Cancer Letters 346 (2014) 24–33

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Circulating tumour cells and circulating free nucleic acid as prognostic and predictive biomarkers in colorectal cancer S.H. Lim a,b,c,d,⇑, T.M. Becker a,c,d, W. Chua b,d, N.J. Caixeiro a,d,e, W.L. Ng b,d, N. Kienzle a,c,d, A. Tognela a,b,d,e, S. Lumba a,b,c,d, J.E.J. Rasko f,g, P. de Souza a,b,c,d,e, K.J. Spring a,d,e a

Medical Oncology Group, Ingham Institute for Applied Medical Research, Liverpool 2170, Australia Department of Medical Oncology, Liverpool Hospital, Liverpool 2170, Australia School of Medicine, University of New South Wales, Kensington 2052, Australia d South West Sydney Translational Cancer Research Unit, Liverpool 2170, Australia e Liverpool Clinical School, University of Western Sydney, Liverpool 2170, Australia f Department of Cell and Molecular Therapies, Royal Prince Alfred Hospital, Camperdown 2050, Australia g Gene and Stem Cell Therapy Program, Centenary Institute, University of Sydney, Camperdown 2050, Australia b c

a r t i c l e

i n f o

Article history: Received 9 September 2013 Received in revised form 7 December 2013 Accepted 13 December 2013

Keywords: Circulating tumour cells Circulating free DNA Circulating free RNA Personalised cancer therapy Colorectal cancer

a b s t r a c t The detection of circulating tumour cells or circulating free tumour nucleic acids can potentially guide treatment and inform prognosis in colorectal cancer using minimally invasive ‘‘liquid biopsies’’. Current literature supports the notion that high circulating tumour cell counts or presence of tumour nucleic acid correlate with inferior clinical outcomes for patients, but they are not yet part of routine clinical care. Future research evolves around the examination of the molecular phenotype of circulating tumour cells. The key unanswered areas include differentiating between circulating tumour cell presence and their proliferative capacity and dormancy, identifying tumour heterogeneity and understanding the epithelial–mesenchymal transition. Ó 2013 Elsevier Ireland Ltd. All rights reserved.

1. Circulating tumour cells and circulating tumour DNA/RNA The ability to detect circulating tumour cells (CTCs) or circulating free tumour DNA (ctDNA) to guide patient treatment and inform prognosis is highly attractive, especially since it has the potential to provide multiple samples by way of serial minimally-invasive ‘‘liquid biopsies’’ [1–6]. In particular, there is increasing evidence for the utility of CTCs in the clinical management of colorectal cancer (CRC), with most studies confirming the association of high CTC counts with worse prognosis. CTCs were first identified by Ashworth in 1869 [7]. However, for over a century CTC research has been hampered by the inability to reliably detect these rare cells. While the normal range of white blood cells in human blood is 7–12  109/L, there may only be a few CTCs. The most widely used CTC enumeration platform, CellSearch™ (Veridex LLC, NJ, USA), was approved for clinical use by the Food and Drug Administration (FDA) in the United States, initially for patients with breast cancer in 2004, then CRC in 2007 ⇑ Corresponding author. Address: Medical Oncology Group, Ingham Institute for Applied Medical Research, 1 Campbell St, Liverpool, NSW 2170, Australia. Tel.: +61 2 9616 4407; fax: +61 2 9616 4499. E-mail address: [email protected] (S.H. Lim). 0304-3835/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.canlet.2013.12.019

and prostate cancer in 2008. To date, the majority of published CRC studies involving CTC analysis have used this instrument. The major limitation of immunomagnetic enumeration platforms utilising epithelial cell adhesion molecule (EpCAM) is the inability to capture non-epithelial CTCs. CellSearch also requires subjective CTC verification and does not permit single cell analysis. There is also an increasing number of CRC studies investigating the clinical utility of ctDNA or circulating RNA (ctRNA) to reflect the circulating tumour burden. ctDNA was first described in 1948 and its importance in cancer in 1977 [8]. It was proposed that for 100 g of tumour, 3.3 g of tumour DNA may enter the bloodstream [9]. ctDNA in cancer patients carry tumour-related genetic and epigenetic alterations, and can include single and double-stranded DNA. These potential biomarkers are still in early development and require further investigation to establish their full clinical utility (Table 1). Further, the definitive identification of the cellular source of these circulating tumour nucleic acids (ctNA) is currently not possible and there is no single validated platform or standardised method for ctDNA/RNA extraction and analysis. Currently, the most common downstream analysis are either specific mutation screening or quantitative reverse transcription PCR (qRT-PCR). Assays of free ctDNA/ctRNA do not permit analysis at the single cell

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S.H. Lim et al. / Cancer Letters 346 (2014) 24–33 Table 1 ctNA investigated as biomarkers in colorectal cancer. ctNA

Main potential clinical utility

Molecular alterations

References

ctDNA mutation

Prognostic, diagnostic

Kras TP53, APC

[64,65] [65]

ctDNA integrity

Diagnostic, correlation with CTCs Diagnostic and monitoring

DNA fragment size distribution ALU repeats

[47] [66]

ctDNA methylation

Prognostic, diagnostic

SEPT9 HLTF, HPP1 p16 Gene promoter hypermethylation RASSF2A promoter methylation

[67,68] [69] [64] [70]

ctRNA expression

Prognostic, predictive

CD133 hTERT, CK 19 CK 20 c-MET, MAGE-A3, GalNAc-T CEA EphB4, LAM, MAT

[21,36] [34,36,40] [34,36–40] [37] [34,36,39,40] [39]

Abbreviations: APC = adenomatous polyposis coli, ctNA = circulating tumour nucleic acids, CEA = carcinoembryonic antigen, CK = cytokeratin, CTCs = circulating tumour cells, hTERT = human telomerase reverse transcriptase, HLTF = helicase-like transcription factor, HPP1 = hyperplastic polyposis 1, LAM = laminin-5 gamma2 chain, MAT = matrilysin, miRNA = microRNA, SEPT9 = septin 9.

level. Conversely, the molecular analysis of individual CTCs is possible after isolation using platforms such as the C1™ Single-Cell Auto Prep (Fluidigm, CA, USA) system or DEPArray™ (Silicon Biosystems, Bologna, Italy), or a micromanipulator [10,11]. A review of the literature focusing specifically on the utility of CTCs in CRC clearly demonstrates the prognostic and predictive value of CTCs and ctDNA/RNA in both metastatic and non-metastatic settings, using various methods of detection (Tables 2 and 3). The last decade has seen improvements in technologies to capture, enrich and isolate CTCs, as well as measure circulating nucleic acids containing tumour-related molecular alterations, making these analyses easier. Whilst ctDNA/RNA will have the most utility in early disease, as a screening and monitoring tool, CTCs have established prognostic value. The full promise of CTC technology has yet to be realised, but the ability to isolate and interrogate single CTCs to track disease progression and allow us to better understand the biology underlying the changes in CTC presence and numbers is tantalizing.

2. CTC and ctDNA/ctRNA in metastatic CRC 2.1. Prognostic and predictive utility In advanced metastatic CRC, the presence of CTCs in blood appears to be related to overall survival (Table 2). A qualitative analysis of 16 studies of stage IV CRC found a median CTC detection rate of 35%, and a meta-analysis performed on 12 of these studies provides the strongest level of evidence for the prognostic utility of CTCs. These studies confirm the association between CTCs in patients with metastatic disease, and worse progression-free survival and overall survival [12]. This appeared to be related to CTC counts, such that the subgroup of studies with detection rates of more than 35% had inferior outcomes. However, it is important to note that in the majority of cases, the authors estimated the hazard ratio based on the data provided and the studies compared were heterogeneous in terms of sampling site and time, CTC definition and detection method. These variations are significant, and could influence the interpretation of results as illustrated by the individual studies discussed below. The pivotal prospective study by Cohen et al., involving 430 patients with metastatic CRC receiving first, second and third lines of chemotherapy, confirmed CTCs isolated using the CellSearch platform to be prognostic and predictive [13]. At baseline, prior to

the institution of a new therapy, 26% had unfavourable CTC counts, which was defined as P3CTC/7.5 ml. This correlated with worse progression-free survival and overall survival, and was also predictive of worse outcome for all treatment types. Favourable baseline CTCs (

Circulating tumour cells and circulating free nucleic acid as prognostic and predictive biomarkers in colorectal cancer.

The detection of circulating tumour cells or circulating free tumour nucleic acids can potentially guide treatment and inform prognosis in colorectal ...
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