β-Amyloid 25-35 Peptide Reduces the Expression of Glutamine Transporter SAT1 in Cultured Cortical Neurons

β-Amyloid (Aβ) peptides may cause malfunction and death of neurons in Alzheimer’s disease. We investigated the effect of Aβ on key transporters of amino acid neurotransmission in cells cultured from rat cerebral cortex. The cultures were treated with Aβ(25-35) at 3 and 10 μM for 12 and 24 h followed by quantitative analysis of immunofluorescence intensity. In mixed neuronal–glial cell cultures (from P1 rats), Aβ reduced the concentration of system A glutamine transporter 1 (SAT1), by up to 50% expressed relative to the neuronal marker microtubule-associated protein 2 (MAP2) in the same cell. No significant effects were detected on vesicular glutamate transporters VGLUT1 or VGLUT2 in neurons, or on glial system N glutamine transporter 1 (SN1). In neuronal cell cultures (from E18 rats), Aβ(25-35) did not reduce SAT1 immunoreactivity, suggesting that the observed effect depends on the presence of astroglia. The results indicate that Aβ may impair neuronal function and transmitter synthesis, and perhaps reduce excitotoxicity, through a reduction in neuronal glutamine uptake.
Introduction
Alzheimer’s disease (AD) is a neurodegenerative disorder, leading to progressive loss of memory, impaired cognition and debilitation. It affects about 2% of the population in industrialized countries, the risk of having the disease increasing rapidly with age beyond 70, hence the prevalence of AD will increase several fold in the coming decades [1, 2]. AD is characterized by extracellular deposits of β-amyloid (Aβ), associated with dystrophic neuronal processes, degenerating and apoptotic neurons showing neurofibrillary tangles (i.e. aggregates of hyperphosphorylated tau protein filaments).
The neurotoxic effects of Aβ and some of its fragments have been demonstrated in in vitro primary cultures from human and rat cortical neurons, in hippocampal slice cultures and following in vivo injection into various areas of the rat and monkey brain [3]. Aβ peptides cause neuronal cell death and also increase the vulnerability of neurons to excitotoxicity [4]. Aβ(25-35) has been frequently used in investigations of Aβ properties as a less expensive and more easily handled substitute for the native full-length peptide, Aβ(1-42), the latter being implicated in the pathogenesis of AD. Implicit in these experiments is the assumption that the mechanism of action of Aβ(25-35) is similar to that of Aβ(1-42). Indeed, Aβ(25-35) mimics the toxicological and aggregational properties of the full-length peptide, though these characteristics are increased; i.e., the shorter peptide is more toxic to cultured neurons, exhibits toxicity earlier, causes more membrane protein oxidation, and aggregates faster than the native Aβ(1-42) [5].
The amino acids glutamine and glutamate are essential for brain metabolism and function. Glutamate is the major excitatory transmitter of the brain [6], particularly in the cerebral cortex and hippocampus, and therefore has important roles in cognition, learning and memory. After synaptic release, it is taken up mainly into astroglia by glutamate transporters [7]. Perturbed glutamate uptake may cause excitotoxicity and is implicated in the pathogenesis of AD [7]. Once accumulated into astrocytes, glutamate is converted to the non-excitatory amino acid glutamine by the glial enzyme glutamine synthetase. Glutamine is then returned to neurons via glutamine transporters of system N (SNs) and system A (SATs) and used as a source for the synthesis of glutamate through its conversion by phosphate-activated glutaminase [8, 9]. Glutamate is packaged for exocytosis by vesicular glutamate transporters (VGLUTs) [10, 11], or used as precursor of GABA in GABAergic neurons.
The aim of this study was to explore the effects of Aβ(25-35) treatment on main transporters implicated in sustaining synaptic release of amino acids, i.e. the glutamine transporters SN1 and SAT1 and the vesicular glutamate transporters VGLUT1 and VGLUT2, in two different in vitro models: neuronal cell cultures and mixed cell cultures of rat cerebral cortex.
Experimental procedures
Cell cultures
Neuronal cultures were prepared from embryos of Wistar rats at day 18 of gestation (E18) using a modification of an earlier described method [12]. In brief, the neocortices of embryos were dissociated by treatment with 0.25% trypsin/calcium–magnesium-free buffer/EDTA for 10 min at 37○C. The suspension was settled for 2 min at 2,000 × g and then the cells were mixed in DMEM, triturated and passed through a cell strainer. The cells were cultured in DMEM supplemented with 2% B27 and 1% GlutaMAX I. The cells were plated on poly-l-lysine (50 μg/ml) coated glass coverslips at a density 0.5 × 106 cells/ml and the cultures were maintained in a humidified atmosphere of 5% CO2/95% air at 37○C for 5 days in vitro before incubation with peptide.
Primary mixed cell cultures were prepared from the cerebral cortices of 1-day-old (P1) neonatal Wistar rats and plated on poly-L-lysine-treated coverslips (see above). The dissociated cortical cells were suspended in Neurobasal-A medium containing 2% B27 and 1% GlutaMAX I and maintained as described for E18 cells.
Rats were kept according to nationally and internationally approved conditions. Culture media and reagents were from Gibco, Invitrogen Corporation (Carlsbad, CA, USA).
Incubation with Aβ peptide
Neuronal and mixed cell cultures were treated with Aβ(25-35) (Sigma-Aldrich, St.Louis, MO, USA) at final concentrations of 3 and 10 μM, for 12 or 24 h. In this study the B27-containing medium was removed from the cultures at day 6. Cells were washed twice with DMEM or Neurobasal-A medium and then incubated in medium for 12 and 24 h in the absence or presence of Aβ(25-35). Controls received no peptide.
Antibodies
Antibody to SAT1 was raised in rabbit to a fusion protein corresponding to the N-terminal amino acids 1-71 of SAT1 (aka GlnT, [13]; aka SA2, [14]), and affinity purified and characterized (T.T. Solbu & F.A. Chaudhry, to be published) as described for SN1 [15]. A GST-fusion peptide was made by cloning the first 213 base pairs from SAT1 into a pGex3X vector (Amersham Biosciences, GE Healthcare, Oslo, Norway). Using primers 5′- cccgggatccttatgatgcatttcaaa-3′ and 5′-gcggaattcggttgttcctggaat-3′ (Eurogentech, Herstal, Belgium), the 5′ end of SAT1 was amplified by PCR and ligated into pGex3X using BamH1 and EcoR1 (New England Biolabs, Ipswich, MA, USA). GST-SAT1 was purified on Gluthatione Sepharose 4B as prescribed by the manufacturer (Amersham Biosciences; “GST Gene Fusion System Handbook”). Antibodies to other transporters were prepared and characterized as described (VGLUT1 and VGLUT2: [16]; SN1: [15]). Antibodies to marker proteins [glial fibrillary acidic protein (GFAP), anti-microtubule associated protein-2A&B (MAP2)], raised in mice, were from Sigma-Aldrich. Secondary antibodies were Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 555 goat anti-rabbit IgG from Molecular Probes (Eugene, Oregon, USA).
Immunocytochemical staining
Following exposure to peptide, cells attached to 14-mm glass coverslips were fixed with formaldehyde (4% paraformaldehyde, freshly depolymerized, in 0.1 M sodium phosphate buffer pH 7.4) for 20 min at room temperature followed by three washes in phosphate buffered saline (10 mM phosphate buffer in 150 mM NaCl) and then blocked with blocking buffer [10% (v/v) newborn calf serum (NCS), 3% (w/v) bovine serum albumin (BSA), 0.5% (w/v) Triton X-100 in Tris buffered saline (TBS) pH 7.4] for 1 h at room temperature. After blocking, sections were incubated with the primary antibodies (anti-VGLUT1 1:3,000; anti-VGLUT2 1:3,000; anti-SAT1 2 mg/ml; anti-SN1 0.3 mg/ml; anti-MAP2 1:200; GFAP 1:500; dilutions optimized in pilot experiments) in primary antibody solution [3% (v/v) NCS, 1% BSA, 0.5% Triton X-100, 0.05% NaN3 in TBS pH 7.4] overnight at 4°C, and then the cells were washed and incubated with the secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 555 for 1 h at room temperature. The coverslips were then washed three times in fresh antibody solution. Cells on coverslips were sealed to microscopic slides with Fluoromout G water based (Southern Biotechnology Associates, Birmingham, AL, USA).
Quantitative analysis of immunoreactivities
Dual immunofluorescence for VGLUT1, VGLUT2, SAT1, SN1, GFAP and MAP2 was examined and quantified with a Zeiss Axioplan 2 microscope equipped with an LSM 5 PASCAL confocal unit, LSM software and a 40x/1.3 oil PlanApo objective (Carl Zeiss, Heidelberg, Germany). We used standard FICT-rhodamine filter settings. Other settings were optimized initially, and held constant throughout the study so that all sections were digitized under the same conditions of illumination. Care was taken to use similar times for orientation (with conventional fluorescence) and adjustments in all preparations, in order to avoid differences in photobleaching. The cells were selected on the criterion that they showed representative morphology (Fig. 1) and strong immunoreactivity for MAP2 (neurons) or for SN1 (in the case of astroglia). The cell body and the most proximal parts of the processes were outlined for digitalization of area and of fluorescence intensity for two immunoreactivities (MAP2 and a transporter). The analysis was performed without notice of the treatment group; the lack of difference in neuronal area between treatment groups suggests that sampling bias did not interfere with the results obtained. Fluorescence intensity is expressed in arbitrary units, without subtraction of background. For each glass coverslip, five fields were randomly sampled and two cells quantified per field, i.e. 10 cells, to obtain an average value for each immunoreactivity and experiment.
Fig. 1Classification of cell types. (a, c, e) MAP2 positive (green) neuronal cells (arrowheads). (b) GFAP positive (red) astroglial cell (arrow, same field as a). (d, e) SN1 positive (red) astroglial cell (arrow, same field as c); SN1 (in contrast to GFAP) reveals the entire extent of the astrocyte. (e) MAP2 and SN1 immunoreacivities superimposed. (f) DIC image (same field as c–e). Arrowheads and arrows indicate the positions of the nuclei of sample neurons and astrocytes, respectively. Note that all of the 10 cells situated on or next to the astrocyte in (f) are identified as MAP2 positive neurons in (c). Double immunofluorescence observed by confocal microscopy in mixed cell cultures from rat cortex at P1
Data analysis
Data are expressed as mean ± standard error of the mean (SEM) of four different experiments (four different sets of cultures prepared at four different times) for P1 and E18 cultures. Advanced statistical analysis was performed according to a linear mixed model regression analysis, using the function lme of the nlme package [17] in the statistical software R (http://www.r-project.org/). The fundamental assumptions of the model are that the observations are normally distributed with a variance varying only with protein type, and that they are uncorrelated when they come from different experiments. Log (for P1 data) or square root (for E18 data) transformations were performed as quantile–quantile plots showed these transformations to give the best fits to the Gaussian distribution.
Results
Cell types
Cell types were characterized in control cultures incubated without Aβ. In mixed neuronal–glial cultures (from cortex of P1 rat pups), neurons and astroglia were clearly identified by immunoreactivity for the markers MAP2 and GFAP, respectively (Fig. 1a, b). Double labelling for MAP2 and system N glutamine transporter SN1, localized in the membranes of astroglia [15], again showed the two immunoreactivities in separate cells (Fig. 1c–e), SN1 displaying the full extension of protoplasmatic-like astrocytes (i.e. beyond the parts pervaded by GFAP filaments, Fig. 1a, b). The MAP2 expressing cells show the morphological characteristics of neurons and usually grow in close apposition to astroglia (Fig. 1a–f). In neuronal cultures (from E18 rats), MAP2 positive neurons had similar morphology, but no GFAP positive astroglial cells were found (not shown).
Double labelling for MAP2 and different transporter proteins showed the vesicular glutamate transporters VGLUT1 or VGLUT2 to be in neurons. A slight signal for SN1 in neurons was close to background levels. The system A glutamine transporter SAT1 was mainly localized in neurons but slight immunoreactivity was seen in some astroglial cells (Fig. 2a, b). In the neurons, SAT1 staining was strong in central areas of the perikaryon and pervaded the processes.
Fig. 2SAT1 (red) colocalized (yellow-orange) with MAP2 (green) in neurons in P1 cortical mixed cell cultures. Images show sample cells, insets display framed areas separately for SAT1 immunoreactivity. Cultures were exposed to control medium for 12 h (a) or 24 h (b), or to 10 μM of Aβ(25-35) for 12 h (c) or 24 h (d). SAT1 is expressed in the soma and processes of neurons, as revealed by double labelling with antibodies to the somatodendritic neuronal marker MAP2 shown superimposed on SAT1. Foci of strong perikaryal immunoreactivity suggest intracellular stores of SAT1. (Slight SAT1 immunoreactivity occurs also in glia-like perikarya (b, centre right, arrow), immunonegative for MAP2.) The immunoreactive cells were morphologically similar in the different conditions (a–d). Scale bar = 20 μm
Survey of preparations double stained for MAP2 and either VGLUT1 or VGLUT2 indicated that a large proportion of the neurons were VGLUT immunoreactive. Similarly, double staining for MAP2 and SAT1 suggested that most of the neurons in the cultures contained SAT1.
Effects of Aβ in mixed cell cultures
Cortical mixed cell cultures (P1) were exposed to Aβ. After day 5 in vitro cell cultures were incubated with 3 or 10 μM of Aβ(25-35) for 12 or 24 h and then processed for immunohistochemistry and quantitative analyses. Neurons with typical morphology and strong MAP2 staining were seen also after exposure to Aβ (Fig. 2c, d). The cells appeared morphologically similar to those in control cultures (Figs. 1, 2a, b). Transporter immunoreactivities were quantitatively analysed (Fig. 3a, b) in neurons with typical morphology and clear MAP2 immunoreactivity. In addition, SN1 was quantified in cells with typical astroglial morphology according to SN1 immunofluorescence. All immunoreactivities showed a tendency for decline on exposure to Aβ(25-35), at 12 h as well as at 24 h, but this attained statistical significance only for SAT1. (The low SN1 level in neurons is close to background and may not be biologically significant.) The size of the central areas of the cells, over which the fluorescence intensities were recorded, was independent of peptide exposure in the neurons and only slightly reduced (by about 16%) in the glia (Fig. 3c, d). This indicates that, in the neurons recorded, Aβ treatment did not cause general impairment resulting in a reduction of cell size.
Fig. 3Aβ induced a reduction of SAT1 immunoreactivity in neurons in P1 mixed cortical cell cultures. Transporter immunoreactivities (arbitrary units of fluorescence intensity, FL) and areas (μm2) were recorded in individual cells in cultures incubated with 0, 3 or 10 μM Aβ(25-35) for 12 h (a, c, e) or 24 h (b, d, f). Areas (c, d) comprising the perikaryon and proximal neurites were outlinded for recording of an average fluorescence intensity in each cell. To cancel out random differences between cells, transporter immunoreactivity was also expressed relative to MAP2 immunoreactivity in the same cell (e, f). All values are for neurons, except for SN1, which was recorded also in astroglial cells (area and relative immunoreactivity for SN1 in glia were 40 times the displayed numbers). Background was not subtracted; the low values for SN1 in neurons are close to non-immunoreactive background. Columns show mean + SEM of measurements from four independent experiments (n = 4), in each experiment 10 cells were averaged. Asterisks indicate a statistically significant difference compared with control (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001) in a linear mixed model regression analysis (statistical software R). Only SAT1 showed a robust effect of Aβ
To adjust for differences in the condition of the individual cell, transporter immunoreactivity was expressed relative to the simultaneously recorded MAP2 immunoreactivity of the same cell (Fig. 3e, f). In this analysis, the effect of Aβ on SAT1 stood out even more clearly, showing the largest percentage changes, whereas there were no statistically significant effects of Aβ on VGLUT1, VGLUT2 or SN1 in neurons, neither on SN1 in astroglia. After both 12 and 24 h incubation, SAT1 was reduced to about two thirds of the control level by 3 μM of Aβ, and about one half by 10 μM of Aβ. As statistical tests showed similar effects at 12 and 24 h, the data were reanalysed statistically with a simplified linear mixed model assuming the same fixed effect at 12 and 24 h. This analysis gave higher statistical significance for the effect on SAT1 (P = 0.0009 at 3 μM, P = 0.0000 at 10 μM) than did the full model reported in Fig. 3. It confirmed the lack of effect on the other transporters (P > 0.2), and the lack of effect on perikaryal size (P > 0.1), except for the size of SN1 positive glia (P < 0.03 for 3 μM, P < 0.01 for 10 μM).
Lack of effect of Aβ in neuronal cell cultures
Cultured cortical neurons (E18) were exposed to Aβ(25-35) in the same way as for P1 mixed cell cultures. MAP2 positive neurons had a similar appearance as in P1 cultures, in controls as well as after exposure to Aβ (Fig. 4a–d). Quantification of the fluorescence intensities (Fig. 5) indicated slight effects of Aβ on SAT1 and SN1 (Fig. 5a, b), but not on the other transporters, or on cell area (Fig. 5c, d). However, when the fluorescence intensity for transporter was normalized to that for MAP2 in the same cell, there were no significant effects of Aβ, except on SN1 (which in neurons is very low and of questionable biological significance). Statistical tests suggested no difference between 12 and 24 h or between 3 and 10 μM. When statistically analysed according to a simplified linear mixed model, assuming the same fixed effect for the different incubation times and concentrations of Aβ, there were no significant effects of Aβ on any of the transporter immunoreactivities (P ≥ 0.07).
Fig. 4SAT1 (red) colocalized (yellow-orange) with MAP2 (green) in neurons in E18 cortical neuronal cell cultures. Sample neurons are shown. The immunoreactive cells were morphologically similar to those in P1 cultures (Fig. 2). No glia-like cells were present. Cells were incubated in the absence (a, b) or presence of 10 μM Aβ(25-35) (c, d) for 12 h (a, c) or 24 h (b, d). Labelling was strong in the perinuclear region and processes of neurons. Scale bar = 20 μmFig. 5Lack of effect of Aβ on SAT1 immunoreactivity in E18 cortical neuronal cells (cultured without glia). Data were obtained and presented as in Fig. 3
Finally, to test more directly whether the results differed between the two types of culture used, the MAP2 normalized data for P1 and E18 cultures were combined into one linear mixed model for regression analysis. The fixed effect was now defined as the expected difference in effect of Aβ exposure between P1 and E18 cultures. Assuming the same fixed effect at 12 and 24 h, the effect on SAT1 was significantly different between neurons in P1 and E18 cultures (P = 0.016 for 3 μM, P = 0.010 for 10 μM of Aβ) but those on the other transporters were not (P > 0.3). Assuming similar effect at the two concentrations of Aβ, the significance level was even higher (SAT1 P = 0.004; other transporters P > 0.2).
Discussion
The main finding of the present study is that Aβ causes a reduction in neuronal SAT1, a transporter thought to be essential for neuronal uptake of glutamine [9, 13, 14] to support neuronal function including the formation of neurotransmitter in glutamatergic as well as GABAergic neurons. The transmitter of the neurons loosing SAT1 was not directly identified, but as double labelling for MAP2 with VGLUTs and MAP2 with SAT1 both showed major proportions of double labelled cells, they are likely to comprise glutamatergic neurons.
The concentrations of Aβ were selected on the basis of literature data to be effective but with low to moderate toxicity levels [12]. To further minimize confounding influence of indiscriminate toxic damage, the immunoreactivities were measured in individual cells that showed near normal morphology and near normal immunoreactivity for the neuronal marker MAP2. Therefore, unlike in biochemical studies of whole cultures, cells dying or dead due to toxic effects of Aβ [5] were excluded from analysis. Further, random variation in the condition of cells was corrected for by expressing the transporter immunoreactivities relative to that of MAP2, measured in the same cell by double immunofluorescence. Aβ peptides have been found to induce proteolysis of MAP2 in cultured mouse cortical neurons [18], but in the cells sampled in the present study such proteolysis must have been minor, as the relation between the values for the different transporters and conditions were similar whether expressed as fluorescence units or relative to MAP2 fluorescence units.
The effect of Aβ on neuronal SAT1 was observed in cultures containing neurons in close apposition to astroglial cells, but not in neuronal cultures without glia. This suggests that Aβ does not produce the effect by acting directly on the neurons, but via the glial cells, an idea gaining some support from the observed reduction in astroglial cell size. However, as the astrocytes had only slightly reduced size and normal levels of their marker protein SN1, the changes in neuronal SAT1 cannot be ascribed simply to toxic damage in the glia.
The findings are consistent with previous reports that the presence of astrocytes enhances Aβ induced neurotoxicity in hippocampal cell cultures [19] and that the presence of reactive astrocytes is a characteristic feature of the lesions in AD brain [20]. Notwithstanding, the effect on neuronal SAT1 cannot be ascribed simply to an indiscriminate toxic influence on the neurons from the Aβ exposed glia, because there was no similar effect on VGLUTs, and because the effect was observed in neurons with apparently normal morphology and normal expression of the neuronal marker MAP2. Could the reduced level of neuronal SAT1 contribute to the impaired resistance of neurons to Aβ when cultured in the presence of glia? While this does not appear likely, the observed 50% reduction in glutamine transporter level might possibly be critical in certain conditions.
The mechanism by which astroglial cells cause changes in neuronal expression of SAT1 must be determined by future research. As free radicals and oxidative stress play important parts in AD [21], it is interesting that crotonaldehyde accumulates in reactive astrocytes in AD brain [22]. Astrocytes mediate Aβ induced neuronal death through oxidative stress [23]. One possible mediator of the observed glia-neuron influence is BDNF signalling, which is impaired in cortical neuron cultures exposed to Aβ [24, 25], although Aβ may affect this signalling cascade without the participation of glia [24]. An attractive possibility is offered by the observation that Aβ(25-35) causes astrocytes to produce nerve growth factor (NGF), thereby inducing tau hyperphosphorylation and reduced survival in hippocampal neurons [26]. As the effect is exacerbated by NO, this mechanism may offer a link with the effects of free radicals in AD pathology. It fits with the recent observation that reduction of endogenous tau protects against Aβ toxicity in a mouse model of AD [27].
The mechanism of vulnerability of neurons in AD appears to involve excitotoxicity [4]. The increase in glial glutamate transporters induced by Aβ [28] may represent a measure to limit excitotoxic damage. Similarly, the reduced neuronal levels of SAT1 observed here after exposure to Aβ may limit the production of glutamate, but also of GABA, for synaptic release. It might represent an adaptation to curb excitotoxicity in AD, but might concomitantly impair synaptic function.

glutamine transporter
alzheimer’s disease
cell culture
vesicular glutamate transporter
β-amyloid peptide (25-35)


Erythropoietic protoporphyria without skin symptoms-you do not always see what they feel

Erythropoietic protoporphyria (EPP) is an inherited disorder of the porphyrin metabolism that often remains undiagnosed in children. We report on a 4-year-old girl who had been suffering for 1 year from recurrent painful crises affecting her hands, feet, and nose following sun exposure. Objective skin lesions were absent until the age of 6. Porphyrin analysis revealed elevated free erythrocyte protoporphyrin (FEP) levels confirming the diagnosis of EPP. This illustrates that skin lesions might be completely absent in children affected with EPP, a fact that has only been reported once previously. Because EPP can manifest with few and unspecific cutaneous symptoms or no skin lesions at all, like in this patient, the diagnosis of EPP might be delayed or missed. EPP should be excluded in all photosensitive children, especially when discomfort is disproportionate to the extent of the cutaneous lesions. The clinic, pathophysiology, diagnosis, complications, and therapy of EPP are discussed.
Introduction
Erythropoietic protoporphyria (EPP) (OMIM 177000) results from a partial deficiency of ferrochelatase (FECH), the last enzyme in heme biosynthesis that is located in the mitochondrium [10, 13]. FECH catalyzes the insertion of iron into protoporphyrin IX to form heme (Fig. 1) [10]. Although overall EPP is a rare disease, it is the most common type of porphyria manifesting in childhood with an estimated prevalence of 1 in 130,000 [10, 13, 23].
Fig. 1In the mitochondrium, ferrochelatase catalyzes the formation of heme from protoporphyrin IX. ALA: δ-aminolevulinic acid
The disease has no racial or sex predilection and no precipitating factors have been described to date [10, 20]. The major clinical feature of EPP is cutaneous photosensitivity that usually commences early in infancy or childhood, affects the sun-exposed body sites, and worsens in spring and summer [10]. The symptoms include pain, burning, itching, erythema, and swelling, which can develop within minutes of sun exposure [4, 10, 13, 14]. Petechiae, purpura, and vesicles may be seen, but are uncommon [4]. Typically, children experience relief when cooling the skin with cold water or wet towels [11]. Chronic UV (ultraviolet) exposure can lead to lichenification, postinflammatory hyperpigmentation, and scarring, particularly over the knuckles and on the nose [13, 23]. Of note, the cutaneous symptoms can be discrete and barely notable in early childhood despite significant subjective discomfort including burning and stinging sensations in the skin.
Here, we describe a girl with EPP with an unusual clinical course, demonstrating that parents and physicians cannot always recognize how much the patients suffer.
Case report
An otherwise healthy, 4-year-old Dutch Caucasian girl presented with a 1-year history of suddenly occurring severe itching and pain in the hands, feet, and nose after sunlight exposure. These sensations lasted up to 3 days, and she reported on three such incidents during the last 12 months. The symptoms developed either immediately or up to 8 h after UV exposure. Erythema or other skin manifestations were not seen. The condition worsened in the summer months and during vacations in sunny climates. Itching commenced immediately after sunlight exposure and reached a maximum at night. After a few hours, the itching changed into pain. Application of wet towels or cold baths led to partial relief, and the symptoms usually vanished spontaneously after a couple of cloudy days without intense UV exposure. Looking back, her parents also remembered that at the age of 1 year, during a summer vacation, she had suffered from an unexplained acute swelling of the hands and feet. At that time no specific diagnosis was made, besides the suspicion of an allergy. During that vacation at the sea side, the overall family life was disturbed because she was continuously agitated, cried a lot without an apparent reason, and did not sleep well.
Upon physical examination in our outpatient clinic at the age of 4, she showed no skin symptoms except for few minimal excoriations on the dorsal aspects of the feet. The family history revealed eczema in the mother and atopy in the paternal grandmother, but was unremarkable regarding increased photosensitivity. Eventually, a pediatrician suggested the differential diagnoses polymorphic light eruption, warmth intolerance, or hypochondria, and the girl was treated unsuccessfully with antihistamines.
Due to continuation of her complaints, a pediatric dermatologist was consulted who suspected EPP despite the absence of cutaneous manifestations. Blood porphyrin analysis revealed elevated free erythrocyte protoporphyrin (FEP) levels of 9,821 μg/l (normal 0–300 μg/l), confirming the diagnosis of EPP. Affirmative phototesting showed maximal cutaneous sensitivity at a wavelength of 400 nm (violet light). Full blood count and liver enzymes were normal.
Therapeutically, strict UV light avoidance, protective sunscreens, and annual follow-up visits were advised. Still, her photosensitivity did not improve, and mild hyperpigmentation and sores at her nose and upper lip as well as pronounced knuckles and progressive lichenification developed at the age of 6. To date, she has not shown liver enzyme alterations.
Discussion
Here we present a girl with EPP who suffered from a significant decrease of quality of life due to severe itching and pain of the skin. Clinically, EPP is characterized by cutaneous photosensitivity manifesting early in life. Acute photosensitivity episodes include burning, stinging, and pruritus in sun-exposed skin, particularly on the nose, cheeks, and dorsal aspects of the hands, followed by erythema, edema, and wax-like scarring. Skin symptoms can occur within minutes of sun exposure, often starting early in spring time, continuing through the summer, and diminishing in fall and winter.
Interestingly though, our patient did not show any visible skin lesions until the age of 6. Over the last 30 years there have been ten reports on children with EPP who developed mild to severe cutaneous photosensitivity and specific skin manifestations [1, 2, 11, 15, 19–21, 23, 30, 31]. With mild or absent visible skin symptoms, however, diagnosis may be delayed for several years or is not made at all because the discomfort experienced by the young patients is disproportionate to the objective cutaneous manifestations [22, 23]. Thus, children with EPP are often labeled as hysterical, hypochondriac, or even malingering, a distressing situation for both the patients and their parents [2, 22]. To the best of our knowledge the complete absence of visible skin lesions in early childhood EPP has only been described once to date [12].
The symptoms in EPP are associated with an overproduction and accumulation of lipophilic protoporphyrin as a result of FECH deficiency [28]. Excess deposition of protoporphyrin in the skin leads to cutaneous photosensitivity upon UV light exposure with maximum susceptibility at a wavelength around 400 nm (Soret band) [22]. Upon excitation, protoporphyrin exerts its phototoxic effects through the generation of reactive oxygen species (ROS). ROS can induce lipid peroxidation, oxidation of amino acids, and mediator release from mast cells, contributing to the acute photosensitivity, and protein cross-linking leading to cell membrane damage and cell death [3, 7, 13, 17, 25].
The diagnosis of EPP is based on the clinical symptoms and verified by a significant increase of FEP (more than five times the normal level of 0 to 300 μg/l ) in peripheral erythrocytes [15, 18, 28]. Since protoporphyrin is a hydrophobic metabolite, it is not renally excreted. Hence, urinary porphyrin values are usually normal [15, 23]. Another diagnostic modality includes fluorescence microscopy of erythrocytes [2, 17].
Histological changes are predominantly seen in the upper dermis and include deposition of amorphous material containing immunoglobulins, complement components, glycoproteins, acid glycosaminoglycans, and lipids around blood vessels [5, 21, 24, 26, 27, 29]. However, these alterations are not specific. Regardless of the aforementioned finding, we emphasize that it is unnecessary to take a skin biopsy if one of the cutaneous porphyrias is suspected because of two reasons. Firstly, simple non-invasive biochemical laboratory techniques can easily prove or exclude the presumptive diagnosis of porphyria. Secondly, any kind of external trauma, such as a biopsy or excision, inevitably constitutes an unnecessary risk for delayed or disturbed wound healing in porphyria patients.
The differential diagnoses of EPP include solar dermatitis, solar urticaria, polymorphous light eruption, lipoid proteinosis, hydroa vacciniforme, and lupus erythematosus [2, 22]. Elevated FEP levels can also be found in lead poisoning, anemia, renal failure, cholestasis, and liver failure. However, these conditions are not associated with photosensitivity [13, 18, 23].
Beside the cutaneous manifestations, the most important concern in EPP patients is the development of cholestasis with rapid accumulation of protoporphyrin in hepatobiliary structures resulting in severe liver damage and hepatic failure. Although rarely occurring, progressive liver failure is a well-recognized complication in EPP, and about one third of EPP patients show biochemical liver abnormalities [6, 13]. Therefore, liver function tests should be performed annually [13, 28]. In our patient, no liver dysfunction has been detected so far.
Although in rare instances an autosomal recessive inheritance pattern has been described, EPP is a predominantly autosomal dominantly transmitted disorder with incomplete penetrance resulting from mutations in the FECH gene on chromosome 18q21.3 [31, 32]. To date, more than 65 different FECH mutations have been reported, reflecting the genetic heterogeneity encountered in EPP [13, 28]. Recently, the genetic mechanisms underlying the cutaneous symptoms in EPP have been uncovered. It is clear now that only those individuals will develop skin symptoms who not only inherit a heterozygous FECH gene mutation on one parental allele, but also a specific intronic FECH polymorphism on the other parental allele [8]. Thus, clinically overt EPP results from a marked deficiency of FECH activity below a certain threshold due to co-inheritance of a specific FECH gene mutation with a low-penetrance IVS3-48C allele. Recent studies indicate that the frequency of the IVS3-48C allele shows a high degree of variability in different ethnic groups [9]. The identification of the molecular mechanisms underlying the manifestation of photosensitivity in EPP certainly has to be considered a milestone in porphyria research. Still, the development of protoporphyrin-induced hepatic disease and the molecular mechanisms governing the phenotype with severe liver injury are not well understood. It seems that other as yet unidentified factors may contribute to the pathogenesis of severe liver failure in EPP.
The current treatment modalities for EPP are limited and not effective in all patients [13]. In an ideal scenario, the harmful effects of exposure to visible light should be prevented. This can partially be achieved with sunscreens containing titanium oxide or zinc oxide. Common sunscreens, even those with high UV absorption capacity, do not block visible light and, thus, have no protective effect [2, 28].Consequent avoidance of UV light exposure and sun protection by protective clothing remains the most important measure in preventing acute photosensitivity and was also advised to our patient [13, 14, 18, 28]. The effectiveness of other modalities such as antioxidants (e.g., beta-carotene), cysteine, narrow-band UVB-phototherapy, and oral antihistamines remains doubtful and is certainly not of benefit in all patients. In the future, gene therapy approaches might play a role in the treatment of EPP because, e.g., normal ferrochelatase activity could be restored in vitro by transferring a wild-type copy of the FECH gene into cultured fibroblasts of EPP patients [16].
In conclusion, the diagnosis of EPP is often delayed or missed. Therefore, EPP should always be considered in photosensitive children, especially when the subjective discomfort is disproportionate to the extent of visible skin manifestations because we obviously do not always see what these patients feel. If cutaneous symptoms are completely absent, an accurate diagnosis is difficult and requires all diagnostic abilities of the attending physicians.

erythropoietic protoporphyria
porphyrias
photosensitivity
ferrochelatase


Antibody therapy in renal cell carcinoma

The treatment of metastasized renal cell carcinoma (RCC) still represents a formidable challenge, despite the development of small molecule, tyrosine kinase inhibitors (TKI) that have made a major impact on the disease. Although the percentage of patients achieving a partial response or stabilization of disease has been impressive, these effects are mostly non-durable. Additionally, drug-related side effects can be quite severe. Alternative treatment modalities might be monoclonal antibodies (mAbs). mAbs against RCC-associated antigens have been developed and have shown promise. Additionally, current efforts focus on Bevacizumab that recognizes vascular endothelial growth factor (VEGF). VEGF overexpression in RCC provides the opportunity to inhibit this proangiogenic pathway. Also with Bevacizumab, promising results have been obtained, particularly in combination with other treatment modalities. It is likely that mAbs, either as single agents or in combination with other agents, may become useful additions to the armamentarium to diagnose and treat RCC.
Introduction
The hypothesis by Ehrlich in early 1900s that malignant cells express unique structures that can be used to guide cytotoxic therapy to tumors [1] followed by the development of the hybridoma technique by Kohler and Milstein almost 70 years later [2] has led to the development of anti-cancer reagents with unique characteristics. One of the most distinguishing factors is the possibility to select monoclonal antibodies (mAbs) recognizing target molecules with very restricted expression in normal tissues. To date, tumor-specific antigens (antigens expressed on all tumor cells of a particular tumor type not expressed by normal cells) have not been identified. The members of the so-called cancer-testis family do exhibit highly tissue-restricted expression, but are considered promising target molecules for cancer vaccines, less for antibody therapy, particularly in view of the extreme intra- and inter-tumor heterogeneity [3].
Similar to other malignancies, monoclonal antibodies (mAbs) targeting renal cell carcinoma (RCC) associated molecules were developed without understanding the molecular events underlying RCC [4–8]. The increased understanding of molecular events important in the carcinogenesis of RCC led to the recognition that these aberrations can be used to target RCC. Specifically, aberrant von Hippel-Lindau (VHL) gene expression has been identified as a general event in clear cell RCC (ccRCC) [9], which represents 80–85% of localized cases and 90–95% of metastatic RCC (mRCC). The loss of a functional VHL gene product leads to accumulation of the transcription factor HIF-1α that is an obligatory element for the transcription of several genes. This includes vascular endothelial growth factor (VEGF) and carbonic anhydrase 9 (CA9), targets for which most clinical experience with mAbs in RCC has been generated (Bevacizumab and G250, respectively).
The rationale and effects of Bevacizumab and G250-directed therapy are fundamentally different: Bevacizumab treatment leads to VEGF-depletion and consequently to diminished neovascularization followed by tumor cell death, mainly due to loss of vascularization. In contrast, G250 treatment targets the cell surface of RCC cells where it must exert toxic effects. Both approaches have advantages and disadvantages. Bevacizumab treatment has the advantage that VEGF depletion can be achieved in the circulation, and homing to all tumor vessels is not necessary. However, other regulatory pathways can also lead to neovascularization and small, non-vascularized tumor loci will not be affected. G250 treatment has the advantage that RCC cells can be targeted, irrespective of tumor size. However, in view of the generally poor perfusion rate and high interstitial fluid pressure in RCC, deep penetration of tumors may be difficult. Also, since G250-binding alone does not confer a lytic signal to RCC cells, tumor cell kill requires effector cells or coupling of G250 to toxic agents.
Bevacizumab
Bevacizumab is a humanized mAb against VEGF that binds and neutralizes all of the major isoforms of VEGF [10]. This prevents VEGF from interacting with its receptors and activation of downstream signaling pathways. This mode of action is thought to lead to regression of existing microvasculature, normalization of mature vasculature, and inhibition of the production of new vasculature [11]. Whether all these effects are true for RCC is unclear at the moment.
Significant protein dose levels are needed to maintain sufficiently high Bevacizumab levels to trap VEFG, the target of Bevacizumab. The first Bevacizumab trial in metastatic RCC (mRCC) patients addressed whether Bevacizumab treatment could lengthen the time to progression of disease and the response rate [12]. Survival was a secondary end point. In this randomized phase II trial, 116 patients with metastatic, refractory clear cell RCC were randomized to placebo, low-dose (3 mg/kg) Bevacizumab, or high-dose (10 mg/kg) Bevacizumab given intravenously every 2 weeks. All patients had prior disease progression while on systemic treatment; the vast majority had received prior interleukine-2. Patients with disease progression on placebo crossed over to receive low-dose Bevacizumab. Bevacizumab treatment resulted in a significant prolongation of the time to progression of disease in the high-dose antibody group (4.8 months as compared with 2.5 months). Possibly, the low-protein dose was inadequate to sufficiently deplete circulating VEGF levels in-between injections, explaining the poor outcome in this group. The study was inadequately powered to show a significant difference in overall survival between groups. Based on this encouraging result, Bevacizumab has been combined with other treatment modalities to augment the therapeutic index.
Bevacizumab in combination
The AVOREN trial investigated the effects of standard therapy of interferon alfa-2a plus placebo or interferon alfa-2a plus Bevacizumab, administered every 2 weeks at a dose of 10 mg/kg [13]. In this randomized, double blind phase III trial, 649 patients with first-line mRCC were enrolled. The primary analysis endpoint was assessment of improvement in progression-free survival (PFS), defined as the length of time the tumor did not grow or patient death did not occur. Other endpoints of the study included overall survival, time to progression, time to treatment failure, overall response rate, and safety profile. The addition of Bevacizumab to IFN-α2a significantly increased PFS (10.2 vs. 5.4 months) and objective tumor response rate (30.6 vs. 12.4%; P < 0.0001). Additionally, the combination treatment showed a trend toward improved overall survival (P = 0.0670), which leads to the conclusion that the combination of Bevacizumab with IFN-α2a is superior to either of the single treatment regimens in mRCC. The working mechanism explaining the superiority of this combination treatment has not been defined yet, but most likely the superiority is the net effect of the reduction of the immunosuppressive effects due to decreased VEGF levels combined with the immunomodulatory effects of IFN.
Considering that the epidermal growth factor receptor (EGFR) is also overexpressed in RCC, a multicenter, phase II study evaluated the addition of erlotinib (Tarceva), an EGFR inhibitor, to Bevacizumab in metastatic RCC patients [14]. Treatment consisted of 10 mg/kg Bevacizumab given intravenously every 2 weeks and 150 mg erlotinib given orally each day. With 15 (25%) patients showing objective responses, and an additional 36 patients (61%) with stable disease after 8 weeks of treatment, a randomized phase II trial was performed evaluating Bevacizumab + placebo versus Bevacizumab + erlotinib. Disappointingly, identical response rates and PFS rates for the two arms were observed [15], and it is doubtful that EGFR-targeting is of any benefit.
The effect of Bevacizumab and low-dose interleukine-2 (IL-2) in mRCC was evaluated in a phase II trial in previously untreated, good and intermediate risk, mRCC patients. Patients received 8-week cycles of IL-2 (250,000 U/kg per day s.c. Day 1–5 during week 1 and 125,000 U/kg per day s.c. Day 1–5 during weeks 2–6, followed by a 2 week break), and Bevacizumab 10 mg/kg was administered i.v. every 2 weeks starting on day-14. With 16 of the planned 35 patients enrolled, and 11 evaluable patients for response, 1 partial response (PR) and 3 stable disease (SD) lasting >3 months were observed [16]. All patients with SD demonstrated some degree of tumor shrinkage. Similar to the working mechanism of the Bevacizumab/IFN combination, the anti-tumor effects are possibly the result of the reduction of the immunosuppressive effects due to decreased VEGF levels combined with the general immune activating effects of IL-2. Interestingly, treated patients demonstrated an increase in the number of regulatory T cells without effect on DC activation. Larger, randomized studies will be necessary to address the value of this combination treatment.
In a phase I trial, Bevacizumab has also been combined with sunitinib (Sutent®), a tyrosine kinase inhibitor, with the hypothesis that this combination may increase antitumor efficacy by maximizing inhibition of the VEGF pathway. The Bevacizumab dose was kept constant (10 mg/kg) while the sunitinib dose was escalated starting at 25 mg (escalation with 12.5 mg increments). Of 13 patients evaluated for best response, 4 had partial responses, 7 had stable disease, and 2 had PD [17].
Similarly, the combination of Bevacizumab and the mTOR inhibitor CCI-779 (Temsirolimus®) has been investigated. Patients received 25 mg/week Temsirolimus and 5 or 10 mg/kg Bevacizumab. In 12 evaluable patients, 7 PR and 3 SD were observed [18]. The encouraging results certainly deserve further testing of these combinations in phase II trials.
Monoclonal antibody G250
G250 is a mAb against CA9, a molecule which is ubiquitously expressed in ccRCC [19]. CA9 expression in non-ccRCC has also been documented, and there, it is most likely a reflection of (sustained) hypoxia [20]. Clinical efforts with mAb G250 in RCC have focused on radioimmunotherapy and passive immunotherapy. This mAb was described as a mAb recognizing an RCC-associated antigen, absent in normal kidney and homogeneously expressed in most RCC [4], most notably clear cell RCC [19]. In 2000, the G250 antigen molecule was identified and shown to be CA9 [21]. The molecular characterization allowed transcriptional regulation studies that revealed a strict dependence of G250 expression on HIF-1α [22]. Thus, the molecular mechanism responsible for CAIX expression in ccRCC is similar to VEGF, namely due to non-functional VHL protein leading to HIF-1α accumulation and gene expression.
The first clinical trials with mAbG250 were already performed and published before the molecular characterization of G250 antigen was achieved. The combined data from the immunohistochemical tissue distribution, animal experiments and ex vivo perfusion of tumor bearing kidneys had provided sufficient evidence to initiate a biopsy-based phase I protein dose escalation trial with murine mAbG250. The rationale of G250-directed therapy obviously differs from Bevacizumab: Bevacizumab treatment leads to VEGF-depletion and consequently to diminished neovascularization whereas G250 treatment targets RCC cells directly. This first mAbG250 clinical trial demonstrated various pivotal aspects: most notably, virtually no uptake in other tissues resulting in excellent tumor visualization, and very high tumor uptake [23].
The G250 antibody uptake that was observed was up to 10-fold higher than any other mAb uptake in solid tumors, which led to the design of a phase I/II radioimmunotherapy (RIT) trial with murine mAbG250. RIT led to stabilization of disease in 17 of 33 patients, with tumor shrinkage observed in two patients. Transient liver toxicity was observed, quite likely the result of mAbG250 liver uptake, although there was no correlation between the amount of 131I administered or hepatic absorbed radiation dose and the extent and nature of hepatic toxicity [24].
Because the murine G250 antibody was highly immunogenic, restricting multiple injections, mAbG250 was chimerized. The results of the phase I protein dose escalation trial with chimeric G250 (cG250) basically duplicated the results from the murine G250 trial: virtually no uptake in other tissues resulting in excellent tumor visualization, and very high tumor uptake. The half-life of the antibody was extended, as was to be expected, but, more importantly, the chimerized from of G250 was almost immunosilent [25]. Thus, multiple injections became possible. Various phase I and phase II trials have been performed with cG250 aimed at therapeutic intervention. Based on the very high uptake levels, several RIT trials were performed. In the first phase I trial with 131I-cG250, one patient showed a partial response (>9 months) [26] which set the stage for phase II RIT trials in metastatic RCC patients. RIT studies with single high dose 131I-G250, rapid fractionated dose 131I-G250 [27], and sequential high dose 131I-G250 [28] have resulted in only occasional therapeutic responses, although dosimetric analyses suggest that tumor-sterilizing levels can be reached. Even two sequential high-dose treatments with 131I-G250 did not result in objective responses, but in stabilization of previously progressive disease in a few patients. RIT with G250 has been accompanied by bone marrow toxicity similar to mAb RIT in other tumor types and considering the minimal benefit, 131I-based RIT with cG250 have been abandoned. Since RCC is a radiotherapy resistant tumor, possibly even higher radiation doses are necessary to achieve tumor-sterilizing levels. Current G250 RIT efforts are directed to 177-lutetium and 90-yttrium labeled G250. It is hypothesized that the use of more powerful radionuclides that are also better retained in the tumor cells may lead to clinical responses. Animal experiments have demonstrated the superiority of 177Lu- and 90Y-labeled G250 over 131I-G250 [29]. Importantly, stabilization of previously progressive disease has been observed in almost all 177Lu-G250 treated patients, although the maximum tolerable 177Lu dose has not been achieved. Dosimetric analyses of the first patients treated with 177Lu-G250 suggest that indeed tumor-sterilizing levels may be achieved. Figure 1 illustrates targeting of 177Lu-G250 in a patient with metastatic renal cancer.
Fig. 1Anterior (left) and posterior (right) whole body scans acquired 7 days post-injection of patient injected with177Lu-cG250. Please note high uptake in both pulmonary lesions and in contralateral kidney lesions. Uptake in liver is due to the conjugation methodology and is not related to G250 antigen expression
In view of the obvious tumor-specific accumulation of cG250, passive immunotherapy of RCC patients has also been studied extensively. In vitro mAbG250 can elicit antibody-dependent cellular cytotoxicity (ADCC), which can be enhanced by low dose IL-2 [30]. Various (non-randomized) clinical trials have now been completed with cG250 alone, and in combination with IL2 or interferon [31, 32]. Thus far, these treatments appear to lead to extended survival time. The apparent clinical benefit appears to be quite substantial with a documented median survival of 22 months in patients with metastatic RCC who have progressive disease at study entry. Nevertheless, it is difficult to judge the value of this treatment. Clearly, randomized trials are necessary to unequivocally demonstrate whether passive immunotherapy with cG250 is of benefit for metastatic RCC patients.
The largest trial, which is currently ongoing, is the adjuvant ARISER trial (adjuvant Rencarex immunotherapy phase III trial to study efficacy in nonmetastatic renal cell carcinoma). In this phase III randomized, double blind, placebo-controlled trial, patients with ECOG performance status of 0 with completely resected primary clear cell RCC and no evidence of remaining local or distant disease, are treated. The study is designed to detect a significant difference between the two treatment arms with respect to disease-free survival; patients will be followed-up long-term to determine overall survival statistics.
Recently, the potential utility of mAbG250 as a diagnostic imaging agent was investigated [33]. The excellent imaging capability had been noted in almost all patients, but this line of research was not pursued mainly because detection of suspect renal masses and occult metastatic RCC was not deemed advantageous. Additionally, treatment modalities for metastasized RCC were poor, and, therefore, efforts focused on treatment. However, with a steady increase of incidentally discovered renal masses and new therapeutic modalities becoming available, imaging might become of importance to distinguish more potentially malignant tumours from less aggressive variants. In the first prospective clinical trial with 124I-labeled cG250, a very high specificity and sensitivity to identify ccRCC in patients with suspect renal masses was demonstrated, a clear indication of the potential clinical utility. Whether this imaging modality can be used to follow therapy effects remains to be determined.
In conclusion, it is reasonable to assume that Bevacizumab and G250 monoclonal antibodies either as single agents or in combination with other agents may become useful additions to the armamentarium to diagnose and treat (cc)RCC. Several trials evaluating the combination of G250 or Bevacizumab with registered RCC treatments are currently in progress and will further define the role of these mAbs in RCC.

therapy
renal
rcc
monoclonal antibody
vegf
g250
caix


A Highly Active and Tolerable Neoadjuvant Regimen Combining Paclitaxel, Carboplatin, 5-FU, and Radiation Therapy in Patients with Stage II and III Esophageal Cancer

Background The present phase II study aimed to assess the feasibility and efficacy of a new paclitaxel-based neoadjuvant chemoradiation regimen followed by surgery in patients with stage II–III esophageal cancer.
The prognosis of patients with esophageal cancer who undergo surgery with a curative intention is poor, with a 5-year survival rate of 5–20%.1 In recent decades, several efforts have been made to improve outcome. Improved and standardized surgical techniques and supportive care as well as concentration of care have contributed to a better outcome; however, further improvement of survival from a single modality approach seems unlikely.2,3
Neoadjuvant chemoradiation has become the focus of interest to improve long-term survival and reduce recurrence rates. Despite a large number of phase II and several phase III trials, the role of neoadjuvant chemoradiation remains unclear. Until now, only one phase III trial demonstrated a significant survival advantage.4 All neoadjuvant regimens used in these phase III trials were cisplatin-based, which leads to frequent toxicity-related dose reductions and delays and relatively low pathologic complete response rates (10–28%).
New neoadjuvant chemoradiation regimens including drugs such as paclitaxel have shown encouraging complete response rates (29–39%) and R0 resection rates.5–7 Since pathologic complete response and R0 resection have been shown to be associated with improved survival,8,9 it can be expected that these new regimens lead to better survival rates. Therefore, the optimal neoadjuvant treatment regimen with high pCR rates and minimal toxicity has to be determined.
Paclitaxel interferes with mitotic spindle function by enhancing the rate and yield of microtubule assembly and preventing microtubule depolymerization.10 Paclitaxel is a potent radiosensitizer because it synchronizes cells at G2/M, the phase of the cell cycle that is particularly sensitive to radiotherapy.11,12 Furthermore, paclitaxel radiosensitization persists well after the period of G2/M synchronization, suggesting that other factors may also relate to its ability to enhance radiotherapy.13 The p53 gene is required for initiation of apoptosis in response to most chemotherapeutic agents and radiotherapy. In vitro, paclitaxel-mediated blockade at the G2/M has shown to activate cell-cycle control pathways that induce apoptosis independent of p53.14
Therefore, paclitaxel-based neoadjuvant chemoradiation seems a rational treatment approach for esophageal cancer. A recent study by Meluch et al.6 has reported an encouraging complete response rate of 38% with a paclitaxel-based regimen. However, toxicity was severe as only 54% of the patients received a full dose of the neoadjuvant regimen, because of complicating esophagitis and leucopenia. The goal of this study was to assess the feasibility and efficacy of a new paclitaxel-based neoadjuvant chemoradiation protocol in which we applied a reduced chemotherapy dose compared with the protocol of Meluch et al.6
PATIENTS AND METHODS
Patients
Referral to our institute was accomplished as a result of agreements between hospitals affiliated to the Comprehensive Cancer Centre South in the Netherlands to concentrate surgical care of patients with esophageal cancer. The protocol of this prospective clinical phase II trial was approved by the Medical Ethics Committee, and from all patients written informed consent was obtained. All consecutive patients with a potentially resectable stage II and III esophageal cancer who were referred to our Centre between January 2002 and November 2004 were found eligible.
Further eligibility criteria included: patient age between 18 and 75 years, WHO performance status ≤2, written informed consent, central venous catheter, and adequate hematological, renal, and hepatic functions defined as white blood cell count > 3.5 × 109/L, platelet count > 100 × 109/L, creatinine level < 120 μmol/L and/or creatinine clearance > 60 mL/min, and normal hepatic enzyme levels. Patients with distant metastatic disease or tumor positive supraclavicular lymph nodes were excluded from entry into the protocol. However, M1a disease in the regional lymph nodes was not.
Pretreatment staging evaluation included clinical examination, upper gastrointestinal endoscopy with histological biopsies of the tumor, computed tomography (CT) scan of chest and upper abdomen, and ultrasound of the neck. Endoscopic ultrasound (EUS) was performed when a T1 tumor could not be excluded by conventional techniques. More recently, PET imaging was introduced to exclude distant metastases.
Neoadjuvant Treatment
The neoadjuvant regimen consisted of paclitaxel 175 mg/m2 intravenously and carboplatin AUC 5 intravenously (IV) on day 1 and 22, and 5-FU 200 mg/m2 continuous infusion on day 1–42. All patients were treated prophylactic with dexamethasone 20 mg and a 5HT3 antagonist IV followed by ranitidine 50 mg IV and clemastine 2 mg IV.
Radiotherapy was performed with a linear accelerator with a minimal photon energy of 6 MV. The total dose of 45 Gy was given in 25 fractions of 1.8 Gy once daily, starting on day 1. The radiation fields encompassed the primary tumor and enlarged lymph nodes, if any, defined by endoscopy, CT scan, and EUS, surrounded by 5-cm proximal and distal margins and a 2-cm radial margin. Neoadjuvant treatment was given on a fully outpatient base.
Dose modifications were made for a toxic reaction as defined according to the criteria of the World Health Organization (WHO).15
Surgery
Surgery was planned 6–8 weeks after completion of neoadjuvant treatment. For tumors in the distal third of the esophagus, a transhiatal approach was used whenever possible. A thransthoracic Ivor-Lewis technique was used for tumors in the proximal and middle third of the esophagus. Esophagogastric continuity was reestablished using the stomach with a cervical anastomosis in all patients. A feeding jejunostomy was placed, and enteral feeding was started 24 hours after surgery.
Data Collection
The following data were prospectively collected: age, sex, WHO performance status, location of the tumor, histology of the tumor, comorbidity, toxicity of neoadjuvant treatment, type of resection, and postoperative course and complications.
Histological Examination
All pathology reports and specimens were centrally reviewed by one pathologist (GL). Pathologic assessment was performed after standard hematoxylin-eosin (H&E) staining. The assessment included a determination of the histological type of the tumor, the depth of invasion of the tumor, whether there was nodal involvement, and the status of circumferential, proximal, and distal resection margins. The tumor stage was defined according to the classification of the American Joint Committee on Cancer.16 Patients with no residual viable tumor cells in the surgical specimen were classified as having a pathologic complete response (pCR). In addition, response after chemoradiation of the primary tumor was classified according to the Tumor Regression Grade (TRG) as described by Mandard et al.17 Categories according to the TRG ranged from TRG1, complete regression, to TRG 5 with no regressive changes. TRG 1–3 scores were defined as a major response to chemoradiation.
Statistical Analysis
The analyses were performed on the basis of intention-to-treat. Survival time was calculated as the duration from the day of start of chemoradiation therapy until death or last day of follow-up, and recurrence free interval was calculated from the day of surgery until the day of diagnosis of recurrence. Overall survival and disease-free survival were estimated using the Kaplan-Meier method. Median survival time was obtained from the time corresponding to 50% survival based on the Kaplan-Meier survival curve. The log-rank test was used to assess survival differences. Significance was defined at a confidence level of P < .05. Statistical analyses were performed with SPSS version 15.
RESULTS
Patient Characteristics
Fifty-two patients with stage II–III esophageal carcinoma were treated in the period between January 2002 and November 2004. Two patients were found ineligible. One patient had a history of Hodgkin’s disease and squamous cell carcinoma of the tongue. Previous treatment of this patient included external beam radiotherapy on the chest. One patient had intellectual disability (mental retardation) and was extremely anxious. Therefore, our study population consisted of 50 patients. All 50 patients signed the written informed consent and were included. Patient characteristics are detailed in Table 1.
TABLE 1.Patient characteristicsNo. of patients n = 50Age (years)  Median60  Range34–75Sex  Male44  Female6WHO performance status  Median1  Range0–2ASA classification  I24  II24  III2  IV–Tumor location  Upper third2  Middle third1  Lower third47Histology  Adenocarcinoma42  Squamous cell carcinoma8
Toxicity Related to Neoadjuvant Treatment
Forty-two patients (84%) completed full neoadjuvant treatment. Three patients discontinued chemotherapy prematurely due to grade II hand-foot syndrome, four patients due to esophagitis, and one due to grade IV leucopenia. Forty-seven patients (94%) received at least 5 weeks chemotherapy; the remaining three patients discontinued chemotherapy in week 4. Full dose of radiotherapy was received by 49 patients (98%). Grade III leucopenia occurred in 23 patients (46%), without febrile episodes. No other hematologic toxicity was recorded. All 50 patients experienced esophagitis, usually mild (≤ grade 2). A total of 13 patients (26%) needed nasogastric enteral feeding. Infection of the indwelling central venous catheter occurred in two patients. There were no neoadjuvant treatment related deaths. However, one patient died of myocardial infarction 3 weeks after completing neoadjuvant treatment.
Postoperative Complications
Surgery was performed after a median of 8.1 weeks (range 6.0–17.9 weeks). No significant difference was observed between responders (TRG 1–3) and nonresponders (TRG 4–5) with respect to the time between the end of chemoradiation and surgery. Two patients showed metastatic disease at the time of surgery; hence, 47 patients underwent surgery with a curative intention. Transhiatal esophageal resection was performed in 44 patients; a transthoracic Ivor-Lewis resection was performed in three patients.
The postoperative course was uneventful in 23 patients (49%).There were four postoperative deaths (8.5%). Two patients died of necrosis of the gastric tube. One patient developed a fistula between the gastric conduit and the trachea and died of pulmonary sepsis. One patient died of a progressive chylothorax. Other postoperative complications included major anastomotic leakage (n = 5), pulmonary complications (n = 15), recurrent nerve palsy (temporary n = 4, permanent n = 1), and cardiac arrhythmias (n = 3). An overview is outlined in Table 2.
TABLE 2.Postoperative complicationsType of complicationNo. of patients (%)Major anastomotic leakage5 (11)Pulmonary15 (32) Pneumonia7 Emphysema4 ARDS1 Mediastinal infection1 Trachea-esoph fistula1 Chylothorax1Recurrent nerve palsy  Permanent1 (2)  Temporary4 (9)Cardiac arrhythmias3 (6)Wound infection2 (4)Diaphragmatic hernia1 (2)Hospital stay (days)  Median15  Range9–83ICU/MCU stay (days)  Median2  Range2–18Ventilation time (days)  Median1Range0–9
Pathologic Response to Neoadjuvant Treatment
Pathologic complete regression in the primary tumor was achieved in 18 of 47 operated patients (38.3%). However, in two patients the primary tumor showed complete response, but microscopic residual tumor was detected in locoregional lymph nodes. The ypTNM stages of the remaining patients were: pT1N0M0 in six patients (12.8%), pT2-3N0M0 in 12 patients (25.5%), pT1-2N1M0 in four patients (8.5%), and pT3N1M0/pT4N0-1M0 in seven patients (14.9%). Subsequent postoperative tumor stages are outlined in Table 3. Postoperative tumor regression grades (TRG) scored by an independent pathologist are outlined in Table 4. Eighteen patients (38.3%) showed a complete tumor regression (TRG1), and 38 patients (80.9%) displayed a major response (TRG1-3) after neoadjuvant treatment. Two patients had positive margin involvement of the resected specimen; therefore, a R0 resection was achieved in 45 of 47 patients (95.7%).
TABLE 3.Pathological stage of patients treated with neoadjuvant chemoradiation and surgeryStage, ypTNMNo. of patients (%)018 (38.3)I6 (12.8)II a12 (25.5)II b4 (8.5)III7 (14.9)TABLE 4.Tumor regression grade (TRG) of patients treated with neoadjuvant chemoradiation and surgeryTRGaNo. of patients (%)1—Absence of histologically identifiable residual cancer and fibrosis extending through the different layers of the esophageal wall18 (38.3)2—Presence of rare residual cancer cells scattered through the fibrosis11 (23.4)3—Increase in the number of residual cancer cells, but fibrosis still predominated9 (19.1)4—Residual cancer outgrowing fibrosis7 (14.9)5—Absence of regressive changes2 (4.3)a TRG according to Mandard et al.17
Survival and Pattern of Failure
All 50 patients were included in the survival analysis. The median follow-up for surviving patients was 41.5 months (range 21–59 months). The median overall survival and disease-free survival were 44 and 50 months, respectively. The estimated 3- and 5-year overall survival rates were 55.6 and 47.8%, respectively, whereas the estimated 3- and 5-year disease-free survival rates were 52.2 and 41.8%, respectively. The Kaplan-Meier curves for overall and disease-free survival are shown in Fig. 1 and 2.
FIG. 1.Kaplan-Meier curve of overall survival of all patients (n = 50).FIG. 2.Kaplan-Meier curve of disease-free survival (n = 50).
Patients with TRG 1–3 showed a significant improved overall and disease-free survival compared with patients with TRG 4–5. Median survival time in TRG 1–3 patients was not yet reached and 23 months in patients with TRG 4–5. The estimated 3- and 5-year overall survival rates were 61.0 and 55.9% for the patients with a TRG 1–3 and 33.3 and 16.7% for the patients with a TRG 4–5. Median disease-free survival in TRG 1–3 patients was not yet reached and 11 months in patients with TRG 4–5. The estimated 3-year disease-free survival rates for patients with TRG 1–3 was 58.6 vs 25.0% for patients with TRG 4–5. Comparing the TRG 1–3 patients with TRG 4–5 patients, the log-rank test of equality of the survival curves generated a P value of .03 for overall survival and .02 for disease-free survival time in favor of patients with TRG1–3. The Kaplan-Meier curve for disease-free survival according to tumor regression grade is shown in Fig. 3.
FIG. 3.Kaplan-Meier curve of disease-free survival by tumor regression grade (TRG). TRG1–3 (n = 38): major response, TRG 4–5 (n = 9): minor or no response (P = .0183).
Recurrent disease was found in 20 patients surviving postoperatively (20 of 43, 47%) Five of them were still alive at the time of the analysis. Recurrence was only locoregional in three patients. Distant metastases occurred in 16 patients, and in one patient recurrence presented with locoregional and pulmonary metastases.
To date 22 patients have died. Of these 22 patients, three died from nondisease-related causes and were presumed disease free. Of the 19 patients who had disease-related deaths, four patients died of postoperative complications, and three patients died of complications from local recurrence, whereas 12 patients died due to systemic failure that included metastases to lung, liver, brain, and bone.
DISCUSSION
This study shows the results of a phase II efficacy study, with a modified chemoradiation regimen according to Meluch et al.6 A reduction of the dose of chemotherapy components of this regimen resulted in a decrease of toxicity with preservation of its high efficacy rate. The pathologic complete tumor regression rate of 38.3% and R0 resection rate of 95.7 % are among the highest described in the literature. Toxicity and morbidity of this regimen compared with the original scheme were mild and acceptable.
Numerous phase II trials using neoadjuvant chemoradiation showed marked downstaging and suggested survival benefits. In most trials, chemotherapy regimens included cisplatin and 5-FU. However, randomized controlled trials (RCT) comparing chemoradiation followed by surgery with surgery alone have yielded inconclusive results.4,18–22 One of these trials showed significant improvement in survival in the patients who received combined-modality therapy.4 The relevance of this study was disputed because the 3-year survival rate of 6% in the control arm was exceptionally low. Two additional trials showed a possible benefit of neoadjuvant chemoradiation without reaching a statistical significance.19,22 Furthermore, the power of these trials to detect modest but clinically relevant differences was limited by small patient numbers. In a meta-analysis report by Fiorica et al.23 six RCTs were pooled comparing neoadjuvant chemoradiation and surgery versus surgery alone. A significant difference in 3-year mortality favoring neoadjuvant chemoradiation with surgery versus surgery alone was detected (OR 0.53; 95% CI 0.31–0.93; P = .03 NNT = 10). It was concluded that neoadjuvant chemoradiation and surgery significantly improved 3-year survival compared with surgery alone in patients with resectable esophageal cancer. However, a careful interpretation of these results is warranted, because this meta-analysis was based on summarized patient data.
Neoadjuvant chemoradiation induces acute treatment-related toxicity. These effects include myelotoxicity, esophagitis, nausea, and vomiting. The regimen used in this phase II trial produced moderate toxicity and is comparable with reported literature on neoadjuvant chemoradiation regimens including paclitaxel.5–7,24,25 The highly effective regimen used by Meluch et al.6 was modified to reduce toxicity. The modification consisted of dose reduction of 5-FU 200 mg/m2 instead of 225 mg/m2, paclitaxel 175 mg/m2 instead of 200 mg/m2 and carboplatin AUC 5.0 instead of AUC 6.0. This modification resulted in a reduction of toxicity. Forty-two patients (84%) received full doses of the planned 6 weeks chemotherapy, whereas in the group of Meluch et al. only 54% of patients received full dose chemotherapy. A relative large proportion of the patients (26%) received preoperative nasogastric enteral feeding. This was partly due to our liberal use of nasogastric enteral feeding, since in our experience optimizing preoperative nutritional status in patients treated with a neoadjuvant chemoradiaton protocol is critical. The neoadjuvant treatment was delivered on a fully outpatient basis. Further improvement of our regimen may be obtained by replacing 5-FU for novel oral fluoropyrimidines. In colorectal cancer, the novel fluoropyrimidines such as capecitabine and UFT show equal if not superior efficacy and improved patient convenience.
The reported postoperative mortality in the literature for neoadjuvant chemoradiation followed by surgery ranges from 0 to 29 % (median 9%). A nationwide study in the United States demonstrated in-hospital mortality rates of high-volume versus low-volume centers of 8.4 vs 20.3%.2 A comparative Dutch study found in-hospital mortality rates of 4.9 vs 12.1%.26 Therefore, the in-hospital mortality rate of 8.5% in our trial is within these boundaries. Several studies reported an increase in postoperative mortality after neoadjuvant chemoradiation. The aforementioned recent meta-analysis of six randomized trials confirmed a significant effect of chemoradiation on postoperative mortality (OR 2.10 95% CI 1.18–3.73, P < .01, NNT = 25).23 A relatively short interval between completion of neoadjuvant therapy and surgery of 2–4 weeks is noted in these trials and might be of influence in the relative high mortality rates described in these trials.
We decided to perform surgery after at least 6 weeks to achieve an adequate downstaging and permit maximal recovery; in addition, surgery was postponed in some patients due to a slow recovery from the chemoradiation. We believe that the timing of surgery is important. Performing an esophagectomy too soon after neoadjuvant chemoradiation may lead to increased morbidity due to prolonged myelosuppression and hence an increased risk for developing postoperative complications.
Until recently, neoadjuvant chemoradiation studies have been performed using cisplatin/5-FU based regimens with a variation in dose and a variation in radiotherapy schemes. Moderate complete response rates of 8–26 % and R0 resection rates of 55–84% have been described in these studies.4,19,20,22,27–29 With the implementation of regimens including paclitaxel, 5-FU, and cisplatin, complete response rates of 32–38% and R0 resection rates of 78–95% have been documented.6,7,25,30 Our study reports a complete pathologic response of 38.3%, which is high. Furthermore, a R0 resection rate of 95.7% is among the highest described in the literature. The dismal prognosis of esophageal cancer has discouraged many clinicians. For many patients, the prognosis was supposed to be dependent on the presence of indolent distant metastasis despite the type of operative treatment given. In a recent phase III trial, however, extended radical transthoracic esophagectomy showed a trend to a better survival when compared with a less radical transhiatal esophagectomy, indicating that an adequate locoregional treatment may have a beneficial effect on survival.3 However, in both groups a R0 resection rate of only 72% was noted. This observation is an important rationale for neoadjuvant combined multimodality treatment. One of the goals of neoadjuvant chemoradiation therapy is increasing curative R0 resection rates by downsizing the tumor and theoretically eradicating micrometastasis. Achieving R0 resection in locally advanced (stage II–III) esophageal cancer has been identified as a prognostic factor.8,9 Furthermore, complete pathologic response has been associated with an improved survival and local control.30,31 In the present study a major response, tumor regression grade 1–3, resulted in a significant increase in overall and disease-free survival. Since the time between surgery and the end of the chemoradiation did not significantly differ between the responders and nonresponders, a difference in response could not be explained by a lag-time difference. Thus, a regimen with high complete pathologic response and R0-resection rates will be very likely to improve survival. However, with our relatively short follow-up we cannot report definitive long-term survival data yet.
In conclusion, although the discussion regarding the value of neoadjuvant therapy is ongoing, the neoadjuvant treatment regimens used for the last 20 years produced only modest benefit, at best. Improving chemoradiation regimens as in our study, by incorporating modern chemotherapeutics such as paclitaxel in combination with modern 3-D conformal radiotherapy seems to be promising. Further improvement of the systemic therapy seems to be critical as survival is mainly determined by recurrences at metastatic sites. Data of ongoing phase III trials including novel chemoradiation protocols have to be awaited in the near future.

paclitaxel
esophageal cancer
neoadjuvant chemoradiation
pathologic complete response
tumor regression grade