Comparison of Biocartis IDYLLA ™ cartridge assay with Qiagen GeneReader NGS for detection of targetable mutations in EGFR, KRAS/NRAS, and BRAF genes.

Lung and colorectal cancers (CRC) have two of the highest mortality rates among all cancer types, and their occurrence and the need for personalized diagnostics and subsequent therapy were not influenced by the COVID-19 pandemics. However, due to the disruption of established delivery chains, standard assays for in vitro diagnostics of those cancers were temporarily not available, forcing us to implement alternative testing methods that enabled at least basic therapy decision making. For this reason, we evaluated rapid testing on the Biocartis Idylla™ platform (Biocartis, Mechelen, Belgium) for four important genes commonly mutated in lung and colorectal cancers, namely EGFR, NRAS, KRAS, and BRAF. Clinical specimens from which the mutation status has previously been determined using Next Generation Sequencing (NGS), were retested to determine whether Idylla™ can offer accurate results. To compare the results, the sensitivity, specificity, positive predictive values (PPV) and negative predictive values (NPV) are calculated for each of the mutation types and then combined to determine the values of the Idylla™ system in total, while setting NGS as the gold-standard basis the assays were compared with. Idylla testing thereby displayed acceptable sensitivity and specificity and delivered reliable results for initial therapy decisions.

Differential cytology profiles in bronchoalveolar lavage (BAL) in COVID-19 patients: A descriptive observation and comparison with other corona viruses, Influenza virus, Haemophilus influenzae, and Pneumocystis jirovecii.

ABSTRACT: Brochoalvelolar lavages (BALs) from patients suffering from hospitalized infections with SARS-CoV-2, other corona viruses (human coronavirus (HCoV)-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1), Influenza virus type A and B, Haemophilus influenzae and Pneumocystis jirovecii were compared cytopathologically.The aim of the study was to evaluate if the cellular profile detectable in BAL may be specific for the respective pathogens and could lead to diagnosis of COVID-19 even in the absence of PCR results.Differential cytology and flow cytometry datasets of 62 patients were observed and compared.We observed a significant association between individual cell pattern changes and the causing pathogen, but no general cell distribution pattern.The cytology pattern of the BAL fluid in COVID-19 is not specific enough to use it as a sole diagnostic criterion, although it may support clinical decision making.

Detailed overview on the mutations detected by and the sensitivity of the GeneReader NGS sequencing platform.

This article presents additional next generation data from our pre-clinical validation study. In total 121 samples (clinical specimen and interlaboratory test samples) were tested successfully with next generation sequencing. 38 different mutations in six different genes were detected. Next to the detection of different mutations, the reproducibility of the NGS test was analyzed. Three samples were analyzed five times and the results were compared. Several mutations classified as non-pathogenic so far, have been detected repeatedly.

Pre-clinical validation of a next generation sequencing testing panel.

OBJECTIVE: Next Generation Sequencing (NGS) has become a useful tool for gene mutation testing which is required for targeted therapies. The aim of this study was to validate the GeneRead QIAact Actionable Insights Tumor Panel (Qiagen) on the GeneReader System in a diagnostic laboratory setting. METHODS: The GeneRead QIAact Actionable Insights Tumor Panel allows the analysis of 773 variant positions in 12 genes (ALK, BRAF, EGFR, ERBB2, ERBB3, ESR1, KIT, KRAS, NRAS, PDGFRA, PIK3CA and RAF1). For the validation of the panel we used a commercial available multiplex reference standard carrying 11 mutations in defined positions, samples from interlaboratory tests, and FFPE tumor samples from patients which were tested previously for mutations in KRAS, NRAS, BRAF, EGFR, KIT, and/or PDGFRA with pyrosequencing. RESULTS: Among the 122 tested samples, 121 samples (99.2%) were successfully sequenced. The sensitivity and specificity for detecting variants was 100% and results proved to be reproducible and precise. 119 (98.3%) results were concordant to the expected results. The differences between NGS and pyrosequencing observed in two samples were due to a wrong analysis by the pyrosequencing software which did not cover the present mutations. CONCLUSION: Overall, the GeneRead QIAact Actionable Insights Tumor Panel was specific and sensitive for mutation analysis for targeted therapies and can be incorporated into laboratory diagnostics' daily practice.

Low frequency of EGFR mutations in pleural mesothelioma patients, Cologne, Germany.

EGFR mutations were previously found in patients suffering from peritoneal mesothelioma but have not yet been described in pleural mesothelioma. The aim of the present study was the identification of EGFR mutations in patients suffering from pleural mesothelioma. Pleural mesothelioma tissue from 31 patients was used to analyze possible mutations in the EGFR gene comprising the exons 18-21 with the codons 719, 768, 790, 858+861, 731+734, 785, 797+801, 831, and 868 with pyrosequencing. The results indicate that 31 pleural mesothelioma patients show a wild-type EGFR gene when analyzing the codons D19, 768, 790, 858+861, 731+734, 785, 797+801, 831, and 868, whereas 2 patients have a mutation in the EGFR gene in codon 719. Sanger sequencing of the EGFR codon 785 was used for the determination of a polymorphism in the sequencing of tumor-free patients and pleural mesothelioma patients with a distribution of a wild-type homozygous sequence with guanine, a wild-type heterozygous sequence having guanine and adenine, a wild-type homozygous sequence with adenine, and a wild-type heterozygous sequence with adenine and guanine. Next, the identification of less EGFR mutations in the EGFR gene of the pleural mesothelioma an up to this time unknown polymorphism in the EGFR gene was identified which could be wrongly interpreted as a mutation.

Combined point mutation in KRAS or EGFR genes and EML4-ALK translocation in lung cancer patients.

A total of three cases with novel constellations regarding mutation patterns in non-small-cell lung cancer (NSCLC) are reported. The mutation patterns that are observed are novel and unexpected. First, a combined simultaneous KRAS mutation and EML4-ALK translocation, both in the main tumor and a bone metastasis, were observed, these mutations are assumed to mutually exclude each other. A further two cases include a father and a daughter, both of whom are suffering from NSCLC with different EGFR mutation patterns. A common cause was assumed; however, could not be deduced to mutations in the KRAS, BRAF and EGFR genes. The aforementioned cases are important, as it must be taken into account that mutations previously assumed to be exclusive can occur in combination, may influence the clinical outcome and may require different therapy compared with single mutated tumors. It has to be discussed whether diagnostic algorithms need to be adapted. The cases of father and daughter show that further unknown factors can influence development of NSCLC.

The Human Bocavirus Is Associated with Some Lung and Colorectal Cancers and Persists in Solid Tumors.

Human bocavirus is the second autonomous human parvovirus with assumed pathogenic potential. Other parvoviruses are known to persist and even integrate into the host genome, eventually contributing to the multi-step development of cancer. Human bocavirus also persists in an unknown percentage of clinically asymptomatic patients in addition to those with primary infection. The aim of the present study was to analyze the role of Human bocavirus in lung and colorectal cancers. Therefore, formalin-fixed, paraffin-embedded, archived tumor samples were screened for Human bocavirus DNA by PCR, Southern blotting, and sequencing. Positive tissues were further subjected to fluorescence in situ hybridization analysis to specifically detect human bocavirus DNA in the infected cells. In total, 11 of the 60 (18.3%) lung and 9 of the 44 (20.5%) colorectal tumors tested positive for human bocavirus DNA by PCR and were confirmed by sequencing and fluorescence in situ hybridization analysis. Thus, human bocavirus DNA is present in the nuclei of infected cells, in either single or multiple copies, and appears to form concatemers. The occurrence of these human bocavirus DNA structures supports the existence of the postulated σ- or rolling-hairpin replication mechanism. Moreover, the fluorescence in situ hybridization patterns inspired the hypothesis that human bocavirus DNA either persists as cccDNA or is integrated into the host genome. This finding suggests that this virus may indirectly contribute to the development of some colorectal and lung cancers, as do other DNA viruses, such as the human hepatitis B virus, or may play an active role in cancer by interacting with the host genome.

Combination of Pyrosequencing® and Sanger sequencing reveals alleged novel mutation in exon 18 of EGFR.

BACKGROUND: EGFR sequencing is crucial for therapeutic decisions in treatment regimens of lung cancer patients. Several mutations have been identified in the past, of which some were confirmed as activating or resistance mutations by in vitro phenotyping. In this study, novel mutations of so far unknown relevance were identified by a combination of Sanger and Pyrosequencing®. MATERIALS & METHODS: Formalin-fixed paraffin-embedded lung biopsies from 20 randomly selected patients suffering from non-small-cell lung cancer with previous external EGFR mutation analyses were reanalyzed by Sanger sequencing according to a previous study, and Pyrosequencing with the EGFR Pyro Kit (Qiagen, Hilden, Germany). Sensitivity and specificity were determined in comparison with the results of an external supplier for all relevant mutations in exons 18, 19, 20 and 21. RESULTS: Our analyses revealed that Pyrosequencing is faster and more sensitive for the common mutations compared with Sanger sequencing and the results of the external supplier. A new mutation, c.2160delC, in exon 18 in 40% of patients leading to a frameshift was identified. Another two frameshift mutations were detected in exon 18 in 10% of patients; c.2168delT in combination with c.2160delC, and c.2163insG alone. CONCLUSION: Divergences regarding the detection of the common mutations could be traced back to inhomogeneous or insufficient tumor material. Surprisingly, none of the newly identified mutations have been previously described, although they occurred in total in up to 40% of randomly selected cases. A possible explanation may be that commercial assays did not cover these deletions that are located nearby but not in the known mutation hotspot of exon 18, or that Sanger sequencing produced serious artifacts.

Detection of head-to-tail DNA sequences of human bocavirus in clinical samples.

Parvoviruses are single stranded DNA viruses that replicate in a so called "rolling-hairpin" mechanism, a variant of the rolling circle replication known for bacteriophages like φX174. The replication intermediates of parvoviruses thus are concatemers of head-to-head or tail-to-tail structure. Surprisingly, in case of the novel human bocavirus, neither head-to-head nor tail-to-tail DNA sequences were detected in clinical isolates; in contrast head-to-tail DNA sequences were identified by PCR and sequencing. Thereby, the head-to-tail sequences were linked by a novel sequence of 54 bp of which 20 bp also occur as conserved structures of the palindromic ends of parvovirus MVC which in turn is a close relative to human bocavirus.

Respiratory infections by HMPV and RSV are clinically indistinguishable but induce different host response in aged individuals.

BACKGROUND: Human metapneumovirus and respiratory syncytial virus can cause severe respiratory diseases, especially in infants, young children, and the elderly. So far it remains unclear why infections in the elderly become life threatening despite the presence of neutralizing antibodies in the serum, and to which extent double infections worsen the clinical course. METHODS: Young and aged BALB/c-mice were infected with RSV or/and HMPV. Appearance of the mice was observed during course of infection. On day 5 p.i. animals were dispatched by cervical dislocation and levels of TNF-α and NF-κB were determined. RESULTS: The observation of activity, weight and appearance of the different mice showed no differences among the tested groups. Despite this, the immunologic response depends on the animals' age and the virus they were infected with. In young animals, NF-κB levels were elevated if infected with HMPV and HMPV/RSV but remained low in RSV infections, whereas in aged animals the opposite was observed: solely RSV-infected animals showed elevated levels of NF-κB. TNF-α was slightly elevated in HMPV-infected young and old animals, but only in young animals this elevation was significant. CONCLUSIONS: Contrary to other studies, no weight loss or change in activity despite productive lung infection with the different viruses were observed. This may be due to the weaker anaesthesia or the lesser volume of virus solution used, leading to less stress in the animals. The observed differences in TNF-α and NF-κB elevation lead to the assumption that young and old individuals have different mechanisms to react against the viruses.

High seroprevalence of neutralizing capacity against human metapneumovirus in all age groups studied in Bonn, Germany.

Human metapneumovirus (hMPV) infections occur frequently despite high rates of perpetual seroprevalence for all age groups. Analyses of approximately 2,000 archived, randomly selected serum samples demonstrated that neutralizing capacities remain high, with a minor decrease for individuals over 69 years of age, leading to the hypothesis that reinfections occur because humoral immune responses play minor roles in the clearance of hMPV infections.

Polyomaviruses KI and WU in children with respiratory tract infection.

The polyomaviruses KI (KIPyV) and WU (WUPyV) have recently been discovered in specimens from patients with respiratory tract infections. To analyze the frequency and clinical impact in a cohort of pediatric patients in a German University Children's Hospital. Nasopharyngeal aspirates or bronchoalveolar lavage specimens of 229 children with acute respiratory tract infection were screened for KIPyV and WUPyV using polymerase chain reaction-based methods. KIPyV was detected in 2 (0.9%) and WUPyV in 1 (0.4%) patients, without co-infections with other respiratory viruses but with co-detection of CMV, EBV and HHV 6 in one immunocompromised patient. Only a very small proportion (1.3%) of positive samples for KIPyV and WUPyV was documented in this study; the clinical relevance of these viruses remains unclear and requires further evaluation.

Low prevalence of human metapneumovirus and human bocavirus in adult immunocompromised high risk patients suspected to suffer from Pneumocystis pneumonia.

BACKGROUND: Novel respiratory viruses were discovered in the last years predominantly in children. Until now information on newly identified respiratory viruses in immunosuppressed adult patients is limited. Here we investigated immunocompromised adults with suspected Pneumocystis jirovecii pneumonia (PCP) for new respiratory viruses. METHODS: Bronchoalveolar lavage (BAL) samples of 128 patients with atypical pneumonia (HIV-infected n=50, hematological malignancy n=51, immunosuppressive treatment n=27) were prospectively evaluated for P. jirovecii and retrospectively for new respiratory viruses (HMPV, HBoV, HCoV-NL63/SARS/HKU1). RESULTS: P. jirovecii was detected in 26/128, bacteria in 10, fungi in four, Influenza A in two patients. Novel respiratory viruses were found in only two/128 patients with hematological malignancy, of those one patient with HBoV-infection and one with HMPV-infection, respectively. No pathogens were detected in 82/128 patients. The one patient with detection of hMPV and clinical diagnosis of atypical pneumonia died of pulmonary failure. CONCLUSION: Human bocavirus and human metapneumovirus are rarely involved in atypical pneumonia in immunocompromised adult patients with suspected PCP, but may contribute to severe respiratory failure.

Sensitive commercial NASBA assay for the detection of respiratory syncytial virus in clinical specimen.

The aim of the study was to evaluate the usability of three diagnostic procedures for the detection of respiratory syncytial virus in clinical samples. Therefore, the FDA cleared CE marked NOW(R) RSV ELISA, the NucliSENS EasyQ RSV A+B NASBA, and a literature based inhouse RT-PCR protocol were compared for their relative sensitivities. Thereby, NASBA turned out to be the most sensitive method with a total number of 80 RSV positive samples out of a cohort of 251 nasopharyngeal washings from patients suffering from clinical symptoms, followed by the inhouse RT-PCR (62/251) and ELISA (52/251). Thus, NASBA may serve as a rapid and highly sensitive alternative for RSV diagnostics.

Detection of bocavirus DNA in nasopharyngeal aspirates of a child with bronchiolitis.

We describe a case of bronchiolitis associated with the newly detected human bocavirus (hBoV) in a child with a suspected Noonan syndrome. This is the first report of a bronchiolitis probably linked to hBoV that required intensive care while being accompanied by a congenital heart disease and a history of several episodes of severe respiratory symptoms.