Evaluating Breast Cancer Biomarkers Using Contrast-enhanced Ultrasound and Shear Wave Elastography

Summary

This protocol demonstrates the combined application of contrast-enhanced ultrasound and shear wave elastography for evaluating biological prognostic factors, including Ki-67, HER2, and ER in breast cancer patients.

Abstract

The purpose of this protocol was to evaluate the relationship between quantitative parameters derived from shear wave elastography (SWE) and contrast-enhanced ultrasound (CEUS) and biological prognostic markers in breast cancer. The study included 68 patients with breast cancer and 68 healthy controls. All patients underwent SWE and CEUS imaging, and surgical specimens from the breast cancer group were analyzed using immunohistochemistry to assess the expression of estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2), and Ki-67.

Results demonstrated that breast cancer tissue exhibited significantly different elastic and perfusion characteristics compared to normal breast tissue. Specifically, the cancer group showed lower E_mean values and higher E_max, E_min, peak intensity, and mean transit time (MTT) values. Correlation analysis revealed that Ki-67 expression was positively correlated with E_mean, E_max, peak, and MTT, while ER was negatively correlated with all parameters except peak intensity. HER2 expression showed positive correlations with all measured imaging parameters. These findings suggest that SWE and CEUS can provide non-invasive, complementary insights to support molecular characterization and clinical evaluation of breast cancer.

Introduction

Breast cancer ranks as one of the most prevalent cancers affecting women worldwide, with incidence rates steadily rising¹. Recent epidemiological studies show that breast cancer is surpassed only by cervical cancer in incidence and remains the leading cause of cancer-related mortality in women overall^2,3. The pathogenesis of breast cancer involves multiple interacting factors, including hereditary traits, endocrine influences, and environmental exposures⁴. With advances in precision medicine, there is a growing interest in understanding the relationships between imaging phenotypes and molecular features in breast cancer management⁵.

The purpose of this study was to investigate whether shear wave elastography (SWE) and contrast-enhanced ultrasound (CEUS) can serve as non-invasive imaging biomarkers for characterizing key molecular markers of breast cancer, including the estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2), and Ki-67. This investigation sought to determine whether imaging can correlate with these biomarkers, which guide treatment decisions and carry prognostic significance, and potentially serve as an adjunct or alternative to biopsy. Validating this correlation could provide an accessible, reproducible method to evaluate breast cancer at the molecular level through advanced imaging technologies.

The clinical presentation typically begins with the discovery of a painless breast mass, which occurs in approximately 80% of women⁶. As the disease progresses, additional signs may include nipple discharge, skin dimpling caused by traction from Cooper's ligaments, peau d'orange due to lymphatic obstruction, and, in advanced cases, nipple retraction accompanied by skin changes such as pruritus, vesiculation, and ulceration⁷.

Current imaging modalities used in breast cancer diagnosis include ultrasonography, mammography, magnetic resonance imaging (MRI), and nuclear medicine techniques^8,9,10. Among these, ultrasonography remains the most prevalent because it is non-invasive, does not expose patients to radiation, and offers high reproducibility and resolution for distinguishing between fatty and dense glandular tissues. SWE enhances traditional ultrasound by providing quantitative stiffness measurements based on Young's modulus, derived from the velocity of shear waves¹¹. It reduces operator dependence by using acoustic radiation force to induce shear waves, improving reproducibility¹². CEUS visualizes tumor vasculature and perfusion patterns, contributing to lesion detection and characterization, and aiding in the differentiation of benign and malignant tumors¹³.

The utility of molecular immunohistochemistry markers is critical for determining prognosis and guiding treatment in breast cancer¹⁴. ER, progesterone receptor (PR), Ki-67, and HER2 are key markers with established roles in clinical decision-making¹⁵. Previous studies suggest that ultrasound imaging characteristics may reflect the expression of these molecular markers, supporting the idea that imaging phenotypes mirror underlying tumor biology^16,17,18. This study aimed to apply an appropriate protocol to assess correlations between CEUS and SWE imaging parameters and a range of biological prognostic markers, contributing to the evolving field of radiogenomics.

Protocol

The research was completed in accordance with the Declaration of Helsinki and institutional guidelines for clinical investigations. The protocol for this research was submitted to and approved by the Institutional Ethical Review Committee of The Third Affiliated Hospital of Jinzhou Medical University.

Written informed consent was obtained from all participants prior to enrollment, including permission to utilize de-identified clinical, imaging, and pathological information for future publication. Throughout this research, patient confidentiality was maintained at all times.

The recruitment and assessment process for patient participation also adhered to the 2021 Edition of the Chinese Anti-Cancer Association Guidelines.

1. Patient selection and preparation

1. Include patients with a definitive diagnosis of breast cancer, adequate ultrasound image quality, and complete clinical data.
2. Exclude patients with any of the following: recent breast surgery, breast augmentation, mastitis, recurrent breast cancer, lesions adjacent to scars, cardiovascular or hepatorenal disease, pregnancy, or if pathology examinations occurred more than 30 days prior to imaging.
3. Record each participant's age, body mass, and clinical history prior to imaging.
4. Position the patient supine with both arms raised above the head.
5. Apply coupling gel liberally to the breast to ensure optimal acoustic transmission and image clarity.

2. Shear wave elastography (SWE) examination

NOTE: See the Table of Materials for the ultrasound system and probe.

1. Begin with a routine B-mode ultrasound to locate and characterize the lesion.
2. Activate SWE mode and position the probe perpendicular to the skin directly above the lesion, without applying external pressure.
3. Adjust the sampling frame to fully include the lesion and an adequate margin of surrounding tissue.
4. Monitor the system's quality control map to ensure sufficient shear-wave propagation.
5. After the image is stabilized, instruct the patient to hold their breath for 3-5 s.
6. Hold the probe steady during this time to stabilize the image.
7. Save the color-coded elastography image and outline the lesion using the system's quantitative analysis tool.
8. Record the maximum (Emax), minimum (Emin), and mean (Emean) elasticity modulus values for the lesion.
9. Repeat each measurement three times and calculate the average.
10. Follow published international guidelines for shear wave elastography acquisition and quality control to ensure measurement consistency and reproducibility¹⁹.

3. Contrast-enhanced ultrasound (CEUS) assessment

1. Identify the optimal imaging plane showing the lesion's maximal cross-sectional area.
2. Reconstitute the contrast agent according to the manufacturer's instructions: add 5 mL of saline and withdraw 2.4 mL into a syringe.
3. Establish venous access in the antecubital vein with an appropriate cannula.
4. Set the ultrasound system to contrast imaging mode with the mechanical index adjusted for microbubble visualization.
5. Administer 2.4 mL of contrast agent via rapid bolus, followed by 5 mL of saline flush.
6. Begin continuous digital recording immediately to capture the wash-in and wash-out phases.
7. Record for a minimum of 3 min to document complete enhancement dynamics.
8. Identify peak enhancement and place four measurement points (superior, inferior, medial, lateral) around the lesion at intervals of approximately 3-4 mm.
9. Analyze time-intensity curves using the quantification software.
10. Generate peak Intensity and mean transit time (MTT) metrics from the quantification analysis.
11. Follow validated contrast-enhanced ultrasound (CEUS) acquisition and quantitative analysis methodologies established in prior clinical studies to ensure consistency and reproducibility¹⁵.
12. Use CEUS imaging and quantitative analysis to complement standard breast imaging by assessing vascularity and perfusion.

4. Tissue sampling and immunohistochemical analysis

1. Obtain tissue samples via core needle biopsy or surgical excision as per clinical standards.
2. Fix samples in 10% neutral-buffered formalin for 6-24 h at room temperature.
3. Dehydrate, clear, and embed the samples in paraffin according to standard histopathological processing protocols²⁰.
4. Slice the paraffin-embedded tissue blocks into 4 µm sections using a microtome.
5. Mount the sections on positively charged glass slides.
6. Deparaffinize the slides in xylene (2 x 10 min each) at room temperature, followed by rehydration through a graded ethanol series (100%, 95%, 85%, and 70% ethanol; 5 min per step) and rinse in distilled water.
   NOTE: All steps were performed at room temperature without agitation.
7. Perform antigen retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0).
8. To block endogenous peroxidase activity, incubate the tissue sections in a 3% hydrogen peroxide solution for 10 min.
9. Apply primary antibodies against ER, HER2, and Ki-67 diluted in PBS containing 1% BSA (ER 1:100; HER2 1:200; Ki-67 1:200) and incubate overnight at 4 °C in a humidified chamber without agitation, in accordance with manufacturers' instructions.
10. Incubate with horseradish peroxidase-conjugated secondary antibodies diluted in PBS containing 1% BSA (1:200) for 30 min at room temperature in a humidified chamber without agitation.
11. Visualize antibody binding using 3,3'-diaminobenzidine (DAB) chromogen and counterstain with hematoxylin for 30 s.
12. Evaluate staining under 400× magnification using standard criteria: ER positivity >10% nuclear staining; HER2 assessed according to ASCO/CAP guidelines; and Ki-67 expressed as the percentage of positive tumor nuclei.
13. Perform immunohistochemistry using a standardized protocol, including heat-induced antigen retrieval (citrate buffer, pH 6.0, or EDTA buffer, pH 9.0, 95-98 °C for 20 min), endogenous peroxidase blocking with 3% hydrogen peroxide for 10 min at room temperature, incubation with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies, 3,3′-diaminobenzidine (DAB) chromogenic detection, hematoxylin counterstaining, dehydration through graded ethanol, and coverslipping, applied identically to all samples, in accordance with established immunohistochemical methodology^21,22.

5. Statistical analysis

1. Express continuous variables as mean ± standard deviation (SD).
2. Use independent samples t-tests to compare SWE and CEUS parameters between cancer and control groups after assessment of normality.
3. Perform Spearman's correlation analysis to assess associations between ultrasound parameters and immunohistochemical markers (ER, HER2, Ki-67).
4. Generate receiver operating characteristic (ROC) curves for each ultrasound parameter using cancer status as the classification variable.
5. Calculate the area under the curve (AUC) values, and derive sensitivity and specificity at the optimal cutoff determined by Youden's index (J = sensitivity + specificity − 1).
6. Calculate positive predictive value (PPV) and negative predictive value (NPV). based on the selected cutoff values.
7. Consider a two-sided P value < 0.05 to be statistically significant. Present results in tables and figures.

Results

Patient characteristics

Implementation of this protocol in 68 breast cancer patients and 68 healthy controls demonstrated significant differences in elastographic and perfusion parameters between the groups. All breast cancer cases presented as unilateral lesions, with a mean diameter of 20.78 ± 5.37 mm. Histopathological analysis revealed 49 cases of invasive ductal carcinoma, 9 cases of lobular carcinoma, 6 cases of intraductal carcinoma, 3 cases of mucinous carcinoma, and 1...

Discussion

This protocol clearly demonstrates the relationship between quantitative ultrasound parameters and molecular prognostic markers in breast cancer, providing a well-defined framework for non-invasive disease characterization. The correlations between imaging parameters and immunohistochemical markers reflect underlying biological changes that affect both the mechanical properties (tissue stiffness) and perfusion characteristics observed during imaging.

Epidemiological studies show that breast ca...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Citrate Buffer (pH 6.0)	12364	Thermo Fisher Scientific	www.thermofisher.com
EDTA Buffer (pH 9.0)	88763	Thermo Fisher Scientific	www.thermofisher.com
Hydrogen Peroxide (3%)	998765	Sigma-Aldrich	www.sigmaaldrich.com
L11-3 Probe (Ultrasound)	ABC987	Myriad Ultrasound	www.myriad.com, 4-15 MHz
SonoVue (Contrast Agent)	12345	Bracco Diagnostics	www.bracco.com
SPSS Statistical Software	N/A	IBM	www.ibm.com