MD Inge R H M Konings
email: i.konings@erasmusmc.nl

Stefan Sleijfer

Ron H J Mathijssen

Peter Bruijn

Inge M Ghobadi Moghaddam-Helmantel

Linda M Dam

Erik A C Wiemer

Jaap Verweij

Walter J Loos

Peter De Bruijn

Linda M Van Dam

Increasing tumoral 5-fluorouracil concentrations during a 5-day continuous infusion: a microdialysis study

Keywords: microdialysis, pharmacokinetics, target site, 5-fluorouracil 2

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

Traditionally, most oncological treatment regimens and doses are based on safety profiles. It is generally assumed that the obtained pharmacokinetic profile in blood is directly related to the mentioned safety and the magnitude of antitumor response. However, the efficacy of anticancer drugs will likely depend on the maintenance of adequate unbound drug concentrations in tumor tissue rather than plasma. Furthermore, the pharmacokinetic profile of a drug in blood may not per definition resemble its pharmacokinetic profile in tumor or normal tissue, as was demonstrated in a mouse model [START_REF] Zamboni | Tumor, tissue and plasma pharmacokinetic studies and antitumor response studies of docetaxel in combination with 9nitrocamptothecin in mice bearing SKOV-3 human ovarian xenografts[END_REF][START_REF] Zamboni | Plasma, tumor, and tissue disposition disposition of STEALTH liposomal CKD-602 (S-CKD602) and nonliposomal CKD-602 in mice bearing A375 human melanoma xenografts[END_REF][START_REF] Zamboni | Relationship between tumor extracellular fluid exposure to topotecan and tumor response in human neuroblastoma xenograft and cell lines[END_REF]. More detailed information about drug levels at the tumor site itself would therefore be helpful in optimizing oncological treatment.

In clinical oncology, evaluation of tumor and tissue disposition of drugs has become possible with the technique of microdialysis. This minimally invasive sampling method is based on the exchange of compounds from the extracellular fluid into perfusate, which runs through an inserted catheter, by diffusion through a semipermeable membrane following their concentration gradient. No net fluid exchange occurs during microdialysis, so prolonged continuous sampling is possible without interfering with the pharmacokinetic behavior of the drug [START_REF] Müller | Science, medicine, and the future: microdialysis[END_REF]. The exiting solution, the so-called microdialysate, can be used for both pharmacokinetic as well as pharmacodynamic analyses.

The clinical use of microdialysis to evaluate drug disposition in tumors is of considerable interest with the recognition that insufficient drug penetration into the interstitium of solid tumors may be involved in resistance to anti-tumor drugs [START_REF] Trédan | Drug resistance and the solid tumor microenvironment[END_REF][START_REF] Presant | Association of intratumor pharmacokinetics of fluorouracil with clinical response[END_REF]. One of the problems associated with efficient drug delivery is the presence of increased interstitial fluid pressure, a phenomenon occurring in most solid tumors [START_REF] Heldin | High interstitial fluid pressure -an obstacle in cancer therapy[END_REF]. To date, several studies have been performed applying the microdialysis technique in clinical oncology with several anticancer drugs [START_REF] Müller | 5-Fluorouracil kinetics in the interstitial tumor space: clinical response in breast cancer patients[END_REF][START_REF] Mader | Penetration of capecitabine and its metabolites into malignant and healthy tissues of patients with advanced breast cancer[END_REF][START_REF] Tegeder | Cisplatin tumor concentrations after intra-arterial cisplatin infusion or embolization in patients with oral cancer[END_REF][START_REF] Blochl-Daum | Measurement of extracellular fluid carboplatin kinetics in melanoma metastases with microdialysis[END_REF][START_REF] Joukhadar | Penetration of dacarbazine and its active metabolite 5-aminoimidazole-4-carboxamide into cutaneous metastases of human malignant melanoma[END_REF][START_REF] Ekström | Continuous intratumoral microdialysis during high-dose methotrexate therapy in a patient with malignant fibrous histiocytoma of the femur: a case report[END_REF][START_REF] Müller | Interstitial methotrexate kinetics in primary breast cancer lesions[END_REF]. In most of these studies the collected data covered only a relatively short period of time, i.e. up to a maximum of 4 hours (h), which might not be adequate to obtain useful pharmacokinetic profiles for many drugs. However, recently we showed the feasibility of prolonged microdialysis sampling up to 48h in ambulant cancer patients treated with intravenous (iv) bolus carboplatin [START_REF] Konings | Application of prolonged microdialysis sampling in carboplatin-treated cancer patients[END_REF].

In the present study, we investigated the feasibility of microdialysis for even a longer period of time using another drug, namely 5-fluorouracil infused continuously for 5 days. The aim was to accomplish a model which could be used in further studies to monitor the influence of co-administration of other agents on tumoral 5-FU concentrations, each patient being its own control. 5-Fluorouracil belongs to the group of the antimetabolites and is a widely used agent for the treatment of colorectal, breast and head and neck cancer [START_REF] Wolpin | Systemic treatment of colorectal cancer[END_REF][START_REF] Seidman | Systemic treatment of breast cancer. Two decades of progress[END_REF][START_REF] Pan | Pharmacotherapy of head and neck squamous cell carcinoma[END_REF]. After entering the tumor cell, 5-fluorouracil is converted into several metabolites that exert anti-tumor activity by interfering with DNA synthesis and DNA repair, and by incorporation in RNA. The majority of 5-FU is inactivated by the enzyme dihydropyrimidine dehydrogenase (DPD), which is mainly found in the liver but also in other tissues such as the gastrointestinal mucosa, peripheral blood cells and tumor tissue [START_REF] Kubota | 5-Fluorouracil and dihydropyrimidine dehydrogenase[END_REF]. Numerous dose regimens exist, continuous administration at 1000 mg/m 2 /daily for 5 days being one of the most commonly used. Given the fact that 5-fluorouracil rapidly reaches a steady state in plasma during continuous infusion, it was expected that 5-fluorouracil concentrations could be quantitated in the microdialysates during the whole 5 times 24 hours period.

The aims of the present study were to explore the feasibility of continuous microdialysis sampling over a 5-day period, to assess differences in plasma and tumor pharmacokinetics of 5-fluorouracil during a continuous 5-fluorouracil infusion and to set up a clinical model, with 5-fluorouracil as model drug, for future drug delivery modulating studies.

Patients and Methods

Patient Selection Criteria

Patients who had a (sub)cutaneous primary tumor or metastasis and for whom 5-fluorouracil-based therapy was considered a viable treatment option were eligible for the study. The (sub)cutaneous lesion had to be of sufficient size (i.e. ≥ 20 mm) and readily accessible for catheter implantation. Other inclusion criteria included: no use of therapeutic doses of anti-coagulants (prophylactic use was allowed); WHO performance status ≤ 1; age ≥ 18 years; absolute neutrophil count ≥ 1.5 x 10 9 /L; absolute platelet count ≥ 100 x 10 9 /L; creatinine clearance ≥ 60 mL/min.; no other serious illness or medical unstable condition requiring treatment or a history of psychiatric disorder that would prohibit the understanding and giving of informed consent. This single-center study was approved by the ethical committee of the Erasmus Medical Center (Rotterdam, the Netherlands) and was performed in accordance with standards laid down in the 1964 Declaration of Helsinki and its later amendments. All patients gave written informed consent. (EudraCT number: 2006-001070-24).

Administration of 5-fluorouracil

5-Fluorouracil (Ebefluoro, EBEWE Pharma, Belgium) was started as a 5-day continuous intravenous infusion on Day 1, at a dose of 1000 mg/m 2 /day. To prevent allergic reactions, nausea and vomiting a premedication schedule consisting of dexamethasone and granisetron was administered intravenously prior to the start of the 5-fluorouracil infusion. In case 5-fluorouracil was combined with another cytotoxic, this second agent was administered only on the last day of treatment.

Plasma pharmacokinetic and microdialysis sampling were performed during a single treatment course of five days.

Microdialysis procedures

The microdialysis catheters (CMA 63 catheters, membrane length 10 mm, molecular weight cut-off level of ~20,000 Da), pumps (CMA 107, flow rate adjustable) and microdialysis consumables were purchased from CMA/Microdialysis AB (Solna, Sweden). On Day 0, each patient had one microdialysis catheter inserted in a suitable (sub)cutaneous tumor lesion and a second one into healthy abdominal subcutaneous adipose tissue. Catheter insertion procedures have previously been described in detail [START_REF] Müller | Application of microdialysis to clinical pharmacokinetics in humans[END_REF].

The intratumoral location of the probe tip was verified by ultrasound.

As microdialysis is not performed under equilibrium conditions, the concentration of the drug in the dialysate may be different from that in the fluid surrounding the probe [START_REF] Kitzen | The relevance of microdialysis for clinical oncology[END_REF]. To correct for this difference, the relative recovery of each probe was assessed the morning after the implantation of the probes by retrodialysis with 1 µg/mL of 5-FU in Ringer's solution as described recently [START_REF] Konings | Application of prolonged microdialysis sampling in carboplatin-treated cancer patients[END_REF], at a flow rate of 0.5 µL/min (i.e., the flow rate as used during the pharmacokinetic sampling).

Prior to the clinical study, in vitro recovery experiments (data not shown) were conducted to demonstrate that 5-fluorouracil does not irreversibly bind to the catheters, the probe recovery is independent of the concentration and is equal in both directions over the probe, similar to previous studies [START_REF] Konings | Application of prolonged microdialysis sampling in carboplatin-treated cancer patients[END_REF].

During the pharmacokinetic experiment, samples were collected during the following sample periods: 0-2h, 2-5h following the start of infusion and subsequently every 5h till the end of infusion at day 5. The 5-hour interval was chosen as at a flow-rate of 0.5 µL/min the vials could not be overfilled during this period. Samples were stored directly at the bed-side of the patient at T < -20°C. The dialysates were collected by centrifugation of the microvials upside down in micro-centrifuge reaction vials for 1 min at 500 x g, after which they were stored at T < -20°C until analysis.

Blood sampling

For 5-fluorouracil plasma pharmacokinetic analysis, blood samples were collected in the presence of lithium heparin as anticoagulant at the following time points: before start of the infusion, 1, 2, 4, 7 and 12h after the start of infusion and subsequently every 12h till the end of infusion at day 5. Within 15 minutes after collection, plasma was separated by centrifugation at 3,000 g for 10 min and stored at T < -20°C until analysis.

Unbound fraction in plasma

Although the free fraction of 5-fluorouracil has been reported to be > 90% [START_REF] Czejka | The binding of 5-fluorouracil to serum protein fractions, erythrocytes and ghosts under in vitro conditions] Bindung von 5-Fluorouracil an Serumproteinfraktionen, Erythrozyten und Ghosts unter in vitro Bedingungen[END_REF], and plasma protein binding will have a minor influence on the tissue distribution, the percentage binding may be different between patients. Therefore, we estimated the free fraction of 5-fluorouracil in each individual patient.

Aliquots of 250 µL of plasma samples collected each 24 hour during the infusion-period were transferred into Microcon Ultracel YM-30 Centrifugal Filter Devices (Millipore Corp., Bedford, MA, USA) with a molecular weight cut-off level of 30,000 Da. The samples were centrifuged in a fixed angle rotor at 18,000

x g for 30 min. The ultrafiltrate was stored at T < -20°C until analysis. The individual observed concentration in each sample was divided by the total concentration. The mean free fraction was subsequently used to estimate the free fraction in all samples collected.

5-Fluorouracil analysis

5-Fluorouracil (molecular weight 130.1) in the dialysate (and ultrafiltrate) was quantitated using liquid chromatography (LC) with tandem mass-spectrometric detection (MS/MS). Briefly, a volume of 25 µl of the internal standard solution containing 500 ng/mL 5-chlorouracil in water was added to 25 μL of dialysate, from which aliquots of 25 µL were injected into the LC (Model 2795 XC chromatograph, Waters Alliance, Mildford, MA). Chromatographic separations were achieved on a Hypercarb column (100 × 3 mm internal diameter, 5 µm particle size, Thermo, Breda, the Netherlands), which was held at T=40°C. The mobile phase was composed of water and acetonitrile and was delivered using a linear gradient setting. The initial flow rate was 0.75 mL/min where the composition changed from 10% to 90% acetonitrile in 1 minute. In the subsequent minute, the flow rate was linearly decreased to 0.25 mL/min and kept at this rate for 6 min at this rate. Subsequently, the flow rate was linearly increased to 1.0 mL/min in 1 min and kept at 1.0 mL/min for 2 min. Hereafter the composition changed from 90% to 10% acetonitrile in 1 minute. The overall run time was 12 min. Detection was performed with a Waters MicroMass Quatro Micro triplequadropole mass spectrometer in the negative ion mode. The detector was programmed to allow the [MH] - ions of 5-fluorouracil (m/z 129) and 5-chlorouracil (m/z 145) to pass through the first quadropole and into the collision cell. The collision energy for collision-induced dissociation of 5-fluorouracil and 5chlorouracil was set at 20 eV and 18 eV, respectively, with argon used as collision gas at a pressure of 6e -3 mbar. The daughter ions of 5-FU (m/z 86) and 5-chlorouracil (m/z 102) were monitored through the third quadropole. The dwell time per channel for data collection was 0.150 seconds. Retention times of fluorouracil and 5-chlorouracil were 2.5 and 3.3 min respectively. Calibration curves were linear from 10.0 to 1,000 ng/mL. The accuracy ranged from 94.0% to 103.7%, the within-run precisions were ≤ 12.9% and the between-run precisions were ≤ 2.7% at five tested concentrations, including the lower limit of quantitation of 10.0 ng/mL. Plasma 5-fluorouracil concentrations were quantitated likewise. Aliquots of 30 µL plasma were extracted, after the addition of 10 µL of 500 ng/mL 5-chlorouracil, with 1.5 mL ethyl acetate. Following vigorous vortex mixing for 5 min and centrifugation for 5 min at 18,000 x g, the clear supernatant was evaporated to dryness at T=70°C under nitrogen. The residue was resuspended in 100 µL aliquots of Ringer's solution, from which an aliquot of 50 µL was injected into the system as described above. LC-MS/MS system settings were as described for dialysate. Calibration curves were linear from 20.0 to 2,000 ng/mL. The accuracy ranged from 94.4% to 107.5%, the within-run precisions were ≤ 7.1% and the between-run precisions were ≤ 4.1% at five tested concentrations, including the lower limit of quantitation of 20.0 ng/mL.

During the analysis of the dialysates of the first 3 patients, a significant matrix effect was observed for 5-chlorouracil. The observed peak areas of 5-chlorouracil were significantly lower in dialysate samples compared to the Ringer's solution in which the calibration curve standard and quality control samples were prepared. This results in an overestimation of the 5FU concentrations in the ECF. Replacing the internal standard 5-chlorouracil by stable 13 C/ 15 N 2 labeled 5-fluorouracil, which elutes at the same time from the analytical column as 5FU, this to mass spectrometry related potentially occurring issue was resolved.

Multiple reaction monitoring of 13 C/ 15 N 2 5-fluorouracil was applied at m/z 132>88, with the cone voltage and collision energy set at 30 and 18 eV, respectively. Individual pharmacokinetic parameters were estimated using non-weighted noncompartmental analysis using the software program WinNonLin 5.2 (Pharsight, CA, USA). As an increase in the ECF/plasma ratios was observed over time, partial AUCs were then calculated over 24-h intervals.

Although sampling time points were not based on a 24-h cycle, the use of WinNonLin 5.2 allows for interpolation of AUCs.

Results

Patients

Seven patients were included in the study between April 2008 and November 2009, of which six patients were assessable for pharmacokinetic analysis of 5-fluorouracil in the extracellular fluid of tumor tissue. One patient (No 2) was not evaluable as there was not enough microdialysate left for re-analysis following the switch of the internal standard in the bioanalytical methodology. Only one patient (No 4) was evaluable for analysis of extracellular fluid of normal tissue as probe recovery in the other patients was variable. In two patients (No 1 and 6) treatment of 5-FU was combined with carboplatin, which was administered on the last day of treatment after termination of the 5-FU infusion. A summary of demographic characteristics of the six patients assessable for pharmacokinetic analysis in the extracellular fluid of tumor tissue is presented in Table 1.

Probe recovery

The in vitro recovery was independent of the concentrations tested and the method applied (i.e., retrodialysis or extraction recovery). Furthermore, the in vitro recovery was constant over up to 10 studied days and showed that 5-fluorouracil does not bind to the microdialysis catheter. For instance, at a flow rate of 1.0 µL/min the recovery was 91 (± 2.2), 82 (± 0.5) and 91 (± 0.2) % on days 1, 4 and 10, respectively. In addition, as increased interstitial fluid pressure is a well known characteristic of several tumors [START_REF] Heldin | High interstitial fluid pressure -an obstacle in cancer therapy[END_REF][START_REF] Jain | Delivery of molecular and cellular medicine to solid tumors[END_REF], we tested if increased pressures influenced the recovery. Pressures up to 15 mm Hg (i.e., 20 cm of Ringer's solution above the probe) had no effect on the recovery of 5-fluorouracil.

At a flow rate of 0.5 µL/min, the in vivo recovery in the six patients evaluable for tumor tissue drug levels varied from 40-92 % (Table 2) and was constant in each individual patient.

Pharmacokinetics

A summary of the pharmacokinetic data is presented in Table 2. As also depicted in Figure 1 and2, the 5-fluorouracil concentrations in the extracellular fluid of tumors are lower than the concentrations of unbound 5-fluorouracil in the plasma compartment. Over time however, the difference between tumor and plasma concentrations decreased, as a result of increased tumor concentrations.

Figure 3 depicts the 5-fluorouracil concentrations in tumor extracellular fluid according to the time of sampling during the day. The tumoral 5-fluorouracil concentrations were higher during the night than during daytime. Of note, tumor ECF was not available from patient 3 at later time-points due to the significant matrix effect observed for 5-chlorouracil during the initial analysis and the inability for reanalysis of the samples (all used).

Discussion

Optimization of cancer therapy by enhancing drug delivery has become of major interest as many barriers to drug delivery are being identified, underlining the need for new therapeutic strategies increasing drug delivery to tumors. Obviously, this requires a model that can easily be applied in clinical practice and with the ability to monitor intratumoral drug concentrations. The present study addresses this need and proposes the use of microdialysis to obtain detailed information on drug concentrations at the tumor site.

Earlier studies explored the applicability of microdialysis in clinical oncology with microdialysate collection with a maximum of up to 48 hours after iv bolus administration of carboplatin [START_REF] Konings | Application of prolonged microdialysis sampling in carboplatin-treated cancer patients[END_REF]. In this study, we showed that microdialysis is also feasible for longer periods of time in cancer patients treated with continuous 5-fluorouracil infusion during a 5x 24h period.

Our results evidently show lower concentration-time curves at the tumor site in comparison with plasma. This is in line with the results of an earlier microdialysis study by Müller et al. where the obtained area under the curve (AUC) in tumor after a single iv administration of 5-fluorouracil was 61% of the AUC in plasma [START_REF] Müller | 5-Fluorouracil kinetics in the interstitial tumor space: clinical response in breast cancer patients[END_REF]. A remarkable observation in our study is the fact that the difference between plasma and tumor concentrations diminished over time, as a result of increasing 5-fluorouracil concentrations in the tumor. A possible explanation for this phenomenon may be a change in interstitial fluid pressure in the tumor. Many solid malignancies display a high interstitial fluid pressure, which causes poor uptake of anticancer drugs and has been associated with poor clinical response to treatment [START_REF] Trédan | Drug resistance and the solid tumor microenvironment[END_REF][START_REF] Heldin | High interstitial fluid pressure -an obstacle in cancer therapy[END_REF]. Mechanisms involved yielding a high interstitial fluid pressure in tumors are a high vessel permeability, inadequate lymphatic drainage, fibrosis and contraction of the interstitial matrix and a high cell density around the blood vessels [START_REF] Heldin | High interstitial fluid pressure -an obstacle in cancer therapy[END_REF]. Reduction of tumoral interstitial fluid pressure is an attractive means to augment efficacy of anti-tumor drugs. In pre-clinical models, several agents have been revealed to decrease interstitial fluid pressure including dexamethasone [START_REF] Stuhr | High-dose, shortterm, anti-inflammatory treatment with dexamethasone reduces growth and augments the effects of 5-fluorouracil on dimethyl-α-benzanthracene-induced mammary tumors in rats[END_REF][START_REF] Kristjansen | Dexamethasone reduces the interstitial fluid pressure in a human colon xenograft[END_REF], imatinib [START_REF] Pietras | Inhibition of platelet-derived growth factor receptors reduces interstitial hypertension and increases transcapillary transport into tumors[END_REF][START_REF] Pietras | Inhibition of PDGF receptor signaling in tumor stroma enhances antitumor effect of chemotherapy[END_REF], and prostaglandin E1 [START_REF] Salnikov | Lowering of tumour interstitial fluid pressure specifically augments efficacy of chemotherapy[END_REF]. Also several cytotoxic agents, including 5-fluorouracil, have shown to lower tumor interstitial fluid pressure within days [START_REF] Stuhr | High-dose, shortterm, anti-inflammatory treatment with dexamethasone reduces growth and augments the effects of 5-fluorouracil on dimethyl-α-benzanthracene-induced mammary tumors in rats[END_REF][START_REF] Feretti | Tumor interstitial fluid pressure as an early-response marker for anticancer therapeutics[END_REF][START_REF] Griffon-Etienne | Taxane-induced apoptosis decompresses blood vessels and lowers interstitial fluid pressure in solid tumors: clinical implications[END_REF]. Although the decrease in tumor interstitial fluid pressure following 4 days of 5-fluorouracil treatment in female Sprague-Dawley rats with induced mammary tumors was not significantly different, a reduction of 35% in interstitial fluid pressure was noted on day 5 compared to day 1 (7.2 vs. 4.7 mmHg; [START_REF] Stuhr | High-dose, shortterm, anti-inflammatory treatment with dexamethasone reduces growth and augments the effects of 5-fluorouracil on dimethyl-α-benzanthracene-induced mammary tumors in rats[END_REF]). The increasing 5-fluorouracil concentration at the tumor site as observed in our study might also be due to the effect of 5-fluorouracil on the interstitial fluid pressure. Assuming that 5-fluorouracil enhanced its own tumor concentration due to its antitumor and thereby lowering effect on interstitial fluid pressure, our data would be in line with a recent study showing that modulation of convection currents by lowering interstitial fluid pressure by treatment with collagenase within tumors enhances the delivery of 5fluorouracil approximately 1.5 fold [START_REF] Gade | Imaging intratumoral convection: pressure-dependent enhancement in chemotherapeutic delivery to solid tumors[END_REF]. The transport of low-molecular weight compounds, such as 5fluorouracil, from the vascular space into the interstitial space is thus also driven by convection besides by diffusion. Though only available for one patient, 5-fluorouracil uptake in normal tissue does not seem to increase over time. In the patient with evaluable pharmacokinetic data in extracellular fluid of normal tissue, the tumor exposure increased, while normal tissue exposure was constant over time (Figure 1 and2). This is also in agreement with the above mentioned study [START_REF] Gade | Imaging intratumoral convection: pressure-dependent enhancement in chemotherapeutic delivery to solid tumors[END_REF], that did not show a difference in 5fluorouracil accumulation in normal tissue of mice treated with or without collagenase, i.e. with or without lowering of interstitial fluid pressure.

The interstitial fluid pressure and its effect on convention currents are only two of the multiple factors influencing drug delivery at the target site. Tumor vasculature differs significantly from that of normal tissue, with great heterogeneity in density, length and diameter, thereby impeding blood flow.

Tumor pH gradients are of importance too, as membrane transport and accumulation of weakly acidic 5fluorouracil in tumors is pH dependent [START_REF] Presant | Association of intratumor pharmacokinetics of fluorouracil with clinical response[END_REF][START_REF] Peters | Prolonged retention of high concentrations of 5fluorouracil in human and murine tumors as compared with plasma[END_REF][START_REF] Stubbs | Causes and consequences of tumour acidity and implications for treatment[END_REF]. DPD activity may be of influence as well, since its activity is known to be variable in tumor tissue and dysregulation of DPD is more frequent in malignant tissue [START_REF] Kubota | 5-Fluorouracil and dihydropyrimidine dehydrogenase[END_REF][START_REF] Milano | Clinical pharmacokinetics of 5-fluorouracil with consideration of chronopharmacokinetics[END_REF]. These tumor specific characteristics may explain the differences observed in ECF tumor /plasma unbound ratio's between the patients. Similar to the findings of our previous microdialysis study in carboplatin-treated patients measurement of 5-fluorouracil in extracellular fluid of normal adipose tissue was troublesome because of fluctuating recovery assessments [START_REF] Konings | Application of prolonged microdialysis sampling in carboplatin-treated cancer patients[END_REF]. It is well confirmed now that subcutaneous adipose tissue is not the best site to use as control, most probably due to its different and poorer vascularisation. Other normal tissues should be chosen as site control in future studies, e.g. muscular tissue or preferably, tissue surrounding the tumor.

Another interesting observation was the typical pharmacokinetic pattern of 5-fluorouracil, with higher 5-fluorouracil tumor extracellular fluid concentrations observed during the night compared to daytime (Figure 3). Circadian rhythmicity of 5-fluorouracil pharmacokinetics in plasma has been an issue of investigation for decades. During continuous 5-fluorouracil infusion, 5-fluorouracil plasma concentrations turned out to be higher during the night than during daytime. Many factors such as age, diet, physical activity, high serum alkaline phosphatase and length of drug infusion were previously identified as patient variables affecting 5-fluorouracil clearance [START_REF] Milano | Clinical pharmacokinetics of 5-fluorouracil with consideration of chronopharmacokinetics[END_REF][START_REF] Block | Making circadian cancer therapy practical[END_REF]. Also, dihydropyrimidine dehydrogenase activity has been found to change during the day, with its highest activity occurring during daytime [START_REF] Milano | Clinical pharmacokinetics of 5-fluorouracil with consideration of chronopharmacokinetics[END_REF]. In order to achieve the highest efficacy and least toxicity chronomodulated 5-fluorouracil therapy was developed, albeit not regarded standard of care yet [START_REF] Block | Making circadian cancer therapy practical[END_REF][START_REF] Lévi | Implications of circadian clocks for the rhythmic delivery of cancer therapeutics[END_REF]. Our study is the first to suggest the presence of a circadian rhythm of 5-fluorouracil pharmacokinetics in the tumor itself.

In summary, we showed the feasibility of microdialysis in cancer patients treated with a continuous 5-fluorouracil-infusion during a 5x 24h period. Plasma levels of 5-fluorouracil were higher compared to tumor, but there was a distinct rise of 5-fluorouracil at the tumor site over time. Since 5-fluorouracil is a widely used cytotoxic agent and high concentrations at the tumor site are directly linked with a better response [START_REF] Presant | Association of intratumor pharmacokinetics of fluorouracil with clinical response[END_REF][START_REF] Müller | 5-Fluorouracil kinetics in the interstitial tumor space: clinical response in breast cancer patients[END_REF], evaluation of its disposition in tumor tissue is of notable importance to further optimize individual cancer patient treatment. The technique of microdialysis can be used to improve insight into processes involved in tumor pharmacokinetics, such as e.g. interstitial fluid pressure. In a rat model the coadministration of imatinib resulted in a significant decrease in interstitial fluid pressure with as a consequence a 2-4 fold increase of the ECF tumor /plasma unbound ratio's of 51 Cr-EDTA and paclitaxel respectively, and a significant tumor volume reduction of 44% in rats treated with 5-FU and imatinib [START_REF] Pietras | Inhibition of platelet-derived growth factor receptors reduces interstitial hypertension and increases transcapillary transport into tumors[END_REF][START_REF] Pietras | Inhibition of PDGF receptor signaling in tumor stroma enhances antitumor effect of chemotherapy[END_REF]. Clinical trials investigating the impact of co-administration of drugs thought to (in)directly effect tumor drug uptake in cancer patients are warranted and this microdialysis model, with 5-fluorouracil as representative drug for other small molecules, might be a useful tool to study pharmacokinetics and pharmacodynamics in more detail. The ongoing development of newer microdialysis probes that can be used in deeper-lying (tumor) tissue, e.g. the liver, will add to the clinical use of this technique. AUC area under the concentration time curve, ECF extracellular fluid, NA not available 1 Mean ± SD, 2 calculated using data observed during a timeframe after start of the infusion with subscript numbers referring to start and endpoint of timeframe, 3 (AUC ratio ECF tumor /Plasma unbound )

Legends for Figures

Figure 1 :

 1 Figure 1: Concentration time curves of the unbound fraction of 5-FU in plasma (open circles) and

Figure 2 :

 2 Figure 2: Exposure ratio ECF tumor /plasma unbound during the infusion of 5-FU. Each symbol represents a single patient. Patient 4 (i.e., the patient with evaluable normal tissue PK) is represented

Figure 3 :

 3 Figure 3: Concentration time curves of the unbound fraction of 5-FU in tumor ECF during day and

  Figure 2

Table 2 :

 2 Summary of pharmacokinetic data of 5-FU in plasma and ECF of tumor tissue

	Parameter	Patient 1	Patient 3	Patient 4	Patient 5	Patient 6	Patient 7
	Dose 5-fluorouracil (mg/day)	2300	1890	1700	2000	1650	1500
	Plasma unbound
	Fraction unbound (%) 1	87 ± 3.2	93 ± 11	97 ± 9.1	86 ± 8.5	83 ± 4.7	88 ± 2.7
	AUC 0-120 (µg*h/mL) 1	50.8	62.7	54.6	33.6	38.6	NA
	AUC 0-24 (µg*h/mL) 2	8.79	11.1	7.39	7.22	6.25	6.56
	AUC 24-48 (µg*h/mL) 2	10.6	11.4	9.83	7.05	6.58	7.94
	AUC 48-72 (µg*h/mL) 2	10.2	11.8	12.4	6.74	7.39	NA
	AUC 72-96 (µg*h/mL) 2	10.8	14.4	13.2	5.75	8.71	NA
	AUC 96-120 (µg*h/mL) 2	10.4	14.1	11.8	6.79	9.62	NA
	ECF tumor tissue
	Probe recovery (%) 1	40 ± 4.5	89 ± 3.4	89 ± 2.7	92 ± 2.4	63 ± 1.2	92 ± 1.2
	AUC 0-120 (µg*h/mL) 2,3	11.0 (0.22)	NA	13.0 (0.24)	13.6 (0.41)	6.38 (0.17)	19.3 (NA)
	AUC 0-24 (µg*h/mL) 2,3	1.31 (0.15)	1.69 (0.15)	0.92 (0.12)	2.09 (0.29)	0.94 (0.15)	2.61 (0.40)
	AUC 24-48 (µg*h/mL) 2,3	1.70 (0.16)	2.59 (0.23)	2.03 (0.21)	2.43 (0.35)	1.13 (0.17)	3.51 (0.44)
	AUC 48-72 (µg*h/mL) 2,3	2.23 (0.22)	2.60 (0.22)	2.49 (0.20)	2.96 (0.44)	1.24 (0.17)	5.05 (NA)
	AUC 72-96 (µg*h/mL) 2,3	2.73 (0.25)	NA	3.82 (0.29)	2.85 (0.50)	1.50 (0.17)	4.28 (NA)
	AUC 96-120 (µg*h/mL) 2,3	3.03 (0.29)	NA	3.69 (0.31)	3.26 (0.48)	1.58 (0.16)	3.87 (NA)

Conflict of Interest

The authors declare no conflicts of interest.


Dr Cosetta Minelli
email: cosetta.minelli@eurac.edu

John Thompson

Meta-analysis of genetic association studies: magic tool or dangerous black box?

In the field of genetic epidemiology, where primary studies are often underpowered to detect small genetic effects and where conflicting results and non-replication of initial findings are often encountered [START_REF] Colhoun | Problems of reporting genetic associations with complex outcomes[END_REF] , the importance of meta-analysis has been widely recognised, as demonstrated by the steep increase in the number of published genetic meta-analyses over time [START_REF] Minelli | The quality of meta-analyses of genetic associations: a review with recommendations[END_REF] . What has not received sufficient attention is the fact that the results of a meta-analysis, as with any other study design, are only valid if the meta-analysis has been conducted following appropriate methodology. Therefore, the paper by Boccia and colleagues published in this issue [START_REF] Boccia | A systematic review evaluating the methodological aspects of meta-analyses of genetic association studies in cancer research[END_REF] , which reports the findings of a systematic review on the quality of meta-analyses of genetic association studies in the field of cancer research, represents an important contribution to a rather neglected area.

Boccia and colleagues reviewed 169 papers published up to January 2009 and report a substantial methodological improvement over time. However, such improvement seems limited to more general aspects, such as the search for articles published in different languages and the assessment of statistical heterogeneity and publication bias, while other aspects specific to genetics, such as the assessment of Hardy-Weinberg equilibrium and the justification for the choice of a genetic model, still appear to be largely overlooked. Similar findings were reported in a previous review of 120 genetic meta-analyses, randomly sampled among those published between 2005 and 2007, where methodological issues specific of genetic meta-analyses were those most neglected 2 . This is not surprising, given that the use of metaanalysis in genetic epidemiology is still relatively new compared with its use in other fields of medicine.

The Human Genome Epidemiology Network (HuGE Net), founded in 1998 with the aim of developing and disseminating population-based human genome information, provides some guidance on how to perform meta-analyses of genetic association studies. HuGE reviews, that is systematic reviews and metaanalyses performed in accordance with the HuGE Review Handbook [START_REF]The HuGENet™ HuGE Review Handbook, version 1.0[END_REF] and with their methods prespecified in a protocol submitted to HuGE Net, have shown better quality compared with the rest of published genetic meta-analyses, particularly for those methodological aspects specific to genetics [START_REF] Minelli | The quality of meta-analyses of genetic associations: a review with recommendations[END_REF] . This suggests that the positive influence of HuGE Net on the quality of genetic meta-analyses performed within its sphere has not yet extended to the broader circle of investigators working in this field. Boccia and colleagues conclude in their article that there is a need for consensus guidelines for the conduct and reporting of meta-analyses of genetic association studies. Producing and disseminating such guidelines may not suffice, though, and it could be argued that the most effective way of improving the quality standard of any type of research is through enforcement of stricter journal policies on the requirements for publication. The adoption of the Consolidated Standards for Reporting of Trials (CONSORT) statement by high quality journals has been shown to improve the quality of published randomised clinical trials [START_REF] Moher | Consolitdated Standards for Reporting of Trials). Use of the CONSORT statement and quality of reports of randomized trials: a comparative before-and-after evaluation[END_REF] , with similar effects having been suggested for the Quality of Reporting of Meta-analyses (QUOROM) statement on the quality of published meta-analyses [START_REF] Delaney | A systematic evaluation of the quality of meta-analyses in the critical care literature[END_REF] . It is interesting that the quality of published metaanalyses of genetic association studies does not seem to be influenced by the type of publication journal (general medicine, genetics or specialty journal) or its quality (crudely approximated by its impact factor) [START_REF] Minelli | The quality of meta-analyses of genetic associations: a review with recommendations[END_REF] , which suggests that guidance is indeed urgently needed to help journal editors achieve a quality standard for these papers similar to that of other research articles.

Guidelines for quality assessment of meta-analyses of genetic association studies could make a dramatic difference for a journal's reviewers, who are sometimes unaware of the specific methodological issues involved and their possible implications for the validity of the findings. The fact that meta-analyses published together with a genetic association study show poorer quality than meta-analyses published on their own 2 , suggests that people who are not familiar with this study design may sometimes see metaanalysis as a "nice addition" to a primary study, even when it is performed "quick-and-dirty". This is clearly not true and a poor quality meta-analysis may be very misleading, since it could provide biased results and misleadingly narrow confidence intervals. The development of guidelines requires good knowledge of the methods available and their assumptions, and also an appreciation of the practical problems involved in their application. Collecting empirical evidence on the quality of published metaanalyses requires a large amount of work, and yet such effort is undoubtedly worthwhile if it helps improve the quality of future meta-analyses by highlighting areas where more methodological guidance is needed. Articles such as the one by Boccia and colleagues are therefore to be welcomed.

In interpreting the findings from reviews on the quality of published meta-analyses such as the article by Boccia and colleagues, one needs to remember that quality assessment can only be based on what is reported in the paper, so that the assessor cannot disentangle quality of conduct from quality of reporting.

Obviously it is the investigator´s responsibility to guarantee that important information on aims, methodology and findings of the meta-analysis are clearly reported, with discussion of the assumptions underlying the methods and their implications on the results, but limitation of space for publication may sometimes be a constraint on reporting, particularly when a meta-analysis is published alongside a primary genetic association study, where much of the paper needs to cover methods and results of that study. In practice, however, the opportunity to post additional information on journals' websites is becoming increasing common, and even when this is not possible, investigators can often use their own institution's website. Reviewers and journal editors can play an important role in improving the transparency of the reporting of genetic meta-analyses by encouraging and facilitating the posting of detailed information on the web.

While everybody agrees on the need for more guidance in the conduct and reporting of meta-analyses of genetic association studies, we believe that a "recipe-like approach" would not be appropriate. There are ways of conducting and reporting a meta-analysis of genetic association studies that should always be discouraged, but unfortunately there is not a single recipe for producing valid results that applies in all situations. This has direct implications for the quality assessment of such meta-analyses, where no checklist can avoid the need from the assessor to critically evaluate the appropriateness of a given approach in the light of the specific scenario and aims of that meta-analysis. One example will illustrate the limitations of excessive simplification. Boccia and colleagues stress the importance of a comprehensive literature search which can identify all possible studies performed on a given topic, and they use the absence of language restriction to English as a marker of good quality. Given the increasingly large contribution of countries such as China to current genetic research, coverage of other languages is not a minor decision and could mean a substantial difference in the number of studies to be included. As Boccia and colleagues point out, language restrictions can be associated with a geographical form of publication bias, known as "language bias" or "local literature bias", whereby studies performed in a non-English speaking country are more likely to be published in international journals (in English) if their results are statistically significant, and in national journals if they are not [START_REF] Pan | Local literature bias in genetic epidemiology: an empirical evaluation of the Chinese literature[END_REF] . However, there are situations in which comprehensiveness of the literature search may not be necessary or even desirable.

For example, imagine one has performed a genetic association study and wish to conduct in parallel a systematic review and meta-analysis. There are two possible reasons for doing so: 1) the findings of the primary study are not "convincing" because of limited sample size, and the aim is to address this by pooling the study with other similar studies to increase statistical power; 2) the findings of the primary study are conclusive, and the aim is to evaluate whether the same association is observed across different populations, as this would suggest that the overall genetic effect is not heavily influenced by gene-gene or gene-environment interactions. It is obvious that identifying studies performed in all countries is much more important in the second scenario, where the primary aim of the meta-analysis is to assess generalisability of results and therefore the presence of between-study heterogeneity is crucial. In the first situation, where the aim is to increase statistical power, one might be better off by restricting the literature search to facilitate identification of studies performed in similar populations, in order to reduce betweenstudy heterogeneity.

In conclusion, a meta-analysis cannot be conducted as it were a black box, with data from a number of studies being fed into it and a fixed set of rules adopted to process them and provide a pooled estimate.

Performing a meta-analysis requires as much methodological knowledge and experience as any other study design, and the investigator needs to understand the underlying assumptions and implications of the use of alternative methods in order to choose the appropriate one in any given situation. On the other hand, the assessor of the quality of a meta-analysis, be it the reader of a journal or the reviewer and journal editor judging the study's suitability for publication, needs to evaluate critically the appropriateness of the methods chosen in light of the stated aims of the meta-analysis. The availability of checklists for quality assessment would greatly help, but these would still need to be used with judgment.

It is certainly easier to assess the quality of reporting rather than conduct, where a golden rule is to ensure that the methods and results are transparently reported.


Professor Raj ; Bhopal
email: raj.bhopal@ed.ac.uk

Colin ; <none> Fischbacher

Ganka Mueller

Markus ; Steiner

C Fischbacher

C Povey

J Chalmers

H Brown

D H Brewster

Bansal C Addresses

Scottish Health and Ethnicity Linkage Study (SHELS) of 4.65 million people exploring ethnic variations in disease in Scotland: cohort profile

Keywords: ethnicity, cohort, linkage, Ethnic Group, Outcomes

Many countries require health services to show that they are meeting the needs of ethnic minority populations. [START_REF] Bhopal | Ethnicity, race, and health in multicultural societies; foundations for better epidemiology, public health, and health care[END_REF] This requires data on health status, health care uptake and outcomes and population denominators. Weaknesses in routine data collection often make such requirements difficult to meet. Routine data sources in Scotland, as in most countries, may not include a patient's ethnicity. In Scotland, the need for such data is driven by both policy and legislation responding to rapidly increasing ethnic diversity. Fair For All (2003), Scotland's policy, provides a strategic approach to improve the health of minority ethnic groups [START_REF] Executive | Fair for All: Working Together Towards Culturally-Competent Services[END_REF]. The UK Race Relations (Amendment) Act (2000) placed a duty on public bodies to promote racial equality [START_REF]Race Relations (Amendment) Act 2000: New Laws for a Successful Multi-Racial Britain[END_REF]. These mandates are reflected in guidance on ethnic monitoring [START_REF]The Duty to Promote Race Equality: A Guide for Public Authorities in Scotland. 2002. UK, Commission for Racial Equality (CRE)[END_REF][START_REF]Practical guide to ethnic monitoring in the NHS and social care[END_REF][START_REF]Ethnic Monitoring Toolkit & Communication Guideline[END_REF].

Appropriate service and research is undermined by the lack of data. Ethnic variations occur in all Scotland's national health priority areas, including coronary heart disease/stroke (7;8), cancer [START_REF] Bahl | Cancer and ethnic minorities--the Department of Health's perspective[END_REF][START_REF] Bhopal | Cancer in minority ethnic populations: priorities from epidemiological data[END_REF][START_REF] Wild | Mortality from all cancers and lung, colorectal, breast and prostate cancer by country of birth in England and Wales, 2001-2003[END_REF], maternal and child health (12;13) and mental health [START_REF] Sashidharan | Inside Outside: Improving Mental Health Services for Black and Minority Ethnic Communities in England[END_REF].

In view of the mismatch between need for, and availability of, data by ethnic group Bhopal proposed a demonstration project to explore retrospective approaches. [START_REF] Bhopal | Ethnicity and health in Scotland: can we fill the information gap? A demonstration project focusing on coronary heart disease and linkage of census and health records[END_REF] The project tested proposals including name search methods, analyses by country of birth, modelling/extrapolation from other nations' datasets, and linkage methods. The demonstration project concluded that census-health records linkage methods -in the context of this project first mooted by Povey -held most promise. To our knowledge attempting matching of a national health dataset to a complete national census using demographic identifiers rather than national identity numbers had not been reported F o r R e v i e w O n l y 3 though health data linkage is well established in the UK and internationally, [START_REF] Newcombe | Handbook od record linkage: methods for health and statistical studies, administration and business[END_REF] including exploring ethnicity and health. (17;18) Who set it up, how, and why, and how was it funded

The study was set up in collaboration by the University of Edinburgh (Bhopal and Fischbacher), the Information Services Division (ISD) of NHS National Services Scotland, and the General Register Office for Scotland (GROS). ISD maintains hospital episode data (the Scottish Morbidity Record) that has already been linked to GR0S mortality data. A permanently linked database joins together mortality to general hospital discharge records (SMR01), maternity and birth records (SMR02), psychiatric inpatient records (SMR04), and cancer registrations (SMR06). In addition we have accessed child immunisation and health records (SIRS, and CHSP-PS) and Mental Welfare Commission records which are not in the ISD 'linked' dataset. We have linked the census, which contains ethnic codes, to all these data using methods reported in detail elsewhere. (15;19) The Registrar General of Scotland judged that under present legislation, namely the Census Act 1920 and the Census Confidentiality Act 1991, the linkage described here was acceptable if the confidentiality of individual personal information was maintained during linkage and analysis. We used both one-way encryption methods ("hashing") and organisational procedures already published (19) e.g. GROS required that the linkage was performed on a stand-alone computer in a locked 'safe haven' within GROS premises. Modifications were made to the computer operating system to monitor activity and all peripheral devices were disabled. The room at GROS was Date of birth, surname (using soundex codes to allow for variations in spelling), forename, address and full postcode, which were available in both data sets albeit not always recorded identically, were used to link the census number to the CHI. At this stage, other data fields in the two datasets were disconnected from identifying variables. CHI and the census unique number were encrypted prior to linkage. A oneway cryptographic ("hashing") algorithm (currently impossible to reverse) was used to encrypt the CHI number. The census number was encrypted using an algorithm developed by GROS. For the records deemed to be matches, 73.6% were exact matches. For the remainder, a probability matching process was performed. Here, the rate of false positives is critical. Methods have been developed to identify how false positives occur and what kind of strategies a human checker employs to decide whether a pair match is "good". ( 16) These decision strategies were built into a "partitioning" computer algorithm. These "partitions" then allow the allocation of effort to the most profitable "partitions" which yield the lowest false positive rates and highest true positive rates.

Once the linkage was completed personal identifying variables (such as names, address, postcode and dates of birth) were removed leaving a file with an encrypted CHI number and its corresponding encrypted census number (look up file). A census extract containing ethnic code (and limited other data including age, sex and indicators of socio-economic status (see Table 1) and an encrypted census number was joined to the above look up file using the encrypted census number. The encrypted census numbers were then discarded leaving the ethnicity code, some other variables from the census and the encrypted CHI number. The relevant parts of the ISD linked database were linked via the encrypted CHI numbers. The encrypted CHI was replaced with an unrelated serial number (to keep together the multiple records on the same people, known as hospital admission spells), resulting in depersonalised clinical health records carrying census ethnicity codes. Using methods previously described we estimated an upper limit to the false positive linkage rate of 0.08 %.( 19)

F o r R e v i e w O n l y

What does it cover?

For phase 1 (demonstration project) we studied myocardial infarction, since it is one of the most important conditions in Scotland, in terms of both mortality and morbidity, is common in ethnic minority groups, and likely to be recorded in hospital morbidity records. (19;21) For phase 2 we are studying cardiovascular disease, maternal and child health, cancer and mental health -see Table 2. We are currently planning phase 3, studying a wider range of conditions and linkage to primary care records. In theory we can study long-term outcomes for any health problem coded in NHS records (and in the records of any other organisations that are able and willing to provide data for linkage). In practice, our work is heavily governed and controlled by data privacy and other ethical considerations, and practical matters of resource, expertise and interest.

Who is in the sample?

The objective was to link all 5 million or so people resident in Scotland in April 2001, the time of the census, but we set an arbitrary standard of 80% or more in each ethnic group, on the basis of a judgement that potential biases would be too great for linkages less than that. About 94% overall were linked in the phase 1 demonstration project using the methods described below, and 95% in phase 2, with the minimum standard of 80% linkage met for every ethnic group (see Table 3 The cohort focuses on ethnicity (as reported on the census form completed by the head of household) as the primary exposure variable. Other census information is included, and is listed in table 1. Religion (both current and that of upbringing) and country of birth is added to the study database and used to provide a more comprehensive assessment of the relationship between an individual's health and their ethnic group. Socio-economic status is an important co-variable that could potentially explain some aspects of ethnic variation. Accordingly, a careful analysis of the influence of socio-economic status is being built into the analysis. Census variables relating to employment and educational attainment, both of the individual and the head of household, together with area-based socio-economic indicators, are used to build up a comprehensive picture of socio-economic status. The Scottish Index of Multiple Deprivation (SIMD), based on area of residence, and included in many Scottish health databases, is used. This will enable the complex relationships between health, ethnicity and socio-economic status to be elucidated. In addition, data on selfreported long-standing illness are available from the census. We also have travel times from home to the nearest hospital.

The outcomes currently under study are given in table 2. The number of events varies, obviously, by ethnic group, disease outcome, and number of years of follow-up.

Scotland's non-White ethnic minority population is small at about 2%. Table 3 gives the number of people linked by ethnic group. For White minority populations e.g. other British and Irish the population size and hence number of events is large. In phase 2, we are analysing seven years of follow-up for most events. In phase 3,

F o r R e v i e w O n l y 8
currently planned, we anticipate nine years of follow-up. For most analysis, given the degree of ethnic inequality, our analyses are statistically robust. For many endpoints, ethnic inequalities are large, with two-fold plus risks being common. Differences of interest (e.g. a 50% difference) can be detected for most end-points. For example, asthma is a relatively important but rare cause of hospitalisation and Indians comprised 0.3% of the Scottish population in 2001 (compared with Pakistanis who were 1%). There are more than 3500 hospital discharges and deaths for asthma per year. With 8 years of outcomes we accrue about 28,000 admissions. Assuming 2% of them are in non-White populations, and that is conservative given previous studies showing a relative excess of hospitalisation in minority populations,( 22) that makes 560. If 0.3% of the total hospitalisation/deaths are in Indians, that is about 84 admissions. With 84 expected events, we can detect a difference of 44% in the rate, with about 80% power and 0.05 statistical significance. In phase 2, we have already demonstrated that robust statistical analyses is possible within this data set for many of the cardiovascular and maternal/child health outcomes in the table (analyses on mental health and cancer are underway but we cannot comment at this point).

What is attrition like?

The 5% of the Scottish population that is not linked is being characterised to assess potential biases by comparing with the 95% that was linked. It is not possible to compare with the additional 4% of the Scottish population that was not enumerated at the 2001 census. People who migrate, or are hospitalised or die, outside Scotland are poorly characterised in our databases and, to date, we have not developed a sound strategy for estimating the potential biases. We particularly welcome collaborations on this challenge. among non-South Asians. The incidence rate ratio was 1.45 (95% CI 1.17, 1.78) for South Asian compared to non-South Asian men and 1.80 (95% CI 1.31, 2.48) for South Asian women. After adjustment for age, sex and any previous admission for diabetes the hazard ratio for death following AMI was 0.59 (95% CI 0.43, 0.81), reflecting better survival among South Asians [START_REF] Fischbacher | Record linked retrospective cohort study of 4.6 million people exploring ethnic variations in disease: myocardial infarction in South Asians[END_REF]. Phase 2 analyses of cardiovascular diseases are underway and will be submitted for publication shortly. They seem to both corroborate and extend phase 1 findings e.g. confirming increased incidence rather than increased case fatality in explaining the high CHD mortality rate in the South Asian population and showing heterogeneity between Indians and Pakistanis.

For example, directly standardised rate ratios for first MI (in the period May 2001 to April 2008) in males were highest for Pakistanis, followed by Other South Asians, and Indians. For females, rates were higher for Other South Asians, followed by Pakistani and Indian. Pakistani males had significantly higher rate ratios of first MI than their White Scottish counterparts. After adjustment for diabetes and the Scottish index of social and economic deprivation (SIMD), Pakistani males and females had significantly better survival 28 days after first MI than the White Scottish population.

Analyses relating to phase 2 goals on cardiovascular disease, cancer, maternal and child health and mental health will be reported in 2010. We had to balance individuals' right to data privacy (individual consent was impossible) and the potential benefits to society of producing information derived from potentially sensitive data. [START_REF] Boyd | Ethnicity and the ethics of data linkage[END_REF] We and a professional ethicist [START_REF] Boyd | Ethnicity and the ethics of data linkage[END_REF] judge that our methods strike the balance. The proportion of records successfully linked was only slightly lower than that normally achieved within ISD in internal linkages (typically around 98%), even although we linked data held by two separate agencies. As the non-White ethnic minority population (about 2%) is comparatively small the false positive rate is critical. A rigorous matching methodology appropriate for administrative matching was, therefore, used.

Census forms are completed by the public and processed electronically using optical recognition and keying from images. The success of these processes depends on legibility. The detailed spelling of a name, particularly if it is associated with a minority ethnic group, is more likely to be prone to error when transcribed by a third party onto NHS records, than in the census where the census informant writes it. Such errors could lead to varying accuracy of linkage by ethnic group. Nevertheless, we met our prior stated standard of 80% for every ethnic group, lower in non-White than White populations. People who are not matched are not included in our analyses. At present we do not know about the characteristics of those matched compared with those not matched, but work on this is underway.

The major strength of this analysis is the population coverage with the inclusion of community and hospital deaths as the SMR database contains generally validated and ISD, personal communication, 2006). Possible, as opposed to probable or definite, AMIs are coded as chest pain and are excluded fromAMI figures, so the AMI figures could be underestimates. The quality of data on AMI in the database has been validated as reliable [START_REF] Fischbacher | Record linked retrospective cohort study of 4.6 million people exploring ethnic variations in disease: myocardial infarction in South Asians[END_REF]. Information about the quality of SMR01, SMR04 and SMR06 is available.

Validation has not, however, been done by ethnic group. For example, the diagnosis of AMI was based on criteria used by clinicians to make the diagnosis, which would be inconsistent over time. This should not, however, affect ethnic groups differently.

If misclassification of diagnosis is non-differential then the differences would probably be underestimated, but if they were differential, differences would be exaggerated. We do not have data to assess these options, and the anonymised methods precluded validity studies. We do know that South Asian populations in the UK, and clinicians looking after them, are sensitised to the high rate of CHD, and early admission and treatment, (23;24) concordant with excellent survival,( 19) is likely.

Linkage rates were slightly lower for South Asians than non-South Asians. If nonlinkage occurred at random, this would reduce the power of the study, but not bias the results. Factors reducing the power of the study are particularly important for small ethnic minority populations and rare outcomes. If those not matched were at different risk of hospitalisation or death from those who were matched, which seems quite likely, this would bias the results. We intend to explore this in future research. Quantifying the potential impact of deaths abroad, to account for the possibility that minority ethnic groups may be more likely to move abroad when they are elderly or ill with chronic conditions (the so-called "salmon bias") is proving problematic. This bias would improve apparent survival. Such an effect is unlikely to be large, as reflected in mortality by country of birth analyses in England and Wales showing that mortality is not uniformly low in most ethnic minority groups, but we will scrutinise our results to detect such bias (e.g. as reflected in relatively good long-term compared to short-term survival).

The statistical power is governed by the size of the population and event rate which determine number of cases, and the size of the differences between comparison groups. At the 2001 Census about 2% of the Scottish population was not White (about 100,000 people) and most of them were South Asian (Pakistani and Indian) or Chinese. These groups will be our main foci for analysis, although where there is sufficient statistical power for other ethnic groups we will examine them e.g. of African ethnicity. We are also paying attention to White minorities in Scotland e.g. the other UK (mainly English and Irish) populations. In our phase 2 analysis and interpretation we are examining heterogeneity of broadly defined ethnic groups e.g. South Asian. For these non-White ethnic groups we are finding major differences in event rates in relation to comparison populations. One limitation is that we are unable Use of the data files requires the use of specialist software and the expertise of research team members, not least in obtaining necessary approvals. Collaboration will be pursued if the prime objectives of the project are not impaired or delayed in the four areas under study in phase 2. Collaborators would be primarily responsible for funding the additional research, including the cost of extra work for the core research team. We are committed to seek ways of increasing access to the database.

Conclusion

Our approach, potentially, has international applicability. It demonstrates how the glaring absence of cohort studies reporting by ethnic group in Europe can be overcome. [START_REF] Ranganathan | Exclusion and Inclusion of Nonwhite Ethnic Minority Groups in 72 North American and European Cardiovascular Cohort Studies[END_REF] There is considerable potential in linking databases that have previously been considered too sensitive for record linkage or where linkage is restricted by data protection legislation. The methods described here and in more detail in our report and publication (19;21) have the potential to fill the information gap on data by ethnic group. This gap will persist until we have high quality prospective ethnic group coding systems in health-care databases and the inclusion of a valid ethnic code on birth and death registration -both formidable long-term challenges, hitherto unachieved in either Europe or North America [START_REF] Bhopal | Ethnicity, race, and health in multicultural societies; foundations for better epidemiology, public health, and health care[END_REF]. It is noteworthy, however, that prospective ethnic coding will not provide the detailed socio-demographic data available in the census, so will not replace our method.

  agreement of GROS, ISD and the PI(Bhopal). GROS maintains a register of visits. We obtained approval from the Scottish Multi-centre Research Ethics Committee (MREC) and the Privacy Advisory Committee (PAC) which advises ISD and GROS on the use of data. This work was supported by the Commission for Racial Equality, NHS Health Scotland (through The National Resource Centre for Ethnic Minority Health), ISD, the Scottish Executive Health Department and the Chief Medical Officer. An ethical opinion, subsequently published, was given by a Professor of Medical Ethics.(20) For funding see acknowledgements. The figure, republished from open access reference number 19, (19) illustrates in concept how record linkage was based on the use of subsets of information from three datasets: health care records, which include personal identifiers and clinical information; the Community Health Index (CHI) dataset which contains personal identifiers and the CHI number; and the census file which contains personal identifiers and details of individuals' ethnicity. The CHI dataset lists everyone registered with a General Practitioner or eligible for NHS screening services and forms a unique identifier for NHS use. More than 99% of the Scottish population is listed on the CHI.

  ), thus creating a retrospective cohort study of about 4.65 million people. Taking into account the census enumeration rate (96%), this represents about 91% of the population resident in Scotland in April 2001.

  How often have they been followed up and what has been measured? How many events related to the health outcomes are there?

  What has it found? Key findings and publicationsOverall, 94% in phase 1 and 95% in phase 2 of the 4.9 million census records were matched to a CHI record with an estimated false positive rate of less than 0.1 %.Phase 1 data showed that between April 2001 and December 2003 there were 126 first episodes of acute myocardial infarction (AMI) among South Asians and 30,978

  e.g. over 99% of hospital admissions for AMI in Scotland (A Redpath,

  bias in the analyses have not yet been assessed systematically. A separate data linkage evaluation exercise is planned in collaboration with NHS Central Register to provide alternative linkage outcomes for a small sample of records that can help assess the accuracy of the main data linkage project. A comparison of census characteristics of matched and non-matched individuals is also planned.

14 •

 14 Europeans and any other ethnic groups which were not separately identified in 2001. (This kind of work may be a focus for future work based on census 2011.) Power calculations (80% power at the 5% level of significance) have shown that differences of a size that is common and of interest (e.g. a 50% increase) can be detected for most end-points, for the three largest non-White minority populations, namely Pakistanis, Indians and Chinese. Our calculations assume 5 years of follow-up data but for most outcomes we have 7-years, and this will increase in future linkages. Can I get hold of the data? Where can I find out more? This project has created a retrospective cohort of about 4.6 million people living in Scotland at the time of the 2001 Census. Phase 2 work is focusing on cardiovascular disease, cancers, mental health and maternal and child health. Subject to ethical approval, future work will have potential to examine health care procedures and adjust for other variables that available in the census. Research using the datasets requires approval from GROS, ISD, a multicentre research ethics committee, Privacy Advisory Committee and the Directors of Public Health. Approvals have been given only to examine ethnic variations in health and healthcare.The researchers appreciate the benefits of collaboration and sharing of data, and are in principle open to proposals for relevant collaborative analysis of datasets. There are two products of this data linkage project that may be of value to other researchers:• The CHI-census linkage file, containing the encrypted census identifier numbers, and encrypted CHI numbers (dataset #1) and The matched, anonymised dataset containing census variables and health records (dataset #2).

  made many contributions to the linkage component, and Duncan Macniven of GROS gave helpful advice, particularly on dissemination of the findings. Professor Phil Hanlon acted enthusiastically and promptly to help make this study possible when it was first mooted in 2002. Dr Rafik Gardee ensured the study received the full backing of the National Resource Centre for Ethnic Minority Health. Mr Hector Mackenzie of the Scottish Executive, Dr Mac Armstrong (CMO), and Chris Oswald (CRE) also played influential roles in maintaining the directions and funding of the work. Kate Macintyre advised on analytic methods and interpretation of data, using experience in the SLIDE project, under the direction of Simon Capewell. Finally, we thank our employing organisations for their support of our work in this project. Phase 1 was funded under a special grant from the Scottish Executive and phase 2 with a project research grant (CZH/4/432). The researchers acted independently of the funding body at all stages of the work.The Corresponding Author has the right to grant an exclusive licence for publication.

  Figure 1Overview of Record Linkage Process

Table )

 )

		Personal Identifiers
	CHI Number
	Health Information	Ethnicity Information
	Health Database	Census Database
	CHI Number Personal Identifiers Record Linkage Census Number Personal Identifiers CHI Number F o r	Census Number
	(Look-up Table)
	Health Information R e Ethnicity Information v
		i e
		w
		O n l
			y
		20

Table 1 Baseline 2001 variables extracted from census: Phase 2

 1

	Ethnic group
	Religion, current
	Religion of upbringing
	Country of birth
	Age
	Sex
	Long term illness
	Self assessed health
	Marital status
	Labour force status
	Socioeconomic status
	Highest qualification
	Scottish Index of Multiple Deprivation decile
	Car ownership
	Housing tenure
	Household size
	Number of rooms
	Urban/rural indicator
	Health board (Glasgow, Lothian, Tayside, Other)
	Mobile (temporary) accommodation
	Self-contained accommodation
	Central heating
	Moved within last year
	Activity last week

Table 3

 3 Linkage Rates by Ethnic group

	Number	Percentage

Acknowledgements, Copyright and Funding

We thank the Scottish Executive for supporting the phase 1 study and the organisation that assisted in managing the grant and the work -the Public Health Institute for Scotland (now part of NHS Health Scotland). Numerous people offered their expertise, guidance and support (too many to list comprehensively). David Orr of GROS was intimately involved in the phase 1 study from an early stage and served the Steering Committee of the project until 2004 when his place was taken by Peter Scrimgeour, Kirsty McLachlan and Ganka Mueller. Joe Fuchs, Ian Maté and Susan

Authors' Contributions

RB was the originator of the project and the principal applicant for the phase 1 grant with JC and CF as co-applicants. RB and CF are joint PI's for phase 2. All authors are coinvestigators or collaborators for either phase 1 or 2, except NB who is the research fellow in phase 2. CP had the idea of linking the census data to the data held by ISD and he performed most of the linkage work in phase 1 and phase 2 including the development of the linkage methods. JC had the original plan for the use of oneway encryption. GM was the main contributor from GROS, making both practical and intellectual contributions. Helen Brown (HB) was the statistical research associate on phase 2 of the project from August 2007-December 2009. HB, NB, CF, RB, and MS (MS for phase 1) led the analytic plan and CF, HB, NB and MS did the analysis. RB and CF are guarantors for this paper. All authors contributed to the design of the study, and contributed to, read and approved the final version of this manuscript which was prepared by RB.

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