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Cancer

Immunotherapy TOP ARTICLES SUPPLEMENT

CONTENTSREVIEW: Immune checkpoint therapy for non-small-cell lung cancer: an update Immunotherapy Vol. 8 Issue 3

RESEARCH ARTICLE: A pooled analysis of nivolumab for the treatment of advanced non-small-cell lung cancer and the role of PD-L1 as a predictive biomarker Immunotherapy Vol. 8 Issue 9

REVIEW: Beyond melanoma: inhibiting the PD-1/PD-L1 pathway in solid tumors Immunotherapy Vol. 8 Issue 5

REVIEW: Checkpoint inhibitors for renal cell carcinoma: current landscape and future directions Immunotherapy Vol. 8 Issue 7

REVIEW: Checkpoint inhibition for colorectal cancer: progress and possibilities Immunotherapy Vol. 8 Issue 6

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http://www.futuremedicine.com/doi/full/10.2217/imt.15.123

http://www.futuremedicine.com/doi/full/10.2217/imt.15.123

http://www.futuremedicine.com/doi/full/10.2217/imt-2016-0032









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279Immunotherapy (2016) 8(3), 265–277 ISSN 1750-743X10.2217/imt.15.123 © 2016 Future Medicine Ltd

Immunotherapy

Review 2016/02/288

3

2016

The role of immunotherapy in treatment of non-small-cell lung cancer (NSCLC) has been gaining interest over the past few years. This has been driven primarily by promising results from trials evaluating antagonist antibodies that target co-inhibitory immune checkpoints expressed on tumor cells and immune cells within the tumor microenvironment. Immune checkpoints exist to dampen or terminate immune activity to guard against autoimmunity and allow for self-tolerance. However, tumors can take advantage of these immune checkpoint pathways to evade destruction. Antibodies that block inhibitory checkpoints, such as anti-CTLA-4, anti-PD1 and anti-PD-L1 antibodies have demonstrated delayed tumor growth and increased survival. Novel therapies are now investigating combining checkpoint inhibitors with chemotherapy, targeted therapy, radiation and vaccines to produce synergistic antitumor activity.

First draft submitted: 1 July 2015: Accepted for publication: 16 December 2015: Published online: 10 February 2016

Keywords: anti-CTLA4 • anti-PD1 • anti-PD-L1 • atezolizumab (MPDL 3280A) • checkpoint inhibitors • durvalumab (MEDI4736) • immunotherapy • ipilimumab • nivolumab • pembrolizumab

Lung cancer is the leading cause of cancer-related deaths in the United States and worldwide [1]. Non-small-cell lung cancer (NSCLC) accounts for about 85% of all lung cancers and is often diagnosed at an advanced stage with poor overall prognosis [2]. Main-stay treatment for metastatic NSCLC is platinum-based doublet chemotherapy with a median survival of about 10 months [3]. With the addition of bevacizumab, an anti-angiogenesis agent, to chemotherapy, median survival has improved to 1 year [3,4].

A subgroup of patients with NSCLC have specific mutations in the epidermal growth factor receptor (EGFR) or anaplastic lym-phoma kinase (ALK ) gene that can serve as potential targets for therapy. Somatic activat-ing mutations in the kinase domain of EGFR have been identified in 10–15% of patients of Caucasian ethnicity and 30–50% of patients

in Asia [2,5,6]. The ALK gene rearrangement is found in approximately 4–5% of all NSCLC, including both Caucasian and Asian popula-tions [7–10]. Tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib, and small molecule ALK inhibitors such as crizotinib, have resulted in higher response rates and prolonged survival. However, the vast major-ity of patients will eventually relapse within 1 year, illustrating the need for additional, more effective therapies [2,7].

Immune checkpoint overviewRecently, there has been a renewed interest in immunotherapies for the treatment of NSCLC, especially with promising results in patients with advanced disease who have progressed on multiple lines of treatment.

T-cell immunity is mediated by inter-actions between antigen-presenting cells

Immune checkpoint therapy for non-small-cell lung cancer: an update

Bing Xia*,1 & Roy S Herbst1

1Yale Comprehensive Cancer Center, Yale School of Medicine, 333 Cedar Street WWW221, New Haven, CT 06520, USA *Author for correspondence: [email protected]

part of

Review

For reprint orders, please contact: [email protected]

280 Immunotherapy (2016) 8(3) future science group

Review Xia & Herbst

(APCs) and T cells, regulated by co-stimulatory and inhibitory signals. For T-cell response to occur, co-stimulatory signals must be engaged by their cognate ligands as illustrated in Figure 1. Co-stimulatory and inhibitory receptors often bind to the same ligand with varying kinetics [11]. Co-stimulatory receptors are expressed on naive and resting T cells while inhibi-tory receptors are upregulated after T-cell activation to maintain self-tolerance and limit tissue damage during immune responses [11,12].

Immune checkpoints are crucial for downregulation of the immune system to prevent autoimmunity [13,14]. However, the tumor microenvironment has been shown to be composed of dysfunctional immune cells and stromal host cells that exploit immunoregulatory pathways to evade immunity [15]. Inhibitory ligands and receptors are commonly overexpressed on tumor or in the tumor microenvironment and are targetable with antagonist antibodies [11]. Major mechanisms of immune evasion by tumors that can be clinically targeted include the CTLA-4 and PD-1 pathways [15]. Properties of the most common anti-CTLA-4, PD-1, and PD-L1 antibodies are shown in Table 1, includ-ing type of antibody and binding affinity. Inhibition of these pathways has been shown in preclinical models to improve intratumoral immune responses [13,16,17]. Better understanding of these pathways and immune

invasion is essential for development of effective treatments.

Anti-CTLA-4 pathwayCTLA-4 and CD28 are immunoglobulin (Ig) co-receptors that bind to CD80 (B7–1) and CD86 (B7–2) [18–20]. CD80 binds CTLA-4 with greater affinity than CD28, Kd values of approximately 12 and 200 nM, respectively [21]. CD28 co-stimulation is required for T cell activation to most peptide antigens and results in activation of NFAT and NF-κB [18,22–27].

In contrast, CTLA-4 opposes the actions of CD28-mediated co-stimulation and functions as an inhibi-tor of T-cell response to regulate T cell proliferation, allowing for self-tolerance. [28,29] CTLA-4 has been shown to downregulate AKT phosphorylation through recruitment of two phosphatases, SHP2 and PP2A, to dampen T cell activation and decrease production of NF-κB, IL-2 and cytokines as shown in Figure 2 [30–33]. CTLA-4 has also been shown to regulate tumor immunity through regulatory T cells (Tregs), which express high levels of surface CTLA-4, and there-fore suppressing activation and expansion of effec-tor cells [34]. Blocking CTLA-4 or depletion of Tregs have been found to increase immune response, with increase in antigen-specific T follicular helper (Tfh) cells, plasma and memory B cells after vaccination and

Figure 1. Immune priming occurs in the lymph node and requires a secondary co-stimulatory signal to activate T-cell response, resulting in production of granzymes, perforin, IFN-γ and activation of the PI3 kinase pathway, to allow for T-cell proliferation and destruction of pathogens [101].

TCRMHC

Virus-infected cell

Dendritic cell

Processing of antigen

CD80/86 CD28

T cell

(+) Activation

Co-stimulatorysignal

Antigen

Signal 2

Signal 1

GranzymesPerforin IFN-γ

inflammatorycytokines

NF-κB

PI3Kactivation

Immune response

Lymph nodes

www.futuremedicine.com 281future science group

Immune checkpoint therapy for non-small-cell lung cancer: an update Review

enhance secondary immune responses [35,36]. Tregs have been described to be on tumor cells, and block-ing Treg function through anti-CTLA-4 antibodies may be an effective mechanism to enhance antitumor activity [34].

IpilimumabIpilimumab is a fully human immunoglobulin G1 anti-CTLA-4 monoclonal antibody [34]. Activity of ipiliu-mumab was described in melanoma in a Phase III trial evaluating ipilimumab plus glycoprotein 100 (gp100) peptide vaccine, ipilumumab alone, or gp100 alone in patients with stage III or IV melanoma. They described improved overall survival (OS) with ipilimumab alone compared with gp100 alone (HR for survival = 0.66, p = 0.003) and also an improved OS in ipilimumab plus gp100 compared with gp100 alone (HR = 0.68, p < 0.001) [34,37]. Ipilumumab was US FDA approved for treatment of late stage melanoma in 2011.

Ipilimumab has also been shown to be effec-tive in NSCLC. A Phase II study evaluated chemo-therapy-naive patients with NSCLC (n = 204) who were randomly assigned to 1:1:1 to receive paclitaxel (175 mg/m2) and carboplatin (area under the curve, 6) with concurrent ipilimumab (four doses of ipilimumab plus paclitaxel and carboplatin followed by two doses of placebo plus paclitaxel and carboplatin), phased ipilimumab (two doses of placebo plus paclitaxel and carboplatin followed by four doses of ipilimumab plus paclitaxel and carboplatin), or placebo control (up to 6 doses of placebo plus paclitaxel and carboplatin). The study showed improved immune-related progression free survival (irPFS) for phased ipilimumab verses the control (hazard ratio [HR]: 0.72; p = 0.05), but not for concurrent ipilimumab (HR: 0.81; p = 0.13). Median irPFS for phased ipilimumab, concurrent ipilimumab and control groups were 5.7, 5.5 and 4.6 months, respectively; median progression free survival (PFS) were 5.1, 4.1 and 4.2 months, respectively; immune-related best overall response rate were 32, 21 and 18%, respectively; best overall response rate were 32, 21 and

14%, respectively, and a median OS of 12.2, 9.7 and 8.3 months. Overall rates of grade 3 and 4 immune related adverse events were 15% for phased ipilim-umab, 20% for concurrent ipilimumab and 6% for the control group. The most common adverse events were liver function enzyme abnormalities, hematologic abnormalities (thrombocytopenia, anemia and neutro-penia), fatigue, alopecia, nausea, peripheral neuro-pathy, rash, pruritus and diarrhea. This trial showed that combination of phased ipilimumab with pacli-taxel and carboplatin, but not concurrent, improved irPFS. Interestingly, subset analysis appeared to show improved efficiency with phased ipilimumab for squa-mous histology, without apparent benefit for nonsqua-mous histology, indicating the need for additional investigation in a larger clinical trial [38].

TremelimumabTremelimumab is a fully human IgG2 anti-CTLA4 antibody, and has been most well studied in mela-noma. A Phase III study examined tremelimumab at 15 mg/kg every 3 months compared with standard chemotherapy in patients with metastatic melanoma without brain metastases. At interim analysis, there was a nonstatistically significant improvement in survival favoring tremelimumab (p = 0.14) [39,40].

Tremelimumab in NSCLC was studied in a ran-domized Phase II clinical trial comparing tremelim-umab as maintenance therapy with best supportive care in patients with good performance status who previ-ously received platinum-based chemotherapy. Patients assigned to the treatment arm received tremelimumab 15 mg/kg IV every 90 days until disease progression. Results showed that treatment with tremelimumab did not result in a superior PFS. However, there were 2 (4.8%) partial responses seen only in the investigational arm which may support additional studies [41].

PD-1/PDL-1 pathwayA major mechanism of immune escape has been described by the B7-H1/PD-1 pathway [15]. PD-1 is

Table 1. Properties of the six most commonly used anti-CTLA4, PD-1 and PD-L1 antibodies, including the antibody type, manufacturer and binding affinity.

Antibody Type Manufacturer Affinity

Ipilimumab IgG1 anti-CTLA-4 Bristol-Myers Squibb KD= 5.25 nM

Tremelimumab IgG2 anti-CTLA-4 AstraZeneca KD = 0.28 nM

Pembrolizumab IgG4 anti-PD-1 Merck KD=0.028 nM

Nivolumab IgG4 anti-PD-1 Bristol-Myers Squibb KD=2.60 nM

Atezolizumab IgG1 anti-PD-L1 Genentech/Roche KD=0.4 nM

Durvalumab IgG1 anti-PD-L1 MedImmune N/A

Data taken from [39,64,94–100].


Review Xia & Herbst

expressed by activated T cells and is a key immune checkpoint receptor that mediates immunosuppres-sion. It attenuates T cell receptor signaling by binding to its ligands PD-L1 (B7-H1) and PD-L2 (B7-DC) [42].

PDL-1 expression on tumor and tumor infiltrat-ing immune cells is thought to be primarily induced by Th1 cytokines (in particular, interferon-γ) released by infiltrating tumor immune cells [43,44]. PD-1 signaling has been found to recruit SHP-2, which mediates decreased T-cell receptor signaling possibly through inhibition of the AKT pathway [30,45–48].

Clinical trials have produced promising results eval-uating antagonist antibodies that target the co-inhib-itory immune checkpoint, PD-1 expressed on acti-vated antitumor T cells and its primary ligand, PD-L1 expressed on tumor cells and immune cells within the tumor microenvironment. Targeting this pathway can be categorized as directed against PD-1 such as nivolumab and pembrolizumab, or targeted against PD-L1, such as atezolizumab (MPDL3280A) and durvalumab (MEDI4736). Response rates for these compounds have been consistently over 20% across all trials, which has led to large ongoing Phase III trials, and recent FDA approval of nivolumab for metastatic squamous cell lung cancer [49].

Anti-PD1NivolumabNivolumab is a fully human IgG4 monoclonal antibody against PD-1 [50]. Nivolumab was investigated with the CheckMate -017 trial, which was a Phase III, open-label, randomized, multicenter study of nivolumab compared with docetaxel as second-line in patients with squamous cell lung cancer (NCT01642004). This trial was stopped early after interim analysis showed supe-rior overall survival of patients in the nivolumab arm compared with control. In this study, patients with metastatic squamous cell lung cancer, who progressed on platinum doublet-based chemotherapy, were ran-domized to receive nivolumab (3 mg/kg every 2 weeks) (n = 135) or standard of care, docetaxel (75 mg/m2 IV every 3 weeks; n = 137). During interim analysis in January 2015, the median OS was 9.2 months in the nivolumab arm (95% CI: 7.3–13.3) and 6 months in the docetaxel arm (95% CI: 5.1–7.3). The hazard ratio was 0.59 (95% CI: 0.44–0.79; p = 0.00025), translat-ing to a 41% lower risk of death with nivolumab com-pared with docetaxel. One year overall survival rate was 42% (95% CI: 34–50) with nivolumab, compared with 24% (95% CI: 17–31) with docetaxel. Response rate in the nivolumab arm was 20% compared with 9% in the docetaxel arm (p = 0.008). Median pro-gression free survival was 3.5 months with nivolumab

Figure 2. Downregulation of T cells occurs through the CTLA-4 and PD-1 pathways, through activation of two phosphatases, SHP2 and PP2A, resulting in inhibition of the PI3 kinase pathway, downstream AKT and Bcl-xL activation, as well as decrease of NF-κB, IL-2 and inflammatory cytokines. Blockade of this pathway with checkpoint inhibitors restores T-cell activity [30–33,102].

MHC TCR

CD86 CTLA4

T cell

PI3KAKTBcl-xLNF-κBIL-2

Inhibition of immune andantitumor response

TCR MHC

PD1PDL1/PDL2

Anti-CTLA4IpilimumabTremelimumab

Anti-PD1NivolumabPembrolizumab

Anti-PDL1Atezolizumab(MPDL3280A)Durvalumab(MEDI 4736)

PP2A

SHP2

P

Tumor

SHP2

APC



and 2.8 months with docetaxel, resulting in a hazard ratio for death or disease progression of 0.62 (95% CI: 0.47–0.81; p < 0.001). The improved overall survival led to early termination of the trial and FDA approval of nivolumab as second line therapy for metastatic squamous lung cancer. This trial also evaluated tumor PD-L1 expression and quantified levels as ≥1, <1, ≥5, <5 and ≥10% and found that tumor PD-L1 status was not prognostic or predictive of efficacy [51,52].

A Phase III, open-label, randomized study (Check-Mate-057) examined 582 patients with nonsquamous NSCLC, who had disease recurrence or progression during/after one prior line of platinum based dou-blet chemotherapy (NCT01673867). Patients were randomized to receive nivolumab 3 mg/kg IV every 2 weeks (n = 292) or docetaxel 75 mg/m2 IV every three weeks (n = 290). The primary endpoint was over-all survival with secondary endpoints being objective response rate (ORR) and PFS. The study was ended early in April 2015 due to survival advantage observed in the nivolumab arm. Nivolumab showed better OS and response rate. Median OS in the nivolumab group was 12.2 months (95% CI: 9.7–15.0) compared with 9.4 months (95% CI: 8.1–10.7) in the docetaxel group (HR = 0.73; 96% Cl: 0.59–0.89; p = 0.002), and response rate was 19.2% with nivolumab verses 12.4% with docetaxel (p = 0.02335). One year overall survival was 51% (95% CI: 45–56) with nivolumab compared with 39% (95% CI: 33–45) with docetaxel. At 18 months, the overall survival was 39% (95% CI: 34–45) with nivolumab, compared with 23% (95% CI: 19–28) with docetaxel. PD-L1 expression was quantified as ≥1, <1, ≥5, <5 and ≥10%, but in contrast to the CheckMate-017 study in squamous NSCLC, this study showed that PD-L1 expression was associ-ated with improved efficacy across all predefined cut-points of 1, 5 and 10%. Grade 3–5 drug-related adverse events were less common in the nivolumab arm, 10.5% (30/387) compared with 53.7% in the docetaxel arm. This study showed that nivolumab also has a survival benefit in nonsquamous cell lung cancer [53–55].

Updated data from the Phase II study (Check-Mate-063) of nivolumab in patients with advanced squamous NSCLC demonstrated 1-year OS rate of 41% (95% Cl: 32–50), 1-year PFS rate of 20% (95% Cl: 13–29) and median PFS of 1.9 months (95% Cl: 1.8–3.2). Objective responses were seen across patient subgroups regardless of PD-L1 expression. Elevation of peripheral IFN-γ stimulated cytokines, CXCL9 and CXCL10 were observed. 17% of patients experienced grade 3–4 treatment related adverse events, including fatigue (4%), diarrhea (3%) and pneumonitis (3%). This study demonstrates that nivolumab is clinically efficacious with acceptable safety profile [56].

Nivolumab as monotherapy in treatment-naive patients with NSCLC is also being evaluated in an ongoing Phase I study (NCT01454102). As of interim analysis, 52 patients with squamous or nonsquamous advanced NSCLC received nivolumab 3 mg/kg IV every 2 weeks until progression of disease or unaccept-able toxicity. ORR was 21% with 3 confirmed com-plete responses. Response was also evaluated by tumor PD-L1 expression (PD-L1+ was defined as ≥5% tumor cells expressing PD-L1). Responses were observed in patients who were both PD-L1+ and PD-L1-, with ORR higher in patients with PD-L1+ tumors, 31% compared with 10% [57].

PembrolizumabPembrolizumab (MK-3475) is a humanized mono-clonal IgG4 antibody that is potent and highly selec-tive against PD-1. It was approved by the FDA in 2014 for treatment of metastatic melanoma and was granted accelerated approval on 2 October 2015 as second line treatment for PD-L1 positive metastatic NSCLC. The FDA recently approved a PD-L1 immu-nohistochemistry 22C3 pharmDx test developed by Dako to aid in identifying patients for treatment with pembrolizumab [58].

The safety and tolerability of pembrolizumab in NSCLC was evaluated in a Phase I study (KEY-NOTE-001) that enrolled 495 patients, including 394 patients who had progressed on prior lines of treat-ment and 101 patients who have never received treat-ment. Patients were treated with pembrolizumab at dose of either 2 mg or 10 mg per kilogram of body weight every 3 weeks or 10 mg per kilogram every 2 weeks (NCT01295827). The overall response rate was 19.4% (95% CI: 16.0–23.2). Of the previ-ously treated patients, response rate was 18.0% (95% CI: 14.4–22.2). Response rate was 24.8% (95% CI: 16.70–34.3) in patients who had never been treated. Response rates were similar regardless of dose, schedule and histologic analysis. Median duration of response was 12.5 months (range: 1.0–23.3) in all patients with median duration of progression-free survival of 3.7 months (95% CI: 2.9–4.1). Median progression-free survival for all patients was 3.7 months (95% CI: 2.9–4.1), with 3.0 months (95% CI: 2.2–4.0) for pre-viously treated patients, and 6 months (95% CI: 4.1–8.6) for previously untreated patients. The most com-mon drug related adverse events were fatigue (19.4%), pruritus (10.7%), decreased appetite (10.5%), rash (9.7%), arthralgia (9.1%), diarrhea (8.1%), nausea (7.5%) and hypothyroidism (6.9%) [59–61].

This study further examined PD-L1 as a potential biomarker of response and showed that tumor cells with at least 50% PD-L1 expression had improved


Review Xia & Herbst

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response to pembrolizumab. Membranous PD-L1 expression in at least 50% of tumor cells (proportion score ≥50%) was selected as the cut-off. The response rate was 45.2% (95% CI: 33.5–57.3) in the 73 patients with proportion score of at least 50%, which included 50.0% (95% CI: 24.7–75.3) of patients who had not received prior treatment and 43.9% of patients who had progressed on prior lines of treatment. The response rate of patients with proportion score of at least 50% was statistically significantly better than the group with proportion score 1–49% and <1% in both patients who had previous treatment (p < 0.001) and for patients who never received prior lines of treatment (p = 0.01). The prevalence of PD-L1 expression was examined in 1143 patients who were screened, 824 samples were available for analysis. 23.2% of patients had a proportion score of at least 50%, 37.6% of patients had a score of 1–49% and 39.2% of patients had a score <1% [59].

Another pivotal study of pembrolizumab was Key-note-010, which examined pembrolizumab at the 2 mg/kg FDA-approved dose and higher 10 mg/kg inves-tigational dose, each given every 3 weeks, compared with docetaxel, in PD-L1 positive patients who have progressed on prior lines of therapy (NCT01905657). PD-L1 positivity was defined as tumor proportion score (TPS) of 1% or greater, and strong PD-L1 posi-tivity was classified as TPS of 50% or greater. Pem-brolizumab at both doses was associated with longer OS and superior PFS compared with docetaxel in TPS score >50% and greater than 1% [62].

The role of pembrolizumab in treatment of CNS metastasis was evaluated in an ongoing Phase II trial with two separate arms, one for advanced NSCLC and one for metastatic melanoma (NCT02085070). Patients with ≥1 untreated or progressing brain metas-tases with pretumor biopsy showing PD-L1 expression were enrolled to receive pembrolizumab 10 mg/kg IV every 2 weeks. As of December 2014, ten patients with NSCLC were enrolled, with nine patients evaluable for response. Brain metastasis response rate was 44% (4/9 partial responses). 34% (3/9) patients had progres-sion of disease. Systemic response rate was 34% (3/9). One patient responded systemically but had progres-sion of disease in the brain. This study showed that pembrolizumab has promising clinical activity in CNS disease [63].

Anti-PDL-1Atezolizumab (MPDL 3280A)MPDL3280A, most recently named atezolizumab, is a high-affinity specific human IgG1 antibody against PD-L1 and therefore prevents its binding to ligands PD-1 and B7–1 [64]. However, unlike PD-1 inhibi-

tors, PD-L1 inhibition will allow PD-1 to bind to its alternative ligand PD-L2, which has been described to maintain tolerance in the lung, and may decrease risk for pneumonitis [61,64–66].

In a single arm Phase I study, a total of 277 patients with advanced incurable cancer (NSCLC, melanoma, renal cell carcinoma, colorectal cancer, gastric cancer and head and neck squamous cell carcinoma) were enrolled to receive atezolizumab every 3 weeks intra-venously (NCT01375842). One hundred and seventy-five patients were evaluable. Confirmed responses (complete and partial responses) was 18% (32 of 175 patients) across all tumor types. Response rate of 21% (11 of 53 patients) was observed for patients with NSCLC. Patients with melanoma had the highest response rate of 26% (11 of 43 patients). Median PFS of all patients in the study was 18 weeks. Exploratory analysis revealed a potential trend of better response to atezolizumab in former/current smokers versus never smokers (46% [11 of 26] versus 10% [1 of 10] respec-tively; p = 0.422). Most drug-related adverse events were grade 1–2 and did not require treatment. Thirty-five patients (13%) experienced treatment related grade 3–4 toxicities, and 3 patients (1%) experienced immune-related grade 3–4 adverse events. There were no cases of grade 3–5 pneumonitis. The most common treatment related adverse events were fatigue (24%), decreased appetite (12%), nausea (12%), pyrexia (11%), diarrhea (11%) and rash (11%) [64].

This study showed that atezolizumab has acceptable drug-related toxicity and robust antitumor activity. An association between expression of PD-L1 in pretreat-ment samples and response to treatment with atezoli-zumab was described, with greater efficacy observed in tumor-infiltrating immune cell PD-L1 expression IHC score of 3. In the study, pretreatment specimens were scored based on their PD-L1 positivity. Specimens were scored IHC of 0, 1, 2, or 3 if <1%, 1–5%, 5–10%, or ≥10% of cells per area were PD-L1 positive, respec-tively. Tumor-infiltrating immune cell PD-L1 expres-sion was associated with response to atezolizumab treatment, reaching statistical significance in NSCLC, p = 0.015. However, tumor cell PD-L1 expression was not associated with statistically significant response to atezolizumab across all tumors (p = 0.079) or NSCLC (p = 0.920) [64].

Eighty-three percent of patients with tumor-infil-trating immune cell IHC score of 3 responded to treatment with atezolizumab, with only 17% progress-ing, whereas in patients with IHC score of 2 (tumor-infiltrating immune cell), only 43% responded to treatment. However, the correlation was not observed between response to atezolizumab and expression of PD-L1 in tumor cell. In patients with tumor cell IHC 3,


Review Xia & Herbst Ta

ble

3. o

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Tab

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t in

hib

ito

rs (

con

t.).


Review Xia & Herbst

Ag

ent

NC

T n

um

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Ad

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CT0

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Res

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y, r

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n, o

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t in

hib

ito

rs (

con

t.).



Ag

ent

NC

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um

ber

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l nam

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pu

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Review Xia & Herbst

ORR was 46% whereas ORR in IHC 2 patients was 17%. Paired biopsy studies showed increase in PD-L1 expression on both tumor-infiltrating immune cells and tumor cells after treatment with atezolizumab. In patients with >30% reduction in tumor size, 5/6 (83%) patients had increase in PD-L1 observed on tumor-infiltrating immune cells and 3/6 (50%) patients had increase in PD-L1 in tumor cells. PD-L1 increase dur-ing treatment correlated with changes in tumor IFNγ expression (Pearson correlation coefficient = 0.70) and activation of CD8 and TH1 responses [64].

The study also showed that expression of CTLA-4 in pretreatment tumors correlated strongly with response whereas fractalkine, a transmembrane protein and che-mokine involved in adhesion and migration of leuko-cytes, identified in pretreatment tumors correlated with progression [67]. Elevated expression of IFNγ as well as IFNγ-inducible genes (IDO1 and CXCL9) in pretreat-ment tumors correlated with response in melanoma whereas these associations were weaker in NSCLC and renal cell carcinoma tumors. Evaluation of post-treatment biopsies showed lesions that regressed after treatment showed dense immune infiltrate and tumor cell necrosis. Sterilization of cancer cells occurred in some cases [64].

In tumors that failed to respond to treatment, little or no tumor-infiltrating immune cell infiltration were observed and minimal to no expression of PD-L1 were observed on intratumoral immune filtrate. Biopsies obtained from tumors that progressed also showed presence of immune infiltrate present only around the outer edge of the tumor cell mass. Chip analysis showed that nonresponders failed to provide T cell activation and T-effector cell activity compared with those who responded to atezolizumab [68]. Foxp3, a transcription factor that plays at least a partial role in regulating Treg cell signature, was neither increased nor decreased in lesions that responded to treatment with atezolizumab [64,68].

Immune biomarkers were also examined in the blood, but did not significantly correlate with progres-sion or response to treatment with atezolizumab. Dur-ing cycle 1 of treatment with atezolizumab, increases in IL-18, ITAC (also known as CXCL11 or IP-9), pro-liferating CD8+ T cells (CD8+HLA-DR+Ki-67+) and IFN-γ were observed. Downtrend of IL-6 expression was observed by cycle 2, day 1 [64].

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immune cell (IC) 0, 1, 2, or 3 and tumor cell (TC) 0, 1, 2, or 3. 205 patients with TC 2/3 and/or IC 2/3 tumors were enrolled. The highest ORR was observed in patients with PD-L1 TC3 or IC3 [69]. Archival and fresh tumor specimens demonstrated high agreement in T3 or IC3 PD-L1 expression [70].

The POPLAR study examined 287 patients who were randomized to receive 1200 mg IV q3w atezolizumab or 75 mg/m2 IV q3w docetaxel (NCT01903993). Patients were scored as TC and IC 0, 1, 2, or 3 based on PD-L1 expression. Again, patients with increas-ing PD-L1 expression were observed to have increased efficacy with improved OS, PFS and ORR. Patients with TC3 or IC3 PD-L1 appeared to have most ben-efit from atezolizumab with OS HR 0.47 (0.20–1.11), PFS HR 0.56 (0.28–1.11) and ORR 38% versus 13% compared with docetaxel. Patients with TC0 and IC0 PD-L1 levels did not appear to benefit from atezoli-zumab with OS HR 1.22 (0.69–2.14), PFS HR 1.15 (0.72–1.82) and ORR 8% versus 10% compared with docetaxel [71].

Atezolizumab is also being evaluated in the BIRCH and OAK trials. The BIRCH trial is a single arm Phase II study evaluating atezolizumab in patients who have PD-L1+ locally advanced or metastatic NSCLC. Target enrollment is about 300 patients, who will be divided into three cohorts: patients who are chemo-therapy-naive, patients who have progressed after single prior platinum-based regimen, and patients who have received ≥2 prior lines of therapies. The OAK trial is a global Phase III randomized, open-label, multicenter trial evaluating atezolizumab compared with docetaxel (75 mg/m2 IV) after progression on platinum-contain-ing chemotherapy in patients with locally advanced or metastatic NSCLC. Both trials are ongoing with interim results pending [72].

Durvalumab (MEDI4736)MEDI4736, recently named durvalumab, is a human IgG1-κ PD-L1 inhibitor that prevents binding to PD-1 and CD80. The safety data for MEDI4736 is from an ongoing Phase I multicenter, open-label study (NCT01693562), evaluating its safety, pharmacokinet-ics (PK) and antitumor activity in patients with advanced solid tumors. MEDI4736 was given 10mg/kg IV every 2 weeks up to 12 months, or until unacceptable toxicity/disease progression. As of 31 October 2014, 198 patients were enrolled with 149 patients evaluable for response with ≥24 weeks of follow up. ORR was 14% (23% in PD-L1+) and disease control rate (DCR) at 24 weeks was 24%. Seventy-six percent of responses were durable (duration of response range 0.1+ to 35+ weeks). ORR was found to be higher in squamous (21%) compared with nonsquamous histology (10%).

Adverse events were observed in 48% of patients. The most frequently reported drug-related adverse events were fatigue (14%), decreased appetite (9%) and nausea (8%). Grade ≥3 drug-related AEs were reported in 6% of patients, with 2% of patients hav-ing drug-related AEs that led to discontinuation of the study [73,74].

Combination of checkpoint inhibitorsCheckpoint inhibitors have provided remarkable clini-cal benefit for patients with advanced NSCLC, but they are not effective in all patients. Clinical trials are now evaluating the use of checkpoint inhibitors in combination to improve response rates. Combinations of CTLA-4 and PD-1 inhibitors are being studied in Phase I/II clinical trials in hopes that combination therapy will produce additive or synergistic activity. The use of checkpoint inhibitors are also being evalu-ated for use in combination with targeted therapy in patients who harbor activating EGFR mutations or as second line therapy in patients who have progressed on initial EGFR TKIs. Additional trials are examining combination of immune checkpoint inhibitors with chemotherapy.

Interim Phase I results were reported for nivolumab and ipilimumab as first line in patients with advanced NSCLC (NCT01454102). The study included four cohorts of chemotherapy naive patients, both squamous and nonsquamous lung cancer who received 3 mg/kg nivolumab + 1 mg/kg ipilimumab or 1 mg/kg nivolumab + 3 mg/kg ipilimumab followed by nivolumab 3 mg/kg IV every 2 weeks until dis-ease progression or unacceptable toxicity. Forty-eight patients were evaluated at time of interim analysis. Overall ORR was 22% with stable disease rate of 33%. Of the seven squamous cell patients who received 1 mg/kg nivolumab + 3 mg/kg ipilimumab, there was 1 ongoing confirmed response (14%), 1 unconfirmed response (14%) and 2 patients with stable disease (29%). Of the 15 patients with nonsquamous NSCLC who received nivolumab 1 mg/kg + ipilimumab 3 mg/kg, there was 1 ongoing confirmed response (7%), 2 unconfirmed responses (13%) and 6 patients with stable disease (40%). Eight squamous cell NSCLC patients were treated with nivolumab 3 mg/kg + 1 mg/kg ipimumab. Two patients had confirmed response (25%), none of whom were ongoing respond-ers; three patients had unconfirmed response (38%), and stable disease was observed in four patients (50%). The last cohort of patients consisted of 16 patients with nonsquamous disease who received nivolumab 3 mg/kg + ipilimumab 1 mg/kg. Confirmed responses were observed in two patients (13%), both of whom were ongoing responders; four patients had unconfirmed


Review Xia & Herbst

responses (25%), and stable disease was observed in three patients (19%). Treatment related adverse events were reported in 39 patients (85%). Grade 3–4 toxici-ties were reported in 22 patients (48%) which led to 16 patients to discontinue treatment. 2 patients were observed to have unconventional “immune related” responses. ORR was reported to not be correlated with PD-L1 status in the 29 tumor samples that was available for analysis at time of interim data. The results showed that combination of nivolumab and ipi-limumab had acceptable toxicity and activity in both PD-L1+ and PD-L1- patients [75].

Another Phase I study examined the safety and tolerability of MEDI4736 with tremelimumab in patients with advanced tumors, including NSCLC, cervical, head and neck, colorectal, ovarian and renal cell carcinomas who were not eligible for, declined, or failed standard treatment (NCT01975831). The study design included dose escalation phase using a 3+3 design, with MEDI4736 administered at escalating doses starting at 0.3 mg/kg every 2 weeks; tremelim-umab was administered at escalating doses starting at 3 mg/kg up to 10 mg/kg, every 4 weeks for the first six cycles and then every 12 weeks. Primary end points were safety/tolerability of MEDI4736 and tremelim-umab when administered together. Preliminary data is still pending [76].

Combination of pembrolizumab plus ipilimumab as second line in stage IIIB/IV NSCLC is being studied in KEYNOTE-021 (NCT02039674). As of December 2014, 17 patients were enrolled to receive pembro 10 mg/kg + ipi 3 mg/kg, pembro 10 mg/kg + ipi 1 mg/kg, or pembro 2 mg/kg + ipi 1 mg/kg. Responses were seen at all dose groups without drug limiting toxicities or dose modifications. Eleven patients were on treatment for ≥6 weeks with 1 CR (9%) and 5 PRs (45%) [77].

PD-1 Inhibition combined with targeted therapyPreclinical evidence suggests that PD-L1 tumor expres-sion may be constitutively driven by EGFR signaling in EGFR mutant NSCLC. Treatment with anti-PD1 antibodies in preclinical EGFR mutant lung cancer models have demonstrated delayed tumor growth and increased survival [43,44].

Encouraging early activity has also been seen in the clinical setting when combining the anti-PD-1 antibody, nivolumab with erlotinib in patients with EGFR mutant NSCLC [78–80]. The Phase I study examined patients with Stage IIIB/IV EGFR mutant NSCLC who were chemotherapy naive or experienced progression after prior TKI therapy. Patients received nivolumab 3 mg/kg IV Q2W + erlotinib 150 mg PO daily until progression of disease or unacceptable toxic-

ity. Interim analysis included 21 patients who received treatment for >10 months prior to analysis. Only one patient did not receive prior treatment with EGFR TKI. Of the 20 patients, who had acquired erlotinib resistance, 3 patients were observed to have partial responses, all ongoing. Duration of response were 6.1+, 16.3+ and 27.1+ weeks. Nine patients (45%) had sta-ble disease with three patients (33%) having ongoing responses. One patient was observed to have uncon-ventional ‘immune-related’ response that was ongoing at the time of interim analysis. Treatment related grade 3–4 adverse events were reported in four patients, which included increased AST or ALT, weight decrease and diarrhea. Two patients were discontinued from the study due to treatment related grade 3 AST increase and grade 2 nephritis. No cases of pneumonitis were reported at time of interim analysis [80].

Checkpoint inhibitors & chemotherapyThere are several ongoing studies examining the role of adding immune checkpoint inhibitors to standard che-motherapy regimens for treatment of NSCLC. From the 2015 ASCO meeting, preliminary data are avail-able for pembrolizumab combined with chemotherapy and atezolizumab combined with chemotherapy in treatment naive patients.

Pembrolizumab is being investigated in combina-tion with carboplatin AUC 6 + paclitaxel 200 mg/m2 (cohort A: any histology) or carboplatin AUC 5 + pemetrexed 500 mg/m2 (cohort C: nonsquamous without EGFR mutation or ALK translocation) in patients with treatment naive stage IIIB/IV NSCLC (NCT02039674). After four cycles, patients in cohort A received maintenance pembro and patients in cohort C received maintenance pembro + peme-trexed. As of interim analysis, Dec 2014, 44 patients were treated with preliminary ORR (confirmed and unconfirmed) of 30% in cohort A and 58% in cohort C. Grade 3–4 treatment related adverse events were observed in 15% of patients in cohort A and 38% in cohort C. One patient in cohort C discontinued the study due to grade 3 rash. This study showed prom-ising ORR with pembrolizumab plus carboplatin and pemetrexed, which will be further evaluated in a larger cohort [81].

Atezolizumab was combined with chemother-apy in patients with previously untreated NSCLC (NCT01633970). The study combined atezolizumab with carboplatin + either paclitaxel (Arm C), peme-trexed (arm D), or weekly nab-paclitaxel (Arm E). At time of interim analysis on 29 September 2014, ORR was 67% (95% CI: 48–82%) across all arms, 60% (95% CI: 19–92%) in Arm C with 3 PRs, 75% (95% CI: 45–93%) in arm D with 9 PRs, and 62% (95% CI:



33–83%) in arm E with 6 PRs and 2 CRs. The most common adverse events across all arms were nausea (Arms C and D, 50%; Arm E 73%), fatigue (Arm C, 38%; Arm D, 36%; Arm E, 73%) and constipa-tion (Arm C, 25%; Arm D, 71%, Arm E, 27%). The responses across all arms were independent of PD-L1 expression. Results showed that adding atezolizumab to standard first line chemotherapy produced promis-ing clinical activity with acceptable drug related toxici-ties. Phase III studies evaluating this combination are still ongoing [82].

Combination of Nivolumab with platinum-based doublet chemotherapy as first line in NSCLC was evalu-ated in a Phase I multicohort study (NCT01454102). Preliminary data included 56 patients with advanced NSCLC who were assigned to four cohorts based on his-tology: nivolumab 10 mg/kg + gemcitabine/cisplatin for squamous cell, nivolumab 10 mg/kg + pemetrexed/cispl-atin for nonsquamous cell, nivolumab 10 mg/kg + pacli-taxel/carboplatin and nivolumab 5 mg/kg + paclitaxel/carboplatin for both squamous and nonsquamous cell lung cancer. ORR was 33–50% across all arms with no drug limiting toxicities observed during the first 6 weeks of treatment. Overall survival rates at one year were 59–87%. 45% of patients reported grade 3–4 treatment related adverse events (25–73% across all arms), includ-ing 4 patients with pneumonitis. This study is ongoing and currently demonstrates encouraging antitumor activity and 1-year overall survival [83].

Future perspectivePD-1/PD-L1 and CTLA-4 pathways appear to play dominant roles in T-cell immunosuppression. Block-ade of these targets have been shown to produce antitumor activity. However, pre-existing immunity may be necessary, which is further amplified dur-ing treatment [64]. The tumor microenvironment has been shown to have four distinct subtypes: presence of both PD-L1 and TILs; presence of TILs without PD-L1; presence of PD-L1 with absence of TILs; absence of both PD-L1 and TILs [84,85]. Each sub-group likely has a different mechanism of immune resistance comprised of unique suppressive factors and inhibitory ligand-receptor interactions that need to be further characterized. On-treatment biopsies of nonresponders to PD-L1 therapy showed tumors with three patterns: an immunologic ignorance pattern displaying little or no tumor-infiltrating immune cell infiltration; nonfunctional immune response with presence of intratumoral immune infiltrate with min-imal to no expression of PD-L1; excluded infiltrate response with immune filtrate residing solely on the outer edge of tumor cell mass [64]. Additional stud-ies are needed to better understand immune regula-

tory pathways that may be involved in these nonre-sponders and identify treatment strategies to amplify immune response.

Identifying molecular markers to treatment response remains a challenge. Although some studies have shown that PD-L1 and CTLA-4 expression in tumor or immune microenvironment may correlate with response to therapy [53,64], other studies suggest that tumor PD-L1 status is not prognostic nor predictive of efficacy [51]. This discrepancy may be explained by heterogeneity of PD-L1 expression on biopsy samples leading to reporting inconsistencies, and technical dif-ficulties with PD-1/PD-L1 analysis due to the harsh conditions required for antigen retrieval progress [3]. Table 2 summarizes the major biomarker driven trials described in this article, highlighting the different ways PD-L1 expression has been quantified. Further investigation is needed to develop improved diagnos-tic assays and identification of blood-based immune biomarkers.

The role of immunotherapy is also being explored in early stage lung cancer. Despite optimal treatment, recurrence rates remain high with 5-year survival of less than 50% [86–88]. Unpublished preliminary data have suggested that perhaps tumors at an earlier stage of disease may have higher PD-L1 levels compared with late stage disease [88]. Therefore, there may be a role for checkpoint inhibitors to improve recurrence rate and survival in early stage lung cancer. A Phase II trial is evaluating the role of nivolumab in the neo-adjuvant setting in patients with resectable NSCLC (NCT0225962) and will further explore changes to cellular and molecular characteristics in the tumor microenvironment of early stage, potentially curable disease.

Novel multimodality treatment strategies are explor-ing combining checkpoint inhibitors with tumor vac-cines (NY-ESO-1) and other new molecules. Table 3 summarizes current ongoing clinical trials involving immune checkpoint inhibitors. Nivolumab is being combined with lirilumab, a monoclonal antibody against killer cell immunogolublin-like receptors (KIR) on NK cells, which has shown promising pre-clinical activity in lymphoma [89]. A Phase I/II study is exploring combination of nivolumab with varlilumab, an anti-CD27 antibody in patients with advanced solid tumors. Additional early phase clinical trials are evaluating immune check point inhibitors in combi-nation with an antibody against B7-H3 (MGA271), with an inhibitor of Indoleamine 2,3-dioxygenase 1 (IDO1) which is a tryptophan-catabolizing enzyme overexpressed in cancer cells that suppresses T-cell response, and with pegylated recombinant human IL-10 (AM0010) [13,90–93].


Review Xia & Herbst

Financial & competing interests disclosureRS Herbst is on the Scientific Advisory Boards of Biothera,

Diatech, Kolltan, N of 1; and is a consultant for Eli Lilly,

Genentech/Roche, Gilead, Merck, Pfizer; he has received

clinical trials support/grant from Genentech and Merck.

The authors have no other relevant affiliations or financial

involvement with any organization or entity with a finan-

cial interest in or financial conflict with the subject matter

or materials discussed in the manuscript apart from those

disclosed.

No writing assistance was utilized in the production of this

manuscript.

Executive summary

Immune checkpoint inhibition in lung cancer• Inhibition of immune checkpoints with anti-CTLA-4, PD-1 and PDL-1 agents have produced promising results

demonstrating delayed tumor growth and increased survival. The durability of effect is most striking in those who respond.

Anti CTLA-4• CTLA-4 binds CD86 and suppresses activation and expansion of effector cells through regulation of Tregs.

Inhibition of the CTLA-4 pathway with ipilimumab has improved immune-related progression free survival when given with standard chemotherapy, paclitaxel and carboplatin, compared with chemotherapy alone.

Anti-PD-1/PD-L1• PD-1 is expressed by activated T cells and is a key immune checkpoint receptor that mediates

immunosuppression. Inhibition of the interaction between PD-1 and PD-L1 has produced durable antitumor activity in NSCLC. Nivolumab has been FDA approved as second line treatment for metastatic squamous cell lung cancer and has now been shown to improve survival in patients with nonsquamous cell lung cancer as well. Phase I and II studies for anti-PD-L1 agents, atezolizumab (MPDL3280A) and durvalumab (MEDI4736), have shown these drugs have acceptable toxicity profile and have promising activity.

Biomarkers• Tumor infiltrating immune cell PD-L1 expression and expression of CTLA-4 were associated with response

to atezolizumab. However, there was not an association between tumor cell PD-L1 expression and response to atezolizumab. Fractalkine identified in pretreatment tumors was correlated with disease progression on atezolizumab treatment. Other smaller studies have shown increased efficiency of atezolizumab with high PD-L1 expression, IC or TC of 3.

• Higher response rate to pembrolizumab has been shown with tumor cell PD-L1 expression of ≥50%.• There has been conflicting data on PD-L1 status and response to nivolumab. The CheckMate-017 study showed

that PD-L1 status was not prognostic or predictive of efficacy, while CheckMate-057 showed that PD-L1 status correlated with response to treatment. These differences could be due to differences in the tumor types (i.e., smoking status) or speak to the need for better developed biomarker studies.

Current limitations & future trends• Immune checkpoint inhibitors are being studied in the neoadjuvant setting for resectable disease to better

characterize the tumor and immune microenvironment in early stage disease. Identification of molecular markers in the tumor, immune microenvironment and peripheral blood that correlate with treatment response or disease progression is needed. Further characterization of the immune profile in nonresponders and in those who progress on treatment will provide valuable information to help develop novel strategies to overcome resistance. New trials are now combining checkpoint inhibitors with chemotherapy, targeted therapy, radiation and other new antibodies in hopes of producing more durable response.

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1011Immunotherapy (2016) 8(9), 1011–1019 ISSN 1750-743X10.2217/imt-2016-0032 © 2016 Future Medicine Ltd

Immunotherapy

Research Article 2016/07/288

9

2016

Background: Recent studies with nivolumab (a monoclonal antibody against programmed cell death 1 [PD-1] receptor) have shown promise non-small-cell lung cancer (NSCLC) treatment. Methods: To review available clinical trials data in order to assess nivolumab efficacy and the role of tumoral PDL-1 expression as a biomarker. Results: Nine eligible studies included 2102 patients. In the second line setting, nivolumab achieved a 1-year survival rate of 41%; and in the first line, a 1-year survival rate of 76%. For those with PD-L1 expression <1%, nivolumab showed a trend for improved survival compared with docetaxel. Conclusions: The available data reinforce nivolumab activity against NSCLC in first-line or subsequent lines. Although PD-L1 expression is related to greater response, PD-L1 negative patients had also some benefit.

First draft submitted: 21 February 2016; Accepted for publication: 26 May 2016; Published online: 3 August 2016

Keywords: lung cancer • meta-analysis • nivolumab

Most cancer cells emerge and develop through the acquisition and accumulation of several genetic alterations, mainly mutations [1,2]. These mutations can produce aberrant pro-teins that can serve as neoepitopes, which are recognized by the immune system [3]. The immune system is able to recognize and destroy tumor cells as well as pathogens. Nev-ertheless, one of the hallmarks of cancer is its ability to evade the immune system [1].

Programmed cell death 1 (PD-1) is a T-cell surface receptor that inhibits immune response when bound to its ligands (PD-L1 or PD-L2) present in normal human cells as well as in cancer cells. PD-1 is the target of immunotherapeutic agents such as nivolumab. Moreover, several other agents target PD-L1 [4–6]. Nivolumab is a fully human IgG4 antibody that enhances the immune response against cancer. Immune checkpoint inhibitors, such as nivolumab, have already been studied in many neoplasms and numerous other studies are ongoing [7,8].

Non-small-cell lung cancer (NSCLC) is among the main causes of cancer-related deaths worldwide [9]. Data from Surveillance, Epidemiology, and End Results (SEER) Pro-gram database of the American National Cancer Institute showed that 57% of the cases are diagnosed at stage IV (metastatic disease) and the 5-year survival rate in this group is only 1–4% [10]. In the last decade, several improvements in NSCLC therapy with the development of novel targeted can-cer therapies, including monoclonal anti-bodies [11] and oral tyrosine kinase inhibitors have emerged [12]. Nevertheless, these agents are active only in a subset of patients whose tumors harbor specific genetic alterations [2]. There are few options to treat NSCLC, e specially in the second-line setting [13].

The first studies with nivolumab have been conducted exactly in this setting and showed promising results [5,14–16]. The USFDA has already approved nivolumab for the second-line treatment of squamous-cell

A pooled analysis of nivolumab for the treatment of advanced non-small-cell lung cancer and the role of PD-L1 as a predictive biomarker

Pedro N Aguiar Jr1, Ilka Lopes Santoro1, Hakaru Tadokoro1, Gilberto de Lima Lopes3, Bruno Andraus Filardi1, Pedro Oliveira4, Pedro Castelo-Branco5, Giannis Mountzios6 & Ramon Andrade de Mello*,5,7,8

1Division of Medical Oncology, Federal

University of São Paulo, São Paulo, Brazil 2Oncoclinicas do Brasil group, São Paulo,

Brazil 3Department of Medical Oncology, Johns

Hopkins University, Baltimore,

MD 21218, USA 4Department of Population Studies, Abel

Salazar Biomedical Institute, University of

Porto, Porto, Portugal 5Division of Oncology, Department

of Biomedical Sciences & Medicine,

University of Algarve, Faro, Portugal 6Department of Medical Oncology,

University of Athens Medical School,

Athens, Greece 7Department of Medicine, Faculty of

Medicine, University of Porto, Porto,

Portugal 8Research Center, Cearense School of

Oncology, Instituto do Câncer do Ceará,

Fortaleza, CE, Brazil

*Author for correspondence:

Tel.: +351 289 800 094

Fax: +351 289 800 076

[email protected];

[email protected]

part of

Research Article



Research Article Aguiar, Santoro, Tadokoro et al.

and non-squamous cell NSCLC based upon results of two large Phase 3 trial [15,16].

Several studies regarding immune checkpoint inhibi-tors assessed the tumor PD-L1 expression as a predic-tive biomarker of such treatments with heterogeneous results [17]. Furthermore, PD-L1 expression assessment remains a controversial topic of debate in the scientific community. Taking under consideration all those para-mount features, we conducted a pooled analysis to assess the efficacy and safety of nivolumab and the role of the tumor PD-L1 expression as a biomarker to predict tumor response and other parameters of clinical benefit.

MethodsData sourcesWe searched for clinical trials in electronic data-bases (Pubmed, Lilacs and Cochrane Library) from 2000 to June 2015. MeSH terms included as follows: ‘non-small-cell lung cancer’, ‘anti PD-1’, ‘anti PD-l1’, ‘nivolumab’ and their synonyms. Second, we searched conference papers (American Society of Clinical Oncology (ASCO), European Society for Medical Oncology (ESMO) and International Association for the Study of Lung Cancer (IASLC)) in order to update included studies and search for additional eligible tri-als. Finally, we assessed references of included studies for potentially eligible studies.

Study selectionStudy inclusion criteria were as follows: manuscript published in English; and prospective clinical trial that assessed efficacy and safety of nivolumab in the treat-ment of NSCLC, regardless of the number of previous treatments, tumor histology and study regimen.

Data extractionWe used The Cochrane Collaboration Guideline to assess study’s quality, collect data, evaluate the risk of bias and perform analyses. The authors analyzed the randomization process, the presence or absence of masking and the outcomes report in order to evaluate the risk of bias. Two independent reviewers assessed each eligible study [18]. All disagreements were resolved by an authors’ presential meeting and by a videoconfer-ence with a third reviewer. The data of feasible studies were extracted according to Cochrane’s protocol [19]. Study results were interpreted with caution regard-ing the risk of potential bias. All issues were assessed according to the findings published in the studies. Therefore, we did not ask for individual data.

Main outcomes & measuresThe Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 guided image assessment for

overall response rate (ORR) in all studies. Although immunotherapy may produce a heterogeneous response pattern that is not better evaluated by RECIST, all studies used this method because it is the most con-solidated in the literature and it is easily audited or reproduced. The interval between each assessment was defined by each author; however, in the majority of cases, computed tomography or magnetic resonance imaging assessed the clinical activity at baseline and at 6, 12 and 16 weeks, and every 8 weeks thereafter. We considered either the investigator’s assessment or a central assessment.

Progression-free survival (PFS) was defined as the time, in months, from study inclusion until the first evidence of disease progression or death for any cause, whichever occurred first. In cases of short follow-up, we assessed the 24-week PFS rate. Overall survival (OS) was the time, in months, from study inclusion to death. Besides PFS, some trials did not evaluate the entire survival curve, and thus we considered the 1-year survival rate.

Studies that assessed tumor PD-L1 expression used immunohistochemistry with Dako North America antibodies (Antibody Dako 28–8). First trials estab-lished 5% as the optimal cut-off value but subsequent studies reported different results according to each cut-off value ranging from 1, 5 to 10%. In this article, we considered a cut-off value of 5%.

Nivolumab as well as other immune checkpoint inhibitors are generally well tolerated. Because of this, we did not include detailed adverse events analysis. Only highlights of the safety were cited in this manu-script.

Statistical analysisAuthors used Review Manager Version 5.3 in this assess-ment, developed and freely available from the Cochrane Collaboration. Pooled analysis of ORR, 24-week PFS rate and 1-year OS rate were assessed using descrip-tive statistics. Authors performed exploratory analysis according to the line of treatment and tumor histology. In this meta-analysis, we used the risk ratio (RR) for statistical efficacy analysis. Survival variables were eval-uated using the hazard ratio (HR). We assessed hetero-geneity using the I2 statistic. A value less than 25% was minimal and from 25% to 50% was moderate. In these cases, the authors adopted the fixed model. Otherwise, I2 higher than 50% indicated substantial heterogeneity and the random model was used. p-values <0.05 were considered statistically significant.

ResultsSearch resultAn initial search identified 82 studies. Only three


Nivolumab for the treatment of advanced NSCLC & the role of PD-L1 as a predictive biomarker Research Article

c omplied with the inclusion criteria [14,15,20]. Six addi-tional studies were identified after the conference papers review [7,16,21–25]. A total of 2102 patients were included in the nine eligible studies. Figure 1 illustrates the search process.

Eight studies assessed nivolumab in previously treated patients; two of them were randomized Phase 3 trials comparing nivolumab with docetaxel in the second-line setting. In the first-line setting, there was only one trial with different regimens in each arm [21,23–25]. One of these arms included ipili-mumab in combination with nivolumab. Ipilimumab is a monoclonal antibody against another recep-tor that inhibits T-cells death. This different path-way may confuse the results, particularly regarding PD-L1 as a biomarker. However, we included this study arm because of the paucity of first-line setting studies. Table 1 summarizes the characteristics of the included studies. The number of patients treated with nivolumab among all included trials was 1643 (459 patients were treated with docetaxel). Table 2

describes the main characteristics of patients treated with nivolumab.

Efficacy & safety: first-line settingA large Phase I trial assessing the safety and efficacy of nivolumab in the first-line setting is ongoing. It has eight arms; in this analysis, we assessed four of them separately. Despite a substantial heterogeneity among treatment regimens, nivolumab showed activity in the first-line setting. Among 146 patients included in these reports, 29% achieved tumor response and the 1-year survival rate was 76%.

Overall, nivolumab was well tolerated in the first-line setting. Reports described 40% of adverse events of any grade; however, they were manageable, espe-cially if detected early. There were few cases of treat-ment interruption and treatment-related three deaths were reported. Although immune-mediated adverse events, mainly pneumonitis, are a worrying side effect, they occurred in a minority (9% of patients; 5% were grade 3 or 4).

Figure 1. PRISMA statement of the search result.

Records identi�ed through databasesearching (n = 82)

Records screened (n = 3)Records excluded:• Not clinical trials (n = 79)

Full text article assessed for eligibility (n = 9)

Studies included in qualitative synthesis (n = 9)

Studies included in quantitative synthesis(meta-analysis) (n = 9)

Conference paper assessment• Studies updated (n = 3)• Recent studies included (n = 6)



Table 1. Characteristics of included studies.

Study First author

Year Phase Population Dose n Antibody Sample Cutoff Response assessment

Ref.

CheckMate 063

Rizvi 2015 II SQ NSCLC after failure

3 mg/kg q2w 117 Dako Tumor 5% RECIST 1.1 [14]

CheckMate 003

Brahmer 2014 I NSCLC after failure


3 mg/kg q2w 37

10 mg/kg q2w 59

CheckMate 017

Brahmer 2015 III SQ NSCLC after failure


Docetaxel 75 mg/m² q3w

137

CheckMate 057

Paz-Ares 2015 III non-SQ NSCLC after failure


Docetaxel 75 mg/m² q3w

290

Bauer 2015 III/IV NSCLC after failure

3 mg/kg q2w 824 NA NA NA RECIST 1.1 [7]

CheckMate 012

Antonia 2014 I NSCLC chemonaive

10 mg/kg q3w + gem/cis

12 NA NA NA RECIST 1.1 [23]

10 mg/kg q3w + pem/cis

15

10 mg/kg q3w + pac/carb

15

5 mg/kg q3w + pac/carb

14

Rizvi 2014 I NSCLC chemonaive, EGFR +

3 mg/kg q2w + erlotinib

21 NA NA NA RECIST 1.1 [25]

Gettinger 2015 I NSCLC chemonaive


Antonia 2014 I NSCLC chemonaive

1 mg/kg q3w + ipi 3 mg/kg

24 Dako Tumor 5% RECIST 1.1 [21]

3 mg/kg q3w + ipi 1 mg/kg

25

Efficacy & safety: second-line setting & beyondIn the second-line setting and beyond, the ORR was 17% among 1204 patients assessed. Analyzing by histology, squamous cell NSCLC achieved higher response (21% among 451 patients) than nonsqua-mous cell NSCLC (15% among 752 patients; p = 0.01). Interestingly, the 1-year survival rate was lower among patients with squamous cell NSCLC (32 vs 48%; p < 0.01). For the entire population, four studies with 673 patients were analyzed showing a 1-year survival rate of 41%.

Nivolumab was well tolerated in second- or third-line: in five studies with 1488 patients, 25% expe-rienced grade 3 or 4 adverse events. Pneumonitis occurred in 29 (2%) patients 11 (0.7%) of which were severe (grade 3 or 4).

Nivolumab vs docetaxel in the second-line settingTwo Phase 3 trials addressed this issue. In their meta-analysis, nivolumab reached a statistically sig-nificant higher ORR (RR: 1.74; 95% CI: 1.25–2.43; Figure 2A), as well as a better prognosis (PFS HR: 0.82; 95% CI: 0.71–0.95; Figure 2B and OS HR: 0.69; 95% CI: 0.58–0.81; Figure 2C) compared with docetaxel in the second-line setting.

PD-L1 expression as a predictive biomarkerSix studies with 576 patients assessed the PD-L1 expression as a biomarker of clinical benefit. Among 237 PD-L1 ‘positive’ patients (>5% of expression), the ORR was 27%. Among 339 PD-L1 ‘negative’ patients (<5% of expression), the ORR was 13%. H eterogeneity



was minimal and this difference was statistically sig-nificant (RR: 2.04; 95% CI: 1.37–3.04; Figure 3A). PD-L1 positive patients also had a non-statistically significant higher 24-week PFS rate (RR: 0.93; 95% CI: 0.79–1.11; Figure 3B) and 1-year survival rate (RR: 0.90; 95% CI: 0.65–1.26; Figure 3C).

Nivolumab was numerically superior to docetaxel even among patients with a PD-L1 expression <1%. ORR was 13 versus 12% (RR: 1.04; 95% CI: 0.58–1.87) and there was a trend towards better OS (HR: 0.78; 95% CI: 0.60–1.01). The role of PD-L1 as a predictive biomarker for overall survival was more rel-evant for non-squamous tumors (patients with PD-L1 <1% HR: 0.90; 95% CI: 0.66–1.24) than for squa-mous tumors (patients with PD-L1 < 1% HR: 0.58; 95% CI: 0.37–0.92).

Risk of bias assessmentThe authors considered that the high proportion of undefined bias in the study’s patient randomization was because many trials did not have a control arm. The high risk of bias in the blinding was not harm-ful to analysis results, and it is possible to allow this because many studies were not Phase 3 trials and had to assess the safety of these novel agents. In general, another type of bias (outcomes report) was considered low.

DiscussionNivolumab showed activity in the treatment of NSCLC regardless of tumor histology and number of previous treatments. The agent was slightly more active among patients with squamous-cell histol-ogy. It has been hypothesized that in squamous cell NSCLC, tumor cells have a high mutation burden, and consequently, they can produce more aberrant proteins as well as neoepitopes recognized by the immune system [3]. On the other hand, patients with squamous-cell NSCLC had a worse 1-year survival rate. We presume that tumor biology and availability of few postprogression t reatment options may explain this finding.

Although in the first-line setting the number of patients was small and the heterogeneity of regimens was substantial, we feel these results may stimulate the development of Phase 2 or 3 trials assessing this issue. The design of these novel trials presents a major challenge. First, there are many regimens as well as targeted therapies to combine and/or to compare. Second, it is important to consider that nivolumab as well as other immune checkpoint inhibitors has a specific pattern of antitumor activity and may not be the best option for all patients in this setting. Fur-thermore, tumor response criteria using the RECIST

v1.1 may not be the most appropriate marker of c linical activity and may lead to an under-estimation of the immunotherapeutic drug efficacy [5]. Studies described unusual tumor responses compared with those currently observed for cytotoxic agents as fol-lows: lesions that grow first and then start to reduce in size, lesions that reduce while new lesions develop, or lesions stable for a long period. This promoted the development of new response assessment criteria [26]. However, this criteria was not commonly used in clinical trials. Therefore, patient inclusion criteria and study end points should be chosen carefully.

Despite the aforementioned constraints regarding tumor response with nivolumab, we argue that the results of this analysis in the second-line and beyond are an important contribution to the current state of the art. The FDA currently approved nivolumab for the treatment of patients with either squamous or non-squamous NSCLC who were previously treated with chemotherapy or targeted therapy. Of note, previous studies in patients with advanced melanoma with lon-ger follow up showed tumor response sustained for a

Table 2. Baseline characteristics of the patients.

n %

Gender

Male 948 58

Female 695 42

Age Median 66 years

Tumor histology

Squamous NSCLC 576 35

Non-squamous NSCLC 1066 65

Unknown or other 1 <1

Number of prior treatments

0 137 8

1 646 39

2 317 19

3 or more 460 28

Unknown 83 5

Performance status

ECOG 0 or 1 1557 95

ECOG 2 67 4

Unknown or other 19 1

Tobacco history

Current or past 1181 72

Never 179 11

Unavailable 283 17

n: Number of patients, ECOG: Eastern Cooperative Oncologic Group Performance Status Criteria.



long time period [27]. Nivolumab also showed activity and survival superior to docetaxel regardless of tumor histology. In the current meta-analysis, nivolumab had a statistically significant better PFS compared with docetaxel. We believe these results enhance the evi-dence regarding nivolumab superiority over docetaxel for the second-line treatment of NSCLC regardless of histology.

In general, nivolumab was well tolerated and adverse events were manageable. The high suspicion for immune related adverse events and a quick response to them reduces the number of patients with severe adverse events. The PD-L1 expression was assessed as a predictive biomarker for immune checkpoint inhib-itors because of its relationship with the agents’ tar-get. However, there is currently no standard approach to assess this biomarker in routine clinical practice and the optimal cut-off value remains to be defined. Nevertheless, all studies used immunohistochemistry with the same antibody and the same assessment cri-teria. The PD-L1 expression was not a reliable pre-dictive biomarker, despite the higher ORR observed among PD-L1 positive patients. Among patients with a PD-L1 expression < 1% nivolumab also had

c onsiderable activity for non-squamous histology and was superior to docetaxel. For nonsquamous tumors, PD-L1 seems to be more reliable. We conclude that the study of other biomarkers is warranted to improve prediction of clinical activity in this setting.

ConclusionNivolumab shows activity in advanced NSCLC in both first- and second-line settings and it was supe-rior to docetaxel with respect to ORR, PFS and OS in both squamous cell and nonsquamous cell NSCLC. Tumor PD-L1 overexpression was related to higher ORR; however, nivolumab was also active among patients with PD-L1 expression <1% and nonsqua-mous histology.

Future perspectiveFurther studies are warranted to identify a reliable bio-marker and to assess the role of nivolumab in the first-line setting.

An extended follow-up may emphasize the long-term benefits of nivolumab and may help to identify predictive features for patients with response sustained for a long time.

Figure 2. Clinical outcomes regarding nivolumab versus docetaxel. (A) Overall response rate – nivolumab vs docetaxel, (B) progression-free survival – nivolumab vs docetaxel, (C) overall survival – nivolumab vs docetaxel.

A

B

C



Author contributionsDr de Mello had full access to all of the data in the study and

takes responsibility for the integrity of the data and the accu-

racy of the data analysis. Study concept and design: de Mello.

Acquisition, analysis, or interpretation of data: Aguiar Jr, de

Mello, Mountzios, Santoro, Oliveira, Castelo-Branco, Lopes

Jr. Drafting of the manuscript: Aguiar Jr, Lopes Jr, Tadokoro,

Mountzios, de Mello. Critical revision of the manuscript for

important intellectual content: Statistical analysis: Adminis-

trative, technical, or material support: Study supervision: de

Mello.

AcknowledgementsThe authors thank Professor Rachel Riera, a member of Brazil-

ian Cochrane Center, who helped us to search the literature

and strictly follow the Cochrane Guideline.

Financial & competing interests disclosureAguiar Jr has received speaker honoraria from Bristol Meyers

Squib and received travel funding from Bristol Meyers Squib,

Roche, and Pfizer. Lopes received research funds and honor-

ary from MSD, Lilly, Roche, Astra Zeneca, BMS, and Sanofi.

Mountzios has received honoraria, and travel funding from Bris-

tol Meyers Squib, Roche, Amgen, and Novartis. de Mello has

received honoraria from Astra Zeneca, Zodiac, Pfizer Advisory

Board, National Science Centre, Krakow, Poland, and educa-

tional grant from Pierre Fabre. Mello is ad hoc consultant at the

Ministry of Health, Brasília, Brazil. The authors have no other

relevant affiliations or financial involvement with any organi-

zation or entity with a financial interest in or financial conflict

with the subject matter or materials discussed in the manuscript

apart from those disclosed. No writing assistance was utilized

in the production of this manuscript.

Figure 3. Clinical outcomes according to the PDL1 status. (A) Overall response rate by PD-L1 expression status, (B) 24-weeks progression-free survival rate by PD-L1 expression status, (C) 1-year survival rate by PD-L1 expression status.

A

B

C



Ethical conduct of researchThe authors state that they have obtained appropriate institu-

tional review board approval or have followed the principles

outlined in the Declaration of Helsinki for all human or animal

experimental investigations. In addition, for investi¬gations

involving human subjects, informed consent has been ob-

tained from the participants involved.

Executive summary

• Few treatment options are available for squamous non-small-cell lung cancer despite recent advances, especially in the second-line setting.

• Nivolumab has been studied in several neoplasms such as lung cancer. The antibody is currently being assessed in the first-line and subsequent lines of treatment. The aim of this study is to assess the efficacy of nivolumab and the role of PD-L1 as a predictive biomarker.

Methods• A pooled analysis of all available data regarding nivolumab for the treatment of advanced lung cancer.• Nine trials with 2102 patients were included.Results• Nivolumab has shown activity in the first-line and subsequent lines of treatment.• In second-line setting, nivolumab achieved an overall response rate (ORR) of 17% (five studies, 1204 patients)

and a 1-year survival rate of 41% (four studies, 673 patients).• PD-L1 expression was a predictive biomarker for tumor response.• Patients with PD-L1 < 1% and squamous histology had also significant activity with nivolumab.• PD-L1 overexpression was related to higher ORR; however, nivolumab provided superior activity to docetaxel

even among PD-L1 negative patients.Discussion• Nivolumab is active in the treatment of advanced lung cancer, but further studies should find a more reliable

predictive biomarker and better evaluate nivolumab in the first-line of the treatment.

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Immunotherapy

Review 2016/04/288

5

2016

Immune checkpoint inhibitors have been identified as breakthrough treatment in melanoma given its dramatic response to PD-1/PD-L1 blockade. This is likely to extend to many other cancers as hundreds of clinical trials are being conducted or proposed using this exciting modality of therapy in a variety of malignancies. While immune checkpoint inhibitors have been extensively studied in melanoma and more recently in lung cancer, little is known regarding immune checkpoint blockade in other cancers. This review will focus on the tumor immune microenvironment, the expression of PD-1/PD-L1 and the effect of immune modulation using PD-1 or PD-L1 inhibitors in patients with head and neck, prostate, urothelial, renal, breast, gastrointestinal and lung cancers.

First draft submitted: 19 December 2015; Accepted for publication: 4 March 2016; Published online: 3 May 2016

Keywords: atezolizumab • immune checkpoint blockage • immune checkpoint inhibitors •immunotherapy • nivolumab • PD-1 • PD-L1 • pembrolizumab • solid tumors • tumor infil-trating lymphocytes

The discovery of immune checkpoints repre-sents a new era in cancer treatment. Recently, it became evident that tumors can hijack the immune tolerance mechanism by overex-pressing these immune checkpoints placing a brake on the activation of immune system [1]. Inhibiting these immune checkpoints or their ligands can lift the break on the immune sys-tem leading to a powerful immune response against tumor cells. Not surprisingly, immune checkpoints were first investigated in melanoma given the well-known immu-nogenic nature of this malignancy [2]. The CTLA-4 antibody ipilimumab was the first immune checkpoint inhibitor that showed activity in melanoma treatment [2] followed by PD-1 inhibitors [3,4]. Since then many studies have been conducted to expand the indication of immune checkpoints beyond melanoma. This review will focus on these studies targeting PD-1/PD-L1 pathway in cancers other than melanoma.

The Programmed Death Pathway (PD-1) is an inhibitory receptor that belongs to the B7-receptor family and binds to its ligands PD-L1 (also known as B7-H1) and PD-L2. When T cells are activated they start to express PD-1 which interacts with PD-L1 on tumor cells leading to T-cell deactivation and apoptosis, therefore, downmodulating the immune response against tumors [5,6]. PD-1 is expressed on a variety of tumor infiltrating lymphocytes (TILs) such as activated T- and B-lymphocytes, natural killer (NK) cells and myeloid cells while PD-L1 is expressed not only on tumor cells but also on stroma, antigen presenting cells and tumor-associated macro-phages [5,6]. The downregulation of the T-cell response is thought to allow the unchecked growth of tumor cells and also represents one of the many tumor immune escape mecha-nisms that regulate the immune response against cancer cells [1,7]. More recently, a new stratification of tumors was proposed

Beyond melanoma: inhibiting the PD-1/PD-L1 pathway in solid tumors

Ryan Gentzler‡,1, Richard Hall‡,1, Paul R Kunk1, Elizabeth Gaughan1, Patrick Dillon1, Craig L Slingluff Jr2 & Osama E Rahma*,1

1Division of Hematology-Oncology,

Department of Medicine, University of

Virginia Health System, Charlottesville,

VA, USA 2Departement of Surgery, University of

Virginia Health System, Charlottesville,

VA, USA


[email protected]‡Authors contributed equally

part of

Review


Review Gentzler, Hall, Kunk et al.

based on PD-L1 status and presence or absence of the TILs [8]. Based on this classification, tumors with PDL-1 and TILs positive were called ‘immune resis-tant’ since the expression of PD-L1 was thought to be driven by TIL-induced INF-γ, while tumors with both PD-L1 and TILs negative were called ‘immune ignorant’. On the other hand, the induction of PD-L1 in PD-L1 + TILs – tumors was defined as ‘intrinsic induction’ that may be related to oncogenic induction of PD-L1 rather than TILs driven. Finally, the pres-ence of TILs in PD-L1- tumors was blamed on other suppressors in a type called ‘tolerant tumors’. This clas-sification may help to better understand the biology of the PD-1/PD-L1 pathway in order to choose an appro-priate immunotherapy [9]. Although the expression of PD-L1 was reported to correlate with poor survival in many malignancies including NSCLC, GU and GI cancers [10–12] this correlation remains unclear given the retrospective nature of this data. Considerably less is known about PD-L2. Compared to the expression of PD-L1 in most cancers, constitutive PD-L2 expression is low but can increase in response to Th2 cytokines leading to the suppression of CTL reactivity [13]. The overexpression of PD-L2, however, has been found in a variety of cancers including primary mediastinal B-cell and Hodgkin lymphoma [14] but its correlation to survival remains unclear.

Squamous cell carcinoma of the head & neckMuch research has elicited the risk factors of alcohol, tobacco use and the human papillomavirus (HPV) in head and neck cancer, suggesting a role of chronic inflammation that results in immune system escape and tumor growth [15]. Despite this, little is known regarding the lack of immune response in squamous cell carcinoma of the head and neck (SCCHN). The expression of PD-L1 is higher in SCCHN patients with HPV infection compared with those without (70 vs 29%), possibly due to the inflammatory reac-tion that is driven by HPV infection [15]. Targeting the PD-1/PD-L1 pathway is an active area of investiga-tion, primarily resulting from abstracts at international conferences, although multiple trials are ongoing to define the role of these agents in the management of SCCHN. The KEYNOTE-012 multi-cohort Phase Ib study of the anti-PD-1 antibody pembrolizumab included patients with advanced SCCHN. In 2014, an analysis of 60 selected PD-L1-positive patients from this group with recurrent/metastatic SCCHN was presented [16]. PD-L1 expression in stroma or ≥ 1% tumor cells was required for inclusion. These patients received pembrolizumab 10 mg/kg every 2 weeks. The overall response rate (ORR) was 20% and was similar in HPV positive and negative patients. 58%

of patients experienced a drug-related adverse effect (AE). Subsequently, at the 2015 ASCO Annual Meet-ing, results from a different SCCHN cohort from the KEYNOTE-012 trial including PD-L1 unselected patients were presented [17]. 132 patients with recur-rent/metastatic SCCHN were treated with a fixed dose of pembrolizumab 200 mg IV every 3 weeks. ORR was 18.2% and 47% of patients had a drug-related AE. Furthermore, early studies of the anti-PD-L1 antibody durvalumab (formerly MEDI4736) reported tumor shrinkage in treated patients with recurrent/metastatic SCCHN [18]. CTLA-4 blockade is under investigation as single agent therapy and in combination with anti-PD-1 therapy for recurrent/metastatic SCCHN.

Glioblastoma multiformeGlioblastoma multiforme (GBM) is another aggressive malignancy with little known about its relationship to the immune system. Recent data from the 2015 ASCO Annual Meeting evaluated TILs infiltration as well as PD-1 and PD-L1 expression in GBM tumor samples from 117 patients [19]. Dense immune cell infiltration was not found, but spare infiltration of CD8+ cells was found in 44% of tumors with spare PD-1 expression on TILs seen in 29% of tumors. PD-L1 expression on tumor cells was observed in 38% of tumor samples (defined as >5%). No correlation was found between PD-1 or PD-L1 and TILs density or patient outcome. Interestingly, 22% of tumors were PD-L1+ initially but lost PD-L1 expression at the time of recurrence. In the same meeting, preliminary results were presented on cohort 1 of the Phase I CHECKMATE-143 trial [20]. Twenty patients with recurrent GBM were treated with single agent nivolumab 3 mg/kg every 2 weeks or com-bination nivolumab 1 mg/kg + ipilimumab 3 mg/kg every 3 weeks followed by nivolumab 3 mg/kg every 2 weeks. All patients were in their first recurrence, had no prior bevacizumab and a Karnofsky Performance Score >70 with primary endpoint of safety/tolerability. No grade 3–4 AEs were seen in the nivolumab alone group, whereas nine out of ten patients in the combi-nation group had a grade 3–4 AE (most commonly lipase elevation, fatigue and diarrhea). Overall survival at 6 months in both groups was 75%. Pembrolizumab is currently under investigation in this patient popula-tion (NCT02337491 and NCT02337686).

Urothelial carcinomaAdvanced urothelial carcinomas (transitional cell car-cinoma of the bladder, renal pelvis, ureter and urethra) are currently treated with cisplatin-based chemother-apy, with significant challenges in this patient popu-lation due to advanced age, comorbid conditions and poor tolerance. For nonresponders, there is l imited


Inhibiting the PD-1/PD-L1 pathway in solid tumors Review

benefit of second and subsequent lines of cytotoxic chemotherapy, highlighting the need for effective therapy. Urothelial cancers have been shown to have a high rate of somatic mutations and possible neo-anti-gens, suggesting the potential success of targeted- and immuno-therapies [21]. In one study of 65 urothelial cancer cases, PD-L1 was found to be expressed in all tumor samples and tumors with expression of PD-L1 ≥ 12.2% were found to have higher grade patho-logic features and worse prognosis [10]. Accordingly, targeting this pathway is an active area of research, with promising early results. Two multicohort Phase I trials of checkpoint inhibitor monotherapy have included patients with recurrent/metastatic urothelial cancer. In 2014, data from the Phase I urothelial can-cer expansion cohort evaluating the safety and anti-tumor activity of atezolizumab (formally known as MPDL3280A, an anti-PD-L1) was reported [22]. All patients had immunohistochemical (IHC) analysis of tumor-infiltrating immune cells for PD-L1. Patients had either high PD-L1 expression (IHC 2/3) or low-expression (IHC 0/1). Initially, only PD-L1-positive (IHC 2/3) patients were included, but the cohort was ultimately expanded to include all patients regardless of IHC result. 67 patients who received atezolizumab at a dose of 15 mg/kg or 1200 mg IV every 3 weeks for up to 1 year were evaluated for safety and efficacy. Over 93% of these patients had received prior plat-inum-based therapy and many had poor prognostic features. The ORR for patients with a minimum of 6 weeks of follow-up was 43% for IHC 2/3 tumors including a 7% complete response (CR). For patients with IHC 0/1 tumors, the ORR was 11%. Half of the patients reported a drug-related AE. Based on this, the FDA granted atezolizumab breakthrough status in urothelial bladder cancer. Updated results from this trial were presented at the 2015 ASCO Annual Meeting [23]. Out of the 85 patients with at least 12 weeks of follow-up for efficacy evaluation, 46 were IHC 2/3, 38 were IHC 0/1 and 1 had unknown IHC status. The ORR for the IHC 2/3 group was 46% including six CR. The ORR for the IHC 0/1 group was 16% and all were partial response (PR). Pembrolizumab has also been tested in patients with recurrent/metastatic urothelial carcinoma as part of the multicohort Phase I KEYNOTE-012 trial. Initial results of the urothelial cohort were reported in 2014 and updated at the 2015 ASCO Annual Meeting. 33 patients with ≥ 1% of tumor cells expressing PD-L1 by IHC received 10 mg/kg pembrolizumab IV every 2 weeks until complete response, progression or unac-ceptable toxicity. 76% of the patients had prior ther-apy and 66% had visceral or osseous metastases. The ORR for evaluable patients was 24% including three

CR. 64% of patients treated with pembrolizumab had decrease in the size of target lesions. The rate of grade 3 or 4 toxicity was 12% [24].

Renal cell carcinomaWith the recent US FDA approval of nivolumab for metastatic renal cell carcinoma (RCC), there is much excitement about the role of PD-1 inhibition in this malignancy. PD-L1 expression of ≥1% has been shown in roughly 50% of RCC and has been cor-related to a more advanced stage, higher grade and worse overall survival [25,26]. Interestingly, one study showed similar expression levels of PD-L2 [27]. While early studies were primarily in clear cell histology, this high level of expression seems to exist regard-less of histology type, with an even higher level of expression (89%) in patients with sarcomatoid his-tology [28]. PD-1 inhibition was first studied in 33 patients with pretreated metastatic RCC who received nivolumab at a dose of 0.1–10 mg/m2 every 2 weeks as a part of the Phase 1 nivolumab study. This cohort of RCC had an ORR of 27% [29]. This led to Phase II [30] and III [31] trials which recently led to nivolum-ab’s FDA approval in RCC after progression on anti-angiogenic therapy. This is based on demonstrating an overall survival (OS) of 25 months compared with 19.6 months with everolimus (HS: 0.73, p = 0.002). OS was not statistically different based on PD-L1 expression suggesting PD-L1 may not be a useful biomarker in RCC and further studies are needed to explain this lack of correlation. ORR was also improved compared with everolimus (25 vs 5%, p <0.001), including four CRs in the nivolumab group. Grade 3 or higher AEs were reported in fewer patients receiving nivolumab compared with everolimus (19 vs 37%) and quality of life scores were improved in the nivolumab group [31]. PD-L1 inhibition has also been shown to have a promising activity in renal cell carcinoma. In the multicenter Phase I trial, BMS-936559 (PD-L1 inhibitor) was given to a cohort of patients with metastatic renal cell carcinoma at a dose of 10 mg/kg every 2 weeks. ORR was 12%, with 41% having stable disease [32]. Grade 3 or higher AEs were 5% and PFS was 53% at 24 weeks. Recently at the 2015 ASCO Annual Meeting, combination therapy of nivolumab and ipilimumab in pretreated patients with metastatic RCC was presented. ORR was found to be 39% in the group treated with nivolumab 1 mg/kg and ipilimumab 3 mg/kg compared with 29% when treated with nivolumab 3 mg/kg and ipilim-umab 1 mg/kg. 43 patients reported grade 3 or higher AEs [33]. Pembrolizumab is currently being studied in the neoadjuvant setting in patients with resectable disease (NCT02212730).



Prostate cancerThe FDA-approval of sipuleucel-T, an autologous dendritic cell-based cancer vaccine against pros-tatic acid phosphatase (PAP), in 2010 was the first evidence of immunotherapy improving survival in this patient population [34]. Since then, other forms of immune therapy including anti-CTLA-4 therapy and PD-1 therapy have been tested without the suc-cess seen in other disease sites. While there were early studies showing activity of ipilimumab in castration-resistant disease [35], a Phase III trial was unable to show a survival benefit [36]. In one of the few studies evaluating the PD-1/PD-L1 pathway, no tumor sam-ples of prostate cancer showed expression of PD-L1 by IHC [29]. A cohort of men with castration-resistant prostate cancer (CRPC) were included in the initial Phase I study with nivolumab (a PD-1 inhibitor) [29], however no objective responses were observed in the 17 patients. Optimizing immunotherapy for pros-tate cancer remains an area of active investigation. With the lack of response to date, various combina-tions with chemotherapy, varying patient popula-tions and predictive biomarkers are currently being i nvestigated.

Colorectal cancer (CRC) & anal cancerThe ‘immunoscore’ was first described in colorec-tal cancer and found to outweigh the TNM staging system in predicting prognosis [37–40]. This ‘immu-noscore’ is calculated based on the density and the location (in the center of the tumor and the invasive margins) of CD3+ T cells and CD8+ T cells [41] and is currently being validated globally. In addition, there is a mounting evidence to suggest that the host develops spontaneous humoral and cellular immune responses against CRC tumor antigens [42]. On the other hand, CRC may have a direct immunosuppressive effect at the molecular and cellular level with suppression of cell-mediated immunity. Strong PD-L1 (defined as ≥5 cells per 0.0625 mm2) expression was observed in 30% of CRC patients and was found to correlate with infil-tration by CD8+ lymphocytes which did not express PD-1 [43]. However, targeting the PD-L1/PD-1 path-way in CRC resulted in disappointing outcomes. No response was reported in 19 CRC patients treated on the first-in-human Phase I trial of nivolumab [29]. Dur-ing 3 years follow-up, one patient achieved an objective response on an intermittent dosing regimen following discontinuation of therapy [44]. In another study using a PD-L1 inhibitor, none of the 18 CRC patients treated on this dose escalation Phase I study had a clinical response [32]. More recently, a subset of CRC tumors with mismatch repair deficiency (MRD) was reported to show a response rate of 40% to PD-1 inhibition

compared with 0% in mismatch repair-proficient (MRP) colorectal cancers [45]. This was due to the high somatic mutation loads that mismatch repair-deficient tumors have leading to an immune response against their coded antigens. Further studies are investigating the efficacy of PD-1 inhibition in a larger cohort of CRC patients with MRD (NCT02563002). In addi-tion, we are investigating the combination of anti-PD-1 and chemoradiation therapy (CRT) in patients with rectal cancer in the neoadjuvant setting, based on preliminary data showing that CRT can lead to an increase in TILs [46] which could be further enhanced and activated by blocking the PD-1/PD-L1 pathway (NCT02586610).

In a recent abstract at the European Cancer Con-gress 2015, Ott et al.; presented results from KEY-NOTE-028, an ongoing Phase Ib trial of pembro-lizumab in patients with previously treated anal cancer [47]. All patients were required to have PD-L1 positivity of at least 1% by IHC. Forty-seven patients were screened, with 38% having PD-L1 expression. Out of the 25 patients enrolled, ORR was 20% to pembrolizumab given in a dose of 10 mg/kg every 2 weeks up to 2 years or until progression or unaccept-able toxicity. One complete response and 40% stable disease were observed. Grade 3 or more AEs were reported in 8% of the patients.

Pancreatic cancerIt had been speculated that pancreatic cancer may not be responsive to immunotherapy due to the unique pancreatic cancer tumor microenvironment (PCTM). TILs rarely infiltrate the PCTM, although their den-sity in the PCTM had been reported to correlate with better clinical outcomes [48]. On the other hand, the PCTM is associated with a massive infiltration of sup-pressive immune cells that contribute to a worse prog-nosis [49]. More importantly, pancreatic cancer is asso-ciated with a dense desmoplastic stroma that forms a barrier for immune cells and interacts with cancer cells leading to tumor progression and invasion [50]. The PD-1/PD-L1 pathway have been targeted in pancre-atic cancer given the fact that pancreatic cancer tumors that express PD-L1 were found to have worse progno-sis [12,51]. However, no clinical response was reported in 14 pancreatic cancer patients who received BMS-936559 (a fully human IgG monoclonal antibody tar-geting PD-L1) [32]. At the ASCO 2015 Annual Meet-ing, results from 24 patients with pancreatic cancer treated with another PD-L1 antibody (durvalumab) were presented. While the majority of patients pro-gressed through PD-L1 inhibition, 2/24 (8%) showed a partial response [18]. The ongoing KEYNOTE-28 study is investigating pembrolizumab (anti-PD-1) in a



variety of malignancies including pancreatic cancer. In addition, we are currently testing whether the addition of CRT to anti-PD-1 could overcome the resistance of pancreatic cancer to anti-PD-1 therapy by creating an inflammatory microenvironment that could lead to a better immune response against pancreatic cancer (NCT 02305186).

CholangiocarcinomaThe knowledge regarding the immune profile of cholangiocarcinoma is very limited due to the low incidence of the disease and its poor prognosis. Chol-angiocarcinoma patients with intraepithelial tumor-infiltrating CD4+, CD8+ TILs had a significantly longer overall survival compared with patients with low TILs [52]. Recently, Rosenberg et al., demon-strated a complete response in a patient with meta-static cholangiocarcinoma after adoptive transfer of TILs containing about 25% mutation-specific poly-functional T(H)1 cells [53]. Not only were the effec-tor T cells found to correlate with survival in cholan-giocarcinoma but also the expression of the immune checkpoints on tumor cells. Expression of PD-1 and PD-L1 was found to be upregulated in intrahepatic cholangiocarcinoma (ICC) tissues compared with the cancer adjacent tissues and significantly correlated with both poor tumor differentiation and advanced pathological stage and was inversely correlated with CD8+ TILs [54]. Therefore, the PD-1/PD-L1 path-way may be linked to malignant potential of ICC and may contribute to tumor immune evasion by pro-moting CD8+ TILs apoptosis. Thus, this pathway may indeed be a potential therapeutic target in the t reatment of cholangiocarcinoma.

Hepatocellular carcinoma (HCC)The immune system has been shown to play an impor-tant role in the course of HCC. HCC is considered an inflammation-associated cancer since hepatitis B and C virus infection are known to be the major risk factors. In addition, few immune cell subsets have been associated with poor outcome in HCC including tumor-infiltrating CD4+ regulatory T cells [55,56] and myeloid-derived suppressor cells (MDSC) [57]. The overexpression of PD-L1 had been also associated with poor prognosis in HCC [58]. This observation had led to a Phase I/II study using nivolumab in patients with advanced HCC who failed sorafenib but have a Child-Pugh score of 7 or less. The preliminary result of this study was recently presented at the 2015 ASCO annual meeting indicating a promising effect of nivolumab in HCC with an overall response rate of 23% including two complete responses in all disease subsets whether they had hepatitis B, C or no infection [59].

Gastric & gastro-esophageal junction cancersSimilar to other malignancies in the GI tract, there is mounting evidence to suggest that the unique immune microenvironment of gastric and gastro-esophageal junction (GEJ) cancers includes an abundance of suppressive immune cells compared with activating immune cells [60,61]. The density of PD-1 positive CD4+ and CD8+ T cells in gastric cancer samples were found to be higher than normal healthy controls and a higher density correlated to worse disease pro-gression, poor tumor differentiation and lymph node metastases [60]. PD-1 and PD-L1 expression has cor-related not only with worse overall survival, but also with malignant transformation [60,62–64]. Recently at the 2015 ASCO Annual Meeting, a study of 105 tumor samples from patients with metastatic gastric cancer found that PD-1 positive TILs were present in 26% of samples and disease-free survival at 3 years was worse in patients with PD-1 positive TILs compared with tumors that did not (36 vs 65%, p = 0.022) [65].

The KEYNOTE-012 trial included 162 patients with gastroesophageal cancer. All were assessed by IHC for PD-L1 expression, with 40% having at least 1% expression or more. In this single arm Phase Ib study, 10 mg/kg pembrolizumab (anti-PD-1) was given every 2 weeks for up to 24 weeks and showed an RR of 22% and 6-month PFS and OS of 24 and 69%, respectively [66]. There was a nonsignificant trend toward an association between higher levels of PD-L1 expression and ORR and OS (p = 0.102 and 0.124, respectively). Subsequently, at the 2015 ASCO Annual Meeting, data correlating four prespecified multigene immune response signatures (IFN-γ, TCR signaling, Expanded immune and Denovo) from 33 patients with advanced gastric cancer treated with pembrolizumab on KEYNOTE-012 was presented [67]. Analysis sug-gested a low gene signature score correlated with a lack of response. This may be of significant benefit to pre-dict patients who will respond to PD-L1 therapy and those who will not. In another study, one gastric cancer patient was treated with atezolizumab (anti-PD-L1) every 3 weeks, as a part of Phase I study. This patient’s tumor sample was 3+ for PD-L1 by IHC and clinically showed partial response at 6 weeks with eventual pro-gression at week 51 [68].

Breast cancerOut of the major subtypes of breast cancer, triple-neg-ative breast cancer (TNBC) and HER2 positive are most consistently associated with the presence of TILs and are currently thought to be the most amenable to anti-PD-1 and other checkpoint inhibitor-based thera-pies. Clinical studies suggest that the presence of TILs



may predict for effectiveness of therapy in HER2+ and triple-negative breast cancer [69,70]. In contrast, for luminal A, estrogen receptor positive, breast cancer, CD8 infiltrates were correlated with increased risk of death in a 12,000 tumor series. Clinical response to immunotherapy in ER-positive breast cancer has been disappointing to date in early phase studies [71]. There are several explanations for poor response to immu-notherapy in ER-positive breast cancers, including the relatively scarcity of neo-antigens, the relative fre-quency of myeloid-derived suppressor cells (MDSC’s), the unfavorable cytokine milieu, and the relatively lower levels of lymphocytic infiltrates compared with other solid tumors [72,73]. There are biologic reasons to expect TNBC and HER2+ breast cancer to be ame-nable to PD-1-based therapies. The rationale includes strong immunogenicity of the HER2 protein, the ele-vated mutational loads of these subtypes, the purported high levels of neo-antigen formation and the reli-ance on nonestrogenic signaling pathways. Recently, there are hints that relative TIL infiltrates may have predictive potential for response to adjuvant HER2-based therapies [74]. Despite identification of PD-L1 on HER2+ breast cancer [75] and despite encourag-ing preclinical data [76], there have been no published prospective clinical trials of checkpoint inhibitors in HER2+ disease; an anti-PD-1 trial is currently accru-ing (NCT02129556). In TNBC, there is a little more clinical data to suggest a role for anti-PD-1-based ther-apy. For example, a study of 116 breast cancers found PD-1 expression on the surface of 50% of TILs, while PD-L1 expression was detected in 45% of breast cancer tumor cells. Concurrence between PD-1 on TILs and PD-L1 on tumors was observed 29% of the time [77]. Not surprisingly, PD-1+ TILs were more frequent in TNBC samples (70%) than in ER+ and HER2+ sub-types (25–44%, p <0.001) [77]. Likewise, PD-L1 was observed on the triple-negative cancer cells more than ER+ and HER2+ types (59 vs 33%, p = 0.017). RNA sequencing from The Cancer Genome Atlas found that TNBC mRNA expression of PD-L1 was nearly twice as high as other subtypes (p <0.001) [78]. Likewise, a tissue microarray found PD-L1 expression in 19% of TNBC samples [78]. Finally, a comparative genomic hybridization study of 326 tumors found PD-L1 and PD-L2 high level amplification in 29% of TNBC [79]. Completed clinical trials of PD-1 targeting agents include a pembrolizumab study in TNBC reported at the 2014 San Antonio Breast Cancer meeting by Nanda et al. [80]. The Nanda Phase Ib study enrolled 32 heavily pretreated patients with recurrent or meta-static TNBC and with any level of PD-L1 expression in their tumor. Based on their assay methods, only 58% of all screened patients had PD-L1+ tumors. In

the study, 10 mg/kg of pembrolizumab was given every 2 weeks, and treatment remained ongoing for patients whose disease was not clearly progressing as assessed by RECIST v1.1 every 8 weeks. Treatment was tol-erable with only 16% of patients experiencing grade 3–5 toxicity, but one treatment-related death from dis-seminated intravascular coagulation was reported. The overall RR was 19% with one (4%) complete response, four (15%) partial responses and seven patients (26%) with stable disease. In this population, the median time to response was 18 weeks (range, 7–32). A second trial of a PD-L1 inhibitor was presented at AACR 2015 by Leisha Emens [81]. In that Phase I trial, atezolizumab was given to 12 TNBC patients with metastatic dis-ease. Grade 3–4 toxicity occurred in one patient with renal insufficiency to date and there were no treatment-related deaths. The overall RR was 33% in the nine patients evaluable for efficacy, with one CR and two PRs. All responses were seen within the first 6 weeks of treatment. Another 17 PD-1 studies in breast cancer are currently listed at clinicaltrials.gov.

Non-small-cell lung cancer (NSCLC)With the recent FDA approvals of immune checkpoint inhibitors for adenocarcinoma and squamous lung cancers, interest in the tumor microenvironment and immune interaction has increased dramatically over the past several years. In one study, 56% of tumor samples with lung adenocarcinoma and squamous cell histology showed PD-L1 positivity ≥1% and correlated with worse prognosis [11]. Efforts to engage this interac-tion for the treatment of lung cancer have progressed at a feverish pace, particularly with the development of checkpoint inhibitors.

Single agent immunotherapyPD-1 inhibitionThe initial report of the CHECKMATE 001 study of nivolumab was promising because it demonstrated single agent immunotherapy activity in NSCLC and suggested that the responses were prolonged in some patients [29]. CHECKMATE 001 ultimately enrolled 129 previously treated NSCLC patients (54% had received at least three lines of prior therapy) [82]. AEs were observed in 41.1% of patients; six patients experi-enced grade 3 or greater AEs, four of which were due to pneumonitis (one of which was grade 5) [82]. The ORR was 17.1% with median duration of response of (DOR) 17.0 months (range 1.4 to 36.8 months) [82]. Responses were observed in both squamous (9 out of 54 patients) and nonsquamous histology (11 out of 74 patients) and across all dose levels studied [82]. PD-L1 status was not predictive of responses, though archived tumor tissue was not available for all patients and pretreatment biop-



sies were not required prior to starting nivolumab. As noted in the initial publication of the CHECKMATE 001 study, the impressive ORR and DOR in patients with heavily pretreated NSCLC caught many oncolo-gists by surprise [29]. Another PD-1 inhibitor, pembro-lizumab, was the subject of the large Phase I trial KEY-NOTE 001 to examine the safety and efficacy of this drug in both previously treated and treatment-naive NSCLC patients [83]. Among the total study popula-tion (n = 495, 394 previously treated and 101 treat-ment naive) the ORR was 19.4% (18.0% among prior treated and 24.8% in treatment naive) with stable dis-ease (SD) in 21.8% [83]. Patients with a PD-L1 propor-tion score ≥ 50%, defined as a proportion of cells with ≥1% over all cells (n = 73), had an RR of 45.2% and median PFS of 6.3 months for all patients, 6.1 months for previously treated patients and 12.5 months for treatment-naive patients [83]. Among patients with PD-L1 proportion scores less than 1% or between 1 and 49%, the median PFS and OS were similar in both groups, but comparatively less than those with a score ≥ 50% [83]. As with nivolumab, there were low rates of treatment-related AEs (fewer than 10% with grade ≥ 3). The KEYNOTE 001 trial demonstrated that response rates, PFS, and OS were increased in those patients with high PD-L1 staining; however there does not seem to be a scientific basis for choosing a cut point of ≥ 50%, a seemingly arbitrary selection. Neverthe-less, pembrolizumab is now FDA approved for patients with PD-L1-positive NSCLC whose disease has pro-gressed after other treatments. After the first report of responses to nivolumab in NSCLC, multiple Phase II and Phase III clinical trials were opened to further investigate checkpoint inhibitors in NSCLC. Final results were first reported from trials that focused on patients with squamous cell NSCLC (SC-NSCLC), a histology that had little progress compared with nonsquamous NSCLC (NS-NSCLC) in recent years. CHECKMATE 063 was a single arm, Phase II study of nivolumab 3 mg/kg in 6117 patients, 65% of who had received at least three prior lines of therapy [84]. The ORR was 14.5% and the SD rate was 26% among a population of patients with best response (CR or PR) of 4% to the most recent line of therapy [84]. Thirteen of 17 patients with a CR or PR had ongoing responses at the time of the analysis, and the median OS in this group had not been reached [84]. CHECKMATE 017 was a randomized, Phase III trial that compared nivolumab to docetaxel for second-line treatment in 272 patients with SC-NSCLC [85]. Treatment with nivolumab led to a 3.2-month improvement in median OS compared with docetaxel (9.2 vs. 6.0 months; p <0.001). ORR was also improved with nivolumab compared with docetaxel (20 vs 9%; p = 0.008) [85].

Based on the CHECKMATE 063 and CHECK-MATE 017 studies, the FDA approved nivolumab for the treatment of second-line SC-NSCLC. A simi-lar randomized Phase III trial of nivolumab versus docetaxel, CHECKMATE 057, was conducted for patients with NS-NSCLC [86]. This trial randomized 582 patients after progression following first-line plati-num doublet chemotherapy to nivolumab (n = 292) or docetaxel (n = 290). As in the CHECKMATE 017 trial, nivolumab showed a significant improvement in median OS (12.2 vs 9.4 months; p = 0.001) and ORR (19 vs 12%; p = 0.02) over docetaxel. The investigators reported improved safety and tolerability of nivolumab over docetaxel with all grade and high grade (grade 3–4) toxicities higher with docetaxel when compared with nivolumab (88 vs 69% and 54 vs 10%, respec-tively). Based on this study, nivolumab was also FDA approved for patients with NS-NSCLC. Most recently, the KEYNOTE 010 trial, a randomized Phase II/III trial of two doses of pembrolizumab versus docetaxel chemotherapy, also showed an OS improvement with pembrolizumab [87]. The CHECKMATE 017, 057 and KEYNOTE 010 trials have convincingly dem-onstrated that anti-PD-1 therapy improves response rates and survival for patients with NSCLC and has a better toxicity profile than docetaxel chemotherapy in second-line therapy.

PD-L1 inhibitionPD-L1 inhibition has shown efficacy in advanced SC- and NS-NSCLC as well as second-line therapy. BMS-936559, a fully human IgG monoclonal anti-body toward PD-L1, was studied in 207 patients with a variety of solid tumors [32]. Among the 75 patients with NSCLC, five patients experienced a PR with three patients experiencing a PR at 24 weeks [32]. Another PD-L1 antibody, atezolizumab (formerly MPDL3280A) was studied in a Phase I dose esca-lation study that enrolled 88 patients with NSCLC and compared responses according to PD-L1 expres-sion in tumor cells and tumor infiltrating immune cells [88]. Patients with the highest PD-L1 expression in tumor or immune cells had an ORR of 45 versus 14% for patients with low or absent PD-L1 expres-sion [88]. Interim results of a larger Phase II study, POPLAR, that compared atezolizumab to docetaxel after prior platinum doublet therapy were reported at the ASCO Annual Meeting in 2015 [89]. OS was improved in the group with high PD-L1 expres-sion with the median not reached in those receiving atezolizumab compared with patients who received docetaxel (median 11.1 months) [89]. Interim OS was not significantly different for the entire inten-tion to treat (ITT) analysis that contained a total



of 288 patients in both cohorts, though this may change with longer follow-up [89]. Atezolizumab is also the subject of an ongoing study in chemother-apy-naive NSCLC patients (NCT01846416). Dur-valumab is another anti-PD-L1 IgG1 antibody that has been studied in patients with advanced NSCLC after first-line therapy and in novel combinations. An update to the ongoing Phase I/II study of dur-valumab in advanced NSCLC was presented at the

ASCO Annual Meeting in June 2015 [90]. Among the 198 patients treated, 149 were evaluable for response with ≥ 24 weeks of follow-up [90]. The ORR was 14% among all evaluable patients, though it was higher in PD-L1-positive patients (23%) and in SCC com-pared with NS-NSCLC (21 vs 10%, respectively) [90]. Avelumab (also known as MSB0010718C) is another anti-PD-L1 IgG1 antibody that is currently being evaluated in NSCLC. In a Phase I expansion cohort

Figure 1. PD-1/PD-L1 inhibition & response rate. (A) PD-1 inhibition based on cancer, drug and overall response rate. (B) PD-L1 inhibition based on cancer, drug and overall response rate. (C) Combination therapy based on cancer, drug and overall response rate. **Microsatellite-instability-high tumors only. CRC: Colorectal carcinoma; HCC: Hepatocellular carcinoma; NSCLC: Non-small-cell lung carcinoma; ORR: Overall response rate; OS: Overall survival; RCC: Renal cell carcinoma; SCLC: Small-cell lung carcinoma; uro: Urothelial.

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Pembrolizumab

Pembrolizumab + lpilimumab

Nivolumab

Nivolumab + lpilimumab

Atezolizumab

Atezolizumab + Platinum doublet

Avelumab

Durvalumab

Durvalumab + Tremelimumab

**



treating 184 NSCLC patients, there was a 13.5% RR and 50.5% disease control rate. Only 12.5% of patients had grade 3 or higher treatment-related

adverse events [91]. A Phase III randomized trial of avelumab versus docetaxel is ongoing (JAVELIN Lung 200, NCT02395172).

Figure 2. Response rate based on PD-L1 expression. (A) NSCLC, all histologies. (B) Urothelial cancer. (C) Other cancers. *≥50% tumor cells PD-L1+; **1–49% tumor cells PD-L1+. NSCLC: Non-small-cell lung carcinoma, all histologies; ORR: Overall response rate; OS: Overall survival; PD-L1+: ≥1% expression; PD-L1-: ≤1% expression; RCC: Renal cell carcinoma; SCCHN: Squamous cell carcinoma of head/neck; SCLC: Small-cell lung carcinoma; Uro: Urothelial.

Pembrolizumab

Pembrolizumab

Pembrolizumab

Gastric (Pembrolizumab)

SCCHN (Pembrolizumab)

Pembrolizumab

Nivolumab

Nivolumab

Atezolizumab

Atezolizumab

Breast (Atezolizumab)

Breast (Pembrolizumab)

Atezolizumab

Atezolizumab

Avelumab

Durvalumab

Durvalumab



0 10 20 30 40 50

0 10 20 30 40 50

Uro

NSCLC

**

*

0 10 20 30 40

Other

ORR

PD-L1 +

PD-L1 -



Combination therapyPD-1 inhibition in combination with chemotherapy or targeted therapyIn addition to monotherapy trials in previously treated patients, checkpoint inhibitors have been studied in the first-line setting and in combination with other check-point inhibitors or chemotherapy agents. The CHECK-MATE 012 trial (NCT01454102) is an ongoing Phase I trial with multiple arms (currently A through S) evalu-ating nivolumab as monotherapy (Arm F) or in com-bination with various agents. Results from nivolumab in combination with platinum doublet chemotherapy have been presented on multiple occasions [92,93]. Fifty-six patients treated on these arms of the study received nivolumab concurrently with cisplatin/gemcitabine (A), cisplatin/pemetrexed (B) or carboplatin/paclitaxel (C and C5). Treatment was well tolerated and there were no dose-limiting toxicities (DLT) in the first 6 weeks. At the time of recent report, the median OS in each of these arms was (A) 50.5 weeks, (B) 83.4 weeks, (C) 64.9 weeks and (C5) not reached [93]. For patients treated on Arm F with single-agent nivolumab, 11 of 52 (21%) had an objective response, three with complete response [94]. Median OS was 98.3 weeks and 1-year OS rate presented was 74%. Notably, 15% of patients in this arm had EGFR mutations. Additional cohorts of patients divided by histology are being enrolled for nivolumab first-line treatment. Another combination trial, KEYNOTE 021 (NCT02039674), is a Phase I/II trial evaluating pem-brolizumab in combination with platinum doublet che-motherapy, targeted therapy (erlotinib or gefitinib) or immunotherapy (ipilimumab). Results from 44 patients treated on cohorts A and C, carboplatin/paclitaxel and carboplatin/pemetrexed respectively, were recently reported [95]. As with the CHECKMATE 012 study, these results also demonstrated safety with these PD-1 plus platinum doublet combinations as only one patient experienced a DLT. ORR was 30 and 56% in Arms A and C, respectively. Cohort G, an open-label Phase II portion of this trial is currently accruing and randomiz-ing patients to carboplatin/pemetrexed with or without pembrolizumab. Pembrolizumab in combination with carboplatin and nab-paclitaxel is currently being inves-tigated in a Phase I/II clinical (NCT02382406).

PD-1 inhibition in combination with immunotherapyTrials evaluating combination immune checkpoint inhibition have also been conducted and are ongoing in lung cancer. The Phase I/II CHECKMATE 032 trial (NCT01928394) is enrolling patients on multiple cohorts of nivolumab monotherapy or in combination with ipilimumab (anti-CTLA-4 antibodies) for solid tumors including NSCLC and small-cell lung cancer

(SCLC). Interim data from this trial have been pre-sented from patients with NSCLC and SCLC [96,97]. In NSCLC, 49 chemotherapy-naive patients were treated in cohorts of 3 + 1 mg/kg or 1 + 3 mg/kg ipilimumab and nivolumab, respectively. Grade 3–4 treatment-related AEs occurred in 24 patients (49%). Combina-tions of ipilimumab and pembrolizumab are also being studied in KEYNOTE 021, Cohort D. This trial is evaluating the combination in the second-line meta-static setting in contrast to the first-line CHECKMATE 032 trial described above. Data from the first 17 patients were presented at ASCO 2015 [98]. The combination appears to be safe, and additional patients are currently being enrolled. Durvalumab combined with tremelim-umab (anti-CTLA-4) has been studied in 61 patients with 64% of patients experiencing an AE (31% grade 3 or higher) [99].

PD-L1 inhibition in combination with chemotherapy or targeted therapyFewer studies have investigated PD-L1 inhibition in combination compared with PD-1 inhibition. Atezoli-zumab has been studied in first-line treatment of NSCLC in combination with platinum doublet che-motherapy, and as with the CHECKMATE 012 and KEYNOTE 021 studies, the combination anti-PD-L1 therapy with chemotherapy was safe and tolerable [100]. Durvalumab is also the subject of a multicohort study combined with an EGFR inhibitor (osimertinib) with or without selumetinib (MEK inhibitor) in EGFR mutant NSCLC (NCT02143466) and in combina-tion with the anti-CTLA-4 antibody tremelimumab (NCT02000947).

Small-cell lung cancerIn the SCLC arm of the CHECKMATE 032 trial [97], 90 patients with progressive SCLC after platinum doublet chemotherapy were treated with nivolumab alone (n = 40) or in combination with ipilimumab (n = 50) [97]. Grade 3–4 treatment-related toxicity in the nivolumab and ipilimumab combination arms was 34%, with only 2.1% developing grade 3–4 pneumo-nitis. Response rates were 17 and 18% with median duration of response not reached and 6.9 months in the nivolumab and combination cohorts, respectively. Three patients on this study developed limbic enceph-alitis. Although the numbers of cases of this autoim-mune toxicity are small, this may be a new safety signal specific to patients with SCLC.

ConclusionThe use of anti-PD-1 and anti-PD-L1 antibodies has generated significant excitement in the oncology community with numerous ongoing studies in variety



of cancers (summarized in Tables 1 & 2 & Figures 1 & 2). Without doubt, the benefits of PD-1/PD-L1 inhibition outweigh their adverse effects, particu-larly compared with traditional chemotherapy. While prolonged and durable responses were achieved using immune checkpoints inhibitors, only a relatively small number of patients experience such clinical responses. Although there are emerging data that some malignancies have very high response rates, for example, Merkel cell carcinoma (RR of 80%) [101], significant efforts are still needed for why certain malignancies respond to immunotherapy while oth-ers seem untouched by its use.

Moreover, the predictive value of PD-L1 as a bio-marker that may determine which patients are more likely to respond to PD-1/PD-L1 blockade remains debatable given the fact that a subset of patients with PD-L1-negative tumors still responds to PD1/PD-L1

blockade. The variability of response based on PD-L1 expression could be related to many factors includ-ing: using different cut-off to define PD-L1 positiv-ity (1% vs 5 or 50%); the variation of the staining method (different antibodies, manual vs automatic count, pathologist training); the variation in cellular distribution of the staining (membrane vs cytoplas-mic); the location of PD-L1 expression (tumor cells vs immune cells); tissue acquisition (fresh vs archived); the heterogeneity of tumors and the dynamic changes in the tumor microenvironment. Indeed, substantial efforts are underway to address this debate with FDA calling for a validated method of staining and phar-maceutical companies teaming up to launch prospec-tive trials to answer these questions [102].

However, the expression of PD-L1 may be use-ful in making clinical decision as shown recently in melanoma. Particularly, in melanoma patients with

Table 1. Expression of PD-L1 and its correlation with pathological features and survival.

Cancer type PD-L1 expression on tumor cells

PD-1 expression on TILs reported

Associated pathologic features Survival Ref.

Head/neck 70% (HPV+)§ + ↑ IFN-γ NR [15]

29% (HPV-)§

Genitourinary

Urothelial 21%‡ + ↑ grade and tumor size ↓ [10]

RCC 24–50%‡ + ↑ grade, ↑ pathological stage ↓ [25,26,31]

Prostate 0% - N/A N/A [29]

Gastrointestinal

Colorectal 30%§ - ↓ CD8+ infiltration, ↑IFN-γ ↑ if MMR proficient

[43]

Pancreatic 39%§ - ↓ CD8+ & CD4+ infiltration ↓ [12,51]

CC 5.45† + ↑ grade, ↑ pathological stage, ↓ CD8+ infiltration

↓ [54]

HCC 33% (defined as ≥10%) - ↑ recurrence rate postresection, ↑ FoxP3+ infiltration

↓ [58]

Gastric 40%‡ + ↑ grade, ↑ pathological stage, ↓ CD8+ infiltration, ↑ metastases

↓ [62,65]

Esophageal 52% (defined as ≥10%) - ↑ grade, ↑ pathological stage, ↑ metastases

↓ [64]

Breast 45%§(59% in TNBC; 100% in metaplastic)

+ ↑ CD8+ infiltration ↓ [75,77,78]

Lung

NSCLC, adeno 65%‡ - ↑ in adeno (compared with SCC) ↓ [11,85]

NSCLC, squamous

44%‡ + NR ↓ [11]

†Staining intensity score.‡≥1%.§≥5%.CC: Cholangiocarcinoma; HCC: Hepatocellular carcinoma; HPV: Human papilloma virus; MMR: Mismatch repair; N/A: Not applicable; NR: Not reported; NSCLC: Non-

small-cell lung cancer; RCC: Renal cell carcinoma; SSC: Squamous cell carcinoma; TILs: Tumor infiltrating lymphocytes; TNBC: Triple-negative breast cancer.



Table 2. Clinical outcomes of PD-1/PD-L1 inhibition in solid tumors other than melanoma.

Cancer type Drug Patient population

ORR DCR Drug-related AE (grade ≥3)

Survival benefit Ref.

Head/neck Pembrolizumab PD-L1+ ≥1% 20% (regardless of HPV status)

NR 17% NR [16]

Genitourinary

Urothelial Atezolizumab Unselected 43% (PD-L1≥5%)

NR 3.2% NR [22]

11% (PD-L1-)

Pembrolizumab PD-L1+ ≥1% 24% NR 12% NR [24]

RCC Nivolumab Unselected 25% 59% 19% ↑ OS by 5.4 mos vs everolimus (HR 0.73, p = 0.002)

[31]

BMS-936559 Unselected 12% 53% 5% 53% PFS at 24 weeks [32]

Nivolumab + Ipilimumab

Unselected 39% 78% 43% NR [33]

Prostate Nivolumab Unselected 0% 0% 14% N/A [29]

Gastrointestinal

Colorectal Nivolumab Unselected 0% 0% 14% N/A [29]

BMS-936559 Unselected 0% 0% 5% N/A [32]

Pembrolizumab Unselected 40% (if MRD) 90% 41% 78% PFS rate @ 20 weeks

[45]

Anal Pembrolizumab PD-L1+ ≥1% 20% 60% 8% NR [47]

Pancreatic Pembrolizaumab Unselected 0% 0% 5% N/A [32]

Durvalumab Unselected 8% 25% 13% NR [18]

HCC Nivolumab Unselected 23% 69% 17% 72% OS rate @ 6 months [59]

Gastric Pembrolizumab PD-L1+ ≥1% 22% 36% 10% 24% OS rate at 6 months

[66]

Breast Pembrolizumab PD-L1+ ≥1% 16% 26% 16% NR [80]

Atezolizumab PD-L1+ >5% 33% 44% 8% NR [81]

NSCLC, squamous

Nivolumab Unselected 20% regardless of PD-L1 expression

45% 7–14% ↑ OS by 3.2 mos vs docetaxel (HR: 0.59; p <0.001)

[85]

Lung

NSCLC, nonsquamous

Nivolumab Unselected 19.2% 44% 10.5% ↑ OS by 2.8 mos vs docetaxel (HR: 0.73; p <0.001)

[86]

NSCLC, all histologies

Pembrolizumab Unselected 19.4% (45% if PD-L1+ ≥50%)

46% 9.5% 3.7 months PFS, 12 months OS

[83]

Atezolizumab Unselected 45% PD-L1+ (14% PD-L1-)

56% (47% PD-L1-)

11% 89% OS @ 1 year if PD-L1+

[88]

Atezolizumab Unselected 38% PD-L1+ NR 43% OS NR in PD-L1+ (vs 11 months with docetaxel)

[89]

Durvalumab Unselected 14% (23% PD-L1+)

42% (48% PD-L1+)

6% 76% ongoing response at 35 weeks

[90]

AE: Adverse event; DCR: Disease control rate; MRD: Mismatch repair deficient; N/A: Not applicable; NR: Not reported; NSCLC: Non-small-cell lung carcinoma;

ORR: Overall response rate; OS: Overall survival, PFS: Progression-free survival; RCC: Renal cell carcinoma; SCLC: Small-cell lung carcinoma.



PD-L1-negative tumors, the combination of PD-1 and CTLA-4 blockade was more effective than either agent alone [103]. Moreover, the presence of pre-exist-ing CD8+ T cells, that are negatively regulated by PD-1/PD-L1-mediated adaptive immune resistance in melanoma, were found to be essential for tumor response to PD-1 blockade [104]. Whether these obser-vations are applicable to other malignancies such as lung cancer remains to be determined.

It has been speculated recently that the number of somatic mutations, leading to a large repertoire of neo-antigens, could explain the clinical responses in some tumors compared with others [105]. This is likely to be one piece in this complex puzzle, given the varying responses to immunotherapy in tumors with similar numbers of somatic mutations. As such, a new form of clinical trials has been created to identify and treat malignancies based on their immune profile, such as the density of TILs and mutational status rather than organ origination (NCT02054806). These ‘basket trials’ may be able to identify predictive biomarkers for response to immunotherapy by analyzing unique T-cell receptor (TCR) phenotypes, immune gene signature and mutational load. Hopefully, this will increase the number of patients who may derive a durable and prolonged response to immunotherapy, such that ‘remission’ may be a more commonly used word.

Future perspectiveWithout question, immunotherapy will continue to create a paradigm shift in the treatment of various malignancies. The indication of immune checkpoint inhibitors targeting PD-1 and PD-L1 is expected to expand to other disease settings beyond melanoma, lung and renal cancers, as single agents and in com-bination with other modalities such as chemotherapy and radiation therapy. In addition, it is expected that the next wave of immune checkpoint inhibitors, currently under investigation, will make their way to clinic in the near future. Indeed, cancer immu-notherapy will continue to evolve in the next decade with better-defined mechanism of action and novel biomarkers that could be predictive for response.

Financial & competing interests disclosureCL Slingluff Jr: Supported in part by two research grants from

the Investigator-Initiated Studies Program of Merck Sharp &

Dohme Corp. OE Rahma: Supported in part by two research

grants from Investigator-Initiated Studies Program of Merck

Sharp & Dohme Corp. The authors have no other relevant

affiliations or financial involvement with any organization or

entity with a financial interest in or financial conflict with the

subject matter or materials discussed in the manuscript apart

from those disclosed.


manuscript.

Table 2. Clinical outcomes of PD-1/PD-L1 inhibition in solid tumors other than melanoma (cont.).

Cancer type Drug Patient population

ORR DCR Drug-related AE (grade ≥3)

Survival benefit Ref.

Avelumab Unselected 12% (16% PD-L1+)

50% 12% PFS 11.7 months PD-L1+ (vs 5.9 months PD-L1-)

[91]

Pembrolizumab + Ipilimumab

Unselected 55% 100% 11% NR [98]


Unselected 27% 41% 31% NR [99]

Atezolizumab + platinum doublet

Unselected 67% 87% 7–73% (all grades)

NR [100]

SCLC Nivolumab + Ipilimumab

Unselected 25% (15% nivolumab alone)

55% (37% nivolumab alone)

34% ↑ duration of response (NR vs 6.9 months with nivo alone)

[97]

AE: Adverse event; DCR: Disease control rate; MRD: Mismatch repair deficient; N/A: Not applicable; NR: Not reported; NSCLC: Non-small-cell lung carcinoma;

ORR: Overall response rate; OS: Overall survival, PFS: Progression-free survival; RCC: Renal cell carcinoma; SCLC: Small-cell lung carcinoma.




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Executive summary

Squamous cell carcinoma of head & neck (SCCHN)• HPV-associated squamous cell carcinoma of head and neck (SCCHN) have a higher expression of PD-L1

compared with HPV-negative tumors. ORR to anti-PD-1 is 20% regardless of PD-L1 expression or HPV status based on small samples.

Urothelial cancer• PD-L1 expression correlates to worse overall survival and high-grade pathologic features. In heavily pretreated

patients, overall response rate (ORR) was 24% in PD-L1+ tumors treated with PD-1/PD-L1 blockade, with a small number having complete responses.

Renal cell carcinoma• PD-L1 expression correlates with a more advanced stage, higher grade tumor and worse overall survival. In the

second-line setting, anti-PD-1 therapy demonstrated an ORR of 25% with prolonged overall survival compared with everolimus. Combination immunotherapy of anti-PD-1 and anti-CTLA-4 increased ORR but also adverse events (AEs).

Prostate cancer• Limited data for immune checkpoint expression or inhibition. No response to PD-1 inhibition to date.Colorectal cancer (CRC) & anal cancer• PD-L1 and CD8+ T lymphocytes correlate with survival. In patients with metastatic CRC, RR is 2% but increases

to 40% if mismatch repair deficient tumors. In anal cancer, ORR of 20% was reported.Pancreatic cancer• Suppressive tumor-infiltrating lymphocytes (TIL) and PD-L1 expression correlate to worse prognosis. There are

limited data to support the activity of PD-1 and PD-L1 inhibitors in pancreatic cancer.Cholangiocarcinoma• Increased TILs correlate with better prognosis. PD-L1 expression correlates with worse survival, advanced

pathologic grade and tumor stage. Immune checkpoint inhibitors have not been studied.Hepatocellular carcinoma (HCC)• PD-L1 expression correlates to a poor prognosis. In one small study, PD-1 inhibition showed an ORR of 20%

with a small number of patients having a complete response.Gastric cancer• PD-1 and PD-L1 expression correlates with worse overall survival. PD-1 inhibition has an RR of 20%, with an

immune gene signature predicting benefit in a small number of patients.Breast cancer• PD-L1 expression is more common in triple negative (TNBC) than ER+ breast cancer. PD-1 inhibition has an RR

of 20–30% with rare complete responses in TNBC.Non-small-cell lung carcinoma (NSCLC)• PD-L1 expression correlates with worse survival and is predictive of clinical response to PD-1 inhibition in most

studies, particularly for adenocarcinoma histology. Response rates range from 15 to 20%, with some studies showing higher PD-L1 expression correlating to a higher response rate of 45%. PD-1 inhibition increases PFS and OS compared with chemotherapy in the second-line setting.



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71 Ali HR, Provenzano E, Dawson SJ et al. Association between CD8+ T-cell infiltration and breast cancer survival in 12,439 patients. Ann. Oncol. 25(8), 1536–1543 (2014).

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76 Stagg J, Loi S, Divisekera U et al. Anti-ErbB-2 mAb therapy requires type I and II interferons and synergizes with anti-PD-1 or anti-CD137 mAb therapy. Proc. Natl Acad. Sci. USA 108(17), 7142–7147 (2011).

77 Gatalica Z, Snyder C, Maney T et al. Programmed cell death 1 (PD-1) and its ligand (PD-L1) in common cancers and their correlation with molecular cancer type. Cancer Epidemiol. Biomarkers Prev. 23(12), 2965–2970 (2014).

78 Mittendorf EA, Philips AV, Meric-Bernstam F et al. PD-L1 expression in triple-negative breast cancer. Cancer Immunol. Res. 2(4), 361–370 (2014).

79 Barrett MT, Anderson KS, Lenkiewicz E et al. Genomic amplification of 9p24.1 targeting JAK2, PD-L1, and PD-L2 is enriched in high-risk triple negative breast cancer. Oncotarget 6(28), 26483–26493 (2015).

80 Nanda RC, Laura Q, Dees EC, Berger R et al. A Phase Ib study of pembrolizumab (MK-3475) in patients with advanced triple-negative breast cancer. Presented at: Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium. San Antonio, TX, 9–13 December 2014.

81 Emens LAB, Fadi S Cassier, Philippe DeLord et al. Inhibition of PD-L1 by MPDL3280A leads to clinical activity in patients with metastatic triple-negative breast cancer. Presented at: Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium. San Antonio, TX, 9–13 December 2014.

82 Gettinger SN, Horn L, Gandhi L et al. Overall survival and long-term safety of nivolumab (anti-programmed death 1 antibody, BMS-936558, ONO-4538) in patients with previously treated advanced non-small-cell lung cancer. J. Clin. Oncol. 33(18), 2004–2012 (2015).


•• PhaseIIItrialcomparingpembrolizumabtodocetaxelinmetastaticNSCLC.

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85 Brahmer J, Reckamp KL, Baas P et al. Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer. N. Engl. J. Med. 373(2), 123–135 (2015).

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88 Horn L, Spigel DR, Gettinger SN et al. Clinical activity, safety and predictive biomarkers of the engineered antibody MPDL3280A (anti-PDL1) in non-small cell lung cancer (NSCLC): update from a Phase Ia study. ASCO Meeting Abstracts 33(Suppl. 15), 8029 (2015).

89 Spira AI, Park K, Mazieres J et al. Efficacy, safety and predictive biomarker results from a randomized Phase II study comparing MPDL3280A vs docetaxel in 2L/3L NSCLC (POPLAR). ASCO Meeting Abstracts 33(Suppl. 15), 8010 (2015).

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Immunotherapy

Review 2016/06/288

7

2016

Immunotherapy with checkpoint inhibitors has arrived and begun to change the landscape of clinical oncology, including for patients with renal cell carcinoma. Specifically, drugs targeting the programmed death 1 and cytotoxic T-lymphocyte associated antigen pathways have demonstrated remarkable responses for patients in clinical trials. In this article, we review the most recent available data for immune checkpoint inhibitors for patients with renal cell carcinoma. We discuss potential strategies for rational combination therapies in these patients, some of which are currently being studied, and address important future considerations for use of these novel agents in the years to come.

First draft submitted: 10 February 2016; Accepted for publication: 19 April 2016; Published online: 28 June 2016

Keywords: cytokine • cytotoxic T-lymphocyte associated protein 4 • immune checkpoint blockade • programmed death 1 • renal cell carcinoma • immunotherapy • VEGF

In 2015 alone, about 61,560 new cases of kidney cancer will be diagnosed in the USA, with an estimated 14,080 kidney-cancer related deaths [1]. These numbers largely reflect cases of renal cell carcinoma (RCC), the most common primary cancer of the kidney. While many tumors are found early and can be cured surgically, approximately 20% present with de novo metastatic disease and about a third of patients diagnosed with localized RCC will ultimately develop metas-tases [2,3]. The management of metastatic renal cell carcinoma (mRCC) has under-gone substantial changes in the past decade, with novel systemic strategies fundamentally altering the approach to this disease.

Prior to 2005, treatment options for mRCC were limited, but evidence existed that RCC might be particularly sensitive to manipulations of the immune system. Ever-son and Cole were among the first to describe spontaneous remissions in patients with mRCC when they reported cases of tumors exhibiting shrinkage without any substantial

treatment in the 1960s [4,5]. IL-2, a cytokine important for regulating circulating lympho-cytes, was found to result in durable com-plete remissions in about 5–7% of clear cell mRCC patients, leading to the approval of aldesleukin by the US FDA for treatment of mRCC in 1992. Unfortunately, most patients did not respond and treatment was extremely toxic, limiting use to experienced, high vol-ume centers and reserved for patients with a good performance status and acceptable comorbid conditions [6,7]. A second cytokine, IFN-α, was also used for this disease, with a modest response rate (RR) of approximately 15% as a single agent, but overall the initial era of immunotherapy for mRCC was char-acterized by low rates of disease control and high rates of toxicity.

Between 2005 and 2012, seven new drugs garnered approval for the treatment of mRCC. This ushered in the VEGF tyro-sine kinase inhibitor (TKI) era, reflecting an enhanced understanding of the biology of mRCC. With the exception of bevacizumab,

Checkpoint inhibitors for renal cell carcinoma: current landscape and future directions

Matthew Zibelman1, Pooja Ghatalia2, Daniel M Geynisman1 & Elizabeth R Plimack*,1

1Department of Medical Oncology, Fox

Chase Cancer Center, Temple Health,

333 Cottman Avenue, Philadelphia,

PA 19111, USA 2Temple/Fox Chase Hematology

Oncology Fellowship Program, Temple

University School of Medicine, 3401

North Broad Street, Philadelphia,

PA 19140, USA


[email protected]

part of

Review



Review Zibelman, Ghatalia, Geynisman & Plimack

these new agents are oral, small molecule TKIs target-ing the VEGF. In addition, two drugs targeting the mammalian target of rapamycin (mTOR) pathways to disrupt angiogenesis and intrinsic cell proliferation signals were also approved [8]. By interfering with sys-tems essential for tumor growth and metastasis, com-binations of these drugs used in sequence improved the overall survival of mRCC to >28 months [9]. Unfor-tunately, while initial RRs are encouraging, most patients ultimately develop resistance and few have durable disease control.

Consequently, the impetus to harness the immune system to combat this disease remained strong. A meta-analysis evaluating tumor response in patients receiving placebo in randomized trials revealed high rates of spontaneous remissions in RCC, validating this approach [10]. More recently, new agents known as immune checkpoint inhibitors that unlock the anti-tumor abilities of the immune system have entered the clinic with promising durable responses in patients across a variety of tumor types, including RCC. The term immune checkpoint refers to the idea that cer-tain pathways inherent to the immune system regulate a sustained immune response and can be stimulated to shut down immune activation. This is in place to protect the host from overzealous immune activity that can result in harm after an insult (be it infectious, malignant or other) is recognized and dealt with. Many tumors have evolved the ability to exploit these pathways to evade immune recognition and maintain undisturbed growth and proliferation. Pharmacologic inhibition of these pathways can restore antitumor control in some patients. Two checkpoint pathways have been successfully targeted in Phase III oncol-ogy trials and have led to the approval of new drugs. The programmed death 1 (PD-1) pathway functions peripherally in the tumor microenvironment, while the cytotoxic T-lymphocyte associated protein (CTLA-4) pathway controls early T-cell activation [11]. In this review, we will discuss the agents being used to modify these pathways in the context of RCC, as well as other targets, combinations and strategies being explored to enhance efficacy and advance the treatment paradigm for RCC, commencing a new era of immunotherapy. We will focus on clear cell RCC (ccRCC), the predom-inant histologic subtype of this disease.

Programmed death 1 pathway blockadePD-1 is an immunoinhibitory receptor that belongs to the CD28/CTLA-4 family and is inducibly expressed on CD4+ and CD8+ T cells, natural killer (NK) cells, B cells and monocytes within 24 hours from their immunological activation [12]. Usually, activated T-cells, B-cells, NK cells, dendritic cells (DCs) and

monocytes express PD-1 in order to restrict autoim-munity during inflammatory states such as infec-tions. However, tumors have evolved the ability to express the main PD-1 ligand (PD-L1) to exploit this mechanism, thus downregulating the antitumor T-cell response [13]. PD-L1 is not expressed on normal kid-ney tissues, but is expressed in a significant proportion of both primary and metastatic RCC specimens. [14] Therefore, inhibiting this pathway with monoclonal antibodies (mAbs) targeting either PD-1 or PD-L1 can reenergize exhausted T-cells downregulated by tumor-directed PD-L1 expression, culminating in innate anti-tumor detection and coordinated tumor cell death.

Releasing this restraint on the immune system is not without consequences. The PD-1 checkpoint protects the immune system from overactivation and precipi-tating autoimmunity. Thus, side effects from check-point blockade have been characterized by organ-specific inflammatory reactions such as dermatitis and colitis, clinically and pathologically mimicking site-specific autoimmune diseases, and termed immune-related adverse events (irAEs). Serious and unusual toxicities rarely seen with any other agents, such as endocrinopathies and autoimmune pneumonitis, have been described [15]. These toxicities have proven to be a class effect, with severe adverse events (AEs) occurring at a relatively consistent 10–20% rate across clinical trials of PD-1/PD-L1 inhibitors regardless of tumor type. In the landmark Phase III trial evaluating the PD-1 inhibitor nivolumab in patients with previously treated mRCC, grade 3–4 AEs occurred in 19% of patients, but not all of these would be considered irAEs [16]. None of the most common irAEs, such as diarrhea/colitis, occurred as grade 3 or higher in >1% of patients. Nearly all acute autoimmune events have been reversible with prompt initiation of corticoste-roids and/or other immunosuppressive agents, and chronic endocrine deficiencies can be managed effec-tively with replacement therapy [15].

Immune checkpoint inhibitor monotherapy in mRCCPD-1 blockadeThe first immune checkpoint inhibitor to gain FDA approval for patients with mRCC was nivolumab (BMS-936558), a fully human IgG4 monoclonal anti-body (mAb) targeting PD-1. Nivolumab has also been FDA approved for the treatment of patients with meta-static melanoma and advanced non-small-cell lung can-cer (NSCLC) [17]. Preclinical work by Thompson et al. evaluated PD-L1 expression in ccRCC. In a series of 306 resected tumors, PD-L1 was expressed by 23.9% of the tumors analyzed. Patients whose tumors expressed PD-L1 had a significantly higher rate of metastatic


Checkpoint inhibitors for renal cell carcinoma Review

cancer progression as well as death from RCC [18]. In 2012 results of a Phase I trial with nivolumab were first reported. The trial included patients with various tumor types, including 33 patients with mRCC who had failed conventional agents such as TKIs. There was a 27% objective response rate (ORR) in these patients [19]. Since then several trials studying check-point inhibitors specifically in RCC have been reported (Table 1). Notably, the Phase III CheckMate-025 trial demonstrated superior median overall survival (mOS) with nivolumab (n = 406) in patients with advanced RCC who progressed on antiangiogenic therapy com-pared with patients receiving everolimus (n = 397; 25 vs 19.6 months, respectively; HR: 0.73; p = 0.002) [16]. Nivolumab also showed a trend toward improved pro-gression-free survival (PFS; HR: 0.88; p = 0.11) and fewer patients experienced grade ≥ 3 adverse events (19 vs 37%, respectively). Based on this trial, nivolumab earned approval by the FDA as a second-line therapy following antiangiogenic treatment failure in patients with advanced RCC [20].

Although nivolumab displayed a respectable ORR of 25% in the Checkmate-025 trial, the majority of patients did not respond. There are currently no validated biomarkers for selection of patients who may benefit from nivolumab therapy, but second-ary analyses in Checkmate-025 demonstrated that PD-L1 expression on tumor cells was prognostic but not predictive of benefit from checkpoint inhibition. Similarly, an exploratory Phase I study in patients with mRCC receiving three different doses of nivolumab analyzed potential biomarkers of response using four distinct methods: PD-L1 expression, soluble factors from peripheral blood, gene expression profiling and T-cell receptor sequencing [21]. As reported at the 2015 American Society of Clinical Oncology annual meeting, median OS appeared longer in PD-L1 posi-tive patients, but data were not yet mature and PD-L1 negative patients clearly showed responses. While this study suggested PD-L1 expression level may be associ-ated with the probability of prolonged survival, it did not uncover a definitive predictive biomarker. Final efficacy and survival data from this study is expected in early 2016. On the other hand, a study by Sekar et al. showed no correlation between PD-1 expression and survival in patients with RCC [22].

Pembrolizumab (MK-3475) is a humanized IgG4 PD-1-blocking mAb that has received FDA approval for the treatment of patients with metastatic mela-noma and advanced NSCLC after progression on platinum-based chemotherapy [23]. Pembrolizumab was investigated in a Phase I trial enrolling patients with advanced solid tumors, however no patients with mRCC were included [24]. Despite this, and given the

robust activity of nivolumab in this disease, pembro-lizumab is currently being studied further as a mono-therapy in RCC in the neoadjuvant setting (Clinical Trial: NCT02212730) but has not been explored as a single agent in the metastatic setting. As discussed later in this review, pembrolizumab is being studied extensively in combination with other agents in RCC. Table 2 includes ongoing clinical trials of single agents for RCC.

PD-L1 blockadeGiven the direct interaction of PD-1 on immune effec-tor cells with PD-L1 on tumor cells, both have proven to be viable targets for pharmacoinhibition. A Phase I trial of the PD-L1 inhibitor BMS-936559 was con-ducted in patients with varied tumor types, including mRCC, who had failed conventional agents. Seventeen patients with mRCC were enrolled. Objective response was seen in two (12%) and stable disease was seen in seven (41%) patients over 24 weeks [25]. Though this agent is not currently being studied further in mRCC, it demonstrated the potential of PD-L1 inhibition in RCC.

Atezolizumab (atezolizumab, MPDL3280A) is a humanized mAb targeting PD-L1, and results of an expansion cohort of patients with mRCC from its ini-tial Phase I trial have been reported [26]. This trial pre-dominantly enrolled previously treated (57% ≥ 2 prior lines) patients, and also included both clear cell and non-clear cell RCC (nccRCC). Amongst all ccRCC patients assessed for survival (n = 63), the mOS was 28.9 months and the mPFS was 5.6 months. The ORR (n = 62 for this outcome) was 15% amongst ccRCC patients, with no responses by RECIST in the patients with nccRCC (n = 7). Biomarker analysis found no clear association to PD-L1 expression on tumor cells and response. Grade 3 treatment-related adverse events (AEs) occurred in 17% of patients, which was compa-rable to similar studies of other agents.

At least two other PD-L1 inhibitors have been tested in Phase I studies of patients with advanced solid tumors and have included patients with mRCC, however, efficacy results in mRCC patients have not yet been made available. Recruitment is ongoing for a Phase I trial of the selective human IgG1 anti-PD-L1 mAb durvalumab (MEDI4736, NCT01693562). In this trial, patients with numerous tumor types, includ-ing mRCC, are being enrolled. This agent has reported single-agent activity in other tumors, but further stud-ies specific to RCC are noted below with discussion of combination trials. Avelumab (MSB0010718C), a fully human mAb targeting PD-L1, had results of safety and pharmacokinetic data reported at the 2015 American Society of Oncology (ASCO) Annual Meet-



ing [27,28]. Grade ≥3 AEs were reported in 12.3% of wpatients, with irAEs reported in 11.7%, both compa-rable to agents in other trials [28]. Further study of this agent in mRCC is currently limited to c ombination approaches.

Importantly, PD-1 actually has two main ligands, PD-L1 and PD-L2 [29]. PD-L1 has been more exten-sively studied and appears to be more diffusely expressed in the immune environment, thus it has been extensively targeted in clinical trials. PD-L1 also binds CD-80 on activated immune cells to impart inhibitory signals, though the clinical relevance of this interac-tion remains unknown. PD-L2 similarly engages other targets with unclear clinical significance. As drugs inhibiting these targets on both sides of the pathway are tested, these important escape mechanisms need to be considered as sources of differential response k inetics, toxicities and resistance pathways.

CTLA-4 blockadeAnother prominent immune checkpoint that is expressed on activated T-cells and has been targeted successfully with antineoplastic agents is CTLA-4. This type I transmembrane protein is upregulated on T-cells and functions largely in the lymphatic system as a physi-ologic safeguard against over activation of the immune system by outcompeting the T-cell co-stimulatory recep-tor CD-28 for its ligands CD80 and CD86 to dampen the overall immune response. However, like with PD-1/PD-L1, many tumor types have developed the ability to exploit this system to circumvent immunosurveillance and avoid a robust antitumor response [30].

Two CTLA-4 directed monoclonal antibodies (mAbs) have been assessed in clinical trials. Ipilimumab was the first immune checkpoint inhibitor made avail-able for clinical use when it received FDA-approval for the treatment of patients with metastatic melanoma in 2011 based on demonstration of a survival benefit in a Phase III trial [31]. It has continued to be studied as a viable single agent in other cancer types, as well as in various combinations which will be covered later in this review. Published data are available from a Phase II trial that investigated two separate dosing schedules of ipi-limumab as a single agent in patients with mRCC [32]. This study yielded an ORR of 12.5% in the higher dosed cohort, with six durable PRs reported across the dosing schedules. In a separate analysis of this study focusing on patients who developed enterocolitis as an irAE, the ORR in the mRCC cohort was 35% for patients who developed e nterocolitis, compared with 2% in those who did not [33].

Tremelimumab is the other CTLA-4 inhibitor that has been tested in clinical trials, but it has not gained FDA-approval for commercial use, likely due to multiple

Tab

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factors engrained in the design of the definitive Phase III trial in melanoma patients (dosing schedule, criteria used for tumor assessments, patient selection, subsequent treatment options) which failed to meet its primary end point [34,35]. For patients with mRCC, tremelimumab has been evaluated in a Phase I trial in combination with the oral tyrosine kinase inhibitor (TKI) sunitinib [36]. The study enrolled 28 patients, but it was stopped due to excess toxicity (one death and four patients with renal failure) and this combina-tion was aborted. Of 21 patients evaluable for response, 43% achieved a PR.

Although the data in mRCC is limited, it is clear from the melanoma population that CTLA-4 blockade yields a lower ORR and higher toxicity as compared with PD-1 pathway blockade. Thus, with nivolumab now available to patients, there is little experimen-tal appetite for single agent CTLA-4 inhibitor stud-ies to continue in mRCC. However, combinations of CTLA-4 inhibitors with PD-1 pathway blockers are leading the way in the nascent combination stage of immunotherapy for RCC.

Immunotherapy combinationsThe capacity for achieving durable responses with checkpoint inhibitors is what drives much of the excite-ment surrounding these agents. However, the ORR of the nivolumab arm in the Phase III Checkmate 025 trial was a modest 25% [16]. Although ORR is an underestimation of the proportion of patients ben-efiting from these agents since it cannot account for pseudoprogression, a substantial proportion of mRCC

patients do not attain a significant benefit from PD-1 inhibition. Therefore, strategies to increase the number of patients who benefit from checkpoint inhibition are being explored and rational combinations are viewed as the most viable path to success.

Dual checkpoint blockadeAgain extrapolating from studies in the metastatic melanoma population, combined PD-1 and CTLA-4 blockade set the bar for what is possible with immuno-therapy. In a three arm, double-blind, Phase III trial, patients with metastatic melanoma were randomized to receive ipilimumab alone, nivolumab alone, or the combination [37]. The trial met its co-primary end point of improving PFS, which was 11.5 months in the combination arm, compared with 6.9 and 2.9 months, respectively, in the nivolumab and ipilimumab arms (p < 0.001). The ORR in the combination arm was an impressive 57.6%. Unfortunately, with the improved efficacy comes increased toxicity. The incidence of grade 3–4 adverse events was 55.0% in the combina-tion arm, while in the single agent arms grade 3–4 events occurred in 16.3% (nivolumab) and 27.3% (ipi-limumab) of patients. More than a third of patients discontinued combination therapy due to toxicity. Notably, responses were noted to endure after cessation of therapy. Accordingly, this combination has gained FDA-approval for patients with metastatic melanoma and is being evaluated in patients with other immuno-genic tumors, including RCC.

Checkmate 016 is a Phase I study evaluating vari-ous combination regimens in patients with mRCC,

Table 2. Ongoing clinical trials with single agent checkpoint inhibitors in renal cell carcinoma.

Trial number Drug Setting Arms Phase Expected completion

NCT02595918 Nivolumab Neoadjuvant, nonmetastatic Single-arm Pilot September 2017

NCT02446860 (ADAPTeR)

Nivolumab Perioperative therapy, first-line metastatic

Single-arm II November 2016

NCT02596035 (Checkmate 347)

Nivolumab Second- to fourth-line, metastatic

Single-arm postmarketing IIIB/IV October 2017

NCT02575222 Nivolumab Neoadjuvant, nonmetastatic Single-arm I December 2017

NCT02212730 Pembrolizumab First-line neoadjuvant Prior to cytoreductive nephrectomy

Two arms, randomized to pembro or no presurgical treatment

I February 2017

NCT02599779 Pembrolizumab Second-line or above, metastatic

Pembrolizumab + SBRT given on progression vs SBRT with concurrent Pembro

II January 2019

NCT02626130 Tremelimumab First- or second-line, metastatic

Tremelimumab +/- cryoablation

Pilot February 2021

NCT01772004 Avelumab First-line metastatic Single-arm dose escalation with RCC expansion cohort

I April 2017



including three dosing combinations of ipilimumab/nivolumab (arm I1: nivolumab 3 mg/kg + ipilimumab 1 mg/kg; arm I3: nivolumab 1 mg/kg + ipilimumab 3 mg/kg; arm IN-3: nivolumab 3 mg/kg + ipilim-umab 3 mg/kg). Both treatment naive and previously treated (cytokine therapy only) patients were eligible for enrollment. Updated results from the two arms that progressed to the expansion cohorts (arm IN-3 stopped for excess toxicity) were reported at the 2015 ASCO Annual Meeting [38]. With 47 patients evalu-able in each arm, the ORR ranged from 38–43%, with another 40% achieving stable disease (SD) in each arm, thus resulting in a disease control rate (complete and partial responses plus stable disease) approaching 80%. Grade 3–4 toxicities were comparable to those reported in the melanoma study, with 16% of patients discontinuing therapy. The rate of toxicity was high-est in the arms with higher ipilimumab dosing. Based on these results, an open-label, randomized, Phase III trial (NCT02231749, Checkmate 214) comparing the ipilimumab/nivolumab combination to sunitinib for patients with previously untreated mRCC has c ompleted accrual.

Combined checkpoint blockade is not restricted to the ipilimumab/nivolumab combination. Several combinations of agents targeting PD-1, PD-L1 and CTLA-4 are being studied in RCC (see Table 3). Pembrolizumab is being evaluated in combination with ipilimumab or the cytokine pegylated IFN-α in a Phase I/II study in patients with mRCC and mela-noma (NCT02089685). The dose escalation portion will establish recommended Phase II doses (RP2D) of pembrolizumab with each combination, followed by randomization to each combination or pembrolizumab alone. The PD-L1 inhibitor durvalumab (MEDI4736) is being combined with tremelimumab in a Phase I study of patients with select, advanced solid tumors, including RCC (NCT01975831). Durvalumab is also being studied in combination with MEDI0680 (AMP-514), a PD-1-targeted mAb that also triggers internal-ization of PD-1 by contacted T-cells, in a Phase I study including all solid tumors (NCT02118337) [39].

Early studies have also begun exploring the inhi-bition of novel checkpoints in addition to the PD-1 pathway. One example of this is LAG-3, which is an immune checkpoint found on multiple immune cells, including CD8+ T-cells, and binds major histocom-patibility complex class II to suppress T-cell prolif-eration and function [40]. IMP321 is a soluble LAG-3 fusion protein that was tested in a Phase I dose escala-tion study in patients with mRCC [41]. 21 patients were treated at varying doses, and the drug was well toler-ated with grade 1 local reactions being the only clinical side effects and reduced tumor growth noted at higher

doses. A Phase I study of the anti-LAG-3 drug BMS-986016 with or without nivolumab in patients with select solid tumors, including RCC, is currently recruit-ing patients (NCT01968109). Indoleamine 2,3-dioxy-genase-1 (IDO1) is not a true immune checkpoint, but is an intracellular enzyme that serves as the rate lim-iting step in the kynurenine/tryptophan degradation pathway and has been shown to play an important role in immune tolerance in the tumor microenvironment of many cancers [42,43]. Epacadostat (INCB024360) is an IDO1 inhibitor that is being studied in a Phase I/II study in combination with pembrolizumab in multi-ple advanced solid tumors (NCT0217822), including mRCC. Preliminary results reported have shown the combination to be well tolerated. Of 19 patients evalu-able for response, 15 were reported to have reductions in tumor burden, including an ORR of 40% in the mRCC subset with a disease control rate of 80% [44].

Combinations of checkpoint inhibitors with VEGF receptor inhibitorsThe standard of care for first-line treatment of mRCC for the better part of the past decade has been with oral TKIs that target the VEGFR, as well as other pathways, to inhibit angiogenesis and suppress tumor growth [9,45,46]. These drugs achieve about a 25–30% response rate in treatment naive patients, with a dis-ease control rate >50%, but rarely result in durable responses, with median PFS ranging from 8.5–11 months [9,45,46]. The TKIs have a manageable and predictable safety profile, with little overlap with the checkpoint inhibitors, thus interest existed to com-bine approaches. The Checkmate 016 trial, in addition to combining nivolumab with ipilimumab as noted above, also included arms combining nivolumab with a TKI (either sunitinib or pazopanib) in both previ-ously treated and treatment-naive patients. Data for these cohorts was most recently presented at the 2014 ASCO Annual Meeting [47]. In 20 previously treated patients in the nivolumab/pazopanib group, the ORR was 45%. However, 60% of patients experienced a grade 3–4 adverse event, and significant hepatotoxicity led to closure of the nivolumab/pazopanib arm after the first cohort. The nivolumab/sunitinib arms com-bined (varying by dose of nivolumab) included both previously treated and untreated patients and yielded an ORR of 52%. 73% of patients on this combina-tion experienced a grade 3–4 AE, and though initially this combo showed less hepatotoxicity, effects on liver enzymes were prominent. The significant increase in toxicity of these regimens has discouraged further studies utilizing these combinations. Final results for both toxicity and efficacy are awaited to better under-stand the value of this combination. Whether a ltering


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the checkpoint inhibitor or the TKI will make a dif-ference is still a source of interest. Pembrolizumab is being studied in combination with pazopanib in a Phase I/II study in patients with previously untreated patients with mRCC (NCT02014636). The design includes a standard dose, followed by a three arm, ran-domized Phase II portion looking at the combination versus either drug alone. In a Phase Ib dose finding study, pembrolizumab is also being combined with axitinib, a TKI associated with much less hepatotoxic-ity, in previously untreated patients (NCT02133742). Preliminary results of the first 11 patients were pre-sented at the 2015 Kidney Cancer Symposium [48]. In regards to toxicity, two patients discontinued treat-ment due to related AEs and 73% of patients expe-rienced grade 3 AEs, but most were reversible and no patients discontinued treatment due to hepatotoxicity. 6 patients had confirmed PRs and all 11 experienced at least some degree of tumor shrinkage. Enrollment of 55 total patients is now completed with results awaited. Other planned combination trials are listed in Table 3.

Bevacizumab, a mAb that also works to inhibit angiogenesis via inhibition of the circulating ligand VEGF-A, is approved for use in mRCC combined with interferon-alfa based on data from Phase III tri-als [49,50]. Bevacizumab is not known to be hepatotoxic, so given the initial experience combining the oral TKIs with nivolumab, this combination is attractive. Bevaci-zumab was investigated in combination with the anti-PD-L1 inhibitor atezolizumab in a Phase I trial with a cohort of patients with mRCC [51]. As presented at the 2015 Genitourinary Cancers Symposium, 12 patients were evaluable for toxicity, 10 for response, with 83% of patients receiving this combination as their first line of systemic therapy in the metastatic setting. No grade 3–4 AEs were attributed to atezolizumab. The ORR was 40%. Currently, atezolizumab is being studied in combination with bevacizumab as an initial treatment for patients with mRCC in Phase II and Phase III stud-ies. In a three-armed, randomized Phase II study, treat-ment naive patients will receive atezolizumab with or without bevacizumab, or sunitinib as a single agent, until disease progression, with the option to cross-over to the combination arm for patients progressing after sunitinib (NCT01984242). This trial has com-pleted accrual with analysis ongoing. A randomized Phase III trial comparing the combination of atezoli-zumab/bevacizumab to sunitinib is currently open and recruiting (NCT02420821). Combining bevacizumab with pembrolizumab is also currently being tested for safety in patients with mRCC in a Phase I/II trial (NCT02348008).

Checkpoint blockade along with bevacizumab is being explored in a presurgical setting combined with

nivolumab. For some patients with mRCC, a cytore-ductive nephrectomy is performed based on data from the pre-TKI era demonstrating an overall survival ben-efit for patients receiving surgery prior to systemic ther-apy with IFN-α [52,53]. While the utility of this inter-vention is unproven during the TKI era (prospective trials ongoing), interest may be further re-energized with the emergence of checkpoint inhibition. A three-arm, randomized, Phase II study is currently accruing patients to evaluate the role of nivolumab with or with-out bevacizumab or ipilimumab prior to planned cyto-reductive nephrectomy in patients s urgically eligible (NCT02210117).

Combinations of checkpoint inhibitors with cytokines & vaccinesGiven the dynamic nature of the immune system, there is interest in exploring exogenous ways of priming or manipulating the immune milieu to favor a synergistic response when combined with checkpoint inhibition. One strategy being explored in multiple studies harks back to a previous iteration of treatments for mRCC. IFN-α is a type I cytokine previously in wide use for systemic therapy in patients with mRCC; however, its role has largely been replaced by the TKIs, and now by newer immunotherapies as well. IFN-α has been eval-uated in several large trials for patients with mRCC and as a single agent yields an ORR ranging from 7.5–16% [54–56]. This cytokine has also been shown to play an integral role in recruitment and immune recognition during the antitumor response [57]. IFN-γ, the only type II interferon, has a less triumphant role as an anticancer agent, failing to improve survival as a single agent in randomized Phase III trials for vari-ous malignancies [58,59]. Nonetheless, its importance in the antitumor immune response is well studied. Dunn and colleagues outlined its many important functions in fostering immunosurveillance in their description of the cancer immunoediting process [60]. Notably, IFN-γ is also known to be a key regulator of PD-L1 expres-sion, and Taube and colleagues have demonstrated that IFN-γ is an essential component of highly PD-L1 enriched tumors [61]. Consequently, combinations of these cytokines with PD-1 pathway inhibitors offer intriguing opportunities to prime the tumor micro-environment and potentially work synergistically to increase efficacy.

As mentioned previously, pembrolizumab is being combined with pegylated-IFN-α or ipilimumab in a randomized Phase I/II study of patients with mRCC or melanoma (NCT02089685). This study is closed to accrual, but not yet reported in RCC. Atezolizumab is being studied combined with conventional IFN-α in a Phase I dose-finding study (NCT02174172).



While this study also includes other tumor types and a combined atezolizumab/ipilimumab arm, the mRCC patients will only be accrued to the arm including IFN-α, and accrual is ongoing. One study is under-way utilizing IFN-γ in combination with nivolumab (NCT02614456). This is a Phase I dose-finding study exploring a short course of IFN-γ as induction, fol-lowed by combination therapy, in select, advanced tumors with an expansion cohort for patients with mRCC.

Another strategy being employed to prime the immune system includes the use of various cell and vec-tor-based vaccines that attempt to sensitize the immune response to a specific tumor antigen and generate a targeted antitumor response [62]. One current combi-nation involves pidilizumab (CT-011), a mAb against PD-1 derived using an IgG1 base to enable antibody-dependent cellular cytotoxicity, along with a dendritic cell-RCC (DC-RCC) fusion vaccine. DCs function as potent antigen presenting cells and play an integral role in stimulating a primary immune response. The vac-cine is generated by fusing autologous tumor cells with host DCs to create a population of DCs that express the entire tumor antigen repertoire, as opposed to one antigen which can more easily be downregulated by the tumor as a defense mechanism [63]. This combina-tion and the PD-1 inhibitor alone are being assessed in a Phase II trial in patients with mRCC during any line of therapy (NCT01441765). The study has completed accrual, but has not yet reported results.

Adjuvant & neoadjuvant settingsMost of the trials discussed thus far have been eval-uating patients with mRCC. While the durability of responses seen in some patients in this setting is encouraging, there is little expectation that most, if any, of these patients will be truly ‘cured’ of their cancer. On the other hand, the potential to apply the theories of checkpoint inhibition to the setting of early stage disease, with the goal of initiating and maintaining a lifelong antitumor response after resec-tion of a localized mass, is intriguing as a strategy to increase the cure fraction among patients with l ocalized high-risk RCC.

Currently, no FDA-approved therapy exists for the adjuvant treatment of patients with early stage RCC. Despite their success in the metastatic setting, the efficacy of oral TKIs has not translated to improved outcomes when administered after primary surgery, most notably highlighted by the disappointing results of the ASSURE trial evaluating adjuvant sorafenib and sunitinib [64]. With the advent of checkpoint inhibi-tors, the logical next step is to determine whether these agents could have a role in this setting. However, there

are important biological implications to consider. First, the mechanism of action with checkpoint inhibition relies on the presence of tumor antigens to be recog-nized and targeted by immune cells. After a resection of a primary tumor mass, there may be little, if any, tumor antigen remaining, which may subject patients to the toxicity of therapy without a clear mechanism for benefit. Furthermore, there is evidence that after surgical resection of a kidney tumor, PD-1 expression on immune cells is rapidly and significantly reduced, potentially eliminating the target of a PD-1 inhibi-tor [65]. These issues have created uncertainty regard-ing the optimal design of purely adjuvant studies using checkpoint inhibitors, and thus far none have been opened for accrual.

A rational strategy to circumvent some of these concerns is neoadjuvant administration instead of, or in addition to, adjuvant use. This design allows for increased tumor antigen burden at the initiation of treatment to maximize desired immune recognition. It enables a more objective measure of response radiologi-cally, while also enriching pathologic assessments pre and post treatment to support correlative biomarker exploration. Accordingly, multiple trials are cur-rently planned using this design. Nivolumab is being evaluated in two separate, small, single arm trials in slightly different patient populations (NCT02575222, NCT02595918). Pembrolizumab is also being stud-ied in one trial in the neoadjuvant setting, and this trial has begun accruing patients (NCT02212730). Data from these trials will be mostly explorative and hypothesis-generating to support further randomized designs as questions clearly remain. Some of these include the optimal number of doses and time planned preoperatively, whether postoperative treatment should be offered, and whether it could be administered in a brief or extended maintenance fashion. However, since (neo)adjuvant trials take a long time to produce results, the combined Eastern Cooperative Oncology Group and American College of Radiology Imaging Network (ECOG-ACRIN, EA) is in the final stages of planning a large, multicenter, two-armed, randomized, Phase III perioperative trial, The EA8143 (PROSPER trial) has been granted National Cancer Institute approval and will compare two doses of nivolumab followed by surgery and post-op nivolumab for nine months, ver-sus surgery followed by observation alone, in high-risk RCC patients.

Future perspective & questionsWhile the FDA-approval of nivolumab marks the beginning of what may prove to be the new immuno-therapy era in the treatment of mRCC, it does inject some uncertainty into the evolving treatment dogma.



The approval of nivolumab in mRCC is in the second-line metastatic setting, after progression on a first line TKI. This establishes a new standard of care for most patients with mRCC to receive either sunitinib or pazopanib as their initial treatment, followed by nivolumab in the second line, as it has demonstrated a clear OS benefit compared with everolimus in this set-ting [16]. Based on the path of prior oncologic agents, one might expect the next logical step would be a com-parison of nivolumab against a TKI in the first line setting. However, that study has not been planned. Instead, the dual checkpoint blockade combination of ipilimumab/nivolumab is currently the immunother-apy regimen being evaluated in comparison to suni-tinib for treatment-naive patients. If this study meets its primary endpoint, where does that leave us? Does ipilimumab/nivolumab become the de facto treatment of choice for all patients in the first line setting? Impor-tantly, we know from the preliminary results from the Checkmate 016 trial, as well as from the melanoma population, that ipilimumab/nivolumab significantly increases the toxicity compared with PD-1 inhibition alone. How much excess toxicity are we willing to accept in the name of improved ORR, especially when some patients may achieve similar benefit, at a fraction of the risk, with single agent nivolumab?

This is where biomarkers may truly become indis-pensable. Early hope was high that PD-L1 expression on tumors would identify tumors that would respond to PD-1 inhibition, but subsequent studies have clearly demonstrated that PD-L1 expression, whether on the tumor or stromal cells, is not by itself sufficient to predict benefit from PD-1 inhibition in RCC. RCC trials to date have consistently illustrated that while high PD-L1 expression can enhance the likelihood of response, PD-L1 deficient tumors can still respond to treatment. In fact, the Checkmate 025 trial was the first immunotherapy trial to find no difference in effi-cacy between the PD-L1 positive and negative popula-tions. While much of the focus of biomarker discovery in modern immunotherapy trials has focused on cor-relating with response, the true power may come from being able to predict which patients can derive ben-efit from PD-1 pathway inhibition alone, and which patients need combination therapy. Additionally, as it is likely that several combination strategies will dem-onstrate some clinical benefit, meticulous and rational biomarker assessments embedded in ongoing trials is crucial to be able to categorize patients by the strategy most likely to benefit them. It is our opinion that both single agent and dual checkpoint blockade strategies will ultimately develop a niche in this disease, with biomarkers utilized to help identify patients that can ‘get away with’ single agent therapy versus those that

require dual therapy and the associated increased toxic-ity risks. The progress of some of the alternative com-bination strategies discussed may further segregate the current homogeneity of the mRCC patients towards more personalized approaches based on biomarkers and tumor and/or host immune biology.

As more checkpoint inhibitors yield benefits in patients, both alone and in combination, having more options for patients is undoubtedly an advantage. But questions regarding sequencing, optimal dosing, dura-tion and treatment schedules, maintenance versus intermittent treatment strategies, and patient selection remain. The potential emergence of checkpoint inhi-bition in early stage disease may have a trickle-down effect as well. If neoadjuvant or adjuvant therapy dem-onstrates clinical benefit, what options will we have for those that do relapse? Will they still respond to immunotherapy upon retreatment in a recurrent meta-static setting, or will they need a “boost” from another agent? What if a patient develops a significant irAE in the adjuvant setting necessitating treatment discontin-uation? Are they forever excluded from potentially life-altering therapy upon relapse, or can they be rechal-lenged? These are difficult questions without clear answers at this early stage, but ones that need to be considered and addressed. Supporting innovative clini-cal trial designs may be needed to help answer some of these, and many other, important questions. These could include sequential therapy trials, designs com-paring adjuvant immunotherapy versus immunother-apy upon relapse, continuous versus intermittent PD-1 blockade with redosing at relapse, and rechalleng-ing responders who stop drugs for toxicity and later relapse. Schematic examples of some of these designs are represented in Figure 1.

Summary & conclusionCheckpoint inhibition has made its mark in the treat-ment of RCC and the future is bright. Nivolumab is now FDA-approved and available to patients as a stan-dard of care, with more drugs undoubtedly on the way. PD-1 inhibitors offer the chance for durable remissions in a meaningful subset of patients and with a gener-ally manageable toxicity profile. However, the major-ity of patients still do not achieve a clinical response and more work needs to be done for this resistant/refractory group. Rational combination approaches with TKIs, novel checkpoint inhibitors, and other immune modulators, offer some hope for improved efficacy. Additionally, extending the benefits we have begun to see in the metastatic setting to early stage disease presents a new opportunity for possible cures. We must continue to support clinical trials in mRCC, particularly those with strong preclinical foundations,



and we must safely incorporate high yield correlatives when possible to maximize our knowledge for future trial designs and biomarker candidates. It is an exciting time to study and treat RCC, but the excitement may have really just begun.

Financial & competing interests disclosureDr Zibelman has received institutionally directed clinical trial

support from Horizon Pharma. Dr Geynisman has served on

advisory boards for Pfizer, Novartis and Prometheus, and

has received institutionally directed clinical trial support from

M illennium and Pfizer. Dr Plimack has served on advisory

boards and as consultant for Novartis, Acceleron, Pfizer and

Bristol-Myers Squibb and has received institutionally directed

clinical trial support from Acceleron, Pfizer, Bristol-Myers

Squibb, AstraZeneca, GlaxoSmithKline, and Merck. The au-

thors have no other relevant affiliations or financial involve-

ment with any organization or entity with a financial interest

in or financial conflict with the subject matter or materials

discussed in the manuscript apart from those disclosed.


manuscript.


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2 Flanigan RC, Campbell SC, Clark JI, Picken MM.

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Figure 1. Schematic examples of potential future clinical trial designs to investigate emerging issues with checkpoint blockade in renal cell carcinoma. (A) Adjuvant study where patients are randomized to checkpoint blockade or observation. At relapse, the observation patients receive dual checkpoint blockade, while the patients who received checkpoint blockade postoperatively are randomized to dual blockade or a TKI. (B) Sequencing study for patients with previously untreated mRCC to investigate checkpoint blockade before and after TKI. (C) Study of continuous versus finite checkpoint blockade in mRCC, with dual checkpoint blockade added at relapse. mRCC: Metastatic RCC; PD-1: Programmed death 1; RCC: Renal cell carcinoma.

Stage I–III RCCs/p nephrectomy

Treatment-naivemRCC

Previously TKI-treated mRCC

Biomarker+

Biomarker+

Biomarker+

Biomarker+

Biomarker+PD-1 blockadefor 6 months

Observation

PD-1 blockade

PD-1 blockade

Relapse

Relapse

Progression

VEGF TKI

VEGF TKI

VEGF TKI

Dual checkpointblockade

Dual checkpointblockade

Re-initiate PD-1blockade

PD-1 blockadefor 1 year or

until best response

ContinuousPD-1 blockade

A

B

C



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future science group



Immunotherapy

Review 2016/05/288

6

2016

Colorectal cancer (CRC) remains the third most common cause of cancer death in the USA. Despite an increase in the repertoire of treatment options available for CRC, median overall survival has plateaued at approximately 2.5 years. Strategies that engage the patient’s native immune system to overcome checkpoint inhibition have proven to be promising in subsets of CRCs, specifically those with mismatch repair deficiency. Further studies are required to determine combinations of standard therapies with immunotherapy drugs and to discover the best biomarkers to predict response. This review provides insight into the progress made in treating patients with advanced CRC with immunotherapeutics and the areas that demand further research to make these drugs more effective in this patient population.

First draft submitted: 19 January 2016; Accepted for publication: 23 March 2016; Published online: 20 May 2016

Keywords: checkpoint inhibition • colorectal cancer • immunotherapy

BackgroundColorectal cancer (CRC) is the third most commonly diagnosed cancer in the USA with an estimated 50,000 deaths in 2015 [1]. Approximately 20% of patients present with advanced metastatic disease that is treatable but not curable. Advances in both cyto-toxic chemotherapeutics and targeted agents have resulted in improved outcomes with median survivals reaching almost to 3 years. Regardless, less than 10% of patients experi-ence long term survival and resistance to all known therapies eventually develops. There-fore, new strategies to prolong survival in these patients are still warranted and lessons can be learned from the success of alternative approaches in other tumor types.

The body’s immune system consists of a balanced milieu of stimulatory and inhibitory cells that works to both prevent tumorigen-esis and destroy active cancer cells (Figure 1). In CRC, the presence of tumor-infiltrating lymphocytes (TILs) has for many years been associated with improved overall survival

(OS) [2]. TIL-containing tumors are more likely to demonstrate microsatellite insta-bility, with recent data revealing strikingly high response rates to immune c heckpoint in hibitors [3].

The pivotal role of the immune system in both the surveillance and destruction of tumors has been exploited to produce new treatment options that have garnered much success in melanoma and non-small-cell lung cancer. Specifically, monoclonal antibodies that target and block immune checkpoint proteins allow for the activation of the patient’s innate immune system to fight tumors internally. Despite promising preclinical data that support harnessing the immune system to fight CRC, gaps of knowl-edge remain in terms of which patients will demonstrate clinical benefit when treated with immunotherapies, and how to incite an immune response in the majority of CRCs where current immune-based therapies seem to be inactive. This article will review the current landscape of immune checkpoint

Checkpoint inhibition for colorectal cancer: progress and possibilities

Barry Paul1, Bert H O’Neil2 & Autumn J McRee*,3

1School of Medicine, University of North

Carolina, Chapel Hill, NC, USA 2Indiana University Melvin & Bren Simon

Cancer Center, Indianapolis, IN, USA 3Lineberger Comprehensive Cancer

Center, University of North Carolina,

Chapel Hill, NC, USA


Tel.: +1 919 966 5902

Fax: +1 919 966 6735

[email protected]

part of

Review



Review Paul, O’Neil & McRee

inhibition in treating advanced CRC with a focus on further research that is required for increased efficacy of these strategies in CRC.

Immune surveillance in CRCThe concept of immune surveillance of cancer cells was originally hypothesized by Paul Erhlich in 1909 [4]. Unfortunately, subsequent studies failed to support this hypothesis. For example, athymic nude mice (entirely lacking T-cells) bred in the 1970s failed to generate increased levels of carcinogen-induced or spontaneously arising tumors relative to mice with intact immune systems [5,6]. Later studies, however, elucidated that the nude mice used in these experi-ments still had a small population of functional T-cells as well as a normal population of NK cells that likely explained the previous results [7,8], keeping the idea of immune surveillance alive. Finally, in 2001 the

immune surveillance concept was demonstrated in a mouse model by Shankaran et al. using Rag-2-null mice, which lack the recombination activating gene and are thus unable to undergo V(D)J rearrangement. He obtained mice completely deficient of T and B lym-phocytes and with more unstable and less functional NK cells [9,10]. In these mice, he noted significant increases in methylcholanthrene-induced tumors that were rejected when transplanted into immunocompe-tent wild-type mice. Rag-2-null animals also showed a significant increase in spontaneous malignant carcino-mas. Interestingly, the majority of spontaneous tumors (4 of 6) were colonic in origin [9]. This effect was even more evident when Rag-2-null x γ

c null mice were

examined. These mice lacked all lymphocytes and consequently had no adaptive immune system and a diminished innate immune system. Increased methyl-cholanthrene-induced tumor burden was seen in these

Figure 1. The pivotal balance of the immune milieu in both stimulating and inhibiting the immune system to both prevent cancer formation and destroy tumor cells.

IL-12

NO

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CSF-1IL-10

IL-12Stress ligand(in MHC I)

NK KIR

NKG2Dreceptor

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VEGF, EGF, MMPs(act on tumor micro-environment to promoteinvasion and metastasis)

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IL-2IFN-γTNF-α

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TGF-β, IL-6

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IL-4IL-5IL-10IL-13

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Checkpoint inhibition for colorectal cancer Review

animals when compared with Rag-2-nulls and there was a higher frequency of rejection when cells from the Rag-2-null x γ

c null mice were injected into wild-

type mice [8]. Finally, several studies have observed increased rates of cancers (many of which were nonvi-rally mediated) among severely immunocompromised humans [11,12]. Similarly, registries of solid-organ trans-plant patients have also shown a small but statistically significant increased risk of CRC [12–14].

Understanding immune surveillance in CRC requires appreciation of the interaction between the tumor and the immune system in a process referred to as immunoediting [15]. Current theories propose that under constant selective pressure from the immune system some tumors develop heterogeneous resistance mechanisms to escape immune destruction [16]. The mechanisms by which this process occurs relies on a complex interplay between the cells of the immune sys-tem and tumor cells. The so called ‘Three E’s’ model of immunoediting theorized that this process occurs in three phases: the elimination phase during which the immune system destroys cancer cells; the equilib-rium phase during which growth of the tumor cells and immune-mediated tumor cell death exist in bal-ance manifested by a latency period; and finally the escape phase where tumor progression and hence clini-cal evidence of cancer occurs [17]. Increasing evidence supports an important role of both the innate immune system as well as the adaptive immune system in CRC immune surveillance. Given the complex interplay between immunomodulators it is helpful to examine each cell type individually.

MacrophagesMacrophages are present at all stages of tumor progres-sion. They develop from circulating monocytes and are recruited to tumors by chemokines such as CCL2, as well as the growth factors CSF-1 and VEGF [18]. They are referred to as tumor associated macrophages (TAMs) and are present within the tumor itself and classically exist in two distinct phenotypes: the so-called M1 and M2 phenotypes (although transcrip-tome analysis suggests there may be more overlap between the two classes than previously thought) [19,20]. The M1 cells secrete proinflammatory cytokines (IL-6, IL-12, IL-23 and TNF-α), are directly tumoricidal through nitric oxide generation and promote adaptive immunity by increasing MHC1 expression and T-cell costimulatory molecules (CD86 and CD40) [21,22]. M2 cells work primarily to scavenge cellular debris, induce angiogenesis, and hence have an increased tendency to promote tumorigenesis. In human CRC samples, TAMs are often found around necrotic areas of tumor and the advancing margin. Their prognostic

significance in CRC has been controversial but seems to portend a positive prognosis [21,23]. Zhang et al. conducted a meta-analysis of 55 studies represent-ing several cancer types and found that only in the five CRC studies included (representing 1149 patient samples) did a high density of TAMs correlate with increasing overall survival [24]. Also Zhou et al. exam-ined a series of 160 cases of stage IIIB and stage IV CRC and found that infiltration of macrophages at the invasive front of the tumor edge was associated with an improved 5-year overall survival and lower levels of hepatic metastases [25]. While the ratio of M1/M2 populations does not seem to differ based on survival or stage, a study of about 450 CRC samples did observe a significant correlation between M1 and M2 markers and improved prognosis. Additionally, a statistically significant inverse correlation with stage was seen for both M1 and M2 expression [26].

Natural killer cellsNatural killer (NK) cells also play an important role in innate immunosurveillance. NK cells have two classes of receptors that work in concert to screen cells for potential malignant transformation: the NKG2D receptor that binds to ligands expressed on tumor cells, and the human killer cell immunoglobulin-like recep-tor (KIR) that recognizes MHC-1 molecules on cells. The receptor binds to cells with stress-induced ligands presented in their MHC-1. These ligands are thought to function as ‘immuno-alerters’ and are often present in cells that have undergone malignant transforma-tion. Studies have shown that there are a wide variety of ligands that bind to this receptor with significant redundancy, which is thought to help counter tumor immunoevasion, but the affinities of the ligands to the receptor vary widely [27,28]. KIR receptors exist on the surface of NK cells and recognize MHC-1 molecules. Cancer cells often downregulate expression of surface MHC-1 molecules and NK mediated killing of cells with low levels of MHC-1 is thought to be another form of immune surveillance [29]. In humans, increased levels of intratumoral NK cells have been associated with increased overall survival [30].

Tumor-associated neutrophilsRecently, the role of neutrophils in the immune sys-tem’s surveillance of CRC has been investigated. Simi-lar to TAMs these so-called tumor-associated neutro-phils (TANs) seem to exist in two subsets: the tumor inhibiting N1 subtype and the tumor promoting N2 subtype with elevated levels of peritumoral TGF-β lev-els appearing to select for the N2 phenotype [31]. These cells are recruited to sites of tumorigenesis through IL-8, and has been shown to be induced by mutant



KRAS [32,33]. In clinical studies of CRC, higher levels of TANs are correlated with more advanced clinical stage and decreased overall survival [34,35]. Additionally, several studies have shown that an elevated peripheral blood neutrophil to lymphocyte ratio (NLR) is associ-ated with poor prognosis [36]. Specifically, Chua et al. showed that an NLR > 5 was an independent predictor of poor overall survival in a study of 171 patients with mCRC receiving first-line palliative chemotherapy.

Myeloid-derived suppressor cellsThe myeloid-derived suppressor cells (MDSCs) are a group of immature macrophages, granulocytes and dendritic cells present in lymphoid and tumor cells [37]. They have been shown to inhibit NK and T-cells through production of reactive oxygen species and nitric oxide (NO), induction of regulatory T-cells (Treg) and enhanced TGF-β secretion [38]. In vitro models of MDSCs indicate direct cell-to-cell contact as an important step in this process [38,39]. In CRC tumor samples an increased proportion of MDSCs in the tumor was correlated with increased metastasis and more advanced tumor stage [37,38].

T-cellsLikely the most critical step in inducing the adaptive immune response to tumor is T-cell activation, a pro-cess that is tightly regulated and better understood in recent years. Activation of cytotoxic CD8+ T-cells (CTLs) is dependent on at least three signals: anti-genic stimulation through the T-cell receptor (TCR); co-stimulation through molecules such as CD28, CD40, 4–1BB, CD27, ICOS and/or OX40; and stimulation through receptors for inflammatory cyto-kines, especially IL-12 and IFNα [40]. Once activated, CTLs clonally expand and mediate cellular destruc-tion through direct lysis or apoptosis using perforin and granzyme B [41]. CTLs are critical in detecting the so-called neoantigens that arise as a consequence of tumor-specific mutations as well as tumor-associ-ated antigens (TAAs), classified as normal self-anti-gens expressed in abnormal concentrations in tumor cells [42,43]. Intracytoplasmic staining combined with cytokine release assays have demonstrated that CTLs found in tumors (TILs) do indeed bind to TAAs in human CRC [44]. Several studies have shown a positive correlation between increased quantity of TILs in the tumor and improved prognosis [45–47]. Mlecnik et al. examined 599 CRC specimens and found that high levels of memory CD8 T-cells (CD8+/CD45RO+) in the tumor epicenter and invasive margin were more accurate at predicting disease-free survival than tra-ditional TNM classifications [46]. Efforts are ongoing to translate these data clinically into a companion to

standard staging practices, named the Immunoscore, which can be used to better prognosticate cancers that have been resected [48]. In another study of 959 CRC samples, increased TILs were associated with lower levels of vascular, lymphatic or perineural invasion. Further analysis of these samples showed the TILs from tumors without evidence of local invasion were enriched for mature CD8 cells compared with TILs from tumors where local invasion had occurred [49]. The improved prognosis in patients with tumors that demonstrate increased TILs (which often include tumors with mismatch repair deficiency) is thought to result from suppression of micrometastases through the adaptive immune response, accounting for their improved prognosis.

CD4+ T-cells bind to antigens presented in the MHC-II molecule and further differentiate into helper T-cells (Th1, Th2 or Th17) or Tregs. The CD4+ T-cell differentiation pathway is directed by cytokines. Spe-cifically IL-12 induces Th1 phenotype, IL-4 induces Th2 phenotype, TGF-β and IL-6 induce Th17 pheno-type, and TGF-β induces Tregs [50]. Th1 cells secrete IL-2, IFNγ and TNF-α and have been shown to promote CTL proliferation and antibody-dependent cell-mediated cytotoxicity (ADCC) in CRC [45]. IL-4 induces the Th2 phenotype, which promotes tumor growth through secretion of IL-4, IL-5, IL-10 and IL-13. Notably, secretion of Th1 cell cytokines sup-presses Th2 cell production and vice versa and this so-called ‘Th1/Th2 shift’ is considered a major cause of escape from immune response [45,51,52]. In CRC, higher expression of genes associated with a Th1 phenotype correlated with improved survival whereas the Th17 cell signature (proinflammatory cells that produce IL-17) portended a poorer prognosis [53].

Tregs are induced by TGF-β inhibited by IL-6, and express CD4, CD25 and Foxp3 [54]. Functionally they serve to diminish the activation of other T-cell populations through direct cell contact and secretion of adenosine, IL10 and TGF-β resulting in net immu-nosuppression [54,55]. In mouse models, removal of Tregs results in rejection of transplanted tumors [56]. In patients with CRC, increased numbers of Tregs have been demonstrated both in peripheral blood, tumor-draining lymph nodes and tumor tissue [57,58]. Higher stages of CRC and larger tumor size have also been shown to correlate with increased intratumoral expres-sion of Foxp3 [59].

Modes of immune escape in CRCAs mentioned previously the constant selection pressure placed on transformed cells may result in expansion of clones resistant to immunoediting. The mechanisms by which CRC cells accomplish this is an area of active



research but generally focus on tumor adaptations to allow decreased host defense immunomodulation.

Presentation of antigens in MHC molecules is required for T-cell activation. In studies of CRC reg-istries, HLA-1 has been shown to be reduced in 63% of samples and absent in 7% of samples [60]. Complete loss of HLA-1 is driven by β2-microglobin in microsat-ellite unstable tumors and LMP7/TAP2 downregula-tion in microsatellite stable tumors [61,62]. Watson et al. examined MHC-1 expression in 462 CRC patients and reported that 76% had normal levels of MHC-1 and that these patients had the highest disease-specific survival (68 months). Only 9.9% of patient samples had decreased MHC-1, which resulted in the lowest disease-specific survival (41 months). Notably, 13.6% of patient tumors had absent levels and these patients had similar disease-specific survival (60 months) to the patients with normal MHC 1 levels, which is thought to be due to higher NK-induced immunoediting in this population [29].

HLA-G is a nonclassical MHC-1 molecule that has recently been implicated in immune escape given its ability to provide tolerance from NK cell recognition and confer resistance to IFN therapies in melanoma patients [63–65]. In several CRC studies, increased expression of HLA-G is significantly correlated to poor overall survival in patients [66,67]. This is thought to be due to direct interaction of HLA-G1 with the inhibi-tory receptors ILT2, ILT4 or KIR2DL4 on NK cells. Additionally, the secreted isoform of HLA-G5 can exert its suppressive effect by binding with the CD8 molecule, leading to the Fas/FasL-mediated apoptosis of activated CD8 T-cells [68].

B7-H3 is a costimulatory molecule present on APCs that belongs to the B7 family. Interaction of B7-H3 and its T cell counter-receptor (TLT-2) induces pro-liferation of both CD4+ and CD8+ T cells, enhances the induction of CTLs and increases IFNγ produc-tion [69]. In preclinical models of CRC, increased lev-els of B7-H3 inhibited apoptosis and were associated with increased cancer cell survival and drug resis-tance. This effect was shown to be driven by activa-tion of the JAK/STAT3 pathway [70]. In patient CRC samples, high levels of B7-H3 correlate with advanced tumor grade and decreased TILs [71]. Taken together these data implicate B7-H3 as a possible effector of immune escape and hence of potential drug target.

Recent evidence has also suggested that CD73 may play a role in tumor-associated immune suppression. In CRC, high expression of CD73 is significantly asso-ciated with increased risk of metastasis and decreased overall survival [72,73]. Levels of CD73 have also been shown to be higher in CRC liver metastases then in primary tumor specimens. This has led to the theory

that CD73 interacts with both stromal and inflamma-tory cells to create a metastatic niche that is immune-privileged and predisposes to metastasis [74].

Clinical data on checkpoint inhibitorsCTLA-4Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is a cell surface receptor found on T-cells that, when bound to its ligands (CD80 and CD86) on antigen-presenting cells, works to downregulate the immune system [75]. In addition, crosslinking of CTLA-4 also results in apoptosis of activated T cells, which further impairs their innate function [76]. While CTLA-4 exhibits homology with the co-stimulatory receptor CD28 and binds the same ligands (albeit at much higher affinity), the expression of CTLA-4 raises the activation threshold of T cells, reducing the potency of CD28 activity and hence paving the way for tumori-genesis to take place [77]. Therefore, targeting CTLA-4 as a means of enhancing T-cell-mediated tumor responses represents an attractive treatment strategy.

Polymorphisms within the CTLA-4 gene have been identified, which may dampen the inhibitory effects of the receptor and increase cancer susceptibility. A meta-analysis of 1180 cases of CRC paired with 2110 controls found a significant association between the +49A/G polymorphism and risk of developing CRC with an odds ratio of 1.69 [78]. This amino acid substi-tution is theorized to result in decreased mRNA levels and thus less CTLA-4 protein production.

The inhibitory effects of CTLA-4 on immune sys-tem function are best illustrated in CTLA-deficient mice, in which a rapid lymphoproliferative process occurs within weeks resulting in severe myocarditis and pancreatitis and early death [79]. Subsequent stud-ies performed by Allison et al. showed that blocking CTLA-4 activity with monoclonal antibodies led to decreased tumor growth in mouse models of CRC [80]. Additionally, mice initially treated with anti-CTLA-4 antibodies exhibited either delayed tumor growth or no new tumors at all upon re-challenge of tumor cells, compared with untreated controls that developed sig-nificant tumor burden at a rapid rate. Splenocytes iso-lated from mice treated with anti-CTLA-4 antibodies show an increase in cytotoxic tumor-specific T-cells with an increase in interferon (IFN)-γ secretion sug-gestive of a Th1 response [81]. Synergy has also been illustrated with anti-CTLA-4 antibodies and chemo-therapy, specifically ixabepilone and paclitaxel, in a subcutaneous cell line xenograft model of CRC, with a similar induced memory response [82].

Tremelimumab is a fully human monoclonal antibody (mAb) targeting CTLA-4 that in Phase I studies resulted in complete responses in melanoma



patients and has since been studied alone and in combination with chemotherapy in multiple tumor types [83]. Given strong preclinical rationale support-ing a role for CTLA-4 blockage in CRC, a single-arm Phase II trial of tremelimumab in refractory meta-static CRC was conducted in 47 patients [84]. Of the 45 evaluable patients, 95% had disease progression with only one patient exhibiting a partial response. While this result was not considered promising, stud-ies are ongoing with both tremelimumab and ipilim-umab (another antibody antagonist of the interaction between CTLA-4 and its ligands) in combination with PD-1 inhibitors in this disease (NCT02408861 and NCT01975831).

PD-1/PD-L1The programmed cell death protein 1 (PD-1) is a receptor found on the surface of T-cells, B-cells and macrophages. PD-1 binds two ligands, PD-L1 and PD-L2. The role of PD-1 in negatively regulating the immune system has been solidified in PD-1-deficient mice, in which a lupus-like proliferative syndrome spontaneously develops with evidence of arthritis and glomerulonephritis [85]. CD8+ T-cells harvested from the spleens of mice lacking PD-1 demonstrate signifi-cantly increased activation and proliferation. While the ligands for PD-1 are constitutively expressed on T and B cells, as well as antigen presenting cells, mul-tiple cancers have been reported to upregulate PD-L1. This results in decreased susceptibility of tumor cells to T-cell-mediated attack and hence increased tumor development [86].

Approximately 35% of mismatch-repair (MMR) proficient and 30% of MMR deficient CRC samples have been reported to demonstrate strong expression of PD-L1 via immunohistochemical staining [87]. In MMR proficient CRC, the presence of PD-L1 was associated with lower T-stage, lower histologic grade, lack of lymph node involvement and less vascular invasion. Additionally, improved OS in univariate analysis (HR 0.84, p = 0.003) was demonstrated with a 5-year survival rate of 35% in tumors lack-ing PD-L1 expression compared with 62% in strong PD-L1 expressing tumors. A direct correlation was also noted between tumors that expressed PD-L1 and the presence of CD8+ T-cells within the tumor infil-trate, suggesting that activation of the innate immune system results in improved outcomes.

Preclinical data exist that suggest a role for target-ing the PD-1 pathway in CRC. In a mouse model of CRC in which Colon-26 cells were subcutaneously injected into the flank, intraperitoneal treatment with anti-PD-1 mAb resulted in decreased tumor growth compared with control [88]. Additionally, an increase

in the expression of activating cytokines, includ-ing IFN-γ and tumor necrosis factor (TNF)α, was observed in mice treated with anti-PD-1 mAb com-pared with controls. Interestingly, a synergistic effect was seen with dual inhibition of the vascular endo-thelial growth factor receptor 2 (VEGFR2) compared with either VEGFR2 or PD-1 inhibition alone with a decrease in tumor vascularization but no conflicting effect on T-cell infiltration. A similar additive effect was seen in mice harboring CRC tumors treated with MEDI4736, an anti-PD-L1 mAb, in combination with oxaliplatin, a chemotherapeutic used widely in the treatment of metastatic CRC [89]. This led to an increase in high mobility group box 1 (HMGB1), a marker of immune-mediated cell death and in increase in intratumoral T-cell infiltration.

Phase I trials of both anti-PD-1 and anti-PD-L1 mAbs have been published, and lend credence to the benefit of immune check point inhibition in mul-tiple tumor types [90,91]. A total of 37 patients with CRC were included in these studies with objective responses in patients with non-small-cell lung can-cer (NSCLC), melanoma and renal cell carcinoma but none in CRC patients. A larger Phase I study of the anti-PD-L1 mAb MPDL3280A, which mainly enrolled NSCLC, RCC and melanoma patients, did include six patients with refractory CRC with one partial response noted in a patient whose tumors dem-onstrated increased PD-L1 expression [92]. The com-bination of MPDL3280A and the anti-VEGF mAb bevacizumab was also evaluated both as a doublet (Arm A) and in combination with 5-FU and oxali-platin (FOLFOX, Arm B) in a trial of 44 patients with mCRC [93]. Patients in Arm A with refractory disease demonstrated an overall response rate (RR) of 8% whereas treatment-naive patients in Arm B had responses of 44%. The combination was well toler-ated with no unexpected toxicities.

While inhibitors of the PD-1/PD-L1 axis have been largely unsuccessful in CRC, data are emerg-ing that patients whose tumors possess deficient MMR proteins with microsatellite instability (MSI) have increased response rates to immune checkpoint inhibitors. This was first noted in a Phase I study of pembrolizumab in 23 CRC patients presented at European Society of Medical Oncology (ESMO) 2015 in which one patient exhibited a partial response and was known to harbor a tumor with MSI [94]. Indeed, a retrospective review of primary CRC tumors found that tumors with MSI, compared with MSS tumors, demonstrated higher expression of five checkpoint proteins (PD-1, PD-L1, LAG-3, IDO and CTLA-4) [95]. Based on the observation that MMR-deficient CRCs harbor TILs far more frequently than



MMR-proficient tumors, the KEYNOTE-164 study evaluated pembrolizumab in three groups of patients: one with MSI-H mCRC, one with MSS mCRC and a third with MSI-H noncolorectal cancers [3]. Validat-ing the investigators’ hypothesis, patients with MMR deficient CRC showed a partial RR to treatment of 40% compared with 0% in patients with MMR pro-ficient CRC with a hazard ratio for disease progres-sion or death of 0.10 (p < 0.001) that favored those with MSI. These tumors possessed a significantly increased number of mean somatic mutations (1782 vs 73) with the authors suggesting that the enhanced presence of neoantigens results in improved respon-siveness to PD-1 inhibition. Trials are ongoing inves-tigating the effects of combining immune checkpoint inhibitors with both cytotoxic agents and targeted small molecules in CRC, specifically a study of FOLFOX plus pembrolizumab and a trial combining MPDL3280A with the MEK inhibitor cobimetinib, both in r efractory CRC (Table 1).

CD40CD40 is a transmembrane receptor that is expressed on the surface of antigen presenting cells and is involved in the stimulation of the immune system [96]. The CD40 ligand (CD154) is a member of the TNF recep-tor family and is found primarily on activated T-cells. Upon binding, increased expression of the MHC results in the leakage of cytokines that both prime activated T-cells as well as recruit activated NK cells. Small studies of human CRC samples have shown CD40 expression rates of about 35% with the infiltra-tion of CD40+ macrophages in CRC demonstrating an advantageous prognosis [97,98]. In mouse models of CRC, treatment with a CD40 agonistic antibody resulted in improved survival with NK cells isolated from these mice demonstrating increased cytotoxic activity against tumor cells [99]. Phase I studies have been completed that evaluated the safety and MTD of the CD40 agonist CD-870,893 in combination with carboplatin and paclitaxel, of which one patient with CRC was enrolled [100]. Four other compounds (ADC-

Table 1. Ongoing clinical trials of checkpoint inhibitors in colorectal cancer.

Drug name Trial number Phase Therapy Patient population

AVX701 NCT01890213 Phase I CEA VRP vaccine Resected stage III CRC

MEDI4736 (anti-PD-L1 mAb)

NCT02227667 Phase II Single agent MEDI4736 Advanced refractory CRC

NCT02261220 Phase I + tremeliumumab (anti-CTLA mAb) Solid tumors

NCT02586987 Phase I + selumetinib (MEK inhibitor) Solid tumors

NCT02318277 Phase I + INCB024360 (IDO inhibitor) Solid tumors

NCT02503774 Phase I + MEDI9447(anti-CD 73 mAB) Solid tumors

NCT02617277 Phase I + AZD1775 (wee 1 inhibitor) Solid tumors

NCT02118337 Phase I + MEDI0680 (anti-PD1 mAb) Solid tumors

MPDL3280A (anti-PD-L1 mAb)

NCT01988896 Phase I expansion + combimetinib (MEK inhibitor) Advanced refractory CRC

NCT02350673 Phase I + RO6895882 (IL-2v targeting CEA) CEA positive solid tumors

NCT02471846 Phase I + GDC-0919 (IDO1 inhibitor) Solid tumors

RO7009787 Phase I + RO7009787 (CD40 agonist) Solid tumors

NCT02410512 Phase I + MOXR0916 (anti-OX40 mAb) Solid tumors

NCT02323191 Phase I + RO5509554 (CSF1-R inhibitor) Solid tumors

Pembrolizumab (anti-PD-1 mAb)

NCT02437071 Phase II + ablation Advanced CRC

NCT02375672 Phase II + FOLFOX Advanced CRC

NCT02260440 Phase II + azacitadine Advanced CRC

NCT02298959 Phase I expansion + aflibercept (anti-VEGF mAb) Advanced CRC

NCT02528357 Phase I + GSK3174998 (OX40 agonist) Solid tumors

NCT02432963 Phase I + p53 vaccine Solid tumors

NCT02179918 Phase I + PF-05082566 (4–1BB agonist) Solid tumors

Nivolumab (anti-PD-1 mAb)

NCT02335918 Phase I/II + varlilumab (anti-CD27 mAb) Advanced refractory CRC

NCT02060188 Phase II + ipilimumab (anti-CTLA-4 mAb) Advanced refractory CRC

Carcinoembryonic antigen NCT02327078 Phase I + INCB24360 (IDO1 inhibitor) Solid tumors



1013, RO7009789, SEA-CD40 and ChiLob 7/4) are currently in trial development either as single agents or in combination with other checkpoint inhibitors.

ConclusionOur understanding of how the immune system surveil-lances the body for cancer and how ultimately cancer cells successfully evade the immune system has allowed for the development of a new promising class of drugs that has made great strides in tumors such as melanoma and lung cancer. Early Phase I studies of drugs that tar-get pivotal immune checkpoints, such as CTLA-4 and

PD-1/PD-L1, have enrolled only a handful of patients with CRC and results as single agents have been less than robust. Newer data on the role of these agents in MMR deficient CRC, where the mutational burden is higher and hence a more advanced repertoire of neo-antigens is present, appear more promising. Trials are ongoing that evaluate whether combining checkpoint inhibitors with cytotoxic chemotherapy or with other targeted agents will prove more effective. Additionally, more data are urgently required to determine the most accurate biomarker to predict response to these agents in CRC.

Executive summary

Colorectal cancer (CRC) remains a common cause of cancer death despite advances in treatment paradigms• Long-term life expectancies in patients with advanced disease is rare, begging the question as to what other

strategies exist to prolong a plateaued overall survival of approximately 30 months.The role of immunosurveillance in the development of CRC• The three E’s model of immunoediting theorizes how the immune system shifts from destroying cancer cells,

to a latency period in which tumor growth and immune-mediated tumor cell death exist in balance, to an escape phase in which tumor progression occurs.

Various immune cells contribute to the immunomodulation of CRC• Tumor-associated macrophages (TAMs) portend a positive prognosis in CRC with the M1 phenotype secreting

proinflammatory cytokines and increasing T-cell co-stimulatory molecules.• The role of tumor-associated neutrophils (TAN) in CRC is less well defined, but some data suggest a skew

toward a protumor N2 phenotype.• Intratumoral natural killer (NK) cells are associated with increased overall survival.• Myeloid-derived suppressor cells (MDSCs) induce regulatory T-cells (Tregs) and hence are associated with

more advanced tumor stage.• Cytotoxic T-cells (CTLs) are critical for recognizing neoantigens present due to tumor-specific mutations and

binding tumor associated antigens in CRC.Modes of immune escape in CRC• Reduced HLA-1 expression, which encodes the α chain of the MHC-1 complex, results in decreased disease

specific survival.• The presence of HLA-G, implicated in immune escape in melanoma, leads to apoptosis of activated CD8

T-cells in CRC and portends a poor prognosis.• In preclinical models, the co-stimulatory molecule B7-H3 inhibits apoptosis of tumor cells and is associated

with drug resistance.CTLA-4 downregulates the immune system & impairs T-cell activation• CTLA-4 polymorphisms increase the risk of developing CRC.• Tremelimumab is an anti-CTLA-4 mAb that in a Phase II study of refractory CRC patients had less than ideal

response. Studies are ongoing with combinations of tremelimumab and other checkpoint inhibitors.The PD-1/PD-L1 pathway as a promising target for deficient MMR CRC• 35% of mismatch repair (MMR) proficient and 30% of MMR deficient CRC tumors express PD-L1, which is

associated with good risk features.• Phase I studies of both anti-PD-1 and anti-PD-L1 mAbs included 37 CRC patients, none of which demonstrated

meaningful response.• However, in patients with MMR deficient CRC treated with pembrolizumab, a marked increase in response

rate of 40% was reported compared with 0% in patients whose tumors were MMR stable.Conclusion• Further studies are forth coming to better evaluate combinations of chemotherapy with immunotherapy

drugs in CRC.• Sequencing these drugs with targeted agents is also a promising approach.• The future will likely bring more data on the role of peptide vaccines and adoptive T-cell transfer strategies

for CRC.



Future perspectiveWe exist on the cusp of fully understanding the par-ticular subset of patients with CRC that will benefit from immunotherapy and in exactly what sequence these drugs should be given with other agents, includ-ing vaccines, targeted drugs, cytotoxics and even adoptive T-cell transfer. It is now being recognized that targeted drugs, including anti-EGFR and VEGF antibodies, prime dendritic cells for antigen presen-tation and may represent potential synergistic com-binations with immunotherapy [101]. Alternatively, while whole cell vaccines proved to elicit a less than ideal immune response in CRC, vaccines that target specific TAAs appear more promising. An adenovirus based vaccine used to target carcinoembryonic anti-gen (CEA) in advanced CRC patients demonstrated a median overall survival in the refractory setting of 11 months with no long-term adverse events [102]. Chi-meric antigen receptor therapy, in which T-cells are collected from patients and genetically engineered to

express specific receptors on their surface, allowing them to recognize tumor specific antigen, is actively being studied in refractory CRC (NCT01218867). Therefore, the promise of immunotherapy existing as a relevant and reliable therapy option for advanced CRC holds true with further understanding of the mechanisms that drive immune escape and the ways in which these drugs can best be manipulated to har-ness the most effective results.

Financial & competing interests disclosureB Paul discloses that as a previous employee of Bristol-Myers

Squibb, he has been granted a retirement package as well as

stock options. BH O’Neil has received travel funds from Mer-

ck. The authors have no other relevant affiliations or financial

involvement with any organization or entity with a financial

interest in or financial conflict with the subject matter or ma-

terials discussed in the manuscript apart from those disclosed.


manuscript.


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•• PivotalPhaseIstudyofanti-PD-1antibodyinsolidtumorswithlittletonoresponseinthosepatientswithCRC.


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101 Ribas A, Wolchok JD. Combining cancer immunotherapy and targeted therapy. Curr. Opin. Immunol. 25(2), 291–296 (2013).

• Thisreviewhighlighttheeffectsofmanyapprovedtargetedagentsontheimmunesystemandhowcombiningthemwithcheckpointinhibitorsmayresultinsynergydependingonthesequence.

102 Balint JP, Gabitzsch ES, Rice A et al. Extended evaluation of a Phase 1/2 trial on dosing, safety, immunogenicity, and overall survival after immunizations with an advanced-generation Ad5 [E1-, E2b-]-CEA(6D) vaccine in late-stage colorectal cancer. Cancer Immunol. Immunother. 64(8), 977–987 (2015).

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