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.

Quantification of microRNA-21 and microRNA-125b in melanoma tissue.

Although microRNAs (miRNAs) have emerged as potent mediators of melanoma development and progression, a precise understanding of their oncogenic role remains unclear. In this study, we analysed formalin-fixed and paraffin-embedded tissues from two separate melanoma cohorts and from a series of benign melanocytic nevi. Using three different quantification methods [array analysis, quantitative PCR (qPCR) and in-situ hybridization (ISH) quantified by digital image analysis], we found considerable miRNA dysregulation in tumours. Using array analysis, samples mainly clustered according to their biological group (benign vs. malignant) and 77 miRNAs differed significantly between nevi and melanoma samples. Increase of miR-21 and miR-142, and decrease of miR-125b, miR-211, miR-101 and miR-513c in the melanomas were verified in both cohorts using qPCR, whereas the decrease of miR-205 observed with array analysis could not be confirmed using qPCR. ISH with digital quantification showed expression of miR-21 and miR-125b in the melanocytic lesions. miR-21 ISH was increased in melanomas, whereas quantification of miR-125b showed uniform ISH expression across nevi and melanomas. Our results support the important involvement of different miRNAs in melanoma biology and may serve as solid basics for further miRNA investigations in melanoma formalin-fixed and paraffin-embedded tissue. In particular, there is increased expression of miR-21 in melanomas compared with benign nevi.

Automated quantification of Ki67/MART1 stains may prevent false-negative melanoma diagnoses.

BACKGROUND: Inability to distinguish melanomas from benign nevi is the most frequent reason for malpractice lawsuits in surgical pathology. Reliable diagnostic tools to support hematoxylin and eosin (H&E) stains and induce diagnostic vigilance are thus highly needed. Because high diagnostic performance recently was showed using automated image analysis, the immunohistochemical proliferation marker Ki67 seems a potential candidate. This study aimed to investigate if this previously presented automated algorithm could have prevented 10 false-negative melanoma diagnoses. In addition, diagnostic utility of another, but narrower, immunohistochemical proliferation marker, phosphohistone H3 (PHH3), was explored. METHODS: A total of 10 formalin-fixed paraffin-embedded melanocytic tumors, initially classified as benign or dysplastic but revised as melanomas at metastatic debut, were dual-stained for Ki67/MART1 and PHH3/MART1. A Ki67 index was automatically calculated in epidermis, dermis, a combination of such, and a dermal hot spot. Dermal PHH3/MART1 scores were established semi-automatically. RESULTS: The dermal Ki67 index identified all 10 melanomas, the hot-spot index 8 and the epidermal and combined indices only 2 and 5, respectively. Nine melanomas were PHH3 positive and scores correlated with Ki67. CONCLUSIONS: PHH3 added limited information, but supplemental automated Ki67 assessment could possibly have prevented the misdiagnosis of most melanomas had the algorithm been available at the time of diagnosis.

Fatal haemolytic crisis with microvascular pulmonary obstruction mimicking a pulmonary embolism in a young African man with glucose-6-phosphate dehydrogenase deficiency.

We report a fatal case of haemolytic crisis mimicking a pulmonary embolism in a previously healthy 42-year-old African man. The patient was admitted to hospital with fatigue, shortness of breath and jaundice lasting for 2 days. Laboratory tests were consistent with haemolysis and inflammation. The patient was treated as having a mycoplasma pneumonia. His condition deteriorated rapidly, with respiratory distress and circulatory failure. Echocardiography showed pulmonary hypertension and right heart dilation. Despite the fact that he was given fibrinolysis for suspected pulmonary embolism, he developed cardiac arrest and died after a long-lasting resuscitation attempt. Postmortem examinations revealed that the patient had a glucose-6-phosphate dehydrogenase deficiency and disseminated intravascular coagulation with pulmonary microthrombi. To the best of our knowledge, this is the first case of death caused by right heart failure due to microvascular obstruction resulting from multiple microvascular thrombosis in a patient with acute haemolysis due to glucose-6-phosphate dehydrogenase deficiency.

Ethanol exerts anti-inflammatory effects in human adipose tissue in vitro.

Moderate alcohol consumption is suggested to be associated with reduced inflammation and morbidity. Human adipose tissue (AT) and obesity is characterised by low-grade inflammation, so the present study wanted to investigate the effects of ethanol on inflammation in human AT in vitro. Subcutaneous human AT was incubated with ethanol [11-88 mM] under non- or LPS-stimulated [50mg/mL] conditions. Protein and mRNA levels of adiponectin, IL-6, IL-8, TNF-alpha, MCP-1, and CD68 were assessed using ELISA and real-time RT-PCR, respectively. Non-stimulated, ethanol incubations up to 24h increased adiponectin release and mRNA expression (p<0.01) and decreased IL-6 release in both short-term [1.5h] (p<0.05) and long-term [24h] (p<0.01) incubations. Ethanol decreased LPS-stimulated IL-6, IL-8, TNF-alpha, and MCP-1 dose-dependently (all p<0.01). Ethanol decreased CD68 mRNA (p<0.001), which correlated with the investigated adipokines (p<0.05) but not adiponectin (p>0.05). In conclusion, ethanol exerts anti-inflammatory effects in human AT, suggesting that ethanol may attenuate whole-body inflammation.