|Ahead of print publication
Comparison of p63 immunohistochemistry and shear wave elastography in the diagnosis of indeterminate breast lesions: A prospective study
Ranjith Kumar Sridhar1, R Chandru1, V Pavithra2, D Rajiv Raj1, Bhawna Dev3, Sandhya Sundaram2, R Ramya1
1 Department of General Surgery, Sri Ramachandra Medical College and Research Institute, Chennai, Tamil Nadu, India
2 Department of Pathology, Sri Ramachandra Medical College and Research Institute, Chennai, Tamil Nadu, India
3 Department of Radiology and Imaging Sciences, Sri Ramachandra Medical College and Research Institute, Chennai, Tamil Nadu, India
|Date of Submission||11-Nov-2021|
|Date of Decision||05-May-2022|
|Date of Acceptance||06-May-2022|
|Date of Web Publication||13-Oct-2022|
D Rajiv Raj,
Sri Ramachandra Medical College and Research Institute, No. 1, Ramachandra Nagar, Porur, Chennai - 600 116, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Purpose: To evaluate the accuracy of breast shear wave elastography (SWE) and p63 immunohistochemistry (IHC) in the diagnosis of indeterminate breast lesions.
Methods: Based on detailed clinical examination and a combination of X-ray mammography/B-mode ultrasound with SWE, a total of 40 patients with breast lumps (BI-RADS 4) were included. Patients with previous diagnosis of breast cancer and a previous history of surgery, chemotherapy, or radiotherapy in the same breast as the present lesion were excluded. Core needle biopsy of the breast lesion was performed, and p63 IHC staining was performed. A final histopathological report of the definitive procedure was considered as the gold standard. The sensitivity, specificity, positive (PPV) and negative predictive values (NPV), and accuracy were calculated for each modality.
Results: The mean age of the patients included in the study was 50.85 ± 13.53 years. Of the 40 patients recruited, 23 were clinically malignant and 17 were benign. The sensitivity, specificity, PPV, NPV, and accuracy of SWE were 91.3%, 94.1%, 95.5%, 88.9%, and 92.5% and those of p63 IHC were 95.7%, 100%, 100%, 94.4%, and 97.5%, respectively. Overall, the parametric values were higher for p63 IHC as compared to clinical examination and elastography. The area under the ROC curve (AUC) for p63 IHC (.978) was higher than those for SWE (.927) and clinical examination (.898).
Conclusion: SWE and p63 IHC are highly reliable novel modalities that demonstrate enhanced diagnostic accuracy of indeterminate breast lesions aiding in the early initiation of appropriate treatment and reducing the number of women subjected to biopsy or short-term follow-up for benign-appearing solid breast lesions.
Keywords: BI-RADS, elastography, indeterminate breast lesion, p63 immunohistochemistry
|How to cite this URL:|
Sridhar RK, Chandru R, Pavithra V, Raj D R, Dev B, Sundaram S, Ramya R. Comparison of p63 immunohistochemistry and shear wave elastography in the diagnosis of indeterminate breast lesions: A prospective study. J Can Res Ther [Epub ahead of print] [cited 2022 Dec 9]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=358581
| > Introduction|| |
Breast cancer accounts for 29% of all newly diagnosed cancers in females and is seen responsible for 14% of the cancer-related deaths in women aged between 20 and 59 years. The incidence rates in India begin to rise in the early thirties and peak at the age of 50–64 years. For every two women newly diagnosed with breast cancer, one woman dies of it in India. With the upward trend in the incidence of breast cancer, early detection and characterization of the breast lesion are of utmost importance from both the diagnostic viewpoint and the viewpoint of the patient's psychological well-being. The widespread adoption of screening mammography over the past decade has led to an epidemic of diagnosis of carcinoma of the breast. Mammography is restricted in patients with dense breast tissues, thereby reducing its diagnostic performance. Hence, ultrasound has become an important adjunct to mammography. Breast elastography is an imaging modality that can quantify tissue stiffness or hardness and is analogous to a clinical examination which allows only the subjective judgment of the stiffness of a lesion.
Shear wave elastography (SWE) allows the measurement of the propagation speed of shear waves within the tissue to locally quantify its stiffness in kilopascals (kPa) or meters per second (m/sec). Within a given region of interest (ROI), a variety of stiffness parameters can be measured, including the mean stiffness (Emean), maximum stiffness (Emax), and standard deviation (SD). Several studies have shown that those quantitative parameters using Young's modulus of elasticity (kPa) in SWE improved the diagnostic accuracy of breast ultrasound. Benign lesions tend to have lower elasticity values than malignant lesions. However, the elasticity value of adenosis or stromal fibrosis mimicked a malignant range, whereas that of ductal carcinoma in situ (DCIS) and apocrine cancer showed lower kPa values. The functional information provided by elasticity evaluation is much useful for atypical benign or malignant lesions (BI-RADS 3 or 4a). Some studies show that the biopsy rate could be reduced in the case of BI-RADS 3 or 4a lesions after the application of SWE. According to the BI-RADS categorization, BI-RADS 2 and 3 lesions are considered benign, whereas BI-RADS 5 lesion suggests malignancy. It is the BI-RADS 4 lesions that present a diagnostic dilemma requiring intensive clinico-radio-pathological analysis for arriving at the correct diagnosis and appropriate treatment.
Although both the ductal and lobular structures of the breast are made up of a double-cell layer (an inner luminal cell and an outer myoepithelial cell), it has been conclusively demonstrated in a series of studies over the past 2 decades that the presence of an intact peripheral myoepithelial cell layer distinguishes all normal and benign breast lesions as well as DCIS. Invasive carcinoma is defined by the loss of the outer myoepithelial layer, and the demonstration of this loss has been documented by immunohistochemical (IHC) techniques. In normal breasts, p63 (a homolog of p53) is only expressed in myoepithelial cells. The advantage of employing p63 is that it is nuclear localized and does not stain smooth muscle cells such as myofibroblasts or blood vessels. As a result, it possesses about 100% specificity, although its sensitivity has been reported to be around 90%. The so-called “focal gaps” in staining in the myoepithelial layer, which are partly attributable to the plane of section, reveal this.
With this background, our study was performed to evaluate the accuracy of breast SWE as an adjunct to mammography and p63 IHC in the diagnosis of indeterminate BI-RADS 4 breast lesions.
| > Materials and Methods|| |
It was a prospective study conducted at the Department of General Surgery after obtaining due ethical clearance from the Institutional Ethics Committee (CSP-MED/18/APR/43/77). The cases were collected between June 2017 and October 2019 after obtaining informed consent from the patient. Women presenting with a lump in the breast with X-ray mammogram/ultrasound showing BI-RADS 4 lesions were included in the study. Patients already diagnosed to have carcinoma breast or phyllodes tumor, patients with a previous history of surgery in the same breast as the present lesion, and those with a previous history of chemotherapy or radiotherapy to the breast were excluded from the study.
A detailed clinical history and demographic data were collected from all the patients, and a thorough physical examination of the breasts was performed.
The X-ray mammogram of bilateral breasts was performed using Fujifilm Amulet Innovality with tomosynthesis. Ultrasound of the breasts with SWE was performed using a Toshiba APLIO 500, 7-14 MHz linear transducer by a single expert radiologist. After B-mode ultrasound, the operator placed the transducer perpendicular to the lesion without pressure, while maintaining only slight contact with the skin in order to minimize compression artifacts. A mechanical impulse from the probe generated shear waves in the breast tissue, and their propagation was analyzed in a real-time color map. The breast lesion stiffness was assessed by imposing an ROI in the lesion. The ROI for the measurement was positioned including as much nodule area as possible, avoiding signal loss corresponding to an artifactual effect. At least three measurements for each lesion in the stiffest portion or in the adjacent 3–5 mm of the surrounding tissue were taken, and the median value was taken as the most reliable [Figure 1.1] and [Figure 1.2]. The values were expressed in kPa. The ultrasound images and the most appropriate SWE frames and cine loops were transferred to the local picture archive and communication system (PACS).
Core needle biopsy of the breast lesion was performed with ultrasound guidance using automated spring-loaded biopsy devices, Trucut Needle 14 G, 10 cm [Figure 1.3]. Tissue samples collected were sent to the Department of Pathology for histopathological examination. p63 IHC staining was performed using the p63 (Clone - 4A4) Mouse Monoclonal Antibody (PathnSitu Biotechnologies, CA, USA) as the primary stain and a Biogenex (Fremont, CA, USA) Super Sensitive Polymer-HRP IHC Detection System (HRP/DAB) as the secondary stain on the biopsy specimen. Positive staining was evidenced by the presence of myoepithelial cells turning brown as in benign lesions [Figure 2.1], and negative staining was evidenced by the absence of the brown color as in malignant lesions [Figure 2.2]. However, in DCIS, there was only peripheral positivity as depicted [Figure 2.3].
Based on the core needle histopathological examination report, the patients were taken up for the definitive procedure of surgical excision for benign lesions and mastectomy/breast-conserving surgery for malignant lesions, and the final histopathological examination report was considered the gold standard.
The collected data were analyzed with the SPSS v17 statistics software. To describe the data, descriptive statistics frequency analysis was used. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of each modality to be analyzed were calculated. To find the significance of the modalities, the Pearson Chi-Square test was used. To correlate the modalities applied in the diagnosis of the breast lump, a receiver operating characteristic (ROC) curve was used to identify the ideal modality.
| > Results|| |
A total of 40 patients fulfilled the inclusion criteria and were included in the study. A total of 23 patients were clinically diagnosed as malignant and 17 as benign. The youngest patient in the study population was 24 years old, and the oldest patient was 78 years of age with a mean age of 50.85 ± 13.53 years. On correlating the BI-RADS score with the final histopathological examination report, there were 14 benign and three malignant cases in the 4a category (% malignancy 17.6), three benign and six malignant cases in the 4b category (% malignancy 66.6), and 14 malignant cases in the 4c category [comparison shown in [Table 1]]. The mean elasticity value (in kPa) for benign and malignant lesions was 62.17 ± 30.20 and 112.36 ± 15.32, respectively. The comparison of performance and accuracy among the modalities is listed in [Table 2].
The area under the ROC curve (AUC) for p63 IHC (0.978) is higher than that of SWE (0.927) and clinical examination (0.898). This was statistically significant with a P value of 0.000. This shows that p63 IHC is the most reliable modality in comparison to SWE in the diagnosis of indeterminate breast lesions.
| > Discussion|| |
With an increase in the incidence of breast cancer in the present era, it is vital to accurately diagnose a breast lesion and initiate early appropriate treatment, thereby leading to a positive outcome. Hence, clinical examination alone is not sufficient in the diagnosis of a breast lump. Our study demonstrated the usefulness of SWE combined with mammography/ultrasound BI-RADS and p63 IHC as an adjunct to clinical examination in the diagnosis of BI-RADS 4 indeterminate breast lesions.
On extensive reviewing of the literature, the study by Feldmann A et al. showed that adding SWE to the BI-RADS score helped downgrade especially the BI-RADS 4a and 4b breast lesions to benign and accurately detecting the malignant BI-RADS 4 lesions. This increased the specificity of SWE, while preserving the sensitivity and NPV as compared to the BI-RADS ultrasound test alone. Similarly, a study by Youk J H et al. performed only on BI-RADS category 4 breast lesions showed that combined B-mode ultrasound and SWE had a significantly higher AUC than stand-alone B-mode ultrasound or SWE, thereby increasing its diagnostic performance. In the study by Berg et al., they considered using SWE features to influence treatment of BI-RADS category 3 and 4a lesions as the SWE feature would not change the treatment of clearly benign (BI-RADS 2) and moderate to high suspicion (BI-RADS 4c or 5) breast lesions. Similarly, in this study, we utilized SWE features to influence the management of BI-RADS category 4a, 4b, and 4c lesions. The study by Zhi H et al. highlights the diagnostic efficacy of the combination of SWE and B-mode ultrasound, which was superior to mammography or B-mode ultrasound alone. The statistical data of the combined modalities showed a sensitivity of 89.7%, a specificity of 95.7%, a PPV of 89.7%, and an NPV of 95.7% with an accuracy of 93.9%, which are comparable to our study having a sensitivity of 91.3%, a specificity of 94.1%, a PPV of 95.5%, an NPV of 88.9%, and an accuracy of 92.5%. The study by Evans A et al. showed that the sensitivity of SWE was 95%, the specificity was 77%, the PPV was 88%, and the NPV was 90% with an accuracy of 89%. When combined with BI-RADS grayscale, their study showed a superior sensitivity (100%) and NPV (100%).
In our study, p63 IHC showed a sensitivity of 95.7%, a specificity of 100%, a PPV of 100%, and an NPV of 94.4% with an overall accuracy of 97.5%, which is comparable to a study by Kim S K et al., which had a sensitivity of 93.8%, a specificity of 99%, a PPV of 94.1%, an NPV of 94.1%, and an accuracy of 96.4%. Other studies by Stefanou D et al., Wang X et al., Barbareschi M et al., and Werling R W et al. showed p63 as a very specific myoepithelial marker which is positive in all benign lesions and negative in malignant lesions, and the peripheral rim is positive (to a lesser extent compared to benign lesions) in DCIS.,,,
In our study, we observed one false positive p63 staining. This can be attributed to sampling error despite ultrasound guidance in which the malignant region was not sampled. Because there was a high clinical suspicion and the elasticity value in the malignant range, the patient underwent wide local excision of the breast lump with a frozen section and then proceeded to modified radical mastectomy.
A diagnostic dilemma occurs in DCIS lesions in which the peripheral positivity of p63 can be misinterpreted for positivity in benign lesions, and hence, a careful examination in such lesions is imperative. This shows the importance of triple assessment in a breast lesion and the usefulness of elastography as an adjunct to histopathological examination in the diagnosis of suspicious breast lesions (BI-RADS 4). However, histopathological examination remains the gold standard investigation for the exact diagnosis.
Limitations of the study
The study population was relatively small, and a larger cohort will strengthen the study. p63 IHC reacts with a small but significant subset of breast carcinoma tumor cells such as metaplastic carcinomas with spindle cell or squamous differentiation; however, this aberrant reactivity rarely causes diagnostic difficulty. Multi-variate analysis for the evaluation of confounding factors was not performed in our study. Lesion depth and breast thickness, which are known to be able to influence diagnostic performance, were also not considered. More clinical studies on how best to combine SWE and p63 IHC to enhance benign and malignant differentiation of indeterminate breast lesions are necessary.
| > Conclusion|| |
In conclusion, p63 is a sensitive and specific myoepithelial marker and may be included in IHC panels aiming to identify myoepithelial cells in indeterminate breast lesions. SWE is also a very helpful adjunct to B-mode ultrasound of the breast in differentiating the malignant from benign BI-RADS 4 breast lesions. From our study, SWE and p63 IHC are two novel modalities that show enhanced diagnostic accuracy of indeterminate BI-RADS 4 breast lesions and help in the early initiation of appropriate treatment and also reduce the number of women subjected to biopsy or short-term follow-up for benign-appearing solid breast lesions.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]