ALK-rearranged NSCLC Patients Research Articles

Risk factors and clinical characteristics of pneumonitis among patients with ALK-rearranged non-small cell lung cancer who received alectinib and thoracic radiotherapy.

e20591 Background: Alectinib and thoracic radiotherapy (TRT) are important modalities in the management of ALK-rearranged non-small cell lung cancer. We therefore aimed to assess the incidence and risk factors of pneumonitis when these two treatments were combined. Methods: Patients with ALK-rearranged non-small cell lung cancer, receiving alectinib and TRT from January 2018 to December 2020 were reviewed, and the clinical and dosimetry data were collected. Logistic regression analyses were performed to evaluate risk factors associated with treatment-related pneumonitis (TRP). The prediction ability of dosimetry parameters for pneumonitis were examined by receiver-operating characteristic (ROC) curve analyses. Results: Of the 25 enrolled patients, 16 (64%) developed pneumonitis, and 12 (48%) developed grade 2 or higher pneumonitis. Logistics regression analyses revealed chemotherapy (OR = 8.750, 95% CI 1.207 - 63.428, p = 0.032), higher ipsilateral V5 (OR = 1.060, 95%CI 1.002 - 1.122, p = 0.043) and total lung V5 (OR = 1.083, 95%CI 1.000 - 1.174, p = 0.05) to be risk factors for TRP. Total lung V10, total lung V5, and ipsilateral V5 were strong predictors of TRP among the dosimetry parameters (AUC = 0.800, 0.786 and 0.781, respectively). After developing pneumonitis, 52% of patients recovered or improved, but one patient died due to respiratory failure. Conclusions: The combined use of alectinib and TRT significantly increased the risk of pneumonitis. Clinicians should consider the elevated risks and related dosimetric factors when deciding on combination treatment for ALK-rearranged NSCLC patients.

P84.06 Alectinib in ALK-Rearranged NSCLC Patients Following Crizotinib. Final Results and Biological Outcomes - Phase II ATALK Study

P84.06 Alectinib in ALK-Rearranged NSCLC Patients Following Crizotinib. Final Results and Biological Outcomes - Phase II ATALK Study

P84.07 Distribution and Therapeutic Outcomes of Intergenic Sequence-ALK Fusion and Coexisting ALK Fusions in Lung Adenocarcinoma Patients

ALK-rearranged NSCLC patients show survival benefits from ALK inhibitor. Studies about outcome were mostly focused on EML4–ALK fusion. Here, we performed next-generation sequencing (NGS) of baseline specimens from ALK-positive patients to examine the rearrangement distribution and possible correlation to therapeutic benefit. ALK-positive NSCLC patient(determined by immunohistochemistry) were screened at the West China Hospital, and NGS was performed on baseline samples. Clinical information and therapeutic outcomes were collected and retrospectively analyzed. Among the 89 patients with 22 ALK rearrangements partners, fusions of intergenic sequences with ALK were found in 15 (16.85%). Non-EML4–ALK fusions were present in 18 patients (20.22%), while EML4–ALK fusions in 71 (79.76%). Coexisting rearrangements were present in 16 patients (17.98%). Intergenic–ALK and non EML4–ALK fusions occurred at higher rates in patients with coexisting fusions (62.5% versus 6.85%, and 62.5% versus 10.96%). First-line crizotinib was administered in 41 patients, and the median progression-free survival (mPFS) was 9.7 months. No significant difference in mPFS was found between patients with or without intergenic ALK fusion (12 versus 9.6 months, p = 0.989). In the seven patients who had at least two fusions each, the mPFS was 11.9 months, compared with 9 months among the 24 patients with single (p = 0.336). No significant difference in mPFS was observed between patients with and without EML4–ALK fusions (9.6 versus 12 months, p = 0.924). The hazard ratio(HR) of gender, age and three rearrangements (EML4-, non EML4-, intergenic-ALK) were 0.47, 1.18, 1.09, 1.31, 0.96, respectively.Table 1Characteristics of enrolled patients.CharacteristicTotal (n = 90)TreatmentP valueSurgery (n = 46)Systemic therapy (n = 44)Age (median [range], years)51 (29-78)52.5 (30-78)49 (29-70)0.396Gender (cases, %)0.404Male47 (52.2)26 (56.5)21 (47.7)Female43 (47.8)20 (43.5)23 (52.3)Smoking status (cases, %)0.185Smoker6 (6.7)1 (2.2)5 (11.4)Non-smoker84 (93.3)45 (97.8)39 (88.6)Histology (cases, %)/Adenocarcinoma90 (100)46 (100)44 (100)Non-adenocarcinoma0 (0)0 (0)0 (0)ECOG performance status (cases, %)0.6890-179 (87.8)41 (89.1)38(86.4)2-411 (12.2)5 (10.9)6 (13.6)Stage (cases, %)<0.001I-III49 (54.4)46 (100)3 (6.8)IV41 (45.6)0 (0)41 (93.2)Metastasis (cases, %)<0.001Absent49 (54.4)46 (100)3 (6.8)Present41 (45.6)0 (0)41 (93.2)Brain metastasis13 (14.4)0 (0)13 (29.5)Bone metastasis24 (26.7)0 (0)24 (54.5)Multi-organ metastases20 (22.2)0 (0)20 (45.5)ALK aberrations (cases, %)0.489Rearrangement free Uncertain aberration1 (1.1)0 (0)1 (2.3)ALK rearrangement89 (98.9)46 (100)43 (97.7)Distribution of rearrangements (cases, %)Intergenic sequence-ALK fusion15 (16.9)9 (19.6)6 (14.0)0.480Non-EML4-ALK fusion18 (20.2)10 (21.7)8 (18.6)0.713EML4-ALK fusionVariant 126 (29.2)12 (26.1)14 (32.6)0.502Variant 24 (4.5)3 (6.5)1 (2.3)0.658Variant 330 (33.7)14 (30.4)16 (37.2)0.499Other variants11 (12.4)8 (17.4)3 (7.0)0.136Status of coexisting ALK fusions (cases, %)Absent73 (82.0)37 (80.4)36 (83.7)0.687Intergenic sequence-ALK fusion alone5 (5.6)2 (4.3)3 (7.0)0.938Non-EML4-ALK fusion alone8 (9.0)5 (10.9)3 (7.0)0.787EML4-ALK fusion alone60 (67.4)30 (65.2)30 (69.8)0.647Present16 (18.0)9 (19.6)7 (16.3)0.687Intergenic sequence-ALK fusion + EML4-ALK fusion6 (6.7)4 (8.7)2 (4.7)0.736Intergenic sequence-ALK fusion + non-EML4-ALK fusion1 (1.1)1 (2.2)0 (0)1.000EML4-ALK fusion + non-EML4-ALK fusion3 (3.4)1 (2.2)2 (4.7)0.953Intergenic-ALK fusion + EML4-ALK fusion+ non-EML4-ALK fusion2 (2.2)2 (4.3)0 (0)0.495Intergenic-ALK fusion + intragenic-ALK fusion + non-EML4-ALK fusion1 (1.1)0 (0)1 (2.3)0.483non-EML4-ALK fusion + non-EML4-ALK fusion3 (3.4)1 (2.2)2 (4.7)0.953 Open table in a new tab This is the first to report the occurrence rates and distribution of intergenic–ALK and coexisting of ALK fusions. Intergenic–ALK and non-EML4–ALK were more likely to coexist with other fusions. Neither the type nor number of rearrangements had a significant effect on the therapeutic benefit of treatment with crizotinib.

Tumor volume dynamics and tumor growth rate in ALK-rearranged advanced non-small-cell lung cancer treated with crizotinib

PurposeThe purpose of the study is to investigate volumetric tumor burden dynamics and tumor growth rates in ALK-rearranged advanced NSCLC patients during crizotinib monotherapy. MethodsThe study included 44 ALK-rearranged advanced NSCLC patients treated with crizotinib monotherapy as their initial ALK-directed therapy, who had at least one measurable lung lesion and at least two follow-up CT scans, and experienced tumor volume increase while on crizotinib. The tumor volume (in mm3) of the dominant lung lesion was measured on serial CT scans during therapy for analysis of tumor growth rates after the volume nadir. ResultsA total of 231 volume measurements from the nadir to the end of crizotinib therapy or the last follow-up in 44 patients were analyzed in a linear mixed-effects model, fitting time (in months since baseline) as a random effect. When measured from the volume nadir, the tumor growth rate of the logarithm of tumor volume (logeV) was 0.04/month (SE = 0.012, P = 0.0011) in the unadjusted model. When adjusted for the baseline volume (logeV0), the growth rate was again 0.04/month (SE = 0.011, P = 0.0004). When adjusted for clinical variables and logeV0, the growth rate was 0.045/month (SE = 0.012, P = 0.0002), indicating that the tumor growth rate after nadir in this cohort remains very close to 0.04/month regardless of logeV0 or clinical factors. ConclusionsTumor volume growth rate after nadir in ALK-rearranged NSCLC patients treated with crizotinib was obtained, providing objective reference values that can inform physicians when deciding to keep their patients on ALK directed therapy with slowly progressing lung cancer.

1544P - The safety assessment of crizotinib and alectinib from real-world data of 840 ALK-inhibitor naïve patients with NSCLC harboring ALK-rearrangement (WJOG9516L)

1544P - The safety assessment of crizotinib and alectinib from real-world data of 840 ALK-inhibitor naïve patients with NSCLC harboring ALK-rearrangement (WJOG9516L)

Diagnosis and Treatment of ALK Aberrations in Metastatic NSCLC.

There has been rapid progress in the use of targeted therapies for ALK-positive which has led to improve dramatically PFS and OS in the metastatic ALK-rearranged NSCLC patients. There are several molecules now available (crizotinib, ceritinib, brigatinib, alectinib, and lorlatinib) and others in development. Such an improvement in treatment efficacy has even more highlighted the importance of an adequate identification of ALK alterations. Efficient and easily accessible testing tools are required to identify eligible patients in a timely fashion. Different methods for detecting ALK+ NSCLC patients are now available, with fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) currently representing validated diagnostic techniques for the initial assessment of ALK status. Furthermore the widespread use of next-generation sequencing to detect other possible different activating mutations has allowed to identify individual ALK fusion variants. Several more expensive and time-consuming methods are also available nowadays which have the advantage to detect even rarer uncommon ALK fusion variants and mutations in tumour or blood samples. A review of the evolving testing-treatment landscape is needed to highlight the importance of properly diagnosing and treating this group of patients.

The impact of sequential therapy of crizotinib followed by alectinib: Real-world data analysis of 840 ALK-inhibitor naïve patients with NSCLC harboring ALK-rearrangement (WJOG9516L).

9038 Background: Previous clinical trials demonstrated that alectinib (ALEC) had a longer time-to-progression than crizotinib (CRZ) in 1st-line settings. Information on long-term overall survival (OS), however, is still limited with a few studies having reported that the sequential strategy of “CRZ followed by other ALK-inhibitor” can provide extended OS. In Japan, ALEC was approved for a 1st-line setting earlier than in other countries. Methods: We reviewed the clinical data of ALK-rearranged NSCLC patients who received CRZ or ALEC between May 2012 and Dec 2016. Patients were divided into two groups according to the first-administered ALK inhibitor, the CRZ or ALEC group. In order to evaluate the efficacy of the sequential strategy of “CRZ followed by ALEC”, the combined time to treatment failure (TTF) was calculated in the CRZ group as defined by the sum of the “TTF of CRZ” plus the “TTF of ALEC” if patients were treated with ALEC followed by CRZ. In the ALEC group, the “TTF of ALEC” was calculated. The primary endpoint is the comparison between the combined TTF in the CRZ group with the TTF in the ALEC group. Results: Of 864 patients enrolled from 61 institutions, 840 patients were analyzed. Median age was 61 (range, 20-94); 56% were female; and 95% had adenocarcinoma. There were 535/305 patients in the CRZ/ALEC group. In the CRZ group, 282 patients received ALEC after CRZ failure. The combined TTF in the CRZ group was significantly longer than TTF in the ALEC group; median, 34.4 vs 27.2 months (mo); hazard ratio (HR), 0.709 [95%CI;0.559- 0.899]; P= 0.0044. However, there was no significant difference in OS between the patients who received ALEC after CRZ in the CRZ group and the patients in the ALEC group; median, 88.4 months vs. not reached; HR 1.048 [95%CI;0.758-1.451]; P= 0.7770. In the whole population, the CRZ group had a significantly shorter OS than the ALEC group; median, 53.6 mo vs not reached HR, 1.821 [95%CI;1.372-2.415]; P&lt; 0.0001. Conclusions: The combined TTF in the CRZ group was significantly longer than TTF in the ALEC group, however, OS benefit of sequential therapy of CRZ followed by ALEC was not shown. Clinical trial information: UMIN000028605.

Analysis of single circulating tumor cells (CTCs) to identify resistance mutations to ALK-inhibitors in both ALK-gene and bypass oncogenic pathways.

12038Background: Non-invasive methods including CTCs are crucial to develop for the implementation of precision medicine in the treatment of NSCLC. ALK-rearranged NSCLC patients develop resistance ...

P3.02-050 Mechanisms of Acquired Resistance to the ALK Inhibitor Lorlatinib in ALK-Rearranged NSCLC Cell Lines

Lorlatinib is a potent third-generation ALK tyrosine kinase inhibitor, which has demonstrated promising efficacy in ALK-rearranged NSCLC patients and excellent preclinical activity against ALK resistance mutations. However, as with all targeted treatments, resistance inevitably emerges. The molecular mechanisms underlying lorlatinib resistance remain largely undescribed, except for the L1198F mutation in ALK that was presented in a case study involving a single patient. Thus, the present study is performed to discover novel mechanisms of resistance to lorlatinib treatment.

Evaluating First-Line Ceritinib Therapy in ALK-Rearranged NSCLC Patients

Evaluating First-Line Ceritinib Therapy in ALK-Rearranged NSCLC Patients

Abstract 856: Number of ALK-amplified circulating tumor cells predicts progression-free survival in ALK-rearranged non-small cell lung cancer patients treated by crizotinib

Abstract The duration of clinical response is unpredictable in ALK-rearranged NSCLC patients treated by crizotinib but all patients invariably develop resistance. Using filter-adapted fluorescence in situ hybridization (FA-FISH) we previously reported ALK-rearrangement detection in circulating tumor cells (CTCs) from ALK-rearranged patients. Here we report the monitoring of ALK-rearranged (≥1×3’/5’, ≥1x(3’ and 5’)) and ALK-amplified (&amp;gt;2×3’/5’) CTCs at baseline and at an early time point under crizotinib therapy in an extended cohort of ALK-rearranged patients. The correlation between CTC subsets harboring distinct FISH patterns and clinical parameters including progression-free survival (PFS) and overall survival (OS) is presented. Forty ALK-rearranged patients were recruited. Blood samples were collected at baseline to crizotinib and at 1-3 months. Abnormal ALK-FISH patterns were examined in CTCs using immunofluorescence staining (DAPI/CD45) combined with FA-FISH after isolation by size of epithelial tumor cells (ISET) filtration. CTCs were defined into five distinct subsets according to the number of FISH ‘break-apart’ signals (3’ and 5’) or isolated red signals (3’) and/or native copies (3’/5’). All ALK-abnormal cells were validated by a cytogenetician. Clinical data was collected retrospectively. Confirming our previous data, ALK rearrangement was detected in CTCs: 32/39 patients (82%) had ≥4 ALK-rearranged CTCs per 1 ml of blood. Age &amp;gt;60 years and smoking status &amp;gt;15 pack years were associated with a higher mean number of ALK-amplified CTCs (p = 0.0423 and p = 0.0407 respectively). No statistically significant correlation was observed between the different CTC subsets with abnormal FISH patterns at baseline and PFS or OS. However we observed a statistically significant correlation (p = 0.0196) between the evolution under crizotinib of the numbers of ALK-amplified CTCs and PFS. An increase in the numbers of ALK-amplified CTCs during treatment is associated with a PFS of 6.5 months, while a decrease in this subset is associated with a PFS of 25 months. There was no corresponding correlation with OS. The evolution under crizotinib therapy of numbers of ALK-amplified CTCs is significantly correlated with PFS. Although not dominant, amplification of the ALK gene has been reported to be one mechanism of acquired resistance to crizotinib therapy in tumor biopsies. CTCs with an amplification of ALK do not express the oncogenic ALK fusion protein and are therefore not targeted by crizotinib. FISH patterns suggested these CTCs may have a high degree of ploidy and chromosomal instability which may contribute to promote the emergence of CTC subclones with a high metastatic potential. Our data suggest that number of ALK-amplified CTCs may be a predictive biomarker allowing prediction of ALK-rearranged NSCLC patients who are at risk of early resistance to crizotinib. Citation Format: Emma Pailler, Marianne Oulhen, Kirsty Ross, Fanny Billiot, Nathalie Auger, Isabelle Borger, Maud NgoCamus, Jordi Remon-Masip, David Planchard, Jean-Charles Soria, Benjamin Besse, Françoise Farace. Number of ALK-amplified circulating tumor cells predicts progression-free survival in ALK-rearranged non-small cell lung cancer patients treated by crizotinib. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 856.

ALK-amplified circulating tumor cells as a surrogate marker for crizotinib benefit in ALK-rearranged non-small cell lung cancer patients.

11515Background: Duration of response in ALK-rearranged NSCLC patients treated by crizotinib is in the range of 10 months but all patients invariably acquire resistance. Using filter-adapted fluore...

Abstract 698: CPI-based Crizotinib systemic study on discovery of phenotypic off-target

Abstract Crizotinib, a first ever ALK/MET multi-targeted receptor tyrosine kinase inhibitor for the treatment of ALK-rearranged NSCLC patients. We hypothesize that crizotinib acts on other molecular targets in addition to tyrosine kinases ALK/MET and may play critical role given that there is a complex network of kinases that work together to regulate a number of important cellular processes and different disease signature. Approaching with a comprehensive docking method with our established chemical-protein interactome (CPI) and crizotinib, we have discovered 301 PDB-deposited proteins corresponding to 353 ligand binding pockets among a total of 1,780 PDB-deposited human protein entries. Systemic pharmacology approach also applied including validation the molecular target(s) of TKIs in vitro. Especially multiple human cellular models (including NSCLC and melanoma) and signaling study, luc-reporter, autophagy/apoptosis, proteomics study and NGS analysis applied. Interestingly, crizotinib besides had a high CPI binding score (ZZ_score) of -2.2778 against tyrosine kinase Lck, also had -1.4672, -1.1242, -0.7033 and -1.5384 against MAPK3, S6K, GSK-3β and HDAC 7A, respectively, which indicated crizotinib had potential broad range interaction with both TK and ser/thr kinases. Our data have showed that the crizotinib induced autophagy by activated LC3 in vitro and inhibited oncogenic Akt/mTOR signaling in human multiple melanoma cells resulting in G2/M cell cycle arrest. Furthermore, crizotinib increased p21Waf1/cip1 and Foxo3A expression and tumor suppressor miR-146a in human melanoma cells. Taken together, crizotinib induces miR-146a, autophagy and p21waf1/cip1 may potentially treat other type of human cancer by concurrently targeting tyrosine kinase, Akt/mTOR/Foxo axis and nuclear receptor, shedding a light for future both anti-cancer and anti-metabolic disorder drug discovery and development. This is the first CPI-based systemic study on leading discovery of crizotinib off-target phenotypic importance. These findings suggest that non-tyrosine kinase target of crizotinib may play critical role(s) for drug repositioning may represent a new and effective approach to expanding the application of existing drugs. Citation Format: Jiazhi Sun, Lun Yang, Minghua Li, Shimin Zhang, Ian E. Heller, Johnathan Samuelson, Steven Cindric, Kunal Mishra, Eslam Mohamed, Feng Cheng, Kevin B. Sneed, Lin He, Shu-Feng Zhou. CPI-based Crizotinib systemic study on discovery of phenotypic off-target. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 698. doi:10.1158/1538-7445.AM2015-698

Abstract 5122: In vivo imaging models of bone and brain metastases and pleural carcinomatosis developed using a novel human EML4-ALK lung cancer cell line, A925LPE3

Abstract Introduction:EML4-ALK lung cancer accounts for approximately 3%-7% of non-small cell lung cancer cases. Crizotinib, an ALK tyrosine kinase inhibitor, shows dramatic clinical efficacy with a response rate of approximately 60%-80% in EML4-ALK NSCLC patients. However, almost all patients who strongly responded to crizotinib acquired resistance over time. Recently, new generation ALK-TKIs such as alectinib and ceritinib have been approved for treatment of ALK-rearranged NSCLC patients, and it is now clear that resistance may also develop against this class of inhibitors. To investigate the molecular mechanism underlying tumor progression and targeted drug sensitivity/resistance in EML4-ALK lung cancer, clinically relevant animal models are indispensable. Methods: A human lung adenocarcinoma cell line A925L, which expresses an EML4-ALK gene fusion (variant 5a, E2:A20), established from a surgical specimen obtained from a Japanese male patient. We further established highly tumorigenic A925LPE3 cells with luciferase gene transfection, which also have the EML4-ALK gene fusion. A925LPE3 cells were injected into the right thoracic cavity for the pleural carcinomatosis model, into the tibia for the bone metastasis model, and into the right striatum for the brain metastasis model. After inoculation, the quantity of tumors and the quality of bones were tracked in live mice by repeated noninvasive optical imaging of tumor-specific luciferase activity using the IVIS Lumina XR Imaging System. Results: In this study, we found that A925L and A925LPE3 cell lines are sensitive to the ALK inhibitors crizotinib and alectinib. By using A925LPE3 cells we established in vivo imaging models for pleural carcinomatosis, bone metastasis, and brain metastasis, all of which are significant clinical concerns of advanced EML4-ALK lung cancer. Interestingly, crizotinib caused tumors to shrink in the pleural carcinomatosis model, but not in bone and brain metastasis models, while alectinib showed remarkable efficacy in all three models, indicative of the clinical efficacy of these ALK inhibitors. Conclusions: Our in vivo imaging models of multiple organ sites may provide useful resources to analyze further the pathogenesis of EML4-ALK lung cancer and its response and resistance to ALK inhibitors in various organ microenvironments. Citation Format: Shigeki Nanjo, Shinji Takeuchi, Kenji Kita, Koji Fukuda, Mitsutoshi Nakada, Hiroshi Nishihara, Seiji Yano. In vivo imaging models of bone and brain metastases and pleural carcinomatosis developed using a novel human EML4-ALK lung cancer cell line, A925LPE3. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5122. doi:10.1158/1538-7445.AM2015-5122

Abstract 1842: Synergistic inhibition of ALK inhibitor-resistant lung cancer cells by dual-targeting EGFR and ALK pathways

Abstract Background: Despite the excellent initial responses of ALK-rearranged lung cancer to the ALK tyrosine kinase inhibitors (TKIs), most patients develop acquired resistance through several mechanisms, including ALK amplification, mutation or activation of alternative signaling pathways. In this study, we investigated the effect of dual-targeting EGFR and ALK pathways in NSCLC cells that are resistant to ALK inhibitors. Methods: The cell proliferation, apoptosis and cell cycle analysis were measured by spectrophotometry and flow cytometry. The combination effect of EGFR TKI (afatinib or dacomitinib) and ALK TKI (crizotinib or LDK378) was evaluated using a combination index (CI). We analyzed EGFR ligand levels by ELISA and expression of ALK, EGFR and its downstream molecules were detected by Western blot assay after exposure to TKIs. Results: NCI-H3122CR1 that was resistant to crizotinib and NCI-H3122 cell lines became resistant to LDK378 through the step-wise increase of the drug, resulting in H3122CR1LR1 and H3122LR1 cells, respectively. The resistant cell lines exhibited 10-14-fold higher IC50 values for crizotinib or LDK378 compared with parental cells. The genetic alterations of ALK or EGFR genes were not found in H3122CR1, H3122LR1, and H3122CR1LR1 cells. We analyzed EGFR signaling pathways in these cells to determine their roles in mediating ALK inhibitor resistance. We observed that H3122CR1, H3122LR1 and H3122CR1LR1 cells had higher phospho-EGFR than parental cells. In these cells, mRNA and protein levels of amphiregulin and EGF were up-regulated. Combined treatment with EGFR TKI and LDK378 synergistically inhibited cell growth (CI&amp;lt;1) and induced apoptosis in H3122LR1 and H3122CR1LR1 cells. Similar results were observed in H3122CR1 and SNU-2535 harboring ALK G1269A mutation cells, when exposed to afatinib and crizotinib. The combination of EGFR and ALK TKIs decreased the phosphorylation of several downstream molecules in H3122LR1 and H3122CR1LR1 cells, whereas EGFR or ALK TKI alone did not reduce the phosphorylation levels. Conclusion: Our results suggest that dual inhibition of ALK and EGFR effectively abrogate ALK and its downstream signals and induce apoptosis in ALK TKIs-resistant, ALK-rearranged NSCLC cells. Therefore, dual-targeting of EGFR and ALK might be a promising strategy for ALK-rearranged NSCLC patients who showed acquired resistance to crizotinib and/or LDK378. Citation Format: Soyeon Kim, Tae Min Kim, Yong-Oon Ahn, Bhumsuk Keam, Se-Hoon Lee, Dae Seog Heo, Dong-Wan Kim. Synergistic inhibition of ALK inhibitor-resistant lung cancer cells by dual-targeting EGFR and ALK pathways. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1842. doi:10.1158/1538-7445.AM2014-1842

Abstract 3738: Crizotinib induces tumor suppressor miR-146a, autophagy and p21 waf1/cip1 by alternative targeting non-TK target Akt-mTOR-Foxo axis: A CPI-based systemic study on discovery of phenotypic off-target

Abstract Crizotinib, a first ever ALK /MET multi-targeted receptor tyrosine kinase inhibitor developed by Pfizer and fast approved by FDA in 2011 for the treatment of ALK-rearranged NSCLC patients. We hypothesize that crizotinib acts on other molecular targets in addition to tyrosine kinases ALK/MET and may play critical role given that there is a complex network of kinases that work together to regulate a number of important cellular processes and different disease signature. Approaching with a comprehensive docking method with our established chemical-protein interactome (CPI) and crizotinib, we have discovered 301 PDB-deposited proteins corresponding to 353 ligand binding pockets among a total of 1,780 PDB-deposited human protein entries. Systemic pharmacology approach also applied including validation the molecular target(s) of TKIs in vitro. Especially multiple human cellular models (including NSCLC and melanoma) and signaling study, luc-reporter, autophagy, apoptosis assay, FACS and NGS analysis applied. Interestingly, crizotinib besides had a high CPI binding score (ZZ_score) of -2.2778 against tyrosine kinase Lck, also had -1.4672, -1.1242, -0.7033 and -1.5384 against MAPK3, S6K, GSK-3β and HDAC 7A, respectively, which indicated crizotinib had potential broad range interaction with both TK and ser/thr kinases. In addition, those crizotinib achieved high ZZ scores against PPAR-γ (-1.6145) and VitD3R (0.9479), suggesting a high binding affinity of crizotinib with these nuclear proteins. Our preliminary studies have showed that the crizotinib induced autophagy by activated LC3 in vitro and inhibited oncogenic Akt/mTOR signaling in human multiple melanoma cells resulting in G2/M cell cycle arrest. Furthermore, crizotinib increased p21Waf1/cip1 and Foxo3A expression and tumor suppressor miR-146a in human melanoma cells. Taken together, crizotinib induces miR-146a, autophagy and p21 waf1/cip1 may potentially treat other type of human cancer by concurrently targeting tyrosine kinase, Akt/mTOR/Foxo axis and nuclear receptor, shedding a light for future both anti-cancer and anti-metabolic disorder drug discovery and development. This is the first CPI-based systemic study on leading discovery of crizotinib off-target phenotypic importance. These findings suggest that non-tyrosine kinase target of crizotinib may play critical role(s) for drug repositioning may represent a new and effective approach to expanding the application of existing drugs. Citation Format: Jiazhi Sun, Lun Yang, Minghua Li, Ian E. Heller, Issac Raplee, Steven Guzman, John P. Pasciak, Feng Cheng, Kevin B. Sneed, Lin He, Shu-Feng Zhou. Crizotinib induces tumor suppressor miR-146a, autophagy and p21 waf1/cip1 by alternative targeting non-TK target Akt-mTOR-Foxo axis: A CPI-based systemic study on discovery of phenotypic off-target. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3738. doi:10.1158/1538-7445.AM2014-3738

Anti-Tumor Activity of Alectinib in Crizotinib Pre-Treated Alk-Rearranged Nsclc in Jp28927 Study

ABSTRACT Aim: Alectinib is a CNS-penentrant, highly selective ALK inhibitor with a novel scaffold. JP28927 is a clinical pharmacological study to evaluate the bioequivalence of alectinib 300 mg b.i.d. with 20 and 40 mg capsules (caps) vs. 150 mg caps and food effect with 150mg caps in ALK-rearranged NSCLC patients (pts), regardless of history of previous ALK inhibitor treatment. Results for bioequivalence, food effect, efficacy and safety were reported at ASCO2014. ALK-rearranged NSCLC pts had to discontinue treatment from crizotinib, a first generation ALK inhibitor, because of drug resistance or intolerance. Methods: 35 ALK-rearranged NSCLC pts were enrolled into JP28927 study. Pts continued alectinib 300 mg b.i.d. with 150 mg caps until the investigator determined lack of clinical benefit. This report describes the updated efficacy and safety data for alectinib in 28 crizotinib pre-treated NSCLC pts included in JP28927. Results: As of Jan 11, 2014, median follow-up duration was 141 days (range: 35-166) and 21 pts continued treatment with alectinib without progressive disease (PD). Among 24 pts with target lesions, tumor shrinkage of more than 30 % was observed in 18 pts. Confirmed response rate was 58.3% (95%CI: 36.6-77.9) and disease control rate was 83.3 % (95%CI: 62.6-95.3) by investigator assessment. 19 of 28 pts had brain metastases at baseline, and 6 pts had no prior brain irradiation. 13 pts with brain metastases, including 4 pts without prior brain irradiation, were still on study treatment without PD. The safety profile was favorable, and continued the same trend previously reported. No pts discontinued study treatment for a safety reason. Gastrointestinal and visual disorders, characteristic of crizotinib treatment, were mild and not so frequent with alectinib. Conclusions: Alectinib showed promising response, including in brain metastases, and good tolerability in crizotinib pre-treated pts. These findings suggest that alectinib is a novel therapeutic option for crizotinib pre-treated ALK-rearranged NSCLC. Disclosure: T. Seto: Other substantive relationships: Honoraria (Chugai Pharmaceutical Co., Ltd., Pfizer Japan Inc., Novartis Pharma K.K.), Research funding(Chugai Pharmaceutical Co., Ltd., Pfizer Japan Inc., Novartis Pharma K.K., Astellas Pharma Inc.); T. Hida: Corporate sponsored research: Chugai, Pfizer, Novartis; K. Nakagawa: Advisory board: Abbott Japan Co., Ltd. Astellas Pharma Inc. Chugai Pharmaceutical Co.,Ltd. Pfizer Japan Inc.Novartis Pharma K.K. Corporate sponsored research:Chugai Pharmaceutical Co.,Ltd.Pfizer Japan Inc.Novartis Pharma K.K.; M. Satouchi: Corporate sponsored research:Chugai, Pfizer, Novartis Other substantive relationships:Honoraria; Chugai, Pfizer, Novartis; M. Nishio: Corporate sponsored research:Chugai, Novartis, Pfizer Other substantive relationships:Honoraria; Chugai, Novartis, Pfizer; K. Hotta: Corporate sponsored research: Chugai Other substantive relationships:Honoraria; Chugai, Pfizer; H. Murakami: Corporate sponsored research:Chugai, Novartis, Astellas; Y. Ohe: Corporate sponsored research: Chugai, Pfizer Other substantive relationships: Honoraria; Chugai, Pfizer; K. Takeda: Other substantive relationships: Chugai, Pfizer, Novartis; M. Tatsuno: Chugai employee; T. Shimada: Chugai employee; T. Tanaka: Chugai employee; T. Tamura: Corporate sponsored research: Chugai, Astellas, AbbVie (Abbott) Other substantive relationships: Honoraria; Chugai, Astellas, Novartis.

SY14-4 - Acquired Resistance to ALK Tyrosine Kinase Inhibitors

ABSTRACT The ALK tyrosine kinase inhibitor crizotinib has demonstrated clinical efficacy in ALK-rearranged NSCLC patients and is currently under additional phase III clinical development as both initial and second-line therapy for advanced ALK-rearranged NSCLC. However, their efficacy will ultimately be limited by the development of acquired drug resistance. Recent studies have identified and studied two mechanisms of ALK TKI resistance based on evidence from a crizotinib-treated ALK-positive lung cancer patient and in a cell line generated from the resistant tumor. To date four secondary mutations, all identified from crizotinib-treated ALK-rearranged tumor patients, have been reported. These mutations either involve the ‘gatekeeper’ residue (L1196) or sites away from crizotinib binding (F1174L, L1152R and C1156Y). The mechanistic basis for how the different mutations lead to crizotinib resistance is not fully understood. The L1196 mutation may create a steric hindrance for crizotinib binding, whereas the F1174L mutation likely promotes the active conformation of ALK, thus disfavoring crizotinib binding which preferentially binds the inactive conformation of ALK. The other mechanism was activation of EGFR signaling as a bypass signaling mechanisms that contributes to ALK inhibitor resistance. Finally, we discuss the strategies that are underway to clinically overcome acquired drug resistance.