Longitudinal stability of molecular alterations and drug response profiles in tumor spheroid cell lines enables reproducible analyses.

The utility of patient-derived tumor cell lines as experimental models for glioblastoma has been challenged by limited representation of the in vivo tumor biology and low clinical translatability. Here, we report on longitudinal epigenetic and transcriptional profiling of seven glioblastoma spheroid cell line models cultured over an extended period. Molecular profiles were associated with drug response data obtained for 231 clinically used drugs. We show that the glioblastoma spheroid models remained molecularly stable and displayed reproducible drug responses over prolonged culture times of 30 in vitro passages. Integration of gene expression and drug response data identified predictive gene signatures linked to sensitivity to specific drugs, indicating the potential of gene expression-based prediction of glioblastoma therapy response. Our data thus empowers glioblastoma spheroid disease modeling as a useful preclinical assay that may uncover novel therapeutic vulnerabilities and associated molecular alterations.

MiR-16-5p is frequently down-regulated in astrocytic gliomas and modulates glioma cell proliferation, apoptosis and response to cytotoxic therapy.

AIMS: Aberrant expression of microRNAs (miRNAs) is frequent in various cancers including gliomas. We aimed to characterize the role of miR-16-5p as a candidate tumour suppressor miRNA in gliomas. METHODS: Real-time PCR-based approaches were used for miRNA and mRNA expression profiling of glioma and non-neoplastic brain tissues as well as glioma cell lines. Protein levels were determined by Western blotting. In vitro analyses were performed following overexpression of miR-16-5p, trichostatin A (TSA) treatment, and siRNA-mediated knock-down of HDAC3 in glioma cells. Effects of miR-16-5p on glioma cell viability, apoptosis and response to irradiation and temozolomide (TMZ) were assessed. RESULTS: Expression of miR-16-5p was reduced relative to control brain tissue in isocitrate dehydrogenase (IDH)-mutant astrocytomas of World Health Organization (WHO) grades II, III and IV, and a subset of IDH-wildtype glioblastomas WHO grade IV. MiR-16-5p expression was lower in IDH-mutant than in IDH-wildtype gliomas, and down-regulated in IDH-wildtype glioma lines. MiR-16-5p overexpression reduced expression of important cell cycle and apoptosis regulators in glioma cells, including CDK6, CDC25A, CCND3, CCNE1, WEE1, CHEK1, BCL2 and MCL1. In line, CDK6, WEE1, CHEK1, BCL2 and MCL1 transcript levels were increased in WHO grade III or IV gliomas. TSA treatment and HDAC3 knockdown in glioma cells induced miR-16-5p up-regulation and reduced expression of its targets. Moreover, miR-16-5p overexpression inhibited proliferation and induced apoptosis in various glioma cell lines and increased sensitivity of A172 glioma cells to irradiation and TMZ. CONCLUSION: Reduced expression of miR-16-5p contributes to glioma cell proliferation, survival and resistance to cytotoxic therapy.

Low incidence of heparin-induced skin lesions in orthopedic surgery patients with low-molecular-weight heparins.

BACKGROUND: Heparins are widely prescribed for prevention and therapy of arterial and venous thromboembolic diseases. Heparin-induced skin lesions are the most frequent adverse effect of subcutaneous heparin treatment in non-surgical patients (7.5%-39.8%); no data exist on surgical patients. Commonly, they are due to a delayed-type hypersensitivity reaction (DTH), but may also be a manifestation of life-threatening heparin-induced thrombocytopenia (HIT). Lesions of both entities resemble initially. The risk of HIT is highest among heparin-anticoagulated orthopedic surgery patients. OBJECTIVE: To determine incidence and causes of heparin-induced skin lesions in major orthopedic surgery patients. METHODS: In a prospective cohort study, consecutive patients with subcutaneous low-molecular-weight heparin (LMWH) treatment were examined for cutaneous adverse effects. Further diagnostics (skin biopsy, clinical/laboratory assessment for thrombosis, bleeding, HIT, cross-allergies) were performed. RESULTS: Six of 316 enrolled patients (1.9%; 95% CI: 0.4%-3.4%) developed heparin-induced skin lesions. All were caused by a DTH reaction, and none was due to HIT or other rare heparin-associated skin diseases. Therapeutic use (dosage) of LMWH was identified as only risk factor (odds ratio: 3.1, 95% CI: 1.4-4.9; P = .00141). In addition to DTH, 5 thromboembolic, 4 major bleeding complications but no cases of HIT or cross-allergies were observed. CONCLUSIONS AND CLINICAL RELEVANCE: Orthopedic surgery patients have-unlike non-surgical patients-a low risk for heparin-induced skin lesions during LMWH treatment; all lesions were due to a DTH reaction. The risk for DTH differs considerably between individual patient cohorts. No association with HIT was observed. These data help to tailor anticoagulatory treatment individually and to increase patient safety.

Unusual acute and delayed skin reactions during and after whole-brain radiotherapy in combination with the BRAF inhibitor vemurafenib. Two case reports.

BACKGROUND: Besides radiotherapy (RT) and surgery, the introduction of BRAF inhibitors like vemurafenib has provided new opportunities for treatment of patients with metastasized malignant melanomas. RT and vemurafenib are being increasingly used concurrently, although little is known about the potential side effects of this combination. Vemurafenib is known to cause severe cutaneous skin reactions such as phototoxicity and evidence is accumulating that RT may further enhance these side effects. PATIENTS AND METHODS: We report two cases of unusual skin reactions occurring during and after treatment with a combination of vemurafenib and whole-brain irradiation in patients with cerebral metastases arising from malignant melanomas. RESULTS: One case report describes excessive acute radiodermatitis which arose during whole-brain irradiation in combination with vemurafenib. The second describes a late skin reaction occurring approximately 30 days after completion of RT. CONCLUSION: These two case reports show that combination of both treatment modalities is possible, but requires close monitoring of patients and good interdisciplinary collaboration.

[Bitemporal scalp necrosis : a very rare manifestation of giant cell arteritis]. / Bitemporale Skalpnekrose : Eine sehr seltene Manifestation der Riesenzellarteriitis.

A 71-year-old woman developed progressive spreading of bitemporal scalp necrosis within 4 weeks accompanied by headaches, myalgia of the shoulder girdle and muscle weakness that had started a few months previously. No additional diseases were reported. The suspected temporal giant cell arteritis could be confirmed by temporal artery biopsy. Therapy with glucocorticoids led to a rapid resolution of clinical symptoms and was tapered over 18 months. Recovery of the scalp necrosis emerged following second intention healing and split-skin transplantation of necrotic areas after successful wound conditioning. The case study demonstrates a rare and serious complication of temporal arteritis which is often accompanied by a poor prognosis.

Downregulation of PRDX1 by promoter hypermethylation is frequent in 1p/19q-deleted oligodendroglial tumours and increases radio- and chemosensitivity of Hs683 glioma cells in vitro.

Deletions of chromosomal arms 1p and 19q are frequent in oligodendroglial tumours and linked to radio- and chemotherapy response as well as longer survival. The molecular mechanisms underlying this clinically important association are as yet unknown. Here, we studied the peroxiredoxin 1 (PRDX1) gene at 1p34.1 for promoter methylation and expression in primary gliomas and investigated its role in radio- and chemosensitivity of glioma cells in vitro. In total, we screened primary glioma tissues from 93 patients for methylation of the 5'-CpG island of PRDX1 by sodium bisulfite sequencing. PRDX1 mRNA and protein expression levels were determined in subsets of the tumours by quantitative PCR and western blot analysis, respectively. PRDX1 hypermethylation and reduced expression were frequently detected in oligodendroglial tumours and secondary glioblastomas, but not in primary glioblastomas. In oligodendroglial tumours, both PRDX1 hypermethylation and reduced mRNA expression were significantly associated with 1p/19q-deletion. Stable knockdown of PRDX1 by lentiviral transduction of short-hairpin (sh)RNA constructs significantly increased apoptosis and reduced cell viability of Hs683 glioma cells exposed to ionizing irradiation or temozolomide in vitro. Taken together, our findings indicate that epigenetic silencing of PRDX1 is frequent in 1p/19q-deleted oligodendroglial tumours and likely contributes to radio- and chemosensitivity of these tumours.

De-repression of CTGF via the miR-17-92 cluster upon differentiation of human glioblastoma spheroid cultures.

All-trans retinoic acid is a potent promoter of cellular differentiation processes, which is used in cancer therapy. Glioblastoma spheroid cultures are enriched in tumor-initiating cells, and provide a model to test new treatment options in vitro. We investigated the molecular mechanisms of response to exposure to differentiation-promoting conditions in such cultures. Microarray analyses of five independent cultures showed that after induction of differentiation, inhibitors of transforming growth factor-beta/bone morphogenetic protein, Wnt/beta-catenin and IGF signaling were upregulated, whereas expression of several microRNAs decreased, particularly that of the miR-17-92 cluster. In primary astrocytic gliomas (n=82), expression of several members of miR-17-92 was significantly higher relative to those of normal brain (n=8) and significantly increased with tumor grade progression (P<0.05). A high-level amplification of the miR-17-92 locus was detected in one glioblastoma specimen. Transfection of inhibitors of miR-17-92 induced increased apoptosis and decreased cell proliferation in glioblastoma spheroids. Mir-17-92 inhibition was also associated with increased messenger RNA (mRNA) and/or protein expression of CDKN1A, E2F1, PTEN and CTGF. The CTGF gene was shown to be a target of miR-17-92 in glioblastoma spheroids by luciferase reporter assays. Our results suggest that miR-17-92 and its target CTGF mediate effects of differentiation-promoting treatment on glioblastoma cells through multiple regulatory pathways.

Dermatofibrosarcoma protuberans: wide local excision vs. Mohs micrographic surgery.

BACKGROUND: Dermatofibrosarcoma protuberans (DFSP) is an uncommon tumor of the skin with high rates of local recurrence. It is debated whether Mohs micrographic surgery (MMS) involves lower recurrence rates than wide local excision (WLE). Recent preliminary reports indicate more consistently favorable cure rates with MMS. We report comparative observational data on 41 patients who underwent MMS and 38 who underwent WLE. Their data were then pooled with those available in the medical literature to obtain more precise estimates of recurrence rates with MMS and WLE. METHODS: The records of 79 patients with DFSP who underwent WLE (n=38) or MMS (n=41) in 1990-2005 were reviewed retrospectively. The primary endpoint was tumor recurrence rate. The PubMed database was searched for DFSP case series treated with WLE or MMS, and the recurrence proportions reported for the two separate procedures were pooled. RESULTS: Five of the 38 WLE patients (follow-up=4.8 years) had recurrences (13.2%, 95% CI 4.4-28.1%) as opposed to none (95% CI 0-8.6%) of the 41 MMS patients (follow-up=5.4 years). Pooling of these data with those from the literature yielded 6/463 recurrences for MMS (1.3%, 95% CI 0.5-2.8%) and 288/1394 recurrences for WLE (20.7%, 95% CI 18.6-22.9%). The relative risk of recurrence for WLE vs. MMS patients was 15.9 (95% CI 7.2-35.5). CONCLUSIONS: Significantly lower recurrence rates were recorded in our patients subjected to MMS compared with those treated with WLE. The pooled data also indicated a clear advantage of MMS. There is inconclusive evidence for any advantage of MMS in non-primary cases, while MMS was most effective in treating head and neck tumors. These data may be useful to guide clinicians in the choice of the more appropriate surgical treatment for DFSP patients.

[Postoperative pyoderma gangrenosum of Cullen]. / Postoperatives Pyoderma gangraenosum Cullen.

The clinical picture in pyoderma gangrenosum varies but a typical medical history with resistance to antimicrobial treatment and worsening or first manifestation of disease because of surgical procedures are indications of this diagnosis. We describe the course of a woman patient who had a pyoderma gangrenosum for more than 1.5 years. After confirming the diagnosis an immunomodulating therapy was initiated until complete remission of the ulcers. Differential diagnosis and different clinicopathologic forms of pyoderma gangrenosum are discussed and an overview of the association with internal diseases is provided.

Sodium thiosulphate as a promising therapeutic option to treat calciphylaxis.

A 35-year-old haemodialysis-dependent woman with chronic renal failure developed large, very painful necrotic ulcers and necrosis on the thighs, buttocks and the abdomen with signs of fast progression. The skin biopsy specimens showed a broad necrosis of the epidermis and thrombosed dermal vessels with focal calcium deposits within the wall. In addition, laboratory findings presented an increased product of serum calcium and phosphate concentrations. Thus, we diagnosed calciphylaxis on the basis of clinical, biochemical and histopathological criteria. We initiated a therapy in which our patient was treated with intravenous sodium thiosulphate 3 times weekly. Already after 2 weeks of treatment, no new lesions were detectable and there was a dramatic pain relief. In the following 4 weeks, a successive decline of the ulcers and the healing of individual tissue defects could be seen. Four months after the start of the therapy, the patient underwent successful renal transplantation. Thus, the intravenous therapy of calciphylaxis with sodium thiosulphate might be a new effective alternative in the treatment of this life-threatening disease.

Comprehensive genomic analysis of desmoplastic medulloblastomas: identification of novel amplified genes and separate evaluation of the different histological components.

Desmoplastic medulloblastoma (DMB) is a malignant cerebellar tumour composed of two distinct tissue components, pale islands and desmoplastic areas. Previous studies revealed mutations in genes encoding members of the sonic hedgehog pathway, including PTCH, SMOH and SUFUH in DMBs. However, little is known about other genomic aberrations. We performed comparative genomic hybridization (CGH) analysis of 22 sporadic DMBs and identified chromosomal imbalances in 20 tumours (91%; mean, 4.9 imbalances/tumour). Recurrent chromosomal gains were found on chromosomes 3, 9 (six tumours each), 20, 22 (five tumours each), 2, 6, 7, 17 (four tumours each) and 1 (three tumours). Recurrent losses involved chromosomes X (eight tumours), Y (six of eleven tumours from male patients), 9, 12 (four tumours each), as well as 10, 13 and 17 (three tumours each). Four tumours demonstrated high-level amplifications involving sequences from 1p22, 5p15, 9p, 12p13, 13q33-q34 and 17q22-q24, respectively. Further analysis of the 9p and 17q22-q24 amplicons by array-based CGH (matrix-CGH) and candidate gene analyses revealed amplification of JMJD2C at 9p24 in one DMB and amplification of RPS6KB1, APPBP2, PPM1D and BCAS3 from 17q23 in three DMBs. Among the 17q23 genes, RPS6KB1 showed markedly elevated transcript levels as compared to normal cerebellum in five of six DMBs and four of five classic medulloblastomas investigated. Finally, CGH analysis of microdissected pale islands and desmoplastic areas showed common chromosomal imbalances in five of six informative tumours. In summary, we have identified several novel genetic alterations in DMBs and provide genetic evidence for a monoclonal origin of their different tissue components.

[Herpetic neonatal hepatitis]. / Les hépatites herpétiques néonatales.

Herpes simplex virus (HSV) infection can affect various organs-systems in the neonatal period. Herpetic hepatitis was seldom reported in the literature. We report on 2 cases. Firstly, a 16 day-old newborn infant was admitted because of haemorrhagic syndrome and shock. Biological assessment showed a severe hepatic insufficiency. Antibiotic and aciclovir therapy was started as HSV infection was suspected. Five days later, the herpetic attack was confirmed by polymerase chain reaction (PCR) in blood and cerebrospinal fluid (CSF). The genotye of the virus in the CSF was HSV1. Treatment included aciclovir for 21 days intravenously and 2 months orally. At 10 months, the clinical and biological examinations were normal. Secondly, a 4 day-old newborn was hospitalised because of fever and polypnea. Pulmonary X rays showed heterogeneous opacities of the right base. Serum C reactive protein was 30 mg/l. Antibiotic therapy was started. Two days later, the fever persisted while a severe hepatic insufficiency developed. The diagnosis of herpetic hepatitis was evoked and the child was given aciclovir. Forty-eight hours later, the PCR confirmed a HSV in blood, while viral culture of a mouth swab found HSV 2. Evolution was favourable after 21 days of specific and symptomatic treatment. Aciclovir treatment was continued orally for six months. Herpetic hepatitis is rare in the neonatal period. Diagnosis must be evoked early when facing severe neonatal hepatic insufficiency. Provided specific treatment, prognosis is good.

[Silica-containing urinary stones--clinical issues to keep in mind]. / Silikathaltige Harnsteine im Klinikalltag. Was gibt es zu beachten?

Formation of calculi in efferent urinary passages is always due to supersaturation of urinary calculi substances and associated increased crystallization. Apart from the typical calculi, consisting of calcium oxalate, inorganic phosphates, uric acid or cystine, there are occasional signs of rare substance classes. Although more than 50 silicate stones have already been reported internationally, this stone entity remains relatively unknown. In particular, the occurrence of silicate stones in the absence of magnesium trisilicate abuse is extremely rare. A medium-sized left-sided ureterolith was removed from a 54-year-old male patient using a ureteroscope. X-ray diffraction showed it to be a compound stone consisting of 40% silicate. The patient, who in 1986 was living close to the nuclear reactor accident in Chernobyl, showed no signs of a constant uptake of magnesium trisilicate. However, he had undergone partial (2/3) gastrectomy 4 months before for a drug-refractory gastric ulcer, which had been diagnosed at the end of the 1980s and treated with excessive dosages of a magnesium trisilicate antacid preparation until the time of the operation. The patient had also been suffering from unstable angina pectoris since 1986 and treated with Pentalong (pentaerythrityltetranitrate) for 17 years. We were also able to detect silicium dioxide in components of this drug using X-ray diffraction. Silicate uroliths are extremely rare but they can be clearly identified by X-ray diffraction or infrared spectroscopy and distinguished from artifacts or quartz pebbles. Formation of calculi can be prevented by increasing diuresis as well as switching to a different drug and reducing the dosage.

Somatic mutations in the PTCH, SMOH, SUFUH and TP53 genes in sporadic basal cell carcinomas.

BACKGROUND: Basal cell carcinoma (BCC) of the skin is the most common human cancer. The genetic alterations underlying BCC development are only partly understood. OBJECTIVES: To investigate further the molecular genetics of sporadic BCCs, we performed mutation analyses of 10 skin cancer-associated genes in 42 tumours. METHODS: Single-strand conformational polymorphism analysis followed by DNA sequencing was used to screen for mutations in the sonic hedgehog pathway genes PTCH, SMOH, SUFUH and GLI1, in the TP53 tumour suppressor gene, and in the proto-oncogenes NRAS, KRAS, HRAS, BRAF and CTNNB1. Microsatellite markers flanking the PTCH, SUFUH and TP53 loci at 9q22, 10q24 and 17p13, respectively, were studied for loss of heterozygosity (LOH). RESULTS: PTCH mutations were found in 28 of 42 tumours (67%). Microsatellite analysis revealed LOH on 9q22 in 20 of 38 tumours investigated (53%), including 14 tumours with and six tumours without PTCH mutations. SMOH mutations were identified in four of the 42 BCCs (10%) while two tumours demonstrated mutations in SUFUH, including one missense mutation and one silent mutation. None of the BCCs showed LOH at markers flanking the SUFUH locus. Seventeen BCCs (40%) carried TP53 mutations, with only three tumours showing evidence of biallelic TP53 inactivation. TP53 mutations were present in BCCs with and without mutations in PTCH, SMOH or SUFUH. Interestingly, 72% of the TP53 alterations were presumably ultraviolet (UV)-induced transition mutations. In contrast, only 40% of the PTCH and SMOH alterations corresponded to UV signature mutations. No mutations were identified in GLI1, NRAS, KRAS, HRAS, BRAF or CTNNB1. CONCLUSIONS: Our data confirm the importance of PTCH, SMOH and TP53 mutations in the pathogenesis of sporadic BCCs. SUFUH alterations are restricted to individual cases while the other investigated genes do not appear to be important targets for mutations in BCCs.