Ye, Kenny

Abstract Lung diseases, including lung cancer, are rising causes of global mortality. Despite novel imaging technologies and the development of biomarker assays, the detection of lung cancer remains a significant challenge. However, the lung communicates directly with the external environment and releases aerosolized droplets during normal tidal respiration, which can be collected, stored and analzsed as exhaled breath condensate (EBC). A few studies have suggested that EBC contains extracellular vesicles (EVs) whose microRNA (miRNA) cargos may be useful for evaluating different lung conditions, but the cellular origin of these EVs remains unknown. In this study, we used nanoparticle tracking, transmission electron microscopy, Western blot analyses and super resolution nanoimaging (ONi) to detect and validate the identity of exhaled EVs (exh‐EVs). Using our customizable antibody‐purification assay, EV‐CATCHER, we initially determined that exh‐EVs can be selectively enriched from EBC using antibodies against three tetraspanins (CD9, CD63 and CD81). Using ONi we also revealed that some exh‐EVs harbour lung‐specific proteins expressed in bronchiolar Clara cells (Clara Cell Secretory Protein [CCSP]) and Alveolar Type II cells (Surfactant protein C [SFTPC]). When conducting miRNA next generation sequencing (NGS) of airway samples collected at five different anatomic levels (i.e., mouth rinse, mouth wash, bronchial brush, bronchoalveolar lavage [BAL] and EBC) from 18 subjects, we determined that miRNA profiles of exh‐EVs clustered closely to those of BAL EVs but not to those of other airway samples. When comparing the miRNA profiles of EVs purified from matched BAL and EBC samples with our three tetraspanins EV‐CATCHER assay, we captured significant miRNA expression differences associated with smoking, asthma and lung tumor status of our subjects, which were also reproducibly detected in EVs selectively purified with our anti‐CCSP/SFTPC EV‐CATCHER assay from the same samples, but that confirmed their lung tissue origin. Our findings underscore that enriching exh‐EV subpopulations from EBC allows non‐invasive sampling of EVs produced by lung tissues.

Abstract Chronic idiopathic myelofibrosis (MF) is a clonal hematopoietic disorder that leads to progressive marrow fibrosis and peripheral cytopenias. Very little is known about the role of aberrant DNA methylation in the pathobiology of this disease. Whole genome methylation was analyzed by a recently described novel method, the HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR; Khulan et al, Genome Res. 2006 Aug;16(8)) that uses differential methylation specific restriction digestion by HpaII and MspI followed by amplification, two color labeling and cohybridization to quantitatively determine individual promoter methylation. A whole genome human promoter array (Nimblegen) was used to determine the level of methylation of 25626 gene promoters by calculating HpaII/MspI cut fragment intensity ratio. Peripheral blood leucocytes from 9 patients with IMF were compared to 9 age-matched normal and anemic controls. Gene expression analysis was performed using 37K oligo maskless arrays using cDNA from the same samples. Methylation analysis showed that myelofibrosis samples clustered separately from normal and anemic controls when grouped by unsupervised clustering based on Pearson’s correlation coefficient. On the other hand, global gene expression demonstrated no clear cut differences between myelofibrosis and control samples suggesting that methylation profiling has greater discriminatory power when compared to conventional gene expression profiling. A high correlation (r=0.88–0.96) was observed between whole genome methylation profiles of matched sets of bone marrow and peripheral blood leucocyte samples from selected patients demonstrating that peripheral blood leucocytes can act as a valid surrogates for epigenomic analysis. Further analysis showed that genes aberrantly methylated in all myelofibrosis samples included v-myc, histone 2A, TNF, TNF Receptor1, FGF14 and others. Functional pathway analysis by Ingenuity showed that pathways involved in Inflammation and Cell signaling were the most affected by epigenetic silencing. Most interestingly, a large proportion of gene promoters were also aberrantly hypomethylated. These included genes for chemokine CXCL13, APC, IL-3, STAT2 and others. The pathways most activated by hypomethylation were involved in hematopoiesis and cell growth and proliferation demonstrating the biological validity of our analysis. Thus, our data demonstrates that myelofibrosis is characterized by distinct epigenetic aberrations that are preserved in peripheral blood leucocytes. These can be the basis of future studies on pathogenesis and diagnosis for this disease and lead to translational studies with agents targeting DNA methylation.

Formalin-fixed paraffin-embedded (FFPE) specimens, when used in conjunction with patient clinical data history, represent an invaluable resource for molecular studies of cancer. Even though nucleic acids extracted from archived FFPE tissues are degraded, their molecular analysis has become possible. In this study, we optimized a laboratory-based next-generation sequencing barcoded cDNA library preparation protocol for analysis of small RNAs recovered from archived FFPE tissues. Using matched fresh and FFPE specimens, we evaluated the robustness and reproducibility of our optimized approach, as well as its applicability to archived clinical specimens stored for up to 35 years. We then evaluated this cDNA library preparation protocol by performing a miRNA expression analysis of archived breast ductal carcinoma in situ (DCIS) specimens, selected for their relation to the risk of subsequent breast cancer development and obtained from six different institutions. Our analyses identified six miRNAs (miR-29a, miR-221, miR-375, miR-184, miR-363, miR-455-5p) differentially expressed between DCIS lesions from women who subsequently developed an invasive breast cancer (cases) and women who did not develop invasive breast cancer within the same time interval (control). Our thorough evaluation and application of this laboratory-based miRNA sequencing analysis indicates that the preparation of small RNA cDNA libraries can reliably be performed on older, archived, clinically-classified specimens.