Norihiro Yamaguchi

Structural, Biochemical, and Clinical Characterization of Epidermal Growth Factor Receptor (EGFR) Exon 20 Insertion Mutations in Lung Cancer.

Crystal structure and detailed analysis of different EGFR mutants explain why some mutations in exon 20 make lung cancers resistant to EGFR inhibitors and others make them more sensitive. A Crystal Clear Cause of Drug Resistance Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are used to treat a variety of cancers, including non–small cell lung cancer. EGFR mutations have a wide range of effects on the success of TKI treatment in this cancer type, with some sensitizing the tumors to TKI inhibitors and others making them resistant to targeted therapy. For example, most of the mutations in exon 20, a relatively common mutation site, prevent cancer cells from responding to EGFR inhibitors. Here, Yasuda and co-workers determined the crystal structure of EGFR with an exon 20 mutation and used a combination of kinetic studies and structural analysis to elucidate the mechanism for these mutants’ differential sensitivity to TKIs. The findings of Yasuda et al. clarify the reasons for the drug resistance of most exon 20 mutations and show the mechanism for the rare mutation in the same exon that increases tumors’ sensitivity to treatment. In addition to explaining which of the mutants are resistant to targeted inhibition of EGFR and the reasons for this phenomenon, this work could help with the development of future therapeutics. By taking advantage of the crystal structure and detailed insights into the function of mutant EGFR, researchers may be able to design drugs that exploit the unique structural features of resistant mutants and specifically target them for treatment. Epidermal growth factor receptor (EGFR) gene mutations (G719X, exon 19 deletions/insertions, L858R, and L861Q) predict favorable responses to EGFR tyrosine kinase inhibitors (TKIs) in advanced non–small cell lung cancer (NSCLC). However, EGFR exon 20 insertion mutations (~10% of all EGFR mutations) are generally associated with insensitivity to available TKIs (gefitinib, erlotinib, and afatinib). The basis of this primary resistance is poorly understood. We studied a broad subset of exon 20 insertion mutations, comparing in vitro TKI sensitivity with responses to gefitinib and erlotinib in NSCLC patients, and found that most are resistant to EGFR TKIs. The crystal structure of a representative TKI-insensitive mutant (D770_N771insNPG) reveals an unaltered adenosine triphosphate–binding pocket, and the inserted residues form a wedge at the end of the C helix that promotes the active kinase conformation. Unlike EGFR-L858R, D770_N771insNPG activates EGFR without increasing its affinity for EGFR TKIs. Unexpectedly, we find that EGFR-A763_Y764insFQEA is highly sensitive to EGFR TKIs in vitro, and patients whose NSCLCs harbor this mutation respond to erlotinib. Analysis of the A763_Y764insFQEA mutant indicates that the inserted residues shift the register of the C helix in the N-terminal direction, altering the structure in the region that is also affected by the TKI-sensitive EGFR-L858R. Our studies reveal intricate differences between EGFR mutations, their biology, and their response to EGFR TKIs.

Brain metastases in patients with EGFR-mutated or ALK-rearranged non-small-cell lung cancers

INTRODUCTION Brain metastases (BM) are common in non-small-cell lung cancer (NSCLC). However, the baseline incidence and evolution of BM over time in oncogene-driven NSCLCs are seldom reported. In this study, we evaluated the frequency of BM in patients with epidermal growth factor receptor (EGFR)-mutated or anaplastic lymphoma kinase (ALK)-rearranged NSCLC. METHODS The presence of BM, clinicopathologic data, and tumor genotype were retrospectively compiled and analyzed from a cohort of 381 patients. RESULTS We identified 86 EGFR-mutated (90.7% with metastatic disease; 85.9% received an EGFR inhibitor) and 23 ALK-rearranged (91.3% with metastatic disease; 85.7% received an ALK inhibitor) NSCLCs. BM were present in 24.4% of EGFR-mutated and 23.8% of ALK-rearranged NSCLCs at the time of diagnosis of advanced disease. This study did not demonstrate a difference in the cumulative incidence of BM over time between the two cohorts (EGFR/ALK cohort competing risk regression [CRR] coefficient of 0.78 [95% CI 0.44-1.39], p=0.41). In still living patients with advanced EGFR-mutated NSCLC, 34.2% had BM at 1 year, 38.4% at 2 years, 46.7% at 3 years, 48.7% at 4 years, and 52.9% at 5 years. In still living patients with advanced ALK-rearranged NSCLC, 23.8% had BM at 1 year, 45.5% at 2 years, and 58.4% at 3 years. CONCLUSIONS BM are frequent in advanced EGFR-mutated or ALK-rearranged NSCLCs, with an estimated >45% of patients with CNS involvement by three years of survival with the use of targeted therapies. These data point toward the CNS as an important unmet clinical need in the evolving schema for personalized care in NSCLC.

Compound EGFR Mutations and Response to EGFR Tyrosine Kinase Inhibitors

Background: Non–small-cell lung cancers (NSCLCs) containing EGFR mutations are exquisitely sensitive to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). This is the case of the most common EGFR mutations affecting exon 18 (G719X), 19 (inframe deletions), and 21 (L858R and L861Q). However, the frequency of compound (i.e., double or complex) EGFR mutations—where an EGFR TKI sensitizing or other mutation is identified together with a mutation of unknown clinical significance—and their pattern of response/resistance to EGFR TKIs are less well described. Methods: We analyzed the EGFR mutation pattern of 79 cases of NSCLC harboring EGFR mutations and compiled the genotype-response data for patients with NSCLCs with compound EGFR mutations treated with EGFR TKIs. Results: Of the 79 EGFR-mutated tumors identified, 11 (14%) had compound mutations. Most involved the EGFR TKI–sensitizing G719X (n = 3, plus S768I or E709A), L858R (n = 4, plus L747V, R776H, T790M, or A871G), L861Q (n = 1, plus E709V), and delL747_T751 (n = 1, plus R776H). Eight patients received an EGFR TKI: three cases with G719X plus another mutation had partial responses (PRs) to erlotinib; of three cases with L858R plus another mutation, two displayed PRs and one (with EGFR-L858R+A871G) progressive disease (PD) to erlotinib; one NSCLC with EGFR-L861Q+E709A and one with delL747_T751+R776S had PRs to EGFR TKIs. Conclusion: Compound EGFR mutations comprised 14% of all mutations identified during routine sequencing of exons 18–21 of EGFR in our cohort. Most patients with an EGFR TKI–sensitizing mutation (G719X, exon 19 deletion, L858R, and L861Q) in addition to an atypical mutation responded to EGFR TKIs. Reporting of the genotype-response pattern of NSCLCs with EGFR compound and other rare mutations, and the addition of this information to searchable databases, will be helpful to select the appropriate therapy for EGFR-mutated NSCLC.

Success and failure rates of tumor genotyping techniques in routine pathological samples with non-small-cell lung cancer

INTRODUCTION Identification of some somatic molecular alterations in non-small-cell lung cancer (NSCLC) has become evidence-based practice. The success and failure rate of using commercially available tumor genotyping techniques in routine day-to-day NSCLC pathology samples is not well described. We sought to evaluate the success and failure rate of EGFR mutation, KRAS mutation, and ALK FISH in a cohort of lung cancers subjected to routine clinical tumor genotype. METHODS Clinicopathologic data, tumor genotype success and failure rates were retrospectively compiled and analyzed from 381 patient-tumor samples. RESULTS From these 381 patients with lung cancer, the mean age was 65 years, 61.2% were women, 75.9% were white, 27.8% were never smokers, 73.8% had advanced NSCLC and 86.1% had adenocarcinoma histology. The tumor tissue was obtained from surgical specimens in 48.8%, core needle biopsies in 17.9%, and as cell blocks from aspirates or fluid in 33.3% of cases. Anatomic sites for tissue collection included lung (49.3%), lymph nodes (22.3%), pleura (11.8%), bone (6.0%), brain (6.0%), among others. The overall success rate for EGFR mutation analysis was 94.2%, for KRAS mutation 91.6% and for ALK FISH 91.6%. The highest failure rates were observed when the tissue was obtained from image-guided percutaneous transthoracic core-needle biopsies (31.8%, 27.3%, and 35.3% for EGFR, KRAS, and ALK tests, respectively) and bone specimens (23.1%, 15.4%, and 23.1%, respectively). In specimens obtained from bone, the failure rates were significantly higher for biopsies than resection specimens (40% vs. 0%, p=0.024 for EGFR) and for decalcified compared to non-decalcified samples (60% vs. 5.5%, p=0.021 for EGFR). CONCLUSIONS Tumor genotype techniques are feasible in most samples, outside small image-guided percutaneous transthoracic core-needle biopsies and bone samples from core biopsies with decalcification, and therefore expansion of routine tumor genotype into the care of patients with NSCLC may not require special tissue acquisition or manipulation.

Adequacy of lymph node transbronchial needle aspirates using convex probe endobronchial ultrasound for multiple tumor genotyping techniques in non-small-cell lung cancer.

Introduction: Adequate tumor acquisition is essential to identify somatic molecular alterations in non–small-cell lung cancer (NSCLC), such as epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) translocations. The success and failure rates for tumor genotyping of tissue obtained from fine-needle aspirates of nodal tissue using a convex probe endobronchial ultrasound (CP-EBUS) and other diagnostic modalities in routine NSCLC care have not been described. Methods: Clinicopathologic data, tumor genotype success and failure rates were retrospectively compiled and analyzed from 207 patient-tumor samples sent for routine tumor genotype in clinical practice, including 42 patient-tumor samples obtained from hilar or mediastinal lymph nodes using CP-EBUS. Results: The median age of the patients was 65 years, 62.3% were women, 77.8% were white, 26.6% were never smokers, 73.9% had advanced NSCLC, and 84.1% had adenocarcinoma histology. Tumor tissue was obtained from CP-EBUS–derived hilar or mediastinal nodes in 42 cases (20.2% of total). In this latter cohort, the overall success rate for EGFR mutation analysis was 95.2%, for Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation 90.5%, and for ALK fluorescence in situ hybridization 90.5%. In the complete 207 tumors, the success rate for EGFR was 92.3%, for KRAS 91.8%, and for ALK 89.9%. The failure rates were not significantly different when comparing CP-EBUS–derived nodal tissue versus all other samples or versus surgical biopsies of mediastinal nodes, but were significantly lower than image-guided percutaneous transthoracic core-needle biopsies. Conclusions: The success rate of multiple tumor genomic analyses techniques for EGFR, KRAS, and ALK gene abnormalities using routine lung cancer tissue samples obtained from hilar or mediastinal lymph nodes by means of CP-EBUS exceeds 90%, and this method of tissue acquisition is not inferior to other specimen types. Tumor genotype techniques are feasible in most CP-EBUS–derived samples and therefore further expansion of routine tumor genotype for the care of patients with NSCLC may be possible using targeted sample acquisition through CP-EBUS.

Smoking status and self-reported race affect the frequency of clinically relevant oncogenic alterations in non-small-cell lung cancers at a United States-based academic medical practice

INTRODUCTION The identification of somatic genomic aberrations in non-small-cell lung cancer (NSCLC) is part of evidence-based practice guidelines for care of patients with NSCLC. We sought to establish the frequency and correlates with these changes in routine patient-tumor sample pairs. METHODS Clinicopathologic data and tumor genotype were retrospectively compiled and analyzed from an overall cohort of 381 patient-tumor samples. RESULTS Of these patients, 75.9% self-reported White race, 13.1% Asian, 6.5% Black, 27.8% were never-smokers, 54.9% former-smokers and 17.3% current-smokers. The frequency of EGFR mutations was 23.9% (86/359), KRAS mutations 34.2% (71/207) and ALK FISH positivity 9.1% (23/252) in tumor samples, and almost all had mutually exclusive results for these oncogenes. In tumors from White, Black and Asian patients, the frequencies of EGFR mutations were 18.4%, 18.2% and 62%, respectively; of ALK FISH positivity 7.81%, 0% and 14.8%, respectively; and of KRAS mutations 41.6%, 20% and 0%. These patterns changed significant with increasing pack-year history of smoking. In White patients, the frequencies of EGFR mutations and ALK FISH positivity decreased with increasing pack-year cohorts; while the frequencies of KRAS mutations increased. Interestingly, in Asian patients the frequencies of EGFR mutations were similar in never smokers and in the cohorts with less than 45pack-year histories of smoking and only decreased in the 45pack-year plus cohort. CONCLUSIONS The frequencies of somatic EGFR, KRAS, and ALK gene abnormalities using routine lung cancer tissue samples from our United States-based academic medical practice reflect the diverse ethnicity (with a higher frequency of EGFR mutations in Asian patients) and smoking patterns (with an inverse correlation between EGFR mutation and ALK rearrangement) of our tested population. These results may help other medical practices appreciate the expected results from introduction of routine tumor genotyping techniques into their day-to-day care of NSCLC.

β-catenin contributes to lung tumor development induced by EGFR mutations.

The discovery of somatic mutations in EGFR and development of EGFR tyrosine kinase inhibitors (TKI) have revolutionized treatment for lung cancer. However, resistance to TKIs emerges in almost all patients and currently no effective treatment is available. Here, we show that β-catenin is essential for development of EGFR-mutated lung cancers. β-Catenin was upregulated and activated in EGFR-mutated cells. Mutant EGFR preferentially bound to and tyrosine phosphorylated β-catenin, leading to an increase in β-catenin-mediated transactivation, particularly in cells harboring the gefitinib/erlotinib-resistant gatekeeper EGFR-T790M mutation. Pharmacologic inhibition of β-catenin suppressed EGFR-L858R-T790M mutated lung tumor growth, and genetic deletion of the β-catenin gene dramatically reduced lung tumor formation in EGFR-L858R-T790M transgenic mice. These data suggest that β-catenin plays an essential role in lung tumorigenesis and that targeting the β-catenin pathway may provide novel strategies to prevent lung cancer development or overcome resistance to EGFR TKIs.

The Impact of Japan's 2004 Postgraduate Training Program on Intra-Prefectural Distribution of Pediatricians in Japan

Objective Inequity in physician distribution poses a challenge to many health systems. In Japan, a new postgraduate training program for all new medical graduates was introduced in 2004, and researchers have argued that this program has increased inequalities in physician distribution. We examined the trends in the geographic distribution of pediatricians as well as all physicians from 1996 to 2010 to identify the impact of the launch of the new training program. Methods The Gini coefficient was calculated using municipalities as the study unit within each prefecture to assess whether there were significant changes in the intra-prefectural distribution of all physicians and pediatricians before and after the launch of the new training program. The effect of the new program was quantified by estimating the difference in the slope in the time trend of the Gini coefficients before and after 2004 using a linear change-point regression design. We categorized 47 prefectures in Japan into two groups: 1) predominantly urban and 2) others by the definition from OECD to conduct stratified analyses by urban-rural status. Results The trends in physician distribution worsened after 2004 for all physicians (p value<.0001) and pediatricians (p value = 0.0057). For all physicians, the trends worsened after 2004 both in predominantly urban prefectures (p value = 0.0012) and others (p value<0.0001), whereas, for pediatricians, the distribution worsened in others (p value = 0.0343), but not in predominantly urban prefectures (p value = 0.0584). Conclusion The intra-prefectural distribution of physicians worsened after the launch of the new training program, which may reflect the impact of the new postgraduate program. In pediatrics, changes in the Gini trend differed significantly before and after the launch of the new training program in others, but not in predominantly urban prefectures. Further observation is needed to explore how this difference in trends affects the health status of the child population.

Identification and Characterization of ALK Kinase Splicing Isoforms in Non-Small-Cell Lung Cancer

Introduction: Anaplastic lymphoma kinase (ALK) rearrangements are present in an important subset of non–small-cell lung cancer (NSCLC) and predict for response to the tyrosine kinase inhibitor crizotinib. In this study, we evaluated the yet unknown frequency and functional role of ALK splicing isoforms in NSCLC. Methods: We analyzed 270 cases of NSCLC for ALK kinase domain splicing aberrations and in addition generated constructs with full-length echinoderm microtubule-associated protein-like 4 (EML4)-ALK (E13;A20) and a splicing isoform. Results: Splicing isoforms of the kinase domain of ALK—including complete skipping of exon 23 (ALKdel23, ALK p.I1171fs*42) and exon 27 (ALKdel27, ALK p.T1312fs*0)—were identified in 11.1% (30 of 270 cases) of NSCLC, and these changes coexisted with ALK rearrangements, KRAS mutations, and EGFR mutations. ALK splicing isoforms were observed with full-length EML4-ALK in crizotinib-naive and treated NSCLCs. ALK T1312fs*0 was unable to render cells solely dependent on ALK signaling. Unlike EML4-ALK and EML4-ALK p.L1196M, EML4-ALK T1312fs*0 did not autophosphorylate ALK or other phosphotyrosine sites. Coexpression of equal amounts of EML4-ALK T1312fs*0 and EML4-ALK did not result in resistance to crizotinib, whereas coexpression of EML4-ALK L1196M with EML4-ALK resulted in resistance to inhibition of ALK by crizotinib. Conclusions: ALK kinase splicing isoforms were present in NSCLC and even if translated seemed to be nonfunctional variants of ALK.

Abstract 968: β-catenin plays an important role in lung tumor development induced by EGFR mutations

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The discovery of somatic mutations in epidermal growth factor receptor (EGFR) and the development of EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib, have revolutionized treatment for non-small cell lung cancer (NSCLC). Resistance to TKIs emerges in almost all patients, but currently no effective treatment is available.Therefore, novel strategies to either prevent or overcome resistance are sorely needed. Here we show that β-catenin is essential for development of EGFR mutated lung cancers. We found that β-catenin was upregulated, translocated to the nucleus, and subsequently activated in both EGFR mutated lung cancer cell lines and EGFR mutation driven lung tumors. We demonstrated that mutant EGFR preferentially bound to β-catenin and caused tyrosine-phosphorylation of β-catenin. Tyrosine-phosphorylation of β-catenin led to stabilization, nuclear translocation, and transcriptional activity particularly in cells harboring EGFR-L858R (LR)-T790M (TM). Pharmacological β-catenin inhibition using ICG-001, which specifically blocks the CBP-β-catenin interaction, suppressed growth of both H1975 cells harboring EGFR-LR-TM and lung tumors in EGFR-LR-TM transgenic mice. To further examine whether β-catenin plays an important role in lung tumorigenesis, we generated an EGFR-LR-TM lung cancer mouse model in which the β-catenin gene (Ctnnb1) can be conditionally deleted. Genetic deletion of Ctnnb1 dramatically reduced lung tumor formation and showed significantly longer survival. Taken together, our data suggest that β-catenin plays an important role in mutant EGFR-driven lung tumorigenesis and that targeting the β-catenin pathway may provide novel strategies to prevent lung cancer development and/or overcome resistance to EGFR TKIs. Citation Format: Sohei Nakayama, Natasha J. Sng, Julian Carretero, Robert Welner, Yuichiro Hayashi, Mihoko Yamamoto, Tan J. Alistair, Norihiro Yamaguchi, Hiroyuki Yasuda, Li Danan, Kenzo Soejima, Soo A. Ross, Costa B. Daniel, Kwok-Kin Wong, Susumu S. Kobayashi. β-catenin plays an important role in lung tumor development induced by EGFR mutations. [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 968. doi:10.1158/1538-7445.AM2014-968