Application of 99mTc-3PRGD2 Imaging for Early Prediction of Pathological Response to Neoadjuvant Chemotherapy in Breast Tumors and Axillary Lymph Nodes

Background and Purpose Technetium 99m-dimeric cyclic RGD peptides with three polyethylene glycol spacers ( 99m Tc-3PRGD 2 ) had a good performance for diagnosing breast cancer. The prospective study was to assess the performance of 99m Tc-3PRGD 2 tumor imaging for predicting pathological complete response (pCR) outcomes to neoadjuvant chemotherapy (NAC) in breast cancer patients. Forty-one patients were examined using both 99m Tc-3PRGD 2 and 18 F-uoro-deoxy-glucose ( 18 F-FDG) imaging before NAC (baseline), and after the rst and fth NAC cycle. The tumor-to-background (T/B) ratios for 99m Tc-3PRGD 2 imaging and the maximum standardized uptake values (SUV max ) from the 18 F-FDG imaging in breast tumors and axillary lymph node (ALN) metastases were separately calculated and analyzed—based on receiver operating characteristic (ROC) analysis. imaging, our study shows that use of 99m Tc-3PRGD 2 imaging offered a similar level of predictive performance for breast cancer pCR to NAC, and early T/B 1 trends of ALN showed an higher performance for predicting pCR.


Introduction
Breast cancer is one of the most common malignant tumors in females worldwide [1,2]. Neoadjuvant chemotherapy (NAC) is an important therapeutic option for use with advanced stage patients and allows for more conservative surgeries to be performed and can facilitate the resection of tumors which are initially deemed inoperable [3,4]. Several studies have demonstrated that the responses to chemotherapy in breast cancer and metastatic axillary lymph nodes (ALN) are factors that apparently affect tumor prognosis, with especially pronounced associations in patients who achieve a pathological complete response (pCR) [5][6][7].
Previous studies used 18 F-uoro-deoxy-glucose ( 18 F-FDG) positron-emission-tomography/computedtomography (PET/CT) to monitor the therapy response during NAC [8][9][10][11]. However, the performance in predicting a pCR have to date been inconsistent among diverse breast cancer subtypes and between different monitoring time points. Study suggested that the relative changes of the maximum standardized uptake values (SUV max ) after one cycle of NAC can accurately predict responsiveness in human epidermal growth factor receptor 2 (HER2) positive tumors [9], whereas a similar second study reported that PET/CT is useful for predicting pCR in estrogen receptor (ER) positive/HER2-negative and triple-negative tumors [10].
There were contradictory ndings among different studies, thus, we aimed to explore another peptide probe to evaluate the pathological response of breast cancer. Integrin αvβ3 is widely expressed in the cell-cell and cell-matrix, may have an essential function in contact that promotes tumor metastatic ability [12]. Tumor imaging study has shown that the HYNIC-3PEG4-E[c (RGDfK) 2 ] (3PRGD 2 ) peptide speci cally bound to tumor αvβ3 and had a noticeable performance for diagnosing breast cancer when compared with 18 F-FDG imaging [13]. It is notable that 99m Tc-3PRGD 2 employs a lower radiation dose and more convenient labeling procedure than 18 F-FDG; the 99m Tc-3PRGD 2 technique can be used to assesses tumor vascularization without access to PET equipment; even 99m Tc-3PRGD 2 can accurately recognized tumors with low 18 F-FDG uptake [14][15][16][17][18].
Several studies have reported that RGD peptides can be used to monitor early response of malignancies to chemoradiotherapy or to anti-angiogenesis therapy [19][20][21][22]. The changes of tumor integrin α v β 3 expression and tumor to normal background ratio (T/B) on 99m Tc-3PRGD 2 imaging in the squamous cell tumors signi cantly decreased in treatment group [22]. A study showed that 99m Tc-3PRGD 2 imaging can monitor the early pathological response of primary breast tumors to NAC [23]. However, the de nition of the pathological response of that study included minimal residual tumors, and it examined breast tumor pathological response only (i.e., no assessment of potential metastasis sites). Previous study showed that the 99m Tc-3PRGD 2 uptake rate was higher in HER2-positive breast patients than in luminal patients and triple-negative patients [13]. The combination of HER1/EGFR and HER2 is the most potent inducer of VEGF expression and tumor vascularity [24,25]. Therefore, we want to explore whether 99m Tc-3PRGD 2 imaging is a promising functional imaging modality for molecular phenotypes in breast cancer patients.
So, this study investigated the performance of 99m Tc-3PRGD 2 and 18 F-FDG imaging technologies in predicting pCR to NAC in both breast tumors and axillary nodes in advanced breast cancer patients. And the relationship between breast cancer subtype and pathological was also taken into consideration.

Materials And Methods
Ethics and institutional review board approval This study was approved by the institutional ethics committee of hospital. It has been registered online at NIH ClinicalTrails.gov (NCT 02742168). Written informed consent was obtained from all participants. The authors had full control of the data and information submitted for publication.

Study participants
This prospective study recruited 46 women who were rst diagnosed with locally advanced stage II-III breast cancer from October 2015 to October 2018. The ''gold standard'' for diagnosis of the primary tumors and axillary lymph nodes were based on histopathological ndings of ne needle biopsy (FNA) before treatment. The stage of cancer was determined by 18 F-FDG PET/CT, ultrasound, MR, and/or whole-body bone imaging according to the TNM classi cation.
Exclusion criteria were: 18 year's old, pregnancy, and lactation period and the patients with distant metastatic lesions.
Five patients were excluded because two patients were diagnosed with distant metastases during NAC (a brain metastasis and a lumbar vertebrae metastasis diagnosed by MRI or PET/CT), and three patients missed followed up checks during NAC. Finally, forty-one patients were included in the nal statistical analysis (mean age 61.50 ± 7.84 years, range from 25 to 65; mean weight 56.64 ± 7.53 kg, range from 40 to 69).

Histopathological analysis and treatment
Immunohistochemical (IHC) staining was used to determine the histological subtypes. ER or progesterone receptor (PR) status was de ned as positive when the number of positive nuclei was > 1%. HER2 expressed on the membrane was scored from 0 to 3. Scores of 3 + or 2 + and uorescence in situ hybridization positive detection was de ned as HER2-positive. The breast cancer subtypes were categorized into luminal, HER2-positive and triple-negative.
All patients were treated with four cycles of NAC (epirubicin 90 mg/m 2 on day 1 and cyclophosphamide 600 mg/m 2 on day 1 every 14 days) followed by four cycles of T (taxane 175 mg/m 2 on day 1 every 21 days). In HER2-positive patients, trastuzumab was given with the last fourth cycle of taxane.

Radiopharmaceutical preparation
A 3PRGD 2 kit was supplied by the Medical Isotope Research Center of Peaking University. 99m Tc-labled RGD peptide was a noninvasive imaging of integrin α v β 3 expression via SPECT and the preparation process of 99m Tc-3PRGD 2 have been fully characterized in previous studies [13,26] The 18 F-FDG was purchased from Advance Medical Systems Limited, Nanjing, China.

Imaging
All patients underwent 99m Tc-3PRGD 2 imaging and then 18 F-FDG imaging within three days. Each imaging series included three single-photon-emission-computed-tomography/computed-tomography (SPECT/CT) scans followed by three PET/CT scans. Note that baseline SPECT/CT 0 and PET/CT 0 imaging ( rst scan) were performed before the start of NAC, and after the rst (second scan) and fth (third scan) NAC cycles. 99m Tc-3PRGD 2 Imaging For patients at SPECT/CT: after intravenous injection of 11.1 MBq/kg 99m Tc-3PRGD 2 , whole-body planar (10cm/min) and SPECT/CT (30s/frame/6 ) scans for thorax were performed using a double-headed γ camera equipped with low-energy high-resolution collimators (In nia Hawkeye4, GE Healthcare). The matrix was 128 128 pixels, and the photo-peak was centered on 140 keV with a 20% energy window. 18 F-FDG Imaging Patients were prepared via a 6-hour fasting period with blood glucose levels <150mg/dl. An FDG dose of 170-230 MBq (0.1 mCi/kg) was given intravenously. PET/CT (Biography mCT (64), Siemens) scans were acquired at 60 minutes after 18 F-FDG injection. A PET scan (2.00 min per bed position) was performed for each patient, and PET acquisition was followed by low-dose CT (3 mm slices). The standard supine PET/CT was from the skull to the middle femur region.

Imaging reading
The three experienced nuclear medicine reading physicians were blinded to the pathological results, and could refer to other imaging results. A consensus decision was required for any discordant initial assessments. The readers evaluated the images visually for the focal tracer uptake compared with the uptake in surrounding normal tissue and the maximum radioactive counts of the primary breast tumors and prominent ALNs were measured in the two imaging modalities. If the patient without visible lymph node trace uptake for both of the imaging modalities at baseline would not undergo the subsequent pathological response predicting analysis. 99m Tc-3PRGD 2 imaging analysis Analysis of 99m Tc-3PRGD 2 uptake into tumors was based on region of interest (ROI) analyses. Seeking to avoid nonspeci c 99m Tc-3PRGD 2 uptake into benign lesions or normal breast tissue which were found in the previous studies [12], the blood pool of the aortic arch was used as a control for the primary tumor. For the controls in the for ALN image data, contralateral normal axillary areas were used. The mean counts for control regions of interest were measured and used as the background values for calculating the tumor-tobackground (T/B) ratios. Note that if an apparent tumor remission yielded no obvious tracer uptake, we analyzed the same region of interest location as in SPECT/CT 0 . We calculated T/B ratios for three SPECT/CT scans: T/B 0 , T/B 1 , and T/B 2 , with each T/B ratio calculated as follow:

F-FDG imaging analysis
Nuclear medicine physicians used a standard analytical pipeline identi ed the mostly focal 18 F-FDG uptake of tumors and ALN. The ROIs were drawn manually in the primary tumors and ALNs and measured the SUV max (SUV max0 , SUV max1 , SUV max2 ) obtained by generating a 3D region of interest [21].
The relative changes of SUV max were calculated as follows: Response assessment Surgery with ALN dissection was performed after 8 cycles of NAC routinely. We de ned pathological complete response (pCR) as the simultaneous absence of any residual invasive tumor cells from all of the breast and axillary node specimens.

Statistical analysis
Statistical analyses were performed using SPSS 24.0 software. Student's t-tests, Fisher's exact test, and binary logistic regression were used for analysis of parameters comparisons. The ROC curve analysis was used to evaluate the predictive performance of pathological responders vs. pathological non-responders. Z tests were used to compare the areas under curve (AUCs). P < 0.05 was considered to be statistically signi cant.  Table 1. Analysis of these data indicated that tumor size, tumor stage, and HER2-positive status were signi cantly associated with pCR (P = 0.004, 0.037, and 0.014, respectively) in this cohort. Further, a logistic regression demonstrated that breast tumor size and HER2-positive status were predictive factors for pCR, with odds ratio values (OR) of, respectively, 2.81 (P = 0.014, 95% CI 1.237-6.388) and 0.129 (P = 0.020, 95% CI 0.023-0.722).

Initial analyses with both imaging modalities
There were 13 of 41 NAC pCR for primary breast tumors with 99m Tc-3PRGD 2 and 18 F-FDG uptake. Only one patient (HER2-positive) had no de nite metastatic lymph node before treatment and nally reached pCR (Fig. 1). Forty of the patients were diagnosed with metastatic ALNs prior to NAC, and 12 of 40 patients with ALN metastases achieved pCR by ALN dissection. There were 2 of 3 HER2-positive patients with ALN metastases did not initially exhibit 99m Tc-3PRGD 2 and 18 F-FDG tracer uptake during the baseline scans, as well as 10 of 37 patients with tracer uptake were eventually divided into pCR group.

Comparison early trends between the two imaging methods
In the ROC analysis, AUCs of the ΔT/B 1 on 99m Tc3PRGD 2 imaging (0.827) and the ΔSUV max1 on 18 F-FDG imaging (0.859) for breast tumors showed comparable predictive value for pCR after one cycle (Z = 0.33, P = 0.74). However, for ALN metastases, the AUC for the ΔT/B 1 data from the 99m Tc-3PRGD 2 imaging data (AUC = 0.859) after rst NAC cycle was greater than the AUC for the ΔSUV max1 data from 18  17.82 8.60; all P < 0.05) ( Table 3). The result were observed among luminal patients, but it was notable that the trends (both ΔT/B 1 and ΔSUV max1 ) in primary breast cancer and ΔSUV max1 in ALN metastases were additionally evident based on the SUV max data (25. Fig. 3 showed the images at three time-points scanning in a HER2-positive patient with pCR. Further, the Fig. 4 showed a non-pCR images in a luminal patient. Because only one patient achieved pathological complete responder in triple-negative, the subtype analysis was only performed in HER2-positive and luminal patients.

Discussion
The integrin α v β 3 played important roles in tumor angiogenesis, invasion, and metastasis [27,28]. The integrin α v β 3 expression can be noninvasively observed in vivo by 99m Tc-3PRGD 2 imaging. This study showed that the primary tumors were found by two images, in addition, we found that some small metastatic lymph nodes exhibited no tracer uptake both in the initial scans of two modalities. Our previous work found that 99m Tc-3PRGD 2 imaging and 18 F-FDG imaging had a low-to-moderate sensitivity in small lymph nodes due to the low spatial resolution of SPECT and the size of lymph nodes [13]. Studies reported that micro-metastases with few malignant cells exhibit very weak tracer uptake levels and even need to be con rmed by pathology [13,29].
A pCR in both primary breast tumor and axillary node shows a favorable outcome than breast pCR only [29,30]. It bears emphasis that the de nition used for pCR in our study was a lack of any invasive tumors in breasts and in lymph nodes. The pCR rate (13/41 31.7%) in this study was similar to that reported by previous studies [8,9]. The ΔT/B 1 of primary tumors showed a ROC-AUC (0.827) which was slightly lower than in a previous study (0.89) [23]. However, both that study and the present study found that the ΔT/B ratios for breast tumors in pCR group were higher than non-pCR group. Despite these similarities, there are some notable differences between the two studies, including the number of patients and the de nition of pathological response, as well as the time interval for the chemotherapy. The proportion of patients of pCR in our study was only 31.7%, as there were patients that presented with a small number of scattered tumor cells, thereby classifying them as non-pCR status. 99m Tc-3PRGD 2 imaging performed similarly with the 18 F-FDG imaging for pCR prediction. When compared with 18 F-FDG, 99m Tc-3PRGD 2 can be imaged even in practices without accessing to PET equipment.
Moreover, the RGD peptide may offer biologically informative data relevant to antiangiogenic therapies and to anti-integrin α v β 3 therapies. Previous studies have reported that micro-vessels are important factors that can affect breast cancer prognosis [31,32]. The RGD peptide can be used to monitor the response of malignancies to antiangiogenic therapies [19][20][21]. A clinical trial showed that 18 F-galacto-RGD offered an advantage over 18 F-FDG PET for evaluating the anti-angiogenesis or anti-integrin α v β 3 e cacy of cancer treatments [33]. At present, the targeted drugs for anti-angiogenesis therapy in breast cancer have been used in clinical research [19]. Imaging based on integrin α v β 3 found that 18 F-ALT-NOTA-PRGD 2 may predict a better response to apatinib anti-angiogenesis therapy in breast cancer [34]. A receptor imaging by combing diagnosis and monitoring drug response may be a valid instrument to select breast cancer patients who could bene t from targeted therapies [35].
Additionally, we found that the ΔT/B1 ratios of ALN metastases in pCR group were higher than in the non-pCR group; however, there were no statistically signi cant differences in the early FDG uptake changes (ΔSUV max1 ). This can potentially be explained by the expected decrease in the integrin α v β 3 expression level upon the initiation of tumor cell necrosis and/or apoptosis during NAC [22]. Integrin α v β 3 is widely expressed in the cell-cell and cell-matrix, may have an essential function in contact that promotes tumor metastatic ability [12]. Previous study reported that both the migration and metastasis capacity of tumor cells was signi cantly inhibited upon α v knockdown [36]. We found that the AUC of ΔT/B 1 (0.859) in ALNs was greater than that of ΔSUV max1 (0.572) for the early prediction of pCR (P = 0.035). Xiaonan Jin et al.
also reported that 99m Tc-3PRGD 2 SPECT/CT imaging outperformed 18 F-FDG PET/CT in terms of higher speci city for the diagnosis of lung cancer lymph node metastasis [37]. It was thus notable that we were able to detect early 99m Tc-3PRGD 2 uptake changes in ALN metastases when monitoring the pCR. However, the aim was just to investigate the value of 99m Tc-3PRGD 2 imaging for predicting the breast tumors and ALNs pathological response but not to provide the monitor guidelines.
This study also indicated that HER2 status is associated with pCR: HER2-positive group obtained a higher pCR rate (9/13) than the HER2-negative group. Trastuzumab may increase the sensitivity of breast tumors to chemotherapy [38,39]. We found in the present study that the pCR group showed higher ΔT/B than the non-pCR group among HER2-positive patients. Our previous study demonstrated that the 99m Tc-3PRGD 2 uptake rate of breast tumors was higher in HER2-positive patients than in luminal patients and triplenegative patients [23]. A clinical pathology study found a signi cant correlation between HER2 expression and microvessel density (MVD) in breast tumor specimens [40]. Another study reported a signi cant positive correlation between the RGD PET parameters and the HER2/CEP17 ratio in advanced invasive ductal carcinoma patients [41]. When compared with previous study [8], a similar result was observed in our study that 18 F-FDG PET/CT may predict pathological response in luminal tumors, but may be less accurate for HER2-positive tumors. As 5 triple-negative patients with non-pCR mentioned in the study, the chemoresistant may be predicted by imaging.
A limitation of our study was its limited number of patients, though the number was larger than previous study with 99m Tc-3PRGD 2 imaging [23]. Further multi-center clinical studies with larger patient numbers would be informative: for example, the tumor subtype analyses and cut-off points could be given further consideration if the patient cohort size permitted in more hospital. Furthermore, different NAC regimes should be included in future comparative studies.

Conclusion
Our study shows that analysis of primary tumors and ALNs with either 99m Tc-3PRGD 2 imaging or 18 F-FDG imaging offers comparable performance levels for predicting pCR to NAC. Moreover, we found that monitoring of early changes in ALN metastases using 99m Tc-3PRGD 2 imaging offered a more meaningful predictive information than 18 F-FDG imaging. 99m Tc-3PRGD 2 may be a promising functional imaging modality for prediction pathological response in HER2-positive patients.