Abstract B09: Multiplexed mass spectrometry-based assay to quantify translocation markers from non-small cell lung cancer (NSCLC) FFPE tissue.
Authors: Liao Wei-Li , Thyparambil Sheeno , An Eunkyung , Hartley Christopher P. , Ma patrick , Rodriguez Jaime , Wistuba Ignacio , Burrows Jon , Hembrough Todd , Tafe Laura J. 
Published: August 2014

Abstract
Introduction: Translocations in ALK, ROS1 and RET have been shown to be oncogenic in NSCLC. Lung cancers having ALK or ROS1 rearrangements represent unique subpopulations that are seen in only 2-5% and 1-2% of NSCLC, respectively. ALK fusions lead to constitutive activation of ALK signaling involved in cell proliferation. Crizotinib has significant anti-tumor activity in ALK rearranged NSCLC and break-apart FISH is the approved diagnostic test to determine treatment eligibility. However, FISH is laborious, expensive and low throughput, and thus is not ideal for the detection of oncogenic drivers of low frequencies. In patients with advanced disease, a small tissue biopsy is often the only material available so yielding as much information as possible from a limited sample is necessary. The aim of this study was to develop a multiplexed quantitative Liquid-Tissue-selected reaction monitoring (LT-SRM) assay for assessing ALK, ROS1, and RET expression within our “Lung OncoPlex” MS test. The LT-SRM platform quantitates these translocation markers along with several diagnostic and potentially targetable biomarkers, e.g. TTF1, K7, p63, K5, EGFR, HER2, HER3, MET, KRAS and IGF1R, in NSCLC.
Methods: We used trypsin digestion mapping of recombinant proteins specific for ALK, ROS1, and RET to identify optimal quantitative peptides. Stable isotope-labeled peptides were synthesized as internal standards, and standard curves were generated in Pyrococcus complex matrix to determine LOD, LLOQ, accuracy, precision and linearity of the assays. The ALK assay was pre-clinically validated in an EML4-ALK rearrangement positive cell line-H3122. ALK and ROS1 were screened in 87 archived FFPE sections from NSCLC.
Results: We identified at least two optimal peptides for each target. At least one peptide from each protein had acceptable technical assay performance and was used for assay development. H3122 cell expressed 396 amol ALK/ug cell protein, while 11 ALK translocation positive NSCLC tissues expressed ALK from 107 to 437 amol/ug protein. ALK peptides were not detected in ALK negative control NSCLC tissues or in a single ALK translocation positive case. ROS1 was detected in 2 of 87 NSCLC samples at levels of 659 amol/ug in a case of unknown translocation status and 377 amol/ug in a ROS1 translocation positive case. Finally, the Lung OncoPlex assay successfully subtyped lung adenocarcinoma and quantified the other potentially targetable biomarkers.
Conclusions: The Lung OncoPlex assay was able to detect ALK protein in 11/12 ALK rearranged samples. In the one proteomically negative/FISH+ case, we are performing ALK IHC to assess ALK protein expression, as well as DNA sequencing to evaluate for potential mutations within the MS targeted peptides. Of the two cases positive for ROS1 by the MS assay, one is known to be FISH positive and the other is undergoing FISH verification. RET protein expression has not yet been assessed in any known RET translocation positive cases; however, the RET technical performance suggests this is a promising assay and we are continuing to screen for RET positive control samples. While additional studies are needed to validate the clinically utility of the ALK, ROS1, and RET assay; multiplexed proteomic screening of patient tissue could be performed at the time of initial biopsy, allowing for simultaneous assessment of multiple clinically actionable gene rearrangements and biomarker targets.
Citation Format: Wei-Li Liao, Sheeno Thyparambil, Eunkyung An, Christopher P. Hartley, patrick Ma, Jaime Rodriguez, Ignacio Wistuba, Jon Burrows, Todd Hembrough, Laura J. Tafe. Multiplexed mass spectrometry-based assay to quantify translocation markers from non-small cell lung cancer (NSCLC) FFPE tissue. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B09.