Name: Sequencing Analysis of Non-Squamous Non-Small Cell Lung Cancer Using Integrated Genomic Viewer Software
Uploaded: 2023-09-08T14:20:00
Description: Watch this Scientific Journal Video about Sequencing Analysis of Non-Squamous Non-Small Cell Lung Cancer Using Integrated Genomic Viewer Software at JoVE.com

sequencing analysis of non-squamous non-small cell lung cancer using integrated genomic viewer software 

הערכת שינוי גנומי בחולי סרטן ריאה מסוג תאים לא קטנים שאינם קשקשיים

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.

זרקור המחבר: התקדמות בבדיקות סמנים ביולוגיים מולקולריים לסרטן ריאה של תאים לא קטנים שאינם קשקשיים 

ריצוף הדור הבא מבוסס אמפליקון מהיר במיוחד בסרטן ריאות של תאים לא קטנים שאינם קשקשיים

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 Sequencing Analysis of Non-Squamous Non-Small Cell Lung Cancer Using Integrated Genomic Viewer Software at JoVE.com

Sequencing Analysis of Non-Squamous Non-Small Cell Lung Cancer Using Integrated Genomic Viewer Software   

ניתוח ריצוף של סרטן ריאה מסוג תאים לא קטנים שאינם קשקשיים באמצעות תוכנת Integrated Genomic Viewer 

כדי להתחיל, פתח את תוכנת המציג הגנומי המשולב. בתפריט תוצאות, לחץ על תוצאות לדוגמה. לאחר מכן לחץ על שם המדגם של עניין מעמודת שם המדגם כדי להציג תוצאות רצף.כדי לסקור את תוצאות תוסף ניתוח הכיסוי המולקולרי, בפינה השמאלית העליונה, לחץ על הורד קבצים. לאחר מכן, בסיכום ההפעלה, לחץ על הכרטיסיה הפעל דוח כדי להציג מדדי בדיקה והפעל דוח. כדי להציג את התוצאה SNV ו- INDEL, לחץ על הכרטיסיה שונות ולאחר מכן לחץ על SNV ו- INDELs.לחץ על ערוך מסננים ובחר ללא מסנן. לאחר מכן, לחץ על הכרטיסייה שונות ולאחר מכן על היתוך כדי להציג תוצאות היתוך בגרסאות אקסון RNA. בפינה השמאלית העליונה, לחץ על תצוגה חזותית ו- RNA exon variant.לאחר מכן סקור את תרשים השונות אקסון RNA. לחץ/י על הכרטיסיה ״שונות״ ולאחר מכן בחר/י ״פיוזים״ כדי להציג את חוסר האיזון של איחוי אריח האילון של RNA. בפינה השמאלית העליונה, לחץ על תצוגה חזותית ולאחר מכן על RNA Exon Tile Fusion Imbalance וסקור את התרשימים RNA Exon Tile Fusion Imbalance.בכרטיסייה שונות, לחץ על CNV כדי להציג תוצאות CNV. לבסוף, צור דוח וריאנט על ידי לחיצה על הקישור להורדת ה- PDF. מחקר זה מייצג את הניתוח המולקולרי שבוצע ב -259 חולים עם NS-NSCLC כולל סוגי ביופסיה שונים ודגימות כירורגיות.הגנים המוטנטיים המניעים ואיחוי הגנים נצפו גם בניתוח DNA-RNA. רגישות השיטה לאיתור החדרות ומחיקות של שונות נוקלאוטידים בודדים, שונות מספרי העתקה ואיחוי הייתה גבוהה ונעה בין 91.67% ל-100%

Research

JoVE Journal

Biology

Sequencing Analysis of Non-Squamous Non-Small Cell Lung Cancer Using Integrated Genomic Viewer Software 

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 ...