Combined CHK1 and PD-L1 blockade as a novel therapeutic strategy against stemness and immunosuppression in ovarian cancer

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

ABSTRACTDespite its rare incidence worldwide, penile squamous cell carcinoma (PeSCC) still presents with significant morbidity and mortality due to the limited treatment options for advanced patients, especially those in developing countries. The program death-1 (PD-1)/PD-1 ligand (PD-L1) axis has been demonstrated to play an important role in tumor immune escape, and immunotherapies targeting this pathway have shown great success in certain cancer types. Here, we analyzed the expression pattern of PD-L1 in tumor cells and tumor-infiltrating lymphocytes (TILs) in PeSCC with a multi-center cohort. We found that the majority of PeSCCs (53.4%) were PD-L1-positive and that high PD-L1 expression in tumor cells was associated with a poor prognosis. Notably, PD-L1 expression in tumor cells was significantly associated with the extent of TILs and CD8+ TILs. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) showed that PD-L1 was positively correlated with interferon-gamma (IFNγ) and CD8+ gene expression. Moreover, we defined the constitutive and inducible surface expression of PD-L1 in newly established primary PeSCC cell lines. Interestingly, two PeSCC cell lines had high intrinsic PD-L1 expression. Another cell line showed low PD-L1 expression, but the PD-L1 expression could be induced by IFNγ stimulation. Overall, our data showed that high PD-L1 expression in penile tumor cells indicated a poor prognosis. The upregulation of PD-L1 in PeSCC included both extrinsic and intrinsic mechanisms. These findings indicated that the PD-1/PD-L1 axis might be a potential therapeutic target for patients with penile squamous cell carcinoma.

Background: Invasive lobular carcinoma (ILC) is the second most common type of invasive breast cancer and accounts for 10-15% of all cases. Though ILC has distinct clinical, prognostic and molecular features, studies are limited and include few patients. ILCs show a decreased response to neoadjuvant chemotherapy and an increased resistance to endocrine therapy. Thus, there is a great need to identify alternative therapies, such as immunotherapy, that could improve overall survival. Success of immunotherapy largely depends on tumor immunogenicity which varies with histologic type. Determination of predictive and prognostic biomarkers for ILC will help determine who can benefit the most. Our study investigates canonical markers of immunogenicity - PD-L1 expression and Tumor Mutational Burden (TMB) - in patients with ILC compared to invasive ductal carcinoma (IDC). We further correlate these markers in different subtypes i.e. hormone receptor positive (HR+), HER2 positive (HER2+), and in triple negative breast cancers (TNBC). We also analyze differences in immune cell profiles constituting the tumor microenvironment (TME) and differences in genomic alterations between ILC and IDC. Methods: A retrospective data analysis was performed to identify breast cancer tumors with ILC or IDC histology profiled at Caris Life Sciences (Phoenix, AZ) that were tested for PD-L1 by SP142 assay and had whole transcriptome sequencing data available. ILC and IDC cases were further subtyped as HR+ and HER2+ and TNBC. PD-L1 expression in immune cell was assessed using Ventana PD-L1 (SP142) histochemical assay and PD-L1 expression in tumor cell was assessed by laboratory developed test using SP142 clone with staining higher than 2+ considered positive. TMB was measured by counting somatic non-synonymous missense mutations on the 592 gene panel (Nextseq) next generation sequencing (NGS) assay, and ≥ 10 mutations/megabase (mut/Mb) was considered high. Using the whole transcriptome RNA sequencing (NovaSeq) data we analyzed the difference in immune cell profiles constituting the TME using a computational RNA deconvolution approach. NGS was used to identify significant differences in genomic alterations between ILC and IDC. Results: We identified 1,359 breast cancer patients (ILC: 194 vs IDC: 1,165). Among ILC, 79% were HR +, 11% TNBC, <2% HER2+ and the rest were of unclear subtype, compared to 55% HR +, 31% TNBC, 10% HER2+ in the IDC group. PD-L1 expression in immune cells was lower in ILC (PD-L1≥ 1% ILC: 13% vs. IDC: 35% p <0.0001; PD-L1>10% ILC: 1% vs. IDC: 5% p=0.017). PD-L1 expression in tumor cells was also lower in ILC (ILC 1% vs IDC 5%; p =0.0136). All subtypes of ILC had lower PD-L1 expression in immune and tumor cells when compared to IDC. TMB was similar and comparable in IDC and ILC (median TMB 7 mut/Mb). Assessment of TME showed a significantly increased abundance of cytotoxic lymphocytes and monocytic cells in IDC, and stromal endothelial cells in ILC. Androgen receptor (AR) expression, and mutations in CDH1 (ILC 76% vs IDC 2%) and PIK3CA (ILC 44% vs IDC 30%) were more common in ILC. CDH1 mutation was significantly higher in both TN ILC (ILC 60% vs IDC 1%; p<0.05) and HR+ ILC (77% vs IDC 2%; p<0.05) subtypes of ILC. TP53 mutation was lower in ILC (ILC 26% vs IDC 65%) irrespective of subtype. Conclusion: In summary, PD-L1 expression in immune cells and tumor cells was lower in ILC, however TMB was comparable between ILC and IDC. Immune cell profiling supports a cold or less immunogenic TME for ILC. A composite immune biomarker may be able to better characterize immunogenicity of ILC. Citation Format: Upama Giri, Joanne Xiu, Tatiana Karadimitriou, Sofi Castanon, Foluso Ademuyiwa, Paula Pohlmann, Elias Obeid, Jasgit Sachdev, Elisa Krill-Jackson, Michael Simon, Antoinette Tan, Neelima Denduluri, Lee Schwartzberg, Michael Korn, Evanthia T Roussos Torres. Molecular evaluation of immunogenicity and genomic alterations in invasive lobular breast cancer [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 PS17-27.

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.

Background: High PD-L1 expression has been shown to be associated with improved clinical outcomes to anti-PD-1/L1 therapies in NSCLC and other indications. However, only a fraction of the PD-L1 high patients (pts) respond. PD-L1 can be induced by IFNG and expressed in tumor cells (TC) and inflammatory cells (IC). Improving our ability to predict patient benefit from anti-PD-1/L1 therapies requires a better understanding of associations between PD-L1 expression in TC and/or IC and outcome. Here we explore the relationship between patterns of IHC PD-L1 expression in TC and IC, gene expression, and clinical outcome. Methods: CP1108/NCT01693562 was a nonrandomized phase 1/2 trial evaluating durvalumab in pts with advanced NSCLC or other solid tumors. As of 29 APR 2016, 368 previously treated NSCLC pts received durvalumab ≤12 months with a median 18.8 months follow up. Pts with ≥25% TC or IC were scored TC+ and/or IC+, respectively. Results: TC+ PD-L1 pts (includes IC+ and IC- PD-L1 pts) had improved survival compared to TC- PD-L1 pts. TC+ and IC+ PD-L1 pts had: improved survival compared to TC+/IC-, TC-/IC+, or TC-/IC- PD-L1 pts. However, prevalence of TC+/IC+ was lower than TC+. Twenty-one genes significantly differed between TC+/IC+, TC+/IC-, TC-/IC+, and TC-/IC- PD-L1 patient subsets, the vast majority being well-known IFNG-inducible genes and mostly over-expressed in the TC+/IC+ subset. Conclusions: TC+ and IC+ PD-L1 pts had the highest levels of IFNG-inducible gene expression, a key biological feature that distinguishes PD-L1 IHC positive from negative pts. Thus, in addition to PD-L1 IHC, the predictive value of IFNG should be investigated in additional relevant studies Selection CriteriaPrevalence of Biomarker Positive BiomarkerPositive/negative median OSlog-rank pHRcox pTC+158/276 (57%)15.67/7.730.00910.690.046TC+/IC+70/276 (25%)25.63/8.377.46E-060.345.24E-05TC+/IC-88/276 (32%)10.5/13.970.0941.50.032TC-/IC+50/276 (18%)9.07/13.230.830.960.84TC-/IC-68/276 (25%)5.77/14.030.000841.660.012 All comers: median OS: 11.2 months (N=304) Citation Format: Carlos Bais, Chris Morehouse, Brandon W. Higgs, Rebelatto Marlon, Keith Steele, Xiaoping Jin, Li Shi, Susana Korolevich, Ashok Gupta, Koustubh Ranade. Biological and clinical relevance of PD-L1 expression in tumor and inflammatory cells in NSCLC [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 3720. doi:10.1158/1538-7445.AM2017-3720

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.

50 Background: Although immune checkpoint inhibitors have demonstrated promising results in melanoma and lung cancer, their effects on gastrointestinal cancer are still under investigation. Understand the prognostic effect of PDL1 expression, infiltration by CD8+ and CD3+ T cells in gastric cancer (GC) might help the identification of candidate patient who might benefit from immunotherapy. Methods: Immunohistochemistry were performed on a tissue microarray including 1,014 GC specimens with PD-L1 antibodies. Immune cell markers CD3 and CD8 were also stained and the quantitative differences were calculated using automated image analysis. Results: 37.8% of the cases showed a membranous PD-L1 expression in tumor cells and 74.9% in tumor infiltrating immune cells. GC patients with high level PDL1 expression in tumor cells or in immune cells were both associated with better survival compared with those with negative expression (5-year OS, 61% vs. 48%, P = 0.000, and 55% vs. 45%, P < 0.0001, respectively). As continuous variables, higher CD3+ and CD8+ cell density were associated with patients’ overall survival and the associations remained as the significant prognostic factors that predict favorable outcomes even after adjusting other unknown confounding factors.(HR: 0.974, 95%CI: 0.963-0.985, P = 0.000; and HR: 0.973, 95%CI: 0.960-0.986, P = 0.000 for CD3 and CD8 respectively). We also observed the close relationship between CD3+, CD8+ cell density and PDL1 expression both in tumor cells and in infiltrating immune cells. Conclusions: This study indicates that PDL1 expression, and TILs in particular, are important prognostic biomarkers. Patients with higher CD8 and CD3 T cell densities also have higher PD-L1 expression, indicating an adaptive immune resistance mechanism may be occurring. The counterintuitive observation that higher PD-L1 expression associating with better patients’ outcome highlights the need to assess both PDL1 expression in all tumor compartments and the characterization of the GC immune microenvironment.

Relevance. Prognostic value of PD-L1 expression in oral cavity squamous cell carcinoma (OCSCC) and its effect on survival is still controversial. It should be to determine the prognostic role of PD-L1 expression on tumor and immune cells of OCSCC and assess their effect on overall survival (OS) and progression-free survival (PFS).Materials and methods. A prospective study included 145 patients, first diagnosed with OCSCC. PD-L1 expression on tumor and immune cells, infiltrating tumor and its microenvironment, was assessed in all tumor samples by IHC, CPS was calculated. Cut-off values were determined by ROC analysis for identification of PD-L1 expression effect on OS and PFS.Results. Most patients with oral mucosa squamous cell carcinoma showed positive expression of PD-L1 on tumor (77.2%) and immune cells (92.4%). The median PD-L1 expression on tumor cells was 13.5% [1.0-40.0], the median PD-L1 expression on immune cells was 5.0% [1.0-11.0], and the median CPS – 18.0 [3.0-7.8]. Univariate and multivariate analyses revealed a significant negative effect of PD-L1 expression on immune cells ≤ 7% on OS (HR 0.66; 95% CI 0.45-0.93; p = 0.0498); PD-L1 expression in tumor cells ≤ 15% (HR 0.65; 95% CI 0.43-0.98; p = 0.0416) and CPS ≤ 21 (HR 0.62; 95% CI 0.44-0.92; p = 0.0183) for PFS. PD-L1 expression in tumor cells ≤ 6% (HR 0.71; 95% CI 0.47-1.08; p = 0.1096) and CPS ≤ 7 (RR 0.67; 95% CI 0.44-1.01; p = 0.0575) had a confident tendency to negative impact on OS.Conclusion. Positive PD-L1 expression in tumor and immune cells as well as CPS are effective additional factors in the prognosis of the disease course, OS and PFS in patients with OCSCC.

PurposeThe present study aimed to investigate the prognostic effect of PD-L1 expressing in tumor and immune cells among patients with esophageal squamous cell carcinoma.Patients and MethodsWe performed a retrospective cohort study by consecutively recruiting 142 patients. The clinicopathological features and PD-L1 expression on tumor and immune cells were independently evaluated by two pathologists.ResultsThe median expression rate of PD-L1 was 5% and 30% in tumor and immune cells, respectively. Patients with higher expression of PD-L1 in tumor cells had shorter disease-free and overall survival, and the HRs were 1.52 for relapse (95% CI: 0.88, 2.60) and 1.48 for death (95% CI: 0.82, 2.69). There was no significant association between the PD-L1 expression in immune cells and survival. However, among the patients with PD-L1 expression rate ≤30% in immune cells, the high expression rate of PD-L1 in tumor cells was significantly associated with the relapse and death, with HRs of 2.51 (95% CI: 1.25, 5.06) and 3.51 (95% CI: 1.57, 7.85), respectively. Among patients with PD-L1 expression rate >30% in immune cells, the PD-L1 expression in tumor cells did not show any association with the disease-free and overall survival.ConclusionOur study demonstrates that the integration of PD-L1 expression in tumor and immune cells could be used to predict the relapse and survival among patients with esophageal squamous cell carcinoma.

Objective: To study the effect of MYD88 L265P mutation on the expression of PD-L1 in tumor cells and tumor microenvironment in diffuse large B-cell lymphoma (DLBCL), and to provide theoretical basis for immunotherapy for patients. Methods: Multiplex ligation-dependent probe amplification (MLPA) was used to detect the frequency of MYD88 L265P mutation in 72 cases of DLBCL diagnosed by pathologists in Cancer Hospital of Chinese Academy of Medical Sciences from August 2008 to May 2010. Expression of PD-L1 in tumor cells and tumor microenvironment in all samples was evaluated using PD-L1 (22C3) and PD-L1 (SP142) with Ventana automatic immunohistochemical (IHC) platform. The relationship between MYD88 L265P mutation and the expression of PD-L1 in DLBCL tumor cells and tumor microenvironment was assessed. Results: Of the 72 cases of DLBCL, MYD88 L265P mutation was detected in 15 (20.8%) cases. Nine cases with JAK2 amplification were excluded, and the remaining 63 cases of DLBCL were divided into MYD88 L265P mutant group (n=14) and MYD88 L265P wild-type group (n=49). IHC results showed that among the 14 cases of MYD88 L265P mutant groups, PD-L1 (22C3) was positive in 7 cases (7/14) of tumor cells and PD-L1 (SP142) was positive in 4 cases (4/14) of tumor microenvironment. Among the 49 cases of MYD88 L265P wild-type group, 9 cases (18.4%) were positive for PD-L1 (22C3) in tumor cells, and 38 cases (77.6%) were positive for PD-L1(SP142) in tumor microenvironment. In addition, among the 16 cases with PD-L1(22C3) expression in tumor cells, only 2 of the 7 cases with MYD88 L265P mutation were positive for PD-L1 (SP142) in tumor microenvironment. All 9 cases with wild-type MYD88 L265P were positive for PD-L1 (SP142) in tumor microenvironment. Statistical analysis showed that the expression level of PD-L1 (22C3) in tumor cells in the MYD88 L265P mutant group was significantly higher than that in the MYD88 L265P wild-type group (P=0.017). The expression level of PD-L1 (SP142) in tumor microenvironment in the MYD88 L265P mutant group was significantly lower than that in the MYD88 L265P wild-type group (P=0.001). Conclusions: MYD88 L265P mutation may play an important role in the regulation of PD-L1 expression in DLBCL tumor cells and tumor microenvironment. Further studies will provide a theoretical basis for immunotherapy of DLBCL patients with MYD88 L265P mutation.

Conjunctival melanoma (CM) iss a rare and aggressive tumour that is increasing in frequency. The prognostic value of PD-L1 expression, alone or in combination with CD8 and PD-1 expression and the BRAF and NRAS status, has not been determined in CM to date. We evaluated the expression of PD-L1, CD8, PD-1 in CM and investigated whether there was an association between the expression of these markers and the BRAF and NRAS molecular profile as well as some clinico-pathological criteria. A total of sixty-five CM were assessed for PD-L1, PD-1, and CD8 expression by immunohistochemistry (IHC) and for BRAF and NRAS genomic alterations using molecular biology techniques and anti-BRAF and anti-NRAS antibodies. PD-L1 expression in tumour cells (TC) was very low or absent but detected in tumour-infiltrating immune cells (IC). A correlation was observed between the expression of PD-L1, CD8, and PD-1 in IC. No correlation between PD-L1 expression (in tumour and/or immune cells) and BRAF or NRAS mutations was observed. PD-L1 expression in IC correlated with a higher pTNM stage and PD-L1 expression in TC with worse disease-specific survival. PD-L1 expression is a potential prognostic biomarker that correlates with poor prognosis in CM patients.

Objective: To study and correlate PDL-1 expression with Tumour budding and Tumour Infiltrating Lymphocytes in Colorectal Carcinoma. Background: Colorectal cancer (CRC) is third most common cancer with a high mortality. Many attempts have been made to raise overall survival of CRC patients. The immune system plays an important role in clearing the unhealthy cancer cells. Programmed death 1 (PD1) is a regulatory molecule which dampens the immune response when bound to one of its complementary ligands (PDL1). Its expression is related to the response of immunotherapy in CRC treatment which has been exploited in recent times. However, its prognostic value is still controversial, and the distribution of PD-L1 on tumour Cells or Immune Cells has not been comprehensively analysed. Method: A total of 30 patients diagnosed with CRCs were included who underwent surgical intervention. Cases who took preoperative neoadjuvant chemotherapy or radiotherapy were excluded. IHC analyses of PDL1 was done and was correlated with tumour budding and Tumour Infiltrating Lymphocytes (TILs) and statistical significance was assessed. Results: 11 cases showed low bud count at the invasive front out of which only 3 cases showed PDL1 positivity. The rest 19 cases had high bud count out of which 18 were PDL 1 positive. This difference was highly significant (p = 0.002). In Low Bud / High TILs, 75% cases showed no PDL1 expression in tumour cells, whereas 62.5% cases showed PDL1 positivity in TILs whereas in High Bud / Low TILs group, all the cases (100%) showed PDL1 expression in tumour cells whereas only 75% cases showed PDL1 positivity in TILs, again being statistically significant (p <0.001). Conclusion: This study showed an inverse correlation between PDL1 in tumour buds and immune cells, thus emphasising the role of tumour microenvironment. Our study reiterates the fact that high expression of PDL1 in tumour cells suppresses antitumor response whereas its high expression in TILS correlates with a better prognosis.

<div>Abstract<p><b>Purpose:</b> With recent approval of inhibitors of PD-1 in melanoma, non–small cell lung cancer (NSCLC) and renal cell carcinoma, extensive efforts are under way to develop biomarkers predictive of response. PD-L1 expression has been most widely studied, and is more predictive in NSCLC than renal cell carcinoma or melanoma. We therefore studied differences in expression patterns across tumor types.</p><p><b>Experimental Design:</b> We used tissue microarrays with tumors from NSCLC, renal cell carcinoma, or melanoma and a panel of cell lines to study differences between tumor types. Predictive studies were conducted on samples from 65 melanoma patients treated with PD-1 inhibitors alone or with CTLA-4 inhibitors, characterized for outcome. PD-L1 expression was studied by quantitative immunofluorescence using two well-validated antibodies.</p><p><b>Results:</b> PD-L1 expression was higher in NSCLC specimens than renal cell carcinoma, and lowest in melanoma (<i>P</i> = 0.001), and this finding was confirmed in a panel of cell lines. In melanoma tumors, PD-L1 was expressed either on tumor cells or immune-infiltrating cells. The association between PD-L1 expression in immune-infiltrating cells and progression-free or overall-survival in melanoma patients treated with ipilimumab and nivolumab was stronger than PD-L1 expression in tumor cells, and remained significant on multivariable analysis.</p><p><b>Conclusions:</b> PD-L1 expression in melanoma tumor cells is lower than NSCLC or renal cell carcinoma cells. The higher response rate in melanoma patients treated with PD-1 inhibitors is likely related to PD-L1 in tumor-associated inflammatory cells. Further studies are warranted to validate the predictive role of inflammatory cell PD-L1 expression in melanoma and determine its biological significance. <i>Clin Cancer Res; 23(15); 4270–9. ©2017 AACR</i>.</p></div>

Purpose: With recent approval of inhibitors of PD-1 in melanoma, non-small cell lung cancer (NSCLC) and renal cell carcinoma, extensive efforts are under way to develop biomarkers predictive of response. PD-L1 expression has been most widely studied, and is more predictive in NSCLC than renal cell carcinoma or melanoma. We therefore studied differences in expression patterns across tumor types.Experimental Design: We used tissue microarrays with tumors from NSCLC, renal cell carcinoma, or melanoma and a panel of cell lines to study differences between tumor types. Predictive studies were conducted on samples from 65 melanoma patients treated with PD-1 inhibitors alone or with CTLA-4 inhibitors, characterized for outcome. PD-L1 expression was studied by quantitative immunofluorescence using two well-validated antibodies.Results: PD-L1 expression was higher in NSCLC specimens than renal cell carcinoma, and lowest in melanoma (P = 0.001), and this finding was confirmed in a panel of cell lines. In melanoma tumors, PD-L1 was expressed either on tumor cells or immune-infiltrating cells. The association between PD-L1 expression in immune-infiltrating cells and progression-free or overall-survival in melanoma patients treated with ipilimumab and nivolumab was stronger than PD-L1 expression in tumor cells, and remained significant on multivariable analysis.Conclusions: PD-L1 expression in melanoma tumor cells is lower than NSCLC or renal cell carcinoma cells. The higher response rate in melanoma patients treated with PD-1 inhibitors is likely related to PD-L1 in tumor-associated inflammatory cells. Further studies are warranted to validate the predictive role of inflammatory cell PD-L1 expression in melanoma and determine its biological significance. Clin Cancer Res; 23(15); 4270-9. ©2017 AACR.

<div>Abstract<p><b>Purpose:</b> With recent approval of inhibitors of PD-1 in melanoma, non–small cell lung cancer (NSCLC) and renal cell carcinoma, extensive efforts are under way to develop biomarkers predictive of response. PD-L1 expression has been most widely studied, and is more predictive in NSCLC than renal cell carcinoma or melanoma. We therefore studied differences in expression patterns across tumor types.</p><p><b>Experimental Design:</b> We used tissue microarrays with tumors from NSCLC, renal cell carcinoma, or melanoma and a panel of cell lines to study differences between tumor types. Predictive studies were conducted on samples from 65 melanoma patients treated with PD-1 inhibitors alone or with CTLA-4 inhibitors, characterized for outcome. PD-L1 expression was studied by quantitative immunofluorescence using two well-validated antibodies.</p><p><b>Results:</b> PD-L1 expression was higher in NSCLC specimens than renal cell carcinoma, and lowest in melanoma (<i>P</i> = 0.001), and this finding was confirmed in a panel of cell lines. In melanoma tumors, PD-L1 was expressed either on tumor cells or immune-infiltrating cells. The association between PD-L1 expression in immune-infiltrating cells and progression-free or overall-survival in melanoma patients treated with ipilimumab and nivolumab was stronger than PD-L1 expression in tumor cells, and remained significant on multivariable analysis.</p><p><b>Conclusions:</b> PD-L1 expression in melanoma tumor cells is lower than NSCLC or renal cell carcinoma cells. The higher response rate in melanoma patients treated with PD-1 inhibitors is likely related to PD-L1 in tumor-associated inflammatory cells. Further studies are warranted to validate the predictive role of inflammatory cell PD-L1 expression in melanoma and determine its biological significance. <i>Clin Cancer Res; 23(15); 4270–9. ©2017 AACR</i>.</p></div>