Utilizing CD44v6 and V600EBRAF-mutation for in vitro targeted combination therapy of thyroid carcinomas.

Aim: The aim of this study was to assess the feasibility of targeted therapy of thyroid carcinoma, first exploring potential targets BRAF, EGFR and CD44v6 in patient material through immunohistochemistry and mutation analysis. Materials and methods: A patient cohort (n = 22) consisting of seven papillary (PTC), eight anaplastic (ATC) and seven follicular (FTC) thyroid carcinomas were evaluated. Additionally, eight thyroid carcinoma cells lines were analyzed for CD44v6-expression and sensitivity to the multi-kinase inhibitor sorafenib (Nexavar®), which targets numerous serine/threonine and tyrosine kinases, including the Raf family kinases. Targeted therapy using 131I-AbN44v6, a novel anti-CD44v6 antibody, and/or sorafenib was evaluated in 3D multicellular tumor spheroids. Results: Of the two cell surface proteins, EGFR and CD44v6, the latter was overexpressed in >80 % of samples, while EGFR-expression levels were moderate at best in only a few samples. BRAF mutations were more common in PTC patient samples than in ATC samples, while FTC samples did not harbor BRAF mutations. CD44v6-expression levels in the thyroid carcinoma cell lines were more heterogenous compared to patient samples, while BRAF mutational status was in line with the original tumor type. Monotherapy in 3D multicellular ATC tumor spheroids with either 131I-AbN44v6 or sorafenib resulted in delayed spheroid growth. The combination of 131I-AbN44v6 and sorafenib was the most potent and resulted in significantly impaired spheroid growth. Conclusion: This "proof of concept" targeted therapy study in the in vitro ATC 3D multicellular tumor spheroids indicated applicability of utilizing CD44v6 for molecular radiotherapy both as a monotherapy and in combination with sorafenib.

Selection, characterization and in vivo evaluation of novel CD44v6-targeting antibodies for targeted molecular radiotherapy.

Molecular radiotherapy combines the advantages of systemic administration of highly specific antibodies or peptides and the localized potency of ionizing radiation. A potential target for molecular radiotherapy is the cell surface antigen CD44v6, which is overexpressed in numerous cancers, with limited expression in normal tissues. The aim of the present study was to generate and characterize a panel of human anti-CD44v6 antibodies and identify a suitable candidate for future use in molecular radiotherapy of CD44v6-expressing cancers. Binders were first isolated from large synthetic phage display libraries containing human scFv and Fab antibody fragments. The antibodies were extensively analyzed through in vitro investigations of binding kinetics, affinity, off-target binding, and cell binding. Lead candidates were further subjected to in vivo biodistribution studies in mice bearing anaplastic thyroid cancer xenografts that express high levels of CD44v6. Additionally, antigen-dependent tumor uptake of the lead candidate was verified in additional xenograft models with varying levels of target expression. Interestingly, although only small differences were observed among the top antibody candidates in vitro, significant differences in tumor uptake and retention were uncovered in in vivo experiments. A high-affinity anti-CD44v6 lead drug candidate was identified, mAb UU-40, which exhibited favorable target binding properties and in vivo distribution. In conclusion, a panel of human anti-CD44v6 antibodies was successfully generated and characterized in this study. Through comprehensive evaluation, mAb UU-40 was identified as a promising lead candidate for future molecular radiotherapy of CD44v6-expressing cancers due to its high affinity, excellent target binding properties, and desirable in vivo distribution characteristics.

Epidemiology and prognosis in children treated for intracranial tumours in Denmark 1960-1984.

A total of 911 Danish children under 15 years of age were treated for an intracranial tumour in the 25-five year period 1960-1984. All cases were followed up to the end of 1994 or to emigration or death if one of these came sooner. The mean annual incidence was 32.5 per million children with a slight increase over the 25 years. The male/female ratio was 1.15 and close to the M/F ratio for the entire Danish population of children. Of the tumours, 46% were located in the supratentorial and 54% in the infratentorial compartment, and 94% were verified histologically. In order of frequency the most common types were astrocytomas (all grades, 35%), medulloblastomas (20%), ependymomas (14%), and craniopharyngiomas (5%). Total removal of the tumour was performed in 277 and partial removal, including biopsy, in 490 children. In 57 patients a shunt operation only was performed, and 87 children did not have an operation or died before the correct diagnosis was established. Radiotherapy was administered in 55%. The outcome depended on extent of removal, radiation, location and histology of the tumour. Most (784 or 86%) of the children survived more than 1 month after diagnosis or operation, and 353 children (39% of the whole series, 47% of those alive more than 1 month after diagnosis) were alive at follow-up. Of the survivors 29% had a tumour in the supratentorial midline, 26% one in the lateral part of the supratentorial area, 31% a cerebellar tumour and 13% a IV ventricle tumour. It was possible for 66% of the survivors with supratentorial and 90% of those with infratentorial tumours to lead a normal life. The long-term prognosis was especially good for children with cerebellar and supratentorial astrocytomas and optic chiasma tumours. Children with juvenile cerebellar astrocytoma had the best prognosis: 90% were alive at the end of the follow-up period, as against 20% of those with medulloblastoma and 6% of those with glioblastoma. A comparison of the data from the present series and from a similar Danish series of intracranial tumours in 533 children seen in the years 1935-1959 shows no significant differences in location or histology, a slight increase in annual incidence, and improved survival rates during the 50 years in question.

Phosphorous and proton spectroscopy in relation to near incarceration and incarceration of the human brain.

We report 3 cases of 31P and 1H MR spectroscopy (MRS) performed at different stages on patients with clinical signs of near or fulminant incarceration of the brain. The measurements were made on a whole body, 1.5 T scanner. 1H-MRS was obtained with the STEAM sequence and 31P-spectra were obtained using the chemical shift imaging technique. Medical treatment including controlled ventilation and sedation of the patients was carried out during the examination. The first patient was evaluated on days 6 and 10 after evacuation of an acute subdural haematoma. An intracranial pressure of 35 mm Hg was registered during the first examination. The 2nd patient had suffered a spontaneous intracerebral haemorrhage and showed clinical signs of imminent incarceration during the examination. The 3rd patient showed clinical signs of incarceration just prior to the examination. In the 1st patient 1H-MRS showed a 3-fold increase in the concentration of choline-containing compounds and a small decrease in N-acetyl aspartate from the 1st to the 2nd examination, which we interpret as a loss of neurones. In case 2 only small changes in metabolism could be detected, indicating that, despite signs of imminent clinical incarceration, the energy supply to the brain was substantial. 1H-MRS of the 3rd patient showed massive lactate concentration, and 31P-MRS revealed the total absence of high-energy phosphorous compounds leaving only one single peak of inorganic phosphate, indicating irreversible brain death.

Carcinomas following Grenz ray treatment of benign dermatoses.

During the years 1977-1986 five patients were referred to the Department of Plastic Surgery. University Hospital, Arhus, for treatment of carcinomas occurring in the skin on the sites previously treated with Grenz rays for benign dermatoses. Grenz ray treatment had been given for eight to nineteen years. The dose given, in one case, was very extensive. Otherwise a dose between 10,000 rad (100 Gy) and 29,300 rad (293 Gy) was given. The tumours were squamous cell carcinomas in four cases and basal cell carcinoma in one case. One person developed both squamous and basal cell carcinomas. The tumours behaved aggressively and in one young patient the dura and the parietal bone had to be replaced by tensor fasciae latae graft and a free latissimus dorsi flap. None of the patients had been exposed to other known carcinogens. Caution in applying Grenz ray treatment is stressed.

Transverse divergent dislocation of the elbow. A report of two cases.

Two children with transverse divergent dislocation of the elbow had quick and complete recovery following closed reduction and immobilisation.

Human corneal endothelial cell density after an in vitro imitation of elevated intraocular pressure.

Normal human cadaver corneas were mounted in test chambers 10-52 h post mortem, regenerated for 24 h in a modified tissue culture medium, and exposed to a laboratory imitation of elevated intraocular pressure. The endothelial cell density was determined before application of the pressure and after 24 h of culture without a pressure difference between the anterior and the posterior corneal surfaces. Pressure differences of 160 and 80 mmHg maintained for 8 and 72 h did not lead to decreased endothelial cell densities.