Clinical trial NCT02873975
Conditions: Advanced Cancers
Keywords: Advanced Cancers Solid tumors MYC amplification CCNE1 amplification Rb loss FBXW7 mutation BRCA1 mutation BRCA2 mutation PALB2 mutation RAD51C mutation RAD51D mutation ATR mutation ATM mutation CHK2 mutation Fanconi anemia
Sponsor: Dana-Farber Cancer Institute
Start date: 2016-10-12; Completion date: 2021-07-01
Enrollment: 27 participants; Allocation: NON_RANDOMIZED; Intervention model: SINGLE_GROUP

Brief summary:
This research study is studying a checkpoint kinase 1 (CHK1) inhibitor as a possible treatment for advanced solid tumors that harbor genetic alterations in the homologous repair (HR) pathway, genetic alterations that indicate replication stress, or with CCNE1 amplification.

Detailed description:
This is an open label, phase II, two-arm study exploring the anti-tumor activity of the CHK1 inhibitor prexasertib (LY2606368) in patients with advanced solid tumors exhibiting one of the following:

1. Replicative stress, including MYC amplification, CCNE1 amplification, Rb loss, or an FBXW7 mutation
2. An HR deficiency, including tumors with genomic or somatic mutations of BRCA1, BRCA2, PALB2, RAD51C, RAD51D, ATR, ATM, CHK2, or the Fanconi anemia pathway genes
3. A CCNE1 amplification

Arms: arm 1: Prexasertib (LY2606368) will be administered as an IV infusion in patients with homologous repair (HR) deficiency on day 1 and day 15 of every 28 day cycle. Prexasertib will be given at the recommended phase 2 dose of 105 mg/m2 administered over approximately 1 hour. arm 2: Prexasertib (LY2606368) will be administered as an IV infusion in patients with advanced solid tumors exhibiting replicative stress on day 1 and day 15 of every 28 day cycle. Prexasertib will be given at the recommended phase 2 dose of 105 mg/m2 administered over approximately 1 hour. arm 3: Prexasertib (LY2606368) will be administered as an IV infusion in patients with CCNE1 amplification on day 1 and day 15 of every 28 day cycle. Prexasertib will be given at the recommended phase 2 dose of 105 mg/m2 administered over approximately 1 hour.
Interventions: intervention 1: LY2606368
Intervention descriptions: intervention 1: None

Protocol documents:
Prot_SAP_000.pdf: 
 
 
Page 1 
 
NCI Protocol #: Not Applicable 
 
DF/HCC Protocol #: 16-281 
 
TITLE:  A phase II study of the CHK1 inhibitor LY2606368 in patients with advanced solid 
tumors exhibiting replicative stress or homologous recombination repair deficiency 
 
Coordinating Center: 
 
 
Dana-Farber Cancer Institute 
 
Principal Investigator (PI): 
Geoffrey Shapiro M.D., PhD 
 
Director, Early Drug Development Center 
 
Dana-Farber Cancer Institute 
 
Geoffrey_Shapiro@dfci.harvard.edu 
 
Statistician: 
 
Project Manager:  
Alona Muzikansky 
 
Adrienne Anderson 
Dana-Farber Cancer Institute  
Dana-Farber Cancer Institute 
amuzikansky@partners.org 
 
Telephone: 617-632-6594 
 
 
Fax: 617-632-5152 
 
 
Aanderson10@partners.org 
 
Study Coordinator: 
Ketki Bhushan 
Dana-Farber Cancer Institute 
Telephone: 617-632-4270 
Fax: 617-582-7922 
Ketki_Bhushan@dfci.harvard.edu 
 
Agent:  LY2606368/Prexasertib – Eli Lilly and Company 
 
IND #:  130,512 
IND Sponsor: Investigator-Initiated Study, Geoffrey Shapiro MD, PhD 
 
Protocol Type / Version # / Version Date: Revised, Version #5, 26 January 2018 
 
 
 
 
 
 


 
 
Page 2 
 
SCHEMA 
Replicative Stress 
Cohort: enroll 9 patients 
HR Deficiency Cohort: 
enroll 9 patients 
Informed consent 
and confirmation 
of trial eligibility 
Response assessment: 
number of patients 
progression-free 4 months 
following initiation of 
prexasertib 
≥ 2 patients are 
progression-free 4 
months following 
initiation of 
prexasertib  
Cohort enrollment 
stops 
Expand cohort to 
enroll an additional 10 
patients 
STAGE 1 
CCNE1 Cohort: enroll 9 
patients 
< 2 patients are 
progression-free 4 
months following 
initiation of 
prexasertib  
STAGE 2 


 
 
Page 3 
 
TABLE OF CONTENTS 
 
1. 
OBJECTIVES ......................................................................................................................6 
1.1 
Study Design ............................................................................................................6 
1.2 
Primary Objectives...................................................................................................6 
1.3 
Secondary Objectives...............................................................................................6 
1.4 
Exploratory Objectives ............................................................................................6 
2. 
BACKGROUND .................................................................................................................6 
2.1 
LY2606368 (Prexasertib).........................................................................................6 
2.2 
Study Disease .........................................................................................................16 
2.3 
Replicative Stress and HR Deficiency Cohort Rationale ......................................17 
2.4 
CCNE1 Cohort Rationale ......................................................................................18 
2.5 
Correlative Studies Background ............................................................................21 
3. 
PARTICIPANT SELECTION ...........................................................................................22 
3.1 
Eligibility Criteria ..................................................................................................22 
3.2 
Exclusion Criteria ..................................................................................................23 
3.3 
Inclusion of Women and Minorities ......................................................................24 
4. 
REGISTRATION PROCEDURES ...................................................................................25 
4.1 
General Guidelines for DF/HCC Institutions ........................................................25 
4.2 
Registration Process for DF/HCC Institutions .......................................................25 
5. 
TREATMENT PLAN ........................................................................................................25 
5.1 
Treatment Regimen ................................................................................................25 
5.2 
Pre-Treatment Criteria ...........................................................................................25 
5.3 
Agent Administration.............................................................................................26 
5.4 
General Concomitant Medication and Supportive Care Guidelines ......................27 
5.5 
Criteria for Taking a Participant Off Protocol Therapy .........................................28 
5.6 
Duration of Follow Up ...........................................................................................28 
5.7 
Criteria for Taking a Participant Off Study ...........................................................29 
6. 
DOSING DELAYS/DOSE MODIFICATIONS ...............................................................29 
6.1 
Expected Toxicities ................................................................................................29 
6.2 
Specific Toxicity Management ..............................................................................30 
6.3 
Dose Modifications/Delays ....................................................................................32 
7. 
ADVERSE EVENTS:  LIST AND REPORTING REQUIREMENTS ............................33 
7.1 
Expected Toxicities ................................................................................................33 
7.2 
Serious Adverse Events .........................................................................................34 
7.3 
Expedited Adverse Event Reporting ......................................................................35 
7.4 
Expedited Reporting to the Food and Drug Administration (FDA) ......................36 
7.5 
Expedited Reporting to Hospital Risk Management .............................................36 
7.6 
Routine Adverse Event Reporting .........................................................................36 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
4 
8. 
PHARMACEUTICAL INFORMATION ..........................................................................36 
8.1 
LY2606368/Prexasertib .........................................................................................36 
9. 
BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES ........................................41 
9.1 
Biomarker Studies ..................................................................................................42 
10. 
STUDY CALENDAR .......................................................................................................43 
11. 
MEASUREMENT OF EFFECT........................................................................................47 
11.1 
Antitumor Effect ....................................................................................................47 
12. 
DATA REPORTING / REGULATORY REQUIREMENTS ...........................................54 
12.1 
Data Reporting .......................................................................................................54 
12.2 
Data Safety Monitoring..........................................................................................55 
13. 
STATISTICAL CONSIDERATIONS...............................................................................55 
13.1 
Study Design/Endpoints.........................................................................................56 
13.2 
Sample Size, Accrual Rate and Study Duration ....................................................56 
13.3 
Analysis of Primary and Secondary Endpoints .....................................................57 
13.4 
Analysis of Exploratory Endpoints ........................................................................57 
13.5 
Reporting and Exclusions ......................................................................................57 
14. 
PUBLICATION PLAN .....................................................................................................58 
REFERENCES ..............................................................................................................................59 
APPENDIX A 
PERFORMANCE STATUS CRITERIA ..................................................63 
APPENDIX B 
List of drugs known to predispose to torsade de pointes ...........................64 
 
 
LIST OF TABLES 
 
Table 1: Summary of Prexasertib Derived Radioactivity Pharmacokinetic Parameters in Rat and 
Dog Plasma ................................................................................................................................... 10 
Table 2: Toxicokinetic Parameters of Prexasertib in Rats Following a Single and Repeated 1-
Hour IV Infusion Administered as Daily Doses on 3 Consecutive Days per Week for 3 Weeks 10 
Table 3: Mean ± SD Toxicokinetic Parameters of Prexasertib in Dogs Following a Single and 
Repeated IV Infusion Administered on 3 Consecutive Days per Week for 3 Weeks .................. 11 
Table 4: Toxicities Considered Expected ..................................................................................... 29 
Table 5: Management of Neutropenia .......................................................................................... 30 
Table 6: Management of Thrombocytopenia ................................................................................ 31 
Table 7: Dose Modifications ......................................................................................................... 33 
Table 8: Expedited AE Reporting Exclusions .............................................................................. 35 
Table 9: Compatible Materials for Prexasertib Administration .................................................... 40 
Table 10: Study Calendar.............................................................................................................. 45 
Table 11: RECIST Criteria for Evaluation of Response ............................................................... 53 
Table 12: RECIST Criteria for Patients with Non-Measurable Disease....................................... 53 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
5 
Table 13: Accrual Targets ............................................................................................................. 56 
 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
6 
 
1. OBJECTIVES 
 
 
1.1 
Study Design 
 
This is an open label, phase II, three-arm study exploring the anti-tumor activity of the 
checkpoint kinase 1 (CHK1) inhibitor prexasertib (LY2606368) in patients with advanced solid 
tumors exhibiting replicative stress, a homologous repair (HR) deficiency, or CCNE1 
amplification. 
 
 
1.2 
Primary Objectives 
 
1) To evaluate the activity of the CHK1 inhibitor prexasertib in patients with tumors 
exhibiting either replication stress, HR deficiency, or CCNE1 amplification; specifically, 
to determine the proportion of patients alive and progression-free at 4 months in these 
cohorts.  
 
 
1.3 
Secondary Objectives 
 
1) To assess toxicity following CHK1 inhibition among participants in these cohorts.   
2) To examine the objective response and overall survival rate following CHK1 inhibition in 
these patient cohorts. 
 
 
1.4 
Exploratory Objectives 
 
1) Pre-treatment biopsies will be obtained from patients in the first stage of the study in both 
the replicative stress and HR deficiency arms, and will be subjected to whole exome 
sequencing (WES) to explore potential genomic determinants of sensitivity and resistance 
to CHK1 inhibition.   
2) Cell-free DNA (cfDNA) will be collected from patients on study and analyzed for 
mutations associated with the replicative stress and HR deficiency pathways.  
 
 
2. BACKGROUND 
 
 
2.1 
LY2606368 (Prexasertib) 
 
Please refer to the prexasertib investigator’s brochure (IB) for comprehensive background 
information. LY2606368 monomesylate monohydrate (LY2606368 or prexasertib) is an 
adenosine triphosphate (ATP)-competitive selective inhibitor of CHK1. Prexasertib is being 
developed as a treatment for patients with advanced cancer and may have utility both as a single 
agent and in combination with deoxyribonucleic acid (DNA)-damaging agents or targeted 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
7 
agents. 
 
CHK1 is a multifunctional protein kinase and regulator of cell-cycle progression1. During cell 
cycle progression, the integrity of DNA is continuously monitored, with DNA damage or 
perturbation of DNA metabolism activating cell cycle checkpoints. Activation of these 
checkpoints leads to cell cycle arrest in order to provide the cell with the time necessary to repair 
DNA damage and complete DNA replication prior to progressing to mitosis. CHK1 is a key 
component of the checkpoint response and whose activation in response to DNA damage (e.g. 
from cytotoxic chemotherapeutic agents like cisplatin) arrests the cell in the S or G2 phase of the 
cell cycle. Inhibition of CHK1 leads to abrogation of cell cycle checkpoints, thereby preventing 
DNA repair and resulting in premature entry into mitosis2. Following early entry into mitosis, the 
cell is rapidly killed by mitotic catastrophe attributable to failed chromosomal segregation due to 
a damaged or incompletely replicated genome. 
 
In addition to its role in cell cycle checkpoint control, CHK1 contributes to the DNA damage 
response through its role in coordinating DNA repair. Specifically, CHK1 is essential for HR 
repair of DNA double strand breaks (DSBs) 3 and CHK1 phosphorylates FANCE and 
FANCD24,5, proteins involved in the Fanconi Anemia DNA repair pathway, which serves to 
remove DNA cross-links induced by DNA damaging agents like cisplatin. As such, CHK1 
inhibition may increase the activity of DNA-damaging cytotoxic chemotherapeutics by 
interfering with DNA repair. CHK1 also plays a role in the correction of erroneous kinetochore-
microtubule attachments and optimal spindle checkpoint signaling, suggesting that CHK1 
inhibition may enhance chromosome mis-segregation and increase the activity of anti-mitotic 
drugs6. 
 
CHK1 also phosphorylates multiple downstream targets that regulate DNA replication, 
chromosome alignment, spindle checkpoints, and exit from cytokinesis7. In normal cell cycle 
progression, CHK1 prevents the activation of late-stage origins until near the end of S phase and 
stabilizes active replication forks. When CHK1 is inhibited, the number of active replication 
origins increases, resulting in stalled replication forks and causing DNA strand breakage and 
replication catastrophe8-12. Cells deficient in CHK1 have increased spontaneous chromosome 
mis-segregation and lose the ability to recruit the spindle checkpoint protein BUBR1 to 
kinetochores and fail to activate the spindle checkpoint in response to misaligned 
chromosomes13. This finding suggests that CHK1 is essential for stable attachment of mitotic 
spindles to metaphase chromosomes. It is therefore postulated that CHK1 inhibition alone can 
generate DNA damage and mitotic catastrophe.  
 
As of 13 January 2017, there were 7 Eli Lilly-sponsored studies assessing prexasertib. In these 
studies, prexasertib has been administered to 260 patients as monotherapy and to 113 patients in 
combination with other treatments. Please refer to the investigator’s brochure (IB) for full 
information. The first-in-human phase I study, I4D-MC-JTJA (JTJA), evaluated monotherapy 
prexasertib in patients with advanced cancer. Study I4D-MC-JTJF (JTJF) evaluated prexasertib 
in combination with chemotherapy or targeted agents in patients with advanced cancer.  
 
2.1.1 
Prexasertib Pre-Clinical Studies 
 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
8 
2.1.1.1 
In Vitro Biology 
 
Prexasertib is a potent ATP-competitive inhibitor of CHK1. In an in vitro enzyme assay, 
prexasertib inhibits the activity of CHK1 with an IC50 of 1 nM and an inhibition binding constant 
(Ki) of 0.5 nM. The compound is a selective protein kinase inhibitor demonstrating an IC50 of 
less than 100 nM against only 8 out of 223 kinases in the Upstate Biotechnology kinase 
screening panel. Only CHK2 and the p90S6 kinases (RSKs) were inhibited by prexasertib at an 
IC50 of less than 10 nM. 
  
As a single agent, prexasertib has potent in vitro effects on cells which reflects the essential 
protective role that CHK1 plays in DNA replication. In this regard, CHK1 functions as a 
negative regulator of replication origin activation; specifically, keeping late replication origins 
silent until late S phase. When CHK1 protein is depleted, more replication origins are activated 
in early S phase than the replication apparatus can tolerate, resulting in slowed and arrested DNA 
replication forks and DNA double-strand breakage. Chemical inhibition of CHK1 with 
prexasertib results in a similar outcome, namely, an initial accumulation of cells in S-phase, 
extensive DNA damage, premature entry into mitosis with unrepaired DNA, and induction of 
apoptosis. Prexasertib’s activity is dependent upon inhibition of CHK1 and subsequent increase 
in CDC25A activation of CDK2, which results in an increase in the number of replication forks 
while reducing their stability. Following prexasertib treatment, cells demonstrate a rapid 
appearance of terminal deoxynucleotidyl transferase nick end labeling (TUNEL) and pH2AX-
positive double-stranded DNA breaks in the S-phase cell population. The majority of treated 
mitotic nuclei consist of extensively fragmented chromosomes, indicating that prexasertib causes 
replication catastrophe12. Additionally, treatment with prexasertib causes a cellular phenotype 
identical to that reported in knockdown experiments utilizing CHK1 selective ribonucleic acid 
(RNA) interference (RNAi)14; HeLa cells treated with prexasertib show a clear defect in 
chromatin condensation and failure of the mitotic spindle to attach to chromosomes. Taken 
together, these results indicate that in vitro, prexasertib as a single agent behaves mechanistically 
as a DNA-damaging agent, a checkpoint inhibitor, an inhibitor of DNA replication and mitosis, 
and as an inducer of replication catastrophe. 
 
The broad anti-proliferative effects of single agent prexasertib were demonstrated in various 
tissue culture studies whereby growth inhibition or clonogenic survival in soft agar served as 
phenotypic endpoints. Specifically, the evaluation of prexasertib in 395 cell lines derived from 
multiple cancer types and various histologies showed widespread anti-proliferative effects, with 
growth of 64% of these lines inhibited at IC50 concentrations of 50 nM or below. 
 
Similarly, a subset of the 49 cell lines derived from squamous cell cancers (SCC) have been 
evaluated for response to prexasertib and these studies showed that 61% of these lines had IC50 
values of 50 nM or below. Related studies evaluating clonogenic survival in soft agar showed 
that prexasertib potently inhibited survival in 44 of 54 (81%) models derived from orthotopically 
grown human tumors representing 16 different tumor types. The mean IC50 values determined in 
the 44 most sensitive models ranged from 2 to 33 nM. 
 
2.1.1.2 
Prexasertib Pharmacokinetic/Pharmacodynamic Models 
 
An indirect response pharmacokinetic (PK)/pharmacodynamic (PD) model was developed in 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
9 
order to link prexasertib plasma concentrations with the inhibition of phosphorylated CHK1 
(pCHK1) and tumor growth response in Calu-6 xenografts following IV administration of 
prexasertib (monotherapy). The IC50 determined from the nonclinical single agent PK/PD model 
is 14.1 ng/mL. The PK/PD model predicts that in Calu-6 xenograft tumor models, an average 
pCHK1 inhibition of 49.7% (90% confidence interval [CI]: 45-54.7%) and 70.5% (90% CI: 
67.5-74.5%) over the first 72-hours following prexasertib administration is correlated with the 
minimum and maximum tumor responses, respectively. The minimum and maximum tumor 
responses are defined as the smallest and largest statistically significant tumor regressions, 
respectively, when compared to controls. 
 
The predicted human systemic prexasertib exposure that correlates with the minimum and 
maximum clinical tumor response with prexasertib monotherapy is an area under the plasma 
concentration-time curve from 0 – 72 hours (AUC(0-72)) of 664 ng•hr/mL (90% CI: 353-1236 
ng•hr/mL) and 1896 ng•hr/mL (90% CI: 1008-3533 ng•hr/mL), respectively. 
 
2.1.1.3 
Pre-Clinical Pharmacokinetics and Toxicokinetics 
 
The plasma toxicokinetics of prexasertib were evaluated in Sprague-Dawley rats and beagles 
following single and repeated intravenous (IV) infusions administered as daily doses on 3 
consecutive days per week for 3 weeks. In both rats and dogs, increases in exposures were 
approximately dose-proportional following single and repeat doses. No plasma accumulation 
was noted in either rats or dogs, and there were no sex-related differences in PKs.  
 
Pharmacokinetic parameters of prexasertib were estimated in rats and dogs following a single IV 
administration of [14C]LY2606368 formulated in Captisol. Prexasertib was rapidly cleared in 
both species and had high volumes of distribution suggesting good tissue distribution.  The major 
metabolic routes associated with the clearance of prexasertib were based on oxidative 
deamination and O-demethylation in dogs and on O-demethylation in rats.  
 
Radiolabeled studies show that hepatobiliary excretion plays a major role in the elimination of 
prexasertib in both rats and dogs. In rats, drug-derived radioactivity crossed both the blood brain 
barrier and the blood–testis barrier but at very low levels. Overall, radioactivity was selectively 
associated with melanin-containing tissues in rats. 
 
2.1.1.4 
Single Dose Pharmacokinetics 
 
The PK of prexasertib and prexasertib-derived radioactivity were evaluated in Sprague-Dawley 
rats and beagles following an IV administration of [14C]LY2606368 and summarized in Table 1 
below: 
 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
10 
Table 1: Summary of Prexasertib Derived Radioactivity Pharmacokinetic Parameters in 
Rat and Dog Plasma 
 
 
Concentrations of prexasertib declined rapidly in a monoexponential fashion following a single 
1-hour IV infusion of [14C]LY2606368 in both rats and dogs. At Cmax, prexasertib accounted for 
approximately 37% and 69% of the plasma radioactivity in rats and dogs, respectively. However, 
the exposure (AUC(0-∞)) of prexasertib accounted for approximately 16% and 2% of the 
radioactivity exposure in plasma in rats and dogs, respectively, indicating the presence of 
circulating metabolites. Total radioactivity in plasma was followed by a biexponential decline 
with a steep alpha phase observed during the first 8 hours post start of infusion, and a relatively 
longer terminal elimination half-life (t1/2) for the residual low levels of radioactivity. Metabolites 
appear to contribute to the longer t1/2 of radioactivity when compared with that of prexasertib. 
 
2.1.1.5 
Multi-Dose Toxicokinetics 
 
Prexasertib was administered as a 1-hour IV infusion to male and female Sprague-Dawley rats in 
doses ranging from 1 to 40 mg/kg/day on 3 consecutive days per week for 3 weeks (dosed on 
days 1, 2, 3, 8, 9, 10, 15, 16, and 17). Toxicokinetic parameters were calculated and are 
summarized in Table 2 below:  
 
Table 2: Toxicokinetic Parameters of Prexasertib in Rats Following a Single and Repeated 
1-Hour IV Infusion Administered as Daily Doses on 3 Consecutive Days per Week for 3 
Weeks 
 
 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
11 
Prexasertib was also administered as a 1-hour IV infusion to male and female beagles in doses 
ranging from 1 to 10 mg/kg/day and as a 2-hour IV infusion at a dose of 40 mg/kg/day on 3 
consecutive days per week for 3 weeks (dosed on days 1, 2, 3, 8, 9, 10, 15, 16, and 17). 
Toxicokinetic parameters were calculated and are summarized in Table 3:  
 
Table 3: Mean ± SD Toxicokinetic Parameters of Prexasertib in Dogs Following a Single 
and Repeated IV Infusion Administered on 3 Consecutive Days per Week for 3 Weeks 
 
 
For both rats and dogs, systemic exposures to prexasertib were generally similar for male and 
female animals on days 1 and 17. No accumulation of prexasertib in plasma was observed 
following multiple doses. Increases in maximum observed plasma concentration (Cmax) and AUC 
from time 0 to 24 hours post-dose [AUC(0-24)] were approximately dose-proportional on days 1 
and 17. 
 
2.1.1.6 
Pre-Clinical Enzyme Induction 
 
No indications of in vivo CYP induction were observed during the 3-week repeat-dose studies in 
rats and dogs. A slight decrease (ranging from approximately 1% to 24% in rats and 4% to 29% 
in dogs) in total CYP content was observed in prexasertib-treated groups compared with the 
vehicle-treated group. 
 
2.1.1.7 
Prexasertib Distribution 
 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
12 
Radioactivity was extensively distributed in tissues and organs following a 1-hour IV infusion of 
[14C]LY2606368 to pigmented male and female and non-pigmented male rats. In most tissues, 
peak radioactivity concentrations were observed at the end of infusion in both male and female 
pigmented rats. In pigmented male rats, the tissues showing the highest peak concentrations were 
the eye uveal tract, adrenal gland, pancreas, stomach mucosa, kidney medulla, and myocardium. 
In pigmented female rats, the tissues showing the highest peak concentrations were adrenal 
gland, pancreas, liver, stomach mucosa, exorbital lacrimal gland, and eye uveal tract.  
 
Radioactivity concentrations in central nervous system tissues protected by the blood–brain 
barrier (ie, cerebellum, cerebrum, medulla, and spinal cord) were low, measurable only in brain 
olfactory lobe (males) and spinal cord (females) up to 2 hours and 1 hour post start of infusion, 
respectively. Low levels of [14C]LY2606368-related radioactivity were observed in testes of 
pigmented and non-pigmented male rats, suggesting that low levels of drug-derived radioactivity 
crossed the blood–testis barrier. Furthermore, [14C]LY2606368-related radioactivity was 
selectively associated with melanin-containing tissues.  
 
There was some association of [14C]LY2606368-related radioactivity with the cellular 
component of blood in dogs (blood to plasma [B:P] ratio ≥1 up to 8 hours post-dose) and a 
minimal association to the cellular component of blood in rats (B:P ratio <1 up to 24 hours post-
dose). 
 
2.1.1.8 
Pre-Clinical Metabolism 
 
Metabolism of prexasertib has been evaluated in both male beagle dogs and male Sprague-
Dawley rats following a single 1-hour IV administration of [14C]LY2606368. Overall, 
prexasertib and a total of 15 metabolites were detected in dogs and 13 metabolites were detected 
in rats.  
 
In dogs, prexasertib was the major drug-related entity circulating in the plasma up to the 4-hour 
time point, and radiolabeled thiocyanate was the predominant circulating entity at all subsequent 
time points up to 120 hours, and is the likely contributor for the long t1/2 illustrated in Table 1. 
Oxidative and glucuronide metabolites were also observed in plasma at the early sampling times. 
Oxidative metabolites along with prexasertib were the predominant excreted entities in both 
feces and urine. Oxidative deamination of the propylamine side chain and O-demethylation to 
form the corresponding phenol appear to be predominantly responsible for clearance of 
prexasertib. Overall, oxidative metabolism was the path of clearance of prexasertib followed by 
smaller contributions from renal and biliary excretion.  
 
In rats, no quantifiable radioactive peaks were reported at or beyond the 4-hour time point, 
demonstrating rapid clearance of both prexasertib and related radioactivity from plasma. A 
glucuronide metabolite of prexasertib was the predominant drug-related circulating entity 
followed by prexasertib. Oxidative metabolites along with prexasertib were the major excreted 
entities in feces, urine, and bile. Additionally, glucuronides and acetylated metabolites were 
detected in both urine and bile. Formation of the O-demethylated phenol appears to be 
predominantly responsible for the clearance of prexasertib. Overall, oxidative metabolism and 
glucuronidation seems to be the major routes responsible for the clearance of prexasertib 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
13 
followed by smaller contributions from renal and biliary excretion in rats. 
 
2.1.1.9 
Pre-Clinical Excretion 
 
Following a single IV infusion in dogs, approximately 72.9% and 16.3% of the administered 
radioactivity was recovered in feces and urine, respectively. Radioactivity was eliminated 
rapidly, with approximately 84% of the administered dose recovered in the initial 48 hours post 
start of infusion. However, the remaining radioactivity was eliminated slowly, with measurable 
radioactive concentrations in excreta at all the collection intervals through 168 hours post start of 
infusion.  
 
In rats with intact bile ducts, mean recoveries in feces and urine accounted for 58.6% and 34.1% 
of the administered dose, respectively. Approximately 85% of the dose was eliminated in the 
first 24 hours post start of infusion. In bile duct–cannulated rats, radioactivity recoveries in bile 
and urine represented approximately 45% and 37% of the administered radioactivity, 
respectively.  
 
The data show that hepatobiliary excretion plays a major role in the elimination of prexasertib 
both in rats and in dogs. 
 
2.1.1.10 
Summary of Pre-Clinical Safety Pharmacology and Toxicology 
 
To support human clinical studies, the toxicity profile of prexasertib has been characterized in 
rats and dogs through repeat-dose toxicology, safety pharmacology, and genetic toxicology 
studies. These studies demonstrate a toxicity profile that is considered to be monitorable and 
reversible.  
 
In repeat-dose toxicity studies in rats and dogs, treatment-related findings associated with 
prexasertib were generally similar to those of a cytotoxic chemotherapeutic agent, with dose-
limiting effects primarily related to bone marrow suppression and gastrointestinal (GI) injury. 
These effects were dose dependent, with hematologic effects also being shown to be schedule-
dependent. In rats, treatment-related neutropenia was schedule-dependent, with more severe 
effects associated with 3 consecutive days compared with a single dose. On the basis of these 
collective studies in animals, the dose-limiting toxicity in humans was expected to be one or 
more of the following: myelosuppression, GI toxicity, tachycardia, and/or hypotension.  
 
Results from the nonclinical toxicology, safety pharmacology, and genetic toxicology studies for 
prexasertib demonstrate a toxicity profile that is considered to be monitorable and reversible. 
 
 
2.1.2 
Prexasertib (LY2606368) Human Studies 
 
As of 13 January 2017, 373 patients have been exposed to prexasertib. Please refer to the IB for 
complete information. 
 
 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
14 
2.1.2.1 
Pharmacokinetics 
 
Prexasertib systemic exposure increased in a dose-dependent manner with consistent PK profiles 
across the dose range 10 to 130 mg/m2 after single-dose and multiple-dose administration across 
2 different schedules of administration in cancer patients. A population PK analysis has been 
conducted across all doses and days of administration in Cycles 1 and 2 (N=146). The population 
PK analysis determined that a linear, 3-compartment PK model best described the prexasertib PK 
profile. The population median CL was 82.2 L/hr, with a volume of distribution at steady-state 
(Vss) of 1276 L, and median terminal half-life of 27 hours. The population model–predicted post-
hoc individual CL values after single-dose and multiple-dose administration from both schedules 
of administration in Cycles 1 and 2 were consistent across the dose range 10 to 130 mg/m2, 
indicating dose-independent and time-independent PK behavior. The interpatient variability 
varied across PK parameters of the model (percentage coefficient of variation [CV%]=27% to 
73%). Please refer to the IB for complete information. 
 
2.1.2.2 
Pharmacodynamics 
 
Blood, hair, and tissue samples were collected and analyzed for various biomarker assessments 
in a Lilly-sponsored trial of prexasertib. These assessments included circulating plasma DNA 
concentrations, circulating tumor cells, phosphohistone 2AX, and cytokeratin 18. However, due 
to variability in the clinical pharmacodynamic (PD) data and the corresponding lack of 
statistically significant changes, these assessments were not used to inform dose selection. 
Because the magnitude and duration of pCHK1 inhibition associated with the recommended 
phase 2 dose of 105 mg/m2 in humans is not known, human PD profile simulations after 
administration of 105 mg/m2 were generated by linking the human population PK model to the 
murine single-agent PK/PD indirect response model (that is, they assume a similar PD response 
in humans as that characterized in murine xenografts). Simulation results of the predicted median 
human percentage pCHK1 profile demonstrate that the average percentage pCHK1 inhibition 
over the first 72 hours of Cycle 1 after 105 mg/m2 is 41.7% (90% CI: 25.0% to 59.5%). The 
predicted average percentage pCHK1 inhibition over the first 72 hours following administration 
of 105 mg/m2 once every 14 days approaches the average percentage pCHK1 inhibition of 
49.7% (90% CI: 45.0% to 54.7%) over the first 72 hours following prexasertib administration 
described for the minimum tumor response in nonclinical xenograft models. However, the 
predictive capability of this approach has limitations given the differences between animal 
models and humans (e.g., protein binding, tumor size/location, target expression, immune 
system, and prior therapies in humans). As a result, the specific magnitude and duration of 
pCHK1 inhibition required for optimal clinical activity are currently not known. 
 
2.1.2.3 
Clinical Metabolism 
 
Exploratory analysis (non-radiolabeled) of plasma and urine samples collected from 3 patients 
following the administration of a single 50 mg/m2 dose of prexasertib showed the presence of 
prexasertib as the major entity (prexasertib appeared as the peak with the most intense mass 
spectrometric response) in both plasma as well as urine, along with multiple minor oxidative and 
glucuronide metabolite peaks. 
 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
15 
2.1.2.4 
Clinical Drug Interactions 
 
The potential for drug interactions was evaluated in human liver microsomes. Prexasertib 
(LY2606368) exhibited in vitro potential for competitive inhibition of CYP1A2 (Ki = 1.6 μM = 
767 ng/mL) and of CYP2D6 (Ki = 2.8 μM = 1343 ng/mL). There was no inhibition of CYP2C8, 
CYP2C9, CYP2C19, CYP2B6, or CYP3A4 over a range of concentrations (0.103 - 25 μM). 
Furthermore, potential time-plus nicotinamide adenine dinucleotide phosphate (NADPH)-
dependent inhibition by prexasertib was not observed for any CYPs over the concentration range 
tested (0.103 to 25 μM).  
 
Simulations of human PK profiles that were performed using a dynamic physiologically based 
pharmacokinetic (PBPK) model (SimCYP®), suggest that the potential for a clinical drug 
interaction is minimal. More specifically, simulations of drug-drug interaction (DDI) studies 
with prexasertib and sensitive CYP2D6 probe substrates (desipramine and dextromethorphan) 
did not predict any clinically significant DDIs with either substrate. The increase in CYP2D6 
substrate AUC and Cmax ratios were < 1.2 for both substrates at the recommended prexasertib 
phase II dose of 105 mg/m2, when administered either as a single dose or following daily 
administration over three consecutive days.  
 
Similarly, human simulations of DDI studies with prexasertib and sensitive CYP1A2 probe 
substrates (phenacetin and theophylline) did not predict any clinically significant DDIs with 
either one of the substrates. The increase in CYP1A2 substrate AUC and Cmax ratios were < 1.2 
for both substrates at the highest proposed prexasertib phase II dose of 105 mg/m2, when 
administered either as a single dose or following daily administration over three consecutive 
days. 
 
The effect of prexasertib on CYP mRNA levels was evaluated using cryopreserved human 
hepatocytes from a single donor. There was no induction of CYP1A2, CYP2B6 or CYP3A4 
mRNA observed at concentrations corresponding to current clinical Cmax concentrations. 
However, concentration dependent down regulation of CYP3A4 mRNA was observed in vitro 
with no loss or change in CYP3A4 activity. The mechanism of this modulation and its clinical 
relevance has not been evaluated. The likelihood of this finding manifesting in vivo is further 
minimized given the bi-weekly dosing regimen.  
 
The relative contributions of microsomal CYP-mediated clearance of prexasertib was studied in 
vitro using a panel of recombinant human cytochrome P450s (rCYPs): rCYP1A2, rCYP2B6, 
rCYP2C8, rCYP2C9, rCYP2C19, rCYP2D6, rCYP2E1, rCYP2J2, rCYP3A4, and rCYP3A5. 
Based on a substrate depletion approach, CYP1A2, CYP2D6, and CYP2J2 are responsible for 
88%, 9.6% and 2.8% of hepatic CYP-mediated clearance of prexasertib, respectively, with other 
CYPs not appearing to be involved in the metabolism of prexasertib. Although CYP1A2 seems 
to be the major CYP involved in the in vitro clearance of prexasertib, the relative contributions 
of other non-CYP elimination pathways as well as the in vivo clearance pathways have not been 
evaluated.  
 
Overall, in vitro data showed that prexasertib was 63.1 to 65.3% protein-bound in human plasma 
over the concentration range of 0.5 to 50 μM and 48.8 to 54.6% protein-bound in human liver 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
16 
microsomes over the concentration range of 1 to 10 μM. 
 
2.1.2.5 
Clinical Safety Evaluation of Prexasertib 
 
Please refer to the prexasertib IB for complete safety background information. Neutropenia has 
been the most frequent and severe toxicity after treatment with 105 mg/m2 prexasertib every 14 
days. Grade ≥4 decreases in neutrophils deemed related to prexasertib treatment have been 
observed in 57.7% of patients. In general, neutrophil counts reach nadir on approximately Day 8, 
and Grade 4 decreases are often transient (e.g., less than 5 days). Febrile neutropenia has been 
reported in 10.8% of patients, but no patients have discontinued or died because of febrile 
neutropenia. Related Grade ≥4 decreases in platelets have been observed in 5.2% of patients, and 
3 patients (1.4%) had clinically significant bleeding (epistaxis and hematochezia) in the setting 
of thrombocytopenia. No patients experienced Grade ≥4 anemia deemed related to prexasertib 
treatment. 
 
Non-hematologic toxicity occurs at a lower frequency than hematologic toxicity and has been 
predominantly Grade 1 or 2 in severity. Fatigue and nausea are the only non-hematologic AEs 
that are considered to be related to study drug treatment occurring in more than 10% of patients. 
The non-hematologic treatment-emergent AEs (TEAEs) occurring in greater than 5% of patients 
treated at 105 mg/m2 every 14 days and deemed by the investigators to be related to prexasertib 
treatment were fatigue (25.8%), nausea (11.7%), decreased appetite (8.5%), diarrhea (7.0%), and 
headache (5.6%). 
 
2.1.2.6 
Serious Adverse Events 
 
Cumulatively, 360 serious adverse events (SAEs) have been reported in 193 patients in 6 Lilly-
sponsored and 3 Investigator-sponsored monotherapy or combination studies up to 14 January 
2017. In the 373 patients treated in the Lilly-sponsored studies, 289 SAEs in 159 patients have 
been reported. Out of 289 SAEs, 117 events were assessed as being possibly related to study 
drug treatment. The majority of drug-related events (76 events [65%]) were hematologic 
toxicities including neutropenia, leukopenia, anemia, febrile neutropenia, thrombocytopenia, or 
pancytopenia. Of the remaining related events, 14 were complications occurring in the setting of 
hematologic toxicity such as infections (10 events), bleeding events (3 events), or pyrexia (1 
event). 
 
2.1.2.7 
Deaths 
 
As of 13 January 2017, no patients had died due to adverse events (AEs) deemed related to study 
treatment. Please refer to the IB for comprehensive prexasertib background information. 
 
 
 
2.2 
Study Disease 
 
In cancer cells, uncontrolled cell growth and sustained proliferative signaling can result in 
replication stress.  Replication stress is associated with several genomic alterations that drive 
unbridled S phase entry, resulting in error prone replication, nucleotide or replication factor 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
17 
depletion and the slowing or stalling of replication forks 15-17. These genomic alterations include 
MYC amplification 15, cyclin E gene (CCNE1) amplification 18, and loss of the retinoblastoma 
protein (Rb) 19.  Additionally, mutations in the E3 ubiquitin ligase FBXW7 can stabilize MYC, 
cyclin E, and other replication stress-inducing oncoproteins 20,21. 
 
MYC amplification is involved broadly in many cancers; MYC over-expression results in 
increased proliferation, cellular survival and angiogenesis, promoting tumor growth22. High 
levels of MYC have been linked to both aggressive prostate and triple-negative breast cancer 
(TNBC)23. CCNE1 amplification is the most frequent amplified gene in ovarian serous 
carcinomas and is correlated with poor prognosis and primary treatment resistance. CCNE1 
amplification is also associated with disease progression and poor clinical outcome in breast, 
bladder, and colorectal carcinoma24. Rb loss is associated with approximately 40% of TNBCs 
that harbor a p53 mutation (estimated to occur in > 80% of all TNBC patients)25. 
  
In addition to replication stress, uncontrolled cell cycle progression in cancer cells results in 
double strand DNA breaks.  HR repair is essential for repair of this type of DNA damage and the 
maintenance of genomic stability and deficiency in HR repair is implicated in tumorigenesis. 
Mutations in HR associated genes have been identified in a number of cancers26-30. Germline or 
somatic mutations in HR genes, including BRCA 1/2, RAD51C, RAD51D, and ATM, are present 
in nearly 1/3 of all ovarian carcinomas (including both serous and non-serous histologies). 
Germline mutations among this group are frequently associated with familial forms of breast and 
pancreatic cancers30,31.  
 
2.3 
Replicative Stress and HR Deficiency Cohort Rationale 
 
Inhibitors of CHK1 have been shown to sensitize cells to DNA damaging agents and have been 
under development in combination with chemotherapy2. However, there is recent interest in 
defining the monotherapy vulnerabilities of these agents. Emerging data suggest that when 
undergoing severe replication stress, cells require CHK1 for stabilizing replication forks32-35 and 
to promote homologous recombination (HR) repair via the phosphorylation and activation of HR 
associated proteins (e.g., RAD51 and Fanconi anemia proteins) for repair of DNA strand breaks 
during replication3,36-38, therefore inhibition of CHK1 may be lethal to these cells. In cells with 
HR deficiency, there is increased intrinsic DNA damage that activates the ATR-CHK1 pathway 
for checkpoint control. Inhibition of CHK1 in HR deficient cells will disrupt checkpoint control, 
allowing for continued cell cycle progression without DNA repair and resulting in mitotic 
catastrophe. Inhibition of CHK1 activity itself also leads to replication stress, with an increase in 
DNA replication initiation and an associated increase in RPA protein, single-stranded DNA 
formation, and DNA strand breaks9,39. HR-deficient cells cannot faithfully repair such strand 
breaks, resulting in cell death. In addition, it has been shown that severe replication stress and 
HR repair deficiency are synthetically lethal40. These observations suggest that cancer cells 
under replication stress or those with HR deficiency may be susceptible to CHK1 inhibitor 
monothera
py: 
 
 
 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
18 
 
 
 
 
 
 
 
Clinical and preclinical studies support this hypothesis that CHK1 inhibition may be clinically 
relevant in patients with tumors exhibiting replication stress or HR deficiency. 
 
In a retrospective analyses performed during assessment of prexasertib in anal cell carcinoma 
and head and neck cancer populations, patients with tumors harboring FBXW7 mutations 
achieved partial response or stable disease (Lilly Data on File). Additionally, breast, ovarian and 
endometrial cancer cell lines harboring CCNE1 amplification have been found to be sensitive to 
prexasertib, with the most sensitive cell line (KLE endometrial cancer) harboring both CCNE1 
amplification and FBXW7 mutation. Taken together, these data suggest that cells with replication 
stress will have vulnerability to CHK1 inhibitor monotherapy, with CHK1 inhibition leading to 
destabilization of replication forks and disrupted HR, events critical to the survival of cells that 
with severe replication stress. 
 
In the clinical setting, patients with anal and head and neck carcinomas harboring HR deficiency 
associated mutations (e.g., mutations in BRCA1/2, MRE11, or FANCC) were among those 
responding to treatment. Prior pre-clinical work performed in the D’Andrea laboratory examined 
the effect of CHK1 inhibition in model systems exhibiting defects in the Fanconi Anemia (FA) 
DNA repair pathway (which includes BRCA1/2). These studies found that FA-deficient cell lines 
were hypersensitive to CHK1 inhibition by siRNAs targeting CHK1 and pharmacologic 
inhibition by the CHK1 inhibitors Gö6976 and UCN-0141. An siRNA screen designed to identify 
genes synthetically lethal with CHK1 inhibition was also carried out, identifying genes required 
for FA pathway function. These findings were confirmed in vivo, with whole zebrafish embryos 
depleted for FANCD2 demonstrating hypersensitivity to Gö6976. There was also an increased 
accumulation of DNA strand and chromosomal breaks when CHK1 was inactivated in FA-
deficient cell lines. These results suggest that CHK1 and the FA pathway mutually compensate 
in maintaining genome integrity.    
 
2.4 
CCNE1 Cohort Rationale 
 
As of 5 December 2017, a total of 15 participants have been enrolled to the trial. Ten participants 
were enrolled to the replicative stress cohort during the first enrollment stage (one participant 
was replaced after failing to complete the first cycle of dosing). Of these ten, one participant with 
CCNE1 amplified cholangiocarcinoma exhibited stable disease per RECIST 1.1 criteria and has 
successfully initiated his 14th cycle of therapy. The patient’s tumor was found to harbor 113 
copies of CCNE1. 
 
No other participants met the 4 month progression-free target for expansion, and the replicative 
stress cohort is now closed to enrollment. Only one other participant with CCNE1 amplification 
was enrolled to the cohort, a patient with esophageal adenocarcinoma with 25 copies of CCNE1. 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
19 
This participant had a mixed response on their first restaging scan but was ultimately determined 
to have progressed. The remaining participants had MYC amplifications (n=4), FBXW7 
mutations (n=3), and a SETD2 deletion (n=1).  
 
CCNE1 plays a key role in cell proliferation by binding to cyclin dependent kinases (CDKs), 
chiefly CDK2, in late G1 phase of the cell cycle42. Cyclin E1/CDK2 facilitates G1/S phase 
transition through phosphorylation of downstream targets including Rb protein. In normal cells, 
cyclin E1 accumulates at the G1/S phase boundary and is degraded as the cell passes through S 
phase. This periodicity is regulated by cell-cycle dependent transcription and post-translational 
protein turnover by ubiquitin-dependent proteolysis. Cyclin E1/CDK2 is frequently dysregulated 
in cancer, including ovarian, breast, colorectal, and bladder cancers, where overexpression of 
cyclin E is associated with a poor prognosis43-47.  
  
In ovarian cancer, high level CCNE1 expression is associated with increased tumor grade48. Dr. 
David Bowtell’s group at Peter MacCallum Cancer Center49,50 and others48,51-53 have shown that 
CCNE1 amplification and overexpression are associated with reduced overall survival in high 
grade serous ovarian cancers (HGSOC). Preclinical work performed in the Center for DNA 
Damage and DNA Repair (CDDR) at Dana-Farber Cancer Institute has confirmed that 
prexasertib exhibits monotherapy anti-tumor activity across both ovarian cell lines (Figure 1) 
and patient-derived xenograft (PDX) models that exhibit increased CCNE1 copy number (Figure 
2) (unpublished data). The Cancer Genome Atlas (TCGA) project reported that CCNE1 
amplification is mutually exclusive with BRCA1/2 germline mutations, suggesting that patients 
with CCNE1 amplification are less likely to benefit from treatments such as poly (ADP-ribose) 
polymerase (PARP) inhibitors and will require novel therapeutic approaches40,47. 
 
The observed clinical response to therapy combined with the preclinical data gives justification 
to examine prexasertib in a small cohort of CCNE1 amplified patients. Although increased 
CCNE1 copy number can lead to increased cyclin E protein expression, this is not a universal 
finding. As Figure 3 demonstrates, we can expect that 50% of tumors will have high cyclin E 
protein levels in the presence of any degree of CCNE1 amplification (unpublished data).  Our 
collaborators have further evaluated the CCNE1 amplified group to see if a certain degree of 
gene amplification correlates with high cyclin E protein expression. Based on this data, we are 
proposing to establish 6-fold CCNE1 amplification as the minimum required to be enrolled in the 
CCNE1 amplified cohort as it will give the highest probability of treating tumors with high 
cyclin E protein expression, but will allow consideration of borderline participants following 
consultation with the overall principal investigator. 
 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
20 
 
 
 
 
 
 
 
Figure 2: Monotherapy LY2606368 activity in luciferized PDX models. Implantation of ascites derived 
cells, ~5 x 106 for each of the PDX types in NSG mice, 5 mice per each PDX. DF-172luc: mixed serous and 
endometroid histologic subtype post 2 prior lines of chemotherapy, with high cyclin E copy number, and 
TP53, RET, and RAD51C mutations. DF-106luc: HGSOC histologic subtype post 1 prior line of 
chemotherapy, with high cyclin E copy number, TP53 mutation, and CDKN2A loss. Shapiro and D’Andrea, 
CDDR, unpublished data. 
Figure 1: Cyclin E protein levels are low in prexasertib-resistant ovarian cancer cell lines. Cells 
exposed to prexasertib or DMSO for 72 hours in 96 well plates and survival was determined using 
CellTiter Glo reagent. IC50 values for prexasertib were high in ovarian cancer cell lines with low levels 
of cyclin E proteins. Shapiro and D’Andrea, CDDR, unpublished data. 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
21 
 
 
 
2.5 
Correlative Studies Background 
 
Use of targeted therapies in patients with genomically complicated tumors presents a variety of 
confounding variables that could contribute to success or failure of the therapy.  Correlative 
studies are designed to both enable better understanding of the possible reasons that prexasertib 
(LY2606368) may prove to be effective or ineffective in individual patients and to enable us to 
develop more effective targeting strategies in the future.   
 
2.5.1 
Collection of Fresh Tumor Tissue – Replicative Stress and HR Cohorts 
 
In order to retrospectively explore the determinants of response and resistance to prexasertib, a 
mandatory pre-treatment fresh tumor biopsy will be obtained from the patients during Stage 1 of 
the 2-stage trial. The nine biopsies from both cohorts will be sufficient to demonstrate 
meaningful results, however will allow patients who are not amenable to biopsy or for whom a 
biopsy would pose significant risk to enroll on trial at the later stage.  
 
Analysis of biopsy samples taken at distinct points enables addressing of key questions including 
the genomic lesions present in samples studied and their potential impact upon response 
following prexasertib therapy. 
 
It is the anticipation that most patients on this study will have had their tumor genomically 
characterized by either the OncoPanel test at DFCI/BWH (a custom hybrid capture panel which 
via next-generation sequencing will identify mutations and copy-number alterations in cancer 
associated genes) or via another CLIA-certified method at the time of enrollment. A pre-
Figure 3: Six-fold CCNE1 amplification is associated with high cyclin E expression within tumor 
cells. Degree of cyclin E protein expression for non-amplified, moderately amplified and highly 
amplified CCNE1.  80% of tumors with 6-fold or greater CCNE1 gene amplification were highly 
expressed cyclin E protein, compared with 25% and 50% of tumors with low or moderate amplification 
respectively. 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
22 
treatment tumor tissue sample will be subjected to WES at the Broad Institute Center for Cancer 
Precision Medicine. WES enables a comprehensive analysis of DNA mutation in human tumor 
samples. Gene expression profiling has been demonstrated to predict disease-free and overall 
survival in multiple cancers and guide future treatment decisions and biologic discovery54-56. 
WES will be performed to profile the HR proficiency of the tumor, and will involve up to 100 
genes of interest in DNA repair in order to obtain the entire HR state. This will allow 
identification of further genomic alterations that may occur outside of the pre-set enrollment 
genetic alterations of these patients.  The data obtained via WES will enable us to identify 
secondary alterations that may impact clinical response. 
 
2.5.2 
Collection of Blood – All Cohorts 
 
Cell-free DNA (cfDNA) will be collected and analyzed for mutations associated with the 
replicative stress and HR deficiency pathways. Levels of tumor DNA will decline longitudinally 
over time if there is a response to the treatment administered. To further assess this concept, 
plasma will be collected from participants to evaluate whether circulating free plasma DNA 
declines with the administration of prexasertib and this information will be compared to clinical 
outcome. 
 
 
3. PARTICIPANT SELECTION 
 
 
3.1 
Eligibility Criteria  
 
Baseline evaluations are to be conducted within 14 days prior to start of protocol therapy, 
with the exception of the informed consent and baseline tumor imaging which may be 
obtained up to 28 days prior to the start of protocol therapy. Participants must meet the 
following criteria to be eligible to participate in the study: 
 
3.1.1 Participants must have a pathologically confirmed advanced solid tumor for which 
standard therapy proven to provide clinical benefit does not exist or is no longer 
effective. 
 
3.1.2 Participants must have one of the following (confirmed via targeted NextGen Sequencing 
using the DFCI/BWH OncoPanel or another CLIA-certified method): 
• For the replicative stress cohort: MYC amplification, CCNE1 amplification, Rb 
loss, FBXW7 mutation, or another genomic abnormality indicative of replicative 
stress as agreed upon with the principal investigator. 
-OR- 
• For the HR deficiency cohort: genomic or somatic mutation in BRCA1, BRCA2, 
PALB2, RAD51C, RAD51D, ATR, ATM, CHK2, the Fanconi anemia pathway 
genes, or another genomic or somatic mutation in a known HR gene as agreed 
upon with the principal investigator.  
-OR- 
• For enrollment to the CCNE1 cohort: CCNE1 amplification of 6-fold or greater. 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
23 
Patients with borderline amplification levels may be considered following 
approval from the overall principal investigator. 
 
3.1.3 Participants must have measurable disease, defined as at least one lesion that can be 
accurately measured in at least one dimension (longest diameter to be recorded for non-
nodal lesions and short axis for nodal lesions) as ≥ 20 mm with conventional techniques 
or as ≥ 10 mm with spiral CT scan, MRI, or calipers by clinical exam.  See Section 11 for 
the evaluation of measurable disease. 
 
3.1.4 Age ≥ 18 years. As no dosing or adverse event data are currently available in participants 
< 18 years of age, children are excluded from this study but will be eligible for future 
pediatric trials. 
 
3.1.5 ECOG performance status < 2 (see Appendix A). 
 
3.1.6 Participants must have adequate organ and marrow function as defined below: 
 
 
Absolute neutrophil count 
≥ 1.5 K/uL 
 
Platelet count 
≥ 100 K/uL 
 
Hemoglobin 
≥ 9 g/dL (with or without transfusion support) 
 
Total bilirubin 
≤ 1.5 × institutional upper limit of normal (ULN) 
 
AST(SGOT)/ALT(SGPT) 
≤ 2.5 × institutional ULN, unless liver metastases are  
present and then ≤ 5 × institutional ULN is acceptable 
 
Serum creatinine 
≤ 1.5 × institutional ULN 
 
3.1.7 Participants enrolling to the HR or replicative stress cohorts during Stage 1 must have 
disease that is amenable to biopsy and be willing to undergo a pre-treatment tumor 
biopsy.  
 
3.1.8 The potential effects of prexasertib (LY2606368) use during pregnancy and lactation are 
not known. Nonclinical studies of prexasertib on pregnancy and fetal development have 
not been performed. To minimize any potential risks, men and women with reproductive 
potential should use medically approved contraceptive precautions during treatment and 
for 3 months following the last dose of prexasertib. Should a woman become pregnant or 
suspect she is pregnant while she or her partner is participating in this study, she should 
inform her treating physician immediately.  Men treated or enrolled on this protocol must 
also agree to use adequate contraception prior to the study, for the duration of study 
participation, and for 3 months after completion of prexasertib administration. 
 
3.1.9 Ability to understand and the willingness to sign a written informed consent document. 
 
3.1.10 QTcF value of ≤ 470 msec on screening electrocardiogram (EKG). 
 
 
3.2 
Exclusion Criteria 
 
Participants who exhibit any of the following conditions will not be eligible to participate 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
24 
in the study: 
 
3.2.1 Participants who have had chemotherapy, other investigational or biologic therapy, major 
surgery, or radiotherapy within 3 weeks (6 weeks for nitrosoureas or mitomycin C) prior 
to the planned first dose of prexasertib (LY2606368) therapy. 
 
3.2.2 Participants who have not recovered to eligibility levels from prior toxicity or adverse 
events as a result of previous treatment prior to the study. 
 
3.2.3 Participants who have received prior treatment with a CHK1 inhibitor. 
 
3.2.4 Participants who have received prior radiation therapy to > 25% of the bone marrow. 
 
3.2.5 Participants with known untreated brain metastases should be excluded from this clinical 
trial because of their poor prognosis and because they often develop progressive 
neurologic dysfunction that would confound the evaluation of neurologic and other 
adverse events. Participants with a history of brain metastases that have been treated, are 
no longer taking corticosteroids, and have been stable on imaging for at least one month 
following the end of treatment are permitted. 
 
3.2.6 History of allergic reactions attributed to compounds of similar chemical or biologic 
composition to prexasertib. 
 
3.2.7 Participants with a personal or family history of long QT syndrome. 
 
3.2.8 Uncontrolled intercurrent illness including, but not limited to, ongoing or active infection, 
NYHA Class III/IV heart failure, unstable angina pectoris, cardiac arrhythmia, 
myocardial infarction within 3 months of enrollment, or psychiatric illness/social 
situations that would limit compliance with study requirements. 
 
3.2.9 Pregnant or breastfeeding females. The potential effects of prexasertib use during 
pregnancy and breastfeeding are not known and prexasertib has the potential for 
teratogenic or abortifacient effects. 
 
3.2.10 Known HIV-positive participants are ineligible because these participants are at increased 
risk of lethal infections when treated with marrow-suppressive therapy.  Appropriate 
studies will be undertaken in HIV-positive participants when indicated. 
 
3.2.11 Participants enrolling to the HR or replicative stress cohort during Stage 1 may not be on 
anticoagulant therapy unless the treating investigator has deemed it safe to temporarily 
hold to facilitate the pre-treatment tumor biopsy. 
  
 
3.3 
Inclusion of Women and Minorities 
 
Both men and women of all races and ethnic groups are eligible for this trial. 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
25 
 
 
4. REGISTRATION PROCEDURES 
 
 
4.1 
General Guidelines for DF/HCC Institutions 
 
Institutions will register eligible participants in the Clinical Trials Management System (CTMS) 
OnCore. Registrations must occur prior to the initiation of protocol therapy. Any participant not 
registered to the protocol before protocol therapy begins will be considered ineligible and 
registration will be denied. 
 
An investigator will confirm eligibility criteria and a member of the study team will complete the 
protocol-specific eligibility checklist. 
 
Following registration, participants may begin protocol therapy. Issues that would cause 
treatment delays should be discussed with the Overall Principal Investigator (PI). If a participant 
does not receive protocol therapy following registration, the participant’s registration on the 
study must be canceled. Registration cancellations must be made in OnCore as soon as possible.  
 
 
4.2 
Registration Process for DF/HCC Institutions 
 
DF/HCC Standard Operating Procedure for Human Subject Research Titled Subject Protocol 
Registration (SOP #: REGIST-101) must be followed.   
 
5. TREATMENT PLAN 
 
 
5.1 
Treatment Regimen 
 
Prexasertib (LY2606368) will be administered on day 1 and day 15 of a 28 day cycle. 
Prexasertib will be given at the recommended phase II dose of 105 mg/m2. Treatment will be 
administered on an outpatient basis.  Reported adverse events and potential risks are described in 
Section 7.  Appropriate dose modifications are described in Section 6.  No other investigational 
or commercial agents or therapies may be administered with the intent to treat the participant’s 
malignancy, with the exception of palliative radiation therapy to non-target lesions which may be 
administered with the principal investigator’s agreement. Maintenance hormonal therapy for 
breast or prostate cancer patients will be permitted as long as the therapy was started prior to the 
initiation of the study medication. Bisphosphonate use is permitted. 
 
 
5.2 
Pre-Treatment Criteria 
 
5.2.1 
Cycle 1, Day 1 Infusion 
 
Patients who completed screening assessments > 72 hours prior to cycle 1 day 1 should 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
26 
have cycle 1 day 1 laboratory values that re-meet eligibility criteria. If screening 
assessments were completed ≤ 72 hours prior to cycle 1 day 1, laboratory tests do not 
need to be repeated on cycle 1 day 1 and the screening laboratory values can be used as 
the cycle 1 day 1 values. 
 
5.2.2 
Subsequent Infusions  
 
Please refer to Section 6 for toxicity management guidelines. 
 
 
5.3 
Agent Administration 
 
5.3.1 
Administration Instructions 
 
Prexasertib (LY2606368) will be given at the recommended phase II dose of 105 mg/m2 
and should be administered IV over 1 hour (+/- 5 minute infusion window) using a 
central or free flowing peripheral IV line with an in-line, sterile, non-pyrogenic, 0.22-μm 
filter. Prexasertib should not come in contact with normal saline or Lactated Ringer’s 
solution; the infusion line should be flushed with 5% dextrose injection (D5W) before 
and after prexasertib administration. The infusion should be completed within 4 hours of 
priming the infusion set. 
 
Prexasertib should be handled according to standard procedures and precautions 
consistent with a cytotoxic anticancer drug. 
 
Body surface area (BSA) should be calculated per institutional standards. Dose 
recalculation for changes in participant weight should be made in accordance with 
institutional standards. 
 
5.3.2 
Hypersensitivity Reactions 
 
Prexasertib mesylate monohydrate is formulated in Captisol® (sulfobutylether-β-
cyclodextrin [SBECD]), which has been associated with allergic reactions. The etiology 
of the hypersensitivity reactions observed with prexasertib mesylate monohydrate is not 
known.  
 
The patient should be monitored for signs and symptoms indicative of an infusion-related 
reaction for the duration of the infusion (including nausea, dizziness, chest pain, 
diaphoresis, flushing, decreased blood pressure, shortness of breath, hypoxia, and 
tachycardia). In the event of a suspected infusion reaction, institutional guidelines for 
managing infusion reactions should be followed. For subsequent infusions, premedication 
with diphenhydramine hydrochloride (dosing per investigator discretion) and/or other 
premedication as per institutional standards or investigator discretion may be 
administered. The infusion rate may also be decreased by 50 percent. 
 
 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
27 
5.3.3 
Serotonin Syndrome 
 
In non-clinical assessments, prexasertib interacts with multiple 5-hydroxytryptamine (5-HT) 
receptors. Patients should be monitored for symptoms of serotonin syndrome, including 
autonomic changes (tachycardia, mydriasis, diaphoresis, or body temperature increases), 
mental status changes (restlessness or anxiety), and neurological hyperexcitability (tremor, 
hyperreflexia, or myoclonus).  
 
If observed, appropriate treatment (e.g., cyproheptadine or octreotide) may be used as a 
treatment. As of 13 January 2017, serotonin syndrome had not been reported for any patient 
receiving prexasertib. 
 
 
5.4 
General Concomitant Medication and Supportive Care Guidelines 
 
Investigators should use appropriate supportive medications to address toxicities that arise 
during the study, including but not limited to antiemetics, antidiarrheals, and blood product 
transfusion.  
 
5.4.1 
Antibiotic and Granulocyte Colony Stimulating Factor Administration 
 
Prophylactic antibiotics should be considered for use in patients that have experienced 
febrile neutropenia or patients deemed at higher risk for febrile neutropenia by the 
treating investigator.  
 
Prophylactic Granulocyte Colony Stimulating Factor (G-CSF) such as filgrastim (or 
equivalent), as well as pegfilgrastim (or equivalent), are permitted in accordance with the 
American Society of Clinical Oncology (ASCO) guidelines. Primary or secondary 
prophylactic G-CSF support is permitted.  
 
5.4.2 
Medications Known or Suspected to cause Prolonged QTc Intervals or Torsades de Pointes 
 
On days when prexasertib (LY2606368) is administered, patients should avoid taking 
multiple concomitant medications that are known or suspected to cause prolonged QTc or 
Torsades de Pointes (see Appendix B). If possible, alternative agents should be 
considered.  
 
 
5.4.3 
Other Anticancer Therapy 
 
No other investigational or commercial agents or therapies may be administered with the 
intent to treat the participant's malignancy, with the exception of palliative radiation 
therapy to non-target lesions which may be administered with the principal investigator’s 
agreement. Bisphosphonates are permitted. Maintenance hormonal therapy for breast or 
prostate cancer patients will be permitted as long as the therapy was started prior to the 
initiation of the study medication. Consideration of other medications for pre-existing 
conditions may be approved on a case-by-case basis by the overall principal investigator. 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
28 
 
5.4.4 
Other Concomitant Medication Guidelines 
 
The potential risk of a clinical DDI with either CYP1A2 or CYP2D6 substrates is 
minimal. Therefore, there are no recommended restrictions on CYP1A2 or CYP2D6 
substrates. 
 
 
5.5 
Criteria for Taking a Participant Off Protocol Therapy 
 
Duration of therapy will depend on individual response, evidence of disease progression, and 
tolerance. In the absence of treatment delays due to adverse event(s), treatment may continue 
indefinitely or until one of the following criteria applies: 
 
• 
Disease progression (unless the participant is exhibiting clinical benefit as agreed 
upon with the principal investigator) 
 
• 
Intercurrent illness that prevents further administration of treatment 
 
• 
Unacceptable adverse event(s) 
 
• 
Pregnancy 
 
• 
Participant demonstrates an inability or unwillingness to comply with the trial 
regimen 
 
• 
Participant decides to withdraw from the protocol therapy 
 
• 
General or specific changes in the participant's condition render the participant 
unacceptable for further treatment in the judgment of the treating investigator 
 
Participants will be removed from the protocol therapy when any of these criteria apply.  The 
reason for removal from protocol therapy, and the date the participant was removed, must be 
documented in the case report form (CRF). Alternative care options will be discussed with the 
participant. 
 
The research team will update the relevant Off Treatment information in OnCore. 
 
In the event of unusual or life-threatening complications, treating investigators must immediately 
notify the Overall PI, Geoffrey Shapiro, MD, PhD at (617) 632-4942. 
 
 
5.6 
Duration of Follow Up 
 
Participants will be followed for serious adverse events (SAEs) for 30 days after removal from 
protocol therapy or until death, whichever occurs first. Participants will be followed for survival 
status only until death after removal from protocol therapy. Survival follow up will be performed 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
29 
by review of the medical record, contact with care providers, and/or telephone contact as needed 
every 3 to 4 months.  
 
 
5.7 
Criteria for Taking a Participant Off Study 
 
Participants will be removed from study when any of the following criteria apply: 
• Lost to follow-up 
• Withdrawal of consent for data submission 
• Death 
 
The reason for taking a participant off study, and the date the participant was removed, must be 
documented in the CRF. 
 
The research team will update the relevant Off Study information in OnCore.  
 
 
6. DOSING DELAYS/DOSE MODIFICATIONS 
 
Dose delays and modifications will be made as indicated in the following sections. The 
descriptions and grading scales found in the revised NCI Common Terminology Criteria for 
Adverse Events (CTCAE) version 4.0 will be utilized for dose delays and dose modifications. A 
copy of the CTCAE version 4.0 can be downloaded from the CTEP website at:  
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm.  
 
 
6.1 
Expected Toxicities 
 
The table below summarizes treatment-emergent AEs considered expected for prexasertib 
(LY2606368) serious adverse reaction reporting. 
 
Table 4: Toxicities Considered Expected 
System Organ Class 
Event Preferred Term 
Blood and lymphatic system 
disorders 
Neutropenia (including 
neutrophil count decreased) 
Thrombocytopenia (including 
platelet count decreased) 
Anemia (including 
hemoglobin and hematocrit 
decreased) 
Leukopenia (including white 
blood cell count decreased) 
Febrile neutropenia 
Lymphopenia (including 
lymphocyte count decreased) 
Infections and infestations 
Sepsis (including urosepsis) 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
30 
 
 
6.2 
Specific Toxicity Management 
 
6.2.1 
Hematologic Toxicity Management 
 
6.2.1.1 
Neutropenia 
 
Neutropenia has been the most frequent and severe toxicity following treatment with prexasertib 
(LY2606368) every 14 days, with grade 3 or greater decreases in neutrophil count deemed 
related to prexasertib treatment having been observed in approximately three-quarters of patients. 
In general, neutrophil counts nadir on day 8 and grade 4 decreases are often transient (e.g., less 
than 5 days). Febrile neutropenia has been reported in approximately 11% of patients, but no 
patients have died due to febrile neutropenia. Please refer to Section 5.4.1 for G-CSF guidelines. 
 
 
Table 5: Management of Neutropenia 
Toxicity 
Prexasertib Dose 
Management 
Uncomplicated Grade 3 
Neutropenia (ANC < 1 K/uL) 
Hold infusion until recovery to 
grade 2, resume at same dose 
level. 
Grade 3 Neutropenia 
Associated with a Fever or 
Infection (ANC < 1 K/uL) 
Hold infusion until recovery to 
grade 2, resume with one dose 
level reduction. 
Uncomplicated Grade 4 
Neutropenia < 7 Consecutive 
Days in Duration (ANC < 0.5 
K/uL) 
Hold infusion until recovery to 
grade 2, resume at same dose 
level. 
Grade 4 Neutropenia ≥ 7 
Consecutive Days in 
Duration (ANC < 0.5 K/uL) 
Hold infusion until recovery to 
grade 2, resume with one dose 
level reduction. 
 
6.2.1.2 
Thrombocytopenia 
 
Grade 3 or greater thrombocytopenia has been observed in approximately one fifth of patients. 
 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
31 
 
 
 
 
 
 
Table 6: Management of Thrombocytopenia 
Toxicity 
Prexasertib Dose 
Management 
Grade 3 Platelet Count 
Decrease - Not Associated 
with Bleeding (Platelet count 
25 -  50 K/uL) 
Hold infusion until recovery to 
grade 2, resume at same dose 
level. 
Grade 3 Platelet Count 
Decrease - Associated with 
Bleeding (Platelet count 25 -  
50 K/uL)  
OR 
Grade 4 Platelet Count 
Decrease (Platelet count < 25 
K/uL) 
Hold infusion until recovery to 
grade 2, resume with one dose 
level reduction. 
 
6.2.1.3 
Anemia 
 
Patients should receive blood transfusions as deemed necessary in the judgment of the treating 
investigator. Participants may be dose reduced for anemia ≥ grade 2 and considered at least 
possibly related to prexasertib if the treating investigator believes it to be in the best interest of 
the participant. 
 
6.2.2 
Non-hematologic Toxicity Management 
 
6.2.2.1 
Fatigue 
 
Participants experiencing ≥ grade 3 fatigue should have their prexasertib (LY2606368) infusion 
held until recovery to ≤ grade 2. Upon resuming, the prexasertib dose should be reduced to the 
next lowest dose level unless the fatigue was attributed to anemia and has resolved following 
transfusion. In that case, the participant may be maintained at their current dose level if the 
treating investigator feels it is in the participant’s best interest. 
 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
32 
Participants with intolerable grade 2 fatigue may have their prexasertib held and/or reduced to 
the next lowest dose level if it is considered to be in their best interest by the treating 
investigator. 
 
 
6.2.2.2 
Nausea/vomiting 
 
Patients experiencing nausea or vomiting should be offered appropriate supportive care, 
including the use of antiemetic therapy and IV hydration if needed. Participants experiencing ≥ 
grade 3 nausea or vomiting (or intolerable grade 2) despite optimal medical management should 
have prexasertib dose-reduced to the next lowest dose level. 
 
6.2.2.3 
Management of Other Non-hematological Toxicity 
 
For any other ≥ grade 3 non-hematological toxicity which cannot be managed despite optimal 
supportive care, prexasertib should be held until recovery to ≤ grade 1 or baseline. Upon 
resuming, prexasertib should be dose-reduced to the next lowest dose level. Exceptions include: 
• Photosensitivity, rash, or neuropathy, in which cases prexasertib may be resumed upon 
resolution to grade 2 if deemed appropriate by the treating investigator. 
• Non-clinically significant laboratory abnormalities. Prexasertib may be continued 
without interruption at the current dose level if deemed appropriate by the treating 
investigator, or may be held and/or dose reduced at the treating investigator’s discretion. 
• Asymptomatic laboratory abnormalities that resolve to ≤ grade 1 or baseline within 48 
hours of repletion. Prexasertib may be resumed at the current dose level if deemed 
appropriate by the treating investigator, or may be dose reduced at the treating 
investigator’s discretion. Repletion and treatment may occur on the same day. 
 
Patients experiencing intolerable grade 2 toxicities may have their infusion of prexasertib 
delayed at the treating investigator’s discretion. Upon resolution, the dose of prexasertib may be 
reduced if the treating investigator considers a dose reduction appropriate. 
  
6.3 
Dose Modifications/Delays 
 
Participants who are benefiting from prexasertib (LY2606368) in the opinion of the treating 
investigator may have their infusion held for up to 4 weeks to allow for recovery of toxicity. 
Participants requiring a longer hold should be removed from the trial. Exceptions to this 
requirement are possible should the principal investigator agree that the patient may continue 
despite the length of time off drug. 
 
 
6.3.1 
Dose Modifications 
 
A maximum of two dose reductions are allowed. If a patient requires more than two dose 
reductions, they should be removed from the trial.  Once a patient’s dose has been reduced, it 
may be re-escalated to the previous dose level if the toxicity has resolved to baseline or ≤ grade 1 
and with agreement from the principal investigator. 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
33 
 
 
 
Table 7: Dose Modifications 
Dose Reduction Level 
Prexasertib Dose 
-1  
80 mg/m2 
-2 
60 mg/m2 
 
If the study medication is placed on hold for toxicity, the counting of cycle days and assessment 
schedule will continue without interruption. For example, a patient who does not receive their 
prexasertib (LY2606368) infusion on cycle 2 day 15 due to toxicity will proceed with their next 
scheduled visit as previously planned (cycle 3 day 1) with additional interim visits as needed for 
clinical management. The cycle will not restart for dosing delays due to toxicity. 
 
6.3.2 
Overdose 
 
There is no known antidote for over-dosage of prexasertib (LY2606368). In the case of 
suspected overdose, monitor hematologic parameters, serum chemistry, vital signs, cardiac 
function, and provide supportive care as necessary. 
 
 
7. ADVERSE EVENTS:  LIST AND REPORTING REQUIREMENTS  
 
Adverse event (AE) monitoring and reporting is a routine part of every clinical trial.  The 
following list of reported and/or potential AEs (Section 7.1) and the characteristics of an 
observed AE (Sections 7.2 and 7.3) will determine whether the event requires expedited 
reporting in addition to routine reporting. 
  
 
7.1 
Expected Toxicities 
 
A list of the expected adverse events associated with the investigational agent administered in 
this study can be found in Section 6.1.  
 
7.1.1 
Adverse Event Characteristics 
 
• CTCAE term (AE description) and grade:  The descriptions and grading scales found 
in the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) version 
4.03 will be utilized for AE reporting.  All appropriate treatment areas should have access 
to a copy of the CTCAE version 4.03.  A copy of the CTCAE version 4.03 can be 
downloaded from the CTEP web site 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
34 
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm. 
 
• For expedited reporting purposes only:   
- 
AEs for the agent that is listed above should be reported only if the AE varies in 
nature, intensity or frequency from the expected toxicity information which is 
provided in Section 7.1. 
- 
Other AEs for the protocol that do not require expedited reporting are outlined in 
section 7.4 - Expedited Adverse Event Reporting, under the sub-heading of Protocol-
Specific Expedited Adverse Event Reporting Exclusions. 
 
• Attribution of the AE: 
- 
Definite – The AE is clearly related to the study treatment. 
- 
Probable – The AE is likely related to the study treatment. 
- 
Possible – The AE may be related to the study treatment. 
- 
Unlikely – The AE is doubtfully related to the study treatment. 
- 
Unrelated – The AE is clearly NOT related to the study treatment. 
 
 
7.2 
Serious Adverse Events 
 
A serious adverse event (SAE) is any adverse event that occurs after the initial dose of study 
treatment, during treatment, or within 30 days of the last dose of treatment that results in one of 
the following outcomes: 
 
• Death 
• Hospitalization for greater than 24 hours 
• Prolonging an existing inpatient hospitalization 
• A life-threatening experience (that is, immediate risk of dying) 
• Persistent or significant disability/incapacity 
• Congenital anomaly/birth defect 
• Considered significant by the investigator for any other reason 
 
Previously planned (prior to signing the informed consent form) surgeries, and non-disease 
related elective surgeries planned during the course of the study, should not be reported as SAEs 
unless the underlying medical condition has worsened or appeared during the course of the 
study. Events that occur prior to the first administration of study medication should not be 
reported as SAEs. 
 
Preplanned hospitalizations or procedures for preexisting conditions that are already recorded in 
the patient’s medical history at the time of study enrollment should not be considered SAEs. 
Hospitalization or prolongation of hospitalization without a precipitating clinical AE (e.g., for 
the administration of study therapy or other protocol-required procedure) should not be 
considered SAEs. 
 
Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered SAEs when, based on appropriate medical judgment, they 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
35 
may jeopardize the patient and may require medical or surgical intervention to prevent one of the 
outcomes listed in this definition. 
 
Death due to disease progression should not be reported as an SAE unless the investigator deems 
it to be related to the use of study drug. 
 
Study site personnel must alert Eli Lilly of any SAE as soon as possible and no later than 24 
hours of the investigator and/or institution receiving notification of the SAE experienced by a 
patient participating in the study. The SAE reports should be on a standard reporting form (e.g. 
CIOMS, MedWatch 3500a, or similar) and are to be sent to Eli Lilly via fax at 1-866-644-1697. 
 
 
7.3 
Expedited Adverse Event Reporting  
 
7.3.1 Investigators must report to the Overall PI any SAE that occurs after the initial dose of 
study treatment, during treatment, or within 30 days of the last dose of treatment.  
 
7.3.2 
DF/HCC Expedited Reporting Guidelines 
 
Investigative sites within DF/HCC will report AEs directly to the DFCI Office for 
Human Research Studies (OHRS) per the DFCI IRB reporting policy.  
 
7.3.3 
Protocol-Specific Expedited Adverse Event Reporting Exclusions 
 
For this protocol only, the AEs/grades listed below do not require expedited reporting to 
the Overall PI or the DFCI IRB.  However, they still must be reported through the routine 
reporting mechanism (i.e. case report form). 
 
Table 8: Expedited AE Reporting Exclusions 
CTCAE 
SOC 
Adverse 
Event 
Grade 
Resulting in 
Hospitalization/ 
Prolongation of 
Hospitalization 
Attribution 
Comments 
Investigations Neutrophil 
count 
decreased 
4 
No 
Definite, 
probable, or 
possible 
Only applies if the AE does 
not involve a 
hospitalization/prolongation 
of hospitalization (otherwise 
needs to be reported) 
Investigations White blood 
cell 
decreased 
4 
No 
Definite, 
probable, or 
possible 
Only applies if the AE does 
not involve a 
hospitalization/prolongation 
of hospitalization (otherwise 
needs to be reported) 
Investigations Platelet 
count 
decreased 
4 
No 
Definite, 
probable, or 
possible 
Only applies if the AE does 
not involve a 
hospitalization/prolongation 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
36 
of hospitalization (otherwise 
needs to be reported) 
Investigations Lymphocyte 
count 
decreased 
4 
No 
Definite, 
probable, or 
possible 
Only applies if the AE does 
not involve a 
hospitalization/prolongation 
of hospitalization (otherwise 
needs to be reported) 
 
 
7.4 
Expedited Reporting to the Food and Drug Administration (FDA) 
 
The Overall PI, as study sponsor, will be responsible for all communications with the FDA. The 
Overall PI will report to the FDA, regardless of the site of occurrence, any SAE that meets the 
FDA’s criteria for expedited reporting following the reporting requirements and timelines set by 
the FDA. 
 
 
7.5 
Expedited Reporting to Hospital Risk Management 
 
Participating investigators will report to their local Risk Management office any participant 
safety reports or sentinel events that require reporting according to institutional policy.  
 
 
7.6 
Routine Adverse Event Reporting 
 
All Adverse Events must be reported in routine study data submissions to the Overall PI on the 
toxicity case report forms.  AEs reported through expedited processes (e.g., reported to the 
IRB, FDA, etc.) must also be reported in routine study data submissions. 
 
 
8. PHARMACEUTICAL INFORMATION 
 
A list of the AEs and potential risks associated with the investigational agent administered in this 
study can be found in Section 6.1. 
 
 
8.1 
LY2606368/Prexasertib 
 
8.1.1 
Description 
 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
37 
 
Chemical name: 2-Pyrazinecarbonitrile, 5-[[5-[2-(3-aminopropoxy)-6-methoxyphenyl]-
1H-pyrazol-3-yl]amino] monomesylate monohydrate 
 
Molecular formula: C19H25N7O6S  
 
Molecular weight: 479.51 
 
pKa: 10.0 (basic) and 3.23 (basic) 
 
pH: 5.51 (water slurry 10 mg/mL) 
 
Specific Rotation, [α]20D: Not applicable, achiral 
 
pI: Not applicable 
 
Solubility: Methanol 8.44 mg/mL (slightly soluble)  
 
 
8.1.2 
Form 
 
Prexasertib (LY2606368) for injection is supplied for clinical trial use as a lyophilized, 
yellow to white solid, in glass vials, and is composed of prexasertib mesylate 
monohydrate and the inactive ingredient sulfobutylether-β-cyclodextrin (Captisol). The 
drug product is available in two vial strengths containing either 20 mg or 40 mg of the 
base compound prexasertib. The 40 mg strength vial contains a 3% excess whereas the 20 
mg strength vial contains a 5% excess to facilitate the withdrawal of the label amount (40 
mg/vial and 20 mg/vial, respectively) for application with an appropriate device, such as 
an infusion set.  
 
Reconstituting the 40 or 20 mg vial contents with 19  mL or 10 mL of water, 
respectively, yields a clear yellow solution with a concentration of 2 mg/mL of 
prexasertib, pH 4 to 6, and an osmotic pressure ratio of 344 mOsm.  
 
The reconstituted solution may be diluted with dextrose 5% injection (D5W) prior to 
administration. 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
38 
 
 
8.1.3 
Storage and Stability 
 
The drug product is stable when stored at room temperature at 10˚C - 30˚C (50˚F to 
86˚F). Do not freeze or refrigerate prexasertib (LY2606368). 
 
The reconstituted formulation is stable for at least 24 hours at room temperature; 
however, since the reconstituted drug product does not contain a preservative, the unused 
solution must be discarded after 6 hours. The infusion should be completed within 4 
hours of priming the infusion set. 
 
The investigator or pharmacist will ensure that all investigational products are stored in a 
secure area, under recommended storage conditions and in accordance with applicable 
regulatory requirements. The site should monitor the minimum and maximum 
temperature and maintain temperature logs to confirm the correct storage of all 
investigational products used in the trial. 
 
 
8.1.4 
Compatibility 
 
Prexasertib (LY2606368) is incompatible with solutions containing saline or Lactated 
Ringer’s and must not be mixed or administered simultaneously with other drugs through 
the same infusion line. 
 
 
8.1.5 
Handling 
 
Qualified personnel, familiar with procedures that minimize undue exposure to 
themselves and the environment, should undertake the preparation, handling, and safe 
disposal of the chemotherapeutic agent in a self-contained and protective environment.  
 
 
8.1.6 
Availability 
 
Prexasertib (LY2606368) is an investigational agent that will be supplied by Eli Lilly and 
Company. 
 
 
8.1.7 
Preparation 
 
Prexasertib (LY2606368) diluted in D5W is compatible with standard IV solution bags 
constructed of polyolefin or polyvinyl chloride (PVC). Contact with PVC bags should be 
limited to not more than 6 hours. Administration sets constructed of PVC (with DEHP or 
TOTM), polyethyelene-lined (paclitaxel) may be used. Administration sets with 
integrated filters or filter extension sets MUST be used for all patients. Compatibility 
with filters fabricated from polyether sulfone has been demonstrated. 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
39 
 
Table 9 illustrates the compatible materials for prexasertib administration. 
 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
40 
Table 9: Compatible Materials for Prexasertib Administration 
 
 
8.1.7.1 
Prexasertib 20 mg: 
 
Prexasertib for injection, 20 mg: Reconstituted by adding 10 mL of water for injection 
and shaking. The resulting solution contains 2 mg/mL of prexasertib for injection.  
 
Multiple vials of prexasertib will be required to prepare most dosage strengths. Obtain the 
number of vials needed to prepare the desired dose and reconstitute each vial with 10 mL 
of water for injection.  
 
Transfer the total volume of solution required into an IV bag for subsequent dilution. 
Reconstituted drug product from the vials should be diluted with dextrose 5% injection 
(D5W) to a final volume of no more than 250 mL. If less than 250 mL is used, the 
concentration of prexasertib must be maintained within a range of 0.08 mg/mL - 1.6 
mg/mL of prexasertib.  
 
Reconstituted or diluted prexasertib should NOT be used if it appears turbid or contains 
foreign particles. 
 
8.1.7.2 
Prexasertib 40 mg: 
 
Prexasertib for injection, 40 mg: Reconstituted by adding 19 mL of water for injection 
and shaking. The resulting solution contains 2 mg/mL of prexasertib for injection.  


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
41 
 
Multiple vials of prexasertib will be required to prepare most dosage strengths. Obtain the 
number of vials needed to prepare the desired dose and reconstitute each vial with 19 mL 
of water for injection.  
 
Transfer the total volume of solution required into an IV bag for subsequent dilution. 
Reconstituted drug product from the vials should be diluted with D5W to a final volume 
of no more than 250 mL. If less than 250 mL is used, the concentration of prexasertib 
must be maintained within a range of 0.08 mg/mL - 1.6 mg/mL of prexasertib. 
 
Reconstituted or diluted prexasertib should NOT be used if it appears turbid or contains 
foreign particles. 
 
 
8.1.8 
Administration 
 
Prexasertib (LY2606368) should be administered IV over 1 hour (+/- 5 minute infusion 
window) using a central or free flowing peripheral IV line with an in-line, sterile, non-
pyrogenic, 0.22-μm filter. Reconstituted or diluted prexasertib should NOT be used if it 
appears turbid or contains foreign particles. 
  
 
8.1.9 
Ordering 
 
Drug supply will be ordered from Eli Lilly by site pharmacy personnel. 
 
 
8.1.10 Accountability 
 
The investigator, or a responsible party designated by the investigator, should maintain a 
careful record of the inventory and disposition of the agent using the NCI Drug 
Accountability Record Form (DARF) or another comparable drug accountability form.  
(See the NCI Investigator’s Handbook for Procedures for Drug Accountability and 
Storage.) 
 
8.1.11 Destruction and Return 
 
Expired supplies of prexasertib (LY2606368) should be destroyed according to 
institutional policies. Destruction will be documented in the Drug Accountability Record 
Form. At the end of the study, unused supplies of prexasertib should be destroyed 
according to institutional policies. Destruction will be documented in the Drug 
Accountability Record Form. 
 
 
9. BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES 
 
 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
42 
9.1 
Biomarker Studies 
 
9.1.1 
Exploratory Tumor Biopsy – Replicative Stress and HR Cohorts 
 
Mandatory fresh tumor biopsies will be obtained from the patients enrolled to the replicative 
stress or HR cohorts during Stage 1 of the 2-stage trial. The biopsies will be obtained at baseline 
(any time prior to the first infusion). 
 
Core biopsy samples should be obtained for analysis. Three-to-four biopsy passes utilizing a 16-
18 gauge needle are preferable, but a 20 gauge core needle biopsy is also acceptable. Less than 
the goal amount of tissue is acceptable for the biopsy procedure, and should be based upon the 
clinical judgment of the treating investigator and the clinician performing the procedure. 
 
Biopsy samples should be fresh frozen. A plastic cryomold should be labeled with the protocol 
number, study subject number, and the exact date and time of collection.  Several drops of OCT 
compound will be placed into the cryomold and at time of biopsy the sample will be placed into 
OCT, covered with additional drops of OCT and then placed upon dry ice immediately.  The 
specimen will then be stored at -80ºC until used. 
 
The frozen samples should be shipped to the Broad Institute Center for Cancer Precision 
Medicine. The baseline tumor tissue sample should be shipped at the same time as the germline 
blood sample as described below in Section 9.1.3. 
 
Biopsy samples should be sent on dry ice to: 
 
Broad Institute Center for Cancer Precision Medicine  
320 Charles Street  
ATTN: Samples Lab 
Cambridge, MA 02141   
 
 
9.1.2 
Blood sample collection – All Cohorts 
 
For all patients enrolling on trial, blood samples will be collected to be analyzed for cell-free 
DNA (cfDNA). A sample will be collected at baseline (prior to the first infusion), at visits 
immediately following restaging scans (i.e., cycle 3 day 1, cycle 5 day 1, cycle 7 day 1, etc.) and 
at the off study visit. Samples collected on days where a prexasertib infusion will be given can be 
collected anytime pre-infusion. 
 
For the patients enrolling during Stage 1 of the trial in the replicative stress or HR cohorts only, a 
germline blood sample will be collected at baseline (any time prior to the first infusion) to be 
paired with the pre-treatment fresh tumor biopsy obtained for WES.  
 
 
9.1.2.1 
Germline Blood Sample for WES Collection Procedure (HR and Replicative Stress Cohorts): 
 
Blood sample should be labeled with the protocol number, study subject number, and the date of 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
43 
collection.  
 
• One 10 mL sample will be drawn into an EDTA containing test tube. 
• Freeze immediately upright at -80ºC until shipping. 
• Both the germline blood sample and pre-treatment fresh tumor biopsy samples 
should be shipped to the Broad Institute at the same time for processing. 
• The samples should be sent to the following address to be processed: 
Broad Institute Center for Cancer Precision Medicine 
320 Charles Street  
ATTN: Samples Lab 
Cambridge, MA 02141  
 
 
9.1.2.2 
cfDNA Sample Collection Procedure (All Cohorts): 
 
1. Draw two 9 mL blood samples into Streck Tubes. 
2. Blood samples should be labeled with: cfDNA, protocol number, study subject number, 
and the date of the draw. 
3. Within 2 hours of the draw time, cfDNA samples should be processed by the DF/HCC 
Clinical Trials Core laboratory as follows: 
1. Immediately centrifuge for 10 minutes at 1500 (+\- 150) x g. NOTE: Brake switch 
must be off so the cell/plasma interface is not disturbed. 
2. Pipette the plasma layer into a 15 mL tube labeled “CFDNA/with patient #”. Do 
not ship. NOTE: Do not dip the tip of the pipette into the plasma/cell interface.  
Leave a thin plasma layer intact over the interface. 
3. Centrifuge the 15 mL tube containing the plasma only for 10 minutes at 3000 (+/- 
150) x g. 
4. Transfer using a fresh pipette, the supernatant into a second 15 mL tube labeled 
“CFDNA super.do not ship”. NOTE: Leave about 0.3 mL of supernatant in the 
centrifuged 15 mL tube. This leftover 0.3 mL contains cellular debris. 
5. Using a fresh pipette, transfer 1 mL of plasma from the “super.do not ship” tube 
into max four (4) 2 mL cryovials labeled CFDNA.ship.patient#/ 
6. Freeze immediately upright at -70ºC or colder until shipping. 
 
4. Samples should then be sent to the Broad Institute at: 
Broad Institute of MIT and Harvard 
75 Ames Street 
Lab 75A-4045 
Cambridge, MA 02142 
 
 
10. STUDY CALENDAR 
 
Baseline evaluations are to be conducted within 14 days prior to start of protocol therapy, with 
the exception of the informed consent and baseline tumor imaging which may be obtained up to 
28 days prior to the start of protocol therapy. Assessments must be performed prior to 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
44 
administration of any study agent. 


 
 
Page 45 
 
 
 
 
Table 10: Study Calendar 
Screeninga 
Cycle 1 Day 1 
Cycle 1 Day 8h 
Cycle 1 Day 15i 
Cycle 1 Day 22h 
Cycle 2+ Day 1j 
Cycle 2+ Day 15i 
Off 
Treatmentk 
Every 3 Months 
After Discontinuingl 
Prexasertib 
(LY2606368)b 
 
X 
 
X 
 
X 
X 
 
 
Informed consent 
X 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
Medical history 
X 
 
 
 
 
 
 
 
 
Physical exam 
X 
X 
X 
X 
X 
X 
X 
X 
 
Vital signsc 
X 
X 
X 
X 
X 
X 
X 
X 
 
Height 
X 
 
 
 
 
 
 
 
 
Weight 
X 
X 
 
X 
 
X 
X 
X 
 
ECOG performance 
status 
X 
X 
 
X 
 
X 
X 
X 
 
CBC w/diff & Plt 
X 
X 
X 
X 
X 
X 
X 
X 
 
Serum chemistryd 
X 
X 
X 
X 
X 
X 
X 
X 
 
β-HCGe 
X 
 
 
 
 
 
 
X 
 
EKGf 
X 
X 
 
 
 
X 
 
 
 
Adverse Events 
 
X 
X 
X 
X 
X 
X 
X 
 
Radiologic evaluation 
X 
CT or MRI imaging of any disease-involved site. Radiologic measurements should be performed at the end of cycle 2 
(cycle 2 day 28) and at the end of every 2 cycles of treatment thereafter (i.e. cycle 4 day 28, cycle 6 day 28, and so 
on). There is a ± 7 day window on imaging evaluations. 
X 
 
Fresh Tumor Biopsyg 
X 
 
 
 
 
 
 
 
 
Research Germline 
Blood Sample for 
X 
 
 
 
 
 
 
 
 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
46 
 
Table 10: Study Calendar 
Screeninga 
Cycle 1 Day 1 
Cycle 1 Day 8h 
Cycle 1 Day 15i 
Cycle 1 Day 22h 
Cycle 2+ Day 1j 
Cycle 2+ Day 15i 
Off 
Treatmentk 
Every 3 Months 
After Discontinuingl 
WESg 
Research Blood 
Samples for cfDNA 
 
X 
cfDNA samples to be collected at the visit immediately following a restaging scan (i.e., cycle 3 day 
1, cycle 5 day 1, cycle 7 day 1, and so on) as described in Section 9. 
X 
 
Telephone or Care 
Provider Contact 
 
 
 
 
 
 
 
 
X 
a) 
Screening evaluations are to be performed within 14 days of cycle 1 day 1, with the exception of the informed consent and baseline tumor imaging which may be obtained up to 28 days prior to 
cycle 1 day 1. 
b) 
Prexasertib (LY2606368) dose as assigned; see administration guidelines in Section 5. 
c) 
Heart rate, respiratory rate, blood pressure, temperature, and oxygen saturation (O2 sat). On infusion days, vital signs are to be collected anytime pre-infusion (exact time of vital signs should be 
recorded). May be repeated as clinically indicated. 
d) 
Sodium, potassium, chloride, CO2, blood urea nitrogen (BUN), creatinine, glucose, albumin, total protein, alkaline phosphatase, total bilirubin, SGOT [AST], SGPT [ALT], globulin, and 
magnesium. Other tests may be ordered as clinically indicated. 
e) 
Serum pregnancy test only required for women of childbearing potential. Childbearing potential defined as any female who has experienced menarche and who has not undergone successful 
surgical sterilization (hysterectomy, bilateral tubal ligation or bilateral oophorectomy) or is not postmenopausal (defined as amenorrhea >12 consecutive months; or women with a documented 
plasma follicle-stimulating hormone level >35µlU/mL). 
f) 
To be performed at screening, at any time pre-dose on cycle 1 day 1, upon completion of the first infusion (+15 minute window from the end of the infusion), and 1 hour after the end of the 
infusion (+30 minute window). EKG to be completed pre-dose (any time prior to the infusion) on the first day of each subsequent cycle. Additional EKGs may be performed as clinically 
indicated at the treating investigator’s discretion. If QTcF increases > 30 msec from screening EKG at any time point, EKG should be repeated at 10 minutes after the end of the infusion (± 5 
minute window), 30 minutes after the end of the infusion (± 10 minute window), and 1 hour after the end of the infusion (+30 minute window). The exact time of all EKGs should be recorded. 
g) 
Baseline fresh tumor biopsy and germline blood sample for WES required for the patients enrolled during Stage 1 of the HR and replicative stress cohorts. Please see Section 9 for further detail.  
h) 
Cycle 1 day 8 and cycle 1 day 22 may be shifted by ± 3 days for scheduling issues (vacations, holidays, adverse weather, etc.). 
i) 
Day 15 assessments and infusion may shifted ± 1 day for scheduling issues (vacations, holidays, adverse weather, etc.). 
j) 
The start of a subsequent cycle may be delayed by up to 7 days for scheduling issues (vacations, holidays, adverse weather, etc.).  
k) 
Off-treatment evaluation. Note: follow up visits or other contact is required in order to identify SAEs during the 30 days following the end of study treatment. 
l) 
Participants will be followed until death or withdrawal of consent after removal from protocol therapy for survival status only. This follow up will be performed by review of the medical record, 
contact with care providers, and/or telephone contact as needed every 3 to 4 months.   


 
 
Page 47 
 
 
11. MEASUREMENT OF EFFECT 
 
 
11.1 
Antitumor Effect 
 
For the purposes of this study, participants should be re-evaluated for response at the end of 
every 2 cycles of treatment (every 8 weeks) as described in the Study Calendar.  In addition to a 
baseline scan, confirmatory scans should also be obtained not less than 4 weeks following initial 
documentation of objective response. 
 
Response and progression will be evaluated in this study using the new international criteria 
proposed by the Response Evaluation Criteria in Solid Tumors (RECIST) guideline (version 1.1) 
[Eur J Ca 45:228-247, 2009].  Changes in the largest diameter (unidimensional measurement) of 
the tumor lesions and the shortest diameter in the case of malignant lymph nodes are used in the 
RECIST criteria. 
 
11.1.1 Definitions 
 
Evaluable for Target Disease response.  Only those participants who have measurable 
disease present at baseline, have received at least one cycle of therapy, and have had their 
disease re-evaluated will be considered evaluable for target disease response.  These 
participants will have their response classified according to the definitions stated below.  
(Note:  Participants who exhibit objective disease progression prior to the end of cycle 1 
will also be considered evaluable.) 
 
Evaluable Non-Target Disease Response.  Participants who have lesions present at 
baseline that are evaluable but do not meet the definitions of measurable disease, have 
received at least one cycle of therapy, and have had their disease re-evaluated will be 
considered evaluable for non-target disease.  The response assessment is based on the 
presence, absence, or unequivocal progression of the lesions.  
 
11.1.2 Disease Parameters 
 
Measurable disease.  Measurable lesions are defined as those that can be accurately 
measured in at least one dimension (longest diameter to be recorded) as ≥ 20 mm by 
chest x-ray or ≥10 mm with CT scan, MRI, or calipers by clinical exam.  All tumor 
measurements must be recorded in millimeters (or decimal fractions of centimeters). 
 
Note: Tumor lesions that are situated in a previously irradiated area are not considered 
measurable unless there has been demonstrated progression in the lesion.  
 
Malignant lymph nodes.  To be considered pathologically enlarged and measurable, a 
lymph node must be ≥15 mm in short axis when assessed by CT scan (CT scan slice 
thickness recommended to be no greater than 5 mm).  At baseline and in follow-up, only 
the short axis will be measured and followed. 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
48 
 
Non-measurable disease.  All other lesions (or sites of disease), including small lesions 
(longest diameter <10 mm or pathological lymph nodes with ≥10 to <15 mm short axis), 
are considered non-measurable disease.  Bone lesions, leptomeningeal disease, ascites, 
pleural/pericardial effusions, lymphangitis cutis/pulmonitis, inflammatory breast disease, 
abdominal masses (not followed by CT or MRI), and cystic lesions are all considered 
non-measurable. 
 
Note: Cystic lesions that meet the criteria for radiographically defined simple cysts 
should not be considered as malignant lesions (neither measurable nor non-measurable) 
since they are, by definition, simple cysts. 
 
‘Cystic lesions’ thought to represent cystic metastases can be considered as measurable 
lesions, if they meet the definition of measurability described above. However, if non-
cystic lesions are present in the same participant, these are preferred for selection as 
target lesions. 
 
Target lesions.  All measurable lesions up to a maximum of 2 lesions per organ and 5 
lesions in total, representative of all involved organs, should be identified as target 
lesions and recorded and measured at baseline.  Target lesions should be selected on the 
basis of their size (lesions with the longest diameter), be representative of all involved 
organs, but in addition should be those that lend themselves to reproducible repeated 
measurements. It may be the case that, on occasion, the largest lesion does not lend itself 
to reproducible measurement in which circumstance the next largest lesion which can be 
measured reproducibly should be selected. A sum of the diameters (longest for non-nodal 
lesions, short axis for nodal lesions) for all target lesions will be calculated and reported 
as the baseline sum diameters. If lymph nodes are to be included in the sum, then only the 
short axis is added into the sum. The baseline sum diameters will be used as reference to 
further characterize any objective tumor regression in the measurable dimension of the 
disease.  
 
Non-target lesions.  All other lesions (or sites of disease) including any measurable 
lesions over and above the 5 target lesions should be identified as non-target lesions and 
should also be recorded at baseline. Measurements of these lesions are not required, but 
the presence, absence, or in rare cases unequivocal progression of each should be noted 
throughout follow up.  
 
11.1.3 Methods for Evaluation of Disease 
 
All measurements should be taken and recorded in metric notation using a ruler, calipers, 
or a digital measurement tool.  All baseline evaluations should be performed as closely as 
possible to the beginning of treatment and never more than 28 days before the beginning 
of the treatment. 
 
The same method of assessment and the same technique should be used to characterize 
each identified and reported lesion at baseline and during follow-up. Imaging-based 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
49 
evaluation is preferred to evaluation by clinical examination unless the lesion(s) being 
followed cannot be imaged but are assessable by clinical exam.  
 
Clinical lesions. Clinical lesions will only be considered measurable when they are 
superficial (e.g., skin nodules and palpable lymph nodes) and ≥10 mm in diameter as 
assessed using calipers (e.g., skin nodules).  In the case of skin lesions, documentation by 
color photography, including a ruler to estimate the size of the lesion, is recommended.  
 
Chest x-ray.  Lesions on chest x-ray are acceptable as measurable lesions when they are 
clearly defined and surrounded by aerated lung; however, CT is preferable.  
 
Conventional CT and MRI. This guideline has defined measurability of lesions on CT 
scan based on the assumption that CT thickness is 5mm or less. If CT scans have slice 
thickness greater than 5 mm, the minimum size of a measurable lesion should be twice 
the slice thickness. MRI is also acceptable in certain situations (e.g. for body scans).   
 
Use of MRI remains a complex issue.  MRI has excellent contrast, spatial, and temporal 
resolution; however, there are many image acquisition variables involved in MRI, which 
greatly impact image quality, lesion conspicuity, and measurement.  Furthermore, the 
availability of MRI is variable globally.  As with CT, if an MRI is performed, the 
technical specifications of the scanning sequences used should be optimized for the 
evaluation of the type and site of disease.  Furthermore, as with CT, the modality used at 
follow-up should be the same as was used at baseline and the lesions should be 
measured/assessed on the same pulse sequence.  It is beyond the scope of the RECIST 
guidelines to prescribe specific MRI pulse sequence parameters for all scanners, body 
parts, and diseases.  Ideally, the same type of scanner should be used and the image 
acquisition protocol should be followed as closely as possible to prior scans.  Body scans 
should be performed with breath-hold scanning techniques, if possible. 
 
FDG-PET. While FDG-PET response assessments need additional study, it is sometimes 
reasonable to incorporate the use of FDG-PET scanning to complement CT scanning in 
assessment of progression (particularly possible 'new' disease).  New lesions on the basis 
of FDG-PET imaging can be identified according to the following algorithm:  
(a) Negative FDG-PET at baseline, with a positive FDG-PET at follow-up is a sign of 
PD based on a new lesion. 
(b)  No FDG-PET at baseline and a positive FDG-PET at follow-up:  If the positive 
FDG-PET at follow-up corresponds to a new site of disease confirmed by CT, this is 
PD.  If the positive FDG-PET at follow-up is not confirmed as a new site of disease 
on CT, additional follow-up CT  scans are needed to determine if there is truly 
progression occurring at that site (if so, the date of PD will be the date of the initial 
abnormal FDG-PET scan).  If the positive FDG-PET at follow-up corresponds to a 
pre-existing site of disease on CT that is not progressing on the basis of the anatomic 
images, this is not PD. 
(c)  FDG-PET may be used to upgrade a response to a CR in a manner similar to a 
biopsy in cases where a residual radiographic abnormality is thought to represent 
fibrosis or scarring.  The use of FDG-PET in this circumstance should be 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
50 
prospectively described in the protocol and supported by disease-specific medical 
literature for the indication.  However, it must be acknowledged that both approaches 
may lead to false positive CR due to limitations of FDG-PET and biopsy 
resolution/sensitivity. 
 
Note:  A ‘positive’ FDG-PET scan lesion means one which is FDG avid with an uptake 
greater than twice that of the surrounding tissue on the attenuation corrected image. 
 
PET-CT. At present, the low dose or attenuation correction CT portion of a combined 
PET-CT is not always of optimal diagnostic CT quality for use with RECIST 
measurements.  However, if the site can document that the CT performed as part of a 
PET-CT is of identical diagnostic quality to a diagnostic CT (with IV and oral contrast), 
then the CT portion of the PET-CT can be used for RECIST measurements and can be 
used interchangeably with conventional CT in accurately measuring cancer lesions over 
time.  Note, however, that the PET portion of the CT introduces additional data which 
may bias an investigator if it is not routinely or serially performed.   
MIBG (meta-iodobenzylguanidine).The following is recommended, to assure high 
quality images are obtained.  
Patient preparation: Iodides, usually SSKI (saturated solution of potassium iodide), 
are administered to reduce thyroidal accumulation of free radioiodine, preferably 
beginning the day prior to injection and continuing for 3 additional days (4 days 
total). For infants and children, one drop t.i.d. is sufficient, for adolescents 2 drops 
t.i.d., and for adults 3 drops t.i.d. Participants and/or parents are asked about exposure 
to potential interfering agents. If none is noted, an indwelling intravenous line is 
established. The dose of MIBG is administered by slow intravenous injection over 90 
seconds.  
Images from the head to the distal lower extremities should be obtained.  
I-123MIBG scintigraphy is performed to obtain both planar and tomographic images.  
Planar: Anterior and posterior views from the top of the head to the proximal lower 
extremities are obtained for 10 minutes at 24 hours and occasionally at 48 hours 
following injection of 10 mCi/1.7 square meters of body surface area (~150 μCi/kg, 
maximum 10 mCi). Anterior views of the distal lower extremities are adequate. A 
large field of view dual head gamma camera with low energy collimators is preferred.  
 
SPECT: Most participants receiving I-123 MIBG also undergo SPECT at 24 hours, 
using a single or multi-headed camera with a low energy collimator. The camera is 
rotated through 360 degrees, 120 projections at 25 seconds per stop. Data are 
reconstructed using filtered back projections with a Butterworth filter and a cut off 
frequency of 0.2-0.5. SPECT/CT may be performed at institutions with this capacity. 
 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
51 
Ultrasound. Ultrasound is not useful in assessment of lesion size and should not be used 
as a method of measurement. Ultrasound examinations cannot be reproduced in their 
entirety for independent review at a later data and, because they are operator dependent, it 
cannot be guaranteed that the same technique and measurements will be taken from one 
assessment to the next. If new lesions are identified by ultrasound in the course of the 
study, confirmation by CT or MRI is advised. If there is concern about radiation exposure 
from CT, MRI may be used instead of CT in selected instances.  
 
Endoscopy, Laparoscopy. The utilization of these techniques for objective tumor 
evaluation is not advised. However, such techniques may be useful to confirm complete 
pathological response when biopsies are obtained or to determine relapse in trials where 
recurrence following complete response (CR) or surgical resection is an endpoint. 
 
Tumor markers. Tumor markers alone cannot be used to assess response. If markers are 
initially above the upper normal limit, they must normalize for a participant to be 
considered in complete clinical response.  Specific guidelines for both CA-125 response 
(in recurrent ovarian cancer) and PSA response (in recurrent prostate cancer) have been 
published [JNCI 96:487-488, 2004; J Clin Oncol 17, 3461-3467, 1999; J Clin Oncol 
26:1148-1159, 2008].  In addition, the Gynecologic Cancer Intergroup has developed 
CA-125 progression criteria which are to be integrated with objective tumor assessment 
for use in first-line trials in ovarian cancer [JNCI 92:1534-1535, 2000]. 
 
Cytology, Histology. These techniques can be used to differentiate between partial 
responses (PR) and complete responses (CR) in rare cases (e.g., residual lesions in tumor 
types, such as germ cell tumors, where known residual benign tumors can remain). 
 
The cytological confirmation of the neoplastic origin of any effusion that appears or 
worsens during treatment when the measurable tumor has met criteria for response or 
stable disease is mandatory to differentiate between response or stable disease (an 
effusion may be a side effect of the treatment) and progressive disease. 
 
 
11.1.3.1 
Evaluation of Target Lesions 
 
Complete Response (CR):  Disappearance of all target lesions.  Any pathological 
lymph nodes (whether target or non-target) must have reduction in short axis to <10 
mm. 
 
Partial Response (PR):  At least a 30% decrease in the sum of the diameters of target 
lesions, taking as reference the baseline sum diameters. 
 
Progressive Disease (PD):  At least a 20% increase in the sum of the diameters of 
target lesions, taking as reference the smallest sum on study (this includes the 
baseline sum if that is the smallest on study).  In addition to the relative increase of 
20%, the sum must also demonstrate an absolute increase of at least 5 mm.  (Note:  
the appearance of one or more new lesions is also considered progressions). 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
52 
 
Stable Disease (SD):  Neither sufficient shrinkage to qualify for PR nor sufficient 
increase to qualify for PD, taking as reference the smallest sum diameters while on 
study. 
 
11.1.3.2 
Evaluation of Non-Target Lesions 
 
Complete Response (CR):  Disappearance of all non-target lesions and normalization 
of tumor marker level.  All lymph nodes must be non-pathological in size (<10 mm 
short axis). 
 
Note:  If tumor markers are initially above the upper normal limit, they must 
normalize for a patient to be considered in complete clinical response. 
 
Non-CR/Non-PD:  Persistence of one or more non-target lesion(s) and/or 
maintenance of tumor marker level above the normal limits. 
 
Progressive Disease (PD):  Appearance of one or more new lesions and/or 
unequivocal progression of existing non-target lesions.  Unequivocal progression 
should not normally trump target lesion status.  It must be representative of overall 
disease status change, not a single lesion increase.     
 
Although a clear progression of “non-target” lesions only is exceptional, the opinion 
of the treating physician should prevail in such circumstances, and the progression 
status should be confirmed at a later time by the review panel (or Principal 
Investigator). 
 
11.1.3.3 
Evaluation of New Lesions 
 
The finding of a new lesion should be unequivocal (i.e. not due to difference in 
scanning technique, imaging modality, or findings thought to represent something 
other than tumor (for example, some ‘new’ bone lesions may be simply healing or 
flare of pre-existing lesions). However, a lesion identified on a follow-up scan in an 
anatomical location that was not scanned at baseline is considered new and will 
indicate PD. If a new lesion is equivocal (because of small size etc.), follow-up 
evaluation will clarify if it truly represents new disease and if PD is confirmed, 
progression should be declared using the date of the initial scan on which the lesion 
was discovered. 
 
11.1.3.4 
Evaluation of Best Overall Response 
 
The best overall response is the best response recorded from the start of the treatment 
until disease progression/recurrence (taking as reference for progressive disease the 
smallest measurements recorded since the treatment started).  The patient's best 
response assignment will depend on the achievement of both measurement and 
confirmation criteria. 
 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
53 
For Participants with Measurable Disease (i.e., Target Disease) 
 
Table 11: RECIST Criteria for Evaluation of Response 
Target 
Lesions 
Non-Target 
Lesions 
New 
Lesions 
Overall 
Response 
Best Overall Response when 
Confirmation is Required* 
CR 
CR 
No 
CR 
>4 wks Confirmation** 
CR 
Non-CR/Non-
PD 
No 
PR 
>4 wks Confirmation** 
CR 
Not evaluated 
No 
PR 
PR 
Non-CR/Non-
PD/not 
evaluated 
No 
PR 
SD 
Non-CR/Non-
PD/not 
evaluated 
No 
SD 
Documented at least once >4 
wks from baseline** 
PD 
Any 
Yes or No 
PD 
no prior SD, PR or CR 
Any 
PD*** 
Yes or No 
PD 
Any 
Any 
Yes 
PD 
 
See RECIST 1.1 manuscript for further details on what is evidence of a new lesion. 
**        Only for non-randomized trials with response as primary endpoint. 
***      In exceptional circumstances, unequivocal progression in non-target lesions may be 
            accepted as disease progression. 
 
Note:  Participants with a global deterioration of health status requiring discontinuation of 
treatment without objective evidence of disease progression at that time should be 
reported as “symptomatic deterioration.”  Every effort should be made to document the 
objective progression even after discontinuation of treatment. 
 
For Participants with Non-Measurable Disease (i.e., Non-Target Disease) 
 
Table 12: RECIST Criteria for Patients with Non-Measurable Disease 
Non-Target Lesions 
New Lesions 
Overall Response 
CR 
No 
CR 
Non-CR/non-PD 
No 
Non-CR/non-PD* 
Not all evaluated 
No 
not evaluated 
Unequivocal PD 
Yes or No 
PD 
Any 
Yes 
PD 
 
‘Non-CR/non-PD’ is preferred over ‘stable disease’ for non-target disease since SD is 
increasingly used as an endpoint for assessment of efficacy in some trials so to assign 
this category when no lesions can be measured is not advised 
 
11.1.4 Duration of Response 
 
Duration of overall response:  The duration of overall response is measured from the time 
measurement criteria are met for CR or PR (whichever is first recorded) until the first 
date that recurrent or progressive disease is objectively documented (taking as reference 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
54 
for progressive disease the smallest measurements recorded since the treatment started, or 
death due to any cause. Participants without events reported are censored at the last 
disease evaluation). 
 
Duration of overall complete response: The duration of overall CR is measured from the 
time measurement criteria are first met for CR until the first date that progressive disease 
is objectively documented, or death due to any cause. Participants without events 
reported are censored at the last disease evaluation. 
 
 
Duration of stable disease:  Stable disease is measured from the start of the treatment 
until the criteria for progression are met, taking as reference the smallest measurements 
recorded since the treatment started, including the baseline measurements.  
 
11.1.5 Progression-Free Survival 
 
Overall Survival: Overall Survival (OS) is defined as the time from randomization (or 
registration) to death due to any cause, or censored at date last known alive. 
 
Progression-Free Survival: Progression-Free Survival (PFS) is defined as the time from 
randomization (or registration) to the earlier of progression or death due to any cause. 
Participants alive without disease progression are censored at date of last disease 
evaluation. 
 
Time to Progression: Time to Progression (TTP) is defined as the time from 
randomization (or registration) to progression, or censored at date of last disease 
evaluation for those without progression reported. 
 
11.1.6 Response Review 
 
Evaluation of scans will be done centrally at the DFCI using the Tumor Metrics Core. 
 
 
12. DATA REPORTING / REGULATORY REQUIREMENTS 
 
Adverse event lists, guidelines, and instructions for AE reporting can be found in Section 7.0 
(Adverse Events: List and Reporting Requirements). 
 
12.1 
Data Reporting 
 
12.1.1 Method 
 
The Office of Data Quality (ODQ) will collect, manage, and perform quality checks on 
the data for this study. 
 
12.1.2 Responsibility for Data Submission 
 
Investigative sites within DF/HCC or DF/PCC are responsible for submitting data and/or 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
55 
data forms to the ODQ according to the schedule set by the ODQ. 
 
 
12.2 
Data Safety Monitoring 
 
The DF/HCC Data and Safety Monitoring Committee (DSMC) will review and monitor toxicity 
and accrual data from this study. The committee is composed of clinical specialists with 
experience in oncology and who have no direct relationship with the study. Information that 
raises any questions about participant safety will be addressed with the Overall PI and study 
team. 
 
The DSMC will review each protocol up to four times a year or more often if required to review 
toxicity and accrual data. Information to be provided to the committee may include: up-to-date 
participant accrual; current dose level information; DLT information; all grade 2 or higher 
unexpected adverse events that have been reported; summary of all deaths occurring with 30 
days of intervention for Phase I or II protocols; for gene therapy protocols, summary of all deaths 
while being treated and during active follow-up; any response information; audit results, and a 
summary provided by the study team. Other information (e.g. scans, laboratory values) will be 
provided upon request.  
 
 
13. STATISTICAL CONSIDERATIONS 
 
This is a three-arm phase II study exploring the anti-tumor activity of prexasertib (LY2606368) 
in patients with advanced cancer exhibiting either replicative stress, HR deficiency, or CCNE1 
amplification. The primary goal of the study will be to examine the progression-free rate at 4 
months in all arms separately. Secondary efficacy measures will include assessment of objective 
response rate by RECIST 1.1 as well as toxicity using CTCAE version 4.0 criteria, overall 
survival (OS), and progression free survival (PFS). 
 
Each arm will be conducted as a Simon’s 2-stage design and will initially enroll 9 participants. If 
there are less than 2 subjects that are alive and progression-free at 4 months after initiation of 
prexasertib, that arm will be terminated. If 2 or more subjects are progression free by 4 months, 
then enrollment for that arm will be expanded to 19 subjects.  A minimum of 6 patients must be 
alive and progression-free at 4 months for the cohort to be considered meaningful for further 
evaluation. If at any point a participant does not complete Cycle 1 dosing of the study agent, that 
participant may be replaced at the principal investigator’s discretion.  
 
Progression status by 4 months will be determined for all patients. We expect all patients to have 
this status documented. PFS will be defined as the time from study registration to documented 
disease progression per RECIST 1.1 criteria or death from any cause, whichever occurs first. OS 
will be defined as the time from study registration until death. Patients who have not experienced 
an event of interest by the time of analysis will be censored at the date they are last known to be 
progression-free for PFS and alive for OS.  
 
This design provides 80% power to detect a progression-free rate at 4 months of 40% tested 
against a historical control rate of ≤ 15%57 using a one-sided significance of 0.05.  


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
56 
 
13.1 
Study Design/Endpoints 
 
Primary Efficacy Measures: 
1) Investigate the progression-free rate at 4 months in three cohorts of patients with tumors 
exhibiting:  
a. 
Replication stress, as defined by tumors with MYC amplification, CCNE1 
amplification, Rb loss, or FBXW7 mutation  
 
b. 
HR deficiency, as defined by patients with either genomic or somatic alteration in 
a known HR gene, including BRCA1, BRCA2, PALB2, RAD51C, RAD51D, ATR, 
ATM, CHK2, or Fanconi anemia pathway genes  
 
c. 
CCNE1 amplification, defined as tumors with 6-fold amplification or greater.  
 
Secondary Efficacy Measures: 
1) Investigate the toxicity and objective response rates to prexasertib in these cohorts. 
 
Exploratory Endpoints: 
1) WES will be performed on the pre-treatment biopsies collected from participants enrolled 
to the HR and replicative stress cohorts during Stage 1 to examine possible genomic 
determinants of sensitivity or resistance to CHK1 inhibition. 
 
2) Clinical outcome will also be correlated to the tumor DNA levels obtained on the serial 
plasma samples submitted for cfDNA. 
 
 
13.2 
Sample Size, Accrual Rate and Study Duration 
 
The planned sample size is a maximum of 19 patients in each arm. The replicative stress cohort 
is now closed and will not expand, having enrolled 10 participants. Allowing for replacement of 
participants who do not make it through one cycle of dosing, the estimate for the maximum 
sample size is 50 participants (with 35 yet to enroll as of 5 December 2017).  
 
As of 5 December 2017, a total of 15 participants have been accrued. Adjusting for the current 
accrual level and slower estimated accrual for the CCNE1 cohort, we anticipate accruing 
approximately 3-4 patients per quarter. An additional year of follow-up will be required on the 
last participant accrued to observe the patient’s response following study therapy, for a total 
study length of about 4.5 years. Participants will be identified via targeted NextGen sequencing 
using the DFCI/BWH OncoPanel or another CLIA-certified method for genotyping. 
 
 
Table 13: Accrual Targets 
Ethnic Category 
Sex/Gender 
Females 
 Males 
 Total 
Hispanic or Latino 
3 
+ 
0 
= 
3 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
57 
Not Hispanic or Latino 
25 
+ 
22 
= 
47 
Ethnic Category: Total of all 
subjects 
28 
+ 
22 
= 
50 
Racial Category 
 
American Indian or Alaskan Native 
0 
+ 
0 
= 
0 
Asian 
0 
+ 
0 
= 
0 
Black or African American 
2 
+ 
0 
= 
2 
Native Hawaiian or other Pacific 
Islander 
0 
+ 
0 
= 
0 
White 
26 
+ 
22 
= 
48 
Racial Category: Total of all 
subjects 
28 
+ 
22 
= 
50 
 
 
  
  
 
13.3 
Analysis of Primary and Secondary Endpoints 
 
The primary and secondary analyses will include all eligible patients who completed at least one 
cycle of dosing. The exception to this includes the planned analysis of toxicity data, which will 
include all patients who received study drug regardless of eligibility and regardless of whether 
they completed a full cycle. Progression-free rate will be estimated as binomial proportion and 
overall survival and progression-free survival estimates and curves will be generated using the 
Kaplan-Meier method. 
 
 
13.4 
Analysis of Exploratory Endpoints 
 
WES will be retrospectively performed on the pre-treatment biopsies obtained during the first 
stage of the HR and replicative stress study arms. The WES will profile the entire HR 
proficiency of the tumor, and will involve up to 100 genes of interest in DNA repair in order to 
obtain the entire HR state for each participant. The results of the tissue analyses will be 
compared to the clinical outcomes of the patients, and will hopefully help to further identify the 
genetic characteristics of patients who will respond to treatment with prexasertib (LY2606368) 
versus patients who will not. This data may help with future selection of patient populations 
appropriate for treatment with CHK1 therapy. 
 
Clinical outcome will also be correlated to the tumor DNA levels obtained on the serial plasma 
samples submitted for cfDNA. 
 
Data from correlative studies will be summarized using descriptive statistics and may be 
explored graphically. 
 
 
13.5 
Reporting and Exclusions 
 
13.5.1 Evaluation of Toxicity 
 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
58 
All participants will be evaluable for toxicity from the time of their first treatment. 
 
13.5.2 Evaluation of the Primary Efficacy Endpoint 
 
All eligible participants included in the study who complete at least one cycle of dosing 
will be assessed for response, even if there are major protocol therapy deviations. Each 
participant should be assigned one of the following categories: 1) complete response, 2) 
partial response, 3) stable disease, 4) progressive disease, 5) early death from malignant 
disease, 6) early death from toxicity, 7) early death because of other cause, or 9) 
unknown (not assessable, insufficient data). By arbitrary convention, category 9 usually 
designates the "unknown" status of any type of data in a clinical database. 
 
 
14. PUBLICATION PLAN 
 
The results should be made public within 24 months of reaching the end of the study. The end of 
the study is the time point at which the last data items are to be reported, or after the outcome 
data are sufficiently mature for analysis, as defined in the section on Sample Size, Accrual Rate 
and Study Duration. If a report is planned to be published in a peer-reviewed journal, then that 
initial release may be an abstract that meets the requirements of the International Committee of 
Medical Journal Editors. A full report of the outcomes should be made public no later than three 
(3) years after the end of the study.  


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
59 
 
REFERENCES 
 
1. 
Dai Y, Grant S. New insights into checkpoint kinase 1 in the DNA damage response 
signaling network. Clin Cancer Res. 2010;16(2):376-383. 
2. 
Bucher N, Britten CD. G2 checkpoint abrogation and checkpoint kinase-1 targeting in the 
treatment of cancer. Br J Cancer. 2008;98(3):523-528. 
3. 
Sorensen CS, Hansen LT, Dziegielewski J, et al. The cell-cycle checkpoint kinase Chk1 
is required for mammalian homologous recombination repair. Nat Cell Biol. 
2005;7(2):195-201. 
4. 
Wang X, Kennedy RD, Ray K, Stuckert P, Ellenberger T, D'Andrea AD. Chk1-mediated 
phosphorylation of FANCE is required for the Fanconi anemia/BRCA pathway. Mol Cell 
Biol. 2007;27(8):3098-3108. 
5. 
Zhi G, Wilson JB, Chen X, et al. Fanconi anemia complementation group FANCD2 
protein serine 331 phosphorylation is important for fanconi anemia pathway function and 
BRCA2 interaction. Cancer Res. 2009;69(22):8775-8783. 
6. 
Petsalaki E, Zachos G. Chk1 and Mps1 jointly regulate correction of merotelic 
kinetochore attachments. J Cell Sci. 2013;126(Pt 5):1235-1246. 
7. 
McNeely S, Beckmann R, Bence Lin AK. CHEK again: revisiting the development of 
CHK1 inhibitors for cancer therapy. Pharmacol Ther. 2014;142(1):1-10. 
8. 
Feijoo C, Hall-Jackson C, Wu R, et al. Activation of mammalian Chk1 during DNA 
replication arrest: a role for Chk1 in the intra-S phase checkpoint monitoring replication 
origin firing. J Cell Biol. 2001;154(5):913-923. 
9. 
Syljuåsen RG, Sørensen CS, Hansen LT, et al. Inhibition of human Chk1 causes 
increased initiation of DNA replication, phosphorylation of ATR targets, and DNA 
breakage. Mol Cell Biol. 2005;25(9):3553-3562. 
10. 
Maya-Mendoza A, Petermann E, Gillespie DA, Caldecott KW, Jackson DA. Chk1 
regulates the density of active replication origins during the vertebrate S phase. EMBO J. 
2007;26(11):2719-2731. 
11. 
Petermann E, Woodcock M, Helleday T. Chk1 promotes replication fork progression by 
controlling replication initiation. Proc Natl Acad Sci U S A. 2010;107(37):16090-16095. 
12. 
King C, Diaz HB, McNeely S, et al. LY2606368 Causes Replication Catastrophe and 
Antitumor Effects through CHK1-Dependent Mechanisms. Mol Cancer Ther. 
2015;14(9):2004-2013. 
13. 
Zachos G, Black EJ, Walker M, et al. Chk1 is required for spindle checkpoint function. 
Dev Cell. 2007;12(2):247-260. 
14. 
Carrassa L, Damia G. Unleashing Chk1 in cancer therapy. Cell Cycle. 2011;10(13):2121-
2128. 
15. 
Zeman MK, Cimprich KA. Causes and consequences of replication stress. Nature cell 
biology. 2014;16(1):2-9. 
16. 
Dobbelstein M, Sorensen CS. Exploiting replicative stress to treat cancer. Nat Rev Drug 
Discov. 2015;14(6):405-423. 
17. 
Bester AC, Roniger M, Oren YS, et al. Nucleotide deficiency promotes genomic 
instability in early stages of cancer development. Cell. 2011;145(3):435-446. 
18. 
Jones RM, Mortusewicz O, Afzal I, et al. Increased replication initiation and conflicts 
with transcription underlie Cyclin E-induced replication stress. Oncogene. 
2013;32(32):3744-3753. 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
60 
19. 
Tort F, Bartkova J, Sehested M, Orntoft T, Lukas J, Bartek J. Retinoblastoma pathway 
defects show differential ability to activate the constitutive DNA damage response in 
human tumorigenesis. Cancer Res. 2006;66(21):10258-10263. 
20. 
Welcker M, Clurman BE. FBW7 ubiquitin ligase: a tumour suppressor at the crossroads 
of cell division, growth and differentiation. Nat Rev Cancer. 2008;8(2):83-93. 
21. 
Davis RJ, Welcker M, Clurman BE. Tumor suppression by the Fbw7 ubiquitin ligase: 
mechanisms and opportunities. Cancer Cell. 2014;26(4):455-464. 
22. 
Cascón A, Robledo M. MAX and MYC: a heritable breakup. Cancer Res. 
2012;72(13):3119-3124. 
23. 
Dang CV. MYC on the path to cancer. Cell. 2012;149(1):22-35. 
24. 
Nakayama N, Nakayama K, Shamima Y, et al. Gene amplification CCNE1 is related to 
poor survival and potential therapeutic target in ovarian cancer. Cancer. 
2010;116(11):2621-2634. 
25. 
Knudsen ES, McClendon AK, Franco J, Ertel A, Fortina P, Witkiewicz AK. RB loss 
contributes to aggressive tumor phenotypes in MYC-driven triple negative breast cancer. 
Cell Cycle. 2015;14(1):109-122. 
26. 
Consortium BCL. Cancer risks in BRCA2 mutation carriers. J Natl Cancer Inst. 
1999;91(15):1310-1316. 
27. 
Ford D, Easton DF, Bishop DT, Narod SA, Goldgar DE. Risks of cancer in BRCA1-
mutation carriers. Breast Cancer Linkage Consortium. Lancet. 1994;343(8899):692-695. 
28. 
Jones S, Hruban RH, Kamiyama M, et al. Exomic sequencing identifies PALB2 as a 
pancreatic cancer susceptibility gene. Science. 2009;324(5924):217. 
29. 
Hiramoto T, Nakanishi T, Sumiyoshi T, et al. Mutations of a novel human RAD54 
homologue, RAD54B, in primary cancer. Oncogene. 1999;18(22):3422-3426. 
30. 
Pennington KP, Walsh T, Harrell MI, et al. Germline and somatic mutations in 
homologous recombination genes predict platinum response and survival in ovarian, 
fallopian tube, and peritoneal carcinomas. Clin Cancer Res. 2014;20(3):764-775. 
31. 
Lord CJ, Ashworth A. The DNA damage response and cancer therapy. Nature. 
2012;481(7381):287-294. 
32. 
Petermann E, Caldecott KW. Evidence that the ATR/Chk1 pathway maintains normal 
replication fork progression during unperturbed S phase. Cell Cycle. 2006;5(19):2203-
2209. 
33. 
Petermann E, Woodcock M, Helleday T. Chk1 promotes replication fork progression by 
controlling replication initiation. Proc Natl Acad Sci U S A. 2010;107(37):16090-16095. 
34. 
Zachos G, Rainey MD, Gillespie DA. Chk1-dependent S-M checkpoint delay in 
vertebrate cells is linked to maintenance of viable replication structures. Mol Cell Biol. 
2005;25(2):563-574. 
35. 
Enders GH. Expanded roles for Chk1 in genome maintenance. The Journal of biological 
chemistry. 2008;283(26):17749-17752. 
36. 
Bahassi EM, Ovesen JL, Riesenberg AL, Bernstein WZ, Hasty PE, Stambrook PJ. The 
checkpoint kinases Chk1 and Chk2 regulate the functional associations between hBRCA2 
and Rad51 in response to DNA damage. Oncogene. 2008;27(28):3977-3985. 
37. 
Wang X, Kennedy RD, Ray K, Stuckert P, Ellenberger T, D'Andrea AD. Chk1-mediated 
phosphorylation of FANCE is required for the Fanconi anemia/BRCA pathway. Mol Cell 
Biol. 2007;27(8):3098-3108. 
38. 
Zhi G, Wilson JB, Chen X, et al. Fanconi anemia complementation group FANCD2 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
61 
protein serine 331 phosphorylation is important for fanconi anemia pathway function and 
BRCA2 interaction. Cancer Res. 2009;69(22):8775-8783. 
39. 
Zachos G, Rainey MD, Gillespie DA. Chk1-deficient tumour cells are viable but exhibit 
multiple checkpoint and survival defects. EMBO J. 2003;22(3):713-723. 
40. 
Etemadmoghadam D, Weir BA, Au-Yeung G, et al. Synthetic lethality between CCNE1 
amplification and loss of BRCA1. Proceedings of the National Academy of Sciences of 
the United States of America. 2013;110(48):19489-19494. 
41. 
Chen CC, Kennedy RD, Sidi S, Look AT, D'Andrea A. CHK1 inhibition as a strategy for 
targeting Fanconi Anemia (FA) DNA repair pathway deficient tumors. Mol Cancer. 
2009;8:24. 
42. 
Caldon CE, Musgrove EA. Distinct and redundant functions of cyclin E1 and cyclin E2 
in development and cancer. Cell Div. 2010;5:2. 
43. 
Keyomarsi K, Tucker SL, Buchholz TA, et al. Cyclin E and survival in patients with 
breast cancer. N Engl J Med. 2002;347(20):1566-1575. 
44. 
Del Pizzo JJ, Borkowski A, Jacobs SC, Kyprianou N. Loss of cell cycle regulators 
p27(Kip1) and cyclin E in transitional cell carcinoma of the bladder correlates with tumor 
grade and patient survival. Am J Pathol. 1999;155(4):1129-1136. 
45. 
Li JQ, Miki H, Ohmori M, Wu F, Funamoto Y. Expression of cyclin E and cyclin-
dependent kinase 2 correlates with metastasis and prognosis in colorectal carcinoma. 
Hum Pathol. 2001;32(9):945-953. 
46. 
Natrajan R, Mackay A, Wilkerson PM, et al. Functional characterization of the 19q12 
amplicon in grade III breast cancers. Breast Cancer Res. 2012;14(2):R53. 
47. 
Network CGAR. Integrated genomic analyses of ovarian carcinoma. Nature. 
2011;474(7353):609-615. 
48. 
Rosen DG, Yang G, Deavers MT, et al. Cyclin E expression is correlated with tumor 
progression and predicts a poor prognosis in patients with ovarian carcinoma. Cancer. 
2006;106(9):1925-1932. 
49. 
Etemadmoghadam D, deFazio A, Beroukhim R, et al. Integrated genome-wide DNA 
copy number and expression analysis identifies distinct mechanisms of primary 
chemoresistance in ovarian carcinomas. Clin Cancer Res. 2009;15(4):1417-1427. 
50. 
Etemadmoghadam D, George J, Cowin PA, et al. Amplicon-dependent CCNE1 
expression is critical for clonogenic survival after cisplatin treatment and is correlated 
with 20q11 gain in ovarian cancer. PLoS One. 2010;5(11):e15498. 
51. 
Mayr D, Kanitz V, Anderegg B, et al. Analysis of gene amplification and prognostic 
markers in ovarian cancer using comparative genomic hybridization for microarrays and 
immunohistochemical analysis for tissue microarrays. Am J Clin Pathol. 
2006;126(1):101-109. 
52. 
Gorringe KL, Jacobs S, Thompson ER, et al. High-resolution single nucleotide 
polymorphism array analysis of epithelial ovarian cancer reveals numerous 
microdeletions and amplifications. Clin Cancer Res. 2007;13(16):4731-4739. 
53. 
Nakayama K, Nakayama N, Jinawath N, et al. Amplicon profiles in ovarian serous 
carcinomas. Int J Cancer. 2007;120(12):2613-2617. 
54. 
van 't Veer LJ, Dai H, van de Vijver MJ, et al. Gene expression profiling predicts clinical 
outcome of breast cancer. Nature. 2002;415(6871):530-536. 
55. 
van de Vijver MJ, He YD, van't Veer LJ, et al. A gene-expression signature as a predictor 
of survival in breast cancer. N Engl J Med. 2002;347(25):1999-2009. 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
62 
56. 
Van Allen EM, Wagle N, Stojanov P, et al. Whole-exome sequencing and clinical 
interpretation of formalin-fixed, paraffin-embedded tumor samples to guide precision 
cancer medicine. Nat Med. 2014;20(6):682-688. 
57. 
Hong D, Infante J, Janku F, et al. Phase I Study of LY2606368, a Checkpoint Kinase 1 
Inhibitor, in Patients With Advanced Cancer. J Clin Oncol. 2016;34(15):1764-1771. 
58. 
Do K, Wilsker D, Ji J, et al. Phase I Study of Single-Agent AZD1775 (MK-1775), a 
Wee1 Kinase Inhibitor, in Patients With Refractory Solid Tumors. J Clin Oncol. 
2015;33(30):3409-3415. 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
63 
 
APPENDIX A 
PERFORMANCE STATUS CRITERIA 
 
ECOG Performance Status Scale 
Karnofsky Performance Scale 
Grade 
Descriptions 
Percent 
Description 
0 
Normal activity.  Fully active, able 
to carry on all pre-disease 
performance without restriction. 
100 
Normal, no complaints, no evidence 
of disease. 
90 
Able to carry on normal activity; 
minor signs or symptoms of disease. 
1 
Symptoms, but ambulatory.  
Restricted in physically strenuous 
activity, but ambulatory and able 
to carry out work of a light or 
sedentary nature (e.g., light 
housework, office work). 
80 
Normal activity with effort; some 
signs or symptoms of disease. 
70 
Cares for self, unable to carry on 
normal activity or to do active work. 
2 
In bed <50% of the time.  
Ambulatory and capable of all 
self-care, but unable to carry out 
any work activities.  Up and about 
more than 50% of waking hours. 
60 
Requires occasional assistance, but 
is able to care for most of his/her 
needs. 
50 
Requires considerable assistance and 
frequent medical care. 
3 
In bed >50% of the time.  Capable 
of only limited self-care, confined 
to bed or chair more than 50% of 
waking hours. 
40 
Disabled, requires special care and 
assistance. 
30 
Severely disabled, hospitalization 
indicated.  Death not imminent. 
4 
100% bedridden.  Completely 
disabled.  Cannot carry on any 
self-care.  Totally confined to bed 
or chair. 
20 
Very sick, hospitalization indicated. 
Death not imminent. 
10 
Moribund, fatal processes 
progressing rapidly. 
5 
Dead. 
0 
Dead. 
 
 
 
 
 
 
 
 
 


DF/HCC Protocol #: 16-281 
Protocol Version Date: 26 January 2018 
 
64 
 
APPENDIX B 
LIST OF DRUGS KNOWN TO PREDISPOSE TO TORSADE DE POINTES