Shinzo Takamori

Association of PD-L1 overexpression with activating EGFR mutations in surgically resected nonsmall-cell lung cancer

BACKGROUND Recent clinical trials have shown that immune-checkpoint blockade yields a clinical response in a subset of individuals with advanced nonsmall-cell lung cancer (NSCLC). We examined whether the expression of programmed death-ligand 1 (PD-L1) is related to clinicopathologic or prognostic factors in patients with surgically resected NSCLC. PATIENTS AND METHODS The expression of PD-L1 was evaluated by immunohistochemical analysis in 164 specimens of surgically resected NSCLC. Cell surface expression of PD-L1 in NSCLC cell lines was quantified by flow cytometry. RESULTS Expression of PD-L1 in tumor specimens was significantly higher for women than for men, for never smokers than for smokers, and for patients with adenocarcinoma than for those with squamous cell carcinoma. Multivariate analysis revealed that the presence of epidermal growth factor receptor gene (EGFR) mutations and adenocarcinoma histology were significantly associated with increased PD-L1 expression in a manner independent of other factors. Cell surface expression of PD-L1 was also significantly higher in NSCLC cell lines positive for activating EGFR mutations than in those with wild-type EGFR. The EGFR inhibitor erlotinib downregulated PD-L1 expression in the former cell lines but not in the latter, suggesting that PD-L1 expression is increased by EGFR signaling conferred by activating EGFR mutations. A high level of PD-L1 expression in resected tumor tissue was associated with a significantly shorter overall survival for NSCLC patients. CONCLUSIONS High expression of PD-L1 was associated with the presence of EGFR mutations in surgically resected NSCLC and was an independent negative prognostic factor for this disease.

Induction of PD-L1 Expression by the EML4–ALK Oncoprotein and Downstream Signaling Pathways in Non–Small Cell Lung Cancer

Purpose: Therapies targeted to the immune checkpoint mediated by PD-1 and PD-L1 show antitumor activity in a subset of patients with non–small cell lung cancer (NSCLC). We have now examined PD-L1 expression and its regulation in NSCLC positive for the EML4–ALK fusion gene. Experimental Design: The expression of PD-L1 at the protein and mRNA levels in NSCLC cell lines was examined by flow cytometry and by reverse transcription and real-time PCR analysis, respectively. The expression of PD-L1 in 134 surgically resected NSCLC specimens was evaluated by immunohistochemical analysis. Results: The PD-L1 expression level was higher in NSCLC cell lines positive for EML4–ALK than in those negative for the fusion gene. Forced expression of EML4–ALK in Ba/F3 cells markedly increased PD-L1 expression, whereas endogenous PD-L1 expression in EML4–ALK–positive NSCLC cells was attenuated by treatment with the specific ALK inhibitor alectinib or by RNAi with ALK siRNAs. Furthermore, expression of PD-L1 was downregulated by inhibitors of the MEK–ERK and PI3K–AKT signaling pathways in NSCLC cells positive for either EML4–ALK or activating mutations of the EGFR. Finally, the expression level of PD-L1 was positively associated with the presence of EML4–ALK in NSCLC specimens. Conclusions: Our findings that both EML4–ALK and mutant EGFR upregulate PD-L1 by activating PI3K–AKT and MEK–ERK signaling pathways in NSCLC reveal a direct link between oncogenic drivers and PD-L1 expression. Clin Cancer Res; 21(17); 4014–21. ©2015 AACR.

Identification of a Gene Coding for a New Squamous Cell Carcinoma Antigen Recognized by the CTL

Peptide-based specific immunotherapy has resulted in tumor regression in some melanoma patients. However, tumor Ags and peptides for specific immunotherapy, except for treatment of melanomas, have not yet been well identified. In this study, we report a gene encoding a new squamous cell carcinoma (SCC) Ag recognized by cells of the HLA-A24-restricted and tumor-specific CTL line. This gene with 3958-bp length was transcribed from the chromosome 6q22 with six exons, and its mRNA was ubiquitously expressed in both SCCs and normal tissues, and partly expressed in adenocarcinomas. The deduced 958-aa sequence encoded by this gene showed no similarity to any known amino acid sequences. This gene product had a characteristic of an endoplasmic reticulum-resident protein. A 100-kDa protein was detected in the vast majority of SCCs from various tissues, in majority of renal cell carcinomas and brain tumors, and in about one-third of melanomas and adenocarcinomas from various organs other than the breast. In contrast, it was not expressed at all in any of the normal cells or tissues tested, including the testis and fetal liver. Three different peptides at positions 93–101, 161–169, and 899–907 of this Ag were recognized by this CTL line, and all of them induced HLA-A24-restricted and tumor-specific CTLs from PBMCs of SCC patients. Therefore, these peptides may be useful for peptide-based specific immunotherapy of HLA-A24+ patients with SCC in various organs, as well as for treatment of other cancer.

Dynamic MRI of Solitary Pulmonary Nodules: Comparison of Enhancement Patterns of Malignant and Benign Small Peripheral Lung Lesions

OBJECTIVE The purpose of this study was to compare the dynamic contrast-enhanced MRI enhancement characteristics of malignant and benign solitary pulmonary nodules. MATERIALS AND METHODS The characteristics of 202 solitary pulmonary nodules (diameter, 1-3 cm; 144 cases of primary lung cancer, 31 cases of focal organizing pneumonia, 15 tuberculomas, 12 hamartomas) were reviewed retrospectively. In all cases dynamic MR images were obtained before and 1, 2, 3, 4, 5, 6, and 8 minutes after bolus injection of gadopentetate dimeglumine. Maximum enhancement ratio, time at maximum enhancement ratio, slope of time-enhancement ratio curves, and washout ratio were assessed. Statistical analyses were performed with the Kruskal-Wallis test with Bonferroni correction, chi-square test, and receiver operating characteristic curves. RESULTS For 122 (85%) of 144 primary lung cancers, time at maximum enhancement ratio was 4 minutes or less. For all tuberculomas and hamartomas, time at maximum enhancement ratio was greater than 4 minutes or gradual enhancement occurred without a peak time (p < 0.0001). Lung cancers had different maximum enhancement ratios and slopes than benign lesions (all p < 0.005). With 110% or lower maximum enhancement ratio as a cutoff value, the positive predictive value for malignancy was 92%; sensitivity, 63%; and specificity, 74%. With 13.5%/min or greater slope as a cutoff value, sensitivity, specificity, positive predictive value, and negative predictive value for malignancy were 94%, 96%, 99%, and 74%, respectively. CONCLUSION Dynamic contrast-enhanced MRI is helpful in differentiating benign from malignant solitary pulmonary nodules. Absence of significant enhancement is a strong predictor that a lesion is benign.

Clinicopathologic characteristics of adenosquamous carcinoma of the lung.

Fifty‐six cases of surgically resected adenosquamous carcinoma of the lung were studied clinicopathologically, and their outcome was compared with that of adenocarcinomas and squamous cell carcinomas of the lung. The frequency rate of adenosquamous carcinoma was 2.6% of 2160 primary lung cancers resected in the National Cancer Center Hospital (Tokyo, Japan). The survival curves of patients with adenosquamous carcinomas, adenocarcinomas, and squamous cell carcinomas indicated that the outcome of adenosquamous carcinoma was poorer than that of adenocarcinomas and squamous cell carcinomas, particularly in Stages I and II. The amount of adenocarcinoma component did not affect the survival rate, although the histologic features of metastatic lymph nodes was somewhat influenced by the histologic type of the primary tumors. The histologic subtype of adenosquamous carcinoma was one of the independent prognostic determinants.

Identification of non-small-cell lung cancer with activating EGFR mutations in malignant effusion and cerebrospinal fluid: Rapid and sensitive detection of exon 19 deletion E746-A750 and exon 21 L858R mutation by immunocytochemistry

BACKGROUND Recently, we have reported that EGFR mutation-specific antibodies performed well in immunohistochemical analysis, with good sensitivity. We investigated whether this method could detect non-small-cell lung cancer (NSCLC) carrying EGFR mutations in malignant effusions and cerebrospinal fluid (CSF), comparable to the peptide nucleic acid-locked nucleic acid (PNA-LNA) PCR clamp assay. Furthermore, we compared activating EGFR mutations between primary and recurrent NSCLC. PATIENTS AND METHODS Twenty-four patients with NSCLC effusions and CSF were examined by immunocytochemistry using antibodies specific for the E746-A750 deletion mutation in exon 19 and the L858R point mutation in exon 21. The PNA-LNA PCR clamp assay was used to detect the E746-A750 deletion at exon 19, L858R mutation at exon 21, and T790M mutation at exon 20. RESULTS We were able to identify EGFR mutations in NSCLC effusion and CSF with a sensitivity of 100% (5/5) using the anti-delE746-A750 antibody and 100% (8/8) using the anti-L858R antibody. Furthermore, in samples without these EGFR mutations, immunocytochemistry with the two specific antibodies identified 91% (10/11) as negative for both the deletion and the point mutations in EGFR. Activating EGFR mutations decreased in recurrent NSCLC compared with primary NSCLC, and the T790M mutation was detected in recurrent NSCLC of patients receiving gefitinib treatment. CONCLUSIONS Identification of EGFR mutations is important for patients with primary and recurrent NSCLC. Rapid and sensitive immunocytochemistry using mutation-specific antibodies to detect EGFR mutations will be useful for diagnosing responsiveness to EGFR-targeted drugs.

Nuclear Y-Box Binding Protein-1, a Predictive Marker of Prognosis, Is Correlated with Expression of HER2/ErbB2 and HER3/ErbB3 in Non-small Cell Lung Cancer

Introduction: Nuclear expression of Y-box binding protein-1 (YB-1) is closely associated not only with global drug resistance and expression of several growth factor receptors in various human malignancies but also with overall patient survival. Methods: The effect of YB-1 knockdown on expression of epidermal growth factor receptor (EGFR) family proteins was examined by Western blot using human lung cancer cell lines. Immunohistochemistry was used to evaluate the expression of nuclear YB-1 and EGFR family proteins in patients with non-small cell lung cancer (NSCLC) (n = 104). Results: In the five NSCLC cell lines, expressions of EGFR, human epidermal growth factor receptor 2 (HER2), HER3, and hepatocyte growth factor receptor (c-Met) in PC-9 cells; of HER2 and c-Met in EBC-1 cells; and of HER3 in QG56 cells were down-regulated by YB-1 knockdown. By immunohistochemical analysis, we observed that HER3 expression was significantly negatively correlated with nuclear YB-1 expression in squamous cell carcinoma (p = 0.038). HER2 expression was positively correlated with nuclear YB-1 expression in adenocarcinoma (p = 0.052). Nuclear expression of YB-1 correlated with overall survival of all patients (p = 0.028) and of patients with adenocarcinoma (p = 0.007). Furthermore, there was a significant difference in therapeutic efficacies of gefitinib between patients with nuclear YB-1 expression and those with non-nuclear YB-1 expression in patients with NSCLC (p = 0.004, n = 26) but not between those with high and those with low expression of EGFR, HER2, HER3, and c-Met. Conclusion: Nuclear YB-1 expression might be essential for the malignant phenotype in lung cancer patients and might be an important biomarker for the development of therapeutic strategy against NSCLC.

Intraoperative intercostal nerve blockade for postthoracotomy pain.

BACKGROUND Epidural analgesia is widely employed as a means to control postthoracotomy pain, but is sometimes inadequate. The purpose of this study is to evaluate the effectiveness of intraoperative, temporary, intercostal nerve blockade in addition to epidural analgesia for control of postthoracotomy pain. METHODS Forty patients undergoing elective lobectomy through antero-axillary thoracotomy were randomized to receive epidural analgesia only (group A, n = 20) or epidural analgesia plus temporary, intraoperative intercostal nerve blockade using 0.25% bupivacaine (group B, n = 20). Postoperative pain was assessed using a subjective analogue visual scale, and with the Prince Henry pain scale. Food intake and nonsteroidal analgesic consumption were also investigated. Serum ACTH and cortisol in each group were measured before and after the operation. RESULTS The analogue visual scale scores were significantly higher in group A than group B (p < 0.001), and were significantly higher on the day of operation and postoperative days 1, 2, and 3 (p < 0.001, p < 0.005, p < 0.005, p < 0.05, respectively). Prince Henry pain scale scores were significantly higher on the day of operation and postoperative day 1 (p < 0.05, p < 0.005, respectively). Food intake was significantly lower in group A than in group B (p < 0.05), and nonsteroidal analgesic consumption was not significantly different between groups. There was no significant difference between group A and group B in serum ACTH or in cortisol levels. CONCLUSIONS Additional intraoperative intercostal nerve blockade provides an additive benefit for postthoracotomy pain relief, especially early after operation.

Personalized peptide vaccination in patients with refractory non-small cell lung cancer.

Since the prognosis of non-small cell lung cancer (NSCLC) remains poor, the development of novel therapeutic approaches, including cancer vaccines, is highly desirable. In the current study, we conducted a phase II study of personalized peptide vaccination (PPV), in which a maximum of 4 peptides were selected based on pre-existing humoral immune responses and administered subcutaneously (weekly for 6 consecutive weeks and bi-weekly thereafter) in refractory NSCLC patients. Forty-one refractory NSCLC patients (4 stage IIIb, 22 stage IV and 15 recurrent), who had failed to respond to chemotherapy and/or targeted therapy (median number of regimens, 3; median duration, 10 months), were enrolled. Median overall survival (OS) was 304 days with a one-year survival rate of 42% in the enrolled patients. The main toxicity of PPV was skin reactions at the injection sites, but no serious adverse events were observed. In order to identify potential biomarkers for predicting OS, pre-vaccination and post-vaccination clinical findings and laboratory data were retrospectively assessed and evaluated by multivariate Cox regression analysis. Among the pre-vaccination factors examined, high C-reactive protein (CRP) level was a significant predictor of unfavorable OS [hazard ratio (HR)=10.115, 95% confidence interval (CI)=2.447-41.806, P=0.001]. Among the post-vaccination factors, high CRP level and low frequency of CD3⁺CD26⁺ cells were significant predictors of unfavorable OS (HR=23.127, 95% CI=2.919-183.233, P=0.003; HR=0.952, 95% CI=0.917-0.989, P=0.012). Taken together, our results suggest the feasibility of PPV for the treatment of refractory NSCLC. Evaluation of the identified factors before or at an early stage of vaccination could be potentially useful for selecting NSCLC patients who would likely have better prognosis following PPV.

Association of the Expression of Mutant Epidermal Growth Factor Receptor Protein as Determined with Mutation-Specific Antibodies in Non-small Cell Lung Cancer with Progression-Free Survival after Gefitinib Treatment

Introduction: Somatic mutations in the epidermal growth factor receptor (EGFR) gene are associated with an increased response to EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib in patients with non-small cell lung cancer (NSCLC). Although most NSCLC patients with EGFR mutations benefit from EGFR-TKI treatment, the efficacy of such treatment varies among individuals. Molecular markers for prediction of EGFR-TKI treatment efficacy in EGFR mutation-positive NSCLC have not been well defined. Methods: The expression of mutant EGFR proteins was quantitated by immunohistochemical analysis with mutation-specific antibodies in tumor specimens from 47 NSCLC patients with postoperative recurrent disease who harbored activating EGFR mutations. The expression score was determined from both the staining intensity and the proportion of tumor tissue expressing the mutant EGFR. Results: The median progression-free survival after the start of gefitinib treatment was significantly longer in patients with a high score for mutant EGFR expression than in those with a low score (12.2 versus 3.4 months, p < 0.001), whereas no significant difference in median overall survival was apparent between the two groups (24.9 versus 17.7 months, respectively, p = 0.144). This association between the expression score for mutant EGFR and progression-free survival was apparent both in patients with deletions in exon 19 of EGFR and in those with the L858R mutation in exon 21. Conclusions: Quantitative analysis of mutant EGFR expression by immunohistochemical analysis with mutation-specific antibodies may predict the efficacy of gefitinib treatment for EGFR mutation-positive NSCLC.