Radiotherapy and Immunotherapy—A Future Partnership towards a New Standard

Abstract

The impressive results in terms of survival brought by immune checkpoint inhibitors (ICI) in metastatic malignant melanoma and the transformation of this disease with a poor prognosis into a chronic disease even with long-term survival cases have opened horizons for a new era in cancer treatments. Later, therapy with CTLA-4 and PD-1/PD-L1 inhibitors became standard in other solid tumors, especially in relapsed and metastatic settings. The PACIFIC clinical trial revolutionized the concept of consolidation immunotherapy after the favorable response to curative chemoradiotherapy in non-small cell lung carcinoma (NSCLC). Two new effects will govern the future of the immunotherapy–radiotherapy association: the local “in situ” vaccination effect and the systemic remote “abscopal” response. Even if stereotactic body irradiation (SBRT) or stereotactic radiosurgery (SRT) seems to be more effective in generating the synergistic effect, the PACIFIC trial demonstrates the role of conventional irradiation in combination with chemotherapy in modulating the host’s immune response. Thus, the radiotherapy–chemotherapy–immunotherapy triad may become the future standard in locally advanced disease. The different mechanisms of producing immune-mediated cell death and the indirect role of augmenting the immune effect induced by radiotherapy make the old theories related to the therapeutic sequence, fractionation, doses, and target volumes as well as the protection of healthy tissues to be re-evaluated. The new concept of immuno-radiotherapy in synergistic association has as its physiopathological substrate the dual immunosuppressive and enhancement of antitumor response to irradiation, including the activation of the immune effectors in the tumor microenvironment (TME). The choice of sequential treatment, a hypofractionated irradiation regime, and the possible omission of lymph node irradiation with the limitation of lymphopenia could tilt the balance in favor of the activation and potentiation of the antitumor immune response. The selection of therapeutic targets chosen for the combination of immunotherapy and associated radiotherapy can be conducted based on the classification of tumors in the three immune phenotypes that characterize “cold” and “hot” tumors from the point of view of the response to therapy.

1. Introduction

The development of immunotherapy based on the use of immune checkpoint inhibitors (ICI) was implemented in clinical practice more than 10 years ago. Metastatic malignant melanoma, a disease with a dismal prognosis (sometimes a median survival less than 6 months after diagnosis), is the first beneficiary of therapy with ipilimumab and nivolumab, which modulate the immune anti-tumoral response, the prognosis being considerably improved in some cases. Dual inhibition with inhibitors of cytotoxic T-lymphocyte associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) is associated with a 58% survival at 3 years in cases with metastatic/recurrent malignant melanoma. In this context, it is easy to understand why cancer immunotherapy can be considered to have opened a new era in oncological treatment. Tolerance and a favorable safety profile are other arguments in favor of this revolutionary treatment. An abscopal effect related to metastatic malignant melanoma was reported early and frequently in the literature, a systemic response being induced by an association of immunotherapy with hypofractionated palliative radiotherapy [1,2,3,4]. Currently, ICI therapies have been applied to an increasing number of cancer types, becoming a standard in the first line, not only for metastatic/recurrent disease but also in consolidation or adjuvant treatment. However, a favorable response is identified in only 2–40% of cases, but it should be mentioned that in these situations the tumor response can be a long-lasting one. The presence of CD8+ T cells is associated with prolonged survival in cancer and the absence of lymphocytes can be associated with immunosuppression and an unfavorable response to immunotherapy. The presence of the tumor infiltrate with T cells is essential in obtaining a durable response to ICI [1,5].

2. Tumor Immune Phenotype—A Classification with Therapeutic Implications

The presence and distribution of immune cells in the tumor microenvironment (TME) can be a factor that classifies a tumor into three basic immune phenotypes: desert, excluded, and inflamed. Three characteristics make a tumor “hot” from an immunological point of view: a large amount of infiltrated T lymphocytes, interferon-γ signaling, a tumor mutation burden (TMB), and expressions of Programmed death-ligand 1 (PD-L1). From this last class of tumors are selected the most responsive to ICI treatment. The other two immune phenotypes place the tumors in the “cold” category, these cases being much less or non-responsive to immunotherapy. Both of these categories show a reduced mutational load and a lower expression of PL-L1. The differences between the “immune-excluded” and “desert” subtypes are generated by the presence of T lymphocytes. In the first case, CD8+ T are located at the periphery of the tumor, but do not infiltrate it, and in the case of tumors with a “desert” phenotype, T lymphocytes are absent or present in reduced number. Both “cold” immune phenotypes are also characterized by the presence of some categories of cells with immunosuppressive potential. Among these, the most important are tumor-associated macrophages (TAM), derived from myeloid suppressor cells (MDSC). The presence of a larger number of cells with immunosuppressive potential counterbalances the effect of CD8+ and makes tumors belonging to these phenotypes inert to the action of CTLA-4 and PD-1/PD-L1 inhibitors [1,6]. Tumor cells express PD-L1 (also called B7-H1 or CD274) and the action of inhibiting immunity is produced by the interaction of PD-1/PD-L1 and the inhibition of T-lymphocyte action. The blockage of T-lymphocyte action and the release of cytotoxic mediators limiting the killing of cancer cells are mechanisms that normally tilt the balance in favor of immunosuppressive action, promoting tumor progression. For this reason, blocking this PD-1/PD-L1 interaction was conceived as a strategy to inhibit immunosuppressive mechanisms [7,8,9].

T cell-inflamed TME shows increased activity of type 1 interferon and chemokines, the end result being the recruitment of activated CD8+ effector T cells. Endogenous T cell priming against tumor antigens is strongly correlated with the tumor response to ICI, and type 1 interferon is a bridge to adaptive immunity. It should also be mentioned the involvement of the STING pathway in the innate immune sensing of cancer. Modification of TME metabolism by some enzymes including indoleamine-2,3-dioxygenase, but also the recruitment of FOXP3+ Treg can still have a negative effect on the response to immunotherapy [10,11].

CTLA-4 is a cell surface molecule expressed especially in CD8+ and CD4+ T lymphocytes, having an essential role in the homeostasis of the T lymphocyte and in the immune response mediated by the T lymphocyte, being essential both in the early and late stages of its activation [12]. In the case of resting naive T cells, CTLA-4 is located in the intracellular compartment. Stimulatory signals can lead to exocytosis and expression of CTLA-4 on the cell surface. Due to the competitive competition for these ligands (CD80 and CD86) on antigen-presenting cells APCs, CD28, and CTLA-4 activate feedbacks of T lymphocyte activation and inhibition, respectively [13,14]. Tregs have the role of suppressing abnormal/excessive immune response and in the case of tumors, it has the role of stimulating progression by inhibiting the anti-tumor immune response. There is a relationship between Tregs and CTLA-4, CTLA-4 deficiency on the surface of Tregs able to affect their suppression capacity [14,15,16]. PD-1 binding to its ligand PD-L1 has an inhibitory effect similar to CTLA-4, being associated with the reduction of the production of IL-2, interferon-γ, tumor necrosis factor-α, and with the inhibition and limitation of T lymphocyte survival. PD-1 binding to its ligand PD-L1 has an inhibitory effect similar to CTLA-4, being associated with the reduction of the production of IL-2, interferon-γ, tumor necrosis factor-α, and with the inhibition and limitation of T lymphocyte survival. Considered a true hallmark of exhausted T cells, PD-1 is thus associated with this state of T lymphocytes identified in infections and cancer. “Exhausted T cells” are associated with a suboptimal antitumor effect [14,17].

3. Radiotherapy and Immunotherapy: Abscopal and “In Situ” Vaccination Effect

The importance of the host’s immune response in relation to the benefit of radiotherapy was highlighted more than 60 years later by Luka and colleagues, finding the need for a 50% escalation of the irradiation dose in order to obtain the same tumoricidal response in the cases of immune-compromised mice. The concept of “in situ” vaccination represents an approach in which the antitumor vaccine is generated in vivo without being prepared and processed outside the host. The advantage is conferred by the exploitation of tumor-associated antigens (TAA) available at the site of the tumor without the need for their identification and isolation. The much higher expression of TAA in tumor tissue than in normal tissue opens perspectives for the use of this method as an anti-cancer strategy. Through the processing of TAA by antigen-presenting cells (APC), the antitumor response mediated by T lymphocytes is activated [18]. Radiotherapy has the potential to stimulate the release of TAA and the presentation of antigens to the dendritic cell (DC) with an immune stimulatory effect. Thus, radiotherapy acts locally at the level of the tumor like “in situ” vaccines. DC are APCs with a role not only in inducing an anti-tumor immune response but also in maintaining T cell tolerance to self-antigens. Additionally, the induction of a long-lasting antitumor response depends on the ability of the radiotherapy to maximize the absorption of the APC antigen. In this way, activated T cells will not need co-stimulatory signals to activate tumor destruction mechanisms. The release of the destroyed DNA fragments also results in the release of new tumor antigens that generate an immune response via the major histocompatibility complex (MHC). The release of natural killer (NK) cells and the alteration of the rigidity of the extracellular matrix are other phenomena involved in the potentiation of the antitumor immune response. However, alteration of the extracellular matrix is generally reported at high doses per fraction, possibly more associated with SBRT. Damage to the vascular endothelium by inducing apoptosis and limiting blood flow by limiting the recruitment of cytotoxic T cells is also reported at higher doses per fraction irradiation. However, at typical doses of conventional irradiation, there is an additional generation of adhesion molecules that favor the trafficking of T lymphocytes and the potentiation of the immune response. However, it should be noted that radiotherapy also has effects on some components with an immunosuppressive role, among which we mention regulatory T cells (T-regs), mesenchymal-derived suppressor cells (MDSCs), and M2 macrophages [19,20].

The abscopal effect produced by irradiation refers to a clinical phenomenon induced by localized irradiation, a systemic response being produced in a metastatic site located in a non-irradiated region. In the absence of one or two ICIs systemic treatment, the abscopal phenomenon is considered rare. For the first time, the abscopal effect is reported by Mole et al. in 1953 [21,22]. Guo et al. mention the case of a 70-year-old male patient, who was diagnosed with a left cervical tumor formation and an abdominal tumor mass, the diagnosis is considered an unknown primary squamous cell carcinoma. In this case, irradiation with a total dose of 60 Gy in 30 daily fractions on the left cervical mass induced a favorable response in the case of the abdominal tumor [23]. The systematic literature review proposed by Abuodeh et al. identified 46 cases over a period of 45 years, in all cases no systemic treatment was associated with irradiation [24].

The preclinical data highlights an essential aspect of combining radiotherapy with immunotherapy: the different effects of dose/fractionation regimes and irradiated volumes on the synergistic potential of radiotherapy-immunotherapy, the balance being fragile between immunosuppression and potentiation of the antitumor immune effect [19,23]. Morisada et al. mention a possible detrimental antitumor effect when administering a standard fractionation regimen that can induce lymphopenia as in the case of head and neck cancers, but hypofractionated irradiation regimens seem to have an augmentation effect of antitumor immunity. Comparing the hypofractionated regimen 8 Gy × 2 with the standard fractionation regimen 2 Gy × 10, the study evaluated the immune correlations, the antitumor effect on the primary tumor, and the possibility of inducing the Abscopal effect. The activation and maintenance of peripheral and tumoral CD8+ T lymphocyte level, but also the reduction of peripheral and tumoral accumulation of MDSC was superior for the hypofractionated regimen. In addition, the hypofractionated regimen enhanced the expression of MHC class I and PD-L1 sensitivity to IFN using the 8 Gy × 2 regimen, but the Treg level was not influenced by any of the irradiation regimens. IFN responses of CD8+ T lymphocytes in draining lymph nodes were also improved by the 8 Gy × 2 regimen. Worth mentioning is the effect of limiting the responses of CD8+ T lymphocytes when the 2 Gy × 10 regimen is used. The study demonstrates the ability of hypofractionated irradiation to maintain the antitumor immune response, and in the case of the association of anti-PD-1 therapy, hypofractionation, but not the standard irradiation regimen, can generate the reversal of adaptive immune resistance and the augmentation of the response to local and abscopal ICI dependent on CD8+ T lymphocytes. The results could be the basis for the design of new stages that exploit the synergistic potential of immunotherapy—radiotherapy association [25].

The role of lymph nodes in mediating the abscopal antitumor response mediated by the association of radiotherapy with anti-PD-1 immunotherapy was investigated with the help of a modified subcutaneous B16F10 flank tumor model that allows the dynamic monitoring of tumor-specific T cells. Using irradiation of the primary tumor or of the tumor and lymph nodes the study evaluated both the response to treatment and the dynamics of tumor-specific T that infiltrate the primary tumor and lymph nodes. The authors also identify stem-like CD8+ T cells that populate the lymph nodes, which may infiltrate the primary tumor, much poorer in tumor-specific T. The effect of irradiation on this category of cells is still less known [26].

In most of the cases, the abscopal effect is induced by stereotactic body radiation therapy (SBRT), with lower doses ≤3 Gy per fraction up to a total dose ≤30 Gy not being considered immune stimulatory [1,17,27,28]. The preclinical study by Rejmen and collaborators demonstrated that four daily fractions of 3.2 Gy reduced the number of CD8+ T cells and APC lymphocytes in lung tumors, thus identifying potential risk to reduce the immune response associated with radiation therapy. For this reason, a new target volume for radiotherapy theory proposed the avoiding of the tumor-draining lymph nodes (TDLN) in order to reduce the destruction of CD8+ cells. The TDLN was of particular attention in the case of the use of CTLA-4 inhibitors, being considered an anatomical structure involved in the priming of T lymphocytes. Recent studies have also demonstrated the involvement of proliferating stem cell-like and progenitor-exhausted T cells in the TDLN in the response to therapy with PD inhibitors -1 [28,29]. Analyzing 24 cases aged between 24 and 74 reported in 15 scientific papers, Dagoglu et al. identified median total doses between 18–58 Gy. Using the linear quadratic (LQ) formalism, the biologically effective dose (BED) 10 for the median tumoricidal effect was 49.65 Gy. The effect was identified at different time intervals after irradiation, ranging from more than one month to 12 months. All patients received immunotherapy as a systemic treatment, but the therapeutic sequence differed from case to case [30].

Hammerich et al. report a case of the abscopal effect associated with ICI therapy and radiotherapy in non-small cell lung carcinoma (NSCLC). In this case, the abscopal effect was associated with the reactivation of the immune response to pembrolizumab after the cessation of the synergistic remote effect of immunotherapy and radiotherapy. Thus, the abscopal and the “in situ” vaccination effects induced by the association of immunotherapy with radiotherapy were successively established. The authors mention the need for detailed evaluation in clinical trials of this synergistic association of radiotherapy with PD-L1 inhibitors to be able to understand all the mechanisms involved, the identification of some biomarkers being one of the priorities in order to increase the percentage of “responders” [9,20]. Preclinical data mention the detrimental effect on the abscopal effect of growth factor-β (TGF-β) in the TME by converting cytotoxic T lymphocytes (CTLs) into exhausted phenotypes. The absence of the thymus in mice was also associated with the lack of effect, demonstrating- thus the involvement of the innate immune host status and also the role of T lymphocytes in the immunotherapy-radiotherapy synergy. Exhaustion is considered to be a state of T lymphocytes associated with a reduction in the production of active cytokines, low cytotoxic activity, and an upregulation of inhibitory receptors [9,31,32,33].

Due to the lymph opening effect, chemotherapy should theoretically have an immunosuppressive effect, detrimental to the tumor response to ICI therapy. However, depending on the cytotoxic agent, dose, regimen, and therapeutic sequence, chemotherapy can induce “immunogenic cell death” (ICD) by releasing tumor antigens that will stimulate an immune response sensitizing the surviving cells to the cell-killing mechanisms of the immune system. Apoptosis induced by chemotherapy, but also by endoplasmic reticulum stress of tumor cells, triggers signaling mechanisms that affect anti-tumor antigen-specific T cells and thus induce an “in situ vaccination” effect. At the same time, chemotherapy can relatively avoid the destruction of CD8+ and CD4+ T cells and of NK cells, but it contributes to the depletion of stromal components and MDSC, through this TME modulation effect, increasing the tumor response to immunotherapy [34,35,36,37].

Radiotherapy and the ICD mechanism could decide the “repositioning” of chemotherapy in order to obtain a maximal immune-mediated antitumoral effect. In the case of carboplatin and paclitaxel, there is evidence of ICD potentiation through synergistic use. 5-fluorouracil and gemcitabine have a dual effect and for this reason, their association with concurrent irradiation is controversial. The positive effect of eliminating MDSCs and enhancing CD8 T cell-dependent immune responses can be counteracted by caspase-1 activation in MDSCs that promotes tumor growth [38].

Six mechanisms of ICD induced by irradiation are mentioned by Sia et al., mitotic catastrophe, mitotic death, necrosis, senescence, necroptosis, and ferroptosis, explaining the heterogeneity of the immune response mediated by irradiation but also the multitude of factors involved in the synergistic effect [39]. The expression of DAMPs, HMGB1, and Hsp70 are the molecules released after the lesions induced by spot irradiation that can be detected and can trigger the activation of innate immune response. Even if the results are contradictory, these molecules are currently being evaluated as possible biomarkers of ICD induced by irradiation [40]. Calreticulin a is among the most frequently evaluated DAMPs in relation to the response to radiotherapy. If in non-small cell lung carcinoma (NSCLC) patients treated with radiotherapy, calreticulin expression was identified as prognostic, in the case of esophageal cancer treated with chemoradiotherapy, calreticulin expression was not superior in the group treated with neoadjuvant treatment [40,41]. In head and neck cancers, Hsp70 expression after radiotherapy was identified as prognostic, and in breast cancer, the Hsp70 level was associated with the risk of recurrence and metastasis 2 years after treatment [40,42,43]. HMGB1 was identified as a prognostic and predictor of pathological response after chemoradiotherapy in rectal cancer, with higher values of HMG1 being correlated with either poor prognosis or unfavorable pathological response [44].

4. Radiotherapy and Immunotherapy—From Research to Clinical Practice

An analysis that evaluated 20 clinical trials including 2027 patients with NSCLC concluded that compared to uncombined therapy, treatment that combined PD-1/PD-L1 inhibitors and radiotherapy improved overall survival (OS) and objective response rate (ORR) without significantly increasing the risk of ≥grade 3 toxicities but with an increased risk of mild pneumonitis. The therapeutic sequence in which PD-1/PD-L1 inhibitors followed radiotherapy offered superior results in relation to the sequence that include immunotherapy delivered before radiotherapy. Stereotactic radiosurgery (SRT) and SBRT also demonstrated superior synergistic potential compared to conventional radiotherapy [45].

The necessity to use a double combination of immunotherapy is justified by a low number of TILs and low expression of immune markers. One such case is triple-negative breast cancer (TNBC) which, although considered an immunogenic tumor, will not generate an immune-mediated antitumor response using a single inhibitory checkpoint, thus falling into the “cold” phenotype. Two other types of cancer, malignant melanoma and kidney cancer, are considered “hot” due to the infiltration of TILs, being responsive both to single-agent immunotherapy and double ICI [46,47].

The phase 2 and phase 1/2 trials PEMBRO-RT (NCT02492568) and MDACC (NCT02444741) (phase 1/2) evaluated the benefit of adding radiotherapy to immunotherapy for patients with metastatic small cell lung cancer. Three phenomena (T cells infiltration, tumor antigen release, and antigen presentation) are the basis of the study concept proposed by Theelen et al. in the PEMBRO-RT trial. Even if the results were not statistically significant in the arm that combined radiotherapy and pembrolizumab, a benefit of “abscopal” irradiation was observed. The inclusion criteria were similar in the two studies, PEMBRO-RT including cases previously treated with chemotherapy, and MDACC cases treatment-naive or previously treated. In the PEMBRO-RT study, immunotherapy with pembrolizumab was administered in a dose of 200 mg every 3 weeks, the initiation of ICI being less than a week after the last dose of stereotactic body radiotherapy (SBRT) in a dose of 24 Gy in 3 fractions. The regimen proposed by MDACC included the concurrent administration of pembrolizumab with radiotherapy (50 Gy in 4 fractions or 45 Gy in 15 fractions). The abscopal response rate was significantly higher (41.7%) and was obtained at 12 weeks in cases that combined radiotherapy with ICI, compared to only 19.7% in cases treated only with pembrolizumab. A more than double PFS (9 months vs. 44.4 months) was associated with the administration of the combination of immunotherapy and radiotherapy, and the median OS of 19.2 months vs. 8.7 months in favor of the combined regimen are arguments for evaluating the regimen in phase III trials [48]. Without intending to carry out a detailed review of all the cases of abscopal effect reported in clinical practice, we briefly presented some examples of associations of some radiotherapy protocols in combination with immunotherapy as inducers of the synergistic abscopal effect in different types of cancer (

Table 1. Abscopal effect in clinical practice.

Type of Cancer	Clinical Trial/Case Report	Immunotherapy Agent/Regimen	Radiotherapy Regimen	Results
Lung	Case report [30]	Atezolizumab	Whole-brain radiotherapy (WBRT) 30 Gy in 10 fractions	Primary tumor and lung metastases remission
Lung	ABSCOPAL-1 Clinical Trial [9]	Nivolumab one a month	hypofractionated brachytherapy single 30 Gy fraction Stereotactic body radiotherapy (SBRT) 40–50 Gy in 5 fractions	plasma levels of interleukin IL-6, IL-10, and IL-17A were significantly reduced
Lung	NCT04238169 [31]	Toripalimab 240 mg once every three weeks/bevacizumab + toripalimab	SBRT 30–50 Gy in 5 fractions	Ongoing
Renal cell carcinoma	4 case reports [32]	Interferon + IL-2/Thalidomide (antiagiogenic agent) 50 mg daily or no systemic treatment	SBRT (32 Gy in 4 fractions) Stereotactic radiosurgery (SRT) 30 Gy in 2 fractions	Metastases response or no new lesions
Intrahepatic cholangiocarcinoma	Case report [33]	Nivolumab at a dose of 200 mg every 2 weeks for 15 cycles	SBRT 55 Gy in 5 fractions	recurrent intrahepatic lesion and the lymph node metastases
Melanoma and squamous cell carcinoma of the left ear, neck, and forehead Melanoma Squamous cell cervical cancer	Case series [9]	Nivolumab 3 mg/kg every 2 weeks 4 cycles of ipilimumab Nivolumab	Not specified Not specified 27 Gy (no fractionation regimen specified)	diminish in size in the absence of any treatment
metastatic cutaneous squamous cell carcinoma (SCC) SCC of larynx	2 case reports [34]	Combimetinib and atezolizumab Panitumumab and pembrolizumab	Postoperative SBRT 45 Gy in 5 fractions for primary tumor Radiochemotherapy (70 Gy) and SBRT on axillary lymph node 21 Gy in 3 fractions	Abdomen and chest tumor regression Complete metabolic response to therapy in the larynx, bilateral neck, and right axilla lymph nodes
advanced sinonasal squamous cell carcinoma	Case report	Nivolumab 480 mg every 4 weeks	Chemoradiotherapy 66 Gy in 33 fractions on primary tumor SRT 30 GY in 5 fractions for intraorbital lesion	significant tumor response for intraorbital mass and metastatic lesions
Malignant melanoma	National Cancer Institute “Fondazione G.Pascale” study	Ipilimumab	Extracranial radiotherapy	11 cases of abscopal effect
Melanoma, non-small cell lung cancer (NSCLC) and renal cancer	Antônio Ermírio de Moraes Oncology Center, Brazil study	Nivolumab and pembrolizumab	Median total dose of 24 Gy (1–40 Gy) in 3 fractions (1–10 fractions)	18.7% asbscopal effect (25% among melanoma patients)

), [48,49,50,51,52,53,54,55,56].

The combination of pembrolizumab with chemoradiotherapy in advanced unresectable stage II-III esophageal and gastroesophageal junction cancer were evaluated in the phase III study KEYNOTE-975. The trial proposes 3 treatment sequences including platinum-fluorouracil chemotherapy protocols and radiotherapy in a total dose of 50 Gy or 60 Gy or the FOLOFOX chemotherapy regimen. Immunotherapy with pembrolizumab vs. placebo is proposed up to 13 cycles in 1:1 randomization, the endpoints being OS and event-free survival PD-L1 with a combined positive score ≥10. The trial could provide new data regarding the role of radiotherapy and the preferred dose in generating a synergistic response [57].

The randomized, double-blind, phase 3 trial CheckMate-577 study evaluated the role of nivolumab in stage II-III esophageal and gastroesophageal junction cancer with residual disease after chemoradiotherapy. With an OS of 22.4 months vs. 11 months in favor of the group treated with immunotherapy, the study demonstrates not only the benefit of nivolumab in association with radio-chemotherapy but also identifies PD-L1 status, tumor histology, and lymph node status as prognostic factors [58].

The non-randomized phase 2 study KEYNOTE-799 enrolled eligible patients with previously untreated unresectable stage IIIA/IIIB/IIIC NSCLC. Depending on the squamous/non-squamous histology, the chemotherapy regimen included carboplatin and paclitaxel, respectively, and cisplatin pemetrexed to which pembrolizumab and thoracic radiotherapy were associated in both groups. After the concurrent treatment by radio-chemo-immunotherapy, consolidation treatment with 14 additional cycles of pembrolizumab was administered. The results have a relevant ORR of 70.5% and 70.6% for the group with squamous and non-squamous histology, respectively. Even if the median duration of response was not obtained, a proportion of more than 75% of response at 12 months and an average rate of grade 3 or higher pneumonia of 8% and 6.9% in the group with squamous and non-squamous histology, respectively, the study demonstrates the feasibility of combining pembrolizumab with radiochemotherapy, the adverse effects being considered manageable [59]. A significantly longer PFS and OS for lung patients who previously received radiotherapy is demonstrated by a secondary analysis of the results of the KEYNOTE-001 trial [60].

The efficacy and safety of pembrolizumab are currently evaluated by the KEYNOTE-992 trial in urothelial bladder cancer in association with trimodal treatment including maximal transurethral resection of the bladder followed by radio-chemotherapy [61].

The addition of avelumab (anti-PD-L1) to chemoradiotherapy, the standard treatment in locally advanced unresectable head and neck squamous cell carcinoma (HNSCC) was evaluated in a randomized phase 3 study including 907 cases for the potential to improve the prognosis of these patients. An amount of 10 mg/kg intravenous avelumab every 2 weeks was added to the standard treatment (concurrent chemoradiotherapy with cisplatin 100 mg·m² every 3 weeks and radiotherapy using the intensity-modulated radiotherapy (IMRT) technique in a dose of 70 Gy in 35 daily fractions over 7 weeks. Even if the rate of treatment-related toxicities was not significant, the primary objective of median progression-free survival was not achieved, and the study was discontinued. CD8+ T depletion due to elective nodal irradiation in HNSCC, but also the concurrent therapeutic sequence used in the Javelin Head Neck 100 trial were possible causes of the failure of this trial [62]. Selective depletion of host immune cells, mentioning here the 60% risk 3–4 lymphopenia reported in a trial with a similar design by Wiess et al., but also the surprising flow cytometry results that demonstrated a reduction of CD4 T cells and B cells in the case of the concurrent association of pembrolizumab, but not CD8 T cells, are data that must be taken into account in the design of trials that associate immunotherapy with radiotherapy [63]. Adjuvant nivolumab in resected esophageal or gastroesophageal junction cancer demonstrated benefit in PFS compared to the standard treatment of chemoradiotherapy followed by surgery. A nivolumab dose of 240 mg every 2 weeks for 16 weeks, followed by a dose of 480 mg every 4 weeks was administered for up to one year. A 22.4-month median disease-free survival in the active treatment arm compared to 11 months in the placebo arm represents a net benefit of the adjuvant immunotherapy regimen, but adverse events of 13%, more than double compared to the placebo arm, led to the discontinuation of the trial [58,64,65].

However, the results of the PACIFIC trial are clear evidence of the benefit brought by conventional radiotherapy and standard fractionation, or more precisely, proof of the advantage brought by the triple combination of chemotherapy and radiotherapy. Analyzing the data from the PACIFIC trial, Guo and collaborators note the value of this study that opens a new ERA, but at the same time opened a series of questions and unknown concepts that the future will have to answer. If, regarding the therapeutic sequence, the data supporting the advantage of “consolidation” immunotherapy treatment are confirmed, the study also suggests the optimal time interval, a real “window of opportunity” for immunotherapy administration. From the point of view of the time factor, a “too soon” or “too late” after radio-chemotherapy can tilt the advantage in favor of immunosuppressive factors in the TME, leading to an unfavorable response of the tumor to immunotherapy [61]. Modern radiotherapy techniques, such as three-dimensional conformal radiotherapy (3D-CRT), intensity-modulated radiotherapy (IMRT), and, more recently, volumetric modulated arc therapy (VMAT) and helical tomo-therapy (HT) have brought superior results in term of target volumes coverage with a significant reduction of severe toxicities. In the context created by the inclusion of immunotherapy with durvalumab in the consolidation treatment of NSCLC after the favorable response to the curative treatment with radiation therapy and chemotherapy, a possible future new therapeutic standard for different locally advanced cancers, the spreading effect of small doses associated with modulated intensity techniques deserves increased attention. Thus, static field IMRT and HT were associated with a significant increase in the volumes of healthy tissue receiving between 5 and 10 Gy. The cutoff value of 63 Gy at which the benefit brought by IMRT irradiation in lung cancer was lost suggests that the benefit of IMRT up to this dose was brought exclusively by the reduction of toxicities [66,67]. The volume of healthy tissue receiving doses between 5 Gy and 10 Gy (V5–10) has been correlated with an increased risk of pneumonitis in patients receiving concurrent chemotherapy and radiotherapy, but the study by Tang et al. also draws attention to an associated lymphocyte nadir with high values of these dosimetric parameters [68]. T cell priming phenomenon in draining lymph nodes (DLNs) is mentioned by Darragh et al. as a factor associated with immunosuppression, elective nodal irradiation being a possible factor in the failure of the immunotherapy–radiotherapy association [69]. A reduction in antigen-specific T cells and epitope spreading after ENI justifies the theory of the authors who consider that the failure of the antitumor response to immuno-radiotherapy is counterbalanced by the eradication of micrometastases by radiotherapy in the irradiation of the sentinel node. The use of particle radiotherapy, the new concept of involved field irradiation (IFI), and the omission of the clinical target volume (CTV) may be future strategies to demonstrate therapeutic benefit in these new therapeutic settings [70]. The implications of identifying some predictors of radiation-induced lymphopenia (RIL) are highlighted by the studies that analyze the correlations between the parameters of the multimodal treatment including radiation dosimetry and the decrease in absolute lymphocyte count. (

Table 2. Dosimetric predictors of RIL.

Target Organ/Organ at Risk (OAR)	Radiation Dose/Regimen	Toxicity Reported
Whole brain	24–25 Gy	Long term RIL [71]
Breast	Effective dose to the circulating immune cells (EDIC) of 1.2, 2.1 and 3.7 Gy, respectively	50% of grade 1, grade 2, and grade 3 RIL [72]
Esophagus	Larger planning target volume (PTV), higher heart and body dose, higher EDIC	Lymphocyte nadir after 4-6 weeks of radiotherapy [73]
Brain (glioma), lung, pancreas	Field size, dose per fraction, and fraction number SBRT, Proton therapy	Increased risk of RIL [74] Reduction of RIL risk [74]
Spleen	Volumes receiving at least 5 Gy (V15) and maximum dose (Dmax) an increase of 1 Gy in mean splenic dose	Nadir lymphocyte count [75] 1% decrease in absolute lymphocyte count at nadir [75]
Esophagus	EDIC < 2, 2 to 4, and >4 Gy, respectively	2-year OS rates were 66.7%, 42.7%, and 16.7% [76]

) [71,72,73,74,75,76].

5. Conclusions

Two new effects will govern the future of the immunotherapy–radiotherapy association: the local “in situ” vaccination effect and the systemic remote “abscopal” response. Even if irradiation by stereotactic body irradiation (SBRT) or stereotactic radiosurgery (SRT) seems to be more effective in generating the synergistic effect, the PACIFIC trial demonstrates the role of conventional irradiation in combination with chemotherapy in modulating the host’s immune response. Thus, the radiotherapy–chemotherapy–immunotherapy triad may become the future standard in locally advanced diseases. The different mechanisms of producing immune-mediated cell death and the indirect role of augmenting the immune effect induced by radiotherapy make the old theories related to the therapeutic sequence, fractionation, doses, and target volumes as well as the protection of healthy tissues to be reevaluated. The new concept has as a substrate the possibilities of modulating the immunosuppressive and activating factors of the immune system so as to amplify the anti-tumor immune-mediated tumoricidal response by suppressing the immunosuppressive factors and activating the immune effectors in the TME. The selection of therapeutic targets chosen for the combination of immunotherapy and associated radiotherapy can be conducted based on the classification of tumors in the three immune phenotypes that characterize “cold” and “hot” tumors from the point of view of the response to therapy. The complexity of the synergistic mechanisms that could enhance but also suppress the anti-tumor immune response makes it necessary to translate the preclinical data for each histological subtype of the tumor and combination regimen of immunotherapy with radiotherapy and chemotherapy, as it is obvious that there is no universal solution. Omitting elective irradiation of draining lymph nodes is one of the most promising strategies, but must be approached with caution in order not to compromise loco-regional control.