Koji Haratani

LIS1 and NDEL1 coordinate the plus-end-directed transport of cytoplasmic dynein

LIS1 was first identified as a gene mutated in human classical lissencephaly sequence. LIS1 is required for dynein activity, but the underlying mechanism is poorly understood. Here, we demonstrate that LIS1 suppresses the motility of cytoplasmic dynein on microtubules (MTs), whereas NDEL1 releases the blocking effect of LIS1 on cytoplasmic dynein. We demonstrate that LIS1, cytoplasmic dynein and MT fragments co‐migrate anterogradely. When LIS1 function was suppressed by a blocking antibody, anterograde movement of cytoplasmic dynein was severely impaired. Immunoprecipitation assay indicated that cytoplasmic dynein forms a complex with LIS1, tubulins and kinesin‐1. In contrast, immunoabsorption of LIS1 resulted in disappearance of co‐precipitated tubulins and kinesin. Thus, we propose a novel model of the regulation of cytoplasmic dynein by LIS1, in which LIS1 mediates anterograde transport of cytoplasmic dynein to the plus end of cytoskeletal MTs as a dynein‐LIS1 complex on transportable MTs, which is a possibility supported by our data.

Tumor immune microenvironment and nivolumab efficacy in EGFR mutation-positive non-small-cell lung cancer based on T790M status after disease progression during EGFR-TKI treatment

Background The efficacy of programmed death-1 blockade in epidermal growth factor receptor gene (EGFR) mutation-positive non-small-cell lung cancer (NSCLC) patients with different mechanisms of acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) is unknown. We retrospectively evaluated nivolumab efficacy and immune-related factors in such patients according to their status for the T790M resistance mutation of EGFR. Patients and methods We identified 25 patients with EGFR mutation-positive NSCLC who were treated with nivolumab after disease progression during EGFR-TKI treatment (cohort A). Programmed death-ligand 1 (PD-L1) expression and tumor-infiltrating lymphocyte (TIL) density in tumor specimens obtained after acquisition of EGFR-TKI resistance were determined by immunohistochemistry. Whole-exome sequencing of tumor DNA was carried out to identify gene alterations. The relation of T790M status to PD-L1 expression or TIL density was also examined in an independent cohort of 60 patients (cohort B). Results In cohort A, median progression-free survival (PFS) was 2.1 and 1.3 months for T790M-negative and T790M-positive patients, respectively (P = 0.099; hazard ratio of 0.48 with a 95% confidence interval of 0.20-1.24). Median PFS was 2.1 and 1.3 months for patients with a PD-L1 expression level of ≥1% or <1%, respectively (P = 0.084; hazard ratio of 0.37, 95% confidence interval of 0.10-1.21). PFS tended to increase as the PD-L1 expression level increased with cutoff values of ≥10% and ≥50%. The proportion of tumors with a PD-L1 level of ≥10% or ≥50% was higher among T790M-negative patients than among T790M-positive patients of both cohorts A and B. Nivolumab responders had a significantly higher CD8+ TIL density and nonsynonymous mutation burden. Conclusion T790M-negative patients with EGFR mutation-positive NSCLC are more likely to benefit from nivolumab after EGFR-TKI treatment, possibly as a result of a higher PD-L1 expression level, than are T790M-positive patients.

Association of Immune-Related Adverse Events With Nivolumab Efficacy in Non–Small-Cell Lung Cancer

Importance Immune-related adverse events (irAEs) have been associated with the efficacy of PD-1 (programmed cell death protein 1) inhibitors in patients with melanoma, but whether such an association exists for non–small-cell lung cancer (NSCLC) has remained unknown. Objective To evaluate the relation of irAEs to nivolumab efficacy in NSCLC. Design, Setting, and Participants In this study based on landmark and multivariable analyses, a total of 134 patients with advanced or recurrent NSCLC who were treated with nivolumab in the second-line setting or later between December 2015 and August 2016 were identified from a review of medical records from multiple institutions, including a university hospital and community hospitals. Data were updated as of December 31, 2016. Exposures The absence or presence of any irAE before the landmark date. Main Outcomes and Measures Kaplan-Meier curves of progression-free survival (PFS) according to the development of irAEs in 6-week landmark analysis were evaluated with the log-rank test as a preplanned primary objective. Overall survival (OS) was similarly evaluated. Multivariable analysis of both PFS and OS was performed with Cox proportional hazard regression models. Results In a cohort of 134 patients (median [range] age, 68 [33-85] years; 90 men [67%], 44 women [33%]), irAEs were observed in 69 of the 134 study patients (51%), including 12 patients (9%) with such events of grade 3 or 4, and 24 patients (18%) requiring systemic corticosteroid therapy. In 6-week landmark analysis, median PFS was 9.2 months (95% CI, 4.4 to not reached [NR]) and 4.8 months (95% CI, 3.0 to 7.5) (P = .04) whereas median OS was NR (95% CI, 12.3 to NR) and 11.1 months (95% CI, 9.6 to NR) (P = .01) for patients with or without irAEs, respectively. Multivariable analysis also revealed that irAEs were positively associated with survival outcome, with hazard ratios of 0.525 (95% CI, 0.287 to 0.937; P = .03) for PFS and 0.282 (95% CI, 0.101 to 0.667; P = .003) for OS. Conclusions and Relevance Development of irAEs was associated with survival outcome of nivolumab treatment in patients with advanced or recurrent NSCLC. Further studies are needed to confirm our findings.

Peripheral blood biomarkers associated with clinical outcome in non–small cell lung cancer patients treated with nivolumab

Objective The aim of this study was to identify baseline peripheral blood biomarkers associated with clinical outcome in patients with NSCLC treated with nivolumab. Methods Univariable and multivariable analyses were performed retrospectively for 134 patients with advanced or recurrent NSCLC treated with nivolumab to evaluate the relationship between survival and peripheral blood parameters measured before treatment initiation, including absolute neutrophil count (ANC), absolute lymphocyte count (ALC), absolute monocyte count, and absolute eosinophil count (AEC), as well as serum C‐reactive protein and lactate dehydrogenase levels. Progression‐free survival, overall survival, and response rate were determined. Results Among the variables selected by univariable analysis, a low ANC, high ALC, and high AEC were significantly and independently associated with both better progression‐free survival (p = 0.001, p = 0.04, and p = 0.02, respectively) and better overall survival (p = 0.03, p = 0.03, and p = 0.003, respectively) in multivariable analysis. Categorization of patients according to the number of favorable factors revealed that those with only one factor had a significantly worse outcome than those with two or three factors. A similar trend was apparent for patients with a programmed death 1 ligand tumor proportion score less than 50%, whereas all patients with a score of 50% or higher had at least two favorable factors. Conclusions A baseline signature of a low ANC, high ALC, and high AEC was associated with a better outcome of nivolumab treatment, with the number of favorable factors identifying subgroups of patients differing in survival and response rate.

Phase 1 study of new formulation of patritumab (U3-1287) Process 2, a fully human anti-HER3 monoclonal antibody in combination with erlotinib in Japanese patients with advanced non-small cell lung cancer

BackgroundThis phase 1 study evaluated the safety, tolerability, pharmacokinetics and efficacy of patritumab (U3-1287) Process 2, a new formulation of fully human anti-HER3 monoclonal antibody in combination with erlotinib, an epidermal growth factor receptortyrosine kinase inhibitor (EGFR-TKI) in prior chemotherapy treated Japanese patients with advanced non-small cell lung cancer (NSCLC).MethodsPatients received intravenous patritumab Process 2 formulation at 9 mg/kg every 3 weeks after initiation of 18 mg/kg loading dose combined with continuous daily dose of erlotinib (150 mg QD) until any of the withdrawal criteria are met. Adverse events (AEs) were assessed using CTCAE v4.0 and tumor response was assessed using RECIST v1.1. Full pharmacokinetic sampling and serum biomarker analyses were mainly performed during cycle 1 and 2.ResultsTotal of six EGFR-mutant NSCLC patients including one EGFR-TKI naïve patient received patritumab Process 2 formulation combined with erlotinib. No dose-limiting toxicities were observed. The most frequent AEs were gastrointestinal or skin toxicities, which were generally mild and manageable. One patient discontinued from study due to reversible grade 3 interstitial lung disease. The mean area under the curve (AUC) value was 2640 μg/day/mL; the Cmax value was 434 μg/mL, respectively. The median progression-free survival (95% confidence interval) was 220.0 (100.0–363.0) days. HER3 ligand heregulin was detected in serum from only a patient that maintained most durable stable disease.ConclusionsPatritumab Process 2 formulation in combination with erlotinib was well tolerated compatible with favorable PK profile in Japanese patients with advanced NSCLC.

Heregulin-expressing HER2-positive breast and gastric cancer exhibited heterogeneous susceptibility to the anti-HER2 agents lapatinib, trastuzumab and T-DM1

Background Overexpression of heregulin, a HER3 ligand, is one mechanism that confers resistance to the anti-HER2 agents trastuzumab and lapatinib. We investigated the impact of heregulin expression on the efficacy of HER2-targeted therapeutic agents, including trastuzumab, trastuzumab emtansine (T-DM1) and lapatinib, in vitro and in vivo and evaluated the heregulin messenger RNA (mRNA) levels in specimens from patients with HER2-positive breast or gastric cancer. Results Cell proliferation and apoptosis assays demonstrated that heregulin conferred robust resistance to lapatinib and trastuzumab via HER3-Akt pathway activation followed by survivin overexpression; however, heregulin conferred minimal or no resistance to T-DM1 and paclitaxel. The heregulin mRNA level of one of 10 patients was up-regulated after the acquisition of resistance to trastuzumab-based therapy. Materials and Methods SK-BR-3, NCI-N87, BT-474, MDA-MB-453, HCC1954, SNU-216 and 4-1ST cells were pharmacologically treated with recombinant heregulin or transfected with the heregulin gene. We also assessed the expression of heregulin mRNA in HER2-positive breast or gastric cancer samples before and after trastuzumab-based therapy using a RT-PCR-based method. Conclusions mRNA up-regulation of heregulin was observed in clinical breast cancer specimens during trastuzumab-based treatment, but heregulin overexpression had a limited effect on the sensitivity to T-DM1 in vitro and in vivo.

B7-H3 Negatively Modulates CTL-Mediated Cancer Immunity

Purpose: Anti-programmed-death-1 (PD-1) immunotherapy improves survival in non–small cell lung cancer (NSCLC), but some cases are refractory to treatment, thereby requiring alternative strategies. B7-H3, an immune-checkpoint molecule, is expressed in various malignancies. To our knowledge, this study is the first to evaluate B7-H3 expression in NSCLCs treated with anti-PD-1 therapy and the therapeutic potential of a combination of anti-PD-1 therapy and B7-H3 targeting. Experimental Design: B7-H3 expression was evaluated immunohistochemically in patients with NSCLC (n = 82), and its relationship with responsiveness to anti-PD-1 therapy and CD8+ tumor-infiltrating lymphocytes (TILs) was analyzed. The antitumor efficacy of dual anti-B7-H3 and anti-programmed death ligand-1 (PD-L1) antibody therapy was evaluated using a syngeneic murine cancer model. T-cell numbers and functions were analyzed by flow cytometry. Results: B7-H3 expression was evident in 74% of NSCLCs and was correlated critically with nonresponsiveness to anti-PD-1 immunotherapy. A small number of CD8+ TILs was observed as a subpopulation with PD-L1 tumor proportion score less than 50%, whereas CD8+ TILs were still abundant in tumors not expressing B7-H3. Anti-B7-H3 blockade showed antitumor efficacy accompanied with an increased number of CD8+ TILs and recovery of effector function. CD8+ T-cell depletion negated antitumor efficacy induced by B7-H3 blockade, indicating that improved antitumor immunity is mediated by CD8+ T cells. Compared with a single blocking antibody, dual blockade of B7-H3 and PD-L1 enhanced the antitumor reaction. Conclusions: B7-H3 expressed on tumor cells potentially circumvents CD8+-T-cell–mediated immune surveillance. Anti-B7-H3 immunotherapy combined with anti-PD-1/PD-L1 antibody therapy is a promising approach for B7-H3–expressing NSCLCs. Clin Cancer Res; 24(11); 2653–64. ©2018 AACR.

Clinical characteristics of non-small cell lung cancer harboring mutations in exon 20 of EGFR or HER2

Unlike common epidermal growth factor receptor gene (EGFR) mutations that confer sensitivity to tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC), mutations in exon 20 of either EGFR or the human EGFR2 gene (HER2) are associated with insensitivity to EGFR-TKIs, with treatment options for patients with such mutations being limited. Clinical characteristics, outcome of EGFR-TKI or nivolumab treatment, and the presence of coexisting mutations were reviewed for NSCLC patients with exon-20 mutations of EGFR or HER2 as detected by routine application of an amplicon-based next-generation sequencing panel. Between July 2013 and June 2017, 206 patients with pathologically confirmed lung cancer were screened for genetic alterations including HER2 and EGFR mutations. Ten patients harbored HER2 exon-20 insertions (one of whom also carried an exon-19 deletion of EGFR), and 12 patients harbored EGFR exon-20 mutations. Five of the 13 patients with EGFR mutations were treated with EGFR-TKIs, two of whom manifested a partial response, two stable disease, and one progressive disease. Among the seven patients treated with nivolumab, one patient manifested a partial response, three stable disease, and three progressive disease, with most (86%) of these patients discontinuing treatment as a result of disease progression within 4 months. The H1047R mutation of PIK3CA detected in one patient was the only actionable mutation coexisting with the exon-20 mutations of EGFR or HER2. Potentially actionable mutations thus rarely coexist with exon-20 mutations of EGFR or HER2, and EGFR-TKIs and nivolumab show limited efficacy in patients with such exon-20 mutations.

A case of ALK -rearranged non–small cell lung cancer that responded to ceritinib after development of resistance to alectinib

The second-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) alectinib and ceritinib are standard treatment options for patients with non–small cell lung cancer (NSCLC) positive for ALK fusion genes. However, almost all patients eventually develop resistance to these drugs. We here report a case of ALK-rearranged NSCLC that developed resistance to alectinib but remained sensitive to ceritinib. The L1196M mutation within the ALK fusion gene was detected after failure of consecutive treatment with crizotinib and alectinib, but no other mechanism underlying acquired resistance to ALK-TKIs was found to be operative. Given the increasing application of ALK-TKIs to the treatment of patients with ALK-rearranged NSCLC, further clinical evaluation is warranted to provide a better understanding of the mechanisms of acquired resistance to these agents and to inform treatment strategies for such tumors harboring secondary mutations.

An HER3-targeting antibody–drug conjugate incorporating a DNA topoisomerase I inhibitor U3-1402 conquers EGFR tyrosine kinase inhibitor-resistant NSCLC

EGFR tyrosine kinase inhibitors (TKIs) are standard therapy for EGFR-mutant non-small cell lung cancer (NSCLC); however, these tumours eventually acquire chemoresistance. U3-1402 is an anti-HER3 antibody–drug conjugate with a novel topoisomerase I inhibitor, DXd. In the current study, we evaluated the anticancer efficacy of U3-1402 in EGFR-mutant NSCLC cells with acquired resistance to EGFR-TKIs. HCC827GR5 and PC9AZDR7 are EGFR-TKI-resistant clones for gefitinib and osimertinib, respectively. U3-1402 alone or in combination with the EGFR-TKI erlotinib demonstrated potent anticancer efficacy in HCC827GR5 cells using an in vitro growth inhibition assay and in vivo xenograft mouse model. U3-1402 induced apoptosis in HCC827GR5 cells accompanying phosphorylation of histone H2A.X, a marker of DNA damage, but did not block HER3/PI3K/AKT signalling. Further, we found using flow cytometry that the cell surface HER3 expression level in HCC827GR5 cells was twice that found in HCC827 cells, indicating internalization of U3-1402 was increased in resistant cells. In addition, administration of U3-1402 notably repressed growth of EGFR-TKI osimertinib-resistant PC9AZDR7 xenograft tumours, and that PC9AZDR7 cells expressed five times greater cell surface HER3 than PC9 cells. Furthermore, using immunofluorescent microscopy, HER3 was observed predominantly in the nucleus of PC9 cells, but was localized in the cytoplasm of PC9AZDR7 cells. This finding indicates that altered trafficking of the HER3-U3-1402 complex may accelerate linker payload cleavage by cytoplasmic lysosomal enzymes, resulting in DNA damage. Our results indicate that administration of U3-1402 alone or in combination with an EGFR-TKI may have potential as a novel therapy for EGFR-TKI-resistant EGFR-mutant NSCLC.