A case-control study of the local and systemic ...

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Title A prospective case-control study of the local and systemic cytokine response after laparoscopic versus open colonic surgery. Running head A prospective case-control study Authors and Affiliations Dr Tarik Sammour, MBChB Research Fellow Department of Surgery South Auckland Clinical School Faculty of Medical and Health Sciences University of Auckland Auckland, NZ Dr Arman Kahokehr, MBChB Research Fellow Department of Surgery South Auckland Clinical School Faculty of Medical and Health Sciences University of Auckland Auckland, NZ Dr Kamran Zargar-Shoshtari, MD Department of Surgery Rotorua Hospital Rotorua, NZ Assoc Prof Andrew G Hill, MD, FRACS Associate Professor of Surgery Department of Surgery South Auckland Clinical School Faculty of Medical and Health Sciences University of Auckland Auckland, NZ Correspondence addressed to: Dr Tarik Sammour Research Fellow, Department of Surgery, South Auckland Clinical School Private Bag 93311, Middlemore Hospital, Otahuhu, Auckland, NZ Tel no. +6421317417 Fax no. +6496264558 Email: [email protected]

Sources of funding: Dr Tarik Sammour is supported by a Surgeon Scientist Scholarship administered by the Royal Australasian College of Surgeons. Dr Arman Kahokehr is supported by the Ruth Spencer Fellowship administered by the Auckland Medical Research Foundation.

Acknowledgments The authors would like to thank Mr Sofian Tijono, Auckland Cancer Society Research Centre for his assistance with cytokine analysis.

Disclosures Drs. Sammour, Kahokehr, Zargar-Shoshtari and Hill have no conflicts of interest or financial ties to disclose.

Presentations 5th Annual Academic Surgical Congress, San Antonio, Texas, Feb 3rd – 5th, 2010. Surgical Research Society Annual Meeting, Adelaide, Australia, Nov 20th 2009.

Word count Text: 2844, Abstract: 255.

Abstract

Background There is a sequential, high concentration cytokine response after major abdominal surgery. The magnitude of this response has been directly linked to post-operative metabolic derangement, ileus, adhesions, and oncological outcomes. We aimed to compare the local and systemic cytokine response in laparoscopic and open colonic surgery and relate this to postoperative recovery parameters.

Methods Using a prospectively collected patient database, we compared a Study Group (n = 50) of patients undergoing elective laparoscopic colonic resection with a Control Group (n = 25) of patients undergoing equivalent open colonic surgery within an ERAS program. Patients were matched for age, sex, BMI, ASA, Cr Possum, side of resection, diagnosis, and histological stage. Plasma and peritoneal fluid concentrations of IL-6, IL-8, IL-10, and TNFα were measured at 20 – 24 hours after surgery. The Surgical Recovery Score was determined pre-operatively and at 3, 7, 30, and 60 days post-operatively. All data were prospectively collected, and a priori definitions were used for discharge parameters, complications, and complication severity.

Results Peritoneal fluid IL-6 concentration was lower after laparoscopic surgery. There were no significant differences in the other cytokines measured, or in any post-operative recovery outcomes. Significant correlations were found between cytokine levels and discharge criteria achievement, day stay, post-operative complications, and the Surgical Recovery Score.

Conclusion With the exception of a lower peritoneal IL-6 level, the systemic and peritoneal cytokine response at 20-24 hours is similar after laparoscopic versus open colonic resection within an ERAS program, with corresponding equivalent rates of post-operative recovery.

Keywords Cytokine, laparoscopy, colectomy, interleukin, pneumoperitoneum, colon

A prospective case-control study of the local and systemic cytokine response after laparoscopic versus open colonic surgery. Sammour T, Kahokehr A, Zargar-Shoshtari K, Hill AG

Introduction Cytokines are polypeptides that function by means of paracrine and autocrine mechanisms. They form a subgroup of humoral mediators that are directly responsible for the inflammatory processes that define the injury response.(1-5) There is a sequential, high concentration cytokine response after major abdominal surgery.(6-8) This contributes to the systemic hemodynamic instability and global metabolic derangement that is observed post-operatively, (9-11) and is thought to be a determinant of post-operative fatigue which can last for up to 3 months after surgery.(12)

The systemic humoral response may be reduced after laparoscopic surgery compared with equivalent open procedures.(13-18) A recent meta-analysis of randomised controlled trials demonstrated significantly lower serum interleukin 6 (IL-6) levels on day 1 after laparoscopic colorectal resection.(19) However, conclusions were limited by heterogeneity in surgical indication, humoral factor measured, measurement timing, method of sample analysis, and lack of correlation of cytokine levels with clinical outcomes. In addition, despite randomisation, small subject numbers and exclusion of laparoscopic cases converted to laparotomy resulted in poor matching between the study and control groups in several included trials.(19)

Furthermore, the local peritoneal response may be the more important determinant of postoperative recovery.(20, 21) In addition local peritoneal cytokines also have a specific role to play in post-operative ileus,(22-26) the formation of peritoneal adhesions,(27-32) and tumour promotion.(33-37) Only 2 studies identified in the meta-analysis above measured levels of local peritoneal humoral factors, and their results were conflicting.

The aim of this paper is to compare the local and systemic cytokine response in laparoscopic and open colonic surgery and relate this to post-operative recovery parameters. Our hypothesis is that

the local cytokine response, and hence post-operative recovery measures, will be similar after laparoscopic and open colonic surgery.

Materials and Methods Study Group The study group consisted of a subset of consecutive patients recruited as part of a double-blinded randomized controlled trial investigating humidification of insufflation gas in laparoscopic colonic surgery (registered ClinicalTrials.gov identification no. NCT00642005). All patients undergoing elective laparoscopic colonic resection for any indication at any of the three public hospitals in the Auckland region (Auckland City Hospital, North Shore Hospital, and Middlemore Hospital) between April 2008 and June 2009 were screened for inclusion. Exclusion criteria were: Patients under 15 years of age, acute colonic resections, hand-assisted colonic resection, decision to perform open surgery pre-operatively (intra-operative conversions were included as intention to treat), surgery for rectal lesions defined as within 15 cm of the anal verge, stoma formation (preoperative or intraoperative decision), pre-operative steroid dependence, inability to consent due to cognitive impairment or language barrier, patients with ASA > 4, and deviation from anaesthetic protocol (patients who had an epidural or spinal anaesthesia were excluded).

Study group patients were matched to patients in the control group for age, sex, Body Mass Index (BMI), American Society of Anaesthesiologists Score (ASA), Colorectal Physiological and Operative Severity Score (Cr Possum), side of surgery (right versus left sided resection), diagnosis, presence or absence of malignancy, and Dukes’ histological stage.

Control Group The historical control group consisted of consecutive patients recruited as part of a completed double-blinded randomized controlled trial investigating the use of intravenous Dexamethasone (Dexamethasone Sodium Phosphate Injection, 4mg/ml, Hospira NZ Limited, Wellington, New Zealand) in open colonic surgery.(38) All patients undergoing elective open colonic resection within an Enhanced Recovery After Surgery (ERAS) program at the Manukau Surgical Centre between

June 2006 and March 2008 were screened for inclusion. Exclusion criteria were equivalent to the study group. Only patients in the intervention arm of the Dexamethasone study were included in the comparison because Dexamethasone administration results in an attenuated early peritoneal cytokine response.(38) Similarly, all patients in the Study Group (above) had also received a single dose of intravenous Dexamethasone after anaesthesia induction as part of the study protocol.

Sample collection and storage Patients in both arms of the study had a size 15F Blakes drain (Blake Silicone Drain, Ethicon inc, Somerville, New Jersey 08876-0151) inserted into the peritoneal cavity at the conclusion of surgery. This was brought out through the abdominal wall, and sutured to the skin. The drain was attached to a low vacuum drain bottle (Low vacuum wound drainage system 80kPa / neg 150mmHg, Leur-lock replacement bottle, Van Straten Medinorm, 66583 Spiesen, Germany). At 20 - 24 hours post-operatively, 4 mL of fluid from the drain bottle as well as a simultaneous sample of 4 mL of venous blood were collected into two separate vacutainer tubes containing either ethylenediamine tetra-acetic acid or sodium citrate to prevent clotting (BD Vacutainer, BD Diagnostics Franklin Lakes, NJ 07417 USA). The timing of sample collection was chosen because peritoneal IL-6 levels peak at 20 – 24 hours after abdominal surgery.(6) All collected samples were sent immediately on ice to the Middlemore Hospital Laboratory, where they were centrifuged for 10 minutes at 1000 x g and the supernatant removed. Samples were then aliquoted and stored in polypropylene tubes at -80 °C for future batch analysis.

Cytokines studied Median concentrations of IL-6, IL-8, IL-10, TNFα and were compared between groups. IL-6 is considered to be a major mediator of the acute phase protein response following injury(39, 40) with levels rising in proportion to the extent of tissue injury.(3) Post-operatively, an exaggerated IL-6 response precedes the clinical onset of complications by 12-48 h.(39, 41) Interleukin-8 (IL-8) directs the transmigration of neutrophils(42) and has been linked to tumour development, promoting tumour cell growth in colon cancer cells.(43) IL-10 in an anti-inflammatory cytokine. It inhibits the production of several of the pro-inflammatory mediators outlined above, and down-

regulates the functional properties of immunocompetent cells.(44) TNFα is one of the first cytokines to be produced in the peritoneum after injury. There is evidence that an increase in plasma TNFα is deleterious in septicaemia.(45, 46) Conversely, local production of TNFα in the abdominal cavity during peritonitis may have a protective function.(39, 47, 48)

Cytokine analysis At the end of recruitment of the Study Group patients, all samples from both groups were retrieved, thawed, and assayed together (analysis of Control Group samples was repeated to ensure equivalent analysis technique). Median concentrations of IL-6, IL-8, IL-10, TNFα and were compared between groups. Cytokine assays were carried out by multiplexed cytometric bead immunoassays using the Milliplex™ human cytokine kit (#MPXHCYTO-60K 96 well plate assay, Millipore Corporation, Billerica, MA 01821, USA). All samples were assayed in duplicate, and peritoneal fluid samples were re-assayed for IL-6 in 1:5 dilution, as peritoneal fluid levels of this cytokine are frequently above the detection limit of the assay (> 10000 pg/ml). The minimum assay detection limits for TNFα, IL-6, IL-8, and IL-10 were 0.1, 0.3, 0.2, and 0.3 pg/ml, respectively. Data were acquired using Luminex 100™ laser-based fluorescent analytical test instrumentation (Luminex 100™ IS, Luminex Corporation, Austin, TX 78727, USA). The Median Fluorescent Intensity (MFI) data obtained using Luminex IS 2.3 software were saved and analysed using a weighted 5-parameter logistic curve-fitting method.

Post-operative assessment Data on post-operative course was prospectively collected in both Study and Control groups. Day that discharge criteria were achieved and actual day of discharge were recorded. Discharge criteria were defined as return of bowel function (days to passage of flatus or bowel motion, resumption of full solid oral diet, and the absence of nausea and vomiting), independent mobilisation (or mobilisation back to baseline function), and adequate pain relief using oral analgesia only. Readmission was defined as return to hospital within 30 days post-discharge requiring hospital stay of 24 hours or more. Total hospital stay (day stay on index admission plus hospital stay on readmission) was derived and recorded. Functional recovery was measured using the Surgical

Recovery Score (SRS), a comprehensive recovery assessment questionnaire derived from the previously validated multi-dimensional Identity-Consequence Fatigue Scale.(49, 50) This score assesses 5 categories of post-operative recovery: Fatigue, Vigor, Mental Function, Impact on Patient Activity and Impact on Activities of Daily Living and is expressed as a percentage of maximum possible score. The SRS questionnaire was filled out by all patients pre-operatively (baseline) and at 3, 7, 30, and 60 days post-operatively. Further details regarding the SRS can be found in Appendix A.

Post-operative complications up to 30 days after surgery were recorded prospectively using predefined criteria. Complications were defined as per the standardised “definitions of operation and / or disease related complications” proposed by Buzby et al.(51) In addition to this, ileus was defined as post-operative obstipation and vomiting requiring nasogastric tube insertion, but without radiological evidence of bowel obstruction. All complications were also graded as per the ClavienDindo classification.(52, 53)

Power calculation Power calculation was performed using a two tailed Mann–Whitney U test for non-parametric data. An estimate of peritoneal fluid IL-6 concentration for the Control Group patients was based on the concentrations measured in the intervention arm of the Dexamethasone trial.(38) Twenty five patients had complete peritoneal fluid IL-6 concentrations measured in that study (mean 3800 pg/ml, standard deviation 2300 pg/ml). We estimated that matching 50 patients in the Study Group in 2:1 design would be able to detect a 35% reduction in peritoneal fluid IL-6 concentration between groups with an alpha of 0.05 and power of 0.8.(54)

Statistical analysis Results were analysed using SPSS® for Windows® version 17.0 (SPSS, Chicago, Illinois, USA). Continuous variable parametricity was tested using the Shapiro-Wilk test. Groups were compared using the Fisher’s Exact or χ2 test for categorical variables, the Mann–Whitney U test for nonparametric continuous variables, and the t test for parametric continuous variables. Spearman's

rho was used for correlations. Statistical significance was accepted at the 0.05 level.

Results Baseline and intra-operative parameters Groups were very well matched at baseline, with the exception of operation time which was significantly longer in the study group (Table 1). There were two conversions to laparotomy in the Study Group, one due to extensive adhesions to the inferior aspect of the liver, and one due to inadvertent damage to a gonadal vessel which resulted in bleeding that could not be controlled laparoscopically.

Post-operative recovery There were no significant differences between groups in days to meeting discharge criteria, actual discharge day, and total day stay (Table 2). Post-operative recovery, as measured by the SRS score, was not significantly different at any time point. The readmission rate, post-operative complication rate (percentage of patients with any complication), and complication severity were similar in both groups. There were no deaths in either group at 30 day follow-up.

Cytokine response The peritoneal fluid IL-6 level was significantly lower in the study group than the open group, with no other significant differences in the peritoneal fluid or plasma (Table 2). Cytokine concentrations are depicted graphically in Figures 1 and 2.

There were several significant correlations between cytokine concentrations and post-operative recovery parameters (Table 3). Plasma and peritoneal fluid TNFα correlated positively with days to meeting discharge criteria, plasma IL-6 correlated positively with actual discharge day and total day stay, and plasma IL8 correlated positively with actual discharge day and negatively with the SRS score at baseline and day 30.

Patients who developed a complication had a significantly higher concentration of IL-10 and TNFα in the peritoneal fluid than those who did not (510.92 + 390.00 vs. 365.08 + 372.61, P = 0.031 pg/ml and 18.61 + 25.40 vs. 9.61 + 13.23 pg/ml, P = 0.038, respectively). There were no significant correlations between cytokine concentrations and complication severity.

Discussion We conducted a case-control study comparing patients undergoing laparoscopic colonic surgery with patients undergoing open colonic surgery, with the primary endpoint of peritoneal fluid and plasma cytokine concentrations. Patients were very well matched at baseline, and all the data were prospectively collected using standardised definitions. The peritoneal fluid IL-6 concentration was significantly lower at 20 – 24 hours after laparoscopic surgery, but no differences were detected in any of the other cytokines measured in peritoneal fluid or plasma. There were no other differences between the groups in any of the post-operative recovery outcomes measured. Significant positive correlations between cytokine levels and discharge criteria achievement, actual day stay, postoperative complications, and negative correlations with the Surgical Recovery Score were demonstrated.

This is the largest study so far undertaken evaluating the peritoneal cytokine response after colonic surgery. The lower level of peritoneal fluid IL-6 after laparoscopic colonic surgery is a novel finding in the literature. In addition we directly correlated cytokine levels measured with post-surgical recovery, and the results support previously postulated relationships.(12) Hewitt et al randomised 16 patients to laparoscopic and open colonic resection for neoplasia, and sampled peritoneal fluid at the end of surgery. No difference was found in peritoneal IL-6 between groups, but the study was weakened by small numbers and exclusion of conversions to laparotomy.(55) Wu et al similarly randomised 26 patients and also did not detect a difference in peritoneal fluid IL-6 at day 1 or day 4 post-operatively. A higher concentration of IL-8 at day 4 in the laparoscopic arm was reported; however, only 16 patients (5 in the laparoscopic arm, and 11 in the open arm) actually had peritoneal fluid measurements.(56, 57) To the authors knowledge, only one other small case-

control study measured IL-6 concentrations in peritoneal drain fluid at 24 hours in 13 patients undergoing laparoscopic or open colectomy and once again no difference was demonstrated.(58)

In the systemic circulation, IL-6 is considered to be a major mediator of the acute phase protein response,(1, 40) with levels appearing to be proportional to the extent of tissue injury.(3) The role of peritoneal fluid IL-6 in the post-surgical course is uncertain. An association between peritoneal IL-6 on day 1 and anastomotic leakage after mesorectal excision for rectal cancer has been demonstrated in one study, but the impact of pre-operative radiation may have been significant, and applicability to colon resection is limited.(59) It is notable that in our study we did not find a correlation between peritoneal fluid IL-6 and post-operative complications or any measures of postoperative recovery. Therefore, the clinical relevance of the higher level of peritoneal IL-6 in the study group is unclear, particularly since there were no differences between the study and control groups in terms of discharge parameters, complication rates, or the SRS score. With the exception of the lower peritoneal IL-6 response, the local cytokine response was otherwise similar after laparoscopy compared with laparotomy, with no difference in the 3 other cytokines measured. This may be because of the known local immunological effects of desiccation and acidification caused by the formation of carbonic acid during carbon dioxide pneumoperitoneum.(20, 60-69)

The equivalent plasma cytokine response after laparoscopic and open surgery is consistent with the results of a recent meta-analysis conducted by the authors in which a lower IL-6 on day 1 after laparoscopic colorectal resection was demonstrable only in serum (and not in plasma).(19) Centrifugation of samples after blood is collected in plain tubes and allowed to clot yields serum, whereas centrifugation of samples collected in anticoagulant tubes provides plasma. It is thought that plasma rather than serum assays are more reflective of the in vivo state,(70) because humoral factors stored in WBCs and platelets are released when blood clots, resulting in overestimation.(71) Overall, our results suggest an equivalent systemic inflammatory response after colonic surgery regardless of the access incision. We attribute this to the fact that, in colonic surgery, the trauma caused by the intra-peritoneal dissection is considerably more extensive than the access incision, perhaps dwarfing its systemic impact by comparison.(56, 72-74) As can be

seen in this study, plasma cytokine concentrations were much lower than peritoneal concentrations. This has been noted in several earlier publications and is thought to be due incomplete absorption into the portal circulation, hepatic metabolism, and dilution in plasma, causing an apparent independence of the peritoneal response from the systemic one.(21, 56, 75)

This study has several limitations. Patients in the two groups were recruited from different hospitals, with their surgery and care undertaken by different clinicians.(76) Secondly, patients in the Control Group were managed within a formal enhanced recovery after surgery program, whereas patients in the Study Group were not. As a result, all patients in the Control Group were given spinal afferent blockade via a thoracic epidural peri-operatively. Epidural blockade has been shown to blunt the systemic humoral response.(77) While this means that there was significant cointervention bias in this study, it is generally accepted standard practice in our hospital to use an epidural for open colonic resections and not for laparoscopic resections, due to the differences in post-operative analgesia requirement. This co-intervention is therefore present in current clinical practice. Another limitation is that samples were collected at a single time point only. This time interval was chosen because peritoneal IL-6 levels peak at 20 – 24 hours after abdominal surgery. However, due to financial constraints we could not sample at multiple intervals to coincide with different peaks, and it is possible that we may have missed peak differences in other cytokines.

Conclusion With the exception of a lower peritoneal IL-6 response, the systemic and peritoneal cytokine response at 20-24 hours is similar after laparoscopic versus open colonic resection within an ERAS program, with corresponding equivalent rates of post-operative recovery. There are significant correlations between cytokine concentrations and post-operative complications, discharge parameters, and the Surgical Recovery Score.

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Tables Study Group (n = 50)

Control Group (n = 25)

P Value

73.5 (21-94)

71 (37-92)

0.218

17 (34%) 33 (66%)

8 (32%) 17 (68%)

1.000**

Mean BMI (SD)

26.0 (5.6)

27.1 (4.0)

0.331

ASA score I II III

7 (14%) 28 (56%) 15 (30%)

8 (16%) 26 (52%) 16 (32%)

18 (3)

17(3)

0.206

34 (68%) 16 (32%)

17 (68%) 8 (32%)

1.000**

172.2 (45.1)

109.6 (30.3)