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Purpose: We evaluated plasma cell-free DNA (cfDNA) and tissue-based sequencing concordance for comprehensive oncogenic driver detection in non–small cell lung cancer (NSCLC) using a large-scale prospective screening cohort (LC-SCRUM-Liquid). Experimental Design: Blood samples were prospectively collected within 4 weeks of corresponding tumor tissue sampling from patients with advanced NSCLC to investigate plasma cfDNA sequencing concordance for alterations in 8 oncogenes (EGFR, KRAS, BRAF, HER2, MET, ALK, RET, and ROS1) compared with tissue-based next-generation targeted sequencing. Results: Paired blood and tissue samples were obtained in 1,062/1,112 enrolled patients with NSCLC. Oncogenic alteration was detected by plasma cfDNA sequencing and tissue assay in 455 (42.8%) and 537 (50.5%) patients, respectively. The positive percent agreement of plasma cfDNA sequencing compared with tissue DNA and RNA assays were 77% (EGFR, 78%; KRAS, 75%; BRAF, 85%; HER2, 72%) and 47% (ALK, 46%; RET, 57%; ROS1, 18%; MET, 66%), respectively. Oncogenic drivers were positive for plasma cfDNA and negative for tissue due to unsuccessful genomic analysis from poor-quality tissue samples (70%), and were negative for plasma cfDNA and positive for tissue due to low sensitivity of cfDNA analysis (61%). In patients with positive oncogenic drivers by plasma cfDNA sequencing but negative by tissue assay, the response rate of genotype-matched therapy was 85% and median progression-free survival was 12.7 months. Conclusions: Plasma cfDNA sequencing in patients with advanced NSCLC showed relatively high sensitivity for detecting gene mutations but low sensitivity for gene fusions and MET exon 14 skipping. This may be an alternative only when tissue assay is unavailable due to insufficient DNA and RNA. See related commentary by Jacobsen Skanderup et al., p. 1381

Purpose:We evaluated plasma cell-free DNA (cfDNA) and tissue-based sequencing concordance for comprehensive oncogenic driver detection in non–small cell lung cancer (NSCLC) using a large-scale prospective screening cohort (LC-SCRUM-Liquid).Experimental Design:Blood samples were prospectively collected within 4 weeks of corresponding tumor tissue sampling from patients with advanced NSCLC to investigate plasma cfDNA sequencing concordance for alterations in 8 oncogenes (EGFR, KRAS, BRAF, HER2, MET, ALK, RET, and ROS1) compared with tissue-based next-generation targeted sequencing.Results:Paired blood and tissue samples were obtained in 1,062/1,112 enrolled patients with NSCLC. Oncogenic alteration was detected by plasma cfDNA sequencing and tissue assay in 455 (42.8%) and 537 (50.5%) patients, respectively. The positive percent agreement of plasma cfDNA sequencing compared with tissue DNA and RNA assays were 77% (EGFR, 78%; KRAS, 75%; BRAF, 85%; HER2, 72%) and 47% (ALK, 46%; RET, 57%; ROS1, 18%; MET, 66%), respectively. Oncogenic drivers were positive for plasma cfDNA and negative for tissue due to unsuccessful genomic analysis from poor-quality tissue samples (70%), and were negative for plasma cfDNA and positive for tissue due to low sensitivity of cfDNA analysis (61%). In patients with positive oncogenic drivers by plasma cfDNA sequencing but negative by tissue assay, the response rate of genotype-matched therapy was 85% and median progression-free survival was 12.7 months.Conclusions:Plasma cfDNA sequencing in patients with advanced NSCLC showed relatively high sensitivity for detecting gene mutations but low sensitivity for gene fusions and MET exon 14 skipping. This may be an alternative only when tissue assay is unavailable due to insufficient DNA and RNA.See related commentary by Jacobsen Skanderup et al., p. 1381

Supplementary Figure S1. Consort diagram. Supplementary Figure S2. Frequency of the targetable gene alterations detected by plasma cfDNA sequencing (A) and tissue assay (B) in squamous cell carcinoma Supplementary Figure S3. Positive percent agreement of plasma cfDNA sequencing compared to tissue assay according to patient characteristics. PPA, positive percent agreement. Supplementary Figure S4. Positive percent agreement of plasma cfDNA sequencing compared to tissue assay according to metastatic site. PPA, positive percent agreement.

Supplementary Table S1 Patient characteristics in patients with 8 oncogene drivers and squamous cell carcinoma Supplementary Table S2 Detectability of eight oncogenic alterations by plasma cfDNA sequencing compared to tissue assay Supplementary Table S3 Detectability of eight oncogenic alterations by plasma cfDNA sequencing compared to tissue assay in patients only with brain metastasis Supplementary Table S4 Clinical outcomes of patients treated with a genotype-matched therapy who had oncogenic alterations detected by only plasma cfDNA sequencing.

Supplementary Figure from A Large-Scale Prospective Concordance Study of Plasma- and Tissue-Based Next-Generation Targeted Sequencing for Advanced Non–Small Cell Lung Cancer (LC-SCRUM-Liquid)

Supplementary Data from A Large-Scale Prospective Concordance Study of Plasma- and Tissue-Based Next-Generation Targeted Sequencing for Advanced Non–Small Cell Lung Cancer (LC-SCRUM-Liquid)

Supplemental Experimental Procedures

supplemental legend

Purpose: Although large-cell neuroendocrine carcinoma (LCNEC) of the lung shares many clinical characteristics with small-cell lung cancer (SCLC), little is known about its molecular features. We analyzed lung LCNECs to identify biologically relevant genomic alterations.Experimental Design: We performed targeted capture sequencing of all the coding exons of 244 cancer-related genes on 78 LCNEC samples [65 surgically resected cases, including 10 LCNECs combined with non–small cell lung cancer (NSCLC) types analyzed separately, and biopsies of 13 advanced cases]. Frequencies of genetic alterations were compared with those of 141 SCLCs (50 surgically resected cases and biopsies of 91 advanced cases).Results: We found a relatively high prevalence of inactivating mutations in TP53 (71%) and RB1 (26%), but the mutation frequency in RB1 was lower than that in SCLCs (40%, P = 0.039). In addition, genetic alterations in the PI3K/AKT/mTOR pathway were detected in 12 (15%) of the tumors: PIK3CA 3%, PTEN 4%, AKT2 4%, RICTOR 5%, and mTOR 1%. Other activating alterations were detected in KRAS (6%), FGFR1 (5%), KIT (4%), ERBB2 (4%), HRAS (1%), and EGFR (1%). Five of 10 cases of LCNECs combined with NSCLCs harbored previously reported driver gene alterations, all of which were shared between the two components. The median concordance rate of candidate somatic mutations between the two components was 71% (range, 60%–100%).Conclusions: LCNECs have a similar genomic profile to SCLC, including promising therapeutic targets, such as the PI3K/AKT/mTOR pathway and other gene alterations. Sequencing-based molecular profiling is warranted in LCNEC for targeted therapies. Clin Cancer Res; 23(3); 757–65. ©2016 AACR.

Supplementary Table 1- List of the 244 cancer related genes of the target capturing panel Supplementary Table 2 - The coverage statistics for each sample of large cell neuroendocrine carcinoma (LCNEC) Supplementary Table 3 - The coverage statistics for each sample of advanced small cell lung cancer (SCLC) Supplementary Table 4 - Validation of the mutations detected in this study using the Oncomine® Cancer Research Panel (OCP) Supplementary Table 5- Primer Sequences: Sanger Sequencing Supplementary Table 6 - Antibody for immunohistochemical staining Supplementary Table 7 - Candidate somatic mutations of large cell neuroendocrine carcinoma (LCNEC) Supplementary Table 8 - Candidate somatic mutations in advanced small cell lung cancer (SCLC) Supplementary table 9 - Hotspot mutations detected in small cell lung cancer (SCLC) Supplementary Table 10- Variant frequency of candidate somatic mutations of large cell neuroendocrine carcinoma (LCNEC) combined with non-small cell lung cancer (NSCLC) Supplementary Table 11- Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) analysis for the top six most frequently mutated genes in large cell neuroendocrine carcinoma (LCNEC)

Supplementary figure legends

Supplementary Fig. S1. The mean baseline Levels of PIP3 (38:4) per mg tissue in xenograft models transplanted with SCLC cells Supplementary Fig. S2. Metabolic alteration in SCLC cells treated with gedatolisib in vitro and in vivo Supplementary Fig. S3. Comparative metabolic profiling in LAD and SCLC cell lines Supplementary Fig. S4. Gene expression alteration after treatment with PI3K/mTOR inhibitor Supplementary Fig. S5. Schematic diagram of metabolic linkage Supplementary Fig. S6. Addition of 5''-deoxy-5''-(methylthio)-adenosine (MTA) reversed drug sensitivity from a resistant to a sensitive phenotype Supplementary Fig. S7. HPRT1 expression was downregulated by an HPRT1-specific siRNA in SCLC cells

Supplementary Table S1. The pathway classification of metabolites used for the MSEA Supplementary Table S2. Genetic profiles in 42 SCLC cell lines Supplementary Table S3. Quantitative phospho-peptide analysis in H1048 cells with or without Gedatolisib treatment Supplementary Table S4. Quantitative phosphatidyl lipids analysis in SCLC cell Supplementary Table S5. Quantitative phosphatidyl lipids analysis in H1048 cells Supplementary Table S6. 116 metabolites in SCLC cells with or without Gedatolisib treatment Supplementary Table S7. 116 metabolites in H1048 and SBC5 with or without Gedatolisib treatment Supplementary Table S8. Metabolite set enrichment analysis (MSEA) to identify significantly enriched metabolic pathways related to gedatolisib Supplementary Table S9. Amount of 116 metabolites in SCLC tissues

Supplementary methods for Sanger sequencing, copy number analysis with quantitative PCR, and Immunohistochemical staining

Supplemental Tables 1-4. Supplemental Table 1: Characteristics of primary adenocarcinoma from which CAFs were obtained. Supplemental Table 2: Primary tumor characteristics(N=106). Supplemental Table 3: Impact of predictive factors on progression free survival of EGFR-TKI in patients with EGFR activating mutation by univariate and multivariate analysis (n=106). Supplemental Table 4: The correlation between PDPN expression status and clinical response to EGFR-TKIs in patients with EGFR common mutations, exon 19 deletion and L858R (N=102).

Comprehensive genomic analysis has revealed that the PI3K/AKT/mTOR pathway is a feasible therapeutic target in small-cell lung carcinoma (SCLC). However, biomarkers to identify patients likely to benefit from inhibitors of this pathway have not been identified. Here, we show that metabolic features determine sensitivity to the PI3K/mTOR dual inhibitor gedatolisib in SCLC cells. Substantial phosphatidyl lipid analysis revealed that a specific phosphatidylinositol (3,4,5)-trisphosphate (PIP3) subspecies lipid product PIP3 (38:4) is predictive in assessing sensitivity to PI3K/mTOR dual inhibitor. Notably, we found that higher amounts of purine-related aqueous metabolites such as hypoxanthine, which are characteristic of SCLC biology, lead to resistance to PI3K pathway inhibition. In addition, the levels of the mRNA encoding hypoxanthine phosphoribosyl transferase 1, a key component of the purine salvage pathway, differed significantly between SCLC cells sensitive or resistant to gedatolisib. Moreover, complementation with purine metabolites could reverse the vulnerability to targeting of the PI3K pathway in SCLC cells normally sensitive to gedatolisib. These results indicate that the resistance mechanism of PI3K pathway inhibitors is mediated by the activation of the purine salvage pathway, supplying purine resource to nucleotide biosynthesis. Metabolomics is a powerful approach for finding novel therapeutic biomarkers in SCLC treatment.Significance: These findings identify features that determine sensitivity of SCLC to PI3K pathway inhibition and support metabolomics as a tool for finding novel therapeutic biomarkers. Cancer Res; 78(9); 2179–90. ©2018 AACR.

Supplemental Figures 1-6. Supplemental Figure 1: Fluorescence microscopy showing mRFP-labeled PC-9 (red) and Venus-labeled CAFs (green) with or without gefitinib treatment. Supplemental Figure 2: mRNA levels of podoplanin (PDPN) in CAF-Ctrl and CAF PDPN-WT. Supplemental Figure 3: Co-culture with other CAFs induces gefitinib resistance. mRFP-labeled PC-9 and HCC-827 cells were co-cultured with CAFs (CAF-Ctrl, PDPN-WT, or CAF-PDPN-Del.IC) in the presence or absence of gefitinib treatment. The total cell numbers of PC-9 or HCC-827 cells treated for 72 hours with gefitinib in the presence or absence of Venus-labeled CAF-Ctrl, PDPN-WT, or CAF-PDPN-Del.IC are shown. Supplemental Figure 4: The number of the cases according to the PDPN-positive CAF area/stromal area (%). Supplemental Figure 5: PFS according to podoplanin expression in patients with EGFR common mutations (exon 19 deletion and L858R). Supplemental Figure 6: mRNA levels of HGF in CAF-Ctrl and CAF PDPN-WT.

Supplementary Figure 1: Representative example of tissue microarray (TMA) punched out from a combined large-cell neuroendocrine carcinoma (LCNEC) with adenocarcinoma. Supplementary Figure 2: Validation of five mutations on the PI3K/AKT/mTOR pathway by Sanger sequencing (LCNEC 68c: PIK3CA E545K, LCNEC 9c: PTEN K342*, LCNEC 13c: PTEN S59*, LCNEC 4c: RICTOR R910H, LCNEC 11p: MTOR E2419K). Supplementary Figure 3: Correlation of gene copy number, calculated using the total depth on the covered region of each of 244 targeted genes and those obtained from the Oncomine® Cancer Research Panel (OCP). Supplementary Figure 4: Validation of the copy number gains or amplifications of 13 samples (8 LCNEC samples, 1 SCLC biopsy samples and 4 SCLC surgically resected cases) for MYCL1, MYC and FGFR1 by a quantitative real-time polymerase chain reaction (qPCR). Supplementary Figure 5: Overview of the key driver alterations and other activating alterations in small cell lung cancer (SCLC). Genetic alterations in the PI3K/AKT/mTOR pathway were detected in 24 (17%) of the tumors: PIK3CA (4%), PTEN (6%), AKT2 (2%) and RICTOR (6%). Copy number gains of each MYC family member were mutually exclusive. Supplementary Figure 6: Relationship between RB1 mutation and IHC staining of RB and p16. The mutual exclusivity of the protein expression between RB and p16 was distinct. Supplementary Figure 7: Relationship between genetic alteration and protein expression in large-cell neuroendocrine carcinoma (LCNEC) of the lung. Comparison of the over expression of receptor tyrosine kinases (RTKs) with the genetic alterations in resected LCNEC specimens (51 of 65 resected cases) revealed no significant relationship between strongly positive RTKs (KIT, EGFR, IGF1R, KDR, ERBB2) expression and genetic alterations.

Supplemental Figure legends

Purpose: The biologic characteristics of microenvironmental constituents, especially cancer-associated fibroblasts (CAF), can be key regulators of the cellular sensitivity to molecular-targeted therapy. Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) have marked therapeutic effects against non–small cell lung cancer (NSCLC) with EGFR mutations, but some patients have exhibited primary resistance to EGFR-TKIs. We recently reported that podoplanin-positive fibroblasts are associated with a tumor-promoting phenotype of CAFs in lung adenocarcinoma. The aim of this study was to evaluate whether the susceptibility of NSCLC to EGFR-TKIs could be affected by podoplanin-expressing CAFs.Experimental Design: We evaluated the EGFR-TKI sensitivity of EGFR-mutant lung adenocarcinoma cell lines cocultured with podoplanin-expressing CAFs. We also examined the association between the expression of podoplanin in CAFs in surgical specimens and EGFR-TKI response of postoperative recurrent patients with EGFR mutations (N = 106).Results: Lung adenocarcinoma cell lines became more resistant to EGFR-TKI when cocultured with podoplanin-expressing CAFs, compared with control CAFs in vitro. The knockdown of podoplanin expression on CAFs cancelled the resistance to EGFR-TKIs in cancer cells. Compared with control CAFs, the cancer cells that were cocultured with podoplanin-positive CAFs continued to exhibit significantly higher p-ERK levels after treatment with gefitinib. Furthermore, postoperative recurrent patients with podoplanin-positive CAFs had a significantly lower overall response rate to EGFR-TKIs compared with those with podoplanin-negative CAFs (53% vs. 83%; P < 0.01).Conclusions: Podoplanin-positive CAFs play an important role in primary resistance to EGFR-TKIs and may be an ideal therapeutic target for use in combination therapy with EGFR-TKIs. Clin Cancer Res; 21(3); 642–51. ©2014 AACR.

Background Strategies to implement early specialized palliative care have not yet been established. The present study investigated the feasibility of a nurse-led, screening-triggered early specialized palliative care intervention programme and obtained data to design a randomized controlled trial. Methods Patients with metastatic lung cancer undergoing first-line platinum-based chemotherapy were eligible. The intervention consisted of (1) a questionnaire-based screening programme and (2) advanced-level nurse counselling and care coordination with interdisciplinary team approach. The primary endpoint was the completion rate of the assessment questionnaire after the second course of first-line chemotherapy (T2). Secondary endpoints included changes in Functional Assessment of Cancer Therapy-Lung scores, depression and anxiety rates based on the Patient Health Questionnaire 9 and the Hospital Anxiety and Depression Scale, and the contents of specialized palliative care. Results A total of 50 patients were enrolled between August 2012 and March 2014. Median age was 66 years (range, 40–78 year) and 84% were male. A total of 38 patients had stage IV non-small cell lung carcinoma and 12 had extensive disease small-cell lung carcinoma. The completion rate was 70% (95% confidence interval 56.0–81.0). The median duration between baseline and T2 was 53 days. Improvement from baseline were observed at T2 in Functional Assessment of Cancer Therapy-Lung scores (86.0 ± 18.1 vs 94.9 ± 18.2, P = 0.057), depression (16.0 vs 5.7%; P = 0.26) and anxiety (32.0 vs 22.9%; P = 0.65); however, these results were not statistically significant. Conclusions This early specialized palliative care intervention is feasible and could be useful in improving patients’ quality of life. The present results justify the initiation of a randomized control trial.

BACKGROUND: Thymic epithelial tumours (TETs) are rare tumours comprised of thymomas and thymic carcinoma. Novel therapies are needed, especially in thymic carcinoma where the 5-year survival rate hovers at 30%. Mesothelin (MSLN), a surface glycoprotein that is cleaved to produce mature MSLN (mMSLN) and megakaryocyte potentiating factor (MPF), is expressed in limited tissues. However, its expression is present in various cancers, including thymic carcinoma, where it is expressed in 79% of cases. METHODS: We utilised flow cytometry, in vitro cytotoxicity assays, and an in vivo xenograft model in order to demonstrate the ability of the MSLN targeting antibody–drug conjugate (ADC) anetumab ravtansine (ARav) in inhibiting the growth of thymic carcinoma. RESULTS: Thymoma and thymic carcinoma cell lines express MSLN, and anetumab, the antibody moiety of ARav, was capable of binding MSLN expressing thymic carcinoma cells and internalising. ARav was effective at inhibiting the growth of thymic carcinoma cells stably transfected with mMSLN in vitro. In vivo, 15 mg/kg ARav inhibited T1889 xenograft tumour growth, while combining 7.5 mg/kg ARav with 4 mg/kg cisplatin yielded an additive effect on inhibiting tumour growth. CONCLUSIONS: These data demonstrate that anetumab ravtansine inhibits the growth of MSLN positive thymic carcinoma cells in vitro and in vivo.

This prespecified subanalysis of the global, randomized controlled phase III KEYNOTE‐024 study of pembrolizumab vs chemotherapy in previously untreated metastatic non‐small‐cell lung cancer without EGFR/ALK alterations and a programmed death ligand 1 (PD‐L1) tumor proportion score of 50% or higher evaluated clinical outcomes among patients enrolled in Japan. Treatment consisted of pembrolizumab 200 mg every 3 weeks (35 cycles) or platinum‐based chemotherapy (four to six cycles). The primary end‐point was progression‐free survival; secondary end‐points included overall survival and safety. Of 305 patients randomized in KEYNOTE‐024 overall, 40 patients were enrolled in Japan (all received treatment: pembrolizumab, n = 21; chemotherapy, n = 19). Median progression‐free survival was 41.4 (95% confidence interval [CI], 4.2‐42.5) months with pembrolizumab and 4.1 (95% CI, 2.8‐8.3) months with chemotherapy (hazard ratio [HR], 0.27 [95% CI, 0.11‐0.65]; one‐sided, nominal P = .001). Median overall survival was not reached (NR) (95% CI, 22.9‒NR) and 21.5 (95% CI, 5.2‐35.0) months, respectively (HR, 0.39 [95% CI, 0.17‐0.91]; one‐sided, nominal P = .012). Treatment‐related adverse events occurred in 21/21 (100%) pembrolizumab‐treated and 18/19 (95%) chemotherapy‐treated patients; eight patients (38%) and nine patients (47%), respectively, had grade 3‐5 events. Immune‐mediated adverse events and infusion reactions occurred in 11 pembrolizumab‐treated patients (52%) and four chemotherapy‐treated patients (21%), respectively; four patients (19%) and one patient (5%), respectively, had grade 3‐5 events. Consistent with results from KEYNOTE‐024 overall, first‐line pembrolizumab improved progression‐free survival and overall survival vs chemotherapy with manageable safety among Japanese patients with metastatic non‐small‐cell lung cancer without EGFR/ALK alterations and a PD‐L1 tumor proportion score of 50% or higher. The trial is registered with Clinicaltrials.gov: NCT02142738., This prespecified subanalysis of the global, randomized controlled phase III KEYNOTE‐024 study of pembrolizumab vs chemotherapy in previously untreated metastatic non‐small‐cell lung cancer without EGFR/ALK alterations and a PD‐L1 tumor proportion score of 50% or higher evaluated clinical outcomes among patients enrolled in Japan. Consistent with results from KEYNOTE‐024 overall, first‐line pembrolizumab improved progression‐free survival and overall survival vs chemotherapy with manageable safety among 40 Japanese patients in the study.