PhD; Alfonso Gautieri

PhD Simone Vesentini
email: simone.vesentini@polimi.it

PhD Mr Simone Vesentini

PhD Alberto Redaelli

Keywords: coarse grain, molecular dynamics simulation, multi scale models, diffusion, polymeric matrices, nanofiltration Coarse grain  Molecular dynamics simulation  Multi scale models  Diffusion  Polymeric matrices  Nanofiltration

published or not. The documents may come

How to predict diffusion of medium-sized molecules in polymer matrices. From atomistic to coarse grain simulations

Introduction

Molecular dynamics (MD) simulations are a powerful tool in the material science field as they provide material's structural and dynamics details that are difficult, cost-or time-consuming to be assessed with experimental techniques. In particular, MD simulations are a valid tool for the design of polymeric membranes with tuned permeability properties. Up to now, however, the design of barrier materials is based on trial and error experimental procedures, in which a large part of the effort is spent to synthesize and characterize materials and blends which finally turn out to be unsatisfactory.

Atomistic simulation have been successfully applied in the past to obtain the diffusion coefficients of small molecules (like oxygen, carbon dioxide or water) in polymeric membranes [START_REF] Gestoso | Molecular simulation of the effect of temperature and architecture on polyethylene barrier properties[END_REF][START_REF] Chiessi | Water and polymer dynamics in chemically cross-linked hydrogels of poly(vinyl alcohol): A molecular dynamics simulation study[END_REF][START_REF] Zhang | Microstructure dependent diffusion of water-ethanol in swollen poly(vinyl alcohol): A molecular dynamics simulation study[END_REF][START_REF] Hofmann | Detailed-atomistic molecular modeling of small molecule diffusion and solution processes in polymeric membrane materials[END_REF][START_REF] Hofmann | Molecular simulation of small molecule diffusion and solution in dense amorphous polysiloxanes and polyimides[END_REF][START_REF] Tocci | A molecular simulation study on gas diffusion in a dense poly(ether-ether-ketone) membrane[END_REF][START_REF] Pavel | Molecular dynamics simulation of diffusion of O-2 and CO2 in amorphous poly(ethylene terephthalate) and related aromatic polyesters[END_REF], polymeric blends [START_REF] Fermeglia | PET/PEN blends of industrial interest as barrier materials. Part 1. Many-scale molecular modeling of PET/PEN blends[END_REF][START_REF] Pavel | Molecular dynamics simulation of diffusion of O-2 and CO2 in blends of amorphous poly(ethylene terephthalate) and related polyesters[END_REF], biopolymers [START_REF] Ionita | Diffusion of small molecules in bioartificial membranes for clinical use: molecular modelling and laboratory investigation[END_REF][START_REF] Prathab | Molecular modeling study on surface, thermal, mechanical and gas diffusion properties of chitosan[END_REF] and organic-inorganic hybrid membranes [12]. However, despite the increasing computational power available to researchers and the improvements in the MD codes, atomistic simulations are still able to handle only systems with tens or hundreds of thousands of atoms and in the nanoseconds time scale. Several phenomena of interest at the material scale, however, cover time and space scales larger than those affordable with atomistic modeling. This is the case of normal diffusion regime. The diffusion coefficient D can be directly calculated from the motion of the particles extracted during a MD simulation, in particular from the mean square displacement (MSD) of the particles, using the Einstein equation [START_REF] Hofmann | Molecular simulation of small molecule diffusion and solution in dense amorphous polysiloxanes and polyimides[END_REF]. This equation holds only in the case that the observation time (i.e., the simulation time) is large enough to allow the particles to show uncorrelated motion. This means that the MSD is linear with time, i.e. MSD ~ t n where n=1. Conversely, if n < 1 then the diffusion is in the anomalous diffusion regime. Therefore, in order to assess an accurate diffusion coefficient, MD simulations must reach the normal diffusive regime which, depending on the membrane and diffusive molecule, can be on time scales higher than few nanoseconds. This regime is therefore often difficult to reach for molecules larger than diatomic molecules, like water or benzene, hindering molecular simulations to assess an accurate diffusion coefficient [12,[START_REF] Entrialgo-Castaño | An Atomistic Modeling and Quantum Mechanical Approach to the Hydrolytic Degradation of Aliphatic Polyesters[END_REF].

Recently, the use of coarse grain (CG) modeling, in which a number of atoms is condensed into beads or interacting particles, has proven to be a suitable option to model large systems and long time scales, providing realistic results. The methods, assumptions and level of resolution greatly vary depending on the scope of the models and the properties of interests [START_REF] Tozzini | Coarse-grained models for proteins[END_REF][START_REF] Chng | Coarse-Grained Models Reveal Functional Dynamics -II. Molecular Dynamics Simulation at the Coarse-Grained Level -Theories and Biological Applications[END_REF]. CG models have been developed whit particular focus on biomolecular systems, since biomolecules are often too large and their characteristic times too long to be treated with full atomistic simulations. However, there are no theoretical impediments to the application of CG methods to polymeric materials.

In this view, of particular interest is the coarse grain force field developed by Marrink and coworkers and called MARTINI force field. This CG force field was originally developed to model the lipid bilayers forming the cellular membranes and then extended to proteins [START_REF] Marrink | Coarse grained model for semiquantitative lipid simulations[END_REF][START_REF] Marrink | The MARTINI force field: Coarse grained model for biomolecular simulations[END_REF][START_REF] Monticelli | The MARTINI coarsegrained force field: Extension to proteins[END_REF]. Unlike other CG models, which focus on accurate modeling of a particular state or a particular molecule, the philosophy of the MARTINI force field is to accurately parameterize the basic building blocks of the system (e.g., the single amino acids for proteins), thus allowing a broad spectrum of applications without the need of reparameterization.

Relying on the same philosophy, in this work we present an atomistic-informed parameterization of the MARTINI force field for the modeling of penetrants diffusion in polymeric membranes. We used atomistic simulations to calculate the interaction free energy between the basic building blocks of the system, i.e. the penetrant molecule and the polymer monomers, and then we performed CG molecular dynamics simulations to assess the diffusion behavior. As test case, we investigated the diffusion behavior of benzene in a matrix of Poly (vinyl alcohol), PVA. Benzene, an important industrial solvent and precursor in the production of drugs, plastics, synthetic rubber, and dyes, was chosen since it is a well known representative of medium-sized permeant molecules. On the other hand, the choice of PVA as the polymer matrix is based on the fact that this polymer is widely used in several fields and finds applications as membrane material due to its excellent chemical stability, film forming capacity, barrier properties and high hydrophilicity.

Methods

Coarse grain mapping

The original MARTINI mapping scheme is based on the four-to-one rule, i.e. on average four heavy (non-hydrogen) atoms are grouped into a bead or interaction center. Here we used a similar mapping, where the benzene molecule is represented by one bead and the vinyl alcohol (VA) monomers are represented by a different bead (see Fig. 1). The mass of the two types of beads are calculated as the sum of the masses of the atoms grouped into the bead.

Bonded and nonbonded interactions

The chemically bonded beads interact through a bond interaction modeled as a harmonic potential V bond (r):

0 1 ( ) ( ) 2 bond bond V r k r r  ( 1
)
where r is the distance between two bonded beads, k bond is the force constant of the bond interaction and r 0 is the equilibrium distance. In the system under investigation, only VA beads representing chemically bonded monomers along a PVA chain are subject to bond interactions.

The beads i and j that are not chemically connected interact via nonbonded interactions, which are described by a Lennard-Jones 12-6 potential:

12 6 00 ( ) 2 ij ij LJ ij rr Vr rr                   (2)
where r is the distance between the two nonbonded beads, ε ij is the energy minimum depth (the strength of the interaction) and r 0ij is the distance at the minimum of the potential.

Parameterization

Given the mapping scheme and the type of interaction, we needed to calculate one set of bonded parameters (for bonded VA-VA beads) and three sets of nonbonded parameters (VA-VA, benzene-benzene and VA-benzene). For each set we run a full atomistic simulation of the two groups involved in the interaction and we calculated the free energy of interaction between the two groups. The free energy calculations are performed using the Adaptative Biasing Force (ABF) framework [START_REF] Darve | Calculating free energies using average force[END_REF] as implemented in the NAMD code [START_REF] Darve | Calculating free energies using a scaled-force molecular dynamics algorithm[END_REF][START_REF] Phillips | Scalable molecular dynamics with NAMD[END_REF]. The atomistic MD simulations are carried out using the NAMD program and the all-atom CHARMM force field [START_REF] Mackerell | All-atom empirical potential for molecular modeling and dynamics studies of proteins[END_REF], for a simulation time of 40 ns (time step of 1 fs) at a temperature of 300 K. Nonbonded interactions are computed using a switching function between 20 and 22 Å. The free energy of interaction, computed via the ABF framework, is monitored between the two groups of atoms as a function of their center-of-mass distance, in the range 2-20 Å using windows of 0.01 Å.

Generation of the CG models

We generated three different molecular models: pure benzene, pure PVA and PVA with a small amount of benzene as penetrant molecule. All the three systems were generated in the atomistic form using the Amorphous Cell construction tool of Materials Studio 4.4 (Accelrys, Inc.). The pure benzene system contained 500 benzene molecules in a cubic periodic box with initial density of 0.88 g cm -3 . For the pure PVA system we considered 6 atactic PVA molecules (consisting of 200 repeat units) in a cubic periodic box with initial density of 1.25 g cm -3 . Finally, the third system contained 4 PVA chains (of 200 repeat units) and 12 benzene molecules in a cubic periodic box with initial density of 1.25 g cm -3 . The three atomistic systems are then converted into CG systems using the Coarse Grainer tool of Materials Studio according to the mapping scheme described above (see Fig. 2).

CG simulations

CG molecular dynamics simulations are carried using the Mesocite module and the MARTINI force field implemented in Materials Studio. Prior to the simulations, we modified the original force field including the parameters for bonded and nonbonded interactions between benzene and VA beads, as obtained from the ABF atomistic simulations. The CG systems are minimized for 1000 steps, then equilibrated for 1 ns (using a time step of 20 ps) at 300 K. Finally, production simulations are run for a simulated time of 10 ns (for pure benzene

0 N i i D D N    (4)
In this work the diffusion of all 500 benzene beads (for benzene self diffusion) and the 12 benzene beads (for benzene in PVA) was investigated during the CG simulations.

Results

The parameters of the CG force field, i.e. the bonded and nonbonded interactions between the beads, are calculated through full atomistic simulations and by applying the ABF framework.

The ABF calculations provide the free energy profile as a function of the distance between the group of atoms, as shown in Fig. 3. The free energy profile of two bonded VA monomers (Fig. 3A) is interpolated with a harmonic potential, giving the equilibrium distance and the force constant (see Table 1). On the other end, from the interaction free energy profiles between nonbonded VA monomers, benzene molecules and VA-benzene molecules (Fig. 3B-D) we obtained the three sets of r 0 (energy minimum) and ε (energy minimum depth), used to feed the Lennard-Jones 12-6 potential of the CG force field (see Table 1).

We used the CG approach to investigate three different systems: pure benzene, pure PVA and benzene molecules in a PVA matrix (see Fig. 2). The pure benzene system consisted of 500 benzene molecules, which were coarse grained into benzene beads. We measured the density of the system during the 1 ns equilibration obtaining a realistic value of 0.881±0.001 g cm -3 (where the experimental value is 0.876 g cm -3 ). After the equilibration we carried a 10 ns simulation in which we monitored the MSD of the benzene molecules (see Fig. 4A) and, by applying Eq. 3, we determined the self diffusion coefficient of benzene, obtaining a value of 2.273±0.588•10 -9 m 2 s -1 , very close to the experimental value of 2.203±0.004 •10 -9 m 2 s -1 [START_REF] Mills | Search for isotope effects in the self-diffusion of benzene and cyclohexane at 25.deg[END_REF].

In the case of pure PVA the system consisted of a periodic box with 6 chains of 200 monomers each. The coarse grained system was equilibrated for 1 ns and the density of the CG PVA box was measured, giving a value of 1.305±0.011 g cm -3 , which rely in the experimental range (1.232-1.329 g cm -3 ) [START_REF] Mark | Polymer Data Handbook[END_REF]. Finally, the third system consisted of 12 benzene molecules diffusing in a PVA matrix. The density of the coarse grained system, measured at the end of 1 ns equilibration, was 1.293±0.003, similar to that of pure PVA. The MSD of the benzene beads was monitored during a 200 ns MD simulation (see Fig. 4B) and, from the derivative of the curve, we calculated the diffusion coefficient of benzene in PVA.

We obtained a diffusion coefficient of 0.263±0.035•10 -12 m 2 s -1 which is in good agreement with the experimental value, that is 0.25•10 -12 m 2 s -1 [12]. The 200 ns simulation of the 4 nm x 4 nm x 4 nm CG box (with 812 beads representative of ≈ 6000 atoms) took 36 hours on a single CPU. The results of the simulations as well as the experimental data are shown in Table 2.

Discussion

In this work we present a multi-scale method for the parameterization of the MARTINI CG force field and its application for the calculation of the diffusivity of medium sized molecules in polymeric membranes. In the past, atomistic simulations have been already successfully applied for the calculation of diffusivity of small molecules in polymeric membranes.

Nonetheless, the computational costs can hinder the ability of MD simulations to predict such parameter. Indeed, the Einstein relation (Eq. 3) applied for the assessment of the diffusion coefficient can only be used when the simulation is in the regime of normal diffusion and this realm is reached when the slope of the function log[MSD(t)] = f [log(t)] equals 1. In the case of very small molecules, like oxygen or hydrogen, the normal diffusion regime can be reached within few nanoseconds [START_REF] Hofmann | Molecular simulation of small molecule diffusion and solution in dense amorphous polysiloxanes and polyimides[END_REF] while, in the case of medium sized molecules like benzene [12] or even water [START_REF] Entrialgo-Castaño | An Atomistic Modeling and Quantum Mechanical Approach to the Hydrolytic Degradation of Aliphatic Polyesters[END_REF], it cannot be reached within the limit of atomistic simulations. In this view, the use of CG simulations, where a number of atoms is condensed into beads or interacting particles, can be a useful approach to overcome the limitations of atomistic simulations.

In order to test the feasibility of the CG approach, we investigated the self diffusion coefficient of benzene and its diffusivity in PVA matrix using an atomistic-informed CG force field (see Fig. 1). As shown in Table 2, the coarse graining reduces the number of interacting particles by a factor of ≈10. Furthermore, since the CG interactions are much smoother compared to atomistic interactions [START_REF] Marrink | Coarse grained model for semiquantitative lipid simulations[END_REF], it is possible to use a time step of 20-40 fs, much larger than that typical of classical MD (1-2 fs). Thus, the CG approach leads to a total speedup factor of 200-400 with respect to atomistic simulations.

In this work we did not relied on the standard MARTINI bead types but rather we defined two ad hoc bead types (one for benzene molecule and one for VA monomer) and we estimated the bonded and nonbonded parameters using atomistic free energy calculations.

The predicted parameters are then used to feed the MARTINI force field. The extended force field is then used to perform CG molecular dynamics simulations of three different systems:

pure benzene, pure PVA and benzene in PVA (see Fig. 2). The coarse grain systems underwent to a 1 ns equilibration dynamics, during which they reached a stable density very close to the experimental values (Table 2), thus confirming that the outcome of the atomistic free energy calculations are reasonable.

As final validation of our approach, we run longer CG simulations in order to assess the self diffusion coefficient of benzene and the diffusion coefficient of benzene in PVA. For the pure benzene, we run a 10 ns simulations from which we estimated the diffusion coefficient. The calculated and the experimental values are shown in Table 2, and the comparison confirms that the CG model is able to predict the experimental value with good approximations. In the case of benzene diffusing in a PVA matrix, 10 ns of simulation were not enough to reach the normal diffusion regime, since the derivative of the function log[MSD(t)] = f [log(t)] was lower than 1. This result is in agreement with the observations of Pan et al. [12], which showed that MD simulations of few nanoseconds are not long enough to reach the normal diffusion regime and provide a good estimation of benzene diffusivity in PVA. For this reason, we run a longer simulation of 200 ns, in order to reach the realm of normal diffusivity, as shown in Fig. 5. Indeed, the trajectory of this long simulation permitted to obtain a diffusion coefficient very close to the experimental value (see Table 2). This result confirms the feasibility of the CG approach to reach the normal diffusion regime of medium sized molecules in polymer matrices and that our multi-scale approach is a valid method to treat this kind of problems.

The major limitation of atomistic computational techniques applied in the literature to solve diffusive problems is related to the restricted time scale and sample size which can be simulated, which are few nanoseconds and few nanometers, respectively. Thus, when the phenomena under investigation exceed these limits MD simulations fail to provide reliable values of the diffusivity. In order to overcome these limitations we developed a novel method, which combine combining atomistic and coarse grain simulations in a multi-scale paradigm, where the parameters for the meso-scale model are derived from atomistic MD simulations.

-9 -Similar techniques are increasingly applied for the study of biological problems but, to the best of our knowledge, have not been used for the investigation of diffusion problems. Here we showed that this technique can be successfully applied to investigate the diffusion of penetrant molecules in polymer matrices, reliably predicting experimental data. A similar method as used in this paper could be applied to study the diffusion of larger molecules, which require a longer time to reach the normal diffusion regime, or the study of complex polymeric materials, for which representative volumes are larger than few cubic nanometers.

Conclusions

In conclusion, the main focus of this work has been to develop and validate a novel multiscale method for the prediction of diffusivity in polymer matrices. We demonstrated that atomistic-informed CG simulations can be a valid approach to treat problems where the computational limits of classical MD simulations are too restrictive while, at the same time, strictly atomistic details are not mandatory. Thus, the multi-scale approach presented in this work extends the application range of computational modeling and provide a useful tool to investigate phenomena at the micro-scale which determine macroscopic physical properties of polymeric materials. In this view, multi-scale paradigm here discussed can further help computation aided molecular modeling to reduce the extend of the experimental trial-anderror approach during the design and investigation of new materials, thus resulting in a more cost and time efficient process.

Tables

Figure captions

2 (

 2 and pure PVA) and 200 ns (for benzene in PVA matrix). The diffusion coefficient D i of a single permeant molecule i is calculated by the Einstein relation, starting from the diffusion trajectory () i rt  which is determined during the production MD simulations: 0) r t r   represents the root mean square displacement (MSD) of the permeant molecule i averaged over all possible time origins and t represents the time. The -6computationally derived diffusion coefficient D for a given kind of permeant molecule is then obtained as average over the diffusion coefficients for N permeant molecules:

Fig. 1 Fig. 2 Fig. 3 Fig. 4

 1234 Fig. 1Overview of the CG modeling approach. Panel A shows the initial atomistic model of benzene in PVA matrix. The system is then coarse grained using the mapping scheme depicted in panel B: in the case of benzene each molecule is represented by one CG bead, while for PVA each monomer is modeled with a different bead. This leads to the CG packing model used for the subsequent molecular simulations (panel C)

Fig. 5

 5 Fig. 5 Log(MSD) vs. log(t) plots (straight lines) and linear interpolation (dashed lines) for the self diffusion of benzene (panel A) and for the diffusion of benzene in PVA (panel B) obtained from CG simulations. The plots show that in the case of pure benzene the normal diffusion regime is reached, as indicated by the slope very close to 1, already in the range from 10 -1 to 10 1 ns, while in the case of benzene in PVA (panel B) a longer simulation time is required since the normal diffusion regime is reached in the range from 10 1.7 to 10 2.3 ns (i.e., from 50 to 200 ns)

Figure 1

 1 Figure 1 Click here to download colour figure: Fig1.doc

Table 1

 1 Parameters of the CG force field, obtained from full atomistic free energy calculations. Within this approach bonded interactions (i.e., VA beads covalently connected)

	are modeled through a harmonic potential, while nonbonded interactions are approximated
	with a Lennard-Jones 12-6 potential
	Interaction	Bonded		Nonbonded
		k bond (kcal/mol/Å 2 )	r 0 (Å)	ε (kcal/mol)	r 0 (Å)
	VA -VA	40.36	2.76	1.306	4.115
	VA -Benzene	-	-	0.957	5.51
	Benzene -Benzene	-	-	0.9634	5.62

Table 2

 2 Main results of the CG simulations. The conversion of the three systems under study from atomistic to coarse grain reduces the interacting particles by a factor between 7 and 12, depending on the specific system. Despite the loss of atomistic details, the CG models feature realistic densities and are able to predict benzene diffusion coefficient very close to the experiments

	Systems	Total atoms	Total beads	Final density (g/cm 3 )	Experimental density (g/cm 3 )	Predicted D benzene (m 2 /s)	Experimental D benzene (m 2 /s)
	500 benzene molecules	6000	500	0.881	0.876	2.273•10 -9	2.203•10 -9 [23]
	6 PVA chains	8400	1200	1.305	1.23-1.32 [24]	-	-
	4 PVA chains + 12 benzene	5744	812	1.293	-	0.263•10 -12	0.25•10 -12 [12]

Acknowledgments

This research was partially supported by the Italian Institute of Technology (IIT). The authors declare no conflict of interest of any sort.

(B) (C)


C Sturesson

I Keussen

K.-G Tranberg

Prolonged chemotherapy impairs liver regeneration after portal vein occlusion -an audit of 26 patients Short title: Portal vein occlusion

Keywords: portal vein embolisation, liver, chemotherapy, regeneration

Aim: The aim of the present retrospective study was to investigate the influence of neoadjuvant chemotherapy on liver regeneration after portal vein occlusion before major hepatectomy.

Method

: Between 2003 and 2007, 26 patients underwent portal vein occlusion, of whom 22 had portal vein embolisation and 4 portal vein ligation. 15 of 23 patients with colorectal liver metastases had neoadjuvant chemotherapy.

Results: After portal vein occlusion, the ratio of the future liver remnant volume to total parenchymal liver volume (FLR%) was reduced in patients receiving neoadjuvant chemotherapy (27 ± 1 % vs 32 ± 1 %, p=0.03). A smaller future liver remnant before portal vein occlusion resulted in a greater degree of hypertrophy (r 2 =0.18, p=0.04). Patients with tumour size greater than 60 mm showed a decreased degree of hypertrophy (7 ± 1) % as compared to patients with smaller tumours (13 ± 1 %, p=0.01). There was one death shortly after portal vein embolisation. 19/26 patients were resected with zero operative mortality.

Conclusion:

Prolonged neoadjuvant chemotherapy has a small negative effect on liver regeneration induced by portal vein occlusion. Liver regeneration induced by portal vein occlusion is relatively large when tumour burden is small.

Introduction

Surgical resection is used with increasing frequency for hepatobiliary malignancies in order to provide a chance for cure. One major limitation to the extent of resection is the remnant liver volume. In patients with normal liver parenchyma, a remnant volume of >25% of initial liver volume is considered sufficient to allow resection with an acceptably low risk of postoperative liver failure [START_REF] Shoup | Volumetric analysis predicts hepatic dysfunction in patients undergoing major liver resection[END_REF].

When the future liver remnant, FLR, is considered too small, portal vein occlusion (PVO) can be used. Portal vein occlusion, which includes embolisation and ligation, is performed to increase the volume of the future liver remnant before major hepatectomy. After occlusion, the liver is allowed to regenerate during approximately three-four weeks, followed by resection [START_REF] Madoff | Portal vein embolization in preparation for major hepatic resection. Evolution of a new standard of care[END_REF][START_REF] Ribero | Portal vein embolization before major hepatectomy and its effects on regeneration, resectability and outcome[END_REF].

For colorectal liver metastases, there is growing evidence showing that neoadjuvant chemotherapy before hepatectomy prolongs survival [START_REF] Nordlinger | Final results of the EORTC Intergroup randomized phase III study 40983 [EPOC] evaluating the benefit of peri-operative FOLFOX4 chemotherapy for patients with potentially resectable colorectal cancer liver metastases[END_REF][START_REF] Tanaka | Role of neoadjuvant chemotherapy in the treatment of multiple colorectal metastases to the liver[END_REF]. Preoperative chemotherapy can be used to downsize tumors to become resectable [START_REF] Adam | Rescue surgery for unresectable colorectal liver metastases downstaged by chemotherapy. A model to predict long-term survival[END_REF]. In the following, chemotherapy administered before hepatectomy will be referred to as neoadjuvant chemotherapy.

Extensive neoadjuvant chemotherapy for colorectal cancer may induce hepatic damage [START_REF] Rubbia-Brandt | Severe hepatic sinusoidal obstruction associated with oxaliplatin-based chemotherapy in patients with metastatic colorectal cancer[END_REF] and increase morbidity [START_REF] Karoui | Influence of preoperative chemotherapy on the risk of major hepatectomy for colorectal liver metastases[END_REF] and even short-term mortality [9] after hepatectomy.

The lower limit of liver remnant volume to ensure safe resection after chemotherapy has not been established, but a minimal liver remnant of 30-40% has been proposed [START_REF] Azoulay | Resection of nonresectable liver metastases from colorectal cancer after percutaneous portal vein embolization[END_REF]. If preoperative portal vein occlusion is required, neoadjuvant chemotherapy is also frequently used to minimize the risk of tumour progression during the time interval between occlusion and resection. Previous clinical studies have shown no or only modest impact by neoadjuvant chemotherapy on liver regeneration after portal vein occlusion [START_REF] Ribero | Portal vein embolization before major hepatectomy and its effects on regeneration, resectability and outcome[END_REF][START_REF] Goéré | Chemotherapy does not impair hypertrophy of the left liver after right portal vein obstruction[END_REF][START_REF] Beal | Portal vein embolisation prior to hepatic resection for colorectal liver metastases and the effect of periprocedure chemotherapy[END_REF]. On the other hand,

M A N U S C R I P T A C C E P T E D

ACCEPTED MANUSCRIPT 4 adjuvant chemotherapy has been shown to cause suppression and delay of regeneration after partial hepatectomy in rats [START_REF] Tanaka | Effect of doxorubicin on liver reneneration and host survival after two-thirds hepatectomy in rats[END_REF].

The aim of the present retrospective study was to evaluate the significance of preoperative chemotherapy and tumour size on regenerated volume after portal vein occlusion.

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

5

Methods

Patients

Between June 2003 and July 2007, the records of 26 consecutive patients who underwent portal vein occlusion in preparation for extended right hepatectomy, i.e., a planned resection of Couinaud's segments 4-8, with or without resection of the caudate lobe (segment 1), were reviewed. Portal vein occlusion was considered to be indicated when the future liver remnant volume to total parenchymal liver volume (FLR%) was <20% in patients without chemotherapy and <30%, preferably <35%, in patients with chemotherapy.

Patient characteristics are shown in Table 1. No patient had any known liver parenchymal disease and all had normal values of bilirubin and international normalized ratio (INR) prior to intervention. Indications for neoadjuvant chemotherapy in patients with colorectal liver metastases were multiple (>5) liver tumours in 13 patients and downsizing in 2.

Volumetric assessment

Liver volumetry was made in 19 cases by the MeVis institute (Bremen, Germany) using computed tomography images with 1 mm slices, giving the volume of the left lateral segment, total liver volume and tumour volume. In seven patients volumetry was manually assessed at our institution using computed tomography or magnetic resonance images. The contours of the left lateral segment, the whole liver and liver tumours were traced manually on each 5 mm slice, the areas were automatically calculated and volumes were obtained by summation of areas and multiplying by the distance between slices. Liver parenchymal volume was calculated by subtracting tumour volume from total liver volume. The future liver remnant percentage, FLR%, was calculated as the quotient between the volume of future liver

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 6
remnant and liver parenchymal volume. Initial evaluation was made using the first volume measurements after portal vein occlusion. Final evaluation employed the last measurements before the decision to resect or not. The degree of hypertrophy, DH, was defined as the difference in FLR% before portal vein occlusion and after initial or final evaluation.

Portal vein embolisation

Right portal vein embolisation was performed on 22 patients, including 3 patients who, in addition, received embolisation to the medial segment (segment 4). Patients received a single dose of intravenous antibiotic prophylaxis before the procedure. In general or local anaesthesia with i v sedation, percutaneous access to the portal system was gained under sonographic and/or fluoroscopic guidance with a 20 G needle. The approach was ipsilateral in 20 patients, and contralateral in 2, including one where puncture from the right side failed.

Using the Seldinger technique, a 5 Fr introducer system was inserted after portography with a

Portal vein ligation

Right portal vein ligation was performed in 4 patients, 2 of whom had neoadjuvant chemotherapy. These patients were scheduled for right hepatectomy (segments 5-8) but had unexpected intraoperative findings of tumour extension necessitating an extended right hepatectomy (segments 4-8 ±1) for tumour clearance. Ligation was performed by dissecting the right portal branch at the hilum and dividing it with a vascular stapler. During the study time period an additional four patients underwent portal vein ligation as part of a two-stage operation. As these procedures included resection of a substantial portion of the left lateral segment (segments 2-3), these patients were excluded from the present analysis.

Tumour size and response

In patients with colorectal liver metastases, tumour response evaluation was based on liver imaging before and after PVO according to Response Evaluation Criteria in Solid Tumors (RECIST) [START_REF] Therasse | New guidelines to evaluate the response to treatment in solid tumors[END_REF]. Patients received 9 (4-29) (median, range) cycles of neoadjuvant chemotherapy during 23 weeks before portal vein occlusion. Chemotherapy was stopped 3 [START_REF] Shoup | Volumetric analysis predicts hepatic dysfunction in patients undergoing major liver resection[END_REF][START_REF] Madoff | Portal vein embolization in preparation for major hepatic resection. Evolution of a new standard of care[END_REF][START_REF] Ribero | Portal vein embolization before major hepatectomy and its effects on regeneration, resectability and outcome[END_REF][START_REF] Nordlinger | Final results of the EORTC Intergroup randomized phase III study 40983 [EPOC] evaluating the benefit of peri-operative FOLFOX4 chemotherapy for patients with potentially resectable colorectal cancer liver metastases[END_REF][START_REF] Tanaka | Role of neoadjuvant chemotherapy in the treatment of multiple colorectal metastases to the liver[END_REF][START_REF] Adam | Rescue surgery for unresectable colorectal liver metastases downstaged by chemotherapy. A model to predict long-term survival[END_REF][START_REF] Rubbia-Brandt | Severe hepatic sinusoidal obstruction associated with oxaliplatin-based chemotherapy in patients with metastatic colorectal cancer[END_REF][START_REF] Karoui | Influence of preoperative chemotherapy on the risk of major hepatectomy for colorectal liver metastases[END_REF](9)[START_REF] Azoulay | Resection of nonresectable liver metastases from colorectal cancer after percutaneous portal vein embolization[END_REF][START_REF] Goéré | Chemotherapy does not impair hypertrophy of the left liver after right portal vein obstruction[END_REF] weeks before the first portal vein occlusion. It was restarted after 2 (0-8) weeks, and 1.5 (0-5) cycles were given during 2.5 (0-13) weeks before CT-based decision of surgery or palliative treatment, or 2 (1-3) weeks before the second portal vein occlusion. The interval between the second portal vein occlusion and chemotherapy was 2 (1-4) weeks and a further 3 (2-7) cycles were given during 4 (1-15) weeks before final CT. Chemotherapy was administered in the time interval between portal vein occlusion and final volume evaluation in all of the 15 patients receiving neoadjuvant chemotherapy in addition to one patient without prior chemotherapy in order to halt tumour progression after PVO.

Chemotherapy

Liver resection

Hepatectomy was performed using standard technique. In brief, the liver was mobilized through a bilateral subcostal incision with upwards midline extension.

Intraoperative ultrasound was used in all patients. The right portal vein and right hepatic artery were divided extrahepatically. Parenchymal transection was made using an ultrasound aspiration dissector (CUSA ® , Valleylab Inc, Boulder, CO) with selective use of Pringle's manoeuvre.

Complications

In-hospital postoperative complications were recorded. Hepatic failure was defined as a bilirubin value > 50 µmol/l or an INR >1.7 at postoperative day 5 [START_REF] Balzan | The "50-50 criteria" on postoperative day 5. An accurate predictor of liver failure and death after hepatectomy[END_REF].

Results

Procedures

Portal vein embolisation was technically successful in 22 of 23 patients. In one patient two attempts failed to provide access to the portal system, and portal vein ligation was subsequently performed. There was one death occurring the day after embolisation. The patient, a 66 year-old male, had received extensive neoadjuvant chemotherapy for downstaging of liver metastases of colorectal origin prior to embolisation. Autopsy revealed circulatory collapse as cause of death, with no evidence of direct treatment-related causes. No other complications were noted and the median hospital stay after the procedure was 1 day, (range 1-4 days).

Portal vein ligation was performed on 4 patients, including the one patient in whom the embolisation failed. No complications occurred. The median hospital stay was 7 days (range 6-13 days).

Efficacy

The effect on liver volume after PVO was first evaluated after a median time of 22 days after occlusion (range 6-34 days). Ten patients with inadequate hypertrophy (see below) showed radiological signs of patent segmental branches of the right portal vein, which were judged accessible to repeat portal vein embolisation. These patients were subjected to a second right portal vein embolisation. Two patients with inadequate regeneration and an obliterated right portal venous system underwent a second portal vein embolisation to

Influence of chemotherapy

Before PVO, there was no significant difference in FLR% between groups with and without chemotherapy (18 ± 1 % and 20 ± 1 %, respectively; p=0.11). After PVO, FLR% was lower in patients with (27 ± 1 %) than in patients without chemotherapy (32 ± 2 %) (p=0.03, Fig. 1). Adequate hypertrophy, as judged by radiological evaluation, was obtained after 59 (median; range 6-157) days in the chemotherapy group, and after 30 (median; range days in the group without chemotherapy. The future liver remnant volume increased from 268 ± 22 cm 3 to 393 ± 24 cm 3 in patients with chemotherapy, and from 337 ± 23 cm 3 to 490 ± 35 cm 3 in patients without chemotherapy.

Influence of tumour size and future liver remnant volume before occlusion

There was a negative correlation between FLR% before portal vein occlusion and the degree of hypertrophy, i.e., the degree of hypertrophy was larger the smaller the FLR% before portal vein occlusion (r 2 =0,18, p=0.04; Fig. 2). FLR% before portal vein occlusion was related to tumour size (r 2 =0.22, p=0.02; Fig. 3), i.e., the larger the tumour the larger the FLR% before portal vein occlusion. For instance, patients with tumours larger than 60 mm had a larger FLR% before PVO than patients with tumours less than 60 mm (21 ± 1 % vs 17 ± 1 %; p=0.03). As compared to patients with tumours larger than 60 mm, patients with tumours less than 60 mm had a greater degree of hypertrophy (13 ± 1 % vs 7 ± 1 %; p=0.01; Fig. 4).

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In patients with colorectal cancer metastases tumour size did not differ between patients with and without chemotherapy, neither before nor after portal vein occlusion (p>0.05 in both situations).

Rate of resection

The total number of resected patients was 19 out of 26. Four patients were excluded without surgical exploration due to death after portal vein embolisation (n=1), bad general condition (n=1), and inadequate growth of future liver remnant (n=2). Three patients were excluded at exploration because of unexpected peritoneal carcinomatosis (n=1) and unanticipated massive tumour extension in the liver precluding resection (n=2). Of these 7 patients, 4 had had chemotherapy and 3 had not.

Operative outcome

Seventeen patients underwent extended right hepatectomy (segments 4-8) with or without resection of segment 1, together with an atypical resection in the left lateral segment in four patients. Another two patients had a right hepatectomy (segments 5-8), because of intraoperative findings of a more limited liver tumour distribution than expected. Pathological examination revealed 15 cases of R0 resection (resection line free of tumour), and 4 cases of R1 resection. After resection, there were no deaths within 30 days of surgery. In-hospital complications are shown in Table 2. Median hospital stay was 10 days (range 7-28 days) in the neoadjuvant chemotherapy group and 11 days (range 8-18 days) in the group without chemotherapy, respectively.

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Discussion

Suppressed liver regeneration after chemotherapy

In the present study neoadjuvant chemotherapy led to less hypertrophy of the future liver remnant. This contrasts with previous studies that have failed to show an effect on liver regeneration by concomitant chemotherapy for colorectal liver metastases [START_REF] Ribero | Portal vein embolization before major hepatectomy and its effects on regeneration, resectability and outcome[END_REF][START_REF] Tanaka | Remnant liver regeneration after two-stage hepatectomy for multiple bilobar colorectal metastases[END_REF][START_REF] Giraudo | Preoperative contralateral portal vein embolization before major hepatic resection is a safe and efficient procedure: a large single institution experience[END_REF], although the extent of treatment was not accounted for in the latter 2 studies. In addition, chemotherapy between embolisation and surgery has been suggested not to influence the regenerating capacity of the liver [START_REF] Goéré | Chemotherapy does not impair hypertrophy of the left liver after right portal vein obstruction[END_REF]18]. It is possible that the prolonged chemotherapy used in the present study, where the number of chemotherapy cycles pre PVO was, for instance, double the number the cycles reported in the study by Ribero et al [START_REF] Ribero | Portal vein embolization before major hepatectomy and its effects on regeneration, resectability and outcome[END_REF], explains the difference in outcome.

Rationale for portal vein occlusion

It has been shown that a FLR% less than 20% and/or a degree of hypertrophy less than 5% is associated with an increased morbidity [START_REF] Ribero | Portal vein embolization before major hepatectomy and its effects on regeneration, resectability and outcome[END_REF]. In the present study, no resected patient had a FLR% less than 20% after portal vein occlusion.

Both embolisation and ligation was used for portal vein occlusion in the present study. Results from previous clinical investigations indicate that the two methods are equal in inducing hypertrophy of the future liver remnant [START_REF] Bouzari | Hypertrophy of the future liver remnant volume following portal vein occlusion. Portal vein embolization vs portal ligation[END_REF][START_REF] Aussilhou | Right portal vein ligation is as efficient as portal embolization to induce hypertrophy of the left liver remnant[END_REF], although one study has shown ligation to be inferior to embolisation [START_REF] Broering | Portal vein embolization vs. portal vein ligation for induction of hypertrophy of the future liver remnant[END_REF].

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Influence of tumour size on regeneration

In livers with normal parenchyma and without tumours, the left lateral segment constitutes less than 20% of total liver volume in 75% of patients [START_REF] Abdalla | Total and segmental liver volume variations: implications for liver surgery[END_REF]. Therefore, in absence of compensatory hypertrophy by tumour growth, preoperative portal vein occlusion is indicated in many patients planned for an extended right hepatectomy (segments 4-8 ± 1). We found that large tumours before occlusion resulted in less hypertrophy of the future liver remnant. Since the FLR% before occlusion was larger for patients with large tumours, one explanation could be that the process of hypertrophy had already begun in patients with larger tumours. This process could be analogous to the reduced liver regeneration after hepatectomy in patients who have undergone preoperative portal vein embolisation [START_REF] Nagino | Liver regeneration after major hepatectomy for biliary cancer[END_REF]. The negative correlation between FLR% before PVO and degree of hypertrophy, as shown in Fig. 2, has been described previously [START_REF] Imamura | Preoperative portal vein embolization: an audit of 84 patients[END_REF], but we are unaware of previous results connecting tumour size to the degree of hypertrophy (Figs. 34). One previous study on mostly hepatocellular carcinomas failed to shown any association between hypertrophy and tumour size [START_REF] Nanashima | Parameters associated with changes in liver volume in patients undergoing portal vein embolization[END_REF].

Morbidity and mortality

Death after portal vein embolisation has not been previously reported. The diseased patient had been treated with chemotherapy (FOLFOX, followed by FOLFIRI and FOLFIRI + cetuximab) for 29 months (51 cycles) due to colorectal metastases confined to the liver.

Eventually, this resulted in downstaging of the disease and portal vein embolisation was performed. Post-mortem examination could not reveal any cause of death directly related to the embolisation procedure. No other complications were noted after embolisation. Portal vein embolisation is generally considered safe with a morbidity rate of approximately 10%, mainly consisting of transient abdominal pain and fever [START_REF] Madoff | Portal vein embolization in preparation for major hepatic resection. Evolution of a new standard of care[END_REF].

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15 Chemotherapy induces hepatic injury [START_REF] Rubbia-Brandt | Severe hepatic sinusoidal obstruction associated with oxaliplatin-based chemotherapy in patients with metastatic colorectal cancer[END_REF] and some [START_REF] Karoui | Influence of preoperative chemotherapy on the risk of major hepatectomy for colorectal liver metastases[END_REF]9] but not all [START_REF] Welsh | Safe liver resection following chemotherapy for colorectal metastases is a matter of timing[END_REF] studies suggest that this augments the morbidity and short-term mortality after hepatectomy. The effect of combining preoperative chemotherapy and portal vein occlusion on morbidity after hepatectomy has not yet been specifically addressed. It appears that the addition of chemotherapy to portal vein occlusion did not increase the rate of complications after hepatectomy (Table 2).

Repeat portal vein embolisation

In comparison to previously reported results, we had a high proportion of patients which we considered would gain from repeated portal vein embolisation. Twelve of 25 patients were embolised twice. The decision was based on inadequate hypertrophy coupled with radiological signs of patent segmental branches, as displayed on follow-up computed tomography. These branches were considered recanalised as the portograms obtained at the end of the first embolisation in all cases revealed complete stasis of right portal branches.

After the repeat procedure, the hypertrophy was equal to what was seen in patients embolised once. Our standard technique was right portal embolisation since this has been shown to give a similar volume increase of the left lateral segment as right portal vein embolisation with inclusion of segment 4 branches [START_REF] Capussotti | Extension of right portal vein embolization to segment IV portal branches[END_REF]. We used polyvinyl particles only, which has been reported to provide efficient and durable portal vein occlusion

Conclusions

In conclusion, the present study has shown that prolonged neoadjuvant chemotherapy decreases liver regeneration after portal vein occlusion. Liver regeneration after portal vein occlusion is relatively large when the FLR% before embolisation is small and when liver tumour burden is small.

5

  Fr Omniflush catheter (Cordis, Johnson & Johnson) in the main trunk of the portal vein to delineate the portal system. Selective catheterization of the target segmental branches of the portal vein was made using Sidewinder or Cobra catheters (Cordis, Johnson & Johnson). Polyvinyl particles 250-750 µm in size (Contour, Boston Scientific) were used for embolisation until cessation of flow. After occlusion of all segmental branches, a final venogram was obtained with the flush catheter in the main portal vein to evaluate the completeness of embolisation. If needed, additional embolisation was performed until satisfactory results were achieved. At the end of the procedure, the percutaneous tract was embolised with gelatine sponge (Spongostan-Standard, Cook Europe). After portal vein embolisation, repeat volumetry based on computed tomography was scheduled after approximately 3 weeks. If recanalisation of segmental portal branches to the right liver was M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 7 found, and the liver regeneration was judged insufficient, a second portal embolisation was attempted.

8 FOLFOX

 8 Chemotherapy was administered to 15 of 23 patients, all with colorectal liver metastases, prior to portal vein occlusion. The regimens, the type of which was decided by the referring oncologist, included oxaliplatin in combination with 5-fluorouracil (FOLFOX) in 8 patients. One patient received irinotecan in combination with 5-FU (FOLFIRI), one patient was treated with FOLFOX in combination with bevacuzimab and one patient had FOLFIRI in combination with cetuximab. Some patients was given two lines of chemotherapy, including M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT followed by FOLFIRI (2 patients), FOLFOX followed by FOLFIRI+cetuximab (1 patient) and capecitabine in combination with oxaliplatin followed by FOLFOX (1 patient).

  Data are expressed as means ±SEM unless stated otherwise. Mann-Whitney U-test or Wilcoxon test was used to compare continuous data between groups. Fisher's exact test was used for 2x2 categorical data. All tests were two-sided. A p value less than 0.05 was considered statistically significant.M A N U S C R I P T A C C E P T E DACCEPTED MANUSCRIPT 10

segment 4 .

 4 Thus, 12 of 25 patients were embolised twice. Three of 11 patients without chemotherapy received 2 embolisations as compared to 9 of 14 patients with neoadjuvant chemotherapy. In the group of patients who underwent ligation or were embolised once, open M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 11 segmental branches were occasionally seen, but as regeneration was considered sufficient to allow a safe resection, no further embolisation attempts were made.

  [28], although other investigators add microcoils deployed in segmental portal branches [29]. Recent experimental results indicate that reversible portal vein occlusion also induces liver hypertrophy [30], but the present results indicate that the goal should be complete and permanent occlusion of the target portal branches to maximize regeneration. M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 16

Fig. 1 Fig. 2 Fig. 3 Fig. 4

 1234 Figure legends


E S Van Der Zaag
email: e.vanderzaag@gelre.nl

N Kooij

M J Van De Vijver

W A Bemelman

H M Peters

C J Buskens

Diagnosing occult tumour

Keywords: Occult tumour cells, Sentinel lymph node, colorectal cancer

come

INTRODUCTION

Sentinel lymph node (SN) mapping is an increasingly popular technique to improve staging accuracy for patients with colorectal cancer by identifying nodes with the highest likelihood to harbour metastatic disease. This technique has shown promising results for colon carcinomas with SN mapping accurately predicting nodal status in 80-100%. An advantage of SN mapping is the potential to use this technique for a more accurate and costeffective pathologic assessment of lymph node status. The additional stepwise sectioning combined with immunohistochemical analysis of the SN has been demonstrated to detect occult tumour cells (OTC) in up to 40% of histologically negative patients with colorectal cancer , possibly identifying a patient group that may benefit from adjuvant systemic therapy.

However, previously reported results are difficult to interpret because most studies publishing SN results include immunohistochemical analysis of the SN only. This might result in flattering negative predicting values and sensitivity rates. Moreover, most studies published so far have not subdivided the finding of OTC into micrometastases and isolated tumour cells (ITC) on the basis of their dimensions as recommended by the American Joint Committee on Cancer (AJCC) staging manual. [START_REF] Green | AJCC cancer staging manual[END_REF] This possibly results in an overestimation of the number of upstaged patients and makes the clinical significance of the detected OTC difficult to evaluate. The widespread use of SN immunohistochemical analysis in patients with breast cancer has demonstrated that stage migration due to SN mapping harbours the danger of potential misleading effects and artefacts in prognostication. [START_REF] Singletary | Revision of the American Joint Committee on Cancer staging system for breast cancer[END_REF] This highlights the need to differentiate correctly between clinically relevant (micro)metastases (0.2-2 mm)(pN1mi+) and immunohistochemically detected isolated tumour cells without prognostic significance (<0.2mm)(pN0itc+).

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This is particularly so since the main purpose of SN mapping in patients with colorectal cancer is to establish a more accurate staging of patients at risk of recurrence due to lymfogenic spread and who could benefit from adjuvant chemotherapy.

For clinical application with therapeutical consequences, an immunohistochemical marker for OTC has to be both highly sensitive (i.e. detect the majority of tumour cells), and specific (i.e. absence of false positive staining of cells). Since most studies use different antibodies without a gold standard and uniform definition of OTC, the antibody of first choice for the detection of clinically relevant micrometastases in patients with colorectal cancer is still difficult to determine on the basis of published data.

We performed this study to evaluate the real diagnostic accuracy of upstaging N0 patients with colorectal cancer by SN-procedure with immunohistochemical analysis. Therefore the incidence of OTC in SNs was compared to the presence of these cells in all histologically negative lymph nodes of these patients. Two experienced pathologists independently assessed the presence of lymph node metastases. The detection of OTC was analysed by three different antibodies to determine the sensitivity and specificity of these antibodies. In addition, the presence of OTC was correlated to clinicopathological parameters.

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5

PATIENTS AND METHODS

Patients

Between November 2006 and July 2007, an ex vivo SN procedure was performed in 100 patients with colorectal carcinoma who were operated on with curative intent. Exclusion criteria for SN mapping were invasion of other organs (T4 carcinomas), or two simultaneous colorectal carcinomas. Patients with locally advanced rectal cancer who underwent neoadjuvant chemoradiotherapy were excluded because of stage migration before surgical treatment. Of the 100 patients 42 patients were shown to have macrometastases in one or more lymph nodes. The remaining 58 patients were histologically staged as N0 (Dukes A or B), and they comprise the population of the current study. All patients with colon cancer underwent an oncological resection, including the mesocolon of the vascular trunk. Patients with rectal carcinoma underwent total mesorectal excision (TME) after preoperative shortcourse radiotherapy comprising five fractions of five Gray. In addition, the mesenteric lymph nodes of 6 patients were included who underwent colonic resection for benign disease (e.g. Crohn's disease or diverticulosis) as a negative control group.

The study was done in accordance with the guidelines of the local ethics committee.

Sentinel lymph node mapping

In this study, the ex vivo lymph mapping was used since only this technique can also be performed easily in patients with rectal carcinomas. The ex vivo technique has been demonstrated to be as accurate as the in vivo technique in identifying SNs [START_REF] Teuch | Sentinel node mapping in coloncarcinoma : invivo versus ex-vivo approach[END_REF][START_REF] Wood | Validation of lymphatic mapping in colorectal cancer: in vivo, ex vivo, and laparoscopic techniques[END_REF], and it facilitates standardised, uniform specimen processing and assessment, recognising that the principal limitation of this technique is its inability to detect a small percentage (1-10%) of cases with aberrant lymphatic drainage. [START_REF] Kelder | The sentinel node procedure in colon carcinoma: a multi-centre study in The Netherlands[END_REF][START_REF] Fitzgerald | Ex vivo sentinel lymph node biopsy in colorectal cancer: a feasibility study[END_REF][START_REF] Bilchik | Prognostic impact of micrometastases in colon cancer: interim results of a prospective multicenter trial[END_REF] After standard resection, 0.5-2 ml (depending on the volume of the tumour) patent blue V (Guerbet, Gorinchem, The

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Netherlands) was injected around the tumour with the colonic specimen left intact. [START_REF] Van Der Zaag | Improving staging accuracy in colon and rectal cancer by sentinel lymph node mapping: a comparative study[END_REF] For colonic carcinomas, the mesocolon was inspected and the first one to four blue lymph nodes were identified as SNs and either dissected or marked with a suture. For rectal carcinomas the specimen was sent to the department of pathology immediately after resection in fresh state.

The identification of blue nodes was performed at the department of pathology immediatly, to keep the circumferential resection margin intact.

Immunohistochemical staining

The surgical resection specimens were analysed at the pathology department using a standardised protocol. All lymph nodes (SNs and non-SNs) were collected in separate boxes and marked according to location, then cut in two with both sides stained with hematoxylin and eosin and evaluated for tumour involvement. Of the 58 histologically N0 patients, serial sectioning was performed at 500μm intervals of all lymph nodes from formalin-fixed and paraffin-embedded archival tissue blocks. Three serial sections at three separate levels were immunohistochemically stained with three different monoclonal antibodies: the anti-epithelial cell antibody Ber-EP4 (DAKO, The Netherlands), was combined with two anti-cytokeratin antibodies: the anti-CK20 antibody, with its expression limited to gastrointestinal epithelial cells (Euro Diagnostica, Arnhem, The Netherlands), and the anticytokeratin marker Cam5.2 directed against cytokeratin 7 and 8 expressed in all epithelial cells (Becton and Dickinson, Alphen aan den Rijn, The Netherlands). Appropriate positive and negative controls were added on each automated run, to confirm the sensitivity and specificity of the antibodies (sections of colonic carcinoma tissue served as positive controls; negative controls were obtained by omitting the primary antibody). The staining procedures have been described in detail previously. [START_REF] Van Der Zaag | Improving staging accuracy in colon and rectal cancer by sentinel lymph node mapping: a comparative study[END_REF]

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7

Microscopic evaluation and definitions

All slides were reviewed by two independent experienced pathologists who were unaware of the clinical data. The type of involvement (single tumour cells or clusters), topography (lymph-angio invasion and/or lymph sinuses and/ or parenchyma) and extension of the lesion was assessed in each case (Figure 1).

Occult tumour cells were classified as either an ITC (diameter < 0.2 mm) or a micrometastasis (diameter between 0.2 mm and 2 mm). Patients were then restaged according to the AJCC classification. [START_REF] Green | AJCC cancer staging manual[END_REF] OTC comprised of micrometastases (0.2-2 mm)(pN1mi+) and ITC (<0.2mm)(pN0itc+). In case of disagreement, the slides were re-evaluated in a consensus meeting. False-positive non-neoplastic haematopoeitic cells (e.g. reticular cells and plasma cells which can also show staining for cytokeratins), were discriminated from ITCs on the basis of histopathologic features.

Statistical analyses

The following definitions were used for calculations (Figure 2). Identification rate is the number of patients with one or more SN identified (b) divided by the total number of procedures (a) x 100%. Sensitivity is the number of patients with an immunohistochemically positive SN (c) divided by the total number of patients with detected OTC (c+e) x 100%.

Negative SNs were considered false negative if one of the other regional lymph nodes (non-SNs) demonstrated micrometastases or ITCs (e). The accuracy of the immunohistochemical SN analysis suggests a conformity of the SN status and the regional nodal status, i.e. the total number of patients with an immunohistochemically positive SN (c) plus the number of patients with a true-negative SN (f)/ the number of patients with an identified SN (b) x 100%.

RESULTS

Restaging patients by detection of occult tumour cells

Three serial sections of all 908 lymph nodes from 58 patients with N0 carcinomas were examined with three different antibodies (mean 15.7 lymph nodes per patient, range 6-42). In 27 patients none of the lymph nodes had metastases after immunohistochemical staining and in 8 patients one or more lymph nodes demonstrated false positive staining unanimously recognised by both pathologists. There was consensus on 6 patients with micrometastases and 9 patients with ITCs; overall agreement 86% (50 of 58 patients). For the remaining 8 patients there was discrepancy in the diagnosis by the two pathologists. In these 8 patients, one pathologist diagnosed one micrometastasis and 10 ITCs in 11 lymph nodes, whereas the other pathologist classified 2 lesions as micrometastases, 6 lesions as ITC and 3 lesions as false positive staining. It was noted that the discrepancy between micrometastases and ITCs was mainly due to differences in measuring multiple foci. According to the AJCC 6 th edition of TNM classification of malignant tumours, clusters of tumour cells are considered as one focus if the nests are separated by a few cells, and when in doubt the lower category should be selected to prevent upstaging. In a consensus meeting the following was agreed upon: 52 lymph nodes with OTC were found in 19 of the 58 patients (33%); seven patients had micrometastases (12% pN1mi+) and 12 patients had ITCs (21% pN0itc+)(Table 1).

The presence of OTC (micrometastases and ITCs combined) was not related to any clinicopathological characteristic (Table 2). Although micrometastases and ITCs were found more frequently in tumours infiltrating the serosa and in tumours larger than 5cm, this was not statistically significant. Since micrometastases are thought to represent a different level of clinical significance, a correlation between pN1(mi+) versus pN0 (itc+/-) and clinicopathological characteristics was also analysed. The presence of micrometastases was

Sensitivity and specificity of three different antibodies

No epithelial cells with atypical nuclear features characteristic of carcinoma cells were identified in lymph nodes from six control patients operated upon for benign disease, although occasional staining for keratin was observed in stellate cells located in the interfollicular regions of the nodes consistent with staining of reticulum cells. However, this pattern was easily distinguishable from the staining of carcinoma cells.

Each micrometastasis was detected with all three antibodies, as were 39 of 45 ITC-positive lymph nodes. All ITCs were detected with BerEP4. Six OTC were not detected with CK20 and two were missed with Cam5.2. It was noted that the intensity of staining with Cam5.2 and CK20 was related to differentiation of the primary tumour, and the undetected ITCs were found in lymph nodes of patients with a dedifferentiated tumour.

In 18% of the patients false-positive staining was detected with BerEP4 (e.g. reticulocytes, fibroblasts and mastcells) and this antibody yielded the most discordant classifications of ITCs and false-positive stained cells between the two pathologists. False-positive staining was less frequently encountered with Cam5.2 (although there was occasional staining of dendritic cells) and was hardly seen with CK20 immunohistochemical staining. These results would make Cam5.2 the antibody of first choice since it demonstrated the most consistent staining with high sensitivity and acceptable specificity.

Diagnostic value of SN mapping

The SN (median two SNs, range 1-4) was found in 53 of the 58 N0 patients (identification rate 91%). Of the five patients in which the SN could not be identified, two patients demonstrated OTC in lymph nodes; these cells were all ITC . The remaining 53 patients were

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of 88% (15/17) and a negative predictive value of 94% (34/36)(Figure 2). OTC were found in 18 of the 105 SNs (17%), compared to 34 of 803 non-SNs (4%); this difference was statistically significant (p<0.001). Because all seven micrometastases were found in the SNs exclusively, the accuracy of the SN procedure in upstaging patients is 100%.

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DISCUSSION

Value of SN procedure in refining staging of colorectal patients

Our study emphasises the potential value of SN procedure for optimising staging of patients with colorectal cancer. The examination of 908 lymph nodes by stepwise sections and immunohistochemical analysis revealed OTC in 33% of histologically N0 patients (12% micrometastases and 21% ITCs). OTC were predominantly found in SNs with an overall sensitivity of SN mapping for OTC of 88%.

A large number of studies already addressed the value of SN mapping for colorectal carcinomas (Table 3). In reviewing the literature, most studies demonstrate identification rates and accuracy rates varying from 80 to 100%, and conclude that this technique can predict lymph node status with good accuracy rates. However, in these studies the percentage of histologically N0 patients is often more than 50% which makes the presented accuracy rates difficult to interpret. [1-7, 9-17, 19-23] This is merely because accuracy rates up to 90% may be accompanied by sensitivity rates and false negative rates of 50%. [START_REF] Bembenek | Sentinel lymph node biopsy in colon cancer: a prospective multicenter trial[END_REF][START_REF] Thomas | Use of sentinel node mapping for cancer of the colon: 'to map or not to map[END_REF][START_REF] Fitzgerald | Ex vivo sentinel lymph node biopsy in colorectal cancer: a feasibility study[END_REF][START_REF] Bilchik | Prognostic impact of micrometastases in colon cancer: interim results of a prospective multicenter trial[END_REF][START_REF] Bertagnolli | Sentinel node staging of resectable colon cancer: results of a multicenter study[END_REF][START_REF] Faerden | Sentinel node mapping does not improve staging of lymph node metastasis in colonic cancer[END_REF] For clinical use the SN procedure should be both highly sensitive and specific. Evaluating upstaging percentages in studies lacking immunohistochemically analysis of non-SNs harbours the danger of an underestimation of sensitivity rates and the presentation of flattering negative predictive values. The primary role of SN procedure in patients with colorectal cancer is to increase the accuracy of staging N0 patients by identifying and analysing those nodes with the highest likelihood to harbour metastatic disease in a cost-effective manner. Therefore, it is of great importance that OTC are predominantly found in SNs. We demonstrate that the incidence of OTC is more than four times higher in SNs compared to non-SNs. Our results are in agreement with two previous studies that showed SNs to have the highest probability of harbouring OTC. [START_REF] Bembenek | Detection of lymph node micrometastases and isolated tumor cells I sentinel and nonsentinel lymph nodes of colon cancer patients[END_REF][START_REF] Turner | Frequency and nature of cytokeratinpositive cells in sentinel and nonsentinel lymph nodes[END_REF]

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Classification and detection of occult tumour cells

However, our study also emphasises that the classification of OTC remains difficult. Reliable and reproducible discrimination between micrometastases (pN1) and ITCs (pN0) is an essential component of adjuvant treatment planning. In analysing discrepant diagnoses for clinically relevant micrometastases between the two pathologists, we observed that the most challenging issue was whether multiple tumour clusters should be measured as one. A useful guideline in the AJCC system is that when controversy exists, the lower category should be selected. This recommendation was used during the consensus meeting. Another problem was the distinction between ITCs and false positively staining (non-tumour) cells. Although this has no impact on treatment recommendations, the inter-observer variation highlights the need for well defined antibodies. [START_REF] Redston | Analysis of micrometastatic disease in sentinel lymph nodes from resectable colon cancer: results of cancer and leukaemia group B trial 80001[END_REF] Our data suggest that Cam5.2 would be the antibody of first choice for the detection of OTC. The anticytokeratin-marker BerEP-4 is more sensitive but has an almost 20% false positive rate with high inter-observer variation. The use of the highly specific anti-CK20 with its expression limited to gastrointestinal epithelial cells can not be recommended due to its loss of expression in dedifferentiated tumours, resulting in low specificity.

True upstaging rate of SN mapping and predictive value

Comparing the incidence of clinically relevant upstaging by SN mapping with immunohistochemical analysis to literature is difficult. Before the revised TNM classification was introduced in 2003 micrometastases were not distinguished from ITCs. [START_REF] Redston | Analysis of micrometastatic disease in sentinel lymph nodes from resectable colon cancer: results of cancer and leukaemia group B trial 80001[END_REF] Only three studies discriminated micrometastatic disease from ITCs, [START_REF] Bembenek | Sentinel lymph node biopsy in colon cancer: a prospective multicenter trial[END_REF][START_REF] Thomas | Use of sentinel node mapping for cancer of the colon: 'to map or not to map[END_REF][START_REF] Bilchik | Prognostic impact of micrometastases in colon cancer: interim results of a prospective multicenter trial[END_REF] with low incidence of micrometastatic disease varying from 5 to 10%. Other studies reporting higher upstaging rates all included ITCs in their findings. [START_REF] Van Schaik | Ex vivo sentinel lymph node "mapping" in colorectal cancer[END_REF][START_REF] Codignola | Is there any role for sentinel node mapping in colorectal cancer staging? Personal experience and review of the literature[END_REF][START_REF] Cox | Sentinel lymph node evaluation is technically feasible and may improve staging in colorectal cancer[END_REF][START_REF] Smith | Sentinel nodes are identifiable in formalinfixed specimens after surgeon-performed ex vivo sentinel lymph node mapping in colorectal cancer[END_REF][START_REF] Bembenek | Detection of lymph node micrometastases and isolated tumor cells I sentinel and nonsentinel lymph nodes of colon cancer patients[END_REF][START_REF] Turner | Frequency and nature of cytokeratinpositive cells in sentinel and nonsentinel lymph nodes[END_REF] This variation in definition of micrometastases makes it difficult to reach well-founded conclusions on the role of OTC and

Conclusion

Occult tumour cells are predominantly found in the SN with a true upstaging rate of 12% in patients with pN0 colorectal cancer. For immunohistochemical staining Cam5.2 seems the antibody of choice with an overall agreement of pathologist of 88% in staging patients. The SN procedure has the potential to refine the staging system for patients with colorectal cancer. Further survival analyses should be awaited to reveal the real prognostic relevance of these findings.

  M A N U S C R I P T A C C E P T E D ARTICLE IN PRESS 8 All statistical analyses were performed using the Statistical Software Package version 14.0 (SPSS INC., Chicago, IL, USA). Associations between clinicopathological features and the presence of micrometastases were analysed using Student's t-test (continuous variables) or Chi-squared test (categorical variables). P-values of 0.05 or less were considered statistically significant. M A N U S C R I P T A C C E P T E D ARTICLE IN PRESS

9

 9

  to tumour infiltration depth, differentiation grade, and extent of tumour spread, but a significant relation with lymphangio-invasion could be demonstrated (p<0.001).

  controversial prognostic significance of OTC. So far, only two studies have analysed the prognostic significance of micrometastatic disease in SNs with a decreased disease-specific survival in the pN1(mi+) group.[START_REF] Codignola | Is there any role for sentinel node mapping in colorectal cancer staging? Personal experience and review of the literature[END_REF][START_REF] Braat | Sentinel node detection after preoperative short-course radiotherapy in rectal carcinoma is not reliable[END_REF] All other studies commenting on prognostic significance of OTC did not perform SN mapping.[33] Most studies failed to demonstrate a relation between the presence of OTC and tumour recurrence or survival. Messerini et al. demonstrated that the inclusion of ITCs should probably be regarded as a confounding factor in these studies.[33] Although our study was not designed to comment on the prognostic significance of OTC, the relation between lymphangio-invasion of tumour cells and micrometastases is suggestive of clinical relevance. Especially since this relation could not be demonstrated between lymphangio-invasion and ITCs.

  , Wicherts DA, Hobbelink MG, Borel Rinkes IH, Schipper ME, Van der Zee JA, Van Hillegersberg R. Sentinel lymph node mapping in colon cancer: current status. Ann Surg Oncol 2007; 14:1070-80. 33. Messerini L, Cianchi F, Cortesini C, Comin CE. Incidence and prognostic significance of occult tumor cells in lymph nodes from patients with stage IIA colorectal carcinoma. Hum Pathol 2006; 37:1259-67.

Figure 1 Figure 2 Flowchart.

 12 Figure 1

Table 1

 1 Classification of immunohistochemical staining results by two independent pathologists of 58 histologically negative patients with colorectal cancer.

					Pathologist 1
			Micrometastasis Isolated tumour	False positive	Absent
				cells	staining
		Micrometastasis	6	1	0	0
	Pathologist 2	cells Isolated tumour	1	9	4	0
		False positive staining	0	5	8	3
		Absent	0	0	2	27
	Consensus		7	12	10	29

Table 2

 2 The presence of occult tumour cells correlated to tumour characteristics of 58 patients

	Occult tumour cells

Table 3

 3 Predictive value of blue dye SN mapping in patients with colorectal cancer including immunohistochemical analyses (comparison of existing literature until may 2008; excluding duplicate series and princeps studies with updated publications).

		N	No LNs	N0	Identification	Accuracy	False	Sensitivity	Upstaging	ITC	Antibody
	Study		(mean)	(%)	(%)	(%)	neg	(%)	(%)	a
							(%)
				Immunohistochemical analysis of SN only
	Matter 1	52	30 c	63	92	-	25 d	44 d	23	N	CK11/CEA /Ca19-9
	Kelder 2	69	11	73	97	96	30 d	89 d, f	18	N	CK7/8/18
	Bembenek 3	315	20	69	85	86	46	54	5/16 g	Y	MNF16
	Stojadinovic 4	162	18	65	98	90 d	31 d	69 d, f	11	N	AE1/AE3 Cam5.2
	Van Schaik 5	44	10.5	52	94	100	0	100	30	N	LU-5
	Bianchi 6	22	22 c	73	100	95	17	83	13	N	CK?
	Thomas 7	69	16	62	93	78	54	46 f	5/-e, g	Y	CEA/ CK
	Smith 8	17	16	41 e	94	100	0	100	20 d	N	MNF116
	Saha 9	500	15	50 d	98	96	10	90	26	N	AE1/AE3
	Codignola 10	56	21	59	100	89 e, f	11 d	86 e, f	38 d	N	Cam5.2
	Braat 11	91	7.7 e	62 e	90 e	90 e	26 e	74 e, f	12 e	N	CK7/8
	Cox 12	17	18	82	100	100	0	100 f	29	N	AE1/AE3
	Smith 13	40	17	56	98	95 e	12	88 f	29 d	N	Cam5.2
	Bell 14	58 b	30	57	97	84 e	16 d	64 e	4	N	CK
	Paramo 15	55	12	67 e	82	98	3 d	93 e	11 d	N	Cam5.2
	Fitzgerald 16	26	15	87 e	88	91 d	9 d	33 e	10	N	Cam5.2
	Bendavid 17	20	-	55	90	-	-	-	25	N	AE1/AE3
	Joosten 18	50	14 c	48	70	66 e	60	40	13	N	LU-5/ CK19
	Bilchik 19	141	17	73	99	91	39	61 e	8/22 g	Y	CK
	Bertagnolli 20	72	17	66	92	80	54	46	-	N	AE1/AE3 CEA
				Immunohistochemical analysis of all lymph nodes
	Faerden 21	199	13 c	68	93	85	47	53	30	N	Cam5.2
	Yagci 22	47	19	57	98	91 e	20	80	15	N	AE1/AE3 / CEA
	Bembenek 23	55	26 c	70	85	-	4	96	39	N	MNF 116
	Turner 24	51	14 c	55 e	100	88 e	26 e	74 e	29	N	AE1/AE3
	a Discrimination between micrometastasis and isolated tumour cells (N=no, Y=yes)
	b Specimens
	c Median
	d Discrepancy between reported value and calculated value (reported values are mentioned)

e Calculated value f Immunohistochemistry results of SN included in sensitivity g % micrometastases (true upstaging)/ % isolated tumour cells

evaluated for the calculation of the diagnostic value of the SN. Overall, OTC were found in

Acknowledgements

The authors greatly acknowledge J. van Benthem and dr E. Weltevreden from the Gelre Hospital Department of Pathology for excellent technical assistance in staining and reviewing all immunohistochemically stained slides. Dr. W. Bouma is greatly acknowledged for supporting this study and critically reading the manuscript.

Conflict of interest statement:

All authors disclose any financial and personal relationships with other people or organisations that could inappropriately influence our work.

Role of the funding source:

All authors state there is no role of funding sources.


W Van Gijn

P Krijnen

V E P P Lemmens

M Den Dulk

H Putter

MD Cornelis J H Van De Velde
email: c.j.h.van_de_velde@lumc.nl

Quality assurance in

come

Introduction

In the last two decades, treatment of rectal cancer has been substantially improved by the introduction of the Total Mesorectal Excision (TME) surgical technique in combination with preoperative treatment. In the Netherlands, TME surgery was implemented in a structural way within the framework of the TME trial (1996-1999) [START_REF] Kapiteijn | Preoperative radiotherapy combined with total mesorectal excision for resectable rectal cancer[END_REF] . This trial was performed to study the effects of preoperative radiotherapy in patients who underwent TME surgery. All participating surgeons were trained in the TME technique by workshops and videotapes. At least five procedures of each participating surgeon were supervised by an instructor surgeon. Pathologists were trained to examine the specimens according to the protocol of Quirke et al. regarding the circumferential resection margin (CRM), lymph nodes and dissection plane [START_REF] Quirke | Local recurrence of rectal adenocarcinoma due to inadequate surgical resection. Histopathological study of lateral tumour spread and surgical excision[END_REF] . This is of importance for adequate staging and adjuvant treatment as well as for essential feedback to the surgeons. The TME trial resulted in a five-year local recurrence rate of 5.6% with and 10.9% without preoperative radiotherapy and a five-year overall survival of 64% in both groups [START_REF] Peeters | The TME trial after a median follow-up of 6 years: increased local control but no survival benefit in irradiated patients with resectable rectal carcinoma[END_REF] .

The transition from conventional surgery to a quality-controlled multidisciplinary treatment regimen was not limited to the trial population. For all rectal cancer patients treated in the Comprehensive Cancer Centres South and West in the Netherlands survival increased substantially. Before the TME trial (1990-1995), five-year overall survival after rectal cancer was 56%, during the trial (1996-1999) 62%, and after the TME trial (2000-2002) 65% [START_REF] Den Dulk | Improved overall survival for patients with rectal cancer since 1990: the effects of TME surgery and pre-operative radiotherapy[END_REF] . The majority of patients in this study did not participate in the TME trial proving the lasting positive effects which quality assurance in surgical oncology can have.

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Nevertheless, not only the operative treatment of rectal cancer improved. For instance, postoperative chemotherapy was introduced in the treatment schedule of colon cancer in the late 1990s. While there are similarities between colon and rectal cancer, there are also striking differences in treatment and tumour characteristics. For example, local recurrence is an important complication for rectal cancer patients, whereas it is less an issue for colon cancer. Traditionally, survival after rectal cancer was inferior compared to survival after colon cancer [START_REF] Gatta | Survival of colorectal cancer patients in Europe during the period 1978-1989[END_REF] . Although there were improvements in the treatment of colon cancer, surgery for colon cancer is not standardised or quality controlled as it is for rectal cancer. The aim of this study is to examine the effects of quality assurance measures on survival for patients treated for rectal cancer compared to survival of patients treated for colon cancer before, during and after the TME trial.

Patients and methods

Patients

The Comprehensive Cancer Centres South and West register all newly diagnosed malignancies in the southern and western part of the Netherlands with a population of approximately 4 million inhabitants. Trained registration clerks extract primary and follow-up data from hospital records while survival data are synchronized with the municipal personal records databases. The regional cancer registries have shown to have a coverage exceeding 95% [START_REF] Schouten | Completeness of cancer registration in Limburg, The Netherlands[END_REF] . We selected all patients with primary invasive colon or rectal cancer diagnosed between 1990 and 2005. All patients have a complete follow-up until 1 January 2008. Patients with clinical or pathological distant metastases (TNM stage IV) were excluded as well as patients that did not undergo surgery. Patients were

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classified in three groups depending on their year of diagnosis relative to the inclusion period of the TME trial: 1990-1995 (pre-trial period), 1996-1999 (trial period) and 2000-2005 (post-trial period). Rectosigmoid tumours, including proximal rectal cancers and distal sigmoid cancers, were categorised as a separate group, as these tumours form a heterogeneous group with respect to treatment.

Statistical analysis

Data were analysed using SPSS software (SPSS 16.0 for Windows; SPSS Inc., Chicago, IL). Univariate comparisons between the three study periods were performed by ANOVA for age and by Chi-square test for categorical variables. Five-year survival and median follow-up time were calculated with the Kaplan Meier method. Log rank tests were used to calculate significance. In a multivariate analysis, a Cox proportional hazards model for survival was used to calculate Hazard Ratio's adjusted for age, gender and tumour stage.

Interaction was tested to compare improvements in survival between colon cancer and rectal cancer. Two-sided p-values ≤ 0.05 were considered statistically significant.

Results

Patient characteristics

A total of 23 078 patients were included in the study: 15 266 colon cancer patients, 1973 rectosigmoid cancer patients and 5839 rectal cancer patients. Patient characteristics are shown in table 1. The median follow-up is 15.13 years (range 12-18 yr) for the pre-trial period, 9.93 years (range 8-12 yr) for the trial period and 4.87 years (range 2-8 yr) for the post-trial period. For colon and rectal cancer, a clear increase in patient numbers is

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noticeable when comparing the first time period (1990-1995) with the last period (2000-2005). Rectal cancer patients were significantly younger than colon cancer patients (66.6 yrs vs 69.9 yrs, p< 0.01). Female / male ratio changed to a more equal distribution for colon cancer while for rectal cancer, the proportion of males increased over time. For rectal cancer, there is an evident switch from postoperative to preoperative radiotherapy while for colon cancer patients there is an increase in the use of chemotherapy. The use of both radiotherapy and chemotherapy in the 'rectosigmoid' group shows the mix of colon and rectal cancer present in this category.

Survival

In table 2, overall five-year survival is given per tumour stage and period of diagnosis. In the pre-trial period, uncorrected five-year survival was 56.2% for colon cancer and 56.1% for rectal cancer whereas in the post-trial period both colon and rectal cancer improved significantly to 59.1% for colon cancer and 64.8% for rectal cancer. Notwithstanding the improvements for colon and rectosigmoid cancer, the greatest and most significant survival rise in all stages was made for rectal cancer. In a Cox proportional hazards model adjusting for age, gender and tumour stage, rectal cancer had a significant lower survival in the pre-trial period (1990-1995) compared to colon cancer: HR 1.248, 95% confidence interval 1.17-1.33, P<0.01 (Table 3, figure 1). However, in the post-trial period (2000-2005), survival after rectal cancer was similar to colon cancer: HR 0.987, 95% confidence interval 0.91-1.07, n.s. (Table 4, figure 1). After testing of interaction, the magnitude of improvement showed significant greater for rectal cancer than for colon cancer (P<0.01) which is visualized in figure 1.

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Discussion

In the past 15 years, survival improved markedly for both colon and rectal cancer.

Remarkably, the substantial survival backlog of rectal cancer compared to colon cancer has been completely caught up through the introduction of standardised TME surgery combined with preoperative radiotherapy. In our opinion, this is an example of the lasting effects that structural surgical training and quality assurance can have on survival outcome. Similar results were found for training and quality assurance measures within the Dutch gastric cancer D1-D2 trial (1986-1989) which have led to lasting improvements in surgical quality in the region of Comprehensive Cancer Centre West [START_REF] Krijnen | Improved survival after resectable non-cardia gastric cancer in The Netherlands: the importance of surgical training and quality control[END_REF] .

Quality assurance can be summarized as "the complete set of systematic actions that is required to achieve a treatment result that meets a certain standard" 8 . Compared to other fields such as chemotherapy and radiotherapy, quality assurance in surgical oncology is relatively new. For a long time, surgery was thought to have too much unexpected variation to be feasible for standardisation and quality control. However, the conception of surgery steadily shifted from being a non-definable craftsmanship to a transparent and well-defined skill. Nowadays many quality assurance programs have been successfully enrolled in surgical oncology with encouraging results, in particular for rectal cancer. The succeeding Eurocare studies match with the European efforts to improve rectal cancer care. Between 1978 and 1989, five-year relative survival in Europe was 43% (95% CI: 42-44) for rectal cancer and 47% (95% CI: 46-48) for colon cancer [START_REF] Gatta | Survival of colorectal cancer patients in Europe during the period 1978-1989[END_REF] . Between 1995 and 1999, European five-year relative survival for rectal cancer (53.6%) was similar to colon

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cancer (54.3%) 9 . While for both rectal and colon cancer survival increased, rectal cancer treatment caught up from a survival backlog compared to colon cancer. Although our study lacks data on cost-effectiveness, it is plausible that surgical quality assurance is a cost-effective instrument to improve quality of care. In the Nordic countries, rectal cancer care is monitored by national audit registrations that provide casemix-adjusted feedback to all hospitals performing rectal cancer surgery. All these audit projects are successful in improving nationwide quality of care 10 . The Norwegian Rectal Cancer Project includes more than 99% of patients operated for rectal cancer and showed to be very cost-effective with the costs of every saved life being less than €700 11;12 . Compared to most chemotherapy regimes these costs are extraordinary. For instance, for colon cancer patients, every life year gained with fluorouracil, levamisole and folinic acid costs more than € 50.000 13 .

Study limitations

It might be possible that more emergency surgery is performed for (obstructive) colon cancer than for rectal cancer. This could have an effect on short-term survival because emergency surgery is associated with higher post-operative mortality. Unfortunately, we lacked data on emergency status of primary surgery, making it impossible to further analyse or correct for urgency.

In the multivariate Cox regression model, we did not analyse the 'rectosigmoid' group. Because of the uncertain mix of colon and rectal cancer patients we decided to leave this group out of consideration.

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Future improvements of surgical quality

In a recent study of West et al, only 32% of the resections for colon cancer were performed in the appropriate surgical plane (mesocolic plane) whilst in the recently published MRC CR07 rectal cancer study the percentage of resections in the appropriate surgical plane (mesorectal plane) was 52% 14;15 . Though in both study populations no quality assurance measures were taken, their results clearly show the improvements that can be made in surgical oncology and this especially applies to colon cancer. Although our study shows that positive effects of quality assurance within a clinical trial reaches much further than the trial population alone, a continuous nationwide audit program such as the Nordic countries provides the best data for adequate casemix-adjusted feedback and quality control. Therefore, nationwide rectal cancer audit programs are set up in several European countries (Norway, Sweden, Denmark, UK and Belgium). In the Netherlands, the Dutch Surgical Colorectal Audit started in January 1 st 2009 and in the first 7 months 3200 patients were included 16 . Despite the positive results of quality control initiatives from clinical trials and audit programs on a national level, there remain striking differences in treatment outcome in Europe. For colorectal cancer, the Eurocare IV study showed five-year relative survival varying between 32% and 64% between European registrations 9 . The conclusion is that a European audit registration is needed to ameliorate the treatment of cancer even further and decrease variation in the quality of not only rectal cancer care but all solid malignancies. Urged by these arguments, the European Society of Surgical Oncology (ESSO) initiated an international, multidisciplinary, outcome-based quality improvement program which is fully embraced by the European CanCer Organisation (ECCO).

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In this study, quality assurance in surgical oncology has shown to be an excellent, instrument to raise quality of care and decrease outcome variation. A European audit could advance future improvements and spread these to every cancer patient in Europe.

The ECCO has recognised the importance of quality assurance and has created a framework to develop a European audit. All information about this project can be found on the webpage: www.canceraudit.eu.

Table 1 :

 1 Patient Characteristics by period of diagnosis.

	M A N U S C R I P T

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