Aafke Meerveld-Eggink

O Weerdt

R M Voer

Guy A M Berbers

H Velzen-Blad

Bart J Vlaminckx

Douwe H Biesma

Ger T Rijkers

Okke De Weerdt

Richarda M De Voer

Heleen Van Velzen-Blad

Impaired antibody response to conjugated meningococcal serogroup C vaccine in asplenic patients Running head: MenC conjugated vaccine in asplenia

Keywords: meningococcal serogroup C, conjugated vaccines, asplenia, avidity, IgG subclasses

Impaired antibody response to conjugated meningococcal serogroup C vaccine in asplenic patients

Introduction

Functional or anatomical asplenic patients are at increased risk of a severe overwhelming post splenectomy infection (OPSI) with encapsulated bacteria. After Streptococcus pneumoniae as the major cause, one of the other prevalent causative organisms is Neisseria meningitidis. [START_REF]Prevention of pneumococcal disease: recommendations of the Advisory Committee on Immunization Practices (ACIP)[END_REF][START_REF]Guidelines for the prevention and treatment of infection in patients with an absent or dysfunctional spleen. Working Party of the British Committee for Standards in Haematology Clinical Haematology Task Force[END_REF][START_REF] Davies | Working Party of the Haematology/Oncology Task Force. Update of guidelines for the prevention and treatment of infection in patients with an absent or dysfunctional spleen[END_REF] To reduce the incidence of these infections in asplenic patients, a vaccination schedule including N. meningitidis is strongly recommended. [START_REF] Bilukha | Prevention and control of meningococcal disease. Recommendations of the Advisory Committee on Immunization Practices (ACIP)[END_REF] Of the known meningococcal groups, 5 groups (A, B, C, Y and W135) are responsible for more than 95% of invasive disease. [START_REF] Granoff | Protective activity of group C anticapsular antibodies elicited in two-year-olds by an investigational quadrivalent Neisseria meningitidis-diphtheria toxoid conjugate vaccine[END_REF] In 2002, after an outbreak of MenC infection, a national vaccination campaign was performed in the Netherlands in which all persons 1-18 years of age were vaccinated against MenC. Since then, due to vaccination and herd immunity, the circulation of MenC is very low. [START_REF] De Greeff | Protection from routine vaccination at the age of 14 months with meningococcal serogroup C conjugate vaccine in the Netherlands[END_REF] Data on the ability of asplenic individuals to establish an adequate antibody response to vaccination with polysaccharide vaccines are conflicting. The response depends among others on the reason for asplenia and on the vaccine that is used (polysaccharide versus conjugated). [START_REF] Giebink | Serum antibody and opsonic responses to vaccination with pneumococcal capsular polysaccharide in normal and splenectomized children[END_REF][START_REF] Sullivan | Immune response after splenectomy[END_REF][START_REF] Eigenberger | Antibody responses to pneumococcal and hemophilus vaccinations in splenectomized patients with hematological malignancies or trauma[END_REF][START_REF] Smets | Randomised revaccination with pneumococcal polysaccharide or conjugate vaccine in asplenic children previously vaccinated with polysaccharide vaccine[END_REF] Immunisation with meningococcal polysaccharide vaccines was not recommended for patients with asplenia due to the short duration of protection and the absence of protection against the most common serogroup B. [START_REF]Guidelines for the prevention and treatment of infection in patients with an absent or dysfunctional spleen. Working Party of the British Committee for Standards in Haematology Clinical Haematology Task Force[END_REF][START_REF] Melles | Prevention of infections in hyposplenic and asplenic patients: an update[END_REF] With the introduction of conjugated vaccines, inducing long-term immunity, vaccination of asplenic patients with meningococcal conjugated vaccines now is recommended. [START_REF] Melles | Prevention of infections in hyposplenic and asplenic patients: an update[END_REF][START_REF] Davidson | Prevention and management of infections in patients without a spleen[END_REF] Beside increase in antibody concentration as a surrogate marker of protection offered by the MenC vaccine, antibody avidity can be determined. Antibody avidity is a biomarker, reflecting the generation of immunological memory after immunisation. [START_REF] Wuorimaa | Avidity and subclasses of IgG after immunization of infants with an 11-valent pneumococcal conjugate vaccine with or without aluminum adjuvant[END_REF][START_REF] Harris | Avidity of the immunoglobulin G response to a Neisseria meningitidis group C polysaccharide conjugate vaccine as measured by inhibition and chaotropic enzyme-linked immunosorbent assays[END_REF][START_REF] Goldblatt | Antibody avidity as a surrogate marker of successful priming by Haemophilus influenzae type b conjugate vaccines following infant immunization[END_REF] Antibody avidity is defined as the overall antigen-binding capacity of polyclonal antibodies with different affinities. [START_REF] Wuorimaa | Avidity and subclasses of IgG after immunization of infants with an 11-valent pneumococcal conjugate vaccine with or without aluminum adjuvant[END_REF][START_REF] Harris | Avidity of the immunoglobulin G response to a Neisseria meningitidis group C polysaccharide conjugate vaccine as measured by inhibition and chaotropic enzyme-linked immunosorbent assays[END_REF][START_REF] Kelly | Immunological memory: the role of B cells in long-term protection against invasive bacterial pathogens[END_REF][START_REF] Balmer | Serologic correlates of protection for evaluating the response to meningococcal vaccines[END_REF] Polysaccharide conjugate vaccines have the ability to elicit higher avidity antibodies with increased functional activity than do plain polysaccharide vaccines. [START_REF] Harris | Avidity of the immunoglobulin G response to a Neisseria meningitidis group C polysaccharide conjugate vaccine as measured by inhibition and chaotropic enzyme-linked immunosorbent assays[END_REF] The magnitude of the avidity index is dependent on the type of conjugate vaccine. [START_REF] Schlesinger | Avidity and bactericidal activity of antibody elicited by different Haemophilus influenzae type b conjugate vaccines. The Vaccine Study Group[END_REF] In this study, the efficacy of a MenC conjugated vaccine in terms of increase in antibody concentration after vaccination (quantity) and the avidity of the antibodies (quality) is determined in adult, asplenic patients.

Patients and methods

Patients

In 2006 and 2007, patients were included in a study to evaluate the vaccination coverage of adult asplenic patients in the Utrecht area of the Netherlands. [START_REF] Meerveld-Eggink | Vaccination coverage and awareness of infectious risks in patients with an absent or dysfunctional spleen in the Netherlands[END_REF] This study was approved by the local institutional ethics committee and all patients gave written informed consent.

When applicable, patients were vaccinated with conjugated pneumococcal, Hib and meningococcal vaccines according to the institutions protocol and serum samples were taken pre-and post-vaccination to determine antibody concentrations. Results of the pneumococcal and Hib vaccines will be reported separately (Meerveld et al., manuscript submitted).

Vaccination protocol

Patients were vaccinated against N. meningitidis when they were never vaccinated before, when vaccination status against the meningococcus could not be retrieved or when vaccination with a polysaccharide meningococcal vaccine occurred more than three years ago. A first dose of conjugate meningococcal group C vaccine was given, consisting of MenC polysaccharide covalently coupled to tetanus toxoid (NeisVac-C ® , Baxter AG, Vienna, Austria). Blood samples were drawn pre-immunization, 3 weeks and 35 weeks postimmunization. Serum samples were stored at -20°C until use. When antibody concentrations after the first vaccination remained below 2 μg IgG/mL in patients (so-called hyporesponders), a second dose of conjugate MenC vaccine was given.

Laboratory determinations

Meningococcal antibody concentrations were measured against serotype C by multiplex immunoassay (MIA) as described previously. [START_REF] De Voer | Simultaneous detection of Haemophilus influenzae type b polysaccharide-specific antibodies and Neisseria meningitidis serogroup A, C, Y, and W-135 polysaccharide-specific antibodies in a fluorescent-bead-based multiplex immunoassay[END_REF][START_REF] De Voer | Kinetics of antibody responses after primary immunization with meningococcal serogroup C conjugate vaccine or secondary immunization with either conjugate or polysaccharide vaccine in adults[END_REF][START_REF] Lal | Development and evaluation of a tetraplex flow cytometric assay for quantitation of serum antibodies to Neisseria meningitidis serogroups A, C, Y, and W-135[END_REF] All antibody concentrations were logtransformed and geometric mean concentrations (GMC) were calculated. Seroconversion was defined as a ≥ fourfold increase in antibody concentration after vaccination. The chosen anti-meningococcal antibody threshold for correlation with long-term protection was ≥ 2.0 µg/mL. MenC-specific IgG1 and IgG2 subclasses were measured by a modification of the MIA as described above. [START_REF] De Voer | Development of a fluorescent-bead-based multiplex immunoassay to determine immunoglobulin G subclass responses to Neisseria meningitidis serogroup A and C polysaccharides[END_REF] Previously assigned concentrations of meningococcal IgG1, and IgG2 antibodies were used in this assay. [START_REF] Holder | Assignment of Neisseria meningitidis serogroup A and C class-specific anticapsular antibody concentrations to the new standard reference serum CDC1992[END_REF][START_REF] Joseph | Assignment of Neisseria meningitidis serogroups A, C, W135, and Y anticapsular total immunoglobulin G (IgG), IgG1, and IgG2 concentrations to reference sera[END_REF] Avidity of MenC-specific IgG antibodies was assessed by using a modification of the MIA for determining MenC-specific IgG. [START_REF] De Voer | Simultaneous detection of Haemophilus influenzae type b polysaccharide-specific antibodies and Neisseria meningitidis serogroup A, C, Y, and W-135 polysaccharide-specific antibodies in a fluorescent-bead-based multiplex immunoassay[END_REF][START_REF] De Voer | Kinetics of antibody responses after primary immunization with meningococcal serogroup C conjugate vaccine or secondary immunization with either conjugate or polysaccharide vaccine in adults[END_REF] Serum samples with an IgG concentration of ≥ 0.25 µg/mL were diluted to an antibody concentration of 25 ng/mL in a buffer composed of 25% antibody depleted human serum (Valley Biomedical, USA). Ammonium thiocyanate (NH 4 SCN; Sigma-Aldrich, St. Louis, MO) was added to a final concentration of 0.5 M in order to dissociate low-avidity antigen-antibody binding. [START_REF] Anttila | Avidity of IgG for Streptococcus pneumoniae type 6B and 23F polysaccharides in infants primed with pneumococcal conjugates and boosted with polysaccharide or conjugate vaccines[END_REF] After incubation of polysaccharideconjugated-beads with serum, 0.5M NH 4 SCN was added for 10 minutes exactly. Beads were washed, R-phycoerythrin(RPE)-conjugated goat anti-human IgG (gamma chain specific) (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) was added and incubated for a further 30 minutes. After a final wash, the plate was read on a Luminex-100 system (Bioplex; Bio-Rad Laboratories, Hercules, CA). Data are expressed as the avidity index (AI) which is the percentage of antibodies that remained bound to the MenC polysaccharideconjugated beads after treatment with NH 4 SCN and was calculated as follows: AI = (amount of IgG bound in the presence of 0.5 M NH 4 SCN)/ (amount of IgG bound in PBS) x 100. Sera were categorized into high, intermediate and low avidity, based on the AI: high, 100-67%; medium, 66-34%; and low, 0-33%.

Statistical analysis

Comparisons of GMC before and after vaccination (paired samples) and comparisons of antibody avidity before and after vaccination were performed using the paired samples Ttest. Independent sample T-test and Chi-square were used when appropriate. A p-value of ≤ 0.05 was considered statistically significant. In case of subset analysis, in small groups the Wilcoxon signed rank test was used.

For statistical analysis, the Statistical Package for the Social Sciences (SPSS) for Windows ® software, version 15.0 (SPSS Inc., Chicago, IL) was used.

Results

We included 184 asplenic patients in this study. Previously, we have described the characteristics of the majority of this group. [START_REF] Meerveld-Eggink | Vaccination coverage and awareness of infectious risks in patients with an absent or dysfunctional spleen in the Netherlands[END_REF] In 117 patients a MenC conjugated vaccine was indicated. Samples after vaccination, of all but one patient, were available and these 116 sera were analysed in this study. Baseline characteristics of the patients are given in Table 1.

All but one patient underwent a splenectomy, the prevalent reason being a trauma (30%).

One patient was considered to be functional asplenic, because of irradiation of the spleen for Hodgkin's disease.

Seventeen (15%) patients had previously received a meningococcal vaccination, either

MenA,-C,-W135 and-Y polysaccharide vaccine (n=6), MenA,-C polysaccharide vaccine (n=4) or an otherwise not-specified meningococcal vaccine (n=7). The median time since the last vaccination was 4 years (range 2-6 years).

Meningococcal antibody concentrations

In asplenic patients, MenC geometric mean antibody concentrations (GMC) rose from 0.16 μg/mL prior to vaccination to 3.69 μg/mL three weeks post-vaccination (Figure 1). For MenC, trauma patients had significantly higher post-vaccination antibody concentrations than the other asplenic patients (p<0.05). We observed no statistically significant influence of age at splenectomy, age at vaccination or time from splenectomy to vaccination on the antibody concentrations of MenC-specific IgG. Previous meningococcal vaccination had a statistically significant influence on the MenC GMC's preand post-vaccination (p<0.05). GMC prevaccination in previously vaccinated patients was 1.6 μg/mL (95% CI 0.5-5.7) versus 0.1 μg/mL (95% CI 0.1-0.2) in patients not vaccinated before. For GMC post-vaccination, these data were 10.7 μg/mL (95% CI 4.8-24.0) and 3.1 μg/mL (95% CI 2.1-4.8), respectively. GMC's post-vaccination were 10% higher in patients receiving the MenC vaccine alone compared to patients receiving concomitant pneumococcal and/or Hib vaccines. This difference was not statistically significant.

Post-vaccination, 67% of the patients reached the threshold of 2.0 μg/mL for MenC (Table 2).

Patients who were splenectomised because of trauma reached this threshold more often than other patients (p<0.05). Time since splenectomy did not influence seroconversion of MenC.

In 38 patients, we were able to determine meningococcal antibody concentrations up to approximately eight months after the first conjugate MenC vaccine. Mean time since vaccination was 8.3 months (range 6,5 -10,5). GMC's for serogroup C are given in Figure 2. In this group of 38 patients, for MenC GMC pre-vaccination was 0.21 µg/mL and rose to 3.71 µg/mL post-vaccination (p<0.05). After eight months, a decrease in GMC to 3.10 µg/mL was observed. This decrease was not significant compared to the GMC 3 weeks postvaccination.

Thirty eight patients (33%) did not reach adequate antibody concentrations after one dose of conjugate vaccine. Characteristics of this group of hypo-responders are given in Table 3. These hypo-responders had an adequate response to Hib vaccination (98% reached the threshold) and pneumococcal conjugate vaccination (93,5% reached protective antibody concentrations of 1.0 µg/mL for 5 out of 7 vaccine-serotypes after one vaccination). Of the 38 patients, 58% (n=22) did reach seroconversion for serogroup C after the first vaccinaton.

Because of their subnormal antibody response, a revaccination with the MenC conjugate vaccine was performed. Sera after revaccination were available of 22 patients. In this patient group, GMC pre-vaccination was 0.05 µg/mL, rising after the first vaccination to 0.29 µg/mL.

After the second vaccination, the GMC of MenC PS-specific IgG rose significantly to 1.12 µg/mL (Figure 3). Thirteen (59%) patients reached the threshold of ≥ 2.0 µg/mL.

Antibody avidity

The avidity index (AI) of the MenC antibodies was measured using a grading system to discriminate between sera with low AI (0-33%), intermediate AI (34-66%) and high AI (67-100%). Only sera that ≥ 0.25 µg/mL of MenC-specific IgG were selected for AI determination.

In the overall cohort, the geometric mean AI (GMAI) was 27% (95% CI 19-35%) prevaccination (n=48), 25% (95% CI 20-30%) 3 weeks post-vaccination (n=104) and 32% (95% CI 23-41%) 35 weeks post-vaccination (n=34), respectively. In patients who were revaccinated, the mean AI rose from 14% after the first dose of vaccine to 26% after the second vaccination (n=13; p<0.01). No associations were found between the AI pre-and post-vaccination and the underlying disease resulting in asplenia or previous vaccination with meningococcal vaccines.

IgG subclasses

In Figure 4a, the concentrations of MenC IgG1 and IgG2 are given before, three weeks and 35 weeks after vaccination in patients with ≥ 0.25 µg/mL of MenC PS-specific IgG prevaccination. In Figure 4b the development of the IgG1/IgG2 ratio in this patient group is given in relation to the changes in avidity indices. Great inter-individual differences between antibody responses were seen, with IgG1/IgG2 ratio's increasing in 7 patients and decreasing in 26 patients. In only 7 patients the IgG1/IgG2 ratio after one conjugated vaccination was >1 and in only 2 patients the ratio changed from <1 to >1 post-vaccination.

The mean IgG1 concentration after vaccination was 1.17 µg/mL versus 9.52 µg/mL IgG2.

The ratio of IgG1/IgG2 did not change by vaccination (0.23 pre-vaccination, 0.25 3 weeks post-vaccination, and 0.20 35 weeks post-vaccination).

After revaccination, the mean IgG1 concentration rose from 0.93 to 3.25 µg/mL (Figure 4c).

The concentration of IgG2 rose from 0.76 to 1.72. The ratio of IgG1/IgG2 was 1.43 postvaccination, which is significantly higher (p< 0.01) than after primary vaccination.

Discussion

The immune response to the MenC conjugate vaccine in a group of 116 asplenic patients was found to be of poor quality and quantity. After a single dose of MenC conjugate vaccine, only 67% of the asplenic patients reached the chosen threshold antibody concentration of ≥ 2.0 μg/mL, with an increase in GMC from 0.16 μg/mL to 3.69 μg/mL post-vaccination. In healthy children and adults, including our own series, 5-10 times higher GMC's were reached after one dose of this MenC conjugate vaccine. [START_REF] Zonneveld-Huijssoon | Safety and efficacy of meningococcal c vaccination in juvenile idiopathic arthritis[END_REF][START_REF] Burrage | Effect of vaccination with carrier protein on response to meningococcal C conjugate vaccines and value of different immunoassays as predictors of protection[END_REF][START_REF] Richmond | Safety and immunogenicity of a new Neisseria meningitidis serogroup C-tetanus toxoid conjugate vaccine in healthy adults[END_REF][START_REF] Richmond | Ability of 3 different meningococcal C conjugate vaccines to induce immunologic memory after a single dose in UK toddlers[END_REF] Previously, Balmer et al., using a different MenC conjugated vaccine, found three times higher GMC's in asplenic patients after vaccination compared to our asplenic patients. [START_REF] Balmer | Immune response to meningococcal serogroup C conjugate vaccine in asplenic individuals[END_REF] In that study, no difference was found between IgG GMC's in asplenic patients and an age-matched control group. However, functionality of the antibodies (based on bactericidal antibody in serum (SBA)) was significantly lower in the asplenic patient group. In our asplenic patient group, the functionality of the antibodies in terms of antibody avidity was also low. As indicated above we used a different MenC conjugated vaccine (MenC coupled to tetanus toxoid (TT)) than Balmer et al. (CRM 197 conjugated). The vaccine we used has turned out to be more immunogenic in healthy subjects than other MenC conjugated vaccines. [START_REF] Richmond | Ability of 3 different meningococcal C conjugate vaccines to induce immunologic memory after a single dose in UK toddlers[END_REF] Yet, in our patients lower antibody concentrations were observed than in the study of Balmer et al. [START_REF] Balmer | Immune response to meningococcal serogroup C conjugate vaccine in asplenic individuals[END_REF] Balmer et al. recommend a second dose of vaccine offered to the patients if their postvaccination concentration is below the proposed protective threshold, or routinely offering two doses of MenC conjugate vaccine. In our study, after revaccination still 41% of the revaccinated patients did not achieve protective serum levels. This percentage is in accordance with the percentage of hypo-responders found by Balmer et al. [START_REF] Balmer | Immune response to meningococcal serogroup C conjugate vaccine in asplenic individuals[END_REF] We therefore can conclude that hypo-responsiveness to MenC remained in a considerable fraction of patients and cannot be overcome by a more potent conjugate MenC vaccine.

In healthy adults, IgG GMC's fell by 50% from 33.3 to 16.7 ug/mL at six months after a single dose of MenC-TT vaccine. [START_REF] Richmond | Safety and immunogenicity of a new Neisseria meningitidis serogroup C-tetanus toxoid conjugate vaccine in healthy adults[END_REF] In the asplenic patients of whom sera were available eight months after vaccination, GMC dropped only slightly from 3.69 to 3.10 µg/mL. The asplenic state of the patients may be a factor which determines the only marginal decrease in GMC in the 8 months post-vaccination. In accordance with our findings, in the study of Smets et al.,[START_REF] De Greeff | Protection from routine vaccination at the age of 14 months with meningococcal serogroup C conjugate vaccine in the Netherlands[END_REF] months after pneumococcal conjugate vaccination in asplenic children, GMC's had not decreased significantly. [START_REF] Smets | Randomised revaccination with pneumococcal polysaccharide or conjugate vaccine in asplenic children previously vaccinated with polysaccharide vaccine[END_REF] High antibody concentrations months after vaccination can be explained by ongoing production of antibodies and/or lower rate of removal from the circulation. In asplenic individuals, alternative homing of peripheral B-cells takes place, with B-memory-cells homing in peripheral organs and bone marrow in stead of in the spleen.

These specific niches may be favourable for long lived plasma cells. Another argument for longer persistence of antibodies in asplenic patients is the absence of the reticulo-endothelial system (RES) function of the spleen. Immune complexes can longer remain in circulation and cause prolonged B-lymphocyte activation.

Antibody persistence following pneumococcal polysaccharide or conjugate vaccination of healthy adults (50-80 years) with an intact spleen has been studied by Goldblatt et al. [START_REF] Goldblatt | The immunogenicity of 7-valent pneumococcal conjugate vaccine versus 23-valent polysaccharide vaccine in adults aged 50-80 years[END_REF] One year after one dose of pneumococcal conjugate vaccine, GMC's for 4 out of the 7 pneumococcal serotypes were at least 80% of the GMC's at the peak of the response, i.e. 1 month after vaccination. It should therefore be concluded that polysaccharide (conjugated) vaccines have the ability to induce antibody levels which persist for periods up to 1 year after vaccination, in the presence or absence of a functional spleen.

Avidity and IgG subclasses

It is thought that long term protection after immunisation with conjugate vaccines depends on herd immunity, persistence of anti-capsular antibodies in serum and immunological memory. [START_REF] Blanchard Rohner | The magnitude of the antibody and memory B cell responses during priming with a proteinpolysaccharide conjugate vaccine in human infants is associated with the persistence of antibody and the intensity of booster response[END_REF] As a marker of immunological memory, avidity of the MenC-specific antibodies was determined.

High antibody avidity indicates high functional activity of the meningococcal antibodies. [START_REF] Wuorimaa | Avidity and subclasses of IgG after immunization of infants with an 11-valent pneumococcal conjugate vaccine with or without aluminum adjuvant[END_REF] In the asplenic patient group, the GMAI rose from 19% 3 weeks post-vaccination to 29% eight months after vaccination. The rise in antibody avidity is consistent with earlier studies on affinity maturation of antibodies after conjugated vaccines. [START_REF] Wuorimaa | Avidity and subclasses of IgG after immunization of infants with an 11-valent pneumococcal conjugate vaccine with or without aluminum adjuvant[END_REF][START_REF] Goldblatt | Antibody avidity as a surrogate marker of successful priming by Haemophilus influenzae type b conjugate vaccines following infant immunization[END_REF][START_REF] Richmond | Safety and immunogenicity of a new Neisseria meningitidis serogroup C-tetanus toxoid conjugate vaccine in healthy adults[END_REF][START_REF] Southern | Effects of prior polysaccharide vaccination on magnitude, duration, and quality of immune responses to and safety profile of a meningococcal serogroup C tetanus toxoid conjugate vaccination in adults[END_REF] However, in our study, most patients do not develop antibodies with intermediate or high avidity; most antibodies are of low avidity.

In adults, natural exposure to meningococci and exposure to cross-reactive agents can lead to the development of memory B-cells (priming). These memory B-cells will be stimulated after polysaccharide and conjugate vaccination, leading to higher avidity indices in adults compared to children. [START_REF] Balmer | Serologic correlates of protection for evaluating the response to meningococcal vaccines[END_REF][START_REF] Goldblatt | Natural and vaccine-induced immunity and immunologic memory to Neisseria meningitidis serogroup C in young adults[END_REF] For this reason, measurement of AI's in adults may be considered to be of less importance. [START_REF] Balmer | Serologic correlates of protection for evaluating the response to meningococcal vaccines[END_REF][START_REF] Goldblatt | Natural and vaccine-induced immunity and immunologic memory to Neisseria meningitidis serogroup C in young adults[END_REF][START_REF] Hutchins | Elderly immune response to a TI-2 antigen: heavy and light chain use and bactericidal activity to Neisseria meningitidis serogroup C polysaccharide[END_REF] In our adult asplenic group however, most adults had low antibody avidity indices before vaccination. In this patient group therefore, an antibody AI measurement may be an adequate way of determining the development of immunological memory.

In general, IgG1 antibodies have a higher affinity than IgG2. [START_REF] Southern | Effects of prior polysaccharide vaccination on magnitude, duration, and quality of immune responses to and safety profile of a meningococcal serogroup C tetanus toxoid conjugate vaccination in adults[END_REF] The ability to induce a IgG1 T-cell dependent (TD) response is among others dependent on age at vaccination, type of vaccine and type of adjuvant. [START_REF] Hutchins | Elderly immune response to a TI-2 antigen: heavy and light chain use and bactericidal activity to Neisseria meningitidis serogroup C polysaccharide[END_REF][START_REF] Findlow | Immunoglobulin G subclass response to a meningococcal quadrivalent polysaccharidediphtheria toxoid conjugate vaccine[END_REF][START_REF] Makela | Isotype concentrations of human antibodies to Haemophilus influenzae type b polysaccharide (Hib) in young adults immunized with the polysaccharide as such or conjugated to a protein (diphtheria toxoid)[END_REF] In the asplenic patients studied, after one meningococcal conjugate vaccination, both IgG1 and IgG2 increase, with higher concentrations of IgG2 (mean concentration 9.52 µg/mL 3 weeks after vaccination) than IgG1 (mean concentration 1.17 µg/mL). There is a large variability in the IgG1/IgG2 ratio in individual patients. This is consistent with the findings of Findlow et al., who detected significant increases in IgG1 and IgG2 with large differences in IgG1/IgG2 ratio's after vaccination with one dose of quadrivalent meningococcal conjugated vaccine in healthy adults. [START_REF] Findlow | Immunoglobulin G subclass response to a meningococcal quadrivalent polysaccharidediphtheria toxoid conjugate vaccine[END_REF] After revaccination, the amount of IgG1 in the asplenic population rises more than IgG2, suggesting that after revaccination, the T-cell dependent response is more adequate.

Influence of immunisation schedule

In contrast with the study of Balmer et al., in the current study a number of patients received Hib and pneumococcal vaccines concomitant with the MenC vaccine, dependent on the vaccination status before start of the study. [START_REF] Balmer | Immune response to meningococcal serogroup C conjugate vaccine in asplenic individuals[END_REF] The response to Hib vaccine was much better than to MenC vaccine; 97% of the patients reached a protective antibody concentration after vaccination (Meerveld-Eggink, manuscript submitted).Theoretically, the diminished response to MenC could be due to the simultaneous administration of different vaccines, thereby overloading the immune system. Although the MenC-specific IgG GMC post-vaccination in patients receiving concomitant vaccines was 10% lower than in patients solely immunised with the MenC vaccine, this difference was not statistically significant.

In both the current study in asplenic patients and the study of Balmer et al., 15-30% of patients had received prior MenC polysaccharide vaccination. [START_REF] Balmer | Immune response to meningococcal serogroup C conjugate vaccine in asplenic individuals[END_REF] These previously vaccinated patients had higher MenC GMC's pre-vaccination than patients not vaccinated with meningococcal vaccines before. In contrast to Balmer et al., we found that previous MenC vaccination also lead to higher GMC's post-vaccination.

Conclusions

We conclude that a single dose of MenC conjugated vaccine does not result in an adequate antibody response in 33% of asplenic patients. A second dose of MenC vaccine leads to an additional increase in antibody concentration and avidity, suggesting that asplenic patients benefit from a second dose of vaccine. This conclusion should be limited to older, asplenic patients, who did not receive MenC conjugated vaccines as part of the Dutch National Vaccination Programme. The relevance of a second dose of vaccine in countries where all children are vaccinated against MenC and circulation of MenC therefore is low, should be considered.

Figure 1 .Figure 2 .Figure 3 . 27 Figure 4a .

 123274a Figure 1. Geometric mean concentrations of MenC IgG before and after MenC conjugate

Table 1 .

 1 Baseline characteristics of asplenic patients vaccinated with MenC conjugate

	vaccine
	Patients (n)

Table 2 .

 2 Neisseria meningitidis serogroup C: response to vaccination

	≥ 2.0 µg IgG/mL pre-vaccination: n (%)	14 (12)
	≥ 2.0 µg IgG/mL post-vaccination: n (%)	78 (67)
	Seroconversion: n (%)	93 (80)
	Data are given in numbers of patients with percentages in parentheses
	Seroconversion: a ≥ fourfold increase in antibody concentration after vaccination

Table 3 .

 3 Characteristics of responders and hypo-responders to one dose of MenC MenC-specific IgG < 2.0 µg IgG/mL post-vaccination For gender, reason for Sx and previously vaccinated, data are given in numbers of patients with percentages recorded in parentheses. For age at Sx, age at vaccination and time from Sx to vaccination, data are given in mean number of years, with the range in parentheses

	conjugated vaccine
		responders	hypo-responders
	Patients (n)	78	38
	Male gender	39 (50)	20 (53)
	Reason for Sx
	Trauma	31 (40)	4 (11)
	Haematological malignancy	12 (15)	6 (16)
	Other reasons	35 (45)	28 (74)
	Age at Sx in years	27 (5-76)	30 (3-78)
	Age at vaccination in years	52.0 (23-86)	53.5 (29-83)
	Time from Sx to vaccination in years	25 (0-55)	23 (2-50)
	Previously vaccinated	14 (18)	3 (8)
	Sx= splenectomy
	Hypo-responder =

Acknowledgements

The authors thank Mrs. A van Heugten-Roeling (Department of Medical Microbiology and Immunology of the St. Antonius Hospital Nieuwegein) for her contribution to this study.

Conflict of interest

The authors declare that they have no conflict of interest.


Vanderstraeten Eva

Bogaert Lies

Ignacio G Bravo

Martens Ann
email: ann.martens@ugent.be

Prof. Dr Ann Martens

EcPV2 DNA in equine squamous cell carcinomas and normal genital and ocular mucosa

Keywords: horse, squamous cell carcinoma, EcPV2, papillomavirus, mucosa

Squamous cell carcinoma (SCC) represents the most common malignant tumour of the eye and external genitals in horses. Comparable to humans, papillomaviruses (PV) have been proposed as etiological agents of cancer in horses and recently, Equine papillomavirus type 2 (EcPV2) has been identified in genital SCCs. Hitherto it had never been demonstrated in ocular SCCs .

The first goal of this study was to determine the prevalence of EcPV2 DNA in tissue samples from equine genital and ocular SCCs, genital papillomas and penile intraepithelial neoplasia (PIN) lesions, using EcPV2-specific PCR. The second goal was to investigate the possibility of latent EcPV2 infection in the genital and ocular mucosa of healthy horses on swabs obtained from the eye, penis, vulvovaginal region and cervix. EcPV2 DNA was detected in all genital SCCs (17/17), genital papillomas (8/8), PIN lesions (11/11) and ocular SCCs (9/9).

In healthy horses, EcPV2 DNA was detected in 43% (17/40) of penile swabs, 53% (9/17) of vulvovaginal swabs, 47% (8/17) of cervical swabs and 57% (32/56) of ocular swabs. This study confirms the presence of EcPV2 DNA in equine genital SCCs. Moreover, we demonstrate for the first time its involvement in other genital lesions and in ocular SCCs and latent EcPV2 infections in normal genital (including cervical) and ocular equine mucosa. The close relatives of EcPV2 are associated to cutaneous lesions, and this virus is not related to high-risk human papillomaviruses causing cervical cancer. Thus, similar viral tropism does not imply close evolutionary relationship.

A c c e p t e d M a n u s c r i p t

Introduction

Squamous cell carcinoma (SCC) represents 20% of all equine tumours, making it the second most common neoplasm in horses. SCC is a malignant epithelial tumour and is most often associated with the eyes (Fig. 1) and the external male genitals (Fig. 2), but it can develop in any epithelial tissue of the body [START_REF] Macfadden | Clinical Manifestations of Squamous-Cell Carcinoma in Horses[END_REF]. In many horses, penile SCCs are accompanied by large, confluent, pink to yellowish plaques (Fig. 2), often referred to as "precancerous lesions" [START_REF] Brinsko | Neoplasia of the male reproductive tract[END_REF]. Similar to the frequently described penile intraepithelial neoplasia (PIN) in men, the same term can be used in horses. Furthermore, according to our own clinical observations, many genital SCCs are accompanied by genital papillomas, which can also be considered as precancerous lesions.

Papillomaviruses (PVs) are small, epitheliotropic, non-enveloped double-stranded circular DNA viruses. In humans, more than 100 different PV types have been fully sequenced, some of them associated with both benign and malignant clinical conditions, ranging from spontaneously regressing cutaneous and genital warts, to invasive anogenital and skin cancer [START_REF] De Villiers | Classification of papillomaviruses[END_REF]. Comparable to humans, PVs have been proposed as etiological agents of cancer in horses. Bovine papillomavirus type 1 (BPV1) and less commonly type 2 (BPV2) are associated with equine sarcoids, a common fibroblastic skin tumour in horses.

The presence of PVs in equine SCCs was first investigated in 1984, but could not be demonstrated at that time [START_REF] Junge | Papillomas and squamous cell carcinomas of horses[END_REF]. In 2004 the complete nucleotide sequence of the first equine PV, Equus caballus papillomavirus 1 (EcPV1), isolated from a cutaneous papilloma, was determined [START_REF] Ghim | Equine papillomavirus type 1: complete nucleotide sequence and characterization of recombinant virus-like particles composed of the EcPV-1 L1 major capsid protein[END_REF]. In 2007 EcPV1 DNA was demonstrated in a high proportion of cutaneous papillomas but could not be isolated from genital papillomas [START_REF] Postey | Evaluation of equine papillomas, aural plaques, and sarcoids for the presence of Equine papillomavirus DNA and Papillomavirus antigen[END_REF]. However, the existence of a different PV already suggested in 1986 by Obanion et al., was strongly supported since both PCR and immunohistochemical analysis respectively indicated the presence of PV DNA and PV antigens in genital lesions (Postey et , 2007). Recently a novel equine papillomavirus (EcPV2) has been identified in genital SCCs and papillomas, but it could not be demonstrated in ocular SCCs yet [START_REF] Scase | Is papillomavirus infection associated with genital and ocular neoplasia in horses?[END_REF][START_REF] Scase | Papillomaviruses and squamous cell carcinoma[END_REF]. The first goal of this study was therefore to determine the prevalence of EcPV2 DNA in tissue samples from equine genital and ocular SCCs, genital papillomas and PIN lesions.

Humans are exposed since early life to PV infection, and most of these PV infections in humans are asymptomatic [START_REF] Antonsson | The ubiquity and impressive genomic diversity of human skin papillomaviruses suggest a commensalic nature of these viruses[END_REF]. The same appears to be true in several animal species, including virtually all mammals [START_REF] Antonsson | Healthy skin of many animal species harbors papillomaviruses which are closely related to their human counterparts[END_REF]. Since latent PV infections have also been shown in cattle and horses [START_REF] Campo | Latent papillomavirus infection in cattle[END_REF][START_REF] Antonsson | Healthy skin of many animal species harbors papillomaviruses which are closely related to their human counterparts[END_REF][START_REF] Bogaert | High prevalence of bovine papillomaviral DNA in the normal skin of equine sarcoid-affected and healthy horses[END_REF] the second goal of this study was to investigate the possibility of latent EcPV2 infections in the genital and ocular mucosa of healthy horses.

Finally, although cervical cancer has never been described in horses, we also investigated the presence of EcPV2 in the cervix of clinically normal horses, regarding the importance of high-risk HPVs in cervical cancer in women.

Methods

Study population and sample collection

Affected horses

This group consisted of 26 horses, including 19 geldings, one stallion and six mares. The median age was 14 years (range 3-24 years). There were six ponies, seven Haflingers, ten nine ocular SCCs (O1-O9). Additional samples included one nasal SCC (N1) from a 26 th patient, a mouth lesion (M1) in one of the horses with a penile SCC (patient #2), as well as two superficial inguinal lymph nodes (L1-L2) with metastases from a patient with a penile SCC (patient #1). Table 1 summarizes the samples, sex, age and breed of the patients.

Samples were collected after surgical tumour excision or after debulking of the tumoural mass before cryosurgery, immunotherapy or chemotherapy, except for the samples of the inguinal lymph nodes, which were obtained after euthanasia of the horse. All samples were collected by excising a representative part of the mass using a sterile scalpel and forceps. When several samples were obtained from the same horse, each sample was processed with a different set of instruments. Samples were stored dry at -18 °C.

Healthy horses

The stallions (N=22) and geldings (N=18) were patients referred to the Department of Surgery and Anaesthesiology of Domestic Animals of Ghent University (Belgium) for nononcological surgery between August 2007 and April 2008. .From each of them an ocular and a penile swab were obtained under general anaesthesia, except from one stallion in which only a penile swab was available. A single cotton-tipped swab was used to sample the third eyelid, cornea and conjunctiva of both eyes. A second swab was used to sample the genital region: the penis was protruded and the distal part of the urethra, the sinus urethralis and fossa glandis, the glans penis, the penile shaft and the preputium were sampled with the same swab.

All precautions were taken to avoid cross contamination and samples were stored dry at -18 °C. Age, breed or colour were not recorded in six stallions. The remaining stallions had a median age of three years (range 1-12 years); there were two ponies, ten Warmbloods, two From each of them an ocular, a vulvovaginal and a cervical swab were taken. Ocular swabs were taken as described for stallions and geldings. Before obtaining the genital swabs, the vulva was scrubbed with povidonum iodinatum 7.5% soap. A single cotton-tipped swab was used to sample the vulva, the vestibulum vagina, the glans clitoris and the fossa clitoridis.

To sample the cervix, a tube speculum was inserted and the sample was taken using a uterus biopsy forceps with a small sterile tampon between the tips. The portio vaginalis, the ostium uteri externum and the canalis cervicalis were sampled with this single tampon. Samples were stored dry at -18 °C. The age and breed of 5 mares were not recorded. The remaining mares had a median age of 6.5 years (range 4-19 years); there were four Warmbloods and eleven Standardbreds, with a colour distribution of 15 bay and two chestnut horses.

DNA isolation and PCR

DNA was isolated using the Puregene Genomic DNA isolation kit (Gentra Systems) as described previously [START_REF] Bogaert | High prevalence of bovine papillomaviral DNA in the normal skin of equine sarcoid-affected and healthy horses[END_REF]. For detection of EcPV2 DNA, PCR was performed using an EcPV2 specific primer set: EcPV2 forward primer 5'-

GCGGACTGCGCGTCACAAGAGGGGC

-3' and reverse primer 5'-ACGCAAGCACCACCCACTGCTTGGCA -3'. This primer set amplified a 679 base pair PCR products were separated by electrophoresis on a 2% agarose gel and visualised by ethidium bromide staining.

Sequencing of PCR products

In order to confirm the identity of the PCR products, amplicons of one penile, vulval, ocular and nasal SCC and amplicons of one ocular, penile, vulvovaginal and cervical swab from healthy horses were purified using the Geneclean II kit (Bio 101 Systems), ligated into pCR® 2.1 vector and transformed in Escherichia coli bacteria (One shot® INVαF' Chemically Competent E. coli, Invitrogen) using the TA Cloning kit (Invitrogen). Bacteria were incubated for blue-white colony screening on agar plates containing ampicilline and X-gal. Three white colonies of each sample were amplified and the plasmid DNA was purified using the Qiagen Plasmid Maxi Kit. Sequencing was performed at least twice per sample with universal T7 primers, using a Big Dye® Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems).

Cycle sequencing reaction products were purified using ethanol precipitation and sequenced with an ABI prism 310 genetic analyzer (Applied Biosystems).

Amplicon sequences were aligned using CAP3 (freely distributed by Xiaoqiu Huang at http://seq.cs.iastate.edu/), using the published EcPV02 sequence as a reference. All amplified chosen, and the E1, E2 and L1 genes concatenated, as described (Gottschling et al., 2007a).

Briefly, the sequences were aligned at the amino acid level with MUSCLE (http://www.drive5.com/muscle/), visualised for manual correction with Se-Al (freely distributed by Andrew Rambaut at http://tree.bio.ed.ac.uk/software/seal/) and back-translated to the nucleotide level using PAL2NAL (http://www.bork.embl.de/pal2nal). Maximum Likelihood (ML) phylogenetic analysis was performed with RAxML [START_REF] Stamatakis | RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models[END_REF] using the GTR+4 model of evolution and the CAT approximation of rate heterogeneity, introducing three partitions that corresponded to each of the codon positions. After 1,000 bootstraps the final tree topology was optimised without resorting to the CAT approximation. using TREEPRUNER (freely distributed by Olaf Bininda-Emonds at http://www.unioldenburg.de/molekularesystematik/33997.html).

Statistical analysis

The data obtained from the healthy horses were statistically analysed using SPSS 15.0. The positivity from the swabs depending of the sample location was analysed by Fisher's exact tests (2x2 tables). The ²-test for an RxC contingency table was used to examine differences in presence of EcPV2 between sexes, breeds, coat colours and ages. Differences were considered statistically significant if P-values were below 0.05.

Results

EcPV2 DNA analysis

Affected horses

Amplicons of the expected size were detected in all penile, preputial, vulval and anal SCCs (17/17), in all PIN lesions (11/11), in all genital papillomas (8/8) and in all ocular SCCs (9/9) (Fig. 3) The two metastatic lymph nodes, the mouth lesion and the nasal SCC also tested positive. DNA of equine papillomas, equine sarcoids and H 2 O, were successfully used as negative controls.

Healthy horses

In total, 56 ocular swabs, 40 penile swabs, 17 vulvovaginal swabs and 17 cervical swabs were obtained from the healthy horses. 3. There was no significant effect of sex, breed, coat colour or age on positivity.

Sequencing results

Primers designed using the EcPV2 sequence could amplify in all cases a DNA fragment of ca.

680 bp, which after sequencing could be identified without ambiguity as belonging to this PV type. The EcPV2 sequence deposited in the databases (GenBank EU503122) lacks the Nterminus of the E1 open reading frame, which is otherwise highly conserved among PVs. In all of the amplicons sequenced in this study there was a consistent mismatch with the deposited EcPV2 sequence, which restored the E1 frame. This corrected sequence has been deposited under GenBank accession number GU809241.

Phylogenetic reconstruction

The equine EcPV2 belonged unequivocally to the delta-epsilon-zeta PV superclade (Gottschling et al., 2007a;[START_REF] Bravo | Phylogeny and evolution of papillomaviruses based on the E1 and E2 proteins[END_REF] 4b). However, PV that infect horse and also PV that infect cow are not monophyletic respectively. Finally, there is also a global disagreement between the topology of PV infecting Cervidae and the topology of the hosts they infect (Fig. 4c).

Discussion

In the present study EcPV2 DNA was detected in all (para-)genital SCCs, PIN lesions and genital papillomas. This suggests an etiological role of EcPV2 in the development of these genital lesions, comparable to the role of high-risk HPVs in anogenital cancers such as cervical cancer. However, we assume that an EcPV2 infection is not sufficient to induce tumoral transformation, since we observed 75% of healthy EcPV2 carriers. In humans, highrisk-HPV infection is a necessary but not sufficient cause of cervical cancer. Other cofactors, such as long term use of oral contraceptives, high parity, smoking, immunosuppression, or other sexually transmitted infections, are necessary for progression from high-risk HPV infection to cancer [START_REF] Munoz | HPV in the etiology of human cancer[END_REF]. Known cofactors in the development of SCCs in horses are repeated trauma, retention of smegma [START_REF] Burney | Identifying and treating squamous cell carcinoma of horses[END_REF], solar radiation, breed, hair colour and age [START_REF] Valentine | Survey of equine cutaneous neoplasia in the Pacific Northwest[END_REF]. The EcPV2 mRNA and protein expression pattern as well as functional pathways should be studied in order to establish the precise role of EcPV2 in tumour development.

Our study is the first to demonstrate PV DNA in equine lymph nodes, as EcPV2 DNA was detected in two metastatic lymph nodes. In humans, the relationship between HPV DNA in lymph nodes and metastases has not yet been elucidated. The presence of HPV DNA in histologically tumour-free lymph nodes could be a sign of (early) tumoral involvement and therefore an important prognostic factor [START_REF] Lukaszuk | Predictive value of HPV DNA in lymph nodes in surgically treated cervical carcinoma patients-A prospective study[END_REF]. Other researchers hypothesise that HPV DNA detected in histologically tumour-free lymph nodes originates A c c e p t e d M a n u s c r i p t either from immune cells that have taken up HPV particles or from free migrating HPV particles, and therefore do not attribute a prognostic value to HPV in lymph nodes [START_REF] Fule | Prognostic significance of high-risk HPV status in advanced cervical cancers and pelvic lymph nodes[END_REF]. The significance of EcPV2 DNA in the lymph nodes in horses with SCCs cannot be explained on the basis of the available data, since only two lymph nodes obtained from one single patient have been analysed in this study.

EcPV2 DNA was also detected in a mouth lesion of a horse with a penile SCC. Histological examination of this mouth lesion showed focal epithelial hyperplasia. According to the owners of the horse, the horse was often licking and biting his penile SCC and after a few months the mouth lesion appeared. The detection of EcPV2 DNA in this lesion suggests a broad tropism of EcPV2, able to productively infect histologically different mucosal tissues in different anatomical locations, as has been also reported for COPV, infecting dogs and causing both oral and ocular lesions [START_REF] Brandes | Detection of canine oral papillomavirus DNA in conjunctival epithelial hyperplastic lesions of three dogs[END_REF]. The possibility of transmission from infected to non-infected horses is unknown. In women, cervical cancer is strongly associated with genital high-risk HPV infections in their male sexual partners and vice versa [START_REF] Barrasso | High Prevalence of Papillomavirus-Associated Penile Intraepithelial Neoplasia in Sexual Partners of Women with Cervical Intraepithelial Neoplasia[END_REF]. In horses, sexual transmission could also be possible since EcPV2

DNA was detected in both penile and vaginal SCCs. However, also horses that never had been used as breeding animals were included in this study, which suggests transmission routes other than the sexual route. In this sense, the possibility of both intra-and inter-individual PV inoculation among different anatomical locations (e.g. penis, scrotum, anus, cervix or hand) has also been demonstrated in humans [START_REF] Hernandez | Transmission of human papillomavirus in heterosexual couples[END_REF]). An alternative hypothesis is that the virus could be spread by insects, which is a suggested transmission route of BPV in the pathogenesis of equine sarcoids [START_REF] Finlay | The detection of Bovine Papillomavirus type 1 DNA in flies[END_REF]. Finally, vertical transmission from mare to foal, before, during of directly after delivery, could also be an alternative transmission route, as has also been described in humans and suggested in cows [START_REF] Dos Santos | Bovine papillomavirus transmission and chromosomal aberrations: an experimental model[END_REF][START_REF] Rombaldi | Transplacental transmission of Human Papillomavirus[END_REF]. of the study of [START_REF] Scase | Papillomaviruses and squamous cell carcinoma[END_REF], where EcPV2 DNA was only demonstrated in genital but not in ocular lesions.

A nasal SCC analyzed in the present study also tested positive for EcPV2. Known predisposing factors in equine nasal and paranasal tumours are epithelial changes due to chronic inflammatory processes [START_REF] Head | Equine nasal and paranasal sinus tumours. Part 1: Review of the literature and tumour classification[END_REF]. In humans, risk factors in the development of head and neck cancer, including tumours of the nose and paranasal sinuses, are smoking and alcohol consumption and in addition there is a relationship between certain HPVs and these cancers [START_REF] Syrjanen | Human papillomavirus (HPV) in head and neck cancer[END_REF].

In healthy horses, EcPV2 DNA was detected in 57% (32/56) of ocular swabs, 43% (17/40) of penile swabs, 53% (9/17) of vulvovaginal swabs and 47% (8/17) of cervical swabs, which indicates the existence of latent EcPV2 infections. This elevated prevalence is comparable to that in humans where the majority of the population undergoes subclinical HPV infections at least once in their life, while only a small part shows progression to clinical lesions [START_REF] Koutsky | Epidemiology of Genital Human Papillomavirus Infection[END_REF]. We interpret therefore that, as in humans, spontaneous clearance will happen in most horses, while evolution to precarcinogenic lesions will only occur in a limited number of horses, of which only a small percentage will evolve to SCCs. A long-term follow-up study of infected but clinically normal horses is required to gain insight in the evolution of latency.

In order to determine whether the presence of EcPV2 DNA in one location is related to its presence in a second location, a statistical analysis was done to verify whether EcPV2 frequently infects several locations in the same horse. This could reflect either an inherent susceptibility of certain horses, or be instead an indication of self-inoculation, as has been suggested in humans [START_REF] Hernandez | Transmission of human papillomavirus in heterosexual couples[END_REF]. However, no significant correlation was found.

Since geldings, certain breeds (draft horses, Appaloosas, Paints and Pintos), pale coloured A c c e p t e d M a n u s c r i p t

horses and older horses are more likely to develop SCCs according to several authors [START_REF] Burney | Identifying and treating squamous cell carcinoma of horses[END_REF][START_REF] Valentine | Survey of equine cutaneous neoplasia in the Pacific Northwest[END_REF], the effect of sex, breed, coat colour or age on positivity was also statistically analyzed but no significant correlation was found. However, it should be taken into account that only small groups of horses were sampled.

In the present study, EcPV2 DNA has been detected in the cervix of eight mares. This is the first study demonstrating PV DNA in the equine cervix. Probably EcPV2 can reach the cervix by sexual transmission or by using EcPV2-infected material during artificial insemination or other gynaecological interventions. The fact that EcPV2 DNA is detectable in the cervix of mares, but that cervical cancer has never been described in this species can have several explanations. It is possible that the local immune milieu of the mare differs from the human one and facilitates spontaneous clearance. In humans most women are exposed to at least one HPV type during their sexual life, and spontaneous clearance of genital infection by high-risk

HPVs occurs within two years and only in less than 10% cervical (pre)cancer arises [START_REF] Schiffman | Human papillomavirus and cervical cancer[END_REF]. Additional factors could be the limited age reached by horses in comparison to humans and the differential exposure to carcinogenic cofactors. In women the evolution of high-risk HPV infection to cervical cancer takes many years to even decades.

The peak incidence of HPV infections occurs in their twenties, that of high grade cervical intraepithelial lesions in their thirties and cervical cancer occurs mostly in their forties [START_REF] Kitchener | Achievements and limitations of cervical cytology screening[END_REF]. Another possibility is that mares do not develop cervical cancer because of a different anatomohistological cervical structure in comparison with women. In women cervical cancer occurs typically in the transformation zone of the cervix, which is located at the transition from the multilayered squamous epithelium of the ectocervix to the glandular epithelium of the endocervix [START_REF] Schiffman | Human papillomavirus and cervical cancer[END_REF]. To date no investigation about the transformation zone in mares has been carried out. Finally, an evolutionary explanation for the non-existence of cervical cancer in mares despite cervical EcPV2 infection EcPV2. PVs in the delta-epsilon-zeta superclade, where EcPV2 belongs, are associated mainly to cutaneous papillomas and fibropapillomas. This is the case of EcPV1, which was isolated and characterized from cutaneous lesions [START_REF] Ghim | Equine papillomavirus type 1: complete nucleotide sequence and characterization of recombinant virus-like particles composed of the EcPV-1 L1 major capsid protein[END_REF][START_REF] Obanion | Cloning and Characterization of An Equine Cutaneous Papillomavirus[END_REF] but also that of PV infecting Bovidae and Cervidae, and also classified in this superclade (Gottschling et al., 2007b;[START_REF] Bravo | Phylogeny and evolution of papillomaviruses based on the E1 and E2 proteins[END_REF]. Thus, the most parsimonious explanation is that the tropism of the common ancestor to the whole clade was cutaneous, whereas the mucosal tropism of EcPV2 is derived, appeared and developed after viral speciation, and is not shared by the rest of the members of the superclade. On the other hand, high-risk HPVs belong to the alpha-omicron PV superclade, where the predominant tropism is mucosal [START_REF] Bravo | Phylogeny and evolution of papillomaviruses based on the E1 and E2 proteins[END_REF], and only a few PV, such as HPV3 or SsPV1, have subsequently developed a cutaneous tropism. The last common ancestor to both superclades could have existed before the radiation within mammals, around 95 millions of years ago [START_REF] Garcia-Vallve | Papillomaviruses: different genes have different histories[END_REF]. The ancestors of both superclades specialised in different tropisms, and only later, EcPV2 colonised separately the genital mucosal niche, in an event of convergent evolution.

Conclusion

The present study confirms the presence of EcPV2 DNA in equine genital SCCs and is the first to demonstrate its involvement in precancerous genital lesions, in ocular SCCs, in a nasal SCC, in lymph nodes and in a mouth lesion. Moreover, latent EcPV2 infection in normal genital (including cervical) and ocular equine mucosa could be demonstrated in healthy patients. A c c e p t e d M a n u s c r i p t

  Warmbloods, one Standardbred, one Appaloosa and one Arabian horse. Forty-five tissue samples derived from 25 horses with one or more genital or ocular SCC, PIN lesion or papillomas were included in the study. These horses were patients referred to the Department of Surgery and Anaesthesiology of Domestic Animals of Ghent University (Belgium) for treatment of the SCC, PIN lesion or papilloma. The samples enclosed twelve penile and two A c c e p t e d M a n u s c r i p t 5 preputial SCCs (P1-P14), two vulval SCCs (V1-V2), one anal SCC (A1), eleven PIN lesions (PIN1-PIN11), seven penile papillomas (PPA1-PPA7), one vaginal papilloma (VPA1) and

  Paint Horses, one Lusitano and one Thoroughbred, with a colour distribution of two black, eight bay, four chestnut, four grey and three skewbald horses. The gelding group had a median age of eleven years (range 2-19 years). There were 13 Warmbloods, two Standardbreds, one Quarter horse, one Andalusian horse and one Anglo-Arabian horse, with a colour distribution of two black, three bay, six chestnut, six grey and one skewbald horse. The mares (N=17) were patients referred to the Department of Obstetrics, Reproduction and Herd Health of Ghent University for artificial insemination between August 2007 and April 2008.

  fragment of the E1 gene (position 215-893) in the genomic sequence of EcPV2, (GenBank accession number EU503122). PCR was performed in a 10 µL reaction mixture, containing 200 µM of each dNTP, 0.5 µM of forward and reverse primer, 0.5 U Fast Start Taq DNA polymerase (Roche) and 1.5 mM MgCl with 2.5 µl of template DNA. Amplification was performed as follows: 95°C for 5 min, 40 cycles of 95°C for 1 min, 65°C for 1 min, 72°C for 1 min and finally 72°C for 10 min. Negative controls with DNA from two equine papillomas and two equine sarcoids (with confirmed EcPV-1 and BPV-1 infections respectively) as well as a non-template control with H 2 O, were included in each experiment.

  EcPV2, as determined by Basic Local Alignment Search Tool analyses. For phylogenetic analyses, a representative selection of 84 PV sequences was

  EcPV02 could be unambiguously ascribed to the delta+epsilon PV superclade, including viruses infecting Carnivora (Canis familiaris, CPV4 and CsPV3), Perissodactyla (Equus caballus, EcPV1) and Cetartiodactyla (Bos taurus BPV1, BPV2, BPV5 and BPV8; Ovis aries, OvPV1 and OvPV2; Rangifer tarandus, RPV; Odocoileus virginianus, DPV; Alces alces, EEPV; and Capreolus capreolus, CcPV). An additional phylogenetic search was then performed with these sequences and the Rousettus aegyptiacus PV, RaPV, as outgroup.Maximum likelihood was computed with the above-described settings. Bayesian phylogenetic analysis was performed with BEAST v1.4.8 (http://beast.bio.ed.ac.uk)[START_REF] Drummond | BEAST: Bayesian evolutionary analysis by sampling trees[END_REF] with the GTR+4 model of evolution, and for both strict clock and uncorrelated log normal relaxed clock, introducing three partitions that corresponded to each of the codon positions, and unlinking parameters across codon positions. Two independent chains of 50 million steps were calculated, writing every 1,000 steps, and analysed with a burn-in of ten million steps. Compatibility of both chains was assessed by calculating the corresponding Bayes factor, and both chains were combined into one. The reference A c c e p t e d M a n u s c r i p t 9 cladogram for the animal species included in the taxon sample was pruned out from a mammalian tree constructed after the supertree methodology (Bininda-Emonds et al., 2007),

  geldings, EcPV2 DNA was detected in 71% and 67% respectively (15/21 and 12/18) of ocular swabs, compared to 29% (5/17) of positive ocular swabs in mares. Forty-five percent (10/22) of penile swabs tested positive in stallions, compared to 40% (7/18) in geldings. Regarding the 17 vulvovaginal and cervical samples, 53% (9/17) and 47% (8/17) tested positive, respectively. Results are summarized in Table2and a typical gel image is provided in Fig.3. Positivity in one body location was not significantly correlated to positivity in another location. Results are summarized in Table

  (Fig. 4a), which encompasses PVs infecting different hosts within Laurasiatheria: dog (CsPV3 and CPV4), horse (EcPV1 and EcPV2), cow (BPV1, BPV2, BPV5 and BPV8), sheep (OvPV1 and OvPV2), European elk (EEPV), roe deer (CcPV), deer (DPV), and white-tailed deer (RPV). Within this superclade, PV infecting Canidae and infecting Cetartiodactyla were respectively monophyletic, i.e., they A c c e p t e d M a n u s c r i p t 11 share a recent common ancestor (Fig.

  detected in 100% of ocular SCCs and the PCR amplified sequence of this ocular PV DNA was identical to the genital PV DNA. This finding is in contrast to the results

  the virus-host interaction mechanisms between high-risk HPVs and those of EcPV2 may be essentially different, even though they infect anatomically equivalent host cells. High-risk HPVs involved in cervical cancer are phylogenetically very distant from

  Overview of the samples, sex, age and breed of affected horses. node; M: Mouth lesion; N: Nasal SCC; O: Ocular SCC; P: Penile SCC; PIN: Penile 487 Intraepithelial Neoplasia lesion; PPA: penile papilloma; V: Vulval SCC; 488 VPA: Vulval papilloma 489

Fig. 1 .Fig. 2 .

 12 Fig. 1. Limbal squamous cell carcinoma involving the cornea and sclera in a 6 years old

Fig. 4 .

 4 Fig. 4. Phylogenetic relationships of EcPV2 among the Papillomaviridae family.

  Overview of the samples, sex, age and breed of affected horses. node; M: Mouth lesion; N: Nasal SCC; O: Ocular SCC; P: Penile SCC; PIN: Penile Intraepithelial Neoplasia lesion; PPA: penile papilloma; V: Vulval SCC; VPA: Vulval papilloma

  Figure 2 Click here to download high resolution image

Table 2 .

 2 Summary of the EcPV2 DNA analysis of samples obtained from healthy horses.

	Sample group	Penile swabs	Vulvovaginal	Cervical swabs Ocular swabs
				swabs
		N	Pos (%)	N	Pos (%)	N	Pos (%)	N	Pos (%)
	Stallions	22	10 (45%)					21	15 (71%)
	Geldings	18	7 (40%)					18	12 (67%)
	Mares			17	9 (53%)	17	8 (47%)	17	5 (29%)
	N: number of samples examined
	Pos (%): number (percentage) of samples positive for EcPV2 DNA

Table 3 .

 3 Positivity in the different locations analyzed in healthy horses.

	Sample group	N	n(1)	n(2)	n(3)	n
	Stallions	22	9	8		17 (77%)
	Geldings	18	11	4		15 (83%)
	Mares	17	3	5	3	11 (65%)
	Total	57	23	17	3	43 (75%)
	N: number of horses examined
	n(1), n(2), n(3): number of horses positive in respectively one, two or three locations
	n: total number of positive horses

Table 1

 1

Table 3 .

 3 Positivity in the different locations analyzed in healthy horses.

	Sample group	N	n(1)	n(2)	n(3)	n
	Stallions	22	9	8		17 (77%)
	Geldings	18	11	4		15 (83%)
	Mares	17	3	5	3	11 (65%)
	Total	57	23	17	3	43 (75%)
	N: number of horses examined
	n(1), n(2), n(3): number of horses positive in respectively one, two or three locations

n: total number of positive horses

Acknowledgements

We thank Cindy De Baere (Ghent University, faculty of Veterinary Medicine, Belgium) for helping with the PCR reactions. We are grateful to Dr. Jan Van Doorsselaere (KATHO, campus Roeselare, Belgium) for performing the sequencing reactions.

This research was funded by the Special Research Fund, Ghent University, grant no. 01D26308. Lies Bogaert is a Fulbright Research Scholar at the University of Southern California. I.G. Bravo is funded by the Spanish MICINN (CGL2009-08350) and the Ramón y Cajal Program.

A c c e p t e d M a n u s c r i p t


Abeer El Wakil

Enzo Lalli

The Wnt/beta-catenin pathway in adrenocortical development and cancer

Keywords: Adrenal cortex, Wnt, -catenin, development, cancer Conflict of interest: None declared

Signaling by the Wnt family of secreted glycolipoproteins plays key roles in embryonic development of organisms ranging from nematodes to mammals and is also implicated in several types of human cancers. Canonical Wnt signaling functions by regulating the translocation of -catenin to the nucleus, where it controls key gene expression programs through interaction with Tcf/Lef and other families of transcription factors. Wnts can also act through non-canonical pathways that do not involve -catenin activation, but implicate small GTPases/JNK kinase and intracellular calcium.

Here we review recent studies that have revealed the expression of several components of Wnt/-catenin signaling in the adrenal cortex and discovered a key role for this pathway in the regulation of proliferation/differentiation of progenitor cells and in tumorigenesis of that endocrine organ.

Introduction

The adrenal gland contains a neuroendocrine medulla within a steroidogenic cortex. The medulla produces catecholamines in response to sympathetic inputs, while the cortex produces essential steroids in response to hormonal inputs.

Aldosterone secretion from the adrenal cortex regulates sodium retention and plasma volume [START_REF] Takeda | Vascular synthesis of aldosterone: role in hypertension[END_REF][START_REF] Whaley-Connell | Aldosterone: role in the cardiometabolic syndrome and resistant hypertension[END_REF], and has been classically considered to be regulated by the renin-angiotensin system. Glucocorticoid output, which is an essential component of the stress response [START_REF] Sapolsky | How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions[END_REF], is modulated by the hypothalamic-pituitary-adrenal (HPA) axis. Although adrenocortical function requires precise control of the differentiation of steroidogenic cell lineages, the developmental and postnatal mechanisms that regulate their production and maintenance have only recently started to be defined [START_REF] Kim | Adrenocortical cells with stem/progenitor cell properties: recent advances[END_REF].

Wnt signaling has been identified as one of the key signaling pathways in both normal development (regulating cell growth, motility and differentiation) and tumorigenesis. Deregulation of this pathway is in fact often present in human cancers [START_REF] Kikuchi | Tumor formation by genetic mutations in the components of the Wnt signaling pathway[END_REF]. Here, we will review what is known about the role of Wnt signaling in normal adrenal gland development and its alterations in adrenocortical tumors. Wnt-Fz-LRP6/5 complex together with the recruitment of the scaffolding protein Dishevelled (Dvl), which antagonizes the action of GSK-3 through an unknown mechanism, results in the inhibition of -catenin phosphorylation. At that stage catenin dissociates from the Axin complex and is no longer degraded, accumulating in the cytoplasm and also being translocated to the nucleus, where it competes with Groucho/TLE corepressors for binding to Tcf/Lef [START_REF] Daniels | Beta-catenin directly displaces Groucho/TLE repressors from Tcf/Lef in Wnt-mediated transcription activation[END_REF], thereby stimulating the expression of various Wnt target genes [START_REF] Huang | Wnt/beta-catenin signaling: new (and old) players and new insights[END_REF][START_REF] Macdonald | Wnt/beta-catenin signaling: components, mechanisms, and diseases[END_REF][START_REF] Mulholland | Interaction of nuclear receptors with the Wnt/beta-catenin/Tcf signaling axis: Wnt you like to know?[END_REF][START_REF] Van Amerongen | Towards an integrated view of Wnt signaling in development[END_REF].

Wnt signaling in development and function of the adrenal cortex

Mechanisms of Wnt signaling

Signaling by the

This constitutes the most widely accepted current model of Wnt/-catenin signaling (Figure 1). To increase the complexity of the system, Wnts can also act through "noncanonical" pathways that do not involve -catenin activation, but implicate small GTPases/JNK kinase and intracellular calcium [START_REF] Rao | An updated overview on Wnt signaling pathways: a prelude for more[END_REF]. Additionally, natural antagonists of Wnt signaling exist, which can bind either to 1) Wnt proteins or to 2) their receptors. Some of the most prominent members of the first class are secreted Frizzled-related proteins (sFrp), which display a high sequence homology with the extracellular Wnt binding domain of Frizzled in their N-terminal region and function as Wnt antagonists for both canonical and non-canonical signaling [START_REF] Kawano | Secreted antagonists of the Wnt signaling pathway[END_REF], while Dickkopf family members encode secreted proteins which antagonize Wnt signaling by inhibiting the Wnt coreceptors LRP5 and 6 [START_REF] Niehrs | Function and biological roles of the Dickkopf family of Wnt modulators[END_REF].

Zonal patterns of expression of Wnt-related genes in the adrenal cortex

A role for Wnt signaling in the development of the adrenal cortex is suggested by the zonal expression of several of its component elements. Wnt4 is a signaling A c c e p t e d M a n u s c r i p t 5 factor with multiple roles in organogenesis [START_REF] Brisken | Essential function of Wnt-4 in mammary gland development downstream of progesterone signaling[END_REF][START_REF] Jordan | Wnt4 overexpression disrupts normal testicular vasculature and inhibits testosterone synthesis by repressing steroidogenic factor 1/beta-catenin synergy[END_REF][START_REF] Vainio | Female development in mammals is regulated by Wnt-4 signaling[END_REF]. Its deficiency leads to abnormal development of the kidney, pituitary gland, female reproductive system, and mammary gland. Wnt4 is expressed in the cortical region of the developing adrenal gland from embryonic d11.5 onward, especially in the outermost part, the zona glomerulosa. Its expression correlates temporally with that of the transcription factor Sf1 (see below). Wnt4-deficient mice have a smaller number of glomerulosa cells, which leads to a decrease in the production of aldosterone. Such a defect in aldosterone biosynthesis is associated with an abnormal distribution of aldosterone synthase, Cyp11b2, in the adrenal [START_REF] Heikkila | Wnt-4 deficiency alters mouse adrenal cortex function, reducing aldosterone production[END_REF]. Cyp11b2 is the terminal enzyme in the pathway of aldosterone biosynthesis and is normally confined to the zona glomerulosa. [START_REF] Eberhart | Wnt signaling in human development: beta-catenin nuclear translocation in fetal lung, kidney, placenta, capillaries, adrenal, and cartilage[END_REF]. Knockout (KO) mice that are globally deficient in β-catenin undergo embryonic lethality during gastrulation and lack mesoderm, precluding analysis of potential defects in adrenal development [START_REF] Haegel | Lack of beta-catenin affects mouse development at gastrulation[END_REF]. To get further insight into the role of β-catenin in the adrenal cortex, recent studies shutting down -catenin expression through the use of a Sf1

promoter-Cre transgene in adrenocortical cells produced mice with either complete adrenal aplasia or defects in maintenance of the adult cortex resulting in depletion of adrenocortical cells [START_REF] Kim | Targeted disruption of beta-catenin in Sf1-expressing cells impairs development and maintenance of the adrenal cortex[END_REF]. These results show the essential role of βcatenin in mouse adrenocortical development. At least part of this phenotype may be attributed to the lack of -catenin function in cell-cell adhesion and be independent of Wnt signaling, since this defect is considerably more severe than that seen in Wnt4deficient mice [START_REF] Heikkila | Wnt-4 deficiency alters mouse adrenal cortex function, reducing aldosterone production[END_REF]. However, Wnt4 may have additional roles in noncanonical Wnt signaling [START_REF] Brisken | Essential function of Wnt-4 in mammary gland development downstream of progesterone signaling[END_REF][START_REF] Vainio | Female development in mammals is regulated by Wnt-4 signaling[END_REF], the canonical pathway may be activated by additional Wnt ligands in the adrenal gland, or βcatenin activation may be the target of other, Wnt-independent signaling pathways [START_REF] Hino | Phosphorylation of betacatenin by cyclic AMP-dependent protein kinase stabilizes beta-catenin through inhibition of its ubiquitination[END_REF][START_REF] Taurin | Phosphorylation of beta-catenin by cyclic AMP-dependent protein kinase[END_REF]. is differentially expressed with a higher expression in the zona glomerulosa than in the zona fasciculata or the zona reticularis [START_REF] Suwa | Zonal expression of dickkopf-3 and components of the Wnt signaling pathways in the human adrenal cortex[END_REF]. Studies screening for expression of other gene families involved in the Wnt pathway, the frizzled (Fz) and dishevelled (Dvl) families, in the adrenal cortex have reported that Fz1, Fz2 and Dvl3 transcripts are found in human, whereas only Fz2 and Dvl3 are present in the mouse [START_REF] Suwa | Zonal expression of dickkopf-3 and components of the Wnt signaling pathways in the human adrenal cortex[END_REF]. Recently, Bernichtein et al. have also reported the presence of Sfrp1 mRNA expression in mouse adrenal gland and assessed its expression by immunohistochemistry, showing an intense staining for the sFrp1 protein throughout the adrenal cortex [START_REF] Bernichtein | Adrenal gland tumorigenesis after gonadectomy in mice is a complex genetic trait driven by epistatic loci[END_REF].

To

Effect of -catenin on SF-1-mediated transcription

Steroidogenic Factor-1 (SF-1) is a critical regulator of adrenogonadal development and function. Its overexpression also has an important role in the pathogenesis of adrenocortical tumors (reviewed in [START_REF] Schimmer | Minireview: Steroidogenic Factor 1: its roles in differentiation, development, and disease[END_REF][START_REF] Lalli | Adrenocortical development and cancer: focus on SF-1[END_REF]. -catenin has been shown to physically associate with SF-1 and increase its transactivation of several gene promoters [START_REF] Hossain | Synergistic cooperation between the beta-catenin signaling pathway and steroidogenic factor 1 in the activation of the Mullerian inhibiting substance type II receptor[END_REF][START_REF] Jordan | Wnt4 overexpression disrupts normal testicular vasculature and inhibits testosterone synthesis by repressing steroidogenic factor 1/beta-catenin synergy[END_REF][START_REF] Mizusaki | Dax-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1) gene transcription is regulated by wnt4 in the female developing gonad[END_REF]. One report also showed that in the testis Wnt4

represses steroidogenesis by antagonizing SF-1/-catenin synergy [START_REF] Jordan | Wnt4 overexpression disrupts normal testicular vasculature and inhibits testosterone synthesis by repressing steroidogenic factor 1/beta-catenin synergy[END_REF]. Thes -catenin also plays important roles in adrenocortical function through the modulation of SF-1 action. Dkk3 inhibits aldosterone and cortisol biosynthesis in primary human adrenocortical cells, both in basal conditions or in the presence of cyclic AMP (cAMP) [START_REF] Chen | Adenovirus-delivered DKK3/WNT4 and steroidogenesis in primary cultures of adrenocortical cells[END_REF]. Conversely, Wnt4 increases steroidogenesis, as evidenced by the increase of both Cyp17 and Cyp21 when added alone, but decreases it in the presence of cAMP [START_REF] Chen | Adenovirus-delivered DKK3/WNT4 and steroidogenesis in primary cultures of adrenocortical cells[END_REF]. Dkk3 also decreases cAMPstimulated Cyp17 expression. Both Dkk3 and Wnt4 increase the level of Cyp11b2.

Overall, these data indicate that the overlap of Dkk3 expression with Wnt4 and catenin in the zona glomerulosa and the stimulation of Cyp11b2 by Dkk3 and Wnt4

might imply an action on glomerulosa-specific function.

Wnt signaling in adrenocortical tumors

Adrenocortical tumors (ACTs) in the literature refer to both adrenocortical adenoma (ACA) and adrenocortical carcinoma (ACC), even though they are different entities. ACA are frequent in the general population and nowadays they are most often found incidentally, while ACC is a rare tumor with 2 peaks occuring mostly in the first decade of life as well as in the fourth and fifth decades, with an incidence of 4-12 per million per year in adults [START_REF] Bertherat | Pathogenesis of adrenocortical cancer[END_REF]. They are more common in women. ACTs have been classified as either functional (75%) or nonfunctional (25%) tumors, depending on hormone hypersecretion syndromes. In contrast to adult tumors, over 95% of pediatric tumors are functional and are frequently associated with congenital abnormalities and secondary tumors.

Functional tumors produce symptoms depending on their production of an excess of glucocorticoid (Cushing's syndrome), androgen (virilization) or mineralocorticoid (Conn's syndrome) hormones. It is difficult to distinguish between benign and malignant ACTs. However, a number of criteria can be applied, including size: benign tumors of the adrenal cortex are generally smaller than malignant tumors. The presence of distant metastases is an absolute indication of malignancy. et al., 2009;[START_REF] Giordano | Distinct transcriptional profiles of adrenocortical tumors uncovered by DNA microarray analysis[END_REF][START_REF] Giordano | Molecular classification and prognostication of adrenocortical tumors by transcriptome profiling[END_REF][START_REF] West | Gene expression profiling of childhood adrenocortical tumors[END_REF] the precise sequence of events involved in their pathogenesis remains to be elucidated. Adrenocortical tumorigenesis in rodents and humans involves both genetic and epigenetic alterations, with accumulation of chromosomal alterations being a key mechanism during tumor progression.

Mutations of APC and adrenocortical tumors

Genetic alterations of the Wnt signaling pathway were initially identified in patients with familial adenomatous polyposis coli (FAP) and have since then been extended to a variety of cancers. Patients with FAP have constitutive activation of the Wnt/β-catenin signaling pathway due to germline loss-of-function mutations of the APC gene. They are predisposed to both colonic and extra-colonic tumors, among which adrenocortical tumors. Interestingly, most of these ACT in patients with FAP are benign and reveal no mutations in the -catenin gene [START_REF] Schinner | Pathophysiological aspects of Wnt-signaling in endocrine disease[END_REF][START_REF] Smith | Adrenal masses are associated with familial adenomatous polyposis[END_REF].

-catenin and adrenocortical tumors

It has been reported that activating mutations of exon 3 of the -catenin gene (CTNNB1), encoding a specific serine/threonine rich domain which is phosphorylated by GSK-3β and is essential for the targeted degradation of β-catenin, are found in a wide variety of human cancers including adrenocortical tumors [START_REF] Gaujoux | Wnt/beta-catenin and 3',5'cyclic adenosine 5'-monophosphate/protein kinase A signaling pathways alterations and somatic beta-catenin gene mutations in the progression of adrenocortical tumors[END_REF][START_REF] Polakis | Wnt signaling and cancer[END_REF]Tadjine et al., 2008a;Tadjine et al., 2008b;[START_REF] Tissier | Mutations of beta-catenin in adrenocortical tumors: activation of the Wnt signaling pathway is a frequent event in both benign and malignant adrenocortical tumors[END_REF]. These mutations were reported with similar frequencies in ACAs and ACCs. However, abnormal cytoplasmic or nuclear immunolocalization of -catenin was observed in 85% of adrenocortical carcinomas compared to 38% of ACAs [START_REF] Tissier | Mutations of beta-catenin in adrenocortical tumors: activation of the Wnt signaling pathway is a frequent event in both benign and malignant adrenocortical tumors[END_REF].

Similarly, a somatic mutation of -catenin has been observed in a heterogeneous

ACT with an apparently benign part adjacent to a malignant one. The catenin mutation was localized only in the malignant part of this tumour, suggesting that its occurrence would be associated with a more aggressive ACT [START_REF] Gaujoux | Wnt/beta-catenin and 3',5'cyclic adenosine 5'-monophosphate/protein kinase A signaling pathways alterations and somatic beta-catenin gene mutations in the progression of adrenocortical tumors[END_REF]. reported in colon cancer cell lines [START_REF] He | Identification of c-MYC as a target of the APC pathway[END_REF][START_REF] Tetsu | Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells[END_REF].

-catenin

Moreover, knock-down of -catenin blocks colon cancer cells in G1, providing proof of principle that interference with aberrant Wnt signaling could be of therapeutical value (van de Wetering et al., 2003). In addition, β-catenin immunoreactivity has also been observed in the nuclei of neoplastic cells that accumulate in the adrenal cortex of gonadectomized inbred mice, suggesting that the Wnt signaling pathway may participate in gonadectomy-induced adrenocortical neoplasia [START_REF] Bielinska | Gonadotropin-induced adrenocortical neoplasia in NU/J nude mice[END_REF].

Berthon et al. have recently developed transgenic mice expressing a constitutively active -catenin in their adrenal cortex. These mice show adrenal hyperplasia and dysplasia and develop malignant tumors over a 17-month time course. Moreover, profound changes in cellular and zonal adrenocortical identity are present. They manifest as ectopic differentiation of Cyp11b2-expressing cells at the expense of fasciculata cells, leading therefore to primary hyperaldosteronism. These data demonstrate that constitutively active -catenin is an adrenal oncogene which can trigger aberrant differentiation of aldosterone-secreting cells and promotes malignancy [START_REF] Berthon | Constitutive betacatenin activation induces adrenal hyperplasia and promotes adrenal cancer development[END_REF]. Of note, the H295R adrencortical cancer cell line harbors a -catenin-activating mutation. This leads to constitutive -catenindependent transcription. Treatment of this cell line with the Tcf/β-catenin antagonist PKF115-584 inhibits the transcriptional effect of -catenin and decreases cell proliferation [START_REF] Doghman | The T cell factor/beta-catenin antagonist PKF115-584 inhibits proliferation of adrenocortical carcinoma cells[END_REF]. Altogether these findings establish the Wnt signaling pathway as a potential therapeutic target in patients with adrenocortical tumors displaying abnormal -catenin accumulation.

Secreted Frizzled-related proteins (sFrps) and adrenocortical tumorigenesis

Alterations in the levels of sFrps expression caused by hypermethylation of their promoter region are among the most frequently occurring epigenetic alterations in different pathological conditions including tumor formation and make these molecules relevant to investigations in diverse fields of biology and biomedical sciences. Such epigenetic alterations are associated with transcriptional gene silencing [START_REF] Baylin | DNA hypermethylation in tumorigenesis: epigenetics joins genetics[END_REF][START_REF] Feinberg | The history of cancer epigenetics[END_REF]) and put the spotlight on the study of the correlations between sFrps and human cancer development.

Sfrp1 and/or Sfrp2 hypermethylation seems to occur in a wide variety of tumor types [START_REF] Dahl | Frequent loss of SFRP1 expression in multiple human solid tumours: association with aberrant promoter methylation in renal cell carcinoma[END_REF][START_REF] Huang | Down-regulation of SFRP1 as a putative tumor suppressor gene can contribute to human hepatocellular carcinoma[END_REF][START_REF] Lodygin | Functional epigenomics identifies genes frequently silenced in prostate cancer[END_REF][START_REF] Suzuki | Epigenetic inactivation of SFRP genes allows constitutive WNT signaling in colorectal cancer[END_REF]; et al., 2008). It has been therefore proposed, at least for bladder carcinomas, that the status of Sfrp1 promoter methylation could serve as an epigenetic biomarker for cancer detection and progression [START_REF] Lee | Autocrine/paracrine secreted Frizzled-related protein 2 induces cellular resistance to apoptosis: a possible mechanism of mammary tumorigenesis[END_REF][START_REF] Veeck | Aberrant methylation of the Wnt antagonist SFRP1 in breast cancer is associated with unfavourable prognosis[END_REF]. Although

Sfrp1 re-expression in breast, hepatocellular and prostate tumors, where Sfrp1 had been inactivated, induces apoptosis and suppresses cell proliferation, the incidence of spontaneous tumor formation is not increased in Sfrp1 null mice [START_REF] Trevant | Expression of secreted frizzled related protein 1, a Wnt antagonist, in brain, kidney, and skeleton is dispensable for normal embryonic development[END_REF]. These data suggest that Sfrp1, and possibly the related Sfrp2 gene, act as tumor suppressor genes. Their inactivation may predispose to neoplastic progression rather than to tumorigenesis and other additional genetic alterations would in this case be necessary for tumor onset. It is remarkable that, in the gonadectomized mice adrenocortical tumor model, linkage studies have evidenced Sfrp1 as a candidate gene positively influencing tumorigenesis [START_REF] Bernichtein | Adrenal gland tumorigenesis after gonadectomy in mice is a complex genetic trait driven by epistatic loci[END_REF]. In these mice, Sfrp1 levels are lower in the tumor tissue compared to the adjacent normal adrenal cortex. Given the oncogenic potential of constitutive Wnt signaling, it has been generally accepted that antagonism of Wnt/-catenin signaling constitutes the mechanism by which Sfrp1 inhibits tumor cell growth and prevents metatastatic invasion [START_REF] Esteve | The advantages and disadvantages of sfrp1 and sfrp2 expression in pathological events[END_REF]. In contrast to these anti-tumorigenic effects of Sfrp1, other groups suggested that it can also act as an oncogene that is responsible for the metastatic properties of renal carcinoma and promotes prostate carcinogenesis [START_REF] Joesting | Identification of SFRP1 as a candidate mediator of stromal-to-epithelial signaling in prostate cancer[END_REF]. This dual function suggests that the cellular context is an important determinant of Sfrp1 activity. Also, studies in colorectal tumors showed that another Sfrp gene, Sfrp4, may not act as a tumor suppressor gene and have quite different biological properties compared to other Sfrp family members [START_REF] Feng Han | Expression of sFRP-4 and beta-catenin in human colorectal carcinoma[END_REF].

In summary, the fact that several oncogenes are Wnt responsive genes argues for the involvement of constitutive Wnt signaling in the development of cancer after the shutting down of Sfrp1 expression. It is thus evident that studies of the interactions between the different sFrp molecules and Wnt-related proteins will be of key importance to clarify the role of this family of soluble factors in tumor pathogenesis. These studies will also open the possibility for development of novel therapeutic approaches that target this pathway.

Conclusions and perspectives

A c c e p t e d M a n u s c r i p t 11

The Wnt/-catenin pathway plays an important role in the regulation of the stem/progenitor cell pool and of the differentiation process in several tissues [START_REF] Katoh | WNT signaling pathway and stem cell signaling network[END_REF]. Remarkably, in the gonads, which share a common embryological origin with the adrenal gland, activation of the Wnt/-catenin pathway has an essential role in regulating ovarian differentiation [START_REF] Chassot | Activation of beta-catenin signaling by Rspo1 controls differentiation of the mammalian ovary[END_REF][START_REF] Liu | Sex-specific roles of betacatenin in mouse gonadal development[END_REF][START_REF] Maatouk | Stabilization of beta-catenin in XY gonads causes male-to-female sexreversal[END_REF][START_REF] Tomizuka | Rspondin1 plays an essential role in ovarian development through positively regulating Wnt-4 signaling[END_REF]. Studies in the mouse have shown that -catenin is involved in the regulation of proliferation and differentiation of progenitor cells in the adrenal cortex and that its constitutive activation may lead to tumor formation [START_REF] Berthon | Constitutive betacatenin activation induces adrenal hyperplasia and promotes adrenal cancer development[END_REF][START_REF] Kim | Targeted disruption of beta-catenin in Sf1-expressing cells impairs development and maintenance of the adrenal cortex[END_REF]. We believe that now among the most relevant questions to be addressed are the following:

-What is the nature of the Wnt factors responsible for-catenin activation in the subcapsular area of the adrenal cortex? Are other, Wnt-independent mechanisms involved?

-How does -catenin crosstalk with other signaling pathways (growth factors, hedgehog, Notch) to regulate the size of the progenitor cell population and their differentiation?

-What is the precise relationship of Wnt signaling with adrenal cell differentiation into aldosterone-producing cells?

-An interesting study reported regulation of downstream components of Wnt signaling at the post-transcriptional level by miRNAs in PPNAD, a benign neoplasm of the adrenal cortex [START_REF] Iliopoulos | MicroRNA signature of primary pigmented nodular adrenocortical disease: clinical correlations and regulation of Wnt signaling[END_REF]. Can miRNAs regulate Wnt signaling also during normal development and in other pathological settings?

It is easy to predict that in the years to come we shall witness a burgeoning of studies concerning the Wnt/-catenin pathway in the adrenal cortex that will considerably increase our understanding of its role in adrenocortical development and cancer.

  Wnt family of secreted glycolipoproteins plays key roles in embryonic development of organisms ranging from nematodes to mammals. A critical and most studied Wnt pathway is the canonical Wnt signaling, which functions by regulating the amount of the transcriptional co-activator -catenin that controls essential developmental gene expression programs (for an exhaustive overview www.stanford.edu/%7Ernusse/wntwindow.html). In the absence of Wnt, casein kinase 1 (CK1) and glycogen synthase kinase-3 (GSK-3) sequentially phosphorylate the amino terminal region of -catenin in the Axin complex, which is composed of the scaffolding protein Axin, the tumor suppressor adenomatous A c c e p t e d M a n u s c r i p t 4 polyposis coli gene product (APC), CK1, and GSK-3 horylated -catenin is subsequently ubiquitinated, resulting in its proteasomal degradation. This continual elimination of -catenin by the action of the Axin complex keeps its level low, preventing it from reaching the nucleus, and Wnt target genes are thereby repressed by the DNA-bound T cell factor/lymphoid enhancer factor (Tcf/Lef) family of proteins complexed with Groucho/TLE corepressors. The Wnt/-catenin pathway is activated when a Wnt ligand binds its cell-surface receptor consisting of a seven-pass transmembrane Frizzled (Fz) receptor and its co-receptor, low-density lipoprotein receptor related protein 6 (LRP6) or its close relative LRP5. The formation of a likely

  The -catenin, a downstream effector of Wnt signaling, can serve as a marker for Wnt pathway activation. Nuclear -catenin expressing cells are localized in a subcapsular position in the mouse adrenal cortex (Figure 2). Eberhart and Argani have published a survey of -catenin nuclear localization in normal human fetal and adult tissues in which they have shown its localization in the glomerulosa of the adrenal cortex

6

  Gene expression profiling studies on human and mouse adrenal glands have shown that among the Dickkopf factors only Dkk3 was expressed at a detectable level. In situ hybridization on adult human adrenal gland sections showed that Dkk3

  summarize, Wnt4 and -catenin expression in mice and DKK3 expression in human are confined to the zona glomerulosa, whereas Sfrp1 is distributed throughout the whole cortical region of the mouse adrenal. The colocalization of Wnt4 and catenin in the glomerulosa implies an action of the canonical Wnt signaling pathway on glomerulosa-specific function, which is probably locally limited by a potential inhibitor like DKK3. Altogether, these data show that a variety of genes of the Wnt signaling pathways are expressed in the adrenal cortex and suggest a role for Wnt signaling in the development and/or maintenance of functional zonation in the adrenal cortex.

7

  Action of Wnt signaling components on steroidogenesisDkk3 and Wnt4 have multiple actions on steroidogenesis in normal human adrenocortical cells, including effects on overall steroidogenesis (aldosterone and cortisol biosynthesis) and distinct effects on steroidogenic enzyme mRNA levels.

8

  Although our understanding of ACT biology has increased after the results obtained by analysis of their gene expression profiles (de Reyniès

  nuclear accumulation produces consequent activation of Tcf/Lef transcription factors, which induce expression of c-myc and/or cyclin D genes, as A c c e p t e d M a n u s c r i p t

Figure 1 .

 1 Figure legends

Figure 2 .

 2 Figure 2. Immunostaining of -catenin in the mouse adrenal cortex. Nucleocytoplasmic accumulation of -catenin is found in the subcapsular zone and in the glomerulosa. 40x magnification.

Figure 2

 2 Figure 2Figure2

Acknowledgements

We thank F. Aguila for artwork. Research in E.L. laboratory is supported by INCa, ANR GENOPAT and CNRS (LIA).