As heterogenous diseases, either at cellular and histopathological perspective, with distinct diagnostic, prognostic and therapeutic features , ADC and SQC keep being the most prevalent bronchopulmonary carcinomas, responsible for approximately 50% and 30% of cases, respectively .
With 5-year survival rate still under 20% , near 30% of patients with tumors in non-surgical stages have mutations amenable to targeted therapy , and PD-1/PD-L1 inhibitors have been prolonging patients survival with acceptable toxicity, proving undoubted superiority over chemotherapy and targeted therapy in terms of efficacy [9, 28].
Durable host immune anti-neoplasm responses and long-term remissions of several tumor types proved favorable benefit-to-risk of anti-PD therapy [17, 29]. For advanced carcinomas without EGFR/ALK mutations, European Medicines Agency (EMA) and FDA approved pembrolizumab monotherapy after PD-L1 score ≥ 50% or in combination with pemetrexed and platinum chemotherapy in carcinomas other than SQCs as first-line treatment, and as monotherapy for ADCs with PD-L1 expression between 1% and 50% after at least one prior chemotherapy regimen. [5, 17, 27].
Relationship between PD-L1 expression and gender remains contradictory [30, 31]. Concerning a limited series, our findings demonstrated that PD-L1 expression was significantly associated with gender, as 48 of 56 positive PD-L1 cases belonged to male patients.
It is becoming evident that histopathological subtyping relates to PD-L1 expression in TCs, particularly among solid ADC subgroup, still with worse prognosis. Driver et al. and Mandarano et al. demonstrated that lung ADCs defined by PD-L1 expression in TCs and tumor-infiltrating immune cells correlated with solid pattern, while acinar, mucinous and papillary patterns presented lower PD-L1 expression in TCs [4, 32]. As in literature [26, 30], our study confirmed that positive PD-L1 expression was relevant in solid pattern of ADCs (21/23), defining a clear relationship, while among acinar and mucinous subtypes, less than 50% of cases showed PD-L1 positive TCs.
EGFR mutations and ALK rearrangements keep being rare in advanced lung SQC, while immunotherapeutic strategies have been particularly effective [33, 34]. In CheckMate 017 trial, nivolumab improved survival, PFS and response rate versus docetaxel . Following progression after first-line chemotherapy, PD-L1 inhibitors are the preferred treatment, according to U.S. National Comprehensive Cancer Network (NCCN) guidelines .
Cytokeratin 7, complementary gut differentiation marker present in normal glandular and transitional epithelium but not in squamous epithelium and expressed in 60-100% of ADCs [36, 37], is used to subclassify lung SQC into two groups: pure SQC (CK7-negative), and non-pure SQC with CK7 expression, according to WHO 2015/2020 criteria for lung tumors. In considered pure SQC subgroup, EGFR and ALK mutations are almost absent and targeted therapy is much more limited, while for non-pure SQCs, molecular pathology may define therapy .
In our preliminary results, 8 cases out of 13 CK7-negative SQCs expressed PD-L1, with 4 cases over 50% of positive TCs. This tendency to high PD-L1 expression in pure lung SQC cases (CK7-negative) needs to be further characterized in future for a more personalized application of PD-1/PD-L1 immune checkpoint inhibitors in so-called pure SQCs.
The applicability of tumor microenvironment (TME) as a diagnostic, prognostic or predictive biomarker in bronchopulmonary carcinomas seems to correlate with tumorigenesis, heterogeneity, resistance to immunotherapy and tumor progression [2, 39], and also stromal cells may express the ligand PD-L1, with still unclear meaning .
The tendency of high PD-L1 expression in TCs among cases with lymphocytic-predominant/immune-inflamed stroma was 9/10 samples. As in previous studies , induction of TCs PD-L1 expression by interferon-γ produced by T lymphocytes present in the TME corroborates our results. Tumor-infiltrating lymphocytes (TILs) have also been proposed as biomarker of response for PD-1/PD-L1 inhibition therapy [41, 42]. Anti-PD therapy seems less effective in non-inflamed tumors (low lymphocyte infiltration/PD-L1 expression) with increased levels of transforming growth factor-β (TGF-β), inducer of resistance to anti-PD-L1 therapy, and fusiform cells rich stroma .
Epithelial-mesenchymal transition (EMT) with epithelial cells becoming mesenchymal cells by losing cell-cell adhesion and polarity and acquiring invasive/migratory properties, might contribute to drug resistance and hence poor prognosis [2, 39, 44, 45]. E-cadherin downregulation and overexpression of vimentin, N-cadherin, α-actin and fascin depending on EMT genes, such as snail family transcriptional repressor 2 (Slug), twist family bHLH transcription factor 1 (TWIST), zinc finger E-box binding homeobox 1 (ZEB1), nuclear-translocated β-Catenin and TGF-β, have been associated with TKi resistance, namely to EGFR-TKis [31, 42, 46].
To Kim et al., PD-L1 expression may be responsible for EMT oncogenesis and immune evasion during tumor development , contradictory with EMT-induced PD-L1 expression in pulmonary carcinomas . PD-L1 and EMT bidirectional cross-talk has since then been proposed to promote tumor aggressiveness [47, 49]. Neurotrophic tyrosine receptor kinase (NTRK) gene rearrangements present in 0.1–1% of lung carcinomas, assessed by NGS and with effective targeted therapy [50, 51], are associated with microscopically high grade features and less differentiated phenotype in mesenchymal tumors , needing further studies to be correlated with EMT phenotype in carcinomas, where vimentin and other EMT markers expression might become relevant.
A significant association was found between PD-L1 expression and high vimentin expression in TCs, with 24 of 32 vimentin-positive cases expressing PD-L1, versus 33 of 41 PD-L1-negative samples without vimentin expression. This result was consistent with previous observations that PD-L1 expression was positively correlated with vimentin expression and EMT phenotype in lung ADC, extrahepatic cholangiocarcinoma, breast carcinoma and head/neck/esophageal squamous carcinoma, suggesting that tumors with EMT status stand as potential targets for immunotherapy agents [47, 49]. In fact, Ancel et al. proposed vimentin as canonical marker and actor of EMT , verified in this series in 24 vimentin-positive/PD-L1-positive cases where 14 had PD-L1 expression in over 50% TCs, becoming a frequent event upon increasingly higher PD-L1 expression, significantly leading the risk of 3.85 times higher expression among cases with more than 50% PD-L1 stained TCs, versus PD-L1 negative samples.
Proliferation marker ki-67 keeps being associated with tumor aggressiveness and metastization in solid tumors . A significant association was present between ki-67 LI and both positive PD-L1 expression and stratified PD-L1 score, as 49 of 54 PD-L1-positive cases had ki-67 LI>30% and 37 from 38 samples with more than 5% of PD-L1 stained TCs showed ki-67 LI>30%. Association between PD-L1 status and tumor cell proliferation was also confirmed by the tendency of PD-L1 positivity in the solid pattern of ADC samples, in accordance with literature [21, 53], still contradictory regarding association between PD-L1 expression and ki-67 LI in SQCs . Similarly to vimentin, the risk of ki-67 LI>30% is 9.90 times higher in samples with more than 50% of PD-L1 stained TCs, versus PD-L1 negative specimens.
To the best of our knowledge, our study was the first to investigate the relationship between PD-L1 expression and EMT status, evaluated by vimentin included in IHC panel applied in Pathology practice, with perspective of risk analysis. Similarly to previous investigations [45, 47], EMT status by vimentin staining can be relevant in selecting patients more likely to have higher TCs PD-L1 expression. Favorable response to PD-1/PD-L1 immune checkpoint blockade in bronchopulmonary carcinomas would be then more accurate based on two biomarkers expressed in TCs, namely in follow-up of targeted therapy acquired resistance . Also, as 10-20% of unselected patients with advanced carcinomas benefit from anti-PD therapy [18, 29, 41, 43], combined therapy of PD-1/PD-L1 inhibitors with EMT targeted therapies might become an ultimate therapeutical option and vimentin might work as a fundamental biomarker.
Combination of mitogen-activated protein kinase kinase (MEK) inhibitors with PD-L1 inhibitors improved tumor regression, where MEK inhibitors may sensitize TCs to immunotherapy agents . Mechanistic target of rapamycin (mTOR) promotes EMT phenotype and immune evasion through upregulation of PD-L1 expression and the effect of mTOR inhibition combined with PD-L1 blockade was also reported in preclinical lung cancer trials . Finally, combination of PD-1 inhibitors with EGFR TKis in PD-L1-positive carcinomas with EGFR activating mutations raised promising results in preclinical trials, as EGFR activation up-regulated PD-L1 expression, probably making these tumors more susceptible to PD-1/PD-L1 blockade therapy [27, 44, 49].
Limitations of this study, concerning 97 biopsies, and the utility of the applied IHC panel, deserve replication with additional EMT markers, to support EMT-phenotype as a new potential predictive biomarker to define immunotherapy in pulmonary carcinomas. Research on combined therapies with EMT targeted agents and PD-L1 inhibitors to improve patients outcome and survival deserve to be implemented.