Maria A Pascual

Betlem Graupera

Lourdes Hereter

Anna Rotili

Ignacio Rodriguez

Juan Luis Alcazar

Keywords: adnexal mass, three-dimensional ultrasound, agreement, ultrasonography dimensional ultrasound: intra-inter-observer agreement adnexal mass, three-dimensional ultrasound, agreement, and ultrasonography adnexal mass, three-dimensional ultrasound, agreement, and ultrasonography 21 22

published or not. The documents may come

Intra-and inter-observer variability of 2D and 3D transvaginal sonography in the diagnosis of benign

versus malignant adnexal masses.

INTRODUCTION

Adnexal masses encompass a wide variety of ovarian disorders including benign lesions, malignant tumors and masses affecting the para-ovarian region. The use of 2D US allows discriminating benign from malignant lesions by the subjective evaluation of the gray scale ultrasound features, the so called "pattern recognition" analysis) 1-6 1-4 . However, conventional twodimensional ultrasound (2D US) has several limitations. One of its major disadvantages is that it relies on operator's ability. In fact, recent studies have shown that reproducibility and confidence in making a specific diagnosis of 2D US depends on examiner's experience 7. This technique has the potential to overcome some of the limitations of 2D US because it enables a cross-sectional imaging from data obtained by a single sweep of the US beam across the involved organ. Furthermore, with 2D US a "static" anatomic section is displayed on a video monitor while 3D US allows displaying anatomical features in planes not possible with conventional 2D US [START_REF] Alcázar | Three-dimensional sonographic morphologic assessment of adnexal masses: a reproducibility study[END_REF][START_REF] Guerriero | Diagnosis of the most frequent benign ovarian cysts: is ultrasonography accurate and reproducible?[END_REF] . [START_REF] Nj | The interobserver reliability of three-dimensional power Doppler data acquisition within the female pelvis[END_REF] Although the benefits of 3D US have been emphasized 118 , its role in assessing adnexal masses has not been elucidated and more studies are required to demonstrate if it improves differentiation between benign and malignant ovarian lesions and if it adds any real benefit compared to conventional 2D US. Notwithstanding, before to be introduced definitively in clinical practice any diagnostic method should be demonstrated as reproducible. As a matter of fact, one study has shown that 3D US seems to be reproducible for assessing adnexal masses 129 . However, to the best of our knowledge, no study has assessed whether 3D US is more reproducible or not than 2D US The purpose of our study was to compare the intra-observer and inter-observer agreement between two experienced observers using 2D US and 3D US for assigning a diagnosis of malignancy or benignity in the same set of adnexal masses.

MATERIALS AND METHODS

Although this is a retrospective study, Institutional review board approval was asked for and obtained before performing the study. All images were obtained with the same type of scanner (Voluson 730 Expert, GE Healthcare, Milwaukee, USA), equipped with a transvaginal multi-frequency (2.9-10 MHz) transducer. Each patient was first examined using conventional 2D US. Representative images from the adnexal mass were digitally stored. After 2D US was performed 3D volume box was activated and a 3D volume was acquired. 3D box was adjusted trying to include the whole adnexal mass within it.

When the volume acquisition was completed the data file was sent using a DICOM (Digital Imaging and Communication in Medicine) connection to a personal computer. Representative 2D images and 3D volumes were selected by one investigator (BG) to be analyzed by two other observers (MAP, LH) who were blinded to 2D-image and 3D-volume selection. All selected 2D images were retrieved from the database and showed without the possibility of being modified, whereas the 3D stored volumes were analyzed off-line in a dedicated workstation by using multiplanar reformatting with the 4D-view™ software, version 5.0 (GE Healthcare, Milwaukee,

USA).

Two observers A and B (MAP and LH with, 25 and 20 years experience of gynecologic US, respectively) were required to analyze 2D images in a random order. One week after 2D images review they reviewed 3D volumes in a different order of cases than 2D images were to avoid recall bias (referencia). The observers were blinded to each other's findings, they were unaware of the diagnostic report. They were also unaware of the physical examination's findings and histopathology results. The only information they had was the age and hormonal status of the patient. For "static" 2D images only one representative gray-scale sonogram was available. 3D volumes were analyzed using multiplanar rendering (Figures 1 and2). According to patter recognition the observer had to provide a diagnosis of benignity or malignancy. Criteria for each type of lesion were defined in previous publications [START_REF] Guerriero | Diagnosis of the most frequent benign ovarian cysts: is ultrasonography accurate and reproducible?[END_REF] Patients with suspected functional and hemorrhagic cysts were managed with follow-up scans at three and six months until spontaneous resolution. Patients with persistent or suspicious masses were submitted to surgery and mass removal with pathological confirmation (gold standard).

Statistical analysis was performed using the SAS 9.1 Institute Inc Software (Cary, NC, USA.). The intra-observer and inter-observer agreement analysis for classifying the mass as benign or malignant were evaluated with Cohen's kappa index (κ) at the 95% confidence interval (CI). A kappa value of <0 indicates no agreement, ≤ 0.20 indicates poor agreement, 0.21-0.39 indicates fair agreement, 0.40-0.59 indicates moderate agreement, 0.60-0-79 indicates substantial agreement, and 0.80-1.00 indicates almost perfect agreement [START_REF] Pascual | Role of Color Doppler Ultrasonography in the Diagnosis of Endometriotic Cyst[END_REF][START_REF] Pascual | Borderline cystic tumors of the ovary: Gray-scale and color Doppler sonographic findings[END_REF] . 11,12 .

Intra-observer agreement refers to the agreement between 2D US and 3D US diagnosis of each examiner. Inter-observer agreement refers to the agreement between examiners when providing a diagnosis using 2D US or 3D US. It was considered that an statistical significant existed (p value less 0.05) when 95% CI of kappa values did not overlap.

RESULTS

The mean age of patients was 38.8 ±8.3. Among the 41 adnexal masses included in the study, there were 34 benign masses and 7 malignancies. Table 1 summarizes the histological diagnoses.

Thirty-one patients underwent surgery with a histopathology result. Ten patients with a diagnosis of functional or hemorrhagic cyst were followed up with 2DUS at three and six months that showed a resolution of the lesion. The intra-observer agreement between 2D US and 3D US was of 0.96 (95% CI: 0.91-1.0) for observer A and of 0.77 (95% CI: 0.60-0.99) for observer B.

The intra-observer agreement between 2D US and 3D US was of 1.00 (95% CI: 1.00-1.00) for observer A and of 0.69 (95% CI: 0.35-1.00) for observer B, respectively. (Tables 3 and4)

The inter-observer agreement for 2D US was 0.73 (95% CI: 0.57-0.89) and 0.92 (95% CI: 0.84-0.99) for 3D US.

The inter-observer agreement for 2D US was 0.69 (95% CI: 0.35-1.00) and 1.00 (95% CI: 1.00-1.00) for 3D US (Tables 5 and6). This difference was not considered statistically significant.

Discussion

The diagnostic efficacy of conventional 2D US in the diagnosis of adnexal masses is well documented 19,20 13 .However, this technique is highly operator-dependent and based mainly on the subjective assessment of mass characteristics. In spite of this fact, few studies have addressed the issue of reproducibility of this technique for classifying adnexal masses as benign or malignant.

Timmerman et al showed that 2D US is reproducible among observers for providing a diagnosis of malignancy or benignity in adnexal masses 21 14 .However, in this study anamnestic data and color Doppler findings were also included in the evaluation and final diagnosis. Guerriero et al demonstrated that 2S US was reproducible among experienced examiners for diagnosing ovarian cancer 75 and for assigning specific diagnosis of some benign ovarian cysts [START_REF] Valentin | Use of morphology to characterize and manage common adnexal masses[END_REF] . [START_REF] Guerriero | Diagnosis of the most frequent benign ovarian cysts: is ultrasonography accurate and reproducible?[END_REF] Similar results have bee recently reported by Yazbek et al 86 . This latter study also emphasized that the level Three-dimensional ultrasound allows the storage of a 3D volume of the area of interest, in this case an adnexal mass, and an increased number of post-processing imaging tools, not available for 2D static images, that could provide a more accurate anatomic evaluation 23,24 8 To date, no previous study has compared the reproducibility of 2D and 3D ultrasound in evaluating adnexal masses. In the present study we have analyzed the intra-observer and interobserver agreement between two experienced observers using 2D US and 3D US in making a diagnosis of malignancy or benignity in adnexal masses. Analyzing intra-observer agreement we could conclude that 2D US and 3D US evaluation of adnexal masses by the same examiner provide similar diagnostic information in most of the cases when applying same criteria for malignancy suspicion in both techniques. This may be explained by the fact that both examiners are highly experienced in gynecologic ultrasound and adnexal masses evaluation. As a matter of fact, this may be a bias in our study, because intra-observer reproducibility could be lower in less experienced examiners, as shown by Yazbek et al 8 6 On the other hand, our data show that the agreement between observers with respect to the characterization of an adnexal mass using 2D US images is slightly inferior to that attained with 3D US data. We explained this result because 3D technique allows the possibility to elaborate the acquired volumes that may display better the pathologic features of the lesion. We also believe that the present study may be biased against 2D technique since real time scan is better that static image evaluation for predicting malignancy in adnexal masses [START_REF] Maymon | Threedimensional vaginal sonography in obstetrics and gynaecology[END_REF] In conclusion, our data suggest that 3D US is slightly more reproducible, but not significantly, than 2D US. These findings, although need to be validated with further investigations, seem to be relevant in order to suggest the use of 3D US in routine clinical practice for the evaluation of adnexal masses. The purpose of this study was to compare the intra-observer and inter-observer agreement in 3 assigning a diagnosis of malignancy or benignity to adnexal masses using 2-dimensional 4 ultrasonography (2D US) and 3-dimensional ultrasonography (3D US). 5

Methods 6

Two experienced observers (Observers A and B) performed a retrospective review of digitally 7 stored 2D images and 3D data from a sample of 41 consecutive patients with a diagnosis of adnexal 8 mass. Each observer independently, and blinded each other, evaluated the 2D static images of each 9 adnexal mass and then the 3D volumes one-week later. The observers were required to classify the 10 adnexal lesion as benign or malignant according to pattern recognition analysis. Intra-observer and 11 inter-observer agreement were assessed by calculating the kappa index (κ). 12

Results 13

Intra-observer agreement between 2DUS and 3DUS for the observer A was 1.00 and for the 14 observer B was 0.69. Inter-observer agreement was 0.69 for 2D US and 1.00 for 3D US (p >0.05) 15

Conclusions 16

Our results indicate that 3D US for assessing adnexal masses is reproducible, but not significantly, 17 than 2D US. 18

Running Head: Three and Two-dimensional ultrasound: intra-inter-observer agreement 19

INTRODUCTION 23

Adnexal masses encompass a wide variety of ovarian disorders including benign lesions, 24 malignant tumors and masses affecting the para-ovarian region. The use of 2D US allows 25 discriminating benign from malignant lesions by the subjective evaluation of the gray scale 26 ultrasound features, the so called "pattern recognition" analysis) 1-4 . However, conventional two-27 dimensional ultrasound (2D US) has several limitations. One of its major disadvantages is that it 28 relies on operator's ability. In fact, recent studies have shown that reproducibility and confidence in 29 making a specific diagnosis of 2D US depends on examiner's experience 5, [START_REF] Yazbek | Confidence of expert ultrasound operators in making a diagnosis of adnexal tumor: effect on diagnostic accuracy and interobserver agreement[END_REF] . 30

Three-dimensional ultrasound (3D US) has recently become available for clinical practice. 31

This technique has the potential to overcome some of the limitations of 2D US because it enables a 32 cross-sectional imaging from data obtained by a single sweep of the US beam across the involved 33 organ. Furthermore, with 2D US a "static" anatomic section is displayed on a video monitor while 34 3D US allows displaying anatomical features in planes not possible with conventional 2D US [START_REF] Nj | The interobserver reliability of three-dimensional power Doppler data acquisition within the female pelvis[END_REF] . 35

Although the benefits of 3D US have been emphasized 8 , its role in assessing adnexal masses has not 36 been elucidated and more studies are required to demonstrate if it improves differentiation between 37 benign and malignant ovarian lesions and if it adds any real benefit compared to conventional 2D 38 US. Notwithstanding, before to be introduced definitively in clinical practice any diagnostic method 39 should be demonstrated as reproducible. As a matter of fact, one study has shown that 3D US seems 40 to be reproducible for assessing adnexal masses [START_REF] Alcázar | Three-dimensional sonographic morphologic assessment of adnexal masses: a reproducibility study[END_REF] . However, to the best of our knowledge, no study 41 has assessed whether 3D US is more reproducible or not than 2D US 42 The purpose of our study was to compare the intra-observer and inter-observer agreement 43 between two experienced observers using 2D US and 3D US for assigning a diagnosis of 44 malignancy or benignity in the same set of adnexal masses. respectively) were required to analyze 2D images in a random order. One week after 2D images 68 review they reviewed 3D volumes in a different order of cases than 2D images were to avoid recall 69 bias (referencia). The observers were blinded to each other's findings, they were unaware of the 70 diagnostic report. They were also unaware of the physical examination's findings and 71 histopathology results. The only information they had was the age and hormonal status of the 72 patient. For "static" 2D images only one representative gray-scale sonogram was available. 3D 73 volumes were analyzed using multiplanar rendering (Figures 1 and2). According to pattern 74 recognition the observer had to provide a diagnosis of benignity or malignancy. Criteria for each 75 type of lesion were defined in previous publications 5, [START_REF] Guerriero | Diagnosis of the most frequent benign ovarian cysts: is ultrasonography accurate and reproducible?[END_REF]

76

Patients with suspected functional and hemorrhagic cysts were managed with follow-up scans 77 at three and six months until spontaneous resolution. Patients with persistent or suspicious masses 78 were submitted to surgery and mass removal with pathological confirmation (gold standard). 79

Statistical analysis was performed using the SAS 9.1 Institute Inc Software (Cary, NC, 80 USA.). The intra-observer and inter-observer agreement analysis for classifying the mass as benign 81 or malignant were evaluated with Cohen's kappa index (κ) at the 95% confidence interval (CI). A 82 The mean age of patients was 38.8 ±8.3. Among the 41 adnexal masses included in the study, 91 there were 34 benign masses and 7 malignancies. Table 1 summarizes the histological diagnoses. 92

kappa
Thirty-one patients underwent surgery with a histopathology result. Ten patients with a 93 diagnosis of functional or hemorrhagic cyst were followed up with 2DUS at three and six months 94 that showed a resolution of the lesion. Table 2 summarizes the agreement between histopathologic 95

result and both observers (A and B) 96

The intra-observer agreement between 2D US and 3D US was of 1.00 (95% CI: 1.00-1.00) for 97 observer A and of 0.69 (95% CI: 0.35-1.00) for observer B, respectively. (Tables 3 and4) 98

The inter-observer agreement for 2D US was 0.69 (95% CI: 0.35-1.00) and 1.00 (95% CI: 99 1.00-1.00) for 3D US (Tables 5 and6). This difference was not considered statistically significant.

Discussion

The diagnostic efficacy of conventional 2D US in the diagnosis of adnexal masses is well documented 13 . However, this technique is highly operator-dependent and based mainly on the subjective assessment of mass characteristics. In spite of this fact, few studies have addressed the issue of reproducibility of this technique for classifying adnexal masses as benign or malignant.

Timmerman et al showed that 2D US is reproducible among observers for providing a diagnosis of malignancy or benignity in adnexal masses 14 . However, in this study anamnestic data and color Doppler findings were also included in the evaluation and final diagnosis. Guerriero et al demonstrated that 2S US was reproducible among experienced examiners for diagnosing ovarian cancer 5 and for assigning specific diagnosis of some benign ovarian cysts [START_REF] Guerriero | Diagnosis of the most frequent benign ovarian cysts: is ultrasonography accurate and reproducible?[END_REF] . Similar results have bee recently reported by Yazbek et al [START_REF] Yazbek | Confidence of expert ultrasound operators in making a diagnosis of adnexal tumor: effect on diagnostic accuracy and interobserver agreement[END_REF] . This latter study also emphasized that the level of confidence for providing a diagnosis affects agreement among observers. All these studies used static 2D images for analyzing intra-and inter-observer agreement. observers with different level of expertise on 3D ultrasound. However, intra-observer agreement was higher for experienced examiner 9 . Three-dimensional ultrasound allows the storage of a 3D volume of the area of interest, in this case an adnexal mass, and an increased number of post-processing imaging tools, not available for 2D static images, that could provide a more accurate anatomic evaluation 8 .

To date, no previous study has compared the reproducibility of 2D and 3D ultrasound in evaluating adnexal masses. In the present study we have analyzed the intra-observer and interobserver agreement between two experienced observers using 2D US and 3D US in making a diagnosis of malignancy or benignity in adnexal masses. Analyzing intra-observer agreement we could conclude that 2D US and 3D US evaluation of adnexal masses by the same examiner provide similar diagnostic information in most of the cases when applying same criteria for malignancy suspicion in both techniques. This may be explained by the fact that both examiners are highly experienced in gynecologic ultrasound and adnexal masses evaluation. As a matter of fact, this may be a bias in our study, because intra-observer reproducibility could be lower in less experienced examiners, as shown by Yazbek et al [START_REF] Yazbek | Confidence of expert ultrasound operators in making a diagnosis of adnexal tumor: effect on diagnostic accuracy and interobserver agreement[END_REF] On the other hand, our data show that the agreement between observers with respect to the characterization of an adnexal mass using 2D US images is slightly inferior to that attained with 3D US data. We explained this result because 3D technique allows the possibility to elaborate the acquired volumes that may display better the pathologic features of the lesion. We also believe that the present study may be biased against 2D technique since real time scan is better that static image evaluation for predicting malignancy in adnexal masses 15 In conclusion, our data suggest that 3D US is slightly more reproducible, but not significantly, than 2D US. These findings, although need to be validated with further investigations, seem to be relevant in order to suggest the use of 3D US in routine clinical practice for the evaluation of adnexal masses. Has not been correlated.

when reviewing 3DUS was 2DUS available ? This would occur in the reality

The reviewers analyzed 3DUS cases without knowledge of the diagnosis of the lesion in 2DUS or access to the 2D images. 6. did reviewers report yes or no, or did they report on a scale (e.g. 1 to 5) with further grouping ? The latter would allow for studying different cut offs We evaluated two options: Benign/Malignant 7. A table with final outcome (cancer = 7 -no cancer = 34) listing the original and the reviewers' 2D and 3D report should be provided: the less reproducible the original report, the less reliable is the review scenario. With the limitation of the considered series sensitivity and PPV (orginal, review A and B, 2D and 3D shoudl be provided) Table 2 is added , and text in Results (page 5). 8. statistical analysis of the significance of interobserver reproducibility in2D and 3D shoudl be provided, given the small sample size The statistical significance of interobserver reproducibility has been evaluated with the CI of 95% k values (page 5). Reviewer 3 1. The conclusion in the abstract states "... 3D US ... is reproducible, even more than 2D US." This is repeated at the end of the paper. The authors do not show a statistically significant difference in the kappas and the confidence intervals reported overlap. Thus, we cannot conclude that 3D is more reproducible. Abstract (page 1) and Discussion ( page 6 ) has been amended according to the observation 2. Please present the tables showing the data from which the kappas were computed.

Tables 3456has been added, and text in Results (pages 5-6).

Replay to the Editor:

We have made changes according to the suggestions

2 2

 2 Three-dimensional ultrasound (3D US) has recently become available for clinical practice.

A

  retrospective review of transvaginal sonograms obtained between May 2006 and January 2007 and stored in the PACS at our institution from 41 consecutive patients diagnosed as having an adnexal mass evaluated and treated at our institution was performed. Patients with ectopic pregnancy, lack of 3D volume, absence of surgery or ultrasound follow-up and an unquestionable ultrasound diagnosis of tubal disorder were excluded.

  providing a diagnosis affects agreement among observers. All these studies used static 2D images for analyzing intra-and inter-observer agreement.Regarding 3D US, only one study has assessed the reproducibility of the technique. Alcazar et al showed that evaluation of 3D volumes from adnexal masses is reproducible among two different observers with different level of expertise on 3D ultrasound. However, intra-observer agreement was higher for experienced examiner 129 .

15

 15

  the diagnosis of ovarian cancer. Ultrasound Med Biol. 2008;34:1711-6 6 Yazbek J, Ameye L, Testa AC, Valentin L, Timmerman D, Holland TK, Van Holsbeke C, Jurkovic D. Confidence of expert ultrasound operators in making a diagnosis of adnexal tumor: effect on diagnostic accuracy and interobserver agreement. Ultrasound Obstet Gynecol. 2010;35:89-93. 7 Raine-Fenning NJ, Campbell BK, Clewes JS, Kendall NR, Johnson IR. The interobserver reliability of three-dimensional power Doppler data acquisition within the female pelvis. Ultrasound Obstet Gynecol 2004;23:501-508. 8 Alcazar JL, Rodriguez D, Royo P, Galvan R, Ajossa S, Guerriero S. Intraobserver and Interobserver reproducibility of 3-Dimensional power Doppler ultrasound indices in assessment of solid and cystic-solid adnexal masses. J Ultrasound Med 2008;27:16. 8 Coyne L, Jayaprakasan K, Raine-Fenning N. 3D ultrasound in gynecology and reproductive medicine. Women's Health (Lond Engl). 2008 Sep;4(5):501-16. 9 Alcazar JL, Garcia-Manero M, Galvan R. Three-dimensional sonographic morphologic assessment of adnexal masses. J Ultrasound Med 2007;26:1007-1011. 10 Pascual MA, Carreras O, Hereter L, Gomez MT, Barri PN. Diagnóstico ecográfico del quiste dermoide de ovario. Prog Obst Gin 1993;36:508-512. 10Guerriero S, Alcazar JL, Pascual MA, Ajossa S, Gerada M, Bargellini R, Virgilio B, Melis GB. Diagnosis of the most frequent benign ovarian cysts: is ultrasonography accurate and reproducible? J Womens Health. 2009 Apr;18(4):519-27. 11 Cohen J. Weighted kappa: Nominal scale agreement with provision for scaled disagreement or parcial credit. Psychol Bull 1968;70:213-220.

3 3

 3 Two observers, A and B (MAP and LH with, 25 and 20 years experience of gynecologic US, 67

  value of <0 indicates no agreement, ≤ 0.20 indicates poor agreement, 0.21-0.39 indicates fair 83 agreement, 0.40-0.59 indicates moderate agreement, 0.60-0-79 indicates substantial agreement, and 84 0.80-1.00 indicates almost perfect agreement 11,[START_REF] Landis | The measurement of observer agreement for categorical data[END_REF] . 85 Intra-observer agreement refers to the agreement between 2D US and 3D US diagnosis of 86 each examiner. Inter-observer agreement refers to the agreement between examiners when 87 providing a diagnosis using 2D US or 3D US. It was considered that an statistical significant existed 88 (p value less 0.05) when 95% CI of kappa values did not overlap. 89

  Regarding 3D US, only one study has assessed the reproducibility of the technique. Alcazar et al showed that evaluation of 3D volumes from adnexal masses is reproducible among two different

Figure 1b .

 1b Figure 1a. Transvaginal 2DUS showing a septate complex cyst mass, in a 48-years old women. 169x116mm (120 x 120 DPI)

Table 2 summarizes the agreement between histopathologic result and both observers (A and B)

	5
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	5

Page 20 of 36 John Wiley & Sons Journal of Clinical Ultrasound

	1		7 8
	2
	3 4	187	the preoperative assessment of adnexal masses. Ultrasound Obstet Gynecol.
	5 6	188	2008;32:828-31.
	7
	8 F o r F o r F o r F o r
			P P P P
			e e e e
			e r e r e r e r
			R R R R
			e e e e
			v i e v i e v i e v i e
			w w w w

Table 1 .

 1 Histologic diagnosis of adnexal masses.

	Final diagnosis	N	%
	Dermoid cyst	5	12.20
	Endometrioma	9	21.95
	Ovarian fybroma	3	7.32
	Hemorrhagic cyst	5	12.20
	Mucinous cystoadenoma	4	9.76
	Malignant tumor (Borderline tumors)	7	17.07
	Paraovarian cyst	3	7.32
	Functional cyst	5	12.20

Table 2 .

 2 Histopathologic result and 2D/3D-US diagnostic provided by both observers

	(A and B)
		A-2D		A-3D		B-2D		B-3D
		M	B	M	B	M	B	M	B
	Malignant (M)	7	0	7	0	4	3	7	0
	Benign (B)	0	34	0	34	0	34	0	34

Table 3 .

 3 Intra-observer agreement between 2D-US and 3D-US for examiner A

				3D-US
			Benign	Malignant
	2D-US	Benign	34	0
		Malignant	0	7

Table 4 .

 4 Intra-observer agreement between 2D-US and 3D-US for examiner B

	3D-US
	Benign	Malignant

Table 5 .

 5 Inter-observer agreement for 2D-US between examiners

	Examiner A
	Benign	Malignant

Table 6 .

 6 Inter-observer agreement for 3D-US between examiners

	Examiner A
	Benign	Malignant

limitation of the study. but unfortunately we cannot relate our data set with a tumor registry

  2. had the two reviewers seen the study cases before ? Strange and peculiar US features could have been remembered…It

may be that some of the cases had been diagnosed by one of the reviewers, however the paper material is from the years 2006-2007 and were reviewed for the paper in 2009

  .3. had 3DUS been used for reporting in the reality or was only considered for review ? Some

pathologies are currently performed off-line 3DUS report. In the period in which these ovarian lesions were diagnosed (2006-2007) 3DUS report was not implemented in our Unit.

	4. It would be interesting to know how the reviewer's report correlated with the
	original. It's the original we are interested in (with other clinical information,
	knowledge of Ca125….)

Journal of Clinical Ultrasound

Acknowledgements

The authors are grateful to Mrs Natalia Garrofé for her secretarial support.

MATERIALS AND METHODS 46

Although this is a retrospective study, Institutional review board approval was asked for and 47 obtained before performing the study. 48


M Mihalkovič
email: mihalkovic@savba.sk

M Widom

C L Henley

Cell-constrained melt-quench simulation of d-AlCoNi: Ni-rich versus Co-rich structures

Keywords: modelling, molecular dynamic simulations, Monte-Carlo, quasicrystalline alloys molecular dynamics, effective Hamiltonian, Al-TM, ab-initio energies 1. introduction

Complex crystalline ground state structures may be obtained by direct (simulated) quenches from the melt for systems of up to a few hundred atoms, given the constraint of a fixed unit cell, coupled with use of (i) replica-exchange Monte Carlo and (ii) realistic empirical interaction pair potentials. We applied this to decagonal approximants of Al 72 Co 8 Ni 20 and Al 73 Co 27 , obtaining the best energies seen to date for d-AlCoNi models (respectively +20 meV/atom and +9 meV/atom above the tie-plane of competing phases). We elucidated the reasons that different decagonal structures are associated with the Ni-rich and Co-rich compositions. We find a cell doubling to c=8 Å due to layer puckering not only in the Co-rich structure, but (locally) in the nominally c = 4 Å Ni-rich structure.

This work is part of an effort to understand the structure and stability of decagonal d-AlNiCo, the highest quality decagonal quasicrystal. Due to the complexity of the structure, and averaging-over-disorder effects, even good diffraction datasets [1,2] cannot reliably provide the precise structure -not well enough to predict the well-known fact that d-AlCoNi is thermodynamically stable with respect to the competing crystal phases owing to their excess energy relative to competing crystals.

Previous simulation studies derived low-energy structural models on the Nirich [3,4] and Co-rich [5][6][7] compositions, or on the whole range [8] by first Monte Carlo annealing a lattice-gas on discrete sites. (augmented by tile-reshuffling moves). The prerequisites for that method were (i) realistic pair potentials; (ii) "minimal experimental input" in the form of the layer spacing and quasilattice constant (i.e tile size); (iii) most importantly the fact of layering, and the assumption of near-ideal candidate atomic positions.

In agreement with experiment, these studies arrived at similar but distinct structure types in the Ni-rich and Co-rich cases, which we will call "Ni type" and "Co type" for short. The Ni-type structure is based on hexagon/boat/star HBS) tiles (at a hierarchy of length scales [3]) and, according to experiment, has a stacking period of 4 Åalong the 10-fold axis; the HBS tiles group into (possibly overlapping) decagonal clusters with diameter 20 Å, having central motifs with (only) mirror symmetry, appearing as triangles of TM atoms in high-resolution electron microscope images. The Co-type structure, although it contains HBS tiles also, is dominated by 20 Å non-overlapping decagon clusters which we shall call "pentagonal decagons" (PD). Other prominent features are clusters similar to pentagonal bipyramids (PB), centered on the perimeter of the 20 Å decagons and forming columns in which some Al atoms pucker out of the layers in alternate directions, causing a cell doubling to c =8 Å in the stacking direction.

We believe the key reason for the excess energy is the displacement of certain Al atoms pucker out of the main atom layers, called "puckering". This is explained in terms of "channels": [4][5][6] linear potential wells for Al atoms that are aligned transverse to the atom layers, located between two columns of TM atoms. The Al atoms, when packed tightly into the channels, must be spaced ∼2.6 Å, i.e. 1.3 layers apart,so that three Al atoms fit in four layers. Hence locally in any channel, the stacking period gets doubled from two to four layers; only one of the three atoms aligns with a layer, which becomes the local mirror plane of that channel. Puckerings occur, as a form of local order, even in alloys whose average structure is just two layers. Consequently, diffuse scattering [9][10][START_REF] Frey | Disorder in decagonal quasicrystals[END_REF][START_REF] Kobas | Structural disorder in the decagonal Al-Co-Ni. I. Patterson analysis of diffuse x-ray scattering data[END_REF] is observed corresponding to the 4-layer local order, and this diffuse scattering shows elaborate modulations within the 10-fold layer due to correlations between the phases of puckering in nearby "channels". Thus a successful understanding of those correlations will also give the first theory for this diffuse scattering.

Structure models without puckering at all are many tens of meV/atom too high in energy. Allowing puckering locally can capture half of this energy but is still not sufficient to obtain the lowest energies. For that, we must incorporate the correct correlations between the signs of puckering of Al atoms in nearby channels. since these atoms interact strongly. (A weakness of our past approach [3,5] is that the discrete site list used initially does not incorporate puckering displacements.) No simple recipe is known for the global puckering pattern. Note that d-AlNiCo has a well-known zoo of structural modifications [19] which depend on tiny differences in composition, and (we suspect) go with different puckering patterns.

Cell-constrained melt-quench method

One of the most challenging tasks of contemporary material science, and the basic question in our studies of quasicrystal structures is: given a composition, what is the optimum structure? A general method was put forward using ideas of genetic algorithms and including variation of the cell, as implemented in the program US-PEX [13], but apparently this cannot yet handle systems with complex structure.

We have developed an approach we call "cell-constrained melt-quench" which is surprisingly successful at predicting low-temperature structures of complex alloys with hundred(s) of atoms per cell. This simply means cooling the system under brute-force molecular dynamics using periodic B.C.'s in a small cell, while fixing the composition, atomic density, and the unit cell of the system to the experimentally known values. Quite often those are available experimentally from electron diffraction or powder diffraction, when single-crystal structure refinement is impossible. The "cell constraint" seems to limit the ensemble of possible states so we can shepherd the system to the global energy minimum.

One's expectation is that, in a multi-species system described by oscillating in- AlCoNi from DOE08 3

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teratomic potentials [3], such a quench should get caught in a glassy or highly defective metastable state rather than the true energy minimum. But we found it was sufficient to apply the trick of "replica exchange" ("parallel tempering") [14], wherein a set of simulations is run, each at a different temperature, and the entire configuration may be exchanged between temperatures according to a Metropolis step. (Note that within each loop, we perform not only MD but also MC steps that swap the species of two atoms.) Of course, it is also crucial to have realistic pair potentials. These may be constructed systematically (as with Moriarty's GPT potentials [26] which we used in this project). Alternatively, for Al-transition metal (Al-TM) and many other alloys, one does just as well with empirical potentials including Friedel oscillations that can quickly be fitted to an appropriate database of ab-initio energies and forces. [START_REF] Mihalkovič | Empirical oscillating potentials for alloys from ab-initio fits[END_REF] Empirical potentials (specified by a few parameters) may be fitted to data sets of ab-initio forces and energies. Alternatively, analytical potentials are available for certain systems, in particular GPT potentials [26] were developed for Al-rich Al-TM systems.

The general philosophy of this method -taking advantage of lattice and composition information to moderately constrain the simulation -also inspired our earlier work based on quasi lattice-gases [3,7]. In the present approach, we need fewer assumptions , and the method directly finds good answers for the (intricate) patterns of atoms deviating out of the layers, which turns out to be the crucial unresolved aspect of the structure model.

The cell-constrained method has been successful for a variety of intermetallics [START_REF] Mihalkovič | [END_REF]. For example, in the i-CaCd family of structures, this method successfully gave the starting point for a Rietveld refinement of the "2/1-1/0-1/0" AlCuSc approximant [15].

Set up for Al-Co-Ni melt-quench study

Our simulations used the realistic GPT pair potentials [26] as we used before [3,5,7]. The cell sizes for decagonal approximants are all based on tilings with lattice constants a q = 2.44 Å in-plane, and c = 4.08 Å for the minimum stacking period.

The melt-quench (MQ) simulation was set up with 20 annealing temperatures distributed between 1000K-2500K. One tempering cycle consisted of 1000 MD steps with time increment ∆T =0.2 fs, followed by a Metropolis Monte Carlo (MC) loop of 7800 attempted species swaps (the acceptance rate was 0.03-0.05). Typically, for a system of this size the simulation needs ∼100 steps until first metastable lowenergy configurations appear. Occasionally, we take the current lowest-temperature sample and quench it to monitor the energy of the underlying zero-temperature state.

To validate our method, we first verified that cell-constrained quenching with GPT potentials [26] reproduced all Al-rich binary structures in the Al-Co and Al-Ni systems, as well as the ternary phase X-AlCoNi.

With Al-Ni, we verified that 12Al+4Ni in the orthorhombic unit cell of Al 3 Ni (16 atoms 6.6 × 7.4 × 4.8 Å) easily reproduced that structure after just 10 loops. On the Co-rich side, the essential test was the orthorhombic Al 13 Co 4 phase; the monoclinic (fully occupied) version m-Al 13 Co 4 with 51 atoms per primitive cell was exactly recovered after 119 loops. We also reproduced Al 9 Co 2 .

Finally, we checked the ternary X(AlCoNi) phase [23] which has ratio 18:5:3 Al:Co:Ni in a monoclinic unit cell: the structure was observed after 71 loops. The longest of the test simulations (m-Al 13 Co 4 ) needed just a few minutes on a standard quad-core PC, demonstrating the efficiency of the method for system sizes up to ∼ 50 atoms per simulation box.

First we show the results of simulations in a larger unit cell we call the "2B+H cell" since it accommodates two Boat tiles and one Hexagon tile of edge 6.5 Å per primitive cell; the cell is centered orthorhombic with cell parameters (in a monoclinic setting) a = b = 19.78 Å, γ=108 • , and c = 8.06 Å. With composition Al 0.75 Co 0.25 , we obtain a decagonal approximant [Fig. 1(a)] On the other hand, using our standard Ni-rich composition Al 148 Co 16 Ni 41 in this cell, we obtain the B2 structure [Fig. 1(b)].

The chief technical hindrance to obtaining decagonal approximants is competition from the B2 phase (Fig. 1(b), which has ideal composition Al(Co,Ni) in which Co and Ni substitute each other. Since B2 has a small unit cell, it fits with moderate distortion into almost every one of our simulation cells. B2 is a thermodynamically stable phase at high temperatures, and is probably more accessible than the desired phases. We ascribe both properties to the very large configurational entropy of Al atoms substituting on TM sites (as necessitated by our Al-rich compositions.)

Due to their unrealistically deep Al-TM nearest-neighbor wells, the GPT pair potentials exaggerate the stability of B2. Still, it is known experimentally that B2-Al(Co,Ni) (with TM site disorder) does coexist with decagonal phases in AlCoNi samples carefully annealed at high T [START_REF] Grushko | [END_REF].

Results

To avoid formation of the spurious B2 phase, we adopted a simulation cell smaller in linear dimensions by a factor τ ≡ (1 + √ 5)/2 ≈1.618 that we believe matches the B2 phase poorly. It is centered orthorhombic with lattice constant a = b = 12.3 Å, γ = 108 • in monoclinic setting, and c = 8.20 Å (so the layer spacing is 2.05 Å). Each primitive cell accommodates one τ 2 -inflated Boat tile of edge 6.4 Å.

[In orthorhombic setting the cell is a=14.36 Å, b=19.69 Å and c=8. [START_REF] Mihalkovič | Empirical oscillating potentials for alloys from ab-initio fits[END_REF] Atomic density is a key parameter; experimentally reported values in d-AlCoNi vary from 0.066 atoms/ Å3 according to [22] up to 0.070 atoms/ Å3 (see References in [21]). Our runs explored this whole range which corresponds to 77-80 atoms per primitive cell. We call a structure "high density" when it has 58 or 57 Al atoms in the "Boat" cell (see Fig. 2)

As expected, we could obtain both Ni-type and Co-type decagonal structures in different compositions. What was unclear a priori was (i) are these two discrete possibilities, or is there a gradual continuum of types interpolating between them? (ii) what determines which of the types is seen? For our chosen Ni-rich composition, depending whether we used a low or high atom density, we obtained either the Ni-type structure or the Co-type structure, corresponding to the the respective low and high densities of these phases. (Specifically, the Co-type structure occurred with 58 or 57 Al atoms/cell.) By contrast, for Al-Co at any density (and composition), the optimum structure (as found by the simulation) is always Co-type. If a lower density is used, Al-Co will incorporate vacancies, rather than switch to the lower-density Ni-type structure.

We find that only the discrete Ni-type and Co-type optimal low-temperature structures are found. A prominent motif found in both structure types is the "Dring" of diameter 12 Å, consisting of ten TM atoms alternating from one layer to the other, spaced by 4.0 Å in-layer, or actually 4.5 Å between atoms (which is very favorable for the TM-TM potentials). Remarkably, D-rings have even been observed superimposed on the disordered B2 type structure It is likely that the D-rings form at temperatures where the Al atoms are still relatively disordered (we know from other simulation studies that TM-TM order precedes Al order [28].

The Co-type structure seemed essentially unique, since (with long annealing times) several independent quench runs arrived at the identical sites (apart from some variation between Ni and Co on TM sites.). On the other hand, annealing the Ni-type structure gave a different HBS tilings every time. What was robust in the Ni-type structure was the (often overlapping) D-rings.

Puckering

We find not only Co-rich but Ni-rich structures are locally 8 Å periodic. A convenient summary of the puckering behavior is seen in the distribution function of the atoms' z coordinates, as shown by insets in each figure .  The Co-type structures all contain four 2 Å layers: two perfectly flat (mirror) layers alternating with two puckered layers. The reasons for this kind of ordering were laid out in Ref. [6]; a long range order can propagate because there is a well connected network of channels across the system. The Al 58 Ni 22 sample (in a Co-type structure) shows the same puckering behavior as the Co-rich sample.

By contrast, in the Ni-type structures found at lower atomic densities all four layers have some puckered atoms: Fig. 2(a), inset). That means each layer has a mixed nature: for some "channels" it is a (local) mirror layer, but for others it is a (local) puckered layer. Presumably the Ni-type system has statistical 4 Å period despite its local 8 Å ordering, because the puckered motifs are rarer and less correlated.

In both types of decagonal structures, one finds certain columns of TM atoms surrounded by five neighboring TM columns 4 Å away. In the Ni-type structure this happens at the center of every Star tile of the small HBS tiling in Fig. 2(a). In the Co-rich type structure [6], it occurs on every other vertex of the D-ring which bounds the 12 Å Decagon (Fig. 2(b). Such columns are surrounded by five "channels", which have strongly correlated puckering patterns due to steric constraints. In Ref. [6] we identified a characteristic motif of such "puckering centers", called the "crooked cross", and it is in fact visible in every puckering center in Figs. 1(a in the well-known Al 13 Co 4 structure. Some puckered atoms are an intrinsic part of the "pentagonal bipyramid" motif [20,21]; on top of the same cluster there is an atom pair, as in a "crooked cross", but the other arm of the cross is missing, because an Al atom appears right on the local 5-fold axis at the same z (a mirror-layer) that the second two atoms would have been (and excludes them sterically).

To demonstrate how essential the local puckering is, we ran the same Ni-rich composition while replacing the 8 Å cell constraint by 4 Å. The resulting structure contains no clearly pentagonal motifs; instead, it somewhat resembles the 4 Å periodic phase X(AlCoNi) [23]. Although its pair-potential energy is practically degenerate with the (8 Å) decagonal's , we never observed the 4 Å phase in the 8 Å box: presumably its entropy is too small.

Ab-initio calculations of phase stability

Having obtained structures from cell-constrained simulations with pair potentials, we check their (relaxed) energy a posteriori using the ab initio program VASP [27]. Our criterion for stability is a zero-temperature energy of at most 10 meV/atom above the tie-plane of other phases. Note that a small excess energy is often compensated in quasicrystals and approximants by a higher vibrational entropy not found in other alloy structures, meaning the quasicrystal, or approximant, is stable as a high-temperature phase.

For the Al-Co alloy system [21] and the Al-Ni binary and also the Al-Co-Ni ternary system, we evaluated the total energies of all experimentally stable Alrich phases (except -Al 3 Co, which at 342 atoms/cell is too large to handle). We constructed the convex hull of coexistence tie-planes on the plot of energy as a function of composition; our energies are quoted relative to the convex hull defined by Al 3 Ni, Al 3 Ni 2 , Al 9 Co 2 , and Al 5 Co 2 .

We pause for a moment to sketch the "energy landscape" of these competing phases. X-Al 9 Co 2 Ni 2 had energy 8.5 Å above the tie-line [23] (+6.1 meV for composition Al 9 Co 2.5 Ni 1.5 ), and W -AlCoNi had energy +12.0 meV [25]. Al 9 Co 2 remains stable in a ternary composition (replacing Co→Ni) up to 9% Ni. Note that [21], Al 13 Co 4 is a high-temperature phase, stabilized by vibrational and vacancy/substitutional entropy; at full occupancy, it is unstable by 10 meV/atom.

The decagonal approximant structures we obtained had the best energies seen to date for any d-AlCoNi models with the Ni-rich and Co-rich composition respectively +20 meV/atom (Fig. 2(a)) and +19 meV/atom (Fig. 1(c A different viewpoint explains the difference within the decagonal phase itself. When we examine the electronic density of states [Fig. 2(c)] we see a prominent pseudogap as in most Al-TM quasicrystal-like phases. The optimum stability occurs when the Fermi level coincides with the pseudogap. But since Ni has a different (effective) valence than Co, we need different compositions in order to achieve this matching of the Fermi energy. Clearly, the "high-density" Co-type Ni-rich structure of Fig. 2(b) is disfavoured by this effect (thin line in the same figure, (c)).

Discussion

In summary, we obtained excellent structural energies from direct quenching molecular dynamics with pair potentials, when constrained by cell parameters and aided by realistic potentials as well as replica-exchange and other Monte Carlo steps. The size limit on our unit cell was not from the computer time needed to anneal it, but rather from the need to ensure that unwanted high-temperature phases are mismatched with the lattice parameters. We found there are two discrete structure types, and explained (Sec. 6) why one goes with Ni-rich compositions and the other with Co-rich compositions. Ultimately we believe the physical parameter controlling the phase is electron density (which tracks atom density): higher or lower electron densities are optimal for Co-rich or Ni-rich compositions, respectively.

There is no hope to realistically model any decagonal without (at least local) 8 Å period due to "puckering" of Al atoms out of their layers, as emphasized by the failure to obtain a decagonal in a 4 Å cell. Note that the best energy for Nirich d-AlCoNi found here is comparable to the best energy found by hand-built variations and/or MD-relaxations applied to tile-decoration models [29] approach. Thus, each structure includes some feature that the other one lacks. The cellconstrained quench finds the large-scale feature of the D-ring, as well as local puckerings, but probably does not find the absolute best global correlations of the puckerings. The decoration variations are based on a hierarchical pattern of tilings [3] with edges ∼ 2.5 Å, ∼ 4.0 Å, and ∼ 6.5 Å, which may be superior to the quench results (which have well defined TM positions but some Al variability); furthermore, due to a more systematic testing of puckering patterns, they may have achieved better puckering correlations.

The cell-constrained quench, thus, may ultimately not be a direct shortcut to good structures. Rather, it might replace lattice-gas Monte Carlo as a better method to use in the early stages of our recipe [3,5], where one is discovering basic decoration rules which permit further simulations at larger length scales. It will be interesting to see if decorations which ensure a high density of D-rings can reach energies as low as the melt-quench structures.

Figure 1 .

 1 Figure 1. Various outcomes of constrained-cell melt-quench simulation, (a). The "2B+H" unit cell is tractable on the Co-rich side, here Al 158 Co 52 . Here and in Fig. 2, the view is along the short (c) axis; atoms are marked by circles, with larger sizes denoting larger z coordinate. Lines show bonds between atoms in different layers heavy and light for TM-TM and Al-Al, with projected lengths 4.1 ± 0.3 Å and 2.6 Å, respectively. (b). A B2-like structure found in the same cell for Ni-rich composition Al 148 Co 16 Ni 41 viewed along the (1,1,0) direction) (c axis is horizontal). (c). Co-rich "Co-type" MQ structure Al 58 Co 14 Ni 9 , "Boat" cell. Composition similar to W-phase [24].

5 Figure 2 .

 52 Figure 2. (a,b) Ni-type or Co-type structures emerge in the "Boat" cell, for (respectively) lower density (Al 56 Co 6 Ni 16 ) or higher density (Al 58 Co 6 Ni 16 ). (c). Electronic density of states e(DOS) for three cases: Co-type Co-rich (Al 58 Co 14 Ni 9 in Fig.1c), Co-type Ni-rich (Fig.2b) and Ni-type Ni-rich (Fig.2a).

  ) and 2(a,b). This consists (in projection) of a pair of puckered Al atoms at the same z and on opposite sides of the central TM; a second such pair lies at a different z and (in projection) roughly at right angles to the first. (These atoms stand out in the figures because they are the only ones whose projections do not lie on ideal quasilattice sites.)Finally, the Al 0.75 Co .25 structure of Fig.1(a) has the same puckering pattern as

  Å.] Also, the approximate composition of the Ni-rich decagonal is reported to be Al 72 Co 8 Ni 20 . So we studied various cell contents, most importantly Al 56 Co 6 Ni 16 at the Ni-rich end (with variations having 57 Al and/or 15 Ni), and Al 58 Co 14 Ni 9 at the Co-rich end.

  )) above the convex hull of low-temperature competing phases. It turns out that Al-Co has quite good energies in typical Al-Ni structures (e.g. nearly stable in the Al 3 Ni structure), but not vice versa: structures favorable for Al-Co e.g. (Al 13 Co 4 or Al 5 Co 2 ) are highly unfavorable in the Al-Ni system. The well ordered Co-type structures are unstable with Ni-rich compositions, by +53 meV/atom (Al 58 Co 6 Ni 16 ). For lower densities, optimal structures were always Ni-type: unstable by +36 meV/atom (Al 57 Co 6 Ni 16 ), optimally +20 meV/atom (Al 56 Co 6 Ni 16 ), and by +24 meV/atom at even lower density cell content (Al 56 Co 6 Ni 15 ). For Al-Co the best decagonallike structure is always Co-type; in particular, Al 0.75 Co 0.25 in Fig. 1(a) is just 7.5 meV/atom unstable against decomposition to Al 9 Co 2 and Al 3 Co. We now come to the point, why (in reality) the (nearly) stable decagonal-type structures are Ni-type for Ni-rich compositions and Co-type for Co-rich compositions. For Co fraction above ∼25% the Al-Co binary is destabilized by competition with Al 5 Co 2 : nothing breaks structurally in the decagonal, but the tie-line is simply pulled down by the very good Al 5 Co 2 energy. Conversely, in the Al-Ni system, decagonals with low Ni content are destabilized by competition with Al 3 Ni. From this viewpoint, the fact that Al-Ni phases are low-density and Al-Co phases are high-density is an accident of the different landscape of competing non-quasicrystal phases.

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Acknowledgments

Supported by U.S. Dept of Energy grant DE-FG02-89ER-45405 (CLH) and by Slovak VEGA 2/0157/08. CLH acknowledges a travel fellowship from the Slovak Academic Information Agency (2010).