• Serum TK1 activity was measured using the highly sensitive DiviTum™ assay (Fig 4)

Background and Rationale

DiscussionDi

SABCS 2016Abstract P5-04-02

This presentation is the intellectual property of the author/presenter. Contact shana.thomas@wustl.edu for permission to reprint and/or distribute.

Serum Thymidine Kinase 1 Activity as a Pharmacodynamics Marker of Cyclin-Dependent Kinase 4/6 Inhibition in Patients with Early Stage Breast Cancer Receiving Neoadjuvant Palbociclib

Shana Thomas1, Ning Liu1, Rosy (Jingqin) Luo1, Jeremy Hoog1, Mattias Bergqvist2, Magnus Neumüller2, Edward M. Suh2, Zhanfang Guo1, Kiran Vij1, Souzan Sanati1, Matthew Ellis3, Cynthia X. Ma1

1Washington University School of Medicine, St. Louis, MO, 63110; 2 Biovica International AB, Uppsala, Sweden, 3Baylor College of Medicine, Houston, TX, 77030

• Thymidine kinase 1 (TK1) is a fundamental enzyme in DNA synthesis.• TK1 expression is E2F-dependent and peaks in the S-phase of the cell cycle. • CDK4/6 inhibition suppresses E2F-dependent transcription of TK1 (Fig 1).

• In preclinical studies, inhibition of cyclin-dependent kinase (CDK) 4/6 led to dose dependent reduction of TK1 activity in cancer cells measured by DiviTumTM (Fig 2).

Table 1 Patient Characteristics (n=50)Age, median (range) yrs 58 (34-79)RaceWhiteBlack

47 (94%)3 (6%)

Menopausal StatusPremenopausalPostmenopausal

18 (36%)32 (64%)

PgRNegPos

3 (6%)47 (94%)

Tumor grade123

12 (24%)31 (62%)7 (14%)

Clinical StageIIIII

36 (72%)14 (28%)

• At C1D15, TK activity was below the detection limit of 20 Du/L in 44 of 48 pts, and was at low levels (24, 26, 26, and 58 Du/L) in the remaining 4 pts, indicating a profound effect by palbociclib.

• Palbociclib significantly reduced serum TK activity 2 weeks post initiation in all patients (p<0.001, Fig 5) and in the anastrozole or palbociclib sensitive group by Ki67 (Figs 6). Palbo washout led to a significant increase in serum TK at surgery time point (Fig 5, 6).

• The subject-level bivariate correlation coefficient (BCC) correlation coefficient between serum TK and tumor Ki67 was 0.46 by the Bland-Altman method [1], 0.67 by the Meta-analysis approach and 0.456 using a bivariate linear mixed effects model [2], indicating a median correlation (Fig 6).

Method

Results

Fig 6 Serum TK and Tumor Ki67 by Timepoint and Ki67 Response Category for Individual Patient

Results (cont.)

Conclusion• CDK4/6 inhibition significantly reduced serum TK1 levels in patients with early stage ER+

HER2- breast cancer 2 weeks post treatment.• Changes in serum TK1 was significantly correlated to that in tumor Ki67 proliferation

index and tumor TK1 mRNA levels. • Discordance between changes in TK and in Ki67 were associated with low baseline TK.• Serum TK1 may serve as a non-invasive pharmacodynamics marker and early response

indicator of the anti-tumor effect of CDK4/6 inhibitors. Further investigation is warranted.References:1 Bland, J. M. and Altman, D. G. (1995a, Feburary). Calculating correlation coefficients with repeated observations: Part 1--correlation within

subjects. BMJ, 310, 446.2 Luo, J, Gao, F, Ding, J, Xiong, C. Bivariate Correlation Coefficients in Family-type Clustered Studies. The Biometrical Journal. 2015.57 (6):

1084-1109 PMCID: PMC4741284.

Acknowledgement: The clinical trial was funded by Pfizer Pharmaceuticals, Susan G. Komen Promise Grant,NCI Cancer Clinical Investigator Team Leadership Award, St. Louis Men’s Group Against Cancer, and SitemanCancer Center. The TK1 activity was analyzed by Biovica. We appreciate patients who participate in this study.

Ki67 ↓ (N) Ki67 ↑ (N) Total (N)TK ↓ (N) 32 4* 36TK ↑ (N) 2* 21 23Total (N) 34 25 59

*Discordant cases are detailed in Fig 7

ObjectiveTo test whether reduction in serum TK1 activity could serve as a surrogate marker for CDK4/6 inhibition in tumor cells, we conducted the following experiments: • Compare TK1 activity in serum collected before and after palbociclib in patients

with early stage ER+ HER2- breast cancer enrolled on a neoadjuvant trial (Fig 3)• Correlate serum TK1 activity with tumor cell proliferation Ki67 and TK1 mRNA level

before and after palbociclib in patients with early stage ER+ HER2- breast cancer enrolled on a neoadjuvant trial (Fig 3).

• Similar to tumor Ki67, serum TK was significantly lower on C1D15, 2 week post adding palbociclib.• Unlike tumor Ki67 and TK mRNA, serum TK did not differ between baseline C0D1 and C1D1, with AI alone. • Similar to tumor Ki67, significant rebound increase in serum TK level was observed at surgery with palbo

withdraw, which was suppressed when an additional C5 palbo was added before surgery.• Table 1 shows the median (IQR) of TK, Ki67 and TK mRNA over time for these patients.

Fig 5 Box Plot of Serum TK1, Tumor Ki67, and Tumor TK1 mRNA Levels

Results (cont.)Table 2 Concordance Between Changes in Serum TK and Changes in Ki67 by Palbo

Comparing data on C1D15 vs C1D1 and C1D15 vs surgery

• The overall concordance rate between TK change and Ki67 change in the same direction by palbociclib was 89.83% (53/59, 95% CI: 79.17%~96.18%).

• Assessing serum TK activity reduction by palbo as a marker of tumor Ki67 response:Ø Sensitivity was 94.12% (32/34, 95% CI: 86.21%~100%)Ø Specificity was 84% (21/25, 95% CI: 69.63%~98.37%)

CDK4/6 inhibitor

Fig 1 DiviTum™ - Scientific Rationale of TK1 as a Biomarker Assay for CDK4/6 Inhibitors

Fig 2 Dose Dependent Reduction of TK1 Activity in Response to CDK4/6 InhibitionTK (top panel) and Ki67 (lower panel) levels in log scale over time for individual patients are shown in line graphs. TK and Ki67 from the same patient are denoted by the same line color. *p<0.05, ** p<0.01, ***p<0.001

� denotes the average of TK or Ki67 from different patients at the corresponding time point.

CDK4/6 inhibitors induce cell cycle arrest at the G1/S checkpoint. Since TK is expressed downstream of the G1/S checkpoint, successful inhibition of CDK4/6 can be detected as changed levels of TK, measured with DiviTum™.

CDK-inhibitor

Thymidine Kinase Expression

Cell cycle transition

Cell cycle arrestE2F

E2F

E2FRbRb

E2F E2F

E2F

P

p53

Cyclin D

CD

K4/6

p16

p21WntER/PgR/AR

MAPKsSTATs

P13K/AKT/mTOR

NF - KB

Gene transcription

G1

S-phase

G2

Mitosis

G1/Scheckpoint

K562s cells were treated for 6h with palbociclib and harvested after 24 hours.

Cellular TK activity was measured by DiviTumTM.

TK activity was reduced in a linear and dose dependent manner in response to palbociclib.

Fig 3 Study Population and Study Schema (NCT01723774): A neoadjuvant phase II trial of palbociclib and anastrozole in

clinical stage II or III ER+ (Allred 6-8) HER2- breast cancer

C5: 10-12 days of palbociclib before surgery

Fig 4 DiviTumTM (Biovica) : A Highly Sensitive Assay to Measure Serum TK Activity

Fig 7 Discordant Cases (n=3 comparing C1D15 to C1D1; n=3 comparing C1D15 to Surg)

Discordance between changes in TK and in Ki67 were associated with low baseline TK at baseline

Anastrozole Sensitive (n=11) Palbo Resistant (n=6) Palbo Sensitive (n=26)

C0D1 C1D1 C1D15 Surgery C0D1 C1D1 C1D15 Surgery C0D1 C1D1 C1D15 Surgery

Ki67 ≤ 2.7% on C1D1 Ki67 > 2.7% on C1D1; ≤ 2.7% on C1D15Ki67 > 2.7% on C1D15

**

****

***

*

***

***

**

Table 1. Serum TK1, Tumor Ki67, and Tumor TK1 mRNA Levels Over Time for All Patients Time point Serum TK Tumor Ki67 Tumor TK mRNA

Median (IQR) (Du/L) N Median (IQR) (%) N Median (IQR) (%) NBaseline (C0D1) 46 (25-73) 48 24.34% (12.91%-34.87%) 45 0.315 (-0.178-1.128) 16

Cycle 1 day 1 42.55 (29-94.6) 49 5.37% (2.52%-13.51%)*** 45 -0.42 (-1.19-0.47)* 33

Cycle 1 day 15 20 (20-20)*** 48 0.78% (0.24%-1%)*** 45 0.98 (-1.89-0)* 29

Surgery without C5 143.96 (90.9-306.4)*** 31 10.63% (4.59%-23.67%)*** 27 0.07 (-0.81-0.69) 17

Surgery with C5 20 (20-21.5) 6 0.52% (0.16%-1.66%) 7 -0.35 (-1.98~ -0.11) 6

*p<0.05, ***p<0.001, compared to the preceding time point using the Wilcoxon signed rank test.

Step 1 Reaction

Step 2 Detection

Mic

rotit

er p

late

• When serum is mixed with the reaction mixture in a microtiter plate, BrdU-monophosphate is generated by TK reaction, which is further phosphorylated to BrdU-triphosphate and incorporated into a DNA-strand bound to the bottom of the well in the microtiter plate.

• The BrdU incorporation is detected by ELISA-technique using an anti-BrdU antibody conjugated to alkaline phosphatase and a chromogenic substrate, which produces the optical density of the color. The conversion of the absorbance readings to DiviTum-units per litre (Du/L) is calculated using the values from standards with known TK-activity.

DIviTumTM