Jaemoon Koh

Clinicopathological analysis of PD-L1 and PD-L2 expression in pulmonary squamous cell carcinoma: Comparison with tumor-infiltrating T cells and the status of oncogenic drivers.

PURPOSE Programmed cell death-1 (PD-1)/programmed cell death-ligand-1 (PD-L1) pathway-targeted immunotherapy has beneficial therapeutic effects in pulmonary squamous cell carcinoma (SqCC) patients. However, the expression patterns of PD-1 and PD-1 ligands (PD-Ls) in pulmonary SqCC remain unclear. Moreover, the association between the PD-1/PD-Ls pathway and the status of oncogenic drivers in pulmonary SqCC is unknown. METHODS PD-L1 and PD-L2 expression in tumor cells and the numbers of PD-1(+) and CD8(+) tumor-infiltrating lymphocytes (TILs) were examined in 331 resected SqCC tumors along with matched lymph node metastases from 77 cases using immunohistochemistry. EGFR and FGFR1 and MET expression and genetic status were also examined. RESULTS PD-L1 and PD-L2 expression was detected in 26.9% and 23.9% of the pulmonary SqCC samples, respectively. PD-L1 and PD-L2 expression was maintained or increased in the metastatic lymph node tumors in 81.1% and 93.5% of the 77 cases, respectively. The numbers of PD-1(+) and CD8(+) TILs were significantly positively correlated (P<0.001). Cases displaying high PD-L1 expression exhibited consistently high CD8(+) T cell infiltration (P<0.001), even in subgroup analyses according to age, smoking status, tumor size, lymph node metastasis, stage, and the EGFR, MET and FGFR1 status. Moreover, MET expression in the tumors was significantly correlated with high PD-L2 expression and increased PD-1(+) TILs (P=0.001 for both). Increased numbers of CD8(+) or PD-1(+) TILs were significantly associated with prolonged disease-free survival of these patients, whereas PD-L1 and PD-L2 expression had no significant prognostic implications. CONCLUSION PD-L1 and PD-L2 expression in pulmonary SqCC is associated with an increased number of CD8(+) TILs and increased MET expression, which might provide therapeutic insight into targeting the PD-1/PD-Ls pathway in pulmonary SqCC.

Clinicopathologic analysis of programmed cell death-1 and programmed cell death-ligand 1 and 2 expressions in pulmonary adenocarcinoma: comparison with histology and driver oncogenic alteration status

Immunotherapies targeting the programmed cell death-1/programmed cell death-ligand 1 pathway have emerged as promising therapeutic strategies for lung cancer. However, the expression pattern and prognostic implications of programmed cell death-ligand 1 and 2 and programmed cell death-1 in comparison with the histology and genetic alterations in pulmonary adenocarcinomas remains unclear and thus were addressed here. Programmed cell death-ligand 1 and 2 expression in tumor cells and the quantities of programmed cell death-1+ and CD8+ tumor-infiltrating lymphocytes were immunohistochemically evaluated in 497 resected pulmonary adenocarcinomas and analyzed according to clinicopathological and genetic statuses. Programmed cell death-ligand 1 and 2 expression were observed in 59% and 64% of pulmonary adenocarcinomas, respectively, and showed a strong positive correlation with each other (P<0.001). Programmed cell death-ligand 1 expression was higher in nodal metastasis cases (P=0.006), smokers (P=0.056), poorly differentiated tumors and histologic subtypes of solid and micropapillary patterns (P<0.001). There was no significant difference in programmed cell death-ligand 1 and 2 expression according to EGFR mutation status. However, programmed cell death-ligand 1 expression was correlated with ALK translocation (P=0.054) and expression of EGFR and MET (P<0.001). Meanwhile, programmed cell death-ligand 2 expression was correlated with ALK translocation (P=0.052), and expression of MET (P<0.001) and ERBB2 (P=0.013). The numbers of CD8+ and programmed cell death-1+ lymphocytes were higher in smokers (P=0.012 and 0.016) and MET-expressing adenocarcinomas (P<0.001). Patients expressing programmed cell death-ligand 1 and/or high ratios of programmed cell death-1+/CD8+ lymphocytes showed shorter disease-free survival (P=0.001). Our study demonstrated that programmed cell death-ligand 1 and 2 expression varied with histology, EGFR, ALK, MET, and ERBB2 statuses, and activation of the programmed cell death-1/programmed cell death-ligand 1 pathway may be a poor prognostic factor in pulmonary adenocarcinomas.

Concomitant ALK translocation and EGFR mutation in lung cancer: a comparison of direct sequencing and sensitive assays and the impact on responsiveness to tyrosine kinase inhibitor

BACKGROUND Epidermal growth factor receptor (EGFR) mutation and anaplastic lymphoma kinase (ALK) translocation are considered mutually exclusive in nonsmall-cell lung cancer (NSCLC). However, sporadic cases having concomitant EGFR and ALK alterations have been reported. The present study aimed to assess the prevalence of NSCLCs with concomitant EGFR and ALK alterations using mutation detection methods with different sensitivity and to propose an effective diagnostic and therapeutic strategy. PATIENTS AND METHODS A total of 1458 cases of lung cancer were screened for EGFR and ALK alterations by direct sequencing and flourescence in situ hybridization (FISH), respectively. For the 91 patients identified as having an ALK translocation, peptide nucleic acid (PNA)-clamping real-time PCR, targeted next-generation sequencing (NGS), and mutant-enriched NGS assays were carried out to detect EGFR mutation. RESULTS EGFR mutations and ALK translocations were observed in 42.4% (612/1445) and 6.3% (91/1445) of NSCLCs by direct sequencing and FISH, respectively. Concomitant EGFR and ALK alterations were detected in four cases, which accounted for 4.4% (4/91) of ALK-translocated NSCLCs. Additional analyses for EGFR using PNA real-time PCR and ultra-deep sequencing by NGS, mutant-enriched NGS increased the detection rate of concomitant EGFR and ALK alterations to 8.8% (8/91), 12.1% (11/91), and 15.4% (14/91) of ALK-translocated NSCLCs, respectively. Of the 14 patients, 3 who were treated with gefitinib showed poor response to gefitinib with stable disease in one and progressive disease in two patients. However, eight patients who received ALK inhibitor (crizotinib or ceritinib) showed good response, with response rate of 87.5% (7/8 with partial response) and durable progression-free survival. CONCLUSIONS A portion of NSCLC patients have concomitant EGFR and ALK alterations and the frequency of co-alteration detection increases when sensitive detection methods for EGFR mutation are applied. ALK inhibitors appear to be effective for patients with co-alterations.

Programmed death-1 ligand 1 and 2 are highly expressed in pleomorphic carcinomas of the lung: Comparison of sarcomatous and carcinomatous areas.

Pleomorphic carcinoma (PC) of the lung is a rare type of poorly differentiated non-small cell lung carcinoma (NSCLC) that belongs to sarcomatoid carcinoma (SC). It exhibits aggressive behaviour and resistance to chemotherapy and radiotherapy. Recently, immunotherapy targeting the programmed death-1 (PD-1)/PD ligand 1 (PD-L1) pathway has demonstrated favourable clinical outcomes in NSCLC. However, the expression patterns of PD-1-related molecules in pulmonary PC remain elusive. PD-L1 and PD-L2 expression was estimated in 41 cases of PC using immunohistochemistry. CD8(+) and PD-1(+) tumour-infiltrating lymphocytes (TILs) were also evaluated. PD-L1 and PD-L2 were highly expressed in pulmonary PCs (90.2% [37/41)]; 87.8% [36/41]). The amount of CD8(+) or PD-1(+) TILs and the ratio of PD-1(+)/CD8(+) TILs in PC were higher in males, smokers and older patients. PD-L1-positive PCs were infiltrated by higher numbers of CD8(+) TILs compared to PD-L1-negative cases (P=0.006). Of note, PD-L1 expression in pulmonary PCs was significantly higher in sarcomatous areas than in the carcinomatous portion (P=0.006). PC patients with a high ratio of PD-1(+)/CD8(+) TILs showed a shorter progression-free survival (P=0.036), whereas PD-L1 and PD-L2 expression had no prognostic implications. Our study demonstrates that pulmonary PCs very frequently express PD-L1 and PD-L2. Moreover, their expression is higher in sarcomatous cells than in carcinomatous areas. Thus, targeting the PD-1/PD-L1 pathway may represent a potential therapeutic candidate for this aggressive tumour.

PD-L1 expression is associated with epithelial-to-mesenchymal transition in adenocarcinoma of the lung.

PD-1/PD-L1-targeted immunotherapy has emerged as a promising therapeutic strategy for pulmonary adenocarcinoma (pADC). Epithelial-to-mesenchymal transition (EMT) is involved in the progression and immune evasion of cancers. Therefore, we investigated the association between PD-L1 expression and EMT phenotype in pADC. Immunohistochemistry for E-cadherin (epithelial marker), ZEB1, SNAIL, SLUG, vimentin (mesenchymal markers), PD-L1, CD8, and PD-1 was performed on 477 cases of pADC. Cases were classified into epithelial, mesenchymal, epithelial-mesenchymal, and unspecified types based on immunohistochemical results. PD-L1 expression was scored as 0 in 14.0% (n=67), 1 in 26.4% (n=126), 2 in 51.2% (n=244), and 3 in 8.4% (n=40). PD-L1 score was positively correlated with SNAIL and vimentin H scores (P<.001, both). After dichotomizing patients into PD-L1-negative and PD-L1-positive groups, PD-L1 positivity was significantly higher in patients with mesenchymal (71.2%; 84/118) and epithelial-mesenchymal (62.7%; 84/134) phenotypes compared with those with epithelial (50.6%; 44/87) and unspecified (50.0%; 35/70) phenotypes (P=.005). The significant association between PD-L1 expression and EMT phenotype was maintained in EGFR-mutated pADCs. Moreover, cases with EMT phenotype (ie, mesenchymal and epithelial-mesenchymal) were infiltrated by higher numbers of CD8+ and PD-1+ cells than those with epithelial and unspecified phenotypes in EGFR-mutated pADCs (P=.043 for CD8+ cells and P<.001 for PD-l+ cells). Particularly, cases with EMT phenotype and PD-L1 expression showed the greatest amount of CD8+ and PD-1+ cells in EGFR-mutated cases (P=.043 for CD8+ cells and P=.005 for PD-1+ cells). This study demonstrates that EMT phenotype is related to PD-L1 overexpression in pADC cells and patients with EMT-phenotype pADC may benefit from PD-1/PD-L1-blocking immunotherapy.

Prognostic implications of intratumoral CD103+ tumor-infiltrating lymphocytes in pulmonary squamous cell carcinoma

CD103 is the αE subunit of αEβ7 integrin that is expressed in tissue-resident memory T cells, where it promotes cytotoxic T cell responses against tumors. However, little is known about its expression or clinicopathological implications in non-small cell lung cancer (NSCLC). This study investigated the prognostic implications of CD103+ tumor-infiltrating lymphocytes (TILs) in NSCLC. We established two cohorts: patients with resected NSCLC (n = 132) and patients with pulmonary squamous cell carcinoma (pSCC), a subset of NSCLC (n = 378), to estimate the prognostic significance of CD103+ TILs. The numbers of CD103+ TILs in the intratumoral (i.e., intraepithelial) and stromal regions of NSCLC were estimated using immunohistochemistry and automated image analysis. In the NSCLC cohort, high numbers of intratumoral CD103+ TILs were significantly associated with prolonged disease-free survival (DFS) and overall survival (OS) in patients with pSCC but not in those with pulmonary adenocarcinoma. In the pSCC cohort, a positive correlation was observed between the numbers of intratumoral CD103+ and CD8+ TILs (correlation coefficient = 0.736, P < 0.001). The ratio of intratumoral/stromal CD103+ TILs was higher in pSCC with high compared to low E-cadherin expression (P = 0.021). According to Kaplan-Meier analysis, high intratumoral but not stromal CD103+ TILs were associated with prolonged DFS and OS in patients with resected pSCC (P = 0.021 and 0.002, respectively). Multivariate analysis revealed that a high number of intratumoral CD103+ TILs is an independent predictor of a more favorable DFS (P = 0.021). Thus, a high number of intratumoral CD103+ TILs is a favorable prognostic indicator in patients with pSCC.

Napsin A is a useful marker for metastatic adenocarcinomas of pulmonary origin

To address whether napsin A is useful for identifying metastatic adenocarcinomas of pulmonary origin.

A comprehensive immunohistochemistry algorithm for the histological subtyping of small biopsies obtained from non‐small cell lung cancers

Need for accurate histologic subtyping of non‐small cell lung carcinomas (NSCLCs) is growing. IHC patterns may be ambiguous in some cases, rendering it difficult to determine subtypes.

High ratio of programmed cell death protein 1 (PD-1)+/CD8+ tumor-infiltrating lymphocytes identifies a poor prognostic subset of extrahepatic bile duct cancer undergoing surgery plus adjuvant chemoradiotherapy

BACKGROUND AND PURPOSE This study investigated the prognostic role of PD-L1 expression, PD-1(+) tumor-infiltrating lymphocytes (TILs), and the ratio of PD-1(+)/CD8(+) TILs in extrahepatic bile duct (EHBD) cancer. MATERIALS AND METHODS We analyzed 83 patients with EHBD cancer who underwent curative surgery plus fluoropyrimidine-based chemoradiotherapy (CRT). Expressions of PD-L1, PD-1, and CD8 were assessed by immunohistochemistry. RESULTS Fifty-six (68%) patients were PD-L1-positive, and its lower expression level was associated with hilar tumor location (P=0.044). A higher ratio of PD-1(+)/CD8(+) TILs was associated with poorer overall survival (OS) (P=0.032), relapse-free survival (RFS) (P=0.024), and distant metastasis-free survival (DMFS) (P=0.039) in Kaplan-Meier analyses, but survival differences were not observed according to the PD-L1 expression level. With Cox proportional hazards models, the ratio of PD-1(+)/CD8(+) TILs was the independent prognostic factor in OS (HR 2.47, 95% CI 1.04-5.86), RFS (HR 2.41, 95% CI 1.08-5.41), and DMFS (HR 2.67, 95% CI 1.00-7.11) after adjusting for other significant clinicopathologic variables. CONCLUSION A strong survival impact of the ratio of PD-1(+)/CD8(+) TILs was observed in EHBD cancer. In the poor prognostic subgroup, the blockade of the immune checkpoint in combination with conventional multimodality treatment needs to be considered.

Pellino-1 promotes lung carcinogenesis via the stabilization of Slug and Snail through K63-mediated polyubiquitination

Pellino-1 is an E3 ubiquitin ligase acting as a critical mediator for a variety of immune receptor signaling pathways, including Toll-like receptors, interleukin-1 receptor and T-cell receptors. We recently showed that the Pellino-1-transgenic (Tg) mice developed multiple tumors with different subtypes in hematolymphoid and solid organs. However, the molecular mechanism underlying the oncogenic role of Pellino-1 in solid tumors remains unknown. Pellino-1-Tg mice developed adenocarcinoma in the lungs, and Pellino-1 expression was higher in human lung adenocarcinoma cell lines compared with non-neoplastic bronchial epithelial cell lines. Pellino-1 overexpression increased the cell proliferation, survival, colony formation, invasion and migration of lung adenocarcinoma cells, whereas Pellino-1 knock-down showed the opposite effect. Pellino-1 overexpression activated PI3K/Akt and ERK signaling pathways and elicited an epithelial–mesenchymal transition (EMT) phenotype of lung adenocarcinoma cells. Pellino-1-mediated EMT was demonstrated through morphology, the upregulation of Vimentin, Slug and Snail expression and the downregulation of E-cadherin and β-catenin expression. Notably, Pellino-1 had a direct effect on the overexpression of Snail and Slug through Lys63-mediated polyubiquitination and the subsequent stabilization of these proteins. Pellino-1 expression level was significantly correlated with Snail and Slug expression in human lung adenocarcinoma tissues, and lung tumors from Pellino-1-Tg mice showed Snail and Slug overexpression. The Pellino-1-mediated increase in the migration of lung adenocarcinoma cells was mediated by Snail and Slug expression. Taken together, these results show that Pellino-1 contributes to lung tumorigenesis by inducing overexpression of Snail and Slug and promoting EMT. Pellino-1 might be a potential therapeutic target for lung cancer.