Virtual Library
Start Your Search
J. Castellví
Author of
-
+
MINI 22 - New Technology (ID 134)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
-
+
MINI22.01 - Detecting ALK, ROS1 and RET Fusion Genes in Advanced Non-Small Cell Lung Cancer (NSCLC) Using a Novel Multiplexed NCounter-Based Assay (ID 2254)
16:45 - 16:50 | Author(s): J. Castellví
- Abstract
- Presentation
Background:
Gene fusions of anaplastic lymphoma kinase (ALK), ROS1, and RET are targetable oncogenes present in approximately 9% of advanced NSCLC. Current assays for detecting gene fusions are based on FISH (FDA-approved companion diagnostic test for ALK), immunohistochemistry (IHQ) and qRT-PCR. These tests, however, are complex and have disadvantages in terms of turnaround, sensitivity, cost and throughput. The nCounter platform allows joint detection, in a single tube, without any enzymatic reaction and in 72 hours, of multiple fusion genes by transcript-based method from formalin-fixed paraffin-embedded (FFPE) samples.
Methods:
A custom set consisting of 5´and 3´ probes and/or fusion-specific probes to detect ALK, ROS1 and RET fusion transcripts was evaluated. A panel of ALK-ROS-RET positive cell lines (H2228, H3122 [EML4-ALK], SU-DHL-1 [NPM-ALK], HCC78 [SLC34A2-ROS], BaF3 pBABE [CD74-ROS], LC2/ad [RET]) and control fusion negative cell lines (PC9, H1975 [EGFR mut], H460, H23 [KRAS mut]) were used for nCounter validation. To determine the minimum of tumor surface area for detection, ALK translocated cell line H2228 was tested in FFPE at increasing cell numbers (2500, 5000, 10.000, 25000, 50000) corresponding to a surface area of 0.27, 0.55, 1.1, 2.75 and 5.5 mm2, respectively, in the FFPE block. A total of 38 FFPE samples positive by FISH, IHC and/or qRT-PCR for ALK (n=30), ROS (n=7) and RET (n=1) were also analyzed. Total RNA was isolated from cell lines and FFPE and < 225 ng were used for hybridization. Raw counts were normalized using positive controls, negative controls and 4 house-keeping genes (GAPDH, GUSB, OAZ1 and POLR2A) as described in Lira et al. J Mol Diagn 2013. Positive and negative ALK fusion translocation was defined by a 3’/5’ ratio score of > 2.0 and ≤ 2.0 respectively. Response to crizotinib by RECIST criteria was retrospectively collected in patients with ALK-positive NSCLC.
Results:
nCounter sensitivity to predict fusion transcripts ALK, ROS and RET in cell lines by using both methods (3’/5’ and direct reporter probes) was 100%. Results indicate that samples containing as few as 10% positive tumor cells and a 2.75 mm2 tumor surface area were sufficient for adequate gene fusion detection. The accuracy of prediction (AUC) of ALK 3’-5’ ratio score in 45 independent samples was 82.6% (95% CI 69.3-95.6) with a kappa coefficient score of 0.637. Among 28 samples ALK-FISH-positive, ALK 3’-5’ scoring was positive in 27 samples (96%). One sample was non-evaluable by ALK 3’-5’ scoring. Among the 17 samples ALK-FISH-negative, ALK 3’-5’ score was negative and positive in 10 (59%) and 7 (41%) samples, respectively. All patients with ALK-FISH-negative samples but ALK 3’-5’ score positive (n=7) were positive for ALK IHC and 5 of them were treated with crizotinib. Response assessment was available in 3 of these patients and response rate was 100%. One patient non-evaluable by FISH but positive 3’-5’ scoring also responded to crizotinib.
Conclusion:
The ALK/ROS1/RET nCounter-based assay is a highly sensitive screening modality that might identify FISH-negative/non-evaluable NSCLC patients who could benefit from ALK inhibitors.
Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
