Introduction

Previous studies have shown that trauma, surgery, and anesthesia induce a state of immunosuppression [1, 2]. A downward regulation of the immune response and host defense against tumor cells may occur in the perioperative period. As a consequence, patients are more susceptible to infections and sepsis [1]. The immunological response to surgery has been studied more since the introduction of minimally invasive techniques. Laparoscopic surgery reduces the magnitude of operative trauma. If alterations of the systemic immune response are proportional to the extent of injury, then the response to minimally invasive techniques will be reduced when compared with open surgery. A key factor in postoperative morbidity is the surgical stress response with ensuing increased demand on the patient’s reserves and the immunological competence. Increased demands on organ functions are thought to be mediated by trauma-induced endocrine and metabolic changes. The stress response has been studied by monitoring levels of hormones such as cortisol, prolactin, and growth hormones, whereas the acute-phase response has been measured by means of various cytokine and acute-phase response protein levels. Of these, interleukin-6 (IL-6), interleukin-8 (IL-8), and C-reactive protein (CRP) are among the most frequently studied after surgical trauma [3–9]. Each of the two cytokines represents a different component of the inflammatory response: IL-6 is a mediator of the acute phase, inducing synthesis of proteins by the liver, whereas IL-8 has chemoattractant activity and is able to activate and degranulate neutrophils [10]. Immune status, inflammatory response, and stress response have been analyzed in different studies comparing conventional open surgery to minimally invasive surgery, and the minimally invasive approach has been favored [3–5].

Currently, for patients with advanced esophageal cancer, the best curative treatment is surgical resection. This extensive surgical procedure involves a thoracotomy and laparotomy with gastric tube reconstruction and an esophagogastric anastomosis. A high morbidity rate, mostly due to respiratory complications, and a long intensive care unit and hospital stay are consequences of this procedure.

In recent years, many surgical techniques have been developed to reduce morbidity rates after esophageal resection. Minimally invasive approaches are likely to be favorable over conventional open procedures, as observed in previous studies [11]. This study was performed as a substudy of a randomized trial (TIME trial: traditionally invasive versus minimally invasive esophagectomy) that compares conventional open esophagectomy by thoracotomy and laparotomy [open surgical technique (OE)] with minimally invasive esophagectomy [minimally invasive procedure (MIE)] by thoracoscopy and laparoscopy [12]. Different parameters, i.e., stress response hormone levels, acute-phase response protein levels, and immunological status, were studied during different postoperative stages in both groups.

Methods

Study design

Data were collected of patients who had been included in a randomized, multicenter trial comparing conventional esophagectomy with minimally invasive esophagectomy (TIME trial, NTR TC 2452). Details of the trial design have been published elsewhere [12, 13]. From 2009 to 2010, a total of 27 consecutive patients who participated in the TIME trial in the VU University Medical Center were included into this substudy.

Patient selection

Patients were eligible for this trial if they had a histologically proven squamous cell carcinoma, adenocarcinoma, or undifferentiated carcinoma of the intrathoracic esophagus and gastroesophageal junction tumors that were considered surgically resectable (T1-3, N0-1, M0). Moreover, the eligible patients must have had an eastern cooperative oncology group (ECOG) performance status of 0, 1, or 2. All patients in this trial received neoadjuvant chemoradiotherapy, as defined in the CROSS protocol [14].

Surgical technique

The OE involves a right thoracotomy with lung blockade and laparotomy with either a cervical or a thoracic anastomosis. The MIE involves a right thoracoscopy with the patient in the prone position with a single lumen tube and laparoscopy with either a cervical or thoracic anastomosis.

End points

A choice was made of the most standardized markers to study the end points. End points were stress response measurements of cortisol, prolactin, and growth hormone levels; immune status as manifested by the preservation of HLA-DR on monocytes; white blood cell count; and acute inflammatory response measured by means of IL-6, IL-8, and CRP level determinations. All measurements were performed preoperatively and on postoperative day 1, 3, 4, and 7.

Material and methods

Peripheral blood and serum (BD Vacutainer Systems, Plymouth, UK) were collected preoperatively (baseline) and 24, 72, 96, and 168 h after surgery. Serum IL-6 and IL-8 samples were obtained by centrifugation for 10 min at 3,000 rpm at 4 °C. All samples were stored in aliquots at −80 °C until tested.

Immunological response

White blood cell count and phenotype were determined from fresh heparinized venous blood (within 2 h after obtainment). Phenotyping was performed by using CD14-PE and HLA-DR-FITC moAbs (Becton Dickinson, Franklin Lakes, NJ, USA), lysis of erythrocytes, and fixation with paraformaldehyde. Evaluation of monocyte HLA-DR expression was performed by FACS analysis (FACS Calibur, Becton Dickinson), quantified by using calibration beads (Quantum™ 26, Flow Cytometry Standards Corp, Bangs Laboratories Inc, Fishers, IN, USA), and expressed as the ratio of the mean fluorescence intensity after surgery/before surgery.

Acute inflammatory response

Concentrations of IL-6 and IL-8 were measured using commercially available enzyme-linked immunosorbent assay kits (PeliKine compact ELISA kit, Sanquin, Amsterdam, The Netherlands). CRP was measured in blood serum by immunoturbidimetric testing using the BM/Hitachi 705 (Boehringer, Mannheim, Germany).

Stress response

Cortisol and growth hormone concentrations were measured by competitive immunoassay (Bayer Diagnostics, Mijdrecht, The Netherlands). Prolactin was measured by immunometric assays (DPC, Los Angeles, CA, USA).

Statistical analysis

Statistical analysis was performed using the SPSS software package 15 (SPSS Inc., Chicago, IL, USA). The results for the two different groups were compared by means of Mann–Whitney U test. Categorical parameters were analyzed with Fisher’s exact test. Significance was set at p < 0.05.

Results

A total of 27 patients were included in this substudy of the TIME trial, 14 patients in the minimally invasive group and 13 patients in the open group.

Clinical characteristics

Demographic parameters, surgical data, and pathological tumor indices are given in Table 1. No significant differences were observed between groups with respect to gender, age, tumor location, and operation time. There was significantly less blood loss in the MIE group (450 vs. 275 ml, p < 0.05). General outcome and postoperative morbidity are depicted in Table 2.

Table 1 Patient characteristics
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Table 2 General outcome and morbidity
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Immunological response

At baseline, white blood cell count was comparable in both groups (Table 3) At 24, 72, and 96 h after surgery, there were no significant differences between the open and minimally invasive groups. However, at 168 h (1 week) after surgery, there was a significant difference in white blood cell count due to an increase in the OE group as opposed to a sustained decrease in the MIE group (Fig. 1) The change in leukocyte count after 1 week is also significantly different between the groups (p = 0.011).

Table 3 Postoperative immune, inflammatory, and stress responses
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Fig. 1
figure 1

Concentration of leukocytes in blood. *Significant difference at p = 0.004. Leukocyte OE, concentration of leukocytes in blood in the open esophagectomy group; Leucocyte MIE, concentration of leukocytes in blood in the minimally invasive esophagectomy group

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The expression of HLA-DR on monocytes showed a decrease of 60 % or more in both groups at all measured moments (p < 0.05). This was lowest for the open group at 72 h after surgery (23.8 %), although no statistical significance between the groups was reached (Fig. 2).

Fig. 2
figure 2

Preservation of HLA-DR on monocytes. %HLA-DR OE percentage of HLA-DR on monocytes in blood in the open esophagectomy group, %HLA-DR MIE percentage of HLA-DR on monocytes in blood in the minimally invasive esophagectomy group

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Acute inflammatory response

IL-6, IL-8, and CRP levels were comparable at baseline (Table 3). There were no significant differences between the groups in the postoperative IL-6 and CRP levels. However, CRP levels peaked at 72 h after surgery for both groups (Fig. 3), and both IL-6 and IL-8 levels showed an increase at 24 h compared to baseline measurements (Figs. 4, 5). Remarkably, at 168 h after surgery, the IL-8 level in the OE group was significantly higher than that in the MIE group (p = 0.047) (Fig. 5).

Fig. 3
figure 3

Concentration of C-reactive protein in blood. CRP OE, CRP concentration in blood in the open esophagectomy group; CRP MIE, CRP in concentration in blood in the minimally invasive esophagectomy group

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Fig. 4
figure 4

Concentration of IL-6 in blood. IL 6 OE, IL-6 concentration in blood in the open esophagectomy group; IL-6 MIE, IL-6 concentration in blood in the minimally invasive esophagectomy group

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Fig. 5
figure 5

Concentration of IL-8 in blood. *Significant difference at p = 0.047. IL8 OE, IL-8 concentration in blood in the open esophagectomy group; IL8 MIE, IL-8 concentration in blood in the minimally invasive esophagectomy group

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Stress response

Cortisol, prolactin, and growth hormone at baseline in both groups were comparable (Table 3). Cortisol levels upon surgery were elevated at all time measurements for both groups as growth hormone peaked at 24 h for the OE group but showed no significant differences between the groups (data not shown). Prolactin levels in the OE group, however, fluctuated in time, being significantly elevated at 168 h (1 week) after surgery compared to the MIE group (Fig. 6).

Fig. 6
figure 6

Concentration of prolactin in blood. *Significant difference at p = 0.49. PROL OE, prolactin concentration in blood in the open esophagectomy group; PROL MIE, prolactin concentration in blood in the minimally invasive esophagectomy group

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Discussion

Immunological reaction after surgery may contribute to infectious complications, sepsis, and tumor growth [1, 2]. Minimally invasive surgery has been shown to preserve immunological function better than conventional open surgery in different surgical procedures, including cholecystectomy, Nissen fundoplication, and laparoscopic colorectal surgery [3–5]. Scheepers et al. [8] described the immunological consequences of laparoscopic versus open transhiatal resection for malignancies of the esophagus. That nonrandomized study compared six patients in the laparoscopic group with 11 patients in the open group. They found an increase in all markers, with significantly higher levels of IL-6 for the open group, suggesting that the surgical trauma in the minimally invasive group was less extensive. This increase of IL-6, as an expression of the extent of trauma and predictor for postoperative complications, is also seen in this study. A peak is observed 24 h after surgery, as in other studies [3, 6, 8]. However, no significant differences were found in IL-6 and CRP levels between the OE group and the MIE group. Remarkably, the leukocyte count was significantly higher in the OE group at 7 days, whereas the expression of HLA-DR on monocytes was decreased postoperatively but without a significant difference between the groups. A possible explanation is that the extent of trauma was so predominant in both groups of transthoracic esophagectomy that differences, measured by IL-6 and CRP, were masked. However, it must be acknowledged that this study includes a small number of patients. Additional differences would probably be seen with a larger number of patients.

There was a significant increase in IL-8 and white blood cell counts in the OE group 1 week after surgery. Not only was the difference in white blood cell count at 1 week significant, but the change in leukocyte count was also significantly different in both groups, in favor of the minimally invasive group. IL-8 is thought to play an important role in the development of pneumonia [7, 10, 15, 16]. Fujimori et al. [15] investigated the role of IL-8 in interstitial pneumonia and found that an increased level is associated with fibrosis and injury of the lung. Yamada et al. [7] evaluated serum IL-6 and IL-8 in patients who underwent conventional thoracic surgery and found a significant increase in IL-8 levels until the postoperative day 7 in patients who developed postoperative pulmonary infections. The authors suggest that this increase in IL-8 may reflect the severe surgical stress due to reperfusion of ischemic lung tissue as a result of one-lung ventilation during thoracotomy. In our study we also found increased IL-8 levels in the early postoperative period for both groups, with a significant difference at postoperative day 7 in favor of the MIE group. This could explain the trend of fewer respiratory infections observed in the MIE group in comparison to the OE group (seven in the OE group and three in MIE group). This trend actually was confirmed in the main study, the TIME trial, which was recently published [12]. Other studies that compared open to minimally invasive techniques used in cholecystectomy, Nissen fundoplication, and colorectal surgery have shown better immunological outcomes in favor of the minimally invasive techniques [3–5]. However, an important difference between esophagectomy and the other interventions, e.g., cholecystectomy, is the amount of surgical trauma. It is probably more difficult to demonstrate significant differences in immunosuppression in interventions with a huge wound surface like the esophagectomy. For example, expression of HLA-DR was decreased at all times for both groups in our study, whereas after Nissen fundoplication and even after colorectal surgery the expression of HLA-DR on monocytes returned to preoperative levels within a week [4, 5].

Since the increase in IL-8 levels has been identified both during and after surgery, the possibility has risen to block this increase intraoperatively to minimize respiratory infections after esophagectomy [7, 9]. Kawahara et al. [9] introduced the administration of a neutrophil elastase inhibitor in patients undergoing esophageal resection by thoracoscopy. They showed that this inhibitor seems to partially suppress the postoperative increase in IL-8 levels and shorten the duration of systemic inflammatory response in a randomized trial. As a result, acute lung injury and respiratory infections could be prevented.

The increase of prolactin, cortisol, and growth hormone levels seems to be less intense and of shorter duration after laparoscopic gynecologic pelvic surgery [17, 18]. An increase in these levels with open surgery might not be due to surgery alone, but also to the administration of anesthetic drugs such as morphine [19]. All patients in our study received epidural anesthesia for at least 3 days. At 1 week after surgery a significant difference in prolactin levels between the two groups was observed. This might be attributable to the use of analgesics. At 1 week after surgery, the use of morphine was increased in the open group (11 vs. 5 patients).

In conclusion, in this substudy of a randomized trial comparing minimally invasive with conventional esophagectomy for cancer, a significantly better preserved leukocyte count and IL-8 level were observed in the MIE group than in the open group. Though the differences are small, both values might be related to fewer respiratory infections found postoperatively in the MIE group. Moreover, stress response was also better preserved in the MIE group, as expressed by the slightly different prolactin values at 1 week postoperatively. These findings indicate that less surgical trauma could lead to better preserved acute-phase response and fewer respiratory infections. Overall, further studies will be needed to subscribe to our findings.