Giulia Chinetti-Gbaguidi

Morgane Baron

Mohamed Amine Bouhlel

Jonathan Vanhoutte

Corinne Copin

Yasmine Sebti

Bruno Derudas

Thérèse Mayi

Gael Bories

Anne Tailleux

Stephane Haulon

Christophe Zawadzki

Brigitte Jude

Bart Staels
email: bart.staels@pasteur-lille.fr

Human atherosclerotic plaque alternative macrophages display low cholesterol handling but high phagocytosis due to distinct activities of the PPARγ γ γ γ and LXRα α α α pathways

Keywords: Atherosclerosis, macrophages, nuclear receptors, cholesterol Non-standard Abbreviations and Acronyms: None

Rationale -A crucial step in atherogenesis is the infiltration of the sub-endothelial space of large arteries by monocytes where they differentiate into macrophages and transform into lipid-loaded foam cells. Macrophages are heterogeneous cells which adapt their response to environmental cytokines. Th1 cytokines promote monocyte differentiation into M1 macrophages, while Th2 cytokines trigger an "alternative" M2 phenotype. Objective -We previously reported the presence of CD68+MR+ M2 macrophages in human atherosclerotic plaques. However, the function of these plaque CD68+MR+ macrophages is still unknown. Methods and Results -Histological analysis revealed that CD68+MR+ locate far from the lipid core of the plaque and contain smaller lipid droplets compared to CD68+MR-macrophages. IL-4 polarized CD68+MR+ display a reduced capacity to handle and efflux cellular cholesterol due to low expression levels of the nuclear receptor Liver X Receptor (LXR)α and its target genes, ABCA1 and ApoE, caused by the high 15-lipoxygenase activity in CD68+MR+ macrophages. By contrast, CD68+MR+ highly express opsonins and receptors involved in phagocytosis resulting in high phagocytic activity. In M2 macrophages, Peroxisome Proliferator-Activated receptor (PPAR)γ activation enhances the phagocytic, but not the cholesterol trafficking pathways. Conclusions -These data identify a distinct macrophage sub-population with a low susceptibility to become foam cells, but high phagocytic activity due to different regulatory activities of the PPARγ-LXRα pathways.

INTRODUCTION

A crucial step in atherogenesis is the infiltration of monocytes within the sub-endothelial space of large arteries and their differentiation into macrophages. In early plaques, recruited macrophages play reparatory roles via the phagocytosis of oxidized lipids and apoptotic cells. However, during atherosclerosis progression, macrophages contribute to foam cell formation, lesion growth, plaque rupture and thrombosis by secreting immune-inflammatory factors, growth factors, proteolytic enzymes and tissue factor [START_REF] Wilson | Macrophages: promising targets for the treatment of atherosclerosis[END_REF] . One of the most important functions of macrophages in the context of atherosclerosis is the handling of lipids, especially cholesterol. The maintenance of macrophage cholesterol homeostasis is of critical importance in the pathogenesis of atherosclerosis, since an imbalance between cholesterol influx and efflux leads to an excessive accumulation of cholesterol in macrophages and their transformation into foam cells [START_REF] Libby | Inflammation in atherosclerosis[END_REF][START_REF] Libby | Cholesterol and atherosclerosis[END_REF] . Macrophage scavenger receptors, including scavenger receptor A (SR-A), CD36 and lectin-like oxidized LDL receptor-1 (LOX-1), mediate the uptake of modified LDL lipoproteins, including oxidized (Ox)LDL. Within the macrophages, modified LDL-derived cholesteryl esters are hydrolysed in lysosomes by the lysosomal acid lipase (LAL). The released unesterified cholesterol traffics to and integrates in the plasma membrane. The excess of cholesterol is transported back to the endoplasmic reticulum where it is re-esterified by acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) and stored as lipid droplets [START_REF] Buhman | Mammalian acyl-CoA:cholesterol acyltransferases[END_REF] . Cellular cholesterol at the plasma membrane is available for efflux, a highly regulated process mediated by specific proteins, including ATP-binding cassette (ABC) transporters (ABCA1 and ABCG1/ABCG4) [START_REF] Remaley | Human ATP-binding cassette transporter 1 (ABC1): genomic organization and identification of the genetic defect in the original tangier disease kindred[END_REF] and apolipoprotein (apo) E [START_REF] Zhang | Apolipoprotein E produced by human monocytederived macrophages mediates cholesterol efflux that occurs in the absence of added cholesterol acceptors[END_REF] . Besides their role in lipid handling and the immune-inflammatory response, macrophages are also involved in phagocytosis of opsonized bacteria and pathogens and efferocytosis, the clearance of apoptotic cells and debris, since they express receptors and adaptors which recognize the "eat-me" signals at the surface of dying cells [START_REF] Thorp | Mechanisms and consequences of efferocytosis in advanced atherosclerosis[END_REF] . Ingestion of apoptotic cells generally triggers an antiinflammatory response [START_REF] Henson | Dampening inflammation[END_REF] . Hence, atherosclerosis may be caused not only by a sustained proinflammatory reaction, but also by the failure of anti-inflammatory control mechanisms. Macrophages are heterogeneous cells, which adapt their response to environmental cytokines and microbial products. While Th1 cytokines (IFNγ and IL1-β) or bacterial LPS induce a "classical" activation profile (M1), the Th2 cytokines IL-4 and IL-13 induce an "alternative" activation (M2) of macrophages. Macrophages are plastic cells because they can switch from the M1 to M2 state and vice versa, upon specific signals [START_REF] Gordon | Alternative activation of macrophages[END_REF] . M1 macrophages produce pro-inflammatory cytokines, such as tumor necrosis factor α (TNF), IL-6 and IL-12 [START_REF] Gordon | Alternative activation of macrophages[END_REF] , whereas M2 macrophages dampen this inflammatory Th1 response by producing anti-inflammatory factors (IL-10, TGF-β, IL-1 receptor antagonist (IL-1Ra)), thus promoting angiogenesis and tissue repair [START_REF] Gordon | Alternative activation of macrophages[END_REF][START_REF] Mantovani | Decoy receptors: a strategy to regulate inflammatory cytokines and chemokines[END_REF] . Macrophage functions are under the control of several transcription factors, among which the Liver X Receptors (LXR) and the Peroxisome Proliferator-Activated Receptors (PPAR). LXRα, LXRβ and PPARγ are ligand-activated nuclear receptors controlling cholesterol distribution and efflux, the inflammatory response [START_REF] Rigamonti | Regulation of macrophage functions by PPAR-alpha, PPAR-gamma, and LXRs in mice and men[END_REF] and, in the case of PPARγ, macrophage polarization state [START_REF] Bouhlel | PPARg activation primes human monocytes into alternative M2 macrophages with anti-inflammatory properties[END_REF][START_REF] Odegaard | Macrophage-specific PPARgamma controls alternative activation and improves insulin resistance[END_REF] . We have previously shown that, besides classically activated M1 macrophages, human atherosclerotic plaques contain also macrophages expressing the mannose receptor (MR), an alternative macrophage marker, suggesting heterogeneity of plaque macrophage populations [START_REF] Bouhlel | PPARg activation primes human monocytes into alternative M2 macrophages with anti-inflammatory properties[END_REF] . However, the phenotypic characteristics and functions nor the regulatory pathways operative in these alternative M2 macrophages are still unknown. The objective of this study was to characterize the macrophage subpopulations in human atherosclerotic plaques and to study their functions.

METHODS

Endarterectomy tissue samples

Human atherosclerotic plaques were removed from 30 patients eligible for surgical carotid endarterectomy according to the European Carotid Trialists' Collaborative Group [START_REF]Carotid Surgery Trial: interim results for symptomatic patients with severe (70-99%) or with mild (0-29%) carotid stenosis. European Carotid Surgery Trialists' Collaborative Group[END_REF] , recruited at the Cardiovascular Surgery Department, Hospital of Lille, France [START_REF] Zawadzki | Dyslipidemia shifts the tissue factor/tissue factor pathway inhibitor balance toward increased thrombogenicity in atherosclerotic plaques: evidence for a corrective effect of statins[END_REF] . Informed consent was obtained from all patients.

Cell culture

Human peripheral blood mononuclear cells were isolated from healthy donors by Ficoll density gradient centrifugation. Monocytes were differentiated into resting (RM), alternative (M2) or classical (M1) macrophages (see online supplemental file). Where indicated, T0901317 (T09, 1 µmol/L), 22(R)-hydroxycholesterol (22OH, 10 µmol/L), GW1929 (600 nmol/L), cinnamyl-3,4-dihydroxy-acyanocinnamate (CDC, 5 µg/ml) and R04508159 (10 µmol/L) compounds were added for 24h.

Immunohistochemical analysis and laser capture microdissection (LCM)

Immunohistochemical analysis and laser capture micro-dissection were performed on frozen sections (see the online supplemental file).

RNA extraction and analysis

Total cellular RNA was extracted from macrophages using Trizol (Life Technologies, France). RNA extraction from LCM-isolated samples was performed using the Picopure RNA extraction kit (MDS Analytical Technologies). Gene expression was analysed by quantitative PCR (Q-PCR) (see the online supplemental file).

Protein extraction and western blot analysis

Cells were harvested in lysis buffer containing PBS, 4% Triton X100, 10% Na Deoxycholate and protease inhibitors. Proteins (20 µg) were separated by SDS-PAGE, transferred to Hybond-C Extra membranes (Amersham) and immunoblotted using antibodies against human LXRα (abcam), human LOX-1 or β-actin (SantaCruz Biotechnology).

Cellular triglyceride and cholesterol loading

Macrophages were lipid-loaded by incubation with AcLDL or VLDL (50 µg/ml) for 48h or with native LDL (1 mg/ml) or OxLDL (50 µg/ml) for 24h. Intracellular lipids were extracted with hexane/isopropanol (3v:2v) and triglycerides and total cholesterol measured using an enzymatic assay (Boehringer) and cholesterol distribution were measured (see the online supplemental file). In certain experiments, cholesterol-loaded macrophages were stained with Oil red O. Where indicated cells were incubated with fluorescent DiI-LDL (0.2 mg/ml) for 24h [START_REF] Pitas | Acetoacetylated lipoproteins used to distinguish fibroblasts from macrophages in vitro by fluorescence microscopy[END_REF] .

Measurement of cholesteryl ester formation

Cholesteryl ester formation was assessed by measuring the incorporation of [ 14 C]-oleate into cholesteryl esters. Human macrophages were cholesterol-loaded by incubation with AcLDL (50 µg/ml) for 48 hours. After the cholesterol-loading period, cholesteryl ester formation was measured (see the online supplemental file).

Cholesterol efflux

Macrophages were cholesterol-loaded by incubation with [ 3 H]cholesterol-AcLDL (50 µg/mL) for 48h. HDL 3 and ApoAI-mediated cholesterol efflux was measured (see the online supplemental file).

Small interfering (si)RNA-mediated macrophage RNA interference siRNA oligonucleotides corresponding to human LXRα and LXRβ (Dharmacon), PPARγ (Ambion) sequences and scrambled control RNA oligonucleotides (Ambion) were used. RM and M2 macrophages were transfected using Dharmafect4 reagent (Dharmacon) for 16h and then treated for a further 24h with T0901317 (1 µmol/L) or GW1929 (600 nmol/L).

Adenovirus generation

The recombinant adenovirus (Ad)-GFP (Green Fluorescent Protein) and Ad-PPARγ were obtained as described [START_REF] Rigamonti | Induction of CXCR2 Receptor by Peroxisome Proliferator-Activated Receptor {gamma} in Human Macrophages[END_REF] . Viral titers were determined on HEK 293 cells and defined as plaque-forming units/ml. 2x10 6 macrophages were infected for 16h at a multiplicity of 100 viral particles/cell and subsequently incubated for 24h in the absence or presence of GW1929 (600 nmol/L).

In vitro phagocytosis assay

Phagocytosis tests were performed using fluorescent apoptotic cells or latex beads (see the online supplemental file).

Statistical analysis

Statistical differences between groups were analyzed by Student t-test and considered significant when p <0.05.

RESULTS

Identification of distinct macrophage sub-populations in human atherosclerotic plaques.

We previously reported the presence of MR-expressing macrophages in human atherosclerotic lesions [START_REF] Bouhlel | PPARg activation primes human monocytes into alternative M2 macrophages with anti-inflammatory properties[END_REF] . However, their morphology, phenotype and tissue localization has not been investigated yet. Therefore, macrophage-rich areas were identified by immunostaining with the pan-macrophage marker CD68, a glycoprotein located on the lysosomal membrane [START_REF] Martinez-Pomares | Macrophage membrane molecules: markers of tissue differentiation and heterogeneity[END_REF] (fig. 1A). Distinct macrophage sub-populations were then discriminated on the basis of co-expression of MR, an endocytic and phagocytic receptor whose expression is strongly enhanced during alternative M2 macrophage differentiation [START_REF] Stein | Interleukin 4 potently enhances murine macrophage mannose receptor activity: a marker of alternative immunologic macrophage activation[END_REF] . As expected, CD68+MR-macrophages were abundantly found in the lipid core of the plaque and very rarely in the intimal space. By contrast, CD68+MR+ macrophages were predominantly observed in the area of the plaque overlying the lipid core (fig. 1A). Q-PCR analysis of areas infiltrated by CD68+MR+ macrophages and isolated by laser capture-microdissection (LCM) showed a higher expression of IL-4, a cytokine primarily produced by Th2 lymphocytes and inducer of macrophage alternative differentiation, compared to CD68+MR-rich areas (fig. 1B). Thus, based on morphology, localization and differential expression of the MR, human atherosclerotic lesions contain at least two macrophage sub-populations present in distinct, discrete zones of the plaque.

Alternative macrophages are less susceptible to transform into foam cells. Interestingly, Oil Red O staining revealed a different pattern of lipid droplet accumulation in CD68+MR-and CD68+MR+ macrophages in vivo (fig. 1A). Morphological comparison revealed that while CD68+MR+ macrophages are smaller and their cytoplasm contain several small lipid droplets (mean diameter of the lipid droplet: 1-1.5 µm), CD68+MR-macrophages contain fewer and bigger lipid droplets (mean diameter approximately 15 µm) (fig. 1A). To investigate the mechanism behind the lower lipid accumulation observed in CD68+MR+ macrophages, in vitro studies were performed using human primary monocytes polarized to the M2 alternative phenotype with IL-4. As expected, the expression of the M2 macrophage markers MR, AMAC-1 and IL-1Ra was strongly induced by IL-4, compared to resting macrophages (RM) (data not shown). By contrast, the expression of the specific dendritic cell marker CD1a was almost undetectable in M2 macrophages, showing that dendritic cell differentiation is not induced in our model (data not shown). Expression of SR-A and CD36, two scavenger receptors, was similar in RM and M2 macrophages (fig. 2A,B). In line, alternative differentiation of macrophages did not modify AcLDL or VLDL-induced cholesterol or triglyceride accumulation (fig. 2C,D). Interestingly, M2 macrophages displayed a lower gene expression level of LOX-1 and caveolin-1 compared to RM (fig. 2E,G). Both the pro-(approx. 50KDa) and mature (approx. 30KDa) LOX-1 protein forms were lower expressed in M2 than in RM macrophages (fig. 2E). In line, M2 macrophages exhibited a reduced accumulation of oxidized and native LDL (fig. 2F,H Macrophage foam cell formation induced by native LDL might occur through receptor-independent fluid phase pinocytosis, a process highly stimulated by the PKA activator PMA [START_REF] Kruth | Macrophage foam cell formation with native low density lipoprotein[END_REF] . However, LDL accumulation in M2 macrophages was similar as in RM upon PMA activation, thus excluding a defective fluid phase pinocytosis (online fig.II). Altogether, M2 alternative macrophages accumulate less oxidized and native LDL, an effect likely contributing to the lower accumulation of cytoplasmic lipid droplets in CD68+MR+ macrophages observed in vivo.

Human alternative macrophages display a lower cholesterol efflux capacity.

To determine whether alternative differentiation may influence the reverse cholesterol transport pathway, the impact of M2 differentiation on the expression of key genes involved in cholesterol efflux such as ABCA1 and ApoE, was studied (fig. 3A,B). The expression of both genes was significantly lower in M2 macrophages compared to RM and pro-inflammatory M1 macrophages (online fig.II), suggesting that M2 macrophages may have lower cholesterol efflux capacities. CD68+MR+ macrophage-rich areas of human atherosclerotic plaques exhibit lower levels of ABCA1 mRNA (fig. 3C), protein (fig. 3E) and ApoE mRNA (fig. 3D) compared to CD68+MR-rich zones.

In line, apoAI-and HDL 3 -mediated cholesterol efflux was significantly lower in [3H]cholesterol-AcLDL-loaded M2 macrophages compared to RM macrophages (fig. 3F), indicating that alternative differentiated macrophages display impaired cholesterol efflux capacities.

Alternative macrophage differentiation leads to an increased cholesteryl ester content and cholesteryl ester formation.

Increased macrophage cholesterol esterification capacity is a protective mechanism to control free cholesterol toxicity in conditions in which the cholesterol efflux pathway is defective [START_REF] Warner | Cell toxicity induced by inhibition of acyl coenzyme A:cholesterol acyltransferase and accumulation of unesterified cholesterol[END_REF] . We sought to determine whether the reduced cholesterol efflux capacity in M2 macrophages can, in turn, affect cholesterol esterification. Expression of LAL was higher in M2 macrophages compared to RM or TNFα and IL-1β-activated macrophages (fig. 4A, online fig. III,B), suggesting enhanced lysosomal cholesteryl ester hydrolysis in M2 macrophages and higher unesterified cholesterol availability for intracellular trafficking. In line, the expression of the cholesterol transporter Niemann Pick type C 1 (NPC-1) [START_REF] Carstea | Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis[END_REF] was higher in M2 compared to RM (data not shown). Moreover, intracellular cholesterol esterification (fig. 4D,E) was higher in M2 macrophages, which correlated with increased ACAT-1 mRNA levels in M2 macrophages, both in vivo and in vitro (fig. 4B,C, online fig. III,C). By contrast, CPT-1, a key enzyme in mitochondrial fatty acid catabolism and the neutral cholesteryl ester hydrolase (NCEH) were expressed at similar levels in RM and M2 macrophages (fig. 4F and data not shown).

LXRα α α

α expression and activity is decreased in alternative macrophages. Since ABCA1 and ApoE are well-known LXR target genes [START_REF] Rigamonti | Regulation of macrophage functions by PPAR-alpha, PPAR-gamma, and LXRs in mice and men[END_REF] , we investigated the expression of LXRα and LXRβ. LXRα mRNA (fig. 5A) and protein (fig. 5C), but not LXRβ expression (fig. 5B) were significantly lower in M2 macrophages compared to RM or IL-1β and TNFα pro-inflammatory M1 macrophages (online fig. III,F). A lower expression level of LXRα mRNA and protein was also observed in vivo in CD68+MR+ macrophage-rich areas of human atherosclerotic plaques (fig. 5D,E). To determine the capacity of M2 macrophages to respond to LXR activation, differentiated macrophages were incubated with the highly active synthetic dual LXRα/β agonist T0901317 [START_REF] Schultz | Role of LXRs in control of lipogenesis[END_REF] . Pharmacological LXR activation in M2 macrophages enhanced LXRα, ABCA1 and ApoE gene expression to levels comparable to untreated RM macrophages, but much lower than in T0901317activated RM macrophages (fig. 6). In line, M2 macrophages were less sensitive than RM to the activation by the natural LXR agonist 22-hydroxycholesterol (22OH) [START_REF] Lala | Activation of the orphan nuclear receptor steroidogenic factor 1 by oxysterols[END_REF] (online fig.V). LXR activation did not affect the macrophage alternative phenotype, since the expression of MR was unchanged (online fig.IV). To determine the role of LXRα and LXRβ in M2 macrophage function, their expression was silenced by siRNA in M2 macrophages resulting in about 70% reduction (in comparison with control siRNA-transfected cells; fig. 6E,L). LXRβ-silencing did not affect T0901317induced expression of LXRα, ABCA1 or ApoE, indicating that the observed responses are LXRαdependent (fig. 6F,G,H). LXRα-silencing affected the basal as well as the T0901317-induced expression of LXRα itself, ABCA1 and ApoE, without affecting the LXRβ expression (fig. 6I,L,M,N). These results indicate that LXRα is the LXR isoform controlling M2 macrophage cholesterol metabolism and that its low expression in these cells is involved in the decreased cholesterol handling properties.

IL-4 decreases LXRα α α

α expression via a mechanism involving 15-lipoxygenase. 15-lipoxygenase (15-LOX) is a lipid peroxidating enzyme responsible for the conversion of arachidonic and linoleic acid into 13-hydroxyoctadecadienoic (13-HODE). 15-LOX expression is strongly induced by IL-4 [START_REF] Sigal | Human 15-lipoxygenase: induction by interleukin-4 and insights into positional specificity[END_REF] . Since 12/15-lipoxygenase transgenic mouse macrophages display reduced ABCA1 expression and cholesterol efflux [START_REF] Nagelin | Murine 12/15-lipoxygenase regulates ATP-binding cassette transporter G1 protein degradation through p38-and JNK2-dependent pathways[END_REF] , a phenotype similar to the one observed in human M2 macrophages, the potential involvement of human 15-LOX in the regulation of LXRα signalling in M2 macrophages was assessed. Interestingly, 15-LOX gene expression was elevated in M2 macrophages in vitro (online fig.VI) and 15-LOX protein was higher in CD68+MR+ macrophages compared to CD68+MR-macrophage-rich areas in atherosclerotic plaques (fig. 7A). Expression of 15-LOX (blue) colocalized with MR (red), leading to purple staining (fig. 7A). Inhibition of 15-LOX activity in M2 macrophages with two different chemical inhibitors, CDC and R04508159 [START_REF] Cho | Novel caffeic acid derivatives: extremely potent inhibitors of 12-lipoxygenase[END_REF][START_REF] Klein | Regulation of bone mass in mice by the lipoxygenase gene Alox15[END_REF] , resulted in an increased expression of LXRα and its target genes ABCA1 and ApoE (fig. 7B-G). Taken together, these results indicate that the decreased LXRα expression and activity in M2 macrophages is likely related to an enhanced 15-LOX activity.

Expression of genes involved in phagocytosis is altered in alternative macrophages.

To determine whether other macrophage functions, such as phagocytosis, are operative in CD68+MR+ macrophages, the expression of several key genes involved in this pathway was analyzed both in vivo and in vitro. The expression of C1qa, C1qb, C1qc, GAS-6, and TSP-1 was higher in vivo when comparing CD68+MR+ macrophages to CD68+MR-macrophage-rich areas of atherosclerotic plaques (online fig. VII,A-F). Interestingly, this gene expression profile is comparable to the one observed in vitro in M2 macrophages, compared to RM, with the exception of MERTK, which is drastically lower upon M2 polarization (online fig. VII,G-M). Amongst these, TSP-1 gene was the highest expressed in CD68+MR+ macrophage-rich areas in vivo (online fig. VII,F). These results suggest that CD68+MR+ macrophages likely have enhanced phagocytic activities.

PPARγ γ γ γ activation enhances phagocytic activity of alternative macrophages.

To assess functional phagocytic activities, engulfment of fluorescent apoptotic cells or beads was determined. M2 macrophages displayed an increased capacity of phagogytosis compared to RM macrophages (fig. 8A,B) and this effect was enhanced by PPARγ activation (fig. 8C,D). Since TSP-1 is highest expressed in vivo (median value=3.25), and given its role in phagocytosis [START_REF] Brechot | Modulation of macrophage activation state protects tissue from necrosis during critical limb ischemia in thrombospondin-1-deficient mice[END_REF][START_REF] Moodley | Macrophage recognition and phagocytosis of apoptotic fibroblasts is critically dependent on fibroblast-derived thrombospondin 1 and CD36[END_REF] , we investigated whether it is regulated by LXRα and PPARγ. Whereas TSP-1 expression was not altered upon LXR activation of IL-4 driven-alternative macrophages in vitro (data not shown), its expression was enhanced by PPARγ activation with GW1929 (fig. 8E), an effect which was abolished upon PPARγ siRNA-silencing (fig. 8F). Moreover, infection of macrophages with an adenovirus coding for PPARγ (Ad-PPARγ) increased TSP-1 expression 3.5-fold, which was further enhanced by GW1929 (fig. 8G). Expression of the TSP-1 receptor CD47 was higher in alternative macrophages, but not regulated by PPARγ (fig. 8H). Inhibition of TSP-1 in M2 macrophages using a blocking antibody inhibited the PPARγ-enhanced phagocytosis activity (online fig.VIII). Altogether, these results indicate that alternative macrophages display enhanced phagocytic capacity compared to RM macrophages, an effect amplified by PPARγ activation.

DISCUSSION

Human atherosclerotic lesions are highly heterogeneous structures containing a variety of cell types contributing to a complex inflammatory environment. The local cytokine environment can promote differentiation of infiltrated monocytes to alternative or to classical macrophage sub-populations. We have been the first to report the presence of alternative macrophages expressing the MR within human carotid atherosclerotic plaques [START_REF] Bouhlel | PPARg activation primes human monocytes into alternative M2 macrophages with anti-inflammatory properties[END_REF] . However, the phenotypic characterization of macrophage subpopulations within the plaques and their role in the pathogenesis of atherosclerosis has not yet been elucidated. Immunohistological analysis shows that the macrophage sub-populations positive for CD68 and MR expression are abundant in stable cell-rich areas of the atherosclerotic plaque. In contrast, CD68+MRmacrophages predominate in the lipid core of the atherosclerotic plaque and can occasionally be detected in the intimal space. We also found that CD68+MR+ macrophage-rich areas express higher levels of IL-4 than CD68+MR-rich zones, thus providing the appropriate cytokine environment necessary to orient macrophage polarization. Considering that the cytokine milieu is complex and might vary during the different stages of atherosclerotic plaque progression, it is likely that intermediate spectra of differentiation states exist, with CD68+MR-and CD68+MR+ being the extreme situations, similar as reported for human adipose tissue macrophages [START_REF] Zeyda | Human adipose tissue macrophages are of an anti-inflammatory phenotype but capable of excessive pro-inflammatory mediator production[END_REF] . However, it is of note that CD68+MR-macrophages are filled more abundantly with lipid droplets which are larger than those observed in CD68+MR+ macrophages. Our results using in vitro differentiated M2 macrophages indicate that these cells are less competent to take up native and oxidized lipoproteins, indicating the existence of cell-intrinsic functional differences in terms of lipid accumulation and storage. The macrophage CD68+MR+ sub-population identified in vivo shares the same features with in vitro differentiated primary monocytes in the presence of the Th2 cytokine IL-4. Moreover, these IL-4 driven alternative macrophages display a lower capacity of cholesterol efflux to extra-cellular acceptors, associated with reduced expression of ABCA1 and ApoE. Interestingly, the reduced cholesterol efflux capacity of alternative macrophages is probably independent of ABCG1 expression, since alternative macrophages express 2.5-fold more ABCG1 mRNA than RM macrophages (data not shown). These data are in agreement with a previous report showing that cholesterol efflux from human primary macrophage-foam cells to HDL is independent of ABCG1, but specifically requires ABCA1 expression [START_REF] Larrede | Stimulation of cholesterol efflux by LXR agonists in cholesterol-loaded human macrophages is ABCA1-dependent but ABCG1-independent[END_REF] . Our results also show that, as a consequence of a defective cholesterol efflux, IL-4-induced alternative human macrophages instore a protective mechanism against free cholesterol excess by increasing cholesterol esterification capacities, due to an induction of LAL and ACAT-1 gene expression. This latter observation is in agreement with previous results obtained in IL-4-stimulated mouse macrophages [START_REF] Vats | Oxidative metabolism and PGC-1beta attenuate macrophage-mediated inflammation[END_REF] . Our results show that LXRα, but not LXRβ expression, is much lower in alternative macrophages, both in vitro and in vivo in CD68+MR+ macrophages. Previously, we reported that LXR activation in RM macrophages increases cholesterol trafficking to the plasma membrane leading to a reduced cholesteryl ester formation [START_REF] Rigamonti | Liver X receptor activation controls intracellular cholesterol trafficking and esterification in human macrophages[END_REF] . The increased esterification in alternative macrophages could be the consequence of the low LXRα activity. Indeed, pharmacological treatment of alternative macrophages in vitro with the highly active synthetic LXR ligand T0901317 and the natural ligand 22-OH cholesterol restored the expression of LXRα and its target genes to a level comparable to those observed in untreated RM macrophages, albeit still much lower than in T0901317-activated RM macrophages indicating that endogenous LXRα is limiting. Interestingly, siRNA mediated LXRα and LXRβ silencing demonstrated that the responses to T091317 stimulation are mediated by LXRα, thus identifying a specific regulatory role of the LXRα isoform in alternative human macrophages. Silencing of LXRα expression in alternative macrophages further supports this conclusion. These results are in agreement with a recent publication [START_REF] Bischoff | Non-redundant roles for LXR{alpha} and LXR? in atherosclerosis susceptibility in low density lipoprotein receptor knockout mice[END_REF] reporting a distinct non-redundant role for LXRα and LXRβ in atherosclerosis susceptibility in mice and that LXRα is required for a robust response to LXR ligands, such as T0901317. Of note, these effects of LXRα occur without affecting the alternative phenotype, since MR expression did not change upon T091317 treatment. This phenotype, characterized by reduced expression of ABCA1 and cholesterol efflux, resembles the one of macrophages isolated from 12/15-LOX transgenic mice. 12/15 LOX is the homolog of human 15-LOX. However, human 15-LOX is a unique lipoxygenase, since it can oxygenate polyenoic fatty acids esterified to membrane lipids and hence may have biological roles distinct from its action on free arachidonic acid. The expression of 15-LOX is highly induced by Th2 cytokines such as IL-4 in vitro [START_REF] Conrad | Specific inflammatory cytokines regulate the expression of human monocyte 15-lipoxygenase[END_REF] . Moreover, we show that in vivo CD68+MR+ macrophages express higher levels of 15-LOX compared to CD68+MR-macrophages. Using two distinct chemical inhibitors we demonstrate that inhibition of 15-LOX in M2 macrophages restores the expression of LXRα and its target genes. Further studies are required to determine how and which 15-LOX-derived lipid mediators inhibit LXRα. Whereas CD68+MR-macrophages display a lower cholesterol handling capacity, they appear competent for phagocytosis. In line, they express higher levels of genes coding for opsonins and receptors that bridge apoptotic cells to macrophages. Notably, alternative macrophages express higher levels of C1qa, C1qb, C1qc, GAS-6 and TSP-1 than RM macrophages. Since C1q-deficiency leads to defective clearance of apoptotic cells [START_REF] Taylor | A hierarchical role for classical pathway complement proteins in the clearance of apoptotic cells in vivo[END_REF] , the high levels of several opsonins in alternative macrophages likely provides the molecular basis for their high phagocytotic capacity. Interestingly, while the cholesterol handling properties are likely due to the reduced expression of LXRα, the phagocytosis appears to be directly controlled by PPARγ, whose expression is induced by IL-4 in macrophages [START_REF] Huang | Interleukin-4-dependent production of PPAR-γ ligands in macrophages by 12/15 lipoxygenase[END_REF] . Moreover, TSP-1, which is involved in phagocytosis, is induced by PPARγ in macrophages. By contrast, treatment of alternative macrophages with GW1929 did not result in the induction of the expression of ABCA1 (data not shown), whose expression is indirectly induced via LXRα induction in RM [START_REF] Chinetti | PPARα and PPARγ activators induce cholesterol removal from human macrophage foam cells through stimulation of the ABCA1 pathway[END_REF] . Thus, in alternative macrophages PPARγ activities mediated via LXRα induction are deactivated, whereas the phagocytosis pathway is activated, at least in part, through the regulation of TSP-1.

In conclusion, we identified a macrophage sub-population in human atherosclerotic plaques which presents a different morphology and localization, a functional heterogeneity related to the deactivation of the LXRα signalling pathway, being less susceptible to handle lipids and more competent for phagocytosis, an activity enhanced by PPARγ activation. It is tempting to speculate that these alternative macrophages may exert beneficial activities in atherosclerotic lesion development since they are less prone to transform into foam cells and more competent for cell engulfment, in addition to their anti-inflammatory properties.

Novelty and Significance

What is known? -Monocytes differentiate in functionally distinct pro-inflammatory "classical" M1 macrophages and anti-inflammatory "alternative" M2 macrophages depending on the cytokine-environment. -M1 macrophages contribute to atherogenesis by generating inflammatory foam cells. -M2 macrophages have only recently been identified in human atherosclerotic lesions.

What new information does this article contribute? -M2 macrophages in human atherosclerotic plaques display lower lipid-handling capacities and reduced foam cell transformation capacity.

-This phenotype is due to low expression levels of the nuclear receptor Liver X Receptor alpha (LXRα) resulting in decreased Peroxisome Proliferator-Activated Receptor (PPAR)γ-LXRα regulation of cholesterol metabolism.

-By contrast, M2 macrophages display an enhanced apoptotic cell phagocytosis capacity which is enhanced by PPARγ activation.

During atherogenesis, monocytes infiltrate the sub-endothelial space of large arteries where they differentiate into macrophages which turn into lipid-loaded foam cells. Macrophages are functionally heterogeneous cell populations which adapt their phenotype depending on the cytokine environment. Th1 cytokines promote M1 macrophage differentiation, while Th2 cytokines trigger an "alternative" M2 phenotype. We previously reported the presence of M2 macrophages in human atherosclerotic plaques. However, their function was still unknown. We now show that atherosclerotic lesion M2 macrophages contain smaller lipid droplets. In vitro IL-4 polarized M2 macrophages display a reduced capacity to handle and efflux cellular cholesterol due to low expression levels of the nuclear receptor LXRα and its target genes. By contrast, M2 macrophages express opsonins and receptors involved in phagocytosis resulting in high phagocytic activity, which is enhanced by PPARγ activation. We thus identified a distinct macrophage sub-population with a low susceptibility to become foam cells, but high phagocytic activity due to de-activation of the PPARγ-LXRα and activation of alternative PPARγregulatory pathways. We speculate that these alternative M2 macrophages may exert beneficial activities in atherosclerotic lesion development since they are less prone to transform into foam cells and more competent to clear apoptotic cells.

  , online fig.I), as measured by ORO staining and by a reduced accumulation of DiI-native LDL (online fig.I). In line, cholesterol content was lower in M2 macrophages loaded with native LDL compared to RM (online fig.I).

AFigure 1 .

 1 Figure 1. Identification of distinct macrophage sub-populations in human atherosclerotic plaques. Panel A. Immunostaining (top row) and higher magnification (bottom rows) of representative stainings for CD68, MR and Oil red O in human carotid atherosclerotic lesions. Scale bars are shown. Panel B. Q-PCR analysis of IL-4 performed on RNA from LCM isolated CD68+MR-and CD68+MR+ macrophage-rich areas. mRNA levels were normalized to cyclophilin mRNA and expressed relative to the levels in CD68+MR-area set at 1. Each point corresponds to a single atherosclerotic plaque. The median value is shown. Statistically significant differences are indicated (t-test; *p< 0.05).

Figure 1 .Figure 2 .Figure 2 .Figure 3 .Figure 3 .Figure 4 .Figure 4 .

 1223344 Figure 1. Identification of distinct macrophage sub-populations in human atherosclerotic plaques. Panel A. Immunostaining (top row) and higher magnification (bottom rows) of representative stainings for CD68, MR and Oil red O in human carotid atherosclerotic lesions. Scale bars are shown. Panel B. Q-PCR analysis of IL-4 performed on RNA from LCM isolated CD68+MR-and CD68+MR+ macrophage-rich areas. mRNA levels were normalized to cyclophilin mRNA and expressed relative to the levels in CD68+MR-area set at 1. Each point corresponds to a single atherosclerotic plaque. The median value is shown. Statistically significant differences are indicated (t-test; *p< 0.05).

Figure 5 .Figure 5 .Figure 6 .

 556 Figure 5. Decreased LXRα α α α expression in alternatively differentiated macrophages. Panels A,B. Q-PCR analysis of LXRα (A) and LXRβ (B) in RM and M2 macrophages. mRNA levels were normalized to cyclophilin mRNA and expressed as means ± SD relative to RM set at 1 from three independent experiments. Statistically significant differences are indicated (t-test; *p< 0.05, **p< 0.01). Panel C. LXRα protein expression analyzed by western blot in RM and M2 macrophages isolated from 4 different donors. Panel D. Q-PCR analysis of LXRα performed on RNA from LCM-isolated CD68+MR-and CD68+MR+ macrophage-rich areas isolated from 7 samples. mRNA levels were normalized to cyclophilin mRNA and expressed relative to the levels in CD68+MR-area set at 1. Each point corresponds to a single atherosclerotic plaque. The median value is shown. Statistically significant differences are indicated (t-test; **p< 0.01). Panel E. MR and LXRα immunostaining performed in human carotid atherosclerotic lesions. Scale bar is shown.

Figure 6 .

 6 Figure 6. Decreased LXRα α α α activity in M2 macrophages. Panels A-D. Q-PCR analysis of LXRα (A), LXRβ (B), ABCA1 (C) and ApoE (D) in RM or M2 macrophages treated or not with T0901317 (T09). mRNA levels were normalized to cyclophilin mRNA and expressed as means ± SD relative to RM set at 1 from three independent experiments. Statistically significant differences are indicated (t-test; RM vs M2 § p< 0.05, § § p< 0.01, § § § p< 0.001; and T09 treated vs control **p< 0.01, ***p< 0.001). Panels E-N. Q-PCR analysis of LXRβ, LXRα, ABCA1 and ApoE in M2 macrophages transfected with non-silencing control (scrambled), LXRβ siRNA (E-H) or LXRα siRNA (I-N) and treated or not with T0901317 (T09). Results were normalized to cyclophilin mRNA and expressed relative to the levels in control-siRNA transfected cells set at 1 (mean ± SD of two independent experiments). Statistically significant differences are indicated (t-test; siRNA vs scrambled §p<0.05, § §p<0.01, T09-treated vs control *p<0.05, **p<0.01, ***p<0.001).

Figure 7 . 15 -Figure 7 . 15 -Figure 8 .

 7157158 Figure 7. 15-LOX inhibition restores the expression of LXRα α α α and its target genes in M2 macrophages. Panel A. MR and 15-LOX immunostaining in human carotid atherosclerotic lesions. Bottom: Double staining of MR (red) and 15-LOX (blue). Most cells present double staining (purple). Scale bar is shown. Panels B-G. Q-PCR analysis of LXRα (B & E), ABCA1 (C & F) and ApoE (D & G) performed in M2 macrophages in the absence or in the presence of the CDC or the R04508159 compounds. mRNA levels were normalized to cyclophilin mRNA and expressed as means ± SD relative to untreated cells set at 1 from three independent experiments. Statistically significant differences are indicated (t-test; **p< 0.01, ***p< 0.001).

Figure 8 .

 8 Figure 8. Alternative macrophage phagocytic activity is enhanced by PPARγ γ γ γ activation. Panels A,B. Phagocytosis of apoptotic cells (A) and fluorescent beads (B) in RM and M2 macrophages. Panels C,D. Phagocytosis of apoptotic cells (C) and fluorescent beads (D) in M2 macrophages in the absence or in the presence of GW1929 (600 nmol/L) for 24h. White histogram: isotype control. Panels E-H. Q-PCR analysis of TSP-1 (E-G) and CD47 mRNA (H) in RM and M2 macrophages 24h-tretaed with GW1929 (600 nmol/L) (E,H), or in RM macrophages transfected with control or human PPARγ siRNA (F) or infected with Ad-GFP or Ad-PPARγ (G) and subsequently treated or not with GW1929 (600 nmol/L) during 24h. TSP-1 and CD47 mRNA levels were normalized to cyclophilin mRNA and expressed as means ± SD relative to control set at 1, from three independent experiments. Statistically significant differences are indicated (t-test; M2 vs RM, §p<0.05, § § §p<0.001; Ad-PPARγ vs Ad-GFP, † †p<0.01; GW1929-treated vs control, *p<0.05, **p<0.01, ***p<0.001).

ACKNOWLEDGMENTS

A. Blondy, C. Duhem and A. D'huysser are acknowledged for technical contribution. We thank Genfit SA (Loos, France) for providing the T0901317 and R04508159 compounds.

SOURCES OF FUNDING

Grants from the Région Nord-Pas de Calais/FEDER (CPER N. 1449), COST action BM0904, the Agence Nationale de la Recherche, France (AlMHA project), the transatlantic Leducq HDL Network, and the Fondation Coeur et Artères are acknowledged.

DISCLOSURES None


Mathieu Hatt

Dimitris Visvikis

Nidal M Albarghach

Florent Tixier

Olivier Pradier

Catherine Cheze-Le Rest

Hatt Mathieu

Latim

Prognostic value of 18 F-FDG PET image-based parameters in esophageal cancer and impact of tumor delineation methodology

Keywords: PET, tumor volume, tumor segmentation, esophageal cancer, survival

Background: 18 F-FDG PET image-derived parameters, such as standardized uptake value (SUV), functional tumor length (TL) and volume (TV) or total lesion glycolysis (TLG) may be useful for determining prognosis in patients with esophageal carcinoma. The objectives of this work were to investigate the prognostic value of these indices in esophageal cancer patients undergoing combined chemoradiotherapy treatment and the impact of TV delineation strategies. Methods: 45 patients were retrospectively analysed. Tumors were delineated on pretreatment 18 F-FDG scans using adaptive threshold and automatic (FLAB) methodologies. SUV max, SUV peak , SUV mean , TL, TV, and TLG were computed. The prognostic value of each parameter for overall survival was investigated using Kaplan-Meier and Cox regression models for univariate and multivariate analyses respectively. Results: Large differences were observed between methodologies (from -140% to +50% for TV). SUV measurements were not significant prognostic factors of overall survival, whereas TV, TL and TLG were, irrespective of the segmentation strategy. After multivariate analysis including standard tumor staging, only TV (p<0.002) and TL (p=0.042) determined using FLAB were independent prognostic factors.

Conclusions: Whereas no SUV measurement was a significant prognostic factor, TV, TL and TLG were significant prognostic factors of overall survival, irrespective of the delineation methodology. Only functional tumor volume and length derived using FLAB were independent prognostic factors, highlighting the need for accurate and robust PET tumor delineation tools for oncology applications.

Introduction

The incidence of esophageal cancer is increasing and despite advances in therapy, the diagnosis still carries a poor prognosis [START_REF] Falk | Risk factors for esophageal cancer development[END_REF]. As with all tumors, the outcome for patients is strongly associated with the stage at initial diagnosis. The TNM (Tumor, Node, Metastasis) system currently in use for the staging of esophageal cancer does not take into account non-anatomical factors such as histopathologic type, grade or various biomarkers that may be important determinants of prognosis. The pathologic stage is given by surgery but this procedure is not possible for all patients because it is associated with a high risk of mortality and morbidity. Therefore a non-invasive staging method would be of great interest, and within this context the primary role of 18 F-FDG positron emission tomography (PET) in esophageal cancer is the detection of distant metastases [START_REF] Flamen | Utility of positron emission tomography for the staging of patients with potentially operable esophageal carcinoma[END_REF][START_REF] Heeren | Detection of distant metastases in esophageal cancer with (18)F-FDG PET[END_REF][START_REF] Vliet | Staging investigations for esophageal cancer: a meta-analysis[END_REF].

This modality is also gaining acceptance in esophageal cancer for the assessment of therapy response [START_REF] Kim | Multimodality assessment of esophageal cancer: preoperative staging and monitoring of response to therapy[END_REF][START_REF] Chuang | The evolving role of PET-CT in the management of esophageal cancer[END_REF] or for radiotherapy treatment planning [START_REF]Use of PET and PET/CT for radiation therapy planning: IAEA expert report 2006-2007[END_REF][START_REF] Macmanus | PET-based treatment planning in radiotherapy: a new standard?[END_REF][START_REF] Grégoire | The value of PET/CT in gross tumor volume delineation in lung and esophagus cancer[END_REF]. Lately, some authors also suggested that different parameters derived from initial 18 F-FDG PET images could have a role as independent prognostic factors [START_REF] Choi | 18F-FDG PET in patients with esophageal squamous cell carcinoma undergoing curative surgery: prognostic implications[END_REF][START_REF] Mamede | FDG-PET/CT tumor segmentation-derived indices of metabolic activity to assess response to neoadjuvant therapy and progression-free survival in esophageal cancer: correlation with histopathology results[END_REF][START_REF] Hyun | Prognostic Value of Metabolic Tumor Volume Measured by 18F-Fluorodeoxyglucose Positron Emission Tomography in Patients with Esophageal Carcinoma[END_REF][START_REF] Hong | Value of baseline positron emission tomography for predicting overall survival in patient with nonmetastatic esophageal or gastroesophageal junction carcinoma[END_REF][START_REF] Blackstock | A prospective evaluation of the impact of 18-F-fluoro-deoxy-D-glucose positron emission tomography staging on survival for patients with locally advanced esophageal cancer[END_REF]. Studied parameters include standardized uptake value (SUV max as the maximum uptake in the primary tumor or in the combined primary and regional area), tumor functional longitudinal length (TL), tumor functional volume (TV), nodal uptake or FDG avid metastases [START_REF] Choi | 18F-FDG PET in patients with esophageal squamous cell carcinoma undergoing curative surgery: prognostic implications[END_REF][START_REF] Mamede | FDG-PET/CT tumor segmentation-derived indices of metabolic activity to assess response to neoadjuvant therapy and progression-free survival in esophageal cancer: correlation with histopathology results[END_REF][START_REF] Hyun | Prognostic Value of Metabolic Tumor Volume Measured by 18F-Fluorodeoxyglucose Positron Emission Tomography in Patients with Esophageal Carcinoma[END_REF][START_REF] Hong | Value of baseline positron emission tomography for predicting overall survival in patient with nonmetastatic esophageal or gastroesophageal junction carcinoma[END_REF][START_REF] Blackstock | A prospective evaluation of the impact of 18-F-fluoro-deoxy-D-glucose positron emission tomography staging on survival for patients with locally advanced esophageal cancer[END_REF]. Although a few studies have demonstrated the interest of these indices for determining prognosis, there are conflicting results concerning the independent prognostic value of each of these indices. For example, Hyun et al [START_REF] Hyun | Prognostic Value of Metabolic Tumor Volume Measured by 18F-Fluorodeoxyglucose Positron Emission Tomography in Patients with Esophageal Carcinoma[END_REF], analyzing results from 151 patients with principally squamous cell carcinoma (SCC), have recently suggested that primary tumor SUV max is not an independent prognostic factor, in agreement with other studies [START_REF] Choi | 18F-FDG PET in patients with esophageal squamous cell carcinoma undergoing curative surgery: prognostic implications[END_REF][START_REF] Van Westreenen | Prognostic value of the standardized uptake value in esophageal cancer[END_REF][START_REF] Omloo | Importance of fluorodeoxyglucose-positron emission tomography (FDG-PET) and endoscopic ultrasonography parameters in predicting survival following surgery for esophageal cancer[END_REF]. On the other hand, Kato et al [START_REF] Kato | The clinical application of 18FDG positron emission tomography to predict survival in patients with operable esophageal cancer[END_REF] based on the analysis of 184 patients with esophageal SCC have shown that SUV max of the primary tumor is an independent prognostic factor of overall survival, in agreement with other studies [START_REF] Rest | Prognostic value of initial fluorodeoxyglucose-PET in esophageal cancer: a prospective study[END_REF][START_REF] Cerfolio | Maximum standardized uptake values on positron emission tomogr aphy of esophageal cancer predicts stage, tumor biology, and survival[END_REF][START_REF] Rizk | Preoperative 18FDG positron emission tomography standardized uptake values predict survival after esophageal adenocarcinoma resection[END_REF]. These conflicting results can be potentially caused by differences in the methodology used for the analysis of the PET images.

Although SUV max is less sensitive to tumor size, the conflicting results considering its value as an independent prognostic factor may also be due to variability in the tumor sizes considered in the different studies.

Pathological TL has been shown to be an independent prognostic factor in esophageal carcinoma [START_REF] Yendamuri | Esophageal Tumor Length Is Independently Associated with Long-term Survival[END_REF]. Therefore determining the functional TL in 18 F-FDG PET images may be a good surrogate. The use of different thresholds for the determination of the functional TL in the existing studies may be responsible for the conflicting results regarding its value as a predictor of response to chemoradiotherapy [START_REF] Mamede | FDG-PET/CT tumor segmentation-derived indices of metabolic activity to assess response to neoadjuvant therapy and progression-free survival in esophageal cancer: correlation with histopathology results[END_REF][START_REF] Roedl | Assessment of treatment response and recurrence in esophageal carcinoma based on tumor length and standardized uptake value on positron emission tomography-computed tomography[END_REF], while it has been shown as an independent predictor in patients undergoing surgery [START_REF] Choi | 18F-FDG PET in patients with esophageal squamous cell carcinoma undergoing curative surgery: prognostic implications[END_REF]. On the other hand, functional TV may be more representative of overall tumor burden. The value of the functional TV has been only recently explored in a single study of patients with esophageal carcinoma considering a heterogeneous treatment regime (76% and 24% treated by surgery and combined radio-chemotherapy respectively) [START_REF] Hyun | Prognostic Value of Metabolic Tumor Volume Measured by 18F-Fluorodeoxyglucose Positron Emission Tomography in Patients with Esophageal Carcinoma[END_REF]. In this study both the presence of metastases and the TV were found to be independent prognostic factor of patient overall survival.

Tumors were delineated based on a three fixed threshold scale depending on the tumor SUV max . Although such an approach may be simple to implement in clinical practice the use of fixed threshold for functional TV determination suffers from multiple shortcomings which have been highlighted in different studies [START_REF] Nestle | Comparison of Different Methods for Delineation of 18F-FDG PET-Positive Tissue for Target Volume Definition in Radiotherapy of Patients with Non-Small Cell Lung Cancer[END_REF][START_REF] Hatt | Accurate automatic delineation of heterogeneous functional volumes in positron emission tomography for oncology applications[END_REF]. In addition, the proposed scale is not universally applicable to the different clinical settings spanning from the acquisition protocols to the scanning systems used and variable associated image qualities.

Therefore despite early evidence that functional TL and TV may be useful parameters in predicting survival and response to therapy there is a clear need in assessing the influence of the methodology used in obtaining these indices. Finally, the determination of functional TV may allow the calculation of alternative image derived indices such as the total glycolytic lesion index (TLG) (g), defined as the product of the TV (ml) and its associated mean activity (SUV mean ) (g/ml) [START_REF] Larson | Tumor treatment response based on visual and quantitative changes in global tumor glycolysis using PET-FDG imaging. The visual response score and the change in total lesion glycolysis[END_REF], whose value has not as yet been explored in predicting response to therapy or as prognostic factor of survival using 18 F-FDG in esophageal carcinoma.

The objective of this study was therefore to retrospectively investigate the prognostic value of 18 F-FDG PET in 45 esophageal cancer patients treated with concomitant radio-chemotherapy, considering for the first time in a single study all of the commonly-used PET-derived parameters such as functional TL, TV, SUV measurements (max, peak, mean) and TLG. In addition the impact of different tumor delineation strategies was assessed.

Materials and Methods

Patients

45 patients with a newly diagnosed esophageal cancer treated between 2004 and 2008 with concomitant radio-chemotherapy and without surgery were included in this study. The characteristics of the patients are given in table 1. 41 patients were male (91%), and the mean age at the time of diagnosis was 66±10 years. Most of the tumors were squamous cell carcinoma (73%) and originated from the middle and lower esophagus (76 %). All patients were referred before treatment for an 18 F-FDG PET study as part of a routine procedure for the initial staging in esophageal cancer.

The treatment included three courses of 5-fluorouracil/cisplatin and a median radiation dose of 60Gy given in 180cGy daily fractions delivered once daily, 5 days a week for 6-7 weeks. Follow-up data were prospectively collected in a database for further analysis and overall survival was calculated. The current analysis was carried out after an approval by the institutional ethics review board. F-FDG PET studies were carried out prior to the treatment. Patients were instructed to fast for a minimum of 6h before the injection of 18 F-FDG. The administered dose was 5 MBq/kg, and static emission images were acquired (2min per bed position) from head to thigh beginning 60 minutes after injection on a Philips GEMINI PET/CT system (Philips Medical Systems, Cleveland, OH USA). In addition to the emission PET scan, a low dose CT scan without IV or oral contrast was acquired for attenuation-correction. Images were reconstructed with the RAMLA 3D algorithm using standard clinical protocol parameters: 2 iterations, relaxation parameter of 0.05, a 5mm 3D Gaussian post-filtering, and a 4x4x4mm 3 voxels grid sampling.

18 F-FDG PET acquisitions

PET image analysis

The following parameters were extracted from each PET image: SUV max , SUV peak defined as the mean of SUV max and its 26 neighbors, mean SUV within the delineated tumor (SUV mean ), functional TL in longitudinal direction, functional TV, and TLG. SUV peak , usually defined as a 1cm circle or sphere (26) (we used a 3x3x3 cube of 4x4x4 mm 3 voxels which roughly corresponds to the same ROI), was considered in order to investigate the impact of reducing the potential bias in the SUV max measurements as a result of its sensitivity to noise.

Whereas SUV max and SUV peak are independent on the tumor delineation strategy used, TL, TV, SUV mean and the derived TLG were determined on delineations performed using two strategies. First, an adaptive threshold (23) using a background region of interest (ROI) manually chosen by two experienced nuclear medicine physicians led to two different results T bckgrd 1 and T bckgrd 2

. Observers were instructed to choose the ROI in the mediastinum at a sufficient distance from the lesion to avoid any overlapping. However, they were allowed to choose the size, shape and exact placement of the ROI. Finally the automatic Fuzzy Locally Adaptive Bayesian (FLAB) algorithm (24,27) was considered.

Statistical analysis

All quantitative data were expressed as mean±1 standard deviation (SD) and summary statistics are given in table II.

The correlation between all parameters extracted using the different methodologies was computed with Pearson coefficients. The differences between methodologies were assessed using Bland-Altman analysis [START_REF] Bland | Statistical methods for assessing agreement between two methods of clinical measurement[END_REF] to define bias as the mean error, and agreements intervals (upper and lower limits) as 1.96 times the SD.

Kaplan-Meier methods were used to estimate the survival distributions [START_REF] Kaplan | Non parametric estimation from incomplete observations[END_REF]. Overall survival was calculated from the date of initial diagnosis to the date of death or most recent follow-up in case of patients still alive. For each considered parameter, survival curves were generated. The most discriminating threshold value allowing differentiation of the two groups of patients was selected using receiver operating characteristic (ROC) methodology [START_REF] Metz | Basic principles of ROC analysis[END_REF]. Prognostic value of each parameter in terms of overall survival was assessed by the log-rank test. The significance of the following factors were tested: age, gender, histology type, T, N, M classifications, AJCC (American Joint Committee on Cancer) stage [START_REF] Greene | AJCC cancer staging manual[END_REF], TL, TV, SUV max , SUV peak , , SUV mean , and TLG. Independent prognostic factors of overall survival were determined using multivariate Cox regression analysis (32) by incorporating in the model all parameters that were deemed significant in the univariate analysis.

However, the indices obtained by each delineation (T bckgrd 1 , T bckgrd 2 and FLAB) were incorporated in the multivariate analysis separately since they were found to be highly correlated (Pearson r>0.8, r 2 >0.66 ; see results section 3.1). All tests were carried out using MedCalc TM (MedCalc Software, Belgium). P values <0.05 were considered statistically significant.

Results

All primary lesions were detected by 18 F-FDG PET. The intensity of maximum 18 F-FDG uptake in the primary lesion was quite high with a normally distributed SUV max of 10±3.8. As expected, SUV peak measurements were comparatively lower (8±3). Measurements related to the dimensions of the tumors were less uniformly distributed than SUV measurements, with a larger SD with respect to the mean. For example the TV(FLAB) was 35±33cm 3 .

Correlation between image derived indices and between methodologies

TL measurements were correlated with TV (p<0.0001) although with moderate coefficients (r=0.69, 0.58 and 0.6 for FLAB, T bckgrd 1 and T bckgrd 2 respectively). No significant correlation was found between any SUV measurement (SUV max , SUV peak , SUV mean ) and TV (p>0.2, r<0.3), irrespective of the delineation strategy, in line with results of other studies such as Van Heijl et al [START_REF] Van Heijl | Influence of ROI definition, partial volume correction and SUV normalization on SUV-survival correlation in oesophageal cancer[END_REF].

All SUV mean measurements derived from TV delineation performed using the two different methodologies considered were highly correlated (p<0.0001) with coefficients >0.97. TV (r>0.82), TL (r>0.91) and TLG (r>0.95) results were also highly correlated (p<0.0001) for both methodologies.

Despite high correlation coefficients, large differences were observed for several patients between measurements using the two delineation methodologies considered, and between the two users of the same adaptive thresholding. Figure 1(A-B) illustrates such differences. In the case of adaptive thresholding these differences were the result of the two users placing differently the background ROI.

A summary of the Bland-Altman analysis carried out to compare the delineation methods and highlight potential differences is presented in figure 2(C-D)

and complete results are given in table III. The largest differences between methodologies were observed for TV with a bias of up to 50% between the adaptative thresholding and FLAB: both users resulted in globally smaller volumes (bias of -50%±50% and -21%±54% for T bckd 1 and T bckd 2 respectively). Agreement intervals (upper and lower limits) were large for all parameters and for all comparisons, up to +80% and -140% (see fig. 2B). Even between the two users of the same adaptive thresholding method (see fig. 2A), mean differences of -30±35% were seen and limits of agreement were large, from -100 to +45%. No significant trend was found regarding the correlation between TV and differences between methodologies (r<0.2, p>0.1).

Better agreement was observed for TL and SUV mean , however intervals of agreement were large (-50% to -25% lower limit and +20% to +40% upper limit for TL; -80% to -10% lower limit and +10 to +80% upper limit for SUV mean ).

Due to the combined effect of TV and SUV mean , TLG differences were in between, with moderate bias but still large agreement intervals (upper and lower limits of -120% to -75% and +40% to +90% respectively).

Survival analysis

At the time of last follow-up, 10 patients were alive with no evidence of disease, 9 were alive with recurrent esophageal cancer and 26 had died from the disease. With a median follow-up of 60 months (range 9-82), the overall median survival was 15 months. The 1-year and 2-year survival rates were 63% and 34%

respectively.

The results of the log-rank analysis of significant parameters for overall survival in univariate analysis are given in table IV. Table V summarizes the prognostic value of all the parameters under investigation in this study.

Age, gender, and histology types were not significant prognostic factors in the univariate analysis. Neither were T and N classifications. In the univariate analysis, the presence of metastases (median survival of 26 months (M0) versus 12 months (M1), p=0.01)) and the clinical AJCC stage (p<0.001) were significant prognostic factors.

Although there was a trend observed, neither SUV max nor SUV peak were significant prognostic factors. A SUV max <5 or <8 tend to be a factor of better outcome with a median survival of 14 vs. 7 months (p=0.08) or 21 vs. 13 months (p=0.1) respectively (see fig. 3A).

Mean SUVs in the tumor were not significant prognostic factors in univariate analysis. There was however a trend for shorter survival associated with higher SUV mean . For example the median survival reduced by a factor of 2 for patients with a SUV mean higher than 5 (13 months vs. 21 months, p=0.06). This was however observed only when the FLAB methodology was used to define TV, while no similar trend was observed with SUV mean parameters obtained with adaptive thresholding.

Functional TV was a significant prognostic factor of overall survival, whatever methodology was used (p<0.001 using FLAB, and p=0.004 for both T bckgrd 1 and T bckgrd 2

, see figure 3(B-C)). In addition, using the TV, and independently of the delineation approach used, allowed to separate our population into 3 groups with significantly different outcome (p=0.002, p=0.02 and p=0.004 for FLAB, T bckgrd 1 and T bckgrd 2 respectively). For instance, volumes defined by FLAB less than 14cm 3 , between 14 and 85cm 3 or superior to 85cm 3 were respectively associated with a median survival of 49 (19 patients), 15 (21 patients) and 5.5 (6 patients) months as illustrated in figure 3(D). In figure 4(A-C) three examples of 18 F-FDG PET baseline images of patients belonging to each of these three groups are presented.

Functional TL was also a significant prognostic factor with results similar to TV (p=0.01, p=0.02 and p=0.04 for FLAB, T bckgrd 1 and T bckgrd 2 respectively), apart from not being possible to significantly differentiate 3 groups of patients with different outcome, independently of the strategy.

Similarly, TLG was also a significant prognostic factor whatever methodology was used, while it was equally not possible to significantly differentiate three groups.

Median overall survival was 10 months for patients with a TLG (FLAB) >180g, and increased to 21 months for patients with a TLG<180g (p=0.01). Similar results were obtained with adaptive thresholding (20 versus 8 and 20 versus 10 months for T bckgrd 1 and T bckgrd 2 respectively).

After multivariate analysis, considering each delineation methodology separately only TV obtained using FLAB and AJCC stage were found to be independent significant prognostic factors (p=0.0017 and 0.0021 for TV and AJCC respectively), whereas only AJCC stage was an independent significant prognostic factor (p<0.002) when considering TV obtained by adaptive thresholding.

Similar results were obtained when replacing TV by TL, with both TL and clinical AJCC staging found to be independent significant prognostic factors in the case of FLAB (p=0.017 and p=0.042 for AJCC stage and TL respectively), whereas in the case of adaptive thresholding only AJCC staging was an independent significant prognostic factor (p=0.0021).

On the other hand, in the case of TLG only the AJCC staging was an independent significant prognostic factor (p<0.002), whatever delineation strategy was considered.

Discussion

An accurate staging in esophageal cancer is essential for guiding therapy. The standard conventional modalities are endoscopic ultrasonography and computed tomography even if this combined approach suffers from several shortcomings. 18 F-FDG PET is more and more often included in the initial staging because it allows a more accurate disease assessment, especially regarding the detection of distant metastases [START_REF] Flamen | Utility of positron emission tomography for the staging of patients with potentially operable esophageal carcinoma[END_REF][START_REF] Heeren | Detection of distant metastases in esophageal cancer with (18)F-FDG PET[END_REF][START_REF] Vliet | Staging investigations for esophageal cancer: a meta-analysis[END_REF]. Since no patient underwent surgery in our study, anatomopathology data were not available. Therefore T,N,M classifications and AJCC stages were determined using suboptimal conventional staging and this could explain the poor prognostic value of T or N classification in our population.

As found in our study, 18 F-FDG uptake is always present in esophageal cancer if extended at least to submucosa [START_REF] Himeno | Evaluation of esophageal cancer by positron emission tomography[END_REF]. Some authors suggested that the intensity of 18 F-FDG uptake could be related to prognosis in esophageal cancer, based on the good correlation existing between hexokinase activity or poor differentiation and tumor uptake [START_REF] Fukunaga | Evaluation of esophageal cancers using fluorine-18-fluorodeoxyglucose PET[END_REF] and also because increasing SUV max values seem to correlate with T classification, which is part of the TNM staging [START_REF] Taylor | Correlations between selected tumor markers and fluorodeoxyglucose maximal standardized uptake values in esophageal cancer[END_REF].

In our study, SUV measurements were not significant prognostic factors of overall survival. While various cut-off values of SUV max tend to be associated with a poor prognosis, none led to statistically significant differentiation. Swisher et al. reported similar results in a uniform group of highly selected patients with locally advanced esophageal cancer treated by neoadjuvant radiochemotherapy [START_REF] Swisher | 2Fluoro-2-deoxy-D-glucose positron emission tomography imaging is predictive of pathologic response and survival after preoperative chemoradiation in patients with esophageal carcinoma[END_REF]. On the other hand, these results could appear in contrast with our previous report [START_REF] Rest | Prognostic value of initial fluorodeoxyglucose-PET in esophageal cancer: a prospective study[END_REF], where we initially reported that a SUV max cut-off value of 9 had an independent prognostic value of overall survival, but this difference may be explained by the different patient characteristics considered in the two studies. We previously considered (18) a daily practice population, half of which underwent curative surgery, while we included here only patients with advanced disease exclusively treated by combined radiochemotherapy.

TL established by pathological examination has been demonstrated to be an independent prognostic factor of long term survival [START_REF] Yendamuri | Esophageal Tumor Length Is Independently Associated with Long-term Survival[END_REF]. Some authors proposed estimating TL based on 18 F-FDG PET images using different thresholds [START_REF] Zhong | Using 18F-fluorodeoxyglucose positron emission tomography to estimate the length of gross tumor in patients with squamous cell carcinoma of the esophagus[END_REF].

Functional TL has been studied as a predictor of response to neoadjuvant chemoradiotherapy with conflicting results [START_REF] Mamede | FDG-PET/CT tumor segmentation-derived indices of metabolic activity to assess response to neoadjuvant therapy and progression-free survival in esophageal cancer: correlation with histopathology results[END_REF][START_REF] Roedl | Assessment of treatment response and recurrence in esophageal carcinoma based on tumor length and standardized uptake value on positron emission tomography-computed tomography[END_REF]. In a group of 69 patients with esophageal squamous cell carcinoma undergoing curative surgery, Choi et al.

demonstrated that functional TL was an independent prognostic factor [START_REF] Choi | 18F-FDG PET in patients with esophageal squamous cell carcinoma undergoing curative surgery: prognostic implications[END_REF]. However, one may argue that functional TL is a parameter that does not reflect the real volume of the tumor but only its longitudinal extension and could be therefore considered as only a surrogate of tumor spatial extent. This argument can be supported by the data shown in this work, where only a moderate correlation (r<0.7) was found between TV and TL, suggesting that functional TV may be more accurate in assessing actual tumor burden. In our study we also compared the prognostic value of TL with that of TV. Both parameters were found to be significant prognostic factors irrespective of the functional volume delineation strategy. In addition, both TV and TL were independent prognostic factors of survival in the multivariate analysis. However, this result was found to be dependent on the segmentation algorithm, with both parameters being independent survival prognostic factors only when determined using the automatic FLAB segmentation. This may be related to the higher overall accuracy of FLAB with respect to adaptive thresholding for tumor delineation as previously reported [START_REF] Hatt | Accurate automatic delineation of heterogeneous functional volumes in positron emission tomography for oncology applications[END_REF][START_REF] Hatt | A fuzzy Bayesian locally adaptive segmentation approach for volume determination in PET[END_REF][START_REF] Tylski | Comparative assessment of methods for estimating tumor volume and standardized uptake value in (18)F-FDG PET[END_REF]. Despite the similar prognostic values of TL and TV, only TV allowed a statistically significant stratification of patients into three groups, irrespective of the segmentation methodology. More specifically, two different cut-off values (85 and 14cm 3 ) resulted in significant differentiation of two groups among the patients with median overall survival of 5 to 6 vs 20 months (p=0.0005) and 49 vs 13 months (p=0.036) for 85 and 14cm 3 respectively. Being able to provide such a finer stratification of patient groups could be of value in clinical trials assessing new therapeutic regimes.

SUV mean measured in a volume determined using the different tumor delineation approaches considered was not found to be a prognostic factor of overall survival, although a trend was seen for SUV mean associated with TV defined with FLAB, which tended to differentiate patients with poor and better prognosis (13 vs 21 months, p=0.06).

A fundamental biological question underlying 18 F-FDG PET prognostic value is whether the total volume or the metabolic active portion of the tumor is most important. Intuitively both would seem important and desirable to determine. In our study, both functional TL and TV (representative of the tumor functional spatial extent) were significant prognostic factors compared to SUV mean (representative of the tumor glycolytic metabolism) which was not. Combining both parameters into total lesion glycolysis index (TLG) was a prognostic factor of overall survival whatever methodology was used for tumor delineation. However it was not an independent significant prognostic factor in the multivariate analysis. Only very few data are available on the potential clinical value of TLG in different cancer models. Xie et al reported on prognostic value of TLG in head and neck cancer for long term survival [START_REF] Xie | Prognostic value of (18)F-FDG PET-CT metabolic index for nasopharyngeal carcinoma[END_REF], while Cazaentre et al. demonstrated the usefulness of TLG for predicting response to radioimmunotherapy in lymphoma [START_REF] Cazaentre | Pre-therapy 18F-FDG PET quantitative parameters help in predicting the response to radioimmunotherapy in non-Hodgkin lymphoma[END_REF]. To date, the limited use of TV and TLG in clinical studies could be explained by the poor accuracy, robustness and reproducibility of available tumor delineation tools affecting the clinical value of resulting measurements. The fact that TLG was not an independent prognostic factor whereas TV alone was, suggests that the prognostic value of TLG mainly comes from the volume information and is impaired by the low prognostic value of SUV mean measurements. In addition, the value of TLG might be reduced by a loss of information when combining the TV and the SUV mean into one parameter by simple product, since large tumors with low uptake might result in the same TLG as small tumors with high uptake. Finally, the lack of partial volume effects (PVE) correction might also play a role in the reduced prognostic value of all SUV measurements as well as the resulting TLG, since tumor volumes across the patients range from quite small and significantly affected by PVE (<2cm in diameter) to very large tumors for which PVE have smaller quantitative impact.

As expected, results concerning parameters dependent on the tumor delineation process were well correlated. On the other hand our results also highlighted the potential impact of differences between existing tumor delineation methods, with TV and TL being independent survival prognostic factors only when determined using FLAB. This approach has been previously shown to be both robust and accurate [START_REF] Hatt | Accurate automatic delineation of heterogeneous functional volumes in positron emission tomography for oncology applications[END_REF][START_REF] Hatt | A fuzzy Bayesian locally adaptive segmentation approach for volume determination in PET[END_REF]. At present most commonly used methods are based on fixed or adaptive thresholds. Fixed thresholding has been demonstrated to be both inaccurate and non-robust [START_REF] Nestle | Comparison of Different Methods for Delineation of 18F-FDG PET-Positive Tissue for Target Volume Definition in Radiotherapy of Patients with Non-Small Cell Lung Cancer[END_REF][START_REF] Hatt | Accurate automatic delineation of heterogeneous functional volumes in positron emission tomography for oncology applications[END_REF][START_REF] Hatt | A fuzzy Bayesian locally adaptive segmentation approach for volume determination in PET[END_REF][START_REF] Tylski | Comparative assessment of methods for estimating tumor volume and standardized uptake value in (18)F-FDG PET[END_REF] and was therefore not considered in this study.

Regarding the adaptive thresholding performance, results from one observer (T bckgrd 2

) were closer to these of FLAB compared to the other one (T bckgrd 1

), with however significant differences, as shown in figure 2B ) were independent prognostic factors contrary to TV(FLAB). This can be explained by the behavior of adaptive thresholding (independently on the user) for several tumors. Most of the tumors exhibited simple shapes and homogeneous tracer uptake. However some were more complex and exhibited higher heterogeneity, or were small (<2-3cm) with low contrast. Adaptive thresholding has been demonstrated to provide unsatisfactory delineation for such cases [START_REF] Hatt | Accurate automatic delineation of heterogeneous functional volumes in positron emission tomography for oncology applications[END_REF], because its final threshold is based on the ratio between an isocontour at 70% of the maximum and the background ROI. Such isocontour tends to overestimate (respectively underestimate) the actual value of the entire tumor for heterogeneous uptake (respectively small tumors will low contrast).

Hence the adaptive thresholding led to significant under-evaluation of larger heterogeneous tumors in our study, e.g. a patient with a survival of 6 months had a TV defined by FLAB of almost 97cm 3 , whereas TV(T bckgrd 1

) and TV(T bckgrd 2

) were 38cm 3 (-61%) and 50cm 3 (-50%) respectively, clearly missing parts of the tumor. On the other hand, the dependency on the background ROI is higher regarding small tumors with low contrast, e.g. for a patient with 21 months survival, TV(FLAB) was 5.8cm 3 , whereas TV(T bckgrd 1

) and TV(T bckgrd 2

) were 1.9cm 3 (-67%) and 26.9cm 3 (+364%) respectively. Several patients were therefore incorporated in the wrong survival curve, mostly patients with large volumes that were underestimated by the adaptive threshold.

In addition, adaptive thresholding was found to be highly user dependent, since we observed a bias up to 30% between the two users measuring TV, the agreement interval being too large for clinical applications (-110% to +45%). This seems to be in agreement with results concerning the level of reproducibility in measuring functional TV from 18 F-FDG imaging which can vary from 21% to 90% using automatic and threshold-based approaches respectively [START_REF] Hatt | Reproducibility of 18F-FDG and 18F-FLT PET tumor volume measurements[END_REF]. If advanced segmentation algorithms are not available, the use of adaptive thresholding approaches should be preferred to manual or fixed threshold-based delineation. Automated background ROI determination could reduce the inter observer variability observed in this work.

The limits of this study are its retrospective nature and the limited number of patients. Our results need to be confirmed through a prospective study on a larger cohort of patients. It is finally worth noting that overall survival might have been affected by other factors such as subsequent treatment for patients who relapsed, although this should have minor impact to the results of this study since it applies to all considered parameters. Other outcome measures such as progression-free survival were not investigated in this study.

Conclusion

Our results suggest that the functional tumor volume followed by length has additional value compared to commonly-used SUV measurements (SUV max , SUV peak , SUV mean ) for prognosis in patients with locally advanced esophageal cancer treated with exclusive concomitant radio-chemotherapy. Both parameters were significant prognostic factors of overall survival, independently of the approach used to delineate the tumors. However, only the automatic FLAB algorithm allowed TV and TL to be independent prognostic factors of survival in a multivariate analysis that included standard tumor staging. In addition the total lesion glycolysis index was a statistically significant, but not independent, prognostic factor irrespective of the delineation algorithm used. Our findings confirm the potential value of 18 F-FDG PET to give a useful orientation for patient management purpose in esophageal cancer but they also highlight the influence of the methodology used on the degree of pertinence of these PET image derived parameters of interest as their accuracy and their clinical significance increase if they are computed using more reliable and robust tumor segmentation methods.

Figures captions

Fig. 1: Illustration of differences in tumor delineation depending on the methodology for (A) a small (<8cm 3 ) and low contrast (approximately 2:1) and (B) a larger (30cm 3 ) and higher contrast (approximately 7:1) tumors. 95% confidence intervals for each are given in table III.

18

 18

Fig. 2 :

 2 Fig.2:Bland-Altman analysis of differences between (A) T bckgrd1 and T bckgrd 2 and (B)

Fig. 3 : 1 ,

 31 Fig.3: Kaplan-Meier survival curves obtained using (A) SUV max , TV measured by (B)

Fig. 4 :

 4 Fig.4: 18 F-FDG PET images (axial, coronal and sagittal views from top to bottom) of patients with a (a) small tumor (11cm 3 , 54 months survival), (b) medium size tumor (22cm 3 , 18 months survival) and (c) larger tumor (92cm 3 , 5 months survival).

Figure 1 Figure 2 Figure 3 Figure 4 Parameter # of patients

 1234 Figure 1

Table captions Table I :

 captionsI Patient demographic and clinical characteristics

Table II :

 II Parameters definition and statistics.

Table III :

 III Bland-Altman analysis results comparing delineation strategies for all parameters.

Table IV :

 IV Parameters with significant prognostic value after univariate analysis.

Table V :

 V Prognostic value of all parameters.

Table I

 I

		(%)
	Gender
	Male	41(91)
	Female	4(9)
	Age
	Range	45-84
	Median	68
	Site
	Upper esophagus	11(24)
	Middle esophagus	17(38)
	Lower esophagus	17(38)
	Histology type
	Adenocarcinoma	12(27)
	Squamous cell carcinoma	33(73)
	Histology differentiation
	Well differentiated	12(27)
	Moderately differentiated	11(24)
	Poorly differentiated	4(9)
	Unknown	18(40)
	TNM Stage
	T1	6(13)
	T2	7(16)
	T3	22(49)
	T4	10(22)
	N0	18(40)
	N1	27(60)
	M0	29(64)
	M1	16(36)
	AJCC Stage
	I	3(7)
	IIA	7(16)
	IIB	5(11)
	III	14(31)
	IVa	5(11)
	IVb	11(24)

Conflict of interest:

The authors declare that they have no conflict of interest.

Definition