Abstract 5299: Detection of ALK fusion transcripts in plasma of non-small cell lung cancer patients using a novel RT-PCR based assay
Authors: Heeke Simon , Ilié Marius , Allegra Maryline , Vallée Audrey , Salacroup Carole , Tanga Virginie , Hofman Véronique , Rajamani Jaya , Lee Michael , Ordinario Ellen , Denis Marc G. , Hofman Paul 
Published: September 2020

Abstract
Background and objective: Detection of genomic rearrangements like ALK fusions are of great interest in non-small cell lung cancer (NSCLC) as those alterations can be targeted by an increasing number of drugs. To overcome tissue limitations, detection of these alterations from liquid biopsies is an unmet need, despite the development of novel NGS-based tests. To allow the detection of ALK rearrangements from circulating-free RNA (cfRNA) from NSCLC patients, we have evaluated a novel RT-PCR based assay and compared the results to tissue-based testing using immunohistochemistry (IHC) or fluorescence in-situ hybridization (FISH).
Materials and Methods: Sixty-five patients with late stage NSCLC were included in the study. ALK status was assessed on tissue section using immunohistochemistry and/or FISH. cfRNA was extracted from 2 ml of plasma from EDTA or Streck BCT DNA tubes using a prototype cfRNA Sample Preparation method (Roche Molecular Systems, Pleasanton, CA). For the detection of ALK-rearrangements from plasma, a prototype ALK/RET/ROS1 Fusion Panel assay (Roche) was used.
Results: The analytical sensitivity of the assay was evaluated by testing plasma from healthy individuals spiked with RNA extracted from FFPE tissue section of an ALK positive tumor. Positive results were obtained with samples spiked with as little as 3 ng RNA. Of the forty-five ALK-fusion positive patients, 11 patients were already under anti-ALK therapy and in none of the patients an ALK fusion could be detected. However, of five samples tested at progression, two (40%) were tested positive. For the 29 ALK-fusion positive samples which were included at baseline prior to therapy, 11 samples have been tested positive for ALK fusions (38%). All 20 negative controls were negative using the PCR based assay. Consequently, the key test parameters for samples tested at baseline were: sensitivity = 37.93% [95% CI 20.69% - 57.74%]; specificity = 100.00% [95% 83.16% - 100.00%]; positive predictive value = 100%; negative predictive value weighted for a prevalence of ALK fusions of 4% in metastatic NSCLC patients = 97.48% [95% CI 96.68 - 98.09].
Conclusion: The prototype cobas ALK/RET/ROS1 Fusion Panel assay was able to detect ALK fusion transcripts in the plasma of NSCLC patients at baseline as well as at disease progression. Limited sensitivity could be explained by biological factors influencing nucleic acid shedding by tumours, as well as the presence of fusions not covered by the assay. However, the assay demonstrated high specificity. These data demonstrate that this assay could potentially be used to select patients for an anti-ALK therapy when tissue samples are not available.
Citation Format: Simon Heeke, Marius Ilié, Maryline Allegra, Audrey Vallée, Carole Salacroup, Virginie Tanga, Véronique Hofman, Jaya Rajamani, Michael Lee, Ellen Ordinario, Marc G. Denis, Paul Hofman. Detection of ALK fusion transcripts in plasma of non-small cell lung cancer patients using a novel RT-PCR based assay [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5299.