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Adjuvant chemotherapy has reduced the risk of tumor recurrence and improved survival in patients with resected colorectal cancer. Potential utility of circulating tumor DNA (ctDNA) prior to and post surgery has been reported across various solid tumors. We initiated a new type of adaptive platform trials to evaluate the clinical benefits of ctDNA analysis and refine precision adjuvant therapy for resectable colorectal cancer, named CIRCULATE‐Japan including three clinical trials. The GALAXY study is a prospectively conducted large‐scale registry designed to monitor ctDNA for patients with clinical stage II to IV or recurrent colorectal cancer who can undergo complete surgical resection. The VEGA trial is a randomized phase III study designed to test whether postoperative surgery alone is noninferior to the standard therapy with capecitabine plus oxaliplatin for 3 months in patients with high‐risk stage II or low‐risk stage III colon cancer if ctDNA status is negative at week 4 after curative surgery in the GALAXY study. The ALTAIR trial is a double‐blind, phase III study designed to establish the superiority of trifluridine/tipiracil as compared with placebo in patients with resected colorectal cancer who show circulating tumor–positive status in the GALAXY study. Therefore, CIRCULATE‐Japan encompasses both “de‐escalation” and “escalation” trials for ctDNA‐negative and ‐positive patients, respectively, and helps to answer whether measuring ctDNA postoperatively has prognostic and/or predictive value. Our ctDNA‐guided adaptive platform trials will accelerate clinical development toward further precision oncology in the field of adjuvant therapy. Analysis of ctDNA status could be utilized as a predictor of risk stratification for recurrence and to monitor the effectiveness of adjuvant chemotherapy. ctDNA is a promising, noninvasive tumor biomarker that can aid in tumor monitoring throughout disease management., CIRCULATE‐Japan encompasses both “de‐escalation” and “escalation” trials for circulating tumor DNA–negative and –positive patients, respectively, and helps to answer whether measuring circulating tumor DNA postoperatively has prognostic and/or predictive value. Our circulating tumor DNA–guided adaptive platform trials will accelerate clinical development toward further precision oncology in the field of adjuvant therapy.

FMS‐like tyrosine kinase 3 (FLT3) plays a key role in hematopoiesis. However, the oncogenic role of FLT3 amplification in patients with metastatic colorectal cancer (mCRC) remains unclear. Here, we aimed to evaluate the characteristics, prognosis, and treatment efficacy of an FLT3 inhibitor (regorafenib) in patients with mCRC with FLT3 amplifications. Tumor tissue samples from 2329 patients were sequenced using NGS in the Nationwide Cancer Genome Screening Project in Japan. The effects of clinicopathological features, co‐altered genes, prognosis, and efficacy of regorafenib were investigated. Between April 2015 and June 2018, 85 patients with mCRC with FLT3 amplification were observed. There were no differences in baseline characteristics between patients with or without FLT3 amplification. The frequency of RAS or other gene co‐alterations was inversely correlated with the copy number status. Median survival time in patients with FLT3 amplification was significantly shorter compared with those with non‐FLT3 amplification. Further investigations of FLT3 amplification as a potential treatment target in mCRC are warranted., High FLT3 amplification may function not only as a prognostic factor but a promising treatment target in metastatic colorectal cancer.

Purpose: KRAS is among the most commonly mutated oncogene in cancer including non–small cell lung cancer (NSCLC). In early clinical trials, inhibitors targeting G12C-mutant KRAS have achieved responses in some patients with NSCLC. Possible intrinsic and acquired resistance mechanisms to KRAS G12C inhibitors are not fully elucidated and will likely become important to identify. Experimental Design: To identify potential resistance mechanisms, we defined the sensitivity of a panel of KRAS G12C–mutant lung cancer cell lines to a KRAS G12C inhibitor, AMG510. Gene set enrichment analyses were performed to identify pathways related to the sensitivity, which was further confirmed biochemically. In addition, we created two cell lines that acquired resistance to AMG510 and the underlying resistance mechanisms were analyzed. Results: KRAS expression and activation were associated with sensitivity to KRAS G12C inhibitor. Induction of epithelial-to-mesenchymal transition (EMT) led to both intrinsic and acquired resistance to KRAS G12C inhibition. In these EMT-induced cells, PI3K remained activated in the presence of KRAS G12C inhibitor and was dominantly regulated by the IGFR–IRS1 pathway. We found SHP2 plays a minimal role in the activation of the PI3K pathway in contrast to its critical role in the activation of the MAPK pathway. The combination of KRAS G12C inhibitor, PI3K inhibitor, and SHP2 inhibitor resulted in tumor regressions in mouse models of acquired resistance to AMG510. Conclusions: Our findings suggest that EMT is a cause of both intrinsic and acquired resistance by activating the PI3K pathway in the presence of KRAS G12C inhibitor.

Molecular testing to select the appropriate targeted and standard of care therapies is essential for managing patients with colorectal cancer (CRC). The Japanese Society of Medical Oncology previously published clinical guidelines for molecular testing in CRC. In the third edition published in 2018, RAS and BRAF V600E mutations should be tested prior to first‐line chemotherapy to assess the benefit of anti–epidermal growth factor receptor (EGFR) antibody therapy in patients with unresectable CRC. Microsatellite instability (MSI) testing was recommended in patients with curatively resected stage II CRC because deficient mismatch repair is associated with low risk of recurrence. MSI testing was also recommended in patients with CRC suspected to be Lynch syndrome. The main aim of this fourth edition is to reflect recent advances in comprehensive genomic profiling (CGP) tests and liquid biopsy. Here, CGP tests performed on tumor tissues are strongly recommended to assess the benefit of molecular targeted drugs in patients with CRC. Circulating tumor DNA (ctDNA)‐based CGP tests are also proposed. ctDNA testing is recommended to determine the optimal treatment based on the risk of recurrence for curatively resected CRC and evaluate the suitability and monitor the therapeutic effects of anti–EGFR antibodies in patients with unresectable CRC. While both MSI testing and immunohistochemistry are strongly recommended to determine the indication of immune checkpoint inhibitors in patients with unresectable CRC, next‐generation sequencing‐based tests are weakly recommended because these tests have not been validated in clinical trials., Update to the molecular testing guideline for colorectal cancer defined by Japanese Society of Medical Oncology. The appropriate timing of each test and degree of recommendation are summarized.

Purpose The CKD4/6 inhibitor abemaciclib is related to adverse events such as hematological toxicity and increase in serum creatinine levels associated with abemaciclib pharmacokinetics. Increase in serum creatinine levels is considered a result of competition with abemaciclib via organic cation transporter 2 and multidrug and toxic compound extrusion. Therefore, we evaluated the association among serum creatinine levels, serum abemaciclib concentrations, and adverse events and whether increase in serum creatinine levels is a useful indicator for predicting the onset of the adverse events of abemaciclib. Methods In total, the data of 12 patients with breast cancer who were treated with abemaciclib (150 mg twice daily) were evaluated to determine the association between increased serum creatinine levels and abemaciclib concentrations and hematological toxicity. Results Grade 3 neutropenia, thrombocytopenia, and anemia were observed at 4 weeks in four (33%), two (17%), and one (8%) patients, respectively. A significant association was observed between steady-state abemaciclib concentrations and the rate of decrease in neutrophil and platelet counts (r = - 0.80, P = 0.003 and r = - 0.70, P = 0.016, respectively). Compared with baseline levels (0.61 [0.53-0.82] mg/mL), serum creatinine levels significantly increased and reached a steady state in at least 2 weeks (0.84 [0.61-1.02] mg/mL, P = 0.01). However, we did not find a significant association between increase in serum creatinine levels and abemaciclib concentrations and hematological toxicity. Conclusions Abemaciclib concentrations are associated with neutropenia and thrombocytopenia. However, increase in serum creatinine levels may not be a useful predictor for estimating abemaciclib pharmacokinetics and hematological toxicity.

Although precision oncology is transforming clinical management of patients with cancer, many hospitals face challenges to effectively implement precision oncology. In addition, the cost and time exerted for genomic profiling needs to be balanced with expectations of benefit for each patient. This article summarizes the effort to implement precision oncology in Japan. The most promising development is that tests to profile the genomes of select cancers are now fully covered by the national health insurance system. In May 2019, two gene panels were approved with reimbursement: FoundationOne CDx Cancer Genomic Profile and OncoGuide NCC Oncopanel System, the latter of which was developed in Japan. To make better use of scarce resources, the reimbursement is restricted to patients with solid tumors that have progressed on standard chemotherapy, rare tumors, or tumors of unknown primary. To centralize Japanese precision oncology, the government designated approximately 170 hospitals and stratified them to three layers on the basis of their roles. In addition, Japan’s National Cancer Center launched a Center for Cancer Genomics and Advanced Therapeutics (C-CAT) that collects genomic information and clinical characteristics of patients who received genomic profiling tests. C-CAT is expected to be the central data repository, to match patients with clinical trials, and to assist translational research. The centralized system under the national health insurance system could be a double-edged sword. Although tight regulation may make it hard to keep up with the rapid development of precision oncology, a federated ecosystem for sharing clinical and genomic data will be a precious asset and allow for shared access to data. Access to unapproved drugs and administrative support from C-CAT will be keys for Japanese precision oncology to meet its full potential.

Purpose: While mutations in BRAF in metastatic colorectal cancer (mCRC) most commonly occur at the V600 amino acid, with the advent of next-generation sequencing, non-V600 BRAF mutations are increasingly identified in clinical practice. It is unclear whether these mutants, like BRAF V600E, confer resistance to anti-EGFR therapy. Experimental Design: We conducted a multicenter pooled analysis of consecutive patients with non-V600 BRAF-mutated mCRCs identified between 2010 and 2017. Non-V600 BRAF mutations were divided into functional classes based on signaling mechanism and kinase activity: activating and RAS-independent (class 2) or kinase-impaired and RAS-dependent (class 3). Results: Forty patients with oncogenic non-V600 BRAF–mutant mCRC received anti-EGFR antibody treatment [n = 12 (30%) class 2 and n = 28 (70%) class 3]. No significant differences in clinical characteristics were observed by mutation class. In contrast, while only 1 of 12 patients with class 2 BRAF mCRC responded, 14 of 28 patients with class 3 BRAF responded to anti-EGFR therapy (response rate, 8% and 50%, respectively, P = 0.02). Specifically, in first- or second-line, 1 of 6 (17%) patients with class 2 and 7 of 9 (78%) patients with class 3 BRAF mutants responded (P = 0.04). In third- or later-line, none of 6 patients with class 2 and 7 of 19 (37%) patients with class 3 BRAF mutants responded (P = 0.14). Conclusions: Response to EGFR antibody treatment in mCRCs with class 2 BRAF mutants is rare, while a large portion of CRCs with class 3 BRAF mutants respond. Patients with colorectal cancer with class 3 BRAF mutations should be considered for anti-EGFR antibody treatment. See related commentary by Fontana and Valeri, p. 6896

The applicability of circulating tumor DNA (ctDNA) genotyping to inform enrollment of patients with cancer in clinical trials has not been established. We conducted a phase 2 trial to evaluate the efficacy of pertuzumab plus trastuzumab for metastatic colorectal cancer (mCRC), with human epidermal growth factor receptor 2 (HER2) amplification prospectively confirmed by tumor tissue or ctDNA analysis (UMIN000027887). HER2 amplification was confirmed in tissue and/or ctDNA in 30 patients with mCRC. The study met the primary endpoint with a confirmed objective response rate of 30% in 27 tissue-positive patients and 28% in 25 ctDNA-positive patients, as compared to an objective response rate of 0% in a matched real-world reference population treated with standard-of-care salvage therapy. Post hoc exploratory analyses revealed that baseline ctDNA genotyping of HER2 copy number and concurrent oncogenic alterations adjusted for tumor fraction stratified patients according to efficacy with similar accuracy to tissue genotyping. Decreased ctDNA fraction 3 weeks after treatment initiation associated with therapeutic response. Pertuzumab plus trastuzumab showed similar efficacy in patients with mCRC with HER2 amplification in tissue or ctDNA, showing that ctDNA genotyping can identify patients who benefit from dual-HER2 blockade as well as monitor treatment response. These findings warrant further use of ctDNA genotyping in clinical trials for HER2-amplified mCRC, which might especially benefit patients in first-line treatment., The multicenter phase 2 TRIUMPH trial shows the utility of ctDNA genotyping as a screening platform to select patients with HER2-amplified metastatic colorectal cancer who benefit from dual-HER2 blockade with trastuzumab and pertuzumab

Simple Summary Synovial sarcoma is a soft-tissue sarcoma that lacks effective systemic therapy and carries poor prognosis due to frequent late local recurrence and metastases. The cancer is known to be driven in part by increased expression of the pro-survival protein BCL-2. Surprisingly, synovial sarcoma proved resistant to BCL-2 inhibitors in pre-clinical trials. We identified increased activity of a second pro-survival protein, MCL-1, as responsible for this resistance. We showed that co-targeting both BCL-2 and MCL-1 proves to be an effective therapeutic approach both in cell culture and animal models of synovial sarcoma, supporting translation into clinical trials. Abstract Synovial sarcoma (SS) is frequently diagnosed in teenagers and young adults and continues to be treated with polychemotherapy with variable success. The SS18-SSX gene fusion is pathognomonic for the disease, and high expression of the anti-apoptotic BCL-2 pathologically supports the diagnosis. As the oncogenic SS18-SSX fusion gene itself is not druggable, BCL-2 inhibitor-based therapies are an appealing therapeutic opportunity. Venetoclax, an FDA-approved BCL-2 inhibitor that is revolutionizing care in some BCL-2-expressing hematological cancers, affords an intriguing therapeutic possibility to treat SS. In addition, there are now dozens of venetoclax-based combination therapies in clinical trials in hematological cancers, attributing to the limited toxicity of venetoclax. However, preclinical studies of venetoclax in SS have demonstrated an unexpected ineffectiveness. In this study, we analyzed the response of SS to venetoclax and the underlying BCL-2 family biology in an effort to understand venetoclax treatment failure and find a therapeutic strategy to sensitize SS to venetoclax. We found remarkably depressed levels of the endogenous MCL-1 inhibitor, NOXA, in SS compared to other sarcomas. Expressing NOXA led to sensitization to venetoclax, as did the addition of the MCL-1 BH3 mimetic, S63845. Importantly, the venetoclax/S63845 combination induced tumor regressions in SS patient-derived xenograft (PDX) models. As a very close analog of S63845 (S64315) is now in clinical trials with venetoclax in AML (NCT03672695), the combination of MCL-1 BH3 mimetics and venetoclax should be considered for SS patients as a new therapy.

Purpose: EGFR inhibitors (EGFRi) are effective against EGFR-mutant lung cancers. The efficacy of these drugs, however, is mitigated by the outgrowth of resistant cells, most often driven by a secondary acquired mutation in EGFR, T790M. We recently demonstrated that T790M can arise de novo during treatment; it follows that one potential therapeutic strategy to thwart resistance would be identifying and eliminating these cells [referred to as drug-tolerant cells (DTC)] prior to acquiring secondary mutations like T790M. Experimental Design: We have developed DTCs to EGFRi in EGFR-mutant lung cancer cell lines. Subsequent analyses of DTCs included RNA-seq, high-content microscopy, and protein translational assays. Based on these results, we tested the ability of MCL-1 BH3 mimetics to combine with EGFR inhibitors to eliminate DTCs and shrink EGFR-mutant lung cancer tumors in vivo. Results: We demonstrate surviving EGFR-mutant lung cancer cells upregulate the antiapoptotic protein MCL-1 in response to short-term EGFRi treatment. Mechanistically, DTCs undergo a protein biosynthesis enrichment resulting in increased mTORC1-mediated mRNA translation of MCL-1, revealing a novel mechanism in which lung cancer cells adapt to short-term pressures of apoptosis-inducing kinase inhibitors. Moreover, MCL-1 is a key molecule governing the emergence of early EGFR-mutant DTCs to EGFRi, and we demonstrate it can be effectively cotargeted with clinically emerging MCL-1 inhibitors both in vitro and in vivo. Conclusions: Altogether, these data reveal that this novel therapeutic combination may delay the acquisition of secondary mutations, therefore prolonging therapy efficacy. Clin Cancer Res; 24(22); 5658–72. ©2018 AACR.

Purpose: Epithelial-to-mesenchymal transition (EMT) confers resistance to a number of targeted therapies and chemotherapies. However, it has been unclear why EMT promotes resistance, thereby impairing progress to overcome it. Experimental Design: We have developed several models of EMT-mediated resistance to EGFR inhibitors (EGFRi) in EGFR-mutant lung cancers to evaluate a novel mechanism of EMT-mediated resistance. Results: We observed that mesenchymal EGFR-mutant lung cancers are resistant to EGFRi-induced apoptosis via insufficient expression of BIM, preventing cell death despite potent suppression of oncogenic signaling following EGFRi treatment. Mechanistically, we observed that the EMT transcription factor ZEB1 inhibits BIM expression by binding directly to the BIM promoter and repressing transcription. Derepression of BIM expression by depletion of ZEB1 or treatment with the BH3 mimetic ABT-263 to enhance “free” cellular BIM levels both led to resensitization of mesenchymal EGFR-mutant cancers to EGFRi. This relationship between EMT and loss of BIM is not restricted to EGFR-mutant lung cancers, as it was also observed in KRAS-mutant lung cancers and large datasets, including different cancer subtypes. Conclusions: Altogether, these data reveal a novel mechanistic link between EMT and resistance to lung cancer targeted therapies. Clin Cancer Res; 24(1); 197–208. ©2017 AACR.

Background While the BRAF V600E mutation occurs in 5%–15% of metastatic colorectal cancer (mCRC), BRAF non-V600E mutations were recently reported to range from 1.6% to 5.1%. We have previously reported that BRAF non-V600E mutations could have a negative impact on efficacy outcomes as well as BRAF V600E mutation for antiepidermal growth factor receptor (EGFR) antibody treatment for pretreated patients with mCRC. Recently, simultaneous inhibitions of mitogen-activated protein kinase kinase (MEK), BRAF and EGFR exhibited relevant antitumour activities in patients with BRAF V600E mutant and also in BRAF non-V600E mutant but only in the preclinical model. Trial design The BIG BANG (study is a multicentre, phase II study to assess the efficacy, safety and proof of concept of the combinations of binimetinib+encorafenib+cetuximab in patients with BRAF non-V600E mutated mCRC, identified by either tumour tissue (tumour tissue group) or blood samples (liquid biopsy group). Key eligibility criteria include Eastern Cooperative Oncology Group Performance Status of ≤1, mCRC with BRAF non-V600E mutant and RAS wild type, refractory or intolerant to at least one fluoropyrimidine-based regimen and no prior history of regorafenib, and no prior history of anti-EGFR antibody treatment (primary analysis cohort and liquid biopsy cohort) or refractory to prior anti-EGFR antibody treatment in patients with class 3 BRAF mutations (anti-EGFR antibody refractory class three cohort). Enrolled patients receive binimetinib (45 mg, two times per day), encorafenib (300 mg, once a day) and cetuximab (initially 400 mg/m2 and subsequently 250 mg/m2, once per week). The primary endpoint is the confirmed objective response rate in the primary analysis cohort. Trial registration numbers UMIN000031857 and 000031860.

Ever since Lynch et al . identified that some non-small cell lung cancers (NSCLCs) responded to the EGFR inhibitors gefitinib and erlotinib, and this could be correlated to activating mutations in EGFR, one of the great success stories in the era of targeted therapies was born.

3529 Background: No predictive biomarkers have been validated to determine which patients (pts) with metastatic colorectal cancer (mCRC) benefit the most from angiogenesis inhibitors. Recent studies suggest that plasma angiogenesis factors and their dynamics may have some prognostic or predictive value. Methods: In this prospective longitudinal study, serial plasma sample collections were done at the time points of pre- and post-treatments in mCRC pts receiving biologics in either first- or second-line chemotherapy (chemo). Comprehensive measurements of 17 factors were performed by the multiplex assay with Luminex technology. Statistical analyses were conducted by using the Brunner-Munzel and Jonckheere-Terpstra test. Results: From Sep 2017 to Dec 2020, 789 plasma samples were collected from 498 enrolled pts [first-line chemo plus bevacizumab (BEV, n=102), first-line chemo plus anti-EGFR antibody (aEGFR, n=100), second-line chemo plus BEV (n=100), second-line FOLFIRI plus ramucirumab (RAM, n=99), second-line FOLFIRI plus aflibercept (AFL, n=85) and other treatment (n=7)]. 789 samples were evaluable for this analysis. In the analysis of first-line, level of VEGF-D was significantly higher in both post-BEV and post-aEGFR comparing with pre-first-line [pre-first-line; 264 pg/ml, post-first-line BEV; 354 pg/ml (p

234 Background: Nivolumab (Nivo) is a standard therapy after ramucirumab (RAM)-based second-line chemotherapy in patients (pts) with advanced gastric cancer (AGC). However, the relationship between angiogenesis-related factors and the efficacies of immune-checkpoint inhibitors after anti-angiogenic chemotherapy is unclear. Methods: We retrospectively evaluated pts with AGCs who received Nivo after RAM-based second-line chemotherapy at a single institution from Nov 2017 to Aug 2019. Clinical benefit of Nivo was defined as CR, PR, or >4 months of SD and non-CR/non-PD. In addition, preserved serum samples at time points of pre-RAM, pre-Nivo, and post-Nivo (within 12 weeks from the start of Nivo) were analyzed by using the designated panel, containing vascular endothelial growth factor-A/D (VEGF-A/D), placental growth factor (PlGF), soluble vascular endothelial growth factor receptor-1/2/3 (sVEGFR-1/2/3), Interleukin-6/8 (IL-6/8), soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), tissue inhibitor of metalloproteinase-1 (TIMP-1), angiopoietin-2, hepatocyte growth factor (HGF), interferon gamma (INF-γ), osteopontin (OPN), soluble neuropilin-1, and thrombospondin-2 (TSP-2). We investigated the association between angiogenesis-related factors and clinical outcomes with Nivo. Results: We evaluated 167 samples from 58 pts, including 10 pts (17.2%) exhibiting clinical benefit. Characteristics of pts were as follows: median age, 68; male, 74%; PS, 0/1-2, 17/83%; HER2 positive, 16%; prior gastrectomy, 40%; histology, intestinal/diffuse, 16/84%; disease status, metastatic/recurrent, 69/31%; lung metasstasis, 14%; liver metasstasis, 26%; peritoneum metastasis, 67%; lymph node metastasis, 31%. The median progression-free survival (PFS) and overall survival (OS) of Nivo were 1.7 and 6.2 months, respectively, with a median follow-up of 5.0 months. There were no correlations between pre-Nivo levels of angiogenesis-related factors and clinical benefit; however, the median post-Nivo/pre-Nivo VEGF-A ratio was significantly lower in pts with clinical benefit than in those without clinical benefit (0.44 versus 0.94, p = 0.008). By multivariate analysis using characteristics of pts and data from pre-Nivo samples, VEGF-A above the median level of 1400 pg/mL and sVCAM-1 below the median level of 1640000 pg/mL were independent prognostic factors for poor PFS (hazard ratio, 1.90, p = 0.033) and OS (hazard ratio, 2.02, p = 0.037), respectively. Conclusions: Our study suggests that rapid decline of serum VEGF-A level after Nivo and higher VEGF-A before Nivo were respectively associated with the clinical benefit of Nivo and poor survival in pts with AGC treated with Nivo after RAM-based second-line chemotherapy.

A 76-year-old female in whom a renal cell carcinoma (RCC) lesion was resected 19 years previously presented to our hospital with cognitive dysfunction. Magnetic resonance imaging and computed tomography revealed nodules in the brain, lung, adrenal gland and a pelvic osteolytic lesion. To identify the primary cancer site, the present study performed endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) of the left adrenal lesion. Consequently, the pathological findings of the tissue obtained by EUS-FNA were similar to those of the previous nephrectomy specimen, revealing that the adrenal lesion was the recurrence of RCC. The majority of the metastatic lesions in the patient were reduced in size by the multiple kinase inhibitor, pazopanib. Contralateral adrenal metastasis of RCC is rare and the use of EUS-FNA in the diagnosis of adrenal lesions remains to be elucidated. This is a rare case of adrenal lesion, diagnosed by EUS-FNA. Therefore, EUS-FNA is considered to be a useful diagnostic modality of adrenal metastases from unidentified primary tumor types.

Leptomeningeal carcinomatosis (LMC) remarkably decreases the quality of life of EGFR-mutant lung cancer patients. In contrast to the lesions outside the central nervous system (CNS), molecular mechanisms of EGFR tyrosine kinase inhibitor (TKI) resistance in CNS lesions including LMC are largely unknown. In this study, we established an in vivo imaging model for LMC with EGFR mutant lung cancer cell lines harboring an exon 19 deletion in EGFR and evaluated the effect of first generation EGFR-TKIs, erlotinib, second generation afatinib, and third generation AZD9291. In PC-9/ffluc model, erlotinib treatment slowed the development of LMC. Importantly, treatment with afatinib or AZD9291 apparently delayed the development of LMC. Moreover, treatment with a higher dose of AZD9291, also associated with inhibited phosphorylation of EGFR downstream molecule S6, regressed LMC refractory to the aforementioned EGFR-TKI treatments. These observations suggest that the third generation EGFR-TKI AZD9291 may be an effective treatment for first or second generation EGFR-TKI resistant LMC caused by EGFR-mutant lung cancer.

The combination of CDK4/6 inhibitors with anti-estrogen therapies significantly improves clinical outcomes in ER-positive advanced breast cancer. To identify mechanisms of acquired resistance, we analyzed serial biopsies and rapid autopsies from patients treated with the combination of the CDK4/6 inhibitor ribociclib with letrozole. This study revealed that some resistant tumors acquired RB loss, whereas other tumors lost PTEN expression at the time of progression. In breast cancer cells ablation of PTEN, through increased AKT activation, was sufficient to promote resistance to CDK4/6 inhibition in vitro and in vivo. Mechanistically, PTEN loss resulted in exclusion of p27 from the nucleus, leading to increased activation of both CDK4 and CDK2. Since PTEN loss also causes resistance to PI3Kα-inhibitors, currently approved in the post-CDK4/6 setting, these findings provide critical insight into how this single genetic event may cause clinical cross-resistance to multiple targeted therapies in the same patient, with implications for optimal treatment sequencing strategies. Citation Format: Carlotta Costa, Ye Wang, Amy Ly, Yasuyuki Hosono, Ellen Murchie, Charlotte S. Walmsley, Tiffany Huynh, Christopher Healy, Rachel Peterson, Shogo Yanase, Charles T. Jakubik, Laura E. Henderson, Leah J. Damon, Daria Timonina, Ioannis Sanidas, Christopher J. Pinto, Mari Mino-Kenudson, James Stone, Nicholas J. Dyson, Leif W. Ellisen, Aditya Bardia, Hiromichi Ebi, Cyril H. Benes, Jeffrey A. Engelman, Dejan Juric. PTEN loss mediates clinical cross-resistance to CDK4/6 and PI3Ká inhibitors in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1903.

e23561 Background: While Synovial Sarcoma (SS) only accounts for about 10% of soft tissue sarcomas (STS), or ~10,000 cases/year, ~4,000 of these will be fatal. Little benefit has been seen with newer combination chemotherapies and immune checkpoint inhibitors. SS is a transcription factor-driven cancer characterized by an t(X;18) chromosomal translocation resulting in the SS18-SSX fusion gene. SS is also known to overexpress the anti-apoptotic protein BCL-2, which has been used as a diagnostic marker for SS. Venetoclax, the FDA approved BH3 mimetic which specifically inhibits BCL-2, has been used to treat BCL-2-driven hematological cancers. Yet, preclinical studies of venetoclax, currently in clinical use for BCL-2 related hematologic malignancies, have failed in SS, raising the possibility of other BCL-2 family members playing a role in resistance. Methods: Publicly available gene expression databases of SS were queried for expression of BCL-2 family member expression, including the endogenous MCL-1 inhibitor, NOXA. Six SS cell lines, including ASKA, HS-SY-II, Yamato, SW982, SYO.1, and an ex vivo line from a SS patient-derived xenograft (PDX) were tested in vitro with combinations of venetoclax and the MCL-1 inhibitor S63845, using cell viability assays. In vivo testing employed both a patient-derived xenograft (PDX) model of SS and a cell line derived (SYO.1) xenograft model. Results: Queries of published gene expression data for SS demonstrated that SS expresses low levels of the endogenous MCL-1 inhibitor, NOXA, nominating a mechanism for intrinsic venetoclax resistance in these tumors, and suggesting rational combination of venetoclax and the MCL-1 inhibitor, S63845. In vitro, SS all cell lines possessing the SS18-SSX fusion gene demonstrated synergistic sensitivity to combined S63845 and venetoclax, despite generally insensitivity to either drug as monotherapy. Importantly, in an SS PDX, we demonstrated S63845 and venetoclax synergize to induce tumor regression. In the SYO.1 xenograft model, combination therapy significantly reduced tumor growth compared to no treatment or single agent groups. Conclusions: These findings provide the preclinical rationale for translation of the rational combination of BCL-2 and MCL-1 inhibitors into clinical trials for SS.

The combination of CDK4/6 inhibitors with antiestrogen therapies significantly improves clinical outcomes in ER-positive advanced breast cancer. To identify mechanisms of acquired resistance, we analyzed serial biopsies and rapid autopsies from patients treated with the combination of the CDK4/6 inhibitor ribociclib with letrozole. This study revealed that some resistant tumors acquired RB loss, whereas other tumors lost PTEN expression at the time of progression. In breast cancer cells, ablation of PTEN, through increased AKT activation, was sufficient to promote resistance to CDK4/6 inhibition in vitro and in vivo. Mechanistically, PTEN loss resulted in exclusion of p27 from the nucleus, leading to increased activation of both CDK4 and CDK2. Because PTEN loss also causes resistance to PI3Kα inhibitors, currently approved in the post-CDK4/6 setting, these findings provide critical insight into how this single genetic event may cause clinical cross-resistance to multiple targeted therapies in the same patient, with implications for optimal treatment-sequencing strategies. Significance: Our analysis of serial biopsies uncovered RB and PTEN loss as mechanisms of acquired resistance to CDK4/6 inhibitors, utilized as first-line treatment for ER-positive advanced breast cancer. Importantly, these findings have near-term clinical relevance because PTEN loss also limits the efficacy of PI3Kα inhibitors currently approved in the post-CDK4/6 setting. This article is highlighted in the In This Issue feature, p. 1

The Japanese Society of Medical Oncology (JSMO) previously published 2 editions of the clinical guidelines: "Japanese guidelines for testing of KRAS gene mutation in colorectal cancer" in 2008 and "Japanese Society of Medical Oncology Clinical Guidelines: RAS (KRAS/NRAS) mutation testing in colorectal cancer patients" in 2014. These guidelines have contributed to the proper use of KRAS and RAS mutation testing, respectively. Recently, clinical utility, particularly for colorectal cancer (CRC) patients with BRAF V600E mutation or DNA mismatch-repair (MMR) deficiency, has been established. Therefore, the guideline members decided these genetic alterations should also be involved. The aim of this revision is to properly carry out testing for BRAF V600E mutation and MMR deficiency in addition to RAS mutation. The revised guidelines include the basic requirements for testing for these genetic alterations based on recent scientific evidence. Furthermore, because clinical utility of comprehensive genetic testing using next-generation sequencing and somatic gene testing of analyzing circulating tumor DNA has increasingly evolved with recent advancements in testing technology, we noted the current situation and prospects for these testing technologies and their clinical implementation in the revised guidelines.

The Japanese guidelines for the testing of KRAS mutations in colorectal cancer have been used for the past 5 years. However, new findings of RAS (KRAS/NRAS) mutations that can further predict the therapeutic effects of anti-epidermal growth factor receptor (EGFR) antibody therapy necessitated a revision of the guidelines. The revised guidelines included the following five basic requirements for RAS mutation testing to highlight a patient group in which anti-EGFR antibody therapy may be ineffective: First, anti-EGFR antibody therapy may not offer survival benefit and/or tumor shrinkage to patients with expanded RAS mutations. Thus, current methods to detect KRAS exon 2 (codons 12 and 13) mutations are insufficient for selecting appropriate candidates for this therapy. Additional testing of extended KRAS/NRAS mutations is recommended. Second, repeated tests are not required for the detection; tissue materials of either primary or metastatic lesions are applicable for RAS mutation testing. Evaluating RAS mutations prior to anti-EGFR antibody therapy is recommended. Third, direct sequencing with manual dissection or allele-specific PCR-based methods is currently applicable for RAS mutation testing. Fourth, thinly sliced sections of formalin-fixed, paraffin-embedded tissue blocks are applicable for RAS mutation testing. One section stained with H&E should be provided to histologically determine whether the tissue contains sufficient amount of tumor cells for testing. Finally, RAS mutation testing must be performed in laboratories with appropriate testing procedures and specimen management practices.

The most recent version of the European Society for Medical Oncology (ESMO) consensus guidelines for the treatment of patients with metastatic colorectal cancer (mCRC) was published in 2016, identifying both a more strategic approach to the administration of the available systemic therapy choices, and a greater emphasis on the use of ablative techniques, including surgery. At the 2016 ESMO Asia Meeting, in December 2016, it was decided by both ESMO and the Japanese Society of Medical Oncology (JSMO) to convene a special guidelines meeting, endorsed by both ESMO and JSMO, immediately after the JSMO 2017 Annual Meeting. The aim was to adapt the ESMO consensus guidelines to take into account the ethnic differences relating to the toxicity as well as other aspects of certain systemic treatments in patients of Asian ethnicity. These guidelines represent the consensus opinions reached by experts in the treatment of patients with mCRC identified by the Presidents of the oncological societies of Japan (JSMO), China (Chinese Society of Clinical Oncology), Korea (Korean Association for Clinical Oncology), Malaysia (Malaysian Oncological Society), Singapore (Singapore Society of Oncology) and Taiwan (Taiwan Oncology Society). The voting was based on scientific evidence and was independent of both the current treatment practices and the drug availability and reimbursement situations in the individual participating Asian countries.

BACKGROUND A germline deletion in the BIM (BCL2L11) gene has been shown to impair the apoptotic response to tyrosine kinase inhibitors (TKIs) in vitro but its association with poor outcomes in TKI-treated non-small cell lung cancer (NSCLC) patients remains unclear. We conducted a systematic review and meta-analysis on both aggregate and individual patient data to address this issue. RESULTS In an aggregate data meta-analysis (n = 1429), the BIM deletion was associated with inferior PFS (HR = 1.51, 95%CI = 1.06-2.13, P = 0.02). Using individual patient data (n = 1200), we found a significant interaction between the deletion and ethnicity. Amongst non-Koreans, the deletion was an independent predictor of shorter PFS (Chinese: HR = 1.607, 95%CI = 1.251-2.065, P = 0.0002; Japanese: HR = 2.636, 95%CI = 1.603-4.335, P = 0.0001), and OS (HR = 1.457, 95% CI = 1.063-1.997, P = 0.019). In Kaplan-Meier analyses, the BIM deletion was associated with shorter survival in non-Koreans (PFS: 8.0 months v 11.1 months, P < 0.0005; OS: 25.7 v 30.0 months, P = 0.042). In Koreans, the BIM deletion was not predictive of PFS or OS. MATERIALS AND METHODS 10 published and 3 unpublished studies that reported survival outcomes in NSCLC patients stratified according to BIM deletion were identified from PubMed and Embase. Summary risk estimates were calculated from aggregate patient data using a random-effects model. For individual patient data, Kaplan-Meier analyses were supported by multivariate Cox regression to estimate hazard ratios (HRs) for PFS and OS. CONCLUSIONS In selected populations, the BIM deletion is a significant predictor of shorter PFS and OS on EGFR-TKIs. Further studies to determine its effect on response to other BIM-dependent therapeutic agents are needed, so that alternative treatment strategies may be devised.

金沢大学がん進展制御研究所, Alectinib is a new generation ALK inhibitor with activity against the gatekeeper L1196M mutation that showed remarkable activity in a phase I/II study with echinoderm microtubule associated protein-like 4 (EML4) - anaplastic lymphoma kinase (ALK) non-small cell lung cancer (NSCLC) patients. However, alectinib resistance may eventually develop. Here, we found that EGFR ligands and HGF, a ligand of the MET receptor, activate EGFR and MET, respectively, as alternative pathways, and thereby induce resistance to alectinib. Additionally, the heat shock protein 90 (Hsp90) inhibitor suppressed protein expression of ALK, MET, EGFR, and AKT, and thereby induced apoptosis in EML4-ALK NSCLC cells, even in the presence of EGFR ligands or HGF. These results suggest that Hsp90 inhibitors may overcome ligand-triggered resistance to new generation ALK inhibitors and may result in more successful treatment of NSCLC patients with EML4-ALK.

がん進展制御研究所, Mutations in Kirsten rat-sarcoma (KRAS) are well appreciated to be major drivers of human cancers through dysregulation of multiple growth and survival pathways. Similar to many other non-kinase oncogenes and tumor suppressors, efforts to directly target KRAS pharmaceutically have not yet materialized. As a result, there is broad interest in an alternative approach to develop therapies that induce synthetic lethality in cancers with mutant KRAS, therefore exposing the particular vulnerabilities of these cancers. Fueling these efforts is our increased understanding into the biology driving KRAS mutant cancers, in particular the important pathways that mutant KRAS governs to promote survival. In this mini-review, we summarize the latest approaches to treat KRAS mutant cancers and the rationale behind them. © 2014 The Authors.

Colorectal cancers harboring KRAS or BRAF mutations are refractory to current targeted therapies. Using data from a high-throughput drug screen, we have developed a novel therapeutic strategy that targets the apoptotic machinery using the BCL-2 family inhibitor ABT-263 (navitoclax) in combination with a TORC1/2 inhibitor, AZD8055. This combination leads to efficient apoptosis specifically in KRAS- and BRAF-mutant but not wild-type (WT) colorectal cancer cells. This specific susceptibility results from TORC1/2 inhibition leading to suppression of MCL-1 expression in mutant, but not WT, colorectal cancers, leading to abrogation of BIM/MCL-1 complexes. This combination strategy leads to tumor regressions in both KRAS-mutant colorectal cancer xenograft and genetically engineered mouse models of colorectal cancer, but not in the corresponding KRAS-WT colorectal cancer models. These data suggest that the combination of BCL-2/BCL-XL inhibitors with TORC1/2 inhibitors constitutes a promising targeted therapy strategy to treat these recalcitrant cancers. Significance: Effective targeted therapies directed against colorectal cancer with activating mutations in KRAS remain elusive. We have leveraged drug-screen data from a large panel of human colorectal cancers to uncover an effective, rational targeted therapy strategy that has preferential activity in colorectal cancers with KRAS or BRAF mutations. This combination may be developed for clinical testing. Cancer Discov; 4(1); 42–52. ©2013 AACR. See related commentary by Russo et al., p. 19 This article is highlighted in the In This Issue feature, p. 1

Background FMS-like tyrosine kinase 3 (FLT3) plays a key role in hematopoiesis, and FLT3-associated molecular alterations are an established predictor for the treatment with FLT3 inhibitors in acute myeloid leukemia. However, the oncogenic role of FLT3 amplification (amp) in patients (pts) with metastatic colorectal cancer (mCRC) has not yet been well established. Methods Tumor tissue samples from 2,329 mCRC pts were sequenced using next-generation sequencing (NGS) with Oncomine Comprehensive Assay in the Nationwide Cancer Genome Screening Project in Japan (SCRUM-Japan GI-SCREEN). Clinicopathological features, co-altered genes, prognosis and regorafenib efficacy on FLT3 amp (defined as copy number ≥ 7.0) vs. non-FLT3 amp mCRC were investigated. Results Between Apr 2015 and Jun 2018, a total of 85 pts (3.6%) with mCRC with FLT3 amp were observed. There were no clear differences in baseline characteristics between pts with or without FLT3 amp. The enrichment of TP53 mutation in FLT3 amp mCRC was observed more frequently than non-FLT3 amp (74.1% vs. 64.7%, P = 0.08), but RAS mutation frequency was similar in both (48.2% vs. 42.8%, P = 0.31). In contrast, activating alterations in BRAF (1.1% vs. 7.1%, P = 0.053) and PIK3CA (1.1% vs 7.0%, P = 0.03) mutations were both less frequent in FLT3 amp vs. non-FLT3 amp mCRC. Median OS from 1st-line chemotherapy in FLT3 amp mCRC was significantly shorter than those in non-FLT3 amp (30.2 vs. 43.4 months, P = 0.002). Furthermore, in 20 pts receiving regorafenib, a multikinase inhibitor with a mild inhibitory activity of FLT3, the disease control rate (DCR) was higher in FLT3 amp mCRC pts (n = 7) compared with non-FLT3 amp (57.1% vs. 23.0 %, P = 0.13). Conclusions FLT3 amp was associated with a significantly worse survival and a higher DCR from regorafenib, suggesting it has a distinct oncogenic role in mCRC. Further investigation of the oncogenic role of FLT3 amp in mCRC is warranted in clinical trials. Clinical trial identification UMIN000016344. Legal entity responsible for the study SCRUM-Japan. Funding 17 SCRUM-Japan Collaborating Pharmaceutical Companies, AMED, NCC. Disclosure H. Taniguchi: Research grant / Funding (self): Takeda; Advisory / Consultancy: Chugai; Advisory / Consultancy: Taiho. T. Kato: Honoraria (self): Chugai Pharmaceutical; Advisory / Consultancy: Takeda; Advisory / Consultancy: Eli Lilly; Advisory / Consultancy: Bayer; Advisory / Consultancy: Sanofi; Advisory / Consultancy: Yakult Honsya. S. Yuki: Speaker Bureau / Expert testimony: Chugai Pharma; Speaker Bureau / Expert testimony: Bristol-Myers Squibb; Speaker Bureau / Expert testimony: Bayer Yakuhin; Speaker Bureau / Expert testimony: Ono Pharmaceutical; Speaker Bureau / Expert testimony: Pharma International; Speaker Bureau / Expert testimony: Daiichi Sankyo; Speaker Bureau / Expert testimony: Takeda Pharmaceutical; Speaker Bureau / Expert testimony: Eli Lilly Japan; Speaker Bureau / Expert testimony: Taiho Pharmaceutical; Speaker Bureau / Expert testimony: Sanofi; Speaker Bureau / Expert testimony: Yakult Honsha; Speaker Bureau / Expert testimony: Merck Biopharma. T. Masuishi: Honoraria (self): Eli Lilly; Honoraria (self): Chugai Pharma; Honoraria (self): Merck Serono; Honoraria (self): Taiho Pharmaceutical; Honoraria (self): Yakult Honsya. K. Kato: Research grant / Funding (self): Shionogi; Research grant / Funding (self): Ono Pharmaceutical; Research grant / Funding (self): Merck Serono. N. Izawa: Advisory / Consultancy: Taiho Pharmaceutical; Advisory / Consultancy: Sanofi; Advisory / Consultancy: MSD; Advisory / Consultancy: Bayer; Advisory / Consultancy: Bristol-Myers; Advisory / Consultancy: Lilly; Advisory / Consultancy: Merck Serono. T. Moriwaki: Speaker Bureau / Expert testimony, Research grant / Funding (institution): Taiho Pharmaceutical; Research grant / Funding (institution): MSD; Speaker Bureau / Expert testimony, Research grant / Funding (institution): Takeda; Research grant / Funding (institution): Yakult Honsha; Research grant / Funding (institution): Eisai; Speaker Bureau / Expert testimony: Chugai Pharma; Speaker Bureau / Expert testimony: Yakult Honsha; Speaker Bureau / Expert testimony: Merck Serono; Speaker Bureau / Expert testimony: Sanofi; Speaker Bureau / Expert testimony: Liliy Japan; Speaker Bureau / Expert testimony: Bayer; Speaker Bureau / Expert testimony: Ono Pharmaceutical. Y. Kagawa: Speaker Bureau / Expert testimony: Chugai; Speaker Bureau / Expert testimony: Eli Lilly; Advisory / Consultancy: Sanofi; Speaker Bureau / Expert testimony: Taiho. W. Okamoto: Research grant / Funding (institution): MSD. Y. Nakamura: Research grant / Funding (institution): Ono Pharmaceutical; Research grant / Funding (institution): Taiho Pharmaceutical. K. Yamazaki: Honoraria (self): Daiichi Sankyo; Honoraria (self): Chugai Pharmaceutical; Honoraria (self): Eli Lilly; Honoraria (self): Merck Serono; Honoraria (self), Research grant / Funding (institution): Taiho Pharma; Honoraria (self): Bayer Yakuhin. T. Yoshino: Research grant / Funding (institution): Chugai pharma; Research grant / Funding (institution): Sanofi; Research grant / Funding (institution): Sumitomo Dainippon pharma; Research grant / Funding (institution): Glaxo SmithKline. All other authors have declared no conflicts of interest.

TPS3156 Background: Approximately 7% of advanced solid malignancies have FGFR gene alterations. However, standard treatment for FGFR-altered malignancies has not been established. Moreover, circulating tumor DNA (ctDNA) analysis has a potential to accurately identify FGFR alterations by assessing spatial and temporal intratumoral heterogeneity, which have shown to be associated with a poor prognosis and resistance to anti-cancer therapy. Methods: We are conducting an investigator-initiated multicenter phase II basket-type trial to investigate efficacy and safety of TAS-120, a highly selective covalent pan-FGFR inhibitor, for the patients with advanced solid malignancies with FGFR alterations identified by ctDNA analysis as a part of the Nationwide Cancer Genome Screening Project (GOZILA study, UMIN000029315). Eligibility criteria include histologically confirmed unresectable advanced or recurrent solid tumors regardless of histology of origin; ECOG PS of 0 or 1; refractory or intolerant to the standard therapies; and clonal FGFR alterations ( FGFR1-3 gain-of-function mutations, FGFR1,2 amplifications and FGFR2,3 fusions) identified by a 73-gene sequencing ctDNA panel (Guardant360). Enrolled patients will receive TAS-120 20 mg once daily, orally, in a 21 day-cycle. The primary endpoint is to clarify objective response rate (ORR) assessed by investigators per RECIST v1.1. The secondary endpoints are to evaluate progression-free survival, duration of response, time to treatment failure, disease control rate, overall survival, ORR by central determination, and incidence of adverse events. Target sample size is determined as 26 to test the null hypothesis of ORR as 5% with one-sided alpha level of 2.5% and power of 80% to detect an expected value of ORR as 25%. Furthermore, tumor tissue and ctDNA will be serially collected and analyzed to investigate the resistance mechanisms and provide clinically meaningful biomarker which may be used for identifying and implementing treatment changes. Clinical trial information: 194624.

659 Background: BRAF non-V600 mutations occur in 2-3% of colorectal cancer. These mutants can be classified as RAS independent (class 2) or RAS dependent (class 3). We reported BRAF non-V600 mutations could be a negative predictive factor for anti-EGFR therapy in patients (pts) with pretreated metastatic colorectal cancer (mCRC), while mCRC pts with class 3 BRAF mutations could respond to anti-EGFR therapy because of its dependency on receptors and RAS. Methods: This study evaluates the efficacy to anti-EGFR therapy in a large cohort of pts with BRAF non-V600 mutated mCRC. Pts with mCRC referred to the participating centers from 2010 to 2017 were included. Clinicopathological features, efficacy of anti-EGFR therapy, and survival outcomes were stratified by BRAF mutational class. Results: One hundred seventeen pts with BRAF non-V600 mutated mCRC were identified. Median age was 58 years (range, 27-83), 68 pts (58%) were male, and 38 pts (33%) had right-sided tumors. Mucinous histology was seen in 11 cases (9%); concurrent RAS mutations occurred in 31 cases (27%), and 3 cases (3%) were MSI-H. Also, TP53 mutations were detected in 74 pts among 90 analyzed cases (82%). Regarding BRAF mutation subtype, 25/63/29 pts were classified as class 2/3/not reported (NR), respectively. Median OS in RAS wild-type/mutant were 44.8/34.6 months, respectively (p=0.082). The median OS in RAS wild-type pts with BRAF non-V600 mutations for class 2, 3, and NR were 25.7, 44.2, and 79.1 months, respectively (class 2 vs. 3, p=0.219). Among 40 pts treated with anti-EGFR therapy, response rates were 14%, 44%, and 40% for class 2, 3, and NR, respectively. Median PFS was 4.4, 8.3, 4.0 months for class 2, 3, and NR, respectively. Moreover, in 25 pts receiving anti-EGFR therapy as third or later line, response rate was 0%, 27%, and 50% in class 2, 3, and NR, and median PFS was 2.8, 3.7, and 4.0 months (p=0.762), respectively. Conclusions: Pts with class 2 BRAF mutations tend to have a poor prognosis compared to those with class 3 mutations. While almost half of pts with class 3 BRAF mutations responded to anti-EGFR therapy, response was rare for pts with class 2 BRAF mutations, and none achieved objective response in the third or later line.

KRAS is frequently mutated in lung cancer. Whereas MAPK is a well-known effector pathway of KRAS, blocking this pathway with clinically available MAPK inhibitors is relatively ineffective. Here, we report that epithelial-to-mesenchymal transition rewires the expression of receptor tyrosine kinases, leading to differential feedback activation of the MAPK pathway following MEK inhibition. In epithelial-like KRAS-mutant lung cancers, this feedback was attributed to ERBB3-mediated activation of MEK and AKT. In contrast, in mesenchymal-like KRAS-mutant lung cancers, FGFR1 was dominantly expressed but suppressed by the negative regulator Sprouty proteins; MEK inhibition led to repression of SPRY4 and subsequent FGFR1-mediated reactivation of MEK and AKT. Therapeutically, the combination of a MEK inhibitor (MEKi) and an FGFR inhibitor (FGFRi) induced cell death in vitro and tumor regressions in vivo. These data establish the rationale and a therapeutic approach to treat mesenchymal-like KRAS-mutant lung cancers effectively with clinically available FGFR1 and MAPK inhibitors. Significance: Adaptive resistance to MEKi is driven by receptor tyrosine kinases specific to the differentiation state of the KRAS-mutant non–small cell lung cancer (NSCLC). In mesenchymal-like KRAS-mutant NSCLC, FGFR1 is highly expressed, and MEK inhibition relieves feedback suppression of FGFR1, resulting in reactivation of ERK; suppression of ERK by MEKi/FGFRi combination results in tumor shrinkage. Cancer Discov; 6(7); 754–69. ©2016 AACR. This article is highlighted in the In This Issue feature, p. 681

Tyrosine kinase inhibitors (TKI) that target the EGF receptor (EGFR) are effective in most non–small cell lung carcinoma (NSCLC) patients whose tumors harbor activating EGFR kinase domain mutations. Unfortunately, acquired resistance eventually emerges in these chronically treated cancers. Two of the most common mechanisms of acquired resistance to TKIs seen clinically are the acquisition of a secondary “gatekeeper” T790M EGFR mutation that increases the affinity of mutant EGFR for ATP and activation of MET to offset the loss of EGFR signaling. Although up to one-third of patient tumors resistant to reversible EGFR TKIs harbor concurrent T790M mutation and MET amplification, potential therapies for these tumors have not been modeled in vivo. In this study, we developed a preclinical platform to evaluate potential therapies by generating transgenic mouse lung cancer models expressing EGFR-mutant Del19-T790M or L858R-T790M, each with concurrent MET overexpression. We found that monotherapy targeting EGFR or MET alone did not produce significant tumor regression. In contrast, combination therapies targeting EGFR and MET simultaneously were highly efficacious against EGFR TKI–resistant tumors codriven by Del19-T790M or L858R-T790M and MET. Our findings therefore provide an in vivo model of intrinsic resistance to reversible TKIs and offer preclinical proof-of-principle that combination targeting of EGFR and MET may benefit patients with NSCLC. Cancer Res; 72(13); 3302–11. ©2012 AACR.

Targeted therapies have demonstrated efficacy against specific subsets of molecularly defined cancers1–4. Although most patients with lung cancer are stratified according to a single oncogenic driver, cancers harbouring identical activating genetic mutations show large variations in their responses to the same targeted therapy1,3. The biology underlying this heterogeneity is not well understood, and the impact of co-existing genetic mutations, especially the loss of tumour suppressors5–9, has not been fully explored. Here we use genetically engineered mouse models to conduct a ‘co-clinical’ trial that mirrors an ongoing human clinical trial in patients with KRAS-mutant lung cancers. This trial aims to determine if the MEK inhibitor selumetinib (AZD6244)10 increases the efficacy of docetaxel, a standard of care chemotherapy. Our studies demonstrate that concomitant loss of either p53 (also known as Tp53) or Lkb1 (also known as Stk11), two clinically relevant tumour suppressors6,9,11,12, markedly impaired the response of Kras-mutant cancers to docetaxel monotherapy. We observed that the addition of selumetinib provided substantial benefit for mice with lung cancer caused by Kras and Kras and p53 mutations, but mice with Kras and Lkb1 mutations had primary resistance to this combination therapy. Pharmacodynamic studies, including positron-emission tomography (PET) and computed tomography (CT), identified biological markers in mice and patients that provide a rationale for the differential efficacy of these therapies in the different genotypes. These co-clinical results identify predictive genetic biomarkers that should be validated by interrogating samples from patients enrolled on the concurrent clinical trial. These studies also highlight the rationale for synchronous co-clinical trials, not only to anticipate the results of ongoing human clinical trials, but also to generate clinically relevant hypotheses that can inform the analysis and design of human studies.

EGFR inhibitors (EGFRi) are effective at inducing transient tumor shrinkage in EGFR-mutant lung cancers. The efficacy of these drugs however is mitigated by the outgrowth of resistant cells: this is most often manifested by a secondary mutation in EGFR, T790M, which leads to reactivation of key intracellular signaling despite continued drug treatment. We recently demonstrated that T790M can occur both at low frequencies prior to initiation of EGFR inhibitor therapy, or alternatively arise de novo during treatment (Hata et al., Nat Med 2016). Since some cancers form T790M mutations de novo, one potential therapeutic strategy to thwart resistance is to identify the cells surviving initial therapy (referred to as persister cells or drug-tolerant cells [DTCs]) that eventually acquire the T790M mutation, and eliminate them prior to T790M acquisition. To this end, we hypothesized that some cells were refractory to EGFR inhibitor-induced apoptosis, surviving initial therapy and forming a reservoir of cells that could then eventually acquire T790M. We demonstrate that Western blots of lysates from EGFR-mutant lung cancers surviving initial therapy to the EGFR inhibitor gefitinib detect quick ( Citation Format: Kyung-A Song, Timothy L. Lochmann, Neha U. Patel, Jungoh Ham, Brad E. Windle, Hisashi Harada, Joel D. Leverson, Andrew J. Souers, Aaron N. Hata, Hiromichi Ebi, Anthony C. Faber. A protein synthesis switch underlies initial survival of EGFR-mutant lung cancer to EGFR inhibitors [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr B31.

Sorafenib is a novel oral multikinase inhibitor that targets Raf serine/threonine and receptor tyrosine kinases, and inhibits tumor cell proliferation and angiogenesis. We have conducted a phase I study of sorafenib to determine the safety, tolerability, pharmacokinetics, and potential efficacy of this agent in 31 Japanese patients with advanced refractory solid tumors. Sorafenib (100–600 mg) was given as a single dose followed by a 7-day wash-out period, and then administrated twice daily (bid). The most frequent drug-related adverse events were rash/desquamation (61%), hand–foot skin reactions (39%), diarrhea (36%), and elevations of serum lipase (36%) and amylase (26%) levels. Dose-limiting toxicities (DLTs) were grade 3 diarrhea at 200 mg bid and grade 3 fatigue at 600 mg bid. Grade 3 and 4 pancreatic enzyme elevations were observed at 200–600 mg bid, but they were not deemed dose-limiting because they were asymptomatic and were not associated with pancreatitis or chronic damage to the pancreas. The AUC and Cmax of sorafenib increased linearly with dose up to 400 mg bid. Partial responses were observed in one of 10 patients with non-small cell lung cancer and one of three patients with renal cell carcinoma. In conclusion, sorafenib 400 mg bid was well tolerated in Japanese patients with advanced refractory solid tumors. The recommended dose for future clinical trials is 400 mg bid. (Cancer Sci 2008; 99: 1492–1498)

SummaryBYL719, which selectively inhibits the alpha isoform of the phosphatidylinositol 3-kinase (PI3K) catalytic subunit (p110a), is currently in clinical trials for the treatment of solid tumors, especially luminal breast cancers with PIK3CA mutations and/or HER2 amplification. This study reveals that, even among these sensitive cancers, the initial efficacy of p110α inhibition is mitigated by rapid re-accumulation of the PI3K product PIP3 produced by the p110β isoform. Importantly, the reactivation of PI3K mediated by p110β does not invariably restore AKT phosphorylation, demonstrating the limitations of using phospho-AKT as a surrogate to measure PI3K activation. Consistently, we show that the addition of the p110β inhibitor to BYL719 prevents the PIP3 rebound and induces greater antitumor efficacy in HER2-amplified and PIK3CA mutant cancers.

Background: To evaluate the role of FDG-PET in assessing anti-tumor efficacy of molecular targeted drugs, we prospectively performed FDG-PET and CT for response evaluation in patients treated with lapatinib, a dual inhibitor of ErbB1 and ErbB2 tyrosine kinases. Methods: Lapatinib was given orally once a day at doses ranging from 1200 to 1800 mg in a phase I study. CT and FDG-PET were performed before treatment, and at 1, 2 and 3 months after the initiation of the treatment and every 2 months thereafter. Results: A total of 29 FDG-PET examinations were performed in eight patients with various solid tumors and the metabolic activity in the tumor was evaluated as SUVmax. The best responses, as assessed by CT, were as follows; one partial response, four stable disease and three disease progression. The partial response was observed in a patient with trastuzumabresistant breast cancer, whose SUVmax was decreased by 60% from baseline. In all of the four patients whose best response was stable disease, the SUVmax was decreased by 6-42% one month after the start of treatment. Prolonged stable disease (10 months) was observed in a patient with colon cancer, whose SUVmax was decreased by 42%. In the patient group with disease progression, SUVmax was increased in two out of three patients. Conclusions: FDG-PET detected decreases in the metabolic activity of the tumors in patients who experienced clinical benefits on treatment with lapatinib. Thus, FDG-PET may be useful for the evaluation of molecular targeted drugs, such as lapatinib.

It has been suggested that attenuation of the decatenation G2 checkpoint function, which ensures sufficient chromatid decatenation by topoisomerase II before entering into mitosis, may contribute to the acquisition of genetic instability in cancer cells. To date, however, very little information is available on this type of checkpoint defect in human cancers. In this study, we report for the first time that a proportion of human lung cancer cell lines did not properly arrest before entering mitosis in the presence of a catalytic, circular cramp-forming topoisomerase II inhibitor ICRF-193, whereas the decatenation G2 checkpoint impairment was present independently of the impaired DNA damage G2 checkpoint. In addition, the presence of decatenation G2 checkpoint dysfunction was found to be associated with diminished activation of ataxia-telangiectasia mutated in response to ICRF-193, suggesting the potential involvement of an upstream pathway sensing incompletely catenated chromatids. Interestingly, hypersensitivity to ICRF-193 was observed in cell lines with decatenation G2 checkpoint impairment and negligible activation of ataxia-telangiectasia mutated. These findings suggest the possible involvement of decatenation G2 checkpoint impairment in the development of human lung cancers, as well as the potential clinical implication of selective killing of lung cancer cells with such defects by this type of topoisomerase II inhibitor.