Protein quantitation with cell imaging densitometry

Details Protein quantitation with cell imaging densitometry Protein quantitation with cell imaging densitometry

2001-04-24

2004-06-08

Tate, Christopher R. (Department: 1642)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C435S007210, C435S960000, C435S967000, C436S063000, C436S064000, C436S518000

Reexamination Certificate

active

06746848

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to the fields of microscopy, computerized cell imaging, immunohistochemistry, histopathology, oncology, protein quantitation, and diagnosis and prognosis of cancer and other diseases.
BACKGROUND OF THE INVENTION
1. Immunohistology
The presently universally-accepted method for the diagnosis of all types of solid cancer is the histologic determination of abnormal cellular morphology in surgically biopsied or resected tissue. Once removed, the tissue is preserved in a fixative, embedded in paraffin wax, cut into 5 &mgr;m-thick sections, and stained with two dyes: hematoxylin for the nucleus and eosin for the cytoplasm (“H&E staining”).[1, 2] This approach is simple, fast, reliable, and inexpensive.
Histopathology allows the diagnosis of a variety of tissue and cell types. By providing an estimation of tumor “Grade” (cellular differentiation/tissue architecture) and “Stage” (depth of organ penetration) it also makes prognosis possible.[3, 4] In the Surgical Pathology Departments of larger hospitals histologic H&E staining is generally automated, tissue-processing technique is standardized, and histologic interpretation is well established.
Aside from crude measurements of the tumor diameter, pathologists do not attempt to quantify the area or volume of dysplastic tissue, nor do they perform absolute quantification of the cancer-related proteins present in such specimens. When antibody staining is attempted—commonly called immunohistochemistry (IHC)—the intensity and area of its visible or fluorescent color is ranked in an ordinal fashion. This ordinal ranking by the pathologist is accomplished by to the subjective impression of both the extent (area) and the darkness of the stain, compared to adjacent, morphologically normal tissue. The number of ranked categories and the cutoff points for each is arbitrary and inconsistent among observers. Also, for some organs and cancer proteins, there has been observed a “field-effect” in which abnormal proteins are expressed in adjacent, supposedly negatively-stained, morphologically normal tissue.[5] Furthermore, since there is no matching of cells between the H&E histology slide and the immunostained slide, it is difficult to segregate the immuno-scoring for different histologic classes, e.g., cancer and pre-cancer, within the same section and it is impossible to accurately correlate total immunostaining with histologic area for each tissue class.
The currently available optical techniques of microscope-based cell imaging provide a partial solution to the problem of performing these cellular measurements. This approach uses conventional light microscopy combined with monochromatic light filters and computer software programs. The wavelengths of the light filters are matched to the colors of the antibody stain and the cell counterstain. The filters allow the microscopist to identify, classify and then measure differences in the optical density of specific colors of light transmitted through immunostained portions of tissue sections. See U.S. Pat. Nos. 5,235,522 and 5,252,487, both of which are incorporated herein by reference, for applications of these methods to tumor protein measurement.
More advanced cell imaging systems (image cytometers) permit automated recognition of features, and combine this with automated calculation of feature areas, automated calibration, and automatic calculation of average and integrated (&Sgr;OD) optical density. (See, e.g., U.S. Pat. Nos. 5,548,661, 5,787,189, both of which are incorporated herein by reference, and references therein.) Merely scoring patient tissue immunostaining by ordinal rank, however, even by incorporating the more objective and uniform optical estimation techniques provided by Cell Imaging Densitometry (CID), provides limited information for patient and tissue evaluation. By translating such scoring into common biological units of measurement, oncologists and pathologists can refer to the patient's particular “profile” of tumor suppressor and oncogene protein levels. Thus, the clinician will be able to numerically predict a patient's “relative risk” of relapse or death, probability of chromosomal instability, metastases, response to therapy, or even probable survival duration. The suggested method should also make it possible to sum up a patient's total “body burden” of such proteins, where there are multiple lesions. A fraction of this tissue burden escapes the porous membrane of the cancer cells into the blood stream perfusing the tumor(s), achieving a steady-state concentration over time. Immunoassays e.g., ELISA, can accurately and sensitively measure these volumetric concentrations. Knowing the typical quantitative correlation between blood levels and tissue expression will allow us to more effectively (and less invasively) indirectly monitor residual/recurrent disease.
One reported attempt to improve the accuracy of the measurement of cancer protein in tissue used Western Blotting in combination with CID to create immunohistochemical rankings when measuring HER-2/c-erb&bgr;-2 oncogene protein expression in breast cancer patients.[6] In this attempt, cultured human breast cancer cells were genetically-engineered in order to express different levels of the oncogene protein. HER-2 protein levels (pg/cell) in the cell lysates of these reference cells were estimated with dilutions of a fragment of recombinant purified HER-2, using the Western blot assay and laser densitometry. Cultured cell pellets were snap-frozen in “OCT” (polyethylene glycol-polyvinyl alchohol-trimethylbenzylammonium chloride) embedding media, cut into 4 &mgr;m sections with a cryostat, and then attached to microscope slides, presumably by air drying. Breast cancer tissue was fixed in 95% ethanol, followed by buffered formalin. Alternatively, tissue from the same tumors was either frozen in OCT and cryosectioned, or paraffin-embedded and sectioned with a microtome. A CID/Western blot “standard curve” on the cultured cells was created with a single immunostained CID standard, which was assumed (without testing or reference) to be 1 pg/cell. This “curve” from the frozen reference cells was then applied to the immunostained breast tissue by using a single “correction factor” (~40%) in order to boost the actual optical density scores for the paraffin tissue sections. In the eventual correlation of tumor recurrence with HER-2 overexpression these “quantitative” immunostaining scores were, once again, reduced to ordinal ranks, “Low”, “Medium”, and “High”, which reflected increasing degrees of amplification of the gene's DNA. The authors were able to predict relative differences among the women in their risk of tumor recurrence.
However, fixation conditions of the reference cells and the tissue were different, there were no immunostained paraffin sections for the reference cells, and the frozen tissue stained more intensely than the paraffinized tissue (disproportionately so, depending upon the level of HER-2 protein overexpression). This approach provides no method to summarize the total HER-2 tumor burden per patient or tumor. The reliance upon Western blot for quantitation of the oncoprotein cell is a disadvantage, due to the complexity and slowness of the procedure, plus its modest quantitative accuracy, precision and reproducibility.[7-9] Another approach employs simultaneous measurement of nuclear DNA by cell imaging to provide an internal calibration reference (U.S. Pat. No. 5,252,487). This method is subject to variations in the intensity of the DNA staining and derives its calibration “curves” for staining intensity×pg DNA/cell from a single DNA value.
Another attempted to solve the problem was the Quicgel™ method, described in U.S. Pat. No. 5,610,022, which used immobilized cultured cells as “internal controls” in order to estimate the “pre-processing immunoreactivity level” of individual paraffin tissue sections.[124] The stated goal was to compensate for unpredictable and/or excessi

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