Assessment Of PD-L1 Expression Research Articles

771 Increased PD-L1 expression in human skin acutely and chronically exposed to UV irradiation

Overexpression of the ligand PD-L1 on tumor cells may represent a hallmark of tumor immune evasion, and such overexpression has been documented in several tumors including cutaneous squamous cell carcinoma (cSCC). While PD-L1/PD-1 activity in the skin has been primarily described in inflammatory models, we focus on PD-L1 expression in human keratinocytes exposed to UV irradiation. We first assessed PD-L1 expression in archived samples from acute human studies implementing different UV exposures in sun protected (SP) skin ranging from 2-3 minimal erythema doses (MED). PD-L1 expression was measured by immunohistochemistry (IHC). From the 20 samples evaluated 14 (70%) demonstrated > 5% epidermal PD-L1 expression at 24 hours. PD-L1 in dermal endothelial cells was also noted to be increased at 24 hrs. Thereafter, we determined that HaCaT cells also respond to acute UV by increasing PD-L1 protein expression in a time- and dose-dependent manner (2-4 fold over control at 24-48 hours), an effect which is markedly reduced in the presence of MEK/ERK or JNK inhibitors. Subsequently, we investigated PD-L1 expression in chronically UV exposed human skin using matched samples representing SP skin, sun damaged skin (SD), actinic keratosis (AK) and cSCC using IHC and reverse phase protein microarray technology (RPPA). The RPPA methodology revealed negligible PD-L1 expression in SP, SD and AK, with significantly measurable expression in cSCC. IHC analysis of PD-L1 in another cohort of matched samples revealed a similar profile, with negligible expression of PD-L1 in SP and SD skin, slight staining in AK (mean 4.2%, +/- 9.5), and significant staining in cSCC (mean 19.5%, +/- 26.6). These findings suggest that upregulation of PD-L1 may be induced by UV through established, pharmacologically targetable stress-signaling pathways in keratinocytes.

Assessment of PD-L1 expression in patients with urothelial cancer with contraindications to platinum based therapy

Background. Urothelial cancer ranks 7th and 17th of all the malignant tumors in males and females, respectively. Development of new immunotherapeutic drugs provides new possibilities in treatment of such patients, especially the patient population in whom platinum‑based therapy is contraindicated. Administration of immunooncology drugs requires determination of PD-L1, for which various diagnostic systems are used. The question regarding correlation of results of determination of PD-L1 expression remains of concern.Materials and Methods. The study was performed on 100 samples of surgical and biopsy samples of urothelial cancer. Two clones of 22C3 and SP142 with corresponding detection systems were used for the study. PD-L1 expression was assessed in tumor and immune cells.Results. The study demonstrated a high correlation of negative PD-L1 tumor status determined using both diagnostic agents (92 % and 97 %) and low correlation of results of positive PD-L1 status (67 % and 43 %).Conclusions. Thus, if a negative result of PD-L1 status of urothelial cancer is obtained using any of the diagnostic agents studied, repeated test with the other antibody is not required. If positive status is obtained in one test, the patient may have a negative status in the other test, which allows recommending a repeated testing in borderline cases using a test, recommended for the medicinal product untended for treatment.

MicroRNAs aid the assessment of programmed death ligand 1 expression in patients with non-small cell lung cancer.

The qualification of patients with non-small cell lung cancer (NSCLC) for anti-programmed cell death 1 (PD-1) or anti-programmed death ligand 1 (PD-L1) antibody therapy is based on an immunohistochemistry (IHC) assessment of PD-L1 expression. Immunological checkpoint inhibitors improve the overall survival of patients with expression of PD-L1; however certain PD-L1-negative patients may also benefit from immunotherapy. This indicates the requirement for novel predictive factors for the qualification of immunotherapy. It is also necessary to understand the mechanisms that effect the expression of PD-L1 in tumor cells. The expression of PD-L1 in 47 formalin-fixed, paraffin-embedded, NSCLC specimens was assessed using IHC and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The expression of 8 microRNAs (miRNAs, miRs) complementary to PD-L1-mRNA was also evaluated using RT-qPCR. A positive correlation was revealed between the expression level of PD-L1-mRNA and 2 miRs, miR-141 (R=0.533; P=0.0029) and miR-1184 (R=0.463; P=0.049). There was also a positive correlation between the percentage of PD-L1-positive tumor cells and the expression levels of miR-141 (R=0.441; P=0.0024), miR-200b (R=0.372; P=0.011) and miR-429 (R=0.430; P=0.0028), and between the percentage of the tumor area with immune cell infiltration and the expression levels of miR-141 (R=0.333; P=0.03) and miR-200b (R=0.312; P=0.046). Additionally, the percentage of tumor cells expressing PD-L1 positively correlated with miR-141 expression (R=0.407; P=0.0055). Correlations between the expression of the investigated miRs (particularly miR-141) and PD-L1 indicated that miRs may regulate PD-L1 expression at a post-transcriptional level.

P3.09-04 Assessment of PD-L1 Expression in Cytology Samples: An Anlaysis of 263 Consecutive NSCLC Biopsies.

P3.09-04 Assessment of PD-L1 Expression in Cytology Samples: An Anlaysis of 263 Consecutive NSCLC Biopsies.

MA09.09 EBUS-TBNA in Assessing PD-L1 Expression in NSCLC

MA09.09 EBUS-TBNA in Assessing PD-L1 Expression in NSCLC

P1.04-24 Digital Core Needle-Biopsy to Assess PD-L1 Expression in Non-Small Cell Lung Cancer: Optimal Sampling and Need for Re-Biopsy

P1.04-24 Digital Core Needle-Biopsy to Assess PD-L1 Expression in Non-Small Cell Lung Cancer: Optimal Sampling and Need for Re-Biopsy

Comparative Assessment of PD-L1 Expression in Cytologic and Histologic Samples of Non-small Cell Lung Carcinoma

Comparative Assessment of PD-L1 Expression in Cytologic and Histologic Samples of Non-small Cell Lung Carcinoma

Abstract 4212: PD-L1 expression, EGFR and KRAS mutations in unresectable stage III non-small cell lung cancer (NSCLC) patients

Abstract Background: Anti-PD-L1 therapy may improve prognosis in advanced NSCLC. We examined the relation of PD-L1 expression, KRAS and EGFR mutations, with survival in unresectable stage III NSCLC patients. Methods: We obtained data on unresectable stage III NSCLC patients (defined via TNM AJCC staging and without surgery up to 120 days after diagnosis) diagnosed 2001-2012. We retrieved medical data from Danish population-based registries and paraffin-embedded tumor tissue from pathology archives. We assessed PD-L1 expression using the Ventana IHC (SP263) validated assay. We genotyped KRAS and EGFR using PCR-based kits. Follow-up began at NSCLC diagnosis and continued to death, emigration, or 31/12/2014. We used Cox regression to compute hazard ratios (HRs) and associated 95% confidence intervals (95%CI) for PD-L1, EGFR, and KRAS. Results: Among 305 patients, 183 (60%) were men, 167 (55%) were aged >65 years at diagnosis and none used immunotherapy; 148 (49%) had adenocarcinoma, 117 (38%) squamous histology, 96 (31.5%) had PD-L1 positive tumors (>=25%), 6 (2%) had EGFR mutations, and 69 (23%) had KRAS mutations. Among PD-L1 positive tumors, 55% had stage IIIA, 45% IIIB disease; 1% had EGFR and 34% had KRAS mutations. Among PD-L1 negative tumors, 54% had stage IIIA and 46% IIIB; 3% had EGFR and 16% had KRAS mutations. Median survival was similar in patients with KRAS wild-type and KRAS mutations (Hazards ratio (HR)=1.07; 95% CI=0.76-1.51). Patients with an EGFR mutation had a lower non-statistically significant risk of death (HR=0.75; 95% CI=0.28-2.06). Tumor cell positivity for PD-L1 (>=25% versus <25%) yielded an HR=0.80 (95%CI=0.60-1.07). Immune cell positivity for PD-L1 >=1% and >=25% yielded HRs of 0.50 (95%CI=0.39-0.66) and 0.45 (95%CI=0.30-0.67), respectively. Tumor infiltration by immune cells with PD-L1 measured as a continuous variable yielded an HR=0.96 (95%CI=0.93-0.99). Conclusion: Our findings suggest that PD-L1 expression and EGFR mutations, but not KRAS mutations, are associated with survival in this real-world Danish study of unresectable Stage III NSCLC patients. Citation Format: Deirdre Cronin-Fenton, Tapashi Dalvi, Elizabeth Hedgeman, Mette Norgaard, Lars Pedersen, Hanh Hansen, Jon Fryzek, Jill Walker, Anders Mellemgaard, Torben Rasmussen, Norah Shire, James Riggas, Danielle Potter, Stephen Hamilton-Dutoit, Henrik Sørensen. PD-L1 expression, EGFR and KRAS mutations in unresectable stage III non-small cell lung cancer (NSCLC) patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4212.

Immuno-oncology biomarker study in a large cohort of LC-SCRUM-Japan: Assessment of PD-L1 expression and tumor mutation burden in non-small cell lung cancer patients treated with immune checkpoint inhibitors.

9070Background: PD-L1 high expression and high tumor mutation burden (TMB) are reported to be correlated with high sensitivity to immune checkpoint inhibitor (ICI) in non-small cell lung cancer (NS...

Investigation of PD-L1 expression and response to pembrolizumab (pembro) in gastric cancer (GC) and cervical cancer (CC) using combined positive score (CPS) and tumor proportion score (TPS).

4065Background: TPS, the percentage of viable tumor cells with partial or complete membrane staining at any intensity, has been invaluable for assessing PD-L1 expression in non–small cell lung canc...

4P Assessment of PD-L1 expression by immunohistochemistry in histological and cytological non-small cell lung carcinoma (NSCLC) in the era of immunotherapy: A national Irish study

4P Assessment of PD-L1 expression by immunohistochemistry in histological and cytological non-small cell lung carcinoma (NSCLC) in the era of immunotherapy: A national Irish study

A multicenter round robin test of PD-L1 expression assessment in urothelial bladder cancer by immunohistochemistry and RT-qPCR with emphasis on prognosis prediction after radical cystectomy

BackgroundImmunohistochemical PD-L1 assessment is currently used to identify responders towards checkpoint inhibitors although it is limited by inter-observer effects. Here, we conducted a multi-center round robin test to prove the possibility of assessing the PD-L1 status by gene expression to avoid inter-observer effects.Patients and methodsGene expression of PD-L1 was analyzed in a total of 294 samples (14 cases non-muscle invasive and muscle-invasive bladder cancer; MIBC) in seven centers by a RT-qPCR kit and compared with immunohistochemical scoring of three pathologists (DAKO, 22c3). Both assays were compared towards prognosis prediction in a cohort of 88 patients with MIBC.ResultsPD-L1 gene expression revealed very high inter center correlation (centrally extracted RNA: r = 0.68–0.98, p ≤ 0.0076; locally extracted RNA: r = 0.81–0.98, p ≤ 0.0014). IHC Inter-observer concordance was moderate to substantial for immune cells (IC), fair for combined IC/ tumor cell (TC) (IC: κ = 0.50–0.61; IC + TC: κ = 0.50), and fair for TC scoring (κ = 0.26–0.35). Gene expression assessment resulted in more positive cases (9/14 cases positive vs. 6/14 cases [IHC]) which could be validated in the independent cohort. Positive mRNA status was associated with significantly better overall and disease-specific survival (5-year OS: 50% vs. 26%, p = 0.0042, HR = 0.48; 5 year DSS: 65% vs. 40%, p = 0.012, HR = 0.49). The 1% IHC IC cut-off also revealed significant better OS (5 year OS: 58% vs. 31%, p = 0.036, HR = 0.62).ConclusionGene expression showed very high inter-center agreement. Gene expression assessment also resulted in more positive cases and revealed better prognosis prediction. PD-L1 mRNA expression seems to be a reproducible and robust tool for PD-L1 assessment.

PD-L1 expression in colorectal cancer defines three subsets of tumor immune microenvironments.

ObjectivesWe investigated the PD-L1 expression in colorectal cancer (CRC) and in its microenvironment.ResultsPD-L1 was expressed in neoplastic cells (NCs) and tumor-infiltrating immune cells (IICs). All samples PD-L1+ on NCs were also on IICs. Three types of cancers could be grouped: group A(NCs-/ IICs-); group B (NCs-/ IICs+); group C (NCs+/IICs+). To group A belong tumors characterized by poorly immunogenic competence, poor immune response but massive granulocyte infiltrate, justifying the absence of PD-L1 as an immunoinhibitor receptor. To Group B probably belong more immunogenic CRCs, justifying the strong IICs-mediated immune response, and up-regulation of PD-L1 expression only on IICs. To group C belong CRCs probably characterized by a large amount of tumor neoantigens resulting in a marked infiltration of lymphocytes and PD-L1 upregulation also in NCs.Materials and MethodsSixty-three colorectal cancer specimens from a cohort of 61 patients were retrospectively reviewed. Thirty-seven MSS and 26 MSI-H CRCs enrolled in this study. Immunohistochemical staining to PD-L1 was performed by using MAb E1L3N.ConclusionsOur study calls attention to the importance to assess PD-L1 expression in tumor microenvironment also evaluating type and density of infiltrating immune cells to better stratify CRCs with different immunological patterns.

Gene code CD274/PD-L1: from molecular basis toward cancer immunotherapy.

The programmed death 1 receptor (PD-1) and its ligand (PD-L1) are key molecules of immune checkpoint mechanisms in cancer and actually represent one of the main targets of immunotherapy. The predictive and prognostic values of PD-L1 expression alone in cancer patients is currently under debate due to the methodological assessment of PD-L1 expression and its temporal variations. Better detailed studies about the molecular basis of immunotherapy biomarkers are necessary. Here we summarize the current knowledge of PD-L1 gene modifications at genetic and epigenetic levels in different tumors, thus highlighting their reported correlation with cellular processes and potential impact on patient outcomes.

P2.02-072 Reliability of Small Biopsy Samples for Tumor PD-L1 Expression in Non-Small-Cell Lung Cancer

Programmed death 1 immune checkpoint inhibitor antibody has been effective in patients with PD-L1 positive advanced NSCLC. However, surgically resected specimens or core-needle biopsy samples were used to estimate drug potency in past clinical trials. The aim of this study is to prospectively investigate small sample reliability for NSCLC to determine the PD-L1 expression status. We prospectively enrolled patients who underwent diagnostic biopsy by any procedures (under rigid bronchoscopy, flexible bronchoscopy, CT & US-guided core-needle, excisional method) from March 2017 to March 2018 (ongoing study). Pathologically confirmed non-small cell lung cancer (NSCLC), PD-L1 expression was evaluated in our institution using companion diagnostic PD-L1 immunohistochemistry：PD-L1 IHC 22C3 pharmDx (Daco) with autostainer Link 48, detecting a driver mutation in parallel. We evaluated: 1) the total number of tumor cells and sample size; 2) compared PD-L1 expression for each procedure using tumor proportion score: TPS (<1%, 1～49%, 50%≤), 3) the concordance rate of PD-L1 expression status by biopsy and surgical materials; and 4) the efficacy of administration for PD-1 immune checkpoint inhibitor antibody. During the first three months 44 cases of PD-L1 expression were evaluated. 33 cases were sampled by bronchoscopy (6 under rigid scope with BF 1T260, 17 TBBs using 1T260/P260F in 4/13 cases, 10 TBNAs), and 7 cases were CT-guided core-needle biopsy. The TPS (<1%, 1～49%, 50%≤) was 8/6/10 for TBB, 3/4/2 for TBNA, and 4/0/3 for CT-guided. Four cases harboring EGFR mutation showed a lower PD-L1 expression (<10%). Utilizing smaller samples to evaluate PD-L1 expression, and the frequency of TPS were comparable to past clinical trials using larger samples. Smaller samples might be an accurate alternative to assess PD-L1 expression. Current data will be updated at the conference.

Synthesis and Biologic Evaluation of a Novel 18F-Labeled Adnectin as a PET Radioligand for Imaging PD-L1 Expression.

The programmed death protein (PD-1) and its ligand (PD-L1) play critical roles in a checkpoint pathway cancer cells exploit to evade the immune system. A same-day PET imaging agent for measuring PD-L1 status in primary and metastatic lesions could be important for optimizing drug therapy. Herein, we have evaluated the tumor targeting of an anti-PD-L1 adnectin after 18F-fluorine labeling. Methods: An anti-PD-L1 adnectin was labeled with 18F in 2 steps. This synthesis featured fluorination of a novel prosthetic group, followed by a copper-free click conjugation to a modified adnectin to generate 18F-BMS-986192. 18F-BMS-986192 was evaluated in tumors using in vitro autoradiography and PET with mice bearing bilateral PD-L1-negative (PD-L1(-)) and PD-L1-positive (PD-L1(+)) subcutaneous tumors. 18F-BMS-986192 was evaluated for distribution, binding, and radiation dosimetry in a healthy cynomolgus monkey. Results:18F-BMS-986192 bound to human and cynomolgus PD-L1 with a dissociation constant of less than 35 pM, as measured by surface plasmon resonance. This adnectin was labeled with 18F to yield a PET radioligand for assessing PD-L1 expression in vivo. 18F-BMS-986192 bound to tumor tissues as a function of PD-L1 expression determined by immunohistochemistry. Radioligand binding was blocked in a dose-dependent manner. In vivo PET imaging clearly visualized PD-L1 expression in mice implanted with PD-L1(+), L2987 xenograft tumors. Two hours after dosing, a 3.5-fold-higher uptake (2.41 ± 0.29 vs. 0.82 ± 0.11 percentage injected dose per gram, P < 0.0001) was observed in L2987 than in control HT-29 (PD-L1(-)) tumors. Coadministration of 3 mg/kg ADX_5322_A02 anti-PD-L1 adnectin reduced tumor uptake at 2 h after injection by approximately 70%, whereas HT-29 uptake remained unchanged, demonstrating PD-L1-specific binding. Biodistribution in a nonhuman primate showed binding in the PD-L1-rich spleen, with rapid blood clearance through the kidneys and bladder. Binding in the PD-L1(+) spleen was reduced by coadministration of BMS-986192. Dosimetry estimates indicate that the kidney is the dose-limiting organ, with an estimated human absorbed dose of 2.20E-01 mSv/MBq. Conclusion:18F-BMS-986192 demonstrated the feasibility of noninvasively imaging the PD-L1 status of tumors by small-animal PET studies. Clinical studies with 18F-BMS-986192 are under way to measure PD-L1 expression in human tumors.

Intra- and Interobserver Reproducibility Assessment of PD-L1 Biomarker in Non-Small Cell Lung Cancer.

Purpose: Reliable and reproducible methods for identifying PD-L1 expression on tumor cells are necessary to identify responders to anti-PD-1 therapy. We tested the reproducibility of the assessment of PD-L1 expression in non-small cell lung cancer (NSCLC) tissue samples by pathologists.Experimental Design: NSCLC samples were stained with PD-L1 22C3 pharmDx kit using the Dako Autostainer Link 48 Platform. Two sample sets of 60 samples each were designed to assess inter- and intraobserver reproducibility considering two cut points for positivity: 1% or 50% of PD-L1 stained tumor cells. A randomization process was used to obtain equal distribution of PD-L1 positive and negative samples within each sample set. Ten pathologists were randomly assigned to two subgroups. Subgroup 1 analyzed all samples on two consecutive days. Subgroup 2 performed the same assessments, except they received a 1-hour training session prior to the second assessment.Results: For intraobserver reproducibility, the overall percent agreement (OPA) was 89.7% [95% confidence interval (CI), 85.7-92.6] for the 1% cut point and 91.3% (95% CI, 87.6-94.0) for the 50% cut point. For interobserver reproducibility, OPA was 84.2% (95% CI, 82.8-85.5) for the 1% cut point and 81.9% (95% CI, 80.4-83.3) for the 50% cut point, and Cohen's κ coefficients were 0.68 (95% CI, 0.65-0.71) and 0.58 (95% CI, 0.55-0.62), respectively. The training was found to have no or very little impact on intra- or interobserver reproducibility.Conclusions: Pathologists reported good reproducibility at both 1% and 50% cut points. More adapted training could potentially increase reliability, in particular for samples with PD-L1 proportion, scores around 50%. Clin Cancer Res; 23(16); 4569-77. ©2017 AACR.

Abstract 3736: Characterization of tumor cells and assessment of PD-L1 expression in pleural effusions of metastatic non-small cell lung cancer patients

Abstract Introduction: Malignant pleural effusions (MPE) occur as a frequent complication of advanced non-small cell lung cancer (NSCLC), and denote a poor prognosis. Cytological evaluation of MPEs has a sensitivity of only 40-60%. The CellSearch technology (Janssen Diagnostics, LLC) is an FDA approved method of detecting rare circulating tumor cells (CTCs) in blood. Assessment of pleural fluid samples using CellSearch provides an approach to enrich often undetectable CTCs and phenotype these cells for markers of interest. PD-L1 staining of tumor tissue is essential for determining patient candidacy for checkpoint inhibitor therapy, however, tissue can often be difficult to obtain. Here, we demonstrate the feasibility of applying the CellSearch CTC enrichment platform to measure PD-L1 expression and enumerate tumor cells in MPE of NSCLC patients. Methods: This was a single center, prospective observational study of NSCLC patients with a pleural effusion. The anti-human CD274 (PD-L1) antibody (Biolegend) was used as a marker with the CellSearch CXC CTC kit and was detected in the open 4th channel of the CellTracks Analyzer. Determination of CTC PD-L1 expression was validated through peripheral blood spiking experiments. 7.5ml of pleural fluid was collected from NSCLC patients, preserved in CellSave tubes and processed within 72 hours. Identification and enumeration of CTCs was performed using the CellTracks Analyzer. We then compared CellSearch results to conventional cytological analysis. Results: Pleural effusion samples from 66 patients between the ages of 43 and 80 (median=69) were obtained following thoracentesis. The majority of patients were former (40; 60.6%) or never (21; 31.8%) smokers. At the time of effusion, 23 (34.8%) patients were on a therapeutic regimen: 10 (15.2%) were receiving chemotherapy, 9 (13.6%) were on an EGFR or ALK TKI, and 5 (7.6%) were on a checkpoint inhibitor. CTCs were detected in 63 of 66 samples using the CellSearch system. Among the 66 pleural effusions, cytological evaluation determined 40 samples were malignant and 26 were benign. CellSearch detected more CTCs in patients with a MPE (median 1798, range: 7 to 114,920) compared to patients with a benign effusion (median 8, range: 0 to 953; p=0.005). In the MPE group, 18 (45.0%) samples demonstrated &amp;gt;10% PD-L1 positivity with 9 (22.5%) samples showing &amp;gt;50% PD-L1 positivity. Conclusion: The CellSearch CTC enumeration and characterization platform can be successfully adapted for detection of CTCs in MPE of NSCLC patients. Our preliminary results suggest this approach may be a useful, non-invasive diagnostic adjunct for the determination of a MPE and assessment of PD-L1 expression in NSCLC patients. Further study is warranted to determine what role this test could play in assessing patients for checkpoint inhibitor therapy and its relationship with tissue based tests. Citation Format: Jeffrey C. Thompson, Samantha L. Savitch, Ryan Fan, Stephanie S. Yee, Christian A. Powers, Gordon Yu, Lauren Gebrian, Chandra Rao, Steve Gross, Michael Feldman, Anil Vachani, Erica L. Carpenter. Characterization of tumor cells and assessment of PD-L1 expression in pleural effusions of metastatic non-small cell lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3736. doi:10.1158/1538-7445.AM2017-3736

Abstract 575: PD-L1 positive tumor-infiltrating lymphocytes and mutational load in breast cancer

Abstract Background: PD-1 blockade has emerged as an effective treatment for a subset of cancer patients. Studies have shown that PD-L1 expression is associated with likelihood of response to PD-1 blockade. In order to select the right breast cancer patient for immunotherapy, characterization of the immune landscape of breast tumors is required. Therefore, we assessed PD-L1 expression and tumor-infiltrating lymphocytes (TILs) in different breast tumor subtypes and the link with prognosis. We also sequenced a panel of genes to assess the mutational load in triple negative tumors (TNBC) and investigate the association with PD-L1 positive TILs. Material and methods: We analyzed 438 tumor samples from breast cancer patients of all ages treated between 1986 and 2007 with surgery, with or without adjuvant therapy. PD-L1 was stained using whole slide specimens (E1L3N® antibody) after methodological validation. Pathologists quantified TILs based on International TILs Working Group recommendations and scored PD-L1 based on the percentage of positive (tumor and/or immune) cells; as negative if 0%, positive if ≥1%, and high if &amp;gt;50%. Mutational load was assessed based on DNA kinome sequencing. Associations were measured by Cox/logistic regression model, including pathological variables. Multiplex imaging of 20 immune-infiltrated areas from four ER negative tumors were performed using the Vectra® system based on immunofluorescence staining panel of: CD4, CD68, CD8, FOXP3 and PD-L1. Results: PD-L1 expression and TILs were higher in ductal (compared with lobular), high grade and estrogen receptor (ER)-negative tumors (p&amp;lt;0.001). TILs (density ≥5%) were significantly associated with worse distant metastasis-free survival (DMFS) only in ER-positive tumors (n=204): HR=2.72; 95%CI: 1.07-6.94. PD-L1 positivity (≥1%) followed the same trend: HR=1.66; 95%CI: 0.87-3.15. However, in ER-negative tumors (n=171), high PD-L1 expression (&amp;gt;50%) was significantly associated with better DMFS: HR=0.51; 95%CI: 0.27-0.98. TNBC with high PD-L1 expression of TILs (&amp;gt;50%) showed an association with increased mutation load (p=0.019) and a trend for better DMFS (HR=0.41; 95%CI: 0.16-1.04) compared with tumors lacking TILs. Further characterization of PD-L1 positivity in the immune-infiltrated cells was conducted by a multiplex imaging analysis. Preliminary results indicated that PD-L1 is expressed in CD68+, CD4+, FOXP3+ and CD8+ immune-cells. Conclusion: Our findings suggest that PD-L1 positive TILs are associated with worse prognosis in ER-positive breast cancer and with better outcome in ER-negative group. In TNBC, high mutational load correlates with high PD-L1 positive TILs. Citation Format: Marcelo Sobral-Leite, Koen Van de Vijver, Magali Michaut, Hugo M. Horlings, Tesa M. Severson, Philip C. Schouten, Rianne van der Linden, Kelly Kersten, Anna Marie Mulligan, Nayana Weerasooriya, Joyce Sanders, Ashley Cimino-Mathews, Dennis Peters, Gerrit K. Hooijer, Erik Hooijberg, Annegien Broeks, Rene Bernards, Sabine Linn, Irene L. Andrulis, Marc J. van de Vijver, Lodewyk F. Wessels, Marleen Kok, Karin E. de Visser, Marjanka K. Schmidt. PD-L1 positive tumor-infiltrating lymphocytes and mutational load in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 575. doi:10.1158/1538-7445.AM2017-575

Natural killer cells as a predictive biomarker for response to anti-PD-1 therapy in patients with advanced solid tumors.

e21055 Background: Assessment of PD-L1 expression in tumor biopsy has been widely used for predicting anti-PD-1 therapy. Several factors, including tumor heterogeneity, false negative staining and unavailability of biopsy samples limit the application. Natural killer (NK) cells exert cytotoxicity against cancer cells. Therefore, we explored using NK cells percentage in peripheral blood (PB) as a biomarker to identify pts who are more likely to benefit from anti-PD-1 therapy. Methods: Pts treated with anti-PD-1 antibody (JS001) 1 mg/kg, 3mg/kg or 10mg/kg every 2 weeks in a Phase I clinical trial (ClinicalTrials.gov Identifier: NCT02836795) had PB collected at baseline and at time of radiographic evaluation performed every 8 wks. Disease status (shrinking stable disease, growing stable disease, partial response, complete response, or progressive disease) was characterized using RECIST v1.1 and immune-related response criteria. Frequencies of NK cells (CD3-CD16+CD56+) were measured by flow cytometry. Results: Thirty-six patients were enrolled in this trial, including 22 melanoma, 9 urothelial carcinoma and 5 renal cell carcinoma. As of 1/27/2017, median treatment duration: 3.92 months. Radiographic tumor evaluation has been performed in 32 pts: 1/32 CR, 6/32 PR, 6/ 32 shrinking SD, 4/32 growing SD and 15/32 PD. Pts with high NK cells at baseline observed 60% (6/10) Clinical Benefit Rate (CR/PR/ shrinking SD) versus 40% (13/32) in overall pts. Patients with clinical benefit had higher frequency of NK cells at baseline compared to pts with radiographic growing SD and PD (mean 24.3% vs. 17.9%, P = 0.076). Furthermore, pts with NK cell level consistently above UN or increasing above UN during treatment are more likely to get clinical benefit than those under UN (7/10 vs 3/22, P = 0.013). Conclusions: Baseline high NK cells frequency may predict a favorable response to anti-PD-1 therapy, which provides a non-invasive way to monitor response to PD-1 blockade. These results are being validated in a phase II trial. Clinical trial information: NCT02836795.