either standard radiotherapy or identical radiotherapy with concomitant temozolomide followed by up to six cycles of adjuvant temozolomide. The methylation status of the methyl-guanine methyl transferase gene, MGMT, was determined retrospectively from the tumour tissue of 206 patients. The primary endpoint was overall survival. Analyses were by intention to treat. This trial is registered with Clinicaltrials.gov, number NCT00006353. FINDINGS: Between Aug 17, 2000, and March 22, 2002, 573 patients were assigned to treatment. 278 (97%) of 286 patients in the radiotherapy alone group and 254 (89%) of 287 in the combined-treatment group died during 5 years of follow-up. Overall survival was 27.2% (95% CI 22.2-32.5) at 2 years, 16.0% (12.0-20.6) at 3 years, 12.1% (8.5-16.4) at 4 years, and 9.8% (6.4-14.0) at 5 years with temozolomide, versus 10.9% (7.6-14.8), 4.4% (2.4-7.2), 3.0% (1.4-5.7), and 1.9% (0.6-4.4) with radiotherapy alone (hazard ratio 0.6, 95% CI 0.5-0.7; p<0.0001). A benefit of combined therapy was recorded in all clinical prognostic subgroups, including patients aged 60-70 years. Methylation of the MGMT promoter was the strongest predictor for outcome and benefit from temozolomide chemotherapy. INTERPRETATION: Benefits of adjuvant temozolomide with radiotherapy lasted throughout 5 years of follow-up. A few patients in favourable prognostic categories survive longer than 5 years. MGMT methylation status identifies patients most likely to benefit from the addition of temozolomide. FUNDING: EORTC, NCIC, Nélia and Amadeo Barletta Foundation, Schering-Plough.

Radiation therapy and concurrent plus adjuvant temsirolimus (CCI-779) versus chemoirradiation with temozolomide in newly diagnosed glioblastoma without methylation of the MGMT gene promoter.

Background: Preclinical data indicate activity of mammalian target of rapamycin inhibitors and synergistic activity together with radiotherapy in glioblastoma. The aim of this trial is to assess the therapeutic activity of temsirolimus (CCI-779), an intravenous mTOR inhibitor, in patients with newly diagnosed glioblastoma with unmethylated O6 methlyguanine-DNA-methlytransferase (MGMT)promoter. Methods: Patients (n=257) with newly diagnosed glioblastoma after open surgical biopsy or resection fulfilling basic eligibility criteria underwent a central MGMT promoter analysis using quantitative methylation specific PCR. Patients with glioblastoma harboring an unmethylated MGMT promoter (n=111) were randomized 1:1 between radiotherapy (60 Gy; 5 times 2 Gy per week) plus concomitant and six cycles of maintenance temozolomide or radiotherapy plus weekly temsirolimus at 25 mg flat dose to be continued until progression or undue toxicity. Primary endpoint was overall survival at 12 months (OS12). Sample size of the investigational treatment arm required 54 patients to assess adequacy of temsirolimus activity set at 80%. More than 38 patients alive at 12 months in the per protocol population was considered a positive signal. A control arm of 54 patients treated with the standard of care was implemented to evaluate the assumptions on OS12. Results: Between December 2009 and October 2012, 111 pts in 14 centers were randomized and treated. Median age was 55 and 58 years in the temsirolimus and standard arm, respectively. Most patients (95.5%) had a WHO performance status of 0 or 1. Both therapies were properly administered with a median of 13 cycles of maintenance temsirolimus. In the per protocolpopulation, exactly 38 patients treated with temsirolimus (out of 54 eligible) reached OS12. In the intention to treat population OS12 was 72.2% [95% CI (58.2, 82.2)] in the temozolomide arm and 69.6% [95% CI (55.8, 79.9) in the temsirolimus arm [HR=1.16 95% CI (0.77, 1.76), p=0.47]. Conclusions: The therapeutic activity of temsirolimus in patients with newly diagnosed glioblastoma with an unmethylated MGMT promoter is too low.

A Mutation in CALM1 Encoding Calmodulin in Familial Idiopathic Ventricular Fibrillation in Childhood and Adolescence.

OBJECTIVES: This study aimed to identify the genetic defect in a family with idiopathic ventricular fibrillation (IVF) manifesting in childhood and adolescence. BACKGROUND: Although sudden cardiac death in the young is rare, it frequently presents as the first clinical manifestation of an underlying inherited arrhythmia syndrome. Gene discovery for IVF is important as it enables the identification of individuals at risk, because except for arrhythmia, IVF does not manifest with identifiable clinical abnormalities. METHODS: Exome sequencing was carried out on 2 family members who were both successfully resuscitated from a cardiac arrest. RESULTS: We characterized a family presenting with a history of ventricular fibrillation (VF) and sudden death without electrocardiographic or echocardiographic abnormalities at rest. Two siblings died suddenly at the ages of 9 and 10 years, and another 2 were resuscitated from out-of-hospital cardiac arrest with documented VF at ages 10 and 16 years, respectively. Exome sequencing identified a missense mutation affecting a highly conserved residue (p.F90L) in the CALM1 gene encoding calmodulin. This mutation was also carried by 1 of the siblings who died suddenly, from whom DNA was available. The mutation was present in the mother and in another sibling, both asymptomatic but displaying a marginally prolonged QT interval during exercise. CONCLUSIONS: We identified a mutation in CALM1 underlying IVF manifesting in childhood and adolescence. The causality of the mutation is supported by previous studies demonstrating that F90 mediates the direct interaction of CaM with target peptides. Our approach highlights the utility of exome sequencing in uncovering the genetic defect even in families with a small number of affected individuals.

Photodynamic therapy induces selective extravasation of macromolecules: Insights using intravital microscopy.

Photodynamic therapy (PDT) with Visudyne acts by direct cellular phototoxicity and/or by an indirect vascular-mediated effect. Here, we demonstrate that the vessel integrity interruption by PDT can promote the extravasation of a macromolecular agent in normal tissue. To obtain extravasation in normal tissue PDT conditions were one order of magnitude more intensive than the ones in tissue containing neovessels reported in the literature. Fluorescein isothiocyanate dextran (FITC-D, 2000 kDa), a macromolecular agent, was intravenously injected 10 min before (LK0 group, n=14) or 2h (LK2 group, n=16) after Visudyne-mediated PDT in nude mice bearing a dorsal skin fold chamber. Control animals had no PDT (CTRL group, n=8). The extravasation of FITC-D from blood vessels in striated muscle tissue was observed in both groups in real-time for up to 2500 s after injection. We also monitored PDT-induced leukocyte rolling in vivo and assessed, by histology, the corresponding inflammatory reaction score in the dorsal skin fold chambers. In all animals, at the applied PDT conditions, FITC-D extravasation was significantly enhanced in the PDT-treated areas as compared to the surrounding non-treated areas (p<0.0001). There was no FITC-D leakage in the control animals. Animals from the LK0 group had significantly less FITC-D extravasation than those from the LK2 group (p=0.0002). In the LK0 group FITC-D leakage correlated significantly with the inflammation (p<0.001). At the selected conditions, Visudyne-mediated PDT promotes vascular leakage and FITC-D extravasation into the interstitial space of normal tissue. The intensity of vascular leakage depends on the time interval between PDT and FITC-D injection. This concept could be used to locally modulate the delivery of macromolecules in vivo.