Nuclear Oncology, 1 Ed.



Sibaprasad Bhattacharyya • Aditya Bansal • Manish Dixit • Timothy R. DeGrado


The human epidermal receptor (HER) family consists of four structurally similar cellular receptors: Epidermal growth factor receptor (EGFR or HER1 or ErbB1); HER2 or ErbB2; HER3 or ErbB3; and HER4.1 Each receptor has three components: An extracellular ligand-binding domain, a transmembrane region, and an intracellular or cytoplasmic domain (Fig. 39.1).2 HER1, HER3, and HER4 are associated with one or more specific ligands (proteins). These protein ligands (e.g., growth factors) bind the receptor’s extracellular domain, leading to a conformational change. This conformational change in the HER receptor induces homo- and/or heterodimerization with other HER receptors, resulting in phosphorylation on specific tyrosine residues within the cytoplasmic tail (Fig. 39.1). HER2 does not bind to any known ligand but it can be a heterodimerization partner of other HERs (e.g., HER1/HER2 dimerization). This dimerization stimulates the tyrosine kinase (TK) catalytic activity, leading to multiple downstream cellular signals, which causes numerous cellular effects. HER3 homodimers have no TK domain and, therefore, cannot initiate signal transduction. Among the many signaling pathways that are activated by HERs (Fig. 39.2), the two main pathways are the mitogen-activated protein kinase (MAPK) and the phosphatidylinositol 3-kinase (PI3K)-AKT pathways.3 Activation of MAPK and PI3K-AKT pathways in various cancer cells is associated with resistance to apoptosis, increased cell growth, cell proliferation, metastasis, angiogenesis, and alterations of cellular energy metabolism.3 To exploit the positive effect of HER signaling in carcinogenesis, the HER pathway becomes hyperactivated by a range of mechanisms (Table 39.1): (1) The overproduction of ligands by tumor cells themselves or by surrounding stromal cells, leading to constitutive activation of HERs in tumor cells; (2) the constitutive activation of receptors in tumor cells due to mutation in the extracellular domain of HERs; and (3) overexpression of HERs in tumor cells caused by gene amplification or by increased protein expression.13

HER signaling is important clinically as a strong correlation is observed between HER expression and the overall survival of cancer patients.4 Breast cancer patients whose tumors overexpressed HER1–3 had reduced survival (p ≤ 0.001), whereas those whose tumors overexpressed HER4 had increased survival (p = 0.013) (Fig. 39.3).4 The prognostic value of HER expression has also been shown in a variety of other cancers including non–small-cell lung cancer (NSCLC), ovarian cancer, bladder cancer, colorectal cancer (CRC), endometrial cancer, esophageal cancer, gastric cancer, glioma, head and neck squamous cell cancer (HNSCC), renal cell carcinoma, pancreatic cancer, and prostate cancer.14

HER1 and HER2 are probably the most widely studied members among the HER family of receptors. Abnormal receptor activation or dysregulation through ligand binding to these receptors may lead to the initiation and/or development of human carcinomas. Extensive research in tumor biology reveals that HER1 and HER2 are differentially deregulated, overexpressed, mutated, or amplified in many types of cancers (Table 39.2), making them attractive targets for cancer therapy.5

FIGURE 39.1. A. Members of HER family. B. Conceptualization of HERs conformational change on ligand binding. Ligand binding to HERs seems to induce a conformational change in the folded structure of the molecule that exposes the dimerization domain; this step is required for dimer formation and functional activation of HERs. (Reprinted with permission of the Nature Publishing Group. Baselga J and Swain SM. Novel anticancer targets: Revisiting ERBB2 and discovering ERBB3. Nat Rev Cancer. 2009;9(7):463–475.)

FIGURE 39.2. HER signaling showing activation of complex signaling pathways by activated homo- and heterodimers of HERs permitting regulation of a wide array of biologic processes like apoptosis, migration/invasion, proliferation and differentiation. (Reprinted with permission of the Nature Publishing Group. Yarden Y and Sliwkowski MX. Untangling the ErbB signalling network. Nat Rev Mol Cell Biol. 2001;2(2):127–37.)

Several approaches have been used to block these receptors. Two major strategies to inhibit HER pathway have been developed: (1) Extracellular blocking by antibodies (e.g., trastuzumab, cetuximab, and panitumumab) or (2) intracellular blocking by small-molecule TK inhibitors (e.g., gifitinib, lapatinib, and erlotinib). The mechanism of action of HER1 inhibitors is shown in Figure 39.4.6 Table 39.3summarizes5,7 the compounds which are currently used in the clinic or are in clinical trials for HER1 and HER2.

TABLE 39.1


As an extracellular blocking agent of HER2, the antibody trastuzumab (Herceptin) was the first example that successfully demonstrated the principle of molecular-targeted therapy of human cancer. Trastuzumab acts against HER2 overexpressing tumors, in part, by inducing receptor endocytosis.6 HER2 is overexpressed in approximately 25% to 30% of human breast cancers.8 Trastuzumab improves survival when used in combination with chemotherapy as compared to chemotherapy alone in patients with HER2-overexpressing breast cancers.9

TABLE 39.2


FIGURE 39.3. Survival curves (Kaplan–Meier) demonstrating cumulative survival differences between patients whose tumors were scored as positive for HER1 (A), HER2 (B), HER3 (C), or HER4 (D). The p values represent log–rank differences in cumulative survival. (Reprinted with permission of John Wiley & Sons, Ltd. Witton CJ, Reeves JR, Going JJ, et al. Expression of the HER1-4 family of receptor tyrosine kinases in breast cancer. J Pathol. 2003; 200(3):290–297.)

Pertuzumab (Perjeta), humanized anti-HER2 mAb, binds to a different HER2 epitope than trastuzumab. It has been approved by the U.S. Food and Drug Administration [FDA] (2012) for breast cancer treatment. In contrast to trastuzumab, pertuzumab inhibits the heterodimerization of HER2 with other HER family members. This unique mechanism of action resulted in efficacy against tumor with very low HER2 expression that cannot be the target for trastuzumab treatment.10,11

Several large molecules that can bind the extracellular ligand-binding domain of HER1 have been developed, including cetuximab, which has been approved by FDA for the treatment of HER1-expressing NSCLC, HNSCC, and Colorectal carcinoma.12,13 Preclinical studies show that cetuximab binds to HER1 with an affinity (Kd = 0.1 nmol/L) comparable with that of the neutral HER1 ligand epidermal growth factor (EGF) and transforming growth factor α (TGF-α).5 The high-affinity binding of cetuximab to HER1 prevents ligand binding and subsequent receptor activation.5,12 It has also been approved for the use in combination with radiation therapy (2006) and with platinum-based drug therapy (2011).13 Panitumumab (Vectibix), a fully humanized anti-HER1 mAb has recently been approved by FDA for the treatment of HER1-expressing metastatic colorectal cancer with disease progression.14 Panitumumab is the first mAb to demonstrate the use of K-RAS as a predictive biomarker.

FIGURE 39.4. Mechanisms of action of epidermal growth factor receptor (EGFR) inhibitors. Antibodies prevent ligand access to the EGFR and induce receptor endocytosis. This may result in targeting the receptor to a lysosomal compartment. Small-molecule receptor tyrosine kinase (RTK) inhibitors diffuse into the cell and compete with ATP for binding to the EGFR kinase domain. In turn, both antibodies and small molecules disable EGFR function and block signal transduction downstream the receptor. (Reprinted with permission of the American Society of Clinical Oncology. Arteaga CL. The epidermal growth factor receptor: From mutant oncogene in nonhuman cancers to therapeutic target in human neoplasia. J Clin Oncol. 2001;119:32–40.)

TABLE 39.3


HER-targeted small molecules, usually known as TK inhibitors, act at an intracellular level (Table 39.3),5 competing with ATP for binding to the ATP-binding pocket of the TK domain of the receptor. By inhibiting TK activity these inhibitors disrupt the hyperactive signal transduction pathways in cancer cells. In the early 1980s, some natural products (e.g., quercetin, genistein, herbimycin A) were recognized to have potential as inhibitors of TKs.15Although these natural products tended to be toxic and lacked the necessary selectivity to be clinically useful, they served as the building blocks for the design of more potent, and selective TK inhibitors. In the past few years, over 30 inhibitors have been investigated and are at various stages of development.15,16 Several of these molecules are being investigated in clinical trials.16 To date, three HER1/HER2 low–molecular-weight inhibitors have been approved for clinical use: Lapatinib, gefitinib, and erlotinib. Numerous other TK inhibitors (both reversible and irreversible) are in different stages of development. Gefitinib and erlotinib are more selective for HER1, whereas lapatinib has dual TK activity, targeting both HER1 and HER2.16 Some studies showed that erlotinib also inhibits HER2 phosphorylation and downstream signal transduction in HER2/HER3 overexpressing cells.17

The clinical applications of these drugs are numerous with variable and sometimes contradictory results depending upon tumor types and clinical settings. In some clinical studies, patients who initially responded to the treatment have occasionally developed therapy resistance, for example, because of acquired secondary mutation in the HER1.18 As a result, additional clinical and experimental data must be acquired so as to better predict the response to such therapies.

Though the large amount of clinical data accumulated over the last few years in HER1-positive NSCLC and CRC dramatically improved the understanding of the role of HER1 in solid tumor, the treatment prediction is still an open question in clinical practices.18 Therefore, even though the HER family of receptors, and specifically HER1 and HER2 are widely recognized as promising targets for the development of new drugs in oncology, no conclusive data are available on the role of HERs on cancer progression or the optimal strategy to use the available inhibitors. The questions that arise are as follows: (1) How do we identify patients with the highest chance of benefit from the treatment with these targeted drugs? and (2) which of these targeted therapies is most likely to succeed in a given patient?

It can be argued that the most efficient approach would avoid traditional large-scale randomized clinical trials, but rather adopt the concept of “personalized medicine” by choosing targeted drugs based on the individual’s receptor expression levels. To do so, a suitable method is needed to assess HER status of each patient before the treatment and monitor effects during the treatment process. At present, invasive methods such as tissue biopsy are used to identify HER expression in primary tumor site or metastatic sites in cancer patients. Although useful for tumor characterizations, repeated biopsy for therapy monitoring and disease management is impractical and unreliable.19Moreover, the assessment of HERs in ex vivo tumor specimens is still controversial for both methodologic and biologic points of view.18 Other techniques for HER assessments such as immunohistochemistry, ELISA or western blotting for protein level quantification, FISH for gene amplification still need to be validated.20 Essentially, there are no reliable methods to monitor HER status available for everyday practice. Taking into account these considerations, an in vivo identification and quantitative evaluation of HER receptor expression may be a novel and powerful tool to globally assess the receptor. Functional imaging of tumors via nuclear medicine modalities such as single-photon computed tomography (SPECT) or positron emission tomography (PET) represents the most sensitive methods available.21 These nuclear modalities allow noninvasive in vivo quantitative visualization of receptor levels and enzyme activity/expression with high sensitivity, in subnanomolar concentration range, as opposed to millimolar concentration for other imaging modalities. Thus, these modalities may provide a complementary, noninvasive option to obtain real-time information facilitating the selection of patients for HER-targeted therapy assessment of response to therapeutic interventions.22

In this chapter, the preclinical and clinical applications of radiolabeled imaging agents that are structurally similar to FDA-approved therapeutic HER-targeting drugs are reviewed and a brief discussion is included on HER-targeted radiotherapy. Because HER1 and HER2 are the most important and widely studied among HER family, our discussion is largely limited to these two receptors.


Nuclear imaging in clinical practice and research is based on two well-established imaging modalities, SPECT and PET. These techniques offer high sensitivity to detect and quantify imaging agents in deep tissue. In nuclear imaging, radioactive probes (radiopharmaceuticals) are introduced in living subjects in picomolar to nanomolar range and are not intended to have any pharmacologic effect. In SPECT, single-photon emissions from administered radiopharmaceuticals are collected in a collimated single-photon imaging device that reconstructs the distribution of radioactivity in the area imaged. In PET, radiopharmaceuticals labeled with positron-emitting radionuclides are used. As the radionuclide undergoes decay, it emits positrons that travel for a short distance and interact with an electron giving rise to a pair of high-energy (511 keV) photons that move in nearly the opposite direction (180 degrees) to each other. The coincident photons are detected in opposite regions of a cylindrical array of detectors within the PET scanner, allowing the determination of numerous lines of coincidence throughout the scanner volume (Fig. 39.5). These coincidence events are time-stamped, integrated over relevant imaging frame durations, and utilized for reconstruction of PET images of radioactivity concentration within the tissue. In general, PET offers higher sensitivity and imaging accuracy than SPECT because of its unique use of coincident photon detection (Figure 39.5). Indeed, most of the HER-targeting nuclear imaging agents are based on PET (Table 39.4) radioisotopes.21,22

FIGURE 39.5. Schematic representation of nuclear imaging technique PET. Radiotracer accumulates in target tissue and emits positrons. Each positron interacts with an electron (annihilation) to produce two photons which travel in opposite direction and are detected by a circular array of detectors. Collected photon signals are transferred to image processing unit for image reconstruction. (Reprinted with the permission of the Royal Society of Chemistry. Bhattacharyya S, Dixit M. Metallic radionuclides in the development of diagnostic and therapeutic radiopharmaceuticals. Dalton Trans. 2011;40:6112–6128.)

The two major classes of nuclear imaging probes that have been developed for visualizing HERs are as follows: Radiolabeled small molecules such as TK inhibitors and radiolabeled monoclonal antibodies or antibody derivatives. It should also be mentioned that an alternative indirect approach to monitoring effect of HER therapy is PET imaging with fluorine-18–labeled metabolic substrates such as 2-fluoro-2-deoxy glucose ([18F]FDG) and 3′-deoxy-3′-fluorothymidine ([18F]FLT). [18F]FDG is routinely used to detect glucose metabolism that is often increased in rapidly growing tumor tissues. Reduction of [18F]FDG uptake is the consequence of effective treatment with a kinase inhibitor as a targeted therapeutic agent.22 A similar approach can also be used to measure tumor cell proliferation. [18F]FLT is a radiopharmaceutical used to evaluate cellular proliferation rate22,23 and it has been used to monitor clinical efficacy of the small-molecule inhibitor of HER1 TK, gefitinib.24 Although both [18F]FDG and [18F]FLT (Fig. 39.6) are useful for imaging tumor metabolism and cellular proliferation, imaging HERs with radiolabeled TK inhibitors and monoclonal antibodies (mAbs) may offer a direct and more sensitive approach to assessing HER status and monitoring clinical potential of targeted therapeutics and treatments.25

TABLE 39.4


FIGURE 39.6. Chemical structure of [18F]FDG and [18F]FLT. [18F]FDG is FDA approved the most versatile PET tracer used in clinic to detect glucose metabolism. [18F]FLT is an investigational imaging drug which interrogates the DNA synthesis of growing cells (cell proliferation).

The ideal nuclear imaging probes for HERs should exhibit several specific characteristics related to the target molecules, radioisotopes, and labeling (Table 39.5). For small molecular TK analogs, it is important to minimize differences in their in vivo behavior with that of the parent drug. This can only be accomplished with radiolabeled probes that are structurally very similar to the parent drug molecule. Small-molecule TK inhibitors have typically fast blood clearance and pharmacodynamics properties. Although short-lived, the positron emitters 18F and 11C are ideal for labeling small molecules because they can be incorporated with very little or no change in chemical structure of the parent molecules (Fig. 39.7). On the other hand, large molecules (mAbs, affibodies, large peptides, etc.) need a longer time to reach the target and are usually labeled with longer-lived radioisotopes (Table 39.4).21,26 A bifunctional chelate (BFC) is often used to attach long-lived radiometals (γ-emitters or positron emitters) to the large molecules (Fig. 39.8).26

TABLE 39.5


FIGURE 39.7. Anilinoquinazoline-based FDA-approved small-molecule tyrosine kinase inhibitors (TKIs) and their radiolabeled analogs. Radiolabeled compounds are almost structurally identical to the corresponding TKI.

Preclinical evaluation of the imaging probe is carried out in two major steps. First, in vitro studies should be performed with cell lines which are highly, moderately, and also negatively expressing the targets to define targeting specificity. Next, studies in in vivo tumor-bearing animal models are undertaken to establish the biodistribution, metabolism, and tumor-targeting behavior of the probe. Imaging feasibility can be assessed using small animal SPECT or PET. The novel tracers may also be compared with using standard PET probes such as [18F]FDG and/or [18F]FLT. Finally, to understand the information content and correct interpretation of the images, molecular analyses are undertaken after animal sacrifice to evaluate target expression or activation.

The next phase of the development process is to develop the manufacturing processes needed to produce clinical grade radiopharmaceuticals for human injection. The regulatory requirements for approval of imaging probes for human administration are different in every country. The manufacturing process is defined in detail within a compilation of standard operating procedures (SOPs) that include receiving and qualification of raw materials, materials storage, manufacturing equipment and process validation, in process controls, and quality control of product. To begin a clinical trial in the United States, it is necessary to file an Investigational New Drug (IND) application at the FDA. In general, the IND is composed of pharmacology and toxicology dossier, the chemistry manufacturing and controls (CMC) section, and a plan for conducting clinical studies that will define the safety (Phase I) and efficacy (Phase II–III) of the radiopharmaceuticals. This process entails considerable time and expense. As a consequence, despite the large amount of preclinical studies and despite promising preclinical results, many promising novel imaging probes are not translated to the clinical setting. The Radioactive Drug Research Committee (RDRC) is a special committee27 in the United States which can allow radioactive drugs for clinical trials under its direct supervision without formally going through FDA approval processes.28 For RDRC supervised study, the imaging drug must be generally recognized as safe and effective (GRASE) and the research conducted must be basic in nature.28

FIGURE 39.8. Radiopharmaceutical design with large biomolecules (BMs) and some common bifunctional chelators (BFCs) for holding radiometals. A linker (e.g., PEG) in between BFC and BM is often used to keep the radiometal chelate away from the BM and to facilitate/control radiopharmaceutical movement (pharmacokinetics) in the body, including distribution and elimination.


HER1 Imaging with Radiolabeled Small Molecules

In 2005, gefitinib, an FDA-approved reversible TK inhibitor (IC50 = 0.2 to 0.6 μM) was first radiolabeled with 18F by Seimbille et al.29 In 2006, two reports were published simultaneously on the synthesis of [11C]gefitinib.30,31 A modified synthesis with higher radiochemical yield of [11C]gefitinib was published in 2010 by Zhang et al.32 Both [18F]gefitinib and [11C]gefitinib have been evaluated thoroughly in in vivo preclinical studies using mouse and monkey models. Several human tumor types have been studied (U87, glioblastoma cell line with low HER1 expression; U87-EGFR overexpressing; H3255 and H1975 NSCLC cell line) to evaluate this tracer in mice-bearing human xenografts.33 Unexpectedly, none of the selected xenografts showed increased tracer uptake by PET imaging (Fig. 39.9) and biodistribution analysis. Further studies showed that [18F]gefitinib appeared metabolically stable in mice during PET scans, ruling out the option that metabolism was the reason for lack of tumor uptake.33 Therefore, the lack of HER1-specific tumor uptake of the radiolabeled gefitinibs render them unsuitable as PET probes for determination of HER1 status of the patients. However, radiolabeled gefitinib analogs may be useful to understand the pharmacokinetics of gefitinib by PET imaging.34

Erlotinib, a HER1 TK reversible inhibitor (IC50 = 20 nM) was approved in 2004 as a second-line treatment for patients with advanced NSCLC. Like gefitinib, it is also based on 4-anilinoquinazoline scaffold (Fig. 39.7) and it competes with ATP for its binding site in HER1. However, this drug is only effective for 10% to 20% patient population; therefore, imaging patients with a radiolabeled erlotinib analog probe to select responders would be of clinical important. Erlotinib was radiolabeled with 11C by Memon et al.35 using [11C]methyl iodide as the prosthetic group. [11C]erlotinib was evaluated in three HER1-overexpressing lung cancer cell lines (HCC827, A549, and NCI358).35Only HCC827 was sensitive to erlotinib in vitro as these cells harbored the exon 19 activating mutation in HER1.35 Encouragingly, micro-PET imaging of mice xenograft models showed that [11C]erlotinib uptake was highest in HCC827 tumors whereas erlotinib insensitive NCI358 tumor did not show [11C]erlotinib uptake. These results supported continued investigation of [11C]erlotinib as a PET probe to identify erlotinib sensitive tumors.

Recently, Weber et al.36 reported the first human TK inhibitor PET study in a 32-year-old NSCLC patient who presented with a primary tumor and brain metastasis. The patient was responsive to erlotinib treatment. The metastatic lesion was identified by MRI and a [11C]erlotinib PET scan (Fig. 39.10). This study revealed that [11C]erlotinib PET identified not only in erlotinib sensitive primary tumor but also distant metastases. In another recent study, 13 NSCLC patients were grouped into responders and nonresponders to erlotinib treatment.37 At the beginning of the study a PET/CT scan with [18F]FDG and [11C]erlotinib was performed followed by the treatment with erlotinib for 12 weeks. After the treatment, [18F]FDG PET was performed for response evaluation. In 4 out of 13 patients, PET/CT revealed uptake of [11C]erlotinib in one or more of their lung tumors or lymph node metastases (Fig. 39.11C). More interestingly, [11C]erlotinib PET was capable to visualize some tumor-positive lymph nodes which could not be visualized using [18F]FDG PET. Among the other nine patients with negative [11C]erlotinib accumulation, three died before follow-up and six did not respond adequately to erlotinib treatment. The results from this small study indicated that with [11C]erlotinib PET, it may be possible to identify responders and nonresponders to erlotinib treatment. However, the predictive value of [11C]erlotinib to identify responders to erlotinib treatment for individual patients has yet to be confirmed in larger studies. Moreover, in this study no analysis was done to correlate [11C]erlotinib uptake in tumors with their mutational status. In another recent clinical report, it has been shown that [11C]erlotinib showed significantly higher uptake in tumors with mutated HER1 than in tumors with wild-type HER1.38

FIGURE 39.9. μPET/CT images of tumor-bearing mice at 1-hour post-intravenous injection of [18F]gefitinib. Arrows indicate tumor location showing no significant uptake in tumor tissue. SUV, standardized uptake value. (Reprinted with permission of the European Association of Nuclear Medicine. Su H, Seimbille Y, Ferl GZ, et al. Evaluation of [18F]gefitinib as a molecular imaging probe for the assessment of the epidermal growth factor receptor status in malignant tumors. Eur J Nucl Med Mol Imaging. 2008;35:1089–1099.)

FIGURE 39.10. MRI-PET images of brain metastatic lesions in the cerebellum, showing [11C]erlotinib hot spot in metastatic lesions. (Adapted with permission from Elsevier. Slobbe P, Poot AJ, Windhorst AD, et al. PET-imaging with small-molecule tyrosine kinase inhibitors: TKI-PET. Drug Discovery Today. 2012;17(21–22):1175–1187;

Lapatinib is another FDA-approved orally active drug for the treatment of breast cancer and other HER1- and/or HER2-expressing tumors. It inhibits the TK activity associated with two oncogenes, HER1 and HER2/neu. It has been radiolabeled recently by Basuli et al.39 with 18F but no preclinical data are available so far.

Many other HER1 TK inhibitors (both reversible and irreversible) have been radiolabeled in the last few years and evaluated preclinically to determine their suitability to image HER1 (Table 39.6). Most of these tracers showed very promising in vitro results for imaging; however, subsequent in vivo studies have been generally unfavorable.25 To date almost all the radiotracers that target HER1 TK have shown very low to moderate specific uptake into tumors with insufficient signal to noise for clinical imaging. In many cases, significant nonspecific binding has been observed. Several factors have been suggested for the lack of success. First, tumor characteristics such as vascularity or necrosis may interfere with the tracer’s uptake. Second, rapid hepatobiliary excretion may reduce their bioavailability. Biodistribution studies have shown high accumulation in nontarget organs such as liver, kidney, or intestine. Modification of chemical structure is needed to develop radiotracers that can target HER1 TK effectively to generate high tumor-to-background signals. Some authors have suggested modified methods of administration such as prolonged intravenous infusion to enhance the tumor-specific uptake.18,61

HER1 Imaging with Radiolabeled Intact Antibodies (mAbs)

Imaging HER1 with radiolabeled anti-HER1 mAbs, affibodies, and peptides that bind extracellular domain of HER1 have been generally more successful than small-molecule probes and the subject of intense investigation. It is straightforward to attach a variety of radionuclides, including radiometals and radiohalogens to these large molecules without altering their overall biologic acceptance at HER1.

FIGURE 39.11. Transaxial PET/CT images of erlotinib responsive patient showing [11C]erlotinib-positive lymph nodes (red ) that could not visualized using [18F]FDG PET. Small arrows indicate the lymph nodes. Adapted with permission of the Nature Publishing Group. Memon AA, Weber B, Winterdahl M, et al. British J. Cancer 2011;105(12):1850–1855.

TABLE 39.6


Radiolabeled Cetuximab

In 2004, the FDA approved the chimeric mAb cetuximab under its accelerated approval program for the treatment of patients with HER1-positive metastatic colorectal cancers. It is used either in combination with irinotecan, or alone if patients cannot tolerate irinotecan. It has been shown that cetuximab can shrink tumors in some patients and delay tumor growth in others, when used as a combination treatment.62 It binds the extracellular domain of HER1 and thus provides an opportunity as an imaging probe for the assessment of HER1 expression in tumors. Cetuximab was radiolabeled with indium-111 (111In) using DTPA as BFC and was shown to localize in tumors that overexpress HER1.63 However, a high uptake of radioactivity by liver limited its usefulness. To overcome this problem, polyethylene glycol (PEG) was used as a linker between DTPA and cetuximab. PEG-modified 111In-DTPA-PEG-cetuximab in nude mice showed significant reduction of accumulation in liver.64 To take advantage of superior properties of PET, cetuximab was also labeled with PET isotopes (64Cu, 89Zr). In 2005, Perk et al.53 synthesized 89Zr-DFO-cetuximab and used it as a surrogate marker to evaluate therapy with 90Y and/or 177Lu-labeled DOTA-cetuximab. They showed that the biodistribution of 89Zr-DFO-cetuximub, 88Y-DOTA-cetuximab (88Y as a substitute for 90Y), and 177Lu-DOTA-cetuximab were comparable for all organs, with the exception of differences in bone accumulation. The authors found that 89Zr-labeled mAb was more effective as a PET surrogate marker over 86Y-labeled mAb (t1/214.4 hours) because it has the half-life (78 hours) better matched the half-life of 90Y (64 hours) and the biologic half-life of mAbs (72 to 96 hours). Moreover, 86Y emitted prompt γ-photons which together with 511-keV annihilation photons introduced quantitation artifacts.

Copper-64 (64Cu)-labeled cetuximab (64Cu-DOTA-cetuximab) was first synthesized by Cai et al.54 in 2007 as a potential PET probe for HER1 imaging. The authors evaluated this tracer in seven tumor xenograft models (Fig. 39.12). The study demonstrated high tumor uptake of 64Cu-DOTA-cetuximab in U87GM and PC3 tumor models. Western blots of excised tumors showed very close correlation between tumor HER1 expression and tumor uptake, demonstrating the potential utility of 64Cu-DOTA-cetuximab as biomarker for HER1 expression. However, evaluation of 64Cu-DOTA-cetuximab in mice-bearing A431 tumors showed moderate accumulation in tumor but significant uptake in liver because of the dissociation of 64Cu from DOTA (transchelation). Because this probe is excreted through the hepatobiliary route system, it has limited application in imaging of liver tumors. Because the inability to use such a tracer for evaluation of liver metastases is a serious drawback, more than 70% of patients with CRC develop liver metastases. Recently, several new bifunctional chelators have been synthesized to make more stable Cu-chelates21,26 and biologic evaluation investigations are ongoing.

FIGURE 39.12. Serial micro-PET images of seven xenograft tumor models after intravenous injection of 64Cu-DOTA-cetuximab (n  =  3 per tumor model). Decay-corrected whole-body coronal images that contain the tumor were shown and the tumors are indicated by white arrows. (Reprinted with permission of the European Association of Nuclear Medicine. Cai W, Chen K, He L, et al., Quantitative PET of EGFR expression in xenograft-bearing mice using 64Cu-labeled cetuximab, a chimeric anti-EGFR monoclonal antibody. Eur J Nucl Med Mol Imaging. 2007;34: 850–858.)

Radiolabeled Panitumumab

Another anti-HER1 mAb, panitumumab (Vectibix), is a fully human mAb approved by the FDA for the treatment of HER1-expressing colorectal cancers.58,65 Currently, it is being evaluated in patients with other types of HER1-expressing cancers including breast, lung, head and neck, renal, and ovarian tumors.66 Panitumumab binds to domain III of HER1 and is rapidly internalized, leading to downregulation of cell surface HER1. It also arrests the cell cycle and inhibits tumor growth by suppressing the production of proangiogenic factors (VEGF, IL-8) by tumor cells.67 Moreover, because it is a fully human antibody, panitumumab has minimal immunogenicity when administered intravenously. Recently, panitumumab was radiolabeled and evaluated preclinically in different tumor models to evaluate its potential as a HER1-targeted imaging probe.

In 2009, Ray et al.56 radiolabeled panitumumab with 111In using CHX-A′-DTPA as a BFC. Tumor targeting by 111In-CHX-A′-DTPA-panitumumab was demonstrated in athymic mice-bearing A431 (epidermoid), HT-29 (colon), LS-174T (colon), SHAW (pancreatic), or SKOV-3 (ovarian) xenografts with little uptake in normal tissues. Although panitumumab has a longer elimination phase in patients than does cetuximab (7.5 days versus 5 days), the blood pharmacokinetics of 111In-CHX-A′-DTPA-panitumumab following i.v. injection in tumor-bearing mice was comparable to that reported for 111In-labeled cetuximab.

In 2010, using the same bifunctional ligand panitumumab was labeled with 86Y to develop a HER1-targeted immuno-PET imaging agent.58 The authors noted that the attractive feature of the 86Y PET radionuclide is that it can be used as a surrogate imaging marker for 90Y-based immunotherapy. 86Y-CHX-A′-DTPA-panitumumab was evaluated in mice-bearing LS-174T, PC-3, or A431 tumors. Tumor uptake was correlated with ex vivo HER1 expression levels. However, the highest tumor accumulation was found in LS-174T tumors which showed the lowest ex vivo HER1 expression level among the three tumor models. Discrepancies in tumor uptake and ex vivo HER1 expression levels (determined by immunohistochemistry) were also found in 64Cu-labeled panitumumab68 and 89Zr-labeled cetuximab.69

Most recently (2012), Nayak et al.59 and Bhattacharyya et al.57,65 labeled panitumumab with 89Zr using desferrioxamine (DFO) as a bifunctional ligand (Fig. 39.8). Nayak et al. evaluated this probe to assess the status of HER1 in distant metastatic models of intraperitoneal and pulmonary colorectal cancers. MRI studies were performed for metastatic models to characterize the targeting potential of 89Zr-panitumumab at different lesion sites. Bhattacharyya et al.57 developed 89Zr-DFO-panitumumab with the intention to use it in a clinical trial to monitor the therapeutic efficacy of panitumumab and/or cetuximab in patients with colorectal cancers. Zirconium-89 was chosen as PET radiolabel because its half-life (78 hours) is long enough to get optimal biodistribution of the intact mAb panitumumab and cetuximab. In this study, female athymic nude mice-bearing human breast cancer tumors (n = 5 per tumor group) with variable HER1 expression very low (BT-474), moderate (MDA-MB-231) and very high (MDA-MB-468) were utilized. Micro-PET/CT imaging (Fig. 39.13) was performed at 1 week or more following i.v. injection of 89Zr-DFO-panitumumab. Results showed very strong correlation (R2 = 0.857, p< 0.001) between tumor uptake and ex vivo HER1 uptake (determined by immunohistochemistry on excised tumors after imaging) (Fig. 39.14). The biodistribution in non–tumor-bearing mice revealed very poor blood clearance of 89Zr-panitumumab and accumulation was seen prominently in axillary lymph nodes at all time points. The comparison of organ biodistribution in non–tumor-bearing mice between 111In and 89Zr-labeled panitumumab resulted in an excellent correlation (R2 > 0.94, p < 0.0001) (Fig. 39.15). The authors noted, as there was no significant cross-reactivity between the human antibody panitumumab and mouse HER1 in normal organs, that the mouse data mainly reflected the slower linear lymphatic clearance. However, in human subjects, the dominant clearance route for tracer doses (<1 mg), is expected to be through native HER1 binding, and lymph-node uptake is not expected to be significant. Human dosimetry estimates based on biodistribution data in non–tumor-bearing mice showed that 1 to 1.5 mCi of 89Zr-panitumumab can be safely injected into humans. Organ dosimetry comparison between 111In and 89Zr-labeled panitumumab also showed excellent correlation (Fig. 39.16). This study concluded that because of poor clearance, a very low dose of 89Zr-DFO-panitumumab should show favorable human dosimetry. Because of high tumor uptake, the use of 89Zr-panitumumab is expected to be clinically feasible. Recently (2012), an exploratory IND application was approved by the FDA to use this tracer as an imaging agent to monitor the treatment of colorectal cancer patients with cetuximab and panitumumab.

FIGURE 39.13. Tumor uptake of 89Zr-panitumumab in various subcutaneous athymic nude female xenograft models with very low (A), medium (B) and very high (C) HER1-expression level; 10.18 ± 1.24 MBq of 89Zr-panitumumab were administered intravenously via tail vein, and a 5-minute CT scan followed by a 30-minute static PET scan were performed at 96-hour post injection. Color scale: SUV (g/mL). (Bhattacharyya et al., Zirconium-89 labeled panitumumab: A potential immuno-PET probe for HER1-expressing carcinomas. Nucl Med Biol. 2013;40:451–457.)

FIGURE 39.14. Comparison of the average tumor uptake of 89Zr-panitumumab for (BT-474; MDA-MB-231; and MDA-MB-468) tumor-bearing athymic nude female mice (n = 5 per tumor model) at the 144-hour time point versus the EGFR expression level (HER1/Actin ratio) determined by Western blot analysis for the corresponding tumors resulting in a good correlation of R 2 = 0.857, p < 0.001. (Bhattacharyya et al., unpublished work.)

Radiolabeled Affibodies

Because of their slow clearance from the bloodstream and metabolic tissues intact antibody (mAb)-based radiotracers cannot produce effective imaging contrast early after injection. To overcome this limitation, small molecular weight antibody fragments that is affibodies (∼7 to 15 kDa) and peptides that bind HER1 have been developed. The smaller antibody derivatives are cleared from the circulation faster than the mAbs allowing more rapid imaging sequences. The major drawback of these molecules is their reduced binding affinity in tumors as compared to mAbs. Investigation on cetuximab affibodies (Fab′ and F(ab′)2) showed they have weaker (five times less) affinity to tumor than the mAb.70

FIGURE 39.15. Biodistribution %ID/g comparison in non–tumor-bearing athymic nude mice (n = 2 female and n = 2 male per time point) for radionuclide(s) 111In- and 89Zr-labeled panitumumab for the various time points. Mice were i.v. injected 1.85 and 5.3 MBq via tail vein for 89Zr-panitumumab and 111In-panitumumab, respectively. The %ID/g uptake of panitumumab resulted in a good correlation (R 2 > 0.94, p < 0.0001) between 111In- and 89Zr-labeled panitumumab. (Bhattacharyya S, et al., Zirconium-89 labeled panitumumab: A potential immuno-PET probe for HER1-expressing carcinomas. Nucl Med Biol. 2013;40:451–457.)

FIGURE 39.16. Organ dosimetry (mGy/MBq) comparison in non–tumor-bearing athymic nude mice (n = 2 female and n = 2 male per time point) for radionuclide(s) 111In- and 89Zr-labeled panitumumab. Mice were intravenously injected 1.85 and 5.3 MBq via tail vein for 89Zr and 111In-labeled panitumumab, respectively. The organ dosimetry (mGy/MBq) of radionuclide-labeled pantimumab resulted in a good correlation (R2 > 0.94, p < 0.0001) between 111In and 89Zr, and also illustrating the higher organ dose of 89Zr- than 111In-labeled panitumumab because of the higher energy and emission rate (S-values) of 89Zr. (Bhattacharyya S, et al., Zirconium-89 labeled panitumumab: A potential immuno-PET probe for HER1-expressing carcinomas. Nucl Med Biol. 2013;40:451–457.)

In a recent study, the EGFR-binding affibody, (ZEGFR:955)2, was labeled with 125I and compared with 125I-labeled EGF and 125I-labeled cetuximab.71 An in vitro experiment with A431 cells showed rapid uptake of 125I labeled (ZEGFR:955)2 in comparison to others. It was also observed that 125I-labeled (ZEGFR:955)2 had rapid internalization and greater retention with HER1-positive tumor cells. Later, this affibody was also radiolabeled with 111In72 and in vivo evaluation in A431 tumor-bearing mice showed high tumor uptake, with tumor-to-blood ratios of 9.1 at 4 hours post injection (p.i.). Some other HER1-targeted affibody molecules were radiolabeled and evaluated in A431 tumors. All agents showed tumor uptake between 5% to 7%ID/g at 4 hours p.i. with tumor-to-blood concentration ratios of ∼3.16,73


As previously mentioned, HER2 is a highly significant receptor within HER family. Most of the therapeutic agents that target HER2 are mAb-based (trastuzumab, pertuzumab, or mAb fragment derivatives of these). Examples of small-molecule therapeutic agents are rare. Lapatinib is the only FDA-approved small molecule that can target HER2 as well as HER1. Recently, it has been labeled with 18F radioisotope.

Allan et al.74 were the first to evaluate anti-HER2 rat antibody ICR12 labeled with Technetium-99m [99mTc] (t1/2 = 6 hours) to image breast cancer patients using SPECT. The diagnosis of breast cancer relied on the triple approach of clinical findings, mammography, and positive fine-needle aspiration cytopathology or core biopsy histopathology. In this study, 2 mg of ICR12 labeled with 700 MBq of [99mTc] was injected intravenously into patients; axillary and thoracic regions were imaged at 24 hours after injection. The [99mTc]-labeled antibody showed good tumor localizing properties in patients with tumors that strongly expressed HER2.

Later, Perik et al.75 evaluated an FDA-approved HER2-binding antibody, trastuzumab, labeled with 111In (t1/2= 2.8 days) to image HER2-positive tumor in patients using SPECT–computed tomography (CT) (Fig. 39.17). The combination of CT with SPECT allowed better visualization of tracer uptake than seen with SPECT alone. In a single patient, 5-mg radiolabeled trastuzumab was administered intravenously and planar whole-body image was performed using a two-headed γ-camera equipped with medium-energy collimators at a scan speed of 12 cm/min, at various time points (15 minutes, 1 to 7 days) p.i. 111In-DTPA-trastuzumab was able to identify previously unidentified lesions in 13 of the 15 assessable patients on the first scans. The overall detection rate of tumor lesions was 45% at the single-lesion level.75 The relatively low detection rate may have resulted from the limited spatial resolution of planar single-photon imaging. This issue was resolved by Dijkers et al.76 by adopting PET modality for imaging trastuzumab, which is expected to have a higher spatial resolution than either SPECT or planar single-photon imaging.75

FIGURE 39.17. CT images (top) of a patient with a large liver metastasis. Fusion with 111In-DTPA-trastuzumab SPECT (bottom) shows correspondence of liver metastases (arrows) and SPECT hot spot. (Reprinted with permission of the American Society of Clinical Oncology. Perik PJ, Lub-De Hooge MN, Gietema JA, et al. Indium-111-labeled trastuzumab scintigraphy in patients with human epidermal growth factor receptor 2-positive metastatic breast cancer. J Clin Oncol. 2006; 24(15):2276–2282.)

FIGURE 39.18. Ex vivo tissue uptake after intravenous coinjection of 89Zr-trastuzumab (black) and 111In-trastuzumab (gray) (100 mg of trastuzumab, 2 MBq in total) at day 1 (A and C) and day 6 (B and D). Data are presented as %ID/g of tissue (mean 6 SEM for 5 HER2/neu-positive mice and for 4 HER2/neu-negative mice per group). Significance is indicated (*P, 0.05; **P, 0.01). (Reprinted by permission of the Society of Nuclear Medicine from Dijkers EC, Kosterink JGW, Rademaker AP, et al. Development and characterization of clinical-grade 89Zr-trastuzumab for HER2/neu immunoPET imaging. J Nucl Med. 2009;50(6):974–981. Figure 3)

Because of the advantages of PET, Dijkers et al.75 performed preclinical PET studies with trastuzumab labeled with positron-emitting zirconium-89 (89Zr) (t1/2 = 3.3 days) and showed that it displayed superior image quality compared to 111In-trastuzumab, although the uptake levels of 89Zr-trastuzumab and 111In-DTPA-trastuzumab in the tumors were equivalent (Fig. 39.18).

Later, in a clinical study from the same group, 14 patients with HER2-positive metastatic breast cancer received 37 MBq of 89Zr-trastuzumab at one of the three doses (10 or 50 mg for those who were trastuzumab naive and 10 mg for those who were already on trastuzumab treatment).77 The patients underwent at least two PET scans between days 2 and 5 p.i. The results of the study showed that the best time to assess 89Zr-trastuzumab uptake by tumors in a clinical setup was 4 to 5 days after the injection (Fig. 39.19).

For optimal PET-scan results, trastuzumab-naive patients required a 50-mg dose of 89Zr-trastuzumab, and patients already on trastuzumab treatment were best imaged using a 10-mg dose. The accumulation of 89Zr-trastuzumab in lesions allowed PET imaging of most of the known lesions including some that had been unapproachable by biopsy or undetected by conventional scans (Fig. 39.20).77

Recently in a case study, 89Zr-trastuzumab was used for the noninvasive assessment of the HER2 status of metastatic breast cancer in a patient receiving therapy to design a better treatment plan.78 The primary tumor in the patient in this study was histologically grade III and triple negative (negative for estrogen receptor (ER), progesterone receptor (PgR), and HER2). The treatment plan for this patient consisted of doxorubicin and cyclophosphamide followed by trastuzumab combined with paclitaxel. Thereafter, trastuzumab monotherapy was continued for 1 year. Chemotherapy was followed by treatment with tamoxifen and leuprorelin. Two years after the diagnosis, during a routine checkup, the serum cancer antigen 15.3 level was increased to 70 kU/L. Physical examination of the patient was unremarkable. On CT scan, dissemination analysis showed a large mediastinal mass and a small liver lesion, although no lesions were present on the bone scan. The 89Zr-trastuzumab PET scan showed uptake in a mediastinal mass, lymph nodes in the neck region, and sacral spine (Fig. 39.21). No liver lesions were detected. It was concluded from the 89Zr-trastuzumab PET scan that the metastatic process was HER2-positive. Given the progression during hormonal treatment and the large mediastinal mass, tamoxifen treatment was temporarily discontinued, and chemotherapy was initiated. The patient received three cycles of fluorouracil, epirubicin, and cyclophosphamide followed by three cycles of paclitaxel and trastuzumab. Thereafter, the CT scan showed a partial tumor response, and trastuzumab was continued in combination with the aromatase inhibitor anastrozole.

FIGURE 39.19. Examples of 89Zr-trastuzumab uptake 5 days after the injection. A: A patient with liver and bone metastases, and (B and C) two patients with multiple bone metastases indicated by arrows. (Reprinted with permission of the Nature Publishing Group. Dijkers EC, Oude Munnink TH, Kosterink JG, et al. Biodistribution of 89Zr-trastuzumab and PET imaging of HER2-positive lesions in patients with metastatic breast cancer. Clin Pharmacol Ther . 2010;87(5):586–592.)

In addition to managing the drug treatment plan, HER2 imaging has also been used as a drug response marker.79 HSP90 inhibition is a potentially new-targeted drug modality in the treatment of HER2-positive, trastuzumab refractory breast cancer. Little is known about the pharmacodynamics of HSP90 inhibitors (AUY922) in vivo, but noninvasive PET/CT imaging in a xenograft mouse model could visualize and quantify HER2 reduction after AUY922 treatment by 89Zr-trastuzumab PET imaging.79 Two doses of 50 mg/kg AUY922 resulted in a decrease in HER2 expression of approximately 50%, quantified 6 days after the last administration of AUY922. Visualizing HER2 expression in breast cancer patients before and early following HSP90 inhibition by means of 89Zr-trastuzumab PET may provide insight into the early in vivo effect of HSP90 inhibition and could potentially support patient-tailored therapy.79 In this effort, a clinical study (NCT01081600) has been recently completed as a side study to the multicenter, international phase I–II trial with AUY922. In this study, a minimum of six patients with HER2-positive, trastuzumab refractory breast cancer were to receive an 89Zr-trastuzumab PET scan before (baseline) and during treatment with AUY922. No results have been released from this trial to date.

FIGURE 39.20. Examples of fusion images from HER2 PET and MRI scans. A: In a vertebral metastasis seen on MRI but unapproachable for biopsy, HER2 status was revealed by 89Zr-trastuzumab uptake on PET imaging. B:Example of HER2-positive brain lesion undetected by conventional scans, revealed by 89Zr-trastuzumab PET imaging, and subsequently confirmed by MRI. Arrows indicate lesions. HER2, human epidermal receptor 2; MRI, magnetic resonance imaging; PET, positron emission tomography; 89Zr, zirconium-89. (Reprinted with permission of the Nature Publishing Group. Dijkers EC, Oude Munnink TH, Kosterink JG, et al., Biodistribution of 89Zr-trastuzumab and PET imaging of HER2-positive lesions in patients with metastatic breast cancer. Clin Pharmacol Ther. 2010;87(5):586–592.)

FIGURE 39.21. The PET-CT scan showing uptake in the mediastinal mass, lymph nodes in the neck region level 4 on both sides and level 3 on the right side, and sacral spine. No liver lesions were detected. (Reprinted with permission of the American Society of Clinical Oncology. Gaykema SB, Brouwers AH, Hovenga S, et al. Zirconium-89-trastuzumab positron emission tomography as a tool to solve a clinical dilemma in a patient with breast cancer. J Clin Oncol. 2012;30(6):e74–e75.)

An affibody molecule that targets HER2 has also been evaluated in a clinical trial. The DOTA-chelated version of ZHER2:342 (ABY-002) labeled with 111In80 was the first affibody molecule administered to humans. 111In-ABY-002 was studied in a limited number of patients with recurrent breast cancers. Administration of a microdose (<100 μg) of 111In-ABY-002 resulted in high-quality SPECT images enabling the detection of even small lesions (12 to 14 mm) as early as 2 hours p.i.81,82 This approach was also successful in patients receiving Herceptin.

HER2 imaging of tumors has shown considerable promise. HER2 imaging has been shown to be useful in identifying tumors that were not visible in CT or bone scans. In addition, the knowledge of status of HER2 expression in the tumors allows proper management of the disease in newly diagnosed patients or in patients that are undergoing cancer therapy.


Increasing evidence implicates HER3 and HER4 alterations in carcinogenesis. Overexpression of HER3 receptors has been reported in 20% to 30% in invasive breast carcinomas.83 HER3 signaling relies on the formation of heterodimers with other members of HER family (HER1/HER3, HER2/HER3) because HER3 receptor has no intrinsic kinase activity. The relatively limited activity of HER1 TK inhibitors in tumors is likely to be related to failure to effectively inhibit HER3.84 HER3 expression is substantially increased after long-term trastuzumab exposure of HER2-positive breast cancer cell lines or after development of primary resistance to trastuzumab therapy. There are very few therapeutic agents available which can inhibit HER3. Therefore, examples of HER3- and HER4-targeted nuclear imaging are very rare.

AMG 888 (U3-1287) is a monoclonal antibody with high affinity for HER3. Recently, Sharp et al.85 labeled this monoclonal antibody with 64Cu-DOTA (t1/2 = 12.7 hours) to image cancers. A preclinical study in mice has shown the potential of this tracer to image pancreatic cancer (presented in 2011 World Molecular Imaging Congress). As a result, the Washington University School of Medicine is currently sponsoring a clinical trial (NCT01479023) to evaluate 64Cu-DOTA-U3-1287 in subjects with advanced solid tumors and participants are being recruited. These studies will give insight into the utility of HER3-based imaging in clinical setting.


In general, targeted radionuclide therapy is less advanced than nuclear imaging because destructive doses require much more accurate targeting than tracer doses. For systemic administration a therapeutic radiopharmaceutical should have high tumor uptake, a high tumor-to-background ratio, long residence time, and fast renal clearance.21 Because of their high specificity, mAbs are usually chosen over small molecules, to develop radiotherapeutic agents (e.g., Zevalin, Bexxar). Although mAbs exhibit high specificity, their high metabolic stability and slow clearance represent severe challenges for mAb-based radiotherapeutics because of collateral damage to normal tissues. Radiation toxicity to the kidney, bone marrow, and other internal organs are of primary interest. As a consequence, the development of mAb-based radiotherapeutic agents for clinical use requires tremendous effort to overcome both scientific and regulatory challenges.

Radioimmunotherapy targeting of the HER family (HER1 and HER2) has been extensively studied. Pfost et al.86 radiolabeled anti-HER1 mAb matuzumab with the α-emitter 213Bi and found it very effective to treat bladder cancer when radiolabeled mAb injected intravesically with mice surviving over 300 days compared to 89 and 41 days in unlabeled mAb treated and untreated control groups. Cetuximab labeled with the β-emitter 90Y has been investigated preclinically: Both labeled and unlabeled cetuximab showed efficacy in a mouse model of HNSCC.87

In studying radioimmunotherapy of HER2, several investigators88,89 have shown significant efficacies of anti-HER2 mAbs. Song et al.90 demonstrated efficacy of α-emitting radionuclides 213Bi- and 225Ac-labeled anti-HER2 mAb in treating mice-bearing metastatic breast cancers. In other studies, it was shown that 213Bi- and 225Ac-labeled trastuzumab significantly improved survival of mice with peritoneal ovarian cancer cells.91 Persson et al.92 showed that 177Lu-radiolabeled anti-HER2 mAb pertuzumab significantly delayed cancer progression in an ovarian cancer xenograft model. α-Emitter 211At-labeled trastuzumab has been shown to completely eradicate ovarian cancer xenografts when combined with unlabeled trastuzumab.93

It has been demonstrated that DOTA-conjugated affibody ZHER2:342 (ABY-002) can be stably labeled with 90Y and 177Lu without losing HER2-binding specificity. Biodistribution of the 177Lu-labeled ZHER2:342 in normal nude mice showed rapid clearance from the blood and low uptake in normal organs indicating its potential as a targeting agent for local treatment of bladder carcinoma.94 Breast cancer patients with HER2-expressing tumors also have a risk of developing HER2-expressing metastases in brain, which in principle could be an interesting target for intracerebroventricular (ICV) administration82 of radiolabeled ZHER2:342.

Although many promising results of radioimmunotherapy of the HER receptors have been observed in preclinical models, only one clinical trial of radiolabeled mAb (125I-425) is currently (1985–2012)95 designed to target HER1 in patients with glioblastoma multiforme (GBM). In a large phase II study in which 192 patients with GBM were treated with anti-HER1, either as 125I-mAb-425-RIT or temozolomide + 125I-mAb-425-RIT, the median survivals were 14.5 and 20.2 months, respectively. The treatment was safe and well tolerated.


In the HER family of receptors, HER1 and HER2 are considered the most important molecules with potential for cancer therapy. Over the last few years, several drugs targeting HER1 and HER2 have been included in various preclinical and clinical trials. Among them, a number of mAbs targeting the extracellular domain of HER and also small organic molecules targeting the intracellular TK domain of HER have received FDA approval. It has been observed that only a subset of the patient population is benefited by HER-targeted therapy. The patients that do not respond to HER-targeted therapy either have low uptake of drug in tumors, decreased expression of HERs in tumors, or existence of tumors harboring mutation in HERs that disrupt binding of drug. Thus, for proper treatment planning with HER-targeted drugs, it is imperative to know the expression and mutation status of HERs in tumors along with information about biodistribution of the administered drug in patients. In this regard, nuclear imaging with radiolabeled HER-targeted biomarkers can be a powerful tool assisting therapy. Nuclear imaging coupled with an array of selective radiolabeled probes can not only noninvasively assess the status of HER expression but can also give information about the biodistribution of drug. In addition, nuclear imaging can be used also to characterize the drug–target interaction or indirectly test the existence of primary or secondary mutation in HERs through real-time assessment of target–drug binding and receptor occupancy studies.

Considering the advantages of HER imaging, several preclinical and clinical imaging studies have been performed with radiolabeled HER-specific probes to test their potential to assist HER-targeted treatments. These labeled probes targeting TK or extracellular region of HERs are mainly based on either approved drugs or drugs in process of development. Some preclinical and clinical trials (e.g., 11C-erlotinib and some labeled mAbs) have shown promise whereas some have failed. The primary reason for these inconsistencies is not clear but may be explained by the use of probes with different pharmacokinetic and tumor-binding properties, suboptimal choice of radionuclides, and differences in tumor physiology. As a consequence, many critical questions are still open and the potential to translate these tracers in the clinical setting for HER imaging in cancer patients warrants further efforts. For example, in preclinical trials with mouse xenografts, radiolabeled mAbs (cetuximab, trastuzumab, and panitumumab) have shown encouraging results by exhibiting high specificity. In clinical trials, however, imaging results with HER1-binding analogs of cetuximab and trastuzumab have failed to impress. It is important to note that these mAbs bind only to human HER1 and not to murine HER1. In murine preclinical trials, therefore, these mAbs show predominantly high and selective tumor uptake in HER1-positive human cancer xenografts whereas in clinical trials these mAbs bind to any tissue in patients that expresses HER1 leading to loss of tumor specificity. This is also evident in a recent clinical PET study with HER2-binding 89Zr-trastuzumab that demonstrated (vide supra), where relatively high mAb dose of at least 50 mg was required for reliable HER2 imaging in a breast cancer patient. At low doses, rapid hepatic clearance of the probe was observed, probably because of the presence of high level of HER2 in plasma. This means tumor imaging with labeled mAbs will be difficult when high levels of target receptor are present in readily accessible normal organs that serve as a mAb sink.96

On the other hand, with small organic molecule–based radiotracers, the main hurdles to be overcome include rapid clearance, nonspecific binding, and inadequate pharmacokinetic properties. Although some of these agents were developed based on approved drugs, they did not yield adequate nuclear imaging results. These experiences underscore the general knowledge that radiolabeling successful targeted drugs will not always lead to useful imaging agents. Therefore, making treatment decisions based on nuclear imaging results (predicting and monitoring) and using radiotherapeutics for the treatment of HER-positive cancers require considerable caution.


Considering the limitation of existing radiolabeled HER imaging agents, it is necessary to focus on novel strategies for developing imaging agents. Effort should also be made in the design of preclinical studies to provide data that can be extrapolated to the human situation. In this effort, interdisciplinary collaboration will be crucial to allow design of optimal imaging agents with high target affinity and specificity, stability, optimal blood clearance, and low accumulation in normal tissues.

To target intracellular TK domains of HERs, use of irreversible inhibitors instead of presently used reversible inhibitors shows promise. These irreversible inhibitors show favorable tracer properties such as high solubility, optimal blood clearance, and lower log P in recent studies. Future trends in the field of labeled small molecules will probably also include labeled TK inhibitors directed toward intracellular substrate binding or regulatory sites rather than ATP-binding domain to avoid ATP-binding competition and to obtain higher accumulation and selectivity. On the other hand, to target extracellular domains of HERs, low–molecular-weight affibodies could be preferable to antibodies because of optimized target affinity and specificity, suitable log P, and optimal blood clearance.

Apart from the properties of the targeting molecule, the choice of radionuclide could also improve the utility of imaging agents. To obtain high signal-to-noise ratio with radiolabeled mAbs, image acquisition needs to be performed days after injection to allow wash out of nonspecific binding and optimal blood clearance. Therefore, radionuclides with longer half-life like 89Zr might be preferable although the relatively long half-life results in a radiation-absorbed dose greater than the usual dose from a diagnostic imaging agent.

In radiotherapy, to avoid unwanted radiation exposure to normal tissues and organs, systemic administration should be replaced by localized treatment wherever possible. It is possible to use radiolabeled mAb or affibodies for locoregional treatment, for example, in the treatment of bladder carcinoma by intravesicular administration or for ICV administration in patients with brain tumors. High expressions of some HER receptors (e.g., HER1) on normal organs, like liver and skin, will most likely limit systemic radioimmunotherapy of HER1-expressing tumors and metastases. HER1-targeting affibodies could be used, however, in locoregional treatment of glioma patients after surgery. The small HER1-specific radiolabeled affibody molecule could potentially be a good construct for reaching migrating glioma cells outside the operation cavity. Another possible local treatment modality is the intravesical treatment of urinary bladder cancers.

Radionuclide imaging of HER has the potential to offer crucial assistance to HER-targeted therapy for cancer patients. New chemical entities will likely evolve in near future and have the potential to open new avenues in the development of HER-based nuclear imaging probes and targeted radionuclide therapy.


This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US government.

The authors thank Pragya Sharma of the Mayo Clinic for making all high-resolution figures for this chapter.


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