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Effect of pectoral nerve block type II under general anesthesia on the immune function of patients with breast cancer
The American Journal of Surgery xxx (xxxx) xxx
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Effect of pectoral nerve block type II under general anesthesia on the immune function of patients with breast cancer Xiuling Cui a, *, Cuinv Zhu b, Peng Chen a, Min Qu a, Bowei Zhang c, Hongtao Li d a
Department of Anesthesiology, Cangzhou Central Hospital, No 16 Xinhua Road, Cangzhou, 061000, Hebei, China Department of Anesthesiology, Cangzhou Hospital of Integrated TCM-WM·Hebei, No 31 Huanghe Road, Cangzhou, 061000, Hebei, China c Hubei Medical College, Shiyan, 442000, Hubei, China d Quality Control Office, Cangzhou Central Hospital, No 16 Xinhua Road, Cangzhou, 061000, Hebei, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 January 2020 Received in revised form 28 February 2020 Accepted 4 March 2020
Background: To investigate whether the use of Pecs II block benefit patients with the respect to the immune functions. Methods: Totally 196 patients were included in this study. These patients were randomized to two groups, general anesthesia alone group (G group) and Pectoral nerve (Pecs) II block under general anesthesia group (PG group). Results: It was found that remifentanil consumption was less in PG group than it in G group. PG group showed a higher proportion of NK cells in peripheral blood mononuclear cell (PBMC) and an improved killing activity than G groups after surgery. We also found that postoperative interleukin (IL)-2 concentration in the plasma of PG group was dramatically higher than it of G group. Interestingly, there was even no significant change between preoperative and postoperative IL-2 levels in PG group, suggesting the less inhibitory effect of Pecs II block on immune system of those patients. Conclusions: In conclusion, these results indicate that Pecs II block use in patients may have an enhanced immunity compared with general anesthesia method. © 2020 Elsevier Inc. All rights reserved.
Keywords: Pectoral nerve (Pecs) II block Natural killing (NK) cells Breast cancer Remifentanil
Introduction Breast cancer is a common type of cancer worldwide, as the highest incidence of malignant tumors in women.1 Currently, surgical resection is the primary treatment of breast cancer. However, although surgery aims to eliminate most of tumor tissues, cancer cells could still spread to the blood and lymphatic circulation system,2 which may lead to cancer recurrence especially under surgery and anesthetic induced immunosuppression.3 Therefore, improvement of immunity after surgery may be critical for patient survival. In recent years, pectoral nerve (Pecs) block, a new technique providing analgesia, has been widely applied in breast surgeries. Pecs II block is based on and modified from Pecs I block. These Peck blocks showed relatively less complications during surgeries by blocking the pectoral and intercostal nerves.4,5 Compared to Pecs I block, Pecs II block adds a second injection in the lateral branch of
* Corresponding author. E-mail address: [email protected] (X. Cui).
intercostal nerve.6 Previous studies reported that Pecs blocks were associated with better analgesic effect,7 reduced consumption of opioids such as remifentanil and fentanyl during surgery and reduced chronic pain after surgery by blocking the peripheral nerves.8 Both surgery and anesthetics have adverse impact on immunity.9,10 Previous studies have shown that surgery induced stress are related with enhanced angiogenesis and metastasis and repressed both innate and cell-mediated immunity.3 The immunosuppressive effects were also associated with certain volatile anesthetics and opioids. Both volatile anesthetics and opioids inhibited natural killer (NK) cell activity. It is known that NK cells are important components in innate immunity, assisting the protection of body from the spread of tumor cells.11,12 Therefore, NK cell loss could potentially enhance the metastasis of cancer.13 Evidence in pancreatic mouse model showed that loss of NK cells promoted the progression of tumor.14 A commonly used medicine in breast cancer, trastuzumab, exerts its functions as a monoclonal antibody, which selectively targets Her2. Its lysis ability of Her2positive breast cancer cells primarily dependent on antibodydependent cellular cytotoxicity (ADCC) mediated by NK cells,
https://doi.org/10.1016/j.amjsurg.2020.03.008 0002-9610/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: Cui X et al., Effect of pectoral nerve block type II under general anesthesia on the immune function of patients with breast cancer, The American Journal of Surgery, https://doi.org/10.1016/j.amjsurg.2020.03.008
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X. Cui et al. / The American Journal of Surgery xxx (xxxx) xxx
leading to the critical role of NK cells in this process. With the respect to Pecs II block, we aimed to investigate whether Pecs II block could improve NK cell proliferation or function after breast cancer surgery. In addition to NK cells, other immune cells that are critical in defending cancer including natural killer T (NKT) cells, helper T cells and cytotoxic T cells and related cytokines were assessed and compared between patients treated with and without Pecs II block.
Methods Patients The study was approved by the ethics committee of Cangzhou Central Hospital. All patients received the written and informed consents. A total of 232 patients who were diagnosed with breast cancer and at American Society of Anesthesiologists (ASA) physical status I and II were enrolled in this study. All enrolled participants were prepared to receive surgery (mastectomy or breast conserving surgery). Patients were excluded from the study according to the following exclusion criteria as shown in Fig. 1: 1) under 18 years of age, 2) history of previous surgery and tumors, 3) other types of tumors, 4) history of heart disease, respiratory disease, or neurological disease or other diseases, 5) coagulopathy or allergic to the drugs used in the surgery, 6) recently received radiotherapy or chemotherapy (<8 wks), 7) recently used steroids or opioids, alcohol or other illicit drugs. After exclusion by the criteria, 196 patients were included. These patients were randomized into two groups according to a random set of numbers generated by the computer: general anesthesia alone group (G group) and Pecs II block under general anesthesia group (PG group). During hospitalization, all participants received appropriate anesthesia and surgery. The surgical team and nursing team were blinded to the groups and all patients were anesthetized by one same anesthesiologist.
Surgery procedure All patients were administrated lidocaine (1 mg/kg), propofol, and remifentanil for anesthesia induction and maintenance. Remifentanil dose was adjusted to keep the surgical pleth index between 20 and 50. With the use of a target-controlled infusion (TCI) pump (Orchestra; Fresenius Kabi, Germany), Schnider’s15 and Minto’s16 pharmacokinetic models were respectively applied to calculate the effect-site amount of propofol and remifentanil. Pecs II block was performed according to the published method.17 Firstly, an ultrasound probe was placed under the lateral third of the clavicle to identify the axillary vein and artery. Then the probe was moved to the position between the pectoralis major and minor muscles. After that, under ultrasound guidance, a needle was introduced in-plane view of the ultrasound in an oblique manner between the pectoralis major and minor muscles. Then, ropivacaine (0.5%, 10 mL) was injected. The probe was then moved on from the first rib to the third rib to reach the serratus anterior muscle. The needle was advanced until its tip reached the potential space between the Pmm and SAM, ropivacaine (0.5%, 20 mL) was injected into this area. During the surgery, patients were monitored by electrocardiography, non-invasive blood pressure monitoring and oxygen saturation of pulse oximeter. Additionally, body temperature and the bispectral index (between 40 and 60, BIS vista monitor revision 3.0; Aspect Medical Systems, USA) were also monitored. If the bispectral index was above 60 or less than 40, 0.5 mg/kg with intervals of 1 min or more was applied as the effect-site concentration of propofol. Atropine 0.5 was applied intravenously when the heart rate was declined below 50 beats/min. After surgery, patients confirmed with recovered spontaneous breath and consciousness were moved to postoperative intensive care unit for the next 24 h monitoring. Blood samples collection The blood samples of patients were collected after 24 h of
Fig. 1. Flowchart of the study. G group, general anesthesia alone group. PG group, Pecs II block under general anesthesia group.
Please cite this article as: Cui X et al., Effect of pectoral nerve block type II under general anesthesia on the immune function of patients with breast cancer, The American Journal of Surgery, https://doi.org/10.1016/j.amjsurg.2020.03.008
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surgery in heparin tubes for peripheral blood mononuclear cell (PBMC) isolation and ethylenediaminetetraacetic acid tube for cytokine assessments. The isolation of PBMC was achieved using Ficoll-Paque (GE Healthcare, Sweden) density gradient centrifugation as previously reported,18 and frozen in heat-inactivated human AB serum (Sigma Aldrich, USA) containing 10% dimethyl sulfoxide (Sigma Aldrich, USA) in liquid N2.
between preoperative and postoperative values. A p-value<0.05 was considered as statistically significant. SPSS (version 20.0; SPSS Inc., USA) was used for all calculations.
NK cell isolation and apoptosis analysis
A total of 232 patients who were enrolled in this study (Fig. 1). After exclusion by several conditions, 196 patients were included. These patients were randomized into two groups, general anesthesia alone group (G group, n ¼ 98) and pectoral nerve block type II under general anesthesia group (PG group, n ¼ 98). Their characteristics are shown in Table 1. Patients in the two groups showed no differences in age, height weight, cancer stage, operation type and ASA status. However, during the surgery, the remifentanil consumption in PG groups was significantly lower than it of G group (p ¼ 0.001). No differences of other vital signs between G and PG groups were observed including arterial pressure and heart rate.
CD56þ NK cells were isolated from PBMC immunoselected by anti-CD56 conjugated microbeads (Miltenyi Biotec, UK) according to the protocol provided by the manufacturer using flow cytometry. Cell culture and apoptosis assay For the apoptosis assay in the presence or absence of patient serum or NK cells, we employed a human primary breast cancer cell line, HCC, which were cultured in ATCC-formulated RPMI-1640 Medium containing 10% fetal calf serum (FCS, Sigma). Sample serums were used to replace the FCS in indicated experiment. NK and HCC cells were co-cultured at a ratio of 1:10 in the cell medium with indicated serum for 24 h. Apoptosis rate of HCC cells were assessed using dual staining with FITC-Annexin and propidium iodide (Annexin V-FITC apoptosis detection kit, Biofriend, CN) and sorted by flow cytometry. NK cell cytotoxicity The cytotoxicity of NK cells was detected by incubation with K562 target erythroleukemic cells (American Type Culture Collection, USA). The cells were stained by carboxyfluorescein succinimidyl ester (Sigma Aldrich) and was collected after 4 h incubation. Propidium iodide (Becton Dickinson, USA) was added to determine dead target cells. Isolation of NKT cells, helper T cells (CD4þ) and cytotoxic T cells (CD8þ)
Results Patient characteristics
Effect of pectoral nerve block type II under general anesthesia on NK cell number and function To explore the different immune response in the two groups, we firstly tested the NK cell number. In G group, NK cells occupied about 17.2 ± 3.53% of PBMC before surgery (Fig. 2A) but this percentage was decreased to 13.36 ± 3.07% after general anesthesia (p ¼ 0.023). No significant difference of NK cell number was observed in patients treated with PG. Interestingly, when comparing the NK cell number between the two groups after the surgery, PG group showed a significantly higher occupancy of NK cells in PBMC than G group (p ¼ 0.036). For both G and PG groups, apoptosis rate of NK cells was not affected either before or after the surgery (Fig. 2B). The postoperative cell apoptosis rate of NK cells in PG and G groups also had similar percentage. The killing activities of NK cells in the two groups were dramatically reduced after surgery (p < 0.001, Fig. 2C). However, after the surgery, compared to G group, PG group had more functional NK cells (p ¼ 0.007).
The isolation of NKT cells, CD4þ T cells and CD8þ T cells were achieved using sorting with fluorescence-labeled antibodies by flow cytometry. Phycoerythrin (PE)-conjugated anti-human CD56 monoclonal antibody (cat no.E-AB-F1210D; Elabscience), fluorescein isothiocyanate-conjugated anti-human CD3 monoclonal antibody (cat no. E-AB-F1001C; Elabscience), fluorescein isothiocyanate-conjugated anti-human CD4 monoclonal antibody (cat no. E-AB-F1109C; Elabscience) and phycoerythrin-conjugated anti-human CD8a monoclonal antibody (cat no. E-AB-F1110C; Elabscience) were used.
The cell number of NKT, T helper 1 cells (CD4þ), and cytotoxic T lymphocytes (CD8þ) in PG and G groups
Measurement of cytokines
Cytokine concentrations and neutrophil, lymphocyte counts in the blood of patients in P and PG groups
Enzyme-linked immunosorbent assays were used to detect the plasma levels of interleukin (IL)-2, IL-4, and interferon-g (IFN-g) using a commercially available kit (R&D Systems, Inc.). Each cytokine was measured in triplicate for each blood sample. An automated microplate reader (Molecular Devices, Inc., USA) was used to obtain the results. Statistical analysis Data in patient characteristics and cytokine and neutrophil, lymphocyte counts in blood were indicated as median (interquartile ranges, IQR) or mean (Standard deviation, SD) for normally distributed data. A paired t-test was applied for the comparison
We further tested the effect of the two anesthesia methods on the PBMC percentage of NKT, T helper 1 cells (CD4þ), and cytotoxic T lymphocytes (CD8þ) cells. For both of G and PG groups, after surgery, no significant changes of these cell number were observed compared to their number before surgery (Fig. 3AeC). Additionally, no difference of these cell number percentage in PBMC was shown in the two groups.
The preoperative IL-2 concentration was about 3.9 ng/ml in both G and PG group. Interestingly, the postoperative IL-2 concentration in G group was significantly lower than its preoperative level (Table 2, p < 0.01). This reduction was not observed in PG group. The postoperative IL-4 and IFN-g maintained same concentration as their preoperative levels in G and PG groups. The numbers of neutrophils and lymphocytes were changed by the operation. The neutrophils were significantly increased from 60.5% (preoperational) to 70.1% (post-operative) in G group (p < 0.01) and from 60.8% (preoperational) to 63.1% 70.1% (postoperative) n PG group (p < 0.05). However, the post-operative neutrophils were less in PG group compared to G group
Please cite this article as: Cui X et al., Effect of pectoral nerve block type II under general anesthesia on the immune function of patients with breast cancer, The American Journal of Surgery, https://doi.org/10.1016/j.amjsurg.2020.03.008
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X. Cui et al. / The American Journal of Surgery xxx (xxxx) xxx Table 1 Patient demographic data and perioperative clinical characteristics.
Age (years) Height (cm) Weight (kg) Cancer stage (%) I II III IV Operation type (%) Breast-conserving surgery Modified radical mastectomy ASA status ASA I ASA II During surgery Propofol consumption (mg/kg/h) Remifentanil consumption (mg/kg/h) Use of atropine (n) Use of ephedrine (n) Duration of anesthesia (min) Duration of operation (min) Arterial pressure (mmHg) before induction incision closing ICU arrival Heart rate (bpm) before induction incision closing ICU arrival
G group (n ¼ 98)
PG group (n ¼ 98)
P value
51 (43e57) 160.2 (4.6) 55.7 (7.3)
53 (45e56) 159.4 (4.5) 56.5 (6.9)
0.398 0.455 0.279 0.576
28 (28.6) 43 (43.9) 27 (27.6) 0 (0)
33 (33.7) 35 (35.7) 30 (30.6) 0 (0)
37 (37.8) 61 (62.2)
40 (40.8) 58 (59.2)
60 (61.2) 38 (38.8)
62 (63.3) 36 (36.7)
7.1 (1.3) 11.4 (3.5) 8 7 152 (112e183) 115 (79e141)
6.9 (1.3) 6.3 (2.1) 3 6 149 (109e176) 104 (82e137)
0.493 0.001 0.069 0.542 0.393 0.488
100.1 (13.2) 83.9 (12.7) 84.5 (11.4) 92.6 (8.9)
98.3 83.7 84.8 91.8
0.295 0.286 0.447 0.173
75 66 72 80
72 67 71 81
0.556
0.648
(16) (15) (11) (10)
(14.5) (13.9) (13.2) (10.2)
(17) (15) (10) (8)
0.475 0.522 0.376 0.287
Data are expressed as median (interquartile ranges (IQR)) or mean (SD), or n(%). ASA, American Society of Anaesthesiologists. ICU, intensive care unit.
Fig. 2. Effect of Pectoral nerve block type II use during surgery on NK cells. (A) Mean preoperative and postoperative percentages of natural killer (NK) cells in the peripheral blood in different groups. (B) The apoptosis rate of circulating NK cells in different groups were tested. (C) Preoperative and postoperative NK cell function were examined at an effector: target ratio of 10:1. G group, general anesthesia alone group. PG group, Pecs II block under general anesthesia group. PBMCs, peripheral blood mononuclear cells. P < 0.05 was considered statistically significant.
(p ¼ 0.001). Furthermore, after surgery, lymphocytes were dramatically decreased in G (p < 0.01) and PG (p < 0.05) groups. When comparing the post-operative lymphocyte number, PG group showed a higher percentage of lymphocytes compared to G group (p ¼ 0.001). Accordingly, neutrophil-to-lymphocyte ratio (NLR) was remarkably elevated after surgery in comparison with the ratio before surgery in G (p < 0.001) and PG groups (p < 0.01). However, compared to G group, PG groups showed a significantly lower NLR (p ¼ 0.001). Apoptosis rate of HCC cells in the presence or absence of NK cells and subject serum-supplemented media We next investigated whether the serum of the subject could affect the apoptosis rate of HCC cells. We treated HCC cells with the
serum from normal patients or patients from G and PG groups. As shown in Fig. 4, no difference of HCC apoptosis rate was observed between the three treatments in the absence of NK cells. Intriguingly, in the presence of NK cells, the serum from patients (n ¼ 20) in G groups dramatically promoted HCC cell apoptosis compared to normal serum. HCC cultured in the serum collected from G and PG group patients after surgery showed a significantly lower apoptosis rate compared to those cultured in the serum collected before surgery (G group, p < 0.001; PG group, p ¼ 0.017). However, compared to the post-operative serum from PG group, the postoperative serum from G group significantly repressed HCC apoptosis rate (p ¼ 0.031), demonstrating the post-operative serum from G group may had a stronger ability of inhibition of NK cell function compared to the post-operative serum from PG group.
Please cite this article as: Cui X et al., Effect of pectoral nerve block type II under general anesthesia on the immune function of patients with breast cancer, The American Journal of Surgery, https://doi.org/10.1016/j.amjsurg.2020.03.008
X. Cui et al. / The American Journal of Surgery xxx (xxxx) xxx
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Fig. 3. Mean peripheral blood percentages of (A) natural killer T (NKT) cells, (B) T helper 1 cells (CD4þ), and (C) cytotoxic T lymphocytes (CD8þ) were tested in different groups. G group, general anesthesia alone group. PG group, Pecs II block under general anesthesia group. PBMCs, peripheral blood mononuclear cells. P < 0.05 was considered statistically significant.
Discussion Our study found that Pecs II block use compared to general anesthesia method had less remifentanil consumption during surgery. Pecs II was shown to affect immune system mainly by changing the cell proportion in the blood of patient received breast cancer surgery. In addition, the killing activity of NK cells was also promoted by Pecs II, demonstrating that Pecs II also had an impact on immune cell function. We found that the use of Pecs II during anesthesia significantly reduced the consumption of remifentanil. A similar observation was also found in a recent stud,19 documenting that preoperatiove administration of ropivacaine with Pecs II block significantly reduced the consumption of remifentanil during surgery compared to control (without Pecs II block). It is known that the choice of anesthesia is critical because it may be related to cancer recurrence.3 Although surgery resection may remove most of cancer tissue, a few cancer cells may be still remained in the body.20 In this respect, anesthesia-induced immunosuppression and surgery caused stress could help the spread and survival of these cancer cells. Surgery induces neuroendocrine stress, decreases numerous immune stimulating
Table 2 Perioperative Cytokine Concentrations and neutrophil, lymphocyte counts in blood of patients undergoing Breast Cancer Surgery.
IL-2 (ng/ml) Preop Postop IL-4 (ng/ml) Preop Postop IFN-g(ng/ml) Preop Postop Neutrophil (%) Preop Postop Lymphocyte (%) Preop Postop NLR Preop Postop
G group (n ¼ 98)
PG group (n ¼ 98)
P value
3.92 (0.34) 2.28 (0.36)b
3.87 (0.26) 3.23 (0.33)
0.385 0.002
0.43 (0.13) 0.47 (0.14)
0.45 (0.18) 0.42 (0.15)
0.468 0.313
2.09 (0.24) 1.97 (0.27)
2.12 (0.22) 2.01 (0.32)
0.229 0.263
60.5 (12.7) 70.1 (13.7) 27.0 (7.3) 21.2 (7.0)
60.8 (13.9) 63.1 (14.2)
b
26.4 (6.5) 22.9 (5.9)
b
2.2 (1.6e3.1) 3.3 (2.0e4.3)
c
0.298 0.001
a
0.397 0.001
a
2.3 (1.7e2.8) 2.7 (1.8e3.5)
cytokine production including IFN-g and IL-2 and increases the level of anti-inflammatory cytokines.9 In addition to its inhibitory effects on immune system, surgery was experimentally and clinically found to be associated with increased angiogenesis and promoted metastasis.3 Anesthetics such as opioids were also found to suppress cell-mediated immunity21 and promote tumor development and metastasis.22 In a rat model, remifentanil was found to repress NK cell activity and proliferation of lymphocyte.23 Similar inhibitory role of remifentanil in immunity was also observed in clinical researches.24,25 Therefore, less use of opioids such as remifentanil could benefit the outcomes of the cancer patients. In our study, we found that NK cell percentage in PBMC and its killing activity were dramatically declined after the surgery, implicating the surgery and anesthetic induced immunosuppression. Impressively, the application of Pecs II block showed less inhibitory impact on NK cell number and function. We are the first study finding the supportive effects of Pecs II block on NK cells. This is essential for further clinical application of Pecs II block during surgery as immune regulation could partially determine the recurrence of cancer. We did not observe changes of NKT or T lymphocytes induced by the surgery. However, several previous studies reported that surgery could cause the remarkable loss of T lymphocytes and NKT cells.24,26,27 The controversial results may be due to the differences in the surgery procedure and types of cancer.
b
0.416 0.001
Data are expressed as median (interquartile ranges (IQR)) or mean (SD). IL, interleukin. NLR, neutrophil-to-lymphocyte ratio. Preop, Preoperative, which means immediately before anesthesia induction. Postop, Postoperative, which means 24h postoperatively. a <0.05, Preop vs Postop. b <0.01, Preop vs Postop. c <0.001, Preop vs Postop.
Fig. 4. Apoptosis rate of HCC cells after co-cultured with NK cells in the presence of subject serum-supplemented media. HCC cells were cultured in serum-supplemented media, either alone, or with NK cells. Apoptosis was determined by dual staining with FITC-Annexin V and propidium iodide and tested by flow cytometry. Each experiment was carried out in triplicate. P < 0.05 was considered statistically significant.
Please cite this article as: Cui X et al., Effect of pectoral nerve block type II under general anesthesia on the immune function of patients with breast cancer, The American Journal of Surgery, https://doi.org/10.1016/j.amjsurg.2020.03.008
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A study investigated the effect of propofol on T lymphocytes and NK cells in patients with breast cancer documented that NK cell number was significantly reduced after surgery.28 Lymphocyte count also showed a decreasing trend after surgery with no statistical significance. These observations are same as our results. It was reported that surgery stress could repress the production of immune stimulating cytokines including IL-2 and IFN-g.9 Consistently, here, we observed a significantly diminished level of IL-2 in the blood of patients after surgery compared to its level before surgery but not IFN-g. Interestingly, compared to general anesthetic, Pecs II block use significantly promoted IL-2 production. As IL-2 is primarily produced by activated CD4þ T lymphocytes, Pecs II block may potentially improve the ability of IL-2 expression and production from T lymphocytes as it did not affect T lymphocytes number. It is possible that this is caused by less use of remifentanil because opioids were reported to have adverse effect on IL2 and IFN-g production.29 Further studies are necessary to reveal the how Pecs II block and remifentanil affect the IL-2 production. In addition, since IL-2 is an important regulator of differentiation and homeostasis of T lymphocytes, less IL-2 level may be associated with reduced effector T cells.30 However, although IL-2 level showed higher in PG group than G group after surgery, both CD4þ and CD8þ T lymphocytes had no significant changes. In addition, increase of IL-2 level could be the cause of enhanced NK cell killing ability observed in PG groups compared to G group after surgery because IL-2 was reported to promote the cytolytic activity of NK cells.30 It is possible that surgery and anesthetic suppress other cytokines needed in T cell development and differentiation or affect the expression of critical proteins such as IL-2 receptor. However, further studies are necessary to reveal the detailed mechanism. We also found that PG group showed a reduced NLR compared to G group after surgery, indicating patients in PG group had less inflammation than G group. Since there is an association between high NLR and poor survival rate particularly in breast cancer,31 PG group may have a better prognosis compared to G group, which needs further investigation. We also found that HCC showed more apoptosis rate cultured in the postoperative serum from PG group compared to the postoperative serum from G group in the presence of NK cells. The result suggests that PG group may contain more cytokines supporting the NK cell proliferation or killing activity, which is similar to our aforementioned observations. In addition to these immune cells in blood, it is also important to investigate whether Pecs II block has an impact on the immune cells in the tumor microenvironment. For example, tumor infiltrating lymphocytes (TIL) or PD-1 and PD-L1 levels in tumor tissue and the surrounding stromal area are important parameters to confirm how Pecs II block affects immune functions related to the tumor microenvironment, which are omitted in this study. However, in our further study, it is necessary to investigate the effect of Pecs II block on the immune system in the tumor microenvironment. Conclusion In conclusion, after surgery, elevation of NK cells in PBMC and the killing activity were observed in the group with Pecs II block use compared to the group without it. There was no change of T lymphocytes and NKT between PG group and G group. Neutrophil increase and lymphocyte decrease after surgery in G groups caused a higher level of postoperative NLR compared to the preoperative ratio. However, in PG group, this postoperative NLR was significantly lower than P group, suggesting a less inflammation in PG group. We also observed that IL-2 production was improved in PG group compared to G group after surgery. Taken together, these
results indicate that patients in PG group showed an enhanced immunity compared with those in G group. Funding None. Declaration of competing interest The authors have declared that no conflicts of interest exist. References 1. Siegel RL, Miller KD, Jemal A. Cancer statistics. CA A Cancer J. Clin. 2016;66(1): 7e30, 2016. 2. Ben-Eliyahu S. The price of anticancer intervention. Does surgery promote metastasis? Lancet Oncol. 2002;3(9):578e579. 3. Kim R. Effects of surgery and anesthetic choice on immunosuppression and cancer recurrence. J Transl Med. 2018;16(1):8. 4. Moon E-J, Kim S-B, Chung J-Y, et al. Pectoral nerve block (Pecs block) with sedation for breast conserving surgery without general anesthesia. Ann. Sur. Tret. Res. 2017;93(3):166e169. 5. Freise H, Van Aken H. Risks and benefits of thoracic epidural anaesthesia. Br. J. Anaesth. 2011;107(6):859e868. 6. Morioka H, Kamiya Y, Yoshida T, Baba H. Pectoral nerve block combined with general anesthesia for breast cancer surgery: a retrospective comparison. J. A Clin. Rep. 2015;1(1):15. 7. Neethu M, Pandey RK, Sharma A, et al. Pectoral nerve blocks to improve analgesia after breast cancer surgery: a prospective, randomized and controlled trial. J Clin Anesth. 2018;45:12e17. 8. Macrae W. Chronic pain after surgery. Br. J. Anaesth. 2001;87(1):88e98. 9. Lin E, Calvano SE, Lowry SF. Inflammatory cytokines and cell response in surgery. Surgery. 2000;127(2):117e126. 10. Lim J-A, Oh C-S, Yoon T-G, et al. The effect of propofol and sevoflurane on cancer cell, natural killer cell, and cytotoxic T lymphocyte function in patients undergoing breast cancer surgery: an in vitro analysis. BMC Canc. 2018;18(1): 159. 11. Li Y, Sun R. Tumor immunotherapy: new aspects of natural killer cells. Chin J Canc Res. 2018;30(2):173. 12. Market M, Baxter KE, Angka L, et al. The potential for cancer immunotherapy in targeting surgery-induced natural killer cell dysfunction. Cancers. 2019;11(1): 2. 13. Snyder G, Greenberg S. Effect of anaesthetic technique and other perioperative factors on cancer recurrence. Br. J. Anaesth. 2010;105(2):106e115. 14. Janakiram NB, Mohammed A, Bryant T, et al. Loss of natural killer T cells promotes pancreatic cancer in LSL-KrasG12D/þ mice. Immunology. 2017;152(1):36e51. 15. Schnider TW, Minto CF, Gambus PL, et al. The influence of method of administration and covariates on the pharmacokinetics of propofol in adult volunteers. J. Am. Soc. Anesthesiologists. 1998;88(5):1170e1182. 16. Minto CF, Schnider TW, Egan TD, et al. Influence of age and gender on the pharmacokinetics and pharmacodynamics of RemifentanilI. Model Dev. Anesthesiol. J. Am. Soc. Anesthesiol. 1997;86(1):10e23. 17. Blanco R, Fajardo M, Parras Maldonado T. Ultrasound description of Pecs II (modified Pecs I): a novel approach to breast surgery. Rev Esp Anestesiol Reanim. 2012 Nov;59(9):470e475. PubMed PMID: 22939099. Epub 2012/09/ 04. 18. Jaatinen T, Laine J. Isolation of mononuclear cells from human cord blood by Ficoll-Paque density gradient. Curr. Protoc. Stem Cell Biol. 2007;1(1), 2A. 1.-2A. 1.4. 19. Choi JJ, Jo YY, Kim SH, et al. Remifentanil-sparing effect of pectoral nerve block type II in breast surgery under surgical pleth index-guided analgesia during total intravenous anesthesia. J Clin Med. 2019;8(8):1181. 20. Lloyd JM, McIver CM, Stephenson S-A, et al. Identification of early-stage colorectal cancer patients at risk of relapse post-resection by immunobead reverse transcription-PCR analysis of peritoneal lavage fluid for malignant cells. Clin Canc Res. 2006;12(2):417e423. 21. Gottschalk A, Sharma S, Ford J, et al. The role of the perioperative period in recurrence after cancer surgery. Anesth Analg. 2010;110(6):1636e1643. 22. Cassinello F, Prieto I, del Olmo M, et al. Cancer surgery: how may anesthesia influence outcome? J Clin Anesth. 2015;27(3):262e272. 23. Sacerdote P, Gaspani L, Rossoni G, et al. Effect of the opioid remifentanil on cellular immune response in the rat. Int Immunopharm. 2001;1(4):713e719. 24. Sacerdote P, Bianchi M, Gaspani L, et al. The effects of tramadol and morphine on immune responses and pain after surgery in cancer patients. Anesth Analg. 2000;90(6):1411e1414. 25. Qi Y, Yao X, Zhang B, Du X. Comparison of recovery effect for sufentanil and remifentanil anesthesia with TCI in laparoscopic radical resection during colorectal cancer. Oncol. Lett. 2016;11(5):3361e3365. 26. Angele MK, Faist E. Clinical review: immunodepression in the surgical patient and increased susceptibility to infection. Crit Care. 2002;6(4):298.
Please cite this article as: Cui X et al., Effect of pectoral nerve block type II under general anesthesia on the immune function of patients with breast cancer, The American Journal of Surgery, https://doi.org/10.1016/j.amjsurg.2020.03.008
X. Cui et al. / The American Journal of Surgery xxx (xxxx) xxx 27. Klatka J, Grywalska E, Hymos A, et al. Subpopulations of natural killer-T-like cells before and after surgical treatment of laryngeal cancer. Cent Eur J Immunol. 2017;42(3):252. 28. Woo JH, Baik HJ, Kim CH, et al. Effect of propofol and desflurane on immune cell populations in breast cancer patients: a randomized trial. J Kor Med Sci. 2015;30(10):1503e1508.
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29. Al-Hashimi M, Scott S, Thompson JP, Lambert D. Opioids and immune modulation: more questions than answers. Br. J. Anaesth. 2013;111(1):80e88. 30. Liao W, Lin J-X, Leonard WJ. Interleukin-2 at the crossroads of effector responses, tolerance, and immunotherapy. Immunity. 2013;38(1):13e25. 31. Faria SS, Fernandes Jr PC, Silva MJB, et al. The neutrophil-to-lymphocyte ratio: a narrative review. Ecancermedicalscience. 2016;10.
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Effect of pectoral nerve block type II under general anesthesia on the immune function of patients with breast cancer Xiuling Cui a, *, Cuinv Zhu b, Peng Chen a, Min Qu a, Bowei Zhang c, Hongtao Li d a
Department of Anesthesiology, Cangzhou Central Hospital, No 16 Xinhua Road, Cangzhou, 061000, Hebei, China Department of Anesthesiology, Cangzhou Hospital of Integrated TCM-WM·Hebei, No 31 Huanghe Road, Cangzhou, 061000, Hebei, China c Hubei Medical College, Shiyan, 442000, Hubei, China d Quality Control Office, Cangzhou Central Hospital, No 16 Xinhua Road, Cangzhou, 061000, Hebei, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 January 2020 Received in revised form 28 February 2020 Accepted 4 March 2020
Background: To investigate whether the use of Pecs II block benefit patients with the respect to the immune functions. Methods: Totally 196 patients were included in this study. These patients were randomized to two groups, general anesthesia alone group (G group) and Pectoral nerve (Pecs) II block under general anesthesia group (PG group). Results: It was found that remifentanil consumption was less in PG group than it in G group. PG group showed a higher proportion of NK cells in peripheral blood mononuclear cell (PBMC) and an improved killing activity than G groups after surgery. We also found that postoperative interleukin (IL)-2 concentration in the plasma of PG group was dramatically higher than it of G group. Interestingly, there was even no significant change between preoperative and postoperative IL-2 levels in PG group, suggesting the less inhibitory effect of Pecs II block on immune system of those patients. Conclusions: In conclusion, these results indicate that Pecs II block use in patients may have an enhanced immunity compared with general anesthesia method. © 2020 Elsevier Inc. All rights reserved.
Keywords: Pectoral nerve (Pecs) II block Natural killing (NK) cells Breast cancer Remifentanil
Introduction Breast cancer is a common type of cancer worldwide, as the highest incidence of malignant tumors in women.1 Currently, surgical resection is the primary treatment of breast cancer. However, although surgery aims to eliminate most of tumor tissues, cancer cells could still spread to the blood and lymphatic circulation system,2 which may lead to cancer recurrence especially under surgery and anesthetic induced immunosuppression.3 Therefore, improvement of immunity after surgery may be critical for patient survival. In recent years, pectoral nerve (Pecs) block, a new technique providing analgesia, has been widely applied in breast surgeries. Pecs II block is based on and modified from Pecs I block. These Peck blocks showed relatively less complications during surgeries by blocking the pectoral and intercostal nerves.4,5 Compared to Pecs I block, Pecs II block adds a second injection in the lateral branch of
* Corresponding author. E-mail address: [email protected] (X. Cui).
intercostal nerve.6 Previous studies reported that Pecs blocks were associated with better analgesic effect,7 reduced consumption of opioids such as remifentanil and fentanyl during surgery and reduced chronic pain after surgery by blocking the peripheral nerves.8 Both surgery and anesthetics have adverse impact on immunity.9,10 Previous studies have shown that surgery induced stress are related with enhanced angiogenesis and metastasis and repressed both innate and cell-mediated immunity.3 The immunosuppressive effects were also associated with certain volatile anesthetics and opioids. Both volatile anesthetics and opioids inhibited natural killer (NK) cell activity. It is known that NK cells are important components in innate immunity, assisting the protection of body from the spread of tumor cells.11,12 Therefore, NK cell loss could potentially enhance the metastasis of cancer.13 Evidence in pancreatic mouse model showed that loss of NK cells promoted the progression of tumor.14 A commonly used medicine in breast cancer, trastuzumab, exerts its functions as a monoclonal antibody, which selectively targets Her2. Its lysis ability of Her2positive breast cancer cells primarily dependent on antibodydependent cellular cytotoxicity (ADCC) mediated by NK cells,
https://doi.org/10.1016/j.amjsurg.2020.03.008 0002-9610/© 2020 Elsevier Inc. All rights reserved.
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leading to the critical role of NK cells in this process. With the respect to Pecs II block, we aimed to investigate whether Pecs II block could improve NK cell proliferation or function after breast cancer surgery. In addition to NK cells, other immune cells that are critical in defending cancer including natural killer T (NKT) cells, helper T cells and cytotoxic T cells and related cytokines were assessed and compared between patients treated with and without Pecs II block.
Methods Patients The study was approved by the ethics committee of Cangzhou Central Hospital. All patients received the written and informed consents. A total of 232 patients who were diagnosed with breast cancer and at American Society of Anesthesiologists (ASA) physical status I and II were enrolled in this study. All enrolled participants were prepared to receive surgery (mastectomy or breast conserving surgery). Patients were excluded from the study according to the following exclusion criteria as shown in Fig. 1: 1) under 18 years of age, 2) history of previous surgery and tumors, 3) other types of tumors, 4) history of heart disease, respiratory disease, or neurological disease or other diseases, 5) coagulopathy or allergic to the drugs used in the surgery, 6) recently received radiotherapy or chemotherapy (<8 wks), 7) recently used steroids or opioids, alcohol or other illicit drugs. After exclusion by the criteria, 196 patients were included. These patients were randomized into two groups according to a random set of numbers generated by the computer: general anesthesia alone group (G group) and Pecs II block under general anesthesia group (PG group). During hospitalization, all participants received appropriate anesthesia and surgery. The surgical team and nursing team were blinded to the groups and all patients were anesthetized by one same anesthesiologist.
Surgery procedure All patients were administrated lidocaine (1 mg/kg), propofol, and remifentanil for anesthesia induction and maintenance. Remifentanil dose was adjusted to keep the surgical pleth index between 20 and 50. With the use of a target-controlled infusion (TCI) pump (Orchestra; Fresenius Kabi, Germany), Schnider’s15 and Minto’s16 pharmacokinetic models were respectively applied to calculate the effect-site amount of propofol and remifentanil. Pecs II block was performed according to the published method.17 Firstly, an ultrasound probe was placed under the lateral third of the clavicle to identify the axillary vein and artery. Then the probe was moved to the position between the pectoralis major and minor muscles. After that, under ultrasound guidance, a needle was introduced in-plane view of the ultrasound in an oblique manner between the pectoralis major and minor muscles. Then, ropivacaine (0.5%, 10 mL) was injected. The probe was then moved on from the first rib to the third rib to reach the serratus anterior muscle. The needle was advanced until its tip reached the potential space between the Pmm and SAM, ropivacaine (0.5%, 20 mL) was injected into this area. During the surgery, patients were monitored by electrocardiography, non-invasive blood pressure monitoring and oxygen saturation of pulse oximeter. Additionally, body temperature and the bispectral index (between 40 and 60, BIS vista monitor revision 3.0; Aspect Medical Systems, USA) were also monitored. If the bispectral index was above 60 or less than 40, 0.5 mg/kg with intervals of 1 min or more was applied as the effect-site concentration of propofol. Atropine 0.5 was applied intravenously when the heart rate was declined below 50 beats/min. After surgery, patients confirmed with recovered spontaneous breath and consciousness were moved to postoperative intensive care unit for the next 24 h monitoring. Blood samples collection The blood samples of patients were collected after 24 h of
Fig. 1. Flowchart of the study. G group, general anesthesia alone group. PG group, Pecs II block under general anesthesia group.
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X. Cui et al. / The American Journal of Surgery xxx (xxxx) xxx
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surgery in heparin tubes for peripheral blood mononuclear cell (PBMC) isolation and ethylenediaminetetraacetic acid tube for cytokine assessments. The isolation of PBMC was achieved using Ficoll-Paque (GE Healthcare, Sweden) density gradient centrifugation as previously reported,18 and frozen in heat-inactivated human AB serum (Sigma Aldrich, USA) containing 10% dimethyl sulfoxide (Sigma Aldrich, USA) in liquid N2.
between preoperative and postoperative values. A p-value<0.05 was considered as statistically significant. SPSS (version 20.0; SPSS Inc., USA) was used for all calculations.
NK cell isolation and apoptosis analysis
A total of 232 patients who were enrolled in this study (Fig. 1). After exclusion by several conditions, 196 patients were included. These patients were randomized into two groups, general anesthesia alone group (G group, n ¼ 98) and pectoral nerve block type II under general anesthesia group (PG group, n ¼ 98). Their characteristics are shown in Table 1. Patients in the two groups showed no differences in age, height weight, cancer stage, operation type and ASA status. However, during the surgery, the remifentanil consumption in PG groups was significantly lower than it of G group (p ¼ 0.001). No differences of other vital signs between G and PG groups were observed including arterial pressure and heart rate.
CD56þ NK cells were isolated from PBMC immunoselected by anti-CD56 conjugated microbeads (Miltenyi Biotec, UK) according to the protocol provided by the manufacturer using flow cytometry. Cell culture and apoptosis assay For the apoptosis assay in the presence or absence of patient serum or NK cells, we employed a human primary breast cancer cell line, HCC, which were cultured in ATCC-formulated RPMI-1640 Medium containing 10% fetal calf serum (FCS, Sigma). Sample serums were used to replace the FCS in indicated experiment. NK and HCC cells were co-cultured at a ratio of 1:10 in the cell medium with indicated serum for 24 h. Apoptosis rate of HCC cells were assessed using dual staining with FITC-Annexin and propidium iodide (Annexin V-FITC apoptosis detection kit, Biofriend, CN) and sorted by flow cytometry. NK cell cytotoxicity The cytotoxicity of NK cells was detected by incubation with K562 target erythroleukemic cells (American Type Culture Collection, USA). The cells were stained by carboxyfluorescein succinimidyl ester (Sigma Aldrich) and was collected after 4 h incubation. Propidium iodide (Becton Dickinson, USA) was added to determine dead target cells. Isolation of NKT cells, helper T cells (CD4þ) and cytotoxic T cells (CD8þ)
Results Patient characteristics
Effect of pectoral nerve block type II under general anesthesia on NK cell number and function To explore the different immune response in the two groups, we firstly tested the NK cell number. In G group, NK cells occupied about 17.2 ± 3.53% of PBMC before surgery (Fig. 2A) but this percentage was decreased to 13.36 ± 3.07% after general anesthesia (p ¼ 0.023). No significant difference of NK cell number was observed in patients treated with PG. Interestingly, when comparing the NK cell number between the two groups after the surgery, PG group showed a significantly higher occupancy of NK cells in PBMC than G group (p ¼ 0.036). For both G and PG groups, apoptosis rate of NK cells was not affected either before or after the surgery (Fig. 2B). The postoperative cell apoptosis rate of NK cells in PG and G groups also had similar percentage. The killing activities of NK cells in the two groups were dramatically reduced after surgery (p < 0.001, Fig. 2C). However, after the surgery, compared to G group, PG group had more functional NK cells (p ¼ 0.007).
The isolation of NKT cells, CD4þ T cells and CD8þ T cells were achieved using sorting with fluorescence-labeled antibodies by flow cytometry. Phycoerythrin (PE)-conjugated anti-human CD56 monoclonal antibody (cat no.E-AB-F1210D; Elabscience), fluorescein isothiocyanate-conjugated anti-human CD3 monoclonal antibody (cat no. E-AB-F1001C; Elabscience), fluorescein isothiocyanate-conjugated anti-human CD4 monoclonal antibody (cat no. E-AB-F1109C; Elabscience) and phycoerythrin-conjugated anti-human CD8a monoclonal antibody (cat no. E-AB-F1110C; Elabscience) were used.
The cell number of NKT, T helper 1 cells (CD4þ), and cytotoxic T lymphocytes (CD8þ) in PG and G groups
Measurement of cytokines
Cytokine concentrations and neutrophil, lymphocyte counts in the blood of patients in P and PG groups
Enzyme-linked immunosorbent assays were used to detect the plasma levels of interleukin (IL)-2, IL-4, and interferon-g (IFN-g) using a commercially available kit (R&D Systems, Inc.). Each cytokine was measured in triplicate for each blood sample. An automated microplate reader (Molecular Devices, Inc., USA) was used to obtain the results. Statistical analysis Data in patient characteristics and cytokine and neutrophil, lymphocyte counts in blood were indicated as median (interquartile ranges, IQR) or mean (Standard deviation, SD) for normally distributed data. A paired t-test was applied for the comparison
We further tested the effect of the two anesthesia methods on the PBMC percentage of NKT, T helper 1 cells (CD4þ), and cytotoxic T lymphocytes (CD8þ) cells. For both of G and PG groups, after surgery, no significant changes of these cell number were observed compared to their number before surgery (Fig. 3AeC). Additionally, no difference of these cell number percentage in PBMC was shown in the two groups.
The preoperative IL-2 concentration was about 3.9 ng/ml in both G and PG group. Interestingly, the postoperative IL-2 concentration in G group was significantly lower than its preoperative level (Table 2, p < 0.01). This reduction was not observed in PG group. The postoperative IL-4 and IFN-g maintained same concentration as their preoperative levels in G and PG groups. The numbers of neutrophils and lymphocytes were changed by the operation. The neutrophils were significantly increased from 60.5% (preoperational) to 70.1% (post-operative) in G group (p < 0.01) and from 60.8% (preoperational) to 63.1% 70.1% (postoperative) n PG group (p < 0.05). However, the post-operative neutrophils were less in PG group compared to G group
Please cite this article as: Cui X et al., Effect of pectoral nerve block type II under general anesthesia on the immune function of patients with breast cancer, The American Journal of Surgery, https://doi.org/10.1016/j.amjsurg.2020.03.008
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X. Cui et al. / The American Journal of Surgery xxx (xxxx) xxx Table 1 Patient demographic data and perioperative clinical characteristics.
Age (years) Height (cm) Weight (kg) Cancer stage (%) I II III IV Operation type (%) Breast-conserving surgery Modified radical mastectomy ASA status ASA I ASA II During surgery Propofol consumption (mg/kg/h) Remifentanil consumption (mg/kg/h) Use of atropine (n) Use of ephedrine (n) Duration of anesthesia (min) Duration of operation (min) Arterial pressure (mmHg) before induction incision closing ICU arrival Heart rate (bpm) before induction incision closing ICU arrival
G group (n ¼ 98)
PG group (n ¼ 98)
P value
51 (43e57) 160.2 (4.6) 55.7 (7.3)
53 (45e56) 159.4 (4.5) 56.5 (6.9)
0.398 0.455 0.279 0.576
28 (28.6) 43 (43.9) 27 (27.6) 0 (0)
33 (33.7) 35 (35.7) 30 (30.6) 0 (0)
37 (37.8) 61 (62.2)
40 (40.8) 58 (59.2)
60 (61.2) 38 (38.8)
62 (63.3) 36 (36.7)
7.1 (1.3) 11.4 (3.5) 8 7 152 (112e183) 115 (79e141)
6.9 (1.3) 6.3 (2.1) 3 6 149 (109e176) 104 (82e137)
0.493 0.001 0.069 0.542 0.393 0.488
100.1 (13.2) 83.9 (12.7) 84.5 (11.4) 92.6 (8.9)
98.3 83.7 84.8 91.8
0.295 0.286 0.447 0.173
75 66 72 80
72 67 71 81
0.556
0.648
(16) (15) (11) (10)
(14.5) (13.9) (13.2) (10.2)
(17) (15) (10) (8)
0.475 0.522 0.376 0.287
Data are expressed as median (interquartile ranges (IQR)) or mean (SD), or n(%). ASA, American Society of Anaesthesiologists. ICU, intensive care unit.
Fig. 2. Effect of Pectoral nerve block type II use during surgery on NK cells. (A) Mean preoperative and postoperative percentages of natural killer (NK) cells in the peripheral blood in different groups. (B) The apoptosis rate of circulating NK cells in different groups were tested. (C) Preoperative and postoperative NK cell function were examined at an effector: target ratio of 10:1. G group, general anesthesia alone group. PG group, Pecs II block under general anesthesia group. PBMCs, peripheral blood mononuclear cells. P < 0.05 was considered statistically significant.
(p ¼ 0.001). Furthermore, after surgery, lymphocytes were dramatically decreased in G (p < 0.01) and PG (p < 0.05) groups. When comparing the post-operative lymphocyte number, PG group showed a higher percentage of lymphocytes compared to G group (p ¼ 0.001). Accordingly, neutrophil-to-lymphocyte ratio (NLR) was remarkably elevated after surgery in comparison with the ratio before surgery in G (p < 0.001) and PG groups (p < 0.01). However, compared to G group, PG groups showed a significantly lower NLR (p ¼ 0.001). Apoptosis rate of HCC cells in the presence or absence of NK cells and subject serum-supplemented media We next investigated whether the serum of the subject could affect the apoptosis rate of HCC cells. We treated HCC cells with the
serum from normal patients or patients from G and PG groups. As shown in Fig. 4, no difference of HCC apoptosis rate was observed between the three treatments in the absence of NK cells. Intriguingly, in the presence of NK cells, the serum from patients (n ¼ 20) in G groups dramatically promoted HCC cell apoptosis compared to normal serum. HCC cultured in the serum collected from G and PG group patients after surgery showed a significantly lower apoptosis rate compared to those cultured in the serum collected before surgery (G group, p < 0.001; PG group, p ¼ 0.017). However, compared to the post-operative serum from PG group, the postoperative serum from G group significantly repressed HCC apoptosis rate (p ¼ 0.031), demonstrating the post-operative serum from G group may had a stronger ability of inhibition of NK cell function compared to the post-operative serum from PG group.
Please cite this article as: Cui X et al., Effect of pectoral nerve block type II under general anesthesia on the immune function of patients with breast cancer, The American Journal of Surgery, https://doi.org/10.1016/j.amjsurg.2020.03.008
X. Cui et al. / The American Journal of Surgery xxx (xxxx) xxx
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Fig. 3. Mean peripheral blood percentages of (A) natural killer T (NKT) cells, (B) T helper 1 cells (CD4þ), and (C) cytotoxic T lymphocytes (CD8þ) were tested in different groups. G group, general anesthesia alone group. PG group, Pecs II block under general anesthesia group. PBMCs, peripheral blood mononuclear cells. P < 0.05 was considered statistically significant.
Discussion Our study found that Pecs II block use compared to general anesthesia method had less remifentanil consumption during surgery. Pecs II was shown to affect immune system mainly by changing the cell proportion in the blood of patient received breast cancer surgery. In addition, the killing activity of NK cells was also promoted by Pecs II, demonstrating that Pecs II also had an impact on immune cell function. We found that the use of Pecs II during anesthesia significantly reduced the consumption of remifentanil. A similar observation was also found in a recent stud,19 documenting that preoperatiove administration of ropivacaine with Pecs II block significantly reduced the consumption of remifentanil during surgery compared to control (without Pecs II block). It is known that the choice of anesthesia is critical because it may be related to cancer recurrence.3 Although surgery resection may remove most of cancer tissue, a few cancer cells may be still remained in the body.20 In this respect, anesthesia-induced immunosuppression and surgery caused stress could help the spread and survival of these cancer cells. Surgery induces neuroendocrine stress, decreases numerous immune stimulating
Table 2 Perioperative Cytokine Concentrations and neutrophil, lymphocyte counts in blood of patients undergoing Breast Cancer Surgery.
IL-2 (ng/ml) Preop Postop IL-4 (ng/ml) Preop Postop IFN-g(ng/ml) Preop Postop Neutrophil (%) Preop Postop Lymphocyte (%) Preop Postop NLR Preop Postop
G group (n ¼ 98)
PG group (n ¼ 98)
P value
3.92 (0.34) 2.28 (0.36)b
3.87 (0.26) 3.23 (0.33)
0.385 0.002
0.43 (0.13) 0.47 (0.14)
0.45 (0.18) 0.42 (0.15)
0.468 0.313
2.09 (0.24) 1.97 (0.27)
2.12 (0.22) 2.01 (0.32)
0.229 0.263
60.5 (12.7) 70.1 (13.7) 27.0 (7.3) 21.2 (7.0)
60.8 (13.9) 63.1 (14.2)
b
26.4 (6.5) 22.9 (5.9)
b
2.2 (1.6e3.1) 3.3 (2.0e4.3)
c
0.298 0.001
a
0.397 0.001
a
2.3 (1.7e2.8) 2.7 (1.8e3.5)
cytokine production including IFN-g and IL-2 and increases the level of anti-inflammatory cytokines.9 In addition to its inhibitory effects on immune system, surgery was experimentally and clinically found to be associated with increased angiogenesis and promoted metastasis.3 Anesthetics such as opioids were also found to suppress cell-mediated immunity21 and promote tumor development and metastasis.22 In a rat model, remifentanil was found to repress NK cell activity and proliferation of lymphocyte.23 Similar inhibitory role of remifentanil in immunity was also observed in clinical researches.24,25 Therefore, less use of opioids such as remifentanil could benefit the outcomes of the cancer patients. In our study, we found that NK cell percentage in PBMC and its killing activity were dramatically declined after the surgery, implicating the surgery and anesthetic induced immunosuppression. Impressively, the application of Pecs II block showed less inhibitory impact on NK cell number and function. We are the first study finding the supportive effects of Pecs II block on NK cells. This is essential for further clinical application of Pecs II block during surgery as immune regulation could partially determine the recurrence of cancer. We did not observe changes of NKT or T lymphocytes induced by the surgery. However, several previous studies reported that surgery could cause the remarkable loss of T lymphocytes and NKT cells.24,26,27 The controversial results may be due to the differences in the surgery procedure and types of cancer.
b
0.416 0.001
Data are expressed as median (interquartile ranges (IQR)) or mean (SD). IL, interleukin. NLR, neutrophil-to-lymphocyte ratio. Preop, Preoperative, which means immediately before anesthesia induction. Postop, Postoperative, which means 24h postoperatively. a <0.05, Preop vs Postop. b <0.01, Preop vs Postop. c <0.001, Preop vs Postop.
Fig. 4. Apoptosis rate of HCC cells after co-cultured with NK cells in the presence of subject serum-supplemented media. HCC cells were cultured in serum-supplemented media, either alone, or with NK cells. Apoptosis was determined by dual staining with FITC-Annexin V and propidium iodide and tested by flow cytometry. Each experiment was carried out in triplicate. P < 0.05 was considered statistically significant.
Please cite this article as: Cui X et al., Effect of pectoral nerve block type II under general anesthesia on the immune function of patients with breast cancer, The American Journal of Surgery, https://doi.org/10.1016/j.amjsurg.2020.03.008
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X. Cui et al. / The American Journal of Surgery xxx (xxxx) xxx
A study investigated the effect of propofol on T lymphocytes and NK cells in patients with breast cancer documented that NK cell number was significantly reduced after surgery.28 Lymphocyte count also showed a decreasing trend after surgery with no statistical significance. These observations are same as our results. It was reported that surgery stress could repress the production of immune stimulating cytokines including IL-2 and IFN-g.9 Consistently, here, we observed a significantly diminished level of IL-2 in the blood of patients after surgery compared to its level before surgery but not IFN-g. Interestingly, compared to general anesthetic, Pecs II block use significantly promoted IL-2 production. As IL-2 is primarily produced by activated CD4þ T lymphocytes, Pecs II block may potentially improve the ability of IL-2 expression and production from T lymphocytes as it did not affect T lymphocytes number. It is possible that this is caused by less use of remifentanil because opioids were reported to have adverse effect on IL2 and IFN-g production.29 Further studies are necessary to reveal the how Pecs II block and remifentanil affect the IL-2 production. In addition, since IL-2 is an important regulator of differentiation and homeostasis of T lymphocytes, less IL-2 level may be associated with reduced effector T cells.30 However, although IL-2 level showed higher in PG group than G group after surgery, both CD4þ and CD8þ T lymphocytes had no significant changes. In addition, increase of IL-2 level could be the cause of enhanced NK cell killing ability observed in PG groups compared to G group after surgery because IL-2 was reported to promote the cytolytic activity of NK cells.30 It is possible that surgery and anesthetic suppress other cytokines needed in T cell development and differentiation or affect the expression of critical proteins such as IL-2 receptor. However, further studies are necessary to reveal the detailed mechanism. We also found that PG group showed a reduced NLR compared to G group after surgery, indicating patients in PG group had less inflammation than G group. Since there is an association between high NLR and poor survival rate particularly in breast cancer,31 PG group may have a better prognosis compared to G group, which needs further investigation. We also found that HCC showed more apoptosis rate cultured in the postoperative serum from PG group compared to the postoperative serum from G group in the presence of NK cells. The result suggests that PG group may contain more cytokines supporting the NK cell proliferation or killing activity, which is similar to our aforementioned observations. In addition to these immune cells in blood, it is also important to investigate whether Pecs II block has an impact on the immune cells in the tumor microenvironment. For example, tumor infiltrating lymphocytes (TIL) or PD-1 and PD-L1 levels in tumor tissue and the surrounding stromal area are important parameters to confirm how Pecs II block affects immune functions related to the tumor microenvironment, which are omitted in this study. However, in our further study, it is necessary to investigate the effect of Pecs II block on the immune system in the tumor microenvironment. Conclusion In conclusion, after surgery, elevation of NK cells in PBMC and the killing activity were observed in the group with Pecs II block use compared to the group without it. There was no change of T lymphocytes and NKT between PG group and G group. Neutrophil increase and lymphocyte decrease after surgery in G groups caused a higher level of postoperative NLR compared to the preoperative ratio. However, in PG group, this postoperative NLR was significantly lower than P group, suggesting a less inflammation in PG group. We also observed that IL-2 production was improved in PG group compared to G group after surgery. Taken together, these
results indicate that patients in PG group showed an enhanced immunity compared with those in G group. Funding None. Declaration of competing interest The authors have declared that no conflicts of interest exist. References 1. Siegel RL, Miller KD, Jemal A. Cancer statistics. CA A Cancer J. Clin. 2016;66(1): 7e30, 2016. 2. Ben-Eliyahu S. The price of anticancer intervention. Does surgery promote metastasis? Lancet Oncol. 2002;3(9):578e579. 3. Kim R. Effects of surgery and anesthetic choice on immunosuppression and cancer recurrence. J Transl Med. 2018;16(1):8. 4. Moon E-J, Kim S-B, Chung J-Y, et al. Pectoral nerve block (Pecs block) with sedation for breast conserving surgery without general anesthesia. Ann. Sur. Tret. Res. 2017;93(3):166e169. 5. Freise H, Van Aken H. Risks and benefits of thoracic epidural anaesthesia. Br. J. Anaesth. 2011;107(6):859e868. 6. Morioka H, Kamiya Y, Yoshida T, Baba H. Pectoral nerve block combined with general anesthesia for breast cancer surgery: a retrospective comparison. J. A Clin. Rep. 2015;1(1):15. 7. Neethu M, Pandey RK, Sharma A, et al. Pectoral nerve blocks to improve analgesia after breast cancer surgery: a prospective, randomized and controlled trial. J Clin Anesth. 2018;45:12e17. 8. Macrae W. Chronic pain after surgery. Br. J. Anaesth. 2001;87(1):88e98. 9. Lin E, Calvano SE, Lowry SF. Inflammatory cytokines and cell response in surgery. Surgery. 2000;127(2):117e126. 10. Lim J-A, Oh C-S, Yoon T-G, et al. The effect of propofol and sevoflurane on cancer cell, natural killer cell, and cytotoxic T lymphocyte function in patients undergoing breast cancer surgery: an in vitro analysis. BMC Canc. 2018;18(1): 159. 11. Li Y, Sun R. Tumor immunotherapy: new aspects of natural killer cells. Chin J Canc Res. 2018;30(2):173. 12. Market M, Baxter KE, Angka L, et al. The potential for cancer immunotherapy in targeting surgery-induced natural killer cell dysfunction. Cancers. 2019;11(1): 2. 13. Snyder G, Greenberg S. Effect of anaesthetic technique and other perioperative factors on cancer recurrence. Br. J. Anaesth. 2010;105(2):106e115. 14. Janakiram NB, Mohammed A, Bryant T, et al. Loss of natural killer T cells promotes pancreatic cancer in LSL-KrasG12D/þ mice. Immunology. 2017;152(1):36e51. 15. Schnider TW, Minto CF, Gambus PL, et al. The influence of method of administration and covariates on the pharmacokinetics of propofol in adult volunteers. J. Am. Soc. Anesthesiologists. 1998;88(5):1170e1182. 16. Minto CF, Schnider TW, Egan TD, et al. Influence of age and gender on the pharmacokinetics and pharmacodynamics of RemifentanilI. Model Dev. Anesthesiol. J. Am. Soc. Anesthesiol. 1997;86(1):10e23. 17. Blanco R, Fajardo M, Parras Maldonado T. Ultrasound description of Pecs II (modified Pecs I): a novel approach to breast surgery. Rev Esp Anestesiol Reanim. 2012 Nov;59(9):470e475. PubMed PMID: 22939099. Epub 2012/09/ 04. 18. Jaatinen T, Laine J. Isolation of mononuclear cells from human cord blood by Ficoll-Paque density gradient. Curr. Protoc. Stem Cell Biol. 2007;1(1), 2A. 1.-2A. 1.4. 19. Choi JJ, Jo YY, Kim SH, et al. Remifentanil-sparing effect of pectoral nerve block type II in breast surgery under surgical pleth index-guided analgesia during total intravenous anesthesia. J Clin Med. 2019;8(8):1181. 20. Lloyd JM, McIver CM, Stephenson S-A, et al. Identification of early-stage colorectal cancer patients at risk of relapse post-resection by immunobead reverse transcription-PCR analysis of peritoneal lavage fluid for malignant cells. Clin Canc Res. 2006;12(2):417e423. 21. Gottschalk A, Sharma S, Ford J, et al. The role of the perioperative period in recurrence after cancer surgery. Anesth Analg. 2010;110(6):1636e1643. 22. Cassinello F, Prieto I, del Olmo M, et al. Cancer surgery: how may anesthesia influence outcome? J Clin Anesth. 2015;27(3):262e272. 23. Sacerdote P, Gaspani L, Rossoni G, et al. Effect of the opioid remifentanil on cellular immune response in the rat. Int Immunopharm. 2001;1(4):713e719. 24. Sacerdote P, Bianchi M, Gaspani L, et al. The effects of tramadol and morphine on immune responses and pain after surgery in cancer patients. Anesth Analg. 2000;90(6):1411e1414. 25. Qi Y, Yao X, Zhang B, Du X. Comparison of recovery effect for sufentanil and remifentanil anesthesia with TCI in laparoscopic radical resection during colorectal cancer. Oncol. Lett. 2016;11(5):3361e3365. 26. Angele MK, Faist E. Clinical review: immunodepression in the surgical patient and increased susceptibility to infection. Crit Care. 2002;6(4):298.
Please cite this article as: Cui X et al., Effect of pectoral nerve block type II under general anesthesia on the immune function of patients with breast cancer, The American Journal of Surgery, https://doi.org/10.1016/j.amjsurg.2020.03.008
X. Cui et al. / The American Journal of Surgery xxx (xxxx) xxx 27. Klatka J, Grywalska E, Hymos A, et al. Subpopulations of natural killer-T-like cells before and after surgical treatment of laryngeal cancer. Cent Eur J Immunol. 2017;42(3):252. 28. Woo JH, Baik HJ, Kim CH, et al. Effect of propofol and desflurane on immune cell populations in breast cancer patients: a randomized trial. J Kor Med Sci. 2015;30(10):1503e1508.
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29. Al-Hashimi M, Scott S, Thompson JP, Lambert D. Opioids and immune modulation: more questions than answers. Br. J. Anaesth. 2013;111(1):80e88. 30. Liao W, Lin J-X, Leonard WJ. Interleukin-2 at the crossroads of effector responses, tolerance, and immunotherapy. Immunity. 2013;38(1):13e25. 31. Faria SS, Fernandes Jr PC, Silva MJB, et al. The neutrophil-to-lymphocyte ratio: a narrative review. Ecancermedicalscience. 2016;10.
Please cite this article as: Cui X et al., Effect of pectoral nerve block type II under general anesthesia on the immune function of patients with breast cancer, The American Journal of Surgery, https://doi.org/10.1016/j.amjsurg.2020.03.008