Immunohistochemical double nuclear staining for cell-specific automated quantification of the proliferation index - A promising diagnostic aid for melanocytic lesions

AIMS: Pathologists often use immunohistochemical staining of the proliferation marker Ki67 in their diagnostic assessment of melanocytic lesions. However, the interpretation of Ki67 can be challenging. We propose a new workflow to improve the diagnostic utility of the Ki67-index. In this workflow, Ki67 is combined with the melanocytic tumour-cell marker SOX10 in a Ki67/SOX10 double nuclear stain. The Ki67-index is then quantified automatically using digital image analysis (DIA). The aim of this study was to optimise and test three different multiplexing methods for Ki67/SOX10 double nuclear staining.METHODS: Multiplex immunofluorescence (mIF), multiplex immunohistochemistry (mIHC), and multiplexed immunohistochemical consecutive staining on single slide (MICSSS) were optimised for Ki67/SOX10 double nuclear staining. DIA applications were designed for automated quantification of the Ki67-index. The methods were tested on a pilot case-control cohort of benign and malignant melanocytic lesions (n = 23).RESULTS: Using the Ki67/SOX10 double nuclear stain, malignant melanocytic lesions could be completely distinguished from benign lesions by the Ki67-index. The Ki67-index cut-offs were 1.8% (mIF) and 1.5% (mIHC and MICSSS). The AUC of the automatically quantified Ki67-index based on double nuclear staining was 1.0 (95% CI: 1.0;1.0), whereas the AUC of conventional Ki67 single-stains was 0.87 (95% CI: 0.71;1.00).CONCLUSIONS: The novel Ki67/SOX10 double nuclear stain highly improved the diagnostic precision of Ki67 interpretation. Both mIHC and mIF were useful methods for Ki67/SOX10 double nuclear staining, whereas the MICSSS method had challenges in the current setting. The Ki67/SOX10 double nuclear stain shows potential as a valuable diagnostic aid for melanocytic lesions.</p

TGF-beta 1 and TNF-alpha differentially regulate Twist1 mediated resistance towards BRAF/MEK inhibition in melanoma

Summary: Resistance to BRAF and MEK inhibition is a common phenomenon in melanoma. Cytokines and transcription factors have been attributed to contribute to the loss of sensitivity towards these inhibitors. Here, we show that transforming growth factor (TGF)-β1 if combined with PLX4032, a BRAF inhibitor, or GSK1120212, a MEK inhibitor, substantially increased cell death in BRAF-mutant melanoma cell lines. This increase was based on the combined regulatory decrease in Twist1, an antiapoptotic protein. Overexpression or silencing of Twist1 attenuated or aggravated induction of apoptosis through PLX4032 or GSK1120212, respectively. Exposure to tumour necrosis factor (TNF)-α, however, led to increased Twist1 levels and oppositional decrease in cell death if exposed to PLX4032 or GSK1120212. This increase in drug resistance again depended on Twist1 levels. Our studies suggest that Twist1 as a common downstream target of multiple signalling cascades plays a crucial role in mediating drug resistance to BRAF- and MEK-targeted molecular inhibitors

Pulmonary cystic echinococcosis acquired during a short-term tourist travel

Background: Cystic echinococcosis is non-endemic in Denmark and primarily diagnosed in migrants from endemic areas. Here, we report a case of pulmonary cystic echinococcosis in a Danish woman with no history of longer-term stays abroad, only holiday travelling to tourist destinations. This is the first case reported in international literature from Denmark where the causative parasite was identified to species and genotype level. Case: A 27-year-old pregnant Danish woman was admitted for examination because of haemoptysis for three months.Chest X-ray and computed tomography revealed a cystic structure in the left lung and a left-sided thoracotomy was performed to remove the cyst. Postoperative histopathological examination revealed a hyaline membrane and protoscoleces. Subsequently, infection with Echinococcus granulosus was confirmed by molecular methods. The causative agent was further characterised as E. granulosus sensu stricto G1, which is not known to have an established life cycle in Denmark. It was concluded that the infection was most likely acquired during a tourist travel to an endemic country. The patient was treated with albendazole for four weeks. Conclusion: This case of pulmonary cystic echinococcosis in a person who had lived in Denmark and had history of only short-term tourist travelling abroad highlights that the disease may be acquired during tourist travelling. Thus, a diagnosis of cystic echinococcosis should be considered not only in migrants from endemic countries but also in travellers upon incidental findings of a lung or liver cysts. The case also exemplifies the importance of reaching a diagnosis at species and genotype level