IMPORTANCE OF MOLECULAR PROFILING BREAST CANCER

Breast cancer shows marked clinical diversity and extreme variability in prognosis and response to currently employed therapeutic modalities.  The existing histological classification systems are far from accurate in predicting disease course or selecting the appropriate treatment for a given cancer patient.  This can be attributed to molecular differences that exist within histologically identical subtypes.   Histologic features, when combined with regulatory protein markers (ER, PgR, Her-2, Ki67, EGFR and CK5/6), have provided a classification of 6 different primary cancer subtypes which differ markedly in prognostic and treatment outcomes.  Much more needs to be done in this area of research, molecular profiling, to provide a “wiring diagram” of proteins and pathways amenable to precise chemical intervention.  This approach promises to provide the right medicine to the right patient with the lowest toxicity.

VALUE OF THE AUTOMATED GRACE ANALYTICAL PLATFORM FOR MOLECULAR PROFILING

Ultimately what is needed is a set of molecular tests which are companion to anatomic pathology and immunocytochemistry.  This set of tests, made preferentially at the time of first detection, will be to classify the disease in terms of aggressiveness and sites for chemical intervention.  The tests can be of the tumor, sensitive enough to be conducted on a small fraction of an FNA, or in bodily fluids, where numerous markers are known to circulate for a number of chronic diseases.  Blood is a rich source of chronic disease markers, and methods are constantly developing to facilitate antibody-based assays directly on proteins in whole blood.

The ELISA method offers a sensitive and specific means to detect a wide variety of protein analytes in complex biological materials like tissue and blood.   The efficiency of ELISA assay is greatly enhanced by multiplexing (detecting more than one analyte in one pass), but multiplexing is largely incongruous with ELISA’s standard plate technology, even with fluorescent end-points.  The evolution of microarray technology has overcome this limitation and created immense opportunities to improve the effectiveness of ELISA assays by combining multiplexing with microarrays.

Microarrays are well suited to companion diagnostics in the sense that we can look at complex wiring diagrams in one assay on a small tissue or blood volume – hundreds of antibodies can be employed on a small fraction of a patient sample, FNA for example.    There are generally two relevant formats, one is an ELISA-based capture format; the other is “reverse phase protein arrays” (RPPA).  RPPA is an excellent tool for bio-marker discovery, and at present, this technology is used primarily to accelerate target marker identification.

Grace has developed a protein binding surface that is excellent for both ELISA and RPPA.   Porous nitrocellulose (PNC) binds 500X more protein than 2D planar surfaces.   Binding on PNC occurs non-covalently, preserving both antibody and antigen activity.   The increased binding of capture antibody results in a nearly one-hundred fold increase in assay sensitivity as compared to other surfaces.  To overcome the endogenous fluorescent background of PNC in the visible spectrum (which has limited its usefulness for multiplexing), we have developed a nitrocellulose-based thin Film with no background in the near infrared (NIR) wavelengths.   Furthermore, the porosity of this Film has been selected to create a unique fluorescent signal amplification that we call “resonance signal amplification” or RSA.

While a limited number of digitizing scanners for NIR detection are commercially available, they are prohibitively expensive, extremely slow to acquire data and not widely used.    Grace has developed a rapid and affordable imaging technology called ArrayCAM for use with RPPA and ELISA based microarrays.   ArrayCAM capitalizes on the high binding capacity of PNC and the brightness and stability of quantum nanocrystal-labeled detection to increase the linear dynamic range, and lower the limit of detection (LOD) of assays at a fraction of the time and cost compared to other platforms.

The ArrayCAM technology is extremely flexible and configurable, and provides bench top imagers using both colorimetric and fluorescent detection in a single instrument.   Interchangeable filters provide an array of options for multiplex analysis on a single spot allowing users to incorporate more sensitive and relevant standardization within each sample.   The ArrayCAM family of instruments includes a small, portable, single slide imager suitable as a point of care reader, a versatile 4-slide / MTP imager for higher throughput applications, and as an on-board, integrated component of a robotic liquid handling assay system for maximum throughput.   Quality controlled reagent kits, and user friendly operation will make the ArrayCAM platform the method of choice for the standardized testing of clinically relevant biomarkers.