Keywords

Human epidermal receptor type 2 (HER2; also known as ErbB2) is a member of the HER family that is encoded by the HER2 gene (also known as HER2/neu or ErbB2 gene). The HER2 pathway promotes cell growth and division when it functions normally; however, when it is overexpressed, cell growth accelerates beyond its normal limits. In cancer cells, HER2 protein can be expressed up to 100 times more than in normal cells (2,000,000 vs. 20,000 per cell). This overexpression leads to strong and constantly proliferative signaling and hence tumor formation. Overexpression of HER2 also causes deactivation of checkpoints, allowing for even greater increases in proliferation.

HER2-targeted treatments with trastuzumab and its derivates or analogues can improve the overall survival of patients with HER2-overexpression tumors. HER2 is overexpressed in about 30% of breast cancer and 7% to 34% of advanced gastric cancer patients, respectively. Researches have contributed great efforts on the development of noninvasive, whole-body HER2-targeted imaging in both HER2 overexpressed mice models and patients, by applying the advanced positron emission tomography (PET) molecular imaging technique.

38.1 HER2 Overexpression Tumor Model Construction

Currently, cell derivatives xenografts (CDX) models are the most commonly used models in preclinical studies. CDX models are established by injecting human tumor cells, typically from an immortalized cell line, into immunodeficient animals, which are likely to develop the same type of lesions. The high HER2-expressing (BT474 human breast cancer cells, SK-BR3 human breast cancer cells, SK-OV3 human ovarian cancer cells) and low HER2-expressing (MCF7 tumor and MDA-MB-231) cell lines are widely used for developing HER2-targeted drugs and molecular probes. Almost all cell lines have been subcultured many circles and may have lost most of patient’s original characteristics. More seriously, the microenvironments of these tumor models were completely different from the in situ scenario, due to the lack of blood vessel supply, tumor-associated stroma, and so on.

Patient-derived tumor xenograft (PDX) models have become much more popular in the last several years and have more advantages than CDX models. Nowadays, most PDX models are established by subcutaneously transplanting tumor tissues of patients into NOD/SCID (Non-obese Diabetic/Severe Combined Immunodeficiency) mice, and PDX models form various tumors have been established, such as non-small-cell lung carcinoma, breast cancer, and gastric cancer. PDX models can not only faithfully preserve the molecular phenotype and genotype changes of the tumor focus of the patient, but also reproduce the heterogeneity of the tumor of the source patient. Therefore, it is gradually applied to the research of tumor drug resistance mechanism and antitumor drug screening, and plays an irreplaceable role in the research of clinical tumor therapy and translational medicine. We previously reported the generation of gastric cancer-based PDX models using gastroscopic biopsies technology. We built HER2-positive (case 176) and HER2-negative (case 168) PDX tumor models based on two gastric cancer patients. The HER2 expression of both patient tumor tissues and PDX mice model tumor tissues were confirmed by H&E, IHC, FISH, DNA amplification, and/or autoradiography [1]. 64Cu-NOTA-Trastuzumab noninvasive PET imaging makes the monitoring of gastric cancer progresses in PDX models feasible. There were barely signals in tumor tissues that could be found using 18F-FDG PET, while brain showed very high uptake. This means 18F-FDG cannot indicate the tumor tissues of case 176 PDX mice models and may get false negative PET/CT results in this patient. Stronger PET signals in tumor tissues could be detected by 64Cu-NOTA-Trastuzumab in HER2 overexpression PDX models, whereas the weaker uptake could be detected in low HER2-expressing PDX models. According to ROI-based quantification analysis, the 64Cu-NOTA-Trastuzumab tumor accumulation was about 3.5 times higher than the contrast probe 64Cu-NOTA-Trastuzumab at 36 h p.i., further underlining the clinical relevance of these tumor models. In conclusion, we confirmed that 64Cu-NOTA-Trastuzumab could make noninvasive, specific detection of HER2 overexpression lesions in gastric cancer PDX models.

38.2 HER2 Targeting Immune-PET Imaging

High sensitivity, high spatial resolution, and proven quantification ability make PET the modality of choice for applying molecular imaging to the clinical setting. HER2 targeting immune-PET imaging uses radioactive molecular probes to specifically and noninvasively detect and evaluate HER2 expression information of systemic lesions of cancer patients, which enables patient screening, therapeutic monitoring, drug response evaluation, and early warning of recurrence and metastasis in the treatment process of HER2 high-expression tumors.

Many of antibody-based HER2-targeted probes have been used to image HER2-positive breast cancer over the past 20 years. The monoclonal antibody is labeled with long physical half-life isotopes such as 124I (T1/2 = 100.2 h) or 89Zr (T1/2 = 78 h), which matches the biological half-life of monoclonal antibody (T1/2 about 72 h). For example, Prof. Dijrkes et al. were the first to label clinical-grade trastuzumab with 89Zr and use it for immune-PET imaging in HER2-positive breast cancer patients. This radiotracer can detect HER2 status not just in primary tumor, but in patients with non-accessible metastases. The best imaging time of 89Zr-trastuzumab was 4–5 days after the injection.

Our preliminary researches demonstrated that clinical-grade novel 64Cu-NOTA-Trastuzumab can be formulated with good stability, immune activity, and specificity. The gastric cancer PDX models were successfully established, validated, and evaluated by 64Cu-NOTA-trastuzumab. It was highly consistent with gastric cancer patients regarding the expression of HER2, which made this model a superb tumor model for clinical translational study. He reported that co-injection of nonspecific hIgG1 antibody with 64Cu-NOTATrastuzumab appeared to enhance the tumor uptake in PDX models.

Based on these researches, his team successfully translated 64Cu-NOTA-trastuzumab into clinical gastric cancer patient PET/CT imaging. It exhibited comparable lesion detection ability compared with 18F-FDG, even in liver metastases. All those results guaranteed the further clinical application of 64Cu-NOTA-Trastuzumab in patients.

124I-trastuzumab has been developed by Prof. Zhu et al. as a PET imaging reagent for assessing HER2 expression status preclinically and clinically [2]. 124I-trastuzumab gives higher imaging contrast than 64Cu-NOTA-tratuzumab because of lower nonspecific uptake and better tumor-to-soft tissue ratios. In animal studies, PET imaging of 124I-tratuzumab shows significant higher tumor uptake than that of 124I-IgG1 in HER2-positive PDX mouse models at 24 h. The low tumor uptake of 124I-tratuzumab in HER2-negative PDX models further confirmed the specificity. In human clinical studies, the PET images showed significant difference in tumor uptake between HER2-positive and HER2-negative lesions at 24 h. Quite striking difference in tumor uptake was observed between 124I-trastuzumab and 18F-FDG in HER2-negative lesions, further confirming the specific binding of 124I-trastuzumab in HER2-positive lesions. No toxicities or adverse effects were observed in any of the patients. The PET imaging indicated that the use of 124I-trastuzumab to detect HER2-positive lesions in primary and metastatic gastric cancer patients to differentiate HER2-positive and HER2-negative lesions quantitatively was feasible.

38.3 HER2 Targeting Affibody PET Imaging

However, the clinical application of antibodies is limited because of their high molecular weight (MW = 150 kDa), resulting in low tumor penetration and slow clearance. To improve the imaging performance, alternative ligands have been developed over the past few years, such as F(ab′)2, F(ab′), single-chain Fv, and affibodies. Among them, the HER2 affibody is extensively studied preclinically and clinically. Affibody adapts the short half-life isotopes such as 68Ga (T1/2 = 68 min) or 18F (T1/2 = 110 min). Medium half-life nuclides such as 64Cu (T1/2 = 12.7 h) can also be used to label monoclonal antibodies and affibodies. The first clinical study using radiolabeled affibody was performed by Richard Baum and the team, 68Ga-labeled ABY-002 for molecular imaging in breast cancer patients. The initial attempt established the validity of the strategy, yet further research is need to solve the problem caused by the high background level in surrounding nonmalignant liver tissue that might prevent the detection of liver metastases. Then, Prof. Sörensen and his team produced a 68Ga-labeled ABY-025 for the diagnosis of HER2-positive breast cancer tumors. The results showed that HER2-positive primary and distant metastases were clearly visualized at 4 h after the injection.

Apart from trastuzumab modification, his team also pursued fancier HER2 targeting PET imaging. As we all known, HER2-binding affibody 68Ga-ABY-025 accurately quantified whole-body HER2-receptor status in patients with metastatic breast cancer at 2 to 4 h postinjection. Recent progress has been made on labeling novel HER2 affibody (NOTA-MAL-MZHER2) with 18F for micro-PET scans in nude mice bearing HER2-positive tumors (SKOV-3). Prof. Yang and his team translated this 68Ga-ZHER2 in clinical PET imaging [3]. As shown in Fig. 38.1, the high 68Ga-ZHER2 uptake is compatible with HER2 overexpression of the primary tumor. Compared with 18F-FDG, 68Ga-ZHER2 PET/CT showed better image contrast, especially in the bone lesions (with SUVmax, 66.0).

Fig. 38.1
5 Scanned images of the human upper body. A 1 and B 1 are P E T slash C T images of the axial views have arrows pointing to the left supraclavicular lymph node. A 2, A 3, and B 2 are P E T slash C T images of the frontal view depicting the lesions in the bone.

68Ga-ZHER2 affibody PET/CT was performed 1 h and 2 h after 218 MBq 68Ga-ZHER2 injection. The axial PET/CT image (A1) showed the left supraclavicular lymph node, and maximum intensive projection (MIP) PET images (A2-A3) showed multiple 68Ga-ZHER2-avid lesions in bones. B1 and B2 exhibit the 18F-FDG PET images in the same patient

Full size image

This novel PET/CT reveals the whole-body lesions at 1 h postinjection in patients with recurrent HER2-positive gastric cancer, which is much earlier than 64Cu-labeled (2 days) or 89Zr-labeled (5–8 days) intact antibody.

38.4 HER2 Targeting Therapy

Continuous low-dose irradiation from a tumor-targeted radiolabeled mAb produces tumoricidal effects. For therapy, α- and β-emitters are of practical relevance. There have been numerous investigations with a number of these radionuclides; however with intact antibodies, the most promising and practical radionuclides are the β-emitters such as 90Y, 177Lu, and 225Ac. Currently, the research on HER2-targeted therapy is still in preclinical stage, and no clinical research report has been published.

Trastuzumab can be labeled with 90Y and 177Lu using DOTA, DTPA, or 3p-C-NETA as the chelator. Clearance of 177Lu-DTPA-Trastuzumab in Swiss mice was predominantly through the hepatobiliary route with minimal bone uptake. Prof. Nasir Abbas et al. compared the bio-distribution, normal tissue toxicity, and therapeutic effect of the α-emitting 227Th-trastuzumab and the β-emitting 177Lu-trastuzumab in mice with HER2-expressing SKBR-3 breast cancer xenografts. The result showed that the relative biological effect (RBE) was higher for 227Th-trastuzumab than for 177Lu-trastuzumab, while the therapeutic index of 177Lu-trastuzumab was superior to that of 227Th-trastuzumab.

Recently, Prof. Tolmachev V et al. prepared 177Lu-CHX-A”-DTPA-F(ab′)2-trastuzumab and 177Lu-CHX-A”-DTPA-trastuzumab for nuclide therapy of HER2-positive SKOV3 models. The findings of this study indicate that both 177Lu-CHX-A”-DTPA-F(ab′)2-trastuzumab and 177Lu-CHX-A”-DTPA-trastuzumab are equally effective under in vitro conditions and could be employed with other apoptosis-inducing chemotherapeutic drugs for combinational therapy. The cellular toxicity exhibited by both 177Lu-CHX-A”-DTPA-trastuzumab and 177Lu-CHX-A”-DTPA-F(ab′)2-trastuzumab was similar in triggering membrane damage, inducing apoptosis, and causing cell death particularly at high radiation doses of 177Lu-CHX-A”-DTPA-trastuzumab and its 177Lu-CHX-A”-DTPA-F(ab′)2-trastuzumab. These in vitro results indicate that 177Lu-CHX-A”-DTPA-F(ab′)2-trastuzumab could be a potential theranostic agent; however, its in vivo efficacy needs to be studied extensively.

38.5 HER2 Using Affibody Receptor Radionuclide Therapy

Based on the mentioned molecular imaging technology, especially the HER2-affibody-based PET image, SUVmax reaches to incredible high 66.0. This initial study strongly shows that 68Ga-ZHER2 PET/CT can supply a whole-body vision of tumor load and HER2 expression including the heterogeneous as early as 1 h postinjection.

Inspired by recently most popular peptide receptor radionuclide therapy with lutetium-177 dotatate (177Lu-DOTATATE) for advanced gastroenteropancreatic neuroendocrine tumors (GEP-NETs) and 177Lu-PSMA-617 for metastatic castration resistant prostate cancer therapy (mCRPC), the establishment of affibody receptor radionuclide therapy (ARRT) has the potential to provide an alternative treatment option for HER2 positive resistant patients. As shown in Fig. 38.1, the high uptake of 68Ga-HER2 affibody in systemic bone metastases indicated a high affinity of this probe for the lesions, and it is expected that these patients would benefit from the 177Lu labeled HER2 affibody therapy.

38.6 Clinical Significance of HER2 ARRT

HER2 PET molecular probe can provide a whole-body view of the tumor load and HER2 expression status, and since lesions with very high uptake indicated that the drug had a very high affinity to the lesion, this method also reveals what the response to anti-HER2 drug will be, if de novo resistance occurs. Combined HER2 PET examinations and ctDNA sequencing is promising and may conquer the heterogeneity of HER2-positive cancer. These results of these indicators have a great impact on the overall prognosis of patients. The treatment plan of HER2-positive patients is different from that of HER2-negative patients, which can be intervened from the early stage of patients’ treatment and affect the whole course of patients. During the treatment, some of patients may develop trastuzumab resistance; monitoring HER2 expression by PET imaging could help us to adjust the treatment plan in time [4].

Radionuclide-targeted therapy provides a new therapeutic method. These biological missiles can be fired with pinpoint accuracy, more in line with our concept of individualized and precise treatment for patients. When patients have intolerance to chemotherapy, targeted therapy can be used as a supplementary means.