The Grand Challenge in Circulating Tumor Cell Isolation: The need for a Gold Standard Platform

The urgent need for a Standardized Technology for Tumor Cell Isolation

In recent years, a number of technologies have claimed the ability to isolate tumor cells for clinical cancer management. These technologies can be broadly classified as falling into the following categories with regards to their value proposition,

a)      Using specific antibodies (either single-EpCAM or combination) in a conventional magnetic isolation assay or a microfluidics assay (Eg., Veridex, Biocept, OnQity, Biofluidica, Adnagen)

b)      Using size and biomechanical properties as a differentiator between tumor cells and normal blood cells (Eg., Clearbridge, CellSeivo, Rare cells, Screen Cell, Creative Microtech, Filtini, Parsortix)

c)       Technologies whose value proposition is in automated enumeration and identification such as automated microscopy  (Eg., Bioview, Ikonisys, eDAR, Epic Sciences)

d)      Technologies that use electrical properties (Silicon biosystems, Apocell)

The advantages and disadvantages of various techniques are tabulated extensively in various reviews [1 -8]. It’s also widely noted that most of these technologies remain to be independently validated and not considered optimal for tumor cell isolation [9-15]. Some criterion were recently (2012) suggested by a group of KOLs, titled “Considerations in the development of circulating tumor cell technology for clinical use” [16]. A list of technological requirements of an ideal CTC platform were previously suggested in a Lab on a chip paper in 2011 titled “Circulating Tumor Cells: The Grand Challenge” [17].

The pain due to lack of robust and standardized tumor cell isolation technology:

So far CTCs have only been FDA approved for cancer prognosis, which is of limited clinical utility. The real clinical impact of CTCs will be realized when their relevance is shown for personalized therapy & monitoring and early diagnosis. The clinical community is now investigating this relevance. However, the present technology platforms are inherently biased and may lead to poor clinical interest. For example, “A recent study revealed that “normal-like” breast cancer cells, which usually display an aggressive phenotype, express low expression of EpCAM and are not detected by the CellSearch® test [65]. Moreover, a retrospective study that involved 292 patients with metastatic breast cancer has shown that 36% of them showed an undetectable CTC status, which could be due, at least in part, to an underestimation of CTCs by the Cell-Search® test due to CTC undergoing epithelial-mesenchymal transition (EMT) ” [1]

Another paper noted that, “ The challenge of CTC detection is related to the requirement of both high sensitivity and specificity. A wrong labeling of ‘‘non-tumor cells’’ (epithelial non tumor cells or normal hepatocytes, for instance) as ‘‘tumor cells’ could generate poor clinical interest” [8]

This problem is well summarized by the following passage in recent literature “The true potential of CTCs has yet to be realized because of limits in technology used to capture these cells and our lack of a complete understanding of metastasis. This is complicated by the fact that our understanding of CTCs is subject to the techniques available to identify and isolate CTCs, and the biases inherent to them.” [6]

From the above passage it is very clear that what is urgently needed now in CTC field is not “cheap” or “fast” technology but the “most reliable” platform to accurately reflect the patient physiology. 

The problem with existing technologies:

“Despite the recent technological advances, the development of a single device capable of simultaneously achieving high throughput, high target cancer cell recovery, high purity, and high cell viability remains challenging.” [5]

As pointed out by the numerous review papers, all technology platforms existing today suffer from one trade-off or another. For example, antibody-based technologies are limited by the expression of specific antibodies. A universal CTC marker is yet unknown and given the heterogeneity of cancer [18], it is doubtful that a single universal cancer marker will be found.

The problem with Size and biomechanical properties based isolation:   “The size range of different tumor cells is highly variable and does overlap with that of normal blood cells” [19]. Also, “CTC may not be always .8 mm making the sensitivity of these assays questionable.” [20]. Moreover, “ these methods suffer from low cell viability resulting from potential damage incurred as the cells pass through narrow filter pores, which renders the use of microfilters less compatible for live cell interrogations (e.g., cell suspensions were partially fixed before being passed through a membrane micro-filter)” [4]

The assessment of technologies based on size is well summed up in this review “Unfortunately, large leukocytes can be trapped by the filter as well, therefore contaminating the CTC fraction or small CTC can pass through the pores therefore depleting the CTC population. This is why this technique is generally considered not highly sensitive and poorly specific” [8]

There are several companies based on the principle of size and biomechanical properties and this is the most crowded sub-space within CTC. A reason for this is because of low technological barrier to entry and wide availability of hole making technology in various materials such as polycarbonate, silicon nitride and other polymers. As it can be readily inferred by-passability is easy and patentability is low for technologies operating on this principle. 

The Solution:

The clinical need and technology requirements are well documented [16, 17]. Briefly, the assay must be highly sensitive, well characterized with analytical and clinical samples, standardized for cross reference studies at various clinical sites, should be free of operator bias, should use standard blood collection and processing techniques and should be time stable over a period to allow for stable sample transportability. Above all, the assay should isolate viable, pure tumor cells with high efficiency in a simple and cost effective manner. 

Neegative depletion approach has been recommended over the positive selection method [2, 21]. In negative depletion approach, all normal cells are eliminated, leaving behind “abnormal cells”. These cells are then identified by immunohistochemical staining or molecular methods.

Conclusion: Despite the presence of several technologies, the need for a gold standard tumor cell isolation platform remains unmet. 

References:

1.       Am. J Cancer Res 2011;1(6):740-751

2.       Nature reviews, clinical oncology, December, 2010

3.       Clinical Chemistry 58:5 (2012)

4.       Lab Chip, 2012, 12, 1753–1767

5.       Review article, Frontiers in oncology, vol 2, article 69, july 2012

6.       J Cancer Res Clin Oncol DOI 10.1007/s00432-011-0988-y

7.       International Journal of Hepatology, Volume 2012 (2012),

8.       Methods 50 (2010) 289–297

9.       Circulating tumor cells: approaches to isolation, and characterization, JCB review, 2011

10.   New technologies for the detection of circulating tumour cells, British Medical Bulletin 2010; 1–16

11.   Let Me Do More Than Count the Ways: What Circulating Tumor Cells Can Tell Us about the Biology of Cancer  VOL. 6, NO. 5, 1307–1310 MOLECULAR PHARMACEUTICS, 2009

12.   Disseminated Tumor Cells in Bone Marrow and Circulating Tumor Cells in Blood of Breast Cancer Patients: Current State of Detection and Characterization
 Pathobiology 2008;75:140–148

13.   Circulating tumor cells (CTC) detection: Clinical impact and future directions Patrizia Paterlini-Brechot *, Naoual Linda Benali  Cancer Letters 253 (2007) 180–204

14.   Clin Cancer Res; 16(20) October 15, 2010

15.   Cancer Letters 253 (2007) 180–204

16.   Journal of Translational Medicine 2012, 10:138 

17.   Lab Chip, 2011,11, 375-377

18.   PLoS ONE 7(5): e33788

19.   Current Opinion in Genetics & Development 2010, 20:96–99

20.   British Medical Bulletin 2010; 1–16

21.   Cancer Res; 71(18), 2011