L M Brown

An Interpretation of the Haasen-Kelly Effect

Keywords: dislocations, aluminium, nickel user supplied): flow stress

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1. Introduction: A simplified description of the work-hardened state envisages three idealised types of dislocation sources: (1) segments in small-angle boundaries which are mainly found in the 'carpets' or regions roughly parallel to glide planes, but also in the incidental dislocation boundary (IDB) structure within the geometrically necessary boundary structure (GDB) encasing slip bands (Kuhlmann-Wilsdorf and Hansen 1992); (2) primary screw dislocation dipoles which limit slip in a direction perpendicular to the Burgers vector; (3) primary edge dislocation dipoles which limit slip parallel to it, as sketched by [START_REF] Hirsch | Chapter 5 in Physics of Metals 2[END_REF]. The dipole arrays act as staging posts for dislocations, exchanging with the mobile segments to produce ever finer and more numerous dipoles, but allowing the mobile segments to penetrate the array with a certain probability. The arrays are thus both sources and sinks for mobile dislocations. Edge dipoles are the most numerous sources.

In thinking about the dipole arrays, it is important to recognise1 that the local resistance they pose to primary dislocations is not proportional to the square root of their dislocation density, nor even to their dipole density, but varies from dipole to dipole depending only on the height 2 of each dipole. The dipoles are spaced about ten times further apart than their average height. Because the dipole arrays consist of a distribution of heights, the arrays are 'statistically deformable', meaning that they have a mixture of weak and strong points. For screw arrays, there is a minimum height of dipole which can survive without crossslipping to extinction. For edge arrays, the smallest dipoles transform to faulted dipoles, which can act as sources of deformation twinning (Steeds 1967, Niewcas andSaada, 2002). As the applied stress increases, the maximum height of stable dipole diminishes, until at the onset of Stage III hardening, there are no screw dipoles remaining in the crystal, only edge dipoles Within the dipole arrays there is opportunity to exchange partners. In the case of the edge dipoles, screw dislocations connecting one dipole with another can move to transfer partners. In general, higher (coarser) dipoles have more energy per unit length than finer (narrower) ones, and because of nonlinear effects, interstitial dipoles (half-planes pointing inwards) have greater energy than vacancy dipoles. As deformation proceeds, the dipole array thus not only becomes finer, it tends to contain more narrow vacancy dipoles than interstitial ones. Niewcas (2002) presents beautiful detailed observations of the dipole debris produced by the monotonic deformation of Cu. Although the screw dislocations are not retained in the thin foils, the distribution of edge dislocation dipoles is broadly consistent with this simplified description. [START_REF] Haasen | [END_REF] Effect: When their classic paper on yield phenomena in face-centred cubic single crystals was published, what was at issue was whether or not the small yield points observed after ageing deformed crystals were due to the formation of Cottrell atmospheres at dislocations. Haasen and Kelly showed that the mere act of unloading the crystals produces a small but measurable yield point. Fig 1 shows a schematic diagram, redrawn from their paper. The crystals must be unloaded by more than 50% to produce the full effect. The effect is observed at temperatures too low for any deformationinduced defect to migrate, but it is enhanced at room temperature. The presence of impurities seems to reduce it. Upon reloading after unloading, the size of the yield point as defined in Fig. 1 is approximately proportional to the applied stress above the level at the end of stage I of the hardening curve. However, the effect is very small, the yield point at the lowest temperature amounting to about 1.5% of the applied

The

A simple interpretation:

The stress required to split an edge dislocation dipole is given by Brown and Nabarro (2004) as

( ) ( )( ) ( )         ∆ ± =         - - + ± - ≈ A A A G C h b σ σ σ ν π ν ν γ ν π µ σ 1 1 2 1 1 6 1 1 8 2 2 3 .
(1)

Here, µ is the shear modulus, b the magnitude of the Burgers vector, h is the dipole height, υ is Poisson's ratio, and G γ is the Grüneisen constant, about equal to two for most materials, and a measure of non-linear effects. The term outside the bracket is the usual splitting stress in linear elasticity, as derived for example by [START_REF] Kelly | Crystallography and Crystal Defects[END_REF]. Under such a stress, linear elasticity shows that a dipole can be flipped from one stable configuration to another and split entirely, each dislocation then becoming mobile, free to travel separately to another obstacle. For the dipole to act as a source of dislocations, the splitting stress equals the applied stress, A σ . The term inside the bracket is an estimate of the correction due to non-linear elasticity. It increases the flipping stress but decreases the splitting stress for interstitial dipoles, and does the opposite for vacancy dipoles.

One can imagine that compressive regions are elastically harder than tensile ones, so it becomes more difficult to force dislocation half-planes over one another when they point toward each other, but easier when they point away from each other.

Brown and Nabarro also show that screw dislocations connecting dipoles of opposite sign are subject to an effective stress tending to convert interstitial dipoles into vacancy ones. The magnitude of the stress is about 10% of the stress required to split the dipoles, if the dipoles are spaced about ten times their height. But the stress required to split the dipoles is equal to the current level of the work-hardened flow stress, so the screws cannot respond to the small conversion forces unless the crystal is unloaded.

One has here a possible basis for an interpretation of the Haasen-Kelly effect: upon unloading, screw dislocations can move so as to convert interstitial dipoles into vacancy ones, thereby increasing slightly the stress required to split them so as to release the dislocations. The magnitude of the effect is just proportional to the flow stress. The process requires slip alone, no diffusion is necessary.

Equation (1) predicts a surprisingly large effect. The fractional change in splitting stress is predicted to be about 13% if Poisson's ratio3 is taken to be about 1/3. The experimental value is much less than this. In order to achieve agreement between theory and experiment, a slightly more complicated analysis is required.

A more realistic interpretation:

According to [START_REF] Nabarro | [END_REF], the action of the mobile screw dislocations is to convert dipole arrays with equal sizes of vacancy and interstitial dipoles into arrays with smaller vacancy dipoles and larger interstitial ones, whilst conserving both the number of dipoles and total vacancy/interstitial content of the array. Only if the array connects with external surfaces can material be extruded or intruded from the slip bands. This happens in persistent slip bands in cyclic plasticity, but in unidirectional deformation most slip bands do not connect with outer surfaces. The unloading impact on interstitial and vacancy dipoles of equal size is to create smaller vacancy dipoles, harder to split on reloading, as well as larger interstitial dipoles, easier to split on reloading. Not all dipoles can be modified in this way, only some of the edge dipoles -those connected to screw dislocations -whereas the screw dipoles (if any) and the links of dislocations in the small-angle boundaries are likely to be unmodified on unloading. Fig. 2 shows schematically what can happen. On the left is sketched ( ) σ S n , the number of dipoles per unit area which can act as a source of mobile dislocations under the stress σ . Under load, all the dipoles which can be split at stresses lower than A σ are wiped out, whereas the stronger dipoles remain. On unloading, the step-function discontinuity in dipole density is modified: some narrower vacancy dipoles are created, which increases the density above the step, but also some wider interstitial dipoles, which increases the density below it. At the step itself, the density is reduced by an appreciable fraction. The non-linear forces producing the rearrangement of the dipoles are small, so only dipoles very close to those at the step are affected. The dipole distribution is modified without external plastic strain, only the motion of the short segments of screw dislocations can produce some reverse strain

The relationship between the dipole density and the plastic strain increment P de caused by a stress increment σ d is

( ) σ σ λ d bn de S P 2 = , ( 2
)
where λ is the mean free path of each of the two liberated dislocations split from the dipole. For strains very near to the unloading and reloading event, we can assume a constant source density and a constant mean free path, so

( ) σ λ σ S P n b de d 1 2 ≈ .
(3)

On the right-hand side of Fig. 2 is sketched the stress-strain curve upon reloading. We see that the 'premature yielding' of Fig. 1 results from the extra interstitial dipoles produced by the rearrangement, and the 'yield point' is produced by the extra vacancy dipoles. The shape of the observed stress-strain curve is roughly reproduced if about half the sources at the step undergo rearrangement. We see that the magnitude of the yieldpoint, as defined in Fig. 1, is lower than the fractional change in splitting stress estimated in Eq. ( 1) by a factor of about 6, bringing it much more in line with the experimental observations. The sketch of Fig. 2 is the outcome of a crude attempt to reproduce the experimental curve. It is based upon the assumption that no macroscopic instability occurs upon yielding. However, the curve of Fig. 1 shows a short range of strain in which the stress-strain curve shows zero or even negative slope. In this range, the Considère condition for plastic stability is clearly violated. Although Haasen and Kelly make no mention of it, some sort of Lüders band probably propagates through their specimens at yield. The reduced slope of the curve produced by modelling in Fig. 2 can produce plastic instability and result in an apparent zero or negative slope in the output of the tensile testing machine. A more sophisticated model must take this into account before one can reliably derive the source density distribution from the experimental data.

At temperatures where cross-slip of the screw dislocations can occur, the slip required for the rearrangement will be facilitated because the screws will be more flexible, not constrained to one glide plane. Rearrangement of more pairs of dipoles will occur. Cross-slip is accomplished by the thermally activated motion of jogs, with an activation energy around 0.1eV, that is, cross-slip becomes fast between 20K and 77K, the temperature range where an enhancement of the Haasen-Kelly effect is observed.

The action of impurities is to anchor the jogs, greatly slowing the cross-slip, so impeding the rearrangement. Thus one finds a simple semi-quantitative explanation for the temperature and impurity dependence of the effect.

There is one further point worth mentioning: for edge dipoles in the shape of elongated isolated loops, there may be a bowing stress to overcome, as well as a passing stress. Bowing under stress forces the dislocations to be curved at the point of splitting, so it reduces the nominal splitting stress for an infinite straight dipole. The bowing stress is equal to the nominal passing stress if the dipole is about ( ) 17 1 8 ≈ -ν π times longer than its height. Under these circumstances, it is easy to use the calculations of Brown (2006) to estimate the reduction in passing stress: it is reduced by about 50%. However, the diagram of Fig. 2 is unchanged. All that happens is that the contribution of the passing (splitting) stress to the overall flow stress is reduced by a factor that depends upon the shape of the dipole loops. Such a factor will further reduce the size of the yield point. The interpretation of the Haasen-Kelly effect proposed here is unlikely to be unique. However in the voluminous literature on work-hardening I can find no other attempt at an explanation more detailed than that given by Haasen and Kelly in their original paper. Their explanation, quoted in the abstract, is succinct and correct!

Conclusions:

The assumption that a major component of dislocation sources in unidirectional deformation comprises edge dislocation dipoles leads to a simple semi-quantitative explanation of the Haasen-Kelly effect.

Acknowledgements:

It is a pleasure to acknowledge the many years of discussions and encouragement from Tony Kelly. I am grateful to a referee for helpful criticism, and to the Cavendish Laboratory and to Robinson College for support. [START_REF] Haasen | [END_REF]). The definition of the observed yield point, σ ∆ , is shown. The plastic strain interval is estimated from another diagram in their paper. Fig. 2. On the left hand side is sketched the density of dislocation sources (abscissa) as a function of the applied stress under load (ordinate). There is a step discontinuity at the current stress level. On unloading, screw dislocations move to create larger interstitial dipoles and smaller vacancy dipoles, thereby modifying the distribution in the immediate neighbourhood of the discontinuity. The corresponding stressstrain curve is sketched in the right-hand diagram. The slope of the stress-strain curve is proportional to the reciprocal of the source density. If no rearrangement occurs, it follows the dashed line. Rearrangement produces a yield point at the onset of plastic flow. The slopes of the straight-line segments on the reloading curve have been estimated from equation (3), using the known slope of the basic work-hardening curve in Fig. 1.

  angle boundaries are left. Full confinement of the slip bands is then lost.

  appears that similar effects are observed in zonerefined iron (Wray and Horne 1966).

Fig. 1 (

 1 Fig. 1 (adapted from Haasen and Kelly (1957)). The definition of the observed yield point, σ ∆ , is shown. The plastic strain interval is estimated from another diagram in their paper.

Fig. 2 .

 2 Fig. 2. On the left hand side is sketched the density of dislocation sources (abscissa) as a function of the applied stress under load (ordinate). There is a step discontinuity at the current stress level. On unloading, screw dislocations move to create larger interstitial dipoles and smaller vacancy dipoles, thereby modifying the distribution in the immediate neighbourhood of the discontinuity. The corresponding stressstrain curve is sketched in the right-hand diagram. The slope of the stress-strain curve is proportional to the reciprocal of the source density. If no rearrangement occurs, it follows the dashed line. Rearrangement produces a yield point at the onset of plastic flow. The slopes of the straight-line segments on the reloading curve have been estimated from equation (3), using the known slope of the basic work-hardening curve in Fig. 1.

  Fig.1(adapted from[START_REF] Haasen | [END_REF]). The definition of the observed yield point, σ ∆ , is shown. The plastic strain interval is estimated from another diagram in their paper.

  Fig.1(adapted from[START_REF] Haasen | [END_REF]). The definition of the observed yield point, σ ∆ , is shown. The plastic strain interval is estimated from another diagram in their paper.

This seems first to have been stated byFriedel (Dislocations, 1964, Pergamon Press, p

248): '. . .in many cases the distance h between slip planes. . .is so small that the maximum elastic interaction. . .between dislocations in neighbouring planes is large compared with the applied stress. The whole Taylor model then breaks down.' 2 The 'height' of a dipole is the distance h measured perpendicular to the slip plane between the two dislocations of opposite sign making it up.

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The value of Poisson's ratio to be inserted here is not the value one uses to produce accurately the ratio between the line energies of edge and screw dislocations, but it is the value which gives the ratio between the shear modulus and the bulk modulus. Using the tables given by Hirth and Lothe (1992) one finds that for all the metals discussed here the value can be taken to be


Konstantin B Borisenko

Yixin Chen

Guoqiang Li

David J H Cockayne Frs

Applications of electron reduced density function analysis to the study of amorphous materials: an example of Zr 64.13 Cu 15.75 Ni 10.12 Al 10.0 and Zr 55 Cu 30 Ni 5 Al 10 metallic glasses

Keywords: electron diffraction, metallic glasses, atomic structure, plasticity

    Applications of electron reduced density function analysis to the study of amorphous materials: an example of Zr64.13Cu15.75Ni10.12Al10.0 and Zr55Cu30Ni5Al10 metallic glasses

Introduction

Accurate reduced density functions (RDF) can now be routinely obtained from thin films of materials using the electron diffraction technique developed by Cockayne et al [1][2][3]. These functions are closely related to the radial distribution functions, taking into account the different scattering factors of different elements in the material. The technique provides unique information about distribution of interatomic distances in the material, on the basis of which atomistic models can be built and refined, and then related to the observed properties of the films.

The models can be refined using the observed experimental diffraction data in a Reverse Monte Carlo (RMC) routine [4,5] and also checked for minimum energy conditions using density functional theory (DFT) energy calculations. A combined refinement of the models with constraints obtained from DFT calculations leading to an agreement with both the experimental data and theoretical DFT models can also be applied.

One of the examples is in application to the study of atomic structures of metallic glasses.

Metallic glasses have promising industrial potential due to their high strength and resistance to corrosion [6,7]. However, a serious limiting factor in their applications is their poor ductility.

Recently, novel metallic glasses of general composition Zr-Cu-Ni-Al with very good ductility have been prepared [8][9][10]. Such properties in metallic glasses are unusual and to understand them, obtaining reliable information on local atomic structure is very important.

There have been several recent structural studies of the related Zr-Cu-Al or Zr-Ni-Al metallic glasses by either experimental X-ray diffraction [11,12] or theoretical techniques [13,14]; however additional and more reliable structural information can be obtained by combination of the two approaches.

In this paper, we describe application of the RDF technique using electron diffraction combined with the DFT calculations to study atomic structures of two metallic glasses with compositions Zr 64.13 Cu 15.75 Ni 10.12 Al 10.0 and Zr 55 Cu 30 Ni 5 Al 10 . The metallic glasses with the nominal compositions (in at. %) Zr 64.13 Cu 15.75 Ni 10.12 Al 10.0 (Zr64CuNiAL) and Zr 55 Cu 30 Ni 5 Al 10 (Zr55CuNiAl) were prepared by arc melting the constituents under a Ti-gettered argon atmosphere in a water-cooled copper crucible. The alloys were cooled and remelted several times to ensure compositional homogeneity. In the final stages of the preparation they were cast into copper molds to form ingots of 3 mm diameter.

Electron diffraction experiment

To prepare samples for transmission electron microscopy (TEM), the metallic glass ingots were first sliced into 3 mm discs, mechanically ground to a thickness of ~40 µm, and then ionmilled in a precision ion polishing system (PIPS) operating at 3.0 kV (Gatan, Pleasanton, CA) until appearance of a tiny hole in the sample. For final thinning the samples were moved to a low angle ion milling system with a liquid nitrogen cooling stage working at a low voltage of 300 V (Fischione 1010 Low Angle Ion Milling & Polishing System) to prevent or remove any structural alteration possibly caused by the high-energy ion beam of PIPS. The electron diffraction experiments for both glasses were carried out on a JEOL 3000F field-emission gun TEM operating at 300 kV. Selected area diffraction (SAD) was used to collect the electron diffraction patterns from different thin areas in the samples. The selected area was, in each case, a disc of diameter about 100 nm on the sample. To avoid contribution from multiple scattering in the diffraction data, only very thin areas were used for SAD. The condition for the sample to be sufficiently thin to give only single scattering was that the total scattered intensity, I(q), would oscillate about the sum of scattering intensities from independently scattering atoms, over the full range of q. Larger contribution of multiple scattering in thicker samples, to our experience, results in that a good fit can only be achieved in the q range from usually 15 to 70 nm -1 , while even adjusting the number of atoms as a fitting parameter will not produce a good fit in the higher q The diffraction patterns were recorded on a 1024×1024 pixel CCD camera and corrected for dark current and pixel sensitivity. All the diffraction patterns were accurately calibrated by using constant lens settings for the TEM and a polycrystalline gold sample assuring a better than 1% accuracy in interatomic distances. This corresponds to the error in all obtained distances of ±0.02 Å. Using a camera length of 15 cm, diffraction patterns were collected up to a scattering vector of q ~ 220 nm -1 , where q = 4πsin(θ)/λ. Here θ is the half scattering angle and λ  is the wavelength of electrons. The physical limit imposed by the size of the detector of about 2.5 cm would correspond to the maximum q of about 530 nm -1 . However, usable data for refinements extended up to q = 130 nm -1 due to low signal to noise ratio at higher q. From the obtained total scattering intensity, I(q), the experimental reduced scattering intensity, φ(q), and then the reduced density function can be obtained as described in Refs. 1-3. As the studied materials are expected to contain no interatomic distances shorter than 2.0 Å, the contribution to the reduced scattering intensities that produced peaks in the RDF below 2.0 Å was removed by a Fourier filter. The Reverse Monte Carlo refinements were performed by fitting the theoretical reduced scattering intensity computed from an atomistic model to that obtained experimentally as described above by minimizing the sum of the squared differences between the two data sets, In this procedure, first, an atomistic model was constructed by random packing of atoms with periodic boundary conditions in three dimensions with the density close to the experimental density of similar studied materials, 6.6 g cm -3 [8]. From this model the theoretical reduced scattering intensity was computed utilizing the Debye approximation,

Reverse Monte Carlo refinements

∑ - = Φ i i theor i i i q q 2 exp )] ( ) ( [ φ φ . An additional cost function, ∑ - = Ε i AB i AB i c r
ij i i j ij ij i B qr qr q f q ∑ ∑∑ + = sin ) ( ) ( 2 φ
, where

2 2 2 q u ij ij e B - =
is the Debye-Waller type probabilitydensity factor to account for the phonon vibrations. The atomic scattering amplitudes, f(q), compiled by Kirkland [START_REF] Kirkland | Advanced computing in electron microscopy[END_REF] were used. The vibrational amplitudes, u ij , in the Debye-Waller factor were assumed to be 0.02 Å and equal for all interatomic distances. The additional cost function E was also evaluated. An atom was then selected randomly in the model and moved by a random amount within the specified distance from its initial position. The change in the reduced scattering intensity and its agreement with the corresponding experimental data as well as change in the cost function E was then calculated. If the move improved the total agreement, expressed as

2 2 E w w F E + Φ = Φ
, where Φ w and E w are weights, it was accepted; if not, the move was accepted with the probability

M F e / ∆ -
, where M is a parameter. The typical value of M used in the refinements was 0.002. This procedure was repeated until a minimum in the total agreement, with good agreement in experimental data and low cost function values, was achieved. To prepare initial models for energy optimizations, models of 100 atoms were first refined using the described RMC procedure to agree with the experimental diffraction data. These RMCrefined models were then used as starting points for energy optimizations with full relaxation of the atomic positions within a fixed unit cell with periodic boundary conditions. The energy optimizations of the models were performed using the VASP code [START_REF] Kresse | [END_REF]17]. Projector-augmented wave potentials were employed for Zr, Cu, Ni and Al within the local density approximation (LDA) described by the Perdew and Zunger functional [18]. The plane-wave basis set energy cut off was 230 eV. The relaxation of the structures continued until the change in energy for ions was less than 1x10 -5 eV. The convergence criterion for maximum force on ions was -0.01 eV/Å. An 2 x 2 x 2 grid of k points, automatically generated by the Monkhorst and Pack algorithm [19],

Density functional theory energy optimisations

was used for sampling of the electron density. This scheme produced k-points with their origin offset from the gamma point by 1/4.

Results and discussion

Comparison of the experimental RDFs obtained for the two metallic glasses is presented in Figure 1. It is seen that changing the composition of the glass results in a prominent shift of the first three peaks in the RDFs to shorter distances. The main experimental peak positions for the two glasses are summarized in Table 1. The RDFs obtained from the models from the DFT calculations for the two metallic glasses show qualitative agreement with the experimental RDFs in the number and shape of the peaks, Figure 2, although the bond lengths in the energy-optimised models contributing to the first peak are shorter, resulting in a shift of the peak to shorter values. The peaks of the RDFs obtained from the energy-optimized models are shown in Table 1. The difference in position between the observed and computed peaks is about 4%, which can be regarded as satisfactory.

The experimentally observed trend of shortening first neighbour distances when going from Zr64CuNiAl to Zr55CuNiAl metallic glass is well reproduced in the energy-optimized models.

Using the average bond length obtained in the DFT calculations of the models of the metallic glasses as constraints, atomic models were prepared by random packing of 1000 atoms.

The RDFs computed from these models are compared to the experimental ones in Figure 3.

Although the randomly packed models do not agree quantitatively with the experimental RDF curves, their shapes appear to be similar. This suggests that there is certain amount of local ordering in the materials, although it is not particularly strong and the structure resembles a randomly packed model to a considerable degree.

Using the same bond-length constraints obtained from the DFT calculations, the models of the metallic glasses were refined against the experimental data in the RMC procedure. For these refinements the weights for the experimental data and theoretical constraints were selected to obtain a compromise model where the constraints are satisfied as much as possible while maintaining a good fit to the experimental diffraction data. The resulting RDFs from the refined models are compared to the experimental ones in Figure 4. Using the constraints for bond lengths from the DFT calculations has resulted in a somewhat worse fit in the first peak of the RDFs. The average bond lengths and coordination numbers from the RMC refinements together with those from the DFT calculations are shown in Table 2.

The structures of both glasses have similar average bond lengths, some of which are considerably different from the corresponding bond lengths from the DFT calculations, as they had to be adjusted to fit the experimental diffraction data. While Zr-Zr bond lengths agree relatively well, there is, for example, considerable lengthening of the Zr-Cu bond lengths in the refined structure as compared to that from the DFT energy optimizations, Table 2. In addition, the absolute values of the coordination numbers from the two techniques differ considerably, although the general trends in the change of the coordination number when going from the Zr64CuNiAl to Zr55CuNiAl metallic glass do agree. For instance, the coordination number of Zr, N Zr , is the same in the two metallic glasses, while that of the copper, N Cu , increases.

Analysing the structures from the DFT energy calculations and RMC refinements, it is seen that the energy optimisations lead to small but noticeable changes in the structures, reflected in bond type, bond angle and dihedral angle distribution histograms. In the energy-optimised models of both glasses a decrease in the probability of finding Zr-Zr and Cu-Cu bonds is observed compared to the refined structure, while the probability of formation of Zr-Cu, Zr-Ni and Zr-Al bonds increases, Figure 5. In addition, in the bond angle distributions for both glasses, Figure 6, the probability to find angles at about 60 and 120 degrees increases in the energy-optimised models, while the probability of the angles to be at about 90 degrees decreases notably. This may indicate that fragments of hexagonal or body centered type packing are energetically preferred in local coordination polyhedra to fragments of face centered or cubic type close packing. The improved energetic stability appears to increase the probability of finding a dihedral angle around 40, 80 and 150 degrees for the energy optimized models as compared to the randomly packed or refined models, Figure 7.

In general, however, there is a considerable degree of randomness in the packing of the studied metallic glass, as models with the bond type, bond angle and dihedral angle distributions close to those observed in the randomly packed models fit the diffraction data well. According to both the DFT-energy optimized model and the randomly packed model of the Zr64CuNiAl glass, Zr-Zr bonding predominates, while with increase in the Cu content in the Zr55CuNiAl glass, the number of Zr-Zr bonds decreases while the number of Zr-Cu bonds increases. This can explain the shortening of the first neighbour distances in the Zr55CuNiAl glass, as longer Zr-Zr are replaced by shorter Zr-Cu bonds.

A randomly packed structure of the studied glasses with a small degree of local ordering suggested in the present study agrees with a previous investigation of enthalpy of mixing in Zr-Cu-Al and Zr-Cu-Ni-Al alloys [20]. In that study, using an association model with various binary and ternary stable associates representing local ordering, good agreement of the experimental enthalpy of mixing and temperature dependence of the heat capacity of the alloys with corresponding model parameters was obtained. It was, however, shown that the molar contributions of the associates were very small [20]. We note that the energy-optimized structures discussed here were arrived at starting from randomly packed models. It may therefore happen that this is just one of the possible energetically stable models of the material, and other energetically stable models may also be feasible. To investigate this, extensive reliable molecular dynamics simulations of the systems may be needed, which are a goal of future work.

It has been suggested that ZrCu(Ni)Al metallic glasses with more densely packed local coordination polyhedra (having higher average coordination numbers) are less plastic, with Zr 64 Cu 26 Al 10 being more plastic than Zr 45 Cu 45 Al 10 [14]. The variation of the coordination number with composition agrees well with the results of the present structural study for the ZrCuNiAl glasses. The coordination numbers of both Cu and Al are higher in the Zr55CuNiAl than those in the Zr64CuNiAl metallic glass, while coordination numbers of Zr and Ni are the same.

Conclusions

Refined structures for both studied glasses have indicated a considerable similarity with the corresponding randomly packed models. Although the DFT calculations have suggested the presence of some local ordering, several of the main bond lengths from the RMC refinements disagree considerably with those computed in the DFT energy optimizations. Local ordering reflected in the formation of preferred bond angles and dihedral angles in the material found in the DFT calculations, however, still cannot be excluded. The largely random structure of the glasses with a degree of local ordering agrees with previous thermodynamic studies of similar systems and can be important in understanding the unusually high plasticity of the materials.

  to 200 nm -1 . The energy filtered TEM (EFTEM) elemental mapping has found no clustering of atoms and no crystallites were seen in the high resolution images of the materials.

  first nearest neighbours in the model, AB i r , from values used as constraints, AB i c , for example, from the DFT calculations or the sum of the covalent radii of the elements,

Figure 1 .

 1 Figure 1. Comparison of the experimental RDFs from the two metallic glasses.

Figure 2 .

 2 Figure 2. Comparison of the experimental (Exp) RDFs with those obtained from the energy-

Figure 3 .

 3 Figure 3. Comparison of the experimental (Exp) RDFs with those obtained from randomly

Figure 4 .

 4 Figure 4. Comparison of the experimental (Exp) RDFs with those obtained from RMC

Figure 5 .

 5 Figure 5. Normalised bond type distribution histograms from refined models (RMC) as compared

Figure 6 .Figure 7 .

 67 Figure 6. Normalised bond angle distribution histograms from refined models (RMC) as

Table 1 .

 1 Peak positions (Å) in the experimental RDFs of Zr64CuNiAl and Zr55CuNiAl metallic glasses as compared with the RDFs from DFT calculations

					Page 14 of 30
	Peak	Zr64CuNiAl		Zr55CuNiAl
		Exp (±0.02 Å) DFT	Exp (±0.02 Å) DFT
	1	3.18	3.04	3.06	2.94
	2	5.64	5.40	5.51	5.12
	3	8.13	-	7.91	-
	4	10.80	-	10.75	-

Table 2 .

 2 Main average bond distances (Å) and coordination numbers from the DFT energy optimisations as compared to those obtained in the constrained RMC refinements for

	Zr64CuNiAl and Zr55CuNiAl metallic glasses
	Parameter	Zr64CuNiAl		Zr55CuNiAl
		DFT	RMC	DFT	RMC
			(±0.02 Å)		(±0.02 Å)
	Zr-Zr	3.17	3.13	3.18	3.12
	Zr-Cu	2.84	2.96	2.89	2.96
	Zr-Ni	2.74	2.69	2.78	2.71
	Zr-Al	2.98	2.67	2.99	2.65
	Cu-Cu	2.67	2.78	2.61	2.80
	Cu-Ni	2.61	2.52	2.59	2.54
	Cu-Al	2.53	2.52	2.63	2.52
	N Zr	9.8	7.8	9.8	7.8
	N Cu	8.7	7.0	9.1	7.4
	N Ni	8.2	5.8	8.5	5.8
	N Al	8.0	6.2	8.5	6.5

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Acknowledgements

We thank Professor Y. Zhang (University of Science and Technology, Beijing) for the provision of samples of the metallic glasses. KBB was supported by the EPSRC Grant (EP/F048009/1).


Katharina Effenberger-Neidnicht

Rainer Schobert
email: rainer.schobert@uni-bayreuth.de

Katharina Effenberger

Combinatorial effects of thymoquinone

Keywords: Anti-tumour agents, Apoptosis, Conjugates, Doxorubicin, Multidrug resistance, Thymoquinone

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Introduction

A good deal of naturally occurring quinones have pharmacologically interesting properties including anti-tumoural activity [START_REF] Cragg | Impact of natural products on developing new anti-cancer agents[END_REF][START_REF] Colucci | Natural and synthetic quinones and their reduction by the quinone reductase enzyme NQO1: from synthetic organic chemistry to compounds with anticancer potential[END_REF][START_REF] Bolton | Role of quinones in toxicology[END_REF]. The anthraquinone doxorubicin (DOX, 1) is a Streptomyces metabolite that is clinically used in the treatment of human tumours such as soft tissue sarcomas, lymphomas and various types of carcinomas. However, the clinical application is hampered by an early onset of resistance and by dose restrictions necessary to prevent cumulative cardiac toxicity [START_REF] Robert | Anthracyclines[END_REF]. The para-benzoquinone thymoquinone (TQ, 2) is the active principle of thyme essential oil and of the volatile oil of black seed (Nigella sativa), responsible for the anti-oxidant and anti-neoplastic effects of these spices. It has a low general toxicity, but a promising anti-tumour activity [START_REF] Badary | Inhibitory effects of thymoquinone against 20-methylcholanthrene-induced fibrosarcoma tumorigenesis[END_REF][START_REF] Gali-Muhtasib | Thymoquinone: A promising anti-cancer drug from natural sources[END_REF][START_REF] Gali-Muhtasib | Thymoquinone reduces mouse colon tumour cell invasion and inhibits tumour growth in murine colon cancer models[END_REF][START_REF] Gali-Muhtasib | Thymoquinone extracted from black seed triggers apoptotic cell death in human colorectal cancer cells via p53-dependent mechanism[END_REF]. It induces apoptosis in cancer cells in p53-dependent and -independent ways and it was found to delay tumour growth by induction of cell cycle arrest in certain xenograft models [START_REF] El-Mahdy | Thymoquinone induces apoptosis through activation of caspase-8 and mitochondrial events in p53-null myeloblastic leukemia HL-60 cells[END_REF].

As part of a project aimed to improve the efficacy of anti-cancer drugs in resistant tumour cells we previously prepared covalent conjugates of both DOX and TQ with fatty acid and terpene carriers [START_REF] Breyer | Effects of thymoquinone-fatty acid conjugates on cancer cells[END_REF][START_REF] Effenberger | Terpene conjugates of the nigella sativa seed oil constituent thymoquinone with enhanced efficacy in cancer cells[END_REF][START_REF] Effenberger | Modulation of doxorubicin activity in cancer cells by conjugation with fatty acyl and terpenyl hydrazones[END_REF]. In some cases we saw dramatically greater anti-proliferative effects of the conjugates when compared to those of the parent quinones. For instance, 6-(hencosahexaenyl)thymoquinone, derived from the reaction of TQ with docosahexaenoic acid (DHA), was efficacious in multidrug resistant MCF-7/TOPO breast carcinoma cells at an IC 50 (72 h) < 50 nM while TQ itself required an IC 50 (72 h) ≈ 30 µM [START_REF] Breyer | Effects of thymoquinone-fatty acid conjugates on cancer cells[END_REF]. Conjugation to betulinic acid resulted in a compound with IC 50 (72 h) ≈ 130 nM in these cancer cells [START_REF] Effenberger | Terpene conjugates of the nigella sativa seed oil constituent thymoquinone with enhanced efficacy in cancer cells[END_REF].

In the case of DOX similar conjugates with improved efficacy and a potential to overcome multi-drug resistance were developed. Some of them had a mechanism of action different from that of DOX [START_REF] Effenberger | Modulation of doxorubicin activity in cancer cells by conjugation with fatty acyl and terpenyl hydrazones[END_REF].

Inspired by studies proving that TQ can indeed mitigate or even prevent doxorubicin-induced cardiomyopathy [START_REF] Oa | Thymoquinone protects against doxorubicin-induced cardiotoxicity without compromising its antitumor activity[END_REF][START_REF] Nagi | Protective effect of thymoquinone against doxorubicin-induced cardiotoxicity in rats: a possible mechanism of protection[END_REF][START_REF] Badary | Inhibition of benzo(a)pyrene-induced forestomach carcinogenesis in mice by thymoquinone[END_REF], we now investigated the influence of TQ on the magnitude and mechanism of the various effects exerted by DOX in human cancer cells. We tested both substances and an equimolar mixture of them for growth inhibition on the human cancer cell lines HL-60 leukaemia and 518A2 melanoma, HT-29 colon, KB-V1 cervix, MCF-7 breast carcinomas as well as in their multi-drug resistant variants and non-malignant fibroblasts (HF). The impact of the test compounds and their mixture on the mitochondrial membrane potential of HL-60 and 518A2 cells and on the cellular levels of various apoptosis-relevant species such as caspases, bax and bcl-2 mRNA, and reactive oxygen species (ROS) was also scrutinised.

Materials and methods

Chemicals:

Thymoquinone was purchased from Sigma Aldrich, Munich (Germany), doxorubicin was a gift from Ribosepharm GmbH, Gräfelfing (Germany). Both drugs were kept at 4 °C as 10 mM stock solutions in DMSO. Appropriate test concentrations were obtained by dilution with cell culture medium immediately before use.

Cell lines and culture conditions:

The HL-60 leukaemia cells were obtained from the German Collection of Biological Material (DSMZ), Braunschweig (Germany), the 518A2 melanoma penicillin G, 100 µg mL -1 streptomycin sulfate, 0.25 µg mL -1 amphotericin B and 250 µg mL -1 gentamycine (all from Gibco, Egenstein, Germany). The 518A2 and the KB-V1 cells as well as the HF were cultured in D-MEM medium (Gibco) containing 10% FCS, 100 IU mL -1 penicillin G, 100 µg mL -1 streptomycin sulfate, 0.25 µg mL -1 amphotericin B and 250 µg mL -1 gentamycine. The MCF-7 cells were grown in E-MEM medium (Sigma) supplemented with 2.2 g L -1 NaHCO 3 , 110 mg L -1 sodium pyruvate and 5% FCS. The cells were maintained in a moisture-saturated atmosphere (5% CO 2 ) at 37 °C in 75-mL culture flasks (Nunc, Wiesbaden, Germany). They were serially passaged following trypsinisation by 0.05% trypsin / 0.02% EDTA (PAA laboratories, Cölbe, Germany). Mycoplasma contamination was routinely monitored, and only mycoplasma-free cultures were used.

Inhibition of cell growth and metabolic activity (MTT assay):

MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (ABCR, Karlsruhe, Germany) was used to identify viable cells which reduce it to a violet formazan. HL-60 leukaemia cells (5 10 5 mL -1 ), and cells (5 10 4 mL -1 ) of 518A2 melanoma, KB-V1 cervix, MCF-7 breast and HT-29 colon carcinoma, and HF foreskin fibroblasts were seeded out in 96-well tissue culture plates and cultured for 24 h [START_REF] Mosmann | Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays[END_REF]. Inhibitors of P-pg, BCRP or MRP 1 (P-gp overexpressing KB-V1/VBL cells: 24 µM verapamil hydrochloride, BCRP-rich MCF-7/TOPO cells:

1.2 µM fumitremorgin C, MRP 1-rich HT-29/COLC cells: 10 µM MK571) were optionally added. Incubation (5% CO 2 , 95% humidity, 37 °C) of the cells following treatment with the test compounds was continued for up to 72 h. Blank and solvent controls were treated identically. MTT in phosphate buffered saline (5 mg mL -1 ) was added to a final concentration of 0.05% (HL-60, 518A2, HF) or 0.1% (KB-V1, MCF-7, HT-29). After 2 h the formazan precipitate was dissolved in 10% sodium dodecylsulfate in DMSO containing 0.6% acetic acid in the case of the HL-60 cells. For the adherent 518A2, KB-V1, MCF-7, HT-29 and HF cells the microplates were swiftly turned to discard the medium before adding the solvent mixture. The microplates were gently shaken in the dark for 30 min and the absorbance at 570 nm and 630 nm (background) was measured with a Tecan F200 plate reader. All experiments were carried out in quadruplicate; the percentage of viable cells was calculated as the mean ± SD with controls set to 100%.

Caspase activity assay:

Caspase activities were determined by luminometric assays (Caspase-Glo 3/7, 8 and 9, Promega, USA), according to the manufacturer's instructions. Cellular proteins were extracted from HL-60 and 518A2 cells following treatment with 5 µM of the test compounds or a combination of both compounds at equal 5 µM concentrations in a lysis buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton-X-100 and 1×EDTA-free protease inhibitor mix (Calbiochem).

Cell lysates were incubated at 4 °C for 15 min, centrifuged at 800 g for 10 min and the precipitates were discarded. Protein concentrations were measured using the Bradford Reagent (Sigma) and bovine serum albumin as a standard. Cell lysates (15 µg cellular protein) were analysed for caspase activities using the homogeneous luminescent assay. After cleavage of the luminogenic substrate containing a caspase-specific peptide sequence, the luminescence was quantified with a Tecan Genios Plus plate reader and assumed to be proportional to the caspase activities in the cell lysates [START_REF] Earnshaw | Mammalian caspases: Structure, Activation, Substrates, and Functions During Apoptosis[END_REF].

Real-time PCR for detection of bcl-2 and bax mRNA expression:

HL-60 and 518A2 cells were treated with 5 µM of the test compounds or a combination of both compounds at equal concentrations (5 µM) for up to 24 h. The total RNA was extracted using peqGOLD RNAPure (peqLab, Erlangen, Germany) according to the manufacturer's instructions. The synthesis of cDNA was conducted in a 21 µL reaction mixture starting with 80 ng total RNA using the reverse transcription system (Promega, Madison, WI, USA). Quantitative PCR was performed using the SYBR GREEN PCR-Kit LightCycler fast start DNA Master for a LightCycler 2.0 System (Roche Diagnostic, Mannheim, Germany) and threshold numbers were determined using the LightCycler Software, version 3.5. All genes examined were normalized to a house-keeping gene encoding GAPDH [START_REF] Bas | Utility of the housekeeping genes 18S rRNA, beta-actin and glyceraldehyde-3-phosphatedehydrogenase for normalization in real-time quantitative reverse transcriptasepolymerase chain reaction analysis of gene expression in human T-lymphocytes[END_REF]. The primers were obtained from Qiagen: GAPDH (Hs_Gapd_2_SG QuantiTect Primer Assay, Cat.No.: QT01192646), Bax (Hs_Bax_2_SG QuantiTect Primer Assay, Cat.No.: QT00031192), Bcl-2 (Hs_Bcl2_1_SG QuantiTect Primer Assay, Cat.No.: QT00025011).

Mitochondrial membrane potential:

Changes in mitochondrial membrane potential were determined by the Mitochondrial Membrane Detection Kit (Stratagene, La Jolla, CA, USA) according to the manufacturer's procedure. Following treatment, cell samples were centrifuged at 400 g for 5 min. The pellets were resuspended in 500 µL JC-1 solution (0.1×), incubated at 37 °C for 15 min (HL-60) or 35 min (518A2) and then centrifuged again for 5 min at 400 g. After washing, the pellets were resuspended in 100 µL PBS and transferred into a well of a black 96-well plate.

The red (λ ex = 585 nm, λ em = 590 nm) and green (λ ex = 510 nm, λ em = 527 nm) fluorescence intensities were measured with a Tecan F200 plate reader and their ratio was calculated [START_REF] Desagher | Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis[END_REF].

Generation of ROS (NBT assay):

HL-60 cells (5 10 5 mL -1 ) were plated in 96-well tissue culture plates, and test compounds were added after 24 h incubation at 37 °C to achieve a final concentration of 5 µM. Incubation (5% CO 2 , 95% humidity, 37 °C) of cells following treatment with the test compounds was continued for 24 and 72 h. After removal of the cell medium by centrifugation, the cells in each well were resuspended in 100 µL 0.1% NBT, and the plates were placed into the incubator for 1 h. The reduced NBT was solubilised with 100 µL 2M KOH and 130 µL DMSO for 30 min. The absorbance was measured for each well at 630 and 405 nm (background) using an ELISA plate reader. The adherent 518A2 cells (5 10 4 mL -1 ) were seeded out in 96-well tissue culture plates after trypsinisation and incubation for 24 h at 37 °C to allow attachment, then treated similarly, only the medium was removed prior to incubation with NBT for 4 h. All experiments were carried out in quadruplicate [START_REF] Rook | A simple method for the solubilisation of reduced NBT, and its use as a colorimetric assay for the activation of human macrophages by gamma interferon[END_REF].

Mathematical analysis of combined drug effects:

The combined effects of DOX and TQ on the 518A2, HL-60, HT-29/COLC, KB-V1/VBL and MCF-7/TOPO cells were quantified using a known "multiple-drug effect analysis" based on the median effect principle and isobologram technique by Chou and Talalay [START_REF] Chou | Generalized equations for the analysis of inhibitors of Michaelis-Menten and higher order kinetic systems with two or more mutually exclusive and non-exclusive inhibitors[END_REF][START_REF] Chou | Quantitative analysis of dose effect relationships: the combined effects of multiple drugs or enzyme inhibitors[END_REF] This model allows the determination of CI values (combination indices) based on the median dose-effect and combination index equation. We interpreted the combination of DOX and TQ as synergistic when CI < 1, additive when CI = 1 and antagonistic when CI > 1.

Statistical analysis of measurement results:

The results are expressed as means ± standard deviation (SD). The Student-Newman-Keuls test was used to determine statistical significance with P value < 0.05 considered significant (*).

Results and discussion

Inhibition of cell growth

Compounds 1 (DOX), 2 (TQ), and an equimolar mixture of both were tested for their growth inhibition in cells of human HL-60 leukaemia and 518A2 melanoma, P-gp-rich KB-V1/VBL cervix carcinoma, BCRP-rich MCF-7/TOPO breast carcinoma and MRP 1-rich HT-29/COLC colon carcinoma as well as their parental, sensitive variants (KB-V1, MCF-7 and HT-29) and in non-malignant human foreskin fibroblasts (HF). The IC 50 values after 72 h exposure to the test compounds are summarised in Table 1 [START_REF] Mosmann | Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays[END_REF].

((Table 1 here))

In sensitive HL-60 cells DOX was efficacious at IC 50 (72 h) = 80 nM [START_REF] Effenberger | Modulation of doxorubicin activity in cancer cells by conjugation with fatty acyl and terpenyl hydrazones[END_REF]. The equimolar mixture of DOX and TQ was even fourfold more active in these cells.

An opposite activity lowering effect of adding TQ to DOX was found for the 518A2 melanoma cells.

Somewhat surprising, the non-malignant foreskin fibroblasts (HF) were affected most by the drug mixture which was growth inhibiting at an IC 50 value ca. 20times lower than that of DOX alone. A possible explanation for this could be developed along the following line. There were reports that DOX induces apoptosis in normal and tumour cells via different mechanisms [START_REF] Wang | Doxorubicin induces apoptosis in normal and tumor cells via distinctly different mechanisms[END_REF]. While a transcriptional activation of p53 is crucial for a DOX-induced apoptosis in tumour cells, the apoptosis of normal cells is initiated mainly by a rise in caspase-3 and in ROS. Should a mixture of 1 and 2 lead to a significantly higher ROS level in the fibroblasts they would be hit harder than the tumour cells (see below).

For multi-drug resistant MCF-7/TOPO cells a doubling of growth inhibition by the mixture of the two quinones over that by DOX alone was observed. The multidrug resistant KB-V1/VBL cells responded equally poorly to DOX and to the mixture of DOX and TQ. Furthermore, a co-incubation of multi-drug resistant HT-29/COLC colon carcinoma cells with DOX and TQ resulted in a decrease of growth inhibition compared to DOX alone.

To quantitatively analyse the combined drug effects of DOX and TQ against the above-mentioned cells the multiple-drug effect analysis based on the median effect principle and the isobologram technique by Chou and Talalay was used [START_REF] Chou | Generalized equations for the analysis of inhibitors of Michaelis-Menten and higher order kinetic systems with two or more mutually exclusive and non-exclusive inhibitors[END_REF][START_REF] Chou | Quantitative analysis of dose effect relationships: the combined effects of multiple drugs or enzyme inhibitors[END_REF]. DOX and TQ exhibited synergy (CI < 1) for HL-60 and MCF-7/TOPO cells, they were additive (CI = 1) for HT-29/COLC cells and antagonistic (CI > 1) for KB-V1/VBL and 518A2 cells at 72 h.

In order to check whether TQ can help overcome the DOX related multi-drug resistance of cancer cells, we measured the IC 50 (72 h) values of the individual quinones and a combination of both in HT-29/COLC, KB-V1/VBL and MCF-7/TOPO cells, in the absence and in the presence of specific inhibitors of their respective ABC-transporters. To this end 10 µM MK571 was added to the MRP 1-rich HT-29/COLC cells, 24 µM verapamil hydrochloride was added to the P-gp overexpressing KB-V1/VBL cells and 1.2 µM fumitremorgin C was added to the BCRP-rich MCF-7/TOPO cells. The interference of the ABC-transporters with the applied drugs was specified by the ratio of the IC 50 (72 h) values with and without addition of the specific inhibitors (R +/-) in MTT assays. This ratio is typically close to unity for drugs that are not substrates of these ABC-efflux pumps. This was approximately the case for TQ when applied to KB-V1/VBL (R +/-= 0.75) and to MCF-7/TOPO cells (R +/-= 0.87). In contrast and as expected, DOX was a good substrate for the transporters MRP 1, P-gp and BCRP, respectively, of these three cell lines, apparent from ratios R +/-< 0.15. For the breast and colon carcinoma cells MCF-7/TOPO and HT-29/COLC this ratio could be distinctly increased to R +/-= 0.28 by incubating them with an equimolar mixture of the two quinones.

In addition, the ratio (R r/s ) of the IC 50 (72 h) values in the multi-drug resistant cells and the parental sensitive cells was determined. R r/s could be improved by adding TQ to DOX by a factor of nearly 5 in the case of MCF-7 and at least by a factor of 3 for the KB-V1 cells. In HT-29 cells no difference was observed between resistant and sensitive cells.

We also tested DOX and TQ individually and in combination against KB-V1, MCF-7 and HT-29 cells that had been desensitised beforehand with clinical relevant doses of DOX (MCF-7/DOX: 55 nM, KB-V1/DOX: 200 nM, HT-29/DOX: 75 nM). We found a 1.2-fold higher efficacy of the mixture of DOX and TQ in the HT-29 cells desensitised for DOX.

Thus, the addition of TQ can improve the growth inhibitory effect of DOX in various cancer cells, including DOX-refractory ones.

Induction of apoptosis

The induction of apoptosis by the quinones 1 and 2 had already been visualised via DNA fragmentation [START_REF] El-Mahdy | Thymoquinone induces apoptosis through activation of caspase-8 and mitochondrial events in p53-null myeloblastic leukemia HL-60 cells[END_REF][START_REF] Effenberger | Modulation of doxorubicin activity in cancer cells by conjugation with fatty acyl and terpenyl hydrazones[END_REF][START_REF] Mizutani | Mechanism of apoptosis induced by doxorubicin through the generation of hydrogen peroxide[END_REF]. Additionally, we now probed the involvement of the apoptosis-relevant caspases-3, -8 and -9. HL-60 and 518A2 cells were treated with 5 µM of DOX or TQ or an equimolar mixture of both, which is a clinically relevant concentration resembling the peak plasma concentrations observed in patients after standard bolus infusions of DOX [START_REF] Gewirtz | A critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin[END_REF][START_REF] Minotti | Anthracyclines: Molecular advances and pharmacologic developments in antitumour activity and cardiotoxicity[END_REF]. When applied at markedly higher concentrations (> 20 µM), the cytotoxicity of DOX is no longer correlated to its cardiotoxicity and its generation of reactive oxygen species [START_REF] Effenberger | Modulation of doxorubicin activity in cancer cells by conjugation with fatty acyl and terpenyl hydrazones[END_REF]. The changes in caspase activities were then analysed at regular intervals between 1 h and 24 h by a substrate-cleaving luminometric assay based on specific dye-tagged oligopeptides (Caspase-Glo Assay, Promega) [START_REF] Earnshaw | Mammalian caspases: Structure, Activation, Substrates, and Functions During Apoptosis[END_REF].

Incubation of 518A2 cells with DOX and co-incubation with DOX and TQ gave similar caspase kinetics, while for HL-60 cells different kinetics were observed (Fig. 1). The levels of initiator caspase-8 and effector caspase-3 in HL-60 cells treated with Dox alone both reached a maximum after 12 h, while application of TQ caused only a maximum of caspase-3 after 2 h. Treating HL-60 cells with a mixture of TQ and DOX led to two maxima for the activities of either caspase.

There was a simultaneous first maximum of caspases-3 and -8 after 2 h and a second maximum of caspase-8 activity after 12 h and of caspase-3 activity after 18 h. In effect, that means that mixtures of DOX and TQ give rise to two waves of pro-apoptotic effector caspase-3 activity in HL-60 leukaemia cells. This effect should contribute to the efficacy boost observed in the MTT tests with HL-60 cells upon addition of TQ to DOX.

((Figure 1 here))

Next, we measured the mRNA expression of anti-apoptotic bcl-2 and proapoptotic bax proteins in 518A2 and HL-60 cells upon incubation with 5 µM of DOX, TQ or an equimolar mixture of these quinones for up to 18 h (Figure 2) [START_REF] Bas | Utility of the housekeeping genes 18S rRNA, beta-actin and glyceraldehyde-3-phosphatedehydrogenase for normalization in real-time quantitative reverse transcriptasepolymerase chain reaction analysis of gene expression in human T-lymphocytes[END_REF]. Exposure of 518A2 cells to either DOX or TQ for 18 h led to a significant increase of the bax to bcl-2 mRNA ratio while their incubation with an equimolar mixture of DOX and TQ caused a sharp drop in this ratio due to a greater mRNA expression of anti-apoptotic bcl-2 (P < 0.05). HL-60 cells had earlier been reported to react by an immediate rise of the bax/bcl-2 ratio to a short-term treatment (2-6 h) with either TQ [START_REF] El-Mahdy | Thymoquinone induces apoptosis through activation of caspase-8 and mitochondrial events in p53-null myeloblastic leukemia HL-60 cells[END_REF] or DOX [START_REF] Mizutani | Mechanism of apoptosis induced by doxorubicin through the generation of hydrogen peroxide[END_REF]. This is in line with the caspase kinetics for TQ and its mixture with DOX as described above. Upon exposure of HL-60 cells to the test compounds for more than 6 h, the bax/bcl-2 ratio was now found to decrease again (Fig. 2). In summary, both 518A2 melanoma and HL-60 leukaemia cells respond to TQ and DOX in a pro-apoptotic manner, the former more slowly than the latter. The combination of TQ and DOX seems to accelerate the rise and subsequent fall of the pertinent bax/bcl-2 mRNA ratio.

((Figure 2 here))

Mitochondrial membrane potential and generation of ROS

The intrinsic pathway of apoptosis is associated with a temporal change not only of the caspase levels but also of the mitochondrial membrane potential ∆Ψ m which we analysed using a kit from Stratagene that is based on the fluorescent cationic dye JC-1 [START_REF] Desagher | Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis[END_REF]. 518A2 and HL-60 cells were again treated with 5 µM DOX or TQ or their equimolar mixture for 6 h and 18 h and then stained with JC-1. The ratio of red (JC-1 aggregates in intact mitochondria) to green fluorescence (JC-1 monomers in the cytosol) is decreased in apoptotic cells. Incubation of HL-60 cells with a mixture of DOX and TQ for 6 h resulted in a decrease of intact mitochondria relative to untreated cells which was more pronounced than that observed for incubation with DOX alone. However, this difference dwindled upon prolonged incubation: DOX (6 h: 83%; 18 h: 28%), TQ (6 h: 77%; 18 h: 63%), DOX + TQ (6 h: 64%; 18 h: 32%), P < 0.05. In 518A2 cells TQ exerted no additional effect.

((Table 2 here normal and tumour cells were established for DOX [START_REF] Wang | Doxorubicin induces apoptosis in normal and tumor cells via distinctly different mechanisms[END_REF]. However, unlike TQ which is nontoxic in normal tissues, DOX-initiated ROS generation seems to contribute both to the desired anti-cancer effect and to the unwanted cardiotoxicity [START_REF] Wang | Doxorubicin induces apoptosis in normal and tumor cells via distinctly different mechanisms[END_REF][START_REF] Minotti | Anthracyclines: Molecular advances and pharmacologic developments in antitumour activity and cardiotoxicity[END_REF]. Table 2 also lists the results of the NBT assays for incubation periods of 24 h and 72 h as in the MTT assays. This allows a correlation of ROS generation and cell growth inhibition. In both cancer cell lines the mixture of DOX and TQ caused an increase in ROS generation when compared to the effect of DOX alone (P < 0.05). In 518A2 cells we noticed a doubling of the ROS level, for the HL-60 cells the increase was even more than 7fold.

This over-additive effect is remarkable. It might be due to a coupled redox pair of both para-quinones that can possibly tap other or more NAD(P)Hoxidoreductases than DOX alone and so generate intermediate semiquinones and eventually ROS more effectively and in higher concentrations [START_REF] Minotti | Anthracyclines: Molecular advances and pharmacologic developments in antitumour activity and cardiotoxicity[END_REF]. Anyway, there is some coherence between the growth inhibitory effects of the test compounds and their ROS initiation in the two cancer cell lines examined.

Interestingly, for the non-malignant HF foreskin fibroblasts no enhanced generation of ROS was observed upon treatment with either DOX, or TQ or the mixture of both for 24 h and 72 h. Thus, the stronger growth inhibition of HF observed in the MTT assay for the mixture of TQ and DOX remains inexplicable on the basis of the different impact of ROS on normal and cancer cells.

Protective effects of administration of high doses of TQ, i.e. 10 mg kg -1 d -1 [START_REF] Nagi | Protective effect of thymoquinone against doxorubicin-induced cardiotoxicity in rats: a possible mechanism of protection[END_REF],

against DOX-induced cardiotoxicity have been observed when starting several days before application of DOX. The potential of TQ to act as a superoxide radical scavenger [START_REF] Badary | Thymoquinone is a potent superoxide anion scavenger[END_REF] and to inhibit lipid peroxidation was believed to be responsible. However, so far, the superoxide scavenging properties of TQ were established only in cell free model systems. Further experiments, especially with cardiomyocytes are necessary to elucidate the precise role of ROS mediation by TQ in its cardioprotective effect when administered in combination with DOX.

This work is already under way.

Conclusion

We showed that the in vitro efficacy and selectivity of doxorubicin can be improved by adjuvant administration of the black seed constituent thymoquinone.

In multi-drug resistant MCF-7/TOPO breast carcinoma cells the growth inhibition by DOX could be doubled by adding equimolar amounts of TQ (CI < 1). In HL-60 cells the efficacy of DOX was even quadrupled in this way (CI < 1). In to be two consecutive pro-apoptotic impacts that prevent the recovery of shattered cells. In 518A2 cells DOX and its mixture with TQ gave rise to similar caspase kinetics, and no beneficial effect of TQ on the mitochondrial membrane potential ∆Ψ m was seen when applied in combination with DOX. Either of the two quinones gave a pro-apoptotic effect in 518A2 cells when applied alone, whereas their mixture evoked an anti-apoptotic effect due to a higher level of antiapoptotic bcl-2. 518A2 melanoma cells are known to have a bcl-2-related blockade in their mitochondrial pathway of apoptosis resulting in caspase-8 inhibition [START_REF] Benimetskaya | Relative bcl-2 independence of drug-induced cytotoxicity and resistance in 518A2 melanoma cells[END_REF][START_REF] Budihardjo | Biochemical pathways of caspase activation during apoptosis[END_REF]. When apoptosis eventually occurs it mainly relies on caspase-9 that can be activated by a cytochrome c translocation which is independent of a mitochondrial transmembrane depolarisation [START_REF] Bossy-Wetzel | Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independent of mitochondrial transmembrane depolarization[END_REF]. As in HL-60 cells the combination of DOX and TQ caused an increase of reactive oxygen species (ROS) in 518A2 melanoma cells, eventually leading to growth inhibition.

In summary, thymoquinone is a potential booster for the in vitro anti-cancer effect of doxorubicin. Depending on the cancer cell line distinct improvements of efficacy, selectivity, and breach of multi-drug resistance were observed for equimolar mixtures of doxorubicin and thymoquinone. Values represent means of four independent experiments ± standard deviations.

c Values taken from [START_REF] Breyer | Effects of thymoquinone-fatty acid conjugates on cancer cells[END_REF]. d Values taken from [START_REF] Effenberger | Modulation of doxorubicin activity in cancer cells by conjugation with fatty acyl and terpenyl hydrazones[END_REF]. * Significant decrease of intact mitochondria / ROS generation vs. untreated controls (P < 0.05).

25

((Figure 1))

  cells from the Department of Oncology and Haematology of the Martin-Luther University, Halle (Germany), the KB-V1 cervix and the MCF-7 breast carcinoma cells from the Institute of Pharmacy of the University of Regensburg (Germany), and the HT-29 colon carcinoma cells as well as the human foreskin fibroblasts (HF) from the University Hospital of Erlangen (Germany). The multi-drug resistant cells were bred by repeated treatment with vinblastine sulfate (KB-V1/VBL: 340 nM), topotecan hydrochloride (MCF-7/TOPO: 550 nM), colchicine (HT-29/COLC: 62.5 nM) and doxorubicin (KB-V1/DOX: 200 nM, MCF-7/DOX: 55 nM, HT-29/DOX: 75 nM). The HL-60 and HT-29 cells were grown in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), 100 IU mL -1

  )) Finally, we assessed the ability of the test compounds to initiate the generation of ROS in p53-negative HL-60 and p53-mutated 518A2 cells by means of the colorimetric nitroblue-tetrazolium (NBT) assay which is based on the selective reduction of a yellow, water-soluble tetrazolium chloride to an insoluble violet diformazan by superoxide (O 2 -• ) [20]. The p-quinones DOX and TQ can act either as anti-oxidants or pro-oxidants depending on the given redox environment. The anti-proliferative and apoptotic effects of TQ in human colon cancer cells but not in normal intestinal cells were shown by El-Najjar et al. to be induced via the generation of ROS [27]. Similary, different apoptosis-triggering mechanisms in

  contrast, a diminished growth inhibitory effect of the 1:1-mixture of the two quinones was observed for 518A2 melanoma and HT-29/COLC colon cancer cells. In multi-drug resistant KB-V1/VBL cervix carcinoma cells no difference between DOX and its mixture with TQ was noticed. The interference of DOX with the BCRP transporter of the MCF-7/TOPO cells, as characterised by the ratio (R +/-) of IC 50 (72 h) values in the presence and in the absence of the specific inhibitor fumitremorgin C, was attenuated by a factor of ca. 3.5 upon coincubation with equimolar amounts of TQ. In the case of the HT-29/COLC cells, similar experiments with the specific MRP 1 inhibitor MK-571 revealed a less pronounced effect of TQ. The mixture of DOX and TQ was also more efficacious than DOX alone against HT-29 cells that had been desensitised by repeated exposure to DOX (CI < 1). This is remarkable since TQ itself exhibited the same low activity in DOX-sensitive and DOX-resistant HT-29 cells.The mode of action of both drugs and of their equimolar mixture was mainly apoptotic. Treatment of HL-60 cells with DOX, TQ or its equimolar mixture caused an induction of apoptosis by activating caspase-3 and -8. The mixture of the two quinones led to a greater decrease of intact mitochondria in HL-60 cells than the pure compounds. These beneficial effects of TQ could explain the observed quadruplication of growth inhibition and the rise of ROS in HL-60 cells treated with a mixture of TQ and DOX. Judged by the caspase kinetics there seem

2 106 ±

 106 10 94 ± 9 77 ± 1 * 63 ± 8 * 0.8 ± 0.2 c 1.2 ± 0.1 c 1.0 ± 0.0 c 1.0 ± 0.0 c 1+2 99 ± 14 71 ± 12 64 ± 4 * 32 ± 8 * 6.2 ± 0.4 * 26 ± 5 * 12 ± 2 * 40 ± 6 * a Ratio of red to green fluorescence relative to untreated controls (100%) after 6 h and 18 h exposure of 518A2 melanoma and HL-60 leukaemia cells to 5 µM of the test compounds. Determined with the Mitochondrial Membrane Detection Kit (Stratagene, La Jolla, CA, USA). Values represent means of four independent experiments ± standard deviations. b NBT reduction as determined from percent absorbance of formazan relative to untreated controls (1%) after 24 and 72 h exposure of 518A2 melanoma and HL-60 leukaemia cells to the test compounds.

Table 1 .

 1 Inhibitory concentrations IC 50 a in µM of DOX[START_REF] Cragg | Impact of natural products on developing new anti-cancer agents[END_REF], TQ[START_REF] Colucci | Natural and synthetic quinones and their reduction by the quinone reductase enzyme NQO1: from synthetic organic chemistry to compounds with anticancer potential[END_REF] and an equimolar mixture of 1 and 2 when applied for 72 h to 518A2, HL-60, KB-V1, MCF-7, HT-29 cancer cells and foreskin fibroblasts (HF). VBL 18.3±7.6 d 1.0±0.04 d 32.3±6.0 c 24.1±3.5 c 20.9±3.5 0.44±0.16 TOPO 1.0±0.4 d 0.08±0.01 d 26.7±5.6 c 23.1±2.5 c 0.64±0.22 0.18±0.01 Values are derived from concentration-response curves obtained by measuring the percentual absorbance of viable cells relative to untreated controls (100%) after 72 h exposure of 518A2 melanoma, HL-60 leukaemia, KB-V1 cervix carcinoma, MCF-7 breast carcinoma and HT-29 colon carcinoma cells as well as human foreskin fibroblasts (HF) to the test compounds in the MTT assay. Values represent means of four independent experiments ± standard deviations. b With 24 µM verapamil hydrochloride added for KB-V1/VBL, with 1.2 µM fumitremorgin C added for MCF-7/TOPO and with 10 µM MK-571 added for HT-29/COLC cells. c Values taken from [11]. d Values taken from [12].

	Compd	1	1 b	2	2 b	1+2	1+2 b
	518A2	0.12±0.04 d	-	28.3±9.2 c	-	0.33±0.03	-
	HF	6.9 ±3.7	-	32.6±19.6 c	-	0.29±0.09	-
	HL-60	0.08±0.01 d	-	27.8±6.0 c	-	0.02±0.01	-
	HT-29	0.85±0.11	-	46.8±18.7	-	1.1±0.4	-
	HT-29/COLC 0.69±0.10 0.10±0.03 57.2±27.9 77.5±14.1 0.83±0.22 0.17±0.03
	HT-29/DOX 0.42±0.12	-	39.0±12.6	-	0.35±0.21	-
	KB-V1	0.05±0.01	-	25.8±13.4	-	0.18±0.02	-
	KB-V1/KB-V1/DOX 1.8±0.6	-	18.7±1.6	-	5.6 ±1.3	-
	MCF-7	0.09±0.01	-	20.1±2.0	-	0.26±0.02	-
	MCF-7/MCF-7/DOX 1.7±0.4	-	40.2±2.3	-	5.6±1.2	-

a

Table 2 .

 2 Intact mitochondria [%] a and increase of ROS [%] b upon treatment of 518A2 melanoma and HL-60 leukaemia cells with 5 µM of DOX (1) or TQ(2) or an equimolar mixture of 1 and 2.

			Mitochondria [%]			ROS [%]
	Comp		518A2	HL-60	518A2	HL-60
		6 h	18 h	6 h	18 h	24 h	72 h	24 h	72 h
	1	98 ± 10 d 74 ± 19 d 83 ± 8 d,* 28 ± 4 d,*	2.5 ± 0.3 d,*	15 ± 2 d,*	2.8 ± 0.8 d,*	5.6 ± 0.8 d,*

((Figure2))

((picture for graphical abstract))

Acknowledgements

We thank the Deutsche Forschungsgemeinschaft for financial support (grant Scho 402/8-2), Ribosepharm GmbH, Gräfelfing (Germany) for a free batch of doxorubicin, and Prof. M. Ocker (Marburg) for providing the luminometric caspase kit and the facilities for measuring the bax and bcl-2 mRNA expression.

((Caption to Scheme 1))

Two biologically active natural products with para-quinone motif: doxorubicin (DOX, 1) isolated from Streptomyces peucetius var. caesius and thymoquinone (TQ, 2) a constituent of the essential oil of black seed (Nigella sativa).

((Caption to


Jgh G H Van Nes

H Putter

M Van Hezewijk

Etm T M Hille

H Bartelink

L Collette

Professor Cjh J H Van De Velde
email: c.j.h.van_de_velde@lumc.nl

Tailored follow-up for early breast cancer patients

Keywords: breast cancer, locoregional recurrence, prognostic index, follow-up

Tailored follow-up for early breast cancer patients: A prognostic index that predicts locoregional recurrence

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

Index for LRR

INTRODUCTION

Background

Early breast cancer patients are enrolled into a follow-up programme after treatment. The key elements of this programme are periodic visits for history taking, physical examination and annual surveillance mammograms. [START_REF] Khatcheressian | American Society of Clinical Oncology 2006 update of the breast cancer follow-up and management guidelines in the adjuvant setting[END_REF] This follow-up schedule is intensive and stressful for patients and the cost-effectiveness is low. 2;3 Besides, it has led to considerable workload for physicians, especially surgeons: worldwide more than 7.5 million patients are seen at outpatient clinics yearly. These problems will increase as a result of a number of trends. First, the majority of patients currently have early-stage disease and therefore more than 80% are expected to survive five years or longer. [START_REF] Jemal | Cancer statistics[END_REF] Second, better survival results are obtained due to new systemic therapies and accordingly more patients will live longer. Third, only a minority of patients are discharged of follow-up; it was shown that only 15% of patients are discharged at five years and 43% at 10 years. 5

Aims of follow-up

Follow-up of breast cancer patients has several aims. The first year is particularly important for quality of life of patients and monitoring of treatment and side-effects. After this first year, the main goal of follow-up is early detection of second primary breast tumours and locoregional recurrences (LRR) at an early stage in order to begin immediate potentially curative therapy. 6;7 Early detection of distant metastases is not an aim because these cannot be cured. It was shown that a more intensive follow-up strategy including additional investigations to detect distant metastasis did not result in a survival benefit. 8;9 Early detection of distant metastasis will only result in a poorer quality of life for patients because of the knowledge of having an incurable disease and the side effects of earlier treatment that will not result in longer lifespan.

Tailoring

In contrast to local and systemic treatment regimens, follow-up is not based on patient or tumour characteristics. All patients follow the same regimen in spite of different risk profiles. Risk of LRR

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

ACCEPTED MANUSCRIPT Index for LRR differs between patients; patients treated with breast conserving surgery (BCS) are at higher risk for LRR than patients treated with a radical mastectomy. [START_REF] Van Dongen | Long-term results of a randomized trial comparing breast-conserving therapy with mastectomy: European Organization for Research and Treatment of Cancer 10801 trial[END_REF] A better definition of what constitutes a patient at high or low risk of LRR would be valuable to improve follow-up. Different indices were developed that integrate combination of factors to identify high risk patients, like Adjuvantonline! and the Nottingham Prognostic Index. [START_REF] Haybittle | A prognostic index in primary breast cancer[END_REF] However, these indices emphasise at overall survival, not at LRR.

One of the gene-expression profiles identified subgroups of patients at increased risk of local recurrence after BCS. [START_REF] Nuyten | Predicting a local recurrence after breast-conserving therapy by gene expression profiling[END_REF] However, to construct and validate this profile, very young breast cancer patients (80% <40 years old) were used and this does not reflect common breast cancer patients.

Therefore, a prognostic index for LRR is lacking. To address this issue, we developed and validated a prognostic model for LRR. For extensive clinical use of such an index, our aim was not to derive a precise risk indicator but a simple model which can be determined without difficult calculations and which can be applied for all early breast cancer patients treated according to general guidelines. To establish this, we used clinicopathological data from four different international trials with almost 10000 patients with an adequate follow-up.

METHODS

Patients

Data of four large international trials with a long follow-up were used, the European Organisation of The EORTC trial 10854 (the so-called "Peri-operative chemotherapy, POP-trial") included patients with early breast cancer followed by surgery with curative intent from 1986 to 1991 to study whether one course of peri-operative chemotherapy yields better therapeutic results than surgery alone. [START_REF] Clahsen | Improved local control and disease-free survival after perioperative chemotherapy for early-stage breast cancer. A European Organization for Research and Treatment of Cancer Breast Cancer Cooperative Group Study[END_REF] The patients from the surgery alone arm were included for the current analysis (n=1 395/2 793). Surgery consisted of either modified radical mastectomy or BCS, both followed by axillary clearance. Axillary lymph node positive premenopausal patients were recommended to receive five courses of CMF.

Radiotherapy was given after BCS to the whole breast (50 Gy) followed by a boost on the initial tumour site (16 Gy). Besides, radiotherapy was given in all cases in which surgery was considered not to be radical. Prolonged adjuvant systemic treatment was up to the discretion of local physicians.

The EORTC trial 10902 (the so-called "Preoperative chemotherapy in operable breast cancer, POCOB-trial") included early breast cancer patients (T1c, T2, T3, T4b, N0-1 and M0) from 1991 to 1999 to evaluate the value of preoperative chemotherapy. [START_REF] Van Nes | Preoperative chemotherapy is safe in early breast cancer, even after 10 years of follow-up; clinical and translational results from the EORTC trial 10902[END_REF] The patients from the postoperative chemotherapy arm were included (n= 348/698) for this analysis. Chemotherapy consisted of four cycles of fluorouracil, epirubicin, and cyclophosphamide (FEC).

The EORTC trial 22881, the so-called "boost versus no boost trial", included patients (n=5569) from

1989 to 1996. [START_REF] Bartelink | Impact of a Higher Radiation Dose on Local Control and Survival in Breast-Conserving Therapy of Early Breast Cancer: 10-Year Results of the Randomized Boost Versus No Boost EORTC 22881-10882 Trial[END_REF] The aim of this trial was to evaluate the value of a boost dose after primary BCS. All patients were treated with BCS and axillary dissection, followed by irradiation of the whole breast ( 50Gy). Patients with microscopically complete excisions were randomised to a boost (16 Gy) to the tumour bed and no boost. Patients with microscopically incomplete excisions were randomised to receive a boost (10 or 26 Gy) to the tumour bed versus no boost.

Statistical analysis

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

Index for LRR

To use only early breast cancer patients, all patients with T3 and T4 tumours and/or with more than 10 positive nodes were excluded. Patients with preoperative chemotherapy were also excluded because of the influence on the pathological tumour and nodal status. Finally, patients treated with perioperative chemotherapy and patients treated with BCS without radiotherapy were excluded because this is not the standard anymore. In our analysis, systemic treatment (endocrine and chemotherapy) was not included as explanatory variables because the effects of such treatment are mainly on distance recurrence and these drugs and schedules have been subject to change over the last decades.

Time from randomisation to the first occurrence of local recurrence was considered. Patients were censored at time of last follow-up, distant recurrence or death. Univariable and multivariable models were fitted using Cox proportional hazards regression, stratified by the different EORTC studies. The bootstrap was used for internal validation to assess stability of prognostic variables selected. [START_REF] Sauerbrei | A bootstrap resampling procedure for model building: application to the Cox regression model[END_REF] The cross-validation procedure described by Verweij et al was used. [START_REF] Verweij | Cross-validation in survival analysis[END_REF] After deriving the prognostic index, cumulative incidence of LRR, accounting for distant recurrences and death as competing risk was calculated. [START_REF] Putter | Tutorial in biostatistics: competing risks and multi-state models[END_REF] Harrell's c index was used to quantify the predictive value of the index. [START_REF] Harrell | Multivariable prognostic models: issues in developing models, evaluating assumptions and adequacy, and measuring and reducing errors[END_REF] Harrell's c index ranges from 0.5 to 1.0 with 0.5 indicating no predictive value and 1.0 perfect discrimination.

However, if the index is not continuous but divided into groups, the maximum possible value of the Harrell's c index is <1.0. 20

RESULTS

Patients, tumour and treatment characteristics are shown in Table 1. A total of 9 964 patients were included in the four EORTC trials. Exclusion of patients not treated according to current guidelines (n=2 285) and with incomplete covariable information on the covariables (n=1 163) considered left 6 516 patients for our analysis. No significant differences in LRR rates were found between the 6 516 patients considered and the 1 163 patients with incomplete covariables (p=0.86).

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

ACCEPTED MANUSCRIPT Index for LRR

Constructing of the index

Since mastectomy and BCS followed by radiotherapy (BCT) constitute different (surgical) techniques with different prognostic effects, multivariable Cox regressions were first performed for mastectomy and BCT separately. The results in Table 2 show that the effect of most prognostic factors is similar for mastectomy and BCT, with two exceptions. The first is the boost, which is only defined for BCT.

The second is nodal status. For BCT, the effect of nodal status was not significant, but for mastectomy it was highly significant (p<0.0001). The interactions between age, tumour stage and surgical margins on the one hand and type of surgery (mastectomy versus BCT) on the other were not significant (p=0.74, 0.79, and 0.41, respectively). The interaction between nodal status and type of surgery was highly significant (p=0.003), suggesting different effects of nodal status for BCT and mastectomy.

Therefore, an overall model was considered with identical effects of age, tumour stage and surgical margins across types of surgery, and with separate effects of nodal status and radiotherapy/boost. The effect of nodal status for BCT was again observed to be virtually zero, so the final model was obtained by deleting the nodal status effect for BCT. The estimated regression coefficients and hazard ratios of this final model are shown in Table 2. The internal bootstrap validation showed that selection of variables was quite stable; each of the variables of the final model was selected in more than 80% of the 500 bootstrap models, with the exception of surgical margins, which was selected in 79% of the bootstrap models. A continuous index can be constructed by adding the regression coefficients, depending on the covariable values of the patient. A histogram of the values of this index in our population is given in Figure 1. The univariable hazard ratio of this continuous prognostic index was estimated as 2.72 per unit increase of the index with a 95% confidence interval from 2.31 to 3.20 (P<0.0001). The corresponding regression coefficient is 1 by definition. A cross-validated prognostic index was calculated for each individual following the procedure of Verweij and van Houwelingen. [START_REF] Verweij | Cross-validation in survival analysis[END_REF] The estimated regression coefficient of such a cross-validated prognostic index is typically <1 and can be regarded as a shrinkage factor, with values well below 1, say <0. For an individual patient, the value of the continuous prognostic index can be calculated by adding the regression coefficients of Table 2, depending on the patient's covariable values. The patient can then be assigned to a risk group, depending on whether the value of this prognostic index is below or above 1.4 and 2.1. To facilitate implementation into clinical practise, a decision tree was constructed, one for patients treated with mastectomy and one for patients treated with BCT (figure 3). The tree is stopped whenever taking further covariables into account will not change the resulting risk group. The number within brackets indicates the value of the continuous prognostic index so far.

DISCUSSION

Prognostic factors

The goal of this analysis was to develop and validate a simple prognostic index for LRR for all patients with early breast cancer who are treated according to acceptable standards. Well-defined variables were used that can be combined to produce a prognostic index. This index is simple and the decision tree can be used immediately after surgery/local therapy and gives very good discrimination, as shown by the Kaplan Meier curve.

The first distinction was local therapy: mastectomy versus BCT. The overall survival of patients treated with BCT is comparable to the one of patients treated with mastectomy. However, their chance on LRR is 4 times higher after twenty years of follow-up. [START_REF] Veronesi | Twenty-year follow-up of a randomized study comparing breast-conserving surgery with radical mastectomy for early breast cancer[END_REF] Radiotherapy after mastectomy and a boost in patients treated with BCT have previously shown to decrease the chance of a LRR in patients and were included in our index. 15;22 Age is the second factor in this prognostic index. Significance of age was already recognised in other studies; young age is independently associated with unfavourable outcome in patients with early breast cancer. 23;24 In the EORTC 22881 trial, disease recurrence in the ipsilateral breast or in the axilla as first event was more seen in younger patients. [START_REF] Bartelink | Impact of a Higher Radiation Dose on Local Control and Survival in Breast-Conserving Therapy of Early Breast Cancer: 10-Year Results of the Randomized Boost Versus No Boost EORTC 22881-10882 Trial[END_REF] A boost reduced the rate of LRR, however, this effect was mainly seen in younger patients (≤ 40 years) and was less prominent in older patients (≥ 51 years). Tumour stage was also an important factor. Unfortunately,

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

ACCEPTED MANUSCRIPT Index for LRR Version EJSO10 not all pathological tumours stage were known, this was especially the case in the EORTC 10854 trial, therefore, unfortunately, the majority of patients of this trial were excluded for this analysis. The pathological nodal status was only important for LRR in patients treated with mastectomy. The last factor of influence in the index are the surgical margins: positive margins are correlated with a higher chance on LRR.

Limitations

Information about histological grade was not available, this factor was not used in our analyses.

Several studies investigating predictors of LRR demonstrated that grade was an important factor, other studies did not demonstrated it. 25;26 We do not have information concerning relatively new prognostic and predictive factors like ER, PgR and HER2. If we want to include these factors in the index we need information of newer studies with a shorter follow-up than the current used studies. We prefer to have a long follow-up. In the future, new factors can be included, like hormone receptor status, HER2, grade and gene expression arrays.

Several factors limit accuracy of the index to predict the exact percentage of LRR. Firstly, the trials included patients over different time periods. Over time, local therapies and administration of systemic therapies have been improved. Consequently, patients will live longer and are therefore longer exposed to risk of LRR. Secondly, treatment options differed between trials; they included different kinds of patients. Mastectomy was compared to BCT in the EORTC trial 10801 while all patients in the EORTC trial 22881 were treated with BCT. However, because this index was developed using different trials, heterogeneity in prognostic risk in early breast cancer patients was realistically reflected. And thirdly, factors considered important in our prognostic index were not routinely collected in all trials. In the EORTC trial 10854, the pathological T stage was not recorded for all patients. In spite of all differences mentioned above, an index was constructed and validated. Also, the fact that data from four different trial populations were used enabled us to study the internal validity in a natural way. This indicates that this index can be useful in different clinical settings, for example to improve the value of follow-up.

Follow-up

Doubts about the value of routine follow-up have been expressed for many years and the rationale of follow-up is still subject of discussion. 27;28 It was shown that routine follow-up confers little survival benefit and increases distress in patients. 8;29 Moreover, it was suggested that clinical examinations do not improve clinical outcome. [START_REF] Montgomery | Changing pattern of the detection of locoregional relapse in breast cancer: the Edinburgh experience[END_REF] Consequently, research was performed to examine both the burden and health gains of different follow-up programmes. However, in all these studies, different risks of LRR were not taken into account. LRR are potentially curable and therefore should be detected early.

When follow-up is risk based for LRR using our index, it could be more efficient.

In the Netherlands, an implementation study will be performed to evaluate a new follow-up schedule based on our index. We do not aim to modify the first year visits for any patient, as optimising quality of life after local therapy is equally relevant to all patients following treatment for breast cancer. This physical and psychosocial rehabilitation of patients will in general take one year. Therefore, we will tailor the follow-up after the first year. Patients with an intermediate risk on LRR according to the prognostic index will follow the routine follow-up programme (the second year twice and thereafter yearly, www.oncoline.nl). Patients at low risk can be seen less frequently (visit once every two years)

and patients at high risk should be seen more often (visit twice every year). Mammographies will be taken yearly in every group and questionnaire will be filled in by patients and physicians to evaluate the new schedule and quality of life of patients.

In conclusion, we constructed and validated a continuous prognostic index that divided patients in three groups according to risk of LRR. We will incorporated this index in our follow-up schedule to make it (more) evidence based.

ACKNOWLEGDEMENTS

We are grateful to the EORTC Datacentre and all the institutions collaborating in the EORTC trials for their help and data, without which this work would not have been possible. We thank Jan Bogaerts, Jérôme Rapion, for their support with the databases and Richard Sylvester for his advice. And of course we are in debt to all patients participating in these trials.

  Research and Treatment of Cancer (EORTC) trials 10801, 10854, 10902 and 22881. The EORTC trial 10801 included patients (n=902) from 1980 to 1986 in order to assess the safety of breast conserving treatment (BCT). 10 Eligible patients had clinical stage I or II early breast cancer and were randomised between radical mastectomy and BCS followed by radiotherapy. Radiotherapy consisted of irradiation to the breast (50 Gy), with an additional booster dose (25 Gy) directed to the lumpectomy site. For all patients of ≤55 years with pathological nodal positive disease, six cycles of M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT Index for LRR chemotherapy with cyclophosphamide, methotrexate, and 5-fluorouracil (CMF) were indicated. No information was available on endocrine therapy.

8 ,

 8 indicating overfitting. Applying this procedure resulted in an estimated regression coefficient of 0.94 (standard error 0.08), indicating that the predictive value of the proposed index is almost equally good on new data as on the present data.

Figure 1 :

 1 Figure 1: Histogram of LRR prognostic index. On the x-axis the value of the added regression

Figure 2 :

 2 Figure 2: Estimated cumulative incidences of locoregional recurrence for each of the three proposed

Figure 3 .

 3 Figure 3. The decision tree for patients treated with breast conserving and with mastectomy. The tree

  Abbreviations: BCT: breast conserving therapy; LRR: locoregional recurrence; RT: radiotherapy

Figure 1 :Figure 2 :Figure 3 .

 123 Figure 1: Histogram of LRR prognostic index. On the x-axis the value of the added regression coefficients. On the y-axis, the number of patients.

Table 1 :

 1 Patient and tumour treatment characteristics of patients in the different EORTC trials. Data are shown in amount (grey row) and percentages (white row).

Table 2 :

 2 Estimated regression coefficients and associated hazard ratios for the penalized multivariable Cox regression. Last column: deriving the simple score

Table 1 :

 1 Patient and tumour treatment characteristics of patients in the different EORTC trials. Data are shown in amount (grey row) and percentages (white row).

		EORTC		EORTC		EORTC		EORTC		Total
		10801		10854,		10902, post	22881
				standard		operative
				arm		chemo arm
		N	%	N	%	N	%	N	%	N	%
	Patients in trials	902	100 1395/	100 348/	100	5569	100 9964
				2795		698
	Patients used in analysis	752	100	190	100	205	100	5369	100 6516
	Age at randomisation
	<40	64	9	48	25	3913	19	453	8	604
	40-60	479	64	141	74	144	70	3179	59	3943
	≥60	209	28	1	1	22	11	1737	32	1969
	Tumour stage
	pT1	363	48	91	48	82	40	4293	80	4829
	pT2	389	52	99	52	123	60	1076	20	1687
	Nodal stage
	pN-	455	61	190	100	79	39	4237	79	4961
	pN+	297	39	0	0	126	61	1132	21	1555
	Local treatment
	Mastectomy -RT	209
	Mastectomy + RT	154
	BCT -boost	0
	BCT + boost	389

Table 2 :

 2 Estimated regression coefficients and associated hazard ratios for the penalized multivariable Cox regression. Last column: deriving the simple score

		All patients	Mastectomy	Breast conserving therapy
	Age at	Coefficient	Hazard ratio	Coefficient	Hazard ratio	Coefficient	Hazard ratio
	randomisation	(SE)	(95%CI)	(SE)	(95%CI)	(SE)	(95%CI)
	≥60		1.00		1.00		1.00
	40-60	0.368 (0.092)	1.44 (1.21 -1.73)	0.656 (0.447)	1.93 (0.80 -4.63)	0.357 (0.094)	1.43 (1.19 -1.72)
	<40	1.062 (0.119)	2.89 (2.29 -3.64)	1.089 (0.563)	2.97 (0.99 -8.96)	1.062 (0.122)	2.89 (2.28 -3.67)
	Tumour stage
	pT1		1.00		1.00		1.00
	pT2	0.317 (0.083)	1.37 (1.17 -1.61)	0.412 (0.302)	1.51 (0.84 -2.73)	0.315 (0.087)	1.37 (1.16 -1.62)
	Nodal stage
	pN-		1.00		1.00		1.00
	pN+ (only Mast)	0.880 (0.310)	2.41 (1.31 -4.43)	0.889 (0.328)	2.43 (1.28 -4.63)	-0.080 (0.096)	0.92 (0.77 -1.11)
	Local treatment
	Mastectomy + RT		1.00		1.00
	Mastectomy -RT	0.960 (0.328)	2.61 (1.37 -4.97)	1.019 (0.355)	2.77 (1.38 -5.55)
	BCT + boost	1.217 (0.361)	3.38 (1.66 -6.85)				1.00
	BCT -boost	1.574 (0.366)	4.83 (2.36 -9.90)			0.353 (0.083)	1.42 (1.21 -1.67)
	Surgical margins
	Negative		1.00				1.00
	Positive	0.426 (0.145)	1.53 (1.15 -2.03)	NA*		0.438 (0.146)	1.55 (1.16 -2.06)

* Could not be estimated

Abbreviations: BCT: breast conserving therapy; RT: radiotherapy

Version EJSO10

Different risk groups

The LRR index is to be used for tailored follow-up. We are aiming for a low-risk group, with a 10years LRR probability of at most 7.5%, a medium-risk group, with a 10-years LRR probability between 7.5% and 12.5%, and a high-risk group with a 10-years LRR probability of at least 12.5%.

The low-risk group will get less follow-up, the high-risk group more. The corresponding cut-off values of the prognostic index were found to be 1.4 and 2.1, for a 10-years LRR probability of 7.5% and 12.5%, respectively. With a low-risk group defined as having a prognostic index ≤1.4, a high-risk group defined as having a prognostic index >2.1, and the medium-risk group with a prognostic index between 1.4 and 2.1, in our study population 1 027, 4 389, and 1 100, patients were low risk, medium risk, and high risk, respectively. Figure 2 shows the estimated cumulative incidence functions for each of these three risk or follow-up intensity groups. The hazard ratios (95% confidence interval) of medium risk and high risk with respect to low risk were estimated as 2.00 (1.53 -2.62) and 3.91 (2.94 -5.21), respectively. The 10-years estimated probabilities of LRR (95% confidence interval) were 0.05 (0.04 -0.07), 0.11 (0.10 -0.12), and 0.19 (0.16 -0.22), for low risk, medium risk and high risk groups respectively. When the index was executed in another breast cancer population (a ten year cohort of our own centre), the same distinction was seen, however, there were more patients included in the low risk group compared to the high risk group (data not shown).

Harrel's c-index was estimated as 0.61 for the continuous prognostic index and 0.59 for the three prognostic groups, indicating a moderate loss of predictive accuracy as a result of categorising the prognostic index. These values indicate only modest predictive accuracy, however, such values are quite common in the context of survival analysis. For the continuous index, Harrel's c-index was estimated as 0.65 for patients treated with a mastectomy and 0.61 for patients treated with BCT. For the three prognostic groups, Harrel's c-index was estimated as 0.60 for patients treated with a mastectomy and 0.58 for patients treated with BCT.