Quantification of Anaplastic Lymphoma Kinase Protein Expression in Non–Small Cell Lung Cancer Tissues from Patients Treated with Crizotinib

Abstract

BACKGROUND Crizotinib has antitumor activity in ALK (anaplastic lymphoma receptor tyrosine kinase)-rearranged non–small cell lung cancer (NSCLC). The current diagnostic test for ALK rearrangement is breakapart fluorescence in situ hybridization (FISH), but FISH has low throughput and is not always reflective of protein concentrations. The emergence of multiple clinically relevant biomarkers in NSCLC necessitates efficient testing of scarce tissue samples. We developed an anaplastic lymphoma kinase (ALK) protein assay that uses multiplexed selected reaction monitoring (SRM) to quantify absolute amounts of ALK in formalin-fixed paraffin-embedded (FFPE) tumor tissue. METHODS After validation in formalin-fixed cell lines, the SRM assay was used to quantify concentrations of ALK in 18 FFPE NSCLC samples that had been tested for ALK by FISH and immunohistochemistry. Results were correlated with patient response to crizotinib. RESULTS We detected ALK in 11 of 14 NSCLC samples with known ALK rearrangements by FISH. Absolute ALK concentrations correlated with clinical response in 5 of 8 patients treated with crizotinib. The SRM assay did not detect ALK in 3 FISH-positive patients who had not responded to crizotinib. In 1 of these cases, DNA sequencing revealed a point mutation that predicts a nonfunctional ALK fusion protein. The SRM assay did not detect ALK in any tumor tissue with a negative ALK status by FISH or immunohistochemistry. CONCLUSIONS ALK concentrations measured by SRM correlate with crizotinib response in NSCLC patients. The ALK SRM proteomic assay, which may be multiplexed with other clinically relevant proteins, allows for rapid identification of patients potentially eligible for targeted therapies.

Quantitative proteomics in lung cancer

“…Furthermore, crizotinib is a kinase inhibitor for multiple lung cancer oncogene including ALK, c-Met, and ROS1, especially for ROS1-rearranged NSCLC [ 60 ]. Current diagnostic test for ALK arrangement is based on low throughput fluorescence in situ hybridization (FISH), Hembrough et al developed an ALK protein assay that could save time and the expense of multiple FISH testing to detect different biomarkers [ 61 ]. They used SRM approach to quantify absolute amounts of ALK in 188 formalin-fixed paraffin-embedded NSCLC tissues and the results were correlated with patients response to crizotinib.…”

Data-Independent Acquisition Mass Spectrometry To Quantify Protein Levels in FFPE Tumor Biopsies for Molecular Diagnostics

“…Cellular expression levels of clinically relevant proteins in patient tumor samples can guide selection of cancer therapies. While antibody-based immunohistochemistry (IHC) assays are most widely used in the clinic, mass spectrometry (MS)-based quantitative proteomics has been increasingly applied to clinical samples to objectively measure tumor expression levels of treatment-related protein biomarkers 1–2 . A targeted strategy using selected reaction monitoring (SRM) has been a method of choice for clinical proteomics due to its high analytical performance including exceptional sensitivity from a fixed quadrupole setting, and inherent selectivity achieved by paired selection of precursor and fragment ions defined as transitions.…”

Serum SP70 is a sensitive predictor of chemotherapy response in patients with advanced nonsmall cell lung cancer

“…Recently, the availability of companion biomarkers could improve drug efficacy, decrease toxicity, and lead to a more individualized approach to cancer treatment. Anaplastic lymphoma receptor tyrosine kinase ( ALK ) translocations are used for identifying patients with NSCLC likely to benefit from crizotinib . However, existing biomarkers are not sensitive enough to provide real‐time information on the effectiveness of chemotherapy.…”

“…Anaplastic lymphoma receptor tyrosine kinase (ALK) translocations are used for identifying patients with NSCLC likely to benefit from crizotinib. 19,20 However, existing biomarkers are not sensitive enough to provide real-time information on the effectiveness of chemotherapy. Anticancer drugs can induce cancer cell apoptosis.…”