Ruth Cr Meex

Esther Phielix

Vera B Schrauwen-Hinderling

Esther Moonen-Kornips

Gert Schaart

Patrick Schrauwen

Matthijs Kc

Ruth C R Meex

Esther Moonen- Kornips

Matthijs K C Hesselink
email: matthijs.hesselink@bw.unimaas.nl

la diffusion de documents scientifiques de niveau

Keywords: 2 diabetes, statin, exercise training, cardiometabolic risk-factors, insulin sensitivity, mitochondrial function

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The use of statins potentiates the insulin sensitizing effect of exercise training in obese males with and without type 2 diabetes.

Introduction

Hyperlipidemia and hyperglycemia are frequently reported in obese subjects and are highly associated with the development of cardiovascular disorders, the primary cause of death in obesity. Whereas treatment with HMG-CoA reductase inhibitors like statins is often prescribed in case of hyperlipidemia [START_REF] Snow | Lipid control in the management of type 2 diabetes mellitus: a clinical practice guideline from the American College of Physicians[END_REF][START_REF] Downs | Design & rationale of the Air Force/Texas Coronary Atherosclerosis Prevention Study (AFCAPS/TexCAPS)[END_REF], implementing routine physical exercise in daily life is advocated in treatment of hyperglycemia [START_REF] Sigal | Physical activity/exercise and type 2 diabetes: a consensus statement from the American Diabetes Association[END_REF]. Hyperlipidemia and hyperglycaemia, however, often present concurrently. Hence, combining the current guidelines would imply combining statin treatment with physical exercise. Recently released guidelines from the American Diabetes Association state that in diabetic individuals >40 years of age, statin use should be added to lifestyle therapy in order to reach target levels of LDL cholesterol < 2.6 mmol/l in patients with type 2 diabetes (T2D) or < 1.80 mmol/l in T2D with established CVD [START_REF] Eldor | American Diabetes Association indications for statins in diabetes: is there evidence?[END_REF]. The use of statins, however, has been associated with myotoxicity, mitochondrial dysfunction [START_REF] Ghirlanda | Evidence of plasma CoQ10-lowering effect by HMG-CoA reductase inhibitors: a double-blind, placebo-controlled study[END_REF][START_REF] Paiva | High-dose statins and skeletal muscle metabolism in humans: a randomized, controlled trial[END_REF][START_REF] Phillips | Statin-associated myopathy with normal creatine kinase levels[END_REF][START_REF] Schick | Decreased skeletal muscle mitochondrial DNA in patients treated with highdose simvastatin[END_REF][START_REF] Sirvent | New insights into mechanisms of statinassociated myotoxicity[END_REF] and muscular damage [START_REF] Phillips | Statin-associated myopathy with normal creatine kinase levels[END_REF]10]. In addition, it has been suggested that statin use may blunt the adaptive response to exercise training [11,[START_REF] Thompson | Lovastatin increases exercise-induced skeletal muscle injury[END_REF].

To examine the role of statins in exercise training-mediated improvements in cardiometabolic risk-factors, markers of in vivo mitochondrial function and insulin sensitivity, we here evaluate these effects after 12 weeks of training in a group of elderly male obese healthy and T2D subjects, stratified to the use of statins.

Research design and methods

Subject recruitment and stratification Initially, we recruited 38 sedentary elderly male obese subjects of which 18 subjects had been diagnosed with T2D for at least one year [START_REF] Meex | Restoration of muscle mitochondrial function and metabolic flexibility in type 2 diabetes by exercise training is paralleled by increased myocellular fat storage and improved insulin sensitivity[END_REF]. Diabetic patients had well-controlled diabetes (HbA1c = ± 7.2%) and were using oral anti-diabetic agents (metformin only, or in combination with SU derivatives). The other 20 subjects were categorized as normoglycemic. Glycemic control was checked for by measuring HbA1c, and glucose tolerance by performing an oral glucose tolerance test. Subjects were informed about the nature and risks of the experimental procedures before their written informed consent was obtained. The study has been carried out in accordance with the Declaration of Helsinki (2000) of the World Medical Association and was approved by the institutional medical ethical committee.

Retrospectively, the entire group of 38 subjects was stratified according to the use of statins. Two subjects on fibrates were excluded from analysis. Statin use was defined as using any type of statins, for over a year at the onset of the study, irrespective of dosage. Individual data on statin use can be found in the supplementary material. Stratification resulted in 14 subjects on statins (ST) and 22 subjects without statin treatment (C) with 8 T2D subjects in both groups. None of the baseline characteristics was significantly different between the C and ST group, except for LDL cholesterol which was lower in the patients using statins (subject characteristics see table 1). To examine if the type of statin and the dosage used may have affected the outcome of the present study, we ranked the statins on their reported LDL lowering potency, taking into account the dosage used. Using this marker of potency as an independent variable we performed correlative analysis with all parameters under investigation.

Exercise training protocol

All subjects were engaged in an exercise program for 12 weeks, consisting of a combination of aerobic and resistance exercise. Subjects were monitored extensively during the training period and attention has been paid to follow up conversations with respect to their motivation. Aerobic exercise was carried out on a cycling ergometer twice a week for 30 minutes at 55% of their previously determined maximal work load. Training sessions took place in small groups of 3 to 4 persons and as a measure of exercise intensity, heart rate was monitored and registered in a Resistance exercise was performed once a week for 40 minutes. The training involved a "circuit" of eight exercises, focussing on large muscle groups (i.e., chest press, leg extension, lat pull down, leg press, triceps curl, biceps curl, crunches and horizontal row). A first series of 8 repetitions was performed at 55% of their pre-determined maximal voluntary contraction (MVC), followed by 2 series of 8 repetitions at 75% MVC [START_REF] Meex | Restoration of muscle mitochondrial function and metabolic flexibility in type 2 diabetes by exercise training is paralleled by increased myocellular fat storage and improved insulin sensitivity[END_REF]. Resistance training was given individually and MVC was re-assessed every 4 weeks.

Hyperinsulinemic-euglycemic clamp Dietary habits were stable, physical exercise was avoided the 2 days prior to the clamp procedure and anti-diabetic medication was withdrawn 7 days prior to the clamp. The use of statins was continued throughout the study. Insulin sensitivity was measured by a 3-hour hyperinsulinemiceuglycemic clamp, before and after the training period. A primed constant infusion of glucose tracer ([6,6-2H2]glucose) was initiated at t=0 minutes to determine non-insulin stimulated rates of glucose appearance (Ra) and disposal (Rd). At t=180, the actual clamp procedure was started with a primed constant infusion of insulin (40mU/m 2 /min) [START_REF] Meex | Restoration of muscle mitochondrial function and metabolic flexibility in type 2 diabetes by exercise training is paralleled by increased myocellular fat storage and improved insulin sensitivity[END_REF]. Endogenous glucose production (EGP) was calculated as Ra minus exogenous glucose infusion rate. Non-oxidative glucose disposal was calculated as Rd minus carbohydrate oxidation. In the non-insulin stimulated period (t=150-180) and under steady clamp conditions (t=330-360), blood samples and indirect calorimetry measurements (ventilated hood) were obtained. A needle muscle biopsy was obtained from the m. vastus lateralis before starting the tracer infusion.

Blood sample analysis

Blood was sampled and analysed as described previously [START_REF] Meex | Restoration of muscle mitochondrial function and metabolic flexibility in type 2 diabetes by exercise training is paralleled by increased myocellular fat storage and improved insulin sensitivity[END_REF]. [START_REF] Frayn | Calculation of substrate oxidation rates in vivo from gaseous exchange[END_REF].

MRS-based measurement of mitochondrial function

Maximal muscle strength during one-legged knee-extension exercise was assessed a few days prior to the actual 31 P-MRS measurement with an incremental protocol in an MRS compatible exercise device. During this test subjects were asked to perform knee-extensions at 0.5 Hz, while increasing the load to be lifted every 30 seconds by 500 g, with an initial weight of 5 kg. The test was performed until exhaustion or until the frequency of 0.5 Hz could no longer be maintained (maximal load). The phosphocreatine (PCr) lowering exercise session during the test-day was performed at 60% of this pre-determined load.

Mitochondrial density

Five different structural components of the electron transport chain were measured at the protein level as a reflection of mitochondrial density [START_REF] Meex | Restoration of muscle mitochondrial function and metabolic flexibility in type 2 diabetes by exercise training is paralleled by increased myocellular fat storage and improved insulin sensitivity[END_REF]. The ND6 subunit of complex I, the 30-kDa Ip subunit of complex II, the 47-kDa core protein 2 of complex III, subunit II of cytochrome c oxidase (COXII), and the subunit of the F1F0 ATP synthase (complex V) were measured using a monoclonal antibody cocktail of 5 monoclonal antibodies directed to the subunits mentioned (MitoSciences,Eugene, OR) as previously described [START_REF] Schrauwen | Reduced skeletal muscle uncoupling protein-3 content in prediabetic subjects and type 2 diabetic patients: restoration by rosiglitazone treatment[END_REF]. As these subunits differ considerably in molecular mass, this antibody gives 5 distinct bands in human samples. Each individual band reflects one of the mitochondrial subunits. In short, muscle biopsies were homogenized in icecold Tris-EDTA buffer at pH 7.4, and then the homogenates were sonicated for 15 sec. Subsequently, two volumes of each skeletal muscle homogenate and one volume of SDS-sample buffer were boiled for 4 min. Next, 13% polyacrylamide gels containing 0.1% SDS were loaded with equal amounts of protein from each sample, and electrophoresis was performed using a Mini-Protean 3 Electrophoresis Cell (Bio-Rad Laboratories, Hercules, CA). After gel electrophoresis, the gel was scanned and the optical density of all individual bands (per subject per subunit) was measured. To adjust for inter-gel variation, the optical density of the band of interest per subject was normalized to the mean optical density of the complete gel. Protein content is expressed as arbitrary units (AU).

Statistics

Statistical analyses were performed two-sided using SPSS for Windows 15.0 software (SPSS, Chicago,IL. Statistical significance was set at p<0.05. A two-way ANOVA model for repeated measures was applied using control and statin users as between subject variables and pre-and post training data as repeated within subject variables. Differential responses between statin users and non statin users were examined using the interaction terms. Also, in case of a significant training effect, differential responses of T2D and healthy subjects within statin users and non statin users were examined.

Results

Clinical A c c e p t e d M a n u s c r i p t effects were detected between healthy subjects and diabetic subjects, indicating that differences were not specific for healthy or diabetic subjects solely.

Pre-training pleiotropic effects of statins

After stratification to the use of statins, both groups were comparable for all subject characteristics, except for LDL cholesterol (table 1). Also markers for insulin sensitivity, substrate metabolism and mitochondrial function were similar (table 2). Interestingly, this is in contrast with previous studies in rodents [START_REF] Wong | Atorvastatin induces insulin sensitization in Zucker lean and fatty rats[END_REF]21] and humans, [22] showing improved insulin sensitivity on whole body level and in liver. Another study report a decrease in mtDNA copy number [START_REF] Schick | Decreased skeletal muscle mitochondrial DNA in patients treated with highdose simvastatin[END_REF], suggesting that statins may affect these parameters somehow. In a more recent study, however, a high dose of statins (80 mg/day simvastatin) did not affect insulin-mediated glucose disposal, hepatic glucose production and myocellular lipid deposition [START_REF] Szendroedi | Effects of high-dose simvastatin therapy on glucose metabolism and ectopic lipid deposition in nonobese type 2 diabetic patients[END_REF].

Exercise tolerance on statins

Classical measures of physical fitness were similar in C as in ST, which is in line with previous reports [START_REF] Traustadottir | High-dose statin use does not impair aerobic capacity or skeletal muscle function in older adults[END_REF]. A 12-week interval cycling training combined with resistance exercise did not provoke statin-related muscle pain or stiffness and was equally well tolerated in C as in ST. Selfreported muscle-stiffness due to unfamiliar exercise occurred predominantly in the first 1.5 weeks and rapidly disappeared after familiarization to the exercise program. Training-mediated improvements in exercise capacity were similar in both groups. Hence, the data of the present study do not support the hypothesis that the use of statins provokes muscle pathology or may blunt the adaptive responses to exercise training, as suggested previously [25]. However, the present study applied a tailored progressive exercise training program to avoid the development of muscle soreness, whereas other investigations applied eccentric exercise and exercise bouts on maximal intensity [11,[START_REF] Thompson | Lovastatin increases exercise-induced skeletal muscle injury[END_REF], types of exercise prone to the development of muscle soreness. Also, none of the ST subjects in the present study reported muscle related complaints under resting conditions after starting their statin treatment. We can hence not exclude that the subjects in the present study represent a statin-tolerating subgroup and that this may contribute to the fact that none of them experienced muscle related problems whilst exercising.

Post-training pleiotropic effects of statins

In contrast to previous reports in humans [START_REF] Paiva | High-dose statins and skeletal muscle metabolism in humans: a randomized, controlled trial[END_REF] we did not detect any (negative) effects of statin use on mitochondrial content and function pre-training and showed similar improvements upon training. It should be noted though, that in the present study the dosage of statins used was in general lower [START_REF] Paiva | High-dose statins and skeletal muscle metabolism in humans: a randomized, controlled trial[END_REF].

Intriguingly, exercise training in ST improved insulin stimulated glucose disposal, both by an increase in oxidative as well as in non-oxidative glucose disposal, without an effect on hepatic insulin sensitivity. The improvement in non-oxidative glucose disposal is of particular interest, as we previously identified non-oxidative glucose disposal as the non-responsive culprit for trainingmediated improvements in type 2 diabetic patients [START_REF] Meex | Restoration of muscle mitochondrial function and metabolic flexibility in type 2 diabetes by exercise training is paralleled by increased myocellular fat storage and improved insulin sensitivity[END_REF]. In contrast, in C, insulin-stimulated nonoxidative glucose disposal did not improve significantly after training.

One of the characteristics of insulin resistant muscle is metabolic inflexibility (delta RER). Here we observed that metabolic flexibility improved upon training in ST, indicating that statin use does not interfere with training-induced metabolic improvements. The improvement in metabolic flexibility was less pronounced (and non-significant) in C.

Putative mechanisms of statin-mediated potentiation of the beneficial effect of exercise training

We strikingly observed that the insulin sensitizing effects of exercise training were potentiated by the use of statins, but the precise mechanism include a statin mediated reduction in superoxide-induced cell damage [START_REF] Wassmann | Cellular antioxidant effects of atorvastatin in vitro and in vivo[END_REF], statin mediatedreduction of low-grade inflammation [START_REF] Forrester | The inflammation hypothesis and its potential relevance to statin therapy[END_REF], or a reduction in FFA acid flux and hepatic fat storage [START_REF] Isley | The effect of high-dose simvastatin on free fatty acid metabolism in patients with type 2 diabetes mellitus[END_REF] resulting in improved hepatic insulin sensitivity. The present study does not allow conclusions on the putative effects of statins on superoxide production or superoxide mediated cell damage. Using hsCRP as a marker for low-grade inflammation, we report no differences in low-grade inflammation pre-training between C and ST and did not detect any training effects on hsCRP. Recently it has been shown that even a high-dose of simvastatin (80 mg/daily) does not affect hepatic lipid stores or insulin sensitivity significantly [START_REF] Szendroedi | Effects of high-dose simvastatin therapy on glucose metabolism and ectopic lipid deposition in nonobese type 2 diabetic patients[END_REF]. This is in line with the present study showing similar insulin mediated suppression of hepatic glucose output pre-training in C and ST. Many patients taking statins also are taking other drugs targeting cardiovascular risk control. The most clear design to examine the effects of the use of statins on exercise mediated improvements in glucose homeostasis and cardiovascular risk would be a double blind, placebo controlled, randomized clinical trial. A disadvantage, however, would be that only subjects can be included who were not on statins before the start of the study while in daily life patients usually been taking statins for quite some time even before they are advocated to physical exercise. Hence, we feel that the design of the present study provides a valuable and valid representation of what happens when subjects implement regular exercise in their daily life, on top of any medication they are taking already. The drawback of our design is that the statin group is not homogeneous for some of the output parameters examined. The fact that we indeed do observe significant differences for the key outcome parameters, however, indicates the effects reported are strong enough to reach significance, even with the relatively small group size shown.

In conclusion, the present study shows in elderly males with moderate obesity that statin treatment did not inhibit exercise-induced improvements with respect to mitochondrial density and function. Rather, improvements in total cholesterol, insulin stimulated lipolysis, insulin sensitivity, non-oxidative glucose disposal, metabolic flexibility, and substrate oxidation were more pronounced and/or even exclusively detectable when exercise and statin use was combined. This study suggests that combining physical exercise training in subjects who well-tolerate statin treatment under resting conditions may be at least as beneficial, and for some parameters even more beneficial, than exercise training exclusively. Thus, the present findings indicate that the recently published guidelines of the ADA to prescribe statins to all type 2 diabetic subjects over the age of 40 can be safely combined with previous guidelines on lifestyle interventions.

ClinicalA c c e p t e d M a n u s c r i p t 3 training

 3 Science Immediate Publication. Published on 13 May 2010 as manuscript CS20100153 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100153 diary every 5 minutes. Maximal workload was re-evaluated after 6 weeks of training and training load was adjusted accordingly.

ClinicalA c c e p t e d M a n u s c r i p t 4

 4 Science Immediate Publication. Published on 13 May 2010 as manuscript CS20100153 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100153Baseline 31 P-MRS measurements were performed on a 1.5 T whole body scanner (Intera, Philips Health Care, Best, the Netherlands). A 6 cm surface coil was used for localization and was fixed in the middle of the vastus lateralis muscle. A series of partially saturated spectra (free induction decays) was acquired (TR = 4 seconds, 1 measurement, spectral bandwidth 1500 Hz, adiabatic pulse). Knee extension exercise was performed at 0.5 Hz to an acoustic cue on a home-built MR compatible ergometer with a pulley system. The PCr lowering exercise was performed with identical weights before and after training. The acquisition time during time series was structured as follows: 40 sec of rest, 5 minutes of knee-extension exercise and 5 minutes of recovery. After application of phase correction, linebroadening and DC correction, spectra were fitted in the time domain with the AMARES algorithm [16] in the jMRUI software (http:// www.mrui.uab.es)[START_REF] Naressi | Javabased graphical user interface for MRUI, a software package for quantitation of in vivo/medical magnetic resonance spectroscopy signals[END_REF] using prior knowledge. Five peaks were fitted with gaussian curves (Pi, PCr, and 3 ATP peaks) in the partially saturated spectra of the time-serie. To determine the pH during the exercise period, five consecutive spectra of the time-series were added and the pH was calculated in jMRUI from the frequency shift between PCr and Pi peak. The time-course of the PCr amplitude (PCr(t)) during the last 20 seconds of exercise (steady state) and during the recovery period was fitted with MATLAB software (The Mathworks. Inc.), as described earlier [18], asssuming a monoexponential PCr recovery. Post-exercise PCr resynthesis rate is an almost exclusively oxidative process and is hence a good reflection of mitochondrial oxidative function. The rate constant of the monoexponential recovery is given. The higher the rate constant (s -1 ), the better in vivo mitochondrial function.

  (s) remains to be established. Candidate mechanisms Clinical Science Immediate Publication. Published on 13 May 2010 as manuscript CS20100153 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100153 A c c e p t e d M a n u s c r i p t

  55 ± 0.06* Data are expressed as mean ± SE., #ST significantly different from control group, *post-training significantly different from pre-training, § significant interaction effect between C and ST. . Clinical Science Immediate Publication. Published on 13 May 2010 as manuscript CS20100153 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100153 A c c e p t e d M a n u s c r i p t

Figure legends

 legends Figure legends

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

Table 1

 1 Science Immediate Publication. Published on 13 May 2010 as manuscript CS20100153Before training, maximal oxygen uptake (VO 2 max/kg BW) and maximal workload was comparable in both groups (VO2max/kgBW 28.6±1.0 ml/kg and 27.7±1.5 ml/kg in C and ST respectively, p=0.63; workload 208±9 Watt 201±10 Watt in C and ST respectively, p=0.61).Exercise training improved VO 2 max/kg body weight and maximal workload significantly and to the same extent in C and ST (VO 2 max +7.0±2.2% and +11.7±3.2% respectively, p<0.01; workload +14.6±2.7% and +16.1±2.3% in C and ST respectively, p<0.01). Muscle strength was comparable before training (83.5±3.2 kg and 87.4±4.0 kg in C and ST respectively, p=0.46) and improved significantly and to the same extent in both groups (+24.7±1.9% and +21.6±2.1% in C and ST respectively, p<0.01). In the rare occasion of missing a training session, this session was rescheduled within a week of the original training date. As maximally achieved workload during the maximal test was similar in both groups and the number of training sessions performed was identical, total workload performed during the training sessions across groups was comparable.

	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100153	A c c e p t e d M a n u s c r i p t

5 Subjects

Before training, body weight (BW) (94.9±2.8 and 93.2±2.9 kg), BMI (30.1±0.8 and 30.0±0.9 kg/m2), and age (59.2±1.0 and 58.4±0.9 years) were comparable in C and ST respectively. Training did not alter body weight and BMI in C while body weight but not BMI tended to decline in ST (from 93.2±3.2 to 91.9±3.1 kg, p=0.09 and from 30.0±0.9 to 29.6±0.8 kg/m 2 ). Fat mass declined in C (from 31.2±1.8 to 30.4±1.8 kg after training, p=0.02) whereas there was a tendency towards a decreased fat mass in ST (from 27.9±2.3 to 26.4±2.2 kg after training; p=0.10). Fat free mass was similar between both groups and was unaffected by training.

Glucose and insulin profile Fasting glucose levels (7.2±0.4 mmol/l and 7.5±0.5 mmol/l in C and ST) and HbA1c levels (6.4±0.2% and 6.6±0.3% in C and ST) were similar at the onset of the training program. Twelve week of exercise training did not change HbA1c or fasting glucose levels in either group. Fasting plasma insulin levels did not differ significantly before onset of the training program between C and ST (17.2±2.0 vs. 17.9±2.0mU/l in C and ST) but decreased significantly in both groups after training (from 17.2±2.0 mU/l to 15.7±1.7 mU/l in C and from 17.9±2.0 mU/l to 15.0±1.7 mU/l in ST, p<0.05 in both groups).

  DiscussionWe examined the effect of training on cardiometabolic risk-factors, markers of in vivo mitochondrial function and insulin sensitivity in male obese elderly with and without type 2 diabetes who were on lipid lowering drugs (statins, ST group) and compared these effects with a control group (C) not taking statins. Maximal exercise capacity and markers of mitochondrial content and function were not negatively impacted by statins. In addition, training-induced improvements in markers of cardiometabolic risk, insulin sensitivity and fuel selection were more prominent, and sometimes even exclusively detectable in ST compared to C. Exercise training mediated improvements in mitochondrial density and function were as prominent in ST as in C. The present study hence shows that exercise prescription on top of statin treatment is safe and at least as beneficial -and for some parameters even more beneficial-for cardiometabolic health, glucose homeostasis and mitochondrial function than exercise training without statins.

	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100153	M Mitochondrial density was evaluated by measuring protein content of 5 structural subunits of the Markers of mitochondrial density distinct complexes of the electron transport chain. Neither the individual complexes, nor the mean protein content of these complexes revealed a difference in mitochondrial density between C and a n u s c r i p t ST pre-training (0.56±0.10 and 0.71±0.10 AU in C and ST, NS). Training resulted in increased mitochondrial density in both groups after training (0.56±0.10 to 1.29±0.16 AU in C, p<0.01 and 0.71±0.10 to 1.22±0.16 AU in ST, p=0.03) (Figure 1 panel a). MRS measurement Pre-training in vivo mitochondrial function in C was similar to ST (rate constant: 0.034±0.002 and 0.034±0.003 s -1 in C and ST respectively) while after training in vivo mitochondrial function improved with 34% and 46% (from 0.034±0.002 to 0.044±0.003 s -1 in C, p<0.01 and from 0.034±0.003 to 0.049±0.006 s -1 in ST, p=0.02) (Figure 1 panel b). Metabolic flexibility Prior to training, metabolic flexibility (∆RER 0.071±0.008 and 0.062±0.012, p = 0.51), insulin stimulated glucose oxidation (∆CHOox 4.7±0.7 and 4.7±0.9 umol/kg/min, p=0.99) and suppressed fat oxidation (∆FATox -0.41±0.05 and -0.34±0.07 umol/kg/min, p=0.47) was comparable between C and ST. Exercise training did not affect metabolic flexibility in C (∆RER from 0.071±0.008 to 0.083±0.011, p=0.22) whereas metabolic flexibility improved significantly in ST (∆RER from 0.062±0.012 to 0.089±0.008, p=0.01), reflecting a concomitant increase in insulin stimulate glucose oxidation (from 4.7±0.9 to 6.5±0.7 umol/kg/min, p=0.01) (Figure 1 panel c) and suppression of fat oxidation (from -0.34±0.07 to -0.55±0.06 umol/kg/min, p=0.01) A c c e p t e d (Figure 1 panel d).

).

Whole-body insulin-stimulated glucose uptake Insulin-stimulated glucose uptake, as determined by the delta glucose disposal rate during the hyperinsulinemic euglycemic clamp (delta Rd), was not different between C and ST pre-training (12.7±2.1 and 9.5±2.5 umol/kg/min in C and ST, p=0.33). C and ST responded differentially to training as indicated by significant interaction (p<0.01). Delta Rd did not change after training in Clinical Science Immediate Publication. Published on 13 May 2010 as manuscript CS20100153 6 C (from 12.7±2.1 to 13.6±1.9 umol/kg/min), but improved significantly in ST (from 9.5±2.5 to 16.3±2.3 umol/kg/min, p<0.01). Non-oxidative glucose disposal (from 4.7±2.1 to 10.0±2.1 umol/kg/min, p<0.01) as well as oxidative glucose disposal (from 4.7±0.9 to 6.5±0.7 umol/kg/min, p<0.01) improved significantly in ST upon training, but did not change in C (tabel 2, with a significant interaction effect between groups for NOGD; p<0.01). Insulin mediated suppression of endogenous glucose production (delta EGP) was comparable pre-training (-6.8±0.8 and -7.1±1.5 umol/kg/min in C and ST respectively) and was not significantly affected by training, despite a significant interaction effect (Table

2

). Data of insulin and glucose disposal is presented in table 2. We did not find correlations between statin potency and other parameters, suggesting that the findings do not scale with the dosage or potency of the statin used. Furthermore, no interaction Clinical Science Immediate Publication. Published on 13 May 2010 as manuscript CS20100153 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100153

Table 1 :

 1 Data are expressed as mean ± SE., #ST significantly different from control group, *post-training significantly different from pre-training, Clinical Science Immediate Publication. Published on 13 May 2010 as manuscript CS20100153

	subject characteristics
		C		ST
	Subject characteristics	Pre-training	Post-training	Pre-training	Post-training
	Age (years)	59.2 ± 1.0		58.4 ± 0.9
	Subjects diagnosed with diabetes	8		8
	Weight (kg)	94.9 ± 2.8	94.4 ± 2.9	93.2 ± 3.2	91.9 ± 3.1
	Height (cm)	177.6 ± 1.3		177.2 ± 1.5
	BMI (kg/m 2 )	30.1 ± 0.8	30.0 ± 0.9	30.0 ± 0.9	29.6 ± 0.8
	Body fat (%)	32.4 ± 1.3	31.6 ± 1.3*	29.6 ± 1.8	28.4 ± 1.8
	FM (kg)	31.2 ± 1.8	30.4 ± 1.8*	27.9 ± 2.3	26.4 ± 2.2
	FFM (kg)	64.5 ± 1.9	64.9 ± 1.9	65.3 ± 2.1	65.5 ± 2.1
	VO2max (ml/min/kg)	28.6 ± 1.1	30.5 ± 1.2*	28.5 ± 1.4	31.7 ± 1.7*
	Wmax (Watt)	208 ± 9	236 ± 9*	201 ± 10	231 ± 10*
	Average strength (kg)	83.5 ± 3.2	102.9 ± 3.7*	87.4 ± 4.0	105.3 ± 4.4*
	Fasting glucose (mmol/l)	7.2 ± 0.4	6.9 ± 0.4	7.5 ± 0.5	7.3 ± 0.6
	HbA1c (%)	6.4 ± 0.2	6.3 ± 0.2	6.6 ± 0.3	6.7 ± 0.3
	Triglycerides (mmol/l)	1.6 ± 0.1	1.6 ± 0.2	1.7 ± 0.2	1.6 ± 0.1
	Total Cholesterol (mmol/l)	5.4 ± 0.1	5.2 ± 0.2	5.0 ± 0.2	4.6 ± 0.2*
	Low density lipoproteins (mmol/l)	3.5 ± 0.1	3.3 ± 0.2	2.9 ± 0.2#	2.7 ± 0.2#
	High density lipoproteins (mmol/l)	1.2 ± 0.0	1.2 ± 0.1	1.3 ± 0.1	1.3 ± 0.1
	HsCRP (mg/l)	2.0±0.3	1.8±0.3	1.6±0.5	1.6±0.5

Table 2 :

 2 Substrate kinetics pre-and post training

		C		ST
		Pre-training	Post training	Pre-training	Post training
	Plasma insulin (mU/l)
	Basal	17.2 ± 2.0	15.7 ± 1.7*	17.9 ± 2.0	15.0 ± 1.7*
	Clamp	109.4 ± 5.4	107.8 ± 4.2	111.2 ± 4.5	107.9 ± 5.1
	Plasma FFA (µmol/l)
	Basal	498.6 ± 18.3	498.7 ± 31.1	473.3 ± 31.0	421.4 ± 27.9
	Clamp	90.2 ± 7.7	81.5 ± 8.2	103.9 ± 10.0	71.1 ± 8.3* §
	Rd glucose (µmol/kg/min)
	Basal	10.0 ± 0.8	8.7 ± 0.7	10.9 ± 0.9	8.7 ± 0.7*
	Clamp	22.7 ± 2.0	22.3 ± 2.1	20.3 ± 2.2	25.0 ± 2.1* §
	Delta	12.7 ± 2.1	13.6 ± 1.9	9.5 ± 2.5	16.3 ± 2.3* §
	EGP (µmol/kg/min)
	Basal	9.7 ± 0.6	9.3 ± 0.6	9.7 ± 0.7	7.9 ± 0.8
	Clamp	2.8 ± 0.4	0.8 ± 0.5*	2.7 ± 1.0	1.8 ± 1.3
	Delta	-6.8 ± 0.8	-8.5 ± 0.7	-7.1 ± 1.5	-6.5 ± 1.5 §
	CHO oxidation (µmol/kg/min)
	Basal	6.8 ± 0.5	7.3 ± 0.5	7.8 ± 0.8	7.0 ± 0.5
	Clamp	11.5 ± 0.7	12.9 ± 0.8	12.6 ± 1.1	13.5 ± 0.7
	Delta	4.7 ± 0.7	5.2 ± 0.

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2010 The Authors Journal compilation © 2010 Portland Press Limited

© 2010 The Authors Journal compilation © 2010 Portland Press Limited

Acknowledgements

The authors gratefully acknowledge the valuable discussions with Prof Dr R.P. Mensink and Dr J. Plat. E. Phielix was supported by a grant of the Dutch Diabetes Research Foundation (grant 2004.00.059). A VICI (grant 918.96.618) and a VIDI (grant 917.66.359) for innovative research from the Netherlands Organization for Scientific Research (NWO) supports the work of P. Schrauwen and M. Hesselink, respectively.


Mark Van De Velde

The Alleged Class 2a Prefix b in Eton: A Plural Word

a plural word.

Introduction

Since the beginnings of Bantu linguistics, the extended gender system of the Bantu languages has received a lot of attention. 1 The focus of this attention has been mainly comparative-historical, sometimes to the detriment of careful synchronic analysis. This paper focuses on the morpheme b in the Cameroonian Bantu language Eton (A71), which should be identified as the nominal prefix of class 2a according to the traditional criteria and terminology. It will be shown that this morpheme is not a prefix but a word, probably a proclitic. Its function is to pluralize a following genderless word. Since the most typical genderless words are proper names and deictically restricted kinship terms (e.g. ta da 'my father'), the result is usually an associative plural. This explains how the succession of the locative preposition a and the plural word could grammaticalise into the complex preposition a b 'chez'. The behaviour of b in Eton confirms some observations that Matthew Dryer (1989) made in his typological study on plural words. The end of this paper presents a brief comparative overview of the class 2a marker in other Bantu languages.

1.

The Noun Class System Eton is a previously undescribed Narrow Bantu language spoken just north of the Cameroonian capital Yaoundé. It belongs to the Beti part of the Beti-Bulu-Fang dialect cluster (also called Pahouin). Its closest relatives are Ewondo and 1 I interpret noun class and gender as alternative terms for the same phenomenon. In Bantu studies the term noun class is usual, but I follow [START_REF] Greville | Gender[END_REF] in using the term gender, except when explicitly referring to the Bantuist tradition. Moreover, I wil use the term class 1a for the set of nouns that is traditionally analysed as a subgender of gender 1, but that I analyse as a group of genderless words in Eton (idem for class 2a). I wish to thank Dmitry Idiatov and my informants Pie-Claude Ondobo and Désiré Essono for their help. Vertical bars mark morphophonological notation. In this notation accentuated syllables are underlined and all morpheme boundaries are marked as follows: # word boundary, = clitic boundary,affix boundary. The following glosses are used: AU augment; CON connective; DEM demonstrative; DIM diminutive; IMP imperative; PL plural; PRES present; SF suffix. Arabic numbers are used for nominal gender prefixes in the glosses, roman numbers for agreement prefixes.

Mengisa. Eton has a typical gender system with ten genders, but lacks locative genders. Table 1 gives an overview. The numbers in the first column refer to the genders reconstructed for Proto-Bantu of which the current Eton genders are the reflex. The second column gives the nominal gender prefix (NPr), the third column the verbal prefixes (VPr) and the fourth column the pronominal prefixes (PPr). The double accent  symbolises the morphotoneme D, which is represented by a high tone, except if preceded by a high tone. In the latter case D is represented by a low tone. The gender prefixes have variable forms depending on the form of the following morpheme. In gender 1 and 3 the nominal prefix is a syllabic homorganic nasal. Before a vowel the homorganic nasal is represented by the phoneme /m/, as in the gender 1 noun m-i na 'woman'. The prefix is not syllabic in that case. Gender 9 and 10 historically had a homorganic nasal nominal prefix. This nasal is only preserved before voiced stops, but there are no morphological arguments for treating it as a prefix in present day Eton. The CVprefixes of gender 2, 4, 6 and 8 are reduced to C-or CG-before morphemes that begin in a vowel. The preconsonantic and prevocalic forms of the gender 5 and gender 7 prefix cannot be reduced to one morphophoneme and must be described as allomorphs. The forms in the two rightmost columns of Table 1 are suppletive agreement targets. The connective morpheme (Con) is a proclitic that relates a head noun to a modifying noun, as in (1). The choice between the segmental and the tonal form of the connective depends on the syllable structure of the modifying word.

( Note that three types of nouns belong to gender 3: nouns with a nasal prefix (3n), nouns with the prefix u -(3u) and nouns that take the prefix a -(3a). The plurals of gender 1 nouns belong to gender 2. Gender 9 words can form their plurals freely in gender 10 or 6. Other common gender pairings are 3n/4, 5/6, 7/8, 3a/6 and 3u/5.

One set of nouns is not represented in Table 1. These are the nouns that have no gender prefix and that trigger the same agreement pattern as gender 1 nouns, as in (4). Their plurals are marked by b and trigger agreement pattern II, the agreement pattern that is also associated to gender 2, as in ( 5). ( 4) dwa bd a -te kwa n doctor 1-PRES be.ill 'The doctor is ill.' ( 5) b dwa bd be -te kwa n PL doctor 2-PRES be.ill 'The doctors are ill.'

These sets of nouns are traditionally called class 1a and class 2a respectively in Bantu studies. Class 1a is treated as a subgender of gender 1, because it has the same agreements, and class 2a as a subgender of gender 2. These subgenders were first signalled by [START_REF] Doke | The Significance of Class 1a of Bantu Bouns[END_REF], who observed that they typically contain kinship terms, proper names, the question word 'who' and borrowings from European languages. This is also the case in Eton.

( It appears to be impossible to reconstruct a form for the class 2a marker in Proto-Bantu. Meeussen (1967:100) tried baa-, but could not decide on the tone. Some scholars (e.g. [START_REF] Maho | A Comparative Study of Bantu Noun Classes[END_REF]:156, Poulos & Louwrens 1994:16) distinguish between class 2a and class 2b based on the form of the marker. Class 2b has a marker with a back vowel ( or o) and is found in most languages of zone S (roughly South-Africa, Botswana, Lesotho and parts of Zimbabwe and Mozambique), in some zone K languages and in some northwestern Bantu languages, such as Eton. The marker of class 2a has the vowel a and is found elsewhere (see the map in Maho 1999:155). I will not make this distinction here for two reasons. First, there are many more lines along which the form of the class 2a/2b marker differs among languages than just vowel quality. Second, the crosslinguistic difference in vowel quality does not reflect any difference in function and morphosyntactic status of the class 2a marker.

2.

bç› is a Plural Word in Eton Contrary to what one expects from a Bantu gender marker, b turns out to be a word in Eton, not a prefix. Its word status can be easily established by means of phonological criteria. This is because the first syllable of every word stem is accentuated. Accentuated syllables have a prominent initial consonant, i.e. a consonant that is longer than the others in intervocalic position and that is not subject to lenition rules. Moreover, they can carry two structural tones, whereas non-accentuated syllables can carry only one. The plural word b clearly differs from prefixes in this respect. In (10-11) two plural nouns are preceded by the locative preposition a , which, according to the general tone rules, copies its high tone onto the following word. Since b is accentuated, it can carry two structural tones, so that the copied high tone can simply add to the original low tone and form a falling tone (10b). The gender 8 prefix bi -, on the other hand, is not accentuated. It is not the first syllable of a stem. Therefore it cannot carry both the copied high tone and the original low tone. Consequently, the copied high tone pushes the low tone of the nominal prefix to the right, where it downsteps the high tone of the noun stem le 'tree' (11b). The phonotactic generalisation that mid back vowels never occur in prefixes provides additional evidence for the word status of b . The important formal differences between b and prefixes such as biraise the question of whether both have the same function. The answer is no. In Van de Velde (2005) I argue that the nouns commonly referred to as class 1a are outside of the gender system. 2 Gender and number are intimately linked. Whether a noun is singular or plural is determined by its gender membership. Therefore, genderless nouns need independent number marking. Their singular is zeromarked, their plural by means of b , which is not a gender marker, but a plural word.

The argument is most easily illustrated by means of proper names. These can be derived from nouns, noun phrases or even clauses by means of the suffix -A. The harmonic vowel |A| is represented by a vowel phoneme identical to that of the preceding stem vowel, except if the latter is close. In that case the harmonic vowel is represented by /a/. If the stem has a structural succession of |u| and |a|, the harmonic vowel is represented by //, according to the regular rules of vowel coalescence in Eton. During the derivation, the source noun is also lifted out of the gender system. The original prefix is incorporated into the stem, the original agreement pattern is replaced by agreement pattern I and the original plural formation by change in gender is replaced by the preposition of b . This is true for all proper names, not only names for persons.

2 Developing the arguments would lead us too far here. The claim is made for Eton only, but might prove to be valid in other Bantu languages as well. Note that I do not use the absence of a gender prefix on class 1a words as an argument. As has been said, gender 9 and 10 nouns do not have a prefix either. In order to accept the possibility of genderless nouns, it is important to acknowledge that agreement patterns can have multiple functions. In Van de Velde (forthcoming) I show that agreement pattern I marks agreement either with nouns of gender 1 or with nouns that are less in need of referential disambiguation. Proper names are a prototypical example of the latter, because they are strictly referential.

3 This peculiar tonal behaviour is historically explainable. Final stem vowels eroded in Eton and the surrounding languages, except in proper names, which tend to be conservative. The final vowel of proper names has been morphologically reinterpreted as a suffix. Rising tones on monosyllabic stems are the result of the historical loss of the final vowel of cvcv-stems with a low-high tone pattern. The examples in (16-18) of proper names for plant species are for different reasons morphosyntactically challenging, but they all confirm the analysis of b as a separate word used to pluralize a genderless element. The name for mimosa in (16a) is a phrase containing an imperative and its object. The plural of this phrasal name is formed by b in (16b). The high tone on the gender prefix of the object is due to high tone copy from the previous verb form. The following downstep shows that (16a) is not a compound. If there were no word boundary between wu and u swa n , then the copied high tone would have deleted the low tone on the following prefix instead of pushing it to the right. The names in (17-18) are noun phrases. In (17) the gender 5 noun  -sa  'hand of bananas' is followed by the numeral 'one', which should normally agree in gender, but which here takes a form of agreement pattern I instead of V. Interestingly, the pluralization of the phrasal name involves the pluralization of the head noun and the addition of the plural word before the entire phrase. The numeral remains in its singular form. Something similar occurs in the name for a kind of chilli pepper (18). This is a connective construction with a head noun expressing a property followed by the connective proclitic (H=) plus a dependent noun. In the plural the head noun is pluralized and the connective morpheme still agrees with it. It is not clear whether the second noun is pluralized, since the plural form of zu d does not differ from the singular. Anyhow, the pluralization of its constituents does not suffice to derive the plural of the phrasal name. The plural word must again be added. There is one word in Eton that behaves similarly, viz. the diminutive proclitic m- H, plural b- H. This word can be put in front of any noun in order to form a diminutive or simulative. A combination of the diminutive word and a noun triggers gender 1 agreement in the singular and gender 2 agreement in the plural. Thus, the plural word forms a minor parts-of-speech category with the diminutive word. These words have in common with nouns that they determine concord, i.e. they belong to a gender and that they occupy the initial position in the noun phrase. They differ from nouns in their limited distribution and syntactic dependence and in their grammatical meaning. 4The Eton data confirm some observations that Matthew Dryer makes in a typological study on plural words (Dryer 1989). In a sample of 307 languages, Dryer found 48 languages in which the category plural is indicated by means of a separate word. These languages lack number as an inflectional category on the noun. Only two of these 48 languages are spoken in Africa, viz. Gbeya (Adamawa-Ubangi) and Yoruba (Benue Congo). The grammatical category of these plural words varies. In some languages the plural word is a numeral or an article. In others, plural words belong to a minor category or constitute a oneword category of their own. Dryer found two languages in his sample in which the plural word belongs to a minor category of noun modifiers: Hixkaryana (a Carib language) and Gbeya. In

The honorific meaning can be found for instance in Venda, as in ( 24), where vho is the class 2a prefix.

(24) Vho-Vele vha khou d a 'Vele is coming.' (Poulos 1990:20) Sometimes the class 2a marker spread to become a general plural marker, replacing or reinforcing markers of plural genders. This happens when the noun class system is disintegrating, as in Kinshasa Lingala (Aikhenvald 2000:388). It can also occur to some extent in language acquisition, as is illustrated by the data in Table 2. These are the results of an experimental study by Kunene (cited via Demuth 1985:311) on SiSwati in which children from 4 1/2 to 6 years old were given novel word forms and SiSwati nouns out of context and asked to provide the corresponding plural form of the noun. The right column show that young children overgeneralise the class 2a marker bo as a plural marker. It replaces the gender 2 and gender 4 prefix and adds to the prefixes of gender 14 and 15.

(25) The forms and uses of the class 2a marker in the Bantu languages are in need of a thorough comparative study. In the mean time this brief overview suggests that the plural word analysis forwarded for Eton might be valid for many other Bantu languages as well. A comparative study should also clarify the origin of the class 2a markers. Denis Creissels (p.c.) suggests that in Tswana the class 2a marker bó is the result of the coalescence of ba and ga, where ba is the connective morpheme of gender 2 and ga the connective of gender 17. The original meaning of bó N was 'the people at N's place'. This is a very attractive hypothesis for Tswana. However, it is not valid for Eton, since the plural word has a low tone, whereas all connectives have a high tone, except that of gender 1.

Conclusion

Based on phonological criteria and the insight that there is a group of genderless nouns in Eton, the morpheme b could be recognised as a plural word. According to traditional criteria, however, it would be incorrectly analysed as the prefix of class 2a, which is said to be a subgender of gender 2. The plural word analysis explains how b was able to combine with the preposition a in order to form a new independent preposition a b 'chez' and how it can pluralize idioms such as the phrasal names in (16)(17)(18). It is likely that a plural word analysis of the class 2a marker works for other Bantu languages as well. In that case, plural words are not as rare in Africa as Dryer's typological survey suggests. The fact that the plural word belongs to a minor parts of speech category together with a diminutive word in Eton confirms some of Dryer's typological observations.

(

  10) a. b ka la da 'books' b. a b ka la da 'in the books' (11) a. bi -le 'trees' b. a bi -le 'in the trees' The phonetic notation in (12) shows that the initial /b/ of b is not subject to lenition in intervocalic position, contrary to the initial /b/ of the gender prefix bi -. (12) a. [a b ka la ra ] 'in the books' b. [a i l ] 'in the trees'

  (13) a. |u a | → /wa / 'stone' [9]stone b. |u a -A | → /wa  / 'Stone (proper name)' stone-SF Usually the suffix has a low tone, but when it is attached to a monosyllabic noun stem with a rising tone, the high part of the stem tone attaches to the suffix. 3 (14) a. |d-u a d| → /dwa d/ 'furuncle' 5-furuncle b. |du a d-A| → /dwa d / 'Furuncle (proper name; elicited, not attested)' furuncle-SF

(

  15) a. |m-u a d| → /mwa d/ 'furuncles' 6-furuncle b. |b # du a d-A| → /b dwa d / 'Furuncle and his family/friends/people' PL furuncle-SF

  (16) a. /wu u swa n / | wu -H # u -su a n-A | die-IMP 3-shame-SF 'plant (sp.), Mimosa Pudica' (lit.: 'die of shame') b. b wu u swa n 'mimosa plants'

(

  17) a. / sa  m mwa  / |  -sa  # N -vu a -A | 5-hand I-one-SF 'Horn plantain (sp.)' b. /b me sa  m mwa  / | b # me -sa  # N -vu a /i b ka la da ba / |i -b # ka la da # ba | AU-PL book 2.DEM 'these books' Since bis the prefix of gender 2 before vowel initial stems, b might be analysable as b- .

(

  21) a. /m  l n a te ku / | m- H= -l n # a -Lte # Lku | 1-DIM=5-palm.tree I-PR fall 'The small palm tree falls.' b. /b me l n be te ku / | b- H=me -l n # be -Lte # Lku | 2-DIM=6-palm.tree 2-PR fall 'The small palm trees fall.' c. /b me jwa / | b- H=me -ju a | 2-DIM=6-wine 'some wine'

Table 2 .

 2 Gender markers in the acquisition of SiSwati.

	class Siswati Experiment
	1/2	umu/ba umu/bo
	1a/2a Ø/bo	Ø/bo
	3/4	umu/imi umu/bo
	5/6	li/ema	li/ema
	7/8	si/ti	si/ti
	9/10 in/tin	i/ti
	11/10 lu/tin	li/ema
	14	bu	bu/bo-bu
	15	ku	ku/bo-ku

The plural word might be a proclitic as well, but the difference between words and proclitics is only visible in Eton if they carry a final high tone.

As seen in the translation of (15b), the plural word usually has an associative meaning when combined with the name of a person. Via this associative meaning b could combine with the general locative preposition a to form the preposition a b , which means 'chez (at or to somebody's place)'. In present day Eton the locative preposition a is incompatible with nouns or pronouns that have human reference, whether classless or not. If b were a gender prefix, it could not have combined with a preceding preposition.

(19) zu a e e y e a b ma , me ya  e come with him to me 1SG.heal him 'Bring him to me, so that I heal him.'

Interestingly, the origin of this preposition is reflected in Cameroonian French.

When speakers tell me that a certain form is used in the dialect of a person we both know, for instance somebody called Désiré, he would tell me "that is how they say it chez les Désiré(s)", with a plural article before the first name of that person.

A Minor Parts-of-Speech Category

This section discusses the grammatical status of the plural word and provides a brief comparison with plural words in other languages. The plural word is always immediately followed by a genderless noun, which may be a phrasal name. It cannot be used in isolation, e.g. deictically or anaphorically. Nothing can be inserted between the plural word and the noun it pluralizes. This is because the order in nominal constituents is strictly head-before-dependent in Eton. From a syntactic point of view the plural word is the head of the noun it pluralizes. It is the plural word that determines gender agreement, not the following noun, which is genderless. Moreover, the augment is prefixed to the plural word, not to the following noun. The augment is a morpheme that is prefixed to the head of a nominal constituent if this head is modified by a demonstrative or a relative clause or if the head is normally a nominal modifier.

Hixkaryana, there are four other words in the category: a diminutive txko meaning 'small' or 'good'; an augmentative, meaning 'big' or 'bad'; a word indicating 'loss of value', and a word which means either 'dead' or 'set of'. In Gbeya there are three other words in the category that also contains the plural word (which is o ): 'even, also, just', 'big, real', and 'a, some, certain, few'. Thus, as in Eton, these minor parts-of-speech categories contain a word for 'small' and/or 'big'. Dryer further notes that in a number of other languages in his sample the category to which the plural word belongs (either articles or numerals) also contains a diminutive word.

Dryer also raises the issue of word order. He compared the order in the pair Plural word -Noun to that in the pair Verb -Object and found that in all VO languages the plural word precedes the noun (as in Eton), whereas the plural word follows the noun in the great majority of OV languages. Dryer proposes two possible explanations for this pattern: either plural words usually are the head of the noun phrase in which they occur, or this pattern confirms his claim that there is no general tendency for modifiers to follow the noun in VO languages and to precede in OV languages. The former explanation, i.e. that the plural word is the head of the following noun (in terms of dependency grammar), turns out to be correct in Eton.

4.

The Class 2a Marker in Other Bantu Languages A superficial glance at descriptions of Bantu languages learns that the so-called class 2a prefix differs very often from the other nominal gender prefixes. In many cases the differences are formal, e.g. a high tone, as in Lega (Botne 2003:427) or a long vowel, as in Mongo [START_REF] Hulstaert | Grammaire du lomongo. Deuxième partie: Morphologie[END_REF]. In Yao the class 2a marker atšiis bisyllabic and in Karanga there are several forms, including madziand vadzi-. In Luganda the class 2a marker is a clitic, rather than a prefix (Hyman and Katamba to appear). In Myene-Nkomi, finally, the class 2a marker is the only gender prefix that lacks an augment [START_REF] Rekanga | Le système myene-nkomi (groupe B10)[END_REF]. Often also the class 2a marker has specific meanings such as honorific or associative plural. Examples of the latter can be found in Mongo (22) and Xhosa (23).

(22) a. baa Byeka 'Byeka and his family/pupils/followers...' b. baa mésá 'tables and similar things; tables, for instance' [START_REF] Hulstaert | Grammaire du lomongo. Deuxième partie: Morphologie[END_REF]:145) (23) a. oontlanzi < oo + iintlanzi 'fish and similar things' b. oozingwe < oo + izingwe 'leopards and similar things' c. oomaRhini < oo + amaRhini 'Grahamstown and environment' d. ookulamba < oo + ukulamba 'hunger and similar feelings' (Hendrikse 1990:391)


MD, PhD)* Yan Ansquer
email: yan.ansquer@sat.aphp.fr

MD)* Sarah Delaney

MD Pietro Santulli

MD Laurence Salomon

MD)* Bruno Carbonne

MD Remy Salmon

Risk of invasive breast cancer after lobular intra-epithelial neoplasia: Review of the literature

Keywords: lobular intraepithelial neoplasia, lobular carcinoma in situ, atypical lobular hyperplasia, breast cancer, review

Objective: Lobular intraepithelial neoplasia (LIN) is a rare breast disease that has been regarded alternately as a risk factor for invasive breast cancer in both breasts or a true breast cancer precursor. The controversy is largely dependent on the estimation of the IBC (Invasive Breast Cancer) risk after LIN; however a systematic review of the published data has not been previously performed. We aimed to review the IBC after LIN and the characteristics of those cancers.

Methods: a PubMed search was performed to identify the published articles in English addressing the breast cancer risk after LIN.

Results: There was a wide range in the figures estimating the risk of the breast cancer among the 22 studies that form the basis of this review. The cumulative average risk of invasive breast cancer (IBC) was 8.7% (range 0-33). It was 4.7% (range 0-25) for the ipsilateral and 4.2% (range 0-16) for the contralateral breast. 52% of the breast cancers occurred more than 10 years after the initial LIN. A lobular histotype was present in 30% (range 0 to 67%) of all IBC.

Conclusions: LIN should be considered both as a risk factor (low and similar level of IBC risk for both breasts, long delay between LIN and IBC) and a precursor for IBC (overrepresentation of lobular histotype).

Introduction

Lobular intra-epithelial neoplasia (LIN), which comprises atypical lobular neoplasia and lobular carcinoma in situ, is a rare disease characterised by non invasive lobular breast proliferation [START_REF] Tavassoli | Lobular Neoplasia[END_REF].

The incidence of LIN, has increased dramatically in recent years, at least partly due to the widespread uptake of mammographic screening [START_REF] Li | Age specific incidence rates of in situ breast carcinomas by histologic type, 1980 to 2001[END_REF].

Since the first description of LIN in 1941 [START_REF] Foote | Lobular carcinoma in situ. A rare form of mammary cancer[END_REF], its treatment has remained controversial; ranging from simple biopsy [START_REF] Ottesen | Carcinoma in situ of the female breast. 10 years follow-up results of a prospective nationwide study[END_REF][START_REF] Goldstein | Clinicopathologic implications of E-cadherin reactivity in patients with lobular carcinoma in situ of the breast[END_REF][START_REF] Page | Atypical lobular hyperplasia as a unilateral predictor of breast cancer risk: a retrospective cohort study[END_REF][START_REF] Fisher | Pathologic findings from the National Surgical Adjuvant Breast and Bowel Project: twelve-year observations concerning lobular carcinoma in situ[END_REF] to ipsilateral mastectomy and contralateral biopsy [START_REF] Rosen | Lobular carcinoma in situ of the breast: preliminary results of treatment by ipsilateral mastectomy and contralateral breast biopsy[END_REF],

or even bilateral mastectomy [START_REF] Leis | The second breast[END_REF]. The treatment choice has largely been dependent on the estimation of the invasive breast cancer risk and on whether LIN was considered as a marker of increased risk of cancer for both breasts (the "risk factor theory") [START_REF] Chuba | Bilateral risk for subsequent breast cancer after lobular carcinoma in situ[END_REF] or a true breast cancer precursor (the "precursor theory") [START_REF] Acs | Differential expression of Ecadherin in lobular and ductal neoplasms of the breast and its biologic and diagnostic implications[END_REF].

This review addresses the evaluation of the risk of invasive breast cancer (IBC) and ductal carcinoma in situ (DCIS) following a diagnosis of LIN. It also examines whether the characteristics of the cancers diagnosed following LIN favour "the risk factor" or "the precursor" theory.

Methods

A PubMed search was carried out on January 31, 2010. The search strategy consisted of terms "lobular intra-epithelial neoplasia", "lobular carcinoma in situ", "atypical lobular hyperplasia", combined with "breast cancer", "breast carcinoma". The search was limited to articles published in English from 1969 to the search date and resulted in an output of 1761 articles. The available abstracts were reviewed for relevance to the topic of breast cancer risk of original series of patients after LIN diagnosis.

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4 Since LIN was in most cases an incidental finding, it was often associated with various benign breast disease conditions that had led to the breast biopsy. However patients were only included when LIN was considered the most risk-associated pathological finding. Patients with associated DCIS and invasive breast cancers were excluded.

Where several articles referred to the same patient's series, only the last publication was taken into account. The 22 articles which met these criteria form the basis of this report.

The publications of Chuba et al [START_REF] Chuba | Bilateral risk for subsequent breast cancer after lobular carcinoma in situ[END_REF] and Li et al [START_REF] Li | Risk of invasive breast carcinoma among women diagnosed with ductal carcinoma in situ and lobular carcinoma in situ, 1998-2001[END_REF] referred both to the SEER (Surveillance, Epidemiology, and End Results Program) with an overlapping in inclusion dates (respectively 1973 to 1998 and 1988 to 2002). The overlapping time frame prevented us from using both studies in the risk estimations. For the estimations of breast cancer risk after LIN (Tables 1 and2) we chose to present as "Total 1" the results of all studies including Chuba et al but

Results

Cumulative risk of breast cancer after LIN

For a women diagnosed with LIN, the cumulative average risk of metachronous breast cancer (IBC or DCIS) was 9.6% (range 0-32). The average risk of IBC was 8.7% (range 0-33) whereas the average risk of DCIS was 2.9% (range 0-13) (Table 1).

The average risk for IBC for the ipsilateral breast was 4.7% (range 0-25) which was (although slightly greater) near the level of risk for the contralateral breast that was 4.2% (range 0-16).

The difference in risk between the two breasts was more pronounced for the risk of DCIS that M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 5 was 2.3% (range 0-10) for the ipsilateral breast and 0.9% (range 0-4) for the contralateral breast (Table 2).

Relative risk of invasive breast cancer after LIN

To answer the question whether LIN modifies the IBC risk in a woman it is necessary to compare the observed IBC incidence in LIN patients with that of the general population.

Table 3 summarizes the studies, which compared the risk of IBC in LIN patients to the risk of a referent population. Only studies with available 95% confidence intervals are reported. The relative risk was calculated by the ratio of the IBC rates observed in the LIN patients to the expected rates if IBC incidence was the same in LIN patients as it is in the population at large. The expected number of females developing IBC after LIN was calculated per year of diagnosis and age group [START_REF] Chuba | Bilateral risk for subsequent breast cancer after lobular carcinoma in situ[END_REF][START_REF] Bodian | Lobular neoplasia: long term risk of breast cancer and relation to other factors[END_REF]. All studies were concordant to demonstrate an excess of IBC risk in LIN patients, with a relative risk ranging from 2.4 to 11.9.

Modifiers of invasive breast cancer risk after LIN

Various qualitative and quantitative pathological features were evaluated for association with IBC risk after LIN [START_REF] Ottesen | Carcinoma in situ of the female breast. 10 years follow-up results of a prospective nationwide study[END_REF][START_REF] Goldstein | Clinicopathologic implications of E-cadherin reactivity in patients with lobular carcinoma in situ of the breast[END_REF][START_REF] Fisher | Pathologic findings from the National Surgical Adjuvant Breast and Bowel Project: twelve-year observations concerning lobular carcinoma in situ[END_REF][START_REF] Bodian | Lobular neoplasia: long term risk of breast cancer and relation to other factors[END_REF][START_REF] Wheeler | Lobular carcinoma in situ of the breast. Long term follow-up[END_REF][START_REF] Haagensen | Lobular neoplasia (so called lobular carcinoma in situ) of the breast[END_REF][START_REF] Rosen | Lobular carcinoma in situ of the breast[END_REF]. Higher IBC risk was only found to be associated with intense lymphocytic reaction in lobules with LIN [START_REF] Rosen | Lobular carcinoma in situ of the breast[END_REF], larger nuclear size [START_REF] Ottesen | Carcinoma in situ of the female breast. 10 years follow-up results of a prospective nationwide study[END_REF], and pathological grade 3 or 2 according a three tired grading system [START_REF] Fisher | Pathologic findings from the National Surgical Adjuvant Breast and Bowel Project: twelve-year observations concerning lobular carcinoma in situ[END_REF].

Age at LIN diagnosis also modified the IBC risk in the study by Bodian et al, with an increased risk for women younger than 40 years and a decreased risk for women older than 55 years [START_REF] Bodian | Lobular neoplasia: long term risk of breast cancer and relation to other factors[END_REF]. However this result was not confirmed in other studies [START_REF] Chuba | Bilateral risk for subsequent breast cancer after lobular carcinoma in situ[END_REF][START_REF] Li | Risk of invasive breast carcinoma among women diagnosed with ductal carcinoma in situ and lobular carcinoma in situ, 1998-2001[END_REF][START_REF] Andersen | Lobular carcinoma in situ of the breast: an approach to rational treatment[END_REF][START_REF] Levi | Invasive breast cancer following ductal and lobular carcinoma in situ of the breast[END_REF].

No association could be demonstrated between IBC risk after LIN and ethnicity, reproductive factors, or family history [START_REF] Chuba | Bilateral risk for subsequent breast cancer after lobular carcinoma in situ[END_REF][START_REF] Li | Risk of invasive breast carcinoma among women diagnosed with ductal carcinoma in situ and lobular carcinoma in situ, 1998-2001[END_REF][START_REF] Bodian | Lobular neoplasia: long term risk of breast cancer and relation to other factors[END_REF][START_REF] Rosen | Lobular carcinoma in situ of the breast[END_REF].

Characteristics of invasive breast cancers after LIN

Fifty-two percent of breast cancer (BC) occurred more than 10 years after the initial diagnosis of LIN was made (Table 4). Compared to ipsilateral BC, contralateral BC were more patients was not dependent on the follow-up [START_REF] Chuba | Bilateral risk for subsequent breast cancer after lobular carcinoma in situ[END_REF][START_REF] Li | Risk of invasive breast carcinoma among women diagnosed with ductal carcinoma in situ and lobular carcinoma in situ, 1998-2001[END_REF][START_REF] Bodian | Lobular neoplasia: long term risk of breast cancer and relation to other factors[END_REF][START_REF] Haagensen | Lobular neoplasia (so called lobular carcinoma in situ) of the breast[END_REF][START_REF] Andersen | Lobular carcinoma in situ of the breast: an approach to rational treatment[END_REF][START_REF] Levi | Invasive breast cancer following ductal and lobular carcinoma in situ of the breast[END_REF]. The excess of risk was almost identical up to 20 years after the LIN diagnosis [START_REF] Chuba | Bilateral risk for subsequent breast cancer after lobular carcinoma in situ[END_REF][START_REF] Bodian | Lobular neoplasia: long term risk of breast cancer and relation to other factors[END_REF][START_REF] Andersen | Lobular carcinoma in situ of the breast: an approach to rational treatment[END_REF]. Beyond twenty years of follow-up there were too few patients to provide valid estimations.

A lobular histotype was present in 30% (range 0 to 67) of all IBC recurrences (Table 1). A lobular histotype was more frequent for ipsilateral IBC (51% range 0 -100) than for contralateral IBC (41% range 0 -60).

The topography of ipsilateral IBC recurrences were only reported in 3 studies. In the study by Carson et al, one out of three recurrences were in the same quadrant as the initial LIN [START_REF] Carson | Lobular carcinoma in situ: observation without surgery as an appropriate therapy[END_REF]. In the study by Cutuli et al [START_REF] Cutuli | Breast conserving surgery and radiotherapy : a possible treatment for lobular carcinoma in situ ?[END_REF], the sole ipsilateral recurrence was in the same quadrant as the initial LIN and in the series reported by Fisher et al, all the nine ipsilateral recurrences were in the same quadrant [START_REF] Fisher | Pathologic findings from the National Surgical Adjuvant Breast and Bowel Project: twelve-year observations concerning lobular carcinoma in situ[END_REF].

Discussion

In this review the cumulative average risk of metachronous IBC was 8.7% but with a wide variation from study to study, ranging from 0 to 33%. The risk was slightly greater for the ipsilateral as compared to the contralateral breast. Tumors characteristics supported arguments both for considering LIN as a non obligatory breast cancer precursor and as a risk factor for bilateral breast cancer.

Before interpreting results of this study, it is important to acknowledge its limitations. Most of the follow-up studies of women with LIN are both small and retrospective. Studies have differed with respect to several factors including criteria for patient selection, length of follow-up, treatment of LIN and periods of inclusion (spanning from 1940 [START_REF] Hutter | Lobular carcinoma in situ: long term follow up[END_REF] to 2002 [START_REF] Li | Risk of invasive breast carcinoma among women diagnosed with ductal carcinoma in situ and lobular carcinoma in situ, 1998-2001[END_REF] ). were inconsistently reported [START_REF] Wheeler | Lobular carcinoma in situ of the breast. Long term follow-up[END_REF][START_REF] Andersen | Lobular carcinoma in situ of the breast: an approach to rational treatment[END_REF][START_REF] Chuba | Bilateral risk for subsequent breast cancer after lobular carcinoma in situ[END_REF][START_REF] Li | Risk of invasive breast carcinoma among women diagnosed with ductal carcinoma in situ and lobular carcinoma in situ, 1998-2001[END_REF], and in some series it was not possible to distinguish DCIS and IBC [START_REF] Bodian | Lobular neoplasia: long term risk of breast cancer and relation to other factors[END_REF]. We only recorded metachronous breast cancers, however the definition differed slightly from study to study using the term metachronous to define breast cancers occurring either more than 6 or 12 months after the initial LIN diagnosis [START_REF] Fisher | Pathologic findings from the National Surgical Adjuvant Breast and Bowel Project: twelve-year observations concerning lobular carcinoma in situ[END_REF][START_REF] Li | Risk of invasive breast carcinoma among women diagnosed with ductal carcinoma in situ and lobular carcinoma in situ, 1998-2001[END_REF].

Histological diagnoses were oftently made by community pathologists at different periods of

Another limitation of the current study was the impossibility due to the study design to control for the confounding factors in the estimation of IBC risk, particularly the length of follow-up and the initial treatment of the LIN.

However, the greatest limitation of the LIN series that form the basis of our review is the rarity of the disease. In our study the estimations of IBC risk are based on more than 6000 patients diagnosed with LIN. When Foote and Stewart described LIN it was viewed as a malignancy requiring a mastectomy [START_REF] Foote | Lobular carcinoma in situ. A rare form of mammary cancer[END_REF]. However, retrospective studies of archived breast biopsy specimens originally read as benign and diagnosed as LIN on review showed that most women had not developed breast cancer even after long follow-up and that invasive breast cancer did not necessarily arise in the breast where the LIN was initially diagnosed [START_REF] Haagensen | Lobular neoplasia (so called lobular carcinoma in situ) of the breast[END_REF]. This results were confirmed by series of patients treated with excisional biopsy alone. In those series the 20 years cumulative risk was up to 33%, and women with LIN were equally likely to be diagnosed with ipsilateral and contralateral invasive breast cancers [START_REF] Wheeler | Lobular carcinoma in situ of the breast. Long term follow-up[END_REF][START_REF] Haagensen | Lobular neoplasia (so called lobular carcinoma in situ) of the breast[END_REF][START_REF] Rosen | Lobular carcinoma in situ of the breast[END_REF][START_REF] Mcdivitt | In situ lobular carcinoma: a prospective follow-up study indicating cumulative patient risks[END_REF]. The LIN was then considered as a marker of breast cancer risk. The only logical approaches under this "risk factor theory" were either a close follow-up or a bilateral mastectomy. However, bilateral mastectomy was unnecessary for the vast majority of patients, and this procedure never achieved great popularity even by the time when conservative procedures were developed for IBC [START_REF] Goldschmidt | Lobular carcinoma in situ of the breast[END_REF].

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8 More recently the "precursor theory" has gained popularity since it was demonstrated that LIN and invasive lobular neoplasia share the lack of expression of the E-cadherin adhesion cell molecule. This latter finding suggests a possible transformation of LIN into invasive lobular neoplasia [START_REF] Acs | Differential expression of Ecadherin in lobular and ductal neoplasms of the breast and its biologic and diagnostic implications[END_REF].

In our study the relatively low level of risk for invasive breast cancer and the time distribution of the recurrences with more than 50% of BC occurring more than 10 years after the initial LIN were in favour of the risk factor theory. Another major argument was the risk of invasive contralateral breast cancer that was near the level of risk for ipsilateral invasive breast cancer. Some authors have argued that contralateral breast cancers risk was explained by the frequent bilaterality of LN. However, similar levels of risk were observed in series were contralateral breast biopsies were systematically performed at the time of the LIN diagnosis [START_REF] Rosen | Lobular carcinoma in situ of the breast: preliminary results of treatment by ipsilateral mastectomy and contralateral breast biopsy[END_REF]. Another argument for the "risk factor theory" was that the majority of IBC following LIN do not contain any invasive lobular histotype (Table 1).

On the other hand some results of our study clearly advocate the precursor theory. Although the average level of IBC risk was low, there was a wide range in the risk between studies. The highest risks were observed in studies where no surgical procedure other than the initial excision biopsy were performed. It has been demonstrated that residual LIN was present in the majority of cases after excisional biopsy [START_REF] Warner | Lobular carcinoma of the breast[END_REF]. This results suggested that the removal of the initial LIN may be beneficial. As previously stated the risk of IBC for the ipsilateral breast was near the level observed for the contralateral breast. However, in most series it was slightly lower (Table 2). This could be in keeping with an evolution of the LIN into IBC in some women. Although not predominant, lobular invasive breast cancer was over represented in IBC recurrences since it was observed in 30% of cases, whereas it only represents 16% of IBC in the general population [START_REF] Tavassoli | Lobular Neoplasia[END_REF]. Moreover lobular invasive breast cancers were more frequent in the ipsilateral breast (51%) as compared to the contralateral (41%). Finally the M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 9 topography of the ipsilateral IBC recurrences brought another argument for the precursor theory since there were in most cases in the same quadrant as the initial LIN [START_REF] Fisher | Pathologic findings from the National Surgical Adjuvant Breast and Bowel Project: twelve-year observations concerning lobular carcinoma in situ[END_REF].

In this review LIN patients had a relative risk of IBC that was 2 to 4 folds that of the general population, except in the study of Andersen et al [START_REF] Andersen | Lobular carcinoma in situ of the breast: an approach to rational treatment[END_REF]. Since the majority of LIN patients will ultimately not develop an IBC, the identification of subgroups of patients at increased risk would be helpful to tailor the initial treatment. Studies that have attempted to identify IBC risk modifiers in LIN patients have however yielded either inconclusive or conflicting results.

An exception is exemplified by the pathological grading system proposed by Fisher et al [START_REF] Fisher | Pathologic findings from the National Surgical Adjuvant Breast and Bowel Project: twelve-year observations concerning lobular carcinoma in situ[END_REF], which however requires confirmation in further studies.

Conclusions

This review of the current data on LIN presents strong arguments for LIN both as a precursor and a risk factor for IBC. In the author's opinion, consideration of the precursor argument makes it logical to attempt to remove the LIN. However, the risk factor aspect with a low level of risk and a risk for contralateral breast cancer argue in our opinion for a lumpectomy rather than a mastectomy, and a close follow-up. Further evaluations are required to evaluate the potential benefit of the removal of the LIN, the role of free margins, and to identify IBC risk modifiers among LIN patients.

excluding

  Li et al, and as "Total 2" the results of all studies including Li et al but excluding Chuba et al. In the results and discussion sections we chose to refer to the results of all studies including Chuba et al and excluding Li et al, since the risk estimations are greater than those obtained with Li et al (except for the risk of ipsilateral IBC).

  M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 6 frequently delayed. Contralateral BC were less frequent during the first five years and more frequent after 10 years. Compared to the general population the relative risk of IBC in the LIN

  of histologic types is likely to have occurred. DCIS following LIN

Table 3 :

 3 Relative risk of invasive breast cancer (IBC) after lobular intraepithelial neoplasiaLegend: The ratio observed to expected (number of IBC observed in the study group of women with a past history of LIN to the number of IBC expected in this group on the basis of incidence rates in the general population) was expressed either as the Relative Risk (*), the hazard ratio (**) or the Standardized Incidence Ratio ( §), according to the study design.

	(LIN)
	author, year	number of	number of	Ratio observed to	95% confident
		observed IBC	expected IBC	expected	interval
	Andersen, 1977	11	0.9	11.9*	6.3 -19.2
	[17]	-	-	2.6**	1.7 -3.9
	Page, 2003 [6]	8	1.7	4.2 §	2.1 -7.5
	Levi, 2005 [18]	-	-	2.4 §	2.1 -2.3
	Chuba, 2005 [10]

Table 4 .

 4 Time interval until subsequent carcinoma after lobular intraepithelial neoplasia (LIN)

	Author,		Overall cancers		Ipsilateral cancers		Contralateral cancers
	year	N	1 to	6 to	>10	N	1 to	6 to	>10	N	1 to	6 to	>10
			≤5 yrs	≤10	yrs		≤5 yrs	≤10	yrs		≤5 yrs	≤10	yrs
				yrs				yrs				yrs
	Hutter,	20	6	4	10	14	5	2	7	6	1	2	3
	1969 [21]
	Wheeler,	4	0	2	2	1	0	0	1	3	0	2	1
	1974 [14]
	Andersen,	12	3	3	6	8	3	1	4	4	0	2	2
	1977 [17]
	Haagensen,	35	15	6	14	19	6	5	8	16	9	1	6
	1978 [15]
	Rosen,	35	3	7	25	19	2	3	14	16	1	4	11
	1978 [16]
	Ringberg,	1	1	0	0	1	1	0	0	0	0	0	0
	1991 [27]
	Graham,	1	1	0	0	1	1	0	0	0	0	0	0
	1991 [28]
	Ciatto,	-	-	-	-	4	3	1	0	-	-	-	-
	1992 [29]
	Carson,	3	2	1	0	3	2	1	0	0	0	0	0
	1994 [19]
	Zurrida,	10	5	5	0	4	3	1	0	6	2	4	0
	1996 [30]
	Bodian,	62	10	19	33	-	-	-	-	-	-	-	-
	1996 [13]
	Goldstein,	21	2	5	14	-	-	-	-	-	-	-	-
	2001 [5]
	Fisher,	19	8	7	4	9	5	4	0	10	3	3	4
	2004 [7]
	Cutuli,	2	1	0	1	1	0	0	1	1	1	0	0
	2005 [20]
	Chuba,	265 34	83	148 99	13	34	52 166 21	49	96
	2005 [10]
	Total	490 91	144	257	183 44	52	87	228 38	67	123
			(19%)	(29%)	(52%)		(24%)	(28%)	(48%)		(17%)	(29%)	(54%)

Conflict of interest statement

The authors state that they have no conflict of interest M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 14 30.Zurrida S, Bartoli C, Galimberti V, Raselli R, Barletta L. Interpretation of the risk associated with the unexpected finding of lobular carcinoma in situ. Ann Surg Oncol 1996; 3: 57-61.

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-Carson, 1994 [START_REF] Carson | Lobular carcinoma in situ: observation without surgery as an appropriate therapy[END_REF] 7* 60 9

7* 51 -Expression of follow-up:*median, §mean, **minimal and maximal follow-up -"Women at risk" for the ipsilateral breast, or for the contralateral breast, are women who at the time of LIN diagnosis had no mastectomy of the ipsilateral breast or of the contralateral breast, respectively.

-rec: recurrence


MD F Derkx

A J G Maaskant-Braat

M J C Van Der Sangen

G A P Nieuwenhuijzen

L V Van De Poll-Franse

R M H Roumen

A C Voogd

Staging and management of axillary lymph nodes in patients with local recurrence in the breast or chest wall after a previous negative sentinel

Keywords: Breast carcinoma, Axilla, Sentinel lymph node biopsy, Lymph node dissection, Local recurrence, staging

Staging and management of axillary lymph nodes in patients with local recurrence in the breast or chest wall

after a previous negative sentinel node procedure

Introduction

The axillary lymph node status is an important prognostic factor for disease recurrence and survival in patients with primary breast cancer. During the last decade sentinel lymph node biopsy (SNB) has become a routine procedure in the management of patients with early breast cancer. Numerous follow-up studies have shown that it is safe to omit axillary lymph node dissection (ALND) in patients with tumour-negative sentinel lymph nodes (SLNs). [START_REF] Roumen | Treatment of 100 patients with sentinel nodenegative breast cancer without further axillary dissection[END_REF][START_REF] Naik | The risk of axillary relapse after sentinel node biopsy for breast cancer is comparable with that of axillary lymph node dissection: a follow-up study of 4008 procedures[END_REF][START_REF] Veronesi | Sentinel-lymph-node biopsy as a staging procedure in breast cancer: update of a randomised controlled study[END_REF] However, during follow-up 5 to 15 percent of these patients will ultimately develop a local recurrence on the chest wall or in the breast after mastectomy or breast-conserving therapy. [START_REF] Voogd | Differences in risk factors for local and distant recurrence after breast-conserving therapy of mastectomy for stage I and II breast cancer: pooled results of two large European randomized trials[END_REF][START_REF] Jacobsen | Ten-year results of a comparison of conservation with mastectomy in the treatment of stage I and II breast cancer[END_REF] The number of patients with local recurrence (LR)

appearing after a previous negative SNB is still small, but will increase with the expansion of the cohort of patients who undergo SNB. Currently, little is known about the best clinical approach of the axillary lymph nodes in these patients, especially when there is no clinical suspicion of tumour involvement of the axilla. We postulate that in the absence of evidence-based treatment guidelines, different strategies are in use. Although several studies have shown that a repeat SNB is technically feasible and can provide useful information, [START_REF] Roumen | Lymphatic mapping and sentinel node harvesting in patients with recurrent breast cancer[END_REF][START_REF] Port | Reoperative sentinel lymph node biopsy: a new frontier in the management of ipsilateral breast tumor recurrence[END_REF] it remains questionable whether it is a safe alternative to ALND.

The aim of this study was to evaluate axillary staging and management in patients who developed a local recurrence after a previous negative SNB, performed in 14 hospitals in the southern part of The Netherlands.

Patients and methods

Patients were identified by making use of the population based Eindhoven Cancer Registry, which records data on all patients newly diagnosed with cancer in the southern part of The Netherlands, an area with approximately 2.4 million inhabitants. Patient data were derived from 14 hospitals, consisting of large non-university teaching hospitals and community hospitals, two radiotherapy departments and 6 pathology laboratories.

SNB was introduced in the south of The Netherlands in 1995. In 1997, surgeons started performing SNB as a routine staging procedure and since the year 2000 indications for SNB have been described in national guidelines. [START_REF]Oncoline Oncological Guidelines[END_REF] During the period of 1997-2006, 6800 patients with an invasive breast carcinoma underwent SNB in the South of the Netherlands. Of these, 4094 had negative SLNs and underwent no ALND.

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In 1989 the Eindhoven Cancer Registry started collecting follow-up information on all patients with breast cancer diagnosed in the 7 hospitals in the eastern part of the region, including the data and site of local, regional and distant recurrence. The information is provided by the pathologists, radiotherapists and the majority of the surgeons. Since 2004 follow-up information has also been collected for patients diagnosed in the other 7 hospitals in the western part of the region covered by the Eindhoven Cancer Registry.

Until September 2008, 130 women with a locoregional recurrence had been recorded by the cancer registry among the 4094 patients with a negative SNB. After examination of clinical records, 60 patients were excluded. Thirty-six patients had distant metastasis in stead of or prior to LR, 15 patients had axillary metastasis only and medical records of 9 patients could not be retrieved. Eventually 70 patients with a LR remained available for analysis. The medical records were used to collect detailed information on the treatment of the primary tumour and the characteristics of the local recurrence, including the diagnostic procedures and different treatment strategies used for the axilla.

Results

Patient characteristics

Between 2000 and 2008 seventy patients were treated for an isolated local recurrence after negative SLB in the Southeast region of The Netherlands. Characteristics of these patients are presented in Table 1. Diagnosis of the primary tumour took place between 1999 and 2006. The median age at diagnosis of the primary tumour was 58 years (range 29-84 years). The median interval between diagnosis of the primary tumour and diagnosis of local recurrence was 40 months (range 7-99 months). Fifty-two patients underwent breast-conserving surgery at time of primary tumour and 49 of them received adjuvant radiotherapy of the breast. Chemotherapy and/or hormonal therapy were given to 22 patients.

Management of the axilla

The different staging and treatment strategies of the axilla are presented in Table 2. Thirty-seven patients underwent ALND, followed by axillary radiotherapy in 9 cases. In 26 of these 37 patients no

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT
positive axillary lymph nodes were found. Patients received axillary radiotherapy because of tumour involvement of the lymph node at the most cranial site of the axillary surgical specimen or because of an irradical resection, according to current guidelines. In one patient, the nodal status could not be given, because the axillary tissue had been completely invaded by tumour tissue and separate lymph nodes could not be identified.

In 13 patients a second SNB was attempted (Table 3). Twelve of the 13 patients had undergone breast conserving therapy (BCT) for their primary tumour. The SNB was successful in 5 patients and one of them was positive. Eight patients underwent a complementary ALND, including the patient with a positive SLN. Two of these patients turned out to have positive axillary lymph nodes.

One patient, who presented without clinically suspicious findings in the axilla and refused to undergo surgery of the axilla, received locoregional radiotherapy without any surgical intervention. Nineteen patients received no treatment of the axilla. Of those, 9 were older than 70 years of age at diagnosis of LR. Distant metastases were detected simultaneously with local recurrence in three of them. In one, ALND was attempted, but was not successful because of fibrosis, probably originated at the time of the primary tumour.

Findings of axillary staging

Pre-operative staging of the axilla by using ultrasound was performed in 14 patients and 5 of them had suspicious findings. Two other patients had suspicious findings in the axilla at clinical examination and one at both clinical examination and ultrasound. All 7 patients with suspicious findings underwent ALND, except for one patient, who already had distant metastasis at time of diagnosis of LR. Positive lymph nodes were found in 3 of the patients who underwent ALND. Overall, positive lymph nodes were detected in 13 of the 50 patients who underwent axillary lymph node staging, either by SNB or ALND.

Follow-up

The median length of follow-up of the 70 patients following their diagnosis of LR was 24 months (range 2-81 months). During this follow-up period one patient developed an axillary recurrence. This was the patient who refused to undergo ALND but was given locoregional radiotherapy instead.

Discussion

Our results show that, in the absence of guidelines, different treatment strategies are currently being used for patients with LR of the breast or chest wall after an initial negative SNB, ranging from second SNB and ALND with or without locoregional radiotherapy to no treatment at all. Despite the relatively short follow up, the size of our patient series is quite unique and clearly demonstrates the need for consensus in the management of the axilla at diagnosis of LR of the breast or chest wall.

Reasons to stage the axilla

In our study 13 of the 50 patients who underwent axillary staging appeared to have positive lymph nodes. This is only slightly lower than the percentage of patients showing positive axillary lymph nodes at the time of diagnosis of the primary tumour, 9,10 for whom there is no discussion about the value of axillary staging. To decide if axillary staging should also be a routine procedure in patients with local recurrence following previous negative SNB, we must first consider why staging is done.

Staging can provide useful prognostic information which may help to decide about the need for systemic treatment. In case of a positive oestrogen and/or progesterone receptor status however, patients with local recurrence are candidates for hormonal treatment anyway, irrespective of their axillary nodal status. This decision is supported by results from a randomized controlled trial, which showed an increase of 5-year disease-free survival for patients with local recurrence treated with tamoxifen compared to observation alone (Borner 1994). [START_REF] Borner | First isolated locoregional recurrence following mastectomy for breast cancer: results of a phase III multicenter study comparing systemic treatment with observation after excision and radiation. Swiss Group for Clinical Cancer Research[END_REF] Until now, the effectiveness of chemotherapy in patients with local recurrence has remained unclear, [START_REF] Haylock | Locoregional first recurrence after mastectomy: prospective cohort studies with and without immediate chemotherapy[END_REF][START_REF] Rauschecker | Systemic therapy for treating locoregional recurrence in women with breast cancer[END_REF] and a randomized study is ongoing to determine the value of chemotherapy in this setting. [START_REF] Aebi | A randomized clinical trial of adjuvant chemotherapy for radically resected locoregional relapse of breast cancer[END_REF] So, at this moment, the possible need for adjuvant systemic treatment is not a convincing reason to re-stage the axilla in patients with local recurrence.

The second reason to treat the axilla is to improve locoregional disease control. Several trials studying the effects of additional locoregional therapy in patients with high-risk breast cancer have shown an improvement in survival, together with a decrease in the risk of locoregional recurrences.

15-

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Derkx et al. 7 [START_REF] Orr | The impact of prophylactic axillary node dissection on breast cancer survival -a Bayesian meta-analysis[END_REF] So, the primary reason to re-stage the axilla in patients with local recurrence is to improve locoregional disease control, which in turn could contribute to the reduction of breast cancer mortality.

Axillary staging in the elderly

Almost half of patients not receiving any axillary treatment at time of LR was older than 70 years of age. Different opinions exist about the influence of age on the need to perform axillary surgery at time of breast cancer. Several randomized trials compared ALND with no ALND in elderly women with oestrogen-receptor-positive and clinically node-negative primary breast carcinoma. There were no differences in axillary recurrence rate, distant metastases or survival after 5 to 15 years of follow up. [START_REF] Martelli | Elderly breast cancer patients treated by conservative surgery alone plus adjuvant tamoxifen: fifteen-year results of a prospective study[END_REF][START_REF] Hughes | Lumpectomy plus tamoxifen with or without irradiation in women 70 years of age or older with early breast cancer[END_REF] Furthermore, ALND does not usually affect systemic treatment choice in the elderly. [START_REF] Wildiers | Management of breast cancer in elderly individuals: recommendations of the International Society of Geriatric Oncology[END_REF] This might implicate that it is appropriate to omit axillary staging in the majority of elderly patients, especially in those with a proven receptor-positive tumour.

Axillary dissection versus sentinel node biopsy

Once the decision to re-stage the axilla has been made, the next question is how this should be done. ALND is generally considered a safe procedure, but it is also associated with significant morbidity. Up to 50% of the patients undergoing ALND have been reported to suffer from complications, such as seroma formation, lymphoedema and shoulder dysfunction with a subsequent negative impact on quality of life. [START_REF] Ververs | Risk, severity and predictors of physical and psychological morbidity after axillary lymph node dissection for breast cancer[END_REF] SNB is associated with a significant reduction in physical and psychological morbidity, as compared to ALND. [START_REF] Schijven | Comparison of morbidity between axillary lymph node dissection and sentinel node biopsy[END_REF] For patients with primary breast cancer and clinically negative lymph nodes SNB has been proven to be a safe and valid alternative to ALND. However, in patients with LR after negative SNB at time of primary tumour adequate lymph drainage might be obscured by previous surgery and radiotherapy, and for them the role of repeat SNB is not clear yet. In our study a SLN was identified in only 5 of 13 patients undergoing repeat SNB. Other studies have reported much higher identification rates, ranging from 65-97%. [START_REF] Karam | Reoperative sentinel lymph node biopsy after previous mastectomy[END_REF][START_REF] Cox | Use of reoperative sentinel lymph node biopsy in breast cancer patients[END_REF][START_REF] Intra | Second axillary sentinel node biopsy for ipsilateral breast tumour recurrence[END_REF] This difference might be due to sampling error because of the small number of patients undergoing SNB in our study. It could also be related to the level of experience of the various surgeons from the different hospitals or to different techniques used for injection of radioactive material and blue dye.

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

Pre-operative ultrasound of the axilla Only 7 of 70 patients had clinically suspicious findings in the axilla. A remarkable finding was that the large majority of the patients did not undergo standard axillary ultrasound investigation. In the absence of radiology reports we relied on information provided by surgeons and radiotherapists and therefore some underreporting may have resulted. In the latest version of the Dutch breast cancer guidelines, ultrasound of the axilla is recommended for all patients with pathologically confirmed breast carcinoma. [START_REF]Oncoline Oncological Guidelines[END_REF] We can not think of any good reason not to perform a routine ultrasound of the axilla as part of a re-staging procedure in patients with a LR.

Conclusion

The rate of positive axillary lymph nodes among patients with local recurrence after breast surgery and negative SNB is too high to be ignored. Therefore repeat surgical staging is appropriate in these patients. Whether SNB is accurate enough in these patients has to be evaluated in further, larger studies.

  in 15 patients. In 5 patients distant metastases were discovered at the time of local recurrence. Seven patients died from breast cancer, after a median follow-up time of 34 months (range 3-42 months) after diagnosis of LR.

  -Braat AJ, Roumen RM. Sentinel Node And Recurrent Breast cancer (SNARB). BOOG 2009-02, NTR1450 (SNARBgroup@gmail.com).

Table 1

 1 Characteristics of 70 patients with local recurrence (LR) following a negative sentinel lymph node biopsy (SNB) at the time of diagnosis of the primary tumour

	Characteristics

Table 2

 2 Overview of different treatment strategies of the axilla in 70 patients with local recurrence and a negative sentinel lymph node biopsy (SNB) at the time of diagnosis of the primary tumour.

	ALND	SNB alone	SNB + ALND Axillary RT	No treatment
			alone

Conflict of interest: the authors state that they have no conflict of interest.