Masaki Shimoji

Clinical and pathologic features of lung cancer expressing programmed cell death ligand 1 (PD-L1).

BACKGROUND Programmed cell death 1 (PD-1) negatively regulates antigen receptor signaling upon binding by either of its ligands, programmed cell death ligand 1 or 2 (PD-L1/2). Blockade of this interaction with either PD-1 or PD-L1 antibodies has been successful in the treatment of human cancer, especially melanoma and non-small cell lung cancer. PD-L1 expression has been proposed as a predictor of tumor response. However, the relationships between PD-L1 expression and various clinicopathological characteristics remain unclear. MATERIALS AND METHODS PD-L1 expression was examined in 220 non-small cell lung cancer specimens that were consecutively resected at our hospital after validating the E1L3N antibody immunohistochemical assay by comparing IHC and RT-PCR data for lung cancer cell lines. We evaluated the relationships between PD-L1 positivity, several clinical factors and the immunohistochemical expression of epithelial-mesenchymal transition (EMT), cancer stem cell and proliferative markers. RESULTS PD-L1 was expressed in 22% of lung adenocarcinomas and 60% of squamous cell lung cancers. There was no significant association between PD-L1 expression and clinicopathological features in squamous cell lung cancer. However, in patients with lung adenocarcinoma, PD-L1 expression was significantly correlated with solid subtype histology, vimentin expression, increased Ki-67 labeling index and poor prognosis by multivariate analysis. CONCLUSION PD-L1 expression was associated with high proliferative activity and the EMT phenotype in adenocarcinoma but not in squamous cell carcinoma of the lung. PD-L1 expression was a significant poor prognostic factor in patients with lung adenocarcinoma.

EGFR Exon 18 Mutations in Lung Cancer: Molecular Predictors of Augmented Sensitivity to Afatinib or Neratinib as Compared with First- or Third-Generation TKIs.

Purpose: Lung cancers harboring common EGFR mutations respond to EGFR tyrosine kinase inhibitors (TKI), whereas exon 20 insertions (Ins20) are resistant to them. However, little is known about mutations in exon 18. Experimental Design: Mutational status of lung cancers between 2001 and 2015 was reviewed. Three representative mutations in exon 18, G719A, E709K, and exon 18 deletion (Del18: delE709_T710insD) were retrovirally introduced into Ba/F3 and NIH/3T3 cells. The 90% inhibitory concentrations (IC90s) of first-generation (1G; gefitinib and erlotinib), second-generation (2G; afatinib, dacomitinib, and neratinib), and third-generation TKIs (3G; AZD9291 and CO1686) were determined. Results: Among 1,402 EGFR mutations, Del19, L858R, and Ins20 were detected in 40%, 47%, and 4%, respectively. Exon 18 mutations, including G719X, E709X, and Del18, were present in 3.2%. Transfected Ba/F3 cells grew in the absence of IL3, and NIH/3T3 cells formed foci with marked pile-up, indicating their oncogenic abilities. IC90s of 1G and 3G TKIs in G719A, E709K, and Del18 were much higher than those in Del19 (by >11–50-fold), whereas IC90s of afatinib were only 3- to 7-fold greater than those for Del19. Notably, cells transfected with G719A and E709K exhibited higher sensitivity to neratinib (by 5–25-fold) than those expressing Del19. Patients with lung cancers harboring G719X exhibited higher response rate to afatinib or neratinib (∼80%) than to 1G TKIs (35%–56%) by compilation of data in the literature. Conclusions: Lung cancers harboring exon 18 mutations should not be overlooked in clinical practice. These cases can be best treated with afatinib or neratinib, although the currently available in vitro diagnostic kits cannot detect all exon 18 mutations. Clin Cancer Res; 21(23); 5305–13. ©2015 AACR.

Oncogene swap as a novel mechanism of acquired resistance to epidermal growth factor receptor‐tyrosine kinase inhibitor in lung cancer

Mutant selective epidermal growth factor receptor‐tyrosine kinase inhibitors (EGFR‐TKIs), such as rociletinib and AZD9291, are effective for tumors with T790M secondary mutation that become refractory to first‐generation EGFR‐TKI. However, acquired resistance to these prospective drugs is anticipated considering the high adaptability of cancer cells and the mechanisms remain largely obscure. Here, CNX‐2006 (tool compound of rociletinib) resistant sublines were established by chronic exposure of HCC827EPR cells harboring exon 19 deletion and T790M to CNX‐2006. Through the analyses of these resistant subclones, we identified two resistant mechanisms accompanied by MET amplification. One was bypass signaling by MET amplification in addition to T790M, which was inhibited by the combination of CNX‐2006 and MET‐TKI. Another was loss of amplified EGFR mutant allele including T790M while acquiring MET amplification. Interestingly, MET‐TKI alone was able to overcome this resistance, suggesting that oncogenic dependence completely shifted from EGFR to MET. We propose describing this phenomenon as an “oncogene swap.” Furthermore, we analyzed multiple lesions from a patient who died of acquired resistance to gefitinib, then found a clinical example of an oncogene swap in which the EGFR mutation was lost and a MET gene copy was gained. In conclusion, an “oncogene swap” from EGFR to MET is a novel resistant mechanism to the EGFR‐TKI. This novel mechanism should be considered in order to avoid futile inhibition of the original oncogene.

Effect of dasatinib on EMT-mediated-mechanism of resistance against EGFR inhibitors in lung cancer cells

OBJECTIVE The epithelial to mesenchymal transition (EMT) is associated with acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in certain non-small cell lung cancers that harbor EGFR mutations. Because no currently available drugs specifically kill cancer cells via EMT, novel treatment strategies that overcome or prevent EMT are needed. A recent report suggested that dasatinib (an ABL/Src kinase inhibitor) inhibits EMT induced by transforming growth factor (TGF)-beta in lung cancer cells (Wilson et al., 2014). In this study, we analyzed effects of dasatinib on the resistance mechanism in HCC4006 cells, which tend to acquire resistance to EGFR-TKIs via EMT. MATERIALS AND METHODS Sensitivity to dasatinib in HCC4006 and HCC4006 erlotinib-resistant (ER) cells with an EMT phenotype was analyzed. HCC4006 cells acquired resistance against the combination of erlotinib and dasatinib (HCC4006EDR) following chronic treatment with these drugs. The expression of EMT markers and the resistance mechanism were analyzed. RESULTS Short-term or long-term treatment with dasatinib did not reverse EMT in HCC4006ER. In contrast, HCC4006EDR cells maintained an epithelial phenotype, and the mechanism underlying resistance to erlotinib plus dasatinib combination therapy was attributable to a T790M secondary mutation. HCC4006EDR cells, but not HCC4006ER cells, were highly sensitive to a third-generation EGFR-TKI, osimertinib. CONCLUSIONS Although dasatinib monotherapy did not reverse EMT in HCC4006ER cells, preemptive combination treatment with erlotinib and dasatinib prevented the emergence of acquired resistance via EMT, and led to the emergence of T790M. Our results indicate that preemptive combination therapy may be a promising strategy to prevent the emergence of EMT-mediated resistance.

Characterization of EGFR T790M, L792F, and C797S mutations as mechanisms of acquired resistance to afatinib in lung cancer

Lung cancers harboring common EGFR mutations respond to EGFR tyrosine kinase inhibitors (TKI). We previously reported that tumors with exon 18 mutations are particularly sensitive to irreversible second-generation (2G) afatinib compared with first-generation TKIs (1G-TKI). However, data on the mechanisms of acquired resistance to afatinib are limited. We established afatinib-resistant cells by transfecting Ba/F3 cells with common or exon 18 (G719A and Del18) mutations and subjecting them to chronic exposure to increasing concentrations of afatinib. Afatinib-resistant clones were separately established through N-ethyl-N-nitrosourea (ENU) mutagenesis and exposure to fixed concentrations of afatinib. Rebiopsy samples from patients whose tumors acquired resistance to afatinib were analyzed. Afatinib-resistant cells with Del19, L858R, or G719A developed T790M, whereas those with Del18 acquired novel L792F mutation. ENU mutagenesis screening established 84 afatinib-resistant clones. All Del19 clones and most of the other clones acquired only T790M. However, C797S occurred in subsets of L858R, G719A, and Del18 clones. In addition, subsets of Del18 clones acquired L792F. C797S-acquired cells were sensitive to 1G erlotinib. L792F demonstrated intermediate resistance between T790M and C797S to both 1G- and 3G-TKIs, whereas L792F was the least resistant to 2G-TKIs, particularly dacomitinib. Chronic exposure of Del18 + L792F cells to dacomitinib induced additional T790M. T790M was detected in one of four clinical samples. In conclusion, L792F and C797S, in addition to the major T790M, can develop in afatinib-resistant cells particularly using a low dose of afatinib, and these minor mutations appear to exhibit sensitivity to dacomitinib and erlotinib, respectively. These secondary mutations should be tested in clinical practice. Mol Cancer Ther; 16(2); 357–64. ©2016 AACR. See related article by Talbert et al., p. 344

Recent evidence, advances, and current practices in surgical treatment of lung cancer

In the last 10-15 years, strategies and modalities of lung cancer treatment have changed dramatically. Meanwhile, the treatment objectives, the lung cancers themselves, have also changed, probably owing to early detection by computed tomography and aging of the population. In particular, the proportions of smaller lung cancers, lung adenocarcinomas with ground-glass opacity, and lung cancers in older patients are increasing. Along with these changes, surgeons have innovated and evaluated novel procedures for pulmonary resection. These include the application of minimally invasive surgical techniques, such as video-assisted thoracoscopic surgery (VATS) and robotic surgery, and sub-lobar resection, such as wedge resection and segmentectomy, for small peripheral lung cancers. Currently, VATS has gained wide acceptance and several institutions in Japan have started using robotic surgery for lung cancers. Two important clinical trials of sub-lobar resection for small peripheral lung cancers are now underway in Japan. In addition, surgery itself is of growing importance in lung cancer treatment. In particular, recent evidence supports the use of surgery in strictly selected patients with locally advanced disease, lung cancers with N2 lymph node metastases, small cell lung cancers, recurrent oligo-metastasis after pulmonary resection, or relapsed tumors after drug treatment. Surgical treatment also provides abundant tumor samples for molecular analysis, which can be used for drug selection in the adjuvant setting or after disease relapse. In the era of personalized treatment, surgery is still one of the most important treatment modalities to combat lung cancer.

A clinicopathological and immunohistological re-evaluation of adenosquamous carcinoma of the lung

Since the World Health Organization histological criteria were published in 1999, several studies have focused on adenosquamous carcinoma of the lung. Therefore, we aimed to clinicopathologically re‐evaluate this tumor using immunohistochemical methods. In our hospital, there have been 21 surgically resected adenosquamous carcinomas. The frequency of adenosquamous carcinoma was 1.9% and the clinical data including the patient prognosis data obtained in this study were similar to those reported previously. A fluorodeoxyglucose positron emission tomography study first revealed that the median maximum standardized uptake value of adenosquamous carcinoma was 9.3 and ranged from 2.0 to 24.5. According to the results of immunohistochemical staining for thyroid transcription factor‐1 (TTF‐1) and p63, adenosquamous carcinomas were divided into four subgroups: group 1, TTF‐1+ and p63+ (10 cases); group 2, TTF‐1‐ and p63+ (six cases); group 3, TTF‐1+ and p63‐ (three cases); and group 4, TTF‐1‐ and p63‐ (two cases). Of the six group 2 tumors, three were composed of unique solid nests with mucin‐filled cysts and showed characteristic p63 expression, which might suggest a special type of adenosquamous carcinoma. Immunohistochemical analysis of TTF‐1 and p63 expression shows that adenosquamous carcinoma is composed of diverse tumor groups, for which the biological and histogenetic nature further needs to be clarified.

Small non‐mucinous bronchioloalveolar carcinoma with anaplastic lymphoma kinase immunoreactivity: A novel ALK fusion gene?

Echinoderm microtubule‐associated protein‐like 4 and anaplastic lymphoma kinase (EML4–ALK) and kinesin family member 5B (KIF5B)–ALK are newly identified transforming fusion oncogenes causing non‐small‐cell lung cancers. These molecular abnormalities have become detectable using not only molecular biological methods, but also highly sensitive immunohistochemistry. During the immunohistochemical study of ALK expression in adenocarcinoma of the lung, we unexpectedly discovered that a small bronchioloalveolar carcinoma (BAC) showed strong ALK immunoreactivity. However, FISH studies failed to reveal EML4–ALK and KIF5B–ALK fusion genes in this BAC. These findings suggest the possibility that a novel or unknown ALK fusion gene plays a crucial role in BAC development. (Cancer Sci 2012; 103: 390–392)

Heterogeneity of EGFR Aberrations and Correlation with Histological Structures: Analyses of Therapy-Naive Isogenic Lung Cancer Lesions with EGFR Mutation

Introduction: EGFR gene somatic mutation is reportedly homogeneous. However, there are few data regarding the heterogeneity of expression of mutant EGFR protein and EGFR gene copy number, especially in extrathoracic lesions. These types of data may enhance our understanding of the biology of EGFR‐mutated lung cancer and our understanding of the heterogeneous response patterns to EGFR TKIs. Methods: An 81‐year‐old never‐smoking female with lung adenocarcinoma could not receive any systemic therapy because of her poor performance status. After her death, 15 tumor specimens from different sites were obtained by autopsy. Expression of mutant EGFR protein and EGFR gene copy numbers were assessed by immunohistochemical analysis and by silver in situ hybridization, respectively. Heterogeneity in these EGFR aberrations was compared between metastatic sites (distant versus lymph node) or histological structures (micropapillary versus nonmicropapillary). Results: All lesions showed positive staining for mutant EGFR protein, except for 40% of the papillary component in one of the pulmonary metastases (weak staining below the 1+ threshold). Expression of mutant‐specific EGFR protein, evaluated by H‐score, was significantly higher in the micropapillary components than in the nonmicropapillary components (Mann‐Whitney U test, p = 0.014). EGFR gene copy number was quite different between lesions but not correlated with histological structure or metastatic form. However, EGFR gene copy numbers were similar between histological structures in each lesion. Conclusion: These data indicate that expression of EGFR mutant protein and EGFR gene copy number do not change as a consequence of tumor progression. This also justifies using the biopsy specimens from metastases as a surrogate for primary tumors.

Pulmonary adenosquamous carcinoma with mucoepidermoid carcinoma-like component with characteristic p63 staining pattern: Either a novel subtype originating from bronchial epithelium or variant mucoepidermoid carcinoma

BACKGROUND Our previous study found unique adenosquamous carcinomas (ADSQs) containing a mucoepidermoid carcinoma (MEC)-like component and a characteristic p63 staining pattern. This study focused on these unique ADSQs. METHODS Thirty ADSQ cases were studied histologically and by immunohistochemistry for TTF-1 and p63. Of these 30 ADSQs, eight were selected as unique ADSQs. The clinicopathological characteristics of these ADSQs were further studied, and the gene rearrangement of mammalian mastermind-like 2 (MAML2) was investigated by fluorescence in situ hybridization (FISH) for differentiation from pulmonary MEC. RESULTS The clinicopathological characteristics between the eight ADSQs and the other ADSQ cases showed no statistically significant differences, except for serum CEA level. Histologically, the eight ADSQs contained varying degrees of the MEC-like component, which consisted of solid nests with mucin-filled cysts or a cribriform-like structure. Immunohistochemically, p63-positive nuclei characteristically encircled the tumor nests, although TTF-1 was completely negative. All unique ADSQs not only had a variable degree of squamous cell carcinoma component in addition to the MEC-like component, but also contained a small tubular adenocarcinoma component in three tumors. FISH analysis revealed no MAML2 gene rearrangement in the eight ADSQs. CONCLUSIONS Of the 30 ADSQs investigated in this study, eight contained a MEC-like component with a characteristic p63 basilar staining pattern similar to that of bronchial basal cells. These unique ADSQs shared clinical characteristics with ordinary ADSQs, but clinicopathologically differed from pulmonary ordinary MEC. Therefore, these unique ADSQs may be either a novel ADSQ subtype originating from bronchial epithelium or variant-type MEC.