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Supplementary Information

Combination therapy of ALK and β3 inhibitors shows promising results in vivo and in ALK-rearranged NSCLC

Treatment information of patients who received crizotinib

Purpose:In early-stage, EGFR mutation–positive (EGFR-M+) non–small cell lung cancer (NSCLC), surgery remains the primary treatment, without personalized adjuvant treatments. We aimed to identify risk factors for recurrence-free survival (RFS) to suggest personalized adjuvant strategies in resected early-stage EGFR-M+ NSCLC.Experimental Design:From January 2008 to August 2020, a total of 2,340 patients with pathologic stage (pStage) IB–IIIA, non-squamous NSCLC underwent curative surgery. To identify clinicopathologic risk factors, 1,181 patients with pStage IB–IIIA, common EGFR-M+ NSCLC who underwent surgical resection were analyzed. To identify molecular risk factors, comprehensive genomic analysis was conducted in 56 patients with matched case–controls (pStage II and IIIA and type of EGFR mutation).Results:Median follow-up duration was 38.8 months (0.5–156.2). Among 1,181 patients, pStage IB, II, and IIIA comprised 577 (48.9%), 331 (28.0%), and 273 (23.1%) subjects, respectively. Median RFS was 73.5 months [95% confidence interval (CI), 62.1–84.9], 48.7 months (95% CI, 41.2–56.3), and 22.7 months (95% CI, 19.4–26.0) for pStage IB, II, and IIIA, respectively (P < 0.001). In multivariate analysis of clinicopathologic risk factors, pStage, micropapillary subtype, vascular invasion, and pleural invasion, and pathologic classification by cell of origin (type II pneumocyte-like tumor cell vs. bronchial surface epithelial cell–like tumor cell) were associated with RFS. As molecular risk factors, the non-terminal respiratory unit (non-TRU) of the RNA subtype (HR, 3.49; 95% CI, 1.72–7.09; P < 0.01) and TP53 mutation (HR, 2.50; 95% CI, 1.24–5.04; P = 0.01) were associated with poor RFS independent of pStage II or IIIA. Among the patients with recurrence, progression-free survival of EGFR-tyrosine kinase inhibitor (TKI) in those with the Apolipoprotein B mRNA Editing Catalytic Polypeptide-like (APOBEC) mutation signature was inferior compared with that of patients without this signature (8.6 vs. 28.8 months; HR, 4.16; 95% CI, 1.28–13.46; P = 0.02).Conclusions:The low-risk group with TRU subtype and TP53 wild-type without clinicopathologic risk factors might not need adjuvant EGFR-TKIs. In the high-risk group, with non-TRU subtype and/or TP 53 mutation, or clinicopathologic risk factors, a novel adjuvant strategy of EGFR-TKI with others, e.g., chemotherapy or antiangiogenic agents needs to be investigated. Given the poor outcome to EGFR-TKIs after recurrence in patients with the APOBEC mutation signature, an alternative adjuvant strategy might be needed.

Supplementary Data from Prediction of Recurrence-Free Survival in Postoperative Non–Small Cell Lung Cancer Patients by Using an Integrated Model of Clinical Information and Gene Expression

Purpose: One of the main challenges of lung cancer research is identifying patients at high risk for recurrence after surgical resection. Simple, accurate, and reproducible methods of evaluating individual risks of recurrence are needed.Experimental Design: Based on a combined analysis of time-to-recurrence data, censoring information, and microarray data from a set of 138 patients, we selected statistically significant genes thought to be predictive of disease recurrence. The number of genes was further reduced by eliminating those whose expression levels were not reproducible by real-time quantitative PCR. Within these variables, a recurrence prediction model was constructed using Cox proportional hazard regression and validated via two independent cohorts (n = 56 and n = 59).Results: After performing a log-rank test of the microarray data and successively selecting genes based on real-time quantitative PCR analysis, the most significant 18 genes had P values of CALB1, MMP7, SLC1A7, GSTA1, CCL19, and IFI44). Two pathologic variables, pStage and cellular differentiation, were developed. Validation by two independent cohorts confirmed that the proposed model is significantly accurate (P = 0.0314 and 0.0305, respectively). The predicted median recurrence-free survival times for each patient correlated well with the actual data.Conclusions: We have developed an accurate, technically simple, and reproducible method for predicting individual recurrence risks. This model would potentially be useful in developing customized strategies for managing lung cancer.

Purpose: Anaplastic lymphoma kinase (ALK)-positive cancers are sensitive to small-molecule ALK kinase inhibitors, but most cases experience failure following treatment. Hence, additional drug targets and combination therapeutic treatments are needed. We investigated gene expression that is regulated by the expression of ALK and explored its roles in cancer progression and therapeutic implication.Experimental Design: We screened ALK-rearranged non–small cell lung cancer (NSCLC) cases using immunohistochemistry and fluorescence in situ hybridization and then conducted multiplex gene expression analysis. We also performed a clinicopathologic analysis to validate the findings. Additional cellular experiments, including inhibition and migration assays, and in vivo lung cancer model studies were performed.Results: Among patients with ALK-rearranged NSCLC, integrin β3 (ITGB3) was one of the overexpressed genes in comparison with that in ALK-negative NSCLC (P = 0.0003). ALK and integrin β3 expression were positively correlated, and we discovered that high integrin β3 mRNA expression was associated with metastasis and more advanced tumor stages (P < 0.005; P < 0.05). Furthermore, we found that inhibition of both ALK and integrin β3 led to increased drug sensitivity in vitro and in vivo (both P < 0.05).Conclusions: We discovered a positive correlation between ALK and integrin β3 expression levels in ALK-rearranged NSCLC. Our findings suggest that high integrin β3 expression in ALK-rearranged NSCLC is associated with tumor progression and a worse prognosis. This finding demonstrates the prognostic value of integrin β3 and provides a rationale for combination treatment with ALK and integrin β3 inhibitors in patients with ALK-rearranged NSCLC. Clin Cancer Res; 24(17); 4162–74. ©2018 AACR.

Supplementary Data from Clinical, Pathologic, and Molecular Prognostic Factors in Patients with Early-Stage EGFR-Mutant NSCLC

Functional role and regulation of integrin β3 in ALK-rearranged cell lines

Clinical characteristics of ALK-rearranged and ALK-negative NSCLC patients

Clinical characteristics in ALK-rearranged NSCLC based on integrin β3 immunohistochemistry score

Sequence of siRNAs and primers

ALK and integrin β3 show strong correlation in expression in ALK-rearranged NSCLC patient cases and in vitro

Clinical characteristics in ALK-rearranged NSCLC based on integrin β3 mRNA expression level

Purpose: Anaplastic lymphoma kinase (ALK)-positive cancers are sensitive to small-molecule ALK kinase inhibitors, but most cases experience failure following treatment. Hence, additional drug targets and combination therapeutic treatments are needed. We investigated gene expression that is regulated by the expression of ALK and explored its roles in cancer progression and therapeutic implication. Experimental Design: We screened ALK-rearranged non–small cell lung cancer (NSCLC) cases using immunohistochemistry and fluorescence in situ hybridization and then conducted multiplex gene expression analysis. We also performed a clinicopathologic analysis to validate the findings. Additional cellular experiments, including inhibition and migration assays, and in vivo lung cancer model studies were performed. Results: Among patients with ALK-rearranged NSCLC, integrin β3 (ITGB3) was one of the overexpressed genes in comparison with that in ALK-negative NSCLC (P = 0.0003). ALK and integrin β3 expression were positively correlated, and we discovered that high integrin β3 mRNA expression was associated with metastasis and more advanced tumor stages (P < 0.005; P < 0.05). Furthermore, we found that inhibition of both ALK and integrin β3 led to increased drug sensitivity in vitro and in vivo (both P < 0.05). Conclusions: We discovered a positive correlation between ALK and integrin β3 expression levels in ALK-rearranged NSCLC. Our findings suggest that high integrin β3 expression in ALK-rearranged NSCLC is associated with tumor progression and a worse prognosis. This finding demonstrates the prognostic value of integrin β3 and provides a rationale for combination treatment with ALK and integrin β3 inhibitors in patients with ALK-rearranged NSCLC. Clin Cancer Res; 24(17); 4162–74. ©2018 AACR.

Purpose: This study was aimed at analyzing the recurrence-related prognostic significance of 12 candidate molecular biomarkers in node-negative stage I–II non–small cell lung cancer (NSCLC). Experimental Design: We retrospectively analyzed promoter methylation of eight genes using methylation-specific PCR in formalin-fixed and paraffin-embedded tissues from 328 node-negative stage I–II NSCLCs. The expression of Bcl-2, E-cadherin, p53, and p63 proteins was also assessed by immunohistochemistry. Results: Recurrence was found in 145 (44%) of 328 node-negative stage I–II NSCLCs with a median follow-up period of 6.2 years. No association was found between recurrence and alteration of individual biomarker in univariate analysis. We defined recurrently divergent groups on the basis of recursive partitioning analyses for 12 biomarkers and found a significant association of co-alteration of RASSF1A and p63 with poor recurrence-free survival (RFS). Cox proportional hazards analysis showed that hypermethylation of RASSF1A and negative expression of p63 was associated with poor RFS [HR, 1.93; 95% confidence interval (CI), 1.13–5.47; P = 0.009] compared with those without co-alteration of RASSF1A and p63, after adjusting for age, adjuvant therapy, histology, and tumor size. Random forest classifier including RASSF1A and p63 showed best performance in the prediction of recurrence in node-negative stage I–II NSCLCs: area under receiver operator characteristic curve for random forest was 0.91 and error rate for the model was 17%. Conclusion: The present study suggests that RASSF1A and p63 may be independent prognostic indicators for RFS in node-negative stage I–II NSCLCs. Clin Cancer Res; 19(5); 1204–12. ©2012 AACR.

Background: Patients with EGFR activating mutation in non-small cell lung cancer (NSCLC) eventually develop AR to EGFR tyrosine kinase inhibitors (TKI) after durable and high response. Several mechanisms of AR to EGFR TKIs identified include T790M gatekeeper mutation, PIK3CA mutation, C-met amplification, transformation into small cell carcinoma or epithelial-mesenchymal transition (EMT). In the present study, to identify other genetic alterations, we performed targeted deep sequencing for over 700 hotspots in 50 cancer-related genes using next generation sequencing (NGS) technology. Methods: Genomic DNA was extracted from FFPE tissue or pleural/pericardial fluid or primary culture cells of 57 NSCLC patients who developed AR to EGFR TKIs. Amplicons NGS libraries for 50 oncogenes included in Ion AmpliSeqTM Cancer Hotspot Panel v.2 were generated and sequenced in the Ion PGM Sequencer. Variant caller included in Torrent Suite Software was utilized to identify mutations in the samples, annotation was performed with IonTorrentTM Software. Results: All 57 patients were treated with EGFR TKIs, gefitinib (n=39), erlotinib (n=17), or afatinib (n=1) as first-line or later line of therapy. 65% were female, 74% never smokers, median age of 58 years (range 25-77) and 98% were adenocarcinoma. 31 patients have exon 19 deletion and 14 with L858R at baseline. The median PFS for EGFR TKIs was 11 months (range 4-63). Targeted deep sequencing of second biopsy identified recurrent coexisting mutations of T790M in 52.6%. Other frequently altered genes include mutation of CTNNB1 (n=9), c-KIT (n=7), c-met (n=7), SMARCB1 (n=5), PDGFRA (n=5), PTEN (n=2), NRAS (n=2), and PIK3CA (n=1). However, in order to investigate whether these recurrent mutations observed in second biopsy represent genes associated with AR or pre-existing genes, targeted deep sequencing of available paired baseline samples will be performed and updated. Conclusions: We found that NGS technology is feasible in FFPE tissues. Although T790M mutation confers the most common resistant mechanism to EGFR TKI, several detected genetic mutations are novel observations which might provide potential candidates for AR mechanisms. Further validation results will be discussed. Citation Format: Myung-Ju Ahn, Youngwook Kim, Kyoung-Mee Kim, Yoon-la Choi, Joungho Han, EunHye Jho, Joung-Mu Sun, Jin-Sock Ahn, Keunchil Park. Targeted deep sequencing of second biopsy tissues from patients with acquired resistance (AR) to EGFR tyrosine kinase inhibitors harboring EGFR mutations: Preliminary results. [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 A02.

Oncogenic fusions involving ALK, ROS1 and RET define a small subpopulation (∼7%) of non-small cell lung carcinomas (NSCLC). Tumors harboring ALK and ROS1 rearrangements are highly sensitive to crizotinib whereas RET inhibitors may be of potential benefit for the treatment for RET-fusion positive tumors1. Current methods for detection of ALK, ROS1, and RET rearrangements involve fluorescence in situ hybridization (FISH), immunohistochemistry (IHC) or RT-PCR, each test offering its own advantages and disadvantages, and making it difficult for screening large numbers of patient samples for all three targetable fusions. To explore screening modalities for the simultaneous detection of ALK, ROS1 and RET fusions from a single analyte, we designed a multiplexed, transcript-based assay to detect for presence or absence of fusions. Utilizing a combined 3’ over-expression and fusion-specific detection strategy, we developed a single-tube multiplex assay with a quantitative scoring modality that is highly sensitive, reproducible and capable of detecting low abundant ALK, ROS1 and RET fusion transcripts. We successfully validated the assay in 273 NSCLC specimens. For ALK, our results were highly concordant (97.4%) to prior results obtained by IHC (n=189). We confirmed ROS1 (n=7) and RET (n=11) fusion-positive tumors by RT-PCR plus sequencing. There is a significant enrichment for ROS1 and RET fusions in triple negative NSCLC (i.e. KRAS/EGFR/ALK wildtype) (Table 1). ALK, ROS1, RET, KRAS and EGFR mutations are mutually exclusive in this study. Our assay offers an easy to perform, high-throughput, and FFPE-compatible screening method for detection of ALK, ROS1 and RET fusions. Table 1. Incidence of ALK, ROS1 and RET fusions cohorts clinical/molecular feature # cases ALK+ ROS1+ RET+ Set 1 ALK IHC+, Kras/EGFR wt 95 91 0 0 Set 2 ALK IHC−, Kras/EGFR wt 94 1 5 11 Set 3 never smokers 84 10 2 1 Citation Format: Maruja Lira, Yoon-La Choi, Shibing Deng, Donghui Huang, Mark Ozeck, Joungho Han, Ji Yun Jeong, Keunchil Park, Jhingook Kim, Myung-Ju Ahn, Mao Mao. A single-tube, multiplexed, transcript-based assay to detect ALK, ROS1 and RET fusions in lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3493. doi:10.1158/1538-7445.AM2013-3493

Background: Focal gene amplification is a potential target for diagnosis and therapy in cancer. Fibroblast growth factor receptor-type 1 (FGFR1) may play a role in the pathogenesis of lung squamous cell carcinoma (LSCC) and amplification of the FGFR1 gene is considered a candidate target for treatment of LSCC. The aim of this study was to simultaneously explore copy number changes of FGFR1 and 23 other genes by multiplex ligation-dependent probe amplification (MLPA), to compare the results with fluorescent in situ hybridization (FISH) data, and determine the clinicopathological significance of gene amplification. Methods: This study included 81 resected LSCC specimens obtained between 2008 and 2010, and 14 lung cancer cell lines. MLPA was performed using fresh tumor samples and cell lines and FISH was applied to formalin-fixed, paraffin-embedded tissues. Results: MLPA showed amplification/gain of MYC (48.1% of LSCCs), AURKA (44.4%), MET (29.6%), KIT (21.0%), and FGFR1 (14.8%). FISH showed FGFR1 amplification in 22 LSCCs (27.2%) using a cut-off value of ≥5. There was high concordance between FISH and MLPA (85.2%). No association between FGFR1 amplification and any clinicopathological variables was observed; however, associations were identified between EGFR and FGFR1 FISH-amplified tumors; PDGFRA and poor differentiation; KDR, ABL1, and MET and age; and SMO and CCND2 and pT stages. Conclusions: Our MLPA and FISH data showed that FGFR1 amplification is a common event, occurring in 15-27.2% of LSCCs. Other amplified genes may be also candidate targets for the treatment of LSCC. Citation Format: Mi Jung Kwon, Seung Eun Lee, Joungho Han, Jin Seok Ahn, Keunchil Park, Jhingook Kim, Myung-Ju Ahn, Yoon-La Choi. FGFR1 amplification as a druggable target in lung squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1913. doi:10.1158/1538-7445.AM2013-1913