UNASSIGNED: We herein report the case of a patient with advanced lung adenocarcinoma who presented a heterogeneous distribution of EGFR mutation.
UNASSIGNED: A 74-year-old Moroccan male former smoker was diagnosed with advanced lung adenocarcinoma, harboring S768I exon 20 substitution mutation confirmed by Real Time PCR and Pyrosequencing, but not detected by direct sequencing despite 70% of tumor cells. The present report describes a case of minor histologic intratumoral heterogeneity with heterogeneous distribution of EGFR mutation.
UNASSIGNED: Both sensitivity and specificity of molecular methods can provide evidence of intratumoral heterogeneity, which may explain the mismatch between the validation of oncology biomarkers and predicting therapeutic response to targeted therapy.

OBJECTIVE: One of the most common sequelae of tympanostomy tube (TT) placement is post-tympanostomy tube otorrhea (PTTO). Granulation tissue formation has been reported in 5-13.8% of patients with TT placement. The purpose of this study is to determine the biodistribution of microorganisms on TTs and middle ear fluid obtained from patients with PTTO.
METHODS: This was a prospective observational study of subjects (6-months-16-years) who underwent standard of care treatment for chronic PTTO. Data was collected on diagnosis, existence of antibiotic resistance, duration of tube placement, and evidence of recurrent infection and/or PTTO. TTs and middle ear fluid were subjected to pyrosequencing; additionally, ear fluid samples were sent for culture-based laboratory diagnostics.
RESULTS: DNA-pyrosequencing analysis of bacteria from fluid and TTs of pediatric subjects with PTTO revealed a mixture of both aerobic and anaerobic populations. Retained tubes with minimal otorrhea revealed a predominance of Staphylococcus species, normal external auditory canal (EAC) microbiome, within middle ear fluid as well as on TTs. However, TTs with active mucopurulent otorrhea and granulation tissue unveiled prominence of Gram-negative facultative anaerobes such as Pseudomonas and Eikenella. Discrepancies in prominent bacteria were seen between standard culture-based techniques versus pyrosequencing.
CONCLUSIONS: Retained tympanostomy tubes are colonized primarily with normal flora of the EAC. However, mucopurulent otorrhea associated with granulation tissue formation revealed a prominence of Gram-negative facultative anaerobes. Standard culture-based diagnostics may identify bacteria, which are not predominant species of infection. Future studies are necessary to assess the association of Gram-negative facultative anaerobes with granulation tissue formation.

BACKGROUND: Histopathological diagnosis is important for prognostication and choice of treatment in patients with cancer in the lung. Metastases to the lungs are common and need to be distinguished from primary lung cancer. Furthermore, cases with synchronous or metachronous primary lung cancers (although infrequent) are often handled differently than cases with lung cancer with intrapulmonary metastasis or relapse, respectively. In some cases, morphology and immunohistochemical staining is not sufficient for certain diagnosis.
METHODS: The present study included six cases where molecular genetic analysis in form of pyrosequencing or targeted next-generation sequencing was of value for certain diagnosis of selected tumours in the lung.
RESULTS: Two of the included cases were rare metastases to the lung; colorectal cancer with IHC profile consistent with primary lung cancer and malignant adenomyoepithelioma of the breast, respectively, where molecular genetic analysis was of aid for proving the relationship to the primary tumour. The other four cases were multiple lung adenocarcinomas where molecular genetic analysis was of aid to distinguish between intrapulmonary metastasis and synchronous tumour.
CONCLUSIONS: Comparison of molecular genetic profile may be an important tool for determination of relationship between tumours in some situations and should always be considered in unclear cases. Further studies on concordance and discordance of molecular genetic profiles between spatially or temporally different tumours with common origin may be helpful for improved diagnostics of pulmonary tumours.

BACKGROUND: Pyrosequencing Allele Quantification (AQ) is a cost-effective DNA sequencing method that can be used for detecting somatic mutations in formalin-fixed paraffin-embedded (FFPE) samples. The method displays a low turnaround time and a high sensitivity. Pyrosequencing suffers however from two main drawbacks including (i) low specificity and (ii) difficult signal interpretation when multiple mutations are reported in a hotspot genomic region.
RESULTS: Using a constraint-based regression method, the new AdvISER-PYRO-SMQ algorithm was developed in the current study and implemented into an R package. As a proof-of-concept, AdvISER-PYRO-SMQ was used to identify a set of 9 distinct point mutations affecting codon 61 of the NRAS oncogene. In parallel, a pyrosequencing assay using the Qiagen software and its AQ module was used to assess selectively the presence of a single point mutation (NRAS[Formula: see text] - Q61R-1) among the set of codon 61 mutations, and to analyze related pyrosequencing signals. AdvISER-PYRO-SMQ produced a lower limit of blank (0 %) than the AQ module of Qiagen software (5.1 %) and similar limit of detection were obtained for both software (5.6 vs 4.8 %). AdvISER-PYRO-SMQ was able to screen for the presence of 9 distinct mutations with a single pyrosequencing reaction whereas the AQ module was limited to screen a single mutation per reaction.
CONCLUSIONS: Using a constraint-based regression method enables to analyze pyrosequencing signal and to detect multiple mutations within a hotspot genomic region with an optimal compromise between sensitivity and specificity. The AdvISER-PYRO-SMQ R package provides a generic tool which can be applied on a wide range of somatic mutations. Its implementation in a Shiny web interactive application (available at https://ucl-irec-ctma.shinyapps.io/Pyrosequencing-NRAS-61/) enables its use in research or clinical routine applications.

In this study, the first survey of microbial community in thermophilic anaerobic digester using swine manure as sole feedstock was performed by multiple approaches including denaturing gradient gel electrophoresis (DGGE), clone library and pyrosequencing techniques. The integrated analysis of 21 DGGE bands, 126 clones and 8506 pyrosequencing read sequences revealed that Clostridia from the phylum Firmicutes account for the most dominant Bacteria. In addition, our analysis also identified additional taxa that were missed by the previous researches, including members of the bacterial phyla Synergistetes, Planctomycetes, Armatimonadetes, Chloroflexi and Nitrospira which might also play a role in thermophilic anaerobic digester. Most archaeal 16S rRNA sequences could be assigned to the order Methanobacteriales instead of Methanomicrobiales comparing to previous studies. In addition, this study reported that the member of Methanothermobacter genus was firstly found in thermophilic anaerobic digester.

BACKGROUND: In this report, we discuss the case of a 39-year-old woman presenting with a case of chronic maxillary sinusitis.
METHODS: Dialister pneumosintes, Staphylococcus epidermidis, and Peptostreptococcus spp. were isolated from endosinusal samples obtained during surgery. The patient showed extensive periodontopathy and had undergone prior endodontic treatment for endodontic infection of teeth #13, #14, and #15, which failed and presumably acted as a bridge for the sinusal infection. After nasosinusal surgery, consisting of opening and toilet of the maxillary sinus, combined with extraction of the 3 previously mentioned teeth and antibiotic treatment, the patient showed complete healing.
RESULTS: S. epidermidis and Peptostreptococcus spp. were identified with a traditional biochemical test and confirmed by pyrosequencing. Conversely, D. pneumosintes could not be identified with the conventional method, but it was identified using DNA pyrosequencing. In addition, to better understand the role and the virulence of this bacterium in odontogenic sinusitis, we have evaluated the ability of D. pneumosintes to produce biofilms onto inert surfaces. D. pneumosintes is a known endodontic and periodontal pathogen found in necrotic pulp, subgingival plaque, and deep periodontal pockets.
CONCLUSIONS: To our knowledge, the pathogenic role of D. pneumosintes in odontogenic sinusitis has never been evidenced. Thus, its detection in endosinusal specimens may provide a significant insight into the pathogenesis of this relevant medical condition.

BACKGROUND: Hippocampal sclerosis is the most common lesion in patients with mesial temporal lobe epilepsy. Recently, there has been growing evidence on the involvement of mitochondria also in sporadic forms of epilepsy. In addition, it has been increasingly argued that mitochondrial dysfunction has an important role in epileptogenesis and seizure generation in temporal lobe epilepsy. Although mtDNA polymorphisms have been identified as potential risk factors for neurological diseases, the link between homoplasmy and heteroplasmy within tissues is not clear. We investigated whether mitochondrial DNA (mtDNA) polymorphisms are involved in a case report of a patient with mesial temporal lobe epilepsy-hippocampal sclerosis (MTLE-HS).
METHODS: We report the whole genome mtDNA deep sequencing results and clinical features of a 36-year-old woman with MTLE-HS. We used pyrosequencing technology to sequence a whole mitochondrial genome isolated from six different regions of her brain and blood. To assess the possible role of mitochondrial DNA variations in affected tissues, we compared all specimens from different regions of the hippocampus and blood.
RESULTS: In total, 35 homoplasmic and 18 heteroplasmic variations have been detected in 6 different regions of the hippocampus and in blood samples. While the samples did not display any difference in homoplasmic variations, it has been shown that hippocampus regions contain more heteroplasmic variations than blood. The number of heteroplasmic variations was highest in the CA2 region of the brain and accumulated in ND2, ND4 and ND5 genes. Also, dentate and subiculum regions of the hippocampus had similar heteroplasmic variation profiles.
CONCLUSIONS: We present a new rare example of parallel mutation at 16223 position. Our case suggests that defects in mitochondrial function might be underlying the pathogenesis of seizures in temporal lobe epilepsy.