Original ArticlesLongitudinal monitoring reveals dynamic changes in circulating tumor cells (CTCs) and CTC-associated miRNAs in response to chemotherapy in metastatic colorectal cancer patients
Introduction
Colorectal cancer is the third most common cancer and the fourth most common cause of cancer-related death worldwide [1]. First-line chemotherapy for metastatic colorectal cancer includes the combinations of 5-fluorouracil, leucovorin, and irinotecan (FOLFIRI), 5-fluorouracil, leucovorin, and oxaliplatin (FOLFOX), capecitabine and oxaliplatin (XELOX), and the combination of 5-fluorouracil, leucovorin, oxaliplatin, and irinotecan (FOLFOXIRI) [2]. The use of these combination therapies for metastatic colorectal cancer have led to response rates of >50% and median survival of up to 2 years [3,4]. However, practically all metastatic colorectal cancers eventually become resistant to chemotherapy [5]. Therefore, the development of biomarker assays to predict resistance and the identification of alternative strategies to overcome chemotherapeutic resistance are important in reducing the morbidity and mortality. Candidate predictive biomarkers for chemotherapy include mutant TP53, thymidylate synthase (TS) expression, amplified ERCC1, microsatellite instability (MSI), CpG island methylator phenotype (CIMP), and mutant BRAF [6]. However, none of these are recommended in clinical guidelines as companion diagnostics for colorectal cancer treatment [7] and require further validation studies.
Recently, targeted biologic therapies such as bevacizumab targeting the vascular endothelial growth factor (VEGF) and cetuximab and panitumumab targeting the endothelial growth factor receptor (EGFR) have demonstrated significant survival benefits in metastatic colorectal cancer alone or in combination with cytotoxic chemotherapy [8,9]. However, patients respond differently with respect to efficacy and toxicity, therefore it is important to identify biological biomarkers that can predict response to therapy and hence identify patients that would benefit from the therapy. KRAS and NRAS mutation status are clinically useful to predict response to anti-EGFR therapy [7]. Other molecular markers such as PIK3CA and BRAF are not yet established for prediction of response to therapy [7].
Circulating tumor cells (CTCs) are cancer cells dislodged from the primary tumor or its metastases and disseminated in the blood stream [10]. CTCs can be isolated directly from peripheral blood, obviating the need for invasive tumor biopsies [11]. Many studies have shown that CTCs may be used to predict disease progression and survival in metastatic cancer [12,13]. Molecular profiles obtained from isolated CTCs can be correlated with treatment outcomes [14] and may reveal druggable candidates. Despite advances in technology that have allowed the development of assays for using CTCs as biomarkers of disease progression and therapeutic response, the kinetics of the changes in numbers and molecular characteristics of CTCs over time remain poorly understood [15]. Because of these limitations, despite much evidence for their prognostic value, CTCs have not yet been recommended clinically to guide decisions on therapy as data demonstrating the clinical utility of CTC-based tests are lacking [7,16].
MicroRNAs (miRNAs) are short (18-22-nucleotides) RNA that bind to complementary sequences in the 3′ untranslated region of multiple target messenger RNAs [17]. By either blocking translation or inducing target mRNA degradation, miRNAs regulate multiple biological processes, and are implicated in pathological processes [17]. Specific miRNA expression patterns are associated with cancer, and miRNAs have been suggested as diagnostic and predictive clinical biomarkers [18]. Few studies have examined the expression of miRNA in CTCs [13,19], and to the best of our knowledge, there is currently no published study on longitudinal CTC-specific miRNA expression in metastatic colorectal cancer.
We previously reported a paper-based method to efficiently extract miRNAs from CTCs [20]. Using this digital-PCR inspired method, background miRNA expression was excluded from contaminating blood cells, and CTC-specific miRNA expression profiles were derived [20]. In this study, we evaluated the changes in CTC count and CTC-associated miRNAs during the course of chemotherapy in patients with metastatic colorectal cancer by comparing with CEA levels and imaging results. We hypothesized that CTC count and miRNA expression changes during the course of treatment reflect response to therapy and may be used to assess the efficacy of ongoing treatment.
Section snippets
Patients
This prospective single-institution study enrolled patients with the following inclusion criteria:
- 1.
Histologically proven metastatic colorectal carcinoma where XELOX/mFOLFOX-6/XELIRI/FOLFIRI/XELOX + bevacizumab/mFOLFOX-6 + bevacizumab/XELIRI + bevacizumab/FOLFIRI + bevacizumab regimen is indicated and planned for chemotherapy;
- 2.
Patient age ≥21 years;
- 3.
Measurable disease according to RECIST criteria or evaluable disease;
- 4.
A life expectancy of at least 3 months;
- 5.
Patient signed informed consent.
A patient
Clinical characteristics of patients
All patients were diagnosed with colorectal cancer at clinical stage IV with distant metastases and were treated with oxaliplatin-based chemotherapy. A summary of the clinical characteristics of the patients is shown in Table 2. Three patients had partial response, four patients had stable disease and two patients had progressive disease as determined by Response Evaluation Criteria In Solid Tumors (RECIST) criteria [23].
Longitudinal monitoring of CTCs
We measured CTC counts and CTC-associated miRNA expression in patients
Discussion
CTCs have been represented as “liquid biopsies” of solid tumors and may potentially be used as biomarkers to assess the patient's disease progression and response to therapy [10,11]. In order to validate CTCs as biomarkers of prognostic significance, we investigated how CTC counts and CTC-associated miRNAs correlate with established clinical parameters over the course of chemotherapy in metastatic colorectal cancer patients. In this study we observed that the changes in CTC counts reflected the
Conclusion
In this pilot study, we have studied the changes in CTC counts and CTC-associated miRNAs over the course of chemotherapy in 9 metastatic colorectal cancer patients. Our results suggest that longitudinal monitoring of the dynamic CTC changes over the course of the disease and treatment in metastatic colorectal cancer may be useful in predicting patient response and disease progression. These results need to be validated in larger studies.
Conflicts of interest
All authors have no conflicts of interest to declare.
Funding
This study was supported by a National Medical Research Council (NMRC) Clinician New Investigator Grant (CNIG) (NMRC/CNIG/1119/2014) and a National University Health System (NUHS) Bench to Bedside Grant (NUHSRO/2014/019/BBP/04).
Author contributions
KT, SML, ESCK, WPY, and ASLP conceptualized and designed the study. MVMB, WKC, TS, WPY, ASLP recruited the patients for the study. KT, PVC, JT, ZZK performed the CTC and miRNA experiments and data analyses. KT, MVMB, WPY and ASLP obtained clinical-pathological variables for correlation. KT and ASLP prepared the manuscript. SML, ESCK, WKC, and WPY reviewed the manuscript. All authors approved of the final version to be submitted.
Acknowledgements
We thank all patients who have consented to the study and donated their blood for this study. We thank all the oncologists who recruited the patients and all the research coordinators and nurses who helped obtain blood from the patients for this study.
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