Keywords

Introduction

Molecular imaging allows visualization of tumor biology in vivo [1]. Different processes can be visualized, such as glucose metabolism, proliferation, and hypoxia. But also numerous ligands for receptors and other relevant targets in the tumor microenvironment have been labeled to be used as a tracer for molecular imaging. For positron emission tomography (PET) and single photon emission computerized tomography (SPECT) imaging, ligands are labeled with a radioactive nuclide, while for optical imaging ligands are labeled with a fluorescent dye. Also for magnetic resonance imaging (MRI), computerized tomography (CT), and ultrasound, specific contrast agents are available for molecular imaging [2–4]. Head and neck squamous cell carcinoma (HNSCC) is diagnosed in more than 500,000 patients worldwide per year. Many patients present with locally advanced disease, which has a poor prognosis with around 50 % 5-year survival. Adequate staging and tumor delineation could enhance precision of surgery and radiotherapy, which may lead to a reduction of recurrences. PET imaging has great potential to improve staging, while optical imaging is investigated for its ability to improve tumor delineation. Furthermore, molecular imaging can be used to visualize specific tumor characteristics that can be used for targeted treatment. Therefore, this chapter will focus on PET imaging and optical imaging in HNSCC.

PET Imaging

Next to a role in diagnosis, staging, and response evaluation when combined with CT or MRI, PET imaging maybe useful for prognostication and radiotherapy treatment planning. Furthermore, PET imaging can be used during drug development by demonstrating the distribution of a drug or treatment target [5]. The role of PET in the management of head and neck cancer patients has been summarized in an excellent review by Cammaroto et al. [6]. Radionuclides that are frequently used for PET imaging in patients are fluor-18 (18F), copper-64 (64Cu), zirconium-89 (89Zr), and iodine-124 (124I), which differ among others in half-life (1.8 h, 12.7 h, 78.4 h, and 100.2 h, respectively). The half-life of the SPECT tracer indium-111 (111In) is 67.4 h. Antibodies have long half-lives of 1–3 weeks, which requires the use of radionuclides that also have long half-lives [7].

18F-Fluorodeoxyglucose PET

In HNSCC patients, many PET tracers have been tested but only 18F-fluorodeoxyglucose (FDG)-PET is incorporated in management guidelines. FDG-PET enables visualization and quantification of glucose metabolism which is enhanced in most tumors, but also in areas of inflammation. The National Comprehensive Cancer Network (NCCN) Clinical Practice Guidelines for head and neck cancer (version 1, 2015) recommend the use of FDG-PET/CT for patients with lymph node metastasis in the neck of a squamous cell carcinoma (SCC), adenocarcinoma, and anaplastic or undifferentiated epithelial tumors of an unknown primary site [8]. For patients with stage III and IV HNSCC, FDG-PET is considered optional because it may alter treatment decisions by upstaging patients. For response evaluation after chemoradiotherapy or radiotherapy alone, FDG-PET is recommended 12 weeks after completion of treatment in patients with a clinical response, to guide the decision on neck dissection. A meta-analysis of mainly single center small studies demonstrated a high negative predictive value of FDG-PET/CT after chemoradiotherapy for persistent/recurrent disease [9]. A recent phase III trial compared FDG-PET/CT guided active surveillance with planned neck dissection for HNSCC patients with locally advanced disease treated with primary radical chemoradiotherapy. The study showed that overall survival was equivalent. Moreover, in the surveillance arm only 20 % of the patients underwent a neck dissection, which resulted in fewer complications, better cost effectiveness, and similar quality of life [10].

Quantification of tumor FDG uptake may also have prognostic value. A meta-analysis suggested that a low tumor standardized uptake value (SUV) is associated with a better disease free survival, a better overall survival, and improved local control [11]. In a large retrospective study from Denmark, tumor FDG uptake was shown to be an independent prognostic factor in patients who received radiotherapy as primary treatment, with high tumor SUVmax corresponding to a worse failure free survival [12].

Finally, FDG-PET is under investigation as a tool to improve radiotherapy (RT) treatment planning. The observation that local recurrence after radiotherapy frequently occurs in the area with the most intense FDG uptake has boosted trials that investigate FDG-PET based radiotherapy dose painting [13]. Two different strategies are applied: dose painting by contours, where a higher uniform RT dose is delivered to a target volume based on PET imaging; and dose painting by numbers, where on a voxel scale SUV is used to calculate RT dose.

Non-FDG PET Tracers

Apart from FDG, more than 20 PET tracers have been tested in HNSCC for imaging hypoxia, proliferation, amino acid metabolism, and other cellular processes and tumor characteristics (see Table 5.1). Especially imaging of tumor hypoxia is an area of active research.

Table 5.1 Examples of studies with non-FDG PET tracers in HNSCC patients
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Hypoxia Imaging

Tumor hypoxia is associated with poor prognosis and resistance to treatment. Tumor hypoxia can be analyzed directly by measuring oxygen tension with an electrode, but this is an invasive procedure which does not take into account tumor heterogeneity. Hypoxia imaging on the other hand allows serial noninvasive assessment of tumor hypoxia, both of the primary tumor and of lymph node metastases. Multiple hypoxia PET tracers have been developed, mostly based on a nitroimidazole structure. These molecules freely diffuse through cell membranes but get trapped into cells in the presence of a low oxygen level [34, 35]. The most used hypoxia PET tracer is 18F-fluoromisonidazole (18F-FMISO) which has recently been reviewed by Rajendran and Krohn [36]. Several, generally small single center, 18F-FMISO PET studies have been published in HNSCC patients over the last 10 years (Table 5.2). In these studies, different parameters for quantification were used, but also different reference tissues, different treatment schedules, and different timing of follow up imaging. This complicates interpretation and hampers robust conclusions. However, several studies showed that patient with more hypoxic tumors had a worse outcome [14, 37, 41, 44, 46, 50]. Furthermore, early reoxygenation during chemoradiotherapy appears to be associated with a lower risk of recurrence [46, 50].

Table 5.2 18F-FMISO PET studies in HNSCC patients
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Where prognostic markers provide information about outcome of patients independent of treatment, predictive markers give information on the effect of a specific treatment strategy [51]. The prognostic value of hypoxia PET can potentially be used to guide treatment de-escalation in patients with nonhypoxic tumors with favorable prognosis and/or treatment escalation in patients with hypoxic tumors (Fig. 5.1). Currently a treatment de-escalation study is ongoing in patients with human papillomavirus (HPV) positive oropharynx cancers that are nonhypoxic at baseline or show early re-oxygenation on repeat imaging (ClinicalTrials.gov Identifier: NCT00606294). On the other end of the spectrum, in silico studies have demonstrated the feasibility of increasing radiotherapy dose to hypoxic tumor subvolumes [53–57]. Currently two randomized studies are comparing standard chemoradiotherapy with chemoradiotherapy using an increased radiation dose to hypoxic tumor subvolumes (ClinicalTrial.gov. Identifiers: NCT02352792 and NCT01212354).

Fig. 5.1
figure 1

Study design using hypoxia PET as prognostic marker. LA-HNSCC locally advanced head and neck squamous cell carcinoma, PET positron emission tomography, R randomization. Patients with LA-HNSCC undergo hypoxia PET imaging before start of treatment. Patients with nonhypoxic tumors are randomized between standard of care and an experimental treatment de-escalation regimen. Patients with hypoxic tumors are randomized between standard of care and an experimental treatment intensification regimen. Double enrichment design (After Freidlin et al. [52])

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Several therapeutic strategies have been developed to reduce tumor hypoxia during radiotherapy, including carbogen and nicotinamide, tirapazamine, and nimorazol [58–60]. Hypoxia PET may have predictive value by identification of patients who benefit from hypoxia targeting treatment. This could be investigated by using a biomarker stratified study design (Fig. 5.2). Data from a sub study using 18F-FMISO PET suggested that patients with hypoxic tumors derived benefit from treatment with tirapazamin, a cytotoxic drug with selective toxicity towards hypoxic cells [38]. Currently an international randomized phase III trial comparing chemoradiotherapy plus nimorazol with chemoradiotherapy plus placebo in patients with locally advanced HNSCC uses a hypoxic gene signature as stratification factor but also tests predictive value of hypoxia PET in a subset of the patients (ClinicalTrials.gov Identifier: NCT01880359). However, nimorazole is a cheap drug with limited side effects, therefore it is doubtful if hypoxia PET is going to be implemented as predictive marker even if the positive and negative predictive values are high.

Fig. 5.2
figure 2

Study design testing hypoxia PET as predictive marker. LA-HNSCC locally advanced head and neck squamous cell carcinoma, PET positron emission tomography, R randomization, SOC standard of care. Patients with LA-HNSCC undergo hypoxia PET imaging before start of treatment. Patients with nonhypoxic tumors as well as patients with hypoxic tumors are randomized between standard of care plus a hypoxia targeting drug and standard of care plus placebo. Biomarker stratified design (After Freidlin, et al. [52])

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PET Imaging Using Radiolabeled Antibodies

PET imaging with radiolabeled monoclonal antibodies, also called immuno-PET, can potentially be used to select patient for targeted treatment and for drug development [5]. For HNSCC, the epidermal growth factor receptor (EGFR) blocking antibody cetuximab is the only targeted therapy shown to be effective, in combination with radiotherapy and in combination with chemotherapy [61, 62]. EGFR, also known as human epidermal growth factor receptor 1 (HER1), is a member of the human EGFR (HER) family that further consists of HER2, HER3, and HER4.

Preclinical research has shown that activation of HER3 after dimerization with HER2 limits activity of EGFR inhibition in HNSCC and that dual inhibition of EGFR and HER3 can overcome resistance to radiation and to EGFR inhibition [63, 64]. Several agents targeting HER3 are currently under investigation in clinical trials, including monoclonal antibodies directed against HER3, dual inhibitors of EGFR and HER3, and pan-HER monoclonal antibody mixtures and tyrosine kinase inhibitors. Immuno-PET using radiolabeled antibodies against EGFR and HER3 could be useful to provide information on availability of the drug target and distribution of therapeutic antibodies in HNSCC patients.

EGFR Imaging

EGFR expression determined by immunohistochemistry has prognostic value in HNSCC, but is not a predictive biomarker for efficacy of cetuximab [65]. This may be related to heterogeneity in EGFR expression but also to accessibility of the tumor to EGFR inhibitors. Tumor drug delivery is not solely dependent on expression of the target, but also determined by perfusion, permeability, interstitial pressure, and drug characteristics including size [66, 67]. Two preclinical studies investigating 64Cu-cetuximab PET imaging in xenograft models reported a correlation between tumor uptake of 64Cu-cetuximab and EGFR expression [68, 69]. A third study with 89Zr-cetuximab PET imaging in tumor bearing mice showed tracer uptake in EGFR positive tumors, but no correlation between tracer uptake and EGFR expression was found [70]. This may however be related to the tracer dose used [71].

Antibodies have a long half-life which implicates that in order to achieve a good tumor-to-background ratio and tumor-to-blood ratio, the optimal timing of imaging is around 7 days after tracer injection. To allow imaging within 24 h and repeat imaging early after start of treatment, antibody fragments of cetuximab (cetuximab-F(ab’)2) have been developed and radiolabeled for SPECT and PET imaging [72, 73]. Imaging studies in head and neck cancer xenograft models using 111In-cetuximab-F(ab’)2 SPECT have shown that localization of the tracer correlates with EGFR expression and that the model with the highest uptake was the most sensitive to cetuximab treatment [72, 74]. Furthermore, increased tumor tracer uptake was found after radiotherapy in a cetuximab sensitive HNSCC xenograft model, which was accompanied by translocation of EGFR to the tumor cell membrane [75]. On the other hand, in a cetuximab resistant tumor model, no increase in tumor tracer uptake after radiotherapy occurred. Finally, treatment of human HNSCC xenograft models with radiotherapy alone, cetuximab alone, or the combination demonstrated reduced tracer uptake in responding tumors while in resistant tumors an increase in tumor tracer uptake was found [75]. Therefore, translation of this molecular imaging technique to the clinic offers a promising tool for selecting patients who will benefit from treatment with cetuximab but it could also be useful as an early read-out of treatment efficacy. Three clinical studies have started using 89Zr-cetuximab PET imaging, one in HNSCC and two in colorectal cancer patients (ClinicalTrials.gov Identifiers: NCT01504815, NCT02117466, NCT01691391) [31, 76]. The head and neck cancer trial was initiated as a randomized phase II study comparing cisplatin with cetuximab and standard radiotherapy with redistributed radiotherapy in a two by two factorial design. One of the objectives was to evaluate the predictive value of 89Zr-cetuximab tumor uptake on a pretreatment PET scan [31]. Unfortunately the trial design has been changed and cetuximab treatment and cetuximab imaging are no longer part of the protocol (https://clinicaltrials.gov/archive/NCT01504815/2014_08_21/changes).

HER3 Imaging

The HER3 antibodies lumretuzumab and patritumab have been labeled for PET imaging [77, 78]. In a phase I study, 13 patients with solid tumors expressing HER3, determined by immunohistochemistry, underwent imaging with 89Zr-lumretuzumab PET before start of treatment with the same antibody [79]. Two patients with HNSCC were included in this study. The aim of the imaging part was to determine in vivo biodistribution and the ability of the antibody to target the tumor. In all patients, tracer uptake in tumor lesions was seen. Metastases in the bone and brain that were unknown were detected in three patients. Results of serial imaging during treatment to assess HER3 saturation are awaited. In another phase I study, dosimetry of 64Cu-DOTA-patritumab and receptor occupancy after a therapeutic dose patritumab were investigated [78]. Three out of six patients in the receptor occupancy cohort had a negative PET scan, likely because patients were not preselected for HER3 tumor expression. In the remaining patients, receptor occupancy was ~42 %. Larger studies are needed to assess predictive value of immuno-PET for efficacy of antibody therapy and/or for selecting the right treatment dose.

PET Imaging of Tumor Immunity

Cancer immunotherapy with immune checkpoint inhibitors has been a great breakthrough for melanoma, non–small cell lung cancer and other tumor types and has shown very promising early results in head and neck cancer [80, 81]. Antibodies directed at programmed death 1 (PD-1) and its ligand PD-L1 are currently investigated in phase III trials in HNSCC. To date there are no biomarkers that predict efficacy of immune checkpoint inhibition, although in some tumor types expression of PD-L1 using immunohistochemistry is associated with a higher response rate. Expression of relevant targets for immunotherapy may vary between and within tumor lesions, and over time. Molecular imaging offers a noninvasive platform for serial assessment of whole body target expression and antibody distribution. PET imaging of tumor immunity is in an early phase of development with no clinical data available yet. One imaging study is currently ongoing in patients with triple negative breast cancer, bladder cancer and non–small cell lung cancer using 89Zr-atezolizumab (PD-L1 antibody) PET before treatment with the same antibody (ClinicalTrials.gov Identifier: NTC02453984). Two preclinical studies already demonstrated feasibility and specificity of radiolabeled antibodies for PD-L1 imaging in tumor bearing mice [82, 83].

Another interesting strategy is to use molecular imaging as an early read-out of treatment response. PD1 and PD-L1 antibodies are supposed to act by augmenting the activity of tumor infiltrating cytotoxic T lymphocytes. Activated T lymphocytes express interleukin-2 (IL-2) receptor. SPECT imaging with radiolabeled IL-2 (99mTc- IL2) has successfully been used in patients for visualization of activated T lymphocytes in atherosclerotic plaques and in melanoma [84, 85]. For IL-2 PET imaging, which allows more sensitive and more precise quantification than SPECT, 18F-IL2 has been developed and validated preclinically [86, 87].

The subset of lymphocytes that can eliminate tumor cells are CD8 expressing cytotoxic T cells. Antibody fragments against murine CD8 have successfully been labeled with 64Cu and showed specific uptake in lymph nodes and spleen of antigen positive mice [88]. If this technique can successfully be translated to the clinic, it would allow to study in vivo tumor T cell infiltration which appears to be a prerequisite for immunotherapy to be effective.

Another important class of immune cells affecting tumor behavior are the tumor associated macrophages (TAMs). The subset of M2 macrophages appears to have a tumor promoting and cytotoxic T cell suppressive effect [89]. Macrophage depleting drugs are currently investigated in clinical trials. M2 macrophages specifically express the macrophage mannose receptor (MMR). Radiolabeled antibody fragments targeting MMR have been developed for PET imaging and showed specific uptake in tissues and tumors expressing MMR in mice [90]. MMR imaging could be helpful in the process of drug development, for patient selection, and as a read out for treatment efficacy.

Optical Imaging

Optical molecular imaging is a much more recent field of research, and evidence from clinical studies is still scarce. Optical imaging techniques use illumination with light of different wave lengths, ranging from safe ultraviolet range (350–400 nm), and the visible spectrum (400–750 nm) to infrared regions (750–1000 nm). Penetration of light is limited due to scattering and absorption, which vary substantially in different target tissues. Generally, in the range of 350–1000 nm, light penetration is deeper with increasing wavelength. Near-infrared (NIR) fluorescence imaging can visualize structures up to 8 mm below the surface, depending on the optical properties of the target tissue [91]. Several optical spectroscopy and imaging technologies have been and are currently investigated in HNSCC, including Raman spectroscopy, narrow band imaging, autofluorescence and exogenous fluorophore imaging, optical coherence tomography, confocal laser endomicroscopy, and confocal reflectance microscopy [92]. Optical imaging is currently investigated for its potential to differentiate malignant lesions from normal tissue, and from benign and premalignant lesions. Optical imaging can be used during endoscopy but also intraoperatively to guide surgical resection margins. However, in cancer diagnosis, most optical techniques are either difficult to apply in vivo (i.e., Raman spectroscopy) or have not shown to yield sufficient specificity to support routine clinical use. Only in certain fields optical imaging techniques have shown to aid the clinician in diagnostic procedures. For instance, narrow band imaging helps to identify (pre)malignant lesions in head and neck cancer [93]. Here we present examples of molecular optical imaging using fluorescently labeled molecules that target specific tumor characteristics to improve the contrast between cancer and non-cancer tissue.

EGFR Imaging

In the first clinical trial on molecular optical imaging in HNSCC patients, cetuximab labeled with the NIR fluorescent dye IRDye800 was systemically injected 3–4 days before surgery in a dose finding study [94, 95]. Wide field NIR imaging was performed at day 0, day 1, and immediately before surgery, and closed field NIR imaging of fresh tissue slices of 4–5 mm was done. After histologic preparation, a corresponding slide was analyzed with a fluorescence scanning system for comparison with immunohistochemistry. Cetuximab-IRDye800 specifically accumulated in tumor lesions with a sharp demarcation of the tumor border. The mean fluorescence intensity signal was highly correlated with EGFR expression (Fig. 5.3) [94]. However, in tumor areas with necrosis and in areas with mature, differentiated keratinizing cancer cells, fluorescence was low despite high EGFR expression. The latter might be explained by loss of ligand binding affinity of EGFR during maturation [94]. Of interest, the highest tracer dose (62.5 mg/m2) suggested receptor saturation according to the authors, because the tumor-to-background ratio seemed to have reached a plateau. This raises the question whether the standard therapeutic cetuximab loading dose of 400 mg/m2 followed by weekly doses of 250 mg/m2 might be too high, and imposes unnecessary off-target effects. However, tumor-to-background ratio may also be reduced due to a higher background fluorescence which occurs by increasing the cetuximab-IRDye800 dose. The study also revealed cetuximab-IRDye800 localization in sebaceous glands and basal cells which might be related to the skin toxicity that is frequently seen during cetuximab therapy. This novel imaging technique is an interesting tool for intraoperative use to lower the rate of involved or close surgical margins. Next to this, studying the localization of the cetuximab-IRDye800 in histological slides of the excised tumor may add another dimension to the pathology report which could be useful in determining postoperative strategies.

Fig. 5.3
figure 3

Co-localization of fluorescence signals of cetuximab-IRDye800CW and epidermal growth factor receptor (EGFR) expression. Representative hemotoxylin/eosin (H&E) image indicating tumor (T) and normal (N) with corresponding EGFR expression immunohistochemistry stain and fluorescence image [94]

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In a xenograft study of human oral cavity squamous cell carcinoma, fluorescent optical imaging was used to investigate whether tumor uptake of the cetuximab-IRDye800 could be improved by pretreatment with bevacizumab, a monoclonal antibody targeting human vascular endothelial growth factor A (VEGF-A) [96]. Neoadjuvant bevacizumab administration but not simultaneous bevacizumab increased cetuximab-IRDye800 tumor accumulation. This was accompanied by a higher pericyte coverage of tumor blood vessels compared to mice that did not receive bevacizumab, which suggests vascular normalization. Translating such a study design to the clinic could provide important information on effective treatment combinations and schedules.

Quantum dots (QDs) are semiconductor nanocrystals with a wide excitation and a small emission spectrum that can be conjugated to antibodies and peptides for molecular optical imaging. The size of QDs determines emission wave length, which can vary from the UV to the NIR range. A NIR QD800-EGFR antibody has been used for in vivo imaging of mice with a human orthotopic oral cavity squamous cell carcinoma [97]. Specific binding to tumor cells with a high signal-to-noise ratio up to 6 h after intravenous injection was demonstrated.

Another interesting development is topical application of a fluorescently labeled EGF peptide (EGF-Alexa 647) for early detection of oral neoplasia [98]. Immediately after excision, oral neoplastic lesions and paired normal tissue biopsies were incubated with EGF-Alexa 647 showing a consistently higher fluorescent signal in lesions which corresponded with EGFR immunohistochemistry. Clinically applicable conjugates are under development.

Integrin αvβ3 Imaging

Integrin αvβ3 is expressed by endothelial cells during angiogenesis in many cancers, including HNSCC, and by some tumor cells. Peptides containing an arginine-glycine-aspartic acid (RGD) sequence bind to αvβ3 integrin. A tetravalent RDG peptide labeled with a NIR fluorescent molecule (AngioStamp800) is commercially available for preclinical optical imaging. Using this probe in an αvβ3 expressing orthotopic HNSCC xenograft model, Atallah et al. operated 12 mice with the use of integrin αvβ3 imaging and 12 mice with visual guidance only [99]. In the first group, after visual complete resection, tumor beds contained fluorescent spots in all mice, and 35 out of 37 specimens of these fluorescent spots contained tumor foci. Furthermore, recurrence free survival after 2 months was 75 % in mice that had αvβ3 integrin imaging guided surgery compared to 25 % in mice resected without optical imaging. In a second study by the same group, mice were followed for lymph node recurrence after resection of orthotopic HNSCC [100]. Intraoperative integrin αvβ3 imaging correctly identified clinical and subclinical lymph node metastases in these mice.

Quantum dots conjugated with RGD (QD800-RGD) have also been used for integrin αvβ3 imaging in mice bearing HNSCC. The xenografted human oral squamous cell carcinoma cell line did not express integrin αvβ3 but specific targeting of tumor vessels in this mouse model resulted in clear tumor fluorescence with the highest tumor-to-background ratio 6 h after intravenous injection of QD800-RGD [101].

Other Optical Imaging Targets

Cancer cells display aberrant glycosylation of cell surface proteins and lipids with increased sialic acid content. This has been exploited for optical imaging using topical application of wheat germ agglutinin (WGA) conjugated with fluorophores in the UV range (Alexa Fluor 350) and NIR range (Alexa Fluor 647)[102]. Ex vivo imaging of tumor and normal mucosa biopsies of patients with HNSCC demonstrated a satisfactory signal-to-noise ratio.

Another characteristic of many cancers is overexpression of cyclooxygenase-2 (COX-2). Fluorocoxib, a COX-2 targeted NIR probe, has been developed for optical imaging [103]. Specific uptake in COX-2 overexpressing human HNSCC xenografts was demonstrated with an optimal signal-to-noise ratio at 7 days post injection in mice.

Interestingly, also a NIR dye conjugated PD-L1 antibody was successfully tested in tumor bearing mice [83].

Finally, also tumor M2 macrophage recruitment has been visualized with optical imaging using an antibody against MMR (αCD206) conjugated with NIR dyes in a murine breast cancer model [104, 105].

Future Perspectives

Molecular imaging with radiolabeled ligands for PET imaging provides whole body information on distribution of targets and/or drugs with low resolution. Optical imaging gives local information with very high resolution but with limited penetration. These complementary techniques can be used simultaneously by injecting molecules labeled with a fluorescent dye and a radionuclide [106].

Furthermore, for optical imaging, several tumor characteristics have already been analyzed simultaneously by using probes with different excitation wavelengths in preclinical studies [107].

Molecular imaging cannot replace anatomical imaging or biopsies but can potentially provide additional information to improve diagnosis and treatment of HNSCC. Before implementation, large well powered clinical studies are needed to assess its added value. Alternatively, data from multiple small studies can be combined which could be facilitated by creating ware houses with imaging data. The currently publicly available databases for genomics could serve as a role model in this respect. However, standardization of techniques and endpoints is critical for combined analysis.