Evaluation of the optimal provision of formalin-fixed, paraffin-embedded material for reverse transcription-PCR in soft-tissue tumour diagnosis

Abstract

AimsMolecular genetic analysis is now a routine ancillary diagnostic modality to the histopathological diagnosis of soft-tissue neoplasms, many of which harbour characteristic gene fusions detectable by reverse transcription-PCR (RT-PCR). As the final diagnosis often depends on the molecular result, it is important to obtain the optimal yield of patient RNA.MethodsWe assessed the most reliable method of providing formalin-fixed, paraffin-embedded material for optimal RNA yield by comparing three consecutive periods in which different preparations (5×10 μm scrolls, 5×5 μm sections and 1×10 μm sections) were used for RNA extraction for RT-PCR, with its technical success rate.ResultsFor ‘2011’, ‘2012’ and ‘2013’, RT-PCR technical failure rates were 13.4%, 4.4% and 7.9%, respectively. The percentage of failed referral cases was 71.4%, 85.7% and 31.3%, and the proportion of core biopsy to excision specimens was 3:15, 2:5 and 13:3.ConclusionsThis study shows that the effectiveness of RNA extraction and purification is dependent on both specimen type and the tissue sectioning strategy. The failure rate has improved over recent years, particularly for large specimens as large numbers of thick 10 μm scrolls can saturate RNA extraction columns. In contrast, recent technical fails are more frequent in core biopsies, where 1×10 μm sections are insufficient for adequate RNA extraction. While previous technical fails occurred mostly in referred cases, this appears no longer the case due to the better fixation and processing of specimens in external surgical pathology departments because of the widespread recognition of the importance of molecular diagnostics as an important part of the patient pathway.

The spectrum of EWSR1-rearranged neoplasms at a tertiary sarcoma centre; assessing 772 tumour specimens and the value of current ancillary molecular diagnostic modalities

“…Amplification of B2M was performed to assess the quality of the RNA as described previously (Thway et al , 2016). RQ–PCR reactions were performed to assess for any or all of the following fusion transcripts: EWSR1-NR4A3, TAF15-NR4A3, EWSR1-FLI1, EWSR1-ERG, EWSR1-WT1, EWSR1-ATF1 and EWSR1-CREB1 ).…”

Development of a targeted sequencing approach to identify prognostic, predictive and diagnostic markers in paediatric solid tumours

“…13 paediatric cancer cell lines were tested harbouring a total of 30 known SNVs, deletions and amplifications previously identified by the Cancer Cell Line Encyclopaedia using Target Enrichment Sequencing (Agilent Technologies, Santa Clara, CA) and other published data [ 45 – 50 ]. Furthermore 33 samples (FF=14, FFPE=19) had a total of 65 known genetic alterations including i) SNVs detected by Sanger Sequencing ( H3F3A, TP53, CTNNB1, HIST1H3B, ALK, BRAF ) [ 51 – 53 ] and RNA-Seq ii) copy number changes by FISH ( MYCN )[ 54 ] and 450k array and iii) rearrangements by Real-Time Quantitative PCR involving ESWR1 as previously described [ 55 , 56 ] (Refer to Supplementary Materials ).…”

Impacto de PH del fijador, tiempo de fijación, tiempo previo sin fijación en la expresividad de marcadores moleculares de inmunohistoquímica

“…El formaldehido actúa en distintos puntos de las cadenas proteicas, generando grupos hidroximetil, que desencadena una reacción de enlaces cruzados con formación posterior de puentes de metileno transformando al tejido en insoluble, esta reacción es estable y prolongada (Dey, 2018;Thway et al, 2017). Ha sido usada por más de 50 años, normalmente en concentraciones al 10% bufferado, para mantener un pH neutro y una presión osmótica similar al medio externo de las células, evitando una contracción y endurecimiento excesivos (Lester, 2014;Dey, 2018).…”