Journal of Men's Health. 2024; 20(5): 48-56. doi: 10.22514/jomh.2024.070
Original Research

A nomogram for predicting extraprostatic extension in prostate cancer based on extraprostatic extension grade and clinical characteristics

Quan Ma1,†, Wei Tian1,†, Shasha Lv2, Yongna Fu2, Hui Wang3, Jiansong He1, Yongliang Chen1,*,

1Department of Urology, Shaoxing Central Hospital, 312000 Shaoxing, Zhejiang, China

2Department of Radiology, Shaoxing Central Hospital, 312000 Shaoxing, Zhejiang, China

3Department of Pathology, Shaoxing Central Hospital, 312000 Shaoxing, Zhejiang, China

*Corresponding Author(s): sxcentral@stu.hebmu.edu.cn (Yongliang Chen)

† These authors contributed equally.

History Submitted: 06 October 2023 | Accepted: 21 November 2023 | Published: 30 May 2024
Copyright:  ©2024 The Author(s). Published by MRE Press.
This is an open access article under the CC BY 4.0 license (https://creativecommons.org/licenses/by/4.0/).

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Abstract

To assess the efficacy of a nomogram model derived from extraprostatic extension (EPE) grade on magnetic resonance imaging (MRI) and clinical features in forecasting pathological EPE in prostate cancer. We conducted a retrospective analysis of the clinical data from 232 prostate cancer patients. Patients were categorized into EPE and non-EPE groups based on the presence of pathological EPE. Subsequently, they were randomly allocated into a training set (162 cases) and a validation set (70 cases) at a 7:3 ratio. We gathered clinical attributes and EPE grades for all patients. Three predictive models—clinic, magnetic resonance (MR) and clinic + MR—were developed within the training set. The clinic + MR model was visualized through a nomogram. The models’ performance was assessed using the receiver operating characteristic (ROC) curve, calibration curve, and decision curve analysis (DCA). Both univariate and multivariate logistic regression analyses identified the biopsy International Society of Urological Pathology (ISUP) category and biopsy maximum unilateral positive percentage as independent risk factors for EPE within the training set. The EPE grade exhibited consistent inter-observer agreement, evidenced by weighted Kappa values of 0.72 and 0.71 in the training and validation sets, respectively. Compared to the clinic and MR models, the clinic + MR model was the most effective in predicting pathological EPE, boasting area under the curves (AUCs) of 0.85 and 0.82 in the training and validation sets, respectively. Calibration curves from both sets demonstrated that the clinic + MR model provided accurate predictions for pathological EPE. Within the DCA, the clinic + MR model surpassed the clinic and MR models in terms of clinical net benefit in both sets. The clinic + MR model excels in predicting the pathological EPE of prostate cancer. Its superiority over the clinic model underscores its clinical relevance and the potential for broader implementation.

Keywords:Extraprostatic extensionProstate cancerMagnetic resonance imagingNomogramDiagnostic performance
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Cite this article

Quan Ma, Wei Tian, Shasha Lv, Yongna Fu, Hui Wang, Jiansong He, Yongliang Chen. A nomogram for predicting extraprostatic extension in prostate cancer based on extraprostatic extension grade and clinical characteristics. Journal of Men's Health. 2024; 20(5): 48-56. doi: 10.22514/jomh.2024.070

1. Introduction

Prostate cancer is a common malignant tumor that affects the male genitourinary system. Globally, its incidence and mortality rates rank second and fifth, respectively, among male neoplasms [1]. Compared to localized prostate cancer, extraprostatic extension (EPE) is associated with higher incidences of positive surgical margins and biochemical recurrence, often requiring additional adjuvant therapy. Furthermore, EPE is intrinsically linked to disease progression and adverse prognosis [2, 3]. Radical prostatectomy (RP) is a primary treatment option for prostate cancer [4]. While RP effectively manages prostate cancer, it comes with notable complications, such as erectile dysfunction and urinary incontinence [5, 6], which profoundly impact patients’ quality of life. For those without EPE, nerve-sparing RP offers an avenue to enhance sexual function and urinary continence without compromising the integrity of surgical margins [7]. Thus, accurate preoperative determination of EPE is critical for informed clinical decision-making and optimal surgical planning.

Magnetic resonance imaging (MRI) serves as the foremost technique for the preoperative prediction of EPE. The Prostate Imaging Reporting and Data System (PI-RADS) provides a comprehensive framework for assessing the imaging characteristics of EPE [8]. Yet, its diagnostic efficacy is limited by the lack of a quantitative evaluation of these characteristics [9]. The Likert scale amalgamates the imaging traits of EPE, as detailed in PI-RADS, classifying the likelihood of EPE into five categories. Nevertheless, due to its lack of objective criteria, this scale exhibits considerable variability in diagnostic accuracy [10, 11, 12]. In 2019, Mehralivand et al. [13] introduced a standardized, streamlined EPE grade system that amalgamates both qualitative and quantitative MRI indicators. At present, validation studies focusing on the EPE grade remain sparse, especially concerning the Chinese demographic.

Consequently, this research undertook a retrospective analysis of the clinical data from prostate cancer patients treated at Shaoxing Central Hospital between January 2018 and October 2022. Our objectives were to ascertain the diagnostic precision of the EPE grade in predicting pathological EPE and to gauge inter-observer concordance. In tandem, we crafted a clinic + MR model by amalgamating the EPE grade with pertinent clinical and pathological data, aiming to evaluate its diagnostic prowess in predicting pathological EPE.

2. Materials and methods

2.1 Study population

Clinical and pathological data were retrospectively gathered from patients who underwent laparoscopic radical prostatectomy for prostate cancer at the Department of Urology, Shaoxing Central Hospital, between January 2018 and October 2022. Inclusion criteria were: (1) Pathological diagnosis of prostate cancer; (2) Preoperative prostate MRI examination. Exclusion criteria included: (1) Preoperative MRI for more than 6 months or MRI at an outside institution (n = 12); (2) Adjuvant therapy such as chemotherapy and endocrine therapy before surgery (n = 5); (3) Presence of artifacts on prostate MRI (n = 3); (4) Inadequate clinical information (n = 15). A total of 232 prostate cancer patients were considered for this study. Out of them, 103 exhibited a pathological EPE post-surgery, translating to an EPE incidence rate of 44.40%. These patients were then randomly assigned into a training set (n = 162) and a validation set (n = 70) at a 7:3 ratio (Fig. 1).

Flow diagram of patient selection and randomization grouping for the study. EPE: extraprostatic extension; MRI: magnetic resonance imaging.

Fig. 1.Flow diagram of patient selection and randomization grouping for the study. EPE: extraprostatic extension; MRI: magnetic resonance imaging.

2.2 Clinical information

Collected data encompassed clinical, laboratory, MRI and pathological details, which included age, Total Prostate Specific Antigen (TPSA), Free Prostate Specific Antigen (FPSA), prostate volume, Prostate Specific Antigen Density (PSAD), biopsy Gleason score, biopsy International Society of Urological Pathology (ISUP) category, biopsy maximum unilateral positive percentage, biopsy total positive percentage, and EPE grade of prostate MRI.

2.3 EPE grade on MRI

All MRIs were conducted using a 1.5T MRI scanner (Philips, Amsterdam, Netherlands). The imaging protocols incorporated axial and sagittal T1-weighted imaging (T1WI), T2-weighted imaging (T2WI), and diffusion weighted imaging (DWI). Corresponding apparent diffusion coefficient (ADC) maps were computed by b-values of 0 and 800 mm2/s. The EPE grade system, based on Mehralivan et al. [13], was employed as follows: grade 0, no suspicion for EPE; grade 1, either curvilinear contact length (CCL) ≥1.5 cm or capsular irregularity and bulge; grade 2, both CCL ≥1.5 cm and capsular irregularity and bulge; grade 3, frank EPE visible at MRI or invasion of adjacent anatomic structures. Two experienced radiologists, with 10 and 5 years of prostate MRI expertise respectively, assigned the EPE grades without prior knowledge of the prostate pathology. Consensus EPE grades were documented, in instances of disagreement, a mutually agreed-upon grade was finalized.

2.4 Pathological diagnosis of prostate cancer

Following radical prostatectomy, pathological specimens were appropriately preserved in a 10% neutral formaldehyde solution and subsequently stained using hematoxylin and eosin for both the surgical margins and prostate tissues. Sections were consistently taken at intervals of 2–3 mm, aligned perpendicular to the gland’s long (apical-basal) axis. An adept pathologist assessed these sections, identifying the lesion and confirming the presence or absence of EPE. Both the Gleason score and ISUP category for the lesion were documented in accordance with the 2019 edition of the ISUP guidelines [14].

2.5 Statistical analysis

Statistical evaluations were conducted using SPSS (version 19.0, IBM Corporation, Armonk, NY, USA) and R software (version 4.2.3, R Development Core Team, Vienna, Austria). Data following a normal distribution were denoted as x̄ ± s, while data with a skewed distribution were presented as M (Q1, Q3). To compare between groups, the independent samples t-test or the Mann-Whitney U-test was employed. For count data group comparisons, the χ2 test or Fisher’s exact probability method was utilized. The consistency between observation groups was determined using the weighted Kappa test, with weighted Kappa values categorized as: <0.21 (poor), ≥0.21–0.41 (fair), ≥0.41–0.61 (moderate), ≥0.61–0.81 (good), and ≥0.81–1.00 (very good). Both univariate and multivariate logistic regression analyses were employed to identify clinical risk factors associated with pathological EPE in post-surgery prostate cancer patients within the training set. Based on these identified risk factors, a clinic model was established. A separate MR model, reliant on the MRI-determined EPE grade, was also constructed for the training set. By integrating the clinical risk factors with the EPE grade, a combined clinic + MR model was developed to predict pathological EPE in the training set. This integrated model was visualized as a nomogram. The respective ROC curves of the three models were graphed, the AUC was calculated, and the DeLong test discerned differences among these models. Calibration curves evaluated the clinic + MR model’s calibration, and DCA gauged the models’ net clinical benefits. A p-value of less than 0.05 was considered indicative of statistical significance.

3. Results

3.1 Clinical characteristics

A comparison of clinical characteristics between EPE and non-EPE groups within the training set is detailed in Table 1. Distinct variables including age, TPSA, PSAD, biopsy Gleason score, biopsy ISUP category, biopsy maximum unilateral positive percentage, biopsy total positive percentage were statistically significant (p < 0.05). Within the training set, EPE patients exhibited notably elevated values for age, PSA and PSAD in contrast to non-EPE patients (p < 0.05). The biopsy Gleason score, biopsy ISUP category, biopsy maximum unilateral positive percentage, and biopsy total positive percentage also demonstrated significant variations between EPE and non-EPE groups (p < 0.05) (Table 1).

Table 1.Characteristics of EPE and non-EPE groups in the training set.
ParametersEPE group (n = 65)Non-EPE group (n = 97)Statistical valuep-value
Age (year)*75.91 ± 5.6073.68 ± 6.01−2.375b0.019
TPSA (ng/mL)‡15.47 (8.99, 32.88)8.73 (6.66, 14.00)−4.294c<0.001
FPSA (ng/mL)‡1.43 (1.06, 3.08)1.37 (0.92, 2.28)−1.093c0.274
Prostate volume (cm3)*36.42 ± 15.3041.80 ± 18.371.949b0.053
PSAD (ng/mL2)‡0.58 (0.28, 0.93)0.22 (0.16, 0.41)−5.371c<0.001
Biopsy Gleason score n (%)
611 (16.92%)51 (52.58%)29.114a<0.001
721 (32.31%)30 (30.93%)
819 (29.23%)11 (11.34%)
911 (16.92%)3 (3.09%)
103 (4.62%)2 (2.06%)
Biopsy ISUP category n (%)
111 (16.92%)51 (52.58%)29.751a<0.001
214 (21.54%)24 (24.74%)
37 (10.77%)6 (6.19%)
419 (29.23%)11 (11.34%)
514 (21.54%)5 (5.15%)
Maximum unilateral positive percentage at biopsy n (%)
<34%13 (20%)5936.421a<0.001
34–67%13 (20%)22
>67%39 (60%)16
Total positive percentage at biopsy n (%)
<34%197334.97a<0.001
34–67%3019
>67%165
EPE grade n (%)
0187539.567a<0.001
12011
2166
3115
Abbreviations: TPSA: Total Prostate Specific Antigen; FPSA: Free Prostate Specific Antigen; PSAD: Prostate Specific Antigen Density; ISUP: International Society of Urological Pathology; EPE: extraprostatic extension. *Data are mean ± standard deviation. ‡Data are mean, and data in parentheses are the interquartile spacing. aData are chi-square. bData are t-value. cData are z-value.

3.2 Univariate and multivariate logistic regression analyses of pathological EPE after prostate cancer surgery

Both clinical and pathological features (age, TPSA, FPSA, prostate volume, PSAD, biopsy Gleason score, biopsy ISUP category, biopsy maximum unilateral positive percentage and biopsy total positive percentage) were incorporated into the univariate and multivariate logistics regression analyses. The biopsy ISUP category and biopsy maximum unilateral positive percentage emerged as independent clinical risk factors for pathological EPE post-prostate cancer surgery, as shown in Table 2.

Table 2.Univariate and multivariate logistic regression analysis of pathological EPE after prostate cancer surgery.
ParametersUnivariate logistic regression analysisMultivariate logistic regression analysis
OR (95% CI)p valueOR (95% CI)p value
Age1.068 (1.010–1.129)0.021--
TPSA1.030 (1.006–1.054)0.013--
FPSA1.031 (0.973–1.093)0.306--
Prostate volume0.981 (0.962–1.001)0.057--
PSAD4.796 (1.895–12.142)0.001--
Biopsy Gleason score2.291 (1.621–3.239)<0.001--
Biopsy ISUP category1.910 (1.489–2.451)<0.0011.588 (1.209–2.085)0.001
Biopsy maximum unilateral positive percentage3.335 (2.192–5.076)<0.0012.606 (1.666–4.075)<0.001
Total positive percentage at biopsy4.216 (2.463–7.215)<0.001--
TPSA: Total Prostate Specific Antigen; FPSA: Free Prostate Specific Antigen; PSAD: Prostate Specific Antigen Density; ISUP: International Society of Urological Pathology; OR: odds ratio; CI: confidence interval; -: Data is not measured.

3.3 EPE grade on MRI

Statistically significant discrepancy in the EPE grade was observed between the EPE and non-EPE sets in the training set (p < 0.05) (Table 1). Both evaluators displayed commendable consistency in the EPE grade, boasting weighted Kappa values of 0.72 (95% CI: 0.65–0.80) and 0.71 (95% CI: 0.58–0.83) in the training and validation sets respectively.

3.4 Predictive models

In the training set, two clinical features (biopsy ISUP category and biopsy Maximum unilateral positive percentage) were leveraged to devise a clinic model predicting pathological EPE, resulting in AUC values of 0.80 and 0.76 for the training and validation sets respectively (Table 3). Utilizing the EPE grade on MRI from the training set, the MR model was formulated, producing AUCs of 0.75 and 0.77 in the training and validation sets respectively (Table 3). The clinic + MR model was established incorporating three parameters: biopsy ISUP category, biopsy maximum unilateral positive percentage, and EPE grade from the training set; this model yielded AUCs of 0.85 and 0.82 for the training and validation sets respectively (Table 3). The ROC curves for the trio of models are illustrated in Fig. 2. Relative to the clinic model, the clinic + MR model markedly augmented diagnostic efficacy in both the training set (clinic vs. clinic + MR, p = 0.029) and validation set (clinic vs. clinic + MR, p = 0.035). A corresponding nomogram for the clinic + MR model is depicted in Fig. 3. Calibration curves confirmed the clinic + MR model’s adept calibration for predicting pathological EPE (Fig. 4), with the Hosmer-Lemeshow test rendering non-significant results (p = 0.472 for training set, p = 0.669 for validation set). In the decision curve analysis, the clinic + MR model outperformed both the clinic and MR models in terms of net clinical benefit (Fig. 5).

Table 3.Statistical indicators of predictive models.
modelsAUCSensitivitySpecificityAccuracy
Clinic model in training set0.800.650.870.78
Clinic model in validation set0.760.820.660.74
MR model in training set0.750.720.770.75
MR model in validation set0.770.760.780.77
Clinic + MR model in training set0.850.890.730.80
Clinic + MR model in validation set0.820.820.780.80
AUC: area under the curve; MR: magnetic resonance.
ROC curves of models in the training and validation sets. (A) ROC curves of the clinic model, MR model, and clinic + MR model in the training set. (B) ROC curves of the clinic model, MR model, and clinic + MR model in the validation set. AUC: area under the curve; MR: magnetic resonance.

Fig. 2.ROC curves of models in the training and validation sets. (A) ROC curves of the clinic model, MR model, and clinic + MR model in the training set. (B) ROC curves of the clinic model, MR model, and clinic + MR model in the validation set. AUC: area under the curve; MR: magnetic resonance.

The nomogram of the clinic + MR model. EPE: extraprostatic extension; ISUP: International Society of Urological Pathology.

Fig. 3.The nomogram of the clinic + MR model. EPE: extraprostatic extension; ISUP: International Society of Urological Pathology.

Calibration curves of the clinic + MR model. (A) Calibration curves of the clinic + MR model in the training set. (B) Calibration curves of the clinic + MR model in the validation set.

Fig. 4.Calibration curves of the clinic + MR model. (A) Calibration curves of the clinic + MR model in the training set. (B) Calibration curves of the clinic + MR model in the validation set.

Decision curve analysis of models. (A) Decision curve analysis of the clinic model, MR model, and clinic + MR model in the training set. (B) Decision curve analysis of the clinic model, MR model, and clinic + MR model in the validation set. MR: magnetic resonance.

Fig. 5.Decision curve analysis of models. (A) Decision curve analysis of the clinic model, MR model, and clinic + MR model in the training set. (B) Decision curve analysis of the clinic model, MR model, and clinic + MR model in the validation set. MR: magnetic resonance.

4. Discussion

In this study, we developed an MR model based on the EPE grade derived from MRI, demonstrating both robust diagnostic efficacy and consistent inter-observer agreement. This clinic model was formulated to predict pathological EPE, drawing on biopsy ISUP category and biopsy maximum unilateral positive percentage. Using the biopsy ISUP category, biopsy maximum unilateral positive percentage, and EPE grade from MRI, we crafted the clinic + MR model. Subsequently, we employed nomogram visualization. This clinic + MR model surpasses both the pure MR model and the clinical model in terms of diagnostic efficacy and clinical net benefit.

MRI provides added value in predicting pathological EPE. A meta-analysis indicated that while MRI boasts a specificity of 0.91, its sensitivity is a mere 0.57 for predicting pathological EPE [15]. Although the European Society of Urogenital Radiology (ESUR) scores and Likert scales are utilized to assess pathological EPE, their absence of objective indicators compromises reproducibility. Current studies validate that CCL stands as an autonomous predictor of pathological EPE, showcasing impressive sensitivity and inter-observer agreement [16, 17, 18]. Accordingly, the EPE grade introduced by Mehralivand et al. [13] omits intricate qualitative descriptors, saving for capsular irregularity or bulge. It also integrates quantitative attributes for a CCL ≥15 mm. Park et al. [16] reinforced the diagnostic efficacy of the EPE grade, noting a sensitivity between 77.5% and 79.8%, an AUC between 0.77 and 0.81, and the most potent association with pathological EPE relative to ESUR scores and Likert scales. The EPE grade, encompassing both qualitative and quantitative factors, minimizes reliance on observer expertise. Its weighted kappa, ranging from 0.647 to 0.71 for inter-observer agreement, eclipses that of the ESUR scores and Likert scales, which are strictly qualitative [16, 17]. A recent inquiry deduced that the EPE grade possesses marked diagnostic efficacy in anticipating the biochemical recurrence of prostate cancer [19]. Within our study, the weighted kappa scores for the EPE grade in the training and validation groups stood at 0.72 and 0.71, respectively. The participating radiologists, representing both junior and senior levels, achieved remarkable inter-observer congruence. This suggests that the EPE grade, demanding minimal MRI reading experience, is apt for both reporting and instruction. Echoing prior research, our study’s AUC values for the training and validation sets were 0.75 and 0.77, with sensitivities of 0.72 and 0.76, respectively, further externalizing the diagnostic efficacy of the EPE grade.

Several clinical models, including the Partin tables, Cancer of the Prostate Risk Assessment (CAPRA) score, and the Memorial Sloan Kettering Cancer Center (MSKCC) nomogram [20, 21], have been employed to forecast pathological EPE. However, these models primarily draw on data from European and American demographics and notably exclude the biopsy ISUP category. The ISUP category boasts superior prognostic accuracy relative to the Gleason score [22]. Moreover, both the biopsy ISUP category and biopsy positive percentage are recognized as distinct risk determinants for pathological EPE, reflecting AUC values of 0.796 and 0.762, respectively [23]. In our research, we adopted the highest grade for the biopsy ISUP category due to its enhanced predictive capacity concerning the stage and grade of prostate cancer [24]. Additionally, we separately quantified the positive percentages for the left and right lateral lobes, selecting the greater value as the biopsy maximum unilateral positive percentage. A clinical model was designed around the biopsy ISUP category and biopsy maximum unilateral positive percentage, presenting AUC values of 0.8 and 0.76 in the training and validation sets, respectively—aligning with prior studies. Yet, we discerned significant volatility in the sensitivity and specificity across the training and validation sets, which might be attributed to the clinical model’s diminished reliability during internal validation [25]. Thus, there’s a pressing need to devise a more potent and consistent prediction model, potentially through the incorporation of additional markers.

In recent years, numerous researchers have sought to amalgamate the EPE grade with clinical and pathological characteristics to craft a composite model for predicting pathological EPE, endeavoring to refine predictive effectiveness. Mehralivand et al. [13] formulated a clinical model utilizing log PSA and biopsy ISUP category, achieving an AUC of 0.77. Yet, when merged with the EPE grade, the AUC ascended to 0.81. Xu et al. [26] similarly validated that merging the EPE grade with clinical traits considerably elevated the diagnostic proficiency of the clinical model. Furthermore, this integrated model exhibited superior calibration prowess and clinical net advantage. A contemporaneous study inferred that the model incorporating the EPE grade potentially surpasses the efficacy of PI-RADS V2.1, with AUC values standing at 0.879 and 0.802, respectively [27]. In our endeavor, we fashioned a clinic + MR model for predicting pathological EPE by assimilating the EPE grade and biopsy pathological metrics. This model’s nomogram visualization displayed an AUC of 0.85, a value significantly higher than that of the standalone clinical model. Our internal validation yielded an AUC of 0.82 for the validation set. Moreover, the clinic + MR model demonstrated enhanced calibration and clinical net benefit in both training and validation sets.

This study presents several limitations. First, it is retrospective in nature, introducing potential selection bias. Second, it is confined to a single centre and encompasses a limited sample size. Lastly, the study lacks external validation data, necessitating further data acquisition to ascertain the predictive model’s robustness.

5. Conclusions

The EPE grade showcases commendable diagnostic efficacy and inter-observer consistency, hinting at its augmented predictive value for pathological EPE. The clinic + MR model, integrating biopsy ISUP category, biopsy maximum unilateral positive percentage, and EPE grade, exhibits pronounced predictive acumen for pathological EPE. As delineated by the clinic + MR model’s nomogram, it facilitates the estimation of pathological EPE likelihood, making it an invaluable tool for clinical decision-making and meriting broader adoption.

Abbreviations

EPE, extraprostatic extension; ROC, received operating characteristic; AUC, area under the curve; DCA, decision curve analysis; ISUP, International Society of Urological Pathology; MRI, Magnetic resonance imaging; MR, magnetic resonance; PI-RADS, Prostate Imaging Reporting and Data System; TPSA, Total Prostate Specific Antigen; FPSA, Free Prostate Specific Antigen; PSAD, Prostate Specific Antigen Density; T1WI, T1-weighted imaging; T2WI, T2-weighted imaging; DWI, diffusion weighted imaging; ADC, apparent diffusion coefficient; CCL, curvilinear contact length; ESUR, European Society of Urogenital Radiology; CAPRA, Cancer of the Prostate Risk Assessment; MSKCC, Memorial Sloan Kettering Cancer Center.

Availability of data and materials

The data presented in this study are available on reasonable request from the corresponding author.

Author contributions

YLC—guarantor of the article. YLC and QM—conception and design. WT, SSL, YNF and HW—collection and assembly of data. QM, WT, SSL, YNF, HW and JSH—data analysis and interpretation. QM and HW—writing manuscript. All authors have collaboratively contributed to and endorsed the final manuscript.

Ethics approval and consent to participate

Research involving human participants were reviewed and approved by Institutional Review Board of Shaoxing Central Hospital (2023-037). The Medical Ethics Committee of Shaoxing Central Hospital approved this research, and informed consent from patients was waived.

Acknowledgment

Not applicable.

Funding

This research received no external funding.

Conflict of interest

The authors declare no conflict of interest.

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