Proefschrift

CLINICAL RESEARCH IN SOLID CANCERS From biology to prognostic and predictive biomarkers Debbie G.J. Robbrecht

CLINICAL RESEARCH IN SOLID CANCERS From biology to prognostic and predictive biomarkers Klinisch onderzoek in solide tumoren Van biologie naar prognostische en voorspellende factoren Debbie Germaine Joseph Robbrecht

Cover illustration: Loiss Mangnus Cover design: Ilse Modder | www.ilsemodder.nl Layout design: Ilse Modder | www.ilsemodder.nl Printed by: Gildeprint Enschede | www.gildeprint.nl ISBN: 978-94-6419-440-1 © 2022 D.G.J. Robbrecht. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior permission in writing from the proprietor.

CLINICAL RESEARCH IN SOLID CANCERS From biology to prognostic and predictive biomarkers Klinisch onderzoek in solide tumoren Van biologie naar prognostische en voorspellende factoren 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 het besluit van het Collega voor Promoties. De openbare verdediging zal plaatsvinden op 08-04-2022 Debbie Germaine Joseph Robbrecht geboren te Hulst, Nederland

Promotiecommissie Promotor: Prof. dr. R. de Wit Overige leden: Prof. dr. A.C. Dingemans Prof. dr. A.J. Gelderblom Prof. dr. A.J.M. van den Eertwegh Copromotor: Dr. F.A.L.M. Eskens

“Vooral doorgaan!” Barrie Stevens

Contents Chapter 1 General introduction and outline of the thesis PART I Chapter 2 A first-in-human phase 1 and pharmacological study of TAS-119, a novel selective Aurora A kinase inhibitor in patients with advanced solid tumours. Chapter 3 First-in-human phase 1 dose escalation study of CANO4, a first in class interleukin-1 receptor accessory protein (il1rap) antibody in patients with solid tumours. Chapter 4 ModraDoc006, an oral docetaxel formulation in combination with ritonavir (ModraDoc006/r), in metastatic castration- resistant prostate cancer patients: A phase Ib study. PART II Chapter 5 Impact of a dedicated multidisciplinary research and treatment network on outcomes of muscle-Invasive bladder cancer patients. Chapter 6 Impact of progression at baseline and on-treatment progression events in three large prostate cancer trials. Chapter 7 Pain progression at initiation of cabazitaxel in metastatic castration-resistant prostate cancer (mCRPC): A post hoc analysis of the PROSELICA study. Chapter 8 Outcomes of treatment choices in poor prognosis prostate cancer: not against all odds. PART III Chapter 9 Genome wide aneuploidy detected in circulating DNA predicts for poor response to pembrolizumab in advanced urothelial cancer patients. Chapter 10 A blood-based immune marker for resistance to pembrolizumab in patients with metastatic urothelial cancer. PART VI Chapter 11 Summary, general discussion and future perspectives 11 23 25 45 69 87 89 105 127 145 153 155 175 189 191

APPENDICES Nederlandse samenvatting Curriculum vitae PhD portfolio List of publications Dankwoord 215 216 226 228 232 236

General introduction and outline of the thesis Chapter 1

This thesis discusses research with variable approaches but with one unique goal; to contribute to the improvement of treatment and outcomes in cancer patients. The paradigm of anticancer drug development is traditionally based on a sequence of clinical study phases; phase I, II, and III. Based on pre-clinical findings, a novel antitumour compound or new treatment combination will be evaluated within the setting of a clinical phase I study. These studies aim to assess toxicity, traditionally in association with increasing doses of the study drug, the pharmacokinetic and pharmacodynamic behaviour of the compound or combination and, if possible, evaluate evidence and early signs of clinical activity. Defining the recommended phase II dose (RP2D) and/or schedule is the primary objective of such a phase I clinical study, which is typically pursued following a dose escalation design that allows for escalations up to the maximum tolerated dose based on the observed toxicity in, often end-stage, patients. The 3+3 dose-escalation algorithms still is by far the most used design(1, 2), which comprises a set of rules that dictate doseescalation decisions based on the observed safety outcomes. A dose expansion phase following the dose escalation phase has several intentions; Assessment of the RP2D in an additional group of patients with sometimes pre-specified tumour types to further assess safety and/or anti-tumour activity, evaluate alternative doses or schedules of administration(3). Typically, an anticancer compound or combination treatment that is defined to be safe and tolerable based on the results of a phase I study, is being evaluated (in its RP2D if applicable) in a phase II study to further define safety and toxicity, to estimate antitumour activity and to determine relationships between both these elements. These studies involve a few dozen to several hundreds of patients. Progression-free survival (PFS) and measures of anti-tumour activity, such as Overall Response Rate (ORR) or reduction of a tumour marker (e.g. β-HCG, PSA), are frequently used endpoints in phase II studies. When safety is being confirmed and, in particular, sufficient evidence of antitumour activity has been acquired, a phase III study can be undertaken (typically in several hundred up to thousands of patients). The outcomes (e.g. PFS, overall survival (OS), health quality of life) in these patients will be used to evaluate whether the new treatment option is to become the new standard of care. Both in the context of phase II and III studies, biomarker research can be part of such studies. In fact, already within the setting of phase I studies there is an increasing tendency to evaluate the potential role of biomarkers. Depending on the type of biomarker (e.g. a genomic signature, expression of a certain protein, imaging-based biomarker), whether it concerns a prognostic or a predictive biomarker, the phase of a 12 | CHAPTER 1 1

study and its objectives, and whether a biomarker is already validated or not, biomarker research can be implemented in studies in multiple ways. For example; - Exploratory phase II studies to unravel potential new predictive biomarkers, - Phase II studies evaluating the use of a biomarker in relation to the decision to stop or continue a certain treatment, - Studies using pre-screening for the presence of a specific biomarker to be eligible for participation in the study, - Studies that evaluate biomarkers as surrogates for risk of recurrent disease, - Phase III studies evaluating biomarkers in association to superiority of one treatment over another treatment. The other way round is also possible; Positive results from phase II or III studies leading to (provisional) approval by the authorities of a new treatment in cancer patients, could also inspire to perform new studies evaluating prognostic or predictive biomarkers. Performing a new prospective study or conducting a retrospective study by using data that are available from the already performed phase II or III studies is both possible. Combining data from various prospectively conducted studies in retrospective analyses is also feasible in case the studies reflect a comparable patient population. Nevertheless, one should always be aware of biases as a consequence of the retrospective nature of the study. This thesis presents various types of clinical research. On the one hand it presents the results of two early clinical studies with novel compounds within the setting of so-called all-comer phase I studies. In addition it presents data from a phase I clinical study in a specific patient population, be it men with advanced prostate cancer, assessing a novel oral formulation of docetaxel ; and it presents several studies to unravel prognostic and predictive markers in prostate and urothelial cancer. In PART I, results of early clinical studies performed in a patient population with various solid tumour types are being presented in chapters 2, 3 and 4. Chapter 2 presents data from the first-in-human phase I study of TAS-119, a selective Aurora A kinase (AurA) inhibitor in patients with advanced solid tumours(4). The primary objective of this study was to assess safety of this compound, to describe its toxicity up to the maximum tolerated dose and to define the recommended phase II dose using a dose-finding escalation and expansion phase. The assessment of the pharmacokinetic profile of TAS-119 and the search for target engagement in both surrogate and tumour tissue were part of the study. Concluding from this study, the RP2D and preferred schedule of administration were CHAPTER 1 | 13 1

established for TAS-119 (200 mg BID; 4 days on/3 days off, 3 out of 4 weeks) and TAS119 appeared to have a favourable safety profile that is remarkably different from other AurA inhibitors. Chapter 3 presents data from the first-in-human phase I study of CAN04, a first in class, IgG1 Interleukin-1 Receptor Accessory Protein (IL1RAP) monoclonal antibody in patients with solid tumours. Data on safety and tolerability from the dose-escalation part are being accompanied by an extensive analysis of the pharmacodynamic properties of CAN04 and a thorough description and interpretation of the frequently observed infusion related reactions (IRRs). It was concluded from this study that CAN04 can be safely administered to patients up to 10.0 mg/kg weekly, which was defined as the RP2D. Careful further evaluation of the frequency and symptoms of IRRs is important, and additional research into the underlying mechanism was felt to be indicated. Chapter 4 presents results from the phase I study of ModraDoc006 (5), an example of research evaluating the possibility to improve standard-care treatment by using an oral formulation of docetaxel instead of the broadly used intravenous formulation. Aims of the study were to evaluate safety and pharmacokinetics of ModraDoc006 and to establish the RP2D in men with metastatic castration-resistant prostate cancer (mCRPC). In this study, ModraDoc006 was evaluated with co-administration of ritonavir, a substrate for cytochrome P450 (CYP)3A4 with an inhibitory effect on the drug efflux pump P-glycoprotein (P-gp). This combination was referred to as ModraDoc006/r. The rationale behind this combination is the inhibitory effect of ritonavir on CYP3A4 and P-gp which is used to improve the bioavailability of oral docetaxel (6-8). The study led to the establishment of the RP2D of ModraDoc006/r (once weekly ModraDoc006/r 30-20/200-100 mg) and based on the favourable safety and tolerability data, a phase II study was initiated comparing weekly ModraDoc006/r with 3-weekly IV docetaxel in men with mCRPC. In PART II, three studies are presented covering separate analyses on potential prognostic factors in a setting of standard of care treatment, focussed on a urological oncology population. In Chapter 5, we evaluated treatment-related factors in association with survival outcomes in patients with muscle-invasive urothelial cancer. The data describe the differences between Dutch hospitals that were involved in a national study-group network (Dutch Uro-Oncology Study Group) versus Dutch hospitals that were not involved in this 14 | CHAPTER 1 1

network(9). Survival appeared to be superior in those patients who had their treatment, consisting of neo-adjuvant chemotherapy (NAC) followed by radical cystectomy and lymph node dissection, in a network-involved hospital compared to patients who were not treated in a network-involved hospital. Moreover, surgery was more often radical (R0) and more pelvic lymph nodes (LNs) were collected by the urologist and identified by the pathologist in the network-involved hospitals. These two variables significantly correlated with improved overall survival. Chapter 6 describes a retrospective analysis based on data from three large prospectively conducted phase III studies (VENICE(10), TAX327(11) and FIRSTANA(12)) from 3076 prostate cancer patients. The impact of type of progression, which can be divided into clinical or pain progression, radiological progression or biochemical / PSA progression, before the initiation of first-line treatment with either docetaxel or cabazitaxel was evaluated in association with survival outcomes (13). Survival appeared to be significantly shorter (~ 1 year) in patients who started treatment with first-line chemotherapy based on pain progression in comparison to patients who started because of PSA progression, also after correction for lead time bias. Moreover, type of progression during treatment was evaluated and pain or radiological progression without rising PSA was observed as the first signal for progressive disease in more than 50% of the patients. In chapter 7, similar analyses as described above were performed on data available from 1200 prostate cancer patients prospectively collected in the phase III PROSELICA study(14); a study that evaluated the non-inferiority of a lower dose of cabazitaxel (20 mg/m2) vs. the standard dose at that time (25 mg/m2), as second or third line treatment option. Our analysis revealed that an increase in pain prior to initiation of cabazitaxel was associated with worse overall survival (~ 6 months) in comparison to that of patients who only had PSA progression before initiation of cabazitaxel(15). Comparable to what has been found in the first-line setting (described in chapter 6), the difference in OS persisted after correction for lead time bias. Inpatients with pain progression, cabazitaxel dosed at 25 mg/m2 had a numerically greater activity than cabazitaxel dosed at 20 mg/m2 in terms of PSA response, radiological PFS, and OS. And comparable to what had been observed in the post-hoc analysis based on data from the VENICE, TAX327 and FIRSTANA studies, also in PROSELICA most patients (39.4%) had pain progression without a rising PSA as a first progression event during treatment. Part II ends with an editorial (chapter 8) regarding decision-making when it comes CHAPTER 1 | 15 1

to treatment choices in men with ‘poor prognostic metastatic castration-resistant prostate cancer’ (poor prognostic mCRPC)(16). The impetus to write this editorial was a study (17) reporting on the outcome of a randomized phase II study in patients with poor prognostic mCRPC, treated with cabazitaxel versus an androgen receptor pathway inhibitor (ARPI) (abiraterone or enzalutamide) in first-line. With respect to defining ‘poor prognostic mCRPC’, this study used a definition comprising all well-known prognostic factors in mCRPC(18-21). Since treatment choices in this specific population is fuel for ongoing discussion, the editorial covers this discussion by describing available literature that can support clinicians in their treatment decisions in this specific population. Although ARPIs do have a prominent place in the treatment armamentarium for men with mCRPC, the overall conclusion of the editorial is to prefer treatment with cabazitaxel in men with ‘poor prognostic mCRPC’. PART III covers two sub-studies of the RESPONDER study (ClinicalTrials.gov Identifier: NCT03263039) (chapter 9 and chapter 10). The RESPONDER is a phase II biomarker discovery study in patients with advanced or metastatic urothelial cancer (UC). Between September 2017 and December 2019 patient were entered in this study and treated with pembrolizumab in first or second line (200 mg intravenously, 3-weekly). The main aim of the study was to identify blood-based and tissue-based biomarkers that can prospectively distinguish patients that will or will not derive benefit from pembrolizumab. Patients were classified as responder when there was ongoing complete or partial response, or stable disease at 6 months after the initiation of pembrolizumab according to response evaluation criteria in solid tumors (RECIST) v1.1(22). When this was not the case, patients were classified as non-responder. Chapter 9 describes the evaluation of the modified Fast Aneuploidy Screening TestSequencing System (mFast-SeqS) in prospectively collected baseline plasma samples from patients who were treated with pembrolizumab within the context of the RESPONDER study. Since the value of circulating cell-free DNA (cfDNA) and more specifically, the fraction of circulating tumour DNA (ctDNA) within the pool of ctDNA has been demonstrated in UC before(23-28), research is focusing on its predictive usefulness as a biomarker as well as on different approaches how to investigate ctDNA. The results showed that the mFast-SeqS is an assay that can reliably estimate ctDNA abundance in plasma and has the advantage of being affordable and easily applicable in clinical practice. Moreover, the results of the mFast-SeqS analyses significantly correlated with response to treatment with pembrolizumab. 16 | CHAPTER 1 1

In Chapter 10, a sub-study of the RESPONDER study focusing on immune cell populations in blood is presented. As previous studies already showed that baseline numbers of circulating T-cells, and dynamic changes in specific T-cell subsets were associated with response to immune checkpoint inhibition (ICI) in cancer patients (29-32), this study focused on 18 different immune cell populations by using multiparameter flow cytometry. The analyses showed no differences in numbers of lymphocytes, T-cells, granulocytes, monocytes or their subsets between responders and non-responders. The most important findings however, were a high baseline neutrophil-to-lymphocyte ratio (NLR ≥ 4.5) and a high baseline mature neutrophil-to-T-cell ratio (MNTR ≥ 11.5) in association to non-response and to worse survival outcomes in comparison to patients with low NLR and low MNTR, respectively. In PART IV; Chapter 11 discusses the studies described in this thesis and future perspectives are addressed. CHAPTER 1 | 17 1

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Cabazitaxel in Metastatic Castration-Resistant Prostate Cancer (mCRPC): A Post Hoc Analysis of the PROSELICA Study. Cancers. 2021;13(6):1-13. 16. Robbrecht DGJ, Buck SAJ, de Wit R. Outcomes of treatment choices in poor prognosis prostate cancer: not against all odds. Ann Oncol. 2021;32(7):831-2. 17. Annala M, Fu S, Bacon JVW, Sipola J, Iqbal N, Ferrario C, et al. Cabazitaxel versus abiraterone or enzalutamide in poor prognosis metastatic castration-resistant prostate cancer: a multicentre, randomised, open-label, phase II trial. Ann Oncol. 2021;32(7):896-905. 18. Chi KN, Kheoh T, Ryan CJ, Molina A, Bellmunt J, Vogelzang NJ, et al. A prognostic index model for predicting overall survival in patients with metastatic castration-resistant prostate cancer treated with abiraterone acetate after docetaxel. Ann Oncol. 2016;27(3):454-60. 19. Halabi S, Kelly WK, Ma H, Zhou H, Solomon NC, Fizazi K, et al. Meta-Analysis Evaluating the Impact of Site of Metastasis on Overall Survival in Men With Castration-Resistant Prostate Cancer. J Clin Oncol. 2016;34(14):16529. 20. Hussain M, Goldman B, Tangen C, Higano CS, Petrylak DP, Wilding G, et al. Prostate-specific antigen progression predicts overall survival in patients with metastatic prostate cancer: data from Southwest Oncology Group Trials 9346 (Intergroup Study 0162) and 9916. J Clin Oncol. 2009;27(15):2450-6. 21. Khalaf DJ, Aviles CM, Azad AA, Sunderland K, Todenhofer T, Eigl BJ, et al. A prognostic model for stratifying clinical outcomes in chemotherapy-naive metastatic castration-resistant prostate cancer patients treated with abiraterone acetate. Can Urol Assoc J. 2018;12(2):E47-E52. 22. Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer. 2009;45(2):228-47. 23. Chalfin HJ, Glavaris SA, Gorin MA, Kates MR, Fong MH, Dong L, et al. Circulating Tumor Cell and Circulating Tumor DNA Assays Reveal Complementary Information for Patients with Metastatic Urothelial Cancer. Eur Urol Oncol. 2019. 24. Vandekerkhove G, Todenhofer T, Annala M, Struss WJ, Wong A, Beja K, et al. Circulating Tumor DNA Reveals Clinically Actionable Somatic Genome of Metastatic Bladder Cancer. Clin Cancer Res. 2017;23(21):6487-97. 25. Raja R, Kuziora M, Brohawn PZ, Higgs BW, Gupta A, Dennis PA, et al. Early Reduction in ctDNA Predicts Survival in Patients with Lung and Bladder Cancer Treated with Durvalumab. Clin Cancer Res. 2018;24(24):6212-22. 26. Birkenkamp-Demtroder K, Nordentoft I, Christensen E, Hoyer S, Reinert T, Vang S, et al. Genomic Alterations in Liquid Biopsies from Patients with Bladder Cancer. Eur Urol. 2016;70(1):75-82. 27. Christensen E, Birkenkamp-Demtroder K, Nordentoft I, Hoyer S, van der Keur K, van Kessel K, et al. Liquid Biopsy Analysis of FGFR3 and PIK3CA Hotspot Mutations for Disease Surveillance in Bladder Cancer. Eur Urol. 2017;71(6):961-9. 28. Birkenkamp-Demtroder K, Christensen E, Nordentoft I, Knudsen M, Taber A, Hoyer S, et al. Monitoring Treatment Response and Metastatic Relapse in Advanced Bladder Cancer by Liquid Biopsy Analysis. Eur Urol. 2018;73(4):535-40. 29. Mazzaschi G, Facchinetti F, Missale G, Canetti D, Madeddu D, Zecca A, et al. The circulating pool of functionally competent NK and CD8+ cells predicts the outcome of anti-PD1 treatment in advanced NSCLC. Lung Cancer. 2019;127:153-63. 30. Nabet BY, Esfahani MS, Moding EJ, Hamilton EG, Chabon JJ, Rizvi H, et al. Noninvasive Early Identification of CHAPTER 1 | 19 1

Therapeutic Benefit from Immune Checkpoint Inhibition. Cell. 2020;183(2):363-76.e13. 31. Kamphorst AO, Pillai RN, Yang S, Nasti TH, Akondy RS, Wieland A, et al. Proliferation of PD-1+ CD8 T cells in peripheral blood after PD-1–targeted therapy in lung cancer patients. Proceedings of the National Academy of Sciences. 2017;114(19):4993-8. 32. Huang AC, Postow MA, Orlowski RJ, Mick R, Bengsch B, Manne S, et al. T-cell invigoration to tumour burden ratio associated with anti-PD-1 response. Nature. 2017;545(7652):60-5. 20 | CHAPTER 1 1

CHAPTER 1 | 21 1

PART I

Debbie G.J. Robbrecht Juanita Lopez Emiliano Calvo Xiaomin He Hirai Hiroshi Nital Soni Natalie Cook Afshin Dowlati Angelica Fasolo Victor Moreno Ferry A.L.M. Eskens Johann S. de Bono Br J Cancer, 2020;124(2):391-398 A First-in-Human Phase 1 and Pharmacological study of TAS-119, a novel selective Aurora A kinase inhibitor in patients with advanced solid tumors Chapter 2

Abstract Purpose TAS-119 is a selective Aurora A kinase (AurA) inhibitor. Methods In this 3+3 dose escalation and expansion phase 1 study, patients (pts) with advanced solid tumors without standard treatment options were enrolled into 5 dose escalation cohorts (70-300 mg BID 4 days on/3 days off, 3 out of 4 weeks or 4 out of 4 weeks). In the expansion part the RP2D was evaluated in pts with small-cell lung cancer (SCLC), breast cancer, MYC-amplified/B-catenin mutated tumors, as well as in pts with other solid tumors. Results Dose limiting toxicity (DLT) in the dose escalation part (34 pts) was observed at various dose levels in 5 pts (1 grade 3 nausea, 1 grade 2 and 2 grade 3 ocular toxicity, 1 pt with fatigue, ocular toxicity and nausea, all grade 2). The MTD was 250 mg BID, however the RP2D was set at 200 mg BID due to additional grade 1 ocular toxicity. The most frequent related adverse events were fatigue (52.9%), diarrhea (41.2%), cough (29.4%), dyspnea (26.5%), and anorexia (26.5%). Toxicity grade ≥ 3 occurring in ≥ 10% of pts were diarrhea (14.7%) and asymptomatic increased lipase (11.8%). Grade 3 ocular toxicity occurred in two pts. Evidence for target modulation was acquired. 40 pts were enrolled in the expansion part (10 SCLC, 9 HER2 negative breast cancer, 13 MYC-amplified/B-catenin mutated tumors, 8 other). Overall, stable disease was reported in 37.8% of pts. Conclusions TAS-119 is safe with a distinct toxicity profile compared to other AurA inhibitors. 26 | CHAPTER 2 2

Introduction Mitosis in cells is strictly regulated, mostly by serine/threonine kinases. The Aurora (Aur) kinase family, classified as Aur kinase A, B and C, plays an important role in mitosis, spindle assembly checkpoint and regulation of transition from G2 to M phase1–3. AurA is overexpressed in cancer cell lines and is often amplified in human cancers 4–6. Increased AurA protein expression in cancer cells is linked to resistance to cytotoxic agents targeting the mitotic spindle checkpoint7,8. Therefore inhibiting AurA could serve as a target for the development of anticancer drugs. Multiple Aur kinase inhibitors have been developed to date2,9–11. Toxicity, in particular neutropenia and mucositis, has hampered the clinical application of these compounds. This toxicity may be, at least in part, a consequence of cross-inhibition of other kinases of these Aur kinase inhibitors. TAS-119 is a selective and orally AurA inhibitor. Preclinical data showed selectivity with a half-maximum inhibitory concentration (IC50) of 1.04 (± 0.09 nM) for AurA, an IC50 of 95 (± 11 nM) for AurB and an IC50 of 36.5 (± 6.2 nM) for AurC. TAS-119 also has some inhibitory activity on tropomyosin related kinases A (TRK) (TRK-A IC50 1.46 ± 0.16 nM; TRK-B IC50 1.53 ± 0.12 nM, TRK-C IC50 1.47 ± 0.04 nM), RET (IC50: 25.8 ± 1.5 nM) and ROS (IC50: 29.3 ±0.8 nM)12. TAS-119, and other Aur kinase inhibitors, demonstrated more potent growth inhibitory effects on cancer cells with MYC oncogene amplifications and/or mutations in the Wnt/beta-catenin pathway13,14. We conducted a first-in-human phase 1 study with TAS-119 to assess safety and tolerability, and to determine the maximum-tolerated dose (MTD) and recommended phase 2 dose (RP2D) using a ‘4-days on 3-days off’ intermittent schedule administered 3 out of every 4-weeks. Other objectives of this study included the assessment of pharmacokinetics (PK), pharmacodynamics (PD) and preliminary antitumor activity of TAS-119. In the expansion part of this study, pts with specific tumor types and tumors known to harbor either MYC oncogene amplifications or beta-catenin mutations were enrolled to further explore antitumor activity. Patients and methods Study design This study consisted of a dose escalation part with a 3+3 design to explore safety and tolerability and to establish the MTD and RP2D. For this part pts were enrolled into predefined dose levels (70, 150, 200, 250, 300 mg BID) utilizing an intermittent schedule. The rationale for this intermittent schedule (3 out of 4 weeks) was based on data showing a more favorable toxicity profile in animal studies, compared to continuous dosing regimens while maintaining antitumor activity. In addition to this intermittent CHAPTER 2 | 27 2

schedule, a prespecified continuous dosing (200 mg BID) schedule with the same weekly schedule administered 4 out of 4 weeks, was possible to explore in the escalation phase. In the dose-escalation part, a minimum of 3 pts were to be treated at each dose level (DL), and at least 6 pts were planned to be enrolled at the MTD level. The MTD was defined as the highest DL at which <33% of pts experienced a DLT during cycle 1. The RP2D was defined as a dose below or equal to the MTD based upon the evaluation of all available information (tolerability in cycles after cycle 1, PK, PD or other safety information). The RP2D was used in the expansion part of this study. Based upon the safety profile assessed in the dose escalation part of this study, either the intermittent or the continuous dosing schedule could be selected. The dose escalation part of this study enrolled pts with unselected advanced solid tumors for which no standard treatment options were available. Enrollment in the expansion part of the study was restricted to patients with either small-cell lung cancer (SCLC), HER2 negative breast cancer, or MYC-amplified/B-catenin mutated (MT) tumors, as well as pts with other solid tumors in a basket cohort. The study was approved by the local ethics committees of the participating centers and was performed according to the principles defined by the Declaration of Helsinki and Good Clinical Practice guidelines. All pts gave written informed consent prior to any study related procedure. Additional inclusion criteria (full description in the Supplementary text) were age ≥ 18 years; Eastern Cooperative Oncology Group (ECOG) performance status 0-1; adequate bone marrow, renal and hepatic function. Pts in the dose expansion part had to undergo a core tumor biopsy, as well as pretreatment sampling of non-tumor tissue (skin biopsies), for pharmacodynamic assessments if considered clinically safe and appropriate (this was optional in the dose escalation part of this study). For the expansion part of this study pts with tumors harboring MYC-amplification/Bcatenin mutations were selected, based on preclinical evidence that MYC amplification as well as beta-catenin mutation could sensitize to Aur A inhibition13–15. Treatment, starting dose and dose-escalation TAS-119 was administered orally BID as tablets of 25 and 100 mg strength, on an empty stomach. Based on rodent toxicology data, 70 mg BID was determined as the starting dose after conversion from the severely toxic dose in 10% of exposed animals (STD10) (63 mg/kg BID) to one tenth of the human equivalent dose. Predefined dose escalation cohorts were 70, 100, 150, 200, 250 and 300 mg BID. Non-hematological dose-limiting 28 | CHAPTER 2 2

toxicity (DLT) consisted of any toxicity grade ≥3 (excluding nausea/vomiting lasting < 48 hours and controlled by antiemetic therapy, diarrhea lasting < 48 hours and responsive to antidiarrheal medication, or hypersensitivity reactions), whereas hematological DLT consisted of any grade 4 neutropenia lasting > 7 days, any febrile neutropenia (documented ANC < 1000/mm3) lasting > 1 hour, any grade 4 thrombocytopenia or grade 3 thrombocytopenia associated with bleeding and requiring blood transfusion. In addition, any grade ≥3 drug-related toxicity (excluding hypersensitivity reactions) that prevented administration of ≥ 80% of the assigned dose of cycle 1 or resulted in a delay of >14 days in starting cycle 2 was considered DLT. Pretreatment and study evaluations Vital signs assessment, blood cell count, serum biochemistry, coagulation parameters, urinalysis, a 12-lead electrocardiogram (ECG) and if applicable a pregnancy test were performed at baseline. In addition, after the amendment of the protocol (Amendment 2: 31-March-2015) an ophthalmologic assessment, including visual acuity, pupil shape and pupillary reflexes, extraocular motility (eye movement) and alignment, tonometry, visual field, external examination, slit-lamp examination, and fundoscopy, was performed and repeated in all pts on day 8 and 22 during cycle 1 and on day 1 of every subsequent cycle, beginning with cycle 3. This was a consequence of ocular toxicity seen in 2 out of 26 pts until that moment. Adverse events at baseline and during the study were recorded and graded based on the Common Terminology Criteria for Adverse Events v4.03. Tumor measurements were done at the end of every 2nd cycle or as per the Institutional standard of care in case of clinical indications. Response were assessed using Response Evaluation Criteria in Solid Tumors (RECIST) v1.116. Blood samples for PK analysis were collected in Cycle 1, on days 1, 4, and 18 pre-dose and at 0.5, 1, 2, 3, 5, 8, and 12 hours post-dose. Urine samples were collected on day 1 of Cycle 1 before dosing and from 0-12 hours after dosing. Plasma and urine concentrations of TAS-119 were determined by validated LC-MS/MS method. PK parameters included the peak plasma concentration (Cmax), time to reach maximum concentration in plasma (Tmax), area under the plasma concentration-time curve up to the last observable concentration (AUC0-last) and up to infinity (AUC0-inf), terminal phase elimination halflife (T1/2), clearance (CL/F), apparent volume of distribution (Vd/F), renal clearance (CLr) and oral clearance (CL/F). Blood and tissue samples were taken for PD on-target effects during mitosis of TAS119. Firstly, the rate of phosphorylated histone H3 (pHH3) immunohistochemistry (IHC) positive cells to total cells were measured in skin biopsies and tumor samples collected CHAPTER 2 | 29 2

from pts prior to TAS-119 administration and after receiving TAS-119 administered on day 4 of cycle 1. In case of target engagement an increase in pHH3 is expected because of cells will stagnate in mitosis. Secondary, pre- and post-dose mRNA expression of genes involved in mitosis; BORA, SGOL2, KIF20A and DEPDC-1, was analyzed in tissue samples by reverse transcriptase-polymerase chain reaction (RT-PCR). The influence of polymorphisms of SLCO1B1 encoding the drug influx transporter OATP1B1 was examined in a blood sample obtained pre-dose on Day 1 of Cycle 1 for all pts during the Dose Escalation part. MYC amplification and beta-catenin mutation were assessed in archival FFPE tumor samples obtained after enrollment of the patient into the study. Optional blood samples were collected pre-dose on Day 1 and Day 22 of Cycle 1 and at disease progression, in order to do measurement of CTC and future molecular analysis of circulating tumor DNA (ctDNA). Statistical analysis Planned enrollment in the dose escalation part included 18 to 30 evaluable pts, with 3 to 6 DLT evaluable pts in each DL. To further assess the feasibility as well as preliminarily efficacy of the RP2D, approximately 40 pts were planned to be enrolled into the expansion part (approximately 10 to 15 pts in each of the expansion cohorts). An additional 20 pts were pre-planned to be enrolled in an extension of the expansion part for each specific indication in case of an overall response rate of ≥20%or ≥10% for pts with MYC amplification or beta-catenin mutation. The addition of 20 pts in the indication which demonstrates the most promising response, provides a reasonable number of pts (n=30) to be explored. Descriptive statistics were used to summarize safety data (adverse events, vital signs, and clinical laboratory results) overall response based on RECIST, PK and PD data. PK parameters were calculated by standard non-compartmental methods using PhoenixTMWinNonlin®(Ver 6.3 or later, Certara L.P.). Dose proportionality of TAS-119 was evaluated with a power and a linear regression models using logarithmic values of PK parameters such as Cmax and AUCs , as well as a one-way analysis of variance (ANOVA) using dose-normalized parameters such as Cmax, AUCs, CL/F, and apparent volume of distribution (Vd/F). The Student’s t-test was used to test statistical significance of the PD data and calculate the mean ratio for AUC0-last at RP2D or MTD. Influence of SLCO1B1 genotypes on PK parameters was tested by ANOVA. Results 34 pts were enrolled in the dose escalation part of this study and received at least one dose of TAS-119. 4 pts did not receive ≥80% of the assigned dose in cycle 1 and 30 | CHAPTER 2 2

were deemed unevaluable. In the expansion part 40 pts were enrolled and received at least one dose of TAS-119. One patient was ongoing as of the data cutoff (beta-catenin mutated NSCLC). Patient baseline characteristics for the both the dose escalation and expansion population are summarized in Table 1. TABLE 1. Baseline patient characteristics in escalation and expansion phase escalation phase expansion phase (total n = 34) (total n = 40) Age (years) Mean (SD) 66.0 (7.65) 59.4 (12.84) Median (range) 67.0 (42-77) 60.5 (20-79) Gender, n (%) Male 30 (88.2) 16 (40.0) Female 4 (11.8) 24 (60.0) Race, n (%) Caucasian 26 (76.5) 32 (80.0) Black or African-American 1 (2.9) 2 (5.0) Other 2 (5.9) 6 (15.0) Not collected 5 (14.7) Baseline ECOG, n (%) Score 0 6 (17.6) 14 (35.0) Score 1 28 (82.4) 26 (65.0) Score >1 0 0 Primary tumor type, n (%) Breast 9 (22.5) Lung 4 (11.8) 10 (25.0) Mesothelioma 13 (38.2) 6 (15.0) Other solid tumor* 17 (50) 15 (37.5) MYC-amp/B-cat mutated tumors, n (%) 13 (32.5) Number of prior regimens, n (%) 0 1 (2,9) 0 1 10 (29.4) 3 (7.5) 2 11 (32.4) 8 (20.0) >2 12 (35.3) 29 (72.5) Prior radiation therapy, n (%) Yes 17 (50.0) 26 (65.0) Palliative 10 (29.4) 17 (42.5) Therapeutic 6 (17.6) 13 (32.5) N/A 3 (8.8) 1 (2.5) No 17 (50.0) 14 (35.0) SD = standard deviation; ECOG = Eastern Cooperative Oncology Group; N/A = not evaluable. * Colorectal, renal, ovarian, pancreatic, stomach/esophageal/GE junction, prostate, head and neck, urothelial, endometrial, metastatic (adeno-) carcinoma of unknown primary, adrenal cortical carcinoma, choroidea melanoma, metastatic neuro-endocrine carcinoma, extra-skeletal chondrosarcoma CHAPTER 2 | 31 2

Briefly, for the dose escalation population the median age was 67 years (range 42-77), 30 (88%) were male, and 35.3% had received more than 2 prior therapies. Five dose levels were investigated and the median treatment duration was 57-days (range 28651). The median delivered dose intensity was 91% (range 33.3-101.5) (Table 2). For the expansion cohort there were 10 pts with SCLC (25.0%), 9 with HER2 negative breast cancer (22.5%), 6 with mesothelioma (15.0%), and 15 with other cancer types (37.5%). Of these, 13 had either MYC-amplified/B-catenin mutated tumors. Median age was 60 years (range 20-79), 16 (40%) were male, and 72.5% received more than 2 prior therapies. The median treatment duration was 56 days (range 28-616) and the median delivered relative dose intensity was 85% (range 47.9-100) (Table 2). TABLE 2. Study Drug Administration escalation phase expansion phase (total n = 34) (total n = 40) Total Dosage of TAS-119 (mg) Mean (SD) 14947.6 (18113.7) 14430.0 (18998.3) Median 10200.0 (range 630-100800) 9600.0 (range 4600105600) Treatment Duration (Days) Mean (SD) 102.1 (113.9) 96.9 (113.0) Median 57.0 (range 28- 651) 56.0 (range 28-616) Cycles Initiated Mean (SD) 3.5 (4.0) 3.4 (3.9) Median 2.0 (range 1-23) 2.0 (range 1-22) Cycles Completed n 34 40 Mean (SD) 3.1 (4.1) 2.9 (3.9) Median 2.0 (range 0-23) 2.0 (range 1-21) Relative Dose Intensity (%) n 34 38 Mean (%) (SD) 82.7 (19.8) 85.4 (16.3) Median (%) 91 (range 33.3- 101.5) 85 (range 47.9- 100) SD = standard deviation. Note: The overall treatment duration is defined as the first dose date of last cycle minus first dose date + 28. If a patient died within 28 days after the first dose day of the last cycle, the overall treatment duration is defined as death date minus first dose date + 1 Safety and tolerability Five pts in the dose escalation part experienced DLTs (table S1); one grade 3 serious adverse event of nausea (worsening nausea) 2-weeks after the first dose of study drug (DL 2 150 mg BID), one grade 2 adverse event of dry eyes 4-days after the first dose of study drug (DL 2.1 200 mg BID), one patient with grade 2 adverse events of fatigue, corneal epithelial microcysts, and nausea 8-days after the initiation of the study drug (DL 32 | CHAPTER 2 2

2.2 250 mg BID) and two pts at DL 3 (300 mg BID) experienced a serious adverse event of corneal microcysts, grade 2 and 3 resp. (5- and 4-days after the initiation of study drug, respectively). All events resolved without sequelae after the TAS-119 was stopped. 97% of pts experienced adverse events (AEs) (77% treatment-related). The most frequently reported AEs (≥ 10%) were fatigue (32%), diarrhea (24%), nausea (15%), corneal microcysts (15%), vomiting (12%), blurred vision (12%), and decreased appetite (12%). Serious adverse events (SAEs) and treatment-related SAEs were reported for 38.2% and 14.7% of patients, respectively. Four (12%) pts had AEs leading to discontinuation of study drug; 3 (9%) were considered treatment-related. One (3%) patient at DL 2 (150 mg BID) experienced an AE with the outcome of death. This patient developed disease progression, which was initially reported as a serious grade 3 adverse event that worsened in intensity to grade 5 on the day of death. The sequelae in this patient were deemed not to be study treatment related. Based on the 2 DLTs reported at DL 3 (300 mg BID), the MTD was determined to be 250 mg BID. As a result of one patient with grade 1 treatment related eye toxicity at this dose level, the RP2D moving forward in the study was set at DL 200 mg BID. In the expansion part (using the RP2D), 98% experienced AEs (80% treatment related). The most frequently reported AEs in the expansion part (≥ 10%) were diarrhea (33%), decreased appetite (18%), ALT increased (15%), AST increased (15%), blurred vision (15%), anemia (15%), fatigue (15%), nausea (13%), lipase increased (13%), and vomiting (13%) (Table 3). Toxicity grade ≥ 3 in ≥ 10% of pts were diarrhea and increased lipase. SAEs and treatment-related SAEs were reported for 40% and 3% of the pts, respectively. Three (8%) pts had adverse events leading to discontinuation of study drug; none were considered treatment-related. No adverse events with the outcome of death were reported. In total, ocular side effects were seen in 9 pts (blurred vision, dry eyes, corneal epithelial microcysts, corneal punctate epithelial erosion, punctate epitheliopathy, punctate keratitis). Of them four had an ocular DLT; two pts were treated with TAS-119 preceding the amended obligatory ophthalmologic examinations (DL 3; 300 mg BID). The ocular toxicity was dose-dependent and only seen at doses of 200 mg BID and above. The 4-week continuous dosing regimen was evaluated in the dose escalation part, in parallel with conducting the expansion part and the continuous dosing schedule has never been initiated in the expansion part based on the preliminary results from the dose escalation part, showing no significant differences between the intermittent and continuous schedule of 200 mg BID. No DLT was observed in 6 pts treated at 200 mg BID in the 4-week continuous dosing regimen. CHAPTER 2 | 33 2

TABLE 3. AEs and treatment related AEs occurring in ≥10% of all patients, by grade escalation phase expansion phase (total n = 34) (total n = 40) AE any grade (n, %) grade ≥ 3 (n, %) any grade (n, %) grade ≥ 3 (n, %) fatigue 18 (52.9) 14 (35.0) diarrhoea 14 (41.2) 5 (14.7) 18 (45.0) pain 13 (38.2) 14 (35.0) ocular symptoms* 14 (41.2) 14 (35.0) cough 10 (29.4) 5 (12.5) dyspnoea 9 (26.5) 9 (22.5) decreased appetite 9 (26.5) 13 (32.5) nausea 7 (20.6) 12 (30.0) constipation 6 (17.6) 7 (17.5) vomiting 6 (17.6) 8 (20.0) abdominal pain 5 (14.7) weight loss 4 (11.8) hypotension 5 (14.7) urinary tract infection 6 (15.0) upper respiratory tract infection 5 (12.5) headache 5 (12.5) pruritis 4 (10.0) anemia 14 (35.0) alanine aminotransferase increase 10 (25.0) aspartate aminotransferase increase 9 (22.5) lipase increase 4 (11.8) 4 (11.8) 7 (17.5) 5 (12.5) alkaline phosphatase increase 7 (17.5) gamma-glutamyltransferase increase 7 (17.5) hyperglycemia 5 (12.5) hypoalbuminaemia 5 (12.5) amylase increase 4 (10.0) hypokalaemia 4 (10.0) * corneal epithelial microcysts, blurred vision, keratitis, eye irritation, vitreous floaters, vitreous haemorrhage, blepharitis Pharmacokinetics and pharmacodynamics A total of 34 pts were evaluable for PK data. Mean plasma concentrations over time showed a dose-proportional increase of plasma exposure which did not significantly change after multiple doses on day 4 and on day 18, both being on-treatment days (Figure 1). Dose-proportionality analyses by power model, linear model, and one-way analysis of variance (ANOVA) confirmed dose-proportionality of TAS-119 PK (figure 2). 34 | CHAPTER 2 2

FIGURE 1. Mean (± SD) Plasma Concentration vs. Time Curves (Linear Scales) for TAS-119 1A. Cycle 1 Day 1 Figure 1. Mean (± SD) Plasma Concentration vs. Time Curves (Linear Scales) for TAS-119 1A. Cycle 1 Day 1 1B. Cycle 1 Day 4 1C. Cycle 1 Day 18 Legend: A total of 34 pts were evaluable for PK data. In 2 patients, parameters from one pre-dose 0 1 2 4 6 8 10 12 Time (hr) 0 5000 10000 15000 20000 Mean (±SD) TAS-119 Plasma Concentration (ng/mL) DL2.1 (Continuous) 200 mg BID (n=6) DL3 300 mg BID (n=2) DL2.2 250 mg BID (n=5) DL2.1 200 mg BID (n=7) DL2 150 mg BID (n=10) DL1 70 mg BID (n=4) 1B. Cycle 1 Day 4 Figure 1. Mean (± SD) Plasma Concentration vs. Time Curves (Linear Scales) for TAS-119 1A. Cycle 1 Day 1 1B. Cycle 1 Day 4 1C. Cycle 1 Day 18 Legend: A total of 34 pts were evaluable for PK data. In 2 patients, parameters from one pre-dose resp. were unavailable. Parameters collected on a non0 1 2 4 6 8 10 12 Time (hr) 0 5000 10000 15000 20000 Mean (±SD) TAS-119 Plasma Conce tration (ng/mL) (Continuous) 200 mg BID (n=6) 3 30 2) 2.2 250 mg BID (n=5) DL2.1 200 mg BID (n=7) DL2 150 mg BID (n=10) DL1 70 mg BID (n=4) 1C. Cycle 1 Day 18 Figure 1. Mean (± SD) Plasma Concentration vs. Time Curves (Linear Scales) for TAS-119 1A. Cycle 1 Day 1 1B. Cycle 1 Day 4 1C. Cycle 1 Day 18 Legend: A total of 34 pts were evaluable for PK data. In 2 patients, parameters from one pre-dose resp. were unavailable. Parameters collected on a nonor dose reductions (1 patient) were excluded. 0 1 2 4 6 8 10 12 Time (hr) 0 5000 10000 15000 20000 Mean (±SD) TAS-119 Plasm Concentration (ng/mL) L2.1 (Continuous) 200 mg BID (n=6) L3 300 mg BID (n=2) DL2.2 250 mg BID (n=5) DL2.1 200 mg BID (n=7) DL2 150 mg BID (n=10) DL1 70 mg BID (n=4) A total of 34 pts were evaluable for PK data. In 2 patients, parameters from one pre-dose sample and one day 4 sample resp. were unavailable. Parameters collected on a non-predefined day (2 pts), or collected from pts with dose omissions (2 pts) or dose reductions (1 patient) were excluded. CHAPTER 2 | 35 2

Figure 2. Scatterplots of TAS-119 Parameters (Cmax and AUC0-last) versus Dose on Cycle 1 Day 1 2A. Cmax versus dose in cycle 1 day 1 Figure 2. Scatterplots of TAS-119 Parameters (Cmax and AUC0-last) versus Dose on Cycle 1 Day 1 2A. Cmax versus dose in cycle 1 day 1 2B. AUC0-last versus dose in cycle 1 day 1 Legend: AUC0-last = Area under the plasma concentration-time curve from time 0 to the time point of last observable concentration; Cmax=maximum observed plasma concentration; PK=pharmacokinetics Note: Each symbol represents individual PK parameters. The regression curve was provided by the linear model. The shaded area indicates 90% confidence band. 2B. AUC0-last versus dose in cycle 1 day 1 Figure 2. Scatterplots of TAS-119 Parameters (Cmax and AUC0-last) versus Dose on Cycle 1 Day 1 2A. Cmax versus dose in cycle 1 day 1 2B. AUC0-last versus dose in cycle 1 day 1 Legend: AUC0-last = Area under the plasma concentration-time curve from time 0 to the time point of last observable concentration; Cmax=maximum observed plasma concentration; PK=pharmacokinetics Note: Each symbol represents individual PK parameters. The regression curve was provided by the linear model. The shaded area indicates 90% confidence band. AUC0-last = Area under the plasma concentration-time curve from time 0 to the time point of last observable concentration; Cmax=maximum observed plasma concentration; PK=pharmacokinetics Note: Each symbol represents individual PK parameters. The regression curve was provided by the linear odel. The shaded area indicates 90% confidence band. There was a median Tmax of 1.2 hours (range 0.5 – 2.0 hours); blood concentrations declined with a mean half-life between 2.8 and 6.0 hours. The main renal clearance (CLr) was much lower (median 0.25 L/hr, range 0.15 – 0.31 L/hr) than the main oral clearance (CL/F) (median 9.79 L/hr, range 2.72 – 13.94 L/hr) in plasma. No trends were observed between dose and urinary PK parameters. The accumulation ratios throughout 36 | CHAPTER 2 2

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