PD-L1 In Non-small Cell Lung Cancer Research Articles

Abstract 1373: Gene expression correlation of immune checkpoint molecules Siglec-15 and PD-L1 varies widely by cancer indication

Abstract Siglec-15 is a novel target for immunotherapy in cancer and exhibits low mRNA expression in normal tissues but broad expression across cancer indications, specifically on tumor-associated macrophages and tumor cells [1]. Expression of Siglec-15 and the immune checkpoint molecule PD-L1 have previously been reported to be mutually exclusive [1][2]. However, a large-scale meta-analysis of this claim has not been undertaken across cancer indications. Here, RNA-Seq analyses were performed across single cells, cell lines, and bulk tumor samples for Siglec-15 and PD-L1 mRNA expression. Data and meta-data from TCGA, CCLE, and primary tumor samples from 62 studies were obtained through OmicSoft OncoLand and single cell analysis was performed on the BioTuring Talk2Data platform. Analyses were limited to non-small cell lung cancer (NSCLC), cholangiocarcinoma, breast, thyroid, head and neck, colon, rectal, bladder, kidney, and endometrial cancers. Expression values were binned separately into percentiles across all indications and samples in increments of 10%. These analyses revealed individual gene expression levels of Siglec-15 and PD-L1 in bulk tumor samples varied substantially by indication, while co-expression of Siglec-15 and PD-L1 varied across indications. Analysis of gene expression of Siglec-15 and PD-L1 from the TCGA database showed a positive correlation in several indications including NSCLC, head and neck, kidney, and thyroid cancers. Analyses from additional studies in primary tumor samples confirmed co-expression of Siglec-15 and PD-L1 in NSCLC and head and neck cancer. Cell lines showed a similar trend of positive correlation with varying levels of Siglec-15 and PD-L1 expression. However, when examining expression at the single cell level (not bulk tumor), in 30 single cell studies spanning >1.3M cells, only 731 cells in 20 studies were found to co-express Siglec-15 and PD-L1 on the same cell while each gene was expressed individually on other cells. The majority of the cells co-expressing the genes were macrophages (n=~350), followed by CD4 (n=~115) and CD8 T cells (n=~50). This study demonstrates that at the bulk tumor level, Siglec-15 and PD-L1 mRNA expression are not broadly mutually exclusive across cancer indications and instead expression of each gene varies broadly within a given indication. Additionally, their expression is frequently positively correlated, including in NSCLC, head and neck, kidney, and thyroid cancers. These results also demonstrate that Siglec-15 and PD-L1 are rarely found co-expressed on same cell. These associations enable a better understanding of the landscape of target expression in patients across a wide variety of cancer indications and can inform combination strategies with anti-Siglec-15 therapies. [1] Sun J, et al. Clin Cancer Res, 2021;27(3):680-688. [2] Wang J, et al. Nat Med, 2019;25(4):656-666. Citation Format: Krystal Watkins, Liyang Diao, Marsha Crochiere, Jan Pinkas. Gene expression correlation of immune checkpoint molecules Siglec-15 and PD-L1 varies widely by cancer indication [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1373.

Ezrin regulates the progression of NSCLC by YAP and PD-L1.

To determine whether ezrin regulates Yes-associated protein (YAP) and programed cell death ligand-1 (PD-L1), which are involved in the invasion and metastasis of non-small cell lung cancer (NSCLC). Immunohistochemistry of 164 NSCLC and 16 para-cancer tissues was performed to detect ezrin, YAP, and PD-L1 expression. Further, H1299 and A549 cells were transfected with lentivirus, and then colony formation, CCK8, transwell, and wound-healing assays were used to assess cell proliferation, migration, and invasion. RT-qPCR and western blotting were used for quantitative analysis of ezrin, PD-L1, and YAP expression. Moreover, the role of ezrin in tumor growth was assessed in vivo, and immunohistochemistry and western blotting were performed to evaluate changes in ezrin expression in mouse samples. The positive protein expression rates of these molecules in NSCLC were as follows: ezrin, 43.9% (72/164); YAP, 54.3% (89/164); and PD-L1, 47.6% (78/164); these were higher than those in normal lung tissues. Moreover, YAP and ezrin expression positively correlated with PD-L1 expression. Ezrin promoted proliferation, migration, invasion, and expression of YAP and PD-L1in NSCLC. Inhibition of ezrin expression reduced the effects of ezrin on cell proliferation, migration, invasion, inhibited the expression of YAP and PD-L1, and obviously reduced experimental tumor volume in vivo. Ezrin is overexpressed in NSCLC patients and correlates with PD-L1 and YAP expression. Ezrin regulates YAP and PD-L1 expression. Inhibition of ezrin delayed NSCLC progression.

Real-world multicentre cohort of first-line pembrolizumab alone or in combination with platinum-based chemotherapy in non-small cell lung cancer PD-L1 ≥ 50%

IntroductionPembrolizumab alone (IO-mono) or in combination with platinum-based chemotherapy (CT-IO) is first-line standard of care for advanced non-small cell lung cancer (NSCLC) patients with PD-L1 ≥ 50%. This retrospective multicentre study assessed real-world use and efficacy of both strategies.MethodsPatients with advanced NSCLC PD-L1 ≥ 50% from eight hospitals who had received at least one cycle of IO-mono or CT-IO were included. Overall survival (OS) and real-word progression-free-survival were estimated using Kaplan–Meier methodology. Cox proportional hazards regression models were used to estimate hazard ratios (HRs) and 95% CIs, and a Cox model with inverse propensity treatment weighting was carried out.ResultsAmong the 243 patients included, 141 (58%) received IO-mono and 102 (42%) CT-IO. Younger patients, those with symptomatic disease and brain metastases were more likely to be proposed CT-IO. With a median follow-up of 11.5 months (95% CI 10.4–13.3), median OS was not reached, but no difference was observed between groups (p = 0.51). Early deaths at 12 weeks were 11% (95% CI 4.6–16.9) and 15.2% (95% CI 9.0–20.9) in CT-IO and IO groups (p = 0.32). After adjustment for age, gender, performance status, histology, brain metastases, liver metastases and tobacco status, no statistically significant difference was found for OS between groups, neither in the multivariate adjusted model [HR 1.07 (95% CI 0.61–1.86), p = 0.8] nor in propensity adjusted analysis [HR 0.99 (95% CI 0.60–1.65), p = 0.99]. Male gender (HR 2.01, p = 0.01) and PS ≥ 2 (HR 3.28, p < 0.001) were found to be negative independent predictive factors for OS.ConclusionYounger patients, those with symptomatic disease and brain metastases were more likely to be proposed CT-IO. However, sparing the chemotherapy in first-line does not appear to impact survival outcomes, even regarding early deaths.

Immunohistochemical expression of CD8, CTLA4, and PD-L1 in NSCLC of smokers versus non smokers and its effect on prognosis

ABSTRACT Background Identifying hig- risk non-small cell lung cancer (NSCLC) patients is the most contentious area in lung pathology for reducing cancer-related morbidity and mortality. Aim of the work Was to investigate the immunohistochemical expression of CD8, CTLA4, and PD-L1 among different NSCLC histopathological variants and it’s correlation with different clinicopathological variables. Material and Methods Expression of CD8, CTLA4, & PD-L1 was evaluated immunohistochemically in 45 NSCLC cases. Results Higher expression of CD8 tumor infiltrating lymphocytes (TILs) was significantly associated with better progression-free survival (PFS). The expression of CTLA4&PD-L1 on tumor cells was significantly associated with lower PFS. However, smoking status of the studied cases showed no statistically significant correlation with expression of any of the studied immunohistochemical markers. Conclusions Immunostaining for CD8, CTLA4, and PD-L1 could have a major role in the anticipation of PFS of NSCLC cases regardless of their smoking status.

PD-L1 Over-Expression Varies in Different Subtypes of Lung Cancer: Will This Affect Future Therapies?

Programmed death-ligand (PD-L) 1 and 2 are ligands of programmed cell death 1 (PD-1) receptor. They are members of the B7/CD28 ligand-receptor family and the most investigated inhibitory immune checkpoints at present. PD-L1 is the main effector in PD-1-reliant immunosuppression, as the PD-1/PD-L pathway is a key regulator for T-cell activation. Activation of T-cells warrants the upregulation of PD-1 and production of cytokines which also upregulate PD-L1 expression, creating a positive feedback mechanism that has an important role in the prevention of tissue destruction and development of autoimmunity. In the context of inadequate immune response, the prolonged antigen stimulation leads to chronic PD-1 upregulation and T-cell exhaustion. In lung cancer patients, PD-L1 expression levels have been of special interest since patients with non-small cell lung cancer (NSCLC) demonstrate higher levels of expression and tend to respond more favorably to the evolving PD-1 and PD-L1 inhibitors. The Food and Drug Administration (FDA) has approved the PD-1 inhibitor, pembrolizumab, alone as front-line single-agent therapy instead of chemotherapy in patients with NSCLC and PD-L1 ≥1% expression and chemoimmunotherapy regimens are available for lower stage disease. The National Comprehensive Cancer Network (NCCN) guidelines also delineate treatment by low and high expression of PD-L1 in NSCLC. Thus, studying PD-L1 overexpression levels in the different histological subtypes of lung cancer can affect our approach to treating these patients. There is an evolving role of immunotherapy in the other sub-types of lung cancer, especially small cell lung cancer (SCLC). In addition, within the NSCLC category, squamous cell carcinomas and non-G12C KRAS mutant NSCLC have no specific targetable therapies to date. Therefore, assessment of the PD-L1 expression level among these subtypes of lung cancer is required, since lung cancer is one of the few malignances wherein PD-L1 expression levels is so crucial in determining the role of immunotherapy. In this study, we compared PD-L1 expression in lung cancer according to the histological subtype of the tumor.

Andrographolide suppresses non-small-cell lung cancer progression through induction of autophagy and antitumor immune response

Despite recent advances in diagnosis and therapeutic strategies, treatment of non-small-cell lung cancer (NSCLC) remains unsatisfactory in terms of prognosis. Andrographolide (AD), a principal active component of Andrographis paniculata (Burm.f.) Nees, exerts anti-cancer therapeutic properties. AD has been used for centuries in China for clinical treatment of viral infections. However, the pharmacological biology of AD in NSCLC remains unknown. In this study, AD regulated autophagy and PD-L1 expression in NSCLC. Molecular dynamics simulations indicated that AD bound directly to signal transducer and activator of transcription-3 (STAT3) with high affinity. Proteomics analysis indicated that AD reduced the expression of tumour PD-L1 in NSCLC by suppressing JAK2/STAT3 signalling. AD modulated the P62-dependent selective autophagic degradation of PD-L1 by inhibiting STAT3 phosphorylation. In vivo study revealed that AD suppressed tumour growth in H1975 xenograft mice and Lewis lung carcinoma cell models, and better efficacy was obtained at higher concentrations. AD prolonged the survival time of the mice and enhanced the treatment efficacy of anti-PD-1 mAb immunotherapy by stimulating CD8+ T cell infiltration and function. This work elucidated the specific mechanism by which AD inhibited NSCLC. Treatment with the combination of AD and anti-PD-1 mAb immunotherapy could be a potential strategy for patients with NSCLC.

The impact of a pathologist’s personality on the interobserver variability and diagnostic accuracy of predictive PD-L1 immunohistochemistry in lung cancer

ObjectivesProgrammed death-ligand 1 (PD-L1) is the only approved predictive biomarker for immunotherapy in non-small cell lung cancer (NSCLC). However, predictive PD-L1 immunohistochemistry is subject to interobserver variability. We hypothesized that a pathologist’s personality influences the interobserver variability and diagnostic accuracy of PD-L1 immunoscoring. Materials and MethodsSeventeen pathologists performed PD-L1 immunoscoring on 50 resected NSCLC tumors in three categories (<1%;1–49%;≥50%). Also, the pathologists completed a certified personality test (NEO-PI-r), assessing five personality traits: neuroticism, extraversion, openness, altruism and conscientiousness. ResultsThe overall agreement among pathologists for a series of 47 tumors was substantial (kappa = 0.63). Of these, 23/47 (49%) tumors were entirely negative or largely positive, resulting in a kappa value of 0.93. The remaining 24/47 (51%) tumors had a PD-L1 score around the cutoff value, generating a kappa value of 0.32.Pathologists with high scores for conscientiousness (careful, diligent) had the least interobserver variability (r = 0.6, p = 0.009). Also, they showed a trend towards higher sensitivity (74% vs. 68%, p = 0.4), specificity (86% vs. 82%, p = 0.3) and percent agreement (83% vs. 79%, p = 0.3), although not significant. In contrast, pathologists with high scores for neuroticism (sensitive, anxious) had significantly lower specificity (80% vs. 87%, p = 0.03) and percent agreement (78% vs. 85%, p = 0.03). Also, a trend towards high interobserver variability (r = -0.3, p = 0.2) and lower sensitivity (68% vs. 74%, p = 0.3) was observed, although not significant.Pathologists with relatively high scores for conscientiousness scored fewer tumors PD-L1 positive at the ≥ 1% cut-off (r = −0.5, p = 0.03). In contrast, pathologists with relatively high scores for neuroticism score more tumors PD-L1 positive at ≥ 1% (r = 0.6, p = 0.017) and ≥ 50% cut-offs (r = 0.6, p = 0.009). ConclusionsThis study is the first to demonstrate the impact of a pathologist’s personality on the interobserver variability and diagnostic accuracy of immunostaining, in the context of PD-L1 in NSCLC. Larger studies are needed for validation of these findings.

MA08.05 High Concordance in Scoring PD-L1 in NSCLC Using the Roche Digital Pathology uPath system and PD-L1 Assessment Algorithm

Accurate and reproducible assessment of PD-L1 expression is crucial in guiding the use of anti PD-1/PD-L1 immunomodulatory therapy. Interpretation can be challenging and both intra- and inter-pathologist discordance has been shown to be potentially significant in affecting clinical outcomes. Digital pathology and algorithmic assessment tools provide an opportunity to increase accuracy and concordance when making this important assessment in non-small cell lung cancer (NSCLC). Sections of 198 NSCLCs, (84 EBUS aspirates or small tissue biopsies and 114 resections) immunolabelled for PD-L1 using the Roche-Ventana SP263 antibody were scanned with a Roche DP200 digital image scanner and analysed using uPath slide viewer and the Roche PD-L1 interpretative algorithm. Sections were assessed by three ‘blinded’ pathologists for expression of PD-L1 which was given as an absolute percentage, the tumour proportion score (TPS) in four ways; (1) unassisted (by pathologist using conventional microscopy), (2) automated whole slide (unsupervised uPath assessment of whole slide), (3) automated annotated (uPath assessment of slide annotated by pathologist) and (4) assisted (by pathologist using digital image assisted by uPath assessment of annotated slide). The last method of assessment was repeated after a six-week ‘washout’ period. Intraclass correlation co-efficient (ICC) and Cohen’s Kappa (for clinical categories of <1%, 1-49% or ≥50% TPS) were used to compare concordance of scoring assessment methods.Table 1Comparison of TPS scoring methodsTPS assessment methods - Full CohortICCKappap-valueAssisted vs unassisted0.9730.856<0.0001Assisted vs automated whole slide0.9110.475<0.0001Assisted vs automated annotated0.9530.598<0.0001TPS assessment methods - small biopsies/EBUS onlyAssisted vs automated annotated0.9520.836<0.0001 Open table in a new tab Intra-pathologist concordance between repeated assisted reads was very high (ICC 0.991 K 0.989 p<0.0001) as was inter-pathologist concordance for assisted interpretations (ICC 0.986 K 0.910 p<0.0001 respectively) Automated algorithmic assessment guided by pathologists is an accurate approach that helps produce consistent and reliable interpretation of PD-L1 expression in NSCLC by reducing intra- and inter- observer discordance. Digital pathology and automated algorithms are powerful tools that can help optimise the predictive power of PD-L1 expression as a predictor of response to immunomodulatory therapy.

Angiotensin II contributes to intratumoral immunosuppression via induction of PD-L1 expression in non-small cell lung carcinoma

The formation of an immunosuppressive microenvironment and up-regulation of PD-L1 protein are the main causes of tumor immune escape. Previous reports suggest that Angiotensin II (Ang II) can modulate the immune status of tumor microenvironment in non-small cell lung cancer (NSCLC), but the underlying mechanism remains not fully understood. Here we demonstrated that AngII treatment causes the reduction of intratumoral infiltrating CD4 T lymphocytes in tumor-bearing mice, increases the accumulation of immunosuppressive granulocytes and TAMs in tumor tissue, and upregulates the expression levels of immunosuppressive marker genes. In addition, AngII/AGTR1 axis triggers cell PD-L1 expression through a mechanism involving increases in PD-L1 mRNA stability by human antigen R (HuR), an AU-rich element (ARE)-binding protein. Collectively, AngII/AGTR1 signaling promotes the tumor immunosuppressive microenvironment by upregulating PD-L1 in NSCLC, the mechanism of which is largely accounted by HuR-mediated PD-L1 mRNA stabilization.

Non-small cell lung cancer PDL1 &gt;50%—should we go single or combo?

Recent years have witnessed a revolution in the era of immune checkpoint inhibitors (ICIs). It has greatly impacted the oncological field and especially advanced non-small cell lung cancer (NSCLC) treatment. Here, we summarized the 3 main treatment protocols for a specific population of NSCLC with strong PD-L1 staining (PD-L1 ≥50%); which include ICI (e.g., pembrolizumab) monotherapy, ICI in combination with chemotherapy or combination ICIs. To date, it is uncertain whether there is a therapeutic difference amongst the PD-L1 TPS ≥50% group in comparison to other PD-L1 subdivisions. The findings reviewed here show that the combination ICI with chemotherapy yields the best response rate in comparison to other treatment protocols. Crucially, this treatment protocol is highly suggested for patients with high disease burden, especially for those with liver or brain involvement.

High-Plex Predictive Marker Discovery for Melanoma Immunotherapy-Treated Patients Using Digital Spatial Profiling.

Protein expression in formalin-fixed, paraffin-embedded tissue is routinely measured by IHC or quantitative fluorescence (QIF) on a handful of markers on a single section. Digital spatial profiling (DSP) allows spatially informed simultaneous assessment of multiple biomarkers. Here we demonstrate the DSP technology using a 44-plex antibody cocktail to find protein expression that could potentially be used to predict response to immune therapy in melanoma.Experimental Design: The NanoString GeoMx DSP technology is compared with automated QIF (AQUA) for immune marker compartment-specific measurement and prognostic value in non-small cell lung cancer (NSCLC). Then we use this tool to search for novel predictive markers in a cohort of 60 patients with immunotherapy-treated melanoma on a tissue microarray using a 44-plex immune marker panel measured in three compartments (macrophage, leukocyte, and melanocyte) generating 132 quantitative variables. The spatially informed variable assessment by DSP validates by both regression and variable prognostication compared with QIF for stromal CD3, CD4, CD8, CD20, and PD-L1 in NSCLC. From the 132 variables, 11 and 15 immune markers were associated with prolonged progression-free survival (PFS) and overall survival (OS). Notably, we find PD-L1 expression in CD68-positive cells (macrophages) and not in tumor cells was a predictive marker for PFS, OS, and response. DSP technology shows high concordance with QIF and validates based on both regression and outcome assessment. Using the high-plex capacity, we found a series of expression patterns associated with outcome, including that the expression of PD-L1 in macrophages is associated with response.

MTE24.01 Immunohistochemical Assessment of Biomarkers for Immune Checkpoint Inhibitors

Programmed death receptor-1 (PD-1) is a type 1 membrane protein of the immunoglobulin superfamily that has an important role in restricting immune-mediated tissue danage secondary to inflammation and/or infection.1 The clinical advantage of antibodies that target either PD-1 or PD-L1 to block this ligand-receptor interface, allowing cancer killing by T cells became clear when CTLA4, an antagonist against the T-cell, such as ipilimumab, and afterward PD-1, showed an increase survival in patients with metastatic melanoma.2 Clinical investigations in lung cancer have demonstrated the benefit of PD-1 inhibitors pembrolizumab in advanced non–small cell lung cancer (NSCLC) and nivolumab in advanced squamous and nonsquamous NSCLC; both approved as second-line therapies by the US Food and Drug Administration (FDA).3-5 Others PD-L1 inhibitors such as atezolizumab and durvalumab have demonstrated effectiveness in several tumor types6-7 but they were not approved for clinical use until now. PD-1 inhibitors induce around of 20% of complete response frequency in patients with NSCLC, and persistent response in a subgroup of patients treated by immune checkpoint inhibitors. Garon et al.3 showed that tumors with PD-L1 expression ≥ 50% by immunohistochemistry (IHC) were significantly more expected to respond to pembrolizumab than those with less than 50% malignant cell expression. In contrast, response rates to nivolumab are significantly greater in patients with nonsquamous NSCLC, showing ≥ 1% tumor cell positivity.5 These differences are related to the combination of antibody clone and detection system as a companion diagnostic for selecting lung cancer patients for pembrolizumab therapy. Previous investigations reported response taxes in PD-L1–positive tumors of 31% to 52%, but particularly more than 16% of PD-L1–negative tumors also showed treatment response.1 This finding indicates that PD-L1 expression improves for responders but the absence of expression is not a complete indicator of advantage. PD-L1 expression did not predict differential response to nivolumab in lung squamous cell carcinoma as compared with docetaxel.4 PD-L1 in NSCLC is expressed on the membrane of tumor cells, and/or on immune infiltrating cells dendritic cells, antigen-presenting cells and T lymphocyte. PD-1, the PDL1 receptor, is expressed on tumor infiltrating lymphocytes, mainly CD4 T cells, T and B regulatory, NK, monocytes and DC. Concerning PD-L1 binding, PD-1 inhibits kinases involved in T cell activation. Two potential mechanisms are involved in expression of immune checkpoints on tumor cells and their immune stromal component: oncogenic signaling, and response to inflammatory signals.8 Tumor cells express multiple ligands and receptors and antitumor immune response can be enhanced by multi-level blockade of immune checkpoints. PD-1/PD-L1 commitment leads to HSP-2 phosphatase activity which dephosphorylates Pi3K and thus downregulate AKT.8 The positive score on tumor cells has not been evaluated nor enhanced or standardized.3,8 Brambilla and Ming8 assessed a score of positivity for prognosis analysis using E1L3N Cell Signaling antibody commercially available. They found that 20% of lung tumors cell expressed PD-L1 (≥ 20% intensity 2+3+), and 29% the immune stromal cells (T, macrophages, DC) ≥ 10% intensity 2+3+. PD-L1 positivity in both tumor and immune cells were seen in only 9% of NSCLC, 20,7% were both negative. There was no prognostic relevance of PD-L1 (tumor cells or stroma) whatever cut off by 10% increment or linear scoring was used. Only immune PD-L1 expression was correlated with a highly intense immune infiltrations. Previous published evaluations of prognostic value were discordant likely because immune checkpoints modulators play both positive and negative roles in the immune inhibitory pathways with some redundancy, and patients series and assays were not comparable. The two meta-analyzes with different antibodies, cutoffs, patient series, ethnicities and contribution of oncogene driven cancers, initial resection sample or contemporary biopsy rendered their interpretation extremely problematic. Global result was supporting a poor prognosis of “PD-L1 positivity” on tumor cells. Most of phase I trials works with four antibodies targeting PD-1 or its primary ligand PD-L1, response taxes appear higher in patients with increased tumor PD-L1 membrane expression by IHC. However, different antibody assays, absence of standardization, different score to determine PD-L1 positivity, companion test type, and a short number of specimens available for testing, accopled to the variability of the intervals between biopsy and test, has certainly disadvantaged the conclusion and prevent consensus to be reached.10 The best threshold was provided by Garon et al, with ≥ 50% of tumor cells PD-L1 positive to allow the highest response rate of 45% to pembrolizumab.3 In most trial series, biopsies or resected specimen were used and considerable difference between these samples occurs due to tumor heterogeneity. The reliability of small biopsy samples is questioned.10 Indeed lung tumor heterogeneity is characteristic and PD-L1 is typically heterogeneous in its distribution in the tumor majority as is PD-L1 positive immune cells. Multiple questions are still addressed before PD-L1 is considered as a definitive molecular predictor of effectiveness. As for prognostic evaluations, thresholds of ≥ 1%, ≥ 5%, ≥ 10%, ≥ 50% or continuous H score have been used. In addition, in a few trials, PD-L1 expression in TILs was predictive more than PD-L1 on tumor cells but the best cut off was not revealed. PDL1 expression predicts response to immune checkpoint inhibitors. Concordant results showing a better response if PDL1 + in several trials, using drug specific test and for Nivolumab also histology specific. We should evaluate membranous staining in tumor sample with at least 100 tumors cells and immune cells. Perspective for upgrading includes: 1) heterogeneity of the expression of PDL1 within tumor, primitive vs metastases number and size of samples; 2) surgical tissue versus biopsy and 3) archival versus new biopsy and 4) standardize the assays. Published abstracts showed high rates of concordance between primary and metastases (81%). Obtaining multiple biopsies from different areas of the tumor would enhance the validity of the results of IHC evaluation (160 patients=48% discordance).

Abstract 4981: PI3K/AKT/mTOR pathway activation drives expression of the immune inhibitory ligand PD-L1 in NSCLC.

Abstract Despite the development of targeted drug therapies, lung cancer remains the leading cause of cancer-related death worldwide. Targeting immune checkpoints altered in cancer, such as the T cell inhibitory receptors programmed death 1 (PD-1) or CTLA4, represent promising new approaches to lung cancer treatment. Here we investigated the relationship of activation of the PI3K/Akt/mTOR signaling pathway and expression of the immune suppressive ligand, PD-L1, in non-small cell lung cancer (NSCLC). NSCLC cell lines with mutations in KRas, EGFR, BRaf, ALK or RET had high levels of PI3K/Akt/mTOR pathway activation as well as increased expression of PD-L1. Cell lines that lacked these mutations had low PI3K/Akt/mTOR pathway activation and less PD-L1 expression. In murine cell lines derived from tobacco carcinogen (NNK)-induced lung tumors, high levels of active Akt correlated with high PD-L1 expression. Agonists of the PI3K/Akt/mTOR pathway activation such as EGF or the carcinogen NNK, as well as knockdown of PTEN or induction of Kras induced PD-L1 expression. Conversely, treatment of high PD-L1 expressing NSCLC cell lines with inhibitors of PI3K (LY294002), AKT (triciribine) or mTOR (rapamycin) decreased PD-L1 expression. These inhibitors decreased PD-L1 protein levels, but did not alter PD-L1 mRNA. Rapamycin-mediated PD-L1 loss was partially blocked by pre-treatment with a lysosomal, but not a proteosomal inhibitor. In vivo, increased expression of PD-L1 and activation of mTOR was an early event after exposure to NNK and was blocked by rapamycin. To test the efficacy of blocking PD-1 while reducing the expression of PD-L1 in tumor tissue, rapamycin and a murine anti-PD-1 receptor antibody were administered to A/J mice bearing syngeneic lung tumor xenografts. The combination decreased the tumor growth rate to a greater degree than either agent alone and decreased mTOR pathway signaling, PD-L1 expression, and increased cleaved caspase 3. Tumor associated activated CD8+ T cells were also increased in the tumors. These data support a relationship between activation of PI3K/Akt/mTOR signaling, PD-L1 expression and the development of an immune resistant phenotype in lung cancer. Combination regimens using PI3K/Akt/mTOR inhibitors may enhance the efficacy of immune therapies targeting PD-1. Citation Format: Kristin Lastwika, Willie Wilson, Phillip A. Dennis. PI3K/AKT/mTOR pathway activation drives expression of the immune inhibitory ligand PD-L1 in NSCLC. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4981. doi:10.1158/1538-7445.AM2013-4981

High expression of PD-L1 in lung cancer may contribute to poor prognosis and tumor cells immune escape through suppressing tumor infiltrating dendritic cells maturation

The immunohistochemical analysis was used to evaluate the expression of PD-L1 in 109 non-small cell lung cancer (NSCLC) tissues and para-tumor tissues. Associations between expressed PD-L1 and tumor histological types, degree of differentiation, and lymph node metastasis were calculated, and overall survival was assessed. Meanwhile, immunohistochemistry and immunofluorescence double labeling technique were performed to detect the expressions of PD-L1, CD1α, and CD83 on TIDC of 20 lung cancer tissues, and the expression of PD-L1 in CD1α+DCs and CD83+DCs and their significances were also explored. We found that the expression rate of PD-L1 in NSCLC was associated with histological types and overall survival. Patients with either adenocarcinoma or survival time after surgery less than 3 years showed higher expression rate of PD-L1. Furthermore, Cox model analysis indicated that PD-L1 might be regarded as a poor prognostic factor. PD-L1 could be also detected in CD1α+ immature DC in NSCLC, indicating that as a class of key anti-tumor immunocyte in tumor microenvironment, DC expressing PD-L1 itself might play an important role in keeping its immature status and contributing to tumor cells immune escape and disease progression.