While oncologists are becoming more familiar with the idea of using circulating tumor cells (CTCs) to obtain prognostic information on their patients, researchers at Massachusetts General Hospital (MGH) Cancer Center are moving to the next level.

David T. Ting, MD, of the CTC Laboratory at MGH that designed the "CTC-Chip," described what is possible with next-generation CTC capture devices in an invited lecture at the 2011 Gastrointestinal Cancers Symposium.

Why Look for CTCs?

In metastatic cancers, the presence of CTCs has been associated with worse progression-free and overall survival and, in some studies, has also been predictive of treatment response. In the recent phase III CAIRO study in colorectal cancer patients,¹ the 29% of patients with high CTC counts at baseline (≥ 3 per 7.5 mL) had a 1.5-fold increased risk for progression (P = .0003) and a 2.2-fold increased risk for death (P < .0001) vs those with low counts. Baseline and CTC counts at 2 weeks were also predictive of treatment response "and therefore clinically relevant," Dr. Ting said.

CTCs offer a noninvasive means of sampling for genetic markers, monitoring treatment responsiveness, indicating vascular invasion in localized disease, aiding in treatment decision-making, enhancing early detection, revealing metastatic precursors, and indicating novel drug targets.

Next-generation Tools More Efficient

The CellSearch test (Veridex) is the only FDA-approved CTC detection kit. It contains an antibody conjugated to ferromagnetic beads and directed against the epithelial cell adhesion molecular (EpCAM) epitope expressed differentially on CTCs. The system captures and enriches the cells, then stains them for cytokeratin.

"But CellSearch is not perfect. We can do better," Dr. Ting said.

MGH scientists therefore developed the CTC-Chip, a high-throughput microfluidic device that comprises 80,000 "microposts" coated with the EpCAM antibody. With this device, CTCs are captured with "high yield and efficiency," he said.

"We envision that when the patient comes in we run fresh blood, and within 2 hours we process it on the Chip. We capture the cells fresh and therefore can perform many more diagnostic applications than is possible with CellSearch," Dr. Ting said.

Future applications will include immunostaining, fluorescence in situ hybridization (FISH), gene sequencing and expression analysis, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and cell culture. Stains in addition to cytokeratin and CD45 will be used to improve CTC detection, such as prostate-specific antigen (PSA), HER2, mesothelin, S100, and glial fibrillary acidic protein (GFAP), "since not all CTCs will produce cytokeratin," he pointed out.

Some of these stains will be disease-specific.  In prostate cancer, for example, PSA-positive CTCs can discriminate between local and metastatic disease; in a recent MGH study, percent CTC-positive cells was 42% in localized disease and 64% in metastatic disease.²

"What was most interesting in this study was that the mean number of CTCs was about the same in localized prostate cancer (107/mL) as in metastatic (116/mL)," he said. The study also found more robust staining of Ki67 (proliferation) in CTCs in castrate-resistant patients than in castrate-sensitive patients.  "This suggests that CTCs not only occur early and at a time when we can cure patients, but that they are heterogeneous," he said.

Similar findings are being made in other tumors. In non-small cell lung cancer, epidermal growth factor receptor (EGFR) mutations in CTCs were 90% concordant with the primary tumor, and novel secondary mutations were detected in 29%.³

Redesign: The Herringbone Chip

But there are also limitations to the micropost technology: The opaque design poses challenges in imaging, posts are a barrier for releasing and accessing cells for more detailed analysis, and the Chip is challenging to manufacture. The group, therefore, improved upon the design and created a more "scalable" next-generation device called the herringbone chip (HB-Chip). When blood is passed through a chamber lined with a herringbone pattern of grooves, a more chaotic flow is generated and this increases cell capture by several orders of magnitude (Fig. 1).⁴

The platform relies on immunoaffinity capture of CTCs of the EpCAM epitope like the CellSearch system, but has the additional benefit of microvortex mixing that greatly enhances yield. It also has optical and geometric properties that make more sophisticated analytical techniques possible. It is mounted on a glass slide for standard staining and opens to give access to CTCs for additional testing and growth in culture.

Using the HB-Chip Dr. Ting and colleagues have also observed "clusters" of 4 to 12 CTCs, which previous technology had not identified.

This and other next-generation devices will allow for gene expression profiling of CTCs (several profiles are in validation studies) and even culturing of the cells.

The ability to move beyond simple CTC "counts" to fully understand their complexity will yield much more valuable information, according to Dr. Ting. "CTC counts are not what we should focus on," he suggested. "CTC subsets are probably more clinically relevant." ■

Financial Disclosure: Dr. Ting has received grant support from the Pancreatic Cancer Action Network, American Association for Cancer Research, and Andrew L. Warshaw, M.D. Institute for Pancreatic Cancer Research.

References

1. Tol J, Koopman M, Miller MC, et al: Circulating tumour cells early predict progression-free and overall survival in advanced colorectal cancer patients treated with chemotherapy and targeted agents. Ann Oncol 21:1006-1012, 2010.

2. Stott SL, Lee RJ, Nagrath S, et al: Isolation and characterization of circulating tumor cells from patient with localized and metastatic prostate cancer. Sci Transl Med 31:2(25):25ra23, 2010.

3. Maheswaran S, Sequist LV, Nagrath S, et al: Detection of mutations in EGFR in circulating lung-cancer cells. N Engl J Med 359:366-377, 2008.

4. Stott SL, Hsu CH, Tsukrov DI, et al: Isolation of circulating tumor cells using a microvortex-generating herringbone-chip. Proc Natl Acad Sci U S A 107:18392-18397, 2010.