Vilius Dranseika
email: vilius.dranseika@fsf.vu.lt

Eugenijus Gefenas

Asta Cekanauskaite

Kristina Hug

EIMANTAS PEICIUS, VENTS SILIS Signe Mezinska

Eimantas Peicius

Vents Silis

Andres Soosaar

Martin Strosberg

20 Years of Human Research Ethics Committees in the Baltic States

Keywords: research ethics committees, transparency, ethical review, Baltic States Research ethics committees, transparency, ethical review, Baltic States

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

eagerly anticipating the first chance to join international research.2 However, even though the Baltic States share many socio-economic characteristics, all three have developed rather different institutional models of ethical review, despite their common motivation to facilitate local researcher involvement in international research.

In Estonia, ad hoc committees for clinical trials were created as early as 1989. 3 The first permanent RECs were established in 1990 by the University of Tartu and in 1992 by the Institute of Experimental and Clinical Medicine (now National Institute for Health Development) in Tallinn. These two RECs, affiliated with and dependent upon mother institutions, also perform functions of regional RECs since they also review research proposals from outside their own institutions. A national bioethics board, the Estonian Council on Bioethics, was established in 1998. One of its tasks is to The emergence of the Lithuanian ethical review system was similar to that of Estonia. Two RECs were created in the late 1980s as a consequence of local research institution initiative, and not as a realization of some national policy. In 1994, the Law on the Health System 4 was adopted, thus setting the conditions for the official establishment of the national REC, the Lithuanian Medical Ethics Committee (now Lithuanian Bioethics Committee) a year later. The Committee functions as a national bioethics council and also has a subcommittee for ethical review of biomedical research. In 2001, the Law on Ethics of Biomedical Research 5 introduced a two-tier system with regional RECs. The Kaunas Regional Biomedical Research Ethics The situation in Latvia is more complex than in the other two countries.

There are several kinds of RECs with different jurisdictions and scopes of operation. There is a national body, the Central Medical Ethics Committee, and there are other RECs of two kinds: institutional ones and those which are sometimes referred to as 'regional'. 8 However, these so-called 'regional' committees are not assigned to any particular region, all are located in Riga, Currently all three systems of ethical review can be seen as structures that include a national body and local or regional committees established at various institutions that engage in biomedical research. It is only in

Lithuania and Estonia where such local or regional committees are assigned geographically defined jurisdictions although some Latvian committees could be regarded as 'regional committees' in that they review research proposals from outside their founding institution even though there is no defined geographic region.

The relationship between national and local or regional RECs differs a lot across the Baltic States. For example, Estonian RECs simply provide to the INSERT Figure 1 INSERT Figure 2 INSERT Figure 3 Figures A HEAD [CONCLUDING REMARKS

The experience of the Baltic States, as transition societies, may well be relevant to other transition societies. Over the past two decades, the ethical review of human research in the Baltic States has undergone significant legal and institutional development, with each country evolving in a separate direction. In many ways, the resulting systems adhere to international standards. However, problems remain. For example, the limited transparency and procedural clarity of ethical review hinder the creation of a quality research environment. RECs should be encouraged to increase the availability of data on reviewed protocols on their websites.

New methods to manage conflicts of interest need to be created taking into account the influence that the structure of RECs has on those conflicts.

More effort should be made to establish the optimal REC model in terms of composition of committees and motivation of their members. The legal environment should also be improved. Networking among RECs could be a potent tool to encourage the above-mentioned processes.

Acknowledgement HEAD [ACKNOWLEDGMENTS

  twofold:(1) to present a general overview of ethical review of human research in the Baltic States and (2) to identify some of the problems related to the role of ethical review in establishing a high quality research environment. We believe that this overview may provide some important insights into the processes of ethical review and the protection of research participants not only in the Baltic States but also in other transition societies. A HEAD [HISTORICAL AND STRUCTURAL OVERVIEW The emergence of ethical review of human research in the Baltics, which began in late 1980s, can be traced back to certain individuals who were knowledgeable about the workings of ethical review in the West and were by this survey is very recent (report was published in 2007 (EFGCP Ethics Working Party on the Structure and Function of Research Ethics Committees in the European Union. The Procedure for the Ethical Review of Protocols for Clinical Research Projects in the European Union. Int J Pharm Med 2007; 21: 1-113.); updates on the website are correct as of 2009 (EFGCP. 2009. Update of the Report, as of 2009. Brussels: EFGCP. Available at: http://www.efgcp.be/html.asp?what=efgcpreport.htm&L1=5&L2=1 [Accessed 31 Mar 2010].)) and comprehensive, it covers only a limited area of human research because drugunrelated clinical trials as well as other types of biomedical research are not covered by the EU Clinical Trials Directive (European Commission, Directive 2001/20/EC of 4 April 2001, of the European Parliament and of the Council on the approximation of the laws, Regulations and administrative provisions of the Member States relating to implementation of good clinical practice in the conduct of clinical trials on medicinal products for human use. OJ L 121, 1.05.2001, p.34.) that the EFGCP activities are based on.

  regional RECs. A special REC to review research conducted within the Estonian Genome Project was established in 2001 but its functions were later passed to the Ethics Review Committee on Human Research of the University of Tartu. In 2008, a special kind of REC, reviewing only matters related to research with medical data from Estonian e-health databases, was established. It is called the Ethics Committee of the Health Information System.

  reports about main facts and trends of their operation.The RECs themselves have taken the initiative. While in Lithuania, the division of responsibilities and accountability between national and local bodies is clearly defined in different normative documents.All three national bodies (the Estonian Council on Bioethics, the Latvian Central Medical Ethics Committee and the Lithuanian Bioethics Committee) are state institutions. For example, the Lithuanian Bioethics Committee is an institution subordinate to the Ministry of Health. However, national bodies differ in their functions. For example, it is only the Lithuanian committee that supervises the rest of the committees and reviews their decisions upon appeal. The Lithuanian and Latvian committees differ from their Estonian counterpart in that they review research protocols.Before the introduction of the two-tiered system, the Lithuanian committee was the sole reviewer in the country. It is still the only body that issues decisions on clinical drug trials and on biomedical research studies that take place in more than one region. The Latvian committee reviews: (a) research proposals that are related to the National Genome Project and (b) non-drug trials. The Estonian Council on Bioethics does not review research.

4

  Seimas of the Republic of Lithuania. 1995. Republic of Lithuania Law on the Health

	System.	Parliamentary	Record, 12.01.1995, No.	12.	Available	at:
	http://www3.lrs.lt/pls/inter3/dokpaieska.showdoc_e?p_id=23358 [Accessed 31 Mar 2010].
	5 Seimas of the Republic of Lithuania. 2000. Republic of Lithuania Law on Ethics of
	Biomedical		Research.	Available		at:

http://www3.lrs.lt/pls/inter3/dokpaieska.showdoc_e?p_id=148740 [Accessed 31 Mar 2010].

Page 6 of 25 Developing World Bioethics Developing World Bioethics

	Latvia's capital, and the law does not require that applications for ethical
	review be submitted according to region of origin. It would be more
	appropriate, therefore, to distinguish between RECs with a narrow scope of
	review and RECs with a wide scope of review. Three committees review
	only clinical drug trials, one reviews drug trials and other types of human
	biomedical research, while four review all types of biomedical research,
	except clinical drug trials.
		F o r
			P
			e
			e r
				R
				e
				v i e
					w
	6 Latvijas Republikas Saeima (Saeima of the Republic of Latvia). 1998. Ārstniecības likums
	(Medical Treatment Law). Latvijas vēstnesis, 01.07.1997, Nr. 167. Available at:
	http://www.likumi.lv/doc.php?id=44108 [Accessed 31 Mar 2010].
	7 Latvijas Republikas Ministru kabinets (Cabinet of Ministers of the Republic of Latvia).
	1998. Centrālās medicīnas ētikas komitejas nolikums (Statutes of Central Medical Ethics
	Committee).	Latvijas	Vēstnesis,	15.01.1998,	Nr.	10.	Available	at:
	http://www.likumi.lv/doc.php?id=46597 [Accessed 31 Mar 2010].
	8 Eg. Privireal. 2005. Latvia -RECs and Medical Research. Sheffield, UK: Privireal.
	Available at: http://www.privireal.org/content/rec/latvia.php [Accessed 31 Mar 2010].
				7

In all three Baltic States, ethical review of human research developed in bottom-up fashion. Initially RECs were established in particular university hospitals or medical faculties and only then were legal regulations put in place. Consequently the institutional structure of ethical review was reshaped by legal developments.

  the list of approved and rejected research projects), in many cases, is not publicly available. 10 Sometimes this information is not available even if relevant institutions are contacted directly. The limited 10 An exception is the website of the Lithuanian Bioethics Committee (Available at: The Ethics Review Committee on Human Research of the University of Tartu has published an annual list of all approvals since 2008; see Tartu Ülikooli inimuuringute eetika komitee (Ethics Review Committee on Human Research of the University of Tartu). 2009. Tartu Ülikooli inimuuringute eetika komitee menetlus 2008 (Approvals given by the Ethics Review Committee on Human Research of the University of Tartu in 2008). Ethical review by types of human research Procedural clarity and the scope of ethical review may differ for different types of human research. Some types of human research may even escape ethical review altogether. Although, as previously mentioned, clinical drug trials enjoy special status in all three countries, due to the provisions of Directive 2001/20/EC, 21 the situation is less uniform in other cases. It is only in Lithuania where a legal definition of biomedical research, and the requirement to subject such research to review by REC, exists. The Law on Ethics of Biomedical Research defines biomedical research as 'verification of hypotheses of biomedicine by methods of scientific investigation and development of knowledge about characteristics of human health'. 22 This broad definition includes research 'carried out in individuals or their groups,20 This also leads to the issue of rotation of REC members. Lack of potential candidates may be dealt with by having no formal term limits for an REC member. For example, in Estonia, term limits were introduced only in 2007. foetus, tissues, organs, cells and genetic material, cadavers and medical documents'.23 Neither Latvian nor Estonian national law includes a definition of biomedical research or clarifies why, in practice, some kinds of research involving human subjects require ethical review by RECs and others do not. Sometimes specifications can be found in the statutes of particular RECs established at academic institutions, but sanctions for noncompliance with these regulations are limited to the jurisdiction of that institution. For example, it seems that no sanctions could be applied to researchers who are affiliated with institutions that do not have a REC (e.g. any Estonian research institution except the University of Tartu and the

	Page 13 of 25 Page 17 of 25	F o r P e e r R e v i e w the fact that a basic legal and institutional structure exists, does not automatically guarantee high quality ethical review. Although there can be no guarantees, there could be more confidence in a review process that at a minimum required: transparency in reporting REC activities and findings; clear, precise and justifiable rules and principles governing committee processes; specification of the composition and required qualifications for committee members, requirements for reporting of research process and Developing World Bioethics F o r P e e r R e v i e w F o r P e e r R e v i e w research by providing information on approved research projects on their own websites. 12 B HEAD [Conflicts of interest The management of conflicts of interest has received only limited attention in laws and regulations. The Baltic systems of ethical review of human research rely solely on voluntarily disclosure of potential conflicts of at: P e e r R e v i e w B HEAD [Committee composition and motivation of REC members RECs in the Baltic States usually include 7 to 15 members, most of whom have medical qualifications and affiliations. Selection and composition criteria vary significantly among committees in regard to their specificity in P e e r R e v i e w clear. Furthermore, except for being delegated by one of these Committees). Valstyb÷s žinios, 2008-06-17, Nr. 69-2635. Available at: http://www3.lrs.lt/pls/inter3/dokpaieska.showdoc_l?p_id=322379&p_query=&p_tr2= [Accessed 31 Mar 2010]. 16 Lietuvos bioetikos komitetas (Lithuanian Bioethics Committee). 2007. Lietuvos bioetikos komiteto pirmininko įsakymas V-8 D÷l Lietuvos bioetikos komiteto biomedicininių tyrimų ekspertų grup÷s darbo reglamento patvirtinimo (Decree of the Lithuanian Bioethics Committee no. V-8 on the Working Procedures of the Biomedical Research Experts' Group). Valstyb÷s žinios, 2008-02-28, Nr. 24-893. Available at: P e e r R e v i e w P e e r R e v i e w P e e r R e v i e w National Institute for Health Development) for failing to seek ethical review. 24 At the moment, according to law in the Baltic States, non-biomedical human studies do not fall within the scope of REC approval. Ethical review is not required for conducting sociological, anthropological or psychological research outside of healthcare context. When there are no legally binding 23 Ibid: Art 3.1 24 For Estonia, some guidance can be found in the Oviedo Convention (Council of Europe. 1997. Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine. Available at: http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm P e e r R e v i e w Despite differences in REC systems and existing practices, we suggest that closer networking and cooperation of RECs in the Baltic region would help to reduce problems related to the quality of the research environment and open up possibilities for the advancement of research ethics both on the regional and national levels, including the development of common guidelines for ethical review and the joint training of REC members. Baltic 25 Human research conducted by students in educational setting may also involve significant risks to research subjects and therefore should receive ethical scrutiny. However, it may be impracticable to apply stringent procedures to student research. There P e e r R e v i e w interest. Available Developing World Bioethics F o r discussion is whether RECs that are affiliated with research institutions and draw most of their members from that institution (similar to IRBs in the USA) can avoid conflicts of interest. Another potential problem is that funding of RECs in Estonia and Latvia is directly dependent on the number of reviewed protocols. 14 The Lithuanian system follows a redistributive model, where the fees are first paid to the Developing World Bioethics F o r although composition criteria for Lithuanian regional RECs are defined in detail by special ministerial decree, 15 most committees rely on general principles. For example, the only requirement for the composition of the subcommittee for ethical review of biomedical research of the Lithuanian Bioethics Committee is to keep the balance between biomedical and non-biomedical representation. 16 Also, although the membership of the Latvian Central Medical Ethics Committee must represent 11 organizations (Ministry of Health, Council of Science, University of Latvia Institute for Experimental and Clinical Medicine, Latvian Nurses Association, Latvian Pensioners Federation, etc.), 17 the rationale for their membership is not Developing World Bioethics F o r F o r of the education is acquired through practical work on the committee. Some members participate in conferences or workshops, but this is not done in any systematic or coordinated way. Lack of respect by academic researchers for the requirement of ethical review can also prevent qualified scientists from working on RECs. Many perceive the process as slowing down and hindering scientific research. In general, the lack of motivators can result in both poorer quality of ethical reviews and a reduction in the number of interested potential committee candidates. 20 Developing World Bioethics F o r Developing World Bioethics F o r requirements, ethical review may be enforced by what might be called universities or research institutes, requirements for authors wishing to publish in scientific journals and guidelines promulgated by different funding bodies sponsoring human research. At least in one case, provisions to cover non-biomedical human research are included in REC policies. According to the statutes of Ethics Review Committee on Human Research of the University of Tartu, the REC shall also assess the ethical aspects of human research if a danger to the physical or mental health of human(s) may occur while conducting the aforementioned research. 25 Developing World Bioethics F o r RECs have already experienced positive results of such networking over the B HEAD [Page 16 of 25 Page 10 of 25 Page 12 of 25 Page 14 of 25 Page 18 of 25 'softer' social regulations, such as policies of RECs established at the past few years. 26
		http://www.ut.ee/orb.aw/class=file/action=preview/id=595718/Ec_2008_kokkuv6te.pdf defining the qualifications of individual members and in the requirements http://www3.lrs.lt/pls/inter3/dokpaieska.showdoc_l?p_id=315164&p_query=&p_tr2= [Accessed 31 Mar 2010].). This treaty has been ratified by Estonia and Lithuania but not is no definition of student research in Lithuanian, Latvian or Estonian legislation. Most
		[Accessed 31 Mar 2010]. for representation from various bodies and organizations. For example, [Accessed 31 Mar 2010]. Latvia. The Convention states that human scientific research in the field of biology and
		biomedicine cannot be conducted without 'multidisciplinary review of its ethical
		acceptability' (Art 16.3).
		10 Developing World Bioethics 12 Developing World Bioethics 13 Developing World Bioethics 14 Developing World Bioethics 16 Developing World Bioethics Developing World Bioethics 18 Developing World Bioethics 19

1-3 depict the organizational structure of ethical review in the Baltic States. Solid lines show which institutions issue approvals (or recommendations to issue approvals) for particular types of research. Punctuated lines show accountability of institutions. Dashed lines (in Lithuanian figure only) show recommendations to other committee. A HEAD [ETHICAL REVIEW AND QUALITY RESEARCH ENVIRONMENT As reflected in Figures 1-3, the Baltic States have developed the legal and institutional structure for the ethical review of human research. However, 9 European Commission, op. cit. note 1.

findings; reliable funding and facilities for the committee; and legal protection of intellectual property. In this section we will briefly address several problematic areas connected to the quality of ethical review. These include: (a) availability of information, (b) management of conflicts of interest, (c) committee composition and motivation of REC members, and (d) differing stringency of ethical review for different types of studies.

B HEAD [Availability of information

Very few RECs in the region have websites that provide information about their procedures of functioning. Information on statutes, de facto composition, basic statistics on the number of reviewed research protocols (not including http://bioetika.sam.lt

[Accessed 31 Mar 2010]

). This institution also has an official template for information requests and is obliged to answer inquiries. However, even here information sometimes is not regularly updated. For example, at the time of this research, statistics on the number of reviewed protocols had not been updated since

mid-2005. 13

This is not very troubling by itself -voluntarily disclosure is a rather widespread model of managing conflicts of interest in a number of different countries. However, most of the RECs in the Baltic States are units within universities or medical schools, and many or even most of their members are representatives of the same institution that conducts research projects. In addition, biomedical communities are rather small and compact and subject to subtle peer pressure. Therefore it is very difficult to determine the extent to which decisions of RECs are made independently of the interests of researchers and sponsors, how often conflicts of interest occur, how serious they are, and how are they avoided in practice. Open for 12

13

For example, the Statutes of Tallinn MREC [provided by the REC] simply state that members who are not 'independent from the performers of the research' may not vote. budget of a third party (i.e. the state), from which the committees are financed. Arguably, the former system may be less likely to issue negative decisions, especially in cases where researchers are free to choose the REC.

In Lithuania and Estonia, if approval is denied by a REC it is not possible to seek approval for the same protocol from another REC. For example, Estonian RECs, on a quarterly basis, exchange lists of applications to help eliminate 'shopping for RECs'. However, in Latvia, where different RECs are not assigned defined geographical regions, there is no system to exchange information on refused applications. Therefore a protocol refused by one REC can, at least in theory, be submitted to another.

14

In Latvia, it is true only of those RECs that review clinical drug trials. 15 Lietuvos Respublikos Sveikatos apsaugos ministerija (Ministry of Health of the Republic of Lithuania). 2008. Lietuvos Respublikos sveikatos apsaugos ministro įsakymas d÷l regioninių biomedicininių tyrimų etikos komitetų narių skyrimo tvarkos aprašo patvirtinimo (Decree of the Minister of Health of the Republic of Lithuania on the Procedure of Nomination of the Members of Regional Biomedical Research Ethics 17 Latvijas Republikas Ministru kabinets (Cabinet of Ministers of the Republic of Latvia), op. cit. note 7. Art. 3. 21 European Commission, op. cit. note 1. 22 Seimas of the Republic of Lithuania, op. cit. note 5, Art 2.1 a medical schools in the Baltic States have internal regulations concerning the need for ethical review of student research. These internal regulations are based on local initiative not on overarching national policies.

Table 1 :

 1 Workload of RECs in the Baltic States (excluding student research)

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

26

Information on the Baltic research ethics network activities can be found on the website of Lithuanian Bioethics Committee. Available at: http://bioetika.sam.lt/index.php?-809972162 [Accessed 31 Mar 2010]).

Page 25 of 25 Developing World Bioethics Developing World Bioethics

As is indicated by Lithuanian bioethicists, '[t]he process of development of ethical review in Lithuania (and probably many other Central and Eastern European countries) […] was strongly facilitated by foreign pharmaceutical companies.' A. Cekanauskaite, E. Gefenas. 2005. Research Ethics Committees in Lithuania. In Beyleveld et al. eds. op .cit. note 1:140-147, p. 141. Similar views were expressed by Estonian and Latvian experts as well. In the questionnaire received from one of the Estonian RECs, it was indicated that the motive for the establishment was to embrace 'new possibilities to take part in international medical research. [...] The international requirements demanded independent body for the overview of research projects.' One of the Latvian RECs also reported that the main motive was 'the necessity to review the research involving human participants, as well as rapid development of drugs clinical research'.

T. Veidebaum. 2005. Research Ethics in Estonia. In Beyleveld et al. eds., op .cit. note 1, pp.

41-43: 41.

organizations, no formal individual qualifications are defined. In most cases, however, criteria are stated relatively vaguely. For example, the statutes of

The project was supported by Research Grant # R25TW007085 from the Fogarty International Center. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Fogarty International Center or the National Institutes of Health.

We would also like to thank the RECs which kindly agreed to present empirical material via completed questionnaires and an anonymous reviewer for valuable suggestions on how to make this paper clearer.

BIOG [BIOGRAPHY

Vilius

Dranseika (MPhil) is a researcher at the Department of Medical History and Ethics (DMHE) at the Vilnius University, Lithuania. Eugenijus Gefenas (MD, PhD) is the head of DMHE, Vilnius University and is a member of the Lithuanian Bioethics Committee. Asta Cekanauskaite (MPhil) is a researcher at DMHE, Vilnius University and is a member of the Lithuanian Bioethics Committee. Kristina Hug (MA) is a doctoral researcher at the Department of Medical Ethics, Lund University, Sweden. Signe Mezinska (MPhil) is a doctoral candidate in the Riga Stradins University (RSU), Latvia. Eimantas Peicius (PhD) is a lecturer at the Department of Philosophy and Social Sciences, Kaunas University of Medicine, Lithuania and is also a member of the Kaunas Regional Biomedical Research Ethics Committee. Vents Silis (MPhil) is a researcher and lecturer at the Department of Humanities at RSU. Andres Soosaar (MD, PhD) is a lecturer at the Department of Public Health, University of Tartu, Estonia and a member of the University of Tartu Human Research

Developing World Bioethics Developing World Bioethics Developing World Bioethics Developing World Bioethics Developing World Bioethics Developing World Bioethics

availability of information adds to low public and professional awareness of RECs.

In general, there is growing international support for the dissemination of information on clinical trials via publicly available clinical trial registries and databases. 11 However, publicly accessible registries and databases are typically lacking in smaller countries. In their absence, RECs might become vehicles for improving the publicity and transparency of biomedical (Helsinki: 1964; amended Tokyo: 1975; Venice: 1983; Hong Kong: 1989; Republic of South Africa: 1996; Edinburgh: 2000; Washington: 2002; Tokyo 2004; Seoul 2008) The definition of 'necessary expertise' is not further specified.

The RECs themselves have identified the problem of insufficient motivation of members. Reading the protocols and attending meetings is time consuming and usually members are not adequately paid for the work, causing difficulty in recruiting highly qualified professionals. 19 The lack of financial incentives is not outweighed by other types of motivation, such as professional development, prestige or social status. For example, there are no systematic training programs for the members in the Baltic States. Most


Graham ; Betton

Lydia Searchfield

Sally A Price

Bharat Jasani

Daniela Riccardi
email: riccardi@cf.ac.uk

David Fr Griffiths
email: griffithsdfr@cf.ac.uk

Dr F R David

Dr Daniela Griffiths

Riccardi

Glutathione S-transferases as molecular markers of tumor progression and prognosis in clinically confined renal cell carcinoma

Keywords: Glutathione S-transferase, renal cell carcinoma, tissue microarray, prognostic indicators, immunohistochemistry Glutathione S-transferases, renal cell carcinoma, tissue microarray, prognostic indicators, western blotting, immunohistochemistry Deleted: , even after surgical resection. Therefore,

Aims.

Renal cell carcinoma (RCC) often recurs as distant metastasis; there is thus a need for new indicators to identify high-risk patients. Glutathione S-transferases (GST) -α and -π are involved in the renal bioactivation of toxic metabolites. We investigated whether their expression could hold diagnostic and prognostic value.

Methods and Results.

Western blotting of microdissected normal kidney and immunostaining of histological RCC microarrays shows expression of GST-α in proximal tubular cells while GST-π was found in the distal nephron. Of the primary 174 RCC cases examined, GST-α immunoreactivity was restricted to conventional RCC (n = 76, 68% positive) and was not seen in any other RCC sub-types. The cross-tabulation of the GST-α scores with other prognostic indices demonstrated that GST-α immunostaining was significantly more frequent in low grade tumours (χ 2 : p < 0.004), and that conventional GST-α positive RCC patients had a mean disease-free survival of 6.0 years (95% CI 5.33 -6.63), compared with 4.7 years (3.54 -5.90) in GST-α negative tumours (Kaplan-Meier survival analysis, p = 0.011, log rank test).

Conclusions.

GST-α is a highly specific diagnostic marker for primary conventional RCC, where it is a prognostic marker if grade is omitted from the multivariate analysis.

Introduction

Renal cell carcinoma (RCC), the most common adult renal neoplasm accounts for approximately 3% of cancers and 2% of all cancer-related deaths [START_REF] Chuang | Overexpression of Glutathione S-Transferase α in Clear Cell Renal Carcinoma[END_REF]. It has been reported that 50-60% of RCC patients will either present with, or develop metastasis, because RCC is difficult to diagnose in the early stages due to the patients being asymptomatic [START_REF] Llungberg | Prognostic Markers in Renal Cell Carcinoma[END_REF]. Furthermore, stage, grade, presence of vascular invasion and histological subtype are the accepted prognostic indicators, but these parameters are notoriously inaccurate to provide confident prognostication and therapy selection [START_REF] Park | Prognostic Factors in Renal Cell Carcinoma[END_REF]. Therefore, there is the need to identify more accurate molecular markers for the precise diagnosis of RCC and for the assessment of the risk of disease re-occurrence [START_REF] Kane | Renal Cell Cancer Stage Migration[END_REF] to form the basis for future treatments.

Oxidative and electrophilic stress play a key role in RCC growth and progression [START_REF] Lusini | Altered glutathione anti-oxidant metabolism during tumor progression in human renal-cell carcinoma[END_REF][START_REF] Pljesa-Ercegovac | Altered antioxidant capacity in renal cell carcinoma: Role of glutathione associated enzymes[END_REF] and humans have an array of anti-oxidants and enzymes to limit the damage caused by certain xenobiotics and carcinogens. Glutathione S-transferases (GSTs) play an essential role in this detoxification process [START_REF] Zhu | Coordinated upregulation of a series of endogenous antioxidants and phase 2 enzymes as a novel strategy for protecting renal tubular cells from oxidative and electrophilic stress[END_REF]. They belong to a supergene family of ubiquitous dimeric enzymes that conjugate glutathione with electrophilic substrates, including potential alkylating agents where they are eliminated. The resulting S-conjugates are water-soluble, and are transported via the circulation to the kidney [START_REF] Habig | Glutathione S-Transferases -The first enzymatic step in maercapturic acid formation[END_REF]. The resulting glutathione conjugates can be metabolically activated in the kidney by gamma-glutamyltransferase and beta-lyase to create cytotoxic/carcinogenic metabolites, acting on the proximal tubule, the main site of toxicity [START_REF] Dekant | Bioactivation of nephrotoxins and renal carcinogens by glutathione S-conjugate formation[END_REF]. In these circumstances, increased GST expression levels have been associated with a protective effect against the toxicity evoked by certain alkylating agents. However, because the kidney metabolises many of these S-conjugates, higher activity of GSTs have been reported in RCC and is believed to account for the formation of even more reactive glutathione S-conjugates. These conjugates can ultimately cause renal damage directly [START_REF] Karami | Renal cell carcinoma, occupational pesticide exposure and modification by glutathione S-transferase polymorphisms[END_REF]. Indeed, it has been previously shown that GSTs are involved in the renal bio activation of toxic metabolites [START_REF] Lusini | Altered glutathione anti-oxidant metabolism during tumor progression in human renal-cell carcinoma[END_REF][START_REF] Pljesa-Ercegovac | Altered antioxidant capacity in renal cell carcinoma: Role of glutathione associated enzymes[END_REF][START_REF] Savic-Radojevic | Glutathione S-transferase-P1 expression correlates with increased antioxidant capacity in transitional cell carcinoma of the urinary bladder[END_REF] and that selective toxicity and carcinogenicity by the proximal tubular cells can be accounted for the active accumulation of toxic glutathione S-conjugates [START_REF] Dekant | Bioactivation of nephrotoxins and renal carcinogens by glutathione S-conjugate formation[END_REF]. Moreover, GSTs also inactivate several anticancer drugs (i.e., adriamycin, mitoxantrone and melphalan) (12 -14), and their overexpression is believed to exacerbate disease severity and progression. Whether due to direct target of xenobiotics or to their ability to bioactivate While the involvement of GSTs in the pathogenesis or progression of RCC is undisputed, there is discrepancy in the reported levels of expression of the different GST isoforms in renal cell tumours. For instance, some studies have reported over-expression of GSTs in RCC [START_REF] Chuang | Overexpression of Glutathione S-Transferase α in Clear Cell Renal Carcinoma[END_REF][START_REF] Lui | Immunohistological analysis of chromophobe renal cell carcinoma, renal oncocytoma, and clear cell carcinoma[END_REF], while others a decrease in their expression [START_REF] Howie | Glutathione S-Transferase and glutathione peroxidase expression in normal and tumour human tissue[END_REF][START_REF] Klone | Decreased expression of the Glutathione S-transferase alpha and pi genes in human renal cell carcinoma[END_REF]. Thus, it is currently difficult to establish conclusively the possibility of using GST levels as tools for diagnosis, subclassification and disease progression.

The normal human kidney expresses high levels of cytosolic GSTs [START_REF] Sweeney | Glutathione S-tranferase M1, T1 and P1 polymorphisms as risk factors for renal cell carcinoma: a case control study[END_REF]. GST-α is predominantly expressed in the proximal convoluted tubule while GST-π is expressed in the distal convoluted tubule and collecting duct [START_REF] Simic | Isoenzyme profile of glutathione S-transferases in human kidney[END_REF][START_REF] Simić | Glutathione S-transferase isoenzyme profile in non-tumor and tumor human kidney tissue[END_REF]. The proximal tubule is the origin of conventional RCC [START_REF] Van Den Berg | Kidney: Clear cell renal cell carcinoma[END_REF]. It is in this nephron segment that GST-α is specifically and selectively localised, suggesting a potential (although not exclusive) association with this type of renal cancer. In contrast, GST-π is expressed in the distal nephron [START_REF] Simic | Isoenzyme profile of glutathione S-transferases in human kidney[END_REF][START_REF] Simić | Glutathione S-transferase isoenzyme profile in non-tumor and tumor human kidney tissue[END_REF] and it is well established that its expression is increased in the majority of human renal tumours [START_REF] Simić | Glutathione S-transferase isoenzyme profile in non-tumor and tumor human kidney tissue[END_REF][START_REF] Sárváry | Diagnostic value of glutathione-Stransferase[END_REF]. Furthermore, human-derived cell lines selected for studies of resistance to anticancer drugs over express GST-π [START_REF] Hayes | The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance[END_REF][START_REF] Wang | Human renal UOK130 tumour cells: A drug resistant cell line with highly selective over-expression of glutathione S-transferase-π isozyme[END_REF]. All of these observations point to the possibility that GST-α and GST-π may be useful to discriminate the different types of RCC and that they could potentially be used to predict disease outcome.

The aim of this study was, therefore, to evaluate the immunohistochemical distribution profile of GST-α and GST-π in normal human kidney and in different types of RCC, with the view of assessing whether GSTs hold any value as molecular markers for RCC, and to test their ability to predict long-term prognosis. Samples of cortex, medulla and papilla were dissected from non-neoplastic kidney tissue.

Materials and Methods

Tissue

Half of the samples were fixed in buffered formalin and routinely processed into paraffin wax and the other half were frozen in liquid nitrogen and stored at -70 ºC until processed for western blotting.

To study the intra-renal distribution of GSTs, RCC tumour tissue arrays from a series of 174 surgically resected kidney samples from two hospitals were used. The series has been described in detail previously [START_REF] Thomas | Analysis of the prognostic implifications of different tumour margin types in renal cell carcinoma[END_REF]. Briefly, the array is derived from sequential localised (non-metastatic) tumours surgically resected between 1989 and 1998 from patients followed up for up to 10 years. Pathological data on each case included: histological type according to the Heidelberg classification; size, Furhman grade, level of vascular invasion (IVC, renal vein or microvascular) stage (IUCC 1997) and cellular invasion of fat [START_REF] Fuhrman | Prognostic significance of morphological parameters in renal cell carcinoma[END_REF][START_REF] Kovacs | The Heidelberg classification of renal cell carcinomas[END_REF]. The median age of the patients was 65 (34-88) years; 119 of the patients were men and 55 women.

Conventional (clear cell) carcinomas accounted for 119 of the tumours, 23 were papillary, 5 chromophobe and 27 of the tumours were unclassified by conventional histology. There were 12 conventional, 2 papillary and 5 unclassified tumours with sarcomatoid features. Complete clinical follow-up was carried out as previously described [START_REF] Griffiths | Contribution of grade, vascular invasion and age to outcome in clinically localised renal cell carcinoma[END_REF], and the following information being extracted from the patients records: date of birth, sex, date of surgery, date last seen, date of death, cause of death and the date on which recurrent or metastatic disease was first identified.

Western Blot Analysis

The frozen kidney cortex, medulla and papilla samples were dissected by a pathologist immediately after surgery and individually homogenised in RIPA buffer for 30 seconds. The homogenates were placed on ice for 1 minute and homogenised for further 30 seconds. The samples were then centrifuged at 10,000 x g for 5 minutes to remove insoluble debris. The supernatant was aliquoted and kept at -70 °C until used.

Before the electrophoretic separation, the samples were heated at 70 °C for 10 minutes and reduced using 1X NuPage Reducing Agent (Invitrogen Ltd., Paisley, UK). Proteins were resolved by SDS-polyacrylamide gel electrophoresis using NuPage Novex 10% Bis-Tris gels F o r P e e r R e v i e w 6 (Invitrogen Ltd., Paisley, UK). The gels were transferred onto a PVDF membrane (Invitrogen Ltd., Paisley, UK) using an X-Cell sure lock mini-cell and blot module (Invitrogen Ltd., Paisley, UK) set at 40V for 70 minutes. Transfer efficiency was checked by incubating the blots with 0.1% ponceau S (Sigma-Aldrich, Poole, UK). Subsequently, the blots were rinsed in 1% acetic acid for 10 minutes to remove the stain. Membranes were blocked in Trisbuffered saline-Tween 0.05% with 5% non-fat dried milk (NFDM) overnight at 4 °C. The PVDF blots were incubated for 1 hour at room temperature with a 1:1000 dilution of GST-α mouse monoclonal antibody (Argutus Medical, Dublin, Ireland) and a 1:500 dilution of the GST-π mouse monoclonal antibody (Abcam Inc., Cambridge, MA, USA) diluted in 5% NFDM on a rocking platform. After thorough rinsing, the immunoblots were incubated for 1 hour at room temperature on a rocking platform with horseradish peroxidase-conjugated guinea pig anti mouse IgG antibody (Abcam Inc., Cambridge, MA, USA) diluted in 5% NFDM. The protein bands were detected by chemiluminescence using an ECL Plus western blotting system (Amersham International Plc, Buckinghamshire, UK) and developed on Hyperfilm TM ECL (Amersham International Plc, Buckinghamshire, UK).

Tissue Microarray Construction

A tissue microarray (TMA) was constructed using archived paraffin-embedded tumour samples. Representative peripheral regions of each tumour were identified in haematoxylin eosin-stained sections and marked for sampling with the TMAs. Each individual core was 0.6 mm in diameter and was punched from the identified region for each tumour. The cores were precisely arrayed into recipient paraffin blocks using a Manual Arrayer, Type I, Beecher Instruments (Sun Prairie, WI, USA) and the orientation was recorded. Normal renal parenchyma and cores from human placenta were also included as controls and orientation markers respectively. Full sections of normal kidney (from nephrectomy specimens) were used to assess the normal distribution of the antigens. Envision+ system (DAKO, Ely, Cambridgeshire, UK). Endogenous peroxidase activity within the rehydrated tissue was quenched using the supplied peroxidase block for 5 minutes.

The sections were incubated with the primary antibodies [GST-α (mouse IgG; 1:350 -Argutus Medical, Co. Dublin, Ireland) and GST-π (mouse IgG; 1:3000 -Abcam Inc., Cambridge, MA, USA)] for 1 hour and then washed off using 1X phosphate-buffered saline (PBS) added with 0.05% Tween 20 (3 washes for 5 minutes each). Mouse Horse Radish Peroxidase (HRP) labelled polymer (DAKO Cambridgeshire, UK) was applied to the sections for 30 minutes and the immunoreactivity was visualized using 3,3'-diaminobenzidine (DAB) (DAKO, Ely, Cambridgeshire, UK) as the chromogenic peroxidase substrate. The slides were counterstained with haematoxylin and mounted using DPX mounting medium (BioChemika, Sigma-Aldrich Company Ltd. Dorset, England). A mouse isotype control (DAKO, Ely, Cambridgeshire, UK) was used to replace the primary antibody as a negative control. All immunoreactions were carried out at room temperature.

Scoring of Stained RCC Tissue Microarrays

Evaluation of sections was carried out blinded by the research assistant and the histopathologist over a conference microscope; observers were masked to outcome [START_REF] Campbell | Caveolin-1 overexpression predicts poor disease-free survival of patients with clinically confined renal cell carcinoma[END_REF].

Scoring was agreed by consensus; cases were graded semi-quantitatively as follows: negative, 0: [no detectable reaction product (deposit) in tumour cells]; weakly positive, 1+: very light diffuse or focal deposit in tumour cell cytoplasm, moderately positive, 2+: light diffuse or moderate focal deposit (may include very small areas of heavy deposit); strongly positive, 3+: tumour containing areas of heavy deposit in most or all of the tumour cells.

Data and Statistical Analyses

The association of the positive markers with recognised tumour prognostic variables (grade, stage, size, vascular invasion, capsular invasion and tumour type) was examined by crosstabulation and the chi-squared test or Fisher's exact test, as appropriate. Correction for multiple comparisons was made by the Holm-Bonferroni method. Analysis of disease-free survival of patients with tumours showing different scores of staining for each marker was carried out by Kaplan-Meier method using log-rank test, where the first appearance of a metastasis was considered an event. Patients last seen alive without metastasis or who died due to causes other than RCC were considered censored at the date last seen or date of death, Multivariate survival analysis was carried out by Cox regression using the enter function with covariates considered as categorical. We had already determined that the most influential covariates predicting the disease-free survival of these patients are Fuhrman grade (grade 1 & 2 and grades 3 & 4 are pooled for analysis); any degree of vascular invasion (histological correlate of stage T3b), and histological invasion of perinephric tissue (the histological correlate of stage T3a). When these covariates are taken into account then tumour size and type have no influence on disease free survival [START_REF] Peters | Biochemical characterization of resistance to mitoxantrone and adriamycin in Caco-2 human colon adenocarcinoma cells: a possible role for glutathione S-transferases[END_REF]. The statistical package SPSS 11.5 was used for analysis. All tests were two-tailed.

Results

GST-α and GST-π have a molecular weight of approximately 45kD and are dissociable into subunits of approximately 25kD [START_REF] Habig | Glutathione S-Transferases -The first enzymatic step in maercapturic acid formation[END_REF]. Single bands of predicted molecular weight of ~ 25kD were detected for both GST-α (Figure 1A) and GST-π (Figure 1D). Western analysis demonstrates that GST-α expression is confined to the kidney cortex and outer medulla but it is absent from the papilla (Figure 1A). In contrast, western analysis of equal amounts of kidney cortex extracts revealed that GST-π expression was highest in the papilla and, to a lesser extent, in the cortex and medulla (Figure 1D), with immunoreactive species visible at ~25 and 45 kD. These observations are consistent with the immunohistochemical analysis of normal renal parenchyma that revealed GST-α localization in the cytoplasm of proximal tubule cells (Figure 1A-C). GST-π immnunoreactivity was found within the cytosol of the loop of Henle, distal tubule and collecting duct cells (Figure 1E-F). Both glomeruli and proximal tubules were devoid of GST-π immnunoreactivity. These findings indicate stronger GST-π expression in the distal nephron, an observation which is consistent with previous reports [START_REF] Chuang | Overexpression of Glutathione S-Transferase α in Clear Cell Renal Carcinoma[END_REF][START_REF] Simic | Isoenzyme profile of glutathione S-transferases in human kidney[END_REF][START_REF] Simić | Glutathione S-transferase isoenzyme profile in non-tumor and tumor human kidney tissue[END_REF].

TMAs of clinically confined RCC showed that GST-α is exclusively and specifically expressed in the cytoplasm of clear cell RCC (Figure 2A-D appears to be nuclear staining in all the different scores. Vice versa, GST-α is completely absent from papillary (Figure 2E) and chromophobe (Figure 2F) RCC specimens. We confirmed this in staining whole sections of three chromophobe carcinomas which showed no focal staining (not shown).

In contrast to that observed for GST-α, GST-π immunoreactivity was more widespread and of different degree and intensity. For instance, GST-π signal was specifically detected not only in the cytosol and the nuclei of clear cell RCC (Figure 3A-D), but also in those of papillary and chromophobe specimens (Figure 3E and F, respectively). Thus, while GST-α exhibits a high degree of specificity and is confined to proximal tubular cells of clear cellpositive specimens, GST-π appears to be present all along the nephron and its expression pattern does not appear to have any distinctive feature between the different types of RCCS.

Of the 174 RCC samples on the array examined in the current study, 168 were assessable for GST-α and 155 for GST-π immunostaining. The cross-tabulation of the grade of staining of GST-α and GST-π with the conventional histological prognostic covariates are shown on Tables 1 and2, respectively. Of the 112 conventional RCC cases examined, 76 were positive for GST-α (corresponding to 68% of the total); 22 cases of papillary RCC were examined, 100% of these were negative for GST-α; 5 cases of chromophobe were examined, 100% of these were negative for GST-α; 29 cases were unclassified and 13 of those were positive for GST-α, corresponding to 45%.

The striking result is the strong association of GST-α with low grade (p < 0.001) and conventional type RCC (p < 0.001). These associations retain their significance after correction for multiple comparisons (p = 0.006). Indeed, in other specific types of RCC (papillary and chromophobe) GST-α immunoreactivity was completely absent. This finding prompted histological review of the 13 unclassified RCCs, which showed that in all 13 cases with any staining (grade 1+, 2+ and 3+) for GST-α the most likely histological diagnosis was considered conventional RCC.

Table 2 shows that GST-π did not show any significant association with any of the prognostic parameters. Although there was a trend suggesting it was more commonly expressed in Survival analysis for GST-α was restricted to the conventional RCCs as other specific tumours did not show any immunoreactivity for it. On Kaplan-Meier univariate survival analysis, patients with conventional RCCs that were positive for GST-α had a mean diseasefree survival of 6.0 years (95% CI 5.33 -6.63), compared with 4.7 years (95% CI 3.54 -5.90) for those that were GST-α negative. On log-rank testing this difference is significant (p = 0.011) and demonstrates a clear disease-free survival advantage for conventional RCCs expressing GST-α (Figure 4). The result is also significant if conventional and unclassified RCCs are pooled for analysis (p = 0.002). In contrast, tumours expressing GST-π showed no survival difference when compared with tumours without its expression (not shown).

Previously it has been shown that grade, as well as vascular and capsular invasions, are influential predictors of prognostic in this group of patients [START_REF] Griffiths | Contribution of grade, vascular invasion and age to outcome in clinically localised renal cell carcinoma[END_REF][START_REF] Campbell | Caveolin-1 overexpression predicts poor disease-free survival of patients with clinically confined renal cell carcinoma[END_REF]. The current study shows that, by Cox regression analysis, neither GST-α, nor GST-π constitute significantly influential covariates when including the covariates grade, vascular invasion and capsular invasion. "However, if grade is omitted from the analysis, GST-α adds prognostic information (Table 3)"

Discussion

This study of GST expression in normal human kidney tissue and in a large cohort of specimens from patients with RCC has two main results, both of which might have clinical impact: the strong, specific association of GST-α with conventional (or clear cell) RCC and the ability of GST-α to predict disease outcome in patients affected by this type of tumour.

Our study has clarified and added to knowledge of the distribution of GST-α and GST-π in the normal kidney: in addition to using IHC, we have used tissue specifically taken from the cortex, outer medulla and papilla while others have used homogenates of whole kidney for protein or mRNA analysis. Our results add to previous findings in demonstrating that GST- α is specific for the proximal tubule -in both the convoluted and straight sections (pars recta) and GST-π to the loop of Henle and the distal nephron. As GST-α has a sparse distribution in other normal tissues, being found only in the liver in significant quantities, this may indicate GST-α is likely to be a good proximal tubular marker.

The most striking finding of our study is the strong association of GST-α expression with conventional (or clear cell) RCC; this association is to be expected as conventional RCC is postulated to be derived from the proximal tubular cell epithelium. However, the strength and specificity of this association is surprising. Our finding of 13 out of 29 unclassified RCCs staining for GST-α is explained by the fact that our original categorisation of the tumours was carried out on H&E sections alone with no support from IHC. We tried to ensure that no tumours were misclassified: if there was any doubt as the tumour type on morphological grounds, the tumour was designated unclassified; this was to ensure results were not contaminated by misclassified tumours. Subsequent review of the sections of the unclassified tumours that stained for GST-α revealed that in all these tumours the most likely morphological diagnosis was conventional RRC. Our results thus strongly support the findings of Lui et al., 2007 (15) who, in a carefully categorised group of renal tumours, showed 41/45 (90%) conventional RCC staining for GST-α but no chromophobe carcinomas (22 cases) or oncocytomas (17 cases) (although we cannot comment on the expression of GST-α in oncocytomas as no cases were assessed). In contrast, although Chuang et al., 2005 (1) and Takashi et al., 2003 (30), confirmed GST-α immunoreactivity in a high proportion (90%+) of conventional RCCs, there was some reactivity with papillary (11 out of 54) chromophobe (2 out of 62) and oncocytoma (5 out of 40). Neither of these authors specifies how the original classification of these tumours was ascertained, and given the difficulty in classifying many renal tumours, it is possible that some were misclassified. Further differences between the studies include use of a monoclonal antibody at high dilution (1:300) on a tissue array in our study, whereas others have used polyclonal antibodies at low dilution (1:30 to 1:50) on arrays or whole sections. The main limitations of all the studies are the use of tissue arrays rather that whole sections and the lack of genetic evidence to support the diagnosis of the tumour type. Nevertheless, compared with other antibodies commonly used to differentiate these tumours, GST-α appears more highly specific than vimentin, mRCC 12 and CD10 as a marker for primary conventional RCC [START_REF] Allory | Profiling and classification tree applied to renal epithelial tumours[END_REF][START_REF] Martignoni | CD10 is expressed in a subset of chromophobe renal cell carcinomas[END_REF]. This gives this antibody immediate utility in differentiating conventional RCC from other types of RCC, a finding of clinical importance as some of the new treatments available for RCC are licensed for conventional RCC only.

In contrast with our observations made for GST-α, we did not see major differences in GSTπ expression patterns, or levels, for the different types of RCC although a trend towards significance was achieved for papillary tumours. Our observations therefore suggest that GST-π possesses neither the sensitivity, nor the specificity necessary to confer diagnostic utility.

The second important finding is that loss of GST-α immunoreactivity predicts poor survival in conventional RCC. This has not been reported before and appears related to the strong association of GST expression with better differentiated tumours (p=0.005 after correction for multiple analyses) with less well differentiated tumours losing this specific marker of proximal tubular differentiation. However, when tested in multivariate analysis, this loss of proximal tubular phenotype is not an influential predictor of survival if grade is included in the analysis. This limits its value as a prognostic marker. However, it might have some use in small biopsy material. Furthermore, if prognostic models are constructed that include all available information, a strong GST-α staining tends to support the allocation of a low gradetumour, grading being a procedure subject to substantial inter-and intra-observer variation.

The link with differentiation and prognosis also provided clues about the function of GSTs in tumour progression.

The loss of GST-α expression in poor prognosis conventional RCC may have implications in basic cancer biology and in considering treatment options [START_REF] Lusini | Altered glutathione anti-oxidant metabolism during tumor progression in human renal-cell carcinoma[END_REF]. Loss of GST-α would be accompanied with persistence of oxidative stress, which in turn would result in an increase in genetic instability and clonal selection [START_REF] Toyokuni | Persistent oxidative stress in cancer[END_REF], hereby significantly increasing the risk of tumour progression. Furthermore, as the GST pathway is the main pathway to detoxify carcinogens and drugs, it is possible that changes in GST-α expression may alter the sensitivity of tumours to various drugs [START_REF] Asakura | Relationship between expression of drug-resistance factors and drug sensitivity in normal human renal proximal tubular epithelial cells in comparison with renal cell carcinoma[END_REF]. A better understanding of how GST-α expression is regulated Our study also investigated the expression of GST-π, which has been reported to be the predominant GST isoenzyme in RCC [START_REF] Van Den Berg | Kidney: Clear cell renal cell carcinoma[END_REF][START_REF] Shea | Identification of an anionic form of glutathione transferase present in many human tumors and human tumor cell lines[END_REF]. In the current study, GST-π was expressed in the cytosol, and occasionally the nucleus, of all the specimens examined, observations which are consistent with previous reports [START_REF] Van Den Berg | Kidney: Clear cell renal cell carcinoma[END_REF]. GST-π is a known endogenous inhibitor of the MAPK pathway. It binds c-Jun N-terminal Kinase (JNK) [START_REF] Adler | Regulation of JNK signaling by GSTp[END_REF], which is activated in response to oxidative stress. Thus, the high levels of expression of GST-π could account for the increased resistance to apoptotic cell death [START_REF] Townsend | The role of glutathione S-tranferase in anticancer drug resistance[END_REF].

In conclusion, future studies are required to further the intra-renal GST isoenzyme distribution and their relative expression levels. Such information is required to test whether renal GSTs have any power as diagnostic tools to help sub-classify the different types of RCC, to explain differential prognosis and therapeutic responsiveness and as potential drug targets. Finally, given the availability of commercial kits to measure urinary levels of GST-α, further studies are necessary to determine if loss of GST-α in the parenchyma of more aggressive forms of conventional RCC is associated with an increase in its shedding into the urine. Such an intriguing possibility would elect GST-α as a non-invasive, leaky biomarker to assist in the diagnosis of this type of tumour, as well as other benign conditions associated with proximal tubular damage. GST-α specific immunoreactivity is found within the cytoplasm of cells of the proximal convoluted (B, cortex) and straight tubules (C, outer medulla). Note the absence of GST-α immunoreactivity from the glomeruli (g) and the distal convoluted tubule (d). Cellular GST-π immunoreactivity is more widespread and can be observed in the cytoplasm, apically and basolaterally in cells of both the collecting duct (cd) and loop of Henle (lh) of both the renal cortex (E) and outer medulla (F). Note that π-GST is absent in the glomerulus (g). Scale bar: 200 µm (B and E) or 100 µm (C and F).

  these observations suggest that differential GST expression in the kidney could contribute to disease outcome by either affecting the cancer risk and/or the response to chemotherapeutic agents.

  were taken from kidneys surgically resected for RCC. Nephrectomy specimens were delivered to the laboratory within 1 hour of removal and dissected fresh.

  Immunohistochemistry4µm-thick sections were cut from the resulting TMA and from the normal kidney tissue paraffin blocks onto strongly adhesive slides (Superfrost Plus, Thermo Scientific, Waltham, MA, USA). The sections were deparaffinised in xylene (Sigma-Aldrich Company Ltd. Dorset, England) and rehydrated in ethanol. Immunohistochemistry was performed using the

  were converted to a binary simple covariate (positive or negative) by thresholding according to the most informative split on the Kaplan-Meyer using the log-rank statistical test. A score of 0 was recorded as negative, scores of 1+, 2+ or 3+ were considered as positive.

2 Deleted: 2

 22 ). The figure also shows what Deleted: Deleted: with b Comment [t1]: Insert Figure 1.

  this difference did not reach statistical significance after taking into consideration the correction for multiple comparisons (p = 0.49).

  way to explain how certain factors can promote malignant progression and the therapeutic responsiveness to anti-cancer drugs. Manipulation of such an enzyme expression and/or function could contribute to altering disease outcome for conventional RCC.

Figure 1 :

 1 Figure 1: Expression of GST-α and GST-π in normal adult kidney tissue. Representative immunoblots showing 10 µg of kidney homogenate (C: cortex; M: medulla; P: papilla) per lane probed with anti-mouse GST-α (A) or anti-mouse GST-π (D) antisera. Specific immunoreactivities are observed at ~25 kD (GST-α) and at ~45kD and ~25kD (GST-π).

Figure 2 :

 2 Figure 2: GST-α immunostaining in clinically confined RCC. Representative TMA of clinically confined RCC showing GST-α protein pattern of expression and intensity of (A) mouse IgG (negative control); (B) RCC with GST-α score 1+; (C) RCC with GST-α score 2+; (D) RCC with GST-α score 3+. Note that GST-α is completely absent in papillary (E) and chromophobe (F) RCCs, for which GST-α score is 0. Scale bar: 200 µm.

Figure 3 :Figure 4 :

 34 Figure 3: GST-π immunostaining in clinically confined RCC. Representative TMA of clinically confined RCC showing GST-π protein pattern of expression and intensity of (A) mouse IgG (negative control); (B) RCC with GST-π score 1+; (C) RCC with GST-π score 2+; (D) RCC with GST-π score 3+; (E) Papillary RCC with GST-π score 2+; (F) Chromophobe RCC with GST-π score 2+. Scale bar: 200 µm

16 Table 1 :Table 2 :

 1612 Cross tabulation of GST-α grade of staining with the conventional histological prognostic covariates. For contingency tables of the individual prognostic and GST-α covariates, the p values from chi-square testing are as follows: grade, p < 0.001; stage p = 0.88; size > 7cm: p = 0.75; vascular invasion: p = 0.45; capsular invasion: p = 0.84; tumour type: p < 0.001. Cross tabulation of GST-π grade of staining with the conventional histological prognostic covariates. For contingency tables of the individual prognostic and GST-π covariates, the p values from chi-square testing are as follows: grade, p = 0.10; stage p = 0.40; size > 7cm: p = 0.17; vascular invasion: p = 0.77; capsular invasion: p = 0.15; tumour type: p = 0.049.

Figure 1 :Figure 2 :Figure 3 :

 123 Figure 1: Expression of GST-α and GST-π in normal adult kidney tissue. Representative immunoblots showing 10 µg of kidney homogenate (C: cortex; M: medulla; P: papilla) per lane probed with anti-mouse GST-α (A) or anti-mouse GST-π (D) antisera. Specific immunoreactivities are observed at ~25 kD (GST-α) and at ~45kD and ~25kD (GST-π). GST-α specific immunoreactivity is found within the cytoplasm of cells of the proximal convoluted (B, cortex) and straight tubules (C, outer medulla). Note the absence of GST-α immunoreactivity from the glomeruli (g) and the distal convoluted tubule (d). Cellular GST-π immunoreactivity is more widespread and can be observed in the cytoplasm, apically and basolaterally in cells of both the collecting duct (cd) and loop of Henle (lh) of both the renal cortex (E) and outer medulla (F). Note that π-GST is absent in the glomerulus (g). Scale bar: 200 µm (B and E) or 100 µm (C and F). 74x50mm (300 x 300 DPI)

Figure 4 :

 4 Figure 4: Kaplan-Meier disease-free survival plot of GST-α score. Clear cell RCC patients that were positive for GST-α (Score 1) had a mean disease-free survival of 6.0 years (95% CI 5.33 -6.63), compared with those patients whose tumours were negative for GST-α and for which mean diseasefree survival was 4.7 years (3.54 -5.90) (Score 0; p = 0.011, log rank test). 67x50mm (300 x 300 DPI)

Table 3 : Cox regression analysis of GST-α score, vascular invasion and capsular invasion.

 3 GST-α is not influential if grade is included in the Cox regression analysis however, if it is omitted, it has influence at the 0.1 level.

Page 25 of 27 Published on behalf of the British Division of the International Academy of Pathology Histopathology

	1 2
						Total
						Total
	GST-π Score	0	1	2	3
	GST-α Score Tumour grades 1 & 2*	0 21	1 26	2 36	3 30	113
	Tumour grades 1 & 2* Tumour grades 3 &4	44 10	22 3	32 19	22 10	120 42
	Tumour grades 3 &4 TNM pT1	35 15	4 12	4 22	5 11	48 60
	TNM pT1 TNM pT2	33 8	12 10	14 12	11 15	70 45
	TNM pT2 TNM pT3 & pT4	21 31	8 29	10 55	5 40	44 50
	TNM pT3 & pT4 Tumour size <=7cm	25 21	6 14	12 31	11 17	54 83
	Tumour size <=7cm Tumour size> 7cm	46 10	16 15	18 24	14 23	94 72
	Tumour size> 7cm Vascular invasion absent	33 21	10 19	18 31	13 25	74 96
	Vascular invasion absent Vascular invasion present	48 10	18 10	25 24	14 15	105 59
	Vascular invasion present Capsular invasion absent	31 28	8 27	11 42	13 32	63 129
	Capsular invasion absent Capsular invasion present	65 3	23 2	29 13	23 8	140 26
	Capsular invasion present Conventional type	14 21	3 19	7 43	4 22	28 105
	Conventional type Papillary	36 4	23 2	32 4	21 11	112 21
	Papillary Chromophobe	22 2	0 1	0 2	0 0	22 5
	Chromophobe Unclassified	5 4	0 7	0 8	0 7	5 26
	Unclassified Recurrence	16 11	3 8	4 19	6 12	29
	Recurrence	31	8	11	3

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

Page 26 of 27 Published on behalf of the British Division of the International Academy of Pathology Histopathology

	3
	Conventional RCC only (112 cases)	Hazard ratio	p	95.0% C.I.
	Invasion outside the kidney (21)	4.254	< 0.0001	2.003-9.031
	Any vascular invasion (41)	2.780	< 0.01	1.283-6.027
	Negative or +1 GST-α α α α (91)	5.630	< 0.019	1.327-23.881

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

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

Acknowledgements

This project was supported by the Technology Strategy Board (grant K0508: to SAP, GRB, BJ, DR, and DFRG). The Authors would like to thank Wales Cancer Bank for providing the tissue, Argutus Medical for providing the GST-α monoclonal antibody and Dr Mark Pinches and Professor Roz John for valuable input.

Insert Table 2.


Dr Claire Louise Meek
email: claire.meek@nhs.net

Jonathan D Reston

Tim Ramsbottom

Hari Pathmanathan

Adie Viljoen

The Use of High Intensity Statin Therapy with Simvastatin 80mg and Atorvastatin 80mg in Primary Care

come

What is already known about this topic?

The reduction of LDL-cholesterol with statins is well established in the prevention of cardiovascular events. For high risk patients, national guidelines recommend high intensity therapy with simvastatin 80mg but this regimen has been the subject of recent safety concerns. Atorvastatin 80mg is an alternative high intensity regimen with a robust evidence base. The tolerability of high intensity statin therapy is limited by side effects such as muscle aches and rarely myopathy and rhabdomyolysis.

What does this article add?

This study demonstrates the conflict prescribers face between prescribing according to cost and guidelines (simvastatin) and prescribing for proven efficacy (atorvastatin). This study shows that atorvastatin 80mg is being used in general practice for primary prevention. We present the first retrospective observational study comparing these agents and examining their use, tolerability and efficacy in clinical practice in primary care.

Prior Publication

An earlier form of this study was submitted in abstract form to the HEART UK conference and was presented in poster form at the conference on 9/7/2010. disease and stroke is the leading cause of illness and death in the UK and worldwide [START_REF]Top 10 Causes of Death[END_REF][START_REF]Coronary Heart Disease Statistics[END_REF]. The most extensively researched of the pharmaceutically modifiable risk factors is cholesterol; in particular, the lowering of low-density lipoprotein cholesterol (LDL-C) forms the primary target of focus in CVD risk prevention [START_REF]Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III): final report[END_REF]4]. Statins form the cornerstone of pharmaceutical CVD prevention and their efficacy in reducing mortality in both primary and secondary prevention settings is beyond dispute [START_REF] Baigent | Efficacy and safety of cholesterollowering treatment: prospective meta-analysis of data from 90,056 participants in 14 randomised trials of statins[END_REF]. Clinical guidelines including those from the National Cholesterol Education Program (NCEP) [START_REF]Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III): final report[END_REF] Joint British Societies (JBS) [4] and European [START_REF] Graham | European guidelines on cardiovascular disease prevention in clinical practice: full text. Fourth Joint Task Force of the European Society of Cardiology and other societies on cardiovascular disease prevention in clinical practice (constituted by representatives of nine societies and by invited experts)[END_REF] guidelines have recommended progressively lower LDL-C targets based on the evidence of the superiority of more intensive treatments.

When faced with a treatment decision clinicians have to weigh the relative benefits and risks of treatment. This decision is further confounded when the relative drug costs need to be taken into account. Up to a two fold difference in LDL-C lowering ability exists between some statins [START_REF] Jones | STELLAR Study Group. Comparison of the efficacy and safety of rosuvastatin versus atorvastatin, simvastatin, and pravastatin across doses (STELLAR* Trial)[END_REF], however the differences between other statins are more subtle. The two most commonly prescribed statins in the United Kingdom (UK) and most other countries are atorvastatin and simvastatin [START_REF] Kidd | Life after statin patent expiries[END_REF], with the market leader, atorvastatin Effectiveness (NICE) which recommends high intensity statin therapy with simvastatin 80mg for secondary prevention of CVD [START_REF]Lipid Modification[END_REF].

Atorvastatin 80 mg is a well-established treatment option for high risk individuals following the demonstration of its superior efficacy in the PROVE-IT (compared to pravastatin 40 mg) [START_REF] Cannon | Pravastatin or Atorvastatin Evaluation and Infection Therapy-Thrombolysis in Myocardial Infarction 22 Investigators. Intensive versus moderate lipid lowering with statins after acute coronary syndromes[END_REF] and TNT (compared to atorvastatin 10 mg) [START_REF] Larosa | Treating to new Targets (TNT) Investigators. Intensive lipid lowering with Atorvastatin in patients with stable coronary disease[END_REF] studies. In contrast to this, data from the SEARCH trial which compared simvastatin 20mg to 80mg have raised concerns about the riskbenefit ratio of simvastatin 80 mg [START_REF] Wierzbicki | SEARCHing for JUPITER: starry-eyed optimism is not warranted[END_REF][START_REF] Search | SLCO1B1 Variants and statin-induced myopathy -A genomewide study[END_REF]. Most recently, the United States (US) Food and Drug Administration (FDA) issued a warning about increased incidence of myopathy and rhabdomyolysis with simvastatin 80 mg compared with the 20mg dose [START_REF]FDA Drug Safety Communication: ongoing safety review of high-dose Zocor (simvastatin) and increased risk of muscle injury[END_REF]. In the United Kingdom, the Medicines and Healthcare products Regulatory Authority (MHRA) have advised a similarly cautious approach: "There is an increased risk of myopathy associated with high-dose (80 mg) The 80-mg dose should be considered only in patients with severe hypercholesterolaemia and high risk of cardiovascular complications who have not achieved their treatment goals on lower doses, when the benefits are expected to outweigh the potential risks" [START_REF][END_REF]. Subsequently, uncertainty prevails as clinicians who attempt to choose a safe, economic and effective statin medication for their patients are faced with conflicting evidence and guidelines. There is a lack of clinical evidence to guide statin use in current practice. Furthermore, no large-scale studies have compared the highest licensed doses for these two drugs, namely atorvastatin 80 mg and simvastatin 80 mg, although there has been a comparison in familial hypercholesterolaemia [START_REF] Wierzbicki | Comparison of therapy with simvastatin 80mg and atorvastatin 80mg in patients with familial hypercholesterolaemia[END_REF]. The aim of this audit was to examine the use, were based on the Joint British Society guidelines [4] for audit thresholds and were consistent with NICE guidance [START_REF]Lipid Modification[END_REF] that many patients fail to achieve the stricter targets of TC <4 mmol/l and LDL-C <2 mmol/l, and more lenient targets should be employed for audit purposes. Thus, patients with total cholesterol (TC) <5 mmol/l and LDL-C <3 mmol/l were said to be controlled according to audit thresholds. Patients with TC >5 mmol/l or LDL-C >3 mmol/l were said to be uncontrolled. For blood results to be included in the study, the patient had to have received four weeks' treatment with the medication prior to the test being performed. Propensity score matching based on Mahalanobis distances was undertaken using the psmatch2 [START_REF] Leuven | PSMATCH2: Stata Module to Perform Full Mahalanobis and Propensity Score Matching, Common Support Graphing, and Covariate Imbalance Testing[END_REF] command in STATA 11. The propensity score was derived using logistic regression, with the propensity score representing the conditional probability that they had received simvastatin given their age, sex, the surgery they were treated in, and whether they were taking the drug for primary or secondary prevention.

5: Implement change

We have sought to disseminate the information through a series of educational seminars for prescribers to encourage increased discussion of safe prescribing practices for hyperlipidaemia and cardiovascular risk. This information was also presented as a poster at the HEART UK conference annual conference of 2010. and taking a statin for secondary prevention (56.9%).

F

There was evidence that prescribers were using atorvastatin 80mg in primary prevention patients, contrary to recent guidelines. Indeed the proportion of patients on atorvastatin 80mg for primary prevention (34.0%) was statistically similar to the proportion on simvastatin (49.3%; p>0.1). This highlights the value of clinical data and reflects the conflict clinicians face between prescribing according to cost and guidelines, or to superior efficacy.

Patients generally discontinued a medication due to intolerance or a lack of efficacy. The majority of patients discontinuing the medication due to intolerance reported mild muscle aches, fatigue or malaise (n=5 out of 8 more robust evidence at this dosage, particularly for secondary prevention [START_REF] Cannon | Pravastatin or Atorvastatin Evaluation and Infection Therapy-Thrombolysis in Myocardial Infarction 22 Investigators. Intensive versus moderate lipid lowering with statins after acute coronary syndromes[END_REF][START_REF] Larosa | Treating to new Targets (TNT) Investigators. Intensive lipid lowering with Atorvastatin in patients with stable coronary disease[END_REF]. When atorvastatin comes off patent in the next few years the cost difference between these medications will be significantly reduced. Until then, clinicians are faced with a conflict between prescribing for cost or for proven efficacy. This is particularly challenging for clinicians treating high risk patients, for whom the results of further cardiovascular events, loss of earnings and potential disability also have significant adverse economic effects which may justify using a more expensive preventative agent.

Unfortunately, there are no head-to-head randomised controlled studies comparing high intensity atorvastatin and simvastatin. Additionally, there is little evidence about the relative merits of these agents in the setting of current clinical practice.

This study offers some comparison of high intensity atorvastatin with simvastatin in current clinical practice. Prescribers may find this 'real-life' approach useful when large-scale clinical evidence is lacking and clinical guidelines are conflicting. This audit suggests that high intensity atorvastatin is used in primary care, despite national guidelines which specifically recommend simvastatin use [START_REF]Lipid Modification[END_REF]. In this study, patients on atorvastatin were less likely to fail to meet targets of cholesterol and LDL-C control when compared with simvastatin. This is consistent with other evidence supporting the efficacy of atorvastatin [START_REF] Jones | STELLAR Study Group. Comparison of the efficacy and safety of rosuvastatin versus atorvastatin, simvastatin, and pravastatin across doses (STELLAR* Trial)[END_REF][START_REF] Cannon | Pravastatin or Atorvastatin Evaluation and Infection Therapy-Thrombolysis in Myocardial Infarction 22 Investigators. Intensive versus moderate lipid lowering with statins after acute coronary syndromes[END_REF][START_REF] Larosa | Treating to new Targets (TNT) Investigators. Intensive lipid lowering with Atorvastatin in patients with stable coronary disease[END_REF].

There are several limitations to this study. Importantly, it is a retrospective study with no blinding or randomisation of treatment groups. There were very few baseline lipid results available which limits any comparison of relative efficacy of these medications on the basis of current lipid values. This is because many patients had received statin treatment in some form for over a decade, predating notation in current records. However, the comparison of these agents, based on the need to discontinue therapy due to target failure remains valid, although may be affected by differences of prescribing behaviour and risk perception among prescribers. On data analysis, no statistically significant differences among prescribing behaviour and treatment effects were detected between the two general practitioners' surgeries. There has been recent concern about increased incidence of myositis and rhabdomyolysis in patients receiving simvastatin 80mg [START_REF] Search | SLCO1B1 Variants and statin-induced myopathy -A genomewide study[END_REF][START_REF]FDA Drug Safety Communication: ongoing safety review of high-dose Zocor (simvastatin) and increased risk of muscle injury[END_REF]. This study did not identify any patients with myositis or rhabdomyolysis in either treatment group. Interestingly, creatine kinase (CK) was checked in a minority of patients only. There were no statistically significant differences in tolerability between the two treatment groups. Current guidance suggests that patients with muscle-related symptoms on a statin should have a CK checked [START_REF]Lipid Modification[END_REF].

In

  clinical studies comparing these agents are lacking. The present study suggests that atorvastatin 80mg compares favourably to simvastatin in terms of efficacy and has a similar tolerability profile.Conclusion: This retrospective observational study suggests that despite national guidelines, atorvastatin 80mg is used in clinical practice and is more effective and at least as well tolerated as simvastatin 80mg.

  The data was presented to a group of general practitioners locally on 21/4/2010. CVD) which manifests primarily as coronary heart

  demonstrating superior efficacy. The patent for simvastatin expired in 2004 (UK) and 2006 (USA) making it a vastly cheaper treatment option compared to atorvastatin. This cost merit has been incorporated into the national guideline from the United Kingdom (UK) National Institute of Clinical

8 efficacy 1 : 2 :

 812 and tolerability of high intensity statin treatment with simvastatin 80mg and atorvastatin 80mg in patients in primary care.Patients who had ever been treated with atorvastatin 80 mg or simvastatin 80 mg were identified at two local general practitioners' surgeries in Hertfordshire, UK. The first surgery is a semi-rural practice of 16,000 patients based in an affluent village with 7 partners. It is a dispensing practice which is also involved in training. The second practice is based in a large town with 5 partners and is non-dispensing. The practice serves 9,300 patients in a lower demographic group and has a slightly larger proportion than average of patients who are elderly or with ischaemic heart disease or diabetes. Data was obtained through computer-based medical records and use of the hospital pathology database.The Caldicott guardians for each of the surgeries were aware of the study and were satisfied with the methodology. According to the guidelines of the local ethics committee, this study was an audit and did not require formal research ethics approval. This methodology follows the audit cycle[START_REF] Derry | Auditing audits: the method of Oxfordshire Medical Audit Advisory Group[END_REF][START_REF] Gnanalingham | An audit of audits: are we completing the cycle?[END_REF]. This is a first cycle audit. Identify audit topic or the problem to be addressed The audit question was, 'Does local prescribing in primary care follow the NICE guidance?' Set standards for acceptable care As a measure of the adequacy of treatment, patients were allocated to groups depending on their serum lipid concentrations. Thresholds for lipid control

  3: Data collectionData was collected from the surgeries by a single investigator and was anonymised. Encrypted devices and secure NHS-based email accounts were used to protect the data.4: Data analysisStatistical analysis was performed using STATA 11 [19; Boston College Department of Economics]. Categorical variables were described using number and percentage and analysed for significance using Pearson's Chi (X 2 ) test. Continuous variables were described using medians and lower/upper quartiles, and were analysed for significance using the Mann-Whitney U test.In order to ensure treatment effect estimates were not biased because of differences in the demographic and clinical makeup of the treatment score matching was employed to adjust for pre-treatment differences between the groups.

16 Discussion

 16 , ALT and GGT. There were no significant differences between level of control in treatment groups.Inthe UK, NICE seeks to guide clinicians towards clinically effective and economic choices for patient management. The NICE guidance (May 2008) on lipid modification recommends simvastatin 80mg for secondary prevention and highlights the cost difference between a year's treatment with simvastatin 80mg (£64.53) compared with atorvastatin 80mg (£364.67) [9]. According to the September 2010 British National Formulary the cost difference has remained relatively unchanged, with a year's treatment with simvastatin 80mg now costing £35.28 compared with £338.52 for atorvastatin 80mg. However, atorvastatin is supported by

A further limitation to this study involves the significantly increased usage of other

  lipid-lowering treatments in the atorvastatin group (p=0.002). One must interpret the lipid concentrations with caution in light of this confounding factor, and regarding the absence of pretreatment lipid values.

Table 1 :

 1 summary, this retrospective observational study describes routine clinical practice and highlights the conflict prescribers face with regard to high Characteristics of all patients ever treated with atorvastatin 80mg compared with simvastatin 80mg.

	intensity statin use. Clinical trial evidence favours atorvastatin while
	simvastatin remains the economic option and is favoured by national
	guidelines. Patients receiving atorvastatin 80mg were significantly more likely
	to remain on the medication, achieving adequate cholesterol and LDL-C
	control, with no increase in intolerance when compared with patients treated
	with simvastatin 80mg.

Table 4 :

 4 Characteristics of patients currently treated with atorvastatin 80mg and simvastatin 80mg.

			Current	Current	Significance
			Atorvastatin 80mg	Simvastatin 80mg
			n=43 (%)	n=35 (%)
	Age -<60 years	18 (40.9)	11 (31.4)	-
	>60 years	26 (59.1)	24 (68.6)
	Sex -male		28 (63.6)	23 (65.7)	-
	Primary prevention	16 (36.4)	18 (51.4)	-
	Secondary prevention	28 (63.6)	17 (48.9)
	BMI kg/m	2
	<25		8 (18.2)	2 (5.7)	-
	>25		29 (66.0)	27 (77.1)
	Current smoker	8 (18.2)	10 (28.6)	-
	Family history of IHD	26 (59.1)	15 (42.

Acknowledgements

No funding was sourced for this study or for its publication. We gratefully acknowledge the staff of Bridge Cottage and Nevells Road general practitioners' surgeries who kindly permitted us access to their database for data collection.

Dr Viljoen has received lecturing honoraria and⁄or travel support and/or are involved in clinical research trials funded by AstraZeneca, Novo Nordisk, Eli Lilly, GlaxoSmithKline, Pfizer, Bristol-Myers Squibb, Merck-Sharp & Dohme, Sanofi-Aventis and Takeda pharmaceuticals. Dr Pathmanathan is a member of the Pfizer East of England Advisory board on lipid guidelines. Dr Meek, Dr Ramsbottom and Mr Reston have no conflicts of interest to declare.

Ethical Approval

The study was approved by the audit department of our department. The local research ethics committee is aware of the study.