Rikard Landberg
email: rikard.landberg@lmv.slu.se

Afaf Kamal-Eldin

PER ÅMAN a Jane Christensen

Kim Overvad C

Anne Tjønneland

Anja Olsen

Determinants of plasma alkylresorcinol concentration in Danish postmenopausal women 1,2,3

Keywords: Alkylresorcinols, determinant, biomarker, whole grain, wheat, rye

Background: Alkylresorcinols (AR), a group of phenolic lipids present in the outer parts of wheat and rye grain kernels, have been suggested as biomarkers of whole-grain wheat and rye intake. In this study we investigated potential determinants of plasma AR concentration in a free-living population.

Subjects/Methods: Non-fasting samples from postmenopausal women enrolled in the Danish Diet, Cancer and Health study (n=360) were selected. Diet was assessed using a semiquantitative FFQ and the association between food items likely to contain AR as well as relevant non-dietary factors were studied by ANCOVA-models.

Results:

The median AR concentration was 78 nmol/l (interquartile range (IQR) = 106.9 nmol/l). Intake of rye bread, identified as the main determinant, was associated with 87% higher plasma total AR concentration per 100 g bread (95% CI= 46-139%). About 8-12 % of the total variation (depending on AR homologue) in plasma AR concentration was explained by the selected dietary variables. At a nutrient level, total dietary fiber and cereal fiber were significantly associated with plasma total AR concentration (P=0.05), but only ≈2 % of the total plasma AR concentration was explained by the dietary fiber or cereal fiber intake.

Conclustion:

In the studied population, AR plasma concentration was mainly affected by rye bread intake among investigated determinants.

INTRODUCTION

Epidemiological studies have consistently shown a reduction in risk of coronary heart disease and Type 2 diabetes [START_REF] Mellen | Whole grain intake and cardiovascular disease: A meta-analysis[END_REF][START_REF] Priebe | Whole grain foods for the prevention of type 2 diabetes mellitus[END_REF], and some studies have shown a reduction in risk for certain cancers [START_REF] Jacobs | Whole-grain intake and cancer: an expanded review and meta-analysis[END_REF]. The mechanisms for the protective effect(s) are poorly understood, but components within the dietary fibre complex are believed to act together [START_REF] Slavin | Why whole grains are protective: biological mechanisms[END_REF]. A major obstacle in nutritional epidemiology, is the relative large errors in the estimation of dietary and nutrient intake, which are often derived from semi-quantitative food frequency questionnaires (FFQs) [START_REF] Kaaks | Biochemical markers of dietary intake[END_REF].

Whole grain intake estimation is particularly likely to be subjected to measurement errors, due to lack of uniform whole grain definition across studies (Koh-Banerjee et al 2004) and the fact that consumers might have difficulties in discerning whole grain from non-whole grain foods [START_REF] Adams | Helping consumers achieve recommended intakes of whole grain foods[END_REF]. Moreover, FFQs usually contain few whole-grain food items listed, and whole grain content in whole grain foods may vary considerably in different products and hence, misclassification is likely [START_REF] Fung | Whole-grain intake and the risk of type 2 diabetes: a prospective study in men[END_REF]. A selective biomarker of whole grain intake would have the potential to provide a more valid ranking tool of intake than traditional dietary assessment methods since it is most likely independent of errors associated with these methods [START_REF] Kaaks | Biochemical markers of dietary intake[END_REF]. Before use, the biomarker needs to be evaluated and validated in both controlled studies and in randomly selected free living subjects in order to get insights of its potential limitations [START_REF] Hunter | Biochemical indicators of dietary intake[END_REF][START_REF] Landberg | Short-term reproducibility of plasma alkylresorcinols as biomarker for whole grain/bran wheat and rye intake[END_REF][START_REF] Van Dam | Are alkylresorcinols accurate biomarkers for whole rain intake?[END_REF]. In controlled studies, the pharmacokinetics (revealing the time course of reflection) and response to changes in intake can be studied in detail, whereas population-based samples can be used to estimate variation, determine long term reproducibility, and to explore determinants of the biomarker concentration [START_REF] Johnsen | Intake of whole grains and vegetables determines the plasma enterolactone concentration of Danish women[END_REF][START_REF] Kaaks | Biochemical markers of dietary intake[END_REF], Landberg et al 2009a[START_REF] Montonen | Reliability of fasting plasma alkylresorcinol concentrations measured 4 months apart[END_REF][START_REF] White | Effects of biomarker measurment error on epidemiological studies[END_REF] Alkylresorcinols (AR), a group of phenolic lipids, have recently been suggested as biomarkers of whole grain wheat and rye intake, because they are exclusively present in the outer parts of these cereals (Landberg et al 2008a, Landberg et al 2008c[START_REF] Linko-Parvinen | Wholegrain alkylresorcinols are transported in human plasma lipoproteins, and their intake corresponds to plasma concentrations[END_REF], Ross et al 2004). Results from whole grain intervention studies have shown that AR concentration increases with increased whole-grain intake in a dose-dependent manner and that the concentration remains stable over weeks in people with frequent whole grain intake [START_REF] Landberg | Human plasma kinetics and relative bioavailability of alkylresorcinols after intake of rye bran[END_REF], Landberg et al 2008a[START_REF] Landberg | Short-term reproducibility of plasma alkylresorcinols as biomarker for whole grain/bran wheat and rye intake[END_REF], Landberg et al 2008d[START_REF] Linko-Parvinen | Wholegrain alkylresorcinols are transported in human plasma lipoproteins, and their intake corresponds to plasma concentrations[END_REF][START_REF] Linko | Whole-grain rye bread consumption by women correlates with plasma alkylresorcinols and increases their concentration compared with low-fiber[END_REF]. One recent study showed a modest correlation (r= 0.4) between cereal fibre intake assessed by 3 day-weighed food records and plasma AR concentration in selected Finnish free living subjects [START_REF] Aubertin-Leheudre | Plasma alkylresorcinols and urinary alkylresorcinol metabolites as biomarkers of cereal fiber intake in Finnish women[END_REF].

Moreover, a recent small study (n=33) compared whole grain intake (g/d) assessed by a FFQ designed specifically for whole grain products and plasma total AR concentration and found a good correlation (r=0.57) (Ross et al 2009). The reproducibility over a period of 4 months was shown to be modest-good (ICC=0.55) for women in a subsample of the EPIC-Potsdam cohort suggesting that plasma AR concentration is rather stable over time within an individual [START_REF] Montonen | Reliability of fasting plasma alkylresorcinol concentrations measured 4 months apart[END_REF]. However, no study has explored possible determinants of plasma AR concentration in a broad group of free living subjects. Such a study is an important step in the evaluation of the selectivity of a biomarker. The aim of the present study was to estimate the variation in plasma AR concentration and to determine dietary and non-dietary factors affecting the plasma AR concentration in free living Danish postmenopausal women.

MATERIALS AND METHODS

Study population, data-and sample collection

Between December 1993 and May 1997, 79 729 women who lived in the greater Copenhagen and Aarhus area were invited to participate in the prospective cohort study "Diet, Cancer and Health," (DCH). A total number of 29 875 women that fulfilled the criteria were enrolled. [START_REF] Tjønneland | Study design, exposure variables and socioeconomic determinants of participation in Diet, Cancer and Health: a population-based prospective cohort study of 57 053 men and women in Denmark[END_REF]. At baseline, participants attended one of two established centers, where 30 mL of blood (collected in citrated and plain Venojects) were drawn from each participant. For practical reasons, subjects were not instructed to provide fasting samples time since last meal was recorded at blood draw. Samples were centrifuged (2000 G for 10 minutes) and plasma was divided into 1 mL tubes which were stored in liquid nitrogen vapour until analysis (maximum temperature -150°C).

Subjects filled in a 192-item FFQ and a lifestyle questionnaire, which they received by mail before visiting the clinic to donate blood. The development and validation of the FFQ has been described elsewhere [START_REF] Haraldsdóttir | Validity of individual portion size estimates in food frequency questionnaire[END_REF], Overvad et al 1991, Tjønneland et al 1991[START_REF] Tjønneland | Influence of individually estimated proportion size data on the validity of semi-quantitative food frequency questionnaire[END_REF]. The participants were instructed to report their intake of foods and beverages during the past 12 months, within 12 possible categories ranging from never to ≥ 8 times /d. Daily intakes of specific foods and nutrients were calculated for each participant by the software program Food Calc (http://www.ibt.ku.dk/jesper/foodcalc/) using specially developed standardized recipes and portion sizes [START_REF] Johnsen | Intake of whole grains and vegetables determines the plasma enterolactone concentration of Danish women[END_REF]. Anthropometrical measurements were carried out by professionals and the BMI was calculated as weight (kg) divided by height squared (m 2 ).

A total number of 360 postmenopausal women (177 cases and 184 subcohort members) from a nested case-cohort study on AR in relation to endometrial cancer [START_REF] Olsen | Plasma levels of alkylresorcinols and incidence of endometrial cancer[END_REF] were included in the present study. Because there were no statistically significant differences between cases and the sub-cohort with regard to plasma AR concentrations [START_REF] Olsen | Plasma levels of alkylresorcinols and incidence of endometrial cancer[END_REF], both groups were pooled and included in the present cross-sectional analysis. Base-line blood samples from all 360 women were analyzed for plasma AR concentration. The DCH study and the present sub-study were approved by the regional ethical committees on human studies in Copenhagen and Aarhus and by the Danish Data Protection Agency. All participants provided written informed consent.

Identification of potential plasma AR determinants

Alkylresorcinols are only present in significant amounts in whole grain/bran of wheat and rye and in Denmark a large proportion of the whole-grain intake comes from very specific food items, particularly rye bread [START_REF] Mejborn | Definition og vidensgrundlag for anbefaling af fuldkornsindtag i Danmark [Wholegrain-Mejborn H[END_REF]. We therefore selected to investigate potential determinants of plasma AR concentration among foods known to contain AR and foods reflected by FFQ questions that have been used before to reflect whole grain intake in this cohort [START_REF] Egeberg | Intake of wholegrain products is associated with dietary, lifestyle, anthropometric and socio-economic factors in Denmark[END_REF]. The foods included rye bread, "other-bread" (breads that contained whole grain wheat, were not purely refined and did not contain rye). crisp-bread and refined bread. Danish rye bread, "other-bread" and rye crisp bread (which contributes to about 50% of the total crisp bread intake) typically contains 32, 7.5 and 83% whole grain on fresh weight basis, respectively [START_REF] Mejborn | Definition og vidensgrundlag for anbefaling af fuldkornsindtag i Danmark [Wholegrain-Mejborn H[END_REF]. However, the variation in whole grain content and, hence, also AR content within these food items is high. Refined bread was included because it was recently shown to contain very small amounts of AR (Ross and Kochhar 2009). We also investigated beer, which contains traces (<30 µg/g on dry matter basis) of AR (unpublished results) and total fat derived from animal sources as predictors of AR concentration. We recently showed that AR homologues are accumulated in adipose tissue in pigs and humans [START_REF] Jansson | Accumulation of a potential whole grain biomarker (alkylresorcinols) in human adipose tissue[END_REF]. Due to a short apparent elimination half-life (about 5 h) and potential accumulation of AR in adipose tissue in case dietary sources are frequently consumed, time since last meal and BMI were tested as predictors as well. Serum cholesterol was included because AR are transported in lipoproteins [START_REF] Linko-Parvinen | Wholegrain alkylresorcinols are transported in human plasma lipoproteins, and their intake corresponds to plasma concentrations[END_REF].

Analytical methods

Plasma samples (300 µL) were analyzed for AR homologues C17:0-C25:0 according to a recently published GC-MS-SIM method, using molecular ions for quantification (Landberg et al 2009b). Samples were analyzed randomly in singlicates in 17 batches by 3 different people.

In each batch, five replicates of a control sample were included randomly in the sequence.

The within-and between day batch variation determined as the coefficient of variation (CV) was <7% and <10%, respectively.

Statistical analysis

For the statistical evaluation, ANCOVA models with AR as the dependent variable were used and results where P<0.05 were considered significant. Since plasma AR concentration was skewed, data were transformed by natural logarithm before analysis and estimates were then back transformed to illustrate percentage change in AR concentration. Statistical analysis was combined for cases and controls (n=360) since no significant differences were observed (P=0.76 (total AR), P=0.54 (C17:0), P=0.85 (C19:0), P=0.53 (C21:0), P=0.67 (C23:0) and P=0.96 (C25:0)). All quantitative variables were entered linearly in the model [START_REF] Greenland | Avoiding power loss associated with categorization and ordinal scores in dose-response and trend analysis[END_REF]). The women were grouped in quartiles according to their plasma total AR concentrations and the distribution of selected dietary and non-dietary variables was calculated. The linearity of the associations was evaluated graphically by linear splines with the boundaries placed at the quartiles (Greenland 1995a). The analysis was carried out separately for cereal products known to contain AR (rye bread, crisp bread, refined bread, beer) and for dietary fiber (total-and cereal fiber). Associations were expressed per 100g/d for cereal food components and per 10 g/d for dietary fiber. Spearman's rank correlation coefficients were calculated for selected variables. The GLM procedure in SAS version 9.1 for Unix (SAS institute, NC, USA) was used for statistical analysis.

RESULTS

Base-line characteristics of women were calculated according to quartiles of plasma total AR (Table 1). The plasma AR homologue concentration for combined cases and controls were skewed to the right for all individual homologues and their sum (Figure 1). The median total plasma AR concentration was 78 nmol/l and inter quartile range was 106.9 nmol/l. The mean±SD in the same variable was 115±112 nmol/l.

The average intake of products identified as likely to contain AR was 133±62 g/d (range 0.5-368 g/d) and included only bread. Among these product categories, rye bread was the dominant source, accounting for 51±24 % whereas 13±17% was refined bread, 4.6± 8.9 % crisp bread and 31±25% "other bread". Additionally, daily intake of beer and fat derived from animal carcasses were investigated. The intake of beer was 77±146 g/d and animal fat intake was 15±6 g/d. The FFQ contained one question on intake of oats/muesli, but since this question is likely to reflect oats to a large extent and because oats do not contain AR, this question was omitted to avoid misclassification.

All plasma AR homologues were correlated to rye bread intake (r= 0.21-0.31, P<0.0001) whereas only C17:0 and total plasma AR concentration was correlated to crisp bread intake (r= 0.11, P=0.03 and r= 0.12, P<0.03, respectively) (Table 2). Some AR homologues were significantly correlated to total dietary fiber intake and all were correlated to cereal fiber intake (r= 0.16-0.20, P<0.001) (Table 2). No significant correlation between non-fasting serum cholesterol and plasma AR concentration was found (Table 2).

Three different regression models (univariate, multivariate with dietary predictors, multivariate with dietary and non-dietary predictors) were tested in order to find the one that explained most of the variation in plasma AR concentration. In most cases, inclusion of potential predictors other than AR containing foods, such as BMI, serum cholesterol (nonfasting), time since last meal and energy intake did not affect the estimates or the variation explained by the model and hence, these were omitted in the final models. When testing food items likely to contain whole grain in the final model, rye bread was the only significant determinant of plasma total AR concentration (P<0.0001) (Table 3). An increase of rye bread intake of 100 g/d was associated with a raise of plasma total AR concentration of 87 % (46-139%, 95% CI) (P<0.0001). Results were the same for the individual AR homologues with an associated increase of plasma AR homologue concentration of 69-114 % (P<0.0001) for homologues C17:0-C25:0 per 100 g/d increment of rye bread intake. The final models, including all identified potential food sources of AR simultaneously, explained 8-12 % of the total variation of plasma concentration of individual AR homologues and 9% of total AR concentration (Table 3).

Total dietary fiber was a significant predictor of total plasma AR concentration (P=0.04), but not for individual AR homologues (Table 4). Only about 1 % of the total plasma AR concentration was explained by the model. When dietary fiber was divided into cereal fiber and non-cereal fiber, a 10 g/d increase in cereal fiber intake was associated with a 36% increase in plasma total AR concentration (P=0.011) (Table 4). The associated increase was of the same magnitude for the individual AR homologues, and was significant for all homologues except for C25:0. About 2% of the total variation in plasma AR concentration was explained by the models.

DISCUSSION

This is the first study where determinants of plasma AR concentration were investigated.

Samples used were from an endometrial cancer case-cohort study conducted within the Danish "Diet, Health and Cancer" cohort [START_REF] Olsen | Plasma levels of alkylresorcinols and incidence of endometrial cancer[END_REF].. Plasma total AR concentration in the present study, where non-fasting samples were used, was considerably higher and showed a wider range than what was reported in a recent Finnish study (n=54) where fasting samples in free-living subjects were analyzed (115±112 nmol/l compated to 65±34 nmol/l) (Aubertin-Leheudre et al 2008).

Among commonly consumed foods in the Western diet, AR are mainly present in whole grain and bran products of wheat and rye [START_REF] Chen | Alkylresorcinols as markers of whole grain wheat and rye in cereal products[END_REF][START_REF] Landberg | Alkylresorcinols in durum wheat (Triticum durum) kernels and pasta products[END_REF][START_REF] Mattila | Contents of phenolic acids, alkyl-and alkenylresorcinols, and avenanthramides in commercial grain products[END_REF][START_REF] Ross | Alkylresorcinols in cereals and cereal products[END_REF]. Small amounts are also found in barley and in very small amounts in some refined products due to contamination or a high extraction rate in the milling process (Landberg et al 2008a, Ross and Kochhar 2009[START_REF] Zarnowski | Alkylresorcinols in barley (Hordeum vulgare L. distichon) Grains[END_REF]. From the FFQ used in the study, rye bread, white bread, crisp bread, and "other bread" were identified as food categories likely to contain AR. Rye was expected to be the main predictor because of the high whole grain content typically found in Danish rye bread [START_REF] Mejborn | Definition og vidensgrundlag for anbefaling af fuldkornsindtag i Danmark [Wholegrain-Mejborn H[END_REF] and due to about twice as much AR in whole grain rye compared to whole grain wheat [START_REF] Chen | Alkylresorcinols as markers of whole grain wheat and rye in cereal products[END_REF][START_REF] Mattila | Contents of phenolic acids, alkyl-and alkenylresorcinols, and avenanthramides in commercial grain products[END_REF][START_REF] Ross | Alkylresorcinols in cereals and cereal products[END_REF] . The spearman rank correlation coefficient between rye bread intake and plasma AR concentration in the present study (r=0.25) was lower compared to what was found when comparing total whole grain intake and plasma AR concentration in a recent study in free-living subjects in Switzerland (Ross et al 2009) and about the same as what was found between rye bread intake and plasma concentration in a Finish intervention study (r=0.34) [START_REF] Linko | Whole-grain rye bread consumption by women correlates with plasma alkylresorcinols and increases their concentration compared with low-fiber[END_REF]. The higher correlation coefficient in the Swiss study is likely due to the fact that the FFQ used in that study was designed to capture whole grain intake specifically whereas a general FFQ was used in the present study. Neither crisp bread nor "Other bread" were independent predictors of plasma AR concentration, which is probably due to the large variation in whole grain content in these categories and a small contribution of AR intake from these foods. The observed correlation between crisp bread and plasma C17:0 concentrations is probably due to confounding by rye bread intake because the association disappeared when mutually adjusting for the other food variables. As expected, white bread was not associated with an increase in plasma AR since it contains no, or only very small amounts of [START_REF] Chen | Alkylresorcinols as markers of whole grain wheat and rye in cereal products[END_REF]Kochhar 2009). Additionally, beer and fat from animal sources were tested, because small amounts of AR have been detected in beer samples as well as in adipose tissue from pigs and humans [START_REF] Jansson | Accumulation of a potential whole grain biomarker (alkylresorcinols) in human adipose tissue[END_REF] (unpublished data), but without showing any association with plasma AR concentration.

In an earlier experimental study, it was suggested that plasma AR concentration should be adjusted for serum cholesterol value in order eliminate variation caused by differences in carriers, since AR are transported in lipoproteins [START_REF] Linko-Parvinen | Wholegrain alkylresorcinols are transported in human plasma lipoproteins, and their intake corresponds to plasma concentrations[END_REF]. However, in the present study, no correlation between serum cholesterol and AR homologues or the sum of them was observed, giving no support to this adjustment. The lack of a correlation may be due to the fact that serum cholesterol was determined in non-fasting samples or that plasma AR concentration is unaffected by difference in serum-cholesterol concentrations. In the present study, no significant correlation between plasma AR concentration and total energy intake, BMI, or time since last meal was found (Table 2) and hence, in most cases, models adjusting for these variables did not affect associations between intake and AR concentrations (Table 3- 4). The finding of no association between plasma AR concentration and energy intake in the present study is probably due to fact that AR are present in foods with low energy density (products mainly classified in the rye bread category).

It was possible to explain about ≈10 % of the variation in plasma AR concentration by the final models with cereal product intake, whereas only about 1-2 % of the variation was explained by the models with dietary fibre. The amount of explained variation was in the same range as found for serum ENL in different studies [START_REF] Horner | Dietary determinants of plasma enterolactone[END_REF][START_REF] Johnsen | Intake of whole grains and vegetables determines the plasma enterolactone concentration of Danish women[END_REF], Kilkkinen et al 2001[START_REF] Milder | Relation between plasma enterodiol and enterolactone and dietary Intake of lignans in a Dutch endoscopy-based population[END_REF]. In the present study, a large proportion of the unexplained variation is probably due to the use of non-fasting samples. The half-life of plasma AR has been estimated to about 5 h [START_REF] Landberg | Human plasma kinetics and relative bioavailability of alkylresorcinols after intake of rye bran[END_REF], Landberg et al 2008d), and hence the within-subject plasma concentration over the day will be highly affected by the intake frequency. However, a rather long absorption time will dampen fluctuations around steady state, given a frequent intake. There was no improvement by including time since last meal in the models, probably since time since last meal did not efficiently correct for the time since last AR containing food intake. Another likely source of unexplained variation is the inter-individual differences in absorption and elimination kinetics. Moreover, the FFQ used in the present study was designed with the focus on the ability to rank subjects according to intake of foods potentially important for cancer aetiology [START_REF] Tjønneland | Validation of a semi-quantitative food frequency questionnaire developed in Denmark[END_REF]. However, only 4 (6 if including beer and animal fat) food-variables were used for the intake estimation of foods including whole grain likely to contain AR. Since both the whole grain content in products and AR concentration in whole grain varies considerably [START_REF] Chen | Alkylresorcinols as markers of whole grain wheat and rye in cereal products[END_REF][START_REF] Mattila | Contents of phenolic acids, alkyl-and alkenylresorcinols, and avenanthramides in commercial grain products[END_REF], the estimate is likely to be subjected to a considerable measurement error. Additionally, subjects may not have been able to distinguish between artificially coloured dark bread and dark bread containing whole grain leading to additional measurement errors with resulting underestimation of the value of plasma AR as biomarkers of whole grain intake.

Cereal fiber intake was significantly correlated to plasma total AR in the present study (Spearmans' r= 0.19, P<0.001). This correlation was weaker than observed in a recent survey in 56 free living Finnish subjects (Pearsons'r=0.41, P<0003) in which fiber intake was assessed by 3-day food record (Aubertin-Leheudre et al 2008) and plasma AR was analysed in a sample taken during the food record period. Since plasma AR have a rather short half-life, a single sample is expected to reflect short-to medium term intake (Landberg et al 2008d), and hence reflects the same time period as the food records. The FFQ, on the other hand, is expected to reflect long-term average intake. The underlying true correlation between plasma AR concentration and intake estimated by FFQ is probably stronger than the observed, since no adjustment for measurement errors in the biomarker was undertaken [START_REF] Rosner | Interval estimates for correlation coefficients corrected for within-persona variation-Implications for study design and hypothesis testing[END_REF].

In conclusion, the results of the present study demonstrate that the rye bread intake was the major determinant of plasma AR concentration in the studied population, among foods and non-dietary variables tested. At a nutrient level, both total-and cereal fiber were recognized as significant determinants. However, these variables only explained a small proportion of the total variation in plasma AR concentration, probably due to misclassification of whole grain intake by the FFQ and because of poor precision in the biomarker due to measurement error inherent to short half-life and the use of non-fasting samples. Future studies to evaluate the usefulness of AR as biomarkers in epidemiological studies should focus on determination of long.-term reproducibility and to what extent plasma concentration reflect whole grain intake in fasting and non-samples in different free-living populations.

Figure 1 :

 1 Figure 1: Frequency distributions of plasma AR homologues C17:0, C19:0, C21:0,

Table 2 :

 2 Spearman's rank correlation 1 between plasma AR homologue concentration and dietary and non-dietary factors among 360 Danish women, of which 176 were diagnosed with

	endometrial cancer.
			Plasma AR homologue concentration
				(nmol/l)
	Variable	C17:0	C19:0	C21:0	C23:0	C25:0	Total
	Rye bread (g/d)	0.31*** 0.26*** 0.21*** 0.25*** 0.26*** 0.25***
	White bread (g/d)	-0.07	-0.06	-0.05	-0.06	-0.07	-0.06
	Crisp bread (g/d)	0.11*	0.09	0.08	0.08	0.05	0.12*
	Other bread (g/d)	0.02	0.11*	0.16**	0.11*	0.06	0.09
	Beer (mL/d)	0.06	-0.06	-0.08	-0.11*	0.04	-0.09
	Animal fat 2 (g/d)	0.05	0.02	0.01	0.01	-0.10	0.02
	BMI (kg/m 2 )	-0.02	-0.02	-0.01	-0.04	-0.01	-0.01
	Time since last meal	0.04	0.04	0.03	0.06	0.06	0.05
	(h)
	Total S-	0.05	0.07	0.05	0.07	0.09	0.07
	cholesterol(mmol/L)
	Total energy intake	0.06	0.08	0.08	0.08	0.06	0.08
	(KJ/d)
	Total dietary fiber	0.10*	0.14**	0.14**	0.13**	0.11	0.14**
	(g/d)
	Cereal fiber (g/d)	0.17**	0.20**	0.20*** 0.19***	0.16	0.19***
	Other fiber (g/d)	0.00	0.04	0.05	0.05	0.03*	0.05

Table 3

 3 Food determinants of plasma AR homologue C17:0, C19:0, C21:0, C23:0, C25:0 and total AR concentration (nmol/l) for 360 postmenopausal women 1 .

		Univariate analysis	Full model 2	Final model 3	P 4
	Whole grain
	food items 5
	Rye bread
	C17:0	106 (62;164)	124 (72;193)	114 (67;173)	<0.0001
	C19:0	88 (46;141)	105 (57;168)	96 (52;151)	<0.0001
	C21:0	62 (26;109)	74 (33;129)	69 (31;117)	<0.0001
	C23:0	85 (43;141)	96 (48;161)	91(47;149)	<0.0001
	C25:0	106 (54;175)	117 (59;198)	110 (57;181)	<0.0001
	Total AR	81 (41;131)	94 (-0.1;153)	87 (46;139)	<0.0001
	Crisp bread
	C17:0	0.07 (-9;8)	2 (-19;11)	-19 (-36;2)	0.91
	C19:0	-5 (-13;4)	-3 (-11;6)	-4 (-11;5)	0.39
	C21:0	-7 (-14;102)	-5 (-13;4)	-5 (-13;3)	0.20
	C23:0	-5 (-13;4)	-3 (-11;7)	-4 (-12;5)	0.38
	C25:0	-3 (-12;7)	-0.1 (-10;10)	-2 (-11;8)	0.68
	Total AR	-5 (-12;4);	-3 (-11;38)	-3 (-11;5)	0.40
	White bread
	C17:0	-21 (-45;14)	-11 (-38;29)	-19 (-43;14)	0.23
	C19:0	-16 (-41;21)	-3 (-34;41)	-13 (-39;24)	0.44
	C21:0	-11 (-0.4;29)	2 (-30;49)	-6 (-34;34)	0.72
	C23:0	-11 (-39;30)	0.1 (-32;49)	-8 (-36;33)	0.66
	C25:0	-18 (-46;24)	-10 (-42;40)	-18 (-45;24)	0.36
	Total AR	-15 (-40;22)	-4 (-33;40)	-12 (-38;24)	0.47
	Other bread 6
	C17:0	-11 (-28;9)	-1 (-21;23)	-7 (-24;12)	0.43
	C19:0	4 (-15;27)	15 (-8;44)	7 (-12;31)	0.48
	C21:0	12 (-9;36)	21 (-4;52)	15 (-6;40)	0.17
	C23:0	2 (-17;26)	12 (-12;42)	-4 (-12;5)	0.57
	C25:0	-6 (-26;19)	4 (-20;35)	-2 (-22;23)	0.84
	Total AR	3.6 (-15;26)	13 (-9;41)	7 (-12;30)	0.48
	R 2
	C17:0	-	0.12	0.12	-
	C19:0		0.11	0.10
	C21:0		0.09	0.08
	C23:0		0.09	0.09
	C25:0		0.09	0.08
	Total AR		0.11	0.09

Table 4 :

 4 Fiber determinants of plasma AR homologues C17:0, C19:0, C21:0, C23:0, C25:0 and total AR concentration (nmol/l) for 360 postmenopausal women 1

		Univariate	Full model 2	Final model 3	P 4
		analysis
	Nutrient
	Total dietary fiber
	C17:0	8 (-6;23)	12 (-8;36)	-	0.26
	C19:0	13 (-1;29)	19 (-2;45)	-	0.08
	C21:0	15 (-0.5;31)	21 (-1;47)	-	0.07
	C23:0	15 (-0.2;32)	22 (-0.4;51)	-	0.05
	C25:0	13 (-3; 33)	25 (-1;57)	-	0.11
	Total AR	14 (0.3;30)	22 (1;49)	-	0.04
	Cereal Fiber
	C17:0	36 (7;73)	62 (17;127)	38 (8;76)	0.01
	C19:0	41 (11;79)	70 (22;139)	41 (11; 80)	0.01
	C21:0	39 (10;77)	64 (18;29)	39 (9;77)	0.01
	C23:0	36 (6;75)	61 (14; 128)	35 (5; 74)	0.02
	C25:0	31 ( -1;73)	63 (10-139)	30 (-2; 72)	0.07
	Total AR	37 (9;74)	65 (19;128)	36 (8; 73)	0.01
	Non-cereal fiber
	C17:0	-21 (-45;14)	1 (-18;25)	-6 (-22; 12)	0.46
	C19:0	3 (-14;23)	8 (-12;34)	-0.6 (-17; 19)	0.94
	C21:0	7 (-11;27)	11 (-10;37)	3 (-14; 23)	0.75
	C23:0	8 (-10;30)	14 (-9; 42)	4 (-13; 26)	0.65
	C25:0	8 (-12;32)	16 (-9;48)	5 (-15; 29)	0.66
	Total AR	7 (-10;27)	13 (-8; 39)	3 (-13; 23)	0.72
	R 2
	C17:0	-	0.031	0.019	-
	C19:0		0.039	0.021
	C21:0		0.035	0.021
	C23:0		0.022	0.016
	C25:0		0.028	0.011
	Total AR		0.023	0.019

Significant correlation coefficients are indicated with stars, where * P<0.05, ** P<0.01 and *** P<0.001.

Fat from animal sources

Estimated change (%) of plasma total AR concentration corresponding to a 100 g/d

Estimated change (%) of plasma total AR concentration corresponding to a 10 g/d increase in intake of fiber component. Within parentheses, the 95% CIs of the estimates are presented. Data from both cases and controls were included, after testing for any differences.

Model included cereal-and non-cereal fiber and were adjusted for BMI, serum cholesterol (non-fasting), total energy intake, time since last meal. R 2 are reported for models where cereal fiber and non-cereal fiber were added in the same model.

Model included cereal-and non-cereal fiber but excluded non-dietary factors. R 2 are reported for models where cereal fiber and non-cereal fiber were added in the same model.

P-values are shown for variables in the final model except for total dietary fiber, where P-values are from the univariate analysis.

ACKNOWLEDGEMENTS

BSc Erika Jansson is acknowledged for skilled technical assistance. The author's contributions were as follows: RL, AK-E, PÅ and AO planned the study. RL was responsible for the analysis of AR in plasma and drafted the manuscript. JC was main responsible for the statistical analysis. AT and KO were the founders of the cohort. All authors critically evaluated the manuscript before submission and RL was primarily responsible for the final content. None of the authors have any have any advisory board affiliation, financial agreements or other relationships that might have led to a conflict of interest.

Swedish Governmental Agency for Innovation Systems (VINNOVA) and the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS), The Nordic Centre of Excellence (NCoE): HELGA 2 No potential or actual conflicts of interest. This work has never been published before alkylresorcinol(s), DCH (Diet, Cancer and Health study


Niklas Thon

Sabina Eigenbrod

Eva Maria Grasbon-Frodl

Juergen Lutz

Simone Kreth
email: fkreth@med.uni-muenchen.de

Gabriele Poepperl

Claus Belka

Joerg Hans A Kretzschmar

Christian Tonn

MD Friedrich-Wilhelm W Kreth

Gabriele Popperl

Joerg-Christian Tonn

Predominant influence of MGMT methylation in non-resectable GBM after radiotherapy plus temozolomide

Keywords: GBM, glioblastoma, MGMT, temozolomide, biopsy, epigenetic silencing

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

Introduction

The incidence of malignant gliomas has increased over the last two decades especially in the elderly subpopulation and currently lies in the range of 5 cases per 100,000. [START_REF] Wen | Malignant gliomas in adults[END_REF] Glioblastoma account for approximately 60-70% of these tumours. Recently, important progress has been achieved in the treatment of this most malignant tumour: 1. Radiotherapy plus concomitant and adjuvant temozolomide (XRT/TMZ TMZ) has resulted in significant prolongation of both time to progression and survival as compared to XRT alone; This was demonstrated by the results of the prospective randomized trial performed by the European Organisation for Research and Treatment of Cancer (EORTC) and the National Cancer Institute of Canada (NCIC) Clinical Trials Group.[2;3] 2. Epigenetic silencing of the promoter of the gene that encodes O 6 -methylguanine-DNA methyltransferase (MGMT) in tumour specimens has been identified as a favourable predictive factor of benefit from XRT/TMZ TMZ; This was shown in a companion retrospective study. [START_REF] Hegi | MGMT gene silencing and benefit from temozolomide in glioblastoma[END_REF] The impact of these findings on clinical practice, however, is still under debate: Subgroup analysis suggests that relative young and/or fit patients harbouring a tumour with a methylated MGMT promoter and undergoing resection rather than biopsy benefit most from XRT/TMZ TMZ. [START_REF] Stupp | Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial[END_REF] It remains unclear, to which extent unfavourable prognostic factors such as increased age, and/or a deep seated tumour location might outweigh potential benefit resulting from MGMT methylation. [START_REF] Gorlia | Nomograms for predicting survival of patients with newly diagnosed glioblastoma: prognostic factor analysis of EORTC and NCIC trial 26981-22981/CE.3[END_REF] The important subgroup of patients with nonresectable glioblastoma is particularly concerned: Patients having undergone biopsy only experienced the worst outcome in the EORTC/NCIC trial, which was irrespective of treatment [START_REF] Stupp | Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial[END_REF] -however, no stratification by the MGMT methylation status was done, because this biomarker could not be analyzed from small sized biopsy specimens. [START_REF] Hegi | MGMT gene silencing and benefit from temozolomide in glioblastoma[END_REF] In this situation oncologist are confronted with unawareness and uncertainty concerning appropriate treatment strategies for those patients not suitable for open tumour resection. The current prospective study was conducted to elucidate for the first time the prognosis of patients with nonresectable glioblastoma undergoing XRT/TMZ TMZ after adjustment for the effects of MGMT promoter methylation and other relevant prognostic factors. Characterization of the tumour was performed by molecular stereotactic biopsy technique which combines state of the art histopathological evaluation with small sample size adjusted molecular genetic analysis. [START_REF] Kreth | Identification of valid endogenous control genes for determining gene expression in human glioma[END_REF][START_REF] Thon | Novel Molecular Stereotactic Biopsy Procedures Reveal Intratumoral Homogeneity of Loss of Heterozygosity of 1p/19q and TP53 Mutations in World Health Organization Grade II Gliomas[END_REF][START_REF] Grasbon-Frodl | Intratumoral homogeneity of MGMT promoter hypermethylation as demonstrated in serial stereotactic specimens from anaplastic astrocytomas and glioblastomas[END_REF] The current study should not be regarded as an appendix to the EORTC/NCIC trial. Both studies differ in terms of selection criteria and applied treatment strategies.

Methods

Patients

Adult patients (age 18 to 85 years) were eligible if they had i) a supratentorial glioblastoma not suitable for gross total tumour resection (because of an eloquent tumour location and/or significant co-morbidity) with histology being proven by stereotactic biopsy, ii) no severe mass effect of the tumour demanding debulking surgery because of brain stem compression and/ or midline shift, iii) no prior history of surgery, XRT, and/or chemotherapy, iv) a Karnofsky performance score (KPS) ≥ 60, [START_REF] Karnofsky | The use of the nitrogen mustards in the palliative treatment of carcinoma: with particular reference to bronchogenic carcinoma[END_REF] and v) adequate hematologic, renal, and hepatic function [as being defined before]. [START_REF] Stupp | Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma[END_REF] All enrolled patients gave informed consent. The study protocol was approved by the institutional review board (AZ 216/14) of the Ludwig Maximilians University Munich, Germany.

Molecular stereotactic biopsy technique

The still new technique of molecular stereotactic biopsy has been described previously. [START_REF] Kreth | Identification of valid endogenous control genes for determining gene expression in human glioma[END_REF][START_REF] Thon | Novel Molecular Stereotactic Biopsy Procedures Reveal Intratumoral Homogeneity of Loss of Heterozygosity of 1p/19q and TP53 Mutations in World Health Organization Grade II Gliomas[END_REF][START_REF] Grasbon-Frodl | Intratumoral homogeneity of MGMT promoter hypermethylation as demonstrated in serial stereotactic specimens from anaplastic astrocytomas and glioblastomas[END_REF] In brief, co-registration of computerized tomography (CT), and magnetic resonance imaging (MRI; including T1-and T2-weighted sequences) served for 3-dimensional (3D) visualization (i-plan stereotaxy ® , BrainLAB ® , Feldkirchen, Germany) of the tumour and the simulation of the best trajectory. Serial biopsies were taken along a trajectory representative of the solid tumour. Using micro forceps the maximum amount of tissue per biopsy specimen was 1 mm 3 . The number of specimens taken in 1mm steps along the chosen trajectory was in the range of 10-18 samples per tumour. The tissue sampling procedure was guided by intraoperative smear preparations, which were routinely performed by the attending neuropathologist: Only tumour probes next (i.e. 1mm distance) to smear preparations exclusively showing solid vital tumour tissue were used for molecular genetic analysis; A corresponding sample (level +1mm) was taken for paraffin embedding and histopathological examination using standard protocols. [START_REF] Grasbon-Frodl | Intratumoral homogeneity of MGMT promoter hypermethylation as demonstrated in serial stereotactic specimens from anaplastic astrocytomas and glioblastomas[END_REF] Samples chosen for molecular genetic analysis were snap frozen immediately after withdrawal. The described biopsy technique was chosen to minimize the risk of tissue contamination (e.g. by non-neoplastic or necrotic tissue) and more importantly, to recognize contamination, if it occurs. For determination of the MGMT promoter methylation status, at least 2 tissue samples were collected from different sites throughout each tumour to test for both the intra-tumoural distribution of the biomarker and the reproducibility of the molecular genetic analysis. The histological diagnosis of all tissue specimens was made according to the 2007 World Health Organization (WHO) classification of tumours of the central nervous system. [START_REF] Louis | The 2007 WHO classification of tumours of the central nervous system[END_REF] A CT-scan was done one day after biopsy in all patients and any sequels attributed to the biopsy procedure were classified as morbidity. [START_REF] Kreth | The risk of haemorrhage after image guided stereotactic biopsy of intra-axial brain tumours--a prospective study[END_REF]

Methylation-specific PCR (and sequencing analysis)

Isolation of nucleic acids, bisulfite modification of DNA, methylation-specific PCR (MSP) and sequencing analyses were done as being published in detail before. [8;12] In brief, DNA isolation from tumour specimens was performed using the QIAamp ® DNA Micro Kit (Qiagen, Hilden, Germany), DNA recovery from each stereotactic biopsy sample amounted to around 30-60 ng/µl. This step was followed by purification and bisulfite-modification of DNA according to Mollemann et al.. [START_REF] Mollemann | Frequent promoter hypermethylation and low expression of the MGMT gene in oligodendroglial tumors[END_REF] MSP and sequencing analyses were performed using the specific primer pairs as described before. [START_REF] Esteller | Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents[END_REF][START_REF] Clark | DNA methylation: bisulphite modification and analysis[END_REF][START_REF] Frommer | A genomic sequencing protocol that yields a positive display of 5methylcytosine residues in individual DNA strands[END_REF] "Unmethylated" and "methylated" tumours were defined according to Grasbon-Frodl et al.: [START_REF] Grasbon-Frodl | Intratumoral homogeneity of MGMT promoter hypermethylation as demonstrated in serial stereotactic specimens from anaplastic astrocytomas and glioblastomas[END_REF] Sequencing of bisulfite-modified DNA indicated a methylated promoter when more than half of the CpG sites (13 of 25 CpG sites) were found to be methylated; partial methylation was defined as the cytosine and thymine peaks being equally sized or the cytosine peak being twice as high as compared to the corresponding thymine peak.

Study design

Patients were enrolled in the period from March 2006 to August 2008. Patients were categorized according to the Radiation Therapy Oncology Group (RTOG)-recursive partitioning analysis (RPA) classes. [START_REF] Curran | Recursive partitioning analysis of prognostic factors in three Radiation Therapy Oncology Group malignant glioma trials[END_REF] The KPS was used for clinical evaluation. [START_REF] Karnofsky | The use of the nitrogen mustards in the palliative treatment of carcinoma: with particular reference to bronchogenic carcinoma[END_REF] Rating on the KPS was done independently by two investigators (NT and FWK). In case of discordantly rated performance scores a third investigator (JCT) was introduced and the most matching score was used thereafter. Histopathological diagnosis and determination of the MGMT methylation status were obtained within 5-7 working days. Within 3 weeks after stereotactic biopsy, patients were assigned to receive XRT/TMZ TMZ: Treatment included XRT (60 gray in 30 fractions)/ TMZ [daily dose of 75 mg/m 2 ] TMZ (150 to 200mg/m 2 per day for 5 days of every 28-day cycle). In case of long term compliance, however, TMZ was continued (at the same dose) until tumour progression occurred. At baseline evaluation, 4-6 weeks after XRT/TMZ and every 3 cycles during TMZ maintenance therapy, clinical and neuroradiologic examinations were performed (until last follow up). MRI interpretation was independently done according to the Macdonald criteria [START_REF] Macdonald | Response criteria for phase II studies of supratentorial malignant glioma[END_REF] by an experienced neuroradiologist (JL), who was blinded for the MGMT status and the follow up data of the patient. Treatment response was evaluated after the completion of 3 TMZ cycles or earlier in case of clinical deterioration. Tumour progression had to be confirmed by further clinical and neuroradiological follow up to exclude any bias by pseudoprogression. [START_REF] Brandes | MGMT promoter methylation status can predict the incidence and outcome of pseudoprogression after concomitant radiochemotherapy in newly diagnosed glioblastoma patients[END_REF] Additional metabolic imaging [START_REF] Popperl | Analysis of 18F-FET PET for grading of recurrent gliomas: is evaluation of uptake kinetics superior to standard methods?[END_REF] and/or stereotactic re-biopsy were considered necessary, when further (modified) invasive treatment strategies were under consideration. Haematology was performed weekly. Adverse events were defined according to the National Cancer Institute (NCI) Common Toxicity Criteria, version 3.0. The minimum follow up after inclusion of the last patient had to be 6 months.

Statistical analysis

The hazard ratio for tumour progression was in the range of 0.48 in favour of the MGMT promoter methylated group in the study of Hegi et al.. [START_REF] Hegi | MGMT gene silencing and benefit from temozolomide in glioblastoma[END_REF] In the absence of cytoreductive surgery we expected an even more pronounced outcome difference; this assumption was indirectly supported by the study of Chinot et al. [START_REF] Chinot | Correlation between O6-methylguanine-DNA methyltransferase and survival in inoperable newly diagnosed glioblastoma patients treated with neoadjuvant temozolomide[END_REF] who have found highly divergent outcome scores as a function of MGMT expression after neoadjuvant TMZ of non-resectable glioblastoma [median progression free survival (PFS): 5.5 versus 1.9 months]. In the current series a hazard ratio of 0.4 or even less in favour of the MGMT promoter methylated group was expected. Assuming exponential survival, we estimated that a sample size in the range of 27 patients or more in each group would be sufficient to have a power of 80% to demonstrate a significant difference in PFS in favour of glioblastoma with a methylated MGMT promoter.

The reference point of this study was the date of the diagnosis. Primary endpoint was PFS; Secondary endpoints were treatment response (TR) and overall survival (OS). PFS and OS were analyzed by the Kaplan-Meier method [START_REF] Kaplan | Non parametric estimation for incomplete observation[END_REF] and compared with the two-sided log-rank test. Logistic regression models were used to analyze the association between TR and other variables. The Cox model was fitted to asses the prognostic value of the MGMT methylation status and other potential prognostic factors. [START_REF] Cox | Regression model and life-tables[END_REF] First, the importance of each variable was tested univariately. Second, all variables were fitted together. The "BEST" model contained only variables associated with PFS or OS after the adjustment for the effects of the other variables. The distribution of patient-and tumour-related variables between MGMT promoter methylated and unmethylated subgroups was analyzed by the chi-squared statistics (for dichotomized variables) and the Wilcoxon test (for continuously scaled variables). P≤ 0.05 was considered significant. All calculations were performed using the SAS software package (version 9.1).

Results

Patient characteristics

Out of an overall number of 136 patients that have been newly diagnosed with a glioblastoma was 53.6% (30/56) including one patient with a partially methylated promoter. Methylated and unmethylated subpopulations did not differ with respect to patients' characteristics even though patients with a methylated MGMT promoter were somewhat younger (Table 1).

Clinical outcome

The median follow up time was 48 weeks (range: 24-144) for the survivors. Forty-one out of 56 patients exhibited tumour progression and 34 patients had died. Death was tumour-related in all patients. Kaplan-Meier estimates for PFS and OS of the whole study population are presented in Figure 1. Both primary and secondary end points were in favour of MGMT promoter methylated tumours (Table 1): MGMT promoter methylated tumours exhibited superior median PFS (56 versus 20 weeks) and superior median OS (104 versus 28 weeks);

PFS after 6 ( 12) months was 83.3% (54.4%) for MGMT promoter methylated tumours and 34.6% (0%) for those with an unmethylated MGMT promoter (p<0.0001) (Figure 2); The corresponding 6 (12) months survival rates were 90.0% (64.8%) and 57.7% (13.2%), respectively (p<0.0001) (Figure 2). Among patients with MGMT promoter methylation the unadjusted hazard ratio for disease progression and death was 0.15 (95% confidence interval: 0.07-0.33) and 0.18 (95% confidence interval: 0.08-0.38), respectively.

Treatment responders showing partial response or stable disease (three months after treatment) were more often seen in case of a methylated MGMT promoter (28/30 MGMT methylated tumours versus 12/26 unmethylated tumours; p=0.0008). Treatment responders were younger and experienced a longer OS than non-responders (median: 72 versus 18 weeks; p<0.0001) (see Table 3). The survival advantage, however, was less pronounced for those with an unmethylated MGMT promoter (median: 44 weeks versus 104 weeks, p<0.001). At the time of tumour progression, treatment concepts included second-line chemotherapy (n=16), re-irradiation (n=4), tumour debulking due to significant space occupying effects (n=2), stereotactic brachytherapy (n=3), a combination of these treatment modalities (n=8), and palliative care (n= 23). Median survival after progression was longer for methylated tumours (3.7 months versus 1.9 months); the difference, however, was not significant.

Prognostic and predictive factors

Univariately, MGMT promoter methylation, younger age, higher KPS scores, and RTOG-RPA classes III+IV were positively correlated with both increased PFS and OS. In the "BEST" prognostic Cox models only MGMT promoter methylation status (OS: p<0.0001; PFS: p<0.0001), younger age (PFS: p= 0.02), and higher KPS scores (OS: p=0.02) remained statistically significant (Table 4). [START_REF] Hegi | MGMT gene silencing and benefit from temozolomide in glioblastoma[END_REF]; the difference between these studies was statistically not significant and might have occurred by chance. However, the exact assignment of each biopsy specimen within the stereotactically defined tumour space, the determination of its relation to tumour necrosis or non-neoplastic tissue by alternating histopathological and molecular evaluation within 1mm-steps, the simultaneous application of two molecular genetic methods at different sites of each tumour, and the exclusive use of cryopreserved specimens indicate important differences to the study of Hegi et al. [START_REF] Hegi | MGMT gene silencing and benefit from temozolomide in glioblastoma[END_REF].

We further demonstrate that the MGMT status after XRT/TMZ TMZ remained unchanged, which was seen in 13 patients undergoing stereotactic re-biopsy because of tumour progression. Both the detected intra-tumoural homogeneous distribution of the MGMT promoter methylation status and its unchanged pattern before and after XRT/TMZ TMZ suggests that MGMT promoter methylation may be a rather early event in tumour genesis.

In summary, the current series describes for the first time the powerful treatment effects of XRT/TMZ TMZ for patients with non-resectable glioblastoma in case of a methylated

  in our department between March 2006 and August 2008 a total of 56 consecutive patients (33 men, 23 women) fulfilled inclusion criteria and were enrolled in this study. Patients were categorized into RTOG-RPA class III (4 patients, 7.1%), class IV (12 patients, 21.4%), class V (28 patients, 50.0%), and class VI (12 patients, 21.4%). The median of the age distribution was 62.5 years (range, 23-85 years); 22 (39%) patients were 65 years of age or older. The median KPS was 70; 24 patients (42.9%) experienced a KPS of 70 and 13 a KPS of 60 (23.2%). Forty-eight patients (85.7%) exhibited multi-focal (6 patients) or eloquent (42 patients) tumour locations including involvement of the primary motor/ sensory cortex (10 patients), speech area (8 patients), visual cortex (3 patients), basal ganglia (16 patients), and the corpus callosum (5 patients). Stereotactic biopsy procedures were associated with a transient morbidity of 1.8% (1 patient with transient aphasia). MSP and sequencing analyses always exhibited concordant and reproducible results throughout each tumour (mean number of samples per tumour: 3; range 2-5). The overall frequency of MGMT methylated tumours

  cycle of TMZ, respectively. MRI-based suspicion of tumour progression was additionally verified by metabolic imaging [O-(2-[18F]fluoroethyl)-L-tyrosine (FET) positron emission tomography (PET)] in 20 patients and/or re-biopsy in 13 patients; the latter always revealed an unchanged MGMT promoter methylation status.

  MGMT promoter; It underscores that non-resectable tumours should not be associated per se with a worse prognosis as compared to those undergoing open tumour resection. The demonstrated high diagnostic sensitivity and specificity of MSP and sequence analysis in combination with stereotactic biopsy technique might help to encourage the further development and implementation of molecular genetic analysis into biopsy procedures of daily clinical practice.

Figure 1 :

 1 Figure 1:

Figure 2 :

 2 Figure 2:

Table 1 .

 1 Characteristics and Clinical Outcome of Patients with Glioblastoma according to the Methylation Status of the MGMT Promoter

	Unmeth
	Meth (N=30)	P-value
	(N=26)

after XRT/TMZ, which was associated with a decline in the mental status (disorientation and confusion). TMZ maintenance therapy caused grade 1/2 toxicity in 12 patients. As a

Table 2 .

 2 Toxicity Symptoms*

	Number of patients (%)
	(N=56)

Table 3 .

 3 Characteristics of Patients with Glioblastoma stratified for Treatment

	Response
	Non-responder	Responder
		P-value
	(progression)	(regressive + stable disease)

Table 4 .

 4 Prognostic Factors (univariate and multivariate models) TMZ), was a strong predictor for a more favourable outcome and not unequivocally related to the MGMT promoter status: 93.3% of the MGMT methylated and, noteworthy, 46.2% of the unmethylated tumours exhibited partial response/stable disease after XRT/TMZ TMZ. Survival, however, was significantly longer in case of a methylated MGMT promoter. Thus, for treatment responders with an unmethylated MGMT promoter, the benefit of XRT/TMZ TMZ as compared to XRT alone continues to be unclear. Future studies are required that further elucidate the molecular network of epigenetic silencing of the MGMT promoter and MGMT gene expression in correlation with clinical course and prognosis.[START_REF] Everhard | Identification of regions correlating MGMT promoter methylation and gene expression in glioblastomas[END_REF] Molecular stereotactic biopsy turned out to be a safe (transient morbidity of as low as 1.8% in this series) and precise diagnostic procedure. The highly controlled sampling technique ensured the use of only vital tumour tissues for subsequent molecular analyses, and excluded bias of the results by non-neoplastic tissue contaminations. Handling of nucleic acids as well

	PFS	OS
	P-value (Hazard ratio/95% CI)

Apparently, pseudoprogression seems to be not a frequent event in non-resectable glioblastoma patients undergoing XRT/TMZ. Partial response or tumour control (three months after XRT/

Acknowledgements

Michael Ruiter is gratefully acknowledged for help with MSP and sequencing analysis. We further thank Barbara Stetter, Margit Deschner, and Anja Ewert for their support with management of the patient database. Funding did not influence collection, analysis and interpretation of the data, writing of the manuscript, and the decision to submit the paper for publication.

Licence for Publication:

The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd to permit this article (if accepted) to be published in JNNP and any other BMJPGL products and sublicences such use and exploit all subsidiary rights, as set out in our licence.

Competing Interest: None declared.


F I Arrigoni

M Matarin

P J Thompson

M Michaelides

Clements

E Redmond

L Clarke

E Ellins

S Mohamed

I Pavord

D M Hunt

N Klein

J Halcox

S M Sisodiya
email: sisodiya@ion.ucl.ac.uk

Extended extraocular phenotype of PROM1 mutation in kindreds with known autosomal dominant macular dystrophy

Mutations in prominin 1 (PROM1) have been shown to result in retinitis pigmentosa, macular degeneration and cone-rod dystrophy. Because of the putative role of PROM1 in hippocampal neurogenesis, we examined two kindreds with the same R373C PROM1 missense mutation using our established paradigm to study brain structure and function. As the protein encoded by PROM1, known as CD133, is used to identify stem/progenitor cells that can be found in peripheral blood and reflect endothelial reparatory mechanisms, other parameters were subsequently examined that included measures of vascular function, endothelial function and angiogenic capacity. We found that aspects of endothelial function assayed ex vivo were abnormal in patients with the R373C PROM1 mutation, with impaired adhesion capacity and higher levels of cellular damage. We noted also renal infections, haematuria and recurrent miscarriages possibly reflecting consequences of abnormal tubular modelling. Further studies are needed to confirm these findings.

Introduction

The effects of a single human gene mutation may be widespread and, in their breadth, often unsuspected. We have previously explored the cerebral structural and functional effects of mutations in genes expressed in the eye and the brain, in individuals ascertained by their mutant eye phenotype, in whom extraocular phenotypes were often not appreciated. Using this paradigm, we have demonstrated roles for the genes PAX6, PITX2, SOX2, OTX2 and RIMS1 in human brain development, and cognitive function [START_REF] Sisodiya | PAX6 haploinsufficiency causes cerebral malformation and olfactory dysfunction in humans[END_REF][START_REF] Idrees | A novel homeobox mutation in the PITX2 gene in a family with Axenfeld-Rieger syndrome associated with brain, ocular, and dental phenotypes[END_REF][START_REF] Sisodiya | Role of SOX2 mutations in human hippocampal malformations and epilepsy[END_REF][START_REF] Henderson | Inherited PAX6, NF1 and OTX2 mutations in a child with microphthalmia and aniridia[END_REF].

Mutations in prominin 1 (PROM1) have been shown to result in retinitis pigmentosa [START_REF] Zhang | Severe retinitis pigmentosa mapped to 4p15 and associated with a novel mutation in the PROM1 gene[END_REF][START_REF] Maw | A frameshift mutation in prominin (mouse)-like 1 causes human retinal degeneration[END_REF] macular degeneration [START_REF] Yang | Mutant prominin 1 found in patients with macular degeneration disrupts photoreceptor disk morphogenesis in mice.[see comment][END_REF][START_REF] Permanyer | A novel mutation in PROM1 that results in nonsense mediated decay causes autosomal recessive Retinitis Pigmentosa with early macular affectation[END_REF] and cone-rod dystrophy [START_REF] Michaelides | The PROM1 mutation p.R373C causes an autosomal dominant bull's eye maculopathy associated with rod, rod-cone and macular dystrophy[END_REF]. PROM1 encodes prominin-1, a 5-transmembrane glycoprotein also known as CD133 and AC133. CD133 was originally identified as a cell surface antigen present on hematopoietic stem cells and early progenitor cells in the bone marrow, including endothelial progenitor cells (EPC) [START_REF] Yin | AC133, a novel marker for human hematopoietic stem and progenitor cells[END_REF][START_REF] Miraglia | A novel five-transmembrane hematopoietic stem cell antigen: isolation, characterization, and molecular cloning[END_REF].

Emerging evidence suggests that EPCs are able to differentiate into mature endothelial cells, contributing to neovascularization and re-endothelialization during both embryonic and postnatal physiological processes [START_REF] Urbich | Endothelial progenitor cells: characterization and role in vascular biology[END_REF]. Consequently, CD133 is used to identify stem/progenitor cells that can be found in peripheral blood and thereby identify endothelial reparatory mechanisms and also to identify tumour stem cell populations in a variety of blood and solid cancers [START_REF] Singh | Identification of human brain tumour initiating cells.[see comment[END_REF][START_REF] Zhu | Prominin 1 marks intestinal stem cells that are susceptible to neoplastic transformation[END_REF]. Consistent with a role in tissue repair, increased levels of circulating early EPCs marked by CD133 have also been found in response to brain injury [START_REF] Liu | Changes in circulating human endothelial progenitor cells after brain injury[END_REF]. CD133 is also involved in murine and human adult hippocampal neurogenesis [START_REF] Kempermann | Natural variation and genetic covariance in adult hippocampal neurogenesis[END_REF], murine brain myelin genesis and maintenance [START_REF] Corbeil | Expression of distinct splice variants of the stem cell marker prominin-1 (CD133) in glial cells[END_REF] Because of the putative role of PROM1 in hippocampal neurogenesis, we examined two kindreds with the same known PROM1 mutation using our established paradigm to study brain structure and function. We subsequently examined other parameters including measures of vascular function, endothelial function and angiogenic capacity.

Methods

The study included two unrelated kindreds that were already reported as having autosomal dominant macular degeneration mapped to 4 p15.2-16.3 [START_REF] Zhang | Severe retinitis pigmentosa mapped to 4p15 and associated with a novel mutation in the PROM1 gene[END_REF][START_REF] Michaelides | An autosomal dominant bull's-eye macular dystrophy (MCDR2) that maps to the short arm of chromosome 4[END_REF]. From kindred A, the proband (A-V:1), her mother (A-IV:2), aunt (A-IV:4) and grandmother (A-III:6) were studied. In kindred B, the proband (B-II:1) and her mother (B-I:1) were included in the study (Table 1). In both probands, the same R373C PROM1 mutation had been identified. Clinical history was obtained from all participants.

Healthy control groups were recruited from staff and colleagues from the Institute of Child Health.

Brain imaging

Subjects were studied on a 3T scanner. Details of the high-resolution MRI, cerebral and hippocampal volumetry are provided in the Supplementary material.

Cognitive tests applied to kindreds

Neuropsychological tests employed were as described previously [START_REF] Sisodiya | Genetic enhancement of cognition in a kindred with cone-rod dystrophy due to RIMS1 mutation[END_REF]. All of the standardised tests selected require verbal interaction only. Intellectual level, executive and memory function were tested. Details are given in the Supplementary Material.

Clinical testing

Proband A-V:1 had renal and pulmonary assessments previously undertaken due to relevant clinical symptoms. Olfactory testing was undertaken using the UPSIT [START_REF] Sisodiya | PAX6 haploinsufficiency causes cerebral malformation and olfactory dysfunction in humans[END_REF] (Sensonics Inc., Haddon Heights, NJ), with age-matched control data based upon North American subjects, shown to be applicable to British populations [START_REF] Gibberd | Smell testing: an additional tool for identification of adult Refsum's disease[END_REF][START_REF] Silveira-Moriyama | The use of a color coded probability scale to interpret smell tests in suspected parkinsonism[END_REF].

Blood and vascular investigations

After a 12-hour overnight fast, venous blood was taken from three subjects from kindred A, and one from kindred B. After subjects had rested for >15 minutes, recumbent right brachial artery blood pressure was recorded using an automated sphygmomanometer (Dinamap, Critikon, Florida, USA). Five readings were taken and mean systolic and diastolic blood pressure calculated.

The isolation of peripheral blood mononuclear cells (PBMCs), flow cytometric analysis for detection of EPCs and cellular microparticles, circulating endothelial cell extraction and analyses, growth factor detection, the ability of specific cells to form colonies, microparticle assays and in vitro colony forming and matrigel angiogenesis assays were performed as detailed in the Supplementary Material. For kindred A, vascular structure (carotid artery intima-media thickness, IMT) and function (flow-mediated dilatation of brachial artery, FMD) were also assessed (Supplementary Material).

Statistics

Nonparametric tests (U-Mann-Whitney) were used with SPSS, version 14 (SPSS Inc).

Data were expressed as median and interquartile range.

Results

Clinical findings

The ocular phenotypic data have been published [START_REF] Zhang | Severe retinitis pigmentosa mapped to 4p15 and associated with a novel mutation in the PROM1 gene[END_REF][START_REF] Michaelides | An autosomal dominant bull's-eye macular dystrophy (MCDR2) that maps to the short arm of chromosome 4[END_REF]. A-V:1 had steroid-resistant asthma. She had a clinical assessment for microscopic haematuria, with a normal renal ultrasound and intravenous pyelogram. A-IV:2 also had microscopic haematuria, with recurrent renal infection, and renal scarring on intravenous pyelography. She had eight pregnancies, and four children. A-III:6 had recurrent renal infections, seven pregnancies with three miscarriages, and four children. A-IV:4 had two unprovoked seizures.

In kindred B, B-I:1 had high blood pressure and high cholesterol.

According to North-American normative data UPSIT A-IV:4 and B-II:1 had mild microsmia and A-III:6 had moderate microsmia.

Neuroimaging

Subject A-III:6 had many small high-intensity lesions in the cerebral white matter (Fig 1B). Subjects A-IV:4 and B-I:1 also had a few (1-2) non-specific white matter (WM) lesions. The presence of WM hyperintensities on the FLAIR images for subject A-III:6 was scored by a radiologist blind to the clinical data, using the Scale of Tarvonen-Schroder and Scheltens semiquantative rating scale [START_REF] Scheltens | A semiquantative rating scale for the assessment of signal hyperintensities on magnetic resonance imaging[END_REF]. This scale scores the severity of hyperintensities separately in different anatomical areas of the brain including periventricular regions, areas of deep white matter , basal ganglia and different infratentorial regions. The total score was 19.

Lesions were focused on deep white matter (score=16/24) and pons (score=3/6). Lesions in frontal brain area were considered as severe and mild to moderate in the other brain areas.

An empty sella turcica was noted in A-III:6, A-IV:2 and A-IV:4 (Fig 1A). Baseline pituitary function (TSH, T4, cortisol, FSH and LH) was normal in all three. No other changes were noted. MRI brain scans in kindred B were normal. Hippocampal and cerebral volumes were within normal ranges for all patients from both kindreds.

Cognitive function

No distinctive pattern of cognitive deficit was noted (Table 1). All subjects in kindred

A except A-IV:4 showed average to superior verbal memory scores. A-IV:4 showed poor performance for delayed recall on the auditory verbal learning test with an average performance on the remaining tests of memory. In kindred B, B-I:1 had impaired verbal learning for trials ,reduced performance on tests of immediate recall and delayed verbal learning. All subjects except A-IV:4 and B-I: 1 showed average executive functions.

Vascular and endothelial function

Vascular profiles

Subjects A-V:1, A-IV:2 and A-III:6 underwent vascular investigation. Each subject was ranked into an age group, (1, 2 or 3), in which 12 healthy controls, with a median age similar to that of the subject, were included (see Table 2). Patients and controls were all female and had no known cardiovascular risk factors with the exception of high blood pressure and high cholesterol in subject B-1:1.

Brachial artery FMD and carotid artery IMT

Detailed descriptions of procedures are in Supplementary Material. Briefly the diameter of the brachial artery was measured at rest, during reactive hyperaemia to induce flow-mediated endothelium-dependent dilation (FMD), again at rest, and after administration of GTN, an endothelium-independent dilator. FMD and the ratio FMD:GTN-induced dilatation provide the best measures of endothelial function.

Flow-mediated dilatation did not alter with age in the controls (Table 1). There was no difference in median FMD when comparing grouped controls and patients (8.28 (5.68-11.40) and 4.88 (4.06-12.27)). The ratio FMD:GTN did not alter with age in controls, but fell with age in the patients. The median FMD:GTN ratio for the grouped patients did not differ from the controls (0.95 (0.90-0.97) and 0.98 (0.96-1.02)). Intima-media thickness (IMT) increased significantly with age in the controls (Table 2). Increasing IMT values were also observed in the patients. However, there was no difference in the median values for IMT between grouped controls and PROM-1 patients (0.65 (0.58-0.72) and 0.52 (0.48-0.75)).

Determination of Growth Factor Concentration

Angiopoietin-2 levels and vascular endothelial growth factor (VEGF) levels were within the normal range according to established clinical ranges.

Isolation and quantification of EPC

In controls, circulating endothelial cell (CEC) number significantly increased between the younger groups 1 and 2, but not between either of the other age groups (Table 2). Increase of CECs with age is less evident in PROM1 patients but falls within ranges for healthy agematched controls (Table 2). No change in the expression of surface markers was observed with age in controls or PROM1 patients. Calculated as a percentage of the lymphocyte gate, there was no significant difference between groups in the expression of any of the surface markers alone or in combination (Table 2).

Cultivation of EPC

Colony-forming unit endothelial cells (CFU-EC) fell with age in controls. This trend was also observed in the few PROM1 patients. Colony number tended to be low but values fell within the normal range for their ages (controls, 20.37 (7.05-32.57) and PROM1 patients, 11.9 (4.6-41)).

Relationship Between Cellular and Vascular Measures

Numbers of CFU-EC correlated with FMD:GTN ratio in the controls (r=0.42, p<0.05). CFU-EC from PROM1 patients demonstrated a similar trend (Supplementary Material ).

Microparticle number.

Total microparticle number, which consisted of endothelial microparticles (CD144, D105 and CD62e), platelet microparticles (CD42a) and monocyte-derived microparticles (CD14), fell within the upper range in PROM1 patients in relation to age-matched controls.

Comparisons between patients and controls independently to age, showed higher number of total microparticles in PROM-1 patients ((2.606(1.399-2.97); 0.3023(0.14-1.35) for PROM-1 patients and healthy age matched controls respectively, median ± interquartile range, p= 0.0028). Furthermore endothelial microparticles defined by CD144 expression were also found to be significantly elevated in the PROM-1 progeny (Table 3).

Angiogenic capability

A Matrigel assay was used to determine the effect of colony-forming unit endothelial cells labelled with acetylated low-density lipoprotein (DiI-acLDL) on HUVEC growth. DiI-acLDL-labelled cells from PROM1 patients did not adhere to or incorporate into the human umbilical endothelial cells (HUVEC) as well as their age-matched controls (5.6 (0-18); 26.7 (15.5-44.3), p=0.03; Figure 3). Furthermore, a lack of DiI-acLDL-labelled cell incorporation into the HUVEC tubules tended to produce lower tubule length in PROM1 subjects when compared to their age-matched controls (0.8 (0.74-1.2) and 1.38 (0.9-1.46), p=0.06). No difference in tubule number was observed between the two groups (1 (1-1.14); 0.88(0.52-1.22), p=0.57; Figure 3).

Discussion

We describe vascular and endothelial function in addition to cerebral structure and function in two independent families with the same R373C mutation in the PROM1 gene.

Carriers of the mutation were already reported to present clinical features of autosomal dominant macular dystrophy [START_REF] Yang | Mutant prominin 1 found in patients with macular degeneration disrupts photoreceptor disk morphogenesis in mice.[see comment][END_REF][START_REF] Michaelides | An autosomal dominant bull's-eye macular dystrophy (MCDR2) that maps to the short arm of chromosome 4[END_REF]. Human PROM1 is expressed by various stem and progenitor cells originating from diverse sources [START_REF] Wu | CD133 as a marker for cancer stems cells: progresses and concerns[END_REF]. Due to the role of EPC in vascular and endothelial function [START_REF] Werner | Influence of cardiovascular risk factors on endothelial progenitor cells: limitations for therapy?[END_REF], we investigated these characteristics in five carriers of the R373C mutation. We found that the number of circulating endothelial cells, endothelial markers such as CD133, as well as the ability of EPC to form endothelial colonies fell within the normal range in the tested patients.

Endothelium-dependent and endothelium-independent function assessed by FMD and the FMD:GTN ratio did not differ from controls. There was also no difference in intima-media thickness. The basic function and EPC regenerative resources would seem unaffected by the R373C mutation.

However the number of circulating microparticles (MPs) was raised in patients compared to controls. MPs are membrane fragments shed by cells which have been activated by a variety of stimuli including serine proteases, inflammatory cytokines, growth factors and stress inducers [START_REF] Doeuvre | Cell-derived microparticles: a new challenge in neuroscience[END_REF]. Clinical studies have revealed elevated plasma levels of MPs in multiple sclerosis, thrombotic thrombocytopenic purpura, coronary artery disease, hypertension, preeclampsia, and diabetes [START_REF] Morel | Procoagulant microparticles: disrupting the vascular homeostasis equation?[END_REF][START_REF] Chironi | Endothelial microparticles in diseases[END_REF]. Such data have led to the concept that endothelial MP are key factors at the crossroads between inflammation, coagulation, proteolysis and vascular repair [START_REF] Sabatier | Circulating endothelial cells, microparticles and progenitors: key players towards the definition of vascular competence[END_REF]. Elevated circulating levels of MPs in our patients could be an indicator of either platelet, endothelial or leukocyte activation and could reflect a persistent state of endothelial dysfunction.

We looked also at the contribution of immature ECFCs to tubule growth, of immature ECFCs. A significantly decreased ability of these cells to incorporate or adhere to the HUVEC was observed in carriers of the mutation. As this assay evaluates multiple cellular processes involved in blood vessel growth, EPC functional activity would appear impaired by the mutation. In turn, vascularization might be affected in the patients, at least under certain circumstances, perhaps those requiring neogenesis or repair.

Notably, the mutation carriers had microhaematuria and/or renal infections and a high number of miscarriages. Although there is no study reporting endothelial dysfunction in patients with hematuria and/or renal infections, several studies have reported that EPCs are numerically and functionally impaired in patients with acute [START_REF] Westerweel | End-stage renal disease causes an imbalance between endothelial and smooth muscle progenitor cells[END_REF] or chronic renal failure [START_REF] Herbrig | Endothelial progenitor cells in chronic renal insufficiency[END_REF], as compared to healthy subjects and mechanisms such as inflammation or uraemic toxins have been given as potential explanations for the observed EPC dysfunction [START_REF] Herbrig | Endothelial progenitor cells in chronic renal insufficiency[END_REF].

More clear and direct is the relationship between miscarriages and possible endothelial dysfunction, given that development, maturation and maintenance of a neovascular network are necessary for successful haemochorial placentation as well as normal embryonic development and growth [START_REF] Demir | Vasculogenesis and angiogenesis in the early human placenta[END_REF]. Impairment in EPC functionality could thus affect placental angiogenesis and contribute to history of recurrent miscarriages in our patients. This requires further study in a larger group of patients, and might merit study in women with unexplained recurrent miscarriages.

CD133 has been also reported to mark several cell types with a variety of putative roles, including involvement in hippocampal neurogenesis [START_REF] Kempermann | Natural variation and genetic covariance in adult hippocampal neurogenesis[END_REF][START_REF] Corbeil | Expression of distinct splice variants of the stem cell marker prominin-1 (CD133) in glial cells[END_REF] and diverse haematopoietic and extrahaematopoietic progenitive and regenerative functions [START_REF] Urbich | Endothelial progenitor cells functional characterization[END_REF][START_REF] Zeppernick | Stem cell marker CD133 affects clinical outcome in glioma patients[END_REF][START_REF] Cantley | Adult stem cells in the repair of the injured renal tubule[END_REF].

Carriers of the PROM1 mutation showed hippocampal and cerebral volumes within normal ranges for all patients from both kindreds. Three patients had small lesions in white matter.

Interestingly the presence of MPs has been also reported to be positively associated with contrast-enhancing lesions on brain MRI in subjects with multiple sclerosis [START_REF] Minagar | Elevated plasma endothelial microparticles in multiple sclerosis[END_REF] and furthermore endothelial dysfunction is thought to play an important role in the pathogenesis of cerebral small-vessel disease especially in those patients with concomitant silent lacunar infarcts and ischemic white matter lesions [START_REF] Knottnerus | Endothelial dysfunction in lacunar stroke: a systematic review[END_REF]. In line with this possible accelerated aging effect, the older member from the kindred B and one carrier of the PROM1 mutation in family A showed some memory disturbance and impairment in measures of executive functions in addition to small WM lesions. The remaining carriers in both families had average IQs and unimpaired performance on the other cognitive measures.

The three (of four) members of family A showed an empty sella turcica. The familial empty sella together with an eye phenotype could suggest a developmental defect of mesenchymal origin caused by mutation in the same gene [START_REF] Asahara | Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization[END_REF]. Familial empty sella has not, to our knowledge, been reported previously. Three patients had impaired olfaction.

All together, the elevated plasma levels of MPs, decreased ability of EPC to incorporate into or adhere to tubules, the presence of empty sella turcica as well as leucoaraiosis and cognitive decline in the oldest mutation carriers suggest endothelial dysfunction that could be more pronounced or more evident with age. The R373C mutation results in a stable mutant protein. However, the mutant protein is not only mislocalized in retina, but it also interferes with the action of the normal protein [START_REF] Yang | Mutant prominin 1 found in patients with macular degeneration disrupts photoreceptor disk morphogenesis in mice.[see comment][END_REF] thereby accounting for its dominant mode of inheritance. Though we found no difference in the number of circulating EPCs or numbers of cells expressing CD133, the mutation may impair the function. For example the mutation could render EPCs less effective in the process of angiogenesis or repair of damaged endothelium. Carriers of the R373C mutation showed no obvious greater risk of vascular dysfunction, but structural and functional microcirculation alterations tend to occur during ageing. Functional vascular examination was only undertaken in one of the two older patients as the second declined to take part in this test. Moreover the presence of high blood pressure and high cholesterol, as well as leucoaraiosis [START_REF] Wr | Microvascular changes in the white mater in dementia[END_REF] could be a sign of vascular alteration. Longitudinal study of more individuals with PROM1 mutation might be informative.

To our knowledge this is the first study reporting a family with eye phenotype together with empty sella turcica, presence of microhematuria and possible endothelial dysfunction. In the case of the eye phenotype (macular dystrophy) the penetrance was complete although showing differences in severity. Penetrance in the other phenotypes was incomplete. Notable is the fact that none of the members in family B presented extra-ocular features, nor MRI findings (other than a few WM lesions), besides the cognitive decline in the older member, raising the possibility of organ-specific penetrance differences, perhaps related to organ-specific splicing differences of the PROM1 transcript. PROM1 transcription regulation is rather complicated and poorly understood: it exhibits extensive splice variation [START_REF] Fargeas | Nomenclature of prominin-1 (CD133) splice variants -an update[END_REF] with tissue-specific distribution [START_REF] Pleshkan | Methylation of the prominin 1 TATAless main promoters and tissue specificity of their transcript content[END_REF], and in addition transcripts can be regulated by epigenetic factors, as suggested by experiments with artificial in vitro methylation. However we cannot exclude the possibility that mutation in a second gene could be influencing the extra-ocular phenotype in family A, especially the presence of empty sella turcica.

The role of PROM1 in brain is still unclear. The gene, by itself or in combination with other genes, seems to have a role in the development of the brain, and/or cerebral recovery and repair [START_REF] Corbeil | Expression of distinct splice variants of the stem cell marker prominin-1 (CD133) in glial cells[END_REF][START_REF] Hermann | Mesodermal cell types induce neurogenesis from adult human hippocampal progenitor cells[END_REF]. The heterozygous mutation studied here results in a stable protein [START_REF] Yang | Mutant prominin 1 found in patients with macular degeneration disrupts photoreceptor disk morphogenesis in mice.[see comment][END_REF] and is perhaps insufficient to completely inhibit its function in the brain. In contrast, recessive mutations arise from either frameshift or nonsense mutations [START_REF] Zhang | Severe retinitis pigmentosa mapped to 4p15 and associated with a novel mutation in the PROM1 gene[END_REF][START_REF] Maw | A frameshift mutation in prominin (mouse)-like 1 causes human retinal degeneration[END_REF] that result in the generation of a premature stop codon and truncated protein [START_REF] Zhang | Severe retinitis pigmentosa mapped to 4p15 and associated with a novel mutation in the PROM1 gene[END_REF][START_REF] Maw | A frameshift mutation in prominin (mouse)-like 1 causes human retinal degeneration[END_REF][START_REF] Pras | Cone-rod dystrophy and a frameshift mutation in the PROM1 gene[END_REF]. In these cases the homozygous patients display a severe eye phenotype, and polydactyly was also reported in one of the carriers, an additional symptom that confirms the existence of variable penetrance associated with PROM1. No other documentation about vascular, endothelial or brain structures was reported.

The nature and size of our study does not permit definitive conclusions but leads to the suggestion that endothelial function may be affected in patients with the PROM1 R373C mutation, despite the apparently normal levels of EPC. Further studies are needed to confirm these interesting findings that broaden the phenotype of PROM1 mutation, and inform our understanding of CD133 function.

  Running Head: Extended PROM1 phenotype Key words: PROM1, CD 133, endothelial, tubule, sella turcica Total Word Count: 3,019 (max 4,000) Abstract text: 146 (max 250) Tables and Illustrations: 3 figures and 3 tables (max 6) Table1: Cognitive performance and plus

Figure 1 :

 1 Figure 1: Leucoaraiosis and empty sella turcica

Figure 2 :Figure 3 :

 23 Figure 2: Representative micrograph of HUVEC Figure 3: Angiogenesis assay between HUVEC and ECFCs. References: 42 plus 10 in supplementary material (max 50) Supplementary material: 1,733

  The study was approved by the Joint Research Ethics Committees of the Institute of Neurology/ National Hospital for Neurology and Neurosurgery, and Moorfields Eye Hospital and the Institute of Child Health/Great Ormond Street Hospital for Children NHS Foundation Trust. Subjects provided informed written consent for all parts of the study.

Titles and legends to figuresFigure 1A .

 1A Figure 1A. The white arrows show empty sella turcica in three PROM1 patients (A-IV:4, A-III:6 and A-IV:2) and one normal sella turcica (A-V:1). Figure 1B. The black arrows show White Matter Hyperintensities in one PROM1 patient (A-III:6).

Figure 2 .

 2 Figure 2. Representative micrograph of HUVEC in Matrigel for 18 hours with EGM-2 grown cells from PROM1 patients and age-matched controls (24, 47 and 61 years old). Cells labelled red are DiI-labelled EGM-2 grown cells after seven days in culture. Fewer adherent (red-coloured) cells and a tendency for shorter tubule formation are apparent in patients compared to controls. The adhesion of the (red-labelled) ECFCs contributes to endothelial tubule formation. Following culture of ECFCs with HUVEC, fewer ECFCs were adherent or incorporated into the tubules from the PROM1 subject when compared to the age matched control. Magnification x100, 8 random fields studied in A-V:1, A-IV:2 and A-III:6 and B-I:1.

Figure 3 .

 3 Figure 3. Angiogenesis assay for HUVEC and ECFCs. Alteration in growth of HUVEC tubules with ECFCs from PROM1 subjects. A. Number of cells adherent/incorporated into endothelial tubules on Matrigel in PROM1 subjects and healthy age-matched controls; B. Number of tubules formed on Matrigel plate, averaged over 8 random fields between PROM1 patients and controls. C. Average tubule length of HUVEC formed on Matrigel, averaged over 8 random fields, between PROM1 and controls, *p<0.05 significant difference between PROM1 and control group.

Table 2 :

 2 Baseline characteristics

Table 3 :

 3 Microparticles

Table 1 :

 1 Summary of age -corrected cognitive performance (in cursive poor or impaired

	R373C PROM1 mutation		Family A		Family B
	Subject	A-III:6	A-IV: 4	A-IV:2	A-V:1	B-I:1	B-II:1
	Relationship to proband	Maternal Grandmother	Maternal Aunt	Mother	Proband	Mother	Daughter
	Age	65	41	44	21	65	32
	VIQ	114	90	96	110	106	110
	Verbal Learning Trials	46 (50th)	46 (25th)	51 (50th)	50 (25th)	22 (<1st)	56 (50th)
	Verbal learning delay	7 (25th)	9 (10th)	13 (75th)	12 (50th)	6 (10th)	14 (75th)
	Verbal Recall Immediate	38 (75th)	31 (25th)	43 (90th)	49 (90th)	17 (10th)	48 (90th)
	Verbal Recall %	105 (90th)	90 (50th) 102 (90th)	98 (90th)	88 (50th)	92 (50th)
	Fluency phonemic "s"	12	19	16	18	15	24
	Fluency animals	19	15	26	24	20	28
	Hayling total score	14	16	13	18	11	17
	Cognitive estimates	5	12	8	7	4	5
	Extraocular medical history	RI, M,MC	M	MH,RI,MC	SRA,MH,ne	normal	M
	MRI findings	WMH,EST	WMH,EST	EST	normal	WMH	normal
	Vascular profiles	EX	ne	EX	EX	ne	ne
	Endothelial function	EX	ne	EX	EX	ne	EX

scores) and extraocular findings. VIQ: Verbal Intelligence Quotient EX: Examined; ne: not examined; RI: Renal Infections; MC: Miscarriages; M= Microsmia; MH: Microscopic Haematuria; SRA: Steroid Resistant Asthma; WMH: White Matter Hyperintensities; EST: Empty Sella Turcica.

Acknowledgments

We thank the patients who participated. We are grateful to the Big Lottery Fund, Wolfson Trust and the National Society for Epilepsy for supporting the NSE MRI scanner. The work was supported by a joint grant from the Biomedical Research Centres at UCLH/UCL, Great Ormond Street Hospital for Children/UCL Institute of Child Health, and Moorfields Eye Hospital/UCL Institute of Ophthamology. This work was undertaken at UCLH/UCL who received a proportion of funding from the Department of Health's NIHR Biomedical