P3.04-009: Evaluation of RT-PCR Methodology for ALK Assessment in Patients with NSCLC in Europe: Results from the ETOP Lungscape Project


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Inproceedings: an article in a conference proceedings.
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Poster: Summary – with images – on one page of the results of a researche project. The summaries of the poster must be entered in "Abstract" and not "Poster".
P3.04-009: Evaluation of RT-PCR Methodology for ALK Assessment in Patients with NSCLC in Europe: Results from the ETOP Lungscape Project
Title of the conference
16th World Conference on Lung Cancer
Letovanec I., Peters S., Tsourti Z., Finn SP, Soltermann A., Bubendorf L., Speel EJM, Marchetti A., Nonaka D., Hager H., Martorell M., Monkhorst K., Sejda A., Chenev R., Sansano I., Verbeken EK, Tischler V., Savic S., Di Lorito A., Calabuig MC, Felip E., Adjei A., Warth A., Baas P., Meldgaard P., Blackhall F., Dingemans AM, Dienemann H., Dziadziusko R., Vansteenkiste J., Kammler R., Dafni U., Kerr K., Thunissen E., Stahel R.
Denver, USA, September 6-9, 2015
Publication state
Issued date
Journal of Thoracic Oncology
Background: ALK rearrangement is documented in 2%-7% of NSCLC, depending on the population studied and detection method used. Although the reverse transcriptasepolymerase chain reaction (RT-PCR) was the first used and published method, fluorescence in situ hybridization (FISH) has become the primary standard diagnostic method. Recently, immunohistochemistry (IHC) has also proven to be a reproducible, faster and sensitive technique. This is one of the first studies concurrently comparing all three techniques in resected lung adenocarcinomas from the large ETOP Lungscape cohort. Methods: 95 cases from the ETOP Lungscape iBiobank, selected based on any degree of IHC staining (clone 5A4 antibody, Novocastra, UK), were examined by ALK FISH (Abbott Molecular, Inc.; Blackhall, JCO 2014) and central RT-PCR. For the latter, formalin-fixed, paraffin-embedded (FFPE) unstained slides were collected from participating centers. Slides were de-paraffinized, Toluidine Blue stained, and tumors macro-dissected. Tissue digestion and RNA extraction were performed (Qiagen RNeasy FFPE Kit). Using primers described in the literature covering most of ALK known translocations, RT-PCR (Superscript One-Step RT-PCR with Platinum Taq - 40 loops) was performed, followed by capillary electrophoresis in two separate mixes. Co-amplification of B-actin was done to validate the procedure and RNA quality. All tests were duplicated. Results: 76 of 95 RT-PCR had adequate RNA quality (B-actin co-amplification present). Among these, 18 were FISH positive, 16 were RT-PCR positive, including EML4-ALK V3a/b in 7, V1 in 5, V2 in one, and undetermined variants in 3 cases. 53 of 54 FISH negative cases were also RT-PCR negative (98%). 15 of 18 FISH positives harbored a translocation by RT-PCR (83%). Among the 4 discrepant cases, 2 FISH+/ RT-PCR- cases had IHC H-scores of 180 and 260, and 98.3% and 95% of rearranged cells by FISH, probably corresponding to variants not covered by the RT-PCR. One had an IHC H-score of 5, and 16% cells rearranged on FISH, most probably corresponding to a FISH false positive case. The last had an IHC H-score of 200, 13% rearranged cells by FISH, and, thus is defined as a false negative FISH result. Provided IHC is defined as positive by an H-score above 120, all but one case (H-Score 20, FISH and RT-PCR positive) gave concordant results by a combination of FISH and RT-PCR. Overall, using as true negative or true positive the concordant result of two of the methods, the third method is characterized by high specificity and sensitivity with corresponding values of 100/98/100% and 94/94/89% for IHC/FISH/RT-PCR, respectively. Conclusion: RTPCR is a very good tool for sorting discordant IHC/FISH cases, however, we do not recommend using this technique as single method due to the lower sensitivity of RT-PCR, as not all variants are covered, and also due to the limitations with RNA preservation.
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10/08/2016 7:32
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