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After a median follow-up of 18·3 months, the third-generation anaplastic lymphoma kinase (ALK) tyrosine-kinase inhibitor, lorlatinib, improved progression-free survival in patients with treatment-naive, ALK-positive non-small-cell lung cancer in the phase 3 CROWN study. Here we report updated efficacy data, including intracranial activity, from an unplanned analysis after 3 years of follow-up.CROWN is an ongoing, international, randomised, open-label phase 3 trial done in 104 centres in 23 countries worldwide. Eligible participants were aged 18 years and older or aged 20 years and older (depending on local regulations) with advanced, ALK-positive non-small-cell lung cancer, had received no previous systemic treatment for metastatic disease, had at least one extracranial measurable target lesion (according to the Response Evaluation Criteria in Solid Tumours [RECIST], version 1.1), and had an Eastern Cooperative Oncology Group performance status score of 0-2. Patients were randomly assigned (1:1) to oral lorlatinib 100 mg daily or oral crizotinib 250 mg twice daily in 28-day cycles. Randomisation was stratified by the presence or absence of brain metastasis, and by ethnicity. Since the primary endpoint of the study had been met at the planned interim analysis, no further formal analysis of progression-free survival was planned, per protocol. The current unplanned analysis was done to further characterise tumour-related endpoints with a longer follow-up and is presented descriptively. For the planned study, the primary endpoint was progression-free survival assessed by blinded independent central review. Secondary endpoints included progression-free survival (investigator), objective response rate, intracranial objective response rate, time to intracranial progression, duration of response, intracranial duration of response, and safety. Efficacy endpoints were also assessed by the presence or absence of baseline brain metastases. This study is registered with ClinicalTrials.gov, NCT03052608.Between May 11, 2017, and Feb 28, 2019, 425 patients were screened for eligibility, of whom 296 were enrolled and randomly assigned to the lorlatinib (n=149) or crizotinib (n=147) group. At data cutoff for this unplanned analysis (Sept 20, 2021), median duration of follow-up for progression-free survival was 36·7 months (IQR 31·3-41·9) for lorlatinib and 29·3 months (10·8-35·0) for crizotinib. Median progression-free survival by blinded independent central review was not reached (95% CI not reached-not reached) for lorlatinib and was 9·3 months (7·6-11·1) for crizotinib (hazard ratio [HR] 0·27 [95% CI 0·18-0·39]). 3-year progression-free survival was 64% (95% CI 55-71) in the lorlatinib group and 19% (12-27) in the crizotinib group. Progression-free survival (investigator), objective response rate, intracranial objective response rate, time to intracranial progression, and duration of response were improved with lorlatinib versus crizotinib. In patients with baseline brain metastases (n=37 lorlatinib; n=39 crizotinib), the HR for time to intracranial progression for lorlatinib versus crizotinib was 0·10 (95% CI 0·04-0·27); in patients without baseline brain metastases (n=112 lorlatinib; n=108 crizotinib), the HR was 0·02 (95% CI 0·002-0·14). In patients without brain metastases, one (1%) in the lorlatinib group and 25 (23%) in the crizotinib group had intracranial progression. Grade 3-4 adverse events occurred in 113 (76%) of 149 patients (most commonly due to altered lipid levels) with lorlatinib and in 81 (57%) of 142 patients with crizotinib. Adverse events led to treatment discontinuation in 11 (7%) patients in the lorlatinib group and 14 (10%) patients in the crizotinib group. There were no new safety signals.These updated, long-term data from CROWN show the durable benefit of lorlatinib over crizotinib in patients with treatment-naive, ALK-positive non-small-cell lung cancer and support the use of first-line lorlatinib in patients with and without baseline brain metastases.Pfizer.

Duration of PFS in the patient subgroup with MET amplification

Treatment-related adverse events in treated patients with MET amplification and/or MET IHC 2+/3+

Representativeness of study population

Outcomes of treatment for the patient subgroup with MET overexpression

Plain language summary

Outcomes of treatment for all individuals enrolled

Purpose:The final analyses of the INSIGHT phase II study evaluating tepotinib (a selective MET inhibitor) plus gefitinib versus chemotherapy in patients with MET-altered EGFR-mutant NSCLC (data cut-off: September 3, 2021).Patients and Methods:Adults with advanced/metastatic EGFR-mutant NSCLC, acquired resistance to first-/second-generation EGFR inhibitors, and MET gene copy number (GCN) ≥5, MET:CEP7 ≥2, or MET IHC 2+/3+ were randomized to tepotinib 500 mg (450 mg active moiety) plus gefitinib 250 mg once daily, or chemotherapy. Primary endpoint was investigator-assessed progression-free survival (PFS). MET-amplified subgroup analysis was preplanned.Results:Overall (N = 55), median PFS was 4.9 months versus 4.4 months [stratified HR, 0.67; 90% CI, 0.35–1.28] with tepotinib plus gefitinib versus chemotherapy. In 19 patients with MET amplification (median age 60.4 years; 68.4% never-smokers; median GCN 8.8; median MET/CEP7 2.8; 89.5% with MET IHC 3+), tepotinib plus gefitinib improved PFS (HR, 0.13; 90% CI, 0.04–0.43) and overall survival (OS; HR, 0.10; 90% CI, 0.02–0.36) versus chemotherapy. Objective response rate was 66.7% with tepotinib plus gefitinib versus 42.9% with chemotherapy; median duration of response was 19.9 months versus 2.8 months. Median duration of tepotinib plus gefitinib was 11.3 months (range, 1.1–56.5), with treatment >1 year in six (50.0%) and >4 years in three patients (25.0%). Seven patients (58.3%) had treatment-related grade ≥3 adverse events with tepotinib plus gefitinib and five (71.4%) had chemotherapy.Conclusions:Final analysis of INSIGHT suggests improved PFS and OS with tepotinib plus gefitinib versus chemotherapy in a subgroup of patients with MET-amplified EGFR-mutant NSCLC, after progression on EGFR inhibitors.

Demographic data in the patient subgroup with MET overexpression

Demographic data of all individuals enrolled

PDF file, 22K, Activation of JAK family by treatment of afatinib.

Cisplatin inhibits colony formation of lung squamous cell carcinoma (SCC) cells.

MEK inhibition triggers G1 arrest, while cisplatin induced S-phase arrest, in lung SCC cells.

PDF file, 104K, Effects of afatinib on cell cycle and cell death.

MEK inhibition improves in vivo efficacy of cisplatin in SCC xenografts.

PDF file, 29K, Effect of JAK2 knockdown on afatinib-induced cell cytotoxicity.

Cisplatin-resistant H596 sustained activation of p-ERK after cisplatin treatment.

PDF file, 20K, Effect of Src inhibitor on afatinib-induced STAT3 activation.

PDF file, 53K, Anti-proliferative effect of dacomitinib in NSCLC cells harboring EGFR T790M.

Lack of significantly altered genes in lung squamous cell carcinoma (SCC) tumors.

Silencing of ERK1/2 enhances cisplatin sensitivity and stabilizes Bim in lung squamous cell carcinoma (SCC) cells.

PDF file, 14K, Inactivation of c-MET by treatment of afatinib.

Supplementary Methods Supplementary Table Captions and legends for Supplementary Figures S1 -S11; Supplementary Table S1: Differentially regulated genes in both cisplatin-resistant and -sensitive phenotypes after cisplatin treatment (fold change > 1.5, P < 0.05); Supplementary Table S2: Baseline characteristics of the head and neck squamous cell carcinoma (HNSCC)population, classified according to the p-ERK1/2 scoring.

PDF file, 19K, Enhancement of afatinib-induced apoptosis by STAT3 knockdown.

PDF file, 92K.

MEK inhibition enhances colony inhibition in lung squamous cell carcinoma (SCC) cells.

Src phosphorylation does not mediate MAPK/ERK signaling in lung SCC cells.

The secondary T790M mutation in epidermal growth factor receptor (EGFR) is the major mechanism of acquired resistance to EGFR tyrosine kinase inhibitors (TKI) in non–small cell lung cancer (NSCLC). Although irreversible EGFR TKIs, such as afatinib or dacomitinib, have been introduced to overcome the acquired resistance, they showed a limited efficacy in NSCLC with T790M. Herein, we identified the novel de novo resistance mechanism to irreversible EGFR TKIs in H1975 and PC9-GR cells, which are NSCLC cells with EGFR T790M. Afatinib activated interleukin-6 receptor (IL-6R)/JAK1/STAT3 signaling via autocrine IL-6 secretion in both cells. Inhibition of IL-6R/JAK1/STAT3 signaling pathway increased the sensitivity to afatinib. Cancer cells showed stronger STAT3 activation and enhanced resistance to afatinib in the presence of MRC5 lung fibroblasts. Blockade of IL-6R/JAK1 significantly increased the sensitivity to afatinib through inhibition of afatinib-induced STAT3 activation augmented by the interaction with fibroblasts, suggesting a critical role of paracrine IL-6R/JAK1/STAT3 loop between fibroblasts and cancer cells in the development of drug resistance. The enhancement of afatinib sensitivity by inhibition of IL-6R/JAK1/STAT3 signaling was confirmed in in vivo PC9-GR xenograft model. Similar to afatinib, de novo resistance to dacomitinib in H1975 and PC9-GR cells was also mediated by dacomitinib-induced JAK1/STAT3 activation. Taken together, these findings suggest that IL-6R/JAK1/STAT3 signaling can be a potential therapeutic target to enhance the efficacy of irreversible EGFR TKIs in patients with EGFR T790M. Mol Cancer Ther; 11(10); 2254–64. ©2012 AACR.

Cisplatin resistance in head and neck squamous cell carcinoma (HNSCC) cells is associated with activation of MAPK/ERK pathway.

Effects of cisplatin treatment on levels of phosphorylated kinases are assessed in H596 and Calu-1 cells.

Gene expression levels in H3122, LR, and panobinostat treated LR cells and the promoter or enhancer H3K27 ac gain or loss

Gene set enrichment analysis of previously published EMT gene signatures in LR cells compared with parental cells

Purpose:RET is an estrogen response gene with preclinical studies demonstrating cross-talk between the RET and estrogen receptor (ER) pathways. We investigate the role of lenvatinib, a multikinase inhibitor with potent activity against RET, in patients with metastatic breast cancer.Patients and Methods:Patients with advanced ER+/HER2− breast cancer were treated with lenvatinib plus letrozole in a phase Ib/II trial. Primary objectives included safety and recommended phase II dose (RP2D) determination in phase Ib, and objective response rates (ORR) in phase II dose expansion.Results:Sixteen patients were recruited in dose finding, where deescalating doses of lenvatinib from 20 to 14 mg were investigated. Lenvatinib 14 mg plus letrozole 2.5 mg daily was determined as RP2D. Thirty-one patients with 5 median lines of prior therapy in the metastatic setting (range, 0–11) were recruited in dose expansion. In this cohort, ORR was 23.3% [95% confidence interval (CI) 9.9%–42.3%], with median duration of response (DoR) of 6.9 months [interquartile range (IQR) 5.9 to 13.1]. Clinical benefit rate ≥6 months (CBR) was 50.0% (95% CI, 31.3%–68.7%). Similar efficacy was observed in the subgroup of 25 patients who progressed on prior CDK4/6 inhibitor therapy [ORR 20.0% (95% CI, 6.8%–40.7%), median DoR 6.9 months (IQR 5.9–13.1), and CBR 52.0% (95% CI, 31.3%–72.2%)]. Pharmacodynamic studies showed target modulation, with paired tumor biopsies indicating downregulation of RET/pERK and improved vascular normalization index.Conclusions:Lenvatinib plus letrozole had manageable toxicity, with target engagement and preliminary antitumor activity observed, supporting further assessment in randomized studies.

Figure S1- Study schema Figure S2 - Example of complete metabolic response after 3 cycles of induction therapy in a representative Arm C patient treated with bevacizumab at 7.5mg/kg in combination with cisplatin and gemcitabine. Figure S3- Relapse free survival (RFS) in months by study arms of patients with locally advanced disease who underwent concurrent chemoradiation after induction chemotherapy. Figure S4 - Relapse free survival in months by metabolic response to induction therapy. Figure S5 - (A) Spaghetti Plot of EBV viral load at baseline (BL), C2D1 and C3D1; (B)Circulating VEGF levels in plasma samples collected at BL, C1D1, C2D1 and end of induction therapy, EOT. Figure 6a- Figure S6 - Induction of mature blood vessel in the NPC tumour microenvironment 7 days following initiation of sunitinib and bevacizumab exposure. Figure S6b - Quantification of blood vessel densities Figure S7- Immune cell infiltration in the NPC tumour microenvironment 7 days following initiation of sunitinib or bevacizumab exposure

Supplementary methods and supplementary tables

Supplementary Table from Phase Ib/II Dose Expansion Study of Lenvatinib Combined with Letrozole in Postmenopausal Women with Hormone Receptor–Positive Breast Cancer

Supplementary Data from Phase Ib/II Dose Expansion Study of Lenvatinib Combined with Letrozole in Postmenopausal Women with Hormone Receptor–Positive Breast Cancer

Supplementary Figure from Phase Ib/II Dose Expansion Study of Lenvatinib Combined with Letrozole in Postmenopausal Women with Hormone Receptor–Positive Breast Cancer

Supplementary Figure S1. Viability of H3122-LR and H2228-LR cells Supplementary Figure S2. Gene set enrichment analysis and western blot Supplementary Figure S3. Effects of AXL inhibition on EMT gene expression in LR cells, related to Fig. 1E. Supplementary Figure S4. Mice with established H3122-derived tumors were treated with four doses (50, 75, 87.5, or 100 mg/kg) of ceritinib to derive ceritinib-resistant tumors Supplementary Figure S5. Enhancer remodeling during ceritinib resistance, related to Fig. 2. Supplementary Figure S6. Genes with H3K27ac loss or gain in their promoters during ceritinib resistance, related to Fig.2.

Supplementary Materials and Methods that contain detailed experimental procedures

Purpose:Induction cisplatin and gemcitabine chemotherapy is a standard treatment for locally advanced nasopharyngeal carcinoma (NPC). Inhibition of VEGF axis has been shown to promote maturation of microvasculature and improve perfusion. We conducted a four-arm study to assess the effect of two doses of either sunitinib or bevacizumab with chemotherapy in NPC.Patients and Methods:Patients with treatment-naïve locally advanced NPC were treated with three cycles of 3-weekly cisplatin and gemcitabine preceded by 1 week of anti-VEGF therapy for each cycle, followed by standard concurrent chemoradiation: arm A patients received 7 days of 12.5 mg/day sunitinib; arm B 7 days of 25 mg/day sunitinib; arm C bevacizumab 7.5 mg/kg infusion; arm D bevacizumab 2.5 mg/kg infusion. Patients with metastatic NPC were treated with up to six cycles of similar treatment without concurrent chemoradiation.Results:Complete metabolic response (mCR) by whole body 18FDG PET was highest in arm C (significant difference in four groups Fisher exact test P = 0.001; type 1 error = 0.05), with 42% mCR (95% confidence interval, 18–67) and 3-year relapse-free survival of 88% in patients with locally advanced NPC. Significant increase in pericyte coverage signifying microvascular maturation and increased immune cell infiltration was observed in posttreatment tumor biopsies in Arm C. Myelosuppression was more profound in sunitinib containing arms, and tolerability was established in arm C where hypertension was the most significant toxicity.Conclusions:Bevacizumab 7.5 mg/kg with cisplatin and gemcitabine was well tolerated. Promising tumor response was observed and supported mechanistically by positive effects on tumor perfusion and immune cell trafficking into the tumor.

Anaplastic lymphoma kinase (ALK) inhibitors are highly effective in patients with ALK fusion–positive lung cancer, but acquired resistance invariably emerges. Identification of secondary mutations has received considerable attention, but most cases cannot be explained by genetic causes alone, raising the possibility of epigenetic mechanisms in acquired drug resistance. Here, we investigated the dynamic changes in the transcriptome and enhancer landscape during development of acquired resistance to ALK inhibitors. Histone H3 lysine 27 acetylation (H3K27ac) was profoundly altered during acquisition of resistance, and enhancer remodeling induced expression changes in both miRNAs and mRNAs. Decreased H3K27ac levels and reduced miR-34a expression associated with the activation of target genes such as AXL. Panobinostat, a pan-histone deacetylase inhibitor, altered the H3K27ac profile and activated tumor-suppressor miRNAs such as miR-449, another member of the miR-34 family, and synergistically induced antiproliferative effects with ALK inhibitors on resistant cells, xenografts, and EML4-ALK transgenic mice. Paired analysis of patient samples before and after treatment with ALK inhibitors revealed that repression of miR-34a or miR-449a and activation of AXL were mutually exclusive of secondary mutations in ALK. Our findings indicate that enhancer remodeling and altered expression of miRNAs play key roles in cancer drug resistance and suggest that strategies targeting epigenetic pathways represent a potentially effective method for overcoming acquired resistance to cancer therapy.Significance: Epigenetic deregulation drives acquired resistance to ALK inhibitors in ALK-positive lung cancer. Cancer Res; 78(12); 3350–62. ©2018 AACR.

Differentially Expressed miRNAs in Ceritinib Resistant Cells

Supplementary figure legends S1 to S12

Supplementary Figure S7. Supplementary Figure S8. Transfection efficiency of miR-34a and miR-449a mimics and inhibitors in cell lines, related to Fig. 3. Supplementary Figure S9. The effects of miR-34a and miR-449a inhibitors on the viability of parental H3122 cells. Supplementary Figure S10. Genes with H3K27ac gain or loss in their promoters induced by the panobinostat treatment, related to Fig. 4. Supplementary Figure S11. H3122 and H3122-LR cells treated with ceritinib alone (1 Ã,µM), panobinostat alone (100 nM), or the combination, related to Fig. 5. Supplementary Figure S12.

Clinical information of IHC samples.

Effect of ICG-001 in EGFR-LR-TM transgenic mice.

Generation of conditional, lung-specific Ctnnb1 knockout/EGFR-L858R-T790M (EGFRTL) transgenic mice.

Incomplete deletion of β-catenin gene leads to tumor formation in EGFRTL/CCSP-rtTA/Cre/Ctnnb1F/F mice.