IMPaCT Investigator Highlights (Text Version) - Dr. Richard J. Lee

Title: Analysis of Circulating Prostate Cancer Tumor Cells Using the CTC-Chip Microfluidic Device

Investigator: Richard J. Lee, MD, PhD; Massachusetts General Hospital Cancer Center

This particular project is looking at circulating tumor cells and through a collaboration of engineers, cancer geneticists and molecular biologists and clinicians, we were able to come up with a very interesting project that allows us to look at cells from cancer patients.

The basis of this work is that it uses the—the CTC chip, the circulating tumor cell chip. Which is a device that’s about the size of a microscope slide on which were affixed 78,000 microposts and on those microposts were coded antibodies. These antibodies were designed to be able to identify proteins that are expressed on prostate cancer cells, so if those cells make contact with the posts and stick to the posts then all the cells that don’t express that particular protein float on by, and by doing this we’re able to enrich the population of circulating cancer cells and by capturing them in this manner we can do some protein staining. We can count these cells; we can do molecular analyses on these cells in order to assess the numbers of cells that we can find in the blood of these patients.

I should say that this isn't the only technology out there that’s looking at CTCs. But what we have found is that the yield, the numbers of cells that we capture, in addition to the purity of cells that we have seen, appears to be a great improvement over certain other techniques that are available.

This particular analysis device allows for the computer to do the analysis. This program will identify, for example, two cells in an image; there’s staining for DNA, which stains in blue, and there’s another stain for PSA, the prostate-specific antigen, which is what we use to identify these cells and you can see here that of the two cells that are identified by their nuclei, only one of them stains positively for PSA. And through a computer algorithm, is able to merge these images, to then count which of the cells have nuclei and stain positively for PSA. This takes away the subjectivity of the human eye and allows us to make this a much more automated process.

Here is an example of some high magnification images of exactly the types of output that you see with this program, where again we see cells here that stained positively for DNA, positively for PSA, and when you merge these images you see this green and blue. It’s all one cell.

This figure we looked at different molecular markers that we might be able to detect in the CTCs. Here we’re using three different colors for staining. One is for DNA, one is for PSA to identify the prostate cancer cells and another is for Ki67, which is a proliferative marker to see how many of those cells are actually dividing.

Here you can see that when we merged the three different stains that I’ve shown you here, the DNA, PSA, and Ki67, you can see that is a cell that we would consider proliferating. This is another set of cells in which you can see two cells identified by DAPI, both express PSA but only one of them is proliferating. So this staining process can distinguish the Ki67 positive cells versus Ki67 negative cells.

Suggesting that maybe we can find something out from the very beginning before we start treating these patients with any therapies that may give us a clue about the clinical course that these patients are going to undergo.

This image is looking at other markers for prostate cancer cells. This is a stain for PSMA, which is a membrane antigen. And this is a FISH analysis, a fluorescence in situ hybridization assay, looking at the androgen receptor. We know from many studies that the androgen receptors are critical components to prostate cancer proliferation. We know the androgen receptor can be amplified in patients with metastatic prostate cancer and this allows us to demonstrate that they’re—in this case, there are at least seven copies of androgen receptor identified in that cell.

And the last figure here is looking at the translocation—TMPRSS2:ERG. And what we know now is that this translocation is seen in about 50% of men with prostate cancer. So it’s the most common translocation that we see in carcinomas. Can we see these translocations in CTCs of men with prostate cancer? Well in fact we can.

And here is a PCR analysis of a VCaP cell line, which is known to have this translocation, and then two separate patients, one of whom had the translocation and the other did not. This is a sequencing analysis that shows the typical break point for TMPRSS2:ERG.

So what we’re seeing is that these cells harbor the exact same translocation that we see in prostate cancer specimens that are removed from patients. This is the translocation that defines a prostate cancer cell. So we know that these CTCs are not just some bizarre PSA expressing epithelial cell; they actually harbor the translocation and this identifies them as prostate cancer cells.

To date the preliminary studies has been a validation that this is a technique that—that can identify circulating prostate cancer cells. Ultimately, what we hope to do is to be able to use the circulating tumor cells as a—in a sort of point of care device where we’re able to use the information gleaned from the number of circulating tumor cells or the molecular characteristics of the circulating tumor cells to—to be able to make decisions, clinical therapeutic decisions whether to change therapies or whether a particular type of targeted therapy may be best for a patient based on the CTC analyses.