Abstract
Purpose:
In both the early and advanced settings, CDK4/6 inhibitors are approved for use in hormone receptor-positive/HER2− breast cancer (BC) in combination with endocrine therapy and increase the duration of invasive disease-free survival (IDFS) and progression-free survival (PFS). The duration of response to these treatments is variable between individual patients, supporting the use of biomarkers to inform treatment. Here, we investigated plasma thymidine kinase activity (TKa) as a continuous prognostic and predictive biomarker of response to CDK4/6 inhibitor-based therapy in early and advanced hormone receptor-positive/HER2− BC.
Patients and Methods:
TKa levels were assessed longitudinally at baseline, on treatment, and post-treatment on plasma samples from 80 metastatic and 28 high-risk patients receiving CDK4/6 inhibitor-based therapy as standard-of-care. Patients were enrolled in the prospective observational Roswell Park Ciclib Study (NCT04526587).
Results:
In the metastatic setting, TKa levels in baseline samples were inversely associated with the duration of PFS (HR=1.91, P=0.03). There was also a reduction in TKa levels from baseline to on-treatment in metastatic disease that displayed longer PFS (≥20 months) (120.7 DiviTum units of activity, DuA vs. 30.6 DuA, P=0.028). Metastatic disease with shorter PFS (≤8 months) displayed higher TKa values on treatment (220.2 DuA vs. 30.6 DuA, P=0.008) compared to patients with longer PFS. In early-stage high-risk cases, TKa values were lower on treatment compared to baseline (41.4 DuA vs 114.8 DuA, P=3.55e-08).
Conclusion:
These findings support the further investigation of circulating TKa levels as a continuous prognostic and predictive biomarker of response to CDK4/6 inhibitor-based therapy. Future studies are well suited to assess definitive TKa cutoff values to inform treatment decisions.
Keywords: thymidine kinase, biomarker, breast cancer, CDK4/6 inhibitor, metastatic
Context Summary
Key Objective:
Does thymidine kinase activity (TKa) in plasma samples associate with outcomes and track response in real time to CDK4/6 inhibitors in early and advanced BC?
Knowledge Generated:
Baseline TKa levels prior to CDK4/6 inhibitor treatment associate with likelihood of disease progression in the metastatic setting. Dynamic evaluation of TKa levels while on CDK4/6 inhibitors mirrors the patient’s response to therapy in early and advanced disease.
Relevance:
Plasma TKa analysis can help inform BC treatment decisions before and during CDK4/6 inhibitor-based therapy in both high-risk adjuvant and metastatic patients.
Introduction
Breast cancer (BC) remains the most common malignancy in women, with most cases presenting as hormone receptor-positive, human epidermal growth factor receptor 2-negative (HER2-) disease.1,2 While 5-year survival is promising for early-stage disease at over 90%, approximately 20–30% of individuals will eventually develop metastatic disease, which carries a significantly lower 5-year survival rate of around 25%.3,4 The addition of cyclin-dependent kinase 4/6 (CDK4/6) inhibitors as standard-of-care treatment for advanced hormone receptor-positive/HER2− disease, along with endocrine therapy (ET), has substantially improved progression-free survival (PFS)5–12 and overall survival (OS)13–15 for these patients. However, ~20% of patients display resistance to CDK4/6 inhibitors and progress rapidly.16,17 Despite substantial work around biomarker development,18,19 there is a dearth of markers that can inform treatment in hormone receptor-positive/HER2− metastatic BC (mBC).
There has been success in the adjuvant use of the CDK4/6 inhibitors abemaciclib and ribociclib in the recent monarchE20,21 and NATALEE trials,22,23 respectively. These trials treated early-stage BC patients with high risk of recurrence, leading to FDA approval of abemaciclib with ET in the adjuvant setting. However, a subset of patients do not benefit from CDK4/6 inhibitor inclusion. Following the ASCO update, which now recommends the use of abemaciclib or ribociclib in combination with ET for high-risk patients in the adjuvant setting,24 there is an urgent need for improved methods to evaluate which patients are most likely to respond to treatment.
CDK4/6 inhibitors act through the RB tumor suppressor, inhibiting its phosphorylation and subsequent deactivation, which enables the expression of E2F target genes and cell cycle progression. One such gene, thymidine kinase 1 (TK1), plays an essential role in DNA synthesis by converting thymidine to deoxythymidine monophosphate, which is further converted to deoxythymidine triphosphate before being incorporated into the growing DNA strand.25,26 Since TK1 is regulated downstream of the E2F pathway, is most active in cycling cells, and is released into the bloodstream, it represents a promising biomarker for assessing CDK4/6 inhibitors efficacy.
Previous studies demonstrated that circulating TK1 levels are elevated in cancer patients compared to healthy individuals.27,28 Similarly, TK1 levels have shown prognostic value in several cancer types, including breast, lung, kidney, prostate, pancreas, skin, and blood.29–38 Thus, we hypothesized that the DiviTum® TKa assay, which measures thymidine kinase activity in the blood, could be used on plasma samples as a continuous prognostic and predictive biomarker of response to CDK4/6 inhibitor-based therapy in early and advanced hormone receptor-positive/HER2− BC. We hypothesized that patients with mBC that displayed longer PFS would display lower TKa values in response to CDK4/6 inhibitor-based therapy and that TKa dynamics could monitor the response of early-stage patients to CDK4/6 inhibitors. The DiviTum® TKa assay is FDA-approved for monitoring serum TKa levels in postmenopausal women with hormone receptor-positive mBC and has been used extensively as both a prognostic and predictive biomarker on both serum28,39–47 and plasma48–50 samples to predict outcomes and monitor therapeutic disease control in hormone receptor-positive BC.
Materials & Methods
Patient Selection:
Informed consent was received from 80 metastatic and 28 early-stage BC patients being seen at the BC Clinic of Roswell Park Comprehensive Cancer Center (RPCCC) receiving CDK4/6 inhibitor-based therapy with concurrent ET as standard-of-care to enroll in the prospective observational RPCCC Ciclib Study (NCT04526587) from July 2020 to July 2024. The end of patient follow-up was either death or transfer to a different institution. This study assessed TKa levels from blood sampling that was available based on clinical testing for the Ciclib study, limiting the ability to proactively define study sample sizes and collection timepoints. All study participants were female except for one male study participant in the metastatic cohort. Patients ≥18 years of age with early-stage or advanced hormone receptor-positive/HER2− BC were considered eligible if treated with a CDK4/6 inhibitor after being diagnosed with metastatic or early disease with high-risk of recurrence. Patient race/ethnicity was self-reported.
Ethics Approval:
All patient data, samples, and protocols of this study were conducted in accordance with the Declaration of Helsinki, approved by the RPCCC Institutional Review Board, and were obtained after written informed consent was received from the patient.
Sample Collection:
All samples were collected in accordance with the DiviTum® TKa Test Requisition (Biovica, San Diego, CA) instructions. The only amendment to their protocol was that plasma was used instead of serum. Blood samples were collected in K2 EDTA tubes, processed for plasma extraction, and stored at −80°C. Patient samples were collected beginning July 2020, and while this study includes those through July 2024, blood sample collection is ongoing. Timepoints of blood collection for metastatic patients included pre-CDK inhibitor treatment, Cycle 1, Cycle 2, on-treatment past Cycle 4 (averaging 6 months into treatment), and post-progression. Timepoint of blood collection for adjuvant patients included pre-treatment, Cycle 1, Cycle 2, every 6 months on treatment, and post-treatment/progression. Some samples were unavailable due to a variety of factors, such as samples being drawn off-site, patient declining having blood taken, patient being dehydrated or providing difficult venous access, and the sample being unattainable after having clinic labs done. Patient samples were chosen for inclusion in the current study if TKa analysis was conducted at least twice and included pre- and post-treatment and/or on- and post-treatment measurements. To measure TKa levels, samples were shipped overnight on dry ice to Biovica’s CLIA-certified, CAP-accredited laboratory for analysis with the DiviTum assay.
Sample Assessment:
DiviTum is analytically and clinically validated and is FDA cleared (510(k) #K202852) as a monitoring tool for hormone receptor-positive mBC. DiviTum is a multi-step end-point ELISA assay. A patient’s plasma sample is combined with a reaction mixture containing the substrate bromodeoxyuridine (BrdU). Since BrdU is a substrate analog to thymidine, TK from the sample phosphorylates BrdU to form BrdUMP, which is then phosphorylated to BrdUTP and incorporated into a DNA/RNA hybrid, and bound to the 96-well microplate using a reverse transcriptase DNA polymerase. An alkaline phosphatase-conjugated anti-BrdU antibody is added to the reaction product after washing. The amount of phosphatase conjugate bound to the DNA is determined by a colorimetric reaction, with absorbance indicating the TKa level in DiviTum Units (DuA). TKa clinical reference values of 250 and 50 DuA were used for baseline and on-therapy analysis, respectively. The 250 DuA clinical reference value is based on the 95th percentile of the distribution of TKa levels in a healthy blood donor population.40 The 50 DuA reference value reflects the lower limit of quantification of the assay (data on file, www.divitum.com). Due to the nature of the study, we have ensured compliance with the REMARK criteria for assessment of tumor biomarkers.51
Statistical Analysis:
PFS was calculated as the time from initiation of CDK4/6 inhibitor to scan or biopsy-proven progression, as determined by the RPCCC clinicians. In this study, patients that had a PFS ≥20 months were deemed as displaying a durable response to therapy and were placed in the Longer PFS cohort. Patients that had a PFS ≤8 months were deemed as displaying resistance to therapy and were placed in the Shorter PFS cohort. These cutoffs were chosen based on the definition of intrinsic resistance to CDK4/6 inhibitors in previous studies and time to disease progression on ET alone,9,11,14,52,53 in addition to analysis of the distribution of responses of the patients in the current study. Each collection timepoint was analyzed separately to control for any potential confounding. Clinical features were tested by multivariate analysis using the Cox proportional hazards model. Missing data was automatically excluded from these models. Cox regression analysis was conducted to assess baseline TKa values with PFS in the metastatic setting. Kaplan-Meier curves were created using the log-rank test and were plotted using the survival (version 3.6.4) and survminer (version 0.4.9) package from base R (version 4.3.0). The Wilcoxon rank sum test with Bonferroni correction was used to compare continuous TKa values between the different cohorts since normal distribution of the data was not assumed and multiple hypothesis testing was conducted, with P value <0.05 deemed as being significant. * P<0.05; ** P<0.01; **** P<0.0001.
Results
Study design
A total of 80 hormone receptor-positive/HER2− mBC patients receiving CDK4/6 inhibitor-based therapy at RPCCC had blood collections at baseline and on treatment or on treatment and post-progression and were included in this study (Figure 1). Patients ranged from 26 to 87 years of age at initial enrollment in the study, with the average patient being 61.1 (Table 1). The end of patient follow-up was either death or the patient transferring to a different institution. Most patients (82.5%) in the study self-identified as White/Hispanic. Of the 80 patients, 40 displayed longer PFS (≥20 months), 15 displayed shorter PFS (≤8 months) and 25 had intermediate PFS. The median PFS of patients in the longer and shorter PFS cohorts were 32.7 months and 4.7 months, respectively (Supplemental Figure 1). Since patients with matched samples that included post-progression timepoints were selected for given the study criteria, the included patients displayed shorter median PFS than the excluded patients at 21.04 and 45.96 months, respectively (Supplemental Figure 1). Similarly, a higher percentage of the included patients were treated with fulvestrant and had larger tumor sizes than patients that were excluded from the study (Table 1). While metastatic site (visceral or non-visceral) and metastatic status (de novo or recurrent) were assessed, no significant differences were observed as associating with longer or shorter PFS. Similarly, whether the patient had received prior ET or chemotherapy, the type of ET used in combination with the CDK4/6 inhibitor, mutation status (variant of known significance identified in somatic or germline testing), estrogen receptor/progesterone receptor (ER/PR) positivity, histological subtype, time from diagnosis to metastasis/recurrence or last follow-up, the number of positive nodes, and tumor size had no significant association with the patient displaying longer or shorter PFS. While type of CDK4/6 inhibitor used was significantly different between the longer and shorter PFS cohorts, this is likely a spurious finding due to the limited sample size of the given study, as previous studies of larger cohorts observed no such association.54,55 Finally, Scarff-Bloom Richardson (SBR) grading of disease aggressiveness56 displayed no significant difference between longer and shorter PFS cohorts (Table 1).
Figure 1. TKa study participants.
Consort diagram of TKa study participants at Roswell Park Comprehensive Cancer Center receiving CDK4/6 inhibitor-based treatment for metastatic or early-stage, high-risk BC.
Table 1. Demographics of study participants.
Patient demographics separated by longer (≥ 20 months), intermediate (8–20 months), and shorter (≤ 8 months) progression-free survival (PFS) and excluded patients without blood samples that matched the study criteria for the metastatic cohort and the early-stage patients at high risk of recurrence with (included) and without (excluded) blood samples that matched the study criteria. P value displays comparison between metastatic longer and shorter PFS cohorts.
| Patient Demographics and Clinical Characteristics | Excluded Metastatic Patients No. (%) | Included Metastatic Patients No. (%) | Longer PFS No. (%) | Intermediate PFS No. (%) | Shorter PFS No. (%) | Long vs Short P value | Included/Excluded Metastatic Patients Pvalue | Excluded Adjuvant Patients No. (%) | Included Adjuvant Patients No. (%) | Included/Excluded Adjuvant Patients Pvalue |
|---|---|---|---|---|---|---|---|---|---|---|
| ≥ 20 months | 8–20 months | ≤ 8 months | ||||||||
| 177 (68.9) | 80 (31.1) | 40 (50.0) | 25 (31.2) | 15 (18.8) | 27 (49.1) | 28 (50.9) | ||||
| Age at CDK Start (years) | ||||||||||
| Range | [27, 84] | [26, 87] | [33, 80] | [34, 87] | [26, 83] | 0.33 | 0.47 | [34, 72] | [28, 81] | |
| Mean | 62.43 | 61.1 | 63.1 | 59.6 | 58.4 | 52.6 | 53 | 0.90 | ||
| Race/Ethnicity | ||||||||||
| White/Hispanic | 157 (88.7) | 66 (82.5) | 30 (75.0) | 21 (84.0) | 15 (100.0) | 0.13 | 0.21 | 26 (96.3) | 26 (92.9) | |
| Not White | 17 (9.6) | 12 (15.0) | 8 (20.0) | 4 (16.0) | 0 (0.0) | 1 (3.7) | 2 (7.1) | 1 | ||
| Unknown | 3 (1.7) | 2 (2.5) | 2 (5.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | |||
| Metastatic Sites | ||||||||||
| Visceral | 77 (43.5) | 39 (48.8) | 16 (40.0) | 13 (52.0) | 10 (66.7) | 0.14 | 0.50 | |||
| Non-Visceral | 100 (56.5) | 41 (51.2) | 24 (60.0) | 12 (48.0) | 5 (33.3) | - | - | - | ||
| Metastatic Status | ||||||||||
| De Novo | 67 (37.9) | 26 (32.5) | 13 (32.5) | 8 (32.0) | 5 (33.3) | 1 | 0.48 | |||
| Recurrent | 110 (62.1) | 54 (67.5) | 27 (67.5) | 17 (68.0) | 10 (66.7) | - | - | - | ||
| Prior Endocrine Therapy | ||||||||||
| Yes | 108 (61.0) | 52 (65.0) | 24 (60.0) | 18 (72.0) | 10 (66.7) | 0.89 | 0.58 | 1 (3.7) | 4 (14.3) | |
| No | 69 (39.0) | 28 (35.0) | 16 (40.0) | 7 (28.0) | 5 (33.3) | 22 (81.5) | 24 (85.7) | 0.36 | ||
| Prior Chemotherapy | ||||||||||
| Yes | 102 (57.6) | 43 (53.8) | 19 (47.5) | 15 (60.0) | 9 (60.0) | 0.60 | 0.59 | 25 (92.6) | 24 (85.7) | 0.67 |
| No | 75 (42.4) | 37 (46.3) | 21 (52.5) | 10 (40.0) | 6 (40.0) | 2 (7.4) | 4 (14.3) | |||
| Therapy Used w/ CDK | ||||||||||
| AI | 147 (83.1) | 57 (71.3) | 28 (70.0) | 20 (80.0) | 9 (60.0) | 0.65 | 0.02 | 26 (96.3) | 28 (100.0) | |
| Fulvestrant | 29 (16.4) | 22 (27.5) | 11 (27.5) | 5 (20.0) | 6 (40.0) | 0 (0.0) | 0 (0.0) | 0.49 | ||
| Tamoxifen | 0 (0.0) | 1 (1.3) | 1 (2.5) | 0 (0.0) | 0 (0.0) | 1 (3.7) | 0 (0.0) | |||
| Unknown | 1 (0.6) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | |||
| CDK Therapy Used | ||||||||||
| Abemaciclib | 6 (3.4) | 1 (1.3) | 1 (2.5) | 0 (0.0) | 0 (0.0) | <0.001 | 0.64 | 27 (100.0) | 28 (100.0) | |
| Palbociclib | 134 (75.7) | 60 (75.0) | 36 (90.0) | 17 (68.0) | 7 (46.7) | 0 (0.0) | 0 (0.0) | 1 | ||
| Ribociclib | 37 (20.9) | 19 (23.8) | 3 (7.5) | 8 (32.0) | 8 (53.3) | 0 (0.0) | 0 (0.0) | |||
| Mutation Status | ||||||||||
| Yes | 102 (57.6) | 63 (63.8) | 30 (75.0) | 21 (84.0) | 12 (80.0) | 1 | 0.11 | 4 (14.8) | 7 (25.0) | |
| No | 33 (18.6) | 11 (13.8) | 7 (17.5) | 1 (4.0) | 3 (20.0) | 9 (33.3) | 16 (57.1) | 1 | ||
| Not Tested/Uknown | 42 (23.7) | 6 (7.5) | 3 (7.5) | 3 (12.0) | 0 (0.0) | 14 (51.9) | 5 (17.9) | |||
| ER/PR Positivity | ||||||||||
| ER+/PR+ | 109 (61.6) | 46 (57.5) | 24 (60.0) | 14 (56.0) | 8 (53.3) | 1 | 0.55 | 20 (74.1) | 27 (96.4) | |
| ER+/PR– | 53 (29.9) | 27 (33.8) | 13 (32.5) | 9 (36.0) | 5 (33.3) | 4 (14.8) | 1 (3.6) | 0.17 | ||
| Unknown | 15 (8.5) | 7 (8.8) | 3 (7.5) | 2 (8.0) | 2 (13.3) | 0 (0.0) | 0 (0.0) | |||
| SBR | ||||||||||
| 1 | 22 (12.4) | 3 (3.8) | 1 (2.5) | 1 (4.0) | 1 (6.7) | 0.22 | 0.06 | 0 (0.0) | 3 (10.7) | |
| 2 | 99 (55.9) | 54 (67.5) | 30 (75.0) | 16 (64.0) | 8 (53.3) | 18 (66.7) | 13 (46.4) | |||
| 3 | 32 (18.0) | 16 (20.0) | 6 (15.0) | 6 (24.0) | 4 (26.7) | 8 (29.6) | 11 (39.3) | 0.14 | ||
| Unknown | 24 (13.6) | 7 (8.8) | 3 (7.5) | 2 (8.0) | 2 (13.3) | 1 (3.7) | 1 (3.6) | |||
| Histological Subtype | ||||||||||
| IDC | 136 (76.8) | 58 (72.5) | 31 (77.5) | 18 (72.0) | 9 (60.0) | 0.286 | 0.44 | 21 (77.8) | 19 (67.9) | |
| ILC | 22 (12.4) | 14 (17.5) | 5 (12.5) | 6 (24.0) | 3 (20.0) | 5 (18.5) | 8 (28.6) | |||
| Mixed | 8 (4.5) | 5 (6.3) | 2 (5.0) | 1 (4.0) | 2 (13.3) | 0 (0.0) | 1 (3.6) | 0.44 | ||
| Unknown | 11 (6.2) | 3 (3.8) | 2 (5.0) | 0 (0.0) | 1 (6.7) | 1 (3.7) | 0 (0.0) | |||
| Months Diagnosis to Metastasis/Recurrence | ||||||||||
| Range | [0, 377.3] | [0, 351.6] | [0, 351.6] | [0, 238.5] | [0, 201.0] | 0.587 | 0.20 | [249.0, 347.1] | [15.0, 18.2] | |
| Mean | 71.7 | 58.9 | 58.9 | 60.6 | 66.2 | 298.1 | 16.6 | 0.33 | ||
| No Recurrence | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 25 (92.6) | 26 (92.9) | |||
| Months Diagnosis to Last Follow-Up | ||||||||||
| Range | [0.07, 451.6] | [12.6, 454.4] | [34.3, 454.4] | [17.4, 262.12] | [12.6, 358.0] | 0.57 | 0.16 | [9.7, 359.1] | [0.8, 49.8] | 0.18 |
| Mean | 130.54 | 112.9 | 123.3 | 100 | 106.4 | 46.2 | 26.1 | |||
| Number of Positive Nodes | ||||||||||
| <5 | 43 (24.3) | 20 (25.0) | 7 (17.5) | 8 (32.0) | 5 (33.3) | 0.3 | 0.13 | 16 (59.3) | 13 (46.4) | |
| ≥5 | 11 (6.2) | 12 (15.0) | 8 (20.0) | 3 (12.0) | 1 (6.7) | 8 (29.6) | 13 (46.4) | 0.27 | ||
| Unknown | 123 (69.5) | 48 (60.0) | 25 (62.5) | 14 (56.0) | 9 (60.0) | 3 (11.1) | 2 (7.1) | |||
| Tumor Size (cm3) | ||||||||||
| Range | [0.1, 10.0] | [0.1, 13.0] | [0.1, 10.7] | [1.4, 6.5] | [0.6, 13.0] | 0.91 | 0.02 | [0.4, 10.5] | [0.2, 11] | |
| Mean | 2.3 | 3.3 | 3.5 | 3 | 3.4 | 3.0 | 4.6 | 0.06 | ||
| Unknown/No Surgery | 84 | 33 (41.3) | 18 (45.0) | 9 (36.0) | 6 (40.0) | 2 (7.4) | 0 (0.0) | |||
| TKa at Baseline | ||||||||||
| Range | - | [17, 1912] | [17, 486] | [62, 1912] | [153, 1196] | 0.02 | - | [44, 400] | ||
| Mean | - | 335.7 | 120.7 | 759.7 | 628.3 | - | - | 114.8 | - | |
| No Baseline Measurement | - | 64 (80.0) | 30 (75.0) | 22 (88.0) | 12 (80.0) | - | 0 (0.0) |
CDK, cyclin dependent kinase 4/6 inhibitor; TX, treatment; AI, aromatase inhibitor; ER, estrogen receptor; PR, progesterone receptor; SBR, Scarff-Bloom Richardson score; IDC, invasive ductal carcinoma; ILC, invasive lobular carcinoma.
TKa dynamics mirror disease control in the metastatic setting
Metastatic patient TKa values at baseline (prior to starting CDK4/6 inhibitor therapy) had a significant negative association with duration of PFS (HR=1.91) (Figure 2A, Table 2). Patients with a baseline TKa under 250 DuA displayed an average PFS duration of 36.1 months, whereas patients with a baseline TKA of at least 250 DuA displayed an average PFS duration of 12.3 months (Figure 2B). Despite being underpowered, this baseline TKa comparison approached significance (HR=3.07, P=0.13) and is consistent with prior studies using a TKa value above or below 250 DuA to predict the risk of progression.40 Baseline TKa values of patients that displayed longer PFS (120.7 DuA) were significantly lower than patients that displayed shorter PFS (628.3 DuA) (Table 1), but significance was lost when the analysis was corrected for multiple comparisons (Figure 2C, Table 3). Similarly, TKa values from samples taken while on Cycle 1/2 of CDK4/6 inhibitor therapy from patients that displayed longer PFS were significantly lower than TKa values from the patients that displayed shorter PFS (30.6 DuA vs. 220.2 DuA, respectively) (Figure 2C, Table 3). Bifurcation of all metastatic patients with Cycle 1/2 TKa values under or at least 50 DuA (the limit of detection, LOD) revealed significant separation in median PFS at 21.0 and 6.8 months, respectively (Figure 2D, Supplemental Figure 2). This cutoff is consistent with previous studies using the DiviTum LOD for cutoff of on CDK4/6 inhibitor-based therapy TKa levels to predict response to treatment.42,44 However, TKa values from plasma samples taken after metastatic patients were removed from CDK4/6 inhibitor-based therapy were elevated as compared to values while on therapy (Figure 2E, Table 3). These dynamics were consistent for individual (Figure 2F) and all metastatic patients (Figure 2G).
Figure 2. Plasma TKa levels predict and mirror disease control and progression in BC patients with metastatic disease before, on, and post-CDK4/6 inhibitor-based therapy.
A, Cox regression analysis of continuous plasma TKa levels and months progression-free survival (PFS) among metastatic BC patients for baseline samples collected. B, Kaplan-Meier plot of all metastatic patients with baseline TKa values under 250 DuA (blue) or at least 250 DuA (red). C, Patient plasma TKa values at baseline and on CDK4/6 inhibitor-based therapy (“Cycles1/2”) for metastatic patients. Data reported separately for patients that displayed longer (≥ 20 months) and shorter (≤ 8 months) PFS. D, Kaplan-Meier plot of all metastatic patients with Cycle 1/2 TKa values under 50 DuA (blue) or at least 50 DuA (red). E, Patient plasma TKa values on CDK4/6 inhibitor-based therapy (“On CDK”) and post-therapy (“Post CDK”) for metastatic patients. Data reported separately for metastatic patients that displayed longer (≥ 20 months) and shorter (≤ 8 months) PFS. F, TKa value timelines for individual and G, all metastatic BC patients that displayed shorter (red) and longer PFS (blue) for baseline, on CDK4/6 inhibitor-based treatment (“Cycle 1/2”, “On-CDK”), and post-CDK4/6 inhibitor-based therapy (“Post-CDK”). HR, hazard ratio.
Table 2. Baseline TKa multivariate analysis in the metastatic patient cohort.
TKa values assessed as a continuous variable.
| Baseline Samples | Coxph Pvalue | Coxph Pvalue | Coxph Pvalue |
|---|---|---|---|
| 16 (%) | Univariate | Univariate with log(TKa) | Multivariate |
| 0.0006 | |||
| [2.83, 7.56] | 0.03 | - | |
| 5 | |||
| [33, 76] | 0.2 | 0.01 | |
| 61.6 | |||
| 14 (87.5) | 0.4 | 0.2 | |
| 1 (6.3) | |||
| 1 (6.3) | |||
| 8 (50.0) | 0.7 | 0.1 | |
| 8 (50.0) | |||
| 8 (50.0) | 0.8 | 0.1 | |
| 8 (50.0) | |||
| 8 (50.0) | 0.8 | 0.1 | |
| 8 (50.0) | |||
| 4 (25.0) | 0.8 | 0.09 | |
| 12 (75.0) | |||
| 14 (87.5) | |||
| 2 (12.5) | 0.02 | 0.007 | |
| 0 (0.0) | |||
| 0 (0.0) | |||
| 14 (87.5) | 1 | 0.05 | |
| 2 (12.5) | |||
| 9 (56.3) | |||
| 4 (25.0) | 0.002 | 0.003 | |
| 3 (18.8) | |||
| 12 (75.0) | |||
| 2 (12.5) | 0.9 | 0.2 | |
| 2 (12.5) | |||
| 1 (6.3) | |||
| 12 (75.0) | 0.6 | 0.4 | |
| 2 (12.5) | |||
| 1 (6.3) | |||
| 10 (62.5) | |||
| 4 (25.0) | 6.00E-04 | 7.00E-04 | |
| 1 (6.3) | |||
| 1 (6.3) | |||
| [0, 351.6] | 0.9 | 0.1 | |
| 65.61 | |||
| [30.11, 454.42] | 0.7 | 0.09 | |
| 110.59 | |||
| 3 (18.8) | |||
| 2 (12.5) | 0.2 | 0.1 | |
| 11 (68.8) | |||
| [0.1, 13] | |||
| 3.9 | 0.7 | 0.3 | |
| 7 (43.8) |
CDK, cyclin dependent kinase 4/6 inhibitor; TX, treatment; AI, aromatase inhibitor; ER, estrogen receptor; PR, progesterone receptor; SBR, Scarff-Bloom Richardson score; IDC, invasive ductal carcinoma; ILC, invasive lobular carcinoma. Coxph, Cox proportional hazards.
samples included in multivariate analysis.
Table 3.
Summary of average TKa values in the mBC patient cohort before, on, and post-CDK4/6 inhibitor-based therapy.
| Group Comparison | n | TKa value (Mean) | P value | Adjusted P value |
|---|---|---|---|---|
| All PreCDK All Cycle1/2 |
16 45 |
335.7 118.0 |
0.006 | 0.056 |
| Longer PreCDK Longer Cycle1/2 |
10 14 |
120.7 30.6 |
0.003 | 0.028 |
| Shorter PreCDK Shorter Cycle1/2 |
3 19 |
628.3 220.2 |
0.069 | 0.688 |
| Shorter PreCDK Longer PreCDK |
3 10 |
628.3 120.7 |
0.022 | 0.221 |
| Shorter Cycle1/2 Longer Cycle1/2 |
19 14 |
220.2 30.6 |
0.0008 | 0.008 |
| All OnCDK All PostCDK |
66 70 |
109.9 987.7 |
2.29E-13 | 2.29E-12 |
| Longer OnCDK Longer PostCDK |
38 33 |
111.4 851.9 |
1.10E-07 | 1.10E-06 |
| Shorter OnCDK Shorter PostCDK |
5 15 |
207.6 746.8 |
0.23 | 1.00 |
| Shorter OnCDK Longer OnCDK |
5 38 |
207.6 111.4 |
0.049 | 0.487 |
| Shorter PostCDK Longer PostCDK |
15 33 |
746.8 851.9 |
0.664 | 1.00 |
All, all metastatic patients; Longer, longer progression-free survival (PFS) cohort (≥ 20 months); Shorter, shorter PFS cohort (≤ 8 months); PreCDK, pre-CDK4/6 inhibitor treatment; Cycle1/2 and OnCDK, on CDK4/6 inhibitor treatment; PostCDK, post-CDK4/6 inhibitor treatment; TKa, thymidine kinase activity.
Early-stage disease participants display similar TKa dynamics
A total of 28 early-stage BC patients deemed to be at high risk of recurrence that received adjuvant CDK4/6 inhibitor-based therapy were included in this study (Figure 1, Table 1). Patients ranged from 28–81 years of age with an average age of 53. Most of the high-risk patients identified as White/Hispanic (92.9%) but unlike the metastatic cohort, most high-risk patients were ET naïve (85.7%). However, most high-risk patients had previously received chemotherapy. A total of 27 high-risk patients were excluded from the study due to unavailability of blood samples that met the study’s inclusion criteria (Table 1). No differences were observed in terms of demographics, clinical variables, or invasive disease-free survival data between the included and excluded high-risk patients (Table 1, Supplemental Figure 3).
Similar to the metastatic cohort, high-risk patients displayed a significant reduction in average TKa values while on therapy as compared to baseline (Figure 3A, Table 4). TKa level dynamics mirrored response to treatment, as TKa values on average were lower and remained lower in a high-risk patient that displayed durable disease control (blue) as compared to a patient that displayed progression to invasive disease (red) (Figure 3B). Of note, on treatment TKa values from metastatic patients were higher than the corresponding average TKa values from high-risk patients (Table 4).
Figure 3. Plasma TKa levels mirror disease control and progression in BC patients with early-stage disease on and off CDK4/6 inhibitor-based therapy.
A, Patient plasma TKa values at baseline and on CDK4/6 inhibitor-based therapy (“Cycles1/2”) for early-stage patients. B, TKa value timelines for individual early-stage, high-risk BC patients that displayed resistance (red) or sensitivity (blue) to CDK4/6 inhibitor-based therapy for baseline, on treatment (“Cycle 1/2”), and post-CDK4/6 inhibitor-based therapy.
Table 4.
Summary of average early-stage BC patient cohort TKa values before and on CDK4/6 inhibitor-based therapy and as compared to the metastatic cohort.
| Group Comparison | n | TKa value (Mean) | P value |
|---|---|---|---|
| Adjuvant PreCDK Adjuvant Cycle 1/2 |
28 34 |
114.8 41.4 |
3.55E-08 |
| Adjuvant PreCDK Metastatic PreCDK |
28 16 |
114.8 335.3 |
1.000 |
| Adjuvant Cycle 1/2 Metastatic Cycle 1/2 |
34 45 |
41.4 117.7 |
0.039 |
Adjuvant, early-stage, high risk patient cohort; PreCDK, pre-CDK4/6 inhibitor treatment; Cycle1/2, on CDK4/6 inhibitor treatment; TKa, thymidine kinase activity.
Discussion
Prognostic and predictive biomarkers that can better inform treatment decisions are needed for patients with hormone receptor-positive/HER2− BC. While Ki67 expression determined by immunohistochemical analysis of tumor biopsies has shown to be highly informative, tumor biopsies are invasive, infrequent, and can be logistically complicated.57 Similarly, gene expression-based biomarker panels have shown efficacy in predicting tumors that are likely or not likely to respond to treatment.18,19 But the same limitations exist, as these analyses require tumor tissue and can be costly. Thus, the efficacy of the more cost and time-effective blood-based TKa assessment is well-positioned for further investigation and implementation.
In 1990, it was first reported that TKa associates with disease state and reflects the response to BC therapy.58 Findings from several groups since are in agreement that TKa values at baseline and in response to ET both with42,44,47,49,50 and without CDK4/6 inhibitors17,40,41,43,46,48 are prognostic and predictive of outcomes in mBC patients. While our study retrospectively compared relatively sensitive (≥20 months PFS) and resistant (≤8 months PFS) patients, Bergqvist and colleagues demonstrated that a definitive DiviTum TKa score <250 DuA was predictive of longer PFS and OS in hormone receptor-positive mBC patients treated with ET alone.40 This is in agreement with the bulk of the previous data using the average baseline cutoff of 245.9 DuA (after converting from the former DiviTum units of Du/L using the equation DuA=134+0.53x DuL provided by Bergvist et al.40) as a prognostic biomarker of PFS and OS in hormone receptor-positive/HER2− mBC patients.28,41–50 In the current study, a baseline TKa value of ≥250 DuA carried a HR of 3.07 despite not reaching significance (P=0.13). And while most prior analyses were conducted using serum for which the test is currently approved, our study builds upon the limited number of prior studies that have shown prognostic value of baseline TKa values in plasma for predicting PFS.48,49 This is important to show the robustness of the test and expand its application to both serum and plasma samples.
As a predictive biomarker of response to CDK4/6 inhibitor-based therapy, TKa values have shown to be efficacious when bifurcating at a cutoff of 205 DuA,49 240 DuA,47 as increasing or decreasing from baseline values,50 or above or below the DiviTum TKa assay’s LOD.42,44 Since the expression of TK1 is restricted to S/G2 phases of the cell cycle, combined with the mechanism of action of CDK4/6 inhibitors being G1 arrest, TKa measurement is an ideal pharmacodynamic biomarker to assess a tumor’s response to CDK4/6 inhibitor-based therapy. The data from this study is the first to show that patients receiving a CDK4/6 inhibitor with plasma TKa levels <50 DuA during Cycle 1/2 had significantly longer PFS than those who had higher levels. With additional confirmatory studies, this target value could be used to make drug selection and dosing decisions to improve treatment outcomes. Further, serial TKa measurements during therapy could be used to detect the development of resistance before disease progression is detected by imaging and/or clinical symptoms.
While well-established as being informative in the metastatic setting, TKa levels have also been shown to be effective at predicting and tracking response to CDK4/6 inhibitor-based therapy39 and other therapies for patients with early-stage BC.28 It’s surprising that baseline levels in adjuvant patients were found to be as high as they were, given that these patients theoretically have no dividing tumor cells as they have already undergone surgical resection. However, given the fact that these patients all had clinical and pathological features that correlate with an increased likelihood of disease recurrence, it is possible that measurable TKa levels after surgery reflect the presence of residual disease. Data from the PENELOPE-B study showed that high baseline levels of TKa (>250 DuA) in hormone receptor-positive early BC patients at high risk of relapse were prognostic for disease recurrence within the first year of adjuvant therapy.59 Also interesting in this study was the on-therapy increase in TKa levels that preceded disease recurrence in two of the adjuvant patients with documented relapse. This suggests that TKa could potentially be used as a non-invasive monitoring tool for detecting disease recurrence in early BC patients ahead of clinical symptoms.
Although not assessed here, comparison of TKa performance in the metastatic and adjuvant contexts to other circulating biomarkers such as cancer antigen (CA 15–3), circulating tumor cells (CTCs), and circulating tumor DNA (ctDNA) is warranted. In previous studies, CA 15–3 was shown to be predictive of response to chemotherapy treatment, but not prognostic of outcome in patients with locally advanced or mBC, underperforming TKa’s predictive power at matched timepoints.41 High TKa levels at baseline have been shown to correlate with CTC positivity in patients with hormone receptor-positive/HER2− mBC.48 Further, data from the BioItaLEE trial demonstrated that both TKa and ctDNA were independently prognostic at baseline and predictive of PFS in patients with hormone receptor-positive/HER2− mBC treated with CDK4/6 and aromatase inhibitors, with inclusion of both biomarkers improving prediction of outcome.60 Thus, future studies would benefit from evaluation of TKa in conjunction with other informative biomarkers to predict and track responses to CDK4/6 inhibitor-based therapy.
One limitation of this real-world study was the relatively small number of patients and samples available given the nature of this assessment being embedded in the overarching Ciclib study. Further recruitment of patients under a prospective study dedicated to TKa assessment would better allow for collection of samples from all timepoints of interest and association of TKa with clinical variables and potential confounders. Previously, number and site of metastases, de novo metastases, tumor size, tumor grade, tumor necrosis, ER/PR status, vascular invasion, baseline ECOG performance status, treatment type, and primary endocrine resistance were found to associate with TKa levels at baseline, despite findings varying significantly between studies.28,41,43–46,48–50 Additionally, a larger study population would enable further distinction of race and ethnicity beyond White and Non-White and its association with TKa, as this has yet to be assessed. Data on individual gene mutational status should also be examined in larger cohorts to assess any association with TKa levels. While several analyses in the current study assessed all data and TKa as a continuous variable, future studies would benefit from external validation and larger cohorts to interrogate definitive cutoff values and important interactions between clinical variables.
Assessment of previously established cutoffs of 250 DuA at baseline and 50 DuA on-treatment proved to be informative in the current study and warrant further validation. Additionally, our study importantly builds on the relatively sparse assessment of TKa and its application on plasma samples and in the context of early-stage disease. Thus, findings from the current study corroborate previous findings and support the use of plasma TKa levels as a prognostic and predictive biomarker to monitor and inform treatment in real-time in both the metastatic and adjuvant settings.
Supplementary Material
Supplemental Figure 1. Kaplan-Meier plot of metastatic patients. A, Progression-free survival (PFS) of patients displaying longer (≥ 20 months) or shorter (≤ 8 months) PFS. B, PFS of patients that were either included or excluded from the current study based on blood sample availability. HR, hazard ratio.
Supplemental Figure 2. Progression-free survival (PFS) of all metastatic patients with plasma TKa levels under or at least 50 DuA during Cycle 1 and 2 of CDK4/6 inhibitor therapy. A, Kaplan-Meier plot of all metastatic patients with Cycle 1 and B, Cycle 2 TKa values under 50 DuA (blue) or at least 50 DuA (red). HR, hazard ratio.
Supplemental Figure 3. Kaplan-Meier plot of adjuvant patients with and without available blood samples that were either included in or excluded from the study. HR, hazard ratio; iDFS, invasive disease-free survival.
Acknowledgements:
We would like to thank the patients for their participation in this study and the consultants at Biovica International AB for their helpful feedback throughout the study.
Competing Interests:
ESK has sponsored research funded by Blueprint Medicine and Bristol Myers Squibb and is a member of the Cancer Cell Cycle-LLC consulting enterprise. AKW has sponsored research funded by Blueprint Medicine and Bristol Myers Squibb. SK serves as a consultant for Daiichi Sankyo, Gilead, and AstraZeneca and has received compensation for speaking engagements with OncLive, Curio-Science, and Dava Oncology.
Funding Sources:
This study was supported by funding to ESK and AKW from the NCI (CA247362 and CA247362-S1) and from the Roswell Park Alliance Foundation. This work was also supported by RPCCC and the NCI (P30CA016056).
Footnotes
Data from this study was presented as a poster at SABCS from 12/10–14/2024 in San Antonio, TX.
Disclaimer: The views expressed in this article are each authors’ own and do not reflect the views or positions of their institution or funder(s).
Data Availability:
Data is available from the corresponding author upon reasonable request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Supplemental Figure 1. Kaplan-Meier plot of metastatic patients. A, Progression-free survival (PFS) of patients displaying longer (≥ 20 months) or shorter (≤ 8 months) PFS. B, PFS of patients that were either included or excluded from the current study based on blood sample availability. HR, hazard ratio.
Supplemental Figure 2. Progression-free survival (PFS) of all metastatic patients with plasma TKa levels under or at least 50 DuA during Cycle 1 and 2 of CDK4/6 inhibitor therapy. A, Kaplan-Meier plot of all metastatic patients with Cycle 1 and B, Cycle 2 TKa values under 50 DuA (blue) or at least 50 DuA (red). HR, hazard ratio.
Supplemental Figure 3. Kaplan-Meier plot of adjuvant patients with and without available blood samples that were either included in or excluded from the study. HR, hazard ratio; iDFS, invasive disease-free survival.
Data Availability Statement
Data is available from the corresponding author upon reasonable request.