Immune checkpoints, such as programmed death ligand 1 (PD-L1) or its receptor, programmed death 1 (PD-1), appear to be Achilles’ heels for multiple tumor types. PD-L1 not only provides immune escape for tumor cells but also turns on the apoptosis switch on activated T cells. Therapies that block this interaction have demonstrated promising clinical activity in several tumor types. In this review, we will discuss the current status of several anti–PD-1 and anti–PD-L1 antibodies in clinical development and their direction for the future.

Programmed death 1 (PD-1) is an immune inhibitory receptor expressed on several immune cells, particularly cytotoxic T cells.[1] It interacts with two ligands, programmed death ligand 1 (PD-L1) (B7-H1, CD274) and PD-L2 (B7-DC). While PD-L2 is expressed primarily on macrophages and dendritic cells, PD-L1 is expressed on tumor cells, as well as other immune cells. The interaction of these ligands with PD-1 inhibits T-cell activation and cytokine production. Their ligation with PD-1 during infection or inflammation in normal tissue is critically important in maintaining homeostasis of immune response to prevent autoimmunity. Their interaction in tumor microenvironments, however, provides an immune escape for tumor cells by turning off cytotoxic T cells.[2] Thus, blocking these interactions may subject the tumor cells to attack from cytotoxic T cells.

How Is PD-L1 Expression Determined? What Is Its Significance?

PD-L1 expression is measured most commonly by immunohistochemistry (IHC). Tumoral PD-L1 expression status has been shown to be prognostic in multiple tumor types, including melanoma (MEL), renal cell carcinoma (RCC), and non–small-cell lung cancer (NSCLC). In addition, tumoral PD-L1 expression appears to correlate closely with response to anti–PD-1 antibodies.[3] However, no test is uniformly accepted as the standard for quantitating PD-L1 expression. The IHC tests used in clinical trials are proprietary; data on similarities between and among the antibodies used and the assay conditions, staining pattern, threshold for signal detection, and assessment of positivity are not published. The protein expression patterns of PD-L1 on tumor cells, dendritic cells, and tumor-infiltrating immune cells differ, and exact cell type and degree of expression vary between assays. A different methodology for evaluating PD-L1 messenger RNA (mRNA) expression, using an antibody-independent in situ hybridization assay coupled with quantitative fluorescence, showed that increased PD-L1 mRNA transcript was associated with elevated tumor-infiltrating lymphocytes and better clinical outcomes in patients with breast cancer[4] and NSCLC.[5] The role of PD-L1 expression as a biomarker is discussed in this supplement to ONCOLOGY, in the review “Prognostic and Predictive Markers for the New Immunotherapies,” by Drs. Kathleen M. Mahoney and Michael B. Atkins.[6]

Several PD-1 and PD-L1 antibodies are in clinical development (Table 1). Overall, they are very well tolerated; most did not reach dose-limiting toxicity in their phase I studies. As listed in Table 2, no clinically significant difference in adverse event profiles has been seen between anti–PD-1 and anti–PD-L1 antibodies. Slightly higher rates of infusion reactions (11%) were observed with BMS-936559 (anti–PD-L1) than with BMS-96558 (nivolumab). In an early stage of a nivolumab phase I study, there was concern about fatal pneumonitis.[7] It has been hypothesized that PD-1 interaction with PD-L2 expressed on the normal parenchymal cells of lung and kidney provides unique negative signaling that prevents autoimmunity.[8] Thus, anti–PD-1 antibody blockage of such an interaction may remove this inhibition, allowing autoimmune pneumonitis or nephritis. Anti–PD-L1 antibody, however, would theoretically leave PD-1–PD-L2 interaction intact, preventing the autoimmunity caused by PD-L2 blockade. With implementation of an algorithm to detect early signs of pneumonitis and other immune-related adverse events, many of these side effects have become manageable. However, it does require discerning clinical attention to detect potentially fatal side effects. In terms of antitumor activity, both anti–PD-1 and anti–PD-L1 antibodies have shown responses in overlapping multiple tumor types. Although limited to a fraction of patients, most responses, when observed, were rapid and durable.

MEL is among the first types of solid tumors in which nivolumab has shown promising antitumor activity. In a phase I study across all dose levels, nivolumab resulted in an objective response rate (ORR) of 28% (95% confidence interval [CI], 19–38) in patients with advanced MEL who had no prior ipilimumab treatment.[7] Responses were adjudicated according to Response Evaluation Criteria in Solid Tumors (RECIST), version 1.0, with modification. A recently updated analysis in 107 MEL patients confirmed these responses and suggested potential survival benefit.[9,10] The ORR was 32% (95% CI, not reported [NR]). The median duration of response was 99.4 weeks (range, 17.0+ to 117.0+). More than half (11/21) of responding patients who discontinued therapy for reasons other than progressive disease responded for more than 24 weeks. The 1-, 2-, and 3-year overall survival (OS) rates were 63% (95% CI, 53–71), 48% (95% CI, 38–57), and 41% (95% CI, 31–51), respectively. Median OS was 17.3 months (95% CI, 12.5–36.7). Survival analysis by type of response suggested that patients who had immune-related responses, such as reduction in the target lesion in the presence of new lesions or following initial progression, had similar OS to patients with RECIST responses.[9,10]

Nivolumab in patients who were pretreated with ipilimumab was tested as part of a “sequential cohort” of an ongoing phase I trial. Nivolumab was given at two dose levels (1 mg/kg and 3 mg/kg every 2 weeks). Of 33 patients, ORR by RECIST criteria was 31% (95% CI, NR). The 1-year survival rate was 70% (95% CI, NR). The residual plasma concentration of prior ipilimumab at the start of nivolumab appears to have an influence on response to nivolumab. An ipilimumab concentration ≥ 7.255 Î¼g/mL (the median) was associated with greater ORR to nivolumab (57.1% vs 14.3%).[11]

Pembrolizumab (MK-3475, lambrolizumab) is a humanized immunoglobulin G (IgG)-4 kappa antibody against PD-1. In KEYNOTE-001, the safety and antitumor activities of pembrolizumab were tested in the largest-ever phase I study of patients with metastatic MEL (N = 411).[12] This study began with ipilimumab-naive (IPI-N) and ipilimumab-treated (IPI-T) patients, and later implemented a randomized dosing comparison of 2 mg/kg vs 10 mg/kg in ipilimumab-refractory (IPI-R) and IPI-N patients. Among patients evaluable by RECIST 1.1, overall ORR was 34% (95% CI, 29–39). The ORRs were 40% (95% CI, 32–48) in IPI-N patients and 28% (95% CI, 22–35) in IPI-T patients. The 1-year OS rate was 69%; median OS has not yet been reached. A subgroup analysis suggested that a tumor size smaller than the median of 90 mm was an independent predictor of response.[13]

In the randomized comparison of 2 mg/kg vs 10 mg/kg of pembrolizumab in IPI-R and IPI-N patients,[14] there was no statistically significant difference between the ORRs for the two cohorts by either RECIST 1.1 (independent central review) or immune-related response criteria (investigator review). In the IPI-N cohort, the ORRs by RECIST 1.1 were 33% (95% CI, 20–49) vs 40% (95% CI, 26–56) in the 2-mg/kg and 10-mg/kg arms, respectively (

 = .4835). In the IPI-R cohort, the ORRs were 26% in both arms (95% CI, 17–37 in the 2-mg/kg arm and 95% CI, 17–36 in the 10-mg/kg arm). According to Kaplan-Meier estimates of OS in IPI-N patients, 1-year OS rates were 72% (95% CI, 15–NR) and 64% (95% CI, 10–NR), and in IPI-R patients, they were 58% (95% CI, 11–NR) and 63% (95% CI, NR)-in the 2-mg/kg and 10-mg/kg arms, respectively. Given favorable benefit-risk profiles at both doses, 2 mg/kg every 3 weeks was recommended for future investigation. The most common immune-mediated adverse event of any grade was hypothyroidism (~8%). There was a < 1% incidence of grade 3 or 4 pneumonitis, hepatitis, colitis, or hypophysitis reported.

Pidilizumab is a humanized anti–PD1 IgG1 kappa with binding affinity at 20 nM. A phase II study in MEL patients showed ORR (by investigator review) of 7% in IPI-N patients and 5% in IPI-T patients.[15] The rate of immune-related stable disease was 37%. The 12-month survival rate was 64% (95% CI, 55.6–72.0). Pidilizumab is being investigated in hematologic malignancies.

MPDL3280A is an IgG1-engineered, anti–PD-L1 antibody. A phase I study in 38 patients with metastatic MEL reported a 39% ORR (95% CI, NR), with 43% achieving a 24-week progression-free survival (PFS).[16] Both patients with cutaneous (33%) and mucosal (25%) MEL responded. Patients with PD-L1–positive status by IHC had a 27% ORR (95% CI, NR). Similarly, patients with a negative status had a 20% ORR (95% CI, NR). An additional 60% of patients with PD-L1–positive tumors had stable disease, suggesting that PD-L1–positive patients are more likely to derive clinical benefit. No grade 3 or 4 pneumonitis or treatment-related deaths occurred. Based on these findings and a potential synergy with vemurafenib, a phase Ib study is currently testing the safety and tolerability of this combination in patients with 

 V600E mutations (National Cancer Institute ClinicalTrials.gov ID NCT01656642).

BMS-936559 is a fully human, anti–PD-L1, IgG4 monoclonal antibody. In a study of 1-, 3-, and 10-mg/kg doses in metastatic MEL patients, 9 of 52 (17%; 95% CI, 8–30) achieved objective responses.[17] Three patients achieved a complete response (CR). Five of 9 responding patients had an objective response lasting beyond 1 year. In addition, 14 of 52 patients (27%; 95% CI, 16–41) had stable disease lasting at least 24 weeks.

MEDI4736 is an engineered IgG1 kappa monoclonal antibody with triple mutations in the Fc domain to remove antibody-dependent, cell-mediated cytotoxic activity. It has shown high affinity and selectivity for PD-L1 and, reportedly, no binding to PD-L2.[18] In its dose escalation study of doses ranging from 0.1 mg/kg every 2 weeks to 15 mg/kg every 3 weeks, no dose-limiting toxicity or drug-related deaths were seen. Tumor regression was seen in multiple tumor types, including MEL, NSCLC, RCC, and colorectal cancer (CRC). Segal et al reported the results of a multiarm expansion study using a dose of 10 mg/kg every 2 weeks in 44 patients with MEL (uveal and cutaneous).[19] Although an analysis of antitumor activity in MEL patients has not been reported, preliminary data suggest that several patients with both cutaneous and uveal MEL remained in the study beyond 12 weeks, and no serious adverse events have been reported.

In light of their promising activity as single agents, several combinations of anti–PD-1 and anti–PD-L1 antibodies have been studied. A preclinical study has shown that dual immune checkpoint blockade of cytotoxic T-lymphocyte antigen 4 (CTLA-4) and PD-1 resulted in more pronounced antitumor activity than either single blockade alone.[20] Wolchok et al reported the clinical activity of concurrent administration of nivolumab and ipilimumab in 53 patients with advanced MEL; the resulting ORR was 40% (95% CI, 27–55) (by modified World Health Organization [WHO] criteria), and responses were “rapid and deep.”[21] In an updated survival analysis, Sznol et al reported 1- and 2-year OS rates of 85% and 79% (95% CI, NR), respectively; median OS was 40 months, and median PFS was 27 weeks.[11] This study added another cohort of 40 patients who received nivolumab 1 mg/kg and ipilimumab 3 mg/kg every 3 weeks for 4 cycles, followed by nivolumab 1 mg/kg every 3 weeks for 4 more cycles, then 3 mg/kg every 2 weeks for up to 48 cycles. Results confirmed clinical activity, with an ORR of 43% (95% CI, NR). Responses were seen regardless of PD-L1 tumor expression status or 

Nivolumab in combination with multiple peptide vaccines in IPI-R or IPI-N MEL patients was well tolerated.[22] This study also found that increased peripheral blood regulatory T cells and decreased antigen-specific T cells were associated with progression.The clinical benefit of adding a vaccine remains to be seen.

A phase I/II study of MEDI4736 in combination with a BRAF inhibitor (dabrafenib) and a MEK inhibitor (trametinib) or with trametinib alone is ongoing (NCT02027961). Other immune modulatory agents, such as interferon (IFN) alfa-2b, are combined with various anti–PD-1 and anti–PD-L1 antibodies in ongoing clinical trials.

Metastatic RCC (mRCC) has been a target of immunotherapy for several decades; high-dose interleukin-2 has shown an ORR of approximately 20%, with long-term remission in a small subset of patients,[23] and ipilimumab has shown an ORR of 12.5% (95% CI, 4–27).[24] Over the past decade, the US Food and Drug Administration (FDA) has approved several antiangiogenic agents for mRCC on the basis of improvement in PFS; however, none have demonstrated survival benefits.

The activity of nivolumab in mRCC was reported in its first phase I study, in which 9 of 33 patients (27%; 95% CI, 13–46) achieved objective responses and 56% (95% CI, 39–73) were alive without disease progression at 24 weeks. At the time of data analysis, five of these responding patients had a response lasting for more than a year.[7] The clinical activity of nivolumab in mRCC was confirmed in a dose-ranging phase II study, in which patients were randomized to receive either 0.3, 2, or 10 mg/kg every 3 weeks.[25] Clinical activities were seen across all dose levels, with ORRs of 20%, 22%, and 20% (95% CI, NR), respectively. On survival analysis, median OS outcomes were 18.2 months (80% CI, 16.2–24.0), 25.5 months (80% CI, 19.8–28.8), and 24.7 months (80% CI, 15.3–26.0), respectively. These survival data appeared very promising in the context of other published median OS data of 12 to 16 months for mRCC in second-line settings.[26,27] In a phase III randomized controlled trial, nivolumab is being compared with everolimus in patients previously treated with antiangiogenic therapy; the primary outcome measure is OS (NCT01668784).

In patients with mRCC, 2 of 17 (12%; 95% CI, 2–36) had an objective response to 10 mg/kg of BMS-936559, with responses lasting 4 and 17 months.[17] Seven additional patients (41%; 95% CI, 18–67) had stable disease lasting at least 24 weeks.

Cho et al reported that in 55 patients with mRCC, the anti–PD-L1 antibody MPDL3280A was very well tolerated, with no dose-limiting toxicities.[28] The overall ORR was 13% (95% CI, NR). Responses were seen in both clear-cell and non–clear-cell histology and in both PD-L1–positive and –negative patients. An additional 60% of patients had stable disease. At 24 weeks, 53% of the patients were alive without disease progression. In a phase II study, MPDL3280A is being assessed either as monotherapy or in combination with antiangiogenic agents (bevacizumab or sunitinib) in untreated mRCC (NCT01984242).

To assess combination strategies, investigators have launched a large phase Ib multiarm combination study, CheckMate-016. Nivolumab plus ipiliumab was tested in a randomized study with different induction doses (nivolumab 3 mg/kg plus ipilimumab 1 mg/kg [N3+I1] or nivolumab 1 mg/kg plus ipilimumab 3 mg/kg [N1+I3], followed by nivolumab 3 mg/kg every 2 weeks for both arms. N1+I3 had a higher frequency (26%) of treatment-related adverse events leading to discontinuation, including elevations in lipase and alanine transaminase, diarrhea, and pneumonitis. The ORR was 43% (95% CI, 21.8–66.0) in the N3+I1 cohort and 48% (95% CI, 26.8–69.4) in the N1+I3 cohort[29]; these results appear to be more than additive effects. Approximately 80% of these responses were ongoing at the time of data analysis. An additional 24% of patients given N3+I1 and 36% given N1+I3 have achieved stable disease. The 24-week PFS rates were 64% to 65%. In 44 available samples, PD-L1 expression status did not correlate with response. With 1% tumor membrane staining as a cutoff, similar percentages of patients with PD-L1–positive and –negative tumors achieved objective responses (55% and 50%).

Based on data supporting immune-suppressive effects of vascular endothelial growth factor (VEGF) and FDA-approved antiangiogenic agents for mRCC, nivolumab was tested in combination with either sunitinib (n = 33) or pazopanib (n = 20).[30] Both pretreated and treatment-naive patients responded. Overall confirmed ORRs were 52% (95% CI, 33.5–69.2) in the sunitinib combination arm and 45% (95% CI, 23.1–68.5) in the pazopanib combination arm. As seen with ipilimumab combinations, PD-L1 expression status, regardless of the staining cutoff, did not predict response to combination therapy with either sunitinib or pazopanib. The most common side effects were consistent with known toxicity profiles of these tyrosine kinase inhibitors and included diarrhea, fatigue, and hypertension. Two grade 3 pneumonitis events (3%) were observed.

No pembrolizumab monotherapy data have been reported yet in patients with mRCC. Pembrolizumab is under investigation in combination with pazopanib in a phase I/II study (NCT02014636). In patients treated with pazopanib, increased PD-L1 expression was associated with shorter PFS.

The PD-1/PD-L1 pathway appears to be a critical therapeutic target for advanced NSCLC. Multiple PD-1 and PD-L1 antibodies have demonstrated antitumor activities in both pre- and postsystemic therapy settings. The activity appears to be more pronounced in earlier disease settings. As shown in Table 5, in chemotherapy-naive, PD-L–positive patients, nivolumab[31] and pembrolizumab[32] demonstrated ORRs of 50% (95% CI, NR) and 26% (95% CI, 14–42), respectively. In pretreated patients, nivolumab,[33] pembrolizumab,[34] MPDL3280A, [35] and MEDI4736[36] have demonstrated ORRs ranging from 16% to 23%.

Brahmer et al presented survival data of a phase Ib trial of nivolumab that included 129 heavily pretreated patients with advanced NSCLC, more than half of whom received > 3 prior therapies.[33] At a median follow-up of 27 months across all dose levels, 1- and 2-year OS rates were 42% (95% CI, 34–51) and 24% (95% CI, 16–32), respectively, and median OS was 9.9 months (95% CI, 9.8–12.4). Patients given the 3-mg/kg dose appeared to have superior survival data, with 1- and 2-year OS rates of 56% (95% CI, 38–71) and 45% (95% CI, 27–61), respectively, and a median OS of 14.9 months (95% CI, 7.3–NR). Unlike cytotoxic agents-and as seen in other settings, such as in mRCC[25]-nivolumab did not show a clear dose-response relationship in advanced NSCLC. The survival data are certainly encouraging in a disease where the median OS has been no more than 7 to 8 months.

PD-L1 expression on NSCLC tumor cells or tumor-infiltrating immune cells was associated with higher response rates. Although not statistically powered to show this, some degree of correlation has been observed with most of the agents, including pembrolizumab, MPDL3280A, and MEDI-4736. As noted in Table 5, across all these agents, except nivolumab in pretreated patients, PD-L1 expression was associated with higher response rates. ORRs were 23% to 46% vs 3% to 15% in PD-L1–positive vs PD-L1–negative groups, respectively.

As also noted, while PD-L1 expression was highly predictive of response to nivolumab in treatment-naive patients (50% in PD-L1–positive patients vs 0% in PD-L1–negative patients),[31] this was not the case for pretreated patients (15% in PD-L1–positive vs 14% in PD-L1–negative), using 5% as a PD-L1 expression cutoff.[33] This difference, despite the use of the same assay and the same drug, raises several questions, including questions of assay sensitivity, of archival tissue vs “fresh” pretreatment biopsy, and of whether the complexity of immune-mediated cytolysis is underestimated. Several ongoing phase III trials of nivolumab in advanced NSCLC require mandatory tissue samples and will explore the role of PD-L1 as a predictive biomarker for nivolumab, with its prespecified evaluation correlated to OS (see Table 7).

Another important potential predictive biomarker is smoking status. Both MPDL3280A[35] and pembrolizumab[34] have demonstrated higher response rates among current or former smokers than among never-smokers. Among current/former smokers, ORRs by RECIST 1.1 were 25% (95% CI, NR) and 26% (95% CI, 19–35) with MPDL3280A and pembrolizumab, respectively. Among never-smokers, ORRs were 16% (95% CI, NR) and 8% (95% CI, 3–18) with MPDL3280A and pembrolizumab, respectively. It has been hypothesized that because lung cancers in smokers are associated with more genetic mutations, they generate new tumor-associated or tumor-specific antigens, which are rendered more susceptible to recognition by immune cells. Thus, when an immune checkpoint such as PD-L1 is blocked, these patients’ immune cells may be more likely to respond and induce immune-mediated tumor cell killing.

Several combination strategies are being tested in large, multicohort phase I trials. Nivolumab, pembrolizumab, MEDI-4736, and MPDL3280A are being evaluated in combination with chemotherapies, oral epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, anti–CTLA-4 antibodies, or other targeted therapies.

Nivolumab and ipilimumab in combination were tested in chemotherapy-naive advanced NSCLC in the ongoing CA209-012 study.[37] At the time of data analysis, 8 of 29 patients (16%) had objective responses. Six of these patients (75%) had ongoing responses at the time of analysis. Most of the responses were seen by the time of the first tumor assessment (10 weeks). In an exploratory analysis based on PD-L1 status, responses were seen in both PD-L1–positive and PD-L1–negative patients, with ORRs of 19% (3/16) and 14% (3/22), respectively.

The CA209-012 study also tested nivolumab in combination with various platinum-doublet chemotherapy regimens, including gemcitabine/cisplatin, pemetrexed/cisplatin, and paclitaxel/carboplatin. The safety data showed an adverse event profile reflecting the additive toxicities of nivolumab and chemotherapy. No dose-limiting toxicities were seen during the first 6 weeks of treatment.[38] ORRs were 33% (95% CI, 10–65) to 47% (95% CI, 21–73) across all arms. One-year OS rates were 50% (95% CI, 21–74) to 87% (95% CI, 56–96).

Nivolumab plus erlotinib was tested in 21 patients with chemotherapy-naive, 

-mutant, advanced NSCLC.[39] All except one patient had acquired resistance to the EGFR tyrosine kinase inhibitor. The overall ORR was 19% (95% CI, 5.4–41.9). Of 20 patients who had erlotinib resistance, 3 (15%) achieved a partial response. One erlotinib-naive patient achieved a near CR, and all responses were durable beyond 78+ weeks. Nine patients (45%) had stable disease. The most common treatment-related adverse events were rash, fatigue, paronychia, diarrhea, and skin fissures. No pneumonitis of any grade was observed.

Bladder cancer is another disease primed for PD-1 and PD-L1 blockade. Bladder cancer has been known to be an immune-responsive disease since the first use of bacillus Calmette-GuÃ©rin immunotherapy several decades ago.[40] Mullane et al reported PD-L1 expression status on 160 bladder tumor samples and found PD-L1 expression on mononuclear cells (37%) and tumor cells (20%).[41] Only PD-L1 expression on mononuclear cells was significantly associated with longer OS in patients who developed metastatic disease (

 = .04). Sharma et al showed that the presence of CD8+ tumor-infiltrating lymphocytes in muscle-invasive bladder cancer was associated with longer disease-free survival (median, 13 months vs > 80 months; 

 < .001).[42] Furthermore, like NSCLC and MEL, bladder cancer has a high rate of somatic mutations,[43] which can potentially make it more susceptible to immune cell recognition and attack when the immune checkpoint is blocked.

The first clinical data were reported from a phase I study of MPDL3280A in 67 patients with metastatic urothelial bladder cancer.[44] Approximately 80% of the patients had received cisplatin-based therapy, and 75% had visceral metastases. The overall ORR was 26%. The ORRs in patients with PD-L1–positive and PD-L1–negative status were 43% (95% CI, 26–63) and 11% (95% CI, 4–26), respectively. Median time to first response was 42 days (range, 38 to 85 days). Median duration of response was not reached. Sixteen of 17 responses were ongoing at the time of data cutoff. Based on these data, a single-arm phase II trial of MPDL3280A in patients with metastatic urothelial bladder cancer is enrolling patients into two cohorts: treatment-naive/cisplatin-ineligible and platinum-pretreated. The primary outcome measure is ORR (NCT02108652). Preliminary data from the phase Ib study of pembrolizumab were presented at the 2014 European Society for Medical Oncology Congress. Plimack et al reported that 7 of 33 (24%) patients achieved an objective response. Three patients (10%) achieved a CR.[45] A randomized phase III study is underway comparing pembrolizumab against standard second-line chemotherapies (docetaxel, paclitaxel, or vinflunine), with OS as a primary endpoint (NCT02256436). Other ongoing phase I trials of MEDI4736 and of nivolumab with or without ipilimumab are enrolling patients with urothelial tract cancer (NCT01693562, NCT01928394).

Squamous Cell Carcinoma of the Head and Neck

Head and neck squamous cell carcinoma (HNSCC) is another tumor well suited for PD-1 and PD-L1 blockade. Similar to lung and bladder cancers, it carries high mutation rates.[43] PD-L1 expression has been well documented, and the PD-1 pathway has been implicated in immune resistance in human papillomavirus (HPV)-associated HNSCC.[46] A gene expression signature study has shown that about one-third to one-half of the examined HNSCC samples had an immune cell phenotype similar to that seen in MEL. PD-L1 expression and the presence of tumor-infiltrating lymphocytes were strongly associated with the mesenchymal phenotype of HNSCC, suggesting a potential benefit for PD-1 blockade.[47] Indeed, several PD-1 and PD-L1 antibodies have induced impressive objective responses. Preliminary data on the antitumor activity of pembrolizumab from the HNSCC dose expansion cohort of the phase Ib KEYNOTE-012 trial were recently presented.[48] Patients had to have at least 1% PD-L1 expression in their tumor samples to participate. Sixty eligible patients were treated with pembrolizumab. At the time of data cutoff in May 2014, the best overall response rate was 19.6% (95% CI, 10.2–32.4). Both HPV-positive and HPV-negative patients responded in equal percentages. One patient had pseudoprogression followed by partial response on treatment.

MEDI4736 has also demonstrated activity in metastatic HNSCC. At 10 mg/kg every 2 weeks, it showed an overall ORR of 14% (3/22), including 2 of 4 PD-L1–positive and 1 of 16 (6%) PD-L1–negative patients.[19]There was impressive shrinkage of a fungating mass of metastatic HNSCC following two doses of MEDI4736 in a 96-year-old woman. Similarly, Herbst et al reported on a patient with metastatic HNSCC with inflamed cutaneous lesions over the chest wall who had remarkable tumor regression after the first infusion and over the following course of MPDL3280A.[49]

A phase III trial of pembrolizumab vs standard of care as second-line therapy is underway for metastatic recurrent HNSCC (NCT02252042).

Metastatic Colorectal Cancer and Gastric Carcinoma

An early report of a CR involved a patient with advanced metastatic CRC.[50,51] The patient was a 71-year-old man with microsatellite instability (MSI)-high metastatic CRC (mCRC), who developed metastatic disease 1 year after completion of adjuvant chemotherapy and who had received multiple lines of chemotherapies. He received a single dose of an anti–PD-1 antibody at 3 mg/kg in mid-2007. His first two restaging scans, obtained at 8 and 12 weeks after the dose, confirmed a partial response. He subsequently received additional doses, up to 5 over the following 9 months, and achieved a CR at 6 months. IHC studies of his primary tumor showed PD-L1 expression by infiltrating immune cells and by tumor cells, associated with infiltrating PD-1–positive, CD3+ T cells. Based on this observation and others, it has been hypothesized that MSI-high tumors with a high propensity for mutation would generate more neoantigens and make them more susceptible to immune recognition and attack. Llosa et al evaluated the immune tumor microenvironment of MSI-high and microsatellite-stable (MSS) CRC tumor samples.[52] The MSI-high specimens (n = 8) were characterized by higher infiltration of CD3+, CD4+, and CD8+ cells vs MSS samples (n = 7). Furthermore, the tumor microenvironment of MSI-high CRC was characterized by a high expression of IFN-gamma and genes associated with cytotoxic T cells, as well as greater expression of IFN-gamma–driven immune checkpoints, such as PD-1 and PD-L1.

Gatalica et al[53] reported that MSI-high tumors were significantly more frequently infiltrated by PD-1–positive intraepithelial lymphocytes than MSS tumors (72% vs 39%). Similarly, PD-L1–positive tumor cells were more common in MSI-high than in MSS samples (56% vs 21%; 

 = .007). Concurrent PD-1–positive intraepithelial lymphocytes and PD-L1 tumor cells were seen more frequently in MSI-high vs MSS samples (30% vs 5%; 

While few official data are available in this disease group, MPDL3280A reportedly has shown confirmed responses in mCRC and gastric carcinoma.[54]

Currently, a phase II trial of nivolumab vs nivolumab plus ipilimumab is recruiting patients with recurrent and metastatic MSI-high colon cancer (NCT02060188), with a primary outcome measure of ORR. Another ongoing phase I study of nivolumab with or without ipilimumab is evaluating safety and antitumor activity in patients with pancreatic or gastric cancers (CA209-032).

PD-1/PD-L1 pathway inhibitors have a high potential to impact cancer therapy. Although antitumor activity was seen more frequently in patients with positive PD-L1 expression status, activity in patients with unknown or “negative” status in multiple tumor types should not be discounted. There may be limitations to the use of PD-L1 as a predictive biomarker, for reasons that include the dynamic nature of the immune response and the change in immunologic phenotype over the course of a therapy.[55] Powderly et al demonstrated that PD-L1 expression is an adaptive change, and its expression status can change over time with anti–PD-L1 antibody therapy.[55] Thus, expression status determined from an archival tumor tissue may not reflect the tumor’s present immunologic phenotype. Another facet of this complex immune-mediated cytotoxicity is the role of tumor-infiltrating immune cells. At the end of the day, it is the immune cells in the tumor microenvironment that mediate cytotoxicity. Given the available data regarding PD-L1 status as a biomarker, clinical investigators have taken different approaches in selecting patients for larger phase II/III trials. For instance, whereas the pivotal trials of nivolumab (CheckMate 026) and pembrolizumab (KEYNOTE-024) for patients with untreated advanced NSCLC are enriched for patients with PD-L1–positive status, the phase III nivolumab trial in a second-line setting accepts all eligible patients regardless of PD-L1 status but randomizes them with their expression status as a stratification factor (CheckMate 057 and 017) (Table 7).

PD-1-targeted antibodies and autoimmune toxicities

Immune checkpoint–targeted agents have given rise to a new spectrum of toxicity for clinicians to manage. Because of the role of the PD-L1/2–PD-1 axis in modulating the immune response under normal conditions, adverse events with potential immune-related causes would be expected. In the first published phase I report of nivolumab, drug-related adverse events of special interest (those with potential immune-related causes) were observed in 122/269 patients (44%), 18 (6%) > grade 3.[7] These events included pneumonitis, vitiligo, colitis, hepatitis, hypophysitis, and thyroiditis. Hepatic or gastrointestinal adverse events, predominantly diarrhea, were managed with treatment interruption and administration of glucocorticoids. These events were all reversible. Endocrine disorders were managed with replacement therapy. Drug-related pneumonitis occurred in nine patients (3%) and was grade 3/4 in three (1%). No clear relationship between the occurrence of pneumonitis and tumor type, dose level, or number of doses was noted. Early-grade pneumonitis in six patients was reversible with treatment discontinuation, glucocorticoid administration, or both. In three patients with pneumonitis, infliximab, mycophenolate, or both were used for additional immunosuppression. Treatment with an anti–PD-1 antibody was reinitiated once the adverse event had been successfully managed, if the treating physician concurred. There were three drug-related deaths (1%) due to pneumonitis (two patients with NSCLC and one patient with CRC). Brahmer et al observed possible immune-related events of interest in 81 of 207 patients (39%); these included rash, hypothyroidism, hepatitis, and one case each of sarcoidosis, endophthalmitis, diabetes mellitus, and myasthenia gravis in a trial of BMS936559 (anti-PD-L1 antibody).[17] These events were predominantly grade 1/2 and were managed with treatment interruption or discontinuation.

Although anti–PD-1/PD-L1 antibodies have shown promising activity, benefits are limited to a fraction of patients. In other words, a significant percentage of patients have primary resistance to immune-checkpoint inhibitors.[2,56,57] Even after responses are achieved, a patient’s tumors will eventually acquire a resistance mechanism that will make it possible to re-escape the immune attack.

An observation of two major phenotypes of MEL metastases, “inflamed” and “noninflamed,” provides a good conceptual framework for understanding immune escape mechanisms. Inflamed tumors are characterized by tumor-infiltrating immune cells; noninflamed tumors are characterized by the lack thereof. While the tumor-infiltrating cytotoxic T lymphocyte is the key player in mediating cytotoxicity, in a tumor microenvironment there are multiple potential barriers to T-cell infiltration. These include, among others: (1) immune-suppressive cytokines (VEGF, interleukin [IL]-6, IL-8, etc), and (2) immune-suppressive cells (regulatory T cells, myeloid-derived suppressive cells, and tumor-associated macrophages). High levels of VEGF have been shown to delay dendritic cell migration and maturation, as well as adaptive immune response.[30] Additionally, the tumor cells themselves may also downregulate antigen presentation or costimulatory molecules, or upregulate additional immune checkpoint molecules to turn off activated T cells.

A biomarker biopsy study of the ongoing phase I study of MPDL3280A indicated that patients who responded to therapy had tumor-infiltrating lymphocytes and tumoral PD-L1 expression at baseline.[55] By contrast, in a PD-L1–negative patient not responding to therapy, very few CD8+ T cells were noted at the tumor periphery at baseline. Following therapy, there was minimal infiltration of T cells, and a gene expression study showed lack of cytotoxic T-cell marker expression.

Similar observations have been reported with anti–CTLA-4 antibodies,[58,59] implying that noninflamed tumors may have a greater tendency to be resistant to this class of therapy. Several combination approaches are currently under investigation to overcome this mechanism of resistance. A preclinical study demonstrated that PD-1 and CTLA-4 combination blockade expanded infiltrating cytotoxic T cells and reduced immune-suppressive cells.[20] This finding serves as a rationale for many ongoing clinical investigations. Dual blockade of CTLA-4 and PD-1 has resulted in dramatic clinical responses in MEL patients,[60] as well as in the other tumor types we have reviewed here.

Several other combination approaches using immune-modulatory agents, including IFN-alfa, IL-2, antiangiogenic agents, and tyrosine kinase inhibitors, are underway. Among these agents, colony-stimulating factor 1 receptor (CSF1R) inhibitor is of interest. Colony-stimulating factor is a chemokine that recruits tumor-infiltrating myeloid cells, such as M2 macrophages and myeloid-derived suppressor cells. In a preclinical study using a syngeneic mouse model of 

-driven MEL, inhibition of CSF1/CSF1R led to effective adoptive T-cell transfer by inhibiting intratumoral accumulation of immunosuppressive macrophages.[61]

Another way to overcome this immune suppression is by adding an immune “accelerator,” such as immune-stimulatory agonistic immunoglobulins; OX40 (CD134), GITR, 4-1BB (CD137), and CD40 are in clinical development as monotherapies and in combination with immune checkpoint inhibitors.[62] As an example, OX40 is a potent T-cell costimulatory receptor.[63] OX40 ligands are expressed on antigen-presenting cells. The engagement of OX40 by its ligand leads to enhanced T-cell survival, proliferation, effector function, and cytokine release, and inhibits regulatory T-cell function. An anti-OX40 monoclonal antibody (9B12) induced tumor regression in patients with MEL in a phase I study.[64]

Cancer immunotherapy is entering a renaissance. Anti–PD-1/PD-L1 antibodies, in particular, have shown early but high promise in multiple tumor types. However, meticulous artistry in clinical trial design is essential to carve out the clinical benefits for carefully selected patients. The clinical benefits of this class of therapy may be expanded to a broader range of patients when “immunomodulatory” agents are shrewdly selected for combination studies. When this class of therapy becomes available to community oncologists, an algorithm will be implemented for treatment of immune-related adverse events. More importantly, heightened clinical acumen will be mandatory not only for detection of early signs of immune-mediated adverse events, but also for judicious use of corticosteroids to maximize clinical benefits.

The authors have no significant financial interest in or other relationship with the manufacturer of any product or provider of any service mentioned in this article.

 Keir ME, Butte MJ, Freeman GJ, Sharpe AH. PD-1 and its ligands in tolerance and immunity. Annu Rev Immunol. 2008;26:677-704.

 Taube JM, Anders RA, Young GD, et al. Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape. Sci Transl Med. 2012;4:127ra37.

 Taube JM, Klein A, Brahmer JR, et al. Association of PD-1, PD-1 ligands, and other features of the tumor immune microenvironment with response to anti-PD-1 therapy. Clin Cancer Res. 2014;20:5064-74.

 Schalper KA, Velcheti V, Carvajal D, et al. In situ tumor PD-L1 mRNA expression is associated with increased TILs and better outcome in breast carcinomas. Clin Cancer Res. 2014;20:2773-82.

 Velcheti V, Schalper KA, Carvajal DE, et al. Programmed death ligand-1 expression in non-small cell lung cancer. Lab Invest. 2014;94:107-16.

 Mahoney KM, Atkins MB. Prognostic and predictive markers for new immunotherapy. Oncology (Williston Park). 2014;28(suppl 3):39-48.

 Topalian SL, Hodi FS, Brahmer JR, et al. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med. 2012;366:2443-54.

 Nishimura H, Honjo T. PD-1: an inhibitory immunoreceptor involved in peripheral tolerance. Trends Immunol. 2001;22:265-8.

 Hodi FS, Sznol M, Kluger HM, et al. Long-term survival of ipilimumab-naive patients (pts) with advanced melanoma (MEL) treated with nivolumab (anti-PD-1, BMS-936558, ONO-4538) in a phase I trial. J Clin Oncol. 2014;32(suppl 5S):abstr 9002.

 Topalian SL, Sznol M, McDermott DF, et al. Survival, durable tumor remission, and long-term safety in patients with advanced melanoma receiving nivolumab. J Clin Oncol. 2014;32:1020-30.

 Sznol M, Kluger HM, Callahan MK, et al. Survival, response duration, and activity by BRAF mutation (MT) status of nivolumab (NIVO, anti-PD-1, BMS-936558, ONO-4538) and ipilimumab (IPI) concurrent therapy in advanced melanoma (MEL). J Clin Oncol. 2014;32(suppl 5S):abstr LBA9003.

 Ribas A, Hodi FS, Kefford R, et al. Efficacy and safety of the anti-PD-1 monoclonal antibody MK-3475 in 411 patients (pts) with melanoma (MEL). J Clin Oncol. 2014;32(suppl 5S):abstr LBA9000.

 Joseph RW, Elassaiss-Schaap J, Wolchok JD, et al. Baseline tumor size as an independent prognostic factor for overall survival in patients with metastatic melanoma treated with the anti-PD-1 monoclonal antibody MK-3475. J Clin Oncol. 2014;32(suppl 5S):abstr 3015.

 Hamid O, Robert C, Ribas A, et al. Randomized comparison of two doses of the anti-PD-1 monoclonal antibody MK-3475 for ipilimumab-refractory (IPI-R) and IPI-naive (IPI-N) melanoma (MEL). J Clin Oncol. 2014;32(suppl 5S):abstr 3000.

 Atkins MB, Kudchadkar RR, Sznol M, et al. Phase 2, multicenter, safety and efficacy study of pidilizumab in patients with metastatic melanoma. J Clin Oncol. 2014;32(suppl 5S):abstr 9001.

 Hamid O, Sosman JA, Lawrence DP, et al. Clinical activity, safety, and biomarkers of MPDL3280A, an engineered PD-L1 antibody in patients with locally advanced or metastatic melanoma (mM). J Clin Oncol. 2013;31(suppl):abstr 9010.

Articles

In this review, we will discuss the current status of several anti–PD-1 and anti–PD-L1 antibodies in clinical development and their direction for the future.

Prospects for Targeting PD-1 and PD-L1 in Various Tumor Types 

Blocking the programmed cell death 1 (PD-1) pathway with monoclonal antibodies has shown promising antitumor responses in clinical trials, with less toxicity than has been seen with prior immune therapies such as interleukin 2 and ipilimumab. Pembrolizumab, an anti–PD-1 antibody, recently gained US Food and Drug Administration (FDA) accelerated approval for the treatment of patients with ipilimumab-refractory melanoma, while nivolumab, another anti–PD-1 antibody, and MPDL3280A, an anti–programmed cell death 1 ligand (PD-L1) antibody, have been granted FDA “breakthrough designation” for treatment of subsets of patients with refractory Hodgkin lymphoma and metastatic bladder cancer, respectively. Encouraging antitumor activity has also been seen with these agents in patients with other malignancies, including non–small-cell lung cancer and head and neck cancer, tumors not previously thought to be immune-responsive. PD-L1 expression has emerged as a potential predictive biomarker for PD-1–directed therapy. Multiple, distinct, companion assays for PD-L1 positivity have been developed, but there is as yet no comparison, standardization, or prospective validation of these assays. PD-L1 expression on tumor cells and/or the tumor-immune infiltrate is likely only part of the predictive model necessary for selecting patients predisposed to respond to monotherapy. Additional predictive biomarkers are necessary to identify patients most likely to benefit from PD-1–based combination therapy, since tumor cell PD-L1 expression appears to have limited predictive value in this setting.

Historically, active immune therapies such as interleukin 2 (IL-2) produced durable responses in only a small minority of patients while being associated with significant, nearly universal toxicity. This has limited their applicability to selected patients, usually with either melanoma or renal cell cancer (RCC) treated at experienced centers. The immune checkpoint inhibitor ipilimumab, while being better tolerated than high-dose (HD) IL-2, also exposes patients to a risk of significant toxicity. Therefore, there has been considerable effort over the past decade to identify patients most likely to benefit from these agents in order to enhance their therapeutic index. The discovery and availability of alternative treatment options for patients with advanced melanoma (BRAF inhibitor therapy) and RCC (vascular endothelial growth factor [VEGF]-targeted therapy) in the last decade have intensified the need to identify those patients who will not benefit from immunotherapy, so as not to delay the use of these more generally accessible alternative therapies, particularly in symptomatic patients. Programmed cell death 1 (PD-1) pathway–blocking antibodies appear to exhibit an improved therapeutic index, relative to HD IL-2 and ipilimumab, and efficacy against an expanded array of tumor types (eg, non–small-cell lung cancer (NSCLC), bladder cancer, head and neck cancer), each of which has a distinct array of standard treatments. This has further highlighted the need for clinically useful biomarkers that can help determine how best to incorporate these new agents into treatment algorithms for patients with specific diseases. Finally, identifying factors that predict the subpopulations of patients and tumor types that respond to immunotherapy is critical to both understanding the mechanism of action of immunotherapy and the efficient development of novel immunotherapy combinations.

A biomarker is a biologic molecule, such as a protein or gene, that is measureable in tissue, blood, or other body fluids, and is an indicator of some clinically significant condition. Biomarkers can be diagnostic, surrogate, prognostic, or predictive. While the gold standard of diagnosis in oncology is a pathologic tissue review, a highly elevated prostate-specific antigen (PSA) level in the right clinical setting can be diagnostic of prostate cancer. Although the value of PSA level as a diagnostic biomarker is limited by its sensitivity and specificity, it can be an excellent surrogate biomarker for monitoring prostate cancer response to treatment. Prognostic biomarkers refer to markers that correlate with the natural progression or aggressiveness of a disease. Prognostic biomarkers are useful for informing patients about the risk of recurrence or median survival for their particular type of malignancy and for minimizing confounding factors when analyzing clinical trial cohorts or when prospectively stratifying patients in randomized clinical trials. Predictive biomarkers are defined by their role in predicting a response to a given treatment. Therefore, these are most useful if they can be assessed before the initiation of treatment. This review will focus on predictive biomarkers, as these are most relevant to decision making regarding immunotherapy selection; we will only mention prognostic or surrogate markers to the extent that such discussion sheds potential light on the mechanisms underlying the value of a predictive biomarker.

The immune system is controlled by a delicate balance of immune-stimulatory and immune-inhibitory forces that enable the rapid destruction of cells expressing foreign antigens while preventing uncontrolled destruction of normal tissues. The inhibitory forces include regulatory T cells (Tregs), immunosuppressive cytokines, and immune checkpoints, such as the cytotoxic T-lymphocyte antigen 4 (CTLA-4)/B7 and the PD-1/programmed cell death 1 ligand (PD-L1) pathways. For example, IL-2 not only activates CD8+ effector T cells and subsets of natural killer cells, but also CD4+CD25+ and CD4+Foxp3+ Tregs,[1] which constitutively express the IL-2 receptor alpha chain. Studies in viral models of acute and chronic infections shed light on this phenomenon. PD-1 expression is induced by activation of T lymphocytes, and after a successful immune response has eliminated the foreign antigen, PD-1 expression decreases.[2,3] If the immune response is unsuccessful in eliminating the antigen-expressing cells, as in cancer or chronic infections, prolonged antigen stimulation leads to elevated PD-1 expression and induction of the expression of immune-inhibitory ligands, such as PD-L1 or PD-L2, on tumor cells and infiltrating immune cells. The binding of PD-1–expressing T cells to PD-L1 or PD-L2 leads to T-cell dysfunction and “exhaustion,” and results in eventual escape from immune elimination. In addition to this model of adaptive immune inhibition or resistance, as illustrated in melanoma,[4] other tumors may evade immune surveillance by overexpression of PD-L1 secondary to genetic amplification[5] or as a result of aberrant signaling.[6]

Some tumors have an inflamed phenotype, while others have only a sparse immune infiltrate. While the extent of the immune infiltrate can be a good prognostic indicator in some cancers,[7,8] the antitumor response is clearly insufficient to prevent disease progression. In inflamed tumors, negative immune-regulatory factors tend to dominate due to the chronic nature of the immune infiltrate, as illustrated in Gajewski et al (Figure 1A).[9] Therefore, it is hypothesized that patients with tumors containing T-cell infiltrates might be induced to respond to immunotherapy if the immune cells within the tumor microenviroment can be reactivated.[10]

Predictive Biomarkers for Response to Cytokine Therapy-a Proof of Principle

IL-2 received US Food and Drug Administration (FDA) approval for the treatment of patients with RCC or melanoma in the 1990s based on its ability to produce durable antitumor responses in a small minority of patients, an effect that was largely restricted to the high-dose regimen (HD IL-2). While tumor responses were seen in only 15% to 25% of patients, the 6% to 10% of patients who exhibited tumor responses lasting more than 30 months were likely cured. In retrospective analyses of patients with advanced melanoma, IL-2 response has been associated with normal serum lactate dehydrogenase levels, or low plasma VEGF and fibronectin levels,[11] as well as with tumors containing mutations in 

. Prospective validation of these largely clinical features of response is ongoing. In patients with RCC, tumor response to IL-2 was largely restricted to patients with clear-cell tumors.[12,13] Further, while initial studies suggested that high levels of carbonic anhydrase 9 (CA9) expression by RCC cells predicted for response to IL-2, a prospective clinical trial failed to confirm this finding, supporting a modicum of caution regarding the value of inherent tumor features in predicting response to immunotherapy.

One of the more impressive surrogate markers for the benefit of cytokine therapy is induced or increased autoimmunity. IL-2 responses and the benefit of adjuvant interferon (IFN) appeared more common in patients who exhibited autoimmune phenomena such as thyroiditis or vitiligo. These entities frequently were accompanied by enhanced titers of autoantibodies, suggesting that responses were both autoimmune in nature and more common in patients who were predisposed to develop autoimmunity.[14,15] More recent studies have suggested that response to IL-2 is associated with enhancement of a preexisting gene expression pattern within the tumor that is associated with immune-mediated, tissue-specific destruction under the control of the T helper 1 (Th1)-type cytokine, IFN-Î³.[16] Benefit from vaccination has also been linked to tumors expressing an IFN-driven chemokine signature.[9,17] In addition, preliminary results suggest that IL-2 response in patients with RCC may be correlated with tumor cell surface expression of PD-L1,[18] which, as mentioned previously, is frequently associated spatially with tumor-reactive CD8+ T cells in the microenvironment.[4] Taken together, these data suggest that effective immunotherapy may require the preexistence of tumor-specific immunity within the microenvironment and the use of agents, such as HD IL-2 or IFN, in a subset of patients in whom these agents drive effector T-cell function more potently than immune-regulatory signals. While prospective validation of this hypothesis is necessary, it suggests that approaches that could block immunoregulatory signals might also alter the balance within the tumor microenvironment in such predisposed patients, leading to a shift from immune tolerance of a tumor to an active antitumor immune response. This concept has foreshadowed the effectiveness of checkpoint blockade and informed the predictive biomarker work with such agents.

Clinical Results of Checkpoint Blockade-Efficacy and Toxicity

CTLA-4 was the first of the immune checkpoints to be therapeutically targeted in cancer. Ipilimumab, a fully human immunoglobulin (Ig) G-1 monoclonal antibody blocking CTLA-4, improved overall survival in patients with metastatic melanoma and received FDA approval in 2011.[19,20] As with HD IL-2, the great majority of responders alive at 2 years were still alive long after therapy was completed. Although acute side effects from ipilimumab administration were minimal, patients did sometimes develop immune-related adverse events weeks into therapy that required aggressive immunosuppression in order to control.[21] Similar to the autoimmunity described with IL-2 therapy, baseline presence and induction of NY-ESO-1 antibodies have been associated with clinical benefit from ipilimumab.[22] Specific ipilimumab-associated, immune-mediated adverse effects, such as hypophysitis, have been associated with the development of autoantibodies targeting the affected organ.[23]

PD-1 and its ligands, PD-L1 and PD-L2, are also members of the B7-CD28 superfamily and form an immune-checkpoint pathway. Blocking the binding site of the PD-1 receptor and the ligand PD-L1 with monoclonal antibodies also has shown impressive antitumor effects in patients in multiple tumor types,[24-26] including those not previously thought to be immune-responsive. The PD-1/PD-L1 pathway appears to induce peripheral tolerance of the adaptive immune system.[27] Unlike in CTLA-4 knockout mice, which exhibit lethal autoimmunity within weeks of birth,[28] mice in which PD-1 or PD-L1 is knocked out require some additional initiator to develop an autoimmune phenotype.[27,29-31] Clinically, blockade of the PD-1/PD-L1 pathway appears to produce fewer immune-related adverse events than ipilimumab. This improved side-effect profile and broad span of antitumor efficacy have resulted in extremely active drug development in recent years. Pembrolizumab is the first of this anti–PD-1 pathway family of checkpoint inhibitors to gain FDA–
accelerated approval for the treatment of ipilimumab-refractory melanoma. The phase III clinical trial comparing dacarbazine with nivolumab, an anti–PD-1 monoclonal antibody, recently was stopped early due to improved overall survival for the patients who received anti–PD-1 therapy, and nivolumab has already been approved in Japan. In the United States, nivolumab has received breakthrough status for Hodgkin lymphoma. MPDL3280A, an anti–PD-L1 monoclonal antibody, has received breakthrough status for the treatment of bladder cancer. Development of PD-1/PD-L1 pathway–blocking agents in many other tumors is underway, given the responses seen in early trials.

Initial single-arm trials of combination checkpoint blockade (ipilimumab and nivolumab) have shown potential to significantly improve response rates.[32,33] However, they also appear to increase the number of grade 3 and 4 side effects. The ipilimumab + nivolumab combination is currently being further investigated, including in phase III trials in which the efficacy and toxicity of the combination can be compared with either nivolumab monotherapy or standard of care. While the increased antitumor efficacy of combination immune-modulatory therapies is encouraging, such combinations may not only significantly increase the toxicity but also the cost of therapy. Thus, the development of predictive biomarkers for the routing of patients to the most appropriate therapies is essential to maximize clinical benefit and minimize toxicity. Predictive biomarkers may also facilitate use of checkpoint inhibitors in patients with high-risk resected disease, since a percentage of patients treated in the adjuvant setting may already be cured, and thus the toxicity and treatment expense may be harder to justify.

Clearer insight into the biology of a therapeutic target facilitates its clinical development. Therefore, it may prove easier to define predictive biomarkers for individual immune-checkpoint inhibitors than was the case for pleiotropic cytokines such as IL-2 or IFNs. Despite the role of autoimmunity as a surrogate for response to cytokine therapy, its role in CTLA-4 and PD-1 immune-checkpoint pathway–blocking therapy is less clear. Both CTLA-4 and PD-1/PD-L1 play important roles in regulating T-lymphocyte tolerance, and both have been implicated in human autoimmune disease.[34] In addition, both ipilimumab and, to a lesser extent, PD-1–pathway inhibitors induce immune-related toxicities that appear to be autoimmune in nature. The relationship of these toxicities to tumor response with these agents is unclear, and because trials to date have generally excluded patients with a history of autoimmune disease, the extent to which a predisposition to autoimmunity predicts for antitumor activity remains to be established. Studies are actively investigating not only predictive biomarkers for efficacy but also for toxicity. Moving forward, a patient’s germline genetics also may play a critical role in predicting the clinical effects of various immune therapies, as has already been proposed in the case of ipilimumab.[35,36]

While pretreatment gene expression profiling of tumors has suggested that those with high baseline expression levels of immune-related genes are more likely to respond to ipilimumab,[37] few biomarkers in ipilimumab studies have been truly predictive in nature. For example, the appearance of ICOS+CD4+ T lymphocytes after ipilimumab treatment has been proposed as a pharmacodynamic biomarker that can be used to assess adequate biologic response to treatment.[38] While early reports suggested that an absolute lymphocyte count > 1,000/Î¼L after two doses of ipilimumab[39] correlated with clinical benefit and overall survival,[39] Postow et al reported that nearly all patients who received ipilimumab had a significant increase in their absolute lymphocyte count, potentially mitigating the value of this observation.[40] In addition, investigators have reported that absence of tumor-infiltrating lymphocytes at 4 weeks into therapy or an increase in myeloid-derived suppressor cell (MDSC) numbers in the peripheral blood over the first 24 weeks were both negatively associated with clinical benefit from ipilimumab therapy.[41] In any event, since these studies measured pharmacodynamic markers of treatment effect, they do not inform the choice of patients for ipilimumab therapy.[38] Of note, in a small cohort of 26 patients receiving ipilimumab at 10 mg/kg, a lower number of MDSCs in the baseline peripheral blood was predictive of overall survival.[41] However, much like the negative association of pretreatment serum VEGF level with clinical benefit and overall survival after ipilimumab,[42] baseline MDSC count and serum VEGF level may be functioning as prognostic rather than predictive markers.[37]

Some uncertainty about the mechanism of action of ipilimumab in the tumor microenvironment may be confounding the ability to develop predictive biomarkers of benefit. Given that tumor-infiltrating lymphocytes likely express many immune checkpoints and are already deactivated, it has been postulated that the mechanism of action of ipilimumab in tumors is to target Tregs, as suggested by mouse studies.[43] Thus, ipilimumab’s primary antitumor effect may not be unblocking T-cell activation at initial tumor-antigen presentation but targeting Tregs for destruction within the tumor microenvironment. In support of this concept, Hamid et al reported that elevated Foxp3 and IDO expression in baseline tumor biopsies was associated with clinical benefit from ipilimumab.[44] This and other reports suggest that, as with IL-2, tumors with an inflamed microenvironment may respond better to ipilimumab, since the presence of immunosuppressive factors such as Tregs may not be as much of an obstacle to its efficacy.[10]

Biomarkers for the PD-1/PD-L1–Pathway Inhibitors

PD-L1 expression in tumors is one of a number of different immunosuppressive factors proposed in the Gajewski model (Figure 1) of chronically inflamed tumors with an immunosuppressed microenvironment. In preclinical models, expression of PD-L1 allows tumors to evade tumor-specific effector T-cell cytotoxicity.[45] Clinically, PD-L1 is expressed by many tumor types and is associated with worse prognosis in several, including lung adenocarcinoma and RCC.[46,47] PD-L1 expression is IFN-Î³–inducible and can be on either the immune infiltrate or the tumor cells (Figure 2). Patients whose kidney tumors had ≥ 10% tumor cell expression with the murine anti–human PD-L1 antibody (clone 5H1) had a threefold increased risk of dying of their disease. By contrast, PD-L1 expression on some tumors, most notably melanoma, appears to be associated with a good prognosis.[48] The mechanisms underlying this prognostic diversity and the relative importance of PD-L1 expression in various cell populations within the tumor have yet to be fully elucidated. Nonetheless, the critical role played by PD-L1 expression in tumor immunobiology has clearly identified this pathway as an important target for checkpoint-inhibitor therapy.

Early clinical trials with PD-1 pathway–blocking agents have produced exciting clinical responses in multiple tumor types, including some previously believed to be unresponsive to systemic immunotherapy (Figure 3).[49] Tumor PD-L1 expression was initially explored as a potential predictive biomarker in the single-dose pilot phase I study of nivolumab.[24] This exploratory analysis consisted of tumor specimens from nine patients. PD-L1 expression on tumors by immunohistochemical analysis was performed with clone 5H1 mentioned above.[50] While three of four patients with PD-L1 expression on the tumor cell surface responded to nivolumab (75% response rate), all of the five tumors that did not express any membranous PD-L1 did not respond to treatment. In a follow-up study performed in conjunction with the multidose, phase I nivolumab trial, 9 of 25 patients with PD-L1–expressing tumors responded to treatment, while none of 18 patients with PD-L1–nonexpressing tumors responded (Figure 3). These preliminary data suggest that tumor cell expression of PD-L1, using the clone 5H1 assay, could be a strong predictor of response to nivolumab treatment.

Unfortunately, several subsequent observations have suggested that the predictive value of PD-L1 expression may not be as clear-cut as initially reported. As previously mentioned, PD-L1 could also be expressed on infiltrating lymphocytes, monocytes, and macrophages (Figure 2).[47] Taube et al explored the predictive function of the expression of PD-L1 and PD-L2 on tumor cells and of PD-1 on the immune infiltrate.[51] They found that the expression of PD-L1 on tumor cells and immune cells was highly associated with PD-1 expression on infiltrating lymphocytes-yet PD-L1 expression on tumor cells had the strongest association with response to nivolumab. Based on these data, Bristol-Meyers Squibb developed an automated assay (using the 28-8 clone) that defined > 5% tumor cell membranous expression of PD-L1 to be positive.[47,52,53] However, the other two companies (Genentech/Roche and Merck) leading in the development of PD-1–pathway blockers have developed distinct companion assays for PD-L1 expression, each with its own anti–PD-L1 antibody. The Genentech/Roche assay measures PD-L1 expression on immune-infiltrating cells, which investigators claim best enriches for responders to MPDL3280A.[54-57] This assay predicted for benefit from MPDL3280A in patients with bladder cancer, contributing to this antibody receiving breakthrough status for study in patients with PD-L1–positive metastatic bladder cancers. The Merck PD-L1 assay, developed in conjunction with pembrolizumab, defined PD-L1 positivity in melanoma specimens as tumor surface expression (> 1%).[58,59] However, variations in this assay and its application have emerged across tumor types. For example, in NSCLC patients, both immune and tumor cells were included in the cut-off (Figure 3, Gandh et al),[60] while in the head and neck cancer study, only patients with some PD-L1 expression on tumor cells were enrolled (Figure 3, Seiwert et al).[61] Since these assays have not been compared, it is impossible to ascertain whether these differences in definitions are a function of the assay (antibody specificity), of the biologic differences between anti–PD-1 and anti–PD-L1 therapy, or of the nature of the patient samples analyzed. Thus, it is hard to determine whether there is any consistency in the tumors that are declared to be PD-L1–positive. Clearly, some sort of standardized definition of PD-L1 positivity that links these various assays is necessary to facilitate the study of PD-L1 as a predictive biomarker for PD-1–pathway blockade. Lacking this, comparing clinical results across studies in biomarker-defined subsets of patients will be especially hazardous.

Further complicating matters, analyses with these different companion assays (such as an automated PD-L1 antibody assay used in conjunction with nivolumab) have suggested that, in contrast to earlier results, many patients with PD-L1 negative tumors can respond to PD-1–pathway blockade, including some who have exhibited a complete response (Figure 3).[49] Nonetheless, despite the significant number of false negatives, it is notable that across multiple tumor types, with four different therapies and four different assays for PD-L1 expression, there was a clear trend for PD-L1–positive tumors to respond better than PD-L1–negative tumors. In addition, the degree of PD-L1 expression, by whatever assay, may be positively correlated with response. Therefore, establishing a more stringent cut-off for PD-L1 expression would better enrich for responding patients but fail to identify a larger absolute number of potential responders, while a less stringent PD-L1 expression cut-off might capture more of the responding patients but reduce the predictive power of the assay. An example of this dynamic in patients with melanoma is shown in the Table.[52] Also, tumors are heterogeneous, and PD-L1 expression is inducible on both immune cells and tumor cells. Therefore, the sample used for the assay may not be representative of the whole tumor. Further, PD-L1 expression may be discordant between the primary tumor and the metastatic lesions that are being targeted by the immune therapy. For example, preliminary data suggest moderate discordance in RCC PD-L1 expression between primary and metastatic lesions. Whether this discordance has been a result of tissue preparation and storage issues or biologic differences is uncertain. However, it is perhaps notable that higher PD-L1 expression was associated with higher Fuhrman grade, a factor that was more variable in the primary than the metastatic lesions. Not surprisingly, therefore, the metastatic lesions were typically more homogeneous for PD-L1 expression than the primary tumors.[62] Finally, tumor PD-L1 expression may be less relevant as a predictive biomarker for combination immunotherapy. For example, the combination of ipilimumab and nivolumab actually produced similar response rates for PD-L1–positive and –negative tumors.[32,33] Further, tumor objective response rates for combinations of PD-1–blocking agents with non–immune-based therapies may also be less likely to be associated with PD-L1 expression.[63] For all of these reasons, PD-L1 expression is not an optimal biomarker for patient selection, and lack of PD-L1 expression cannot be reliably used to exclude patients from treatment with PD-1–pathway blockade.

Nonetheless, PD-L1 expression can be a useful predictive biomarker in several capacities. The data in Table 1 clearly suggest that PD-L1 expression, by whatever assay, can enrich for patients who can respond to single-agent PD-1–pathway blockade. Thus, PD-L1 expression could serve as a stratification factor in randomized trials comparing different types of therapies. Further, data suggest that tumor types lacking a consistent percentage of PD-L1 expression are less likely to benefit from single-agent PD-1–pathway blockade.[45]

Therefore, the extent of PD-L1 expression in a tumor type might encourage the inclusion of such tumors in clinical trials. Such an approach has predicted the responsiveness of head and neck cancer and bladder cancer,[56,61] and has fostered investigation of PD-1–pathway blockade in other highly PD-L1–positive tumors, such as Merkel cell carcinoma and Hodgkin lymphoma.[64,65]

Selecting a stringent cut-off for PD-L1 expression might identify a subset of patients with sufficiently high benefit from PD-1–pathway blockade to justify the exploration of single-agent treatment either compared with standard therapy in randomized trials or in single-arm trials in a setting where there is no curative treatment approach. Such strategies have been employed for the development of PD-1–pathway inhibitors in bladder cancer, head and neck cancer, and NSCLC.

Finally, a low level of PD-L1 expression might identify a group of patients who would be best treated with combination immunotherapy. While a prospective trial will be necessary to validate a role for PD-L1 as a predictive biomarker, the data in patients with metastatic melanoma suggest that this may be a reasonable criterion for selecting patients who should be treated with the ipilimumab/nivolumab regimen. Optimal agents to combine with a PD-1–pathway blocker might be those capable of inducing immune infiltration and PD-L1 expression within the tumor. However, given the heterogeneity of many tumors, particularly primary specimens, and technical issues related to tissue processing and storage, if PD-L1 expression is to be used for any of these purposes, it would seem prudent to analyze pretreatment biopsy specimens from metastatic lesions whenever possible.

In addition, understanding the biology of PD-L1 expression in tumors may contribute to the development of better predictive markers and models for assessing response to checkpoint inhibitor therapy. This may be particularly useful in determining why some patients with PD-L1–positive tumors fail to respond to therapy. In some cases this could be because the PD-L1 expression is constitutive, driven by activation of a pathway within the tumor (eg, PI3-kinase), rather than induced by an adaptive response to tumor-specific CD8+ lymphocyte infiltration. Indeed, if PD-L1 is overexpressed on a tumor lacking an appropriate immune infiltrate, blockade may have no effect because the tumor is lacking the effector cells that fight the cancer. Therefore, some measure of immune infiltration (whether assessment of immune phenotypes, as suggested by the Immunoscore research, or gene expression profiles linked to chemokine expression) together with PD-L1 expression might be a better predictor of tumor response.[7,66] Such a model for a dual biomarker has been proposed by Sznol et al (Figure 4).[67]

Alternatively, resistance to treatment in this population might suggest that other immune-suppressive factors may be downregulating the immune response. Understanding the factors involved in a particular tumor type might be the key to the development of novel PD-1/PD-L1–based combination therapies, such as those targeting additional immune checkpoints (eg, TIM-3 [T-cell immunoglobuilin and mucin domain–containing protein 3] or LAG-3 [lymphocyte activation gene 3 protein]) or other immunosuppressive factors (eg, Tregs, IDO, or MDSCs).

Assessing Potentially Immune-Responsive Tumors by Genomics

Mutational burden varies greatly between tumors. Many tumors that respond to PD-1–pathway inhibitors, such as melanoma, NSCLC, and bladder cancer, are those with a high mutation load.[68] While the mutational load in melanoma was not sufficient to predict response to ipilimumab, it was significantly associated with clinical benefit (

 = .028, complete response vs no response, Fisher’s exact test).[69] It is interesting that PD-1–pathway inhibitors also have shown responses in tumors-such as bladder cancer, NSCLC, and head and neck cancer-that are associated with smoking, a known exogenous mutagen. Assaying the neoantigens created within these tumors that might be presented by the patient’s major histocompatibility complex may better identify tumors able to induce immune responses that can be restored by immune-checkpoint inhibition.[70] Further, it is conceivable that the presence of such recognizable neoantigens may also one day form a component of a predictive biomarker model for response to checkpoint blockade.

Immune-checkpoint inhibition clearly has a significant role in the future of oncology. Multiple agents in this class have shown remarkable antitumor effects in a wide variety of clinical trials. While intratumoral PD-L1 expression appears to predict better responses to monotherapy with PD-1 pathway–blocking agents, it is unclear whether this will translate into a clinically useful predictive biomarker in isolation. Characterizing tumors by PD-L1 expression, immune infiltration, chemokine signature, and tumor mutational frequency may be a means of creating an integrated model for determining which patients may benefit from which immune-checkpoint inhibitors, either alone or in combination. Further, the identification of tumors lacking such biomarkers might prove useful in selecting patients for alternative therapies, including adoptive transfer of genetically modified T cells. Taken together, the identification and application of such markers are critical to the rational development and application of immune therapies.

Dr. Atkins serves as a consultant to Amgen, Bristol-Myers Squibb, Genentech/Roche, Merck, and NeoSTem. Dr. Mahoney has no significant financial interest in or other relationship with the manufacturer of any product or provider of any service mentioned in this article

 van der Vliet HJ, Koon HB, Yue SC, et al. Effects of the administration of high-dose interleukin-2 on immunoregulatory cell subsets in patients with advanced melanoma and renal cell cancer. Clin Cancer Res. 2007;13:2100-8.

 Vibhakar R, Juan G, Traganos F, et al. Activation-induced expression of human programmed death-1 gene in T-lymphocytes. Exp Cell Res. 1997;232:25-8.

 Zajac AJ, Blattman JN, Murali-Krishna K, et al. Viral immune evasion due to persistence of activated T cells without effector function. J Exp Med. 1998; 188:2205-13.

 Taube JM, Anders RA, Young GD, et al. Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape. Sci Transl Med. 2012;4:127-37.

 Green MR, Monti S, Rodig SJ, et al. Integrative analysis reveals selective 9p24.1 amplification, increased PD-1 ligand expression, and further induction via JAK2 in nodular sclerosing Hodgkin lymphoma and primary mediastinal large B-cell lymphoma. Blood. 2010;116:3268-77.

 Parsa AT, Waldron JS, Panner A, et al. Loss of tumor suppressor PTEN function increases B7-H1 expression and immunoresistance in glioma. Nat Med. 2007; 13:84-8.

 Ascierto PA, Capone M, Urba WJ, et al. The additional facet of Immunoscore: immunoprofiling as a possible predictive tool for cancer treatment. J Transl Med. 2013;11:54.

 Galon J, Mlecnik B, Bindea G, et al. Towards the introduction of the ‘Immunoscore’ in the classification of malignant tumours. J Pathol. 2014;232:199-209.

 Gajewski TF, Fuertes M, Spaapen R, et al. Molecular profiling to identify relevant immune resistance mechanisms in the tumor microenvironment. Curr Opin Immunol. 2011;23:286-92.

 Gajewski TF, Chesney J, Curriel TJ. Emerging strategies in regulatory T-cell immunotherapies. Clin Adv Hematol Oncol. 2009;7:1-10.

 Sabatino M, Kim-Schulze S, Panelli MC, et al. Serum vascular endothelial growth factor and fibronectin predict clinical response to high-dose interleukin-2 therapy. J Clin Oncol. 2009;27:2645-52.

 Cangiano T, Liao J, Naitoh J, et al. Sarcomatoid renal cell carcinoma: biologic behavior, prognosis, and response to combined surgical resection and immunotherapy. J Clin Oncol. 1999;17:523-8.

 Upton MP, Parker RA, Youmans A, et al. Histologic predictors of renal cell carcinoma response to interleukin-2-based therapy. J Immunother. 2005;28:488-95.

 Gogas H, Ioannovich J, Dafni U, et al. Prognostic significance of autoimmunity during treatment of melanoma with interferon. N Engl J Med. 2006;354: 709-18.

 Koon H, Atkins M. Autoimmunity and immunotherapy for cancer. N Engl J Med. 2006;354:758-60.

 Weiss GR, Grosh WW, Chianese-Bullock KA, et al. Molecular insights on the peripheral and intratumoral effects of systemic high-dose rIL-2 (aldesleukin) administration for the treatment of metastatic melanoma. Clin Cancer Res. 2011;17:7440-50.

 Ulloa-Montoya F, Louahed J, Dizier B, et al. Predictive gene signature in MAGE-A3 antigen-specific cancer immunotherapy. J Clin Oncol. 2013;31:2388-95.

 Bailey AS, Cheng S, Kwon ED, et al. PDL-1/PDL-3 (programmed death ligand-1/3) tissue expression and response to treatment with IL2 and antiangiogenic therapies. J Clin Oncol 2013;31(suppl):abstr 4521.

 Hodi FS, O’Day SJ, McDermott DF, et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med. 2010;363:711-23.

 Robert C, Thomas L, Bondarenko I, et al. Ipilimumab plus dacarbazine for previously untreated metastatic melanoma. N Engl J Med. 2011;364:2517-26.

 Weber JS. Practical management of immune-related adverse events from immune checkpoint protein antibodies for the oncologist. Am Soc Clin Oncol Educ Book. 2012;174-7.

 Yuan J, Adamow M, Ginsberg BA, et al. Integrated NY-ESO-1 antibody and CD8+ T-cell responses correlate with clinical benefit in advanced melanoma patients treated with ipilimumab. Proc Natl Acad Sci USA. 2011;108:16723-8.

 Iwama S, De Remigis A, Callahan MK, et al. Pituitary expression of CTLA-4 mediates hypophysitis secondary to administration of CTLA-4 blocking antibody. Sci Transl Med. 2014;6:230ra45.

 Brahmer JR, Drake CG, Wollner I, et al. Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors: safety, clinical activity, pharmacodynamics, and immunologic correlates. J Clin Oncol. 2010;28:3167-75.

 Brahmer JR, Tykodi SS, Chow LQ, et al. Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med. 2012;366:2455-65.

 Segal NH, Antonia SJ, Brahmer JR, et al. Preliminary data from a multi-arm expansion study of MEDI4736, an anti-PD-L1 antibody. J Clin Oncol. 2014;32(suppl 5S):abstr 3002.

 Wang J, Yoshida T, Nakaki F, et al. Establishment of NOD-Pdcd1-/- mice as an efficient animal model of type I diabetes. Proc Natl Acad Sci USA. 2005;102: 11823-8.

 Khattri R, Auger JA, Griffin MD, et al. Lymphoproliferative disorder in CTLA-4 knockout mice is characterized by CD28-regulated activation of Th2 responses. J Immunol. 1999;162:5784-91.

 Okazaki T, Tanaka Y, Nishio R, et al. Autoantibodies against cardiac troponin I are responsible for dilated cardiomyopathy in PD-1-deficient mice. Nat Med. 2003;9:1477-83.

 Nishimura H, Nose M, Hiai H, et al. Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor. Immunity. 1999;11:141-51.

 Kasagi S, Kawano S, Okazaki T, et al. Anti-programmed cell death 1 antibody reduces CD4+PD-1+ T cells and relieves the lupus-like nephritis of NZB/W F1 mice. J Immunol. 2010;184:2337-47.

 Wolchok JD, Kluger H, Callahan MK, et al. Nivolumab plus ipilimumab in advanced melanoma. N Engl J Med. 2013;369:122-33.

 Hammers H, Plimack E, Infante J, et al. Phase I study of nivolumab in combination with ipilimumab in metastatic renal cell carcinoma (mRCC). J Clin Oncol. 2014; 32(suppl 5S):abstr 4504.

 Sharpe AH, Wherry EJ, Ahmed R, Freeman GJ. The function of programmed cell death 1 and its ligands in regulating autoimmunity and infection. Nat Immunol. 2007;8:239-45.

 Breunis WB, Tarazona-Santos E, Chen R, et al. Influence of cytotoxic T lymphocyte-associated antigen 4 (CTLA4) common polymorphisms on outcome in treatment of melanoma patients with CTLA-4 blockade. J Immunother. 2008;31:586-90.

 Adaniel C, Rendleman J, Polsky D. Germline genetic determinants of immunotherapy response in metastatic melanoma. J Clin Oncol 2014;32(suppl 5S):abstr 3004.

 Ji RR, Chasalow SD, Wang L, et al. An immune-active tumor microenvironment favors clinical response to ipilimumab. Cancer Immunol Immunother. 2012;61:1019-31.

 Ng Tang D, Shen Y, Sun J, et al. Increased frequency of ICOS+ CD4 T cells as a pharmacodynamic biomarker for anti-CTLA-4 therapy. Cancer Immunol Res. 2013;1:229-34.

 Ku GY, Yuan J, Page DB, et al. Single-institution experience with ipilimumab in advanced melanoma patients in the compassionate use setting: lymphocyte count after 2 doses correlates with survival. Cancer. 2010;116:1767-75.

 Postow MA, Chasalow SD, Yuan J, et al. Pharmacodynamic effect of ipilimumab on absolute lymphocyte count (ALC) and association with overall survival in patients with advanced melanoma. J Clin Oncol. 2013;31(suppl):abstr 9052.

 Kitano S, Postow MA, Cortez C, et al. Myeloid-derived suppressor cell quantity prior to treatment with ipilimumab at 10mg/kg to predict for overall survival in patients with metastatic melanoma. J Clin Oncol.2012;30(suppl):abstr 2518.

 Yuan J, Zhou J, Dong Z, et al. Pretreatment serum VEGF is associated with clinical response and overall survival in advanced melanoma patients treated with ipilimumab. Cancer Immunol Res. 2014;2:127-32.

 Selby MJ, Engelhardt JJ, Quigley M, et al. Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells. Cancer Immunol Res. 2013;1:32-42.

 Hamid O, Chasalow, SD, Tsuchihashi Z, et al. Association of baseline and on-study tumor biopsy markers with clinical activity in patients (pts) with advanced melanoma treated with ipilimumab. J Clin Oncol. 2009;27:15(suppl):abstr 9008.

 Mu CY, Huang JA, Chen Y, et al. High expression of PD-L1 in lung cancer may contribute to poor prognosis and tumor cells immune escape through suppressing tumor infiltrating dendritic cells maturation. Med Oncol. 2011;28:682-8.

 Thompson RH, Gillett MD, Cheville JC, et al. Costimulatory B7-H1 in renal cell carcinoma patients: indicator of tumor aggressiveness and potential therapeutic target. Proc Natl Acad Sci USA. 2004; 101:17174-9.

 Gadiot J, Hooijkaas AI, Kaiser AD, et al. Overall survival and PD-L1 expression in metastasized malignant melanoma. Cancer. 2011;117:2192-201.

 Callahan MK. Understanding the biology behind responses to immunotherapy. Presented at ASCO Annual Meeting; 2014 May 30–June 4; Chicago, IL. Available from: hjttp://meetinglibrary.asco.org/content/94863?media=vm. Accessed October 8, 2014.

 Thompson RH, Kuntz SM, Leibovich BC, et al. Tumor B7-H1 is associated with poor prognosis in renal cell carcinoma patients with long-term follow-up. Cancer Res. 2006;66:3381-5.

 Taube JM, Klein AP, Brahmer JR, et al. Association of PD-1, PD-1 ligands, and other features of the tumor immune microenvironment with response to anti-PD-1 therapy. Clin Cancer Res. 2014;20:5065-74.

 Grosso J, Horak CE, Inzunza D, et al. Association of tumor PD-L1 expression and immune biomarkers with clinical activity in patients (pts) with advanced solid tumors treated with nivolumab (anti-PD-1; BMS-936558; ONO-4538). J Clin Oncol. 2013;31(suppl):abstr 3016.

 Weber JS, Kudchadkar RR, Yu B, et al. Safety, efficacy, and biomarkers of nivolumab with vaccine in ipilimumab-refractory or -naive melanoma. J Clin Oncol. 2013;31:4311-8.

 Herbst RG, Gordon MS, Fine GD, et al. A study of MPDL3280A, an engineered PD-L1 antibody in patients with locally advanced or metastatic tumors. J Clin Oncol. 2013;31(suppl):abstr 3000.

 Powles T, Vogelzang NJ, Fine GD, et al. Inhibition of PD-L1 by MPDL3280A and clinical activity in pts with metastatic urothelial bladder cancer (UBC). J Clin Oncol. 2014;32(suppl 5S):abstr 5011.

 Soria JC, Cruz C, Bahleda R, et al. Clinical activity, safety and biomarkers of PD-L1 blockade in non-small cell lung cancer (NSCLC): additional analyses from a clinical study of the engineered antibody MPDL3280A (anti-PDL1). Eur Cancer Congr 2013 (ECCO-ESMO-ESTRO); September 27–October 1, 2013; Amsterdam, The Netherlands. Abstr 3408.

 Daud AI, Hamid O, Ribas A, et al. Antitumor activity of the anti-PD-1 monoclonal antibody MK-3475 in melanoma (MEL): correlation of tumor PD-L1 expression with outcome. American Association for Cancer Research Annual Meeting 2014; April 5–9, 2014; San Diego. Abstr CT104.

 Ribas A, Hodi FS, Kefford, R, et al. Efficacy and safety of the anti-PD-1 monoclonal antibody MK-3475 in 411 patients (pts) with melanoma (MEL). J Clin Oncol 2014;32(suppl 5S):abstr LBA9000.

Characterizing tumors by PD-L1 expression, immune infiltration, chemokine signature, and tumor mutational frequency may be a means of creating an integrated model for determining which patients may benefit from which immune-checkpoint inhibitors, either alone or in combination.

Prognostic and Predictive Markers for the New Immunotherapies  

Immune checkpoint–blocking drugs such as ipilimumab, pembrolizumab, and nivolumab have demonstrated clinical efficacy as anticancer agents. Through modulation of immunoregulatory molecules, these novel therapeutics can produce durable cancer remissions in a variety of tumor types. As these medications are administered to an increasing number of patients, clinicians must be able to recognize and treat the associated immune-related side effects. This review summarizes the unique mechanisms of toxicity associated with immune checkpoint–blocking drugs, appropriate steps in patient evaluation, and strategies for mitigating risk and optimizing patient outcomes. Although the management of each patient receiving immune checkpoint–blockade therapy must be individualized, a conceptual framework upon which to base a multidisciplinary approach to best practices will help oncology practitioners deliver safe and effective care.

Recent advances in the clinical application of agents that block immunoregulatory molecules have led to unprecedented optimism about the potential of these novel therapies for bringing about durable antitumor responses in patients with various advanced malignancies.[1] However, enhancing immune responses to cancer via modulation of these immune checkpoints is associated with drug-related toxicities that are distinct from those associated with traditional chemotherapeutic agents and molecularly targeted therapies.[2] Because the use of checkpoint blockade agents will likely be widely expanded in the near term, it is critical that healthcare practitioners caring for oncology patients have a basic familiarity with these immune-related adverse events (irAEs), their variable presentations, and recommendations regarding their evaluation and management.

To understand the mechanism of checkpoint blocker–related autoimmune toxicities, it is helpful to consider their mechanism of action. At a basic level, the role of the human immune system, and of T lymphocytes in particular, is to activate against non-self–antigens (eg, viral proteins or cancer neoantigens resulting from tumoral genetic and epigenetic alterations) and to tolerate self-antigens. Antigen recognition by a T-cell receptor is followed by interactions between a tightly regulated cadre of immunoregulatory molecules that either activate or inhibit T-cell activation.[3] Some of the best studied of these checkpoints are cytotoxic T-lymphocyte antigen 4 (CTLA-4) and the pathway composed of programmed death 1 (PD-1) and one of its major ligands, programmed death ligand 1 (PD-L1).

When they are engaged with their respective binding partners, CTLA-4 and PD-1 promote immune tolerance via downregulation of T-cell activation.[4,5] The application of antibody antagonists of these immune checkpoints promotes T-cell activation, which can lead to tumor destruction as well as clinically relevant decreases in self-tolerance. This immune dysregulation is among the mechanisms underlying the autoimmune toxicities related to immune checkpoint blockers.

Incidence of Immune Checkpoint–Blockade Toxicities

The adverse event (AE) profiles of checkpoint-blocking drugs (ie, incidence of toxicities and organ systems most frequently affected) vary depending on agent and target. In clinical studies, toxicity severity is described using the Common Terminology Criteria for Adverse Events (CTCAE), which grades toxicities on a scale of 1 (mildest) to 5 (death related to that toxicity).[6]

The CTLA-4 antibody ipilimumab is the most widely studied checkpoint-blocking agent. In 2011, after extensive clinical testing, it was approved by the US Food and Drug Administration (FDA) for use in patients with advanced melanoma. One seminal study that led to its approval was a phase III trial involving patients with advanced melanoma.[7] Ipilimumab was administered at 3 mg/kg with or without a peptide vaccine to 511 patients. Grade 3 or 4 immune-related toxicities were observed in approximately 15% of patients, and 7 patient deaths were associated with irAEs. A retrospective review of safety data from 1,498 patients treated with ipilimumab on any of 14 phase I–III clinical trials found that drug-related AEs of any grade occurred in 85% of patients.[8] About 25% of patients experienced a grade 3 or 4 drug-related toxicity, and drug-related death was observed in < 1% of patients.

Clinical trials using ipilimumab in tumor types other than melanoma have demonstrated similar AE rates. For example, in a phase III study of ipilimumab administered after patients were treated with radiotherapy for metastatic prostate cancer, 63% of patients had immune-related toxicity of any grade.[9] Similarly, in a phase II study of ipilimumab plus chemotherapy for metastatic non–small-cell lung cancer, the rate of grade 3/4 irAEs was 15% to 20%, depending on the sequence of drug administration.[10]

In a phase II trial of 245 patients with advanced melanoma, study participants were randomly assigned to ipilimumab plus granulocyte macrophage colony-stimulating factor (GM-CSF) or ipilimumab alone.[11] Of note, ipilimumab was administered at 10 mg/kg, which is higher than the FDA-approved dose of 3 mg/kg. Patients who received combination therapy experienced a significantly lower rate of high-grade AEs (45% vs 58%; two-tailed 

 = .038). The lower rate of high-grade AEs was accompanied by significantly improved overall survival (OS) in the GM-CSF arm (median, 17.5 vs 12.7 months; hazard ratio, 0.64; 

 = .014), although further study is required to determine whether the increased OS is linked to improvements in AE rates.

Another CTLA-4 antibody, tremelimumab (CP-675,206; formerly ticilimumab) has been similarly studied in patients with metastatic melanoma and other advanced cancers. In a phase II clinical trial testing tremelimumab in patients with advanced melanoma, 19% of participants had a ≥ grade 3 adverse event.[12] When tested in patients with metastatic colorectal adenocarcinoma, a similar rate of high-grade toxicities was seen, with diarrhea/colitis representing the majority of cases.[13]

Although clinical data are still emerging, anti–PD-1 and anti–PD-L1 agents appear to have toxicity profiles that are different from those of CTLA-4 antibodies (Table 1).

Pembrolizumab is a humanized monoclonal antibody (mAb) blocking PD-1 that was approved by the FDA in September 2014 for use in treatment-refractory unresectable or metastatic melanoma. In a phase I clinical trial involving 135 patients with advanced melanoma, grade 3/4 AEs were reported in 13% of subjects.[14] An expansion cohort comprising patients whose disease was refractory to ipilimumab (and, if BRAF-mutant, refractory to BRAF inhibition) was reported separately.[15] In 173 patients, at a median follow-up of 8 months, toxicity rates were similar to those in previous reports.

Nivolumab is a genetically engineered, fully human immunoglobulin (Ig) G-4 mAb specific for human PD-1. In a phase I dose-escalation study conducted in 296 patients with multiple tumor types, grade 3/4 treatment-related toxicities occurred in 41 patients (14%). Treatment-related pneumonitis was observed in 9 patients (3%), 3 cases of which were fatal.[16] Longer-term follow-up of the entire study cohort (306 patients) demonstrated that exposure-adjusted toxicity rates were not cumulative.[17]

BMS-936559 (MDX-1105), a PD-L1–blocking antibody, was tested in a 207-patient phase I trial.[18] A 9% rate of grade 3/4 drug-related irAEs was reported. Similar findings were reported in 140 patients who received MPDL3280A, a human monoclonal IgG1 antibody engineered to block PD-L1 binding.[19] Drug-related grade 3/4 AEs were observed in 14% of patients.

Preclinical evidence suggests that blockade of multiple immune checkpoints can achieve synergistic antitumor activity.[20] Several clinical trials of combinatorial regimens are underway. In a trial of ipilimumab plus nivolumab in patients with metastatic melanoma, a 53% rate of treatment-related AEs of grade 3 or 4 was reported when the drugs were administered concurrently.[21] When ipilimumab was followed sequentially by nivolumab, the rate dropped to 18%, which is closer to observed rates for the single agents.

While theoretically any immune checkpoint–blockade toxicity can occur at any time, certain toxicities have been seen more frequently earlier in the treatment course, while others generally manifest as later complications. Commonly encountered irAEs include rash/dermatitis, diarrhea/colitis, hepatitis, and endocrinopathies. However, because essentially any tissue in the body can be affected, clinicians must consider patient signs and symptoms to be autoimmune in origin until proven otherwise. The timing of adverse events from ipilimumab has been well described[22]; recently released data about the onset of irAEs in 411 patients who received pembrolizumab are illustrated in Figure 1. Familiarity with these patterns is helpful in successfully anticipating and treating these toxicities.

In the case of ipilimumab, the most common early toxicity is dermatologic toxicity.[22] The window for the highest risk for rash and other skin toxicity starts approximately 3 weeks into treatment, although late and long-lasting dermatologic toxicities have been observed. Enterocolitis most frequently occurs between weeks 5 and 10 of treatment, although gastrointestinal symptoms occurring at any time after initiation of ipilimumab could represent immune-related drug toxicity. Hepatitis is often seen slightly later than colitis, with the period of highest risk occurring between weeks 6 and 14 of treatment. Endocrinopathies generally are more unpredictable, with the risk period starting around 7 weeks after initiation of therapy. Even with optimal management, the effects of autoimmune endocrinopathies can be long-lasting, and late manifestations of hypophysitis can occur.

Patients with a history of autoimmunity are potentially at greater risk for irAEs. In clinical trials of checkpoint-blocking drugs, patients with autoimmunity were typically excluded, and a history of autoimmunity is considered a relative contraindication for immune checkpoint–blockade therapy. Although post–FDA-approval experience with ipilimumab has identified instances of successful treatment in patients with autoimmunity and other high-risk comorbidities (eg, history of organ transplant[23]), close monitoring is warranted in this patient population.

One active area of investigation is the pretreatment identification of patients who are more likely to experience irAEs. Two studies have suggested a correlation between specific genetic changes detectable in the blood and the development of colitis, the most common serious side effect observed with the anti–CTLA-4 agents.[24,25] Of note, a strategy of prophylaxis with budesonide to prevent colitis in patients being treated with ipilimumab was ineffective.[26] In that study, 115 patients were randomly assigned to budesonide at 9 mg daily or placebo at the initiation of therapy. Rates of grade 2 and higher diarrhea/colitis were similar between groups.

Identification and Characterization of Toxicities

The identification of irAEs requires effective patient-provider communication, familiarity with the potential manifestations of irAEs, and a high index of suspicion that patient complaints may reflect drug toxicity. Perhaps because irAEs have been reported to be associated with antitumor effects of therapy,[27] patients often minimize their symptoms in the hope of remaining on therapy. It is important for providers to impress upon patients that toxicities can lead to significant morbidity or even death unless they are promptly recognized and treated.

Grade 1 and 2 dermatologic toxicities from checkpoint-blocking drugs are among the most commonly seen irAEs. Typically, mild dermatitis manifests as a maculopapular rash or erythroderma (Figure 2). Pruritus can accompany the rash or, less commonly, can present as an isolated complaint in the absence of skin findings. Rarely, these processes can be severe, manifesting as ulceration, bullous dermatitis, or Stevens-Johnson syndrome. Pathologic evaluations of biopsy specimens of affected skin often demonstrate eosinophilic infiltration or leukocytoclastic vasculitis; or, they may reveal a lymphocytic predominance characterized by CD8+ T cells, sometimes with tropism for melanin-containing cells. Specifically for patients with metastatic melanoma, the rash associated with immune-checkpoint inhibitors may be indicative of immune response to melanocytes and may progress to vitiligo in some cases.[28,29]

Autoimmune enterocolitis is common in patients receiving ipilimumab and generally manifests as watery diarrhea, abdominal pain, nausea/vomiting, or hematochezia. About 30% of 511 patients who received ipilimumab in a large phase III trial experienced diarrhea of any grade.[7] In severe cases, patients may experience significant dehydration, fever, peritoneal signs, bowel perforation, or ileus. Stool cultures are negative. Endoscopy performed in these cases may identify an inflamed mucosa with ulceration, which can involve any part of the bowel but most commonly the descending colon.[30] Biopsy specimens of the colon typically reveal an inflammatory cell infiltrate (neutrophilic, lymphocytic, or mixed) with cryptitis.[31] Even in the setting of a relatively mild colitis seen grossly during endoscopy, tissue biopsies can demonstrate significant inflammation. Deaths secondary to bowel perforation have occurred.[7]

Endocrinopathies are perhaps the most elusive irAEs, due to their nonspecific presentations. A patient may have vague symptoms yet be at risk for serious morbidity or death if the endocrinopathy is not promptly identified and treated. For example, hypophysitis, which can cause pituitary inflammation with meningeal irritation, often presents as headache refractory to nonsteroidal anti-inflammatory drugs or other analgesics. Compression of the optic nerve from an inflamed pituitary may also lead to visual complaints. However, hypophysitis may manifest more nonspecifically, with symptoms secondary to hypopituitarism, such as hypotension, electrolyte disturbances, abdominal pain, weakness, behavioral changes, loss of libido, or fatigue.

Magnetic resonance imaging (MRI) of the brain typically demonstrates enlargement of the pituitary with variable contrast enhancement characteristics.[32] Patients will generally have abnormal endocrinologic laboratory test results; assessment of basic chemistries, adrenocorticotropic hormone, cortisol, thyroid-stimulating hormone (TSH), free thyroxine (T

), prolactin, follicle-stimulating hormone, luteinizing hormone, and testosterone/estradiol is indicated.[33] Biopsy should only be considered in rare cases in which metastatic disease of the pituitary must be ruled out.

In the appropriate clinical setting, other endocrine irAEs that should be considered include thyroiditis (ie, hyper- or hypothyroidism), adrenal insufficiency, and hypogonadism. Because patients who experience irAEs often require prolonged courses of corticosteroids, resultant temporary secondary adrenal insufficiency is not uncommon.

Hepatic toxicity often manifests as asymptomatic elevated levels of hepatic transaminases. Rarely, patients may complain of nonspecific symptoms, including fever, fatigue, nausea, and abdominal pain. In these cases, one must distinguish between checkpoint drug–related toxicity and other etiologies of hepatic injury, such as neoplastic disease progression in the liver, infections, and effects of other medications or alcohol intake. Titers for antibodies associated with autoimmune hepatitis, including antinuclear antibodies, anti–smooth-muscle antibodies, and anti-mitochondrial antibodies, are nonspecific and of questionable utility. Serologic testing should be performed for viral hepatitis (including hepatitis A and B), cytomegalovirus, and Epstein-Barr virus. Ultrasonograms of the liver can appear normal or may demonstrate homogenous hepatomegaly, edema, or enlarged perihepatic lymph nodes. Biopsy of the liver most commonly shows a diffuse T-cell infiltrate. Other reported pathology patterns include inflammation focused around either hepatocytes or bile ducts.[34]

As immune checkpoint–blocking medications are administered to an increasing number of patients, clinicians must be able to recognize and treat the associated immune-related side effects.

Oncology Vol 28 No 11_Suppl_3

Prospects for Targeting PD-1 and PD-L1 in Various Tumor Types

Prognostic and Predictive Markers for the New Immunotherapies

Identification and Management of Toxicities From Immune Checkpoint–Blocking Drugs