Epidermal growth factor receptor mRNA expression: A potential molecular escape mechanism from regorafenib

Abstract Regorafenib has improved the survival of patients with refractory metastatic colorectal cancer (mCRC), yet the mechanisms of inherited or acquired resistance are not well understood. A total of 50 patients with refractory mCRC were enrolled. Circulating tumor cell (CTC) enumeration was carried out at baseline, day 21 after initiation of regorafenib, and at the time of progression of disease (PD) using the CellSearch System (Veridex LLC, NJ, USA). Poly(A) mRNA was extracted from CTCs, and gene expression of epithelial and epithelial‐mesenchymal transition markers was analyzed by a multiplex‐PCR based DNA Chip. Patients with fewer than 3 CTCs at baseline and day 21 had a longer progression‐free survival than those with 3 or more CTCs (3.3 vs 2.0 months, P = .008 and 3.3 vs 2.0 months, P = .004, respectively). Patients with fewer than 3 CTCs at baseline and day 21 had a longer overall survival (OS) than those with 3 or more CTCs (10.0 vs 4.6 months, P < .001 and 8.7 vs 3.8 months, P = .003, respectively). In multivariable analysis, CTC counts remained significantly associated with OS at baseline and day 21 (P = .019 and P = .028). Circulating tumor cell EGFR gene expression was upregulated at day 21 and/or PD in 64% of patients. Patients had significantly increased EGFR expression at PD compared to baseline (P = .041) and at day 21 and/or PD compared to baseline (P = .004). Our findings suggest that CTC count and EGFR expression could be useful markers of regorafenib efficacy and outcomes. Upregulation of CTC EGFR expression might be a molecular escape mechanism under regorafenib therapy.


| INTRODUC TI ON
Regorafenib, an oral multikinase inhibitor, blocks the activity of several protein kinases, including v-raf murine sarcoma viral oncogene homolog B1 (BRAF), 1  system is one such method and is the only FDA-approved assay for the enumeration of CTCs in peripheral blood. [3][4][5] In a study by Cohen et al using CellSearch, the presence of 3 or more CTCs at baseline and follow-up was an independent prognostic marker of inferior survival in mCRC patients. [5][6][7] However, it is unclear whether CTC enumeration at baseline and over time is predictive or prognostic in mCRC patients specifically treated with regorafenib.
In addition to enumeration, protein expression and molecular profiling of CTCs might serve as more refined biomarkers and help inform a more personalized treatment approach. Under the pressure imposed by chemotherapy and mAbs, clonal selection and genomic instability emerge and provoke eventual treatment resistance. The ability to characterize such intratumoral heterogeneity could help to identify novel predictive and prognostic biomarkers and improve treatment decision-making. To this end, our group has previously examined the prognostic role of epithelial-mesenchymal transition (EMT) gene (PI3K, Akt-2, and Twist1) expression within CTCs of patients with mCRC (Ning Y et al). 8 In this study, we examined the ability of CTC enumeration and molecular characterization, using mRNA levels of epithelial (EGFR, EpCAM, and CEA) and EMT (PI3K, Akt-2, and Twist1) markers, to predict outcomes and identify potential molecular escape mechanisms in mCRC patients undergoing regorafenib therapy.

| Isolation and enumeration of CTCs
Circulating tumor cell enumeration was carried out at baseline prior to initiation of regorafenib, day 21 after initiation of therapy, and at the time of treatment intolerance or progression of disease (PD) using the CellSearch system. A total of 7.5 mL whole blood was collected on a CellSave Preservative Tube, and the CellSearch Circulating Tumor Cell Kit (Veridex) was applied for CTC enrichment and enumeration. Immunomagnetic enrichment was undertaken using anti-EpCAM ferrofluid.
Enriched cells were then fluorescently stained with the nucleic acid dye DAPI and labeled with mAbs specific for epithelial cells (anti-CK8, 18, and 19) and leukocytes (anti-CD45). A CTC was defined as an EpCAM, isolated, intact, round to oval cell, with a visible nucleus (DAPI-positive), positive staining for CK, and negative staining for CD45. Results of CTC enumeration were then expressed as the number of cells per 7.5 mL blood. 9
Visualization of PCR fragments was carried out with the Agilent 2100 Bioanalyzer, with a limit of detection of 0.01 ng/μL or more.

| Statistical analysis
The primary outcome measure was PFS, defined as the date of randomization to the date of first documented disease progression or death from any cause. If disease progression or death was not observed, PFS was censored on the day of the last computed tomography scan. Secondary end-points were response rate and OS. Overall

| Patient and tumor characteristics
Clinicopathologic characteristics are presented in Table 1

| Clinical outcomes by CTC count and gene expression in patients receiving regorafenib
The distribution of CTC count and CTC gene expression at baseline and day 21 is outlined in Table 2 Associations between CTC count and outcomes are analyzed (   (Table 3). Additionally, patients with less than 3 CTCs at baseline and day 21 had a significantly higher tumor response rate compared to those with 3 or more CTCs (Fisher's exact test, P = .007 and P = .03, respectively) ( Table 3).
We then examined associations between CTC gene expression levels and outcomes. We used the optimal cut-off value method as previously described. 10

| Changes in CTC count and CTC gene expression at day 21
Next, we assessed CTC count and CTC gene expression levels at day 21 and at the time of disease progression as compared to baseline.
Circulating tumor cell EGFR expression was significantly increased at day 21 and/or PD (P = .004) in 64% of patients (Table 4). More specifically, CTC EGFR expression was significantly increased at PD compared to baseline (P = .041) in 48% of patients. No significant associations were seen with the other genes that were analyzed.

| Associations between changes in CTC EGFR and EMT marker expression
We then hypothesized that increased CTC EGFR expression could result in an increase in the expression of CTC EMT markers. 13 However, there were no significant associations between changes in CTC EGFR and EMT marker gene expression.   It has been reported that CTCs often exist in a state of EMT or could coexpress both epithelial and mesenchymal markers. During the EMT, epithelial cells downregulate epithelial-related genes, acquire mesenchymal gene expression, and undergo major changes in their cytoskeleton that result in loss of cell-cell contact and cell polarity, leading to increased motility and invasiveness. 19 Here, we investigated the expression of CTC EMT markers, including TWIST1, Akt2,

| D ISCUSS I ON
and PI3Kα, as well as associations between CTC EGFR and EMT gene expression, and found a nonsignificant trend towards an inverse association. The lack of a significant association could reflect challenges associated with CTC characterization using the CellSearch technique, including the presence of an excess of leukocytes, which express EMT marker genes, in CTC-enriched blood fractions.
Certain limitations of our study should be acknowledged. Our data are limited by the small sample size and are only exploratory.
The lack of a control group of patients not receiving regorafenib preclude any conclusions that can be drawn regarding the use of CTC count or associated EGFR expression as predictive markers.
In summary, we identified CTC count as a prognostic marker in mCRC patients treated with regorafenib. Circulating tumor cell EGFR expression was significantly increased with regorafenib at the time of disease progression, suggesting this to be a mode of resistance and lending further mechanistic evidence for the synergistic effects seen with regorafenib and anti-EGFR mAbs. 20 Our findings warrant further investigation and, if replicated in future investigations, could inform the selection of mCRC patients best suited for regorafenib in the chemorefractory setting.

ACK N OWLED G EM ENTS
The project described was supported in part by award number P30CA014089 from the NCI. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NCI or the NIH. This project was also supported in part by the