Dual time point 18F-fluorodeoxyglucose positron emission tomography/computed tomography fusion imaging (18F-FDG PET/CT) in primary breast cancer

Background To evaluate the clinicopathological and prognostic significance of the percentage change between maximum standardized uptake value (SUVmax) at 60 min (SUVmax1) and SUVmax at 120 min (SUVmax2) (ΔSUVmax%) using dual time point 18F-fluorodeoxyglucose emission tomography/computed tomography (18F-FDG PET/CT) in breast cancer. Methods Four hundred and sixty-four patients with primary breast cancer underwent 18F-FDG PET/CT for preoperative staging. ΔSUVmax% was defined as (SUVmax2 − SUVmax1) / SUVmax1 × 100. We explored the optimal cutoff value of SUVmax parameters (SUVmax1 and ΔSUVmax%) referring to the event of relapse by using receiver operator characteristic curves. The clinicopathological and prognostic significances of the SUVmax1 and ΔSUVmax% were analyzed by Cox’s univariate and multivariate analyses. Results The optimal cutoff values of SUVmax1 and ΔSUVmax% were 3.4 and 12.5, respectively. Relapse-free survival (RFS) curves were significantly different between high and low SUVmax1 groups (P = 0.0003) and also between high and low ΔSUVmax% groups (P = 0.0151). In Cox multivariate analysis for RFS, SUVmax1 was an independent prognostic factor (P = 0.0267) but ΔSUVmax% was not (P = 0.152). There was a weak correlation between SUVmax1 and ΔSUVmax% (P < 0.0001, R2 = 0.166). On combining SUVmax1 and ΔSUVmax%, the subgroups of high SUVmax1 and high ΔSUVmax% showed significantly worse prognosis than the other groups in terms of RFS (P = 0.0002). Conclusion Dual time point 18F-FDG PET/CT evaluation can be a useful method for predicting relapse in patients with breast cancer. The combination of SUVmax1 and ΔSUVmax% was able to identify subgroups with worse prognosis more accurately than SUVmax1 alone.

fluorodeoxyglucose positron emission tomography/computed tomography ( 18 F-FDG PET/CT) has come to play an increasing role in the diagnosis of biological properties as well as staging, treatment monitoring of residual disease, and detection of disease recurrence in breast cancer patients [4,5]. For that purpose, the maximum standardized uptake values (SUV max ) of 18 F-FDG has been shown to be correlated with tumor size, nuclear grade (NG), and Ki-67 labeling index (LI) [6,7]. Furthermore, several studies [8][9][10][11] have shown that the SUVmax of primary tumor, that reflects its metabolic activity, on 18 F-FDG PET/CT can predict patients' poor prognosis.
In malignant tumors, glucose metabolism is usually enhanced, and the uptake of 18 F-FDG increases. Therefore, a higher level of 18 F-FDG accumulation in PET/CT should reflect higher proliferative activity of the tumor cells. Recently, several studies and meta-analyses have been performed on the relationships between PET/CT and histopathological findings in the field of diagnostic oncology [6,[12][13][14][15][16]. Especially, the uptake of 18 F-FDG was shown to be correlated with expressions of histopathological markers, e.g., Ki-67 LI, vascular endothelial growth factor, and hypoxia induced factor 1α, in head and neck cancer, lung cancer, and lymphoma [12][13][14][15][16].
Because the SUV max is usually measured at a single time point, such as 1 h after 18 F-FDG administration, the dynamic index of the tumor is not included in routine examination. Some articles reported the utility of measurement of 18 F-FDG uptake levels at dual time points [17,18]. The 18 F-FDG uptake level at a later point has a tendency to increase in malignant lesions but to decrease in benign lesions, such as inflammatory reactions [19]. Therefore, the measurement of 18 F-FDG uptake in dual time point 18 F-FDG PET/CT may be able to estimate biological properties and predict patient prognosis more accurately.
The aim of this study was to investigate the clinicopathological significance of dual time point 18 F-FDG PET/CT in patients with primary breast cancer. In addition, we assessed the prognostic significance of the measurement of dynamic 18 F-FDG uptake levels.

Methods
This was a retrospective study in a single institute.

Ethics approval and consent to participate
This study was performed in accordance with the Declaration of Helsinki and was approved by the institutional review board of National Defense Medical College (registration number: 2695). All patients agreed to participate in this study, and written informed consent was obtained from all these patients.

Eligible patients
Between September 2008 and December 2017, 18 F-FDG PET/CT was performed for 820 consecutive preoperative patients with primary breast carcinoma. Of these, 356 patients were excluded from the study because of (1) history of malignant diseases other than breast cancer within 5 years, (2) preoperative medication therapy, (3) diabetes mellitus, (4) previous treatment of ipsilateral or contralateral breast cancer, (5) presence of distant metastases, (6) acquisition of only single time point data of 18 F-FDG PET/CT, and/or (7) difficulty in measuring SUV max due to low 18 F-FDG accumulation. Finally, 464 female patients were eligible.
In all cases, diagnosis of breast cancer was made based on cytopathological and/or histopathological examination before surgery. 18 F-FDG PET/CT was performed before surgery, and the interval between the PET/CT examination and surgery was 42 days on an average, ranging from 7 to 71 days. Postoperative surveillance for 5 years was performed through examination every 3 months and mammography every year. After 5 years, patients underwent mammography every year and were followed up to 10 years after surgery. If relapse was suspected in these tests, it was confirmed using CT or PET/ CT.

Quantification of 18 F-FDG uptake in primary breast cancer
All 464 patients underwent 18 F-FDG PET/CT at the Tokorozawa PET Diagnostic Imaging Clinic (Tokorozawa, Japan). Patients fasted for at least 4 h before the examination. One hour after intravenous administration of 3.7 Mbq/kg 18 F-FDG, the first scanning was performed. The first examination involved whole-body imaging from the head to thigh, and the second scanning involved the chest only, within 50-60 min of the first examination.
After image reconstruction, the region of interest (ROI) was placed in primary breast cancer. The SUV is defined as decay-corrected tissue activity divided by the injected dose per patient body and is calculated using the formula, The SUV max 1 and SUV max 2 were obtained at dual time points, and the ΔSUV max % was calculated using the formula, where the SUV max 1 and SUV max 2 were the SUV max at the initial phase (60 min) and SUV max at delayed phase (120 min), respectively.

Histological study
Two observers (H.T., Y.Y.) performed pathological diagnosis. NG was defined according to the General Rules for Clinical and Pathological Recording of Breast Cancer, 17th edition [20]. NG was determined by the sum of the nuclear atypia score and the mitosis count score. Estrogen receptor (ER) and progesterone receptor (PgR) expression was assessed by immunohistochemistry and defined as positive if ≥1% of carcinoma cells were immunoreactive [21]. Human epidermal growth factor receptor 2 (HER2) positivity was determined according to the American Society of Clinical Oncology/College of American Pathologists guideline 2013 [22]. According to the recommendation of the Breast Cancer Working Group, Ki-67 LI was defined as high if 14% or higher of constituent carcinoma cells were immunoreactive [23,24]. Pathological stage was determined by the clinical and pathological recording of breast cancer, 8th edition, by Union for International Cancer Control (UICC).

Evaluation of 18 F-FDG PET/CT results as prognostic factor
Receiver operating characteristic (ROC) curves were drawn to determine the optimal cutoff values of SUVmax 1 and ΔSUV max %. Furthermore, the Youden index [= sensitivity -(1specificity)] of each cutoff value was calculated, and the highest value was taken as the optimal cutoff point.

Statistical analysis according to clinicopathological factors and prognosis
The correlations between SUV max parameters (SUV max 1, SUV max 2, and ΔSUV max %) and clinicopathological factors were evaluated using the non-parametric Wilcoxon and the Kruskal-Wallis tests. All statistical analyses were two-sided, with significance defined as P value of < 0.05. The Kaplan-Meier curves for relapse-free survival (RFS) and overall survival (OS) were drawn, and their differences were tested by the log-rank test. A Cox proportional hazards model was used for univariate and multivariate analyses for RFS. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and the accuracies of SUV max 1, ΔSUV max %, and their combination for RFS were calculated. Statistical analyses were performed using JMP® 13 (SAS Institute Inc., Cary, NC, USA).

Patient characteristics between high and low groups divided by SUV max 1 and ΔSUV max %
The correlations between high and low SUV max 1 groups and clinicopathological parameters are presented in Table 2. Tumor size, pathological T factor, NG, lymphatic invasion, pathological N factor, pathological stage, and SUV parameters (SUV max 1, SUV max 2, ΔSUV max %) were significantly different between high and low SUVmax 1 groups. High Ki-67 LI was more frequent in the high SUV max 1 group than in the low SUV max 1 group (P < 0.0001) whereas ER, PgR, HER2, and subtype were not correlated with SUV max 1. The correlations between the high and low ΔSUV max % groups and clinicopathological parameters are presented in Table 3. The factors correlated with SUV max 1 were significantly different between the high and low ΔSUV max % groups. High Ki-67 LI was more frequent in the high ΔSUV max % group than in the low ΔSUV max % group (P = 0.0336) whereas ER, PgR and subtype were not correlated with ΔSUV max %. HER2 status was significantly different between high and low ΔSUV max % groups (P = 0.0304). Two patients with HER2-positive ductal carcinoma in situ (DCIS) were classified into the low ΔSUV max % group. Therefore, when these DCIS cases were excluded from the analysis, HER2 status showed no significant difference between these two groups.

Comparison of survival curves
The RFS curves for the high and low SUV max 1 groups were significantly different between these curves (P = 0.0003) ( Fig. 2A). Although there was no significant difference in OS curves for the high and low SUV max 1 groups, the high SUV max 1 group tended to show worse prognosis (P = 0.0553) (data not shown). The RFS curves for the high and low ΔSUV max % groups were significantly different (P = 0.0151) (Fig. 2B). Although, there was no significant difference in OS curves between the high and low ΔSUV max % groups, the former groups tended to show worse prognosis (P = 0.141) (data not shown). Because the correlation of SUV max 2 with RFS was weaker than that of SUV max 1 (P = 0.0012), we did not use SUV max 2 for prognostic analysis (data not shown).
Prognostication by the combination of SUV max 1 and ΔSUV max % The 464 patients were classified into three subgroups (group A, B, and C) by the combination of SUV max 1 and ΔSUV max %. Group A was SUV max 1 ≥ 3.4 and ΔSUVmax % ≥ 12.5 (n = 179), group B was SUV max 1 ≥ 3.4 and ΔSUV max % < 12.5 (n = 62), and group C was SUV max 1 < 3.4 (n = 223). Although group C could also be subclassified into the high ΔSUV max % (n = 83) and low ΔSUVmax % subgroups (n = 140), no significant difference in RFS was observed between these two subgroups (P = 0.625, data not shown).
There were significant differences in RFS curves between these three subgroups (P = 0.0006), and between groups A and C (P = 0.0001). On the other hand, there were no significant differences between groups A and B (P = 0.285), and between groups B and C (P = 0.146) (Fig. 3A). The 10-year RFS rates were 90.6% in group B and 89.0% in group C, whereas the rate was 78.8% in group A. Furthermore, RFS curves were significantly different between group A and group "B + C" (P = 0.0002) (Fig. 3B). By the combination of the ΔSUV max % and the SUV max 1, it was possible to predict a group with the worse prognosis more sensitively than SUV max 1 or ΔSUV max % alone.
In the subgroup analyses, there were significant differences in RFS between group A and group B/C in nodenegative patients (n = 334) and in node-positive patients (n = 130) (P = 0.0126 and P = 0.0455, respectively). In the pTis/pT1 (n = 297) and pT2/pT3 groups (n = 167), there were no significant differences in RFS between group A and group B/C (P = 0.120 and P = 0.131, respectively). With regard to subtype, group A showed a significantly lower RFS than group B/C in the ER-positive/HER2-negative group (P = 0.0008, n = 345), but such a relationship was not found in the ER-positive/HER2-positive, ER-negative/ HER2-positive, and ER-negative/HER2-negative patient groups (P = 0.0614, P = 0.358, P = 0.823, respectively).

Univariate and multivariate analyses
By Cox's univariate analyses to estimate relapse risk, five clinicopathological parameters, invasive tumor size, lymph node metastasis, NG, lymphatic invasion, and Ki-67 LI, as well as SUV max 1 and ΔSUV max % were statistically significant factors. The combined SUV max 1 and ΔSUV max % was also a significant prognostic factor in RFS (P = 0.0007) ( Table 4). Because SUV max 1 and ΔSUV max % were correlated with together, we performed the Cox's multivariate analyses including these five clinicopathological parameters with either SUV max 1, ΔSUVmax %, or the combination of SUV max 1 and ΔSUV max %. In the multivariate analyses, SUV max 1 or the combination of SUV max 1 and ΔSUV max % was an independent prognostic factor (P = 0.0267 and P = 0.0283, respectively, Table 4). As the test to detect relapse, the combined measurement of SUV max 1 and ΔSUV max % showed higher specificity, PPV, and accuracy than the measurement of SUV max 1 or ΔSUV max % alone (Table 5).

Discussion
In malignant tumors, glucose metabolism is usually enhanced, and the extent of increase in glucose consumption was shown to be correlated with higher proliferation rates of cancer cells. Therefore, a higher level of accumulation of 18 F-FDG in PET/CT is a sign of the primary breast cancer with high proliferative activities [8][9][10]25], and 18 F-FDG PET/CT has been used not only for cancer diagnosis but also for functional assessments of breast cancer, i.e., clinical aggressiveness and higher sensitivity to neoadjuvant therapies [26,27]. In fact, Deng et al. and Surov et al. summarized that the uptake of 18 F-FDG was associated with Ki-67 LI in their meta-analyses [6,7]. We were able to confirm their results in this study. For the evaluation of PET/CT images, the most commonly used parameter is the SUV max , which is usually measured 60 min after the injection of 18 F-FDG. It has also been believed that the addition of information of the later phase can be used to determine the biological properties of the examined cancers in more detail. Some reported that 18 F-FDG uptake in malignancy continued to increase until approximately 4-5 h after injection, but the uptake decreased in the benign lesion 30 min after the injection [18,28]. Furthermore, the ΔSUV max % was correlated with the grade of malignancy in lung cancer and lymphoma [29,30]. Although the usefulness of ΔSUV max % was generally considered acceptable, few reports have been published on its relationship with the prognosis of breast cancer.
In this report, we confirmed that SUV max 1 was an independent prognostic factor for RFS. Furthermore, we showed that ΔSUV max % was a significant prognostic indicator of RFS and that the combination of SUV max 1 and ΔSUV max % was possible to predict a group with poorer prognosis more sensitively than SUV max 1 alone. With the optimal cutoff value (12.5 of ΔSUV max %), the subgroup with better prognosis can be detected among from the high SUV max 1 (≥ 3.4) group. In contrast, the effectiveness of SUV max 1 and ΔSUV max % for OS could not be demonstrated. In the present patient cohort, follow up period is still short, and the number of events appears too small to analyze the effectiveness of ΔSUVmax % for OS prediction.
The RFS rate of patients with breast cancers of the ER-positive/HER2-negative subtype was significantly lower in the high-SUV max /high-ΔSUV max % group than in the other groups (P = 0.0008). SUV max was shown to be correlated with 21-gene recurrence score in ERpositive/HER2-negative breast cancer [31]. Therefore, SUV-related parameters might be clinically useful, in addition to the 21-gene recurrence score, for the selection of high-risk node-negative luminal breast cancers, although a larger-scale study is necessary. Furthermore, the combination of SUV max 1 and ΔSUV max % would be able to increase the accuracy of preoperative diagnoses of lymph node metastasis and therapeutic response to neoadjuvant therapies.
In this study, patients with previous treatment were excluded. In these patient groups, 24 ER-negative/HER2positive patients and 54 ER-negative/HER2-negative patients were included. Therefore, only 10.8% (50/464) were HER2-positive type and 11.6% (56/464) were ERnegative/HER2-negative type. These types of breast cancers were reported to have a higher SUV value than ERpositive types and to have worse prognosis than the ERpositive types [32][33][34]. Furthermore, we excluded the    (b) RFS curves for the patients of subgroup A and subgroup "B + C". RFS curves were significantly different between these two groups (P = 0.0002). Ten-year RFS rates were 78.8% in group A and 89.0% in group "B + C"  109 patients whose 18 F-FDG accumulation was not visible and SUV max was not measurable from the study. These cases appear to show very low SUV max values and accordingly, were also expected to have a good prognosis. For these reasons, it seemed that the true efficacy of ΔSUV max % and combined measurement of SUV max 1 and ΔSUV max % as prognostic indicators might be higher than the present results. The pN factor was a very strong prognostic factor in the univariate analysis but did not have an independent prognostic power in the multivariate analysis. In these analyses, pN was divided into pN0 and pN1-3. Because pN1 was shown to reveal relatively good prognosis and a majority of pN-positive patients showed pN1 in this study, the impact of node-positivity might have been diluted by the good-prognosis effect in pN1 cases. Lymphatic invasion and pT might also have been confounding factors along with pN.
The limitations of this study include its retrospective nature, single center data, and a relatively small number of events. A multicenter, prospective study is needed to highlight the effectiveness of ΔSUV max % in the prognostication of primary breast cancer.
Nonetheless, the strength of the present study involves the large number of images reviewed, the correlation between relevant clinicopathological and prognostic data, and exclusion of patients with diabetes. Furthermore, SUV max parameters were easy to compute and reproducible, and dual time point imaging could be performed in a relatively short time with minimal inconvenience to the patient and be readily performed at most centers.

Conclusions
In conclusion, dual time point 18 F-FDG PET/CT can be a useful modality for prediction of relapse in patients with breast cancer. The combination of SUV max1 and ΔSUV max% was able to identify the patient groups with worse prognosis more accurately than SUV max 1 alone.