7^thAnnual NIH SBIR/STTR Conference

Drug Response Indicators in Cancer Cells Provide the Basis of Personalized Anticancer Chemotherapy

Personalized anticancer chemotherapy based on measurement
of drug response indicators (DRI) expressed by cancer cells. Ts’o, P.O.P.1,
Lesko, S.A.1, Lum, Z.P.1 , Deamond, S.F.1, Shan, E.1, Zhou, D.B.1, Daniel,
J.R.1. 1 CCC Diagnostics, LLC, Baltimore, MD.

Targeted therapy in cancer patients coupled with the diagnostic
tools to measure the target status in individual patients form the basis
of personalized anticancer chemotherapy (PAC). The targeted therapies for
cancer are designed against specific cellular components (receptors, enzymes)
or processes (repair, mitotic division). The targets, termed drug response
indicators (DRI) can now be evaluated by immunochemical staining since
fluorescent dye labeled monoclonal antibodies (MAB) are available to stain
these cellular targets. Quantitative numbers can be derived from fluorescence
microscopy measurements and normalized to a reference standard for inter-laboratory
comparison. For interpretation of the fluorescence data, particularly for
the establishment of a cut off level distinguishing the responsive tumor
cells from the resistant tumor cells, we utilized a battery of cancer cell
lines with different sensitivities to various anticancer drugs. The inhibition
of in-vitro cell growth by each drug was correlated to expression of the
related DRI. This report describes an in-vitro experimental system for
generating drug response data from breast cancer tumor cells, and correlating
it with the level of expression of respective DRI.

Methods: Linear standard curves of fluorescence intensity
vs. exposure time were generated from digital images for each microscope
filter cube utilizing 6 um fluorescent microspheres. Human breast cancer
cell lines, fixed on microscope slides, were incubated simultaneously with
3 different MAB (Herceptin, anti-estrogen receptor and anti-beta tubulin
III) labeled with different fluorescent dyes, plus anti-pancytokeratin
(CK) for epithelial cell visualization. Digital images of the same field
of cells were acquired for each of the 4 fluorescent markers using exposure
times that produce a fluorescence of 2000 with standard microspheres as
reference. The spatial area of the cells, outlined using CK fluorescence,
was overlaid on an identical field of cells in a DRI image in order to
quantify DRI expression. The effect of DRI related drugs (Tamoxifen, Paclitaxel,
Herceptin) on the proliferation of human cancer cell lines in culture was
determined spectrophotometrically by measuring the bioreduction of a tetrazolium
salt.

Results: The cytotoxic response of various breast cancer
cell lines to Herceptin correlated inversely with the level of HER-2/neu
expression (Herceptin binding), evidenced by a Spearman rank coefficient
of (-1). There was an inverse correlation between the cytotoxic response
of breast cancer cell lines treated with Tamoxifen and level of estrogen
receptor expression (specific MAB), shown by a Spearman rank correlation
coefficient of (-.995). The cytotoxic response of various breast cancer
cells to paclitaxel corresponds with expression of beta-tubulin III. (Spearman
rank correlation coefficient of +.995). Cell lines displaying a lower level
of binding beta-tubulin III were sensitive to treatment with paclitaxel.
In contrast, cell lines displaying a higher level of binding beta-tubulin
III displayed a slight response to higher doses of paclitaxel. Measurement
of DRI in the circulating cancer cells in breast cancer patients is currently
in progress.

Paul OP Ts'o
CCC Diagnostics LLC
Caton Research Center
3918 Vero Rd
Suite B
Baltimore, MD 21227

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