Expression of this receptor occurs in around 20% of patients with breast cancer; it is currently determined by means of biopsy and immunohistochemistry and is considered a negative prognostic factor (8). It is located on the outer cell surface where it is involved in the regulation of cell growth and differentiation. Tumours with HER2 expression tend to be more aggressive and are associated with a relatively high mortality (8, 9).

Trastuzomab (Herceptin™) is a recombinant G1 immunoglobulin monoclonal antibody that targets and blocks the extracellular domain of the HER2 receptor. It is an FDA-approved humanized monoclonal antibody that targets HER2/neu cancer cells, and which causes an inhibitory effect on cell growth (10, 11). It follows then that trastuzomab would make for an attractive pharmaceutical to label with both imaging and therapeutic isotopes.

Non-representative tissue biopsies may unfortunately result in false negatives on immunohistochemistry in up to 20% of cases (12). Considering the potential impact this has on patient management, there is a strong case for PET/CT imaging in determination of HER2 expression.

In the pre-clinical setting, studies have made use of the following radiopharmaceuticals, namely ⁶⁴Cu-trastuzomab, ²¹¹At-trastuzomab, ¹¹¹In-DOTA-trastuzomab and ¹⁷⁷Lu/⁹⁰Y-trastuzomab, amongst others. These studies (performed on mouse models) have shown mostly promising results with a significant increase in median survival demonstrated (13, 14).

A group from Chandigarh, India demonstrated the feasibility of in-house labelling of trastuzomab with Lu-177 via the DOTA chelator. They reported 12 h of stability, and image confirmation of the targeted primary and metastatic lesions was obtained at 5 days post-injection. More recently, the same group shared their findings in 6 patients with HER2 positive metastatic breast cancer compared to four patients with HER2 negative breast cancer (15).

SPECT/CT imaging confirmed uptake in the primary and metastatic lesions in contrast to the HER2 neg group that did not demonstrate any abnormal tracer accumulation. ¹⁷⁷Lu-trastuzumab administration was well tolerated in nine patients and one patient experienced mild fever. Fever is a known side effect that is associated with trastuzumab administration, and the patient was managed with simple antipyretics. No unexpected toxicities were observed with ¹⁷⁷Lu-trastuzumab administration and biochemistry, and cardiac function was not affected. Intense liver accumulation is a limiting factor as expected, and further studies are needed. Feasibility of this form of targeted therapy in metastatic HER2 breast cancer was demonstrated (15).

One of the first studies to be published on the use of trastuzomab imaging in patients with breast cancer was by Dijkers and colleagues and involved the use of ⁸⁹Zr-trastuzomab in 14 patients. Normal biodistribution of Zr-89-trastuzumab was reported in the liver, kidneys, spleen, and brain apart from localization in metastatic lesions (16).

Other investigators included Gebhart et al. and Bhusari et al. (15, 17). The latter proceeded to substitute ⁸⁹Zr with Lu-177 in a group of 10 patients with breast cancer to demonstrate feasibility and the possibility of SPECT/CT imaging for target confirmation and dosimetry. They established an optimal imaging time point of 4–5 days post-injection (allowing for sufficient background clearance) and confirmed feasibility of this imaging and therapeutic approach. HER2 positive breast lesions demonstrated sufficient uptake with 10 mCi of ¹⁷⁷Lu-trastuzumab whilst no uptake was noted on HER2 negative lesions. The authors highlighted the possibility of a therapeutic approach with ¹⁷⁷Lu-trastuzumab in patients with HER2 positive lesions that are refractory to standard forms of therapy (15).

As a complete antibody, trastuzomab is a large protein with a high molecular weight, a long half-life, relatively poor tumour infiltration and inadequate renal filtration. This may lead to limited internalisation and tumour effects, off-target binding with haemato- and hepatotoxicity and inhomogeneous tumour distribution. Most of these problems could, however, be eliminated by making use of an antibody fragment with resultant increased target to background ratio, rapid elimination, and reduced toxicity. The issue of tumour resistance to trastuzomab remains however, and the intense liver accumulation remains problematic (18, 19).

Trastuzomab-directed radionuclide therapy appears feasible and allows for labelling to multiple radioisotopes with promising pre-clinical- and clinical results. The inability to visualise liver lesions due to intense physiological uptake is a limitation.

Cancer-associated fibroblasts (CAF) fulfil an integral role within the tumour micro-environment, where they support and facilitate malignant growth and metastatic spread. These fibroblasts express a protein, FAP-alpha, which provides an attractive target for both imaging and therapeutic translation as it is expressed in the stromal tissues of many malignancies, whilst remaining largely absent from normal tissues. Considering that the stroma can account for up to 90% of the total tumor mass, this may increase tumor cell accessibility for pharmacologic, immunologic, or cell-based therapies. Simultaneous irradiation of CAFs and surrounding tumor cells may also be achieved through the crossfire effect. Additionally, since FAP-expressing CAFs are known to be immunosuppressive, a combination with immunotherapy may produce a synergistic therapeutic effect (20, 21).

Fibroblast Activation Protein is a type II integral membrane glycoprotein from the serine protease family that plays a role in fibrogenesis and in the remodelling of the extracellular matrix. FAP-inhibitors (derived from quinoline peptidomimetics) are used to target these receptors and several forms of FAPI are available, such as FAPI-04, FAPI-02, FAPI-46, FAPI-2286. Major advantages offered by this target include its superior target to background ratios, the lack of any required patient preparation and its potential for therapeutic translation (22–24).

Fibroblast Activation Protein as a theranostic target has enjoyed significant attention in recent times with a 2023 review article including 35 publications on targeted radioligand therapy in both the pre-clinical and clinical settings and stating that over 100 patients have been treated with different FAP targeted radionuclide therapies (including ¹⁷⁷Lu-FAPI-04, ⁹⁰Y-FAPI-46, ¹⁷⁷Lu-FAP-2286, ¹⁷⁷Lu-DOTA.SA.FAPI and ¹⁷⁷Lu-DOTAGA.(SA.FAPi)2. These studies have reported objective responses in difficult to treat end-stage cancer patients with manageable adverse events (25).

Ballal and colleagues shared their experience in a 2020 publication on the compassionate use of ¹⁷⁷Lu-DOTA-FAPi in a 31-year-old female with breast cancer and a metastatic brain lesion. The primary breast tumour was characterised as ER-, PR- and HER 2+, and she had disease progression on conventional therapy. They administered 3.2 GBq of ¹⁷⁷Lu-DOTA-FAPi with target lesion confirmation on dosimetry images, which resulted in symptomatic relief.

A later study with SA.FAPI reported on a total of ten patients, which included four with breast cancer (26).

Baum and colleagues also reported on the use of 5.8 =/− 2 GBq of ¹⁷⁷Lu-FAPI-2286 in eleven patients with a variety of malignancies that included four patients with breast cancer. Treatment in this group was well tolerated with acceptable dosimetry and toxicity in addition to resulting in significant pain relief. Post-therapy images revealed significant uptake of ¹⁷⁷Lu-FAP-2286 and long retention of the radiopharmaceutical in all patients (27). In contrast, FAPI-02 and FAPI-04 demonstrated earlier washout with a corresponding shorter tumour retention time (28).

Lindner et al. used 2.9 GBq of ⁹⁰Y-FAPI-04 to treat a patient with metastatic breast cancer, which resulted in significant pain reduction without significant side effects. The authors postulated that higher treatment doses could potentially be used to increase the tumoricidal effect. Bremsstrahlung images confirmed the intended target lesion delivery up to one day post-injection (28).

Assadi et al. treated 21 patients (including 5 breast cancer patients) with advanced stage malignancies with 1.85–4.44 GBq of ¹⁷⁷Lu-FAPi-46 per cycle. Feasibility and an acceptable side effect profile was demonstrated in this compassionate use setting (29).

The LUMIERE trial is a phase I/II study to evaluate the safety, dosimetry, pharmacokinetics, and preliminary anti-tumor activity of ¹⁷⁷Lu-FAP-2286 in patients with advanced or metastatic solid tumours (30).

A dosimetry study by Kuyumcu et al. following administration of ¹⁷⁷Lu-FAPI-04 to four patients (which included one patient with breast cancer) also highlighted the short tumour retention time of FAPI-04 whilst confirming acceptable dosimetry (31).

These initial experiences highlight the safety and feasibility of using radiopharmaceuticals such as ⁹⁰Y-FAPI-04 and ¹⁷⁷Lu-DOTA.SA.FAPI in settings where patients are refractory to conventional forms of therapy.

Several FAP-based tracers have been reported with most data referring to FAPI-04, FAPI-46, FAP-2286, and DATAGA.(SA.FAPI)2, and promising clinical data reported in patients with breast cancer.

Studies have reported acceptable side effect and toxicity profiles, with the most important reported adverse events related to bone marrow toxicity (anaemia, leukopenia, and reduced number of platelets), similar to the now known side effect profiles of ¹⁷⁷Lu-PSMA and ¹⁷⁷Lu- DOTATATE. Based on the inconsistent data from the various small and non-standardised studies, it is not yet possible to conclusively determine which FAP inhibitor has the best efficacy and toxicity profile. The reported absorbed dose to the tumor are comparable to those of ¹⁷⁷Lu-DOTATATE and ¹⁷⁷Lu-PSMA-617 and range from 0.62 ± 0.55 Gy/GBq, 2.81 ± 1.25 Gy/Gbq, 3.0 ± 2.7 Gy/GBq, and 6.70 (IQR: 3.40–49) Gy/GBq, for ¹⁷⁷Lu-FAPI-04, ⁹⁰Y-FAPI-46, ¹⁷⁷Lu-FAP-2286, and ¹⁷⁷Lu-DOTAGA.(SA.FAPi)2, respectively (32, 33).

Most malignancies express FAP in the tumour micro-environment, while some cancers express FAP on their cellular membrane (e.g., sarcoma, certain ovarian, and pancreatic cancers). Crucial to the success of FAP-based targeted radionuclide therapy is the issue of tumour retention time, which should be long enough to achieve a tumoricidal effect, without increasing radiation to non-target lesions and organs. To date the most promising results have been reported with ¹⁷⁷Lu-FAP-2286, ¹⁷⁷Lu-DOTAGA.(SA. FAPi)2, ¹⁷⁷Lu-PNT6555, and the albumin-binding tracers (e.g., ¹⁷⁷Lu-EB-FAP) which demonstrates uptake up to 168-h post injection. Attempts to increase tumour retention time have focused on labelling FAPi to radio-isotopes with shorter half-lives (such as Y-90, ¹⁸⁸Re, ¹⁵³Sm, ²¹³Bi, or ²¹²Pb) in an attempt to better match the peptide half-life to that of the isotope. Other strategies to improve on dose delivery to the tumour include development of FAPI-derivatives with better pharmacokinetics and substituting b-emitters with alpha emitters (34).

A publication by Mori summarizes the most important challenges to be addressed with FAPi-based radioligand therapy as follows: (1) increasing tumor retention time to achieve sufficient cumulative activity in the tumor to facilitate biologically effective radiation doses, (2) selection of the most appropriate radionuclide to match the physiological and biological half-life of the tracer, (3) heterogeneity, and (4) minimizing radiation toxicity (35).

Prostate-specific membrane antigen (PSMA) is a type 2 transmembrane protein that is encoded by the FOLH1 (folate hydrolase 1) gene and is overexpressed not only in prostate cancer cells but also in the neo-vasculature of several solid tumours such as breast cancer. Preclinical data suggest that PSMA may be involved in cancer-related angiogenesis through degradation of the extracellular matrix and by participating in integrin signal transduction. PSMA expression has been closely linked both to enhanced angiogenesis and tumour aggressiveness. Interestingly, estrogen and progesterone-negative breast tumours tend to exhibit a higher PSMA expression than receptor-positive phenotypes. In the setting of triple negative breast cancer, this becomes especially useful as therapeutic receptor targets are absent and expression has been demonstrated to increase in hypoxic conditions (36, 37).

A 2023 in vitro study publication by Heesch and colleagues analysed PSMA and its isoform expression in Triple Negative Breast Cancer cells, breast cancer stem cells, and tumor-associated endothelial cells. They found PSMA expression in 91% of the investigated Triple Negative Breast Cancer cell lines and highlighted the possible role of therapeutic strategies in this clinical setting. The researchers demonstrated that TNBC cells strongly induce PSMA in tumor-associated vasculature, which makes it a promising candidate to target tumor angiogenesis. Interestingly, they also found that hypoxia strongly increased PSMA expression in breast cancer cells, which could be approached by administering PSMA therapy in a hypo-fractionated way, with the first fraction targeting PSMA-expressing endothelial cells and, in this way, decreasing the oxygen supply (38).

Morgenroth et al. used a mouse model of triple negative breast cancer to evaluate the potential of prostate-specific membrane antigen (PSMA) as a target for radio-ligand therapy. The researchers used ¹⁷⁷Lu-PSMA-617 and their results demonstrated that it strongly impaired the vitality and angiogenic potential of human umbilical vein endothelial cells. Target lesion localisation was confirmed and the authors concluded that ¹⁷⁷Lu-PSMA-617 could potentially be used in a theranostic approach (39).

In tumours other than prostate cancer, PSMA expression happens mainly in the endothelial cells of tumor-associated neo-vasculature, with no endothelial expression under physiological conditions (40).

In a 2013 study by Wernicke et al, the researchers set out to evaluate PSMA expression in tumour-associated vasculature in 106 patients with invasive breast cancer ranging from stage I–IV. Representative tumour tissue from each participant was stained for PSMA expression together with CD31 for confirmation of vasculature presence. The level of PSMA expression detected was scored from 0 to 2 in the following way: 0 = no detectable expression up to 2 = presence of PSMA expression in >50% of micro vessels. PSMA expression was present in 74% of primary breast cancer tumours and in all (n = 14) of those with brain metastases. Furthermore, its expression was absent in 98% of normal breast tissue and associated vasculature. Their results further demonstrated that patients with the highest PSMA expression (score of 2) demonstrated higher median tumour size, higher Ki-67, and poorer overall survival. Patients in whom Estrogen receptors were negative were more likely to have a PSMA score of 2 (41).

In a study consisting of 19 patients with breast cancer, Sathekge et al. demonstrated significant PSMA uptake in 84% of the 81 malignant and metastatic lesions with ⁶⁸Ga-PSMA-HBED-CC PET/CT. Metastatic lesions demonstrated higher SUV mean values compared to the primary tumours and no specific association with the presence or absence of progesterone receptors could be demonstrated. The authors highlighted the potential role in anti-angiogenic therapeutic strategies (42).

In one of the biggest studies to date, Tolkach and colleagues evaluated PSMA expression in the tumour endothelium (via immunohistochemistry) of 315 breast cancer patients with invasive non-special type carcinoma (NST) and lobular carcinoma. This group demonstrated PSMA expression in 60% of their study population, with higher expression noted in higher grade tumours and those with HER-2 expression and the highest expression in those with triple negative breast cancers (43).

A 2021 review article by Uijen et al, summarizes the use of PSMA-based radioligand therapy in solid tumours other than prostate cancer, which included breast cancer. The authors summarised the PSMA expression (based on SUVmax values) as low to moderate in breast cancer and did not highlight breast cancer as an ideal candidate for treatment with PSMA-based radioligand therapy (40).

In one of the few published cases of PSMA-based targeted radionuclide therapy in breast cancer, Tolkach et al. shares their experience in treating a young woman with an aggressive triple negative breast cancer that was unresponsive to several conventional lines of therapy. Two cycles of Lu-177-PSMA-RLT at a dose of 7.5 GBq was administered based on sufficient PSMA expression on ⁶⁸Ga-PSMA PET/CT. Post-injection SPECT imaging confirmed delivery of the radiopharmaceutical to the targeted lesions and no adverse effects were reported. Treatment was discontinued after the second cycle of therapy based on disease progression (43).

Considering the beta particle tissue range (+/−2 mm) of radioligand therapy with ¹⁷⁷Lu-PSMA, it could potentially still reach nearby tumour cells in well-perfused malignancies, even if PSMA is exclusively over-expressed in the endothelial cells of neo-vasculature, with resultant abscopal effect. Other concerns with ¹⁷⁷Lu-PSMA therapy that targets PSMA expression only on the neo-vasculature relate to the expected short tumour retention time and reduced efficacy.

Dalm et al. targeted SSTR2 receptors in a mouse model of breast cancer with both an SSTR agonist (¹⁷⁷Lu-DOTATATE) and an antagonist (¹⁷⁷Lu-DOTA-JR11). Results demonstrated a five times higher tumour accumulation of ¹⁷⁷Lu-DOTA-JR11 compared to the agonist target and this uptake translated to better tumour control and survival rates in mice (47).

Savelli and colleagues evaluated the use of ⁹⁰Y-DOTATOC in a patient with metastatic breast cancer with neuroendocrine differentiation and liver metastases. They administered 2.57 GBq of ⁹⁰Y-DOTATOC over a course of three cycles and used bremsstrahlung to confirm the intended target with SPECT/CT at 4 days post-administration. They reported a significant CT and biochemistry response in the absence of any adverse effects (48).

In a case study published by Liu et al., the authors administered 6 cycles of ¹⁷⁷Lu-DOTATOC in a patient with metastatic invasive ductal breast carcinoma and primary large-cell neuroendocrine breast cancer. Intense tracer accumulation was confirmed in the target lesions, which led to a partial treatment response in the absence of any significant side effects or toxicity (49).

The current level of evidence is unfortunately mostly limited to pre-clinical studies and isolated clinical case reports.

A first in human study evaluated the use of ¹⁷⁷Lu-RM2 in the setting of metastatic castration-resistant prostate cancer. Tumour absorbed doses were found to be therapeutically relevant, whereas rapid clearance from normal GRPR-rich organs, such as the pancreas, was confirmed. (The latter is considered to be the dose-limiting organ due to its high radioligand uptake). Despite the encouraging dosimetry results, certain derivatives demonstrate poor metabolic stability, which limits theranostic use (56). Lu-177-AMTG was identified as the therapeutic candidate with the most potential, but further studies are needed. ⁶⁸Ga/¹⁷⁷Lu-NeoBOMB1 provides another promising theranostic approach which has been evaluated in prostate cancer patients with good detection of primary tumours and liver and bone metastases (58).

Combination therapies with mTOR-inhibitors, PRRT, immunotherapy and hormone therapy in breast cancer may provide another promising route toward higher therapeutic responses.

Sankaranarayanan et al, investigated a theranostic approach in a triple negative breast cancer xenografted mouse model by radiolabelling the PARPi-derivative Olaparib to the Auger emitters ^123/125I. SPECT/CT images were acquired at 4 h or 24 h post injection of ¹²³I-PARPi-01, followed by a therapeutic approach with ¹²⁵I-PARPi-01 administered in 4 doses of 10 MBq/dose injected 10 days apart. A significant delay in tumour growth and doubling time was demonstrated on weekly CT scans, in the absence of significant radiotoxicity to the thyroid and liver. A significantly higher apoptosis ratio was demonstrated in the ¹²⁵I-PARPi-01-treated tumour tissues (62).

This presents a potential targeted radionuclide therapy approach for patients with triple negative breast cancer, although improvements to the pharmacokinetics are necessary prior to its potential clinical application.