Tumor-infiltrating Lymphocytes PD-L1 Expression Research Articles

Retrospective immunophenotypical evaluation of MET, PD-1/PD-L1, and mTOR pathways in primary tumors and pulmonary metastases of renal cell carcinoma: the RIVELATOR study addresses the issue of biomarkers heterogeneity.

In renal cell carcinoma (RCC), tumor heterogeneity generated challenges to biomarker development and therapeutic management, often becoming responsible for primary and acquired drug resistance. This study aimed to assess the inter-tumoral, intra-tumoral, and intra-lesional heterogeneity of known druggable targets in metastatic RCC (mRCC). The RIVELATOR study was a monocenter retrospective analysis of biological samples from 25 cases of primary RCC and their paired pulmonary metastases. The biomarkers analyzed included MET, mTOR, PD-1/PD-L1 pathways and the immune context. High multi-level heterogeneity was demonstrated. MET was the most reliable biomarker, with the lowest intratumor heterogeneity: the positive mutual correlation between MET expression in primary tumors and their metastases had a significantly proportional intensity (P = 0.038). The intratumor heterogeneity grade was significantly higher for the mTOR pathway proteins. Combined immunophenotypical expression patterns and their correlations with the immune context were uncovered [i.e., mTOR expression in the metastases positively correlated with PD-L1 expression in tumor-infiltrating lymphocytes (TILs), P = 0.019; MET expression was related to PD-1 expression on TILs (P = 0.041, ρ = 0.41) and peritumoral lymphocytes (RILs; P = 0.013, ρ = 0.49)], suggesting the possibility of predicting drug response or resistance to tyrosine kinase, mTOR, or immune checkpoint inhibitors. In mRCC, multiple and multi-level assays of potentially predictive biomarkers are needed for their reliable translation into clinical practice. The easy-to-use immunohistochemical method of the present study allowed the identification of different combined expression patterns, providing cues for planning the management of systemic treatment combinations and sequences in an mRCC patient population. The quantitative heterogeneity of the investigated biomarkers suggests that multiple intralesional assays are needed to consider the assessment reliable for clinical considerations.

PD-L1, CD4+, and CD8+ Tumor-Infiltrating Lymphocytes (TILs) Expression Profiles in Melanoma Tumor Microenvironment Cells.

(1) Background: Because melanoma is an aggressive tumor with an unfavorable prognosis, we aimed to characterize the PD-L1 expression in melanomas in association with T cell infiltrates because PD-1/PD-L1 blockade represents the target in treating melanoma strategy. (2) Methods: The immunohistochemical manual quantitative methods of PD-L1, CD4, and CD8 TILs were performed in melanoma tumor microenvironment cells. (3) Results: Most of the PD-L1 positive, expressing tumors, have a moderate score of CD4+ TILs and CD8+TILs (5-50% of tumor area) in tumoral melanoma environment cells. The PD-L1 expression in TILs was correlated with different degrees of lymphocytic infiltration described by the Clark system (X2 = 8.383, p = 0.020). PD-L1 expression was observed often in melanoma cases, with more than 2-4 mm of Breslow tumor thickness being the associated parameters (X2 = 9.933, p = 0.014). (4) Conclusions: PD-L1 expression represents a predictive biomarker with very good accuracy for discriminating the presence or absence of malign tumoral melanoma cells. PD-L1 expression was an independent predictor of good prognosis in patients with melanomas.

Clinicopathological correlation and prognostic value of PD-L1 expression in renal cell carcinoma

Introduction: The expression of programmed cell death-ligand 1 (PD-L1) and its correlation with the prognosis of renal cell carcinoma (RCC) remains controversial. Objectives: This study aimed to study PD-L1 expression in tumor cells and tumor-infiltrating lymphocytes (TILs) in patients with RCC and its association with clinicopathological factors and survival outcomes. Patients and Methods: PD-L1 expression in tumor cells and TILs was analyzed using immunohistochemistry (IHC) from patients with histologically proven RCC. Results: PD-L1 was positive in tumor cells for 55.8% of patients. PDL-1 expression in TIL was reported in 31.2 % of patients. Patients with PDL1 positive tumor cells had higher median tumor size (P=0.07), higher nuclear grade (P=0.56), and higher lymphovascular invasion (LVI) (P=0.23). Patients with PDL1 positive TILs were significantly associated with larger median pathological tumor size (P=0.02), higher probability of renal fat invasion (P=0.001), higher nuclear grade (P=0.05), higher probability of positive margin (P=0.02), positive LVI (P=0.03), higher pathological T stage (P=0.0004); whereas patients with PDL-1 negative TILs had earlier stage at presentation (stage I-II) (P=0.004). There was no statistically significant difference in disease-free survival (DFS), progression-free survival (PFS), or overall survival (OS) for PD-L1 expression of tumor cells and TILs. Conclusion: PDL1 positivity in TILs and not in tumor cells was significantly associated with more aggressive features, and higher stage. No association was found with DFS, PFS, or OS. These data suggest that PD-L1 expression of TILs in RCC tumors contributes to cancer aggressiveness.

Прогностическая значимость экспрессии лиганда рецептора программируемой клеточной гибели PD-L1 в хордомах

Introduction. Chordomas are rare malignant neoplasms that are highly recurrent and with limited treatment options. Invasion of the vital structures of the nervous system complicates their treatment. The search for new methods of drug therapy for chordomas is an urgent problem. The aim of the research was to study PD-L1 expression in tumor cells and tumor infiltrating lymphocytes (TILs) in chordoma samples and its interrelation with tumor characteristics and prognosis. Materials and methods. We analyzed PD-L1 expression in 30 primary chordomas using immunohistochemistry and differences in independent groups using Chi-square and Kruskal–Wallis tests. Survival analysis was performed by constructing Kaplan–Meier survival curves. We compared the significance of differences with the logarithmic rank test. Differences were considered significant at p<0.05. Results. PD-L1 expression was detected in tumor cells in 43% of samples and in TILs in 80%. PD-L1 expres-sion in tumor cells and TILs was not associated with the clinical features of the disease. However, PD-L1 expression in tumor cells in all samples correlates positively with the content of this marker in TILs (r=0.409, p=0.028). Different histological variants of chordomas were shown to differ in the content of PD-L1 in both tumor cells and TILs. PD-L1 expression in tumor cells is not a predictor of the disease, while its expression in TILs tends to be a marker of a favorable prognosis (HR=0.1429; p=0.0570). Conclusion. We found the lowest level of PD-L1 expression in both tumor cells and TILs in dedifferentiated chordoma. PD-L1 expression in TILs is associated with a favorable prognosis of chordomas and indicates the potential for the use of checkpoint inhibitors in therapy. Keywords: chordoma, PD-L1, lymphocytes, prognosis, survival

PD-L1 expression in tumor-infiltrating lymphocytes (TILs) as an independent predictor of prognosis in patients with pN0 bladder cancer undergoing radical cystectomy

ObjectivesCheckpoint inhibitors have led to a paradigm shift in urothelial carcinoma (UC) treatment. However, the relationship between PD-L1 expression status and oncological outcomes in UC patients remains uncertain. Here, we investigated the prognostic value of PD-L1 expression status in patients with UC of the bladder (UCB) who underwent radical cystectomy (RC). Materials and methodsWe retrospectively analyzed pathological specimens from 97 UCB patients treated with RC from 1990 to 2015 at Kitasato University Hospital. Immunohistochemical staining using SP263 was performed to evaluate PD-L1 expression in tumor cells (TCs) and tumor-infiltrating lymphocytes (TILs). Kaplan-Meier plots and proportional Cox hazard ratios were examined to assess the relationship between PD-L1 expression and clinicopathological parameters and survival outcomes. ResultsOf the 97 specimens, 19.5% contained PD-L1-positive TCs, and 35.0% contained PD-L1-positive TILs. Regarding clinicopathological factors, PD-L1-positive TCs and TILs were significantly associated with high-grade tumors (TCs, P = 0.01; TILs, P = 0.003). Kaplan-Meier analyses showed that PD-L1-positive TCs were not correlated with survival rates. However, PD-L1-positive TILs were significantly associated with better recurrence-free survival (RFS; P = 0.03) and better cancer-specific survival (CSS; P = 0.02). Univariate analysis, but not multivariate analysis, CSS indicated that PD-L1-positive TILs were significant predictors of patient prognoses. Multivariate analysis showed that PD-L1-positive TILs independently predicted CSS in patients without lymph node metastasis (pN0). ConclusionPositive PD-L1 expression is associated with high-grade tumors. PD-L1-positive TILs are independent predictors of favorable survival outcomes in surgically resected UCB patients at stage pN0.

The Expression of Programmed Death Ligand-1 (Pd-L1) in Testicular Seminoma as Potential Prognostic Factor

BackgroundThe prognosis of testicular seminoma cases which failed to be cured with conventional therapy is poor. Several recurrent eventsafter chemotherapy were also found. PD-L1 is expressed in various types of malignancy and tumor infiltrating lymphocytes (TILs)and its role is known as a prognostic factor. This study was conducted to determine the role of PD-L1 expression seminoma indetermining overall survival (OS) and progression free survival (PFS).MethodsThis is a retrospective cohort study with survival analysis. Clinical data were obtained from medical record in RSUPN CiptoMangunkusumo since January 2011-December 2016 and observed for 2 years. Histopathological data were obtained fromAnatomical Pathology Department and PD-L1 immunohistochemical staining were performed.ResultsA significant correlation between PD-L1 expression in tumor cells with 2-year OS (p=0.023) and PFS (p=0.002) in testicularseminoma was found. No significant correlation between PD-L1 expression in TILs with 2-year OS (p=0.235) and PFS (p=0.111).We also found significant correlations between PD-L1 expression of tumor cells and TILs with PFS (p=0.019). A significantcorrelation between stage with 2-year OS (p=0.010) and PFS (p=0.000) and lymph node metastases with 2-year OS (p=0.010) andPFS (p=0.000).ConclusionPD-L1 expression of testicular seminoma cells were significantly correlated with OS and PFS. There were no statistically significantassociations between PD-L1 expression in TILs with OS and PFS.

Abstract P4-10-30: PD-L1 expression in tumor infiltrating lymphocytes (TILs) inversely correlates with androgen receptor (AR) expression in HER2-positive breast cancer

Abstract Background: Human epidermal growth factor receptor 2 (HER2)-positive breast cancer accounts for 20-30% of breast carcinomas and is associated with a poor prognosis. While anti-HER2 targeted therapies have improved outcomes within this group, a subset of these tumors eventually develop resistance urgently requiring novel therapeutic options. In addition to classic HER2+ subtype, estrogen receptor (ER)-positive and HER2-positive tumors (luminal B) are another subtype of HER2 expressing breast carcinomas which could also benefit from additional therapies. Targeting the androgen receptor (AR) pathway and the PD-L1 axis may represent potential new therapeutic strategies in breast cancer. Our group previously reported that AR expression is seen in ¾ of HER2+ carcinomas and is more common in luminal B compared to classic HER2+ tumors. In this report, we evaluated expression of PD-L1 in tumor infiltrating lymphocytes (TILs) in both classic HER2 + and Luminal B subtypes of breast carcinomas and correlated the expression of PD-L1 with AR expression in these tumors. Methods: The study population consisted of 114 patients with HER2-positive invasive breast carcinoma diagnosed from 2009-2014 at Northwestern Memorial Hospital. Electronic medical records were reviewed for patient demographics (mean age 54, range 23-80). Pathologic tumor characteristics (histologic type, size, and grade) and tumor marker profile (ER and HER2) were evaluated. Tissue microarrays were constructed (3 cores from each case to account for tumor heterogeneity) for immunohistochemical evaluation of AR and PD-L1. AR was graded as positive when at least 1% of tumor cells showed nuclear immunoreactivity. For PD-L1, the extent of staining (0%=0, 1-10%=1, 11-50%=2, and 51-100%=3) and staining intensity (0, 1+, 2+, 3+) were assessed in TILs. Then a composite score (CS) was calculated by multiplying extent and intensity results (range 0-9; 0-3=negative, 4-9=positive). Results: Of the 114 studied breast carcinoma cases, 54 were classic HER2+ tumors (47%) and 60 were luminal B (53%) tumors. Approximately 69% of classic HER2+ and 87% of luminal B cases were positive for AR. Of interest, in classic HER2+ cases, the expression of PD-L1 in TILs was inversely correlated with the expression of AR, with 82% (14/17) of AR- tumors being positive for PD-L1 in TILs compared to only 46% (17/37) of AR+ tumors with PD-L1 expression in TILs (p<0.01). No association of expression of PD-L1 in TILs with AR expression was observed in luminal B cases (62% in AR+ versus 50% in AR- tumors; p=0.70). Moreover, in classic HER2+/AR- group, grade 3 tumors were seen almost exclusively in the PD-L1 expressing group (13 versus 3), in contrast to the equal number of grade 3 tumors in classic HER2+/AR+ group regardless of PD-L1 expression in TILs (14 versus 14). No association of the PD-L1 expression with age, race, and tumor histologic types was observed in classic HER2+/AR+ or classic HER2+/AR- cases. Conclusions: Our results demonstrated that the expression of PD-L1 in TILs was inversely correlated with the expression of AR in classic HER2 positive tumors but not in luminal B tumors. We also found that in classic HER2+/AR- group, grade 3 tumors were seen almost exclusively in the PD-L1 expression group. Our findings add to the body of knowledge of the molecular mechanisms that drive the different molecular subtypes of breast carcinomas and raise the interesting possibility that new treatment strategies such as targeting the PD-L1 axis may show more benefit against the AR negative subtype of the classic HER2 positive carcinomas. Citation Format: Tiansheng Shen, Jennifer L. Pincus, Kalliopi P. Siziopikou. PD-L1 expression in tumor infiltrating lymphocytes (TILs) inversely correlates with androgen receptor (AR) expression in HER2-positive breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-10-30.

PD-L1 expression in tumor infiltrated lymphocytes predicts survival in triple-negative breast cancer

ObjectiveTriple-negative breast cancer (TNBC), a complex and highly aggressive subtype of breast cancer, generally has the poorest clinical outcome, there is a pressing need for more effective therapeutic strategies. Immune checkpoint inhibitors against programmed death 1/programmed death ligand 1 (PD1/PDL1) have revolutionized treatment of several solid tumours, such as non-small cell lung carcinoma (NSCLC), renal, malignant melanoma. However, no checkpoint inhibitors were previously approved for the treatment of TNBC. So far, very limited data have reported PDL1 (SP142) expression and its relationship with clinicopathological behaviors and survival in TNBC. MethodsPD-L1(SP142) immunohistochemistry was performed on 223 TNBC cases and assessed in tumour cells(TC) as well as tumor-infiltrating lymphocytes(TILs).The relationships between PD-L1 expression and clinicopathological characteristic both in TC and TILs. Futhermore,we also explored the effect of PD-L1 expression on prognosis as illustrated by overall survival(OS). ResultsPD-L1 expression was detected in both tumor cells and TILs at a ratio of 8.5 % and 25.1 % respectively. PD-L1 expression in TILs was related to histological grade and abundance of TILs. Tumor cell expression of PD-L1 was not associated with outcome. While PD-L1 expression in TILs and lymphnode transfer were associated with a poor outcome, and PD-L1 expression was an independently prognostic of overall survival (OS) (HR = 0.867, P = 0.029). ConclusionPD-L1 expression in TILs, but not in tumor cells, was a poor prognostic factor in TNBC. These data provide further impetus for assessing immunotherapy in TNBC, in view of the clinical significance of the expression of PD-L1 (SP142) in TNBC.

Circulating Lymphocytes, PD-L1 Expression on Tumor-infiltrating Lymphocytes, and Survival of Colorectal Cancer Patients with Different Mismatch Repair Gene Status.

Clinical outcomes of checkpoint blockade immunotherapy on colorectal cancer (CRC) are influenced by mismatch repair (MMR) gene status, which is associated with distinct tumor immune infiltrates and systemic inflammatory response status. However, the prognostic value of PD-L1 expression and the systemic inflammatory response for patients with MMR deficiency has not been fully investigated. In this study, we examined the association of systemic inflammatory markers, PD-1/PD-L1 pathway expression, microsatellite instability (MSI) status, and clinicopathological characteristics of CRC with patient survival between MMR-deficient (dMMR) group (N=168) and MMR-proficient (pMMR) group (N=169). We found a large proportion of dMMR CRC patients displayed increased level of systemic inflammatory markers such as C-reactive protein, Neutrophil/Lymphocyte Ratio (NLR), Glasgow Prognostic Score (GPS), and low expression of PD-L1 in tumor stroma. Several systemic inflammatory markers were associated with AJCC stage only in dMMR patients. Similarly, Tumor infiltrating lymphocyte (TIL) PD-L1 or stroma PD-L1 expression was associated with AJCC stage only in dMMR patients. Circulating serum lymphocytes and TIL PD-L1 expression are both independent prognosis predictors for CRC patients. Overall, we found that dMMR CRC displayed a comprehensively distinct tumor immune microenvironment and systemic inflammatory response makers. PD-L1 expression at different location has different impacts on CRC patient survival, and the TIL PD-L1 expression might be a potential predictor for dMMR CRC patient response to anti-PD-1 therapy.

Abstract P2-03-11: The immune microenvironment of HER2-positive ductal carcinoma in situ of the breast

Abstract Background: Ductal carcinoma in situ (DCIS) consists of a heterogeneous group of tumors that can be subclassified based on their molecular profile similar to invasive breast carcinomas. DCIS ducts are often surrounded by an inflammatory infiltrate, yet few studies have focused on this inflammatory response in the different subtypes of DCIS. The role of the immune microenvironment in tumor progression and response to therapy has led to a number of effective immune-based therapies, particularly targeting the PD-1/PD-L1 axis, and these approaches may extend to breast cancer subtypes and their precursor DCIS. In this study we evaluated the inflammatory response and PD-1/PD-L1 expression in both tumor-infiltrating lymphocytes (TILs) and tumor cells in HER2-positive (HER2+) and HER2-negative (HER2-) DCIS. Design: Our population consisted of 85 cases of pure DCIS treated with surgical excision or mastectomy at our institution from 2008 to 2012. The molecular subtypes of DCIS were determined based on ER and HER2 expression. Tissue microarrays (TMAs) were constructed with 3 cores from each case to account for tumor heterogeneity. The presence of tumor-associated inflammation (TAI) was graded as absent, mild (<10%), moderate (10-50%) or severe (>50%) and the extent and intensity of PD1 and PD-L1 in the TILS and in the tumor cells were also assessed. A composite score was calculated by multiplying extent and intensity (range 0-12 with 9-12=strong). Results: Of the 85 cases, 51 were classified as Luminal A (ER+/HER2-), 15 as Luminal B (ER+/HER2+), 13 as HER2+ (ER-/HER2+) and 6 as basal-like (ER-/HER2-). Moderate/severe inflammation around DCIS correlated with HER2 expression (20/28 HER2-expressing cases (71.4 %) compared to 21/36 HER2-non-expressing cases (58.3%), p=0.005). Of interest, severe inflammation was seen almost exclusively around HER2-expressing cases (7/28, 25% vs 1/57, 1.7 %, p=0.002). Furthermore, over half of the TILs around HER2+ cases expressed PD-L1 (7/13, 54%), while only 6% of Luminal A (3/47) and none of Luminal B (0/15) or basal-like (0/6) cases did (p=0.00001). In addition, about 1/3 of the TILs around HER2+ cases expressed PD-1 (4/13, 31%), while only 8% of Luminal A (4/49) and none of Luminal B (0/15) or basal-like (0/6) cases did (p=0.004). None of the DCIS tumor cells expressed PD-1. Of interest, 15% of HER2+ cases (2/13) and 7% of Luminal B cases (1/15) expressed PD-L1 in the DCIS tumor cells. Conclusions: 1. Moderate/severe inflammation around DCIS foci correlates with HER2 expression. 2. PD-L1 expression in TILs is seen almost exclusively in HER2+ DCIS cases 3. About 1/3 of the TILs around HER2+ DCIS cases also express PD-1. 4. None of the DCIS tumor cells express PD-1. 5. Small subgroups of HER2+ and Luminal B DCIS cases show PD-L1 expression in the tumor cells themselves. The results of our study add to the understanding of the role of the immune microenvironment in DCIS, especially in the HER2+ subtype. The complex interactions between DCIS tumor cells and the local immune system may play a role in breast cancer progression and may be further exploited for immune-based therapeutic strategies. Citation Format: Ubago JM, Blanco, Jr. LZ, Siziopikou KP. The immune microenvironment of HER2-positive ductal carcinoma in situ of the breast [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-03-11.

PD-L1 and PD-1 and characterization of tumor-infiltrating lymphocytes in high grade sarcomas of soft tissue – prognostic implications and rationale for immunotherapy

ABSTRACTTherapies targeting programmed death 1-(PD-1) or its ligand (PD-L1), promoting antitumor T-cell activity have been successfully introduced into clinical practice. Clinical response correlates with PD-L1 expression by tumor cells or immune cells within the tumor microenvironment. The PD-L1/PD-1 axis and tumor microenvironment has been rarely studied in high-grade sarcomas of soft tissue (hSTS), a group of rare, genetically heterogenous and clinically aggressive tumors. We examined PD-L1 protein and CD274/PD-L1 gene copy number variations in 128 primary resected, therapy-naive hSTS using immunohistochemistry and fluorescence-in-situ hybridization. Frequency of tumoral PD-L1 expression varied widely in different disease subentities, with highest rates of positivity (40%) seen in undifferentiated pleomorphic sarcomas (UPS) and rare positivity detected in synovial sarcomas (6%). Amplification of the CD274/PD-L1 gene occurred in 14% of UPS and was rare in other subtypes. PD-L1 protein expression was significantly more frequent in CD274/PD-L1 amplified cases (p = 0.015). The subgroup of UPS was further characterized regarding the interaction between PD-L1 and the immunologic tumor microenvironment. High density of CD3+ and CD8+ tumor infiltrating lymphocytes (TILs) was significantly correlated with the presence of PD-L1 expression and seen more frequently in tumors with lower TNM stage (p = 0.024). Both, PD-L1 expression and high density lymphocytic infiltration were independent prognostic factors for a favorable overall (p = 0.001, HR 6.105 (2.041–8.258)), disease-specific (p = 0.003, HR 10.536 (2.186–50.774)) and disease-free survival (p = 0.020, HR 3.317 (1.209–9.106); values for CD8) in this particular subgroup of hSTS, whereas PD-L1 expression in TILs or CD274/PD-L1 gene amplification were not associated with outcome. These findings represent novel insights into the immune landscape of soft tissue sarcomas, in particular UPS and strengthen the rationale for immunotherapy, including targeting the PD-1/PD-L1 axis in these tumors.

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 >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<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 (>50%) was significantly associated with better DMFS: HR=0.51; 95%CI: 0.27-0.98. TNBC with high PD-L1 expression of TILs (>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

Case-control study of PD-1, PD-L1, and B7-H3 expression in human immunodeficiency virus infected (HIV+) and uninfected patients with head and neck cancers.

e23194 Background: HIV+patients are typically excluded from immunotherapy trials but there are limited data on co-signaling molecule expression in these patients, including in head and neck squamous cell carcinoma (HNSCC). Methods: A case control study was conducted to evaluate tumor tissue sections for PD-L1, PD-1, and B7-H3 expression in HIV+HNSCC patients (n = 12) and HNSCC controls (n = 12). Cases and controls were matched for age at diagnosis, race, gender, TNM stage, and primary site. Smoking status, HIV RNA viral load (VL), and CD4 count were also recorded. Immunostained tumor sections were analyzed for percent of tumor cells expressing PD-L1 (PD-L1%) and B7-H3(B7-H3%) (Abcam), and percent of tumor infiltrating lymphocytes (TIL) expressing PD-1 (TIL-PD-1%) and PD-L1 (TIL-PD-L1%) (Abcam). Statistical analysis used the non-parametric Mann-Whitney test, chi-square test and Spearman’s rank correlation, Rs. Results: The 12 HIV+ HNSCC cases were predominantly male (67%), black race (67%) and cigarette smokers (100%), with a median age of 50.4 years, median viral load (VL) of 52399 copies/mL, median CD4 count (CD4) of 236cells/uL, predominantly locally advanced (75% Stage III/IVa/IVb), and oropharyngeal primary site (42%). Defining positive expression as > 5%, 42% of HIV+ HNSCC patients tumors were positive for PD-L1, 100% for B7-H3, and 92% had TILs that expressed PD-1 and PD-L1. HIV+ patients had significantly higher B7-H3% (Median 60% vs. 20%, p = 0.03) and TIL-PD-L1% (Median 15% vs. 10%, p = 0.045) compared to controls. There were no differences in PD-L1% or TIL-PD-1%. In HIV+ cases, increased VL correlated with increased TIL-PD-1% (Rs = 0.73, p = 0.011) and increased CD4 count correlated with increased tumor PD-L1% (Rs = 0.62, p = 0.04). There were no other significant correlations between CD4 count or VL and co-signaling molecule expression. Conclusions: HIV+ HNSCC patients had significantly higher tumor B7-H3 and TIL PD-L1 expression, with similar tumor PD-L1 and TIL PD-1 expression, compared to HIV negative HNSCC control patients. These findings support inclusion of HIV+ HNSCC patients in immunotherapy trials with checkpoint inhibitors.

Temporal and spatial discordance of programmed cell death-ligand 1 expression and lymphocyte tumor infiltration between paired primary lesions and brain metastases in lung cancer

The dynamics of PD-L1 expression may limit its use as a tissue-based predictive biomarker. We sought to expand our understanding of the dynamics of PD-L1 expression and tumor-infiltrating lymphocytes (TILs) in patients with lung cancer-related brain metastases. Paired primary lung cancers and brain metastases were identified and assessed for PD-L1 and CD3 expression by immunohistochemistry. Lesions with 5% or greater PD-L1 expression were considered positive. Agreement statistics and the χ(2) or Fisher's exact test were used for analysis. We analyzed 146 paired lesions from 73 cases. There was disagreement of tumor cell PD-L1 expression in 10 cases (14%, κ = 0.71), and disagreement of TIL PD-L1 expression in 19 cases (26%, κ = 0.38). Most paired lesions with discordant tumor cell expression of PD-L1 were obtained 6 or more months apart. When specimens were categorized using a proposed tumor microenvironment categorization scheme based on PD-L1 expression and TILs, there were significant changes in the classifications because many of the brain metastases lacked either PD-L1 expression, tumor lymphocyte infiltration or both even when they were present in the primary lung cancer specimens (P = 0.009). We identified that there are significant differences between the tumor microenvironment of paired primary lung cancers and brain metastases. When physicians decide to treat patients with lung cancer with a PD-1 or PD-L1 inhibitor, they must do so in the context of the spatial and temporal heterogeneity of the tumor microenvironment.

PD-L1 expression in the Merkel cell carcinoma microenvironment: association with inflammation, Merkel cell polyomavirus and overall survival.

Merkel cell carcinoma (MCC) is a lethal, virus-associated cancer that lacks effective therapies for advanced disease. Agents blocking the PD-1/PD-L1 pathway have demonstrated objective, durable tumor regressions in patients with advanced solid malignancies and efficacy has been linked to PD-L1 expression in the tumor microenvironment. To investigate whether MCC might be a target for PD-1/PD-L1 blockade, we examined MCC PD-L1 expression, its association with tumor-infiltrating lymphocytes (TILs), Merkel cell polyomavirus (MCPyV), and overall survival. Sixty-seven MCC specimens from 49 patients were assessed with immunohistochemistry for PD-L1 expression by tumor cells and TILs, and immune infiltrates were characterized phenotypically. Tumor cell and TIL PD-L1 expression were observed in 49% and 55% of patients, respectively. In specimens with PD-L1(+) tumor cells, 97% (28/29) demonstrated a geographic association with immune infiltrates. Among specimens with moderate-severe TIL intensities, 100% (29/29) demonstrated PD-L1 expression by tumor cells. Significant associations were also observed between the presence of MCPyV DNA, a brisk inflammatory response, and tumor cell PD-L1 expression: MCPyV(-) tumor cells were uniformly PD-L1(-). Taken together, these findings suggest that a local tumor-specific and potentially MCPyV-specific immune response drives tumor PD-L1 expression, similar to previous observations in melanoma and head and neck squamous cell carcinomas. In multivariate analyses, PD-L1(-) MCCs were independently associated with worse overall survival (hazard ratio 3.12; 95% CI, 1.28-7.61; p=0.012). These findings suggest that an endogenous immune response promotes PD-L1 expression in the MCC microenvironment when MCPyV is present, and provide a rationale for investigating therapies blocking PD-1/PD-L1 for patients with MCC.