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Effect of Propofol and Desflurane on Immune Cell Populations in Breast Cancer Patients: A Randomized Trial

Several factors can affect the perioperative immune function. We evaluated the effect of propofol and desflurane anesthesia on the surgery-induced immune perturbation in patients undergoing breast cancer surgery. The patients were randomly assigned to receive propofol (n = 20) or desflurane (n = 20) anesthesia. The total and differential white blood cell counts were determined with lymphocyte subpopulations before and 1 hr after anesthesia induction and at 24 hr postoperatively. Plasma concentrations of interleukin (IL)-2 and IL-4 were also measured. Both propofol and desflurane anesthesia preserved the IL-2/IL-4 and CD4+/CD8+ T cell ratio. Leukocytes were lower in the propofol group than in the desflurane group at 1 hr after induction (median [quartiles], 4.98 [3.87-6.31] vs. 5.84 [5.18-7.94] 103/µL) and 24 hr postoperatively (6.92 [5.54-6.86] vs. 7.62 [6.22-9.21] 103/µL). NK cells significantly decreased 1 hr after induction in the propofol group (0.41 [0.34-0.53] to 0.25 [0.21-0.33] 103/µL), but not in the desflurane group (0.33 [0.29-0.48] to 0.38 [0.30-0.56] 103/µL). Our findings indicate that both propofol and desflurane anesthesia for breast cancer surgery induce a favorable immune response in terms of preservation of IL-2/IL-4 and CD4+/CD8+ T cell ratio in the perioperative period. With respect to leukocytes and NK cells, desflurane anesthesia is associated with less adverse immune responses than propofol anesthesia during surgery for breast cancer. (Clinical trial registration at https://cris.nih.go.kr/cris number: KCT0000939) Graphical Abstract


INTRODUCTION
Breast cancer is the most commonly diagnosed cancer in the developed and developing countries and the leading cause of cancer mortality among women, most of which is attributable to recurrence and metastasis (1). Curative surgical resection re mains the main treatment of breast cancer, but it is recognized that surgical resection may paradoxically create a critical win dow for cancer recurrence (2). Cancer surgery can induce an acute inflammatory response, related to local tissue damage and the shedding of malignant cells into the blood and lympha tic circulation despite meticulous surgical manipulation (3). Pre existing micrometastasis may be undetectable even in localized breast cancer (4). Accumulating evidence suggests that several factors during the perioperative period can affect the immune system (2,57). These include the surgery per se, pain, anxiety, hypothermia, blood transfusion, anesthetic technique, and an esthetic drugs. Impairment in the immune system may allow malignant cells to escape immunosurveillance and metastasize in the perioperative period (8) as well as increase the risk of post operative complications, such as systemic inflammatory response syndrome, sepsis, and multiorgan failure (9).
Propofol and desflurane are widely used anesthetics for gen eral anesthesia. Desflurane is the most recently introduced in halation agent and facilitates a fasttrack recovery when com pared with other inhalation agents. It allows the patient to re sume earlier normal activities after surgery (10,11). It has been reported that intravenous and volatile anesthetics produce dif ferent immune system effect. Several studies have demonstrat ed that propofol has a more favorable immunomodulating pro file than other anesthetic agents (12,13). On the other hand, isoflurane and sevoflurane, volatile anesthetics, have been re ported to suppress natural killer (NK) function and increase proinflammatory cytokines more than propofol (1316). More over, there have been few investigations of the effect of desflu rane on the immune system (17,18). Leukocytes, including granulocytes and lymphocytes, are the cellular mediators of immune function. CD4 + T cells include type1 helper T (Th1) and type2 helper T (Th2) cells. Th1 cells secrete cytokines such as interferon γ, interleukin (IL2), and tumor necrosis factor, which cooperate with cytotoxic CD8 + T cells. In contrast, Th2 cells are associated with IL4, IL6, IL10, and IL13 and promote a humoral, protumorigenic response (19). A decrease in the balance of the Th1/Th2 ratio is associat ed with suppression of cellmediated immunity (CMI) and has been the focus of many previous studies on perioperative im  (17,20,21).
The objective of this prospective, randomized clinical study was to compare the effect of propofol and desflurane on peri operative immune cell populations in patients undergoing breast cancer surgery. We examined perioperative total and differen tial white blood cell counts, lymphocyte subsets, including CD4 + T cells, CD8 + T cells, and NK cells, and plasma concentrations of IL2 (Th1 cytokine) and IL4 (Th2 cytokine) in patients un dergoing primary breast cancer surgery. We hypothesized that propofol would induce less adverse immune effects, as indicat ed by preservation of the Th1/Th2 ratio and attenuation of the NK cell count decrease.

MATERIALS AND METHODS
We studied female patients aged 18 to 85 yr, American Society of Anesthesiologists (ASA) physical status IIII, undergoing mas tectomy, axillary node dissection or wide local tumor excision at Ewha Womans University Hospital. Exclusion criteria were previous breast cancer surgery (except diagnostic biopsy), in flammatory breast cancer, evidence of systemic infections (clini cally defined or elevated Creactive protein levels, leukocytosis, body temperature ≥ 37.8°C) and any contraindication to drugs used in this study.
On arrival at the operating room, standard ASA monitors were used throughout the surgery. The patients were assigned ran domly to receive propofol (n = 20) or desflurane (n = 20) anes thesia using a computergenerated randomization table. In the propofol group, anesthesia was induced with propofol 1.52 mg/kg and fentanyl 12 µg/kg and maintained with a continu ous infusion of propofol 48 mg/kg/h. In the desflurane group, anesthesia was induced with thiopental sodium 45 mg/kg and fentanyl 12 µg/kg and maintained with inhalation of desflurane at an endtidal concentration of 3%7%. Propofol and desflu rane were titrated to maintain the bispectral index (BIS) value between 40 and 60.
In all patients, tracheal intubation was facilitated by adminis tration of rocuronium 0.6 mg/kg and the lungs were ventilated with a mixture of 1:1 O2/N2O. A continuous infusion of vecuroni um 0.02 mg/kg/hr was performed for muscle relaxation and a bolus of fentanyl 0.51 µg/kg was given as needed. Postopera tive analgesia was provided with nonsteroidal antiinflamma tory drugs and tramadol on request. Pain scores at 12 and 24 hr postoperatively were evaluated. The standardized protocol for the surgical procedure and postoperative patient care was used to minimize any difference in surgical impact between the ran domized groups.

Sampling
Venous blood samples were collected before and 1 hr after an esthesia induction and at 24 hr postoperatively. The blood sam ples were collected in ethylenediamine tetraacetic acid (EDTA) tubes for blood cell counting and lymphocyte analysis and in a serum separator tube (SST) for cytokine concentration testing. Samples in SSTs were centrifuged (3,000 g, 10 min) and the re sulting serum was stored at 20°C until analysis.

Blood cell counts
A complete blood count was performed on all blood samples using an automated hemoanalyzer. Total and differential leu kocyte counts were determined.

Cytokine measurements
Plasma concentrations of IL2 and IL4 were measured with an enzymelinked immunosorbent assay (ELISA) using a commer cially available kit (Human IL2 and IL4 Quantikine Elisa Kit, R&D Systems, Inc., USA) according to protocols provided. The assays were read using an ELISA microplate reader (Spectra Max 190, Molecular Devices, Sunnyvale, CA, USA). We deter mined the Th1/Th2 balance by calculating the IL2/IL4 ratio.

Statistical analysis
SPSS (ver. 18.0, Chicago, IL, USA) was used for the statistical analysis. The primary endpoint was the Th1/Th2 ratio (IL2/IL 4) on the first postoperative day. Using a twosided design at a significance level of 5% with a probability of 85%, an estimated 19 patients per group were needed to detect an effect size based on previous data with SD approximately 0.8 (22). Continuous variables were analysed by student's ttests or Mann Whitney Utest after assessment for normality. Discrete variables were analysed using the chisquared test or Fisher's exact test. Data of white blood cells, lymphocyte subsets counts and cytokines were analyzed by repeated measures analysis of variance (AN OVA) followed by Bonferroni correction. Mann Whitney Utest was used to analyse a difference between groups. Results are presented as the means ± standard deviations (SD) or as medi ans with interquartile ranges (IQR) as appropriate. It was con sidered to be statistically significant if P < 0.05.

RESULTS
The numbers of participants screened, enrolled, and who dropp ed out are shown in Fig. 1. This study was performed between September and December 2013. In total, 40 patients completed the study, 20 in each group. There was no significant difference between the groups in terms of age, height, weight, ASA class, anesthesia, and operation time, type of surgery, and TNM stage according to the American Joint Committee on Cancer criteria. Total consumption of fentanyl and pain score by numeric rat ing scale at 12 and 24 hr postoperatively were similar between the groups (Table 1). There was no difference in analgesic use during 24 hr postoperative between groups (data not shown). Plasma concentrations of IL2 and IL4 were not changed significantly after anesthesia induction. The IL2/IL4 ratio also showed no significant change between the groups ( Table 2).
Leukocytes revealed a decrease at 1 hr after induction only in the propofol group, with borderline significance (P = 0.05) and were lower in the propofol group than in the desflurane group at 1 hr after induction and 24 hr postoperatively. They increased in all patients at 24 hr postoperatively, largely as a result of in creased neutrophils, which was statistically significant only in the desflurane group. Lymphocytes in the propofol group were lower than those in the desflurane group 1 hr after induction (P = 0.019). Monocytes at 24 hr increased versus baseline val ues in the desflurane group, with a significant difference be tween the groups. Neutrophils at 24  with baseline values, in both groups with a difference between groups ( Table 3).
As shown in Table 4, CD4 + and CD8 + T cells showed no statis tically significant change after anesthesia induction in either group. The CD4 + /CD8 + T cell ratio remained unchanged at 1 hr after induction and then increased at 24 hr in both groups (P < 0.001). NK cells decreased significantly 1 hr after induction in the propofol group (P = 0.040), but not in the desflurane group. At 24 hr postoperative, NK cell counts decreased further in all patients (P < 0.001) with no difference between groups. There was no adverse event related to anesthesia or surgery during the study period.

DISCUSSION
Whether anesthesia and surgery affect immune function is an issue for breast cancer patients undergoing cancer surgery, and the least possible immunosuppressive regimen would be ben eficial for them. Our results show that surgery with both anes thetic agents preserve the IL2/IL4 ratio in the perioperative period, although desflurane anesthesia is even less associated with a decrease in leukocyte and NK cell counts at 1 hr after in duction, when compared to propofol anesthesia.
IL2 and IL4 are associated with Th1 and Th2 cells, respec tively (21,23). As a shift from a Th1 to Th2 response appears to be related with infectious complications and cancer progres sion, the balance between Th1 and Th2 cell has the significance in those patients with cancer (2426). An increased Th1/Th2 cell ratio indicates enhanced CMI and would be potentially benefi cial for cancer elimination, while a decreased ratio would raise the risk of cancer progression. In an animal study using mice, Hiroki et al. (27) demonstrated that combined spinal and sevo flurane general anesthesia preserved the Th1/Th2 balance with a resulting reduction in liver metastasis, whereas general anes thesia alone reduced the Th1/Th2 ratio. In another study in cra niotomy patients, propofol anesthesia preserved the Th1/Th2 ratio, while isoflurane anesthesia reduced the Th1/Th2 ratio significantly (22). The authors concluded that propofol anes thesia attenuated the adverse immune effects related with sur gical stress when compared to isoflurane anesthesia. Our re sults show ed that the IL2/IL4 ratio was not significantly chang ed after propofol or desflurane anesthesia induction. The CD4 + / CD8 + T cell ratio has been regarded as an important index of CMI (28). In our patients, it remained unchanged at 1 hr after induction and then even increased, compared with baseline values. A significant decrease in lymphocytes and monocytes, which are usually found in changes of the immune system after surgery, also did not occur in our patients. From this, desflu rane is considered to provide a favorable effect on the immune system, at least as much as propofol anesthesia, which is known not to have major adverse effect on immune system.
With respect to NK cells and leukocytes, desflurane anesthe sia showed more favorable effect on the immune system than propofol anesthesia. CMI consists of two immune system com ponents, the innate and adaptive systems (19). The innate im mune system is a nonspecific system, including macrophages, NK cells, and dendritic cells. NK cells play a key role in destroy ing tumors as a firstline of defense against cancer cells. NK cell activity in the perioperative period has been reported to be in versely related to the development of metastasis (26). In the pres ent study, NK cells decreased significantly 1 hr after induction only in the propofol group, suggesting the possibility of some what impaired innate immunity. Leukocytes decreased only in the propofol group at 1 hr after induction and were lower in pro pofol group than in the desflurane group at 1 hr after induction and 24 hr postoperatively. Values are expressed as median (interquartile range). *P < 0.05 vs. preoperative value; † P < 0.05 vs. propofol group. NK cells decreased significantly in all patients at 24 hr post operatively. This finding is consistent with Liu et al. (23) regard ing immune cell populations in craniotomy patients. They re ported that a decrease in immune cell populations was most obvious early after anesthesia induction. Most immune cells, such as leukocytes, total lymphocytes, T cells, and B cells de creased to their minimum levels after anesthesia induction and recovered by the end of surgery, while NK cells remained at a decreased level.
Several studies have reported markedly different impacts of propofol and desflurane after surgery. Schilling et al. (17) inves tigated proinflammatory reactions using bronchoalveolar lavage during onelung ventilation (OLV) and concluded that desflu rane anesthesia alleviated alveolar inflammatory responses to OLV, compared with propofol anesthesia. Another study show ed that desflurane anesthesia produced less inflammatory re sponse than propofol anesthesia, in terms of lower plasma con centrations of IL6 and IL8, after cardiopulmonary bypass (18).
The present study has several limitations. First, although the anesthetic drugs may have caused complex effects in cancer cell biology, in terms of cancer proliferation, migration, inva sion, and interference with the CMI system (6), we did not eval uate actual longterm clinical outcomes of the cancers, the in teractions between immune cells and surrounding factors in these individuals. Second, we used fentanyl to provide analge sia, which is known to have immunomodulating effects, which might also have influenced our findings. Experimental studies concerning the effects of opioids on immune function have shown conflicting results (2931) and clinical evidence is lack ing. Third, we may be criticized for the use of nitrous oxide with respect to hematologic, myocardial, neurological, and immu nological concerns (32). A randomized controlled trial in color ectal cancer patients demonstrated that cancer recurrence risks were similar in those who were assigned to 65% nitrous oxide or nitrogen during cancer surgery (33). We used nitrous oxide because of familiarity and ease of use with regard to low blood/ gas partition coefficients, its own anesthetic properties, and second gas effects. We sought to compare the effects of propo fol and desflurane in the same condition so we used nitrous ox ide in both groups. Further work without the use of nitrous ox ide might show different results.
In conclusion, surgery with both propofol and desflurane re veals favorable effect on immune system in terms of IL2/IL4 and CD4 + /CD8 + T cell ratio in the perioperative period. With re spect to leukocytes and NK cells, desflurane anesthesia is even less associated with adverse effect on immune system than pro pofol anesthesia during surgery for breast cancer. Further stud ies are needed to determine the clinical implications of anes thetic drug choices.

DISCLOSURE
The authors declare that no potential conflicts of interest exist with any companies. Our research including data analysis was not influenced by a pharmaceutical company offering research grants.