Radiofrequency Ablation in Cancer Therapy: Tuning in ... - Springer Link

Chapter 3

Radiofrequency Ablation in Cancer Therapy: Tuning in to in situ Tumor Vaccines Stefan Nierkens, Martijn H. den Brok, Theo J. Ruers and Gosse J. Adema

Abstract Radiofrequency ablation (RFA) is a minimally invasive therapy for the local destruction of primary tumors and unresectable metastases, primarily in the liver. The clinical efficacy of RFA is mainly determined by the destruction of tumor mass. However, after ablation tumor antigens become instantly available for leucocytes, and the ablation procedure creates an inflammatory environment that may contribute to stimulate innate and adaptive anti-tumor immunity. Unfortunately, immune responses induced by RFA are only occasionally strong enough to lead to spontaneous regression of tumors. Combination of tumor debulking by RFA with immune stimulatory approaches that increase antigen presentation and induction of anti-tumor T cell reactivity is a promising strategy to prevent local recurrences and to induce long-term systemic protection against residual disease. Keywords Radiofrequency ablation · Cancer · Immunotherapy · In situ tumor destruction · Combination therapy · Danger associated molecular patterns · Antigen presentation · Immunogenic cell death · Dendritic cell · T cell · Hepatocellular carcinoma · Tumor-associated antigen · Immune regulation

G. J. Adema () · S. Nierkens · M. H. den Brok Department of Tumor Immunology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB, Nijmegen, The Netherlands e-mail: [email protected] T. J. Ruers Department of Surgery, The Netherlands Cancer Institute, Amsterdam and MIRA Research Institute, Twente, The Netherlands S. Nierkens Department of Immunology, University Medical Center Utrecht, Utrecht, The Netherlands

Y. Keisari (eds.), Tumor Ablation, The Tumor Microenvironment 5, DOI 10.1007/978-94-007-4694-7_3, © Springer Science+Business Media Dordrecht 2013

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3.1

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Introduction

Radiofrequency ablation (RFA) is a minimally invasive therapy for the local destruction of primary and metastatic tumors, primarily of the liver. This technique encompasses low morbidity and mortality rates and causes tumor destruction with no significant damage to normal liver parenchyma. In patients with hepatocellular carcinoma (HCC), RFA is still inferior to surgical resection concerning tumor recurrence rates and the establishment of disease-free and overall survival [1]. RFA is however the preferred alternative treatment option when surgical resection of HCC is not possible due to number or location of tumors, a poor general condition of the patient, or in patients with limited liver function. It has shown superior efficacy in comparison with other alternative treatments, such as percutaneous ethanol injections [1]. In a selected group of patients (child–pugh class B, multiple HCC, or in patients with HCC≤ 3 cm), RFA seems similarly effective when compared to surgical resection [2], emphasizing that the careful selection of eligible patients is imperative. The reported 3- and 5-year survival rates after RFA of HCC are 45–62 % and 33–40 %, respectively [3–5]. Giovanni et al. reported a remarkably successful cumulative 3-year survival rate of 94 % and a 3-year disease-free survival of 70 % [6], emphasizing the potential of this treatment in selected patients. Besides its relevance for the treatment of HCC patients, RFA is also increasingly applied as a primary treatment option for patients suffering from unresectable liver metastasis from colorectal cancer. In a retrospective analysis of these patients, Abdalla and colleagues [7] found a statistically significant better survival rate for patients treated with RFA alone or in combination with resection versus chemotherapy only. Another study reported an overall survival at 3 and 5 years of 42 and 30.5 %, respectively [8] and concluded that RFA can contribute to encouraging long term survival and appears to confer a survival benefit over systemic therapy alone. In addition, RFA increased the percentage of curative local treatments for liver recurrence after hepatectomy [9]. Beneficial responses of RFA have also been observed in other metastatic liver tumors, such as neuroendocrine tumors [10] and primary intrahepatic cholangiocellular carcinoma [11], as well as in other cancers, such as lymphoma [12], head and neck cancer [13], prostate cancer [14], primary and metastatic tumors of the lung [15], breast cancer [16], bone metastases [17], and small renal cell carcinoma [18]. The clinical efficacy of RFA is not only derived from the destruction of tumor mass. After ablation tumor antigens become instantly available for antigen presenting cells (APCs), and the procedure itself creates an inflammatory environment that may further the initiation of anti-tumor immunity. The involvement of immunological phenomena after RFA suggests that combining this technique with immunotherapy may be promising to prevent local recurrences and induce long-term systemic protection against residual disease.

3 Radiofrequency Ablation in Cancer Therapy: Tuning in to in situ Tumor Vaccines

3.2

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RFA Techniques/Parameters and Tumor Cell Death

RFA uses electromagnetic energy sources to generate heat. Electrode probes are placed within tumors percutaneously or during open or laparoscopic surgery. The target tissue receives high-frequency (375–500 kHz) electromagnetic waves that displaces molecules within the tissues in alternating directions causing ionic agitation and friction that locally generates heat. Tissue injury following this procedure can be subdivided in two distinct phenomena, resulting in direct and indirect effects.

3.2.1

Direct Effects

Temperatures higher than 100–300 ◦ C immediately next to the electrode results in carbonized tissue, which (if applied) represents the first (application zone) of four zones indicated by morphological changes in the affected tissue. Carbonization may limit tumor dissemination by creating a heat trap. It is therefore essential to achieve and maintain cytotoxic temperatures (below 100 ◦ C) throughout the entire target volume [19]. Between 60 and 100 ◦ C cell membranes fuse, proteins coagulate and coagulative necrosis is induced: the so-called central zone. The transition zone surrounding the central zone contains cells in the earliest stages of death, showing a loss of enzymatic activity, impaired membrane function and structural changes in mitochondria. This zone is morphologically hemorrhagic and infiltrated with leukocytes. These ‘direct effects’ of focal hyperthermia depend on the temperature attained in the tissue, which is determined by the total thermal energy applied, the decline rate of heat, and the specific thermal sensitivity of the tissue (reviewed in [20]). The surrounding normal unaffected tissue is dedicated as the reference zone [21]. Tumor cells are more susceptible to heat and are destroyed at lower temperatures than normal cells (for detailed information we refer to the excellent review by Nirfarjam et al. [20]). At temperatures approaching 45 ◦ C oxygen consumption rises and tumor cells suffer from metabolic stresses due to lower mitochondrial levels, reduced ATP and lower oxygen intake compared to normal tissue. In addition, tumor vessels show reduced ability to augment blood flow by vasodilatation, which limits heat dissipation away from the tumor by absorption to hemoglobin in erythrocytes, and are thus more likely to undergo irreversible injury. Furthermore tissue hypoxia and a low pH have been attributed to the increased thermal sensitivity of tumor cells. Insufficient vascular drainage of the thermo-induced increasing levels of metabolites and cellular injury causes a drop in pH, which in turn increases thermosensitivity. The increased susceptibility of tumor cells to heat treatment seems independent of the proliferation rate, absorbance or benign/malignant nature of the cells.

3.2.2

Indirect Effects

Tissue damage progresses after the cessation of focal RFA treatment resulting in ‘indirect heat injury’. These processes appear to be independent of the initial thermal

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effect and may be a major determinant of completeness of tumor ablation [22]. RFA creates a temperature gradient that progressively decreases away from the site of probe insertion. In areas where temperatures remain below 60 ◦ C, the beforementioned transition zone, cells do not undergo direct hyperthermia-induced death, but they suffer from thermal stress. This initiates increased lysosymal activation and mitochondrial damage, which can lead to apoptosis of the tumor cells. The peak of apoptosis in this zone is observed two hours following hyperthermia [23]. According to the definition, apoptosis includes the elimination of the apoptotic cell by heterolytic degradation following phagocytosis by APCs. An alternative and largely underestimated outcome of apoptosis is secondary necrosis. In this process the cell disintegrates with release of degraded cellular components without intervention of scavengers. Secondary necrosis is a mode of cell elimination with specific molecular and morphological features and can be considered the natural outcome of cells that complete the full apoptotic program [23]. The distinct spatial distribution of each type of cell death in an RFA lesion is difficult to determine histochemically and critically relies on demonstrating ceased enzyme activity. RFA treatment most likely results in a patchwork of apoptosis and primary or secondary necrosis. It has been postulated that RFA may result in live single circulating tumor cells, able to disseminate to other locations. Circulating tumor cells in patients with hepatic metastases from colorectal cancer have been identified before and after resection. It is however controversial whether the numbers of cells after ablative strategies are higher compared to other conventional techniques [24–26]. The technical development of probe design is a fast evolving field. In addition, visualization methods for needle guidance, as well as real-time monitoring and controlling of the thermal ablation progress are subject of intensive research [27–29]. The incorporation of newly developed treatment tools and modalities into clinical practice requires controlled trials in which the treatment responses and side effects are carefully monitored. It would be interesting to see whether clinical responses could be linked to a specific range of direct and indirect effects induced by the treatment. Besides the analyses of tumor cell death—directly important for the prevention of local recurrence, the mode of tumor cell death may be imperative for the induction of tumor protection in the long run. In addition to the elimination of large tumor masses by the induction of direct or indirect cell death, the ablation process itself produces tumor debris that remains in situ, eliciting specific immune responses that may prove valuable in the post-treatment surveillance for potential recurrences. Local and systemic inflammatory responses have been extensively studied for almost all the in situ tumor destruction techniques available.

3.3

Release of Immune-Mediating Factors and ‘Immunogenic’ Cell Death

The distinct type of injury or cell death, apoptotic or necrotic, is one of the important elements in Matzinger’s immunological “danger” model [30, 31]. This theory explains why the immune system develops an immune response against one antigen,


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but remains tolerant to others. In the classical view, apoptotic cell death is a phenomenon that is not regarded as harmful by the immune system, while necrotic cell death is seen as a dangerous event alarming the immune system. In recent years, however, it has been recognized that this concept is oversimplified, and that more factors related to cell death determine whether death is tolerogenic or immunogenic (as reviewed in [32]). Both apoptosis and necrosis can be accompanied by release of “danger signals” triggering adaptive T cell responses, provided that at the same time an antigen is adequately presented to the immune system. Especially in the complex situation within an RFA lesion where apoptosis and (secondary) necrosis exist side by side, tight regulation by the immune system is essential.

3.3.1

Release of Tumor-Antigens

The local destruction of a tumor mass results in a huge amount of dead tumor material that stays in situ. After ablation, this mass forms a depot from which antigens potentially enter the local circulation and lymphatics, available for uptake by the immune system. Following RFA of colorectal liver metastases patients show an initial rise in carcinoembryonic antigen values (CEA, a tumor antigen), while in conventional surgical resection it is known that values fall rapidly, reflecting the elimination of the tumor load. Following RFA, CEA values also drop to background levels much slower, suggesting a slow release of immune reactive antigens from the tumor debris [33]. We formerly showed that the ablated tumor depot is essential for in vivo induction of anti-tumor immunity. We additionally demonstrated that DCs residing in the tumor-draining lymph node readily internalized antigens from the tumor microenvironment during the first 2 days after RFA [34]. In a similar model for cryoablation, these lymph node DCs were able to cross-present tumor-derived antigens to T cells ex vivo [35, 36]. These observations suggest that the ablated tumor provides an antigen depot that releases antigens systemically, which become available for professional antigen-presenting cells (APCs) and subsequent initiation of anti-tumor immunity (see below).

3.3.2

Release of Immune Stimulating Mediators

Following RFA, many immune-related factors could be detected in the serum of patients. Most of these factors merely show a temporary rise after which values go back to baseline levels. Potentially, these factors could play a key role in modulating the immune responses towards the in vivo released tumor antigens. In patients with primary or metastatic lung tumors, increased plasma levels of proinflammatory chemokines (MIP-1a, MIP-1b, eotaxin, interleukin-8) and acute phase proteins (complement C3 and C4, serum amyloid P, C-reactive protein) were found [37]. Also in hepatocellular carcinoma patients treated with combined ethanol injection

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and RFA treatment elevated levels of serum TNF-α and IL-1β could be observed [38]. Although many studies describe the release of (soluble) mediators in serum or ablated tissue, the exact immunological correlates needed for strong anti-tumor immune responses are less well defined. Although the importance of pathogen associated molecular patterns (PAMPs) in the balance between tolerance or immunity has long-standing evidence [39], the role of endogenous factors in the induction of inflammation is the subject of more recent work. Endogenous damage associated molecular patterns (DAMPs) are distinct molecules released or exposed following an injury or cellular stress [40]. They alarm and activate the immune system and recruit professional cells to the site of injury, just like PAMPs do. At low concentration DAMPs seem to have a regulatory role, while at high concentrations they alarm and activate the immune system and recruit professional cells to the site of injury, just like PAMPs do. DAMPs like HMGB1, uric acid, HSPs, IL1α/β and the S100 family of proteins have all been shown to be released upon cell death and to modulate the immune system. Heat shock proteins (HSPs) are highly conserved cell chaperones involved in the repair or elimination of proteins affected in various stress conditions. Oxidative stress, irradiation or chemotherapeutic drugs all lead to transcriptional and translational activation of HSPs in most cells. HSPs are mostly expressed in the cytosol or organelles (e.g. mitochondria), but some inducible HSPs, like HSP70 and HSP90, may translocate to the plasma membrane. Intracellular expression of HSPs in cancer cells is associated with cancer progression (as it provides a survival mechanism for cancer cells under oxidative stress), while extracellular expression attracts and activates immune cells and thus results in cancer regression [41–43]. Extracellular HSPs, like expressed on tumor cell surfaces or tumor exosomes, determine the immunogenicity of stressed/dying cells via their ability to interact with a number of APC receptors like CD40, TLR receptors, LOX1 and CD91, thereby enhancing APC function [44, 45]. In a murine experimental model using B16 melanoma, Liu et al. [46] demonstrated that while untreated tumor lesions showed hardly any staining, RFAtreated tumors became highly positive for HSP70 and gp96. Immunochemistry and RT-PCR analysis in other species revealed that various HSPs are expressed in the transition zone surrounding coagulated liver tissue up to 5 days following RFA [47–49]. Haen et al. [50] recently demonstrated a significant increase in serum levels of HSP70 in patients undergoing RFA for the treatment of malignancies of the liver, kidney, and lung. After classifying the patients for low or high HSP expression, they found that 61 % in the low HSP group suffered from progressive disease, compared to only 11 % in the high HSP group. Due to the large variety and small size of this cohort, a firm correlation between HSP levels and clinical outcome still needs to be established. The high mobility group box 1 protein (HMGB1, amphoterin) is a non-histone chromatin-binding protein very abundant in the nucleus of all cells. It assists in the assembly of nucleoprotein complexes and has other functions in transcription. HMGB1 is secreted actively from immune cells (e.g. monocytes and macrophages), or passively from necrotic cells [51–53]. In contrast, apoptotic cells modify their chromatin so that HMGB1 binds irreversibly and therefore is not released. HMGB1


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protein released from dying cells acts as a cytokine with potent pro-inflammatory properties. It has chemotactic and activating effects on monocytes, macrophages, neutrophils and DCs [54, 55]. Moreover, the ability to attract muscle, endothelial and stem cells suggests that HMGB1 is an important regulator in tissue regeneration responses [51, 56]. HMGB1 has been shown to direct the release of proinflammatory cytokines like IL-1, IL-6, IL-8 and TNF by binding to several PRRs (TLR2, TLR4, RAGE) [52, 57]. It remains to be established whether the interaction of HMGB1 with TLR receptors involves a direct binding or depends on the formation of specific complexes with TLR-ligands like LPS or immunogenic DNA [58, 59]. RFA treatment of murine melanoma was shown to induce translocation of nuclear HMGB1 into the cytoplasm and intercellular space, leading to active release of this protein in the tumor tissue [46]. This study also describes mice receiving RFA treatment with previous injection of DCs. DCs loaded with in vitro heat-shocked tumor lysate followed by RFA yielded a bigger anti-tumor immune response compared to RFA after DCs loaded with normal tumor lysate. Unfortunately, the authors do not provide evidence that released HMGB1 is causally related to the immune responses observed. This relationship was more intensively studied by Apetoh et al. [52] in the context of other in situ tumor destruction techniques. They found that various dying tumor cells released HMGB1 following anthracycline treatment or irradiation. Interference with HMGB1 release strongly impaired the immunogenicity of cell death in vitro. Nevertheless, the exact consequences of HMGB1 release in vivo, especially in response to in situ tumor destruction techniques remains to be determined in further detail. Calreticulin (CRT) is a highly conserved protein of approx. 55–60 kDa, predominantly located in the endoplasmatic reticulum (ER). Next to its role as a chaperone it functions as a regulator of calcium homeostasis [60, 61]. Within the ER, CRT interacts with various molecules like calnexin in order to coordinate the proper folding of proteins. Of immunological importance is the role CRT plays in assembly and antigenic peptide loading of the MHC class I molecule. Outside the ER, CRT is also exposed at the cell surface, where it is involved in the regulation of phagocytosis. Extracellular CRT was shown to interact with the plasma membrane of phagocytes, thereby forming a functional receptor complex that drives the phagocytosis of opsonized apoptotic bodies [62]. Interestingly, cancer cells that died in response to specific lethal stimuli (e.g., anthracyclines, oxaliplatin, ionizing irradiation and photodynamic therapy) were shown to have elevated levels of CRT at the outer leaflet of the plasma membrane [63, 64]. This generated a specific phagocytosis signal for APCs like DCs, and correlated with tumor-specific immune responses in vivo. To our knowledge, the release of CRT following RFA and the consequences for the immune system, have not been studied yet. Other DAMPs (e.g. uric acid and the S100 protein family) have all been shown to be released from dying cells and have pro-inflammatory potential. However, their actual role in cell death-induced inflammation in vivo is for the most part unclear. The release of these DAMPs in vitro or in vivo in response to RFA has not been demonstrated so far, but forms an interesting research subject. Determining the molecular identity of DAMPs and their contribution to inflammation in actual in vivo

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models is important because these molecules could be instrumental in the induction of systemic immunity following RFA.

3.4

Induction of Immune Responses by RFA: The Tumor Microenvironment

Mutations that alter the function of only a few key genes may contribute to malignant transformation. The subsequent genetic instability of developing tumor tissue permits accumulation of further mutations, and deregulates the expression of other normal cellular components. These mutations have the potential to create novel antigens for recognition by cells of the adaptive immune system. Indeed, the discovery of (mutated) tumor-associated antigens (TAA) specifically recognized by T cells provided proof of concept and opened the possibility to perform cancer immunotherapy, e.g. adoptive T cell transfer.

3.4.1 Antigen Presentation after Radio Frequency Ablation Dendritic cells control the initiation of stimulatory and regulatory immune responses. They are strategically located within tissues and continuously sample the microenvironment, displaying their internalized cargo on their cell surface. We have previously demonstrated that CD11c+ DCs in the tumor-draining lymph node internalize radioand fluorescently-labeled antigens that were injected intra-tumorally just prior to RFA [34]. In addition, we found CD11c+ cells with internalized apoptotic tumor material in the draining lymph nodes after cryoablation of EG7 tumors that were stably transfected with a FRET-based fluorescent probe monitoring caspase-3 activity (Nierkens unpublished observation). Together, these data suggest that in situ released tumor-derived antigens end up in antigen-presenting cells in the draining lymph nodes and thus may be presented to tumor antigen-specific CTLs. RFA increased the influx of CD11c+ cells into the ablated area [65] and in the tumor-draining lymph nodes [34]. We also demonstrated that the lymph node DCs expressed higher levels of co-stimulatory molecules than DCs in untreated mice. Interestingly, especially the cells that took up tumor antigens were the ones that matured [34]. Zerbini et al. [66] showed that necrotic material from HCC after thermal ablation (in contrast to untreated HCC and non-tumor liver tissue) enhanced the expression of co-stimulatory molecules, chemokine receptors, antigen presentation, and cytokine secretion by monocyte-derived DCs in vitro. Moreover, CD1c+ DCs taken after RFA of HCC showed increased expression of co-stimulatory molecules and were able to stimulate CD4+ T cell proliferation in a mixed lymphocyte reaction (MLR) [38]. Although CD4+ T cells may contribute to anti-tumor immune responses, the main effector arm of the immune system to fight cancers is represented by CD8+ CTLs that, in contrast to CD4+ T cells, recognize antigen in complex with MHC class I. For


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the functional priming of CTL responses against tumor antigens released by RFA, APCs need to possess the capacity to internalize exogenous antigens and process them for presentation in MHC class I, a process known as cross-presentation. The ability to cross-present has been attributed to specific subsets of DCs. Studies in mice originally identified the CD8α-expressing cDCs as the most efficient crosspresenting DCs of cell-associated material [67]. Translation of these data to a human setting has always been hampered due to the fact that CD8α is not expressed on human DCs. Importantly, a recent microarray analyses showed that BDCA3+ (CD141) human DCs show a transcriptional signature resembling that of mouse CD8α+ DCs [68]. Moreover, both BDCA3+ human DCs [69–71] and their CD8α+ murine counterpart [72] are endowed with the cross-presenting capacity of both soluble and cell-associated antigens, IL-12 production and CTL activation. Whether tumorspecific T cell responses initiated after RFA are primarily directed against soluble antigens released or merely against antigens packed in cell-associated debris remains to be established. BDCA3+ DCs selectively express the surface molecule Clec9A, a sensor of necrotic cells [73] implying a specialized role for these DCs in crosspresentation of antigens derived from dead or dying cells. We [74] and others [75] have however recently challenged the exclusivity of cross-presentation within one DC subset in mice. This newly described population of CD8α-CD11b- DCs potently primed both CD8+ and CD4+ T cells to cell-associated antigens upon uptake of apoptotic cells. They were named merocytic DCs after their uptake of small particles that are stored in non-acidic compartments for prolonged periods, resulting in sustained antigen presentation, and the induction of type I IFN [74]. A thorough comparison of the T cell priming potential of different DC subsets after RFA has not been performed up to date. It would be interesting to elucidate whether the before-mentioned mediators (e.g. HMGB1, HSPs, calreticulin, uric acid, etc.) are directly responsible for the activation of a particular DC subset and subsequent increased antigen-presentation and T cell responses in vivo.

3.4.2

Immunity after Radiofrequency Ablation

One of the first studies to show adaptive immune responses after RFA monitored transplanted VX2 carcinomas in the liver of rabbits. Twenty-four hours after ablation, CD3+ T cells infiltrated the hemorrhagic margin in the periphery of the tumor and were present in the center of the tumor after 2 weeks. Increased levels of tumorspecific T cells were detected in peripheral blood [76]. We confirmed the presence of tumor-specific CTLs in RFA-treated mice and showed that protection could be transferred to naïve mice by splenocytes. Serum transfer only resulted in a minor delay in tumor growth, indicating that protection is primarily provided by cell-mediated responses [77]. In cancer patients, only few studies have described the induction of specific immune responses after RFA. Napoletano et al. [78] reported that naïve and memory CD62L+ T cells translocate to the tissues and that T cells produced IFN-γ in response

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to the tumor-associated MUC1 antigen, while humoral immune responses were unaffected by RFA treatment. The latter is in contrast to a study by Widenmeyer et al. [79] who concluded that TAA-specific antibodies increased within weeks to months after ablation in 6 out of 49 treated patients. In patients with HCC and colorectal liver metastases IFN-γ production (directed against autologous tumor tissue) was observed in both CD8+ and CD4+ T cells after RFA [80, 81]. The CTLs possessed highly elevated cytotoxic activity as indicated by adenylate cyclase release [81]. Interestingly, cross-recognition between ablated and non-ablated tissue was observed, but responses to autologous non-tumor tissue were absent or weak [80]. These results suggest specificity for tumor antigens that are absent in normal tissue thereby limiting autoimmune reactivity. Univariate analyses of parameters in 20 patients identified the number of TAA-specific CD8+ T cells as a significant prognostic factor for recurrence-free survival after RFA [82]. In contrast, Zerbini et al. [80] reported the absence of a correlation between enhancement of anti-tumor T cell responses and disease progression, which may be due to tumor escape mechanisms. These authors were also able to show increased IFN-γ production and cytotoxic activity of NK cells 4 weeks after RFA. By dividing the patients into high and low responders these parameters gained predictive value on the efficacy of the ablative treatment. These data suggest the involvement of NK cells in tumor control after RFA [83].

3.4.3

Regulation after Radiofrequency Ablation

The T cell stimulatory potential of DCs is determined by a multitude of receptors that react to changes in the environment. The tumor microenvironment generally lacks activating signals and contains high levels of immune modulating factors, such as IL-10 and TGF-β that prevent proper activation of DCs and limit effector functions of lymphocytes. These factors also attract regulating immune cells (Treg and myeloidderived suppressor cells) that actively suppress the function of DCs and T cells. Eliminating active immune suppression by tumor-derived factors therefore seems an important tool in the design of more powerful therapies. Thirty days after RFA the levels of peripheral CD25+ Foxp3+ Tregs in patients undergoing radiofrequency thermal ablation for lung cancer were decreased to levels found in controls [37]. Conversely, in irradiated tumors, increased numbers of Tregs were demonstrated in the ablated area, but these cells lost their suppressive capacities, thereby allowing functional immune responses to occur [84]. From findings in a mouse model for breast carcinoma it was also suggested that the levels of myeloidderived suppressor cells (analyzed here as CD11b+ Gr1+ cells, spleen) were reduced following RFA, but statistics for this small difference were lacking [85]. Although above-mentioned studies suggest that RFA causes a reduction of suppressive cell numbers in the periphery, the drop may simply reflect the elimination of the tumor burden, and thereby the tumor-derived factors that induced peripheral Treg expansion and/or survival. From a tissue-regeneration perspective the immune response to self-antigens following RFA needs to be regulated, which requires the


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local help of suppressive cells. Interestingly, when we depleted Treg using anti-CD25 antibodies prior to ablation, the efficacy of RFA was drastically increased [34]. It is not known whether these antibodies interfered with the regulatory response after ablation. It needs to be investigated how the immune system makes use of suppressive cells during the healing phase post-ablation and how this knowledge can be used to provoke anti-tumor immunity. Future research should therefore take into account the differences in numbers of the suppressive cells, location (e.g. peripheral blood or ablated area) and suppressive status.

3.5

RFA Combinational Strategies

RFA induces (weak) immune responses that are only occasionally strong enough to lead to spontaneous regression [86]. Clinical data additionally shows that it does poorly protect against secondary growth, local recurrence, or (intra-hepatic) metastasis and thus encompasses a high rate of (intra-hepatic) metastases and recurrent disease [87]. Combinational therapies of RFA with other debulking strategies or immune stimulatory approaches are therefore of great interest.

3.5.1

Tumor Cytotoxicity

Combination of intratumoral injection of liposomal doxorubicin with RFA has been shown to be superior in tumor destruction to either treatment modality alone [88]. The possible synergistic effect of these two therapies may include increased agent deposition secondary to changed vascular permeability in tumor tissue and cytotoxic activity of the drugs. In addition, the beneficial effects of combinational therapies may be derived from effects on immune parameters. For instance chemotherapeutics may significantly affect immune cells [89]. We recently described that exposure to platinum-based chemotherapeutics markedly reduced expression of the T cell inhibitory molecule programmed death receptor-ligand 2 (PD-L2) not only on human tumor cells but also on DCs. Down-regulation of PD-L2 by STAT6 knockdown resulted in enhanced antigen-specific proliferation and Th1 cytokine secretion as well as enhanced recognition of tumor cells by T cells [89]. The induction of hyperthermia by RFA at lower temperatures as used for direct ablation of tumor masses also has a radio-sensitizing effect [90]. When the target lesion is heated up to around 42 ◦ C vasculature changes seem to enhance the cancer-killing effect of radiation [91]. While radiation alone was effective in controlling 80 % of small (< 10 cm3 ) tumors and only 30 % of big tumors (> 10 cm3 ), the combination with RF affected 80 % local tumor control regardless of the tumor volume [92]. Although appropriate control groups were not included, RF hyperthermia administered simultaneously with irradiation and chemotherapy (5-fluorouracil in combination with cis-platinum or methotrexate) showed local tumor control [93].

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3.5.2

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Immunotherapy

RFA provides a feasible strategy to administer immunomodulatory compounds in close proximity to the released antigens. Immunotherapy is currently one of the most studied novel cancer treatments. The rationale of immunotherapy is based on the observation that cells from the adaptive immune system recognize TAAs as their target. In most cancers TAA are self-antigens that are mutated or over-expressed in tumor cells compared to healthy cells. Immune reactivity against self-antigens is however highly regulated, which limits the generation of productive immune responses against cancerous cells. Immunotherapeutic strategies to overcome suppression and further stimulate DC and T cell activation are therefore of great interest. The application of these strategies as a monotherapy has so far resulted in meager responses in clinical trials. This may be due to the employment in patient groups that did not respond to standard therapy and thus had a lower chance on clinical responses. Moreover, the stimulation of the immune system may particularly be of value in combination with standard therapies, like RFA, to reduce the tumor mass and inhibit the immunosuppressive tumor microenvironment (Fig. 3.1).

3.5.2.1

Stimulation of Antigen Presentation

The instantaneous release and availability of tumor antigens for antigen-presenting cells founded interest in strategies to increase antigen presentation to T cells. Increasing the numbers of DCs by transfer of ex vivo-generated DCs is the most straightforward approach in this respect. Although DC vaccination indeed increased the efficacy of radiotherapy [94–96], intratumoral ethanol injections [97], cryoablation [98, 99], and photodynamic therapy [100, 101], the intratumoral injection of ex vivo-engineered bone marrow-derived DCs failed to improve the outcome of RFA in a murine model for urothelial carcinoma [65]. Unfortunately a trial by Engleman [102] to study the effects of DC injection after RFA was prematurely terminated. In a model of cryoablation of melanoma tumors we found that the ablative treatment combined with peri-tumor injections of the TLR9 agonist CpG resulted in superior protection against tumor re-challenges [35]. The clinical efficacy of CpG was highly dependent on the timing of administration relative to the ablation [103] and was also directly correlated with the timing of injection and subsequent in vivo co-localization of antigens and CpG within DCs [36]. The immune adjuvant efficacy of CpG was founded specifically upon TLR9 function in plasmacytoid DCs that subsequently stimulated the CD8α+ DCs and merocytic DCs to cross-prime tumorspecific CTLs [104]. The administration of CpG in close proximity to the ablated area is a simple procedure in RFA where the insertion of RF probes enables an immediate entry for the injection of immune stimulants. A clinical trial is currently in progress in patients with colorectal liver metastases evaluating the safety and efficacy of intrahepatic CpG injections in combination with RFA.

Fig. 3.1 Induction of anti-tumor immunity by RFA combined with immunotherapy


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3.5.2.2

Stimulation of T Cell Reactivity

Saito et al. [105] established a murine orthotopic model of head and neck squamous cell carcinoma (HNSCC) and additionally treated the mice with IL-2 gene transfer. The combinational treatment was most successful in reducing tumor growth, which coincided with increased influx of macrophages and DCs, enhanced CTL activity and protection against tumor re-challenges. Habibi et al. [85] performed RFA on mammary carcinomas in FVBN202 transgenic or BALB/c mice and coadministered IL-7 and IL-15 intra-lesionally. These cytokines were selected based on their supporting role in proliferation and homeostasis of effector and memory T cells. In contrast to IL-2, they do not induce activation-associated cell death, nor are they needed for the maintenance of regulatory T cells. Only combinational treatment resulted in tumor-specific IFN-γ responses in splenocytes and protected the mice following a tumor re-challenge. Similarly, combining RFA of murine hepatoma with three (every 2 days) subcutaneous injections of an active variant of CC chemokine ligand 3/macrophage inflammatory protein-1alpha (ECI301) eliminated the RFA-treated primary tumors and retarded the growth of contralaterally injected (non-RFA-treated) tumors, accompanied by CCR1-dependent T cell infiltration [106]. We previously established a mouse model in which B16 melanoma tumors were destructed by RFA and showed that 10–40 % of the RFA-treated mice were able to reject a re-challenge with an otherwise lethal dose of B16. Additional systemic administration of anti-CTLA4 mAb (clone 9H10) increased the survival rate to 70 % and drastically elevated the levels of tumor-specific T cells [34]. These data implicated that blocking the transmission of T cell inhibitory signals is a potent strategy to increase the clinical efficacy after RFA. It would be interesting to study the infiltration of CTLs into the ablated tumor area after these treatments. Recently, T cell transfer approaches have been tested in combination with RFA. Transfer of autologous mononuclear cells stimulated with anti-CD3 and human fibronectin fragment (CH-296) in the presence of IL-2, to prepare so-called RetroNectin activated killer (RAK) cells, after RFA was found to be safe and induce increased IFN-γ levels in five out of six patients. No clinical data is yet available.

3.6

Conclusions and Future Perspective

Percutaneous RFA has emerged as a promising treatment alternative for cancer patients, offering several advantages over conventional treatments; the technique is minimally invasive, encompasses low morbidity and mortality rates and has shown superior efficacy in comparison with other alternative treatments, such as percutaneous ethanol injections. Although very diverse in their ways to induce cell death, ablative techniques share one key feature: the in situ availability of the ablated material. During the efforts of the body to clear this dead material there is a time frame in which the immune


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system is actively controlling immune responses directed towards antigens from this antigen ‘depot’. The ability to stimulate the immune system with antigens from dead tumor cells has led to the idea that RFA can be used to achieve ‘in vivo vaccination’ against tumors. Many studies have demonstrated antigen-specific T cells or antibodies following RFA, but results show that this does not necessary dictate effectiveness of the induced immune response. Even activation of APCs by the cell-death associated mediators, may not be enough to generate immune responses that clear entire tumors. The possibility that not only intact tumors employ mechanisms to suppress immune responses, but also the body itself when its facing massive cell death of self-based tissue, is unfortunately less well studied. Most studies, including ours, report only low numbers of specific T cells following RFA as a stand-alone treatment. It therefore seems fair to conclude that tumor-directed immune responses exist, but that they are relatively weak. The fact that only very rarely spontaneous regression of metastases after RFA of the primary tumor is observed is illustrative for this. Currently, data is lacking about which ablation technique might result in the most effective release of antigens and the most immunostimulatory environment. Unlike RFA, techniques like for instance cryoablation will not denature a large part of the proteins and cause release of different antigens. Future research will have to compare the distinct types of ablation in a context of immune stimulation. The weak immune responses following ablation suggest that local enforcement by immune interventions can be helpful to boost immunity. As discussed in this review, strategies that employ or activate DCs in the tumor area seem to be most effective in this. Additional stimulation with TLR ligands or immune stimulating antibodies might be valuable tools to create solid immune responses. The immune modulating antibody CTLA4 is an example of a tool that is now available for clinical use and could be easily combined with RFA treatment [107]. It remains a challenge to link the quality of the antigens generated and the potency of the endogenous or exogenous immune modulation to clinical results. Of importance here is the question which immunological correlate is predictive for clinical outcome and should therefore be used to monitor the effects of ablation. De Vries et al. [108] reported that the presence of tumor-specific T cells in delayed-type hypersensitivity skin biopsies, rather than levels of specific T cells found in peripheral blood, after DC vaccination of melanoma patients correlated with clinical outcome. In line with these results, we predict that the presence of activated effector CD8+ T cells in the ablated tumor area and/or the intra-tumor ratio of CD8+ T cells versus Treg ratios may be a better prognostic marker for treatment efficacy than the presence in peripheral blood. In conclusion, immunomodulatory approaches are likely to become an important part of the armamentarium in the treatment of cancer, as mono-therapies, but especially in combination approaches together with in situ tumor destruction techniques. Immunotherapy will benefit from the primary function of radiofrequency ablation, which is debulking of tumor mass, as immunotherapy itself will most likely not be sufficient to cure end-stage cancers. Learning to ‘shape’ the initial ablation-induced immunity by external enhancements will provide us with robust treatment modalities for combination with in situ tumor destruction techniques.

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Radiofrequency ablation instantly releases tumor antigens from the tumor mass, which may flow to the draining lymph through lymphatic drainage or may be taken up by phagocytic cells, such as DCs, and transported to the lymph node. RF-induced cell damage will release endogenous danger signals as well as (pro-) inflammatory mediators that further DC maturation, resulting in the (specific) stimulation of immune effector cells. Nevertheless, RFA induces (weak) immune responses that are only occasionally strong enough to lead to spontaneous regression and does poorly protect against secondary growth, local recurrence, or metastasis. Combinational therapies of RFA with immune stimulatory approaches may further stimulate tumor-specific immunity leading to enhanced local and systemic tumor control. For example, local DC vaccinations may increase the level of antigen presentation. Furthermore, immune adjuvants can be administered to ensure full DC maturation, and the administration of immune stimulating compounds, such as anti-CTLA-4, will result in profound T cell proliferation. Such approaches may be best positioned in a regimen together with strategies to abrogate immune regulatory networks.

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