Florian B Lagler

Søren W Gersting

Clemens Zsifkovits

Alice Steinbacher

Anna Eichinger

Marta K Danecka

Michael Staudigl

Ralph Fingerhut

Hartmut Glossmann

Ania C Muntau
email: ania.muntau@med.lmu.de

New insights into tetrahydrobiopterin pharmacodynamics

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This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. BTBR, black and tan brachyuric mouse strain; DTT, dithiothreitol; K e , elimination constant; c 0 , initial concentration; c max , peak concentration; t max , time to peak concentration; AUC, area under the concentration vs. time curve at 0 to 180 minutes; t 1/2 , elimination half-life; 13 C-phenylalanine, L-[1-13]C-phenylalanine; DOB, delta over baseline; DOB BH4 , delta over baseline after BH 4 treatment; DOB Placebo , delta over baseline after Placebo; Phe 0 , initial phenylalanine concentration; Phe 180 , phenylalanine concentration at 180 minutes.

A c c e p t e d M a n u s c r i p t 3 ABSTRACT Phenylketonuria (PKU), an autosomal recessive disease with phenylalanine hydroxylase (PAH) deficiency, was recently shown to be a protein misfolding disease with loss-of-function. It can be treated by oral application of the natural PAH cofactor tetrahydrobiopterin (BH 4 ) that acts as a pharmacological chaperone and rescues enzyme function in vivo. Here we identified Pah enu1/2 bearing a mild and a severe mutation (V106A/F363S) as a new mouse model for compound heterozygous mild PKU. Although BH 4 treatment has become established in clinical routine, there is substantial lack of knowledge with regard to BH 4 pharmacodynamics and the effect of the genotype on the response to treatment with the natural cofactor. To address these questions we applied an elaborate methodological setup analyzing (i) blood phenylalanine elimination, (ii) blood phenylalanine/tyrosine ratios, and (iii) kinetics of in vivo phenylalanine oxidation using 13 C-phenylalanine breath tests. We compared pharmacodynamics in wild-type, Pah enu1/1 , and Pah enu1/2 mice and observed crucial differences in terms of effect size as well as effect kinetics and dose response.

Results from in vivo experiments were substantiated in vitro after overexpression of wild-type, V106A, and F263S in COS-7 cells. Pharmacokinetics did not differ between Pah enu1/1 and Pah enu1/2 indicating that the differences in pharmacodynamics were not induced by divergent pharmacokinetic behavior of BH [START_REF] Gersting | Loss of function in phenylketonuria is caused by impaired molecular motions and conformational instability[END_REF] . In conclusion, our findings show a significant impact of the genotype on the response to BH 4 in PAH deficient mice. This may lead to important consequences concerning the diagnostic and therapeutic management of patients with PAH deficiency underscoring the need for individualized procedures addressing pharmacodynamic aspects. Introduction Phenylketonuria (PKU; [MIM 261600]) is an autosomal recessive inborn error of metabolism caused by deficiency of hepatic phenylalanine-4-hydroxylase (PAH; EC 1. 14.16.1). PAH catalyzes the rate-limiting step in phenylalanine catabolism and is regulated by binding of its L-phenylalanine substrate and the cofactor 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH 4 ) and by phosphorylation [START_REF] Shiman | Regulation of rat liver phenylalanine hydroxylase. I. Kinetic properties of the enzyme's iron and enzyme reduction site[END_REF][START_REF] Shiman | Regulation of rat liver phenylalanine hydroxylase. II. Substrate binding and the role of activation in the control of enzymatic activity[END_REF][START_REF] Xia | Regulation of rat liver phenylalanine hydroxylase. III. Control of catalysis by (6R)-tetrahydrobiopterin and phenylalanine[END_REF]. Mutations in the PAH gene lead to loss of function of the PAH protein, which is often induced by protein misfolding [START_REF] Gersting | Loss of function in phenylketonuria is caused by impaired molecular motions and conformational instability[END_REF]. The clinical phenotypes of hyperphenylalaninemia due to PAH deficiency are classified from classic PKU (blood phenylalanine concentrations > 1,200 µM) to mild PKU (600 to 1,200 µM) and mild hyperphenylalaninemia (MHP, 120 to 600 µM). Patients with classic and mild PKU need lifelong treatment to prevent mental retardation, whereas the necessity of MHP treatment is under debate [START_REF] Bonafé | Treatable neurotransmitter deficiency in mild phenylketonuria[END_REF][START_REF] Weglage | Normal clinical outcome in untreated subjects with mild hyperphenylalaninemia[END_REF]. In the last sixty years the only treatment available for patients with PAH deficiency was dietary phenylalanine restriction, a burdensome treatment associated with significant risks of malnutrition. A paradigm change occurred ten years ago when researchers observed that pharmacological doses of the natural PAH cofactor, BH 4 , can reduce blood phenylalanine concentrations in a significant number of patients that do not display one of the rare forms of BH 4 deficiency [START_REF] Kure | Tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency[END_REF][START_REF] Muntau | Tetrahydrobiopterin as an alternative treatment for mild phenylketonuria[END_REF][START_REF] Steinfeld | Tetrahydrobiopterin monotherapy for phenylketonuria patients with common mild mutations[END_REF]. This led to definition of a new clinical phenotype, BH 4 -responsive PAH deficiency. Efficacy and safety of sapropterin dihydrochloride, the synthetic form of BH 4 , was subsequently demonstrated in clinical trials [START_REF] Trefz | Efficacy of sapropterin dihydrochloride in increasing phenylalanine tolerance in children with phenylketonuria: a phase III, randomized, double-blind, placebocontrolled study[END_REF][START_REF] Burton | The response of patients with phenylketonuria and elevated serum phenylalanine to treatment with oral sapropterin dihydrochloride (6R-tetrahydrobiopterin): a phase II, multicentre, open-label, screening study[END_REF][START_REF] Levy | Efficacy of sapropterin dihydrochloride (tetrahydrobiopterin, 6R-BH4) for reduction of phenylalanine concentration in patients with phenylketonuria: a phase III randomised placebo-controlled study[END_REF][START_REF] Lee | Safety and efficacy of 22 weeks of treatment with sapropterin dihydrochloride in patients with phenylketonuria[END_REF] and the Food and Drug Administration (FDA) and the European Medicines Agency (EMEA) approved Kuvan ® (Merck Serono) as an orphan drug to treat BH 4 -responsive PAH deficiency. However, at the time of market approval the molecular mode of action of the new drug was not well understood. By now, analyses of the pharmacological BH 4 effect in vitro performed In addition, an animal model with the specific clinical and biochemical phenotype of BH 4 -responsive PAH deficiency was not available for drug approval. PAH deficiency in mice has previously been generated by germline mutagenesis [START_REF] Mcdonald | The use of N-ethyl-N-nitrosourea to produce mouse models for human phenylketonuria and hyperphenylalaninemia[END_REF][START_REF] Shedlovsky | Mouse models of human phenylketonuria[END_REF]. The V106A mutation in Pah enu1/1 leads to a MHP phenotype [START_REF] Mcdonald | The use of N-ethyl-N-nitrosourea to produce mouse models for human phenylketonuria and hyperphenylalaninemia[END_REF], whereas Pah enu2/2 , harboring the null mutation F263S, shows classical PKU (www.pahdb.mcgill.ca) [START_REF] Shedlovsky | Mouse models of human phenylketonuria[END_REF][START_REF] Mcdonald | Characterization of mutations at the mouse phenylalanine hydroxylase locus[END_REF][START_REF] Sarkissian | A heteroallelic mutant mouse model: A new orthologue for human hyperphenylalaninemia[END_REF]. We recently showed that mice homozygous for the V106A mutation display a molecular phenotype of protein misfolding with loss-of-function and that Pah enu1/1 is an animal model for the clinical phenotype of BH 4 -responsive MHP [START_REF] Gersting | Pahenu1 is a mouse model for tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency and promotes analysis of the pharmacological chaperone mechanism in vivo[END_REF]. However, patients that most benefit from treatment with BH 4 are those with mild PKU [START_REF] Muntau | Tetrahydrobiopterin as an alternative treatment for mild phenylketonuria[END_REF][START_REF] Lambruschini | Clinical and nutritional evaluation of phenylketonuric patients on tetrahydrobiopterin monotherapy[END_REF][START_REF] Bélanger-Quintana | Spanish BH4-responsive phenylalanine hydroxylase-deficient patients: evolution of seven patients on long-term treatment with tetrahydrobiopterin[END_REF]. Moreover, the majority of patients (87 %) with BH 4 -responsive PAH deficiency are compound heterozygous carrying two different mutations on the maternal and the paternal allele [START_REF] Zurflüh | Molecular genetics of tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency[END_REF] with about 26 % of the alleles being putative null mutations. Recent studies showed a marked influence of the patient's genotype on the intensity and time frame of response to BH 4 [START_REF] Nielsen | Tetrahydrobiopterin responsiveness after extended loading test of 12 Danish PKU patients with the Y414C mutation[END_REF][START_REF] Fiege | Extended tetrahydrobiopterin loading test in the diagnosis of cofactor-responsive phenylketonuria: a pilot study[END_REF][START_REF] Karacić | Genotypepredicted tetrahydrobiopterin (BH4)-responsiveness and molecular genetics in Croatian patients with phenylalanine hydroxylase (PAH) deficiency[END_REF].

Previous clinical studies mostly analyzed the effect of BH 4 on the patients' blood phenylalanine level, an endpoint with limited functional value. We showed that the effect of BH 4 on blood phenylalanine concentrations in humans is accompanied by an increase in PAH function in vivo [START_REF] Muntau | Tetrahydrobiopterin as an alternative treatment for mild phenylketonuria[END_REF] and recently replicated this in the mouse model Pah enu1/1 [START_REF] Gersting | Pahenu1 is a mouse model for tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency and promotes analysis of the pharmacological chaperone mechanism in vivo[END_REF]. However, comprehensive pharmacodynamic and pharmacokinetic studies in a specific mouse model displaying the clinical phenotype of BH 4responsive PAH deficiency are still not available.

Thus, the aims of this study were (i) to investigate whether the compound heterozygous strain Pah enu1/2 is a model for BH 4 -responsiveness in mild PKU and (ii) to characterize pharmacodynamics and pharmacokinetics of BH 4 treatment in the A c c e p t e d M a n u s c r i p t 6 mouse models Pah enu1/1 and Pah enu1/2 . Our results show that Pah enu1/2 is a model for compound heterozygous mild PKU with BH 4 -responsiveness. Pharmacodynamics of BH 4 , in particular with respect to effect size and effect kinetics differed significantly in both strains, while pharmacokinetics was congruent.

Materials and Methods

Animals

BTBR, Pah

Combined phenylalanine-BH 4 loading tests and 13 C-phenylalanine breath tests

In order to minimize the influence of naturally occurring 13 C from food and to standardize phenylalanine supply, the experiments were performed after an overnight deprivation of food, whereas free access to water was allowed.

Combined phenylalanine-BH

 Y  Y0  Plateau    e KX    Plateau 2.2.2 13 C-phenylalanine breath tests L-[1-13]C-phenylalanine ( 13 C-phenylalanine, Eurisotop, Saint-Aubin Cedex, France)
was injected in a dose of 15 µg/g bw i.p. simultaneously with BH 4 or placebo.

Preparation and application of the compounds were performed as described above.

Mice were placed in individual 100 ml breath chambers with a continuous flow of CO 2 -free air. Breath samples were collected in 6.5 minute intervals over a 104 minute period. For each sample the airflow was discontinued for 90 seconds to allow CO 2 levels to accumulate above 0.8 %. At the end of accumulation the sample was insufflated into an infrared spectrometer (IRIS Wagner Analysen Technik, Bremen, Germany) for online isotope analysis. The cumulative recovery of 13 C was calculated based on the ratio of 13 CO 2 to 12 CO 2 as previously described [START_REF] Treacy | Analysis of phenylalanine hydroxylase genotypes and hyperphenylalaninemia phenotypes using L-[1-13C]phenylalanine oxidation rates in vivo: a pilot study[END_REF], assuming a total CO 2 production rate of 94 ml per min per g bw x m 2 body surface area [START_REF] Leijssen | Recovery of 13CO2 and 14CO2 in human bicarbonate studies: a critical review with original data[END_REF]. For dose response studies, BH 4 was given in doses of 5, 10, 20, 30, and 40 µg/g bw, respectively. To analyze the effect duration, 40 µg/g bw of BH 4 were given 90, 24, 18,

A c c e p t e d M a n u s c r i p t 8 9, 6, 4.5 or 3 hours before or simultaneously to 13 C-phenylalanine. Subsequently, the cumulative recovery of 13 CO 2 was determined at 104 minutes.

BH 4 pharmacokinetics

In order to determine pharmacokinetics, BH 4 at a dose of 20 µg/g bw was prepared and injected as described above. Venous blood samples (approximately 40 µl) were collected on filter cards before and 20, 40, 60, 120, and 180 minutes after injection.

Total biopterin (BH 4 , dihydrobiopterin, and biopterin) was quantified by High Pressure Liquid Chromatography (HPLC) as previously described [START_REF] Zurflüh | Screening for tetrahydrobiopterin deficiencies using dried blood spots on filter paper[END_REF][START_REF] Curtius | Pterins[END_REF]. Biopterin clearance was determined by non-linear regression of a double exponential Bateman function as described by Koch et al. [START_REF] Koch | The bi-exponential pharmacokinetic equation is suited to characterize lactate and ammonia concentration versus time data of the ischemic forearm exercise test[END_REF].

Transient expression of PAH in COS-7

COS-7 cells were maintained in basic RPMI 1640 medium (PAA Laboratories, Pasching, Austria) with stable glutamine supplemented with 10 % fetal bovine serum (PAA) and 1 % antibiotics (Antibiotic-Antimycotic; PAA). For transient expression of the murine wild-type and variant PAH pEF-DEST51 (Invitrogen, San Diego, USA) cDNA constructs coding for wild-type, V106A, and F263S PAH were used in single transfection (wild-type, Pah enu1/1 , Pah enu2/2 ) or co-transfection (Pah enu1/2 ). A total amount of 3 µg DNA per 1 million cells was applied using the Amaxa electroporation system (Lonza, Basel, Switzerland). Cells were cultured for 24 and 72 hours under two different conditions: i) basic medium (as described above), ii) basic medium with 43 µM BH 4 , 5 µg/ml ascorbic acid, and 1 mM L-phenylalanine. Culture medium was changed every 24 hours. The cells were harvested and lysed by three freeze-thaw cycles in a lysis buffer containing 1 % Triton X-100 and proteinase inhibitors, followed

PAH activity assay

PAH enzyme activity was determined as previously described [START_REF] Gersting | Loss of function in phenylketonuria is caused by impaired molecular motions and conformational instability[END_REF][START_REF] Martínez | Expression of recombinant human phenylalanine hydroxylase as fusion protein in Escherichia coli circumvents proteolytic degradation by host cell proteases. Isolation and characterization of the wild-type enzyme[END_REF][START_REF] Miranda | The active site residue tyrosine 325 influences iron binding and coupling efficiency in human phenylalanine hydroxylase[END_REF] with modifications. 20 µl of total lysates obtained from cell culture were preincubated with

Statistics

Group mean values were compared by Student's unpaired two-tailed t-test. Statistical analyses were performed using GraphPad Prism 4.0c (GraphPad Software, San Diego, USA).

Results

Pah enu1/2 is a model for compound heterozygous BH 4 -responsive PAH deficiency

The heteroallelic Pah enu1/2 was previously reported as an orthologue for human hyperphenylalaninemia [START_REF] Sarkissian | A heteroallelic mutant mouse model: A new orthologue for human hyperphenylalaninemia[END_REF] with plasma phenylalanine levels of 147 to 200 µM and a +/-54.2 µM for Pah enu2/2 , in ascending order (Fig. 1A). Phenylalanine oxidation, which is a measure of in vivo PAH enzyme activity, was assessed by a 13 C-phenylalanine oxidation test [START_REF] Kure | Tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency[END_REF][START_REF] Muntau | Tetrahydrobiopterin as an alternative treatment for mild phenylketonuria[END_REF][START_REF] Gersting | Pahenu1 is a mouse model for tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency and promotes analysis of the pharmacological chaperone mechanism in vivo[END_REF]. The cumulative recovery of 13 CO 2 (the product of 13 Cphenylalanine oxidation) after 104 minutes reached 38 +/-2.8 % for the wild-type, whereas Pah enu1/1 (12 +/-1.0 %), Pah enu1/2 (9 +/-0.8 %), and Pah enu2/2 (5 +/-0.6 %)

showed lower values in descending order (Fig. 1B).

Results obtained from 13 (Fig. 1E). In wild-type mice, however, BH 4 led to a reduction in the cumulative 13 CO 2 recovery, which is in line with the known inhibitory effect of the cofactor on enzyme activity [START_REF] Phillips | Ligand effects on the phosphorylation state of hepatic phenylalanine hydroxylase[END_REF].

In COS-7 cells, treatment with BH 4 over 24 hours led to an increase in enzyme activity in cells overexpressing wild-type PAH as well as V106A and V106A/F263S (Fig. 1F) with the effect being most pronounced in cells expressing V106A-PAH.

Taken together, determination of phenylalanine oxidation mirrored the biochemical phenotype and allowed for sensitive discrimination between all genotypes tested.

Moreover, combined analysis of the biochemical and molecular phenotype showed that Pah enu1/2 displays more severe PAH deficiency than Pah enu1/1 and identified Pah enu1/2 as a compound heterozygous model for human BH 4 -responsive PAH deficiency.

Pharmacodynamic characterization of BH 4 treatment in wild-type and PAH deficient mice

To characterize the pharmacodynamic effects of BH 4 on wild-type and variant PAH in vivo we selected a combined set of three different endpoints: (i) blood phenylalanine elimination, (ii) blood phenylalanine/tyrosine ratios, and (iii) kinetics of in vivo phenylalanine oxidation.

In wild-type mice a phenylalanine challenge (15 µg/g bw i.p.) led to no significant changes in blood phenylalanine concentrations (Fig. 2A) or the A c c e p t e d M a n u s c r i p t 12 phenylalanine/tyrosine ratio (Fig. 2B). Accordingly, the 13 C-phenylalanine applied was almost completely oxidized to 13 CO 2 within the first hour (Fig. 2C).

In Pah enu1/1 and Pah enu1/2 the phenylalanine challenge induced a marked increase in both blood phenylalanine concentrations (Fig. 2A) and blood phenylalanine/tyrosine ratios (Fig. 2B) that did not recover to initial values within 3 hours (Table 1). In agreement with the respective biochemical (blood phenylalanine concentrations) and functional (phenylalanine oxidation) phenotypes, Pah enu1/2 showed more severe alterations of blood phenylalanine elimination and phenylalanine/tyrosine ratio elevations than Pah enu1/1 .

The effect of BH 4 on phenylalanine elimination and on the phenylalanine/tyrosine ratio was different from that on phenylalanine oxidation, where both Pah enu1/1 and Pah enu1/2 reached wild-type levels after treatment (Fig. 1E). Only Pah enu1/1 showed normalization of phenylalanine elimination and the phenylalanine/tyrosine ratio within 120 and 60 minutes, respectively. Both parameters did not significantly decrease in Pah enu1/2 and the blood phenylalanine concentration and the phenylalanine/tyrosine ratio remained elevated 3 hours after BH 4 administration (Table 1).

In both animal models the rates of phenylalanine oxidation, assessed by determination of delta over baseline, exceeded half maximum values as early as 6.5 minutes post BH 4 injection. However, Pah enu1/1 reached maximum oxidation rates at 13 minutes, whereas Pah enu1/2 displayed a delayed maximum at 26 minutes (Fig. 2C).

The resulting elimination constants (K e ) for blood phenylalanine again showed lower response to the drug for Pah enu1/2 (0.014) than for Pah enu1/1 (0.023) (Table 1).

In summary, the application of the three endpoints presented here allowed for a clear discrimination in pharmacodynamics between the two mouse strains. Differences in the response to the drug were not only confined to the effect size but also observed in terms of effect kinetics.

A c c e p t e d M a n u s c r i p t 13

Pharmacokinetics of BH 4

In order to determine, whether differences in pharmacodynamics were induced by discrepant pharmacokinetic behavior, we quantified total biopterin blood concentrations over time after intraperitoneal injection of 20 µg/g bw of BH 55.6 +/-6.7) at 180 minutes. None of the pharmacokinetic parameters (c 0 , c max , t max , AUC, t 1/2 ) significantly differed between Pah enu1/1 and Pah enu1/2 (Table 2).

Effect duration of BH 4

We subsequently aimed to analyze, whether BH 4 effect duration diverges between Pah enu1/1 and Pah enu1/2 . For this purpose we monitored the time course of 13 Cphenylalanine oxidation after a single dose of BH 4 and analyzed the effect of BH 4 on PAH activity in cultured cells over 72 hours.

In both PAH deficient strains phenylalanine oxidation reached its maximum immediately after BH 4 application (Fig. 2C). In order to compare effect durations we normalized the data defining the highest values as 100 % and the placebo level as 0 %. A single dose of 40 µg/g bw BH 4 induced enhanced phenylalanine oxidation for more than 48 hours in Pah enu1/1 , while in Pah enu1/2 phenylalanine oxidation decayed to placebo levels within 18 hours (Fig. 3A).

A c c e p t e d

M a n u s c r i p t 14 These results were confirmed by assessing the long-term effect of BH 4 on PAH enzyme activity in COS-7 cells (Fig. 3B). In cells expressing V106A, BH 4 treatment normalized enzyme activity, while cells expressing V106A/F263S reached 75 % of wild-type activity. BH 4 did not affect enzyme activity in cells transfected with F263S.

In addition, we compared the fold increase of enzyme activity over the control (no BH 4 treatment) after 24 hours and 72 hours of cell incubation with BH 4 . At overexpression of V106A the major part of the response occurred within 24 hours

(1.54-fold increase) and cultivation for additional 48 hours did not substantially further increase enzyme activity (1.75-fold). By contrast, cells expressing V106A/F263S

showed their main increase in enzyme activity (1.9-fold) after prolonged cultivation of 72 hours and not after 24 hours (1.15-fold) (Fig. 3C).

In conclusion, Pah enu1/1 and Pah enu1/2 showed pronounced differences in effect duration of BH 4 treatment. The in vivo effect of BH 4 was shorter in Pah enu1/2 than in Pah enu1/1 . This was demonstrated by showing that the functional effect of the drug on phenylalanine oxidation leveled off more rapidly. In vitro, a prolonged treatment was needed to reach the maximum treatment effect in Pah enu1/2 .

Dose effects of BH 4

To further characterize genotype-specific pharmacodynamics we studied the dose response to BH 4 . Analysis of the cumulative recovery of 13 C-phenylalanine oxidation at 104 minutes upon single dose BH 4 treatment revealed a non-dose-dependent inhibitory effect in the range of 5 to 40 µg/g bw i.p in wild-type mice (Fig. 4A).

Pah enu1/1 showed a positive dose response starting at 5 µg/g and reached a plateau at 20 µg/g with no further increase in phenylalanine oxidation at higher doses (Fig. 4B). In the compound heterozygous mouse Pah enu1/2 we also identified a positive

A c c e p t e d M a n u s c r i p t dose effect. However, BH 4 doses of 30 and 40 µg/g resulted in a lower activity as compared to the peak effect at 10 to 20 µg/g (Fig. 4C). With respect to the effect size Pah enu1/2 revealed a stronger response as compared to Pah enu1/1 at 5, 10, and 20 µg/g, respectively (Fig. 4B,C).

In conclusion, Pah enu1/1 and Pah enu1/2 showed a dose dependent and increasing response to BH 4 at low doses up to 10 µg/g with no further benefit at higher dosages.

Moreover, the results observed for Pah enu1/2 are in line with a negative dose effect for concentrations above 20 µg/g BH 4 .

Discussion

The approval of BH 4 marked a paradigm change in the management of phenylketonuria treatment. Clinical studies performed to analyze BH 4 -responsiveness in patients often focused on the surrogate marker blood phenylalanine concentration as an endpoint. However, this method produced high intraindividual variability and discrepant findings among similar genotypes [START_REF] Zurflüh | Molecular genetics of tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency[END_REF] and therefore proved to be of limited value. Moreover, an animal model with the specific clinical and biochemical phenotype of BH 4 -responsive PAH deficiency was not available during the development of the drug. Thus, important insights into pharmacodynamics of BH 4

were still lacking, in particular with regard to pharmacogenetics as well as effect size and effect kinetics.

We recently characterized Pah enu1/1 (V106A/V106A) as the first animal model for BH 4 -responsive MHP [START_REF] Gersting | Pahenu1 is a mouse model for tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency and promotes analysis of the pharmacological chaperone mechanism in vivo[END_REF]. This opened up the opportunity for first in vivo investigations in order to elucidate the BH 4 mode of action and to substantiate the view of the cofactor being a pharmacological chaperone. The genetic alteration in this mouse does not lead to changes in PAH affinity to the cofactor [START_REF] Gersting | Pahenu1 is a mouse model for tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency and promotes analysis of the pharmacological chaperone mechanism in vivo[END_REF] reflecting the human situation where only few K m variants were found [START_REF] Gersting | Loss of function in phenylketonuria is caused by impaired molecular motions and conformational instability[END_REF][START_REF] Erlandsen | Correction of kinetic and stability defects by tetrahydrobiopterin in phenylketonuria patients with certain phenylalanine hydroxylase mutations[END_REF][START_REF] Pey | Mechanisms underlying responsiveness to tetrahydrobiopterin in mild phenylketonuria mutations[END_REF][START_REF] Knappskog | Structure/function relationships in human phenylalanine hydroxylase. Effect of terminal deletions on the oligomerization, activation and cooperativity of substrate binding to the enzyme[END_REF][START_REF] Leandro | The V388M mutation results in a kinetic variant form of phenylalanine hydroxylase[END_REF][START_REF] Sanford | Sapropterin: a review of its use in the treatment of primary hyperphenylalaninaemia[END_REF]. Thus, the Here we present Pah enu1/2 (V106A/F263S) as a second mouse model for BH 4responsive PKU. This strain displays compound heterozygosity representing the genotype of the large majority of PKU patients (87 %) [START_REF] Zurflüh | Molecular genetics of tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency[END_REF] that are dealt with in daily clinical routine. Amino acid residues V106 in the regulatory domain and F263 in the catalytic core are conserved between mouse and human but neither V106A nor F263S have so far been identified in human PKU patients. However, the F263L amino acid substitution was described to be also associated with a severe phenotype. A comparison of specific activities of murine and human PAH variants V106A, F263S, and F263L confirmed that amino acid substitutions at the respective loci result in similar effects in both species (Supplementary Table 1). Thus, murine V106A and F263S variants are good models for human BH 4 -responsive PAH deficiency.

As previously shown [START_REF] Sarkissian | A heteroallelic mutant mouse model: A new orthologue for human hyperphenylalaninemia[END_REF] and confirmed in our current study, Pah enu1/2 exhibits an intermediate phenotype between Pah enu1/1 and Pah enu2/2 . Hyperphenylalaninemia in vivo is more severe than in Pah enu1/1 and less severe than in Pah enu2/2 , whereas PAH activity in vivo and in vitro is lower than in Pah enu1/1 but higher than in Pah enu2/2 .

Analyses of phenylalanine oxidation by 13 C-phenylalanine breath tests mirrored these differences in severity and were in good agreement with biochemical (blood are often referred to as slow responders [START_REF] Muntau | Tetrahydrobiopterin as an alternative treatment for mild phenylketonuria[END_REF][START_REF] Bélanger-Quintana | Spanish BH4-responsive phenylalanine hydroxylase-deficient patients: evolution of seven patients on long-term treatment with tetrahydrobiopterin[END_REF][START_REF] Fiege | Extended tetrahydrobiopterin loading test in the diagnosis of cofactor-responsive phenylketonuria: a pilot study[END_REF][START_REF] Zurflüh | Pharmacokinetics of orally administered tetrahydrobiopterin in patients with phenylalanine hydroxylase deficiency[END_REF]. Our results indicate that this may be rather due to a limited pharmacodynamic effect than to a delayed onset of drug action. Cell culture experiments pointed to a beneficial effect of prolonged treatment for slow responder genotypes and hence suggest appreciation of this fact when testing for and treating BH 4 -responsive PAH deficiency. Third, both animal models bear the same mutation responsible for BH 4 -responsiveness (V106A), however, the null-mutation on the second allele in the compound heterozygous genotype Pah enu1/2 had substantial impact on pharmacodynamics. Thus, response to treatment is not exclusively related to the putative milder mutation [START_REF] Zurflüh | Molecular genetics of tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency[END_REF][START_REF] Nielsen | Tetrahydrobiopterin responsiveness after extended loading test of 12 Danish PKU patients with the Y414C mutation[END_REF][START_REF] Zurflüh | Pharmacokinetics of orally administered tetrahydrobiopterin in patients with phenylalanine hydroxylase deficiency[END_REF] and effects induced by interallelic complementation may necessitate careful dose finding procedures in compound heterozygous patients. Fourth, the individual behaviour with regard to effect size, onset of maximum drug action, and effect duration underscores the demand of individual therapeutic regimes for different genotypes. Some patients may for instance benefit from a treatment scheme with several BH 4 administrations to translate the effect of the drug on enzyme activity into a sustained effect on biochemical markers. Moreover, in certain patients, higher dosages may diminish the positive treatment effect at a higher risk of possible adverse effects. The elimination constant (K e ) was determined using a one-phase exponential function. Unpaired two-tailed students t-tests were applied to test the difference between untreated and treated animals (n.s., not significant). The following pharmacokinetic parameters were determined in Pah enu1/1 and Pah enu1/2 : c 0 initial concentration, c max peak concentration, t max time to peak concentration, AUC area under the concentration vs. time curve at 0 to 180 minutes, t 1/2 elimination half-life.

  Abbreviations:

  have provided evidence for structural stabilization of misfolded PAH indicating a pharmacological chaperone mode of action of BH 4[START_REF] Erlandsen | Correction of kinetic and stability defects by tetrahydrobiopterin in phenylketonuria patients with certain phenylalanine hydroxylase mutations[END_REF][START_REF] Pey | Mechanisms underlying responsiveness to tetrahydrobiopterin in mild phenylketonuria mutations[END_REF][START_REF] Gersting | Pahenu1 is a mouse model for tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency and promotes analysis of the pharmacological chaperone mechanism in vivo[END_REF].

A c c e p t e d M a n u s c r i p t 9 by 20

 920 minutes centrifugation at 14,000 rpm, 4 °C. Recovered supernatants were subsequently used for activity assays[START_REF] Gersting | Loss of function in phenylketonuria is caused by impaired molecular motions and conformational instability[END_REF].

  A c c e p t e d M a n u s c r i p t 10 residual enzyme activity of about 5 % as compared to wild-type mice. We revisited this mouse model in order to reevaluate the biochemical phenotype and PAH enzyme function in vivo and in vitro and to test for responsiveness to BH 4 . In our experimental setup mean blood phenylalanine concentrations were 54 +/-2.1 µM for the wild-type, 177 +/-25.7 µM for Pah enu1/1 , 284 +/-24.9 µM for Pah enu1/2 , and 1115

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

 411 C-phenylalanine breath tests were substantiated in vitro by overexpression of wild-type, V106A, F263S, and combined overexpression of V106A and F263S PAH, respectively. COS-7 cells transiently transfected with V106A, the in vitro model for Pah enu1/1 , or V106A/F263S, the in vitro model for Pah enu1/2 , showed decreased PAH activity in comparison to the wild-type (Fig.1C). In line with the in vivo experiments, co-transfection of V106A/F263S resulted in a significantly lower enzyme activity than transfection of V106A only. As expected, transfection of F263S resulted in almost no residual enzyme activity.As a next step, we aimed to determine the response of Pah enu1/2 to pharmacological doses of BH 4 . A single dose of BH 4 (20 µg/g bw i.p.) significantly reduced blood phenylalanine concentrations of Pah enu1/2 from 284 +/-24.9 µM to 135 +/-17.2 µM and of Pah enu1/1 from 177 +/-25.7 µM to 70 +/-17.2 µM, whereas phenylalanine values remained unchanged in wild-type mice and in Pah enu2/2 . Pah enu1/1 reached blood phenylalanine concentrations close to that of wild-type mice, while Pah enu1/2 still displayed mild hyperphenylalaninemia after treatment (Fig.1D). In addition, BH led to an increase in phenylalanine oxidation in Pah enu1/1 and Pah enu1/2 . The cumulative recovery of 13 CO 2 after 104 minutes attained wild-type level in both Pah enu1/2 and Pah enu1/1 . In agreement with the missing effect on blood phenylalanine concentrations, BH 4 treatment did not affect phenylalanine oxidation rates in Pah enu2/2

A c c e p t e d M a n u s c r i p t 16 mode

 16 of action of BH 4 in PAH deficiency was not limited to its cofactor action. We showed that pharmacological doses of BH 4 attenuate the pathophysiological triad of misfolding, aggregation, and accelerated degradation of the PAH enzyme by conformational stabilization augmenting the effective PAH concentration. This led to the rescue of the biochemical phenotype and enzyme function in vivo. Notably, the pharmaceutical action of the cofactor was confined to the pathological metabolic state of hyperphenylalaninemia[START_REF] Gersting | Pahenu1 is a mouse model for tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency and promotes analysis of the pharmacological chaperone mechanism in vivo[END_REF].

A c c e p t e d M a n u

  s c r i p t phenylalanine) and molecular (PAH activity) data allowing for high-sensitivity discrimination of genotypes.We compared pharmacodynamics of BH 4 treatment in the two mouse models and observed crucial differences. Heterogeneous results were obtained with respect to the effect size. In the combined phenylalanine BH 4 -loading test a single load of 20 µg/g bw BH 4 induced a decrease of blood phenylalanine of 67.5 % in Pah enu1/1 and of only 35.5 % in Pah enu1/2 . This value is in close proximity of the threshold of 30 %, an arbitrary but accepted measure for BH 4 -responsiveness in humans considered to be associated with clinical benefit. By contrast, the effect on in vivo phenylalanine oxidation, the direct measure of PAH activity, was striking in this strain. Treatment with BH 4 completely normalized in vivo enzyme activity with the effect being even stronger than in the milder phenotype Pah enu1/1 . Analysis of the effect kinetics further added to dissect the distinct patterns of BH 4response in the two strains. At the biochemical level, Pah enu1/2 showed slower elimination of pathologically elevated phenylalanine concentrations than Pah enu1/1 . This was mirrored by a less pronounced and delayed reduction of the phenylalanine/tyrosine ratio, an important parameter to judge the extent of metabolic derangement. At the functional level, both strains showed an immediate response to BH 4 -loading in the breath test, but the peak phenylalanine oxidation upon treatment again was delayed in Pah enu1/2 in comparison to Pah enu1/1 . Moreover, the duration of the BH 4 effect after a single dose was considerably shorter in Pah enu1/2 than in Pah enu1/1 .In addition, the two animal models displayed important divergences concerning dose response. Both strains attained maximum in vivo phenylalanine oxidation at a dose of 20 µg/g bw BH 4 . Yet, Pah enu1/2 exhibited a response inhibition in the presence of higher dosages, while Pah enu1/1 reached a plateau. This data is in line with well A c c e p t e d M a n u s c r i p t 18 known features of pharmacological chaperones which show inhibitory effects at higher concentrations[START_REF] Fan | A counterintuitive approach to treat enzyme deficiencies: use of enzyme inhibitors for restoring mutant enzyme activity[END_REF].Our findings may lead to important conclusions concerning the diagnostic and therapeutic management of patients with PAH deficiency. First, extending the test procedures to assess BH 4 -responsiveness may allow to draw a more complete picture of the drug response in the single individual. The methods presented in this work can easily and safely be transferred into clinical routine and provide useful endpoints beyond determination of blood phenylalanine concentrations. Second, some patients show a delayed reduction in blood phenylalanine after BH 4 loading, these

Figure captions Fig. 1 .Fig. 2 .

 captions12 Figure captions

  Figure Captions

Fig. 3 .

 3 Fig. 3. In vivo and in vitro characterization of BH 4 effect duration.(A) Effect duration of BH 4 in Pah enu1/1 and Pah enu1/2 mice expressed as relative recovery of 13 CO 2 at 104 minutes. BH 4 was administered 90, 24, 18, 9, 6, 4.5 or 3 hours before or simultaneously to13 C-phenylalanine and recovery of13 CO 2 was

  Figure 1

Table 1 .

 1 Phenylalanine kinetics and phenylalanine/tyrosine ratios Pah enu1/1 and Pah enu1/2 . Phenylalanine concentrations and phenylalanine/tyrosine ratios are given as means ± s.e.m.

			BH4 Phe0 [µmol/l] Phe180 [µmol/l] P value	Ke	Phe/Tyr0	Phe/Tyr180 P value
	Pah	enu1/1	-	109.10 ± 5.4	153.33 ± 19.3	0.0097	-	2.62 ± 0.1 4.79 ± 0.4	0.0035
			+	120.74 ± 16.5	61.64 ± 3.9		0.023 3.65 ± 0.3 1.73 ± 0.1
	Pah	enu1/2	-	187.29 ±19.8	209.50 ± 35.1	n.s.	-	4.71 ± 0.6 5.65 ± 1.0	n.s.
			+	174.94 ± 20.6	136.9 ± 17.3		0.014 5.38 ± 0.8 3.65 ± 0.9
	BTBR	-	53.70 ± 1.8	56.61 ± 5.0	-	0.015 2.15 ± 0.2 2.49 ± 0.2	-
	Phenylalanine kinetics and phenylalanine/tyrosine ratios were determined after
	a phenylalanine load of 15 µg/g bw i.p. with and without simultaneous BH 4 load
	(20 µg/g bw i.p.) to wild-type (BTBR),

Table 1 A

 1

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

Table 2 .

 2 Determination of pharmacokinetic parameters.

		Pah	enu1/1	Pah	enu1/2	P value
	c0 (nmol/g Hb)	19.4 ± 3.1	17.3 ± 2.5	0.5
	cmax (nmol/g Hb)	467.6 ± 47.5	558.8 ± 68.3	0.3
	tmax (min)	17.5 ± 2.5	17.5 ± 2.5	1
	AUC180min	23769 ± 2341	24522 ± 2638	0.8
	t1/2 (min)	26.3 ± 2.8	26.3 ± 2.7	0.98

Table 2 A

 2

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

Acknowledgements

The authors wish to thank Anja Schultze, Heike Preisler, Maria Trieb, and Bernadette Schmid for excellent technical assistance and Georg Wietzorrek for his support. This work was supported by the Bavarian Genome Research Network (BayGene) and the Dr. Legerlotz-Stiftung Liechtenstein. This article is part of an M.D. thesis to be submitted by A.E. at Ludwig-Maximilians-University, Munich, Germany.


V C G Tjan-Heijnen

R Otter

P H B Willemse

L De Munck
email: l.demunck@ikno.nl

M Schaapveld

S Siesling

J Wesseling

A C Voogd

Implementation of trastuzumab in conjunction with adjuvant chemotherapy in the treatment of non-metastatic breast cancer in The Netherlands

Keywords: adjuvant, breast cancer, guideline, HER2, trastuzumab

Implementation of trastuzumab in conjunction with adjuvant chemotherapy in the treatment of non-metastatic breast cancer in the Netherlands

Introduction

Breast cancer is the most common cancer in women in the Netherlands, accounting for about one third of all female cancers [1]. Approximately 10-25% of breast cancers have overexpression of the human epidermal growth factor receptor 2 (HER2) protein and/or amplification of the HER2 gene [START_REF] Slamon | Human Breast Cancer: Correlation of Relapse and Survival with Amplification of the HER-2/neu[END_REF][START_REF] Slamon | Studies of the HER-2/neu Proto-Oncogene in Human Breast and Ovarian Cancer[END_REF][START_REF] Dowsett | Correlation between immunohistochemistry (HercepTest) and fluorescence in situ hybridization (FISH) for HER-2 in 426 breast carcinomas from 37 centres[END_REF][START_REF] Dendukuri | Testing for Her2-positive breast cancer: a systematic review and cost-effectiveness analysis[END_REF]. In May 2005 the results of three large randomised studies were presented, revealing that in women with HER2-positive breast cancer, the risk of developing recurrences or distant metastases and the risk of death decreased by more than 30% by adding one year adjuvant treatment with trastuzumab [START_REF] Romond | Trastuzumab plus Adjuvant Chemotherapy for Operable HER2-Positive Breast Cancer[END_REF][START_REF] Piccart-Gebhart | Trastuzumab after Adjuvant Chemotherapy in HER2-Positive Breast Cancer[END_REF]. Based on these results, recommendations for HER2-testing and the use of adjuvant trastuzumab were rapidly included in national and international guidelines [START_REF]National Breast Cancer Organization of The Netherlands[END_REF].

The aim of this study was to evaluate the level of implementation of the Dutch guidelines for HER2-testing and the use of trastuzumab in clinical practice, analysing regional differences and reasons for withholding trastuzumab.

Patients and Methods

Patients

Patients were selected from the population-based Netherlands Cancer Registry (NCR) of the Association of Comprehensive Cancer Centres (CCCs) in the Netherlands, covering all 16 million inhabitants. The Association of CCCs consisted of 9 regional CCCs, which support the development and implementation of national guidelines by means of multidisciplinary working groups and consultancy services (on local, regional and national level), and monitor guideline compliance after their implementation using Cancer Registry data.

Main source of notification for the NCR is PALGA, the nationwide Dutch network and registry of histo-and cytological pathology, through which all newly diagnosed malignancies are reported to the cancer registry. Case ascertainment is completed by using the national hospital discharge database, which receives the diagnosis of admitted patients from all hospitals. After notification to the NCR, specially trained registrars of the CCCs visit the hospitals to collect information on patient and tumour characteristics, stage and treatment from patient files. Coding of the items is based on international coding rules (IACR). TNM classification was used for the staging of tumours [START_REF] Wittekind | TNM Atlas[END_REF]. Since 2005 HER2 status was recorded in the NCR on a routine basis. HER2-positivity was defined using a widely recommended testing algorithm [START_REF] Bilous | Current Perspectives on HER2 Testing: A Review of National Testing Guidelines[END_REF][START_REF] Yaziji | Her-2 Testing in Breast Cancer Using Parallel Tissue-Based Methods[END_REF]. An immunohistochemistry (IHC) score 3+ was considered positive.

When IHC was inconclusive (2+), an additional fluorescent in situ hybridization (FISH) test was indicated. IHC score 3+ or a positive FISH test was considered indicative for HER2-positivity.

All women diagnosed with invasive breast cancer between September 2005 and January 2007 were selected (n=17,781). Women without surgery or with metastases at diagnosis, and women who received neo-adjuvant chemotherapy were excluded from analysis, leaving 14,934 women for analysis in this study.

Treatment guidelines

In the Netherlands, guidelines are developed by the National Breast Cancer Organization of The Netherlands (NABON). They released the adapted national guideline in September 2005 indicating that for women with HER2positive breast cancer trastuzumab should be given in conjunction with adjuvant chemotherapy. If women with HER2-positive tumours and chemotherapy did not receive adjuvant trastuzumab, reasons to withhold trastuzumab were documented by the registrars from the CCCs.

Statistical analysis

Time of diagnosis was divided into 4 periods of 4 months. Regions in the Netherlands were defined according to the areas covered by the 9 CCCs [1]. Logistic regression was performed to study differences in the use of trastuzumab between regions. The region with the highest use of trastuzumab was coded as reference. Age (10-year categories) and tumour stage (stage I, II and III) were included as covariates. The statistical significance level was set at a pvalue <0.05. Analyses were performed using the STATA software package, version 10.1 for Windows (Stata Corporation LP, College Station, TX, USA).

Results

HER2 status

Between September 2005 and January 2007 14,934 women with invasive breast cancer were included in the study.

Median age at diagnosis was 60 years (range 21-101, Table 1). Of these, 1,928 (13%) women were diagnosed with HER2-positive breast cancer. The percentage of HER2-positive tumours decreased with increasing age from 22% in women younger than 40 years to 10% in women 70 years or older (Figure 1, p<0.001). In 4% of women under 40 years of age HER2-status was unknown or not tested. This percentage increased to 8% in women over 70 (Figure 2).

Both the percentage HER2-positive tumours (range 11%-15%, p=0.008) and the percentage with HER2-status unknown or not tested (range 3%-13%,p<0.001) varied between the CCC regions.

Use of trastuzumab

Of all 1,928 women with HER2-positive breast cancer, 1,114 (58%) received adjuvant chemotherapy. This percentage decreased from 93% among women <40 years to 6% among women over 70 years of age (Figure 3). Of the 1,114 women with HER2-positive breast cancer who received adjuvant chemotherapy, 1,058 (94%) patients did receive trastuzumab as well. Only 6% (66 patients) did not receive trastuzumab; this percentage increased with age from 4% in women younger than 40 years to 11% in women aged 60-69 and varied between 3% and 16% between the CCC regions. Adjusted for age and stage there was a significant difference between CCC regions (p=0.004). The Odds Ratios for the CCC regions ranged from 0.17 to 0.97 compared to the reference region.

Implementation of trastuzumab

At the time the recommendation for the use of trastuzumab was included in the national guideline (September 2005), 10% of the women with HER2 overexpression receiving adjuvant chemotherapy did not receive trastuzumab. This percentage decreased to 4% in the last study period (September-December 2006) (Figure 4, p<0.001). In the period May 2006 -December 2006 the variation between the regions in the proportion of patients receiving trastuzumab had disappeared, after adjusting for age and stage (p=0.901). The Odds Ratios for the CCC regions ranged from 0.32 to 1.56 compared to the reference region.

Reasons to withhold trastuzumab

The most frequently reported reasons for doctors not to give trastuzumab were a history of cardiovascular disease, patient refusal and older age. In 36% no reason to withhold trastuzumab was reported in the patient files (Table 2).

Discussion

Of women with HER2-positve breast cancer who received adjuvant chemotherapy, 94% actually received trastuzumab. For 64% of the women who did not receive trastuzumab, a legitimate reason was reported in the patient files. Keeping in mind that the guideline had only been made public in September 2005 and that the time period immediately after its release was also included in this study, we may conclude that the level and the speed of the implementation of trastuzumab in clinical practice was very satisfactory. The time needed to implement the new guideline in clinical practice varied between CCC regions, but within 8 months the differences between CCC regions had disappeared. These differences in time can partly be explained by different dates of meetings of the multidisciplinary working groups and consultancy services (on local, regional and national level).

In this study, population-based data from the NCR, covering the whole of the Netherlands were used. Of all included women with primary breast cancer, 13% was diagnosed with HER2-positive cancer. In a meta analysis by Dendukuri et al. [START_REF] Dendukuri | Testing for Her2-positive breast cancer: a systematic review and cost-effectiveness analysis[END_REF] about 16% of primary breast cancers was HER2-positive, which is comparable to the percentage found in this study. Other studies have shown a percentage of around 25% [START_REF] Slamon | Studies of the HER-2/neu Proto-Oncogene in Human Breast and Ovarian Cancer[END_REF][START_REF] Dowsett | Correlation between immunohistochemistry (HercepTest) and fluorescence in situ hybridization (FISH) for HER-2 in 426 breast carcinomas from 37 centres[END_REF][START_REF] Bartlett | Human Epidermal Growth Factor Receptor 2 Status Correlates With Lymph Node Involvement in Patients With Estrogen Receptor (ER)-Negative, but With Grade in Those With ER-positive Early-Stage Breast Cancer Suitable for Cytotoxic Chemotherapy[END_REF]. However, those were studies performed in the initial period of HER2 testing, or studies with selected groups of patients, e.g. only including metastatic breast cancer or hormone receptor negative patients. At present, the most widely accepted and well established algorithm to determine HER2 status is to start with IHC, followed by FISH when IHC proves to be inconclusive. HER2 testing was introduced in Dutch reference pathology laboratories before this guideline was issued, as trastuzumab was already common practice for the treatment of metastatic breast cancer. However, in the beginning the quality of HER2 testing varied between laboratories. A number of variables (e.g. tissue handling and fixation) can affect the accuracy of the results and comparative studies were published about the discordance rates between laboratories and tests [START_REF] Bilous | Current Perspectives on HER2 Testing: A Review of National Testing Guidelines[END_REF][START_REF] Yaziji | Her-2 Testing in Breast Cancer Using Parallel Tissue-Based Methods[END_REF][START_REF] Perez | HER2 Testing by Local, Central, and Reference Laboratories in Specimens From the North Central Treament Group N9831 Intergroup Adjuvant Trial[END_REF][START_REF] Paik | Real-World Performance of HER2 Testing -National Surgical Adjuvant Breast and Bowel Project Experience[END_REF]. The pathologists realised that differences in determining HER2 status existed and updated guidelines for HER2 testing were added to the national guideline [START_REF]National Breast Cancer Organization of The Netherlands[END_REF]. Viewing the results of our study in this light, part of the differences between CCC regions could be explained by the differences in pathology laboratories.

IHC and/or FISH-tests are somewhat less often performed in the older patients. The guideline is clear about the conjunction of trastuzumab with chemotherapy in women under 70 [START_REF]National Breast Cancer Organization of The Netherlands[END_REF]. For women above the age of 70 the guideline does not give standard advice for adjuvant chemotherapy due to insufficient data. For these women other factors (e.g. hormone sensitivity, co-morbidity and frailty) play an important role in supplying chemotherapy (and trastuzumab). Therefore, determining HER2 status is of limited relevance in this age group.

Because this study investigated the timelines and the rate of implementation of the new guideline for the use of trastuzumab, patients with metastases or receiving neo-adjuvant chemotherapy were excluded. The use of trastuzumab is coded when treatment with trastuzumab started, but no information was gathered about its duration or if it was temporary interrupted, e.g. for cardiotoxicity. However, the aim of the current study was to study the proportion of patients starting with trastuzumab. Any premature termination of treatment for cardiotoxicity or other reasons and its impact on morbidity, recurrence and survival should be part of another study.

This study is unique because of the setting in which it has been performed. The collaboration between the National Breast Cancer Organization of The Netherlands (NABON), the CCCs and the NCR has resulted in a quality system in which the development, implementation, monitoring and evaluation of the guideline is covered as a whole. This study proves that new tumour characteristics and treatments can be registered by the NCR within reasonable time.

The registry clerks of all CCCs use the same strict definitions and coding rules, which enhances the quality of the data and thus the reliability of the results.

In conclusion, our study shows that of all Dutch women with non-metastatic invasive breast cancer, 13% has HER2 overexpression and this percentage markedly decreases with age. Of the women with HER2-positive breast cancer who received adjuvant chemotherapy, 94% actually received trastuzumab. The implementation of trastuzumab in clinical practice was realized within 8 months after introduction of the new guideline. This study shows that a population-based cancer registry can be of great value when evaluating the implementation of new treatment policies.

Fig. 1

 1 Fig. 1 Percentage of patients with a HER2-positive tumour, by age, in the period September 2005 -December 2006. The vertical lines show the range between CCC regions.

Fig. 2

 2 Fig. 2 Percentage of patients with a HER2 status unknown or not tested, by age, in the period September 2005 -December 2006. The vertical lines show the range between CCC regions.

Fig. 3

 3 Fig. 3 Percentage of patients with HER2-positive breast cancer receiving chemotherapy.

Fig. 4

 4 Fig. 4 Percentage of patients with HER2-positive tumours and adjuvant chemotherapy, receiving trastuzumab, by period. The symbols represent the different CCC regions.

Table 1 -

 1

	Patient characteristics
	All patients (n=14,934)		N	%
	Age	median	60
		range	21-101
		<40	785	5
		40-49	2,806	19
		50-59	4,006	27
		60-69	3,599	24
		70-79	2,448	16
		80+	1,290	9
	HER2 status	negative	11,574	77
	(IHC and/or FISH)	IHC 2+	486	3
		positive	1,928	13
		unknown	946	6
	HER2 positive patients (n=1,928)
	(IHC and or FISH)
	Chemotherapy	no	814	42
		yes	1,114	58
	Trastuzumab	no	871	45
		yes	1,057	55

Table 2 -

 2 Reasons for withholding trastuzumab (n=66)

	N	%

Conflict of interest statement

None declared.


Alexandre Abadie

Benoît Combès

Claire Haegelen

Sylvain Prima

CLARCS, a C++ Library for Automated Registration and Comparison of Surfaces: Medical Applications

published or not. The documents may come   L'archive ouverte pluridisciplinaire

Introduction

In medical image processing, the most widely used methods are voxel-based, which means that their required input data must be (most often) 3D arrays of grey values. Some important issues with this kind of data include the large memory needed to store them, the large run time of standard algorithms to process them, the choice of data type to code the grey value of a voxel or the sometimes problematic orientation of the volumes. Thanks to the increase of 3D rendering and computational capacities of computers in the last few years, new surface based processing methods have emerged as an alternative to standard voxel-based techniques. In this context, the VisAGeS team at INRIA/INSERM (https://www.irisa.fr/visages) has proposed a set of computational tools that have been implemented in a software library called CLARCS (C++ Library for Automated Registration and Comparison of Surfaces).

In this paper, we outline the methods implemented in CLARCS (Section 2) and we provide some potential medical applications of these (Section 3): assessment of dysmorphology in craniosynostosis and plagiocephaly and construction of a statistical shape model of thalami for deep brain stimulation (DBS).

Overview of CLARCS

In the first subsection below, we briefly describe some of the best known libraries or software working on point sets, meshes or surfaces. In the second subsection, we describe the functions implemented in CLARCS.

Related software for surface processing

There exists a bunch of commercial software allowing for some basic (e.g. import/export, visualisation) and higher level (e.g. registration) processing on surfaces, such as Amira (http://www.amira.com), or Rapidform (http://www.rapidform.com), but it is often difficult to know what methods are implemented therein, and always costly to acquire the software.

Some freeware are specifically dedicated to high level geometric computation (e.g. CGAL, http://www.cgal.org) or high level visualisation (e.g. Par-aView, http://www.paraview.org, and VTK, http://www.vtk.org) of surfaces, but we restrict our brief overview here to software which allow surface registration/comparison in a broad sense. The Point Cloud Library (or PCL, http://pointclouds.org) and MeshLab (http://meshlab.sourceforge.net) are two such software/libraries, but they only implement rigid registration. FreeSurfer (http://surfer.nmr.mgh.harvard.edu) allows non-linear registration of surfaces, but is limited to brain (cortical) data (using a specific atlas for this purpose).

Some freely available state-of-the-art software with a larger range of applications include:

-TPS-RPM [START_REF] Chui | A new algorithm for non-rigid point matching[END_REF]: http://noodle.med.yale.edu/~chui/tps-rpm.html -TPS-L2 [START_REF] Jian | A robust algorithm for point set registration using mixture of Gaussians[END_REF]: http://code.google.com/p/gmmreg -CPD [START_REF] Myronenko | Point set registration: Coherent point drift[END_REF]: https://sites.google.com/site/myronenko/research/cpd -SPHARM [START_REF] Shen | Modeling three-dimensional morphological structures using spherical harmonics[END_REF]: http://www.enallagma.com/SPHARM.php -weighted-SPHARM [START_REF] Chung | Tensor-based cortical surface morphometry via weighted spherical harmonic representation[END_REF]: http://www.stat.wisc.edu/~mchung/softwares/software.html

We have experimentally found the first three methods to be most often unable to cope with large (more than 100,000 points) point sets, while the two others impose strong topological constraints on the surfaces to register (they must be closed). The methods implemented in CLARCS allow the processing of large point sets (often needed to encode highly convoluted/complex anatomical structures) without topological constraints, in an efficient and fast way. From a methodological point of view, a strong advantage of these methods over most of the abovementioned ones is the unified computational framework they are based on, as outlined below. Finally, to our knowledge, there is no freely available software allowing the computation of the symmetry plane of approximately bilateral surfaces, and the assessment of asymmetries therof.

CLARCS

CLARCS is a C++ library for surface processing and analysis which has been developed at IRISA (http://www.irisa.fr) since 2007. CLARCS is mainly composed of three basic algorithms allowing (i) the rigid/affine/non-linear registration of two point sets, (ii) the computation of the mid-sagittal plane of one point set, (iii) the computation of a mean point set from several point sets, and the variability around this mean. These algorithms are building bricks that can be combined to allow higher-order surface processing such as computation of pointwise asymmetry fields and statistical analysis thereof within or between populations. The three algorithms rest on a common methodological framework, that we briefly outline here. We refer the reader to the corresponding papers for a deeper insight into these algorithms. Let us first define the following pseudodistance between point sets X 1 and X 2 :

δ 2 (X 1 , X 2 ) = min A,T xi∈X 1 xj ∈X 2 A i,j ||x i -T (x j )|| 2 +2σ 2 i,j A i,j log(A i,j ) + αL(T ) with ∀i j A i,j = 1 and ∀i, j A i,j ≥ 0 Computing δ 2 (X 1 , X 2
) is a minimisation problem involving the unknown transformation T linking the two point sets, and the unknown card(X 1 ) × card(X 2 ) matrix A. The constraints on this matrix allows it to encode the pointto-point correspondences between the point sets in a fuzzy way. The cost function can be seen as the sum of (i) a data-attachment term, (ii) a barrier (smoothing) function, which convexifies the cost function, and (iii) a regularity constraint on the unknown transformation. The relative strength of the three terms is weighted by the positive scalars 2σ 2 and α.

It is enlightening to notice that this minimisation problem is actually the maximum a posteriori (MAP) problem consisting of finding the transformation T best superposing the two point sets, when one makes the hypotheses that (i) each point x i of X 1 is independently drawn from a Gaussian mixture model (GMM) (whose means are the points T (x j ) of T (X 2 ), whose covariance matrices are equal to σ 2 I, I being the identity 3×3 matrix, and whose mixture weights are equal) and (ii) T is a random variable with a distribution of the form ∝ exp(-αL(T )). With this probabilistic view, X 1 can be interpreted as a noised version of T (X 2 ), σ being the standard deviation of this noise.

This MAP problem can typically be solved using the Expectation-Maximisation (EM) algorithm, which leads to the following iterative two-step algorithm:

-E-step: ∀i, j Ãi,j = exp[-||xi-T (xj )|| 2 /(2σ 2 )] k exp[-||xi-T (x k )|| 2 /(2σ 2 )] -M-step: T = arg min T i,j Ãi,j ||x i -T (x j )|| 2 + αL(T )
This EM algorithm can be shown to converge monotonically to an (at least) local maximum of the MAP criterion or, equivalently, to an (at least) local minimum of the aforementioned cost function. Actually, the two steps of the EM algorithm are exactly the same as those obtained when minimising this cost function with respect to A and T in turn.

The formulation of the E-step helps to understand why the matrix A encodes the point-to-point correspondences: when the transformation T is known, Ãi,j is the posterior probability that the point x i has been drawn from the mixture component with centre T (x j ). The E-step simply consists in computing these card(X 1 ) × card(X 2 ) probabilities. On the contrary, solving the M-step is highly dependent on the type of transformations considered.

Below, we show how this generic algorithm can be instantiated to lead to the three basic methods implemented in CLARCS.

Rigid/affine/non-linear registration. When T is set to be rigid and α = 0 (i.e. no prior on the rigid transformation), the MAP problem boils down to a maximum likelihood (ML) problem. Several closed-form solutions exist for the M-step, using typically the unit quaternions or the singular value decomposition. This results in what was termed the EM-ICP algorithm by Granger & Pennec [START_REF] Granger | Multi-scale EM-ICP: A fast and robust approach for surface registration[END_REF]. An earlier variant of this algorithm was devised by Rangarajan et al. and termed the RPM algorithm [START_REF] Rangarajan | A robust point-matching algorithm for autoradiograph alignment[END_REF].

We built on the EM-ICP algorithm to propose some adaptations for nonlinear registration. In previous works, we showed how to use the normals in addition to the point coordinates [START_REF] Münch | A modified ICP algorithm for normal-guided surface registration[END_REF], and how to enforce some constraints on the point-to-point correspondences [START_REF] Combès | Setting priors and enforcing constraints on matches for nonlinear registration of meshes[END_REF]. We also showed how to solve the M-step when considering a model of locally affine transformations [START_REF] Münch | A modified ICP algorithm for normal-guided surface registration[END_REF][START_REF] Combès | Setting priors and enforcing constraints on matches for nonlinear registration of meshes[END_REF] or when using the Reproducing Kernel Hilbert Space (RKHS) theory and the Fourier analysis to build the model [START_REF] Combès | An efficient EM-ICP algorithm for symmetric consistent non-linear registration of point sets[END_REF]. Finally, we showed how to obtain symmetric consistency when using this last transformation model [START_REF] Combès | An efficient EM-ICP algorithm for symmetric consistent non-linear registration of point sets[END_REF].

Symmetry plane computation. When T is set to be a reflection, α = 0 (i.e. no prior on the reflection) and when X 1 = X 2 , the EM algorithm allows to compute the plane best superposing the left and right parts of X 1 = X 2 [START_REF] Combès | Automatic symmetry plane estimation of bilateral objects in point clouds[END_REF]. Needless to say, this assumes that X 1 = X 2 is (at least) approximately bilateral. Our specific contribution was to propose a closed-form for the M-step, relying on the parametrization of the unknown reflection plane using its unit normal and distance to the origin of the coordinate system [START_REF] Combès | Automatic symmetry plane estimation of bilateral objects in point clouds[END_REF]. Non-linear registration of the point set with its flipped image with respect to the approximate symmetry plane allows the pointwise mapping of asymmetries [START_REF] Combès | New algorithms to map asymmetries of 3D surfaces[END_REF].

Atlas construction. The problem here is to find a point set M best representing a set of n point sets X 1 , . . . , X n . We defined it as:

M = arg min X i=1,...,n δ(X, X i )
If T is set to be a similarity (rigid transformation plus uniform scaling), it is possible to devise an iterative algorithm converging to an (at least) optimum of this criterion, in which the point-to-point correspondences, the mean point set (which turns out to be the mean shape in this case where T is defined as a similarity) and the unknown transformations linking the n point sets and the mean point set are estimated in turn. However, in such an approach, point-to-point correspondences are likely to be meaningless; this is why we proposed to establish these correspondences using non-linear transformations while computing the mean shape using similarities. The resulting iterative algorithm can no longer be shown to converge, but behaves well in practice [START_REF] Combès | EM-ICP strategies for joint mean shape and correspondences estimation: applications to statistical analysis of shape and of asymmetry[END_REF]. Once the algorithm has converged, it is straightforward to perform PCA on the residuals. Implementation details. These three algorithms were implemented within a multiscale framework. As previously noted by Granger & Pennec, the σ parameter allows to deal with the correspondences in a fuzzy way, leading to a smoother criterion to minimise. When σ is infinitely small, their EM-ICP algorithm is simply the ICP algorithm of Besl & McKay, hence the name [START_REF] Besl | A method for registration of 3-d shapes[END_REF]. This leads to the idea of devising a scheme in which several EM algorithms are successively run with decreasing σ values, with a large starting value when the point sets to register are far from each other. We also used kd-trees, for increased speed, and a cut-off distance between the points x i and T (x j ), above which they are eliminated from the estimation of the transformation, for increased speed and robustness to outliers.

Applications

Quantification of skull asymmetries in craniosynostoses

Synostosis is the union of two or more separate bones to form a single bone (Merriam-Webster). Cranial synostosis, or craniosynostosis is a rare congenital disease which consists in the premature fusion of one or several cranial sutures. The last medical condition is typically met in conjunction with a hundred of syndromes, among which are Apert or Crouzon. On the contrary, the etiology of isolated (nonsyndromic) craniosynostosis is largely unknown. Early detection and treatment of craniosynostosis is crucial, as hindered skull growth can lead to increased intracranial pressure and thus alteration of normal brain development [START_REF] Tamburrini | Intracranial pressure monitoring in children with single suture and complex craniosynostosis: a review[END_REF].

It is expected that improved characterisation of the dysmorphology of the skull associated with craniosynostosis could help understanding its cause(s), improving its diagnosis (e.g. lambdoid synostosis may be easily confounded with deformational plagiocephaly) and even improving its treatment. In this context, of particular interest are the unilateral coronal synostoses, as it is not clear whether the left and right coronal synostoses are due to the same factors.

We propose to characterise the dysmorphology of one skull with unilateral left coronal synostosis by assessing its pointwise asymmetry. This assessment is based on the computation of an approximate symmetry plane, from which the leftright differences (asymmetries) can be evaluated (cf. Fig. 1). The outer surface of the skull was computed using manual grey-level thresholding, mathematical morphology and the marching cube algorithm from a CT scan. The point set is a triangular mesh of a complete skull made up of about 140,000 cells and 82,000 points.

Fig. 1. Evaluation of pointwise asymmetries on a skull with craniosynostosis. We display the norm of the asymmetry field. Strong asymmetries are visible on the temporal bone, the posterior part of the parietal bone and the supraorbital part of the frontal bone.

Quantification of skull asymmetries in deformational plagiocephaly

Since the inception of the "back to sleep" recommendations in the early 90s to reduce sudden infant death syndrome, the incidence of positional (i.e. nonsynostotic) plagiocephaly has drastically increased [START_REF] Moon | Sudden infant death syndrome[END_REF][START_REF] Kinney | The sudden infant death syndrome[END_REF]. Plagiocephaly consists of the flattening of one side of the head, with aesthetics consequences, and potential altered brain development [START_REF] Kordestani | Neurodevelopmental delays in children with deformational plagiocephaly[END_REF]. Objective assessment of plagiocephaly should help diagnosis and follow-up of this condition [START_REF] Captier | Plagiocephaly: morphometry of skull base asymmetry[END_REF].

We propose to characterise the deformational plagiocephaly of one skull using the same computational tools as in the previous section (cf. Fig. 2). The outer surface of the skull was computed using manual grey-level thresholding, mathematical morphology and the marching cube algorithm from a CT scan. The point set is a triangular mesh of a complete skull made up of about 200,000 cells and 137,000 points.

Building statistical shape models of deep grey nuclei

Deep brain stimulation (DBS) was initially targetted to the ventral intermediate thalamus to reduce tremor in patients with Parkinson's disease [START_REF] Benabid | Long-term suppression of tremor by chronic stimulation of the ventral intermediate thalamic nucleus[END_REF]. Since then, alternative targets have emerged, such as the subthalamic nucleus (STN), globus pallidus interna, which appear to be comparatively efficient in this context [START_REF] Follett | Pallidal versus subthalamic deep-brain stimulation for parkinson's disease[END_REF]. The indications for DBS have also been extended to drug-resistant epilepsy, dystonia, Tourette syndrome or even obsessive compulsive disorders, using the caudate and accumbens nuclei (for instance) as targetted structures. An improved knowledge of the anatomy of these subcortical structures is key to optimise pre-operative planning and to assess treatment efficacy.

In this context, statistical shape models (SSMs) are extremely useful as they allow to help the segmentation of the structures of interest in MR images, in which typical pulse sequences make it hard to distinguish these nuclei based on the grey values alone [START_REF] Cootes | Active shape models-their training and application[END_REF].

As an illustration, we propose to build a statistical shape model of the thalami (Fig. 4) which were manually segmented together with six other deep brain structures (cf. Fig. 3) by a trained neuroanatomist using itk-SNAP (http://www.itksnap.org) in 10 patients with Parkinson's disease. For each patient, the seven pairs of segmented structures were stored in a 3D image with discrete labels. The surfaces of the structures were computed using the marching cube algorithm. The points sets representing the left and right thalami are triangular meshes with sizes of about 10,000 cells and 5,000 points.

Discussion & Conclusion

In this paper, we presented the CLARCS library and its three building bricks, namely: (i) the rigid/affine/non-linear registration of two point sets, (ii) the computation of the mid-sagittal plane of one point set, and (iii) the computation of a mean point set from several point sets. We also gave some possible applications of CLARCS. Some other applications can be found in the full papers describing the methods implemented in CLARCS, for instance:

-the construction of a statistical shape model of the osseous labyrinth [START_REF] Combès | EM-ICP strategies for joint mean shape and correspondences estimation: applications to statistical analysis of shape and of asymmetry[END_REF] and the caudate nuclei [10] -the estimation of the mean pointwise brain asymmetry in male right-handed subjects and the comparison with situs inversus subjects [START_REF] Combès | EM-ICP strategies for joint mean shape and correspondences estimation: applications to statistical analysis of shape and of asymmetry[END_REF] and with chimpanzees [13] -the estimation of the mean pointwise facial asymmetry in males and females, and the comparison thereof [START_REF] Combès | New algorithms to map asymmetries of 3D surfaces[END_REF] With its new, robust and fast methods for surface processing, CLARCS opens interesting perspectives with medical applications. For instance, the non-linear registration of two point sets of about 200,000 points each runs in less than 5 minutes on a recent standard personal computer (3GHz).

The implementation of the framework of CLARCS is compatible with the well-known visualization toolkit VTK (http://www.vtk.org), thus it is possible to insert CLARCS specific methods into more general VTK pipelines.

We plan to distribute CLARCS as an open-source library, but this step will require some code refactoring, thus CLARCS will be first available as an external plugin of the future version of MedInria (http://med.inria.fr) that will be released in early September 2011.

Fig. 2 .

 2 Fig. 2. Evaluation of pointwise asymmetries on a skull with deformational plagiocephaly. We display the norm of the asymmetry field. Strong asymmetries are visible on the posterior part of the parietal bone and on the superior part of the occipital bone.

Fig. 3 .Fig. 4 .

 34 Fig. 3.Front view of seven deep brain structures. Cyan: thalamus, orange: putamen, magenta: amygdala, blue: hippocampus, red: red nucleus, yellow: STN, purple: substantia nigra.

Acknowledgements

We thank Guillaume Captier (CHRU Montpellier, Hôpital Lapeyronie, France) for providing the CT scan of the skull with plagiocephaly, and Gérard Subsol (ICAR Project-Team, LIRMM, CNRS, Montpellier, France) and Gilles Gesquière (LSIS, Université de Provence, Arles, France) for segmenting and reconstructing the outer surface of this skull. We thank Yann Heuzé and Joan T. Richtsmeier (Department of Anthropology, Pennsylvania State University, University Park, USA) for providing the CT scan of the skull with craniosynostosis (through the NIH grant #R01DEO18500), and for segmenting and reconstructing the outer surface of this skull. We thank Pierre Jannin (INSERM/INRIA VisAGeS Unit/Project U746, Rennes, France) for his input on deep brain stimulation through the ACouStiC project (http://anr-acoustic.inria.fr).