Abstract
IHC assay based on anti-ALK D5F3 antibodies clone was reported as reliable, time- and cost-effective diagnostic alternative to FISH 'gold standard' method for detection of ALK aberrations in NSCLC.
We aimed at evaluating the potential applicability of RT-PCR assay for ALK gene rearrangement identification. Reliability of two RT-PCR methods detecting relative expression of 3' ALK mRNA transcript was estimated (method by Wang (I) and/or by Gruber (II) [1,2] and compared to D5F3 antibody-based ALK IHC assay. ALK IHC-positive materials were reassessed with FISH. Total of 89 fresh-frozen and/or FFPE samples from 65 NSCLC patients were analysed.
In FFPE samples, viable results were produced only by RT-PCR (II) assay based on shorter amplicons [2]. Still, 11/24 of FFPE samples (46%) were not eligible for diagnostics due to technical reasons. RT-PCR (II) and IHC presented low conformity of results (Cohen's Kappa=0.262, 95%CI=0.23-0.294; Overall Percent Agreement (OPA) = 81.1% (95%CI=68.5%-93.7%). 37 fresh-frozen and 4 FFPE materials analyzed by both RT-PCR assays presented low Positive Percent Agreement (PPA) =57% (95%CI=20.5%-93.8%) and Cohen's Kappa=0.429 (95%CI=0,395-0.464). There were 7/63 ALK IHC positive results (11%), 4 of which (57%) were confirmed and other 3 technically unevaluable by FISH.
RT-PCR assay presented low reliability and reproducibility, particularly in FFPE materials. Meanwhile, IHC assay with ALK D5F3 antibodies proved robust, provided high conformity with reference method (FISH) and lower rate of unevaluable results in FFPE NSCLC samples.
[1] Wang et al., Clin Cancer Res, 2012
[2] Gruber et al., JTO, 2014.
- Copyright ©ERS 2015
