A Single-Tube Multiplexed Assay for Detecting ALK, ROS1, and RET Fusions in Lung Cancer

Abstract

Approximately 7% of non-small cell lung carcinomas (NSCLCs) harbor oncogenic fusions involving ALK, ROS1, and RET. Although tumors harboring ALK fusions are highly sensitive to crizotinib, emerging preclinical and clinical data demonstrate that patients with ROS1 or RET fusions may also benefit from inhibitors targeting these kinases. Using a transcript-based method, we designed a combination of 3' overexpression and fusion-specific detection strategies to detect ALK, ROS1 and RET fusion transcripts in NSCLC tumors. We validated the assay in 295 NSCLC specimens and showed that the assay is highly sensitive and specific. ALK results were 100% concordant with fluorescence in situ hybridization (FISH) (n = 52) and 97.8% concordant with IHC (n = 179) [sensitivity, 96.8% (95% CI 91.0%-98.9%); specificity, 98.8% (95% CI 93.6%-99.8%)]. For ROS1 and RET, we also observed 100% concordance with FISH (n = 46 and n = 15, respectively). We identified seven ROS1 and 14 RET fusion-positive tumors and confirmed the fusion status by RT-PCR and FISH. One RET fusion involved a novel partner, cutlike homeobox 1 gene (CUX1), yielding an in-frame CUX1-RET fusion. ROS1 and RET fusions were significantly enriched in tumors without KRAS/EGFR/ALK alterations. ALK/ROS1/RET/EGFR/KRAS alterations were mutually exclusive. As a single-tube assay, this test shows promise as a more practical and cost-effective screening modality for detecting rare but targetable fusions in NSCLC.

Comprehensive analysis of RET and ROS1 rearrangement in lung adenocarcinoma

“…Furthermore, psammomatous calcification was seen in three cases. Interestingly, as previously mentioned in the histology of ALK-and ROS1-rearranged lung cancer, 16 the solid signet-ring cell pattern (solid growth pattern containing signet-ring cells; Figure 2a) was at least focally present in four (27%) cases and the mucinous cribriform pattern (cribriform structure associated with abundant extracellular mucus; Figures 2b and g) was identified at least focally in four (27%) cases. The solid signet-ring cell pattern was present in three of six (50%) KIF6B-RETpositive tumors and the mucinous cribriform pattern was present in four of five (80%) CCDC6-RETpositive tumors.…”

“…One tumor carried fusion of CUX1 exon 10 to RET exon 12, which was recently identified as an additional novel fusion partner gene in a previous study. 16 Four tumors carried EZR exon 10-ROS1 exon 34 fusion; three carried SLC34A2 exon 13-ROS1 exon 32 fusions; and one carried CD74 exon 6-ROS1 exon 34 fusion.…”

“…16 In addition, we collected 500 surgically resected lung adenocarcinoma samples from the Samsung Medical Center (SMC) with previous full informed consent from the patient and with approval from SMC. A total of 795 cases were screened for ALK, EGFR, and KRAS mutation status.…”

“…First-strand cDNA was initially synthesized using gene-specific primers. 16 cDNA was subdivided into different PCR reactions using the appropriate fusion variant primers, and PCR products were separated on a 2% E-Gel SizeSelect agarose gel (Invitrogen, Carlsbad, CA, USA). In reactions producing a PCR product of the expected size, the amplicons were gel-purified and sequenced using a 3700 ABI Prism sequencer (Applied Biosystems, Foster City, CA, USA).…”

Actualización de las recomendaciones para la determinación de biomarcadores en el carcinoma de pulmón avanzado de célula no pequeña. Consenso Nacional de la Sociedad Española de Anatomía Patológica y de la Sociedad Española de Oncología Médica

“…Aunque hay que tener cuidado con estos métodos untrasensibles, pues se pueden dar artefactos y aportar falsos positivos con mayor frecuencia 19 . Se asume que, con las tecnologías disponibles, son suficientes cantidades de 250-500 ng de ADN/ARN para realizar los estudios moleculares que exploren diferentes genes simultáneamente 46 . Es crucial que las instituciones establezcan las estrategias necesarias para optimizar los estudios de biomarcadores 47 , y que cada laboratorio determine su propio umbral de sensibilidad analítica, o límite de detección, para validar sus procedimientos.…”