Harnessing radiation to improve immunotherapy: better with particles?

Abstract

The combination of radiotherapy and immunotherapy is one of the most promising strategies for cancer treatment. Recent clinical results support the pre-clinical experiments pointing to a benefit for the combined treatment in metastatic patients. Charged particle therapy (using protons or heavier ions) is considered one of the most advanced radiotherapy techniques, but its cost remains higher than conventional X-ray therapy. The most important question to be addressed to justify a more widespread use of particle therapy is whether they can be more effective than X-rays in combination with immunotherapy. Protons and heavy ions have physical advantages compared to X-rays that lead to a reduced damage to the immune cells, that are required for an effective immune response. Moreover, densely ionizing radiation may have biological advantages, due to different cell death pathways and release of cytokine mediators of inflammation. We will discuss results in esophageal cancer patients showing that charged particles can reduce the damage to blood lymphocytes compared to X-rays, and preliminary in vitro studies pointing to an increased release of immune-stimulating cytokines after heavy ion exposure. Pre-clinical and clinical studies are ongoing to test these hypotheses.

Introduction

Radiotherapy is an effective and largely used treatment to achieve local control of solid tumors.¹ Conventional radiotherapy use X-rays generated by electron accelerators (linacs), but high-energy charged particles produced by larger cyclotrons or synchrotrons improve precision of dose delivery and enable to spare more normal tissue compared to photons.² Around 170,000 patients have been treated worldwide with protons, and 25,000 with C-ions (source: PTCOG, www.ptcog.ch). This remains a small fraction compared to the millions of patients treated with conventional X-rays. The main reason for this difference is the high cost of the particle accelerators compared to conventional linacs.³ Currently, the cost effectiveness of particle therapy remains under scrutiny.⁴

Even with high local control rates achievable with particle therapy, this advantage fails to translate in improved cancer survival in most cancers, because of distant metastasis. Local radiotherapy can trigger an immune response that can immunize the host, leading to immune destruction of distant, unirradiated metastasis.⁵ This phenomenon, described as an abscopal effect, is rare and the mechanisms are unclear.⁶

In recent years the introduction of checkpoint inhibitors has proven a breakthrough strategy, capable to induce powerful tumor rejections and improved survival in metastatic patients.⁷ Excellent results have been obtained with immune checkpoint inhibitors such as anti-CTLA4 and anti-PD1 antibodies in malignant melanoma.⁸ However, severe immune-related side-effects complicate the use of immunotherapy and limits its use in cancer patients.⁹

It is widely acknowledged that the combination of immunotherapy with local therapies can further improve the survival in most solid cancers.¹⁰ Because radiation has the potential to activate an anti tumor immune response,¹¹ it is an optimal candidate for combinations with immunotherapy.^12–18 Animal experiments have shown that the superior activity of radiation and dual immune checkpoint blockade is mediated by non-redundant immune mechanisms in cancer.¹⁹ Following initial positive results of this combination,^20–22 hundreds of trials have been launched to test safety and efficacy of radiation and immunotherapy in different tumors types, including lung cancer,²³ the first cause of cancer-related death in United States for both males and females.²⁴ The PACIFIC trial has shown significant improvements in disease free survival²⁵ and overall survival²⁶ in stage III non-small-cell lung cancer (NSCLC) patients treated with Durvalumab after chemoradiotherapy. Prospective randomized trials in stage IV patients are still missing and will be essential to quantify the benefit of combined radiotherapy and immunotherapy in lung cancer. In the pilot/feasibility trial NCT02221739 at Weill Cornell Medicine, a 30% disease control was achieved in stage IV NSCLC patients refractory to anti CTLA-4 alone or in combination with chemotherapy by combining Ipilimumab with focal hypofractionated radiotherapy of a single metastasis.²⁷ Several strategies are under study to further improve these results, including modifying the dose per fraction²⁸ and the number of metastasis irradiated.²⁹

Considering the success and promise of the combination of X-rays with checkpoint inhibitors, the question is whether particle therapy can present additional advantages, and result in better outcomes.³⁰ This question is relevant to the future of particle therapy, in view of its higher cost. We will discuss here the potential physical and biological advantages of particle therapy in combination with immunotherapy.

Physical advantages

Particle therapy is based on the different depth-dose distribution of charged particles compared to photons.³¹ Using protons or heavier ions, much more normal tissue can be spared in virtually every tumor site, while preserving dose conformality on the tumor (see e.g. Figure 1 for esophageal cancer³²). Sparing of the normal tissue, and in particular reducing the exposure of circulating T-lymphocytes and other immune cells, present advantages to immunotherapy. Non-proliferating peripheral blood lymphocytes are very radiosensitive³³ and during radiotherapy lymphopenia may occur, which is often correlated to a negative prognosis.³⁴ In fact, both radiotherapy and chemotherapy damage circulating immune cells,³⁵ compromising the host’s immunity.³⁶ Consistently, blocking immune response in mice prevents tumor control when radiation therapy is combined with checkpoint immunotherapy.³⁷

Figure 1. .

Coronal and transverse images of 3D-CRT plan (left), IMRT plan (middle), and proton plan (right) for esophageal cancer. The plans clearly show the large tissue sparing using protons. Treatment plans produced at Loma Linda University Medical Center (CA) details in ref.,³² reproduced with permissions.

Damage to peripheral blood lymphocytes can be measured during radiotherapy using the chromosomal aberration assay (Figure 2). When lymphocytes are irradiated in vitro, charged particles induce more chromosomal aberrations than X-rays at the same dose level, and the relative biological effectiveness (RBE) increases by increasing the particle linear energy transfer (LET).³⁸ However, in vivo, the damage mainly depends on the size of the irradiated volume (or integral dose), and on the presence of lymph nodes in the field.³⁹ For sites such as the esophagus, an increased field size leads to a proportional increase of the lymph nodes exposed. In fact, the chromosomal aberrations detected in circulating lymphocytes in esophageal cancer patients are proportional to the radiotherapy target volume.⁴⁰ As a result of the reduced normal tissue volume irradiated with particles (Figure 1), less chromosomal aberrations were measured in patients treated with C-ions than with X-rays for esophageal cancer,.⁴¹ Similar results were reported for patients treated with C-ions or IMRT for prostate cancer.^42,43

Figure 2. .

Damage in peripheral blood lymphocytes of a patient during the course of radiotherapy. The karyotype by mFISH shows a translocation involving chromosomes 4 and 8.

Damaged lymphocytes eventually die and can lead to lymphopenia. The absolute lymphocyte count decreases during the course of radiotherapy. In sites like the esophagus, it can be expected that the use of protons reduce s the risk of severe lymphopenia.⁴⁴ In fact, for the decrease in lymphocyte counts during the radiotherapy course is less pronounced using protons⁴⁵ or C-ions⁴¹ than with X-rays (Figure 3). Even if the data in Figure 3 come from different centers and different chemotherapy drugs, the tendency to spare more effectively lymphocytes using particles is supported for esophageal cancer patients. These results suggest that the physical characteristics of charged particle lead to sparing of immune cells that can then be engaged in the immune response triggered by immunotherapy.

Figure 3. .

Median values of lymphocyte count in esophageal cancer patients during the course of radiotherapy. Data for protons (1.8 Gy RBE/fraction) and IMRT (1.8 Gy/fraction) are from ref.,⁴⁵ data for 3DCRT (1.6–2.0 Gy/fraction) and C-ions (2.7–3.6 Gy RBE/fraction) from ref.⁴¹

While a reduced lymphopenia is certainly an advantage for the patients, whether lymphocyte sparing boosts the immune response remains to be demonstrated. In esophageal cancer patients receiving pre-operative chemotherapy, a significant increases in T-cell receptor diversity was observed in peripheral T-cells but not in tumor-infiltrating lymphocytes.⁴⁶ In general, chemotherapy-induced depletion of immune cells can be followed by a ‘rebound overshoot’ effect, leading to transiently enlarged immune cell numbers, then relaxing to normality.⁴⁷ Whether this transient excess of immune cells supports immunotherapy or not remains to be clarified, and is certainly important to clarify the potential physical advantages of charged particles in radioimmunotherapy.

Biological advantages

In addition to the physical advantages, charged particles have different biological effects compared to X-rays, caused by the different kind of DNA lesions induced by densely ionizing radiation.⁴⁸ High-LET radiation induce more clustered DNA lesions, that are difficult to repair,⁴⁹ and that trigger different DNA damage repair signals.^50–52

DNA repair signaling pathways are strongly related to the immune response. Recently, it has been directly shown that that PD-L1 expression in cancer cells is upregulated in response to DNA double-strand breakage, through the ATM/ATR/Chk1 kinase pathway.⁵³ Similar upregulation of PD-L1 has been recently shown in melanoma cells exposed to UV radiation.⁵⁴ Activation of different DNA damage response pathways at high-LET, such as resection,⁵⁵ may have different effects on the expression of immune receptors. It presently not known whether the radiation-induced upregulation of PD-L1 will actually translate into response to checkpoint inhibitors.⁵⁶

DNA damage eventually leads to cell death through different pathways (such as apoptosis, necrosis, mitotic catastrophe or senescence),^57,58 and to the consequent release of small molecules such as ATP, calreticulin, and HMGB1⁵⁹ that can trigger the immune response (Figure 4).⁶⁰ In vitro studies with different human tumor cell lines have shown that proton radiation induces calreticulin cell-surface expression, increasing sensitivity to cytotoxic T-lymphocyte killing of tumor cells.⁶¹ Increased extracellular concentration of HMGB1 has been measured after irradiation of human cancer cells with heavy ions,⁶² and the release is positively correlated to the particle LET (Figure 5). HMGB1 is upregulated in the serum of esophageal cancer patients following chemoradiotherapy,⁶³ and these results are therefore suggestive of an increased efficiency of densely ionizing radiation compared to X-rays.

Figure 4. .

An illustration of immune-mediated effects of ionizing radiation. The green arrow points to the release of HGMB1, that interacts with the toll-like receptor TLR4 activating the dendritic cell maturation. Cartoon from ref.,⁶⁰ reproduced with permission.

Figure 5. .

Concentration of HGMB1 in the medium following irradiation of HeLa cells with sham (0 Gy) radiation (black), 4 Gy C-ions in the plateau region of the Bragg curve, 13 keV/μm (red) and 2 Gy C-ions in the spread-out-Bragg-peak, 70 keV/μm (green). The two doses were chosen to achieve the same 10% survival in irradiated HeLa cells. Data from ref.⁶²

In addition to the damage to nuclear DNA, recent evidence points to the sensing of cytoplasmic DNA as a key factor in eliciting immune response.⁶⁴ Double stranded DNA (dsDNA) fragments generated by exposure to sparsely ionizing radiation are extruded from the nucleus^65,66 and accumulate in the cytosol where dsDNA sensors, cGAS/STING are activated to transcribe type I interferon genes and jumpstart an immune response. Interferon is a powerful promoter of dendritic cells recruitment and activation, enabling cross-presentation of radiation induced neoantigens and immune response.^67,68 Consistently, metastatic NSCLC patients responding to focal radiation and CTLA4 blockade demonstrated an increased serum level of interferon-βcompared to baseline.²⁷ These results suggest that the cGAS/STING activation may be an essential pathway for a successful combination of radiation and immunotherapy.^69,70 Increased cytosolic DNA is observed after irradiation of human cancer cells, but at high doses per fraction (>10 Gy) cells activate the DNA exonuclease Trex1, that degrades cytosolic DNA thus blocking the STING activation.⁷¹ Type-I interferon activation induced by radiation therefore reflects a balance between sufficient dsDNA induction to stimulate cGAS/STING and Trex1 activation.⁷² How this pathway is affected by charged particles remains unknown, and experiments are needed to clarify whether the production of smaller DNA fragments by densely ionizing radiation can lead to an enhanced response.⁷³

Preliminary animal studies have shown that charged particles effectively induce abscopal responses for instance by reducing lung metastasis after irradiation of the primary tumor.^74–76 However, these results remain inconclusive and a direct comparison of X-rays, protons, and C-ions is warranted.⁷⁷

Conclusions

The potential advantages in using protons or heavy ions in combination with immunotherapy are based on both their physical and biological properties, warranting experimental verification. Since many pre-clinical and clinical trials are ongoing, it is expected that within a few years the role of charged particles for a combined use with modern immunotherapy of cancer will be defined. Several new proton therapy centers are under construction in Europe, and many of these centers will dedicate “beamtime” to radiobiology research.⁷⁸ Often these research efforts are directed to measurements of proton RBE,⁷⁹ but one of the key questions to be answered is whether particles are more effective than X-rays when used in combination with immunotherapy.³⁰ Considering the high cost of the proton or heavy ion therapy facilities and the success of radioimmunotherapy trials, this research is perhaps the most important to decide the future of particle therapy.