Multi-Parametric Analysis of Breast Tissue Utilizing Available Cytometric Systems

Amanda Chargin1,2, Keith Shults2, Lily Chen1, Linda Chen1, Bruce Patterson MD2 1Department of Biology, San Francisco State University, San Francisco, CA

2IncellDx, Menlo Park, CA

ABSTRACT

According to the American Society of Clinical Oncology and the College of American Pathologists, approximately 20% of current HER2/PR/ER breast testing by immunohistochemistry or in situ hybridization may be inaccurate. This inaccuracy in current testing methods for HER2/PR/ER markers in breast cancer highlights the need for an improved method of testing. Currently, there is an absence of tests on the market that have the ability to combine detection of mRNA, protein, DNA content and cell cycle analysis in one assay on intact cells. IncellDx’s proprietary technology has the ability to provide an all-encompassing view of a cancer cell by quantifying multiple biomarkers on a cytometry platform that enables high throughput testing of fine needle aspirates (FNA).

INTRODUCTION • The feasibility of this assay was tested using two breast cancer cell

lines for ability to measure differences in protein and mRNA expression as well as DNA content differences.

• MCF-7 cells express E-cadherin and ER and have a modal chromosome number of 80. SK-BR-3 cells express HER2 and have a modal chromosome number of 84.

• Mixtures of these two cell lines were permeabilized, hybridized with probes to her2 mRNA, and stained with antibodies to E-cadherin, ER and an A-T specific DNA dye.

• Each biomarker is labeled by a fluorophore with a differing emission which makes it possible to differentiate each analyte on multiple cytometric systems.

STUDY AIM & OBJECTIVE

To develop a diagnostic test that combines simultaneous genomic, proteomic, and cell cycle analysis of breast tissue samples utilizing 9 independent variables (ER+/-, PR+/-, her2+/-, E-cadherin expression, DNA proliferation, and DNA content) in a single assay. To determine the clinical performance of this assay, standard pathological parameters and diagnosis will be compared to the cytometric readouts of the derived assay on excised breast tumors.

SFSU Student Demographics in the Stem Cell Program

ACKNOWLEDGEMENT

This internship project is supported by IncellDx, National Science Foundation Science Master’s Program Award (DGE-1011717), Susan G Komen for the Cure®, and Department of Biology at San Francisco State University.

CONCLUSION

• Multiple mixing studies of MCF7, SK-BR-3, and normal human mammary epithelial cells (HMEC) have demonstrated the ability to simultaneously detect multiple protein targets and complete cell analysis on a cell by cell basis.

• Comparisons of oligonucleotide cocktails using HMEC and frozen breast tumor pellets of known her2 immunohistochemical status demonstrate the ability to simultaneously detect differing protein and mRNA levels.

• Combinations of the principles described in this poster produced a proteomic, genomic, morphologic and proliferative readout on multiple cytometric systems.

FUTURE DIRECTION Using the established technology described in this poster, IncellDx has contracted to obtain IRB approved FNA samples excised from 40 breast tumors and 10 cases of normal tissue excised during breast reduction procedures. The study will compare the multi-parametric readout to standard pathological metrics in an effort to derive clinical performance data. This exciting platform promises to be adaptable to standard diagnostic procedures as well as a companion diagnostic for her2 and selective estrogen blockers. Since the core of the assay is single cell analysis, adaptation of circulating tumor cells and xenograft models is feasible.

RESULTS

METHODOLOGY • Proteomic analysis of Estrogen and Progesterone nuclear

hormone receptors as well as E-cadherin surface receptor with antibodies to characterize receptor expression of cell lines.

• Determine optimal DAPI concentration on cell lines for DNA content analysis.

• Analysis of her2 mRNA expression of cell lines by designing and utilizing a fluorescently labeled oligonucleotide cocktail.

• Determine optimal variable mixture on cell lines in combination. • Test optimal variable mixture on characterized clinical samples.

RESULTS The ability to measure both her2 mRNA and protein

simultaneously was confirmed on human tissues on a Sony i-Cyt flow cytometer:

Figure 2: HER2+, ER/PR+ Invasive and Infiltrating Ductal Breast Cancer Tissue

Figure 3: HER2-, ER/PR- Invasive and Infiltrating Ductal Breast Cancer Tissue

Tissue Pathology her2 mRNA MFI HER2 protein MFI HER2+, ER/PR+ 753.16 5717.58 Normal Human Epithelial Mammary Cells 555.55 283.67 HER2-, ER/PR- 119.82 32.17

Figure 1: Output from a sample of MCF-7 cells (depicted in red) and SK-BR-3 cells (depicted in blue) with ER, HER2, E-cadherin antibody staining and DAPI A-T specific

DNA dye combined in suspension.

Results 1: The 3D graph shown above is a 1:1 mixture of MCF-7 and SK-BR-3 cells. This graph has been further broken down to show each metric individually. Both populations are easily resolved in all four graphs and differences in expression levels are characteristic of each cell type.

Figure 1: ER, HER2, E-cadherin, and DNA quantification in combination on mixtures of MCF-7 and SK-BR-3 cells

collected on the Amnis FlowSight Result 2: Fluorescence intensity her2 mRNA vs. protein

E-cadherin Alexa 488

DAPI

HER2 PE

ER Alexa 647