Evaluation of next-generation sequencing performance for in vitro detection of viruses in biological products.

Next-Generation Sequencing (NGS) can detect nucleic acid sequences in a massively parallel sequencing. This technology is expected to be widely applied for the detection of viral contamination in biologics. The recently published ICH-Q5A (R2) draft indicates that NGS could be an alternative or supplement to in vitro viral tests. To examine the performance of NGS for the in vitro detection of viruses, adenovirus type 5 (Ad5), a model virus, was inoculated into Vero cells, which are the most popular indicator cells for the detection of adventitious viruses in the in vitro test. Total RNA extracted from the Vero cells infected with Ad5 was serially diluted with that from non-infected Vero cells, and each sample was analyzed using short- or long-read NGSs. The limits of detection of both NGS methods were almost the same and both methods were sensitive enough to detect viral sequences as long as there was at least one copy in one assay. Although the multiplexing in NGS carries the risk of cross-contamination among the samples, which could lead to false positives, this technology has the potential to become a rapid and sensitive method for detecting adventitious agents in biologics.

Infectivity assessment of porcine endogenous retrovirus using high-throughput sequencing technologies.

Xenogenic cell-based therapeutic products are expected to alleviate the chronic shortage of human donor organs. For example, porcine islet cell products are currently under development for the treatment of human diabetes. As porcine cells possess endogenous retrovirus (PERV), which can replicate in human cells in vitro, the potential transmission of PERV has raised concerns in the case of products that use living pig cells as raw materials. Although several PERV sequences exist in the porcine genome, not all have the ability to infect human cells. Therefore, polymerase chain reaction analysis, which amplifies a portion of the target gene, may not accurately assess the infection risk. Here, we determined porcine genome sequences and evaluated the infectivity of PERVs using high-throughput sequencing technologies. RNA sequencing was performed on both PERV-infected human cells and porcine cells, and reads mapped to PERV sequences were examined. The normalized number of the reads mapped to PERV regions was able to predict the infectivity of PERVs, indicating that it would be useful for evaluation of the PERV infection risk prior to transplantation of porcine products.

A highly sensitive method for the detection of recombinant PERV-A/C env RNA using next generation sequencing technologies.

Several xenogenic cell-based therapeutic products are currently under development around the world for the treatment of human diseases. Porcine islet cell products for treating human diabetes are a typical example. Since porcine cells possess endogenous retrovirus (PERV), which can replicate in human cells in vitro, the potential transmission of PERV has raised concerns in the development of these products. Four subgroups of infectious PERV have been identified, namely PERV-A, -B, -C, and recombinant PERV-A/C. Among them, PERV-A/C shows a high titre and there was a paper reported that an incidence of PERV-A/C viremia was increased in diseased pigs; thus, it would be important to monitor the emergence of PERV-A/C after transplantation of porcine products. In this study, we developed a highly sensitive method for the detection of PERV-A/C using next generation sequencing (NGS) technologies. A model PERV-C spiked with various doses of PERV-A/C were amplified by RT-PCR and the amplicons were analysed by NGS. We found that the NGS analysis allowed the detection of PERV-A/C at the abundance ratios of 1% and 0.1% with true positive rates of 100% and 57%, respectively, indicating that it would be useful for the rapid detection of PERV-A/C emergence after transplantation of porcine products.

Histone deacetylase inhibitor enhances sensitivity of non-small-cell lung cancer cells to 5-FU/S-1 via down-regulation of thymidylate synthase expression and up-regulation of p21(waf1/cip1) expression.

It is desirable to find more appropriate therapeutic opportunities in non-small-cell lung cancer (NSCLC) due to the current poor prognosis of affected patients. Recently, several histone deacetylase (HDAC) inhibitors, including suberoylanilide hydroxamic acid (SAHA), have been reported to exhibit antitumor activities against NSCLC. S-1, a novel oral fluorouracil anticancer drug, has been developed for clinical use in the treatment of NSCLC in Japan. Using an MTT assay, we analyzed the growth-inhibitory effect of 5-fluorouracil (5-FU), S-1, and SAHA against three NSCLC cell lines, as well as the breast cancer cell line MCF7 which is known to be highly sensitive to 5-FU. Combined treatment with low-dose SAHA enhanced 5-FU- and S-1-mediated cytotoxicity and resulted in synergistic effects, especially in 5-FU-resistant cells. Both the mRNA and protein expression levels of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), and orotate phosphoribosyltransferase (OPRT), which are associated with 5-FU sensitivity/response, were analyzed in the cells undergoing treatment. 5-Fluorouracil-resistant lung cancer cells displayed high expression of TS mRNA and protein. Suberoylanilide hydroxamic acid down-regulated TS mRNA and protein expression, as well as repressed the rapid induction of this factor during 5-FU treatment, in all examined cell types. We also examined the status of the Rb-E2F1 pathway, with SAHA up-regulating p21(waf1/cip1) expression via promoter histone acetylation; this, in turn, blocked the Rb-E2F1 pathway. We conclude that combination therapy with SAHA and S-1 in lung cancer may be promising due to its potential to overcome S-1 resistance via modulation of 5-FU/S-1 sensitivity-associated biomarker (TS) by HDAC inhibitor.

Antitumor activity of histone deacetylase inhibitors in non-small cell lung cancer cells: development of a molecular predictive model.

To ascertain the potential for histone deacetylase (HDAC) inhibitor-based treatment in non-small cell lung cancer (NSCLC), we analyzed the antitumor effects of trichostatin A (TSA) and suberoylanilide hydroxamic acid (vorinostat) in a panel of 16 NSCLC cell lines via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. TSA and vorinostat both displayed strong antitumor activities in 50% of NSCLC cell lines, suggesting the need for the use of predictive markers to select patients receiving this treatment. There was a strong correlation between the responsiveness to TSA and vorinostat (P < 0.0001). To identify a molecular model of sensitivity to HDAC inhibitor treatment in NSCLC, we conducted a gene expression profiling study using cDNA arrays on the same set of cell lines and related the cytotoxic activity of TSA to corresponding gene expression pattern using a modified National Cancer Institute program. In addition, pathway analysis was done with Pathway Architect software. We used nine genes, which were identified by gene-drug sensitivity correlation and pathway analysis, to build a support vector machine algorithm model by which sensitive cell lines were distinguished from resistant cell lines. The prediction performance of the support vector machine model was validated by an additional nine cell lines, resulting in a prediction value of 100% with respect to determining response to TSA and vorinostat. Our results suggested that (a) HDAC inhibitors may be promising anticancer drugs to NSCLC and (b) the nine-gene classifier is useful in predicting drug sensitivity to HDAC inhibitors and may contribute to achieving individualized therapy for NSCLC patients.

E-cadherin expression and epidermal growth factor receptor mutation status predict outcome in non-small cell lung cancer patients treated with gefitinib.

It is known that an epidermal growth factor receptor (EGFR) gene mutation(s) is present in a percentage of non-small cell lung cancers (NSCLCs). Gefitinib, an inhibitor of the tyrosine kinase activity of EGFR, is effective on most of them. The EGFR mutation status alone cannot fully predict the response to gefitinib and the prognosis for the patients. We hypothesized that information on the expression levels of phosphorylated-EGFR and -Akt, and E-cadherin, alone or in combination with information on the EGFR mutation, may refine our ability of prediction. We investigated 24 NSCLCs that had recurred after surgery and were treated with gefitinib. Specimens resected by surgery were subjected to the peptide nucleic acid-locked nucleic acid polymerase chain reaction clamp reaction to determine the EGFR mutation status, and to immunohistochemical staining of phosphorylated-EGFR and -Akt, and E-cadherin to determine their expression levels. The EGFR mutation status was predictive of responsive disease (complete response: CR + partial response: PR) and controlled disease (CR + PR + stable disease: SD). Positive E-cadherin staining was predictive of longer time to progression (12.4 vs. 5.9 months, p<0.05) and overall survival (OS) (18.4 vs. 13.0 months, p<0.05). Together the patients with an EGFR mutation and the patients with positive E-cadherin staining defined a patient group with a median OS of 18.4 months and excluded the patient group with the median OS of 3.7 months. Neither p-Akt nor p-EGFR staining was associated with the response and survival. In patients with surgically resected NSCLC tumors, the EGFR mutation status and E-cadherin staining can select patients who will benefit from gefitinib therapy.

PTEN inactivation in lung cancer cells and the effect of its recovery on treatment with epidermal growth factor receptor tyrosine kinase inhibitors.

To understand the mechanisms of PTEN inactivation, which is reported to be involved in tumor progression and drug resistance in lung cancer, we analyzed the expression levels of PTEN at mRNA and protein levels, along with the genetic and epigenetic status of the PTEN gene, in a panel of lung cancer cell lines. Western blot analysis showed that six out of 25 (24%) cell lines displayed low expression of PTEN protein. The level of PTEN mRNA correlated well with corresponding protein expression in each of these six cell lines. In two of the six cell lines genomic analysis revealed homozygous deletions of the PTEN gene. Another two of the six cell lines displayed hypermethylation of the PTEN gene promoter assessed by methylation-specific PCR. The levels of PTEN mRNA and protein expression in PC9/f9 and PC9/f14 cells, which are gefitinib-resistant derivatives of the gefitinib-sensitive cell line, PC9, were reduced compared to the parental line. After treatment with the demethylating agent 5-aza-2'deoxycytidine (5-AZA) and the histone deacetyltransferase (HDAC) inhibitor Trichostatin A (TSA), the expression levels of PTEN mRNA and protein in these four cell lines (PC9/f9, PC9/f14, PC10 and PC14) were actually restored. In summary, reduction in PTEN protein expression was regulated by histone deacetylation and hypermethylation of the gene promoter, as well as homozygous deletion. In addition, we demonstrated that the combination treatment of gefitinib and TSA induced significant growth inhibition in gefitinib-resistant PC9/f9 and PC9/f14 cells. These findings suggest that the combination of the epidermal growth factor receptor tyrosine kinase inhibitor gefitinib with the demethylating agent 5-AZA and the HDAC inhibitor TSA may be a useful strategy for the treatment of some lung cancers.

Gefitinib (IRESSA) sensitive lung cancer cell lines show phosphorylation of Akt without ligand stimulation.

BACKGROUND: Phase III trials evaluating the efficacy of gefitinib (IRESSA) in non-small cell lung cancer (NSCLC) lend support to the need for improved patient selection in terms of gefitinib use. Mutation of the epidermal growth factor receptor (EGFR) gene is reported to be associated with clinical responsiveness to gefitinib. However, gefitinib-sensitive and prolonged stable-disease-defined tumors without EGFR gene mutation have also been reported. METHODS: To identify other key factors involved in gefitinib sensitivity, we analyzed the protein expression of molecules within the EGFR family, PI3K-Akt and Ras/MEK/Erk pathways and examined the sensitivity to gefitinib using the MTT cell proliferation assay in 23 lung cancer cell lines. RESULTS: We identified one highly sensitive cell line (PC9), eight cell lines displaying intermediate-sensitivity, and 14 resistant cell lines. Only PC9 and PC14 (intermediate-sensitivity) displayed an EGFR gene mutation including amplification. Eight out of the nine cell lines showing sensitivity had Akt phosphorylation without ligand stimulation, while only three out of the 14 resistant lines displayed this characteristic (P = 0.0059). Furthermore, the ratio of phosphor-Akt/total Akt in sensitive cells was higher than that observed in resistant cells (P = 0.0016). Akt phosphorylation was partially inhibited by gefitinib in all sensitive cell lines. CONCLUSION: These results suggest that Akt phosphorylation without ligand stimulation may play a key signaling role in gefitinib sensitivity, especially intermediate-sensitivity. In addition, expression analyses of the EGFR family, EGFR gene mutation, and FISH (fluorescence in situ hybridization) analyses showed that the phosphorylated state of EGFR and Akt might be a useful clinical marker of Akt activation without ligand stimulation, in addition to EGFR gene mutation and amplification, particularly in adenocarcinomas.

Mutational analysis of the macrophage scavenger receptor 1 (MSR1) gene in primary lung cancer.

Allelic deletion at chromosome 8p21-25 is an early and frequent event in the carcinogenesis and development of various cancers. To facilitate investigation of alterations of the macrophage scavenger receptor 1 (MSR1), which is located on 8p22, and to determine the role of this gene in human carcinogenesis and tumor progression, we determined intronic primers designed to amplify the coding region. Since frequent deletion of 8p21-23 has been previously reported in lung cancer, we searched for mutations throughout the coding sequence of the MSR1 gene within a panel of genomic DNA samples obtained from 30 primary lung cancers. Our approach, which involved polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis and direct DNA sequencing, revealed nucleotide variants of the MSR1 gene in only one of the 30 cases examined, with this sample displaying both a 6 bp deletion and a thymine-to-cytosine substitution, the latter occurring within intron 7. The 6 bp deletion was located at a DNA microsatellite region and the thymine-to-cytosine substitution seemed to be a polymorphism. These results suggest that the MSR1 gene is not commonly mutated in lung cancer and not important in susceptibility to lung cancer. Further studies may focus on alternative mechanisms through which the MSR1 gene might be inactivated, such as aberrant DNA methylation, and/or pursue analyses of other genes on 8p21-23 for mutational events. Nevertheless, the panel of intronic PCR primer pair sequences presented here will facilitate future studies to determine the full spectrum and frequency of genetic events that may affect expression/activity of the MSR1 gene in human tumors.