Correlation of lymph node metastasis with contrast-enhanced ultrasound features,microvessel density and microvessel area in patients with papillary thyroid carcinoma

Abstract

OBJECTIVE:

This study aims to investigate the relationship of cervical lymph node metastasis (LNM) with contrast-enhanced ultrasound (CEUS) features, microvessel density (MVD) and microvessel area (MVA) in patients with papillary thyroid carcinoma (PTC), and to evaluate the diagnostic value of CEUS for PTC.

METHODS:

A total of 108 patients diagnosed with PTC at the First Affiliated Hospital of Jinzhou Medical University from January 2016 to December 2018 were selected and underwent preoperative CEUS of the thyroid, surgical resection and postoperative histopathological examination of their resected lesion. They were divided into a lymphatic metastasis-positive group (LNM+, n = 61) and a lymphatic metastasis-negative group (LNM–, n = 47) based on their lymph node status. The CEUS quantitative parameters, MVD and MVA, were compared between the two groups, and risk factors for LNM were analyzed by univariate and multivariate logistic regression.

RESULTS:

Compared with patients with in the LNM–group, the tumor diameter and the proportion of capsule contact of patients in the LNM+group were significantly greater and the patients in this group were younger. The rise time (RT), peak intensity (PI), area under the curve (AUC), MVD, and MVA were also significantly higher in the LNM+group than in the LMN–group, while there was no significant difference in time to peak (TP), mean transit time (mTT), velocity of intensity increase (IIV), and velocity of intensity decrease (IDV) between the two groups. Univariate and multivariate correlation analysis indicated that tumor size, RT, PI, AUC, MVD, and MVA were risk factors for LNM, and ROC curves further suggested that RT had the best overall predictive performance.

CONCLUSION:

Tumor size, RT, PI, AUC, MVD and MVA are risk factors for LNM in PTC. In other words, CEUS is an important non-invasive and preoperative tool for evaluating PTC, with MVD and MVA identified as vital postoperative diagnostic indicators.

Keywords

Contrast-enhanced ultrasound (CEUS)Papillary thyroid carcinoma (PTC)Microvessel density (MVD)Microvessel area (MVA)Lymph node metastasis

1 Introduction

Papillary thyroid carcinoma (PTC) is the most common pathological type of thyroid cancer, accounting for more than 80% of all cases. It has a good prognosis, but the risk of lymph node metastasis (LNM) is high; particularly, LNM in the central cervical region shows an incidence of 20% to 90% [1, 2]. Angiogenesis has always been one of the hotspots in cancer research, but the use of immunohistochemical techniques to obtain tumor angiogenesis indicators is invasive and has some lag in clinical guidance [3, 4]. Previous studies [5, 6] reported that PTC tissue often grows slowly due to the lack of blood supply, and neovascularization is essential for its growth, invasion, and metastasis. As a marker of angiogenesis, tumor microvessel density (MVD) reflects the degree of angiogenesis and is the “gold standard” of angiogenic activity [7, 8], closely related to tumor invasion, metastasis and prognosis. Also, the close association of MVD with invasion and cervical LNM of PTC has been widely accepted. However, the determination of MVD is complex. It requires the collection of lesion tissue during surgery and subsequent special staining procedure and thus is difficult to be used for preoperative evaluation [9 –11].

Contrast-enhanced ultrasound (CEUS) can identify small vessels of a diameter < 40μm, display the microcirculatory blood perfusion in the region of interest (ROI) in real-time, and evaluate the hemodynamic characteristics of a lesion. Thus, CEUS serves as an important examination to determine the microvessels of lesions [12, 13]. Contrast agent SonoVue has a microbubble size similar to that of red blood cells, contributing to observing the situation of tissue perfusion and new microvessels in the tumor [14]. CEUS was also shown to have acceptable performance in diagnosing LNM in PTC [15, 16] and in the treatment of thyroid cancer, such as guiding percutaneous radiofrequency ablation [17]. However, there is still no study on the predictive value of various parameters of CEUS for LNM of PTC. Given the lack of related studies, we used standardized CEUS and quantitative analysis methods to investigate the relationship of cervical LNM of PTC with CEUS perfusion parameters, MVD, and microvessel area (MVA) in the present study.

2 Materials and methods

2.1 General information

The data of 108 patients diagnosed with PTC at the First Affiliated Hospital of Jinzhou Medical University from January 2016 to December 2018 were retrieved and analyzed. All patients received thyroidectomy and cervical lymphadenectomy, and based on the absence or presence of LNM, they were divided into a LNM-positive (LNM+) group and LNM-negative (LNM–) group. The study inclusion criteria were: (1) PTC diagnosis confirmed by surgery and pathological examination; (2) Aged≥20 years; (3) CEUS was performed within one week before biopsy or surgery; (4) Nodule diameter > 5 mm; (5) Patients with complete clinical data. Exclusion criteria were: (1) Fluid content in nodules > 50%; (2) Hyperthyroidism; (3) Complicated with underlying metabolic diseases and autoimmune diseases; (4) Complicated with benign/malignant tumors in other parts, nephropathy, diabetes, rheumatoid arthritis, psoriasis and other diseases that affected MVD; (5) Coarse calcification in nodules; (6) Presence of diffuse LNM; (7) Pregnancy and other contraindications for CEUS.

The patients’ baseline characteristics such as age, sex, tumor size, tumor location (left lobe, right lobe, isthmus), tumor border (clear or not), tumor echo (parenchymal, mixed), and capsule contact (yes or no) were recorded. All patients and their families provided signed informed consent. This study was reviewed and approved by the Ethics Committee of the First Affiliated Hospital of Jinzhou Medical University (approval No: KYLL202016).

2.2 Ultrasonic examination

CEUS perfusion analysis is now was off-label when performed as the only method to diagnose PTC. The Siemens Acuson S3000 color Doppler Ultrasound Machine was used in this study. Conventional ultrasound was performed with linear array probe 18 L and frequency 6–18 MHz, while CUES with linear array probe 9L4, frequency 4–9 MHz, pulse-inversion contrast harmonic imaging and Q-LAB quantitative analysis software.

For conventional ultrasound scanning, patients were placed in a supine position and maintained quiet breathing with the head back to fully expose the neck. The location, size, border, echo, blood flow distribution and other basic information of their nodule were observed and recorded.

For CEUS, after a bolus infusion of 2.4 mL contrast agent (SonoVue) through the antecubital vein, 5 mL of normal saline was injected to clean the catheter. During this period, patients were instructed to maintain steady breathing, and dynamic contrast was performed and recorded for a duration≥180 s. An appropriate ultrasound section (the largest section of the lesion, including part of normal thyroid tissue as much as possible around the lesion) was selected, and the examination mode was then converted to the contrast mode. The mechanical index (MI) was set between 0.06 and 0.08, and the preset conditions remained unchanged during the contrast process in all patients. The ROI was determined upon avoidance of organs, thick blood vessels in the nodule, calcified lesion and cystic part, and the boundary of ROI was located in the inner edge of the lesion. Additionally, another ROI in the control area was as deep as possible as the ROI in the nodule. Time-intensity curves and related blood perfusion parameters, including rise time (RT), time to peak (TP), mean transit time (mTT), peak intensity (PI), intensity increase velocity (IIV), intensity decrease velocity (IDV), and area under the curve (AUC) were obtained.

2.3 Detection of microvessel density and microvessel area

Immunohistochemical staining of nodular tissues obtained postoperatively from all patients was performed using the vascular marker CD34. Microvascular staining was assessed using the Weidner method, and their MVD and MVA were calculated.

2.4 Statistical analysis

SPSS 20.0 statistical software was used for data analysis. Measurement data are expressed as mean±standard deviation (SD), and an independent t-test was used for comparison. Enumeration data are expressed as n (%), and the Chi-square test was used for comparison among groups. The influencing factors leading to LNM were analyzed by univariate and multivariate logistic regression analysis. Further, the receiver operating characteristic (ROC) curve was plotted to analyze the relationship between CEUS parameters and cervical LNM. P < 0.05 was considered statistically significant.

3 Results

3.1 Baseline characteristics of patients

In all, 108 patients with PTC were included in this study. Based on the pathological findings of surgically resected lymph nodes, there were 61 and 47 patients in the LNM+group and LMN–group, respectively. There was no significant difference in sex, tumor location, tumor boundary and tumor echo between the two groups (P > 0.05). However, patients in the LNM+group were markedly younger and had larger tumor size and proportion of capsule contact (P < 0.05) (Table 1).

Table 1
Clinical baseline characteristics of patients in LMN+and LMN–group

Variables LNM–group (n = 47) LNM+group (n = 61) X ² P

Age (%) 6.214 0.013

≤45 years 22(46.8) 43(70.5)

>45 years 25(53.2) 18(29.5)

Sex (%) 0.615 0.433

Male 10(21.3) 17(27.9)

Female 37(78.7) 44(72.1)

Tumor size (%) 19.927 0.001

≤10mm 35(74.5) 19(31.1)

>10mm 12(25.5) 42(68.9)

Tumor location (%) 1.840 0.398

Left lobe 6(12.8) 14(23)

Right lobe 31(66.0) 35(57.4)

Isthmus 10(21.3) 12(19.7)

Tumor boundary (%) 1.086 0.297

Clear 20(42.6) 20(32.8)

Not clear 27(57.4) 41(67.2)

Tumor echo (%) 0.111 0.739

Parenchyma 14(29.8) 20(32.8)

Mixed 33(70.2) 41(67.2)

Capsule contact (%) 4.723 0.030

No 35(74.5) 33(54.1)

Yes 12(25.5) 28(45.9)

Variables	LNM–group (n = 47)	LNM+group (n = 61)	X ²	P
Age (%)			6.214	0.013
≤45 years	22(46.8)	43(70.5)
>45 years	25(53.2)	18(29.5)
Sex (%)			0.615	0.433
Male	10(21.3)	17(27.9)
Female	37(78.7)	44(72.1)
Tumor size (%)			19.927	0.001
≤10mm	35(74.5)	19(31.1)
>10mm	12(25.5)	42(68.9)
Tumor location (%)			1.840	0.398
Left lobe	6(12.8)	14(23)
Right lobe	31(66.0)	35(57.4)
Isthmus	10(21.3)	12(19.7)
Tumor boundary (%)			1.086	0.297
Clear	20(42.6)	20(32.8)
Not clear	27(57.4)	41(67.2)
Tumor echo (%)			0.111	0.739
Parenchyma	14(29.8)	20(32.8)
Mixed	33(70.2)	41(67.2)
Capsule contact (%)			4.723	0.030
No	35(74.5)	33(54.1)
Yes	12(25.5)	28(45.9)

Data are expressed as n(%). LMN–group, lymphatic metastasis negative group; LNM+group, lymphatic metastasis positive group.

3.2 Comparison of quantitative parameters of contrast-enhanced ultrasound and pathological examination results between the two groups

CEUS was conducted in PTC patients of the two groups, and the relevant parameters of CEUS blood perfusion in the LNM+and LMN–groups are displayed in Table 2. The study showed that the RT, PI and AUC of PTC patients in the LNM+group were significantly higher than those in the LMN–group (P < 0.05), while there was no significant difference in TP, mTT, IIV and IDV between the two groups (P > 0.05) (Table 2).

Table 2
Comparison of contrast-enhanced ultrasound parameters between LNM+and LNM–groups

Group n RT (s) PI (%) AUC (dB.s) TP (s) mTT (s) IIV IDV

LNM– 47 11.75±2.93 15.32±2.75 857.03±241.99 42.05±9.20 63.22±12.64 1.60±0.58 1.29±0.51

group

LNM+ 61 16.98±3.01 18.91±2.34 1221.06±320.09 43.46±8.8 65.19±14.27 1.77±0.52 1.39±0.49

group

t 9.072 7.313 6.494 0.810 0.747 1.629 1.091

P 0.001 0.001 0.001 0.420 0.457 0.106 0.278

Group	n	RT (s)	PI (%)	AUC (dB.s)	TP (s)	mTT (s)	IIV	IDV
LNM–	47	11.75±2.93	15.32±2.75	857.03±241.99	42.05±9.20	63.22±12.64	1.60±0.58	1.29±0.51
group
LNM+	61	16.98±3.01	18.91±2.34	1221.06±320.09	43.46±8.8	65.19±14.27	1.77±0.52	1.39±0.49
group
t		9.072	7.313	6.494	0.810	0.747	1.629	1.091
P		0.001	0.001	0.001	0.420	0.457	0.106	0.278

RT, rise time; PI, peak intensity; AUC, area under the curve; TP, time to peak; mTT, mean transit time; IIV, intensity increase velocity; IDV, intensity decrease velocity.

The MVD and MVA were further compared, and we observed that the LNM+group had significantly higher MVD and MVA compared with the LNM–group (P < 0.05) (Table 3).

Table 3

Comparison of microvessel density and microvessel area between LNM+and LNM–groups

Group	n	MVD (vessel.HP^-1)	MVA (%)
LNM–group	47	111.39±28.33	7.79±1.91
LNM+group	61	152.50±24.26	10.39±2.11
t		8.114	6.632
P		0.001	0.001

MVD, microvessel density; MVA, microvessel area.

3.3 Analysis of risk factors for cervical lymph node metastasis

Univariate and multivariate analyses were performed to investigate the influencing factors of cervical LNM. Univariate analysis showed that LNM was associated with age, tumor size, capsule contact, RT, PI, AUC, MVD and MVA (P < 0.05), but not with sex, tumor location, tumor boundary, tumor echo, TP, mTT, IIV and IDV (Table 4). Multivariate analysis revealed that tumor size, RT, PI, AUC, MVD and MVA were independent risk factors for LNM (P < 0.05), while age and capsule contact were not independently associated with LNM (Table 5).

Table 4
Univariate analysis of risk factors for lymph node metastasis

Variables B S.E. Wald P OR 95% CI

Lower limit Upper limit

Age –0.999 0.405 6.071 0.014 0.368 0.166 0.815

Sex –0.357 0.457 0.612 0.434 0.700 0.286 1.712

Tumor size 1.864 0.434 18.441 0.001 6.447 2.754 15.094

Tumor location

Left lobe 1.795 0.408

Right lobe –0.726 0.547 1.763 0.184 0.484 0.166 1.413

Isthmus –0.665 0.649 1.049 0.306 0.514 0.144 1.836

Tumor boundary 0.418 0.402 1.081 0.298 1.519 0.691 3.337

Tumor echo –0.140 0.420 0.111 0.739 0.87 0.382 1.980

Capsule contact 0.906 0.422 4.615 0.032 2.475 1.083 5.657

RT 2.056 0.444 21.413 0.001 7.818 3.272 18.680

PI 2.705 0.489 30.603 0.001 14.95 5.734 38.978

AUC 2.474 0.471 27.562 0.001 11.875 4.715 29.911

TP 0.378 0.390 0.939 0.333 1.459 0.679 3.134

mTT 0.075 0.388 0.038 0.846 1.078 0.504 2.308

IIV 0.531 0.392 1.834 0.176 1.700 0.789 3.664

IDV 0.226 0.389 0.339 0.561 1.254 0.585 2.687

MVD 1.864 0.434 18.441 0.001 6.447 2.754 15.094

MVA 2.056 0.444 21.413 0.001 7.818 3.272 18.680

Variables	B	S.E.	Wald	P	OR	95% CI
Age	–0.999	0.405	6.071	0.014	0.368	0.166	0.815
Sex	–0.357	0.457	0.612	0.434	0.700	0.286	1.712
Tumor size	1.864	0.434	18.441	0.001	6.447	2.754	15.094
Tumor location
Left lobe			1.795	0.408
Right lobe	–0.726	0.547	1.763	0.184	0.484	0.166	1.413
Isthmus	–0.665	0.649	1.049	0.306	0.514	0.144	1.836
Tumor boundary	0.418	0.402	1.081	0.298	1.519	0.691	3.337
Tumor echo	–0.140	0.420	0.111	0.739	0.87	0.382	1.980
Capsule contact	0.906	0.422	4.615	0.032	2.475	1.083	5.657
RT	2.056	0.444	21.413	0.001	7.818	3.272	18.680
PI	2.705	0.489	30.603	0.001	14.95	5.734	38.978
AUC	2.474	0.471	27.562	0.001	11.875	4.715	29.911
TP	0.378	0.390	0.939	0.333	1.459	0.679	3.134
mTT	0.075	0.388	0.038	0.846	1.078	0.504	2.308
IIV	0.531	0.392	1.834	0.176	1.700	0.789	3.664
IDV	0.226	0.389	0.339	0.561	1.254	0.585	2.687
MVD	1.864	0.434	18.441	0.001	6.447	2.754	15.094
MVA	2.056	0.444	21.413	0.001	7.818	3.272	18.680

RT, rise time; PI, peak intensity; AUC, area under the curve; TP, time to peak; mTT, mean transit time; IIV, intensity increase velocity; IDV, intensity decrease velocity; MVD, microvessel density; MVA, microvessel area; B, B value; S.E, standard error; CI, confidence interval; OR, odds ratio.

Table 5

Multivariate analysis of risk factors for lymph node metastasis

Variables	B	S.E.	Wald	P	OR	95% CI
						Lower limit	Upper limit
Age	–0.417	0.722	0.334	0.564	0.659	0.160	2.715
Tumor size	1.500	0.702	4.567	0.033	4.481	1.132	17.731
Capsule contact	0.281	0.766	0.135	0.714	1.325	0.295	5.952
RT	1.936	0.739	6.866	0.009	6.929	1.629	29.479
PI	1.581	0.709	4.968	0.026	4.858	1.210	19.500
AUC	1.871	0.747	6.269	0.012	6.497	1.501	28.117
MVD	1.832	0.799	5.265	0.022	6.248	1.306	29.888
MVA	1.567	0.773	4.112	0.043	4.792	1.054	21.791

RT, rise time; PI, peak intensity; AUC, area under the curve; MVD, microvessel density; MVA, microvessel area; B, B value; S.E, standard error; OR, odds ratio.

Further, ROC curves of RT, PI, AUC, MVD and MVA for predicting cervical LNM were drawn (Fig. 1), and the significance of quantitative CEUS parameters in predicting cervical LNM was assessed (Table 6). The ROC curves showed significant statistical correlations between the five parameters (RT, PI, AUC, MVD, and MVA) and LNM in PTC patients. The AUC values of RT, PI, AUC, MVD and MVA for independently identifying LNM in PTC patients were 0.893, 0.853, 0.875, 0.859 and 0.819, respectively. The highest sensitivity and best specificity were observed in RT and PI, respectively.

Fig. 1

Receiver operating characteristic (ROC) curve for the predictive characteristics of lymphatic metastasis.

Table 6

Evaluation of predictive value via quantitative contrast-enhanced ultrasound features for cervical lymph node metastasis

Variables	AUC	95% CI	P	Sensitivity (%)	Specificity (%)
RT	0.893	0.834-0.953	0.000	98.4	68.1
PI	0.853	0.776-0.930	0.000	78.7	83.0
AUC	0.875	0.806-0.943	0.000	83.6	80.9
MVD	0.859	0.791-0.927	0.000	90.2	66.0
MVA	0.819	0.739-0.898	0.000	86.9	68.1

RT, rise time; PI, peak intensity; AUC, area under the curve; MVD, microvessel density; MVA, microvessel area; CI, confidence interval.

4 Discussion

PTC is a vessel-dependent lesion [18] whose growth, invasion and metastasis processes are closely related to neovascularization, which is the basis for rapid tumor growth [19]. With the increase of new vessels with thin walls and incomplete basement membrane in the tumor, tumor cells have access to blood rich in nutrients and oxygen and the contact surface of tumor cells escaping from the blood vessels increases, consequently rising the potential risk for tumor invasion and metastasis. Changes in the perfusion and capillary permeability caused by neovascularization are the pathological basis for imaging abnormalities [20]. Therefore, MVD contributes to determining the biological behavior and prognosis of PTC [21] and plays a vital role in screening high-risk cases and guiding clinical practice, especially when performed by experienced examiners. Unfortunately, clinicopathological measurement of MVD can only be performed postoperatively but not preoperatively.

Ultrasonography has become the first choice of imaging examination for screening thyroid diseases due to its advantages of convenience, low cost, noninvasiveness and no radioactivity. SonVue is a microbubble contrast agent with an average diameter of 2.5μm and hemodynamics similar to red blood cells. This agent can accurately reflect the blood perfusion status of tissues and sensitively show the blood flow status and tissue perfusion of lesions; thus, it is clinically significant for the early detection and diagnosis of thyroid cancer [22]. With the rapid popularization of CEUS in thyroid diseases, its role in the preoperative evaluation of PTC nodules has become increasingly prominent and was found to be markedly superior to conventional ultrasound. Studies [20] have confirmed that CEUS could be used to evaluate the microcirculation in tumors, but related literature is quite limited. In this study, based on the patients’ pathological results of lymph nodes, they were divided into an LNM+(n = 61) or LMN–(n = 47) group. Comparison analysis showed no statistical difference in sex, tumor location, tumor boundary and tumor echo between the two groups. However, patients in the LNM+group were younger, had larger tumor diameter, and greater proportion of capsule contact than those in the LMN–group.

Changes in tissue perfusion induced by differences in anatomical structure, spatial distribution, and function between the microvessels of the lesion and normal thyroid parenchyma in PTC patients are the basis for CEUS diagnosis [23]. In this study, we found that RT, PI and AUC were markedly higher in the LNM+group than in the LMN–group, but no significant difference was observed in TP, mTT, IIV and IDV. Zhou et al. [24] reported that PI could reflect the MVD of PTC, and Tian et al. [25] found that a greater number of effective microvessels in the lesion was associated with higher PI and AUC values. According to the above studies, PI and AUC were able to indirectly and noninvasively reflect the MVD and MVA of a lesion to a certain extent, indicating that these CEUS perfusion parameters could reflect tumor angiogenesis to some degree. Our analysis showed that the MVD and MVA in the LNM+group were significantly higher than those in the LMN–group. This observation could be explained by the fact that lymph node proliferation, growth, invasion, and metastasis mainly depend on angiogenesis; thus, the number of new blood vessels in the LNM+group was considerably greater than that in the LMN–group. New vessels provide a basis for tumor growth and access for the invasion and metastasis of cervical lymph nodes, leading to higher blood perfusion volume and intensity of nodules in the metastatic group [26]. Shi et al. [10] demonstrated that the PI and AUC in the marginal zone of nodules in the PTC patients with cervical LNM were (8.39±2.19) dB and (220.69±83.56) dB s, respectively, which were significantly higher than those in the non-metastasis group (6.52±2.32) dB and (168.29±52.95) dBs. These findings are consistent with the results of this present study, indicating that MVD, MVA, PI and AUC could be used to evaluate cervical LNM. In addition, univariate and multivariate logistic regression analyses were performed on factors such as sex, age, tumor size, number of lesions, capsule contact, calcification, and related contrast parameters, and the results showed that tumor size, RT, PI, AUC, MVD and MVA were independent risk factors for LNM, while age and capsule contact were not independently associated with LNM. ROC was also drawn to further analyze the value of PI, AUC, RT, MVD and MVA in predicting cervical LNM, and the results confirmed that RT had the best overall predictive performance.

Based on the above results, cervical LNM of PTC was deduced to be closely related to CEUS perfusion parameters (RT, PI, AUC), MVD, and MVA; CEUS thus could act as a preoperative evaluation method for PTC. However, there were some limitations in this study that should be clarified. The lack of follow-up, small sample size, and individual differences in blood circulation and tumor development process may have had certain impacts on the CEUS quantitative parameter results. In addition, considering this was a retrospective study, we could not determine inter-observer variability between the examiners when assessing MVD and MVA. Thus, more studies with a large sample, more pathological subtypes, and using prospective settings are needed to confirm our results.

5 Conclusions

Tumor size, RT, PI, AUC, MVD and MVA are identified as risk factors for cervical LNM, of which RT demonstrated the best predictive performance. CEUS is an important non-invasive and preoperative method for evaluating PTC, while MVD and MVA detection are vital parameters to preoperatively determine the LN status of PTC patients.

Funding

This work was supported by Liaoning Provincial Education Department Project Funding (JYTQN2020014).

Conflicts of interest

All the authors certify that there is no actual or potential conflict of interest in this article.

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