P1.14-43 A Novel Patient Derived Synchronous Cell Pair with Different Mutations in an ALK-Rearranged Lung Adenocarcinoma Underlines Tumor Heterogeneity

Abstract

ALK targeted therapy can provide prolonged clinical response rates in ALK-rearranged lung adenocarcinoma (ADC) patients. However, most tumors relapse within a few years of treatment pressure due to a variety of resistance mechanisms, including intratumoral heterogeneity. Understanding these mechanisms is of utmost importance to more precisely tailor future targeted therapies. We established a novel synchronous ALK-translocated lung ADC cell pair from the malignant pleural effusion (PF240-PE) and the pleural carcinosis (PF240-PC) of a 38-year-old female patient following sequential ALK targeted therapy. Immunohistochemistry and mutational analyses were executed in pleural carcinosis tissue specimens and in the tumor cell lines. SRB assays were performed for viability testings following different generations ALK inhibitor treatment alone and combined with SAHA, a pan-HDAC inhibitor. As positive control for all treatment lines we used PF521, another newly established ALK-rearranged but treatment naïve lung ADC cell line. In vivo tumorigenicity was evaluated by performing subcutaneous xenografts. We identified two distinct resistance mutations in both tissue specimens: a so far non-characterized E1161K and the already described L1152R. Strikingly, PF240-PC harbored E1161K and PF240-PE carried L1152R. Immunohistochemistry showed changes from epithelial/carcinomatous to mesenchymal/sarcomatous differentiation following resistance acquisition. In vitro testing revealed that both cell lines were significantly different in morphology and sensitivity to different generation ALK inhibitors including crizotinib, alectinib and lorlatinib. However, the novel tyrosine kinase inhibitor entrectinib was effective in both E1161K and L1152R mutant cells. Importantly, combination treatment of crizotinib or alectinib plus pan-HDAC inhibitor SAHA yielded strong synergism. Of note, both novel cell lines were highly tumorigenic in vivo. In vivo treatment response profiles are currently under evaluation. This is the first evidence of the synchronous establishment of two highly distinct patient-derived ALK translocated lung ADC cell lines carrying different resistant mutations. This concept supports the paramount significance of spatiotemporal intratumoral heterogeneity under targeted therapy. Furthermore, our findings showed that HDAC inhibition could enhance sensitivity of resistant tumor cells to ALK targeted therapy in vitro. Altogether, our findings provide strong evidence for the synchronous emergence of multiple resistance mechanisms and emphasize the importance of multiple site re-biopsies to better identify acquired resistance mechanisms under targeted therapy.