Cell response assay for cancer and methods of producing and using same

A method of monitoring cancer treatment in a cancer patient undergoing said treatment, wherein the treatment is monitored using a multiplexed direct affinity assay, and wherein the method comprises the steps of:

• (A) isolating cells from a first biological sample taken from the cancer patient at a first time point;
• (B) isolating cells from a second biological sample taken from the cancer patient at a second time point, wherein the cancer patient has been exposed to a cancer treatment between the first and second time points;
• (C) reacting the isolated cells from the first biological sample with at least three affinity reagents for at least three biomarkers, wherein the isolated cells are reacted with the at least three affinity reagents in a single reaction, wherein each affinity reagent comprises a different fluorescent label whereby the different fluorescent labels are measured at different excitations and emissions wavelengths, and wherein the at least three biomarkers comprise:
• (i) a cancer cell type biomarker;
• (ii) a metastatic potential biomarker, wherein the metastatic potential biomarker is urokinase plasminogen activator (uPA) and/or plasminogen activator inhibitor (PAI-1);
• and
• (iii) a chemo resistance biomarker, wherein the chemo resistance biomarker is PL2L piwi like;
• wherein biomarkers (i), (ii), and (iii) all co-exist in a cancer disease state, are fundamental to progression or relapse of the cancer disease state, and are all biochemically linked in a biochemical process;
• and
• wherein the presence of markers (ii) and (iii) are linked such that the presence of one facilitates absence of the other;
• (D) reacting the isolated cells from the second biological sample with the at least three affinity reagents of (C), wherein the isolated cells are reacted with the at least three affinity reagents in a single reaction;
• (E) simultaneously measuring, in a multiplexed assay, expression levels of the at least three biomarkers in the isolated cells from the first biological sample;
• (F) simultaneously measuring, in a multiplexed assay, the expression levels of the at least three biomarkers in the isolated cells from the second biological sample;
• (G) calculating a proportion of cells detected by the cancer cell type biomarker that are also detected by the metastatic potential biomarker in the first and second biological samples;
• (H) calculating a proportion of cells detected by the cancer cell type biomarker that are also detected by the chemo resistance biomarker in the first and second biological samples;
• and
• (I) administering a subsequent dose of the cancer treatment if:
• (1) the metastatic potential biomarker is present in a greater proportion of cancer cells detected by the cancer cell type biomarker in the second biological sample when compared to the first biological sample;
• and
• (2) the chemo resistance biomarker is present in a lesser proportion of cancer cells detected by the cancer cell type biomarker in the second biological sample when compared to the first biological sample.