Ligation-dependent RT-PCR: a new specific and low-cost technique to detect ALK, ROS, and RET rearrangements in lung adenocarcinoma

Nicolas Piton , Philippe Ruminy (1) , Claire Gravet , Vinciane Marchand (1) , Élodie Colasse , Aude Lamy (1) , Cécile Le Naoures Mear , Frédéric Bibeau (2) , Florent Marguet , Florian Guisier , Mathieu Salaün (3) , Luc Thiberville (3) , Fabrice Jardin (1) , Jean-Christophe Sabourin (1)

1 GPMCND - Génomique et Médecine Personnalisée du Cancer et des Maladies Neuropsychiatriques
2 IRCM - U1194 Inserm - UM - Institut de Recherche en Cancérologie de Montpellier
3 QuantIF-LITIS - Equipe Quantification en Imagerie Fonctionnelle

Detection of anaplastic lymphoma kinase (ALK), ROS proto-oncogene 1 (ROS1), and rearranged during transfection (RET) gene rearrangements in lung adenocarcinoma is usually performed by immunohistochemistry (IHC) screening followed by fluorescence in situ hybridization (FISH), which is an expensive and difficult technique. Ligation-dependent reverse transcription polymerase chain reaction (RT-PCR) multiplex technique can detect gene rearrangements using probes specifically hybridized to either side of the break point. PCR products are then sequenced by pyrosequencing or high throughput sequencing in order to identify the two genes involved. The reagent cost is <15 dollars per patient and results are available in 2 days. We have developed a 47-probe LD-RT-PCR kit especially for lung adenocarcinomas. Thirty-nine lung adenocarcinomas were studied: 24 ALK+, 14 ROS1+, and 1 RET+. ALK+ and ROS1+ were IHC+ (D5F3 Ventana for ALK and D4D6 Cell Signaling Technology for ROS1) and all cases were FISH+ (Vysis ALK Breakapart Probe Abbott for ALK, Zytolight SPEC ROS1 Dualcolor Breakapart Probe for ROS1 and Zytolight SPEC RET Dual Color Breakapart for RET); 14 wild type samples were included as negative controls. Using LD-RT-PCR, 15 rearrangements (63%) were detected in the ALK cases (gene partner: EML4 in all cases), 9 rearrangements (64%) in the ROS1 cases (gene partners: CD74 in 8 cases and SLC34A2 in 1 case) and 1 (100%) in the single RET case (gene partner: KIF5B). No rearrangement was found in the 14 negative control cases. Negative cases using LD-RT-PCR could be explained by the fact that some partner genes were not included in our assay and therefore could not be detected. Because it is an affordable, fast, and very simple technique, we propose using LD-RT-PCR when ALK immunostaining is positive. For LD-RT-PCR-negative cases, samples should then be analyzed by FISH.

Domaines: Sciences du Vivant [q-bio]

Nicolas Piton, Philippe Ruminy, Claire Gravet, Vinciane Marchand, Élodie Colasse, et al.. Ligation-dependent RT-PCR: a new specific and low-cost technique to detect ALK, ROS, and RET rearrangements in lung adenocarcinoma. Laboratory Investigation, 2017, 98 (3), pp.371-379. ⟨10.1038/labinvest.2017.124⟩. ⟨hal-02054251⟩