Approximately 15%–30% of invasive breast cancer cases demonstrate human epidermal growth factor receptor 2 (HER2) overexpression and/or gene amplification, which is associated with aggressive biological behavior and an unfavorable clinical outcome (1). At the same time, HER2 also serves as a critical therapeutic target in breast cancer, and its status has so far been acknowledged as the best biomarker for predicting response to anti-HER2 therapies. Therefore, the accurate assessment of HER2 expression in breast cancer is essential for clinical diagnosis and treatment decisions.

Current diagnosis of HER2 overexpression or gene amplification relies on the immunohistochemistry (IHC) and/or in situ hybridization (ISH) tests on pathological specimens. However, the spatiotemporal heterogeneity of HER2 expression poses a challenge to the determination of HER2 status in breast cancer using biopsies. The HER2 status in metastases has been observed to be different from that of the primary breast cancer in 3%–10% of patients, or to be even inconsistent within a single lesion (2–5). In addition, HER2 status may sometimes change during anti-HER2 treatment, which can be partially explained by the drug sensitivity of HER2-positive tumor cells (6, 7). These results indicate the necessity of HER2 re-evaluation for breast cancer patients during the disease course, especially for recurrent diseases. However, repeated biopsies are not always clinically feasible in patients. Therefore, alternative noninvasive modalities with high accuracy are urgently needed to determine HER2 status in breast cancer.

Molecular imaging serves as a promising alternative to monitor whole-body HER2 expression quantitatively and dynamically during the management of breast cancer. Radiolabeled HER2 antibody, antibody fragments, and affibody are representative HER2-targeted imaging modalities under current clinical investigations. Positron emission tomography (PET) using HER2 antibodies labeled with radionuclides such as ¹¹¹In, ⁶⁴Cu, and ⁸⁹Zr demonstrates sensitive and specific HER2 uptake in HER2-positive metastatic and primary breast cancer, and it can also identify unsuspected HER2-positive lesions (8–11). However, their clinical use was limited by disadvantages such as slow pharmacokinetics and relatively high radiation dosage. HER2 antibody fragment labeled with ⁶⁸Ga is well-tolerated and allows imaging in a shorter time after injection, but shows a lower sensitivity of lesion detection (12). Affibody molecules are a group of small and robust affinity proteins (6.5 kDa) engineered to mimic antibody based on the immunoglobulin G domain of staphylococcal protein A (13, 14). Due to its higher sensitivity and rapid penetration into target lesions (15, 16), affibody demonstrates superiority in molecular imaging compared with monoclonal antibody and antibody fragments, especially in the detection of metastases inaccessible to biopsies. Affibody also requires lower effective doses and can be rapidly eliminated from blood, which yields a better tolerance and safety profile than other affinity proteins. ⁶⁸Ga-HER2 affibody PET/computed tomography (CT) is one of its imaging applications that has been supported by preclinical and clinical evidence (17–19). ⁶⁸Ga-HER2 affibody PET/CT indicates a high accuracy in the detection of converted HER2 expression in metastatic cancer, which may assist in anti-HER2 treatment modification during patient management (20, 21).

In this prospective pilot study, we aimed to evaluate the clinical value of ⁶⁸Ga-HER2 affibody PET/CT to noninvasively assess the HER2 intensity of lesions using pathologically confirmed HER2 status as the standard and to then explore its value in clinical decision-making regarding the use or non-use of anti-HER2-targeted agents in patients who were otherwise inappropriate candidates for routine use of anti-HER2 agents.

In this prospective study, ⁶⁸Ga-HER2 affibody PET/CT demonstrated a high diagnostic accuracy in differentiating HER2-enriched breast cancer, with a sensitivity of 91.7% and a specificity of 84.6%, regardless of prior lines of anti-HER2 therapies. It is a clinically feasible modality for the whole-body evaluation of HER2 status noninvasively and may complement the current standard HER2 testing approach. Small malignant lesions could not be missed, because they can be found by the morphologic CT component of the PET/CT. In the present study, ⁶⁸Ga-HER2 PET/CT showed a similar performance with ¹⁸F-FDG in assessing metastases. Xu and his colleagues investigated both ¹⁸F- and ⁶⁸Ga-labeled HER2 affibody and found that both of the radiotracers displayed specific binding to the HER2 receptor. However, ⁶⁸Ga-HER2 affibody could be easily prepared in nearly 20 min with good radiochemical purity. Compared with the ¹⁸F-labeled counterpart, the yields and specific activity of ⁶⁸Ga-HER2 affibody were both significantly increased (~80% vs. ~10% and ~23 GBq/μmol vs. ~9 GBq/μmol, respectively) (17). Multiple synthesis steps and low labeling yields may partially limit the use of ¹⁸F-HER2 affibody.

For breast cancer patients who develop recurrent or metastatic lesions, accurate HER2 evaluation is vital for the determination of optimal treatment strategies. Previous studies have found out the spatiotemporal heterogeneity of HER2 expression between or within primary and metastatic tumors, which was also verified in this study. On this account, HER2 re-evaluation of recurrent and metastatic sites has been highly recommended by the ASCO/CAP guideline, even for patients with HER2-negative primary breast cancer (23). Histopathological evaluation based on tissue biopsy is the current standard testing for HER2 status, while repeated biopsy is not always possible, especially for metastatic lesions in locations difficult for biopsies, such as the brain, lung, sternum, and adrenal gland. HER2 PET/CT demonstrates its advantage as a noninvasive modality to detect HER2-positive lesions based on the whole-body scan in these settings.

Higher radioactivity accumulation was found in HER2-high-expressing BT474 cells/xenografts than in HER2-lower-expressing MCF-7 cells/xenografts. Additionally, one breast cancer patient with a HER2 IHC score of 3+ had a higher SUV_max than the patient with a score of 1+ (17). Other previous studies described a good relationship between the FISH ratio and SUV_max, and a significant linear correlation between the HER2 affibody uptake value and relative HER2 expression levels was also found (21, 24). These results implied that the probe might have the capability to accurately diagnose HER2 levels. The surveillance of HER2 status using ⁶⁸Ga-HER2 affibody PET can also provide useful information for patients’ prognosis and treatment modification. Both retrospective and prospective studies have shown that HER2 status can possibly change during anti-HER2 treatment (7, 25, 26). A higher proportion of HER2 discordance is found in patients after neoadjuvant chemotherapy alone than after neoadjuvant chemotherapy combined with anti-HER2 treatment. Loss of HER2 positivity has been reported to be associated with significantly worse disease-free survival and overall survival compared with those with maintained HER2 positivity in breast cancer patients (27, 28). In addition, data have shown that patients who gain HER2 positivity during progression can derive extra benefit from anti-HER2 treatment in the metastatic setting (29). Meanwhile, ⁶⁸Ga-HER2 affibody PET imaging will not be affected by anti-HER2 treatment, since HER2 affibodies bind with different extracellular domains of HER2 from HER2 antibodies. Therefore, ⁶⁸Ga-HER2 affibody PET/CT may be useful as an individualized “image and treat” strategy for the monitoring of changes in receptor expression during treatment, which may facilitate clinicians to optimize treatment decisions promptly.

Our study also suggested the expanded clinical value of ⁶⁸Ga-HER2 affibody PET/CT in the differential diagnosis of breast cancer metastases and synchronous primaries based on the determination of HER2 status. ¹⁸F-FDG PET/CT has been extensively used to detect suspicious multiple metastases of the whole body, and can sometimes identify unexpected lesions, but it cannot always distinguish well between inflammatory lesions and metastases since both may demonstrate high metabolic activity characterized by increased glucose uptake on the scan. The HER2 uptake of suspicious lesions on ⁶⁸Ga-HER2 affibody PET/CT scan can provide an important clue for the diagnosis of metastases from HER2-positive breast cancer and discriminate from inflammation. Previous studies have reported that approximately 12% of breast cancer survivors are diagnosed with second malignant primaries (30), and the incidence of occurring multiple primaries ranges from 4.1% to 16.4% (31). It is critical to discriminate synchronous second or multiple primaries from metastatic lesions in patients with breast cancer since their therapeutic principles are totally different, while invasive diagnostic procedures are always needed in these cases. In this study, a different HER2 uptake was shown on the HER2 PET/CT scan in synchronous primaries confirmed by clinical or pathological diagnosis. ⁶⁸Ga-HER2 affibody PET/CT can serve as a noninvasive approach to assess the HER2 expression of multiple lesions simultaneously, which may extend its use in facilitating the clinical diagnosis of synchronous primaries.

There are also several noteworthy limitations. First, the sample size for analyzing diagnostic accuracy was relatively small. Further validations of its diagnostic ability in a larger population are needed. On top of that, the cutoff values of ⁶⁸Ga-HER2 affibody PET/CT for discriminating HER2-positive breast cancer and defining HER2 low expression are yet to be defined. We look forward to deriving the cutoff values of ⁶⁸Ga-HER2 affibody PET/CT for discriminating HER2-positive/negative breast cancer from further clinical trials. Two clinical trials of ⁶⁸Ga-HER2 affibody PET/CT are currently ongoing in our center: one trial evaluates its ability to differentiate a HER2-enriched subtype of metastatic breast cancer with an exploration of cutoff value (STANDPOINT, NCT: 04758416), and the other trial investigates its predictive value in anti-HER2 treatment through dynamic surveillance (DOLPHIN, NCT: 04769050).

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

The studies involving human participants were reviewed and approved by the Ethics Committee of Fudan University Shanghai Cancer Center. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

HM, XH, SS, and JZ contributed to the conceptualization and design of the study. HM and YS were responsible for the collection and assembly of data. YJ, YS, and HM completed the statistical analyses and drafted the manuscript together. XH, SS, and JZ revised the manuscript with constructive ideas. All authors contributed to the article and approved the submitted version.

This study was supported by the National Natural Science Foundation of China (grant no. 82072915); Project of Shanghai Municipal Health Commission (grant no. 202140397); CSCO-ROCHE Cancer Research Fund 2019 (grant no. Y-2019Roche-171); and Chinese Young Breast Experts Research project (grant no. CYBER-2021-001).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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