Editorial on “Gene expression signatures in circulating tumor cells correlate with response to therapy in metastatic breast cancer”

Breast cancer recurrences, especially in patients with estrogen receptor (ER)-negative disease occur within the first 5 years after diagnosis (1). Several therapeutic options are available for the treatment of metastatic breast cancer, such as surgery, radiotherapy, hormone treatment, chemotherapy as well as targeted therapies (2). However, it would be very important to predict which patients will benefit from each treatment, since several patients experience significant treatment-related adverse events without any clinical benefit.

Analysis of CTC represents a powerful and diagnostic real-time liquid biopsy tool that provides to the physicians the assessment of the tumor burden and the efficacy of treatment (3). The frequency of CTC in whole blood is very low and their isolation and detection still remains a great challenge. Most of the isolation procedures of CTC are mainly based on the expression of EpCAM protein on the surface of the cells. Until now, the Cell Search is the only FDA-cleared technology for the isolation of EpCAM(+) CTCs and their subsequent enumeration (4). Other isolation procedures depend on the expression of specific cancer markers or the size of the cells and their deformability or, finally, based on cellular functions (5).

The published RT-qPCR assays for the quantification of the mRNA of specific cancer genes and, thus the detection of CTCs, suffer from the lack of consensus of their performance (6). According to the MIQE Guidelines of Real-Time PCR Experiments, better experimental practice allows more reliable and unequivocal interpretation of qPCR results (7). However, the availability of guidelines has done very little to improve the quality of published data based on the use of RT-qPCR and raise the question whether the situation must be like for the much more complex technologies based on it (8).

In this issue of Clinical Chemistry, Bredemeier et al. described a 46 multimarker gene PCR assay to characterize CTCs from MBC patients undergoing palliative therapy and predict treatment response on the basis of gene expression at 2 consecutive clinical time points (TP1 and TP2) compared to their expression at baseline. The multimarker RT-PCR assay was developed in order to evaluate whether it might have better prognostic accuracy, since simple RT-PCR may have limitations because the used markers might be expressed not only in CTCs but also in normal blood cells. The pre-analytical and analytical conditions of the assay were carefully controlled by using the AdnaCollectTM tubes (QIAGEN) for the blood collection, which stabilizes blood cells in a blood sample at 4–10 °C for 24 h, allowing the transportation or the storage of the sample as well as 2 different algorithms to find the most optimal normalization gene among 5 candidates control genes and selected the ACTB gene. These are very critical points for the calculation of the relative RNA amount and the validity of an assay (9-11). Bredemeier et al. defined a sample as CTC if at least one of the used markers (EpCAM, ERBB2, MUC1, or KRT19) was expressed. Most of the CTC+ samples expressed two of these genes, while KRT19 was uniquely expressed in some cases. KRT19 is a member of the keratin family, which is intermediate filament protein responsible for the structural integrity of epithelial cells (12). The expression of the mRNA of KRT19 of CTC has been shown to be associated with predictive (13) and prognostic implications (14) in breast cancer patients. Moreover, the authors have shown that KRT19 is highly expressed in patients who failed to respond (NR and ONR) to treatment. These results are consistent with previous findings showing that CTC expressing KRT19 after adjuvant chemotherapy is chemotherapy-resistant (15). Xenidis et al. has shown that the elimination of KRT19 mRNA-positive CTCs during taxane-based chemotherapy seems to be an efficacy indicator of treatment and is related to a better clinical outcome (16). Concerning the specificity of the expression of the mRNA of KRT19, the authors tested 20 healthy donors (HD) and detected KRT19 expression in 2 of them. This fact raises an issue, about the design of the primers used for evaluation of the KRT19-mRNA expression in the RT-qPCR, since KRT19 presents high alignment with 2 pseudo genes. Stathopoulou et al. (17) has shown that the design of the primers and probes is very important for the accurate detection of the KRT19-mRNA (+) CTCs.

An interesting finding of the Bredemeier’s study concerns the observation that both epithelial and EMT or stemness markers were always co-expressed in CTCs. This is in agreement with studies from several research groups, which have demonstrate that the pool of CTCs is consisted from different CTC sub-population which may be hybrids with epithelial and mesenchymal characteristics (18,19). Nevertheless, it should be mentioned that in late metastatic cancers, the isolation of CTC based on the expression of EpCAM protein, could not be feasible, because of the prevalence of the mesenchymal phenotype (20). It has been previously reported that CTCs bearing both an EpCAM- and EMT-associated phenotype have been associated with metastatic disease (21). This clinical correlation clearly validate the importance of the developed 46 multimarker gene PCR assay for the detection of CTCs and emphasizes the need of capturing CTCs not only based on EpCAM expression but also in combination with other markers.

The Bredemeier’s et al. study also revealed that 14 genes (KRT19, FLT1, EGFR, EPCAM, GZMM, PGR, CD24, KIT, PLAU, ALDH1A1, CTSD, MKI67, TWIST1, and ERBB2) were differentially expressed between the CTC+ and CTC− samples but only the ADAM17 and ABCC1genes were differentially expressed in the NR or ONR groups. Although this finding could be related to the fact that some samples were CTC−, based on the expression of the 4 genes, CTCs might expressed other epithelial cancer markers. Therefore, the emerged interesting predictive value of the ADAM17- and ABCC1-expressing CTCs merits to be further evaluated in a prospective and larger cohort of patients with metastatic breast cancer in order to validate their clinical relevance.

The heterogeneity of CTC constitutes the cornerstone of their successful isolation and detection. Molecular characterization of CTC provides their biological specificity and, thus, their characterization based on mRNA gene profile (22), DNA mutation status (23), DNA methylation (24) and chromosomal aberrations (25) of extreme biological and clinical importance. Therefore, the combination of the phenotypical and molecular identity of CTCs could enlighten us about the aggressive and malignant nature of these cells.

Acknowledgments

Funding: None.

Footnote

Provenance and Peer Review: This article was commissioned and reviewed by the Section Editor Dr. Dao-Jun Hu (Department of Clinical Laboratory, Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Chongming Branch, Shanghai, China).

Conflicts of Interest: Both authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/jlpm.2017.12.05). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Colleoni M, Sun Z, Price KN, et al. Annual Hazard Rates of Recurrence for Breast Cancer During 24 Years of Follow-Up: Results From the International Breast Cancer Study Group Trials I to V. J Clin Oncol 2016;34:927-35. [Crossref] [PubMed]
2. Fossati R, Confalonieri C, Torri V, et al. Cytotoxic and hormonal treatment for metastatic breast cancer: a systematic review of published randomized trials involving 31,510 women. J Clin Oncol 1998;16:3439-60. [Crossref] [PubMed]
3. Alix-Panabieres C, Pantel K. Clinical Applications of Circulating Tumor Cells and Circulating Tumor DNA as Liquid Biopsy. Cancer Discov 2016;6:479-91. [Crossref] [PubMed]
4. Coumans F, Terstappen L. Detection and Characterization of Circulating Tumor Cells by the CellSearch Approach. Methods Mol Biol 2015;1347:263-78. [Crossref] [PubMed]
5. Esmaeilsabzali H, Beischlag TV, Cox ME, et al. Detection and isolation of circulating tumor cells: principles and methods. Biotechnol Adv 2013;31:1063-84. [Crossref] [PubMed]
6. Bustin SA, Nolan T. Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction. J Biomol Tech 2004;15:155-66. [PubMed]
7. Bustin SA, Benes V, Garson JA, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 2009;55:611-22. [Crossref] [PubMed]
8. Bustin S, Nolan T. Talking the talk, but not walking the walk: RT-qPCR as a paradigm for the lack of reproducibility in molecular research. Eur J Clin Invest 2017;47:756-74. [Crossref] [PubMed]
9. Liu LL, Zhao H, Ma TF, et al. Identification of valid reference genes for the normalization of RT-qPCR expression studies in human breast cancer cell lines treated with and without transient transfection. PLoS One 2015;10:e0117058 [Crossref] [PubMed]
10. Qin J, Alt JR, Hunsley BA, et al. Stabilization of circulating tumor cells in blood using a collection device with a preservative reagent. Cancer Cell Int 2014;14:23. [Crossref] [PubMed]
11. Luk AWS, Ma Y, Ding PN, et al. CTC-mRNA (AR-V7) Analysis from Blood Samples-Impact of Blood Collection Tube and Storage Time. Int J Mol Sci 2017;18:E1047 [Crossref] [PubMed]
12. Chang TH, Huang HD, Ong WK, et al. The effects of actin cytoskeleton perturbation on keratin intermediate filament formation in mesenchymal stem/stromal cells. Biomaterials 2014;35:3934-44. [Crossref] [PubMed]
13. Chen Y, Zou TN, Wu ZP, et al. Detection of cytokeratin 19, human mammaglobin, and carcinoembryonic antigen-positive circulating tumor cells by three-marker reverse transcription-PCR assay and its relation to clinical outcome in early breast cancer. Int J Biol Markers 2010;25:59-68. [PubMed]
14. Reinholz MM, Kitzmann KA, Tenner K, et al. Cytokeratin-19 and mammaglobin gene expression in circulating tumor cells from metastatic breast cancer patients enrolled in North Central Cancer Treatment Group trials, N0234/336/436/437. Clin Cancer Res 2011;17:7183-93. [Crossref] [PubMed]
15. Xenidis N, Ignatiadis M, Apostolaki S, et al. Cytokeratin-19 mRNA-positive circulating tumor cells after adjuvant chemotherapy in patients with early breast cancer. J Clin Oncol 2009;27:2177-84. [Crossref] [PubMed]
16. Xenidis N, Perraki M, Apostolaki S, et al. Differential effect of adjuvant taxane-based and taxane-free chemotherapy regimens on the CK-19 mRNA-positive circulating tumour cells in patients with early breast cancer. Br J Cancer 2013;108:549-56. [Crossref] [PubMed]
17. Stathopoulou A, Ntoulia M, Perraki M, et al. A highly specific real-time RT-PCR method for the quantitative determination of CK-19 mRNA positive cells in peripheral blood of patients with operable breast cancer. Int J Cancer 2006;119:1654-9. [Crossref] [PubMed]
18. Yu M, Bardia A, Wittner BS, et al. Circulating breast tumor cells exhibit dynamic changes in epithelial and mesenchymal composition. Science 2013;339:580-4. [Crossref] [PubMed]
19. Kallergi G, Papadaki MA, Politaki E, et al. Epithelial to mesenchymal transition markers expressed in circulating tumour cells of early and metastatic breast cancer patients. Breast Cancer Res 2011;13:R59. [Crossref] [PubMed]
20. Gorges TM, Tinhofer I, Drosch M, et al. Circulating tumour cells escape from EpCAM-based detection due to epithelial-to-mesenchymal transition. BMC Cancer 2012;12:178. [Crossref] [PubMed]
21. Bonnomet A, Brysse A, Tachsidis A, et al. Epithelial-to-mesenchymal transitions and circulating tumor cells. J Mammary Gland Biol Neoplasia 2010;15:261-73. [Crossref] [PubMed]
22. Strati A, Markou A, Parisi C, et al. Gene expression profile of circulating tumor cells in breast cancer by RT-qPCR. BMC Cancer 2011;11:422. [Crossref] [PubMed]
23. Beije N, Sieuwerts AM, Kraan J, et al. Estrogen receptor mutations and splice variants determined in liquid biopsies from metastatic breast cancer patients. Mol Oncol 2017; [Epub ahead of print]. [Crossref] [PubMed]
24. Chimonidou M, Strati A, Malamos N, et al. Direct comparison study of DNA methylation markers in EpCAM-positive circulating tumour cells, corresponding circulating tumour DNA, and paired primary tumours in breast cancer. Oncotarget 2017;8:72054-68. [PubMed]
25. Neves RP, Raba K, Schmidt O, et al. Genomic high-resolution profiling of single CKpos/CD45neg flow-sorting purified circulating tumor cells from patients with metastatic breast cancer. Clin Chem 2014;60:1290-7. [PubMed]