MicroRNAs are considered as promising prognostic and diagnostic biomarkers of human cancer since their profiles differ between tumor types. Most of the tumor profiling studies were performed on rarely available fresh frozen (FF) samples. Alternatively, archived formalin-fixed paraffin-embedded (FFPE) tissue samples are also well applicable to larger-scale retrospective miRNA profiling studies. The aim of this study was to perform systematic comparison of the miRNA expression profiles between FF and macrodissected FFPE tonsillar tumors using the TaqMan Low Density Array system, with the data processed by different software programs and two types of normalization methods. We observed a marked correlation between the miRNA expression profiles of paired FF and FFPE samples; however, only 27-38% of the differentially deregulated miRNAs overlapped between the two source systems. The comparison of the results with regard to the distinct modes of data normalization revealed an overlap in 58-67% of differentially expressed miRNAs, with no influence of the choice of software platform. Our study highlights the fact that for an accurate comparison of the miRNA expression profiles from published studies, it is important to use the same type of clinical material and to test and select the best-performing normalization method for data analysis.
- MeSH
- Principal Component Analysis MeSH
- Tissue Fixation MeSH
- Fixatives MeSH
- Formaldehyde MeSH
- Palatine Tonsil metabolism MeSH
- Cryopreservation * MeSH
- Humans MeSH
- MicroRNAs metabolism MeSH
- Microarray Analysis MeSH
- Signal Processing, Computer-Assisted MeSH
- Software MeSH
- Gene Expression Profiling MeSH
- Tonsillar Neoplasms metabolism MeSH
- Paraffin Embedding * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Comparative Study MeSH
Formalin-fixed, paraffin-embedded (FFPE) tissue is the most common tissue specimen available after microscopic examination. Molecular methods, such as polymerase chain reaction (PCR) and gene expression examination, serve as a source of diagnostic and prognostic information but require high-quality RNA. However, the increasing application of RNA extracted from FFPE tissue frequently results in very small and degraded quantities of nucleic acid. This study targets gene expression analysis from FFPE specimens using real-time quantitative PCR. The whole protocol consists of several steps, that is, RNA extraction and its quality control, reverse transcription, and fluorescence detection during real-time quantitative PCR. We compared several methods in each step, chose the most effective, and with that combination we successfully examined 95% (62 from 65) FFPE samples for our genes of interest. We reached the best results with RNA isolation by using a commercial kit, carefully interpreted UV spectrophotometric values, and meticulously chose reverse transcriptase and TaqMan fluorescence detection. Our protocol improves the utility of FFPE tissue for molecular profiling studies.
- MeSH
- Fixatives pharmacology MeSH
- Formaldehyde pharmacology MeSH
- Humans MeSH
- Specimen Handling methods MeSH
- Pathology methods MeSH
- Polymerase Chain Reaction methods MeSH
- RNA genetics isolation & purification MeSH
- Gene Expression Profiling methods MeSH
- Tissue Preservation MeSH
- Paraffin Embedding MeSH
- Check Tag
- Humans MeSH
- Publication type
- Evaluation Study MeSH
- Research Support, Non-U.S. Gov't MeSH
To confirm a diagnosis of malignant lymphomas it is imperative to distinguish between reactive and neoplastic proliferation. The PCR (polymerase chain reaction) is a method that can be used for detection of clonal rearrangements of the immunoglobulin heavy chain (IgH) and T-cell receptor (TCR) genes. This study summarizes the outcomes of PCR analysis of IgH and TCR gene rearrangements in 91 bioptic cases of lymphoproliferative disorders. In the class of B lymphomas we detected clonal IgH rearrangement in nearly 83% of cases and in class of T lymphomas in 81% of cases. We can affirm that PCR analysis of B and T cell clonality on DNA extracted from the whole section of formalin-fixed, paraffin-embedded tissue is very suitable for routinely elaborate this. Its influence on the diagnostics of morphological unclear cases in particular, is crucial and is useful in establishing a diagnosis of lymphoid neoplasias in specimens in which histological and immunophenotypic studies are inconclusive.
- MeSH
- Financing, Organized MeSH
- Fixatives MeSH
- Formaldehyde MeSH
- Gene Rearrangement, T-Lymphocyte MeSH
- Humans MeSH
- Lymphoproliferative Disorders diagnosis genetics MeSH
- Polymerase Chain Reaction MeSH
- Gene Rearrangement, B-Lymphocyte, Heavy Chain MeSH
- Paraffin Embedding MeSH
- Check Tag
- Humans MeSH
- Publication type
- Evaluation Study MeSH
Současně platná molekulárně genetická subklasifikace pacientů s diagnózou difuzního velkobuněčného B lymfom (DLBCL) do tří prognostických podskupin na základě expresního profilu je cílem mnoha genetických studií. V rutinní klinické praxi je však použití expresní čipové technologie pro většinu pracovišť nedostupné. Včetně potřebné technologie, je v některých případech pro molekulárně genetické laboratoře problémem získat kvalitní materiál, např. čerstvou tkáň, pro izolaci RNA k určení exprese genů. Jednou z možností je určovat expresi genů z RNA získané izolací z parafínových bločků. Cílem pilotní studie bylo provést izolaci RNA z archivovaných parafínových bločků z formalinem fixované tkáně (FFPE) nemocných s diagnózou DLBCL a ověřit možnost využití této RNA pro expresní analýzu 7 vybraných genů. Práce ukázala, že izolace RNA a určení exprese vybraných genů z archivovaného materiálu je možná, avšak naměřená relativní exprese některých genů vykazovala v našem souboru značně variabilní hodnoty pro využití pro jednoznačnou prognostickou klasifikaci. Potvrdili jsme, že při získání dostatečného množství materiálu je možné provést retrospektivní analýzy exprese vybraných genů, a že správná archivace i po 8 letech dovoluje molekulárně biologické analýzy.
The currently valid molecular genetic subclassification of patients with diffuse large B-cell lymphoma (DLBCL) into three prognostic subgroups based on expression profiling has been the objective of numerous genetic studies. In routine clinical practice, however, expression profiling technology remains unavailable for the most of centers. Apart from the technology, in some cases molecular genetic laboratories have problems obtaining high-quality material, i.e. fresh tissues, for RNA isolation to determine gene expression. One possibility is to determine the gene expression from RNA obtained by isolation from formalin-fixed, paraffin-embedded (FFPE) tissue. This pilot study aimed at isolating RNA from FFPE in patients diagnosed with DLBCL and verifying the potential use of such RNA for the expression analysis of 7 selected genes. Although the study showed that it is possible to isolate RNA and determine the expression of the selected genes from archival material, the values of relative expression of some genes in the set were too variable to be used for unambiguous prognostic classification. It was confirmed that retrospective analyses of selected genes may be performed with sufficient material obtained, and that properly archived blocks may be used for molecular biology analyses even after 8 years.
- Keywords
- parafínové bločky, genetické změny, BLIMP1,
- MeSH
- Time Factors MeSH
- Lymphoma, Large B-Cell, Diffuse * genetics pathology MeSH
- Adult MeSH
- Gene Expression MeSH
- Tissue Fixation methods MeSH
- Formaldehyde MeSH
- In Situ Hybridization, Fluorescence MeSH
- Immunohistochemistry MeSH
- Humans MeSH
- Specimen Handling utilization MeSH
- Pilot Projects MeSH
- Reverse Transcriptase Polymerase Chain Reaction MeSH
- Predictive Value of Tests MeSH
- Repressor Proteins genetics MeSH
- Reproducibility of Results MeSH
- Retrospective Studies MeSH
- Gene Expression Profiling * MeSH
- Paraffin Embedding * utilization MeSH
- Check Tag
- Adult MeSH
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
Microsatellite instability (MSI) is present in 15-20% of primary colorectal cancers. MSI status is assessed to detect Lynch syndrome, guide adjuvant chemotherapy, determine prognosis, and use as a companion test for checkpoint blockade inhibitors. Traditionally, MSI status is determined by immunohistochemistry or molecular methods. The Idylla™ MSI Assay is a fully automated molecular method (including automated result interpretation), using seven novel MSI biomarkers (ACVR2A, BTBD7, DIDO1, MRE11, RYR3, SEC31A, SULF2) and not requiring matched normal tissue. In this real-world global study, 44 clinical centers performed Idylla™ testing on a total of 1301 archived colorectal cancer formalin-fixed, paraffin-embedded (FFPE) tissue sections and compared Idylla™ results against available results from routine diagnostic testing in those sites. MSI mutations detected with the Idylla™ MSI Assay were equally distributed over the seven biomarkers, and 84.48% of the MSI-high samples had ≥ 5 mutated biomarkers, while 98.25% of the microsatellite-stable samples had zero mutated biomarkers. The concordance level between the Idylla™ MSI Assay and immunohistochemistry was 96.39% (988/1025); 17/37 discordant samples were found to be concordant when a third method was used. Compared with routine molecular methods, the concordance level was 98.01% (789/805); third-method analysis found concordance for 8/16 discordant samples. The failure rate of the Idylla™ MSI Assay (0.23%; 3/1301) was lower than that of referenced immunohistochemistry (4.37%; 47/1075) or molecular assays (0.86%; 7/812). In conclusion, lower failure rates and high concordance levels were found between the Idylla™ MSI Assay and routine tests.
- MeSH
- Tissue Fixation * MeSH
- Fixatives MeSH
- Formaldehyde MeSH
- Immunohistochemistry * MeSH
- Colorectal Neoplasms chemistry genetics pathology MeSH
- Automation, Laboratory MeSH
- Humans MeSH
- Microsatellite Instability * MeSH
- Mutation * MeSH
- DNA Mutational Analysis * MeSH
- Biomarkers, Tumor * analysis genetics MeSH
- Predictive Value of Tests MeSH
- Reproducibility of Results MeSH
- Paraffin Embedding * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Multicenter Study MeSH
- Comparative Study MeSH
Polycythemia vera (PV), esenciálna trombocytóza (ET) a primárna myelofibróza (PMF) patria do skupiny Ph1 negatívnych myeloproliferatívnych neoplázii (MPN), pri ktorých sa často vyskytuje mutácia JAK2V617F. Táto mutácia sa vyskytuje u takmer všetkých pacientov s PV a u približne 60 % pacientov s ET a PMF. V diferenciálnej diagnostike MPN však význam tejto mutácie stále nie je dosť objasnený, preto tu má dôležitú úlohu aj bioptické vyšetrenie kostnej drene. Na našom pracovisku sme sa rozhodli vyšetrovať JAK2V617F mutáciu v DNA izolovanej z parafínovych blokov od pacientov s PV, ET a PMF, keďže túto mutáciu nedávno SZO odporučila využívať ako jeden z markerov v diagnostike uvedených klinických jednotiek. Mutácie V617F sme detekovali pomocou alelovo-špecifickej hot start multiplex PCR. U JAK2V617F negatívnych pacientov s PV sme vyšetrovali sekvenovaním prítomnosť mutácií v exóne 12 JAK2 génu. Dosiaľ sme na našom pracovisku vyšetrili približne 200 pacientov s klinicky verifikovanou diagnózou PV, ET a PMF. Naše výsledky sú u všetkých troch ochorení (ET, PV, PMF) zhodné s dosiaľ publikovanými prácami. Nami implementovaná metodika detekcie mutácie JAK2 z parafínových blokov umožňuje analýzu veľkého množstva archívneho materiálu pre retrospektívne štúdie, a súčasne aj implementáciu analýzy statusu JAK2 ako súčasti bioptického vyšetrenia kostnej drene všetkých pacientov s podozrením na PV, ET alebo PMF.
Polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) are Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) characterized by JAK2 mutation. The exon 14 V617F mutation is present in almost all patients with PV and in approx. 60¨% of patients with ET and PMF. The importance of JAK2V617F in the differential diagnostic considerations is still unclear and here the BM morphology examination still represents an important diagnostic tool. In the WHO classification of Ph1-negative MPNs, the identification of JAK2 mutations represents a major diagnostic criterion of these diseases. Therefore we decided to implement the examination of JAK2V617F mutation in formalin-fixed paraffin-embedded biopsy specimens of patients with Ph1-negative MPN using allele-specific PCR. In addition, in all JAK2 V617F negative patients with PV we sequenced the whole JAK2 exon 12. Until now we examined up to 200 patients with clinically confirmed MPN and our results in all three categories PV, ET and PMF are in agreement with earlier published data. Paraffin embedded tissues represent a valuable source of DNA which can be used in the diagnostics of both JAK2 exon 12 and exon 14 mutations. It is of particular importance if the fresh material is not available and there is a clinical and/or research utility for the performance of PCR on archival bone marrow samples with PV, ET or PMF suspicion.
- MeSH
- Biopsy methods utilization MeSH
- Point Mutation genetics immunology MeSH
- Molecular Diagnostic Techniques utilization MeSH
- DNA genetics isolation & purification MeSH
- Thrombocythemia, Essential diagnosis genetics MeSH
- Financing, Organized MeSH
- Janus Kinase 2 genetics isolation & purification MeSH
- Humans MeSH
- Myeloproliferative Disorders diagnosis etiology genetics MeSH
- Polycythemia Vera diagnosis etiology genetics MeSH
- Polymerase Chain Reaction methods utilization MeSH
- Primary Myelofibrosis diagnosis genetics MeSH
- Retrospective Studies MeSH
- Bone Marrow Examination methods utilization MeSH
- Check Tag
- Humans MeSH
Ewingův sarkom je relativně vzácný nádor reprezentující 6–8 procent nádorů kostí. Cytogeneticky je Ewingův sarkom v 85 procentech případů charakterizován specifickou reciproční chromozomální translokací t(11;22)(q24;q12), která má za následek fúzi genu EWS na chromozomu 22 a genu FLI1 na chromozomu 11. V této studii jsme se zaměřili na porovnání dvou molekulárně diagnostických metod – reverzně transkripční polymerázové řetězové reakce (RT-PCR) a fluorescenční in situ hybridizace (FISH). Z našich výsledků vyplývá, že v případě formalínem fixované, do parafinu zalité tkáně je patrně vlivem degradace RNA, FISH senzitivnější než RT-PCR. Závěrem: molekulárně patologické metody RT-PCR a FISH jsou účinným diagnostickým nástrojem pro diagnostiku nádorů Ewingova typu.
Ewing's sarcoma is relatively uncommon tumor representing 6-8 percent of malignant bone tumors with variable morphology. Cytogenetically, Ewing's sarcomas are characterized by a specific reciprocal chromosomal translocation t(11;22)(q24;q12). The presence of this chromosomal translocation has been detected in approximately 85 percent of the cases. The translocation results in the fusion of EWS gene from chromosome 22 to FLI1 gene at 11q24 which is a member of ETS family of transcription factors. In this study we performed a comparison of two molecular diagnostic strategies, namely RT-PCR and FISH, in fresh, frozen and formalin-fixed paraffin-embedded tissues. We conclude that FISH is a more sensitive technique than RT-PCR for the diagnosis of Ewing's tumors in formalin-fixed paraffin-embedded tissue. In conclusion, molecular pathology techniques, using reverse transcription-polymerase chain reaction (RT-PCR) and/or fluorescence in situ hybridization (FISH) are valuable diagnostic tools for evaluation of undifferentiated small round-cell tumors like Ewing's sarcoma.
- MeSH
- Sarcoma, Ewing diagnosis genetics MeSH
- Financing, Organized MeSH
- Humans MeSH
- Reverse Transcriptase Polymerase Chain Reaction utilization MeSH
- Spectral Karyotyping MeSH
- Check Tag
- Humans MeSH
- Publication type
- Evaluation Study MeSH
