ORIGINAL ARTICLE Randomized Phase II Study of Paclitaxel plus Alisertib versus Paclitaxel plus Placebo as Second-Line Therapy for SCLC: Primary and Correlative Biomarker Analyses Taofeek K. Owonikoko, MD,a,* Huifeng Niu, PhD,b Kristiaan Nackaerts, MD, PhD,c Tibor Csoszi, MD,d Gyula Ostoros, MD,e Zsuzsanna Mark, MD,f Christina Baik, MD, MPH,g Anil Abraham Joy, MD,h Christos Chouaid, MD,i Jesus Corral Jaime, MD,j Vitezslav Kolek, MD, PhD,k Margarita Majem, MD, PhD,l Jaromir Roubec, MD, PhD,m Edgardo S. Santos, MD, FACP,n Anne C. Chiang, MD, PhD,o Giovanna Speranza, MD,p Chandra P. Belani, MD,q Alberto Chiappori, MD,r Manish R. Patel, MD,s Krisztina Czebe, MD,t Lauren Byers, MD,t Brittany Bahamon, BSc,b Cong Li, PhD,b Emily Sheldon-Waniga, PhD,b Eric F. Kong, PhD,u Miguel Williams, MS,b Sunita Badola, MSc,b Hyunjin Shin, PhD,b Lisa Bedford, MSc,b Jeffrey A. Ecsedy, PhD,b Matthew Bryant, BSc,u Sian Jones, PhD,u reports current employment by MaxCyte, Inc., and previous *Corresponding author. employment by Millennium Pharmaceuticals, Inc., Cambridge, MA, Drs. Owonikoko and Niu equally contributed to this work. USA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited. Dr. Byers reports clinical trial research funding and a Disclosure: Dr. Owonikoko reports grants and personal fees from preclinical (laboratory) research grant to her institution from Novartis, BMS, AstraZeneca, and Amgen; personal fees from Takeda, Takeda, as well as personal fees from AstraZeneca, AbbVie, AbbVie, G1 Therapeutics, EMD Serono, and PharmaMar; and grants GenMab, BergenBio, Pharma Mar, SA, Sierra Oncology, Bristol-Myers from Merck and United Therapeutics outside the submitted work. Dr. Squibb, Alethia Therapeutics, Inc., and Merck. Dr. Spigel reports Niu reports employment by Millennium Pharmaceuticals, Inc., Cam- research grants from Acerta Pharma, Aeglea Biotherapeutics, ARMO bridge, MA, a wholly owned subsidiary of Takeda Pharmaceutical Biosciences, Astellas Pharma, Celldex, Clovis Oncology, Daiichi Company Limited. Dr. Nackaerts reports a grant from Millennium Sankyo, G1 Therapeutics, GRAIL, Ipsen, Millennium, Neon Pharmaceuticals (limited funding to institution). Dr. Baik reports Therapeutics, Oncogenex, Tesaro, Transgene, University of Texas grants from Novartis, Loxo, Pfizer, AstraZeneca, Celgene, Roche/ Southwestern Medical center – Simmons Cancer Center; research Genentech, Merck Sharp and Dohme, MedImmune, Mirati, Glax- grants and travel/expense reimbursement from EMD Serono; oSmithKline, Daiichi Sankyo, National Institutes of Health/National consulting/advisory roles and research grants from Abbvie, Amgen, Cancer Institute, and Blueprint Medicines during the conduct of the Foundation Medicine, GlaxoSmithKline, Nektar, Takeda; consulting/ study, as well as grants and personal fees from Novartis and Astra- advisory roles, research grants and travel/expense reimbursement Zeneca, personal fees from F. Hoffman-La Roche AG, and grants from from AstraZeneca, Boehringer Ingelheim, Lilly, Merck, Pfizer; Loxo, Pfizer, Celgene, Roche/Genentech, Merck Sharp and Dohme, consulting/advisory roles for Evelo Therapeutics, Illumina, Moderna MedImmune, Mirati, GlaxoSmithKline, Daiichi Sankyo, the National Therapeutics, PharmaMar, Precision Oncology, TRM Oncology; travel Institutes of Health/National Cancer Institute, and Blueprint and expense reimbursement from Genzyme, Intuitive Surgical, Medicines outside the submitted work; she also reports consultation Purdue Pharma, Spectrum Pharmaceuticals, and Sysmex, outside the for Novartis and AstraZeneca. Dr. Chouaid reports personal fees submitted work. The remaining authors declare no conflict of interest. from AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, Hoffman- la Roche, Sanofi Aventis, Lilly, Novartis, MSD, BMS, and Amgen Trial Registration: Phase 2 Study of Alisertib (MLN8237) in Combination outside the submitted work. Dr. Santos reports speaker bureau with Paclitaxel versus Placebo in Combination with Paclitaxel as service for Genentech, Lilly, Takeda, Celgene, Merck, Pfizer, Second-Line Therapy for Small Cell Lung Cancer. NCT02038647. AstraZeneca, and Boehringer Ingelheim. Dr. Chiang reports personal Presented at the European Society of Medical Oncology 2016 Annual fees from AstraZeneca and AbbVie, as well as grants from Abbvie, Congress, October 7–11, 2016, Copenhagen, Denmark; and the Inter- Lilly, BMS and Amgen outside the submitted work. Dr. Chiappori national Association for the Study of Lung Cancer 17th World Congress reports speaker bureau service for Takeda, Genentech, Merck, on Lung Cancer, December 4–7, 2016, Vienna, Austria. and Celgene; advisory board service for AstraZeneca, BMS, Amgen, and Pfizer; and receipt of research funds from Novartis, BMS, and Address for correspondence: Taofeek K. Owonikoko, MD, Department AstraZeneca. Ms. Bahamon, Dr. Li, Mr. William, Ms. Badola, Dr. Shin, of Hematology and Medical Oncology, Winship Cancer Institute of Emory Ms. Bedford, and Ms. Leonard report employment by Millennium University, 1365 Clifton Rd., NE, Room C3080, Atlanta, GA, 30322. Pharmaceuticals, Inc., Cambridge, MA, USA, a wholly owned E-mail: towonik@emory.edu subsidiary of Takeda Pharmaceutical Company Limited. Dr. Sheldon- ª 2019 International Association for the Study of Lung Cancer. Waniga reports current employment by Bluebird Bio and previous Published by Elsevier Inc. This is an open access article under the employment by Millennium Pharmaceuticals, Inc., Cambridge, MA, CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/ USA, a wholly owned subsidiary of Takeda Pharmaceutical Company 4.0/). Limited. Dr. Ecsedy reports current employment by Kyn Therapeutics and previous employment by Millennium ISSN: 1556-0864 Pharmaceuticals, Inc., Cambridge, MA, USA, a wholly owned https://doi.org/10.1016/j.jtho.2019.10.013 subsidiary of Takeda Pharmaceutical Company Limited. Dr. Ullmann Journal of Thoracic Oncology Vol. 15 No. 2: 274-287 February 2020 Alisertib/Paclitaxel for SCLC 275 John Simmons, PhD,u E. Jane Leonard, MSc,b Claudio Dansky Ullmann, MD, PhD,b David R. Spigel, MD,v on behalf of the C14018 study investigators a Winship Cancer Institute of Emory University, Atlanta, Georgia b Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited, Cambridge, Massachusetts c KU Leuven, Universitaire Ziekenhuizen, Leuven, Belgium d Hetenyi G Korhaz, Szolnok, Hungary e Orszagos Koranyi TBC es Pulmonologiai Intezet, Budapest, Hungary f Tudogyogyintezet Torokbalint, Torokbalint, Hungary g University of Washington Seattle Cancer Care Alliance, Seattle, Washington h University of Alberta, Cross Cancer Institute, Edmonton, Alberta, Canada i CHI de Créteil, Créteil, France j Hospital Universitario Virgen del Rocio, Seville, Spain k Fakultni Nemocnice Olomouc, Olomouc, Czech Republic l Hospital de la Santa Creu i Sant Pau, Barcelona, Spain m Fakultni Nemocnice Ostrava, Ostrava Poruba, Czech Republic n Lynn Cancer Institute/Boca Raton Regional Hospital, Boca Raton, Florida o Yale University School of Medicine, New Haven, Connecticut p Université de Sherbrooke, Centre intégré de cancérologie de la Montéregie, Hôpital Charles Le Moyne, Greenfield Park, Quebec City, Canada q Penn State Cancer Institute, Hershey, Pennsylvania r H. Lee Moffitt Cancer Center, Tampa, Florida s Florida Cancer Specialists/Sarah Cannon Research Institute, Sarasota, Florida t Tudogyogyintezet Torokbalint, Törökbálint, Hungary u University of Texas M. D. Anderson Cancer Center, Houston, Texas v Personal Genome Diagnostics, Baltimore, Maryland Received 4 July 2019; revised 16 October 2019; accepted 17 October 2019 Available online - 23 October 2019 ABSTRACT 0.239–0.654, p ¼ 0.0003]), and overall survival (7.20 versus 4.47 months, respectively [HR ¼ 0.427, 95% CI: 0.259–0.704, Introduction: We assessed the Aurora A kinase inhibitor, p ¼ 0.00085]). A subset of patients with c-Myc expression alisertib, plus paclitaxel (henceforth referred to as alisertib/ showed significantly improved PFS with alisertib/paclitaxel. paclitaxel) as second-line treatment for SCLC. The incidence of grade 3 or higher drug-related adverse Methods: In this double-blind study, patients with relapsed events was 67% (58 patients) with alisertib/paclitaxel or refractory SCLC were stratified by relapse type (sensitive versus 22% (25 patients) with placebo/paclitaxel. Twelve versus resistant or refractory) and brain metastases and patients (14%) versus 11 (12%) died on study, including randomized 1:1 to alisertib/paclitaxel or placebo plus pacli- four versus zero treatment-related deaths. taxel (henceforth referred to as placebo/paclitaxel) in 28-day Conclusions: Efficacy signals were seen with alisertib/ cycles. The primary end point was progression-free survival paclitaxel in relapsed or refractory SCLC. c-Myc expression (PFS). Associations of c-Myc expression in tumor tissue and mutations in cell cycle regulators may be potential (prespecified) and genetic alterations in circulating tumor predictive biomarkers of alisertib efficacy; further pro- DNA (retrospective) with clinical outcome were evaluated. spective validations are warranted. Results: A total of 178 patients were enrolled (89 in each  2019 International Association for the Study of Lung arm). The median PFS was 3.32 months with alisertib/ Cancer. Published by Elsevier Inc. This is an open access paclitaxel versus 2.17 months with placebo/paclitaxel (haz- article under the CC BY-NC-ND license (http:// ard ratio [HR] ¼ 0.77, 95% confidence limit [CI]: 0.557– creativecommons.org/licenses/by-nc-nd/4.0/). 1.067, p ¼ 0.113 in the intent-to-treat population versus HR ¼ 0.71, 95% CI: 0.509–0.985, p ¼ 0.038 with corrected analysis applied). Among 140 patients with genetic alterna- Keywords: Phase II; Aurora A kinase; Alisertib; Paclitaxel; tions, patients with cell cycle regulator mutations (cyclin- SCLC dependent kinase 6 gene [CDK6], retinoblastoma-like 1 gene [RBL1], retinoblastoma-like 2 gene [RBL2], and retinoblas- toma 1 gene [RB1]) had significantly improved PFS with Introduction alisertib/paclitaxel versus with placebo/paclitaxel (3.68 SCLC is an aggressive malignancy, accounting for versus 1.80 months, respectively [HR ¼ 0.395, 95% CI: 13% to 18% of all lung cancer diagnoses.1,2 For many 276 Owonikoko et al Journal of Thoracic Oncology Vol. 15 No. 2 patients with SCLC, the outlook is bleak. In treated primary tumors (Takeda, data on file). The preclinical extensive-stage SCLC, the median overall survival (OS) is and clinical data thus provided justification for this just 7 to 12 months.3,4 The prognosis for relapsed or phase II study of alisertib/paclitaxel as second-line refractory SCLC is more dismal, with a median OS of 4 to therapy for relapsed or refractory SCLC. As part of this 5 months.5 study, analyses were undertaken to assess the impact of There is a strong correlation between the efficacy of c-Myc protein expression and genetic alterations on second-line therapy and the quality and duration of clinical outcomes. response (DOR) to initial treatment (sensitive versus resistant or refractory relapse).6 Response rates are Patients and Methods particularly poor (10%) in patients with resistant or refractory disease who relapse 3 months or less from the Study Design end of initial therapy.5 Patients with platinum-sensitive This multicenter, randomized, double-blind, placebo- disease (relapse >3 months from end of initial ther- controlled phase II trial (NCT02038647) enrolled pa- apy) have a relatively better outcome (response rate tients across 54 sites in the United States (19 sites), w25%).5 Canada (three sites), and Europe (32 sites) from May 7, Because of poor responses to current treatments, 2014, to October 26, 2015. The trial was conducted in there is a critical unmet medical need in patients with accordance with applicable regulatory requirements, SCLC, thereby justifying efforts to evaluate novel tar- International Conference on Harmonization Good Clin- geted agents (with validated predictive biomarkers).7,8 ical Practice guidelines, and the ethical principles foun- One potential target is Aurora A kinase (AAK), a key ded in the Declaration of Helsinki. Study documentation regulator of mitosis.9 AAK is amplified or overexpressed was approved by the institutional review board and/or in several solid tumors, including SCLC, and may play a independent ethics committee at each site. Patients role in tumorigenesis.10,11 Inhibition of AAK leads to provided written informed consent. disrupted mitosis and cell death, reduced proliferation, Patients were randomized 1:1 to receive either ali- and induction of apoptosis in SCLC cells.10,12 sertib (40 mg by mouth twice daily for 3 weeks on days Amplification of v-myc avian myelocytomatosis viral 1–3, 8–10, and 15–17) plus paclitaxel (60 mg/m2 oncogene homolog gene (MYC) in SCLC cell lines is intravenously on days 1, 8, and 15) or placebo (by mouth associated with improved sensitivity to Aurora kinase twice daily as per alisertib) plus paclitaxel (80 mg/m2 inhibitors.13,14 Amplification and overexpression of the intravenously on days 1, 8, and 15) in 28-day cycles. The MYC gene family occurs in 18% to 31% of SCLCs and is dosing schedule permitted maximal overlap of systemic more common in chemorefractory disease.15 The gene exposures between alisertib and paclitaxel while product, c-Myc (a transcription factor), binds directly to providing sufficient treatment-free periods to allow re- AAK, and inhibition of this interaction by AAK inhibitors covery from toxicities associated with both agents. results in c-Myc degradation and cell death.16,17 Thus, Randomization was stratified by type of relapse after the Myc–AAK protein complex represents an actionable primary treatment, based on the common definition for drug target for AAK inhibitors. each type5,6 (with sensitive defined as relapsed >90 but Alisertib (MLN8237) is an investigational, selective, <180 days after primary treatment and resistant or re- oral, small molecule AAK inhibitor that has been fractory defined as relapsed 90 days after primary studied in various solid tumors and hematologic malig- treatment) and presence of brain metastases (yes versus nancies.18-23 Single-agent activity was demonstrated in a no) at study entry. Patients received treatment until phase II study of patients with relapsed or refractory progressive disease, discontinuation because of toxicity, solid tumors, including SCLC (n ¼ 48);23 in patients with loss to follow-up, study termination, protocol violation, SCLC, the objective response rate (ORR) was 21% and or patient withdrawal. The study team and site staff the median progression-free survival (PFS) was 2.1 responsible for assessing patients were blinded to months.23 On the basis of preclinical evidence of synergy, treatment assignment; participating sites were required alisertib plus paclitaxel (henceforth referred to as ali- to designate a non-blinded study pharmacist for dose sertib/paclitaxel) was evaluated in patients with breast preparations. and ovarian cancer19 and showed promise over pacli- taxel alone, with PFS and ORR trending in favor of the Patients combination.19 Preclinical data have also shown ali- The study enrolled patients at least 18 years old with sertib/paclitaxel synergy in SCLC; increased antitumor a pathologically (histologically or cytologically) activity with the combination versus with the single confirmed diagnosis of SCLC. Patients were required to agents was demonstrated in xenograft tumor models have progressed within 180 days of last platinum dose, derived from human SCLC cell lines and human SCLC after receiving a standard platinum-based chemotherapy February 2020 Alisertib/Paclitaxel for SCLC 277 regimen as first-line treatment, and to have measurable safety population included all patients who received at disease per the Response Evaluation Criteria in Solid least one dose of any study drug. Tumors version 1.1 within 2 weeks before randomiza- Full statistical methods are provided in the tion. Other key inclusion and exclusion criteria can be Supplementary Methods. Assuming a median PFS of 3 found in the Supplementary Methods. months for the placebo plus paclitaxel (henceforth referred to as placebo/paclitaxel) arm and also assuming that alisertib/paclitaxel could improve median PFS to 5 End Points and Assessments months (40% reduction of hazard), a minimum of 138 The primary end point was PFS (time from random- PFS events were required for the primary analysis (two- ization to progressive disease or death), with patients sided alpha 0.05, power 85%). PFS was tested by using a stratified by type of relapse after primary treatment two-sided stratified log-rank test to compare arms. (sensitive versus resistant or refractory disease) and Hazard ratios (HRs) and 95% confidence intervals (CI) presence of brain metastases. PFS was also analyzed in were estimated by using a Cox proportional hazard patient subgroups according to baseline characteristics. regression model stratified by type of relapse and Secondary end points were safety and/or tolerability, OS, presence of brain metastases, with treatment arm ORR including complete response, disease control rate included as a factor in the model. Kaplan-Meier survival (DCR: complete response, partial response, or stable curves were provided for each arm. OS and other time- disease 8 weeks), and DOR. Exploratory end points to-event end points were analyzed by using similar included correlative biomarker studies to evaluate the methods. impact of c-Myc expression and genetic alterations on Assessment of c-Myc expression by immunohisto- clinical outcomes (PFS and OS). chemistry was analyzed as (1) percentage of positive Extent of disease was evaluated according to the cells or (2) dichotomized readout converted to a binary Response Evaluation Criteria in Solid Tumors version 1.1 variable (positive versus negative) based on modal in- at screening, after every cycle for the first 6 months, and tensity. For both methods, a Cox proportional hazard subsequently every two cycles (between days 21 and model was used to analyze PFS, with treatment and c- 28) to assess disease response and progression; because Myc expression as the main effects. Interactions between of the aggressiveness of SCLC, disease assessments were NGS-identified genetic alterations and treatments (ali- performed more frequently than in common clinical sertib versus placebo) were tested with a Cox propor- practice. Radiographic images (by contrast-enhanced tional hazards model for PFS and OS. The genetic computed tomography or magnetic resonance imaging) alterations tested included single-gene mutations were assessed locally and submitted for central review if (mutant allele frequency 0.01), pathway gene muta- the results were positive. For biomarker evaluations, c- tions, and mutation load. Myc expression was assessed by immunohistochemical A protocol amendment (January 2015) corrected the analysis of c-Myc expression in tumor tissue, and genetic stratification definition of relapse type after primary alterations were assessed retrospectively by next- treatment so that relapses were recorded “from last generation sequencing (NGS) of circulating tumor DNA administration of platinum-based chemotherapy” rather (ctDNA) from peripheral blood by using a custom than “from initial response.” To maintain balance, the PlasmaSelect-R (Personal Genome Diagnostics, Inc., Bal- primary end point was analyzed by using the original timore, MD) targeted gene NGS panel (Supplementary stratification definition of relapse type. However, a Table 1). sensitivity analysis with use of the corrected stratifica- Details of follow-up for PFS and OS, and the methods tion definition was also included. used for the biomarker assessments, are provided in the Supplementary Methods. Toxicity was graded according to the National Cancer Institute Common Terminology Results Criteria for Adverse Events version 4.03. The use of Patients myeloid growth factors for the management of neu- A total of 178 patients were enrolled and randomized tropenia was not mandated but was permitted at the to receive alisertib/paclitaxel (n ¼ 89) or placebo/ discretion of the investigator if clinically indicated, ac- paclitaxel (n ¼ 89 [Supplementary Fig. 1]). Patient cording to local guidelines and the product label. characteristics at baseline are shown in Table 1. Two patients in the alisertib/paclitaxel arm were randomized Statistical Analysis but did not receive any study drug and were included in The intent-to-treat (ITT) population included all the ITT population analysis only. When the corrected randomized patients and was used for the primary definition of relapse type (sensitive versus resistant or analysis of PFS and all secondary efficacy end points. The refractory disease) was used, 53 (30%) patients had 278 Owonikoko et al Journal of Thoracic Oncology Vol. 15 No. 2 Table 1. Demographics and Baseline Disease Characteristics (Intent-to-Treat Population) Characteristic Alisertib/Paclitaxel (n ¼ 89) Placebo/Paclitaxel (n ¼ 89) Median age, y (range) 62 (37–81) 62 (46–86) Male sex, n (%) 51 (57) 50 (56) Median time since initial diagnosis, mo (range) 7.4 (3–12) 7.8 (3–26) VALG stage at initial diagnosis, n (%) Limited 18 (20) 8 (9) Extensive 61 (69) 62 (70) Unknown 10 (11) 19 (21) ECOG PS, n (%) 0 27 (30) 18 (20) 1 62 (70) 71 (80) Smoking history, n (%) Never 4 (4) 7 (8) Former/current 52 (58)/33 (37) 52 (59)/29 (33) Missinga 0 1 (1) No prior prophylactic cranial irradiation, n (%) 59 (66) 63 (71) No brain involvement at baseline, n (%) 62 (70) 63 (71) Type of relapse after primary treatment (by IVRS), n (%) Sensitiveb 36 (40) 40 (45) Resistant/refractoryb 36 (40)/17 (19) 34 (38)/15 (17) Type of relapse after primary treatment (corrected),c n (%) Sensitive 29 (33) 35 (42) Resistant/refractory 41 (46)/19 (21) 33 (39)/16 (19) Not classifiable or missinga 0 5 (6) a Missing data were treated as missing and no data imputation was applied. b Sensitive disease is defined as progression of disease observed >90 days from the last dose of first-line platinum-based chemotherapy, resistant disease is defined as progression of disease observed 90 days after first-line platinum-based chemotherapy, and refractory disease is defined as no objective response and progression either during or immediately after first-line platinum-based chemotherapy. c Protocol Amendment 2 (January 2015) corrected the stratification definition for time to relapse to follow standard guidance by counting from date of last dose of frontline chemotherapy as opposed to by counting from initial response to first-line platinum-based chemotherapy, as originally set out in the protocol; the stratification factors for the study were corrected accordingly. When the corrected definition of time to relapse for type of relapse after primary treatment (sensitive versus resistant or refractory disease) was used, 53 patients (30%) had their relapse stratification factor changed after initial classification. In all, 15 patients in the alisertib plus paclitaxel arm and 14 in the placebo plus paclitaxel arm were reassigned from “sensitive” to “resistant or refractory”’ relapse, and 8 and 11 patients, respectively, were reassigned from “resistant or refractory” to “sensitive” relapse. ECOG PS, Eastern Cooperative Oncology Group performance status; IVRS, Interactive Voice Response System; VALG, Veterans Administration Lung Study Group. their relapse stratification factor changed after initial PFS between arms based on the presence of metastases, classification, and there was a higher ratio of percentage enrollment region, Eastern Cooperative Oncology Group of sensitive patients in the placebo/paclitaxel arm to that performance status, age category, race, or disease stage in the alisertib/paclitaxel arm (42:33) compared with (data not shown). Although there was no difference in when the original definition (45:40) was used (see PFS for male patients (HR ¼ 1.03, 95% CI: 0.677–1.578, Table 1). In addition, for five patients in the placebo/ p ¼ 0.876), there was a marginal difference for female paclitaxel arm who had been enrolled under the original patients in favor of alisertib/paclitaxel compared with definition, sufficient data had not been provided to allow placebo/paclitaxel (median PFS 4.4 versus 2.6 months classification under the corrected definition, so [HR ¼ 0.60, 95% CI: 0.367–0.992, p ¼ 0.043]). For pa- they were excluded from the corrected stratification tients with resistant or refractory relapse (corrected analysis. definition), the median PFS was 2.86 months with ali- sertib/paclitaxel versus 1.68 months with placebo/ paclitaxel (HR ¼ 0.66, p ¼ 0.037 [Fig. 1B]). For patients Efficacy with sensitive relapse (corrected definition), the median The median PFS was 3.32 months in the alisertib/ PFS was 3.72 months with alisertib/paclitaxel versus paclitaxel arm versus 2.17 months in the placebo/ 3.34 months with placebo/paclitaxel, respectively (HR ¼ paclitaxel arm (HR ¼ 0.77, 95% CI: 0.557–1.067, p ¼ 0.86, 95% CI: 0.493–1.497, p ¼ 0.590). 0.113). In the prespecified sensitivity analysis, when the At data cutoff, there was no significant difference in corrected definition for the stratification factor of OS between arms (Fig. 1C). The median OS was 6.11 relapse type was used, the HR was 0.71 (95% CI: 0.509– months with alisertib/paclitaxel versus 5.42 months 0.985, p ¼ 0.038; [Fig. 1A]). There were no differences in with placebo/paclitaxel. Per protocol, a further OS February 2020 Alisertib/Paclitaxel for SCLC 279 analysis, including data from at least 80% of patients, Exploratory Correlative Biomarker Studies was conducted on October 15, 2016. Overall, 151 pa- In all, 46 tumor tissue samples were evaluable for the tients had died at this time, which was an increase of 23 c-Myc expression by immunohistochemistry analysis; 33 deaths from the previous analysis. The updated median (72%) were positive (a modal intensity of 1þ, 2þ, or OS was 6.86 months with alisertib/paclitaxel versus 5.58 3þ) for c-Myc expression and 13 (28%) were negative months with placebo/paclitaxel (HR ¼ 0.93, 95% CI: (modal intensity ¼ 0). PFS by c-Myc expression is shown 0.652–1.341, p ¼ 0.714; HR with use of the corrected in Fig. 1D and 1E. In c-Myc–positive patients, the median definition for relapse type ¼ 0.73, 95% CI: 0.520–1.021, PFS was 4.64 months with alisertib/paclitaxel (n ¼ 17) p ¼ 0.064). versus 2.27 months with placebo/paclitaxel (n ¼ 16) The ORR was 22% with alisertib/paclitaxel versus (HR ¼ 0.29, 95% CI: 0.12–0.72). In c-Myc–negative pa- 18% with placebo/paclitaxel (Table 2). The DCR was tients, the median PFS was 3.32 months with alisertib/ 58% versus 46%, respectively. For the subgroup of paclitaxel (n ¼ 6) versus 5.16 months with placebo/ resistant or refractory patients when the corrected paclitaxel (n ¼ 7) (HR ¼ 11.8, 95% CI: 1.52–91.2). c-Myc definition for relapse type was used, the DCR was expression was strongly associated with improved PFS significantly higher with alisertib/paclitaxel than with when c-Myc was evaluated as a continuous variable of placebo/paclitaxel (55% versus 33% [odds ratio ¼ 0.40 percentage of cells staining positive (pcontinuous ¼ (range 0.18–0.87), p ¼ 0.020]). The median DOR among 0.0045) or a binary (positive versus negative) mode responders was 3.16 months in the alisertib/paclitaxel (pbinary ¼ 0.0006). arm and 2.79 months in the placebo/paclitaxel arm (see Out of 176 patients, 155 (88%) provided plasma Table 2). The median time to progression was 3.58 samples that were processed for NGS analysis of ctDNA. months with alisertib/paclitaxel versus 2.60 months In all, 142 patient samples (81%) were successfully with placebo/paclitaxel (HR ¼ 0.67, p ¼ 0.038) (see sequenced and genetic alterations were identified from Table 2). 140 patients (80%) (Supplementary Fig. 1). The full A 1.0 0.9 Treatment group: Alisertib + paclitaxel Placebo + paclitaxel C 1.0 0.9 HR: 0.87; 95% CI: 0.612–1.236; p = 0.714 Corrected, HR: 0.79; 95% CI: 0.551–1.141; p = 0.209 0.8 HR: 0.77; 95% CI: 0.557–1.067; p = 0.113 0.8 Corrected, HR: 0.71; 95% CI: 0.509–0.985; p = 0.038 Survival probability 0.7 0.7 PFS probability 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0 0 0 30 60 90 120 150 180 210 240 270 300 0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 Number at risk: Time (days) Number at risk: Time (days) Alisertib + paclitaxel 89 74 55 41 28 13 10 6 3 0 0 Alisertib + paclitaxel 89 82 69 60 52 40 35 25 21 14 6 3 2 1 1 1 0 Placebo + paclitaxel 89 65 45 27 19 12 8 4 3 3 0 Placebo + paclitaxel 89 86 72 58 46 37 24 17 12 9 7 3 2 2 0 0 0 D 1.00 HR: 0.29; 95% CI: 0.12–0.72 B PFS probability 0.75 0.50 1.0 Corrected, HR: 0.66; 95% CI: 0.442–0.983; p = 0.037 0.9 0.25 0.8 0.7 0 PFS probability 0 100 200 300 0.6 Time (days) 0.5 0.4 0.3 E 1.00 HR: 11.8; 95% CI: 1.52–91.2 0.2 PFS probability 0.75 0.1 0 0.50 0 30 60 90 120 150 180 210 240 270 300 Time (days) 0.25 Number at risk: Alisertib + paclitaxel 60 49 36 26 17 7 5 3 1 0 0 0 Placebo + paclitaxel 49 30 18 11 7 4 3 2 1 1 0 0 100 200 300 Time (days) Figure 1. Clinical outcomes for the intent-to-treat population and according to resistant or refractory relapse and c-Myc expression. (A) Progression-free survival (PFS) in all patients. (B) PFS in patients with resistant or refractory relapse. (C) Overall survival in all patients. (D) PFS in patients positive for c-Myc expression. (E) PFS in patients negative for c-Myc expression. (A–C) Hazard ratios (HRs), 95% confidence intervals (CIs), and p values for the comparison of alisertib plus paclitaxel versus placebo plus paclitaxel are shown per protocol and with use of the corrected definition for the stratification factor of relapse type. 280 Owonikoko et al Journal of Thoracic Oncology Vol. 15 No. 2 Table 2. Best Overall Response Rate (ITT Population), Duration of Response, and Time to Progression Alisertib/Paclitaxel Placebo/Paclitaxel OR/HR (95% CI); Variable (n ¼ 89) (n ¼ 89) p Value Response, n (%) ORR (CR þ PR) 20 (22) 16 (18) 0.74 (0.35–1.55); 0.406 CR 1 (1) 0 NE PR 19 (21) 16 (18) NE Stable disease 49 (55) 44 (49) NE DCR (CR þ PR þ stable disease 8 weeks) 52 (58) 41 (46) 0.59 (0.32–1.08); 0.077 PD 13 (15) 23 (26) NE No on-study imaginga 7 (8)b 6 (7)c NE Median DOR (responders), mo (95% CI) 3.16 (2.76–4.64) 2.79 (1.91–5.82) NE Median TTP (ITT), mo (95% CI) 3.58 (2.86–4.41) 2.60 (1.74–3.45) 0.67 (0.462–0.982); 0.038 a Missing data were treated as missing and no data imputation was applied. b Three patients died before on-study imaging, two patients were not assessed, and two patients were not dosed. c Four patients died before to on-study imaging, one patient was not assessed, and one patient was too ill for imaging. CI, confidence interval; CR, complete response; DCR, disease control rate; DOR, duration of response; HR, hazard ratio; ITT, intent-to-treat population; NE, not evaluated; OR, odds ratio; ORR, objective response rate; PD, disease progression; PR, partial response; TTP, time to progression. results from this genetic profiling are provided in the phosphoinositide 3-kinase (KIT proto-oncogene receptor Supplementary Results (Supplementary Figs. 2–6 and tyrosine kinase gene [KIT], phosphatidylinositol-4,5- see also Supplementary Fig. 1). The genetic mutation bisphosphate 3-kinase catalytic subunit alpha gene profiles revealed from the SCLC plasma ctDNA samples [PIK3CA], phosphatase and tensin homolog gene [PTEN], were concordant with those previously identified in tumor protein p73 gene [TP73]) signaling, or histone SCLC primary tumor tissue,24 matching the mutational modifications (CREBBP and E1A binding protein p300 frequency trends in characterized genes, such as tumor gene [EP300]) (see Supplementary Fig. 5). Patients with protein p53 gene (TP53), retinoblastoma 1 gene (RB1), mutations in cell cycle regulators (“mutant”) had formin 2 gene (FMN2), notch receptor 1 gene (NOTCH1), improved median PFS (3.68 versus 1.80 months [HR ¼ collagen type XXII alpha 1 chain gene (COL22A1), spec- 0.395, 95% CI: 0.239–0.654, p ¼ 0.0003]) (Fig. 3A and trin repeat containing nuclear envelope protein 1 gene 3B) and OS (7.20 versus 4.47 months [HR ¼ 0.427, 95% (SYNE1), CREB binding protein gene (CREBBP), ATRX CI: 0.259–0.704, p ¼ 0.00085]) with alisertib/paclitaxel chromatin remodeler gene (ATRX), notch receptor (n ¼ 40) compared with placebo/paclitaxel (n ¼ 47) 3 gene (NOTCH3), regulating synaptic membrane (Fig. 3C and 3D). Conversely, patients without mutations exocytosis 2 gene (RIMS2), and contactin associated in cell cycle regulators (“wild type”) had no improve- protein-like 2 gene (CNTNAP2) (Fig. 2A). The mutation ment in median PFS (2.63 versus 2.60 months [HR ¼ spectrum of the key genes also showed high concor- 1.31, 95% CI: 0.736–2.33, p ¼ 0.359]) (see Fig. 3A and dance (see Supplementary Fig. 3). The correlative anal- 3B) or OS (4.47 versus 5.95 months [HR ¼ 1.70, 95% CI: ysis of clinical outcomes (PFS and OS) was assessed in 0.865–3.33, p ¼ 0.124]) with alisertib/paclitaxel (n ¼ relation to (1) individual gene mutations, (2) biological 28) versus with placebo/paclitaxel (n ¼ 25) (see Fig. 3C pathway gene mutations, and (3) overall mutation load. and 3D). Across the 140 samples sequenced, 2151 mu- At the individual gene mutation level, five of the tations were identified, with a mean mutational load of mutated genes were found to have marginal significance 12.93 mutations per megabase pair of panel sequenced to PFS or OS in the alisertib/paclitaxel arm: RB1, ade- and a median value of 8.52 mutations per megabase pair nylate cyclase 1 gene (ADCY1), CNTNAP2, zinc finger (see Supplementary Fig. 6). Correlation between muta- protein 217 gene (ZNF217), and BCL associated tran- tional load and PFS or OS in the alisertib arm was scription factor 1 gene (BCLAF1) (Fig. 2B). When marginally significant for OS (p ¼ 0.025) but not for PFS grouped by biological pathway (see Supplementary (p ¼ 0.103). Notably, patient samples with mutations in Figs. 4 and 5), mutations in genes involved in cell cycle cell cycle regulation genes had a significantly higher regulation (cyclin-dependent kinase 6 gene [CDK6], mutational load (a mean of 19.03) compared with those retinoblastoma-like 1 gene [RBL1], retinoblastoma-like 2 without cell cycle regulation gene mutations (a mean of gene [RBL2], and retinoblastoma 1 gene [RB1]) were 9.06) (p < 0.0001) (see Supplementary Fig. 6). significantly associated with improved PFS (p ¼ 0.0011) and OS (p ¼ 0.00096) after alisertib/paclitaxel treat- Safety and Tolerability ment. No such association was shown for mutated genes The median numbers of cycles received in the ali- involved in NOTCH (NOTCH1, NOTCH2, and NOTCH3) or sertib/paclitaxel and placebo/paclitaxel arms were 3 February 2020 Alisertib/Paclitaxel for SCLC 281 A 100 Current study (ctDNA), n = 140 90 University of Cologne study (tissue), n = 110 80 Samples with mutation (%) 70 60 50 40 30 20 10 0 CREBBP SPHKAP TP53 RB1 FMN2 NOTCH1 COL22A1 SYNE1 TP73 ATRX ERICH3 KIAA1211 TMEM132D COL11A1 NOTCH3 RIMS2 ZDBF2 AR CNTNAP2 EP300 B 60 50 Samples with mutation (%) 30 20 10 0 RB1 BCLAF1 ZNF217 CNTNAP2 ADCY1 p-value for association with outcome (FDR) Gene PFS OS RB1 0.0028 (FDR = 0.093) 0.0069 (FDR = 0.22) ADCY1 0.020 (FDR = 0.22) 0.088 (FDR = 0.61) ZNF217 0.029 (FDR = 0.24) 0.042 (FDR = 0.46) CNTNAP2 0.020 (FDR = 0.22) 0.092 (FDR = 0.61) BCLAF1 0.074 (FDR = 0.47) 0.019 (FDR = 0.31) Figure 2. Association between gene mutations and clinical outcomes. (A) Gene mutation frequencies identified in this study by using circulating tumor DNA (ctDNA) were compared with those reported previously in primary tumor tissue samples from patients with SCLC.24 Mutation frequencies for the 20 most commonly mutated genes are shown. (B) The interaction between genetic alterations and clinical outcomes (progression-free survival [PFS] and overall survival [OS]) was tested by using a Cox proportional hazards model. The p values and false discovery rates (FDRs) for the association with outcome are shown. Mutations in retinoblastoma 1 gene (RB1), BCL associated transcription factor 1 gene (BCLAF), contactin associated protein- like 2 gene (CNTNAP2), adenylate cyclase 1 gene (ADCY1), and zinc finger protein 217 gene (ZNF217) were independently associated with alisertib plus paclitaxel versus with placebo plus paclitaxel efficacy, as defined by hazard ratios for PFS and OS. Abbreviations: TP53, tumor protein p53 gene; FMN2, formin 2 gene; NOTCH1, notch receptor 1 gene; COL22A1, collagen type XXII alpha 1 chain gene; SYNE1, spectrin repeat containing nuclear envelope protein 1 gene; TP73, tumor protein p73 gene; CREBBP, CREB binding protein gene; ERICH3, glutamate rich 3 gene; KIAA1211, KIAA1211 gene (now known by the gene symbol CRACD); TMEM132D, transmembrane protein 132D gene; COL11A1, collagen type XI alpha 1 chain gene; ATRX, ATRX chromatin remodeler gene; NOTCH3, notch receptor 3 gene; SPHKAP, SPHK1 interactor, AKAP domain containing gene; RIMS2, regulating synaptic membrane exocytosis 2 gene; ZDBF2, zinc finger DBF-type containing 2 gene; AR, androgen receptor gene; CNTNAP2, contactin associated protein 2 gene; EP300, E1A binding protein p300 gene. 282 Owonikoko et al Journal of Thoracic Oncology Vol. 15 No. 2 (range 1–9) and 2 (range 1–11), respectively. Diarrhea including the most common individual grade 3 or higher and fatigue were the most common adverse events AE, neutropenia (Table 3). Drug-related AEs were also (AEs); both were reported more frequently with ali- more common with alisertib/paclitaxel than with pla- sertib/paclitaxel than with placebo/paclitaxel. Grade 3 cebo/paclitaxel (see Table 3); the most common drug- or higher AEs were more common with alisertib/pacli- related grade 3 or higher AEs were neutropenia, febrile taxel (76%) than with placebo/paclitaxel (51%), neutropenia, leukopenia, anemia, diarrhea, and A PFS with or without cell cycle gene mutations C OS with or without cell cycle gene mutations Mutant Wild type Mutant Wild type Median, Median, Median, Median, n n n n months months months months Alisertib + paclitaxel 40 3.68 28 2.63 Alisertib + paclitaxel 40 7.20 28 4.47 Placebo + paclitaxel 47 1.80 25 2.60 Placebo + paclitaxel 47 4.47 25 5.95 HR (95% CI) 0.395 (0.239–0.654) 1.31 (0.736–2.33) HR (95% CI) 0.427 (0.259–0.704) 1.70 (0.865–3.33) p-value 0.0003 0.359 p-value 0.00085 0.124 B Mutant: PFS D Mutant: OS 1.00 1.00 Survival probability Survival probability 0.75 0.75 0.50 0.50 0.25 0.25 0.00 0.00 0.00 3.29 6.57 9.86 0.00 3.29 6.57 9.86 Time (months) Time (months) Alisertib + paclitaxel Alisertib + paclitaxel Placebo + paclitaxel Placebo + paclitaxel Wild type: PFS Wild type: OS 1.00 1.00 Survival probability Survival probability 0.75 0.75 0.50 0.50 0.25 0.25 0.00 0.00 0.00 3.29 6.57 9.86 0.00 3.29 6.57 9.86 Time (months) Time (months) p = 0.0011 p = 0.00096 Figure 3. Clinical outcomes according to treatment and mutations in cell cycle regulation pathway genes. (A) Cox propor- tional hazard regression analysis of progression-free survival (PFS) in patients with (mutant) and without (wild-type) mu- tations in cell cycle pathway genes, respectively. In each subgroup, the hazard ratio (HR) between the two treatment arms (alisertib plus paclitaxel versus placebo plus paclitaxel) was estimated, along with the 95% confidence interval (CI) and p value. (B) Kaplan-Meier plots of PFS in mutant and wild-type patients, respectively. The p value in this panel is the interaction effect between treatment group and mutation status and is calculated by testing the difference between the HRs for the two subgroups. (C) Cox proportional hazard regression analysis of overall survival (OS) in mutant and wild-type patients, respectively. In each subgroup, the HR between the two treatment arms (alisertib plus paclitaxel versus placebo plus paclitaxel) was estimated, along with the 95% CI and p value. (D) Kaplan-Meier plots of OS in mutant and wild-type patients, respectively. The p value in this panel is the interaction effect between treatment group and mutation status and is calculated by testing the difference between the HRs for the two subgroups. February 2020 Alisertib/Paclitaxel for SCLC 283 stomatitis, which were all reported more frequently with paclitaxel; one patient (1%) reported an SAE of alisertib/paclitaxel than with placebo/paclitaxel. Simi- stomatitis. larly, in the alisertib/paclitaxel arm, 38 patients (44%) In the alisertib/paclitaxel and placebo/paclitaxel arms, reported a serious AE (SAE) and 28 (32%) reported a 14 patients (16%) and five (6%) discontinued study drug-related SAE, compared with 28 patients (31%) and treatment because of an AE, 33 patients (38%) and nine six (7%), respectively, in the placebo/paclitaxel arm. The (10%) had a dose reduction because of an AE, and 12 most common (5% of patients in either treatment arm) patients (14%) and 11 (12%) died during the study all-cause SAEs with alisertib/paclitaxel were febrile (within 30 days of last dose). Four of these deaths, all in neutropenia (10%), neutropenia (6%), diarrhea (6%), the alisertib/paclitaxel arm, were assessed as drug related, and stomatitis (5%). There were no SAEs of febrile including one each due to neutropenic sepsis, sepsis, neutropenia, neutropenia, or diarrhea with placebo/ febrile neutropenia, and septic shock (see Table 3). Table 3. Most Frequently Reported All-Cause and Drug-Related Treatment-Emergent AEs, Occurring in at Least 15% (All- Cause) or at Least 10% (Drug-Related) of Patients Overall (Any Grade) in Either Arm, Respectively, with the Corresponding Grade 3 or higher AEs (Safety Population), and All Drug-Related Fatal AEs Alisertib/Paclitaxel (n ¼ 87) Placebo/Paclitaxel (n ¼ 89) AE Any Grade Grade 3 Any Grade Grade 3 All-cause AE, n (%) 86 (99) 66 (76) 85 (96) 45 (51) Diarrhea 51 (59) 14 (16) 18 (20) 1 (1) Fatigue 38 (44) 9 (10) 29 (33) 5 (6) Nausea 29 (33) 2 (2) 30 (34) 4 (4) Anemia 38 (44) 12 (14) 18 (20) 3 (3) Neutropenia 43 (49) 35 (40) 7 (8) 5 (6) Vomiting 28 (32) 2 (2) 21 (24) 3 (3) Decreased appetite 29 (33) 3 (3) 19 (21) 3 (3) Dyspnea 21 (24) 4 (5) 19 (21) 2 (2) Stomatitis 29 (33) 12 (14) 6 (7) 2 (2) Cough 17 (20) 0 17 (19) 0 Constipation 8 (9) 1 (1) 21 (24) 0 Asthenia 14 (16) 3 (3) 11 (12) 0 Dizziness 14 (16) 0 8 (9) 0 Alopecia 14 (16) 0 5 (6) 0 Leukopenia 13 (15) 7 (8) 5 (6) 2 (2) Decreased neutrophil count 14 (16) 11 (13) 4 (4) 1 (1) Weight decreased 13 (15) 0 5 (6) 0 Drug-related AE, n (%) 81 (93) 58 (67) 72 (81) 22 (25) Diarrhea 44 (51) 13 (15) 11 (12) 1 (1) Fatigue 32 (37) 7 (8) 23 (26) 3 (3) Nausea 24 (28) 2 (2) 26 (29) 4 (4) Neutropenia 41 (47) 33 (38) 7 (8) 5 (6) Anemia 31 (36) 9 (10) 14 (16) 1 (1) Vomiting 23 (26) 2 (2) 14 (16) 2 (2) Stomatitis 28 (32) 11 (13) 6 (7) 2 (2) Decreased appetite 21 (24) 3 (3) 13 (15) 1 (1) Leukopenia 13 (15) 7 (8) 5 (6) 2 (2) Alopecia 12 (14) 0 5 (6) 0 Neutrophil count decreased 13 (15) 10 (11) 4 (4) 1 (1) Febrile neutropenia 11 (13) 11 (13) 0 0 WBC count decreased 11 (13) 11 (13) 1 (1) 1 (1) Asthenia 9 (10) 3 (3) 6 (7) 0 Drug-related fatal AE, n (%) Neutropenic sepsis — 1 (1) — 0 Sepsis — 1 (1) — 0 Febrile neutropenia — 1 (1) — 0 Septic shock — 1 (1) — 0 AE, adverse event; WBC, white blood cell. 284 Owonikoko et al Journal of Thoracic Oncology Vol. 15 No. 2 Discussion potentially reflects the more rigorous restaging scan In this study of alisertib/paclitaxel versus placebo/ schedule (every 4 weeks for the first six cycles), which paclitaxel as second-line therapy in patients with enabled more accurate estimation of PFS than in previ- advanced SCLC, the primary end point of PFS in the ITT ous studies. population was not met, but PFS was significantly Although cross-trial comparison should be improved when analyzed on the basis of the corrected approached with caution, the response rate of 22% definition of relapse type after first-line therapy (as observed with the alisertib/paclitaxel combination in the intended by the protocol), with a positive trend favoring study is comparable to the 21% response rate associated alisertib/paclitaxel (p ¼ 0.038). However, this PFS with single-agent alisertib in 48 patients with SCLC in a benefit was not clinically meaningful. Similarly, the phase 1/2 study,23 which may suggest that synergy of subgroup analysis of type of relapse after first-line the combination is limited in this treatment setting. therapy (corrected definition) demonstrated a statisti- Because of the overlapping toxicity profile for ali- cally significant improvement in PFS with alisertib/ sertib and paclitaxel, there was a higher incidence of paclitaxel over that with placebo/paclitaxel in patients grade 3 or higher AEs and drug-related AEs with this with resistant or refractory relapsed SCLC (p ¼ 0.037). combination versus with paclitaxel alone. Increased However, the primary analysis of PFS conducted by us- toxicity is a concern in this treatment setting, in which ing the original stratification definition of relapse type many patients are not clinically robust. Clinically was not significant on statistical testing. Despite a trend manageable hematologic events were among the most in favor of alisertib/paclitaxel, there was no significant frequent drug-related grade 3 or higher treatment- difference in OS between arms; however, the study was emergent AEs in patients who received alisertib/pacli- not powered to show an OS advantage. Notably, the taxel; of particular note, a high rate of grade 3 or higher correlative biomarker studies indicated that both c-Myc neutropenia (38%) was observed. Overall, 44 patients in expression and mutations in cell cycle regulators (CDK6, the study received myeloid growth factor support to RBL1, RBL2, and RB1) showed strong correlation with manage neutropenia (34 patients in the alisertib/pacli- improved clinical outcomes (PFS and OS) in patients taxel arm and 10 patients in the placebo/paclitaxel arm). with SCLC who were receiving alisertib/paclitaxel. These Rates of on-study deaths were similar in both arms, but findings are consistent with the mechanism of action of four AE-related deaths (due to febrile neutropenia, alisertib as a mitotic inhibitor disrupting cell cycle pro- neutropenic sepsis, sepsis, and septic shock) occurred gression through mitosis. Thus, c-Myc expression or cell only with alisertib/paclitaxel. cycle gene mutations may serve as predictive bio- AAK binds to c-Myc and prevents its degradation, markers for alisertib. thereby enhancing its growth-promoting effect in cancer When the results are interpreted, there are points to cells.16,17 Conversely, pharmacologic inhibition of AAK in consider. Although not the primary end point, the preclinical studies results in greater growth inhibition in sensitivity analysis of PFS using the corrected definition cell lines harboring MYC amplification.13,14 We therefore of relapse type may represent a more clinically relevant, anticipated that patients whose tumors harbored high c- scientifically valid approach. In the PFS sensitivity anal- Myc expression would be susceptible to an AAK inhibitor ysis with use of the corrected definition of relapse type, (as seen in preclinical studies).28 Moreover, as c-Myc patients without a stratification factor were excluded, alteration may be correlated with poor response to improving internal validity. There was also a higher ratio chemotherapy, we anticipated that patients with resis- of sensitive patients in the placebo/paclitaxel arm to tant or refractory relapse would derive greater benefit. sensitive patients in the alisertib/paclitaxel arm (42:33), Consistent with this hypothesis, alisertib/paclitaxel as compared with when the original definition was used showed significant benefit over placebo/paclitaxel in (45:40); therefore, the original analysis favored placebo/ patients with resistant or refractory relapse and in pa- paclitaxel. Finally, doses of paclitaxel were different in tients with c-Myc expression. Although the numbers each arm to account for the pharmacodynamic synergy were low and the results need to be reproduced in a of paclitaxel with alisertib. larger independent study, we expect that enrichment The median PFS with placebo/paclitaxel was slightly strategies for these patient subsets could aid further shorter than in a previous study of second-line paclitaxel development of alisertib in SCLC. monotherapy in patients with SCLC (4.76 months),25 With respect to genetic biomarkers, patients treated whereas the median OS was in line with values in pre- with alisertib/paclitaxel who had mutations in cell cycle vious reports (3.3–5.8 months).25-27 A longer interval regulator genes, including CDK6, RBL1, RBL2, and RB1, between restaging scans could lead to overestimation of had significantly improved PFS and OS compared with PFS, and the shorter PFS duration in our study those who received placebo/paclitaxel. Previous genomic landscape studies have implicated cell cycle February 2020 Alisertib/Paclitaxel for SCLC 285 regulation genes as being commonly mutated in topotecan, because of the higher toxicity associated SCLC.24,29 Our findings suggest a predictive value of with topotecan, particularly the overlapping toxicity of these mutations with alisertib/paclitaxel treatment for neutropenia with alisertib, and the existence of pre- SCLC. Interestingly, patients with mutated cell cycle clinical and clinical data for the alisertib/paclitaxel regulators had worse outcomes in response to placebo/ combination.19 Recently, other agents have shown paclitaxel treatment; the implications of these findings similar or improved efficacy compared with topotecan, warrant further investigation. Our study results are including amrubicin8 and cisplatin, etoposide, and iri- consistent with, and provide validation, of the findings notecan,38 and in August 2018, nivolumab received from two preclinical reports in Cancer Discovery, which accelerated approval by the FDA for patients with describe a synthetic lethal relationship between RB1 metastatic SCLC with progression after platinum-based mutations and inhibition of AAK or Aurora B kinase.30,31 chemotherapy and at least one other line of therapy.39 The predictive value of cell cycle mutations for alisertib Approval was based on findings from the open-label, treatment in other diseases is also of interest and worth multicohort CHECKMATE-032 study (NCT01928394), further exploration. Importantly, our data highlight the which reported an ORR of 12% (95% CI: 6.5–19.5) in emerging role of NGS profiling of plasma ctDNA for 109 patients (w65% with platinum-sensitive SCLC, identifying novel predictive biomarkers. defined as progression 90 days after the last dose of No predictive value was demonstrated in genetic al- platinum-containing therapy), with 77% having a DOR terations in other cell cycle progression genes implicated of at least 6 months when treated with nivolumab with in SCLC, such as amplification of Aurora kinase A gene or without ipilimumab.39 Further treatment options (AURKA) and MYC10,17,32,33 (data not shown). Similarly, continue to emerge for SCLC, including immuno- pathway mutations in genes implicated in NOTCH and therapy; in 2019, FDA approvals for have been granted phosphoinositide 3-kinase signaling, and histone modi- for pembrolizumab in relapsed or refractory SCLC, fications, showed no association with PFS or OS in based on the results of the KEYNOTE-028 response to alisertib treatment. As these parameters (NCT02054806) and KEYNOTE-158 (NCT02628067) were established retrospectively, the study was not studies,40,41 and for atezolizumab in combination with optimized for this metric. chemotherapy in the frontline treatment of extensive- A key limitation of our study was the failure to use stage SCLC, based on the IMpower133 trial a validated biomarker for prospective patient selection. (NCT02763579).42 There were seven patients who had Although post hoc biomarker interrogation supported received treatment with immunotherapy before start- c-Myc expression and mutations in cell cycle regulator ing our study (two in the alisertib/paclitaxel arm and genes as promising biomarkers, these observations five in the placebo/paclitaxel arm); postprogression require more rigorous prospective testing and valida- treatment information was not collected. Despite the tion. Additionally, the study failed to demonstrate emerging range of treatment options, topotecan re- survival benefit of alisertib/paclitaxel compared with mains the standard therapy in the second-line setting. placebo/paclitaxel. Patients entering this study were Whether the efficacy signal observed in the present stratified by platinum sensitivity status based on data study is sufficient to support a definitive superiority available at the time, which suggested that platinum trial in comparison with topotecan in unselected pa- sensitivity status was correlated with efficacy out- tients is difficult to ascertain, particularly in light of the comes. However, data from studies in relapsed SCLC negative result of the phase II trial of cabazitaxel published during the conduct of this trial suggest that versus topotecan in a similar population of patients platinum sensitivity may not be strongly associated with relapsed SCLC.7 However, a biomarker-enrichment with efficacy outcomes and that other prognostic sub- strategy using c-Myc expression or mutations in cell groups could be more relevant in study designs.34 cycle regulator genes could enhance the likelihood of Another limitation was that the comparator arm in success of such a comparative study. this study was placebo/paclitaxel. At the time of this In conclusion, alisertib/paclitaxel showed a modest study’s design, topotecan was the only agent approved efficacy signal as second-line therapy for SCLC. Because in the relapsed setting by the U.S. Food and Drug of the overlapping safety profile for alisertib and pacli- Administration (FDA),6,35 having demonstrated symp- taxel, grade 3 or higher AEs and drug-related AEs were tom reduction and less hematologic toxicity than with more frequent with the combination than with paclitaxel cyclophosphamide, doxorubicin, and vincristine,36 and alone. The predictive value of c-Myc expression and cell survival benefit over best supportive care.37 Single- cycle gene mutations for AAK inhibitor susceptibility is agent paclitaxel is frequently used off-label as a stan- promising, but prospective testing and validation are dard treatment for relapsed SCLC, and paclitaxel was required. These results, along with those from a previous chosen as the comparator in this study, rather than phase II study demonstrating activity of alisertib 286 Owonikoko et al Journal of Thoracic Oncology Vol. 15 No. 2 monotherapy in patients with relapsed or refractory References SCLC,23 may warrant further testing in a larger study 1. Alvarado-Luna G, Morales-Espinosa D. Treatment for with predictive biomarker enrichment strategy. small cell lung cancer, where are we now?-a review. Transl Lung Cancer Res. 2016;5:26–38. 2. Sorensen M, Pijls-Johannesma M, Felip E. Small-cell lung Acknowledgments cancer: ESMO Clinical Practice Guidelines for diagnosis, The study was funded by Millennium Pharmaceuticals, treatment and follow-up. Ann Oncol. 2010;21(suppl Inc., Cambridge, MA, USA, a wholly owned subsidiary of 5):v120–v125. Takeda Pharmaceutical Company Limited. Employees 3. Arcaro A. Targeted therapies for small cell lung cancer: where do we stand? 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