Chemistry: analytical and immunological testing – Biological cellular material tested
Reexamination Certificate
2001-05-09
2004-01-13
Witz, Jean C. (Department: 1651)
Chemistry: analytical and immunological testing
Biological cellular material tested
Reexamination Certificate
active
06677157
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to structures involved in the secretory processes of reproductive tissues, including the prostatic secretory processes, and their protein products which may be used as tools for diagnosing reproductive pathology including prostate disease. The present invention also relates to reagents, such as antibodies, other ligands and oligonucleotides, for detecting these structures or their contents and to methods of diagnosing prostate pathology, including prostate cancer and prostatitis. The invention further relates to an improved tissue and cell fixation process for the detection of secretory structures in reproductive tissue. The fixation process is useful for the diagnosis of prostate, testes and renal cancer.
BACKGROUND OF THE INVENTION
The secretory (luminal) cells of normal prostatic glands are separated from the basement membrane by a layer of inconspicuous basal cells (1). This surface layer is characterised by its tall columnar cells with basally orientated nuclei, whose abundant apical cytoplasm synthesises a broad range of secretory proteases including prostate specific antigen (PSA) and prostatic acid phosphatase (PAP). Characteristically, the cytoplasm of surface secreting cells is optically clear to faintly eosinophilic which distinguishes it dramatically from the amphiphilic (dark) cytoplasmic staining of most dysplastic or malignant prostatic epithelial cells (2,3). Optimal tissue staining for diagnostic purposes yields maximal cytoplasmic clarity in benign prostatic secretory cells (2), the abrupt contrast between the cytoplasmic density of cancer and the pallor of the adjacent normal epithelium in well stained sections is often the most striking histologic feature which delineates the boundaries of a carcinoma focus. Conversely, a frequent problem in needle core diagnosis is the “clear cell” glandular atypical focus, in which confirmation of carcinoma is more difficult due to the absence of dark cytoplasm.
The significance of cytoplasmic density in adenocarcinoma is not understood. In the Gleason grading system (4) retention of clear cytoplasm implies a high level of differentiation since it is a requisite feature of all Grade 1 and Grade 2 carcinomas. Furthermore, cytoplasmic clarity is also characteristic of most adenocarcinomas which arise from the transition zone in association with nodular hyperplasia (5,6). Although dark cytoplasm is well described in dysplasia (PIN) and Gleason grade 3 carcinoma, it is not specific for malignant loss of differentiation since it is common in the cells of benign post inflammatory atrophy (7).
The organelles responsible for the appearance of the normal prostatic secretory cell cytoplasm have been described as myriad tiny vesicles which nearly fill the cytoplasm and appear completely devoid of content (1,8). These vesicles or granules are only faintly and variably recognisable by routine light microscopy, depending on the staining intensity of the faintly eosinophilic narrow septa which separate them.
Prostatic corpora amylacea (CA) are extracellular intraluminal structures seen in most adult prostate (9). The protein source of the CA is poorly understood although within these structures a group of sulphur-rich proteins have been previously detected (10). Similar sulphated proteins have also been detected in crystals associated with well-differentiated carcinoma, so-called prostatic crystalloids (11). Further, amyloid possibly related to &bgr;-2 microglobulin has been recognized in CA (12) but, despite these basic observations the origins of these extracellular prostatic structures is yet to be determined.
Round proteinaceous deposits have also been identified in prostatic luminae (9). These deposits (3-8 &mgr;m in diameter) are rare (8 cases of 166 specimens) and when noted are in close association with the luminal surface of the benign secretory cells confined to the central region of prostate in routinely fixed and processed tissues. The nature of this protein deposit is unknown but was noted to be negative for numerous protein fractions including, prostate specific antigen (PSA), prostatic acid phosphatase (PAP), light chain immunoglobulin (&kgr; and &lgr;), &agr;
1
-antitrypsin and &agr;-fetoprotein (9). Stains for mucin and silver stains were also negative but unexpectedly positive for phloxine tartrazine.
Beyond these initial observations, the detailed structure underlying the “clear cell” cytoplasmic appearance of prostatic epithelium as well as the relationships between this appearance and the process of prostatic exocrine secretion have never been systematically studied. Further, common and apparently fundamental alterations of structure and secretory function which must underly the abrupt transition to the dark cytoplasm of most cancers are unknown.
The mammalian oviduct provides an environment that supports the gametes, the process of fertilisation, early embryonic development and the delivery of a viable embryo to the uterus. The lumen of the mammalian oviduct is formed by a complex interdigitating system of longitudinal mucosal folds. These longitudinal mucosal folds are lined by a simple columnar epithelium and the morphological and biochemical characteristics of this epithelium are controlled by ovarian steroids. At the time of ovulation this lining epithelium consists of fully differentiated columnar ciliated and secretory cells. Approximately 40-50% of the epithelial cells are secretory. Secretory glands are observed in the apical regions of the cells. Several secretory products have been identified which enhance sperm motility, viability, binding to zona pellucida and enhance the rate of zona pellucida penetration. (Verhage et al 1997 Characteristics of an oviductal glycoprotein and its potential role in fertility control. J. Reproduction and Fertility Supplement 51:217-226).
SUMMARY OF THE INVENTION
The present inventors have now developed improved techniques for visualising prostatic cytoplasmic structure and the mechanism of cell secretion. These improved techniques have led to the surprising finding that secretory granules which are found in the luminal cells of normal prostate glands are absent in prostate carcinomas. These findings indicate that the prostate secretory granules may provide an important tool in the diagnosis of prostate cancers.
The present inventors have also identified a link between PSG, decapitated cytoplasmic body (DCB), eosinophilic bodies (EB) and corpora amylacea (CA) structures. Briefly, the normal secretory cell cytoplasm is filled with brightly eosinophilic PSG measuring about 1 &mgr;m in diameter and densely concentrated in the apical third of the cell. This apical compartment represents an apocrine secretory bleb. Periodic detachment of blebs carries packages of secretory granules (PSG) into the lumen where the receptacles fragment liberating their contents. The luminal cytoplasmic compartment (bleb), emptied of its protein enzymes, becomes a decapitated cytoplasmic body (DCB), a partly collapsed, faintly basophilic membrane with remnant cytoplasm. The DCB shrinks to form a sphere with a thickened, brightly eosinophilic surface casing, the eosinophilic body (EB). This structure may dissolve in luminal secretions, but it is also observed that it may adsorb to the surface of the corpus amylaceum (CA).
Unexpectedly, the present inventors have found that in prostatic adenocarcinomas, the entire secretory apparatus is substantially absent; neither PSG, DCB nor EB are found. An inability to form corpora amylacea arises from this fact. Prostate carcinomas may therefore be characterised by a significant reduction in levels, or absence, of any one of the structures associated with prostate secretions.
The present inventors have found that PSGs, EBs, DCBs and CAs may be readily visualised in normal prostate tissue which has been fixed in strong glutaraldehyde, or a substance which produces similar cytoplasmic fixation to that produced by strong glutaraldehyde.
Accordingly, in a first aspect the present invention provides a method of p
Marshall & Gerstein & Borun LLP
Uropath Pty Ltd., A.C.N.
Witz Jean C.
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