Sarah Verhoeff



Molecular PET imaging to steer treatment for cancer patients Provided by thesis specialist Ridderprint, Printing Ridderprint | Layout and design Indah Hijmans, Cover design Otura Design | Rianne Koens ISBN 978-94-6483-041-5 The work presented in this thesis was carried out within the departments of Medical Oncology and Medical Imaging (section: Nuclear Medicine), Radboud University Medical Center and the Radboud Institute for Health Sciences, Nijmegen, The Netherlands. The clinical studies were financially supported by: ❖ Radboud Institute for Health Sciences, NWO (91617039, KWF (10099). ❖ The Dutch Cancer Society (KWF): Alpe d’HuZes Grant RUG 2012-5400 ❖ AstraZeneca ❖ Merck KGaA ❖ Paul Speth fonds Copyright © Sarah Relinde Verhoeff, Nijmegen 2023 All rights are reserved. No part of this book may be reproduced, distributed, stored in a retrieval system, or transmitted in any form or by any means, without prior written permission of the author.

MOLECULAR PET IMAGING TO STEER TREATMENT FOR CANCER PATIENTS Proefschrift ter verkrijging van de graad van doctor aan de Radboud Universiteit Nijmegen op gezag van de rector magnificus prof. dr. J.H.J.M. van Krieken, volgens besluit van het college voor promoties in het openbaar te verdedigen op donderdag 11 mei 2023 om 12.30 uur precies door Sarah Relinde Verhoeff geboren op 28 januari 1988 te Lelystad

Promotoren Prof. dr. C.M.L. van Herpen Prof. dr. M.M. van den Heuvel Prof. dr. S. Heskamp Copromotor Dr. E.H.J.G. Aarntzen Manuscriptcommissie Prof. dr. J. Bussink Prof. dr. M.N. Lub-de Hooge (Rijksuniversiteit Groningen) Dr. A.J. de Langen (Antoni van Leeuwenhoek)

TABLE OF CONTENTS Chapter 1 General introduction and outline of this thesis 7 Part 1 Chapter 2 Lesion detection by [89Zr]Zr-DFO-girentuximab and [18F]FDG PET/CT in patients with newly diagnosed metastatic clear cell renal cell carcinoma. EJNMMI 2019 23 Chapter 3 The value of [89Zr]Zr-DFO-girentuximab and [18F]FDG PET/CT to predict watchful waiting duration in patients with metastatic clear cell renal cell carcinoma. Clinical Cancer Research 2023 43 Part 2 Chapter 4 Programmed Cell Death-1/Ligand-1 PET Imaging: A Novel Tool to Optimize Immunotherapy? Pet Clinics 2020 67 Chapter 5 [89Zr]Zr-DFO-durvalumab PET/CT before durvalumab treatment in patients with recurrent or metastatic head and neck cancer. Journal of Nuclear Medicine 2022 81 Chapter 6 [89Zr]Zr-DFO-avelumab PET/CT in early-stage non-small cell lung carcinoma patients to predict neo-adjuvant avelumab treatment response. Manuscript in preparation 111 Chapter 7 Towards a better understanding of immune checkpoint inhibitor in radiolabeled PET-imaging studies. Journal of Nuclear Medicine 2022 129 Chapter 8 Summary, general discussion and future perspectives 137 Chapter 9 Nederlandse samenvatting 151 Addendum List of publications 158 PhD portfolio 160 Research datamanagement 162 Dankwoord 164 Curriculum vitae 168

General introduction and outline of this thesis

8 Chapter 1 GENERAL INTRODUCTION Over the past decades, the landscape of oncology has evolved rapidly. Our knowledge on tumor biology has increased and as a result, many new targeted anticancer agents have been introduced. In daily practice, we encounter difficulties in selecting the right patient for the right drug and vice versa. Patients assigned to an ineffective treatment may experience unnecessary toxicity which impacts the prognosis and quality of life. Moreover, ineffective therapies delay effective treatment strategies that may influence patient’s prognosis and increase the costs for our health care system. Here, we provide an overview of current insight in tumor biology and the challenges we face regarding patient selection to steer anti-cancer treatment. Tumor heterogeneity Tumors are complex and heterogenous. Tumor heterogeneity can be used to describe 1) the differences between tumors of the same type in different patients (inter-patient heterogeneity), 2) between different tumors within one patient (inter-tumoral heterogeneity), and 3) between different cancer cells within one tumor (intra-tumoral heterogeneity). The evolutionary branching of cancer from a primary tumor to distant metastases contributes to heterogenous disease within individual patients1. Genome-sequencing studies have revealed considerable variations in the genetic make-up of tumor cells not only distinct anatomical locations but also distinct regions within the same tumor lesion2. This intra-tumoral heterogeneity is not limited to the genetic level, but encompasses also phenotypic, metabolic and secretory components3. These components are connected and evolve over space and time4. Another factor contributing to heterogeneity is the tumor microenvironment (TME), a dynamic system of interacting stroma cells, immune cells and host factors. Together with the different phenotypes of cancer cells, this creates a unique TME that is believed to explain differences in the course of disease within and between patients5. Drug and biomarker development In recent years, many clinical trials showed unprecedented (durable) response rates of novel targeted and immune therapies. This has led to expanding of the therapeutic arsenal for routine oncological clinical care for various tumor types6. In addition, numerous new drugs are in the pipeline. However, it is unlikely that all these drugs will succeed in getting a place in the current treatment plans. Therefore, it is important to focus on the development of biomarkers before or at least during the development of new drugs. This enables better patient selection to ultimately optimize treatment efficacy for the individual patient and limits the number of trials designed to study many other agents with the same target. A biomarker predictive for treatment response should accurately characterize (the complexity of) the TME, taken the tumor heterogeneity and dynamics into consideration. Conventional laboratory biomarkers of tissue function-based blood samples or tumor size measurements based

9 General introduction and outline of this thesis on clinical anatomical imaging techniques are no longer sufficient. For example, the International Metastatic RCC Database Consortium (IMDC) risk score based on the level of hemoglobin, platelets, calcium, performance score and time from diagnosis to start systemic therapy, predicts survival of patients with metastatic RCC treated with systemic therapy7. This risk assessment is validated for patients treated with vascular endothelial growth factor (VEGF) targeted therapy but does not apply to the newest treatment options including immune checkpoint inhibitors (ICI) targeting programmed cell death 1 or its ligand (PD-1/PD-L1). So far, favorable responses to ICI have been associated with high tumor PD-L1 expression and high T-cell infiltration. Accordingly, the registration of pembrolizumab in e.g., SCCHN and NSCLC has been limited to patients with a high tumor PD-L1 expression as determined by immunohistochemistry8,9. The use of tissue biopsies for molecular diagnostics and evaluation of protein expression such as PD-L1 has been intensified with the introduction of ICI. Despite the advances made in tissue analysis techniques, its use is limited by sampling errors. The previously mentioned intra- and intertumoral heterogeneity and dynamics of the TME could be missed by evaluating only a fraction of the tumor cells10,11. Also, the factor of time needs to be taken into consideration when analyzing an archival biopsy since the target expression may alter during disease12. For molecular purposes, tissue biopsies are still of great value but should be interpreted with caution in heterogeneous tumors. Molecular imaging Molecular imaging can function as a complementary tool to blood and tissue sampling. This technique allows for non-invasive visualization and characterization of tumor lesions, based on biological processes at a molecular and cellular level. Several imaging modalities are available for molecular imaging, including computed tomography (CT), optical imaging (OI), nuclear imaging involving positron emitting tomography (PET) and single photon emission computed tomography (SPECT), ultrasound imaging and magnetic resonance imaging (MRI). Conventional CT and MRI imaging are used for the detection and quantification of tumor size. Both imaging modalities are regularly used in standard patient care and provide accurate anatomical configuration with excellent spatial resolution for tumor imaging. In contrast to MRI, CT is limited in delineating tumor lesions located in bone and soft tissue. These imaging modalities provide details on the anatomical location and lesion size, but not on the underlying tumor biology. This is where nuclear imaging is of additional value. Nuclear imaging Nuclear imaging requires the radiolabeling of a (tumor) targeting agent using a radionuclide that matches the targeting agent’s half-life. Small molecules or peptides with fast pharmacokinetics can be labeled with radionuclides with a short half-life like fluorine-18 (110 minutes). A well-known radiotracer is fluorine-18-fluorodeoxyglucose ([18F]FDG). The uptake of [18F]FDG reflects the increased glucose metabolism in tumors13,14. 1

10 Chapter 1 Larger molecules such as monoclonal antibodies have been used for molecular imaging because of their high specificity and binding affinity towards the target. Additionally, the biodistribution of the antibody can be visualized, and provides the most robust biomarker to correlate tracer uptake or tumor dosing, with treatment response. Their longer half-life requires a longer-lived radionuclides such as zirconium-89 (78.4 hours)15. Zirconium-89 is the radionuclide of preference of imaging with monoclonal antibodies because it remains in cells after internalization of the antibody-receptor complex, resulting in an improved tumor image contrast via accumulation. Additionally, zirconium-89 is a positron-emitting radionuclide and thus suitable for PET imaging, which has improved resolution and quantification compared to Single photon-emission computed tomography (SPECT)16. When radiolabeled, a so-called radiotracer can be visualized by detecting the emitted gamma rays by SPECT or PET-imaging. The result in a whole-body 3-dimensional reconstruction of the distribution of the tracer. The process is depicted in Figure 1. Figure 1. Molecular PET-imaging with radiolabeled antibodies. A monoclonal antibody (1) is radiolabeled to a radionuclide to form a radiotracer (2). This radiotracer is then administered to a patient with e.g., a lung tumor with target expression. Depended on the radionuclide of choice, SPECT or PET-imaging is performed to detect the emitted gamma rays (3). After reconstruction, the radiotracer can be localized at the site of target expression (4). Compared to SPECT, PET has superior resolution and sensitivity, enabling the detection of smaller lesions (±10mm) and accurate quantification. In combination with conventional imaging modalities such as CT and MRI for anatomical reference, tumor lesions can be located, and tracer-uptake can be used to study the biological differences between/within tumor lesions. Combined PET/CT or PET/MRI provides both anatomical and functional/biological information. Currently, the most common used radiotracer is [18F]FDG, which discriminates malignant tissue from benign tissue based on metabolic activity. However, the use of PET is rapidly expanding to other applications to gain non-invasive insight in tumor biology and support drug development. Imaging can be used to visualize different tumor features, such as the expression of tumor cell receptors, tumor-associated antigens, metabolic substrates and immune cells. For example, [89Zr]Zr-girentuximab, antibody targeting carbonic anhydrase IX (CAIX) which is over-expressed in clear cell renal cell carcinomas (ccRCC), can be used to visualize CAIX-expressing ccRCC tumor lesions17. Another application of molecular imaging is the visualization of the in vivo distribution,

11 General introduction and outline of this thesis normal-tissue accumulation and tumor accessibility of radiolabeled therapeutic monoclonal antibodies18. In this way, molecular imaging with radiolabeled antibodies can help to understand the working mechanism of new drugs and to potentially identify responders and non-responders before start of treatment19. Treatment perspectives for patients with solid tumors are changing rapidly. This thesis will focus on metastatic clear cell renal cell carcinoma (mccRCC), squamous cell carcinoma of the head and neck (SCCHN) and non-small cell lung cancer (NSCLC). Metastatic clear cell renal cell carcinoma Renal cell carcinoma (RCC) is the 7th most common type of cancer. Seventy percent of all RCCS have a clear cell component. About one third of the patients with RCC develop metastases, most frequently in the lung, bones, lymph node and the adrenal gland. The overall prognosis of mccRCC patients is highly variable, with a median overall survival ranging from 5 to 30 months7. In recent years, the prognosis has improved with the arrival immune checkpoint inhibitors (ICI). Before their introduction, first-line treatment consisted of monotherapy anti-angiogenic therapy targeted against VEGF or its receptor, e.g., bevacizumab, sunitinib or pazopanib20-22. Recent studies have shown improved survival rates of combination strategies (e.g., ipilimumab and nivolumab or pembrolizumab and axitinib) compared to sunitinib monotherapy, altering the standard first line treatment approach in mccRCC patients23,24. Upon diagnosis, patients with metastatic ccRCC are classified into good, intermediate and poor prognosis groups based on clinical parameters also known as the International Metastatic RCC Database Consortium (IMDC) Risk score7 (Table 1). Table 1. IMDC Criteria25 Karnofsky performance status <80% Time from diagnosis to systemic treatment <1 year Hb <LLN Corrected calcium >ULN Neutrophils >ULN Platelets >ULN Number of criteria Group Median overall survival 0 Favorable 43.2 months (95%CI 31.4-50.1) 1-2 Intermediate 33.5 months (95% CI 18.7-25.1) 3-6 Poor 7.8 months (95% CI 6.5-9.7) LLN= lower limit of normal; ULN= upper limit of normal Patients with rapid progressive disease at presentation need to start systemic treatment immediately. Among patients with a good or intermediate prognosis, a subgroup demonstrates indolent course of disease. In those patients, a period of watchful waiting (WW) can be considered 1

12 Chapter 1 as initial treatment strategy to delay unnecessary treatment toxicity, preserve or improve the quality of life and limit treatment costs. To improve the identification of mccRCC patients with indolent disease eligible for WW, molecular PET imaging could play an important role. The conventional work-up of a patient suspected for mccRCC consists of a contrast-enhanced CT of the thorax and abdomen, and sometimes of the head and neck region. Currently, the [18F]FDG PET/CT has no place as a diagnostic tool in this work-up. However, previous studies have shown that high [18F]FDG-uptake could identify mccRCC patients with a poor prognosis26,27. The characteristic over-expression of Carbonic anhydrase IX (CAIX) because of a mutational loss of Von Hippel Lindau protein, makes CAIX a suitable target for molecular PET-imaging in mccRCC patients. Previous imaging studies with radiolabeled girentuximab, a humanized monoclonal antibody targeting CAIX, have confirmed the potential of this antibody for the visualization of the primary tumor and metastatic ccRCC lesions28-30. In this thesis, we report the results from the IMPACT-RCC study, which assessed the value of [18F]FDG PET/CT and [89Zr]Zr-girentuximab PET/CT in predicting the duration of WW among mccRCC patients with a good or intermediate prognosis according to the IMDC criteria. Head and neck cancer Most cancers of the head and neck derive from the mucosal epithelium in the oral cavity, oropharynx, hypopharynx and larynx, and are collectively known as squamous cell carcinoma of the head and neck (SCCHN). SCCHN is the sixth most common cancer worldwide31. Cancer of the oral cavity, hypopharynx and larynx is generally associated with tobacco consumption, alcohol abuse or both. On the contrary, cancer of the oropharynx is increasingly attributed to an infection with human papillomavirus (HPV), requiring a different treatment strategy with an associated increased survival compared to HPV negative tumors of the head and neck32. Depended on the location and the extent of disease, the treatment of patients with locoregional HPV-/ HPV+ SCCHN consists of surgical resection with or without (chemo)radiation or chemoradiation only. Among patients with recurrent or metastatic disease, some are offered salvage resection or re-irradiation with curative intent33. The remaining patients generally have a poor prognosis34. Until recently, the first-choice palliative systemic treatment consisted of platinum chemotherapy (cisplatin/carboplatin) and 5-fluorouracil, in combination with cetuximab (epidermal growth factor receptor (EGFR) antibody). This intensive scheme results in a median overall survival is only 10.1 months, at the cost of potential high-grade toxicity and health related quality of life35,36. Therefore, there is a great need to optimize treatment approach in R/M SCCHN. Clinical trials evaluating the efficacy nivolumab (anti-PD-1) in in R/M SCCHN patients with disease progression within 6 months after platinum-based chemotherapy, have reported 1-year survival rates of 36.0% (28.5-43.4) compared to 16.6% (8.6-26.8) patients receiving a salvage chemotherapy regimen (HR 0.70 (97.73%CI 0.51-0.96, p=0.01)37. Patients with PD-L1 positive tumors compared to PD-L1 low/negative tumors, showed higher response rates and prolonged survival. As a result,

13 General introduction and outline of this thesis monotherapy pembrolizumab was granted FDA and EMA approval as 1st line treatment option in patients with R/M SCCHN >6 months after primary treatment with a high PD-L1 expression, and in combination with chemotherapy for patients with an intermediate PD-L1 expression8. The HPV status has also been correlated to ICI response, suggesting that patients with HPV+ tumors show a trend towards an improved overall survival with anti-PD-1 therapy38. Despite the advances made in the treatment options for R/M SCCHN patients, still only a small subset of R/M SCCHN patients benefit from ICI treatment. Selecting patients based on the PD-L1 CPS score has important limitations and subsequent consequences for a patient’s treatment options and associated survival chances. Molecular imaging might aid in the selection of patients for ICI treatment, for example by visualizing all accessible PD-(L)1 in a patient39. In this thesis, we report the results of the multi-center PINCH study on PD-L1 PET-imaging with 89Zr-labeled durvalumab in patients with advanced R/M SCCHN to predict durvalumab treatment response. The study design is illustrated in Figure 2. Figure 2. Molecular PET-imaging with 89Zr-labeled antibodies. A patient diagnosed with recurrent or metastatic SCCHN (1), receives an injection with 89Zr-labeled antibody (durvalumab) (2). Five days later, a PET/CT scan is performed (3). Afterwards, the patient initiated durvalumab treatment (4). Non-small cell lung cancer Lung cancer is the 1st and 2nd cause of cancer mortality in men (21.5%) and women (13.7%) in 202040. Most frequently, patients are diagnosed with non-small cell lung cancer (NSCLC) in an advanced stage41. Overall survival rates range from 59 months for patients with stage IA disease to four months for those with stage IV disease when untreated42,43. 1

14 Chapter 1 Patients with early-stage NSCLC up to stage IIIA are considered for surgical resection or stereotactic radiotherapy (stage I) with curative intent. After radical surgical resection, the 5-year survival is best in patient with stage I disease and limited for patients with stage II disease with nodal involvement (N1) (60-80% vs. 35-45%, respectively)44,45. Subsequently, patients with N1 and N2 disease (Stage IIA and IIIA) may benefit from adjuvant chemotherapy, which results in an overall 4-5% absolute 5-years survival improvement46,47. In more advanced stages with no curative treatment options, the introduction of immune checkpoint inhibitors has improved survival significantly. Pembrolizumab has been registered for stage IV NSCLC with a PD-L1 expression of ≥50% of tumor cells and no EGFR mutation or ALK translocation, based on a PFS of 10.3 vs. 6.0 months compared to chemotherapy (p<0.001)48. Also, durvalumab was granted FDA and EMA approval for stage III disease as consolidation treatment after chemoradiation49. These promising results have suggested the benefit of ICI in earlier disease stage. The first publication of neo-adjuvant ICI in NSCLC patients are encouraging, with no important safety issues and major pathological response in 45% of the resected tumors50. In this thesis, we display the results of the PINNACLE study that focused on PD-L1 PET-imaging with [89Zr]Zr-DFO-avelumab (anti-PD-L1) in patients with resectable early-stage NSCLC and irresectable metastatic NSCLC to predict (pathological) response to avelumab treatment.

15 General introduction and outline of this thesis OUTLINE OF THIS THESIS PET-imaging can serve as a complementary tool to guide the development of effective anticancer treatment strategies providing information regarding tumor (glucose) metabolism, tumor biology, tumor heterogeneity (e.g., target expression within and between tumors), tumormicroenvironment, and drug delivery. The aim of this thesis is to explore the potential of molecular PET-imaging to steer treatment of cancer patients. We studied the clinical application of molecular PET-imaging in three clinical trials among patients with consecutively metastatic clear cell renal cell carcinomas (mccRCC), head and neck cancer (SCCHN) and non-small cell lung cancer (NSCLC). IMPACT-RCC study The primary objective of the IMaging PAtients for Cancer drug SelecTion (IMPACT) - RCC study was to assess the added value of [18F]FDG PET and [89Zr]Zr-girentuximab PET to predict the time to progression under watchful waiting (WW) in patients with a good or intermediate prognosis mccRCC eligible for WW. In this study, we first evaluated the performance of baseline [18F]FDG and [89Zr]Zr-girentuximab PET/CT compared to conventional CT, to accurately determine disease status (chapter 2). We report the lesion-detection for all three imaging modalities and describe the observed differences between patients regarding the number of tumor lesions, lesion size and involved organ sites. The heterogeneity of mccRCC patients is reflected by the heterogenous [18F]FDG and [89Zr]Zr-girentuximab tumor-uptake within and between mccRCC patients. These imaging techniques provide unique insight in the tumor biology of the individual patients and may therefore facilitate personalized medicine. In chapter 3, we determined the value of [18F]FDG and [89Zr]Zr-girentuximab PET/CT in predicting the duration of a period of watchful waiting (WW). Moreover, we evaluated a model of clinical parameters to identify patients eligible for WW, and whether this model could be improved with knowledge of the baseline [18F]FDG- and/or [89Zr]Zr-girentuximab-uptake. As an introduction to the studies on PD-L1 PET-imaging, we explored the current available biomarkers for response and evaluated the preclinical and clinical experiences with molecular imaging to predict treatment response of immune checkpoint inhibitors (chapter 4) PINCH study The PINCH (PD-L1 ImagIng to prediCt durvalumab treatment response in SCCHN) trial, is a multicenter phase 1-2 study designed to assess the potential of [89Zr]Zr-DFO-durvalumab PETimaging to visualize tumor PD-L1 expression and predict disease control rate for durvalumab treatment in patients with advanced SCCHN. 1

16 Chapter 1 We performed a dose finding study to determine the feasibility of [89Zr]Zr-DFO-durvalumab PET/CT in SCCHN. Using the optimal dose, additional patients were included to correlate [89Zr]Zr-DFO-durvalumab-uptake to treatment response (chapter 5). PINNACLE study Simultaneously with the PINCH study, the PINNACLE (PD-L1 ImagiNg in Non smAll Cell Lung CancEr) study was designed to perform PD-L1 PET-imaging with [89Zr]Zr-DFO-avelumab in patients with resectable early-stage NSCLC or irresectable metastatic NSCLC. In this study, we focused on both patients with resectable early-stage NSCLC as well as patients with irresectable metastatic NSCLC. In chapter 6, we report the dose finding study and feasibility of [89Zr]Zr-DFO-avelumab PET-imaging to visualize tumor PD-L1 and correlate [89Zr]Zr-DFAavelumab-uptake to (pathological) response to avelumab treatment. Finally, we reflect on the experiences with clinical PD-(L)1 PET-imaging studies that have been performed so far, in chapter 7.

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19 General introduction and outline of this thesis 43. Groome PA, Bolejack V, Crowley JJ, et al. The IASLC Lung Cancer Staging Project: validation of the proposals for revision of the T, N, and M descriptors and consequent stage groupings in the forthcoming (seventh) edition of the TNM classification of malignant tumours. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer. 2007;2(8):694-705. 44. Travis WD, Brambilla E, Nicholson AG, et al. The 2015 World Health Organization Classification of Lung Tumors: Impact of Genetic, Clinical and Radiologic Advances Since the 2004 Classification. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer. 2015;10(9):1243-1260. 45. Postmus PE, Kerr KM, Oudkerk M, et al. Early and locally advanced non-small-cell lung cancer (NSCLC): ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2017;28(suppl_4):iv1-iv21. 46. Goldstraw P, Ball D, Jett JR, et al. Non-small-cell lung cancer. The Lancet. 2011;378(9804):1727-1740. 47. Artal Cortes A, Calera Urquizu L, Hernando Cubero J. Adjuvant chemotherapy in non-small cell lung cancer: stateof-the-art. Translational lung cancer research. 2015;4(2):191-197. 48. Herbst RS, Baas P, Kim D-W, et al. Pembrolizumab versus docetaxel for previously treated, PD-L1-positive, advanced non-small-cell lung cancer (KEYNOTE-010): a randomised controlled trial. The Lancet. 2016;387(10027):1540-1550. 49. Antonia SJ, Villegas A, Daniel D, et al. Overall Survival with Durvalumab after Chemoradiotherapy in Stage III NSCLC. New England Journal of Medicine. 2018;379(24):2342-2350. 50. Forde PM, Chaft JE, Smith KN, et al. Neoadjuvant PD-1 Blockade in Resectable Lung Cancer. N Engl J Med. 2018;378(21):1976-1986. 1

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Lesion detection by [89Zr]Zr-DFOgirentuximab and [18F]FDG PET/CT in patients with newly diagnosed metastatic clear cell renal cell carcinoma Sarah R. Verhoeff, Suzanne C. van Es, Eline Boon, Erik van Helden, Lindsay Angus, Sjoerd G. Elias, Sjoukje F. Oosting, Erik H.J.G. Aarntzen, Adriënne H. Brouwers, Thomas C. Kwee, Sandra Heskamp, Otto S. Hoekstra, Henk Verheul, Astrid A.M. van der Veldt, Elisabeth G.E. de Vries, Otto C. Boerman, Winette T.A. van der Graaf, Wim J.G. Oyen, Carla M.L. van Herpen Eur J Nucl Med Mol Imaging. 2019 Aug; 46(9):1931-1939

24 Chapter 2 ABSTRACT Purpose The main objective of this preliminary analysis of the IMaging PAtients for Cancer drug selecTion (IMPACT)-renal cell cancer (RCC) study is to evaluate the lesion detection of baseline contrastenhanced CT, [89Zr]Zr-DFO-girentuximab-PET/CT and [18F]FDG-PET/CT in detecting ccRCC lesions in patients with a good or intermediate prognosis metastatic clear cell renal cell carcinoma (mccRCC) according to the International Metastatic Database Consortium (IMDC) risk model. Methods Between February 2015 and March 2018, 42 newly diagnosed mccRCC patients with good or intermediate prognosis, eligible for watchful waiting, were included. Patients underwent CT, [89Zr]Zr-DFO-girentuximab-PET/CT and [18F]FDG-PET/CT at baseline. Scans were independently reviewed and lesions of ≥10mmand lymph nodes of ≥15mm at CT were analyzed. For lesions with [89Zr]Zr-DFO-girentuximab or [18F]FDG-uptake visually exceeding background uptake, maximum standardized uptake values (SUVmax) were measured. Results A total of 449 lesions were detected by ≥1 modality (median per patient: 7; ICR 4.25–12.75) of which 42% were in lung, 22% in lymph nodes and 10% in bone. Combined [89Zr]Zr-DFOgirentuximab-PET/CT and CT detected more lesions than CT alone: 91% (95%CI: 87–94) versus 56% (95%CI: 50–62, p=0.001), respectively, and more than CT and [18F]FDG-PET/CT combined (84% (95%CI:79–88, p<0.005). Both PET/CTs detected more bone and soft tissue lesions compared to CT alone. Conclusions The addition of [89Zr]Zr-DFO-girentuximab-PET/CT and [18F]FDG-PET/CT to CT increases lesion detection compared to CT alone in newly diagnosed good and intermediate prognosis mccRCC patients eligible for watchful waiting.

25 Lesion detection by [89Zr]Zr-DFO-girentuximab and [18F]FDG PET/CT in mccRCC patients INTRODUCTION Renal cell carcinoma (RCC) accounts for 2% of all malignancies worldwide, with an estimated 403,262 new cases in 2018. Seventy percent have a clear cell component. Metastatic clear cell (mcc) RCC has a variable course, with a subgroup of patients showing slow disease progression. In those patients, it is safe to observe the course of disease in a period of so-called watchful waiting, avoiding unnecessary side-effects and costs of systemic treatment. To identify patients eligible for watchful waiting, prognostic schemes such as the International Metastatic Database Consortium (IMDC) risk model have been used to differentiate between patients with a good, intermediate or poor prognosis1,2. For staging mRCC, European Society of Medical Oncology (ESMO) guidelines mandate contrast-enhanced computed tomography (CT) of chest, abdomen and pelvis3. Previously, an international phase II study in mRCC patients eligible for watchful waiting showed that higher numbers of IMDC adverse risk factors (p=0.0403) and higher numbers of metastatic disease organ sites (p=0.0414) were associated with a shorter period of watchful waiting4. These results substantiate the clinical value of imaging, which may be further enhanced by molecular imaging with [18F]FDG or emerging radiopharmaceuticals targeting tumor-associated antigens like carbonic anhydrase IX (CAIX) to identify patients in need of urgent systemic or local therapy. CAIX is over-expressed in 94% of ccRCC-tumors due to a mutational loss of Von Hippel Lindau protein5-7. Prognostic implications of immunohistochemically determined CAIX-expression are unequivocal7-12. In-vivo assessment of CAIX-expression can be performed with radiolabeled girentuximab (anti-CAIX antibody) PET-imaging. This technique visualizes primary and metastatic ccRCC lesions13-15. The value of [18F]FDG-PET/CT combined with CT in diagnosing and staging mRCC is not established; however, [18F]FDG-PET/CT may have prognostic value, with a positive scan being unfavourable16,17. The Imaging PAtients for Cancer drug selecTion (IMPACT)-RCC study ( NCT02228954) was designed to assess the added value of [89Zr]ZrDFO-girentuximab-PET/CT and [18F]FDG-PET/CT at presentation in predicting the duration of watchful waiting in patients with good or intermediate prognosis mccRCC. Here, we report the lesion detection of [89Zr]Zr-DFO-girentuximab-PET/CT and [18F]FDG-PET/ CT in mccRCC in addition to CT. We determined the lesion detection yield of the three modalities, assessed inter-observer agreement in [89Zr]Zr-DFO-girentuximab-uptake interpretation, and investigated determinants of quantitative [89Zr]Zr-DFO-girentuximab and [18F]FDG-uptake. 2

26 Chapter 2 MATERIALS AND METHODS Patients In this prospective multi-center cohort study, patients aged 18 years and older with histologically or cytologically proven RCC with a clear cell component, recently (<6 months) diagnosed metastases, and a good or intermediate prognosis according to IMDC score1, were enrolled in the IMPACTRCC study conducted at four Dutch academic medical centers. A period of watchful waiting for 2 months was considered optional according to treating medical oncologist. Patients who received any previous systemic treatment for RCC in any setting were excluded, but previous radiotherapy and surgery (nephrectomy or metastasectomy) was permitted. Furthermore, patients were excluded in the presence of untreated central nervous system metastases or symptomatic intracerebral metastases, pregnant or breast-feeding women. Only patients without prior systemic treatment were enrolled, therefore the IMDC criteria ‘time from diagnosis to treatment <1 year’ was adapted into ‘time from primary diagnosis to diagnosis of metastatic disease <1 year’. Watchful waiting was terminated if radiological disease progression was established, in combination with a clinical need to start systemic treatment. Patient imaging Patients underwent CT, [18F]FDG and [89Zr]Zr-DFO-girentuximab-PET/CT at the start of the watchful waiting period. Further details on the imaging modalities (acquisition and reconstruction protocols) and the conjugation, radiolabeling and quality control of [89Zr]Zr-DFO-girentuximab are provided in the supplementary data. Image assessment All CT and [18F]FDG-PET/CT scans were reported according to standard clinical practice by an experienced local radiologist and nuclear physician, respectively. The assessment of CT lesions was performed according to RECIST 1.118; however, to ensure measurements and documentation of all lesions including non-target lesions of ≥10 mm, CT scans were independently revised by one or two experienced radiologists (E.H.A; T.C.K.). The [89Zr]Zr-DFO-girentuximab PET/CTs were assessed in a central reviewing system to ensure true lesion detection and reproducible inter-observer agreement. All [89Zr]Zr-DFO-girentuximab PET/CTs were assessed by three expert nuclear physicians independently (W.O.; A.H.B.; O.H.) through online central reviewing system designed by CTMM TRaIT. The three reports were harmonized to one final report by one designated reviewer. In case of different findings, a meeting was organized to reach consensus. The treating physician was blinded for the results of either PET/CT; however, for patient safety reasons, the nuclear physician was allowed to communicate findings that required (local) interventions (e.g., brain metastases). A tumor lesion was defined visually positive based on anatomical substrate on low-dose CT in combination with [18F]-FDG and/or [89Zr]Zr-DFO-girentuximab-uptake, or solely on prominent, non-physiological antibody-uptake. Quantification of positive lesions as defined by evaluation

27 Lesion detection by [89Zr]Zr-DFO-girentuximab and [18F]FDG PET/CT in mccRCC patients reports for [18F]FDG and [89Zr]Zr-DFO-girentuximab-PET/CT was performed by drawing regionsof-interest using Inveon Research Workplace software (IRW, version 4.1). The maximum and mean standardized uptake values (SUV) were calculated. SUVmax was used for tumor tracer-uptake; SUVmean for measuring uptake in healthy organs and blood pool. Statistical analysis To compare the agreement in individual lesion detection between observers, we used dependent pair wise or multi-observers kappa-coefficients with the delta method19. Lesion detection rates per imaging modality and combined imaging modalities (CT combined with PET/CT) were estimated and compared (by Wald tests) using mixed effect logistic regression models accounting for within patient and lesion clustering by random intercepts. We evaluated lesion detection rates overall and according to organ sites. Furthermore, we compared the median number of affected organ sites across patients assessed by CT only, or in conjunction with either PET/CT using Wilcoxon signed rank tests. To assess biodistribution of [89Zr]Zr-DFO-girentuximab, we estimated the average SUV mean per organ and compared variability within and between patients (one-sample T-test). SUVmax was evaluated using descriptive methods besides mixed effects linear regression models, taking within patient clustering into account as random intercepts (using intra-class correlation coefficient (ICC) to estimate variation in uptake due to between-patient heterogeneity). These models were also used to assess determinants of tracer-uptake (introduced as fixed effects and compared by Wald tests). SUVmax was natural log-transformed to obtain appropriate model fit, resulting in geometric means or percent changes in SUVmax as interpretation of fixed effects. We fitted these models under restricted maximum likelihood using Satterthwaite approximations to degrees of freedom. We used the marginal R2 to estimate the variance in tracer-uptake explained by the fixed effects of these models20, then fitted under maximum likelihood. We report estimates with 95% confidence intervals (CI), and statistical tests were two-sided with threshold for significance of 5%, without adjusting for multiple testing. Analyses were performed in R (version 3.2.1), particularly using libraries multi-agree (version 2.1), lme4 (version1.1- 11), lmerTest (version2.0-20), and MuMIn (version1.10.0). RESULTS Patients From February 2015 until March 2018, 42 mccRCC patients were included. All patients had a histopathological diagnosis of the primary tumor, either through (partial) nephrectomy or biopsy in 36 and six patients, respectively. A total of 14 patients had a favorable prognosis. Of the remaining 28 patients, 13 had a predicted intermediate prognosis with one risk factor and 15 patients with two risk factors. This was primarily due to the diagnosis of metastases <1 year after the primary diagnosis (80%) and/or the presence of anemia (51%). There was no correlation between histology (e.g., mixed vs. pure clear cell) and the estimated prognosis according to IMDC. 2

28 Chapter 2 All patients without a previous nephrectomy had an estimated intermediate prognosis. In total 57% of all patients presented with metachronous metastases at a median interval of 0.7 (range 0–15) months between primary diagnosis and first metastasis. One patient presented with only sub-centimeter indeterminate lung lesions; therefore, lesions were not included in the analyses. Five others had a negative [18F]FDG-PET/CT, of whom one plus two other patients had a negative [89Zr]Zr-DFO-girentuximab-PET/CT. In two patients, the [18F]FDG-PET/CT and/or [89Zr]Zr-DFOgirentuximab-PET/CT revealed brain metastases warranting local treatment with stereotactic radiotherapy and temporary treatment with corticosteroids. Patient characteristics are shown in Table 1, imaging examples are shown in Figure 1. Lesion detection rates of CT, [18F]FDG and [89Zr]Zr-DFO-girentuximab-PET/CT A total of 449 lesions were identified by at least one modality (median per patient, 7; ICR 4.25–12.75). Lesions were located in lung (42%), lymph nodes (22%), bone (10%), soft tissue (8%), adrenal gland (6%), kidney (4%), pancreas (4%) or elsewhere (4%). Lesion detection rates differed across modalities: 56% was visualized by CT (95%CI 50–62). [18F]FDG-PET/CT detected 59% (95%CI 53–65; p = 0.37). [89Zr]Zr-DFO-girentuximab-PET/CT visualized 70%(95%CI 64–75), which was more than CT alone (p < 0.001) or [18F]FDG-PET/ CT alone (p < 0.005). Nine of 449 (2%) lesions were outside the field of view of CT (brain n = 2; lymph nodes in the neck n = 4, bone (extremities) n = 3). Agreement in detecting lesions between modalities was poor; kappa’s −0.12 (95%CI−0.25;0.01), −0.00 (95%CI −0.13;0.12), and 0.20 (95%CI−0.02;0.37) for CT and [89Zr]Zr-DFO-girentuximab-PET/CT, CT and [18F]FDG-PET/ CT, and [89Zr]Zr-DFO-girentuximab-PET/CT and [18F]FDG-PET/CT, respectively. Agreement between two radiologists in identifying lesions on CT was moderate (kappa 0.51; 95%CI 0.42– 0.59), and substantial for three nuclear physicians assessing [89Zr]Zr-DFO-girentuximab-PET/CTs (kappa 0.71; 95%CI 0.60–0.82). Table 1. Patient demographics and clinical characteristics. Parameter Patients (n =42) Sex Male 31 (74%) Female 11 (26%) Age (years) Median (range) 66.1 (44-86) Nephrectomy Yes 36 (86%) No 6 (14%) Histology Pure clear cell 32 (76%) Mixed 10 (24%)

29 Lesion detection by [89Zr]Zr-DFO-girentuximab and [18F]FDG PET/CT in mccRCC patients Table 1. Patient demographics and clinical characteristics. (continued) Parameter Patients (n =42) Location of first metastases a Lung b 22 (52%) Adrenal gland 4 (10%) Lymph node 9 (21%) Bone 2 (5%) Kidney 2 (5%) Other c 3 (7%) Time from diagnosis to first metastases (median 0.7; range 0-15 months) <1 year 23 (55%) ≥1 year 19 (45%) IMDC risk factors 0 (favorable) 14 (33%) 1 (intermediate) 13 (31%) 2 (intermediate) 15 (36%) * 57% presented with synchronous metastases ** 5 patients had lung-only disease (based on CT only). *** 2 patient presented with soft tissue metastases,1 patient with multiple involved organ sites. Figure 1. On the left are transversal sections of one patient of CT, [89Zr]Zr-DFO-girentuximab and [18F]FDG-PET/CT. The red circle represents an adrenal gland lesion in a patient as visualized by CT (A), [89Zr]Zr-DFO-girentuximab (B) and [18F]FDG-PET/CT (C), respectively. On the right, MIP images of [89Zr]Zr-DFO-girentuximab (D) and [18F]FDG-PET/CT (E) are presented. 2

30 Chapter 2 Combination of modalities for lesion detection With the addition of [89Zr]Zr-DFO-girentuximab-PET/CT and [18F]FDG-PET/CT, lesion detection by CT alone increased from 56% to 91% (95%CI 87–94) and 84% (95%CI 79–88),respectively. Improved lesion detection rate was apparent for all organ sites (Figure 2). The lesion detection of CT-[89Zr]Zr-DFO-girentuximab-PET/CT was better than CT-[18F]FDG-PET/CT (p < 0.005). Largest improvement was seen in the number of bone lesions, with 81% of all bone lesions detected by both [89Zr]Zr-DFO-girentuximab-PET/CT and CT as well as [18F]FDG-PET/CT with CT, compared to 16% by CT alone (p < 0.001). More lung lesions were detected by CT-[89Zr] Zr-DFO-girentuximab-PET/CT compared to CT-[18F]FDG-PET/CT [95% (95%CI 91–98)] versus 84% (95%CI 76–89; p <0.001). Lesion detection approached 100% in pancreas and kidney with combined CT and [89Zr]Zr-DFO-girentuximab-PET/CT. Conversely, detecting enlarged lymph nodes was better with combined [18F]FDG-PET/CT and CT [94% (95%CI 88–97)], compared to [89Zr]Zr-DFO-girentuximab-PET/CT and CT [83% (95%CI 73–89, p <0.05)]. Assessment of affected organ sites The median number of affected organ sites increased with the addition of [89Zr]Zr-DFOgirentuximab-PET/CT or [18F]FDG-PET/CT compared to CT alone in 27 patients (median increased from 2 to 3, range 1–7 (p < 0.005). [89Zr]Zr-DFO-girentuximab-PET/CT and [18F] FDG-PET/CT performed similarly (Table 2). Patients were categorized according to the location of their lesions (e.g., lung only; other organ(s) only and both lung and other organs). With the addition of both PET/CTs, two patients were re-categorized from lung only into ‘both lung and other organs’ based on the additional detected lymph node and bone lesions (Table 1). Figure 2. Lesion detection per imaging modality and per organ. Concordant pairs were lesions that were visualized on all 3 modalities. 9 PET detected lesions were outside the field of view of CT. *p <0∙001 compared to CT only.