RarePlex^® ARv7/SYP CTC Panel Kit

RarePlex Panel and Developer Assays for CTC enumeration and characterization

Presented by Arturo Ramirez, Ph.D., Director of Oncology R&D

Hello my name is Arturo Ramirez and I'm Director of Oncology Research and Development at RareCyte. In this presentation, I'll go over the RarePlex panel and developer assays for CTC enumeration and characterization.

RareCyte platform for CTC enumeration and charaterization

CTCs provide a non-invasive informative window into tumor status and patient response to therapy. However, CTCs are extremely rare and have heterogeneous characteristics which makes them very hard to detect. Thus, CTC analyses demand RareCyte's unique combination of high accuracy in detecting CTCs in patient samples, high precision with low coefficient of variation between experiments, and reproducibility from lab-to-lab, user to user, and day-to-day also very important can be the flexibility to add other biomarkers besides those that allow CTC identification in order to characterize the CTCs phenotypically. This is something that can't be done with cell free DNA. Our platform is quantitative and based on immunofluorescent imaging so biomarker expression can be measured and important information such as biomarker localization and thermal cell morphology is generated. Finally ,our platform is seamlessly integrated from blood sample to result so that it's easy to perform with minimal hands-on steps and can be scaled up so that up to 16 samples can be run at the same time by one user.

RarePlex Staining Kits

RarePlex staining kits are used to enumerate and characterize CTCs. They are characterized into two types: panel kits which are ready to use, optimized and validated kits for CTC enumeration and biomarker characterization and developer kits which when combined with a panel kit, allow users to test and optimize new biomarkers of interest using anti-mouse and anti-rabbit primary antibodies followed by a proprietary amplification scheme that allows for high sensitivity detection of even low abundance targets. Panel kits and developer kits can be combined to create custom assays that are run on automated slide staining systems such as the Ventana Discovery Ultra or the LeicaBond RX auto stainers or alternatively, can be stained manually.

RarePlex Staining Kits

This slide shows a list of the staining kits available from RareCyte. The top five are the panel kits and the bottom line are the developer kits. Enumeration and developer kits can be run on the Leica or Ventana auto stainers.

Patient samples stained with an enumeration CTC Panel Kit

Here are representative images of breast and prostate cancer patient samples stained with the 0900 CTC panel kit which allows for two extra biomarker channels. For each CTC, you can see proper saying of CK/EPCAM in magenta, a nucleus in blue and a lack of CD45 staining in green. All CTC images are automatically found by a computer algorithm and are shown to the reviewer for CTC identification.

Enumeration CTC Panel Kit performance

To characterize the performance of the CTC panel kit, we determine sensitivity specificity and accuracy by spiking in very low numbers of CTCs into seven and a half mls of blood on nine individual blood samples from healthy donors alongside 10 blood samples from 10 different healthy donors without any CTC spiked in. A result of more than one CTC per seven and a half mls was considered positive. Overall, the kit was able to detect 96% spiked in cells over the nine limit of detection samples. No false positives were found in the ten limit of the blank samples for an overall sample accuracy of a hundred percent.

Enumeration CTC Panel Kit performance on clinical samples

One of our collaborators had our CTC panel kit CLIA certified in their lab and compared results to the FDA cleared CellSearch test in a blinded fashion on 50 pair draws from prostate and breast cancer patients. The comparison showed a high correlation between both assays with our assay finding on average a third more CTCs than the CellSearch assay. Note that the graph is linear from 0 to 10 and then logarithmic base 10 from 10 to 1000 in both axes.

RarePlex CTC Panel Kits

As I mentioned earlier, CTC panel kits RarePlex CTC Panel Kits can be combined with a developer kit to stain one or two additional user selected biomarkers with minimal staining optimization. Our platform has a total of five channels available so depending on the panel being used, one or two channels are available to integrate new markers using mouse and rabbit antibodies. With our developer kits, we provide guidelines for the user to optimize staining of their biomarkers of interest using the same procedures we use when optimizing our CTC panel kits.

RarePlex Developer Kit examples

On this slide, we see three custom assays with two biomarkers each that were stained using the developer kits. For prostate cancer, we tested the Androgen Receptor, Androgen Receptor variant 7, and prostate specific membrane antigen. For breast cancer, we tested HER2 and progesterone receptor. A bright white signal on a black background indicate a high signal-to-noise ratio that allows for sensitive and specific biomarker characterization on CTCs.This signal can also be quantified to allow for comparison between different cells and different patients.Because our platform does not depend on size for CTC detection, it is able to find CTCs that are the same size or even smaller than white blood cells like the prostate cancer cell in the first row. Also, the platform only requires one epithelial marker to detect CTCs either CK or EPCAM and not both, which allows us to find CTCs that are undergoing EMT and have lost expression of one of these two markers.

Developer Kit MFI distributions for EGFR control cell lines

When we develop new assays using the developer kit, we require a positive and a negative control cell line for the biomarker of interest. We want to achieve a good separation between both populations with regards to the signal intensity in order to attain high accuracy at determining biomarker positivity in clinical samples. In this slide, I am showing EGFR staining on the positive and negative cell lines based on data reported in the literature. Although there is a range in EGFR expression for each cell line, an intensity cut off can be established that perfectly separates both populations from each other.

Biomarkers tested with the Developer Kit

In my last slide, I wanted to show a non-comprehensive list of biomarkers that have been tested with the developer kit in different disease indications. We have also used them to optimize assays to detect the presence of certain biomarkers as drug targets which could be used as predictive markers. I hope this presentation has allowed you to better understand the suite of products that are available from RareCytefor CTC enumeration and phenotypic characterization. If you have any questions, I urge you to contact us at info@rarecyte.com and we would be happy to work with you to build an assay that addresses your particular needs. Thank you for your attention.