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Introduction

Breast cancer is the second most common type of cancer worldwide and the most common among women. It accounts for approximately 22% of new cancer cases among women each year (1), and the 5-year survival rate among the worldwide population is 61%. This high mortality rate is related to late diagnosis, with the presence of tumors of large dimensions and lymph node metastases.

Locally advanced breast cancer comprises tumors greater than 5 cm in diameter, with wall or skin invasion, metastases to fixed lymph nodes (2,3) and inflammatory carcinoma. Treatment for this is multidisciplinary, consisting of preoperative chemotherapy, surgery, radiotherapy and postoperative chemotherapy (4–6).

Preoperative or neoadjuvant chemotherapy is standard treatment for individuals with locally advanced breast cancer (7–10). It has been found to substantially improve the survival of these patients (6,11,12). It provides better local control over the disease, with increased likelihood that the breast surgery will be conservative (4,5,13). Moreover, this type of chemotherapy treats preexisting microscopic systemic disease and enables the evaluation of tumor resistance in vivo (14).

Reduction in tumor volume has been used as the standard criterion for response evaluation among solid tumors such as breast carcinoma (15). Techniques that measure changes in the molecular biology of the tumor, such as Doppler ultrasonography, functional magnetic resonance and nuclear medicine are promising methods for the identification of tumors that present a favorable response, with great accuracy (16–18).

Breast scintigraphy is a well-established diagnostic imaging technique (19) of relatively low cost compared with positron emission tomography and magnetic resonance imaging. This technique can also be used as a method for evaluating the response of breast carcinoma to chemotherapy treatment (20), thereby providing an in vivo indication of the chemosensitivity of the tumor (8).

Based on the limitations of the clinical examination as well as the main biological characteristics of 99mTc-sestamibi uptake in malignant lesions, an evaluation of the tumor response to neoadjuvant chemotherapy treatment through quantification of 99mTc-sestamibi was proposed using scintigrams from individuals with locally advanced breast cancer.

Materials and methods

This was a cross-sectional study conducted between 2000 and 2008. Prior approval had been obtained from the institution’s research ethics committee. Fifty-five female patients with a diagnosis of locally advanced ductal invasive breast cancer were included. They underwent neoadjuvant chemotherapy and surgical complementation, with evaluation of the response to treatment through clinical measurements and scintigraphy.

Neoadjuvant chemotherapy

The patients underwent chemotherapy consisting of either the AC regimen (doxorubicin hydrochloride, 60 mg/m2 of body surface and cyclophosphamide, 600 mg/m2 of body surface) or the FEC regimen (5-fluorouracil, 500 mg/m2 of body surface; epirubicin, 60 mg/m2 of body surface and cyclophosphamide, 500 mg/m2 of body surface).

Evaluation of tumor response to neoadjuvant chemotherapy by means of clinical measurements

Tumor measurements were made by a clinical oncologist at two points: before starting and after completing the neoadjuvant chemotherapy. The size in cm was calculated as the mean diameter between the two largest axes. The clinical tumor response was based on the change in tumor size before and after the neoadjuvant chemotherapy, expressed as a percentage. The response was classified into two groups; tumor reduction >60% and <60%.

99mTc-sestamibi breast scintigraphy

Breast scintigraphy was performed at two points: before starting and after completing the neoadjuvant chemotherapy, simultaneously with the clinical measurements of the tumor. The images were acquired by a Siemens gamma camera (Orbiter model), coupled to an Icon computer with software version 7.5 and circular detector. A foam pad of 30 cm in thickness was used with openings lateral to the breast projection (21,22). A low-energy high-resolution collimator was used with a matrix of 128×128 pixels, a photopeak of 140 keV and a window of 15%. The image acquisition lasted for 10 min, starting 10 min after injection. Posterior oblique and lateral images with magnification were obtained. The patient was in a prone position with the breasts hanging and the collimator was fitted into the openings. Anterior thoracic and axillary images without magnification were obtained with the patient in a supine position. For all of the examinations 99mTc-sestamibi was used, which has a labeling efficiency of >95%. It was administered through a vein in the foot or the arm contralateral to the compromised breast.

Quantification of 99mTc-sestamibi uptake was carried out on the lateral images of the breasts by creating two identical regions of interest: one on the tumor and the other in the mirror position on the contralateral breast. Pixel counting was performed in these regions. The tumor 99mTc-sestamibi uptake rate was determined as the pixel count ratio between the region of interest in the tumor and the mirror region in the contralateral breast. The pretreatment 99mTc-sestamibi uptake rate (BUR) and post-treatment 99mTc-sestamibi uptake rate (AUR) were obtained from scintigraphic evaluations performed before starting and after completing the neoadjuvant chemotherapy, respectively. The scintigraphic tumor response (STR) was obtained from the following equation: STR = (100 - AUR)/(BUR × 100)%.

Histological evaluation

The results from the diagnostic biopsy and surgical specimen were analyzed by means of histological sections (4–5 μm), cut from paraffin blocks. Hematoxylin and eosin staining was used.

Statistical analysis

To calculate the sample size a correlation coefficient (r) of 0.44 was used in the preliminary analysis. Results showed that a minimum of 45 patients would be needed. The descriptive analysis consisted of calculations of absolute and relative frequencies and central trend and dispersion measurements. The Kolmogorov-Smirnov test was used to evaluate the adherence to the normal distribution curve. Since the variables were found not to present a normal distribution, non-parametric tests were used. The Kruskal-Wallis test was used to compare the mean scintigraphic tumor response and histological type. Dunn’s multiple comparison test was used to detect differences between histological types. The Mann-Whitney test was used to compare means between quantitative and qualitative variables: scintigraphic tumor response vs. clinical tumor response and pretreatment 99mTc-sestamibi uptake vs. scintigraphic tumor response. The Spearman’s test was used to correlate the quantitative variables of clinical reduction in tumor size and scintigraphic tumor response. The level of significance was set at p<0.05.

Results

The patients evaluated (n=55) were aged between 28 and 68 years (mean 50.5; SD, 9.8; median 51). The number of cycles of chemotherapy ranged from 2 to 8 (mean 3.9; SD, 0.9; median 4). The interval between the scintigraphic examinations before starting and after completing neoadjuvant chemotherapy ranged from 2 to 7 months (mean 3.4; SD, 1.1; median 3).

Residual tumor material was observed in 49 surgical specimens, consisting of invasive ductal carcinoma in 21 cases (38.2%) and an association between invasive ductal carcinoma and in situ ductal carcinoma in 28 cases (50.9%). In six cases (10.9%), the tumor response was complete. Table I shows a comparison between the mean scintigraphic tumor response and the different findings from the surgical specimen. The mean scintigraphic tumor response was found to be greatest in tumors that were reduced to zero, followed by those that were purely invasive ductal carcinomas. The mean scintigraphic tumor response was least in tumors in which there was an associated in situ component (p=0.011).

Table I Comparison between mean scintigraphic tumor response (STR) in percentages and the histological findings from the surgical specimen.

Table I

Comparison between mean scintigraphic tumor response (STR) in percentages and the histological findings from the surgical specimen.

Specimen	No. (%)	Mean STR% (SD)	Median	Min-Max	P-valuea
Absence of tumor	6 (10.9)	63.22 (16.83)	71.26	39.05–80.86	0.011
IDC	21 (38.2)	40.58 (20.26)	38.55	7.93–77.28
IDC+ISDC	28 (50.9)	31.28 (24.94)	19.20	−0.83–75.61
Total	55 (100)

a Kruskal-Wallis test.

{ label (or @symbol) needed for fn[@id='tfn2-ol-01-02-0379'] } IDC, invasive ductal carcinoma; IDC+ISDC, invasive ductal carcinoma + in situ ductal carcinoma.

The 99mTc-sestamibi BUR ranged from 1.18 to 10.13 (mean 3.40±0.22), while the AUR ranged from 0.74 to 3.59 (mean 1.88±0.10). Thus, the overall mean scintigraphic tumor response rate was 38.31±3.26 (%). A higher pretreatment 99mTc-sestamibi uptake showed greater response rates (Table II).

Table II Mean 99mTc-sestamibi uptake rate before starting neoadjuvant chemotherapy (BUR) compared with scintigraphic tumor response (STR) groups (>60% and <60%).

Table II

Mean 99mTc-sestamibi uptake rate before starting neoadjuvant chemotherapy (BUR) compared with scintigraphic tumor response (STR) groups (>60% and <60%).

STR	No. (%)	Mean BUR (SD)	Median	Min-Max	P-valuea
<60%	42 (76.4)	2.82 (0.96)	2.55	1.18–5.50	<0.001
>60%	13 (23.6)	5.26 (2.03)	5.22	2.54–10.13
Total	55 (100)

a Mann-Whitney test.

A positive correlation was noted between the clinical and scintigraphic responses (r=0.44; p=0.001). The scintigraphic response was greater in the tumors with a clinical response >60% (p=0.04) (Table III).

Table III Mean scintigraphic tumor response (STR) in relation to groups with reduction in clinical measurement > 60% and <60% (CTR).

Table III

Mean scintigraphic tumor response (STR) in relation to groups with reduction in clinical measurement > 60% and <60% (CTR).

CTR	No. (%)	Mean STR% (SD)	Median	Min-Max	P-valuea
<60%	27 (49)	31.16 (22.7)	23.9	−0.8–75.6	0.040
>60%	28 (51)	45.26 (23.9)	48.3	1.3–80.8
Total	55 (100)

a Mann-Whitney test.

{ label (or @symbol) needed for fn[@id='tfn5-ol-01-02-0379'] } CTR, clinical tumor response.

Discussion

Although clinical examinations are currently standard in evaluating tumor response to chemotherapy treatment, these measurements do not reflect the biological characteristics of tumors, nor do they reflect the real reduction in tumor size. This is because these measurements do not discriminate between inflammatory and healing processes in viable tumors.

On the other hand, the uptake characteristics and efflux dynamics of 99mTc-sestamibi have shown a high correlation with the biological behavior of breast tumors, particularly in relation to aggressiveness and chemoresistance.

In the present study, the focus was on the relationship between the 99mTc-sestamibi uptake in locally advanced breast tumors and the histological type, and between this uptake and the clinically measured response, as a method for evaluating the response to chemotherapy treatment.

Based on the evaluation of the tumor histology in the surgical specimen, we observed that the individuals with invasive ductal carcinoma presented a greater scintigraphic tumor response than those who had an association with an in situ component. This finding may reflect the lower response of in situ carcinomas to chemotherapy, thus suggesting that calculation of the 99mTc-sestamibi uptake has additional usefulness for possible prediction of the chemotherapy response to different types of carcinomas.

With regard to comparing the pretreatment 99mTc-sestamibi uptake with the scintigraphic tumor response, Del Vecchio et al (23) obtained similar results and stated that high early 99mTc-sestamibi uptake in breast carcinomas was related to high rates of cell proliferation. This indicates greater aggressiveness of tumor behavior and correlates directly with a better and faster tumor response to chemotherapy. On the other hand, the tumors with a lower 99mTc-sestamibi uptake showed a lower scintigraphic response. This finding may be related, not only to the histological type, but also to the chemoresistance that was indicated by the inverse correlation between the intensity of 99mTc-sestamibi uptake and Bcl-2 levels (24).

We observed that almost all of our patients presented a reduction in 99mTc-sestamibi uptake (n=54) after chemotherapy. Mankoff et al (20), Marshall et al (25) and Cwikla et al (26) also reported a reduction in the tumor-to-background ratio, thus showing that 99mTc-sestamibi uptake reflects the metabolic activity of the tumor and its reduction consequent to chemotherapy (25). Wilczek et al (27) found a significant reduction in the tumor-to-background ratio after the conclusion of neoadjuvant chemotherapy, which was confirmed by the tumor regression noted in the histological evaluation of the surgical specimens. These findings suggest that calculating the scintigraphic tumor response should be included as a safe parameter when evaluating tumor regression.

Quantitative analysis of breast scintigraphy using 99mTc-sestamibi was shown to be an additional tool for evaluating the preoperative chemotherapy response, given that the variation in 99mTc-sestamibi uptake reflects the biological behavior of the tumor. Thus, a quantitative analysis of breast scintigraphy using 99mTc-sestamibi should be included in the approach towards assessing tumor response related to preoperative chemotherapy.

References