Dr José Félix Lucía
email: jflucia@salud.aragon.es

-José Antonio Aznar

Lydia Abad-Franch

- R Rubio Escuin

-Víctor Jiménez-Yuste

Rosario Pérez

Javier Batlle

Ignacia Balda

Gabriela Alperovich

Rafael Parra

PROPHYLAXIS THERAPY IN HAEMOPHILIA A: CURRENT SITUATION IN SPAIN

Keywords: Haemophilia A, prophylaxis, epidemiology, Spain

The Spanish Epidemiological Study in Haemophilia carried out in 2006 enrolled 2400 patients (2081 -86.7%-with haemophilia A (HA) and 319 -13.3%-with haemophilia B (HB)); 465 of them (19.4%) were on prophylaxis. These rates were higher in patients with severe haemophilia (45.4%) and severe paediatric cases (72.5%). Based on the information recorded in this study we analysed the current situation of prophylaxis therapy administered to patients with HA in Spain, as well as their orthopaedic status.

Prophylaxis was used in 399 (19.2%) patients with HA; such prophylaxis was primary (PP) in 20.3% and secondary (SP) in 75.9% of cases. Among severe HA patients, 313 (45.9%) were on prophylaxis (22.3% on PP and 74.7% on SP). Taking into account the patients' age, 34.7% of severe HA adults were on prophylaxis (6% PP and 92.1% SP) whereas 71.5% of severe HA paediatric patients (40.5% PP and 55.4% SP) received this kind of treatment.

Established haemophilic arthropathy (EHA) was detected in 142 from 313 severe HA patients (45.3%) on prophylaxis, but only in 2.9% of patients under PP versus 59% of patients receiving SP. There was no EHA in adult severe HA patient on PP, whereas 70.4% on SP had joint damage (p<0.00001). Among paediatric severe HA patients, EHA was detected in 3.3% under PP and 37.8% under SP (p<0.00001). In conclusion, our data suggests that an early initiation of prophylaxis avoids EHA in the long term in patients with severe HA. We should emphasize the early onset of prophylaxis regimens.

A and 319 -13.3%-with haemophilia B) was recorded. Illness was severe in 32.3% of patients, moderate in 16.4%, and mild in 51.3%. Adult patients (14 years of age or over) represented 77.2% of cases [START_REF] Aznar | Haemophilia in Spain[END_REF].

We found that 465/2400 (19.4%) were on regular prophylaxis. If we consider patients with severe haemophilia, 45.4% of them were on prophylaxis. Nevertheless, if we consider only paediatric severe patients, 72.5% were on regular prophylaxis treatment.

Data collected during such study were the basis on which the Spanish Haemophilia Registry was built in 2008; this database comprised information on 2905 haemophilia patients (2518 with haemophilia A and 387 with haemophilia B) from 53 haemophilia treatment centres [START_REF] Aznar | The national registry of haemophilia A and B in Spain: results from a census of patients[END_REF].

On the basis of the information from the Spanish Epidemiological Study [START_REF] Aznar | Haemophilia in Spain[END_REF] we aimed to achieve a deeper knowledge of various issues involved in clinical practice like the real current situation of primary and secondary prophylactic treatment provided to both paediatric and adult haemophilia A (HA) patients in our country, as well as their current orthopaedic status according to the Pettersson score [START_REF] Pettersson | A radiologic classification of hemophilic arthropathy[END_REF] and the implementation of central venous access devices (CVAD) insertion often required for an adequate compliance of therapeutic schedules involving frequent intravenous infusions [START_REF] Hacker | Barriers to compliance with prophylaxis therapy in haemophilia[END_REF].

MATERIALS AND METHODS

Data used for our evaluation were obtained from the database made up for the Spanish Haemophilia Epidemiological Study, involving 2081 HA patients registered and managed in 51 haemophilia treatment centres [START_REF] Aznar | Haemophilia in Spain[END_REF].

The degree of severity of haemophilia was sorted according to previously published standard criteria related to FVIII plasma levels as severe (<0.01 U dL -1 ), moderate (≥0.01 U dL -1 to < 0.05 U dL -1 ) and mild (≥0.05 U dL -1 to 0.4 U dL -1 ) [START_REF] White | Definitions in haemophilia. Recommendation of the scientific subcommittee on factor VIII and factor IX of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis[END_REF]. Patients ≥14 years of age were rated as adults and those <14 as paediatric.

Definition of the type of prophylactic treatment was made following the recommendations of the PEDNET group (European Paediatric Network for Haemophilia Treatment) [START_REF] Donadel-Claeyssens | Current co-ordinated activities of the PEDNET (European Paediatric Network for Haemophilia Management)[END_REF] as:

-A primary prophylaxis (APP): Regular continuous (minimum 46 weeks a year) treatment started after the first joint bleed recorded and before the age of 2 years. -A secondary prophylaxis (ASP): Regular continuous (long term) treatment started after 2 or more joint bleeds recorded or at an age >2.

-B secondary prophylaxis (BSP): Intermittent regular (short term) treatment provided in order to cover frequent haemarthroses.

Assessment of joint status was made by visualisation of X-rays of elbows, knees and ankles according to the Pettersson score [START_REF] Pettersson | A radiologic classification of hemophilic arthropathy[END_REF]; scores of ≥3 for any particular joint were consistent with established haemophilic arthropathy (EHA).

Information related to CVAD was focused on particular aspects like catheter duration and incidence of the two most frequent complications associated to their use: infection and thrombosis.

Statistical analysis

A descriptive analysis was done, using absolute and relative frequencies for qualitative variables and centralization and dispersion measures for quantitative variables.

Pearson's Chi-square test (or Fisher's exact test for 2x2 tables, or reason of verisimilitude mxn tables, if necessary) were used to find the relation between qualitative variables. The T Student test or one-factor ANOVA were used for the comparison of continuous variables, if parametric criteria were fulfilled. The statistical significance value has been established at p<0.05.

RESULTS

Information from 2081 HA patients registered in the Spanish Epidemiological Study was analysed; 682 (32.8%) of them were patients with severe haemophilia, whereas 290 (13.9%) and 1109 (53.3%) were moderate and mild cases respectively. Patients classified as adults accounted for 77.2% (1607) of the whole series and children 474 (22.8%).

Prophylaxis in haemophilia A patient population

Evaluation of this group of 2081 HA patients showed that 399 (19.2%) of them were on prophylaxis: primary in 81 cases (20.3 %), secondary in 303 (75.9%) and undetermined in 15 (3.7%). Details are shown in Table 1. In the analysis by groups of age and type of treatment we founded the following results (see Table 2):

-Only a minor proportion of adult HA patients (215; 13.4%) were on prophylactic therapy: primary in 13 (6.1%) and secondary in 196 (92.2%).

-The rate of paediatric patients on prophylaxis was far higher, as expected (184; 38.8%); rates of primary (PP) and secondary prophylaxis (SP) for this age group were 26.9% (68 cases) and 58.1% (107 cases) respectively.

Prophylaxis in severe haemophilia A patient population

Almost half of the 682 severe HA patients (45.9%; 313) were on prophylactic treatment, either PP (70 cases; 22.3%) or SP (234 cases; 74.7%) (Tables 1 &2). Among paediatric patients diagnosed with severe HA (n=207), prophylaxis was administered to 71.5% (148 cases): 40.5% (60 cases) for PP and 55.4% (82 cases) for SP. Adult severe HA patients (475 subjects, 433 on treatment) were far less frequently on prophylaxis (34.7%; 165 cases); the vast majority of them followed a SP treatment scheme (92.1%; 152 cases) and only 6.0% (10 cases) a PP one (Tables 1 &3).

Joint status

Assessment of joint status was possible for 1228 of all patients enrolled in the epidemiological study and evidence of EHA was proved for 555 (45.2%) of them.

Among the 313 severe HA patients receiving prophylaxis, EHA was detected in 142 of them (45.4%), but only in 2.9% of patients under PP versus 59% of patients receiving SP (p<0.001) (Table 4).

For all 433 treated severe HA adult patients recorded, EHA was found in 74.4% (322/433) of them and rates of chronic joint damage were significantly higher for patients receiving on demand treatment (79.4%) than for patients receiving prophylaxis with regular factor VIII infusions (66.1%), p=0.002. There was no EHA in adult severe HA patient on PP, whereas 70.4% on SP had joint damage (p<0.00001) (Table 4). This evaluation was also made in 148 paediatric severe HA patients on prophylaxis.

Pettersson scores consistent with EHA were found in 33 (22.3%) of them, but only in 3.3% (2/60) of paediatric severe HA patients on APP or BPP; however signs of chronic joint damage were found in 37.8% (31/82) of children on ASP or BSP (p<0.00001).

Type of factor VIII concentrate used for prophylaxis

Recombinant factor VIII concentrates were administered for prophylaxis to 71.4% (285/399) of HA patients, and plasma derived products were used in 28.6% of cases (114/399) at the time of data collection. Rates of recombinant concentrate use were higher among the patients on PP (87.7%) than among those on SP (66.7%).

Frequency of infusions and bleeds

During the study period mean number of factor VIII concentrate infusions for the 399 HA patients were 3 (SD: 0.76). The mean annual number of bleeds was 1.3 for subjects belonging to the APP subset (range 0-18, SD: 3.14), 1.2 for those on BPP (range 0-8, SD: 1.88), 3.1 for ASP (range 0-78, SD: 8.14) and 4.3 for BSP (range 0-17, SD: 4.85).

Mean number of annual bleeding episodes for HA patients on prophylaxis was 2.9 (range 0-78, SD: 6.53).

Infusion routes of factor VIII concentrates

Factor concentrate was mostly administered through peripheral veins to HA individuals on regular prophylaxis (87.2%). Only in 9.5% of patients a CVAD was required and in 1.7% of cases both types of venous access were used. Important differences were found in CVAD insertion frequencies between patients on PP (23.4%) and SP (7.3%).

Complications associated to the use of CVAD were reported in 29 of 41 (70.7%) of catheters inserted. Infection was the most common of these complications [START_REF] Windyga | Haemophilia in Poland[END_REF]

DISCUSSION

Our current study aimed to get a real and deep knowledge of the implementation of prophylactic therapy in patients with HA in Spain and to compare this information with that reported in other countries in order to find out any potential differences in clinical practice in this setting which might be susceptible to be improved. However such comparisons are often difficult to make due to heterogeneity in the criteria used for patient definition and classification in the series reported. These differences may largely depend on age cut-offs used, definitions of prophylaxis, dosage intervals prescribed, FVIII:C plasma levels considered for the diagnosis of severity of haemophilia and also type of evaluations used: cross-sectional, retrospective, prospective, etc.

Regarding the rates of HA patients by degree of severity, our series shows a proportion of severe cases of 32.8%, which is similar to that reported in the Canadian (Biss et al; 30.6%), Swedish (Larsson et al; 30%) and Dutch (Plug et al; 39%) studies [START_REF] Biss | The use of prophylaxis in 2663 children and adults with haemophilia: results of the 2006 Canadian national haemophilia prophylaxis survey[END_REF][START_REF] Larsson | Current status of Swedish haemophiliacs. A demographic survey[END_REF][START_REF] Plug | Thirty years of haemophilia treatment in the Netherlands, 1972-2001[END_REF]. These figures are significantly higher in other published series of patients from the USA (Butler et al; 53%) [START_REF] Butler | Practice patterns in haemophilia A therapy-a survey of treatment centres in the United States[END_REF], Poland (Windyga et al; 59.7%) [START_REF] Windyga | Haemophilia in Poland[END_REF] and most recently Japan (Ono et al-69.5%-; Taki et al -73.4 %-) [START_REF] Ono | Assessment of haemophilia treatment practice pattern in Japan[END_REF][START_REF] Taki | Current situation of regular replacement therapy (prophylaxis) for haemophilia in Japan[END_REF].

Our results show that 19.2% of HA patients are following some type of prophylaxis treatment, consistently with data published by Biss et al (20.3%) [START_REF] Biss | The use of prophylaxis in 2663 children and adults with haemophilia: results of the 2006 Canadian national haemophilia prophylaxis survey[END_REF], but not with those reported by Plug et al (29%) [START_REF] Plug | Thirty years of haemophilia treatment in the Netherlands, 1972-2001[END_REF]. Rates of prophylaxis available in Japanese series are very surprisingly heterogeneous, considering that they offer information on patients treated in the same country; thus Ono et al [START_REF] Ono | Assessment of haemophilia treatment practice pattern in Japan[END_REF] report rates of prophylaxis of 67.2%, whereas for Taki et al [START_REF] Taki | Current situation of regular replacement therapy (prophylaxis) for haemophilia in Japan[END_REF] it is of only 11.6%.

When we focused our analysis on severe HA patients (682 altogether) we found out that almost half of them (313; 45.9%) were on some type of prophylactic treatment, a rate similar to that reported by Butler et al (44%) [START_REF] Butler | Practice patterns in haemophilia A therapy-a survey of treatment centres in the United States[END_REF] but lower than in the Dutch (Plug et al; 67.6%) [START_REF] Plug | Thirty years of haemophilia treatment in the Netherlands, 1972-2001[END_REF] or Canadian series (Biss et al; 62.3%). Comparison of our data with those in this latter study is difficult to carry out because prophylaxis as considered in this paper refers to factor VIII treatment administered at least once a week during 45 weeks a year, whereas our patients received factor VIII infusions three times a week.

As expected, a lower proportion of our moderate HA patients were on prophylaxis (22.4%). Our figures are higher than those from the study by Taki et [START_REF] Blanchette | A survey of factor prophylaxis in boys with haemophilia followed in North American haemophilia treatment centres[END_REF] reported joint data from American and Canadian paediatric patients; 77% of their severe HA patients in this group of age (with an age cut-off of 18) were on some sort of prophylaxis treatment consistent with 72.5% from our epidemiological study (using 14 as a top age for patients being considered as paediatric) after exclusion of undetermined cases. Differences in age cut-offs between both groups must be taken into account when comparing results from both studies. Other authors like Plug et al set this cut-off at the age of 16 and report prophylaxis rates of 86% for severe HA patients [START_REF] Plug | Thirty years of haemophilia treatment in the Netherlands, 1972-2001[END_REF]. Great heterogeneity existing in classification of age groups can be an issue when interpreting the results from different studies.

The target prophylaxis rate for paediatric severe HA patients should ideally be 100% but this goal is virtually impossible to achieve for various reasons: regular venipuncture is difficult for children under 1 year of age, prophylaxis start can be delayed in families from a low social and cultural background and also some families can be reluctant to start treatment in children who still remain asymptomatic. Other reasons remain vaguely determined.

Prophylaxis among our 475 adult severe HA patients (>14 years of age) was unfortunately not so commonly used (34.7%; 165/475) as in other studies enrolling similar patients like those by Biss et al (53%) [START_REF] Biss | The use of prophylaxis in 2663 children and adults with haemophilia: results of the 2006 Canadian national haemophilia prophylaxis survey[END_REF] or Plug et al (59.3%) [START_REF] Plug | Thirty years of haemophilia treatment in the Netherlands, 1972-2001[END_REF]; this latter analysis includes severe haemophilia A and B patients over the age of 16.

Among our severe HA adult patients, significant differences in the development of EHA were found between those patients receiving prophylactic treatment versus patients receiving on demand treatment. This data are concordant with those published by Aledort et al in 1994 [START_REF] Aledort | A longitudinal study of orthopaedic outcomes for severe factor-VIII-deficient haemophiliacs. The Orthopaedic Outcome Study Group[END_REF]; they found that after 6 years of observation, patients under prophylaxis obtained better orthopaedic and radiological scores than patients following on demand therapy even though secondary prophylaxis couldn't impede the progression of the EHA.

Moreover, in our study we found that primary prophylaxis resulted strongly protective for the adult population. Although no differences in the degree of joint damage were noticed in the population of severe paediatric patients treated on demand versus those treated with prophylaxis. This results maybe due to the delay on the onset of the secondary prophylaxis in our country.

When we did this analysis in function of the type of prophylactic treatment administered, we found that PP resulted protective for the development of EHA in the paediatric population. Early onset of prophylactic treatment is definitely crucial for the approaches in the late sixties [START_REF] Plug | Thirty years of haemophilia treatment in the Netherlands, 1972-2001[END_REF], long before it became popular in other countries like Spain (where SP began to be used in the late seventies and the PP in the early nineties, approximately) and this may have translated into a steady rate of arthropathy of about 30%, reflected in the Spanish epidemiological study [START_REF] Aznar | Haemophilia in Spain[END_REF]. In our experience the most commonly affected joints were ankles followed by knees and elbows.

Patients for whom a CVAD insertion was decided were more commonly those on PP, which is not surprising considering that patients in this group are more often children under the age of 2 for whom recurrent intravenous infusions via peripheral veins is impractical and may seriously jeopardise compliance with regular prophylaxis.

Data collected from our study disclose that about one in four of our patients on PP (23.5%) had a CVAD inserted for this purpose, which is similar to rates previously reported by Valentino et al [START_REF] Valentino | Central venous access devices in haemophilia[END_REF]; other research groups however are far more active in the usage of such devices in patients on PP who have one inserted in up to 82% of cases according to Blanchette et al [START_REF] Butler | Practice patterns in haemophilia A therapy-a survey of treatment centres in the United States[END_REF][START_REF] Blanchette | A survey of factor prophylaxis in boys with haemophilia followed in North American haemophilia treatment centres[END_REF].

Major complications of CVAD are infrequent [START_REF] Valentino | Central venous access devices in haemophilia[END_REF]. As already mentioned above catheter associated infections are the most frequent amongst these patients for whom frequencies of 0.28 to 1.6 patient days for individuals without inhibitors have been published up to 2003 [START_REF] Ljung | Central venous lines in haemophilia[END_REF]. Thus our infection rate (0.73 per 1000 patient days) lies within the range just mentioned. No cases of catheter-triggered thrombosis have been collected from our study, consistently with data from the paper by Ljung et al [START_REF] Ljung | Port-A-Cath usage in children with haemophilia: experience of 53 cases[END_REF] enrolling a similar number of patients with a CVAD. Malfunctioning frequencies in this study were 18.8% during an average follow-up period of 13 months, not far from our 13.8% in a one-year follow-up time.

CONCLUSIONS

Spanish HA patients and, more specifically severe subjects, are at the same level as most developed countries regarding implementation of therapeutic strategies involving prophylaxis. However information derived from our current research shows some aspects of the management of patients with haemophilia in Spain which might be improved. For instance the reasons leading to a lower rate of prescription of PP or SP in adulthood, as well as the higher frequency of EHA in patients in this group of age must be taken into account. Both issues are probably related to a historic delay in the introduction of PP treatments on a routine basis in our country.

  epidemiological study aimed to update available information on clinical features of haemophilia patients registered in 51 haemophilia treatment centres throughout the country was carried out in Spain in 2006; from these reports relevant information from 2400 patients (2081 -86.7%-with haemophilia

4 -

 4 B primary prophylaxis (BPP): Regular continuous (minimum 46 weeks a year) treatment started before the age of 2 years without any evidence of a previous joint bleed.

Figure 1

 1 Figure 1 reflects the rate of prophylaxis used in the whole Haemophilia A population and in the severe HA patients by age groups.

  . Countries like Holland pioneered wide fostering of such therapeutic

F

  must strongly stress the importance of adopting early PP schedules in order to avoid serious bleeding episodes as well as the triggering and development of degenerative joint damage. The status of EHA noticed in adult patients with haemophilia along with growing age of this population will unavoidably mean a higher need for orthopaedic procedures of joint replacement in the future.

F o r P e e r R e v i e w 14 Table 4 :

 144 Established Haemophilic Arthropathy in relation to age and type of prophylaxis¥ NR: Not recorded in the case report form the type of treatment followed by the patient (on demand or prophylaxis)

FIGURESFigure 1 :

 1 FIGURES

  al (17%) or Plug et al(11.9%). Perhaps physicians involved in haemophilia care should be more aware of the need of prophylactic therapy in some patients affected by moderate haemophilia.

	Blanchette et al
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	8

Table 1 :

 1 Type of treatment in HA patients in relation to the degree of severity of haemophilia and age group

	Haemophilia A		Paediatric patients			Adult patients			Total patients
		Mild	Moderate	Severe	Total	Mild	Moderate	Severe	Total	Mild	Moderate	Severe	Total
		n (%)	n (%)	n (%)	n (%)	n (%)	n (%)	n (%)	n (%)	n (%)	n (%)	n (%)	n (%)
	Total Patients	207	60	207	474	902	230	475	1607	1109	290	682	2081
		(43.7)	(12.7)	(43.7)	(22.8)	(31.4)	(14.3)	(29.6)	(77.2)	(53.3)	(13.9)	(32.8)	(100)
	Total Patients on	80	54	198	332	283	180	433	896	363	234	631	1228
	Treatment	(38.6)	(90)	(95.7)	(67.1)	(31.4)	(78.3)	(91.2)	(55.8)	(32.7)	(80.7)	(92.5)	(59)
	NR ¥	2
		(

Table 2 :

 2 ) Not recorded in the case report form the type of treatment followed by the patient (on demand or prophylaxis) Type of prophylaxis in HA patients in relation to the degree of severity of haemophilia and age group

	Type of
	Treatment
	On demand	68	28	47	143	266	137	267	670	334	165	314	813
		(85)	(51.9)	(23.7)	(43.1)	(94)	(76.1)	(61.7)	(74.8)	(92)	(70.5)	(49.8)	(66.2)
	Prophylaxis	10	26	148	184	11	39	165	215	21	65	313	399
		(12.5)	(48.2)	(74.8)	(55.4)	(3.9)	(21.7)	(38.1)	(24)	(5.8)	(27.8)	(49.6)	(32.5)
	Total	80	54	198	332	283	180	433	896	363	234	631	1228
	¥ NR:

Table 3 :

 3 Type of prophylactic treatment in HA patients of different age groups

			Paediatric patients (<14 years)		Adult patients (≥ ≥ ≥ ≥ 14 years)
	Type	of	Mild	Moderate	Severe	Total	Mild	Moderate	Severe	Total
	prophylaxis		n (%)	n (%)	n (%)	n (%)	n (%)	n (%)	n (%)	n (%)
	Undetermined		2 (20)	1 (3.9)	6 (4.1)	9 (4.9)	1 (9.1)	2 (5.1)	3 (1.8)	6 (2.8)
	APP *		0	2 (7.7)	26 (17.6)	28 (15.2)	0	2 (5.1)	6 (3.6)	8 (3.7)
	BPP #		3 (30)	3 (11.5)	34 (23)	40 (21.7) 1 (9.1)	0	4 (2.4)	5 (2.3)
	ASP ╫		3 (30)	14 (53.9)	71 (48)	88 (47.8) 3 (27.3)	21 (53.9)	94 (57)	118 (54.9)
	BSP ╬		2 (20)	6 (23.1)	11 (7.4)	19 (10.3) 6 (54.6)	14 (35.9)	58 (35.2) 78 (36.3)
	TOTAL		10 (100)	26 (100)	148 (100)	184 (100) 11 (100)	39 (100)	165 (100) 215 (100)

(*) APP: A Primary Prophylaxis (#) BPP: B Primary Prophylaxis (╫) ASP: A Secondary Prophylaxis (╬) BSP: B Secondary Prophylaxis

Haemophilia

Acknowledgements: This study was supported by a Wyeth Spain (now Pfizer) grant.

We thank Dr. Carlos Aguilar for his help in the translation and the critical revision of the manuscript. This work was possible thanks to the collaboration of investigators from the 51 participating hospitals: La Paz (F Hernández, M Quintana, I Fernández), La Fe (S Haya, AR Cid, F Querol), Vírgen del Rocío (R Nuñez), Miguel Servet (F Lucía), N. Sra de las Nieves (MªJ Gutierrez), Cruces (G Iruin), Xeral-Cíes (R González), Vírgen de la Arrixaca (F García), Río Hortega (JI Tortosa), Carlos Haya (E Mingot), Juan Canalejo (F López), N. Sra Candelaria (J García-Talavera), Puerta del Mar (FJ Rodríguez), Dr Negrín (I Balda), Reina Sofía (F Velasco), Univ. Salamanca (JR González), Montecelo (R Vázquez), Donostia (M Uranga), N. Sra Covadonga (I Soto), Virgen del Camino (M Redondo), Torrecárdenas (B Domínguez), S. Pedro de Alcántara (N Bermejo), Materno-Infantil Málaga (A Palomo), General de Jerez (R Campos), Sant Joan de Deu (T Toll), General Ciudad de Jaén (M Nieto), Joan XXIII (MªA Ugarriza), Marqués de Valdecilla (C Sedano), Santa Bárbara (A Foncillas), Infanta Cristina (R Vaca), General Guadalajara (B Pinedo), Juan Ramón Jiménez (A Amian), Puerto Real (M Monje), Materno Infantil Las Palmas (M Negrín), General Yagu¨e (M Prieto), Son Dureta (JF Alvarez), Txagorritxu (R Hernández), N. Sra Cristal (JA Golpe), Santa Ana (F Hernández), Punta de Europa (LA Casaus), San Millán (P Rabasa), Xeral Lugo (A Cobas), Comarcal D. Benito-Villanueva (A Rodríguez), General Insalud Soria (C Aguilar), La Línea (M González), Clínico de Santiago (S Pérez)., N Sra Alarcos (M Tomás), María Inmaculada (AM Navarro), Arquitecto Marcide (C García), General Castellón (IM García) y Santiago Apóstol (E Navarro).


Ian Proctor

Kai Stoeber

Gareth H Williams
email: gareth.williams@ucl.ac.uk

Prof H Gareth

Williams

Invited Review: Biomarkers in Bladder Cancer

Keywords: Bladder cancer, biomarkers, DNA replication licensing Bladder cancer, biomarkers, DNA replication licensing

come

Introduction

Bladder cancer is the sixth most common malignancy in the UK with over 10,000 new cases diagnosed annually and is responsible for the deaths of 4,800 people each year. Like all cancers, the prognosis and clinical management of bladder cancer is strongly dependent upon the stage at which it is first diagnosed. The majority (75%) of patients present with superficial (pTa and pT1) tumours and have a 5-year survival rate of between 88-98%. In contrast the 5year survival rate for patients presenting with muscle invasive disease (pT2-pT4) is 46-63%, and falls to only 15% in those with locally advanced and metastatic disease * . In addition to pathological stage, histological features such as tumour grade are also independent predictors of disease outcome [START_REF] Sylvester | Predicting recurrence and progression in individual patients with stage Ta T1 bladder cancer using EORTC risk tables: a combined analysis of 2596 patients from seven EORTC trials[END_REF] . The accurate histological assessment of non-invasive tumours, commonly divided into 'papillary' and 'non-papillary' subtypes, is especially important. Nonpapillary tumours, more commonly referred to as 'carcinoma in situ' (CIS), are invariably aggressive high grade lesions which frequently progress to invasive cancer. In contrast superficial papillary tumours are typically lower grade lesions which behave less aggressively.

Despite the excellent prognosis associated with superficial tumours, almost 50% of patients will experience recurrence of their disease within 4 years of their initial diagnosis, and 11% will progress to muscle invasive disease [START_REF] Sylvester | High-grade Ta urothelial carcinoma and carcinoma in situ of the bladder[END_REF] . Therefore, the successful management of bladder cancer requires not only early diagnosis, careful histological assessment and accurate staging, but also frequent and long-term surveillance.

The current gold standard for the diagnosis and surveillance of bladder cancer remains direct visualisation of tumours by conventional white light cystoscopy. In addition, urine-based cytology is used to screen symptomatic and high-risk patients and is a useful adjunct in longterm surveillance protocols. The histological assessment of biopsy material taken during cystoscopic examination provides important information about tumour type, size, grade, degree of muscle invasion, and the presence or absence of lymphovascular invasion.

By combining histological, radiological and clinical information, tumours can be staged using the TNM system. This established staging system is an indicator of tumour burden and the extent of spread and is widely used as a predictor of disease outcome and to guide treatment (e.g. local vs systemic; conservative vs aggressive). In bladder cancer it is possible to further refine estimates of disease outcome through the development of prognostic indices, which include clinical variables such as stage, tumour number, grade, presence/absence of CIS and history of previous recurrence [START_REF] Sylvester | High-grade Ta urothelial carcinoma and carcinoma in situ of the bladder[END_REF][START_REF] Kurth | Factors affecting recurrence and progression in superficial bladder tumours[END_REF][START_REF] Allard | The early clinical course of primary Ta and T1 bladder cancer: a proposed prognostic index[END_REF] .

Whilst the widespread use of cystoscopy and urine cytology has contributed to a significant decline in the mortality associated with bladder cancer, both suffer from significant drawbacks as diagnostic and screening tests. Cystoscopy is technically demanding and invasive procedure which exposes the (often elderly) patient to significant iatrogenic risks, including infections, bleeding and perforation as well as complications arising from anaesthesia. Whilst cystoscopic examination identifies nearly all papillary and sessile lesions, small areas of CIS can be easily missed. Furthermore, the histological assessment of tumour grade in biopsies of superficial disease is highly subjective [START_REF] Ooms | Analysis of the performance of pathologists in the grading of bladder tumors[END_REF][START_REF] Maclennan | Histologic grading of noninvasive papillary urothelial neoplasms[END_REF][START_REF] Bol | Reproducibility and prognostic variability of grade and lamina propria invasion in stages Ta, T1 urothelial carcinoma of the bladder[END_REF] . This is reflected in recent changes to the WHO (2004) classification of non-invasive disease which replaces the original division of tumours into grades 1, 2 and 3 (WHO 1973) with two, low and high grade categories [START_REF] Eble | Pathology and genetics of tumours of the urinary system and male genital organs[END_REF][START_REF] Mostofi | Histological typing of urinary bladder tumours[END_REF][START_REF] Epstein | The World Health Organization/International Society of Urological Pathology consensus classification of urothelial (transitional cell) neoplasms of the urinary bladder. Bladder Consensus Conference Committee[END_REF][START_REF] Mostofi | Histological typing of urinary bladder tumours[END_REF] . Whilst this re-classification is likely to improve patient outcome, the elimination of the commonly used grade 2 category has proven contentious and has yet to be Despite a high specificity (78-100%), urine cytology lacks robust sensitivity (12.2-84.6%), especially for low grade tumours, and is subject to considerable inter-observer variability [START_REF] Budman | Biomarkers for detection and surveillance of bladder cancer[END_REF] .

Finally, cystoscopy and cytology are expensive techniques, especially when used in longterm surveillance programmes. In the US it has been estimated that the cost per patient of bladder cancer from diagnosis to death is the highest of all cancers, ranging between $96,000 and $187,000 [START_REF] Botteman | Quality of life aspects of bladder cancer: a review of the literature[END_REF] .

Cancer Biomarkers

The high cost and limitations of the current diagnostic and screening tests have led many to seek alternative markers of bladder cancer. Ideally these cancer 'biomarkers' would allow earlier diagnosis, show higher sensitivity and specificity, be more reliable and easier to perform and reduce overall costs.

A search of the current literature returns a bewildering array of reports relating to biomarker studies in virtually all areas of medicine, and bladder cancer is no exception. Moreover, the terminology used to describe these biomarkers and the context in which they are used can be confusing and often misleading. A widely accepted and comprehensive definition describes a biomarker as "a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention" [START_REF]Biomarkers and surrogate endpoints: preferred definitions and conceptual framework[END_REF] . Within this broad definition, biomarkers specific to cancer are typically classified as either 'diagnostic', 'prognostic' or 'predictive'. Diagnostic cancer biomarkers are those markers which can identify the presence (or absence) of a neoplastic process in a given setting, ideally before there are clinical manifestations. Such biomarkers can also be used as a screening test and to monitor the response to therapy and are already used routinely in the diagnosis and surveillance of other genitourinary malignancies such as prostate (e.g. PSA) and testicular cancers (e.g. βhCG). Prognostic biomarkers are those markers which have an association with clinical outcome, typically overall and recurrencefree survival. Such markers can be exploited as decision-making aids to help identify which patients would benefit most from chemotherapy and radiotherapy, and how aggressive this treatment should be [START_REF] Mcshane | Identification of clinically useful cancer prognostic factors: what are we missing?[END_REF] . In contrast, predictive biomarkers can be used to make more specific choices between treatment options. For example, oestrogen receptor (ER), progesterone receptor (PR) and Her2 receptor testing is now routinely used to select breast cancer patients for treatment with tamoxifen and trastuzumab † 17 , whereas genetic screening of patients with colorectal cancer for K-ras mutations is used to identify individuals who will not respond to cetuximab [START_REF] Karapetis | K-ras mutations and benefit from cetuximab in advanced colorectal cancer[END_REF] .

In contrast to tumour grading and staging, which fail to take account of the molecular diversity of cancer, prognostic and predictive biomarkers offer the ability to draw specific conclusions about individual patients and their tumours, providing so-called 'personalised medicine'. The ability to use such markers to tailor treatment to the individual is both cost effective and avoids unnecessarily exposing patients to the potentially harmful side effects of such treatment. However, despite years of research and almost 100,000 published reports, the number of cancer-specific biomarkers in clinical use remains surprisingly small [START_REF] Ludwig | Biomarkers in cancer staging, prognosis and treatment selection[END_REF] (Figure 1).

Diagnostic Biomarkers

The current paradox in biomarker development is very familiar to specialists involved in the diagnosis and treatment of bladder cancer. However, a handful of diagnostic and screening biomarkers have gained approval from the US Food and Drug Administration (FDA) and are now commercially available (Table 1).

Bladder Tumour Antigen test

The Bladder Tumour Antigen (BTA) test measures levels of complement factor H-related protein in the urine. This protein is similar to factor H and high levels are secreted by tumour cells into the urine [START_REF] Kinders | Complement factor H or a related protein is a marker for transitional cell cancer of the bladder[END_REF] . There are two forms of the test: BTA-Stat, a point-of-care immunoassay, and BTA-Trak, a laboratory-based enzyme-linked immunosorbent assay (ELISA). Whilst both tests outperform cytology on sensitivity (24-89% and 51-79% respectively), the reported specificity falls below that of cytology, and false positive results are common in benign inflammatory and infectious conditions [START_REF] Sharma | Exclusion criteria enhance the specificity and positive predictive value of NMP22 and BTA stat[END_REF][START_REF] Van Rhijn | Urine markers for bladder cancer surveillance: a systematic review[END_REF] .

Nuclear matrix protein 22

Nuclear matrix protein 22 (NMP22) is a regulator of mitosis and its expression is significantly increased in malignant urothelium. NMP22 is released into urine following apoptosis and can be measured by a quantitative immunoassay (NMP22 Test) or a qualitative point-of-care test (NMP22 BladderChek) using monoclonal antibodies. Numerous studies have demonstrated that NMP22 has a superior sensitivity (46-88% and 40-85% respectively) to urine cytology [START_REF] Budman | Biomarkers for detection and surveillance of bladder cancer[END_REF] . Furthermore, in a large prospective study of 668 patients with a history of bladder cancer, the combination of NMP22 and cystoscopy identified 99% of all tumours compared to only 91.3% with cystoscopy alone [START_REF] Grossman | Surveillance for recurrent bladder cancer using a point-of-care proteomic assay[END_REF] . However, the specificity varies widely depending upon the cut-points used and typically falls below that of urine cytology [START_REF] Budman | Biomarkers for detection and surveillance of bladder cancer[END_REF][START_REF] Shariat | Critical evaluation of urinary markers for bladder cancer detection and monitoring[END_REF] .

Furthermore, like BTA Trak, the false positive rate is significantly raised in patients with benign inflammatory conditions [START_REF] Shariat | Variability in the performance of nuclear matrix protein 22 for the detection of bladder cancer[END_REF][START_REF] Chang | Evaluation of nuclear matrix protein-22 as a clinical diagnostic marker for bladder cancer[END_REF] .

ImmunoCyt

ImmunoCyt is an immunofluorescence-based test in which a cocktail of three antibodies are used to detect two mucin-like proteins and a high molecular weight form of carcinoembryonic antigen [START_REF] Bergeron | Identification of a superficial bladder tumor-associated glycoform of the carcinoembryonic antigen by monoclonal antibody 19A211[END_REF] . These molecules are present on the surface of exfoliated malignant urothelial cell. The overall reported sensitivity and specificity rates of this test are good (63-85% and 62-79% respectively), but specificity remains below that offered by conventional cytology [START_REF] Budman | Biomarkers for detection and surveillance of bladder cancer[END_REF][START_REF] Shariat | Critical evaluation of urinary markers for bladder cancer detection and monitoring[END_REF] . Furthermore, the test requires the use of a fluorescence microscope by a trained operator and there are reports of interobserver variability [START_REF] Vrooman | Urinary markers in bladder cancer[END_REF] . As a result, this test is currently recommended only as an adjunct to cytological examination.

UroVysion

UroVysion is also a fluorescence-based assay, but uses fluorescence in situ hybridisation (FISH) to identify common alterations in chromosomal copy number and loss of the 9p21 locus [START_REF] Sarosdy | Clinical evaluation of a multi-target fluorescent in situ hybridization assay for detection of bladder cancer[END_REF] . A number of studies have shown that this test has high sensitivity (69-96%) and a specificity (65-96%) that almost matches urine cytology [START_REF] Budman | Biomarkers for detection and surveillance of bladder cancer[END_REF][START_REF] Shariat | Critical evaluation of urinary markers for bladder cancer detection and monitoring[END_REF] . Furthermore, since this test identifies neoplastic genetic alterations, it is largely unaffected by benign inflammatory conditions, and recent studies suggest that previously reported false positive tests may in fact represent early sub-clinical neoplastic changes [START_REF] Halling | A comparison of BTA stat, hemoglobin dipstick, telomerase and Vysis UroVysion assays for the detection of urothelial carcinoma in urine[END_REF][START_REF] Yoder | Reflex UroVysion testing of bladder cancer surveillance patients with equivocal or negative urine cytology: a prospective study with focus on the natural history of anticipatory positive findings[END_REF] . However, UroVysion is an expensive test requiring specialist equipment and diagnostic expertise. Owing to their inferior specificity and lack of robust prospective testing, none of these tests is currently recommended by international or European guideline panels [START_REF] Lokeshwar | Bladder tumor markers beyond cytology: International Consensus Panel on bladder tumor markers[END_REF][START_REF] Babjuk | EAU guidelines on non-muscle-invasive urothelial carcinoma of the bladder[END_REF] (see also www.uroweb.org). However, improvements in both specificity and sensitivity have been reported using combinations of these biomarkers, and many advocate their use in extending the time between surveillance cystoscopy, especially in low grade non-invasive disease and in cases were cytology was equivocal [START_REF] Horstmann | Combinations of urinebased tumour markers in bladder cancer surveillance[END_REF][START_REF] Bischoff | Bladder cancer[END_REF][START_REF] Schlomer | Prospective validation of the clinical usefulness of reflex fluorescence in situ hybridization assay in patients with atypical cytology for the detection of urothelial carcinoma of the bladder[END_REF][START_REF] Van Der Poel | Can biological markers replace cystoscopy? An update[END_REF] .

Prognostic Biomarkers

Whilst there has been considerable focus on the development of diagnostic biomarkers, clinicians and their patients are becoming increasingly eager to exploit any new test that can provide more accurate prognostic information or better direct the use of both conventional and emerging therapeutic agents. The search for such biomarkers has begun in earnest. Initial studies have focused on the observation that the malignant transformation of urothelial cells appears to arise through alterations in key molecular pathways and that distinct pathways may be involved in the development of invasive and non-invasive forms of the disease [START_REF] Wu | Urothelial tumorigenesis: a tale of divergent pathways[END_REF][START_REF] Knowles | Molecular subtypes of bladder cancer: Jekyll and Hyde or chalk and cheese?[END_REF][START_REF] Mitra | Molecular pathogenesis and diagnostics of bladder cancer[END_REF] (Figure 2).

FGFR/MAPK pathway

Pathways involved in cell growth signalling are frequently dysregulated in low grade, noninvasive tumours. Over 70% of these tumours harbour activating mutations in the fibroblast growth factor receptor 3 (FGFR3) gene compared to only 10-20% of invasive tumours [START_REF] Billerey | Frequent FGFR3 mutations in papillary noninvasive bladder (pTa) tumors[END_REF][START_REF] Van Rhijn | The fibroblast growth factor receptor 3 (FGFR3) mutation is a strong indicator of superficial bladder cancer with low recurrence rate[END_REF][START_REF] Bakkar | FGFR3 and TP53 gene mutations define two distinct pathways in urothelial cell carcinoma of the bladder[END_REF][START_REF] Lindgren | Molecular characterization of early-stage bladder carcinomas by expression profiles, FGFR3 mutation status, and loss of 9q[END_REF] .

The mutations appear to result in overexpression of FGFR3 and constitutive activation of the Ras-MAPK pathway increasing signal transduction to transcription factors in the nucleus.

Interestingly, mutations in FGFR3 and Ras appear to be mutually exclusive but together account for well over 80% of all low grade tumours indicating that activation of the Ras-MAPK pathway may be an obligate event in most low grade tumours [START_REF] Jebar | FGFR3 and Ras gene mutations are mutually exclusive genetic events in urothelial cell carcinoma[END_REF] . Furthermore, overexpression of FGFR3 has also been reported in a significant proportion (42%) of tumours with no identifiable mutations, suggesting that increased FGFR3 signalling can also contribute to malignant transformation in early bladder cancer [START_REF] Tomlinson | FGFR3 protein expression and its relationship to mutation status and prognostic variables in bladder cancer[END_REF] . Although a number of studies have shown promising results, the prognostic value of FGFR3 mutations and protein expression levels remains controversial [START_REF] Mhawech-Fauceglia | FGFR3 and p53 protein expressions in patients with pTa and pT1 urothelial bladder cancer[END_REF][START_REF] Hernandez | Prospective study of FGFR3 mutations as a prognostic factor in nonmuscle invasive urothelial bladder carcinomas[END_REF][START_REF] Burger | Prediction of progression of non-muscle-invasive bladder cancer by WHO 1973 and 2004 grading and by FGFR3 mutation status: a prospective study[END_REF][START_REF] Miyake | Fibroblast growth factor receptor 3 mutation in voided urine is a useful diagnostic marker and significant indicator of tumor recurrence in non-muscle invasive bladder cancer[END_REF] .

p53 and cell cycle regulation

In contrast to superficial tumours, CIS and invasive tumours show frequent alterations in the TP53 tumour suppressor gene [START_REF] Mitra | Molecular pathways in invasive bladder cancer: new insights into mechanisms, progression, and target identification[END_REF] . Loss of heterozygosity (LOH) studies have demonstrated that chromosome 17p, the location of TP53, is lost in 60% of invasive cancers [START_REF] Olumi | Allelic loss of chromosome 17p distinguishes high grade from low grade transitional cell carcinomas of the bladder[END_REF][START_REF] Habuchi | Allelic loss of chromosome 17p in urothelial cancer: strong association with invasive phenotype[END_REF] . Such loss is a rare event in non-invasive tumours. Alterations in related regulatory molecules such as the cyclin-dependent kinase (CDK) inhibitors p21 and p16 have also been linked to increased disease recurrence and progression to invasive disease [START_REF] Stein | Effect of p21WAF1/CIP1 expression on tumor progression in bladder cancer[END_REF][START_REF] Orlow | Deletions of the INK4A gene in superficial bladder tumors. Association with recurrence[END_REF] .Initial studies of mutant p53 expression demonstrated a strong association with disease grade and stage, as well as a significantly higher risk of recurrence and decreased survival [START_REF] Esrig | Accumulation of nuclear p53 and tumor progression in bladder cancer[END_REF] . However, a recent metaanalysis of 168 published reports found such associations to be weak and inconsistent [START_REF] Malats | P53 as a prognostic marker for bladder cancer: a meta-analysis and review[END_REF] . This view was supported by an international panel which concluded that whilst p53 showed promise as a prognostic biomarker, the data remain inconclusive [START_REF] Habuchi | Prognostic markers for bladder cancer: International Consensus Panel on bladder tumor markers[END_REF] . Studies examining alterations in the expression of p16 and p21 also show contradictory findings [START_REF] Stein | Effect of p21WAF1/CIP1 expression on tumor progression in bladder cancer[END_REF][START_REF] Friedrich | Frequent p16/MTS1 inactivation in early stages of urothelial carcinoma of the bladder is not associated with tumor recurrence[END_REF][START_REF] Shariat | p53, p21, pRB, and p16 expression predict clinical outcome in cystectomy with bladder cancer[END_REF][START_REF] Bartoletti | Loss of P16 expression and chromosome 9p21 LOH in predicting outcome of patients affected by superficial bladder cancer[END_REF][START_REF] Liukkonen | Evaluation of p21WAF1/CIP1 and cyclin D1 expression in the progression of superficial bladder cancer. Finbladder Group[END_REF][START_REF] Shariat | Association of p53 and p21 expression with clinical outcome in patients with carcinoma in situ of the urinary bladder[END_REF] , although their prognostic significance did improve significantly when incorporated into a larger panel including p53 [START_REF] Chatterjee | Combined effects of p53, p21, and pRb expression in the progression of bladder transitional cell carcinoma[END_REF][START_REF] Hitchings | Prediction of progression in pTa and pT1 bladder carcinomas with p53, p16 and pRb[END_REF][START_REF] Shariat | Predictive value of cell cycle biomarkers in nonmuscle invasive bladder transitional cell carcinoma[END_REF] . Unfortunately, the prognostic significance of cyclins D1 and E also appears uncertain.

Cyclins D1 and E

Although early studies linked cyclin D1 expression to disease recurrence [START_REF] Sgambato | Cyclin D1 expression in papillary superficial bladder cancer: its association with other cell cycle-associated proteins, cell proliferation and clinical outcome[END_REF] , this observation is not widely supported and a number of studies have demonstrated that there is no association between cyclin D1 expression and prognosis [START_REF] Liukkonen | Evaluation of p21WAF1/CIP1 and cyclin D1 expression in the progression of superficial bladder cancer. Finbladder Group[END_REF][START_REF] Wagner | Cyclin D1 overexpression lacks prognostic significance in superficial urinary bladder cancer[END_REF][START_REF] Shariat | Correlation of cyclin D1 and E1 expression with bladder cancer presence, invasion, progression, and metastasis[END_REF] . Other studies have shown that low cyclin E expression is associated with increased disease recurrence, progression and reduced overall survival [START_REF] Shariat | Correlation of cyclin D1 and E1 expression with bladder cancer presence, invasion, progression, and metastasis[END_REF][START_REF] Richter | High-throughput tissue microarray analysis of cyclin E gene amplification and overexpression in urinary bladder cancer[END_REF][START_REF] Kamai | Decreasing of p27(Kip1)and cyclin E protein levels is associated with progression from superficial into invasive bladder cancer[END_REF] . However, these findings are also widely disputed and appear to be lost on multivariate analysis [START_REF] Richter | High-throughput tissue microarray analysis of cyclin E gene amplification and overexpression in urinary bladder cancer[END_REF][START_REF] Shariat | Association of cyclin D1 and E1 expression with disease progression and biomarkers in patients with nonmuscle-invasive urothelial cell carcinoma of the bladder[END_REF] .

Ki67

Ki67 is a commonly used marker of cell proliferation and has been extensively studied in almost all cancers. Since Ki67 is only expressed by cells actively engaged in the cell cycle, positive immunostaining is considered a good correlate of biological 'aggressiveness'.

Numerous studies have identified Ki67 as an independent prognostic marker of disease recurrence, progression and disease-specific survival [START_REF] Liukkonen | Prognostic value of MIB-1 score, p53, EGFr, mitotic index and papillary status in primary superficial (Stage pTa/T1) bladder cancer: a prospective comparative study. The Finnbladder Group[END_REF][START_REF] Pfister | Prognostic value of the proliferative index determined by Ki-67 immunostaining in superficial bladder tumors[END_REF][START_REF] Wu | The role of bcl-2, p53, and ki-67 index in predicting tumor recurrence for low grade superficial transitional cell bladder carcinoma[END_REF] . Whilst variation in study design and assay interpretation has resulted in a number of contradictory reports, a large multicentre study has confirmed that Ki67 is independently associated with disease recurrence, overall survival and could be used to predict which patients might benefit from preoperative systemic chemotherapy [START_REF] Margulis | Multi-institutional validation of the predictive value of Ki-67 labeling index in patients with urinary bladder cancer[END_REF] . Dysregulation of the apoptotic signalling pathways plays an important role in tumourigenesis, allowing the continued survival of genetically damaged tumour cells. The prognostic significance of the anti-apoptotic gene Bcl-2 in bladder cancer has been studied by a number of groups. Some have shown that increased expression of Bcl-2 is associated with aggressive disease and poor outcome [START_REF] Kirsh | Expression of bcl-2 and bcl-X in bladder cancer[END_REF][START_REF] Ong | Prognostic factors in transitional cell cancer of the bladder: an emerging role for Bcl-2 and p53[END_REF][START_REF] Karam | Use of combined apoptosis biomarkers for prediction of bladder cancer recurrence and mortality after radical cystectomy[END_REF] , whereas others have questioned this association [START_REF] Wu | The role of bcl-2, p53, and ki-67 index in predicting tumor recurrence for low grade superficial transitional cell bladder carcinoma[END_REF][START_REF] Lipponen | Expression of the apoptosis suppressing bcl-2 protein in transitional cell bladder tumours[END_REF] . A number of recent studies have highlighted the potential importance of survivin, which is known to regulate mitotic progression and enhance angiogenesis [START_REF] Ambrosini | A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma[END_REF][START_REF] Altieri | Survivin, versatile modulation of cell division and apoptosis in cancer[END_REF][START_REF] Duffy | Survivin: a promising tumor biomarker[END_REF] . A number of studies have demonstrated a strong association between overexpression of survivin and disease recurrence and progression [START_REF] Karam | Use of combined apoptosis biomarkers for prediction of bladder cancer recurrence and mortality after radical cystectomy[END_REF][START_REF] Shariat | Survivin expression is associated with bladder cancer presence, stage, progression, and mortality[END_REF] , a finding recently confirmed in a large multicentre study [START_REF] Shariat | Survivin as a prognostic marker for urothelial carcinoma of the bladder: a multicenter external validation study[END_REF] .

Apoptosis

However, the association was relatively small and the authors reported that there was no statistical or clinical benefit to be gained from adding survivin to standard histopathological criteria for the prediction of outcome.

Dissection of the molecular pathways involved in the pathogenesis of bladder cancer has provided a rich hypothesis-driven source of potential prognostic biomarkers. These individual markers are quantifiable and show some association with disease behaviour and outcome.

However, few of these markers have proven themselves in the clinical arena and none has been adopted into routine practice. This failure arises in part due to the varied approach to study design (retrospective vs prospective), patient selection and statistical analysis employed by different research groups. In particular, many of the studies described above used immunohistochemical-based assays to quantify protein expression. Whilst this technique has revolutionised diagnostic histopathology in recent years, it is a technique which can be difficult to standardize. Differences in a number of critical variables such as the type of antibodies used, antigen retrieval methods, reagents, pH, temperature, and the quality of the (REporting recommendations for tumour MARKer prognostic studies) [START_REF] Mcshane | REporting recommendations for tumor MARKer prognostic studies (REMARK)[END_REF] . The report lists 20 recommendations outlining specific requirements for study design,, patient and specimen characteristics, assay design and statistical methodology. Many funding organisations and journals have adopted these recommendations and now include them in their reporting criteria. Methodological standardization in immunohistochemistry is also being addressed through the development of national quality assurance schemes such as the UK National External Quality Assessment Service for Immunocytochemistry (NEQAS-ICC, see www.ukneqasicc.ucl.ac.uk) which aim to improve the quality and reproducibility of this technique through education and dissemination of best practice.

In addition to the problems addressed by the REMARK guidelines and national quality assessment schemes, the inability to identify robust prognostic biomarkers also reflects a naive approach to tumourigenesis. Whilst a number of important molecular pathways have been identified in bladder cancer, the mechanisms underlying the malignant transformation and metastatic potential of a tumour cell will in reality depend upon complex and often redundant interconnecting networks of regulatory proteins. The diagnostic waters are further muddied by the diverse genetic background of individual patients, and in such circumstances the identification of lone markers of such complex disease behaviour appears overly optimistic. Indeed, the recent publication of the first cancer genome has demonstrated that a tumour cell might typically carry over 30,000 genetic mutations [START_REF] Pleasance | A comprehensive catalogue of somatic mutations from a human cancer genome[END_REF] .

Genomic Profiling

Fortunately, the emerging field of high-throughput transcript profiling using genomic and proteomic technologies has provided researchers with the means to study the global expression of genes and their related proteins within individual tumours. This approach does not require an initial hypothesis and so is not restricted to the study of specific molecular pathways. Instead characteristic molecular signatures emerge from the 'transcriptome' and are linked to measures of disease outcome.

Whilst this technology has been most comprehensively applied to the study of breast cancer 88, 89 , a growing number of genomic studies have been reported in bladder cancer. These studies have identified unique gene expression signatures associated with disease stage [START_REF] Dyrskjot | Identifying distinct classes of bladder carcinoma using microarrays[END_REF][START_REF] Blaveri | Bladder cancer outcome and subtype classification by gene expression[END_REF][START_REF] Sanchez-Carbayo | Gene discovery in bladder cancer progression using cDNA microarrays[END_REF][START_REF] Modlich | Identifying superficial, muscle-invasive, and metastasizing transitional cell carcinoma of the bladder: use of cDNA array analysis of gene expression profiles[END_REF][START_REF] Dyrskjot | Gene expression in the urinary bladder: a common carcinoma in situ gene expression signature exists disregarding histopathological classification[END_REF] , progression [START_REF] Dyrskjot | A molecular signature in superficial bladder carcinoma predicts clinical outcome[END_REF][START_REF] Wang | Development of a multiplex quantitative PCR signature to predict progression in non-muscle-invasive bladder cancer[END_REF] , recurrence [START_REF] Dyrskjot | Identifying distinct classes of bladder carcinoma using microarrays[END_REF] , nodal metastases [START_REF] Sanchez-Carbayo | Defining molecular profiles of poor outcome in patients with invasive bladder cancer using oligonucleotide microarrays[END_REF] and response to chemotherapy [START_REF] Takata | Predicting response to methotrexate, vinblastine, doxorubicin, and cisplatin neoadjuvant chemotherapy for bladder cancers through genome-wide gene expression profiling[END_REF][START_REF] Als | Emmprin and survivin predict response and survival following cisplatin-containing chemotherapy in patients with advanced bladder cancer[END_REF] .

Notably Dyrskjot and co-workers demonstrated that tumours histologically defined as superficial but which expressed an invasive gene signature displayed a significantly higher probability of subsequent disease progression and poor outcome [START_REF] Dyrskjot | Identifying distinct classes of bladder carcinoma using microarrays[END_REF] . These findings demonstrate the potentially superior prognostic and predictive power that gene expression signatures may have over conventional diagnostic techniques. It is also encouraging that, whilst these technologies identify genes in an unbiased manner, the molecular signatures appear enriched for genes involved in pathways already known to be important in carcinogenesis. One of the most prominent of these relates to cell proliferation and the cell cycle and is observed in many different cancer-specific gene signatures.

However, whilst gene expression profiling has undoubtedly provided new insights into the pathogenesis of bladder cancer and the search for biomarkers, the reliability and reproducibility of gene expression profiling has been widely questioned [100][101][102] . For example, there is little or no correlation between the gene signatures reported in different studies [START_REF] Blaveri | Bladder cancer outcome and subtype classification by gene expression[END_REF][START_REF] Dyrskjot | A molecular signature in superficial bladder carcinoma predicts clinical outcome[END_REF]103 and many fail to provide a significantly improved prognostic classification when compared to conventional clinicopathological criteria. There are several reasons for the apparent failure of gene expression profiling. Many of the studies performed to date have analysed very small patient cohorts, typically drawn from retrospective studies and/or archival tissue banks, and lack sufficient independent validation. Validation across different analysis platforms, methodologies, laboratories and patient groups is essential, as is testing in prospective studies, if this technology is to be adopted into routine clinical use. Expression profiling of resected tumours and biopsy material also suffers from sampling error as RNA/DNA/protein are typically extracted unselectively. This is especially important when considering proliferation signatures since tumours commonly comprise a heterogeneous population of neoplastic cells, many of which will not be actively cycling 104 . The presence of contaminating benign neoplastic cells, lymphoid follicles, inflammatory cells and reactive stroma further distort analysis by adding potentially large numbers of cells with complex cell cycle kinetics. Finally, the cost of genomic-based tests are likely to be considerable. For example, Oncotype DX (Genomic Health) and MammaPrint (Agendia), have recently been introduced as tests which may improve risk assessment in early stage breast cancer, but each costs in excess of $3,500 per test.

DNA Replication Licensing Factors as Diagnostic and Prognostic Biomarkers

Analysis of upstream signalling pathways controlling cell proliferation, differentiation, apoptosis and DNA damage response by conventional hypothesis-driven molecular approaches and genome wide analyses remain unproven as routine tools for clinicopathological assessment of malignant tumours. An alternative approach is to focus on

Cell cycle algorithm

The utility of MCM proteins as cancer biomarkers is further enhanced by combining them with other key cell cycle regulators. Geminin, a repressor of MCM loading at origins of DNA replication, is present only in S, G2 and M phases, and can therefore act as a marker for S-G2-M phase progression 117 . Similarly, protein levels of the mitotic kinases Polo-like kinase 1 (Plk1) and Aurora A are negligible in G1, increase during S phase and reach a peak during G2/M, with rapid degradation occurring at the end of mitosis and therefore also represent biomarkers of S-G2-M phase progression 118 . Phosphorylation of the Aurora kinase substrate Histone H3 on serine 10 (H3S10ph) is restricted to mitosis, enabling its use as a mitosisspecific marker 118,119 .

By combining this panel of G1/S and G2/M regulators into a multiparameter algorithm, it is possible to provide a detailed analysis of the cell cycle kinetics of complex tumour cell populations. For example, in a recent study we have demonstrated that breast tumours can be classified into one of three cell cycle phenotypes: (I) out-of-cycle tumours in which tumour cells lack expression of all markers; (II) slowly cycling tumours in which only Mcm2 is expressed, indicating that the majority of cells reside within a prolonged or arrested G1 phase; (III) rapidly cycling tumours in which the tumour cells express high levels of S-G2-M and M phase markers 120 . Importantly, patients with actively cycling phenotype III tumours have a significantly worse prognosis than those displaying phenotypes I and II. Furthermore, this prognostic readout is superior to both morphological grading and Ki67 on multivariate analysis 118,120 bladder cancer, the application of the multiparameter algorithm to these tumours may provide significant improvements in prognostication. This could be particularly helpful in the assessment of superficial, pTa and pT1 tumours, where the distinction between different grades remains highly subjective and controversial. Whilst many pathologists and clinicians continue to favour the 1973 WHO classification, this approach assigns a significant proportion of lesions to the intermediate or grade 2 category. Interestingly, initial studies of a randomly selected series of grade 2 tumours has demonstrated that all three cell cycle phenotypes are represented, further highlighting the constraints of conventional histological grading (Figure 3). In contrast to Ki67, the multiparameter algorithm also offers the ability to determine where in the cell cycle a tumour predominantly resides. As a result this will enable clinicians to identify those patients most likely to benefit from S and M phase specific chemotherapeutic agents.

In contrast to genomic studies which typically require specialised laboratory facilities as well as fresh tissue, expression levels of cell cycle proteins can be accurately quantified using relatively simple and inexpensive immunohistochemical staining of routinely processed paraffin-embedded material. This not only allows cell cycle dynamics to be easily determined in individual patient samples but also allows tumour-specific biomarker expression to be distinguished from that present within the background stroma. Moreover, since this approach focuses solely on the kinetics of cell proliferation and not specific molecules or pathways, it should be applicable to many different cancers, including bladder cancer.

Future directions

The search for more sensitive and specific biomarkers of bladder cancer continues. Further progress might be achieved by combining existing biomarkers into larger, more powerful Proteomics in particular, is a rapidly developing field of research which offers the opportunity to obtain a real time assessment of global protein expression and has been used to identify tumour-specific proteins secreted into bodily fluids including urine. By comparing protein expression between patients with bladder cancer and healthy controls or proteins expressed by invasive and non-invasive tumours, it is hoped that tumour-specific protein profiles can be identified which have diagnostic and prognostic value. However, like genomics, proteomics is a complex and expensive technique and currently lacks the reproducibility required for routine diagnostic and prognostic testing 122 . Despite numerous published studies, the number and quality of biomarkers available for use in bladder cancer remains disappointingly low. Regardless of the approach taken to identify biomarkers, it is essential that future studies are subjected to rigorous testing which must include prospective multicentre trials. The biopharmaceutical industry has enthusiastically embraced the development of molecular diagnostics and their involvement will certainly help fund improved research and development in this field. However, the introduction of singleuse biomarker tests whose application is restricted to specific cohorts of patients will invariably result in high unit costs in the short term. However, despite these commercial constraints the search for diagnostic biomarkers is likely to continue. Both individual patients and healthcare providers are set to benefit from a new era of 'personalised medicine' in which biomarkers will direct the use of increasingly targeted and potentially more cost effective therapeutic agents.

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

  prospective studies[START_REF] Bostwick | Urothelial papillary (exophytic) neoplasms[END_REF] . The difficulties presented by histological grading are further compounded by tumour heterogeniety and multicentricity.

  Whilst initial reports are often promising, subsequent studies on the same or related markers † Trastuzumab for the adjuvant treatment of early-stage HER2-positive breast cancer. National Institutes for Clinical Excellence (NICE), August 2006. www.nice.org.uk/nicemedia/pdf/TA107guidance.pdf findings. These constraints are discussed below in relation to biomarkers that have undergone clinical evaluation.

  of cyclins D1 and E have also been observed in bladder cancer.Cyclin D1 and cyclin E are both important cell cycle regulators controlling G1 phase progression and the G1/S phase transition respectively. Their dysregulation has been linked to uncontrolled cell proliferation and establishment of autonomous cancer cell cycles.

  likely to contribute to the varied and often contradictory findings reported in prognostic biomarker studies. In an attempt to improve and standardise current prognostic biomarker development, McShane et al. published a set of reporting criteria under the acronym 'REMARK'

  the cell cycle machinery, which act as an integration point for information transduced through upstream signalling pathways. We and others have shown that expression of Mcm2-7 (MCM) proteins, constituents of the DNA replicative helicase and essential for DNA replication initiation 105 , can be used to determine the cell cycle, state in human tissues106 . MCMs are strongly expressed by cells during all stages of the proliferative cell cycle but are absent in quiescent, terminally differentiated and senescent out-of-cycle states, and as such represent new biomarkers of growth 107, 108 .Mcm5Since neoplastic cells are characterized by uncontrolled proliferation, MCM expression has been extensively investigated as a diagnostic marker in a broad range of neoplastic conditions including bladder cancer[109][110][111][112][113][114] . Immunohistochemical staining of normal urothelium with antibodies directed against Mcm5 shows positive staining restricted to the basal stem-transit proliferative compartment 115 . In contrast, both non-invasive and invasive urothelial carcinomas show Mcm5 staining in all layers of the urothelium including superficial epithelial cells, reflecting the arrested maturation of these tumour cell populations and their inability to exit the proliferative cycle. This results in the exfoliation of Mcm5-expressing tumour cells into the urine. Mcm5 protein levels can be accurately measured in urine sediments using a simple automated immunofluorometric assay. Application of this assay in a blinded clinical trial has demonstrated that elevated Mcm5 levels in urine sediments is highly predictive of bladder cancer and superior to urine cytology 116 . Notably, unlike many of the commercially available diagnostic biomarkers discussed earlier, the specificity of the Mcm5 test is not affected by benign proliferative or inflammatory conditions, including bacillus Calmette-Guerin (BCG) induced granulomatous cystitis. Moreover, this study also Mcm5 urine test is able to detect early (organ confined) prostate cancer.

  through the discovery of new biomarkers that robustly outperform current diagnostic tests and clinical indicators. A number of new approaches to biomarker discovery are currently being exploited in bladder cancer. These include molecular techniques such as proteomics, analysis of DNA methylation status and cancer specific microRNAs 121 .

Figure 1 .

 1 Figure 1. Graph showing the increasing number of publications identified in a Medline

Figure 2 .

 2 Figure 2. Diagrammatic representation of a bladder showing proposed pathways involved in

Figure 3 .

 3 Figure 3. Photomicrographs of three different grade 2 bladder cancers (WHO 1973

Figure 1 Page

 1 Figure 1

  . Since grading and Ki67 expression are important prognostic indicators in

	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	18

Table 1 .

 1 Comparison between cytology and commercially available biomarker tests for use in the diagnosis and surveillance of bladder cancer. The broad range of percentages provided for sensitivity and specificity reflect differences in study sizes, assay cut-points, and the grade and stage of tumours studied. † Data obtained from references13 & 24.

	Phenotype I

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

of

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

Acknowledgements

We thank Marco Loddo, Alex Wollenschlaeger and Dr Sara Rodriguez-Acebes for their help with the illustrations and laboratory work.


PhD Katherine Hunt
email: katherine.hunt@pms.ac.uk

PhD BA Helen F Coelho

Barbara Wider

M A Ma

Rachel Perry Mphil

M A Ba

Shao Kang

Hung Msc Msc

PhD BSc Rohini Terry

MD Edzard Ernst

PhD Fmedsci Fsb

Frcp Frcp

Complementary and alternative medicine use in England: Results from a national survey

Keywords: Complementary and alternative medicine, prevalence, survey Complementary and alternative medicine, prevalence, survey

Objectives: In many countries recent data on the use of complementary and alternative medicine (CAM) are available. However, in England there is a paucity of such data. We sought to determine the prevalence and predictors of CAM use in England.

Design: Data were obtained from the Health Survey for England 2005, a national household survey which included questions on CAM use. We used binary logistic regression modelling to explore whether demographic, health and lifestyle factors predict CAM use.

Results: Data were available for 7630 respondents (household response rate 71%). Lifetime and 12-month prevalence of CAM use were 44.0% and 26.3%; 12.1% had consulted a practitioner in the last 12 months. Massage, aromatherapy and acupuncture were the most commonly used therapies. Twenty-nine percent of respondents taking prescription drugs had used CAM in the last 12 months. Women (OR 0.491, 95% CI: 0.419, 0.577), university educated respondents (OR 1.296, 95% CI: 1.088, 1.544), those suffering from anxiety or depression (OR 1.341, 95% CI:

1.074, 1.674), people with poorer mental health (on GHQ: OR 1.062, 95% CI 1.026, 1.100) and lower levels of perceived social support (1.047, 95% CI: 1.008, 1.088), people consuming ≥5 portions of fruit and vegetables a day (OR 1.327, 95% CI: 1.124, 1.567) were significantly more likely to use CAM.

Conclusion:

CAM use in England remains substantial, even amongst those taking prescription drugs. These data serve as a valuable reminder to medical practitioners to ask patients about CAM use and should be routinely collected to facilitate prioritisation of the research agenda in CAM.

• In the UK, 12-month prevalence of CAM use has been estimated between 20-28% but these data are outdated.

• In previous surveys, CAM use has been shown to be more common in females, younger people and people with a higher income and level of education.

What does this article add?

• These nationally representative data come from the largest survey of CAM use in England to date and indicate that the lifetime and 12-month prevalence of CAM use in England is 44% and 26% respectively.

• Our report provides the first predictors of CAM use showing that the presence of anxiety or depression, low levels of perceived social support, eating a healthy diet, being female, and having an income above the national average are independent predictors of 12-month CAM use. • CAM use is common among those taking prescription drugs thus emphasising the importance of asking patients about their use of CAM and routinely collecting CAM prevalence data in future.

Background

Complementary and alternative medicine (CAM) has become an important feature of healthcare. In many countries, CAM use is reported to be substantial and increasing. [START_REF] Barnes | Complementary and alternative medicine use among adults and children: United States[END_REF][START_REF] Xue | Complementary and alternative medicine use in Australia: a national population-based survey[END_REF] In the UK, survey data have identified substantial use of practitioner-provided CAM and over-the-counter CAM remedies. Indeed, in a 1999 random telephone survey of 1204 adults representative of the UK population, 12 month prevalence of CAM use was 20%. [START_REF] Ernst | The BBC survey of complementary medicine use in the UK[END_REF] A survey of 2669 respondents in England in 1998 reported a higher 12 month prevalence of use (28.3%), and found lifetime prevalence of CAM use to be 46.6. [START_REF] Thomas | Use and expenditure on complementary medicine in England: a population based survey[END_REF] In 2001, the 12 month prevalence of seeing a CAM practitioner in the UK was 10.0% [START_REF] Thomas | Use of complementary or alternative medicine in a general population in Great Britain. Results from the National Omnibus survey[END_REF] and 13.6% in England. [START_REF] Thomas | Use and expenditure on complementary medicine in England: a population based survey[END_REF] All of these data are now outdated.

Reliable, nationally representative and up-to-date CAM usage data are of great importance to policy makers. Some CAM treatments are available through the NHS, 6,7 primarily due to patient demand. 8 Prevalence data can also assist in prioritising research into the safety and efficacy of CAM. To address this need we present an analysis of data relevant to CAM which was obtained as part of the 2005 Health Survey for England (HSE). [START_REF]Health Survey for England: The Health of Older People[END_REF] Our primary aim was to provide lifetime and 12 month prevalence data for CAM use and CAM practitioner consultations in England. Our secondary aim was to identify predictors of 12 month CAM use in England.

Methods

The HSE 2005 was carried out by the Joint Health Surveys Unit of the National Centre for Social Research and is based on a representative sample of the civilian non-institutionalised household population in England. Ethical approval for the survey was obtained from the London multi-centre Research Ethics Committee. Data were collected between January and December 2005. Full information on the HSE 2005 methods can be found elsewhere. [START_REF]Health Survey for England[END_REF] The UK Data Archive granted permission to use these data.

Sample

Sampling was conducted using a multi-stage stratified probability sampling method, resulting in 7200 randomly selected addresses from 720 postcode sectors representing the population living in private households in England. This report is based on adults aged at least 16 years from these households. Trained interviewers administered a Household Questionnaire to a representative of the household who provided information on all household members. Additional individual questionnaires were administered to all eligible participants; these included questions related to general health, lifestyle habits and CAM use. Anthropometric measurements and vital statistics were also recorded.

Nurses conducted follow-up visits to collect data on prescribed medication, vitamin supplements, nicotine replacements and eating habits. Built in quality control measures included recalls on 10% of households to check data consistency and monthly interviewer-nurse discussions to check for anomalies.

Variables

CAM variables were derived from the following three questions about each CAM modality: 1) 'Have you used this CAM?' 2) 'Have you used this CAM in the last 12 months?' 3) 'Have you consulted a practitioner of this CAM in the last 12 months?' There is a lack of consensus regarding what constitutes CAM, so a broad range of therapies were covered (included CAM modalities are detailed in Figure 1). Most of these are categories of therapies rather than distinct therapies, of which there would be thousands: for example, specific types of massage therapy were not differentiated. Herbal medicine includes both single and combination preparations but does not include Chinese Herbal Medicine (CHM) which is listed as Traditional Chinese Medicine (TCM). Indeed, in the HSE, the term TCM was used to describe CHM only, with acupuncture being separately rated. Demographic variables included age, sex, ethnicity, education, income, employment status, socioeconomic group and Indices of Deprivation (ID2007) [START_REF]The English Indices of Deprivation[END_REF] [START_REF] Group | EuroQol: a new facility for the measurement of health-related quality of life[END_REF] which covers five dimensions associated with health (mobility, leisure, self-care, main role, family and leisure activities, and pain and mood -although scores can be summed to create a mean score, we analysed these separately) has good reliability [START_REF] Van Agt | Test-retest reliability of health state valuations collected with the EuroQol questionnaire[END_REF] and reasonable validity; [START_REF] Hurst | Validity of EuroQol -a generic health status instrument -in patients with rheumatoid arthritis[END_REF] the General Health Questionnaire (GHQ-12), [START_REF] Goldberg | A scaled version of the General Health Questionnaire[END_REF] a self-administered screening instrument with good reliability and validity, [START_REF] Goldberg | Manual of the General Health Questionnaire[END_REF][START_REF] Mari | A comparison of the validity of two psychiatric screening questionnaires (GHQ-12 and SRQ-20) in Brazil using Relative Operating Characteristic (ROC) analysis[END_REF] widely used to detect current psychiatric disorders (a cut off of 2/3 is Lifestyle variables included smoking history; consumption of five portions of fruit and vegetables a day; membership of a religious organisation; use of vitamins or supplements.

Statistical analysis

Descriptive statistics are used to present the demographic characteristics of the whole sample. For the purpose of the present analyses, it was assumed that those who did not answer the three CAM questions had not used CAM in their lifetime. Lifetime and 12-month prevalence data are presented as frequencies and percentages To assess whether 12 month CAM use differed according to each demographic, health, and lifestyle variable, the following categorical variables were converted to binary: ethnicity, educational attainment, social group, housing tenure, household income, economic status, and fruit and vegetable consumption. Bivariate analyses were performed using chi square tests for categorical data and t-tests for continuous data. In order to determine predictors of 12 month CAM use, variables that met significance were entered into binary logistic regression models. Four logistic regression models are presented. The first three models include demographic factors, healthbased factors, and lifestyle factors respectively. The final model (table 2) was constructed from variables which met significance in the first three models. These data are presented as odds ratios with 95% confidence intervals.

Results

Interviews were carried out at 71% of the 7200 approached households, and 89% of those aged 16 and over in those households were interviewed (N = 7,630). The socio-demographic characteristics of the sample are presented in table 1.

Prevalence of CAM use

The lifetime prevalence of CAM use was 44.0% (n=3355) 12 month prevalence was 26.3% (n=2005), and 12.1% (n=922) had consulted a practitioner in the last 12 months. Of all CAM modalities, massage had the highest lifetime prevalence of use (13.1%), followed by aromatherapy (11.2%) and acupuncture/acupressure (11.2%), relaxation (10.0%), and osteopathy (9.9%). Twelve-month prevalence follows a similar pattern (Figure 1). Massage therapists were the most frequently visited CAM practitioners (Figure 1). Dowsing had the highest proportion of female users (85%) whilst chiropractic had the highest proportion of male users (44.8%) (Figure 1). Users of osteopathy were older and users of Unani younger than users of any other CAM.

Bivariate analyses

Demographic characteristics of CAM users

Females (19.6% male, 31.8% female, χ 2 =144.31, p<.001), white respondents (26.8% white, 21.8% non-white, χ 2 = 6.98, p<.01), those with a university qualification (

Health characteristics of CAM users

Respondents who are obese (28.7% BMI ≥30 kg/m², 25.7% BMI <30 kg/m², χ 2 = 5.90, p<.05), have no mobility problems (27.7% no problems, 24.1% mobility problems, χ 2 = 6.89, p<.01), have pain (29.7% pain, 25.7% no pain, χ 2 = 12.44, p<.001), anxiety or depression (35.0% with anxiety/depression, 25.3% no anxiety/depression, χ 2 = 50.47, p<.001), or a longstanding illness, disability or infirmity (28.5% with longstanding illness, 24.4% no illness, χ 2 = 15.79, p<.001) were more likely to use CAM. CAM users perceived themselves as having reduced levels of perceived social support (CAM users: 19.9 +/-2.2, non-users: 19.6 +/-2.3, t = -4.83, p<.001) and poorer psychiatric health as per the GHQ (CAM users: 1.6 +/-2.8, non-users: 1.1 +/-2.4, t = -6.87, p<.001) compared to those who had not used CAM. No significant differences between CAM users and non-users were noted in medication use (28.9% taking medications, 27.8% not taking, χ 2 = 0.815, p=.867) , in the proportion with problems with self-care (27.0% no problems, 26.9% problems, χ 2 = 0.001, p=.978), problems with usual activities (29.2% problems 27.5% no problems, χ 2 = 1.236, p=.266) or difficulty walking quarter of a mile (17.3% no problems, 15.5% problems, χ 2 = 0.687, p=.407).

Lifestyle characteristics of CAM users

Respondents who are a member of a religious group or organisation (29.2% members, 39.7% nonmembers, χ 2 = 27.35, p<.001), do not smoke (29.4% non-smokers, 24.9% smokers, χ 2 = 9.61, p<.01), consume five or more portions of fruit and vegetables every day (33.2% ≥5 portions, 23.6% <5 portions, χ 2 = 73.69, p<.001), and take vitamins/supplements (39.7% taking supplements, 23.1% not taking, χ 2 = 159.90, p<.001) were significantly more likely to use CAM.

Predictors of 12 month CAM use

A first regression model (R Square 0.082) was used to investigate whether sociodemographic factors predict CAM use. It showed that CAM-users were more likely to be female (OR 2.107, 95% CI: 1.870,2.376), white (OR 1.331, 95% CI: 1.039, 1.704), have a university education (OR 1.441, 95% CI:1.257, 1.651), work in a professional/managerial role (OR 1.336, 95% CI: 1.169, 1.527), have a household income above the 2005 national average (OR 1.162, 95% CI: 1.010, 1.336), and be in active employment (OR 1.449, 95% CI: 1.263, 1.662). In addition, using CAM was associated with decreasing deprivation (OR 0.748, 95% CI: 0.884, 0.970). Age was not a significant independent predictor of 12 month CAM use OR 0.997, 95% CI:0.993, 1.001).

A second regression model (R Square 0.035) was used to investigate whether health factors predict CAM use. It indicated that the presence of mobility problems was associated with a decrease in the odds of using CAM (OR 0.543, 95% CI: 0.453, 0.651) whilst the presence of pain (OR 1.287, 95% CI: 1.117, 1.483), anxiety or depression (OR 1.384, 95% CI: 1.177, 1.627) and longstanding disease or illness (OR 1.230, 95% CI: 1.089, 1.390) was associated with an increase in the odds of using CAM. Users of CAM were more likely have lower levels of perceived social support (OR 1.091, 95% CI: 1.062, 1.120) and poorer mental health as per the GHQ (OR 1.072, 95% CI: 1.046, 1.099).

The next model was performed to assess whether lifestyle factors predict CAM use (R Square 0.063). Members of a religious group were more likely to use CAM (OR 1.490, 95% CI: 1.210, 1.835), as were participants taking vitamins or supplements (OR 2.230, 95% CI: 1.929, 2.577) and individuals who eat five or more portions of fruit and vegetables a day (OR 1.396, 95% CI: 1.202, 1.621). Smoking was not a significant predictor of CAM use (OR 0.877, 95% CI: 0.745, 1.031).

A final model (Table 2) was created to investigate which of the significant socio-demographic, health and lifestyle factors from previous models independently predicted CAM use (R square = 0.133). Variables which were not significant predictors in the first three models were therefore not included in the final model. In this model, women were more likely to use CAM (OR 0.491, 95% CI: 0.419, 0.577), as were participants with a university qualification (OR 1.296, 95% CI: 1.088, 1.544), those in active employment (OR 1.421 95% CI: 1.177, 1.714), suffering from anxiety or depression (OR 1.341, 95% CI: 1.074, 1.674), with lower levels of perceived social support (OR 1.047, 95% CI: 1.008, 1.088), poorer mental health as indicated by the GHQ (OR 1.062, 95% CI 1.026, 1.100), consuming a diet of five or more portions of fruit and vegetables a day (OR 1.327, 95% CI: 1.124, 1.567) and vitamins or supplements (OR 1.327, 95% CI 1.124, 1.567). All other variables were not significant independent predictors of CAM use. The 12 month prevalence of CAM use in England was 26.3%. This is similar to the last 12 month prevalence estimate in England of 28.3%. It is also far lower than CAM use in the US 1 , Germany 18 , Australia 2 or Japan [START_REF] Yamashita | Popularity of complementary and alternative medicine in Japan: a telephone survey[END_REF] where 12 month prevalence was reported to be 40%, 62%, 69% and 76% respectively. Massage therapy was the most frequently used CAM, followed by aromatherapy and acupuncture. Of the 24 CAM therapies assessed, we found herbal medicine to have the sixth highest lifetime prevalence and fourth highest 12 month prevalence of use. Chiropractic had the ninth highest lifetime prevalence and tenth highest 12 month prevalence of use. This is clearly at odds with policy documentation which suggests that acupuncture, herbal medicine, and chiropractic are the most commonly used CAMs. [START_REF]House of Lords Select Committee report on Complementary and Alternative[END_REF][START_REF]Report to ministers from the Department of Health steering group on the statutory regulation of practitioners of acupuncture, herbal medicine, traditional Chinese medicine and other traditional medicine systems practised in the UK[END_REF] Our regression models suggest that individuals experiencing anxiety, depression or other long standing illnesses, with poorer mental health and with lower levels of perceived social support are more likely than those in good health to use CAM. We also found that CAM is used more by females than males, those with a university education, those in active employment, and those who appear to pursue healthy lifestyles (consuming more than five portions of fruit/vegetables per day and using vitamin supplements) compared to those who do not. This confirms previous research suggesting that individuals who are more likely to select healthy lifestyle choices may also be likely to proactively engage in other self-care behaviours including CAM use, and that less risky health behaviours may be associated with CAM use. [START_REF] Nahin | Health behaviors and risk factors in those who use complementary and alternative medicine[END_REF] The HSE [START_REF]Health Survey for England: The Health of Older People[END_REF] data show that different CAMs are not used uniformly. For example, osteopathy, chiropractic and acupuncture have the most even gender spread and the mean age of these users is slightly older than the users of other CAM, whilst dowsing, iridology, kinesiology, crystal therapy and reflexology were used predominantly by younger women. This may in part be due to the referral patterns of health professionals. For example, NHS referrals to osteopathy and chiropractic for back pain treatment may be common while more exotic forms of CAM may be less likely to be endorsed by allopathic health providers and prohibitively costly for many consumers.

Discussion

Prudence is necessary when comparing our findings with previous surveys. First, any differences may be due to sampling methods rather than apparent trends over time or apparent differences between countries. Second, there is no universally accepted definition of CAM. Therefore different surveys include different CAM modalities. For example several surveys have included nutritional supplements, yoga and prayer as CAM [START_REF] Barnes | Complementary and alternative medicine use among adults and children: United States[END_REF][START_REF] Ernst | The BBC survey of complementary medicine use in the UK[END_REF][START_REF] Barnes | Complementary and alternative medicine use among adults: United States[END_REF] which probably greatly increased their prevalence estimates. Further research, of a cross-national nature, would be needed to confirm whether true differences between countries exist, and if so, the reasons for this. These analyses have important limitations. First, all data used were based on self-reports and are thus subject to recall bias although this may be somewhat mitigated by the use of face-to-face interviews rather than questionnaires. Second, although we account for several variables in our regression models, it is possible that other factors that were not measured may better account for CAM use. However, care was taken to include those variables most likely to predict CAM use based on previous evidence. [START_REF] Barnes | Complementary and alternative medicine use among adults and children: United States[END_REF][START_REF] Thomas | Use and expenditure on complementary medicine in England: a population based survey[END_REF][START_REF] Thomas | Use of complementary or alternative medicine in a general population in Great Britain. Results from the National Omnibus survey[END_REF][START_REF] Barnes | Complementary and alternative medicine use among adults: United States[END_REF] In addition, deprivation was based on geographical area which may not reflect all inhabitants in any given area, and may therefore result in false assumptions. We recommend that future data collection programmes on the use of CAM draw upon the limitations of the HSE 2005 dataset. For example, repeating data collection on similar samples (i.e. nationally representative), including children, and using an identical definition of CAM on each occasion.

The prevalence of CAM use is inextricably linked with issues of efficacy and safety: the more CAM is used the more important it is to consider whether the treatments are safe and efficacious. The fact that only a small proportion of CAMs are supported by robust evidence has been widely discussed, [START_REF] Ernst | How much of CAM is based on research evidence? eCAM[END_REF] although it is important to remember that a lack of evidence does not necessarily mean that there is a lack of effect. It should also be noted that CAM treatments are not necessarily safe and, like all treatments, have the potential to bring about direct and indirect adverse effects.

Currently, because CAM practitioners are not required to report adverse effects, [START_REF] Hunt | No obligation to report adverse effects in British complementary and alternative medicine: evidence for double standards[END_REF] estimates on the frequency of adverse effects of CAM are probably inaccurate. [START_REF] Ernst | First, do no harm' with complementary and alternative medicine[END_REF] In addition, herb-drug interactions have not been extensively investigated, a situation which is concerning given that the HSE 2005 data suggest that more than a quarter of those taking medications in England were using CAM in the same 12-month period. To ensure patient safety, healthcare practitioners should routinely ask patients about their use of CAM and policymakers should ensure that CAM prevalence data is regularly collected, as is the case in other countries.

  psychiatric conditions such as depression or anxiety); and perceived social support which was based on seven questions regarding perceptions of contact with people who offer happiness, love, care, acceptance, importance, support and encouragement. A low score demonstrated greater levels of perceived social support.

Figure 1 :

 1 Figure 1: Prevalence of individual CAM use

quintile, a national measure of ecological deprivation based on Lower Layer Super Output Areas (SOAs) with a mean population of 1500 households, determined by postcode. Lower Layer SOAs are ranked nationally from 1 to 32,482

	and are divided into quintiles, each representing 20% of all SOAs in England (1=most deprived
	SOA). ID2007 comprises seven 'Domains of deprivation' which are: Income deprivation;
	Employment deprivation; Health deprivation and disability; Education; Skills and training
	deprivation; Barriers to Housing and Services; Living environment deprivation (includes distance to
	GP surgery, primary school and supermarket); and Crime.
	Health variables included medication use; obesity (BMI≥30kg/m²); the European Quality of Life
	Scale 5-D

  35.3% university education, 22.9% no university education, χ 2 = 14.25, p<.001), those who work in a professional/managerial role (32.9% managerial, 21.7% non-managerial, χ 2 = 119.38, p<.001), own their own property (26.7% own, 22.3% do not own, χ 2 = 29.05, p<.001), have above average income (33.7% ≥£24,711, 22.8% <£24,711, χ 2 = 90.84, p<.001), and in active employment (30.9% in active employment, 20.8% not active, χ 2 = 98.02, p<.001) were more likely to use CAM. CAM-users were also younger (CAM users: 46.8 +/-15.5 years, non-users: 49.1 +/-19.3 years, t = 5.37, p<.001) than non-CAM-users.

Table 1 : Characteristics of the sample Whole sample n=7630 Used CAM in last 12 months n=2005

 1

	Not used
	CAM in last
	12 months
	n=5625

Table 2 : Best predictors of 12 month CAM use Variables Odds ratio (95% CI) for using CAM in last 12 months relative to not using CAM p value

 2

	Male

Acknowledgements

We are grateful to the UK data archive for permission to use the HSE 2005 data. Helen F Coelho is supported by a research fellowship from the Pilkington Family Trusts. Shao Kung Hung is funded by Schwabe. Ethical approval was not required.