Name: Preparation of FFPE DNA and RNA Samples for Sequencing Using an Automated Purification Instrument (API)
Uploaded: 2023-09-08T14:20:00
Description: Watch this Scientific Journal Video about Preparation of FFPE DNA and RNA Samples for Sequencing Using an Automated Purification Instrument API at JoVE.com

preparation of ffpe dna and rna samples for sequencing using an automated purification instrument (api)

Vurdering av genomisk endring hos pasienter med ikke-plateepitel, ikke-småcellet lungekreft

Over the last decade, the development of targeted and immuno-therapies has significantly increased the overall survival (OS) of non-squamous non-small cell lung cancer (NS-NSCLC)1,2. In this regard, the ,number of mandatory genes and molecular targets to analyze when treating NS-NSCLC has increased over the last few years3,4.
Current international guidelines recommend testing EGFR, ALK, ROS1, BRAF, NTRK, RET, and MET at diagnosis of advanced NS-NSCLC5. Moreover, as new drugs have recently given very promising results in clinical trials, additional genomic alterations will shortly be screened in a number of additional genes, notably KRAS and HER2, along with BRAC1/BRAC2, PI3KA, NRG1, and NUT6,7,8,9. In addition, the status of different associated genes, such as STK11, KEAP1, and TP53 may be of strong interest for a better prediction of the response or resistance to some targeted therapies and/or immune checkpoint inhibitors (ICIs)10,11,12.
Importantly, the molecular alterations must be reported without significant delay to ensure careful clinical decision-making. The absence of molecular characterization of a tumor may lead to the initiation of non-targeted therapies such as chemotherapy with/without immunotherapy, leading to a suboptimal treatment strategy, as chemotherapy response is limited in patients with actionable alterations, such as EGFR mutations or gene fusions13.
Moreover, the current development of targeted therapies/immunotherapies in neoadjuvant and/or adjuvant settings could lead to systematically looking for, at least, EGFR and ALK alterations in early-stage NS-NSCLC as ICIs should be administered only in tumors that are wild-type for EGFR and ALK14. It is now also mandatory to test for the presence of EGFR mutations in early-stage NS-NSCLC, since osimertinib (a third-generation EGFR tyrosine kinase inhibitor) can be used as adjuvant therapy in EGFR-mutant NS-NSCLC15.
The strategy for the assessment of the different biomarkers in predicting the response to different targeted therapies and/or immunotherapies in NS-NSCLC patients is moving fast, which makes the identification of these biomarkers sequentially difficult3,16. In this regard, Next-Generation Sequencing (NGS) is now the optimal approach for high throughput parallel assessment of gene alterations in NS-NSCLC5,17.
However, NGS workflow can be difficult to master and may conduct to longer TAT18,19. Thus, many centers still perform sequential approaches (immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) and/or targeted sequencing). However, this strategy is limited in case of small sample size and, above all, because of the increased number of actionable mutations that are required to be tested in NS-NSCLC20. Thus, ultra-fast and straightforward testing methods allowing the rapid assessment of gene alterations have become increasingly important for optimal clinical decision-making. Moreover, approved and accreditated systems for molecular testing are becoming mandatory for the prescription of specific targeted therapies.
Here, we describe an ultra-fast and automated amplicon-based DNA/RNA NGS assay for molecular testing of NS-NSCLC that is used in the Laboratory of Clinical and Experimental Pathology Laboratory (LPCE), Nice University Hospital, France and is accredited according to the ISO 15189 norm by the French Accreditation Committee (COFRAC) (https://www.cofrac.fr/). The COFRAC certifies that the laboratory fulfills the requirements of the standard ISO 15189 and COFRAC rules of application for the activities of testing/calibration in molecular analysis in automated NGS on a sequencer with the panel performed by the laboratory. Accreditation per the recognized international standard ISO 15189 demonstrates the laboratory&#8217;s technical competence for a defined scope and the proper operation of an appropriate management system in this laboratory. The benefits and limitations of this workflow, starting from the preparation of tissue biopsy samples to obtaining the report, are discussed.

Forfatter Spotlight: Fremskritt innen molekylær biomarkørtesting for ikke-plateepitel, ikke-småcellet lungekreft 

Ultra-rask ampliconbasert neste generasjons sekvensering i ikke-plateepitel ikke-småcellet lungekreft

Nowadays, targeted therapies for non-small cell lung cancer require rapid detection of multiple genomic alterations. So it's crucial for an optimal care, as thoracic biopsy are often small and contain few tumor cells. So we present an ultra-fast Next-Generation Sequencing workflow that is implemented for routine non-small cell lung cancer diagnosis in the pathology laboratory.Currently targeted NGS using different panel sizes, one gene sequencing testing using RT-PCR, digital PCR, fluorescence in situ hybridization, and immunohistochemistry are the different methods used to detect molecular alterations. However, most of these methods can be time and tissue material consuming, leading to a delayed diagnosis, and a risk of tissue exertion. The challenge lies in efficiently detecting multiple molecular alterations while saving materials and answering a speeding diagnosis.The workflow provides thoracic oncologist with comprehensive tumor information, including histological diagnosis, PD-L1 status, and targetable genomic aberrations. It enables rapid treatment decision making for optimal therapy selection. Our protocol identify all key molecular targets in thoracic oncology based on ESMO, NCCN, and ASCO recommendations, delivers timely results even with small samples sizes and limited DNA/RNA.And so the automated system reduced technician workload with just three hours of wet lab manipulation. We aim to focus on the rapid and comprehensive genomic profiling characterizations of tumors by developing a large NGS panel for tissue and plasma samples in the future. However, increased strategies to detect numerous molecular alterations in small sample sizes and cytological samples using large panels are urgently needed.

Watch this Scientific Journal Video about Preparation of FFPE DNA and RNA Samples for Sequencing Using an Automated Purification Instrument API at JoVE.com

Preparation of FFPE DNA and RNA Samples for Sequencing Using an Automated Purification Instrument (API)  

Klargjøring av FFPE DNA- og RNA-prøver for sekvensering ved bruk av et automatisert renseinstrument (API)

For å begynne, kutt de formalinfaste parafininnebygde eller FFPE DNA- og RNA-prøvene. Slå på det automatiserte rense- eller API-instrumentet, og logg på med brukernavnet og passordet. Deretter, i Integrated Genomic Viewer-programvaren, under prøver, klikker du Opprett prøver "skriv inn prøvenavnet og velg applikasjonskategorien.Etter å ha skrevet inn prøvedetaljene, klikk på Lagre"Velg deretter løp"klikk på Prøve til resultat"og skriv inn kjørenavnet før du klikker Neste"Velg analysen etterfulgt av en prøve av interesse og klikk Neste"Etter å ha valgt hver prøve, klikk Tildel"sjekk deretter elueringsparametrene og lagre kjøringen. Plasser FFPE vevsprøver og prøvebehandlingsrør. Sentrifuge røret ved 2.000 G i ett minutt.Tilsett Protease Digest-masterblandingen fremstilt fra et nukleinsyrerensingssett i røret. Inkuber prøven ved 60 grader Celsius i en time, etterfulgt av å inkubere den ved 90 grader Celsius i en time. Etter avkjøling av prøvene, løft det indre røret av prøvebehandlingsrøret og lås det ved å vri til venstre.Sentrifuge det indre røret ved 2.000 G i 10 minutter. Deretter låser du opp røret ved å vri til høyre og skille det fra det andre røret. Kast det indre røret og legg det ytre røret på is til det legges på API.Forbered en hovedblanding av DNA-fordøyelse og last i ferdigfylt FFPE DNA og RNA Purification plate to. Legg de forberedte prøvene i FFPE DNA og RNA renseplate en. Klikk på API-skjermen Kjør"velg deretter kjøreplanen og klikk Neste"Følg instruksjonene på skjermen for å laste de nødvendige forbruksvarer og reagenser for rensekjøringen.Når alle reagensene er lastet, klikker du Neste "lukk API-døren og klikk Start"T slutten av kjøringen, klikk Loss"og fjern umiddelbart den 48-brønns nukleinsyrearkivplaten som inneholder det rensede DNA og RNA. Etter at prøverensingen er over, forsegl platen og oppbevar den ved minus 80 grader Celsius.

Research

JoVE Journal

Biology

Preparation of FFPE DNA and RNA Samples for Sequencing Using an Automated Purification Instrument (API)

Ultra-Fast Amplicon-Based Next-Generation Sequencing in Non-Squamous Non-Small Cell Lung Cancer

The number of molecular alterations to be tested for targeted therapy of non-squamous non-small cell lung cancer (NS-NSCLC) patients has significantly ...