Hypoxia and tumor microenvironment in head and neck squamous cell carcinoma Justin E. Swartz
ISBN: 978-94-6473-100-2 Design cover, chapter pages & layout: J.E. Swartz Printed by: Proefschriften.nl Publication of this thesis was financially supported by: ALK, Allergy Therapeutics, BAP Medical, ExamVision, Meditop BV, Rhino Horn Benelux BV © Copyright 2023 Justin Egidius Swartz All rights reserved. No part of this thesis may be reproduced, stored in a retrieval system, or transmitted in any form or by any other means without prior permission of the author.
Hypoxia and tumor microenvironment in head and neck squamous cell carcinoma Hypoxie en tumor micro-omgeving in plaveiselcelcarcinomen in het hoofd-halsgebied (met een samenvatting in het Nederlands) Proefschrift ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, Prof. dr. H.R.B.M. Kummeling, ingevolge het besluit van het college voor promoties in het openbaar te verdedigen op dinsdag 27 juni 2023 des ochtends te 10.15 uur door Justin Egidius Swartz Geboren op 6 september 1989 te Hoorn
Promotoren Prof. dr. R. de Bree Prof. dr. S.M. Willems Beoordelingscommissie Prof. dr. P.J. van Diest Prof. dr. M.R. van Dijk (voorzitter) Prof. dr. J.H.A.M. Kaanders Prof. dr. C.H.J. Terhaard Prof. dr. C.L. Zuur
Contents Chapter 1 General introduction 7 Determining the hypoxic status Chapter 2 Correlation and colocalization of HIF- į DQG SLPRQLGD]ROH staining for hypoxia in laryngeal squamous cell carcinomas: A digital, single-cell-based analysis 33 Hypoxia in relation to immunological and imaging features Chapter 3 Increased HIF-1a expression correlates with high PD-L1 expression and fewer tumor-infiltrating lymphocytes in oropharyngeal squamous cell carcinoma 55 Chapter 4 Influence of tumor and microenvironment characteristics on diffusion-weighted imaging in oropharyngeal carcinoma: A pilot study 79 Effects on clinical outcome Chapter 5 Clinical implications of hypoxia biomarker expression in head and neck squamous cell carcinoma: a systematic review 95 Chapter 6 Poor prognosis in HPV-positive oropharyngeal squamous cell carcinomas that overexpress HIF-1 alpha 131 Chapter 7 HIF-1a expression and differential effects on survival in patients with oral cavity, larynx, and oropharyngeal squamous cell carcinomas. 155 Chapter 8 Summary discussion and possibilities for future research 181 Chapter 9 Nederlandse samenvatting Lijst van publicaties Dankwoord Over de auteur 191 199 200 202
Chapter 1 General introduction
Chapter 1 | 8 Head and neck cancer Head and neck squamous cell carcinoma (HNSCC) comprises carcinomas arising from the epithelium of the upper aerodigestive tract (Figure 1). The most important risk factors for developing HNSCC are smoking and alcohol use.1,2 Recently, the human papillomavirus (HPV) has been established as another, separate risk factor for oropharyngeal squamous cell carcinoma. As the head and neck region is important both functionally and esthetically, the potential effects for the patients by both the tumor and the treatment are considerable. Anatomy Several sites are distinguished within the head and neck region. The oral cavity includes the buccal mucosa, mobile tongue, upper and lower gingiva, the hard palate and the floor of the mouth. The dorsal third of the tongue is considered the base of the tongue and belongs to the oropharynx, along with the vallecula, tonsils, tonsillar fossa and pillars, inferior surface of the soft palate, uvula and posterior from the level of the junction between hard and soft palate cranially until the level of the hyoid bone (or tip of the epiglottis) caudally. 3,4 Figure 1 | The anatomy of the head and neck region. Image reused from the book “Anatomy and Physiology” by OpenStax.3,4
General introduction | 9 Below the oropharynx is the hypopharynx, that includes the piriform sinuses, post-cricoid region and its posterior wall. The voice box, or larynx, separates the airway from the ‘food pipe’ or esophagus and is divided in a supraglottic, glottic and subglottic region. The supraglottis consists of the ventricular folds, the laryngeal ventricle, the arytenoids, epiglottis and aryepiglottic folds. The glottis consists of the vocal cords and the subglottis is the area caudally from 1 cm below the vocal cords. Above the oropharynx is the nasopharynx, and the region anterior to the nasopharynx (separated by the choana) is considered the nasal cavity. Epidemiology With an incidence of around 3000 new cases in the Netherlands, HNSCC is on the 8th place of most common cancers in men and 9th place in women and the incidence has risen in the last years (Figure 2).5 HNSCC is mainly located in the oral cavity (OSCC, 36.5%), followed by larynx (LSCC, 29.4%) and oropharynx (OPSCC, 10.7%).6 The stage at the time of diagnosis can be divided into early, or local disease (stage I-II) or advanced disease, which includes locally advanced disease and locoregional disease (stage III- IVB). OSCC and LSCC are often diagnosed in stages I/II, but OPSCC and hypopharyngeal squamous cell carcinoma (HPSCC) are mostly diagnosed in stages III or IV. In the case of distant metastasis the stage is IVC. 0 500 1000 1500 2000 2500 3000 1989 1991 1993 1995 1997 1999 2001 2003 2005 2007 2009 2011 2013 2015 2017 2019 2021 Patients (n) Year of diagnosis Newly diagnosed patients with a SCC in one of the three major sites Larynx Oropharynx Oral cavity Figure 2 | Incidence of OSCC, LSCC and OPSCC in the Netherlands 1989 – 2021. Data from 2020 and 2021 are still preliminary. Figure based upon data from the Dutch Cancer Registry.5 1
Chapter 1 | 10 The prognosis of patients with HNSCC is relatively poor, and differs per site and disease stage at diagnosis. In recent years, advances in therapy have improved the prognosis and survival of patients only minimally.6 In the Netherlands, 5-year survival rates of the three large subcategories have increased from 57% in the period 1991-2000 to 62% in the period 2011-2019 for patients with OSCC. For OPSCC the increase is from 36% to 52%. The survival of LSCC has remained stable around 68%. Current treatment paradigm Regular treatment options for HNSCC generally include surgery, and organ-preservation treatment in the form of radiotherapy. In some cases of advanced disease, platinum based chemotherapy is added as a radiosensitizer.7 When the patient is not fit for chemotherapy and in HPV-negative disease the monoclonal antibody against the epidermal growth factor receptor (EGFR) cetuximab may be used instead.8 Generally, performing single-modality treatment with surgery or radiotherapy is pursued in stages I and II and selected cases of stage III disease. However, in most advanced cases, multimodality treatment is required. This could be surgery followed by adjuvant (chemo)radiotherapy or primary radiotherapy with platinum-based chemotherapy and salvage surgery in reserve for residual disease. The choice of therapy is based on the disease extent, the disease site and the expected loss of function by the therapy. Several landmark trials, such as the Vetaran Affairs Laryngeal Cancer Study Group trial, have established chemoradiation as an alternative with a higher chance of organ preservation and comparable survival in some, but not all cases.9–11 Novel treatment paradigms Would it not be more logical that the choice of the treatment of cancer patients is not based on the location of the tumor but on its biological properties? In the 1980’s researchers have already tried to culture human tumors in a comparable way to bacteria.12 The goal was to establish in vitro which chemotherapeutic drug would be most successful at eradicating the tumor. Unfortunately, this approach has not found its way into the clinic yet in this exact form as it was hard to culture tumor tissue in vitro.13 Moreover, the correspondence between in vitro and in vivo treatment outcomes was not sufficient. Promising results have been reported by testing chemotherapeutic drugs and irradiation on organoids derived from HNSCC patients, although this technique is not yet ready to predict treatment outcome in clinical practice.14 One important discovery toward tumor biology-based treatment choice concerns the HPVrelated (HPV+) OPSCC. This subgroup of OPSCC is a distinct subgroup of tumors with a favorable prognosis compared to smoking and drinking-related OPSCC.15 In trials and retrospective cohorts, patients with HPV+ OPSCC have a much better prognosis compared
General introduction | 11 to patients with non HPV-related (HPV-) tumors.16 Therefore trials are currently ongoing to see whether treatment de-intensification can be safely performed in patients with HPV+ tumors.17 The distinction in HPV+ from HPV- OPSCC has been recently introduced in the 8th TNM classification as well. As an example, since TNM8 some tumors that may be considered T1N3b (stage IVB) when HPV- are considered T1N1 (stage I) when HPV+, illustrating a beneficial prognosis for HPV+ tumors.18 Another important change concerned the view on tumor biology. In another landmark paper, Hanahan and Weinberg proposed that tumors should not be viewed as a lump of homogenic tumor cells (“The Reductionist View”), but as a heterogeneous microenvironment of tumor cells, cancer stem cells, immune cells, blood vessels, stroma and other cell types (“A Heterotypic Cell Biology”, Figure 3).19,20 Some of the factors in the microenvironment interact to improve survival and therapy resistance and were termed hallmarks. These hallmarks may be exploited singular or in combination with novel therapies (Figure4). One emerging therapy that exploits such a hallmark and has already found its way into clinical practice is immunotherapy, which will be further elaborated later on in this chapter. As hypoxia plays a part in several of these hallmarks, such as resisting cell death, avoiding immune destruction and inducing angiogenesis, targeting hypoxia would result in targeting multiple hallmarks at once. Figure 3 | Comparison of the 'reductionist' view and the ‘heterotypic tumor cell biology’ as proposed by Hanahan and Weinberg. Image from Hanahan and Weinberg.19 1
Chapter 1 | 12 Tumor hypoxia Hypoxia is a characteristic of many solid tumors.21 By definition it is a mismatch between oxygen supply and demand. There is a distinction between acute (or: perfusion-limited) and chronic (or: diffusion-limited) hypoxia, although both forms may co-exist within tumors.22 Perfusion-limited (acute) hypoxia Acute hypoxia, or perfusion-limited hypoxia, is usually a temporary state that occurs because of compromise of blood supply to the tissue. Oxygen homeostasis is an important part of homeostasis in general. Therefore, mechanisms exist to counteract acute and chronic hypoxia. For acute hypoxia, several mechanisms are present in different parts of the body to cause vasodilation in the case of hypoxic circumstances.23Systemic hypoxia may be detected in arterial blood by the glomus cells in the carotid body, leading to systemic vasodilation. Figure 4 | The “Hallmarks of Cancer” model by Hanahan and Weinberg with illustrative examples of therapies directed at these hallmarks. Image from Hanahan and Weinberg. 20
General introduction | 13 Perfusion-limited hypoxia may occur locally for instance when afferent blood vessels are temporarily compressed (due to external pressure, positioning) or clamped (iatrogenic). In a smaller scale within tissues or within a tumor, perfusion-limited hypoxia is thought to arise primarily from vascular stasis (by vascular collapse, occlusion by tumor cells or leukocytes), flow instabilities or increased interstitial fluid pressures. 24–26 Diffusion-limited (chronic) hypoxia Diffusion-limited hypoxia is a state where the distance between cells and the nearest blood vessels is too large for sufficient amounts of oxygen to diffuse to these cells. This may arise because a tumor cell outgrows it vascular supply. Moreover, because tumors often exhibit inefficient, chaotic vascular patterns, the distance of a cell to the nearest blood vessel may also be quite variable. Cells with increased distance to the nearest blood vessel are therefore exposed to increased levels of hypoxia. Transient hypoxia While theoretically this distinction between acute and chronic hypoxia holds true, in vivo, the distinction is less obvious.24 In fact perfusion-limited and diffusion-limited hypoxia may coexist within the same tumor because of the suboptimal vascular patterns.27 Moreover, hypoxia may also be transient and this variant may possibly be most relevant clinically. A simulation study showed that as much as 25% of a tumor region may temporally fluctuate above and below a certain threshold of hypoxia.28 Hypoxia and treatment resistance Hypoxia is a common trait of solid tumors as irregular, exponential growth of tumor cells leads to a tumor outgrowing its own vascular supply causing diffusion-limited hypoxia. As a consequence, the stimulation of angiogenesis by factors such as VEGF lead to the formation of vessels with suboptimal architectures, causing perfusion-limited hypoxia.27,29 Thus, both these forms of hypoxia may co-exist within a single tumor (Figure 5). Clinically, hypoxia has been known to decrease sensitivity to anti-tumor treatment. The most well-known treatment effects are described in radiotherapy.22 It has been established in 1935 that benign or malignant hypoxic tissues are less sensitive to radiation than normoxic tissue and that the decrease in radiosensitivity is around three-fold.30–32 Partly this is attributed to a direct effect of radiation on oxygen: oxygen is required for the formation of free radicals to induce double strand DNA breaks.21 The same mechanism of reduced generation of free radicals leads to treatment resistance for some forms of chemotherapy (including bleomycin and doxorubicin) and photodynamic therapy.33 The critical O2 tension (pO2) below which radiotherapy resistance occurs is around 25-͵Ͳ ͲǤͷ Ǥ ǡ in the human body is 90 +/-5 mmHg, the mean venous is 40+/- 5 mmHg and the 1
Chapter 1 | 14 brain tissue lies between 30 and 48 mmHg.34 ǡ ȋ mucosa) is already below the critical level for radiotherapy resistance with a mean of 8 mmHg. Apart from this direct effect of hypoxia in inducing therapy resistance, there is also an indirect effect by changes in protein expression by hypoxic cells. Hypoxia leads to accumulation of the transcription factor Hypoxia-Inducible Factor 1-alpha (HIF-1a), leading to transcription of its downstream targets. Many of these downstream targets are part of mechanisms to survive under these hypoxic conditions. These include for example proteins involved in glycolysis, cell cycle regulation, angiogenesis and hemopoiesis.35 Measuring tumor hypoxia As hypoxia leads to treatment resistance, there is a need to determine the hypoxic status of a tumor. Measuring hypoxia in solid tumors is challenging and several methods are available. These include measurements using polarographic needles, exogenous and endogenous markers of hypoxia, hypoxic gene signatures, hypoxia-based PET-tracers and diffusion weighted MRI. Eppendorf histography Tissue oxygen tension may be assessed using electrode measurements.36 A needle is placed electrode. The needle is inserted into the tumor and retracted in increments through the tumor, creating several measurements and giving a three- Figure 5 | Perfusion-limited versus diffusion limited hypoxia. Example of perfusion limited hypoxia caused by irregular vessel formation (A), normal vascularization (B) and diffusion limited hypoxia as a result of insufficient vascularity (C). Image from Codony et al.27
General introduction | 15 within the tumor.37 Gatenby and colleagues performed this procedure in a cohort of 31 ͳͲ mmHg were more likely to be non-responders to radiation therapy.38 While this method ǡ invasive nature of the procedure is not preferable for use in clinical practice and not all tumor sites are easily accessible to perform these measurements. Exogenous hypoxia markers When (hypoxia-induced) radiation resistance was identified, several drugs were investigated for their radiosensitizing properties.39 These included antibacterial and antifungal drugs. The class of 2-nitroimidazole antibiotic drugs was shown to have radiosensitizing properties and these drugs were able to diffuse homogeneously through hypoxic tissue while oxygen was not. Other experiments showed that the 2-nitroimidazole drug misonidazole was bound by cells in hypoxic circumstances, specifically to thiolcontaining proteins in cells, in particular when exposed to ͳͲ g.40–43 The hypoxia marker pimonidazole may be administered to patients intravenously prior to biopsy. The presence of pimonidazole is then detected using immunohistochemistry. Endogenous hypoxia markers All nucleated cells in the body respond to hypoxia through a local cellular response.44 As hypoxia induces a cellular and transcriptional response, the proteins that are increased under hypoxia may also be detected as hypoxia markers, using immunohistochemistry, immunofluorescence or alternative methods. As these proteins are endogenous to the body they are considered endogenous hypoxia markers in contrast to exogenous markers that have to be administered to patients externally. This is a great advantage of endogenous hypoxia markers especially for (retrospective) research purposes. The best known endogenous hypoxia markers are discussed. HIF-1a The best described cellular hypoxia response mechanism is the Hypoxia-Inducible Factor 1 (HIF-1) pathway (Figure 6).45 HIF-1 is a transcription factor, existing of an HIF-1 alpha (HIF1a) and HIF-1 beta (HIF-1b or Aryl Hydrocarbon Receptor Nuclear Translocator or ARNT) subunit. The cellular concentration of HIF-1a is oxygen dependent; it is constitutively expressed, but under normoxic circumstances it is quickly degraded by prolyl hydroxylases 1-3 (mostly PHD2) and further ubiquitinated by the Von Hippel-Lindau (VHL) protein.46 The hydroxylation process is O2 dependent and therefore degradation of HIF-1a is reduced under hypoxic circumstances. As a transcription factor, HIF-1 attaches to hypoxia-response elements (HREs) in the DNA leading to increased transcription of its downstream targets. This HRE is characterized by 1
Chapter 1 | 16 the pattern of RCGTG as a constant factor of the binding site.47 Moreover, multiple genes are downregulated in a HIF-dependent manner without binding of HIF to these genes.48 HIF-1a may be detected using immunostaining and is therefore considered an endogenous marker of hypoxia. While HIF-1a accumulation under hypoxia has well been established, there is debate on the speci icity of HIF-1a accumulation for hypoxia. Several studies have shown only weak correlations of HIF-1a to pimonidazole or Eppendorf pO histography.49–51 Moreover, intracellular HIF-1a concentrations may also increase as a result of oncogene gain or tumor suppressor gene loss of function.52 As HIF-1a is a transcription factor, immunohistochemical staining is observed mainly in nuclear cellular compartments. Staining in other compartments such as the cytoplasm and membranous HIF-1a staining have been observed and used in studies as a hypoxia marker. However, the biological relevance of such staining is debatable. The effect of HIF-1a accumulation on clinical outcome has been established in several studies in HNSCC.22 Interestingly, these studies report several different staining patterns for Figure 6 | Regulatory mechanism of HIF-1a. HIF-1a is constitutively expressed, but is hydroxylated and ubiquitinated under normoxic circumstances (top) by prolyl-hydroxylases 1-3 (mostly 2) and VHL, respectively, and degraded. Because this process is oxygen-dependent, under hypoxia (bottom) HIF-1a accumulates and binds to HIF-1b. Together with transcription co-activators such as p300/CBP this HIF1 complex induces transcription of its target genes. O2 O2 O2 O2 O2 HIF-1a Normoxia Hypoxia O2X PHD2 PHD3 PHD1 PHD2 PHD3 PHD1 X HIF-1a OH OH HIF-1a OH OH VHL ubi ubi ubi Proteasomal degradation Proteasomal degradation Transcription of target genes Nucleus HIF-1a HIF-1b HIF-1b HRE (RCGTG) HIF-1 target gene HIF-1a HIF-1b p300/CBP
General introduction | 17 HIF-1a: perinecrotic staining patterns, as well as diffuse staining patterns.53–55 In clear cell renal cell carcinoma degradation of HIF-1a is often inhibited because of a mutation in VHL. This genetic mutation results in diffuse HIF-1a staining.56,57 Because of this, RCC is often used as a positive control in studies with immunohistochemical analysis of HIF-1a. It is argued that perinecrotic staining patterns of HIF-1a may be an indication of tumor hypoxia, while diffuse staining can be induced by oncogenic activation. A perinecrotic staining pattern was associated with poor patient outcomes compared to diffuse HIF-1a staining patterns in a study in breast cancer.53 Interestingly, similar results were not found in a cohort of patients with radiotherapy-treated OPSCC.55 CA-IX Hypoxic circumstances lead to acidic environments, based on the lack of oxygen and a switch to a glycolytic metabolism. Regulation of the acidity is crucial to cellular survival. Carbonic Anhydrases (CAs), specifically the subtype CA-IX, is a membrane protein involved in regulation of acidity under hypoxia.58 It hydrates pericellular CO2 to bicarbonate ions and protons. These bicarbonate ions are then actively transported inward through bicarbonate transporters to neutralize intracellular protons. It has been shown that CA-IX (and to a certain extent also CA-XII) is regulated by hypoxia and that it colocalizes with pimonidazole in some skin and bladder carcinomas.59 In a study in HNSCC this colocalization to pimonidazole expression could not be confirmed.60 Therefore the specificity of CA-IX for hypoxia may also be debated. A positive correlation with HIF-1a staining is not always observed. A possible explanation is that CA-IX expression may be a better reflection of the transcriptional activity of HIF-1a, rather than hypoxic HIF1a accumulation. GLUT-1 Another downstream target of HIF-1a is the glucose transporter 1 (GLUT-1) protein. As mentioned above, the glycolytic metabolism of hypoxic cells increases the need for glucose. HIF-1a increases GLUT-1 and GLUT-3 transcription. Some studies show a correlation between pimonidazole staining and GLUT-1, while others do not.60,61 Osteopontin Osteopontin (OPN) is a protein that is upregulated under hypoxia through a HIFindependent pathway. Overexpression of OPN involves a Ras-activated enhancer and is dependent on Akt.62 Interestingly, OPN has also been shown to increase intracellular HIF-1a through a PI3K-Akt related pathway.63 OPN overexpression in tissue is observed in cytoplasm and also extracellularly secreted.64 In fact, plasma OPN levels are also often investigated as hypoxia markers as an alternative to immunohistochemical staining in tissue.65 1
Chapter 1 | 18 Osteopontin overexpression leads to increased cellular survival, invasion and angiogenesis, which are all properties that are favorable under hypoxic circumstances. Moreover, OPN overexpression is associated with distant metastasis in various tumor types.66 A note on endogenous and exogenous markers While ideally a marker indicates the presence or absence of a certain trait, identifying a single marker for hypoxia is challenging.67 The co-existence of acute, chronic and transient hypoxia within the same tumor is one of the major challenges for the identification of such a marker. While pimonidazole is considered a ‘gold standard’ in many (preclinical) studies, it is of note that pimonidazole binding occurs only after 30 minutes after injection and there is a plateau phase after 6-8 hours in pimonidazole binding.28,68 Moreover, the administration of pimonidazole to patients in a clinical setting in a set time before taking a biopsy may be logistically challenging. To identify transient hypoxia, one study investigated xenograft models where mice were injected with pimonidazole every hour for 8 hours (t1/2 of pimonidazole in mice: 30 minutes).28 In essence, the goal was to ‘saturate’ these mice in pimonidazole to ensure that all hypoxic regions were stained. One hour after the final pimonidazole injection, an injection with another hypoxia marker of the 2-nitroimidazole class (CCI-103F) was administered. There were some tumor cells labeled positive for CCI-103F while negative for pimonidazole. This suggests that the cells were not hypoxic during the 8 hour period of pimonidazole labeling but were hypoxic at the time of CCI-103F labeling. This indicates the presence of areas of transient hypoxia within a tumor. In contrast, HIF-1a concentrations may rise as fast as only two minutes under hypoxia, and may decrease again after 30 minutes of re-oxygenation.69 For pimonidazole binding, the required time of hypoxia is longer and the binding is permanent and therefore maintained even after reoxygenation.70 CA-IX expression will occur even later than HIF-1a accumulation, as it requires the step of transcription under the influence of HIF-1a.71 Even after re-oxygenation and degradation of HIF-1a, CA-IX may still be expressed by the tumor. CA-IX is therefore a sign of HIF-1a’s transcriptional activity, rather than its accumulation or overexpression. This explains that there may be a poor correlation between HIF-1a and CAIX expression.60 Therefore, the challenges in all markers for hypoxia, both endogenous and exogenous, is not only their sensitivity and specificity for hypoxia, but the fact that they may be visualized after different durations of hypoxia. The presence of transient hypoxic areas further complicates this issue. It is therefore necessary to return to the clinical question: which hypoxia markers are able to distinguish patients with a good and a poor prognosis and which hypoxia markers aid in the selection of hypoxia-modified treatment adaptations.
General introduction | 19 Hypoxia gene signatures Alternative to investigating hypoxia on a protein-based level by the use of biomarkers, several hypoxia gene-signatures have been investigated for their ability to classify hypoxic versus non-hypoxic tumors.72 They are based on an altered, hypoxia-induced, transcriptional response not only of HIF-1a target genes but also others. In these studies, gene selection is performed in vitro in tumor cell lines. The gene expression patterns of cell lines cultured under normoxic and hypoxic circumstances are compared. Genes are then included or excluded from the final ‘classifier’ gene signature for their effect on patient prognosis.73,74 While such classifiers carry prognostic value, it does not automatically translate to a classifier able to predict the response to hypoxia modification of treatment. In one study, not only the hypoxic status, but the pH was altered in such an in vitro experiment, as some hypoxia-regulated genes may also be regulated by pH.75,76 This hypoxia gene-signature has also been shown to have predictive value for the benefit from hypoxic modification of therapy in the form of nimorazole addition to radiotherapy. Another genesignature is predictive of the benefit from hypoxic modification of radiotherapy by accelerated radiotherapy, carbogen and nicotinamide (ARCON) therapy in laryngeal cancer patients.77 PET-CT Positron-emission tomography and computed tomography (PET-CT) is an imaging modality used to visualize certain tissue characteristics. The most commonly used PET-tracer is 2deoxy-2-[18F]fluoro-D-glucose ([18F]FDG). Although expression and/or activity levels of GLUT1 contribute to the pattern and intensity of [18F]-FDG expression, PET-CT using this tracer cannot predict expression of clinically relevant histopathological hypoxia biomarkers, e.g. HIF-1a, in HNSCC.78 The use of specific hypoxia tracers is an alternative way to determine tumor hypoxia in vivo. One benefit of the use of hypoxia-PET-CT is that it provides an overview of the hypoxic status throughout a whole tumor, rather than only on a single biopsy and thus only a part of the tumor. This is important particularly because of heterogeneity in oxygen levels within a single tumor that may be present. There are currently several specific hypoxia PET-tracers available.79,80 A number of these tracers are from the same 2-nitroimidazole class as the exogenous hypoxia marker pimonidazole, such as [18F]-FMISO, [18F]-FAZA and third-generation 2-nitroimidazole drug [18F]-HX4. Uptake of these markers in relation to hypoxia has been shown to be reasonable, however there are some issues with PET-CT based hypoxia detection. One of these issues is the spatial resolution: the voxel size of modern clinical PET scanners is around 4-6 mm, while hypoxia may occur on length scales of around 100 μm.80 Also, uptake of these tracers 1
Chapter 1 | 20 is limited to vital cells and is not observed in areas of necrosis. Therefore, areas of necrosis may influence tracer uptake and may hinder an accurate visualization of hypoxic regions. Despite these shortcomings, there is some (albeit limited) evidence of clinical relevance. It has been demonstrated that high uptake of hypoxia-PET tracers is associated with poor survival in HNSCC.81–84 The study by Rischin et al described that hypoxia in [18F]-MISO-PET was a predictive biomarker for response to the hypoxic cytotoxin tirapazamine.85 Other studies on hypoxia PET-CT for response prediction to hypoxia-modified treatments are currently not available. Diffusion-weighted MRI Diffusion-weighted magnetic resonance imaging (DW-MRI) is an MRI sequence that relies on the diffusion of water particles within the tissue.86 By acquiring at least two images with different motion probing gradients, i.e. two different b-values, the apparent diffusion coefficient (ADC) is provided. The ADC is relevant to determine tissue characteristics: a low ADC, corresponding to restricted movement of water particles, is associated with high cellularity and higher nuclear-cytoplasmic ratios.87 Because the DW-MRI describes the microstructure of a tumor, it may also be correlated to hypoxia: hypoxic areas may contain more necrosis, causing less restricted diffusion of water particles. Interestingly, a study comparing ADC to [18F]-MISO uptake showed a lower ADC in hypoxic subvolumes, compared to non-hypoxic subvolumes.88 Another study compared hypoxia determined in biopsies using Blimp-3, a HIF-1a transcription target, as a hypoxia biomarker and also found lower ADCs in more hypoxic pancreatic carcinomas.89 This was also confirmed in a study that investigated ADC versus pimonidazole staining in wholemount sections of prostate cancer.90 The authors assume that the presence of chronic hypoxia leads to more aggressive phenotypes and therefore higher cellularity. The latter was also observed in a preclinical animal study.91 As the ADC reflects the tissue microarchitecture, which is composed of many different variables including cellularity and other features of the tumor microenvironment, such as hypoxia, the relation between these two parameters should be further explored. Hypoxia and immunity in the tumor microenvironment The transcriptional activity of HIF-1a promotes a beneficial environment for cellular survival. An acidic tumor environment and a glycolytic metabolism make an hypoxic microenvironment an excellent environment for survival and thriving of tumor cells. The hypoxic environment also influences the antitumor immune response. This occurs because of a two-fold mechanism: the hypoxic cellular response of tumor cells results in the release of immunosuppressing cytokines and the lack of oxygen itself also causes HIF-1a stabilization in immune cells and adapts their response.
General introduction | 21 Effects of HIF-1a stabilization in immune cells HIF-1a expression occurs in immune cells through oxygen-dependent and oxygenindependent pathways and has both immunosuppressive and pro-immunogenic properties at the same time.92,93 Activation of toll-like-receptors in myeloid cells or T-cell receptors in T-cells lead to increased HIF-1a synthesis through NF-ɈB and PI3K related pathways respectively. For T-cells, the activation of HIF-1a is beneficial when activated, as activated T-cells rely more on glycolytic metabolism than naïve T-cells. In contrast, HIF-1a activation in hypoxic microenvironments also increases shift of naïve T-cells to regulatory T-cells, which will have an immunosuppressing effect. Finally, while expression of HIF-1a increases the lytic capabilities of cytotoxic (CD8+) T-cells, it also delays the T-cell differentiation into CD8+ T-cells.94,95 Immunomodulation by tumor cells Hypoxic tumor cells are also able to attract macrophages (tumor-associated macrophages or TAMs). The hypoxic microenvironment shifts TAMs from an activated M1 phenotype toward an M2 phenotype that suppresses the tumor-immune reactions, causing tumors to evade immune reactions.94,96 Moreover, TAMs increase endothelial and malignant cell proliferation, increase survival under chemotherapy and promote metastasis.97–99 Another transcription target of HIF-1a is the programmed death-ligand 1 (PD-L1), which is part of an immune checkpoint together with the programmed death-receptor 1 (PD-1) on immune cells.100 Increased upregulation of PD-L1 by tumor cells causes inhibition of the antitumor immune response. Immune therapy that blocks the PD-1/PD-L1 binding through antibodies directed against either of these proteins is currently being investigated and used in clinical practice in many solid tumors including HNSCC.101 It has recently been shown that PD-L1 may also be regulated under hypoxia through HIF-1a, causing immune evasion by inhibiting killing by cytotoxic T-cells or natural-killer T-cells.102 In summary, HIF-1a is upregulated under hypoxic circumstances in immune cells, which has both immunosuppressive and pro-immunogenic effects. However, tumor cells also gain immunosuppressive properties under hypoxia that suppress the immune system and prevent tumor cell killing. Overcoming hypoxia: treatments targeting hypoxia or HIF-1a Several strategies have been investigated to overcome tumor hypoxia and increase treatment susceptibility of hypoxic tumors.103 While a review has shown a statistically significant improvement in patient outcome, hypoxia-modified treatments are currently not a part of clinical routine.104 Efforts to improve tumor oxygenation started with application of normobaric oxygen and later hyperbaric oxygen. However, this was abandoned as it was 1
Chapter 1 | 22 difficult to apply logistically in clinical care and a review has shown insufficient evidence of effect.104 Currently other approaches are being investigated. ARCON The concurrent application of radiotherapy in combination with breathing of carbogen is termed ARCON and is a method to increase tumor oxygenation.105The breathing of carbogen gas, a mixture of 2% CO2 and 98% oxygen, increases oxygenation not only by increased oxygen supply, but also by increasing the respiratory drive and vasodilation because of the CO2. Nicotinamide is an amide derivative of vitamin B3 and has radiosensitizing properties, but also decreases intermittent vascular shutdown and therefore decreases perfusionlimited hypoxia.105,106 A randomized trial in stage II-IV LSCC patients of accelerated radiotherapy (AR) versus ARCON has shown an overall benefit in regional control, but not local control or overall survival. 107 Radiosensitizer application The best described radiosensitizing drug as a hypoxia-radiosensitizer is nimorazole, a drug of the 2-nitroimidazole class.108 Drugs of the 2-nitroimidazole class mimic oxygen under hypoxic circumstances, thus enabling the formation of DNA strand breaks during radiotherapy (Figure 7).109 While it has been shown to be effective in a trial in Denmark, an international trial on its effectiveness was shut down early because of recruitment issues.110 Other strategies to overcome hypoxia and HIFs Other current efforts include the application of hypoxia-activated pro-drugs, and drugs that improve the efficiency of the vascularity using angiogenesis-inhibiting agents of vascular disrupting agents.111 Although these agents disrupt tumor vascularity, they are thought to Figure 7 | Example of oxygen-mimicking. Radiation induces reactive oxygen species that bind to the DNA. In order for DNA strand breaks to occur, oxidation of these DNA radicals is required. This may be achieved by binding oxygen or an oxygen mimicking molecule, such as nimorazole (shown in blue). Imaged from Jackson et al.109
General introduction | 23 also improve vascularity by primarily targeting the inefficient vessels involved in areas of perfusion limited hypoxia as shown in Figure 5.112 Another strategy to overcome the effects of hypoxia is by direct inhibition of HIF-1a. Several drugs are currently being investigated in phase II or III trials.27,113 Conclusion In conclusion, hypoxia is a well-known adverse micro-environment factor in a tumor’s response to therapy, tumor aggressiveness and metastasis. Moreover, there is an interaction between hypoxia and other factors within the tumor microenvironment including immunity. There are existing and promising new therapies that may reduce tumor hypoxia or specifically target hypoxic tumor cells, and these therapies have greater effect when patients with hypoxic tumors are preselected. Therefore there is a need to further investigate the role of hypoxia and the role of HIFs in HNSCC, in particular in relation to the tumor microenvironment, and to find a reliable biomarker to determine the hypoxic status of a tumor to assess eligibility for hypoxia-modified or targeted treatment. Aim of this thesis This thesis focuses on the role of hypoxia and more specifically HIF-1a in relation to the tumor microenvironment and patient outcomes in HNSCC. In Chapter 2 we investigate the role of HIF-1a as a hypoxia marker in correlation to pimonidazole as a reference standard. In Chapter 3 we compare the expression of HIF-1a as a hypoxia marker to PD-L1 expression and tumor-infiltrating lymphocytes, as two other factors within the tumor microenvironment in an OPSCC patient cohort. In Chapter 4 we compare tumor and tumor microenvironment factors to imaging features in DW-MRI to see whether this modality is able to determine the hypoxic status in OPSCC patients. In the following chapters, we investigate the effect of hypoxia on outcomes in HNSCC patients. In Chapter 5 we investigate the literature for the clinical relevance of endogenous biomarker expression for patients treated with regular (non-hypoxia modified) therapy. In Chapter 6 we investigate the role of HIF-1a overexpression in patients with HPV-positive versus HPV-negative OPSCC. In Chapter 7 we compare HIF-1a expression and clinical outcome in patients with tumors from different sites in the head and neck. Chapter 8 summarizes the findings of these studies and provides a view on future perspectives. 1
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