**4. Reaction of immune system to renal artery embolization**

RC, with the tumor growth, and then the spread of tumor tissue beyond the original lo‐ cation, begins to affect the activity of the immune system [14]. Nakano et al [5] indicate the importance of cell proliferation inhibitory factor present in the serum of patients with RC. Lymphocytes in RC patients without any therapy, stimulated in vitro with PHA (phytohemagglutinin) in the presence of own serum responded very weakly to this mitogen. After RAE the impact of this inhibiting factor enhanced and proliferation of PHA-stimulated lymphocyte was still lowered [5]. Nephrectomy in patients not treated with RAE before surgery did not influence the ability of cells to stimulation by PHA. In contrary, patients who had RAE prior to nephrectomy the proliferation inhibitory factor quickly disappeared and proliferative response to PHA was normal already 2 months af‐ ter surgery [5]. Catalona et al [34] reported that cell response to Con A (concanavalin A) is impaired in case of urological cancers, including RC, and the cells have a high immu‐ no-suppressor activity. The abolition of the high suppressor activity may be necessary for effective treatment of RC [34]. Osada et al [35] in their study of 50 patients with RC confirmed a significant increase of helper and cytotoxic NK lymphocytes 10-12 months after RAE. This was very impressive, when compared to lowered values of these cells prior to RAE, suggesting that RAE enhanced the immune status [35]. Similar results were obtained by Bakke et al [13], who conducted a study of NK cell activity in patients with RC after RAE. Blood samples of 30 patients were taken before RAE and 24, 48, 72 and 96 hours after surgery. Surgery was performed to remove the kidney from 5 to 7 days after RAE. RAE resulted in increase in NK cells, with peak values observed after 48 hours [13]. RAE in patients with RC is performed in presence of potential existence of immune deficiency caused by cancer itself. Therefore, in this case, the immune responses (still poorly understood and inconsistent) observed at different times after embolization (usually few-several days after RAE) will be the result of the two, often different operat‐ ing mechanisms: 1. response to ischemia and tissue necrosis and inflammation in the area of embolization of a probable stimulation of the macrophage-monocyte system; 2. release from tumor tissues of various factors affecting the immune system, at least some of which appear to have an immunosuppressive effect. The main task of the immune system is to maintain homeostasis. The basic unit, often defined as "immune orchestra conductor" is thymus-dependent T lymphocyte, which, based on the phenomenon of re‐ striction major histocompatibility complex I and II expresses the phenomenon of violence against its own unnormal or changed antigens, and the phenomenon of tolerance to its own antigens [32, 33]. Embolization may lead to stimulation of the immune system in the following mechanism: close off blood supply to the tumor leads to necrosis which gives a chance to enhance antigenicity of cancer cells and evoke the potential amplifica‐ tion of the immune system [14]. This leads in turn to destruction of tumor tissue by infil‐ tration with cytolytic immunocompetent cells.

The overall 5- and 10-year survival for 118 patients embolized before nephrectomy was 62% and 47%, respectively, and it was 35% and 23%, respectively, for the matched group of 116 pa‐ tients treated with surgery alone (p = 0.01). The most important finding of this study was an ap‐ parent importance of preoperative embolization in improving patients' survival. This finding

In conclusion, the available data suggest that RAE is a convenient, relatively tolerable man‐ agement option in patients with unresectable renal tumours and in patients unfit or unwill‐ ing to undergo surgery as a means of palliation of local symptoms and improving clinical status. We believe there is also a role for this procedure in asymptomatic patients who have potentially resectable disease who are unfit or unwilling to undergo surgery, and in asymp‐

RC, with the tumor growth, and then the spread of tumor tissue beyond the original lo‐ cation, begins to affect the activity of the immune system [14]. Nakano et al [5] indicate the importance of cell proliferation inhibitory factor present in the serum of patients with RC. Lymphocytes in RC patients without any therapy, stimulated in vitro with PHA (phytohemagglutinin) in the presence of own serum responded very weakly to this mitogen. After RAE the impact of this inhibiting factor enhanced and proliferation of PHA-stimulated lymphocyte was still lowered [5]. Nephrectomy in patients not treated with RAE before surgery did not influence the ability of cells to stimulation by PHA. In contrary, patients who had RAE prior to nephrectomy the proliferation inhibitory factor quickly disappeared and proliferative response to PHA was normal already 2 months af‐ ter surgery [5]. Catalona et al [34] reported that cell response to Con A (concanavalin A) is impaired in case of urological cancers, including RC, and the cells have a high immu‐ no-suppressor activity. The abolition of the high suppressor activity may be necessary for effective treatment of RC [34]. Osada et al [35] in their study of 50 patients with RC confirmed a significant increase of helper and cytotoxic NK lymphocytes 10-12 months after RAE. This was very impressive, when compared to lowered values of these cells prior to RAE, suggesting that RAE enhanced the immune status [35]. Similar results were obtained by Bakke et al [13], who conducted a study of NK cell activity in patients with RC after RAE. Blood samples of 30 patients were taken before RAE and 24, 48, 72 and 96 hours after surgery. Surgery was performed to remove the kidney from 5 to 7 days after RAE. RAE resulted in increase in NK cells, with peak values observed after 48 hours [13]. RAE in patients with RC is performed in presence of potential existence of immune deficiency caused by cancer itself. Therefore, in this case, the immune responses (still poorly understood and inconsistent) observed at different times after embolization (usually few-several days after RAE) will be the result of the two, often different operat‐ ing mechanisms: 1. response to ischemia and tissue necrosis and inflammation in the area of embolization of a probable stimulation of the macrophage-monocyte system; 2. release from tumor tissues of various factors affecting the immune system, at least some

needs to be interpreted with caution and confirmed in a prospective randomized trial.

**4. Reaction of immune system to renal artery embolization**

tomatic patients with inoperable metastatic disease.

102 Renal Tumor

Recent studies in patients with metastatic RC have shown a small survival advantage in pa‐ tients undergoing radical nephrectomy followed by immunotherapy; however, these studies are biased towards patients with good performance status aqccording to ECOG (Eastern Co‐ operative Oncology Group) scale status 0 or status 1. This small survival benefit should also be viewed in light of the morbidity and mortality associated with a large surgical procedure. The increased morbidity associated with radical nephrectomy may preclude or delay the ad‐ ministration of systemic immunotherapy, which has demonstrated reproducible response rates of 10–20% [15].

In two randomized trials with identical design, patients who underwent nephrectomy fol‐ lowed by interferon alpha (IFN-α) therapy had improved survival (median 13.6 months) compared with those treated with IFN-α alone (median 7.8 months) [15, 16]. The antivascu‐ lar endothelial growth factor (VEGF) antibody; the multityrosine kinase inhibitors, sorafe‐ nib, sunitinib, and pazopanib; and the mammalian target of rapamycin (mTOR) inhibitors, temsirolimus and everolimus, have become the mainstay of therapy for the vast majority of patients with metastatic renal cell carcinoma (mRCC). Large randomized controlled clinical trials have shown improved progression-free survival with these agents and improved sur‐ vival in selected populations, but the majority of these study patients had prior nephrecto‐ my and good performance status [16, 17, 20, 21].

In own studies [14] we examined functional status of immunocompetent cells isolated from peripheral blood of patients with advanced RC treated with RAE. Blood samples were collected by vein puncture and peripheral blood mononuclear cells (PBMC) were isolated on Ficol-Paque gradient, and after determination of cell viability (usually no less than 80% viable cells), the microcultures were set up in triplicates (105 cells/0.2 ml RPMI + 15% autologous inactivated serum) in Nuncoln microplates. Respective triplicates were left without stimulation or stimulated with phytohemagglutinin (PHA, HA16, Murex Bio‐ tech Ltd Dartford U.K., 0.4 μg/cult.) or with concanavalin A (Con A, Sigma, 8 μg/cult.). The plates were placed inside the anechoic chamber in the ASSAB incubator at 37o C and 5% CO2. An identical plate of control cultures was also set up and placed in the AS‐ SAB incubator beyond the chamber. At 24h of incubation, rearrangements of the cultures were performed as described elsewhere [32,33].

As a result of rearrangements of cultures performed at 24 h, the following parameters of T cell and monocyte activities were measured at the end of cultures: T lymphocyte re‐ sponse to PHA and to Con A, saturation of IL-2 receptors, T cell suppressive activity (SAT index), and the index of monocyte immunogenic activity (LM) related to the ratio of produced monokines (IL-1β versus IL-1ra) [32]. For the last 18h of incubation, 3H-thy‐ midine (3HTdR, Amersham, U.K., spec act. 5Ci/mM) was added into the cultures in a dose of 0.4 μCi/cult.

**Investigated parameter RAE-treated RC patients (T 3 and 4)**

PHA Lowering after 2-6 weeks \*

ConA Lowering after 2-6 weeks \*

IL-2 Lowering after 2-6 weeks

CD3+ Increase after 2-6 weks

CD4+ Increase after 2-6 weeks

CD8+ Lowering after 2-6 weeks \*

TGF-β Lowering after 2-6 weeks \*

IL-1ra Lowering after 2-6 weeks \*

IL-10 Lowering after 2-6 weeks \*

**Table 1.** Summary of changes in investigated functional parameters of immune system in a group of 30 patients with

NK No significant differences after 2-6 weeks

CD4+/CD25high Increase after 2-6 weeks

SAT

IL-1β

IL-6

advanced RC treated with RAE (p<0.05).

LM No significant differences after 2-6 weeks

**N=30**

http://dx.doi.org/10.5772/54116

105

Renal Artery Embolization in Treatment of Renal Cancer with Emphasis on Response of Immune System

Increase after 12 weeks

Lowering after 12 weeks

Increase after 12 weeks

Lowering after 12 weeks

Increase after 2-6 weeks No significant differences after 12 weeks

No significant differences after 12 weeks

Increase after 12 weeks

Lowering after 12 weeks

Increase after 12 weeks

No significant differences after 12 weeks

Increase after 2-6 weeks Lowering after 12 weeks

Increase after 2-6 weeks Lowering after 12 weeks

Increase after 12 weeks

Increase after 12 weeks

Increase after 12 weeks

At 72h the cultures were harvested and incorporation of 3HTdR was measured in Pack‐ ard Tri carb 2100 TR scintillation counter. The results were calculated as a mean value of dpm (desintegrations per minute) per triplicate of cultures ± SD. The experiments were repeated 10 times, and the results observed in the exposed cultures were compared with those obtained in the control cultures. The data were analyzed with STATGRAPHICS PLUS 6.0 version. The differences between the mean values were assumed statistically significant if the p values, calculated withthe use of U Mann-Whitney's test, were lower than 0.05.

The results obtained in this study are summarized in Table 1 and described in detail elsewhere [14].

In the analysis of 50 patients with RC treated with RAE, we selected 30 patients where RAE was the only form of treatment. In this group of patients the immune response was studied at different times after the palliative RAE (output test, the test after 2-6 weeks and at 12 weeks after RAE) successive assessment of significant differences in the magni‐ tude and direction of change of parameters characterizing the efficiency of the immune system. It was found that RAE performed in patients with advanced RC exerts immuno‐ modulatory effect on the immune response manifested by the increase of the prolifera‐ tive response to PHA and the percentage of CD4 + cells, and significant increase in the value of saturation of the receptors, IL-2, a cytokine with protrophic properties (Table 1). After RAE significant increase was observed in inflammatory response manifested by the increase of T regulatory cells, which can be a potential source of IL-10, cytokine inhibi‐ tion of the function of the inflammatory response (Table 1).

It was found that RAE lowers the suppressive action of neoplastic cells on the immune sys‐ tem, results in normalization of disordered proportion of lymphocyte subpopulations (CD4, CD8) and enhances the antiinflamatory response (increases levels of certain cytokines- IL-10 and IL-1ra). All together, the result reveal stimulation of certain functions of immunocompe‐ tent cells isolated from blood of RAE-treated RC patients. Clinical relevance of these find‐ ings and concluding whether or not RAE improved immune status of patients needs further studies [1, 14].

The changes in the immune system may, however be heterogeneous and multidirectional and individually changebale. This would indicate that the systemic inflammatory response is not only associated with the release of cytokines from a kidney tumor, and it rather results from the defective immune response in patients with advanced cancer [14].

Renal Artery Embolization in Treatment of Renal Cancer with Emphasis on Response of Immune System http://dx.doi.org/10.5772/54116 105

As a result of rearrangements of cultures performed at 24 h, the following parameters of T cell and monocyte activities were measured at the end of cultures: T lymphocyte re‐ sponse to PHA and to Con A, saturation of IL-2 receptors, T cell suppressive activity (SAT index), and the index of monocyte immunogenic activity (LM) related to the ratio of produced monokines (IL-1β versus IL-1ra) [32]. For the last 18h of incubation, 3H-thy‐ midine (3HTdR, Amersham, U.K., spec act. 5Ci/mM) was added into the cultures in a

At 72h the cultures were harvested and incorporation of 3HTdR was measured in Pack‐ ard Tri carb 2100 TR scintillation counter. The results were calculated as a mean value of dpm (desintegrations per minute) per triplicate of cultures ± SD. The experiments were repeated 10 times, and the results observed in the exposed cultures were compared with those obtained in the control cultures. The data were analyzed with STATGRAPHICS PLUS 6.0 version. The differences between the mean values were assumed statistically significant if the p values, calculated withthe use of U Mann-Whitney's test, were lower

The results obtained in this study are summarized in Table 1 and described in detail

In the analysis of 50 patients with RC treated with RAE, we selected 30 patients where RAE was the only form of treatment. In this group of patients the immune response was studied at different times after the palliative RAE (output test, the test after 2-6 weeks and at 12 weeks after RAE) successive assessment of significant differences in the magni‐ tude and direction of change of parameters characterizing the efficiency of the immune system. It was found that RAE performed in patients with advanced RC exerts immuno‐ modulatory effect on the immune response manifested by the increase of the prolifera‐ tive response to PHA and the percentage of CD4 + cells, and significant increase in the value of saturation of the receptors, IL-2, a cytokine with protrophic properties (Table 1). After RAE significant increase was observed in inflammatory response manifested by the increase of T regulatory cells, which can be a potential source of IL-10, cytokine inhibi‐

It was found that RAE lowers the suppressive action of neoplastic cells on the immune sys‐ tem, results in normalization of disordered proportion of lymphocyte subpopulations (CD4, CD8) and enhances the antiinflamatory response (increases levels of certain cytokines- IL-10 and IL-1ra). All together, the result reveal stimulation of certain functions of immunocompe‐ tent cells isolated from blood of RAE-treated RC patients. Clinical relevance of these find‐ ings and concluding whether or not RAE improved immune status of patients needs further

The changes in the immune system may, however be heterogeneous and multidirectional and individually changebale. This would indicate that the systemic inflammatory response is not only associated with the release of cytokines from a kidney tumor, and it rather results

from the defective immune response in patients with advanced cancer [14].

tion of the function of the inflammatory response (Table 1).

dose of 0.4 μCi/cult.

104 Renal Tumor

than 0.05.

elsewhere [14].

studies [1, 14].


**Table 1.** Summary of changes in investigated functional parameters of immune system in a group of 30 patients with advanced RC treated with RAE (p<0.05).

## **5. Summary and conclusions**

In summary, the present authors conclude that patients with advanced RC benefit from RAE with longer survival. RAE applied prior to nephrectomy facilitates surgery and additionally prolongs survival. Additionally, RAE appears to be a potent immunostimulatory agent. It is our strong feeling that in specialistic urologic centers RAE is a safe procedure which suc‐ cours the complex therapeutic process in patients with RC.

[9] Christensen K, Dyreborg U, Andersen JF, Nissen HM. The value of transvascular em‐

Renal Artery Embolization in Treatment of Renal Cancer with Emphasis on Response of Immune System

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107

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