Molecular Detection Methods in HPV-Related Cancers.

Human papillomavirus (HPV) is responsible for most cervical cancers and some head and neck cancers, including oropharyngeal squamous cell carcinoma and sinonasal carcinoma. Cervical cancer is commonly diagnosed by liquid-based cytology, followed by HPV testing using commercially available DNA polymerase chain reaction (PCR), p16 immunohistochemistry (IHC), or DNA/RNA in situ hybridization. HPV in head and neck cancers is commonly diagnosed by p16 IHC or by RT-qPCR of HPV-16 E6 and E7 oncoproteins. Droplet digital PCR has been reported as an ultrasensitive and highly precise method of nucleic acid quantification for biomarker analysis and has been used to detect oncogenic HPV in oropharyngeal and cervical cancers.

Utility of droplet digital polymerase chain reaction for TERT and BRAF mutational profiling of thyroid nodules.

BACKGROUND: Mutations involving BRAF and TERT are important predictors of disease severity in thyroid cancer, but molecular testing is limited by cost and lack of adequate tissue sample. This study aimed to assess the utility of BRAFV600E and TERT testing using droplet digital PCR (ddPCR) as a diagnostic and prognostic tool for thyroid fine needle aspirate biopsy (FNAB). METHODS: Patients with thyroid nodules were prospectively enrolled from March 2015 to September 2018. Pre-operative FNAB was collected for standard cytology and molecular testing. BRAFV600E and TERT levels were analyzed by ddPCR. Cytology (Bethesda system) and ddPCR results were correlated to surgical pathology. RESULTS: A total of 222 patients were enrolled, of which 124 received thyroid surgery. Pre-operative cytology alone with Bethesda ≥5 was 100% specific and 70% sensitive for malignancy on final surgical pathology. BRAFV600E positivity or TERT overexpression was 100% specific and 60.0% sensitive. Combining cytology (Bethesda ≥5) with BRAFV600E and TERT testing increased the sensitivity of a malignant diagnosis to 80.0%. High TERT levels and/or BRAFV600E was associated with aggressive or advanced stage pathology. CONCLUSIONS: Combining cytology with ddPCR analysis of BRAFV600E and TERT can improve the diagnostic accuracy of thyroid FNAB, and help predict aggressive pathology.

Analytic and clinical validity of thyroid nodule mutational profiling using droplet digital polymerase chain reaction.

BACKGROUND: Recent guidelines for the management of thyroid nodules incorporate mutation testing as an adjunct for surgical decision-making, however current tests are costly with limited accuracy. Droplet digital PCR (ddPCR) is an ultrasensitive method of nucleic acid detection that is particularly useful for identifying gene mutations. This study aimed to assess the analytic and clinical validity of RAS and BRAF ddPCR mutational testing as a diagnostic tool for thyroid fine needle aspirate biopsy (FNAB). METHODS: Patients with thyroid nodules meeting indication for FNAB were prospectively enrolled from March 2015 to September 2017. In addition to clinical protocol, an additional FNAB was obtained for ddPCR. Optimized ddPCR probes were used to detect mutations including HRASG12 V, HRASQ61K, HRASQ61R, NRASQ61R, NRASQ61K and BRAFV600E. The diagnostic performance of BRAF and RAS mutations was assessed individually or in combination with Bethesda classification against final surgical pathology. RESULTS: A total of 208 patients underwent FNAB and mutational testing with the following Bethesda cytologic classification: 26.9% non-diagnostic, 55.2% benign, 5.3% FLUS/AUS, 2.9% FN/SPN, 2.4% SFM and 7.2% malignant. Adequate RNA was obtained from 91.3% (190) FNABs from which mutations were identified in 21.1% of HRAS, 11.5% of NRAS and 7.4% of BRAF. Malignant cytology or BRAFV600E was 100% specific for malignancy. Combining cytology with ddPCR BRAF600E mutations testing increased the sensitivity of Bethesda classification from 41.7 to 75%. Combined BRAFV600E and Bethesda results had a positive predictive value (PPV) of 100% and negative predictive value (NPV) of 89.7% for thyroid malignancy in our cohort. CONCLUSIONS: DdPCR offers a novel and ultrasensitive method of detecting RAS and BRAF mutations from thyroid FNABs. BRAFV600E mutation testing by ddPCR may serve as a useful adjunct to increase sensitivity and specificity of thyroid FNAB.

Development of Lung Epithelium from Induced Pluripotent Stem Cells.

Considerable progress has been made in the field of in vitro development of alveolar epithelium from induced pluripotent stem cells. Patient specific derived alveolar cells could potentially populate tissue engineered lungs, provide a cell source for drug testing or function as a model for research into lung diseases. Induced to pluripotency through a variety of techniques, stem cells can be differentiated to alveolar epithelium through exposure to a variety of different culture conditions and growth media. The ultimate success of differentiated cells for translational medicine applications will depend on further advances in the understanding of the human lung developmental pathway, and successful application to in vitro culture. In this review will focus the major signaling pathways and molecules in lung development and the existing protocol for directed different ion of iPSC and hESC to cells resembling respiratory epithelium in vitro.