**3. Cryoablation**

#### **3.1. Overview**

Cryoablation is a thermal ablative technique that has been used since the nineteenth century. While other thermal ablative techniques add heat to the surrounding tissue, cryoablation removes heat. In its earliest form, a salt and ice solution was applied to breast and skin cancers. This treatment resulted in decreased pain and lesion size. In its current form, cryoablation is performed similarly to other ablative techniques. It can be used in either a percutaneous fashion, with image guidance, or through an open or laparoscopic surgical approach. Cryoablation is used to treat numerous types of cancers, but is most commonly used to treat liver, kidney, lung, prostate, and breast malignancies [47].

forms. This gradient pulls free water into the extracellular space increasing the solute concentration and dehydrating the cells. The high concentration of solutes intracellularly results in damage to enzymes and destabilizes membranes of both intracellular organelles and the cell [47]. Intracellular proteins are denatured as well, but return to their original conformation once thawing is completed [50]. Cells at the periphery of the ablation will remain intact and are not immediately killed by cryoablation. These cells will eventually undergo apoptosis that

**Figure 1.** Hepatic cryoablation. A cryoablation needle was advanced into a focal hepatic lesion (left), with a resultant ice ball visible on CT (asterisk). Follow-up imaging demonstrated a focal defect at the site of the previous lesion (right),

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When cells are frozen rapidly, there is no enough time for fluid shifts to occur, and cell death occurs via physical damage to organelles and the cellular membrane from intracellular ice formation. In both cases, pore formation in the membrane occurs, which allows for fluid shifts during the thaw cycle resulting in swelling and rupture [51]. The intracellular fluid shift during the thaw cycle occurs since extracellular ice melts before intracellular ice creating an osmotic gradient into the cell. It is important to note that intracellular ice will continue to grow during the thaw cycle reaching a maximum at −20 to −25°C. This formation of ice during the thaw cycle occurs due to the influx of free water. Additionally, the rate of thawing determines the amount of cellular death. Rapid thawing will decrease the biocidal effect by reducing the amount of intracellular ice formation. A greater degree of cellular death is seen in passive thawing when compared to active thawing. The highest degree of necrosis is seen

Indirect injury to cells occurs via vascular damage. During the freeze cycle, the endothelium of vessels is damaged, and when thawed, this injury triggers platelet aggregation. This aggregation leads to thrombosis and subsequent ischemia of the tissues [53]. The ischemia is twofold, not only does it lead to cellular death, but it also triggers inflammation. This leads to an influx of neutrophils and macrophages to the ablated zone [54]. The entire process can take months to complete, resulting in a zone of necrosis surrounded by a peripheral band of neutrophils [47].

The one of the best advantages of cryoablation is the ability to monitor the ablation zone in real time. As the ablation proceeds, formation of an ice-ball occurs that is visible on ultrasound (US), magnetic resonance (MR), and CT. This occurs because the water molecules undergo a

is triggered by damage to organelles [47].

consistent with a complete ablation.

with repeated freeze thaw cycles [52].

**3.2. Advantages and disadvantages of cryoablation**

Modern cryoablation requires the use of a specialized cryoprobe that is inserted into the targeted lesion (**Figure 1**). Once in the desired location, the probe is rapidly cooled beginning the freeze cycle for a specified length of time. After the freeze cycle is completed, the probe is warmed up to start the thaw cycle. These freeze/thaw cycles are repeated one or more times depending on the lesion and preference of the clinician [47]. The mechanism of cooling relies on the Joule-Thompson effect that describes how a gas that does not work expands (adiabatic expansion) and results in a decrease in temperature [48]. All gases except hydrogen, helium, and neon will decrease in temperature when expanded through the Joule-Thompson process. Commonly used gases for the freeze cycle are nitrogen and argon. One of these gases is pumped into the cryoprobe, and when the gas reaches the distal tip of the probe, the gas is throttled and then allowed to rapidly expand to atmospheric pressure. The result is a rapid decrease in temperature and cooling of the surrounding tissues via conduction. During the freeze cycles, temperatures can reach as low as −160°C, well below the −20 to −40°C required to cause cell death [49]. As mentioned above, helium does not undergo this effect, rather than cooling when rapidly expanded helium will increase the temperature. For this reason, helium is used in the thaw cycle to heat the surrounding tissues [47].

Cryoablation results in direct and indirect cellular injury and death. When the freeze cycle begins, the tissues are cooled and ice starts to form in the extracellular space. Since the formation of ice occurs in the extracellular space before the intracellular space, an osmotic gradient Minimally Invasive Therapies for Hepatocellular Carcinoma: Mechanisms of Local Control… http://dx.doi.org/10.5772/intechopen.72275 157

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].

Cryoablation is a thermal ablative technique that has been used since the nineteenth century. While other thermal ablative techniques add heat to the surrounding tissue, cryoablation removes heat. In its earliest form, a salt and ice solution was applied to breast and skin cancers. This treatment resulted in decreased pain and lesion size. In its current form, cryoablation is performed similarly to other ablative techniques. It can be used in either a percutaneous fashion, with image guidance, or through an open or laparoscopic surgical approach. Cryoablation is used to treat numerous types of cancers, but is most commonly used to treat

Modern cryoablation requires the use of a specialized cryoprobe that is inserted into the targeted lesion (**Figure 1**). Once in the desired location, the probe is rapidly cooled beginning the freeze cycle for a specified length of time. After the freeze cycle is completed, the probe is warmed up to start the thaw cycle. These freeze/thaw cycles are repeated one or more times depending on the lesion and preference of the clinician [47]. The mechanism of cooling relies on the Joule-Thompson effect that describes how a gas that does not work expands (adiabatic expansion) and results in a decrease in temperature [48]. All gases except hydrogen, helium, and neon will decrease in temperature when expanded through the Joule-Thompson process. Commonly used gases for the freeze cycle are nitrogen and argon. One of these gases is pumped into the cryoprobe, and when the gas reaches the distal tip of the probe, the gas is throttled and then allowed to rapidly expand to atmospheric pressure. The result is a rapid decrease in temperature and cooling of the surrounding tissues via conduction. During the freeze cycles, temperatures can reach as low as −160°C, well below the −20 to −40°C required to cause cell death [49]. As mentioned above, helium does not undergo this effect, rather than cooling when rapidly expanded helium will increase the temperature. For this reason, helium

Cryoablation results in direct and indirect cellular injury and death. When the freeze cycle begins, the tissues are cooled and ice starts to form in the extracellular space. Since the formation of ice occurs in the extracellular space before the intracellular space, an osmotic gradient

liver, kidney, lung, prostate, and breast malignancies [47].

156 Hepatocellular Carcinoma - Advances in Diagnosis and Treatment

is used in the thaw cycle to heat the surrounding tissues [47].

**3. Cryoablation**

**3.1. Overview**

**Figure 1.** Hepatic cryoablation. A cryoablation needle was advanced into a focal hepatic lesion (left), with a resultant ice ball visible on CT (asterisk). Follow-up imaging demonstrated a focal defect at the site of the previous lesion (right), consistent with a complete ablation.

forms. This gradient pulls free water into the extracellular space increasing the solute concentration and dehydrating the cells. The high concentration of solutes intracellularly results in damage to enzymes and destabilizes membranes of both intracellular organelles and the cell [47]. Intracellular proteins are denatured as well, but return to their original conformation once thawing is completed [50]. Cells at the periphery of the ablation will remain intact and are not immediately killed by cryoablation. These cells will eventually undergo apoptosis that is triggered by damage to organelles [47].

When cells are frozen rapidly, there is no enough time for fluid shifts to occur, and cell death occurs via physical damage to organelles and the cellular membrane from intracellular ice formation. In both cases, pore formation in the membrane occurs, which allows for fluid shifts during the thaw cycle resulting in swelling and rupture [51]. The intracellular fluid shift during the thaw cycle occurs since extracellular ice melts before intracellular ice creating an osmotic gradient into the cell. It is important to note that intracellular ice will continue to grow during the thaw cycle reaching a maximum at −20 to −25°C. This formation of ice during the thaw cycle occurs due to the influx of free water. Additionally, the rate of thawing determines the amount of cellular death. Rapid thawing will decrease the biocidal effect by reducing the amount of intracellular ice formation. A greater degree of cellular death is seen in passive thawing when compared to active thawing. The highest degree of necrosis is seen with repeated freeze thaw cycles [52].

Indirect injury to cells occurs via vascular damage. During the freeze cycle, the endothelium of vessels is damaged, and when thawed, this injury triggers platelet aggregation. This aggregation leads to thrombosis and subsequent ischemia of the tissues [53]. The ischemia is twofold, not only does it lead to cellular death, but it also triggers inflammation. This leads to an influx of neutrophils and macrophages to the ablated zone [54]. The entire process can take months to complete, resulting in a zone of necrosis surrounded by a peripheral band of neutrophils [47].

#### **3.2. Advantages and disadvantages of cryoablation**

The one of the best advantages of cryoablation is the ability to monitor the ablation zone in real time. As the ablation proceeds, formation of an ice-ball occurs that is visible on ultrasound (US), magnetic resonance (MR), and CT. This occurs because the water molecules undergo a phase change and subsequent change in density. For example, during a cryoablation, the ablative zone will become hypoattenuating on CT. The leading edge of the ablation marks 0°C, since this region is where the phase change from liquid to solid is occurring [47].

whereas lymphocytes in patients post HR do not. Reintroduced tumor cells are also rendered

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Cryoablation is proven to create a more robust immunological response than hyperthermic ablative techniques such as RFA and MWA. Higher DC antigen loading due to hypothermic cytotoxicity generates the more robust immunologic response. Increased antigen presentation and subsequent heightened immune response are due to two main factors: increased antigen in the native conformation and less coagulation in the ablation zone [47]. The hypothermic mechanism of cryoablation leads to less regional coagulative effect when compared to hyperthermic ablations. This allows the antigens produced by cryoablation to more readily enter circulation and regional lymph nodes for presentation to DCs. The ability of cryoablation to preserve circulation is beneficial for stimulating the immune system but can be detrimental. The spilling of tumor antigens into circulation and subsequent cytokine release is also

Heightened levels of cytokines such as IL-1β, IL-6, TNF-α, and NF-κB are seen after cryoablation [65, 66]. Cryoablation used on hepatic malignancy will increase the levels of these specific cytokines up to 15–25 times more when compared to RFA. Interestingly, Erinjeri et al. found that the changes in WBC count increased linearly with ablation size but the levels of IL-6 did not [65], suggesting that larger ablative zones could trigger higher tumor-specific immune responses without added increased risk for cryoshock. Additionally, this group found that the predominant cytokines that are released post cryoablation, IL-6 and IL-10, stimulate a Th2

According to the Th1/Th2 model, each subset triggers a different type of immunity. Th1 triggers cytotoxic lymphocytes and cellular immunity, whereas Th2 stimulates B-cells and antibody production. Signals that stimulate the Th1 response include IL-2, IL-12, IFN-γ, and TNF, and the cytokines that trigger the Th2 response include IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13 [65, 67]. In addition, IL-6, which activates NF-κB and STAT3, has been implicated in hepatic regeneration and increased tumor regeneration post ablation. Regeneration is mediated by NF-κb and STAT3, both of which are activated by IL-6 [65]. Since, both IL-6 and IL-10 are increased after not only cryoablation but also hyperthermic ablation, they are potential targets for adjuvant immunologic therapy. Decreasing IL-6 could potentially increase the Th1 response (cellular-mediated tumor immunity) and decrease the growth of primary and meta-

Immune checkpoint inhibitor therapy, a new oncologic therapy that uses monoclonal antibody to target and block the T cell surface receptor CTLA-4, has potential use in combination therapy with cryoablation. The function of CTLA-4 is to inhibit self-reactive T-cells in order to prevent autoimmune diseases. However, in the case of cancer, it is beneficial for T cells to be able to recognize specific "self" cells. It has been shown that blockade of CTLA-4 will increase the CD8 T cell response as well as CD4 T cell memory when used as a monotherapy or combination immune therapy [69, 70]. Phase 3 trials using ipilimumab, a CTLA-4 blocking monoclonal antibody, have showed improved recurrence-free survival when used as an adjuvant treatment in patients with high-risk melanoma [71]. Combination cryotherapy with CTLA-4 blockade has been studied in prostate cancer. While cryoablation alone has not been

less effective by the immunologic response [64].

believed to be responsible for cryoshock.

static hepatic malignancy post ablation [68].

response.

In contrast to other modalities, such as RFA, each cryoprobe acts independently from the others and can be used simultaneously to tailor the shape of the ablation to the tumor. This is in sharp contrast to ablating with multiple RF probes where only one probe can be active at a time and they must be operated sequentially. Cryoablation additionally offers better pain control when compared to RFA [47]. The cooling of the tissues can create a level of analgesia not offered by hyperthermic ablative techniques. Cryoablation significantly reduces the amount of opioids used in the 24 hours following the procedure leading to shorter hospital stays [55]. In regards to HCC, it has been shown that RFA can induce ischemia-reperfusion injury of the liver resulting in cancer growth. With cryoablation, there is a lower potential for this type of injury decreasing the risk of cancer growth [56]. Cryoablation can also be used in patients who are candidates for RFA. Patients who are candidates are those with tumors <5 cm, single lesions, or multiple lesions <3 cm with a Child-Pugh class A or B liver function [57].

Cryoablation results in a robust inflammatory response following the procedure. This in combination with the fact that the released proteins return to their native conformation produces a large potential to create beneficial antitumor immunologic responses. The large amount of unaltered tumor antigen coupled with a large inflammatory response creates a scenario in which significant numbers of DCs are able to present a large amount of antigen to T cells [47, 50, 54]. The potential immunologic response will be discussed in detail later. This robust inflammatory response also presents a significant disadvantage, cryoshock [47, 57–59].

Cryoshock is a systemic immune response that leads to hypotension, respiratory distress, multiorgan failure, and disseminated intravascular coagulation. Similar reactions are not seen in patients treated with hyperthermic ablations. Cryoshock occurs in up to 1% of patients who undergo hepatic cryotherapy. Of this 1%, up to 18% of patients can die because of cryoshock [57, 58]. Cryoshock is thought to be mediated by the production of cytokine, such as IL-1β, IL-6, and tumor necrosis factor (TNF), from the robust immune response created by cryoablation [47, 57–60]. These are similar to the mediators found in patients with septic shock [59, 65]. Cryoshock typically occurs when large volume liver ablations are attempted [47]. An additional disadvantage of cryoablation is bleeding complications. Typically, these occur when performing large ablations within the liver. Frozen tissues are extremely brittle and may fracture leading to significant bleeding. For this reason, the user must be careful to not torque or reposition the probes once the ablation has started [61]. Although cryoablation has various disadvantages, it has a similar complication rate compared to RFA and remains a relatively safe and effective procedure for the treatment of HCC [57–60, 62].

#### **3.3. Immunologic response to cryoablation**

For several decades, the immune response to cryoablation has been known. In the 1970s, antitumor antibodies were first seen in humans following cryoablation [63]. Since then, it has been shown that cryoablation will induce specific anti-tumor cytotoxic effects post ablation. Lymphocytes produced post-ablation show specific affinity for tumor cells when rechallenged, whereas lymphocytes in patients post HR do not. Reintroduced tumor cells are also rendered less effective by the immunologic response [64].

phase change and subsequent change in density. For example, during a cryoablation, the ablative zone will become hypoattenuating on CT. The leading edge of the ablation marks

In contrast to other modalities, such as RFA, each cryoprobe acts independently from the others and can be used simultaneously to tailor the shape of the ablation to the tumor. This is in sharp contrast to ablating with multiple RF probes where only one probe can be active at a time and they must be operated sequentially. Cryoablation additionally offers better pain control when compared to RFA [47]. The cooling of the tissues can create a level of analgesia not offered by hyperthermic ablative techniques. Cryoablation significantly reduces the amount of opioids used in the 24 hours following the procedure leading to shorter hospital stays [55]. In regards to HCC, it has been shown that RFA can induce ischemia-reperfusion injury of the liver resulting in cancer growth. With cryoablation, there is a lower potential for this type of injury decreasing the risk of cancer growth [56]. Cryoablation can also be used in patients who are candidates for RFA. Patients who are candidates are those with tumors <5 cm, single

0°C, since this region is where the phase change from liquid to solid is occurring [47].

158 Hepatocellular Carcinoma - Advances in Diagnosis and Treatment

lesions, or multiple lesions <3 cm with a Child-Pugh class A or B liver function [57].

Cryoablation results in a robust inflammatory response following the procedure. This in combination with the fact that the released proteins return to their native conformation produces a large potential to create beneficial antitumor immunologic responses. The large amount of unaltered tumor antigen coupled with a large inflammatory response creates a scenario in which significant numbers of DCs are able to present a large amount of antigen to T cells [47, 50, 54]. The potential immunologic response will be discussed in detail later. This robust inflammatory response also presents a significant disadvantage, cryoshock [47, 57–59].

Cryoshock is a systemic immune response that leads to hypotension, respiratory distress, multiorgan failure, and disseminated intravascular coagulation. Similar reactions are not seen in patients treated with hyperthermic ablations. Cryoshock occurs in up to 1% of patients who undergo hepatic cryotherapy. Of this 1%, up to 18% of patients can die because of cryoshock [57, 58]. Cryoshock is thought to be mediated by the production of cytokine, such as IL-1β, IL-6, and tumor necrosis factor (TNF), from the robust immune response created by cryoablation [47, 57–60]. These are similar to the mediators found in patients with septic shock [59, 65]. Cryoshock typically occurs when large volume liver ablations are attempted [47]. An additional disadvantage of cryoablation is bleeding complications. Typically, these occur when performing large ablations within the liver. Frozen tissues are extremely brittle and may fracture leading to significant bleeding. For this reason, the user must be careful to not torque or reposition the probes once the ablation has started [61]. Although cryoablation has various disadvantages, it has a similar complication rate compared to RFA and remains a relatively

For several decades, the immune response to cryoablation has been known. In the 1970s, antitumor antibodies were first seen in humans following cryoablation [63]. Since then, it has been shown that cryoablation will induce specific anti-tumor cytotoxic effects post ablation. Lymphocytes produced post-ablation show specific affinity for tumor cells when rechallenged,

safe and effective procedure for the treatment of HCC [57–60, 62].

**3.3. Immunologic response to cryoablation**

Cryoablation is proven to create a more robust immunological response than hyperthermic ablative techniques such as RFA and MWA. Higher DC antigen loading due to hypothermic cytotoxicity generates the more robust immunologic response. Increased antigen presentation and subsequent heightened immune response are due to two main factors: increased antigen in the native conformation and less coagulation in the ablation zone [47]. The hypothermic mechanism of cryoablation leads to less regional coagulative effect when compared to hyperthermic ablations. This allows the antigens produced by cryoablation to more readily enter circulation and regional lymph nodes for presentation to DCs. The ability of cryoablation to preserve circulation is beneficial for stimulating the immune system but can be detrimental. The spilling of tumor antigens into circulation and subsequent cytokine release is also believed to be responsible for cryoshock.

Heightened levels of cytokines such as IL-1β, IL-6, TNF-α, and NF-κB are seen after cryoablation [65, 66]. Cryoablation used on hepatic malignancy will increase the levels of these specific cytokines up to 15–25 times more when compared to RFA. Interestingly, Erinjeri et al. found that the changes in WBC count increased linearly with ablation size but the levels of IL-6 did not [65], suggesting that larger ablative zones could trigger higher tumor-specific immune responses without added increased risk for cryoshock. Additionally, this group found that the predominant cytokines that are released post cryoablation, IL-6 and IL-10, stimulate a Th2 response.

According to the Th1/Th2 model, each subset triggers a different type of immunity. Th1 triggers cytotoxic lymphocytes and cellular immunity, whereas Th2 stimulates B-cells and antibody production. Signals that stimulate the Th1 response include IL-2, IL-12, IFN-γ, and TNF, and the cytokines that trigger the Th2 response include IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13 [65, 67]. In addition, IL-6, which activates NF-κB and STAT3, has been implicated in hepatic regeneration and increased tumor regeneration post ablation. Regeneration is mediated by NF-κb and STAT3, both of which are activated by IL-6 [65]. Since, both IL-6 and IL-10 are increased after not only cryoablation but also hyperthermic ablation, they are potential targets for adjuvant immunologic therapy. Decreasing IL-6 could potentially increase the Th1 response (cellular-mediated tumor immunity) and decrease the growth of primary and metastatic hepatic malignancy post ablation [68].

Immune checkpoint inhibitor therapy, a new oncologic therapy that uses monoclonal antibody to target and block the T cell surface receptor CTLA-4, has potential use in combination therapy with cryoablation. The function of CTLA-4 is to inhibit self-reactive T-cells in order to prevent autoimmune diseases. However, in the case of cancer, it is beneficial for T cells to be able to recognize specific "self" cells. It has been shown that blockade of CTLA-4 will increase the CD8 T cell response as well as CD4 T cell memory when used as a monotherapy or combination immune therapy [69, 70]. Phase 3 trials using ipilimumab, a CTLA-4 blocking monoclonal antibody, have showed improved recurrence-free survival when used as an adjuvant treatment in patients with high-risk melanoma [71]. Combination cryotherapy with CTLA-4 blockade has been studied in prostate cancer. While cryoablation alone has not been shown to mediate the rejection of metastatic lesions, when combined with CTLA-4 blockade, it can mediate rejection of metastatic lesions and prevent disease recurrence [72].

**4.2. Advantages and disadvantages of MWA**

regions that are not possible with RFA [88].

active cooling system needs to be employed [86, 89].

ablation (right).

potential use ablating larger lesions than is currently possible.

MWA has the distinct advantage of being able to penetrate through high impedance tissues, meaning that even if charred or desiccated tissues build up near the probe, the field is able to penetrate and continue enlarging the ablation zone. Since MWA does not rely on conduction of tissue, heat is able to penetrate tissues with a high impedance such as lung or bone [86].

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Multiple MWA antennas are able to be used synergistically to enlarge the ablation zone, achieve higher temperatures, or concomitantly ablate multiple lesions [87]. While RFA using multiple probes requires the probes to be used in series, MWA with multiple antennas can be used simultaneously with one power source. Due to the properties of MWA, there is future

The peak temperatures achieved in the central zone can readily exceed 100°C. The ability to achieve higher temperatures and use multiple probes simultaneously means shorter treatment times and larger area of coagulative necrosis and lethal hyperthermia. Higher temperatures and larger central zone lessen the effect of nearby heat sinks. It has been shown that large vessels <10 mm in size will not affect the ablation, making it possible to ablate lesions in

While MWA has many advantages, its ability to deliver a high amount of energy comes with several trade-offs. Coaxial cables have excellent properties for this application and are thus used to connect the antenna to the microwave generator. However, the coaxial cables used have a large diameter in order to avoid dangerous cable overheating. Larger diameter decreases the risk of overheating but becomes cumbersome and inflexible leading to difficulties while manipulating the antennas and performing the procedure [89]. The microwave antennas are likewise made using coaxial cable and also suffer from the same problem. In order for the antenna to handle higher power levels, the diameter must be increased or an

**Figure 2.** Hepatic microwave ablation. Pre-procedure imaging demonstrates a focal HCC lesion at the hepatic dome (left, arrow). Two microwave needles were advanced into the lesion, with gas bubbles developing during the ablation (middle). Follow-up imaging demonstrates a focal defect without enhancing viable tissue consistent with a complete

While cryoablation has been strongly shown to activate the immune system, the opposite has also been seen. Multiple animal models have shown susceptibility to rechallenge and increased metastasis post ablation [73–75]. A possible explanation for these results is variation in the technical factors of cryoablation. Differing animal models, methods of freezing, length, number of freeze-thaw cycles, differences in minimum temperature achieved as well as differing ablation zone size and position all contribute to different clinical outcome and immunologic stimulation or anergy [73]. Sabel et al. established that variation in the technical parameters of cryoablation indeed affect the ratio of apoptosis to necrosis and subsequent immune response. Sabel et al. investigated the rate of freezing in an animal model using either a low or high rate of freezing. They found that a high rate of freezing induced a higher amount of necrosis when compared to a low rate of freezing. The high rate induced more danger signals stimulating a strong anti-tumor response [73]. A high ratio of apoptosis to necrosis has been shown to downregulate the immunologic response and even induce anergy [76]. When apoptotic cells are presented to DCs, a lower amount of TNF-α, IL-1β, IL-8, IL-10, IL-12, and granulocyte macrophage colony-stimulating factor (GM-CSF) produce inhibitory effects on these cells [77, 78]. The ablation zone size and percentage of tumor encompassed may play a role in the immunologic response. An experiment conducted in a murine metastatic liver tumor model demonstrated that smaller volume ablations show a significant decrease in metastasis [79].
