Toward Precision Oncology in Ovarian Cancer: Dual PD-L1 and CD44 Biomarker Profiling for Recurrence Risk Assessment

Background Breast cancer (BC) is considered to be the second highest cause of cancer-related death in women, accounting for 15% of all female cancer-related deaths globally, despite major advancements in early detection and treatment methods. Traditionally, BC has not been considered as an immunogenic tumour, especially when compared to other solid tumours like lung or melanoma. However, a rapidly expanding body of evidence indicates significant immunogenic variability among various BC subtypes, with hormone receptor-negative BC being typically more immunogenic than hormone receptor-positive cancers. Programmed death ligand-1 (PD-L1) is a transmembrane protein, found in a variety of normal tissue cells and also in a wide range of epithelial malignancies, including breast carcinoma. Binding of PD-L1, expressed on tumor cells, to its receptor PD-1, expressed on activated T cells and B cells, disrupts effector immune functions. Antibodies targeting PD-L1 have been approved for treating a wide range of malignancies including breast cancer especially unresectable and metastatic triple negative breast cancer (TNBC). However, PD-L1 expression and its prognostic role in BC is still the target of several researches in order to maximize its therapeutic role in different clinicopathological settings. Objectives to evaluate immunohistochemical expression of PD-L1 in tumour cells and tumour infiltrating lymphocytes (TILs) in different types of breast carcinoma and to correlate between PD-L 1 expression and clinicopathological variables. Material and Methods Ninety cases of breast carcinoma mastectomy specimens were collected and stained immunohistochemically for PD-L1. PD-L1 expression was evaluated in TCs and TILs in the two setting of cases; (Group A) in which the patients didn’t receive preoperative neoadjuvant chemotherapy (NAC) and (Group B) in which the patients received preoperative NAC. The expression of PD-L1 was correlated with clinicopathological parameters. Survival analysis was conducted to correlate disease-free survival (DFS) with PD-L1 expression. Results In cases of Group A (Those didn’t receive preoperative NAC), (31.1%) of cases showed PD-L1 expression by tumor cells [PD-L1 (TCs)] and (47.5%) showed PD-L1 expression by tumor infiltrating lymphocytes [PD-L1 (TILs)]. PD-L1 (TCs) and PD-L1 (TILs) expression were significantly associated with histologic type of invasive duct carcinoma, NST, grade 2, presence of carcinoma in situ, negative hormone receptors, high proliferation index (Ki-67), molecular subtype of TNBC and high level of TILs. In cases received preoperative NAC (Group B), (13.8%) of cases showed PD-L1 expression by tumor cells and (41.4%) showed PD- L1 expression by tumor infiltrating lymphocytes. PD-L1 (TCs) expression was significantly associated with high level of TILs. While PD-L1 (TILs) expression was significantly associated with presence of carcinoma in situ, negative hormone receptors, high proliferation index (Ki-67) and molecular subtype of TNBC. The correlation between PD-L1 expression in TCs and TILs and DFS was statistically insignificant. Conclusion PD-L1 immunohistochemical expression in breast carcinoma is much more prevalent in TILs than TCs and both PD-L1 (TCs) and PD- L1 (TILs) are significantly associated with high proliferation index (K i-67), non-luminal subtypes (TNBC and HER2 Positive) and high TILs level, suggesting that they may be important candidates for anti-PD- 1/PD-L1 therapy.

BACKGROUND: Expression of programmed death ligand 1 (PD-L1) is up-regulated in many cancers and has been found to suppress antitumor immunity through binding to its receptor programmed death 1 (PD-1). Blockade of PD-1/PD-L1 signaling axis enhances antitumor immunity in many cancers including epithelial ovarian cancer (EOC). Despite the importance of PD-L1 in tumor immunity, the epigenetic regulation of PD-L1 expression remains poorly understood. AIMS: To determine the roles of epigenetic components in regulating PD-L1 gene expression and modulating antitumor immunity. METHODS: Toward this goal, I screened a small molecule library of epigenetic inhibitors and examined their inhibition on PD-L1 expression in EOC cells to identify the positive hits. To further confirm PD-L1 inhibition in vivo, C57BL/6 mice are injected with luciferase expressing mouse ovarian cancer ID8 cells and treated with or without epigenetic inhibitors, PD-L1 expression in both tumor cells and immune cells are examined, tumor growth and survival are also monitored. RESULTS: BET inhibitors suppresses both constitutive and IFNγ induced PD-L1 expression in EOC cells through targeting BRD4. Further ChIP-seq and ChIP assay demonstrate that BRD4 directly binds to PD-L1 gene promoter and regulated its transcription. BET inhibitor JQ1 suppresses PD-L1 expression in both tumor cells and immune cells including dendritic cells and macrophages in mice bearing ID8 mouse ovarian cancer cells. Moreover, the BET inhibitor suppresses the tumor growth and improves the survival of EOC bearing mice through activating antitumor T cells. CONCLUSION: Taken together, we demonstrated that BRD4 can directly regulate PD-L1 expression in EOC cells by binding to the promoter of PD-L1 encoding gene, suppressing PD-L1 expression with BET inhibitors blocks PD1/PD-L1 signaling and enhances antitumor immunity in vivo. These findings indicate that pharmacological inhibition of BET proteins represent a new therapeutic strategy for EOC by targeting PD-L1 expression. Citation Format: Hengrui Zhu, Fee Bengsch, Nikolaos Svoronos, Melanie R. Rutkowski, Benjamin G. Bitler, Michael J. Allegrezza, Yuhki Yokoyama, James E. Bradner, Jose R. Conejo-Garcia, Rugang Zhang. TARGETING PD–L1/PD1 PATHWAY THROUGH BET PROTEIN INHIBITION IN EPITHELIAL OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP31.

Differential expression of PD-L1 and IDO1 in association with the immune microenvironment in resected lung adenocarcinomas

Placental trophoblasts contribute to regulatory T (Treg) function via the programmed cell death-1 (PD-1)/PD-1 ligand 1 (PD-L1) pathway during normal pregnancy. Decreased expression of PD-L1 in trophoblasts was closely associated with Treg deficiency in the development of pregnancy failure. Thus, targeting PD-L1 might be a novel therapy to prevent pregnancy loss. However, the mechanisms for modulating the expression of PD-L1 in trophoblasts are an enigma. The proportion of decidual Treg cells, and the profile of decidual macrophages (DMs) sampled from women with normal pregnancy (NP) and recurrent miscarriage (RM) were evaluated by flow cytometry. The expression of Yin and Yang 1 protein (YY1) and PD-L1 in human villous were measured by Immunohistochemistry (IHC), qRT-PCR and western blot. The determination of soluble PD-L1 (sPD-L1) in serum from NP and RM, and trophoblast conditioned media (TCM) was performed by the PD-L1 SimpleStep ELISA kit. Knockdown of YY1 was processed in the human trophoblast derived cell lines, HTR-8 and Bewo, with siYY1 transfection. Peripheral naïve CD4+ T cells were isolated from women with NP for the in vitro culture. The percentages of Treg cells differentiated from peripheral naïve CD4+ T cells were measured by flow cytometry. The interaction between YY1 and CD274 was proved by CHIP. The expression of inducible nitric oxide synthase (iNOS) in decidua was evaluated by IHC. The level of NO in serum from women with NP and RM was determined by the Griess reagent system. The effects of NO on YY1 were determined by the in vitro culture of HTR-8 cells with the NO donor, SNAP. The in vivo model comprising twelve pregnant mice and underwent different treatment. The percentages of Treg cells in murine uterus were measured by flow cytometry. Similarly, Western blot and IHC were performed to determine the expression of YY1 and PD-L1 in murine placenta. Decreased expression of YY1 and PD-L1 in trophoblasts and lower proportion of decidual Treg cells were observed in patients with RM. Knockdown of YY1 contributes to a lower expression of YY1 and PD-L1. Soluble PD-L1 in the supernatant from HTR-8 cells was also decreased with siYY1 administration. Lower Treg differentiation was observed in the presence of supernatant from HTR-8 cells treated with siYY1. CHIP analysis revealed that endogenous YY1 directly occupied the promoter region of the CD274 (PD-L1) gene. Accompanied with increased M1 DMs, higher NO was observed in serum sampled from patients with RM. In the presence of Reduced expression of YY1 and PD-L1 was observed in HTR-8 cells with the treatment of SNAP. Furthermore, less Treg differentiation was observed with SNAP treated TCM. Moreover, our in vivo data found that YY1 deficiency was associated with decreased PD-L1, which further resulting in less Treg differentiation and Treg deficiency at the maternal-fetal interface and increased embryo loss. Our work found the modulatory capacity of YY1 on PD-L1 in trophoblasts during early pregnancy. Furthermore, reduced YY1 was supposed resulting from higher levels of NO produced from the M1 DMs in RM.

Background: Studies pointed out that the tumor-infiltrating lymphocytes (TILs) have considerable importance in canine mammary tumor (CMT). On the other hand, cancer cells sometimes find ways to use immune checkpoint proteins as a shield to avoid being identified and attacked by the immune system as programmed death 1 ligand 1 (PD-L1). In this study, it was investigated the relationship between PD-L1 expression, stromal tumor-infiltrating lymphocytes (TILs) in canine mammary tumor (CMT), and the association with clinical and pathological characteristics of the tumors.Materials, Methods & Results: PD-L1 expression and TILs were assessed in 23 female dogs with CMT. The tumors were grouped into simple carcinoma (CA, n = 8) and complex carcinoma (CC, n = 15). Stromal TILs were assessed using two thresholds as TILs-Low representing < 50% of infiltrate within stromal area and TILs-High representing ≥ 50% of stromal area. Clinicopathological data of CMT was characterized according to key parameters, as well as survival rates. TILs evaluation within tumor stroma revealed that 65.2% (n = 15) of tumors had TILs-Low. PD-L1 expression and stromal TILs were significantly associated (P = 0.009). PD-L1 expression was observed in 39% (n = 9) of all tumors of which 17.4% (n = 4) were from CA group and 21.7% (n = 5) were from CC group. PD-L1 expression within TILs was observed in 39% (n = 9) of the tumors. PD-L1 in malignant epithelium was present in all lymph node metastasis (n = 5). PD-L1 was associated with involvement of regional lymph nodes (P = 0.034). Survival curves demonstrated TILs-Low had higher (P = 0.010) overall survival (OS) compared with TILs-High, and PD-L1+ and PD-L1– (P = 0.06) did not differed. The clinicopathological variables significantly correlated with OS by univariate analysis were the histological grade (P = 0.009), lymph node involvement (P = 0.004), stromal TILs (P = 0.016), and PD-L1+/TILs-High vs. PD-L1–/TILs-Low (P = 0.010). Multivariate analysis revealed that group of tumors with grade II-III was independent and negative prognostic factors for OS.Discussion: In this study, PD-L1 was differently expressed according to the histologic subtypes of TMC. Currently, has been showed the presence of PD-L1 in several canine cancer. Nevertheless, only a few studies have described PD-L1 protein expression in dog tumors and showed PD-L1 was constitutively expressed on canine tumor cell lines, although the levels of basal expression were very variable. This expression can be modulated by IFN-γ exposure. In the present study, it was found a strong PD-L1 expression on TILs. The increase in PD-L1 cell surface expression by tumor cells can lead to decreased T-cell proliferation and increased apoptosis. In human breast cancer (BC) the PD-L1 expression was expressed in TILs and tumor epithelium. It has been reported the association of stromal TILs and PD-L1 expression with aggressive types and stages of BC. In this study, it was detected PD-L1 expression in malignant epithelium in all lymph node metastasis. PD-L1 overexpression was significantly associated with a series of clinicopathological parameters. It was demonstrated that PD-L1+/TILs-High had higher risk of overall survival (OS) than another group of interaction. High PD-L1 expression may be a prognostic indicator for reduced OS, while tumor PD-L1+ was associated with poorer disease-free survival. The presence of TILs has shown to be potentially predictive and a prognostic factor in BC subtypes. In CMT, it has been reported that a high proportion of TILs was correlated to several malignancy characteristics. In relation to PD-L1, further research is necessary to clarify this immune checkpoint as a potential therapeutic target and its application in clinical practice in CMT.

Background: Programmed cell death ligand 1 (PD-L1) is a major immune checkpoint protein that mediates anti-tumor immune suppression and response. Indeed, the importance of PD-L1 based immune suppression is highlighted by the advent of anti-PD-L1 immunotherapies that have moved to the forefront of lung cancer treatment. A better understanding of the “profile” of PD-L1 expression and its interplay with immune cells will provide important insights into lung cancer pathogenesis, and thus, improve immunotherapeutic strategies targeting this important immune checkpoint protein. The goal of this study was to investigate the correlation between immunohistochemical (IHC) expression of PD-L1 and the density and nature of tumor infiltrating immune cells in surgically resected lung adenocarcinomas, and correlate those profiles with clinical and pathological variables including patient outcome. Methods: We examined 146 stages I-III lung adenocarcinomas using whole tissue sections obtained from formalin-fixed and paraffin-embedded tissues. PDL-1 expression on tumor cells and density of inflammatory cells expressing PD-1, CD3, CD4, CD8, CD45RO, CD57, CD68, Granzyme B and FOXP3 were evaluated by automated IHC and quantified using image analysis in intra-tumoral (IT) and peri-tumoral (PT) compartments. PD-L1 expression was scored as positive if at least 5% of tumor cells showed membranous staining, and an immune-score (IMS) using CD8/CD4/CD68 was devised. KRAS and EGFR mutation status were available. Results: Thirty-four (23%) of 146 tumors had positive levels of PD-L1 IHC expression in malignant cells. Higher levels of PD-L1 expression were detected in tumors with solid histology pattern compared with other histologies (P = 0.034), and in lifetime smokers compared with non-smokers (P<0.0001). PDL-1 expression correlated positively with inflammatory cell density in both IT and PT compartments. KRAS mutant tumors with solid pattern showed significantly higher levels of PD-L1 and PD-1 expressions, and IMS, compared with non-solid tumors (P = 0.001). Using PD-L1 and CD8/CD4/CD68 IMS expression levels, we identified 4 groups of tumors: PD-L1high/IMShigh 18%; PD-L1high/IMSlow 5%; PD-L1low/IMShigh 35%; and, PD-L1low/IMSlow 42%. Finally, multivariate Cox proportional hazard regression analysis demonstrated that tumors with high PD-L1 expression and low IMS exhibited significantly poor recurrence-free (HR = 8.975; P<0.0001) and overall survival (HR = 4.220; P = 0.0015). Conclusions: In lung adenocarcinoma, PD-L1 expression correlates with less differentiated tumors and higher level of tumoral immune infiltrate. We developed a lung adenocarcinoma IMS which when combined with PD-L1 expression significantly correlates with patient outcome when analyzed in surgically resected tumors specimens. (Supported by grants UT-Lung SPORE P50CA70907 and CPRIT RP120713). Citation Format: Edwin R. Parra, Carmen Behrens, Jaime Rodriguez-Canales, Heather Lin, Barbara Mino, Jorge Blando, Yanyan Lou, Don L. Gibbons, John V. Heymach, Stephen G. Swisher, Annikka Weissferdt, Neda Kahlor, Julie Izzo, J. Jack Lee, Humam Kadara, Cesar Moran, Ignacio Wistuba. High programmed cell death ligand 1 expression and low immune infiltrate score correlate with worse outcome in patients with lung adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4256. doi:10.1158/1538-7445.AM2015-4256

Shorter Survival in Malignant Pleural Mesothelioma Patients With High PD-L1 Expression Associated With Sarcomatoid or Biphasic Histology Subtype: A Series of 214 Cases From the Bio-MAPS Cohort

Novel Mechanism of Post-Transcriptional Regulation of PD-L1 Expression By 3'-UTR Binding Proteins

BACKGROUNDAnti-programmed death therapy has thrust immunotherapy into the spotlight. However, such therapy has a modest response in hepatocellular carcinoma (HCC). Epigenetic immunomodulation is a suggestive combinatorial therapy with immune checkpoint blockade. Non-coding ribonucleic acid (ncRNA) driven regulation is a major mechanism of epigenetic modulation. Given the wide range of ncRNAs that co-opt in programmed cell-death protein 1 (PD-1)/programmed death ligand 1 (PD-L1) regulation, and based on the literature, we hypothesized that miR-155-5p, miR-194-5p and long non-coding RNAs (lncRNAs) X-inactive specific transcript (XIST) and MALAT-1 are involved in a regulatory upstream pathway for PD-1/PD-L1. Recently, nutraceutical therapeutics in cancers have received increasing attention. Thus, it is interesting to study the impact of oleuropein on the respective study key players.AIMTo explore potential upstream regulatory ncRNAs for the immune checkpoint PD-1/PD-L1.METHODSBioinformatics tools including microrna.org and lnCeDB software were adopted to detect targeting of miR-155-5p, miR-194-5p and lncRNAs XIST and MALAT-1 to PD-L1 mRNA, respectively. In addition, Diana tool was used to predict targeting of both aforementioned miRNAs to lncRNAs XIST and MALAT-1. HCC and normal tissue samples were collected for scanning of PD-L1, XIST and MALAT-1 expression. To study the interaction among miR-155-5p, miR-194-5p, lncRNAs XIST and MALAT-1, as well as PD-L1 mRNA, a series of transfections of the Huh-7 cell line was carried out. RESULTSBioinformatics software predicted that miR-155-5p and miR-194-5p can target PD-L1, MALAT-1 and XIST. MALAT-1 and XIST were predicted to target PD-L1 mRNA. PD-L1 and XIST were significantly upregulated in 23 HCC biopsies compared to healthy controls; however, MALAT-1 was barely detected. MiR-194 induced expression elevated the expression of PD-L1, XIST and MALAT-1. However, overexpression of miR-155-5p induced the upregulation of PD-L1 and XIST, while it had a negative impact on MALAT-1 expression. Knockdown of XIST did have an impact on PD-L1 expression; however, following knockdown of the negative regulator of X-inactive specific transcript (TSIX), PD-L1 expression was elevated, and abolished MALAT-1 activity. Upon co-transfection of miR-194-5p with siMALAT-1, PD-L1 expression was elevated. Co-transfection of miR-194-5p with siXIST did not have an impact on PD-L1 expression. Upon co-transfection of miR-194 with siTSIX, PD-L1 expression was upregulated. Interestingly, the same PD-L1 expression pattern was observed following miR-155-5p co-transfections. Oleuropein treatment of Huh-7 cells reduced the expression profile of PD-L1, XIST, and miR-155-5p, upregulated the expression of miR-194-5p and had no significant impact on the MALAT-1 expression profile.CONCLUSIONThis study reported a novel finding revealing that opposing acting miRNAs in HCC, have the same impact on PD-1/PD-L1 immune checkpoint by sharing a common signaling pathway.

Recurrent PDL1 expression and PDL1 (CD274) copy number alterations in breast implant–associated anaplastic large cell lymphomas

Background: Programmed death ligand 1 (PD-L1) expression on tumor cells (TC), detected by immunohistochemistry (IHC), is associated with response to programmed death-1 (PD-1)/PD-L1 inhibitors in some tumor types. Manual review of PD-L1–positive (PD-L1+) tumors can be subjective, with the potential for misclassification of PD-L1–low tumors as PD-L1–negative due to weak positivity. We compared artificial-intelligence (digital) and manual scoring methods and assessed the association of PD-L1 expression with clinical outcomes in nivolumab (NIVO)-treated patients with urothelial carcinoma (UC) and melanoma (MEL). Methods: PD-L1 expression was determined in baseline samples from NIVO monotherapy-treated patients with UC (CM275, NCT02387996) and MEL (CM067, NCT01844505; CM238, NCT02388906) using the Dako PD-L1 IHC 28-8 pharmDx assay. PD-L1+ TC were scored using digital (PathAI research platform) and manual (LabCorp) methods. Prevalence of tumors with PD-L1+ TC ≥ 1% and ≥ 5% and associations between PD-L1 expression and outcomes with NIVO were evaluated. Results: Prevalence of UC and MEL tumors with ≥ 1% and ≥ 5% PD-L1+ TC was higher for digital vs manual scoring (Table). For all samples, digital and manual scoring was associated with response to NIVO for PD-L1 ≥ 1% and ≥ 5%, and associations were similar between digital and manual scoring (Table). Digital and manual PD-L1 scoring correlated across samples from all trials (Kendall's tau range: 0.57–0.62). TablePrevalence PD-L1+ TC ≥ 1%, n (%)Evaluable samples, nDigitalManualP valueSamples ≥ 1% by digital onlyCM275241166 (69)113 (47)1.61 × 10−658 (24)CM067264173 (66)160 (61)0.27936 (14)CM238377307 (81)259 (69)7.61 × 10−566 (18)PD-L1+ TC ≥ 1% vs < 1%DigitalManualORR, odds ratio (95% CI)CM275a2.15 (0.98–4.70)1.60 (0.82–3.14)CM067b1.99 (1.19–3.35)1.89 (1.12–3.18)Survival, hazard ratio (95% CI)CM275 (OS)a0.67 (0.48–0.92)0.66 (0.48–0.90)CM067 (OS)b0.57 (0.41–0.80)0.71 (0.50–1.00)CM238 (RFS)c0.53 (0.36–0.77)0.83 (0.57–1.21)Prevalence PD-L1+ TC ≥ 5%, n (%)Evaluable samples, nDigitalManualP valueSamples ≥ 5% by digital onlyCM27524190 (37)74 (31)0.14928 (12)CM067264103 (39)76 (29)0.01736 (14)CM238377234 (62)139 (37)7.54 × 10−12104 (28)PD-L1+ TC ≥ 5% vs < 5%DigitalManualORR, odds ratio (95% CI)CM275a3.50 (1.76–6.98)2.37 (1.18–4.73)CM067b2.33 (1.40–3.86)1.77 (1.01–3.09)Survival, hazard ratio (95% CI)CM275 (OS)a0.50 (0.36–0.71)0.58 (0.41–0.83)CM067 (OS)b0.67 (0.47–0.96)0.74 (0.51–1.09)CM238 (RFS)c0.50 (0.35–0.70)0.52 (0.36–0.76)Database lock 2019: CM275, June 14; CM067, January 18; CM238, April 3.aAdjusted for ECOG performance status, liver metastatic status, and hemoglobin.bAdjusted for ECOG performance status, liver metastatic status, lactate dehydrogenase, and BRAF mutation.cAdjusted for ECOG performance status, AJCC stage, lactate dehydrogenase, and BRAF mutation.AJCC, American Joint Committee on Cancer; CI, confidence interval; ECOG, Eastern Cooperative Oncology Group; ORR, objective response rate; OS, overall survival; PD-L1, programmed death ligand 1; RFS, recurrence-free survival; TC, tumor cells. Conclusion: In post-hoc exploratory analyses, digital scoring of PD-L1 expression identified higher prevalence of PD-L1+ tumors and shows good association with response to NIVO in UC and MEL samples compared with manual scoring. Digital quantification demonstrated higher sensitivity at low levels of PD-L1 expression and may identify patients who could benefit from NIVO. Further study of the association with clinical outcomes is warranted and exploratory studies are ongoing to assess the performance of digital scoring in additional tumor types. Citation Format: Chunzhe Duan, Michael Montalto, George Lee, Dimple Pandya, Daniel Cohen, Han Chang, Hao Tang, Nishant Agrawal, Hunter Elliott, Benjamin Glass, Ilan Wapinski, Robin Edwards, Andrew H. Beck, Vipul Baxi. Association of digital and manual quantification of tumor PD-L1 expression with outcomes in nivolumab-treated patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2017.

Approximately 90% of thyroid cancers are differentiated thyroid cancers (DTCs), originating from follicular epithelial cells. Out of these, 90% are papillary thyroid cancer (PTC), and 10% are follicular thyroid cancer (FTC). The standard care procedure for PTC includes surgery, followed by radioiodine (RAI) ablation and thyroid-stimulating hormone (TSH) suppressive therapy. Globally, treating radioiodine-refractory DTC poses a challenge. During malignant transformation, thyroid epithelial cells often lose their ability to absorb radioiodine due to impaired membrane targeting or lack of NIS (sodium/iodide symporter) expression. Recent reports show an increase in PD-L1 (programmed death ligand 1) expression in thyroid cancer cells during dedifferentiation. However, no research exists wherein NIS and PD-L1 expression are analyzed together in thyroid cancer. Therefore, we aimed to investigate and correlate PD-L1 and NIS expression within primary tumor samples of lymph node metastatic PTC. We analyzed the expression of hNIS (human sodium/iodide symporter) and PD-L1 in primary tumor samples from metastatic PTC patients using immunohistochemistry. Immunohistochemistry analysis of PD-L1 and NIS was conducted in 89 and 86 PTC cases, respectively. Any subcellular NIS localization was counted as a positive result. PD-L1 expression was absent in 25 tumors, while 58 tumors displayed PD-L1 expression in 1-50% of their cells; in 6 tumors, over 50% of the cells tested positive for PD-L1. NIS immunohistochemistry was performed for 86 primary papillary carcinomas, with 51 out of 86 tumors showcasing NIS expression. Only in seven cases was NIS localized in the plasma membrane; in most tumors, NIS was primarily found in the intracytoplasmic membrane compartments. In the case of PD-L1 staining, cells showing linear membrane positivity of any intensity were counted as positive. The evaluation of NIS immunostaining was simpler: cells showing staining of any intensity of cytoplasmic or membranous fashion were counted as positive. The number of NIS positive cells can be further divided into cytoplasmic and membrane positive compartments. There was no observed correlation between PD-L1 and NIS expression. We can speculate that the manipulation of the PD-1/PD-L1 axis using anti-PD-L1 or anti-PD-1 antibodies could reinstate the functional expression of NIS. However, based on our study, the only conclusion that can be drawn is that there is no correlation between the percentage of NIS- or PD-L1-expressing tumor cells in the primary tumor of lymph node metastatic PTC.

8551 Background: Similar to programed death ligand 1 (PD-L1), indoleamine 2,3-Dioxygenase 1 (IDO1) is known to exert immunosuppressive effects and be variably expressed in human lung cancer. However, IDO1 expression has not been well-studied in lung adenocarcinoma (ADC). Methods: PD-L1 and IDO1 expression were evaluated in 261 resected ADC using tissue microarrays and H-scores (cutoff 5). We compared IDO1 with PD-L1 expression in association with clinical features, tumor-infiltrating lymphocytes (TILs), HLA class I (β-2 microglobulin; B2M) expression, molecular alterations, and patient outcomes. Results: There was expression of PD-L1 in 89 (34.1%) and IDO1 in 74 (28.5%) cases, with co-expression in 49 (18.8%). Both PD-L1 and IDO1 were significantly associated with smoking, aggressive pathologic features, and abundant CD8+ and T-bet+ (Th1 marker) TILs. PD-L1 expression and abundant CD8+ were inversely associated with a loss of B2M membranous expression (p = 0.0019 and p < 0.001, respectively). Compared to PD-L1+/IDO1+ and PD-L1+ only cases, significantly fewer IDO1+ only cases had abundant CD8+ and T-bet+ TILs (p < 0.001, respectively). PD-L1 expression was significantly associated with EGFR wild-type (p < 0.001) and KRAS mutants (p = 0.021), whereas there was no difference in IDO1 expression between different molecular alterations. As for survival, PD-L1 was significantly associated with decreased progression-free (PFS) and overall survival (OS), while IDO1 was associated only with decreased OS. Interestingly, there was a significant difference in the 5-year PFS and OS (p = 0.004 and 0.038, respectively), where cases without PD-L1 or IDO1 expression had the longest survival, and those with PD-L1 alone had the shortest survival. Conclusions: While PD-L1 +/- IDO1 expression is observed in association with B2M expression, CTL/Th1 microenvironments, EGFR wild-type, and KRAS mutations, isolated IDO1 expression does not demonstrate these associations, suggesting that IDO1 may serve a distinct immunosuppressive role in ADC. Thus, blockade of IDO1 may represent an alternative and/or complementary therapeutic strategy to reactivate anti-tumor immunity.

In this issue of Neuro-Oncology, Berghoff et al present the results of their investigations into the expression of programmed death-ligand 1 (PD-L1) in human glioblastoma specimens and their relationship to other tissue-based and clinical parameters. In specimens from adults with newly diagnosed or recurrent glioblastoma, the investigators reported that diffuse/fibrillary PD-L1 expression of variable extent was found in more than 70% of glioblastoma specimens, with a higher proportion in newly diagnosed cases. Furthermore, more than 70% of cases showed evidence of high tumor-infiltrating lymphocyte (TIL) infiltration, although most were of sparse-to-moderate density. In glioblastoma samples from the Cancer Genome Atlas, the relationships between PD-L1 expression, molecular subtypes, and clinical outcomes were explored. Samples with known molecular subtypes were then classified according to their level of PD-L1 expression (high or low), and a significant difference in the distribution of PD-L1 high and low groups was found across molecular subtypes, with the mesenchymal subtype in particular having a high level of PD-L1 expression and the proneural and G-CIMP subtypes primarily having low expression of PD-L1. The anti-PD-L1 antibody, 5H1, used in this study is not commercially available, although its use has been confirmed in other experiments, whereas some commercially available antibodies have failed to show reliable PD-L1 labeling.1 The authors conclude that PD-L1 expression and TILs were found in most of the glioblastoma samples they evaluated, but a relationship between these parameters and outcome was not observed. Still, the high expression of PD-L1 in these glioblastoma samples suggests that it may be a valid target for further clinical investigation.

The relationship between cancer stem cells (CSCs) in oral squamous cell carcinoma (OSCC) and programmed cell death ligand 1 (PD-L1)/programmed cell death 1 (PD-1) remains unclear. Therefore, the present study aimed to clarify the association between the CD44v3high/CD24low immunophenotype of CSCs in OSCC and PD-L1/PD-1 co-expression, and to assess the prognostic effect of CSCs in terms of immune checkpoint molecules. Formalin-fixed, paraffin-embedded tissue samples and clinicopathological data from 168 patients with OSCC were retrospectively retrieved. Immunohistochemical staining and reverse transcription quantitative polymerase chain reaction were applied to a tissue microarray of the invasive front of each case. Semi-automated cell counting was used to assess CD44v3, CD24, PD-L1 and PD-1 expression by immunohistochemistry (IHC) using a digital image analysis program. Associations between immunological markers and clinicopathological variables were estimated. Patients with the CSC immunophenotype CD44v3high/CD24low, and patients with a high PD-L1/PD-1-positive cell density in the tumor parenchyma and stroma had significantly lower survival rates. Furthermore, patients with the CSC immunophenotype (CD44v3high/CD24low) and high PD-L1/PD-1 co-expression had even lower survival rates (P<0.01, log-rank test). Notably, there was a positive correlation between CD44v3 and PD-L1 expression (τ=0.1096, P=0.0366, Kendall rank correlation coefficient) and a negative correlation between CD24 and PD-1 expression (τ=-0.1387, P=0.0089, Kendall rank correlation coefficient). Additionally, the high CD44v3 expression group, as determined by IHC, exhibited significantly decreased expression of U2 small nuclear RNA auxiliary factor 1 (U2AF1) at the mRNA level compared with that in the low CD44v3 expression group (P<0.001, Mann-Whitney U test), and U2AF1 and PD-L1 mRNA expression exhibited a significant negative correlation (τ=-0.3948, P<0.001, Kendall rank correlation coefficient). In conclusion, CSCs in OSCC may evade host immune mechanisms and maintain CSC stemness via PD-L1/PD-1 co-expression, resulting in unfavorable clinical outcomes.