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Programmed Death Ligand-1 (PD-L1) Expression in the Programmed Death Receptor-1 (PD-1)/PD-L1 Blockade: A Key Player Against Various Cancers.

During the past several decades, the efforts of antitumor immune therapy have been focused on identification of specific tumor antigens to augment antitumor immunity. However, this approach has been constantly challenged by the complexity and diversity of gene mutation and protein modification presented in human cancers, resulting in limited success. (1)

Recently, it has become well known that immune checkpoint pathways, including the programmed death receptor-1/programmed death ligand-1 (PD-1/PD-L1) signaling pathway, (2) which are important in mediating self-tolerance and controlling self-damage, can sometimes be manipulated by cancer cells to evade immune surveillance (as illustrated in Figure 1). (3,4) The fact that PD-1 is highly upregulated in tumor-infiltrating lymphocytes, as well as the correlation between PD-L1 expression and clinical outcome of patients in many types of solid cancers, has resulted in some seminal clinical trials. These trials further demonstrate the efficacy of PD-1/PD-L1-targeted therapy and reveal the new era of cancer immunotherapy. (5)

In this review, we will discuss the mechanism of the PD1/PD-L1 signaling pathway, the regulation of this pathway, PD-1/PD-L1 as a predictive and/or prognostic marker in various cancers, and strategies for measuring PD-L1 expression.

MECHANISM

The PD-1/PD-L1 pathway belongs to the immune checkpoint signaling pathways regulating T-cell-mediated local inflammatory reactions and self-tolerance. (6) The importance of the PD-1/PD-L1 axis in cancer immunity has been demonstrated extensively in both animal models and clinical studies. (7)

PD-1, a cell surface protein belonging to the CD28 family, is encoded by PDCD1 gene located in chromosome 2q37.2 PD-1 is expressed on activated T cells, with particularly high expression by tumor-infiltrating T lymphocytes. (2) PD-1 is also expressed on activated non-T cells, including B cells, natural killer cells, and monocytes, implying that PD-1 may also modulate immunity in a T-cell-independent manner. (8) PD-1 has 2 major ligands: PD-L1/CD274 (encoded by PDCD1LG1 in chromosome 9) and PD-L2/CD273 (encoded by PDCD1LG2 in chromosome 9).

Binding of PD-1 to either PD-L1 or PD-L2 results in the activation of inhibitory kinases involved in T-cell proliferation, adhesion, and cytokine production/secretion via phosphatase SHP2.2 PD-1-PD-L interaction has been shown to play an important role in limiting the initial response of T cells upon antigen exposure and inducing T-cell tolerance. However, there are differences between PDL1 and PD-L2.2 PD-L1 is broadly expressed on many immune cell types, such as T cells, (9) B cells, macrophages, regulatory T (Treg) cells, and dendritic cells, as well as some non-immune cell types, such as vascular endothelial cells and pancreatic cells. On the contrary, PD-L2 is largely limited to antigen-presenting cells, such as macrophages and dendritic cells.

Overexpression of PD-L1 in murine tumor models results in inhibition of T-cell-mediated immune response via the PD-L1/PD-1 axis, indicating that blocking this axis has an important role in immunotherapy against cancers. PD-L1 is expressed in several solid tumors, including melanoma, glioblastoma, lung cancer, renal cancer, gastric cancer, colorectal cancer, pancreatic cancer, breast cancer, and cervical and ovarian cancers (please see Table 1 for percentage of PD-L1 expression in various tumors). PDL1 is also found in hematologic malignancies, such as multiple myeloma, lymphoma, and various leukemia types. In contrast, PD-L2 is preferentially expressed in hematopoietic tumors, like B-cell lymphomas. (10) Additionally, strong association between PD-1/PD-L1 expression and poor prognosis has been reported in several tumor types, including gastric, lung, and renal carcinomas (details in Figure 1).

REGULATION OF PD-L1 EXPRESSION IN CANCERS

Many pathways have been suggested to be involved in PD-L1 upregulation in cancers (as illustrated in Figure 2). (6)

For instance, activation of oncogenic signaling, such as through the phosphatase and tensin homolog-phosphoinositide 3-kinase-protein kinase B (PTEN-PI3K-AKT) pathway, upregulates PD-L1 on glioblastomas. (11) Similarly, activation of the anaplastic lymphoma kinase (ALK) signal transducer and activator of transcription 3 (STAT3) pathway is also able to induce PD-L1 expression in ALK-carrying T-cell lymphoma and chemoresistant non-small cell lung cancer (NSCLC). (12,13) In melanoma, the upregulation of PDL1 is partially mediated by recruitment of histone deacetylase 6 (HDAC6) onto STAT3 to the PD-L1 promoter region. (14) Furthermore, interferons, especially interferon y, are the most common inducer in adaptive immunemediated PD-L1 upregulation on tumor cells or on tumorin-filtrated leukocytes, including melanoma (15) and ovarian cancer. (2,16) Other inflammatory signaling pathways, including interleukin 4, interleukin 10, vascular endothelial growth factor, granulocyte colony-stimulating factor, and bacterial lipopolysaccharide, have also been shown to induce the expression of PD-L1. (17) This may have contributed to the association of chronic inflammation and cancer development.

CURRENT THERAPEUTIC ANTIBODIES, APPROACHES, AND TOXICITIES

Encouraged by the success of preclinical studies in which PD-1 blockade via monoclonal antibodies attenuated the remote metastasis of melanoma cells or colon cancer cells, several agents to block the PD-1/PD-L1 axis have been developed and applied in different types of tumors. The journey started from a small pilot phase 1 study in which nivolumab (a humanized anti-PD-1 antibody; Table 2), manufactured by Bristol-Myers Squibb (New York, New York), was shown to be safe and to have antitumor activity in treatment-refractory patients with solid tumors (melanomas, renal cell carcinomas [RCCs], and NSCLCs) in 2010. A large trial with 296 patients then revealed that nivolumab induced an objective response in 18% to 28% of patients with refractory solid tumors (26 of 94 in melanoma, 14 of 76 in NSCLC, and 9 of 33 in RCC), and the duration of the therapeutic response was greater than 1 year for most of these patients. (5) Nivolumab was shown to be effective in refractory classical Hodgkin lymphoma, with an objective response rate of 87% (20 of 23) reported. (18) Nivolumab soon became the first Food and Drug Administration (FDA)-approved PD-1 inhibitor for metastatic melanoma (2014), NSCLC (2014), and RCC (2015).

Another PD-1 antibody, pembrolizumab (lambrolizumab), manufactured by Merck (Kenilworth, New Jersey), was demonstrated to have antitumor effects in patients with advanced melanoma in an initial phase 1 study. (19) In the KEYNOTE-006 study, the response rate was 33.7% (94 of 279) in patients with advanced melanoma receiving pembrolizumab every 2 weeks. (20) A 19.4% (96 of 495) objective response rate and 1 year of median duration of overall response were also reported in pembrolizumab-treated patients with metastatic NSCLC. (21) Pembrolizumab became the second FDA-approved PD-1 inhibitor for metastatic melanoma and NSCLC. A recent report expanded the application of pembrolizumab further in advanced Merkel cell carcinoma. The objective response rate was as high as 56% (14 of 25) in patients with advanced Merkel cell carcinoma, including patients testing positive and those testing negative for Merkel cell polyomavirus. (22) Another promising PD-1 inhibitor, pidilizumab, manufactured by CureTech (Yavne, Israel), was tested in patients with solid tumors and hematologic tumors, particularly lymphomas. (23) More recent data from a study that evaluated the use of a combination of pidilizumab with rituximab in patients with relapsed follicular lymphoma showed a complete response rate of 52% (15 of 29) and a partial response rate of 14% (4 of 29). (24)

PD-L1-targeted therapy is also currently under investigation for use in both solid and hematologic tumors. (25,26) Atezolizumab, manufactured by Roche/Genentech (Basel, Switzerland), was listed as the Breakthrough Drug of 2014 by the FDA, given its significance in treating metastatic bladder cancer. In a recent phase 1a study in patients with RCC, atezolizumab treatment resulted in a 28.9-month median overall survival, even in some patients previously exposed to anti-vascular endothelial growth factor therapy. (27) Successful results have also been reported using atezolizumab in the treatment of bladder cancer. (28) MEDI4736 is another exciting PD-L1 antibody that is currently being tested in phase 3 trials for stage 3 NSCLC. Another exciting, newly emerged antibody is durvalumab, manufactured by AstraZeneca (London, United Kingdom), which is a more PD-L1-specific inhibitor minimizing the toxicity associated with PD-L2 inhibition. (29) There was a phase 1b study conducted in which a dual-antibody therapy including durvalumab and tremelimumab, manufactured by AstraZeneca, was tested in patients with advanced-stage NSCLC; the therapy demonstrated an objective response of more than 20% (6 of 26) and disease control in more than 30% (9 of 26), in both PD-L1-positive and PD-L1-negative groups. (30) Table 2 summarizes antiPD-1 and anti-PD-L1 agents in clinical use or in the early phases of development.

Furthermore, the synergistic benefits of the combination of PD-1/PD-L1 inhibitors with other agents--such as vaccine, other checkpoint inhibitors, chemotherapy, and targeted inhibitors (epidermal growth factor receptor [EGFR], ALK, BRAF, and vascular endothelial growth factor inhibitors)--have been confirmed by several pre-clinical tests. Recently, these novel drug combinations have been tested in several tumors in various phases of clinical trials.

The most common nonspecific systemic side effects of PD-1/PD-L1-targeted therapy are fatigue and fever. Imbalance of immune tolerance/immunity induced by PD-1/PDL1-targeted therapy sometimes leads to side effects mimicking autoimmune diseases. Toxicity associated with PD-1/PD-L1-targeted therapy occurs with less frequency than the toxicity associated with anti-cytotoxic T-lymphocyte-associated protein 4 (anti-CTL4) therapy, and grades 3 to 4 adverse events occur in only 7% to 12% of patients. The most affected organs/tissues in these immune-related adverse events are the skin, gastrointestinal system, liver, and lungs. Maculopapular rash is the most common skin pathology. Diarrhea/colitis and asymptomatic elevation of alanine aminotransferase/aspartate aminotransferase are the most common digestive system side effects. The incidence of pneumonitis is more common in patients with lung cancer or those treated with combinations of PD-1 inhibitors and conventional chemotherapeutic agents. Patients with pneumonitis may present with shortness of breath, cough, fever, and/or chest pain, as well as the associated radiologic findings mimicking acute interstitial pneumonia or acute respiratory distress syndrome. The onset of pneumonitis is usually seen in the range of 7 to 24 months following the exposure to PD-1/PD-L1 inhibitors. In general, patients with mild symptoms are normally treated by discontinuing the PD-1/PD-L1 inhibitors. Steroids and other immunosuppressants are sometimes necessary for patients with severe symptoms.

PD-1/PD-L1 EXPRESSION AS A PROGNOSTIC MARKER IN VARIOUS CANCERS

The correlation between the PD-L1 expression of cancer cells and prognosis remains to be determined. As shown in Table 1, most of the studies have indicated that expression of PD-L1 in tumor cells served as an adverse prognostic marker. However, a few studies revealed that expression of PD-L1 in tumor cells may imply better prognosis. Additionally, several studies beyond those noted in Table 1 showed that PD-L1 expression in tumor cells and tumor-infiltrating lymphocytes is correlated with poor prognosis in such cancers as NSCLC, (31) melanoma, (32) RCC, (33) and esophageal and gastric cancers. (34) A meta-analysis conducted by Wang et al (31) in 1157 patients with NSCLC showed that PD-L1 expression was significantly associated with poor differentiation of tumors (poor versus well: odds ratio, 1.91; 95% CI, 1.33-2.75; P = .001) and with worse overall survival (pooled hazard ratio, 1.75; 95% CI, 1.40-2.20; P < .001). (31) In another meta-analysis of RCC, high level of PD-L1 expression was a negative prognostic factor that increased the risk of death by 81% (hazard ratio, 1.81; 95% CI, 1.31-2.49; P < .001). (33) In gastric carcinoma, PD-L1 expression by immunohistochemistry was associated with larger tumor size, invasion into the deep muscular layers, lymph node metastasis, and decreased survival time of patients (<2 years). (34) Moreover, multivariate analysis demonstrated that PD-L1 immunodetection could be used as an independent factor to evaluate the prognosis of gastric carcinoma. (34) Additionally, PD-L1 expression has been associated with an aggressive melanoma phenotype characterized by a fibroblast-like morphology and invasive proper ties leading to enhanced aggressiveness and invasiveness. (32) Also, in a meta-analysis of breast cancer patients, PD-L1 expression was associated with aggressive features, such as lymph node metastasis, poor nuclear grade, and negative estrogen receptor status. It is associated with a higher total risk of mortality risk ratio of 1.64 (95% CI, 1.14-2.34) and a higher 10-year risk of mortality risk ratio of 2.53 (95% CI, 1.78-3.59) after surgery. (35) In contrast, there are some studies that showed that PD-L1 upregulation served as a positive prognostic marker in breast cancer and high-grade serous ovarian carcinoma. This is likely due to an increased T-cell cytotoxic immune response in these cancers. (36-38)

The mechanisms leading to these discrepancies are uncertain. However, the current use of nonstandardized immunohistochemistry (IHC) methodologies using different monoclonal antibodies (Tables 1 and 3) for measuring PD Figure 2. Two pathways used by cancer cells to upregulate programmed death receptor-1 (PD-1) ligand and thus avoid immunity. The first one normally involves an innate immune response. The upregulation of programmed death ligand-1 (PD-L1) can be caused by active oncogenic signaling, such as via protein kinase B (AKT), which is independent of inflammatory response. The alternative one is mainly seen in adaptive immune responses. The regulation of PD-L1 is induced by inflammatory response, such as via interferon (IFN). Abbreviations: MHC, major histocompatibility complex; PI3K, phosphoinositide 3-kinase; STAT, signal transducer and activator of transcription.

L1 levels in these studies may have partially contributed to these conflicting results.

PD-1/PD-L1 AS A PREDICTIVE MARKER FOR ANTI-PD-1/ PD-L1 TREATMENT

A predictive marker for predicting treatment response to anti-PD-1/PD-L1 treatment is highly desired because the treatment is associated with certain toxicity as described above, although less common and less severe than conventional chemotherapy. PD-L1 expression has been investigated as a potential predictive biomarker for selecting responders to anti-PD-1/PD-L1 antibody treatment. A study conducted by Topalian et al5 showed an objective response to nivolumab in 36% (9 of 25) of the cancer (advanced melanoma, NSCLC, castration-resistant prostate cancer, RCC, and colorectal cancer) patients who tested positive for PD-L1 (monoclonal antibody 5H1) expression, whereas none of the patients who tested negative for PD-L1 demonstrated a response. Similarly, another recent study showed that about 22% (191 of 824) of the NSCLC tumors (both squamous and nonsquamous) tested expressed PD-L1 in at least 50% of tumor cells, and these patients had a significantly higher response rate to anti-PD-1/PD-L1 treatment (41%), compared with those with PD-L1 expressed in less than 50% of tumor cells. (21) The study data showed a clear link between PD-L1 expression and the efficacy of pembrolizumab for patients with NSCLC, which was also reflected in the indication for use by the FDA. The companion diagnostic PD-L1 IHC 22C3 pharmDx test (Dako North America Inc, Carpinteria, California) is indicated as an aid in identifying NSCLC patients for treatment with pembrolizumab. (32) By definition, a companion diagnostic test is required and essential for the safe and effective use of the corresponding targeted therapy.

Studies have shown that atezolizumab treatment results in a median overall survival of 28.9 months in patients with metastatic RCC, with benefit more obvious in the group with strong PD-L1 IHC staining (Table 3, SP142 assay). (27) Similar results were also reported using atezolizumab in treating bladder cancer, with a 61% overall reduction in tumor burden, a 26% (26 of 100) overall response rate, and a median overall survival of 11.4 months in the IC2/3 subgroup based on an IHC assay, compared with a 45% reduction in tumor burden, an 11% (11 of 107) objective response rate, and a median overall survival of 6.7 months in the IC1 subgroup (Table 3). Additionally, a systemic review and meta-analysis from 20 trials including patients with metastatic melanoma, NSCLC, and RCC receiving anti-PD-1/PD-L1 antibodies (4230 metastatic melanoma, 1417 NSCLC, and 312 RCC patients) showed that PD-L1 expression is associated with lower mortality and better clinical response to anti-PD-1/PD-L1 antibodies in patients with metastatic melanoma and is associated with a better clinical response in patients with nonsquamous NSCLC. (39)

However, a study by Brahmer et al (40) observed that an objective response to nivolumab could still occur in patients with squamous carcinoma of lung who test negative for PDL1 (PD-L1 antibody [clone 28-8, Dako North America]). (40) Consequently, nivolumab has recently received FDA and European Medicines Agency approval for NSCLC, regardless of the PD-L1 expression status (for NSCLC in the United States and in European Union). Of interest, the FDA might have had some reservations about the clinical data submitted for review. The FDA indicated in the premarket approval that patients with PD-L1 expression, as detected by the PD-L1 IHC 28-8 pharmDx assay (Dako North America) in previously treated metastatic nonsquamous NSCLC, may experience enhanced survival when treated with nivolumab. Currently, this assay has been used as a complementary test based on the clinical trial results.

Additionally, in a meta-analysis of cancer patients treated with Nivolumab, pembrolizumab or atezolizumab, Carbognin et al (41) reported a clinical response in 239 of 702 patients (34%) with PD-L1-positive cancers and in 154 of 773 patients (20%) with PD-L1-negative cancers. The significant difference in response based on PD-L1 IHC was observed for NSCLC and melanoma but not for RCC or bladder carcinoma. Thus, using PD-L1 IHC as a predictive marker remains undetermined. Again, the use of different nonstandardized IHC techniques for measuring PD-L1 levels in tissue may have contributed to these differences.

STRATEGIES TO MEASURE PD-L1/PD-1 EXPRESSION

Immunohistochemistry

Assessment of PD-L1 expression through immunohistochemical staining has been advocated as one potential biomarker, as discussed above. Immunohistochemistry has several advantages: (1) the wide availability of formalinfixed, paraffin-embedded tissue; (2) visualization of expression in various cell populations (tumor versus immune/ stromal cells) to some extent based on morphology; (3) relative rapidity of the test; and (4) the test's relatively low cost and widespread use in pathology laboratories, particularly in contrast to molecular pathology-based methods. Different clinical trials have used different IHC assays from different pharmaceutical manufacturers to measure PD-L1 expression (Table 3). These assays use different monoclonal antibody clones recognizing various epitopes of PD-L1. Various systems for amplification and detection of the signal are used for IHC, leading to different thresholds of detecting PD-L1 expression. Additionally, 3 of these assays evaluate the PD-L1 expression in the tumor cells of NSCLC only. The Ventana SP142 assay, manufactured by Spring Bioscience (Pleasanton, California), measures the PD-L1 expression in both tumor cells and tumor-infiltrating immune cells in metastatic uroepithelial cancers. 0f note, studies using different PD-L1 antibodies to evaluate PD-L1 expression of tumor-infiltrating immune cells in various cancer types (head/neck squamous cell carcinoma, melanoma, and bladder cancers) are undergoing further evaluation to discern the impact of tumor-infiltrating immune cells in treatment response.

In 2015 a workshop by the Food and Drug Administration, the American Association for Cancer Research (AACR), and the American Society of Clinical Oncology led to a Blueprint Proposal developed by 4 pharmaceutical companies (Bristol-Myers Squibb Co, Merck & Co Inc, AstraZeneca PLC, and Genentech Inc), 2 diagnostic companies (Agilent Technologies Inc/Dako Corp and Roche/Ventana Medical Systems Inc), 2 professional societies (AACR-International Association for the Study of Lung Cancer), and 2 regulatory agencies (the European Medicines Agency and the FDA) to evaluate the analytic similarities of the 4 PD-L1 assays for use in NSCLC. The goal of this effort is to harmonize companion diagnostics for PD-L1 and to assess the possibility of interchangeable use of these assays. There are 2 phases in this proposal: phase 1 will evaluate analytic components by measuring PD-L1 expression on tumor or immune cells and predefine cutoffs in order to evaluate how these assays would compare using clinical samples; and phase 2 will design a statistically powered study with a large sample size based on the findings of phase 1. The preliminary data of phase 1, using 500 samples, was presented at the recent AACR annual meeting (April 16-20, 2016). (42) The preliminary results have indicated that 3 antibodies (22C3, 28-8, and SP263) have similar analytic performance in measuring the percentage of PD-L1expressing tumor cells. The dynamic range reported for these 3 antibodies was between 1% and 100%. A fourth antibody, SP142, constantly labeled fewer tumor cells. (42) However, there is less precision in analytic performance when labeling immune cells compared with tumor cells. There is also less agreement between observers when evaluating immune cells compared with cancer cells. Additionally, the patient population defined by Ventana SP263, manufactured by Spring Bioscience, at the 25% cutoff point is similar to the group identified by the Dako 28-8 and Dako 22C3, manufactured by Dako, at the 1% cutoff. However, about 37% of the cases studied revealed discrepant results for PD-L1 expression between assays. This suggests the possibility of assignment into different diagnostic categories according to the key clinical cutoffs if assays and algorithms are mismatched. A recent study further suggested that the inherent tumor heterogeneity, or assay- or platform-specific variables may also contribute to the discordant results of these companion diagnostic tests. (43)

These results suggest the challenges of extrapolating the results from one test to that of another test. This is reillustrated by Blueprint Chair Dr Fred Hirsch (professor of medicine at the University of Colorado), who said in an interview, "when pathologists used each assay in combination with its own prescribed cutoff, the assays did sometimes disagree on whether samples were PD-L1-positive." 44 However, these results are preliminary and the planned phase 2 study is needed to further evaluate how to use these antibodies most efficiently in clinical practice.

Additionally, other large-scale projects to investigate harmonization of PD-L1 antibodies are planned. For example, the National Comprehensive Cancer Network will soon begin a harmonization project in collaboration with Bristol Myers Squibb, MD Anderson Cancer Center, and Yale University. These results will be important for the efficient use of PD-L1 assay in pathology laboratories.

Several challenges remain regarding standardization of IHC beyond the Blueprint study. Preanalytically, the time for fixation in formaldehyde can modify the level of expression of PD-L1 and needs to be controlled. Additionally, there is heterogeneous PD-L1 expression in different regions of the same tumor specimen. Therefore, the absence of PD-L1 expression on small biopsies may not reflect the systemic immunologic landscape. This may have contributed to some patients responding to anti-PD-1 or anti-PD-L1 therapy independent of PD-L1 expression. Furthermore, it remains to be clarified whether the PD-L1 expression test should be performed on the primary tumor site or metastatic sites. PDL1 expression in tumor cells is dynamic, influenced by interferon y, hypoxia, and previous treatments, including chemotherapy, radiation therapy, and targeted therapy. (45) Additional comments regarding PD-L1 IHC as a predictive marker have been recently published. (46,47)

Flow Cytometry, Real-Time Quantitative Polymerase Chain Reaction, and Enzyme-Linked Immunosorbent Assay

As shown in Table 4, the first study with human peripheral blood cells demonstrating the feasibility of detecting PD-L1 expression with a flow cytometry method was published in 2009. (48) The authors showed that cryopreservation actually decreased the expression of both PD-1 and PD-L1 in peripheral blood cells. Using a similar approach with different antibodies, a study showed a higher expression of PD-1/PD-L1 in CD4/CD8 T lymphocytes from chronic lymphocytic leukemia patients when compared with age-matched controls. (49) In addition to peripheral blood cells or lymphocytes, Gowrishankar et al (15) have shown that interferon [gamma] induces the upregulation of PD-L1 in a nuclear factor-[kappa]B-dependent fashion in 5 different melanoma cell lines and melanoma patient-derived cells using flow cytometry. (15) Furthermore, Andorsky et al (50) documented that PD-L1 is widely expressed by anaplastic large cell lymphoma, whereas it only has limited expression in diffuse large B-cell lymphoma (DLBCL).

Flow cytometry studies, compared with IHC-based methods, have the advantage of simultaneously measuring the expression of PD-1 and PD-L1 in malignant cells and various types of immune cells. This is accomplished by using combinations of phenotypic markers for immune cells (cytotoxic T cells [CD8, TIA1], natural killer cells [CD56], T-reg cells [CD4, forkhead box P3], and dendritic cells/ macrophages [CD68]), tumor cells (epithelial cell adhesion molecule, epithelial membrane antigen, lymphoma/leukemia markers), and antibodies to PD-1 and PD-L1. This will likely provide a more comprehensive understanding of PD1/PD-L1 interactions between tumor cells and the immune environment. In theory, the efficacy of anti-PD-L1 treatment relies on the blockade of the inhibitory action of anticancer cytotoxic lymphocytes induced by the interaction between PD-1, expressed on immune cells (particularly lymphocytes), and PD-L1, expressed mainly on cancer cells. Therefore, it is reasonable to hypothesize that the higher the expression of PD-1 on immune cells, the more efficient the anti-PD-L1 treatment. This method may prove to be useful for predicting PD-1/PD-L1-targeted therapy, particularly for hematologic malignancies in which flow cytometry studies have been routinely used for diagnostic purposes. Similar to flow cytometry, another method that allows us to analyze PD-1 or PD-L1 expression is immunomagnetic selection using the CellTracks analyzer (Janssen Diagnostics LLC, Raritan, New Jersey). Using this method, researchers were able to identify the expression of PD-L1 in circulating tumor cells from patients with breast cancer with a sensitivity of 68.8%, indicating the promise of this noninvasive rapid screen in the future.5 (1)

Both SYBR dye-based and Taqman probe-based real-time polymerase chain reaction methods have been employed to detect PD-1/PD-L1 mRNA expression in whole-blood samples, cell lines, and surgical specimens. (52,53) In a recent report from Shen et al, (52) it was found that there is no uniform upregulation of PD-L1 expression in cancer cell lines compared with noncancer cell lines. However, authors identified a significant correlation between the expression of PD-L1 in human osteosarcoma and prognosis (median overall survival: 89 months in PD-L1-low patients versus 28 months in PD-L1-high patients).

An enzyme-linked immunosorbent assay-based method aimed at detecting soluble PD-L1 (sPD-L1) was developed in 2011.54 Using this approach, researchers were able to demonstrate that sPD-L1 is a good prognostic marker in multiple myeloma and DLBCL. (55,56) In a small study with 81 multiple myeloma patients, the mean concentration of sPDL1 was 2.851 ng/mL compared with 0.716 ng/mL sPD-L1 in matched controls. (55) Additionally, a lower sPD-L1 in patients with multiple myeloma is associated with higher progression-free survival. (55) In a multicenter, randomized phase 3 clinical trial in patients with DLBCL lymphoma, sPD-L1 was found to be higher in patients with DLBCL compared with healthy individuals, and the levels dropped after complete response. Moreover, cutoff of sPD-L1 at 1.52 ng/ mL can be used to stratify patients with DLBCL into 2 groups with respect to 3-year overall survival. This results in 2 distinct populations: 1 with a 3-year overall survival of 76% and 1 with a 3-year overall survival of 89%.56 These results support the possibility of using PD-1/PD-L1 blockade as a treatment option in these cancers.

Other Surrogate Markers for PD-L1 Expression

Using a fluorescence in situ hybridization probe targeting the gene locus encoding PD-L1 located at 9p24.1, 1 study has shown that 9p24.1 amplification was associated with increased PD-L1 expression via the janus kinase 2 pathway in nodular sclerosing Hodgkin lymphoma and primary mediastinum large B-cell lymphoma. (57) Another study using fluorescence in situ hybridization demonstrated that the incidence of 9p24.1 amplification (at least [greater than or equal to]3 copies) was present in 24% of patients with early-stage Hodgkin lymphoma, compared with a 50% presence in advancedstage (stages 3-4) Hodgkin lymphoma. (58) Additionally, the patients with Hodgkin lymphoma harboring 9p24.1 amplifications had a shorter progression-free survival. These results suggest that fluorescence in situ hybridization studies may be another potential method for predicting the response to PD-1/PD-L1 treatment.

The expression level of PD-L1 has been reported to be associated with other genetic alternations. The NSCLC cell lines with epidermal growth factor receptor (EGFR) mutations tend to have higher PD-L1 expression on the cell surface. (59) In a phase 2 trial studying pembrolizumab in multiple solid metastatic tumor, patients with mismatch repair-deficient (ie, microsatellite instability-high) colorectal cancer are more likely to benefit from PD-1 blockade (pembrolizomab) than those with mismatch repair-proficient tumors. Interestingly, PD-L1 expression is also elevated in mismatch repair-deficient colorectal cancer patients compared with those with mismatch repair-proficient tumors. However, the PD-L1 expression is not significantly associated with progression-free survival or overall survival. (60) In another study, higher nonsynonymous mutation burden was found to be associated with PD-L1-positive tumors and better melanoma-specific survival in patients with stage 3 melanoma. In addition, NF-1 mutation was shown to be limited to PD-L1-positive melanoma, but BRAF and NRAS mutations are distributed equally in PD-L1-positive or PD-L1-negative melanoma. (61)

OUTLOOK

PD-1/PD-L1-targeted therapy has demonstrated the impressive power of cancer immunotherapy. PD-1/PD-L1-targeted therapy either alone or in combination with other treatment modalities will benefit patients with advanced cancers. Because of the complexity of cancer immunity, we still have not yet completely understood the mechanism by which PD-1/PD-L1-targeted therapy improves survival in cancer patients. Many questions remain to be answered, particularly a better prediction system for predicting the response of PD-1/PD-L1-targeted therapy.

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Jian Guan, MD, PhD; Khin Sandar Lim, MD; Tarek Mekhail, MD; Chung-Che Chang, MD, PhD

Accepted for publication December 1, 2016.

Published as an Early Online Release April 18, 2017.

From the Departments of Internal Medicine (Drs Guan, Lim, and Mekhail) and Pathology and Laboratory Medicine (Dr Chang), Florida Hospital, Orlando; and the Department of Pathology, University of Central Florida College of Medicine, Orlando (Dr Chang).

Dr Chang served as a member of the Biomarker Advisory Board Meeting held by Bristol-Myers Squibb in December 2015. The other authors have no relevant financial interest in the products or companies described in this article.

Reprints: Chung-Che Chang, MD, PhD, Department of Pathology and Laboratory Medicine, Florida Hospital, 601 Rollins St, Orlando, FL 32803 (email: c.jeff.chang.md@flhosp.org).

Caption: Figure 1. Immune checkpoint signaling pathway in cancer cells. Activation of immune checkpoint signaling pathway including both cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) and programmed death receptor-1/programmed death ligand-1 (PD1/PD-L1) attenuates the amplitude of T-cell receptor (TCR)-mediated immunity. Abbreviation: MHC, major histocompatibility complex.
Table 1. Prevalence of Programmed Death Ligand/1 (PD/L1) Expression
in Various Cancers by Different Cutoffs and Status of Anti/
Programmed Death Receptor-1/PD-L1 Treatment

Type of             PD-L1+ Cases,            Cutoff Studied
Cancer                 % (No.)

Melanoma          24 (223 of 945)     [greater than or equal to]5%
                                      cancer cells

                  43 (10 of 23)

                  76 (35 of 46)       [greater than or equal to]
                                      1% cancer cells

Non-small cell    23.2 (191 of 824)   [greater than or equal
lung cancer                           to]50% membrane staining

                  Both squamous and   >1% membrane staining
                  nonsquamous
                  44 (119 of 272)

                  Squamous cell       [greater than or equal to]1%
                  54 (455 of 582)     membrane staining

Lung small cell   Nonsquamous         Not defined
carcinoma         71.6 (73 of 102)

Merkel cell       20.6 (24 of 116)    >1% tumor cells
carcinoma
                  56 (14 of 25)       >1% tumor cells

Breast cancers    34 (15 of 44)       Not defined

                  23 (152 of 650)     Modified H-score >100 (a)

Ovarian cancers   19 (20 of 109)      [greater than or equal to]5%
                                      tumor cells

                  68.6 (48 of 70)     [greater than or equal
                                      to]Moderate expression (see
                                      reference)

                  86.9 (20 of 23)

Renal clear       59.1 (153 of 259)   [greater than or equal to]5%
cell carcinoma                        cancer cells

                  24 (181 of 756)     >1% tumor cells

                  23.9 (73 of 306)    [greater than or equal to]5%
                                      tumor cells

Non-clear cell    10.9 (11 of 101)    [greater than or equal to]5%
renal cancer                          tumor cells

Urothelial        20 (10 of 56)       [greater than or equal to]5%
carcinoma                             expression

                  32.2 (100 of 310)   [greater than or equal to]5%
                                      tumor-infiltrating immune
                                      cells

                  20 (32 of 160)      [greater than or equal to]5%
                                      tumor cells

                  70.7 (46 of 65)     >12.2%

Glioblastoma      88 (103 of 117)     [greater than or equal to]5%
                                      tumor cells

                  50                  Not defined

Colorectal        44.8 (64 of 143)    Not defined
carcinoma

Gastric           12 (4 of 34)        [greater than or equal to]5%
carcinoma                             tumor cells

                  30.1 (140 of 465)   [greater than or equal to]1%
                                      tumor cells

Endometrial       83 (24 of 29)       [greater than or equal to]1%
carcinoma                             tumor cells

                  83 (24 of 29)       [greater than or equal to]1%
                                      tumor cells

Classical         74.5 (79 of 106)    Not defined
Hodgkin
lymphoma

                  75 (82 of 109)      [greater than or equal
                                      to]20% Reed-Sternberg cells

Diffuse large     100 (10 of 10)      Not defined
B-cell lymphoma

                  11 (132 of 1253)    >30 tumor cells

                  26 (43 of 163)      >30% tumor cells

Cutaneous         100 (9 of 9)        >2% lymphocytes
T-cell lymphoma

Acute myeloid     37 (22 of 60)       [greater than or equal to]5%
leukemia                              tumor cells

Type of             PD-L1+ Cases,       Clone Name (Manufacturer,
Cancer                 % (No.)               If Applicable)

Melanoma          24 (223 of 945)     Clone 5H1, homemade

                  43 (10 of 23)

                  76 (35 of 46)       Clone 22C3 (Dako North
                                      America Inc, Carpinteria,
                                      California)

Non-small cell    23.2 (191 of 824)   Clone 22C3 (Dako)
lung cancer

                  Both squamous and   Clone 28-8 (Dako)
                  nonsquamous
                  44 (119 of 272)

                  Squamous cell       Clone 28-8
                  54 (455 of 582)

Lung small cell   Nonsquamous         Polyclonal antibody (Abcam,
carcinoma         71.6 (73 of 102)    Cambridge, United
                                      Kingdom)

Merkel cell       20.6 (24 of 116)    Clone 28-8
carcinoma
                  56 (14 of 25)       22C3 (Dako)

Breast cancers    34 (15 of 44)       MIH1 clone (eBioscience, San
                                      Diego, California)

                  23 (152 of 650)     Rabbit anti-human PD-L1
                                      polyclonal antibody
                                      (Abcam)

Ovarian cancers   19 (20 of 109)      5H1

                  68.6 (48 of 70)     27A2

                  86.9 (20 of 23)     5H1

Renal clear       59.1 (153 of 259)   5H1
cell carcinoma

                  24 (181 of 756)     28-8

                  23.9 (73 of 306)    5H1

Non-clear cell    10.9 (11 of 101)    405.9A11
renal cancer

Urothelial        20 (10 of 56)       5H1
carcinoma

                  32.2 (100 of 310)   SP142

                  20 (32 of 160)      5H1

                  70.7 (46 of 65)     MIH1

Glioblastoma      88 (103 of 117)     5H1

                  50                  MIH1 (eBioscience)

Colorectal        44.8 (64 of 143)    Ab58810 (Abcam)
carcinoma

Gastric           12 (4 of 34)        5H1
carcinoma

                  30.1 (140 of 465)   E1L3N (Cell Signaling
                                      Technology)

Endometrial       83 (24 of 29)       Not indicated
carcinoma

                  83 (24 of 29)       Not indicated

Classical         74.5 (79 of 106)    405.9A11
Hodgkin
lymphoma

                  75 (82 of 109)      E1L3N (Cell Signaling
                                      Technology)

Diffuse large     100 (10 of 10)      PD-L1 (405.9A11)
B-cell lymphoma

                  11 (132 of 1253)    EPR1161/ab174838 (Abcam)

                  26 (43 of 163)      E1L3N (Cell Signaling
                                      Technology)

Cutaneous         100 (9 of 9)        Not indicated
T-cell lymphoma

Acute myeloid     37 (22 of 60)       MIH1
leukemia

Type of             PD-L1+ Cases,     Prognostic Marker
Cancer                 % (No.)

Melanoma          24 (223 of 945)     N/A

                  43 (10 of 23)

                  76 (35 of 46)       Good prognostic
                                      marker

Non-small cell    23.2 (191 of 824)   N/A
lung cancer

                  Both squamous and   No correlation
                  nonsquamous
                  44 (119 of 272)

                  Squamous cell       No correlation
                  54 (455 of 582)

Lung small cell   Nonsquamous         Good prognostic
carcinoma         71.6 (73 of 102)    marker

Merkel cell       20.6 (24 of 116)    N/A
carcinoma
                  56 (14 of 25)       N/A

Breast cancers    34 (15 of 44)       Adverse
                                      prognostic
                                      marker

                  23 (152 of 650)     Adverse
                                      prognostic
                                      marker

Ovarian cancers   19 (20 of 109)      N/A

                  68.6 (48 of 70)     Adverse
                                      prognostic
                                      marker

                  86.9 (20 of 23)     N/A

Renal clear       59.1 (153 of 259)   Adverse
cell carcinoma                        prognostic
                                      marker

                  24 (181 of 756)     Adverse
                                      prognostic
                                      marker

                  23.9 (73 of 306)    Adverse
                                      prognostic
                                      marker

Non-clear cell    10.9 (11 of 101)    Adverse
renal cancer                          prognostic
                                      marker

Urothelial        20 (10 of 56)       No correlation
carcinoma

                  32.2 (100 of 310)   NA

                  20 (32 of 160)      No correlation
                                      between tumor
                                      cell PD-L1 and
                                      prognosis.
                                      But PD-L1 in
                                      TIMCs is a good
                                      prognostic
                                      marker.

                  70.7 (46 of 65)     Adverse
                                      prognostic
                                      marker

Glioblastoma      88 (103 of 117)     No correlation

                  50                  N/A

Colorectal        44.8 (64 of 143)    Adverse
carcinoma                             prognostic
                                      marker

Gastric           12 (4 of 34)        Adverse
carcinoma                             prognostic
                                      marker

                  30.1 (140 of 465)   Good prognostic
                                      marker

Endometrial       83 (24 of 29)       N/A
carcinoma

                  83 (24 of 29)       N/A

Classical         74.5 (79 of 106)    Adverse
Hodgkin                               prognostic
lymphoma                              marker

                  75 (82 of 109)      Adverse
                                      prognostic
                                      marker

Diffuse large     100 (10 of 10)      Adverse
B-cell lymphoma                       prognostic
                                      marker

                  11 (132 of 1253)    Adverse
                                      prognostic
                                      marker

                  26 (43 of 163)      N/A

Cutaneous         100 (9 of 9)        N/A
T-cell lymphoma

Acute myeloid     37 (22 of 60)       Adverse
leukemia                              prognostic
                                      marker

Type of             PD-L1+ Cases,     Predictive Marker
Cancer                 % (No.)

Melanoma          24 (223 of 945)     N/A

                  43 (10 of 23)

                  76 (35 of 46)       N/A

Non-small cell    23.2 (191 of 824)   Positive
lung cancer                           predictive marker

                  Both squamous and   N/A
                  nonsquamous
                  44 (119 of 272)

                  Squamous cell       Positive
                  54 (455 of 582)     predictive marker

Lung small cell   Nonsquamous         Positive
carcinoma         71.6 (73 of 102)    predictive marker

Merkel cell       20.6 (24 of 116)    No correlation
carcinoma
                  56 (14 of 25)       No correlation

Breast cancers    34 (15 of 44)       N/A

                  23 (152 of 650)     N/A

Ovarian cancers   19 (20 of 109)      N/A

                  68.6 (48 of 70)     N/A

                  86.9 (20 of 23)     N/A

Renal clear       59.1 (153 of 259)   N/A
cell carcinoma

                  24 (181 of 756)     N/A

                  23.9 (73 of 306)    N/A

Non-clear cell    10.9 (11 of 101)    N/A
renal cancer

Urothelial        20 (10 of 56)       N/A
carcinoma

                  32.2 (100 of 310)   Positive
                                      predictive marker

                  20 (32 of 160)      N/A

                  70.7 (46 of 65)     N/A

Glioblastoma      88 (103 of 117)

                  50                  N/A

Colorectal        44.8 (64 of 143)    N/A
carcinoma

Gastric           12 (4 of 34)        N/A
carcinoma

                  30.1 (140 of 465)   Good predictive
                                      marker

Endometrial       83 (24 of 29)       N/A
carcinoma

                  83 (24 of 29)       N/A

Classical         74.5 (79 of 106)    N/A
Hodgkin
lymphoma

                  75 (82 of 109)      N/A

Diffuse large     100 (10 of 10)      Good predictive
B-cell lymphoma                       marker

                  11 (132 of 1253)    N/A

                  26 (43 of 163)      N/A

Cutaneous         100 (9 of 9)        N/A
T-cell lymphoma

Acute myeloid     37 (22 of 60)       Good predictive
leukemia                              marker

Type of             PD-L1+ Cases,     FDA Approval Status
Cancer                 % (No.)

Melanoma          24 (223 of 945)     Keytruda (pembrolizumab,
                                      Merck, Kenilworth, New
                                      Jersey)

                  43 (10 of 23)       Opdivo (nivolumab, Bristol-
                                      Myers Squibb, New York, New
                                      York)

                  76 (35 of 46)

Non-small cell    23.2 (191 of 824)   Keytruda (pembrolizumab)
lung cancer                           2015

                  Both squamous and   Opdivo (nivolumab) 2015
                  nonsquamous
                  44 (119 of 272)

                  Squamous cell       Opdivo (nivolumab) 2015
                  54 (455 of 582)

Lung small cell   Nonsquamous
carcinoma         71.6 (73 of 102)

Merkel cell       20.6 (24 of 116)
carcinoma
                  56 (14 of 25)

Breast cancers    34 (15 of 44)

                  23 (152 of 650)

Ovarian cancers   19 (20 of 109)

                  68.6 (48 of 70)

                  86.9 (20 of 23)

Renal clear       59.1 (153 of 259)   Opdivo (nivolumab) 2015
cell carcinoma

                  24 (181 of 756)     Opdivo (nivolumab) 2015

                  23.9 (73 of 306)

Non-clear cell    10.9 (11 of 101)
renal cancer

Urothelial        20 (10 of 56)       Tecentriq (atezolizumab,
carcinoma                             Genentech, San Francisco,
                                      California)

                  32.2 (100 of 310)   Tecentriq (atezolizumab)

                  20 (32 of 160)

                  70.7 (46 of 65)

Glioblastoma      88 (103 of 117)

                  50

Colorectal        44.8 (64 of 143)
carcinoma

Gastric           12 (4 of 34)
carcinoma

                  30.1 (140 of 465)

Endometrial       83 (24 of 29)
carcinoma

                  83 (24 of 29)

Classical         74.5 (79 of 106)
Hodgkin
lymphoma

                  75 (82 of 109)

Diffuse large     100 (10 of 10)      Opdivo (nivolumab)
B-cell lymphoma

                  11 (132 of 1253)

                  26 (43 of 163)

Cutaneous         100 (9 of 9)
T-cell lymphoma

Acute myeloid     37 (22 of 60)
leukemia

Type of             PD-L1+ Cases,     Source, y
Cancer                 % (No.)

Melanoma          24 (223 of 945)     Rodic et al, (62) 2015;
                                      and Larkin et al, (63) 2015

                  43 (10 of 23)

                  76 (35 of 46)       Madore et al, (64) 2015

Non-small cell    23.2 (191 of 824)   Garon et al, (21) 2015
lung cancer

                  Both squamous and   Brahmer et al, (40) 2015
                  nonsquamous
                  44 (119 of 272)

                  Squamous cell       Borghaei et al, (65) 2015
                  54 (455 of 582)

Lung small cell   Nonsquamous         Ishii et al, (66) 2015
carcinoma         71.6 (73 of 102)

Merkel cell       20.6 (24 of 116)    Antonia et al, (67) 2016
carcinoma
                  56 (14 of 25)       Nghiem et al, (22) 2016

Breast cancers    34 (15 of 44)       Ghebeh et al, (68) 2006

                  23 (152 of 650)     Muenst et al, (69) 2014

Ovarian cancers   19 (20 of 109)      Mittendorf et al, (70) 2014

                  68.6 (48 of 70)     Hamanishi et al, (71) 2007

                  86.9 (20 of 23)     Dong et al, (72) 2002

Renal clear       59.1 (153 of 259)   Krambeck et al, (73) 2006
cell carcinoma

                  24 (181 of 756)     Motzer et al, (74) 2015

                  23.9 (73 of 306)    Thompson et al, (75) 2006

Non-clear cell    10.9 (11 of 101)    Choueiri et al, (76) 2014
renal cancer

Urothelial        20 (10 of 56)       Faraj et al, (77) 2015
carcinoma

                  32.2 (100 of 310)   Rosenberg et al, (28) 2016

                  20 (32 of 160)      Bellmunt et al, (78) 2015

                  70.7 (46 of 65)     Nakanishi et al, (79) 2007

Glioblastoma      88 (103 of 117)     Berghoff et al, (80) 2015

                  50                  Yao et al, (81 2009

Colorectal        44.8 (64 of 143)    Shi et al, (82) 2013
carcinoma

Gastric           12 (4 of 34)        Thompson et al, (83) 2016
carcinoma

                  30.1 (140 of 465)   Boger et al, (84) 2016

Endometrial       83 (24 of 29)       Liu et al, (85) 2015
carcinoma

                  83 (24 of 29)       Vanderstraeten et al, (86) 2014

Classical         74.5 (79 of 106)    Roemer et al, (58) 2016
Hodgkin
lymphoma

                  75 (82 of 109)      Koh et al, (87) 2015

Diffuse large     100 (10 of 10)      Ansell et al, (18) 2015
B-cell lymphoma

                  11 (132 of 1253)    Kiyasu et al, (88) 2015

                  26 (43 of 163)      Georgiou et al, (89) 2016

Cutaneous         100 (9 of 9)        Kantekure et al, (90) 2012
T-cell lymphoma

Acute myeloid     37 (22 of 60)       Chen et al, (91) 2008
leukemia

Abbreviations: FDA, Food and Drug Administration; H-score,
histo-score; N-A, not applicable; TIMC, tumor-infiltrating
mononuclear cells.

(a) H-score: assigned using the following formula: [1 x (% cells
[1.sup.+]) + 2 x (% cells 2+) + 3 x (% cells [3.sup.+])]. (92)

Table 2. Anti-Programmed Death Receptor-1 (Anti-PD-1) and
Anti-Programmed Death Ligand-1 (Anti-PD-L1) Agents in Clinical Use
or in Early Phase of Development

Target   Name                   Company

PD-1     Pembrolizumab          Merck, Kenilworth, New
         (Keytruda;MK-3475      Jersey
         or Lambrolizumb)

         Nivolumab (Opvido;     Bristol-Myers Squibb,
         DMX1106 or             New York, New York
         BMS-936558)

         AMP-224                GlaxoSmithKline,
                                Brentford, United Kingdom

         AMP-514 (MEDI0680)     AstraZeneca, London,
                                United Kingdom

         CT-011 (pidilizumab)   CureTech, Yavne, Israel

         MDX1105 (BMS936559)    Bristol-Myers Squibb

PD-L1    Atezolizumab           Genentech/Roche, San
         (MPDL3280A)            Francisco, California

         Durvalumab             AstraZeneca,
         (MEDI4736)             Medlmmune, London,
                                United Kingdom

         MESB001078C            Merck

Target   Name                   Characteristic        Approved by FDA/
                                                      Clinical Trial

PD-1     Pembrolizumab          Humanized IgG4        Approved by FDA
         (Keytruda;MK-3475
         or Lambrolizumb)

         Nivolumab (Opvido;     Fully human IgG4      Approved by FDA
         DMX1106 or
         BMS-936558)

         AMP-224                Fusion human PD-L2    Phase 1

         AMP-514 (MEDI0680)     Humanized IgG4. mAb   Phase 1

         CT-011 (pidilizumab)   Humanized IgG1        Phase 2

         MDX1105 (BMS936559)    Fully human IgG4      Phase 1b/2a

PD-L1    Atezolizumab           Fc-modified human     FDA approved
         (MPDL3280A)            IgG1

         Durvalumab             Fully human IgG       Phase 1
         (MEDI4736)

         MESB001078C            Fully human IgG1      Phase 1-2

Target   Name                   Cancer Types

PD-1     Pembrolizumab          Melanoma, NSCLC
         (Keytruda;MK-3475
         or Lambrolizumb)

         Nivolumab (Opvido;     Melanoma, NSCLC,
         DMX1106 or             RCC, and HL
         BMS-936558)

         AMP-224                Solid tumors

         AMP-514 (MEDI0680)     Solid tumors

         CT-011 (pidilizumab)   Lymphoma or
                                solid tumors
         MDX1105 (BMS936559)    Solid tumors

PD-L1    Atezolizumab           Metastatic
         (MPDL3280A)            uroepithelial
                                carcinoma

         Durvalumab             Solid tumors
         (MEDI4736)

         MESB001078C            Solid tumors

Abbreviations: FDA, Food and Drug Administration; HL, Hodgkin
lymphoma; IgG, immunoglobulin G; NSCLC, non-small cell lung
cancer; RCC, renal cell carcinoma.

Table 3. Current Programmed Death Ligand-1 (PD-L1)
Immunohistochemistry (IHC) Assays With Coupled Treatment Agents

               PD-L1 IHC 28-8       PD-L1 22C3 IHC
               pharmDx              pharmDx

Coupled        Nivolumab            Pembrolizumab
treatment      (Bristol-Myers       (Merck,
agent          Squibb, New York,    Kenilworth, New
               New York)            Jersey)

mAb alone      28-8 (Abcam,         22C3 (Dako,
               United Kingdom)      Carpinteria,
               Cambridge,           California)

Diagnostic     Dako                 Dako
platform,
FDA status

               FDA-approved         FDA-approved
               complementary test   companion
               for metastatic       diagnostic
               melanomaa and        test for NSCLC
               nonsquamous          (b)
               NSCLCb

Staining       Membrane             Membrane
location
scored

Cell types     TCs                  TCs
scored

Cutoff(s)      [greater than or     [greater than or
tested         equal to] %1, 5%,    equal to] 1%, or
               or 10% of TCs        50% of TCs

FDA-approved   N/A (c)              [greater than or
thresholds                          equal to] 50% of
                                    TCs

               Ventana SP142        Ventana SP263

Coupled        Atezolizumab         Durvalumab
treatment      (Roche/ Genentech,   (AstraZeneca,
agent          San Francisco,       London, United
               California)          Kingdom)

mAb alone      SP142 (Spring        SP263 (Spring
               Bioscience,          Bioscience)
               Pleasanton,
               California)

Diagnostic     Ventana/Roche        Ventana/Roche
platform,      (Tucson, Arizona)
FDA status

               FDA approved for     Currently
               metastatic           Investigation Use
               uroepithelial        Only (IUO)
               cancer

Staining       Membrane             Membrane
location
scored

Cell types     TCs and TIIC         TCs
scored

Cutoff(s)      TCs: [greater than   [greater than or
tested         or equal to] 1%,     equal to] 25% of
               5%, or 50%; TIICs:   TCs
               [greater than or
               equal to] 1%, 5%,
               or 10%

FDA-approved   N/A (d)              Not FDA-approved
thresholds                          test

Abbreviations: FDA, Food and Drug Administration;mAb, monoclonal
antibody;N-A, not applicable;NSCLC, non-small cell lung cancer;TCs,
tumor cells; TIIC, tumor-infiltrating immune cells.

(a) For treatment-naive patients.

(b) For previously treated patients (ie, second-line therapy).

(c) All patients are eligible for treatment regardless of results.

(d) All patients are eligible for treatment regardless of results;
however, patients with [greater than or equal to] 5% of PD-L1 + TIICs
may be associated with increased objective response rate.

Table 4. Application of Nonimmunohistochemistry Methods to Determine
Programmed Death Ligand-1 (PD-L1) Expression in Human Samples

Methods          Antibodies        Specimen/Cell     Source, y
                                   Types

Flow cytometry   PD-L1             Cryopreserved     Campbell et al,
                 (eBioscience;     PBMCs,            (48) 2009;
                 Biolegend         lymphocytes,      Brusa et al,
                 [clone            macrophages       (49) 2013; Fang
                 29E.2A3];BD                         et al, (53)
                 Pharmingen, San                     2015; Pan et
                 Jose,                               al, (93) 2015;
                 California)                         Rodriguez-
                                                     Garcia et al,
                                                     (94) 2011

Immunomagnetic   PD-L1             Metastatic        Mazel et al,
selection and    (R&D-FAB1561,     breast cancer     (51) 2015
CellTracks       R&D Systems,      cells
analyzer         Minneapolis,
                 Minnesota)

ELISA            PD-L1 (mAb,       Serum             Chen et al,
                 2H11)                               (54) 2011

Real-time PCR    Not applicable    Whole blood;      Shen et al,
                                   osteosarcoma      (52) 2014; Fang
                                   surgical          et al, (53)
                                   specimens         2015

Abbreviations: ELISA, enzyme-linked immunosorbent assay;mAb,
monoclonal antibody;PBMC, peripheral blood mononuclear cells;PCR,
polymerase chain reaction.
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Author:Guan, Jian; Lim, Khin Sandar; Mekhail, Tarek; Chang, Chung-Che
Publication:Archives of Pathology & Laboratory Medicine
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Date:Jun 1, 2017
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