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Cytokines pattern after surgical radiofrequency ablation of liver colorectal metastases
© 2007. Elsevier Masson SAS. Tous droits réservés
25,*,1$/ $57,&/(
Gastroenterol Clin Biol 2007;31:141-145
Cytokines pattern after surgical radiofrequency ablation of liver colorectal metastases Serge EVRARD (1), Christine MENETRIER-CAUX (2), Cathy BIOTA (2), Véronique NEAUD (3), Simone MATHOULIN-PÉLISSIER (4), Jean-Yves BLAY (2), Jean ROSENBAUM (3) (1) Institut Bergonié, Digestive Tumours Unit, Bordeaux ; (2) Centre Léon Bérard, Cytokines Laboratory, Lyon ; (3) INSERM, E362, Université Victor Segalen Bordeaux 2 ; (4) Institut Bergonié, Department of Biostatistics, Bordeaux.
SUMMARY
RÉSUMÉ
Aims — The aim of this study was to evaluate the serum pattern of cytokines evolution after surgical radiofrequency ablation (SRFA) of colorectal metastases.
Profil d’expression de cytokines après destruction peropératoire par radiofréquence de métastases hépatiques colorectales
Methods — Metastases of ten non consecutive patients were destroyed by radiofrequency ablation without concomitant resection after a complete surgical procedure including a laparotomy, a peritoneal examination, liver mobilisation and liver ultrasound. Serum levels of IL-6, TNFα, HGF, VEGF, bFGF, TGFβ1 and CRP were assessed by ELISA assays at different time points.
Serge EVRARD, Christine MENETRIER-CAUX, Cathy BIOTA, Véronique NEAUD, Simone MATHOULIN-PÉLISSIER, Jean-Yves BLAY, Jean ROSENBAUM (Gastroenterol Clin Biol 2007;31:141-145)
Buts — Étudier le profil d’évolution de plusieurs cytokines après ablathermie chirurgicale par radiofréquence de métastases d’origine colorectale.
Results — TNFα and bFGF remained undetectable. IL-6 peaked at 3 hours and remained elevated during the entire study period. HGF increased by three-fold by Day 1 then decreased until Day 7 where it was still twice its baseline level. VEGF level increased from Day 5 onward. TGFβ1 did not show significant variations. CRP was increased throughout the study.
Méthode — Les métastases de dix malades non consécutifs ont été détruites par radiofréquence sans résection parenchymateuse associée au cours d’une intervention par laparotomie incluant une exploration péritonéale, une mobilisation du foie ainsi qu’une échographie peropératoire. Les taux sériques d’IL-6, TNFD, HGF, VEGF, bFGF, TGFE1 et CRP ont été dosés par système ELISA au cours de la première semaine postopératoire.
Conclusions — In contrast with cryotherapy, SRFA does not lead to high serum TNFα suggesting a better tolerance. Nevertheless high IL-6, HGF and VEGF serum levels are characteristic of a general inflammatory stress which should be taken into account.
Résultats — TNFD et bFGF sont restés indétectables tout au long de l’étude. L’IL-6 a présenté un pic à 3 heures et est resté élevé durant toute l’étude. Le taux d’HGF a été multiplié par trois dès le premier jour puis a décru jusqu’au 7e jour où il était encore double de son taux initial. Le taux de VEGF s’est élevé à partir du 5e jour. TGFE1 n’a pas montré de variations significatives. La CRP était élevée durant toute l’étude. Conclusion — Contrairement à la cryothérapie, les radiofréquences à ciel ouvert n’induisent pas d’élévation du TNFD suggérant une meilleure tolérance. Néanmoins, les taux d’IL-6, HGF et VEGF sont caractéristiques d’un stress inflammatoire proche de celui rapporté après hépatectomie.
induce the same circulating level of cytokines without changes in liver volume. Little is known about SRFA which destroys mainly neoplastic tissue and damages only a small margin of surrounding healthy liver. Recently, Ng et al. [11] reported in the swine a systemic responses of RFA significantly less severe than those of cryotherapy but comparable with hepatectomy. The aim of this study was to evaluate the serum pattern of evolution of cytokines after SRFA of colorectal metastases in order to evaluate, in intention-totreat, the inflammatory stress secondary to this procedure in man.
Introduction Radiofrequency ablation is a local ablative technology initially designed to destroy tumours percutaneously [1]. More recently, it has been proposed as a surgical tool. For some teams, surgical radiofrequency ablation (SRFA) is indicated when the tumours are not resectable [2, 3], as a single treatment or as a complement to resection. For others [4-6], it may be applied curatively for small metastases especially when deeply located within the liver parenchyma the latter of which can therefore be spared. Hepatectomy involving from 30 to 80% resected volume [7] and portal embolization [8] increase the blood levels of a panel of cytokines involved in the liver regeneration process. On the other hand, cryoablation [9] and even simple laparotomy [10] also
Patients and methods The protocol of the present study was designed with the collaboration of the Department of Biostatistics of our institution and approved by
Reprints: S. EVRARD, Université Victor Segalen-Bordeaux 2, Institut Bergonié, 229, cours de l’Argonne, 33076 Bordeaux. E-mail : [email protected]
Supported by grants from the Ligue Nationale Contre le Cancer, Pyrénées-Atlantiques Comity, France and by the French Ministry of Health with the QUASAR program.
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Tableau I. – Patients characteristics. Données cliniques. Patient no
Age (years)
Number of Métastases (size in mm)
Total energy Delivered (J)
Duration of procédure (min)
1
69
1 (20)
78 000
2
73
5 (10, 13, 10, 10, 22)
3
54
4
Duration of follow up (months)
Follow up
135
48
Alive without cancer
157 420
255
22
Dead from cancer
3 (6, 6, 6)
58 000
360
21
Dead from cancer
55
3 (35, 40, 40)
304 560
240
+ (77)
11
Dead from cancer
5
56
1 (30)
173 670
240
+ (14)
29
Alive with cancer
6
60
6 (27, 11, 11, 40, 13, 12)
152 241
240
+ (94)
12
Dead from cancer
7
75
3 (5, 45, 25)
276 663
225
+ (50)
25
Alive with cancer
8
75
1 (30)
65 765
225
+ (31)
25
Alive with cancer
9
71
4 (25, 5, 10, 6)
123 565
195
—
24
Alive with cancer
10
67
4 (10,4,3,7)
49 345
195
+ (6)
24
Alive without cancer
Blood sampling and processing
the local Ethics committee. All patients gave written informed consent before participating in the study.
Blood samples from the patients enrolled in this study were collected through of a venous catheter before treatment (Day D-1 and D0 before intervention) and after the end of the treatment (D0+3h, D1, D2, D3, D5 and D7) in the morning to avoid nycthemeral variations. After centrifugation, serum samples were aliquoted and immediately stored at -80 °C until assay. Interleukin 6 (IL-6), tumor necrosis factor α (TNF.α), transforming growth factor β1 (TGFβ1), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and hepatocyte growth factor (HGF) serum levels were determined using specific commercial quantitative sandwich enzyme immunoassay technique (R&D Systems). The sensitivity of these assays, defined as the lowest cytokine concentration significantly different from the zero standard with a probability of 95% are 7 pg/mL, 3 pg/mL, 5 ng/mL, 40 pg/mL, 0.7 pg/mL and 4.4 pg/mL, respectively. C-reactive protein (CRP) determinations were performed on a Synchron Clinical automatized System (CX5 CE Beckman) with CRP microlatex reagent (Sobioda).
Patients Ten non consecutive patients with colorectal cancer aged 18 or over were enrolled. These patients presented with metastases for whom an indication of SRFA was established according to our previous described indications [5] by a multidisciplinary team. The World Health Organisation (WHO) performance status was equal or lower than 2. Chemotherapy was stopped at least 15 days before surgery. Patients were excluded in the event of: extrahepatic metastases, diabetes mellitus, sepsis or had a previous cancer in their medical history. Patient characteristics are described in table I. No patient received transfusion of blood cells.
Surgical procedure A median laparotomy was performed to allow complete exploration of the peritoneal cavity. A second sub-costal incision of Rio Branco was done to allow access to the liver. Palpation of the liver was completed with peroperative echography (Lynx® 3101, B-K MedicalTM, Copenhagen, Denmark). The Pringle manoeuvre (hepatic pedicle clamping) was selectively indicated to treat lesions above 30 mm in diameter or lesions in a para-vascular situation. Bile duct cooling was performed through a choledocotomy to treat a lesion situated in close contact with the portal bifurcation in patient 4. A total vascular exclusion including Pringle manoeuvre plus caval exclusion was required in patient 6 to treat a juxta-sus-hepatic vein lesion. Metastases in all patients were treated only by SRFA, without combined resection. SRFA was conducted using the Elektrotom® 106 HFTT (BerchtoldTM, Tuttligen, Germany) with an electrode needle 1.2 mm in diameter, and 10, 15 or 25 mm in length. Only the tip of the needle was active and was perfused with isotonic saline. During the high frequency application (375 kHz), the electrode saline perfusion was automatically regulated from 30 to 110 mL/h, according to the variation in tissue impedance due to its coagulation. This permanent low infusion prevented the tissues from drying and the electrodes from sticking. Power was between 40 and 60 Watts. According to the duration of cauterisation, the total energy delivered (Joules) per impact was calculated by the computer. The duration of each application and the number of applications depended on the size of the lesion added to a 1 cm margin of normal tissue.
Statistics Serum levels of TGFβ, VEGF, HGF, IL-6 and CRP are presented as box-and-whisker plots showing median value and interquartile range (IQR). Missing data (MD) were replaced by the mean of each serum level (1 MD for D-1; 1 MD for D0+3h; 2 MD for D3). The Levene test was performed to detect inhomogeneity of variance and if detected, the data were log-transformed (base 10) in order to stabilise variances (HGF, VEGF, IL-6, CRP). Then, for each cytokine, statistical analyses were performed using repeated measure one-way ANOVA. Statistical analyses were carried out using the statistics package SPSS v12.0 (SPSS Inc., Chicago, IL, USA). When comparing multiple measures, a P-value less than 0.01 was considered to be statistically significant.
Results Postoperative courses were uneventful excepted for one patient who presented a fever treated by antibiotics. At baseline, VEGF, TNF.β, and HGF concentrations were 231.8 pg/mL (range: 124.7-522.5 pg/mL), 46.2 ng/mL (range: 16.9-98 ng/mL) and 1257 pg/mL (range: 6541612 pg/mL), respectively. For IL-6, TNF. α, and bFGF, basal levels were below detection thresholds. Sera of patients were tested for their cytokines content over 7 days. TNF . α was undetectable at every time point during the follow-up. Values for bFGF
ABBREVIATIONS : SRFA IL6 HGF TNFα TGFβ VEGF bFGF CRP
Pringle manoeuvre (duration in min)
: Surgical radiofrequency ablation : Interleukin 6 : Hepatocyte growth factor : Tumor necrosis factor α : Transforming growth factor β : Vascular endothelial growth factor : basic Fibroblast growth factor : C-reactive protein
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Cytokines pattern after surgical radiofrequency ablation of liver colorectal metastases
500
8000
A
P < 0.0001
B
P < 0.0001
400
300
HGF (pg/mL)
IL6 (pg/mL)
6000
200
4000
100
2000 0 Day-1
Day 0
Day 0 + 3H
Day 1
Day 2
Day 3
Day 5
Day 7
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C
Day -1
P < 0.0001
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Day 3
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Day 7
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VEGF (pg/mL)
VEGF (pg/mL)
600
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0 Day -1
Day 0 Day 0 + 3H Day 1
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0
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Day 0 Day 0 + 3H Day 1
Day 2
Day 3
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TIME 120 250
NS
F P < 0.0001
200
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CRP (mg/L)
TGF-ß1 (ng/mL)
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100
20 50
0 Day -1 Day 0 Day 0 + 3H Day 1
Day 2
Day 3
Day 5
Day 7
0
TIME Day -1
Day 0 Day 0 + 3H Day 1
Day 2
Day 3
TIME
Fig. 1 – Variation of cytokine levels before and after SRFA treatment. The top and bottom of each box represent the 75th and 25th percentiles, respectively. The horizontal line within the boxes represents the 50th percentile (median) and the end of each “whisker” the 10th and 90th percentiles (1a); Serum levels of interleukin 6 (IL- 6) (1b); Serum levels of hepatocyte growth factor (HGF) (1e); Serum levels of vascular endothelial growth factor (VEGF) (1d); Serum levels of transforming growth factor β1 (TGF-β1) (1e); Serum levels of C reactive protein (CRP)(1f). Variation des taux de cytokines sériques avant et après traitement par radiofréquence peropératoire. Le sommet et la base de chaque boîte représentent respectivement le 75e et le 25e percentiles. La ligne horizontale contenue dans chaque boîte représente le 50e percentile (médiane) et les extrémités de chaque « moustache » les 10e et 90e percentiles (1a) ; Taux sériques d’interleukine 6 (IL-6) (1b) ; Taux sériques d’hepatocyte growth factor (1e) ; Taux sériques de vascular endothelial growth factor (1d) ; Taux sériques de transforming growth factor E1 (1e) ; Taux sériques de protéine C réactive (1f).
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were also mostly below the level of detection. Whereas IL-6 levels were below the threshold for detection before surgery, we observed a rapid increase in IL-6 serum concentration in the hours following the end of the treatment (figures 1a et 1b). The IL-6 level decreased progressively but remained detectable until the end of the follow-up, in contrast to that observed at baseline. The changes in IL-6 serum levels were highly significant (P < 0.0001).
We also found increased HGF serum levels following SRFA. This is similar to results reported by De Jong et al. [9] after cryotherapy or partial hepatectomy [10]. Classical sources of HGF in the liver are Kupffer or hepatic stellate cells. In the RFA setting, it could also originate from damaged endothelial cells [21]. The delayed increase in VEGF expression is concordant with results observed following partial hepatectomy in animals where it is known to be important for both neoangiogenesis [22, 23] and hepatocyte regeneration [24]. In the SRFA setting, VEGF expression may be under the direct control of cytokines such as IL-6 [25] or HGF [26] or may be related to the tissue hypoxia generated by RFA. We did not find significant modifications in the serum concentration of another angiogenic factor, bFGF, which is compatible with the fact that its role does not seem to be essential in the regeneration process [27].
SRFA treatment also induced a rapid increase of the HGF serum content that reached three times the baseline level at day 1 (figure 1c). HGF concentration decreased from day 2 until day 7 but remained two fold higher than the baseline value (P < 0.0001). In contrast, VEGF followed different kinetics with a late increase at day 5 that persisted at day 7 (figure 1d). These variations were also highly significant (P < 0.0001). Finally, although TGFβ1 levels tended to progressively decrease until day 3 (figure 1e), variations did not reach statistical significance. CRP levels rose from day 1 and remained very high at the end of the follow-up period (figure 1f).
In conclusion, our results in this intention-to-treat series testify that the overall procedure of SRFA appears to induce less inflammation than cryotherapy performed for the same indications. Given the pattern of serum cytokines observed, surgical use of radiofrequency induces an inflammatory stress near to these reported in the literature for parenchymal resection [10, 13, 14]. Thus, our data can not however exclude the possibility that RFA may also stimulate the growth of dormant metastases. Longitudinal studies are warranted to address this issue.
Discussion All invasive surgical procedures induces the release of a variety of cytokines leading to a general inflammatory stress. This stress response may be implicated in positive processes like the healing of the injured tissues but could also result in the stimulation of tumor cell growth. Sometimes it may lead to severe postoperative disorders like in the case of shock induced by cryotherapy.
REFERENCES
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A high CRP level, a marker of inflammation, was observed at all time points during the study. However, TNF. α, a major proinflammatory mediator remained undetectable. The lack of increase of TNF. α in serum was also observed in another series of patients undergoing SRFA [12] as well as after partial hepatectomy [13-15]. This is in contrast with liver cryotherapy which induced a TNFα increase proportional to the degree of hepatic freezing [16]. As TNFα is known to be a major mediator of shock, these data suggest that SRFA may be safer in this respect than cryotherapy for the treatment of liver tumours.
2. Abdalla EK, Vauthey JN, Ellis LM, Ellis V, Pollock R, Broglio KR, et al. Recurrence and outcomes following hepatic resection, radiofrequency ablation, and combined resection/ablation for colorectal liver metastases. Data validation in an economic evaluation of surgery for colon cancer. Ann Surg 2004;239:818-25.
3. Wood TF, Rose DM, Chung M, Allegra DP, Foshag LJ, Bilchik AJ. Radiofrequency ablation of 231 unresectable hepatic tumors: indications, limitations, and complications. Ann Surg Oncol 2000;7:593-600.
4. Elias D, Baton O, Sideris L, Matsuhisa T, Pocard M, Lasser P. Local
One of the major concerns of surgical procedures for liver metastasis is that the procedure itself may stimulate the growth of dormant metastases through the induction of the liver regeneration process. Liver regeneration is orchestrated by a complex system involving many cytokines and growth factors using both paracrine and endocrine pathways. A simplified scheme [17] suggests that hepatocytes need to be primed for replication through the consecutive action of TNF. α and IL-6. They then become responsive to mitogenic growth factors such as HGF or TGFα. In addition, VEGF also plays a major role in the regeneration of the liver vasculature, and is also involved in hepatocyte replication. On the other hand, TGFβ1 is involved in the arrest of regeneration.
recurrences after intraoperative radiofrequency ablation of liver metastases: a comparative study with anatomic and wedge resections. Ann Surg Oncol 2004;11:500-5.
5. Evrard S, Becouarn Y, Fonck M, Brunet R, Mathoulin-Pelissier S, Picot V. Surgical treatment of liver metastases by radiofrequency ablation, resection, or in combination. Eur J Surg Oncol 2004;30:399-406.
6. Evrard S, Mathoulin-Pelissier S. Controversies between surgical and percutaneous radiofrequency ablation. Eur J Surg Oncol 2006;32:3-5.
7. Masson S, Daveau M, Hiron M, Lyoumi S, Lebreton JP, Teniere P, et al. Differential regenerative response and expression of growth factors following hepatectomy of variable extent in rats. Liver 1999; 19:312-7.
We found a rapid and sustained increase in serum IL-6 in patients undergoing SRFA. IL-6 is a multifunctional cytokine produced as a part of the acute-phase response in tissues exposed to non specific insults. As already mentioned, IL-6 appears to be essential for the priming of hepatocytes as demonstrated in knockout mice for the IL-6 gene where hepatocyte DNA synthesis is impaired, leading to liver failure [18]. An increase in serum IL-6 has been previously reported in humans after partial hepatectomy [13, 19] or cryosurgery [9]. In the single published study dealing with serum cytokines after SRFA [12], IL-6 reached a 3-fold peak at 24 hours and decreased at 48 hours. In experimental liver regeneration, IL-6 induction is dependent on TNFα signalling, as demonstarted using TNF Receptor-1 knockout mice [20]. However, IL-6 supplementation can compensate defects in TNF. α . signalling [20], thus suggesting that in our patients, despite the absence of a TNF. α . serological response, the raised IL-6 concentration is a significant indicator for regeneration.
8. Uemura T, Miyazaki M, Hirai R, Matsumoto H, Ota T, Ohashi R, et al. Different expression of positive and negative regulators of hepatocyte growth in growing and shrinking hepatic lobes after portal vein branch ligation in rats. Int J Mol Med 2000;5:173-9.
9. de Jong KP, von Geusau BA, Rottier CA, Bijzet J, Limburg PC, de Vries EG, et al. Serum response of hepatocyte growth factor, insulin-like growth factor-I, interleukin-6, and acute phase proteins in patients with colorectal liver metastases treated with partial hepatectomy or cryosurgery. J Hepatol 2001;34:422-7.
10. de Jong KP, Hoedemakers RM, Fidler V, Bijzet J, Limburg PC, Peeters PM, et al. Portal and systemic serum growth factor and acutephase response after laparotomy or partial hepatectomy in patients with colorectal liver metastases: a prognostic role for C-reactive protein and hepatocyte growth factor. Scand J Gastroenterol 2004;39:1141-8.
11. Ng KK, Lam CM, Poon RT, Shek TW, To JY, Wo YH, et al. Comparison of systemic responses of radiofrequency ablation, cryotherapy, and surgical resection in a porcine liver model. Ann Surg Oncol 2004; 11:650-7.
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12. Schell SR, Wessels FJ, Abouhamze A, Moldawer LL, Copeland EM,
lacking type I tumor necrosis factor receptor. Proc Natl Acad Sci USA 1997;94:1441-6.
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21. Matsumori A, Ono K, Furukawa Y, Okada M, Sasayama S. Circula-
13. Badia JM, Ayton LC, Evans TJ, Carpenter AJ, Nawfal G, Kinderman H,
ting hepatocyte growth factor as an early marker of arterial thrombus formation. Jpn Circ J 1998;62:311-3.
et al. Systemic cytokine response to hepatic resections under total vascular exclusion. Eur J Surg 1998;164:185-90.
22. Shimizu H, Miyazaki M, Wakabayashi Y, Mitsuhashi N, Kato A,
response to hepatectomy in patients with a healthy or a diseased liver. World J Surg 2003;27:761-4.
Ito H, et al. Vascular endothelial growth factor secreted by replicating hepatocytes induces sinusoidal endothelial cell proliferation during regeneration after partial hepatectomy in rats. J Hepatol 2001; 34:683-9.
15. Schroder J, Gallati H, Kremer B. Increased serum levels of soluble tu-
23. Sato T, El-Assal ON, Ono T, Yamanoi A, Dhar DK, Nagasue N.
14. Lan AK, Luk HN, Goto S, Chen SM, Eng HL, Chen YS, et al. Stress
mor necrosis factor a-receptors in patients undergoing partial liver resection. Hepatogastroenterology 1998;45:1807-12.
Sinusoidal endothelial cell proliferation and expression of angiopoietin/Tie family in regenerating rat liver. J Hepatol 2001;34: 690-8.
16. Seifert JK, Stewart GJ, Hewitt PM, Bolton EJ, Junginger T, Morris DL. Interleukin-6 and tumor necrosis factor-alpha levels following hepatic cryotherapy: association with volume and duration of freezing. World J Surg 1999;23:1019-26.
24. LeCouter J, Moritz DR, Li B, Phillips GL, Liang XH, Gerber HP,
17. Zimmermann A. Liver regeneration: the emergence of new pathways.
25. Cohen T, Nahari D, Cerem LW, Neufeld G, Levi BZ. Interleukin 6 in-
et al. Angiogenesis-independent endothelial protection of liver: role of VEGFR-1. Science 2003;299:890-3.
Med Sci Monit 2002;8:RA53-63.
duces the expression of vascular endothelial growth factor. J Biol Chem 1996;271:736-41.
18. Cressman DE, Greenbaum LE, DeAngelis RA, Ciliberto G, Furth EE, Poli V, et al. Liver failure and defective hepatocyte regeneration in interleukin-6-deficient mice. Science 1996;274:1379-83.
26. Zhang YW, Su Y, Volpert OV, Vande Woude GF. Hepatocyte growth factor/scatter factor mediates angiogenesis through positive VEGF and negative thrombospondin 1 regulation. Proc Natl Acad Sci USA 2003;100:12718-23.
19. Chijiiwa K, Saiki S, Tanaka M. Serum interleukin-6 and hepatocyte growth factor levels in patients after hepatectomy. Hepatogastroenterology 2002;49:467-71.
27. Sturm J, Keese M, Zhang H, Bonninghoff R, Magdeburg R, Vajkoczy P,
20. Yamada Y, Kirillova I, Peschon JJ, Fausto N. Initiation of liver
et al. Liver regeneration in FGF-2-deficient mice: VEGF acts as potential functional substitute for FGF-2. Liver Int 2004;24:161-8.
growth by tumor necrosis factor: deficient liver regeneration in mice
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25,*,1$/ $57,&/(
Gastroenterol Clin Biol 2007;31:141-145
Cytokines pattern after surgical radiofrequency ablation of liver colorectal metastases Serge EVRARD (1), Christine MENETRIER-CAUX (2), Cathy BIOTA (2), Véronique NEAUD (3), Simone MATHOULIN-PÉLISSIER (4), Jean-Yves BLAY (2), Jean ROSENBAUM (3) (1) Institut Bergonié, Digestive Tumours Unit, Bordeaux ; (2) Centre Léon Bérard, Cytokines Laboratory, Lyon ; (3) INSERM, E362, Université Victor Segalen Bordeaux 2 ; (4) Institut Bergonié, Department of Biostatistics, Bordeaux.
SUMMARY
RÉSUMÉ
Aims — The aim of this study was to evaluate the serum pattern of cytokines evolution after surgical radiofrequency ablation (SRFA) of colorectal metastases.
Profil d’expression de cytokines après destruction peropératoire par radiofréquence de métastases hépatiques colorectales
Methods — Metastases of ten non consecutive patients were destroyed by radiofrequency ablation without concomitant resection after a complete surgical procedure including a laparotomy, a peritoneal examination, liver mobilisation and liver ultrasound. Serum levels of IL-6, TNFα, HGF, VEGF, bFGF, TGFβ1 and CRP were assessed by ELISA assays at different time points.
Serge EVRARD, Christine MENETRIER-CAUX, Cathy BIOTA, Véronique NEAUD, Simone MATHOULIN-PÉLISSIER, Jean-Yves BLAY, Jean ROSENBAUM (Gastroenterol Clin Biol 2007;31:141-145)
Buts — Étudier le profil d’évolution de plusieurs cytokines après ablathermie chirurgicale par radiofréquence de métastases d’origine colorectale.
Results — TNFα and bFGF remained undetectable. IL-6 peaked at 3 hours and remained elevated during the entire study period. HGF increased by three-fold by Day 1 then decreased until Day 7 where it was still twice its baseline level. VEGF level increased from Day 5 onward. TGFβ1 did not show significant variations. CRP was increased throughout the study.
Méthode — Les métastases de dix malades non consécutifs ont été détruites par radiofréquence sans résection parenchymateuse associée au cours d’une intervention par laparotomie incluant une exploration péritonéale, une mobilisation du foie ainsi qu’une échographie peropératoire. Les taux sériques d’IL-6, TNFD, HGF, VEGF, bFGF, TGFE1 et CRP ont été dosés par système ELISA au cours de la première semaine postopératoire.
Conclusions — In contrast with cryotherapy, SRFA does not lead to high serum TNFα suggesting a better tolerance. Nevertheless high IL-6, HGF and VEGF serum levels are characteristic of a general inflammatory stress which should be taken into account.
Résultats — TNFD et bFGF sont restés indétectables tout au long de l’étude. L’IL-6 a présenté un pic à 3 heures et est resté élevé durant toute l’étude. Le taux d’HGF a été multiplié par trois dès le premier jour puis a décru jusqu’au 7e jour où il était encore double de son taux initial. Le taux de VEGF s’est élevé à partir du 5e jour. TGFE1 n’a pas montré de variations significatives. La CRP était élevée durant toute l’étude. Conclusion — Contrairement à la cryothérapie, les radiofréquences à ciel ouvert n’induisent pas d’élévation du TNFD suggérant une meilleure tolérance. Néanmoins, les taux d’IL-6, HGF et VEGF sont caractéristiques d’un stress inflammatoire proche de celui rapporté après hépatectomie.
induce the same circulating level of cytokines without changes in liver volume. Little is known about SRFA which destroys mainly neoplastic tissue and damages only a small margin of surrounding healthy liver. Recently, Ng et al. [11] reported in the swine a systemic responses of RFA significantly less severe than those of cryotherapy but comparable with hepatectomy. The aim of this study was to evaluate the serum pattern of evolution of cytokines after SRFA of colorectal metastases in order to evaluate, in intention-totreat, the inflammatory stress secondary to this procedure in man.
Introduction Radiofrequency ablation is a local ablative technology initially designed to destroy tumours percutaneously [1]. More recently, it has been proposed as a surgical tool. For some teams, surgical radiofrequency ablation (SRFA) is indicated when the tumours are not resectable [2, 3], as a single treatment or as a complement to resection. For others [4-6], it may be applied curatively for small metastases especially when deeply located within the liver parenchyma the latter of which can therefore be spared. Hepatectomy involving from 30 to 80% resected volume [7] and portal embolization [8] increase the blood levels of a panel of cytokines involved in the liver regeneration process. On the other hand, cryoablation [9] and even simple laparotomy [10] also
Patients and methods The protocol of the present study was designed with the collaboration of the Department of Biostatistics of our institution and approved by
Reprints: S. EVRARD, Université Victor Segalen-Bordeaux 2, Institut Bergonié, 229, cours de l’Argonne, 33076 Bordeaux. E-mail : [email protected]
Supported by grants from the Ligue Nationale Contre le Cancer, Pyrénées-Atlantiques Comity, France and by the French Ministry of Health with the QUASAR program.
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Tableau I. – Patients characteristics. Données cliniques. Patient no
Age (years)
Number of Métastases (size in mm)
Total energy Delivered (J)
Duration of procédure (min)
1
69
1 (20)
78 000
2
73
5 (10, 13, 10, 10, 22)
3
54
4
Duration of follow up (months)
Follow up
135
48
Alive without cancer
157 420
255
22
Dead from cancer
3 (6, 6, 6)
58 000
360
21
Dead from cancer
55
3 (35, 40, 40)
304 560
240
+ (77)
11
Dead from cancer
5
56
1 (30)
173 670
240
+ (14)
29
Alive with cancer
6
60
6 (27, 11, 11, 40, 13, 12)
152 241
240
+ (94)
12
Dead from cancer
7
75
3 (5, 45, 25)
276 663
225
+ (50)
25
Alive with cancer
8
75
1 (30)
65 765
225
+ (31)
25
Alive with cancer
9
71
4 (25, 5, 10, 6)
123 565
195
—
24
Alive with cancer
10
67
4 (10,4,3,7)
49 345
195
+ (6)
24
Alive without cancer
Blood sampling and processing
the local Ethics committee. All patients gave written informed consent before participating in the study.
Blood samples from the patients enrolled in this study were collected through of a venous catheter before treatment (Day D-1 and D0 before intervention) and after the end of the treatment (D0+3h, D1, D2, D3, D5 and D7) in the morning to avoid nycthemeral variations. After centrifugation, serum samples were aliquoted and immediately stored at -80 °C until assay. Interleukin 6 (IL-6), tumor necrosis factor α (TNF.α), transforming growth factor β1 (TGFβ1), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and hepatocyte growth factor (HGF) serum levels were determined using specific commercial quantitative sandwich enzyme immunoassay technique (R&D Systems). The sensitivity of these assays, defined as the lowest cytokine concentration significantly different from the zero standard with a probability of 95% are 7 pg/mL, 3 pg/mL, 5 ng/mL, 40 pg/mL, 0.7 pg/mL and 4.4 pg/mL, respectively. C-reactive protein (CRP) determinations were performed on a Synchron Clinical automatized System (CX5 CE Beckman) with CRP microlatex reagent (Sobioda).
Patients Ten non consecutive patients with colorectal cancer aged 18 or over were enrolled. These patients presented with metastases for whom an indication of SRFA was established according to our previous described indications [5] by a multidisciplinary team. The World Health Organisation (WHO) performance status was equal or lower than 2. Chemotherapy was stopped at least 15 days before surgery. Patients were excluded in the event of: extrahepatic metastases, diabetes mellitus, sepsis or had a previous cancer in their medical history. Patient characteristics are described in table I. No patient received transfusion of blood cells.
Surgical procedure A median laparotomy was performed to allow complete exploration of the peritoneal cavity. A second sub-costal incision of Rio Branco was done to allow access to the liver. Palpation of the liver was completed with peroperative echography (Lynx® 3101, B-K MedicalTM, Copenhagen, Denmark). The Pringle manoeuvre (hepatic pedicle clamping) was selectively indicated to treat lesions above 30 mm in diameter or lesions in a para-vascular situation. Bile duct cooling was performed through a choledocotomy to treat a lesion situated in close contact with the portal bifurcation in patient 4. A total vascular exclusion including Pringle manoeuvre plus caval exclusion was required in patient 6 to treat a juxta-sus-hepatic vein lesion. Metastases in all patients were treated only by SRFA, without combined resection. SRFA was conducted using the Elektrotom® 106 HFTT (BerchtoldTM, Tuttligen, Germany) with an electrode needle 1.2 mm in diameter, and 10, 15 or 25 mm in length. Only the tip of the needle was active and was perfused with isotonic saline. During the high frequency application (375 kHz), the electrode saline perfusion was automatically regulated from 30 to 110 mL/h, according to the variation in tissue impedance due to its coagulation. This permanent low infusion prevented the tissues from drying and the electrodes from sticking. Power was between 40 and 60 Watts. According to the duration of cauterisation, the total energy delivered (Joules) per impact was calculated by the computer. The duration of each application and the number of applications depended on the size of the lesion added to a 1 cm margin of normal tissue.
Statistics Serum levels of TGFβ, VEGF, HGF, IL-6 and CRP are presented as box-and-whisker plots showing median value and interquartile range (IQR). Missing data (MD) were replaced by the mean of each serum level (1 MD for D-1; 1 MD for D0+3h; 2 MD for D3). The Levene test was performed to detect inhomogeneity of variance and if detected, the data were log-transformed (base 10) in order to stabilise variances (HGF, VEGF, IL-6, CRP). Then, for each cytokine, statistical analyses were performed using repeated measure one-way ANOVA. Statistical analyses were carried out using the statistics package SPSS v12.0 (SPSS Inc., Chicago, IL, USA). When comparing multiple measures, a P-value less than 0.01 was considered to be statistically significant.
Results Postoperative courses were uneventful excepted for one patient who presented a fever treated by antibiotics. At baseline, VEGF, TNF.β, and HGF concentrations were 231.8 pg/mL (range: 124.7-522.5 pg/mL), 46.2 ng/mL (range: 16.9-98 ng/mL) and 1257 pg/mL (range: 6541612 pg/mL), respectively. For IL-6, TNF. α, and bFGF, basal levels were below detection thresholds. Sera of patients were tested for their cytokines content over 7 days. TNF . α was undetectable at every time point during the follow-up. Values for bFGF
ABBREVIATIONS : SRFA IL6 HGF TNFα TGFβ VEGF bFGF CRP
Pringle manoeuvre (duration in min)
: Surgical radiofrequency ablation : Interleukin 6 : Hepatocyte growth factor : Tumor necrosis factor α : Transforming growth factor β : Vascular endothelial growth factor : basic Fibroblast growth factor : C-reactive protein
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Cytokines pattern after surgical radiofrequency ablation of liver colorectal metastases
500
8000
A
P < 0.0001
B
P < 0.0001
400
300
HGF (pg/mL)
IL6 (pg/mL)
6000
200
4000
100
2000 0 Day-1
Day 0
Day 0 + 3H
Day 1
Day 2
Day 3
Day 5
Day 7
TIME 800
0
C
Day -1
P < 0.0001
Day 0 Day 0 + 3H Day 1
Day 2
Day 3
Day 5
Day 7
TIME 800
D
600
400
VEGF (pg/mL)
VEGF (pg/mL)
600
200
400
200
0 Day -1
Day 0 Day 0 + 3H Day 1
Day 2
Day 3
Day 5
Day 7
TIME 140
0
E
Day -1
Day 0 Day 0 + 3H Day 1
Day 2
Day 3
Day 5
Day 7
Day 5
Day 7
TIME 120 250
NS
F P < 0.0001
200
80
CRP (mg/L)
TGF-ß1 (ng/mL)
100
60
40
150
100
20 50
0 Day -1 Day 0 Day 0 + 3H Day 1
Day 2
Day 3
Day 5
Day 7
0
TIME Day -1
Day 0 Day 0 + 3H Day 1
Day 2
Day 3
TIME
Fig. 1 – Variation of cytokine levels before and after SRFA treatment. The top and bottom of each box represent the 75th and 25th percentiles, respectively. The horizontal line within the boxes represents the 50th percentile (median) and the end of each “whisker” the 10th and 90th percentiles (1a); Serum levels of interleukin 6 (IL- 6) (1b); Serum levels of hepatocyte growth factor (HGF) (1e); Serum levels of vascular endothelial growth factor (VEGF) (1d); Serum levels of transforming growth factor β1 (TGF-β1) (1e); Serum levels of C reactive protein (CRP)(1f). Variation des taux de cytokines sériques avant et après traitement par radiofréquence peropératoire. Le sommet et la base de chaque boîte représentent respectivement le 75e et le 25e percentiles. La ligne horizontale contenue dans chaque boîte représente le 50e percentile (médiane) et les extrémités de chaque « moustache » les 10e et 90e percentiles (1a) ; Taux sériques d’interleukine 6 (IL-6) (1b) ; Taux sériques d’hepatocyte growth factor (1e) ; Taux sériques de vascular endothelial growth factor (1d) ; Taux sériques de transforming growth factor E1 (1e) ; Taux sériques de protéine C réactive (1f).
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were also mostly below the level of detection. Whereas IL-6 levels were below the threshold for detection before surgery, we observed a rapid increase in IL-6 serum concentration in the hours following the end of the treatment (figures 1a et 1b). The IL-6 level decreased progressively but remained detectable until the end of the follow-up, in contrast to that observed at baseline. The changes in IL-6 serum levels were highly significant (P < 0.0001).
We also found increased HGF serum levels following SRFA. This is similar to results reported by De Jong et al. [9] after cryotherapy or partial hepatectomy [10]. Classical sources of HGF in the liver are Kupffer or hepatic stellate cells. In the RFA setting, it could also originate from damaged endothelial cells [21]. The delayed increase in VEGF expression is concordant with results observed following partial hepatectomy in animals where it is known to be important for both neoangiogenesis [22, 23] and hepatocyte regeneration [24]. In the SRFA setting, VEGF expression may be under the direct control of cytokines such as IL-6 [25] or HGF [26] or may be related to the tissue hypoxia generated by RFA. We did not find significant modifications in the serum concentration of another angiogenic factor, bFGF, which is compatible with the fact that its role does not seem to be essential in the regeneration process [27].
SRFA treatment also induced a rapid increase of the HGF serum content that reached three times the baseline level at day 1 (figure 1c). HGF concentration decreased from day 2 until day 7 but remained two fold higher than the baseline value (P < 0.0001). In contrast, VEGF followed different kinetics with a late increase at day 5 that persisted at day 7 (figure 1d). These variations were also highly significant (P < 0.0001). Finally, although TGFβ1 levels tended to progressively decrease until day 3 (figure 1e), variations did not reach statistical significance. CRP levels rose from day 1 and remained very high at the end of the follow-up period (figure 1f).
In conclusion, our results in this intention-to-treat series testify that the overall procedure of SRFA appears to induce less inflammation than cryotherapy performed for the same indications. Given the pattern of serum cytokines observed, surgical use of radiofrequency induces an inflammatory stress near to these reported in the literature for parenchymal resection [10, 13, 14]. Thus, our data can not however exclude the possibility that RFA may also stimulate the growth of dormant metastases. Longitudinal studies are warranted to address this issue.
Discussion All invasive surgical procedures induces the release of a variety of cytokines leading to a general inflammatory stress. This stress response may be implicated in positive processes like the healing of the injured tissues but could also result in the stimulation of tumor cell growth. Sometimes it may lead to severe postoperative disorders like in the case of shock induced by cryotherapy.
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