Martine De Herdt

TO SHED OR NOT TO SHED? MET immunoreactivity as biomarker in oral squamous cell carcinoma Maria Jantine De Herdt

To Shed or Not To Shed? MET immunoreactivity as biomarker in oral squamous cell carcinoma Maria Jantine De Herdt

Colofon To Shed or Not To Shed? MET immunoreactivity as biomarker in oral squamous cell carcinoma Author: Maria Jantine De Herdt ISBN: 978-94-6483-115-3 Cover lay-out and design: Erwin Timmerman, Printing: Ridderprint, Copyright © 2023 M.J. De Herdt, Rotterdam, The Netherlands All rights reserved. Save expectations stated by law, no part of this thesis may be reproduced, stored in a retrieval system of any nature, or transmitted, in whole or in part, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, included in a complete or partial transcription, without the written permission of the author, or – when appropriate – of the publishers of the presented publications.

To Shed or Not To Shed? MET immunoreactivity as biomarker in oral squamous cell carcinoma Shedding of geen shedding? MET immunoreactiviteit als biomarker in plaveiselcelcarcinomen van de mondholte Proefschrift ter verkrijging van de graad van doctor aan de Erasmus Universiteit Rotterdam op gezag van de rector magnificus Prof. dr. A.L. Bredenoord en volgens besluit van het College voor Promoties. De openbare verdediging zal plaatsvinden op dinsdag 4 juli 2023 om 13.00 uur door Maria Jantine De Herdt geboren te Naarden.

Promotiecommissie: Promotoren: prof. dr. R.J. Baatenburg de Jong prof. dr. L.H.J. Looijenga Overige leden: prof. dr. R. Fodde prof. dr. C.L. Zuur prof. dr. E.L.J. Smits Copromotoren: dr. J.A. Hardillo prof. dr. S. Koljenović

Contents Chapter 1 General introduction 7 Chapter 2 Context, background, aims, and outline of the thesis 27 Chapter 3 Absent and abundant MET immunoreactivity is associated with poor prognosis of patients with oral and oropharyngeal squamous cell carcinoma 39 Chapter 4 MET ectodomain shedding is associated with poor disease-free survival of patients diagnosed with oral squamous cell carcinoma 83 Chapter 5 A novel immunohistochemical scoring system reveals associations of C-terminal MET, ectodomain shedding, and loss of E-cadherin with poor prognosis in oral squamous cell carcinoma 139 Chapter 6 The potential of MET immunoreactivity for prediction of lymph node metastasis in early oral tongue squamous cell carcinoma 187 Chapter 7 The occurrence of MET ectodomain shedding in oral cancer and its potential impact on the use of targeted therapies 209 Chapter 8 General discussion, conclusion, and future perspective 229 Chapter 9 Summary 251 Chapter 10 Samenvatting 259 Addendum List of used abbreviations, gene symbols, and terms 271 PhD portfolio 274 List of publications 277 Acknowledgements 280 Curriculum vitae 283

CHAPTER 1 General introduction

9 General introduction Head and neck squamous cell carcinoma Head and neck cancers (HNCs) are a diverse group of malignant tumors that arise in the oral cavity, oropharynx, hypopharynx, or larynx (Figure 1A). In 2020, HNC accounted globally for an incidence of approximately 750,000 cases (+/- 40,000 in Western Europe), and over 350,000 deaths (+/- 15,000 in Western Europe). These numbers made HNC the seventh most common cancer worldwide, accounting for around 3.6% of all cancer related deaths that year. Approximately 90% of HNCs are squamous cell carcinomas (HNSCCs) that originate in the epithelial cells forming the mucosal linings of the upper aerodigestive tract. Despite the fact that HNSCCs originate from one cell type residing in one tissue, these cancers behave surprisingly heterogeneous. This can be explained by differences in anatomical localisations, aetiologies, and the variety of molecular changes underlying this disease (1-3). Figure 1: Anatomy of head and neck cancer (HNC). A. Anatomical sites in which HNCs arise. B. Anatomical subsites of the oral cavity in which oral cancers arise. Oral squamous cell carcinoma Over 40% of HNSCC arise in the oral cavity (OSCC), classically presenting with a chronic sore or ulcer (2, 4). The oral cavity encompasses complex functional anatomy that facilitates speech, swallowing, and facial projection. The oral cavity contains the following anatomical subsites: mucosal lip, oral tongue, floor of mouth, mandibular and maxillary gingiva, retromolar trigone, buccal mucosa, and hard-palate subsites (Figure 1B) (5). 1

10 Chapter 1 Overall, 5-year survival for oral cancer (OC) patients lies between 50% and 70% depending on the subsite (Figure 2) (6). Additionally, it varies depending on: disease stage at the time of diagnosis (7), sex, race/ethnicity, and age (Figure 2) (6, 8). Frequently, OCs are diagnosed at an early stage, as patient’s observe the tumor mass and experience symptoms that interfere with eating, speaking. Risk factors associated with environmental carcinogenesis, such as tobacco, alcohol, areca nut, betel quid, and poor dentition, should increase clinical suspicion for OSCC (9, 10). Figure 2: Ten-year relative survival for oral cancer patients per subsite and disease stage according to the Surveillance, Epidemiology, and End Results Program (6).

11 General introduction In contrast to pharyngeal and laryngeal SCC for which concomitant postoperative platinum-based chemo- and radiotherapy (CRT) is the primary treatment approach, the management of OSCC predominantly encompasses surgery with or without adjuvant therapy (11). For this reason, OSCC wil be the main focus of this thesis. Clinical diagnosis Clinical examination – assessing risk factors and co-morbidities – as well as physical examination are part of the initial work-up of patients suspected to have OSCC. Palpation and preoperative imaging are critical to ascertain clinical tumor stage and guide decision making with respect to resection. By evaluating the extent/presence of the primary tumor, and/or regional disease, and/or metastatic disease, and/or synchronous secondary primaries, preoperative imaging helps in determining the feasibility of surgery and whether it can be performed with curative intent. Preoperative imaging involves either computed tomography (CT), magnetic resonance imaging (MRI), or [18F]-Fluorodeoxyglucose positron emission tomography (FDGPET) (3, 5, 12). Final diagnosis of OSCC is established through histopathological evaluation of biopsies (9). Tumor-node-metastasis classification Tumor stage is considered to be the major determinant of prognosis in North America and Western Europe (9). Histopathological diagnosis and tumor-node-metastasis (TNM) classification are necessary prerequisites for the clinical management of OSCC (13). Tumor-node-metastasis staging, evaluates the extent of tumor growth across a patient’s body using size of the primary tumor (T category), involvement of regional lymph nodes (N category), and presence of distant metastasis (M category). Besides recording the anatomical spread of cancer, the TNM staging system is used to stratify cancers into stage groups in view of clinical management (13). The most recent edition (eight) of the American Joint Committee on Cancer (AJCC) Cancer Staging Manual was implemented January 1st 2018. In contrast to the 7th edition it incorporates the depth of invasion (DOI) to determine T category and extranodal extension (ENE) to determine N category for OSCC (14, 15). 1

12 Chapter 1 Management and histopathological evaluation of primary OSCC and regional lymph node metastasis Generally, the management of OSCC includes single modality surgery, or a combination of surgery and adjuvant radiotherapy with or without systemic therapy (11). Primary tumor resection The main goal of surgery is complete resection of the tumor with negative margins, as margin status is one of the most important variables associated with loco-regional control and survival (5, 12, 16-20). The extent of resection is estimated using information on tumor size assessed by preoperative imaging, as well as visual inspection and palpation performed during surgery. Ultimately, the definitive margin status is determined by the pathologist days after surgery (21, 22). This lack in intra-operative communication between surgeon and pathologist, results in inadequate margins in 30-85% of the procedures (23). To reduce these numbers, efforts are made to develop procedures that facilitate intra-operative margin assessment using either the resected specimen or the tumor bed. These techniques include: frozen section analysis, optical techniques (e.g. Raman spectroscopy), fluorescence, conventional imaging techniques (e.g. ultrasound), and cytological assessment (21). Regional lymph node dissection For patients diagnosed with OSCC with clinically positive cervical lymph nodes or cT3T4N0 tumors, primary tumor resection with neck dissection is indicated. The major question is what to do with patients presenting with cT1T2N0 tumors. Although neck dissection is a safe procedure, it is associated with high morbidity (5, 24). Therefore, elective neck dissection (END) is recommended if the risk of occult regional lymph node metastasis (RLNM) is 20% for patients with early-stage tumors that are cN0 (25). In this situation, END is either therapeutic when pN0. However, when pN+, it can help in determining whether adjuvant therapy is necessary (see below). Alternatives for END are observation, elective radiotherapy, or sentinel lymph node biopsy (SLNB) (5). The National Comprehensive Cancer Network recommends the use of DOI in making decisions on END, as it is an established predictor for occult RLNM (11). Since DOI with a cut-off value (> 4 mm) strongly predicts the presence of occult RLNM, this cutoff value is used within the Erasmus MC in making decisions on END (11, 26, 27). Yet, the DOI is determined days after the excision of the primary tumor during final pathological evaluation (15). As such, cancers with DOI of > 4 mm, require a second

13 General introduction stage END having additional morbidity for the patient, inefficient use of resources, time, and extra costs as a consequence. Another disadvantage of DOI is that it has been used intermittently with tumor thickness, which is also a predictor of RLNM (28-31). However, the 8th edition of the AJCC addressed this problem by providing a clear definition for DOI (15). Alternatively, some centers perform SLNB to rule out the presence of occult RLNM. Indeed, detection rates of 95% (32-34), 0.93 sensitivity and NPV of 0.88-1 (33-37), make SLNB a reliable method to detect occult RLNM during surgery. Since the success rate of SLNB depends on the experience and technical expertise of the team performing the procedure, its implementation in routine patient care knows difficulties (11). Histopathological evaluation of the primary tumor and RLNM as gold standard Besides TNM staging, the 7th and 8th edition of the AJCC recommend to collect several histopathological characteristics as they can affect clinical decision making (14, 15). Among them are: the maximum macroscopic diameter of a tumor in mm, which should be measured using the resection specimen. However, when the histological extent of the tumor is greater than macroscopically apparent, the maximum diameter should be measured microscopically (38). The DOI in mm (38), which is measured by finding the “horizon” of the basement membrane of adjacent squamous mucosa and drawing a perpendicular “plump line” from the earlier defined horizon to the deepest point of tumor invasion (15). A DOI > 4 mm is significantly associated with – occult – RLNM in early OSCC (26, 27). The histopathological grade (G) of OSCC is called on the degree of resemblance of the tumor cells to those of the normal epithelium (38). Generally, the following categories are used: cannot be assessed (GX), well differentiated (G1), moderately differentiated (G2), poorly differentiated (G3), and undifferentiated (G4) (14). Grading is performed on the most aggressive area of the tumor and poor differentiation is associated with poor outcome, despite the fact that it is highly subjective (38-40). The presence or absence of perineural, lymphovascular, or bone invasion should be recorded, as their presence is associated with tumor recurrence, nodal metastasis, and/or poor survival (38, 41). The worse pattern of invasion (WPOI) at the deep margin of OSCC can be assessed as: broad cohesive sheets of cells (WPOI type 1), broad pushing “fingers” or separate large stellate like tumor islands (WPOI type 2), large invasive tumor islands of > 15 cells (WPOI type 3), small invasive tumor islands of ≤ 15 cells including single cell invasion (WPOI type 4), or tumor satellites of any size ≥ 1 mm away from the main tumor or next closest satellite (20X objective) (WPOI type 5) (42, 43). Seen its signifi1

14 Chapter 1 cant association with loco-regional recurrence and disease-specific survival in early stage OSCC, it is advised to assess whether WPOI type 5 is present or absent (15, 44). Tumor dispersion in the form of WPOI type 5 goes predominately through soft tissue. However, it can also occur in the form of extratumoral perineural van lymphovascular invasion. It should be mentioned that throughout this thesis tumors have been scored for cohesive (WPOI type 1-3) versus non-cohesive (WPOI type 4, 5) growth pattern, which can be considered as the outdated not precisely defined alternative for WPOI (15, 38, 41, 43). Extranodal extension, being the growth of carcinomatous tissue through the capsule of metastatic lymph nodes (45), occurs in aggressive carcinoma, is associated with poor prognosis (41), and should be recorded as present or not identified (38). The distance between carcinoma and deep resection margin should be measured in mm (15, 38). The margin is clear, when the distance is > 5 mm; close when the margin is 1-5 mm; and involved, when the distance is < 1 mm. Involved margins are significantly associated with any form of recurrence (local, regional and distant metastasis) (20). Post-operative radiation and/or chemotherapy Primary radiotherapy is not routinely used for OSCC, as its high-dose is associated with osteoradionecrosis (46). On the other hand, post-operative radiotherapy is well-established for locally advanced disease and histopathological risk factors – such as pN2-3, ENE, close or involved surgical margins, or perineural invasion – and is known to improve loco-regional control and survival (5, 12, 47-50). Although advances were made with surgery and postoperative radiotherapy, disease control and overall survival remained challenging. Therefore, studies have been performed to examine the efficacy of CRT. It is now established that post-operative CRT is beneficial for patients with ENE and positive margins (5, 12, 16, 17, 51). Management of recurrent and metastatic disease Some patients with recurrent or metastatic OSCC, may be cured by salvage resection, re-irradiation or metastasectomy. However, the majority of this patient population is amendable for systemic therapy (5, 9). Traditionally recurrent and/or metastatic OSCC are treated with cytotoxic chemotherapy consisting of cisplatin with 5-fluorouracill and/or a taxane. For toxicity reasons cisplatin is sometimes replaced with carboplatin, yet this results in reduced tumoricidal activity (52).

15 General introduction Targeted therapy and immunotherapy Besides the classical treatment modalities mentioned above, there is an interest to implement targeted therapies (e.g. cetuximab , a monoclonal antibody (moAb) directed against the epidermal growth factor receptor (EGFR, also known as HER1) (53)) and immune checkpoint inhibitors (e.g. pembrolizumab (a moAb directed against PD-1) (54)), nivolumab (a moAb directed against PD-1 (55)), and ipilimumab (a moAb directed against CTLA-4 (56)) in the management of recurrent and/or metastatic (52), as well as locally advanced OSCC (57, 58). The use of targeted therapy and immunotherapy in the treatment of recurrent and/or metastatic disease Historically, the choice of systemic therapies in the treatment of recurrent and/or metastatic disease was not based on improved overall survival and/or quality of life. This changed in 2008, after publication of the results of the phase III randomized EXTREME trial, showing improved survival after addition of cetuximab, to the traditional cytotoxic doublet platinum/5-fluorouacil while maintaining quality of life in the first line setting for recurrent and/or metastatic disease. As a result, adding cetuximab became standard first-line palliative treatment for patients that are fit enough to withstand this treatment regimen (52, 59, 60). This changed with the advent of the immune checkpoint inhibitors nivolumab and pembrolizumab that showed unprecedented results in the second line (CheckMate 141, KEYNOTE-040) and first line (KEYNOTE-048) treatment or recurrent and/or metastatic disease with objective response rates of 13 to 17% and two-year overall survival rates of 17 to 27% (55, 61-63). Specifically, the KEYNOTE-048 trial illustrated the superiority of pembrolizumab alone or pembrolizumab with platinum and 5-FU compared to cetuximab with platinum and 5-FU in the first line setting, with median overall survival of 11.6 versus 10.7 months (not significant) and median overall survival of 13.0 versus 10.7 months (p=0.0034) respectively (61). This survival benefit increases with PD-L1 combined positive score, defined as ‘the number of PD-L1-positive cells (tumor cells, lymphocytes, and macrophages divided by the total number of tumor cellsx100)’ (61). Based on the results observed in this trial for the entire sample population, pembrolizumab plus chemotherapy is nowadays the preferred first-line treatment for patients with recurrent and metastatic disease with a high disease burden. Pembrolizumab alone is used for patients with small tumors and high PD-L1 expression (9, 60). Patients that are not eligible for first-line immunotherapy, due to e.g. high-comorbidity, should receive cetuximab according to the EXTREME protocol (9, 60). 1

16 Chapter 1 The advent of immunotherapy in the neoadjuvant setting of locally advanced disease Unfortunately, 5-year overall survival after surgery stagnates at 50% (16, 17). Additionally, a substantial part of HNSCC patients that are treated with surgery suffer from problems associated with swallowing, speech, or aesthetics (64). These poor outcomes and high morbidities require new treatment options that not only improve survival, but also de-intensify the current treatment protocols (58). The combination of immune checkpoint inhibitors targeting PD-1 and CTLA-4 , another immune checkpoint protein, specifically nivolumab and ipilimumab, has been proven more effective than either monotherapy in advanced melanomas (65). Although the additive value of adding anti-CTLA-4 (tremelimumab) to anti-PD-L1 (durvalumab), the activating ligand of PD-1, was not observed for recurrent and metastatic HNSCCs (66, 67), there is limited evidence that anti-PD-1 monotherapy has modest effects in the curative neoadjuvant setting of human papilloma virus (HPV)-negative HNSCCs with major pathological tumor responses (pTR), defined as pTR in > 90% observed for the primary tumor, ranging from 7 to 14% (68, 69). Schoenfeld et al. (68) also showed that combining nivolumab and ipilimumab in the neo-adjuvant setting was more effective compared to nivolumab monotherapy. The latter was confirmed by Vos et al. in the IMCISION trial which showed a major pathological response (MPR) rate of 35% for patients treated in the nivolumab and ipilimumab arm, and 17% for those in the nivolumab arm (58). Knowing that none of the MPR patients developed recurrent disease within 24 months, indicates that neoadjuvant immunotherapy is a promising treatment protocol for HNSCC patients that are treated with surgery (58). Notwithstanding the developments in the treatment of OSCC, there is an interest to implement targeted therapies and immune checkpoint inhibitors – other than cetuximab, pembrolizumab, and nivolumab. Among the long list of potentially interesting molecular targets for therapy is the receptor tyrosine kinase (RTK) MET (hepatocyte growth factor receptor) (10, 70, 71). The receptor tyrosine kinase MET, a potential target for therapy in HNSCC? MET and its physiological ligand hepatocyte growth factor/scatter factor (HGF/SF) were discovered in the mid-1980s (72). MET is predominanty expressed on the surface of epithelial cells, HGF/SF is expressed by cells of mesenchymal origin – e.g. fibroblast – residing in the surrounding stroma (73). Signaling via this ligand-receptor

17 General introduction pair facilitates invasive growth, through activation of a complex network of signaling cascades (74). During invasive growth, cancer cells combine proliferation, survival, motility, and epithelial-to-mesenchymal transition (EMT) (Figure 3) (71, 74). The latter process transforms epithelial cells into more mesenchymal derivatives, which is essential for physiological and pathological processes (75). More specifically, processes such as tissue development and regeneration, as well as the dissemination of cancer cells (71-73). Figure 3: HGF - MET signaling, mediated through RAS, PI3K, and STAT, facilitates the invasive growth of cancer cells. The human MET gene has been mapped to chromosome 7, location 7q31.2 (76). Its length spans over 120 kb, consisting of 21 exons, of which 20 coding, and 20 introns (77). The receptor counts 1408 amino acids and is synthesized as a partially glycosylated 170 kDa single-chain intracellular precursor, which is subjective to cleavage and further glycosylation yielding a mature, cell surface-associated 190 kDa disulfide linked single-pass heterodimer that consists of an extracellular NH2-terminal 50 kDa α-chain and a transmembranous 145 kDa β-chain. It is the β-chain that contains intrinsic, ligand-activated tyrosine kinase activity in its intracellular domain (77-79). 1

18 Chapter 1 The ectodomain consists of a large SEMA domain, required for ligand binding and receptor dimerization (80), which is separated from four immunoglobulin-like domains by a small cysteine-rich domain. Next comes the transmembrane domain, followed by a long juxtamembrane sequence, the kinase domain and the COOH-terminal sequence, of which the latter three hold essential motifs for downstream signaling (72). HGF binding induces MET’s kinase activity by facilitating receptor dimerization and trans-phosphorylation of juxtamembrane catalytic residues Tyr1234 and Tyr1235. Subsequent phosphorylation of the carboxy-terminal ‘docking’ residues Tyr1349 and Tyr1356, allows MET to recruit a variety of downstream signal-relay transducers. On the other hand, phosphatases antagonize MET signaling by dephosphorylating either the catalytic or docking tyrosine residues. The majority of MET-mediated downstream signaling modules is transduced through interaction of the receptor with the multi-adaptor protein GAB1 (GRB2-associated-binding protein 1). Ultimately, downstream signaling pathways, including MAPK, PI3K-Akt, and STAT, are effectively activated by MET and its signal transducers (Figure 3) (71, 72). MET is aberrantly activated by mutations and amplifications in approximately 25% of HNSCC. Approximately 4% of HNSCC carry exon 14 deletion. The resulting exon 14 skipping delays MET ubiquitination and down-regulation, having MET overexpression and kinase activation as a consequence (81). Somatic mutations affecting the kinase domain occur at a rate of around 8%, which include Y1230C, Y1235D, and Y1253D (81). Mutations Y1230C and Y1235D constitutively activate MET and were found in RLNMs of HNSCC (82). Interestingly, while transcripts of the Y1235D mutant alleles are highly represented in RLNMs, they are hardly detectable in the corresponding primary tumors. This suggests clonal expansion of cells carrying mutated MET during HNSCC disease progression (82). MET activating point mutation Y1253D has been described to be significantly associated with decreased metastasis-free survival of HNSCC patients treated with radiotherapy or CRT (83). Progression-free and overall survival of HNSCC patients with exon 14 deletions, mutations of the kinase domain (V1110I, H1112Y, V1333I), or juxtamembrane domain mutations (R988C and T1010I), that were included in a phase III randomized trial investigating the effectivity of the EGFR inhibitor gefitinib, was shorter compared to patients with MET wild-type cancers irrespective of the treatment arm (84). Seen differences in used scoring methods and definitions, it has been reported that MET gene amplifications occur in 1-13% of HNSCC. Although known, the biological and clinical consequences of MET amplification in HNSCC need to be further explored (81). If a molecular subtype of HNSCC, and more specifically OSCC, exists that is driven by MET mutation and/or amplification conferring susceptibility to targeted agents, requires further examined

19 General introduction in adequate and well described patient cohorts (81). It should be stressed that the reported mutations and copy number alterations concern all sites of the head and neck region and not OSCC specifically since the number of OSCC specific studies are too low (81). In the majority of cancers including HNSCCs, activation of MET occurs in already transformed cells to increase their proliferative, anti-apoptotic, and migratory potential. In this context, MET is transcriptionally activated by stimuli such as hypoxia, inflammatory cytokines, stromal HGF, and pro-angiogenic factors, often abundantly present in the reactive tumor-associated stroma (71). Notwithstanding that MET expression is associated with poor prognosis in various solid cancers (85)—among which HNSCCs (86)—and numerous targeted therapies are subjective to investigation (72, 87), major survival benefits have not yet been obtained (87, 88). This, together with the fact that the average price of cancer drugs reach approximately $100,000 per year of treatment per patient, hinders the use of MET inhibitors in clinical practice (89, 90). This raises questions as: who to treat or better how to select? 1

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22 Chapter 1 40. Lee LY, Lin CY, Cheng NM, Tsai CY, Hsueh C, Fan KH, et al. Poor tumor differentiation is an independent adverse prognostic variable in patients with locally advanced oral cavity cancer--Comparison with pathological risk factors according to the NCCN guidelines. Cancer Med 2021;10:6627-41. 41. Dolens EDS, Dourado MR, Almangush A, Salo TA, Gurgel Rocha CA, da Silva SD, et al. The Impact of Histopathological Features on the Prognosis of Oral Squamous Cell Carcinoma: A Comprehensive Review and Meta-Analysis. Front Oncol 2021;11:784924. 42. Brandwein-Gensler M, Smith RV, Wang B, Penner C, Theilken A, Broughel D, et al. Validation of the histologic risk model in a new cohort of patients with head and neck squamous cell carcinoma. Am J Surg Pathol 2010;34:676-88. 43. Brandwein-Gensler M, Teixeira MS, Lewis CM, Lee B, Rolnitzky L, Hille JJ, et al. Oral squamous cell carcinoma: histologic risk assessment, but not margin status, is strongly predictive of local disease-free and overall survival. Am J Surg Pathol 2005;29:167-78. 44. Li Y, Bai S, Carroll W, Dayan D, Dort JC, Heller K, et al. Validation of the risk model: high-risk classification and tumor pattern of invasion predict outcome for patients with low-stage oral cavity squamous cell carcinoma. Head Neck Pathol 2013;7:211-23. 45. Carter RL, Barr LC, O’Brien CJ, Soo KC, Shaw HJ. Transcapsular spread of metastatic squamous cell carcinoma from cervical lymph nodes. Am J Surg 1985;150:495-9. 46. Bedwinek JM, Shukovsky LJ, Fletcher GH, Daley TE. Osteonecrosis in patients treated with definitive radiotherapy for squamous cell carcinomas of the oral cavity and naso-and oropharynx. Radiology 1976;119:665-7. 47. Ang KK, Trotti A, Brown BW, Garden AS, Foote RL, Morrison WH, et al. Randomized trial addressing risk features and time factors of surgery plus radiotherapy in advanced head-and-neck cancer. Int J Radiat Oncol Biol Phys 2001;51:571-8. 48. Chinn SB, Spector ME, Bellile EL, McHugh JB, Gernon TJ, Bradford CR, et al. Impact of perineural invasion in the pathologically N0 neck in oral cavity squamous cell carcinoma. Otolaryngol Head Neck Surg 2013;149:893-9. 49. Lundahl RE, Foote RL, Bonner JA, Suman VJ, Lewis JE, Kasperbauer JL, et al. Combined neck dissection and postoperative radiation therapy in the management of the high-risk neck: a matched-pair analysis. Int J Radiat Oncol Biol Phys 1998;40:529-34. 50. Myers JN, Greenberg JS, Mo V, Roberts D. Extracapsular spread. A significant predictor of treatment failure in patients with squamous cell carcinoma of the tongue. Cancer 2001;92:3030-6. 51. Bernier J, Cooper JS, Pajak TF, van Glabbeke M, Bourhis J, Forastiere A, et al. Defining risk levels in locally advanced head and neck cancers: a comparative analysis of concurrent postoperative radiation plus chemotherapy trials of the EORTC (#22931) and RTOG (# 9501). Head Neck 2005;27:843-50. 52. Szturz P, Vermorken JB. Management of recurrent and metastatic oral cavity cancer: Raising the bar a step higher. Oral Oncol 2020;101:104492. 53. Bonner JA, Harari PM, Giralt J, Azarnia N, Shin DM, Cohen RB, et al. Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck. N Engl J Med 2006;354:567-78. 54. Seiwert TY, Burtness B, Mehra R, Weiss J, Berger R, Eder JP, et al. Safety and clinical activity of pembrolizumab for treatment of recurrent or metastatic squamous cell carcinoma of the head and neck (KEYNOTE-012): an open-label, multicentre, phase 1b trial. Lancet Oncol 2016;17:956-65. 55. Ferris RL, Blumenschein G, Jr., Fayette J, Guigay J, Colevas AD, Licitra L, et al. Nivolumab for Recurrent Squamous-Cell Carcinoma of the Head and Neck. N Engl J Med 2016;375:1856-67. 56. Wang Z, Wu VH, Allevato MM, Gilardi M, He Y, Luis Callejas-Valera J, et al. Syngeneic animal models of tobacco-associated oral cancer reveal the activity of in situ anti-CTLA-4. Nat Commun 2019;10:5546.

23 General introduction 57. Elbers JBW, Al-Mamgani A, Paping D, van den Brekel MWM, Jozwiak K, de Boer JP, et al. Definitive (chemo) radiotherapy is a curative alternative for standard of care in advanced stage squamous cell carcinoma of the oral cavity. Oral Oncol 2017;75:163-8. 58. Vos JL, Elbers JBW, Krijgsman O, Traets JJH, Qiao X, van der Leun AM, et al. Neoadjuvant immunotherapy with nivolumab and ipilimumab induces major pathological responses in patients with head and neck squamous cell carcinoma. Nat Commun 2021;12:7348. 59. Mesia R, Rivera F, Kawecki A, Rottey S, Hitt R, Kienzer H, et al. Quality of life of patients receiving platinum-based chemotherapy plus cetuximab first line for recurrent and/or metastatic squamous cell carcinoma of the head and neck. Ann Oncol 2010;21:1967-73. 60. Vermorken JB, Mesia R, Rivera F, Remenar E, Kawecki A, Rottey S, et al. Platinum-based chemotherapy plus cetuximab in head and neck cancer. N Engl J Med 2008;359:1116-27. 61. Burtness B, Harrington KJ, Greil R, Soulieres D, Tahara M, de Castro G, Jr., et al. Pembrolizumab alone or with chemotherapy versus cetuximab with chemotherapy for recurrent or metastatic squamous cell carcinoma of the head and neck (KEYNOTE-048): a randomised, open-label, phase 3 study. Lancet 2019;394:1915-28. 62. Cohen EEW, Soulieres D, Le Tourneau C, Dinis J, Licitra L, Ahn MJ, et al. Pembrolizumab versus methotrexate, docetaxel, or cetuximab for recurrent or metastatic head-and-neck squamous cell carcinoma (KEYNOTE-040): a randomised, open-label, phase 3 study. Lancet 2019;393:156-67. 63. Ferris RL, Blumenschein G, Jr., Fayette J, Guigay J, Colevas AD, Licitra L, et al. Nivolumab vs investigator’s choice in recurrent or metastatic squamous cell carcinoma of the head and neck: 2-year long-term survival update of CheckMate 141 with analyses by tumor PD-L1 expression. Oral Oncol 2018;81:45-51. 64. Rathod S, Livergant J, Klein J, Witterick I, Ringash J. A systematic review of quality of life in head and neck cancer treated with surgery with or without adjuvant treatment. Oral Oncol 2015;51:888-900. 65. Larkin J, Chiarion-Sileni V, Gonzalez R, Grob JJ, Rutkowski P, Lao CD, et al. Five-Year Survival with Combined Nivolumab and Ipilimumab in Advanced Melanoma. N Engl J Med 2019;381:1535-46. 66. Ferris RL, Haddad R, Even C, Tahara M, Dvorkin M, Ciuleanu TE, et al. Durvalumab with or without tremelimumab in patients with recurrent or metastatic head and neck squamous cell carcinoma: EAGLE, a randomized, open-label phase III study. Ann Oncol 2020;31:942-50. 67. Siu LL, Even C, Mesia R, Remenar E, Daste A, Delord JP, et al. Safety and Efficacy of Durvalumab With or Without Tremelimumab in Patients With PD-L1-Low/Negative Recurrent or Metastatic HNSCC: The Phase 2 CONDOR Randomized Clinical Trial. JAMA Oncol 2019;5:195-203. 68. Schoenfeld JD, Hanna GJ, Jo VY, Rawal B, Chen YH, Catalano PS, et al. Neoadjuvant Nivolumab or Nivolumab Plus Ipilimumab in Untreated Oral Cavity Squamous Cell Carcinoma: A Phase 2 Open-Label Randomized Clinical Trial. JAMA Oncol 2020;6:1563-70. 69. Uppaluri R, Campbell KM, Egloff AM, Zolkind P, Skidmore ZL, Nussenbaum B, et al. Neoadjuvant and Adjuvant Pembrolizumab in Resectable Locally Advanced, Human Papillomavirus-Unrelated Head and Neck Cancer: A Multicenter, Phase II Trial. Clin Cancer Res 2020;26:5140-52. 70. Lorch JH, Posner MR, Wirth LJ, Haddad RI. Seeking alternative biological therapies: the future of targeted molecular treatment. Oral Oncol 2009;45:447-53. 71. Trusolino L, Bertotti A, Comoglio PM. MET signalling: principles and functions in development, organ regeneration and cancer. Nat Rev Mol Cell Biol 2010;11:834-48. 72. Gherardi E, Birchmeier W, Birchmeier C, Vande Woude G. Targeting MET in cancer: rationale and progress. Nat Rev Cancer 2012;12:89-103. 1

24 Chapter 1 73. Sonnenberg E, Meyer D, Weidner KM, Birchmeier C. Scatter factor/hepatocyte growth factor and its receptor, the c-met tyrosine kinase, can mediate a signal exchange between mesenchyme and epithelia during mouse development. J Cell Biol 1993;123:223-35. 74. Comoglio PM, Trusolino L, Boccaccio C. Known and novel roles of the MET oncogene in cancer: a coherent approach to targeted therapy. Nat Rev Cancer 2018;18:341-58. 75. Ye X, Weinberg RA. Epithelial-Mesenchymal Plasticity: A Central Regulator of Cancer Progression. Trends Cell Biol 2015;25:675-86. 76. Homo sapiens MET proto-oncogene, receptor tyrosine kinase (MET), transcript variant 1, mRNA. https://www. Accessed: 29-12-2021 2021. 77. Transcript: ENST00000318493.11 MET-201.;g=ENSG00000105976;r=7:116672196-116798377;t=ENST00000318493. Accessed: 29-12-2021 2021. 78. Faletto DL, Tsarfaty I, Kmiecik TE, Gonzatti M, Suzuki T, Vande Woude GF. Evidence for non-covalent clusters of the c-met proto-oncogene product. Oncogene 1992;7:1149-57. 79. Giordano S, Di Renzo MF, Narsimhan RP, Cooper CS, Rosa C, Comoglio PM. Biosynthesis of the protein encoded by the c-met proto-oncogene. Oncogene 1989;4:1383-8. 80. Kong-Beltran M, Stamos J, Wickramasinghe D. The Sema domain of Met is necessary for receptor dimerization and activation. Cancer Cell 2004;6:75-84. 81. Szturz P, Raymond E, Abitbol C, Albert S, de Gramont A, Faivre S. Understanding c-MET signalling in squamous cell carcinoma of the head & neck. Crit Rev Oncol Hematol 2017;111:39-51. 82. Di Renzo MF, Olivero M, Martone T, Maffe A, Maggiora P, Stefani AD, et al. Somatic mutations of the MET oncogene are selected during metastatic spread of human HNSC carcinomas. Oncogene 2000;19:1547-55. 83. Ghadjar P, Blank-Liss W, Simcock M, Hegyi I, Beer KT, Moch H, et al. MET Y1253D-activating point mutation and development of distant metastasis in advanced head and neck cancers. Clin Exp Metastasis 2009;26:809-15. 84. Argiris A, Ghebremichael M, Gilbert J, Lee JW, Sachidanandam K, Kolesar JM, et al. Phase III randomized, placebo-controlled trial of docetaxel with or without gefitinib in recurrent or metastatic head and neck cancer: an eastern cooperative oncology group trial. J Clin Oncol 2013;31:1405-14. 85. Gentile A, Trusolino L, Comoglio PM. The Met tyrosine kinase receptor in development and cancer. Cancer Metastasis Rev 2008;27:85-94. 86. Szturz P, Budikova M, Vermorken JB, Horova I, Gal B, Raymond E, et al. Prognostic value of c-MET in head and neck cancer: A systematic review and meta-analysis of aggregate data. Oral Oncol 2017;74:68-76. 87. Kim KH, Kim H. Progress of antibody-based inhibitors of the HGF-cMET axis in cancer therapy. Exp Mol Med 2017;49:e307. 88. Huang F, Ma Z, Pollan S, Yuan X, Swartwood S, Gertych A, et al. Quantitative imaging for development of companion diagnostics to drugs targeting HGF/MET. J Pathol Clin Res 2016;2:210-22. 89. Kantarjian H, Rajkumar SV. Why are cancer drugs so expensive in the United States, and what are the solutions? Mayo Clin Proc 2015;90:500-4. 90. Prasad V. Do cancer drugs improve survival or quality of life? BMJ 2017;359:j4528.

CHAPTER 2 Context, background, aims, and outline of the thesis

29 Context, background, aims, and outline of the thesis Context Deregulated HGF/SF-MET signaling has been implicated in many human solid cancers (1). Since MET is mutated, overexpressed (at mRNA and protein level), and orchestrates invasive growth in HNSCC (2-4), it is a target of interest (5, 6). This has led to the development of a plethora of MET targeted therapies, which effectivities are under investigation (7, 8). Unfortunately, major survival benefits have not yet been obtained (8, 9). This, together with the high costs that come along with the treatment of cancer patients, hinders the use of MET-based targeted therapies in clinical practice (10, 11). An explanation for the absence of clinical benefit of MET targeted therapies may be found in the absence of suitable companion diagnostics (CDx) (9), defined as “a medical device, often an in vitro device, which provides information that is essential for the safe and effective use of a corresponding drug or biological product” (12). The development of CDx for targeted therapies directed against MET is complicated due to several reasons (9). Some have a technical origin, such as the lack of reliable antibodies and optimal scoring methods (7, 9, 13), while other reasons may be due to biology, such as ectodomain shedding (9). Prior to stating the aims and outline of this thesis some methods and biological concepts, which we consider to be essential for grasping the context of the performed work, are introduced. The need for antibody validation Besides their wide use in scientific research laboratories, antibodies also play a crucial role in molecular pathology and clinical chemistry laboratories. This implies that their use in a diagnostic, prognostic, and/or predictive setting can have a direct effect on clinical decision making (14). Unfortunately, it has been described that almost half of commercially available antibodies do not perform as expected with respect to immunohistochemistry/immunocytochemistry (15). Moreover, the validity of antibodies is dependent on the method used. This implies that antibodies should be extensively validated before design and/or interpretation of scientific experiments, scoring systems, diagnostic tests, CDx, and clinical guidelines (14). To accomplish this, antibody validation decision models and the five conceptual pillars of application specific antibody validation have been developed (14, 16, 17). 2

30 Chapter 2 The use of tissue microarrays in biomarker screening During the production of tissue microarrays (TMAs) (18, 19), a tissue core (0.6 to 3 mm diameter) is sampled from an archival formalin-fixed paraffin-embedded donor block and repositioned in a paraffin acceptor block. Subsequent sectioning of the acceptor block, results in slides representing tens to hundreds of cancer specimens. As such, immunohistochemical staining of TMA sections allows rapid screening of the behavior of biomarkers across large sample population (20). Additional advantages are sparse use of resection specimens, incorporation of internal controls, a decrease in staining variability (no batch effects), possibly automated scoring, lowering in costs, and facilitation of multi-center collaborations (20). Furthermore, when properly annotated, the TMA technology allows examination of the association between the biomarker of interest and patient, tumor, and/or histopathological characteristics, facilitating the identification of prognostic, predictive and therapeutic targets in large independent patient cohort assuring sufficient statistical power (20). Despite the many benefits of using TMAs, the technique also has it downsides (20). Most of them are similar to those involving the use of whole tissue sections (WTSs), such as tissue quality, use of reliable antibodies, standardization of methodologies, antigen loss, and more specifically production and cutting of the actual arrays (20). Besides these practical pitfalls, a major concern in the use of TMAs is spatial tumor heterogeneity (20). For instance, despite hematoxylin and eosin based selection of tumor areas by the pathologist, deeper cuts may not contain any tumor cells, which especially is of concern when analyzing in situ lesions (20). Of major concern is the examination of biomarkers that are heterogeneously expressed across a cancer. Examination of the correlation between homogeneously expressed biomarkers (Pearson r > 0.6) in a breast cancers setting, led to the general consensus that two cores with 0.6 mm diameter are representative for WTSs (20, 21). However, for heterogeneously expressed biomarkers the general notion is that this number should be considerably higher (22, 23). Therefore, it has also been proposed to construct TMAs in such a way that different tumor areas are represented in separate cores, allowing assessment of intratumoral heterogeneity (20). Finally others have argued that for large TMA studies sampling error is compensated by sample size, therefore including only one core per tumor (24).

31 Context, background, aims, and outline of the thesis The use of regression models to establish the prognostic value of a biomarker It is generally accepted in clinical prediction modeling that variable selection should be based on clinical insight and knowledge extracted from the literature (25). Yet, to avoid overfitting (26), it is advised to use the ‘one in ten rule’ when using traditional clinical prediction modelling strategies, such as logistic regression and survival models (27). This rule states that for every 10 events in a dataset one variable can be considered in a model (25, 28, 29). Besides clinical insight, literature knowledge, and experience, candidate variable selection for the final model should be based on the data and data analysis (27). It is advised to combine (30) or exclude highly correlated (31-33) variables, restrict inclusion based on distribution (30, 33), and exclude in case of a large number of missing values (30, 33). Once the candidate variables have been identified, a definitive selection of variables needs to established for the final model (27). This can be done using the full model approach (inclusion of all candidate variables) (33), or formal variable selection methods (e.g. forward and backward elimination) when there is confusion or uncertainty regarding which variables to consider in the final model (27). MET ectodomain shedding In addition to phosphorylation, the activity of MET signaling can be regulated by controlling the number of receptor molecules present on the membrane (34). A process called presenilin-regulated intramembrane proteolysis (PS-RIP) degrades MET through sequential proteolytic cleavage. First, the receptor is cleaved within its juxtamembrane domain by membrane metalloproteases. This process is independent of ligand stimulation and requires no kinase activity. Consequently, a soluble MET N-terminal fragment (MET-NTF) is shed into the extracellular space, an occurrence referred to as ectodomain shedding. ADAM metalloproteases 10 and 17 (ADAM10 and ADAM17) drive MET-NTF shedding, as it is inhibited upon their genetic ablation (35, 36). Immediately after shedding, the remaining membrane bound 55 kDa C-terminal fragment (MET-CTF) undergoes a second cleavage, which is performed by the γ-secretase complex. The resulting 50 kDa intracellular domain of MET (MET-ICD) is degraded by the proteasome upon its release into the cytosol. The MET-CTF fragments that escape γ-secretase cleavage are subjective to lysosomal degradation (Figure 1) (37, 38). 2