Stereotactic Radiotherapy combined with Immunotherapy: Augmenting Radiation’s Role in Local and Systemic Treatment

Introduction and Background

Stereotactic Radiation (SRS and SBRT)

Radiation therapy has a long and established history in the field of oncology[1]. The term stereotactic radiation or stereotaxy refers to the use of a referenced three-dimensional coordinate system to locate targets inside the body and deliver highly focused beams of radiation to that site with millimeter accuracy. Swedish neurosurgeon Dr. Lars Leksell was a pioneer of stereotactic neurosurgery and founder of stereotactic radiosurgery[2]. Dr. Leksell invented a stereotactic frame that used a polar coordinate system and a 'center-of-arc' concept which was more versatile than the Cartesian-coordinate system used by previous stereotactic devices[3]. In 1948 Dr. Leksell used his stereotactic system for the first time in the treatment of a patient with a craniopharyngioma where the frame guided injection of radioactive phosphorus into the cyst. Dr. Leksell then introduced the term stereotactic radiosurgery (SRS) in 1951 to describe the use of multiple beams of radiation incident on a single point as a non-invasive method of ablating tissue in the brain, in lieu of traditional surgery[2]. In 1967 he inaugurated the "Gamma-Knife", as it is known today, which uses hundreds of Cobalt-60 sources all focused on a single point stereotactically referenced to the patient's skull using a fixed frame. This device has been updated and modified over the years and is still an important tool today. Importantly, due to its design the Gamma Knife is essentially limited to targeting intracranial lesions. The "Cyber-Knife" by Accuray was developed by Dr. John R. Adler at Stanford University and uses a miniaturized linear accelerator mounted on a general purpose industrial robot to deliver precision radiation with sub-millimeter accuracy. The Cyber-knife designed to deliver stereotactic radiation to any part of the body[4]. Stereotactic delivery of radiation is usually termed stereotactic body radiation therapy (SBRT), and is also termed Stereotactic Ablative Body Radiation Therapy (SABR), although for this review we will use the acronym SBRT. Importantly, SBRT can also be delivered using standard linear accelerators equipped and commissioned for stereotactic treatment.

Because of the accuracy and ability to precisely modify the radiation dose distribution to minimize radiation to normal tissue, stereotactic radiation allows for "hypofractionation" or delivery of very high doses of radiation during each fraction of treatment. In the United States, clinical stereotactic radiation courses are currently completed in 1 to 5 fractions total. This is a dramatic shortening of total treatment time compared to conventionally fractionated radiation which can range from between 2-8 weeks to complete. The ability to effectively treat patient's tumors anywhere in the body with SBRT using high tumoricidal doses while limiting dose to surrounding normal structures has been a major advance and fundamental shift in the field of radiation oncology[5]. Aside from patient convenience and the ability to resume or transition more rapidly to other therapies such as surgery or systemic therapy, there are also important radio-biologic effects of delivering high doses per fraction which are thought to increase the therapeutic ratio of SBRT. Clinically, stereotactic radiation achieves excellent local control rates, for example SRS for AVMs[6], trigeminal neuralgia[7], pituitary adenoma[8], brain metastases[9]. SBRT for early stage lung cancer has local control rates rivaling those of surgery[10]. SBRT is also actively being studied in the treatment of prostate cancer with excellent early control rates, and is being used in numerous other sites including cancers of the pancreas, liver, kidney and CNS,-CNS among others.

Immune stimulatory effects of radiation

Large radiation fields encompassing significant volumes of bone marrow or blood pool have been observed to result in decreases in white blood cell counts, giving rise to the notion that radiation may be generally immunosuppressive[11, 12]. There are even reports of decreased blood counts after SBRT to the lung, although this effect correlated with the amount of vertebral body irradiated and counts began to recover 4 weeks after therapy[13]. Nonetheless, with the application of SRS and SBRT there is the possibility of significantly limiting the volume of bone marrow and/or blood pool being irradiated thereby minimizing these potentially consequential immunosuppressive effects. This significant advancement in radiation technology and capability calls for a re-evaluation of the effects of focused radiation on the immune system.

There is now an established body of pre-clinical literature demonstrating that radiation can modify anti-tumor immune responses. Radiation has been demonstrated to cause upregulation of Major Histocompatibility Complex (MHC) and increase presentation of antigens on surface of tumor cells [14-19]. The DNA damage and reactive oxygen species induced by radiation have been shown to result in inflammatory tumor cell death and release of damage associated molecular patterns (DAMPs), including high mobility group box chromosomal protein 1 (HMGB1), which can activate antigen presenting cells[20-22]. Radiation induced activation of antigen presenting cells has also been demonstrated in animal models to enhance tumor antigen cross-presentation in the draining lymph node and result in activation and proliferation of tumor specific cytotoxic T-cells[18, 23-28]. Radiation has also been shown to influence expression of cytokines and chemokines, such as IL-1, IL-2, L-6, TNF-alpha, TGF-beta, CXCL-16, as well as Type I and Type II Interferons which may play a critical role in modulating immune responses[29-36]. Undoubtedly due to the culmination of these effects, multiple groups have demonstrated that radiation can cause tumor cells to become more susceptible to immune mediated attack[15, 16, 37]. Indeed some authors have suggested that the therapeutic effects of radiation alone may depend on the status of the host immune system and anti-tumor immune responses in the radiation field[25, 34, 38-40]. Given these effects using radiation alone there has been a significant effort to combine radiation with various immunotherapies with sometimes striking results within the radiation field (radiosensitizing immunotherapy), as well as distantly outside the radiation field (abscopal responses)[15, 18, 24, 36, 37, 40-49]. A number of excellent reviews have also highlighted the potential benefits and ongoing clinical trials of radiation combined with immunotherapy[32, 50-54].

Checkpoint Blockade Immunotherapy (CBI) and CAR T-cells

Over the past 5 years cancer immunotherapy has achieved some major successes, including being named as breakthough of the year in 2013 by Science magazine[55]. Multiple types of immunotherapy have garnered significant attention recently including dendritic-cell vaccines, T-cell adoptive transfer, and checkpoint blockade immunotherapy (CBI). The recent momentum began in 2010 when the FDA granted approval to Sipuleucel-T (Provenge, Dendreon Corp.) for minimally-symptomatic metastatic castrate resistant prostate cancer. Notably Sipuleucel-T was the first therapeutic dendritic-cell based vaccine approved for any cancer type. In 2011 the FDA granted approval to the anti-CTLA-4 drug ipilimumab (Yervoy, Bristol-Myers Squibb) for the treatment of unresectable or metastatic melanoma. In Sept 2014 the FDA granted approval to the anti-PD-1 drug pembrolizumab (Keytruda, Merck Sharp & Dohme Corp.) for the treatment of patients with unresectable or metastatic melanoma. In Dec 2014 the FDA granted accelerated approval for the anti-PD-1 drug nivolumab (Opdivo, Bristol-Myers Squibb) for patients with progressive unresectable or metastatic melanoma. On March 5^th 2014, the FDA expanded the approved use for nivolumab to include treatment of patients with advanced (metastatic) squamous non-small cell lung cancer (NSCLC) with progression on or after platinum-based chemotherapy. Notably Opdivo is the first CBI approved for lung cancer. Additional CBI antibodies in development include: Tremelimumab which targets CTLA-4; MPDL3280A which targets the PD-1 ligand (PD-L1); MEDI4736 targeting PD-L1; Lirilumab which targets the killer inhibitory receptor (KIR); and BMS-986016 which targets the checkpoint molecule Lymphocyte-activation gene 3 (LAG-3).

The significant interest in checkpoint blockade immunotherapy (CBI) stems from the dramatic and durable responses observed in a subset of patients with metastatic disease who have been heavily pre-treated. At its core, CBI functions to inhibit negative regulators of immune responses, or in other words removing the brakes on the immune system. For decades immunotherapy approaches have focused on attempting to positively stimulate the immune responses without necessarily addressing the powerful negative regulatory systems that are in place which dampen or prevent excessive immune responses. It is now understood that disabling these negative regulators or checkpoints can result in robust and clinically efficacious immune responses which in some cases can control widely metastatic disease. CTLA-4 (Cytotoxic lymphocyte antigen 4) is a receptor present on the surface of T-cells which binds the co-stimulatory molecules B7-1 and B7-2 on APCs with a much higher affinity than CD28. Instead of transmitting a positive co-stimulatory signal to the T-cell, ligation of CTLA-4 transmits a powerful inhibitory signal to T cells which can block T-cell activation. Indeed, CTLA-4 is one of the most powerful negative regulatory molecules on the cell surface of T-cells[56]. Similarly, Programmed death receptor 1 (PD-1) is a receptor on T-cells which binds PD-L1 or PD-L2 and recruits SHP phosphatases to impose a powerful inhibitory signal on T-cell activation and proliferation. Inhibiting the CTLA-4 and PD-1 pathways using CBI has demonstrated clinical activity in a variety of tumor types including melanoma, lung cancer, renal cancer, bladder cancer, Hodgkin's lymphoma, and prostate cancer [57-66].

At the same time significant progress and clinical responses have been observed with adoptive T-cell transfer. Adoptive T-cell transfer bypasses the requirement for antigen presentation and development of an adaptive immune response, and instead large numbers of effector T-cells are generated ex-vivo and are directly infused into the patient. This therapy has the capacity to eradicate malignant cells, although it is limited by the ability to isolate and expand endogenous antigen specific anti-tumor T-cells, such as from tumor infiltrating lymphocytes (TIL). One strategy to overcome this limitation is to use Chimeric Antigen Receptor (CAR) T-cells. CAR T-cells are engineered to express a specific antibody binding domain connected to transmembrane and intracellular T-cell activation domains that function as an artificial T-cell receptor and allows for MHC independent T-cell activation to a specified epitope. The activity of CAR therapy was demonstrated as a proof of principle for CD19 positive advanced chronic lymphocytic leukemia (CLL), in which the infused T-cells expanded significantly in-vivo and developed into lasting memory CAR T-cells[67]. Subsequent clinical trials have demonstrated activity in neuroblastoma, chronic lymphocytic leukemia, and B cell lymphoma and additional tumor types are under investigation[68]. By bypassing central tolerance and the requirement for MHC mediated T-cell activation, one significant risk of CAR therapy is the potential for fatal unanticipated off-target side effects, which have been reported. Nevertheless, with clinically validated targets CAR therapy represents a powerful tool in the immunotherapy armamentarium against cancer.

Much of the preclinical work combining radiation with CBI has been performed in animal models of melanoma, breast cancer, and colorectal cancer[18, 36, 43-45, 48]. The initial work by Demaria and Formenti established key models for investigating the effects of radiation outside of the field, or abscopal effects. Their pioneering studies used a bilateral flank model where one side was irradiated and the other flank was shielded allowing for analysis of the systemic effects of radiation combined with CBI. Their studies clearly demonstrated that radiation can augment the systemic effects of CBI, and that the abscopal effect was immune mediated. Recently our work demonstrated that antigen specific immune responses can be induced by stereotactic radiation alone or radiation combined with CBI[18]. We also demonstrated using a knockout model that antigen cross presentation may be required for the immune stimulatory effects of radiation[18]. Overall this body of work in melanoma and other tumor types has been highlighted in a number of excellent reviews[53, 54, 69]. Below we discuss the preclinical and clinical data on additional and upcoming sites of investigation, namely: Glioma, Lung Cancer, and Prostate Cancer.

Radiation combined with Checkpoint Blockade in Glioma

Glioblastoma is one of the most common and highly aggressive forms of adult brain cancer. Life expectancy is very poor with a median overall survival of 14.6 months with an approximate 75% mortality rate within the first 2 years and less than 10% of patients surviving more than 5 years[70]. The current standard of care is maximal safe resection followed by adjuvant radiation combined with temozolomide (Temodar), a treatment regimen founded on a randomized phase III trial testing radiation therapy vs. radiation therapy plus temozolomide. In this study, the addition of temozolomide to adjuvant radiation improved survival by 2.5 months[70]. Of note, O-6-methylguanine-DNA methyltransferase (MGMT) is a protein involved in DNA repair and a companion study showed that patients with methylated MGMT derive a statistically significant benefit from adding temodar to radiation compared to patients with unmethylated MGMT in which there was less of a benefit using temodar [71].

Multiple immunotherapeutic approaches are under investigation for the treatment of GBM including: Anti-EGFRvIII CAR T-cells which target the mutant EGFRvIII peptide which is expressed in around one-third of GBM tumors (NCT01454596); Dendritic cell vaccines, DCVax-L (NCT00045968) and ICT-107 (NCT01280552) which use patient specific tumor lysates and synthetic peptides respectively; the IDO inhibitor Indoximod is in a Phase I/II trial (NCT02052648); as well as Rindopepimut (CDX-110) which is a vaccine in Phase III development also targeting EGFRvIII (NCT01480479). Importantly checkpoint blockade immunotherapy is also being studied in GBM, and below we summarize the pre-clinical and clinical studies using radiation and CBI in Glioma.

Pre-clinical Data in Glioma

Zeng et al., published the first study using the small animal radiation research platform (SARRP—which allows for stereotactic radiation delivery) combined with anti-PD-1 immunotherapy[46]. In this study the authors performed intracranial implantation of mouse glioma cell line GL261 to establish an orthotopic Glioblastoma multiforme (GBM) model to investigate the effects of stereotactic radiosurgery combined with anti-PD-1 immunotherapy. Improved survival was observed in the group which received combined treatment with a median survival of 25 days in the control arm, 27 days in the anti-PD-1 antibody arm, 28 days in the radiation arm, and 53 days in the radiation plus anti-PD-1 therapy arm (P<.05 by log-rank Mantle-Cox). Moreover, analysis on day 21 after implantation revealed increased cytotoxic T-cell tumor infiltration and decreased regulatory T cells in tumors of animals in the combined treatment group compared to controls arms. This pre-clinical data provides ample rationale for clinical trials investigating radiation combined with anti-PD-1 immunotherapy for glioma.

Recently, Belcaid et. al, used a GBM model to investigate the effects of stereotactic radiation combined with an 4-1BB (CD137) agonist antibody and CTLA-4 blocking antibody. The authors first showed that stereotactic brain radiation combined with anti-CTLA-4 Ab could prolong survival of mice with orthotopic GL261-luc gliomas. In an important sequencing experiment the first dose of anti-CTLA-4 was given either prior to, during, or after radiation. While the differences were not statistically significant there appeared to be a trend towards concurrent RT + CBI resulting in better survival than initiating CBI after completion of radiation. The authors then showed that by adding an agonist stimulatory antibody, approximately 50% of tumor bearing mice exhibited long term survival. Interestingly, they also showed that radiation and combined immunotherapy increased TIL populations, and that CD4 T-cells appeared to be required for the improvement in survival, with CD8 T-cells providing a modest benefit in this GBM model. These pre-clinical data provide additional rationale for clinical trials combining stereotactic radiation with CBI, including anti-CTLA-4 Ab, in glioma patients.

Clinical Translation in GBM

Given the promising results of these pre-clinical studies a phase I/II trial of anti-PD-1 therapy in relapsed GBM is currently underway (NCT01952769). In this study Pidilizumab (CT-011, Curetech) will be administered to eligible patients at 6.0mg/kg every other week until disease progression or a serious adverse event. Additionally BMS sponsored Phase III randomized open label trial of Nivolumab Versus Bevacizumab and a Safety Study of Nivolumab or Nivolumab in Combination With Ipilimumab in Adults with Recurrent GBM is currently recruiting (NCT02017717). Clearly additional combinatorial therapies are needed to help improve outcomes in this highly lethal disease.

Finally, there is now a Phase 2 study of MEDI4736 (anti-PD-L1) combined with radiation in patients with Glioblastoma (NCT02336165). This multi-center study sponsored by the Ludwig Institute for Cancer Research aims to enroll over 80 patients with GBM into three cohorts. Cohort A is planned to enroll patients with newly diagnosed unmethylated MGMT GBM who will receive MEDI4736 every 2 weeks in combination with standard radiotherapy; Cohort B will include bevacizumab-naïve patients with recurrent GBM who will receive MEDI4736 every 2 weeks as monotherapy; and Cohort C will enroll bevacizumab-refractory patients with recurrent GBM who will receive MEDI4736 every 2 weeks in combination with continued bevacizumab. The primary endpoint includes overall survival and progression free survival. Secondary objectives include safety, tolerability and biological activity as assessed by immunologic markers. This study will investigate the efficacy of anti-PD-L1 monotherapy in GBM patients and radiation combined with anti-PD-L1 in patients with newly diagnosed GBM with unmethylated MGMT.

Radiation combined with Immunotherapy in Lung Cancer

Lung cancer is the number one cause of cancer related mortality in men and women. The majority of patients present after they become symptomatic and are diagnosed with advanced stage disease. On Feb 2^nd 2015 the Centers for Medicare & Medicaid Services (CMS) issued a final national coverage determination stating that Medicare will now cover annual Low Dose Computed Tomography (LDCT) for Lung Cancer Screening for patients who meet the following criteria: age 55-77, either current smokers or have quit smoking within the last 15 years, and have a tobacco smoking history of at least 30 pack years. This determination was based in part off the results of the National Lung Screening Trial (NLST) which reported a 20% relative reduction in lung cancer mortality with the use of LDCT screening compared to chest x-ray[72]. As with other national screening programs for cancer there is the possibility for a relative increase in the percentage of lung cancer patients diagnosed with early stage disease. The primary treatment for early stage lung cancer is surgical resection, although definitive SBRT is the preferred treatment option for medically inoperable patients or those with poor performance status. Definitive SBRT for early stage lung cancer has excellent local control rates which are similar to those of conventional surgery[10]. Thus, with the use of LDCT it is possible that SBRT will play an even more prominent role in lung cancer treatment in the future.

Systemic treatments for non-small cell lung cancer (NSCLC) have been advancing rapidly with multiple different agents approved for specific molecular subtypes, including four targeted therapies approved since 2011. While traditionally thought to be poorly immunogenic, lung cancer is now one of the most active areas of investigation for immunotherapy, especially CBI[73]. Indeed, Opdivo is now the first CBI approved to treat lung cancer and is FDA approved for advanced (metastatic) squamous non-small cell lung cancer (NSCLC) with progression on or after platinum-based chemotherapy. It is speculated that the high mutational burden induced by the carcinogens in smoke may result in numerous neo-antigens that the immune system can respond to once the tumor mediated immunosuppression is blocked. A search identified at least 26 separate NIH clinical trials currently investigating various CBI antibodies in some phase of investigation for treatment of lung cancer. Interestingly, while radiation plays a key role in the management of lung cancer, very few of these studies incorporate radiation. Below we discuss the pre-clinical data and clinical trials of hypofractionated radiation combined with immunotherapy in lung cancer.

Pre-clinical data in Lung Cancer

Fotin-Mleczek et. al, recently published a study combining mRNA based vaccination with radiation in the syngeneic Lewis lung cancer (LLC) model[74]. The authors reported that the combination of mRNA-based immunotherapy with radiation resulted in a strong synergistic anti-tumor effect. Consistent with prior studies they reported that radiation and radiation combined with immunotherapy induced a number of distinct changes in gene expression at the tumor site, including upregulation of genes involved in antigen presentation, adhesion and immune cell infiltration, as well as activation of the innate immune system[74]. Analysis of LLC tumors after combined treatment with 12Gy x3 demonstrated increases in immune cell infiltrates including CD4 and CD8 positive T-cells, CD8 positive Dendritic cells, and NKT cells[74]. Given the multiple immune stimulatory effects of radiation reported in this study, these results provide mechanistic rationale for clinical trials combining radiation with immunotherapy in lung cancer.

Our group has shown that a combination of PD-L1 blockade and irradiation in an autochthonous model of NSCLC significantly improved local tumor response in the irradiated lung( A. Walker, A. Sharabi, C. Drake, P.T. Tran, unpublished data). We also demonstrated evidence of partial tumor response in the unirradiated lung. An increase in T cells was observed in both irradiated and unirradiated lungs after radiation and checkpoint blockade consistent with an abscopal effect ( A. Walker, A. Sharabi, C. Drake, P.T. Tran, unpublished data). These preclinical findings support clinical trials combining SBRT with CBI in patients with NSCLC.

Clinical translation in Lung Cancer

New York University has developed a phase II study in patients with metastatic non-small cell lung cancer (NSCLC) exploring the combination of radiation and Ipilimumab testing whether this combination can potentiate the immune system to produce an abscopal response (NCT02221739). Patients receive ipilimumab 3mg/kg within 24 hours of starting hypofractionated radiotherapy (RT - 6 Gy x5). Ipilimumab dosing is repeated on days 22, 43 and 64. The primary goal is to evaluate the safety and therapeutic efficacy of anti-CTLA-4 mAb and concurrent local RT in NSCLC patients with metastatic disease. The secondary outcome includes the effects of RT and anti-CTLA-4 mAb on development of anti-tumor immunity.

MD Anderson has also developed a phase I/II Trial of Ipilimumab and Hypofractionated Stereotactic Radiation Therapy in Patients With Advanced Solid Malignancies (NCT02239900). The primary outcome of this study is the maximum tolerated dose (MTD) of Ipilimumab and SBRT. Secondary outcome measures include the effects of RT and anti-CTLA-4 on development of anti-tumor immunity. Patients will receive Ipilimumab (3 mg/kg) combined with SBRT given at 50 Gy in 4 fractions. Importantly, the effect of timing and sequencing will also be investigated with concurrent and sequential treatment groups. These clinical trials and others like them will help to determine whether the synergistic pre-clinical effects observed with radiation combined with CBI can translate into patients and improve clinical outcomes in lung cancer and patients with lung metastases.

Radiation combined with Immunotherapy in Prostate Cancer

Prostate cancer is the most common cancer diagnosed in men in the US and second leading cause of cancer related mortality. The vast majority of patients are diagnosed at early stages when the disease is organ confined. For those men who require therapy, the primary therapeutic treatment approaches are surgical resection or a course of radiation therapy with or without androgen deprivation therapy depending on risk stratification. Although conventionally fractionated external beam radiation or brachytherapy are the standard radiation treatment options, SBRT is under active study for definitive treatment of organ confined prostate cancer. Long term toxicity and outcomes data for Prostate SBRT are being collected and are maturing.

It is notable that the first FDA approved autologous cell-based vaccine to treat any cancer was sipuleucel-T (Provenge, Dendreon Corp.) for treatment of men with hormone-refractory metastatic prostate cancer. Sipuleucel-T is a dendritic cell vaccine generated from autologous peripheral blood mononuclear cells and is designed to induce an immune response against prostatic acid phosphatase (PAP). It was approved by the FDA in 2010 for treatment of minimally-symptomatic metastatic castrate resistant prostate cancer (CRPC) based results of a randomized double blind placebo control phase III trial which showed an average survival improvement 4.1 months along with very minimal side effect profile[75]. Interestingly recent followup data has identified that additional antigen specific immune responses to epitopes not specifically targeted by the vaccine, including PSA and LGALS3, can occur with sipuleucel-T and correlate with improved outcomes[76]. Numerous additional vaccines and combination immunotherapies are being investigated for metastatic CRPC, including: sipuleucel-T (Provenge) with enzalutamide (NCT01981122); sipuleucel-T (Provenge) and indoximod (NCT01560923), adoptive transfer of NY-ESO-1 specific T-cells after chemotherapy (NCT01967823); PROSTVAC-VF vaccina and fowlpox vaccine (NCT01322490), as well as a Neoadjuvant Study of Androgen Ablation Combined With Cyclophosphamide and GVAX Vaccine for Localized Prostate Cancer (NCT01696877). Below we discuss some of the pre-clinical and clinical data combining radiation with immunotherapy in prostate cancer.

Pre-clinical data in Prostate Cancer

Harris, et. al, initially studied the timing and sequencing of immunotherapy and radiotherapy in transgenic mice that develop spontaneous prostate cancer (TRAMP) using an autochthonous model treated with a vaccina based tumor vaccine[24]. In this study Pro-HA-TRAMP mice engineered to express the prostate-restricted hemaglutinin (HA) antigen were used to study the effects of a single high dose of radiation and a recombinant Vaccinia-HA tumor vaccine on adoptive transfer of HA specific CD4 T-cells. In this study, our group reported that radiation combined with vaccine immunotherapy mitigated tumor-mediated T cell tolerance resulting in anti-tumor T cell activation. Importantly, this effect was critically dependent on the timing of RT and immunotherapy with anti-tumor immune responses observed when immunotherapy was administered 3-5 weeks post-RT, but not when immunotherapy was administered either prior to or later than 5 weeks post-RT. This study highlighted many important considerations for sequencing and timing of radiation combined with immunotherapy for prostate cancer.

In a subsequent paper by Wada S., et al, the Pro-HA autochthonous TRAMP mouse model was used to study the effect of stereotactic radiation combined with a granulocyte/macrophage colonystimulating factor-secreting cellular immunotherapy for prostate cancer (T-GVAX)[47]. In this study our group used the SARRP to deliver focused stereotactic radiation of 6Gy x2 fractions to the prostate. We reported the combined therapy resulted in improved overall survival in a preventive metastasis model[47]. This same study used a micrometastasis model to show that combination therapy could mediate a modest but significant systemic antitumor immunity. It was also observed that combined therapy resulted in an increase of the ratio of effector-to-regulatory T cells for both CD4 and CD8 tumor-infiltrating lymphocytes. Similar data have subsequently been reported in numerous studies demonstrating that radiation can enhance immune cell infiltration into tumors. These preclinical data provided ample rational for subsequent clinical trials combining radiation with immunotherapy in prostate cancer.

Clinical Translation in Prostate Cancer

Gully, et al., reported results of a randomized phase II clinical trial designed to determine if a poxviral vaccine encoding prostate-specific antigen (PSA) could induce PSA-specific T-cell responses when combined with radiotherapy in patients with clinically localized prostate cancer[77]. Thirty patients were randomized to radiation alone versus radiation plus vaccine, with the latter group received "priming" vaccines with recombinant vaccinia PSA (rV-PSA) and recombinant vaccinia containing the costimulatory molecule B7.1 (rV-B7.1) followed by monthly booster vaccines with recombinant fowlpox PSA. Standard external beam radiation therapy was given between the fourth and the sixth vaccinations. The authors reported evidence of de novo generation of T cells specific for prostate-associated antigens not present in the vaccine[77]. They argued that this epitope spreading was indirect evidence of immune-mediated tumor killing. The effects of radiation combined with a poxvirus-based vaccine encoding prostate-specific antigen were further analyzed by Nesslinger et al., in patients with localized prostate cancer. In this study, pre- and post-treatment serum samples from a previously reported Phase II clinical trial of patients treated with radiation combined with vaccine or patients treated with radiation alone were evaluated by Western blot and serologic screening. They reported that external beam radiation combined with vaccination against PSA resulted in epitope spreading and immune responses to additional tumor-associated antigens[78].

A Phase I/II study in patients with metastatic castration-resistant prostate cancer (mCRPC) explored ipilimumab treatment alone or in combination with 8 Gy radiation given in a single fraction to metastatic lesions[79]. The study tested the hypothesis that ipilimumab would potentiate antitumor immunity following radiation by converting the tumor into an in situ vaccine via induction of immunogenic cell death and exposure of cryptic tumor antigens[80]. In the highest dose group of 10 mg/kg, 8 of 50 patients, exhibited a PSA decline of at least 50%, and 1 patient experienced a complete response, while 6 patients had stable disease. Kwon, et al, recently reported long term follow up of the large multi-institutional, prospective, phase III randomized trial of radiation combined ipilimumab. This phase III study of men with mCRPC prostate cancer was very close to meeting its primary endpoint of improved median overall survival with a p=0.053 [64]. However, in a subset analysis there appeared to be clinically meaningful benefit in men without good performance status without visceral metastases. A subsequent clinical trial is enrolling men without visceral metastases[81].

Finally the ProstAtak™ trial is a national phase III placebo controlled clinical trial incorporating a replication-defective adenovirus (AdV) combined with radiation in the definitive setting (NCT01436968). This trial employs an intraprostatic injection of AdV to deliver the herpes virus thymidine kinase (HSV-tk) gene directly to prostate cancer cells. The oral anti-herpetic drug, valacyclovir (Valtrex), is then administered as a prodrug which is phosphorylated by HSV-tk to form a cytotoxic nucleotide analog which results in termination of DNA replication and, ultimately, cell death. AdV-tk is given concurrently with standard of care external beam radiation for intermediate to high risk prostate cancer. The primary outcome is disease free survival of the ProstAtak™ arm versus the placebo control arm and secondary endpoints concerned with immune responses are being collected. In part, this trial is supported by a study by Chhikara, et al, in which immunocompetent mice bearing the mouse prostate tumor, RM-1, were treated with direct intratumoral injections of AdV-tk followed by ganciclovir and 5 Gy of radiation. These mice exhibited a significantly greater CD-4 infiltrate in tumors compared to mice treated with AdV-tk alone or radiation alone. Mice also received tail vein injection of RM-1 cells at the same time RM-1 xenografts were established. Xenografts were treated with AdV-tk, radiation, or a combination of both. Mice treated with a combination of AdV-tk and radiation exhibited a significantly reduced number of lung metastasis compared to either unimodal therapy suggesting that the combination therapy resulted in a systemic anti-tumor immune response[82].

Conclusions and Future Directions

Ever accumulating preclinical data have documented that immunotherapy can augment radiation-mediated local tumor response. Similarly, radiation can augment the systemic effects of immunotherapy. Most preclinical data to date have studied high doses of radiation that, when translated to the clinic, may be best delivered with SBRT/SABR. These data also support much of the on-going clinical work including that CBI is demonstrating clinical activity in a wide variety of tumor types as is adoptive T-cell transfer and certain cancer vaccines in tumors with known antigenic targets. One of the primary questions to answer is how to best incorporate immunotherapy into current definitive and palliative radiation treatment regimens. There is now an established body of literature demonstrating that radiation has the potential to enhance local and systemic anti-tumor immune responses and is thus an ideal modality to combine with immunotherapy. Additional questions remain regarding the optimal total dose, fraction size, field size, and scheduling for induction of immune responses. Thus carefully designed studies will be required to investigate the effects of radiation combined with immunotherapy on local tumor control in the definitive setting and on systemic tumor control in the metastatic setting.

Figure 1. Potential immune stimulatory effects of SBRT for lung tumors.

SBRT induces inflammatory cell death, activation of DC and antigen presentation in the draining lymph node, resulting in antigen specific adaptive immune responses.

Figure 2. Potential immune stimulatory effects of stereotactic radiosurgery (SRS).

SRS may result in localized breakdown and/or permability of the blood brain barrier (BBB) enhancing drug delivery and immune cell infiltration. Radiation may also upregulate checkpoints and thus checkpoint blockade may be key to harnessing the immune stimulatory effects of radiation while mitigating any immune-suppressive effects of radiation.