Presence Of PD-L1 Research Articles

Abstract PS7-12: Impact of race/ethnicity on triple negative breast cancer molecular features, treatment response and clinical outcomes in patients receiving neoadjuvant therapy

Abstract Background: Triple negative breast cancer (TNBC) disproportionally impacts Black and Hispanic women and is suggested to be partly responsible for disparities observed in breast cancer mortality. We sought to evaluate the impact of race/ethnicity on TNBC molecular subtype, immune profile, pathologic complete response (pCR) and recurrence free survival (RFS). Methods: The ARTEMIS trial (NCT02276443) uses imaging response and molecular profiling to personalize neoadjuvant chemotherapy in early stage TNBC. After 4 cycles of AC, patients with chemo-sensitive disease receive standard taxane-based therapy, while those with chemo-resistant disease are offered therapeutic trials based upon molecular profiling. Pathologic response was assessed at surgery. Patients self-reported race/ethnicity was categorized as Asian, Black/African-American, Hispanic/Latino and White (non-Hispanic). Patients were excluded if no race or ethnicity was reported. Clinical and pathologic factors were recorded and compared. Gene expression profiling was performed by RNAseq to determine Vanderbilt signature. PD-L1 and Androgen Receptor (AR) were determined by immunohistochemistry. Frequencies at which various factors were observed by race/ethnicity were calculated and Fisher’s exact test performed to determine statistical significance. Kaplan-Meier analysis was used to estimate survival stratified by pCR status and race/ethnicity. Results: Among 321 women enrolled in ARTEMIS, 26 (8.1%) were classified as Asian, 50 (15.6%) as Black, 59 (18.4%) as Hispanic and 186 (57.9%) as White. Demographic and clinical features were similar, except Black women were more likely to have BMI ≥30 (70.0%, p<0.001). Comparing by race/ethnicity, there was no statistically significant difference in tumor histology, tumor size, nodal status, clinical stage, chemo-resistant disease, type of surgery or receipt of radiation. A trend toward higher proportion of N3 disease was observed in Black women (24.0%) compared to other groups (p=0.054). Tumor profiling results are listed in Table 1. No statistically significant difference was observed by race/ethnicity in Vanderbilt signature, presence of PD-L1 or stromal tumor infiltrating lymphocytes (sTIL) status. More AR-positive tumors were identified in Asian women (64%, p=0.007). pCR and RCB status did not significantly vary by race/ethnicity however, higher proportion of RCB-III disease was identified in Black (14%) and Hispanic women (16.9%) as compared to Asian (3.8%) and White (8.6%) women (p=0.164). At median follow up of 23.8 months (range 3.4-51.1), there was no statistically significant difference in RFS by race/ethnicity. Conclusion: Among this population of patients with TNBC treated with neoadjuvant chemotherapy there was no statistically significant difference observed in TNBC Vanderbilt signature, PD-L1 staining, sTIL, pCR or RFS by race/ethnicity. Asian women were found to have a higher incidence of AR-positive subtype. Numerically higher rates of N3 stage in Black women and RCB-III classification in Black and Hispanic women were identified, however not statistically significant. Larger cohorts of patients are needed to further investigate these findings. Table 1. TNBC profiling compared by race/ethnicity.Total n (%)Asian n (%)Black n (%)Hispanic/Latino n (%)White n (%)p valueVanderbilt signature n (%) Basal like (BL1) Basal like 2 (BL2) Immunomodulatory (IM) Luminal androgen receptor (LAR) Mesenchymal (M) Mesenchymal stem-like (MSL) Unstable (UNS)231 50 (21.6) 25 (10.8) 42 (18.2) 22 (9.5) 48 (20.8) 14 (6.0) 30 (13.0)21 (9.1) 0 (0.0) 4 (19.0) 5 (23.8) 3 (14.3) 5 (23.8) 1 (4.8) 3 (14.3)35 (15.2) 9 (25.7) 5 (14.3) 6 (17.1) 2 (5.7) 7 (20.0) 1 (2.9) 5 (14.3)43 (18.6) 14 (32.5) 3 (7.0) 10 (23.2) 5 (11.6) 6 (14.0) 2 (4.7) 3 (7.0)132 (57.1) 27 (20.5) 13 (9.8) 21 (15.9) 12 (9.1) 30 (22.7) 10 (7.6) 19 (14.4)NSAndrogen Receptor n (%) Positive (>=10%) Negative (<10%)311 109 (35.0) 202 (65.0)25 (8.0) 16 (64.0) 9 (36.0)49 (15.8) 11 (22.4) 38 (77.6)57 (18.3) 19 (33.3) 38 (66.7)180 (57.9) 63 (35.0) 117 (65.0)0.007PD-L1 n (%) None >1298 212 (71.1) 86 (28.9)24 (8.1) 15 (62.5) 9 (37.5)47 (15.8) 33 (70.2) 14 (29.8)57 (19.1) 40 (70.2) 17 (29.8)170 (57.0) 124 (72.9) 46 (27.1)NSStromal tumor infiltrating lymphocytes (sTIL) n (%) High (>=20%) Low (<20%)319 107 (33.5) 212 (66.5)26 (8.1) 12 (46.2) 14 (53.8)50 (15.7) 13 (26.0) 37 (74.0)58 (18.2) 21 (36.2) 37 (63.8)185 (58.0) 61 (33.0) 124 (67.0)NS Citation Format: Mediget Teshome, Ryan Sun, Elizabeth E. Ravenberg, Abenaa Brewster, Mariana Chavez-MacGregor, Jason B. White, Stacy Moulder. Impact of race/ethnicity on triple negative breast cancer molecular features, treatment response and clinical outcomes in patients receiving neoadjuvant therapy [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS7-12.

Engineering of a trispecific tumor-targeted immunotherapy incorporating 4-1BB co-stimulation and PD-L1 blockade

ABSTRACT Co-stimulatory 4–1BB receptors on tumor-infiltrating T cells are a compelling target for overcoming resistance to immune checkpoint inhibitors, but initial clinical studies of 4–1BB agonist mAbs were accompanied by liver toxicity. We sought to engineer a tri-specific antibody-based molecule that stimulates intratumoral 4–1BB and blocks PD-L1/PD-1 signaling without systemic toxicity and with clinically favorable pharmacokinetics. Recombinant fusion proteins were constructed using scMATCH3 technology and humanized antibody single-chain variable fragments against PD-L1, 4–1BB, and human serum albumin. Paratope affinities were optimized using single amino acid substitutions, leading to design of the drug candidate NM21-1480. Multiple in vitro experiments evaluated pharmacodynamic properties of NM21-1480, and syngeneic mouse tumor models assessed antitumor efficacy and safety of murine analogues. A GLP multiple-dose toxicology study evaluated its safety in non-human primates. NM21-1480 inhibited PD-L1/PD-1 signaling with a potency similar to avelumab, and it potently stimulated 4–1BB signaling only in the presence of PD-L1, while exhibiting an EC50 that was largely independent of PD-L1 density. NM21-1480 exhibited high efficacy for co-activation of pre-stimulated T cells and dendritic cells. In xenograft models in syngeneic mice, NM21-1480 induced tumor regression and tumor infiltration of T cells without causing systemic T-cell activation. A GLP toxicology study revealed no evidence of liver toxicity at doses up to 140 mg/kg, and pharmacokinetic studies in non-human primates suggested a plasma half-life in humans of up to 2 weeks. NM21-1480 has the potential to overcome checkpoint resistance by co-activating tumor-infiltrating lymphocytes without liver toxicity.

Correlation of DLL3 expression in gastroenteropancreatic neuroendocrine neoplasms with loss of RB1 and prognostic significance.

4611 Background: Neuroendocrine neoplasms (NENs) are a rare subgroup of tumors with challenging management due to their unpredictable and heterogeneous behaviour. The identification of clinically useful biomarkers is a top priority need in this disease. The negative notch regulator DLL3 has gained increasing attention in small cell lung carcinoma, large cell neuroendocrine carcinoma and neuroendocrine prostate cancer, confirming the tumor suppressor function of Notch-1 signaling in neuroendocrine cells. Methods: A retrospective immunohistochemical analysis of DLL3, PD-L1 and RB1 was performed on FFPE samples from 43 patients with gastroenteropancreatic (GEP)-NENs and correlated with clinical characteristics. Results: DLL3 was expressed in high-grade (G3) GEP-NENs. The presence of DLL3 was significantly associated with poorly differentiated NEC (77.8% positive tumors), while none of the patients with well-differentiated NET expressed this marker. Expression of DLL3 was correlated with loss of RB1 and negative 68Ga-PET/CT scan. The 85.7% of DLL3- positive tumors showed loss of RB1 expression, while only 1 out of 35 DLL3- negative tumors had RB1 loss. DLL3 was expressed in 75% of patients with negative 68Ga-PET/CT, while only in 25% of patients with positive 68Ga-PET/CT scan. The presence of DLL3 was negatively associated with PFS and OS. Median PFS was 41.9 months in DLL3-negative patients versus 7.9 months in DLL3-positive patients; median OS was 72.9 months in DLL3-negative patients versus 11.7 months in DLL3-positive patients. No correlation was found with DLL3 and PD-L1 expression. The presence of PD-L1 was not associated with any clinical characteristics. Conclusions: DLL3 is expressed in high grade GEP-NENs and is associated with loss of RB1, negative 68Ga-PET/CT scan and unfavourable clinical outcome. The presence of DLL3 discriminates poorly differentiated NEC from well-differentiated NET. DLL3 could represent the ideal prognostic factor to stratify patients with GEP-NENs and a candidate therapeutic target in NEC patients.

Immunogenomic classification of gastric cancer based on the tumor microenvironments expression of PD-L1 and CD8+ T-cell infiltration.

e16578 Background: Previous data has shown that a positive response to immunotherapy usually relies on active interactions between tumor cells and immunomodulators inside the tumor microenvironment (TME). The aim of this study was to classify gastric cancer (GC) subsets based on the TME immune status according to the expression of PD-L1 and infiltration of CD8+ T cells. Methods: One hundred and eighty-six tumor tissue from gastric cancer patients with a curative D2 gastrectomy were examined for evaluating PD-L1 and CD8+ T cells status using histopathologic analysis. The molecular characteristics of 289 GC samples in TCGA network were further analyzed to distinguish the genetic features of four immune subtypes depending on the presence of PD-L1/CD8+T cell. Results: GC samples were categorized into four types, type I (CD8+/PD-L1+, 60.3%), II (CD8-/PD-L1-, 11.8%), III (CD8-/PD-L1+, 0%), and IV (CD8+/PD-L1-, 27.9%), basing on PD-L1/CD8 expression. The PD-L1 expressing level was geographically associated with the intensity of CD8+ T cell infiltration which was significantly associated with disease-free survival (DFS) and overall survival (OS) (p = 0.003 and p = 0.006). Distinct patterns of genetic profile were described in four types of GC from TCGA database. Type I and III which PD-L1 were overly expressed had comparatively higher MSI and TMB, with EBV mainly enriched in Type I, whereas CIN was more likely to occur in PD-L1 aberrant types II and IV. SNV analysis illustrated higher gene mutations in oncogenes (PIK3CA and ERBB2), and in DNA damage repair related pathway, such as PRKDC, ATM, and SWI/SNF complexes (e.g. ARID1A) in Type I. However, TP53 mutations tend to enrich in Type II and IV. Similar results were obtained by transcriptome analysis. Conclusions: The genetic features of four immune subtypes proof that PD-L1 and CD8+ T cells status are reasonable immunogenomic classification of gastric cancer. SNV analysis prompts a potential mechanism for effectiveness of immunotherapy in Type I patients. Overall, the results may be useful for the development of clinical treatments for the blockade of immune checkpoints.

A phase II trial of PD-1 inhibition with INCMGA00012 in patients with previously treated unresectable or metastatic adenosquamous pancreatic cancer.

TPS4662 Background: Adenosquamous pancreatic cancer (ASQ) constitutes 1-5% of all pancreatic cancers and compared to pancreatic adenocarcinoma (PDAC) has a worse survival. ASQ has glandular and squamous histologic components and given its rarity and aggressiveness, in practice there is no current standard regimens for ASQ. Unfortunately, checkpoint blockade has had an overall disappointing impact on survival in PDAC. In an effort to identify a patient subgroup most likely to respond to immunotherapy, the immune tumor microenviroment (TME) in ASQ was evaluated. Tissue microarrays from archived ASQ samples were first created. Then immunocytochemistry (IHC) staining for immune cells and immune checkpoint proteins was performed. PD-L1 expression and the combined presence of PD-L1+, IDO+, LAG3+, and VISTA+ was seen. All ASQ cases had some degree of tumor infiltrating lymphocytes (TIL, including CD8+ T-cells). Furthermore, PD-L1 and other checkpoint positivity correlated with increased TIL. These findings suggest the presence of adaptive immune resistance. This is in contrast to standard PDAC, in which the expression of immune checkpoints is rarely accompanied by increased effector T-cells. Methods: We are conducting a multiple-center, single arm, phase II clinical trial to evaluate PD-1 inhibition with INCMGA00012 in locally advanced unresectable and metastatic ASQ patients. INCMGA00012 is a humanized monoclonal antibody antagonistic to PD-1. The primary objective is to determine the disease control rate at 4 months using RECIST 1.1. The study is planned with 21 evaluable subjects and allows early termination for lack of efficacy. Patients have a pre-treatment biopsy followed by INCMGA00012 500 mg on Day 1 of each cycle (every 4 weeks). A second biopsy will occur eight weeks later. Eligibility criteria includes histologically- or cytologically-proven adenosquamous carcinoma of the pancreas by central pathologic review and patients must have received (or been intolerant to or ineligible for) at least 1 prior line of cytotoxic chemotherapy and received no more than 2 prior systemic treatments. Patients with known MSI-H/dMMR status are excluded. Exploratory objectives include examining changes in the TME checkpoint expression and immune cell infiltrate in the biopsies via IHC and RNA expression studies. The clinical study was activated in February 2020. Clinical trial information: NCT04116073 .

Development and functional analysis of an anticancer T-cell medicine with immune checkpoint inhibitory ability.

Adoptive cell therapy using patients' own T-cells is expected to be an ideal cancer treatment strategy with excellent antitumor effects and low side effects. However, this therapy targeting solid tumors is unlikely to be effective because tumor tissues have an environment that suppresses T-cell function. In particular, interaction between programmed death-1 (PD-1) and its ligand (PD-L1) inhibits T-cell activation by which T-cells eliminate tumor cells. Here, we attempted to develop T-cells that can exert potent antitumor activity even in tumor tissues by genetically modifying them to express the anti-PD-L1 membrane-anchoring type single chain variable fragment (M-scFv) that can inhibit PD-L1/PD-1 interaction. Anti-PD-L1 M-scFv could be expressed on T-cells while maintaining PD-L1-binding ability. Although T-cell proliferation induced by CD3 stimulation was decreased depending on the PD-L1 stimulation intensity, M-scFv-expressing T-cells showed high proliferative activity even in the presence of PD-L1 by avoiding the PD-L1/PD-1-mediated suppression. Furthermore, M-scFv-expressing T-cells showed higher cytotoxic activity against PD-L1high tumor cells than that of mock T-cells. The effect of PD-L1/PD-1 blockade was more pronounced when the therapeutic target was low-antigenic tumor cells with low major histocompatibility complex expression, presenting only the shared antigen. These results indicated that anti-PD-L1 M-scFv expression was functional in avoiding T-cell dysfunction by PD-L1/PD-1 interaction. Our concept of anti-PD-L1 M-scFv-expressing T-cells is thus expected to improve the efficacy of T-cell therapy and contribute to simplify the treatment system and reduce treatment costs compared with the combination therapy of T-cells and antibodies.

Immune microenvironment in ductal carcinoma in situ: a comparison with invasive carcinoma of the breast

BackgroundThe immune microenvironment in ductal carcinoma in situ (DCIS) and its significance are not well established. This study was conducted to evaluate the immune microenvironment of DCIS including the composition of tumor-infiltrating lymphocyte (TIL) subsets and PD-L1+ immune cells and to compare it with that of invasive breast cancer.Materials and methodsA total of 671 cases including three different disease groups of pure DCIS, DCIS with microinvasion (DCIS-M), and invasive carcinoma were included in this study. CD4+, CD8+, and FOXP3+ TIL subsets and PD-L1+ immune cells were detected with immunohistochemistry using tissue microarrays and were analyzed in relation to clinicopathologic characteristics and different disease groups.ResultsIn pure DCIS, high infiltrations of CD4+, CD8+, and FOXP3+ T cells and the presence of PD-L1+ immune cells were associated with high nuclear grade, comedo-type necrosis, hormone receptor (HR) negativity, and high Ki-67 proliferation index. All immune cell infiltrations were higher in invasive carcinoma than in pure DCIS regardless of the HR status. While CD4+ T cells were more abundant than CD8+ T cells in pure DCIS, CD8+ T cells were dominant in invasive carcinoma, especially in HR-negative tumors. Within individual cases of invasive carcinoma with DCIS component, all immune cell subset infiltration was higher in the invasive component than in the DCIS component; however, CD4+ TIL infiltration did not differ between the two components in HR-negative tumors. Comparing pure DCIS, DCIS-M, and DCIS associated with invasive carcinoma (DCIS-INV), CD4+ TIL infiltration revealed a gradual increase from pure DCIS to DCIS-M and DCIS-INV in the HR-negative group, whereas FOXP3+ TIL infiltration was significantly increased in DCIS-INV than in pure DCIS in the HR-positive group. The high infiltration of FOXP3+ TIL and the presence of PD-L1+ immune cells were associated with tumor recurrence in patients with pure DCIS.ConclusionsOur study showed that the immune microenvironment differs significantly not only between DCIS and invasive carcinoma but also between pure DCIS, DCIS-M, and DCIS-INV depending on the HR status.

Molecular characteristics of renal cell carcinoma (RCC) risk groups from JAVELIN Renal 101.

744 Background: The phase 3 JAVELIN Renal 101 trial in patients (pts) with advanced RCC demonstrated a progression-free survival (PFS) benefit and higher objective response rate (ORR) with avelumab + axitinib (A+Ax) vs sunitinib (S) (Motzer NEJM 2019). PFS and ORR favored A+Ax in all MSKCC risk groups, but median PFS varied. Here, we report results from analyses of baseline tumor samples to define molecular characteristics underlying risk group classifications. Methods: Nephrectomy or tumor samples from pts enrolled in the study were characterized by immunohistochemistry (CD8 and PD-L1), whole exome sequencing, or gene expression profiling (n = 705–850). Gene expression signatures (GES), pathway activation status, and mutational profiles were examined in relation to MSKCC risk groups. Results: Of the 886 total pts enrolled, 23%, 66%, and 11% had favorable (F), intermediate (I), or poor (P) MSKCC risk factors at baseline. In the F, I, and P groups, the ORR was 66%/38%, 50%/24%, and 31%/9%; median PFS was NR/16.7 mo, 13.3 mo/7.9 mo, and 5.6 mo/2.8 mo for the A+Ax/S arms, respectively. Neither the presence of PD-L1+ immune cells nor CD8+ cells differentiated the subgroups; however, the presence of PD-L1+ tumor cells was highest in the P group (p=0.0159). When compared to the I and P groups, the F group was enriched for NOTCH2 mutations (p=0.0002), displayed high FLT1 expression (p=0.007), and showed a trend favoring angiogenesis GES (JAVELIN Renal 101 and IMmotion150). The I group displayed few distinguishing characteristics (low neutrophil GES [p=0.02] and elevated homeobox gene expression [p=0.00052]). Relative to the F group, the P group showed higher cell cycle gene expression (p=0.0057) and PTEN mutation frequency, wild-type NOTCH2 genotype, elevated IMmotion150 Myeloid inflamed GES (p=0.0024), and macrophage-specific GES (p=0.0327), as well as GES for TH2 (p=0.0002), hypoxia (p=0.0199), glycolysis (p=0.0066), and the lowest expression of a dendritic cell GES (p=0.0209). Conclusions: In advanced RCC patients, biological differences within each MSKCC risk category may impact response to treatment and may help explain why these groups perform differently. Clinical trial information: NCT02684006.

Heterogeneity of exhausted T cells in the tumor microenvironment is linked to patient survival following resection in hepatocellular carcinoma

ABSTRACT Despite the success of monotherapies based on blockade of programmed cell death 1 (PD-1) in human melanoma, most patients do not experience durable clinical benefit. T-cell infiltration and/or the presence of PD-L1 in tumors may be used as indicators of clinical response; However, recent studies reported that preexisting tumor-specific T cells may have limited reinvigoration capacity. Therefore, evaluating status of T cells of tumor-adjacent area and its impact on the prognosis are very important. Here, we examined 117 surgical samples from HCC patients for infiltration of exhausted T cell (Tex) including CD4+-Tex, CD8+-Tex and regulatory T cell (FOXP3+-Treg) in tumor and adjacent tissue. CD3+CD45RO+T cells were sorted from adjacent area or tumor core, then the clusters and heterogeneity of T cells were further interrogated by single-cell RNA sequencing. As a result, we suggested that abundance or location of T cell subsets is differentially correlate with long-term clinical outcome of HCC. In contrast with CD4+T or CD4+-Tex, the infiltration of CD8+T or CD8+-Tex cells was closely linked to overall or recurrence-free survival. FOXP3+-Treg is more predictive of early recurrence. Single-cell transcriptional analysis demonstrates the composition of CD4+-Tex, CD8+-Tex, and FOXP3+-Treg is shifted in tumor and adjacent tissue. Molecular profiles including genes coding checkpoint receptors, effector molecules are distinct between CD4+-Tex, CD8+-Tex, though some common features of CD4+ and CD8+ T cell exhaustion are revealed. In conclusion, we underline the heterogeneity and clinical relevance of Tex cells in HCC patients. A better understanding of Tex is critical for HCC monitoring and treatment.

PD-1 Is Expressed at Low Levels on All Peripheral Blood Natural Killer Cells but Is a Significant Suppressor of NK Function Against PD-1 Ligand Expressing Tumor Targets

PD-1 Is Expressed at Low Levels on All Peripheral Blood Natural Killer Cells but Is a Significant Suppressor of NK Function Against PD-1 Ligand Expressing Tumor Targets

PCN378 PHARMACOECONOMIC ANALYSIS OF ATEZOLIZUMAB PLUS NAB-PACLITAXEL IN THE TREATMENT OF TRIPLE-NEGATIVE BREAST CANCER

Breast cancer with a triple negative phenotype (TNBC) is aggressive form of the disease. The survival at stage 4 is 1 year at most. The goal of the study was assessment of a new option for inoperable TNBC (presence of PD-L1 ≥1%): atezolizumab in combination with nab-paclitaxel. A decision analysis model was used followed by Markov modeling. The cost-effectiveness analysis (CEA) with incremental cost-effectiveness ratio (ICER) calculation was performed. The combination of atezolizumab+nab-paclitaxel and nab-paclitaxel monotherapy regimens were evaluated. Overall survival (OS) was taken as an effectiveness criterion. Budget impact analysis (BIA) was performed with a one-year and three-year perspective in order to assess the difference in direct costs between the current therapy practice (excluding the use of the combination under consideration) and the expected practice (taking into account the use of the combination). TNBC therapy has a high cost in both combination therapy and monotherapy regimens. The use of atezolizumab plus nab-paclitaxel option increased the median OS from 15.5 to 25 months. At the same time, the total costs of using the combined regimen were €124,987 for two years, which is 2.6 times more expensive than nab-paclitaxel monotherapy. ICER for the combined regimen amounted to €8,155, which is significantly less than society willingness-to-pay threshold that equals €27,341 (triple GDP per capita). In addition, ICER value for atazodizumab+nab-paclitaxel is significantly lower than the incremental indicators for alternate therapeutic options used for other curable forms of breast cancer, e.g., trastuzumab emtansine in comparison with lapatinib+capecitabine (€272,669 for the LYG, and €329 475 for QALYs). BIA demonstrated the absence of significant impact on the level of costs for providing patients with breast cancer in general. The use of atezolizumab plus nab-paclitaxel for the TNBC treatment is an economically feasible option for patients with a poor prognosis.

Abstract 683: A machine learning based clinical decision support system (CDSS) for anti-PD-1 therapy using non-invasive blood marker and clinical information for lung cancer patients

Abstract Cancer immunotherapy with anti-PD-1 therapy (αPD-1) has greatly improved the survival outcome of patients with non-small cell lung cancer (NSCLC). However, the response rates of αPD-1 are around 20-30% based on clinical trials. The presence of PD-L1, tumor infiltrating lymphocyte or tumor mutation burden may be used as indicators of response, but are still limited to predict αPD-1 response. In this study, we established the machine learning based clinical decision support system (CDSS) to predict the αPD-1 response by comprehensively combining clinical information and blood-based data which are easily assessable in routine practice.We enrolled 126 patients with NSCLC treated with the αPD-1 at Yonsei cancer center. Clinical data including patient characteristics, mutation, treatment outcomes, and adverse events were collected and analyzed. Forty patients additionally had blood-based immune data by flow cytometry. There were two data sets; clinical data set (n=126) with 15 variables, and immune data set (n=40) with 37 variables. We found that 27 variables out of 52 variables are selected by recursive feature elimination. The responders are defined as PR or SD ≥6 months and the non-responders are defined as the others. Supervised learning algorithms such as the LASSO, Ridge, Elastic Net, SVM, ANN, and RF were applied to each data set for predicting the αPD-1 response. The performances of each model were evaluated according to the ROC curve or cross-validation errors. Variable importance was measured by using the random forest and gradient boosting.Patient characteristics included male (69.8%), age ≥ 60 years (66.2%), ECOG 0/1 (77.7%), adenocarcinoma (69.8%), EGFR mutations (15.1%), and PD-L1 positive (61.2%). We classified the patients into responders (38%) and non-responders (62%) in total 126 patients. A result from the clinical data set of 126 patients demonstrated that the Ridge regression model (AUC: 0.78) can more accurately predict the αPD-1 response than others. Of 15 clinical variables, some are considered to be important in the following order; tumor burden,age, PD-L1, ECOG PS, and irAE based on the random forest. When we performed the machine learning process with clinical and immune data, The Ridge regression model (AUC:0.82) showed the good performance to predict αPD-1 response, compared to the single clinical model. The machine learning based CDSS for aPD-1 to NSCLC patients has benefit for predicting aPD-1 responses. Our prediction model could be easily accessible and fast processed in routine practice. The supervised machine learning based non-invasive predictive score (NIPS) demonstrates the rate of aPD-1 response on NSCLC patients. We will validate NIPS in independent patient cohort and currently are establishing the NIPS as web-based software. Citation Format: Kyoung-Ho Pyo, Beung-Chul Ahn, Chun-Feng Xin, Dongmin Jung, Chang Gon Kim, Min Hee Hong, Byoung Chul Cho, Hye Ryun Kim. A machine learning based clinical decision support system (CDSS) for anti-PD-1 therapy using non-invasive blood marker and clinical information for lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 683.

Abstract 541: An unbiased screen identifies a CD137xPD-L1 bispecific IgG1 antibody with unique T cell activation and binding properties

Abstract CD137 (4-1BB) is a transmembrane costimulatory receptor on T and NK cells that enhances adaptive immune responses and is a critical mediator of antitumor immunity. CD137 signaling requires receptor clustering normally facilitated by the trimeric CD137 ligand (CD137L). Alternatively, CD137 signaling can be triggered either directly by agonistic monoclonal antibodies (mAbs) or indirectly via crosslinking of CD137 binding mAbs by Fcγ receptors on neighboring cells. The development of CD137 targeted agents for cancer therapy has been hampered by on-target off-tumor toxicity in the case of agonist, monospecific, bivalent mAbs or limited antitumor activity in the case of crosslinking mAbs. To address the issues of toxicity and efficacy a highly selective and potent CD137xPD-L1 bispecific antibody (bAb) was identified by applying an unbiased functional screening approach. Collections of common light chain Fabs recognizing CD137 and PD-L1 were produced based on antibody panels from immunized MeMo® mice. A large and diverse panel of CD137xPD-L1 bAbs was then produced by combining different CD137 and PD-L1 Fabs based on epitope and sequence diversity in the IgG1 Biclonics® format. The bAbs were screened for activity in reporter cell lines expressing the receptors. This unbiased combinatorial screening identified a CD137xPD-L1 bAb (MCLA-145) for which CD137 mediated activation is dependent on the presence of PD-L1 on a neighboring cell and, as such, the antibody acts in ‘trans’. Flow cytometry experiments demonstrated that MCLA-145 is fully cross-reactive to cynomolgus monkey CD137 and PD-L1. The CD137 Fab arm blocks the interaction of CD137 with CD137L as demonstrated in a competition assay by flow cytometry. The PD-L1 Fab arm blocks the interaction between PD-1 and PD-L1 as demonstrated in ELISA. Binding epitopes were mapped by shotgun mutagenesis using a flow-based screen. In addition, hydrogen-deuterium exchange experiments were performed to map the binding domain on CD137. Data show that MCLA-145 binds the ligand binding domain of CD137 domain (CRDII). The PD-L1 Fab arm binds PD-L1 in the PD-1 binding N-terminal V domain. Both epitope mapping data sets are consistent with the CD137 and PD-L1 ligand blocking activity of MCLA-145. Monovalent binding affinities were measured by surface plasma resonance (SPR) and radioactive iodine labeling and demonstrated affinities in the low nM (CD137) and subnanomolar (PD-L1) range. SPR experiments also confirmed that MCLA-145 was able to bind simultaneously to both CD137 and PD-L1 recombinant proteins. The unique binding properties of MCLA-145 may result in an increased therapeutic window by specifically activating CD137 expressing cells in the tumor niche where PD-L1 is expressed while simultaneously blocking inhibitory input from the PD-1/PD-L1 axis. Citation Format: Cecile A. Geuijen, Paul Tacken, Rinse Klooster, Horacio Nastri, Shaun Stewart, Jing Zhou, Steve Wang, Cheng-Yen Huang, Arjen Kramer, Linda Kaldenberg-Hendriks, John de Kruif, Renate den Blanken-Smit, Vanessa Zondag-van de Zande, Abdul Basmeleh, Willem Bartelink, Patrick Mayes, Gregory Hollis, Reid Huber, Mark Throsby. An unbiased screen identifies a CD137xPD-L1 bispecific IgG1 antibody with unique T cell activation and binding properties [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 541.

Abstract 1532: A novel, monovalent tri-specific antibody-based molecule that simultaneously modulates PD-L1 and 4-1BB exhibits potent anti-tumoral activity in vivo

Abstract The combined immunomodulation of PD-L1/PD-1 and 4-1BB is considered a promising strategy to increase response rates among cancer patients who are eligible to receive PD-L1/PD-1 inhibitors. Unfortunately, encouraging pre-clinical results achieved with such regimens have not yet translated into durable clinical success, due to addition of 4-1BB-agonistic antibodies being either intolerable at effective doses or ineffective, despite tolerability, at all doses. To eliminate this safety/efficacy tradeoff, we engineered a novel, tri-specific immunomodulatory drug candidate. The molecule consists of three monovalent antibody Fvs - specific for PD-L1, serum albumin (SA) and 4-1BB - fused in a single chain (a PD-L1/4-1BB/SA tri-specific scDb-scFv). The monovalent and Fc-less structure of the molecule ensures that 4-1BB agonism is conditional upon drug-mediated formation of an immunological synapse between PD-L1+ cells and 4-1BB+ cells, thereby restricting costimulation of 4-1BB+ cells to the tumor microenvironment (TME). Therefore, the scDb-scFv molecule avoids the extratumoral costimulation of immune cells that is believed to cause the dose-limiting toxicities that arise from therapeutic 4-1BB agonism. Meanwhile, the αSA domain extends the molecule’s serum half-life and is expected to promote delivery to the TME. In the present study, we demonstrate that a novel PD-L1/4-1BB/SA tri-specific scDb-scFv potently blocks PD-L1/PD-1 signaling and elicits T cell costimulation solely in the presence of PD-L1+ cells. In in vitro experiments, the scDb-scFv molecule exhibits a greater capacity to costimulate T cells than combinations of clinical stage α4-1BB and αPD-L1/PD-1 IgGs. Moreover, in contrast to ADCC-enabled αPD-L1 IgGs, the Fc-less scDb-scFv spared CD11c+ monocytes from depletion. We also demonstrate that the carefully balanced relative affinity between the molecule’s component αPD-L1 and α4-1BB Fvs maximizes its pharmacological activity and avoids bell-shaped dose-response curves. Next, in vivo efficacy was demonstrated in two xenograft models - HCC827 NCSLC and HCC1954 breast carcinoma - using humanized mice. While equally effective at slowing tumor progression in vivo, the PD-L1/4-1BB/SA tri-specific scDb-scFv was better tolerated, and was more pro-proliferative vis-à-vis intratumoral CD8+ T cells, than combined αPD-L1 and α4-1BB IgGs. In PK/PD studies in cynomologus monkeys, we confirmed the tolerability, pharmacological activity and extended serum half-life of the scDb-scFv molecule. Finally, the molecule was produced from stable CHO cells at high titers (comparable to IgGs) and exhibits outstanding stability characteristics. In conclusion, our data confirm the successful development of a novel therapeutic that is designed to unlock the full potential of combined immunomodulation and overcome its present limitations. Citation Format: Tea Gunde, Matthias Brock, Stefan Warmuth, Alexandre Simonin, Christian Hess, Eva Oswald, Julia Tietz, Julia Zeberer, Dana Mahler, Simone Muntwiler, Benjamin Küttner, Belinda Wickihalder, Antonia Pölderl, Dania Diem, Teddy Beltrametti, Robin Heiz, Sebastian Meyer, Timothy Egan, David Urech. A novel, monovalent tri-specific antibody-based molecule that simultaneously modulates PD-L1 and 4-1BB exhibits potent anti-tumoral activity in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1532.

Abstract LB-057: Programmed cell death 2 forms coinhibitory microclusters that directly attenuate T cell receptor signaling by recruiting phosphatase SHP2

Abstract Background: Anti-PD-1 antibodies have made tremendous therapeutic effects on advanced or recurrent non-small cell lung cancer. However, the expression level of PD-L1 in tumor tissue which is the biomarker in the clinical setting is not necessarily correlated with their efficacy. In recent reports, some kinds of tumors express PD-L2 which is another ligand of PD-1.It is possible that the binding between PD-L2 and PD-1 is contributing to this mechanism. Because we intended to analyze the microstructural basis of immunological synapse, we used a system of a planar bilayer incorporated with glycophosphatidylinositol (GPI)-anchored intercellular adhesion molecule 1 (ICAM-1) and major histocompatibility complex (MHC) class II (I-Ek) loaded with a moth cytochrome c (MCC) peptide. Using our unique imaging analysis, we found the "TCR microcluster", where TCR proximal signaling molecules are recruited. In addition, PD-1 colocalizes with TCR microclusters in the presence of PD-L1 and SHP2 recruiting to PD-1 suppresses T cell activity by dephosphorylating the activated signaling molecules at TCR microclusters. Here, we investigated the PD-1-PD-L2 pathway using this dynamicimaging technique to reveal the molecular behavior of PD-1 bounded by PD-L2, not PD-L1. Methods: We established tumor cell line (BHK) highly expressing murine PD-L2 (mPD-L2)-GPI and purified mPD-L2-GPI by affinity column with anti-PD-L2. Primary CD4+ T cell isolated from AND-Tg mice in Rag2-/- Pdcd1-/-background and T cell hybridoma expressing AND-TCR (2D12) were infected with retroviral supernatants of PD-1-GFP or GFP-SHP2. We used planar bilayers of mPD-L2-GPI, mICAM-1 and I-Ekloaded with MCC peptideand observed PD-1-GFP-transfected CD4+ T cell and GFP-SHP2-transfected 2D12 by confocal microscopy. Each GPI protein was reconstituted into the planar bilayer as the same density of these ligands expressed by LPS-stimulated B cells. Results: We showed PD-1 is translocated to TCR microclusters and then accumulates at the central region of the immunological synapse in the presence of PD-L2. We also confirmed SHP2 is immediately but transiently recruited to PD-1-PD-L2 microclusters. Conclusions: Our results indicate that both PD-L1 and PD-L2 suppress TCR signaling by the recruitment of SHP2 at the same signaling clusters for the initiation of T cell activation. Further experiments are ongoing to elucidate the mechanism of the individual effect for cancer immunotherapy by PD-1-, PD-L1- and/or PD-L2 blockade and also of the in vivo effects of each blocking antibody. Citation Format: Tomohiro Takehara, Ei Wakamatsu, Hiroaki Machiyama, Hiroyuki Yasuda, Kenzo Soejima, Tadashi Yokosuka. Programmed cell death 2 forms coinhibitory microclusters that directly attenuate T cell receptor signaling by recruiting phosphatase SHP2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-057.

T-bet interferes with PD-1/PD-L1-mediated suppression of CD4+ T cell inflammation and survival in Crohn's disease.

Pathogenic inflammation mediated by overactive type 1 helper T cell (Th1) responses could exacerbate and perpetuate Crohn's disease. Programmed death (PD)-1 and its ligand PD-L1 pathway could be upregulated to suppress inflammation. We wondered why this pathway is ineffective at suppressing pathogenic Th1 inflammation in Crohn's disease patients. Here, we found that overexpression of T-bet via transfection significantly reduced the expression of PD-1. PD-L1 was capable of suppression proinflammatory CD4+ T cells, but T-bet transfection significantly reduced the susceptibility of CD4+ T cells toward PD-L1-mediated suppression, evidenced by the observations that at low PD-L1 concentration T-bet transfected and mock transfected CD4+ T cells presented comparable IL-2 production, but at high PD-L1 concentration, T-bet transfected CD4+ T cells presented significantly higher IL-2 than mock transfected CD4+ T cells. PD-L1 could significantly reduce the survival of CD4+ T cells from Crohn's disease patients, but interestingly, in the absence of PD-L1, the survival was better in mock transfected CD4+ T cells, while in the presence of PD-L1, the survival was better in T-bet transfected CD4+ T cells. Crohn's disease patients with greater severity presented higher T-bet expression and lower PD-1 expression in CD4+ T cells, demonstrating an association between T-bet expression and disease progression. We also discovered that stimulation with bacterial antigens could upregulate the expression of T-bet. Together, this study demonstrated that T-bet overexpression could interfere with PD-1/PD-L1-mediated suppression of CD4+ T cell inflammation and survival, and potentially contributed to the development and persistence of Crohn's disease.

Mathematical Modeling of the Metastatic Colorectal Cancer Microenvironment Defines the Importance of Cytotoxic Lymphocyte Infiltration and Presence of PD-L1 on Antigen Presenting Cells.

Although immune-based therapy has proven efficacious for some patients with microsatellite instability (MSI) colon cancers, a majority of patients receive limited benefit. Conversely, select patients with microsatellite stable (MSS) tumors respond to checkpoint blockade, necessitating novel ways to study the immune tumor microenvironment (TME). We used phenotypic and spatial data from infiltrating immune and tumor cells to model cellular mixing to predict disease specific outcomes in patients with colorectal liver metastases. Formalin fixed paraffin embedded metastatic colon cancer tissue from 195 patients were subjected to multiplex immunohistochemistry (mfIHC). After phenotyping, the G-function was calculated for each patient and cell type. Data was correlated with clinical outcomes and survival. High tumor cell to cytotoxic T lymphocyte (TC-CTL) mixing was associated with both a pro-inflammatory and immunosuppressive TME characterized by increased CTL infiltration and PD-L1+ expression, respectively. Presence and engagement of antigen presenting cells (APC) and helper T cells (Th) were associated with greater TC-CTL mixing and improved 5-year disease specific survival compared to patients with a low degree of mixing (42% vs. 16%, p = 0.0275). Comparison of measured mixing to a calculated theoretical random mixing revealed that PD-L1 expression on APCs resulted in an environment where CTLs were non-randomlylessassociated with TCs, highlighting their biologic significance. Evaluation of immune interactions within the TME of metastatic colon cancer using mfIHC in combination with mathematical modeling characterized cellular mixing of TCs and CTLs, providing a novel strategy to better predict clinical outcomes while identifying potential candidates for immune based therapies.

Abstract PD5-09: Immune parameters associated with survival in triple negative and HER2-positive breast cancer patients with 10 years of follow-up

Abstract The clinical utility of tumor-infiltrating lymphocytes (TIL) is actively being investigated in breast cancer (BC). It is unclear whether TIL spatial location and organization in tertiary lymphoid structures (TLS) have an impact on prognosis. Additionally, the significance of PD-1 and PD-L1 expression is being debated due to conflicting data from several studies. We hypothesize that the presence, extent and spatial location of multiple immune biomarkers, reflecting ongoing immune responses, will be consistently associated with a good prognosis in highly infiltrated BC [triple-negative (TNBC) and HER2+]. The relationship between these immune biomarkers and clinical outcome was examined in the TNBC and HER2+ cohorts of node-positive BC patients enrolled in the BIG 02-98 adjuvant phase III trial with available material for immunohistochemical (IHC) labeling (N=113 and N=136, respectively). HER2+ patients did not receive trastuzumab. Dual IHC staining was performed on full-face consecutive tissue sections. Scoring was independently performed by two pathologists, blinded to the clinical data, and included: global, intratumoral and stromal TIL and TLS, assessed on CD3/CD20 slides; the percentage and location of PD-1 and PD-L1 expression, assessed on PD-1/PD-L1 slides. TIL were considered as a categorical variable with different cut-offs used for each parameter and for each cohort (TNBC and HER2+). Invasive disease-free survival (I-DFS) and overall survival (OS) were analyzed (median follow-up: 10 years). Cox proportional hazard models were used for survival analyses. The TNBC cohort revealed an association between global TIL and outcome [adjusted hazard ratio (HR) for I-DFS: 0.27 (0.15-0.51); OS: 0.26 (0.13-0.53)]. Similar results were observed for stromal and intratumoral TIL. PD-L1 expression within TLS was an independent predictor of OS, after adjustment for tumor size and age [HR: 0.30 (0.09-0.99)]. Multivariate analysis reveals this effect was principally driven by high stromal TIL (>17.5% based on CD3/CD20 assessment) (χ2 OS: p=0.009). In contrast, no significant prognostic associations were found in the overall HER2+ cohort. However high T cell TIL were associated with improved I-DFS and OS in the ER-/HER2+ group [I-DFS: 0.34 (0.14-0.80); OS: 0.32 (0.12-0.86)] and stromal TIL were associated with improved I-DFS in the ER+/HER2+ group [HR: 0.29 (0.09-0.94)] (univariate analyses). No significant associations between the number of TLS nor the expression of PD-1 with outcomes were observed in either cohorts. The presence of PD-L1+ TLS, driven by high baseline TIL, was associated with an excellent prognosis in node-positive TNBC. This observation might reflect specific immune activities taking place in these mini lymph node-like structures adjacent to the tumor bed where specific antitumor memory immune responses could be generated. No different prognostic impact was observed when analyzing TIL spatial location. Although the statistical power of the study might be limited, in line with previous findings our data reveal that, among the immune parameters evaluated, TIL are the strongest predictor of outcome in TNBC, while PD-L1+ TLS could be a new and important parameter that requires further investigation. Citation Format: Solinas C, de Wind A, Van den Eynden G, Ameye L, Garaud S, De Silva P, Boisson A, Noel G, Langouo Fontsa M, Buisseret L, de Azambuja E, Francis PA, Di Leo A, Crown JP, Sotiriou C, Larsimont D, Paesmans M, Piccart-Gebhart M, Willard-Gallo K. Immune parameters associated with survival in triple negative and HER2-positive breast cancer patients with 10 years of follow-up [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD5-09.

Development of a Bifunctional Checkpoint Inhibitory T Cell Engager (CiTE) to Reverse Adaptive Immune Escape in AML

The CD33-targeting bispecific T cell engager (BiTE®) AMG 330 proved to be highly efficient in mediating cytotoxicity of AML cells in vitro and in mouse models (Krupka et al, Blood 2014). Yet, T cell activation is correlated with the upregulation of PD-L1 and other inhibitory checkpoint molecules on AML cells that confer adaptive immune resistance (Krupka et al, Leukemia 2016). PD-1/PD-L1 blocking agents may counteract T cell dysfunction, however, at the expense of broadly distributed immune-related adverse events (irAEs). We developed a checkpoint inhibitory T cell engaging (CiTE) antibody that combines T cell redirection to CD33 on AML cells with locally restricted immune checkpoint blockade.CiTE constructs were generated by first fusing a high-affinity CD33 single-chain variable fragment (scFv) to a CD3ε scFv in one polypeptide chain. Next, this single-chain chain was fused to the extracellular domain of PD-1 (PD-1ex), which naturally holds a low affinity to PD-L1. Antigen binding of CiTE constructs as well as CiTE mediated cytotoxicity of AML cell lines and primary AML cells were done using multiparameter flow cytometry. T cell activation and cytotoxicity assays were complemented by cytometric bead arrays. Murine AML xenograft studies using non-obese diabetic (NOD) scid gamma mice were used for engraftment of primary AML cells and assessment of CiTE mediated cytotoxicity in vivo.CiTE antibody constructs were successfully generated by fusing the bispecific CD33-CD3ε scFv to the endogenous extracellular domain of human PD-1 (PD-1ex). The CiTE was compared to a single chain triplebody (sctb), in which PD-1ex was replaced by a high-affinity PD-L1 scFv. The BiTE-like molecule, PD-1ex.αCD3 and αPD-L1.αCD3, as well as a non-targeting molecule served as controls. When investigating CiTE and sctb as whole molecules, both bound with similar affinities to CD33+PD-L1+ AML cell lines and HD T cells. CiTE- and sctb-induced upregulation of CD69 and CD25 on healthy donor T cells in the presence of MOLM-13-PD-L1 cells. By a synergistic effect of checkpoint blockade and avidity-dependent binding, the PD-1ex attachment increased T cell activation (3.3-fold elevation of IFN-γ release) and lead to efficient and highly selective cytotoxicity of CD33+PD-L1+ cells (EC50 = 2.3 pM to 26.9 pM) as well as primary AML patient samples (n=8). CiTE induced preferential lysis of CD33+PD-L1+ cells and had no activity against CD33-PD-L1+ cells. This was supported by the observation that the CiTE molecule was able to selectively induce elimination of CD33+PD-L1+ cells in the presence of PD-L1+ cells. In a murine xenograft model, the CiTE induced complete AML eradication without causing leukemia-unrelated T cell activation or body weight loss. Notably, murine and human PD-L1 bind with similar affinities to PD-1.We conclude that our molecule preferentially targets CD33+PD-L1+ AML cells, whereas high-affinity blocking agents also address PD-L1+ non-AML cells. Based on these findings, we expect to reverse adaptive immune escape mechanisms of T cell recruiting antibody formats and avoid irAEs associated with systemic checkpoint blockade, suggesting efficient therapeutic potential particularly for patients with relapsed or refractory AML. Future studies will need to further examine efficiency and tolerance in advanced in vivo models before applying the CiTE format into a clinical setting. DisclosuresLindl:Amgen: Research Funding. Metzeler:Celgene: Consultancy, Research Funding; Novartis: Consultancy. Subklewe:Gilead: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Roche: Consultancy, Research Funding; Celgene: Consultancy, Honoraria.

Abstract 1589: Presence of PD-L1+ tumor cells in tumor tissue and blood significantly associated with T/N stage and lymphovascular invasion in colorectal cancer patients

Abstract Background: PD-L1 assessment on tumor tissue is considered as an important predictive marker for anti-PD-1 treatments and has been approved by FDA as companion diagnostics for immunocheckpoint antagonists. Nonetheless, it remains challenging because of the dynamic nature and heterogeneity of PD-L1 expression, and the availability of tumor tissue. Measurement of circulating tumor cells (CTC) serves a form of liquid biopsy and tumor biomarker due to its non-invasiveness and real-time assessment. Here, we analyzed and compared PD-L1 expression in CTCs and tumor cells harvested from surgical specimen from colorectal cancer (CRC) patients. Methods: Mesenteric vein blood (MVB) samples were collected into K2EDTA-containing vacationer tubes and subsequently performed CTC enumeration and PD-L1 analysis by MiSelect R system. Tumor tissues were homogenized via collagenase enzyme digestion to single cell suspension before subjected to MiSelect R analysis. Tumor cells and CTCs are defined as EpCAM+, cytokeratin+ and CD45- with intact nuclei. PD-L1 analysis was performed by anti-PD-L1 primary antibody (clone 28-8). Tumor cells, CTCs and PD-L1+ tumor cells were counted and compared among different clinicopathological parameters. Results: MVB sample were collected intraoperatively from 116 CRC patients across various stages (stage I: 24, II: 38, III:42 and IV:12). CTC presence correlated with T stage and CEA level (P = 0.018 and 0.049, respectively); furthermore, PD-L1+ CTC presence significantly correlated with clinical stage, N stage, microscopic blood vascular and lymphatic vascular invasion (P = 0.0017, 0.013, 0.014 and 0.004, respectively). The percentage of PD-L1+ tumor cells showed positive correlation between tumor tissue and CTC, and both correlated with tumor stage. Conclusion: PD-L1+ cell was detectable in both tumor tissue and CTC in CRC patients and its percentage increased as disease progressed, which might reflect escape of tumor cell from immune surveillance. The percentage of PD-L1 expression on CTCs correlated with that in paired tumor tissues. On the other hand, the expression of PD-L1 on CTC was significantly associated with blood and lymphatic vessel invasion, which might be one of the potential mechanisms for local and distant metastasis of cancer. Further investigation is warranted to better understand the clinical significances of PD-L1-expression on CTCs and its potential utility as prognostic biomarker for cancer immunotherapy. Citation Format: Ju-Yu Tseng, Chwen-Cheng Chen, Yen-Ru Chen, Chia-Ying Lee, Chun-Chi Lin, Shin-Hang Wang, Tzu-Chao Hung, Hong-Ling Wang, Yi Chung, Yen-Lun Tseng, Mu-Yi Chen, Jeng-Kai Jiang. Presence of PD-L1+ tumor cells in tumor tissue and blood significantly associated with T/N stage and lymphovascular invasion in colorectal cancer 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 1589.