Intestinal Wnt in the transition from physiology to oncology.

Adult stem cells are necessary for self-renewal tissues and regeneration after damage. Especially in the intestine, which self-renews every few days, they play a key role in tissue homeostasis. Therefore, complex regulatory mechanisms are needed to prevent hyperproliferation, which can lead in the worst case to carcinogenesis or under-activation of stem cells, which can result in dysfunctional epithelial. One main regulatory signaling pathway is the Wnt/ß-catenin signaling pathway. It is a highly conserved pathway, with ß-catenin, a transcription factor, as target protein. Translocation of ß-catenin from cytoplasm to nucleus activates the transcription of numerous genes involved in regulating stem cell pluripo-tency, proliferation, cell differentiation and regulation of cell death. This review presents a brief overview of the Wnt/ß-catenin signaling pathway, the regulatory mechanism of this pathway and its role in intestinal homeostasis. Additionally, this review highlights the molecular mechanisms and the histomorphological features of Wnt hyperactivation. Furthermore, the central role of the Wnt signaling pathway in intestinal carcinogenesis as well as its clinical relevance in colorectal carcinoma are discussed.

Regulatory Considerations for Clinical Trial Applications with CRISPR-Based Medicinal Products.

Since first proposed as a new tool for gene targeting and genome editing, CRISPR technology has quickly advanced into the clinical stage. Initial studies highlight the potential for CRISPR-Cas9-mediated therapeutic approaches in human medicine to correct incurable genetic diseases and enhance cell-based therapeutic approaches. While acknowledging the opportunities this technology brings for the treatment of patients with severe diseases, timely development of these innovative medicinal products requires regulatory oversight and adaptation of regulatory requirements to ensure the safety and efficacy of medicinal products based on CRISPR technology. We briefly present the current regulatory framework applicable for CRISPR-Cas-based developments as advanced therapy medicinal products. Moreover, scientific- and regulatory-driven considerations relevant for advancing product development toward clinical trial applications in Germany are highlighted by discussing the key aspects of quality and nonclinical and clinical development requirements.

Peritoneal metastasis in gastric cancer: results from the German database.

BACKGROUND: Patients with peritoneal metastases of gastric cancer have a poor prognosis with a median survival of 7 months. A benefit of cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) could be shown in several selected patient cohorts but remains controversial. The aim of this study was, to reflect the results of a national German HIPEC registry initiated by the German Society of General and Visceral Surgery (DGAV). METHODS: The DGAV HIPEC registry StuDoQ|Peritoneum documents patients with peritoneal malignancy contributed from 52 hospitals. All consecutive documented patients from 2011 until 2016 (n = 3078) were treated with CRS and HIPEC and were analysed. A total of 315 (10%) suffered from gastric cancer and were analysed. RESULTS: A complete data set of 235 patients was available for this study, including 113 male (48.1%) and 122 female (51.9%) patients with a median age of 53.4 years (SD ± 11.9). The median PCI was 8.0 (range 1-30). A complete cytoreduction was achieved in 121 patients (71.6%). Postoperative complications (Clavien-Dindo grades 3-4) occurred in 40 patients (17%). The median overall survival (OS) time was 13 months. The 5-year survival rate was 6%. According to the PCI from 0-6 (n = 74); 7-15 (n = 70) and 16-39 (n = 24) the median OS differs significantly (18 months vs. 12 months vs. 5 months; p = 0.002). CONCLUSIONS: CRS and HIPEC in selected patients with gastric cancer and peritoneal spread can improve survival when they are treated in centers. An accurate staging and patient selection are of major importance to achieve long-term survival.

The Human Genome Editing Race: Loosening Regulatory Standards for Commercial Advantage?

Medicinal products based on genome editing must undergo rigorous preclinical testing and are subject to regulatory oversight for proper risk assessment prior to first evaluation in humans. We give a European perspective on the regulatory expectations to translate genome editing to the clinic to ensure their timely progress to market.

Tropism, intracerebral distribution, and transduction efficiency of HIV- and SIV-based lentiviral vectors after injection into the mouse brain: a qualitative and quantitative in vivo study.

Lentiviruses are suitable to transfer potential therapeutic genes into non-replicating cells such as neurons, but systematic in vivo studies on transduction of neural cells within the complete brain are missing. We analysed the distribution of transduced cells with respect to brain structure, virus tropism, numbers of transduced neurons per brain, and influence of the Vpx or Vpr accessory proteins after injection of vectors based on SIVsmmPBj, HIV-2, and HIV-1 lentiviruses into the right striatum of the mouse brain. Transduced cells were found ipsilaterally around the injection canal, in corpus striatum and along corpus callosum, irrespective of the vector type. All vectors except HIV-2SEW transduced also single cells in the olfactory bulb, hippocampus, and cerebellum. Vector HIV-2SEW was the most neuron specific. However, vectors PBjSEW and HIV-1SEW transduced more neurons per brain (means 41,299 and 32,309) than HIV-2SEW (16,102). In the presence of Vpx/Vpr proteins, HIV-2SEW(Vpx) and HIV-1SEW(Vpr) showed higher overall transduction efficiencies (30,696 and 27,947 neurons per brain) than PBjSEW(Vpx) (6636). The distances of transduced cells from the injection canal did not differ among the viruses but correlated positively with the numbers of transduced neurons. The presence of Vpx/Vpr did not increase the numbers of transduced neurons. Parental virus type and the vector equipment seem to influence cellular tropism and transduction efficiency. Thus, precision of injection and choice of virus pseudotype are not sufficient when targeted lentiviral vector transduction of a defined brain cell population is required.

[Mesenchymal stromal cells in the treatment of graft-versus-host disease: where do we stand?]. / Mesenchymale Stromazellen bei der Therapie der Graft-versus-Host-Erkrankung: Wo stehen wir?

Medicinal products based on mesenchymal stromal cells (MSC) are expected to have a therapeutic benefit in a variety of conditions and, accordingly, are being tested in many clinical studies. The treatment and prevention of graft-versus-host disease (GVHD) is one of the world's most widely studied MSC therapy concepts. So far, one MSC medicinal product has been approved for the treatment of GvHD. This article gives an overview of the particular features related to the production of MSC-based medicinal products, the state of non-clinical research, and the clinical development status of MSCs and the associated challenges, especially in the context of GvHD.

Predictive factors for survival and recurrence rate in patients with node-negative gastric cancer--a European single-centre experience.

PURPOSE: Gastric cancer is a common disease with poor prognosis. Lymph node involvement is the strongest prognostic factor regarding survival in curatively (R0) resected patients. The aim of this study was to determine if a subgroup with higher risk for tumour recurrence exists in patients with node-negative gastric cancer. Furthermore, we aimed to identify prognostic factors and recurrence patterns for this subgroup. METHODS: We collected demographical, clinical, pathohistological and follow-up data from 1,074 patients with gastric cancer in a prospectively maintained database. In 228 cases, R0 resections for node-negative gastric cancer were performed. The median follow-up period was 59 months. Statistical analysis was performed using SPSS 19.0. RESULTS: The 5- , 10- and 15-year overall survival was 83, 75.5 and 73 %, respectively, with a disease-free survival of 78, 73 and 73 %. Tumour recurrence was observed in 18.9 % (43 cases), 14 % of which were diagnosed after more than 5 years. Gender, T-category , lymphangioinvasion, tumour differentiation, serosal infiltration, histological growth pattern, tumour size and classification according to Lauren were significant prognostic factors for overall survival in univariate analysis. Multivariate analysis showed tumour size and female sex to be independent prognostic factors. Non-exophytic tumour growth was a relevant factor for the development of local recurrence. Diffuse type gastric cancers as well as signet ring cells were significantly associated with the development of peritoneal carcinomatosis, and male gender with hematogenous metastases. CONCLUSIONS: Multimodal treatment and individual follow-up might be beneficial in patients with higher risk of recurrence after R0 resection of node-negative gastric cancer.

The influence of desmocollin 1-3 expression on prognosis after curative resection of colorectal liver metastases.

PURPOSE: Prognosis after curative resection of colorectal liver metastases is hard to determine based on clinical parameters; biomarkers are therefore needed. The purpose of this study was to determine the value of desmocollins (DSC) as potential biomarkers. Desmocollins are responsible for cell-cell adhesion in epithelial tissue; their loss may lead to reduced cellular adhesion and facilitate cellular migration, enabling tumor cells to form distant metastases. We analyzed DSC expression in colorectal liver metastases with respect to the risk of recurrence following liver resection. METHODS: Tissue microarrays from 257 consecutive patients who underwent R0-resection of colorectal liver metastases were constructed. RESULTS: Low expression of DSC 1, 2, and 3 was observed in 55, 54, and 79 % of liver metastases. There was no correlation between site or stage of the primary tumor, presence of extrahepatic tumor, grading, size or number of metastases, and desmocollin expression. Primary tumor stage I or II (p = 0.005) and no or few lymph node metastases (p < 0.001) were associated with a significantly better disease-free survival on univariate analysis. These parameters reached only marginal significance on multivariate analysis (p = 0.059 and p = 0.052, respectively), as did desmocollin 3 expression (p = 0.050). In the subgroup of patients with stages III-IV primary tumors, however, multivariate analysis showed a significant correlation between DSC 3 expression and disease-free survival after liver resection (p = 0.009). CONCLUSIONS: Reduced expression of DSC3 correlated with an increased risk of developing tumor recurrence after resection of liver metastases. These findings may be helpful in selecting high-risk patients who might benefit from multimodal therapy.

Loss of chromosome 4 correlates with better long-term survival and lower relapse rate after R0-resection of colorectal liver metastases.

PURPOSE: Liver metastases are the major cause of cancer-related death in colorectal cancer patients with a tendency to recur in over 50 % of the cases even after curatively intended surgery. Prognosis after liver resection, however, can neither be based on macroscopic or light microscopic evaluation of the metastases nor on clinical data alone. This is a pilot study in order to determine a potential influence of chromosomal aberrations on overall survival and relapse rate after curative liver resection. METHODS: Twenty randomly selected cases (10 patients with a survival of more and 10 patients with a survival of less than 5 years after resection) were studied by array comparative genomic hybridization. RESULTS: The distributions concerning age, gender, stage and grading of primary tumor, percentage of patients with chemotherapy, number and distribution of the liver metastases, Nordlinger and Fong scores showed no differences between long- and short-term survivors and no correlation to any chromosomal aberration. However, the relapse rate of patients with (partial) monosomy 4 was lower and the long-time survival better than in the other patients. CONCLUSIONS: Loss of chromosome 4 in colorectal liver metastases seems not only to be associated with the progression of the primary tumor as reported in the literature, but also with the long-term survival and the cumulative relapse rate after complete resection of colorectal liver metastases.

Liver transplantation for hilar cholangiocarcinoma--a single-centre experience.

BACKGROUND: Cholangiocarcinoma is an infrequent malignancy, often unresectable at the time of diagnosis. Liver transplantation may offer a chance for cure, but results in the past have been disappointing, prompting transplant centres to adopt multimodal treatment protocols and extreme patient selection. PURPOSE: This study was designed to evaluate the outcome of patients with irresectable hilar cholangiocarcinoma undergoing liver transplantation in order to determine criteria for patient selection. METHODS: We reviewed our prospective cancer registry for patients with hilar cholangiocarcinoma treated by transplantation since 1997. Data were evaluated regarding tumour location, stage, overall survival, recurrence rates and prognostic factors. RESULTS: Liver transplantation with lymphadenectomy was realised in 16 patients with hilar cholangiocarcinoma. Seven patients received a living donor graft. Lymph node metastases were found in eight patients with a median of 13 harvested nodes and had a statistically significant negative impact on overall survival irrespective of tumour size. Only one patient underwent neoadjuvant brachytherapy and developed fatal septic complications; 3- and 5-year survival rates were 63 and 50 % in lymph node-negative patients without neoadjuvant treatment. CONCLUSIONS: Acceptable survival rates can be achieved by transplantation for hilar cholangiocarcinoma with lymph node metastases as the only exclusion criterion. We recommend staging laparotomy with lymphadenectomy along the common hepatic artery prior to liver transplantation.

Long-term results and prognostic factors after resection of hepatic and pulmonary metastases of colorectal cancer.

BACKGROUND: Resection of colorectal liver or lung metastases is an established therapeutical concept at present. However, an affection of both these organs is frequently still regarded as incurable. METHODS: All cancer patients are documented in our prospective cancer registry since 1995. Data of patients who underwent liver and lung resection for colorectal metastases were extracted and analysed. RESULTS: Sixty-five patients underwent surgery for liver and lung metastases. In 33 cases, the first distant metastasis was diagnosed synchronously to the primary tumour. For the remaining patients, median time interval between primary tumour and first distant metastasis was 18 months (5-69 months). Complete resection was achieved in 51 patients (79 %) and was less likely in patients with synchronous disease (p = 0.017). Negative margins (p = 0.002), the absence of pulmonary involvement in synchronous metastases (p = 0.0003) and single metastases in both organs (p = 0.036) were associated with a better prognosis. Five- and 10-year survival rates for all patients are 57 and 15 % from diagnosis of the primary tumour, 37 and 14 % from resection of the first metastasis and 20 and 15 % from resection of the second metastasis. After complete resection, 5- and 10-year survival rates increased to 61 and 18 %, 43 and 17 % as well as 25 and 19 %, respectively. Long-term survivors (≥10 years) were seen only after complete resection of both metastases. CONCLUSIONS: Patients with resectable liver and lung metastases of the colorectal primary should be considered for surgery after multidisciplinary evaluation regardless of the number or size of the metastases or the disease-free intervals. Clear resection margins are the strongest prognostic parameter.

Safety assessment of biolistic DNA vaccination.

DNA-based vector systems have been widely studied as new modalities for the prevention and treatment of human diseases. As for all other medicinal products, safety is an important aspect in the evaluation of such products. In this chapter we reflect on the basic safety issues which have been raised with respect to preventive and therapeutic DNA vaccines, including insertional mutagenesis in case of chromosomal integration, possible formation of anti-DNA antibodies, induction of autoimmune responses and/or immunological tolerance. In addition, local reactions at the site of administration and adverse effects resulting from plasmid DNA spread to nontarget tissues are discussed. Most importantly, however, the benefit-risk profile of a medicinal product is crucial for a decision on providing marketing authorization or not. A product has an acceptable benefit-risk profile if the benefits of the product outweigh its risks for the treated patient.

Epidemiological pattern of hepatitis B and hepatitis C as etiological agents for hepatocellular carcinoma in iran and worldwide.

CONTEXT: Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections constitute a major global health problem. About 60,000 and 350,000 deaths occur as the results of HBV and HCV infections, respectively. Chronic hepatitis B and C infections are leading causes of cirrhosis and hepatocellular carcinoma (HCC) which are considered as the third cancer-associated cause of deaths worldwide. Iran suffers from the same problem but to a lesser extent as it is considered as a low endemic area for HBV and HCV infections and also as a low incidence area of HCC. This study was conducted to assess and provide a clear picture about epidemiology of HBV and HCV infections in Iran and worldwide, with the consequence on HCC distribution all over the world including Iran, and to analyze current literature regarding the modes of transmission and risk factors of HBV and HCV infections. EVIDENCE ACQUISITION: In this review, we performed electronic and manual searches on available databases such as MEDLINE, PubMed, Ovid, Embase, and the Iranian databases such as IranMedex. We also performed a Google search to find related articles. RESULTS: HBV and HCV infections are the most common risk factors of hepatocellular carcinoma. The epidemiology of HCC usually follows that of HBV and HCV infections. With the introduction of HBV national vaccine in Iran and worldwide, there is a noticeable effect on reduction in HBV prevalence in most countries, and we expect that HCV will replace HBV as a major risk factor of HCC in Iran and worldwide. Alcohol plays a minor role as a risk factor for cirrhosis and HCC in Iran, Asia, and Africa, despite its noticeable role in Europe and the USA. CONCLUSIONS: Vaccination against HBV remains the most effective approach against HBV infection with consequence decrease in HBV-related HCC. There is a need to improve the awareness about epidemiology of HBV and HCV infections, modes of transmission, and their complications, specifically HCC among population.

Vascular clips versus ligatures in thyroid surgery--results of a multicenter randomized controlled trial (CLIVIT Trial).

BACKGROUND: New techniques using vascular clips or ultrasonically activated shears have been suggested to shorten operation time without compromising safety. The objective of the CLIVIT Trial was to compare ligatures with vascular clips for hemostasis in elective benign thyroid surgery. METHODS: This multicenter, randomized, controlled, parallel group superiority trial was conducted in 13 German surgical centers. Patients scheduled for at least subtotal resection bilaterally were intraoperatively randomized. The primary endpoint was resection time. Secondary endpoints were the amount of postoperative bleeding, reoperation due to bleeding, wound infection, temporary (reversal within 12 months) and permanent (over 1 year) recurrent laryngeal nerve (RLN) paralysis, length of hospital stay, and safety. REGISTRATION: ISRCTN 96901396. RESULTS: Two hundred fifty patients were treated with ligatures and 241 with vascular clips. No differences in patients' baseline and surgical characteristics were observed. No difference was detected for mean resection time (clip 63.5 min ± 29.6, ligature 66.1 min ± 29.3, P = 0.258). Postoperative bleeding (mean 86 ml ± 93), reoperation due to bleeding (clips 4, ligature 2), wound infections (clips 4, ligature 4), postoperative hospital stay (mean 3.0 ± 1.9), and safety data also did not vary significantly. The rates of temporary and permanent RLN paralysis were 6.9 % (34/491) and 2.9 % (14/491), respectively. Not using a surgical drain (123 patients) was not associated with a higher rate of complications. CONCLUSION: Vascular clips did not reduce the resection time. However, a 2.9 % rate of permanent RLN paralysis is of concern. Drains in elective surgery may be of no benefit.

The European hospital exemption clause-new option for gene therapy?

Gene-therapy medicinal products are currently applied to patients enrolled in authorized clinical trials to demonstrate safety and efficacy. Given a positive outcome, marketing authorization can subsequently be achieved via the centralized procedure coordinated by the European Medicines Agency. With Regulation (EC) No. 1394/2007 in force, advanced therapy medicinal products, including gene- and cell-therapy products, can be excepted from the obligation of obtaining a marketing authorization via the centralized procedure under specific conditions (so-called "hospital exemption"). This hospital exemption allows the application of gene-therapy medicinal products prepared on a non-routine basis for an individual patient and used under the exclusive professional responsibility of a medical practitioner. Here, we explain the requirements to be fulfilled in order to fall under this exemption, the implementation of this regulation into the German national legislation, and its impact on gene-therapy product development in the future.

PKCδ-induced PU.1 phosphorylation promotes hematopoietic stem cell differentiation to dendritic cells.

Human CD34(+) hematopoietic stem cells (HSCs) exhibit the potential to differentiate into a variety of specialized blood cells. The distinct intracellular mechanisms that control cell fate and lineage commitment of these multipotent cells are not well defined. In this study, we investigate and modulate the signaling processes during HSC differentiation toward myeloid dendritic cells (mDCs). DC differentiation induced by the cytokines Granulocyte macrophage colony-stimulating factor (GM-CSF) and Interleukin-4 (IL-4) led to activation of the Extracellular-signal-regulated kinase (ERK), protein kinase C (PKC), and Janus kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) but not the SAPK/c-Jun NH(2) -terminal kinase and p38 mitogen-activated protein kinase signaling pathways. From the activated signaling pathways the PKC isoform δ was found to phosphorylate the transcription factor PU.1, which is described as one of the key factors for myeloid HSC differentiation. On molecular level, PKCδ regulated PU.1 activity by affecting its transactivation activity, whereas its DNA binding activity remained unaffected. This was accompanied by PKCδ-induced phosphorylation of the PU.1 transactivation domain. Furthermore, treatment with PKC- and ERK1/2-specific signaling inhibitors impaired both HSC differentiation toward mDCs as well as phosphorylation-mediated transactivation activity of PU.1. Taken together, these results provide new insights into the molecular mechanisms promoting the differentiation process of HSCs toward mDCs and introduce the PKC isoform δ as critical mediator.

Comparative analysis of transduced primary human dendritic cells generated by the use of three different lentiviral vector systems.

Lentiviral gene transfer vectors are suitable for genetically modifying non-cycling primary human cells. In this study, we analyzed transduced human dendritic cells (DC) generated by the use of three different GFP-encoding lentiviral vectors, HIV-2 ROD A Δenv-GFP (ROD A), SIVsmm PBj ΔE EGFP (PBj), and SIVmac ΔE EGFP (SIVmac). CD14+ monocytes were isolated from buffy coat, transduced, and differentiated to immature and mature DC. Cytofluometric analysis of DC revealed high transduction efficiencies at MOI 1 for simian immunodeficiency virus (SIV)-derived vectors PBj and SIVmac ranging between 80-90 and 70-90%, respectively. In contrast, transduction with ROD A resulted only in approximately 30%-positive DC at the same MOI. Of note, none of the analyzed vectors affected expression of maturation and/or activation markers. Moreover, transduction with PBj or SIVmac did not induce significant cytokine responses whereas ROD A transduction stimulated weak interferon-alpha responses. SIVmac transduced DC showed normal phagocytosis of antigen and normal allo T cell stimulatory capacity when compared with untreated DC. Thus, the SIVmac lentiviral transduction vector is suitable for efficient genetic modification of human DC without affecting phenotype or function and thus qualifies this vector as a versatile tool for use in basic research.