Antoine Venaille
email: antoine.venaille@ens-lyon.org

Julien Le Sommer

Jean-Marc Molines

Bernard Barnier

Stochastic variability of oceanic flows above topography anomalies

We describe a stochastic variability mechanism which is genuinely internal to the ocean, i.e. not due to fluctuations in atmospheric forcing. The key ingredient is the existence of closed contours of bottom topography surrounded by a stirring region of enhanced eddy activity. This configuration leads to the formation of a robust but highly variable vortex above the topography anomaly. The vortex dynamics integrates the white noise forcing of oceanic eddies into a red noise signal for the large scale volume transport of the vortex. The strong interannual fluctuations of the transport of the Zapiola anticyclone (∼ 100 Sv) in the Argentine basin are argued to be partly due to such eddy-driven stochastic variability, on the basis of a 310 years long simulation of a comprehensive global ocean model run driven by a repeated-year forcing.

I. INTRODUCTION

Large-scale oceanic currents show large fluctuations at decadal, centennial and even millennial time scales [START_REF] Ghil | Advanced Spectral Methods for Climatic Time Series[END_REF]. Understanding the nature and the causes of this variability is a major challenge for climate predictions. Some properties of oceanic variability can result from the intrinsic, deterministic non-linear dynamics of oceanic flows [START_REF] Dijkstra | Low-frequency variability of the large-scale ocean circulation: A dynamical systems approach[END_REF]. For instance, this approach has been proven useful to interpret the different peaks arising at decadal time scales in time-series of the Gulf-Stream position [START_REF] Simonnet | Homoclinic bifurcations in the quasi-geostrophic double-gyre circulation[END_REF]. Otherwise, the ubiquity of red noise signals in time series of various oceanic metrics is generally thought to result from the integration of white noise processes by the linearized dynamics [START_REF] Hasselmann | Stochastic climate models, Part I, Theory[END_REF]. Obviously, a source of noise is associated with the rapid fluctuations of atmospheric forcing, therefore leading to low frequency oceanic variability [START_REF] Frankignoul | A Simple Model of the Decadal Response of the Ocean to Stochastic Wind Forcing[END_REF]. However, red noise signals have also been reported in ocean models forced by steady winds but with sufficient spatial resolution to simulate the "oceanic weather system", i.e. mesoscale eddies at scale from 30 to 300 km [START_REF] Berloff | Large-Scale, Low-Frequency Variability in Wind-Driven Ocean Gyres[END_REF][START_REF] Hogg | Mechanisms of Decadal Variability of the Wind-Driven Ocean Circulation[END_REF]. This suggests the existence of intrinsic, stochastic variability of large-scale oceanic currents.

We show in the following that the dynamics of eddydriven flows above closed topography contours provides essential ingredients for supporting such stochastic variability genuinely internal to the ocean. We then argue that this mechanism contributes to the variability of the Zapiola anticyclone in the Argentine Basin.

II. BASIC MECHANISM

Let us consider first a barotropic (depth independent) flow above an axisymmetric topographic bump h(r) such that the total fluid depth h(r) is increasing for r < L h , and constant for r > L h , and surrounded by a "stirring" region, as sketched on figure 1-b. Here, stirring can be due to any mechanism outside the topography anomaly, generating disturbances in the flow field that propagate toward the topography anomaly. This situation is reminiscent of the large scale atmospheric circulation induced by mid-latitude stirring [START_REF] Williams | Ocean and Atmosphere Storm Tracks: The Role of Eddy Vorticity Forcing[END_REF]. Simple arguments relying on Kelvin circulation theorem explain the formation of an anticyclonic flow above the closed topographic contours [35] in the same way as westward flows are formed away from stirring regions in the atmosphere, see e.g. [START_REF] Held | Momentum transport by quasi-geostrophic eddies[END_REF][START_REF] Vallis | Atmospheric and Oceanic Fluid Dynamics[END_REF]. Neglecting other forcing than stirring, and considering that dissipation occurs through bottom friction modeled as a linear drag with coefficient ω b , the dynamics above the topography anomaly reads

∂ t q + u • ∇q = -ω b ζ h , ∇ • (hu) = 0 , q = ζ + f h , (1)
where u is the velocity, ζ = (∇ × u) • e z is the relative vorticity and f is the planetary vorticity, taken here as a constant (so that f /h contours are also topography contours). e z is a unit vector pointing upward.

We call v and u the radial and azimuthal velocities respectively (v is positive when directed outside the topography anomaly, u is positive when anti-clockwise). Defining eddies as deviation from the azimuthal average u ′ = uu and integrating equation (1) inside a circle of constant r yields

∂ ∂t u = - 1 r 2 ∂ ∂r r 2 u ′ v ′ -ω b u. (2)
Multiplying equation ( 2) by the total fluid depth h(r) and integrating from the center r = 0 to the boundary r = L h of the topography anomaly yields the transport equation

dT dt = η eddy -ω b T , (3)
where T = L h 0 dr hu. Assuming that typical variations of h over L h are smaller than h, and assuming that |u ′ v ′ /r| ≪ |∂ r u ′ v ′ | (which corresponds to the limit case of a channel), the eddy term by the eddy momentum fluxes at the boundary of the bump.

η eddy ≈ -h v ′ u ′ | r=L h is given
In the absence of direct external forcing, eddy fluxes tend to reduce large scale potential vorticity gradients, therefore driving anticyclonic flow above topographic bumps [START_REF] Bretherton | Two-dimensional turbulence above topography[END_REF]. One expects then that time-mean of the eddy term η eddy is positive (resp. negative) in the case of a positive (resp. negative) topography anomaly, as for instance predicted by linear theory [START_REF] Held | Momentum transport by quasi-geostrophic eddies[END_REF], and as re-ported in various numerical simulations, see e.g. [START_REF] Holloway | Systematic forcing of large-scale geophysical flows by eddy-topography interaction[END_REF][START_REF] Treguier | Topographically generated steady currents in barotropic turbulence[END_REF]. If η eddy were time independent, the vortex would reach a stationary state and its transport would be inversely proportional to bottom friction [START_REF] Dewar | Topography and barotropic transport control by bottom friction[END_REF]. However, stirring is more generally time varying, and so is the driving term η eddy . In particular, the eddy term η eddies could possibly vary depending on the anticyclone transport T , thus allowing complex feedbacks as in the atmosphere [START_REF] Lorenz | Eddy-Zonal Flow Feedback in the Southern Hemisphere[END_REF]. Assuming here that such feedbacks are negligible, and modelling the eddy term η eddy (t) as a constant plus a gaussian white noise, the transport satisfies a Langevin equation [START_REF] Langevin | Sur la theorie tu mouvement Brownien C[END_REF]. Transport time series are then instances of an auto-regressive process of order one [START_REF] Hasselmann | Stochastic climate models, Part I, Theory[END_REF][START_REF] Von Storch | Statistical Analysis in Climate Research[END_REF]. For frequency ω higher than the cut-off frequency ω b set by bottom friction, the transport power

spectrum | T (ω)| 2 = | η eddy | 2 (ω 2 + ω 2 b ) -1
is a red noise. This shows that oceanic eddies can drive the formation and the low frequency variability of anticyclonic flows above topographic bumps.

We argue in the following that this mechanism is responsible for the massive fluctuations in transport time series of the Zapiola Anticyclone [START_REF] Saraceno | Longterm variation in the anticyclonic ocean circulation over the Zapiola Rise as observed by satellite altimetry: Evidence of possible collapses[END_REF].

III. INTRINSIC VARIABILITY OF THE ZAPIOLA ANTICYCLONE

The Zapiola anticyclone is a strong and robust anticyclonic vortex above a sedimentary bump (the Zapiola drift), located in the Argentine basin, around (45 • S, 45 • W ), see figure 2. This bump is characterized by shoaling bottom topography rising by ∼ 800 m over a distance of ∼ 400 km. The anticyclone appears as a local maximum in sea surface height as shown in figure 2-a. Typical velocities of the anticyclone are about 0.1 m.s -1 over the whole vertical water column (∼ 5000 m depth). The associated transport is of the order of 100 Sv (= 10 6 m 3 .s -1 ) which is comparable with other major oceanic currents including the Gulf-Stream. Remarkably, the Zapiola anticyclone is surrounded by one of the most eddying region of the global ocean, namely the confluence between the Nord-Brazil current and the Malvinas current (see figure 2-b). Perhaps surprisingly, the Zapiola anticyclone has only been discovered rather lately, and almost simultaneously with in situ measurement [START_REF] Saunders | Oceanic Fluxes on the WOCE A11 Section[END_REF][START_REF] Weatherly | On deep-current and hydrographic observations from a mudwave region and elsewhere in the Argentine Basin[END_REF], numerical ocean models [START_REF] De Miranda | On the dynamics of the Zapiola Anticyclone[END_REF], and in theoretical studies [START_REF] Dewar | Topography and barotropic transport control by bottom friction[END_REF]. Also striking is the strong interannual variability of the Zapiola anticyclone transport which has recently been reported from 15 years of altimetric measurements [START_REF] Saraceno | Longterm variation in the anticyclonic ocean circulation over the Zapiola Rise as observed by satellite altimetry: Evidence of possible collapses[END_REF]. High resolution data syntheses also suggest the dominance of interior ocean dynamics as opposed to atmospheric forcing in determining the variability of the Zapiola anticyclone [START_REF] Volkov | The role of vorticity fluxes in the dynamics of the Zapiola Anticyclone[END_REF]. But the strong variability of the Zapiola anticyclone transport remains unexplained.

Here, we report strong interannual fluctuations of the Zapiola anticyclone transport in a 310 year long simulation of the global eddy admitting DRAKKAR model configuration [START_REF] Barnier | Impact of partial steps and momentum advection schemes in a global ocean circulation model at eddy-permitting resolution[END_REF][START_REF] Drakkar | Eddy permitting ocean circulation hindcasts of past decades[END_REF] under a repeated-year forcing. This model configuration is based on the NEMO ocean/seaice modeling framework [START_REF] Madec | Nemo ocean engine. Note du Pole de modelisation[END_REF]. The model grid uses zcoordinates with 46 levels and a partial step representation of bottom topography. The model horizontal resolution at the equator is 1/4th degree and varies with the cosine of the latitude so that at 45 • S the horizontal resolution is about 20km. The model configuration uses a biharmonic viscosity for momentum and an isoneutral harmonic diffusivity for tracers. The bottom friction is a quadratic function of the bottom velocity u b so that the bottom stress τ b is given by

τ b = C d u b 2 + e 1/2 u b ,
where e = 2.5 10 -3 m 2 .s -2 is the bottom turbulent kinetic energy due to tides and other small scale processes. According to common practice, the coefficient C d is calibrated such that for weak barotropic flows, the bottom friction time-scale ω -1 b is about 30 months. For a barotropic flow of about 0.1m.s -1 as the Zapiola anticyclone, the bottom friction time-scale ω -1 b is about 14 months.

The repeated-year forcing is built from the DRAKKAR forcing set DFS4 [START_REF] Brodeau | An ERA40 based atmospheric forcing for global ocean circulation models[END_REF]. The forcing was built by computing a daily climatology of the variables available in DFS4 during 50 years at 6-hourly periods. Notably, the quadratic quantities (wind stress and bulk exchange coefficients) were also averaged in order to guarantee that the climatological forcing is energetically consistent [START_REF] Penduff | Sealevel expression of intrinsic and forced ocean variabilities at interannual time scales[END_REF]. The resulting forcing fields were then low-passed filtered in time (3-point hanning filter) to remove the remaining high-frequency noise. The resulting one year long forcing was applied repeatedly during the 310 years of the simulation.

Figure 3-a,b show strong fluctuations of transport over the last 10 years of the simulation and the associated power-spectrum computed over the first 300 years of the simulation. The model being forced by repeated-year atmospheric fields, the wind forcing is practically identical every year. Therefore, in the model, interannual fluctuations of Zapiola anticyclone transport can only be due to intrinsic ocean dynamics. This experiment supports the idea that the variability of the Zapiola anticyclone can be partly attributed to intrinsic ocean dynamics.

A comparison of the time-series of transport T and the time-series of the eddy momentum flux η eddy is performed over the last 10 years of the simulation, see figure 3. Remarkably, fluctuations of the eddy momentum flux are essentially white at frequency ω lower than ω topo ∼ (20 days) -1 (figure 3-c). This correlation time scale corresponds to the period of a barotropic dipole rotating around the Zapiola drift [START_REF] Fu | 25-Day Period Large-Scale Oscillations in the Argentine Basin Revealed by the TOPEX/Poseidon Altimeter[END_REF][START_REF] Hughes | Three forms of variability in Argentine Basin ocean bottom pressure[END_REF], and interpreted either as a topographic Rossby wave [START_REF] Fu | 25-Day Period Large-Scale Oscillations in the Argentine Basin Revealed by the TOPEX/Poseidon Altimeter[END_REF], or as the superposition of Rossby basin modes [START_REF] Weijer | Multiple Oscillatory Modes of the Argentine Basin. Part I: Statistical Analysis[END_REF]. As expected from equation (3), for frequency ω lower than ω topo , the transport power spectra of figures 3-a and 3-c are well fitted by the function a(ω 2 + ω 2 b ) -1 , where the cut-off frequency ω b is estimated by evaluating the ratio between time-mean transport and time-mean eddy momentum flux η eddy / T ∼ (15 months) -1 , and where the coefficient a is the lowfrequency asymptotic value of the transport power spectrum. Strikingly, the value of the cut-off frequency ω b estimated from the transport and eddy momentum flux time-series is of similar magnitude as the effective bottom friction coefficient used in the model (estimated at about (14 month) -1 , see appendix). According to equation (3), the phase of the cospectrum T η * eddy should be given by -tan -1 (ω/ω b ). This prediction is again consistent with model output presented figure 3-d, despite some discrepancy for frequency of about month -1 . The discrepancy could come either from baroclinic effects or from possible retroaction of the mean flow on the eddies.

The above diagnostics support the idea that the red noise spectrum of the Zapiola anticyclone transport timeseries (at seasonal to interannual time scale, see figure 3-a) results from the integration by the dynamics of the eddy momentum flux fluctuations at the boundary of the anticyclone. Note that in presence of wind fluctuations, this integration mechanism would account for an additional low frequency variability of the anticyclone transport, just as in [START_REF] Weijer | Adjustment of the Southern Ocean to wind forcing on synoptic time scales[END_REF].

IV. CONCLUSION

In this letter, we have described a simple mechanism contributing to low-frequency variability of oceanic flows above large scale (around 500 km) topography anomalies. The key ingredients are closed contours of topography and a source of eddies. Our diagnostics of a comprehensive numerical ocean model suggest that such a mechanism explains at least part of the variability of the Zapiola Anticyclone in the Argentine Basin.

The formation of robust eddy-driven vortices above topography anomalies may provide an effective route for energy dissipation in the ocean. Indeed, because of the unstable nature of large scale oceanic flows, mesoscale eddies are ubiquitous in the ocean. In particular, high values of mesoscale eddy kinetic energy are observed along western boundaries of ocean basins. Here, we have depicted a situation where the mesoscale eddy kinetic energy can be absorbed above some topography anomaly (the Zapiola drift) and transferred to mean kinetic energy (the Zapiola anticyclone). The energy transfer is associated with the excitation, and the propagation of topographic Rossby waves [START_REF] Fu | Interaction of Mesoscale Variability with Large-Scale Waves in the Argentine Basin[END_REF]. A simple estimation of the order of magnitude for this dissipation rate in the case of the Zapiola anticyclone gives a value of about 1. 10 -3 W.m -2 over an area A ∼ 1. 10 6 km 2 , which amounts to a total of 1.10 -3 TW. This amounts to a significant fraction (about one tenth) of the total sink of ocean-eddy energy at the western boundary of the South Atlantic [START_REF] Zhai | Significant sink of ocean-eddy energy near western boundaries[END_REF]. Given the simple ingredients involved in setting the eddy-driven flow presented above, one might conjecture that the mechanism described in this letter could play an significant role in the internal low frequency dynamics and energetics of scale oceanic currents.

Appendix: diagnostic details

The barotropic transport streamfunction ψ is defined according to hu = e z ×∇ψ, where u is the depth averaged velocity. The anticyclone transport is defined as T = ψ(boundary) -ψ(center), where ψ(center) is the value of the streamfunction above the Zapiola drift (45E45S), and ψ(boundary) is the average of the streamfunction along the circle of radius L h ∼ 500 km depicted in figure 2-c.

The term η eddy is estimated by computing the term huv, with azimuthal averages taken along the same circle r = L h . Output data of the 310 years simulation are monthly averaged, except for the last ten years of the simulation, where 5-days averages have been computed. Eddy terms can therefore only be computed over the last 10 years.

FIG. 1 :

 1 FIG. 1: Large scale flows driven by eddies in presence of a mean gradient of potential vorticity f /h. a) Case of mid-latitude atmospheric circulation: the potential vorticity gradient is due to variations of the Coriolis parameter f with latitude (beta effect). Arrows represent Rossby waves that propagate away from the stirring region and imply the formation of a westward flow outside the stirring region and an eastward flow in the stirring region [27, adapted from figures 12.2 and 12.3]. b) Case of oceanic flows above a topographic bump of typical length L h = 600 km: the potential vorticity gradient is due to variations of depth h.

FIG. 2 :

 2 FIG. 2: The Zapiola anticyclone in the Argentine Basin. a) Mean Dynamical Topography (SSH) from CNES-CLS09 dataset. Above the Zapiola drift, SSH is proportional (and opposite) to the transport streamfunction since the flow is mostly depth independent. b) Surface eddy kinetic energy estimated from TOPEX altimeter over 1992-2002. c,d) Same quantities as a,b) in the model output over the last 10 years of the simulation. Isolines of f /h, where h is the local depth, are represented in thin dashed black line. The shaded circle in the lower left panel shows the region over which the diagnostics have been performed.

FIG. 3 :

 3 FIG. 3: Transport fluctuations. a) Transport power-spectrum | T | 2 (ω) from the 300 years time series of monthly mean model output. b) Transport and eddy momentum flux time series from the last 10 years time serie of 5 days mean model output c) Transport and eddy momentum flux power-spectrum from the 10 years time series of 5 days mean model output. d) Phase φ of the cospectrum T η * eddy .

Acknowledgments

The model simulation was performed at CINES (Montpellier, France) as part of the "Grands Challenges GENCI/CINES 2008" and benefited from the technical expertise of Nicole Audiffren. JLS, JMM and BB are sup-ported by the CNRS. AV was supported by DoE grant DE-SC0005189 and NOAA grant NA08OAR4320752 during part of this work. We thank Isaac Held for suggesting the analogy with the atmosphere, as well as David Marshall and Wilbert Weijer for their constructive review. The diagnostics presented in this letter are inspired from a poster presented by Chris W. Hughes at the European Geophysical Union General Assembly 2008 under reference EGU2008-A-06866.


MD Maria Pia Foschini
email: mariapia.foschini@unibo.it

Luca Morandi

Claudio Marchetti

Roberto Cocchi

Leonardo Henry Eusebi

Anna Farnedi

Giovanni Badiali

Davide Bartolomeo Gissi

Maria Gabriella Pennesi

Lucio Montebugnoli

Morandi Luca

Marchetti Claudio

Cocchi Roberto

Eusebi Leonardo Henry

Farnedi Anna

Badiali Giovanni

Bartolomeo

Pennesi Maria

CANCERIZATION OF CUTANEOUS FLAP RECONSTRUCTION FOR ORAL SQUAMOUS CELL CARCINOMA: REPORT OF THREE CASES STUDIED WITH THE mtDNA D-LOOP SEQUENCE ANALYSIS

Keywords: Oral cancer, skin graft, cutaneous flap, mitochondrial DNA D-loop Cutaneous flap cancerization Oral cancer, skin graft, cutaneous flap, mitochondrial DNA D-loop

HAL is

Aims

Tissue defects, resulting from surgical resection of oral squamous cell carcinoma (OSCC), are routinely reconstructed with skin graft. OSCC arising from the grafted skin have been described, however, it is still unclear whether primary and second tumours have a common clonal origin. By screening mitochondrial DNA D-loop region (mtDNA), we evaluated the clonal relationship between the primary OSCC and the second neoplastic features appearing in the skin graft in three patients.

Methods and Results

In all the three cases, the neoplastic lesions arising in the skin graft showed a clonal relationship with the previous OSCC and, on the basis of the results obtained with the mtDNA analysis, could be considered a recurrence of the primary OSCC rather than a second primary OSCC.

Conclusions

Starting from a field of genetically altered cells of the oral mucosa, the spreading of the clonal cell population to the cutaneous flap might be stimulated by cytokines produced by the grafted skin. More studies are needed to evaluate the molecular relationship between primary and second OSCC to identify patients at higher risk of developing a second tumour of the skin graft.

INTRODUCTION

Oral squamous cell carcinoma (OSCC) is the most common type of cancer affecting the oral cavity. Despite its frequency, diagnoses are often delayed and in order to achieve complete tumour removal, major surgical resections are needed. More extensive eradications of OSCC are possible since tissue defects, resulting from surgical resection, are routinely reconstructed with skin flaps obtained from radial forearm including a microvascular anastomosis for attachment at the site of reconstruction. [START_REF] Song | The forearm flap[END_REF] Recurrences and second primary OSCC are not uncommon in the oral cavity with a development rate of 2-3% of new cases per year. [START_REF] Licciardello | Multiple primary cancer in patients with cancer of the head and neck: second cancer of the head and neck, esophagus, and lung[END_REF] Several cases of squamous cell carcinoma or dysplasia arising from the grafted skin have been described (table 1); however, knowledge on behaviour and modification in the grafted skin is still scanty. [START_REF] Sinclair | Histological changes in radial forearm skin flaps in the oral cavity[END_REF][START_REF] Badran | Behavior of radial forearm skin flaps transplanted into the oral cavity[END_REF] In 1953, Slaughter and colleagues introduced the concept of "Field cancerization" as a pathogenic pathway for multiple primary OSCC arising in different areas, associated to pre-neoplastic lesions. [START_REF] Slaughter | Field cancerization in oral stratified squamous epithelium; clinical implications of multicentric origin[END_REF] The Authors 5 based the concept of "Field cancerization" on histological observations from OSCCs and associated pre-neoplastic lesions, such as dysplasia or in situ carcinoma, developed in different areas of a single patients. [START_REF] Slaughter | Field cancerization in oral stratified squamous epithelium; clinical implications of multicentric origin[END_REF] Subsequently, the concept of field cancerization was sustained by further clinical and histological evidence, and in the last decade several studies, based on molecular techniques, evaluated the genetic basis of this concept. Indeed, this observation was considered of crucial importance to differentiate recurrences from second primary OSCC. Furthermore, the oral mucosa undergoes malignant transformation through the development of genetically altered keratinocytes that progressively gain further mutations, most probably as a result of continued exposure to carcinogens, such as tobacco and alcohol. [START_REF] Califano | Genetic progression model for head and neck cancer: implications for field cancerization[END_REF][START_REF] Braakhuis | A genetic progression model of oral cancer: current evidence and clinical implications[END_REF][START_REF] Lumerman | Oral epithelial dysplasia and the development of invasive squamous cell carcinoma[END_REF] Mutated keratinocytes with growth advantage gradually expand and replace normal epithelial cells of the oral mucosa favouring the development of a second primary OSCC. [START_REF] Braakhuis | Expanding fields of genetically altered cells in head and neck squamous carcinogenesis[END_REF][START_REF] Tabor | Multiple head and neck tumors frequently originate from a single preneoplastic lesion[END_REF] Nevertheless, data sustaining the concept of field cancerization are difficult to reconcile with the development of dysplasia and squamous carcinoma in cutaneous skin grafted into the oral cavity.

To date, no molecular studies have been performed on squamous carcinomas arising on the grafted skin; furthermore, the clonal relationship between the primary OSCCs and the second neoplastic features appearing in the skin grafts still needs to be assessed, in order to differentiate recurrences from second primary OSCCs.

The high frequency rate of mtDNA mutations in tumours [START_REF] Fliss | Facile detection of mitochondrial DNA mutations in tumors and bodily fluids[END_REF][START_REF] Coller | High frequency of homoplasmic mitochondrial DNA mutations in human tumors can be explained without selection[END_REF][START_REF] Richard | Nuclear and mitochondrial genome instability in human breast cancer[END_REF] especially those found in the D-loop region, a non coding region, along with numerous mitochondrial genomes present in a single cell, makes mtDNA a reliable marker for clonality assays from microdissected paraffin-embedded tissue samples. [START_REF] Morandi | Intraepidermal cells of Paget's carcinoma of the breast can be genetically different from those of the underlying carcinoma[END_REF][START_REF] Morandi | Genetic similarities and differences between lobular in situ neoplasia (LN) and invasive lobular carcinoma of the breast[END_REF][START_REF] Morandi | Genetic relationship among atypical adenomatous hyperplasia, bronchioloalveolar carcinoma and adenocarcinoma of the lung[END_REF] We evaluated the clonal relationship between the primary cancer affecting the oral mucosa and the second neoplastic features appearing in the skin graft by screening the mtDNA D-loop region in three OSCC patients. The patient has never discontinued smoking. She died of OSCC, 4 years after the first surgery.

Case 2

In February 2004 a 58 year old female patient presented with an ulcerated lesion of the mouth floor (Figure 1A), diagnosed by an incisional biopsy as invasive OSCC (Figure 1B). The patient was a heavy smoker and previous personal and familial anamneses were unremarkable. Radical surgical excision of the neoplastic mass was performed at the time and a cutaneous graft of forearm skin was used to repair the defect (Figure 1C). No radiotherapy was performed. The patient discontinued smoking and was put on followup. of the cutaneous graft, close to the margin with the oral mucosa. An incisional biopsy was performed to define the nature of the ulcer, diagnosing in situ OSCC of the cutaneous graft (Figure 1E). The lesion was then completely removed.

In October 2008, a third lesion was observed, consisting of an erythroplasic area located at the right side of the skin graft, with no spatial relationship with the in situ OSCC. Five years after the first surgical resection, the patient is alive, still on follow up, and free of neoplastic disease.

Case 3

In February 2005, a 52 year old male patient, heavy smoker, presented a leucoplasic area, ulcerated in the centre, in the anterior third of the right margin of the tongue. After the histological diagnosis of invasive OSCC, radical surgical excision of the neoplastic lesion was performed together with bilateral submandibular lymph node dissection. A forearm free flap was used to reconstruct the defect of the right margin of the tongue.

In august 2009, the patient presented an exophytic lesion with focal ulceration of the cutaneous graft used. The lesion was diagnosed as squamous cell carcinoma in incisional biopsy and thereafter, it was surgically removed.

Five months after the last surgical resection the patient is alive, on follow up, and free of neoplastic disease.

MATERIALS AND METHODS

All tissues had been formalin fixed and paraffin embedded. Staging was performed according to the TNM staging system, [START_REF]AJCC. Cancer Staging Handbook[END_REF] grading was performed according to the criteria defined by Kademani et al. [START_REF] Kademani | Prognostic factors in intraoral squamous cell carcinoma: the influence of histologic grade[END_REF] Moreover, in all three cases, the skin flap specimens which included the second tumours were all completely sampled for histological examination.

Microdissection and mtDNA sequencing analysis

Pertinent lesions were microdissected using the laser assisted SL µcut microtest GmbH distributed by Nikon (Firenze, Italy, http://www.mmi-micro.com) as previously described. [START_REF] Kumar | Efficiency of the neighbor-joining method in reconstructing deep and shallow evolutionary relationships in large phylogenies[END_REF][START_REF] Kumar | MEGA2: molecular evolutionary genetics analysis software[END_REF][START_REF] Saitou | The neighbor-joining method: a new method for reconstructing phylogenetic trees[END_REF] Different portions of normal epithelial tissue located far away from the neoplastic lesions were dissected and used as reference DNA sequence. DNA was extracted using the QIAamp® DNA Micro kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. An extraction control, to which no tissue was added, was processed in parallel with each sample extraction. The mtDNA D-loop sequence analysis was performed by amplifying four overlapping segments of about 300 bp, covering the whole region from position 16056 to position 729, according to Anderson and colleagues [START_REF] Anderson | Sequence and organization of the human mitochondrial genome[END_REF] (see www.mitomap.org for revised Cambridge mtDNA reference sequence).

Primers were designed using primer3 (http://www-genome.wi.mit.edu/cgibin/primer/primer3_www.cgi, see Table 2 for sequence details). These primers were selected to avoid amplification of human mitochondrial pseudogenes in the nuclear genome. [START_REF] Woischnik | Pattern of organization of human mitochondrial pseudogenes in the nuclear genome[END_REF] PCR products were directly sequenced using CEQ2000 XL instrument (Beckman Coulter, Inc., Fullerton, CA) following the instructions of the provider.

Phylogenetic and cluster analyses were conducted using MEGA software version 3.1 [START_REF] Kumar | Efficiency of the neighbor-joining method in reconstructing deep and shallow evolutionary relationships in large phylogenies[END_REF][START_REF] Kumar | MEGA2: molecular evolutionary genetics analysis software[END_REF][START_REF] Saitou | The neighbor-joining method: a new method for reconstructing phylogenetic trees[END_REF] (http://www.megasoftware.net ) using the neighbour joining method (NJ) [START_REF] Kumar | MEGA: Molecular Evolutionary Genetics Analysis software for microcomputers[END_REF] and Kimura-2 parameter with Gamma model that corrects for multiple hits taking into account transitional and transversional substitution rates and differences in the site substitution rates. [START_REF] Kimura | A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences[END_REF] Every NJ tree was tested for standard error by the Bootstrap method. [START_REF] Efron | An introduction to the bootstrap[END_REF]

RESULTS

Case 1

Histology: histological examination of the tumour removed from the right mandible (indicated as T1 in the mtDNA analysis) confirmed the diagnosis of OSCC, staged T2N0M0, 17 grade 3. [START_REF] Kademani | Prognostic factors in intraoral squamous cell carcinoma: the influence of histologic grade[END_REF] Resection margins were tumour free, but a small neoplastic nest was present close (< 5 mm) to the medial margin.

The tumour of the right maxilla (indicated as T2 in the mtDNA analysis) was diagnosed as a second primary OSCC as it arose at a distance > 3 cm from the first OSCC and was staged T4N0M0, 17 grade 3. [START_REF] Kademani | Prognostic factors in intraoral squamous cell carcinoma: the influence of histologic grade[END_REF] All resection margins were tumour free (distance >5mm).

The lesion on the skin graft (indicated as T3 in the mtDNA analysis) showed the epidermis filled with atypical keratinocytes appearing as in situ squamous cell carcinoma. [START_REF] Barnes | Pathology and Genetics Head and Neck Tumours, World Health Classification of Tumours[END_REF] In addition, nests of neoplastic cells invaded the superficial dermis.

Therefore, the diagnosis of OSCC with early dermis invasion was reported. Resection margins were free of carcinoma and of dysplastic features. mtDNA analysis: the phylogenetic neighbour joining tree revealed that the two normal epithelial samples (indicated as N1 and N2) clustered together while the three lesions should be considered independent entities with a clonal relationship between T1 (OSCC

Case 2

Histology: the tumour affecting the floor of the mouth (indicated as T1 in the mtDNA analysis) was a conventional type of OSCC, staged T1N0M0, 17 grade 2. [START_REF] Kademani | Prognostic factors in intraoral squamous cell carcinoma: the influence of histologic grade[END_REF] All resection margins were tumour free, with a minimum distance between the tumour and the closest margin > 5 mm.

The lesion appearing in the skin graft (T2) was characterised by the epidermis filled by atypical keratinocytes extending to the follicular infundibula replacing the follicular epithelium. This lesion met the criteria for in situ OSCC. [START_REF] Barnes | Pathology and Genetics Head and Neck Tumours, World Health Classification of Tumours[END_REF] The resection margins were free of tumour and dysplastic lesions.

The oral mucosa adjacent to the skin graft margin (T3) was composed of atypical keratinocytes, arranged in an irregular architecture, replacing the lower two-thirds of the squamous epithelium. Therefore, the diagnosis of moderate dysplasia was performed. [START_REF] Barnes | Pathology and Genetics Head and Neck Tumours, World Health Classification of Tumours[END_REF] mtDNA analysis: the phylogenetic neighbour joining tree showed a close genetic relationship between T1 and T2. On the contrary, the dysplastic lesion appeared in 2008 (T3) had several mutations not in common with the previous ones, a sign of wide genetic distance. As expected, in the tree, normal epithelia (N1, N2 and N3) were located far away from the four lesions (Figure 3). Histology: histological examination of the OSCC removed from the right margin of the tongue (indicated as T1 in the mtDNA analysis) showed features of a conventional well differentiated OSCC and no lymph node metastases were present. Thus, the tumour was staged T1N0M0, [START_REF]AJCC. Cancer Staging Handbook[END_REF] grade 2. [START_REF] Kademani | Prognostic factors in intraoral squamous cell carcinoma: the influence of histologic grade[END_REF] The cancer was surrounded by an area of in situ carcinoma (T1IS). Resection margins were tumour free, however, a small neoplastic nest was present close (distance <5 mm) to the deep margin.

The histological analysis of the lesion developed in the skin graft (T2) showed the epidermis filled by atypical keratinocytes with the appearance of in situ squamous cell carcinoma. [START_REF] Barnes | Pathology and Genetics Head and Neck Tumours, World Health Classification of Tumours[END_REF] In addition, well differentiated invasive OSCC was present. mtDNA analysis: the phylogenetic neighbour joining tree revealed that the two normal epithelial samples (N1 and N2) were clustered together, while the two lesions can be considered independent entities. Furthermore, a moderate relationship between the T1 in situ carcinoma (T1IS) and both the in situ carcinoma (T2IS) and the OSCC (T2) and arising from the skin graft was observed (Figure 4). However, T1 (the OSCC arising in the tongue) appeared to have no clonal relationship and was divergent from the other carcinomas. This might be the consequence of new random acquired mutations during cellular proliferation.

DISCUSSION

To define a second primary OSCC, mainly clinical criteria, such as distance between the first and the second primary tumour >2 cm or time interval between the tumours >3 [START_REF] Braakhuis | Second primary tumors and field cancerization in oral and oropharyngeal cancer: molecular techniques provide new insights and definitions[END_REF] However, increasing evidence suggests that in some cases, regardless of clinical characteristics, primary and second tumours may have a common clonal origin.

Several methods are nowadays available to assess the clonal relationship between two lesions. Clonal assessment by mtDNA analysis [START_REF] Morandi | DNA mitocondriale e CGH array in patologia mammaria[END_REF] is based on the concept that mitochondrial DNA is present in a high copy number in each cell (10 3 -10 4 ) and that the vast majority of these copies are identical at birth (homoplasmic). In other words, neoplastic cells preserve the high copy number but show a high frequency of mutations in the mtDNA especially in the D-loop region. Coller et al. [START_REF] Coller | High frequency of homoplasmic mitochondrial DNA mutations in human tumors can be explained without selection[END_REF] demonstrated that the mtDNA D-loop region is not involved in transformation and disease progression, since it would appear that mutations in this mtDNA segment do not offer a proliferation advantage to the cell. Therefore, these acquired mutations may be considered a reliable marker to assess clonality as previously indicated. [START_REF] Morandi | Intraepidermal cells of Paget's carcinoma of the breast can be genetically different from those of the underlying carcinoma[END_REF][START_REF] Morandi | Genetic similarities and differences between lobular in situ neoplasia (LN) and invasive lobular carcinoma of the breast[END_REF][START_REF] Morandi | Genetic relationship among atypical adenomatous hyperplasia, bronchioloalveolar carcinoma and adenocarcinoma of the lung[END_REF] In all three present cases the neoplastic lesions arising in the skin graft showed a clonal relationship with the previous OSCC. Therefore, on the basis of the results obtained with mtDNA analysis, the present cases of OSCC arising in the skin graft can be considered a recurrence of the primary OSCC rather than a second primary OSCC.

However, these results seem difficult to reconcile with the clinical and histological features of these cases. All lesions arising in the skin graft were characterized by the presence of in situ OSCC. Furthermore, in cases two and three, the skin graft lesions developed more that 3 and 5 years after the removal of the first OSCC, respectively. These observations are difficult to reconcile with the molecular analysis which shows a clonal relationship between the primary OSCC and the neoplastic lesion affecting the graft. However, it should be kept in mind that we are dealing with skin grafted in the oral cavity and, as it is well known, skin tumour cells can spread through the epidermis, as commonly seen in Borst-Jadasson phenomenon, [START_REF] Helm | Borst-Jadassohn phenomenon associated with an undifferentiated spindle cell neoplasm[END_REF] horizontal growth of melanomas and Paget disease of the nipple.

All these phenomena, that are a part of routine surgical pathology, have been explained by demonstrating the capacity of epidermal keratinocytes to produce cytokines that allow and induce the cell movements.

Currently available data indicate that the skin basically maintains its morphology, even when implanted in the oral cavity. Indeed, diseases typically affecting the skin such as psoriasis [START_REF] Dimitrakopoulos | Dermal psoriasis involving an oral split-skin graft. Case report[END_REF] and focal acantholytic dyskeratosis [START_REF] Hurwitz | Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer[END_REF] have been reported in skin grafts. Therefore, it is not implausible to think that skin grafted into the oral cavity may retain its ability to produce cytokines continuing to give its instructive signals. [START_REF] Badran | Behavior of radial forearm skin flaps transplanted into the oral cavity[END_REF] Taken together, our results suggest that single neoplastic cells from the original OSCC can spread laterally, reach the epidermis of the skin graft and implant there giving rise to the OSCC.

The hypothesis of an intra-epithelial spread of neoplastic keratinocytes in the oral cavity has been proposed and described as the "patch-field-carcinoma model" by Braakhius and colleagues. [START_REF] Braakhuis | A genetic progression model of oral cancer: current evidence and clinical implications[END_REF] Starting from a patch of genetically altered cells, through a clonal expansion in a lateral direction, a field lesion develops and gradually grows taking over the normal epithelium. Within this genetically altered field, a transforming event eventually leads to the development of a subclone with invasive growth that turns into a carcinoma.

Within this background, it is possible that after radical resection of the tumour, the genetically altered field is still present in the patient. Therefore, the field expansion In conclusion, primary OSCC and tumours arising from grafted skin might develop from a common genetically altered field. Moreover, the spreading of the clonal cell population to the cutaneous flap might be stimulated by cytokines produced by the grafted skin.

More studies are needed to evaluate the molecular relationship between primary and second OSCC in order to identify those patients who are at higher risk of developing a second tumour and thus require a more accurate long-term follow-up.

F

1

 1 In 2004, a 61 year old female, heavy smoker, presented with an ulcerated mass located in the retromolar trigone of the right mandible. An incisional biopsy diagnosed an invasive OSCC. Thereafter, the right mandible was surgically removed and substituted with a peroneal bone and an osteocutaneous flap comprising forearm skin. In February 2006 the patient presented an exophytic mass of the right maxillary gingiva, which was diagnosed as OSCC by an incisional biopsy. The patient underwent right maxillectomy with reconstruction. In April 2006 the patient presented a polypoid lesion located in the centre of the cutaneous mandibular graft used to reconstruct the defect caused by the surgical resection of the first OSCC. The lesion was surgically removed.

6 Four

 6 years later, in April 2008, an ulcerated lesion appeared at the periphery (Figure1D)

  retromolar trigone of the right mandible) and T3 (the in situ OSCC arising on the skin graft) (Figure2). T2 (the OSCC arising in the maxilla) appears completely separated from T1 and T3 indicating lack of clonal relationship.

  been proposed.

  spread into the skin graft used to reconstruct the tissue defect and be the cause of a new cancer.

Figure 1 :

 1 Figure 1: Macroscopic view (A) and histology (B) of the primary OSCC; skin graft placed to repair the mucosal defect (C); macroscopic view (D) and histology (E) of the SCC on the skin graft.

Figure 2 :

 2 Figure 2: Phylogenetic neighbour joining tree of case 1.

Figure 3 :

 3 Figure 3: Phylogenetic neighbour joining tree of case 2.

Figure 4 :

 4 Figure 4: Phylogenetic neighbour joining tree of case 3.

  Histology of the SCC arised on the skin graft 150x112mm (300 x 300 DPI)Phylogenetic neighbour joining tree of case 1 113x44mm (400 x 400 DPI)Phylogenetic neighbour joining tree of case 2 111x50mm (400 x 400 DPI)

Table 1 -Cases of carcinoma developed from skin grafts Author [ref] Year N. of cases Site of tumour Tumour histology

 1 Nibu K, Sugasawa M, Nakatsuka T, Harii K, Ichimura K. A second primary squamous cell carcinoma arising in a radial forearm flap used for reconstruction of the hypopharynx. ORL J Otorhinolaryngol Relat Spec 1998;60;170-173.38.Scott MJ, Klaassen MF. Squamous cell carcinoma in a tube pedicle forty years after jaw reconstruction. Br J Plast Surg 1992;45;70-71.

	o
	r
	P
	e e r
	R e v i e w

SCC: Squamous Cell Carcinoma; RT: Radiotherapy.

Table 2 -Primers for mtDNA D-loop sequence analysis

 2

	Primer	Sequence
	Primer1 CCAAGTATTGACTCACCCATCAAC
	Primer2 TGTGCGGGATATTGATTTCACG
	Primer3 TGAAATCAATATCCCGCACA
	Primer4 GGATGAGGCAGGAATCAAAG
	Primer5 GAGCTCTCCATGCATTTGGT
	Primer6 TGGTTAGGCTGGTGTTAGGG
	Primer7 CCCTAACACCAGCCTAACCA
	Primer8 AGGGTGAACTCACTGGAACG

Published on behalf of the British Division of the International Academy of Pathology Histopathology

Acknowledgements

Work financed by grants from the Ministry of the University and Research (Fund ex 40% "Fundamental Oriented Research") and Regione Emilia Romagna (Funds: Region -University Research Program 2007-2009, Project Oncology). AF is supported by "Vanda Vanini e Sandro Cavagnino" grants.

Published on behalf of the British Division of the International Academy of Pathology Histopathology

Conflict of interest statement

None declared