Role of immune-checkpoint inhibitors in lung cancer

Introduction

Cancer immunotherapy has its origins in the early 1900s when the concepts of hostimmune defense against cancer and cancer immune surveillance werepostulated.^1,2Since the early 1900s there were attempts at using techniques to stimulate theimmune system against cancer using intratumoral injections of live or an inactivatedmixture of Streptococcus pyogenes and Serratiamarcecsens.¹ Despite some early success with such approaches, lack of understanding of themechanisms of the antitumor immune response and the complexity involved in such anapproach limited the enthusiasm. It took nearly a century to unravel the mysteriesof the immune system and the role of the immune system in cancer.³ In response to an inflammatory signal (secondary to infection or cancer), theantigen-presenting cells (APCs) responding to the antigen are stimulated by theproinflammatory cytokines [interleukin (IL)-1, tumor necrosis factor (TNF)-α] andthe APCs interact with naïve T cells. This interaction between APCs or tumor cellsand T cells allows for the proliferation of the antigen-specific T cells and is thecritical first step in mounting an immune response. Following this interactionbetween APCs or tumor cells with cells in the adaptive immune system (T cells, Bcells) resulting in subsequent immune response via a complexorchestration of immune coregulatory pathways. These intricate coregulatory pathwaysare often redundant mechanisms to avoid immune response against self antigens. Thesecoregulatory pathways, namely immune checkpoints, are coopted by the tumor cells toavoid the immune system.^4–8 Recent advances in ourunderstanding of these key immune regulatory pathways resulted in the development ofpromising new strategies in treating cancer.

Lung cancer is the world’s leading cause of cancer death.⁹ Platinum-doublet chemotherapy has been the standard of care for frontlinetherapy in advanced non-small cell lung cancer (NSCLC) without oncogenic drivers.Five-year survival for these patients is dismal at under 10%. In about 15–20% ofpatients with NSCLC key genomic alterations leading to oncogenic activation, whichis amenable to targeted therapy, can be identified. However, most of these patientsreceiving targeted drugs will have an emergence of resistance to targetedtherapy.^10,11 Recently, understanding the host immune system–tumorinteractions has led to the acknowledgment of immune evasion as an additionalhallmark of cancer.¹² Several immune cell types within the tumor microenvironment serve complex andparadoxical roles from the antitumor response, influence tumorigenesis and immuneevasion. But the key immune regulatory pathways, which serve as the critical immuneevasion interface between the tumor and the immune cells, are promising targets fordrug development.⁸ The recent success of drugs targeting the immune-checkpoint pathways,particularly the programmed cell death 1 (PD-1) pathway, has changed the paradigm ofclinical management of several cancers.⁸ Treatment with immunotherapy has the potential to induce clinicallymeaningful and durable responses.^13–16 Three drugs targeting the PD-1pathway (nivolumab, pembrolizumab, and atezolizumab) have been approved by the USFood and Drug Administration (FDA) for use in both chemotherapy-naïve and previouslytreated advanced stage NSCLC.^17–20 A timeline of FDA approval forcheckpoint inhibitors (CPIs) in lung cancer is presented in Table 1. Immune checkpoint blockade withPD-1/programmed cell death ligand 1 (PD-L1) inhibitors has thus become part of thestandard-of-care treatment option for patients with advanced stage NSCLC; however,only a small subset (20–30%) of patients respond to treatment.^16–25

Table 1.

Timeline for FDA approval of checkpoint inhibitors.

Drug	Manufacturer	FDA approval	Indication	Companion diagnostic
Nivolumab	Bristol-Myers Squibb (Princeton, New Jersey)	March 2015	Second-line advanced stage NSCLC (squamous cell carcinoma)	None required
Nivolumab	Bristol-Myers Squibb	October 2015	Second-line advanced stage NSCLC (nonsquamous cellcarcinoma)	None required
Pembrolizumab	Merck (Kenilworth, New Jersey)	October 2015	Second-line advanced stage NSCLC	PD-L1 IHC >1% TPS*
Atezolizumab	Genentech/Roche (San Francisco, California)	April 2016	Second-line advanced stage NSCLC	None required
Pembrolizumab	Merck	October 2016	First-line advanced stage NSCLC	PD-L1 IHC >50% TPS
Pembrolizumab with carboplatin/pemetrexed	Merck	May 2017	First-line advanced stage NSCLC (nonsquamous cellcarcinoma)	None required

FDA, US Food and Drug Administration; IHC, immunohistochemistry; NSCLC,non-small cell lung cancer; PD-1, programmed cell death 1; PD-L1programmed cell death ligand 1; TPS, tumor proportion score.

Immune checkpoint pathways

Cancer immunotherapy is based on improved tumor antigen presentation and recognition;stimulation or amplification of an immune response; or disinhibition of immune cellsto allow for an improved antitumor immune response.⁸ Immune response begins with antigen presentation by APCs such as dendriticcells that present tumor antigens on the cell surface with major histocompatibilitycomplex (MHC) molecules. APCs present antigens to T cells by MHC peptide complexesto antigen-specific T-cell receptors on the surface. Various regulatory mechanismscheck the proliferation of autoreactive T cells and maintenance of immune tolerancein normal tissues. This intricate balance between immune-stimulatory and inhibitorysignals limit harmful autoimmune responses.²⁶ Tumors utilize multiple mechanisms of immune evasion, such as genetic andepigenetic modifications; expression of immune inhibitory cytokines such as IL-10and transforming growth factor β in the tumor microenvironment; and induction ofT-cell suppressive signaling pathways.⁸ The inhibitory signals to suppress T-cell activity are mediated by‘immune-checkpoint’ molecules (inhibitory ligands and their cognate receptors),including the CD28/cytotoxic T-lymphocyte antigen 4 (CTLA-4) axis, and PD-L1/PD-1which have emerged as promising druggable targets (Figure 1). Other checkpoint molecules such asTIM3, B7H3, VISTA, LAG3, and TIGIT are currently being evaluated as potentialtargets for cancer immunotherapy.

• PD-1/PD-L1 pathway: PD-1 is a coinhibitory surface receptor that is expressedby activated and exhausted T cells. It is also expressed on other immunecells such as B lymphocytes, natural killer (NK) cells, and myeloid derivedsuppressor cells (MDSCs).^27,28 Interaction betweenPD-1 and its ligands, PD-L1 and PD-L2, on tumor cells leads todownregulation of T-cell response in the tumor microenvironment^29,30 (Figure 1). Many lungcancer cells overexpress PD-L1 as a mechanism for suppressing T-cellresponse.^7,29

• CD28/CTLA-4 system of immune modulation: CTLA-4 is expressed mainly on Tcells (CD4+, helper and CD8+, killer T cells) with some expression in otherimmune cells including B lymphocytes and fibroblasts.^31,32 CTLA-4competes with the costimulatory receptor CD28 for binding to the sameligands, B7-1 (CD80) and B7-2 (CD86) on the surface of APCs, resulting indownregulation of immune response^32,33 (Figure 1). CTLA-4 acts early duringthe priming phase of antigen presentation and following T-cellreceptor–peptide complex engagement, it is rapidly mobilized to the cellsurface, allowing feedback inhibition to occur within an hour of antigen presentation.³⁴ Therapeutic anti-CTLA-4 monoclonal antibodies have shown clinicalactivity in advanced melanoma, most likely via disruptingthe CD28 activation on T cells as well as through depletion of regulatory Tcells (T-regs) in the tumor microenvironment.³⁵

Figure 1.

Pathways involved in immune checkpoint regulation.

APC, antigen-presenting cell; PD-1, programmed cell death 1 [co-stimulatorysignals (green)]; PD-L1, programmed cell death ligand 1 [co-inhibitorysignals (red)].

PD-1 blocking antibodies

Anti-PD-1 antibodies block the interaction of PD-1 with PD-L1 and PDL-2, but do notprevent PD-L1 interaction with CD80 (B7.1).

Nivolumab

Nivolumab (BMS-936558) is a fully human immunoglobulin G4 (IgG4) antibody againstPD-1. In an early phase I trial (Checkmate-003 study), nivolumab demonstratedpromising clinical efficacy, particularly in patients with high PD-L1expression.^25,36,37 Results from two landmark studies, CheckMate-017 (squamousNSCLC) and CheckMate-057 (nonsquamous NSCLC), demonstrated benefit inprogression-free survival (PFS) and overall survival (OS) from nivolumab comparedwith docetaxel.^17,18 Checkmate-017 is a randomized phase III clinical trial inpatients with squamous cell lung carcinoma evaluating nivolumabversus docetaxel in patients previously treated with aplatinum-doublet chemotherapy. In this study, nivolumab demonstrated a 1-yearsurvival rate of 42% [95% confidence interval (CI) 34–50] compared with 24% (95% CI17–31) in the docetaxel group. The OS was significantly longer with nivolumab, witha 41% reduction in the risk of death with nivolumab [hazard ratio (HR) 0.59; 95% CI0.44–0.79; p < 0.001]. In addition, overall response rate (ORR)was higher in the nivolumab arm compared with docetaxel [20% (95% CI 14–28)versus 9% (95% CI 5–15); p = 0.008].¹⁸ Checkmate-057 is a randomized phase III clinical trial in patients withnonsquamous cell lung carcinoma evaluating nivolumab versusdocetaxel in patients previously treated with platinum-doublet chemotherapy. In thisstudy, nivolumab demonstrated a 1-year survival rate of 51% (95% CI 45–56%) comparedwith 39% (95% CI 33–45%) in the docetaxel group. The OS was significantly longerwith nivolumab, with a 27% reduction in the risk of death with nivolumab (HR 0.73;96% CI 0.59–0.89; p = 0.002). In addition, ORR was higher in thenivolumab arm compared with docetaxel [19% (95% CI 15–24) versus12% (95% CI 9–17), p = 0.02].

In both the CheckMate-017 and CheckMate-057 trials the predictive role of PD-L1expression was evaluated in the following subgroups: at least 1%, at least 5%, or atleast 10% tumor cell expression using Dako 28-8 assay. In the CheckMate-017 trial,PD-L1 expression at any level was not predictive of clinical benefit. However, inthe CheckMate-057 trial, the was a trend to improve efficacy in patients with higherexpression of PD-L1. There was no statistically significant difference demonstratedin OS in patients lacking PD-L1 expression. Dako 28.8 PD-L1 assay is approved as acomplementary diagnostic test for patients with nonsquamous NSCLC but not forsquamous NSCLC.

These positive trial results led to approval of nivolumab by the FDA in advancedsquamous and nonsquamous NSCLC as second-line systemic therapy after progression onfirst-line chemotherapy, regardless of PD-L1 expression (Table 1).

However, a phase III trial (CheckMate-26) comparing nivolumab with platinum-baseddoublet chemotherapy as first-line treatment for stage IV, recurrent NSCLC with atleast 1% PD-L1 positive, showed no benefit in the primary endpoint, PFS [median PFS4.2 (CI 3.0–5.6) versus 5.9 (CI 5.4–6.9) months; HR 1.15 (CI0.91–1.45); p = 0.2511]. The ORR for nivolumab was 26.1%versus 33.5% for chemotherapy. Nivolumab did not improve PFS orOS in a retrospective subgroup analysis done on patients with at least 5% PD-L1 positivity.³⁸

Pembrolizumab

Pembrolizumab (MK-3475) is a fully humanized IgG4 monoclonal antibody against PD-1.In a phase Ib trial (Keynote-001 study), pembrolizumab demonstrated promisingclinical efficacy, particularly in patients with high PD-L1 expression.¹³ A subsequent open-label randomized phase II/III clinical trial (Keynote-10)compared pembrolizumab with docetaxel in patients with metastatic NSCLC whosedisease had progressed on prior chemotherapy and expressed PD-L1 staining of atleast 1% tumor proportion score. In this study, the median OS and PFS were bothsignificantly improved with pembrolizumab compared with docetaxel [OS: HR 0.71 (95%CI 0.58–0.88, p = 0.0008) with 2 mg/kg; HR 0.61 (95% CI 0.49–0.75,p < 0.0001) with 10 mg/kg] and the benefit was even higherfor the subgroup of patients with PD-L1 of at least 50% tumor proportion score (TPS)[OS: HR 0.54 (95% CI 0.38–0.77, p = 0.0002) in patients receiving 2mg/kg every 3 weeks; HR 0.50 (95% CI 0.36–0.70, p < 0.0001) with10 mg/kg of pembrolizumab every 3 weeks].¹⁹ In the frontline setting, pembrolizumab monotherapy was compared withplatinum-doublet chemotherapy in a phase-randomized open-label clinical trial(Keynote-024) in patients with advanced stage NSCLC with PD-L1 expression (⩾50% TPS).³⁹ In this trial patients were randomized to either pembrolizumab 200 mg every 3weeks or investigator’s choice of platinum-doublet chemotherapy, with patients inthe control arm allowed to cross over to receive pembrolizumab. The study met itsprimary endpoint of PFS, demonstrating improvement in PFS with pembrolizumab (10.3months, 95% CI 6.7–not reached) compared with platinum-doublet chemotherapy (6.0months, 4.2–6.2). PFS was longer in the pembrolizumab-treated patientsversus patients treated with chemotherapy (HR for diseaseprogression 0.50; 95% CI 0.37–0.68; p < 0.001). In addition,80.2% (95% CI 72.9–85.7) of patients in the pembrolizumab arm were alive at 6 monthscompared with 72.4% (95% CI 64.5–78.9) in the chemotherapy arm. OS was significantlyimproved in patients treated with pembrolizumab, with decreased risk of deathcompared with chemotherapy (HR 0.60; 95% CI 0.41–0.89; p = 0.005).³⁹ In a phase II clinical trial (Keynote-21) in patients with chemotherapy-naïveadvanced nonsquamous NSCLC, carboplatin-pemetrexed with continued pemetrexedmaintenance was compared with the same regimen combined with pembrolizumab.⁴⁰

Based on these clinical trial data, pembrolizumab is approved by the FDA in advancedsquamous and nonsquamous NSCLC as first-line systemic therapy for patients withPD-L1 expression [22C3 immunohistochemical staining (IHC) with >50% TPS) or as asecond-line systemic therapy after progression on first-line chemotherapy, with atleast 1% PD-L1 expression on tumor cells. Pembrolizumab was recently also FDAapproved in the first-line setting for metastatic nonsquamous NSCLC in combinationwith pemetrexed and carboplatin independent of PD-L1 expression (Table 1).

PD-L1 blocking antibodies

Anti-PD-L1 antibodies block the interaction of PD-L1 with PD-1 and CD80 (B7.1), butdo not prevent the interaction of PD-L2 with PD-1 and CD80 with CTLA-4.

Atezolizumab

Atezolizumab (MPDL-3280A) is a humanized IgG1 monoclonal antagonistic antibody thattargets PD-L1. It is engineered to bypass antibody-dependent cell-mediatedcytotoxicity (ADCC) of activated T cells that express PD-L1. In a phase I trial withexpansion cohorts in patients with NSCLC, atezolizumab demonstrated promisingclinical efficacy.⁴¹ In an open-label phase II randomized clinical trial (POPLAR), patients withadvanced stage NSCLC whose disease progressed on post-platinum chemotherapy wereassigned to receive either atezolizumab or docetaxel once every 3 weeks.⁴² In this study, patients treated with atezolizumab had an improved median OSof 12.6 months (95% CI 9.7–16.4) versus 9.7 months (8.6–12.0) fordocetaxel [HR 0.73 (95% CI 0.53–0.99); p = 0.04].⁴² A confirmatory phase III (OAK) trial in patients with advanced stage NSCLCfollowing progression on platinum-based chemotherapy compared atezolizumab withdocetaxel. Like the POPLAR results, the OS was significantly improved withatezolizumab in comparison to docetaxel [median OS 13.8 months (95% CI 11.8–15.7)versus 9.6 months (8.6–11.2); HR 0.73 (95% CI 0.62–0.87),p = 0.0003]. PD-L1 expression on tumor cells (TC1/2/3) orimmune cells (IC1/2/3) (⩾1% PD-L1 by VENTANA SP142 assay) was predictive of thebenefit of atezolizumab. In patients with TC1/2/3 or IC1/2/3 PD-L1 expression inadvanced NSCLC, atezolizumab improved OS with a median OS of 15.7 months (95% CI12.6–18.0) versus 10.3 months (8.8–12.0) with docetaxel [HR 0.74(95% CI 0.58–0.93); p = 0.0102]. However, patients lacking PD-L1expression (TC0 and IC0) also had improved survival with atezolizumab [OS 12.6months versus 8.9 months; HR 0.75 (95% CI 0.59–0.96)].²⁰

These results led to approval of atezolizumab by the FDA in the second-line settingfor patients with advanced stage NSCLC and VENTANA SP142 assay was approved as acomplimentary diagnostic (Table1).

Durvalumab

Durvalumab (MEDI4736) is a high-affinity, humanized IgG1κ antagonistic antibody thattargets PD-L1. Results from a phase II study (ATLANTIC) showed preferential activityin tumors with PD-L1 expression.⁴³ PD-L1 positivity was defined as at least 25% of tumor cells with membranousstaining for PD-L1. Response rate was 16.4% in patients with PD-L1-positive tumorsand 7.5% in PD-L1-negative tumors that received durvalumab. The randomized phaseIII, PACIFIC trial [ClinicalTrials.gov identifier: NCT02125461] of durvalumab assequential treatment in patients with locally advanced, unresectable NSCLC whosedisease did not progress following definitive platinum-based concurrentchemoradiation showed median PFS improvement of over 11 months from time ofrandomization [16.8 months versus 5.6 months; HR 0.52 (95% CI0.42–0.65); p < 0.001], regardless of PD-L1 expression.⁴⁴ The OS data are still pending. In another phase III trial (MYSTIC) offrontline therapy in patients with NSCLC, durvalumab monotherapy is being comparedwith either a combination of durvalumab plus the anti-CTLA-4 antibody tremelimumabor standard-of-care chemotherapy [ClinicalTrials.gov identifier: NCT02453282]. Theresults of this trial are still awaited. Combination therapies of durvalumab withother agents, including gefitinib, AZD9291 and other immunotherapies (Table 3), are also beingevaluated in phase I trials.

PD-1 and CTLA-4 blocking antibodies

Combined immune-checkpoint inhibition

Due to distinct mechanisms of activation of immune checkpoints on T cells, such asCTLA-4 stimulation in the lymphatic tissue whereas PD-1/PD-L1 activation occurs inthe tumor microenvironment,⁸ there is rationale for combining CPIs for improved clinical outcomes. Hellmanand colleagues described the results of a phase I study combining nivolumab plusipilimumab as first-line treatment for advanced NSCLC, with patients assigned toeither nivolumab (3 mg/kg) every 2 weeks plus ipilimumab (1 mg/kg) every 12 weeks ornivolumab (3 mg/kg) every 2 weeks plus ipilimumab (1 mg/kg) every 6 weeks (CheckMate 012).⁴⁶ The response rate of 57% was achieved in both arms, with at least 1% PD-L1expression compared with 47% and 38% in the total population. No significantdifference was observed in median PFS, with values of 8.1 (5.6–13.6) and 3.9(2.6–13.2) months, respectively. No previously known toxicities were reported andgrade 3–4 adverse events occurred in 37% and 33% in both arms. A phase III clinicaltrial to evaluate this combination is currently ongoing (CheckMate 227)[ClinicalTrials.gov identifier: NCT02477826].

A combination of durvalumab (anti-PD-L1 antibody) and tremelimumab (anti-CTLA-4antibody) is being evaluated for safety, tolerability and antitumor activity in aphase Ib trial with 102 patients [ClinicalTrials.gov identifier: NCT02000947].⁴⁷ Clinical benefit was observed regardless of PD-L1 expression status andobjective responses were achieved in 23% of patients in the tremelimumab 1 mg/kgcohort. Durvalumab 20 mg/kg every 4 weeks plus tremelimumab 1 mg/kg was defined asthe maximum tolerated dose, and selected for ongoing phase III studies (MYSTIC,NEPTUNE) [ClinicalTrials.gov identifier: NCT02453282, NCT02542293].⁴⁸

Combination with chemotherapy

Chemotherapy can modulate immune responses directly or indirectly byimmunostimulation, increased immunogenicity through increased mutational burden andneo-epitope formation.⁴⁹ Immune CPIs work by reactivating immune responses. The mechanism of action ofchemotherapeutic drugs and CPIs may therefore be complementary.

CheckMate 012 and CheckMate 227 are phase I and phase III trials, respectively,evaluating a combination of nivolumab plus chemotherapy in the frontlinesetting.^50,51 Keynote-021, a phase II study, reported that the addition ofpembrolizumab to carboplatin/pemetrexed in newly diagnosed metastatic nonsquamousNSCLC increased ORR to 55% (42–68%) compared with 29% (18–41%), regardless of PD-L1status. Grade 3–4 adverse events did not differ significantly by addition of pembrolizumab.⁴⁰ This led to the FDA approval of pembrolizumab in combination withcarboplatin/pemetrexed in patients with newly diagnosed stage IV nonsquamous NSCLC.This combination is being further evaluated in phase III trials (Keynote-189,Keynote-407) [ClinicalTrials.gov identifier: NCT02578680, NCT02775435]. Acombination of ipilimumab (in a phased regimen) with carboplatin and paclitaxel infirst-line therapy of advanced or metastatic NSCLC improved PFS (5.1versus 4.2 months) and ORR (32% versus 14%)[ClinicalTrials.gov identifier: NCT01285609]. At present, conclusions fromchemotherapy combinations with CPIs are limited by small numbers of patients andlimited follow-up times. Although response rate and PFS are increased whenpembrolizumab is added to chemotherapy, OS remains unchanged.

Role of biomarkers in patient selection

Cancer immunotherapy has changed conventional treatment paradigms by expanding thetreatment options for patients with cancer. However, despite current success, theresponse rate to CPIs in advanced NSCLC is around 30%. Thus, there is a growing needto identify predictive and prognostic biomarkers for better patient selection. Thebasic principles underlying a good biomarker include analytical validity(reliability and reproducibility), as well as clinical utility. Several studies inNSCLC and melanoma show that tumor response to CPIs is associated with their immuneprofiles. For example, tumors that have high T-cell infiltration and express aninflammatory gene signature and show a ‘T-cell inflamed phenotype’ are more amenableto checkpoint inhibition.⁵²

PD-L1 expression

PD-L1 is the ligand for checkpoint receptor PD-1 expressed on T cells. Tumors withhigh infiltration of T cells may demonstrate higher PD-L1 expression as a form ofadaptive resistance mechanism and are more likely to benefit from PD-1/PD-L1inhibition.^7,25,53–57 At least 50% PD-L1 expressionis approved as a companion biomarker (Dako 22C3 pharmDx) with frontline,single-agent pembrolizumab in patients with NSCLC.^13,39 Other complementary diagnostictests (recommended, but not required for drug prescription) for nivolumab (Dako 28-8pharmDx) and atezolizumab (Ventana SP142) are also FDA approved for use in NSCLC.⁵⁸ Despite approval of IHC assays and evaluation of PD-L1 expression on tumorcells or immune cells to predict the efficacy of PD-1/PD-L1 blockade,^59–61 its clinical utility as anexclusive predictive biomarker remains controversial. While most studies concur thata higher level of tumor cell membrane PD-L1 expression is associated with improvedoutcome/response to PD-1/PD-L1 blockade, there is evidence that a subpopulation ofpatients with PD-L1-negative tumors may also have clinical benefit from CPIs.⁶⁰ The heterogeneity and dynamics of PD-L1 expression confound its use as apredictive biomarker. Different IHC staining assays utilize different antibodyclones and scoring systems for PD-L1 detection, as summarized in Table 2. The VentanaSP263, Dako 22C3 and Dako 28-8 clones have been used most commonly and were found tocluster together when evaluated for the pathologists’ concordance in scoring usingNSCLC specimens.^61,62 The SP142 clone did not cluster with the others and seemed tounderscore PD-L1 expression compared with the other assays.^61,62 Oneexplanation is that this clone was raised against an intracellular epitope of PD-L1,whereas the others target extracellular epitopes of PD-L1.

Table 2.

Diagnostic assays for PD-L1 for anti-PD-1/PD-L1 drugs in non-small cell lungcancer.

	Pembrolizumab			Nivolumab	Atezolizumab
Assay	22C3			28–8	SP142
Indication	1st	1st	2nd	2nd	2nd
PD-L1 required	⩾50%	No	⩾1%	No$	No$
Regimen	Single agent	With chemo*	Single agent	Single agent	Single agent

*With carboplatin and pemetrexed for adenocarcinomas only.

$Response is enriched when positive.

PD-1, programmed cell death 1; PD-L1 programmed cell death ligand 1.

Table 3.

Immune-related adverse events associated with checkpoint inhibition andmanagement.

Manifestation	Severity	Management
Gastrointestinal (GI) • Immune-mediated colitis • Pancreatitis	• Grade 1: Asymptomatic imaging finding of colonthickening • Grade 2: Moderateabdominal symptoms 4–6 stools/day	• Hold immunotherapy for grade ⩾2; work up to rule outinfectious etiology ova, parasites and stool culture. Stoolantigen for Clostridium difficile • American Diet Association colitis diet, loperamide oratropine sulfate • If persistent symptoms over 1 weekstart oral prednisone 1 mg/kg/day or equivalent. Taper over 4weeks if symptoms improve. Start infliximab 5 mg/kg every 2weeks if symptoms do not improve after 3 days on steroidtreatment
	• Grade 3/4: Severe and persistent abdominal pain, fever, ileus andlife-threatening complications >7 stools/day overbaseline	• Strongly recommend GI consult and colonoscopy to rule outnonimmune etiologies • Recommend hospitalization andstart intravenous methyl prednisone 2–4 mg/kg/day or equivalent,taper over 4–6 weeks if resolves to grade 1 or better. If noimprovement after 48–72 h, add alternative immunosuppressiveagents mycophenolate mofetil or infliximab
Liver • Immune-mediated hepatitis	Grade 1: Asymptomatic/mildlysymptomatic AST/ALT <2.5 × ULN Total bilirubin<1.5 × ULN Grade 2: Symptomatic AST/ALT: 2.5–5 × ULN Totalbilirubin: 1.5–3 × ULN	• Delay drug; increase frequency of LFT monitoring untilresolution • Oral prednisone 1 mg/kg/day or equivalent,taper over 4 weeks if symptoms resolve, add alternativeimmunosuppressive agent (tacrolimus, cyclophosphamide ormycophenolate mofetil) if symptoms do not improve after 48 h.Avoid infliximab because of potential forhepatotoxicity. • Consider cautious restarting ofimmunotherapy after LFTs improve to grade 1 or lower
	Grade 3/4: Symptomatic AST/ALT >5 × ULN Totalbilirubin >3 × ULN	• Recommend hospitalization and start intravenous methylprednisone 2–4 mg/kg/day or equivalent, taper over 4–6 weeks ifresolves to grade 1 or better. If no improvement after 48–72 h,add alternative immunosuppressive agents tacrolimus,cyclophosphamide or mycophenolate mofetil. Avoid infliximab dueto potential for hepatotoxicity
Endocrine • Thyroiditis • Hypothyroidism • Hyperthyroidism • Hypophysitis • Hypopituitarism • Adrenalinsufficiency	• Grade 1: Asymptomatic or mild symptoms; clinical or laboratoryfinding only • Grade 2: Moderate;limiting age-appropriate instrumental ADL	Thyroiditis: treat hypothyroidism andhyperthyroidism per standard guidelines: does not requireholding immunotherapy Adrenal insufficiency:physiologic replacement doses of steroids; however, ifpresenting in adrenal crisis/shock, admit to the hospital andstart stress dose steroids, and intravenous fluids. Rule outsepsis. Immunotherapy may be resumed when stable and onphysiologic doses of adrenalreplacement Hypophysitis: prednisone 1–2mg/kg/day or equivalent with a slow taper; consultation withendocrine recommended; patients may require hormone replacementtherapy for life
	• Grade 3/4: Severe or medically significant and life-threatening(grade 4); disabling and limiting ADL and self-care
Skin • Dermatitis	Grade 1: <10% BSA; asymptomatic Grade2: 10–30% BSA Mildly symptomatic	• Administer topical steroids and oral antihistaminicdrugs • If unresolved with above measures consider lowdose systemic corticosteroids and consider treatment break if noimprovement; consider dermatology consultation
	Grade 3/4: >30% BSA Severe: Stevens-Johnsonsyndrome, necrolysis, or rash with dermal ulcerations ornecrotic, hemorrhagic manifestations	• Discontinue drug; administer systemic corticosteroid therapyof 1–2 mg/kg/day of prednisone orequivalent • Dermatology consultation • Holdimmunotherapy until resolved to grade 1
Pulmonary • Immune-mediated pneumonitis	• Grade 1: Asymptomatic imaging finding only • Grade2: Moderate symptoms with limited interferencewith activities of day to day living	• Hold immunotherapy for 3–4 weeks; if asymptomatic monitor forsymptoms closely. • If new symptoms develop, oralprednisone 1 mg/kg/day or equivalent and taper over 4–6 weeksafter symptoms improve
	• Grade 3/4: Severe symptoms limiting activities of day to dayliving; hypoxia and respiratory failure	• Recommend hospitalization and pulmonary consultation and startintravenous methyl prednisone 2–4 mg/kg/day or equivalent, taperover 4–6 weeks if resolves to grade 1 or better. If noimprovement after 48–72 h, consider bronchoscopy withBAL/transbronchial biopsy to rule out other etiology; ifnegative add alternative immunosuppressive agents mycophenolatemofetil or infliximab
Renal • Autoimmune nephritis	• Grade 1: Asymptomatic, increase in creatinine above the baselinebut ⩽1.5 ULN • Grade 2: Increase in creatinine above 1.5 ULN ⩽3	• Continue immunotherapy for grade 1; closely monitor renalfunction and electrolyte imbalances. Rule out other etiology forrenal failure. • Hold immunotherapy for grade 2 andabove; start 0.5–1 mg/kg/day of prednisone or equivalent
	• Grade 3: Increase in creatinine above 3 ULN⩽6 • Grade 4: Increase in creatinine above 6 ULN	• Consult nephrology; renal biopsy. • Hold immunotherapypermanently; start 1.0–2.0 mg/kg/day of prednisone orequivalent

ADL, activities of daily living; ALT, alanine transaminase; AST,aspartate transaminase; BAL, bronchoalveolar lavage; BSA, body surfacearea; LFT, liver function test; ULN, upper limit of normal.

Another caveat with the use of PD-L1 expression as a biomarker is variability inPD-L1 expression (both inter- and intratumor heterogeneity) at different sites ofdisease, such as primary versus metastatic sites, and differenttime points during the treatment course (i.e. before or after chemotherapy). The useof fresh versus archival biopsies may also affect PD-L1expression.^63–66 Differences in PD-L1expression were detected between biopsied specimens and surgically resected tumorsfrom the same patient.⁶⁷ Different biopsies coming from the same lung in patients with multifocal lungcancer showed discordant expression of PD-L1 in about one third of the total patient population.⁵⁴ Nonetheless, PD-L1 expression remains an important factor in achievingresponse to PD-1 blockade. In NSCLC, patients with high levels of PD-L1 tumorstaining achieved an excellent response to PD-1 blockade.

Mutational and neoantigen load

Lung cancer is predicted to be a highly immunogenic tumor, expressing manyneoantigens, and is responsive to checkpoint inhibition.⁶⁸ Rizvi and colleagues showed that higher mutational burden was associated withstable response lasting over 6 months in patients with NSCLC receiving pembrolizumab.⁶⁹ Over 178 nonsynonymous mutations and neoantigen burden were associated withprolonged OS. Several studies later demonstrated that in addition to the highmutational burden, low neoantigen intratumoral heterogeneity might also be animportant factor. McGranahan and colleagues analyzed The Cancer Genome Atlasdatabase on NSCLC adenocarcinoma and showed that a combination of high mutationalburden and low neoantigen intratumoral heterogeneity (<1%) is more significantlyassociated with longer survival time (irrespective of treatment).⁷⁰ High mutation burden is an increasingly important emerging biomarker foridentification of patients for checkpoint immunotherapy. Although promising,prospective studies are warranted to confirm these approaches and otherinvestigational biomarkers for patient selection in routine clinical use. PD-L1status alone is not sufficient to rule in or rule out the use of CPIs and furtherinvestigation to combine two or more methods to capture the immune status might bemore efficient as a composite predictive biomarker for immune CPI therapy.

Spectrum of immune-related toxicities and management

Immune-checkpoint pathways play a critical physiologic role in maintaining selftolerance and preventing autoimmunity. Immune CPIs thus have the potential to alterthe immune homeostasis and result in autoimmune side effects, termed asimmune-related adverse events (irAEs). These side effects can encompass a wide rangeof manifestations, which can affect almost all tissues and organs. IrAEs mostlyaffect the joints (arthritis), colon (colitis), lung (pneumonitis), endocrine glands(endocrinopathies), skin (dermatitis), and liver (hepatitis). In general, mosttoxicities associated with PD-1 and PD-L1 agents are easily managed with a high doseof corticosteroids and are rarely refractory to immunosuppressive treatments.However, in some patients, particularly when not detected early, irAEs can be life threatening.⁷¹

Assessing the severity of irAEs is critical for effective management of these unusualtoxicities that often require a multidisciplinary approach. Toxicities should begraded using the Common Terminology Criteria for Adverse Events developed by theNational Cancer Institute (Table 3). Most irAEs are mild and asymptomatic (grade 1) and patientscan continue treatments in most situations with close monitoring withoutimmunosuppression. However, this is dependent on the specific organ involvement, forexample in patients with grade 1 pneumonitis (asymptomatic radiographic findingsonly), treatment should be stopped, and patients should be monitored closely with arepeat computed tomography scan of the chest within 3 weeks to confirm resolutionprior to restarting treatment. Patients with grade 2 irAEs typically require theirtreatment to be stopped temporarily and they should be monitored closely afterinitiation of oral prednisone of 0.5–1.0 mg/kg/day or the equivalent as anoutpatient. Patients should be evaluated frequently for any worsening symptoms.Patients will need to be tapered off steroids very slowly over 4–6 weeks.Prophylaxis for Pneumocystis jirovecii pneumonia should beconsidered in these patients. Patients with grade 3/4 irAEs have significant risk ofmortality and morbidity and hence hospitalization should be considered for initialmanagement; these patients often require permanent discontinuation of treatment.These patients require 1–2 mg/kg dose of methylprednisolone or the equivalent forinitial management until toxicity resolves to grade 2 or lower; they may requireadditional nonsteroidal immunosuppressive agents like mycophenolate mofetil or TNFinhibitors (infliximab) if no response is seen with the high doses of steroidswithin 72 h. Depending on the clinical situation, additional system-focuseddiagnostic studies like colonoscopies, liver biopsies, bronchoscopy withbronchoalveolar lavage and transbronchial biopsies may be necessary to rule outother etiologies. Patients with irAEs may often require multidisciplinary care.Effective management of irAEs requires heightened awareness of these toxicitiesamong not just the oncologist but also nononcology specialists who are involved inthe care of patients treated with immunotherapy.

In summary, recent approval of immune CPIs in the management of advanced stage NSCLCis a significant advancement in treatment of NSCLC. In addition, the excitingresults from immunotherapy strategies has opened exciting possibilities for futureimmunotherapy combination strategies that will possibly yield even more effectivetreatment strategies and keep our hope alive to achieve a cure for at least a subsetof patients with advanced NSCLC.