International Journal of

Molecular Sciences

Review
Combining Radiotherapy and Immunotherapy in
Lung Cancer: Can We Expect Limitations Due to
Altered Normal Tissue Toxicity?
Florian Wirsdörfer, Simone de Leve and Verena Jendrossek *
Institute of Cell Biology (Cancer Research), University Hospital Essen, 45147 Essen, Germany;
[email protected] (F.W.); [email protected] (S.d.L.)
* Correspondence: [email protected]; Tel.: +49-201-723-3380




Received: 30 November 2018; Accepted: 19 December 2018; Published: 21 December 2018


Abstract: In recent decades, technical advances in surgery and radiotherapy, as well as breakthroughs
in the knowledge on cancer biology, have helped to substantially improve the standard of cancer
care with respect to overall response rates, progression-free survival, and the quality of life of cancer
patients. In this context, immunotherapy is thought to have revolutionized the standard of care
for cancer patients in the long term. For example, immunotherapy approaches such as immune
checkpoint blockade are currently increasingly being used in cancer treatment, either alone or in
combination with chemotherapy or radiotherapy, and there is hope from the first clinical trials that
the appropriate integration of immunotherapy into standard care will raise the success rates of
cancer therapy to a new level. Nevertheless, successful cancer therapy remains a major challenge,
particularly in tumors with either pronounced resistance to chemotherapy and radiation treatment,
a high risk of normal tissue complications, or both, as in lung cancer. Chemotherapy, radiotherapy
and immunotherapy have the capacity to evoke adverse effects in normal tissues when administered
alone. However, therapy concepts are usually highly complex, and it is still not clear if combining
immunotherapy with radio(chemo)therapy will increase the risk of normal tissue complications,
in particular since normal tissue toxicity induced by chemotherapy and radiotherapy can involve
immunologic processes. Unfortunately, no reliable biomarkers are available so far that are suited to
predict the unique normal tissue sensitivity of a given patient to a given treatment. Consequently,
clinical trials combining radiotherapy and immunotherapy are attracting major attention, not only
regarding efficacy, but also with regard to safety. In the present review, we summarize the current
knowledge of radiation-induced and immunotherapy-induced effects in tumor and normal tissue
of the lung, and discuss the potential limitations of combined radio-immunotherapy in lung cancer
with a focus on the suspected risk for enhanced acute and chronic normal tissue toxicity.

Keywords: Irradiation; immune checkpoint inhibition; pneumonitis; fibrosis; T cells; pneumopathy;

adverse effects; PD1; PD-L1; CTLA-4

1. Introduction
Radiotherapy is part of standard care for cancer patients. More than 60% of all cancer patients
receive radiotherapy alone or in multimodal combinations of surgery, radiotherapy and chemotherapy
during the course of their disease, resulting in favorable effects on local tumor regression, long-term
survival, and even tumor cure [1–4]. However, treatment outcome still needs to be improved for cancer
types with high loco-regional failure rates, a high risk for invasive growth or metastatic spread, or a
high risk for normal tissue complications, such as non-small cell lung cancer (NSCLC).
It is widely known that tumor cell intrinsic factors, such as resistance-promoting mutations
and tumor cell plasticity, as well as a pronounced tumor heterogeneity, a resistance-promoting

Int. J. Mol. Sci. 2019, 20, 24; doi:10.3390/ijms20010024 www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2019, 20, 24 2 of 21

microenvironment, and the pronounced radiosensitivity of the normal lung tissue, limit the efficacy of
radiotherapy to the thoracic region or concurrent platinum-based radiochemotherapy [5,6]. Advances
in image-guided radiotherapy and the use of novel radiation techniques such as intensity-modulated
radiotherapy and proton therapy have improved the accuracy and safety of thoracic radiotherapy [7–9],
but the risk for normal tissue complications still limits the application of curative radiation doses to
the thoracic region, whereas tolerable doses might be linked to loco-regional failure, despite accepting
adverse effects that reduce the quality of life [10,11]. Thus, new and biologically optimized strategies
for the radiotherapy of lung cancer with acceptable safety profiles and durable responses are needed
to overcome these limitations.
In an effort to identify novel and effective treatment strategies to combat cancer, in recent decades,
researchers have put a lot of effort into unraveling the principles of the physiological immune response
against cancer and the mechanisms of tumor immune escape. These studies revealed, among other
things, that an efficient immune response against cancer requires the activation of tumor-specific
CD8+ T cells directed against tumor-associated antigens [12,13], and that patients with advanced solid
tumors either present tumors with evidence (“hot” immune-sensitive tumors) or without evidence
(“cold” immune-resistant tumors) for a T-cell inflamed tumor microenvironment [14,15]. Moreover,
it was discovered that, apart from increased production of immunosuppressive mediators such as
transforming growth factor beta (TGF-β) or the propagation of regulatory T cells (Treg), tumor cells and
immune cells up-regulate specific proteins on their surface, namely cytotoxic T-lymphocyte-associated
Protein 4 (CTLA-4), programmed cell death protein 1 (PD1), or indoleamine 2,3-dioxygenase (IDO) on immune
cells, and programmed cell death 1 ligand 1 (PD-L1), as well as CTLA-4 and IDO on tumor cells, that enable
tumor immune escape in tumors with an initial immune response [16–20]. These findings resulted
in the development of several therapeutic strategies aimed at the (re)activation of the antitumor
immune responses in cancer patients. Nowadays, immunotherapies, particularly immune checkpoint
inhibition (ICI) of CTLA4 and PD1/PDL1, are increasingly used as a promising and effective systemic
cancer treatment, boosting the immune response, and thus leading to successful immune recognition
and tumor cell killing [21–23]. However, only a fraction of patients is sensitive to ICI treatment
(responders), some patients fail to ever respond (innate resistance), and some patients even develop
therapy resistance after a short initial response phase (acquired resistance) [24,25]; moreover, patients
may suffer from immune-related adverse effects [26]. Thus, further work is necessary to increase
the efficacy of immunotherapy by optimal combinations with other immunotherapy approaches, or
cytotoxic chemotherapy or radiotherapy.
The use of radiotherapy as a standard treatment option in the therapy of solid human tumors is
based on its ability to locally damage cellular macromolecules, particularly DNA. Thereby, exposure
to ionizing radiation effectively induces growth arrest and cell death in irradiated tumor cells,
resulting in tumor shrinkage and potentially in tumor elimination. However, the discovery that
radiation-induced damage to tumor tissues and normal tissues in the radiation field can trigger the
activation of the immune system via well-known damage-signaling cascades, immunogenic cell death,
or both, has led to a paradigm change in the use of radiotherapy. Preclinical and clinical investigations
revealed a complex interplay between radiotherapy, irradiated cells and tissues, and the immune
system; for example, exposure to radiotherapy was shown to up-regulate major histocompatibility
complex I (MHCI) expression in tumor cells, modulate immunosuppressive barriers in the tumor
microenvironment, activate restrictive tumor vessels, trigger the recruitment of immune effector
cells to the local tumor, and even elicit systemic tumor-specific immune responses leading to the
regression of tumor nodules outside the radiation field (abscopal effects) [27–29]. However, such
abscopal responses to radiotherapy alone are only occasionally observed in patients, presumably
because the tumor microenvironment efficiently shapes tumor immune escape at multiple levels and
thus hampers a beneficial radiation-induced immune activation [30,31]. Because of the limited success
of conventional therapies in patients with resistant and metastatic tumors, current clinical studies focus
on combining radiotherapy with immunotherapy, particularly ICI, to overcome these limitations and
Int. J. Mol. Sci. 2019, 20, 24 3 of 21

harness the combined therapeutic potential of both therapies. The first data of such studies demonstrate
that blockade of the PD-1/PD-L1 immune checkpoint improves progression-free survival in a fraction
of NSCLC patients with an acceptable safety profile when given after radiotherapy or platinum-based
radiochemotherapy [32,33]. Moreover, radiotherapy and CTLA-4 blockade were effective in inducing
a systemic anti-tumor T cell response in chemo-refractory metastatic NSCLC that failed to respond to
anti-CTLA-4 antibodies alone or in combination with chemotherapy [34]. This study also revealed
a rapid expansion of CD8+ T cells recognizing a neoantigen encoded by a radiation-induced gene,
thereby pointing to a contribution of radiation-induced exposure of immunogenic factors to the
systemic antitumor response.
There is hope that the use of T cell-stimulatory immunotherapies or small-molecule inhibitors of
immunosuppressive signaling pathways in the tumor microenvironment in combination with surgery,
radiotherapy and/or chemotherapy will revolutionize the success of standard of care for cancer
patients in the long term. However, radiotherapy can also up-regulate immunosuppressive signaling
cascades in the tumor microenvironment, such as TGF-β signaling or expression of PDL1. Moreover,
radiation-induced immune activation participates in inflammation-associated normal tissue responses
as well as severe, potentially life-threatening acute and chronic inflammatory adverse effects upon
thoracic or total body irradiation such as pneumonitis and pulmonary fibrosis [35]. Thus, interfering
with the immune system by multiple, consecutive therapeutic interventions with immune activating
effects, such as radio(chemo)therapy and immunotherapy, might increase the risk of deregulated and
unwanted adverse (auto)immune responses even when given after a temporal gap. For example, a
recent case report where radiotherapy was followed by ICI treatment revealed severe inflammatory
side effects [36]. The observed inflammatory tissue toxicities suggest potential limitations for combined
radio(chemo)therapy and ICI treatment with respect to long-term safety.
Several recent reviews discussed the potential of combining immunotherapy and radiotherapy to
treat lung cancer with respect to clinical efficacy [37–45]. Therefore, here we will only briefly summarize
the current knowledge of tumor-induced immune escape and the contribution of radiotherapy in
modulating tumor immune responses. Instead, we will discuss in more detail potential risks and
limitations that may occur when combining radiotherapy and immunotherapy in lung cancer with a
focus on the effects of radiation-induced immunomodulation for suppression of efficient anti-tumor
immune responses and normal tissue toxicity in the lung.

2. Radiotherapy in the Thoracic Region—Tumor Control Versus Immune Escape

Lung cancer currently represents the second most common cancer, as well as the leading cause of
death in men and women in the United States (National Cancer Institute, available online: https://seer.
cancer.gov) [46]. It is also one of the most common cancers among both men and women in Germany;
the 1st in men (25% of deaths due to cancer), and the 2nd in women (15% of deaths due to cancer)
(German Centre for Cancer Registry Data, available online: https://www.krebsdaten.de). The majority
of cases comprise localized and locally advanced non-small cell lung cancer (NSCLC). Patients are
typically treated with surgical resection, radiotherapy and chemotherapy. Stereotactic body radiation
therapy (SBRT) is frequently used as an effective treatment in patients with inoperable or early-stage
tumors, whereas inoperable patients with locally advanced tumors receive concurrent chemoradiation
with conventional fractionated radiotherapy [47–49]. The results and outcomes from clinical trials of
the different treatment regimens in lung cancer have been summarized elsewhere [37,49–53]. Thus, we
will focus in this part of the review on how exposure to ionizing radiation shapes the immune response
towards tumor control and the tumor microenvironment towards immune escape, respectively.
Radiation-induced alterations in the tumor microenvironment involve the direct activation of
innate and adaptive immune cells with various effects on tumor growth and tumor cell killing, but also
indirect changes in the tumor microenvironment and the tumor vasculature that alter the recruitment
and activation state of cells from the innate and adaptive immune system (for a review, see [54–60]).
Int. J. Mol. Sci. 2019, 20, 24 4 of 21

Radiotherapy induces damage and cell death in cancer cells, leading to the exposure of
immunogenic molecules such as calreticulin on the cell surface [61]. Moreover, damage-associated
molecular patterns (DAMPs), such as uric acid, S100 proteins, adenosine triphosphate (ATP) or
High-Mobility-Group-Protein B1 (HMGB1), as well as tumor antigens, are released to activate innate
and adaptive immune responses [62,63]. In addition, radiation-induced nuclear DNA release and
subsequent sensing of cytoplasmic dsDNA can activate the cGMP–AMP synthase (cGAS)–stimulator
of interferon genes (STING) pathway, which is closely related to the activation of a type I interferon
response with expression of inflammatory genes and the secretion of cytokines that promote antitumor
immunity. Uptake of tumor antigens by antigen presenting cells, e.g., dendritic cells, as well as sensing
of cancer cell-derived cytoplasmic dsDNA by the cGAS/STING pathway are required for the priming of
tumor-specific T cell responses. To trigger the activation of B and T cells, mature dendritic cells migrate
to and present antigens in secondary lymphoid organs. Activated T cells and B cells can exert systemic
anti-tumor effects by several mechanisms like CD8+ T cell mediated cytotoxicity, antibody-dependent
cell-mediated cytotoxicity, and antibody-induced complement-mediated lysis [56,61,62,64–68].
A complex interaction between the tumor microenvironment and the immune system is
needed to achieve a radiation-induced anti-tumor immunity. Moreover, the induction of tumor
antigen-presenting dendritic cells that are essential for T cell priming largely depends on the
radiation dose and fractionation in a tumor-dependent manner. For example, the radiation dose per
fraction dictates the level of the exonuclease three prime repair exonuclease 1 (TREX1), which degrades
interferon-stimulatory cytosolic dsDNA and thus abrogates the immunogenicity of irradiated cancer
cells [67,69,70]. Finally, tumor cells exert a range of acquired capabilities to escape the immune system,
so that direct anti-tumor immunity in response to radiotherapy alone is a rare event [30,67,71].
Besides radiation-induced anti-tumor immune responses, tumor irradiation can also activate
immune cells with tumor-promoting properties. Pro-inflammatory cytokines that are released after
radiation-induced tissue damage, as well as the humoral immune response from activated B cells,
trigger the recruitment and activation of innate immune cells, such as granulocytes, macrophages and
mast cells [72,73]. By releasing several mediators, these innate immune cells can alter gene expression
programs, thus favoring pro-survival signaling and cell cycle progression, as well as tissue expansion
in tumors [74,75]. Moreover, innate immune cells can release various mediators, which have an impact
on fibroblast activation, angiogenesis, and matrix metabolism. Innate immune cells thus have the
ability to induce repair, regeneration and tissue remodeling in favor of tumor growth [73,76–78].
Finally, radiation-induced stress or damage in the tumor itself results in several phenotypic
changes. By secreting cytokines, chemokines or growth factors, as well as up-regulating specific surface
receptors, e.g., PD-L1, CTLA-4, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1),
and others, tumor cells become competent in dampening and finally escaping the immune system
(Figure 1) [79–84]. Several therapeutic strategies aim at the inhibition of such tumor cell-extrinsic
factors that lead to primary and adaptive resistance and will be addressed in more detail in the
“Immunotherapy in lung cancer” section, but have also been nicely reviewed by others [24,30].

3. Radiation-Induced Normal Tissue Toxicity in the Lung

The lung, with its mucosal barrier, is constantly exposed to foreign particles and pathogens.
Therefore, the mucosal barrier is equipped with means to recognize and eliminate any harmful
exposures, thereby functioning as an immunological organ. Epithelial cells, alveolar macrophages,
and other immune cells in the respiratory tract express pathogen recognition receptors (PRR) to
direct powerful immune responses if needed, for example, during infection, leading to immune cell
recruitment and inflammation [85]. Besides the recognition of foreign harm, the same PRR can identify
endogenous danger signals; the so-called DAMPs.
Despite technological improvements, radiation still directly hits, to some extent,
tumor-surrounding healthy tissues during treatment, e.g., the highly radiosensitive lung tissue.
Radiation-induced stress, damage or cell death to lung resident cells and local immune cells triggers
Int. J. Mol. Sci. 2019, 20, 24 5 of 21

the release of several DAMPs into the extracellular room, immunogenic cell death, or both. Through
recognition of these DAMPs via PRR, the radiation-induced damage induces a sterile inflammation in
sensitive patients that can evolve into a harmful normal tissue inflammation with life-threatening
complications [86,87]. It is noteworthy that the damage response from the malignant and healthy tissue
residing in the radiation field not only contributes to the local and abscopal effects of radiotherapy
against the tumor, but can also induce strong systemic side effects [88–92]. Moreover, as described
before, radiation-induced DNA damage and subsequent detection of cytosolic DNA can activate
the cGAS/Sting pathway, which is important for the activation of an innate and adaptive antitumor
immunity [93,94].
Briefly, the released “danger signals” induce a subsequent chemokine/cytokine secretion, innate
and adaptive immune cell recruitment, and activation of recruited immune cells at the site of radiation
damage leading to tissue inflammation. This inflammatory response is needed to orchestrate tissue
repair and regeneration in order to restore tissue homeostasis. If the inflammation during the acute
phase is too excessive, due to overwhelming release of cytokines and reactive oxygen species (ROS),
the inflammation can exert toxic effects in the normal tissue (pneumonitis), a severe side effect in
patients [95]. Radiation-induced pneumonitis can develop at 4 to 12 weeks after radiotherapy, with
symptoms including fever, dry cough, chest pain and dyspnea, or even failure of the respiratory
system. The reported occurrence of pneumonitis in lung or breast cancer patients ranges from 13
to 36% of patients, depending on the diagnosed severity [34,96]. Patients suffering from severe
radiation-induced pneumonitis are commonly treated with anti-inflammatory corticosteroids, such as
prednisone [35,96–99].
Besides radiation-induced acute inflammatory responses, subsequent repair/regeneration
processes can manifest as a chronic event, where pathologic immunomodulation and altered
microenvironmental changes, e.g., hypoxia, senescence and anti-inflammation, drive excessive tissue
remodeling (fibrosis); moreover, exposure to ionizing radiation can also increase the risk for secondary
tumor formation on the long-term [57,87,92,100–107]. Radiation-induced lung fibrosis mostly develops
6 to 24 months after radiotherapy, and is characterized by breathing difficulties and subsequent volume
loss of the lung [35,108]. The mechanisms of radiation-induced lung disease have been described in
detail elsewhere and will not be addressed here [35,92,106,109].
Both the acute and the chronic events of radiation-induced lung disease are accompanied by
microenvironmental alterations in the lung tissue, as well as local and systemic immune changes.
Current research focuses on the identification of target cells, pathologic signaling molecules and
up-stream regulators to develop novel strategies for prevention or treatment against these adverse
effects. We speculate that tumor-induced and radiation-induced immune changes in tumor and normal
tissues might offer the opportunity to improve immune-mediated tumor killing and to counteract at
the same time radiation-induced chronic adverse effects in the lung tissue [110,111].
This is particularly interesting, as the first promising results have recently been obtained using
immunotherapy with ICI in combination with radiotherapy for the treatment of NSCLC [32–34].
However, as outlined above, radiotherapy modulates the immune repertoire in tumor tissues
and normal lung tissue in various ways. Thus, optimal targeting of the immune system in
combination with radiotherapy will require further work to define therapeutic strategies that balance
pro-immunogenic and immunosuppressive effects of radiotherapy and outweigh the beneficial effects
of radioimmunotherapy between optimal tumor control and normal tissue protection.

4. Immunotherapy in Lung Cancer

As mentioned in the sections above, tumors develop intrinsic or radiation-induced alterations
that shape an immune phenotype with immunosuppressive or even tumor-promoting characteristics.
Major receptors or pathways involved in immunomodulation include vascular endothelial growth factor
receptor (VEGFR), epidermal growth factor receptor (EGFR), as well as PD-L1 on tumor cells and the
receptors PD1 and CTLA-4 on the site of T cells [112]. However, CTLA-4 expression was also detected
Int. J. Mol. Sci. 2019, 20, 24 6 of 21

on multiple tumor cells including NSCLC [19]. Interestingly, a recent study revealed that tumor
cell-intrinsic CTLA-4 expression had an impact on the expression of PD-L1, as well as on tumor cell
proliferation. Inhibition of CTLA-4 induced the upregulation of PD-L1, as well as the activation of the
EGFR pathway in NSCLC cells, highlighting a distinct function of CTLA-4 in tumor cells compared to
T cells [113].
Generally, surface expression of the receptors VEGFR and EGFR on tumor cells and their
interaction with the corresponding growth factors induces tumor-promoting proliferation, expansion,
and malignant conversion of cancer cells, as well as tumor-promoting angiogenesis. VEGF and EGF
are commonly released by innate immune cells, but tumor cells can also secrete VEGF in an autocrine
manner, thereby stimulating cancer stemness [114–116]. Generally, VEGF is a proangiogenic growth
factor and potent inducer of growth and survival of the vascular endothelium. Besides its direct effect
on the tumor vasculature, VEGF can also modulate the immune environment [117]. Several studies
demonstrated that VEGF has the ability to inhibit the differentiation and function of diverse immune
cells like dendritic cells, macrophages, and lymphocytes, respectively [118–120]. More details about
the role of VEGF/VEGFR in tumor and immune cells can be found in the following reviews: [121,122].
Activation of EGFR on tumor cells, including NSCLC, also promotes proliferation, invasion, metastasis,
anti-apoptotic signaling and angiogenesis in malignant tumors [123–125]. Lung cancer patients have
intrinsic or acquire new EGFR mutations during treatment with small-molecule EGFR-inhibitors that
complicate current treatment options highlighting the need for the development of novel strategies for
EGFR-resistant NSCLC, including combined therapies with ICI or radiotherapy [10,126–129].
In contrast to VEGF and EGF, PD1/PD-L1, as well as CTLA-4 are immune checkpoints that
negatively regulate T-cell immune functions, thus indirectly promoting tumor progression via tumor
immune escape [130–132]. Our understanding of the mechanisms and pathways how cancers evade
and inhibit immune responses and their targeting has largely improved over the years and the state of
knowledge has been summarized in recent reviews [23,133–136]. Overall, targeting these structures or
pathways with immunotherapies, alone or in combination, substantially improved the care of patients
with advanced-stage cancers leading to longer survival rates or even long-lasting tumor remissions.
Several of these immunotherapeutic strategies are being evaluated in lung cancer [137–141]. Multiple
strategies using either T cell-stimulating agents (tumor necrosis factor receptor (TNFR) superfamily
antibodies and peptides, e.g., OX-40), genetically modified T cells (Chimeric Antigen Receptor (CAR)-T
cells), bi-specific antibodies, and tumor vaccination or agents that counteract the immunosuppressive
tumor microenvironment (e.g., inhibitors of TGF-β, as well as Toll-like receptor (TLR) agonists) are
currently being tested in preclinical and clinical trials [141–150].
So far, two groups of targeted compounds are FDA approved; these include, on the one hand,
“targeted antibodies” against the VEGF/VEGFR pathway (Bevacizumab), and the EGFR pathway
(Necitumumab), which are being used in a subset of patients with advanced NSCLC [151,152]. Both
target the tumor-promoting effects of growth-factor-induced proliferation, expansion and angiogenesis.
On the other hand, ICI have been approved by the FDA for cancer treatment, and so far, they have
received the most clinical recognition among the current immunotherapeutic agents. ICIs have emerged
as a landmark event for the treatment of lung cancer; among the FDA-approved ICIs, Pembrolizumab
and Nivolumab (both anti-PD1), as well as Atezolizumab and Durvalumab (both anti-PD-L1), and
Ipilimumab (anti-CTLA-4), alone or in combination with each other, were effective against small cell
lung cancer (SCLC), NSCLC, and metastatic lung carcinomas, and improved the prognosis in these
patients [153–158].
Despite the revolutionary efficacy profile of ICIs, which has raised cancer therapy to a new level,
it has to be taken into consideration that unbalancing the immune system with ICI can also generate
adverse events with severe complications in patients [159]. These so-called immune-related adverse
effects (IRAEs) can occur in the gut, skin, endocrine glands, liver, heart and lung, but can potentially
involve any tissue [160–166]. Increasing the knowledge gained over the last years in several trials
with ICI has made oncologists aware of the potential risks, but not all variables are yet understood,
Int. J. Mol. Sci. 2019, 20, 24 7 of 21

e.g., patient predisposition or pretreatment status of the patient. Several recent reviews highlight the
potential risks that could emerge using ICI alone [167–172].
In brief, an ICI-induced immune dysregulation towards an “immune boost” with uncontrolled
pro-inflammatory signaling can trigger toxic effects and normal tissue complications. It is already
known, e.g., from trauma patients that tissue damage can unbalance the immune system with
overwhelming pro-inflammatory responses and the induction of a systemic inflammatory response
syndrome (SIRS). Due to this, severe toxic side effects, multiple organ failure, or even death can occur
in these patients [173]. Therefore, it is not surprising that ICI can also induce SIRS in cancer patients,
including lung cancer [174]. It has to be considered that irradiation also induces tissue damage
(trauma) and inflammation with the potential to induce normal tissue toxicity. Similar to trauma
patients, the extent of induced tissue-damage in irradiated patients will be critical for the extent of the
immune response. Knowing that immunotherapy alone can already induce IRAEs in several tissue
types, including the lung, pre- or post-treatment with radio(chemo)therapy might have additive or
synergistic life-threating effects in patients that are already fragile in health.

5. Combining Radiotherapy and Immunotherapy in Lung Cancer

Several recent reviews have discussed the potential of combining immunotherapy and
radiotherapy, including for lung cancer, with respect to clinical efficacy [37–45]. The recent review
from Ko et al. published in June 2018 nicely summarizes planned and ongoing trials of combined
radioimmunotherapy in patients with NSCLC with respect to present knowledge [37]. Those trials
include phase 1 and 2 studies, with disease stages ranging from early stage NSCLC and locally
advanced stage NSCLC towards the majority of trials investigating stage IV metastatic NSCLC. Among
these trials, all potential settings are under investigation, including the use of different inhibitors and
molecules (durvalumab, atezolizumab, pembrolizumab, tremelimumab, nivolumab, ipilimumab,
granulocyte-macrophage colony-stimulating factor (GM-CSF), thymosin-α1, as well as virus therapy),
different combined inhibitor administrations, and different timing and dosing of radioimmunotherapy.
Thus, ongoing clinical trials will add important knowledge and evidence related to this novel and
promising combinatory cancer therapy.
The majority of reviews highlight the two comprehensive trials, namely the phase I KEYNOTE-001
trial of pembrolizumab (anti-PD1) in patients with metastatic NSCLC [32] and the phase III PACIFIC
trial of durvalumab (anti-PD-L1) in patients with unresectable stage III NSCLC after ≥2 cycles of
platinum-based chemoradiation [33].
Both studies have in common that immunotherapy was administered subsequent to radiotherapy.
The KEYNOTE-001 trial is a secondary analysis of 97 patients with 24 patients that received thoracic
radiotherapy at a median of 11.5 months prior to ICI. Overall pulmonary toxicity was observed in 63%
of all patients that received prior radiotherapy versus 40% in patients that received no radiotherapy.
The incidence of all-grade ICI-related pulmonary toxicities was significantly higher (13% versus 1%, p =
0.046) in patients with previous radiotherapy treatment. Although significant changes were observed,
the study revealed that there are no differences in high-grade (≥3) pulmonary toxicities between the
radiotherapy and non-radiotherapy group (4% versus 1%, p = 0.44).
In the PACIFIC trial, 713 patients were analyzed after sequential ICI treatment starting between
1–42 days after chemoradiation. In the combined radiotherapy/ICI group all-grade pneumonitis was
more frequent than in the radiotherapy alone group (33.9% versus 24.8%, no p-Value). Nevertheless,
the authors reported that the incidence of grade ≥3 pneumonitis was similar in both groups (3.4%
versus 2.6%, no p-value).
Overall, both studies revealed that the risk to develop high-grade pulmonary toxicities is
not enhanced when combining radiotherapy and immunotherapy at least with durvalumab and
pembrolizumab. What has to be considered is that in the KEYNOTE-001 trial the median interval
between radiotherapy and ICI was nearly 1 year. Radiation-induced immunomodulation and
pathologic events in normal pulmonary tissues after 1 year are usually described as chronic
Int. J. Mol. Sci. 2019, 20, 24 8 of 21

inflammation with profibrotic tissue remodeling. It might thus not be surprising that ICI does not
induce high-grade toxicities after that long-time interval.
In contrast, in the PACIFIC trial ICI was given earlier (1–42 day) after radiochemotherapy. In
general, radiation-induced acute inflammatory effects are observed in the lungs of treated patients
during this timeframe shortly after radiotherapy. Consequently, boosting the immune system with ICI
during the acute phase after radiotherapy might bear a higher risk of enhancing all-grade toxicities
in the combined treatment group. This was not the case in the PACIFIC trial, and this is potentially
linked to the type of ICI used. Since durvalumab mainly targets PD-L1, present on tumor cells,
immune-related effects are potentially more likely to be linked to the tumor microenvironment and,
to a lesser extent, to the normal tissue. Instead, targeting PD-1 or CTLA-4 on immune cells might
enhance the immune response in both the tumor and also the normal tissues, thereby increasing the
risk for more frequent or more severe pulmonary toxicities upon combined use with radiotherapy
when compared to a combination of radiotherapy with PD-L1-inhibition.
Two recent reviews about the toxicities of treatment with ICI alone corroborate this hypothesis:
Pillai and colleagues compared 23 studies, including 5744 patients with NSCLC, in a systematic
analysis to investigate potential differences in the toxicities of monotherapies using PD-1 and PD-L1
inhibitors. In fact, the authors revealed that the overall incidence of adverse effects was comparable
between the PD-1 and PD-L1 inhibitors (64% versus 66%, p = 0.8), but in patients treated with PD-1
inhibitors IRAEs (16% versus 11%, p = 0.07) and pneumonitis (4% versus 2%, p = 0.01) increased
compared with patients who received PD-L1 inhibitors [175]. In line with these findings, Khungar
and colleagues reported in a systemic analysis including 19 trials, that there was a higher incidence
of pneumonitis with the use of PD-1 inhibitors compared with PD-L1 inhibitors in a monotherapy in
NSCLC patients [176].
Both the KEYNOTE-001 and the PACIFIC trial show excellent results, as inhibition of PD1 and
PD-L1 in combination with radiotherapy significantly increased the objective response rate in patients
and significantly extended both the median progression free survival, as well as the median time
to death or distant metastasis. However, both trials only tested one potential treatment setting. To
fully understand the potential risks of IRAEs that might occur using combined radioimmunotherapy,
several further aspects have to be considered, as described below.
One important factor might be the treatment sequence. Should ICI and radiotherapy be given
concurrently or sequentially, and does the order of administration have an impact on the outcome?
Since radiotherapy of the thoracic region can induce time-dependent changes in the normal lung tissue
ranging from pro-inflammatory acute effects towards pro-fibrotic chronic side effects, the different
potential treatment settings might also induce distinct effects in the normal lung tissue. Two case
reports have revealed that a setting where radiotherapy was given after ICI had beneficial effects.
Here, the combination immunotherapy followed by radiotherapy had the capacity to influence tumor
responses, overcome resistance to ICI, and even induce abscopal effect with reduced distant metastasis
in the lung, at least when using a PD1-inhibitor (Nivolumab) [177,178]. Thus, radiotherapy can
stimulate immune activation even when immunotherapy has failed.
However, little is known about potential adverse effects in the lung when radiotherapy is delivered
together with ICI. Nevertheless, there are single case reports about adverse pulmonary effects upon
concomitant use of ICI and radiotherapy. Louvel and colleagues recently reported two patients, both
with metastatic melanoma or metastatic colon cancer, where concomitant radiotherapy and PD-1
or PD-L1 blockade induced radiation-pneumonitis [179]. However, the small sample size makes
it difficult to draw firm and clear conclusions, and this was also mentioned by the authors. Thus,
additional studies are needed to clarify the acute and long-term safety of the different settings of
combining radiotherapy and immunotherapy, particularly ICI.
Besides the treatment schedule of ICI plus radiotherapy, the type of inhibitor that is used might
also affect the outcome. PD-1 and PD-L1, but also CTLA-4, are differently expressed in tumor cells
and in diverse subsets of immune cells. Furthermore, as mentioned above, radiotherapy shapes the
Int. J. Mol. Sci. 2019, 20, 24 9 of 21

immune repertoire and tissue microenvironment in the tumor, as well as in the normal tissue, in a
time-dependent, dose-dependent, volume-dependent, and tissue-specific manner. For example, in the
lung, the appearance of distinct immune cells and their phenotypes differs in the acute versus chronic
phase after irradiation [87,106,111,180,181]. Thus, modulating the immune response by targeting PD-1,
PD-L1 or CTLA-4 in lung cancer might cause varying effects in the normal tissue, depending on
the time point (acute versus chronic) of application, especially in a setting where ICI is given after
radiotherapy of the thoracic region.
Finally, the patient itself, especially the immune status and the characteristics of the tumor, plays
an important role. Besides the need for a better understanding how we can optimally combine
radio(chemo)therapy and immunotherapy to best harness the combined potential of both to the benefit
of the patient, we also need to focus on the immune characteristics of the individual patient. The
improved understanding of cancer biology, as well as the identification of predictive and prognostic
biomarkers and of potential therapeutic targets, has supported the development of personalized
therapies over recent years [182–185]. Several case reports highlight the diversity of therapeutic
responses in different patients, especially when the immune system is involved (individual innate
resistance, acquired resistance and normal tissue toxicity). Thus, an individual evaluation of both, the
tumor characteristics and the immune status of a given patient and tumor, are required. Furthermore,
the diverse therapeutic responses in different patients highlight the importance to develop tools for a
reliable prediction of normal tissue toxicity probability in each patient prior to the combined treatment.
Thus, radiotherapy centers should take the opportunity to collect suitable and structured datasets
from the current clinical trials including data about adverse effects. The present technology and
available tools for radiotherapy-dose-fractionation-volume parameter assessment, together with the
available methodology for estimating normal tissue complication probability (NTCP) [186–191] may
provide a reliable background for quantitatively detecting “more-than-expected” lung damage, which
may be related to the ICI combined administration. Such information can be analyzed according
to suspected biological markers to provide suitable hints for prospective trials using radiotherapy
and ICI.

6. Final Remarks
The current trials combining radiotherapy and immunotherapy show promising results. However,
only single possible treatment settings have been tested so far. Moreover, as outlined above, the
complex effects of combining irradiation and unbalancing the immune system with ICI are not
fully understood, and many open questions remain. Figure 1 provides a schematic overview of
the radiation-induced immune changes in tumor and normal tissues. In brief, radiotherapy has
the potential to induce stress responses and cell death in cancer cells with subsequent induction
of a DC/T cell-driven antitumor immunity. Radiation-induced tissue damage also stimulates the
influx of innate immune cells with tumor-promoting potential. These processes are reminiscent of
immune changes observed during tumorigenesis, where tumors upregulate a repertoire of cell surface
receptors and adaptive changes in the tumor microenvironment to escape T cell killing and foster
immunosuppression and tumor-promoting effects such as angiogenesis.
Immunotherapies were initially developed to cope with the processes involved in tumor immune
evasion. ICI interferes with and unbalances the immune response, thereby unleashing an immune
boost, at least in a fraction of tumor patients. However, unbalancing the immune response can also
induce inflammation-induced toxic side effects in normal tissues. Thus, ICI must be considered a
“double-edged sword” that needs careful handling. The same holds true for radiotherapy-induced
immune changes; radiotherapy exerts its effects by efficient tumor killing, but it can also damage
normal lung tissue. The acute and chronic toxic side effects induced in the radiation-sensitive lung
tissue are mediated by a complex interaction between resident lung cells, radiation-induced changes
in the lung environment, and cells from the innate and adaptive immune system. Considering that
Int. J. Mol. Sci. 2019,
Int. 2018, 20,
19, 24
x FOR PEER REVIEW 10
10 of 21
21

chronic) pathologic immune changes in the lung tissue, a combination with ICI might bear the risk of
radiation-induced
synergistic adverselung disease involves time-dependent (acute and chronic) pathologic immune
effects.
changes
Recent clinical trials aonly
in the lung tissue, combination with
represent a ICI might
small bear the
fraction of risk of synergistic
potential adverse
therapeutic effects.of
settings
Recentradiotherapy
combined clinical trials only represent a small fraction
and immunotherapy. of potential
Thus, only therapeutic
additional settings
trials will shed of combined
light on the
radiotherapy and immunotherapy. Thus, only additional trials will shed light
opportunities and risks of combined radioimmunotherapy and will hopefully clarify which of theon the opportunities
and risks
various of combined
potential treatment radioimmunotherapy and will or
settings, e.g., (I) concomitant hopefully
sequentialclarify which
treatment, (II)ofchronology
the various of
potential treatment settings, e.g., (I) concomitant or sequential treatment, (II) chronology
treatment (radiotherapy > ICI, ICI > radiotherapy), (III) the interval between treatments, or (IV) the of treatment
(radiotherapy > ICI,
type of inhibitor used,ICIhas> the
radiotherapy), (III) thefor
highest probability interval between
an effective andtreatments,
safe treatmentor (IV) the type
of patients of
with
inhibitor
NSCLC. used, has the highest probability for an effective and safe treatment of patients with NSCLC.

Figure1.1. Radiotherapy
Figure Radiotherapy of of malignancies
malignancies in in the
the thoracic
thoracic region
region results
results inin stress,
stress, damage
damage and and cell
cell
death inin the
the tumor
tumor tissue,
tissue, and
and to
to some
some extent
extent also
also in
in surrounding
surrounding normal
normal tissue
tissue within
within the
the radiation
radiation
field. The
The release of damage-associated molecular patterns (DAMPs), (DAMPs), cytokines
cytokines and and chemokines
chemokines
leads
leads to the recruitment of immune cells to the site of damage. In the normal lung tissue, thethe
to the recruitment of immune cells to the site of damage. In the normal lung tissue, influx
influx of
of pro-inflammatory
pro-inflammatory immune
immune cellscan
cells caninduce
inducean an acute
acute normal
normal tissue-toxicity
tissue-toxicity (pneumonitis)
(pneumonitis) with with
excessive
excessive inflammation,
inflammation, or or on
on the
the long-term,
long-term, chronic
chronic inflammation,
inflammation, environmental
environmental changes
changes and and
tissue remodeling (fibrosis). In contrast to that, the innate immune system
tissue remodeling (fibrosis). In contrast to that, the innate immune system contributes to tumor contributes to tumor
progression
progressiondue duetototumor-promoting
tumor-promoting activities. Radiation-induced
activities. Radiation-induced cell death of tumor
cell death cells also
of tumor results
cells also
in the release of tumor antigens to activate dendritic cells with subsequent
results in the release of tumor antigens to activate dendritic cells with subsequent antigen antigen presentation and
induction
presentation of Tandcell-mediated
induction oftumor killing, a process
T cell-mediated tumortermed
killing,“in situ vaccination”.
a process termed “in However, several
situ vaccination”.
tumor-intrinsic mechanisms shape the innate immune response towards
However, several tumor-intrinsic mechanisms shape the innate immune response towards tumor tumor promotion and adaptive
immune
promotion responses towards
and adaptive immunosuppression,
immune responses towards so that the tumor cells escape
immunosuppression, sofrom
that Tthe
cell-mediated
tumor cells
cytotoxicity. The use of immune checkpoint inhibition (ICI) boosts
escape from T cell-mediated cytotoxicity. The use of immune checkpoint inhibition (ICI) the immune system, overcoming
boosts the
immunosuppression
immune system, overcoming and immuneimmunosuppression
escape, and thus leading and toimmune
successful eradication
escape, and of tumor
thus cells. In
leading to
normal tissues, ICI might trigger an unbalanced immune activation, thereby
successful eradication of tumor cells. In normal tissues, ICI might trigger an unbalanced immune potentially enhancing
normal tissue
activation, toxicity.
thereby Combined
potentially treatment
enhancing withtissue
normal ICI and radiotherapy
toxicity. Combined bears the potential
treatment with ICI riskand
of
synergistic toxic effects, depending on several variables in the treatment setting of
radiotherapy bears the potential risk of synergistic toxic effects, depending on several variables in the radioimmunotherapy.
treatment setting of radioimmunotherapy.
Author Contributions: F.W., S.d.L. and V.J. equally contributed to the writing of this review article.
Acknowledgements: We acknowledge support by the Open Access Publication Fund of the University of
Funding: The work was supported by grants of the DFG (GRK1739/2; JE275/1) and the BMBF (ZiSStrans
Duisburg-Essen
02NUK047D).
Author Contributions: F.W., S.d.L. and V.J. equally contributed to the writing of this review article.
Int. J. Mol. Sci. 2019, 20, 24 11 of 21

Acknowledgments: We acknowledge support by the Open Access Publication Fund of the University of
Duisburg-Essen.
Conflicts of Interest: The authors declare that the research was conducted in the absence of any commercial or
financial relationships that could be construed as a potential conflict of interest.

Abbreviations
ATP Adenosine triphosphate
CAR Chimeric Antigen Receptor
CEACAM-1 Carcinoembryonic antigen-related cell adhesion molecule 1
cGAS cGMP–AMP synthase
CTLA-4 Cytotoxic T-lymphocyte-associated Protein 4
DAMPs Damage-associated molecular patterns
EGF Epidermal growth factor
EGFR Epidermal growth factor receptor
GM-CSF Granulocyte-macrophage colony-stimulating factor
HMGB1 High-Mobility-Group-Protein B1
ICI Immune Checkpoint Inhibition
IRAEs Immune-related adverse effects
MHCI Major histocompatibility complex I
NSCLC Non-small cell lung cancer
NTCP Normal tissue complication probability
PD1 Programmed cell death protein 1
PD-L1 Programmed cell death 1 ligand 1
PRR Pathogen recognition receptor
ROS Reactive oxygen species
RT Radiotherapy
SBRT Stereotactic body radiotherapy
SCLC small cell lung cancer
SIRS systemic inflammatory response syndrome
STING stimulator of interferon genes
TGF-β transforming growth factor beta
TNFR tumor necrosis factor receptor
TLR TOLL-like receptor
TREX1 three prime repair exonuclease 1
VEGF Vascular endothelial growth factor
VEGFR Vascular endothelial growth factor receptor

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