Sehui Kim

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.

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.

Pan-Cancer Immunogenomic Perspective on the Tumor Microenvironment Based on PD-L1 and CD8 T-Cell Infiltration

Purpose: There is currently no reliable biomarker to predict who would benefit from anti-PD-1/PD-L1 inhibitors. We comprehensively analyzed the immunogenomic properties in The Cancer Genome Atlas (TCGA) according to the classification of tumor into four groups based on PD-L1 status and tumor-infiltrating lymphocyte recruitment (TIL), a combination that has been suggested to be a theoretically reliable biomarker of anti-PD-1/PD-L1 inhibitors. Experimental Design: The RNA expression levels of PD-L1 and CD8A in the samples in the pan-cancer database of TCGA (N = 9,677) were analyzed. Based on their median values, the samples were classified into four tumor microenvironment immune types (TMIT). The mutational profiles, PD-L1 amplification, and viral association of the samples were compared according to the four TMITs. Results: The proportions of TMIT I, defined by high PD-L1 and CD8A expression, were high in lung adenocarcinoma (67.1%) and kidney clear cell carcinoma (64.8%) among solid cancers. The number of somatic mutations and the proportion of microsatellite instable-high tumor in TMIT I were significantly higher than those in other TMITs, respectively (P < 0.001). PD-L1 amplification and oncogenic virus infection were significantly associated with TMIT I, respectively (P < 0.001). A multivariate analysis confirmed that the number of somatic mutations, PD-L1 amplification, and Epstein–Barr virus/human papillomavirus infection were independently associated with TMIT I. Conclusions: TMIT I is associated with a high mutational burden, PD-L1 amplification, and oncogenic viral infection. This integrative analysis highlights the importance of the assessment of both PD-L1 expression and TIL recruitment to predict responders to immune checkpoint inhibitors. Clin Cancer Res; 22(9); 2261–70. ©2016 AACR. See related commentary by Schalper et al., p. 2102

PD-L1 expression is associated with epithelial-mesenchymal transition in head and neck squamous cell carcinoma

Virus-associated malignancies and sarcomatoid cancers correlate with high PD-L1 expression, however, underlying mechanisms remain controversial. We evaluated the correlation between PD-L1 expression and epithelial-mesenchymal transition (EMT) in head and neck squamous cell carcinomas (HNSCC). Tumor tissues from 50 patients with HNSCC were evaluated for PD-L1 by immunohistochemistry, which showed 32 (64.0%) were PD-L1 positive (PD-L1+). Interestingly, PD-L1 expression was significantly associated with EMT (P = 0.010), as assessed by low E-cadherin and high vimentin expression. The overall survival of PD-L1+ patients with EMT features was significantly worse than those without EMT features (P = 0.007). In an independent validation cohort (N = 91), as well as in HNSCC cases of The Cancer Genome Atlas (TCGA) and the Cancer Cell Line Encyclopedia, high PD-L1 expression was also associated with the high probability of an EMT signature, referred from the GEO dataset, GSE4824. Survival analysis confirmed PD-L1+/EMT+ patients had a poorer prognosis than PD-L1+/EMT- patients in the TCGA cohort. PD-L1 positivity can thus be divided into two categories according to the absence or presence of EMT. PD-L1 expression is also independently associated with EMT features in HNSCC.

Clinicopathological analysis of programmed cell death 1 and programmed cell death ligand 1 expression in the tumour microenvironments of diffuse large B cell lymphomas

To investigate the clinicopathological characteristics of programmed cell death ligand 1 (PD‐L1) and programmed cell death 1 (PD‐1) expression in the tumour microenvironments of diffuse large B cell lymphoma (DLBCL).

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.

EML4-ALK enhances programmed cell death-ligand 1 expression in pulmonary adenocarcinoma via hypoxia-inducible factor (HIF)-1α and STAT3

ABSTRACT Programmed cell death (PD)-1/PD-1 ligand-1 (PD-L1)-targeted therapy has emerged as a promising therapeutic strategy for lung cancer. However, whether EML4-ALK regulates PD-L1 expression in lung cancer remains unknown. A total of 532 pulmonary adenocarcinomas (pADCs), including 58 ALK-translocated tumors, were immunohistochemically evaluated for PD-L1 and PD-1. H23 (EGFRWild-typeEML4-ALK−PD-L1Low) and H2228 (EGFRWild-typeEML4-ALK+PD-L1High) cells were transfected with EML4-ALK or ALK short interfering RNAs and used to investigate the alterations in PD-L1 expression. PD-L1 expression was detected in 81% of ALK-translocated pADCs; this value was significantly higher than those of pADCs with EGFR mutation, KRAS mutation or lacking ALK, EGFR or KRAS mutation (p <0.005 for all). Moreover, ALK-translocated pADC with PD-L1 expression showed significantly higher numbers of tumor-infiltrating PD-1+ cells. ALK knockdown or inhibition (crizotinib treatment) in H2228 cells downregulated PD-L1 expression. Transfection of H23 cells with EML4-ALK enhanced PD-L1 expression, which was compromised by crizotinib treatment. This ALK-dependent upregulation of PD-L1 expression was mediated by STAT3 and hypoxia-inducible factor (HIF)-1α under normoxia and hypoxia. Furthermore, EML4-ALK enhanced HIF-1α expression through increasing transcription and decreasing ubiquitination of HIF-1α. In ALK-translocated pADC tissues, significant positive correlations between PD-L1 and nuclear HIF-1α (p < 0.05) or pSTAT3 expression levels (p<0.005) were observed. Among patients with ALK-translocated pADC, strong PD-L1 expression was significantly associated with shorter progression-free (p = 0.001) and overall survival (p = 0.002) after crizotinib treatment. Collectively, our findings demonstrate that ALK-derived pADCs increase PD-L1 expression via HIF-1α and/or STAT3, thus providing a rationale for PD-1/PD-L1 pathway-targeted therapy in ALK-translocated lung cancer.

Prognostic implications of CD30 expression in extranodal natural killer/T-cell lymphoma according to treatment modalities

Abstract Extranodal natural killer/T-cell lymphoma (NKTCL) has aggressive behaviors and poor clinical outcomes. A monomethyl auristatin E-conjugated anti-CD30 antibody (brentuximab vedotin) was recently introduced to treat CD30-positive lymphomas. Thus we investigated the clinicopathological features and prognostic implications of CD30 expression in 72 patients with NKTCL. CD30-positive cases, defined as cases with CD30 expression in more than 1%, 5% and 25% of tumor cells as cut-off values (COVs), accounted for 40 (56%), 27 (38%) and 16 (22%) cases of NKTCL, respectively. CD30 expression was significantly higher in large/anaplastic cell-predominant NKTCL than in small/medium cell-predominant cases. CD30-positive NKTCL showed better responses to non-anthracycline-based therapy. CD30-positive NKTCL with COV of 25% showed a lower rate of relapse. Moreover, in patients treated with non-anthracycline-based chemotherapy, CD30 positivity with COV of 5% was significantly and independently associated with longer overall survival. CD30 may be useful as a prognostic factor and therapeutic target in NKTCL.

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.

MET exon 14 skipping mutation in triple-negative pulmonary adenocarcinomas and pleomorphic carcinomas: An analysis of intratumoral MET status heterogeneity and clinicopathological characteristics

OBJECTIVES MET mutations leading to exon 14 skipping rarely occur in non-small cell lung cancer (NSCLC). Recently, small molecule inhibitors targeting MET mutations showed clinical benefit. However, the clinicopathological characteristics of NSCLC harboring MET mutations, and the correlation among mutations, protein expression, and gene copy number of MET in NSCLC remain unclear. Therefore, we address these issues. MATERIALS AND METHODS MET exon 14 skipping mutations were evaluated using real-time quantitative reverse-transcription-PCR (qRT-PCR) in 102 triple-negative (i.e., EGFR mutation (-)/ALK translocation (-)/KRAS mutation (-)) pulmonary adenocarcinomas, and 45 pleomorphic carcinomas. MET mutation and gene copy were also examined in microdissected tissues obtained from tumor areas with heterogeneous MET immunohistochemical expression. RESULTS MET mutations were detected in 8.8% (9/102) of triple-negative adenocarcinomas and 20% (9/45) of pleomorphic carcinomas of the lung. Patients with MET-mutated adenocarcinomas was significantly older than those without MET mutations (P=0.015). The male to female and ever-to never-smoker ratios were 3:6 and 2:7, respectively, among patients with MET-mutated adenocarcinomas. All (9/9) of the MET-mutated adenocarcinomas showed acinar predominant histology with associated lepidic patterns. In contrast, the male to female and ever- to never-smoker ratios were 8:1 and 7:1, respectively, among patients with MET-mutated pleomorphic carcinomas. The carcinoma component of MET-mutated pleomorphic carcinomas was mostly adenocarcinoma of acinar pattern (8/9). MET mutation was detected by qRT-PCR in all samples with heterogeneous MET expression microdissected from five cases with MET-mutated adenocarcinoma, while MET gene amplification was detected in tumor areas expressing high MET protein levels among MET-mutated adenocarcinomas. CONCLUSION MET-mutated NSCLC is characterized by older age in patients with adenocarcinoma and by an acinar histology and variable MET expression in patients with adenocarcinoma and pleomorphic carcinomas. Moreover, MET gene amplification might occur in the tumor cells harboring the MET mutation.