Phase I Study of GDC-0425, a checkpoint kinase 1 inhibitor, in ...

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Phase I Study of GDC-0425, a checkpoint kinase 1 inhibitor, in combination with gemcitabine in patients with refractory solid tumors Jeffrey R. Infante1, Antoine Hollebecque2, Sophie Postel-Vinay2,3, Todd M. Bauer1, Elizabeth M. Blackwood4, Marie Evangelista4, Sami Mahrus4, Franklin V. Peale4, Xuyang Lu4, Srikumar Sahasranaman4, Rui Zhu4, Yuan Chen4, Xiao Ding4, Elaine R. Murray4, Jennifer L. Schutzman4, Jennifer O. Lauchle4, Jean-Charles Soria2,3, Patricia M. LoRusso5* 1Sarah Cannon Research Institute/Tennessee Oncology, Nashville, TN 2Départemement d’Innovation Thérapeutique et des Essais Précoces (DITEP), Gustave Roussy, Université Paris Saclay, F-94805, VILLEJUIF France 3INSERM, U981, F-94805, VILLEJUIF France 4Genentech, Inc. South San Francisco, CA 5Karmanos Cancer Institute, Detroit, MI *Current: Smilow Cancer Center, Yale University, New Haven, CT Corresponding author: Jeffrey R. Infante, M.D. Tennessee Oncology 250 25th Ave. North, Suite 200 Nashville, TN 37203 Tel: 615-320-5090 Email: [email protected] ClinicalTrials.gov identifier: NCT01359696. Running head: Checkpoint kinase 1 inhibition in refractory solid tumors Clinical Cancer Research http://clincancerres.aacrjournals.org/site/misc/journal_ifora.xhtml Abstract word count (limit 250): 245 Body word count (limit 5000): 4367 Figures and tables (limit 6): 6 References (limit 50): 32

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Disclosure of potential conflicts of interest: JI: None AH: None SP-V: None TB: None EMB: Employee of Genentech, Inc., shareholder of F. Hoffmann La Roche, Ltd. ME: Employee of Genentech, Inc., shareholder of F. Hoffmann La Roche, Ltd. SM: Employee of Genentech, Inc., shareholder of F. Hoffmann La Roche, Ltd. FP: Employee of Genentech, Inc., shareholder of F. Hoffmann La Roche, Ltd. XL: Employee of Genentech, Inc., shareholder of F. Hoffmann La Roche, Ltd. SS: Employee of Genentech, Inc., shareholder of F. Hoffmann La Roche, Ltd. RZ: Employee of Genentech, Inc., shareholder of F. Hoffmann La Roche, Ltd. YC: Employee of Genentech, Inc., shareholder of F. Hoffmann La Roche, Ltd. XD: Employee of Genentech, Inc., shareholder of F. Hoffmann La Roche, Ltd. EM: Employee of Genentech, Inc., shareholder of F. Hoffmann La Roche, Ltd. JS: Employee of Genentech, Inc., shareholder of F. Hoffmann La Roche, Ltd. JL: Employee of Genentech, Inc., shareholder of F. Hoffmann La Roche, Ltd. J-CS: Consultancy fees from Roche-Genentech (myself, compensated, minor <10,000 USD) PL: Advisory board for Genentech.




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Abstract

Purpose: Chk1 inhibition potentiates DNA-damaging chemotherapy by overriding cell cycle

arrest and genome repair. This Phase I study evaluated the Chk1 inhibitor GDC-0425 given in

combination with gemcitabine to patients with advanced solid tumors.

Experimental design: Patients received GDC-0425 alone for a 1-week lead-in followed by 21-

day cycles of gemcitabine plus GDC-0425. Gemcitabine was initially administered at 750 mg/m2

(Arm A), then increased to 1000 mg/m2 (Arm B), on Days 1 and 8 in a 3+3+3 dose escalation to

establish maximum tolerated dose (MTD). GDC-0425 was initially administered daily for 3

consecutive days, however, dosing was abbreviated to a single day based on PK and

tolerability. TP53 mutations were evaluated in archival tumor tissue. On-treatment tumor

biopsies underwent PD biomarker analyses.

Results: Forty patients were treated with GDC-0425. The MTD of GDC-0425 was 60 mg when

administered approximately 24 hours after gemcitabine 1000 mg/m2. DLTs included

thrombocytopenia (n=5), neutropenia (n=4), dyspnea, nausea, pyrexia, syncope, and increased

ALT (n=1 each). Common related adverse events (AEs) were nausea (48%); anemia,

neutropenia, vomiting (45% each); fatigue (43%); pyrexia (40%); and thrombocytopenia (35%).

The GDC-0425 half-life was approximately 15 hours. There were 2 confirmed partial responses

in patients with triple-negative breast cancer (TP53-mutated) and melanoma (n=1 each), and 1

unconfirmed partial response in a patient with cancer of unknown primary origin.

Conclusions: Chk1 inhibition with GDC-0425 in combination with gemcitabine was tolerated with

manageable bone marrow suppression. The observed preliminary clinical activity warrants

further investigation of this chemopotentiation strategy.




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Statement of Translational Relevance

Chemotherapy remains an important standard-of-care treatment for many cancers.

Enhancing the cytotoxic effects of chemotherapy may lead to more durable disease control or

eradication. The most commonly mutated gene in human cancers, TP53 encodes for p53, a key

regulator of the cell cycle in response to DNA damage. Chk1 responds to DNA damage and

replication stress, and regulates cell cycle progression through S and G2 phases. Inhibition of

Chk1 as a therapeutic strategy aims to selectivity potentiate the cytotoxicity of DNA-damaging

chemotherapeutics in cell cycle checkpoint defective tumors cells while minimizing toxicity to

normal cells that are checkpoint competent. Data from this Phase I study demonstrate the

safety, early clinical activity, and pharmacodynamic changes in TP53 mutant and non-mutant

refractory solid tumors treated with the Chk1 inhibitor GDC-0425 in combination with

gemcitabine.




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Introduction

Checkpoint kinase 1 (Chk1) is a serine/threonine kinase that functions as a central

mediator of the S and G2 cell cycle checkpoints. As a consequence of DNA damage or

replication stress, the ATR/Chk1 pathway becomes activated, which leads to phosphorylation

and inhibition of CDK1/2 and a transient delay in cell cycle progression so that DNA can be

properly repaired (1-4). Inhibition of Chk1 results in checkpoint failure and thus an otherwise

transient genotoxic insult becomes a cytotoxic event as cells enter mitosis with unrepaired DNA.

In addition, inhibition of Chk1 or its upstream regulator ATR in the context of replication stress

leads to DNA double-strand breaks and replication catastrophe that result in cell death (5, 6). In

cancer cell lines, Chk1 inhibition preferentially enhances the activity of anti-metabolite-based

DNA-damaging agents, such as gemcitabine, compared with other classes of DNA-damaging

agents (7). Greater chemopotentiation, via inhibition of Chk1 or Wee1, has also been observed

in cells lacking p53 tumor suppressor activity (7, 9). Thus, targeting Chk1 is a strategy for

selectively potentiating the efficacy of chemotherapeutic agents, particularly in tumor cells that

lack functional p53 (7-9).

GDC-0425 is an orally bioavailable, highly selective small molecule inhibitor of Chk1. In

preclinical studies, GDC-0425, when administered in combination with gemcitabine, abrogates

the S and G2 checkpoints, resulting in premature entry into mitosis, and mitotic catastrophe (13-

15). GDC-0425 administration effectively reverses gemcitabine induced cell cycle arrest as

measured by phospho-CDK1/2 (pCDK1/2) and enhances the levels of γH2AX, a marker of

double stranded DNA breaks, above levels observed with gemcitabine alone, both in vitro and in

xenograft models in vivo (15, 16). Additionally, checkpoint abrogation may be more rapid in

tumor cells defective in p53 tumor suppressor function (17, 18). In vivo studies suggest

chemopotentiation may be more effective with a defined lag between gemcitabine

administration and Chk1 inhibition (8, 19).




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Together, these data provided the rationale for investigating GDC-0425 in combination

with gemcitabine for the treatment of patients with refractory solid tumors. The primary

objectives of this study were to evaluate the safety and tolerability of GDC-0425 and to

determine the maximum tolerated dose (MTD) in combination with gemcitabine. We also sought

to characterize the pharmacokinetic (PK) properties of GDC-0425 after single and repeat doses,

and identify a recommended Phase II dose and schedule for GDC-0425 in combination with

gemcitabine. Other objectives included a preliminary assessment of anti-tumor activity, a

correlation of this activity with known TP53 mutation status, and pharmacodynamic (PD)

changes in Chk1 pathway components.

Methods

Study design

This Phase I open-label dose escalation study of GDC-0425 (supplied by Genentech,

Inc.) had 2 treatment arms (Supplemental Figure 1). Initially, patients received oral GDC-0425

alone daily for 3 consecutive days starting on Day -7 during Cycle 0. Beginning with Cycle 1,

patients received intravenous (IV) gemcitabine on Days 1 and 8 and GDC-0425 daily on Days

2-4 and 9-11 of 21 day cycles. In Arm A, patients received gemcitabine 750 mg/m2 and in Arm

B, patients received gemcitabine 1000 mg/m2. Based on validated preclinical models to

establish the starting dose of GDC-0425 in line with standard Phase I oncology clinical trials,

and predicted half-life, the GDC-0425 starting dose was defined as 60 mg QD. Due to

unexpected toxicity likely related to prolonged GDC-0425 exposures, the study was amended to

evaluate a single oral dose of GDC-0425 on Day -7 for PK evaluation during Cycle 0 followed by

21-day cycles of IV gemcitabine on Days 1 and 8 and GDC-0425 on Days 2 and 9 in Cycles ≥ 1.

Upon determination of the MTD, an expansion cohort was planned to enroll 6 additional patients

to confirm safety and tolerability, and to assess PD changes within tumors.

Following a 3+3+3 dose escalation design, MTD was defined as the highest dose at




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which fewer than one-third of at least 6 dose-limiting toxicity (DLT)-evaluable patients had a

dose limiting toxic effect during either Cycle 0 or Cycle 1. A DLT was defined as a study drug-

related toxicity occurring during the first 28 days, including Grade ≥ 4 thrombocytopenia or

anemia, Grade ≥ 4 febrile neutropenia or neutropenia lasting > 7 days, Grade ≥ 3 elevation of

ALT or AST lasting > 7 days, total bilirubin or non-hematologic or non-hepatic major organ

adverse event (AE). In addition, Grade ≥ 3 left ventricular ejection fraction reduction or

asymptomatic LVEF reduction to ≤ 40% were defined as DLTs. The protocol was amended

during enrolment to Arm A so that a delay in Day 8 gemcitabine dosing, for example due to low

grade hematologic toxicities commonly observed with gemcitabine administration alone (e.g.,

Grade 1 or 2 neutropenia or thrombocytopenia), was no longer considered a DLT.

Patients

Eligible patients age ≥ 18 years had locally advanced or metastatic solid tumors for

which standard therapy either does not exist or has proven ineffective or intolerable. Eastern

Cooperative Oncology group (ECOG) performance status of 0-1 and adequate hematologic and

end organ function, and evaluable disease or measurable disease per RECIST v1.1 were

required. Patients with > 2 prior chemotherapy regimens for locally advanced or metastatic

cancer, > 6 cycles of an alkylating or platinum agent, or history of symptomatic congestive heart

failure, myocardial infarction, or serious cardiac arrhythmia were excluded.

The protocol was approved by Institutional Review Boards prior to patient recruitment

and was conducted in accordance with International Conference on Harmonization E6

Guidelines for Good Clinical Practice. Written informed consent was obtained for all patients

prior to performing study-related procedures in accordance with federal and institutional

guidelines. The study was registered on ClinicalTrials.gov (NCT01359696).

Safety assessments




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All patients who received ≥ 1 dose of study treatment were included in the safety

evaluation and underwent physical exam, laboratory assessments, electrocardiography, and

radiographic disease assessments at baseline and throughout the study. ECHO or MUGA

scans were performed on Cycle 1 Day 9 following GDC-0425 administration and at study

treatment discontinuation. Adverse events (AEs) were graded according to the National Cancer

Institute Common Terminology Criteria for Adverse Events, v4.0.

Pharmacokinetic assessments

In the first cohort (single-agent GDC-0425 for 3 consecutive days in Cycle 0), the

following PK sampling scheme for GDC-0425 and thiocyanate (SCN) was used in Cycle 0: pre-

dose and at 0.5, 1, 1.5, 2, 4, and 6 hours after dose on Day 1; pre-dose and at 2 hours after

dose on Day 2; pre-dose and at 0.5, 1, 1.5, 2, 4, 6, 24, and 48 hours after dose on Day 3. In

subsequent cohorts, PK samples for GDC-0425 and SCN in Cycle 0 were pre-dose and at 0.5,

1, 1.5, 2, 4, 6, 24, and 48 hours after dose. During Cycle 1 (combination therapy with

gemcitabine) samples were collected for GDC-0425 and SCN analysis on Days 2 and 9 at pre-

dose, 2 hours and 4 hours after dosing on Days 2 and 9. Additional pre-dose samples for GDC-

0425 and SCN analysis were collected on Day 15 in Cycle 1 and on Day 8 of subsequent

cycles.

Serum samples for gemcitabine PK analysis were collected in Cycle 1 on Day 1 at 15 and

30 minutes, and 1, 2, and 3 hours after start of gemcitabine infusion.

PK parameters were derived from non-compartmental analysis (WinNonlin Professional

version 5.2) from the plasma concentration-time profile of GDC-0425. A validated liquid

chromatographic-tandem mass spectrometry LC/MS-MS method with a lower limit of

quantitation of 1.00 ng/mL was used to measure the concentration of GDC-0425 in plasma

samples (20). A validated LC/MS-MS with a lower limit of quantitation of 50 ng/mL was used to

measure the concentration of gemcitabine in plasma samples. A validated UV/VIS




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spectrophotometry method with a lower limit of quantitation of 25 μM was used to measure the

concentration of SCN in plasma samples (21). GDC-0425 and gemcitabine samples were

analyzed at Covance Laboratories, Madison, WI and SCN samples were analyzed at QPS,

Netherlands.

Activity outcomes

Best overall response, objective response, and time on study were evaluated by arm

and dose level. Objective response was defined as a complete or partial response confirmed ≥

4 weeks after initial documentation, as assessed by the investigator using RECIST v1.1 for

patients with measurable disease. Patients with no post-baseline tumor response assessment

were considered non-responders. Time on study was defined as time from first GDC-0425 dose

to study discontinuation.

Biomarker assessments

TP53 mutations were evaluated in archival tumor tissue by next-generation sequencing

(Asuragen or Expression Analysis). The Asuragen assay is based on 32 amplicons covering all

TP53 coding regions (22). The Expression Analysis assay is based on targeted hybridization

capture covering coding regions of over 200 genes including TP53 (23). The variant frequency

cutoff used for calling TP53 mutations using either assay was 10%.

PD changes in the Chk1 pathway marker pCDK1/2 and Ki-67 were evaluated in

formalin-fixed and paraffin-embedded (FFPE) serial tumor biopsies collected before treatment,

after GDC-0425 alone (approximately 24 hours after administration), after gemcitabine alone

(approximately 48 hours after administration), and after the combination of GDC-0425 and

gemcitabine (approximately 48 hours after gemcitabine and 24 hours after GDC-0425) when

feasible. FFPE biopsy specimens were sectioned at 4 μm onto Superfrost Plus slides. Parallel




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sections were stained on Ventana Benchmark XT and Discovery XT machines with antibodies

to pCDK1/2 (clone EPR2233Y; Epitomics) and Ki-67 (clone SP6; Lab Vision/NeoMarkers).

pCDK1/2 signal intensity in viable tumor cells was evaluated on a 4-point scale (0 to 3+) and

then a pCDK1/2 H-score was calculated based on the fraction of positive cells at each intensity

(24). Ki-67 positivity in viable tumor cells was scored from 0 to 100%. To account for tumors

with different proliferative fractions, pCDK1/2 levels by H-score were normalized by Ki-67

percent positivity.

Clinical simulation study in mouse xenograft tumor model for evaluation of pCDK1/2

A PD biomarker study was conducted in a mouse tumor model under conditions that

simulate GDC-0425 exposure in the clinical setting. TP53 mutant HT-29 colorectal tumor

xenografts were established by subcutaneous injection of 5 x 106 tumor cells (100 uL in 1:1

HEPES buffered saline:Matrigel; BD Biosciences) into the left flank of female NCR nude mice

(Taconic). When tumor volumes reached approximately 300-450 mm3, animals were

randomized into balanced cohorts and administered saline or 120 mg/kg gemcitabine by

intraperitoneal injection. MCT (0.5% w/v methylcellulose/0.2% v/v Tween 80 in reverse osmosis

water) or GDC-0425 (50 mg/kg suspension in MCT) was administered by oral gavage 16 hours

later. FFPE tumor samples were collected at 40 hours (40 hours after gemcitabine and 24 hours

after GDC-0425) for analysis by pCDK1/2 immunohistochemistry as described above. All in vivo

studies were approved by Genentech’s Institutional Animal Care and Use Committee and

adhere to the NIH Guidelines for the Care and Use of Laboratory Animals.

Statistical methods

No formal hypotheses were tested in this study, and all analyses were descriptive and

exploratory. Design considerations were not made with regard to explicit power and type I error,

but to obtain preliminary safety, pharmacokinetic, and pharmacodynamic information. For the




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safety analysis and the activity analysis, all patients who received ≥ 1 dose of GDC-0425 were

included. For PD biomarker analyses, biomarker changes after gemcitabine alone or the

combination of gemcitabine and GDC-0425 were evaluated by using a one-way ANOVA model

for xenograft samples and a linear mixed effect model for patient samples with patient as a

random effect. All statistical analyses were carried out in SAS 9.2 and R 3.1.1. The data cut off

was 29 May 2014.

Results

Patient characteristics

A total of 40 patients were enrolled and received at least one dose of GDC-0425. In Arm

A (n=18; combination with gemcitabine 750 mg/m2), patients initially received 60 mg GDC-0425

for 3 consecutive days during the single-agent run-in and following gemcitabine administration.

GDC-0425 administration was abbreviated to 1 day during the single-agent run-in and following

gemcitabine dosing based on PK and tolerability (see below). In Arm B (n=22; combination with

gemcitabine 1000 mg/m2), patients received 60 mg or 80 mg of GDC-0425 for 1 day during the

single-agent run-in and following gemcitabine administration. Of the 39 patients who received

gemcitabine, 7 patients did not receive Cycle 1 Day 8 gemcitabine and, based on the protocol-

specified dosing schedule, received their second dose of gemcitabine on Day 1 of Cycle 2. An

additional 6 patients discontinued study treatment and did not receive gemcitabine after Cycle 1

Day 1.

The baseline characteristics of the patient population divided by treatment arm are shown

in Table 1. The median age was 56 with slightly more females than males. Triple-negative

breast cancer made up the highest proportion of malignancies (20% of all patients), followed by

non-small cell lung cancer and cancers of unknown primary origin (CUP). Eighty-five percent of

patients had prior systemic treatment with a median of 2 prior therapies, and 43% had prior

radiation. Eight-five percent (n=34) of patients were gemcitabine naïve.




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Dose escalation and Safety

All 18 and 22 patients in Arms A and B, respectively, were evaluable for safety (Table 2).

No DLTs were reported in the Cycle 0 GDC-0425 single-agent run-in (3 consecutive daily doses

or single day dose). In Arm A, 3 patients treated with 3 consecutive daily doses of 60 mg GDC-

0425 in combination with gemcitabine 750 mg/m2 had protocol-defined DLTs of Grade 3 nausea

(n=1), Grade 2 pyrexia with associated Grade 3 syncope (n=1), and a patient with both Grade 2

neutropenia and Grade 4 thrombocytopenia (n=1). Thus, 3 consecutive daily doses of 60 mg

GDC-0425 in combination with gemcitabine 750 mg/m2 was not tolerated.

Following protocol amendments, GDC-0425 administration was changed to 1 day dosing

and subsequently 60 mg GDC-0425 with gemcitabine 750 mg/m2 was tolerated which allowed

evaluation of 60 mg GDC-0425 with gemcitabine 1000 mg/m2. With further dose escalation, 80

mg of GDC-0425 with gemcitabine 1000 mg/m2 was found to exceed the MTD with 3 of 6

patients experiencing DLTs of Grade 4 thrombocytopenia; 1 patient with Grade 4

thrombocytopenia also experienced Grade 3 neutropenia that delayed Cycle 2, a second

protocol-defined DLT. The MTD and recommended Phase II dose (RP2D) was defined as

gemcitabine 1000 mg/m2 on days 1 and 8 followed by 60 mg GDC-0425 on Days 2 and 9 of a

21-day cycle. Of note, all DLTs were reversible, blood counts recovered with treatment

interruption, and no DLTs were attributed to GDC-0425 alone.

In Cycle 0 AEs were Grade 1-2 and the most common were nausea (n=11, 28%),

vomiting (n=8, 20%), and diarrhea (n=6, 15%). Limited hematologic toxicity was observed in

Cycle 0 with only 2 patients experiencing Grade 1-2 anemia. Of the AEs assessed as related to

GDC-0425, gemcitabine, or the combination, across both arms, the most common all grade

non-hematologic toxicities were nausea (n=19, 48%), vomiting (n=18, 45%), fatigue (n=17,

42%), and pyrexia (n=16, 40%). Neutropenia (n=16, 40%) was the most common Grade ≥ 3 AE




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followed by thrombocytopenia (n=6, 15%). Treatment-related ALT and AST elevations ≥ Grade

3 occurred in 10% and 8% of patients respectively.

Overall, half the patients (n=20) experienced at least 1 serious adverse event (SAE)

regardless of attribution. Eight of these patients experienced SAEs assessed by the investigator

as related to study treatment (GDC-0425 and/or gemcitabine), including 2 patients each who

experienced Grade 4 thrombocytopenia and Grade 3 neutropenia assessed as related to GDC-

0425 and gemcitabine. Other SAEs assessed as related to GDC-0425 and/or gemcitabine

occurred in 1 patient each and included pyrexia (Grade 2), dyspnea, gastroenteritis, gastric

ulcer, leukopenia, rash, and ALT, AST, and GGT increased (all Grade 3). No deaths occurred

during this study.

In total, 12 patients (30%) were discontinued from GDC-0425 and gemcitabine treatment

due to AEs. Of these, 6 patients were withdrawn for AEs deemed to be treatment-related by the

investigator (neutropenia concurrent with thrombocytopenia, pyrexia, and dyspnea in Arm A and

elevated liver enzymes [ALT, AST, and GGT], fatigue, and rash in Arm B).

Among the 16 patients treated at the RP2D, 6 patients (38%) had AEs that led to a dose

reduction of gemcitabine, and 3 patients (19%) had AEs that led to a dose reduction of GDC-

0425. Of the 7 patients (44%) who experienced at least one SAE, 2 patients (13%) experienced

SAEs assessed by the investigator as related to study treatment (GDC-0425 and/or

gemcitabine), including 1 patient with Grade 3 rash, and 1 patient with Grade 3 ALT increased,

Grade 3 AST increased and Grade 3 GGT increased. Five patients (31%) were discontinued

from GDC-0425 and gemcitabine treatment due to AEs including 2 patients (13%) who were

withdrawn for AEs deemed related to study treatment by the investigator.

Pharmacokinetic analysis

Pharmacokinetic plasma samples from 39 patients were analyzed for GDC-0425 and

gemcitabine. Following oral administration, GDC-0425 was rapidly absorbed with median Tmax




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ranging from 2−4 hours (Table 3, Figure 1). After reaching peak plasma concentrations,

concentrations decreased with a terminal phase half-life of approximately 15 hours (range

7.52−27.3 hours). Mean Cmax following 3 once-daily doses of 60 mg GDC-0425 was

approximately 1.6 fold higher compared to mean Cmax following a 60 mg single dose. There was

considerable overlap in the range of plasma exposures with 60 mg and 80 mg GDC-0425.

Plasma exposures of 60 mg GDC-0425 given in combination with 750 mg/m2 and 1000 mg/m2

gemcitabine were consistent. Overall, the exposure and half-life observed in patients were

approximately 6 fold and 2 fold higher respectively than what was predicted from nonclinical

models. This higher than expected exposure was a factor that was considered in changing the

dosing schedule from 3 days to 1 day after the first cohort. Additionally, both dosing regimens

exceeded the predicted threshold for instigating checkpoint failure, a binary event, for

approximately 24 hours (with 1 day of dosing) or longer (with 3 days of dosing) (Figure 1).

Gemcitabine plasma concentrations peaked at the end of infusion and declined rapidly

thereafter, with a half-life of approximately 0.25 hours (range 0.172−0.358 hours). Maximum

plasma concentrations and AUC of gemcitabine following 1000 mg/m2 30-min IV infusion were

higher compared to 750 mg/m2 30-min IV infusion (Cmax: 18100 ± 8440 ng/mL vs 11500 ± 5810

ng/mL and AUCinf: 9110 ± 3900 hr•ng/mL vs 4860 ± 1080 hr•ng/mL). Overall, the range of

gemcitabine concentrations in this study was consistent with prior reports (25-27).

There were no clinically significant increases in SCN levels compared with baseline

values (pre-dose on Cycle 0, Day 1) following administration of either 60 mg or 80 mg of GDC-

0425 (21).

Efficacy analysis

Investigator assessments of best overall response were available for 31 of 40 patients

with ≥ 1 post-baseline assessment (n=28 patients with disease measured by RECIST v1.1); 9

patients (23%) did not have post-baseline tumor response assessments and were classified as




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non-responders. A best overall response of stable disease or partial response was observed in

24 (60%) patients. There were 2 confirmed partial responses (5% of patients) by RECIST in

patients with triple-negative breast cancer (TP53 mutated) and melanoma (n=1 each), and 1

unconfirmed partial response in a patient with CUP. CT response and TP53 status by tumor

type is shown in Figure 2A.

The median duration of therapy was 3.5 cycles (range 1-14). Eight (20%) patients

remained on study treatment for over 6 months (> 8 cycles) (cervical cancer, CUP, fallopian

tube cancer, mesothelioma [n=1 each]; melanoma and TNBC [n=2 each]), including 2 patients

with PR (melanoma and TNBC) who received therapy for over 10 months (Figure 2B).

Chk1 pathway modulation in tumor biopsies

HT-29 xenografts were analyzed for pCDK1/2 and Ki-67 expression (Supplemental

Figure 2). Representative images from 2 unique animals in each treatment group (n=5 animals

per group) are shown. Ki-67 expression in surviving tumor cells is relatively consistent among

treatment groups. pCDK1/2 expression increases from baseline after gemcitabine treatment,

whereas GDC-0425 alone had little effect. In contrast, GDC-0425 given 16 hours after

gemcitabine caused marked inhibition of the gemcitabine-induced expression of pCDK1/2.

To assess the impact of GDC-0425 and gemcitabine on Chk1-regulated cell cycle

checkpoints, pCDK1/2 was assayed in tumor tissues collected before study treatment, 24 hours

after GDC-0425 administration, 48 hours after gemcitabine administration, and after the

combination of GDC-0425 and gemcitabine treatment (24 hours after GDC-0425, and 48 hours

after gemcitabine) (Figure 3). Tumor samples were obtained from a total of 8 patients, with 6

patients able to undergo biopsies before study treatment and after gemcitabine administration,

and 3 of these patients also able to undergo biopsies after treatment with the combination of

GDC-0425 and gemcitabine. Representative images from tumor biopsies are shown in

Supplemental Figure 3. Increased pCDK1/2 was observed after gemcitabine administration




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(mean fold change 2.01 [95% confidence interval (CI) 1.25-3.23]), consistent with checkpoint

activation and cell cycle arrest in response to gemcitabine-induced DNA damage. Following

GDC-0425 and gemcitabine administration, pCDK1/2 was decreased (mean fold change 0.54

[95% CI 0.30-0.96]), consistent with checkpoint override following Chk1 inhibition.

Discussion

Our findings in this first-in-human trial show that the Chk1 inhibitor GDC-0425 can be

combined with a standard dose and schedule of gemcitabine when administered as a single

dose approximately 24 hours after gemcitabine 1000 mg/m2 on Days 1 and 8 of a 21 day cycle.

Bone marrow toxicity was manageable but prevented escalating beyond 60 mg GDC-0425.

Consistent with its cell cycle-linked mechanism of action, anti-tumor efficacy of a Chk1

inhibitor is predicted to potentiate the effects on of DNA-damaging chemotherapy. Furthermore,

preclinical models have demonstrated marked synergy when inhibiting Chk1 in combination with

DNA damaging chemotherapy that acts in S phase, including antimetabolites such as

gemcitabine (8, 15, 19). Though gemcitabine is only FDA approved in ovarian, breast, non-small

cell lung, and pancreatic cancers, it is known to have activity across a wide range of solid

tumors pointing to the potential for broad applicability when paired with a Chk1 inhibitor.

Our findings suggest that this strategy is feasible but not without increased toxicity.

Although limited toxicity was observed with brief exposure to single agent GDC-0425,

chemotherapy-related toxicities may be enhanced by the addition of GDC-0425. When GDC-

0425 is given in combination with gemcitabine administered at a standard dose and schedule,

bone marrow toxicity is increased beyond what would be expected with gemcitabine alone.

Neutropenia and thrombocytopenia were manageable but were Grade 3 or 4 and treatment-

related in 40% and 15% of patients, respectively, and appear to be more frequent than what

would be expected with gemcitabine alone (28).




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The addition of GDC-0425 also increased the rates of non-hematologic AEs that can

adversely affect patient quality of life. Although generally Grade 1 or 2, the rates of nausea,

vomiting, fatigue, and pyrexia ranged from 40-48%. Though the number of patients in this trial

was small and patients were treated with different dose levels of both GDC-0425 and

gemcitabine, these observations suggest that long term tolerability may be compromised in

some patients. Fortunately, the rate of Grade ≥ 3 transaminase elevations with the addition of

GDC-0425 were similar to what would be observed with gemcitabine alone (29).

The timing of Chk1 inhibitor administration relative to gemcitabine and how this relates to

maximum chemopotentiation remains unclear. Although some Chk1 inhibitors have been

evaluated with concurrent chemotherapy dosing (30, 31), non-clinical studies suggest that Chk1

inhibitor administration was most effective when dosed with a defined delay after gemcitabine of

approximately 24 hours (8, 32). In our Phase I study, 3 days of GDC-0425 starting 24 hours

after gemcitabine was not tolerable. GDC-0425 exposures in humans were greater than

predicted from the animal models and the half-life of approximately 15 hours was longer than

expected. Furthermore, a single dose of 60 mg in humans exceeded target exposures

associated with checkpoint abrogation and anti-tumor activity in preclinical models. Importantly,

no PK interaction was observed between GDC-0425 and gemcitabine, suggesting that the

observed AEs were not due to unexpected changes in exposures with this combination. Prior to

conducting this clinical study, a mass balance study in rats using radiolabeled GDC-0425 was

performed and one of the drug-related metabolites identified in rat plasma was SCN (21). The

overall contribution of drug-derived (radiolabeled) SCN to the endogenous SCN concentrations

in rats was negligible. However, as part of the safety and PK assessments, the concentrations

of SCN were monitored before and after dosing in this clinical study. There were no clinically

significant increases in SCN levels compared with baseline values following GDC-0425

administration mitigating the concerns for any elevated SCN levels on dosing with GDC-0425.




18

Preliminary signs of clinical activity were observed supporting this chemopotentiation

strategy. Eight of 40 patients (20%) remained on study for more than 6 months and 3 partial

responses were observed in patients with melanoma, TNBC, and CUP. It has been

hypothesized that chemopotentiation with a Chk1 inhibitor which abrogates the S and G2 cell

cycle checkpoint may be more effective in patients whose tumors lack functional p53.

Interestingly, despite the small number of tumor biopsies available for PD biomarker analyses, 2

patients with TP53 mutant tumors showed the predicted decrease in pCDK1/2 after treatment

with the combination of gemcitabine and GDC-0425, whereas a third patient with TP53 wild-type

tumor did not show this effect. Overall, 21 of the 28 patients with RECIST evaluable disease

and on-treatment tumor assessment had adequate archival tumor tissue analyzed for TP53

mutation status. Though 2 of the 3 patients with a partial response and others with minor

radiographic changes had mutations in TP53, there is insufficient data from this dose escalation

trial to understand the correlation between TP53 status and clinical activity.

In summary, this trial provides further evidence that Chk1 inhibition is an attractive

therapeutic strategy to enhance the cytotoxicity of DNA damaging chemotherapeutics.

Preclinical models suggest anti-metabolites such as gemcitabine are a preferred partner to pair

with Chk1 inhibitors to accentuate chemopotentiation. Our findings show that combining a

standard dose and schedule of gemcitabine with GDC-0425 is feasible but the addition of the

Chk1 inhibitor likely adds toxicity that will need to be accounted for in future trials. The

exposures achieved, lack of a PK interaction, pharmacodynamic changes in tumor samples,

and early clinical activity in this diverse solid tumor patient population is encouraging support for

the further development of this chemopotentiation strategy. As future studies are conducted, it

will be important to evaluate tolerability and anti-tumor activity of GDC-0425 and gemcitabine in

a randomized fashion in a less heavily treated patient population and to understand whether

tumors that lack functional p53 may afford greater opportunity to leverage chemopotentiation for

clinical benefit and eventual patient selection.




19

Disclaimer

The authors take full responsibility for the design of the study, the collection of the data, the

analysis and interpretation of the data, the decision to submit the article for publication, and the

writing of the article.

Author’s contributions:

Conception and design: EMB, SS, PL

Development of methodology: EMB, SS, PL

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.):

TB, JCS, AH, SPV, PL

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational

analysis): RZ, FP, XD, XL, EM, SS, JL, JLS, PL

Writing, review, and/or revision of the manuscript: All authors

Administrative, technical, or material support (i.e., reporting or organizing data, constructing

databases):

Study supervision: TB, JL, JLS

Other (study conduction): TB, JCS, AH, SPV, PL




20

Funding: This work was supported by Genentech, Inc.

Acknowledgements

The authors wish many thanks to all of the patients and the investigators who participated in this

study. We thank Shari Lau for IHC support. Writing assistance provided by Genentech, Inc.




21

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25

Table 1. Patient baseline and disease characteristics.

Variable GDC-0425 + gemcitabine

750 mg/m2 (n=18)

GDC-0425 + gemcitabine 1000 mg/m2

(n=22)

All patients (N=40)

Age (yr), median (range) 57 (44−74) 55 (33−82) 56 (33−82) Sex Female Male

10 (56%) 8 (44%)

12 (55%) 10 (45%)

22 (55%) 18 (45%)

ECOG performance status 0 1

10 (56%) 8 (44%)

17 (77%) 5 (23%)

27 (68%) 13 (32%)

Most common tumor types Breast a NSCLC Unknown primary Melanoma

2 (11%)

0 3 (17%) 2 (11%)

8 (36%) 5 (23%) 1 (5%) 1 (5%)

10 (25%) 5 (13%) 4 (10%) 3 (8%)

Prior systemic therapy b 0 1 2-3 >3

4 (22%) 8 (44%) 5 (28%) 1 (6%)

2 (9%)

6 (27%) 12 (55%)

2 (9%)

6 (15%)

14 (35%) 17 (43%)

3 (8%) Prior radiation 6 (33%) 11 (50%) 17 (43%) a Eight of 10 breast cancer patients with triple negative breast cancer (TNBC) b Number of prior lines of therapy (cytotoxic and non-cytotoxic) in neo-adjuvant or adjuvant setting and for locally advanced or metastatic disease.




26

Table 2. Adverse events related to GDC-0425 and/or gemcitabine in ≥ 10% of patients. Includes events that occurred in Cycle 0 (single agent lead-in) and Cycles ≥ 1 (combination treatment).

Adverse Events

Arm A Arm B

All Patients(N=40)

60 mg GDC-0425 (3-day dosing) + 750 mg/m2

gemcitabine(n=5)


gemcitabine(n=13)

60 mg GDC-0425 (1-day dosing) +

1000 mg/m2 gemcitabine

(n=16)



(n=6) Any adverse events All Grade Grade 1/2 Grade 3/4

5 (100%) 1 (20%) 4 (80%)

12 (92%) 8 (62%) 4 (31%)

16 (100%)

6 (38%) 10 (62%)

6 (100%)

0 6 (100%)

39 (98%) 15 (38%) 24 (60%)

Nausea All Grade Grade 1/2 Grade 3/4

2 (40%) 1 (20%) 1 (20%)

6 (46%) 6 (46%)

0

8 (50%) 8 (50%)

0

3 (50%) 3 (50%)

0

19 (48%) 18 (45%) 1 (2%)

Anemia All Grade Grade 1/2 Grade 3/4

4 (80%) 2 (40%) 2 (40%)

7 (54%) 6 (46%) 1 (8%)

4 (25%) 4 (25%)

0

3 (50%) 2 (33%) 1 (17%)

18 (45%) 14 (35%) 4 (10%)

Neutropenia All Grade Grade 1/2 Grade 3/4

1 (20%)

0 1 (20%)

3 (23%)

0 3 (23%)

8 (50%) 2 (12%) 6 (38%)

6 (100%)

0 6 (100%)

18 (45%) 2 (5%)

16 (40%) Vomiting All Grade Grade 1/2 Grade 3/4

5 (100%) 5 (100%)

0

6 (46%) 6 (46%)

0

4 (25%) 4 (25%)

0

3 (50%) 3 (50%)

0

18 (45%) 18 (45%)

0 Fatigue All Grade Grade 1/2 Grade 3/4

2 (40%) 2 (40%)

0

6 (46%) 6 (46%)

0

6 (38%) 5 (31%) 1 (6%)

3 (50%) 3 (50%)

0

17 (42%) 16 (40%) 1 (2%)

Pyrexia All Grade Grade 1/2 Grade 3/4

2 (40%) 2 (40%)

0

7 (54%) 7 (54%)

0

6 (38%) 6 (38%)

0

1 (17%) 1 (17%)

0

16 (40%) 16 (40%)

0 Thrombocytopenia All Grade Grade 1/2 Grade 3/4

1 (20%)

0 1 (20%)

3 (23%) 3 (23%)

0

7 (44%) 5 (31%) 2 (12%)

3 (50%)

0 3 (50%)

14 (35%) 8 (20%) 6 (15%)

Asthenia All Grade Grade 1/2 Grade 3/4

3 (60%) 2 (40%) 1 (20%)

4 (31%) 4 (31%)

0

4 (25%) 3 (19%) 1 (6%)

2 (33%) 2 (33%)

0

13 (32%) 11 (28%) 2 (5%)

Decreased appetite All Grade Grade 1/2 Grade 3/4

2 (40%) 2 (40%)

0

4 (31%) 4 (31%)

0

4 (25%) 4 (25%)

0

3 (50%) 3 (50%)

0

13 (32%) 13 (32%)

0 Diarrhea All Grade Grade 1/2 Grade 3/4

1 (20%) 1 (20%)

0

4 (31%) 4 (31%)

0

5 (31%) 5 (31%)

0

1 (17%) 1 (17%)

0

11 (28%) 11 (28%)

0




27

Rash All Grade Grade 1/2 Grade 3/4

1 (20%) 1 (20%)

0

1 (8%) 1 (8%)

0

4 (25%) 3 (19%) 1 (6%)

2 (33%) 2 (33%)

0

8 (20%) 7 (18%) 1 (2%)

Alanine aminotransferase increased All Grade Grade 1/2 Grade 3/4

0 0 0

1 (8%) 0

1 (8%)

2 (12%) 1 (6%) 1 (6%)

3 (50%) 1 (17%) 2 (33%)

6 (15%) 2 (5%) 4 (10%)

Aspartate aminotransferase increased All Grade Grade 1/2 Grade 3/4

0 0 0

1 (8%) 1 (8%)

0

2 (12%) 1 (6%) 1 (6%)

3 (50%) 1 (17%) 2 (33%)

6 (15%) 3 (8%) 3 (8%)

Leukopenia All Grade Grade 1/2 Grade 3/4

0 0 0

1 (8%)

0 1 (8%)

2 (12%) 2 (12%)

0

2 (33%)

0 2 (33%)

5 (12%) 2 (5%) 3 (8%)

Alopecia All Grade Grade 1/2 Grade 3/4

0 0 0

1 (8%) 1 (8%)

0

2 (12%) 2 (12%)

0

1 (17%) 1 (17%)

0

4 (10%) 4 (10%)

0 Chills All Grade Grade 1/2 Grade 3/4

2 (40%) 2 (40%)

0

1 (8%) 1 (8%)

0

0 0 0

1 (17%) 1 (17%)

0

4 (10%) 4 (10%)

0 Peripheral edema All Grade Grade 1/2 Grade 3/4

1 (20%) 1 (20%)

0

1 (8%) 1 (8%)

0

2 (12%) 2 (12%)

0

0 0 0

4 (10%) 4 (10%)

0 Stomatitis All Grade Grade 1/2 Grade 3/4

0 0 0

2 (15%) 2 (15%)

0

1 (6%) 1 (6%)

0

1 (17%) 1 (17%)

0

4 (10%) 4 (10%)

0




28

Table 3. GDC-0425 pharmacokinetic parameters. Cycle 0 Day 1 Cycle 0 Day

3 Arm A Arm B Arm A 60 mg GDC-

0425 (3-day dosing) + 750 mg/m2

gemcitabine (n=5)


gemcitabine(n=12)

Arm A subtotal(n=17)



(n=16)



(n=6)


gemcitabine(n=5)

Cmax (ng/ml) 106 ± 55.4 100 ± 53.6 102 ±

52.5 97.3 ± 28.6 126 ± 58 174 ± 76.1

Tmax (hr) 2 (0.5-6) 3 (0.5-24) 2 (0.5-24)

4 (0.5-6) 3 (1-4) 2 (1-4)

AUClast (hr*ng/ml) 1170 ± 558 1790 ± 1270 1610 ±

1130 1630 ± 513 1590 ± 594 2650 ± 1180

AUCinf (hr*ng/ml) 1880 ± 702 2320 ± 1950 2200 ±

1690 1910 ± 677 1930 ± 837 4580 ± 2310

CL/F 35.4 ± 12.7 37.1 ± 19.7 36.6 ± 17.7

36.9 ± 18.9 51 ± 28.4 15.7 ± 7.27

Half-life (hr) 14.6 ± 3.62 15.9 ± 5.07 15.5 ±

4.64 14.7 ± 4.76 13 ± 4.64 18 ± 4.08

Cmax = maximum observed plasma concentration; Tmax = maximum time; AUCinf = area under the concentration−time curve from time zero to infinity; AUClast = area under the concentration−time curve from time zero to last observed timepoint; CL/F = apparent clearance. Note: Values are presented as Mean ± SD, except for Tmax presented as median and range. AUClast = 0-24 hours for Arm A Cohort 1 (60 mg GDC-0425 [3-day dosing] + 750 mg/m2 gemcitabine); 0-48 hours for Arm A Cohort 2 (60 mg GDC-0425 [1-day dosing] + 750 mg/m2 gemcitabine), Arm B Cohort 1 (60 mg GDC-0425 + 1000 mg/m2 gemcitabine),and Arm B Cohort 2 (80 mg GDC-0425 + 1000 mg/m2 gemcitabine).




29

Figure legends Figure 1. Cycle 1 pharmacokinetics. Dashed line indicates EC50 defined as free drug target that triggers S and G2 checkpoint failure following S-phase arrest. Figure 2. Efficacy with gemcitabine and GDC-0425. (A) CT responses and TP53 status. All patients with disease measured by RECIST v1.1 who received ≥ 1 dose of study treatment and had ≥ 1 on-treatment tumor assessment (n=28 of 40). (B) Patients were able to remain on study ≥ 6 months. Tumor types are noted for patients on study treatment ≥ 6 months. Figure 3. Modulation of Chk1 pathway by gemcitabine (gem) and GDC-0425 in tumors. (A) HT-29 mouse xenograft tumors evaluated for pCDK1/2 by H-score. (B) Serial tumor biopsies from Phase I study evaluated for pCDK1/2 H-score normalized by Ki-67 percent positivity. A connecting line is not displayed between data points for patients without a post-gem biopsy, including one patient with NSCLC (TP53 unknown) and one patient with fallopian tube cancer (TP53 mutant). Δ denotes mean fold change with 95% confidence intervals in parentheses. Supplemental Figure 1. Study design. Supplemental Figure 2. Preclinical xenograft immunohistochemistry. HT-29 xenografts were analyzed for pCDK1/2 and Ki-67 expression. Representative images from 2 unique animals in each treatment group (n=5 animals per group) are shown. Ki-67 expression in surviving tumor cells is relatively consistent among treatment groups. pCDK1/2 expression increases from baseline (A, B) after gemcitabine treatment (I, J), whereas GDC-0425 alone (E, F) had little effect. In contrast, GDC-0425 given 16 hours after gemcitabine caused marked inhibition of the gemcitabine-induced expression of pCDK1/2. Scale bar=90 µm. Supplemental Figure 3. Clinical trial tumor immunohistochemistry. Representative images from tumor biopsies were analyzed for pCDK1/2 and Ki-67expression. Representative images from a patient with NSCLC and unknown p53 mutation status were obtained 24 hours after administration of GDC-0425 alone (A, B), 48 hours after gemcitabine alone (C, D) and at a time point that was both 24 hours after GDC-0425 and 48 hours after gemcitabine (E, F). Quantitation of percent tumor cell Ki-67 positivity varied minimally between biopsies (G); pCDK1/2 increased from baseline after gemcitabine treatment, whereas the combination of GDC-0425 given 24 hours after gemcitabine suppressed the increase in pCDK1/2. Scale bar=40 µm.




GD

C-0

425

conc

entra

tion

(ng/

mL)

10

100

1000

Nominal time (hours)

0 10 20 30 40 50 60 70 80 90 100 110

60 mg GDC-0425 (3D) + 750 mg/m gem60 mg GDC-0425 + 750 mg/m gem60 mg GDC-0425 + 1000 mg/m gem80 mg GDC-0425 + 1000 mg/m gem

2

2

2

2

EC50

Figure 1




Best

% C

hang

e of

SLD

from

Bas

elin

e

50

50

135 58 13157 67 60 39 183

71 113 176 39 58

67 92319 133 296

141 105 137 7861

223

230 307

Mutant

Wild type (WT)

No sample (NS) /failed sample (F)

Time on study (days)

60 mg GDC-0425 (3D) + 750 mg/m2 gem60 mg GDC-0425 + 750 mg/m2 gem60 mg GDC-0425 + 1000 mg/m2 gem80 mg GDC-0425 + 1000 mg/m2 gem

60

40

20

0

-20

-40

-60

A.

Time on study (months)0 1 2 3 4 5 6 7 8 9 10 11 12

●●

●●

●

●● ●

●

●

●Mesothelioma

TNBC

TNBCFallopian

Melanoma

UnknownCervical

Melanoma

●Partial responseDiscon due to AE

N=40 patients

B.

60 mg GDC-0425 (3D) + 750 mg/m2 gem60 mg GDC-0425 + 750 mg/m2 gem60 mg GDC-0425 + 1000 mg/m2 gem80 mg GDC-0425 + 1000 mg/m2 gem

230 307

TP53

Tum

orW

T

p.H

193R

WT F

p.R

175H

F Fp.

T125

T(s

plic

e re

gion

)N

S

WT

WT

WT

WT

p.R

248W

NS

WT

p.R

333f

s*12

WT

p.R

248L

, p.C

238F

,p.

M23

7Ip.

?(s

plic

e re

gion

)N

S

WT

p.H

193R

p.R

175H

p.W

146X

NS

p.Y1

26C

p.R

337C

Sarc

oma

Panc

reat

ic

NSC

LC

Panc

reat

ic

Rec

tal

Esop

hage

al

Endo

met

rial

Sarc

oma

Kidn

ey

Cer

vica

l

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H&N

Anal

can

al

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ine

Unk

now

n

Mel

anom

a

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Mel

anom

a

NSC

LC

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pian

NSC

LC

Unk

now

n

TNBC

TNBC

Brea

st

Unk

now

n

Mel

anom

a

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Figure 2




pCD

K1/2

H−s

core

75

100

150

200

250

●

●●●

●

●●●

●●

●

●

●

●●

(pC

DK1

/2 H

−sco

re) /

(Ki-6

7+ %

)

0.5

1

2

3456 ●

●60 mg GDC−042580 mg GDC−0425

TP53 MutantWild type

Tumor typeBreastCRCFallopianNSCLCTNBCUnknown

A. B.

Baseline Post- Post- gem combo

Baseline Post- Post- gem combo

∆ = 2.01(1.25–3.23)

∆ = 0.54(0.30–0.96)

∆ = 1.58(1.36–1.83)

∆ = 0.72(0.62–0.83)

Figure 3




Published OnlineFirst November 4, 2016.Clin Cancer Res Jeffrey R. Infante, Antoine Hollebecque, Sophie Postel-Vinay, et al. tumorscombination with gemcitabine in patients with refractory solid Phase I Study of GDC-0425, a checkpoint kinase 1 inhibitor, in

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