Premature birth is a leading cause of childhood morbidity and mortality and often followed by an arrest of postnatal lung development called bronchopulmonary dysplasia. Therapies using exogenous mesenchymal stromal cells (MSC) have proven highly efficacious in term-born rodent models of this disease, but effects of MSC in actual premature-born lungs are largely unknown. Here, we investigated thirteen non-human primates (baboons; Papio spp.) that were born at the limit of viability and given a single, intravenous dose of ten million human umbilical cord tissue-derived MSC per kilogram or placebo immediately after birth. Following two weeks of human-equivalent neonatal intensive care including mechanical ventilation, lung function testing and echocardiographic studies, lung tissues were analyzed using unbiased stereology. We noted that therapy with MSC was feasible, safe and without signs of engraftment when administered as controlled infusion over 15 minutes, but linked to adverse events when given faster. Administration of cells was associated with improved cardiovascular stability, but neither benefited lung structure, nor lung function after two weeks of extrauterine life. We concluded that a single, intravenous administration of MSC had no short- to mid-term lung-protective effects in extremely premature-born baboons, sharply contrasting data from term-born rodent models of arrested postnatal lung development and urging for investigations on the mechanisms of cell-based therapies for diseases of prematurity in actual premature organisms.

The author regret, the correct Table 2 updated as below: The author would like to apologise for any inconvenience caused. Residual gammaH2AX foci in head and neck squamous cell carcinomas as predictors for tumour radiosensitivity: Evaluation in pre-clinical xenograft models and clinical specimensRadiotherapy and OncologyVol. 137PreviewIt is estimated that 50% of patients newly diagnosed with cancer will need to undergo radiotherapy [1]. Tumour response to radiotherapy varies between patients [2,3] and influences the success rate of radiotherapy. Treatment individualisation based on patient characteristics should therefore increase cure rates and reduce normal tissue complications [4]. For that matter, predictive biomarkers are of paramount importance to foresee tumour response ahead of treatment and optimise care for the patient [5]. Full-Text PDF

BACKGROUND AND PURPOSE:Systemic molecular radiotherapy utilizes internal irradiation by radionuclide-labeled tumor-targeting agents with the potential to destroy (micro-)metastases. However, doses that are applicable in solid tumors do not reach the levels nessecary for tumor control. Thus, the combination of molecular and external radiotherapy is a promising treatment strategy, as enhanced tumor doses can be delivered with and without minor overlapping toxicities. Here, we combined a 90Y-labeled anti-EGFR antibody (Cetuximab) with clinically relevant fractionated radiotherapy in a preclinical trial using head and neck squamous cell carcinoma xenograft tumors.MATERIALS AND METHODS:To model 90Y-Cetuximab uptake for treatment schedule optimization, FaDu-bearing mice were injected with near-infrared-labeled-Cetuximab at different time points during radiotherapy with differing doses. Cetuximab uptake was longitudinally followed by in vivo-optical imaging. Tumor control probability experiments with fractionated radiotherapy (30 fx, 6 weeks, 8 dose groups/ arm) in combination with 90Y-Cetuximab were performed to test the curative potential.RESULTS:Imaging of near-infrared-labeled-Cetuximab uptake revealed that low to moderate external beam doses can enhance antibody uptake. Using the optimized schedule, combination of molecular and external radiotherapy using 90Y-Cetuximab at a dose that did not result in permanent tumor inactivation in previous experiments, led to substantially increased tumor control compared to radiotherapy alone.CONCLUSION:Our results indicate that combination of radiolabeled therapeutics with clinically relevant fractionated radiotherapy has a remarkable potential to improve curative treatment outcome. Application of some radiation dose prior to injection may improve drug uptake and enable patient stratification and treatment personalization via a corresponding PET-tracer during therapy.

In kinship analysis, large data sets with estimated haplotype frequencies for marker clusters are very important for the likelihood calculation. Practical use of the X-STRs demonstrated that in some complex kinship cases, the marker set of the Investigator Argus X-12 kit can be insufficient. This study aimed to extend the German data base of the Argus X-12 kit (1037 haplotypes) and for a cluster in Xq21 (806 haplotypes) with additional 700 male haplotypes and to include a further cluster in Xp22.3 to complete the X-STR marker set for complex kinship cases.

Glioblastoma is an aggressive brain tumour with a patient median survival of approximately 14 months. The development of innovative treatment strategies to increase the life span and quality of life of patients is hence essential. This requires the use of appropriate glioblastoma models for preclinical testing, which faithfully reflect human cancers. The aim of this study was to establish glioblastoma patient-derived xenografts (PDXs) by heterotopic transplantation of tumour pieces in the axillae of NMRI nude mice. Ten out of 22 patients' samples gave rise to tumours in mice. Their human origin was confirmed by microsatellite analyses, though minor changes were observed. The glioblastoma nature of the PDXs was corroborated by pathological evaluation. Latency times spanned from 48.5 to 370.5 days in the first generation. Growth curve analyses revealed an increase in the growth rate with increasing passages. The methylation status of the MGMT promoter in the primary material was maintained in the PDXs. However, a trend towards a more methylated pattern could be found. A correlation was observed between the take in mice and the proportion of Sox2+ cells (r = 0.49, p = 0.016) and nestin+ cells (r = 0.55, p = 0.007). Our results show that many PDXs maintain key features of the patients' samples they derive from. They could thus be used as preclinical models to test new therapies and biomarkers.

Background and purpose: Predictive biomarkers can be instrumental to treatment individualisation of cancer patients and improve therapy outcome. Residual gamma H2AX foci represent a promising biomarker to predict tumour radiosensitivity. In this pre-clinical study, the slope of the dose-response curve was evaluated for its predictive relevance in head and neck squamous cell carcinoma xenografts (HNSCC). Additionally, the feasibility of the translated assay was tested in a clinical setting in patient derived HNSCC samples, and associations between residual gamma H2AX foci and clinical parameters were analysed. Materials and methods: Seven HNSCC xenografts models (FaDu, SAS, SKX, UT-SCC-5, UT-SCC-14, UT-SCC-45, XF354) were used. Tumour bearing NMRI nude mice were randomly distributed to five treatment arms (0-8 Gy). Residual gamma H2AX foci (24 h post irradiation) were counted by visual scoring in a micromilieu dependent manner (assessed with BrdU and pimonidazole). The local tumour control values measured as TCD50 (tumour control dose 50%) have previously been published. Patient derived HNSCC biopsies were cultivated ex vivo for 24 h including 4 h of pimonidazole and BrdU treatment, subsequently irradiated with 0-8 Gy and fixed after 24 h. Results: In the pre-clinical study, the dose-response curve slopes negatively correlated with the tumour control dose after fractionated irradiation (TCD50, fx, R-2 = 0.63, p = 0.032) and after single dose irradiation under homogeneous hypoxia (TCD50, SD, clamp, R-2 = 0.66, p = 0.027). The cH2AX assay in clinical HNSCC samples showed a dose-response relationship, with the values of the slopes ranging from 0.099 Gy(-1) to 0.920 Gy(-1) (coefficient of variation = 52.8%). Slopes derived from patients were in the same ranges as the sensitive, moderate and resistant models of the pre-clinical study. Statistical analysis revealed a significant negative correlation between the slope and the patients' age (R-2 = 0.65, p = 0.001). Conclusion: These results further support the promise of the slope of the residual gamma H2AX foci doseresponse as a biomarker for radiosensitivity. In the clinical samples, the variation in the slopes reveals patients' specific repair capacities, which could hold potential value for treatment individualisation. (C) 2019 Elsevier B.V. All rights reserved.

Recent clinical data have linked KRAS/TP53 comutation (mut) to resistance to radiotherapy (RT), but supporting laboratory in vivo evidence is lacking. In addition, the ability of different radiation doses, with/without epidermal growth factor receptor (EGFR)-directed treatment, to achieve local tumor control as a function of KRAS status is unknown. Here, we assessed clonogenic radiation survival of a panel of annotated lung cancer cell lines. KRASmut/TP53mut was associated with the highest radioresistance in nonisogenic and isogenic comparisons. To validate these findings, isogenic TP53mut NCI-H1703 models, KRASmut or wild-type (wt), were grown as heterotopic xenografts in nude mice. A clinical RT schedule of 30 fractions over 6 weeks was employed. The dose that controlled 50% of tumors (TCD50 ) was calculated. The TCD50 for KRASwt/TP53mut xenografts was 43.1 Gy whereas KRASmut/TP53mut tumors required a 1.9-fold higher TCD50 of 81.4 Gy. The EGFR inhibitor erlotinib radiosensitized KRASmut but not KRASwt cells and xenografts. The TCD50 associated with adding erlotinib to RT was 58.8 Gy for KRASmut, that is, a ~1.4-fold dose enhancement. However, the EGFR antibody cetuximab did not have a radiosensitizing effect. In conclusion, we demonstrate for the first time that KRASmut in a TP53mut background confers radioresistance when studying a clinical RT schedule and local control rather than tumor growth delay. Despite the known unresponsiveness of KRASmut tumors to EGFR inhibitors, erlotinib radiosensitized KRASmut tumors. Our data highlight KRAS/TP53 comutation as a candidate biomarker of radioresistance that can be at least partially reversed by dose escalation or the addition of a targeted agent.

BACKGROUND AND PURPOSE:Improvement of the results of radiotherapy by EGFR inhibitors is modest, suggesting significant intertumoural heterogeneity of response. To identify potential biomarkers, a preclinical trial was performed on ten different human squamous cell carcinoma xenografts of the head and neck (HNSCC) studying in vivo and ex vivo the effect of fractionated irradiation and EGFR inhibition. Local tumour control and tumour growth delay were correlated with potential biomarkers, e.g. EGFR gene amplification and radioresponse-associated gene expression profiles.MATERIAL AND METHODS:Local tumour control 120days after end of irradiation was determined for fractionated radiotherapy alone (30f, 6weeks) or after simultaneous EGFR-inhibition with cetuximab. The EGFR gene amplification status was determined using FISH. Gene expression analyses were performed using an in-house gene panel.RESULTS:Six out of 10 investigated tumour models showed a significant increase in local tumour control for the combined treatment of cetuximab and fractionated radiotherapy compared to irradiation alone. For 3 of the 6 responding tumour models, an amplification of the EGFR gene could be demonstrated. Gene expression profiling of untreated tumours revealed significant differences between amplified and non-amplified tumours as well as between responder and non-responder tumours to combined radiotherapy and cetuximab.CONCLUSION:The EGFR amplification status, in combination with gene expression profiling, may serve as a predictive biomarker for personalized interventional strategies regarding combined treatment of cetuximab and fractionated radiotherapy and should, as a next step, be clinically validated.

Purpose: To establish a clinically applicable protocol for quantification of residual gamma H2AX foci in ex vivo irradiated tumour samples and to apply this method in a proof-of-concept feasibility study to patient-derived tumour specimens.Material and methods: Evaluation of gamma H2AX foci formation and disappearance in excised FaDu tumour specimens after (a) different incubation times in culture medium, 4 Gy irradiation and fixation after 24 h (cell recovery), (b) 10 h medium incubation, 4 Gy irradiation and fixation after various time points (double strand break repair kinetics), and (c) 10 h medium incubation, irradiation with graded single radiation doses and fixation after 24 h (dose-response). The optimised protocol was applied to patient-derived samples of seminoma, prostate cancer and glioblastoma multiforme.Results: Post excision or biopsy, tumour tissues showed stable radiation-induced gamma H2AX foci values in oxic cells after >6 h of recovery in medium. Kinetics of foci disappearance indicated a plateau of residual foci after >12 h following ex vivo irradiation. Fitting the dose-response of residual gamma H2AX foci yielded slopes comparable with in situ irradiation of FaDu tumours. Significant differences in the slopes of ex vivo irradiated patient-derived tumour samples were found.Conclusion: A novel clinically applicable method to quantify residual gamma H2AX foci in ex vivo irradiated tumour samples was established. The first clinical results suggest that this method allows to distinguish between radiosensitive and radioresistant tumour types. These findings support further translational evaluation of this assay to individualise radiation therapy. (C) 2015 Elsevier Ireland Ltd. All rights reserved.

Stable haplotypes of closely associated X-STRs have proven to be a powerful tool in kinship analysis especially for cases when father/daughter relationships are to be tested. We present a presumably easy case: A woman wanted to know whether or not her daughters have the same biological father. Initial results with Argus X-12 kit showed only one mismatch at DXS10148, which could have been explained by a paternal one-step mutation. However, comparison between the maternal haplotype and the daughters showed an incompatibility in linkage group III. To explain this, a maternal 3-step mutation in HPRTB or two mutation/recombination events in linkage group III must have occurred. Analysis of 29 autosomal STRs gave a high probability for being full siblings. Analysis of 13 additional X-STRs showed no further exclusion from having the same father. Reconstruction of possible maternal haplotypes demonstrated that two recombination events in linkage group III are very unlikely. We presume that one maternal mutation in DXS10103 and one recombination event in linkage group III must have occurred together with one paternal mutation in DXS10148.This case with 3 rare events occuring within the range of the Argus X-12 kit shows that the use of X-STRs requires not only knowledge of allele and haplotype frequencies but also of mutation rates and rare recombination events of markers within linkage groups. In special cases it would be necessary to involve further X markers. Collaboration of different laboratories will help to solve such problems. (C) 2015 Elsevier Ireland Ltd. All rights reserved.

The Investigator Argus X-12 Kit allows simultaneous amplification of 12 X-chromosomal STR loci which are highly informative for kinship and paternity testing, population genetics and anthropological studies. The markers of the Investigator Argus X-12 Kit are clustered into 4 linkage groups with 3 closely linked markers per group. For genotyping each set of markers should be handled as haplotype. Here we present first haplotype frequency data of a German population involving 1037 unrelated males.

A large number of short tandem repeat (STR) markers spanning the entire human X chromosome have been described and established for use in forensic genetic testing. Due to their particular mode of inheritance, X-STRs often allow easy and informative haplotyping in kinship analyses. Moreover, some X-STRs are known to be tightly linked so that, in combination, they constitute even more complex genetic markers than each STR taken individually. As a consequence, X-STRs have proven particularly powerful in solving complex cases of disputed blood relatedness. However, valid quantification of the evidence provided by X-STR genotypes in the form of likelihood ratios requires that the recombination rates between markers are exactly known. In a collaborative family study, we used X-STR genotype data from 401 two- and three-generation families to derive valid estimates of the recombination rates between 12 forensic markers widely used in forensic testing, namely DXS10148, DXS10135, DXS8378 (together constituting linkage group I), DXS7132, DXS10079, DXS10074 (linkage group II), DXS10103, HPRTB, DXS10101 (linkage group III), DXS10146, DXS10134 and DXS7423 (linkage group IV). Our study is the first to simultaneously allow for mutation and recombination in the underlying likelihood calculations, thereby obviating the bias-prone practice of excluding ambiguous transmission events from further consideration. The statistical analysis confirms that linkage groups I and II are transmitted independently from one another whereas linkage groups II, III and IV are characterised by inter-group recombination fractions that are notably smaller than 50%. Evidence was also found for recombination within all four linkage groups, with recombination fraction estimates ranging as high as 2% in the case of DXS10146 and DXS10134.

The STR markers DXS6795, DXS9907 and GATA144D04 are located on the short arm in the region Xp22.11-Xp11.23. These loci were typed from buccal swab samples from unrelated German individuals ( 161 male, 362 female). Additionally, DNA from 90 males with one ore more daughters and their 135 grandsons were typed to check for mutability of these loci. German population and sequencing data for these three loci as well as statistical parameters of forensic interest are presented. DNA typing patterns of cell lines were used as intra-and inter-laboratory standards to calibrate allelic ladders. We found 8 different alleles in DXS9907 and GATA144D04, whereas DXS6795 exhibited only 6 alleles. Allelic distribution patterns are identical for males and females. There is no evidence for deviation from the Hardy-Weinberg equilibrium in female samples. Furthermore, no mutations were detected within 90 paternal and 135 maternal meioses. The three microsatellites are moderately variable with PIC values of 0.61, 0.56 and 0.70. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

The centromere region of the human ChrX is known as an area with extremely low crossing-over-rates. Since tightly linked and non-recombining STRs are required for kinship testing, we validated a cluster of six STRs for forensic use in this region, where stable haplotypes can be expected. Our multiplex PCR setup comprised the simultaneous amplification of the six markers DXS10161, DXS10159, DXS10162, DXS10163, DXS10164 and DXS10165, which are located between 56 and 64 Mb distanced from the Xp telomere at Xp11.21-Xq11.1. Differences between several populations are expected. We present allele- and haplotype-frequency data of population samples from Germany, Estonia, Latvia, Lithuania, Wroclaw, Vladivostok, Finland, Japan, Ethiopia and Rwanda. Our ChrX centromere multiplex is a suitable supplement of the commercially available ChrX-Kit Argus X12 and can be used as an additional independent haplogroup.

Background. Pancreatic ductal adenocarcinoma is an aggressive tumor; treatment remains a challenge because of the lack of effective therapeutic strategies. Basic research in this field is dependent on the availability of model systems. New pancreatic cancer cell lines are therefore important for the study of its biology. In the present study, we report the establishment and characterization of five new pancreatic cancer cell lines (PaCaDD-43, -60, -119, -135, -137).Material and Methods. All cell lines were derived from pancreatic ductal adenocarcinomas by the Dresden outgrowth protocol. The five cell lines originated from primary pancreatic tumors, lymph node metastases, or malignant pleural effusions. We characterized the cell lines by examining their morphology and their cytostructural and functional profiles.Results. All cell lines grew as adherent monolayers and were cultured in optimized Dresden-medium. The doubling time ranged from 22 to 47 h. v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations were detected in four of the five cell lines. KRAS mutations were identical between each primary tumor and the cell line derived from it. Immunohistochemical staining showed cytoplasmic expression of CK8/18, mostly membrane and partially cytoplasmic expression of E-cadherin and strong expression of ezrin in all cell lines. Three cell lines showed nuclear p53 accumulation and heterogeneous expression of vimentin. SMAD4 was heterogeneously expressed in four of the cell lines.Conclusions. We were able to establish five new primary pancreatic carcinoma cell lines. As applicable tools for basic research, these cell lines might contribute to a better understanding and treatment of this aggressive tumor. (C) 2012 Elsevier Inc. All rights reserved.

In this study a set of 29 X-chromosomal short tandem repeats (STRs) located within the Xq26 region was evaluated. These STRs were found within the 133.14–133.45Mb region around the HPRTB locus. Evaluation of the microsatellites was performed with regard to polymorphism, reliable amplification, and low stutter artefacts. DXS10101, DXS10102, and DXS10103 were identified as those X-STRs with highest diversity; i.e. PIC values of 0.7174–0.8933. The locus DXS10101 was the optimal candidate for the integration in the commercial available test system Mentype Argus X-8 PCR amplification kit.

OBJECTIVE:To investigate the relationships between radiobiological hypoxic fraction (rHF), pimonidazole hypoxic fraction (pHF) as well as other histological parameters of the tumour microenvironment, and local tumour control after fractionated irradiation in human squamous cell carcinomas (hSCCs).MATERIAL AND METHODS:Ten different hSCC cell lines were transplanted into nude mice and rHF was calculated from local tumour control rates after single dose irradiation under normal or clamped blood flow conditions. In parallel, tumours were irradiated with 30 fractions within 6 weeks. Radiation response was quantified as dose required to cure 50% of tumours (TCD(50)). Unirradiated tumours were excised for histological evaluation including relative hypoxic area (pHF), relative vascular area (RVA), and fraction of perfused vessels (PF).RESULTS:A weak but significant positive correlation between rHF (R(2)=0.6, p=0.014) and TCD(50) after fractionated irradiation was found. The pHF did not correlate with rHF but was significantly associated with the TCD(50) after single dose clamp (R(2)=0.8, p=0.003) and showed a trend for an association with TCD(50) after fractionated irradiation (R(2)=0.4, p=0.067). Relative vascular area and fraction of perfused vessels did not show an association with rHF or TCD(50) after fractionated irradiation.CONCLUSIONS:Our data suggest that radiobiological hypoxia contributes to the response after fractionated irradiation but that also other radiobiological mechanisms are involved. In the present study, pimonidazole labelling does not reflect rHF and has a limited value to predict local tumour control after fractionated irradiation. The association between pHF and TCD(50) after single dose clamp warrants further investigation.