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In radiation therapy of pancreatic cancer, tumor alignment prior to each treatment fraction is improved when intratumoral gold fiducial markers (from here onwards: markers), which are visible on computed tomography (CT) and cone beam CT, are used. Visibility of these markers on magnetic resonance imaging (MRI) might improve image registration between CT and magnetic resonance (MR) images for tumor delineation purposes. However, concomitant image artifacts induced by markers are undesirable. The extent of visibility and artifact size depend on MRI-sequence parameters. The authors' goal was to determine for various markers their potential to be visible and to generate artifacts, using measures that are independent of the MRI-sequence parameters.The authors selected ten different markers suitable for endoscopic placement in the pancreas and placed them into a phantom. The markers varied in diameter (0.28-0.6 mm), shape, and iron content (0%-0.5%). For each marker, the authors calculated T2 (∗)-maps and ΔB0-maps using MRI measurements. A decrease in relaxation time T2 (∗) can cause signal voids, associated with visibility, while a change in the magnetic field B0 can cause signal shifts, which are associated with artifacts. These shifts inhibit accurate tumor delineation. As a measure for potential visibility, the authors used the volume of low T2 (∗), i.e., the volume for which T2 (∗) differed from the background by15 ms. As a measure for potential artifacts, the authors used the volume for which |ΔB0|9.4 × 10(-8) T (4 Hz). To test whether there is a correlation between visibility and artifact size, the authors calculated the Spearman's correlation coefficient (Rs) between the volume of low T2 (∗) and the volume of high |ΔB0|. The authors compared the maps with images obtained using a clinical MR-sequence. Finally, for the best visible marker as well as the marker that showed the smallest artifact, the authors compared the phantom data with in vivo MR-images in four pancreatic cancer patients.The authors found a strong correlation (Rs = 1.00, p0.01) between the volume of low T2 (∗) and the volume with high |ΔB0|. Visibility in clinical MR-images increased with lower T2 (∗). Signal shift artifacts became worse for markers with high |ΔB0|. The marker that was best visible in the phantom, a folded marker with 0.5% iron content, was also visible in vivo, but showed artifacts on diffusion weighted images. The marker with the smallest artifact in the phantom, a small, stretched, ironless marker, was indiscernible on in vivo MR-images.Changes in T2 (∗) and ΔB0 are sequence-independent measures for potential visibility and artifact size, respectively. Improved visibility of markers correlates strongly to signal shift artifacts; therefore, marker choice will depend on the clinical purpose. When visibility of the markers is most important, markers that contain iron are optimal, preferably in a folded configuration. For artifact sensitive imaging, small ironless markers are best, preferably in a stretched configuration.

Background— The effect of a mutation in the bone morphogenetic protein receptor 2 ( BMPR2 ) gene on right ventricular (RV) pressure overload in patients with pulmonary arterial hypertension is unknown. Therefore, we investigated RV function in patients who have pulmonary arterial hypertension with and without the BMPR2 mutation by combining in vivo measurements with molecular and histological analysis of human RV and left ventricular tissue. Methods and Results— In total, 95 patients with idiopathic or familial pulmonary arterial hypertension were genetically screened for the presence of a BMPR2 mutation: 28 patients had a BMPR2 mutation, and 67 patients did not have a BMPR2 mutation. In vivo measurements were assessed using right heart catheterization and cardiac MRI. Despite a similar mean pulmonary artery pressure (noncarriers 54±15 versus mutation carriers 55±9 mm Hg) and pulmonary vascular resistance (755 [483–1043] versus 931 [624–1311] dynes·s −1 ·cm −5 ), mutation carriers presented with a more severely compromised RV function (RV ejection fraction: 37.6±12.8% versus 29.0±9%: P −1 ·m −2 ). Differences continued to exist after treatment. To investigate the role of transforming growth factor β and bone morphogenetic protein receptor II signaling, human RV and left ventricular tissue were studied in controls (n=6), mutation carriers (n=5), and noncarriers (n=11). However, transforming growth factor β and bone morphogenetic protein receptor II signaling, and hypertrophy, apoptosis, fibrosis, capillary density, inflammation, and cardiac metabolism, as well, were similar between mutation carriers and noncarriers. Conclusions— Despite a similar afterload, RV function is more severely affected in mutation carriers than in noncarriers. However, these differences cannot be explained by a differential transforming growth factor β, bone morphogenetic protein receptor II signaling, or cardiac adaptation.

The endocrine function of the heart was shown by the identification of a potent inhibitor of renal tubular NaCl reabsorbtion, atrial natriuretic peptide/factor (ANP), in atrial extracts. Since then, Nppa, the gene encoding ANP, has become an important tool for molecular and developmental biologists. Expression of Nppa is an early and specific marker for the differentiating working myocardium of the atria and ventricles of the developing heart. Around the time of birth, ventricular Nppa expression is down-regulated, to be reactivated in response to a variety of cardiovascular disorders. Many aspects of the regulatory pathways that control Nppa gene expression during cardiac development or disease have been revealed. However, several fundamental issues remain to be resolved. Particularly, the regulatory mechanisms underlying the ventricular Nppa expression in the embryonic heart and the reactivation in the failing ventricle remain unclear. Here we review current knowledge on the transcriptional regulation of the Nppa gene.

Bicuspid aortic valve (BAV) is the most common congenital cardiac defect causing serious morbidity including valvular dysfunction and thoracic aortic aneurysms (TAA) in around 30% of BAV patients. Cardiological screening of first-degree relatives is advised in recent guidelines given the observed familial clustering of BAV. However, guidelines regarding screening of family members and DNA testing are not unequivocal. The aim of this review is to provide an overview of the literature on echocardiographic screening in first-degree relatives of BAV patients and to propose a model for family screening. In addition, we provide a flowchart for DNA testing. We performed a PubMed search and included studies providing data on echocardiographic screening in asymptomatic relatives of BAV patients. Nine studies were included. In 5.8-47.4% of the families BAV was shown to be familial. Of the screened first-degree relatives 1.8-11% was found to be affected with BAV. Results regarding a potential risk of TAA in first-degree relatives with a tricuspid aortic valve (TAV) were conflicting. The reported familial clustering of BAV underlines the importance of cardiological screening in relatives. After reviewing the available family history, patient characteristics and the results of cardiological screening in relatives, follow-up in relatives with a TAV and/or DNA testing may be advised in a subset of families. In this study we propose a model for the clinical and genetic work-up in BAV families, based on the most extensive literature review on family screening performed until now.

The development and natural course of lung cysts in patients with Birt-Hogg-Dubé syndrome (BHD) is still unclear, and the relationship between lung cysts and pneumothorax is not fully clarified. Based on the follow-up results of thoracic imaging in six patients with BHD, we hypothesize that decreased potential for stretching of the cysts' wall and extensive contact with the visceral pleura are probably responsible for rupture of the cyst wall resulting in increased risk for pneumothorax.

BACKGROUND Interpretation of genetic screening results in arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) often is difficult. Pathogenicity of variants with uncertain clinical significance may be predicted by software algorithms. However, functional assessment can unambiguously demonstrate the effect of such variants.OBJECTIVE The purpose of this study was to perform functional analysis of potential splice site variants in ARVD/C patients.METHODS Nine variants in desmosomal (PKP2, JUP, DSG2, DSC2) genes with potential RNA splicing effect were analyzed. The variants were found in patients who fulfilled 2010 ARVD/C Task Force Criteria (n = 7) or had suspected ARVD/C (n = 2). Total RNA was isolated from fresh blood samples and subjected to reverse transcriptase polymerase chain reaction.RESULTS An effect on splicing was predicted by software algorithms for all variants. Of the 9 variants, 5 were intronic and 4 exonic. RNA analysis showed a functional effect on mRNA splicing by exon skipping, generation of new splice sites, or activation of cryptic sites in 6 variants. All 5 intronic variants tested severely impaired splicing. Only 1 of 4 exonic potential splice site variants was shown to have a deleterious effect on splicing. The remaining 3 exonic variants had no detectable effect on splicing, and heterozygous presence in mRNA confirmed biallelic expression.CONCLUSION Six variants of uncertain clinical significance in the PKP2, JUP, and DSG2 genes showed a deleterious effect on mRNA splicing, indicating these are ARVD/C related pathogenic splice site mutations. These results highlight the importance of functional assessment of potential splice site variants to improve patient care and facilitate cascade screening.

Mammalian homologues of the Drosophila Iroquois homeobox gene complex, involved in patterning and regionalization of differentiation, have recently been identified (Mech. Dev., 69 (1997) 169; Dev. Biol., 217 (2000) 266; Dev. Dyn., 218 (2000) 160; Mech. Dev., 91 (2000) 317; Dev. Biol., 224 (2000) 263; Genome Res., 10 (2000) 1453; Mech. Dev., 103 (2001) 193). The six members of the murine family were found to be organized in two cognate clusters of three genes each, Irx1, -2, -4 and Irx3, -5, -6, respectively (Peters et al., 2000). As a basis for further study of their regulation and function we performed a comparative analysis of the genomic organization and of the expression patterns of all six Irx genes. The genes are expressed in highly specific and regionalized patterns of ectoderm, mesoderm and endoderm derived tissues. In most tissues the pattern of expression of the clustered genes, especially of Irx1 and -2 and of Irx3 and -5, respectively, closely resembled each other while those of Irx4 and -6 were very divergent. Interestingly, the expression of cognate genes was found to be mutually exclusive in adjacent and interacting tissues of limb, heart and the laryncho-pharyncheal region. The results indicate that the Irx genes are coordinately regulated at the level of the cluster.

Purpose: The first clinical MR-linac will soon become operational, therefore patient plan QA procedures and equipment have to become MRcompatible. Reference dosimetry is affected by the magnetic field, however, relative dosimetry using patient QA systems haven’t been investigated extensively. The purpose of this study was to examine the performance characteristics of the MR-compatible ArcCHECK and Delta4 systems in a transverse 1.5 T magnetic field. Methods: Recently MR-compatible versions of both ArcCheck (Sun Nuclear) and Delta4 (Scandidos) have been developed. To examine the performance characteristics within the magnetic field, the reproducibility, dose linearity, dose rate dependence, field size and angular dependence were evaluated on the MR-linac (8 MV FFF beam, SAD of 142 cm) and a conventional linac (Elekta, 6MV). To allow comparison of the measurements with and without magnetic field, the measurement setup for the conventional linac is adapted to mimic the setup at the MR-linac if possible (e.g. SAD, dose rate). The results from the MR-linac were benchmarked to the results from the conventional linac as being the clinical reference. Results: At the moment of writing, measurements for the Delta4 are still running. Therefore, only the results of the ArcCheck are presented in this abstract. No significant difference was observed in the reproducibility of the ArcCheck (

We present a case of a fatal Epstein-Barr infection in a 17-year-old male patient suspected to be caused by X-linked lymphoproliferative disease. At the time of hospitalization, DNA diagnostics was not available. The suspected diagnosis was confirmed several years later when a SH2D1A missense mutation was identified in stored patient DNA. Extended pedigree analysis showed that this mutation occurred de novo in his mother. In addition, we provide a summary of the currently listed SH2D1A mutations. Conclusion: This case report underlines the importance of DNA storage, pedigree analysis, and multidisciplinary care in patients with rare diseases and their families.

AIMS: We sought to determine the influence of genotype on clinical course and arrhythmic outcome among arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C)-associated mutation carriers.METHODS AND RESULTS: Pathogenic mutations in desmosomal and non-desmosomal genes were identified in 577 patients (241 families) from USA and Dutch ARVD/C cohorts. Patients with sudden cardiac death (SCD)/ventricular fibrillation (VF) at presentation (n = 36) were younger (median 23 vs. 36 years; P < 0.001) than those presenting with sustained monomorphic ventricular tachycardia (VT). Among 541 subjects presenting alive, over a mean follow-up of 6 ± 7 years, 12 (2%) patients died, 162 (30%) had sustained VT/VF, 78 (14%) manifested left ventricular dysfunction (EF < 55%), 28 (5%) experienced heart failure (HF), and 10 (2%) required cardiac transplantation. Patients (n = 22; 4%) with >1 mutation had significantly earlier occurrence of sustained VT/VF (mean age 28 ± 12 years), lower VT-/VF-free survival (P = 0.037), more frequent left ventricular dysfunction (29%), HF (19%) and cardiac transplantation (9%) when compared with those with only one mutation. Desmoplakin mutation carriers experienced more than four-fold occurrence of left ventricular dysfunction (40%) and HF (13%) than PKP2 carriers. Missense mutation carriers had similar death-/transplant-free survival and VT/VF penetrance (P = 0.137) when compared with those with truncating or splice site mutations. Men are more likely to be probands (P < 0.001), symptomatic (P < 0.001) and have earlier and more severe arrhythmic expression.CONCLUSIONS: Presentation with SCD/VF occurs at a significantly younger age when compared with sustained monomorphic VT. The genotype of ARVD/C mutation carriers impacts clinical course and disease expression. Male sex negatively modifies phenotypic expression.

Conclusions: During this feasibility study of a TRUS/CT treatment planning workflow for cervix cancer BT a full treatment could be simulated for one patient, resulting in a clinically acceptable dose distribution. A need for improved applicator localisation in TRUS volumes has been identified. Image registration accuracy could be improved by automatic applicator tracking during US acquisition, thus minimizing reconstruction uncertainties due to insufficient depiction of the whole applicator on US images.

Purpose We investigated radiation-induced changes in the salivary glands, 6weeks after RT, using MRI. Materials and methods Eighteen oropharyngeal cancer patients were treated with salivary gland sparing IMRT. All patients received a 3T MRI exam before and 6weeks after the end of RT, including a T 1 -weighted (T 1 w), a T 2 -weighted (T 2 w), and a dynamic contrast-enhanced (DCE) MRI. For both time points separately, the parotid and submandibular glands were delineated on the MR images. Differences in median signal intensity and signal variation within the glands were tested for significance. Correlations were studied between the MR changes and the planned RT dose. Results The volume of the glands reduced significantly by 25%. The T 1 w signal decreased by 10% and the T 2 w signal increased by 23%. The k ep value decreased, while the v e increased. A correlation of the changes in T 2 w signal with the mean dose was found in both glands. Conclusions Overall radiation-induced changes and volume loss were observed in the parotid and submandibular gland using MR. The observed differences indicated an increased water content such as found in oedema. The overall changes could be related to the mean dose, with a slightly greater impact in the high dose area.

Background— Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is an autosomal dominant inherited disease with incomplete penetrance and variable expression. Causative mutations in genes encoding 5 desmosomal proteins are found in ≈50% of ARVD/C index patients. Previous genotype-phenotype relation studies involved mainly overt ARVD/C index patients, so follow-up data on relatives are scarce. Methods and Results— One hundred forty-nine ARVD/C index patients (111 male patients; age, 49±13 years) according to 2010 Task Force criteria and 302 relatives from 93 families (282 asymptomatic; 135 male patients; age, 44±13 years) were clinically and genetically characterized. DNA analysis comprised sequencing of plakophilin-2 ( PKP2 ), desmocollin-2, desmoglein-2, desmoplakin, and plakoglobin and multiplex ligation-dependent probe amplification to identify large deletions in PKP2. Pathogenic mutations were found in 87 index patients (58%), mainly truncating PKP2 mutations, including 3 cases with multiple mutations. Multiplex ligation-dependent probe amplification revealed 3 PKP2 exon deletions. ARVD/C was diagnosed in 31% of initially asymptomatic mutation-carrying relatives and 5% of initially asymptomatic relatives of index patients without mutation. Prolonged terminal activation duration was observed more than negative T waves in V 1 to V 3 , especially in mutation-carrying relatives Conclusions— Pathogenic desmosomal gene mutations, mainly truncating PKP2 mutations, underlie ARVD/C in the majority (58%) of Dutch index patients and even 90% of familial cases. Additional multiplex ligation-dependent probe amplification analysis contributed to discovering pathogenic mutations underlying ARVD/C. Discovering pathogenic mutations in index patients enables those relatives who have a 6-fold increased risk of ARVD/C diagnosis to be identified. Prolonged terminal activation duration seems to be a first sign of ARVD/C in young asymptomatic relatives.

Item does not contain fulltext AIMS: We investigated the presence of a clinical diagnosis of hypertrophic cardiomyopathy (HCM), risk factors for sudden cardiac death (SCD), and cardiac events during follow-up in predictively tested-not known to have a clinical diagnosis of HCM before the DNA test-carriers of a sarcomeric gene mutation and associations with age and gender to determine the best cardiological screening strategy. METHODS AND RESULTS: One hundred and thirty-six (30%) of 446 mutation carriers were diagnosed with HCM at one or more cardiological evaluation(s). Male gender and higher age were associated with manifest disease. Incidence of newly diagnosed manifest HCM was 10% in older carriers, although numbers were small in carriers /=2 risk factors and manifest HCM) was present in 17 carriers during follow-up (2.4% per person-year). Age but not gender was associated with a high-risk status for SCD. CONCLUSION: Thirty percent of carriers had or developed manifest HCM after predictive DNA testing and risk factors for SCD were frequently present. Our data suggest that the SCD risk is low and risk stratification for SCD can be omitted in carriers without manifest disease and that frequency of cardiological evaluations can possibly be decreased in carriers between 15 and 40 years as long as hypertrophy is absent.

Iroquois (Irx) proteins comprise a family of homeodomain-containing transcription factors involved in patterning and regionalization of embryonic tissues in both vertebrates and invertebrates. The six murine Irx genes are organized in two clusters, each consisting of three genes. Irx1, Irx2 and Irx4 belong to the IrxA cluster on chromosome 13, whereas Irx3, Irx5 and Irx6, comprising the IrxB cluster, are located on chromosome 8 (Peters et al., Genome Res. 10 (2000) 1453). Developmental expression patterns have so far only been reported for five Irx genes. Here, we investigated the expression pattern of Irx6 during mouse morphogenesis and early organogenesis. The pattern was found to be much more restricted compared to the other five Irx genes, and its level of expression was much lower.

Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy (ARVD/C) is a disease characterized by progressive fibrofatty replacement of primarily the right ventricle (RV). 1–3

Purpose Besides sparing the parotid gland, sparing the submandibular gland is considered to be important in preventing xerostomia in head-and-neck cancer patients. Delineation of the submandibular gland at CT, and even on T 1 - and T 2 -weighted MR images, is difficult, due to low contrast with the surrounding tissues. MR sialography might be used for delineation. Methods and materials Sixteen oropharyngeal cancer patients received a CT and MRI exam as part of the standard treatment imaging protocol. Patients were scanned in their five-point RT immobilization mask. The MRI exam included T 1 - and T 2 -weighted MRI scans and an MR sialography scan. Thirty submandibular glands were delineated on only CT, on the combined CT and T 1 - and T 2 -weighted MRI scans and on all MR images. A Wilcoxon signed-rank test was performed to test if the delineated volumes were significantly different. Results The delineated volume of the submandibular gland was 7.3mL in the CT-delineation, 7.1mL in the CT/MRI-delineation and 8.1mL in the MRI-delineation. The MRI-delineation was significantly larger than the other delineations ( p Conclusion The delineation of the submandibular gland was improved in the cranial direction by using T 1 - and T 2 -weighted MRI and MR sialography, compared to the other delineations.

To analyze the combined and updated results from the University of Michigan and University Medical Center Utrecht on normal tissue complication probability (NTCP) of the parotid gland 1 year after radiotherapy (RT) for head-and-neck (HN) cancer.A total of 222 prospectively analyzed patients with various HN malignancies were treated with conventional and intensity-modulated RT. Stimulated individual parotid gland flow rates were measured before RT and 1 year after RT using Lashley cups at both centers. A flow ratio25% of pretreatment was defined as a complication. The data were fitted to the Lyman-Kutcher-Burman model.A total of 384 parotid glands (Michigan: 157; Utrecht: 227 glands) was available for analysis 1 year after RT. Combined NTCP analysis based on mean dose resulted in a TD(50) (uniform dose leading to 50% complication probability) of 39.9 Gy and m (steepness of the curve) of 0.40. The resulting NTCP curve had good qualitative agreement with the combined clinical data. Mean doses of 25-30 Gy were associated with 17-26% NTCP.A definite NTCP curve for parotid gland function 1 year after RT is presented, based on mean dose. No threshold dose was observed, and TD(50) was equal to 40 Gy.

Nppa , encoding atrial natriuretic factor, is expressed in fetal atrial and ventricular myocardium and is downregulated in the ventricles after birth. During hypertrophy and heart failure, Nppa expression is reactivated in the ventricles and serves as a highly conserved marker of heart disease. The Nppa promoter has become a frequently used model to study mechanisms of cardiac gene regulation. Nevertheless, the regulatory sequences that provide the correct developmental pattern and ventricular reactivation during cardiac disease remain to be defined. We found that proximal Nppa fragments ranging from 250 bp to 16 kbp provide robust reporter gene activity in the atria and correct repression in the atrioventricular canal and the nodes of the conduction system in vivo. However, depending on fragment size and site of integration into the genome of mice, the fetal ventricular activity was either absent or present in an incorrect pattern. Furthermore, these fragments did not provide ventricular reactivation in heart disease models. These results indicate that the proximal promoter does not provide a physiologically relevant model for ventricular gene activity. In contrast, 2 modified bacterial artificial chromosome clones with partially overlapping genomic Nppa sequences provided appropriate reactivation of the green fluorescent protein reporter during pressure overload–induced hypertrophy and heart failure in vivo. However, only 1 of these bacterial artificial chromosomes provided correct fetal ventricular green fluorescent protein activity. These results show that distinct distal regulatory sequences and divergent regulatory pathways control fetal ventricular activity and reactivation of Nppa during cardiac disease, respectively.

Background and purpose The submandibular glands are proposed to be important in preventing xerostomia in head-and-neck cancer patients. We investigated the feasibility of sparing the contralateral submandibular gland (cSMG) by reducing the dose to the contralateral planning target volume (PTV) and by reducing the clinical target volume (CTV)-to-PTV margin. Materials and methods Ten oropharyngeal cancer patients with a contralateral elective PTV were included in this planning study, using intensity modulated radiotherapy (IMRT). The effect on the mean dose to the cSMG of reducing the dose coverage to the contralateral elective PTV from 95 to 90% of the prescribed dose (54Gy in 1.8Gy daily fractions) was determined. The influence of reducing the margin for position uncertainty from 5 to 2mm was investigated. Results The mean dose to the cSMG was reduced from 54Gy to approximately 40Gy if the dose coverage to the contralateral PTV was reduced to 90% of the prescribed dose. The estimated normal tissue complication probability (NTCP) was reduced below 50%. Reducing the margin from 5 to 2mm resulted in a decrease in the mean dose to the cSMG of approximately 6Gy. Conclusions Reducing the mean dose to the cSMG below 40Gy is possible with a reasonable dose coverage of the contralateral elective PTV.

Purpose: MRI is increasingly used as a single imaging modality for brachytherapy treatment planning. The presence of a brachytherapy applicator may cause distortions in the images, especially at higher field strengths. Our aim is to develop a procedure to quantify these distortions theoretically for any MR-sequence and to verify the estimated deformations for clinical sequences. Methods: Image distortions due to perturbation of the B0-field are proportional to the ratio of the induced frequency shift and the read-out bandwidth of the applied sequence. By reconstructing a frequency-shift map from the phase data from a multi-echo sequence, distortions can be calculated for any MR-sequence. Verification of this method for estimating distortions was performed by acquiring images with opposing read-out directions and consequently opposing distortions. The applicator shift can be determined by rigidly matching these images. Clinically, T2W-TSE-images are used for this purpose. For pre-clinical tests, EPI-sequences with narrow read-out bandwidth (19.5–47.5Hz), consequently large distortions, were added to the set of clinical MRsequences. To quantify deformations of the Utrecht Interstitial CT/MR applicator (Elekta Brachytherapy) on a Philips Ingenia 3T MRI, pre-clinical tests were performed in a phantom with the applicator in water, followed by clinical validation. Results: Deformations observed in the narrow bandwidth EPI-images were well predicted using the frequency-shift, the latter giving an overestimation up to 30%/up to 1 voxel. For clinically applied MR-sequences distortions were well below the voxel size. In patient setup distortions determined from the frequency-shift map were at sub-voxel level (

Purpose: Pancreatic tumors show large interfractional position variations. In addition, changes in gastrointestinal air volume and body contour take place during treatment. We aim to investigate the robustness of the clinical treatment plans by quantifying the dosimetric effects of these anatomical changes. Methods: Calculations were performed for up to now 3 pancreatic cancer patients who had intratumoral fiducials for daily CBCT-based positioning during their 3-week treatment. For each patient, deformable image registration of the planning CT was used to assign Hounsfield Units to each of the 13—15 CBCTs; air volumes and body contour were copied from CBCT. The clinical treatment plan was used (CTV-PTV margin = 10 mm; 36Gy; 10MV; 1 arc VMAT). Fraction dose distributions were calculated and accumulated. The V95% of the clinical target volume (CTV) and planning target volume (PTV) were analyzed, as well as the dose to stomach, duodenum and liver. Dose accumulation was done for patient positioning based on the fiducials (as clinically used) as well as for positioning based on bony anatomy. Results: For all three patients, the V95% of the CTV remained 100%, for both fiducial- and bony anatomy-based positioning. For fiducial-based positioning, dose to duodenum en stomach showed no discernable differences withmore » planned dose. For bony anatomy-based positioning, the PTV V95% of the patient with the largest systematic difference in tumor position (patient 1) decreased to 85%; the liver Dmax increased from 33.5Gy (planned) to 35.5Gy. Conclusion: When using intratumoral fiducials, CTV dose coverage was only mildly affected by the daily anatomical changes. When using bony anatomy for patient positioning, we found a decline in PTV dose coverage due to the interfractional tumor position variations. Photon irradiation treatment plans for pancreatic tumors are robust to variations in body contour and gastrointestinal gas, but the use of fiducial-based daily position verification is imperative. This work was supported by the foundation Bergh in het Zadel through the Dutch Cancer Society (KWF Kankerbestrijding) project No. UVA 2011-5271.« less

e15579 Background: Birt-Hogg-Dube syndrome (BHD) is an autosomal dominant inherited disorder caused by mutations in the FLCN gene predisposing for renal cell cancer. Generally, surveillance of the ...

Myosin heavy-chain (MHC) isoforms are major structural components of the contractile apparatus of the heart muscle. Their spatio-temporal patterns of expression have been used as a tool to dissect cardiac development and differentiation. Although extensively investigated, controversy still exists concerning the expression patterns of atrial (AMHC), ventricular (VMHC), and cardiac myosin heavy-chain (CMHC) during development in the heart. In this study, we describe that probe length, probe concentration, and staining time in the non-radioactive in situ hybridization procedure seriously influence the observed pattern of MHC expression and the subsequent interpretation, explaining the divergent opinions in the field. Using a variety of external and internal controls for the in situ hybridization procedure, we demonstrate that both AMHC and VMHC are expressed throughout the entire heart tube during early development. During subsequent development, VMHC becomes restricted to the ventricles, whereas AMHC remains expressed in the atria, and, at substantially lower levels, is detected in the ventricles. These results are discussed in the context of methodological constraints of demonstrating patterns of gene expression. This manuscript contains online supplemental material at http://www.jhc.org . Please visit this article online to view these materials.

We identified and characterized the chicken natriuretic peptide precursor gene cluster and found its organization to be highly conserved compared with the mammalian Nppb-Nppa cluster. However, phylogenetic analysis indicated that the putative chicken natriuretic peptide precursor genes are the homologues of CNP-3 and Nppb, respectively. Comparative expression analysis revealed that, in human, mouse, and rat hearts, Nppb is a novel marker for the differentiating working myocardium. Its expression pattern is strikingly similar to that of Nppa before birth, and diverges only after birth. In contrast, whereas the chicken Nppb gene expression profile resembled that of mammalian Nppb, the CNP-3 gene showed very limited expression in the heart, not resembling the pattern of either Nppa or Nppb. These results show that, in chicken, the Nppa gene has been lost from the natriuretic peptide precursor gene cluster, whereas the CNP-3 gene has been retained.

Using degenerated PCR-primers to identify known and novel BMPs that are expressed in the developing chicken heart, we identified not only BMP2, -4, and -7 mRNA, but also the TGFβ superfamily member cvg1. The expression pattern of cVg1 mRNA was determined during chicken development from HH4 to HH44. In early developmental stages, cVg1 mRNA is expressed in the primitive streak, paraxial mesoderm, developing somites, and developing neural tube. Subsequently, cVg1 mRNA is expressed in the developing central and peripheral nervous system, retina, auditory vesicle, notochord, lung alveoli, and olfactory mucosa. In the heart, cVg1 is initially expressed through the linear heart tube, but becomes restricted to the forming chamber myocardium, in an expression domain similar to that of atrial natriuretic factor (ANF) mRNA.

In mouse, atrial natriuretic factor (ANF) gene expression was shown to be a marker for chamber formation within the embryonic heart. To gain insight into the process of chamber formation in the chicken embryonic heart, we analyzed the expression pattern of cANF during development. We found cANF to be specifically expressed in the myocardium of the morphologically distinguishable atrial and ventricular chambers, similar to ANF in mouse, cANF expression was never detected in the myocardium of the atrioventricular canal (AVC), inner curvature, and outflow tract (OFT), which is lined by endocardial cushions. Expression was strictly excluded from the interventricular myocardium and most proximal part of the bundle branches, as identified by the expression of Msx-2, whereas the rest of the bundle branches, trabeculae, and surrounding working myocardium did express cANF. The myocardium that forms de novo within the cushions after looping did not express cANF. At HH9 cANF expression was first observed in a subset of cardiomyocytes, which was localized ventrally in the fused heart tube and laterally in the unfused cardiac sheets. Together, these results show that cANF expression can be used to distinguish differentiated chamber (working) myocardium, including the peripheral ventricular conduction system, from embryonic myocardium. We conclude that differentiation of chamber myocardium takes place already at HH9 at the ventral side of the linear heart tube, possibly preceded by latero-medial signals in the unfused cardiac sheets.

Aim: Birt-Hogg-Dube syndrome (BHD) is an autosomal dominant condition due to germline mutations in the folliculin (FLCN) gene, clinically characterized by skin fibrofolliculomas, lung cysts, (recurrent) spontaneous pneumothorax (SP) and an increased risk of renal cell cancer (RCC). Families with BHD are most often identified through recognition of fibrofolliculomas A less common way to identify a BHD family is through (recurrent) SP. The characteristic pulmonary cystsare generally not visible on standard chest X-ray, and therefore will be missed in the standard – according to British Thoracic Society guidelines - diagnostic work-up for SP cases. We hypothesize that supplementing the diagnostic work-up of SP patients with CT imaging will result in the identification of BHD patients presenting with SP. This provides the opportunity of the identification of (asymptomatic) RCC at an early stage by screening of identified FLCN mutation carriers. Methods: We retrospectively collected clinical and radiological data of 55 families, including 200 BHD patients with a proven pathogenic FLCN mutation. Our database started in 2004; the mean follow-up time of mutation carriers is 5 years (1-10 years). Results: Sixty patients in 33 families had (recurrent) SP. In 14 out of 30 families we detected one or more patients with RCC. In total 19 patients had a history of RCC, 7 of them had a history of (recurrent) SP and 15 patients had a positive familial history for SP. We found in 14 cases RCC at an early stage, 5 patients died due metastases. Histological diagnosis were mainly (a combination of) clear cell carcinoma and chromophobe carcinoma. Conclusions: Based on these results, we suggest that including (low dose) thoracic CT in the standard SP work up should result in the identification of BHD families. In these families annual screening for RCC should be offered to affected relatives, which was shown to result in early detection of BHD associated RCC. We show that a limited period of observation of these families has resulted in detection of 14 patients with RCC at an early stage. Disclosure: All authors have declared no conflicts of interest.

The relative insensitivity of nonradioactive mRNA detection in tissue sections compared to the sensitive nonradioactive detection of single-copy DNA sequences in chromosome spreads, or of mRNA sequences in whole-mount samples, has remained a puzzling issue. Because of the biological significance of sensitive in situ mRNA detection in conjunction with high spatial resolution, we developed a nonradioactive in situ hybridization (ISH) protocol for detection of mRNA sequences in sections. The procedure is essentially based on the whole-mount ISH procedure and is at least equally sensitive. Increase of the hybridization temperature to 70C while maintaining stringency of hybridization by adaptation of the salt concentration significantly improved the sensitivity and made the procedure more sensitive than the conventional radioactive procedure. Thicker sections, which were no improvement using conventional radioactive ISH protocols, further enhanced signal. Higher hybridization temperatures apparently permit better tissue penetration of the probe. Application of this highly reliable protocol permitted the identification and localization of the cells in the developing heart that express low-abundance mRNAs of different members of the Iroquois homeobox gene family that are supposedly involved in cardiac patterning. The radioactive ISH procedure scarcely permitted detection of these sequences, underscoring the value of this novel method.

We isolated cDNAs of mouse Iroquois-related homeobox genes Irx1, -2, -3, -4, and -5 and characterized their patterns of expression in the developing heart. Irx1 and Irx2 were found to be expressed specifically in the ventricular septum from the onset of its formation onward. In fetal stages, the expression of both genes appeared to gradually become confined to the myocardium of the atrioventricular bundle and bundle branches of the forming ventricular conduction system. Irx3 was found to be expressed specifically in the trabeculated myocardium of the ventricles. Irx4 expression was observed in a segment of the linear heart tube and the atrioventricular canal and ventricular myocardium including the inner curvature after looping, resembling the pattern of MLC2V. Transcripts for Irx5 were detected specifically in the endocardium lining the ventricular and atrial working myocardium that also expressed von Willebrand factor, but were absent from the endocardium of the endocardial cushions, i.e., the atrioventricular canal, inner curvature, and outflow tract. The spatiodevelopmental pattern of Irx5 matched that of ANF, a marker for the forming working myocardium of the chambers. Taken together, all members of the Irx gene family were found to be expressed in highly specific patterns in the developing mouse heart, suggesting a critical role in the specification of the distinct components of the four-chambered heart. (C) 2000 Academic Press.

Background Previously, we proposed that familial multiple trichodiscomas (OMIM 190340) is distinct from Birt-Hogg-Dube syndrome (BHD) (OMIM #135150). BHD is characterized by multiple fibrofolliculomas/trichodiscomas, lung cysts, pneumothorax, and renal cell cancer. Germline FLCN mutations can be detected in most but not all BHD families. Objective We sought to evaluate familial multiple trichodiscomas at a clinical and genetic level. We now renamed this condition "familial multiple discoid fibromas" (FMDF) to emphasize the distinction from BHD. Methods In 8 additional families with an autosomal dominant pattern of multiple discoid fibromas we assessed the clinical findings and the histopathological features of skin lesions. FLCN germline mutation analysis was completed in 7 families. In two of these families segregation analysis was performed using polymorphic DNA markers in and around the FLCN locus. Results The clinical findings in FMDF are different from those in BHD with early onset of skin lesions, prominent involvement of the pinnae, and discoid fibromas without the follicular epithelial component characteristic of the fibrofolliculoma/trichodiscoma spectrum of BHD. In addition, there were no evident pulmonary or renal complications. In none of the families were pathogenic FLCN germline mutations identified. Using segregation analysis we could exclude involvement of the FLCN locus in the two kindreds tested. Limitations The prevalence of FMDF is presently unknown. The underlying gene defect has not yet been identified. Conclusions FMDF is clinically distinct from BHD and is not linked to the FLCN locus.

Conclusions: Setup corrections for baseline shifts (and residual errors therein) where shown to result in significantly lower PTV coverage, showing that dose invariance does not hold in this situation. However, the lowest D99 of the GTV observed among all calculated correction scenarios was >53 Gy. Our findings confirm the "dose tracks density" paradigm, which rescues the PTV concept with SBRT of lung cancer.

Radiotherapy is a common treatment for head and neck cancer patients. Unfortunately, it produces serious acute and long-term side effects to the oral cavity. One severe complication is the loss of salivary gland function, which can persists for many years. Saliva has multiple functions relating to speech, taste perception, mastication, and swallowing and bolus formation. Cleansing and dental and mucosal protection are also important functions. In this thesis, a detailed report is given on the effects of radiotherapy on changes in the parotid gland function. Different methods of measuring gland function are described. Moreover, a preventive measure to limit the gland toxicity was investigated in rodents. The radiation field and, in particular, the volume of the parotid gland tissue exposed to radiation is of notable importance with regard to the development of gland damage and hypofunction. Therefore, it is important to achieve detailed information about the size and position of the parotid gland. Great differences in size and position of the parotid glands between the patients were seen. In order to exactly define the localisation of the glands, it is essential to use CT-based simulation of radiation fields. The radiation tolerance of the parotid glands as a function of dose and volume irradiated was accurately described. The best parameter for evaluation of the parotid gland function appeared to be the flow measurement using the Lashley cups. However, if direct flow measurements are not feasible, 99mTc-pertechnetate scintigraphy might be a good alternative. Flow reduction depended on the mean parotid gland dose. For a post treatment parotid flow ratio a 25%, the TD50 (the dose to the whole organ leading to a complication probability of 50%) was found to be 31, 35 and 39 Gy at 6 weeks, 6 months and 1 year after radiotherapy respectively. The consequences of parotid gland injury are still difficult to manage. Prophylactic treatment with sialogogues like the muscarinic receptor agonist pilocarpine has been shown to have radioprotective potential. The effects of preirradiation treatment of pilocarpine on rat parotid gland function were investigated in relation to radiation dose. Pilocarpine induced compensation, which at least underlies the radioprotective effect of the drug. This effect seemed to be dependent on the amount of damage induced. Therefore, the type of fractionation scheme and the volume of the gland that lies within the radiation portal will be crucial for the effectiveness of a prophylactic pilocarpine treatment. With the more detailed knowledge on the dose/volume effects of radiation on parotid function, we can try to focus on sparing the parotid gland function. The prevention of radiation-induced loss of parotid gland function will depend on both optimal sparing radiation therapy techniques and on pharmacological agents, which can selectively interfere with the radiation-induced effects. These preventing strategies will need a joint effort of radiation-oncologists, clinical physicists and radiobiologists.