LAPAROSCOPY AND TUMOUR GROWTH

Umeå University Medical Dissertations New series No 889 – ISBN 91-7305-613-8 ISSN 0346-6612 From the Department of Surgical and Perioperative Sciences...
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Umeå University Medical Dissertations New series No 889 – ISBN 91-7305-613-8 ISSN 0346-6612 From the Department of Surgical and Perioperative Sciences, Surgery, Umeå University Hospital, Umeå, Sweden

LAPAROSCOPY AND TUMOUR GROWTH A clinical and experimental study

Owe Lundberg

Umeå 2004

Copyright © Owe Lundberg ISBN 91-7305-613-8

Printed by Solfjädern Offset Umeå, Sweden, 2004

To my late Grandfather Bertil who never had a chance

CONTENTS ABBREVIATIONS

6

ABSTRACT

7

POPULÄRVETENSKAPLIG SAMMANFATTNING

9

ORIGINAL PAPERS

11

INTRODUCTION

12

History of laparoscopic surgery

13

Carcinoma of the gallbladder

15

The concept and aetiology of port site metastases

20

AIMS

4

Definition

20

Clinical situation

21

Incidence

22

Aetiology

23

-Haematogenous spread

23

-Wound implantation

23

-Surgical technique

24

-Pneumoperitoneum

25

-Immune response

27 30

PATIENTS, MATERIALS AND METHODS

31

Clinical studies – paper I, II and III

31

Statistics and ethics

32

Experimental studies – paper IV, V and VI

33

Laser Doppler blood flow

36

Statistics and ethics

37

RESULTS

38

Clinical studies – paper I, II and III

38

Experimental studies – paper IV, V and VI

43

DISCUSSION

44

Clinical studies - paper I, II and III

44

Experimental studies - paper IV, V and VI

49

SUMMARY AND CONCLUSIONS

53

ACKNOWLEDGEMENTS

54

REFERENCES

55

PAPERS I-VI

72

5

ABBREVIATIONS ACTH ADH CRP DTH EpC GBC GH IFN-γ IL-1,-2,-6 LC LDF NK-cells OC PSM PU SoS Th-cells TSH TNF-α

6

Adrenocorticotropic stimulating hormone Antidiuretic hormone C-reactive protein Delayed type hypersensitivity Epidemiologiskt centrum Gallbladder cancer Growth hormone Interferon gamma Interleukin 1, 2 and 6 Laparoscopic cholecystectomy Laser Doppler flowmetry Natural Killer cells Open cholecystectomy Port site metastases Perfusion units Socialstyrelsen T-helper cells Thyroid stimulating hormone Tumour necrosis factor alpha

ABSTRACT Background and aims: Laparoscopic technique was quickly adopted in general surgery because of less pain, quicker recovery and shorter hospital stay. In the 1990´s several reports on port site metastases restrained the enthusiasm to use laparoscopic surgery in malignant diseases. The numerous reports on port site metastases initiated a debate whether laparoscopic surgery would increase the risk of tumour spread and growth. Personal experience of two patients who developed port site metastases from an incidental gallbladder cancer (GBC) after laparoscopic cholecystectomy (LC), encouraged us to study the incidence of wound metastases from gallbladder cancer after laparoscopic and open cholecystectomy (OC). Experimentally we examined whether pneumoperitoneum would increase the risk of tumour development. Several studies had demonstrated that minimally invasive procedures exert a less negative influence on the immune system and may have beneficial effects for cancer patients.We wanted to compare the long term survival after OC and LC and if the occurrence of port site metastases had any impact on survival. Material and methods: A questionnaire was sent to all major hospitals in Sweden requesting information about the number of port site metastases encountered 1991-1994. Data on all patients with verified GBC were obtained from the Swedish Oncological Centres. Data on all patients with gallbladder cancer registered with surgical codes for cholecystectomy were collected from the National Board of Health and Welfare (EpC). The patient files were scrutinised and long term survival data was achieved (EpC). In the first experiment on Wistar Fu rats, adenocarcinoma cells were injected intraperitoneally in animals insufflated with air, CO2 and in not insufflated controls. In the following studies, rats were similarly insufflated with air, CO2 and compared to not insufflated controls. Laser Doppler blood flow in the abdominal wall was concomitantly measured. To study the effect of reduced blood flow, one rectus muscle was clamped and the other not and laser Doppler blood flow was measured in both rectus muscles. Adenocarcinoma cells were injected into the rectus muscles in all animals at the induction of pneumoperitoneum /clamping. Results: 14 of 55 patients developed wound metastases from gallbladder cancer after LC and 12 of 187 after OC. Gallbladder perforation was overrepresented in patients with wound metastases. Improved survival was noted after LC in patients with T3 tumours. Experimentally, air and CO2 equally increased intraperitoneal tumour development. Insufflation with air, CO2 and clamping decreased blood flow in the abdominal wall and increased tumour growth at the same site.

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Conclusion: Despite a high rate of wound metastases, LC does not seem to worsen the prognosis of GBC and may even have a positive effect on survival. Perforation of the malignant gallbladder seems to be associated with an increased risk of metastatic formation. In the experimental setting, pneumoperitoneum seems to increase tumour development. Other features of laparoscopic surgery such as decreased blood flow in the abdominal wall may contribute to increased risk of tumour progress. Key words: Laparoscopy, tumour growth, metastases, gallbladder cancer, pneumoperitoneum, blood flow.

8

POPULÄRVETENSKAPLIG SAMMANFATTNING (SUMMARY IN SWEDISH). Den snabba utvecklingen och spridningen av den laparoskopiska kirurgin torde sakna motstycke i den moderna kirurgins historia. Fördelarna med mindre smärta, snabbare återhämtning och kortare sjukhusvistelse utgjorde grunden till genombrottet för denna s.k. ”titthålskirurgi”. Tidigt utfördes även cancerkirurgi med laparoskopisk teknik men rapporter om dottersvulster i ärren efter titthålsinstrumenten, s.k. porthålsmetastaser, gjorde att entusiasmen för laparoskopisk cancerkirurgi avtog. En intensiv debatt vidtog huruvida laparoskopisk kirurgi ökade risken för tumörspridning. Ett flertal studier visade samtidigt att laparoskopisk kirurgi var mindre påfrestande för patienterna och sannolikt ett bättre bevarat immunförsvar. Laparoskopisk teknik skulle därmed teoretiskt sett kunna innebära en minskad risk för tumörspridning för cancerpatienter som opererades med denna teknik. En patient av hundra som genomgår gallblåsekirurgi har gallblåsecancer. Inte sällan är detta ett överraskningsfynd då diagnosen oftast är okänd före operationen. Efter att personligen ha opererat ett par patienter som senare utvecklade porthålsmetastaser från gallblåsecancer, väcktes intresset att studera frekvensen av detta nya och okända fenomen. En förfrågan om antalet porthålsmetastaser som påträffats 1991-1994, skickades till alla större sjukhus i Sverige. Med hjälp av register för patienter med gallblåsecancer (Onkologiska Centra) och operationskoder för öppen och laparoskopisk galloperation (SoS; EpC) kunde vi beräkna antalet patienter med gallblåsecancer som opererats med borttagande av gallblåsan. Efter att ha läst igenom samtliga patientjournaler kunde andelen ärrmetastaser utvärderas. Utdrag ur dödsorsaksregistret gav oss senare tillgång till långtidsuppföljning och möjlighet att i efterhand utvärdera om överlevnadstiden påverkades om patienter var opererade med öppen eller laparoskopisk teknik. Resultaten visade att patienter med gallblåsecancer opererade med laparoskopisk teknik utvecklade ärrmetastaser i fler fall än de öppet opererade. Däremot verkade risken vara mindre att dö i sin gallblåsecancer för patienter opererade med laparoskopisk jämfört med öppen teknik. Kirurgisk teknik, avspeglat som instrumentorsakat hål i den canceromvandlade gallblåsan (gallblåseperforation) har ofta föregått uppkomsten av ärrmetastaser. Metastaser från gallblåsecancer till operationsärret är oftast ett uttryck för redan spridd sjukdom och har oftast i sig ingen avgörande inverkan på den för gallblåsecancer redan dåliga prognosen. Dock finns enstaka fall av patienter med tidig gallblåsecancer där gallblåseperforation orsakad av kirurg/instrumenthantering kan ha haft betydelse för spridning av cancersjukdomen. Även om hål i gallblåsan är överrepresenterat vid laparoskopisk 9

kirurgi så finns det inga säkra belägg för att detta skulle innebära en ökad risk för utveckling av metastaser i operationsärret. Kliniskt bör man dock försöka att identifiera patienter med gallblåsecancer före operationen så att den elakartade tumören kan hanteras på ett kirurgiskt adekvat sätt. I de enstaka fall med tidig och potentiellt botbar gallblåsecancer bör sannolikt öppen kirurgi väljas framför allt som den förmodligen är mindre behäftad med risk för perforation av gallblåsan. Djurexperimentellt har vi på råtta studerat hur den gasinblåsning (insufflation) som används vid laparoskopisk kirurgi påverkar tumörtillväxten i bukhålan. Koldioxid (CO2) som är den gas som används vid laparoskopisk kirurgi har använts och jämförts med inblåsning av vanlig rumsluft. Tumörceller injicerades i bukhålan och buken hölls uppblåst i 45 min till ett tryck av 10 mmHg. Efter 12 dagar avlivades djuren och en ökad tumörutveckling hos både luft och CO2 insufflerade råttor jämfört med icke insufflerade kontroller kunde noteras. Även den uppblåsta buken (pneumoperitoneum) och dess påverkan på blodflödet i bukväggen och samtidig effekt på tumörtillväxt har studerats. Då inblåsning av gas (CO2 eller luft) påtagligt minskade blodflödet i bukväggen på råttorna har även blodflödesminskning med mekanisk avstängning studerats. Efter injektion av tumörceller i bukväggen och insufflation som tidigare beskrivits, noterades en ökad tumörtillväxt i bukväggen hos djur insufflerade med CO2 eller luft. Även blodflödesminskning med mekanisk avstängning orsakade en viss ökning av tumörtillväxten. Detta skulle kunna tyda på att blodflödesminskning i bukväggen bidrar till ökad tumörtillväxt. Sammanfattningsvis finns det inga belägg för att laparoskopisk kirurgi vid gallblåsecancer skulle innebära en försämrad prognos även om det finns indikationer på en ökad frekvens ärrmetastaser. Det kan finnas företeelser i den laparoskopiska kirurgin som påverkar tumörspridning och tumörtag/tumörtillväxt i både positiv och negativ riktning. Fortsatt forskning behövs för att vi ska kunna minimera laparoskopiteknikens eventuella negativa effekter och samtidigt utnyttja dess positiva potential även vid kirurgi på patienter med cancersjukdom.

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ORIGINAL PAPERS This thesis is based on the following papers, which will be referred to in the text by their Roman numerals: I.

Lundberg O, Kristoffersson A. Port Site Metastases from Gallbladder Cancer after Laparoscopic Cholecystectomy. Results of a Swedish Survey and Review of Published Reports. Eur J Surg 1999; 165:215-222.

II.

Lundberg O, Kristoffersson A. Wound recurrences from gallbladder cancer after open cholecystectomy. Surgery 2000; 127:296-300.

III.

Lundberg O, Öberg Å, Kristoffersson A. Long term results after open and laparoscopic surgery for gallbladder carcinoma (submitted).

IV.

Lundberg O, Kristofferson A. Effect of pneumoperitoneum induced by carbon dioxide and air on tumor load in a rat model. World J Surg 1998; 22:470-472.

V.

Lundberg O, Kristoffersson A. Pneumoperitoneum impairs blood flow and augments tumor growth in the abdominal wall – an experimental study in rats. Surg Endosc DOI: 10.1007/s00464-0039035-7 29 December 2003 (Epub ahead of print).

VI.

Lundberg O, Kristoffersson A. Reduction of abdominal wall blood flow by clamping or CO2 insufflation increases tumour growth in the abdominal wall – an experimental study in rats (submitted).

Reprints were made with permission of the publishers.

11

INTRODUCTION Since Mouret performed the first laparoscopic cholecystectomy (LC) in 1987, this technique has rapidly replaced conventional open cholecystectomy (OC) and become the standard treatment for surgical management of gallstone disease.1 The widespread adoption of laparoscopic surgery in general and laparoscopic cholecystectomy in particular, was done with the assumption that this technique was beneficial for the patient especially in aspects of less pain, quicker recovery, shorter hospital stay and reduced costs.2 However, the early expansion of LC occurred without scientific evidence to support these assumptions.2 The laparoscopic technique was quickly implemented to other surgical procedures including surgery for malignant disease. In the early nineties several case reports were published on the occurrence of metastases from malignant tumours to the port sites after laparoscopic surgery.3 This new phenomenon raised an intense debate whether the laparoscopic technique was appropriate or not in oncologic surgery.4 In 1994 I personally operated on two patients with gallstone disease who later developed port site metastases from gallbladder cancer (GBC) after laparoscopic cholecystectomy. As in many patients with gallbladder cancer the finding of a malignant specimen was unexpected and therefore the patients were planned for laparoscopic cholecystectomy. In approximately 0.5-2.0 % of all cholecystectomy patients an incidental gallbladder cancer is encountered and the surgeon and the patient are subjected to laparoscopic handling of a possibly malignant disease as laparoscopic cholecystectomy has become the standard treatment for gallstone disease5-7. The clinical findings of port site metastases encouraged us to examine the incidence of metastatic spread from gallbladder cancer to the wound sites created by the laparoscopic working ports and subsequently to study the incidence of wound metastases after open cholecystectomy. Concomitantly, we experimentally wanted to investigate if there could be any features associated with the laparoscopic technique contributing to an increased growth of malignant cells in laparoscopic surgery.

12

History of laparoscopic surgery The first descriptions of endoscopic examinations emanates from the Kos school led by Hippocrates (460-375 BC), who described a rectal speculum, remarkably similar to the ones used today.8,9 Similar speculums, furthermore used for the inspection of the vagina and the inside of nose and ear were also discovered in the ruins of Pompeii.10 In 1585 Aranzi introduced illumination into endoscopy by focusing sun light through a flask of water and projected sun light into the nasal cavity.11 In 1805 Philip Bozzini, an obstetrician from Frankfurt built an aluminium tube with mirrors to reflect images illuminated by a wax candle – the “Lichtleiter”- which could be used to look into various body cavities 12 (figure 1). Fifty years later the French urologist Antoine Jean Desormeaux further developed the Lichtleiter and replaced the candle by a brighter-burning mixture of alcohol and turpentine. His improvements initiated a more common use of the rectoscope in clinical practice 13 and his monograph from 1865, “De l`endoscopie” is said to have stimulated American instrument makers to commence the production of endocopes.14 He is therefore often considered as the “father of endoscopy”. A shift of interest towards the upper gastrointestinal tract began in 1868 when Adolf Kussmaul did the first gastroscopy attempt by passing a rigid metal tube down the oesophagus of a professional sword-swallower.13 Modern endoscopy was born in 1879 when the Dresden physician Maximilian Nitze presented the “Blasenspiegel”, a cystoscope constructed in cooperation with the Viennese instrument maker Josef Leiter.10,15 The first experimental laparoscopy was performed 1901 in Berlin by the German surgeon Georg Kelling who used a cystoscope to peer into the abdomen of a dog after first insufflating with air, a technique he described as kolioskopie when published in 1902.16 Soon after, the Swedish surgeon HC Jacobaeus used this procedure in the thorax and abdomen on patients but without employing a pneumoperitoneum. He was the first to use the term “laparothorakoskopie” when publishing his 115 cases of laparoscopy (with only one serious complication!) in 1911.17 In 1921, a pneumoperitoneum needle for safer introduction into the peritoneal cavity 18 and the insufflator, was developed.19 A few years later, Zollikofer of Switzerland discovered the benefit of CO2 gas for insufflation rather than atmospheric air.20 The still commonly used Veress needle was developed in 1938 by the Hungarian Janos Veress. Veress himself used this needle for the induction of pneumothorax rather than for laparoscopy purposes.21 In 1963 the German gynaecologist Kurt Semm developed the automatic insufflator, capable of monitoring both gas flow and intraabdominal pressure.22 Although his ideas and visions were controversial, the inventions of Semm allowed a safer laparoscopy and had a great impact on the development of modern laparoscopy. His pioneer 13

work was probably also one of the reasons why gynaecologists in the 1970´s thoroughly incorporated the technique into their practice while general surgeons remained confined to traditional open surgery.10 Kurt Semm also performed the first laparoscopic appendectomy in 1980, a procedure at the time regarded as unethical by many prominent surgeons.23 The rod-lens system was invented already in 1953 by the British optical physicist Hopkins, improving image brightness and clarity.24 Together with the development of fiberoptic light sources in the early 1960´s, they revolutionized laparoscopic surgery. In the 70´s a blunt mini-laparotomy technique was proposed by Hasson a method which permits direct visualisation of trocar entrance into the peritoneal cavity.25 The era of video-guided surgery started in 1986 with the development of a computer chip TV camera attached to the laparoscope. Video imaging facilitated the education of other surgeons and house staff. As previously mentioned the laparoscopic cholecystectomy performed by Mouret in Lyon in 1987 started the rapid development of laparoscopic technique in the field of general surgery. Although the credit is given to Mouret, Erich Mühe of Boblingen, Germany performed the first documented laparoscopic cholecystectomy already in 1985.26 In Sweden as well as all over Europe an astonishing development of laparoscopic surgery was seen during the 1990´s. Laparoscopic appendectomy, herniorraphy, fundoplication, adrenalectomy, colorectal surgery and all kinds of procedures in general surgery were done all over the world in a few years. After the initial unrestrained and rapid spread of laparoscopic surgery the situation today seems more controlled. The only laparoscopic procedure in Sweden that has fully replaced the conventional open technique is still laparoscopic cholecystectomy.

Figure 1. The licht leiter endoscope developed by Philip Bozzini (1805).

Picture from “Highlights in the history of laparoscopy” by G.Litynski at www.laparoscopy.com With permission.

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Carcinoma of the gallbladder This neoplasm was first described in 1777 and still more than 200 years later constitutes a major diagnostic as well as therapeutic dilemma.27 Although not a common disorder it is nevertheless the fifth most common cancer of the gastrointestinal tract.28 It is a fatal and often misunderstood diagnosis. The patients usually present with diffuse symptoms and are often beyond surgical cure by the time of discovery. In addition, many patients are diagnosed after being subjected to a “simple” cholecystectomy.29 Patients with gallbladder cancer have a very bad prognosis and 90% of the patients with tumours stage T2-4 have usually succumbed after a few months.30 Aggressive surgical therapy and early diagnosis will hopefully contribute to an improved prognosis for these patients. Incidence GBC is the 5th most common malignancy of the gastrointestinal tract and reveals some interesting ethnical and geographical characteristics. It is a very common malignant disorder in South-America, Poland, Japan and Israel. It affects women 2-3 times more often than men and is usually encountered in elderly patients. The incidence has been reported as high as 7/100 00 for men and 23/100 000 women in native American and South Americans.31 In Sweden, gallbladder carcinoma represents 1% of all malignancies and the incidence is approximately 1.4/100 000 for men and 3.3/100 000 for women.32 Pathogenesis The majority of patients with GBC suffer from cholelithiasis. Although gallstones are present in 65-90% of patients with GBC, only 1% of all patients with gallstones develop carcinoma of the gallbladder.33 Some data indicate that female patients with stones larger than 3 cm are at special risk of developing cancer of the gallbladder.34 The presence of calcifications of the gallbladder in so called “porcelain gallbladder” and larger polyps are possibly premalignant conditions as well as anatomical variations of the bile duct and dearrangement of ratios between bile salts/lecithin and cholesterol.35-38 A range of carcinogenic substances have also been discussed in association to the occurrence of gallbladder cancer. Other factors mentioned are obesity, infectious diseases such as typhoid and chronic inflammation of the gallbladder, colitis and multiple polyposis of the colon.34Patients with gallbladder carcinoma often have a family history of malignancy and a second neoplastic disease is encountered in 15% of the patients.39

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Clinical symptoms Most patients present with diffuse symptoms although some also are encountered as surgical emergencies with intestinal haemorrhage, obstruction or severe jaundice.34 Unfortunately, the presence of these symptoms is usually linked to a noncurable disease in more than 50% of the patients. Common symptoms of gallbladder cancer Symptoms Abdominal pain Weight loss Anorexia Nausea and vomiting Palpable tumour Jaundice Abdominal distention Pruritus

Frequency ( %) 82 72 74 68 65 44 30 20 From Misra, ref 34

Pathology As seen in breast and pulmonary carcinoma, gallbladder carcinoma is probably a progressive disease with a continuum from dysplasia, cancer in situ to invasive carcinoma. The majority of cases are adenocarcinomas, which represents about 85% of all gallbladder malignancies.40 The metastatic pattern includes direct, lymphatic, vascular, neural, intraperitoneal and intraductal spread.41 Tumours are usually spread primarily among lymphatic pathways and venous blood is drained into the 4th segment of the liver which affects surgical strategy. Different staging systems for GBC have been utilized, but today the TNM staging system and stage grouping 41 are most commonly used and have basically replaced the Nevin classification system.42 Descriptions of the different staging systems are presented below and histology of the gallbladder is illustrated in fig 2.

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Nevin classification

TNM staging – T stage

I – Intramucosal II – Tumour extends to the muscularis

IV – Transmural involvment and cystic lymph node involvement

Tis – Carcinoma in situ T1 – Tumour invades lamina propria or muscle layer; T1a – invades lamina propria T1b – invades muscle layer T2 – Tumour invades perimuscular connective tissue; no extension beyond serosa or into the liver T3 – Tumour perforates serosa or directly invades one adjacent organ, or both (extension < 2 cm into the liver)

V – Direct extension into the liver and/or distant metastasis

T4 – Tumour extends > 2 cm into the liver and/or into two or more adjacent organs

III – Tumour extends through serosa

TNM staging – N and M stages Regional lymph nodes (N): NX – Regional lymph nodes can not be assessed N0 – No regional lymph node metastasis N1 – Metastases in lymph nodes close to the primary tumour (cystic duct,pericholedochal etc) N2 – Metastases in lymph nodes not in the immediate proximity of the primary tumour (periduodenal,periportal etc) Distant metastases (M): MX – Distant metastasis can not be assessed M0 – No distant metastasis M1 - Distant metastasis present

17

TNM stage grouping system Stage 0 I II III IVA IVB

T Tis T1 T2 T3 T1-3 T4 anyT anyT

N N0 N0 N0 N0 N1 N0-1 N2 anyN

M M0 M0 M0 M0 M0 M0 M0 M1

Figure 2. Photomicrograph of gallbladder wall a. Mucosa - simple columnar epithelium b. Lamina propria - loose irregular connective tissue c. Muscularis d. Subserosal connective tissue (shared with liver in gallbladder bed) e. Serosa - absent in the attachment between the gallbladder and liver

Diagnostic aspects Ultrasound is usually the first investigation in the work-up of a suspected GBC. In most cases it is also the only necessary investigation.37 However, often a CT is added. The value of MRT and MRCP has still not been fully evaluated.44 Diagnostic laparoscopy has become a common surgical procedure in order to evaluate resectability and peritoneal carcinomatosis.29 Biochemical markers that are specific for GBC are not available.45

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Treatment The only ultimate treatment for gallbladder carcinoma is surgery but only 10-30% of patients with a known diagnosis are candidates for radical surgery due to widespread disease at the time of diagnosis. Surgery implies simple or extended cholecystectomy.40 In cases of incidentally discovered GBC after laparoscopic cholecystectomy, the TNM classification of the tumour often has major impact on the decision of further surgical procedures.46 The value of intra- or postoperative radiation therapy has been discussed and the results are conflicting. 47 Various combinations of chemotherapy have been evaluated but no conclusive results have been reported so far.48 Prognosis As in most neoplastic disorders the prognosis and survival rate is dependent on the stage of disease at discovery. Reported 5-year survival rates differ considerably: for stage I cancer 5-year survival rates range from 40100%, stage II 10-80%, stage III 5-60% and stage IV 1-25%.49 An early diagnosis of gallbladder carcinoma may be even more important than radical surgical approaches, although some promising reports recently haven been published from Japan.50 Patients suspected to have a gallbladder carcinoma or being diagnosed after cholecystectomy should be evaluated for a second operation and may benefit from the referral to oncological and surgical centres with special interest in gallbladder cancer.

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The concept and aetiology of port site metastases Definition Port site metastases has not been strictly defined but is regarded as a tumor deposit in the scar after a laparoscopy port. It is located in the abdominal wall but without discrimination between subcutaneous or muscular tissue. In the literature, there is often no distinction between port site metastases as an isolated phenomenon or in association with widespread metastases or carcinomatosis. It has been proposed that PSM per definition not should be associated with peritoneal carcinomatosis.51 However, PSM have to be present at the site of a port wound or scar but can be found concomitantly with tumor deposits at other locations intraabdominally, in the abdominal wall or elsewhere. PSM are reported as isolated recurrences after curative surgery as well as together with other tumour manifestations indicating a more generalised disease.52 The growth of a primary tumour or local recurrence per continuitatum to the scar is not to be regarded as a port site metastasis. The definition described above is equally applicable to wound metastases after open surgery. In 1978 Dobronte and coworkers were the first to report on a patient with a metastasis in the laparoscopy wound.53 The metastasis occurred two weeks after a diagnostic laparoscopy and was considered to be “extremely rare”. During the rapid expansion of laparoscopic surgery in the 1990´s numerous case reports on port site metastases were presented.54 This started an intense debate whether laparoscopic surgery implied an increased risk of spreading malignant disease. The discussion resulted in a common scepticism among surgeons whether laparoscopic surgery was appropriate to use in malignant diseases.55,56 Furthermore, a number of experimental studies were performed to examine the aetiology of port site metastases and whether tumour development was increased by the laparoscopic technique.57

20

Clinical situation In the early 1990´s laparoscopic surgery for cancer was done outside controlled studies. Without knowledge of long term results laparoscopic surgery for colorectal cancer was then proven to be technically feasible.58,59 The occurrence of port site metastases reduced the initial enthusiasm for this new technique.56 The debate on port site metastases has also mainly been focused on laparoscopic colorectal cancer. The concern that laparoscopic surgery may increase the risk of spreading tumour cells to the surgical wound has urged the design of clinical trials. Several large prospective randomised studies are also in progress in Europe and overseas.60 These studies will hopefully give us the true incidence of port site metastases as well as long term results after laparoscopic versus open surgery for colonic cancer. The situation on port site metastases and gallbladder cancer however, is somewhat different. First, colorectal cancer is more than ten times as common as gallbladder cancer.33 Secondly, as previously mentioned, gallbladder cancer is often found incidentally (i.e. not preoperatively known). Furthermore gallbladder cancer has a very dismal prognosis with a short median survival time.30 Altogether it would be difficult to conduct a prospective randomised trial to study any potential differences between laparoscopic and open cholecystectomy concerning the incidence of port site metastases, survival or any other endpoint. To my knowledge no such studies are in progress or have ever been performed. Most surgeons today agree that laparoscopic surgery for cancer should not be performed outside randomised controlled studies. Although neither the aetiology nor the incidence of port site metastases are known, survival should be the preferable endpoint for such studies. Although PSM is an interesting and new phenomenon, numerous reports of this dismal complication must not evidently imply that the incidence of wound metastases from GBC is higher after LC than after OC. Moreover, a high incidence of port site metastases after LC does not rule out improvement of survival after laparoscopic surgery. A comparable incidence of wound metastases after open surgery is also hard to assess prospectively especially since open cholecystectomy nowadays seldom is performed.

21

Incidence Wound metastases are not unique to laparoscopic surgery. The studies of Reilly and Hughes have reported an incidence of wound metastases following open surgery for colorectal cancer slightly below 1%.61,62 Hence, their results have been commonly accepted and consequently an acceptable PSM rate after laparoscopic surgery for colorectal cancer is regarded to be below 1%.59 A post mortem study on colorectal cancer patients by Welch and Donaldson reported 16% wound metastases, all associated with disseminated disease.63 Small prospective randomised studies from specialised institutions have reported no differences in wound metastases between open and laparoscopic surgery for colonic cancer. The rates of wound metastases in both groups have been equal and well below the presumably acceptable 1% level.64,65 In addition, none of the ongoing large prospective randomized trials (NCI in USA, Conventional Versus Laparoscopic-Assisted Surgery for Colorectal Cancer Trial in U.K and the COLOR-study in Europe) had to be stopped due to an unacceptable PSM rate.60 This would indicate that it is possible to perform laparoscopic colonic surgery with an acceptable number of PSM. Only scattered reports exist attempting to determine the incidence of PSM from GBC after LC and as mentioned above no prospective randomised trials have been conducted comparing open versus laparoscopic surgery for GBC. Paolucci et al have, in a large series including 117 840 LCs out of which 409 had GBC, reported an incidence of 17% PSM and 26 out of the 409 patients with GBC had an early cancer (Tis or T1).54 Z´graggen et al reported 37 patients with GBC of 10 925 patients subjected to LC and found that 14% developed PSM.66 More recently Paolucci and coworkers have reported 142 cases of incidental GBC following LC, 79 following OC and 24 after a converted procedure.67 With a median follow up of 27 months they reported similar wound recurrence rates, 7% after LC and 5.1% following OC as well as similar total recurrence rates.

22

Aetiology Although numerous papers have been published proposing a number of mechanisms the aetiology of port site metastases remains unknown. Despite several case reports, most research in the field of laparoscopic surgery and tumour development has been experimental studies.57 In the clinical situation, especially after studies on laparoscopic colonic cancer, the initial fear of PSM has somewhat faded.68 However, there might still be features of the laparoscopic technique that facilitates tumour spread and growth.69 Haematogenous spread It has been shown that localized tissue trauma increases the ability of implantation of tumour cells 70,71 and metastases predominantly develop in a fresh wound.72,73 Even if only a small number of tumour cells are able to produce a metastasis it is known that malignant cells have been detected in the systemic circulation of patients with cancer.74 In the laparoscopic situation it has been proposed that the increased intraabdominal pressure would facilitate the liberation of tumour cells into the circulation or the passage from lymphatic to venous vessels.75,76 Nevertheless, haematogenous spread has been regarded unlikely to play a central role in the development of PSM and has been sparsely studied. Wound implantation In contrast to haematogenous spread, direct implantation of tumour cells is considered to play a major role in the development of PSM.77 There are several possible mechanisms for tumour cells to deposit in the port site. 1. Extraction of specimen The extraction of a malignant specimen through a narrow access wound represents an obvious risk of tumour cell contamination of the abdominal wall. In patients who developed PSM after LC for unknown GBC, Paolucci and coworkers reported that the port used for retraction of the gallbladder was the most common site of recurrence but 42% of PSM did not occur at the extraction port.52 Consequently this theory does not explain the large number of recurrences seen in other port sites than the extraction wound.

23

2. Instrument contamination Grasping and manipulating a tumour with laparoscopic instruments should clearly be a risk for contaminating the instruments and subsequently port sites with cancer cells. Both clinical and experimental studies have demonstrated tumour cell contamination of both laparoscopic instruments and trocars.78,79 Experimental studies have also shown that increased instrument movement and manipulation of ports augments tumour cell implantation.80,81 Still, PSM have been reported from pancreatic cancer after diagnostic laparoscopy without manipulation of the tumour.82 Free intraperitoneal tumour cells exist, especially but not exclusively in advanced malignancies and could theoretically have contaminated the laparoscopic instruments in such cases.83 Even so, reports of PSM after LC from previously undiagnosed tumours of the colon and occult pancreas cancer indicate that wound contamination may not be the single definitive cause of port site metastases.84,85,86 3. Chimney effect and aerosolization The chimney effect refers to the potential translocation of tumour cells to the port sites caused by leakage of gas along the trocars. This requires free intraperitoneal tumour cells preferably in aerosol formation. Most studies have failed to demonstrate significant aerosolization of tumour cells both in vivo and in vitro studies.81,87-89 Ikramuddin et al studied 37 patients who underwent elective laparoscopy for malignancy and found that tumour cells were aerosolized but only in the presence of carcinomatosis.90 The Dutch group who coined the term “chimney effect” has later presented a study were they conclude that this phenomenon is unlikely to explain the development of port site metastases.91 Surgical technique As previously proposed in open cancer surgery 92, surgical technique is suggested to be the most important factor influencing tumour cell spread and recurrence in laparoscopic surgery.93 Laparoscopic surgery for large bowel cancer is technically demanding and has a long learning curve. This may explain the incidence of PSM as high as 21% reported in the early years 56 compared to more recent studies where a 0.6 % PSM rate was reported from the Colon and Rectal Clinic of Orlando.94 Such a low incidence may also reflect that the operations were conducted at a centre specialised in colorectal cancer surgery and well experienced with the laparoscopic technique.

24

In a murine model with a subcapsular spleen tumour, Lee and coworkers demonstrated that the incidence of PSM after laparoscopically assisted resection of spleen decreased to a level comparable with that of an open operation with increasing experience of the surgeon as the experiment progressed.95 In another study with a similar animal model they also showed that a meticulous resection technique decreased the incidence of PSM and this was independent of whether pneumoperitoneum was used or not.96 In a study in rats, Mutter et al noted an increase of intraabdominal tumour growth and metastatic formation after tumour manipulation in open and laparoscopic surgery but with further enhancement of tumour growth in the open group.97 Another aspect of surgical technique is the high proportion of intraoperative gallbladder perforation among patients with PSM from GBC after LC.66 Doudle et al showed that gallbladder mucosal cells were found on instruments at the end of surgery in 6 of 15 patients undergoing LC.98 This could indicate that “micro perforation” occurs, with a possibility of spreading even an early GBC without macroscopic perforation of the gallbladder. Although surgical technique, skill and experience seem to be the most important factors for tumour spread and growth after laparoscopic surgery, it can hardly explain the development of PSM after diagnostic laparoscopy with no manipulation of the tumour at all.99,100 Pneumoperitoneum The technique of laparoscopic surgery is basically the same as used in conventional open surgery. Although smaller wounds, absence of hands and longer instruments the major dissimilarity between open and laparoscopic surgery is the establishment of pneumoperitoneum. Pneumoperitoneum has in many ways been regarded as responsible for or at least contributing to an increased risk of tumour spread and growth in laparoscopic surgery: 1. Carbondioxide Previous used gases as room air, oxygen and nitrous oxide were all abandoned mainly because of their explosive features and the former ones also had the potential for venous embolism. Other gases as Argon, Xenon and Helium have been used mainly in the research setting. Carbondioxide (CO2) is relatively inexpensive, readily available, colourless, odourless, non-flammable, non-explosive, highly soluble in plasma and therefore suitable to use in laparoscopic surgery. CO2 is also the most commonly used gas for insufflation in laparoscopic surgery.1995, in a hamster model, Jones et al were the first to report that CO2 pneumoperitoneum resulted in “increased 25

implantation of free intra-abdominal cancer cells at wound sites of the abdominal wall or in the abdominal cavity”.101 Soon after, Bouvy and coworkers showed that laparoscopy with CO2 pneumoperitoneum resulted in increased tumour growth and bigger port site metastases compared with gasless laparosocopy.102 Jacobi reported in 1997 that CO2 per se seemed to stimulate the growth of malignant cells compared to helium and controls.103 Several animal studies have afterwards reported promoting effects of CO2 on intraperitoneal tumour development compared with gasless laparoscopy 104-106 or helium pneumoperitoneum.107,108 In contrast, other studies have reported that tumour cell proliferation and growth are independent of the insufflating gas used.109.110 CO2 pneumoperitoneum may decrease subcutaneous pH levels, and it has been debated whether direct exposure of the peritoneum to CO2 or CO2 induced acidosis may promote tumour growth in the abdominal wall.111 2. Intraabdominal pressure It is known that intraabdominal pressure caused by pneumoperitoneum may influence hemodynamics and particularly the splanchnic circulation.112 The effects of intraabdominal pressure have gained new interest in studies of the pathogeneses of port site metastases. Jacobi et al studied the effect of different pressures on tumour growth and port site metastases in a rat model both in vivo and in vitro.113 They found a decrease of in vitro intraperitoneal tumour growth after incubation of tumour cells with CO2 at 10 and 15 mmHg compared with pressures at 0 and 5 mmHg. In vivo, intraperitoneal tumour growth was increased after laparoscopy at 5 and 10 mmHg but decreased at a pressure of 15 mmHg compared with the control group. Subcutaneous tumour growth was increased at laparoscopy at 5, 10 and 15 mmHg compared with controls. Other studies have reported that highpressure CO2 stimulates intraabdominal tumor growth.114,115 Another effect of pneumoperitoneal pressure was demonstrated by Moreira et al in a hamster model. They found that an increase of intraabdominal pressure did not influence aerosolization but significantly increased the rates of both instrument contamination and tumour recurrence.116 The increased rate of wound metastases caused by inadequate surgical technique seems to be aggravated by CO2 insufflation compared with gasless laparoscopy. This was demonstrated by Watson et al who showed that laceration of tumours in the abdominal wall resulted in more wound metastases after laparoscopy with CO2 pneumoperitoneum then after gasless laparoscopy.106 On the other hand a few studies have demonstrated that intraabdominal pressure (i.e. CO2 pnumoperitoneum) does not increase tumour spread or growth compared with gasless controls.117-119 As noted, most of these studies have used CO2 pneumoperiotneum as an “elevator of intra26

abdominal pressure” making it hard to exclude a possible effect on tumour growth by CO2 itself. The suggestion that high intraabdominal pressure and/or CO2 may increase tumour development has promoted the concept of gasless laparoscopy. One argument against this procedure is the number of reported PSM cases after thoracoscopy performed with no insufflation at all.120 An interesting effect of pneumoperitoneum, probably due to the elevated intabdominal pressure and distension, is the injury to the peritoneal layer presumably making it susceptible for metastases formation.121 3. Blood flow Pneumoperitoneum and its effects on blood flow have mainly been studied with focus on general hemodynamic consequences and effects on the splanchnic circulation.112 Especially high pressure pneumoperitoneum is known to impair intraabdominal blood flow.122 The effects of pneumoperitoneum on abdominal wall blood flow, were port site metastases actually occur, have been sparsely studied and conflicting results reported.123,124 The lack of knowledge and the fact that ischemia may promote tumour development 125 encouraged us to further examine the effects of pneumoperitoneum on blood flow and tumour growth in the abdominal wall. Immune response Numerous clinical and experimental studies have addressed the hypothesis that laparoscopic surgery should be less traumatic and thereby less immunosuppressive than open surgery.126 Accordingly, the potential preservation of immune response after laparoscopic surgery for the cure of malignancy has gained much attention. The concern of an increased risk of tumour development after laparoscopic surgery has subsisted in parallel. The immune reaction or stress response triggered by surgery is a very complex and incompletely understood phenomenon. Briefly, surgery produces a local reaction which initiates the coagulation cascade and the accumulation of leucocytes and platelets as well as activation of fibroblasts and endothelial cells. Many of these cells produce cytokines such as IL-6, IL-1 and TNF-α which go on to mediate a variety of systemic effects. Interactions exist with the neural and endocrine response such as catecholamines, aldosterone, ADH, ACTH, TSH, GH, insulin etc resulting in peripheral fat and protein breakdown, insulin resistance, water retention and gluconeogenesis which is in proportion to the degree of surgical injury.127,128 Numerous studies have focused on rates, levels or products of one or more of the parameters mentioned above as well as CRP which is a classic marker of the acute-phase reaction known to be proportional to the extent of injury. Spe27

cific immunity as reflected by NK- and T-cells, as well as their function often measured by DTH, has been commonly studied.129 Even though conflicting data exist in studies regarding some immune parameters, it is clear that major surgery results in a period of cell-mediated immunosuppresion.126 The fact that malignancies were encountered after immunosuppression was noted already in immunosuppressed patients after kidney transplantations.130 Immune function as reflected by studies on DTH response has shown that open surgery is associated with a suppression of cell-mediated immunity.131 Brune et al demonstrated that suppression of T lymphocytes functions, as reflected by decreased production of IFN-γ,TNFα and IL-2, was noted after OC but not after LC. This effect was mediated by downregulation of the specific Th1 cellmediated immune response.132 Clinical studies on immune response mainly derive from comparisons between OC and LC. Most studies on serum levels of IL-6 and CRP demonstrates less increase after LC, indicating greater trauma after OC.129 McMahon et al, found no difference in these markers comparing LC with minilaparotomy cholecystectomy.133 It has been suggested that studies of immunologic functions are more relevant than to assess plasma levels of immunologic parameters.134 In a prospective randomised trial by Ordemann et al the monocyte production of cytokines after ex vivo endotoxin stimulation was increased after laparoscopic colorectal resection compared to open surgery.135 Plasma cytokine levels (IL-6, TNF-α) were significantly higher in patients after open surgery. In a review of laparoscopic surgery and the systemic immune response Vittimberga et al it is suggested that the body´s response to laparoscopy is one of lesser immune activation as opposed to immunosuppression.136 The alterations in the immune system and tumour growth in laparoscopy is well summarized by Hartley and coworkers.129 According to their review an increasing body of evidence from both animal and clinical studies suggest that specific immunity function is better preserved after laparoscopic than after open surgery. Recent clinical studies from laparoscopic colorectal cancer and immune response show more conflicting results.131,137,138 However no clinical trial has been able to demonstrate any good correlation between the changes in immunologic parameters and relevant postoperative clinical endpoints such as significant postoperative complications.134 Recently the interest in immunologic changes has been focused on changes in the local peritoneal immune system after laparoscopic surgery. In a small prospective randomized study after laparoscopic or open colon resection, Wu et al noted much more pronounced elevation of postoperative cytokine levels (Il-6) in peritoneal fluid than in serum, although significant differences in Il-6 levels between the open and laparoscopic groups were 28

noted only in serum and not in peritoneal fluid.139 Several animal studies have demonstrated an impairment of peritoneal macrophage function after exposure to CO2-pneumoperitoneum but results are conflicting.126,140 Laparoscopic surgery seems to reduce the immunologic consequences of the stress response after surgery compared to conventional open technique. Minimally invasive methods may be associated with oncologic advantages but the impact of better preserved immune response in laparoscopic surgery for malignant disease still remains unclear. In conclusion, numerous experimental studies have been performed concerning laparoscopy and tumour spread and growth. Substantial variations in material and methods may reflect the vide range of findings and conflicting results reported. Nevertheless, it is still not clear whether laparoscopic surgery alters the metastatic pattern, increases the risk of tumour spread and growth or if laparoscopic technique may have a beneficial effect in cancer surgery compared with conventional open surgery.

29

AIMS To study the incidence of wound metastases from gallbladder cancer after laparoscopic and open cholecystectomy. To study the long term results after open and laparoscopic surgery for gallbladder cancer. To experimentally study the effects of air and CO2 pneumoperitoneum on intraperitoneal tumour growth. To experimentally study the effects of air and CO2 pneumoperitoneum on blood flow and tumour growth in the abdominal wall. To experimentally study whether a decrease of blood flow by clamping increases tumour growth in the abdominal wall.

30

PATIENTS, MATERIALS AND METHODS Clinical studies – paper I, II and III A written questionnaire was sent to all university (n=8) and central (n=24) hospitals in Sweden requesting information about the number of LC, the number of converted cases and the number of patients who developed port site metastases from GBC after LC done during 1991-94. To assess the incidence of PSM, patient data with histopathologically verified adenocarcinoma or carcinoma in situ of the gallbladder were obtained from the Swedish Oncological Centres. The registry of patients with GBC was matched against the patients with surgical classification codes for cholecystectomy (laparoscopic or open) as reported to The National Board of Health and Welfare (EpC). To identify if patients with completed or converted LC incorrectly were registered as OC, we doublechecked all patients registered as OC with the Swedish Registry of Laparoscopic Cholecystectomy which covered the years 1991-93. The check up for 1994 was done by reviewing the records of all patients with gallbladder cancer recorded as open cholecystectomies. Further information on patients with port site metastases was achieved through a review of their records or information from the operating surgeon. To estimate the incidence of wound metastases from GBC after OC we reviewed the files of all patients with GBC registered as OC. Patient files were scrutinized with respect to surgical procedure, indications for surgery, preoperative suspicion of malignancy as well as the occurrence of wound metastases. Pathological reports were reexamined and classified according to the TNM system.141 In our first study we classified tumours according to the Nevin system 41 but in our second study we used the more commonly used TNM system – descriptions of the staging systems are shown on page 17. In study I no follow up was performed in LC patients where no PSM were reported. In study III we requested and reviewed the files from all GBC patients registered as LC. In addition the National Board of Health and Welfare provided us with data on hospital stay, additional surgical procedures performed and time and causes of death on all patients with GBC operated with OC or LC. Survival data were achieved until 2001 to obtain long term follow up on all patients with GBC operated with open or laparoscopic cholecystectomy.

31

Statistics and ethics Kaplan-Meier curves and log rank test were used to calculate survival. Statistical analyses were performed using a statistical software package (SPSS10.0, SPSS Inc. IL, USA). The study was approved by the Regional Research Ethic Committee at Umeå University.

32

Experimental studies – paper IV,V and VI In all experiments Wistar Fu rats were used. They were anesthetized with a cocktail of midazolam, fentanyl citrate and fluanisone. The cell line used in all experiments was syngenic colonic adenocarcinoma cells. The tumour cells were primarily nitrosoguanidine-induced (NGW) and prepared by homogenizing tumours resected from carrier rats. Pneumoperitoneum in all experiments was maintained for 45 minutes at a pressure of 10 mmHg. In the first experimental study (IV) a cell suspension of 1 ml (105) adenocarcinoma cells was injected intraperitoneally into three groups of rats. 13 rats were insufflated with room air, 13 with CO2 and 13 were not insufflated. After 12 days the rats were killed and the intraperitoneal tumour load was quantified using a modification of the peritoneal cancer index (PCI) scoring system described by Eggermont 142 (table 1).

Table 1. Modification of the peritoneal cancer index (PCI) scoring system described by Eggermont to assess the quantity of intraperitoneal tumour load.

Tumour load

PCI

No intraperitoneal tumour

0

< 4 tumour foci with a diameter < 1 mm

1

4-10 tumour foci

2

10-50 tumour foci

3

Moderate intraperitoneal tumour load (> 50 foci but not replacing most of the peritoneal cavity)

4

Abundant intraperitoneal tumour replacing most of the peritoneal cavity

5

33

In studies V and VI a 2x2 cm pouch was created in the upper midline and abdominal wall blood flow was continuously measured by a 10 mm preperforated laser Doppler probe holder sutured to the rectus muscle (fig 4). Blood flow was measured with laser Doppler probes with a fibre separation of 0.25 mm. Light was generated (780 nm wavelength) and flow measurement was monitored on a Periflux master (Perimed, Sweden) with a time constant of 0.03 sec. Blood flow was registered and presented in arbitrary perfusion units (PU). In study V and VI a tumour cell suspension of 0.1 ml with 50 000 cells was used. After the experiment the probes were removed and the skin was sutured. In study V 40 rats had a laser Doppler probe placed on their left rectus muscle and a tumour cell suspension injected into their right rectus muscle. 20 rats were insufflated with air and 20 were not insufflated and served as controls. One pair of animals, one insufflated and one control, was always studied simultaneously to ensure that tumour cells from the same solution with equal viability were used (fig 4). Blood flow was continuously measured and registrations were made before, during and after insufflation. In study VI 16 rats had a laser doppler probe placed on their right and left rectus muscle. The left rectus muscle was clamped with two forceps, one cranial and one caudal to the laser Doppler probe. The tumour cell suspension was injected into both rectus muscles respectively, but the right rectus muscle was not clamped and served as control. Blood flow was measured simultaneously on both rectus muscles as described above (figure 4). After 9 days the animals were sacrificed and the weight and volume of the tumours were calculated according to the formula V = a x b / 2 as described by Carlsson et al.143 In the second part of the experiment (VI), 11 pairs of rats were studied. 11 animals were insufflated with CO2 and 11 were not insufflated. Tumour injection and blood flow measurements were performed as in study IV.

34

Figure 3. Rat with left rectus muscle clamped with two forceps and right rectus muscle not clamped (control). Probes sutured on each rectus muscle measuring laser Doppler blood flow.

Figure 4. Experimental (insufflated ) and control (not insufflated) rats with laser Doppler probes sutured to the rectus muscles.

35

Laser Doppler blood flow The theory of laser Doppler flowmetry is that a beam of laser light, carried by a fibre-optic probe, is scattered and partly absorbed by the tissue being studied. Light hitting moving blood cells undergoes a change in wavelength (Doppler shift) while light hitting static objects is unchanged (figure 5). The magnitude and frequency distribution of these changes are directly related to the number and velocity of blood cells but unrelated to their direction of movement. The information is picked up by a returning fibre, converted into an electronic signal and analyzed. Laser Doppler perfusion represents the velocity and concentration of moving blood cells.179 Figure 5. Illustration of Laser Doppler flowmetry - transmitted laser light is absorbed and reflected by the studied tissue and picked up by the receiving fibre. Change in wavelength occurs when the light hits moving blood cells (Doppler shift). Reprint with permission of Perimed

A disadvantage of LDF is that measurements can only be expressed in arbitrary Perfusion units (PU) and not in absolute perfusion values (e.g. ml/min/100mg tissue). Thus it is particularly important that the laser Doppler instrument is regularly calibrated, which in our studies was done with a latex suspension motility standard (Perimed, Sweden). Another problem with LDF is that movements of the probe affect the circulation measurements. In our studies the probe was sutured to the rectus fascia to ensure a fixed position of the probe. LDF is a well established method for assessing microcirculatory changes that correlates well with the tissue oxygenation and total blood flow of the tissue under study.180,181 In our setting with a fibre separation of 0.25 mm, a time constant of 0.03 sec and a laser diode with a wave length of 780 nm, the penetration depth is about 0.5-1 mm. The instrument used in our studies (Periflux 4001 master, Perimed, Sweden) was provided with dual channels making simultaneous blood flow measurements of one pair of rats or both rectus muscles in one rat possible.

36

Statistics and ethics Values were expressed as means ± s.e.m. Statistical analyses were performed using a statistical software package (SPSS 10.0, SPSS Inc. IL, USA). In paper IV, tumour weight was compared with the Mann-Whitney U-test. In paper V, blood flow was analysed by means of ANOVA for repeated measurements using the Tukey post-hoc test. The distribution of tumour take between groups were analysed by the McNemar test and comparisons of tumour weight and volume by the Wilcoxon´s signed ranks test. In paper VI blood flow was analysed by means of ANOVA for repeated measurements using the Huynh-Feldt correction procedure. The distribution of tumour take in the two groups was analysed by the McNemar test. Comparisons of tumour weight and volume were analysed by the Wilcoxon signed rank test, using one-sided analysis since the hypothesis according to our previous findings was an increase in tumour weight and volume. Studies were approved by the Animals Ethics committee at the Umeå University hospital, Umeå, Sweden.

37

RESULTS Clinical studies – paper I, II and III Replies were obtained from 30 of the 32 clinics (94%). 11 976 LC were done during 1991-94 with a mean conversion rate of 9.4%. According to the Oncologic Centres 447 patients with histologically verified adenocarcinoma or carcinoma in situ were registered. 270 of these patients had been operated with OC and 55 with LC. In our questionnaire, nine patients were reported to have developed port site metastases after LC from gallbladder cancer (figure 6). When reviewing the files of the 215 patients registered as OC, six patients were excluded because of other malignant diagnoses than GBC. Five patients had incorrect surgical codes and had in fact undergone LC (4 converted and 1 completed LC). Of the remaining 204 patients, clinical files and follow up data were achieved in 186 patients (91%). 12 patients (6.5%) developed wound metastases from GBC after OC (II). Of 55 patients who had been subjected to LC, 3 files were not retrieved and 1 patient was excluded because of incorrect operation code (only biopsy taken). One patient registered as LC had actually undergone open cholecstectomy and was included in the open group (figure 6). When scrutinizing the 46 files of LC patients not previously reported with PSM, another 5 patients had tumour recurrences in the surgical wound. Three patients had recurrences in the port sites (clinical examination) and two in the laparotomy wound after converted procedures. Totally 25% (14/55) of the laparoscopically operated GBC patients developed incisional metastases. Data on patients with wound metastases are presented in table 2. Median interval to the occurrence of wound metastases was 6 months for the open group and 8 months for the laparoscopic group.

38

447 patients with gallbladder cancer

177 no cholecystectomy

270 cholecystectomy Registry data

55 LC

3 files not obtained

52 LC

1 excluded

51 LC

215 OC

Review of files

Review of files 5 LC

55 Laparoscopic Cholecystectomy

46 LC (19 converted)

Questionnaire 9 port site metastases

197 OC

18 files not obtained

6 excluded

191 OC

1 OC

187 Open Cholecystectomy

Review of files

Review of files 5 abdominal wall metastases (3 port site + 2 wound after converted procedures)

14 wound metastases (25%)

12 wound metastases (6.5%)

Figure 6. Study setting, surgical procedures and wound metastases in 447 patients with histopathologically verified adenocarcinoma or carcinoma in situ of the gallbladder.

39

TABLE 2. Tumour stage and intraoperative gallbladder perforation in patients with wound recurrences from gallbladder cancer after laparoscopic and open cholecystectomy. Laparoscopic cholecystectomy (N=55)

Wound recurrences

Open cholecystectomy (N=187)

14 (25%)

12 (6.5%)

2 1 2 7 0 2

0 1 2 8 1 0

9 (65%)

9 (75%)

Tumour stage Tis T1 T2 T3 T4 not stated Gallbladder perforation

The long term follow up of 55 patients subjected to LC and 187 to OC is schematically presented in figure 7 and 8. The median follow up time was nine years and patients not registered as dead in 2001 at the National Board of Health and Welfare are reported as alive.

55 LC

1 lost

12 alive

4 dead other disease

14 wound metastases

25 dead GBC (median 17 months)

1 alive

12 dead (median 18 months )

Figure 7. Long term follow up after laparoscopic cholecystectomy (LC) in 55 patients with gallbladder cancer.

40

187 OC

21 alive

12 wound metastases

1? *

16 dead other disease

137 dead GBC (median 6 months)

12 dead (median 11 months)

Figure 8. Long term follow up after open cholecystectomy (OC) in187 patients with gallbladder cancer. (* Dead in disseminated malignancy of unknown origin.)

Crude 5-year survival in relation to T-stage for OC and LC group is presented in table 3. Survival was significantly shorter in the OC group (figure 9). Patients with T3 tumours operated with LC showed significantly better survival than patients operated with OC (figure 10). 5-year survival in patients with T3 tumours was noted in only 2 patients of the LC and 1 patient of the OC group (table 3).

TABLE 3. Crude 5-year survival in relation to Tumour stage in patients with gallbladder cancer operated with laparoscopic or open cholecystectomy. Tumour stage Tis T1 T2 T3 T4 All Stages

Laparoscopic cholecystectomy

60 % (3/5) 83% (5/6) 37% (6/16) 8% (2/24) 0% (0/2) 30% (16/53*)

Open cholecystectomy

60% (3/5) 72% (13/18) 24% (12/50) 1% (1/80) 0% (0/30) 16% (29/183*)

*Tumour stage not stated in 2 patients from the laparoscopic group and 4 from the open group.

41

Figure 9. Kaplan Meier survival curves for all patients with gallbladder cancer and known T stage operated with open (183 patients)) or laparoscopic cholecystectomy (53 patients). P = 0.001.

Figure 10. Kaplan Meier curves for patients with T3 gallbladder tumours operated with open (80 patients) or laparoscopic cholecystectomy (24 patients). P < 0.001.

42

Experimental studies – paper IV, V and VI In the first experimental study (IV), both pneumoperitoneum with CO2 and air significantly increased intraperitoneal tumour load compared to controls. No difference in tumour growth was noted between the animals insufflated with air or carbondioxide. Animal weights were also significantly increased in the control group but unchanged in the insufflated groups. In study V insufflation with air caused an 82% reduction of blood flow (p < 0.001), while no reduction was registered in the control group. Tumours developed significantly more often in the insufflated group (20/20) compared to the control group (14/20), p= 0.016. Tumour weight (p = 0.003) and volume (p < 0.001) were significantly increased in the insufflated group. In study VI, insufflation of CO2 caused a 69% reduction of blood flow ( p < 0.001) while no reduction was registered in the not insufflated control animals (figure 11). Tumour weight (p = 0.006) and volume ( p = 0.006) were increased in the insufflated group. Clamping decreased blood flow in the rectus muscle with 69% (p < 0.001) while no decrease was registered in the non-clamped rectus muscle. Tumour weight (p =0.028) and volume ( p = 0.030) were increased on the clamped side. 180

160

Blood flow (PU)

140

120

100

80

co ntro ls CO2

60

40

20

0

Figure 11. Abdominal wall blood flow in controls (not insufflated) and insufflated animals ( P < 0.001).

43

DISCUSSION Clinical studies paper I, II and III The proportion of patients with GBC who were not subjected to surgery may seem high (177/447) but is in accordance with previous studies. In a Swedish study on 63 consecutive GBC patients, 48% only had a biopsy taken and 52% underwent cholecystectomy compared to 60% in our study.144 The apparently low number of GBC patients who were subjected to LC compared to OC has several explanations. During the study period 1991-1994 the majority of all cholecystectomies were performed with open technique according to the National Board of Health and Welfare. In the early 1990´s many hospitals in Sweden had just started to perform LC and potentially more difficult cases were probably selected to open surgery. This is reflected by the fact that patients who underwent OC had a suspicion of malignancy, jaundice or acute cholecystitis preoperatively documented in 84% of the patients compared to 25% in the LC group. The findings from our studies seem to indicate that the risk of incisional metastases is higher after LC compared to OC. Previous studies on GBC have also demonstrated a high rate of PSM after LC.54,66 In a retrospective multicentre study by Paolucci and coworkers 117 840 LC were examined and among those 409 patients had an unsuspected GBC. 70 of these patients developed PSM.54 The common view that LC increases the risk of wound recurrences from GBC has persisted despite the lack of comparative studies between open and laparoscopic cholecystectomy. Ricardo et al reported from a tertiary referral centre on 79 patients with GBC. In their study, 26 patients had an open, 21 a laparoscopic and 16 a converted procedure. Independent of the surgical procedure about 30% of the patients developed incisional metastases.145 89% of all patients in their study had an advanced GBC (T3/T4) in contrast to 59% and 47.5% in our OC and LC groups respectively. In a recent German study Paolucci and coworkers reported on 245 patients with postoperative incidental findings of GBC, 79 following OC and 142 following LC and 24 had a converted procedure.67 With a median follow up of 27 months they reported an incidence of wound metastases of 5.1% after OC and 7% after LC. The total incidence of recurrences was similar in both groups and they concluded that the access technique, LC or OC, does not seem to influence the prognosis of GBC. In our studies the rate of port site metastases may be overestimated and the true incidence of PSM after LC from GBC is hard to assess. A possible bias could be that at the time of our questionnaire the awareness was high for the risk of PSM and therefore more likely to be found and reported. Two patients reported with PSM were also poorly documented but as they 44

were reported in our questionnaire, they have not been excluded. On the other hand, when reviewing the files of the LC group another 5 incisional metastases after LC were found. Hence, the incidence of PSM after GBC in all major hospitals in Sweden 1991-1994 seems to be approximately 25%. The incidence of wound recurrences from GBC after OC in our second study may on the other hand be underestimated as many of the open cases may not have lived long enough to develop any obvious recurrent disease. This is reflected by the fact that the median survival of the entire OC group (6 months) was equal to the median interval time for wound recurrences (6 months) for patients subjected to open cholecystectomy. This is in contrast to the LC group with a median survival of 17 months and a median time to port site recurrence of 8 months. Careful clinical follow up of patients with GBC following OC have not been performed and the patients may have developed incisional recurrences that were never detected. Furthermore, as noted by Reilly et al most wound metastases after open surgery on large bowel cancer were incidentally identified at reoperation.61 This may also be the case in GBC after OC. Still, Fahim et al already in 1962 examined 151 patients with GBC who all had surgery and found only 6 patients with abdominal wall metastases, all at the site of a previous cholecystostomy.146 The true incidence of wound metastases from GBC after OC is not known since prospective randomised trials are difficult to design, especially as GBC is quite uncommon and often accidentally encountered. This is in contrast to laparoscopic colorectal cancer were ongoing large prospective randomised trials hopefully will give the answer whether wound metastases after laparoscopic surgery are more common than after open surgery. Even more important is whether survival is affected by the used technique, laparoscopic or open. Although somewhat controversial, but in a prospective randomised study, Lacy et al have reported survival benefits in patients with laparoscopically operated T3 colonic tumours.65 The debate on laparoscopic surgery and tumour growth have also concerned whether the introduction of LC has worsened the prognosis of GBC. Donohue et al compared registry data on 5000 GBC patients operated before (1989-90) and during (1994-95) the LC era and could not find any adverse effects on the general outcome for patients with GBC after the introduction of LC in the United States.49 A similarly designed American study was performed by Whalen et al and they also concluded that LC did not deteriorate the survival of GBC patients.147 They also reviewed the files of half of the patient material (237 patients with GBC) and found that 36% of the patients from the laparoscopic era actually had undergone OC. They also registered the incidence of abdominal wall recurrence in serendipitously treated GBC and found 11% in the laparoscopic group and 3% in the open group. In Ja45

pan, Suzuki and coworkers examined 5027 LC and found 41 GBC and 4 patients developed PSM.148 They compared survival data with previous Japanese studies on OC and found similar 5-year survival rates as in patients operated with LC (92% in Tis/T1, 59% in T2/T3). A similar Japanese survey of 498 patients with laparoscopically removed GBC reported high 5year survival rates (95% in T1, 70% in T2 and 20% in T3 tumours).149 They also concluded that LC was not likely to worsen the prognosis of GBC. In a review on early GBC patients subjected to LC, Weiland et al reported 3-year survival rates (47% in T1, 40% in T2) which they concluded as worse than previous reports for OC.150 In Donohue and coworkers review of 5000 GBC patients in USA, 5-year survival rates of 39% for T1 and 15% for T2 tumours were reported.49 In our studies the overall crude 5-year survival rates were 16% for the OC group and 30% for the LC group (table 3). The median survival was 6 months for the OC group and 17 months for the LC group. These differences may reflect the higher proportion of advanced tumours in the OC group than in the LC group. However, median survival in T3 tumours was 16 months in the LC group and 5 months in the OC group. In the study of Whalen et al they reported a similar proportion in T stage distribution of 154 GBC patients who underwent surgical procedures as in our study.148 Accordingly, most of their patients had a T3 tumour but median survival was similar during the open (6 months) as well as during the LC period (8 months). In our study patients with T3 tumours had a median survival of 5 months in the open group but 16 months in the LC group. Whalen et al could not find any differences, comparing Kaplan Meier survival in patients subjected to OC and LC.148 In our study we found beneficial survival effects for patients operated with LC compared with OC. This is illustrated by the Kaplan Meier curves for patients with T3 tumours in figure 10. Patients with T3 tumours were also most common in both OC (43%) and LC (44%) groups. Although improved survival was noted for patients with T3 tumours subjected to LC only 2 of the patients were alive after 5 years. In our study converted cases were analysed together with patients who underwent complete LC according to the intention to treat principle. Not surprisingly, when comparing Kaplan Meier survival for patients who underwent converted procedures with the OC group, no difference was noted. Despite an apparent higher rate of wound metastases LC does not seem to have a negative impact on the survival in GBC patients. In contrast to other studies, our material on GBC patients subjected to open and laparoscopic cholecystectomy comprises patients from the same hospitals operated during the same period of time.

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We could only identify 15 patients in the OC group (8%) and 6 patients (11%) in the LC group that had extended resections, in contrast to the Japanese studies mentioned above where Ouchi et al reported additional resection in 48% of the cases.149 This could also to some extent explain the high survival rate in their study, as they also report a better survival after additional excision in patients with T2 and T3 tumours compared with LC only. The reason for improved survival in laparoscopically operated GBC patients with T3 tumours in our study is not because of a higher number of extended resections in the LC group as only one T3 patient in the LC group had extended resection compared to 7 patients in the open group. Intraoperative gallbladder perforation is regarded as a risk factor for spreading malignant cells and concomitantly the development of PSM.66 In our material on GBC patients with PSM, 65% had gallbladder perforation compared to 38% in the whole LC group. In the OC group 75% of the patients with wound metastases and 23% in whole OC group were reported by the surgeon to have had intraoperative gallbladder perforation. In the study of Ouchi et al patients with gallbladder perforation were even shown to have a significantly lower survival rate than those without perforated gallbladders,150 which has been confirmed in other studies.151-153 Some authors advocate that LC, with its potential for bile spillage, should be avoided as a initial procedure in suspected GBC patients, especially as this may convert a potentially curable early GBC to a incurable disease.151 In discussing whether LC increases the risk of tumour spread, the risk of intraabdominal tumour dissemination in general should be distinguished from the risk of metastases to the port site. In a recent French review by Fondrinier et al they concluded that laparoscopy had no influence on tumour growth but may affect peritoneal dissemination.154 This also reveals the controversy whether port site metastases are to be regarded as an isolated phenomenon or as a manifestation of disseminated disease. In our study, 75% of the patients with wound recurrences in the LC group and 91% of the patients in the OC group had T2, T3 or T4 tumours. Our review of port site metastases confirms that the overwhelming majority of patients had advanced tumours. Consequently all patients with incisional metastases in our study except one have died from GBC. This would indicate that port site metastases predominantly are associated with advanced disease and poor prognosis. Nevertheless, in our study on PSM after LC, 2 patients developed PSM after a Tis and one after a T1 tumour of the gallbladder. All 3 had intraoperative gallbladder perforation, and all subsequently died from disseminated disease with a mean survival of 51 months. Only scattered reports exist of long time survivors after excision of PSM.65,154 The only patient with PSM reported alive in our study has re-

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cently (February, 2004), 11 years after the initial LC, been subjected to a third reoperation for wound metastases from gallbladder cancer. Our studies seem to indicate that wound recurrences from GBC may be more common after LC than after OC, but do not seem to have any negative influence on survival. The median survival in the LC group is also similar to the median survival of the patients with PSM. Documented intraoperative gallbladder perforation is overrepresented in patients with wound metastases in both LC and OC groups. Gallbladder perforations are also more frequently reported in the LC group than in the OC group which may contribute to an increased risk of PSM. Furthermore our studies indicate that the risk of dissemination of GBC is not generally increased by LC as this most likely should have had a negative influence on survival. Nevertheless, there may be features in laparoscopic surgery that increase the risk of abdominal wall metastases. This increased risk however, does not seem to influence survival possibly because the majority of patients with wound metastases already have an advanced disease and a short expected survival. However, it can not be ruled out that a negative survival effect of PSM may to some extent be compensated by less trauma and better preservation of the immune response seen after laparoscopic cholecystectomy.

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Experimental studies – paper IV, V and VI Although instrumental manipulation and tumour cell spillage seem to play a major role in PSM after laparoscopic cancer surgery, several experimental studies indicate that other features of minimal invasive surgery may be involved in this phenomenon.57 Several studies have demonstrated a promotive effect on intraperitoneal tumour development after CO2 pneumoperitoneum compared with gasless laparoscopy 104-106 or helium pneumoperitoneum.107,156 This encouraged us to perform the first experimental study (IV) where air and CO2 pneumoperitoneum equally increased intraabdominal tumour growth compared to not insufflated controls. The enhancement of intraperitoneal tumour growth after insufflation being independent of the used gas has been confirmed by others,110 indicating that other aspects than the used gas is involved in the enhancement of tumour load in laparoscopic surgery. Tumour cell suspension models have been criticized because of differences in tumour cell viability and for the large number of injected cells.93 In our experiments we studied experimental and control groups simultaneously with the same cell suspension to avoid variations in viability. The number of injected tumour cells was studied by Wu et al who showed that with a decreasing amount of tumor cells injected intraabdominally the tumour promoting effect of CO2 pneumoperitoneum was diminished.157 They noted no enhancement in tumour growth after pneumoperitoneum when 1.6 x 105 human colon cancer cells were injected intraperitoneally in hamsters. Although we used 105 adenocarcinoma cells in study IV, a comparison is difficult because of differences in both metastatic and growth potential of the tumour cells as well as animal features. This difficulty probably also reflects the variety of findings and conclusions presented in animal studies on laparoscopy and tumour growth. In addition, it illustrates the problem to evaluate animal studies in relation to the clinical situation in general. Several studies have examined both abdominal wall and intraperitoneal tumour growth in relation to CO2 pneumoperitoneum and predominantly a promoting effect of CO2 has been noted.101-103,158,159 Some studies have examined tumour growth outside the abdominal cavity and have been unable to demonstrate any promotive effect of CO2 pneumoperitoneum.160162 This may support that the effects of pneumoperitoneum is a local event mainly caused by the intraabdominal distention rather than the gas being used. On the other hand, Jacobi et al found an increase of subcutaneous tumour growth in the back of rats after CO2 pneumoperitoneum independently of intraperitoneal pressure.113 In the same study intraabdominal tumour growth was increased after 5 mmHg and 10 mmHg CO2 pneumoperitoneum but suppressed at 15 mmHg. In another study in rats, Wittich et al found an 49

increase in intraabdominal tumour growth after CO2 insufflation at 16 mm Hg.163 Gutt and coworkers demonstrated that different tumour cell lines reacted disparately to in vitro exposure of CO2 at different pressures but concluded that the insufflation pressure may have additional effects in promoting tumour growth.114 Factors such as lipopolysaccharide in room air have also been proposed to increase peritoneal macrophage cytokine release (TNF-alfa) and thereby enhance the immune response.164 Pidgeon et al further reported increased VEGF levels and metastatic tumour growth in mice exposed to air (lipopolysaccharide) during laparoscopic or open surgery.165 Another negative effect of air is that peritoneal surfaces may dry out causing cell damage. This is even proposed to explain the increased surgical trauma in laparotomy rather than the larger incisions compared to minimal invasive surgery.93 It has also been demonstrated in a rat model that laparotomy in a warm humid environment caused less postoperative intraperitoneal tumour growth than laparotomy in room air.93 Nduka et al reported less intraperitoneal tumour growth and spread with warmed compared to cold CO2.166 Many possible explanations are available for our findings that air and CO2 pneumoperitoneum equally enhances intraperitoneal tumour growth. Nevertheless, a possible enhancement of intraabdominal tumour development after laparoscopy compared to controls does not address whether the risk of abdominal wall metastases is increased after laparoscopic surgery. In our following experimental studies (V, VI) we wanted to investigate whether pneumoperitoneum influenced blood flow and tumour growth in the abdominal wall where port site metastases actually originate. We found a major decrease in abdominal wall blood flow during air as well as during CO2 pneumoperitoneum. Insufflation with air caused a 82% reduction of blood and CO2 decreased the blood flow with 69% as measured with laser Doppler technique. Tumour growth was enhanced in insufflated animals compared to controls but air insufflation also caused an increase in tumour take (i.e. tumours developed more often) which was not seen in the CO2 group. A possible explanation could be that there are different mechanisms involved in tumour growth compared to tumour take. To our knowledge this theory has never been studied or even discussed in the field of laparoscopy and tumour growth. Only a few studies have been published concerning changes in abdominal wall blood flow during pneumoperitoneum and conflicting results are reported. Schilling et al studied laser Doppler blood flow in patients subjected to laparoscopic surgery and found the most pronounced decreases in blood flow in the parietal peritoneum. Yavuz et al, in a study on pigs, registered an increase in the blood flow of the parietal peritoneum but no changes in the rectus muscle during CO2 pneumoperitoneum.167 In another study on 50

CO2 pneumoperitoneum in pigs, Brundell and coworkers noted an increase of peritoneal blood flow and suggested that CO2 induced vasodilatation was responsible.123 In contrast to abdominal wall blood flow, several studies have examined the effect of pneumoperitoneum on intraabdominal circulation. Schaefer et al noted a reduction in hepatic blood flow and assumed that both increased intraabdominal pressure and hypercapnia were the main underlying factors.169 Ishida et al injected tumour cells intraportally in mice and found that the development of liver metastases was enhanced by increased CO2 insufflation pressure.170 In a similar study, Gutt and coworkers compared the oncologic and immunologic effects on the liver of CO2 and helium pneumoperitoneum.171 They found no differences between the two gases and concluded that elevated intraabdominal pressure was responsible for hepatic disadvantages during pneumoperitoneum and not carbon dioxide itself. We demonstrated that insufflation with CO2 and air equally decreased abdominal wall blood flow and increased tumour growth. A similar decrease in blood flow achieved by clamping also increased tumour growth in the abdominal wall but not as significant as after insufflation. Tumour growth in clamped and non-clamped (control) rectus muscle was studied simultaneously in the same animal. Thus, it is possible that systemic effects caused by clamping of one rectus muscle affected tumour growth on the non-clamped side. This design was chosen in order to have similar initial laser Doppler blood flow levels in experimental and control muscle as laser Doppler measurements may show interindividual variations. In contrast, the insufflated animals had another not insufflated animal serving as a control. Robinson and Hoppe already in 1962 reported that ischemia enhances the ability of tumour cells to produce metastases.125 Even though our studies indicate that ischemia itself increases tumour growth, it can not be postulated that the reduction of blood flow is principally responsible. The more pronounced increase in tumour development after insufflation than after clamping may reflect that other factors related to pneumoperitoneum may be involved in the development of abdominal wall metastases. Pneumoperitoneum is also known to cause an increase in the activity of proteolytic enzymes such as matrix metalloproteinases 172,173 with the capability of degrading extracellular matrix allowing invasion of cancer cells.174 Pneumoperitoneum is also known to cause hypoxia 175 which is proposed to cause upregulaion of matrix metalloproteinase activity.176 In our studies blood flow was significantly reduced after insufflation but oxygen tissue partial pressure was not analyzed. This parameter was assessed by Wildbrett et al who found a decrease in partial pressure of oxygen in the abdominal wall after insufflation with both helium and CO2.175 51

Previous to the debate on port site metastases, local trauma was reported to enhance the ability of tumour implantation.72,73 Not only the trocars but pneumoperitoneum itself is known to cause traumatic changes in peritoneum.121 Ziprin et al also demonstrated that the adhesion of tumour cells to the peritoneum in vitro was increased after the mesothelial cell monolayer was exposed to pneumoperitoneum.177 The increase was independent of used gas (CO2, Helium or air) and associated with increased expression of mesothelial intercellular adhesion molecule-1 (ICAM-1). Ziprin et al further suggested that the enhanced adhesion is related to the hypoxic environment generated by pneumoperitoneum and further pronounced by traumatic cytokine release.178 It is possible that a favourable tumour soil produced by ischemia is augmented by local trauma and increased proteolytic activity created by the hypoxic pneumoperitoneum. Enhanced adhesion of tumour cells during pneumoperitoneum may also reflect an increased susceptibility for tumour take rather than tumour growth.

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SUMMARY AND CONCLUSIONS In our clinical studies we have demonstrated that wound metastases from GBC are a common phenomenon after open and laparoscopic cholecystectomy. Incisional metastases may be more common after LC but no negative effect on survival can be demonstrated. There are indications that surgical technique as reflected by intraoperative gallbladder perforation, may contribute to an increased rate of tumour implants in the surgical scar. This may be particularly important in early gallbladder carcinomas, where perforation could convert a curable disease into a disseminated malignancy. A secondary finding was that malignancy of the gallbladder is probably suspected more often than previously assumed. An increased awareness preoperatively may help the surgeon to select a proper surgical approach. This is most likely more important than a dogmatic regimen advocating one surgical procedure before the other. Nevertheless, it may be recommendable at least in cases with a suspected early GBC to perform conventional open surgery to avoid a possible increased risk of gallbladder perforation in LC. Lack of knowledge and experience of handling a malignant gallbladder laparoscopically may also favour a recommendation of open surgery in cases of GBC. The potential benefits of laparoscopic surgery for cancer might well be recognized after further preferably prospective clinical studies. Experimentally we have shown that pneumoperitoneum with air and CO2 increases intraabdominal tumour growth. Pneumoperitoneum also reduces blood flow in the abdominal wall and enhances tumour growth at the same site. Tumour growth was also enhanced by mechanical reduction of blood flow by clamping indicating that impaired blood flow may promote tumour development. Albeit strict clinical conclusions cannot be drawn, our findings indicate that features of pneumoperitoneum may have negative implications in oncologic surgery. Our studies may further help us to identify these potentially negative features so that they can be minimized in the clinical situation. This is important particularly as laparoscopic surgery has potential advantages in surgery for malignant disease as indicated in our clinical studies. More research could further clarify the effects of laparoscopy on tumour growth and hopefully reveal advantages that go way beyond quicker recovery and shorter hospital stay. Our studies have demonstrated interactions between the laparoscopic procedure and tumour development in particular that subsequently may help us to understand aspects of tumour take and growth in general.

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ACKNOWLEDGEMENTS I wish to express my sincere gratitude to: Anders Kristoffersson my tutor and friend for his encouragement, fruitful and enjoyable discussions and particularly for after long nights with scientific discussions providing me the “doctorial room” in his house. Lars-Olof Hafström and Peter Naredi my former and present professor, for scientific support and help in the start and engaged assistance in the finishing part respectively. Karl-Axel Ängquist, former head of Department of Surgery for his support and friendship. Åke Öberg my colleague, travel partner and friend for his unique way and arrangements to manage without me. Anita Westman for skilful assistance and loyal company in the laboratory. My friends, colleagues and staff at the Department of Surgery, especially Örjan Norrgård and Maarku Haapamäki in the Colorectal team for doing the hard work during my absence. Special thanks to my friend Birger Sandzen who confirmed my belief in the importance of surgical technique and also enlightened me in the TME concept and almost never sings out of tune. Gunilla Östberg for help with all kinds of problems. Göran Johansson and Håkan Jonsson for explaining statistics to a surgeon far beyond two standard deviations. All my friends and colleagues at Torsby hospital; the operation staff, Odd Kleppenes who almost forced me into orthopaedics and especially Leif Hoffman who taught me the basics of surgery and gave me unforgettable help and friendship during my first years as a surgeon. My wife Lena for accepting (?) computer work in the bedroom at night. My outstanding children Ida, Anna, Eric and Karin for their mere existence. This work was supported by the Lions Cancer Research Foundation, University of Umeå, Umeå Sweden.

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REFERENCES 1. Filipi, CJ, Fitzgibbons, RJ, Salerno, GM.: Historical review: Diagnostic laparoscopy to laparoscopic cholecystectomy and beyond. In Surgical Laparoscopy, K.A. Zucker, editor. St. Louis: Quality Medical Publishing, 1991, pp. 3–21. 2. Laparoskopisk teknik inom allmän kirurgi. A state of the art document. Medicinska forskningsrådet. Stockholm: 1997. 3. Nduka CC, Monson JR, Menzies-Gow N, Darzi A. Abdominal wall metastases following laparoscopy. Br J Surg 1994;81:648-52. 4. Wibbenmeyer LA, Wade TP, Chen RC, Meyer RC, Turgeon RP, Andrus CH. Laparoscopic cholecystectomy can disseminate in situ carcinoma of the gallbladder. J Am Coll Surg 1995;181:504-510. 5. Suzuki K, Kimura T, Ogawa H. Long-term prognosis of gallbladder cancer diagnosed after laparoscopic cholecystectomy. Surg Endosc 2000;14(8) : 712-6. 6. Kraas E, Frauenschuh D, Farke S. Intraoperative suspicion of gallbladder carcinoma in laparoscopic surgery: what to do? Dig Surg 2002;19(6):48993. 7. Bergdahl L. Gallbladder first diagnosed at microscopic examination of gallbladders removed for presumed benign disease. Ann Surg 1980;191:1922. 8. Rosin, D: History. In Minimal Access Medicine and Surgery, D. Rosin, editor. Oxford, Radcliffe Medical Press, 1993, pp. 1–9. 9. Gorden, A : The history and development of endoscopic surgery. In Endoscopic Surgery for Gynaecologists, Sutton C, Diamond MP, editors. London, Saunders, 1993, pp. 3–7. 10. Lau WY, Leow CK, Li AKC. History of Endoscopic and Laparoscopic Surgery. World J Surg 1997;21:444–453. 11. Gotz, F, Pier A, Schippers E, Schumpelick V: The history of laparoscopy. In Color Atlas of Laparoscopic Surgery, Gotz F, Pier A, Schippers E, Schumpelick V, editors. New York, Thieme, 55

1993, pp. 3–5. 12. Bozzini, PH: Lichtleiter, eine Erfindung zur Anschauung innerer Teile und Krankheiten. J Prak Heilk 1806;24:107. 13. Sircus W. Milestones in the evolution of endoscopy: a short story. JR Coll Physicians Edinb 2003;33:124-134. 14. Desormeaux AJ. De l’Endoscopie, instrument propre a’ ec lairer certaines cavities interieures de l’economie. Compte rendus de L’Academie des Sciences 1855;40:692–3. 15. Nitze M: Beobachtung-und Untersuchungsmethode fur Harnohre, Harnblase und Rectum. Wien Med Wochenschr 1879;29:649. 16. Kelling G: Uber Oesophagokopie, Gastroskopie und Kolioskopie. Munchen Med Wochenschr 1902;49:21. 17. Jacobaeus HC: Kurze Ubersicht uber meine Erfahrungen mit der Laparothorakoskopie. Munchen Med Wochenschr 1911;58:2017. 18. Korbsch R: Die Laparoskopie nach Jakobaeus. Berl Klin Wochenschr 1921;38:696. 19. Goetze O: Die neues Verfabren der Gasfullung fur das Pneumoperitoneum. Munchen Med Wochenschr 1921;51:233. 20. Zollikofer R: Zur Laparoskopie. Schweiz Med Wochenschr 1924;54: 264. 21. Veress J: Neues Instrument zur Ausfuhrung von Brust-Punktmonen und Pneumothorax Behandlung. Dtsch Med Wochenschr 1938;64:1480. 22. Semm, K.: Das Pneumoperitoneum mit CO2. In Endoskopie— Methoden, Ergebnisse. Demling L, Ottenjann R, editors. Munich, Banaschewski, 1967. pp167. 23. Litynski GS. Kurt Semm and the fight against skepticism: endoscopic hemostasis, laparoscopic appendectomy, and Semm´s impact on the “laparoscopic revolution”. JSLS 1998;2:309-313. 24. Hopkins HH: On the diffraction theory of optical images. Proc 56

Soc Lond 1953;A217:408. 25. Hasson HM: Open laparoscopy vs closed laparoscopy: a comparison of complication rates. Adv Planned Parenthood 1978;13:41. 26. Muhe, E: Die erste Cholecystectomy durch das Laparoskope. Langenbecks Arch Chir 1986;369:804. 27. de Stoll M. Rationis Medendi in Nosocomino Practico Unindobonensi (part I). 1777; Vienna: Bernardi. 28. Abi-Rached B, Neugut AI. Diagnostic and management issues in gallbladder carcinoma. Oncology (Huntingt) 1995;9:19-24. 29. Pearlstone DB, Curley SA, Feig BW. The management of gallbladder cancer: before, during and after laparoscopic cholecystectomy. Semin Laparosc Surg 1998;5:121-8. 30. Maibenco DC, Smith JL, Nava HR, Petrelli NJ, Douglass HO Jr. Carcinoma of the gallbladder. Cancer Invest 1998;16:33-9. 31. Pandey M. Risk factors for gallbladder cancer: a reappraisal. Eur J Cancer Prev 2003;12:15-24. 32. Statistics Health and Disease. Cancer incidence in Sweden 2002. Stockholm: The national board of health and welfare; 2003:table 4. 33. Cancer i siffror. Stockholm: Cancerfonden och Socialstyrelsen; 2001:72-73. 34. Misra S, Chaturvedi A, Misra NC, Sharma ID. Carcinoma of the gallbladder. Lancet Oncology 2003;4:167-176. 35. Towfigh S, McFadden DW, Cortina GR, Thompson J E Jr, Tompkins R K, Chandler, C et al. Porcelain gallbladder is not associated with gallbladder carcinoma. Am Surgeon 2001;67:7-10. 36. Aldridge MC, Bismuth H. Gallbladder cancer: the polyp cancer sequence. Br J Surg 1990;77:363-364.

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37. Chijiiwa K, Kimura H, Tanaka M. Malignant potential of the gallbladder in patients with anomalous pancreaticobiliary ductal junction. Int Surg1995; 80:61-64. 38. Ohta T, Nagakawa T, Ueno K, Maeda K, Ueda N, Kayahara M et al. Clinical experience of biliary tract carcinoma associated with anomalous union of the pancraticobiliary ductal system. Jpn J Surg 1990;20:36-43. 39. Perpetuo MD, Valdivieso M, Heilbrun LK, Nelson RS, Connor T, Bodey GP. Natural history of gallbladder cancer: a review of 36 years experience at M.D. Anderson Hospital and Tumor Institute. Cancer 1978; 42: 330-335. 40. Shabo I, Nordensköld K, Svanvik J. The incidence of gallbladder carcinoma in Sweden has decreased. Läkartidningen 2001;98:4584-4589. 41. Sobin LH, Wittekind C. International Union against Cancer. TNM classification of malignant tumours. Fifth ed. New York:Wiley-Liss;1997. p.7879. 42. Nevin JE, Moran TJ, Kay S, King R. Carcinoma of the gallbladder. Staging treatment and prognosis. Cancer 1976;37:141-148. 43. Orth K, Beger HG. Gallbladder carcinoma and surgical treatment. Langenbeck´s Arch Surg 2000;385:501508. 44. Schwartz LH, Coakley FV, Sun Y, Blumgart, L H, Fong Y, Panicek D M. Neoplastic pancreaticobiliary duct obstruction: evaluation with breathhold MR cholangiopancreatography. Am J Roentgenol 1998;170:1491– 1495. 45. Strom BL, Maislin G, West SL, Atkinson B. Herlyn M, Saul S et al. Serum CEA and CA 19-9: Potential future diagnostic or screening tests for gallbladder cancer? Int J Cancer 1990;45:821-824. 46. Bartlett DL, Fong Y, Fortner JG, Brennan MF, Blumgart LH. Long-term results after resection for gallbladder cancer. Implications for staging and management. Ann Surg 1996;224:639-46. 47. Houry S, Haccart V, Huguier M, Schlienger M. Gallbladder cancer: role of radiation therapy. Hepatogastroenterology 1999;46:1578–1584.

58

48. Todoroki T. Chemotherapy for gallbladder carcinoma––a surgeon's perspective. Hepatogastroenterology 2000;47:948–955. 49. Donohue JH, Stewart AK, Menck HR. The National Cancer Data Base report on carcinoma of the gallbladder. Cancer 1998;83:2618-2628. 50. Nimura Y. Extended surgery in bilio-pancreatic cancer: the Japanese experience. Semin Oncol 2002;(6 Suppl 20):17-22. 51. Steinert R, Lippert H, Reymond MA.Tumor cell dissemination during laparoscopy: prevention and therapeutic opportunities. Dig Surg 2002;19: 464-72. 52. Paolucci V. Port site recurrences after laparoscopic cholecystectomy. J Hepatobiliary Pancreat Surg 2001;8:535-543. 53. Dobronte Z, Wittman T, Karacsony G. Rapid development of malignant metastases in the abdominal wall after laparoscopy. Endoscopy 1978;127130. 54. Paolucci V, Schaeff B, Schneider M, Gutt C. Tumor seeding following laparoscopy: international survey. World J Surg 1999;23:989-995. 55. Schaeff B, Paolucci V, Thomopoulos J. Port site recurrences after laparoscopic surgery. A review. Dig Surg 1998;15:124-134. 55. Wexner SD, Cohen SM. Port site metastases after laparoscopic colorectal surgery for cure of malignancy. Br J Surg 1995;85:295-298. 57. Canis M, Botchorishvili R, Wattiez A, Pouly JL, Mage G, Manhes H et al. Cancer and laparoscopy, experimental studies: a review. Eur J Obstet Gynecol Reprod Biol 2000;91:1-9. 58. Franklin Jr ME, Rosenthal D, Abrego-Medina D, Dorman JP, Glass JL, Norem R et al. Prospective comparision of open vs laparoscopic colon surgery for carcinoma. Five-year results. Dis Colon Rectum 1996;39 (10 Suppl):S35-46. 59. Vukasin P, Ortega AE, Greene FL, Steele GD, Simons AJ, Anthone GJ et al. Wound recurrence following laparoscopic colon cancer resection. Results of the American Society of Colon and Rectal Surgeons Laparoscopic Registry. Dis Colon Rectum 1996;39(10 Suppl):S20-23. 59

60. Ziprin P, Ridgway PF, Peck DH, Darzi AW.The theories and realities of port-site metastases: a critical appraisal. J Am Coll Surg 2002;195:395-408. 61. Reilly WT, Nelson H, Schroeder G, Wieand HS, Bolton JB, O´Connel JO. Wound recurrence following conventional treatment of colorectal cancer. Dis Colon Rectum 1996;39:200-207. 62. Hughes ES, Mc Dermott FT, Polglase AL, Johnson WR. Tumor recurrence in the abdominal wall scar tissue after large-bowel cancer surgery. Dis Colon Rectum 1983;26:571-572. 63. Welch JP, Donaldson GA. The clinical correlation of an autopsy study of recurrent colorectal cancer. Ann Surg 1979;189:496-502. 64. Milsom JW, Bohm B, Hammerhofer KA, Fazio V, Steiger E, Elson P. A prospective, randomized trial comparing laparoscopic versus conventional techniques in colorectal cancer surgery: a preliminary report. J Am Coll Surg 1998;187:46-54. 65. Lacy AM, Garcia-Valdecasas JC, Delgado S, Castells A, Taura P, Pique JM et al. Laparoscopy-assisted colectomy versus open colectomy for treatment of non-metastatic colon cancer: a randomised trial. Lancet 2002;359:2224-2229. 66. Z'graggen K, Birrer S, Maurer CA, Wehrli H, Klaiber C, Baer HU. Incidence of port site recurrence after laparoscopic cholecystectomy for preoperatively unsuspected gallbladder carcinoma. Surgery 1998;124:831-8. 67. Paolucci V, Neckell M, Götze T. Unsuspected Gallbladder carcinoma – The CAE-S/CAMIC registry. Zentralbl Chir 2003;128:309-312. 68. Hartley JE, Monson JR. The role of laparoscopy in the multimodality treatment of colorectal cancer. Surg Clin North Am 2002;82:1019-33. 69. Hubens G. Port site metastases: where are we at the beginning of the 21st century? Acta Chir Belg 2002;102:230-7. 70. Alexander JW, Shucart WA, Altemeier WA. Enhancement of tumor growth by growth promoting factor from injured tissues. Surg Forum 1965;16:111-113.

60

71. Fisher B, Fisher ER, Sakai A. Experimental studies of factors influencing hepatic metastases. XV. Effect of neonatal thymectomy. Cancer Res 1965;25:993-6. 72. Murthy SM, Goldschmidt RA, Rao LN, Ammirati M, Buchmann T, Scanlon EF. The influence of surgical trauma on experimental metastasis. Cancer 1989;64:2035-44. 73. Skipper D, Jeffrey MJ, Cooper AJ, Alexander P, Taylor I. Enhanced growth of tumour cells in healing colonic anastomoses and laparotomy wounds. Int J Colorectal Dis 1989;4:172-7. 74. Gutman M, Fidler IJ. Biology of human colon cancer metastasis. World J Surg 1995;19:226-34. 75. Targarona EM, Martinez J, Nadal A, Balague C, Cardesa A, Pascual S et al. Cancer dissemination during laparoscopic surgery: tubes, gas, and cells. World J Surg 1998;22:55-60. 76. Martinez J, Targarona EM, Balague C, Pera M, Trias M. Port site metastasis. An unresolved problem in laparoscopic surgery. A review. Int Surg 1995;80:315-21. 77. Ramirez PT, Wolf JK, Levenback C. Laparoscopic port-site metastases: etiology and prevention. Gynecol Oncol 2003 ;91:179-89. 78. Hewett PJ, Thomas WM, King G, Eaton M. Intraperitoneal cell movement during abdominal carbon dioxide insufflation and laparoscopy. An in vivo model. Dis Colon Rectum 1996;39(10 Suppl):S62-66. 79. Reymond MA, Wittekind C, Jung A, Hohenberger W, Kirchner T, Kockerling F. The incidence of port-site metastases might be reduced. Surg Endosc 1997;9:902-6. 80. Allardyce R, Morreau P, Bagshaw P.Tumor cell distribution following laparoscopic colectomy in a porcine model. Dis Colon Rectum 1996;39(10 Suppl):S47-52. 81. Allardyce RA, Morreau P, Bagshaw PF. Operative factors affecting tumor cell distribution following laparoscopic colectomy in a porcine model. Dis Colon Rectum 1997;40:939-45.

61

82. Jorgensen JO, McCall JL, Morris DL. Port site seeding after laparoscopic ultrasonographic staging of pancreatic carcinoma. Surgery 1995;117:118-9. 83. Hase K, Ueno H, Kuranaga N, Utsunomiya K, Kanabe S, Mochizuki H. Intraperitoneal exfoliated cancer cells in patients with colorectal cancer. Dis Colon Rectum 1998;41:1134-40. 84. Neuhaus S, Hewett P, Disney A. An unusual case of port site seeding. Surg Endosc 200;15:896. 85. Siriwardena A, Samarji WN. Cutaneous tumour seeding from a previously undiagnosed pancreatic carcinoma after laparoscopic cholecystectomy. Ann R Coll Surg Engl 1993;75:199-200. 86. Ugarte F. Laparoscopic cholecystectomy port seeding from a colon carcinoma. Am Surg 1995;61:820-1. 87 Sellers GJ, Whelan RL, Allendorf JD, Gleason NR, Donahue J, Laird D et al. An in vitro model fails to demonstrate aerosolization of tumor cells. Surg Endosc 1998;12:436-9. 88. Mathew G, Watson DI, Ellis T, De Young N, Rofe AM, Jamieson GG. The effect of laparoscopy on the movement of tumor cells and metastasis to surgical wounds. Surg Endosc 1997;11:1163-6. 89. Whelan RL, Sellers GJ, Allendorf JD, Laird D, Bessler MD, Nowygrod R, Treat MR. Trocar site recurrence is unlikely to result from aerosolization of tumor cells. Dis Colon Rectum 1996;39(10 Suppl):S7-13. 90. Ikramuddin S, Lucus J, Ellison EC, Schirmer WJ, Melvin WS. Detection of aerosolized cells during carbon dioxide laparoscopy. J Gastrointest Surg 1998;2:580-3; 91. Wittich P, Marquet RL, Kazemier G, Bonjer HJ. Port-site metastases after CO(2) laparoscopy. Is aerosolization of tumor cells a pivotal factor? Surg Endosc 2000;14:189-92. 92. MacFarlane JK, Ryall RD, Heald RJ. Mesorectal excision for rectal cancer. Lancet 1993;341:457-60.

62

93. Jacobi CA, Bonjer HJ, Puttick MI, O'Sullivan R, Lee SW, Schwalbach P et al. Oncologic implications of laparoscopic and open surgery. Surg Endosc 2002;16:441-5. 94. Patankar SK, Larach SW, Ferrara A, Williamson PR, Gallagher JT, DeJesus S et al. Prospective comparison of laparoscopic vs. open resections for colorectal adenocarcinoma over a ten-year period. Dis Colon Rectum 2003; 46:601-11. 95. Lee SW, Gleason NR, Bessler M, Whelan RL. Port site tumor recurrence rates in a murine model of laparoscopic splenectomy decreased with increased experience. Surg Endosc 2000 Sep;14:805-11. 96. Lee SW, Southall J, Allendorf J, Bessler M, Whelan RL. Traumatic handling of the tumor independent of pneumoperitoneum increases port site implantation rate of colon cancer in a murine model. Surg Endosc 1998;12: 828-34. 97. Mutter D, Hajri A, Tassetti V, Solis-Caxaj C, Aprahamian M, Marescaux J. Increased tumor growth and spread after laparoscopy vs laparotomy: influence of tumor manipulation in a rat model. Surg Endosc 1999;13:365-70. 98. Doudle M, King G, Thomas WM, Hewett P. The movement of mucosal cells of the gallbladder within the peritoneal cavity during laparoscopic cholecystectomy. Surg Endosc 1996;10:1092-4. 99. Nieveen van Dijkum EJ, de Wit LT, Obertop H, Gouma DJ.Port-site metastases following diagnostic laparoscopy. Br J Surg 1996 ;83:1793-4. 100. Pearlstone DB, Mansfield PF, Curley SA, Kumparatana M, Cook P, Feig BW. Laparoscopy in 533 patients with abdominal malignancy. Surgery 1999;125:67-72. 101. Jones DB, Guo LW, Reinhard MK, Soper NJ, Philpott GW, Connett J et al. Impact of pneumoperitoneum on trocar site implantation of colon cancer in hamster model. Dis Colon Rectum 1995;38:1182-8. 102. Bouvy ND, Marquet RL, Jeekel H, Bonjer HJ. Impact of gas(less) laparoscopy and laparotomy on peritoneal tumor growth and abdominal wall metastases. Ann Surg 1996 ;224:694-700;

63

103. Jacobi CA, Sabat R, Bohm B, Zieren HU, Volk HD, Muller JM. Pneumoperitoneum with carbon dioxide stimulates growth of malignant colonic cells. Surgery 1997;121:72-8. 104. Mathew G, Watson DI, Rofe AM, Ellis T, Jamieson GG. Adverse impact of pneumoperitoneum on intraperitoneal implantation and growth of tumour cell suspension in an experimental model. Aust N Z J Surg 1997;67: 289-92. 105. Gutt CN, Riemer V, Kim ZG, Erceg J, Lorenz M. Impact of laparoscopic surgery on experimental hepatic metastases. Br J Surg 2001;88:3715. 106. Watson DI, Mathew G, Ellis T, Baigrie CF, Rofe AM, Jamieson GG. Gasless laparoscopy may reduce the risk of port-site metastases following laparascopic tumor surgery. Arch Surg 1997 ;132:166-8. 107. Neuhaus SJ, Watson DI, Ellis T, Rowland R, Rofe AM, Pike GK et al. Wound metastasis after laparoscopy with different insufflation gases. Surgery 1998;123:579-83. 108. Wenger FA, Jacobi CA, Kilian M, Dressler H, Guski H, Muller JM. The impact of laparoscopic biopsy of pancreatic lymph nodes with helium and carbon dioxide on port site and liver metastasis in BOP-induced pancreatic cancer in hamster. Clin Exp Metastasis 2000;18:11-4. 109. Ridgway PF, Smith A, Ziprin P, Jones TL, Paraskeva PA, Peck DH et al. Pneumoperitoneum augmented tumor invasiveness is abolished by matrix metalloproteinase blockade. Surg Endosc 2002 ;16:533-6. 110. Dorrance HR, Oien K, O'Dwyer PJ. Effects of laparoscopy on intraperitoneal tumor growth and distant metastases in an animal model. Surgery 1999;126:35-40. 111. Kuntz C, Wunsch A, Bodeker C, Bay F, Rosch R, Windeler J et al. Effect of pressure and gas type on intraabdominal, subcutaneous, and blood pH in laparoscopy. Surg Endosc 2000;14:367-71. 112. Schafer M, Krahenbuhl L. Effect of laparoscopy on intra-abdominal blood flow. Surgery 2001;129:385-9.

64

113. Jacobi CA, Wenger FA, Ordemann J, Gutt C, Sabat R, Muller JM. Experimental study of the effect of intra-abdominal pressure during laparoscopy on tumour growth and port site metastasis. Br J Surg 1998;85:141922. 114. Gutt CN, Kim ZG, Hollander D, Bruttel T, Lorenz M. CO2 environment influences the growth of cultured human cancer cells dependent on insufflation pressure. Surg Endosc 2001;15:314-8. 115 Wittich P, Steyerberg EW, Simons SH, Marquet RL, Bonjer HJ. Intraperitoneal tumor growth is influenced by pressure of carbon dioxide pneumoperitoneum. Surg Endosc 2000;14:817-9. 116. Moreira H Jr, Yamaguchi T, Wexner S, Singer L, Zhao R, Baig MK et al. Effect of pneumoperitoneal pressure on tumor dissemination and tumor recurrence at port-site and midline incisions. Am Surg 2001;67:369-73. 117. Iwanaka T, Arya G, Ziegler MM. Mechanism and prevention of portsite tumor recurrence after laparoscopy in a murine model. J Pediatr Surg 1998;33:457-61. 118. Lecuru F, Agostini A, Camatte S, Robin F, Aggerbeck M, Jais JP et al. Impact of pneumoperitoneum on tumor growth. Surg Endosc 2002;16:11704. 119. Agostini A, Robin F, Aggerbeck M, Jais JP, Blanc B, Lecuru F. Influence of peritoneal factors on port-site metastases in a xenograft ovarian cancer model. BJOG 2001;108:809-12. 120. Downey RJ, McCormack P, LoCicero J 3rd. Dissemination of malignant tumors after video-assisted thoracic surgery: a report of twenty-one cases. The Video-Assisted Thoracic Surgery Study Group. J Thorac Cardiovasc Sur. 1996;111:954-60. 121. Volz J,Koster S, Spacek Z, Paweletz N. The influence of pneumoperitoneum used in laparoscopic surgery on an intraabdominal tumor growth. Cancer 1999;86:770-774. 122. Rasmussen IB, Berggren U, Arvidsson D, Ljungdahl M, Haglund U. Effects of pneumoperitoneum on splanchnic hemodynamics: an experimental study in pigs. Eur J Surg 1995 ;161:819-26.

65

123. Brundell SM, Tsopelas C, Chatterton B, Toulomtzoglou J. Experimental study of peritonal blood flow and insufflation pressure during laparoscopy. Br J Surg 2002;89:617-622. 124. Diebel L, Saxe J, Dulchavsky S. Effect of intraabdominal pressure on abdominal wall blood flow. Am Surg 1992;58:573-575. 125. Robinson KP, Hoppe E. The development of Blood-Borne Metastases. Arch Surg 1962;85:40.44. 126. Carter JJ, Whelan RL. The immunologic consequences of laparoscopy in oncology. Surg Oncol Clin N Am 2001;10:655-677. 127. Bessey PQ. Metabolic response to critical illness In: Meakins JL Surgical infections diagnosis and treatment. Scientific American, New York, pp 36-42. 128. Michie HR. Metabolism of sepsis and multiple organ failure. World J Surg 1996;20:460-464. 129.Hartley JE, Mehigan BJ, Monson JRT. Alterations in the immune system and tumor growth in laparoscopy. Surg Endosc 2001;15:305-313. 130. Cole WH. The increase in immunosuppression and its role in the development of malignant lesions. J Surg Oncol 1985;30:139-144. 131. Whelan RL, Franklin M, Holubar SD, Donahue J, Fowler R, Munger C et al. Postoperative cell mediated immune response is better preserved after laparoscopic vs open colorectal resection in humans. Surg Endosc 2003;17: 972-8. 132. Brune IB, Wilke W, Hensler T, Holzmann B, Siewert JR. Downregulation of T helper type 1 immune response and altered pro-inflammatory and anti-inflammatory T cell cytokine balance following conventional but not laparoscopic surgery. Am J Surg 1999;177:55-60. 133. McMahon AJ, O'Dwyer PJ, Cruikshank AM, McMillan DC, O'Reilly DS, Lowe GD et al. Comparison of metabolic responses to laparoscopic and minilaparotomy cholecystectomy. Br J Surg 1993;80:1255-8. 134. Jacobi CA, Wenger F, Opitz I, Muller JM. Immunologic changes during minimally invasive surgery. Dig Surg 2002;19:459-63. 66

135. Ordemann J, Jacobi CA, Schwenk W, Stosslein R, Muller JM.Cellular and humoral inflammatory response after laparoscopic and conventional colorectal resections. Surg Endosc 2001 ;15:600-8. 136. Vittimberga FJ Jr, Foley DP, Meyers WC, Callery MP. Laparoscopic surgery and the systemic immune response. Ann Surg 1998 Mar;227(3): 326-34. 137. Wu FP, Sietses C, von Blomberg BM, van Leeuwen PA, Meijer S, Cuesta MA. Systemic and peritoneal inflammatory response after laparoscopic or conventional colon resection in cancer patients: a prospective, randomized trial. Dis Colon Rectum 2003;46:147-55. 138. Schwenk W, Jacobi C, Mansmann U, Bohm B, Muller JM. Inflammatory response after laparoscopic and conventional colorectal resections results of a prospective randomized trial. Langenbecks Arch Surg 2000;385(1):2-9. 139. Wu FP, Sietses C, von Blomberg BM, van Leeuwen PA, Meijer S, Cuesta MA. Systemic and peritoneal inflammatory response after laparoscopic or conventional colon resection in cancer patients: a prospective, randomized trial. Dis Colon Rectum 2003;46:147-55. 140. Ziprin P, Ridgway PF, Peck DH, Darzi AW. The theories and realities of port-site metastases: a critical appraisal. J Am Coll Surg 2002;195:395408. 141. UICC. TNM classification of malignant tumours. 4th ed. Berlin: Springer –Verlag;1987. 142. Eggermont AM, Steller EP, Marquet RL, Jeekel J, Sugarbaker PH. Local regional promotion of tumor growth after abdominal surgery is dominant over immunotherapy with interleukin-2 and lymphokine activated killer cells. Cancer Detect Prev 1988;12:421-9. 143. Carlsson G, Gullberg B, Hafström L. Estimation of liver tumor volume using different formulas: an experimental study in rats. J Cancer Res Clin Oncol 1983;105:20-23. 144. Jonsson PE, Pettersson BA. Carcinoma of the gallbladder--a natural history type of study. J Surg Oncol 1982;21:215-8. 67

145. Ricardo AE, Feig BW, Ellis LM, Hunt KK, Curley SA, Mac Fayden BV Jr et al. Gallbladder cancer and trocar site recurrences. Am J Surg 1997;174:619-623. 146. Fahim RB, Mc Donald JR, Richards JC, Ferris DO. Carcinoma of the gallbladder: a study of its modes of spread. Ann Surg 1962;156:114-24. 147. Whalen GF, Bird I, Tanski W, Russell JC, Clive J. Laparoscopic cholecystectomy does not demonstrably decrease survival of patients with serendipitously treated gallbladder cancer. J Am Coll Surg 2001;192:18995. 148. Suzuki K, Kimura T, Ogawa H. Long-term prognosis of gallbladder cancer diagnosed after laparoscopic cholecystectomy. Surg Endosc 2000;14:712-716. 149. Ouchi K, Mikuni J, Kakugawa Y . Laparoscopic cholecystectomy for gallbladder carcinoma: results of a Japanese survey of 498 patients. J Hepatobiliary Pancreat Surg 2002;9:256-60. 150. Weiland ST, Mahvi DM, Niederhuber JE, Heisey DM, Chicks DS, Rikkers LF. Should suspected early gallbladder cancer be treated laparoscopically? J Gastrointest Surg 2002;6:50-6. 151. Sarli L, Contini S, Sansebastiano G, Gobbi S, Costi R, Roncoroni L. Does laparoscopic cholecystectomy worsen the prognosis of unsuspected gallbladder cancer? Arch Surg 2000 ;135:1340-4. 152 Wullstein C, Woeste G, Barkhausen S, Gross E, Hopt UT. Do complications related to laparoscopic cholecystectomy influence the prognosis of gallbladder cancer? Surg Endosc 2002;1:828-32. 153. Yoshida T, Matsumoto T, Sasaki A, Morii Y, Ishio T, Bandoh T et al. Laparoscopic cholecystectomy in the treatment of patients with gall bladder cancer. J Am Coll Surg 2000;19:158-63. 154. Fondrinier E, Descamps P, Arnaud JP, Pezet D. Carbon dioxide pneumoperitoneum and peritoneal carcinosis: review. J Gynecol Obstet Biol Reprod 2002 Feb;31(1):11-27.

68

155. Suzuki K, Kimura T, Hashimoto H, Nishihira T, Ogawa H. Port site recurrence of gallbladder cancer after laparoscopic surgery: two case reports of long-term survival. Surg Laparosc Endosc Percutan Tech 2000;10:86-8. 156. Jacobi CA, Ordemann J, Bohm B, Zieren HU, Liebenthal C, Volk HD et al. The influence of laparotomy and laparoscopy on tumor growth in a rat model. Surg Endosc 1997;11:618-21. 157. Wu JS, Jones DB, Guo LW, Brasfield EB, Ruiz MB, Connett JM, Fleshman JW. Effects of pneumoperitoneum on tumor implantation with decreasing tumor inoculum. Dis Colon Rectum 1998;41:141-6. 158. Koster S, Melchert F, Volz J. Effect of CO2 pneumoperitoneum on intraperitoneal tumor growth in the animal model. Geburtshilfe Frauenheilkd 1996;56:458-61. 159. Hubens G, Pauwels M, Hubens A, Vermeulen P, Van Marck E, Eyskens E. The influence of a pneumoperitoneum on the peritoneal implantation of free intraperitoneal colon cancer cells. Surg Endosc 1996;10:809-12. 160. Increased tumor establishment and growth after laparotomy vs laparoscopy in a murine model.Allendorf JD, Bessler M, Kayton ML, Oesterling SD, Treat MR, Nowygrod R et al. Arch Surg 1995 ;130:649-53. 161. Bessler M, Allendorf JDF, Chao JD. Permissive tumour growth after laparotomy versus laparoscopy is associated with altered TNF levels. Surg Forum 1994;45:486-487. 162. Da Costa ML, Redmond HP, Finnegan N. Laparotomy and laparoscopy differentially accelerate experimental flank tumour growth. Br J Surg 1998;85:1439-1442. 163. Wittich P, Steyerberg EW, Simons SH, Marquet RL, Bonjer HJ. Intraperitoneal tumor growth is influenced by pressure of carbon dioxide pneumoperitoneum. Surg Endosc 2000;14:817-9. 164. Watson RW, Redmond HP, McCarthy J, Burke PE, Bouchier-Hayes D. Exposure of the peritoneal cavity to air regulates early inflammatory responses to surgery in a murine model. Br J Surg 1995 ;82:1060-5.

69

165. Pidgeon GP, Harmey JH, Kay E, Da Costa M, Redmond HP, BouchierHayes DJ. The role of endotoxin/lipopolysaccharide in surgically induced tumour growth in a murine model of metastatic disease. Br J Cancer 1999; 81:1311-7. 166. Nduka CC, Puttick M, Coates P, Yong L, Peck D, Darzi A.Intraperitoneal hypothermia during surgery enhances postoperative tumor growth. Surg Endosc 2002;16:611-5. 167. Yavuz Y, Ronning K, Lyng O, Gronbech JE, Marvik R. Effect of carbon dioxide pneumoperitoneum on tissue blood flow in the peritoneum, rectus abdominis, and diaphragm muscles. Surg Endosc 2003;17:1632-5. 168. Schilling MK, Redaelli C, Krhenbuhl L, Buchler Mw. Splanchnic microcirculatory changes during CO2 laparoscopy. J Am Coll Surg 1997;184:378-382. 169. Schafer M, Sagesser H, Reichen J, Krahenbuhl L. Alterations in hemodynamics and hepatic and splanchnic circulation during laparoscopy in rats. Surg Endosc 2001;15:1197-201. 170. Ishida H, Hashimoto D, Nakada H, Takeuchi I, Hoshino T, Murata et al. Increased insufflation pressure enhances the development of liver metastasis in a mouse laparoscopy model: possible mechanisms. Surg Endosc 2002 ;16:331-5. 171. Gutt CN, Gessmann T, Schemmer P, Mehrabi A, Schmandra T, Kim ZG. The impact of carbon dioxide and helium insufflation on experimental liver metastases, macrophages, and cell adhesion molecules. Surg Endosc 2003;17:1628-31. 172. Ridgway PF, Ziprin P, Jones TL, Paraskeva PA, Peck DH, Darzl AW. Laparoscopic staging of pancreatic tumors induces increased invasive capacity in vitro. Surg Endosc 2003;17:306-10. 173. Ridgway PF, Smith A, Ziprin P, Jones TL, Paraskeva PA, Peck DH, et al. Pneumoperitoneum augmented tumor invasiveness is abolished by matrix metalloproteinase blockade. Surg Endosc 2002 ;16:533-6. 174. Pross M Lippert H, Mantke R, Kruger S, Gunther T, Marusch F et al. A proteinase inhibitor decreases tumor growth in a laparoscopic rat model. Surg Endosc 2001;15:882-885. 70

175. Wildbrett P, Oh A, Naundorf D, Volk T, Jacobi CA. Impact of laparoscopic gases on peritoneal microenvironment and essential parameters of cell function. Surg Endosc 2003 ;17:78-82. 176. Canning MT, Postovit LM, Clarke SH, Graham CH. Oxygen-mediated regulation of gelatinase and tissue inhibitor of metalloproteinases-1 expression by invasive cells. Exp Cell Res 2001;267:88-94. 177. Ziprin P, Ridgway PF, Peck DH, Darzi AW. Laparoscopic enhancement of tumour cell binding to the peritoneum is inhibited by antiintercellular adhesion molecule-1 monoclonal antibody. Surg Endosc 2003;17:1812-7. 178. Ziprin P, Ridgway PF, Pfistermuller KL, Peck DH, Darzi AW. ICAM1 mediated tumor-mesothelial cell adhesion is modulated by IL-6 and TNFalpha: a potential mechanism by which surgical trauma increases peritoneal metastases. Cell Commun Adhes 2003;10:141-54. 179. Bakken B. Theory of Laser Doppler at www.perimed.se. 2004-03-10. 180. Kvernebo K, Staxrud LE, Salerud EG. Assessment of human blood perfusion with single-fibre laser Doppler flowmetry. Microvasc Res 1990;39:376-385. 181. Smits GJ, Roman RJ, Lombard JH. Evaluation of laser-Doppler flowmetry as a measure of tissue blood flow. J Appl Physiol 1986;61:666-672.

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