Engineering thermostability in archaebacterial glyceraldehyde-3-phosphate dehydrogenase Hints for the important role of interdomain contacts in stabilizing protein conformation

AbstractConstruction of hybrid enzymes between the glyceraldehyde-3-phosphate dehydrogenases from the mesophilic Methanobacterium bryantil and the thermophilic Methanothermus fervidus by recombinant DNA techniques revealed that a short C-terminal fragment of the Mt.fervidus enzyme contributes largely to its thermostability. This C-terminal region appears to be homologous to the α6-helix of cubacterial and eukaryotie glyceraldehyde-3-phosphate dehydrogenases which is involved in the contacts between the two domains of the enzyme subunit. Site-directed mutagenesis experiments indicate that hydrophobic interaction play an important role in these contacts

Immunophenotyping and oncogene amplifications in tumors of the papilla of Vater

Carcinomas of the ampulla of Vater are rare and assumed to generally arise from preexisting adenomas (adenoma-carcinoma sequence). Histologically, distinct subtypes can be distinguished that were shown to differ significantly in terms of clinical outcome. Since pathologists usually receive bioptic tissue samples of ampullary tumors obtained during endoscopy, accurate classification of carcinoma subtypes can sometimes be difficult on morphological criteria alone. We therefore performed immunohistochemistry using a panel of established marker proteins (CK7, CK20, p21, p27, ESA, bax, and ephrin-B2) on 175 carcinoma, 111 adenoma, and 152 normal mucosa specimens of the ampulla of Vater and identified distinct immunoprofiles for every carcinoma subtype. Fluorescence in situ hybridization analyses of therapeutic target genes (c-myc, EGFR1, CCND1, HER2) found CCND1 to represent the most frequently amplified gene in our series (7.5%

Effect of EpCAM, CD44, CD133 and CD166 expression on patient survival in tumours of the ampulla of Vater

Background: Carcinomas of the Vaterian system are rare and presumably arise from pre-existing adenomas. According to the cancer stem cell (CSC) hypothesis, only a small subset of tumor cells has the ability to initiate and develop tumor growth. In colorectal cancer, CD44, CD133, CD166 and EpCAM have been proposed to represent CSC marker proteins and their expression has been shown to correlate with patient survival. Aims: To evaluate a potential role of these CSC proteins in tumors of the ampulla of Vater, we investigated their expression in 175 carcinoma, 111 adenoma and 152 normal mucosa specimens arranged in a Tissue Microarray format. Materials and methods: Membranous immunoreactivity for each protein marker was scored semi-quantitatively by evaluating the number of positive tumor cells over the total number of tumor cells. Median protein expression levels were used as cut-off scores to define protein marker positivity. Clinical data including survival time were obtained by retrospective analysis of medical records, tumor registries or direct contact. Results: The expression of all evaluated marker proteins differed significantly between normal mucosa, adenoma and carcinoma samples. In all markers, we found a tendency towards more constant expression from normal to neoplastic tissue. EpCAM expression was significantly correlated with better patient survival. The increased expression of CD44s, CD166 and CD133 from normal mucosa samples to adenoma and carcinoma was linked to tumor progression. However, there was no statistically significant correlation with survival. Conclusion: Our findings indicate, that in ampullary carcinomas, loss of expression of EpCAM may be linked to a more aggressive tumor phenotyp

Expression of growth factor receptors and targeting of EGFR in cholangiocarcinoma cell lines

BACKGROUND: Cholangiocarcinoma (CC) is a malignant neoplasm of the bile ducts or the gallbladder. Targeting of growth factor receptors showed therapeutic potential in palliative settings for many solid tumors. The aim of this study was to determine the expression of seven growth factor receptors in CC cell lines and to assess the effect of blocking the EGFR receptor in vitro. METHODS: Expression of EGFR (epithelial growth factor receptor), HGFR (hepatocyte growth factor receptor) IGF1R (insulin-like growth factor 1 receptor), IGF2R (insulin-like growth factor 2 receptor) and VEGFR1-3 (vascular endothelial growth factor receptor 1-3) were examined in four human CC cell lines (EGI-1, HuH28, OZ and TFK-1). The effect of the anti-EGFR-antibody cetuximab on cell growth and apoptosis was studied and cell lines were examined for KRAS mutations. RESULTS: EGFR, HGFR and IGFR1 were present in all four cell lines tested. IGFR2 expression was confirmed in EGI-1 and TFK-1. No growth-inhibitory effect was found in EGI-1 cells after incubation with cetuximab. Cetuximab dose-dependently inhibited growth in TFK-1. Increased apoptosis was only seen in TFK-1 cells at the highest cetuximab dose tested (1 mg/ml), with no dose-response-relationship at lower concentrations. In EGI-1 a heterozygous KRAS mutation was found in codon 12 (c.35G>A; p.G12D). HuH28, OZ and TFK-1 lacked KRAS mutation. CONCLUSION: CC cell lines express a pattern of different growth receptors in vitro. Growth factor inhibitor treatment could be affected from the KRAS genotype in CC. The expression of EGFR itself does not allow prognoses on growth inhibition by cetuximab

Keratinocytic epidermal nevi are associated with mosaic RAS mutations

Background: Activating RAS mutations in the germline cause rare developmental disorders such as Costello syndrome. Somatic RAS mutations are found in approximately 30% of human cancers. Keratinocytic epidermal nevi (KEN) represent benign congenital skin lesions arranged along Blaschko's lines. A subgroup of KEN is caused by hotspot oncogenic FGFR3 and PIK3CA mutations in mosaicism, but the majority lack these mutations. Methods: This study screened 72 KEN for activating mutations in RAS genes and other oncogenes. Results: Activating RAS mutations were identified in 28/72 (39%) of KEN. HRAS was the most commonly affected oncogene (86%), with the HRAS p.G13R substitution representing a new hotspot mutation. Conclusion: These results indicate that activating RAS somatic mutations leading to mosaicism result in benign KEN of the skin. Given the prevalence of KEN, mosaic HRAS mutations appear to be more common in patients than germline ones. These findings identify KEN as a mosaic RASopathy and lend further support to the notion that genetic mosaicism is an important contributor to disease

Особенности транспортной логистики сборных грузов в сфере ВЭД

Перемещение товаров в составе сборного груза может быть выгодным совершенно разным субъектам. Для крупных компаний это удобно для доставки разнородных товаров и пробных образцов, а для более мелких позволяет оптимизировать оборотные средства. Также огромную часть клиентов транспортных компаний составляют ретейловые компании и индивидуальные предприниматели, объемы поставок которых не позволяют экономически выгодно перемещать свои товары иначе. Движение товара в составе сборного груза имеет ряд неоспоримых преимуществ, такие как: упрощение отслеживания товаров и сокращение транспортных издержек, благодаря которым всё больше компаний и физических лиц обращаются к данному способу перемещения товаров, хоть и время доставки увеличивается из-за дополнительных этапов в перемещении товара.The movement of goods as part of a consolidated cargo can be beneficial to completely different entities. For large companies, it is convenient for the delivery of heterogeneous goods and test samples, and for smaller ones, it allows optimizing working capital. Also, a huge part of the customers of transport companies are retail companies and individual entrepreneurs, the volume of supply of which does not allow economically move their goods otherwise. The movement of goods as part of the consolidated cargo has a number of undeniable advantages, such as: simplification of tracking of goods and reduction of transport costs, thanks to which more and more companies and individuals are turning to this method of movement of goods, although the delivery time is increased due to additional step

Aberrant DNA methylation patterns in microsatellite stable human colorectal cancers define a new marker panel for the CpG island methylator phenotype

A distinct group of colorectal carcinomas (CRCs) referred to as the “CpG island methylator phenotype” (CIMP) shows an extremely high incidence of de novo DNA methylation and may share common pathological, clinical or molecular features. However, there is limited consensus about which CpG islands (CGIs) define a CIMP, particularly in microsatellite stable (MSS) carcinomas. To study this phenotype in a systematic manner, we analyzed genome-wide CGI DNA methylation profiles of 19 MSS CRC using methyl-CpG immunoprecipitation (MCIp) and hybridization on 244K CGI oligonucleotide microarrays, determined KRAS and BRAF mutation status and compared disease-related DNA methylation changes to chromosomal instability as detected by microarray-based comparative genomic hybridization. Results were validated using mass spectrometry analysis of bisulfite-converted DNA at a subset of 76 individual CGIs in 120 CRC and 43 matched normal tissue samples. Both genome-wide profiling and CpG methylation fine mapping segregated a group of CRC showing pronounced and frequent de novo DNA methylation of a distinct group of CGIs that only partially overlapped with previously established classifiers. The CIMP group defined in our study revealed significant association with colon localization, either KRAS or BRAF mutation, and mostly minor chromosomal losses but no association with known histopathological features. Our data provide a basis for defining novel marker panels that may enable a more reliable classification of CIMP in all CRCs, independently of the MS status

STRN-ALK Fusion in a Case of Malignant Peritoneal Mesothelioma: Mixed Response to Crizotinib, Mode of Resistance, and Brigatinib Sequential Therapy

ALK fusions were first described by Morris et al1 in 1994. Several studies have reported genetic alterations of the ALK gene in various tumor types since then, consisting of mutations, amplifications, and fusions.1-3 Fusion proteins have an active C-terminal tyrosine kinase domain in common.3 Here, we describe an STRN-ALK fusion in malignant peritoneal mesothelioma (MPM), which has previously been documented in other neoplasms, including thyroid cancer, renal carcinoma, leukemia, lymphoma, colon adenocarcinoma, head and neck adenocarcinoma, pericardial and peritoneal mesothelioma, and cutaneous squamous cell carcinoma.4-6 MPM is a rare disease with an incidence of approximately seven per million people per year.7 Patients' life expectancy is low (on average 12 months) because of the late clinical presentation with abdominal or pelvic pain or lymphadenopathy.8,9 Recently, ALK rearrangements have gained attention, especially in young female patients with MPM. Hung et al10 identified three ALK fusions in 88 consecutively screened patients with MPM. Fusion partners were ATG16L1, TPM1, and STRN. In another study by Mian et al,11 among 32 patients ≤ 40 years old with mesothelioma (of which 25 were MPM), an ALK rearrangement was detected by fluorescence in situ hybridization in two patients (6%). One of the cases harbored an STRN-ALK fusion as described in the current case. Argani et al12 described additional five cases of ALK fusions in pediatric MPM. Subsequently, three more cases of STRN-ALK rearrangements in MPM have been published individually.6,13,14 In non–small-cell lung cancer (NSCLC), the discovery of specific drugs targeting ALK rearrangements led to significant therapeutic advances. Currently, various ALK inhibitors, namely, ceritinib, crizotinib, and alectinib, are used as first-line treatment in adult ALK-positive advanced NSCLC. Although crizotinib as a first-generation ALK inhibitor has already proven superiority over chemotherapy,15 next-generation ALK inhibitors such as ceritinib yielded even better survival rates.16 Moreover, both brigatinib and alectinib demonstrated superior effectiveness when directly compared with crizotinib.17,18 Unfortunately, resistance is frequently observed following an initial response in all these agents.19 Mechanisms of resistance, which often include ALK mutations, are in general universal although variable mutational frequencies are observed depending on the inhibitor.20 Despite this large base of knowledge for lung cancer, the evaluation of ALK fusions in other entities remains challenging because of limited available data

Pitfalls in mutational testing and reporting of common KIT and PDGFRA mutations in gastrointestinal stromal tumors

<p>Abstract</p> <p>Background</p> <p>Mutation analysis of <it>KIT </it>and <it>PDGFRA </it>genes in gastrointestinal stromal tumors is gaining increasing importance for prognosis of GISTs and for prediction of treatment response. Several groups have identified specific mutational subtypes in <it>KIT </it>exon 11 associated with an increased risk of metastatic disease whereas GISTs with <it>PDGFRA </it>mutations often behave less aggressive. Furthermore, in advanced GIST disease with proven <it>KIT </it>exon 9 mutation the doubled daily dose of 800 mg imatinib increases the progression free survival and is now recommended both in the European and the American Guidelines. In Germany, there are still no general rules how to perform mutational analysis.</p> <p>Methods</p> <p>When comparing results from six different molecular laboratories we recognized the need of standardisation. Six German university laboratories with experience in mutation analysis in GISTs joined together to develop recommendations for the mutation analysis of the most common and clinically relevant hot spots, i. e. <it>KIT </it>exons 9 and 11 and <it>PDGFRA </it>exon 18. We performed a three-phased interlaboratory trial to identify pitfalls in performing molecular analysis in GISTs.</p> <p>Results</p> <p>We developed a design for a continuous external laboratory trial. In 2009 this external trial was conducted by 19 laboratories via the initiative for quality assurance in pathology (QuiP) of the German Society of Pathology and the Professional Association of German Pathologists.</p> <p>Conclusions</p> <p>By performing a three-phased internal interlaboratory trial and conducting an external trial in Germany we were able to identify potential pitfalls when performing KIT and PDGFRA mutational analysis in gastrointestinal stromal tumors. We developed standard operation procedures which are provided with the manuscript to allow other laboratories to prevent these pitfalls.</p

Identification of Disparities in Personalized Cancer Care—A Joint Approach of the German WERA Consortium

(1) Background: molecular tumor boards (MTBs) are crucial instruments for discussing and allocating targeted therapies to suitable cancer patients based on genetic findings. Currently, limited evidence is available regarding the regional impact and the outreach component of MTBs; (2) Methods: we analyzed MTB patient data from four neighboring Bavarian tertiary care oncology centers in Würzburg, Erlangen, Regensburg, and Augsburg, together constituting the WERA Alliance. Absolute patient numbers and regional distribution across the WERA-wide catchment area were weighted with local population densities; (3) Results: the highest MTB patient numbers were found close to the four cancer centers. However, peaks in absolute patient numbers were also detected in more distant and rural areas. Moreover, weighting absolute numbers with local population density allowed for identifying so-called white spots—regions within our catchment that were relatively underrepresented in WERA MTBs; (4) Conclusions: investigating patient data from four neighboring cancer centers, we comprehensively assessed the regional impact of our MTBs. The results confirmed the success of existing collaborative structures with our regional partners. Additionally, our results help identifying potential white spots in providing precision oncology and help establishing a joint WERA-wide outreach strategy