The anti-PD1 era – an opportunity to enhance radiotherapy for patients with bladder cancer

Richard C. Walshaw, Jamie Honeychurch, Timothy M. Illidge, and Ananya Choudhury

Abstract | An urgent need exists to improve the outcomes of patients with muscle-invasive bladder

cancer (MIBC), and especially of those with metastatic disease. Treatments that enhance antitumour

immune responses — such as immune-checkpoint inhibition provide an opportunity to do this.

Despite initial success, durable response rates in patients with advanced-stage MIBC treated with

novel inhibitory antibodies targeting programmed cell death protein 1 (PD-1) or its endogenous

ligand programmed cell death 1 ligand 1 (PD-L1) remain low. Radiotherapy forms part of the

management of bladder cancer in many patients. Evidence that radiotherapy has immunogenic

properties is now available, but radiotherapy-induced immune responses are often negated by

immunosuppression within the tumour microenvironment. Anti-PD1 or anti-PD-L1 antibodies might

enhance radiotherapy-induced antitumour immunity. This effect has been demonstrated in

preclinical models of bladder cancer, and early clinical trials are currently recruiting. Combination

treatment strategies provide an exciting opportunity for urological oncologists to not only improve

the chances of cure in patients with radically-treatableMIBC, but also to increase long-term response

rates in those with metastatic disease.

Over 10,000 new cases of bladder cancer are diagnosed each year in the UK, and over 40% of patients have muscle invasive bladder cancer (MIBC) or more advanced disease a diagnosis 1. In the UK, standard treatment for non-metastatic MIBC is radical cystectomy or organ sparing treatment involving radiotherapy with appropriate radiosensitisation to the whole bladder after neo-adjuvant chemotherapy 2. The 5-year survival rate for these patients is 50% 3. If fit enough, those with metastatic disease receive palliative platinum-based chemotherapy 2. With such treatment, patients with advanced disease have a median overall survival of 14.0 months, and a 5-year survival rate of 13% 4. Patients with metastatic disease also often require palliative radiotherapy for alleviation of pain and other local symptoms 2. Currently, second-line systemic options only offer a modest clinical benefit 5.

Characterisation of immune checkpoints has led to the development of a number of novel immunotherapy agents which have shown activity in several different disease sites. Several monoclonal antibodies targeting the PD-1/PD-L1 checkpoint have shown activity in patients with advanced bladder cancer 6-12. For instance, one such agent, pembrolizumab, improved median OS by

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nearly three months compared to standard second-line chemotherapy in patients who had progressed after first-line treatment 9. Despite this success, long-term durable response rates remain relatively low with the majority of patients relapsing after treatment.

Emerging evidence advocating the immunogenic potential of radiotherapy 13-21 has led to the premise that combining it with immune checkpoint blockade may increase durable response rates and ultimately numbers of long-term responders in patients with advanced urothelial cancer. Combination treatment like this may also benefit patients with radically-treatable bladder cancer, improving rates of 5-year survival and cure. This article explores the scientific basis for such combination strategies, as well as the clinical implications these may have for patients with bladder cancer.

[H3] Bacillus Calmette–Guerin (BCG) treatment.

Therapy that stimulates an antitumour immune response has a proven track record in the treatment

of superficial bladder cancer. Following initial use for this indication 40 years ago 22 , intravesical BCG

has been shown to decrease both the risk of recurrence 22-24 and progression to invasive disease 25 in

patients with non-muscle-invasive bladder cancer (NMIBC). Guidelines provided by the European

Association of Urology (EAU) advocate the use of intravesical BCG as the standard-of-care approach

for patients with NMIBC 26.

The immunogenic potential of BCG has long been recognized as the reason for its effectiveness,

although the exact mechanism of action remains the subject of debate. Intravesical BCG treatment is

associated with the activation of several immune-cell populations including CD4+ and CD8+ T

lymphocytes (also known as T cells), natural killer (NK) cells, and granulocytes, orchestrated by a

millieu of different cytokines 27. Internalization of BCG might also lead to upregulated expression of

cell-surface proteins, such as major histocompatibility complex (MHC) class II, therefore increasing

the visibility of bladder cancer cells to the immune system. Ultimately, all of these factors act in

concert to stimulate a localized antitumour immune response.

Intravesical BCG clearly has immunogenic properties, but evidence also exists that this approach can

stimulate a systemic immune response. For example, in a cohort of 62 patients treated with

intravesical BCG, 25 developed a positive response to the purified protein derivative skin test, which

had previously been negative, suggesting that intravesical BCG also has systemic effects28.

Intravesical instillations of BCG might also increase serum IL-2 and IFNγ levels, as well as enhancing

the activity of antitumour peripheral blood mononuclear cells 29.

[H3] Radiotherapy.

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The EAU guidelines on muscle-invasive bladder cancer (MIBC) advocate the use of radical

cystectomy as the treatment of choice for patients with MIBC, although trimodality bladder-

preservation strategies involving radiotherapy are also an important and potentially curative option 2,30. The mechanisms by which radiotherapy induces apoptosis, by damaging DNA, are well described 31. However, emerging evidence suggests that radiotherapy also has immunostimulatory properties

that contribute to the overall anticancer effects of this modality.

Radiotherapy has several immunogenic effects within the local tumour microenvironment (TME).

These include the upregulation and activation of components of the complement pathway, such as

C3a and C5a 20, which is critical for T-cell stimulation, and the release of type-I IFNs, causing

expansion of antigen-specific T-cell populations owing to enhanced cross priming by dendritic cells

(DCs) 13,14. Radiotherapy also improves NK-cell-mediated responses through upregulation of the

activating and costimulatory receptor NKG2-D type II integral membrane protein (NKG2D) 15, and

increases MHC I expression, which facilitates CD8+ T-cell priming by cross presentation of tumour

antigens 18. In animal models, depletion of CD8+ T cells moderates tumour killing, suggesting that

these immune effector cells are crucial for radiotherapy-induced therapeutic effects 16.

[H3] Current treatments of bladder cancer and immunogenic cell death.

Tumour cells undergo apoptosis in response to conventional anticancer treatment modalities.

Apoptosis was previously thought to be nonimmunogenic as it is a highly regulated form of cell

death, but growing evidence now indicates that this is not always the case 32. Several

chemotherapies that are already in clinical use have been shown to induce tumour-cell stress and

apoptosis in vitro that stimulates immune responses, a process referred to as immunogenic cell

death (ICD) 33. ICD is facilitated by the expression of, or release of a combination of molecules known

as damage-associated molecular patterns (DAMPs) by tumour cells undergoing apoptosis.

Specifically, established defining features of ICD include early apoptotic cell-surface exposure of

calreticulin, ATP secretion and release of high mobility group protein B1 (HMGB1) protein during the

later stages of apoptosis. DAMP release in this spatiotemporally specific manner seems to activate

antigen-presenting cells (APCs), therefore priming T-cell-mediated antitumour immunity 34 (FIG. 1).

Furthermore, cells undergoing apoptosis are also a good source of antigens for cross presentation by

dendritic cells 32.

The neurotoxin capsaicin is not a recognized treatment of bladder cancer, although it has been

suggested as a treatment of neurogenic overactive bladder 35. The findings of one study have shown

that capsaicin induces several changes indicative of ICD in human bladder cancer cells 36.

Doxorubicin, cisplatin and mitomycin C are all chemotherapy agents that can be used in the

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treatment of patients with bladder cancer. Mitomycin C and cisplatin reportedly do not induce ICD 33,37. However, doxorubicin, which is commonly used in the treatment of advanced-stage disease 38,39

, was the first drug identified to induce ICD 37. The reasons for these differences in the ability to

induce ICD are unclear, particularly as mitomycin C, like doxorubicin, causes tumour cells to undergo

caspase-dependent apoptosis. Studies involving inoculation of previously treated tumour cells into

an untreated tumour-bearing host animal are the gold-standard approach for demonstrating ICD in

vivo 40, nonetheless, the study in which human bladder cancer cells were exposed to capsaicin 36

provides strong evidence that such cells are able to undergo ICD. In addition to other

immunostimulatory effects, emerging evidence now indicates that radiotherapy has the capacity to

cause ICD, as it induces cell-surface exposure of calreticulin 17 and heat shock protein 70 19 as well as

leading to the release of HMGB1 21.

Despite the availability of promising preclinical evidence demonstrating the immunogenic potential

of conventional treatments of bladder cancer, effects on antitumor immunity are generally not seen

in patients. Indeed, a considerable risk of localized disease relapse exists after treatment for patients

in remission from disease of any stage. For example, after treatment with intravesical BCG, nearly

20% of patients with high-risk NMIBC have disease recurrence and progression within 5 years 41, and

only 48–59% of patients with MIBC survive for 5 years after radical treatment, including

radiotherapy 42,43. In patients with advanced-stage disease receiving palliative radiotherapy, no

reports of immune-mediated abscopal responses outside of the applied radiation field are available 44. One explanation for this lack of an effect might be that immunosuppression within the TME

counteracts the antitumour immunogenic response to radiotherapy.

[H3] Cellular mechanisms of immunosuppression.

A combination of selective pressures and genetic instability causes tumour cells to undergo changes

that often lead to the suppression of localized immune responses 45. Reciprocal interactions

between cancer cells and other constituents of the TME sculpt this response. These changes

facilitate a scenario whereby the growing tumour cells are either not detected, or detected but not

destroyed by the host immune system, referred to as ‘immune evasion’, which is now recognized as

an important feature of malignant transformation 46. Multiple possible mechanisms of

immunosuppression exist within the TME, including recruitment and proliferation of various

immunosuppressive immune-effector cell populations such as regulatory T cells (T reg) 47,48 and type 2

tumour-associated macrophages (TAMs) 49. Indeed, infiltration of tumours with these effector-cell

populations might predict worse survival outcomes, compared with a lack of immune-cell infiltration

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across different cancer types 50,51. Phenotypic changes, including downregulation of MHC I and NK

cell activating ligands, might also enable tumour cells to avoid immunosurveillance 52,53.

[H3] Programmed death protein 1 (PD-1)-mediated immunosuppression.

Much attention is currently focussed on the role of immune checkpoints in regulating T-cell

responses within the TME. Presentation of tumour-associated antigens by MHCs to the T-cell

receptor (TCR) causes T-cell activation. This process is regulated by synchronous signalling through

immune-checkpoint signalling pathways, including the programmed cell death protein 1 (PD-1)

immune checkpoint. PD-1 and/or PD-L1 is expressed on T-cells, B-cells, NK T-cells, activated

monocytes, macrophages and dendritic cells, as well as non-haematopoietic cells. 54 Activation of

this checkpoint on the T-cell surface by programmed death ligand 1 (PD-L1) on tumour cells and

other immune cell populations. Malignant cells often undergo upregulation of PD-L1 in response to

IFNγ released from tumour-infiltrating lymphocytes (TILs), thereby inhibiting the antitumour immune

response 55. Indeed, characterization of the efficacy of this, and other immune-checkpoint inhibitors

has led to the development of several novel agents, which have been used successfully as

monotherapies in different types of cancer, including advanced-stage urothelial carcinoma 11,56-58

(FIG. 2).

[H3] Targeting the PD-1 immune checkpoint in patients with urothelial carcinoma.

Several different monoclonal antibodies targeting either the PD-1 immune checkpoint, or the

endogenous ligand, PD-L1, have now been approved by the FDA (TABLE 1). Atezolizumab was

approved in May 2016 by the FDA for the treatment of patients with advanced-stage urothelial

carcinoma that has progressed during, or after platinum-based chemotherapy. In a phase II trial,

objective response rates of 15% were seen in patients treated with atezolizumab, with 84% of

responders having ongoing responses at a median follow-up duration of 11.7 months 11. Rates of

grade 3-4 treatment-related events were low, occurring in 16% of patients. Unfortunately, although

results are not yet fully described, initial reports indicate that treatment with atezolizumab does not

improve overall survival outcomes compared with those of patients receiving chemotherapy in a

phase III trial with a much larger cohort 59. Treatment with pembrolizumab, an antibody targeting

the PD-1 receptor, improved median overall survival durations by nearly 3 months, relative to

chemotherapy, in a similar cohort of patients (10.3 months versus 7.4 months, respectively; hazard

ratio (HR) for death 0.73; P = 0.002)60. However, in this phase III trial, progression-free survival at 1

year was only 16.8% in patients treated with pembrolizumab. The outcomes of another phase II trial

included a response rate of 27% in patients who received pembrolizumab as a first-line treatment 7.

The anti-PD-1 antibody nivolumab, and the anti-PD-L1-antibodies avelumab and durvalumab have

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also been approved by the FDA, having demonstrated antitumor activity in patients with advanced-

stage urothelial carcinoma 6,10,12. Clearly, although these agents have shown promising initial results,

the rates of durable responses to agents targeting the PD-1/PD-L1 immune checkpoint in patients

with advanced-stage urothelial cancer require improvement in order to provide reliable levels of

benefit to the majority of patients.

Combining immune-checkpoint blockade with conventional therapies that have an immunogenic

component might improve the efficacy of these treatments by reducing tumour-mediated localized

immunosuppressive effects, and therefore improve antitumor immune responses.

[H3] BCG and anti-PD-L1 combination therapy.

Despite intravesical BCG being a standard-of-care immunotherapy for decades, data from preclinical

studies investigating the combination of BCG treatment with concurrent PD-1/PD-L1 blockade are

currently lacking. Interestingly, tumours that recur after treatment with BCG have been reported to

have high levels of PD-L1 expression 61, therefore providing a rationale for the use of BCG in

combination concurrently or sequentially with anti-PD-1 or anti-PD-L1 antibodies. A phase I clinical

trial aiming to determine the safety of combining BCG with pembrolizumab in patients with high-risk

NMIBC following transurethral resection of the bladder tumour (TURBT) is now open 62.

[H3] Radiotherapy and anti-PD-L1 antibodies.

Treatment strategies that combine immune-checkpoint blockade with radiotherapy have the

potential to both improve the extent of local control and also provide systemic abscopal effects by

reversing localized immunosuppression and enhancing radiation-induced, tumour-specific immune

responses (FIGs. 1 and 2). A growing body of preclinical evidence now supports this hypothesis in

several forms of cancer, including urothelial malignancies 63-65. The findings of a study published in

2016 demonstrated the effects of combining radiotherapy with anti-PD-L1 antibodies in a mouse

model of bladder cancer 65. Tumour growth was delayed and tumour cell death was increased in

mice that received a 12-Gy single-fraction dose of radiotherapy, combined with anti-PD-L1

antibodies, compared with those treated with radiotherapy alone. The authors demonstrated that

this synergistic effect was dependent on the presence of CD8+ T cells. Many studies investigating the

mechanisms of radiotherapy-induced tumour cell death in mice use heterotopic models, in which

tumours are implanted subcutaneously; however, this study 65 used orthotopic mouse models of

bladder cancer, with tumours implanted directly into the bladder. These models are recognized as

being more representative of de novo tumours, therefore providing a better simulation of the TME

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while maintaining an intact immune system [66,67]. The findings of another preclinical study

demonstrated that addition of anti-PD-L1 antibodies to radiotherapy might also improve the extent

of the antitumour immune response by reducing the numbers of immunosuppressive myeloid-

derived suppressor cells (MDSCs) in the TME 63.

The findings of several preclinical studies have shown that immune memory can be generated in

response to combined immune-checkpoint inhibition and radiotherapy. For example, mice with

primary tumours that responded successfully to initial treatment are able to reject re-implanted

tumours from the same cell line, although this effect has not been demonstrated in models of

bladder cancer, thus far 63,64. Such demonstrations of immunological memory are an attractive

concept for further investigation as clinically this could translate into immune-mediated rejection of

recurrent micrometastatic disease after radical treatment, and provide protection from disease

recurrence. Furthermore, abscopal effects have been observed in preclinical models, with notable

growth delay in nonirradiated tumours on the contralateral flanks of animals that received

combination treatment 63.

[H3] Scheduling of radiotherapy and anti-PD-1/PD-L1 combination treatment.

Preclinical evidence indicates that the order in which treatments are administered during

combination therapy might be important, with concurrent anti-PD1 or anti-PD-L1 antibodies and

radiotherapy required for a synergistic effect to occur 64. Following these preliminary observations,

prospective trials evaluating the efficacy of combination therapies are focusing on concurrent, rather

than sequential treatment strategies (TABLE 2). For example, the ongoing phase I PLUMMB study,

involving patients with locally advanced or metastatic bladder cancer, aims to test the safety and

tolerability of pembrolizumab given concurrently with hypofractionated radiotherapy delivered to

the bladder 68. Two other early phase clinical trials are also currently evaluating the combination of

anti-PD-1 antibodies with radical radiotherapy and gemcitabine 69, or cisplatin 70. In the future,

investigators attempting to design a practice-changing phase III trial might also need to consider

investigating the efficacy of other commonly used bladder preservation strategies such as

radiotherapy with concurrent carbogen and nicotinamide 71 or 5-fluorouracil and mitomycin C 43 in

combination with immunotherapy.

Advances in image-guided radiotherapy (IGRT) and tumour motion tracking have enabled increased

per fraction doses of radiotherapy to be administered safely. Patients with lung tumours 72, and

those with cancers in other locations are currently able to benefit from hypofractionated

radiotherapy regimens, known as stereotactic ablative radiotherapy (SABR), in which large doses per

fraction of >10 Gy are delivered in a small number of fractions with a high level of precision 73.

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Emerging pre-clinical evidence indicates that radiotherapy treatment schedules, such as those that

use SABR, and involve higher doses per fraction might lead to a greater immune response than

conventionally fractionated regimens 74,75. A phase I clinical trial aiming to determine the safety and

efficacy of combining high-dose SABR with concurrent pembrolizumab in patients with metastatic

MIBC is currently recruiting participants 76. If the phase II RAIDER trial 77 demonstrates positive

results, radiotherapy dose escalation to the primary tumour using larger doses per fraction in

patients with radically treatable bladder cancer might become more common. Such an outcome

might, in future, provide an opportunity to optimize the radiation-induced immune responses

produced by the combination of radiotherapy with immune-checkpoint inhibition. Ultimately,

further work is required to establish the optimal radiotherapy dose and/or fractionation schedule for

priming an antitumour T-cell response in patients with bladder cancer in order to inform the design

of further clinical trials.

[H1] Adverse effects

Synergy between radiotherapy and immune-checkpoint inhibition has the potential to provide

substantial improvements in the efficacy of either approach alone. However, the use of such

treatments in combination might also increase the risk of adverse effects. Diarrhoea is a common

adverse effect of pelvic radiotherapy, and, in a phase III trial, nearly 10% of patients receiving

chemoradiotherapy to the bladder had grade ≥3 acute gastrointestinal toxicities 43 during treatment.

46% of patients treated with radiotherapy alone experienced grade ≥2 diarrhoea in another smaller

series 78. Diarrhoea is also a common adverse effect of anti-PD-1 antibodies, affecting up to 16%

patients, depending on which agent is used 58. Breaks during a course of radiotherapy, owing to

profuse diarrhoea, although uncommon, delay completion of treatment and can have an adverse

effect on patient outcomes. The increased risk of diarrhoea is one concern surrounding the use of

immune-checkpoint inhibition with radiotherapy.

Colitis is a rare immune-mediated complication of treatment with anti-PD-1 antibodies, experienced

by up to 3.6% of patients in a phase III trial. Concerns exist that concurrent irradiation of the bowel

within the pelvis as part of radical treatment could increase the risk of this potentially life-

threatening toxicity, even with use of modern radiotherapy delivery techniques such as intensity-

modulated radiotherapy.

Up to 20% of patients have clinically significant fatigue during treatment with anti-PD-1 antibodies as

monotherapy 58. Fatigue is also often reported by patients during radiotherapy, and the level of

fatigue arising from combination with immune-checkpoint inhibition might be unacceptable.

However, in a preclinical study, combination treatment was well tolerated, with mice that received

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concurrent combination treatment gaining weight at a similar rate to untreated 64. This study used

heterotopic rather than orthotopic mouse models, with tumour cells delivered via subcutaneous

injection rather than to a specific organ, and might not be fully representative of the clinical

situation. Data from a retrospective study with a small cohort of patients with advanced-stage

melanoma have shown that the combination of radiotherapy and anti-PD-1 antibodies does not lead

to excessive levels of toxicity compared to published evidence describing side effects of

immunotherapy alone79. No data on toxicities are available for the combination of radiotherapy with

anti PD-1 antibodies in patients with advanced-stage urothelial carcinoma, despite the approval of

several agents in the past year.

Ultimately, the increased risk of toxicities owing to synergistic effects might only be apparent in

prospective clinical trials. These trials should be carefully designed to detect any early adverse

effects, so that an appropriate intervention can be promptly made, without compromising the

efficacy of treatment.

Predicting a response

Given the increased risk of toxicities associated with use of combination therapy, the selection of

patients who are most likely to benefit from this approach is important. No validated biomarkers

that enable clinicians to predict who might respond to treatment with radiotherapy and anti-PD-1

antibodies are available; however, some progress has been made in understanding markers that

might predict a response to radiotherapy or immune-checkpoint inhibition alone. If validated,

eventually, these biomarkers might also indicate which patients will respond to combination

treatment strategies.

[H3] Immune biomarkers predicting a response to anti-PD-1 antibodies

A considerable research effort has taken place, and is ongoing, in an attempt to establish and

validate biomarkers that enable the prediction of a response in patients receiving anti-PD-1

antibodies. PD-L1 expression within the TME, albeit on tumour cells, immune cells or both is an

attractive potential predictive biomarker. Response rates to anti-PD-1 or anti-PD-L1 antibodies are

enhanced in patients whose tumours express PD-L1 in several different tumour types 80. However,

the findings of studies using immunohistochemical assays to examine this premise in patients with

advanced-stage urothelial carcinoma have come to different conclusions. For example, a higher

response rate to atezolizumab was demonstrated in patients with tumours with ≥5% PD-L1-positive

immune cells, relative to that of those with lower levels of PD-L1 expression 11. This pattern of

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response was not seen in another phase II trial involving patients treated with nivolumab, in which

PD-L1 positivity did not correlate with a response to treatment 12, despite the investigators using the

same immunohistochemical assay. The disparity between these two studies highlighs the need for

better understanding of the biology of immune checkpoint inhibition, particularly the difference

between PD-L1 and PD-1 inhibitors. The investigators of a phase III study comparing the efficacy of

pembrolizumab with that of chemotherapy used a different immunohistochemical assay to form a

PD-L1 score, as determined by the percentage of PD-L1-expressing tumour and immune cells,

relative to the total number of tumour cells 9. In this study, median overall survival duration was

improved in the group of patients treated with pembrolizumab compared with those treated with

chemotherapy, regardless of PD-L1 score. However, within the group that received pembrolizumab,

the median overall survival duration of those in the subgroup with a PD-L1 score >10% was 8.0

months and 10.3 months across all subgroups. These data were not directly compared statistically,

although this observation could imply that a higher PD-L1 score is a negative predictive factor.

These contradictory results highlight the importance of consistency across different trials when

determining ‘PD-L1 positivity’ in terms of both the cutoff level for PD-L1 positivity and the assays

used to assess positivity. This point is further emphasized by the results of a meta-analysis published

in 2015, which included 20 trials involving immune-checkpoint inhibitors targeting the PD-1/PD-L1

axis in several different tumour types, including urothelial carcinoma 80. A significant difference in

overall response rate occurred when 5% (P <0.0001) but not 1% (P = 0.108) of tumour cells having

PD-L1 expression was used as a cutoff level for PD-L1 positivity. The results of the pembrolizumab

study 9r systemic therapy 81.

Ultimately, the majority of patients with PD-L1-positive bladder cancer do not have durable

responses to anti-PD-1 or anti-PD-L1 antibodies. Equally, responses to anti-PD-1 antibodies have

been seen in patients with PD-L1 negative tumours 58, prompting further debate about the validity of

pretreatment PD-L1 expression alone in predicting a response to treatment.

Mutational burden and tumour neoantigen expression, arising as a consequence of genetic

instability, correlates with antitumour T-cell responses 82. Using whole-exome sequencing, the

findings of a study demonstrated that a higher mutational burden is associated with an improved

likelihood of a response, and with increased progression-free survival following treatment with anti-

PD-1 antibodies in patients with non-small-cell lung cancer (NSCLC) 83. Bladder cancers generally

have a high prevalence of somatic mutations, relative to that of various other types of malignancy 82.

Indeed, the findings described in NSCLC 83 have been mirrored in several studies aiming to determine

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the efficacy of agents targeting PD-1 in patients with advanced-stage urothelial carcinoma. For

example, in two studies designed to determine the efficacy of atezolizumab in patients with

advanced-stage disease, tumour mutational load was positively correlated with responsiveness 11 8

and overall survival 8.

Researchers involved in The Cancer Genome Atlas (TCGA) consortium have identified four distinct

subtypes of urothelial carcinoma based on RNA-sequencing data from a total of 129 tumours 84.

Using an adapted TCGA classification approach, investigators from the aforementioned atezolizumab

trials 8,11classified samples from 195 patients into luminal or basal subtypes 11. Response rates were

highest in patients with disease of the ‘luminal II’ subtype (34%), characterized by high levels of CD8 +

effector T-cell-related gene expression.

[H3] Immune biomarkers predicting a response to radiotherapy

Immune biomarkers might have a role in identifying patients who are more likely to respond to

conventional radiotherapy-based treatments. A retrospective analysis of tumour samples from 65

patients with MIBC demonstrated a positive association between high levels of PD-L1 expression and

the development of lymph node metastases and locoregional treatment failure 65. High levels of PD-

L1 expression were also associated with higher locoregional treatment failure rates, and reduced

rates of overall and disease-free survival in patients who underwent concurrent chemoradiotherapy.

Using immunohistochemistry, the authors devised a semiquantitative immunoreactivity score

calculated using staining intensity and percentage of positively stained cells to assess PD-L1

expression, although they did not differentiate between PD-L1 expression on different cell

populations. Similar results were seen in a cohort of patients treated for stage III NSCLC 79. This study

also demonstrated that the density of tumour-infiltrating CD8+ T-cells was an independent and

statistically significant predictor of both progression-free and overall survival after

chemoradiotherapy 85. Tumour levels of HMGB1 might also be predictive of a response to treatment 21, although this association has not yet been demonstrated in patients with bladder cancer. Despite

substantial efforts to establish predictive biomarkers of a response to immunotherapy or

conventional radiotherapy in patients with bladder cancer 86,87, no robust biomarkers are currently

validated for use in clinical practice.

Conclusions

This is an exciting time in the treatment of bladder cancer, with the first new FDA-approved systemic

agent, atezolizumab, an anti-PDL-1 inhibitor, for several decades. Despite this approval, which was

followed by several similar agents, considerable room for improvement of the efficacy of this, and

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similar agents, exists. The optimal roles of these treatments will become defined either as

monotherapies or in combination with chemotherapy, therefore, the possibility of combining

immunotherapy with radiotherapy is of increasing interest. An opportunity exists to reverse the

immunosuppressive nature of the TME, and thus enhance the potency of radiation-induced

antitumour immune responses. The scheduling of combination treatments is likely to be crucial in

optimizing therapeutic synergy. However, the combination of radiotherapy and immunotherapy

could lead to increased and possibly unacceptable levels of toxicity. Adverse effects will have to be

closely monitored in patients receiving such treatments, ensuring minimal disruption so as not to

compromise the delivery of primary radiotherapy.

Given the potential for increased toxicity, the development and validation of biomarkers enabling

the identification of patients who are most likely to benefit from the combination of radiotherapy

and immune-checkpoint inhibition is crucial. Biomarkers predicting a response to single-modality

immune-checkpoint inhibition are currently in their infancy, and are yet to be validated in large

cohorts of patients. Whether such biomarkers remain valid predictors of success with combination

therapy remains to be seen. Clinical trials are currently beginning to provide data on the outcomes

of patients treated with combination therapy, and the examination of tumour biopsies from patients

enrolled in these trials, taken before and after treatment, might yield more-robust immune

biomarkers. Ultimately, the addition of immune-checkpoint inhibition to radiotherapy could provide

benefits to patients with early stage bladder cancer and to those with advanced-stage bladder

cancer by not only improving the level of local control, but also unlocking the potential of

radiotherapy as a systemic therapy.

Richard C. Walshaw, Jamie Honeychurch, and Timothy M. Illidge are at the Targeted Therapy Group, Division of Molecular and Clinical Cancer Sciences, School of Medical Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Sciences Centre, Wilmslow Road, Withington, Manchester M20 4BX, UK.

Richard C. Walshaw, Timothy M. Illidge and Ananya Choudhury are at the Department of Clinical Oncology, The Christie NHS Foundation Trust, Manchester Academic Health Sciences Centre, Wilmslow Road, Withington, Manchester M20 4BX, UK.Ananya Choudhury is at the Translational Radiobiology Group, Division of Molecular and Clinical Cancer Sciences, School of Medical Sciences, Faculty of Biology, University of Manchester, Manchester Academic Health Sciences Centre, Wilmslow Road, Withington, Manchester M20 4BX, UK.

Correspondence to A.C.

Ananya.Choudhury@christie.nhs.uk

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Acknowledgements

Author contributions

R.W. researched data for this article, R.W. and J.H. made a substantial contribution to discussions of content, R.W. wrote the manuscript and J.H., T.I., and A.C. edited and/or reviewed the manuscript prior to submission.

Competing interests

The authors declare no competing interests.

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Figure 1. Radiotherapy leads to immunogenic cell death and immune activation. Radiotherapy to the bladder leads to tumour cell death (1) , causing the release and/or expression of damage-associated molecular patterns (DAMPs) (2). These DAMPs bind to receptors on dendritic cells, resulting in enhanced uptake and processing by dendritic cells, followed by presentation as part of a major histocompatibility complex (MHC, mostly of the MHC II subtype) (3). Antigen presentation is then followed by T-cell maturation and negative selection, which enables the avoidance of autoimmunity. Increased DAMP release also results in the presentation of a wider range of tumour antigens on the tumour cell surface, predominantly in the context of major histocompatibility complex (MHC) class I (4). The MHC class I tumour antigen complex is then recognized by T-cell receptors on mature T cells in the local tumour microenvironment or in peripheral lymph nodes, leading to T-cell activation (5).

Figure 2. Rationale for combining immune-checkpoint inhibition with radiotherapy. Anti-programmed cell death protein 1 (PD1) and/or anti-programmed cell death 1 ligand 1 (PD-L1) antibodies have the potential to reinvigorate T-cell-mediated antitumour immunity after radiotherapy, leading to regression of both the primary bladder tumour and distant metastatic disease. a. | After activation, T-cell-mediated antitumour cytotoxicity might be inhibited owing to coinhibitory signalling through the PD-1 immune checkpoint. b. | Treatment with anti-PD-1 or anti-PD-L1 antibodies can inhibit this interaction, therefore restoring T-cell activation, leading to both localized and systemic anti-tumour immunity.

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Table 1. Clinical studies evaluating the efficacy of anti-PD-1 or anti-PD-L1 antibodies.

Drug Setting Phase n Follow-up duration

Response rate

Survival endpoint

Grade 3–5 toxicities

Atezolizumab 11 Progression on, or after platinum-based chemotherapy

II 310 Median of 11.7 months

15% NR 16%

Atezolizumab 8 As first line therapy and not eligible for cisplatin-based chemotherapy

II 119 Median of 17.2 months

23% Median PFS 2.7 months;median OS 15.9 months

16%

Pembrolizumab 9

Progression on, or after platinum-based chemotherapy

III 542 Median of 14.1 months

21.1% Median PFS 2.1 months;median OS 10.3 months

15.0%

Pembrolizumab 7

As a first-line therapy and in patients not eligible for cisplatin-based chemotherapy

II 370 NR 27% (in patients with ≥4 months of follow-up monitoring; n = 307)

6-month PFS 31%; 6-month OS 67%

16%

Avelumab 6 Progression on, or after platinum-based chemotherapy, or in patients not eligible for platinum-containing therapy

Ib 249 Minimum of 6 weeks

17.4% (in patients with ≥6 months of follow-up monitoring; n = 161)

Post-platinum-containing therapy (n = 242) median OS 7.4 months;median PFS 6.6 weeks;6-month OS 54.9%

8.4%

Durvalumab 10 As first-line therapy, or after progression on, or after first-line therapy

I/II 191 Median of 5.7 months

17.8% Median OS 18.2 months;median PFS 1.5 months

6.8%

Nivolumab 12 Progression on, or after platinum-based chemotherapy

II 270 Median of 7 months

19.6% Median OS 8.7 months

18%

NR, not reported; OS, overall survival; PD-1, programmed cell death protein 1; PD-L1, programmed cell death 1 ligand 1; PFS, progression-free survival.

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Table 2. Ongoing clinical studies evaluating the efficacy of anti-PD-1 or anti-PD-L1 antibodies plus radiotherapy and/or chemotherapy.

Study Phase Disease stage Immunotherapy Radiotherapy schedule and site

Concurrent chemotherapy

NCT02560636 [68] I T2–4, N0–3, M0–1

Pembrolizumab(anti-PD-1)

60 Gy/30 fractions daily to the bladder (conventional EBRT)

None

NCT02621151 [69] II T2–4a, N0, M0 Pembrolizumab 52Gy/20 fractions daily to theBladder(conventional EBRT)

Gemcitabine

NCT02662062 [70] II T2–4a, Nx or N0, M0

Pembrolizumab 64Gy/32 fractions daily to theBladder(conventional EBRT)

Cisplatin

NCT02826564 [76] I M1 Pembrolizumab 24Gy/3 fractions on alternate days to metastatic lesions(SABR)

None

NCT03115801 [88] II M1 Atezolizumab(anti-PD-L1)

30Gy/3 fractions over 1 week to metastatic lesions(conventional EBRT)

None

PD-1, programmed cell death protein 1; PD-L1, programmed cell death 1 ligand 1; EBRT, External beam radiotherapy; SABR, Stereotactic body radiotherapy

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