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Katarzyna Dobruch-Sobczak * / Hanna Piotrzkowska-Wróblewska / Ziemowit Klimoda / Wojciech Secomski / Piotr Karwat / Ewa Markiewicz-Grodzicka / Agnieszka Kolasińska-Ćwikła / Katarzyna Roszkowska-Purska / Jerzy Litniewski
Citation Information : Journal of Ultrasonography. Volume 19, Issue 77, Pages 89-97, DOI: https://doi.org/10.15557/JoU.2019.0013
License : (CC-BY-NC-ND 4.0)
Received Date : 13-February-2019 / Accepted: 10-April-2019 / Published Online: 28-June-2019
Neoadjuvant chemotherapy (NAC) was initially used in locally advanced breast cancer (LABC) and the inflammatory form of cancer to downstage locally inoperable disease. Currently, it is recommended for patients with Stage 3 and with early-stage disease with HER-2 (human epidermal growth factor receptors positive or triple-negative (TNBC) breast cancer(1,2). NAC reduces the tumor mass by inducing intracellular damage, which causes cell death and degeneration. Response to treatment increases chances for surgery, makes breast-conserving surgery more feasible and might lead to eradication of micrometastatic disease and reduction of the risk of dissemination(3,4). This treatment gives us the opportunity to observe the tumor shrink, both palpably and on imaging, enabling an assessment of clinical response. It also provides additional information about chemosensitivity of cancer tissue to different NAC programs, allowing to modify the subsequent treatment. However, the response to NAC is heterogenous, and objective assessment is necessary to distinguish between responders and non-responders and, if necessary, to modify treatment. Response is defined and classified on the basis of changes in cancer cellularity and is divided into two categories: pathological partial response (pPR) subdivided into G1 (<9% reduction), G2 (10–29%), G3 (30–90%) and G4 (>90%), and pathological complete response (pCR)(5). The rate of responding and non-responding patients varies: pCR is seen (depending on the results presented in the literature) in 10–31% of patients, while pPR is seen in 69% to 100% of patients(6–8). (In our study G1 was adopted as non-responding patients).
Currently, in the monitoring of patients treated with NAC, clinical breast examination (CBE), mammography (MMG), traditional B-mode ultrasound imaging (US), magnetic resonance imaging with a contrast agent (CA-MRI), or diffusion-weighted magnetic resonance imaging (DW-MRI) can be used(9). CBE has been shown to be a subjective technique with limited efficacy, especially in tumors smaller than 2 cm: about 60% of residual breast cancers are undetectable by CBE(10).
Ultrasound is considered a more accurate method in assessing tumor size and monitoring residual breast tumors compared to CBE or MMG(11). The literature analyzing the usefulness of an ultrasound examination together with MMG describes an increase in the probability of pCR prediction by up to 80%(12). A promising method for observing regression of the disease is contrast-enhanced ultrasound, but this test is not commonly used and not recommended by EFSUMB(13).
Sonoelastography is another ultrasound technique used to monitor effectiveness of NAC. The published results suggest that a reduction in tumor stiffness observed by shear wave elastography (SWE) and strain elastography allows, with similar performance, to predict the response of the disease to NAC(14–18). Evans et al.(14) have demonstrated, using SWE, that a decrease in breast cancer stiffness evaluated after the 3rd course of NAC was a predictor of pCR with sensitivity of 59% and specificity of 85%. Jing et al. have demonstrated that decreasing stiffness in SWE (relative changes) could effectively predict the response to NAC after 2 cycles(17).
Among the available radiological methods, the monitoring of tumor response during NAC using magnetic resonance imaging (MRI) is more accurate in comparison to CBE, US, or MMG. However, the access to MRI may be limited, and underestimation of residual disease may affect up to 20% of patients(19,20). The limitations of radiological methods may be due to the fact that the tumor size and architecture changes are delayed with respect to cell death which begins several hours or days after the start of the treatment(21). It has been shown that under the influence of NAC, the tumor microstructure, including cell number, changes before its macroscopic dimensions are affected(21,22). In patients with pCR, areas of early tumor invasion display fibrosis and edema of the stroma with increased vascularization and infiltration of inflammatory cells(23). For non-responding patients (pPR G1), tumor cells remain almost unchanged. However, in patients with pPR: G2, G3, G4, there is a change in the percentage of enlarged, multinucleated and neoplastic cells(23,24).
New, easy diagnostic tools are sought that will differentiate patients who respond to treatment from non-responders to NAC with high accuracy and at an early stage of treatment. In the literature, there are publications on the usefulness of quantitative ultrasound (QUS) techniques for monitoring reactions to NAC(24–27). In the case of a classic B-mode examination, the image is created on the basis of an envelope of radio-frequency (RF) signals, which is subjected to intensive downstream processing. Filtration, log-compression, interpolation, and image enhancing algorithms are used to reduce noise and reveal details of the tissues on the ultrasound scanner screen. These procedures, however, reduce the amount of information about the examined tissues, which can be obtained from the analysis of original RF signals. QUS techniques use raw RF echo signals back-scattered on elements included in examined tissues, such as neoplastic cell clusters or elements of fibrous stromal tissue in the mammary gland.
The theory of acoustic scattering in relation to tissue biology and the assessment of the usefulness of various ultrasound techniques for the study of cellular density has been analyzed by, among others, Oelze et al.(28). Since then, many QUS methods have been developed based on the analysis of the scattered echo with the aim to characterize the microstructure and elastic properties of tissues. Ultrasonic parameters determined from the scattered echo that can characterize the microstructure of tissues include statistical parameters of the echo envelope(29,30), texture parameters(31), and scattering parameters. The latter can effectively predict the response of tumor tissue to the used treatment(26,31).
The aim of our study was to evaluate patient responses to NAC using different ultrasound techniques, namely the assessment of tumor size in B-mode imaging, stiffness assessment in elastography examination and using quantitative ultrasound parameter. As a quantitative measure, we applied the integrated backscatter coefficient (IBSC), whose value depends on the quantity, shape, organization and size of the scattering elements.
The study protocol was approved by the institutional review board of the Maria Skłodowska-Curie Memorial Cancer Centre and Institute of Oncology in Warsaw, Poland. All patients gave their written consent to participate in the study. From April 2016 to November 2017, 10 patients aged 32 to 75 (mean age 52.9) with a total of 13 tumors (one bifocal lesion, one trifocal lesion) were deemed eligible for NAC at the Oncology Clinic. AT (doxorubicin, docetaxel), AC (doxorubicin, cyclophosphamide) and paclitaxel were used in the treatment, according to international guidelines. All patients underwent simple mastectomy with lymphadenectomy.
All patients underwent core needle biopsies (CNB) after administration of 2% lidocaine, using a biopsy gun needle (14G diameter – Pro-Mag). Three to five cores were taken from each lesion. After surgery (simple mastectomy), surgical specimens were immediately fixed in 10% buffered formalin. Representative sections from these samples were processed and routinely stained for histopathological (microscopic) examinations (HE). All tumor samples were evaluated by the same pathologist. Based on the pathological assessment of breast tissue the following information was obtained: grade of malignancy, cancer subtype, reduction in cancer cells, and residual tumor size (Tab. 1, Tab. 2, Tab. 3). In order to categorize the tumor’s pathological response to NAC, changes in cellularity of tumors were quantified using the samples obtained from the CNB before treatment and the material obtained after the treatment and surgery, using the Miller-Payne scale(5). In histopathological examination after NAC, tumors were classified into two categories: pathological partial response (pPR) and pathological complete response (pCR). pPR was sub-divided into G1, G2, G3, or G4, according to the extent of the changes observed. The results of the ultrasonic analysis were referred to the pathological verification.
A total of 67 B-mode ultrasound examinations with breast sonoelastography and lymph node assessment were performed in the Department of Ultrasound, Institute of Fundamental Technological Research, Polish Academy of Science in Warsaw.
B-mode images and the corresponding raw RF echoes were recorded using an ultrasound scanner (Ultrasonix SonixTouch, Ultrasonix Medical Corporation, Richmond, BC, Canada) and a linear array transducer L14-5/38, with the transmitted frequency set at 10 MHz, which, as measured by a hydrophone, corresponded to pulses center frequency of approximately 7.5 MHz. The tumor area region of interest (ROI) was determined on the B-mode image by an experienced radiologist.
Each patient underwent at least five ultrasound examinations: baseline recordings were made before the start of the treatment, with subsequent scans conducted a week after each round of chemotherapy.
During each examination, the data from the focal lesion were recorded from four cross-sections (radial, radial+45°, anti-radial, anti-radial+45°). The period of participating patient monitoring was 5–6 months.
The assessment of tumors was based on the guidelines of the American College of Radiology (BI-RADS lexicon) and the standards of the Polish Ultrasound Society(32,33). The RECIST 1.1 classification was adapted to monitor the longest diameter of tumors(34) even though RECIST guidelines state that US is unsuitable for monitoring the tumor size because it is operator-depended (partial response (PR), stable disease (SD), complete response (CR), and progressive disease (PD). Similar RECIST criteria to predict response during NAC were used by Marinovich ML et al.(35).
Tsukuba scale was used in the sonoelastographic assessment of tumors(36). It is a 5-point scale of classification, from Tsukuba 1, denoting strain present in the whole lesion, to Tsukuba 5, denoting no strain detected in the lesion and surrounding tissue (see Fig. 1B, Fig. 2B, Fig. 3B and Fig. 4B).
In order to collect data for quantitative analysis for each B-mode scan, 510 RF signal lines were recorded at the sampling frequency of 40 MHz. The transducer’s focus was always sited in the middle of the lesion. The analysis of the collected data for IBSC determination was performed offline using proprietary programs implemented in the Matlab environment (Mathworks, Natick, MA, USA).
The analysis of RF signals to determine IBSC was carried out using the method proposed by Yao et al.(37). The ROI (tumor area) was analyzed using a sliding window method. The window was moved horizontally with a step corresponding to a distance between successive RF lines (0.08 mm) and vertically with a step of one sample of the analyzed signal (0.02 mm). Parametric maps showing changes occurring due to NAC were built on the basis of IBSC values found in subsequent windows in the tumor area. Higher values of IBSC are represented as red and lower values as blue. In order to determine a single IBSC value characterizing the entire lesion, IBSC values obtained for all windows in a given section of the tumor were averaged, and the mean of four sections was then used.
In this study the 10 patients underwent simple mastectomy with lymphadenectomy after chemotherapy. The largest dimensions in the radial plane (according to RECIST 1.1) were in 4–41 mm range before treatment and in 3–34 mm range after treatment. The dimensions of neoplastic tumors decreased. Partial response (PR – ≥30% reduction of the longest diameter of the primary tumor) was observed in 9 of 13 cases which were histopathologically classified as pCR, G1, G3, or G4. In 3 of 13 lesions, stable disease (SD) was demonstrated (HE indicated pCR, G4, G1). In one case, which was classified in HE as pCR, the ultrasound examination also showed complete response (CR).
The results of ultrasound examinations (B-mode tumor size and elastography, before and after NAC) and the tumor size after pathological examinations are presented in Tab. 1. Elastography assessed after treatment, designated 5 of 13 tumors as non-deformable. These consisted of 3 tumors that did not respond to treatment (pPR G1) and were characterized by high stiffness (T4 and T5), one pCR tumor, and 1 pPR G4 tumor. In 4 of 13 cases, there was a decrease in stiffness (in HE, pPR lesions: G3 and G4). In 2 tumors, an increase in stiffness was observed; these were a pCR tumor and a pPR G4 tumor. In 7 of 13 tumors, stiffness did not change (in HE, pPR lesions: G1, G3 and pCR).
Histopathological examination after NAC and surgery revealed 4 pCR tumors. The remaining lesions were pPR (including 3 G1 cases). Lesions were verified as G2 and G3 invasive carcinoma non-specified type (IC NST) (Tab. 2). Table 3 presents the percentage reduction of cancer cells and histopathological verification performed after NAC.
The images from microscopic verification and the corresponding images from the B-mode examination before and after treatment for two patients with extremely different responses to the treatment (cases 1 and 8) are presented in Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6 below. Patient 1 responded to NAC (pCR), while patient 8 did not respond to the treatment (G1).
Ten tumors which were classified by histopathological examination after NAC as pathological complete response (pCR) and pathological partial response (G2, G3, and G4) were characterized by an increase in the IBSC value, being in the range from 48% to 287% (mean 153%). Three tumors that were designated as pPR G1 (<9% reduction in cancer cellularity) displayed a decrease in IBSC in the range from –20% to –60% (mean –31%). Figure 7 shows the percentage change of IBSC value after NAC. The IBSC value for each lesion prior to the start of the treatment was used as a reference value.
The analysis of B-mode ultrasound images of 13 breast tumors using an adapted RECIST 1.1 methodology indicated partial response in 9, complete response in 1, and stable disease in 3 tumors. This assessment, however, did not correlate with the dimensions and cellularity of tumors obtained in the final histopathological verification. In tumors with partial response and stable disease, pathological response G1, but also G3, G4, and pCR were observed. It is worth noting that the dimensions of tumors obtained in the final histopathological verification were much larger than the dimensions determined on the basis of B-mode imaging.
In our study, there was no correlation of changes in the stiffness of tumors (sonoelastography) with other methods used to demonstrate a response to treatment (QUS or histopathology). The studies published so far have shown that both sonoelastography techniques (relative strains and SWE) are useful in predicting responses to NAC. Ma Y et al. showed in a group of 71 patients that a decrease in mean E and SR (Strain Ratio) after courses of chemotherapy predicted the response to treatment with high accuracy (AUC = 0.93 and 0.90, respectively)(16). They also pointed out that lower initial tumor stiffness correlated with a favorable response after NAC (pCR). Similar results were obtained by Hayashi(18), where the authors concluded that the group with low scores in the Tsukuba scale (1–3) had significantly higher clinical complete response and pCR rates than the high EG group (pCR, low EG group 50 % vs high EG group 14 %, P = 0.003, respectively).
A detailed analysis of pCR tumors using sonoelastography in our study demonstrated no changes or reduced stiffness. In the histopathological evaluation of these pCR lesions, all patients displayed a significant decrease in tumor cellularity compared to CNB (core-needle biopsy). The samples appeared to contain fibrosis and stromal elastosis, which could increase tumor stiffness, but this was demonstrated only in one patient in our study. Different results were obtained for the assessment of tumors using QUS, including tumors with pCR. There were large differences in IBSC values between the group of tumors which responded to the treatment (pCR, pPR G2, G3, and G4) and the group that did not respond to the treatment (pPR G1). In the first group, there was an increase in the IBSC value, while in the second group, there was a slight decrease.
Similar results were published by Sannachi et al.: 30 patients with LABC showed no changes in IBSC values in a group of non-responders. The authors, however, defined non-response as no significant differences in the microscopic assessment of cellularity of lesions and less than 50% reduction in the tumor size(26).
Bearing in mind our results and reports from the literature, we question what constitutes the main source of scattering in breast tumors being examined. Czarnota et al. hypothesized that it is possible to observe cell nucleus defragmentation during apoptosis using high ultrasonic frequencies (>20 MHz)(38). The influence of cell properties on ultrasound scattering has been confirmed by, among others, Czarnota and Kolios who demonstrated an in vitro correlation of the size of an apoptotic cell nucleus with the scattering intensity indicated by IBSC(39,40).
Nevertheless, the hypothesis of an observed increase in ultrasound scattering as a result of defragmentation of cell nuclei cannot be directly extrapolated to our in vivo studies of breast cancer. The length of ultrasonic waves at 7.5 MHz used in our work is relatively large, and thus the wave does not interact directly with individual cells but with larger tissue structures, such as cell clusters or stromal fibroid tissue. The relationship between the change of IBSC and the changes occurring in tissue structure should therefore be considered at the level of reconstruction of whole cell clusters or remodeling of the stroma during chemotherapy.
The interpretation of the alteration in ultrasound backscattered parameters is problematic. The spatial distribution of tissue types and their effect on ultrasonic scattering are not wholly understood. In addition, the results of histopathological examination only provide information about the condition of a part of the tumor tissue before NAC (taken by biopsy) and after the entire treatment cycle, and may not be reflective of the changes taking place after each NAC course.
Early prediction of response to NAC in patients with breast cancer is crucial for planning further therapy and surgery. This is the first report comparing ultra-sound-based tumor size assessment, sonoelastography and quantitative ultrasonography with histopathological findings in patients undergoing neoadjuvant chemotherapy. Preliminary results in a group of 10 female patients with 13 breast cancer tumors confirmed the relationship between the results of post-operative histopathological verification and changes in the IBSC value. In the study group, there were unequivocal differences in IBSC values between patients with pCR lesions, pPR tumors (G2, G3, G4) and patients without changes in cellularity after treatment (pPR G1). The results were not as unambiguous in the assessment of the tumor size in the B-mode study and deformability changes in sonoelastography. The QUS technique, and in particular the IBSC parameter, may supplement the methods of assessing the effectiveness of treatment with NAC. In order to determine whether IBSC extends clinical benefit in the NAC assessment, further observations using a larger cohort of patients are necessary.
This study was supported by the National Science Centre, Poland, grant 2016/23/B/ST8/03391
All the procedures performed in the study that involved human participants were in accordance with the ethical standards of the Maria Sklodowska Curie Memorial Cancer Centre and the Institute of Oncology research committee and followed guidelines set by the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.