Programmed Death-ligand 1 Immunohistochemistry Research Articles

PD-L1 promoter methylation is a prognostic biomarker for biochemical recurrence-free survival in prostate cancer patients following radical prostatectomy.

BackgroundThe rapid development of programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) inhibitors has generated an urgent need for biomarkers assisting the selection of patients eligible for therapy. The use of PD-L1 immunohistochemistry, which has been suggested as a predictive biomarker, however, is confounded by multiple unresolved issues. The aim of this study therefore was to quantify PD-L1 DNA methylation (mPD-L1) in prostate tissue samples and to evaluate its potential as a biomarker in prostate cancer (PCa).ResultsIn the training cohort, normal tissue showed significantly lower levels of mPD-L1 compared to tumor tissue. High mPD-L1 in PCa was associated with biochemical recurrence (BCR) in univariate Cox proportional hazards (hazard ratio (HR)=2.60 [95%CI: 1.50-4.51], p=0.001) and Kaplan-Meier analyses (p<0.001). These results were corroborated in an independent validation cohort in univariate Cox (HR=1.24 [95%CI: 1.08-1.43], p=0.002) and Kaplan-Meier analyses (p=0.029). Although mPD-L1 and PD-L1 protein expression did not correlate in the validation cohort, both parameters added significant prognostic information in bivariate Cox analysis (HR=1.22 [95%CI: 1.05-1.42], p=0.008 for mPD-L1 and HR=2.58 [95%CI: 1.43-4.63], p=0.002 for PD-L1 protein expression).MethodsmPD-L1 was analyzed in a training cohort from The Cancer Genome Atlas (n=498) and was subsequently measured in an independent validation cohort (n=299) by quantitative methylation-specific real-time PCR. All patients had undergone radical prostatectomy.ConclusionsmPD-L1 is a promising biomarker for the risk stratification of PCa patients and might offer additional relevant prognostic information to the implemented clinical parameters, particularly in the setting of immune checkpoint inhibition.

Patterns and prognostic relevance of PD-1 and PD-L1 expression in colorectal carcinoma

AbstractImmune checkpoint blockade targeting the programmed death-1 (PD-1) pathway has shown efficacy in several types of cancers including mismatch-repair-deficient colorectal carcinoma. In some tumor types, programmed death-ligand 1 (PD-L1) expression detected by immunohistochemistry has shown utility as a predictive marker for response to anti-PD-1 therapies. This utility, however, remains to be determined in colorectal carcinoma. In addition, although tumor-infiltrating lymphocytes have been associated with better prognosis in colorectal carcinoma, the prognostic value of PD-1 expression in these lymphocytes and its interaction with PD-L1 expression still await investigation. To address these questions, we performed a pilot study to evaluate the patterns of PD-L1 and PD-1 immunohistochemical expression on colorectal carcinoma cells and their tumor-infiltrating lymphocytes, respectively. Using tissue microarray, we found that 5% (19/394) of colorectal carcinomas exhibited high tumor PD-L1 expression, and 19% (76/392) had elevated numbers of PD-1-positive tumor-infiltrating lymphocytes. PD-L1 levels correlated with PD-1 levels (P<0.001), and mismatch-repair-deficient tumors had significantly higher rates of high PD-L1 and PD-1 expression when compared with mismatch-repair-proficient tumors (18% vs 2% and 50% vs 13%, respectively; P<0.001 for both). Staining intensity was also stronger for both markers in mismatch-repair-deficient tumors. Furthermore, we observed that among patients with mismatch-repair-deficient colorectal carcinoma, PD-1/PD-L1 expression stratified recurrence-free survival in an inter-dependent manner: an association between high PD-1-positive tumor-infiltrating lymphocytes and improved recurrence-free survival (P=0.041) was maintained only when the tumors had low-level PD-L1 expression (P=0.006); patients whose tumors had both high PD-1-positive tumor-infiltrating lymphocytes and high PD-L1 expression had a significantly worse recurrence-free survival (P<0.001). Thus, our results not only provide a foundation for further assessment of PD-L1 immunohistochemistry as a predictive marker for anti-PD-1 therapy in colorectal carcinoma, they also shed light on the prognostic impact of tumor-infiltrating lymphocytes in different subsets of mismatch-repair-deficient colorectal carcinomas.

PD-L1 testing for lung cancer in the UK: recognizing the challenges for implementation.

A new approach to the management of non-small-cell lung cancer (NSCLC) has recently emerged that works by manipulating the immune checkpoint controlled by programmed death receptor 1 (PD-1) and its ligand programmed death ligand 1 (PD-L1). Several drugs targeting PD-1 (pembrolizumab and nivolumab) or PD-L1 (atezolizumab, durvalumab, and avelumab) have been approved or are in the late stages of development. Inevitably, the introduction of these drugs will put pressure on healthcare systems, and there is a need to stratify patients to identify those who are most likely to benefit from such treatment. There is evidence that responsiveness to PD-1 inhibitors may be predicted by expression of PD-L1 on neoplastic cells. Hence, there is considerable interest in using PD-L1 immunohistochemical staining to guide the use of PD-1-targeted treatments in patients with NSCLC. This article reviews the current knowledge about PD-L1 testing, and identifies current research requirements. Key factors to consider include the source and timing of sample collection, pre-analytical steps (sample tracking, fixation, tissue processing, sectioning, and tissue prioritization), analytical decisions (choice of biomarker assay/kit and automated staining platform, with verification of standardized assays or validation of laboratory-devised techniques, internal and external quality assurance, and audit), and reporting and interpretation of the results. This review addresses the need for integration of PD-L1 immunohistochemistry with other tests as part of locally agreed pathways and protocols. There remain areas of uncertainty, and guidance should be updated regularly as new information becomes available.

Correlation of PD-L1 Expression of Tumor Cells with Survival Outcomes after Radical Intensity-Modulated Radiation Therapy for Non-Metastatic Nasopharyngeal Carcinoma

PurposeWe investigated if programmed death-ligand 1 (PD-L1) expression levels were prognostic of survival outcomes after intensity-modulated radiation therapy (IMRT) for non-metastatic nasopharyngeal carcinoma (NPC).Methods and Materials104 patients with non-metastatic NPC treated with radical IMRT were investigated for their PD-L1 expression by immunohistochemistry (IHC) which were correlated with survival endpoints including locoregional failure-free survival (LRFFS), progression-free survival (PFS), distant metastasis-free survival (DMFS) and overall survival (OS).ResultsAfter a median follow-up of 7.6 years, 21 (20.2%), 19 (18.3%) and 31 (29.8%) patients suffered from locoregional failure, distant metastases and overall disease progression, respectively, and 31 (29.8%) patients died. Patients whose tumors had PD-L1 IHC 2+ (moderate to strong membrane staining in ≥ 25% of tumor cells) enjoyed longer LRFFS (5-year 100% vs. 74.4%, Hazard ratio [HR], 0.159, 95% confidence interval [CI], 0.021–0.988; P = 0.042) and marginally longer PFS (5-year 95.0% vs. 65.2%, HR, 0.351, 95% CI, 0.08–0.999, P = 0.067) compared to those whose tumors had PD-L1 IHC 0 (minimal membrane staining with PD-L1 in < 5% tumor cells or no staining with PD-L1) or 1+ (minimal to moderate membrane staining with PD-L1 in between 5–24% tumor cells). PD-L1 IHC 2+ was independently prognostic of both LRFFS (P = 0.014) and PFS (P = 0.045) in multivariable analyses. Only induction chemotherapy followed by concurrent chemoradiation was prognostic of DMFS (P = 0.003) and no prognostic factor for OS was identified.ConclusionPD-L1 expression levels correlated with LRRFS and PFS in non-metastatic NPC treated with radical IMRT. It may play a role in radiosensitivity for NPC, which should be further confirmed in prospective studies using immunotherapy together with IMRT.

Non-Small Cell Lung Cancer, PD-L1, and the Pathologist.

Although most primary cancers of the lung carry a heavy mutational load and will potentially present many "nonself" antigens to the immune system, there are a wide range of possible mechanisms for tumors to avoid so-called immune surveillance. One such mechanism is the adoption of immune checkpoints to inhibit the host immune response. Immune checkpoint inhibitors show great promise in the treatment of advanced non-small cell lung cancer. To discuss the possibility of biomarker selection of patients for these therapies. This is becoming a much debated issue, and the immunohistochemical detection of Programmed Death Ligand 1 (PD-L1), the ligand for the inhibitory Programmed Death receptor 1 (PD-1) checkpoint, is one possible biomarker. Data so far available show some conflicting results, but PD-L1 immunohistochemistry looks likely to be introduced into clinical use for selecting patients for treatment with anti-PD-1 or anti-PD-L1 therapies. Given that there are 4 such drugs rapidly approaching regulatory approval, each with its own independent PD-L1 immunohistochemistry biomarker test, both oncologists and pathologists face some significant challenges. Peer-reviewed literature and meeting proceedings, especially during the last 12 months, were used. The biology of PD-1/PD-L1 is complex, the clinical data for these drugs show considerable variation, the selection performance of the PD-L1 biomarker test is not perfect, and the existence of 4 drug/test combinations adds significantly to the problems faced. This article addresses some of the background to this therapeutic problem and discusses some of the issues ahead.

Programmed Death Ligand-1 Immunohistochemistry--A New Challenge for Pathologists: A Perspective From Members of the Pulmonary Pathology Society.

The binding of programmed death ligand-1 and ligand-2 (PD-L1 and PD-L2) to PD-1 blocks T-cell-mediated immune response to tumor. Antibodies that target programmed death receptor-1 (PD-1) will block the ligand-receptor interface, thereby allowing T cells to attack the tumor and increase antitumor immune response. In clinical trials, PD-1 inhibitors have been associated with an approximately 20% overall response rate in unselected patients with non-small cell lung cancer, with sustained tumor response in a subset of patients treated by these immune checkpoint inhibitors. Facing a proliferation of PD-L1 immunohistochemistry clones, staining platforms, and scoring criteria, the pathologist must decide on the feasibility of introducing a newly approved companion diagnostic assay that may require purchase not only of a specific antibody kit but of a particular staining platform. Given the likely reality that clinical practice may, in the near future, demand access to 4 different PD-L1 antibodies coupled with different immunohistochemistry platforms, laboratories will be challenged with deciding among this variety of testing methods, each with its own potential benefits. Another immediate challenge to PD-L1 testing in lung cancer patients is that of access to adequate tumor tissue, given that non-small cell lung cancer samples are often extremely limited in size. With PD-L1 testing it has become clear that the historically used US regulatory approach of one assay-one drug will not be sustainable. One evolving concept is that of complementary diagnostics, a novel regulatory pathway initiated by the US Food and Drug Administration, which is distinct from companion diagnostics in that it may present additional flexibility. Although pathologists need to face the practical reality that oncologists will be asking regularly for the PD-L1 immunohistochemistry status of their patients' tumors, we should also keep in mind that there may be room for improvement of biomarkers for immunotherapy response. The field is rich with opportunities for investigation into biomarkers of immunotherapy response, particularly in the form of collaborative, multidisciplinary studies that incorporate oncologists, pathologists, and basic scientists. Pathologists must take the lead in the rational incorporation of these biomarkers into clinical practice.

Programmed Death Ligand-1 Immunohistochemistry: Friend or Foe?

The approval of anti-programmed death receptor (PD)-1 therapies for non-small cell lung cancer has directed the spotlight on programmed death ligand-1 (PD-L1) immunohistochemistry as the latest predictive biomarker potentially required in this disease. Several other drugs in this class will likely be approved in the future and each has been developed with a unique anti-PD-L1 immunohistochemistry test. The prospect of 5 drugs competing in the same treatment area, each possibly requiring PD-L1 immunohistochemistry testing, presents a challenge for pathologists unlike any previously faced. The key issue is whether laboratories will attempt to deliver the trial-validated assays for one or more of these treatments, or introduce instead one or more laboratory developed tests, or attempt to provide a single PD-L1 immunohistochemistry assay for all possible anti-PD-1 and anti-PD-L1 treatments that may be used. This paper discusses some of the issues, challenges, hazards, and possible solutions that have recently emerged in this most complex interface between cancer therapeutics and laboratory biomarker testing.

Paucity of PD-L1 expression in prostate cancer: innate and adaptive immune resistance

BackgroundPrimary prostate cancers are infiltrated with PD-1 expressing CD8+ T cells. However, in early clinical trials, men with mCRPC did not respond to PD-1 blockade as a monotherapy. One explanation for this unresponsiveness could be that prostate tumors generally do not express PD-L1, the primary ligand for PD-1. However, lack of PD-L1 expression in prostate cancer would be surprising, given that PTEN loss is relatively common in prostate cancer and several studies have shown that PTEN loss correlates with PD-L1 up-regulation - constituting a mechanism of innate immune resistance. This study tested whether prostate cancer cells were capable of expressing PD-L1, and whether the rare PD-L1 expression that occurs in human specimens correlates with PTEN loss.MethodsHuman prostate cancer cell lines were evaluated for PD-L1 expression and loss of PTEN by flow cytometry and western blotting, respectively. Immunohistochemical (IHC) staining for PTEN was correlated with PD-L1 IHC using a series of resected human prostate cancer samples.ResultsIn vitro, many prostate cancer cell lines up-regulated PD-L1 expression in response to inflammatory cytokines, consistent with adaptive immune resistance. In these cell lines, no association between PTEN loss and PD-L1 expression was apparent. In primary prostate tumors, PD-L1 expression was rare, and was not associated with PTEN loss.ConclusionsThese studies show that some prostate cancer cell lines are capable of expressing PD-L1. However, in human prostate cancer, PTEN loss is not associated with PD-L1 expression, arguing against innate immune resistance as a mechanism that mitigates anti-tumor immune responses in this disease.

Abstract 4303: Programmed death ligand 1 (PD-L1) expression in paired melanoma tumor samples

Abstract Background: There is limited information on changes in PD-L1 expression levels over time in melanoma. Methods: PD-L1 expression was measured in melanoma tumor samples collected at different time points from the same patient using a prototype immunohistochemistry (IHC) assay with the 22C3 antibody. PD-L1 positivity was defined as staining in ≥1% of cells. The concordance of PD-L1 expression in paired samples was analyzed by treating the IHC proportion score as a continuous variable or classifying staining as positive or negative. Results: 48 patients were included. Median age was 65 years (range, 23-85), 73% were male, and 60% had stage I-IIIA disease. All first samples and 85% of second samples were surgical samples. The median interval between sample collection dates was 25.3 months (range, 3.1-203.5). There was no statistically significant correlation of the PD-L1 IHC proportional score between paired samples (Pearson correlation coefficient, 0.13). 40% of patients had an increased proportion score at the second time point, and 10% had a decreased score. The prevalence of PD-L1 positivity was 31% in first samples and 46% in second samples. The concordance rate was 65% (95% CI, 49%-78%) when PD-L1 expression was categorized as positive or negative (Table). The concordance rate was similar in paired samples collected within and beyond 2 years. Concordance of PD-L1 Expression in Paired Melanoma Samples (N = 48)Later Time PointPD-L1 ExpressionPositive, n (%)Negative, n (%)Earlier Time PointPositive10 (20.8)5 (10.4)Negative12 (25.0)21 (43.7) Conclusion: Our preliminary analysis did not suggest a significant correlation of PD-L1 expression in paired melanoma samples collected at different time points. Similar results were observed when samples were categorized as PD-L1 positive or PD-L1 negative. The 35% discordance rate between initial and subsequent tumor samples should be considered if PD-L1 expression is used for selection of enrollment in clinical trials. Citation Format: Torben Steiniche, Allan Vestergaard Danielsen, Zhen (Adelle) Wang, Patricia Switten Nielsen, Lars Bastholt, Henrik Schmidt, Inge Marie Svane, Marisa Dolled-Filhart, Kenneth Emancipator, Dianna Y. Wu, Michael Busch-Sorensen, Wei Zhou. Programmed death ligand 1 (PD-L1) expression in paired melanoma tumor samples. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4303. doi:10.1158/1538-7445.AM2015-4303

Abstract 570: PD-L1 expression in paired non-small cell lung cancer tumor samples

Abstract Background There is limited information on changes over time in programmed death ligand 1 (PD-L1) expression in non-small cell lung cancer (NSCLC). Methods PD-L1 expression was assessed in paired tumor samples collected at different time points from the same patient using a prototype immunohistochemistry (IHC) assay with the 22C3 antibody. PD-L1 strong and weak positivity were defined to be traceable to the 1% and 50% cutoffs used in the clinical trial version of the assay. Concordance of PD-L1 expression between samples was analyzed by treating the IHC proportional score as a continuous and categorical variable. Results 90 patients were included (73 from Korea, 17 from Denmark). 75% were male, 37% had squamous cell carcinoma, and 83% had stage I-IIIA disease. 97% of first samples and 53% of second samples were from lung. 89% and 63%, respectively, were surgical samples, with a median interval of 20.9 months (range, 0.1-94.0) between collection dates; 91% of paired samples were collected &amp;gt;3 months apart. A significant correlation of the PD-L1 IHC proportional score was observed for the paired samples (Pearson correlation coefficient, 0.62; P &amp;lt; 0.001). At the second time point, scores were identical in 39% of paired samples, higher in 32%, and lower in 29%. Of note, 12% of PD-L1-negative cases became positive and 20% of PD-L1-positive cases become negative at the second time point. The concordance rate was 56% (95% CI, 46%-67%) when PD-L1 expression was categorized as strong or weak positive or negative (Table). Conclusion Although a significant correlation of the PD-L1 IHC proportional score was observed, this preliminary analysis suggested a discordance rate of 44% between initial and subsequent NSCLC tumor samples when patients were categorized by PD-L1 expression level. Similar results were observed when analyzing paired melanoma samples. This discordance should be considered if PD-L1 expression is used for selection of enrollment in clinical trials. Table. Concordance of PD-L1 Expression in Paired NSCLC Samples (N = 90) Later Time PointPD-L1 ExpressionStrong positive, n (%)Weak positive, n (%)Negative, n (%)Earlier Time PointStrong positive3 (3)3 (3)1 (1)Weak positive7 (8)20 (22)17 (19)Negative011 (12)28 (31) Citation Format: Jhingook Kim, Steffen Filskov Sorensen, Yoon-La Choi, Zhen (Adelle) Wang, Jong-Mu Sun, Hyejoo Choi, Jeanette Baehr Georgsen, Marisa Dolled-Filhart, Kenneth Emancipator, Dianna Y. Wu, Peter Meldgaard, Wei Zhou, Henrik Hager. PD-L1 expression in paired non-small cell lung cancer tumor samples. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 570. doi:10.1158/1538-7445.AM2015-570

Abstract PD1-6: Inhibition of PD-L1 by MPDL3280A leads to clinical activity in patients with metastatic triple-negative breast cancer

Abstract Background: Metastatic triple-negative breast cancer (TNBC) is associated with a poor prognosis and has no targeted therapy options. Programmed death-ligand 1 (PD-L1) expression is more prevalent in TNBC than in other breast cancer subtypes. PD-L1 may protect cancer cells from immune-mediated destruction by binding to its receptors PD-1 and B7.1. MPDL3280A, a human anti-PD-L1 monoclonal antibody with an engineered Fc-domain designed for optimized efficacy and safety, blocks PD-L1 activity and restores tumor-specific T-cell immunity. Methods: This multicenter Phase I study selectively enrolled a cohort of patients with PD-L1-positive metastatic TNBC. PD-L1 positivity was centrally evaluated based on the PD-L1 immunohistochemistry (IHC) status of tumor-infiltrating immune cells (ICs). Eligible patients received MPDL3280A 15 or 20 mg/kg IV every 3 weeks for up to 1 year. The objective response rate (ORR) was assessed by RECIST v1.1. MPDL3280A immune correlates were evaluated for tumor and circulating biomarkers. The clinical data cutoff was January 1, 2014. Results: Twelve patients were treated with MPDL3280A and evaluable for safety. Patients had ECOG PS 0-1 (33% ECOG PS 1), and the median age was 55 years (range, 29-72 years). All 4 patients (33%) with visceral metastases had liver metastases, and 1 patient (8%) had bone metastases. 92% of patients received ≥ 2 prior therapies. Prior chemotherapies included anthracycline (92%), taxane (75%) and platinum (42%). Grade 3-4 treatment-related adverse events (AEs) occurred in 8% of patients (1 event of Grade 3 adrenal insufficiency). One patient had an immune-related AE (Grade 2 pyrexia). No treatment-related deaths were observed. Nine patients were dosed by November 16, 2013 and evaluable for efficacy. The ORR was 33%, including 1 CR and 2 PRs. Responders included patients with visceral metastases at baseline. At the time of the clinical data cut-off, all responses occurred within 6 weeks of the first dosing of MPDL3280A, and all of the responses were ongoing. The median duration of response had not been reached. One patient achieved stable disease as best response. Two additional patients had tumor shrinkage (-43% and -44% change in target lesions, respectively) but were not counted as RECIST responders due to the appearance of new lesions, which is likely attributable to pseudoprogression. Preliminary pharmacodynamic biomarkers related to MPDL3280A activity, including circulating plasma markers and tumor immunomonitoring with CD8 IHC, will be presented along with updated clinical data. Conclusions: MPDL3280A treatment was well tolerated and was associated with objective clinical activity in patients with pretreated metastatic TNBC. Further evaluation of the safety and clinical activity of MPDL3280A in both PD-L1-positive and PD-L1-negative metastatic TNBC is ongoing. Clinical trial information: NCT01375842. Citation Format: Leisha A Emens, Fadi S Braiteh, Philippe Cassier, Jean-Pierre DeLord, Joseph Paul Eder, Xiaodong Shen, Yuanyuan Xiao, Yan Wang, Priti S Hegde, Daniel S Chen, Ian Krop. Inhibition of PD-L1 by MPDL3280A leads to clinical activity in patients with metastatic triple-negative breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD1-6.

Prevalence of tumor-infiltrating lymphocytes and PD-L1 expression in the soft tissue sarcoma microenvironment

The prognostic and predictive implications of programmed death-ligand 1 (PD-L1) is unknown in sarcoma. We sought to examine the immune milieu in sarcoma specimens. We evaluated PD-L1 expression by immunohistochemistry in sarcoma specimens and quantified tumor-infiltrating lymphocytes (TIL). We correlated expression with clinical parameters and outcomes. Fifty sarcoma patients treated at Memorial Sloan Kettering Cancer Center were selected. Using the DAKO PD-L1 immunohistochemistry assay and archival formalin-fixed paraffin-embedded tissue specimens; PD-L1 expression was examined. Macrophage and lymphocyte PD-L1 status was determined qualitatively. TIL was quantified. Associations between PD-L1 expression in tumor, macrophages and lymphocytes, TIL and clinical-pathological characteristics were performed. The median age was 46 years (range, 22-76), and 66% of patients were men. Tumor, lymphocyte and macrophage PD-L1 expression was noted in 12%, 30% and 58%, respectively, with the highest prevalence in gastrointestinal stromal tumors (29%). Lymphocyte and macrophage infiltration was present in 98% and 90%, respectively. There was no association between clinical features, overall survival and PD-L1 expression in tumor or immune infiltrates. Lymphocyte and macrophage infiltration is common in sarcoma, but PD-L1 tumor expression is uncommon in sarcoma with the highest frequency observed in gastrointestinal stromal tumors. There was no association between PD-L1 expression, TIL and clinicopathological features and overall survival; however, this is limited by the heterogenous patient sample and minimal death events in the studied cohort.