Eiki Kikuchi

Endobronchial ultrasonography with guide-sheath for peripheral pulmonary lesions

The usefulness of endobronchial ultrasonography (EBUS) with guide-sheath (GS) as a guide for transbronchial biopsy (TBB) for diagnosing peripheral pulmonary lesions (PPL)s and for improving diagnostic accuracy was evaluated in this study. EBUS-GS-guided TBB was performed in 24 patients with 24 PPLs of ≤30 mm in diameter (average diameter=18.4 mm). A 20-MHz radial-type ultrasound probe, covered with GS was inserted via a working bronchoscope channel and advanced to the PPL in order to produce an EBUS image. The probe with the GS was confirmed to reach the lesion by EBUS imaging and X-ray fluoroscopy. When the lesion was not identified on the EBUS image, the probe was removed and a curette was used to lead the GS to the lesion. After localising the lesion, the probe was removed, and TBB and bronchial brushing were performed via the GS. Nineteen peripheral lesions (79.2%) were visualised by EBUS. All patients whose PPLs were visible on EBUS images subsequently underwent an EBUS-GS-guided diagnostic procedure. A total of 14 lesions (58.3%) were diagnosed. Even when restricted to PPLs <20 mm in diameter, the diagnostic sensitivity was 53%. In conclusion, endobronchial ultrasonography with guide sheath-guided transbronchial biopsy was feasible and effective for diagnosing peripheral pulmonary lesions.

Efficacy of BET Bromodomain Inhibition in Kras-Mutant Non–Small Cell Lung Cancer

Purpose: Amplification of MYC is one of the most common genetic alterations in lung cancer, contributing to a myriad of phenotypes associated with growth, invasion, and drug resistance. Murine genetics has established both the centrality of somatic alterations of Kras in lung cancer, as well as the dependency of mutant Kras tumors on MYC function. Unfortunately, drug-like small-molecule inhibitors of KRAS and MYC have yet to be realized. The recent discovery, in hematologic malignancies, that bromodomain and extra-terminal (BET) bromodomain inhibition impairs MYC expression and MYC transcriptional function established the rationale of targeting KRAS-driven non–small cell lung cancer (NSCLC) with BET inhibition. Experimental Design: We performed functional assays to evaluate the effects of JQ1 in genetically defined NSCLC cell lines harboring KRAS and/or LKB1 mutations. Furthermore, we evaluated JQ1 in transgenic mouse lung cancer models expressing mutant kras or concurrent mutant kras and lkb1. Effects of bromodomain inhibition on transcriptional pathways were explored and validated by expression analysis. Results: Although JQ1 is broadly active in NSCLC cells, activity of JQ1 in mutant KRAS NSCLC is abrogated by concurrent alteration or genetic knockdown of LKB1. In sensitive NSCLC models, JQ1 treatment results in the coordinate downregulation of the MYC-dependent transcriptional program. We found that JQ1 treatment produces significant tumor regression in mutant kras mice. As predicted, tumors from mutant kras and lkb1 mice did not respond to JQ1. Conclusion: Bromodomain inhibition comprises a promising therapeutic strategy for KRAS-mutant NSCLC with wild-type LKB1, via inhibition of MYC function. Clinical studies of BET bromodomain inhibitors in aggressive NSCLC will be actively pursued. Clin Cancer Res; 19(22); 6183–92. ©2013 AACR.

Epigenetic therapy with 3-deazaneplanocin A, an inhibitor of the histone methyltransferase EZH2, inhibits growth of non-small cell lung cancer cells.

EZH2 (enhancer of zeste homolog 2) is the catalytic subunit of PRC2 (polycomb repressive complex 2), which mediates histone methyltransferase activity and functions as transcriptional repressor involved in gene silencing. EZH2 is involved in malignant transformation and biological aggressiveness of several human malignancies. We previously demonstrated that non-small cell lung cancers (NSCLCs) also overexpress EZH2 and that high expression of EZH2 correlates with poor prognosis. Growing evidence indicates that EZH2 may be an appropriate therapeutic target in malignancies, including NSCLCs. Recently, an S-adenosyl-l-homocysteine hydrolase inhibitor, 3-Deazaneplanocin A (DZNep), has been shown to deplete and inhibit EZH2. The aim of this study was to determine the effect of DZNep in NSCLC cells. Knockdown of EZH2 by small-interfering RNA (siRNA) resulted in decreased growth of four NSCLC cell lines. MTT assays demonstrated that DZNep treatment resulted in dose-dependent inhibition of proliferation in the NSCLC cell lines with a half maximal inhibitory concentration (IC50) ranging from 0.08 to 0.24 μM. Immortalized but non-cancerous bronchial epithelial and fibroblast cell lines were less sensitive to DZNep than the NSCLC cell lines. Soft agarose assays demonstrated that anchorage-independent growth was also reduced in all three NSCLC cell lines that were evaluated using this assay. Flow cytometry analysis demonstrated that DZNep induced apoptosis and G1 cell cycle arrest in NSCLC cells, which was partially associated with cyclin A decrease and p27(Kip1) accumulation. DZNep depleted cellular levels of EZH2 and inhibited the associated histone H3 lysine 27 trimethylation. These results indicated that an epigenetic therapy that pharmacologically targets EZH2 via DZNep may constitute a novel approach to treatment of NSCLCs.

Distinctive expression of the polycomb group proteins Bmi1 polycomb ring finger oncogene and enhancer of zeste homolog 2 in nonsmall cell lung cancers and their clinical and clinicopathologic significance

The polycomb group genes Bmi1 polycomb ring finger oncogene (Bmi1) and enhancer of zeste homolog 2 (EZH2) function as transcriptional repressors involved in gene silencing and in the malignant transformation and biologic aggressiveness of several human carcinomas. In the current study, the authors evaluated Bmi1 and EZH2 protein expression in specimens of human nonsmall cell lung cancer (NSCLC).

HLA class I antigen expression is associated with a favorable prognosis in early stage non-small cell lung cancer

Human leukocyte antigen (HLA) class I displays a repertoire of endogenously processed peptides to CD8+ T lymphocytes. The present study assessed correlations between HLA class I expression, clinicopathologic factors, and tumor‐infiltrating immune cells in human non‐small cell lung cancers (NSCLC). Expression of HLA class I was assessed in 161 resected primary NSCLC by immunohistochemistry using EMR8–5, a novel monoclonal anti‐pan HLA class I heavy chain antibody. Expression of HLA class I was classified into three categories: strongly positive, weakly positive, or negative. Tumor‐infiltrating CD8+ lymphocytes and CD56+ natural killer cells within cancer nests and stroma were also counted. Expression of HLA class I was strongly positive in 50 tumors, weakly positive in 57 tumors, and negative in 54 tumors. Down‐regulation of HLA class I was significantly correlated with male sex, history of smoking, non‐adenocarcinoma histology, and moderate‐/low‐grade differentiation. The density of cancer nest‐infiltrating CD8+ cells in HLA class I‐negative tumors was significantly decreased compared to that in HLA class I strongly positive tumors (P < 0.01). Kaplan–Meier analysis revealed a significant favorable influence on overall survival for patients displaying tumors with strongly positive expression of HLA class I (P < 0.01). Multivariate analysis revealed down‐regulation of HLA class I as an independent factor of poor prognosis in pathological stage I patients, but not in late‐stage patients. These results suggest that down‐regulation of HLA class I expression in NSCLC is a marker of poor prognosis, and this may play a critical role in immune surveillance of patients with NSCLC. (Cancer Sci 2007; 98: 1424–1430)

Clinical Outcomes of Stereotactic Body Radiotherapy for Small Lung Lesions Clinically Diagnosed as Primary Lung Cancer on Radiologic Examination

PURPOSE Image-guided biopsy occasionally fails to diagnose small lung lesions, which are highly suggestive of primary lung cancer. The aim of the present study was to evaluate the outcome of stereotactic body radiotherapy (SBRT) for small lung lesions that were clinically diagnosed as primary lung cancer without pathologic confirmation. METHODS AND MATERIALS A total of 115 patients were treated with SBRT in 12 institutions. Tumor size ranged from 5 to 45 mm in diameter, with a median of 20 mm. RESULTS The 3-year and 5-year overall survival rates for patients with a tumor size < or =20 mm in diameter (n = 58) were both 89.8%, compared with 60.7% and 53.1% for patients with tumors >20 mm (n = 57) (p <0.0005), respectively. Local progression occurred in 2 patients (3.4%) with a tumor size < or =20 mm and in 3 patients (5.3%) with tumors >20 mm. Among the patients with a tumor size < or =20 mm, Grade 2 pulmonary complications were observed in 2 (3.4%), but no Grade 3 to 5 toxicity was observed. In patients with a tumor size >20 mm, Grades 2, 3, and 5 toxicity were observed in 5 patients (8.8%), 3 patients (5.3%), and 1 patient (1.8%), respectively. CONCLUSION In patients with a tumor < or =20 mm in diameter, SBRT was reasonably safe in this retrospective study. The clinical implications of the high local control rate depend on the accuracy of clinical/radiologic diagnosis for small lung lesions and are to be carefully evaluated in a prospective study.

Anti‐OX40 monoclonal antibody therapy in combination with radiotherapy results in therapeutic antitumor immunity to murine lung cancer

The therapeutic effect of agonistic anti‐OX40 (CD134) monoclonal antibody (mAb) in combination with radiotherapy was evaluated in a murine lung cancer model. After intradermal transplantation of ovalbumin (OVA)‐transfected Lewis lung carcinoma, C57BL/6 mice were irradiated locally with a single dose of 20 Gy in combination with an intratumoral injection of anti‐OX40 mAb at 50 µg on day 4 after transplantation, which is when the major axis of the inoculated tumor reached a diameter of 7–9 mm. On days 8, 11, and 14, the tumor‐bearing mice were further treated with the same dose of anti‐OX40 mAb. Anti‐OX40 mAb in combination with radiotherapy prolonged survival and provided greater efficacy than either single treatment against well‐established tumors. An in vivo depletion study suggested that therapeutic immunity was mainly CD8+ T‐cell dependent. OX40+CD8+ T cells were augmented in draining lymph nodes obtained from irradiated mice compared with those from non‐irradiated mice. OVA‐major histocompatibility complex tetramer+ CD8+ T cells had been strongly recruited to the draining lymph nodes obtained from mice treated with anti‐OX40 mAb in combination with radiotherapy, and strong antigen‐specific cytotoxicity was confirmed by a 51Cr‐release assay. Moreover, a tumor‐rechallenge model indicated that this combination therapy induced durable tumor immunity. Thus, anti‐OX40 mAb in combination with radiotherapy may potentially help the management of patients with lung cancer. (Cancer Sci 2008; 99: 361–367)

Minichromosome maintenance (MCM) protein 4 as a marker for proliferation and its clinical and clinicopathological significance in non-small cell lung cancer

BACKGROUND Minichromosome maintenance (MCM) proteins 2-7 form a complex essential for the initiation of DNA replication. In the process to screen expression changes related to growth suppression of non-small cell lung cancer (NSCLC) cells by a cJun dominant-negative mutant, we found that reduced expression of MCM4 was correlated with this growth suppression. METHOD We determined the relevance of MCM4 in proliferation of NSCLC by downregulating its expression with small-interfering RNA in three NSCLC cell lines. We then immunohistochemically analyzed MCM4 expression in 156 surgically resected NSCLCs to correlate clinicopathologic characteristics. RESULTS MCM4 downregulation reduced proliferation in two cell lines. MCM4 expression was higher in cancer cells than in adjacent normal bronchial epithelial cells (p<0.001). High MCM4 expression was correlated with male gender, heavy smoking, poorer differentiation and non-adenocarcinoma histology (p<0.001, respectively). High MCM4 expression was also correlated with proliferation markers, Ki-67 and cyclin E expression (p<0.001, respectively). MCM4 expression was not associated with survival. CONCLUSION MCM4 may play an essential role in the proliferation of some NSCLC cells. Taken together with higher expression in NSCLCs and its correlation with clinicopathologic characteristics such as non-adenocarcinoma histology, MCM4 may have potential as a therapeutic target in certain population with NSCLCs.

Intratumoral Heterogeneity in EGFR-Mutant NSCLC Results in Divergent Resistance Mechanisms in Response to EGFR Tyrosine Kinase Inhibition

Non-small cell lung cancers (NSCLC) that have developed resistance to EGF receptor (EGFR) tyrosine kinase inhibitor (TKI), including gefitinib and erlotinib, are clinically linked to an epithelial-to-mesenchymal transition (EMT) phenotype. Here, we examined whether modulating EMT maintains the responsiveness of EGFR-mutated NSCLCs to EGFR TKI therapy. Using human NSCLC cell lines harboring mutated EGFR and a transgenic mouse model of lung cancer driven by mutant EGFR (EGFR-Del19-T790M), we demonstrate that EGFR inhibition induces TGFβ secretion followed by SMAD pathway activation, an event that promotes EMT. Chronic exposure of EGFR-mutated NSCLC cells to TGFβ was sufficient to induce EMT and resistance to EGFR TKI treatment. Furthermore, NSCLC HCC4006 cells with acquired resistance to gefitinib were characterized by a mesenchymal phenotype and displayed a higher prevalence of the EGFR T790M mutated allele. Notably, combined inhibition of EGFR and the TGFβ receptor in HCC4006 cells prevented EMT but was not sufficient to prevent acquired gefitinib resistance because of an increased emergence of the EGFR T790M allele compared with cells treated with gefitinib alone. Conversely, another independent NSCLC cell line, PC9, reproducibly developed EGFR T790M mutations as the primary mechanism underlying EGFR TKI resistance, even though the prevalence of the mutant allele was lower than that in HCC4006 cells. Thus, our findings underscore heterogeneity within NSCLC cells lines harboring EGFR kinase domain mutations that give rise to divergent resistance mechanisms in response to treatment and anticipate the complexity of EMT suppression as a therapeutic strategy.

Targeting the Oncogenic MUC1-C Protein Inhibits Mutant EGFR-Mediated Signaling and Survival in Non–Small Cell Lung Cancer Cells

Purpose: Non–small cell lung cancers (NSCLC) that express EGF receptor with activating mutations frequently develop resistance to EGFR kinase inhibitors. The mucin 1 (MUC1) heterodimeric protein is aberrantly overexpressed in NSCLC cells and confers a poor prognosis; however, the functional involvement of MUC1 in mutant EGFR signaling is not known. Experimental Design: Targeting the oncogenic MUC1 C-terminal subunit (MUC1-C) in NSCLC cells harboring mutant EGFR was studied for effects on signaling, growth, clonogenic survival, and tumorigenicity. Results: Stable silencing of MUC1-C in H1975/EGFR(L858R/T790M) cells resulted in downregulation of AKT signaling and inhibition of growth, colony formation, and tumorigenicity. Similar findings were obtained when MUC1-C was silenced in gefitinib-resistant PC9GR cells expressing EGFR(delE746_A750/T790M). The results further show that expression of a MUC1-C(CQC→AQA) mutant, which blocks MUC1-C homodimerization, suppresses EGFR(T790M), AKT and MEK→ERK activation, colony formation, and tumorigenicity. In concert with these results, treatment of H1975 and PC9GR cells with GO-203, a cell-penetrating peptide that blocks MUC1-C homodimerization, resulted in inhibition of EGFR, AKT, and MEK→ERK signaling and in loss of survival. Combination studies of GO-203 and afatinib, an irreversible inhibitor of EGFR, further demonstrate that these agents are synergistic in inhibiting growth of NSCLC cells harboring the activating EGFR(T790M) or EGFR(delE746-A750) mutants. Conclusions: These findings indicate that targeting MUC1-C inhibits mutant EGFR signaling and survival, and thus represents a potential approach alone and in combination for the treatment of NSCLCs resistant to EGFR kinase inhibitors. Clin Cancer Res; 20(21); 5423–34. ©2014 AACR.