Serial circulating tumor DNA monitoring of CDK4/6 inhibitors response in metastatic breast cancer

Abstract Cyclin‐dependent kinase 4/6 inhibitors (CDK4/6i) significantly improve progression‐free survival and have become the standard therapy for estrogen receptor‐positive/human epidermal growth factor receptor 2‐negative metastatic breast cancer patients. Treatment surveillance by radiological imaging has some limitations in detection and repeated biopsy genomic profiling is not clinically feasible. Serial circulating tumor DNA (ctDNA) analysis may provide insights into treatment response. Here we performed serial ctDNA analysis (n = 178) on 33 patients. Serial ctDNA analysis identified disease progression with sensitivity of 75% and specificity of 92%. In eight of 12 patients (61%) responding to CDK4/6i who eventually developed progressive disease, serial sampling every 3 or 6 months captured the initial rise of ctDNA with an average lead time of 3 months. In three of eight patients that did not respond to CDK4/6i (progressive disease at first radiological assessment, 3 months), biweekly sequencing within the first cycle of CDK4/6i treatment (1 month) detected sustained ctDNA levels (≥0.2% variant allele frequency), with lead time of 2 months. Serial ctDNA analysis tracked RECIST response, including clinically challenging scenarios (bone metastases or small‐sized target lesions), as well as detecting acquired genetic alterations linked to CDK4/6i resistance in the G1 to S transition phase. Circulating tumor DNA analysis was more sensitive than carcinoembryonic antigen or cancer antigen 15‐3 serum tumor markers at monitoring tumor response to CDK4/6i treatment. Our findings indicated the possible clinical utility of serial ctDNA analysis for earlier progressive disease detection and real‐time monitoring of CDK4/6i response.

tients. Treatment surveillance by radiological imaging has some limitations in detection and repeated biopsy genomic profiling is not clinically feasible. Serial circulating tumor DNA (ctDNA) analysis may provide insights into treatment response. Here we performed serial ctDNA analysis (n = 178) on 33 patients. Serial ctDNA analysis identified disease progression with sensitivity of 75% and specificity of 92%. In eight of 12 patients (61%) responding to CDK4/6i who eventually developed progressive disease, serial sampling every 3 or 6 months captured the initial rise of ctDNA with an average lead time of 3 months. In three of eight patients that did not respond to CDK4/6i (progressive disease at first radiological assessment, 3 months), biweekly sequencing within the first cycle of CDK4/6i treatment (1 month) detected sustained ctDNA levels (≥0.2% variant allele frequency), with lead time of 2 months. Serial ctDNA analysis tracked RECIST response, including clinically challenging scenarios (bone metastases or small-sized target lesions), as well as detecting acquired genetic alterations linked to CDK4/6i resistance in the G 1 to S transition phase. Circulating tumor DNA analysis was more sensitive than carcinoembryonic antigen or cancer antigen 15-3 serum tumor markers at monitoring tumor response to CDK4/6i treatment. Our findings indicated the possible clinical utility of serial ctDNA analysis for earlier progressive disease detection and real-time monitoring of CDK4/6i response.

| INTRODUC TI ON
Cyclin-dependent kinase 4/6 inhibitors have become the standard treatment for ER + /HER2 − advanced breast cancer patients.
Preclinical studies have established the role of a CDK4/6-RB pathway in regulating the cell cycle through the G 1 to S transition phase. 1,2 Multiple clinical trials have reported that patients receiving CDK4/6i showed significantly longer PFS in combination with endocrine therapy in ER + breast cancer. 3,4 Despite improvements in clinical management, a subset of breast cancer patients will develop resistance to treatment.
Treatment response is mainly assessed by serial radiographic images. 5 Radiographic imaging provides assessment of the disease burden but can sometimes be hampered by suboptimal detection limit and inconsistencies in tumor size measurements. 6,7 Carcinoembryonic antigen and CA 15-3 are serum biomarkers mainly used for treatment monitoring in patients with MBC, 8 with elevated levels often associated with poor prognosis. Although clinically useful in some breast cancer patients, CEA and CA 15-3 have limited sensitivity of only 50% 9 and 60%-70%, 8,10 respectively. Tumor genome sequencing remains the gold standard for genomic profiling, providing insights into potential actionable targets for personalized treatment. 11 However, in a metastatic setting, repeated biopsy is not clinically feasible. Taken together, a more sensitive and less invasive approach is needed to provide an earlier measure of treatment response to inform individual treatment decisions.
Many cancers shed DNA into the bloodstream, referred to as ctDNA. Liquid biopsy through serial ctDNA analysis can complement radiographic imaging and CEA and CA15-3 markers in assessing CDK4/6i responses. It is minimally invasive, feasible for repeated sampling, and is able to detect tumor-specific alterations in circulation, reflecting disease burden in real time. 12 Previous studies have investigated the clinical utility of liquid biopsy in predicting response to CDK4/6i. [13][14][15] However, these studies were focused on ctDNA dynamics at early time points 14,15 or on the heterogeneity of the mutational landscapes between baseline and at disease progression. 13 None of the studies used monitoring through a granular time series to catch disease progression at an earlier time point.
In this single center, retrospective, exploratory biomarker study, we evaluated serial ctDNA analysis against radiographic imaging and CEA and CA 15-3 markers as a biomarker for CDK4/6i response monitoring. We show that short interval serial ctDNA analysis was able to catch initial rise or reemergence of ctDNA, indicative of disease progression, well in advance of radiological assessment. This study also identified mutations potentially predictive of CDK4/6i response, tracked tumor response of RECIST-defined nonmeasurable target lesions, and identified acquired genetic alterations linked to CDK4/6i resistance. Our results support the possible clinical utility of serial ctDNA analysis for earlier PD detection and real-time monitoring of CDK4/6i response.

| Sample processing, library preparation, and sequencing
Blood samples were collected at pretreatment, 2 weeks, and 1, 3, 6, 9, 12, 18, and 24 months or until clinical progression, whichever comes first ( Figure 1). For patients with no ctDNA mutations detected at the end of cycle 1 of CDK4/6i therapy (1M), the end of evaluation sample is sequenced to assess for detectable ctDNA mutations. If mutations are present, cfDNA from all sampling intervals are evaluated. Based on our data cut-off on September 30, 2020, we collected 178 plasma samples from 33 patients. Collection and processing of whole blood has been described in previous publications. [16][17][18] Briefly, 14 mL whole blood was collected using EDTA-2Na tubes (Terumo). Whole blood was centrifuged at 2000 g at 4°C for 10 minutes to separate plasma from white blood cells and red blood cells. The plasma layer was further centrifuged at 16 000 g at 4°C for 10 minutes to remove cell debris. Both plasma and white blood cells were stored at −80°C until nucleic acid extraction.
Plasma cfTNA was extracted using MagMAX Cell-Free Total Nucleic Acid Isolation kit (Applied Biosystems) according to the manufacturer's protocol. Genomic DNA from white blood cells was extracted using FlexiGene DNA Kit (Qiagen). Targeted NGS for cfTNA was carried out using Oncomine Pan-Cancer Cell-Free Assay following the manufacturer's protocol (Ion Torrent). Oncomine Pan-Cancer Cell-Free Assay is an amplicon-based ctDNA targeted assay, incorporating short oligonucleotides called UMTs to distinguish PCR/sequencing artifacts from actual variants ( Figure 1). Library construction and subsequent NGS of cfTNA and genomic DNA were undertaken as previously described. [16][17][18] In most samples, 20 ng cfTNA was used for library construction. Sequencing of genomic DNA was carried out using the same methods using 30 ng genomic DNA for library construction.

| Analysis of sequencing data
Sequencing reads were aligned to hg19 and variant calling was carried out using Torrent Suite 5.10.1 and Ion Reporter 5.10.3.0 software, respectively. Briefly, raw sequence files were aligned to hg19 using Torrent Mapping Alignment Program (TMAP) with default analysis parameters. Valid NGS runs have 90% or more of total reads mapped, alignment quality with read error rate of 2% or less (AQ17). The targeted minimum total coverage for each amplicon is at least 20,000 X.
Library conversion rate was evaluated using the theoretical assump-

| Digital droplet PCR analysis
For patient CDK28, plasma cfDNA was insufficient for targeted NGS in 1M, 3M, and 6M samples. Circulating tumor DNA mutations were evaluated focusing only on ESR1 Y537S detected by Oncomine Pan-Cancer Cell-Free Assay at disease progression. Digital droplet PCR was carried out for all seven time points for patient CDK28 from pretreatment to disease progression (10.5M). The assay used was LBx Probe Catalog no. A082 (65117) ESR1 multi probe targeting Y537N/S, D538G (Riken Genesis). All reactions were carried out on a QX200 ddPCR system (Bio-Rad). A total of 10 ng cfDNA was used in each PCR reaction consisting of primers, probes, and ddPCR Supermix for probes (no dUTP). Reactions were divided into approximately 20,000 droplets per cell using the QX200 droplet generator. Emulsified PCR was carried out on a 96-well thermal cycler using the following cycling conditions: (1 cycle of 95°C for 10 minutes; 40 cycles of 94°C for 30 seconds, 60°C for 1 minute, one cycle of 98°C for 10 minutes, and 4°C hold). Samples were analyzed using the QuantaSoft software to assess number of droplets harboring mutant ESR1 or WT ESR1.

| Statistical analysis
The primary outcome was to assess whether granular sampling time were not applicable to our study; as this was a biomarker observational and comparison study, patients were recruited irrespective of the CDK4/6i or combination endocrine therapy they received.
Comparison of ctDNA and detection rates of tumor markers CEA and CA 15-3 were evaluated using Fisher's exact test. All statistical analyses were carried out in R version 4.0.3 using the default statistical package. All statistical tests were two-sided, with P-values less than .05 considered statistically significant.

| Patient characteristics
By the data cut-off date of September 30, 2020, 178 plasma samples from 33 patients (average, 54 years [35-73 years]) had been recruited with a median follow-up period of 12 months (1-24 months; Table 1). Complete response was observed in one patient and partial response in nine patients, giving an objective response rate of 30% (10 of 33 patients). Sixteen patients had stable disease ( Table 1).

| Circulating tumor DNA analysis to monitor small-sized tumors, bone metastasis, and heterogenous tumors
Consistent with previous findings, ctDNA analysis concordantly tracked tumor response of RECIST-measurable disease ( Figure 4).
However, for more challenging scenarios, such as patients showing only bone metastasis or small-sized tumors below RECIST thresholds, the comparisons are less clear. In our study, 11 patients were defined as having nonmeasurable disease at pretreatment due to small tumor sizes that were below RECIST criteria (   (Table S4). We show some key examples. In patients CDK13 and CDK28, acquired ESR1 mutations, undetected in pretreatment samples, appeared prior to disease progression ( Figure S5, Data S1 and S3). For patient CDK13, ESR1 mutation emerged when pretreatment liver target lesion increased in size leading up to disease progression ( Figure S5). As for patient CDK28, ESR1 mutation was detected when a new liver lesion, previously undetected at pretreatment, was observed ( Figure S5).

| DISCUSS ION
In this retrospective, longitudinal monitoring study of CDK4/6i therapeutic response, we show serial ctDNA analysis to be a highly informative biomarker that accurately captures the clonal dynamics of MBC tumors. This study presents comprehensive evaluation of MBC response to CDK4/6i through short interval serial ctDNA analysis, using targeted NGS in every interval (175 samples for 33 patients).
Both DNA and RNA extraction was carried out as the ctDNA assay can detect somatic alterations as well as gene fusions from plasma.
This approach did not impact recovery of cfDNA, achieving an average library conversion rate of 76%.
Our study brings additional insights regarding the biology of Our findings also build on previous studies, [13][14][15]21 providing a more detailed assessment of breast cancer clonal evolution during response to CDK4/6i. Other MBC liquid biopsy monitoring studies applied targeted NGS at pretreatment/baseline followed by personalized ddPCR of NGS alterations in subsequent surveillance [22][23][24] or serial targeted NGS focused on key intervals. 25 Our approach has the added advantage of detecting newly acquired alterations that emerge during CDK4/6i endocrine combination therapy, and precisely documenting the initial rise of ctDNA levels.
The prognostic role of ESR1 mutations is widely recognized as a resistance factor for endocrine therapy, 26