Dr Nermin Serbecic
email: nermin.serbecic@meduniwien.ac.at

S C Beutelspacher

F C Aboul-Enein

K Kircher

A Reitner

U Schmidt-Erfurth

Reproducibility of High Resolution Optical Coherence Tomography Measurements of the Nerve Fiber Layer with the new Heidelberg Spectralis OCT

Keywords: optical coherence tomography, retinal nerve fiber layer, axonal degeneration, reproducibility

True follow-up measurement of identical peripapillary regions may enable clinicians to detect discrete levels of retinal thickness change over time. This constitutes a crucial prerequisite for a reliable monitoring of subtle RNFL changes in neurodegenerative disorders.

all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd to permit this article (if accepted) to be published in BJO and any other BMJPGL products and sublicences such use and exploit all subsidiary rights, as set out in our licence (http://group.bmj.com/products/journals/instructions-forauthors/licence-forms).

Abstract

Purpose

Conventional time-domain OCT technology for detection of retinal nerve fiber layer (RNFL) neurodegeneration suffers from technical inaccuracy due to a lack of exact scan centering around the optic disc as well as a true follow-up possibility. In this study, we evaluated a novel highresolution Spectral-domain OCT device (SD-OCT) with an incorporated eye-tracking feature in its ability to objectively measure the RNFL thickness (RNFLT) by testing intra-observer reproducibility in a series of healthy volunteers.

Methods

Triplicate circumferential RNFL scans of six peripapillary sectors were obtained from both eyes of all 31 participants. We compared the measurements of RNFLT during three separate examination days under miotic (Mi) and mydriatic (My) pupil conditions using high-speed (HS) and high-resolution (HR) scan acquisition mode. To examine the intersession reproducibility of the SD-OCT measurements the mean, standard deviation and the coefficient of variation (COV) were calculated

Results

No significant differences were found in all groups, independent of the mode of image acquisition and examination day (p always > 0,05). Under all conditions, low coefficients of variation (COV) between 0.545% and 3.97% (intrasession COV on baseline) were found. The intersession COV with activated follow-up mode ranged between 0.29% and 1.07%. In both settings the temporal sector showed the highest COV values.

Introduction

Previous studies have almost exclusively used standard timedomain optical coherence tomography (TD-OCT, Stratus OCT; Carl Zeiss Meditec, Dublin, California, USA) as a non-invasive retinal ocular imaging technique to measure structural changes in the axonal integrity of the retinal nerve fiber layer (RNFL) in patients with glaucoma, various optic neuropathies, optic neuritis (ON) and/or multiple sclerosis (MS). [START_REF] Lee | Structure-Function Relationships in Normal and Glaucomatous Eyes Determined by Time Domain and Spectral Domain Optical Coherence Tomography[END_REF][START_REF] Talman | Longitudinal study of vision and retinal nerve fiber layer thickness in multiple sclerosis[END_REF][START_REF] Garcia-Martin | Progressive changes in the retinal nerve fiber layer in patients with multiple sclerosis[END_REF] In most of the MS cases, the discrete progressive axonal changes may be elusive during short monitoring but may become more obvious in long observation periods. [START_REF] Fisher | Relation of visual function to retinal nerve fiber layer thickness in multiple sclerosis[END_REF][START_REF] Gordon-Lipkin | Retinal nerve fiber layer is associated with brain atrophy in multiple sclerosis[END_REF] Before OCT technology can be widely accepted for use in monitoring subtle axonal loss, often interpreted as a sign of progression of neurodegenerative diseases, it is however important that its initial diagnostic accuracy on baseline examinations can be extended to reproducible comparisons of retinal nerve fiber layer thickness (RNFLT) over time. To describe the smallest changes detectable in the context of a rather complex CNS disease, the latest advances in OCT technology have led to the incorporation of high-resolution spectral-domain (SD-OCT) imaging. This technique offers significant advantages in terms of markedly improved image resolution, imaging speed, scan coverage and retinal segmentation algorithms over the conventional TD-OCT. [START_REF] Wojtkowski | Three-dimensional retinal imaging with high-speed ultrahigh-resolution optical coherence tomography[END_REF] Accordingly, in overcoming the problems of prolonged examination time and difficulties in scan centering around the optic disc due to poor fixation and eye movements (in visually impaired or disabled patients) this latest SD-OCT technology provides novel technological features.

For instance, the SD-OCT device offers a unique feature of " freezing " the retinal infrared fundus image, utilizing a real time averaging algorithm. For this purpose the OCT scan can be moved within this stabilized optic disc image, so as to optimize exact scan centering. In conjunction with a built-in eye-tracking mechanism [START_REF] Cettomai | Reproducibility of optical coherence tomography in multiple sclerosis[END_REF][START_REF] Wolf-Schnurrbusch | Morphologic changes in patients with geographic atrophy assessed with a novel spectral OCT-SLO combination[END_REF] image artifacts due to eye movements are consequently ruled out. Additionally, the OCT software can automatically identify previous scan locations and "guide " the OCT broadband light source to scan the same location repeatedly and observer independently during follow-up visits. As a result, the technology markedly facilitates an objective and quantitative evaluation of the RNFLT over time.

We have assessed the ability of the new SD-OCT device to objectively measure RNFL thickness by testing intra-and intersession variability globally around the optic disc and also in the temporal, superior, nasal and inferior peripapillary sectors in a series of healthy volunteers as follows: 1) a comparison of the detection of RNFLT in dilated and undilated eyes either using high-speed (HS) or highresolution (HR) image acquisition mode; 2) a comparison of examinations of the RNFLT with either repeatedly activated automated real-time averaging mode (ART-Mode) and repositioning of the OCT scan target area over the optic disc for each scan or continuously activated ART mode during measurements with locked target area over the fixed optic disc reference fundus image for several image scans; 3) a comparison of automatic RNFLT detection in the follow-up mode using a baseline examination and two consecutive measurements on different days.

Materials and Methods

Patients

RNFL Measurement

Heidelberg Spectralis Optical Coherence tomography (SD-OCT) (Heidelberg Engineering, Heidelberg, Germany, Spectralis software version 5.1.1.0, Eye Explorer Software 1.6.1.0) uses a scanning superluminescence diode to emit a scan beam with a wavelength of 870 nm to provide up to 40,000 A scans/sec with a depth resolution of 7μm in tissue and a transversal resolution of 14 μm of images of ocular microstructures. The instrument combines OCT technology with a confocal Scanning Laser Ophthalmoscope (Heidelberg Engineering, Heidelberg, Germany), which provides a reference infrared fundus image.

An internal nasal fixation light was used to center the disc in the incorporated circle-shaped scanning target area. For OCT scanning, the SD-OCT device offers a unique feature of " freezing " the retinal infrared fundus image, utilizing a real time averaging algorithm (ART-mode). For this purpose the OCT scan can be moved within this stabilized optic disc image, so as to optimize exact scan centering and to adjust the number of obtained frames (=B scans, of which a number of 1-16 frames can be chosen for each peripapillary scan) to average and enhance image quality for each image scan. In the study, this was achieved using a maximum of 16 frames for each scan of the same scanning location during the peripapillary scanning process, thereby optimizing the signal to noise ratio and image quality significantly. Additionally, the device includes a high-speed (HS) and also a high-resolution (HR) mode for the OCT scan acquisition. First, the HS mode allows fast image acquisition; however, the images appear in lower resolution, yet still sufficient for automated layer segmentation. Second, the more time-consuming HR mode delivers high-resolution image quality scans with a clear visualization of each retinal layer. Furthermore, signal strength has been shown to affect RNFLT measurements using conventional Stratus OCT [START_REF] Cheung | Relationship between retinal nerve fiber layer measurement and signal strength in optical coherence tomography[END_REF][START_REF] Wu | Signal strength is an important determinant of accuracy of nerve fiber layer thickness measurement by optical coherence tomography[END_REF]. Therefore, in cases were the OCT software failed to detect the nerve fiber layer due to low image quality (failed automatic segmentation), obtained scans were eliminated and measurements were repeated until excellent quality was achieved. Although a manual correction of the segmentation is provided by the manufacturer, none was performed. Criteria for determining scan quality include a clear fundus image allowing perfect optic disc and scan circle visibility before and during image acquisition, absence of scan or algorithm errors which correspond to a signal strength of 20 units (as shown by the internal signal strength control) or more as suggested by the manufacturer, even and dense grey scale saturation throughout all retinal layers with dense grey visible in the RPE, and the RNFL visible with no irregularities and a continuous scan pattern. The OCT RNFLT scans were performed several times by a single operator (N.S.) during one individual session until at least three highquality scans were obtained and used for further analysis. Scans were acquired in the high-resolution acquisition mode thus allowing a more detailed differentiation of retinal layers, and in the high-speed mode for faster acquisition, but lower image quality, both in miosis followed by a series of scans in mydriasis (Figure 3 B andC). By means of eye tracking (TrueTrac™), each peripapillary OCT scan is registered and locked to a reference image utilized by the ART-mode. The built-in eye tracking modality and the high scanning speed are supposed to reduce image artifacts due to eye movements. In addition, the OCT software can identify previous scan locations (follow-up mode) and " guide " the OCT broadband light source to scan the same location repeatedly during follow-up visits.

In our study, triplicate image scans were obtained within a single session in all eyes for baseline as well as follow-up visits. For each of the three OCT scans, the maximum number of 16 frames, either in HR or HS mode, was utilized. To assess the reproducibility of SD-OCT measurements of the RNFLT we compared a specific set of different OCT scan acquisition modes.

In a first step (corresponding to baseline examination), we addressed the question of whether individuals with clear optic media, dilation of the pupil would be necessary. In addition, we analyzed whether the high resolution (HR) mode would deliver more accurate results than the high speed (HS) mode. Moreover, we performed measurements in repeatedly activated ART-mode (automated real-time averaging mode, either as HS-or HR-mode) with more time-consuming manual disc centering in the scanning area for each of the three peripapillary RNFL measurements. Furthermore, we examined in the same session with continuous ART-mode (either as HS-A-or HR-A-mode) utilizing permanently activated automated real-time averaging during measurements with locked target area over the fixed optic disc reference fundus image for several scans, whilst to avoid detection of the same image reacquisition of 16 frames each time (Figure 2 & 3). These settings were, in all cases, evaluated circumferentially and individually in the temporal, inferior, nasal and superior peripapillary sectors.

In a second step (corresponding to the performed two follow-up visits and based on the results of the baseline examinations), triplicate scans (each scan with the maximum number of 16 frames) for each session, again in the HR,-and HS-modes, were performed in miosis and mydriasis to evaluate intersession variability.

Statistical Analysis

In all cases triplicate high-quality scans were acquired. In the case of the baseline examination, each of the three obtained scans consisted of the maximum achievable number of 16 averaged frames.

The different groups were plotted as box-plots, representing the different image acquisition modes. They were compared a paired, two-tailed t-test, taking a p≤0,05 as statistically significant. The standard deviation was calculated as the square root of the variance. Reproducibility was assessed by calculating the coefficient of variation (COV) for intravisit and intervisit measurements. The intrasession COV (withinsubject variability) was calculated by dividing the squareroot of the within-visit variance component by the average of the measurements and was expressed as a percentage, with a COV < 5% expressing a good reproducibility. As a measure of intersession reproducibility (representing the expected amount of long term variability) we calculated the COV (%) over the parameter values (standard deviation / COVs) in the base, F1 and F2 session as the overall standard deviation (base, F1 and F2) divided by the average.

Results

1.) Baseline examination: comparison of RNFLT measurements in dilated and undilated eyes

In the circumferential frame, we found slightly higher values for the RNFLT in dilated versus undilated eyes, however, without noting statistical significance (Table 1, p>0.05). The measurements (all µm) of the RNFLT were in a range of 97.95 ± 0.64 (undilated, Mi-HR-A-mode) and 101.34 ± 0.685 (dilated, My-HR-A-mode). In the temporal quadrant, we found values between 72.77 ± 2.095 (dilated, My-HR-A-mode) and 76.45 ± 2.985 (dilated, My-HR-mode). Inferiorly, we detected thickness values for the RNFL in a range between 123.11 ± 1.545 (undilated, Mi-HS-A-mode) and 125.46 ± 2.715 (undilated, Mi-HR-mode). In the nasal quadrant, RNFL thickness ranged between 75.975 ± 2.26 (undilated, HR-A-mode) and 79.37 ± 2.22 (undilated, Mi-HR-mode). In the superior quadrant, the measurements for RNFL thickness were between 121.43 ± 2.93 (undilated, Mi-HR-A-mode) and 124.94 ± 2.85 (dilated, My-HR-Amode). In all cases no statistical significance was found within the different sectors (p always > 0,05).

To examine the intergroup variability (regarding the different image acquisition modes) of the Spectralis OCT measurements, the mean, standard deviation and the coefficient of variation (COV) were determined under different conditions as described previously (miosis high resolution (=Mi-HR), miosis high resolution with locked ART mode (=Mi-HR-A), miosis high speed (=Mi-HS), miosis high speed with locked ART mode (=Mi-HS-A), mydriasis high resolution (=My-HR), mydriasis high resolution with locked ART mode (=My-HR-A), mydriasis high speed (=My-HS), mydriasis high speed with locked ART mode (=My-HS-A)) in all peripapillary sectors (global = G, temporal = temp, inferior = inf, nasal = nas, superior = sup). The results are demonstrated in Table 1. Under all conditions, low intrasession coefficients of variation (COV) between 0.545% (G My-HS-A) and 3.97% (temp My-HR) were found. The lowest intrasession COV was documented in the global (G) RNFLT measurement ranging between 0,545% (My-HS-A) and 1.72% (My-HR). Interestingly, the highest COVs were detected in the temporal sector ranging between 2.71% (Mi-HS-A) and 3.97% (My-HR).

2.) Follow-up RNFLT measurements: Comparison of RNFLT in dilated and undilated eyes over 3 different days.

Having discerned that there are no measuring differences between dilated and undilated eyes on baseline (irrespective of the image acquisition mode), we proceeded to investigate whether the measurements of the RNFLT would be reproducible over time, i.e. during three separate days (Table 2). This task was undertaken using the " b aseline / follow-up " setting of the Spectralis device. (Technical explanation: once an obtained image scan is marked as a baseline examination scan, subsequent examinations and OCT scans can be taken at identical positions using the built-in follow-up mode, which is utilised by a second, detecting laser source.) Accordingly, we compared the high-speed with the high-resolution mode.

We used Box-Plots to compare the RNFLT values between the different examination days (base/F1/F2) and modes (HR/HS) (Figure 1). Again, no significant differences were found between the different groups. In none of the sectors analyzed were significant differences in direct comparison found (p>0.05). The comparison of the same parameters in dilated eyes again showed no statistically significant differences (data not shown, p in all cases > 0,05).

We then examined the intersession variability of the Spectralis OCT measurements in regard to the baseline examination and two consecutive examinations under identical conditions on the two following days (F1 and F2). Again, we calculated the mean, standard deviation and the coefficient of variation (COV) using the different conditions described previously. The results are shown in table 2. COV ranged between 0,29% (sup, Mi-HS) and 1,07% (temp, Mi-HS). In dilated eyes, the COV was ranging between 0,35 (sup, My-HS) and 1,12 (temp, My-HS).

Discussion

Among neuroophthalmologists and neurologists who care for MS patients, conventional TD-OCT technology is currently being discussed both as a means of monitoring the progression of the disease and in evaluating the impact of disease-modifying therapy. [START_REF] Sergott | The role of optical coherence tomography in multiple sclerosis: expert panel consensus[END_REF] However, the reproducibility of RNFLT measurements is crucial for its diagnostic precision if the application of OCT is expected to evolve into a reliable outcome metric for the degenerative processes in the central nervous system. [START_REF] Costello | Differences in retinal nerve fiber layer atrophy between multiple sclerosis subtypes[END_REF] In fact, RNFLT anatomically increases as proximity to the optic disc increases. [START_REF] Menke | OCT measurements in patients with optic disc edema[END_REF] Consequently, even a minimal decentering of the target will lead to significantly differing results in RNFLT measurements obtained by TD-OCT. [START_REF] Vizzeri | Effect of improper scan alignment on retinal nerve fiber layer thickness measurements using Stratus optical coherence tomograph[END_REF] An adequate OCT resolution and optimized image quality already at baseline are therefore of paramount concern in patients suffering from optic nerve-and neurodegenerative disordes.

efficacy
Poor fixation -resulting from (ceco-) central scotomas, nystagmus, macular degeneration or progressive optic neuropathy -may be challenging during scan acquisition. For example, with conventional TD-OCT, reproducible RNFL scans during episodes of acute optic neuritis can hardly be achieved [START_REF] Menke | OCT measurements in patients with optic disc edema[END_REF]. The current study provides evidence that SD-OCT technology, by incorporating an eyetracking mode, has the potential to accurately and repeatedly perform RNFLT measurements. For the first time, fixation problems can be overcome using the eye tracking option. As opposed to conventional TD-OCT-devices [START_REF] Vizzeri | Effect of improper scan alignment on retinal nerve fiber layer thickness measurements using Stratus optical coherence tomograph[END_REF] a circle-shaped central target area is incorporated. It can be positioned directly on the rim of the optic disc and viewed on a frozen reference infrared fundus image. This enables improved centering of the optic disc while scanning along the second outer ring of the target area (Figure 2).

We have then addressed the question of whether the examination must be performed in dilated eyes or if undilated eyes would still deliver reproducible data, as all previous, conventional TD-OCT studies in patients with glaucoma or neurodegenerative disorders have been performed in miosis. Moreover, pupil dilation is sometimes refused in a number of MS patients as to avoid a further impairment of gait disturbances or in order to reduce their fitness to drive as a result of blurred vision. [START_REF] Vizzeri | Effect of improper scan alignment on retinal nerve fiber layer thickness measurements using Stratus optical coherence tomograph[END_REF] We also investigated whether or not the ART-mode would need to be permanently activated or if reacquisition of each OCT image with consecutive repositioning of the target area over the optic disc would be more accurate. We found that in direct comparison, the RNFLT measurements did not significantly differ when calculated in dilated or undilated eyes. We also found that -at least for clear optic mediathe HS mode would be equivalent to the HR mode, regarding the image acquisition under conveniently undilated pupil conditions. In severely opaque ocular media, however, this may not hold true, as in such cases a HR image may be needed to gain a reliably segmentable image (Figure 3). [START_REF] Pierre-Kahn | Reproducibility of Peripapillary Retinal Nerve Fiber Layer Thickness and Optic Nerve Head Parameters Measured with CirrusTM HD-OCT in Glaucomatous Eyes[END_REF] As many neurodegenerative diseases constitute chronically progressive disorders, tight follow-up visits are often required to monitor disease progression accurately. Thus, it is important to assess the variability of RNFLT measurements between consecutive follow-up visits. [16,17] For the detection of changes during follow-up, however, it is further crucial to obtain a high quality reliable baseline examination. The registered baseline examination can be stored in the device. For this purpose, any obtained follow-up examinations will be directly compared to the first examination by digitally referencing the initial image (e.g. exactly lining up the OCT circle scan around the optic nerve). Additionally, the OCT software can identify previous scan locations and " guide " the OCT broadband light source to scan the same location repeatedly, thereby enabling, for the first time, a true follow-up measurement while still reducing movement artifacts. smallest denominator in the COV equation.

Whilst not statistically significant, it might still be clinically relevant. This would be of much interest in neurodegenerative disorders (e.g. MS, glaucoma) where only subtle changes below the axial resolution could be expected, as the axial resolution of the device is somewhat limited to approximately 7µm [START_REF] Henderson | A preliminary longitudinal study of the retinal nerve fiber layer in progressive multiple sclerosis[END_REF]. Thus, the "technical component of the error" is fixed in terms of micrometers, regardless on how thick the RNFL is. In the consequence this would mean that the COV in atrophy-affected RNFL would be clearly higher, and would hamper a follow-up capacity in diseased eye. However, long-term longitudinal studies would therefore give more relevance to this technology.

In conclusion, the high level of agreement together with the possibility of utilizing a true follow-up measurement confirm the fact that the presented SD-OCT is a promising technology in terms of identifying localized structural damage, e.g. axonal damage. Scanning identical peripapillary regions may enable clinicians to detect discrete levels of retinal thickness changes over time. This is a crucial condition and a prerequisite for the monitoring of neurodegenerative disorders, including (early) glaucoma. Nevertheless, it remains to be evaluated whether peripapillary SD-OCT analysis can provide enough evidence to follow subtle RNFLT changes in short term. No statistically significant differences were detected. This was independent of the measurement of the RNFLT at baseline (base), first (F1) or second (F2) follow-up visit, as well as the periparpillary sector examined. This was examined in an averaged RNFLT (G) or split into the four sectors (temporal, inferior, nasal and superior). The results are plotted using box-plots. In opaque media (in this case a left eye with a cortical cataract) the automatic segmentation of the RNFL fails altogether (A) in the first place. Upon several tries the segmentation is somewhat successful, however it underestimates the RNFLT, in the case of detection using the HS mode (B). In HR mode (C) the RNFL is successfully detected.

Table 1. Baseline comparison of RNFLT measurements (µm) around the optic nerve either globally (G), temporal (temp), inferior (inf), nasal (nas) or superior (sup).

The modes used were undilated (Mi), dilated (My), High Resolution (HR), High Speed (HS). Usage of the locked ART mode is indicated with -A. We calculated the mean, standard deviation (stdev) and the intrasession coefficient of variation (COV) from the data described. We show, in all conditions, low COV between 0.55% (G, My-HS-A) and 3.97% (temp, My-HR). The highest COVs were detected in the temporal sector ranging between 2.71% (Mi-HS-A) and 3.97% (My-HR) Table 2. Follow-up-comparison of RNFLT measurements (µm) over three different days around the optic nerve either globally (G), temporal (temp), inferior (inf), nasal (nas) or superior (sup). The data are shown in regard to a baseline examination and two consecutive examinations under identical conditions on the two following days (F1 and F2). Again we calculated the mean, standard deviation (stdev) and the intersession COV using the different conditions described previously (all examinations have been performed in undilated (Mi) and dilated (My) eyes using either high resolution (HR) or high speed (HS) modes).

Figure 1 .

 1 Figure 1. Comparison of the RNFLT measurements (µm) using a baseline examination and consecutive follow-up measurements.No statistically significant differences were detected. This was independent of the measurement of the RNFLT at baseline (base), first (F1) or second (F2) follow-up visit, as well as the periparpillary sector examined. This was examined in an averaged RNFLT (G) or split into the four sectors (temporal, inferior, nasal and superior). The results are plotted using box-plots.

Figure 2 .

 2 Figure 2. Exact centering of the peripapillary scan is essential for reproducible data. (A) shows a slightly decentered, peripapillary scan. The RNFLT measurements are different from the (B) centered scan. Additionally the software-based internal control misclassifies the temporal scan as pathological (red segment).

Figure 3 .

 3 Figure 3. Detection of the RNFLT in opaque media. In opaque media (in this case a left eye with a cortical cataract) the automatic segmentation of the RNFL fails altogether (A) in the first place. Upon several tries the segmentation is somewhat successful, however it underestimates the RNFLT, in the case of detection using the HS mode (B). In HR mode (C) the RNFL is successfully detected.

Table 1

 1

			G	temp	Inf	nas	sup
	Mi HR	Mean	99,725 75,25 125,46 79,365 123,33
		Stdev	1,565	2,605	2,715	2,215	3,07
		COV
		(%)	1,555	3,46	2,185	3,055 2,485
	Mi HR-
	A	Mean	97,95 75,255 123,73 75,975 121,43
		Stdev	0,64	2,26	1,8	2,255 2,925
		COV
		(%)	0,655	3,08	1,445	3,19	2,435
	Mi HS	Mean	98,59	73,63 124,00 78,78 122,82
		Stdev	1,17	2,54	2,065	1,985	2,48
		COV
		(%)	1,195	3,465	1,67	2,755	2,05
	Mi HS-
	A	Mean	98,02	73,29 123,11 78,25 121,90
		Stdev	0,76	1,96	1,545	1,55	1,835
		COV
		(%)	0,58	2,71	1,285	2,14	1,535
	My HR	Mean 100,305 76,445 125,235 79,19 124,84
		Stdev	1,75	2,985	3,635	2,065	3,48
		COV
		(%)	1,72	3,97	2,87	2,82	2,825
	My HR-
	A	Mean	101,34 72,77 124,58 78,03 124,94
		Stdev	0,685	2,095	2,49	2,145 2,845
		COV
		(%)	0,67	2,85	1,955	3,06	2,24
	My HS	Mean	99,02	74,28 123,75 78,35 124,09
		Stdev	1,5	2,56	2,19	2,52	2,49
		COV
		(%)	1,505	3,47	1,785	3,42	1,97
	My HS-
	A	Mean	98,91	74,49 123,83 78,22 123,66
		Stdev	0,535	2,36	1,815	1,99	1,73
		COV
		(%)	0,545	3,28	1,475	2,785 1,375

Table 2

 2

	Miosis	G	Temp	Inf	Nas	Sup
	HR
	Mean (µm) 96,73	73,68	117,04	78,42	118,93
	Stdev	0,41	0,655	0,63	0,62	0,41
	COV (%)	0,42	0,885	0,525	0,855	0,535
	HS
	Mean	95,25	73,285	117,54	77,765	116,935
	Stdev	0,29	0,78	0,37	0,63	0,34
	COV (%)	0,31	1,07	0,315	0,895	0,29
	Mydr.	G	Temp	Inf	Nas	Sup
	HR
	Mean (µm) 97,43	75,25	120,06	77,24	118,96
	Stdev	0,05	0,81	0,82	0,73	0,42
	COV (%)	0,53	1,02	0,64	0,95	0,55
	HS
	Mean	95,99	74,39	118,85	78,31	117,83
	Stdev	0,58	0,90	0,29	0,685	0,49
	COV (%)	0,49	1,12	0,45	0,85	0,35

  of longitudinal clinical trials. It should be used to

	precisely	monitor	disease	course	in	terms	of	slowly
	progressive	subtle	axonal	loss	thereby	reflecting	the

Competing Interest: None to declare.

In this study, our results showed agreement between consecutive follow-up measurements independent of the acquisition mode (Table 2). Consecutive follow-up scanning of identical peripapillary RNFL locations (referring to defined baseline scans) demonstrates low intersession variability. Thus, this technology provides follow-up examinations independent of the examiner. [18,19] Intrasession comparison at baseline however, produces slightly higher COVs (Table 1), as this reflects data taken from three independent measurements without activated follow-up mode.

It must be noted that image scan acquisition by eye tracking and activated ART mode only produces higher COVs at baseline. The clue for reducing the COV thus seem to utilize the followup function in parallel with activated ART-mode and TrueTrac™ function. This is a major difference in the concept of image acquisition between eye-tracking guided SD-OCT scanning and other conventional or high-resolution OCT models that perform sweep scans. Here, a large defined area around the optic disc is scanned in the shortest time, without correcting for eye movements. Decentering and image artifacts during the scanning process and decreased reproducibility of retinal thickness measurements are noted over time. Peripapillary scans may as such increasingly differ from visit to visit. [18] Hence, the high level of reproducibility of retinal thickness measurements with the SD-OCT in healthy subjects can be clearly attributed to 1) a combination of optimized disc centering due to the "locking " of an image during the activated ART-mode as well as the integrated correction for eye movements with the TruTrack™ eye tracking capability during the scanning process at baseline; 2) the implementation of a true follow-up scanning of identical RNFL locations at each follow-up visit; and 3) sophisticated retinal segmentation algorithms providing comparable results both under fast (HS) and high-quality image (HR) acquisition conditions, independent of the pupil width.

Interestingly, the highest COV was found in the thinnest (temporal) sectors, which has the smallest mean and hence the


MD Barbro K Linderholm
email: barbro.linderholm@ki.se

Henrik Hellborg

Ulla Johansson

Lambert Skoog

Janne Lehtiö

Hellborg Henrik

Johansson Ulla

Skoog Lambert

Lehtiö Janne

Vascular endothelial growth factor receptor 2 (VEGFR2) and downstream p38 mitogen activated protein kinase (MAPK) are possible candidate markers of intrinsic resistance to adjuvant endocrine treatment in steroid receptor positive breast cancer

Keywords: VEGFR2, p38MAPK, tamoxifen, intrinsic resistance

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

Vascular endothelial growth factor receptor 2 and downstream p38 mitogen-activated protein kinase are possible candidate markers of intrinsic resistance to adjuvant endocrine treatment in steroid receptor positive breast cancer

INTRODUCTION

Extracellular stimuli initiated by mitogens, cytokines and growth factors result in aberrations of normal cellular processes as proliferation, differentiation, neo-angiogenesis and invasion, characteristic for cancer growth [START_REF] Hanahan | The hallmarks of cancer[END_REF]. This is in part mediated through membrane receptors, such as the receptors tyrosine kinases (RTK´s) and downstream mitogen-activated protein kinases (MAPK´s) [START_REF] Guo | Overexpression of vascular endothelial growth factor by MCF-7 breast cancer cells promotes estrogen-independent tumor growth in vivo[END_REF]. Increased angiogenesis has been demonstrated to be a significant negative prognostic factor in several solid tumors including breast cancer. Vascular Endothelial Growth Factor (VEGF) is a potent growth factor for endothelial cells that is produced by tumor cells and mediates its effects by binding to VEGF receptors 1 -3 (VEGFR1-3) [START_REF] Kroll | A novel function of VEGF receptor-2 (KDR): rapid release of nitric oxide in response to VEGF-A stimulation in endothelial cells[END_REF]. These receptors are preferably expressed on endothelial cells, although recent data has shown VEGFR2 expression also on the breast cancer cells suggesting existence of an autocrine stimulation in breast cancer progression [START_REF] Aesoy | An autocrine VEGF/VEGFR2 and p38 signaling loop confers resistance to 4-hydroxytamoxifen in MCF-7 breast cancer cells[END_REF]. Upon ligand binding, VEGFR dimerize and become autophosphorylated resulting in activation of signal transduction cascades including the MAPK´s [START_REF] Folkman | Fundamental concepts of the angiogenic process[END_REF]. This is followed by phosphorylation of several cytosolic substrates which translocation to the nucleus, where they activate transcriptional factors resulting in proliferation and migration of endothelial cells into the surrounding stroma. Three MAPK families have been extensively studied; the extracellular regulated kinase (ERK), c-jun amino terminal kinase (JNK) and the high osmolarity glycerol response kinase (p38) [START_REF] Garrington | Organization and regulation of mitogen-activated protein kinase signaling pathways[END_REF][START_REF] Fanger | Regulation of the MAPK family members: role of subcellular localization and architectural organization[END_REF]. Activation of ERK has been attributed to growth factor signaling while JNK and p38 are activated in response to growth factors and by a large spectrum of stress related stimuli [START_REF] Kyriakis | Mammalian Mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation[END_REF]. The aim with endocrine therapy is to disrupt estrogen stimulation of breast cancer cells which are mediated via estrogen binding to the estrogen receptor (ER). The expression of steroid receptors is correlated to the benefit of endocrine therapy, with a higher effect in ER positive patients while the role of PgR has been debated [START_REF] Rutqvist | The relationship between hormone receptor content and the effect of adjuvant tamoxifen in operable breast cancer[END_REF]. Resistance to endocrine therapy has in part been attributed to abnormalities in growth factor signaling. Preclinical models of tamoxifen resistance have shown that the steroid receptors not only are functional in response to steroid hormones, but also for multiple signal transduction pathways by a ligand-independent phosphorylation of the ER. Several RTK´s i.e., the insulin-like growth factor receptor (IGFR), the epidermal growth factor receptor (EGFR) as well as their downstream cascades has shown to be part of this process [START_REF] Cornier | Decrease in estradiol-stimulated progesterone receptor production in MCF-7 cells by epidermal growth factor and possible clinical implication for paracrine-regulated breast cancer growth[END_REF][START_REF] Cui | Insulin-like growth factor-I inhibits progesterone receptor expression in breast cancer cells via the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin pathway: progesterone receptor as a potential indicator of growth factor activity in breast cancer[END_REF]. Also patients expressing significant levels of ER and/or PgR can have breast cancers that do not respond to endocrine therapy; either at the beginning of treatment (de novo or intrinsic resistance) or after prolonged use (acquired resistance). Data from preclinical model systems investigating initially endocrine sensitive BC cells subjected to long term anti-estrogen therapy has revealed that there are several stages of acquired resistance with an over-time shift in ER as well as growth factor expression [START_REF] Osipo | Reversal of tamoxifen resistant breast cancer by low dose estrogen therapy[END_REF]. Patients with acquired resistance might benefit from other types of endocrine intervention as estrogen withdrawal or therapy with the pure antiestrogen fulvestrant [START_REF] Osborne | Double-blind, randomized trial comparing the efficacy and tolerability of fulvestrant versus anastrozole in postmenopausal women with advanced breast cancer progressing on prior endocrine therapy: results of a North American trial[END_REF][START_REF] Howell | Fulvestrant, formerly ICI 182,780, is as effective as anastrozole in postmenopausal women with advanced breast cancer progressing after prior endocrine treatment[END_REF], contrary to patients likely to develop intrinsic resistance, for which endocrine therapy alone should be avoided. Our aim was to investigate intratumoral levels of VEGFR2 and downstream activated p38 MAPK (referred to as p38) in relation to previously determined intratumoral VEGF levels, routine breast cancer factors, localization of relapses and survival in receptor positive patients subjected to adjuvant endocrine treatment.

We also wanted to determine if expression of these factors had an impact on early and late relapses.

PATIENTS AND METHODS

Patients

A total of 679 consecutive patients with primary operable invasive breast cancer diagnosed from January 1 st 1993 through December 31 st 1996 at two institutions in Stockholm, Sweden had remaining tumor homogenates after determination of steroid receptors in clinical routine.

A total of 404 patients with a steroid receptor positive BC subjected to endocrine therapy as only adjuvant systemic treatment have previously been included in a study investigating VEGF in relation to outcome [START_REF] Linderholm | Shorter survival times following adjuvant endocrine therapy in oestrogen (ER) -and progesterone receptor (PgR) positive breast cancer (BC) overexpressing HER2 and/or with an increased expression of vascular endothelial growth factor (VEGF)[END_REF]. Patients with remaining preserved tumor homogenates were included in the present study. Patients were identified by use of the Breast Cancer registry data base at the Regional Oncologic Centre, Karolinska University Hospital, Stockholm, Sweden, and are in detail described in previous publications [START_REF] Linderholm | Shorter survival times following adjuvant endocrine therapy in oestrogen (ER) -and progesterone receptor (PgR) positive breast cancer (BC) overexpressing HER2 and/or with an increased expression of vascular endothelial growth factor (VEGF)[END_REF][START_REF] Linderholm | Significantly higher levels of vascular endothelial growth factor (VEGF) and shorter survival times for patients with primary operable triple negative breast cancer[END_REF]. Patients with locally advanced breast cancer, presence of distant metastasis at diagnosis, or those subjected to neoadjuvant therapy were not included.

Tumor tissue preparation

Representative tumor tissue was cut out and frozen in liquid nitrogen for later analysis of estrogen-and progesterone levels. Frozen tumor tissue was homogenized and suspended in cold standard receptor buffer (10 mmol/L Tris pH 7.4, 1.5 mmol/L EDTA, 10 mmol/L sodium molybdate, 1.0 mmol/L monothioglycerol). Supernatants were collected after refrigerated centrifugation at 20,000 g and stored at -70C . The pellet fractions were analyzed for DNA content by the method of Burton, in order to evaluate cell concentrations in samples.

ER and PgR analyses

ER and PgR content were determined in tumor homogenates by an enzyme immunoassay (Abbott Laboratories, Diagnostic Division, Abbott Park, IL). Receptor concentrations were expressed as fmol of receptor per microgram of DNA. The cut-off value used to separate ER or PgR positive from negative was 0.05 fmol receptor/ g DNA. Steroid receptor levels were used in clinical routine and performed once or twice weekly pending on number of incoming patients samples. The Department of Cytology, Karolinska University Hospital, Stockholm, Sweden participated in the external quality assessment program arranged by the EORTC-Receptor and Biomarker Group at the Quality Assessment Laboratory, University Medical Centre Nijmegen, the Netherlands [START_REF] Geurts-Moespot | Twenty years of experience with the steroid receptor external quality assessment program -the paradigm for tumour biomarker EQA studies. On behalf of the EORTC Receptor and Biomarker Study Group[END_REF].

VEGFR2 analysis

A VEGFR2 assay was performed by use of a commercial quantitative immunoassay kit for human VEGFR2 (Quantikine, human VEGFR2, R & D Systems, Minneapolis, MN, USA) according to the manufacturers´ recommendations. Tumor homogenates from the primary breast tumor were used. Briefly, a murine monoclonal antibody specific for VEGFR2 had been pre-coated onto a 96-well micro plate. After incubation and washing steps, a secondary polyclonal antibody specific for VEGFR2 was added and used as detection antibody.

VEGFR2 levels in patients´ samples were expressed as pg/ g DNA. Analyses of VEGFR2 were performed in a row by one person.

Activated p38 MAPK analysis

A phospho-p38 MAPK assay was performed by use of a commercial quantitative immunoassay kit for human activated p38 MAPK (Biosource, human phospho-p38 MAPK, Invitrogen, Camarillo, CA, USA) according to the manufacturers´ recommendations. Tumor homogenates from the primary breast tumor were used. In summary, a monoclonal antibody specific for p38 MAPK regardless of phosphorylation state had been pre-coated onto a 96well micro plate. After incubation and washing steps, a detection antibody specific for p38 MAPK phosphorylated at heroine 180 and 182 was added. p38 MAPK protein levels in patients´ samples were expressed as pg/ g DNA. Analyses of p38 were performed in a row by one person.

Statistical methods

Pearson chi-square test was used to investigate associations between intratumoral levels of VEGFR2 and p38 MAPK with VEGF as well as routine clinical breast cancer parameters (tumor size, nodal status, histopathological grade, and age). The median levels of VEGFR2 and VEGF in the tumor tissue of all patients (n=381) was used as cut-off point in the statistical analyses, as we wanted to avoid statistically manipulations. Previous publications from our group, as well as other research groups, have analyzed VEGF as a dichotomized variable in relation to survival [START_REF] Linderholm | Vascular Endothelial Growth Factor Is of High Prognostic Value in Node-Negative Breast Carcinoma[END_REF][START_REF] Linderholm | Correlation of Vascular Endothelial Growth Factor (VEGF) and survival, metastasis sites and responsiveness to adjuvant systemic treatment in primary node-positive breast carcinoma[END_REF][START_REF] Foekens | High tumor levels of vascular endothelial growth factor predict poor response to systemic therapy in advanced breast cancer[END_REF][START_REF] Manders | Vascular endothelial growth factor is associated with the efficacy of endocrine therapy in patients with advanced breast carcinoma[END_REF]. Patients with detectable levels of p38 were considered positive while those without detectable levels were classified as negative. Kruskal-Wallis test was used to test the null hypothesis of equal VEGFR2 and p38 distributions in samples from the two institutions.

The median follow-up time was defined as the median follow-up for those patients still alive and free from distant recurrences at latest follow-up. Survival was estimated using the Kaplan-Meier method and comparison between study groups was performed with the log-rank test for determination of relapse-free survival (RFS) and breast cancer corrected survival (BCCS), the latter as the majority of deaths were unrelated to breast cancer. Survival analyzes were performed in the total patient population and in the steroid receptor positive group. In order to study the impact of the biomarkers on early and later relapses, the cohort was divided in two strata; patients with early relapses (≤ 2 years of follow-up) and patients with late relapses (after 2 years of follow-up). The reason for choosing 2 year as cut-point was that patients included in the study received endocrine therapy, mainly tamoxifen for two or five years. Thereby we were sure that all relapses within the first period occurred early on during adjuvant treatment. Within each group survival was compared between p38 negative versus positive, VEGFR2 negative versus positive and VEGF negative versus positive patients. In order to evaluate the simultaneous effect on different factors on survival, the Cox's proportional hazard model was used. Factors were tested and included in a forward stepwise procedure. For each variable, the patients were classified in two prognostic categories, and the putative best prognosis category was used as the reference. The survival time was measured from date of diagnosis to first documented relapse or death. In all the tests the significance level was set to 0.05, and all tests were two-sided. The study design is done according to the REMARK criteria for tumor marker studies [START_REF] Mcshane | Statistics Subcommittee of the NCI-EORTC Working Group on Cancer Diagnostics (2005) Reporting recommendations for tumor marker prognostic studies[END_REF].

RESULTS

Patients characteristics

Information regarding patients´ characteristics was obtained from the Breast Cancer database at the Regional Oncologic Centre, Karolinska University Hospital, Stockholm, Sweden. The database reveals information on all patients with primary breast cancer within the Stockholm-Gotland region. Parameters registered were: date of diagnosis, age, way of detection (mammogram screening or not), type of surgery, tumor size according to the pathologic report, number of removed axillary lymph nodes, number of axillary metastases, and levels of ER and PgR. The database also includes the adjuvant therapy recommended (radiotherapy, adjuvant systemic therapy), date and type of first relapse, date and cause of death. Patients' records were used to collect data on histopathological type and grade and to control that correct data on the adjuvant therapy actually given were registered. Detailed characteristics of the patients are earlier described [START_REF] Linderholm | Shorter survival times following adjuvant endocrine therapy in oestrogen (ER) -and progesterone receptor (PgR) positive breast cancer (BC) overexpressing HER2 and/or with an increased expression of vascular endothelial growth factor (VEGF)[END_REF][START_REF] Linderholm | Significantly higher levels of vascular endothelial growth factor (VEGF) and shorter survival times for patients with primary operable triple negative breast cancer[END_REF]. A total of 679 consecutive patients with diagnosis of primary operable breast cancer from 1993 through 1996 at two institutions in Stockholm were identified. Of these, 404 steroid receptor positive patients received endocrine therapy as only systemic adjuvant therapy, mainly tamoxifen for 2 or 5 years (Table 1). VEGF levels in relation to routine BC biomarkers and stage are previously published [START_REF] Linderholm | Shorter survival times following adjuvant endocrine therapy in oestrogen (ER) -and progesterone receptor (PgR) positive breast cancer (BC) overexpressing HER2 and/or with an increased expression of vascular endothelial growth factor (VEGF)[END_REF]. Determination of VEGFR2 and p38 were performed in remaining tumor homogenates in 381 and 174 patients respectively. The endocrine therapy given to the 381 patients with data on VEGFR2 were:

Expression of VEGFR2 and p38 MAPK

Wide variances in determined levels of VEGFR2 and p38 were found. We did not find any statistically significantly difference in samples from the two participating institutions; Mann-Whitney U tests VEGFR2 (p=0.59) (Fig 1a) and p38 (p=0.36) (Fig 1b). The median level of VEGFR2 was 0.72 pg/µg DNA (range 0.0-11.66). Higher intratumoral VEGFR2 levels were statistically significant correlated with high VEGF expression (p=0.005), expression of p38 MAPK (p=0.018), negative ER (p=0.008), larger tumors (p=0.001), grade III tumors (p=0.018), premenopausal status (p=0.005) and distant metastasis (p=0.014). No correlation was seen between VEGFR2 and PgR status, tumor size, or age (not shown). Detectable levels of p38 were registered in 113 out of 174 patients (65%) with remaining cytosols for analysis.

Expression of p38 was only statistically significant correlated with negative PgR (p=0.044).

Survival -univariate analysis

Higher intratumoral levels of VEGFR2 were statistically significantly correlated with shorter RFS (HR=1.9; 95%CI 1.1-3.3; p=0.023) and BCCS (HR=2.3; 95%CI=1.044-5.034, p=0.039).

Moreover, high VEGFR2 had a significant impact on survival in both time periods; early relapses, 0-2 years of follow-up (p=0.029) and later relapses in the subsequent period, > 2 years of follow-up (p=0.018) (Fig 2a andb). High VEGFR2 was associated with an increased risk of relapse (HR 1.6; 95%CI =0.901-2.913; p=0.107) and breast cancer corrected survival (HR 2.4; 95%CI =0.955-5.988; p=0.051) also in the receptor positive group, however not reaching statistically significance. Expression of p38 was not correlated with survival in the total patient population when studied during the whole follow-up time; RFS (p=0.774) or BCCS (p=0.960. However, expression of p38 was significantly correlated to early relapses (p=0.021) while no difference was seen after 2 years (p=0.730). The probability of RFS and BCCS at 5 years follow-up is shown in Table 3. Contrary, high expression of VEGF was only significantly associated with an increased risk for relapses after 2 years (p=0.037) while no difference was seen in the early follow-up period (p=0.550) (Fig 3a andb). In the group of patients with a BC positive for both ER and PgR, p38 remained as a predictor of early relapses (p=0.006).

Survival -univariate analysis according to earlier determined VEGF and prognostic factors

Other factors with statistically significant prognostic value in the total patient population were VEGF; RFS (p=0.003), BCCS (p=0.002). ER status; RFS (p=0.002), BCCS (p=0.006), PgR status; RFS (p=0.008), BCCS (p=0.011), tumor size; RFS (p<0.01), BCCS (p=0.002), and nodal status; RFS (p<0.01), BCCS (p<0.01), histological grade; RFS (p=0.001), BCCS (p=0.007) and age (p=0.001), BCCS (p=0.011). The same analyses were performed with the steroid receptor negative patients excluded resulting in the corresponding figures; VEGF;

(RFS p=0.0177, BCCS p=0.0321) tumor size; RFS (p<0.01), BCCS (p=0.009), nodal status; RFS (p<0.01), BCCS (p<0.01), histological grade; RFS (p=0.120), BCCS (p=0.337), and age; (p=0.001), BCCS (p=0.012).

Survival -multivariate analysis

A Cox proportional hazard regression model was used to estimate hazard ratios (HR) for recurrences and deaths. Analysis was performed in the whole patient population. Due to few events during the early follow-up period analyses were not done separately with patients split in the two follow-up periods. Factors tested were those statistically significantly correlated with survival in univariate analyses. Patient's age was also included. HR above 1.0 indicated a greater risk of recurrence or death than for the comparative group set as reference. VEGFR2 was found as an independent statistically significant factor for RFS (HR=1.8; 95%CI 1.1 -2.8, p=0.0016) and BCCS (HR=1.8, 95%CI=1.1-3.2, p=0.030) respectively. Other factors remaining significantly correlated to RFS were tumor size (HR= 2.2, p<0.001), nodal status (HR =2.9, p<0.001), ER (HR=1.9, p=0.016) and age (HR=1.7, p=0.036). The same factors were found as independent predictors of BCCS; tumor size (HR=1.9, p<0.001), nodal status (HR=4.6, p<0.001), ER (HR=1.9, p=0.049) and age (HR=3.0, p=0.001) (Table 4). Duration of tamoxifen treatment for 2 years were correlated with an increased risk of relapse; (HR=1.4, p=0.306), as well as breast cancer corrected survival; (HR=2.0, p=0.122), however not reaching statistical significance.

Multivariate analyses were also calculated with the steroid receptor negative patients excluded with minor changes in the results; tumor size (HR 2.1, p=0.01), nodal status (HR 2.7, p=0.002) and age (HR 3.4, p=0.004) remained statistically significant associated with relapsefree survival, while VEGFR2 were not; HR 1.6, p=0.106. For BCCS, nodal status (HR 11.6, p<0.01) and VEGFR2 (HR 4.2, p=0.027) remained as independent predictors of breast cancer corrected survival, while tumor size, and age did not. Histological grade did not remain as an independent predictor of RFS or BCCS.

DISCUSSION

We investigated the possible effect on survival of VEGFR2 and downstream p38 MAPK in primary breast cancer patients subjected to adjuvant endocrine treatment. The results showed that patients with high intratumoral level of VEGFR2 were more prone to develop distant metastases, and had an impaired survival. A huge amount of publications have shown higher expression of the ligand, VEGF to be correlated with shorter survival times in several tumor types including breast cancer [START_REF] Poon | Clinical implications of circulating angiogenic factors in cancer patients[END_REF]. VEGFR2 is less studied and the data concerning the prognostic and predictive role contradictory. In accordance to our results, a previous study including a large number of premenopausal patients randomized to tamoxifen for two years or no adjuvant endocrine therapy, showed that high VEGFR2 expression were correlated with reduced survival [START_REF] Rydén | Tumor-specific expression of vascular endothelial growth factor receptor 2 but not vascular endothelial growth factor or human epidermal growth factor receptor 2 is associated with impaired response to adjuvant tamoxifen in premenopausal breast cancer[END_REF]. On the contrary, the same group could not demonstrate that VEGFR2 expression was correlated with survival after adjuvant tamoxifen in ER positive postmenopausal patients [START_REF] Rydén | Tumorspecific VEGF-A and VEGFR2 in postmenopausal breast cancer patients with long-term follow-up. Implication of a link between VEGF pathway and tamoxifen response[END_REF]. VEGFR2 expression was in both studies determined by immunohistochemistry on tumor micro arrays excluding technical problems solely as explanation for the different results. The latter study on postmenopausal women included fewer patients which in part may contribute to the discrepant results. Variable proportions of patients with Luminal A and B subtypes in pre-and postmenopausal women may also influence the results. In a recent study investigating the correlation between several potential tumor biomarkers and effect of neo-adjuvant chemotherapy, VEGFR2 did not add predictive information for relapse free survival [START_REF] Guarneri | A prognostic model based on nodal status and Ki-67 predicts the risk of recurrence and death in breast cancer patients with residual disease after preoperative chemotherapy[END_REF].

Examples of pathways involved in intrinsic resistance have been enhanced activity of EGFR or IGFR that result in an impaired estrogen induction of the PgR [START_REF] Cornier | Decrease in estradiol-stimulated progesterone receptor production in MCF-7 cells by epidermal growth factor and possible clinical implication for paracrine-regulated breast cancer growth[END_REF][START_REF] Cui | Insulin-like growth factor-I inhibits progesterone receptor expression in breast cancer cells via the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin pathway: progesterone receptor as a potential indicator of growth factor activity in breast cancer[END_REF][START_REF] Johnston | Changes in estrogen receptor, progesterone receptor, and pS2 expression in tamoxifen-resistant human breast cancer[END_REF]. However, tyrosine kinase receptors have also shown a role in acquired resistance in preclinical studies [START_REF] Knowlden | Elevated levels of epidermal growth factor receptor/c-erbB2 heterodimers mediate an autocrine growth regulatory pathway in tamoxifen-resistant MCF-7 cells[END_REF]. How to define patients with intrinsic and acquired resistance respectively is not clear.

We hypothesized that candidate markers found at early progress after short-time treatment could aid in identification of patients with intrinsic resistance in whom endocrine treatment alone should be avoided up-front. With this aim, we investigated the possible impact of VEGF, VEGFR2 and p38 MAPK on early and later recurrences separately. The time limit was set to two years as all relapses before two years of follow-up occurred when patients still were on treatment.

Intratumoral levels of VEGFR2 had a significant impact on both early and later recurrences. p38 was significantly correlated with an inferior outcome among patients with early relapses also when analyses were restricted to patients with both tumors positive for both ER and PgR.

Our findings suggest the VEGFR2-p38 pathway as involved in intrinsic resistance in accordance with previous results in breast cancer cell lines [START_REF] Aesoy | An autocrine VEGF/VEGFR2 and p38 signaling loop confers resistance to 4-hydroxytamoxifen in MCF-7 breast cancer cells[END_REF][START_REF] Bunone | Activation of the unliganded estrogen receptor by EGF involves the MAP kinase pathway and direct phosphorylation[END_REF]. Expression of p38 was not correlated with later relapses, tumor stage or other breast cancer factors beside negative PgR a marker claimed to be part of intrinsic resistance [START_REF] Cornier | Decrease in estradiol-stimulated progesterone receptor production in MCF-7 cells by epidermal growth factor and possible clinical implication for paracrine-regulated breast cancer growth[END_REF][START_REF] Cui | Insulin-like growth factor-I inhibits progesterone receptor expression in breast cancer cells via the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin pathway: progesterone receptor as a potential indicator of growth factor activity in breast cancer[END_REF]. Preclinical studies in ERexpressing endometrial cancer cells have shown multiple functions of p38 of importance in tamoxifen resistance. This includes promotion of ER nuclear localization, enhancement of ER co-activator interaction, as well as, ability to increase the tamoxifen induced estrogen agonistic activity [START_REF] Lee | Regulation of estrogen receptor nuclear export by ligand-induced and p38-mediated receptor phosphorylation[END_REF]. So far, publications on p38 and other MAPK´s in clinical breast cancer populations has been scare with contradictory results. One publication supporting our findings showed that development of tamoxifen resistance during the short time period of neoadjuvant therapy was accomplished by loss of PgR, and an increase in HER2, as well as activated p38 and ERK in some patients [START_REF] Gutierrez | Molecular changes in tamoxifen-resistant breast cancer: relationship between estrogen receptor, HER-2, and p38 mitogen-activated protein kinase[END_REF]. On the contrary, two retrospective clinical studies have shown higher expression of ERK in smaller breast tumors with lower proliferation rate as well as lower incidence of nodal involvement, and a favorable outcome after adjuvant tamoxifen [START_REF] Svensson | ERK phosphorylation is linked to VEGFR2 expression and Ets-2 phosphorylation in breast cancer and is associated with tamoxifen treatment resistance and small tumours with good prognosis[END_REF][START_REF] Bergqvist | Activated ERK1/2 and phosphorylated oestrogen receptor alpha are associated with improved breast cancer survival in women treated with tamoxifen[END_REF] indicating activation of different intra cellular pathways in endocrine resistance.

The major drawback of the present study is the small patient population, above all regarding p38 analyses, resulting in few events and less power in statistical analyses, unable to fully elucidate the role of p38 in endocrine resistance. Technical aspects on p38 should also be discussed. There are four different isoforms out of which p38α and p38γ have been mostly studied and found to be of pro-apoptotic and proliferative nature, respectively. Since they appear to regulate each other the dominating one will consequently influence which pathway becomes activated [START_REF] Qi | p38alpha antagonizes p38gamma activity through c-Jun-dependent ubiquitin-proteasome pathways in regulating Ras transformation and stress response[END_REF]. The ELISA we used in our study was not constructed to discriminate between different p38 isoforms as specific antibodies at that time not were available. Our hypothesis is that the dominating isoform we detected might be p38γ that induces proliferative pathways, as well as reduce the activation of p38α and thereby also inhibiting its own degradation. Moreover, p38γ can contrary to the p38α be activated without phosphorylation, whereby it might be more likely to be preserved also in clinical samples. Despite that our sample size regarding above all p38 MAPK is small and needs to be enlarged and that we lack a randomized patient population, the results may be considered as hypothesis generating. The role of growth factor receptors as well as MAPK´s in endocrine resistance deserves to be further studied also after proper treatments periods i.e. five years, in patients subjected to newer endocrine compounds as the aromatase inhibitors, as well as in the molecular sub-types Luminal A and B.

Legends to Figures

  tamoxifen 2 years (n=241); tamoxifen 5 years (n=111); GnRH analogue alone or combined with tamoxifen (n=29). Data on clinical stage and routine breast cancer markers are shown in Table 2. The median age of patients included was 63 years (range 29 to 92) and the median follow-up time for patients alive 11.2 years. With this follow-up time a total of 86 recurrences (28 locally, 47 distant and 11 both local and distant) have been registered. The study design was approved by the research ethics board of the Karolinska Institute, Stockholm, Sweden.

Fig 1a and b

  Fig 1a and b Mann-Whitney U tests comparing levels of VEGFR2 (p=0.59) (Fig 1a) and p38

Table 1 .

 1 REMARK diagram showing how patients included in the study were selected

	↓
	679 patients

Table 2 .

 2 Clinicopathological data of steroid receptor positive patients receiving adjuvant endocrine therapy with data on VEGF (n=404), VEGFR2 (n=381) and p38 (n=174).

		VEGF	VEGFR2	p38
	Feature	number (%)	number (%)	number (%)
	Patients enrolled	404	381	174
	Tumor size
	Median	15 mm	15 mm	14mm
	T1	289 (72%)	274 (72%)	128 (74%)
	T2	107 (26%)	101 (26%)	44 (25%)
	T3	6 (2%)	6 (2%)	2 (1%)
	Lymph-node status
	Node-negative	292 (72%)	276 (72%)	123 (71%)
	Node-positive	87 (22%)	83 (22%)	37 (21%)
	Unknown	25 (6%)	22 (6%)	14 (8%)
	Histological grade
	I+II	180 (44%)	156 (41%)	75 (43%)
	III	126 (31%)	137 (36%)	57 (33%)
	Not analyzed	98 (25%)	88 (23%)	42 (24%)
	ER*
	neg	58 (14%)	52 (14%)	29 (17%)
	pos	346 (86%)	329 (86%)	145 (83%)
	PgR *
	pos	306 (76%)	289 (76%)	129 (74%)
	neg	98 (24%)	91 (24%)	45 (26%)
	HER2 positive	45 (11%)	40 (10%)	17 (10%)
	* ER = Estrogen Receptor, PgR = Progesterone Receptor, pos (>0.05 fmol/ g DNA), neg
	(<0.05 fmol/ g DNA)

Table 3 .

 3 Probability of recurrence-free (RFS) and breast cancer corrected survival (BCCS) according to VEGFR2 and p38 MAPK at 5 years follow-up

	Recurrence-free survival (RFS) in all patients
	variable	5year surv 88%CI	HR	95%CI	p-value
	VEGFR2
	0	91.3%	87-95%
	1	82.3%	77-88%	1.9	1.1-3.3	0.022
	p38MAPK
	0	86.5%	77-95%
	1	90.5%	85-96%	0.9	0.4-2.8	0.823
	Breast cancer corrected survival (BCCS) in all patients
	variable	5year surv 88%CI	HR	95%CI	p-value
	VEGFR2
	0	95.6%	93-99%
	1	91.1%	87-95%	2.3	1.05-5.6	0.038
	p38MAPK
	0	94.8%	89-100%
	1	94.4%	90-99%	1.5	0.4-5.65	0.550

Table 4 .

 4 Factors remaining statistically significant in Cox multivariate analysis for recurrencefree survival (RFS) and breast cancer corrected survival (BCCS) in the total patient population regardless of time for relapse. 95% Confidence Interval Estrogen Receptor, pos (>0.05 fmol/ g DNA), neg (<0.05 fmol/ g DNA).

	Variable	Hazard Ratio (HR)	95% CI	p-value
	RFS
	Nodal status
	Negative	1.0
	Not known	1.9	0.8-4.5	0.123
	Positive	2.9	1.9-4.6	<0.001
	Tumor size
	< 20mm	1.0
	20-50 mm	2.2	1.4-3.3	<0.001
	VEGFR2
	< median	1.0
	Not known	2.7	1.3-5.8	0.011
	median	1.8	1.1-2.8	0.016
	ER §
	Positive	1.0
	Negative	1.9	1.1-3.2	0.021
	Age
	< median	1.0
	median	1.7	1.0-2.7	0.036
	Grade
	1-2	1.0
	Not known	1.2	0.6-2.6	0.639
	3+	1.5	1.0-2.3	0.069

Funding:

This study was supported by grants from the Swedish Research Council (K2008-54X-20639-01-3), the Swedish State under the LUA-agreement (Sahlgrenska University Hospital, Gothenburg) (VGFOUREG-75911), the King Gustav V Jubilee Clinic Cancer Research Foundation, Gothenburg (2009:49), and the Gösta Milton Foundation, Stockholm, Sweden.

Declaration of interest:

None of the authors have any conflict of interest that can be perceived as prejudicing the impartiality of the research reported.

Role of the funding sources:

The founding sources were not involved in the design of the study, collection, analysis and interpretation of data, nor the writing of the manuscript or the decision to submit the manuscript for publication.