Eric Kannisto

Evaluation of MicroRNA Expression Profiles That May Predict Recurrence of Localized Stage I Non–Small Cell Lung Cancer after Surgical Resection

Prognostic markers that can predict the relapse of localized non-small cell lung cancer (NSCLC) have yet to be defined. We surveyed expression profiles of microRNA (miRNA) in stage I NSCLC to identify patterns that might predict recurrence after surgical resection of this common deadly cancer. Small RNAs extracted from formalin-fixed and paraffin-embedded tissues were hybridized to locked nucleic acid probes against 752 human miRNAs (representing 82% of the miRNAs in the miRBase 13.0 database) to obtain expression profiles for 37 cases with recurrence and 40 cases without recurrence (with clinical follow-up for at least 32 months). Differential expression between the two case groups was detected for 49% of the miRNAs (Wilcoxon rank sum test; P<0.01). The performance of expression profiles at differentiating the two case groups was assessed by leave-one-out and Monte Carlo cross-validations. In leave-one-out cross-validation using support vector machines- or top-scoring gene pair classifier methods, which looked for six- or two-miRNA-based classifiers, the identified miRNA expression pattern predicted recurrence with an accuracy of 70% and 83%, and hazard ratio of 3.6 [95% confidence interval (95% CI), 1.8-7.1] and 9.0 (95% CI, 4.4-18.2), respectively. Mean accuracy in Monte Carlo cross-validation using 1,000 random 60-17 splits was 69% (95% CI, 68-70) and 72% (95% CI, 71-72), respectively. The specific miRNAs mir-200b*, mir-30c-1*, mir-510, mir-630, mir-657, and mir-146b-3p and mir-124*, mir-585, and mir-708, respectively, represented most commonly among the 1,000 classifiers identified in Monte Carlo cross-validation by the two methods. MiRNAs mir-488, mir-503, and mir-647 were identified as potential reference miRNAs for future studies, based on the stability of their expression patterns across the 77 cases and the two case-groups. Our findings reinforce efforts to profile miRNA expression patterns for better prognostication of stage I NSCLC.

APOBEC3A cytidine deaminase induces RNA editing in monocytes and macrophages

The extent, regulation and enzymatic basis of RNA editing by cytidine deamination are incompletely understood. Here we show that transcripts of hundreds of genes undergo site-specific C>U RNA editing in macrophages during M1 polarization and in monocytes in response to hypoxia and interferons. This editing alters the amino acid sequences for scores of proteins, including many that are involved in pathogenesis of viral diseases. APOBEC3A, which is known to deaminate cytidines of single-stranded DNA and to inhibit viruses and retrotransposons, mediates this RNA editing. Amino acid residues of APOBEC3A that are known to be required for its DNA deamination and anti-retrotransposition activities were also found to affect its RNA deamination activity. Our study demonstrates the cellular RNA editing activity of a member of the APOBEC3 family of innate restriction factors and expands the understanding of C>U RNA editing in mammals.

MicroRNA Expression Profiles of Whole Blood in Lung Adenocarcinoma

The association of lung cancer with changes in microRNAs in plasma shown in multiple studies suggests a utility for circulating microRNA biomarkers in non-invasive detection of the disease. We examined if presence of lung cancer is reflected in whole blood microRNA expression as well, possibly because of a systemic response. Locked nucleic acid microarrays were used to quantify the global expression of microRNAs in whole blood of 22 patients with lung adenocarcinoma and 23 controls, ten of whom had a radiographically detected non-cancerous lung nodule and the other 13 were at high risk for developing lung cancer because of a smoking history of >20 pack-years. Cases and controls differed significantly for age with a mean difference of 10.7 years, but not for gender, race, smoking history, blood hemoglobin, platelet count, or white blood cell count. Of 1282 quantified human microRNAs, 395 (31%) were identified as expressed in the study’s subjects, with 96 (24%) differentially expressed between cases and controls. Classification analyses of microRNA expression data were performed using linear kernel support vector machines (SVM) and top-scoring pairs (TSP) methods, and classifiers to identify presence of lung adenocarcinoma were internally cross-validated. In leave-one-out cross-validation, the TSP classifiers had sensitivity and specificity of 91% and 100%, respectively. The values with SVM were both 91%. In a Monte Carlo cross-validation, average sensitivity and specificity values were 86% and 97%, respectively, with TSP, and 88% and 89%, respectively, with SVM. MicroRNAs miR-190b, miR-630, miR-942, and miR-1284 were the most frequent constituents of the classifiers generated during the analyses. These results suggest that whole blood microRNA expression profiles can be used to distinguish lung cancer cases from clinically relevant controls. Further studies are needed to validate this observation, including in non-adenocarcinomatous lung cancers, and to clarify upon the confounding effect of age.

Overexpression of the lung cancer-prognostic miR-146b microRNAs has a minimal and negative effect on the malignant phenotype of A549 lung cancer cells.

Introduction Expression levels of miR-146b-5p and -3p microRNAs in human non-small cell lung cancer (NSCLC) are associated with recurrence of the disease after surgery. To understand this, the effect of miR-146b overexpression was studied in A549 human lung cancer cells. Methods A549 cells, engineered with lentiviruses to overexpress the human pre-miR-146b precursor microRNA, were examined for proliferation, colony formation on plastic surface and in soft agar, migration and invasiveness in cell culture and in vivo in mice, chemosensitivity to cisplatin and doxorubicin, and global gene expression. miR-146b expressions were assessed in microdissected stroma and epithelia of human NSCLC tumors. Association of miR-146b-5p and -3p expression in early stage NSCLC with recurrence was analyzed. Principal Findings A549 pre-miR-146b-overexpressors had 3–8-fold higher levels of both miR-146b microRNAs than control cells. Overexpression did not alter cellular proliferation, chemosensitivity, migration, or invasiveness; affected only 0.3% of the mRNA transcriptome; and, reduced the ability to form colonies in vitro by 25%. In human NSCLC tumors, expression of both miR-146b microRNAs was 7–10-fold higher in stroma than in cancerous epithelia, and higher miR-146b-5p but lower -3p levels were predictive of recurrence. Conclusions Only a minimal effect of pre-miR-146b overexpression on the malignant phenotype was seen in A549 cells. This could be because of opposing effects of miR-146b-5p and -3p overexpression as suggested by the conflicting recurrence-predictive values of the two microRNAs, or because miR-146b expression changes in non-cancerous stroma and not cancerous epithelia of tumors are responsible for the prognostic value of miR-146b.

Overexpression of MicroRNA miR-30a or miR-191 in A549 Lung Cancer or BEAS-2B Normal Lung Cell Lines Does Not Alter Phenotype

Background MicroRNAs (miRNAs) are small, noncoding RNAs (ribonucleic acids) that regulate translation. Several miRNAs have been shown to be altered in whole cancer tissue compared to normal tissue when quantified by microarray. Based on previous such evidence of differential expression, we chose to study the functional significance of miRNAs miR-30a and -191 alterations in human lung cancer. Methodology/Principal Findings The functional significance of miRNAs miR-30a and -191 was studied by creating stable transfectants of the lung adenocarcinoma cell line A549 and the immortalized bronchial epithelial cell line BEAS-2B with modest overexpression of miR-30a or -191 using a lentiviral system. When compared to the corresponding controls, both cell lines overexpressing miR-30a or -191 do not demonstrate any significant changes in cell cycle distribution, cell proliferation, adherent colony formation, soft agar colony formation, xenograft formation in a subcutaneous SCID mouse model, and drug sensitivity to doxorubicin and cisplatin. There is a modest increase in cell migration in cell lines overexpressing miR-30a compared to their controls. Conclusions/Significance Overexpression of miR-30a or -191 does not lead to an alteration in cell cycle, proliferation, xenograft formation, and chemosensitivity of A549 and BEAS-2B cell lines. Using microarray data from whole tumors to select specific miRNAs for functional study may be a suboptimal strategy.

MiR-205 and MiR-375 MicroRNA Assays to Distinguish Squamous Cell Carcinoma from Adenocarcinoma in Lung Cancer Biopsies

Introduction: Identification of adenocarcinoma (AC) and squamous cell carcinoma (SCC) histology of non–small-cell lung cancer (NSCLC) in biopsies is clinically important but can be inaccurate by routine histopathologic examination. We quantify this inaccuracy at a cancer center, and evaluate the utility of a microRNA-based method to histotype AC/SCC in biopsies. Methods: RNA was extracted from tissue sections with greater than 90% tumor content that were macro- or micro-dissected from formalin-fixed, paraffin-embedded biopsy specimens. MicroRNAs in RNA from the biopsies and from resected tumors were quantified by TaqMan reverse transcription-polymerase chain reaction assays and normalized against the RNU6B housekeeping RNA. Publicly available microRNA expression datasets were examined. Results: NSCLC subtyping of small biopsy specimens by routine histopathologic examination either failed or mistyped the histology of 21% of 190 cases. Using 77 resectates, an reverse transcription-polymerase chain reaction-based assay of microRNAs miR-21, miR-205, and miR-375 was developed to identify AC and SCC subtypes of NSCLC. This method identified the AC/SCC histotypes of 25 biopsies with an accuracy of 96%, and correctly histotyped all 12 cases for which the histology had been mistyped by routine histopathologic examination of the biopsy. Examination of publicly available datasets identified miR-205 and miR-375 as microRNAs with the best ability to histotype AC and SCC, and that levels of the two microRNAs in AC or SCC are unaffected by the pathologic stage of the tumor or the age or race of the patient. Conclusions: Histotypic microRNA assays can aid the subtyping of NSCLC biopsies as AC or SCC by standard histopathologic methods.

Expression of MicroRNAs in the NCI-60 Cancer Cell-Lines

The NCI-60 panel of 60 human cancer cell-lines of nine different tissues of origin has been extensively characterized in biological, molecular and pharmacological studies. Analyses of data from such studies have provided valuable information for understanding cellular processes and developing strategies for the diagnosis and treatment of cancer. Here, Affymetrix® GeneChip™ miRNA version 1 oligonucleotide microarrays were used to quantify 847 microRNAs to generate an expression dataset of 495 (58.4%) microRNAs that were identified as expressed in at least one cell-line of the NCI-60 panel. Accuracy of the microRNA measurements was partly confirmed by reverse transcription and polymerase chain reaction assays. Similar to that seen among the four existing NCI-60 microRNA datasets, the concordance of the new expression dataset with the other four was modest, with mean Pearson correlation coefficients of 0.37–0.54. In spite of this, comparable results with different datasets were noted in clustering of the cell-lines by their microRNA expression, differential expression of microRNAs by the lines’ tissue of origin, and correlation of specific microRNAs with the doubling-time of cells or their radiation sensitivity. Mutation status of the cell-lines for the TP53, PTEN and BRAF but not CDKN2A or KRAS cancer-related genes was found to be associated with changes in expression of specific microRNAs. The microRNA dataset generated here should be valuable to those working in the field of microRNAs as well as in integromic studies of the NCI-60 panel.

Small RNA Sequencing for Profiling MicroRNAs in Long-Term Preserved Formalin-Fixed and Paraffin-Embedded Non-Small Cell Lung Cancer Tumor Specimens

Background The preservation of microRNAs in formalin-fixed and paraffin-embedded (FFPE) tissue makes them particularly useful for biomarker studies. The utility of small RNA sequencing for microRNA expression profiling of FFPE samples has yet to be determined. Methods Total RNA was extracted from de-paraffinized and proteinase K-treated FFPE specimens (15–20 years old) of 8 human lung adenocarcinoma tumors by affinity chromatography on silica columns. MicroRNAs in the RNA preparations were quantified by the Illumina HiSeq 2000 sequencing platform with sequencing libraries prepared with the TruSeq Small RNA Sample Preparation Kit (version 2.0) to obtain unpaired reads of 50 b for small RNA fragments. MicroRNAs were also quantified using Agilent Human miRNA (release 16.0) microarrays that can detect 1,205 mature microRNAs and by quantitative reverse transcription (RT)-PCR assays. Results Between 9.1–16.9 million reads were obtained by small RNA sequencing of extracted RNA samples. Of these, only 0.6–2.3% (mean = 1.5%) represented microRNAs. The sequencing method detected 454–625 microRNAs/sample (mean = 550) compared with 200–349 (mean = 286) microRNAs detected by microarray. In Spearman correlation analyses, the average correlation coefficient for the 126 microRNAs detected in all samples by both methods was 0.37, and >0.5 for 63 microRNAs. In correlation analyses of the sequencing- and RT-PCR-based measurements, the coefficients were 0.19–0.95 (mean = 0.73) and >0.7, respectively, for 7 of 9 examined microRNAs. The average inter-replicate Spearman correlation coefficient for the sequencing method was 0.81. Conclusions Small RNA sequencing can be used to obtain microRNA profiles of FFPE tissue specimens with performance characteristics similar to those of microarrays, in spite of the fragmentation of ribosomal and messenger RNAs that reduces the methods informative capacity. The accuracy of the method can conceivably be improved by increasing sequencing depth and/or depleting FFPE tissue RNAs of ribosomal RNA fragments.

Lung cancer xenografting alters microRNA profile but not immunophenotype

Lung tumor xenografts grown in immunocompromised mice provide a renewable source of tumor tissue for research and a means to study individualized response to chemotherapy. Critical to this utility is verification that the xenograft cells retain core phenotypic characteristics of the original tumor. We compared eight non-small cell lung carcinomas with their corresponding xenografts grown in mice with severe combined immunodeficiency by way of histology, immunohistochemistry, and microRNA expression profiling. Six of the eight xenografts closely resembled their original tumor by light microscopy. The xenografts also largely retained key immunophenotypic features. With expression profiling of human microRNAs, however, xenografts clustered separately from the original tumors. While this may be partly due to contamination by non-neoplastic human and mouse stroma, the results suggest that miRNA expression may be altered in xenografts and that this possibility should be further evaluated.

Lung cancer lymph node micrometastasis detection using real-time polymerase chain reaction: Correlation with vascular endothelial growth factor expression

OBJECTIVES Lymph node staging provides critical information in patients with non-small cell lung cancer (NSCLC). Lymphangiogenesis may be an important contributor to the pathophysiology of lymphatic metastases. We hypothesized that the presence of lymph node micrometastases positively correlates with vascular endothelial growth factors (VEGFs) A, C, and D as well as VEGF-receptor-3 (lymphangiogenic factors) expression in lymph nodes. METHODS Forty patients with NSCLC underwent preoperative positron emission tomography-computed tomography and mediastinoscopy. Real-time polymerase chain reaction (RT-PCR) assays for messenger RNA expression of epithelial markers (ie, cytokeratin 7; carcinoembryonic antigen-related cell adhesion molecule 5; and palate, lung, and nasal epithelium carcinoma-associated protein) were performed in selected fluorodeoxyglucose-avid lymph nodes. VEGF-A, VEGF-C, VEGF-D, and VEGF receptor-3 expression levels were measured in primary tumors and lymph nodes. Wilcoxon rank sum test was run for the association between the RT-PCR epithelial marker levels and VEGF expression levels in the lymph nodes. RESULTS RT-PCR for cytokeratin 7; carcinoembryonic antigen-related cell adhesion molecule 5; or palate, lung, and nasal epithelium carcinoma-associated protein indicated lymph node micrometastatic disease in 19 of 35 patients (54%). There was a high correlation between detection of micrometastases and VEGF-A, VEGF-C, VEGF-D, or VEGF receptor-3 expression levels in lymph nodes. Median follow-up was 12.6 months. CONCLUSIONS RT-PCR analysis of fluorodeoxyglucose-avid lymph nodes results in up-staging a patients cancer. Micrometastases correlate with the expression of VEGF in lymph nodes in patients with NSCLC. This may reflect the role of lymphangiogenesis in promoting metastases.