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Determining the exact type of epidermal growth factor receptor (EGFR) exon 20 insertion (ex20ins) mutation in lung cancer has become important. We found that not all ex20ins mutations reported by cobas EGFR test v2 could be validated by Sanger sequencing even using surgical specimens with high tumor contents. This study aimed to validate the ex20ins results reported by the cobas test and to determine whether there were clinicopathological factors associated with aberrant cobas ex20ins report. In total, 123 cobas-reported cases with ex20ins were retrospectively collected and validated by Sanger sequencing and Idylla assay. Clinicopathological features between ex20ins cobas+/Sanger+ group (n = 71) and cobas+/Sanger− group (n = 52) were compared. The Idylla assay detected ex20ins in 82.6% of cobas+/Sanger+ cases but only in 4.9% of cobas+/Sanger− cases. The cobas+/Sanger− group was significantly associated with higher tumor contents, poorly differentiated patterns, tumor necrosis, and a lower internal control cycle threshold value reported by the Idylla which suggesting the presence of increased EGFR gene copy numbers. EGFR fluorescence in situ hybridization (FISH) revealed the majority of cobas+/Sanger− group had EGFR high copy number gain (16%) or amplification (76%) according to the Colorado criteria. Among cases reported to have concomitant classic EGFR and ex20ins mutations by the cobas, the classic EGFR mutations were all detected by Sanger sequencing and Idylla, while the ex20ins mutations were undetected by Sanger sequencing (0%) or rarely reported by Idylla assay (3%). FISH revealed high EGFR copy number gain (17.9%) and amplification (79.5%) in cases reported having concomitant classic EGFR and ex20ins mutations by the cobas. This study demonstrated an unusually high frequency of EGFR amplification in cases with aberrant cobas ex20ins report which could not be validated by Sanger sequencing or Idylla assay. Ex20ins reported by the cobas test should be validated using other methods especially those reported having concomitant ex20ins and classic EGFR mutations. [ABSTRACT FROM AUTHOR]

Background: Both procalcitonin (PCT) and soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) have been investigated separately as indicators of infection in patients with autoimmune diseases. Our study simultaneously evaluated both PCT and sTREM-1 along with C-reactive protein (CRP) in febrile patients with autoimmune diseases. Methods: Fifty-nine patients were enrolled in the study. The patients were categorized into the infection group (n = 24) or the disease flare group (n = 35). sTREM-1, PCT and CRP concentrations at fever onset were compared between the two groups of patients. Results: sTREM-1 and CRP did not differ between the two groups. PCT [median (range), ng/ml] was higher in the infection group than in the disease flare group [0.53 (0.02–12.85) vs. 0.12 (0.02–19.23), p = 0.001]. The area under the receiver-operating characteristic (ROC) for diagnosis of infection was 0.75 for PCT (p = 0.001), 0.63 for CRP (p = 0.09) and 0.52 for sTREM-1 (p = 0.79). Using 0.2 ng/ml as the cutoff value for PCT, sensitivity was 0.75 and specificity was 0.77. Negative predictive values for PCT were 92%, 87% and 82% for a prevalence of infection of 20%, 30%, and 40%, respectively. Neither immunosuppressants nor biomodulators affected the level of the three biomarkers. However, in patients treated with corticosteroids, the levels of sTREM-1 and CRP were significantly decreased compared with the untreated patients. Conclusions: Setting PCT at a lower cutoff value could provide useful information on excluding infection in febrile patients with autoimmune diseases. The possible effect of corticosteroids on the level of sTREM-1 as an infection marker deserves further study. [ABSTRACT FROM AUTHOR]

The synthesis of the negative-strand [(−)-strand] complement of the ∼30 kilobase, positive-strand [(+)-strand] coronaviral genome is a necessary early step for genome replication. The identification of cis-acting elements required for (−)-strand RNA synthesis in coronaviruses, however, has been hampered due to insufficiencies in the techniques used to detect the (−)-strand RNA species. Here, we employed a method of head-to-tail ligation and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) to detect and quantitate the synthesis of bovine coronavirus (BCoV) defective interfering (DI) RNA (−) strands. Furthermore, using the aforementioned techniques along with Northern blot assay, we specifically defined the cis-acting RNA elements within the 3′-terminal 55 nucleotides (nts) which function in the synthesis of (−)- or (+)-strand BCoV DI RNA. The major findings are as follows: (i) nts from -5 to -39 within the 3′-terminal 55 nts are the cis-acting elements responsible for (−)-strand BCoV DI RNA synthesis, (ii) nts from −3 to −34 within the 3′-terminal 55 nts are cis-acting elements required for (+)-strand BCoV DI RNA synthesis, and (iii) the nucleotide species at the 3′-most position (−1) is important, but not critical, for both (−)- and (+)-strand BCoV DI RNA synthesis. These results demonstrate that the 3′-terminal 55 nts in BCoV DI RNA harbor cis-acting RNA elements required for both (−)- and (+)-strand DI RNA synthesis and extend our knowledge on the mechanisms of coronavirus replication. The method of head-to-tail ligation and qRT-PCR employed in the study may also be applied to identify other cis-acting elements required for (−)-strand RNA synthesis in coronaviruses. [ABSTRACT FROM AUTHOR]

The establishment of persistent viral infection is often associated with the selection of one or more mutant viruses. For example, it has been found that an intraleader open reading frame (ORF) in genomic and subgenomic mRNA (sgmRNA) molecules is selected during bovine coronavirus (BCoV) persistence which leads to translation attenuation of the downstream ORF. Here, we report the unexpected identification of leaderless genomes, in addition to leader-containing genomes, in a cell culture persistently infected with BCoV. The discovery was made by using a head-to-tail ligation method that examines genomic 5′-terminal sequences at different times postinfection. Functional analyses of the leaderless genomic RNA in a BCoV defective interfering (DI) RNA revealed that (1) the leaderless genome was able to serve as a template for the synthesis of negative-strand genome, although it cannot perform replicative positive-strand genomic RNA synthesis, and (2) the leaderless genome retained its function in translation and transcription, although the efficiency of these processes was impaired. Therefore, this previously unidentified leaderless genome is associated with the attenuation of genome expression. Whether the leaderless genome contributes to the establishment of persistent infection remains to be determined. [ABSTRACT FROM AUTHOR]

The positive-strand coronavirus genome of ~30 kilobase in length and subgenomic (sg) mRNAs of shorter lengths, are 5’ and 3’-co-terminal by virtue of a common 5’-capped leader and a common 3’-polyadenylated untranslated region. Here, by ligating head-to-tail viral RNAs from bovine coronavirus-infected cells and sequencing across the ligated junctions, it was learned that at the time of peak viral RNA synthesis [6 hours postinfection (hpi)] the 3’ poly(A) tail on genomic and sgmRNAs is ~65 nucleotides (nt) in length. Surprisingly, this length was found to vary throughout infection from ~45 nt immediately after virus entry (at 0 to 4 hpi) to ~65 nt later on (at 6 h to 9 hpi) and from ~65 nt (at 6 h to 9 hpi) to ~30 nt (at 120-144 hpi). With the same method, poly(U) sequences of the same lengths were simultaneously found on the ligated viral negative-strand RNAs. Functional analyses of poly(A) tail length on specific viral RNA species, furthermore, revealed that translation, in vivo, of RNAs with the longer poly(A) tail was enhanced over those with the shorter poly(A). Although the mechanisms by which the tail lengths vary is unknown, experimental results together suggest that the length of the poly(A) and poly(U) tails is regulated. One potential function of regulated poly(A) tail length might be that for the coronavirus genome a longer poly(A) favors translation. The regulation of coronavirus translation by poly(A) tail length resembles that during embryonal development suggesting there may be mechanistic parallels. [ABSTRACT FROM AUTHOR]

Background: It is important to select appropriate targeted therapies for subgroups of patients with lung adenocarcinoma who have specific gene alterations., Methods: This prospective study was a multicenter project conducted in Taiwan for assessment of lung adenocarcinoma genetic tests. Five oncogenic drivers, including EGFR, KRAS, BRAF, HER2 and EML4-ALK fusion mutations, were tested. EGFR, KRAS, BRAF and HER2 mutations were assessed by MALDI-TOF MS (Cohort 1). EML4-ALK translocation was tested by Ventana method in EGFR-wild type patients (Cohort 2)., Results: From August 2011 to November 2013, a total of 1772 patients with lung adenocarcinoma were enrolled. In Cohort 1 analysis, EGFR, KRAS, HER2 and BRAF mutations were identified in 987 (55.7%), 93 (5.2%), 36 (2.0%) and 12 (0.7%) patients, respectively. Most of these mutations were mutually exclusive, except for co-mutations in seven patients (3 with EGFR + KRAS, 3 with EGFR + HER2 and 1 with KRAS + BRAF). In Cohort 2 analysis, 29 of 295 EGFR-wild type patients (9.8%) were positive for EML4-ALK translocation. EGFR mutations were more common in female patients and non-smokers and KRAS mutations were more common in male patients and smokers. Gender and smoking status were not correlated significantly with HER2, BRAF and EML4-ALK mutations. EML4-ALK translocation was more common in patients with younger age., Conclusion: This was the first study in Taiwan to explore the incidence of five oncogenic drivers in patients with lung adenocarcinoma and the results could be valuable for physicians in consideration of targeted therapy and inclusion of clinical trials.

The synthesis of the negative-strand [(-)-strand] complement of the ∼30 kilobase, positive-strand [(+)-strand] coronaviral genome is a necessary early step for genome replication. The identification of cis-acting elements required for (-)-strand RNA synthesis in coronaviruses, however, has been hampered due to insufficiencies in the techniques used to detect the (-)-strand RNA species. Here, we employed a method of head-to-tail ligation and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) to detect and quantitate the synthesis of bovine coronavirus (BCoV) defective interfering (DI) RNA (-) strands. Furthermore, using the aforementioned techniques along with Northern blot assay, we specifically defined the cis-acting RNA elements within the 3'-terminal 55 nucleotides (nts) which function in the synthesis of (-)- or (+)-strand BCoV DI RNA. The major findings are as follows: (i) nts from -5 to -39 within the 3'-terminal 55 nts are the cis-acting elements responsible for (-)-strand BCoV DI RNA synthesis, (ii) nts from -3 to -34 within the 3'-terminal 55 nts are cis-acting elements required for (+)-strand BCoV DI RNA synthesis, and (iii) the nucleotide species at the 3'-most position (-1) is important, but not critical, for both (-)- and (+)-strand BCoV DI RNA synthesis. These results demonstrate that the 3'-terminal 55 nts in BCoV DI RNA harbor cis-acting RNA elements required for both (-)- and (+)-strand DI RNA synthesis and extend our knowledge on the mechanisms of coronavirus replication. The method of head-to-tail ligation and qRT-PCR employed in the study may also be applied to identify other cis-acting elements required for (-)-strand RNA synthesis in coronaviruses.

Background: The risk of endobronchial ultrasound-guided transbronchial biopsy-related pneumothorax is a major concern and warrants further studies. The aim of our study was to estimate the risk of pneumothorax after this procedure and identify its risk factors., Methods: From 2007 to 2011, 399 patients who underwent endobronchial ultrasound-guided transbronchial biopsy for peripheral lung lesions were included in this study. The variables analyzed included patient factors, lesion factors and procedure factors. Multivariate logistic regression analysis was used to identify independent risk factors for pneumothorax., Results: The incidence of pneumothorax was 3.3% (13/399). Chest tube placement was required for 31% (4/13) of pneumothoraces. Independent risk factors for pneumothorax included pulmonary emphysema (OR, 55.09; 95% CI, 9.37-324.03; p<0.001) and probe position adjacent to the lesion (OR, 17.01; 95% CI, 2.85-101.64; p = 0.002). The number of biopsy specimens, age, sex, history of prior lung surgery and lesion size, location and character did not influence the risk of pneumothorax in our analyses., Conclusions: The risk of pneumothorax after endobronchial ultrasound-guided transbronchial biopsy is low. To further reduce the risk of pneumothorax, every effort should be made to advance the endobronchial ultrasound probe into the bronchus where it is imaged within the target lesion before embarking on transbronchial biopsy.