Multicentre validation of a microRNA-based assay for diagnosing indeterminate thyroid nodules utilising fine needle aspirate smears.

AIMS: The distinction between benign and malignant thyroid nodules has important therapeutic implications. Our objective was to develop an assay that could classify indeterminate thyroid nodules as benign or suspicious, using routinely prepared fine needle aspirate (FNA) cytology smears. METHODS: A training set of 375 FNA smears was used to develop the microRNA-based assay, which was validated using a blinded, multicentre, retrospective cohort of 201 smears. Final diagnosis of the validation samples was determined based on corresponding surgical specimens, reviewed by the contributing institute pathologist and two independent pathologists. Validation samples were from adult patients (≥18 years) with nodule size >0.5 cm, and a final diagnosis confirmed by at least one of the two blinded, independent pathologists. The developed assay, RosettaGX Reveal, differentiates benign from malignant thyroid nodules, using quantitative RT-PCR. RESULTS: Test performance on the 189 samples that passed quality control: negative predictive value: 91% (95% CI 84% to 96%); sensitivity: 85% (CI 74% to 93%); specificity: 72% (CI 63% to 79%). Performance for cases in which all three reviewing pathologists were in agreement regarding the final diagnosis (n=150): negative predictive value: 99% (CI 94% to 100%); sensitivity: 98% (CI 87% to 100%); specificity: 78% (CI 69% to 85%). CONCLUSIONS: A novel assay utilising microRNA expression in cytology smears was developed. The assay distinguishes benign from malignant thyroid nodules using a single FNA stained smear, and does not require fresh tissue or special collection and shipment conditions. This assay offers a valuable tool for the preoperative classification of thyroid samples with indeterminate cytology.

Analytical validity of a microRNA-based assay for diagnosing indeterminate thyroid FNA smears from routinely prepared cytology slides.

BACKGROUND: The majority of thyroid nodules are diagnosed using fine-needle aspiration (FNA) biopsies. The authors recently described the clinical validation of a molecular microRNA-based assay, RosettaGX Reveal, which can diagnose thyroid nodules as benign or suspicious using a single stained FNA smear. This paper describes the analytical validation of the assay. METHODS: More than 800 FNA slides were tested, including slides stained with Romanowsky-type and Papanicolaou stains. The assay was examined for the following features: intranodule concordance, effect of stain type, minimal acceptable RNA amounts, performance on low numbers of thyroid cells, effect of time since sampling, and analytical sensitivity, specificity, and reproducibility. RESULTS: The assay can be run on FNA slides for which as little as 1% of the cells are thyroid epithelial cells or from which only 5 ng of RNA have been extracted. Samples composed entirely of blood failed quality control and were not classified. Stain type did not affect performance. All slides were stored at room temperature. However, the length of time between FNA sampling and processing did not affect assay performance. There was a high level of concordance between laboratories (96%), and the concordance for slides created from the same FNA pass was 93%. CONCLUSIONS: The microRNA-based assay was robust to various physical processing conditions and to differing sample characteristics. Given the assay's performance, robustness, and use of routinely prepared FNA slides, it has the potential to provide valuable aid for physicians in the diagnosis of thyroid nodules. Cancer Cytopathol 2016;124:711-21. © 2016 Rosetta Genomics. Cancer Cytopathology published by Wiley Periodicals, Inc. on behalf of American Cancer Society.

In vivo administration of BL-3050: highly stable engineered PON1-HDL complexes.

BACKGROUND: Serum paraoxonase (PON1) is a high density lipoprotein (HDL)-associated enzyme involved in organophosphate (OP) degradation and prevention of atherosclerosis. PON1 comprises a potential candidate for in vivo therapeutics, as an anti-atherogenic agent, and for detoxification of pesticides and nerve agents. Because human PON1 exhibits limited stability, engineered, recombinant PON1 (rePON1) variants that were designed for higher reactivity, solubility, stability, and bacterial expression, are candidates for treatment. This work addresses the feasibility of in vivo administration of rePON1, and its HDL complex, as a potentially therapeutic agent dubbed BL-3050. METHODS: For stability studies we applied different challenges related to the in vivo disfunctionalization of HDL and PON1 and tested for inactivation of PON1's activity. We applied acute, repetitive administrations of BL-3050 in mice to assess its toxicity and adverse immune responses. The in vivo efficacy of recombinant PON1 and BL-3050 were tested with an animal model of chlorpyrifos-oxon poisoning. RESULTS: Inactivation studies show significantly improved in vitro lifespan of the engineered rePON1 relative to human PON1. Significant sequence changes relative to human PON1 might hamper the in vivo applicability of BL-3050 due to adverse immune responses. However, we observed no toxic effects in mice subjected to repetitive administration of BL-3050, suggesting that BL-3050 could be safely used. To further evaluate the activity of BL-3050 in vivo, we applied an animal model that mimics human organophosphate poisoning. In these studies, a significant advantages of rePON1 and BL-3050 (>87.5% survival versus <37.5% in the control groups) was observed. Furthermore, BL-3050 and rePON1 were superior to the conventional treatment of atropine-2-PAM as a prophylactic treatment for OP poisoning. CONCLUSION: In vitro and in vivo data described here demonstrate the potential advantages of rePON1 and BL-3050 for treatment of OP toxicity and chronic cardiovascular diseases like atherosclerosis. The in vivo data also suggest that rePON1 and BL-3050 are stable and safe, and could be used for acute, and possibly repeated treatments, with no adverse effects.

A continuous delivery system of IL-1 receptor antagonist reduces angiogenesis and inhibits tumor development.

The involvement of interleukin-1 (IL-1) in inflammation, tumor growth, and metastasis makes it an attractive target for therapeutic intervention. Here, we show that a continuous delivery of a low, but steady-state level of the naturally occurring IL-1 receptor antagonist (IL-1Ra) reduced inflammatory responses and inhibited tumor development in mice, phenomena that are induced by IL-1, mainly secretable IL-1beta. The IL-1Ra was delivered from microencapsulated genetically engineered cells, which overexpress and secrete this mediator. For a tumor model, we used fibrosarcoma cell line, which secretes high levels of IL-1beta; when injected s.c. into mice, the cells developed into large tumors characterized by very active angiogenic patterns. The proangiogenic features of IL-1beta were manifested at low levels of the cytokine, and release of 25 ng per day of the IL-1Ra was needed to oppose its effects and inhibit tumor development. The continuous delivery of the IL-1Ra contributed to improved biocompatibility of the microencapsulated cell systems; the fibrotic sac surrounding the systems was much thinner with significantly less blood capillaries and inflammatory cells. Not only do our findings point to the antiangiogenic properties of IL-1Ra in inflammation and tumor growth, but they also provide a more efficient and convenient way for treating diseases involving IL-1.