**2. Radiofrequency ablation**

#### **2.1. Overview**

Radiofrequency ablation (RFA) is a minimally invasive technique used to thermally ablate targeted lesions in a variety of tissues. RFA is performed by percutaneously inserting one or more probes using various forms of image guidance, such as computed tomography (CT) or ultrasound (US). RFA may also be performed through other approaches such as laparoscopy and open surgery. The number of probes used is based on multiple factors such as the size of the lesion, the impedance of the targeted region, and surrounding structures such as blood vessels and lymphatic channels. In addition to the placement of RFA probes, one or more grounding pads are also placed on the patient. These grounding pads are located at a distant site from the probes. For example, a common practice is to place multiple grounding pads on both thighs when using RFA to ablate lesions located in the liver. Once the placement of the RFA probes has been confirmed with image guidance, an alternating current is generated by a power source between the probes and the grounding pads. This alternating current creates the thermal ablative region by causing ions to oscillate, generating frictional heat. RFA can reliably generate temperatures of 60–100°C in the targeted region leading to focal hyperthermic injury to the nearby cells. When temperatures reach 60°C or higher, instant cell death occurs, and at temperatures above 100°C, charring of surrounding tissues occurs. These two temperature points are crucial to the procedure because the operator can be certain that cell death has occurred in the regions >60°C, but it is also important to monitor the temperatures so that they do not increase too quickly or reach >100°C. If the temperature becomes too high, charring of the tissues occurs, which increases the impedance significantly and causes the technique to lose efficacy by diminishing the ablative zone substantially [7]. Additionally, at temperatures above 110°C, vaporization of the tissues occurs. Vaporization also increases the impedance of the tissues limiting the ablative zone [8].

The ablated area with RFA can be divided into three zones: central, transitional, and the unaffected surrounding parenchyma [8]. The central zone is the area directly surrounding the RFA probe. In this zone, the temperatures are the highest, typically >50–60°C, leading to coagulative necrosis of the cells in this region. The cells in this zone immediately undergo irreversible injury through protein denaturation of both the cytosolic, nucleic, and mitochondrial enzymes leading to coagulative necrosis. In addition to protein denaturation, the cell membrane integrity is also compromised. The higher temperatures in the central zone lead to changes in fluid permeability through destruction of the membrane actin filaments. These membrane changes result in an intracellular fluid shift and subsequent cytolysis [5, 8].

Cells in the transitional zone are heated through conductive heat transfer from tissues in the central zone. This conductive heat transfer produces a sharp temperature gradient with average lower temperatures ranging from 40 to 45°C [5]. Cells within the transitional zone experience thermal injury, but since temperatures of 50°C are not reached, these cells do not undergo immediate cellular death [9]. Rather, the cells' metabolic processes and DNA repair mechanisms are impaired, which trigger specific changes that eventually lead to apoptosis or eventual cellular recovery. Other proposed mechanisms of cellular death include ischemia from vascular damage, reperfusion injury, and cytokine release and subsequent immunologic response to the damaged cells. Due to these changes, a complete response to ablation in this region will take several days to fully develop. This region also undergoes reactive hyperemia in response to the damage. The combination of hyperemia and increased cellular susceptibility creates a favorable environment to use liposomal chemotherapeutics. Liposomal chemotherapeutics will accumulate in the region due to the hyperemia and have increased activity on the already susceptible tumor cells. Since very few of the cells in this region are completely denatured, the transitional zone plays a critical role in the immunologic response, which will be discussed in more detail [5, 6, 8].

Surrounding parenchyma is not left totally unaffected by RFA. While the cells within this zone will not undergo cellular changes, necrosis, or apoptosis, there are several processes that will occur. There is an upregulation of various factors, presentation of antigens to antigenpresenting cells (APCs), and stimulation of the immune system, which will be discussed more in depth in later sections. Additionally, hyperemia occurs which can result in reperfusion injury [5, 6].

All of the above processes are dependent on a multitude of different factors such as the tumor composition, the surrounding parenchyma, the rate at which the energy is applied, and surrounding anatomic structures. The majority of the data on the effects of hyperthermia have been generated from literature on low-temperature hyperthermia that was applied uniformly over longer periods.

### **2.2. Patient selection**

and cryoablation (cryo), have been shown to have distinct advantages over traditional treatment methods. These methods are not only able to locally control the malignancy through cellular necrosis and apoptosis but also potentially trigger systemic immune responses [3–6]. Additionally, these minimally invasive techniques offer other advantages such as lower morbidity, preservation of healthy tissues, lower cost, and decreased hospitalization time relative to surgical resection [5]. In this chapter, the mechanisms, advantages, disadvantages, syner-

Radiofrequency ablation (RFA) is a minimally invasive technique used to thermally ablate targeted lesions in a variety of tissues. RFA is performed by percutaneously inserting one or more probes using various forms of image guidance, such as computed tomography (CT) or ultrasound (US). RFA may also be performed through other approaches such as laparoscopy and open surgery. The number of probes used is based on multiple factors such as the size of the lesion, the impedance of the targeted region, and surrounding structures such as blood vessels and lymphatic channels. In addition to the placement of RFA probes, one or more grounding pads are also placed on the patient. These grounding pads are located at a distant site from the probes. For example, a common practice is to place multiple grounding pads on both thighs when using RFA to ablate lesions located in the liver. Once the placement of the RFA probes has been confirmed with image guidance, an alternating current is generated by a power source between the probes and the grounding pads. This alternating current creates the thermal ablative region by causing ions to oscillate, generating frictional heat. RFA can reliably generate temperatures of 60–100°C in the targeted region leading to focal hyperthermic injury to the nearby cells. When temperatures reach 60°C or higher, instant cell death occurs, and at temperatures above 100°C, charring of surrounding tissues occurs. These two temperature points are crucial to the procedure because the operator can be certain that cell death has occurred in the regions >60°C, but it is also important to monitor the temperatures so that they do not increase too quickly or reach >100°C. If the temperature becomes too high, charring of the tissues occurs, which increases the impedance significantly and causes the technique to lose efficacy by diminishing the ablative zone substantially [7]. Additionally, at temperatures above 110°C, vaporization of the tissues occurs. Vaporization also increases the

The ablated area with RFA can be divided into three zones: central, transitional, and the unaffected surrounding parenchyma [8]. The central zone is the area directly surrounding the RFA probe. In this zone, the temperatures are the highest, typically >50–60°C, leading to coagulative necrosis of the cells in this region. The cells in this zone immediately undergo irreversible injury through protein denaturation of both the cytosolic, nucleic, and mitochondrial enzymes leading to coagulative necrosis. In addition to protein denaturation, the cell membrane integrity is also compromised. The higher temperatures in the central zone lead

gism, and immunologic responses to the techniques outlined above are discussed.

**2. Radiofrequency ablation**

150 Hepatocellular Carcinoma - Advances in Diagnosis and Treatment

impedance of the tissues limiting the ablative zone [8].

**2.1. Overview**

Traditionally, hepatic resection (HR) has been regarded as the first-line treatment for HCC, and RFA was typically reserved for patients with non-resectable disease. However, RFA has become a first-line treatment for early-stage HCC in patients with non-resectable disease, metastatic disease, recurrent HCC after HR, and for patients who are unable or are unwilling to undergo surgery [10]. RFA is best used in patients who have a solitary nodule <5 cm measured in the greatest dimension or less than three nodules all measuring <3 cm in the greatest dimension. RFA is most effective when treating HCC lesions that are ≤2 cm measured from the largest dimension. The reason it is more effective in these smaller lesions is that ablation margins of >4–5 mm can be easily obtained [1, 10]. Histologic and prospective studies have shown that the sensitivity of CT for detecting remnant neoplasm is anywhere between 36 and 44% [11, 12]. Thus, the clinician cannot readily rely on imaging to confirm that the lesion has been fully treated during or after the procedure making pre-procedure planning and patient selection crucial. Meta-analysis and systematic reviews have also shown that the efficacy of RFA and HR when used to treat lesions <5 cm is similar. There is no difference in 1-year overall survival; however, there is a difference in the 3- and 5-year survival. HR offers greater 3- and 5-year survival when compared to RFA as well as 1-, 3-, and 5-year disease-free rates. It has been postulated that these findings are due to how each treatment method works. With RFA, the primary lesion is directly targeted with minimal damage to the surrounding tissues, which may leave satellite lesions that would have been removed with HR. Additionally other factors come into play with RFA such as the shape and distribution of the ablation zone. However, RFA has been shown to have fewer complications during and after the procedure, shorter hospital stays, and is considered safer and less invasive than HR. While HR has a significant role in the treatment of HCC, there are limited studies comparing RFA to HR [10, 13, 14]. At the current time, it cannot be confirmed which treatment is superior to the other in treating earlystage HCC. It is up to the treating clinician to determine which treatment is best. RFA should be considered as a first-line treatment in specific patients with small solitary lesions <5 cm; patients with less than three lesions that are <3 cm; patients with non-resectable, metastatic, or recurrent disease; patients who elect for non-operative or a minimally invasive approach; and patients who are non-operative due to medical comorbidities [10].

derived from the hepatic artery, temporary occlusion of this artery with a balloon catheter will reduce the heat sink effect and increase the size of the ablative region. Another method of occlusion is embolization of feeding vessels with gel foam. These methods enable the generation of a larger ablative zone. Risks of hepatic artery occlusion and gel foam embolization do

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Injection of NaCl-containing solutions has also been explored to overcome the heat sink effect [16, 19–21]. Pre-treatment with hypertonic saline, 5–36%, results in significantly higher temperatures and a significantly larger ablation zone when compared to no pre-treatment [19, 20]. The NaCl solutions can be injected at any point during the procedure to expand the size

Transarterial chemoembolization (TACE) combined with RFA is another widely studied technique for overcoming the heat sink phenomenon. Combining these two techniques has multiple benefits that have been shown in both animal and human models [22–24]. First, TACE reduces the amount of conductive cooling by reducing blood flow to the targeted region. The reduction in blood flow increases the size of the central ablation zone, thus increasing the amount of coagulative necrosis. In addition, the amount of tissue that receives sublethal hyperthermia is increased and simultaneously exposed to a chemotherapeutic agent. The synergy is created by increased membrane permeability, intratumoral accumulation of the pharmacologic agent, and increased drug sensitivity of the cells within the transitional zone [5, 6, 8, 24]. Liposomal doxorubicin has been well studied in the setting of liver malignancies and is a good choice of chemotherapeutic agent [22, 24]. The wider ablation margin with increased volume of both the central and transitional zone and high local concentration of chemotherapeutic agent results in destruction of microscopic satellite lesions surrounding the

To adequately discuss the immunologic response to RFA, the basic immunologic response must first be discussed. The immune system is composed of two basic parts, the innate and the adaptive immune systems. Both of these systems work in concert to mount a defense to pathogens, such as viruses and bacteria, and prevent unregulated cell growth. The immune system is able to recognize and eliminate both dangerous self and non-self cells through a system of complex interactions and "danger signals." However, in HCC and other malignancies,

Typically, the first response by the immune system that occurs is by the non-specific or innate immune system. The non-specific immune system is composed of natural killer cells (NK), mast cells, eosinophils, basophils, macrophages, neutrophils, and dendritic cells (DC). These cells are the first to mobilize and produce signals initiating the specific immune response. The specific or adaptive immune system is composed of B and T lymphocytes. With co-stimulation from the innate system, the adaptive system generates a robust and lasting immune response through the formation of antibodies and memory B and T cells. The basic process that must occur to achieve a full immune response is antigen recognition and presentation

lesion consequently improving local control of the tumor [18, 24].

the cells evade the immune system through numerous mechanisms [4, 25].

exist and should be considered [16].

**2.4. Immunologic response to RFA**

of the ablation zone [21].

#### **2.3. Advantages and disadvantages of RFA**

Of all the thermal ablative techniques for treating HCC, RFA has been the most researched. There are many technical advantages using RFA for the treatment of HCC. The most obvious advantage of RFA as well as other thermal ablative techniques is the ability to treat a wide variety of patients while sparing normal liver parenchyma.

However, an important consideration with RFA and other thermal ablative techniques is the "heat sink effect" of surrounding anatomic structures. The heat sink effect is caused when the desired ablative region contains or is abutted by larger vessels that result in heat dissipation. This dissipation can ultimately lead to temperatures not reaching cytotoxic levels in the lesion [15–17]. Animal studies have shown that the heat sink effect is not significant until the vessel diameter is ≥3 mm [15]. Additional studies have shown a clinically significant increase in tumor recurrence when abutted by a vessel at least 3 mm in diameter [16]. Another concern is damage and thrombosis of surrounding vessels. It has been shown that there is minimal damage and thrombosis to surrounding vessels if the size of those vessels is greater than 3 mm [15]. The implication of these studies is that if a lesion contains a vessel greater than 3 mm, the RFA probe should be placed close to, but not in, the vessel to achieve the best outcome [15, 16]. This placement will move the tumor cells surrounding the vessel into the central ablation zone increasing the likelihood of cell death without significantly increasing damage to the vascular structure.

Other methods to mitigate the heat sink effect have also been studied. One method to overcome the heat sink effect is to occlude the blood supply to the region being ablated with a balloon catheter or gel foam [18]. Since the majority of the blood supply to HCC lesions is derived from the hepatic artery, temporary occlusion of this artery with a balloon catheter will reduce the heat sink effect and increase the size of the ablative region. Another method of occlusion is embolization of feeding vessels with gel foam. These methods enable the generation of a larger ablative zone. Risks of hepatic artery occlusion and gel foam embolization do exist and should be considered [16].

Injection of NaCl-containing solutions has also been explored to overcome the heat sink effect [16, 19–21]. Pre-treatment with hypertonic saline, 5–36%, results in significantly higher temperatures and a significantly larger ablation zone when compared to no pre-treatment [19, 20]. The NaCl solutions can be injected at any point during the procedure to expand the size of the ablation zone [21].

Transarterial chemoembolization (TACE) combined with RFA is another widely studied technique for overcoming the heat sink phenomenon. Combining these two techniques has multiple benefits that have been shown in both animal and human models [22–24]. First, TACE reduces the amount of conductive cooling by reducing blood flow to the targeted region. The reduction in blood flow increases the size of the central ablation zone, thus increasing the amount of coagulative necrosis. In addition, the amount of tissue that receives sublethal hyperthermia is increased and simultaneously exposed to a chemotherapeutic agent. The synergy is created by increased membrane permeability, intratumoral accumulation of the pharmacologic agent, and increased drug sensitivity of the cells within the transitional zone [5, 6, 8, 24]. Liposomal doxorubicin has been well studied in the setting of liver malignancies and is a good choice of chemotherapeutic agent [22, 24]. The wider ablation margin with increased volume of both the central and transitional zone and high local concentration of chemotherapeutic agent results in destruction of microscopic satellite lesions surrounding the lesion consequently improving local control of the tumor [18, 24].

#### **2.4. Immunologic response to RFA**

44% [11, 12]. Thus, the clinician cannot readily rely on imaging to confirm that the lesion has been fully treated during or after the procedure making pre-procedure planning and patient selection crucial. Meta-analysis and systematic reviews have also shown that the efficacy of RFA and HR when used to treat lesions <5 cm is similar. There is no difference in 1-year overall survival; however, there is a difference in the 3- and 5-year survival. HR offers greater 3- and 5-year survival when compared to RFA as well as 1-, 3-, and 5-year disease-free rates. It has been postulated that these findings are due to how each treatment method works. With RFA, the primary lesion is directly targeted with minimal damage to the surrounding tissues, which may leave satellite lesions that would have been removed with HR. Additionally other factors come into play with RFA such as the shape and distribution of the ablation zone. However, RFA has been shown to have fewer complications during and after the procedure, shorter hospital stays, and is considered safer and less invasive than HR. While HR has a significant role in the treatment of HCC, there are limited studies comparing RFA to HR [10, 13, 14]. At the current time, it cannot be confirmed which treatment is superior to the other in treating earlystage HCC. It is up to the treating clinician to determine which treatment is best. RFA should be considered as a first-line treatment in specific patients with small solitary lesions <5 cm; patients with less than three lesions that are <3 cm; patients with non-resectable, metastatic, or recurrent disease; patients who elect for non-operative or a minimally invasive approach; and

Of all the thermal ablative techniques for treating HCC, RFA has been the most researched. There are many technical advantages using RFA for the treatment of HCC. The most obvious advantage of RFA as well as other thermal ablative techniques is the ability to treat a wide

However, an important consideration with RFA and other thermal ablative techniques is the "heat sink effect" of surrounding anatomic structures. The heat sink effect is caused when the desired ablative region contains or is abutted by larger vessels that result in heat dissipation. This dissipation can ultimately lead to temperatures not reaching cytotoxic levels in the lesion [15–17]. Animal studies have shown that the heat sink effect is not significant until the vessel diameter is ≥3 mm [15]. Additional studies have shown a clinically significant increase in tumor recurrence when abutted by a vessel at least 3 mm in diameter [16]. Another concern is damage and thrombosis of surrounding vessels. It has been shown that there is minimal damage and thrombosis to surrounding vessels if the size of those vessels is greater than 3 mm [15]. The implication of these studies is that if a lesion contains a vessel greater than 3 mm, the RFA probe should be placed close to, but not in, the vessel to achieve the best outcome [15, 16]. This placement will move the tumor cells surrounding the vessel into the central ablation zone increasing the likelihood of cell death without significantly increasing damage to the

Other methods to mitigate the heat sink effect have also been studied. One method to overcome the heat sink effect is to occlude the blood supply to the region being ablated with a balloon catheter or gel foam [18]. Since the majority of the blood supply to HCC lesions is

patients who are non-operative due to medical comorbidities [10].

variety of patients while sparing normal liver parenchyma.

**2.3. Advantages and disadvantages of RFA**

152 Hepatocellular Carcinoma - Advances in Diagnosis and Treatment

vascular structure.

To adequately discuss the immunologic response to RFA, the basic immunologic response must first be discussed. The immune system is composed of two basic parts, the innate and the adaptive immune systems. Both of these systems work in concert to mount a defense to pathogens, such as viruses and bacteria, and prevent unregulated cell growth. The immune system is able to recognize and eliminate both dangerous self and non-self cells through a system of complex interactions and "danger signals." However, in HCC and other malignancies, the cells evade the immune system through numerous mechanisms [4, 25].

Typically, the first response by the immune system that occurs is by the non-specific or innate immune system. The non-specific immune system is composed of natural killer cells (NK), mast cells, eosinophils, basophils, macrophages, neutrophils, and dendritic cells (DC). These cells are the first to mobilize and produce signals initiating the specific immune response. The specific or adaptive immune system is composed of B and T lymphocytes. With co-stimulation from the innate system, the adaptive system generates a robust and lasting immune response through the formation of antibodies and memory B and T cells. The basic process that must occur to achieve a full immune response is antigen recognition and presentation by antigen-presenting cells (APCs), subsequent recognition of the antigen by T-cells through interaction with APCs, cellular interaction generating costimulatory signals, and the presence of danger signals [4, 6]. It is important to mention the roles of CD4 or T helper cells (Th) and CD8 T cells or cytotoxic T cells (CTLs). In regards to antitumor immunity, the most important role of CD4 cells is to assist in the activation and proliferation of CD8 T cells. It is currently theorized that a high ratio of CD4:CD8 is important to forming lasting immunity to malignancies because of the role of CD4 cells in stimulating CD8 cells [30]. CD8 T cells have been the focus of antitumor immunity due to their ability to recognize MHC I molecules. MHC I molecules are used to display intracellular antigens on the surface of cells infected by viruses and malignant cells. CD8 cells bind to cells expressing specific MHC I molecule complexes and then destroy the targeted cells through the apoptotic cascade [26].

counts of suppressive T cells, but additionally RFA shows increased survival benefit due to improved CD8 T-cell counts. The post ablation increase in CD8 T-cells is strongly associated with decreased recurrence and increased survival in patients with HCC [30]. Patients who will undergo HR or liver transplantation can also benefit from RFA-induced stimulation of CD8 cells. One major issue these patients face long term is disease recurrence. Unitt et al. showed improved survival in patients who demonstrate strong CD4 and CD8 T cell responses after undergoing resection surgery [31]. The response by CD4 and CD8 T cells as well as the

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Most of our knowledge about the immune response to RFA is based on animal models and small human trials. One of the first studies to show a significant immune response to RFA was conducted in 2003. In this animal study, tumors were implanted into rabbits that were then either untouched or treated with RFA. The RFA-treated animals showed at least a threefold increase in specific T-cell infiltration compared to the untreated animals. The treated animals also showed an increase in survival rate. This study suggested that an anticancer immunologic effect could be created through RFA [33]. This study was then further augmented by blocking CTLA-4 with monoclonal antibody at the time of RFA. This strongly enhanced antitumor immunity and provided protection against tumor rechallenge. This demonstrated that a lasting systemic memory response is achievable with combination therapy. Furthermore, a 20-fold increase in specific cytotoxic T-cells (CTLs) was achieved when RFA + blocking antibody was used compared to RFA + control antibody demonstrating that the increased immune response to RFA can be potentiated [34]. Zerbini et al. were the first to demonstrate an increased immunologic response in human subjects. The effect of RFA on 20 patients with HCC was studied and found to have a significant increase in tumor-specific T-cell response. Circulating T and natural killer (NK) cells showed increased activation and expression of specific cytotoxic surface markers. Although an upregulated immune response was demonstrated in these subjects, the effect was not associated with increased protection to HCC relapse [35]. Later, Zerbini and colleagues showed that the immunologic effects post RFA are dependent on maturation of DCs driven by the release of intracellular debris [36]. In a murine urothelial carcinoma model, subtotal RFA was used to induce an immunologic response. In response to subtotal RFA, there was an increase in CD4 and CD8 responses and significant

It is clear that numerous benefits of RFA exist and that there is great potential for targeted stimulation of the immune system using RFA in conjunction with immune modulators. Nevertheless, the possibility of causing rapid growth of metastases exists [38–40]. Recent accounts of RFA and other forms of ablation causing growth of distant metastases have been reported. These reports in conjunction with the fact that RFA will induce mediators such as cytokines, including interleukin-6 (IL-6) and factors such as hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), hypoxia-induced factor-1α (HIF-1α), and HSPs, all have potential to cause tumor growth locally and distantly [38–42]. It is theorized that damage to the surrounding healthy liver parenchyma and following regeneration is the source of the pro-growth factors. Ahmed et al. [40] showed using a rat model that damaging normal hepatic tissue with RFA will induce distant tumor growth, which is mediated by VEGF and the HGF/c-Met pathway. Interestingly, multiple studies have shown that incomplete ablation

of HCC will also promote not only distant growth but also local invasion [41, 43–45].

increase of specific antibodies has been seen weeks to months following RFA [32].

tumor regression with rechallenge [37].

In contrast to HR where the objective is to completely remove the HCC lesion and occasionally remove local lymphoid tissues, minimally invasive techniques such as RFA leave necrosed tumor cells and their spilled intracellular materials behind. The retained intracellular materials, which were previously invisible to the immune system, can now act as antigens that trigger local and systemic immune responses to HCC. In addition to the release of antigenic material from the remaining necrotic tissues, danger signals, such as DNA, RNA, pro-inflammatory cytokines, uric acid, and heat-shock proteins (HSPs), are released. These damage-associated molecular pattern molecules (DAMPs) are then picked up by DCs and presented to T-cells starting the immune cascade. All of these DAMPs are inflammatory mediators and have the potential to trigger a robust tumor suppressing immune response but are only released by cells undergoing necrosis. Cells, which undergo apoptosis, may actually lead to tolerance if the ratio of apoptosis to necrosis within the lesion becomes too high [4, 25].

One class of DAMP of particular importance to RFA and other thermal ablative techniques is the heat shock protein (HSP) family. HSPs have special roles and are involved in protein folding, cellular signaling, cellular transport, and survival. HSPs are produced within cells in response to thermal injury and play a role as chaperones enabling the refolding of denatured proteins. Additionally, HSPs participate in the initiation of the adaptive immune response by presenting antigens to DCs, modulating DAMP-induced immune stimulation, and function as danger signals [25, 27]. HSPs' ability to chaperone peptides and provide maturation signals to dendritic cells causes the ultimate cross-presentation of antigen to CD8+ T cells. Independent of the adaptive response, HSPs induce local necrosis through stimulation of the innate immune system. HSPs' ability to efficiently stimulate both the innate and adaptive immune system holds great potential; therefore, upregulation of HSPs represents a potential approach to eliminate HCC and other malignancies [28].

In patients treated with RFA, there is a decreased response by CD25+ T-regulatory (Treg) cells [29]. Treg cells or T suppressor cells, a specific subset of T-cells, are responsible for downregulating the immune response. The Treg cell's role is to prevent autoimmune disease and create tolerance to self-antigens. Tregs achieve immunosuppression by downregulating CD4 and CD8 T cells, thus decreasing the tumoricidal immunologic response. In fact, high levels of CD25 T cells are associated with poorer outcomes in patients with malignancy [26]. Thus, patients with HCC can benefit from treatments that decrease the number of Treg cells and subsequent decreased immune tolerance of malignant cells. Indeed, RFA results in decreased counts of suppressive T cells, but additionally RFA shows increased survival benefit due to improved CD8 T-cell counts. The post ablation increase in CD8 T-cells is strongly associated with decreased recurrence and increased survival in patients with HCC [30]. Patients who will undergo HR or liver transplantation can also benefit from RFA-induced stimulation of CD8 cells. One major issue these patients face long term is disease recurrence. Unitt et al. showed improved survival in patients who demonstrate strong CD4 and CD8 T cell responses after undergoing resection surgery [31]. The response by CD4 and CD8 T cells as well as the increase of specific antibodies has been seen weeks to months following RFA [32].

by antigen-presenting cells (APCs), subsequent recognition of the antigen by T-cells through interaction with APCs, cellular interaction generating costimulatory signals, and the presence of danger signals [4, 6]. It is important to mention the roles of CD4 or T helper cells (Th) and CD8 T cells or cytotoxic T cells (CTLs). In regards to antitumor immunity, the most important role of CD4 cells is to assist in the activation and proliferation of CD8 T cells. It is currently theorized that a high ratio of CD4:CD8 is important to forming lasting immunity to malignancies because of the role of CD4 cells in stimulating CD8 cells [30]. CD8 T cells have been the focus of antitumor immunity due to their ability to recognize MHC I molecules. MHC I molecules are used to display intracellular antigens on the surface of cells infected by viruses and malignant cells. CD8 cells bind to cells expressing specific MHC I molecule complexes

In contrast to HR where the objective is to completely remove the HCC lesion and occasionally remove local lymphoid tissues, minimally invasive techniques such as RFA leave necrosed tumor cells and their spilled intracellular materials behind. The retained intracellular materials, which were previously invisible to the immune system, can now act as antigens that trigger local and systemic immune responses to HCC. In addition to the release of antigenic material from the remaining necrotic tissues, danger signals, such as DNA, RNA, pro-inflammatory cytokines, uric acid, and heat-shock proteins (HSPs), are released. These damage-associated molecular pattern molecules (DAMPs) are then picked up by DCs and presented to T-cells starting the immune cascade. All of these DAMPs are inflammatory mediators and have the potential to trigger a robust tumor suppressing immune response but are only released by cells undergoing necrosis. Cells, which undergo apoptosis, may actually lead to tolerance if the ratio of apoptosis to necrosis within the lesion becomes too high [4, 25].

One class of DAMP of particular importance to RFA and other thermal ablative techniques is the heat shock protein (HSP) family. HSPs have special roles and are involved in protein folding, cellular signaling, cellular transport, and survival. HSPs are produced within cells in response to thermal injury and play a role as chaperones enabling the refolding of denatured proteins. Additionally, HSPs participate in the initiation of the adaptive immune response by presenting antigens to DCs, modulating DAMP-induced immune stimulation, and function as danger signals [25, 27]. HSPs' ability to chaperone peptides and provide maturation signals to dendritic cells causes the ultimate cross-presentation of antigen to CD8+ T cells. Independent of the adaptive response, HSPs induce local necrosis through stimulation of the innate immune system. HSPs' ability to efficiently stimulate both the innate and adaptive immune system holds great potential; therefore, upregulation of HSPs represents a potential

In patients treated with RFA, there is a decreased response by CD25+ T-regulatory (Treg) cells [29]. Treg cells or T suppressor cells, a specific subset of T-cells, are responsible for downregulating the immune response. The Treg cell's role is to prevent autoimmune disease and create tolerance to self-antigens. Tregs achieve immunosuppression by downregulating CD4 and CD8 T cells, thus decreasing the tumoricidal immunologic response. In fact, high levels of CD25 T cells are associated with poorer outcomes in patients with malignancy [26]. Thus, patients with HCC can benefit from treatments that decrease the number of Treg cells and subsequent decreased immune tolerance of malignant cells. Indeed, RFA results in decreased

and then destroy the targeted cells through the apoptotic cascade [26].

154 Hepatocellular Carcinoma - Advances in Diagnosis and Treatment

approach to eliminate HCC and other malignancies [28].

Most of our knowledge about the immune response to RFA is based on animal models and small human trials. One of the first studies to show a significant immune response to RFA was conducted in 2003. In this animal study, tumors were implanted into rabbits that were then either untouched or treated with RFA. The RFA-treated animals showed at least a threefold increase in specific T-cell infiltration compared to the untreated animals. The treated animals also showed an increase in survival rate. This study suggested that an anticancer immunologic effect could be created through RFA [33]. This study was then further augmented by blocking CTLA-4 with monoclonal antibody at the time of RFA. This strongly enhanced antitumor immunity and provided protection against tumor rechallenge. This demonstrated that a lasting systemic memory response is achievable with combination therapy. Furthermore, a 20-fold increase in specific cytotoxic T-cells (CTLs) was achieved when RFA + blocking antibody was used compared to RFA + control antibody demonstrating that the increased immune response to RFA can be potentiated [34]. Zerbini et al. were the first to demonstrate an increased immunologic response in human subjects. The effect of RFA on 20 patients with HCC was studied and found to have a significant increase in tumor-specific T-cell response. Circulating T and natural killer (NK) cells showed increased activation and expression of specific cytotoxic surface markers. Although an upregulated immune response was demonstrated in these subjects, the effect was not associated with increased protection to HCC relapse [35]. Later, Zerbini and colleagues showed that the immunologic effects post RFA are dependent on maturation of DCs driven by the release of intracellular debris [36]. In a murine urothelial carcinoma model, subtotal RFA was used to induce an immunologic response. In response to subtotal RFA, there was an increase in CD4 and CD8 responses and significant tumor regression with rechallenge [37].

It is clear that numerous benefits of RFA exist and that there is great potential for targeted stimulation of the immune system using RFA in conjunction with immune modulators. Nevertheless, the possibility of causing rapid growth of metastases exists [38–40]. Recent accounts of RFA and other forms of ablation causing growth of distant metastases have been reported. These reports in conjunction with the fact that RFA will induce mediators such as cytokines, including interleukin-6 (IL-6) and factors such as hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), hypoxia-induced factor-1α (HIF-1α), and HSPs, all have potential to cause tumor growth locally and distantly [38–42]. It is theorized that damage to the surrounding healthy liver parenchyma and following regeneration is the source of the pro-growth factors. Ahmed et al. [40] showed using a rat model that damaging normal hepatic tissue with RFA will induce distant tumor growth, which is mediated by VEGF and the HGF/c-Met pathway. Interestingly, multiple studies have shown that incomplete ablation of HCC will also promote not only distant growth but also local invasion [41, 43–45].

While increased local and distant growth of tumor cells is a real possibility, there are multiple studies investigating how to mitigate the pro-growth effects created by RFA. Studies have investigated c-Met and VEGF inhibitors to attenuate the tumorigenic effects [38, 40]. Additionally, studies have looked at non-specific anti-inflammatory drugs to mitigate the effects of RFA-induced inflammation on tumorigenesis. Both aspirin and celecoxib have been investigated to prevent tumorigenesis [41, 42]. In animal models, each of these drugs when used in conjunction with RFA reduced local inflammation and subsequent effects on distant tumor cells. Furthermore, it is crucial to note that while there is information about distant tumorigenesis following RFA, clinically RFA has not been shown to worsen survival compared to untreated patients and remains an effective first-line treatment in appropriate patients [46].
