M.D.; Birgit Linnemann
email: birgit.linnemann@kgu.de

Jan Schwonberg

Ph.D.; Andreas R Rechner

Ph.D.; Helen Mani

M.D. Edelgard Lindhoff-Last

M.D J W Goethe

Assessment of clopidogrel non-response by the PFA-100 ® system using the new test cartridge INNOVANCE® PFA P2Y* *product under development

Keywords: clopidogrel, antiplatelet therapy, non-response, PFA-100® System, INNOVANCE® PFA P2Y clopidogrel, antiplatelet therapy, non-response, PFA-100® System, INNOVANCE® PFA P2Y

Until now, the PFA-100® system has been considered unsuitable for monitoring clopidogrel efficacy. The authors evaluated platelet function in PAOD (peripheral arterial occlusive disease) patients using a new PFA-100® test cartridge (product name: INNOVANCE® PFA P2Y*) specifically designed for this purpose. Twenty-two stable PAOD patients on antithrombotic therapy with clopidogrel alone (n = 22) and 18 patients undergoing a peripheral catheter intervention, preliminarily treated with 100 mg/d aspirin followed by co-administration of clopidogrel (loading dose 300 mg, maintenance dose 75/d), were enrolled in this study. Defining non-responsiveness to clopidogrel as an aggregation response within the reference range (90% central interval), four (18.2%) non-responders using LTA induced by 5 µM ADP and six (27.3%) non-responders using LTA induced by 2 µM ADP (LateAggr >72.1% and >42.9%, respectively) were identified. INNOVANCE® PFA P2Y* determined six (27.3%) non-responders (CT<87s). Agreement between the two aggregometry assays and INNOVANCE® PFA P2Y* on the definition of clopidogrel response and non-response exceeded 70%. Only three patients were uniformly identified as clopidogrel non-responders by all three assays. When clopidogrel was co-administered with aspirin, two (11.1%) non-responders to clopidogrel were detected with INNOVANCE® PFA P2Y*, whereas ADP-induced LTA found all patients to be responsive. INNOVANCE® PFA P2Y* appears to be suitable for monitoring the effect of clopidogrel on platelet function. Its sensitivity in detecting responsiveness or non-responsiveness to clopidogrel is comparable to ADP-induced LTA. Additional prospective studies are needed to clarify the clinical relevance of the test results and classification obtained with INNOVANCE® PFA P2Y*.

Yours sincerely

Dr. Birgit Linnemann, M.D.

Ad reviewer 1: Ad 1: The reasons why we used lower ADP concentration in LTA was added. In a previous work we have shown that late aggregation using 2 μM ADP in non-adjusted PRP provides a good discrimination between clopidogrel treated patients and healthy subjects. However, we were also able to show that in platelet count-adjusted PRP, higher concentrations were necessary to differentiate between patients on clopidogrel treatment and subjects without clopidogrel medication. See reference 22.

Ad 2: We agree that the figures 1-3 are quite similar. So we decided to remove fig. 1 and3 and to keep fig. 2. Ad 3: We discussed the variability of test results and CVs more in detail and added duplicate measurements of patients on dual antiplatelet therapy in figure 2 (formerly 4). Changes in the text are highlighted in blue.

Introduction

Light transmittance aggregometry (LTA) was invented by Born in 1962 and is still regarded as the method of choice for the assessment of platelet function and its pharmacologic inhibition [START_REF] Born | Aggregation of blood platelets by adenosine diphosphate and its reversal[END_REF][START_REF] Harrison | Measuring antiplatelet drug effects in the laboratory[END_REF]. In LTA, the increase in light transmission through platelet-rich plasma following the addition of a platelet agonist is measured. Measurement occurs when platelets become activated by the agonist and form aggregates. However, LTA is time-consuming, not standardized, and its use is limited to specialized laboratories. A number of different agonists are used for LTA, including arachidonic acid (ARA), epinephrine, adenosine diphosphate (ADP), collagen, thrombin, thrombin receptor activating peptide (TRAP), phorbol myristate acetate (PMA), and ristocetin, and different optical aggregometers are available. However, the concentrations of agonists used for testing vary widely, and accepted guidelines on how laboratories should perform LTA are lacking [START_REF] Harrison | Measuring antiplatelet drug effects in the laboratory[END_REF][START_REF] Moffat | Variability in clinical laboratory practice in testing for disorders of platelet function. Results of two surveys of the North American Specialized Coagulation Laboratory Asscociation[END_REF].

There is a growing body of evidence that residual platelet function, despite antithrombotic therapy, identifies patients at risk of future cardiovascular events [START_REF] Snoep | Clopidogrel nonresponsiveness in patients undergoing percutaneous coronary intervention with stenting: a systemic review and metaanalysis[END_REF][START_REF] Matetzky | Clopidogrel resistance is associated with increased risk of recurrent atherothrombotic events in patients with acute myocardial infarction[END_REF][START_REF] Hochholzer | Impact of the degree of peri-interventional platelet inhibition after loading with clopidogrel on early clinical outcome of elective coronary stent placement[END_REF][START_REF] Buonamici | Impact of platelet reactivity after clopidogrel administration on drug-eluting stent thrombosis[END_REF][START_REF] Frere | ADP-induced platelet aggregation and platelet reactivity index VASP are good predictive markers for clinical outcomes in non-ST elevation acute coronary syndrome[END_REF][START_REF] Marcucci | Residual platelet reactivity is an independent predictor of myocardial injury in acute myocardial infarction patients on antiaggregant therapy[END_REF][START_REF] Snoep | Association of laboratory-defined aspirin resistance with a higher risk of recurrent cardiovascular events[END_REF][START_REF] Krasopoulos | Aspirin "resistance" and risk of cardiovascular morbidity: systemic review and meta-analysis[END_REF]. Therefore, interest in whole blood point-of-care methods has increased; these techniques offer the possibility of rapid and reliable identification of patients with antiplatelet "drug resistance" and tests may be routinely performed. Thus, the PFA-100® System, originally introduced as an aid to test for defects of primary hemostasis [START_REF] Hayward | Platelet function analzyer (PFA)-100 closure time in the evaluation of platelet disorders and platelet function[END_REF][START_REF] Jilma | Platelet function analyzer (PFA-100®): a tool to quantify congenital or acquired platelet dysfunction[END_REF], has become one of the most widely-employed tests to assess aspirin efficacy. The PFA-100® System is a global test of high-shear-dependent platelet function. In disposable test cartridges, anticoagulated whole blood is aspirated from the sample reservoir through a capillary and an aperture in a platelet agonist coated membrane, thereby exposing platelets to high shear flow conditions and the platelet agonists of the membrane coating. Two test cartridges are commercially available with a membrane coated either with collagen and epinephrine or collagen and ADP. The closure time depends on different variables, such as von Willebrand factor levels, platelet count, or haematocrit and is usually affected by aspirin intake when using the Collagen/EPI Test Cartridge (CEPI) [START_REF] Hayward | Platelet function analzyer (PFA)-100 closure time in the evaluation of platelet disorders and platelet function[END_REF][START_REF] Jilma | Platelet function analyzer (PFA-100®): a tool to quantify congenital or acquired platelet dysfunction[END_REF].

Theoretically, the Dade® PFA Collagen/ADP Test Cartridge (CADP) should be suitable for the assessment of P2Y12-receptor inhibition. However, activation of the platelets by collagen and the residual activation potential of ADP via the P2Y1-receptor seem to be sufficient to overcome the effect of P2Y12-receptor blockade on platelet function [START_REF] Gachet | Regulation of platelet functions by P2 receptors[END_REF]. In earlier studies, the CADP cartridge was indeed shown to be unsuitable for the detection of the efficacy of clopidogrel therapy [START_REF] Golanski | Limited usefulness of the PFA-100 for the monitoring of ADP receptor antagonistsin vitro experience[END_REF][START_REF] Mueller | Monitoring aspirin 100 mg and clopidogrel 75 mg therapy with the PFA-100 device in patients with peripheral arterial occlusive disease[END_REF][START_REF] Paniccia | Different methodologies for evaluating the effect of clopidogrel on platelet function in high-risk coronary artery disease patients[END_REF][START_REF] Geiger | Monitoring of clopidogrel action: comparison of methods[END_REF][START_REF] Mani | Response to aspirin and clopidogrel monitored with different platelet function methods[END_REF]. A recent in vitro study by Pidcock et al. provided evidence that the P2Y1-receptor is an important contributor to platelet functionality as measured by CADP, despite an inhibition of the P2Y12-receptor [START_REF] Pidcock | Can the PFA-100 be modified to detect P2Y12 inhibition?[END_REF]. The authors showed that a secondary addition of MRS 2179, a P2Y1-receptor antagonist, to a blood sample, already treated with the P2Y12-receptor antagonist cangrelor without effect, resulted in significant prolongation of the CADP CT. They concluded that this method might facilitate the assessment of clopidogrel treatment and related P2Y12-receptor antagonists using the CADP cartridge.

In the present study, we aimed to evaluate a new, but not yet commercially available PFA-100® test cartridge, product name INNOVANCE® PFA P2Y*, which was designed for the assessment of P2Y12 inhibitory effects of clopidogrel, either in monotherapy or in combination with aspirin.

Materials and methods

Study population

Twenty-two subjects (11 males, 11 females, aged 55 to 86 years) with peripheral arterial occlusive disease (PAOD), treated with 75 mg/d clopidogrel as the only antithrombotic drug were enrolled in the study (Group 1). PAOD was defined as an ankle brachial index ≤0.9 as measured with Doppler ultrasound [START_REF] Norgren | Inter-Society Consensus for the Management of Peripheral Arterial Disease (TASC II)[END_REF]. Atherosclerotic disease was required to be stable (i.e., without clinical deterioration within the last 3 months) and patients had to have been treated with clopidogrel for at least 14 days.

We additionally investigated 18 PAOD patients (11 males, 7 females, aged 43 to 84 years) undergoing a peripheral catheter intervention procedure due to intermittent claudication (Group 2). At baseline, these patients were treated with 100 mg/d aspirin. On the day of intervention, a loading dose of 300 mg clopidogrel was administered followed by a maintenance dose of 75 mg/d for at least 4 weeks, while the aspirin treatment was continued. Patients with additional medication known to influence platelet function (e.g., non-steroidal anti-inflammatory drugs (NSAIDs)) were excluded, as well as patients with known coagulation disorders, liver cirrhosis, alcohol abuse, end-stage renal failure, or malignant disease. A platelet count < 100/nl, hemoglobin < 9 g/dl and a haematocrit < 28% were additional exclusion criteria. The study was approved by the local Ethics Committee, and all patients provided written informed consent prior to participation. Using a standardized questionnaire, clinical data detailing vascular and concomitant disease, cardiovascular risk factors, and co-medication were recorded. Compliance with the antithrombotic therapy was determined through patient interviews both at study entry and at follow-up. All patients confirmed that they had taken their medication as directed.

The last dose of antithrombotic medication was administered one to 24 hours before blood sampling.

Blood sampling

Blood sampling was performed once in patients under chronic clopidogrel treatment. In patients undergoing peripheral catheter intervention, blood sampling was performed at baseline when treated with aspirin only and three weeks after intervention when patients were on stable combination therapy of aspirin and clopidogrel. Blood was drawn from an antecubital vein using a 21-gauge butterfly cannula system (Multifly®-Set, 21 G x 1½ TW, 0.8 x 19 mm, Sarstedt, Nümbrecht, Germany). EDTA and citrate (0.129 M (3.8%) and 0.106 M (3.2%) trisodium citrate)-supplemented blood was collected with plastic syringes (Monovette®, Sarstedt, Nümbrecht, Germany). Platelet count was measured on the Sysmex® KX-21 (Roche Diagnostics, Basel, Switzerland), an automatic multi-parameter blood cell counter. Platelet counts between 100 and 500/nl were required for subsequent platelet function testing. Platelet-rich plasma (PRP) was obtained by centrifuging 0.106 M (3.2%) citrated whole blood at room temperature at 140 x g for 5 min. Platelet-poor plasma (PPP, platelet count < 10/nl) was derived from citrated whole blood centrifuged more vigorously at 1,500 x g for 15 min. The time interval between blood sampling and testing was at least 1 h and did not exceed 3 h. All platelet function tests were performed in duplicate using blood or plasma from the same tube.

PFA-100® System

The PFA-100® System (Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany) is a system for assessment of high shear stress-dependent platelet function by a procedure simulating the complex process of primary haemostasis in vitro. The device aspirates 800 µl of whole blood, anticoagulated with 0.129 M (3.8%) trisodium citrate, at high shear rates (5,000-6,000 s The cut-off was statistically derived from the 95 th percentile of the quadruple measurements in blood buffered with 3.8% sodium citrate.

Light transmittance aggregometry (LTA)

The LTA procedure was performed on the BCT* System (Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany). The BCT* System is a fully automatic machine used for routine and special coagulation testing. The BCT* System detects platelet aggregate formation in PRP by changes in light transmission (monochromatic light; wavelength: 620 nm) at 37°C. Platelet agonists (15 µl reagent) were automatically added to PRP (135 µl plasma) and stirred at a velocity of 600 rpm. In this study, adenosine 5-diphosphate (ADP; AppliChem, Darmstadt, Germany), at a final concentration of 2 and 5 µM, was used to induce platelet aggregation. The aggregation response following the addition of the agonist was monitored via the change in light transmission over time relative to PPP derived from the same blood sample. Measurements were performed in native PRP unadjusted for platelet count.

As disaggregation is a frequent phenomenon in clopidogrel-treated patients, we recorded the late aggregation (LateAgg) response after 10 min. Light transmission through PRP relative to PPP was measured prior to the addition of the agonist and at the end of the measurement (10 min). LateAgg was calculated using the formula:

(Aggregation at start -Aggregation at end) LateAgg [%] = 100% * (Aggregation at start -Platelet-Poor Plasma)

In a previous investigation, we demonstrated that LateAgg induced by 2 and 5 µM ADP in nonadjusted PRP offered an accurate index for distinguishing between patients on clopidogrel therapy and healthy volunteers not taking any antithrombotic drug [START_REF] Linnemann | Standardization of light transmittance aggregometry for monitoring antiplatelet therapy: an adjustment for platelet count is not necessary[END_REF]. We determined the within-day precision of LTA by calculating the coefficient of variation (CV) of the aggregation response in blood samples collected from 5 healthy volunteers on 5 consecutive days. The CV, using ADP 2 and 5 µM as agonist in non-adjusted PRP, was 2.72% and 1.40%, respectively. In accordance with the latest recommendations provided at the 53 rd

Fibrinogen and von-Willebrand factor

Fibrinogen was assayed by the Clauss method on the BCT® System, using the Multifibren U reagent (Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany). Von Willebrand Factor (vWF)

was also assayed on the BCT® System, using vWF:Ag medium for determination of vWF antigen and BC von Willebrand Factor Low setting for the measurement of vWF ristocetin cofactor (Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany).

Statistical analysis

Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS version 15.0, Chicago, IL, USA). Aside from descriptive statistics including frequencies, mean and standard deviation, median and range, we calculated the coefficient of variation using the formula CV = standard deviation/mean and performed the Mann-Whitney U-Test for comparison of metric variables.

Cohen's kappa coefficient was calculated as a measure of agreement between LTA and PFA-100® System results. The criterion for statistical significance was a p-value less than 0.05. Results are also presented as box plots with the bar length indicating the interquartile range (25 th -75 th percentile).

Outliers were defined as values differing 1.5-3.0 bar lengths, whereas extreme values are those differing > 3.0 bar lengths from the upper or lower edge of the box. In the figures, outliers are represented as circles and extreme values as stars.

Results

PAOD patients receiving 75 mg clopidogrel daily, either in monotherapy (group 1; n = 22) or in combination with aspirin (group 2; n = 18), were enrolled in this study. Baseline characteristics of the patients are listed in Table 1.

Von Willebrand Factor and fibrinogen

Irrespective of the assay used, no statistically significant differences between defined non-responders and responders to clopidogrel were observed regarding vWF antigen, vWF ristocetin cofactor activity or fibrinogen levels of the blood (Table 2).

Coefficients of variation of duplicates

Discussion

The results of the present study indicate that INNOVANCE® PFA P2Y* for the PFA-100® system is suitable for assessing clopidogrel therapy. The sensitivity of detecting clopidogrel non-responders was comparable to that of ADP-induced LTA.

Following absorption, the thienopyridine clopidogrel is metabolized by the hepatic cytochrome P450 system. Its active metabolite irreversibly binds to and blocks the P2Y12-receptor, one of the two ADPreceptors of platelets. Consequently, laboratory response to clopidogrel is generally determined by measuring the extent of inhibition of ADP-induced platelet aggregation. ADP-induced LTA is currently considered to be the standard platelet function test, but it cannot be practically applied to large numbers of clinical samples due to the labour-intensive sample preparation. A simple, reliable whole blood test system able to assess the antiplatelet effect of clopidogrel administration would be of great value in clinical practice. In addition, the contribution of the P2Y1-receptor to residual platelet function on therapy with P2Y12-receptor inhibitors varies widely. This variable contribution may account for the heterogeneity of the LTA results and indicates that ADP alone may not be appropriate to specifically measure the effect of clopidogrel and other P2Y12-receptor antagonists.

Using INNOVANCE® PFA P2Y*, 16/22 patients with clopidogrel monotherapy (73%) and 16/18 patients on dual therapy of clopidogrel and aspirin (89%) presented with a CT prolongation (> 87 s).

According to the working definition in this study of non-responsiveness to clopidogrel therapy (i.e., values within the reference range), non-responsiveness was observed in 27% of the patients on monotherapy and in 11% of the patients on dual therapy. The corresponding results of LTA performed with 2 or 5 µM ADP were 18% (4/22) and 27% (6/22), respectively, for clopidogrel monotherapy patients, whereas all patients on dual therapy showed full responsiveness to the therapy according to LTA. The most likely reason for this result might be the impact of aspirin on ADP-induced LTA, which is supported by the altered LTA result obtained in patients on aspirin monotherapy, when compared to control values.

Interestingly, the various assays differed in their classification of patients as clopidogrel nonresponders. Only three patients were concordantly identified as non-responders to clopidogrel by INNOVANCE® PFA P2Y* and LTA induced by 2 or 5 µM ADP. To date, most studies utilize higher ADP concentrations (i.e., 5 and 20 μM), and many laboratories still perform platelet count adjustments.

However, in a previous investigation, we demonstrated that late aggregation using 2 μM ADP in nonadjusted PRP provides a good discrimination between clopidogrel treated patients and healthy subjects [START_REF] Linnemann | Standardization of light transmittance aggregometry for monitoring antiplatelet therapy: an adjustment for platelet count is not necessary[END_REF]. A discrepancy between test methods has been observed before when response to aspirin was evaluated by different platelet function tests. Comparing arachidonic acid-induced LTA and the CEPI-CT on the PFA-100® System, Gum et al. observed that the agreement of tests was poor [START_REF] Gum | Profile and prevalence of aspirin resistance in patients with cardiovascular disease[END_REF]. The difference between methods has been attributed to the fact that the two methods measure different aspects of platelet function. First, LTA is performed in platelet-rich plasma, whereas the PFA-100® system utilizes whole blood samples. Secondly, shear stress is low in the context of LTA, whereas the PFA-100® System is a flow-based system with high shear stress. Both factors are known to influence platelet function. It has, for example, been shown that due to the shear forces during measurement, closure time is strongly dependent on the amount of von Willebrand-factor (vWF) [START_REF] Jilma | Platelet function analyzer (PFA-100®): a tool to quantify congenital or acquired platelet dysfunction[END_REF].

High plasma levels of vWF as well as high vWF-ristocetin cofactor activity have been related to shorter CT values for the two commercially available PFA-100® cartridges [START_REF] Lip | Von Willebrand factor: a marker of endothelial dysfunction in vascular disorders?[END_REF][START_REF] Chakroun | In vitro aspirin resistance detected by PFA-100 closure time: pivotal role of plasma von Willebrand factor[END_REF][START_REF] Fuchs | Platelet function in patients with acute coronary syndrome (ACS) predicts recurrent ACS[END_REF]. However, the analysis of vWF-ristocetin cofactor activity, vWF-antigen, and fibrinogen levels in the present investigation did not reveal a statistically significant difference between non-responders and responders to clopidogrel.

Thus, the disagreement of the test results in our patients cannot be attributed to differences in these parameters.

In patients on clopidogrel monotherapy, the mean CV of duplicate measurements with INNOVANCE® PFA P2Y* was calculated to be 11.3% compared to 8.4% and 9.7% with LTA induced by 2 and 5 µM, respectively. Whereas about 50% of patients on clopidogrel consistently showed non-closure with INNOVANCE® PFA P2Y*, there was a substantial number of patients (6/22) with highly varying CT results of duplicate measurement who achieved a > 30% CT variation. In two patients from this subgroup (9%), the variation of the individual results would result in different classifications with respect to responsiveness or non-responsiveness. An even higher variance of test results, i.e., a CV of 32%, has recently been reported by Madsen et al. [START_REF] Madsen | Testing aspirin resistance using the platelet function analyzer-100: some methodological caveats and considerations[END_REF], who performed duplicate measurements of CEPI-CT in patients treated with aspirin. In addition, the authors performed platelet function tests at baseline and after three months, and found that, using a cut-off value of 170 s, about 30% were In studies performed on normal subjects not receiving any medication known to affect platelet function, CVs of duplicates of 9 to 13% have been reported for the commercially available CEPI-and CADPcartridges [START_REF] Heilmann | Comparison of four commercial citrate blood collection systems for platelet function detected by the PFA-100 system[END_REF][START_REF] Marshall | A comparison of the effects of aspirin on bleeding time measured using the Simplate method and closure time measured using the PFA -100, in healthy volunteers[END_REF]. Further investigations revealed a day-to-day variability of 9 to 12% and a diurnal variation as well, with about 30% longer CEPI-CT values in the afternoon than in the morning [START_REF] Homoncik | Monitoring of aspirin (ASA) pharamcodynamics with the platelet function analyzer PFA-100[END_REF][START_REF] Dalby | Diurnal variation in platelet aggregation with the PFA-100 platelet analyser[END_REF][START_REF] Cho | Variables that affect platelet function analyzer-100 (PFA-100) closure times and establishment of reference intervals in Korean adults[END_REF].

Only recently, the College of American Pathologists developed a survey to evaluate platelet function tests. In this survey, the precision of the closure time in normal subjects using the CEPI-and CADPcartridges was also shown to be relatively low (CV 21.1% and 18.6%, respectively) [START_REF] Cunningham | Quality assurance in haemostasis: the perspective from the College of American Pathologists proficiency testing program[END_REF]. Despite this, for normal subjects not on antithrombotic therapy, the results are interpreted correctly as normal in 92.4% and 94.0% of cases. Ingestion of aspirin increases the CV of the CEPI-cartridge in a dosedependent manner [START_REF] Gunnewiek | The influence of acetylsalicylic acid intake by healthy volunteers on duplicate PFA-100 measurements[END_REF], and testing patients with ischemic heart disease instead of healthy volunteers results in higher CVs as well [START_REF] Mortensen | Monitoring aspirin therapy with the Platelet Function Analyzer-100[END_REF]. In two studies, a change in responsiveness to aspirin over time has been described which may at least partly be explained by the variation in CT values [START_REF] Andersen | Aspirin non-responsiveness as measured by PFA-100 in patients with coronary artery disease[END_REF][START_REF] Linnemann | Variability of non-response to aspirin in patients with peripheral arterial occlusive disease during long-term follow-up[END_REF]. Based on these observations duplicate measurements and in case of inconsistent results repetitive platelet function testing should be performed to prevent misclassification. The high variability of test results

after aspirin or clopidogrel intake should also be taken into account when interpreting clinical studies or planning future investigations.

However, in patients prescribed combined use of aspirin and clopidogrel, the results of INNOVANCE® PFA P2Y* seem to be more consistent: two patients presented with duplicate measurements within the reference range, whereas all except for these two yielded two non-closures. In patients with dual antiplatelet therapy, this all-or-none pattern of duplicate measurements resulted in an extremely low mean CV for INNOVANCE® PFA P2Y* of only 0.8%. This value was lower than expected but can at least partially be explained by the fact that measurement on the PFA-100® system stops after 5 minutes resulting in a maximum CT of 300 s. In addition, the new test cartridge was almost independent of any aspirin-related effect, in contrast to ADP-induced LTA. As clopidogrel is usually administered to patients already on chronic aspirin therapy, e.g., patients with cardiovascular disease undergoing catheter interventional procedures, it may be advantageous to differentiate between the effects of aspirin and clopidogrel on platelet function.

Several studies have been published in recent years suggesting an association of a normal CEPI-CT in aspirin-treated patients with an unfavorable cardiovascular outcome; the results of these studies have been summarized in two recent meta-analyses [START_REF] Crescente | Response variability to aspirin as assessed by the platelet function analyzer (PFA)-100. A systemic review[END_REF][START_REF] Reny | Use of the PFA-100® closure time to predict cardiovascular events in aspirin-treated cardiovascular patients: a systemic review and meta-analysis[END_REF]. In addition, on the basis of prospective studies, there is now evidence that high residual platelet reactivity determined by ADP-induced LTA predicts future ischemic events in patients on dual antiplatelet therapy [START_REF] Matetzky | Clopidogrel resistance is associated with increased risk of recurrent atherothrombotic events in patients with acute myocardial infarction[END_REF][START_REF] Hochholzer | Impact of the degree of peri-interventional platelet inhibition after loading with clopidogrel on early clinical outcome of elective coronary stent placement[END_REF][START_REF] Buonamici | Impact of platelet reactivity after clopidogrel administration on drug-eluting stent thrombosis[END_REF][START_REF] Frere | ADP-induced platelet aggregation and platelet reactivity index VASP are good predictive markers for clinical outcomes in non-ST elevation acute coronary syndrome[END_REF][START_REF] Marcucci | Residual platelet reactivity is an independent predictor of myocardial injury in acute myocardial infarction patients on antiaggregant therapy[END_REF]. However, the clinical relevance of INNOVANCE® PFA P2Y* results in assessing clopidogrel efficacy remains to be established. As the PFA-100® system is already widely used, the new test cartridge may be a useful tool for the assessment of clopidogrel effects. Most importantly, it remains to be elucidated which method of platelet function testing provides the best risk estimation for recurrent thrombotic events.

We

Disclosures

The study was supported by Siemens Healthcare Diagnostics Products GmbH, Germany, of which Dr.

Andreas R. Rechner is an employee.

Disclaimer

- 1 )

 1 , first through a stainless steel capillary and then through an aperture in a membrane coated with agonists. The agonists at the membrane trigger platelet adhesion, activation, and aggregate formation at the aperture. The membrane of INNOVANCE® PFA P2Y* is coated with 20 µg ADP, 5 ng prostaglandin E1 and 459 µg calcium chloride. The time required to occlude the central aperture (diameter: 100 µm) resulting in the cessation of blood flow is defined as the closure time (CT); the maximum value for CT is determined by the maximum measurement time of 300 s. If a closure of the aperture was not achieved within the time period of 300 s, the system reports a non-closure. For non-closures, a CT value of 300 s was used in the calculations. A CT within the reference range was considered to indicate non-responsiveness to clopidogrel. This study evaluated a new, as yet commercially unavailable, test cartridge designed to assess the efficacy of clopidogrel treatment. The preliminary cut-off for INNOVANCE® PFA P2Y* was defined as 87 s based on closure times of 134 ostensibly healthy volunteers (83 males, 51 females, aged 19 to 58 years) not taking any antithrombotic drug.

Figure 1

 1 Figure 1 illustrates the variability of responsiveness to the different antithrombotic regimens assessed with INNOVANCE® PFA P2Y* on the PFA-100® System as well as that determined by 2 and 5 µM ADP-induced LTA.

  classified as responsive at one visit, but non-responsive at the other visit. The reasons for this observation are not clear. A possible reason for the high variance observed in some patients in this study as well as in the study of Madsen et al. might be an intermediate response to the antiplatelet therapy with clopidogrel or aspirin for those patients, resulting in either almost normal (normal CT) or insufficient (non-closure) clot formation under high-shear flow conditions. However, due to the high intra-assay variation, it does not seem to be advisable to make a clinical decision about responsiveness to antiplatelet therapy based on a single test result.

  conclude that INNOVANCE® PFA P2Y* is suitable for assessing the effect of clopidogrel on platelet function. Its sensitivity in detecting responsiveness or non-responsiveness to clopidogrel is comparable to ADP-induced LTA although the classification as responder or non-responder was discordant in some patients. The variability of CT values among patients treated with clopidogrel as the only antithrombotic drug is high. However, when used in patients on a combination therapy of aspirin and clopidogrel, INNOVANCE® PFA P2Y* seems to be a useful tool for differentiating between clopidogrel responders and non-responders. Additional prospective studies are needed to clarify the clinical relevance of the test results and classifications obtained with INNOVANCE® PFA P2Y*.

Fig. 1

 1 Fig. 1 Comparison of test results of the different test systems (i.e., the INNOVANCE® PFA P2Y* test cartridge on the PFA-100® System and LTA induced by 2 and 5 µM) in patients on different antiplatelet therapy regimens (i.e., clopidogrel (group 1, n = 22)), aspirin and combined aspirin and clopidogrel therapy (group 2, n = 18)).

Fig. 2AFigure Click here to download high resolution imageFigure

  Fig. 2A und 2B Duplicate measurements (Test 1 and Test 2) and distribution of test results obtained with INNOVANCE® PFA P2Y* and LTA in patients on clopidogrel monotherapy (group 1, n = 22) and patients on dual antiplatelet therapy (group 2, n = 18). (* 11/22 and ** 16/18 with non-closure (CT ≥ 300 s)).

  Annual Scientific and Standardization Committee Meeting during the International Society of Thrombosis and Haemostasis congress in Geneva in 2007, a

	reference range was derived from the 5 th -95 th percentiles of duplicates from a group of healthy
	volunteers (n = 20). Non-responsiveness to clopidogrel was defined as aggregation responses within
	the reference range despite clopidogrel medication (i.e., LateAgg ≥ 42.9% and ≥ 72.1% for ADP 2 and
	5 µM, respectively).

  result were used independently. Interestingly, based on the mean of these highly varying duplicates, all patients are classified as clopidogrel responders based on the preliminary cut-off. The large differences between the two measurements in some samples resulted in a

	coefficient of variation of 11.3% for INNOVANCE® PFA P2Y*, whereas the corresponding values for
	LTA performed with 2 and 5 µM ADP were 8.4% and 9.7%, respectively. However, in the subgroup on
	dual antiplatelet therapy (group 2), the results of INNOVANCE® PFA P2Y* were less variable (Fig
	2B). Only two patients in this subgroup presented with duplicate measurements within the reference
	range, whereas all except for these two yielded a CT ≥ 300 ms. The CVs were 0.8% for
	INNOVANCE® PFA P2Y* and 10.3% and 6.4%, for LTA with 2 and 5 µM ADP, respectively.
	All values of INNOVANCE® PFA P2Y* and LTA were obtained in duplicate from the same collection
	tube. The results of duplicate testing in patients under clopidogrel monotherapy (group 1) are
	presented in Figure 2A. Comparison of the two test results measured with INNOVANCE® PFA P2Y*
	showed a large variation in some patient samples. In 11/22 cases (50%), both measurements were

non-closures (i.e., CT > 300 s), while in 6/22 cases (27%), the difference between the two measurements exceeded 30%. Moreover, 2/22 patients (9%) displayed an ambiguous duplicate with respect to the preliminary cut-off value of 87 s, which would lead to a contrasting classification of responsiveness if each

Table 1

 1 Baseline characteristics in terms of cardiovascular risk factors and comorbidities.

	Clopidogrel monotherapy	Dual antiplatelet therapy
	(group 1, n=22)	(group 2, n=18)

Table 2

 2 Comparison of von Willebrand factor antigen (vWF-Ag), vWF-ristocetin cofactor activity (vWF:RCo), and fibrinogen blood levels in clopidogrel non-responders and responders as defined by the different assays. Ag = vWF antigen; vwF:RCo = vWF ristocetin cofactor activity; NR = Non-responder, R = Responder.

		Non-	vWF-Ag	U-Test	vWF:RCo	U-Test	Fibrinogen	U-Test
		Response	(%)	(p value)	(%)	(p value)	(mg/dl)	(p value)
		N/N	NR vs. R		NR vs. R		NR vs. R
	INNOVANCE® PFA P2Y*	6/22	164 vs. 158 0.693 141 vs. 144	0.641	327 vs. 348	0.494
	LTA with ADP 2 µM	4/22	188 vs. 156 0.538 138 vs. 144	1.000	336 vs. 335	0.837
	LTA with ADP 5 µM	6/22	166 vs. 152 0.641 167 vs. 141	0.407	339 vs. 335	0.914
	vWF-

*Product under developmentnot available for sale

PFA-100® system using the new test cartridge INNOVANCE® PFA P2Y*

The study was supported by Siemens Healthcare Diagnostics Products GmbH, Germany, of which Dr.


Thomas Ripert

Marie Dominique Azemar

Johann Menard

Younes Bayoud

Rabah Messaoudi

François Duval

Frédéric Staerman

Marie-Dominique Azémar

Johann Ménard

Transrectal high-intensity focused ultrasound (HIFU) treatment of localized prostate cancer: Review of technical incidents and morbidity after 5 years of use

Keywords: Localized prostate cancer, minimally invasive therapy, high-intensity focused ultrasound, HIFU, side effects, technical incidents

teaching and research institutions in France or abroad, or from public or private research centers.

INTRODUCTION

High-intensity focused ultrasound (HIFU) is an emerging technology for the treatment of localized prostate cancer. Its efficacy and safety have been demonstrated in several studies [START_REF] Thüroff | High-intensity focused ultrasound and localized prostate cancer: efficacity results from the European multicentric study[END_REF][START_REF] Blana | Eight years' experience with High-Intensity Focused Ultrasonography for treatment of localized prostate cancer[END_REF]. It is considered to provide a level of local control (i.e. negative biopsy rate) similar to that of other treatment options [START_REF] Poissonnier | Control of prostate cancer by transrectal HIFU in 227 patients[END_REF]. Its advantages are manifold according to the investigator centres: it is minimally invasive, it can be repeated if required, and other treatments can be offered in cases of incomplete treatment or recurrence [START_REF] Pasticier | Salvage radiotherapy after high-intensity focused ultrasound for localized prostate cancer: early clinical results[END_REF][START_REF] Zacharakis | The feasibility and safety of high-intensity focused ultrasound as salvage therapy for recurrent prostate cancer following external beam radiotherapy[END_REF]. Currently, it is used in patients who are not eligible for surgery or as salvage therapy for local recurrence after radiotherapy.

Patients are often informed that the treatment is local and precise, can be repeated, requires a short hospital stay and is associated with few side-effects. However, relatively few studies have addressed the complications of HIFU and, to our knowledge, none has described the technical problems that may be encountered. Recently, a British team decided to suspend their HIFU treatment programme because of significant rates of oncological failure and complications [START_REF] Challacombe | High-intensity focused ultrasound for localized prostate cancer: initial experience with a 2-year follow-up[END_REF].

The aim of this retrospective study was to review the technical incidents and the complications encountered by a non-investigator centre in the use of the HIFU procedure in the treatment of localized prostate cancer.

PATIENTS AND METHODS

The files of all patients who underwent HIFU treatment for localized prostate cancer in our centre between April 2004 and November 2008 were reviewed. All HIFU procedures had been performed by two practitioners trained in the technique at the investigator centre where the technique originated (Edouard Herriot Hospital, Lyon, France). The first 4 procedures were performed under the supervision of Professor Albert Gelet, MD, PhD.

A second generation Ablatherm™ device was used. After March 2005, this was a robotassisted Ablatherm™ device with Real Time Integrated Imaging. Transurethral resection of the prostate (TURP), when performed, was carried out during the 3 months before the HIFU procedure and not concomitantly with the procedure. HIFU treatment was performed under general anaesthesia. After insertion of a 18 F Foley catheter for bladder drainage, the patient was transferred to the Ablatherm™ platform and placed in the right lateral position. The ultrasound probe was positioned in the rectum. The volume to be treated was defined by the urologist using the longitudinal and transverse ultrasound imaging system. Treatment was delivered in 4 to 6 treatment blocks depending upon gland size, and was initiated 6 mm from the apex to protect the external sphincter. Lesion size had a constant diameter of 1.7 mm but could be modified in length from 19 to 24 mm. Each shot lasted 5 sec and was followed by a 5-sec interval, for a first HIFU treatment. The central prostate zone was treated separately from the lateral zones and, during treatment of midline blocks containing the urethra, the catheter was removed. It was reinserted upon procedure completion and usually remained in place for 72 hours. Patients were discharged the day after catheter removal. No nerve-sparing surgery was performed and no systematic antibiotic treatment was given before, during or after the HIFU procedure.

The indications for HIFU treatment complied with French Urology Association guidelines: stage T1-T2b tumours, prostate specific antigen (PSA) ≤ 15 ng/ml, <4 positive biopsies, and a Gleason score of ≤7 in patients who did not qualify for or refused surgery. Account was taken of life expectancy. Treatment was reserved for prostates smaller than 50 cc in order to limit treatment time and need for a second session [7,[START_REF] Soulié | Recommandations 2007 en onco-urologie[END_REF].

Data were collected on technical incidents, procedure discontinuations regardless of technical cause, and consequences. Early complications were defined as all complications occurring during the month following the procedure, and late complications as those occurring after 1 month. Stress incontinence was graded as described by Ingelman-Sundberg [START_REF] Ingelman-Sundberg | Urinary incontinence in women[END_REF]. Sexual function before and after HIFU treatment was assessed by posing a set of short questions exploring erectile function, sexual activity, and ability to have sexual intercourse (nonvalidated questionnaire). Postoperative erectile dysfunction (ED) was defined as the de novo inability to have sexual intercourse without pharmacological assistance. Follow-up monitoring included a digital rectal examination and PSA test. Visits took place at 1 month, then every 3 months for the first year, and every 6 months thereafter.

RESULTS

A total of 74 HIFU procedures were performed in 65 patients treated for a prostate adenocarcinoma. Prostate cancer treatment by HIFU was first-line in 55 patients and secondline (salvage therapy for local recurrence after external beam radiotherapy) in 10 patients. The mean age of the 55 patients was 72 ± 4.8 years. Clinical stage was T1 in 38 patients (69.1%) and T2 in 17 patients (30.9%). Five patients (13.1%) had T1a disease and 6 (15.8%) had T1b disease. Median pre-treatment PSA was 7 ng/ml (range: 0.29-13.4). Patient stratification according to Gleason grade was as follows: <6 (10.9%), 6 (67.3%), and 7 (21.8%). The median size of prostates treated (after TURP and prior to HIFU) was 18 cc (± 8.6). On average, 138% of the overall median gland volume was treated after a HIFU session. Median follow-up was 41 months .

Forty-nine patients (89.1%) had undergone preoperative TURP. Of these, 37 underwent TURP during the 3 months before the HIFU procedure (mean, 1.75 ± 0.76 months) and 12 had undergone TURP much earlier (mean, 10.7 ± 8.9 months) for the treatment of prostatic symptoms. The mean weight of resected tissue in the two groups was 14.9 g (range, 1-74 g) and 32.5 g (range, 10-114 g), respectively. Of the 12 "early TURP" patients, 3 (25%) had T1a disease and 5 (41.6%) had T1b disease. None required further resection during the 3 months before HIFU treatment. TURP was not performed in 6 patients with a very low prostatic volume (<15 cc) and no prostatic symptoms. All patients were continent before the HIFU procedure.

All the procedures were well tolerated and no intra-operative or peri-operative medical complications or deaths occurred. Six technical incidents occurred during the 74 HIFU sessions (8.1% rate). A telephone help-line did not enable resolution of these incidents which, in each case, led to treatment discontinuation. Only one of the 6 patients required a repeat session, at 3 months, because of a persistently high PSA level. The other 5 patients underwent close clinical surveillance and biochemical monitoring. Treatment was also discontinued in 6 other cases [START_REF] Soulié | Recommandations 2007 en onco-urologie[END_REF]. Table 1 summarizes results by type of 2 nd generation Ablatherm™ device (early device or robot-assisted device with integrated imaging) and by treatment (first-line or salvage).

Complication rate was calculated for the 55 patients who underwent first-line HIFU treatment for prostate cancer (46/55: 1 session; 9/55: 2 sessions). The 10 patients with localized prostate cancer who had undergone previous radiotherapy were excluded from the calculation to avoid bias [START_REF] Murat | Mid-term results demonstrate salvage High-Intensity Focused Ultrasound (HIFU) as an effective and acceptably morbid salvage treatment option[END_REF][START_REF] Chalasani | Salvage HIFU for recurrent prostate cancer after radiotherapy[END_REF]. Fifteen patients (36.4%) experienced 20 early complications and 7 patients (12.7%) experienced 7 late complications. Overall, 27 treatment-related side effects were reported in 55 patients (49%). Complications are summarized in Table 2. One case of urinary retention was encountered in a patient who did not undergo TURP. There were no cases of rectourethral fistula or of chronic perineal pain syndrome.

Early and late functional outcomes (continence and erectile function) are given in Table 3.

There were 6 cases of early stress incontinence (10.9%) and 11 of late stress incontinence (20%). The patient with late grade III stress incontinence underwent placement of a suburethral sling. Overall, 9 complications (16.4%) required surgical management (Table 2).

Data on pre-and postoperative erectile function were available for 51 patients (92.7%). There were 27 cases (77.1%) of de novo ED. Eight patients (29.6%) received long-term ED treatment, either type 5 phosphodiesterase inhibitor (n =7) or intracavernous injections (n = 1). Table 4 summarizes early and late complications and functional outcomes according to type of Ablatherm™ device.

Among the 10 patients who benefited from salvage HIFU after radiotherapy, one patient experienced early acute prostatitis; another experienced late dysuria and was treated by necrosectomy. No patient undergoing salvage therapy presented urinary incontinence.

DISCUSSION

The technical incidents rate in our study was 8.1% for a total of 74 HIFU procedures. All 6 incidents led to treatment discontinuation but only one patient required a second HIFU session because of a persistently high PSA level 3 months after the interrupted procedure. A single technical incident was reported during the 14 sessions using the pre-2005 Ablatherm™ device with no integrated imaging, highlighting the reliability of the early device. Treatment was also discontinued in a further 8% of patients because of either ultrasound detection problems (2 cases) or an excessive rectal wall thickness (4 cases).

One patient with excessive rectal wall thickness was a case of salvage therapy after radiotherapy. Radiotherapy can increase rectal wall thickness, making prostate contours more difficult to detect. However, thanks to the technical innovations brought to the earlier Ablatherm™ device, a better match can now be obtained between the target zone and the prostate capsule contour. New adjustments, such as variable lesion height and safety features protecting the rectum, mean that more patients with local relapse after external beam radiotherapy may now undergo HIFU treatment. The ease of the procedure has increased for the operator. In our 10 patients, salvage therapy was not associated with a greater number of technical difficulties but this result needs to be confirmed in larger studies.

Like Poissonnier et al., we encountered no cases of rectourethral fistula [START_REF] Poissonnier | Control of prostate cancer by transrectal HIFU in 227 patients[END_REF]. Thüroff et al. reported a 1.2% incidence rate [START_REF] Thüroff | High-intensity focused ultrasound and localized prostate cancer: efficacity results from the European multicentric study[END_REF]. Fistulas may be due to impairment of the rectal wall by cavitation and temperature elevation [START_REF] Colombel | Principles and results of high-intensity focused ultrasound for localized prostate cancer[END_REF]. Five cases of rectal fistula have been reported in patients who had previously undergone brachytherapy and/or radiotherapy [START_REF] Zacharakis | The feasibility and safety of high-intensity focused ultrasound as salvage therapy for recurrent prostate cancer following external beam radiotherapy[END_REF][START_REF] Ahmed | Rectal fistulae after salvage high-intensity focused ultrasound for recurrent prostate cancer after combined brachytherapy and external beam radiotherapy[END_REF]. Rectal fistulas can be prevented by monitoring rectal wall thickness (<6 mm), better knowledge of heat diffusion, and better real-time control of the HIFU shots and of the cooling system [START_REF] Thüroff | High-intensity focused ultrasound and localized prostate cancer: efficacity results from the European multicentric study[END_REF].

Treatment parameters need to be adapted in cases of hypovascularized irradiated tissue as such tissue conducts less heat and generates higher temperatures. The recommendation is to decrease shot duration to 4 sec and increase shot interval to 7 sec. Currently, the risk of rectourethral fistulas is close to zero [START_REF] Poissonnier | Control of prostate cancer by transrectal HIFU in 227 patients[END_REF][START_REF] Blana | First analysis of the long-term result with transrectal HIFU in patients with localized prostate cancer[END_REF].

The most common complication of the HIFU procedure is urinary retention due to an increase in prostate volume and/or the passage of necrotic debris. Our urinary retention rate was higher (20%) than reported rates (0.3% and 8.6% [START_REF] Thüroff | High-intensity focused ultrasound and localized prostate cancer: efficacity results from the European multicentric study[END_REF][START_REF] Lee | High-intensity focused ultrasound therapy for clinically localized prostate cancer[END_REF]) but we observed no urinary retention after the first postoperative month. Our higher retention rate may be due to a short bladder drainage time of 72 hours although short times appear to be feasible if TURP precedes treatment [START_REF] Chaussy | The status of high-intensity focused ultrasound in the treatment of localized prostate cancer and the impact of a combined resection[END_REF].

In our study, 49 patients (89.1%) underwent TURP before the HIFU procedure.

The aim of TURP is to reduce the risk of prolonged HIFU-related urinary retention. Unlike Vallancien et al. and Chaussy et al., we performed TURP before and not concomitantly with HIFU [START_REF] Chaussy | The status of high-intensity focused ultrasound in the treatment of localized prostate cancer and the impact of a combined resection[END_REF][START_REF] Vallancien | Transrectal focused ultrasound combined with transurethral resection of the prostate for the treatment of localized prostate cancer: feasibility study[END_REF]. Vallancien et al. concluded in a non-comparative study that the combination of endoscopic resection or incision of the prostate with HIFU treatment decreased urinary catheterization time and improved post-treatment urinary status without additional morbidity [START_REF] Vallancien | Transrectal focused ultrasound combined with transurethral resection of the prostate for the treatment of localized prostate cancer: feasibility study[END_REF]. Chaussy and Thüroff, who compared the treatment-related morbidity of combined TURP-HIFU treatment and HIFU alone, concluded that the combined treatment significantly reduced morbidity and that patient management did not differ to that after just TURP [START_REF] Chaussy | The status of high-intensity focused ultrasound in the treatment of localized prostate cancer and the impact of a combined resection[END_REF].

Catheter time, incontinence, urinary infection and post-treatment International Prostate Symptom Score (I-PSS) were significantly reduced. In addition, there was less need to resort to additional deobstruction procedures (necrosectomy, urethrotomy) which may cause incontinence.

However, the addition of TURP to HIFU may predispose the patient to an increased incidence of stricture and/or stress incontinence. Bladder neck resection during TURP and disruption of the bladder neck continence mechanism may cause incontinence. However, the primary continence mechanism post-TURP is the intrinsic urethral sphincter which is also the primary determining factor for continence preservation post-HIFU. We performed TURP on average 1.75 months before the HIFU procedure and observed no urinary incontinence before the procedure. However, larger studies would be needed to confirm that there is no difference between a population of patients with an intact bladder neck and a population undergoing combined treatment. A shorter catheter time with TURP may be at the expense of a higher risk of incontinence.

According to a recent study, bladder outlet obstruction after HIFU seems to be associated with older age, and TURP before HIFU was apparently not an independent factor in preventing obstruction [START_REF] Blana | Factors predicting for formation of bladder outlet obstruction after high-intensity focused ultrasound in treatment of localized prostate cancer[END_REF]. However, multiple episodes of bladder outlet obstruction could be reduced by resecting more tissue at TURP before HIFU [START_REF] Blana | Factors predicting for formation of bladder outlet obstruction after high-intensity focused ultrasound in treatment of localized prostate cancer[END_REF]. The necrosectomy rate for urinary retention or dysuria was lower in our study than in Blana et al.'s (7.2% vs 11.7%) [START_REF] Blana | High-intensity focused ultrasound for the treatment of localized prostate cancer: 5-year experience[END_REF]. There were 7 cases of urethral stenosis (12.6%) of which 4 were managed by internal urethrotomy. Published rates for post-HIFU stenosis range from 3.6% to 22% [START_REF] Thüroff | High-intensity focused ultrasound and localized prostate cancer: efficacity results from the European multicentric study[END_REF][START_REF] Uchida | Five years experience of transrectal high-intensity focused ultrasound using the Sonablate device in the treatment of localized prostate cancer[END_REF]. A recent study with a mean follow-up of 6.4 years gave a 13.6% rate [START_REF] Blana | First analysis of the long-term result with transrectal HIFU in patients with localized prostate cancer[END_REF]. Our urinary infection rate was 3.6%. It was lower than the 7.8% rate reported in a 8-year study of 163 patients [START_REF] Blana | Eight years' experience with High-Intensity Focused Ultrasonography for treatment of localized prostate cancer[END_REF] and than the 16% rate in a study patients with high-risk prostate cancers [START_REF] Ficarra | Shortterm outcome after high-intensity focused ultrasound in the treatment of patients with high-risk prostate cancer[END_REF]. Infection responded to appropriate antibiotic treatment. Combined HIFU plus TURP treatment has been shown to be associated with a lower infection rate than HIFU alone (11.4% vs 47.9%) [START_REF] Chaussy | The status of high-intensity focused ultrasound in the treatment of localized prostate cancer and the impact of a combined resection[END_REF].

The stress incontinence rate in our patients was 20% (12.7% for grade I) and was higher than previously reported. Grade I and II incontinence rates of 6.1% and 1.8%, respectively, have been reported after 8 years of follow-up [START_REF] Blana | Eight years' experience with High-Intensity Focused Ultrasonography for treatment of localized prostate cancer[END_REF], and rates of 5% and 0.7% after a median of 6.4 years of follow-up [START_REF] Blana | First analysis of the long-term result with transrectal HIFU in patients with localized prostate cancer[END_REF]. Better identification of the prostate apex and leaving a 4-6 mm treatment margin at the apex have reduced the risk of incontinence [START_REF] Poissonnier | Result of transrectal focused ultrasound for the treatment of localized prostate cancer (120 patients with PSA < or + 10 ng/ml)[END_REF]. None of our 10 patients in the salvage group presented incontinence but their risk of incontinence was higher.

Murat et al. reported an incontinence rate of 49.5% in salvage patients, which included a 31.5% rate of grade 2 or 3 incontinence [START_REF] Murat | Mid-term results demonstrate salvage High-Intensity Focused Ultrasound (HIFU) as an effective and acceptably morbid salvage treatment option[END_REF]. Currently, improvements in imaging and specific post-radiation treatment parameters have reduced this risk [START_REF] Murat | Mid-term results demonstrate salvage High-Intensity Focused Ultrasound (HIFU) as an effective and acceptably morbid salvage treatment option[END_REF].

The de novo postoperative ED rate in our study was 77.1% compared to rates of 20% and 77% in recent studies [START_REF] Murat | High-Intensity Focused Ultrasound (HIFU) for Prostate Cancer[END_REF][START_REF] Rebillard | Highintensity focused ultrasound in prostate cancer: a systematic literature review of the french Association of Urology[END_REF]. We prescribed postoperative ED therapy to a higher proportion of patients (n=8, 29.6%) than another study did (20%) [START_REF] Uchida | Treatment of localized prostate cancer using high-intensity focused ultrasound[END_REF]. However, our study was not a review of a prospectively maintained database, the population size was relatively small, and we did not use a validated potency questionnaire. Moreover, our centre does not perform nerve-sparing HIFU because of the oncological risk and the likelihood of repeat treatment [START_REF] Poissonnier | Control of prostate cancer by transrectal HIFU in 227 patients[END_REF][START_REF] Chaussy | Results and side effects of high-intensity focused ultrasound in localized prostate cancer[END_REF][START_REF] Chaussy | High-intensity focused ultrasound in prostate cancer: results after 3 years[END_REF]. Repeat HIFU carries a significantly higher risk of impotency and incontinence [START_REF] Blana | Morbidity associated with repeated transrectal high-intensity focused ultrasound treatment of localized prostate cancer[END_REF]. Specific criteria have been established for repeat treatment because of the lesions occurring after a first procedure [START_REF] Chen | Influence of ablated tissue on the formation of highintensity focused ultrasound lesions[END_REF][START_REF] Curiel | Experimental evaluation of lesion prediction modelling in the presence of cavitation bubbles: intended for high-intensity focused ultrasound prostate treatment[END_REF] and the problems in detecting prostate boundaries for a repeat procedure.

Our results, albeit in a limited series of patients, thus do not support the early claims of low morbidity and good functional outcomes in patients undergoing HIFU treatment for localized prostate cancer [START_REF] Thüroff | High-intensity focused ultrasound and localized prostate cancer: efficacity results from the European multicentric study[END_REF]. Our overall complications rate of 49%, including a 20% rate of late stress incontinence and a 77.1% de novo ED rate was relatively high. However, this emerging technology is not a morbid procedure but rather a procedure that is not completely complication free. It is less invasive than conventional therapies such as radical prostatectomy, radiotherapy, or brachytherapy. It does not require an incision or puncture, is bloodless, deliverable on an outpatient basis, safe, and repeatable, with a fairly rapid recovery time. The 2009 European Association of Urology guidelines still consider it "investigational or experimental" [START_REF] Heidenreich | EAU guidelines on prostate cancer[END_REF]. Technological advances will hopefully improve the morbidity rates which will need to be assessed in well-designed trials with longer follow-ups.

In our study, we used the Ablatherm™ rather than Sonablate500™ device. The complication rates of these two devices have been compared in a head-to-head, but non randomized, comparison which revealed no significant differences in morbidity [START_REF] Tsakiris | Transrectal high-intensity focused ultrasound devices: a critical appraisal of the available evidence[END_REF].

In conclusion, the Ablatherm™-HIFU procedure seems fairly reliable with a technical incidents rate of 8.1%. The overall complication rate was high (49%) over a median follow-up of 41 months. Most complications were minor, requiring surgical management in 16.4% of cases. Our results confirm that all patients offered HIFU treatment should be properly informed of the risks, in particular with regard to continence and sexual function.

Table 1 .

 1 Technical incidents and treatment discontinuations by type of device and treatment group

	Type of 2	nd generation	Treatment Procedures	Technical incidents	Treatment
	Ablatherm™ HIFU device		(N)	n (%)	discontinuations n (%)
	Early device (Apr. 2004 -	First-line	13	Computer incident	Excessive rectal wall
	Mar. 2005)				1 (7.7)	thickness -1 (7.7)
			Salvage	1	0	0
	Robot-assisted with real	First-line	51	Probe blockage -2 (3.9)	Excessive rectal wall
	time integrated imaging (Mar. 2005 -Nov. 2008)			Abnormal cooling fluid temperature -2 (3.9)	thickness -2 (3.9) Ultrasound detection
					Computer incident -1 (1.9)	problems -2 (3.9)
			Salvage	9	0	Excessive rectal wall
						thickness -1 (11.1)

TABLE 2 .

 2 Complication rates after HIFU treatment (N= 55) Urinary retention: Lack of ability to urinate with a full bladder Dysuria: Difficult, sometimes painful urination, often characterized by straining

	Complications	Urinary	Dysuria	Acute prostatitis	Urethral stenosis	Haematuria
	and treatment	retention		N (%)	N (%)
		N (%)	N (%)			N (%)
	Early (<1 mo)	11 (20)	3 (5.4)	2 (3.6)	2 (3.6)	2 (3.6)
	Treatment Bladder	-	Antibiotics (2) Internal	Bladder
		catheter (9)			urethrotomy (2)	washing
		Necrosectomy				(2)
		(2)
	Late (>1 mo)	0	2 (3.6)	0	5 (9.0)	0
	Treatment	-	Necrosectomy	-	Internal	-
			(2)		urethrotomy (2)
					Autodilatation
					(1)
	Total	11 (20)	5 (9.0)	2 (3.6)	7 (12.6)	2 (3.6)

TABLE 3

 3 graded as described by Ingelman-Sundberg[START_REF] Ingelman-Sundberg | Urinary incontinence in women[END_REF].

	. Functional outcomes
		Preoperative	Postoperative
		N (%)		N (%)
			< 1 month > 1 month
	Stress incontinence (N =55) *
	Grade I	0	4 (7.3)	7 (12.7)
	Grade II	0	2 (3.6)	3 (5.4)
	Grade III	0	0 (0)	1 (1.8)
	Erections (N = 51)	35 (68.6)	8 (15.7)
	Sexual activity (N = 51)	24 (47)		2 (3.9)

*

Table 4 .

 4 Complications and functional outcomes according to device ED: Erectile dysfunction; IU: Internal urethrotomy

	Type of 2 nd	Urinary	Dysuria	Acute	Urethral stenosis	Haematuria	Stress incontinence	ED
	generation	retention	N (%)	prostatitis	N (%)	N (%)		N (%)	N
	Ablatherm™ ™ ™ ™ HIFU	N (%)			N (%)				N + Grade I,II,III
	device		Early Late		Early	Late		Early	Late
	Early device (Apr.	4	0	0	0	0	2	0	1	3
	2004 -Mar. 2005)	(33)					(16.6)		(8.3)	(25)	2
	(N=12)	1 necro-					1 IU		1II	1I, 2II
		sectomy
	Robot-assisted	7	3	2	2	2	3	2	5	8
	with real time	(16.3)	(6.9)	(4.6)	(4.6)	(4.6)	(6.9)	(4.6)	(11.6)	(18.6)
	integrated imaging 2008) (N=48) (Mar. 2005 -Nov.	sectomy 1 necro-				2 IU	1 IU		4I, 1II	6I, 1II,1III