The presence of wild-type RAS alleles, as determined by genotyping codons 12, 13, 59, 61, 117, and 146, is a prerequisite for personalized anti-EGFR treatment of metastatic colorectal cancer (mCRC) patients. Here we describe analytical validation of in-house developed massively parallel sequencing technology (MPS) in comparison to the in vitro diagnostics (IVD) certified qPCR method. DNA extracted from FFPE samples from CRC patients (n=703) and reference standards (n=33) were tested for KRAS and NRAS mutations in 6 codons of exons 2, 3, and 4 using deep amplicon sequencing (DAS) on a MiSeq benchtop sequencer (Illumina). Two different amplicon lengths and two different library preparation methods (long-RAS and short-RAS) were tested in order to evaluate their impact on DAS performance. In parallel, identical tumor DNA was tested by the following IVD assays: therascreen KRAS RGQ PCR Kit (Qiagen), cobas® KRAS Mutation Test (Roche Diagnostics), and SNaPshot assay (Thermo Fisher Scientific). Both DAS assays detected all the mutations present in reference standards and external quality control samples, except for the artificially generated KRAS codon 146 mutation. The DAS assays performed sufficient analytical specificity and sensitivity (≥0.95). The use of shorter amplicons prolonged the preparation steps but significantly improved the sequencing success rate of FFPE-derived DNA. RAS mutation frequencies in the Czech CRC patients were similar to previous reports, although rare mutations were also detected. DAS with short amplicons is a good strategy for routine assessment of somatic mutations in low-quality FFPE-derived DNA.
BACKGROUND Histiocytic sarcoma is a rare malignant hematopoietic neoplasm with morphologic and immunohistochemical features of histiocytic differentiation, usually with unfavorable prognosis. Despite aggressive biological behavior, in subgroup of patients with localized disease, the prognosis can be very good. Few publications are available on localized cases of histiocytic sarcoma. These occur infrequently and continue to be a poorly-recognized morphological entity. CASE REPORT A 73-year old man treated for Parkinson syndrome presented with a tumor resistance on the dorsal surface of the left forearm. This lesion was clinically seen as an organized hematoma and was surgically resected. Histologically, the tumor was situated in the dermis and subcutis and it consisted of multiple neoplastic nodules. Vasoformative growth patterns with the vascular-like spaces containing erythrocytes and hemosiderin pigment presence simulated the morphology of angiosarcoma. Based on the immunohistochemical characteristics, we diagnosed the tumor as cutaneous histiocytic sarcoma. Genetic analysis revealed immunoglobulin heavy-chain gene rearrangement without any concomitant hematological malignancy. The patient demonstrated no systemic disease or impairment associated with diagnosed histiocytic sarcoma, and no recurrence has been found to date. CONCLUSIONS We report a case of primary cutaneous histiocytic sarcoma with an excellent outcome after surgical treatment only. Clinical data and histopathological and immunohistochemical evaluation were essential to rule out other malignant tumors in the differential diagnosis. Genetic analysis together with up-to-date knowledge and understanding of principles of tumorous transformations helped to diagnose this poorly-recognized entity with various clinical behaviors.
Karcinom prsu patří mezi nejčastější zhoubná onemocnění u žen. Signální dráha PI3K/Akt/mTOR hraje důležitou roli v řadě buněčných procesů podílejících se na proliferaci, metabolismu, buněčném růstu a přežití. Alterace této signální dráhy byly nalezeny u celé řady nádorů, včetně nádoru prsu. Konstitutivní aktivace PI3K/Akt/mTOR signální dráhy hraje klíčovou roli v patogenezi nádoru a rezistenci na konvenční terapii. V souvislosti s nově schválenou léčbou pomocí inhibitorů PI3K nabývá na významu i stanovení PIK3CA mutačního statusu jako prediktivního markeru léčebné odpovědi.
Breast cancer is one of the most common malignancies in women. The PI3K/Akt/mTOR signaling pathway plays an important role in several cellular processes involved in proliferation, metabolism, cell growth, and survival. Alterations in this signaling pathway have been found in a variety of tumors, including breast cancer. Constitutive activation of the PI3K/Akt/mTOR signaling pathway plays a key role in tumor pathogenesis and resistance to conventional therapy. In connection with the newly approved treatment with PI3K inhibitors, the determination of PIK3CA mutation status as a predictive marker is also gaining in importance.
- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference MeSH
Mutations in IDH1/2 genes are a marker of good prognosis for glioma patients, associated with low grade gliomas and secondary glioblastomas. Immunohistochemistry and Sanger sequencing are current standards for IDH1/2 genotyping while many other methods exist. The aim of this study was to validate Competitive amplification of differentially melting amplicons (CADMA) PCR for IDH genotyping by comparison with SNaPshot assay and two immunohistochemical methods. In our study, 87 glioma patients (46 from Olomouc and 41 from Ostrava) were analyzed. IDH1/2 mutations in native bioptical samples were analyzed at DNA level by CADMA and SNaPshot while IDH1 mutations in FFPE samples were analyzed at protein level by two IHC methods. CADMA PCR sensitivity for IDH1 was 96.4% and specificity 100% for 86 concluded samples. SNaPshot assay sensitivity was 92.9% and specificity of 100% for 85 concluded samples. IHC in the laboratory no. 2 reached sensitivity 85.7% and specificity 100% for 86 concluded samples. IHC in the laboratory no. 4 reached sensitivity of 96.4% and specificity of 79.7% in 74 concluded samples. Only one IDH2 mutation was found by SNaPshot while CADMA yielded false negative result. In conclusion, CADMA is a valid method for IDH1 p.(R132H) testing with higher sensitivity than SNaPshot assay. Also, molecular genetic methods of IDH1 testing from native samples were more robust than IHC from FFPE.
- MeSH
- glioblastom genetika MeSH
- gliom genetika MeSH
- imunohistochemie metody MeSH
- isocitrátdehydrogenasa genetika MeSH
- lidé MeSH
- mutace genetika MeSH
- mutační analýza DNA metody MeSH
- nádorové biomarkery genetika MeSH
- nádory mozku genetika MeSH
- senzitivita a specificita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Cíl práce: Diagnostika Bartonella henselae metodou polymerázové řetězové reakce (PCR) v exstirpovaných uzlinách 10 pacientů se sérologickým průkazem nemoci z kočičího škrábnutí. Materiál a metody: Soubor z období 2015–2018 zahrnoval 10 pacientů se sérologicky prokázanou nemocí z kočičího škrábnutí, všichni pacienti měli pozitivitu IgG protilátek proti B. henselae, 6 pacientů současně i pozitivitu IgM protilátek. Jednalo se o 4 muže a 6 žen, celkem 7 dětí a 3 dospělé ve věkovém rozmezí 5–52 let. Vyšetřeno bylo 11 exstirpovaných uzlin uložených v parafinových blocích od 10 pacientů, varianty granulomatózního zánětu byly zjištěny u 9 pacientů, chlapec ve věku 13 let měl Hodgkinův lymfom. Pro izolaci DNA byla použita komerční souprava cobas ® DNA Sample preparation Kit (Roche) a pro detekci DNA Bartonella sp. metodou real-time PCR komerční souprava BactoReal® Kit Bartonella spp. (Ingenetix) využívající detekci gltA genu specifického pro rod Bartonella. Výsledky: Průkaz bartonely v histologickém preparátu metodou PCR byl pozitivní či hraničně pozitivní u 4 z 10 pacientů. U jednoho pacienta byly vyšetřeny 2 uzliny s pozitivním výsledkem jen v jedné uzlině. Výsledek byl pozitivní u dospělého muže, slabě pozitivní u 3 dětí. Dva z těchto 4 pacientů měli histologický nález granulomatózně-abscedujícího a 2 pacienti granulomatózně-nekroticko-abscedujícího zánětu. Pozitivita či hraniční pozitivita PCR byla zjištěna u 4 pacientů s pozitivitou protilátek třídy IgM proti B. henselae, ale u žádného pacienta s negativitou IgM protilátek. Rovněž chlapec s lymfomem měl negativní výsledek PCR diagnostiky. Závěr: Kombinace sérologického vyšetření s histologickým vyšetřením uzlin a PCR metodou může zlepšit diagnostiku nemoci z kočičího škrábnutí.
Objective: The diagnosis of Bartonella henselae by polymerase chain reaction (PCR) in lymph nodes removed in 10 patients with serologically confirmed evidence cat-scratch disease. Material and methods: The 2015–2018 group consisted of 10 patients with serologically confirmed cat-scratch disease, all of them having positive IgG antibodies and 6 patients also positive IgM antibodies against B. henselae. The group included 4 men and 6 women, 7 children and 3 adults, aged 5–52 years. Eleven lymph nodes obtained from the 10 patients were formalin-fixed paraffin-embedded. Variants of granulomatous inflammation were found in 9 patients; a 13-year-old boy had Hodgkin’s lymphoma. DNA isolation was per- formed with cobas ® DNA Sample Preparation Kit (Roche). DNA of Bartonella spp. was detected by real-time PCR with BactoReal ® Kit Bartonella spp. (Ingenetix) detecting the gltA gene specific for the genus Bartonella. Results: Four of the 10 patients tested positive or borderline positive for Bartonella when their histological material was analyzed by PCR. One patient with 2 lymph nodes examined showed a positive result for only 1 lymph node. One adult male had a positive result; three children showed borderline positive results. Of those, two patients had suppurative granulomatous and the other 2 patients had necrotizing suppurative granulomatous inflammation as histological findings. All 4 patients had positive IgM antibodies against B. henselae. The boy with lymphoma had a negative PCR result. Conclusion: Serological tests combined with histological examination of lymph nodes and PCR may improve the diagnosis of cat-scratch disease.
- Publikační typ
- abstrakt z konference MeSH
- MeSH
- diagnostické techniky molekulární * MeSH
- DNA virů * MeSH
- infekce papilomavirem * diagnóza genetika MeSH
- lidé MeSH
- messenger RNA MeSH
- nádory děložního čípku diagnóza genetika MeSH
- nádory orofaryngu diagnóza genetika MeSH
- Papillomaviridae genetika patogenita MeSH
- RNA virová MeSH
- Check Tag
- lidé MeSH
BACKGROUND AND OBJECTIVES: Crohn's disease is a multifactorial inflammatory disease affecting mainly the gastrointestinal tract. The genetic factors that are involved in the disease include mainly three mutations of the gene NOD2/CARD15 (R702W, G908R, 3020insC). The aim of this study was to determine the relationship between the presence of these variants and disease phenotype. MATERIAL AND METHODS: 70 patients with Crohn's disease were examined for the presence of the above-mentioned mutations. The researchers used the medical records to retrospectively obtain clinical data and together with the information obtained prospectively according to the protocol they analysed the connection between gene mutations and disease phenotype. RESULTS: At least one mutation was found in 22 patients with Crohn's disease (32%), four patients were found to have two different mutations (composed heterozygotes - 6%) and six patients (9%) were homozygotes for the 3020insC gene. No significant differences were found between the groups with wild-type form and the mutated form of the NOD2 / CARD15 gene with respect to age at the time of diagnosis, form of the disease or localization according to the Montreal classification. CONCLUSION: Mutations of the NOD2 / CARD15 gene did not significantly affect the frequency of reoperations, homozygotes with 3020insC gene mutations, however, represented a high risk group. The phenotype was not related significantly to the presence of the examined mutations.
- MeSH
- Crohnova nemoc genetika chirurgie MeSH
- dospělí MeSH
- fenotyp MeSH
- genetická predispozice k nemoci genetika MeSH
- heterozygot MeSH
- homozygot MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- mutace genetika MeSH
- prospektivní studie MeSH
- reoperace MeSH
- senioři MeSH
- signální adaptorový protein Nod2 genetika MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
