Introduction

Biofuels have been celebrated as an alternative to fossil fuels for their contribution to combating climate change. Recent developments, however, have shown that gaining energy from biomass may not be sustainable due to interference with global food supply. For example, replacing 5.75% of Europe"s fuels with biofuels by 2010, as the European Union has pledged to do, will place enormous demands on existing cropping systems. Up to 12.5% of the world"s corn production is already estimated to be processed to bioenergy [START_REF] Zeddies | Agrarmärkte und Bioenergie[END_REF]). Arguably there is potential for large scale biofuel production to impact food supplies, food prices and food scarcity. The UN (2007) report on biofuels warns about the environmental and food security dangers of the current massive biofuel developments. UN Special Rapporteur on the Right to Food Jean Ziegler, therefore, calls biofuels a "crime against humanity". In 2008, when food prices were dramatically soaring, he demanded an international five-year ban on producing biofuels [START_REF] Ziegler | Report of the Special Rapporteur on the right to food[END_REF]. At the same time, the shift from food to biofuel production affects farmers around the world. Specially, food certification systems have tended to strengthen retail power at the expense of farmers in developing countries [START_REF] Bingen | Agricultural standards: The shape of the global food and fiber system[END_REF][START_REF] Busch | The Moral Economy of Grades and Stand ards[END_REF][START_REF] Humphrey | Private standards, small farmers and donor policy: EUREPGAP in Kenya[END_REF]). Such discrimination and marginalization of, especially, small farmers and subsequently an increase in economic inequality might result from illegitimate systems creating certification standards, and is ultimately linked to the question how certification as a private governance tool can be legitimated.

Biofuel certification is often seen as an institutional arrangement that could counter negative externalities, such as soaring food prices [START_REF] Geibler | Biomassezertifizierung unter Wachstumsdruck; Wie wirksam sind Nachhaltigkeitsstandards bei steigender Nachfrage? Diskussion am Beispiel der Wertschöpfungskette Palmöl[END_REF][START_REF] Lewandowski | Steps towards the development of a certification system for sustainable bio-energy trade[END_REF][START_REF] Mol | Boundless Biofuels? Between Vulnerability and Enviro nmental Sustainability[END_REF][START_REF] Stupak | Sustainable utilisation of forest biomass for energy -Possibilities and problems: Policy, legislation, certification, and recommendations and guidelines in the Nordic, Baltic, and other European countries[END_REF]. Voluntary certification schemes have been proposed by national state authorities in cooperation with major stakeholders (such as the Cramer Commission in the Netherlands; the Low Carbon Vehicle Partnership in the UK and the National Ethanol Vehicle Coalition in the USA). Other initiatives have been initiated by non-state actors themselves (such as the Roundtable on Sustainable Palm Oil or the Roundtable on Sustainable Soy). A global integrated biofuel network is emerging, and these initiatives aim for global process standards. This makes biofuel governance an illustrative case for international relations and agri-food research.

The primary certification schemes for biofuels that are currently in place fall within private governance. Private governance is not just influenced, but "created", by non-state actors. Private standards are of a voluntary nature, i.e. not enforced by state authorities. However, national state authorities often facilitate their creation. The two studies analyzed in this article represent the different cases of privately initiated versus publicly initiated certification systems: the Roundtable on Sustainable Palm O il (RSPO) and the Cramer Commission. These two cases serve well for the study of biofuel governance because they are more advanced than other initiatives in the field and included the formulation of a set of rules at an early stage (2004 and 2006). Both initiatives lack participation of actors from the South, which is argued decreases their legitimacy. In the publicly initiated Cramer Commission, however, representation was more balanced among civil society and businesses, resulting in more concrete and challenging standards for the biofuel industry. The two case studies offer a better understanding of opportunities and challenges which the legitimacy of private governance of biofuel production face. Moreover, the article contributes to an understanding of how private governance in general can be more legitimate.

Fuel versus food

Biofuels are fuels derived from plant matter, e.g., sugarcane, palm or soybean, and other renewable feedstock. The most widely used transport biofuels are ethanol and biodiesel, with ethanol accounting for more than 90 percent of global biofuel production. Global production of ethanol has doubled since 2000, while production of biodiesel, starting from a much smaller base, has expanded nearly threefold [START_REF] Hunt | Cultivating Renewable Alternatives to Oil[END_REF], see chart 1). The biofuel market makes now about 3% of global gasoline consumption, and is predicted to share about 10% of the world fuel use for transport by 2025 [START_REF] Mcneely | Biofuels, Biodiversity and Energy Security: What are the environmental and social impacts? Presentation to SCOPE Conference on Biofuels[END_REF]. Still, less than 10% of global biofuels production is internationally traded. But there is an important expansion in global trade: the European Union, the United States, and Japan which are key consumers will not have the domestic capacity to meet internal demand [START_REF] Mcneely | Biofuels, Biodiversity and Energy Security: What are the environmental and social impacts? Presentation to SCOPE Conference on Biofuels[END_REF]. Fuel derived from the entire US corn crop, with existing technologies, would meet only 15% of domestic light-duty vehicle needs [START_REF] Hunt | Cultivating Renewable Alternatives to Oil[END_REF]). Hence, developed countries are increasingly importing biomass from developing countries in order to process it to fuel.

-chart 1 about here -Brazil, which is the second largest bio-ethanol producer after the USA, is already preparing its industry for large-scale export. A number of other developing countries also plan to increase the production of biofuels. There are several reasons behind this development: Energy gained from biomass is considered a sustainable alternative to energies based on limited fossil resources. Fossil-fuel importing countries (most notably the US and the EU) see biofuels as a way to become more independent from unstable fossil-fuel-producing and exporting regions (notably Russia, the Middle East and Venezuela) and thus increase their national energy security. Moreover, biofuels provide a new market for agricultural commodities. For instance, Malaysia and Indonesia, which already are the largest producers of palm oil, are gearing up biodiesel production with an eye on the growing European market [START_REF] Hunt | Cultivating Renewable Alternatives to Oil[END_REF][START_REF] Mol | Boundless Biofuels? Between Vulnerability and Enviro nmental Sustainability[END_REF].

Meanwhile, many countries have begun to experience the negative consequences of the biofuel boom. China, for instance, curtailed biofuel ambitions because large stockpiles of grain that existed in 1999, when the rapid expansion of ethanol production began, are nearly gone [START_REF] Hunt | Cultivating Renewable Alternatives to Oil[END_REF]. When soaring food prices caused hunger protests around the world in the first half of 2008, biofuels were blamed to raise crop prices [START_REF] Mol | Boundless Biofuels? Between Vulnerability and Enviro nmental Sustainability[END_REF]UN 2007;[START_REF] Ziegler | Report of the Special Rapporteur on the right to food[END_REF]. The biofuel boom caused speculation on food stocks resulting in soaring prices. This way, the transition from fossil fuel to current generation biofuels has intensified problems of world hunger. In addition to social vulnerabilities, NGOs such as the Rainforest Action Network or Organic Consumer Association broached the environmental issue: deforestation (of rain forest, in particular) and a decrease in biodiversity, monocropping, land degradation and water pollution.

Certification schemes are beginning to be established as a proposed solution to such problems. The agri-food governance literature shows that private standards and certification schemes are predominantly driven by the need of companies to control reputational risk (Bingen and Busch 2007;Humphrey 1008). Meanwhile, costs are shifted along the value chain with major consequences for foodand, assumable, biofuel -production systems in developing countries. New certification systems pose challenges for fa rmers, processors and exporters downstream the value chain.

While initially farmers, co-operatives and individual processors were the main players in the local biofuel regions, the market developed during the last decade and the trend is now definitely towards large companies and conglomerates (of major agribusiness such as Cargill and Archer Daniels for the global grain trade, conventional oil companies such as Shell, and auto manufacturer companies such as Daimler-Chrysler and Volkswagen) (Hunt et al. 2006: 75;[START_REF] Mol | Boundless Biofuels? Between Vulnerability and Enviro nmental Sustainability[END_REF]. Already, Archer Daniels Midland produces about one quarter of the ethanol in the US and is the second-largest biodiesel producer in Europe (Hunt et al. 2006: 67). As shown for the agri-food sector, these large, private companies know how to set standards in a way that strengthens their power and, hence, furthers this trend towards concentration. Therefore the question of the legitimacy of biofuel certification needs to be discussed : Who is legitimated to set standards for whom?

Private Governance: Between Effectiveness and Legitimacy

Biofuel certification schemes have been proposed by national state authorities in cooperation with major stakeholders and also by individual stakeholders. The Roundtable on Sustainable Palm Oil (RSPO) is an example of a voluntary certification scheme proposed by stakeholders from the palm oil industry. In this article, it will be compared with the Cramer Commission which was initiated and chaired by a national state authority, the Dutch Environmental Minister Jacqueline Cramer. Both initiatives published a set of criteria for sustainable palm oil or biomass production at a very early stage (see table 1 and2). Thus both were pioneers in the field of biofuel governance and therefore suggest themselves for the study of the legitimacy of biofuel governance.

-table 1 and 2 about here -Proponents of such private governance mechanisms base their arguments on the assumption that, in a globalised world, conventional regulation by the nation-state faces new constraints. Many problems such as the negative effects of biofuel production and associated processes are not able to be solved by unilateralist action. Cause-and-effect chains are highly complex, and controversial interdependences exist. Therefore, the regulative arena has been opened to "new" non-state actors and their financial capital and knowledge capacities [START_REF] Rosenau | Governance, Order and Change in World Politics[END_REF][START_REF] Messner | Das Konzept Global Governance[END_REF].

As key consumer countries will not have the domestic capacity to meet targets set by national or European authorities, state activities (such as the EU target of replacing 5.75% of the fuels with biofuels by 2010) are externalizing production abroad. This could be leading to the negative consequences of biofuel production discussed above, especially if domestic regulation in producing countries is missing or failing to prevent negative impacts. In the long run, such failures can have global impact; for example deforestation and loss of rainforest leading to a decline of carbon sinks and enhanced global warming.

Intergovernmental approaches have thus far failed to solve the problem and there is no relevant global body. Civil society campaigns have revealed a basic demand for regulative action, and private governance initiatives have begun to fill this regulative gap. Proponents of private governance argue that the necessity of solving certain problems can be considered more important than the process leading there [START_REF] Witte | Business as Unusual. Facilitating United Nations Reform Through Partnerships[END_REF][START_REF] Messner | Das Konzept Global Governance[END_REF]. While focusing on effectiveness (e.g. avoiding negative effects of biofuel production), aspects of legitimacy have been neglected by many authors (e.g. Rechkemmer and Schmidt 2006, p. 67-68). However, only state actors have the authority to prescribe behavior of others, and legitimacy is not simply transferable from state to non-state actors. Private governance, i.e. non-state actors prescribing behavior (e.g. how to produce biofuel), demands new sources of legitimacy which will be discussed in the following.

Legitimating Private Governance

Legitimacy deals with normative aspects of how to reason authority, who or what gives authority and why authority is accepted by someone or not [START_REF] Beisheim | Fit für Global Governance[END_REF][START_REF] Cutler | Private international regimes and interfirm cooperation[END_REF][START_REF] Scharpf | Governing in Europe: Effective and Democratic?[END_REF]. Legitimacy can be defined as "a shared expectation among actors in an arra ngement of asymmetric power such that the actions of those who rule are accepted voluntarily by those who are ruled because the latter are convinced that the actions of the former conform to pre-established norms. Put simply, legitimacy converts power into authority -Macht into Herrschaftand, thereby, establishes simultaneously an obligation to obey and a right to rule (Schmitter 2001, p. 2)".

According to democratic conceptions, those who obey are those who rule. Rousseau speaks of the authority of the people for the people -"par le people pour le people". Analytically, Scharpf (1999, among others) distinguishes between the authority of the people, the inputoriented perspective of legitimacy, and the authority for the people, the output-oriented perspective of legitimacy. The output-legitimacy accords to the effectiveness of political measures. Legitimacy is thus composed by a fair process, today based on fundamental democratic norms (input legitimacy), and an effective and equitable performance delivery (output legitimacy) [START_REF] Brozus | Vergesellschaftung des Regierens. Der Wandel nationaler und internationaler politischer Steuerung unter dem Leitbild der nachhaltigen Entwicklung[END_REF][START_REF] Scharpf | Governing in Europe: Effective and Democratic?[END_REF].

According to liberal theory, only state actors are authorized to prescribe behavior to others because only they can be held accountable through the political institutionsin democratic systems this holds true for elected representatives. The transfer of national competencies to an international, intergovernmental organization ignited a debate on the legitimacy beyond the nation-state [START_REF] Scharpf | Governing in Europe: Effective and Democratic?[END_REF][START_REF] Steffek | The Legitimation of International Governance: A Discourse Approach[END_REF][START_REF] Zürn | Regieren jenseits des Nationalstaats[END_REF].

Non-state actors are not foreseen by state-centered concepts of international relations which only know sovereign territorial nation-states and their representatives [START_REF] Messner | Das Konzept Global Governance[END_REF]. Non-state actors are automatically regarded as illegitimate from this view point (per definition). When asking for criteria of non-state actors" legitimacy, we thus dissociate from classic understandings of international relations solely understood as inter-state relations. Private governance is a (new) mode of steering transnational processes that reflects a new relation between state, market and society which cannot strictly be seen as separated spheres, anymore. The same is true for the dichotomy between internal and external politics which appreciably overlap [START_REF] Keohane | Redefining Accountability for Global Governance[END_REF][START_REF] Strange | The Retreat of the State. The Diffusio n of Power in the World Economy[END_REF]. For instance, EU and US "internal" targets for biofuels have major impacts abroad (soaring food prices, rainforest clearing etc.).

Private governance is mostly understood to be legitimate because of the output [START_REF] Witte | Business as Unusual. Facilitating United Nations Reform Through Partnerships[END_REF][START_REF] Rechkemmer | Neue globale Umweltpolitik[END_REF], in the current context for example, the achievement of ending rainforest clearance for palm oil production. According to this pragmatic, output-oriented understanding of legitimacy, political decisions are legitimate if and because they effectively support the common welfare. Consensus is assumed on what needs to be done in order to solve a commonly perceived problem [START_REF] Dobner | Did the state fail? Zur Transnationalisierung und Privatisierung der öffentlichen Daseinsvorsorge am Beispiel der globale n Trinkwasserpolitik[END_REF]. Legitimacy is derived from the fact that these problems necessitate a collective solution, i.e. they can neither be solved by a single nation-state or within an intergovernmental setting nor by the market or volunteer actions of civil society alone. As this may be the case, private forms of governance beyond the nation-state integrating non-state actors are argued to be justified and "de facto" legitimated by their output [START_REF] Keohane | Redefining Accountability for Global Governance[END_REF][START_REF] Majone | The Regulatory State and its Legitimacy Problems[END_REF][START_REF] Scharpf | Governing in Europe: Effective and Democratic?[END_REF].

In democratic theory, legitimacy being derived from the output alone needs to be considered with skepticism. Scharpf (2000, p. 349) denotes non-majoritarian legitimacy concepts such as technical expertise or juridical authority as an "indirect" or a "weak" form of legitimacy. Steffek (2003, p. 257) warns that political results are only accepted if they follow certain objectives and principles generally acquired. Material compensations can help to guarantee acceptance but they do not inherit the prestige of considered binding.

Deliberative democratic theories tell us that deficits of input-legitimacy can be balanced by the participation and inclusion of affected groups, the so called stakeholders [START_REF] Dryzek | Deliberative Democracy and Beyond. Liberals, Critics, Contestations[END_REF][START_REF] Nanz | Legitimation durch Deliberation? Die Rolle der Zivilgesellschaft in der supranationalen Politik[END_REF]. Participation in this sense is essential to "good governance" (e.g. European Commission 2001). The involvement of affected target groups replaces to a certain extent the elected representatives in the process of decision making. This form of legitimacy though participation is described with the term of "throughput-legitimacy" [START_REF] Take | Legitimes Regieren jenseits des Nationalstaats[END_REF]. Throughput-legitimacy is part of the input-legitimacy, following Scharpf, in as far as the process leading to a political result (output) refers to sources of legitimacy "par le people" (input). Therefore, in a way, this legitimacy replaces legitimacy through classic decisions by majority [START_REF] Nanz | Legitimation durch Deliberation? Die Rolle der Zivilgesellschaft in der supranationalen Politik[END_REF].

The question of which groups are "affected" in a particular policy field, i.e. how stakeholder categories are defined and how specific stakeholders are chosen for participation, is the central challenge for legitimacy through participation or stakeholder inclusion. There is no guarantee for representative stakeholder participation. For instance, in private food governance, we observe power asymmetries between participation of retail companies and the rest of the product chain, between North and South, and between representatives of business and of civil society interests [START_REF] Busch | The Moral Economy of Grades and Stand ards[END_REF][START_REF] Fuchs | Power in Global Food Governance. A Basis for Sustainable Consumption and Production?[END_REF]). While state representatives in a democratic setting are the result of general elections, the stakeholder representatives from the private sector and civil society are nominated or offer their participation in private governance through vastly more informal structures [START_REF] Brozus | Vergesellschaftung des Regierens. Der Wandel nationaler und internationaler politischer Steuerung unter dem Leitbild der nachhaltigen Entwicklung[END_REF][START_REF] Kahler | Global Governance in a Global Economy. Political Authority in Transition[END_REF].

The shift from input-legitimacy "par le people" to throughputlegitimacy by stakeholders also changes mechanisms of accountability. According to a democratic understanding, all power emanates from the people. Rousseau (1998 according to 1757) defines the nation or the people as sovereign which appoints the government. The government is thus accountable to the people. The latter exerts political control over the ruling authority by electing the parliament and the government. On this note, the government is the agent and the people are the principal. Political accountability can thus be defined as the central back coupling between those who rule (agents) and those who are ruled (principles) [START_REF] Kahler | Global Governance in a Global Economy. Political Authority in Transition[END_REF][START_REF] Keohane | Redefining Accountability for Global Governance[END_REF].

New throughput-legitimated private governance initiatives do not underlie any democratic control, because stakeholders are not appointed by the sovereign. The stakeholders are not elected but usually selected by the executive authority. Therefore, they suffer from a deficit of input-legitimacy following the understanding of Rousseau and Scharpf respectively. We hence need to find new adequate mechanisms of (legitimacy through) control and accountability for new steering governance modes incorporating non-state actors (e.g. grievance panel; naming and shaming) [START_REF] Cutler | Private international regimes and interfirm cooperation[END_REF][START_REF] Keohane | Redefining Accountability for Global Governance[END_REF][START_REF] Majone | The Regulatory State and its Legitimacy Problems[END_REF]. When analyzing private governance initiatives, we should ask for mechanisms of control and accountability which can improve their legitimacy. In the following, criteria for evaluating will be acuminated.

How to evaluate the Legitimacy of Private Governance

Private governance reflects a fundamental shift from an input-towards an enhanced outputoriented understanding of legitimacy [START_REF] Majone | The Regulatory State and its Legitimacy Problems[END_REF][START_REF] Witte | Business as Unusual. Facilitating United Nations Reform Through Partnerships[END_REF]. Initiatives such as the Roundtable on Sustainable Palm Oil and the Cramer Commission aim to contribute to solving specific problems, such as negative externalities of biofuel production (soaring food prices, deforestation, land degradation, water pollution etc.). The fact that certain problems get solved is considered more important than the modalities how this happens, e.g. if by state or non-state actors, if by public or private means [START_REF] Rechkemmer | Neue globale Umweltpolitik[END_REF]). An informed consensus is assumed on the "solution" or result [START_REF] Dobner | Did the state fail? Zur Transnationalisierung und Privatisierung der öffentlichen Daseinsvorsorge am Beispiel der globale n Trinkwasserpolitik[END_REF], or a "social system" in which ex post acceptance of "the good fight" will be achieved [START_REF] Bernstein | Can non-state global governance be legitimate? An analytical framework[END_REF]. This line of argumentation can be described as "de facto"legitimacy.

In the literature on private forms of (global) governance, including certification, two other lines of argumentation can be identified: legitimacy through stakeholder inclusion (throughput-legitimacy) and through control and accountability. Legitimacy through stakeholder inclusion refers to the process (input) generating a political decision (output). While de facto-legitimacy assumes a "neutral" or commonly agreed "best" solution, those who argue for stakeholder inclusion assume that an output varies always depending on those participating in the decision-making process [START_REF] Dryzek | Deliberative Democracy and Beyond. Liberals, Critics, Contestations[END_REF][START_REF] Nanz | Legitimation durch Deliberation? Die Rolle der Zivilgesellschaft in der supranationalen Politik[END_REF]. This perspective accords with findings from agri-food standards literature which shows the ways in which the construction of standards is bound up in the construction of power. Standards tend to strengthen retailers structural power at the expense of farmers, processors and exporters downstream the value chain [START_REF] Busch | The Moral Economy of Grades and Stand ards[END_REF][START_REF] Humphrey | Private standards, small farmers and donor policy: EUREPGAP in Kenya[END_REF]Reardon et al. 2003).

Those who argue for legitimacy through control and accountability do not deny this fact. However, they turn to the output and modalities for control and accountability in order to guarantee that the political output serves the common welfare, including the possibility to retake decisions [START_REF] Cutler | Private international regimes and interfirm cooperation[END_REF][START_REF] Keohane | Redefining Accountability for Global Governance[END_REF]. Elsewhere I derived these three conditions for the legitimacy of private governance circumstantially and broke them down to analytical questions [START_REF] Partzsch | Global Governance in Partnerschaft. Die EU-Initiative 'Water for Life[END_REF]). In the following, I use this framework to examine the Roundtable on Sustainable Palm Oil (RSPO) and the Cramer Commission. The fact of one or all of these conditions being fulfilled (or not) does not necessarily generate legitimacy. In practice, actions of those who rule must be accepted voluntarily by those who are ruled, as defined above. Accordingly, this set of conditions can only serve as a guideline for analyzing initiatives of private governance and certification.

Roundtable on Sustainable Palm Oil

The Roundtable on Sustainable Palm Oil (RSPO) was founded in Switzerland in 2004 as a result of an informal meeting initiated by the WWF two years earlier with Aarhus United UK Ltd, Golden Hope Plantations Berhad, Migros, Malaysia n Palm Oil Association, Sainsbury's and Unilever. The Statutes state that "RSPO's objectives are to promote the growth and use of sustainable palm oil through co-operation within the supply chain and open dialogue with its stakeholders" (RSPO 2004a, p. 1). Members have agreed to fulfill eight core principles which are further divided into criteria and indicators (RSPO 2007):

Principle 1: Commitment to transparency RSPO members must provide adequate information on environmental, social and legal issues relevant RSPO criteria (except where this is prevented by commercial confidentiality).

Principle 2: Compliance with applicable laws and regulations RSPO member have to comply with all applicable local, national and international legal provisions and regulations. The land rights of local communities with demonstrable [sic!] rights should not be contested.

Principle 3: Commitment to long-term economic and financial viability

A management plan that aims to achieve long-term economic and financial viability, and annual replanting program, projected for a minimum of 5 years, with yearly review are required.

Principle 4: Use of appropriate best practices by growers and millers

Operating procedures are documented, implemented and monitored. Soil, water and biodiversity should be protected and where possible improved through management and monitoring plans, appropriate techniques and trained staff. Agrochemicals should be used according to standards set by the World Health Organisation and Stockholm and Rotterdam Conventions.

Principle 5: Environmental responsibility and conservation of natural resources and biodiversity

Aspects of plantation and mill management that have environmental impacts, including aspects of biodiversity, waste management, energy, fire use and greenhouse gas emissions, are assessed and monitored. Information should be collated that includes both the planted area itself and relevant wider landscape-level considerations. Where the identification of impacts requires changes in current practices in order to mitigate negative effects, timetables for change should be developed.

Principle 6: Responsible consideration of employees and of individuals and communities affected by growers and mills

Social impacts are identified in a participatory way, and plans to mitigate the negative impacts and promote the positive ones are made, implemented and monitored to demonstrate continuous improvement. Participation in this context requires affected parties being able to express their views. There are open and transparent methods for communication and consultation between growers and/or millers, local communities and other affected or interested parties. Documented systems are established for dealing with complaints and grievances and with compensation for loss of legal or customary rights. The employer respects the right of all personnel to form and join trade unions of their choice and to bargain collectively. Children are not employed or exploited. Any form of discrimination based on race, caste, national origin, religion, disability, gender, sexual orientation, union membership, political affiliation, or age, is prohibited.

Principle 7: Responsible development of new plantings

A comprehensive and participatory independent social and environmental impact assessment on new plantings is undertaken, including soil surveys, topographic information and local peoples" agreement and compensation. The results are incorporated into plans and operations. New plantings since November 2005, have not replaced primary forest or any a rea required to maintain or enhance one or more High Conservation Values.

Principle 8: Commitment to continuous improvement in key areas of activity

Growers and millers regularly monitor and review their activities and develop and implement action plans that allow demonstrable continuous improvement in key operations.

In summary, the principles cover a wide range of issues. These are sometimes well specified, for instance, regarding employees" rights and allowance of trade unions (principle 6). Sometimes they are only vague and displayable. The requirement of an annual replanting program, for instance, does not imply any concrete replanting requirements (principle 3). When RSPO members oblige to fulfill the criteria, they basically commit their own particular activities in the field of palm oil to a comprehensive monitoring. In the following the legitimacy of the RSPO will be evaluated according to the conditions defined above (see table 3).

"De facto" Legitimacy

The RSPO criteria define "sustainable" palm oil but are far from being consensus. Various approaches to evaluate the "sustainability" of biomass production exist and compete with each other (see [START_REF] Geibler | Biomassezertifizierung unter Wachstumsdruck; Wie wirksam sind Nachhaltigkeitsstandards bei steigender Nachfrage? Diskussion am Beispiel der Wertschöpfungskette Palmöl[END_REF][START_REF] Lewandowski | Steps towards the development of a certification system for sustainable bio-energy trade[END_REF]Faaij 2006, Stupak et al. 2007). Civil society groups not participating in RSPO such as Friends of the Earth (FoE) groups and La Soja Mata warn that the criteria are flawed and not strict enough. For instance, FoE groups demand an abdication of the use of pesticides and other chemicals on "sustainable" plantations (FoE 2007b). They blame the palm oil industry for not being sustainable at all and pursuing deforestation and greenwash (FoE 2007b): If a plantation is on land cleared before 2005, it could be classed as sustainable by the RSPO, even if the manner in which the deforestation happened was illegal and created land rights conflicts. The same holds true for a company that pushes other farming activities (food or biofuel) into previously unfilled areas of forest (principle 7). When the RSPO presented proposals to label sustainable palm oil, FoE groups staged an installation of "screaming tree stumps outside the meeting in Brussels, representing the current environmental violations caused by producing palm oil" (FoE 2007b).

However, there are also civil society groups in support of the RSPO, such as the WWF which started the initiative. RSPO is composed of ordinary members in seven different sectors: oil palm growers; palm oil processors and/or traders; consumer goods manufacturers; retailers; banks and investors; environmental/nature conservation NGOs; social/developmental NGOs (RSPO 2004a). Non-membership or exit from the RSPO could bear high costs for anyone in the palm oil chain: If the RSPO certification system is successful, access to subsidies and even markets could be hampered for members of these groups being no RSPO members. For instance, the German government is discussing the sustainability of admixed proportion of biofuels and, in this respect, an amendment of the renewable energy law: only certified palm oil should be inserted in future (BMU 2008). Thus constraints to join the RSPO exist while there is no general consensus on what makes palm oil production "sustainable". Such consensus would however be essential for the condition of "de facto" legitimacy which can therefore not be considered as fulfilled.

Legitimacy through Stakeholder Inclusion

Any stakeholders or knowledge holders can request membership in the RSPO (RSPO 2004a, p. 2). The admission request must be addressed to the Executive Board. The Executive Board manages all activities. It is comprised of sixteen members and designated by the General Assembly which consists of all members (RSPO 2004a, p. 3). The Executive Board can reject any admission request without having to inform the candidate of the reasons motivating such decision (RSPO 2004a, p. 2). If accepted, members have to agree to a minimum duration of their membership for a two-year period and an annual fee of 2000 euros (RSPO 2004a, p. 1, 2, 4). These conditions, of course, advantage large companies and retailers and disadvantage or exclude small farmers and civil society actors, especially, from developing countries where incomes are low. Although the decisions within RSPO are taken either by consensus or at the majority of the votes of the ordinary members present (RSPO 2004a, p. 4), these conditions apply only to members, and some stakeholder are not involved or even oppose the RSPO such as FoE (FoE 2007a/b).

Other NGOs and, in particular, WWF and Oxfam are very engaged members and, among other things, permanent members of the grievance panel. Yet, both have their headquarters in Switzerland and the U.K. respectively while no NGOs from the developing countries of production are on the grievance panel. While the RSPO is dominated by the private sector and actors from the North, conflicts exist between members and local actors from the developing countries of production. Hunger protests and demands for a ban on biofuel production indicate conflicting interests of civil society in the South (FAS 2008). Moreover, RSPO member Wilmar, the world"s largest producer of palm oil, is accused of systematic illegal burning of forests to clear land for plantations by Indonesian authorities (FoE 2007a). The company"s palm oil is however still certified as "sustainable". This circumstance illustrates how the north-driven RSPO certification in practice contradicts local legislation and its enforcement in the South (in a way contradicting the RSPO own principles). In consequence, the RSPO cannot be considered legitimized through stakeholder inclusion.

Legitimacy through Control and Accountability

Even though the RSPO could not win opponents to biofuel industry for participation, it established mechanisms of control and accountability within its structures. First, the General Assembly is able to retake unanimously any decision. Second, with the grievance panel the RSPO provides a platformeven for non-membersto address complaints against all RSPO members. "The purpose of the Grievance Panel is to preside and deliberate on grievances that are brought to RSPO and provided detailed recommendations for resolution that would be fine-tuned and adopted by one or both (or more) parties involved in any conflict" (RSPO 2004b, p. 1). However, this process is neither involving state actors nor embedded in any legal system. There is hence neither conceptual guarantee nor empirical proof yet that the grievance system works in reality. Some non-state actors such as FoE groups (non-members) evaluate practices of RSPO member companies, and publish results. It is up to future developments, in how far these results will be taken into account, for example, in the allocation of subsidies.

In summary, the RSPO relies on principles that are arrived at by consensus, at best, among its members. A general consensus or commonly agreed "best" solution on what "sustainable" palm oil is has not been established; i.e. "de facto" legitimacy is not possible. The members are mainly from the private sector and from the North while some civil society actors explicitly oppose the RSPO; i.e. no inclusion of all relevant stakeholder groups. Mechanisms for control and accountability exist, in particular, the grievance panel although we do not know yet how effectively it works and if practices of "unsustainable" palm oil production will be tracked and prevented. Overall, only the condition for legitimacy through control and accountability can be considered partly fulfilled.

Cramer Commission

While the Roundtable on Sustainable Palm Oil has been initiated by non-state actors, the "Cramer Commission" (Project Group Sustainable Production of Biomass) was started by a group of six Dutch ministers that promote energy transition in the Netherlands and chaired by Jacqueline Cramer, the Dutch Environmental Minister. They invited a wide range of stakeholders to formulate criteria for sustainable biomass production and processing. However, the initiative can be considered "private governance" as it is settled outside the legal system of the Netherlands; the parliament is not involved in the creation of rules, for instance. The Commission"s results were planned to be used for the allocation of subsidies to biofuel industry (Cramer Commission 2006, p. 5). Targeting biomass flows in more general, especially, non-food applicants the ir scope was much broader than the RSPO focus on palm oil. Also unlike the RSPO, the Commission"s assignment was limited to the period from January 2006until February 2007(Cramer Commission 2007, p. 2).

Criteria and indicators for the sustainable production of biomass were formulated and devised for two phases 2007-2010and 2011-2020(Cramer Commission 2006, p. 6-7). The criteria for 2007 are minimum requirements which are supposed to be tested in three pilot projects (Cramer Commission 2006, p. 2, 6) while the criteria for 2011 and beyond prescribe active measures of protection. They were classified into six themes (Cramer Commission 2006/2007):

1) Greenhouse gas balance: The minimum requirement is 30% emission reduction for 2007 and 50% emission reduction for 2011 (and 70% emission reduction at the long run), compared with current fossil reference.

2) Competition with food, local energy supply, medicines and building materials should be avoided or decreased through minimum requirements which are supposed to be generated by obligatory reporting from the period 2007-2010.

3) Biodiversity: Plantations must not be located in or in the immediate vicinity of "gazetted protected areas" (areas protected by the government) or areas of "High Conservation Value". Again, minimum requirements are developed on the basis of obligatory reporting from the period 2007-2010.

4) Economic prosperity:

In cases where social and/or economic problems are to be expected by biomass production reporting is required according to the Economic Performance Indicators, as expressed in the Global Reporting Initiative (which is another multi-stakeholder institution).

5) Social well-being:

Compliance is required with the International Labour Organisation, the Universal Declaration of Human Rights and the Business Principles for Countering Bribery of the OECD. Obligatory reporting is required from 2007 on, and minimum requirements are planned for 2011. Active contributions are expected "in co-operation with the local community".

6)

The Environment: Compliance is required with international conventions, EU regulations as well as local and national legislation and regulations on waste management, use of agrochemicals (including fertilizer), prevention of erosion and soil e xhaustion and active improvement of the quality and quantity of surface and ground water. Reporting is required on erosion and soil exhaustion, and on quality and quantity of surface and ground water (2007)(2008)(2009)(2010)(2011). For 2011, again, minimum requirements are planned on the basis of reporting.

These criteria are formulated by the Cramer Commission to pave the way for a broader certification system for biomass production and processing such as the system run by the RSPO (limited to palm oil). The Commission, at first, envisages a certification based on trackand-trace system in which the traceability of biomass is guaranteed (Cramer Commission 2006, p. 18-19) but later preferences changed to a system of negotiable certificates (book and claim) as the latter could be introduced more rapidly (Cramer Commission 2007, p. 32-33). The aim is to certify "sustainable" biomass production around the world, and, potentially, to subsidize only certified biomass/biofuel.

"De facto" Legitimacy

The Cramer Commission is highly output oriented: "[I]t is of importance that the Dutch government together with other EU countries should take the initiative in the setting up of national and/or worldwide monitoring programmes to be able to recognize negative effects [of biomass production and processing] in time" (Cramer Commission 2006, p. 23). A consensus on the formulated criteria to be minimum requirements is assumed not only among the project group "consisting of representatives of the private sector, social organizations, financ ial institutions and the government" (Cramer Commissio n 2006, p. 1) but, beyond, among all stakeholders and general society.

On the one hand, the Cramer criteria are considered as non-binding advice or "recommendations" (Cramer Commission 2007, p. ii, iv). These formulations show that the Commission"s members were aware of their "private governance" status. On the other hand, the Dutch government plans to use these criteria for the allocation of subsidies and plans to take the lead in setting up national and/or worldwide programs (Cramer Commission 2006, p. 4, 5). Thus, strong incentives exist for stakeholders to participate. Exit costs could be high for biofuel industry. However, the criteria formulated so far encompass only demands for reporting; there is no impact "on the ground" yet. "De facto" legitimacy can only be analyzed to a limited extent. So far, as consensus on minimum requirements is only found among (nonrepresentative) members, the "de facto" legitimacy is not fulfilled (see table 3).

Legitimacy through Stakeholder Inclusion

The Cramer Commission encompasses a large number and variety of actors. State actors, in particular the Dutch ministers" group, are in the driver"s seat. As an independent chairperson the Dutch environmental minister Jacqueline Cramer has directed the process. She invited other ministers and non-state actors from the private sector (biofuel industry, financial sector) and civil society to participate (Cramer Commission 2007, p. i). Which particular stakeholders have been involved and consulted is made transparent in reports published on the internet (Cramer Commission 2006, Appendix 4). As among state actors, there is also a Dutch bias among non-state actors. Special expert input was delivered by Dutch private consultancies (Ecofys, CE) and Utrecht University (Cramer Commission 2006, p. ii). Large multinational corporations such as Unilever, Shell and Exxon Mobile (Wilmar did not participate), and firms which specialize in biofuel business such as the BioX Group participated. On the part of civil society, the Dutch sections of Oxfam and WWF and even FoE participated among others.

Different opinions between industry and civil society representatives have become evident in the Commission"s work. For instance, with respect to genetically modified organisms (GMOs), three quarter of the NGOs argued for including this aspect while only a tenth of the companies did so (Cramer Commission 2006, p. 26). In general, NGOs advocated for a stricter framework than state actors and participants from the biofuel industry (Cramer Commission 2006, p. 27). However, the results were also accepted as minimum requirements by the Commission members from civil society.

While interests between industry and NGOs were balanced within the Commission, this is not the case for the North-South divide. The Commission lacks members from the South although the reach of the criteria developed is meant to be "universal" (Cramer Commission 2006, p. 4). Thus, interests of people from the South as well as (business) interests from outside the field of biofuels are not represented in the Commission. Power asymmetries exist between developing countries producing and key consumer countries (US, EU and Japan) of biofuels, and these asymmetries are not perpetuated but aggravated by the composition of the Cramer Commission. Thus, on the one hand, the Cramer Commission (or the Dutch ministers) comes forward and, this way, jumps at the chance to overcome regulative deficits. On the other hand, this advancement goes past actors from affected developing producing countries. While civil society and business were represented in a balanced way, the Commission lacks participants from the South. Thus, the condition for legitimacy through inclusion of stakeholders is only partly fulfilled.

Legitimacy through Control and Accountability

The Cramer Commission repeatedly points to the fact that the proposed sustainability criteria must be integrated into political and policy frameworks at the national, European and global level (Cramer Commission 2006, p. iii). Such integration would allow for control and accountability. Meanwhile, the Commission itself is dominated by state actors from the Dutch government while no parliamentarians or state actors from the European or global level are involved. It can be argued that NGOs (and business) somehow take over the role of parliament as a counterpart to government within the Commission; i.e. there is a system of check and balances. However, the Dutch bias still causes a problem because the Dutch government can only be held accountable by the Dutch voters. Nevertheless, the Commission"s work is made very transparent and could theoretically be challenged by any organization or country opposing it.

In summary, the group of Dutch ministers made a concerted effort in appointing a Commission to formulate criteria that give way to a future worldwide certification system for the "sustainable" production and processing of biomass. A consensus was assumed on the formulated criteria to be minimum requirements. This "consensus" has not been established beyond the Commission, though. Thus the condition of "de facto" legitimacy cannot be fulfilled. However, conditions of legitimacy through stakeholder inclusion and through control and accountability are, at least, partly fulfilled: C ivil society and business" interests were represented equally although the Commission was dominated by Dutch actors. Besides no mechanisms for control and accountability have been established while, however, the Commission"s advance is made transparent and can be challenged by any affected actor.

-table 3 about here -

Conclusion

Certification schemes are beginning to be established as a proposed solution to negative externalities of biofuel production such as soaring food prices, deforestation and a decrease in biodiversity. The agri-food literature shows that certification schemes tend to be driven by retail interests at the expense of farmers, processors and exporters in developing countries. Such findings are ultimately linked to questions of legitimacy. I have analyzed the legitimacy of the completely privately initiated RSPO and of the publicly initiated Cramer Commission. Both initiatives aim to set up a certification scheme, one for palm oil and the other for biomass flows in more general, especially, non-food applicants such as transport in particular. The privately RSPO scheme is much more advanced and already well established: palm oil is certified as "sustainable" by now. Yet, this analysis suggests that the Cramer Commission has actually done more to foster legitimacy by balancing state, business and civil society interests (see table 3). In conclusion, I would like to compare the two initiatives" legitimacy in more detail along the three conditions of my analytical framework ("de facto" legitimacy, legitimacy through stakeholder inclusion, and legitimacy through control and accountability):

1) The publicly initiated Cramer Commission as much as the privately initiated RSPO are highly output-oriented. "De facto" legitimacy assumes a consensus on what needs to be done, in case of biomass production, for example, ending rainforest clearance. There is no general consensus though on what makes biomass or palm oil production (and processing) sustainable. This is most obvious for GMOs: views are divided and both initiatives have not agreed on a position (support or ban) on the use of GMOs in "sustainable" farming. A further illustration is the case of RSPO certified plantations pushing other farming activities into previously unfilled forest areas (leakage effect). Civil society criticizes that even though a plantation indirectly causes further deforestation, it can be certified as "sustainable" by the RSPO. As private governance by the biofuel stakeholders cannot implement an unambiguously consensual output, none of the existing initiatives can be considered "de facto" legitimated. Thus "de facto" legitimacy is not only a weak form of legitimacy; in the case of biofuel production, it is currently impossible to legitimate an initiative only based on the output.

2) In terms of legitimacy through stakeholder inclusion, the publicly initiated Cramer Commission put emphasis on a balanced representation of business and civil society groups whereas the RSPO faces strong opposition from civil society groups which do not participate in the palm oil certification process. However, major business and civil society stakeholders participate in both initiativesa fact that underlines the emergence of a global biofuel network. The RSPO principles, criteria and indicators were formulated in a way that favored the biofuel industry, for example, excluding GMOs. The Cramer criteria are more concrete in some aspects, such as the requirement of 30% greenhouse gas emission reductions (while still at the stage of being tested though). Both initiatives however fail to ensure adequate stakeholder participation from affected developing countries. Thus power asymmetries between key consumer countries (EU and US) and developing countries of production are enhanced by these initiatives of private governance. Consequently the RSPO cannot and the Cramer Commission can only partly refer to legitimacy through stakeholder inclusion.

3) In terms of control and accountability, the privately initiated RSPO has distinctly assigned responsibility to its General Assembly and Executive Board and even set up an impressive grievance system. However, these mechanisms are "private" in the sense that there is no legal suability (outsiders can turn to them but have no enforcement guarantee). The Cramer Commission"s legitimacy will still be challenged as soon as the recommendations formulated so far are put into action and have impact on the ground. As the Cramer criteria do not evolve from the conventional legislative procedure of the Netherlands, they also cannot be changed or retaken by the Dutch parliament or enforced by the Dutch judic ative. While there is no legal accountability, both initiatives however significantly advance the definition of sustainable production criteria. The fact that the allocation of subsidies will depend on certification in the near future gives an idea of their agenda setting power. The certification schemes" impact will not be "private" anymore. Aspects of legitimacy should thus not be faded-out; modalities for control and accountability need to be established in order to guarantee that the political output serves the common welfare.

What scholars and policy makers can learn from the two cases analyzed in this article: Private governance of biofuel production offers opportunities in terms of recommendations from stakeholders. As there is no consensus on what makes biomass flows sustainable though, actors" recommendation need to be considered as partial. A major challenge is to integrate actors from developing countries and adverse groups of civil society in order to balance interests. Again, the Cramer Commission, which was started by state actors, was more successful in balancing state, business and civil society interests than the private sector dominated RSPO. The Dutch government took a lead in agenda-setting due to pressures from Dutch civil society. As national authorities the Dutch ministers are however limited in the scope of their activities. If the inclusion of actors from the South fails, private governance of global scope cannot be considered as legitimate (legitimacy through stakeholder inclusion) as long as no general consensus has been established ("de facto" legitimacy), and as there also is no guaranteed control and accountability (legitimacy through control and accountability). Private authority by the North is unlikely to be accepted by people in the South. Findings from the agri-food certification literature tend to be valid for biofuel certification too: Certification schemes are predominantly driven by the need of companies to control reputational risk while the position, power and security of some of the most vulnerable actors is not likely to change for the better.

  Chart 1; source: http://www.eeb.cornell.edu/howarth/SCOPEBiofuels_home.html, Accessed 9 March 2009

Table 1 : Principles of the Roundtable on Sustainable Palm Oil

 1

	Principle 1	Commitment to transparency
	Principle 2	Compliance with applicable laws and regulations
	Principle 3	Commitment to long-term economic and financial viability
	Principle 4	Use of appropriate best practices by growers and millers
	Principle 5	Environmental responsibility and conservation of natural resources and
		biodiversity
	Principle 6	Responsible consideration of employees and of individuals and communities
		affected by growers and mills
	Principle 7	Responsible development of new plantings
	Principle 8	Commitment to continuous improvement in key areas of activity

Table 2 : Criteria of the Cramer Commission

 2

	Theme 1	Greenhouse gas balance
	Theme 2	Competition with food, local energy supply, medicines and building

 --------------------------------------------------------------------------


Maurizio Callari

Vera Cappelletti

Loris De Cecco

Valeria Musella

Patrizia Miodini

Silvia Veneroni

Manuela Gariboldi

Marco A Pierotti
email: marco.pierotti@istitutotumori.mi.it

Maria Grazia Daidone
email: mariagrazia.daidone@istitutotumori.mi.it

Gene expression analysis reveals a different transcriptomic landscape in female and male breast cancer

Keywords: Male breast cancer, gene expression profile, gender differences, breast cancer, pathway analysis

Purpose -Male breast cancer (MBC) is a poorly characterized disease because of its rarity. Clinical management is based on results obtained from randomized trials conducted in women notwithstanding data in the literature suggesting relevant gender associated differences in terms of biological and clinical behavior. However, a genome wide characterization of MBC on a transcriptional level is lacking.

Methods -In this study, gene expression profiles of 37 estrogen receptor positive (ER+) MBC specimens were compared to that of 53 ER+ Female Breast Cancer (FBC) samples similar for clinical and patho-biological features.

Results -Almost 1000 genes were found differentially expressed (FDR<1%) between female and male patients and biological interpretation highlighted a gender associated modulation of key biological processes ranging from energy metabolism to regulation of translation and matrix remodeling as well as immune system recruitment. Moreover, an analysis of genes correlated to steroid receptors and ERBB2 suggested a prominent role for the androgen receptor in MBC with a minor relevance for progesterone receptor and ERBB2, although, similarly to FBC, a genomic amplification could be observed.

Conclusions -Our findings support the idea that breast cancer is a quite different disease in male and female patients and the underlying gender related biological differences are likely to have clinical implications connected with different susceptibility to treatment.

INTRODUCTION

Male breast cancer (MBC) is a rare disease accounting for less than 1% of all cases of breast cancer diagnosed yearly, but its incidence rate continues to increase by 1.1%

annually. Research on male breast cancer has been limited by its rarity and most information on this disease has been extrapolated from results in female patients.

The etiology of male breast cancer is unclear but, similarly to women, steroid hormones levels may play a role in the development of the disease while a family history confers a relative risk of 2.5. As in women, mutations in BRCA1 and BRCA2

genes increase the risk of developing breast cancer; BRCA1 mutations are relatively rare (occurring in up to 4% of the cases), while those in BRCA2 gene are more frequent accounting for 4-16% of men with breast cancer. The major genetic factors associated with an increased risk of breast cancer in men also include mutations within the DNA-binding domain of androgen receptor (AR), polymorphism in the CYP17 gene coding for an enzyme involved in steroid synthesis, mutation of CHEK2 and PTEN tumor suppressor genes; however none of these genes has been demonstrated to have a causal association with male breast cancer [START_REF] Contractor | Male breast cancer: is the scenario changing[END_REF][START_REF] Nahleh | Male breast cancer: a gender issue[END_REF].

The worse prognosis observed for male compared to female breast cancer (FBC) is mainly due to the delay in diagnosis and to a more advanced stage at presentation, since after adjustment for age at diagnosis and stage of disease, the gender-associated differences disappear [START_REF] Willsher | A comparison outcome of male breast cancer with female breast cancer[END_REF]. The standard management of MBC derives from clinical data obtained in randomized trials conducted in women, and includes radical mastectomy plus axillary lymph node dissection, recently substituted by less invasive surgical procedures with no detectable decline in survival [START_REF] Nahleh | Male breast cancer: a gender issue[END_REF]. As for FBC, adjuvant chemotherapy is used to treat male patients who are at high risk of recurrence while the antiestrogen tamoxifen is considered the standard adjuvant hormonal therapy in male patients with steroid receptor-positive tumors [START_REF] Goss | Male breast carcinoma: a review of 229 patients who presented to the Princess Margaret Hospital during 40 years: 1955-1996[END_REF].

Up to date, the characterization of MBC at molecular level has mainly focused on immunohistochemical analysis of a limited set of well know FBC biomarkers. As summarized in [START_REF] Contractor | Male breast cancer: is the scenario changing[END_REF], a higher positivity rate of estrogen and progesterone receptors (ER and PgR) in MBC respect to FBC is well established. However, Munoz de Toro et al [START_REF] Munoz De Toro | Proliferative activity and steroid hormone receptor status in male breast carcinoma[END_REF] remarkably found that in MBC proliferation rate was higher in ER+/PgR+ compared to ER-/PgR-tumors, an opposite finding respect to FBC which strongly support the hypothesis that these receptors may play a different role in males compared to women. Controversial data were reported for AR positivity as well as for ERBB2 amplification rate, with positivity rates ranging from 0 to 95% among different studies [START_REF] Contractor | Male breast cancer: is the scenario changing[END_REF][START_REF] Nahleh | Male breast cancer: a gender issue[END_REF][START_REF] Curigliano | Recognizing features that are dissimilar in male and female breast cancer: expression of p21Waf1 and p27Kip1 using an immunohistochemical assay[END_REF][START_REF] Bloom | Status of HER-2 in male and female breast carcinoma[END_REF]. Furthermore, an immunohistochemical analysis of ER, AR and functionally related proteins indicates a reduced functionality of ER but a significant role for AR in MBC [START_REF] Weber-Chappuis | Comparison of prognostic markers detected by immunohistochemistry in male and female breast carcinomas[END_REF]. Finally, a higher percentage of BCL2 positive tumors in male was also reported [START_REF] Weber-Chappuis | Comparison of prognostic markers detected by immunohistochemistry in male and female breast carcinomas[END_REF], suggesting a role for this antiapoptotic gene during carcinogenesis.

Globally, these data indicate many differences between the biology of MBC and FBC, an observation which might imply the need of distinct therapeutic approaches and management of the disease according to the gender.

During the last decade, the application of a number of high-throughput genomic technologies refined the taxonomy of breast cancer and identified genomic fingerprints likely to impact future clinical treatment approaches [START_REF] Sotiriou | Gene-expression signatures in breast cancer[END_REF]. However, while FBC has been intensively investigated by comprehensive molecular analysis, at present no gene expression data are available for male breast cancer. Recently, Fassan et al reported a set of microRNA as differentially expressed by comparison of 23 male and 10 female breast cancer samples [START_REF] Fassan | MicroRNA expression profiling of male breast cancer[END_REF].

Taking advantage of the availability in our Tumor Tissue Bank of a series of ER+ primary breast cancer specimens from male patients, we compared their gene expression profiles with the profile of breast cancer from female patients with similar clinical and patho-biological features, to investigate whether a gender-related biological difference does exist at transcriptomic level in breast cancer.

Our data suggest a profound biomolecular difference between male and female breast cancer and highlight a set of differently regulated biological processes, ranging from energy metabolism to regulation of translation and matrix remodeling as well as immune system recruitment. Furthermore, correlation analysis supports a prominent role for AR in MBC with a minor relevance for PgR and ERBB2, although, similarly to FBC, a genomic amplification may be observed for the latter also in males.

MATERIALS AND METHODS

Patients and tumors features

We consecutively collected 37 snap-frozen ER+ primary tumor specimens from men submitted to radical mastectomy or conservative quadrantectomy plus axillary lymph nodes dissection for a primary breast cancer at the Istituto Nazionale dei 1.

ER and PgR content was routinely evaluated at time of diagnosis according to the EORTC recommendations and within national and international quality control programs by a ligand binding assay as previously described [START_REF] Ronchi | A double-labeling assay for simultaneous estimation and characterization of estrogen and progesterone receptors using radioiodinated estradiol and tritiated Org 2058[END_REF] and results were expressed as fmol mg -1 of protein. Tumors with an ER concentration higher than 10 fmol mg -1 of protein or with a PgR concentration higher than 25 fmol mg -1 of protein were defined as ER-positive or PgR-positive, respectively.

RNA isolation and microarray hybridization

Total RNA extraction, probe labeling and hybridization were performed as previously described [START_REF] Cappelletti | Patterns and changes in gene expression following neo-adjuvant anti-estrogen treatment in estrogen receptor-positive breast cancer[END_REF]. Briefly, total RNA was extracted using Trizol reagent (Life Technologies, Inc. Grand Island, NY) following the manufacturer's instructions and treated with DNaseI (Qiagen, Valencia, CA). Each sample was both directly and indirectly labelled with Cy-dCTPs (Amersham Biosciences; GE Healthcare, Piscataway, NJ) and with 3DNA Array900 detection kit (Genisphere, Montvale, NJ), respectively. Samples were co-hybridized together with a common RNA reference (Universal Human Reference RNA, Stratagene, La Jolla, CA) on custom made cDNA microarray slides containing 16457 sequence-verified I.M.A.G.E. clones (Research Genetics/Invitrogen) spotted in triplicate. cDNA microarrays were scanned using the GenePix 4000B microarray scanner at a resolution of 5μm and the images were analyzed using GenePix Pro v.5.0 software (Axon Instruments, Union City, CA).

Data analysis and statistics

Array data -Microarray raw data were processed using marray package (version All class comparisons on gene expression data were performed applying the Significance Analysis of Microarrays (SAM) [START_REF] Tusher | Significance analysis of microarrays applied to the ionizing radiation response[END_REF] to data at clone level, considering as threshold an FDR<1% unless otherwise specified.

To aid the biological interpretation, Gene Ontology Analysis using the TopGO package (version 1.12.0) [START_REF] Rahnenfuhrer | Improved scoring of functional groups from gene expression data by decorrelating GO graph structure[END_REF] using the Fisher's exact test statistics was carried out.

Correlation analysis -Gene expression values of single clones of interest were correlated (Spearman correlation) to gene expression values of all the other clones, separately for MBC and FBC. Only probes with a p<0.01 were considered significantly correlated. After array data filtering, no clones were found referring to the PgR gene, consequently in subsequent analysis PgR expression data were evaluated by qPCR.

Clustering analysis -Hierarchical clustering was performed using Euclidean distance and a complete linkage method. A Fisher's exact test was used to evaluate difference in PgR status distribution in the two clusters identified by considering the first split in the hierarchical three.

All analysis were performed employing the R software (version 2.9.0).

Quantitative RT-PCR

The RNA expression of PgR was evaluated by quantitative RT-PCR on the same stored frozen tissue used for microarray analysis. Total RNA was reverse-transcribed using the High-Capacity cDNA Archive Kit and a TaqMan reaction was carried out in duplicate on an ABI PRISM 7700 machine, using Assays-on-Demand Gene Expression Product (Hs00172183_m1, Applied Biosystems, Foster City, CA). To normalize data, 18S ribosomal subunit was used as housekeeping (4319413E, Applied Biosystems, Foster City, CA, USA). Data analysis was performed using the Sequence Detector v1.9 software.

RESULTS AND DISCUSSION

Biological interpretation of differentially expressed genes

From the class comparison between male and female breast cancer, we obtained 1273 clones differentially expressed at an FDR<1%, corresponding to 920 distinct annotated genes.

Not surprisingly, the most significant gene was XIST, expressed exclusively from the X inactivation center of the inactive X chromosome.

Differentially expressed genes were subjected to Gene Ontology analysis using

TopGO. Sixty-one terms of the Biological Process ontology, 20 of the Molecular Function and 16 of the Cellular Component ontology were found enriched at a significance level of p<0.01 (Supplementary files 1-3).

Using the TopGo results as starting point, we identified a set of biological categories each involving a large number of differentially expressed genes, which were used to attempt an unbiased biological interpretation of the class comparison results. We report below a detailed description of the identified biological functions and of their role in cancer disease, highlighting key genes differentially expressed for each function. An overview of biological differences between genders is reported in Table2. Instead, the complete lists of genes referred to a specific category are available as supplementary file (Supplementary file 4). Genes mapping on the Y chromosome have not been considered.

Energy metabolism

A set of genes (e.g. CYCS, COX6C, NDUFs) coding for enzymes involved in different steps of mitochondrial oxidative phosphorylation (OXPHOS) were found down-regulated in FBC, suggesting a reduced OXPHOS compared to MBC. The observation of an impaired respiration and increased glycolysis in tumor cells dates to many decades ago [START_REF] Warburg | On respiratory impairment in cancer cells[END_REF] and is known as the Warburg's hypothesis. An explanation on why tumor cells switch to a less efficient metabolism has been proposed [START_REF] Vander Heiden | Understanding the Warburg effect: the metabolic requirements of cell proliferation[END_REF],

suggesting that malignant cells have important metabolic requirements that extend beyond ATP. The Warburg's hypothesis was recently verified in breast cancer cells related to normal mammary cells [START_REF] Putignani | Alteration of expression levels of the oxidative phosphorylation system (OXPHOS) in breast cancer cell mitochondria[END_REF], but interestingly our data suggest that this effect is at least reduced in MBC compared to FBC.

Strictly related to OXPHOS is ATP metabolism, a biological function that we also found modulated according to gender. In particular we noticed in MBC an increased expression of subunits of the mitochondrial ATP syntase complex and of the ATPIF1 gene, an inhibitor of the ATPase function of such a complex. Coherently with an increased availability of ATP, some ATPases were overexpressed, among them the vacuolar ATPase complex, which has important physiologic roles, but was also reported to be associated to tumor invasion as it is able to create a pH gradient across the membranes, mediating extracellular matrix acidification [START_REF] Sennoune | Plasmalemmal vacuolartype H+-ATPase in cancer biology[END_REF].

Furthermore, OXPHOS is the major source for ROS production, that is therefore expected to be higher in MBC. It was not surprising to find 4 out of 6 human peroxiredoxins up-regulated in male tumors (PRDX1-4). Peroxiredoxins are antioxidant enzymes which reduce hydrogen peroxide and can indirectly affect cell proliferation: their role in cancer has been in fact extensively studied [START_REF] Neumann | Are peroxiredoxins tumor suppressors?[END_REF]. Similarly, the metabolism of glutathione, a potent reducing agent, seems to be affected. The GSH:GSSG ratio is critical for cell survival and influences S-glutathionylation, a chemical modification catalyzed by glutaredoxins, important for regulation of key cellular processes like the proteasomal protein degradation [START_REF] Mieyal | Molecular mechanisms and clinical implications of reversible protein Sglutathionylation[END_REF]. A rate limiting enzyme of glutathione synthesis (GCLC), a glutaredoxin (GLRX3), two glutathione peroxidase (GPX1, GPX2) and finally the glutathione reductase enzyme (GSR) were among differentially expressed genes in our data.

Translation

Protein synthesis is a fundamental process of eukaryotic cells and translation initiation represents a main regulatory step. Key molecules (e.g. EIF4 family members and poly(A) binding proteins) mediating this step were mostly found upregulated in MBC. Particularly intriguing is EIF4E overexpression in MBC. This protein is frequently up-regulated in cancer and selectively enhances translation of key genes involved in tumorigenesis and cancer progression like MYC and BCL2 [START_REF] Graff | Targeting the eukaryotic translation initiation factor 4E for cancer therapy[END_REF]. Consistently, these two genes are up-regulated in our data. EIF4E is a downstream effector of PI3K/AKT/mTOR mediated signals. Among signals acting on PI3K/AKT/mTOR pathway, there are many growth factors, but also the cell energy status, as a decreased AMP/ATP ratio promotes mTOR activity; mTOR itself, also overexpressed in male tumors, mediate phosphorylation of 4EBP, leading to EIF4E activation [START_REF] Mamane | mTOR, translation initiation and cancer[END_REF] (Fig. 1).

Another result suggesting an increased protein synthesis in MBC is the upregulation of many ribosomal proteins of both the 40S (RPS8, RPS12, RPS16, RPS21) and 60S subunits (RPL18, RPL30, RPL35) and associated proteins.

Globally, an increased protein synthesis has been related to cell growth and proliferation, linking this process with malignancy [START_REF] Caraglia | Modulation of molecular mechanisms involved in protein synthesis machinery as a new tool for the control of cell proliferation[END_REF]. As corollary, a group of DEAD-Box proteins (DDX3X, DDX5, DDX11, DDX12, DDX49, DDX55), involved in many aspects of RNA metabolism are also differentially expressed.

In eukaryotic cells, translation takes place also in mitochondria where 13

polypeptides that constitute the central core of the OXPHOS complex are translated [START_REF] Perez-Martinez | Protein synthesis and assembly in mitochondrial disorders[END_REF]. Up-regulation of an elongation factor (GFM1) and 5 mitochondrial ribosomal proteins (MRPL14, MRPL23, MRPL30, MRPL34, MRPL42), indicates an enhanced translation of these mitochondrial genes in male breast cancer, in agreement with what stated above.

Cytoskeleton and GTPase

Reorganization of the cytoskeleton is the primary mechanism of cell motility, is essential for most types of cell migration and is regulated by Rho family small

GTPase. The tubulin/microtubule system is an important target for anticancer therapy with vinca alkaloids and taxanes, but also with newly developed drugs (e.g.

epothilones [START_REF] Hunt | Discovery of ixabepilone[END_REF] or eribulin [START_REF] Cigler | Eribulin mesylate for the treatment of breast cancer[END_REF]). Therefore, the observed gender divergence at level of cytoskeleton organization is particularly interesting. In fact, genes of both alfa (TUBA1A, TUBA3D, TUBA4A) and beta (TUBB, TUBB2C) tubulin families were up-regulated in MBC while two microtubule-associated proteins (MAP1B, MAP2), able to regulate the assembly-disassembly ratio, were down-regulated.

Actin reorganization is the underlying mechanism of cell migration both in physiological and malignant conditions. In MBC the actin polymerization process was altered by modulation of the ARP2/3 complex components and overexpression of the capping protein CAPZA1. This process is mainly regulated by Rho family small GTPase [START_REF] Etienne-Manneville | Rho GTPases in cell biology[END_REF][START_REF] Vega | Rho GTPases in cancer cell biology[END_REF]. RND2, a member of this family is more expressed in FBC.

Two Rho GTPase activating proteins (SRGAP2, ARHGAP23) and a guanine nucleotide exchange factor, key regulators of Rho activity, are differentially expressed. Additionally, overexpression of TNK2 occurs in MBC. This gene is a tyrosine kinase that binds Cdc42 (a Rho family GTPase) and has been causally related to migration of cancer cells by enhancing cell surface localization of EGFR [START_REF] Howlin | TNK2 preserves epidermal growth factor receptor expression on the cell surface and enhances migration and invasion of human breast cancer cells[END_REF].

Rho GTPase are only one of 5 protein families composing the RAS GTPase superfamily: Rho, Ras, Rab, ARF, RAN family. Members belonging to all of these families showed a different expression pattern in male and female breast cancers. In detail, we observed an increase in MBC of ARF (ARF6, ARL1, ARL8B) and RAB (RAB1A, RAB6A, RAB14, RAB22A, RAB31) GTPase as well as regulator molecules (respectively ARFGEF1 and GDI2, RAB11FIP1). These GTPase are main actors in intracellular trafficking and their role in cancer is well documented as they are implicated in modulating growth factor receptor localization and supporting invadopodia and filopodia formation [START_REF] Chia | Emerging roles for Rab family GTPases in human cancer[END_REF]. Specifically, ARF6, overexpressed in MBC, was shown to mediate MDA-MB-231 breast cancer cells invasive activities [START_REF] Hashimoto | Requirement for Arf6 in breast cancer invasive activities[END_REF].

RAN and the regulatory RANBP1, also up-regulated in MBC, are instead involved in a wide range of cellular processes ranging from DNA synthesis and microtubules organization at mitosis to DNA and protein translocation through the nuclear pore [START_REF] Rensen | The GTPase Ran: regulation of cell life and potential roles in cell transformation[END_REF]. Interestingly RAN is also an important androgen receptor coactivator as later discussed.

Finally also RAS family GTPase (RAP2C, RASL10A) and specific regulators of their activity were found transcriptionally altered comparing the two genders.

Tumor environment

Many recent studies are pointing out the role of the microenvironment during breast cancer development, growth and distant spreading. As a matter of fact, a prognostic role has been reported for some matrix metalloproteinases [START_REF] Kohrmann | Expression of matrix metalloproteinases (MMPs) in primary human breast cancer and breast cancer cell lines: New findings and review of the literature[END_REF] and other ECM genes [START_REF] Helleman | Association of an extracellular matrix gene cluster with breast cancer prognosis and endocrine therapy response[END_REF] as well as for stromal derived signatures [START_REF] Finak | Stromal gene expression predicts clinical outcome in breast cancer[END_REF].

Extracellular matrix is mainly constituted by two major components: the basement membrane and the interstitial matrix. Different types of collagen form a scaffold for both these components, laminins are the most abundant proteins of the basal membrane while among the proteins constituting the interstitial matrix it is worth to cite tenascin, proteoglycans and other glycoproteins. All these molecules strictly interact with a plethora of adhesion molecules expressed on cell surface, not only with a structural function but also to mediate signals regulating primary cell processes. Notably, genes belonging to all of cited extracellular matrix protein families and adhesion molecules as well were found as significantly both up-and down-regulated in our dataset. More in detail, various structural proteins like COL5A3, LAMC1, TNC, ECM2, FBN2, SPON1, THBS1 and the proteoglycans BGN, GPC3 as well as the UGDH, an enzyme which participate in glycosaminoglycan biosyntesis (e.g. hyaluronan) were up-regulated in MBC, although some were more expressed in FBC (FN1, MTN2, MGP, TMBS3).

Furthermore, extracellular matrix is not a static structure and is instead constantly remodeled by well regulated matrix protease [START_REF] Bosman | Functional structure and composition of the extracellular matrix[END_REF]. MMPs (matrix metalloproteinases), ADAMTS (a disintegrin and metalloproteinase with trombospondin motifs), TIMPs (tissue inhibitors of metalloproteinases) along with the SERPIN superfamily of serine protease inhibitors constitute an important proteolitic axis. Many genes from the above families whose disregulation in breast cancer is well documented, were either up-regulated in MBC (MMP11, TIMP3, SERPINA5, SERPINA6, SERPINAB2, SPINT2) or in FBC (ADAMTS3, ADAMTS7, ADAMTSL4, MMP7).

Therefore, while it clearly appears that there are differences in the complex regulation of extracellular matrix remodeling, it is difficult to trace a gender related model using only gene expression data, although it can be suggested that the gender differences at the level of the extracellular matrix may have a relevant impact on cell survival, growth and migration due to the central role of tissue remodeling in tumor biology.

The tumor microenvironment is also strongly affected by effectors of immune response. The myeloid and lymphoid tumor infiltrating cells has been reported for breast cancer with a frequency around 40% of tumors [START_REF] Coronella-Wood | Naturally occurring B-cell responses to breast cancer[END_REF]. Moreover, chronic inflammation has recently been linked with an increased risk of distant metastases [START_REF] De Visser | Paradoxical roles of the immune system during cancer development[END_REF], while other studies suggest a protective role exerted by immune cells [START_REF] Schmidt | The humoral immune system has a key prognostic impact in node-negative breast cancer[END_REF][START_REF] Rody | T-cell metagene predicts a favorable prognosis in estrogen receptor-negative and HER2-positive breast cancers[END_REF] Surprisingly, we found among differentially expressed genes many immune cells specific genes globally indicating a clear reduction of immune response in MBC. For instance, a numerous set of chemokines essentially produced during immune response (CCL25, CXCL1, CXCL9, CXCL10, IL23A, IL17B, IL32) were more expressed in female tumors. A similar pattern was observed for two members of the MHC II complex expressed by APC cells (HLA-DQB1, HLA-DRB1), a lymphocytespecific protein tyrosine kinase (LCK), a member of the T-cell receptor complex (CD3D), a natural killer specific receptor (KLRK1) and a chitotriosidase secreted by activated macrophages (CHIT1). An exception to this is represented by the two B-cell specific genes found in our list (BCAP31, IGHG1) that are more expressed in male samples.

Membrane transport

Genes involved in membrane transport displayed differences between females and males. Two ATP-binding cassette transporters (ABCC2 and ABCC5) which have been linked to chemo-resistance in human cancer [START_REF] Materna | RNA interference-triggered reversal of ABCC2-dependent cisplatin resistance in human cancer cells[END_REF][START_REF] Weaver | ABCC5, ERCC2, XPA and XRCC1 transcript abundance levels correlate with cisplatin chemoresistance in non-small cell lung cancer cell lines[END_REF] are more expressed in FBC.

On the contrary, 21 members of the solute carrier family are either up-or downregulated. This is however a highly heterogeneous family, including active transporters as well as proteins which facilitate transport, ion channels and aquaporins. Here a detailed analysis of single transporters would be useless, but it is worth to remember that they can regulate drug uptake as well as many cancer related processes [START_REF] He | Analysis and update of the human solute carrier (SLC) gene superfamily[END_REF]. For example changed expression of two glucose transporters (SLC2A1 and SLC2A3) could impact on glucose metabolism, consistently with what described about metabolic differences.

Growth factors response and apoptosis

Failure of inhibition of proliferation and overcome of apoptosis signals are crucial for cancer cell growth and survival. These two crucial aspects of breast cancer biology also appeared to be different according to the gender indirectly suggesting that male and female breast tumors adopt different survival strategies. For example, concerning growth signaling, ERBB2 and NRG1, a glycoprotein that enhances ERBB2 phosphorylation [START_REF] Britsch | The neuregulin-I/ErbB signaling system in development and disease[END_REF], GRB2, an EGFR interacting protein and MET, a growth factor receptor with a well established role in breast cancer [START_REF] Lengyel | C-Met overexpression in node-positive breast cancer identifies patients with poor clinical outcome independent of Her2/neu[END_REF] are downegulated in MBC. On the opposite, FGF receptor 2 is more expressed in MBC.

Focusing on apoptosis related genes, we found differentially expressed genes belonging to several apoptotic pathways: BCL2 with its interacting proteins BAG4 and BNIP3L, Fas-related proteins (FAIM3, FASTK), TNF mediated apoptosis genes (TNFRSF21, TNFRSF25, TNFSF13, TRADD). BCL2 was overexpressed in MBC, according to data reported elsewhere by IHC [START_REF] Weber-Chappuis | Comparison of prognostic markers detected by immunohistochemistry in male and female breast carcinomas[END_REF], supporting the role of this antiapoptotic gene in carcinogenesis.

Response to steroid stimulus

As expected, genes related to response to steroid hormones were quite different in MBC and FBC. Some genes coding for proteins functionally related to the androgen receptor as RAN, a GTPase able to enhance AR transactivation [START_REF] Harada | ARA24/Ran enhances the androgen-dependent NH2and COOH-terminal interaction of the androgen receptor[END_REF], RNF14, a coactivator interacting with AR signaling in prostate cancer [START_REF] Kang | Cloning and characterization of human prostate coactivator ARA54, a novel protein that associates with the androgen receptor[END_REF] and PAK6, an AR and ER signaling inhibitor [START_REF] Lee | AR and ER interaction with a p21-activated kinase (PAK6)[END_REF][START_REF] Schrantz | Mechanism of p21-activated kinase 6-mediated inhibition of androgen receptor signaling[END_REF] were overxpressed in MBC.

Correlation and clustering analysis

To further highlight similarities and differences between breast cancer in the two genders, we performed a correlation analysis as described. Given the well established role of steroid hormones in breast cancer, we separately searched in male and female breast tumors profiles for genes significantly correlated with AR expression values obtained from array data and PgR expression value obtained by qPCR. The same type of analysis was carried out for the ERBB2 gene, usually amplified in about 20% of FBC with a strong impact on prognosis and therapeutic strategy.

From the number of correlated genes and the overlap between genes correlated in the two genders, we could indirectly extrapolate some hints on how relevant and how similar the biological functions of these three genes were in the two genders. As shown in Table 3, huge differences were found in the numbers of receptor-correlated genes and few of them were common in the two genders.

2288 clones were significantly correlated with ERBB2 in the female samples dataset and only 243 were so in the male dataset with 45 common genes. This strongly suggests a reduced relevance of ERBB2 in MBC biology compared to FBC.

Moreover, 12 out of the 45 common clones map near the ERBB2 locus (Table S1), with increasing correlation values for genes closer to ERBB2, in both genders (Fig. 2). Hence, similarly to what happen in females, also in male breast tumors ERBB2 overexpression is the consequence of a genomic amplification of its locus, according to literature results of FISH analysis in MBC samples [START_REF] Rudlowski | Her-2/neu gene amplification and protein expression in primary male breast cancer[END_REF][START_REF] Fonseca | Evaluation of ERBB2 gene status and chromosome 17 anomalies in male breast cancer[END_REF].

An inverse behavior was observed for AR correlated genes, where only 86 clones were correlated to AR in FBC, against 441 clones in MBC. Interestingly, two genes up-regulated in MBC, BCL2 and GATA3, which are known to be associated to ER in female breast cancer, were slightly inversely correlated with AR in FBC (S=-0.25, p=0.1486 and S=-0.22, p= 0.1374 respectively), but significantly correlated to AR expression in MBC (S=0.43, p= 0.0194 and S=0.41, p= 0.0347).

More PgR correlated genes were found in FBC (582) than in MBC (159) with a limited overlap (26 genes). Thus the PgR gene, more expressed in FBC (p = 0.0006), also seems to play a more important role in these tumors rather than in MBC. This hypothesis was further confirmed by a clustering analysis exploiting independent public data by Chanrion et al [START_REF] Chanrion | A gene expression signature that can predict the recurrence of tamoxifen-treated primary breast cancer[END_REF]. From this dataset 145 ER+ FBC samples with clinico-pathological features comparable to our FBC samples were isolated. About 4000 clones (common to our data) were searched for genes differentially expressed between PgR+ and PgR-tumors. Fourteen clones were called as differentially expressed at FDR=0. As expected, hierarchical clustering of Chanrion's patients using this gene set revealed two main clusters significantly associated to PgR status (p=0.00001, Fig. 3A). The same clustering in our FBC samples also identified two main clusters significantly associated to PgR status (p=0.002, Fig. 3B). Whereas, clustering of MBC samples identified two clusters not at all associated to PgR status (p=0.413, Fig. 3C). This finding further supports the concept that PgR, similarly to ER, AR and ERBB2, may play a different role in MBC and FBC. The inverse approach searching for differentially expressed genes between PgR+ and PgR-in MBC was also performed. Hierarchical clustering with the 74 clones differentially expressed (FDR<5%) identified a sub-cluster containing 7 out of 8 PgR-tumors, while a quite random distribution of PgR-samples was observed both in our FBC dataset and in the Chanrion FBC dataset (Supplementary Fig. S1).

Conclusion

Male breast cancer is a rare disease poorly characterized and treated similarly to female counterpart. A few studies, reported a different expression in male samples of validated breast cancer biomarkers, but a comprehensive molecular characterization of these tumors is missing. At the best of our knowledge, our study is the first reporting gene expression data of male breast cancer specimens compared to samples with similar clinical and patho-biological features of the female counterpart. The aim of the study was to give a genome wide description of these two tumors focusing on main biological relevant discrepancies in order to generate useful hypothesis to better understand MBC biology and to provide a guide for treatment strategies.

Class comparison uncovered profound differences between MBC and FBC transcriptomes, with around 1000 genes called as differentially expressed despite a stringent FDR. Biological interpretation of our results clearly pointed out the relevance of such a difference, as almost all processes we found to be differentially modulated, have a clear and well known role in tumorigenesis and cancer progression. Furthermore in some cases, the observed differences might have strong implications on treatment choice and outcome. For example, inhibition of translation targeting either EIF4E or mTOR has been proposed as a promising new therapeutic strategy [START_REF] Graff | Targeting the eukaryotic translation initiation factor 4E for cancer therapy[END_REF][START_REF] Mamane | mTOR, translation initiation and cancer[END_REF] and an increased susceptibility to such treatment in MBC could be hypothesized based on our data. Our molecular data also suggest that drugs targeting tubulin/microtubules system like the vinca alkaloids and taxanes might also be more effective in MBC due to their overexpression of various tubulin subunits compared to FBC. It might also be hypothesized that MBC could be a preferred target for the newly developed epothilones [START_REF] Hunt | Discovery of ixabepilone[END_REF].

An interesting difference appeared for ERBB2. In female patients amplification of ERBB2 characterizes a subgroup of tumors with a peculiar behavior in terms of aggressiveness and which is treated in different way from tumors without this amplification [START_REF] Ross | The HER-2 receptor and breast cancer: ten years of targeted anti-HER-2 therapy and personalized medicine[END_REF] The strong biological role played by ERBB2 in FBC is reflected by the high number of correlated genes. In MBC a tenfold lower number of genes was found to be correlated with ERBB2, suggesting a minor role for this gene in males and raising doubts about usefulness of treatment strategies targeting ERBB2 in males.

According to our data, the AR could instead be one of the driving genes in MBC biology as also suggested in previous studies [START_REF] Weber-Chappuis | Comparison of prognostic markers detected by immunohistochemistry in male and female breast carcinomas[END_REF]. Thus, a shift towards hormonal therapy targeting AR rather than ER could be envisioned.

The metabolic features of MBC, where the anaerobic glycolisis pathway does not seem to be the preferential glucose metabolic pathway as for most tumors, is also worth to be further investigated. It has biological implication which may be linked with tumorigenesis, as strong activation of oxidative phosphorylation is responsible for production of ROS, but also represent a warning when using diagnostic procedures as positron emission tomography which relay on 18 F-deoxyglucose uptake.

Research recommendations on MBC collected in a specific multidisciplinary meeting were recently published [START_REF] Korde | Multidisciplinary Meeting on Male Breast Cancer: Summary and Research Recommendations[END_REF] and strongly underline that MBC is a unique disease rather than an illness similar to postmenopausal FBC. As the majority of MBC are endocrine responsive the development of endocrine therapy options designed for MBC in specific clinical trials was considered as a priority. However, while waiting for results from large international multicenter studies to learn more on this disease, treatment cannot be simply derived from FBC, but should rely on single center studies like ours which highlight peculiar features of MBC. In this sense our results which suggest a different role of steroid receptors (and ERBB2) in males are a warning in the design of hormonal treatment in MBC.

In conclusion, our data highlighted the need to investigate the link between molecular characterization and outcome in MBC as done for FBC, in order to personalize the therapy and improve patient survival. These preliminary data may trace a path for future studies, however due to the rarity of MBC, studies with a multicenter setting with a standardized biospecimens handling are essential.

LEGENDS

Fig. 1 Regulation of translation initiation mediated by PI3K/AKT/mTOR pathway.

The translation initiation factor EIF4E specifically promotes translation of cancer associated genes. Its activity is regulated by the PI3K/AKT/mTOR pathway, in turn regulated by AMP/ATP ratio. Genes in red were found up-regulated in MBC compared to FBC, and our data suggest a lower AMP/ATP ratio in MBC.

Fig. 2

 2 Fig.2 Correlation pattern of ERBB2 neighbor genesGenes mapping near the ERBB2 locus on chromosome 17 are reported according to their correlation with ERBB2 in FBC (A) and MBC (B) samples. Genomic coordinates (Mb) are reported on x-axis. Correlation values increase for genes closer to ERBB2, in both MBC and FBC samples.

Fig. 3

 3 Fig.3 Clustering analysis with PgR associated genesUsing a set of clones differentially expressed between PgR+ and PgR-FBC samples in the Chanrion dataset[START_REF] Chanrion | A gene expression signature that can predict the recurrence of tamoxifen-treated primary breast cancer[END_REF], a hierarchical clustering analysis was performed on the Chanrion dataset itself (A) as well as on our dataset, separately for FBC (B) and MBC (C) samples. Significant associations between the two main clusters (red and blue) and the distribution of PgR status were observed in FBC datasets but not in the MBC dataset.

Fig. S1 Tab. 2 -

 S12 Fig.S1 Clustering analysis with PgR associated genes Using a set of clones differentially expressed between PgR+ and PgR-MBC samples, a hierarchical clustering analysis was performed on the MBC dataset itself (A) as well as on our FBC dataset (B) and on the Chanrion FBC dataset (C) samples.A sub-cluster containing 7/8 PgR-tumors was noticed in the clustering of MBC samples, while a quite random distribution of PgR-tumors was observed in FBC datasets.

  Tumori of Milan during the period 1991-1997. All patients signed an informed consent to donate to the Istituto Nazionale dei Tumori the leftover tissue after completing diagnostic procedures. Fifty-three FBC with ER+ primaries for whom

gene expression profiles were available were selected to obtain a distribution of the main clinical and patho-biological variables (age, tumor size, PgR status) comparable to that of MBC samples, limiting in this way confounding effects on comparison of gene expression data between the two genders. Clinical and patho-biological data are summarized in Table

Acknowledgments:

Supported in part by Associazione Italiana per la Ricerca sul Cancro (PIs, MA Pierotti and MG Daidone), Progetto Integrato Oncologia from the Italian Health Ministry (PI, MG Daidone), Alleanza Contro il Cancro (PI, MG Daidone).

Competing interests

The Authors declared that they have no competing interests