Comparison of 18F-fluorothymidine Positron Emission Tomography/Computed Tomography and 18F-fluorodeoxyglucose Positron Emission Tomography/Computed Tomography in Patients with Breast Cancer

The uptake of 18F-fluorothymidine (18F-FLT) depends on cells’ proliferative rates. We compared the characteristics of 18F-FLT positron emission tomography/computed tomography (PET/CT) with those of 18F-fluorodeoxyglucose (18F-FDG) PET/CT for breast cancer. We prospectively diagnosed patients with breast cancer who underwent 18F-FLT PET/CT and 18F-FDG PET/CT. Subsequently, significant differences and correlation coefficients of the maximum standardized uptake value (SUVmax) in primary breast cancer and axillary lymph nodes were statistically evaluated. We enrolled eight patients with breast cancer. In six treatment-naive patients, the SUVmax for primary lesions showed a significant difference (mean, 2.1 vs. 4.1, p = 0.031) and a strong correlation (r = 0.969) between 18F-FLT and 18F-FDG. Further, although the SUVmax for the axillary lymph nodes did not show a significant difference between 18F-FLT and 18F-FDG (P = 0.246), there was a strong correlation between the two (r = 0.999). In a patient-by-patient study, there were cases in which only 18F-FDG uptake was observed in lymph nodes and normal breasts. Bone metastases demonstrated lower accumulation than bone marrow on the 18F-FLT PET/CT. In conclusion, a strong correlation was observed between the 18F-FLT PET/CT and 18F-FDG PET/CT uptake. Differences in the biochemical characteristics of 18F-FLT and 18F-FDG were reflected in the accumulation differences for breast cancer, metastatic lesions, and normal organs.


Introduction
Breast cancer staging aids in determining disease severity, informing treatment planning, and predicting prognosis [1]. 18 F-fluorodeoxyglucose positron emission tomography/computed tomography ( 18 F-FDG PET/CT) can be used to evaluate a lesion's morphology and glucose metabolism [2]. In addition, the maximum standardized uptake value (SUVmax) of 18 F-FDG can inform patients' prognosis [3][4][5]. However, 18 F-FDG accumulates in areas of inflammation and benign/malignant neoplasms [6]; considerable experience and knowledge are necessary when using 18 F-FDG to achieve an accurate diagnosis.
Recently, 3 -deoxy-3 -18 F-fluorothymidine ( 18 F-FLT) was introduced as a stable cell proliferation imaging agent [7]. This pyrimidine analog uses the DNA synthesis salvage 18 F-FLT and 18 F-FDG were administered intravenously to patients at a dose of 3.7 MBq/kg. Fasting for at least 4 h was required before 18 F-FDG administration but not for 18 F-FLT administration. Whole-body images were obtained using a PET/CT system (Cartesion Prime or Celesteion; Canon Medical Systems, Tochigi, Japan). No-contrast CT scans were performed using the following parameters: pitch, 0.938; gantry rotation time, 0.5 s; table time, 30 mm/s; auto-exposure control (SD 20), 120 KVp; and slice thickness, 2.0 mm. After approximately 60 min of 18 F-FDG administration, a whole-body emission PET was performed using the following parameters for the Cartesion Prime and Celesteion, respectively: emission time per bed, 90 s and 2 min; bed position, 6-7 and 9-10; slice thickness, 2.11 and 4.08 mm; and matrix, 336 × 336 and 144 × 144.

PET/CT Analysis
One of four nuclear medicine specialists with 8-12 years of experience prospectively evaluated eligible patients with breast cancer. The nuclear medicine specialists, who were unblinded to each patient's breast cancer diagnosis, evaluated the images and crossreferenced the mammography, ultrasound, and magnetic resonance imaging images as required. Accumulations within primary breast tumors and metastatic lesions were measured in terms of the SUVmax using Vox-base version 2.8 (J-MAC System, Inc., Hokkaido, Japan). For diagnosing the axillary lymph node metastases, we compared the tumor size and 18 F-FDG uptake between the affected and contralateral sides. A mass with an abnormal 18 F-FDG uptake was considered a distant metastasis. Concurrent CT scans were also carefully examined for regions with high physiologic accumulation (i.e., the brain for 18 F-FDG PET/CT and bone marrow for 18 F-FLT PET/CT).

Statistical Analysis
The SUVmax for primary breast cancer and axillary lymph nodes in treatment-naive (no prior surgery or chemotherapy) patients before PET/CT were compared between 18 F-FLT PET/CT and 18 F-FDG PET/CT. Distributions were analyzed using the Shapiro-Wilk test, and p-values of ≥0.05 were considered normally distributed. Significant differences were tested using a two-tailed paired t-test for normally distributed variables and the Wilcoxon signed-rank test for non-normally distributed variables. A p-value of <0.05 was considered statistically significant. In addition, the correlation between the SUVmax of the 18 F-FLT PET/CT and that of the 18 F-FDG PET/CT was assessed using Pearson's correlation coefficient. The correlation coefficients ranged from −1 to +1, with 0 indicating no linear or monotonic associations; the relationship was considered stronger as the coefficient approached an absolute value of 1 [23].

Results
A total of 8 female patients with breast cancer, with a mean age of 64.8 years (standard deviation, 9.8 years; range, 49-73 years), were enrolled. Six patients were treatment-naive before the PET/CT, which included three surgical cases and three inoperable cases. Table 1 shows the number of days from biopsy to 18 F-FLT PET/CT, 18 F-FDG PET/CT, and surgery. All six treatment-naive breast cancers were invasive ductal carcinomas (the luminal type). We compared the 18 F-FLT PET/CT with the 18 F-FDG PET/CT in one of the two patients treated (i.e., non-treatment-naive) before the PET/CT. The interval between the PET/CTs was four days. Further, another patient was assessed solely based on recent 18 F-FLT PET/CT findings.

Surgical Cases
Three patients underwent 18 F-FLT PET/CT for preoperative staging. In all patients, the SUVmax values of the primary lesions and axillary lymph nodes were lower on the 18 F-FLT PET/CT than on the 18 F-FDG PET/CT (Table 2). Patient one had metastases in 5/15 dissected axillary lymph nodes. The axillary lymph node that showed the greatest accumulation differed between the 18 F-FLT PET/CT and 18 F-FDG PET/CT, with SUVmax values of 3.0 and 3.4, respectively ( Figure 1).  Primary FLT, maximum standardized uptake value (SUVmax) of primary breast cancer on 18

Inoperable Cases
Three patients possessed an inoperable disease. The SUVmax of their primary lesions was lower on the 18 F-FLT PET/CT than on the 18 F-FDG PET/CT ( Table 2). In patient four, both the initial 18 F-FLT and 18 F-FDG PET/CTs showed a slight accumulation in the primary lesion but high accumulation in multiple lymph node metastases, as well as in multiple bone metastases. She underwent chemotherapy, and a second 18 F-FLT PET/CT was performed 147 days after the first 18 F-FLT PET/CT. At that time, some bone metastases showed lower 18 F-FLT accumulation than the physiological accumulation within the bone marrow, presumably because the treatment reduced cell proliferation ( Figure 2). treatment-naive patients before PET/CT. Primary FLT, maximum standardized uptake value (SUVmax) of primary breast cancer on 18

Inoperable Cases
Three patients possessed an inoperable disease. The SUVmax of their primary lesions was lower on the 18 F-FLT PET/CT than on the 18 F-FDG PET/CT (Table 2). In patient four, both the initial 18 F-FLT and 18 F-FDG PET/CTs showed a slight accumulation in the primary lesion but high accumulation in multiple lymph node metastases, as well as in multiple bone metastases. She underwent chemotherapy, and a second 18  Patient six experienced a first lumbar vertebra metastasis, and 18 F-FDG PET/CT revealed a clear accumulation. The SUVmax of the first lumbar vertebra was 12.6, whereas that of the second lumbar vertebra was 1.9 ( Figure 3). As for the 18 F-FLT PET/CT, which was performed 25 days before the 18 F-FDG PET/CT, the accumulation in the first lumbar vertebra (SUVmax, 10.8) was similar to the bone marrow uptake (SUVmax in the second lumbar vertebra, 8.5), but the CT showed an osteolytic mass (not shown).

Inoperable Cases
Three patients possessed an inoperable disease. The SUVmax of their primary lesions was lower on the 18 F-FLT PET/CT than on the 18 F-FDG PET/CT (Table 2). In patient four, both the initial 18 F-FLT and 18 F-FDG PET/CTs showed a slight accumulation in the primary lesion but high accumulation in multiple lymph node metastases, as well as in multiple bone metastases. She underwent chemotherapy, and a second 18 F-FLT PET/CT was performed 147 days after the first 18 F-FLT PET/CT. At that time, some bone metastases showed lower 18 F-FLT accumulation than the physiological accumulation within the bone marrow, presumably because the treatment reduced cell proliferation ( Figure 2).  Patient six experienced a first lumbar vertebra metastasis, and 18 F-FDG PET/CT revealed a clear accumulation. The SUVmax of the first lumbar vertebra was 12.6, whereas that of the second lumbar vertebra was 1.9 ( Figure 3). As for the 18 F-FLT PET/CT, which was performed 25 days before the 18 F-FDG PET/CT, the accumulation in the first lumbar vertebra (SUVmax, 10.8) was similar to the bone marrow uptake (SUVmax in the second lumbar vertebra, 8.5), but the CT showed an osteolytic mass (not shown).

Statistical Analysis
When the Shapiro-Wilk test was performed for treatment-naive patients with primary breast cancer (patients 1-6), the p-value for the SUVmax was 0.596 for the 18 F-FLT PET/CT and 0.061 for the 18 F-FDG PET/CT, and both were normally distributed. Regarding the subsequent two-tailed paired t-test, the p-value was 0.031, and the SUVmax of the 18 F-FLT PET/CT was significantly lower than that of the 18 F-FDG PET/CT (mean, 2.1 ± 1.8 vs. 4.1 ± 3.3). The Pearson's correlation coefficient was 0.969, indicating a strong correlation ( Figure 4 and Table 3).
For the axillary lymph node analysis, two patients (patients one and four) with different nodes showed the greatest SUVmax for the 18 F-FLT PET/CT and 18 F-FDG PET/CT; consequently, the SUVmax of the corresponding lymph node was used for the analysis. The Shapiro-Wilk test showed that the SUVmax of the 18 F-FDG PET/CT was normally distributed (p = 0.097) but not that of the 18 F-FLT PET/CT (p = 0.000). The subsequent Wilcoxon signed-rank test showed no significant between-group difference (p = 0.246). The mean and standard deviation were 3.8 ± 6.3 for the 18 F-FLT PET/CT and 4.8 ± 5.1 for the 18 F-FDG PET/CT. The Pearson's correlation coefficient was 0.999, indicating a strong correlation ( Figure 4 and Table 3).

Statistical Analysis
When the Shapiro-Wilk test was performed for treatment-naive patients with primary breast cancer (patients 1-6), the p-value for the SUVmax was 0.596 for the 18 F-FLT PET/CT and 0.061 for the 18 F-FDG PET/CT, and both were normally distributed. Regarding the subsequent two-tailed paired t-test, the p-value was 0.031, and the SUVmax of the 18 F-FLT PET/CT was significantly lower than that of the 18 F-FDG PET/CT (mean, 2.1 ± 1.8 vs. 4.1 ± 3.3). The Pearson's correlation coefficient was 0.969, indicating a strong correlation ( Figure 4 and Table 3).     For the axillary lymph node analysis, two patients (patients one and four) with different nodes showed the greatest SUVmax for the 18 F-FLT PET/CT and 18 F-FDG PET/CT; consequently, the SUVmax of the corresponding lymph node was used for the analysis. The Shapiro-Wilk test showed that the SUVmax of the 18 F-FDG PET/CT was normally distributed (p = 0.097) but not that of the 18 F-FLT PET/CT (p = 0.000). The subsequent Wilcoxon signed-rank test showed no significant between-group difference (p = 0.246). The mean and standard deviation were 3.8 ± 6.3 for the 18 F-FLT PET/CT and 4.8 ± 5.1 for the 18 F-FDG PET/CT. The Pearson's correlation coefficient was 0.999, indicating a strong correlation ( Figure 4 and Table 3).

Patients with Prior Treatment before PET/CT
Patient 7 was a 62-year-old woman diagnosed with right-sided breast cancer who only received chemotherapy. After five years, the initial 18 F-FDG PET/CT showed a large right primary breast tumor, intramammary metastases, and multiple right axillary lymph node metastases ( Figure 5). The SUVmax of the primary lesion was 22.0. Additional chemotherapy was provided. She then underwent 18 F-FLT PET/CT seven months later, followed by a second 18 F-FDG PET/CT four days thereafter. The SUVmax of the primary lesion reduced to 7.0 on the 18 F-FLT PET/CT and 10.1 on the 18 F-FDG PET/CT. On the second 18 F-FDG PET/CT, the accumulation in the right breast was higher than that in the contralateral breast, with an SUVmax of 1.4. However, no increased accumulation was noted on the 18 F-FLT PET/CT, with an SUVmax of 0.6. We concluded that the 18 F-FDG accumulation in the right breast occurred secondary to inflammation. A subsequent biopsy revealed that her right-sided breast cancer was an invasive ductal carcinoma.
Patient 8 was a 51-year-old woman treated for left-sided breast cancer and multiple metastases for 7 years. She underwent multiple 18 F-FDG PET/CTs. Her left breast resection revealed an invasive ductal carcinoma. Thereafter, 18 F-FLT PET/CT was performed as the latest PET/CT ( Figure 6); on the left side of the sacral bone, we found an osteolytic mass, which appeared metastatic. Considering that the lesion had lower 18 F-FLT accumulation than the bone marrow, we concluded that the bone metastasis had less active cell proliferation than the bone marrow. This lesion was not diagnosed histologically, but it had progressed with other lesions on subsequent imaging follow-ups. The left axillary lymph nodes (which exhibited accumulation on the 18 F-FDG PET/CT 10 months prior) displayed coarse calcification; therefore, it was difficult to identify whether the 18 F-FDG accumulation reflected inflammation or viable cancer. Conversely, we observed no accumulation on the 18 F-FLT PET/CT, despite no change in lymph node size. Therefore, cell proliferation was low in the left axillary lymph node with the 18 F-FDG accumulation, thereby indicating inflammation. Patient 8 was a 51-year-old woman treated for left-sided breast cancer and multiple metastases for 7 years. She underwent multiple 18 F-FDG PET/CTs. Her left breast resection revealed an invasive ductal carcinoma. Thereafter, 18 F-FLT PET/CT was performed as the latest PET/CT ( Figure 6); on the left side of the sacral bone, we found an osteolytic mass, which appeared metastatic. Considering that the lesion had lower 18 F-FLT accumulation than the bone marrow, we concluded that the bone metastasis had less active cell proliferation than the bone marrow. This lesion was not diagnosed histologically, but it had progressed with other lesions on subsequent imaging follow-ups. The left axillary lymph nodes (which exhibited accumulation on the 18 F-FDG PET/CT 10 months prior) displayed coarse calcification; therefore, it was difficult to identify whether the 18 F-FDG accumulation reflected inflammation or viable cancer. Conversely, we observed no accumulation on the 18 F-FLT PET/CT, despite no change in lymph node size. Therefore, cell proliferation was low in the left axillary lymph node with the 18 F-FDG accumulation, thereby indicating inflammation.

Discussion
This study revealed that 18 F-FLT accumulation secondary to primary breast cancer was lower than 18 F-FDG accumulation in all cases, and significant differences were found in the SUVmax between these accumulations in treatment-naive patients. In patients with primary breast cancer and axillary lymph nodes, the SUVmax showed a strong correlation between the 18 F-FLT PET/CT and 18 F-FDG PET/CT. In particular, 18 F-FLT PET/CT may be

Discussion
This study revealed that 18 F-FLT accumulation secondary to primary breast cancer was lower than 18 F-FDG accumulation in all cases, and significant differences were found in the SUVmax between these accumulations in treatment-naive patients. In patients with primary breast cancer and axillary lymph nodes, the SUVmax showed a strong correlation between the 18 F-FLT PET/CT and 18 F-FDG PET/CT. In particular, 18 F-FLT PET/CT may be useful for estimating cell proliferation in lymph nodes and bone metastases, distinguishing such changes between inflammation and physiological accumulation. 18 F-FLT is a radiolabeled imaging agent serving as the structural analog of the DNA constituent, thymidine [18,24]. The radiolabeling activity depends on DNA replication within the cells; hence, 18 F-FLT uptake depends on the cells' proliferative rate [18,24]. In contrast, 18 F-FDG accumulation depends on glucose intake and reflects an increased metabolism and the Warburg effect [18]. Few studies have examined the differences between 18 F-FLT and 18 F-FDG accumulation in human breast cancer. For instance, in a study by Smyczek-Gargya et al., six patients with breast cancer received 18 F-FLT PET/CT and 18 F-FDG PET/CT within one week. Five of them possessed primary breast cancers with a lower SUVmax and SUVmean in the 18 F-FLT PET/CT than in the 18 F-FDG PET/CT, whereas the remaining patients demonstrated the opposite result; unfortunately, the reason for this discrepancy was not discussed [19]. In the present study, the SUVmax of 18 F-FLT in the primary lesions averaged 2.1 (range, 0. 6-4.8), whereas that of 18 F-FDG averaged 4.1 (range, 1.5-8.4) among treatment-naive patients (patients 1-6). The SUVmax of 18 F-FLT was approximately half that of 18 F-FDG; this difference was statistically significant in patients 1-6. One esophageal cancer study showed that the 18 F-FLT uptake was significantly lower than the 18 F-FDG uptake, potentially as a result of the difference in biochemical and biological mechanisms between these two radiotracers during cell proliferation and differentiation [25]. Although low 18 F-FLT accumulation might lead to an oversight, studies of head and neck squamous cell cancers have shown that the detection of primary lesions and metastatic lymph nodes was comparable between 18 F-FLT PET/CT and 18 F-FDG PET/CT [12,26]. Our present study also reported a strong correlation of the SUVmax between 18 F-FLT and 18 F-FDG in primary breast cancer and axillary lymph nodes.
Because 18 F-FLT PET/CT reflects the cell proliferation cycle, several reports show an association with the Ki-67 labeling index, a pathological marker of cell proliferation [6,9,13]. The accumulation of 18 F-FLT correlates with the Ki-67 labeling index [6,27]. In a metaanalysis that investigated the relationship between the SUVmax and the Ki-67 labeling index in breast cancer, 18 F-FLT PET showed a higher correlation coefficient (r = 0.54) than 18 F-FDG PET (r = 0.40) [28]. In our study, the higher the Ki-67 labeling index, the higher the 18 F-FLT and 18 F-FDG accumulation in surgical cases (patients 1-3). However, this relationship did not seem relevant in inoperable cases (patients [4][5][6]. This discrepancy could be attributed to the fact that the Ki-67 labeling index was measured in a small number of biopsy specimens obtained from patients with inoperable diseases. In other words, estimating cell proliferation using 18 F-FLT PET/CT might be helpful in measuring the accuracy of the Ki-67 labeling index in patients with inoperable diseases.
Our study yielded interesting results regarding lymph node accumulation. The lymph nodes with the greatest accumulation sometimes differed between the 18 F-FLT PET/CT and the 18 F-FDG PET/CT. In two patients, the SUVmax of 18 F-FLT was higher than that of 18 F-FDG. A study on primary colorectal cancer similarly concluded that the SUVmax of 18 F-FLT was significantly lower than that of 18 F-FDG in primary foci; however, the SUVmax of 18 F-FLT was not always lower, and the SUVmax values of the two tracers were not significantly different in positive metastatic nodes [29]. The authors speculated that this finding was probably due to the different biochemical characteristics of the two tracers. Lymph nodes with higher 18 F-FLT accumulation may have faster cancer cell proliferation. In thoracic esophageal squamous cell carcinoma, 18 F-FLT PET/CT has a significantly higher specificity for diagnosing lymph node metastases than 18 F-FDG PET/CT [16]; and in such cases, 18 F-FLT PET/CT may be deemed more important than 18 F-FDG PET/CT.
A few studies have evaluated distant metastatic lesions using 18 F-FLT PET/CT. In patients four and eight, the bone metastases had a relatively low accumulation because of the high physiological 18 F-FLT accumulation in the bone marrow. Therefore, on 18 F-FLT PET/CT, bone metastases may demonstrate a lower accumulation than bone marrow; however, no similar reports exist. Interestingly, the lumbar metastasis in patient six showed 18 F-FLT accumulation equivalent to that of bone marrow. In this case, concurrent CT might facilitate the detection of bone metastases.
Because physiological 18 F-FLT accumulation in the brain is extremely low, brain metastases should be easily detected [30]; however, the present study had no patients with brain metastases. 18 F-FLT PET can provide high specificity because 18 F-FLT is less likely to accumulate in areas of inflammation secondary to cancer therapy [6,8,17]. Therefore, 18 F-FDG accumulation and scarce 18 F-FLT accumulation in the lymph nodes and breast are presumably the results of inflammation.
This study has several limitations. First, the sample size was small. Second, we did not control for surgical history, disease stage, or prior treatments. However, patients were divided into treatment-naive and non-treatment-naive groups before statistical analysis. Third, one patient did not undergo 18 F-FDG PET/CT concurrently with 18 F-FLT PET/CT. Fourth, the assessments performed by the nuclear medicine specialists were not consistent because the diagnoses were conducted prospectively. Finally, considering that breast cancer diagnoses were conducted before PET/CT, the influence of a prior biopsy was inevitable.

Conclusions
A strong correlation was found between the 18 F-FLT PET/CT and 18 F-FDG PET/CT uptake in primary breast tumors and axillary lymph nodes. Differences in the degree of breast cancer accumulation, lesions with the greatest accumulation, and physiological accumulation in organs reflected the different biochemical characteristics of 18   Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Written informed consent was obtained from the patients to publish this paper.