Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-07-29
2003-07-08
Caputa, Anthony C. (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C435S007210, C435S007220, C435S007230, C435S007700, C435S007710, C435S019000, C435S021000, C435S040500, C435S040510, C435S040520, C436S063000, C436S064000, C536S023500
Reexamination Certificate
active
06589753
ABSTRACT:
STATEMENT OF GOVERNMENTAL INTEREST
None
BACKGROUND OF THE INVENTION
This invention relates generally to medical diagnostics and, more specifically, to a method of diagnosing and staging malignant tumors using an improved method of immunohistochemistry to determine the concentration and distribution of a cytoplasmic tumor marker associated with malignant tumors.
The inventor has previously identified, by cDNA cloning, a gene denoted Metallopanstimulin (MPS-1). The novel DNA sequence which encodes the MPS-1 is disclosed in U.S. Pat. No. 5,243,041 (Re. 35,585), issued Sep. 7, 1993, the disclosure of which is hereby incorporated by reference. The recombinant MPS-1 protein and chemical derivatives can be used to generate polyclonal and monoclonal anti-MPS-1 antibodies. The MPS-1 gene is expressed at high levels in numerous human carcinoma cell lines such as prostate, breast, brain, lung, and particularly melanomas. It is also expressed in human hematological malignancies. MPS-1 is a multifunctional S27 ribosomal protein that is involved in cellular proliferation and oncogenesis in numerous human neoplasms. The MPS-1 gene is expressed at high levels in numerous human carcinoma cell lines such as breast, prostate, colon, brain, lung and melanoma. Table IA indicates the presence of MPS-1 mRNA in cultured human malignant cell lines and in peripheral blood of human patients with hematological malignancies. Cytoplasmic MPS-1 is of particular interest because of the proliferation of cytoplasmic mRNA in proliferative diseases, including cancers. Table 1B indicates the presence of MPS-1 mRNA in human hematological malignancies.
TABLE 1A
PRESENCE OF MPS-1 mRNA IN
CULTURED HUMAN MALIGNANT CELL LINES
Cell type
Cell line
Breast carcinoma
MDA-MB-468, MDA-MB-231, BT-20
Prostrate carcinoma
DU-145, PC-3
Melanoma
SK-MEL-28, RPMI-7951
Colon adenocarcinoma
LoVo
Lung carcinoma
A-549
Vulvar carcinoma
A-431
Fibrosarcoma
HT-1080
Neuroblastoma
LAN-5
Squamous cell carcinoma of skin
SCC-15
TABLE 1B
PRESENCE OF MPS-1 mRNA IN
HUMAN HEMATOLOGICAL MALIGNANCIES
MPS-1 mRNA
Type of Malignancy
Negative
Positive
Presence of Malignant cells in
peripheral blood
Chronic lymphocytic leukemia
0
3
Chronic myelogenous leukemia
0
2
Multiple Myeloma
0
3
Lymphoma
0
3
Table 2 illustrates the results of tests conducted to detect MPS-1 mRNA and protein in human tissues.
TABLE 2
DETECTION OF MPS-1 mRNA AND PROTEIN IN HUMAN TISSUES
No. of samples
Type of Tissue
mRNA
1
/Protein
2
Organ of Origin
A. CARCINOMAS AND
SARCOMAS
Carcinoma
52+++/+++
Vulva, Cervix, Ovary
Endometrium, Colon,
Lung, Bladder, Liver
Metastatic
Squamous Cell
35+++/+++
Vulva, Cervix
Carcinoma
Esophagus, Lung,
Adenocarcinoma
25++/++
Cervix, Ovary,
Endometrium,
Colon, Prostrate
Carcinoma In Situ
9++/++
Vulva
Serous Carcinoma
5+/+
Ovary
Papillary Serous Carcinoma
5++/++
Endometrium
Sarcoma
4++/++
Ovary, Endometrium,
metastasis
Melanoma
2>+++/+++
Vulva, Metastasis
Verrucus Carcinoma
1++/++
Vulva
Retroperitoneal
1+++/+++
Retroperitoneal
Liposarcoma
Mucinous Carcinoma
1+/+
Ovary
Leiomiosarcoma
1++/++
Ovary
Papillary Carcinoma
1++/++
Ovary
Papillary Adenocarcinoma
1++/++
Endometrium
Adenosquamous Carcinoma
1++/++
Endometrium
Clear Cell Carcinoma
1++/++
Endometrium
Mixed Mesodermal
1+/+
Endometrium
Carcinoma
Mixed Mullerian Tumor
1+/+
Endometrium
Ductal Carcinoma
3+++/+++
Breast
Inflammatory Carcinoma
1>+++/+++
Breast
B. BENIGN LESIONS
Lichen Sclerosus atrophicus
1−/−
Vulvar Skin
Benign Cysts
10ND/−
Ovary, Breast
Granuloma
1ND/+
Lung
Leiomyoma
2ND/−
Uterus
Fibroma
1ND/−
Ovary
C. NORMAL TISSUES
3
63−/−
Cervix, Ovary,
Endometrium, Myo-
metrium, Vagina,
Peritoneum, Fallopian
Tubes, Breast, Rectal
Muscles, Skin, Small
Intestine,
Lymph Node
Table 2 should be read as follows:
Signals: (−) negative, (+) weakly positive, (++) positive, and (+++) strongly positive. The stainings recorded refer to that of the cancer cells, since the stroma cells were not significantly stained. Note that although normal tissues are listed as (−), they showed staining (+ to +++) only in areas of normal cell proliferation. ND: Not done. Note that (i) vulvar melanoma, and breast inflammatory carcinoma had the highest levels of MPS-1 mRNA and protein detected; by Northern blot analysis the MPS-1 mRNA levels in these tissues were >80-fold normal levels; and (ii) lichen sclerosus atrophicus, a rare condition characterized by extremely low proliferation rates was negative for MPS-1 mRNA and protein in the usual areas of cell multiplication; these observations are highlighted by bold characters.
The inventor has determined that there is a high correlation between a patient's survival rate and the level of cytoplasmic MPS-1 observed. That is, the patient's survival rate decreases in proportion to increased concentration distribution of cytoplasmic MPS-1 detected. Hence, the method of the present invention is useful as a prognostic indicator of disease progress and survival.
The MPS-1 mRNA was detected using biotynilated single-stranded anti-sense DNA probe. The MPS-1 protein was detected by immunohistochemical staining using anti-peptide A antibodies. As can be observed, there is an excellent correlation between the concentration and distribution of MPS-1 mRNA and protein expression. In contrast, the MPS-1 gene is expressed at low levels in normal cells. The results of experiments indicated that the MPS-1 antigen is a ubiquitous tumor marker which is useful in detection, prognosis and management of various types of neoplastic conditions, particularly when the concentration and distribution of the protein is detected in the cytoplasm, as recently discovered.
Although the detection and management of all forms of cancer is desirable, the detection of malignant melanoma is particularly challenging to the clinician. Often benign lesions are difficult to distinguish from malignant lesions. It is imperative, however, that cancers such as malignant melanoma, breast cancer and prostate be detected early and reliably to improve survival rates.
Recently, immunohistochemical studies were conducted to examine the expression of MPS-1 protein in various types of benign and malignant melanocytic lesions. Protein antigen, detected with anti MPS-1 antibodies was found in both benign and malignant melanocytic lesions. The anti-MPS-1 antibodies directed to the N-terminal portion of the molecule are most useful. In benign lesions, the staining was weak and in a gradient, the most superficial cells with nesting growth patterns were positive, particularly those within the epidermis. The stain intensity decreased as the melanocytes were located deeper in the dermis. Practically speaking, only type A melanocytes stain positive while the B and C types are negative.
Recurrent melanocytic nevi were also studied. MPS-1 was nearly negative in the original untreated nevi. In the recurrent lesions, the regenerating epidermal and dermal melanocytic components were intensely and evenly stained. These findings were very similar to those seen in melanomas. These changes are an example of intense activation of the newly formed melanocyte population and not a sign of malignant transformation.
It is of interest to note that scar tissue generates large amounts of growth factors. Thus, growth factors may be responsible for both activation of melanocytic cells and intense expression of MPS-1 observed in the incomplete biopsy. It will be appreciated that the histological features of these recurrent nevi were indistinguishable from melanomas, a phenomenon that often confounds the diagnostician. The correct d
Caputa Anthony C.
Holleran Anne L.
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