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Bagegni et al. Breast Cancer Research (2017) 19:123 DOI 10.1186/s13058-017-0913-7

RESEARCH ARTICLE

Open Access

Serum thymidine kinase 1 activity as a pharmacodynamic marker of cyclindependent kinase 4/6 inhibition in patients with early-stage breast cancer receiving neoadjuvant palbociclib Nusayba Bagegni1†, Shana Thomas1†, Ning Liu1†, Jingqin Luo1, Jeremy Hoog1, Donald W. Northfelt2, Matthew P. Goetz3, Andres Forero4, Mattias Bergqvist5, Jakob Karen6, Magnus Neumüller5, Edward M. Suh5, Zhanfang Guo1, Kiran Vij1, Souzan Sanati1, Matthew Ellis7 and Cynthia X. Ma1*

Abstract Background: Thymidine kinase 1 (TK1) is a cell cycle-regulated enzyme with peak expression in the S phase during DNA synthesis, and it is an attractive biomarker of cell proliferation. Serum TK1 activity has demonstrated prognostic value in patients with early-stage breast cancer. Because cyclin-dependent kinase 4/6 (CDK4/6) inhibitors prevent G1/S transition, we hypothesized that serum TK1 could be a biomarker for CDK4/6 inhibitors. We examined the drug-induced change in serum TK1 as well as its correlation with change in tumor Ki-67 levels in patients enrolled in the NeoPalAna trial (ClinicalTrials.gov identifier NCT01723774). Methods: Patients with clinical stage II/III estrogen receptor-positive (ER+)/HER2-negative breast cancer enrolled in the NeoPalAna trial received an initial 4 weeks of anastrozole, followed by palbociclib on cycle 1, day 1 (C1D1) for four 28-day cycles, unless C1D15 tumor Ki-67 was > 10%, in which case patients went off study owing to inadequate response. Surgery occurred following 3–5 weeks of washout from the last dose of palbociclib, except in eight patients who received palbociclib (cycle 5) continuously until surgery. Serum TK1 activity was determined at baseline, C1D1, C1D15, and time of surgery, and we found that it was correlated with tumor Ki-67 and TK1 messenger RNA (mRNA) levels. (Continued on next page)

* Correspondence: [email protected] † Equal contributors 1 Division of Oncology, Section of Medical Oncology, Department of Medicine, Siteman Cancer Center, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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Results: Despite a significant drop in tumor Ki-67 with anastrozole monotherapy, there was no statistically significant change in TK1 activity. However, a striking reduction in TK1 activity was observed 2 weeks after initiation of palbociclib (C1D15), which then rose significantly with palbociclib washout. At C1D15, TK1 activity was below the detection limit ( 10%, in which case patients were taken off study owing to inadequate response. Pre- and perimenopausal women received goserelin 3.6 mg subcutaneously every 28 days. Anastrozole was continued until surgery, occurring 3–5 weeks after palbociclib exposure, except in eight patients for whom an additional 10–12 days of palbociclib (cycle 5) was administered until surgery, following the four cycles of combination therapy. Serial

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Study Schema (NCT01723774) Ki67 IHC C0D1

C1D1 C1D15

28-day (C0)

Surgery

28-day cycle x 4 (C1-C4) mRNA microarray DNA sequencing

Anastrozole (A) Palbociclib (P) C5 C0D1

C1D1

C1D15

DiviTum™ Thymidine kinase activity

Fig. 1 Study schema for the NeoPalAna trial. Eligible patients with clinical stage II-III ER+/HER2− breast cancer received anastrozole monotherapy for 28 days in cycle 0, followed by addition of palbociclib on cycle 1, day 1 (C1D1), for four 28-day cycles unless C1D15 Ki-67 was > 10%. Breast surgery occurred after a washout of 3–5 weeks of palbociclib while the patient was continued on anastrozole, except for eight patients who also received cycle 5 of palbociclib immediately prior to surgery. Serial tumor biopsies and blood collections occurred at baseline, C1D1, C1D15, and time of surgery

biopsies and blood collections were obtained at baseline (prior to C0), C1D1, C1D15, and time of surgery. Tumor biopsies were centrally analyzed for tumor Ki-67 level using pathologist-guided image analysis [20]. In this trial, we enrolled 50 patients (18 premenopausal and 32 postmenopausal) (Table 1) with a median age of 58 (range 34–79) years, and demonstrated the potent antiproliferative effect of palbociclib in ER+/HER2− breast cancer, even among patients resistant to anastrozole [19]. This trial provided an appropriate sample set to correlate serum TK1 activity with palbociclib treatment and tumor Ki-67 response. TK mRNA levels were derived from microarray analysis of tumor RNA (Agilent Genomics, Santa Clara, CA, USA) [19]. Serum TK1 activity was determined at study enrollment (baseline, C0D1), C1D1, C1D15, and time of surgery. DiviTumTM assay for serum TK1 activity measurement

The DiviTumTM assay (Biovica International, Uppsala, Sweden) was used for determination of serum enzymatic activity of TK1 according to the manufacturer’s instructions (http://biovica.com/), as previously described [21]. When serum is mixed with the reaction mixture in a 96well enzyme-linked immunosorbent assay (ELISA) titer plate, bromodeoxyuridine (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. BrdU incorporation is then detected by

ELISA using an anti-BrdU monoclonal antibody conjugated to enzyme alkaline phosphatase and a chromogenic substrate, producing the optical density of the color. The absorbance readings to DiviTum units per liter (Du/L) are converted using the values from standards with known TK activity, with a working range from 20 to 4000 Du/L. The analyses were performed at the Biovica laboratory in Uppsala, Sweden, and investigators were blinded to patient or tumor data. In vitro cell culture experiment for effect of palbociclib on intracellular TKA

The human cell line K562S (Sigma-Aldrich, St. Louis, MO, USA) was seeded into T25 flasks (3 million cells/ flask) containing RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (Thermo Fisher Scientific), 100 U/ml penicillin, and 100 U/ml streptomycin (Thermo Fisher Scientific) and treated with palbociclib (0.1 nM to 100 μM; Selleckchem, Houston, TX, USA) for 6 h. Cells were then harvested for determination of cell viability by trypan blue viability assay or lysed for intracellular TK activity by DiviTum assay. Statistical analysis

Box plots were generated to demonstrate tumor Ki-67 and TK1 mRNA by time point in all patients. Line plots displayed the levels of serum TK1 activity and Ki-67 by time point in patients in three tumor Ki-67 response categories. The Wilcoxon signed-rank test was used for

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Table 1 Patient characteristics (n = 50) Characteristics

Data

Age, years, median (range)

58 (34–79)

Race

trypan blue at the same time. As shown in Fig. 2, TK1 activity was reduced in a linear and dose-dependent manner in response to palbociclib with the short duration (6 h) of drug exposure, when the effect on cell viability had not yet become obvious.

White

47 (94%)

Black

3 (6%)

CDK4/6 inhibition reduced serum TK1 activity in NeoPalAna trial

Premenopausal

18 (36%)

Postmenopausal

32 (64%)

To determine whether serum TK1 activity could serve as a surrogate marker for CDK4/6 inhibition and tumor cell proliferation in patients receiving CDK4/6 inhibitors, we analyzed the sample set collected from patients with clinical stage II-III ER+/HER2− breast cancer who received neoadjuvant anastrozole and palbociclib in the NeoPalAna trial [19]. As shown in Fig. 3 and Table 2, there was no statistically significant difference in TK1 activity between baseline and C1D1 following 28 days of anastrozole monotherapy (median serum TK activity was 46 versus 42.55 Du/L, respectively; p = 0.52), despite a significant reduction in tumor Ki-67 index, as well as, a reduction in tumor TK mRNA level. In contrast, a striking decline in TK activity was observed 2 weeks after initiation of palbociclib (C1D15), with a median serum TK activity of less than 20 Du/L, (p < 0.001); the serum TK activity was below the detection limit of 20 Du/L in 92% (44 of 48) of patients. The remaining four participants had serum TK activity of 24, 26, 26, and 58 Du/L, respectively. This indicates a profound on-target inhibitory effect induced by palbociclib. Following palbociclib withdrawal, the median serum TK level increased significantly from C1D15 to surgery (143.96 Du/L at surgery), indicating recovery of CDK4/6 inhibition, with a similar rebound in tumor Ki-67 observed at the time of surgery. When an additional 10–12 days of palbociclib was given (cycle 5) to eight patients prior to surgery ,

Negative

3 (6%)

Positive

47 (94%)

Tumor grade 1

12 (24%)

2

31 (62%)

3

7 (14%)

Clinical stage II

36 (72%)

III

14 (28%)

PgR - Progesterone

comparison between time points of serum TK1 activity, tumor Ki-67 index, or tumor TK1 mRNA level. A value of 20 Du/L was used to impute the measurements of TK1 under the detection limit of 20 Du/L for statistical analysis. The subject-level bivariate correlation coefficient (BCC) between serum TK1 and tumor Ki-67 (in logarithmic scale) was calculated using the BlandAltman method [22], a meta-analysis approach, and the bivariate linear mixed effects model [23]. The concordance of serum TK1 activity change and tumor Ki-67 level change was evaluated by calculating the sensitivity and specificity of decrease in TK1 for predicting decrease in tumor Ki-67 using data at C1D1, C1D15, and time of definitive surgery, excluding the data of the eight patients who were additionally treated with cycle 5. Noncomparable data, such as undetectable TK1 activity at both time points, was also excluded. All tests were two-sided, and significance was set at a 5% α level. All statistical analyses were performed using R version 3.3.2 software (R Foundation for Statistical Computing, Vienna, Austria).

Results Preclinical data indicating CDK4/6 inhibition reduces intracellular TK1 activity in a dose-dependent manner

To assess the effect of CDK4/6 inhibition on intracellular TK1 activity, the human cell line K562S was cultured in the presence of increasing concentrations of palbociclib (0.1 nM to 10 μM) for 6 h and harvested for DiviTum analysis. Cell viability was also examined using

100

7000

90

6000

80 5000

70 60

4000

50 3000 2000

X

Cellular TK Activity Cell Viability

40 30

Cell Viability (%)

PgR

TK Activity (Du/L)

Menopausal status

20 1000

0 0.0001

10

0 0.001

0.01

0.1

1

10

100

Palbociclib (µM)

Fig. 2 Dose-dependent reduction of thymidine kinase 1 (TK1) activity in response to cyclin-dependent kinase 4/6 inhibition in vitro. K562S cells were treated for 6 h with palbociclib at the indicated concentrations, followed by analysis of cell viability and TK1 activity. TK1 activity was reduced in a linear and dose-dependent manner in response to palbociclib prior to changes in cell viability. Du/L DiviTum units per liter

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Serum (log)TK Activity

Tumor Ki67 Level

Tumor TK mRNA Level 3.0

***

7.0

*

***

0.8

*

***

2.0

*** 0.6 6.0

***

1.0

0.0 0.4

5.0

-1.0 0.2

4.0

-2.0

3.0

-3.0

0.0 C0D1

C1D1

C1D15

*** p