Deciphering the immunosuppressive tumor microenvironment in ALK- and EGFR-positive lung adenocarcinoma.

The advent of immune checkpoint blockade (ICB) has led to significantly improved disease outcome in lung adenocarcinoma (ADC), but response of ALK/EGFR-positive tumors to immune therapy is limited. The underlying immune biology is incompletely understood. We performed comparative mRNA expression profiling of 31 ALK-positive, 40 EGFR-positive and 43 ALK/EGFR-negative lung ADC focused on immune gene expression. The presence and levels of tumor infiltration lymphocytes (TILs) as well as fourteen specific immune cell populations were estimated from the gene expression profiles. While total TILs were not lower in ALK-positive and EGFR-positive tumors compared to ALK/EGFR-negative tumors, specific immunosuppressive characteristics were detected in both subgroups: In ALK-positive tumors, regulatory T cells were significantly higher compared to EGFR-positive (fold change: FC = 1.9, p = 0.0013) and ALK/EGFR-negative tumors (FC = 2.1, p = 0.00047). In EGFR-positive tumors, cytotoxic cells were significantly lower compared to ALK-positive (FC =  - 1.7, p = 0.016) and to ALK/EGFR-negative tumors (FC =  - 2.1, p = 2.0E-05). A total number of 289 genes, 40 part of cytokine-cytokine receptor signaling, were differentially expressed between the three subgroups. Among the latter, five genes were differently expressed in both ALK-positive and EGFR-positive tumors, while twelve genes showed differential expression solely in ALK-positive tumors and eleven genes solely in EGFR-positive tumors. Targeted gene expression profiling is a promising tool to read out tumor microenvironment characteristics from routine diagnostic lung cancer biopsies. Significant immune reactivity including specific immunosuppressive characteristics in ALK- and EGFR-positive lung ADC, but not a total absence of immune infiltration supports further clinical evaluation of immune-modulators as partners of ICB in such tumors.

Budczies J ，Kirchner M ，Kluck K ，Kazdal D ，Glade J ，Allgäuer M ，Kriegsmann M ，Heußel CP ，Herth FJ ，Winter H ，Meister M ，Muley T ，Goldmann T ，Fröhling S ，Wermke M ，Waller CF ，Tufman A ，Reck M ，Peters S ，Schirmacher P ，Thomas M ，Christopoulos P ，Stenzinger A ... -  《-》

PD-L1 expression and T cells infiltration in patients with uncommon EGFR-mutant non-small cell lung cancer and the response to immunotherapy.

The efficacy of immunotherapy in epidermal growth factor receptor (EGFR)-activating non-small cell lung cancer (NSCLC) is limited. However, a series of patients with uncommon EGFR alterations was reported to derive clinical benefit from PD-1 blockade. To characterize the tumor immune microenvironment, we retrospectively evaluated tumor PD-L1 expression and T cells infiltration among NSCLC patients with uncommon EGFR mutations. Immunohistochemistry was used to analyze the expression of PD-L1 and the abundance of CD4+ and CD8+ tumor-infiltrating lymphocytes (TILs). Chi-square test and Cox proportional hazards regression were conducted to investigate the correlations between the immune microenvironment features and clinicopathological variables and survival, as well as to explore the potential of immunotherapy in this patient population. Among 600 NSCLC patients with EGFR alterations, we identified 49 (8.2 %) bearing uncommon mutations, including G719X, L861Q, S768I, and Ex20 ins. In total, 49.0 % (24/49) of these patients showed positive PD-L1 expression in tumor cells, markedly higher than the proportion in patients with classic sensitive mutations (19del and L858R, 12.2 % [67/551], P < 0.05). Furthermore, PD-L1 expression was associated with relatively short overall survival (OS; 15.2 vs 29.3 months, P = 0.006) and was identified as an independent predictor of OS (hazard ratio=2.57, 95 % confidence interval: 1.03-7.12, P = 0.045). Additionally, PD-L1 positivity was predominantly observed among tumors with CD8+ TILs infiltration (P = 0.001). Uncommon EGFR-mutant tumors had a high frequency (36.7 %) of concurrent PD-L1 expression and abundant CD8 + TILs infiltration. Moreover, this dual-positive group exhibited the most unfavorable prognosis (P = 0.023). Notably, patients with PD-L1 and CD8 dual positivity showed a favorable response to PD-1 inhibitors. High rates of concomitant PD-L1 expression and CD8 + TILs within the tumor microenvironment were observed in NSCLC patients with uncommon EGFR mutations. Further investigations are needed to confirm the therapeutic sensitivity to PD-1 blockade in this subgroup.

Chen K ，Cheng G ，Zhang F ，Zhu G ，Xu Y ，Yu X ，Huang Z ，Fan Y ... -  《-》

Genomic profiles and their associations with TMB, PD-L1 expression, and immune cell infiltration landscapes in synchronous multiple primary lung cancers.

Diagnosing and treating patients with multiple primary lung cancers (MPLCs) bring challenges to the clinic, and the preliminary evidence has revealed unsatisfying outcomes after targeted therapy and immunotherapy. Therefore, we surveyed genomic profiles of MPLCs and their possible associations with tumor mutation burden (TMB), programmed death-ligand 1 (PD-L1), and the immune cell infiltration landscape. A total of 112 patients with MPLCs with surgically resected 294 tumors were eligible, and 255 tumors were sequenced using a 1021-gene panel. Immunohistochemistry staining was performed to evaluate the levels of PD-L1 and the density of CD3+/CD8+ tumor-infiltrating lymphocytes (TILs), and CD68+/CD163+ tumor-associated macrophages (TAMs) at the central tumor and invasive margin, and immunotypes were generated based on those variables. MPLCs often occur simultaneously in non-smoker women younger than 60 years and manifest as ground-glass opacities, adenocarcinoma, and stage I lung lesions. The most frequently mutated genes in the 255 tumors were EGFR (56%), ERBB2 (12%), TP53 (12%), BRAF (11%), RBM10 (11%), and KRAS (9%). We found 87 (77.7%) patients with diverse genomic profiles, and 61 (54.5%) who shared at least one putative driver gene between different tumors presented more aggressive tumors. The median TMB was 1.92 mutations/Mb, and high-TMB (≥3) lesions often harbored EGFRL858R/KRASG12C/RBM10/TP53/LRP1B mutations or wild-type ERBB2. Only 8.1% of patients and 3.9% of lesions were positive for PD-L1 on tumor cells, and this positivity was more frequent in LRP1B/TP53-mutant tumors. EGFRL858R/RBM10/TP53 mutations were positively associated with specific immune cells and an inflamed immunotype, but ERBB2 mutations were negatively correlated. TMB, CD3+TILs, and CD68+/CD163+ TAMs presented with significant heterogeneity among paired tumors (all kappa <0.2), but PD-L1 and CD8 +TILs were more uniformly present in tumor pairs. MPLCs are driven by different molecular events and often exhibit low TMB, low PD-L1, and a heterogeneous immune infiltration landscape. Specific genomic profiles are associated with TMB and the tumor immune microenvironmental landscape in MPLCs. Our findings can help to guide MPLCs diagnoses and to identify patient populations that may benefit from immunotherapy and targeted therapy.

Hu C ，Zhao L ，Liu W ，Fan S ，Liu J ，Liu Y ，Liu X ，Shu L ，Liu X ，Liu P ，Deng C ，Qiu Z ，Chen C ，Jiang Y ，Liang Q ，Yang L ，Shao Y ，He Q ，Yu D ，Zeng Y ，Li Y ，Pan Y ，Zhang S ，Shi S ，Peng Y ，Wu F ... -  《Journal for ImmunoTherapy of Cancer》

Immune Marker Profiling and Programmed Death Ligand 1 Expression Across NSCLC Mutations.

Programmed death 1/programmed death ligand 1 (PD-L1) axis inhibitors have been proven effective, especially in patients with tumors expressing PD-L1. Their clinical efficacy in patients with EGFR-activating mutations is still unclear, whereas KRAS mutations seem to be associated with good response. We used multiplexed quantitative immunofluorescence to investigate PD-L1 expression and to characterize tumor infiltrating lymphocyte (TIL) populations and their activation status in more than 150 NSCLC patients with known mutation status. PD-L1 expression was significantly lower in EGFR-mutant compared to KRAS-mutant, and EGFR/KRAS wild-type (WT) tumors. KRAS mutant tumors were more inflamed with higher CD4+, CD8+ and CD20+ TILs. Subgroup analysis by TIL activation status revealed that EGFR mutants had a high frequency of inactive TILs even though lymphocytes were present in the tumor microenvironment. In contrast, in KRAS mutants, when TILs were present they were almost always active. Additionally, we found differences between EGFR mutation sites in CD8+ expression and the TIL activation profile. Finally, activated EGFR correlated with increased PD-L1 expression in EGFR mutants but not in EGFR WT, whereas TIL activation was associated with higher PD-L1 only in EGFR/KRAS WT. Our findings show the unique immune profile of EGFR-mutant tumors. The high frequency of inactive TILs could explain the low immunotherapy response rates in these patients, whereas PD-L1 as a predictive biomarker may reflect the constitutive oncogenic signaling rather than immune signaling, which would be associated with high PD-L1 levels and TILs activation.

Toki MI ，Mani N ，Smithy JW ，Liu Y ，Altan M ，Wasserman B ，Tuktamyshov R ，Schalper K ，Syrigos KN ，Rimm DL ... -  《-》

Differences in Immunological Landscape between EGFR-Mutated and Wild-Type Lung Adenocarcinoma.

Recent clinical trials of lung adenocarcinoma with immune checkpoint inhibitors revealed that lung adenocarcinoma patients with EGFR mutations have a poor response to immunotherapy. However, the mechanisms have not been addressed. We performed immunohistochemistry analyses of resected lung adenocarcinoma tissues with and without EGFR mutations to investigate and compare the characteristics of the tumor microenvironment (TME). We retrospectively enrolled a total of 323 lung adenocarcinoma patients (164 had EGFR mutations), and their corresponding tissue samples were analyzed by the EGFR mutation test and immunohistochemistry. We selected the markers of the immune checkpoint molecule (PD1, PD-L1, and LAG-3) and immune cell (CD3, CD4, CD8, and Foxp3) as markers of the tumor microenvironment. Our results revealed that patients had a distinct tumor microenvironment between EGFR-mutant and wild-type lung adenocarcinomas; the expression of CD3, CD4, PD-L1, and Foxp3 in EGFR-mutant tumors was significantly higher than that in wild-type tumors, while the expression of LAG3 and PD-1 showed a positive correlation with EGFR-wild-type tumors. In survival analysis, EGFR-wild-type patients had longer disease-free survival (DFS) than EGFR-mutant patients (P = 0.0065). Our research demonstrates significant differences in tumor microenvironment composition between EGFR-mutant and wild-type patients. Our findings provide novel evidence that contributes to understanding the mechanism underlying the poor efficacy of immune checkpoint inhibitors.

Luo JW ，Guo YH ，Wu FY ，Li XF ，Sun XC ，Wang JL ，Zhou CC ... -  《-》