D Sarkisyan

G Hakhumyan

A Sargsyan

R Mirzoyan

C Leroy

Y Pashayan-Leroy

Laser Spectroscopy with Nanometric Cells Containing Atomic Vapor of Metal: Influence of Buffer Gas

Keywords: Atomic spectroscopy, nano-cell, buffer gas, coherent narrowing, electromagnetically induced transparency

Comparison of absorption and fluorescence in a nano-cell containing Rb vapor with other Rb nano-cells where neon gas is added is presented. The effect of collapse and revival of Dicke-type narrowing is still observable for the Rb nano-cells where Ne gas is added under 6 and 20 Torr pressure for the thickness L=/2 and L=, where  is a resonant laser wavelength 794 nm (D 1 line). Particularly for 6 Torr the line-width of the transmission spectrum for the thickness L=/2 is 2 times narrower than that for L=. For an ordinary Rb cell with L = 0.1 -10 cm with addition of buffer gas, the velocity selective optical pumping/saturation (VSOP) resonances in saturated absorption spectra are fully suppressed when the buffer gas pressure > 0.5 Torr. A spectacular difference is that for L=, VSOP resonances located at the atomic transition are still observable even when Ne pressure is ≥ 6 Torr. Narrowband fluorescence spectra of the nano-cell with L= λ/2 can be used as a convenient tool for online buffer gas pressure monitoring for the conditions when ordinary pressure gauges are unusable. Comparison of electromagnetically induced transparency (EIT) effect in a nano-cell filled with pure Rb with another nano-cell, where buffer gas nitrogen is added, is presented. Use of gas N 2 inside the Rb nanocell strongly extended the region of the coupling laser detuning where EIT resonance is still possible to form.

INTRODUCTION

A number of works published in recent years deal with the laser spectroscopy of the resonance absorption and fluorescence of an atomic vapor column of 10-100 m thickness [START_REF] Briaudeau | Sub-Doppler spectroscopy in a thin film of resonant vapor[END_REF]2] and of an atomic vapor column enclosed in socalled extremely thin cells (ETC), or nano-cells [START_REF] Sarkisyan | Sub-Doppler spectroscopy by sub-micron thin Cs-vapor layer[END_REF][START_REF] Sarkisyan | Optical processes in micro-and nanometric thin cells containing atomic vapor[END_REF] filled with pure metallic Cesium and/or Rubidium. It was shown in the works [START_REF] Dutier | Revisiting optical spectroscopy in a thin vapour cell: mixing of reflection and transmission as a Fabry-Perot microcavity effect[END_REF][START_REF] Dutier | Collapse and revival of a Dicketype coherent narrowing in a sub-micron thick vapor cell transmission spectroscopy[END_REF][START_REF] Sarkisyan | Sub-Doppler Fluorescence on atomic D 2 line of Sub-Micron Rubidium Vapor Layer[END_REF][START_REF] Nikogosyan | Resonant absorption and fluorescence of atomic layer of the thickness of the order of light wavelength[END_REF][START_REF] Sarkisyan | Spectroscopy in an Extremely Thin Vapour Cell: Comparing the Cell Length Dependence in Fluorescence and in Absorption Techniques[END_REF][START_REF] Andreeva | Saturation effects in the sub-Doppler spectroscopy of cesium vapor confined in an extremely thin cell[END_REF][START_REF] Varzhapetyan | Study of atomic transition self-broadening and shift with the help of a nano-cell[END_REF][START_REF] Sargsyan | Dependence of the Cs absorption D 1 linewidth on the thickness of atomic vapor column[END_REF][START_REF] Cartaleva | Sub-Doppler spectroscopy of cesium vapor layers with nanometric and micrometric thickness[END_REF] that an important parameter determining the spectral width, the shape of the line, and the absorption in these nano-cell, is the ratio L/ with L being the thickness of the vapor column (i.e. the distance between the nano-cell windows) and  is the wavelength of the laser radiation resonant with the corresponding atomic transition of Cs or Rb, D 2 and D 1 lines. In particular it was shown that the spectral width of the resonant absorption reaches its minimum value at L = (2n + 1) /2 (n is an integer); this effect has been termed the Dicke coherent narrowing (DCN) effect. Note that the spectral width of the resonant absorption reaches its absolute minimum value for L =  /2 which is more than 4 times narrower than the Doppler width, recorded in the cells of the conventional length (CCL) 0.1-10 cm (note that the Doppler width of hyperfine transition in CCL is about of 350 MHz for Cs and ~500 MHz for the Rb, at room temperature). Relative contrast of spectral width decreases with respect to the broad pedestal which increases proportionally to L = 3 /2, 5 /2 , 7 /2 etc. Simple calculation shows that the the Dicke coherent narrowing should be observable up to the thickness L max = Vt, where V is the mean thermal velocity of Cs /Rb atoms (~250 / 200 m/s), and the t is excited state lifetime (~30 /27 ns), that is L max ~ 7.5 /5.4 m for the Cs/Rb atomic vapor, correspondingly.

It was also demonstrated [START_REF] Nikogosyan | Resonant absorption and fluorescence of atomic layer of the thickness of the order of light wavelength[END_REF][START_REF] Sarkisyan | Spectroscopy in an Extremely Thin Vapour Cell: Comparing the Cell Length Dependence in Fluorescence and in Absorption Techniques[END_REF][START_REF] Andreeva | Saturation effects in the sub-Doppler spectroscopy of cesium vapor confined in an extremely thin cell[END_REF][START_REF] Varzhapetyan | Study of atomic transition self-broadening and shift with the help of a nano-cell[END_REF][START_REF] Sargsyan | Dependence of the Cs absorption D 1 linewidth on the thickness of atomic vapor column[END_REF][START_REF] Cartaleva | Sub-Doppler spectroscopy of cesium vapor layers with nanometric and micrometric thickness[END_REF] that for L = n the spectral width of the resonant absorption reaches its maximum value, close to the Doppler width, recorded in the CCL. This phenomenon was termed collapse of DCN. For relatively high pump intensity (>1 mW/cm 2 ), the VSOP resonances of a reduced absorption (with the line-width close to the natural one), and centered on the hyperfine transitions occurs sharply when L = n, while the narrowest and well-marked VSOP resonance occurs when L = . There are several distinct benefits to use these VSOP resonances as a frequency reference for atomic transitions of alkali atoms in comparison with widely used Saturation Absorption (SA) technique. Particularly, use of the nano-cell with the thickness L=  allows the complete elimination of crossover (CO) resonances when L = . "-Zeeman technique" (LZT) is shown to be efficient for studies of individual transitions between the Zeeman sublevels of hyperfine levels in an external magnetic field of 1 -5000 G [START_REF] Sargsyan | A novel approach to quantitative spectroscopy of atoms in a magnetic field and applications based on an atomic vapor cell with L=λ[END_REF]. LZT is based on nano-cell with thickness L = . Narrow VSOP resonances in the transmission spectrum of the ETC are split into several components in a magnetic field; their frequency positions and probabilities depend on the B-field .

In contrast to the transmission spectra, when L increases, the spectral width of the fluorescence spectra increases monotonically, without oscillations of the line-width versus L, while remaining below the Doppler broadening even when the thickness of the vapor column increased up to a 3-4 μm [START_REF] Sarkisyan | Sub-Doppler spectroscopy by sub-micron thin Cs-vapor layer[END_REF][START_REF] Sarkisyan | Optical processes in micro-and nanometric thin cells containing atomic vapor[END_REF][START_REF] Sarkisyan | Sub-Doppler Fluorescence on atomic D 2 line of Sub-Micron Rubidium Vapor Layer[END_REF][START_REF] Nikogosyan | Resonant absorption and fluorescence of atomic layer of the thickness of the order of light wavelength[END_REF][START_REF] Sarkisyan | Spectroscopy in an Extremely Thin Vapour Cell: Comparing the Cell Length Dependence in Fluorescence and in Absorption Techniques[END_REF][START_REF] Cartaleva | Sub-Doppler spectroscopy of cesium vapor layers with nanometric and micrometric thickness[END_REF]]. The narrowest line-width of the fluorescence spectrum is obtained when L is in the range /4 < L  /2 and is 1.5-1.8 times narrower than the absorption spectral width for L in the same range. Another difference between absorption and fluorescence processes obtained with the nano-cells is that VSOP resonances of a reduced fluorescence appear at much higher intensity (>20 mW/cm 2 ). The"/2-Zeeman technique" (HLZT) is shown to be efficient for studies of individual transitions between the Zeeman sublevels of hyperfine levels in an external magnetic field of 10 -2500 G. HLZT is based on resonant fluorescence spectrum emitted by a nano-cell with L = /2 [START_REF] Hakhumyan | Investigation of Rb D 1 Atomic Lines in Strong Magnetic Fields by Fluorescence from a Half-Wave-Thick Cell[END_REF][START_REF] Sarkisyan | Formation of Narrow Optical Resonances using submicron -thin atomic vapor layer[END_REF].

Considerable interest of coherent population trapping (CPT) and the related electromagnetically induced transparency phenomena is caused by a number of important applications in a variety of fields such as laser cooling, information storage, magnetometry, spectroscopy, atomic frequency references etc [17 -19]. EIT resonance can occurs in -system with a two long-lived states and one excited state coupled by two laser fields (so called coupling and probe lasers). From the application point of view it is important to reduce dimensions of the cell which are containing atomic vapor of metal where an EIT resonance is formed, while keeping resonance parameters good (such as narrow line-width and contrast) [START_REF] Knappe | Dark-line atomic resonances in submillimeter structures[END_REF][START_REF] Sargsyan | Dark-line atomic resonances in sub-micron thin Rb vapor layer[END_REF][START_REF] Pashayan-Leroy | Electromagnetically induced transparency: the thickness of the vapor column is of the order of a light wavelength[END_REF][START_REF] Sargsyan | Efficient technique for measuring laser frequency stability[END_REF][START_REF] Baluktsian | Fabrication method for microscopic cells for alkali atoms[END_REF].

We have developed a new nano-cells filled with the natural Rb where neon gas is added under 6 Torr and 20 Torr pressure [START_REF] Hakhumyan | Essential Features of Optical Processes in Rb submicron Thin Cell Filled with Neon Gas[END_REF]. Also, the nano-cell filled with the natural Rb where nitrogen gas is added is fabricated. Comparison of the above mentioned processes in nano-cell filled with pure Rb is provided both experimentally and theoretically. Multi-region cell consists of two interconnected cells and a side-arm containing metal. The nano-cell region (shown in the upper part of Fig. 1) has a wedged thickness of the gap between the windows that allows one to exploit atomic vapor column thickness in the range of 0.1 -4 m. The first design of the nano-cell (or so called extremely thin cell), which consists of windows and a vertical side-arm (a metal reservoir), was presented in [START_REF] Sarkisyan | Sub-Doppler spectroscopy by sub-micron thin Cs-vapor layer[END_REF]. Later, this design has been somewhat modified; the typical example of the last version is presented in Fig. 1. We use 15 × 25 mm rectangular window wafers, fabricated either from commercial sapphire or garnet (Y 3 Al 5 O 12 ) crystal, which are both chemically resistant to the chemically aggressive vapors of alkali metals. In order to minimize the birefringence effect, the sapphire plates are cut across the c-axis, while the garnet is free of birefringence. The wafers have to be perfectly polished (better than λ/20) before the use. In order to form the wedge gap in the vertical direction (this is needed to have a variable thickness of atomic vapor column-see the inset in Fig. 1), an Al 2 O 3 strip, ~1000 nm thick, 5 mm long, and 1 mm wide, is deposited on the surface of one of the windows in its lower part (in some cases, a platinum strip is used as a spacer: Pt is also resistant to highly corrosive hot Rb atomic vapor). Prior to the deposition, a 2 mm hole is drilled in the bottom of the windows, into which a 25 mm long tube of the same diameter, made of commercial sapphire, is inserted. With the help of this sapphire tube (inside diameter is of 1 mm) the nano-cell is connected with the 1 cm-long cell region (the lower part of Fig. 1) with 2 sapphire windows. The 1 cm-long sapphire cell with sapphire windows is terminated by with a 4-cm long sapphire side-arm. Then, the entire construction is assembled and glued in a vacuum furnace. After the gluing, a glass extension is sealed to the sapphire tube, and the multi-region cell is filled with a natural mixture of the 85 Rb (72%) and 87 Rb (28%) as well as with a buffer gas neon , as is done for an ordinary glass cell. Photograph of the multi-region cell is presented in Fig. 2. The holder of the multi-region cell (which is tied to the side arm and is seen at the bottom in Fig. 2) is made from the short aluminium tube. With the help of this holder MRC is fixed inside the oven.

II. EXPERIMENTAL

II.1. The design of multi-region cells filled with the Rb and neon gas

We have used 2 MRCs, filled with 6 Torr and 20 Torr of neon gas. Such design of MRC allows one to compare absorption and fluorescence signals simultaneously recorded by the nano-cell and 1 cm-long ordinary cell.

The MRC is placed in the oven with openings, which allow passing the laser radiation and registration of fluorescence in the direction perpendicular to the laser beam. The temperature of the MRC was kept at ~ 120 o C at the side-arm (the latter defines Rb atomic vapor pressure), and somewhat (20 o C) higher at the windows in order to prevent Rb vapor condensation at the windows. This regime corresponds to the number density of Rb atoms N ~ 10 13 at/cm 3 . To measure the transmission and fluorescence spectra at different nano-cell thicknesses, the oven with MRC was smoothly translating vertically as indicated by an arrow in Fig. 3.

II.2. Experimental arrangement for spectroscopic measurements

The experimental arrangement for transmission and fluorescence measurements is presented in Fig. 3. Extended cavity diode laser (ECDL) with  = 794 nm, ~30 mW power and spectral width ~1 MHz was used. Faraday isolator (FI) was used in order to avoid optical feedback. Diode laser radiation is focused on the cell (2) nearly perpendicular to its window surface (this is very important in the case of nano-cell use [START_REF] Sarkisyan | Sub-Doppler spectroscopy by sub-micron thin Cs-vapor layer[END_REF][START_REF] Sarkisyan | Sub-Doppler Fluorescence on atomic D 2 line of Sub-Micron Rubidium Vapor Layer[END_REF]), with a spot size of  ~ 0.5 mm. The MRC is placed in the oven made from non-magnetic materials, which has 3 openings: 2 for the laser beam transmission and one for registration of resonance fluorescence in perpendicular direction to the laser beam. The temperature of the ETC is kept at ~ 110 -120 o C on the side arm (this defines the Rb atomic vapor pressure), and 20-30 o C higher at the windows (in order to prevent the Rb vapor condensation at the windows). This regime corresponds to the number density of Rb atoms N ~ 10 13 at/cm 3 . In order to measure the transmission and fluorescence spectra at different nano-cell thicknesses, the oven with the MRC was smoothly moved vertically as indicated by an arrow in Fig. 3.

In order to obtain the transmission or/and fluorescence spectra, the laser radiation frequency is linearly scanned in an appropriate spectral region around the studied transition group of either the 85 Rb or 87 Rb, D 1 line. Part of radiation (~ 50%) is branched to auxiliary reference setup (the upper part in Fig. 3) for comparison with the spectra obtained in the main nano-cell. Depending on task, it could be: i) resonant transmission or fluorescence spectra obtained with 3 cm-long Rb cell; ii) SA spectra based on a room temperature 3 cm-long Rb cell; iii) transmission spectra of hyperfine atomic transitions in an auxiliary nano-cell, which is preferable in some cases. The spectra were recorded by photo-detectors (1) followed by a digital four channel storage oscilloscope, Tektronix TDS 2014B.

II.3. Key features of resonant absorption in nano-cell filled with pure alkali metal (Cs)

We note that in [3 ,7,910,13] the resonance absorption at D 2 lines of Rb and Cs atoms has been studied where the frequency distance between the upper hyperfine sublevels is rather small (several tens of MHz), which hinders practically the consideration of behavior of separate atomic transitions. From this point of view, the D 1 lines of Cs is more convenient because the frequency separation of the upper hyperfine levels reaches 1.167 GHz (see Fig. 4) and hence the atomic transitions are completely resolved. In order to demonstrate the key features of resonant absorption in nano-cell, an auxiliary nano-cell filled with pure Cs was used.

The laser frequency was scanned in the range of 10 GHz covering all four spectral components F g = 3  F e = 3; 4 and F g = 4  F e = 3; 4 of the hfs of the Cs D 1 line (subscripts "g" and "e" denote, respectively, the lower and upper levels of the transition). In order to obtain the transmission spectra for different thicknesses, the oven with the nano-cell was moved vertically as indicated by an arrow in Fig. 3. A small portion of radiation was directed onto a cell of 3 cmlong at room temperature where the well-known saturated absorption (SA) scheme was realized. The filters F were also used for attenuation of the probe (as well as pump) beam in the SA scheme in order to obtain a reference spectrum with a width close to natural.

Depicted in Fig. 5 are the transmission spectra of the nano-cell and of the cell with L = 3 cm for atomic transition F g = 4  F e = 3 and the thickness of the nano-cell L =  /2. Laser intensity  30 W/cm 2 . Curve (1) is the transmission spectrum of the nano-cell at side-arm temperature 110C. The absolute value of absorption amounts to ~2%. The experimental profile 1 of the transmission line for L =  / 2 is best approximated by the curve 2 which is described by the So, for L =  /2 a 4.6 times narrowing of the spectrum is observed. But if we compare the transmission spectra from the nanocell and the usual cell at the temperature of windows of both of them equal 150C, the FWHM is 430 MHz and the spectrum narrowing for L =  / 2 is 5.7 times.

Figure 6 shows the transmission spectra for the same atomic transition and the thickness varying from /2 to 7/2 with the step of  / 2 at the laser intensity  0.1 mW/cm 2 . The phenomena of DCN and collapse of DCN are well seen in Fig. 6: the minimum value of the spectral width is reached at the thicknesses L = (2n+1)  / 2. Note that for L =  / 2 the width is minimal: 75-80 MHz [START_REF] Dutier | Revisiting optical spectroscopy in a thin vapour cell: mixing of reflection and transmission as a Fabry-Perot microcavity effect[END_REF][START_REF] Dutier | Collapse and revival of a Dicketype coherent narrowing in a sub-micron thick vapor cell transmission spectroscopy[END_REF][START_REF] Andreeva | Saturation effects in the sub-Doppler spectroscopy of cesium vapor confined in an extremely thin cell[END_REF], whereas for L = 3 / 2, 5 / 2, and 7 /2, the narrowing of the spectrum occurs in the presence of increasing broad Doppler pedestal). As seen in Fig. 6, for L = n the spectral width is maximal (for L =  it is  300 MHz). So, fourfold narrowing of the transmission spectrum takes place for L =  / 2 as compared to L = . Note that in [START_REF] Dutier | Collapse and revival of a Dicketype coherent narrowing in a sub-micron thick vapor cell transmission spectroscopy[END_REF] the twofold narrowing of the spectrum has been observed which may probably be explained as follows: because of small gaps between the nano-cell windows the evacuation of residual gases (adsorbed at the window surfaces) is strongly hindered and hence many-hours evacuation is needed at the temperature of  400C; work [START_REF] Dutier | Collapse and revival of a Dicketype coherent narrowing in a sub-micron thick vapor cell transmission spectroscopy[END_REF] used the first nano-cell model where the evacuation of residual gases was performed worse than in subsequent models. Residual gases may lead to an additional broadening of the transmission spectrum and this influence is more for the thickness L = /2 than for L = . Note that the peak transmission for the thickness L =  / 2 is practically equal to the peak transmission for L = , whereas at the increase in laser intensity the peak transmission for L =  / 2 becomes higher than for L = . The lower curve in Figs. 6,7 are obtained with the standard SA technique from the cell of usual length.

III. Resonant absorption and fluorescence in nano-cell filled with Rb and neon gas.

III.1Resonant absorption in nano-cell filled with Rb and neon gas.

Transmission spectra for 85 Rb D 1 line 32',3' and 87 Rb, 22' transitions recorded in nano-cell filled with pure Rb (the cases L=λ/2 and L=λ) are shown in Fig. 8a for laser intensity < 0.1 mW/cm 2 . Dicke-type coherent narrowing effect is well seen: the spectral line-width for L=λ/2 (~150 MHz) is narrower by factor of 3 than that for L=λ (~ 450 MHz). Transmission spectra for the nano-cell filled with Rb and 6 Torr of Ne are shown in Fig. 8b for the same thicknesses. . The phenomena of DCN and collapse of DCN are well seen; the lower curve is obtained with the standard SA technique from the cell with L = 3 cm. Spectra are shifted vertically for the convenience. Fig. 7. Transmission spectra. All parameters are the same as in Fig. 6, but the laser intensity is ~5 mW/cm 2 . Theretical model presented in [START_REF] Dutier | Revisiting optical spectroscopy in a thin vapour cell: mixing of reflection and transmission as a Fabry-Perot microcavity effect[END_REF][START_REF] Nikogosyan | Resonant absorption and fluorescence of atomic layer of the thickness of the order of light wavelength[END_REF][START_REF] Sargsyan | Dependence of the Cs absorption D 1 linewidth on the thickness of atomic vapor column[END_REF] well described experimental curves shown in Fig. 6 Dicke-type coherent narrowing effect is still well seen [START_REF] Dutier | Collapse and revival of a Dicketype coherent narrowing in a sub-micron thick vapor cell transmission spectroscopy[END_REF][START_REF] Nikogosyan | Resonant absorption and fluorescence of atomic layer of the thickness of the order of light wavelength[END_REF][START_REF] Sarkisyan | Spectroscopy in an Extremely Thin Vapour Cell: Comparing the Cell Length Dependence in Fluorescence and in Absorption Techniques[END_REF]. The further increase of buffer gas pressure (Fig. 8c) results in broadening and contrast reduction of DCN dips. We should note that transmission for L=λ in Fig. 8b,c is more sensitive to I L as compared with the case of L=λ/2. Particularly, at I L ~ 1mW/cm 2 transmission for L=λ can be higher (the absorption is lower) than for L=λ/2, which is caused by stronger influence of an optical pumping for L=λ . For this reason, in order to get graphs visually comparable with those presented in Fig. 8 we have adjusted I L for L=λ. Fig. 9 presents calculated spectra corresponding to those presented in Fig. 8. An agreement with experimental results is seen. The striking point is that even with Ne pressure is up to 20 Torr the CRDN is still observable (graphs c), though less pronounced as compared with a) and b).

III.2 VSOP formation in nano-cell filled with Rb and neon gas

It is well-known that in the case of alkali metal vapor cells of conventional length (0.1 -10 cm), the addition of > 0.5 Torr of (any) buffer gas leads to disappearance of all the sub-Doppler features in the saturated absorption spectrum. Upper curve in Fig. 10 shows well-known SA spectrum obtained with 3-cm long cell filled with pure Rb. The VSOP and crossover (CO) resonances are well seen. The same spectrum recorded with 1-cm long Rb cell with 6 Torr of neon is shown by the lower curve. One can see that here all the VSOP and CO resonances are completely vanished. The latter is caused by the fact that in the case of buffered cell, the velocity selectivity of Rb atoms reduces due to frequent collisions with Ne, thus deteriorating necessary conditions to form sub-Doppler resonances. Fig. 11 shows transmission spectra for nano-cells with L=λ on 85 Rb F g =3F e =2,3 and 87 Rb F g =2F e =2 transitions (I L ~ 10 mW/cm 2 ) for the following cases: upper curve for nano-cell with pure Rb: the linewidth of VSOP resonances ~25 MHz; middle curve for nano-cell with Rb and 6 Torr Ne: VSOP linewidth is ~90 MHz; lower curve for the nano-cell with Rb and 20 Torr Ne (note, VSOP resonances are absent). The comparison of Fig. 6 and Fig. 7 shows a striking peculiarity: the VSOPs still exist for the nano-cell with Rb + 6 Torr neon when L= λ. This important difference in behavior as compared with vapor cells of the conventional length can be explained as follows. The free path length of Rb atom for 6 Torr Ne is a bit less than 10 m, so the atoms flying parallel to the windows inside the laser beam of 2 mm diameter experience hundreds of collisions with Ne atoms causing just additional broadening of VSOP. Meanwhile collisions of Rb atoms with longitudinal velocity with Ne atoms have a negligible impact since the thickness of nano-cell for L= λ is less than 1 m. It was demonstrated in [START_REF] Sarkisyan | Sub-Doppler Fluorescence on atomic D 2 line of Sub-Micron Rubidium Vapor Layer[END_REF][START_REF] Sargsyan | A novel approach to quantitative spectroscopy of atoms in a magnetic field and applications based on an atomic vapor cell with L=λ[END_REF] that under optimal conditions, the FWHM of the fluorescence spectrum of an individual transition for L= λ/2 can be reduced down to ~70 MHz (in the case of ordinary cm-size cell the value is ~ 500 MHz). This striking feature of the nano-cell offers an important benefit in the case of the nano-cell filled with buffer gas, too. As it is shown in [START_REF] Hakhumyan | Essential Features of Optical Processes in Rb submicron Thin Cell Filled with Neon Gas[END_REF] , a tool for in situ pressure monitoring based on this effect can be developed.

III.3.Resonant fluorescence in nano-cell filled with Rb and neon gas.

IV. Theoretical Consideration and Discussion

We consider a three-level atomic system (see Fig. 9) interacting with a linearly polarized laser radiation of frequency  .

The three levels under the consideration are: one ground hyperfine level 1

F   
  terms, relaxation , 1 0      V H i  ( 1 )
               22 21 * 22 2 ) Im( 2         33 31 * 33 2 ) Im( 2         21 1 * 32 2 22 11 1 21 ) ( ) (               i i i  31 2 32 1 33 11 2 31 ) ( ) (               i i i  32 31 * * 2 32 ) 2 ( 1 21      i i i         where 1 i
 is the rate of the spontaneous decay from the excited state i to the ground state 1 , and

2 31 1 21 2            . Here 1   and 2
  are the rates of population lost from the system, responsible for the optical pumping of the other ground hyperfine level with 2  F denoted as 1 in Fig. 9. The Rabi frequencies of the corresponding transitions are denoted by 1  and 2  , respectively. As the atoms are in thermal motion, we take the average of all ij  values over the range of velocities, weighted by the one dimensional Maxwellian velocity distribution.

To take into account the laser bandwidth we use the phase diffusion model of Wigner-Levy [?], in accordance with which it is assumed that the laser radiation has a Lorentzian spectrum with the Full Width at Half Maximum (FWHM) of (2)

γ L . The bandwidth is incorporated into Eqs. (2) as a relaxation term for the non-diagonal element of the density matrix in accordance to the procedure given in [?]. The main assumptions made in the model are as follows: the atomic number density is assumed to be low enough so that the effect of collisions between the atoms can be ignored; the atoms experience inelastic collisions with the cell walls, i.e. atoms lose completely their optical excitation; the incident beam diameter largely exceeds the cell thickness which allows one to neglect the relaxation of atoms travelling out of the diameter of the laser beam. These assumptions allow us to take into account the collisional relaxation of each atom by solving the temporal equations for the atomic density matrix with proper boundary conditions for each atom separately. The effect of the reflection of the radiation from the highly-parallel windows of the nano-cell behaving as a Fabri-Perot cavity [START_REF] Dutier | Revisiting optical spectroscopy in a thin vapour cell: mixing of reflection and transmission as a Fabry-Perot microcavity effect[END_REF] is also taken into account. The model used here is on the lines of [START_REF] Dutier | Revisiting optical spectroscopy in a thin vapour cell: mixing of reflection and transmission as a Fabry-Perot microcavity effect[END_REF][START_REF] Nikogosyan | Resonant absorption and fluorescence of atomic layer of the thickness of the order of light wavelength[END_REF]. Fig. 14 presents the calculated dependence of the fluorescence line-width and the amplitudes as a function of the neon pressure for Rb, D1 line, Fg = 3Fe = 2;3 ,  = 0.4  , V T =300 m/s. Note that, the FWHM data coincide for 3 2' and 3 3' spectral lines. One of the possible application using this dependence is development of an online pressure gauge which is described in [START_REF] Hakhumyan | Essential Features of Optical Processes in Rb submicron Thin Cell Filled with Neon Gas[END_REF].

V. Electromagnetically induced transparency phenomenon; ordinary cm-size Rb cell; nano-cell filled with pure Rb ; nano-cell filled with Rb and nitrogen gas

The line-width of the EIT resonance in the case of small probe intensity is γ EIT  2Γ 21 + Ω 2 /γ N [START_REF] Wynands | Precision spectroscopy with coherent dark states[END_REF], where Γ 21 is the relaxation rate between ground hyperfine levels (for -system) , with Ω being a Rabi frequency. For the case of the nano-cell [START_REF] Sarkisyan | Optical processes in micro-and nanometric thin cells containing atomic vapor[END_REF][START_REF] Sargsyan | Dark-line atomic resonances in sub-micron thin Rb vapor layer[END_REF], the value of Γ 21 is highly affected by atom-wall (cell's window) collisions. It is known that a unique collision with the dielectric surface of an uncoated vapor cell is sufficient to thermalize the ground hyperfine levels, with depolarization probability 0.5 -1. In order to preserve the atomic coherence in wall collisions either an additional gas or paraffin-coated walls were used [START_REF] Knappe | Dark-line atomic resonances in submillimeter structures[END_REF]. As the size of the vapor cell is reduced, the lifetime of the ground-state coherence becomes shorter because of collisions of the atoms with the windows: Γ 21 = (2π t) -1 , where t=L/u (L is distance between windows and u is the most probable thermal velocity). Also, the EIT resonance contrast (defined as the ratio of the EIT depth to the height of shoulders of the EIT window) strongly depends on Γ 21 . Hence, in the case of L < 1 μm (2Γ 21 >100 MHz), one could expect that the EIT effect will be vanished. Nevertheless, in [START_REF] Sargsyan | Dark-line atomic resonances in sub-micron thin Rb vapor layer[END_REF][START_REF] Pashayan-Leroy | Electromagnetically induced transparency: the thickness of the vapor column is of the order of a light wavelength[END_REF] it was demonstrated that the EIT resonance can be observed in thin cell with thickness as small as ~ 1 μm. The explanation is as follows: when the coupling laser is resonant with the corresponding atomic transition then the atoms moving nearly parallel to the cell windows participates in the EIT resonance formation and collisions with the windows are not essential, meanwhile when the coupling laser is detuned from the corresponding transition by a value of Δ, only those atoms having the velocity projections v z =2 π Δ /k participate in the formation of EIT, and for these atoms the time of flight between the cell windows is small enough τ L = L / v z . This causes a frequent collisions of the atoms with the nano-cell's windows and leads to the increase of Γ 21 and, consequently, to the line-width broadening and contrast reduction of the EIT resonance. One of the most dramatic different in behavior of the EIT resonance formed in ordinary cm-size Rb cell or in nano-cell filled with pure Rb or in nano-cell filled filled with Rb and nitrogen gas is dependence of the EIT resonance line-width and contrast as a function of the coupling laser frequency detuning Δ (from the corresponding atomic transition) which is demonstrated below. The experimental arrangement is sketched in Fig 15 . Both beams ( 3 mm) of two single-frequency extended cavity diode lasers (ECDL) (coupling and probe) with   794 nm (the linewidth is ~ 1 MHz) are well superposed and directed onto the cell (4) at near-normal incidence with the help of the first Glan prism (the coupling and probe beams have linear and perpendicular polarizations).The second Glan 2 prism has been used, which allows to provide separate detection of the spectra of the probe and coupling lasers, (1)-are Faraday isolators. Thin cell (4), of nano-metric thickness, was placed inside the three pairs of mutually perpendicular Helmholts coils (2) providing possibility to cancel laboratory magnetic field as well as to apply homogeneous magnetic field. The optical radiations were recorded by the photodiodes (3) and the signal of the photodiodes was intensified and recorded by a twochannel digital storage oscilloscope Tektronix TDS 3032B, F-are filters. An improved Dichroic-Atomic-Vapor-Laser-Locking (DAVLL) method, realized in a separate nano-cell with the thickness L = λ/2, is used for the coupling laser frequency stabilization; this technique is presented in [START_REF] Sargsyan | Efficient technique for measuring laser frequency stability[END_REF]. The frequency reference spectra formation has been realized with the help of an auxiliary nano-cell with thickness L = λ [START_REF] Sarkisyan | Optical processes in micro-and nanometric thin cells containing atomic vapor[END_REF].

Dependence of the EIT resonance line-width and amplitude (contrast) as a function of the coupling detuning Δ for 6cm-long cell is shown in Fig 16 . The coupling power is ~10 mW ,while the probe power is ~20 W. As it is seen when Δ is increasing : Δ 0,  1,  2, values are 0, 90 and 120 MHz, correspondingly, the line-width of EIT resonances is slightly reduced and line-width values (FWHM)are 13.5, 12.7 and 12.5 MHz , correspondingly, while the amplitude of the EIT resonances remain practically the same. Some reduction of the EIT amplitude with  increasing is caused by Maxwellian velocity distribution. VSOP peak is demonstrating increasing of the absorption, since the coupling laser transfer a number of atoms from the level 3 to level 2, and this causes increase of the probe absorption for 2→ 2',3' transitions (the EIT resonance is superimposed with the VSOP at 2-3'transition ).Note that physical nature of this VSOP is different to the another VSOP presented in Fig. 74 MHz, correspondingly, while the amplitude of the EIT resonances are rapidly reduced. The physical explanation of this fact is presented above.

to study atom-walls collisions, i.e. a wall's material. Note, that that it is expected that a high temperature of the nano-cell wall > 400 o C may causes a reduction of the depolarization probability due to atom-wall collision, i.e. smaller dependence of the EIT line-width versus the coupling detuning could be observed in this case. Thus, there is a dramatic different in behavior of the EIT resonance in nano-cell filled with pure Rb and CCL. Comparison of the EIT resonance line-width for the both cases vs the coupling detuning is shown in Fig. 18. Note, that increasing of the Δ in the case of nano-cell is causes rapid increase of the line-width, meanwhile for CCL even a small reduction of the line-width is observable.

It is expected that addition of a buffer gas (under the appropriate pressure) inside the nano-cell will reduce dramatic behavior of the EIT resonance contrast and the line-width as a function of coupling detuning. If mean free path for Fig. 16.Tansmission spectra of the probe when the coupling frequency is shifted by Δ with the respect of 3 → 3' transition, Δ 0,  1,  2, are equal to : 0 ; 90 and 120 MHz , for the curves 1,2,3, for 6cm-long cell . Lower curve is the reference spectrum which is formed with the help of an auxiliary nano-cell with thickness L= λ. For the VSOP peak see the text. Spectra are shifted vertically for the convenience. Thermocouple is tied to the sapphire side-arm for the T measurement at the top of the Rb metal column [START_REF] Sarkisyan | Sub-Doppler spectroscopy by sub-micron thin Cs-vapor layer[END_REF].The nano-cell is fixing inside the oven with the help of the aluminum holder.

Rb-buffer collisions will be much less than the distance L between nano-cell's windows ( L= ~800 nm) this will prevent direct atom-walls collisions, thus, this will avoid increase of Γ 21 (and, consequently, avoiding the strong linewidth broadening and contrast reduction).In order to check this statement a special nano-cell filled with the Rb and nitrogen (N 2 ) with the pressure ~20 Torr was fabricated and is shown in As it is seen when Δ is increasing ,i.e. Δ 0,  1,  2,  3,  4 are equal to 0 ; 79, 98.5 , 142 and 177 MHz ,correspondingly, the EIT amplitude reduction is much weaker than that in the case of the nano-cell filled with pure Rb vapor. Reference spectrum is formed by the help of an auxiliary nano-cell filled with pure Rb and with thickness L= , thus showing atomic transition position not perturbed by N 2 collisions ,while EIT resonance for  0 =0 is red shifted by 130 MHz which means that atomic transition 3 3' is red shifted due to Rb-N 2 collisions. The comparison of the EIT resonance amplitude for the both cases versus the coupling detuning is shown in Fig. 21. Thus, use of buffer gas N 2 strongly extended the region of the coupling detuning where EIT resonances is still possible to create.

VI Conclusions

Comparison of the resonant absorption in the nano-cell filled with Rb vapor with another nano-cell filled with Rb and neon gas with pressure 6 Torr and 20 Torr shows that the spectra of the resonant absorption demonstrate sub-Doppler narrowing for the thickness L=/2 and broadening for the thickness L=, thus manifesting the effect of collapse and revival of Dicke-type narrowing ( = 794 nm is a resonant laser wavelength). In the Rb cells of the conventional length filled with buffer gas, in the saturated absorption VSOP resonances are washed out when pressure > 0.5 Torr. In contrast, for the nano-cell with L=  the VSOP resonances located at the atomic transition are still observable even when neon pressure is ≥ 6 Torr. Narrowband fluorescence spectra emitted by the nano-cell with L= λ/2 can be used as a convenient tool for online buffer gas pressure monitoring in the MRC in its several centimeter-long part, for the conditions when ordinary pressure gauges are unusable. Developed theoretical model well describes all experimentally observed peculiarities.A strong increasing of the EIT resonance line-width and contrast (amplitude) reduction for a large detuning of the coupling laser frequency with respect to the corresponding transition is revealed in the nano-cell filled with pure Rb and thickness L= . It is shown that this is completely in contradiction to that observable in the case of CCL filled with the Rb. It is demonstrated that addition of buffer gas nitrogen (under the appropriate pressure) strongly extended the region of the coupling detuning where EIT resonance is still possible to create with the help of the nanocell with the thickness L =.

  Fig.1 MRC consisting of 2 interconnected regions: ETC or nano-cell (shown in the upper part) with wedged thickness in the range of 100nm -4 m, and 1 cm-long sapphire cell (shown the lower part) with the sapphire windows (the sapphire plates-windows are cut across the c-axis). MRC is filled with the natural Rb with addition of neon gas at 6 Torr or 20 Torr pressure.

Fig. 2

 2 Fig.2 Photography of the multi-region cell. The nano-cell is seen in the upper part, while ~ 1 cmlong sapphire cell is shown in the middle part. MRC is fixed inside the oven with the help of the aluminum tube-holder which is tied to the side arm (only the top is seen at the bottom of the photo).

Fig. 3 .

 3 Fig.3. Sketch of the experimental setup. ECDL diode laser with  = 794 nm; FI-Faraday isolator, 2multi-region cell (inside the oven), or an auxiliary nano-cell filled with pure Rb or Cs; the upper part -unit for the frequency reference formation, 1photodiodes.

Fig. 4 Fig. 5 .

 45 Fig.4 Relevant energy levels of the Cs, D 1 line.

Fig. 6 .

 6 Fig.6. Transmission spectra, 4'  3' for variation of the column thickness from  / 2 to 7 /2 with the step of  /2; laser intensity  0.1 mW/cm2 . The phenomena of DCN and collapse of DCN are well seen; the lower curve is obtained with the standard SA technique from the cell with L = 3 cm. Spectra are shifted vertically for the convenience.

and Fig. 7 .

 7 Fig.7. Transmission spectra. All parameters are the same as in Fig.6, but the laser intensity is ~5 mW/cm2 . Theretical model presented in[START_REF] Dutier | Revisiting optical spectroscopy in a thin vapour cell: mixing of reflection and transmission as a Fabry-Perot microcavity effect[END_REF][START_REF] Nikogosyan | Resonant absorption and fluorescence of atomic layer of the thickness of the order of light wavelength[END_REF][START_REF] Sargsyan | Dependence of the Cs absorption D 1 linewidth on the thickness of atomic vapor column[END_REF] well described experimental curves shown in Fig.6and Fig.7. Spectra are shifted vertically for the convenience.

Fig. 8 .

 8 Fig.8. Transmission spectra for L= λ/2 and L= λ. a) nano-cell with pure 85 Rb, DCN and its collapse is well seen; b) nano-cell with Rb + 6 Torr Ne, DCN and its collapse is still well seen; c) nano-cell with Rb + 20 Torr Ne, DCN and its collapse is yet seen, though less pronounced compared with a), b).

Fig. 9

 9 Fig.9 Calculated spectra for cases presented in Fig.8. 85 Rb, D 1 line, only 3 2', 3' transitions a) pure Rb, L=λ/2, and λ,  L ~ 0.04 ; b) Rb + 6 Torr Ne for L=λ/2 and λ  L ~ 0.4 ; c) Rb + 20 Torr Ne, for L=λ/2 and λ,  L = 0.8  ( =  N +3 MHz), V T =300m/s, pressure broadening 7.5 MHz/Torr.

Fig. 10 .Fig. 11 . 2 Fig. 12 85

 1011212 Fig.10. Upper curve is well-known SA resonances for 1-cm long cell (pure 85 Rb, D 1 line), CO is crossover resonance; the lower curve is SA spectrum for 1 cmlong cell filled with Rb + 6 Torr Ne showing absence of VSOP resonances. Spectra are shifted vertically for the convenience.

Fig. 12 presents

 12 fluorescence spectrum when L= λ/2 for 3 cases (left-experiment, right-theory): upper curvenano-cell with pure Rb vapor; middle (grey) and lower curves -nano-cell containing Rb vapor + 6 Torr and 20 Torr Ne, correspondingly. For theory (b): Rabi frequency  L =0.4, where  = N (~ 6 MHz) + 3 MHz (the broadening caused by Rb-Rb collisions at 120 o C at the side-arm). For the calculations we used broadening rate of ≈ 7.5 MHz/Torr, thus for 6 Torr, (total) ~ 50 MHz, and for 20 Torr, (total) ~ 150 MHz, and Maxwellian velocity distribution with V T = 300 m/s. Laser intensity is ~ 10 mW/cm 2 . As it is seen, up to 20 Torr Ne the fluorescence spectrum has a sub-Doppler linewidth.

3 .

 3 For simplicity, we consider a one-dimensional situation, where the driving field is in the z  -direction and the atom is moving along z-direction with a velocity z v . The detunings of the laser from the transitions 2 z kv is the shift due to the Doppler effect, with k being the wave vector of the excitation light. We analyze the observed spectra of resonant absorption and fluorescence on the basis of a standard density-matrix approach [?]. The dynamical behavior of the density matrix ρ is given by the Liouville equation of motion

  is the atom-light interaction Hamiltonian in the electric-dipole approximation with d ˆ being the electric dipole operator and ) (t E the electric field of the radiation light.The components of the density-matrix elements of Eq. (1) can be represented by taking rotating-wave-

Fig. 13 .

 13 Fig. 13. The three-level scheme around the D 1 line of 85 Rb. Fig.14. Dependence (theory) of the fluorescence line-width and the amplitudes for L= /2 as a function of the Ne pressure for Rb, D 1 line, 3 2',3' ,  = 0.4  , V t =300m/s 0 1 0 2 0 3 0 4 0 5 0 6 0

7 . 2 Fig. 15

 7215 Fig.15 a) Sketch of the experimental Set-Up. (1)-are Faraday isolators, (2)-Helmholts coils , (3)-photodiodes , (4)-CCL; nano-cell filled with pure Rb; and nano-cell filled with Rb and nitrogen gas.

Fig. 15 b

 15 Fig.15 b) energy levels of the 85 Rb D 1 line involved in EIT process. The coupling is shifted by Δ in blue side from 3-3' transition, while the probe frequency is scanning across 2 → 3' transition.

Fig. 17 .

 17 Fig.17. Nano-cell filled with pure Rb, transmission spectra of the probe when the coupling frequency is shifted by Δ with the respect of 3 → 3' transition , Δ 0 ,  1 ,  2 ,  3 ,  4 values are 0, 15, 40, 50 and 74 MHz, correspondingly , for the curves 1,2,3,4,5, nano-cell thickness L= λ. EIT resonances are indicated by the vertical arrows. For the VSOP peak see the text. Spectra are shifted vertically for the convenience.

Fig. 18 .Fig. 19 .

 1819 Fig.18. Dependence of the EIT resonance line-width vs the coupling detuning Δ, upper curve -for the nano-cell filled with pure Rb, L=794 nm, lower curve -for 6-cm long Rb cell. Dot-lines are for eyes guide.

Fig 19 .

 19 The gap has wedged form in the vertical direction from 100 nm (centum of the ring part) up to 2 m near the bottom. Collisional cross section of Rb-N 2 by several times is larger than that of Rb-Ne, thus providing smaller mean free path for Rb-N 2 collisions (mean free path ~ 300 nm). Dependence of the EIT resonance line-width and amplitude as a function of the blue detuning Δ of the coupling for nano-cell filled with Rb and 20 Torr N 2 with thickness L=λ is shown inFig 20.

Fig. 21 .

 21 Fig.21. Dependence of the EIT resonance amplitude as a function of the coupling-laser detuning Δ, upper curve -the nano-cell, Rb + N 2 gas, L=794 nm, lower curve -nano-cell, pure Rb, L=794 nm. Dot-lines are for eyes guide

Acknowledgements

The authors thank A. Sarkisyan for his valuable participation in the fabrication of the MRC. Research conducted in the scope of the International Associated Laboratory IRMAS. Armenian team thanks for the ANSEF PS 1868 support. Armenian team thanks also for the Grant INTAS 06-1000017-9001 (prolonged by Armenian government).


George Pentheroudakis

Anna Batistatou

Konstantine T Kalogeras

Ralf Kronenwett

Dr Ralph M Wirtz

Evangelos Bournakis

Anastasia G Eleftheraki

Dimitrios Pectasides

Mattheos Bobos

Irini Papaspirou

Sevasti Kamina

Helen Gogas

Angelos K Koutras

Nicholas Pavlidis

George Fountzilas

Prognostic utility of β-tubulin isotype III and correlations with other molecular and clinicopathological variables in patients with early breast cancer: A translational Hellenic Cooperative Oncology Group (HeCOG) study

Keywords: Breast cancer, β-tubulin isotype III, adjuvant chemotherapy, prognostic/predictive factors

Introduction

Breast adenocarcinoma is the most common malignant neoplasm in females.

As many as 60-70% of affected patients present with localised disease and become disease-free following surgical resection of the tumour, thus becoming candidates for adjuvant systemic therapy [START_REF] Ebctc Group | Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival:an overview of the randomised trials[END_REF]. Several clinicopathological parameters, including tumour size, grade, hormonal receptor status, axillary nodal involvement, patient age, and Human Epidermal growth factor Receptor-type 2 (HER2) overexpression are taken into consideration in order to identify which patients are most likely to benefit from administration of adjuvant cytotoxic chemotherapy [START_REF] Trudeau | Selection of adjuvant chemotherapy for treatment of node-positive breast cancer[END_REF]. Still, individual patients may be under-or over-treated. Ultimately, we are unable to predict the true binary outcome of recurrence versus no recurrence at the level of the individual, as this simple clinicopathological profile does not capture the rich molecular heterogeneity of the malignancy.

Taxanes are natural products that disrupt the dynamic equilibrium of microtubule polymerisation and cause mitotic arrest and apoptosis of malignant cells. Data from 21 clinical trials of first-generation taxanes that were randomly allocated over 35,000 women with early-stage breast cancer to taxane-based versus taxane-free adjuvant therapies established a modest improvement in patient outcome [START_REF] Bedard | Taxanes: optimizing adjuvant chemotherapy for early-stage breast cancer[END_REF]. This modest benefit was observed irrespective of the type of taxane, schedule of administration, extent of nodal involvement, and hormone-receptor status. Taxanes are associated with considerable side effects, often require the use of adjunctive medications and are expensive. Accordingly, controversy persists regarding their role in the adjuvant management of early-stage breast cancer, as a result of our inability to identify the subgroup of patients who would truly benefit. Consequently, the quest for tumoural molecular traits that have the potential to be useful predictive factors for taxane activity is justified.

Microtubules are intrinsically dynamic polymers composed of αβ-tubulin heterodimers and constitute essential components of the mitotic spindle and cytoskeleton. Microtubule dynamics control mitosis and cellular motility. Moreover, microtubules serve as scaffolds for signalling molecules and gene transcription regulators, for trafficking of cellular organelles as well as vesicles and for fine-tuning of cell cycle activity [START_REF] Bhat | Microtubule-associated proteins as targets in cancer chemotherapy[END_REF]. This dynamic network should be seen as polymers of tubulin dimers in flux, as it is in equilibrium with a repertoire of modulating factors, such as the α/β tubulin heterodimer ratio, the β-tubulin isotypes being integrated and the microtubule-associated proteins (MAP) attached. Altered tumoural expression of βtubulin isotypes, which constitute the major microtubule proteins, may affect taxane binding and alter microtubule dynamics. At least eight different β-tubulin isotypes (classes I, II, III, IVa, IVb, V, VI and VII) have been identified [START_REF] Mollinedo | Microtubules, microtubule-interfering agents and apoptosis[END_REF]. In particular, it has been suggested that overexpression of class III β-tubulin (TUBB3) may destabilise microtubules and counteract the effects of taxanes. To date, the clinical data that predict taxane resistance in relation to class III β-tubulin expression in advanced breast cancer are unclear and this link has yet to be studied in the adjuvant setting [START_REF] Kavallaris | Microtubules and resistance to tubulin-binding agents[END_REF]. Accordingly, we retrospectively studied the transcription and protein expression of TUBB3 in patients with resected high-risk breast cancer who had been randomised to receive adjuvant chemotherapy in the context of a prospective randomised Hellenic Cooperative Oncology Group (HeCOG) trial. The administration of taxane-based versus taxane-free chemotherapy regimens would allow us to dissect the prognostic versus predictive utility of TUBB3 expression. Moreover, we analysed correlations of TUBB3 expression with clinical, pathological and molecular characteristics of the tumour.

Patients and Methods

Clinical Trial

The HeCOG prospective trial HE10/97 randomised a total of 595 high-risk (T1-3N1M0 or T3N0M0) breast cancer patients in the period 1997-2000, to receive either four cycles of epirubicin followed by four cycles of intensified cyclophosphamide, methotrexate and 5-fluorouracil combination chemotherapy (E-CMF) or three cycles of epirubicin followed by three cycles of paclitaxel and three cycles of intensified CMF (E-T-CMF) [START_REF] Fountzilas | Postoperative dose-dense sequential chemotherapy with epirubicin, followed byCMFwith or without paclitaxel, in patients with high-risk operable breast cancer: a randomised phase III study conducted by the Hellenic Cooperative Oncology Group[END_REF]. Chemotherapy cycles were administered every 2 weeks and patients received granulocyte-colony stimulating factor (GCSF) support. The trial was approved from the Bioethics Committee of the Aristotle University of Thessaloniki and patients provided written informed consent prior to enrolment. All participating patients also gave written informed consent for research use of their biologic material.

Molecular and Immunohistochemical Studies

This was a retrospective translational research study among patients who had been enrolled in a prospective clinical trial ( A REMARK diagram is provided in Figure 1). Accordingly, collection of formalin-fixed paraffin-embedded (FFPE) tumour tissue samples was possible in 317 patients only, due to logistical/organisational barriers. Hematoxylin-eosin sections from all available FFPE tissue specimens were evaluated histologically by a certified pathologist and recorded for the percentage of tumor cell content. Prior to RNA isolation, macrodissection of tumour areas was performed for sections with <50% tumor cell content.

Sufficient RNA was isolated from 314 FFPE specimens. In detail, from each FFPE section or macrodissected tissue fragments (10 μm thick) RNA was isolated using a fully automated isolation method for total RNA from FFPE tissue, based on silicacoated magnetic beads (Versant Tissue Preparation Reagents, Siemens Healthcare Diagnostics, Tarrytown, NY) in combination with a liquid handling robot, as previously described [START_REF] Bohmann | RNA extraction from archival formalin-fixed paraffin-embedded tissue: a comparison of manual, semiautomated, and fully automated purification methods[END_REF]. The method involves DNase I treatment for eliminating DNA contamination in the sample. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was applied for the assessment of the β-tubulin type III (TUBB3) gene, which includes four exons and four introns, by using a gene specific Taq-Man based assay. The TUBB3 gene is situated at chromosome 16 at the genome locus 16q24.3. Forty cycles of nucleic acid amplification were applied and the cycle threshold (CT) value of the target gene was identified. CT values were normalized by subtracting the CT value of the housekeeping gene RPL37A from the CT value of the target gene (ΔCT). RNA results were then reported as 40-ΔCT values, which would correlate proportionally to the mRNA expression level of the target gene.

Expression of the TUBB3 gene, as well as the reference gene RPL37A, was assessed in triplicate by qRT-PCR using the SuperScript III PLATINUM One-Step Quantitative RT-PCR System with ROX (Invitrogen, Karlsruhe, Germany) in an ABI PRISM 7900HT (Applied Biosystems, Darmstadt, Germany) [START_REF] Mueller | Quantitative determination of estrogen receptor, progesterone receptor and HER2 mRNA in formalinfixed paraffin-embedded tissue-a new option for predictive biomarker assessment in breast cancer[END_REF]. The lengths of the amplicons detected by the TUBB3 and RPL37A assays were 91bp and 65bp, respectively.

The Primer/Probe (FAM/TAMRA-labelled) sets used for amplification of the target and reference genes were the following (5' -> 3'): μm thick sections from 17 Tissue Microarray (TMA) blocks, constructed with the use of a manual arrayer (Model I, Beecher Instruments, Sun Prairie, WI, USA), using 2 cores per case of 1.5 mm in diameter. All slides of the study for each antibody were stained in one run. Tumour samples were categorised as ER-or PgR-positive if at least 1% of malignant cells showed nuclear staining for the ER or PgR proteins, respectively [START_REF] Hammond | American Society of Clinical Oncology/College Of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer[END_REF]. Immunohistochemical (IHC) categorisation of tumour cells as HER2+ was done in the presence of 3+ IHC expression in >30% of tumor cells according to proposed criteria or, in the case of 2+ IHC, in the presence of gene amplification by Chromogenic In Situ Hybridisation (CISH) [START_REF] Wolff | American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer[END_REF]. Immunohistochemical staining for TUBB3 and MAPT was homogeneous throughout the TMA sections and was evaluated using a semiquantitative method. In particular, the staining was cytoplasmic and was considered positive if it was of moderate or strong intensity (2+ and 3+ respectively), as previously described by Pusztai et al [START_REF] Pusztai | Evaluation of Microtubule-Associated Protein-Tau Expression As a Prognostic and Predictive Marker in the NSABP-B 28 Randomized Clinical Trial[END_REF]. Cases with absent (0+) or weak (1+) immunostaining were considered negative. The above mentioned criteria were also used for the analysis of the ERCC1 protein. Moderate or strong nuclear staining intensity was considered as positive, whereas cases with absent or mild immunoexpression were classified as negative.

Statistical Methodology

Exploratory analysis was conducted in order to find a prognostic cut-off among the distribution quartiles of TUBB3 mRNA values. The median was chosen as the optimal cut-off in the prognosis of both overall survival (OS) and disease free survival (DFS). The distribution of the mRNA values of TUBB3 is shown in Figure 1.

The median for MAPT and the 25 th percentile for ER and PgR were used to separate mRNA expression as previously described [START_REF] Pentheroudakis | Gene expression of estrogen receptor, progesterone receptor and microtubule-associated protein Tau in high-risk early breast cancer: a quest for molecular predictors of treatment benefit in the context of a Hellenic Cooperative Oncology Group trial[END_REF]. Considering mRNA expression of HER2, the 75 th percentile (a value close to the natural cut-off of the bimodal distribution) was a predefined threshold to separate low and high expressed tumors. OS was measured from the date of randomisation until death from any cause.

Surviving patients were censored at the date of last contact. DFS was measured from the date of randomisation until recurrence of tumour or secondary neoplasm or death from any cause. Time-to-event distributions were estimated using Kaplan-Meier curves [START_REF] Hudis | Proposal for standardized definitions for efficacy end points in adjuvant breast cancer trials: the STEEP system[END_REF]. The non-parametric Mann-Whitney test was performed for comparing continuous variables between two independent groups. To measure the association between the clinicopathological variables and the expression of studied biomolecules, the Fisher's exact test was implemented. Correlations among all genes under study were assessed using the Spearman correlation coefficient (Rho). The degree of agreement corrected by chance was estimated using the Kappa coefficient. Univariate Cox regression analyses, adjusted for group, were performed to assess the relationship of markers with OS or DFS. A multivariate Cox regression analysis, including age (<50 vs. 50 years), group of randomisation (E-T-CMF vs. E-CMF), menopausal status (pre vs. post), involved axillary lymph nodes (0-3 vs. > 4), histological grade (I-II vs. III-Undifferentiated), size (≤2 cm vs. 2-5 cm vs. >5 cm), mRNA expression (low vs. high) of ER, PgR, HER2, TUBB3, MAPT and ERCC1 protein expression (negative vs. positive) was conducted. A backward selection procedure with a removal criterion of P>0.10 was applied and final tests were conducted at the α=0.05 level of significance. Results of this study are reported following the corresponding recommendations for tumour marker prognostic studies [START_REF] Mcshane | Reporting recommendations for tumor marker prognostic studies[END_REF]. The statistical analysis was conducted using SPSS (SPSS for Windows, version 15.0, SPSS Inc.).

Results

Patient and Tumour Characteristics

Three hundred and fourteen patients (median age 50) harboured breast carcinomas, most commonly hormone-receptor positive by IHC (68.8% of patients), T2-3 (69.4%) of moderate to poor differentiation (49.7%), from which neoplastic FFPE material underwent molecular analysis for gene expression of TUBB3, ER, PgR, HER2, and MAPT (Figure 1). After modified radical mastectomy (79%) or breast conserving surgery (21%) and nodal dissection, neoplastic metastases in axillary lymph nodes were present in all but six cases, with four or more involved nodes in 75.5% of patients. Slightly more patients were managed with adjuvant E-CMF chemotherapy (N=170) than E-T-CMF (N=144). With the exception of high-grade and HER2 IHC protein overexpression, which were more common in the E-T-CMF arm, basic clinicopathological characteristics were well balanced when the 314 analysed patients were stratified by adjuvant chemotherapy arm (Tables 1 and2). Following completion of adjuvant chemotherapy, 91.7% of the 314 women received some form of hormonal therapy, mostly tamoxifen (87.2% of patients receiving hormonotherapy). Among women with IHC ER-negative tumours who took tamoxifen, a proportion had PgR-unknown or PgR-positive tumours, while some received hormonal therapy despite having both ER and PgR-negative tumours, a practice that has since been abandoned in all HeCOG participating centres.

The profile of protein expression and gene transcription of studied biomolecules is summarized in Table 2. Among 314 tumours, 64% expressed ER protein, 57% PgR and 17.2% overexpressed HER2. The majority of tumours (71%) expressed ERCC1 protein, while 40% expressed MAPT. Only 36.3% of breast carcinomas stained moderately to strongly for cytoplasmic TUBB3, the definition used for positive cases (Figure 3a and3b). Regarding RT-PCR studies, gene transcription defined as mRNA levels higher than the defined cut offs were seen in 75% of tumours for ER and PgR genes, in 25% for HER2 and in 50% of cases for the MAPT and TUBB3 genes. The distribution of TUBB3 mRNA values is shown in Figure 2. There were no significant differences in protein expression or gene transcription of studied biomolecules between treatment arms, with the exception of HER2 protein overexpression, which was seen in 22.2% of tumours in the E-T-CMF arm versus 12.9% in the E-CMF arm (p=0.045).

Correlation of molecular and clinicopathological parameters

The correlations of TUBB3 mRNA status (high or low) as well as of TUBB3 protein expression with standard clinicopathological and molecular factors are shown in Table 3. In general, high TUBB3 mRNA transcription was significantly associated with tumour characteristics with adverse prognostic impact, such as advanced T stage, high grade, hormone receptor protein and mRNA negativity, HER2 protein and mRNA positivity, and MAPT protein and mRNA negativity. Specifically, tumours larger than 5cm were seen in 21% of TUBB3 mRNA-high versus 12.7% of TUBB3 mRNA-low cases, while high grade was documented in 56% versus 43% of TUBB3 mRNA high versus low tumours, respectively. Absence of ER and PgR protein expression was seen in 35% and 44.4% of TUBB3 mRNA-high tumours versus only 15.6% and 22.2% respectively of TUBB3 mRNA-low tumours. HER2 protein overexpression was observed in 32.8% of TUBB3 mRNA-high versus only 9.4% of TUBB3 mRNA-low breast carcinomas, while absence of MAPT protein expression was documented in 60% of TUBB3 mRNA-high versus 46% of TUBB3 mRNA-low cases. These associations were also observed with robust statistical significance between TUBB3 mRNA expression and mRNA expression status of ER, PgR, HER2, and MAPT genes. TUBB3 mRNA status was correlated to all mRNA markers at p<0.001 significance: negatively with ESR1 mRNA (Rho -0.333), PgR mRNA (Rho -0.291) and MAPT mRNA (Rho -0.250) and positively with ERBB2 mRNA (Rho 0.230).

In contrast, the association of TUBB3 protein expression with adverse clinicopathological variables was less common and considerably weaker. It correlated with HER2 protein overexpression, ERCC1 protein expression and low mRNA status of ER and PgR, as well as high mRNA status of HER2 at p values ranging from 0.01 to 0.047.

Although positive TUBB3 protein expression was associated with higher levels of TUBB3 mRNA (Mann Whitney, p<0.001, Figure 4), there was only poor agreement between mRNA and protein expression status (Kappa statistic 24.6%, p<0.001) (Table 4). In fact, among tumours with higher than median TUBB3 mRNA transcription as many as 45.9% exhibited absent or weak (0+/1+) protein staining. Conversely, among tumours with lower than median TUBB3 mRNA levels, 29.5% had moderate to strong (2+/3+) protein expression. Using the protein expression as reference, overall agreement between the two methods was 62.4%, while positive and negative agreement were 64.0% and 61.2% respectively.

Outcome and Prognostic/Predictive factors

Survival status of the 314 patients was updated in October 2007. At the time of the analysis (July 2010), the median follow up time was 98 months. During this time, 110 patients (35%) had developed a relapse and 80 patients (25.5%) had died.

The median disease-free survival (DFS) was 121 months (95% CI: 105-138, 5-year DFS 71%), while the median overall survival (OS) had not been reached yet (5-year OS 84%).

Malignant relapses occurred in 43.9% and deaths in 30.6% of the TUBB3-mRNA high cases versus 26.1% relapsed and 20.4% dead patients with TUBB3-mRNA low tumours. Univariate Cox regression analysis, adjusted for treatment, showed an increased risk of death in patients with TUBB3-mRNA high tumours [hazard ratio (HR)=1.60, 95% CI: 1.02-2.51, Wald-p=0.039], as well as an increased risk of malignant relapse (HR=1.84, 95% CI: 1.26-2.68, Wald-p=0.002), in comparison to those affected by TUBB3-mRNA low breast carcinomas.

In contrast, neoplastic TUBB3 protein expression did not achieve prognostic significance. Malignant relapses occurred in 28.5% and deaths in 27.2% of the TUBB3-IHC positive cases versus 33.7% relapsed and 24.3% dead patients with TUBB3-IHC negative tumours. Univariate Cox regression analysis, adjusted for treatment, failed to establish distinct risk of death in patients with TUBB3-IHC high tumours (HR=1.17, 95% CI: 0.73-1.87, Wald-p=0.52), or risk of relapse (HR=1.13, 95% CI: 0.76-1.67, Wald-p=0.56), in comparison to those affected by TUBB3-IHC low breast carcinomas. Kaplan-Meier survival curves representing the probability of survival and disease-free survival as a function of TUBB3 mRNA status are presented in Figure 5 (log-rank test for DFS p=0.001, for OS p=0.04). The randomisation of patients to a taxane-free versus a taxane-containing chemotherapy regimen enabled us to screen whether TUBB3 gene transcription carried a prognostic (irrespective of treatment) or predictive (identifying response to therapy) utility. However, neither TUBB3 mRNA nor IHC protein expression status were predictive for benefit from adjuvant treatment with paclitaxel (E-T-CMF arm) for either disease-free survival or overall survival (DFS and OS tests for interaction, Wald-p=0.46 and p=0.96 for TUBB3 mRNA, p=0.95 and p=0.75 for TUBB3 IHC, respectively). Since data on TUBB3 mRNA levels are continuous, we examined whether increasing tumour mRNA levels had a trend for increasing patient benefit from taxane-containing adjuvant chemotherapy. There was no interaction between mRNA levels of TUBB3 and treatment arm (p=0.27 for OS and p=0.15 for DFS). By restricting analysis to patients who received taxanes (E-T-CMF), we observed that increasing transcription of the TUBB3 gene (increasing mRNA levels as a continuous variable) remained non-predictive for benefit from adjuvant treatment with paclitaxel (DFS HR=1.11 and OS HR=1.14, Wald-p=0.14 and p=0.15, respectively).

The impact of paclitaxel on OS and DFS in TUBB3 mRNA-low and high patients are shown in Figures 6a and6b, respectively. Since in the clinical paper, a survival benefit from the addition of paclitaxel was observed in patients harbouring ER IHCnegative tumours and ER status was inversely correlated to TUBB3 gene transcription, we investigated whether TUBB3 gene transcription was able to identify the subgroup of patients with ER IHC negative tumours who might benefit from taxanes. This analysis among ER IHC-negative patients is presented in Figures 7a and7b, respectively and did not show any impact of paclitaxel on either DFS or OS (logrank, p>0.05 in all cases ).

In keeping with data published by our group on MAPT mRNA levels, IHC MAPT expression exhibited a trend for statistical significance as a favourable prognostic factor for OS (HR=0.63, 95% CI: 0.39-1.02, Wald-p=0.065) and DFS (HR= 0.66, 95% CI: 0.44-1.00, Wald-p=0.05). Although, there was a non-significant trend for the interaction of MAPT protein expression and treatment group (p=0.08 for both OS and DFS), there was a favourable prognostic significance for MAPT IHC protein staining when restricting in patients treated in the E-CMF arm (HR for OS=0.42 with Wald-p=0.014 and HR for DFS=0.45 with Wald-p=0.009).

Multivariate analysis for independent prognosticators

Multivariate Cox regression analysis (Table 5), including age, menopausal status, tumour grade, size, number of positive lymph nodes, treatment group, TUBB3, ER, PgR, HER2, and MAPT mRNA expression levels and ERCC1 protein expression revealed that only TUBB3 mRNA, PgR mRNA and number of involved axillary nodes remained significant independent predictors of OS, while TUBB3 mRNA, number of positive nodes and tumour grade independently predicted DFS.

More specifically, high TUBB3 mRNA was associated with an increased risk of death (HR=1.71, 95% CI: 1.03-2.83, Wald-p=0.038) and increased risk of relapse (HR=1.83, 95% CI: 1.25-2.68, Wald-p=0.002). As expected, four or more positive nodes were associated with an increased risk of death (HR=2.62, 95% CI: 1.27-5.38, Wald-p=0.009) and relapse (HR=2.88, 95% CI: 1.66-4.99, Wald-p<0.001). Low PgR mRNA transcription also significantly correlated with increased risk of death (HR=0.49, 95% CI: 0.29-0.84, Wald-p=0.009). High histological grade significantly correlated with increased risk of relapse (HR=1.68, 95% CI: 0.82-1.71, Wald-p= 0.007). Of note, factors such as treatment arm (E-CMF HR=1.57, Wald-p=0.065) and positive ERCC1 protein expression (HR=1.93, Wald-p=0.087) exhibited a trend for adverse prognostic significance in the multivariate model for overall survival.

Discussion

α-and β-tubulins are each encoded by a 9-to 8-member multigene family, respectively that produces conserved gene products in which the sequences are divergent only in their last carboxy-terminal 10-15 aminoacids. This carboxy-terminal sequence has been used to define β-tubulin gene products to seven distinct classes or isotypes [START_REF] Jordan | Microtubules as a target for anticancer drugs[END_REF][START_REF] Cleveland | Molecular biology and genetics of tubulin[END_REF][START_REF] Lopata | In vivo microtubules are copolymers of available h-tubulin isotypes: localization of each of six vertebrate h-tubulin isotypes using polyclonal antibodies elicited by synthetic peptide antigens[END_REF]. Class I and II β-tubulins are the most abundant isotypes, whereas the class III β-tubulin (TUBB3) was initially thought to be restricted to neuronal and testicular tissues. The TUBB3 gene is situated at chromosome 16 at the genome locus 16q24.3. Recently, however, TUBB3 expression has been observed in the mitochondria and cytoplasm of other tissues [START_REF] Cicchillitti | Proteomic characterisation of cytoskeletal and mitochondrial class III b-tubulin[END_REF]. In vitro experiments and retrospective in vivo data implicated TUBB3 as a clinical marker of drug resistance to taxanes and vinca alkaloids [START_REF] Kavallaris | Microtubules and resistance to tubulin-binding agents[END_REF]. These drugs respectively stabilise and depolymerise microtubules at high concentrations, though at concentrations 10-100 times lower, such as the ones achieved in the clinical setting, they kinetically disrupt microtubule dynamics without altering microtubule polymer mass [START_REF] Orr | Mechanisms of taxol resistance related to microtubules[END_REF]. This results in blocking transition from metaphase to anaphase and inducing apoptotic cell death. High levels of TUBB3 were associated with taxane resistance in lung, ovarian, breast, and pancreatic cancer cell lines. TUBB3 has been hypothesized to alter drug binding to tubulin dimers and to reduce the polymerisation rate of microtubules, thus overcoming polymerisation induced by taxanes, or to create aβ ΙΙΙ microtubule dimers with different intrinsic dynamic properties that render them less sensitive to antitubulin agents [START_REF] Mozzetti | Class III btubulin overexpression is a prominent mechanism of paclitaxel resistance in ovarian cancer patients[END_REF][START_REF] Hasegawa | Prediction of response to docetaxel by quantitative analysis of class I and III beta-tubulin isotype mRNA expression in human breast cancers[END_REF]. Molecular modelling experiments showed that Ser277, the aminoacid in the M-loop of microtubules that interacts with taxanes, is replaced by an arginine residue in the class III β-tubulin that does not bind the drug and consequently, does not result in stabilisation of lateral microtubules [START_REF] Magnani | The beta/beta III tubulin isoforms and their complexes with antimitotic agents: Docking and molecular dynamics studies[END_REF]. Several retrospective clinical series of rather small size confirmed the cell line data and correlated protein or mRNA expression of TUBB3 to resistance to taxanes [START_REF] Seve | Is class III b-tubulin a predictive factor in patients receiving tubulinbinding agents?[END_REF]. Still, these series could not discern the prognostic (patient outcome irrespective of therapy) versus predictive (probability of benefit from taxane-based therapy) significance of this biomolecule.

Our retrospective analysis of 314 patients with high-risk resected breast cancer used quantitative mRNA transcription levels of TUBB3. We showed for the first time in the adjuvant setting that TUBB3 RNA expression in breast cancer tissues is significantly correlated to adverse clinicopathological characteristics (advanced T stage, anaplasia, hormone receptor negativity, HER2 overexpression) and inferior patient outcome. Moreover, multivariate analysis revealed that high TUBB3 mRNA transcription retained independent prognostic significance for both OS and DFS.

Finally, the randomisation of patients to a taxane-containing and a taxane-free chemotherapy arm enabled us to show that TUBB3 gene transcription is prognostic for adverse outcome but not predictive for resistance to taxanes. Taking into consideration the high p values for interaction of TUBB3 status with treatment arm and the moderately large sample size, we can rather confidently rule out a clinically significant predictive utility of TUBB3 for taxane efficacy.

Evidence from other investigators seem to confirm our findings and refute the taxane-resistant phenotype hypothesis for TUBB3. In the JBR.10 trial, 482 patients with resected non small cell lung cancer were randomised to observation versus adjuvant cisplatin/vinorelbine chemotherapy [START_REF] Seve | Class III beta tubulin expression and benefit from adjuvant cisplatin/vinorelbine chemotherapy in operable non-small cell lung cancer. Analysis of NCIC BR.10[END_REF]. In 265 patients examined, immunohistochemical TUBB3 protein expression was prognostic for poorer recurrence-free and overall survival in the observation, but not in the chemotherapy arm. Cox regression analysis failed to establish a significant interaction between TUBB3 IHC expression and chemotherapy in predicting outcome. Very recently, Dumontet et al analysed 1350 patients with resected node-positive breast cancer who had been randomised to TAC (docetaxel, adriamycin, cyclophosphamide) versus FAC adjuvant chemotherapy by studying the immunohistochemical protein expression of several biomolecules [START_REF] Dumontet | BCIRG 001 molecular analysis: prognostic factors in node-positive breast cancer patients receiving adjuvant chemotherapy[END_REF]. In multivariate analysis, high TUBB3 protein expression was significantly associated with poorer OS but lacked predictive utility for docetaxel efficacy, as the correlation with outcome was observed in both TAC and FAC arms and interaction testing revealed no differences within treatment subgroups. In our study, we failed to show any TUBB3 predictive significance for taxane benefit even when we restricted our analysis to two patient subgroups: 1) In those patients who received the taxane-containing therapy (E-T-CMF), there was no association of increasing TUBB3 mRNA levels with outcome, rather convincingly establishing the lack of a dose-response relation between gene transcription and taxane effect. 2) Since in the HE10/97 trial a benefit from taxane was seen only in patients with ER IHC-negative tumours, we analysed and failed to find any predictive significance of TUBB3 mRNA status for taxane efficacy in the ER IHC-negative subroup.

The adverse prognostic, but not predictive significance of TUBB3 mRNA may not seem paradoxical in view of emerging in vitro data: the TUBB3 gene is located at 16q24.3 and contains a promoter region, an enhancer sequence and an E-box binding site for transcription factors that all drive its transcription. Hypoxiadependent transcription factors, such as Stimulating Protein-1, Wilms tumour suppressor, p53, and NFkB induce gene expression, in keeping with the observation that TUBB3 is more expressed in hypoxic tumours (breast, ovarian cancer) than in normal mammary and ovarian biopsies [START_REF] Dennis | Cloning and characterisation of 5flanking region of the rat neuron-specific class III beta-tubulin gene[END_REF][START_REF] Raspaglio | Hypoxia induces Class III beta-tubulin gene expression by HIF1a binding to its 3-flanking region[END_REF]. Gan et al have recently shown that TUBB3 is overexpressed not only in taxane-resistant but also in cisplatin-refractory cancer cell lines [START_REF] Gan | Class III b-tubulin mediates sensitivity to chemotherapeutic drugs in non small cell lung cancer[END_REF]. TUBB3 indeed interacts with a plethora of cellular proteins, such as HSP70, FMO4 and Pyruvate Kinase M1. A functional analysis of the network of proteins associated to TUBB3 shows that many of them are involved in oxidative stress, glucose deprivation response and cell cycle control [START_REF] Cicchillitti | Proteomic characterisation of cytoskeletal and mitochondrial class III b-tubulin[END_REF]. Accordingly, TUBB3 seems to be integrated in a survival pathway, which is initiated by hypoxia and effects adaptation of cancer cells to a microenvironment with low oxygen and poor nutrient supply. Increased TUBB3 gene trancription could therefore be a marker of aberrant activation of this «hypoxia lethal phenotype», resulting in cancer proliferation, metastatic dissemination and poor patient outcome. . In addition to increased transcription, other mechanisms such as TUBB3 gene amplification, epigenetic regulation or decreased expression of repressing RNAs, such as microRNAs, might contribute to relatively high TUBB3 mRNA expression. Amplification of the TUBB3 gene seems, in fact, unlikely and has not been reported in breast cancer; the chromosomal region 16q24.3, as well as genes located centromerically or telomerically to TUBB3 have repeatedly been reported as deleted in breast cancer [START_REF] Loo | Array comparative genomic hybridization analysis of genomic alterations in breast cancer subtypes[END_REF][START_REF] Kaur | Functional identification of a BAC clone from 16q24 carrying a senescence gene SEN16 for breast cancer cells[END_REF][START_REF] Chin | Using arraycomparative genomic hybridization to define molecular portraits of primary breast cancers[END_REF]. In regard to the latter two options, TUBB3 gene transcription seems to be induced by hypoxia in cisplatin-sensitive ovarian cancer cells lacking methylation in the 3'-enhancer region of this gene, while the miR-200 family that, among other targets, represses TUBB3 mRNA is downregulated in human cancer cells (Mongroo and Rustgi 2010) [START_REF] Raspaglio | Hypoxia induces Class III beta-tubulin gene expression by HIF1a binding to its 3-flanking region[END_REF][START_REF] Mongroo | The role of the miR-200 family in epithelial-mesenchymal transition[END_REF]. Whether these mechanisms apply to breast cancer tissues warrants further investigation.

The correlation of TUBB3 mRNA levels with protein expression was poor in our study, with a Kappa statistic of only 0.24. Moreover, in contrast to protein, mRNA expression was associated strongly with adverse clinicopathological factors and retained independent prognostic significance for outcome. Among hypotheses that could interpret the finding, we could cite mRNA failing to translate to protein (alternate splicing, aberrant polyadenylation, microRNA-mediated or other form of translational silencing), protein degradation in the tumour block by inadequate fixation, problematic antigen retrieval, protein staining underestimation in old archival material, or finally, RNA amplification of normal tissue along with the tumour [START_REF] Goldstein | Minimum formalin fixation time for consistent estrogen receptor immunocytochemical staining of invasive breast carcinoma[END_REF][START_REF] Rhodes | Quality assurance in immunohistochemistry[END_REF][START_REF] Morris | RNA-mediated transcriptional gene silencing in human cells[END_REF]. Conversely, protein staining in the face of low level gene transcription could be due to the difficulty in capturing short-lived, sensitive to degradation mRNA from FFPE tumour blocks, the different fixation and turnover times of ribonucleic acids in relation to proteins, as well as accumulation of protein due to aberrant catabolism [START_REF] Ravo | Quantitative expression profiling of highly degraded RNA from formalin-fixed paraffin-embedded breast tumour biopsies by oligonucleotide microarrays[END_REF][START_REF] Yasuda | The RNA continent[END_REF]. In any case, TUBB3 mRNA as a continuous, objectively measured variable, was shown to possess correlative and prognostic characteristics not present when evaluating expression of the corresponding protein.

In a report previously published by our group we showed that high-level Tau gene (MAPT) transcription has prognostic significance for favourable patient outcome, though no predictive utility for taxane benefit [START_REF] Pentheroudakis | Gene expression of estrogen receptor, progesterone receptor and microtubule-associated protein Tau in high-risk early breast cancer: a quest for molecular predictors of treatment benefit in the context of a Hellenic Cooperative Oncology Group trial[END_REF]. Our findings were confirmed by the demonstration of prognostic, but not predictive utility for Tau protein expression among breast cancer patients receiving taxane-based adjuvant chemotherapy in the NSABP-B28 (n=1942) and the BCIRG001 (n=1350) randomised trials [START_REF] Dumontet | BCIRG 001 molecular analysis: prognostic factors in node-positive breast cancer patients receiving adjuvant chemotherapy[END_REF][START_REF] Yasuda | The RNA continent[END_REF]. Still other investigators have reported association between low MAPT mRNA or IHC levels with response to taxanes and resistance to tamoxifen [START_REF] Rouzier | Microtubule-associated protein tau: a marker of paclitaxel sensitivity in breast cancer[END_REF][START_REF] Andre | Microtubule-associated protein tau is a bifunctional predictor of endocrine sensitivity and chemotherapy resistance in estrogen-receptor positive breast cancer[END_REF]. In our previous report we speculated that, since all patients who had received adjuvant taxane therapy were also managed with hormonotherapy, in those with low MAPT mRNA levels the positive predictive significance for taxane benefit could have been «cancelled» by the predictive utility for tamoxifen resistance. In this study, MAPT protein expression had strong favourable prognostic significance only in patients receiving E-CMF, but not in those receiving a taxane. It is tempting to speculate that in the E-T-CMF patients, the favourable prognosis afforded by MAPT protein overexpression was diluted by the lack of taxane efficacy. This observation could serve as additional preliminary evidence that high MAPT gene or protein expression serve as both markers of favourable prognosis and markers of resistance to taxanes.

To conclude, our work has established a prognostic significance of the transcription of two gene products related to microtubules, β-tubulin isotype III and MAPT. These biomolecules could serve as useful prognosticators and stratification factors in future trials. Still, their function and relation to other biomolecules should be further investigated, while their prognostic and potentially predictive value remains to be validated. HeCOG plans to validate these findings in over 900 breast cancer blocks from a subsequent adjuvant chemotherapy study (He 10/00).

  The same experimental principles were used in order to study mRNA levels of the following genes: ESR1 (Estrogen Receptor), PgR (Progesterone Receptor), HER2 (Human Epidermal growth factor Receptor type 2, HER2), and MAPT ( Microtubule-Associated Protein Tau). Probes and primers used for the study of these genes are listed in Appendix 1. The quality and quantity of RNA was checked by measuring RPL37A expression as surrogate by qRT-PCR. Human reference total RNA pooled from ten human cell lines (Stratagene, La Jolla, California, USA) was used as a positive control. No-template-controls were assessed in parallel to exclude contamination. ER (clone 6F11, Leica Biosystems, Newcastle Upon Tyne, UK, dilution 1:70) and PgR (clone 1A6, Leica, dilution 1:70) expression, as well as TUBB3 (clone SDL3D10, BioGenex, San Ramon, CA, USA, dilution 1:1250), MAPT (clone T1029, United States Biological, Swampscott, MA, USA, dilution 1:150), HER2 (code Nu. A0485, Dako, Glostrup, DK, dilution 1:200), and ERCC1 [Excision Repair Cross-Complementation group 1 protein (clone 8F1, Thermo, Fremont, CA, USA, dilution 1:450) were processed at the Laboratory of Molecular Oncology of the Hellenic Foundation for Cancer Research, Aristotle University of Thessaloniki School of Medicine and evaluated immunohistochemically at the Pathology department of the Ioannina University Hospital by AB and SK. Immunohistochemical staining was performed according to standard protocols, with slight modifications, on serial 2.5

Figure 2 .Figure 3 .Figure 4 .

 234 Figure 2. Distribution of tumoural TUBB3 mRNA levels in the studied

Figure 5 .

 5 Figure 5. DFS (a) and OS (b) Kaplan-Meier curves by TUBB3 mRNA status.

Figure 6 .

 6 Figure 6. OS (left) and DFS (right) for E-T-CMF and E-CMF in TUBB3 mRNAlow and high patients.

Table 3 . Correlations of TUBB3 gene transcription and protein expression with clinicopathologic and molecular variables.

 3

	TUBB3 mRNA expression		TUBB3 protein expression
		(N=314)			(N=274)
	Low (n=157) N (%)	High (n=157) N (%)	p-value	Negative (n=160) N (%)	Positive (n=114) N (%)	p-value

Table 4 . Concordance of mRNA and IHC expression of TUBB3.

 4

	Protein expression of TUBB3
	Negative	Positive
	N (%)	N (%)

Acknowledgements

The authors wish to thank Ms. Evita Fragou and Ms. Dimitra Katsala for monitoring the study, Ms. Maria Moschoni for coordinating the data management and Ms.

Thalia Spinari for tissue sample collection. . On behalf of the Hellenic Foundation for Cancer Research, Athens, Greece, the senior author (GF) has pending patent applications with Siemens Healthcare Diagnostics, Cologne, Germany.